Discovery and Hit-to-Lead Optimization of Benzothiazole Scaffold-Based DNA Gyrase Inhibitors with Potent Activity against &lt;i&gt;Acinetobacter baumannii&lt;/i&gt; and &lt;i&gt;Pseudomonas aeruginosa&lt;/i&gt;

Davide Benedetto Tiz

pubs.acs.org/jmc Article Discovery and Hit-to-Lead Optimization of Benzothiazole ScaffoldBased DNA Gyrase Inhibitors with Potent Activity against Acinetobacter baumannii and Pseudomonas aeruginosa Andrej Emanuel Cotman, Martina Durcik, Davide Benedetto Tiz, Federica Fulgheri, Daniela Secci, Maša Sterle, Š tefan Možina, Ž iga Skok, Nace Zidar, Anamarija Zega, Janez Ilaš, Lucija Peterlin Mašič, Tihomir Tomašič, Diarmaid Hughes, Douglas L. Huseby, Sha Cao, Linnéa Garoff, Talía Berruga Fernández, Paraskevi Giachou, Lisa Crone, Ivailo Simoff, Richard Svensson, Bryndis Birnir, Sergiy V. Korol, Zhe Jin, Francisca Vicente, Maria C. Ramos, Mercedes de la Cruz, Björn Glinghammar, Lena Lenhammar, Sara R. Henderson, Julia E. A. Mundy, Anthony Maxwell, Clare E. M. Stevenson, David M. Lawson, Guido V. Janssen, Geert Jan Sterk, and Danijel Kikelj* Cite This: J. Med. Chem. 2023, 66, 1380−1425 ACCESS Metrics & More Read Online Article Recommendations sı Supporting Information * ABSTRACT: We have developed compounds with a promising activity against Acinetobacter baumannii and Pseudomonas aeruginosa, which are both on the WHO priority list of antibioticresistant bacteria. Starting from DNA gyrase inhibitor 1, we identified compound 27, featuring a 10-fold improved aqueous solubility, a 10-fold improved inhibition of topoisomerase IV from A. baumannii and P. aeruginosa, a 10-fold decreased inhibition of human topoisomerase IIα, and no cross-resistance to novobiocin. Cocrystal structures of 1 in complex with Escherichia coli GyrB24 and (S)-27 in complex with A. baumannii GyrB23 and P. aeruginosa GyrB24 revealed their binding to the ATP-binding pocket of the GyrB subunit. In further optimization steps, solubility, plasma free fraction, and other ADME properties of 27 were improved by finetuning of lipophilicity. In particular, analogs of 27 with retained anti-Gram-negative activity and improved plasma free fraction were identified. The series was found to be nongenotoxic, nonmutagenic, devoid of mitochondrial toxicity, and possessed no ion channel liabilities. validated antibacterial targets.5−7 Gyrase is a heterotetramer of GyrA and GyrB subunits (A2B2), while topo IV is a heterotetramer of ParC and ParE subunits (C2E2). Whereas fluoroquinolones, which target both gyrase and topo IV, are widely used in the clinic, inhibitors interfering with ATP binding to GyrB/ParE subunits have not found significant clinical application, despite intensive research efforts during the last 70 years since the discovery of novobiocin, the first ATPcompetitive GyrB inhibitor.8 Having worked for several years on the development of benzothiazole scaffold-based GyrB inhibitors,9−17 the intro- 1. INTRODUCTION Carbapenem-resistant Gram-negative bacteria Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacteriaceae, which include Escherichia coli and Klebsiella pneumoniae, are among the antibiotic-resistant bacteria on the WHO global priority list published in 2017 to guide research, discovery, and development of new antibiotics.1 These bacteria are all members of the ESKAPE group of six nosocomial pathogens, which also includes Gram-positive Staphylococcus aureus and Enteroccocus faecium, that exhibit multidrug resistance and high virulence. The increasing frequency of bacterial resistance against currently available antibiotics is a global problem that motivates the development of novel antibacterials with a distinct mode of action.2−4 Bacterial DNA gyrase and DNA topoisomerase IV (topo IV), both type II topoisomerases, responsible for the ATPdriven introduction of negative supercoils into DNA and ATPdriven supercoil relaxation and decatenation, respectively, are © 2023 The Authors. Published by American Chemical Society Received: September 30, 2022 Published: January 12, 2023 1380 https://doi.org/10.1021/acs.jmedchem.2c01597 J. Med. Chem. 2023, 66, 1380−1425 Journal of Medicinal Chemistry pubs.acs.org/jmc Article Figure 1. Evolution of a potent gyrase inhibitor 1 with antibacterial activity as a starting point for hit-to-lead optimization. Figure 2. Cocrystal structures of (a) compound 1 (in cyan sticks, PDB 7P2M) and (b) compound 7 (in magenta sticks, PDB 7P2W) in complex with E. coli GyrB24 (in gray cartoon). For clarity, only amino acid residues that interact with ligands are shown as sticks. Water molecules are presented as red spheres, and hydrogen bonds are shown as dashed black lines. duction of the carboxylic acid group to position 6 and attachment of a benzyloxy substituent to position 4 of the benzothiazole scaffold led us to the discovery of 4-(benzyloxy)2-(3,4-dichloro-5-methyl-1H-pyrrole-2-carboxamido)benzo[d]thiazole-6-carboxylic acid (1, Figure 1), a potent gyrase inhibitor with excellent in vitro and in vivo activities, demonstrated in murine dermal and thigh infection models, against Gram-positive S. aureus as well as against methicillinresistant- (MRSA) and vancomycin-resistant S. aureus (VISA).15 In this paper, we present the promising activity of compound 1 against the main Gram-negative ESKAPE pathogens A. baumannii, P. aeruginosa, and Escherichia coli and provide for it a solid biochemical rationale based on the inhibition of bacterial gyrase and topo IV. Further, we present our efforts to improve the ADMET properties of this frontrunner that resulted in the evolution of 1 to 2-(3,4dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4-(1-phenylethoxy)benzo[d]thiazole-6-carboxylic acid (27) displaying improved activity against ESKAPE pathogens. Finally, the optimization of 27 in several directions to compounds with improved solubility, free fraction, and other ADMET properties is presented. human topoisomerase IIα (IC50 = 1.95 μM). IC50 values for the inhibition of E. coli DNA gyrase-catalyzed supercoiling and topo IV-catalyzed relaxation of 1 measured in gel-based assays are consistent with those determined by high-throughput microtiter-plate-based assays. The compound does not stabilize the gyrase cleavage complex and does not show significant inhibition of ATP-independent relaxation (IC50 > 10 μM). A crystal structure of compound 1 in complex with E. coli GyrB24 (24 kDa amino-terminal subdomain of GyrB) obtained at a 1.16 Å resolution provides evidence of the inhibitor binding to the ATP-binding site of GyrB (Figure 2a). Compound 1 possesses excellent activity against the key Gramnegative pathogens (E. coli: MIC = 4 μg/mL; K. pneumoniae: MIC = 2 μg/mL; P. aeruginosa: MIC = 8 μg/mL; A. baumannii: MIC = 0.5 μg/mL) and also has encouraging activity against MDR P. aeruginosa and A. baumannii strains (MIC90 for A. baumannii = 2 μg/mL, MIC90 for P. aeruginosa = 8 μg/mL measured against 64 recent clinical isolates of each species) (Table S1). The compound is much less affected by efflux in A. baumannii than in the other Gram-negative species. Its frequency of resistance is lower than 10 × 10−10 at 4× and 8× MIC for the wild-type A. baumannii and for an effluxdefective strain of P. aeruginosa. Mutant analysis in efflux-defective P. aeruginosa PA0750 (MH-II liquid MIC < 0.125 μg/mL, MH-II agar MIC < 0.125 μg/mL) identified mutations in seven out of eight mutants selected and showed that only one mutant carried a mutation in the gyrB gene, which encodes for GyrB, a target of 1. Most other resistant mutants are probably associated with the induction of mechanisms to increase the efflux or reduce the influx of the compound. Sequencing of the gyrB gene in selected A. baumannii mutants revealed the mutation of Arg150, which is homologous to Arg136 in E. coli GyrB, to 2. RESULTS AND DISCUSSION 2.1. Frontrunner Compound 1 and Follow-up Compound 27. Compound 1 is a potent inhibitor of DNA supercoiling catalyzed by E. coli DNA gyrase [IC50 = 0.8 nM (gel-based assay), IC50 < 10 nM (microtiter-plate-based assay)], A. baumannii DNA gyrase (IC50 < 10 nM), and P. aeruginosa DNA gyrase (IC50 < 10 nM) as well as of DNA decatenation induced by E. coli topo IV (IC50 = 352 nM), A. baumannii topo IV (IC50 = 64 nM), and P. aeruginosa topo IV (IC50 = 235 nM) and shows selectivity against a related target 1381 https://doi.org/10.1021/acs.jmedchem.2c01597 J. Med. Chem. 2023, 66, 1380−1425 Journal of Medicinal Chemistry pubs.acs.org/jmc Article Figure 3. Plan for optimization of compound 1. cysteine (R150C) and histidine (R150H). Most likely, the two mutants selected in the wild type that had no mutations in gyrB will have mutations affecting the intracellular concentration of the compound. For the tested P. aeruginosa strain as well as for A. baumannii efflux-proficient and efflux-deficient strains, the MIC increase in selected mutants was very large, typically 64to >128-fold. The frontrunner compound 1 is bactericidal in K. pneumoniae (MBC = 1 μg/mL) but not in A. baumannii, in which it reduced the colony counts to <1% but not to <0.1%, the survivors having unchanged MIC. As shown in time-kill assays, 1 is bacteriostatic in A. baumannii. There was no killing of P. aeruginosa up to 8-fold MIC, whereby MICs increased significantly as the concentration of bacterial cells grew (MIC increased to 32 with 5 × 106 CFU/mL). Compound 1 was found to have low cytotoxicity in the HepG2 human liver cell line by LDH15 (IC50 > 100 μM), MTS18 (IC50 = 78 μM), and FMC19 (IC50 = 10.3 μM) assays, having no hERG, CaV1.2, and NaV1.5 ion channel liabilities at a 50 μM concentration (Figure S2) and possessing no in vitro mitochondrial toxicity in HepG2 cells at a 100 μM concentration (Figure S4). It did not show genotoxicity in a micronucleus test at concentrations up to 25 μM (Figure S6) and was found to be nonmutagenic against Salmonella typhimurium TA98 without or with S9 metabolic activation, as confirmed by a negative AMES test (Figure S9). The compound does not inhibit CYP3A4 (IC50 > 50 μM) (Figure S12), is stable in human (t1/2 = 71 min) and mouse hepatocytes (t1/2 = 132 min) as well as in human (t1/2 = 142 min) and mouse liver microsomes (t1/2 = 99 min) (Figure S13), and shows less than 1% hemolysis at a 100 μM concentration. However, despite the promising on-target and antibacterial properties of inhibitor 1, further development of this compound was hampered by its high lipophilicity (clog P = 5.8, clog D = 2.1) that resulted in low kinetic solubility (12 μM; 5.72 mg/L) and low thermodynamic solubility (6.6 μM; 3.14 mg/L). Furthermore, due to high plasma protein binding with less than 0.1% of free fraction in both human and mouse plasma, compound 1 lost its in vitro antibacterial activity against the target Gram-negative pathogens (MIC > 64 μg/ mL) in the presence of 50% human serum. Therefore, we started an intensive optimization campaign aiming at increasing the solubility and free fraction while retaining or improving the excellent antibacterial activity of the initial hit compound 1. 2.1.1. Optimization Plan of Compound 1. To establish the binding modes of novel gyrase inhibitors as a basis for structure-based optimization, we first solved cocrystal structures of compounds 1 and 716 in complex with the 24 kDa fragment of E. coli GyrB (GyrB24) at resolutions of 1.16 Å (PDB code 7P2M) and 1.65 Å (PDB code 7P2W), respectively. Both inhibitors are bound in the ATP-binding site of GyrB24 and form similar interactions with the protein residues (Figure 2). The pyrrole NH group is involved in a hydrogen bond with the Asp73 side chain, while the carboxamide oxygen interacts with the conserved water molecule and Thr165 side-chain hydroxyl group. The 3,4dichloro-5-methylpyrrole moiety forms several hydrophobic interactions with the residues in the lipophilic pocket of the enzyme, namely, Val43, Ala47, Val71, Ile78, Val120, and Val167. The benzothiazole scaffold forms a cation−π stacking interaction with the Arg76 side chain. In addition, a salt bridge is formed between the Arg136 side chain and the aromatic carboxylate group of inhibitors 1 and 7. Some differences were observed in the binding of the substituent at position 4 of the benzothiazole moiety. In 1, the nitrogen of the benzothiazole ring and the oxygen atom at position 4 form hydrogen bonds with a water molecule, while the phenyl ring has hydrophobic contacts with the side chain of Ile94. In 7, the amide nitrogen atom interacts with a water molecule, which is in contact with the Asn46 backbone carbonyl, while the 4-benzyloxy group forms hydrophobic interactions with Ile94 and Val118. The 3carboxylate group on the 4-benzyloxy moiety is not in direct contact with the protein but interacts with two water molecules bound to the Asp49 side chain. The described binding mode of 1 is very similar to the one reported by us for S. aureus GyrB24-1 complex (PDB code 6TCK).15 These and other crystal structures reported herein, together with docking to explore additional interactions with the target, have been used to rationally design the new compounds. Several chemical strategies were planned to increase the solubility and reduce plasma protein binding of the frontrunner 1. Replacement of the phenyl ring by aza-heterocycles or alkyl groups, introduction of polar substituents to the phenyl ring, as well as bioisosteric replacement of the carboxylic group, and modification of the pyrrole moiety were attempted to lower lipophilicity. Carboxamide N-alkylation was aimed to remove the hydrogen bond donor and explore the interaction of the substituents with the lipophilic floor. Finally, substituents were introduced at the benzylic position to increase solubility by disrupting the planarity of the molecules57,58 (Figure 3). 2.1.1.1. Phenyl Group Replacement and Substitution. Replacement of the phenyl ring of 1 with pyridine, pyrimidine, or N-methylpyridinium moiety (compounds 2−6) retained the 1382 https://doi.org/10.1021/acs.jmedchem.2c01597 J. Med. Chem. 2023, 66, 1380−1425 Journal of Medicinal Chemistry pubs.acs.org/jmc Article Table 1. IC50 Values against E. coli, A. baumannii, and P. aeruginosa DNA Gyrase and Topoisomerase IV, MICs, Solubility, and Free Fraction Data of Analogs of the Frontrunner 1 with Replaced or Substituted Phenyl Moiety a E. coli ATCC5922 (wild type), CH3130 (efflux-defective; ΔtolC-mutant isogenic to ATCC25922). bA. baumannii ATCC19606 (wild type), BM4652 (efflux-defective derivative of BM4454). cP. aeruginosa PAO1 (wild type), PAO750 (efflux-defective isogenic to PAO1). dKinetic solubility. eThermodynamic solubility. fGel-based assay. gThe results should be interpreted carefully due to low recovery; TPSA, total polar surface area; Calc., calculated; n.d., not determined; WT, wild type; Δeff., efflux-defective strain. log D values by one unit and introduction of the second nitrogen atom (compound 4) or quaternization (compounds 5 and 6) by an additional unit, the solubility remained low and all compounds were highly bound to plasma proteins (Table single-digit nanomolar DNA gyrase inhibitory activity, but the antibacterial activity was lost. Although the introduction of the first nitrogen atom into the phenyl ring (compounds 2 and 3) decreased the calculated 1383 https://doi.org/10.1021/acs.jmedchem.2c01597 J. Med. Chem. 2023, 66, 1380−1425 Journal of Medicinal Chemistry pubs.acs.org/jmc Article Table 2. IC50 Values against E. coli, A. baumannii, and P. aeruginosa DNA Gyrase and Topoisomerase IV, MICs, Solubility, and Free Fraction Data of the N-Alkylated Analogs a E. coli ATCC5922 (wild type), CH3130 (efflux-defective; ΔtolC-mutant isogenic to ATCC25922). bA. baumannii ATCC19606 (wild type), BM4652 (efflux-defective derivative of BM4454). cP. aeruginosa PAO1 (wild type), PAO750 (efflux-defective isogenic to PAO1). dKinetic solubility. eGel-based assay. fThe results should be interpreted carefully due to low recovery; TPSA, total polar surface area; Calc., calculated; n.d., not determined; WT, wild type; Δeff., efflux-defective strain. 1). We have shown earlier that the substitution of the phenyl ring of 1 with 4-carboxy or 3-carboxy group (compound 7) retains good on-target activity, but again, this modification was detrimental for antibacterial activity against E. coli, A. baumannii, and P. aeruginosa.16 Whereas the same trend was observed also for 4-methylsulfonyl compound 8, the 3-fluoro derivative 916 possessed good on-target activity and activity against A. baumannii (MIC = 4 μg/mL) but poor physicochemical and ADME properties. Replacing the phenyl group of 1 with aminomethyl and morpholinomethyl moieties16 as well as with methoxymethyl moiety in 10 and 1-(dimethylamino)ethyl moiety in 11 was also detrimental for antibacterial activity although the inhibition of gyrase and topo IV was well retained. On the contrary, the gyrase inhibitory activity of 4-(2,2,2-trifluoroethoxy) analog 12 was translated to antibacterial activity, which was most pronounced in A. baumannii with an MIC of 8 μg/mL (Table 1). Compounds 2−12 were all active against the efflux-defective strain of E. coli with MIC ≤ 8 μg/mL, and compounds 2, 3, 9, 10, and 12 were active (MIC ≤64 μg/mL) against efflux-defective P. aeruginosa. This suggests that efflux is a major contributing factor to inactivity against wild-type Gram-negative bacteria. Compounds 2, 3, and 9−12 were all active against wild-type S. aureus (MIC values of all tested compounds are listed in Table S10). 2.1.1.2. Bioisosteric Replacement of the Carboxyl Group. The replacement of a carboxylic acid with an isosteric group to improve the physicochemical properties of a compound, while maintaining the features critical for biological activity, is a wellknown medicinal chemistry strategy.20 We have successfully used 5-oxo-4,5-dihydro-1,3,4-oxadiazol-2-yl,21 5-thioxo-4,5-dihydro-1,3,4-oxadiazol-2-yl,22 and 5-oxo-4,5-dihydro-1H-tetrazol-1-yl22 moieties as carboxyl group surrogates in Nphenylpyrrolamide gyrase inhibitors, and, following the same strategy, the 1H-tetrazol-5-yl (13),16 5-oxo-4,5-dihydro-1,3,4oxadiazol-2-yl (14), and hydrazide (15) derivatives of 1 were prepared. Compounds 13−15 inhibited DNA gyrase but 1384 https://doi.org/10.1021/acs.jmedchem.2c01597 J. Med. Chem. 2023, 66, 1380−1425 Journal of Medicinal Chemistry pubs.acs.org/jmc exhibited weak or no activity against E. coli, A. baumannii, and P. aeruginosa (Supporting Information Table S3). Article carboxamide N-methyl compounds 18, 20, and 26, it can be concluded that a substituent in position 4 does not significantly affect gyrase inhibition but the 4-hydroxyl group is detrimental to antibacterial activity (Table 2). 2.1.1.5. Substitution at the Benzylic Position. Substitution at the benzylic position to disrupt molecular planarity and symmetry has been applied for solubility improvement in several medicinal chemistry optimization programs.24−29 Introduction of a methyl group at the benzylic position of 1 resulted in compound 27. 2.1.1.3. Modification of Pyrrole Substitution. As revealed by the crystal structure of an E. coli GyrB24-1 complex (PDB code 7P2M), a lipophilic pocket that binds the pyrrole 5methyl group would allow lengthening it to an aminomethyl moiety that could interact with a negatively charged area and possibly increase the binding affinity. This modification is in line with the recent assumption that small molecules that are most likely to accumulate in Gram-negative bacteria contain an amine, are amphiphilic and rigid, and have low globularity.23 Guided by these observations and principles, the 5-aminomethylpyrrole derivative 1636 was prepared. Although it was a strong inhibitor of E. coli gyrase (IC50 < 10 nm), its IC50 values for the inhibition of A. baumannii and P. aeruginosa gyrase were only in the micromolar range, and the compound was inactive against the tested Gram-negative bacteria. The methyl ester derivative 17 was devoid of on-target and antibacterial activity, indicating the importance of the carboxylic group in position 6. Notably, the free fraction of 16 in human serum was increased to 0.59% (Supporting Information Table S4). Compound 27 possessed at least 7-fold better kinetic solubility (>80 μM; >39 mg/L) and 14-fold better thermodynamic solubility (92 μM = 45.3 mg/L). Compound 27 was an excellent inhibitor of gyrase from E. coli (IC50 < 10 nM), A. baumannii (IC50 = 15.6 nM), and P. aeruginosa (IC50 < 10 nM) and also inhibited topo IV from E. coli (IC50 = 320 nM), A. baumannii (IC50 < 10 nM), and P. aeruginosa (IC50 = 29 nM). Similarly to 1, compound 27 does not induce cleavage complex formation (IC50 > 100 μM) and very weakly inhibits ATP-independent DNA relaxation (IC 50 = 87 μM). Importantly, compared to 1, compound 27 shows a 12-fold better selectivity against a related human target topoisomerase IIα (IC50 = 25 μM). Overall, compound 27 possesses improved antibacterial activity compared to compound 1 (Table 3). Whereas there is no effluxing of 27 in A. baumannii, efflux occurs in P. aeruginosa but to a smaller extent than for 1. While retaining excellent in vitro activity against A. baumannii, compound 27 also displayed improved activity against P. aeruginosa, possibly due to a 10-fold stronger inhibition of topo 2.1.1.4. N-Alkylation of Carboxamide or Thiazole. Inspection of the crystal structure of 1 in complex with E. coli GyrB24 (PDB code 7P2M) revealed that the carboxamide NH group is not involved in hydrogen bonding and the alkyl group attached to it would point toward the lipophilic floor of the ATP-binding site (Figure 2a) and thus possibly contribute to binding. Bearing in mind that carboxamide N-alkylation would also distort the planarity of the carboxamide group and thus possibly increase the solubility, compounds 18, an Nmethyl derivative of 1, and 19, an N-methyl derivative of 16, were prepared. Whereas strong inhibition of gyrase was kept, the weaker inhibition of topo IV and solid in vitro activity against E. coli, A. baumannii, and P. aeruginosa were retained in 18 and the solubility and free fraction remained low. The amino derivative 19 was a weaker inhibitor of both gyrase and topo IV; it was devoid of antibacterial activity and possessed very low kinetic solubility. Because the carboxamide and benzothiazole N-substituents were expected to interact with the lipophilic floor similarly to the groups bound to oxygen in position 4, we tested the effect of moving a substituent from O4 to the carboxamide N-atom (compound 20) or removing the alkoxy substituent in position 4 and attaching its alkyl part to the thiazole (compounds 22−25) or carboxamide N-atom (compounds 21, 26). All resulting compounds except 21 were low nanomolar inhibitors of E. coli, A. baumannii, and P. aeruginosa gyrase, but only 26 was active against the three pathogens. Compound 26 is also less bound to proteins in human and mouse plasma. From a comparison of the Table 3. Comparison of On-Target and Antibacterial Activities of Compounds 1 and 27a enzyme Cpd. 1 IC50 [nM] E. coli gyrase E. coli topo IV A. baumannii gyrase A. baumannii topo IV P. aeruginosa gyrase P. aeruginosa topo IV human topoisomerase IIα pathogen, strain ID (phenotype) E. coli, ATTC25922 (WT) E. coli, CH3130 (efflux-def.) K. pneumoniae, ATCC13883 (WT) K. pneumoniae, 1161486a (efflux-def.) P. aeruginosa, PAO1 (WT) P. aeruginosa, PAO750 (efflux-def.) A. baumannii, ATCC19606 (WT) A. baumannii, BM4454 (clinical MDR) A. baumannii, BM4652 (efflux-def.) a 1385 Cpd. 27 IC50 [nM] <10 350 <10 64 <10 235 1950 Cpd. 1 MIC [μg/mL] 4 <0.125 2 n.d. 8 <0.125 0.5 <0.125 <0.5 <10 320 16 6.7 <10 29 25,000 Cpd. 27 MIC [μg/mL] 1 <0.125 1 n.d. 1 <0.125 0.5 0.5 0.5 n.d., not determined; WT, wild type; MDR, multidrug-resistant. https://doi.org/10.1021/acs.jmedchem.2c01597 J. Med. Chem. 2023, 66, 1380−1425 Journal of Medicinal Chemistry pubs.acs.org/jmc 65 2 4 1 4 8 4 8 >64 8 8 64 8 8 16 >64 >64 1 1 2 2 2 2 2 >64 4 2 4 2 4 2 >64 >64 51 48 2 1 1 2 2 2 4 >64 4 4 8 2 4 2 >64 >64 1 1 1 1 1 1 4 >64 2 1 4 1 2 2 >64 >64 47 46 2 2 0.5 2 1 2 4 >64 8 8 32 8 8 8 >64 >64 4 2 0.5 2 2 2 4 >64 4 4 8 2 4 2 >64 64 36 35 4 4 0.25 2 2 1 4 >64 8 8 16 8 8 16 >64 >64 8 8 0.125 2 2 2 1 >64 8 8 16 16 8 16 >64 >64 34 31 1 0.5 0.125 0.5 1 0.5 1 >64 1 1 2 0.5 1 1 >64 >64 16 8 0.125 8 64 4 16 >64 16 >64 32 32 16 64 >64 >64 30 27 1 0.5 0.5 1 1 1 1 8 1 1 2 1 1 1 >64 >64 WT WT efflux-defective clinical MDR clinical MDR clinical MDR clinical MDR gyrB R150C clinical MDR clinical MDR clinical MDR clinical MDR clinical MDR clinical MDR gyrB R138C gyrB R136H genotype/phenotype EN7 EN16 EN17 EN273 EN274 EN276 EN277 CH8730 EN0146 EN0147 EN0148 EN0149 EN0340 EN0343 CH9393 CH9389 original no. species A. baumannii A. baumannii A. baumannii A. baumannii A. baumannii A. baumannii A. baumannii A. baumannii P. aeruginosa P. aeruginosa P. aeruginosa P. aeruginosa P. aeruginosa P. aeruginosa P. aeruginosa E. coli strain ID Table 4. MIC Values of Compound 27 and Selected Derivatives against Some Multiresistant Strains of A. baumannii and P. aeruginosa 1386 ATCC19606 BM4454 BM4652 NMI 2692/14 NMI 2699/14 NMI 2704/14 NMI 2715/14 CH8730 NMI 3793/07 NMI 2500/14 RYC 03131390 NMI 2620/03 3947/14 3957/14 CH9393 CH9389 IV and more balanced inhibition of both DNA gyrase and topo IV (Table 3). Compound 27 has a very good activity against MDR A. baumannii strains (MIC90 = 2 μg/mL, based on broth microdilution assay with 61 clinical isolates; Table S2) and seven selected P. aeruginosa clinical isolates (Table 4). However, in the presence of 50% human serum, 27 lost its activity (MIC > 64 μg/mL) against E. coli, P. aeruginosa, and A. baumannii. The frequency of resistance of 27 in A. baumannii and E. coli remained low, beyond the detection limit; but it was higher in wild-type P. aeruginosa (4.1 × 10−8 at 8 × MIC) where the selected mutants were resistant to 27 with MIC increased between 4- and >16-fold compared to the parental. On the contrary, no resistant clones were isolated in the frequency of resistance studies of 27 in A. baumannii. Like 1, compound 27 is bacteriostatic against A. baumannii, but an important difference was observed between both compounds in P. aeruginosa activity. Whereas for 1 there was no killing of P. aeruginosa up to 8-fold MIC, compound 27 was bacteriostatic with an MIC of 2 μg/mL. An important difference between the original hit 1 and compound 27 was observed also in the dependence of MICs on the inoculum size. Whereas in 1 MICs against P. aeruginosa increased significantly, up to 32 μg/mL, as bacterial cell concentration increased, compound 27 did not show any MIC dependence on the inoculum size in P. aeruginosa.59 Insensitivity of 27 and derivatives (see below) to the inoculum effect against P. aeruginosa and its excellent MICs against multidrug-resistant P. aeruginosa (Table 4) are a significant improvement over the initial hit 1. Compound 27 possessed an IC50 of 19.7 μM (20-fold higher than MIC) in a HepG2 fluorometric microculture cytotoxicity assay (FMCA)19 and was thus less cytotoxic than the initial hit 1 (IC50 = 10.3 μM). It had no hERG, CaV1.2, and NaV1.5 ion channel liabilities at a 10 μM concentration (Figure S3) and was neither genotoxic nor mutagenic in both the absence or presence of S9 metabolic activation, as confirmed by micronucleus (Figure S8) and AMES tests (Figure S10). No mitochondrial toxicity of 27 could be observed in HepG2 cells in vitro (Figure S5). Compound 27 is stable in human (t1/2 = 92 min) and mouse hepatocytes (t1/2 = 45 min) and gives rise to less than 1% hemolysis in 1 h at 100 μM. Both stereoisomers of compound 27 were synthesized, and the enantiomer (S)-27 (74% ee) was found more active than (R)-27 (62% ee) against wild-type E. coli, A. baumannii, and P. aeruginosa strains. Interestingly, compound 27 was the only compound in the whole series showing activity against the novobiocin-resistant A. baumannii mutant GyrB R150C (MIC = 8 μg/mL), which resides mainly in the (S)-enantiomer, as confirmed by several independent experiments. On the other hand, 27 was not active against novobiocin-resistant E. coli GyrB R136H mutant and P. aeruginosa GyrB R138C mutants. Comparing the inhibition of gyrase and topo IV, compound 27 as well as its two enantiomers are better inhibitors of gyrase in E. coli and P. aeruginosa, whereas in A. baumannii, the inhibition of topo IV is better. To aid the development of GyrB inhibitors for the treatment of A. baumannii and P. aeruginosa infections, we solved the crystal structures of A. baumannii GyrB23 in complex with the racemic inhibitor 27 (PDB code 7PQL, resolution 1.60 Å) and in complex with its enantiomer (S)-27 (PDB code 7PQM, resolution 1.55 Å) as well as the crystal structure of (S)-27 in complex with P. aeruginosa GyrB24 (PDB code 7PTG) (Figure Article https://doi.org/10.1021/acs.jmedchem.2c01597 J. Med. Chem. 2023, 66, 1380−1425 Journal of Medicinal Chemistry pubs.acs.org/jmc Article Figure 4. (a) Cocrystal structure of the inhibitor 27 [(S)-enantiomer in yellow sticks, (R)-enantiomer in gray sticks] in complex with A. baumannii GyrB23 (in cyan cartoon) (PDB 7PQL, 7PQM). (b) Cocrystal structure of (S)-27 (in yellow sticks) in complex with P. aeruginosa GyrB24 (in green cartoon) (PDB 7PTG). For clarity, only amino acid residues that interact with inhibitor are shown as sticks. Water molecules are presented as red spheres, and hydrogen bonds are shown as dashed black lines. 4). Further on, the crystal structures of A. baumannii GyrB23 and P. aeruginosa GyrB24 subdomains in complex with novobiocin (PDB codes 7PQI and 7PTF) were solved for the first time (Supporting Information Figure S1). This structural information is important because so far there have been no crystal structures of A. baumannii GyrB complexes with small molecules in the PDB, and for P. aeruginosa GyrB only the structures of two complexes (PDB codes 6M1S and 6M1J) have been reported recently.30 The racemic inhibitor 27 was modeled in the electron density of its A. baumannii GyrB23 complex in the (R)- and (S)-configurations with occupancies of 40 and 60%, respectively. The observed binding modes of the (S)configurated inhibitor in 7PQL and (S)-27 in 7PQM were exactly the same, as expected. They formed similar interactions that are characteristic of this structural class of compounds, namely, hydrogen bonds with Asp87 and a conserved water molecule, Thr179 and Arg150, and cation−π interaction with Arg90. Hydrophobic interactions were formed between the pyrrole moiety and Val57, Ala61, Val85, Val181, and Val134, while the hydrophobic α-methylbenzyl group at position 4 of the benzothiazole core interacted with Ile92, Ala104, and Ile108 (Figure 4a). In the crystal structure of (S)-27 in complex with P. aeruginosa GyrB24 (Figure 4b; PDB code 7PTG), the observed inhibitor binding mode is very similar to the one in A. baumannii GyrB23 ATP-binding site with hydrogen bonds to Asp75, conserved water molecule, Thr167 and Arg138, cation−π stacking with Arg78 and the 4-(α-methyl)-benzyloxy moiety hydrophobic contacts with Ile80 and Ile96 side chains. The pyrrolamide moiety is bound in the lipophilic pocket and interacts with Val43, Val73, Met97, Val122, and Val169. The binding modes of novobiocin in the A. baumannii GyrB23 complex 7PQI and in the P. aeruginosa GyrB24 complex 7PTF closely resemble that of E. coli and S. aureus GyrB-novobiocin complexes 1AJ6 and 4URO, respectively. Important interactions are hydrogen bonds formed with Asp87 in A. baumannii (Asp73 in E. coli, Asp75 in P. aeruginosa), the conserved water molecule, Asn60 in A. baumannii (Asn46 in E. coli, Asn48 in P. aeruginosa), Gly 95 in A. baumannii (Gly81 in E. coli, Asp83 in P. aeruginosa), and Arg150 in A. baumannii (Arg136 in E. coli, Asp138 in P. aeruginosa) as well as cation−π stacking with Arg90 in A. baumannii (Arg76 in E. coli, Arg78 in P. aeruginosa). 2.1.2. Optimization of the Follow-Up Compound 27. Improved antibacterial activity and partly ameliorated ADMET properties of the branched derivative 27 in comparison to the original hit 1 stimulated us to further optimize compound 27 toward more soluble and less plasma protein-bound analogs using devised strategies. This resulted in several derivatives, which retained activity against Gram-negative bacteria (Table 5, Tables S5−S9). 2.1.2.1. Introduction of Polar Substituents to the Stereogenic Carbon Atom. To test the effect of increasing the polarity and size of the substituents attached to the stereogenic carbon atom, the compounds compiled in Table S5 were prepared. Replacement of the methyl by a trifluoromethyl group to give 28 slightly reduced the activity against E. coli and A. baumannii, whereas the activity against wild-type P. aeruginosa was lost. Compound 29 bearing a benzocyclobutyl substituent retained excellent activity against A. baumannii (MIC = 1 μg/mL) but was inactive (MIC > 64 μg/mL) against E. coli and P. aeruginosa. Notably, the antibacterial activity against A. baumannii, P. aeruginosa, and E. coli was preserved in compounds 30 and 31 containing oxysubstituents as well as in compounds 34−36 with tertiary amine substituents at the methylene group. The aminomethyl compound 33, its quaternized derivative 37, the N-acetylamino compound 38, and the triazolomethyl derivative 40 were inactive, whereas the N-Boc compound 39 possessed activity against the three Gram-negative bacteria. Whereas the piperidinomethyl and morpholinomethyl compounds 35 and 36 displayed good antibacterial activity, the introduction of the second basic center or substitution in position 4 of piperazine ring (compounds 41−44) was detrimental for antibacterial activity as was quaternization of the amino group (compound 45). Whereas good antibacterial activity of 30, 31, 34−36, and 39 went hand in hand with good inhibition of gyrase and topo IV, the absence of antibacterial activity of the other compounds in Table S5 could not be attributed to lower or absence of on-target activity but rather to efflux issues. All active compounds retained good activity also against multidrug-resistant A. baumannii and P. aeruginosa strains, with compounds 31 and 36 showing the best performance against MDR strains (Table 4). Although for the five active compounds 30, 31, and 34−36 TPSA was increased by 3.2− 20.2 Å2 and calculated log P values were decreased by 0.4−3.2 1387 https://doi.org/10.1021/acs.jmedchem.2c01597 J. Med. Chem. 2023, 66, 1380−1425 Journal of Medicinal Chemistry pubs.acs.org/jmc Article Table 5. IC50 Values against E. coli, A. baumannii, and P. aeruginosa DNA Gyrase and Topoisomerase IV, MICs, Solubility, and Free Fraction Data of Derivatives of Compound 27, Active against Gram-Negative Bacteria 1388 https://doi.org/10.1021/acs.jmedchem.2c01597 J. Med. Chem. 2023, 66, 1380−1425 Journal of Medicinal Chemistry pubs.acs.org/jmc Article Table 5. continued a E. coli ATCC5922 (wild type), CH3130 (efflux-defective; ΔtolC-mutant isogenic to ATCC25922). bA. baumannii ATCC19606 (wild type), BM4652 (efflux-defective derivative of BM4454). cP. aeruginosa PAO1 (wild type), PAO750 (efflux-defective isogenic to PAO1). dKinetic solubility. eThermodynamic solubility. fGel-based assay. gThe results should be interpreted carefully due to low recovery; TPSA, total polar surface area; Calc., calculated; n.d., not determined; WT, wild type; Δeff., efflux-defective strain. 2.1.2.3. Variation of the Pyrrole Ring Substitution. 3,4Dichloro-5-methyl-1H-pyrrole-2-carboxamide moiety of 27 is a major contributor to the compound’s high lipophilicity. Therefore, an array of analogs featuring less lipophilic pyrroles were prepared (Table S7). Removal of chlorine in the pyrrole position 3 of 27 resulted in the substantial loss of antibacterial activity (compound 50), which was regained to a great extent by replacement of the chlorine in 50 by fluorine to give 51. The latter also possessed good activity against clinical multidrug-resistant strains of A. baumannii and P. aeruginosa (Table 4). Because the gyrase and topo IV inhibitory activities of 50 and 51 remained comparable to those of compound 27, a more intensive efflux, poor membrane permeability, and/or unknown off-target activity31 could be responsible for a weaker antibacterial activity of compound 50. On the contrary, the replacement of chlorine in 50 with a cyano group (compound 52) or hydrogen atom (compound 53) further impaired the antibacterial activity (IC50 > 64 μg/mL). Between the two enantiomers of 51, (S)-51 (82% ee) possessed better activity against P. aeruginosa and A. baumannii, possibly due to 10- to 15-fold stronger inhibition of topo IV from both species than (R)-51 (>95% ee). The results for compound (S)-51 are units, the solubility was not improved significantly and the compounds remained almost completely bound to the human and mouse plasma proteins. Compounds 28−45, including quaternary amines, were all active against wild-type S. aureus (MIC < 0.125−64 μg/mL) (Table S10). 2.1.2.2. Phenyl Group Replacements. The contribution of the phenyl substituent to lipophilicity of 27 in terms of clog P is almost 1.5; therefore, its replacement with less lipophilic smaller moieties appeared as a promising approach for the improvement of solubility and plasma protein binding (Table S6). Small alkyl moieties such as methyl (compound 46), isopropyl (compound 47), and cyclopropyl (compound 48) were found to be good phenyl surrogates regarding gyrase inhibition and activity against E. coli, A. baumannii, and P. aeruginosa. However, in comparison to 27, the inhibition of A. baumannii and P. aeruginosa topo IV was decreased. Unfortunately, the lower-calculated log P values of 46−48 did not result in solubility or free fraction improvement. Compound 49 with tetrahydrofuran-3-yl substituent replacing the 1-phenylethyl moiety in 27 retained a single-digit nanomolar inhibition of gyrase but its antibacterial activity against Gram-negative pathogens was almost lost (Table S6). 1389 https://doi.org/10.1021/acs.jmedchem.2c01597 J. Med. Chem. 2023, 66, 1380−1425 Journal of Medicinal Chemistry pubs.acs.org/jmc particularly interesting if considered for development against A. baumannii only. The activity is still good, and the solubility/ free fraction was improved relative to 1. As evident from Table S7, moving the fluorine atom in 51 to position 3 to give compound 54 was again detrimental to antibacterial activity, most likely due to weaker inhibition of A. baumannii and P. aeruginosa topo IV. Finally, the elongation of the pyrrole 5methyl group in 27 to aminomethyl substituent (compound 55) was also detrimental to activity against E. coli, A. baumannii, and P. aeruginosa. The drop in antibacterial activity was more pronounced than in compound 16; however, the introduction of 5-aminomethyl group increased the free fraction of 55 in human plasma to 1.28%. 2.1.2.4. Combined Approaches. The modifications of compound 27 that included a combination of favorable substitution at the stereogenic carbon (see Section 2.1.2.1), phenyl group replacement (see Section 2.1.2.2), and variation of the pyrrole substituents (see Section 2.1.2.3) were also carried out (Table S8). A combination of morpholinomethyl moiety and methyl as a phenyl surrogate (compound 56) was detrimental to antibacterial activity, but the trifluoromethyl analog 57 retained antibacterial activity against A. baumannii (wild type: MIC = 8 μg/mL; clinical MDR strains: IC50 = 8− 16 μg/mL) and was found inactive (MIC > 64 μg/mL) against E. coli and P. aeruginosa. A combination of morpholinomethyl moiety and cyclopropyl group as a phenyl surrogate afforded compound 58. It possessed improved kinetic solubility (57 μM) and plasma protein binding properties (free fraction 0.3 and 1.3% in human and mouse plasma, respectively), retained single-digit nanomolar inhibition of E. coli, A. baumannii, and P. aeruginosa gyrase, and inhibited topo IV of the three species in the 100 nM range. Compound 58 retained activity against wild-type strains of E. coli (MIC = 16 μg/mL) and A. baumannii (MIC = 64 μg/mL) and only against the effluxdefective strain of P. aeruginosa. The excellent antibacterial activity of 36 with morpholinomethyl and phenyl substituents at the branching point decreased or disappeared after the removal of chlorine in position 3 without or with the concomitant variation of halogen in position 4 (59: R = Cl; 60: R = F), although no substantial changes in either gyrase or topo IV inhibition were observed. Notably, compound 60 showed promisingly increased free fraction in human and mouse plasma of 1.5 and 4.4%, respectively. It also possessed improved kinetic solubility and retained borderline antibacterial activity (MIC = 64 μg/mL) against wild-type P. aeruginosa and E. coli. Similarly, the removal of chlorine in position 3 of antibacterially active compound 46 and replacement of chlorine in position 4 with fluorine (compound 61) or cyano group (compound 62) were detrimental to the activity against E. coli, A. baumannii, and P. aeruginosa although the IC50 values of gyrase inhibition were still in the 10−150 nM range. The replacement of 3,4-dichloro-5-methylpyrrole moiety of 46 with 2-methyl-4-chloroimidazole in 63 further lowered log D but gyrase inhibition and antibacterial activity were drastically reduced. 2.1.2.5. Introduction of para Substituents to the Phenyl Ring. 4-Methylsulfonybenzyl derivative 64, although possessing only weak antibacterial activity, suggested that the para substitution of the phenyl ring could be tolerated in the derivatives of compound 27. After inspection of the crystal structures of 27 in complex with A. baumannii GyrB23 (PDB codes 7PQL, 7PQM) and P. aeruginosa GyrB24 (PDB code 7PTG), the 4-morpholinomethyl derivative 65 was prepared Article and found to possess good antibacterial activity against E. coli (MIC = 4 μg/mL), A. baumannii (MIC = 2 μg/mL), and P. aeruginosa (MIC = 8 μg/mL) as well as against some clinical MDR A. baumannii (MIC = 4−8 μg/mL) and P. aeruginosa strains (MIC = 8−64 μg/mL) (Table 4). Its efflux from A. baumannii and P. aeruginosa was much less pronounced than from E. coli. In an attempt to improve the antibacterial activity, solubility, and free fraction, p-morpholinomethyl substitution of the phenyl ring was first combined with different pyrrole substitution patterns. Unfortunately, modifications in the pyrrole ring of 65, including the removal of the chlorine in position 3 to give compound 66 and its replacement with fluorine in 67 or cyano group in 68, reduced antibacterial activity, mostly due to efflux issues. The morpholine moiety of 65 was then replaced by piperazine (69, 70), 1-oxothiomorpholine (71), 1,1-dioxothiomorpholine (72), and 3-oxomorpholine (73), but again all of these variations resulted in the efflux-related reduction or loss of antibacterial activity against E. coli, A. baumannii, and P. aeruginosa. Notably, compounds 64−66, 72, and 73 retained activity against wild-type E. coli (Table S9) and compounds 64−68, 72, and 73 were active against wild-type S. aureus (Table S10). In comparison to 27, compounds bearing the morpholinomethyl moiety and its surrogates in the para position of the phenyl ring retained or slightly increased the inhibition of gyrase from A. baumannii and P. aeruginosa but were weaker inhibitors of topo IV. The inhibition of both E. coli enzymes followed the opposite trend with weaker DNA gyrase inhibition and stronger inhibition of topo IV. To our satisfaction, the free fraction was substantially increased for compounds 64 (mouse: 3.25%), 67 (human: 0.48%; mouse: 1.7%), and 68 (human: 1.7%; mouse: 5.0%), and for compound 71 the thermodynamic solubility was strongly increased to 2760 μmol/L. These results indicate that increasing solubility and free fraction to acceptable levels is possible within the p-morpholinomethyl structural class, although combining good solubility and free fraction with the desired antibacterial activity against P. aeruginosa and A. baumannii remains a challenge. 2.2. Physicochemical and ADMET Properties. 2.2.1. pKa and log D. As a part of compound 27 profiling, its pKa (pKa1 = 4.30 ± 0.10; pKa2 = 8.20 ± 0.01) and log D (4.28 at pH 7.4) were determined. Because the substitution adjacent to the 6-carboxylic acid group was not varied, it can be assumed that also other analogs possess a similar pKa1 value (e.g., compound 59 has experimentally determined pKa1 3.94 ± 0.02 and pKa2 6.38.20 ± 0.06). The measured log D (pH 7.4) for the further 10 compounds was between 1.5 and 4.56 (Table 6). The effect of pyrrole substituents on log D in αmethyl compounds 27, 50−53, and 55, and in the αmorpholinomethyl series (compounds 36, 59, and 60) was examined in more detail. A clear trend was observed that, surprisingly, 3,4-dichloro derivatives are less lipophilic than the respective 4-monochloro derivatives (cf. compounds 27 and 50 in the α-methyl series and compounds 36 and 59 in the αmorpholinomethyl series). The decrease of log D by 1.6 due to the introduction of a morpholinomethyl group to the stereogenic carbon atom is evident by comparing pairs of compounds 27 and 36, 50 and 59 as well as 51 and 60. Introduction of a morpholinomethyl moiety to the para position of the phenyl ring of 52 to obtain 68 decreased the log D value by 0.47. Whereas the decrease in log D by the fluoro and cyano substitutions of the pyrrole ring is well evident, the decrease in log D of 27 coincided with good 1390 https://doi.org/10.1021/acs.jmedchem.2c01597 J. Med. Chem. 2023, 66, 1380−1425 Journal of Medicinal Chemistry pubs.acs.org/jmc Article compounds. The kinetic and thermodynamic solubility data are reported in Tables 1, 2, 5, and S3−S9 and discussed there in relation to the chemical structure of the compounds. Solubility data for most representative compounds with activity against A. baumannii, together with the fraction of compounds not bound to plasma proteins, are compiled in Table 7. Among them, compound 71 has the best kinetic and thermodynamic solubility but, unfortunately, it did not possess significant antibacterial activity. 2.2.3. Plasma Protein Binding. Increasing the fraction of compounds not bound to plasma proteins while maintaining their activity against A. baumannii and P. aeruginosa was the main objective of structural modification of the initial hit 1. Chemical strategies to reduce plasma protein binding included decreasing the lipophilicity, increasing the polar surface area and, in some cases, increasing the basicity of compounds. As demonstrated in Table S11, the free fraction was increased to a few percent in several derivatives of compounds 1 and 27, which all displayed low nanomolar inhibition of gyrase and topo IV of E. coli, A. baumannii, and P. aeruginosa but were, unfortunately, devoid of antibacterial activity. For instance, the comparison of 27 with compounds 32 and 64, both devoid of antibacterial activity but giving a decent free fraction in mouse plasma, demonstrates that the methylsulfonyl group increased the free fraction in mouse plasma by a few percent irrespective of its attachment position. With compounds 27, 36, and 51, which possessed antibacterial activity, we succeeded in decreasing plasma protein binding to some extent but it remained lower than 1%. It seems that there is a narrow window of physicochemical properties within the structural class of our gyrase inhibitors, which is compatible with good permeability and low efflux rate in bacteria as well as with acceptable solubility and plasma protein binding in human plasma. Such properties have not been reached with the compounds synthesized so far, and reaching them would require a comprehensive multiparameter optimization approach. 2.2.4. Genotoxicity and Mutagenic Potential. The initial hit 1 with a 97% purity was found to be genotoxic and able to induce chromosome breaks at a 20 μM concentration in in vitro cell micronucleus test (MNT). The hydrolyzed aromatic amine precursor, 2-amino-4-(phenoxymethyl)benzo[d]thiazole-6-carboxylic acid, which was the main impurity (1.5%) in the final product, was deemed responsible for the genotoxicity of 1, but surprisingly it was found nongenotoxic in the MNT assay at a 500 μM concentration. However, 98.8% pure compound 1 did not show genotoxicity at concentrations up to 25 μM in the micronucleus test, which led to the conclusion that the observed genotoxicity of 1 very likely originated from another unidentified compound present in the Table 6. Experimental log D (pH 7.4) Values of Representative Compounds antibacterial activity only in compound 51 in the α-methyl series and in 36 in the α-morpholinomethyl series. 2.2.2. Kinetic and Thermodynamic Solubility. The kinetic solubility of all final compounds was routinely assessed, and for compounds possessing promising antibacterial activity also thermodynamic solubility was measured. In the kinetic solubility assay, a small volume of DMSO solution of the test compound was added to the aqueous buffer whereupon after an incubation period the concentration was determined before equilibrium was reached.32 The kinetic solubility assay mimics the conditions of in vitro biological tests, and their purpose was to identify compounds that do not have good kinetic solubility even in aqueous buffer containing DMSO, and the results obtained were used to guide the modification of structures to improve solubility. For the determination of thermodynamic solubility that varies with the crystal form of the compound, water or buffer was added directly to solid crystalline material and solubility was measured after equilibrium was established between the dissolved and solid Table 7. Kinetic and Thermodynamic Solubility Data of Representative Compounds with Antibacterial Activity free fraction [%] Cpd. log D Calc. log P Calc. kinetic solub. [μM] thermodyn. solub. [μM] human mouse 1 27 36 51 59 65 71 2.1 2.59 2.26 1.52 1.66 2.56 1.12 5.8 5.94 2.74 4.87 2.14 2.74 2.21 12 >80 35 41 20 30 59 6.6 92.4 3 18 70 25 2760 <0.1 0.016 <0.1 0.13 <0.1 <0.1 0.11 <0.1 0.01 0.3 0.3 0.5 <0.1 0.2 1391 https://doi.org/10.1021/acs.jmedchem.2c01597 J. Med. Chem. 2023, 66, 1380−1425 Journal of Medicinal Chemistry pubs.acs.org/jmc remaining 1.5% impurities. Compound 1 was also found to be nonmutagenic up to 25 μM in the absence or presence of S9 metabolic activation against S. typhimurium TA98, as confirmed by the AMES test (Figure S9). Compound 18, a carboxamide N-methyl derivative of 1, was also found to be nongenotoxic at 37 μM in the MNT assay. (Figure S7), suggesting that the carboxamide N-alkylation could be a viable strategy for structure modification. Compound 27 was found nongenotoxic up to 75 μM, both in a metabolic-activated system and without metabolic activation as concluded from the in vitro MNT assay (Figure S8). In the AMES test, the compound was found nonmutagenic up to 25 μM in both the presence and absence of S9 metabolic activation. With metabolic activation, it showed the possible cytotoxic effect (against S. typhimurium) above 3 μM concentration (Figure S10). Also, the hydrolyzed precursor, 2-amino-4-(1phenoxyethyl)benzo[d]thiazole-6-carboxylic acid, was neither genotoxic in the MNT test (assayed up to 350 μM) nor mutagenic (assayed in the AMES test up to a 25 μM concentration against S. typhimurium TA98 and TA100) in both the absence or presence of S9 metabolic activation. These results went hand in hand with better antibacterial activity and corroborated 27 as an improved derivative of the initial hit 1 worth of further structural optimization. Microbiologically promising derivatives 31, 51, and 59 as well as the hydrolyzed aromatic amine precursor of 31, 2-amino-4-(2-methoxy-1phenoxyethyl)benzo[d]thiazole-6-carboxylic acid, were also found nongenotoxic when tested in the MNT assay with and without metabolic activation. In the AMES test, 31 was found to be nonmutagenic against S typhimurium TA98 without metabolic activation at 25 μM, whereas with metabolic activation a possible cytotoxic effect was observed above 3 μM (Figure S11). Its hydrolyzed aromatic amine precursor did not show mutagenicity up to 25 μM concentration when tested with and without metabolic activation against S. typhimurium TA98 and TA100 in AMES assays. In conclusion, the presented results showed that the benzothiazole-2-amine precursors, as potential impurities in the gyrase inhibitors, presented in this paper do not possess an inherent genotoxic or mutagenic character. 2.2.5. Mitochondrial Toxicity. Parallel to the chemical modification of the original hit 1, the mitochondrial toxicity of the new derivatives and analogs was monitored. Mitochondrial toxicity was tested in vitro using HepG2 cells cultured in either glucose or galactose. When cells are exposed to galactose, they are forced to generate ATP using the mitochondria, rather than glycolysis, which is the case for cells in glucose medium. If a compound inhibits the mitochondrial function, then ATP levels will fall more drastically in galactose- than in glucoseexposed cells and cell death occurs at lower concentrations in galactose-exposed cells.33 Among 30 tested compounds, all were free of mitochondrial toxicity at 100 μM and most of them with the exception of 1, 9, 18, 28, 31, 35, 36, 46, and 47 were not toxic to mitochondria even at 300 μM. Whereas 1 was toxic at 300−1000 μM and showed cytotoxicity in glucose medium below 100 μM, 27 (Figure S5) and its enantiomers (R)-27 and (S)-27, the monofluoro derivative 51 and its both enantiomers (R)-51 and (S)-51, the para-substituted derivatives 69 and 72, as well as N-dimethylamino derivative 34 were not toxic to mitochondria up to 1000 μM and not cytotoxic in glucose medium at 100 μM. In summary, the mitochondrial toxicity testing highlighted compounds 27 and 51 and their enantiomers, para-substituted compounds 69 and 72 as well as Article compound 34 bearing a simple tertiary amine attached to the methine carbon at the branching point as most promising. 2.2.6. Metabolic Stability in Human and Mouse Hepatocytes and Microsomes. In vitro metabolic stability assay in cryopreserved hepatocytes was used to evaluate the metabolic stability of compounds representative of different structure classes in human and mouse hepatocytes. The assay closely resembles the conditions of in vivo liver cells, with all isoforms of metabolizing enzymes, cofactors, cellular components, and membrane permeation mechanisms.34 Compound 1 was stable in human (t1/2 = 71 min) and mouse hepatocytes (t1/2 = 132 min) (Figure S13), and 27 possessing potentially metabolically liable α-methyl group also remained relatively stable in human (t1/2 = 92 min) and mouse hepatocytes (t1/2 = 45 min) (Figure S14). Lower metabolic stability in mouse hepatocytes was observed for monofluoro compound 51 (t1/2 = 11 min) and its enantiomer (S)-51 (t1/2 = 21 min), monochloro derivative 59 (t 1/2 = 14 min) and αhydroxymethyl derivative 30 (t1/2 = 26 min), suggesting that compounds possessing monohalogenated pyrrole moiety or polar substituent on the α-methyl group are more likely to be metabolized in hepatocytes. Microsomal stability assay using human and mouse liver microsomes and NADPH cofactor to assess phase I oxidations by cytochrome and flavin monooxygenases was applied to a broader set of compounds. In the human liver microsome assay, higher metabolic stability (t1/2 > 100 min) was observed for N-alkyl compounds 19, 23, and 24, as well as for compounds 8, 64, and 73 bearing a para substituent on the phenyl ring. Lower metabolic stability was inherent to less substituted pyrroles, e.g., 51, (S)-51, and 53, as well as to α-hydroxymethyl derivative 30. In mouse liver microsomes, no general trends could be observed for the tested compounds. 2.2.7. Caco-2 Permeability Study of Compound 27. Caco2 permeability assay for compound 27 indicated its high permeability in the apical direction [Papp (A−B) = (2.9 ± 0.6) × 10−6 cm/s; Papp (B−A) = 9.9 ± 1.2 × 10−5 cm/s; efflux ratio = 35], which might be caused by P-glycoprotein and may limit its oral bioavailability and permeation through the blood− brain barrier. 2.2.8. Relationship between Collected Physicochemical Data and In Vitro Antibacterial Activity. The analysis of collected lipophilicity, solubility, and plasma protein binding data could provide useful guidance to the physicochemical thresholds required to obtain satisfactory MIC values. Calculated octanol/water partition coefficient (clog P) and total polar surface area (TPSA) were the major molecular descriptors guiding the design of improved analogs of compounds 1 and 27 (Figure 5). Analogs active against A. baumannii lay mostly around two major TPSA values, the initial TPSA of 104 Å2 and the final value of 117 Å2 (after introduction of morpholine moiety). Pushing down clog P at a fixed TPSA resulted in compounds with improved solubility and appreciable plasma free fraction (>0.1% in our case). For example, compound 51 retained good activity against A. baumannii (MIC = 2 μg/mL), and its solubility and free fraction were improved relative to 1. Compounds 57−59 seemingly represent the limit of polarity for this structural class of gyrase inhibitors to retain activity against A. baumannii. Compound 60 is a borderline example with retained activity against P. aeruginosa (Figure S15) but not against A. baumannii. Its kinetic solubility is 67 μM, and its unbound fraction in human and mouse plasma is 1.5 and 4.4%, 1392 https://doi.org/10.1021/acs.jmedchem.2c01597 J. Med. Chem. 2023, 66, 1380−1425 Journal of Medicinal Chemistry pubs.acs.org/jmc Article 5s2, and 5s10−5s12 were obtained following our general protocol using potassium thiocyanate and bromine in glacial acetic acid.35 2-Aminobenzothiazoles obtained via either pathway were N-acylated with 4-dichloro-5-methyl-1H-pyrrole-2-carbonyl chloride. Alkaline hydrolysis of the resulting methyl esters 6s1−6s4, 6s8−6s12, 6s27, and 6s64 afforded the initial hit 1 and analogs 2−4 and 8−12, 27, and 64. Pyridines 2 and 3 were further quaternized with iodomethane to obtain N-methylpyridinium compounds 5 and 6 (Scheme 1). Hydrazide 15 was prepared via a late-stage coupling of the carboxylic acid 1 and tert-butyl carbazate, followed by Bocdeprotection of the obtained 11s15 (Scheme 2). We were not able to convert 15 to oxadiazolone 14 after repeated experiments, so we resorted to a lengthier synthetic route. The oxadiazolone moiety was constructed via hydrazinolysis of ester 3s1, followed by carbonylation using carbodiimidazole (CDI). Intermediate 8s14 was then converted to benzothiazole 14 following the reaction sequence outlined in Scheme 3. Compounds 13,16 a tetrazole isostere of carboxylic acid 1, and 16,36 an aminomethyl derivative of the initial hit 1, were prepared as previously described. Our attempt to alkylate the carboxamide nitrogen atom of 16s with benzyl bromide using NaHCO3 as a base afforded a monoalkylated product for which 1H−1H NOESY experiment revealed that the regioselective benzylation occurred at the thiazole nitrogen, giving rise to 17s22 in which the 2aminobenzothiazole scaffold is locked in its imino tautomer. As demonstrated in a systematic study,37 the observed benzothiazole N-alkylation appears to be general for reactions with benzyl- and phenacyl halides and enables the preparation of thiazole-N-alkylated products 17s22−17s25 that after deprotection under acidic conditions afforded compounds 22−25 (Scheme 4). Using the same approach, alkylation of intermediate 16s with methyl iodide in a pressure tube at 60 °C was not successful. Therefore, we searched for a method that would allow selective access to carboxamide N-alkylated products. Compounds 18, 20, 21, and 26 possessing an N-alkyl carboxamide substituent were prepared according to the reaction sequence presented in Scheme 5. The starting 2- Figure 5. Relationship between clog P, TPSA, kinetic solubility, and MIC against A. baumannii. respectively. On the other hand, targeting the Gram-positive bacteria, for example, S. aureus (Figure S16), with the reported benzothiazole scaffold-based DNA gyrase inhibitors is an easier task. A much broader clog P/TPSA chemical space is tolerated; see, for example, compound 33 with a kinetic solubility of 90 μM and a plasma free fraction of 0.28 and 0.53%. 2.3. Chemistry. 4-Hydroxybenzothiazole 1s35 was successfully alkylated with most benzylic halides of interest to get compounds 5s3, 5s4, 5s8, 5s9, 5s27, and 5s64 together with N- and bis-alkylated products. The desired O-alkylated products were isolated in 13−38% yields by column chromatography and/or trituration with ethyl acetate. Because alkylation of 1s with 4-(bromomethyl)pyridine and some other nonbenzylic halides failed, we opted for alkylation of the more nucleophilic o-nitrophenol 2s either via nucleophilic substitution of alkyl halides or via Mitsunobu reaction to obtain 3s1, 3s2, and 3s10−3s12. These were reduced to anilines 4s1, 4s2, and 4s10−4s12 by catalytic hydrogenation or (in the case of benzylic ether 3s1) by iron/acetic acid. Benzothiazoles 5s1, Scheme 1. Synthesis of 4-Alkoxy Derivativesa a Reagents and conditions: (a) benzylic halide, K2CO3, KI (cat.), MeCN or DMF, 60 °C, 18 h; (b) 3,4-dichloro-5-methyl-1H-pyrrole-2-carbonyl chloride, toluene, 130 °C, 15 h; (c) 2 M NaOH, MeOH, 40 °C, 48 h; (d) ROH, PPh3, DIAD, THF, 22 °C, 18 h; (e) for benzylic ethers: Fe, AcOH, rt, 2 h; (f) for nonbenzylic ethers: H2, Pd/C, MeOH, 22 °C, 5 h; (g) KSCN, Br2, AcOH, rt, 15 h; and (h) iodomethane, THF/DMF, 60 °C, 18 h. 1393 https://doi.org/10.1021/acs.jmedchem.2c01597 J. Med. Chem. 2023, 66, 1380−1425 Journal of Medicinal Chemistry pubs.acs.org/jmc Article Scheme 2. Synthesis of Hydrazide 15a a Reagents and conditions: (a) tert-butyl carbazate, TBTU, DMF, N-methylmorpholine, 22 °C, 8 h; (b) 4 M HCl in 1,4-dioxane, 22 °C, 24 h. Scheme 3. Synthesis of Oxadiazolone Isostere 14a a Reagents and conditions: (a) benzyl bromide, K2CO3, MeCN, 60 °C, 3 h; (b) hydrazine monohydrate, EtOH, reflux, 48 h; (c) CDI, 1,4-dioxane, 100 °C, 15 h; (d) Fe, AcOH, 22 °C, 1 h; (e) KSCN, Br2, AcOH, 0−22 °C, 15 h; and (f) 3,4-dichloro-5-methyl-1H-pyrrole-2-carbonyl chloride, pyridine, CH2Cl2, 22 °C, 15 h. Scheme 4. Synthesis of Thiazole-N-Alkylated Derivativesa a Reagents and conditions: (a) TsCl, t-BuOH, pyridine, 0 °C to 22 °C, 24 h; (b) H2, Pd/C, EtOAc, 2 h; (c) KSCN, Br2, AcOH, 0−22 °C, 24 h; (d) 3,4-dichloro-5-methyl-1H-pyrrole-2-carbonyl chloride, Et3N toluene, 130 °C, 15 h; (e) alkyl halide, KI, NaHCO3, DMF, 60 °C, 14 h; and (f) 1 M HCl in acetic acid, 22 °C, overnight. aminobenzo-thiazoles 18s18, 18s20, 18s21, and 18s26 were first transformed to 2-bromobenzothiazoles 19s18, 19s20, 19s21, and 19s26 via the Sandmeyer reaction. Methylamine or 2-methoxyethylamine was then applied to substitute bromine and give the corresponding N-substituted 2-aminobenzothiazoles, which were transformed into p-methoxybenzyl (PMB) esters 20s18, 20s20, 20s21, and 20s26 and then coupled to 3,4-dichloro-5-methyl-1H-pyrrole-2-carboxylic acid. A switch from methyl/ethyl to PMB esters was required because tertiary amides resulting from the coupling of the alkyl esters were unstable during the final alkaline hydrolysis, as opposed to the analogous secondary amides. After acidolytic cleavage of the resulting PMB esters, the carboxylic acids 18, 21, and 26 were obtained. It is noteworthy that the 1-phenylethyl substituent was cleaved during the acidolysis step affording the 4-hydroxy analog 20 instead of the desired 4-(1-phenylethoxy) derivative. Using the same strategy together with the phthalimido protection, the methylated aminomethyl derivative 19 was obtained from 20s18. For the synthesis of compound 27, building block 5s27 was prepared from phenol 1s and 1-chloro-1-phenylethane. Coupling of 5s27 with the pyrrole moiety and subsequent ester hydrolysis afforded 27 (Scheme 1). Starting from 2s and the commercially available (R)- or (S)-1-phenylethanol, the enantiomerically enriched (S)-27 and (R)-27 (74 and 62% ee, respectively, determined by chiral HPLC analysis) were obtained in a 5-step reaction procedure with the inversion of the absolute configuration in the Mitsunobu reaction step (Scheme 6). Partial racemization most likely occurred during oxidative benzothiazole formation step (step c) or during coupling to acyl chloride (step d) via cleavage−reattachment of the 1-phenylethyl substituent through the carbocation intermediate. Stimulated by the improved antibacterial profile of compound 27, 4-alkoxy analogs 28, 29, and 46−49 with variations at the stereogenic carbon atom were prepared, as outlined in Scheme 7. Alkylation of the phenol 2s was again achieved using the Mitsunobu reaction and the commercially available alcohol building blocks. Either iron/acetic acid or 1394 https://doi.org/10.1021/acs.jmedchem.2c01597 J. Med. Chem. 2023, 66, 1380−1425 Journal of Medicinal Chemistry pubs.acs.org/jmc Article Scheme 5. Synthesis of Carboxamide N-Alkylated Derivativesa a Reagents and conditions: (a) CuBr2, t-BuONO, CH3CN, 22 °C, 15 h; (b) Z-NH2, THF, 22 °C, 15 h; (c) 2 M NaOH, 1,4-dioxane, 50 °C, 24 h (for 20s18, 20s20, 20s26); (d) p-methoxybenzyl chloride, K2CO3, DMF, rt, 15 h (for 20s18, 20s20, 20s26); (e) 3,4-dichloro-5-methyl-1Hpyrrole-2-carbonyl chloride, toluene, 130 °C, 15 h; (f) 1 M HCl in AcOH, 22 °C, 18 h; (g) 3,4-dichloro-5-phthalimidomethyl-1H-pyrrole-2carbonyl chloride,36 toluene, 130 °C, 15 h; (h) (1) hydrazine hydrate, EtOH, 50 °C, 40 min; (2) HCl, MeOH, 22 °C, 15 min; (3) EtOH, reflux, 18 h; and (i) SnCl4, DCM, 22 °C, 2 h. Scheme 6. Synthesis of Compound 27 and its Enantiomersa a Reagents and conditions: (a) for (S)-27: (R)-1-phenylethanol, for (R)-27: (S)-1-phenylethanol, PPh3, DIAD, THF, 22 °C, 18 h; (b) Fe, AcOH, 22 °C, 2 h; (c) KSCN, Br2, AcOH, 22 °C, 15 h; (d) 3,4-dichloro-5-methyl-1H-pyrrole-2-carbonyl chloride, toluene, 130 °C, 15 h; and (e) 2 M NaOH, MeOH, 40 °C, 48 h. Scheme 7. Synthesis of Branched 4-Alkoxy Analogsa a Reagents and conditions: (a) ROH, PPh3, DIAD, THF, 22 °C 18 h; (b) Fe, AcOH, 22 °C, 2 h; (c) H2, Pd/C, MeOH, 22 °C, 5 h; (d) KSCN, Br2, AcOH, 22 °C, 15 h; (e) 3,4-dichloro-5-methyl-1H-pyrrole-2-carbonyl chloride, toluene, 130 °C, 15 h; (f) 2 M NaOH, MeOH, 40 °C, 48 h; and (g) CF3COOH, dichloromethane, 22 °C, 12−30 h. A general strategy for the introduction of polar substituents to the methylene group at the branching point relied on the nucleophilic substitution of α-haloketones 26s and the subsequent NaBH4-mediated reduction of the resulting ketones 27s or anti-Markovnikov opening of epoxides 28s. catalytic hydrogenation was used for the reduction of the nitro group, and standard coupling and deprotection strategies were applied. Protection of the hydroxyl group as a benzoate ester was applied for the synthesis of 30. 1395 https://doi.org/10.1021/acs.jmedchem.2c01597 J. Med. Chem. 2023, 66, 1380−1425 Journal of Medicinal Chemistry pubs.acs.org/jmc Article Scheme 8. Synthesis of Branched 4-Alkoxy Analogs Containing a Polar Groupa a Reagents and conditions: (a) nucleophile, K2CO3, KI, MeCN, 22 °C, 24 h; (b) NaBH4, MeOH, 0−22 °C, 2 h; (c) nucleophile, DMF or neat, heating; (d) 2s, PPh3, DIAD, THF, 22 °C, 18 h; (e) Fe, AcOH, 22 °C, 2 h; (f) H2, Pd/C, MeOH, 22 °C, 5 h; (g) KSCN, Br2, AcOH, 22 °C, 15 h; (h) 3,4-dichloro-5-methyl-1H-pyrrole-2-carbonyl chloride, toluene, 130 °C, 15 h; (i) 2-trichloroacetyl-3,4-dichloro-5-methyl-1H-pyrrole, Na2CO3, DMF, 65 °C, 24 h; (j) 2 M NaOH, MeOH, 40 °C, 48 h; (k) 1 M HCl in 1,4-dioxane, 22 °C, 24 h; (l) MeI, THF, 60 °C, 24 h; (m) 2trichloroacetyl-4-chloro-5-methyl-1H-pyrrole, Na2CO3, DMF, 65 °C, 18 h; (n) 4-fluoro-5-methyl-1H-pyrrole-2-carbonyl chloride, toluene, 130 °C, 18 h; and (o) CF3COOH, dichloromethane, 22 °C, 18 h. The etherification of the obtained alcohols 29s with phenol 2s was achieved via the Mitsunobu reaction, and the target compounds 31−45 and 56−58 were obtained after coupling to 3,4-dichloro-5-methylpyrrole-2-carbonyl chloride and ester cleavage. 2-Aminobenzothiazole 32s36, containing morpholinomethyl moiety, was also coupled to monochloro- and monofluoropyrrole building blocks to obtain compounds 59 and 60, analogs of 36 (Scheme 8). Being aware of the contribution of the dichloropyrrole moiety to the forbiddingly high lipophilicity of compound 27, we prepared analogs 50−55 and 61−63 featuring a less lipophilic pyrrolamide moiety. The required pyrrole building blocks 34s50, 35s51, 35s54, 36s55,36 34s53,38 and 35s5239,40 were prepared according to the published procedures, and the 4-chloro-2-methyl-1H-imidazole-5-carboxylic acid (37s63) was commercially available. Either 2-(trichloroacetyl)pyrroles or pyrrole-2-acyl chlorides were used as acylating agents for coupling to the 2-aminobenzothiazole building block 5s27. Notably, the amide bond formed by the coupling of 2aminobenzothiazole 5s27 and carboxylic acid 37s63 was not stable under the alkaline conditions required for the final-step methyl ester hydrolysis. When the tert-butyl ester protection was used instead, the 1-phenylethyl ether was found unstable under acidolytic conditions required for the tert-butyl ester cleavage. To avoid these restrictions, the 4-isopropoxy analog 63 was prepared from the corresponding tert-butyl ester building block 42s64 (Scheme 9). We found that compounds containing an aliphatic tertiary amine in the side chain, such as 34 and 35, were not accessible via acylation of intermediates 32s34 and 32s35 with 3,4dichloro-5-methyl-1H-pyrrole-2-carbonyl chloride. For example, in an attempted acylation of piperidine-containing intermediate 32s35 using 2.2 equivalents (in two portions) of the aforementioned acyl chloride, merely 10% conversion was achieved. This goes hand in hand with our general observation that the use of tertiary amine or pyridine was detrimental to the yield of coupling of acyl chlorides to 2aminobenzothiazoles. An alternative 2-trichloroacetylpyrrolebased acylating agent 46s was thus prepared, which in analogy to 2-trichloroacetyl-4,5-dibromo-1H-pyrrole11 should operate under milder alkaline reaction conditions. Because efficient direct dichlorination of 2-trichloroacetyl-5-methyl-1H-pyrrole failed using N-chlorosuccinimide or sulfuryl chloride, a twostep protocol from ester 44s was developed. Alkaline hydrolysis accompanied by decarboxylation afforded compound 45s, which was prone to oxidation and was therefore immediately used in the Friedel−Crafts acylation step to afford trichloroacetylpyrrole 46s (Scheme 10). To obtain compounds 65−74, the required building blocks 48s65−48s73 were prepared from 4′-(4-bromomethyl)acetophenone via nucleophilic substitution and subsequent reduction of ketones 47s65−47s73, or they were commercially available. 2-Aminobenzothiazoles 51s65−51s73 were prepared following the usual reaction sequence and were coupled to substituted pyrroles to give esters 52s65, 52s70−52s73, and 1396 https://doi.org/10.1021/acs.jmedchem.2c01597 J. Med. Chem. 2023, 66, 1380−1425 Journal of Medicinal Chemistry pubs.acs.org/jmc Article Scheme 9. Synthesis of Derivatives Containing Less Lipophilic Pyrrole Moietiesa a Reagents and conditions: (a) 24s27, Na2CO3, DMF, 65 °C, 18 h; (b) 2 M NaOH, MeOH, 40 °C, 48 h; (c) oxalyl chloride, CH2Cl2, 22 °C, 24 h; then 24s27, toluene, 130 °C, 24 h; (d) SO2Cl2, 75 °C, 1 h; then 24s27, toluene, 130 °C, 24 h; (e) hydrazine hydrate, EtOH, 40 °C, 40 min; then HCl, MeOH, 22 °C, 15 min; then EtOH, reflux, 24 h; (f) 2 M NaOH, MeOH, 50 °C, 48 h; then HCl, MeOH; (g) SOCl2, DMF (cat.), 75 °C, 1.5 h; then tert-butyl 2-amino-4-isopropoxybenzo[d]thiazole-6-carboxylate (42s63), toluene 130 °C, 18 h; and (h) CF3CO2H, CH2Cl2, 22 °C, 18 h. turned out to be very difficult and would require a comprehensive multiparameter optimization, which could not be performed within this project. However, with over 70 compounds synthesized and tested, promising directions for the optimization of solubility and plasma protein binding were identified. Scheme 10. Synthesis of 2-Trichloroacetyl-3,4-dichloro-5methyl-1H-pyrrolea 4. EXPERIMENTAL SECTION 4.1. Chemistry: General Procedures and Instrumentation. Chemicals were obtained from Acros Organics (Geel, Belgium), Enamine Ltd. (Kyiv, Ukraine), Sigma-Aldrich (St. Louis, MO), TCI (Tokyo, Japan), Fluorochem Ltd. (Derbyshire, U.K.), and Apollo Scientific (Stockport, U.K.) and were used without further purification. Analytical TLC was performed on silica gel Merck 60 F254 plates (0.25 mm), using visualization with UV light and spray reagents. Column chromatography was carried out on silica gel 60 (particle size 240−400 mesh). Analytical reversed-phase HPLC− UV−MS analyses were performed on a 1260 Infinity II LC system (Agilent Technologies Inc., SantaClara, CA). A Waters XBridge C18 column was used (4.6 mm × 150 mm, 3.5 μm), with a sample injection volume of 10 μL and a flow rate of 1.5 mL/min, coupled to an ADVION expression CMSL quadrupole mass spectrometer (Advion Inc., Ithaca). The eluent consisted of acetonitrile (solvent A) and 0.1% formic acid in 1% acetonitrile in ultrapure water (solvent B). The gradient (defined for solvent A) was 0−1.0 min, 25%; 1.0− 6.0 min, 25−98%; 6.0−6.5 min, 98%; 6.5−7.5 min, 98−25%; 7.5− 10.5 min, 25%. 1H NMR (400 MHz, internal Me4Si), 13C NMR (101 MHz, internal CDCl3 or DMSO-d6), and 19F NMR (376 MHz, external CCl3F) spectra were recorded on a Bruker AVANCE III 400 spectrometer (Bruker Corporation, Billerica, MA) in DMSO-d6 or CDCl3 solution. High-resolution mass spectra were obtained using an Exactive Plus Orbitrap mass spectrometer (Thermo Fisher Scientific, a Reagents and conditions: (a) 0.5 M KOH, reflux, 4 h; (b) Cl3CCOCl, 1,2-dichloroethane, 22 °C, 18 h. 53s66−53s68 that afforded para-substituted derivatives 65− 73 after alkaline hydrolysis (Scheme 11). 3. CONCLUSIONS In summary, modification of the initial hit 1 afforded compound 27, which was endowed with excellent activity against A. baumannii, P. aeruginosa, and E. coli, but unfortunately possessed only partially ameliorated physicochemical and ADME properties. Attempts to improve these properties to target values needed for in vivo studies (thermodynamic solubility higher than 0.3 mg/mL, plasma free fraction 0.5−2%) while retaining the antibacterial activity against A. baumannii or P. aeruginosa (MIC < 4 μg/mL) resulted in several derivatives with improved solubility and free fraction but unfortunately with partial or complete loss of promising antibacterial activity against Gram-negative bacteria observed in 27. The optimization of 27 to a lead compound 1397 https://doi.org/10.1021/acs.jmedchem.2c01597 J. Med. Chem. 2023, 66, 1380−1425 Journal of Medicinal Chemistry pubs.acs.org/jmc Article Scheme 11. Synthesis of Derivatives with a para Substituent on the Phenyl Ringa a Reagents and conditions: (a) nucleophile, K2CO3, MeCN, 0−22 °C, 24 h. (b) NaBH4, 0−22 °C, 2 h; (c) 2s, PPh3, DIAD, THF, 22 °C, 18 h; (d) Fe, AcOH, 22 °C, 2 h; (e) KSCN, Br2, AcOH, 22 °C, 15 h; (f) 46s, Na2CO3, DMF, 65 °C, 24 h; (g) 2 M NaOH, MeOH, 40 °C, 48 h; (h) 4 M HCl in dioxane, 22 °C, 1 h; (i) 2-trichloroacetyl-4-chloro-5-methyl-1H-pyrrole, Na2CO3, DMF, 65 °C, 18 h; (j) 4-fluoro-5-methyl-1H-pyrrole-2carbonyl chloride, toluene, 130 °C, 18 h; and (k) 4-cyano-5-methyl-1H-pyrrole-2-carbonyl chloride, toluene,130 °C, 18 h. Waltham, MA). All final compounds were >95% pure by HPLC analysis unless otherwise specified. The tested compounds passed screening against the PAINS filter, as available in CANVAS (Schrödinger Release 2020-3: Canvas, Schrödinger, LLC, New York, NY, 2020). 4.2. Synthesis of Compounds. 4.2.1. 4-(Benzyloxy)-2-(3,4dichloro-5-methyl-1H-pyrrole-2-carb-oxamido)benzo[d]thiazole-6carboxylic Acid (1). The compound was prepared from methyl 3hydroxy-4-nitrobenzoate (2s) according to our published procedure.15 4.2.2. Methyl 4-Nitro-3-(pyridin-4-ylmethoxy)benzoate (3s2): Typical Procedure A for Alkylation of 3-Hydroxy-4-nitrobenzoates with Benzylic Halides. To a solution of methyl 3-hydroxy-4nitrobenzoate (2s) (2.00 g, 10.1 mmol) in acetonitrile (90 mL), K2CO3 (5.60 g, 40.5 mmol), 4-(chloromethyl)pyridine hydrochloride (1.83 g, 11.2 mmol), and KI (0.37 g, 0.2 equiv) were added and the reaction mixture was stirred at 60 °C overnight. The volatiles were removed under reduced pressure, and the residue was partitioned between ethyl acetate and water. The organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated. The crude product was triturated with methanol to get the title compound as a beige solid (1.08 g, 37% yield). 1H NMR (400 MHz, DMSO-d6): δ 8.62 (d, J = 3.7 Hz, 2H), 8.08 (d, J = 8.4 Hz, 1H), 7.87 (d, J = 1.4 Hz, 1H), 7.72 (dd, J = 8.4, 1.5 Hz, 1H), 7.45 (d, J = 5.5 Hz, 1H), 5.50 (s, 2H), 3.91 (s, 3H); MS (ESI): m/z calcd for C14H12N2O5: 288.07. Found: 289.0 [M + H]+. 4.2.3. Methyl 4-Amino-3-(pyridin-4-ylmethoxy)benzoate (4s2): Typical Procedure B for Reduction of 4-Nitro-3-alkoxybenzoates with Iron. Methyl 4-nitro-3-(pyridin-4-ylmethoxy)benzoate (3s2) (500 mg, 1.74 mmol) was suspended in acetic acid (40 mL), and iron powder (486 mg, 8.68 mmol) was then added and the reaction mixture was stirred at 60 °C for 1 h. Water was added to dissolve the white precipitate, and residual iron was removed by filtration through celite. The residue was neutralized with saturated aqueous NaHCO3 and extracted with ethyl acetate. The crude product was purified by flash column chromatography, eluent ethyl acetate/hexane (3:1); colorless oil (380 mg, 85% yield). 1H NMR (400 MHz, DMSO-d6): δ 8.62−8.53 (m, 2H), 7.55 (d, J = 6.0 Hz, 2H), 7.41 (dd, J = 8.2, 1.8 Hz, 1H), 7.36 (d, J = 1.7 Hz, 1H), 6.69 (d, J = 8.2 Hz, 1H), 5.81 (s, 2H), 5.22 (s, 2H), 3.74 (s, 3H); MS (ESI): m/z calcd for C14H14N2O3: 258.10. Found: 258.8 [M + H]+. 4.2.4. Methyl 2-Amino-4-(pyridin-4-ylmethoxy)benzo[d]thiazole6-carboxylate (5s2): Typical Procedure C for Benzothiazole Formation. To a suspension of the above aniline 4s2 (395 mg, 1.52 mmol) in acetic acid (8 mL), KSCN (594 mg, 6.11 mmol) was added and stirred at 22 °C until a clear solution was obtained. The reaction mixture was cooled on an ice bath while a solution of bromine (160 μL, 3.04 mmol) in acetic acid (0.5 mL) was added dropwise. The resulting mixture was stirred at 22 °C overnight; then, it was neutralized with saturated aqueous NaHCO3, and the precipitate was collected, washed with water, and air-dried. The solid was percolated with methanol and then triturated with cold methanol to get the title compound as a white solid (324 mg, 67% yield). 1H NMR (400 MHz, DMSO-d6): δ 8.60 (d, J = 6.0 Hz, 2H), 8.00 (d, J = 1.5 Hz, 1H), 7.95 (s, 2H), 7.49 (d, J = 6.0 Hz, 2H), 7.43 (d, J = 1.5 Hz, 1H), 5.33 (s, 2H), 3.82 (s, 3H); MS (ESI): m/z calcd for C15H13N3O3S: 315.07. Found: 316.0 [M + H]+. 4.2.5. Methyl 2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4-(pyridin-4-ylmethoxy)benzo[d]thiazole-6-carboxylate (6s2): Typical Procedure D for Acylation with Pyrrole-2-carbonyl Chlorides. A suspension of 3,4-dichloro-5-methyl-1H-pyrrole-2carbonyl chloride (prepared from the corresponding carboxylic acid (86 mg, 0.44 mmol) by refluxing in SOCl2 (2 mL) for 1 h and then concentrating under reduced pressure) and the above benzothiazole 5s2 (139 mg, 0.44 mmol) in toluene (9 mL) was refluxed overnight. Upon cooling, the precipitate was collected and washed with toluene to get the title compound as a gray solid (87 mg, 40% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.31 (s, 1H), 12.20 (s, 1H), 8.73 (d, J = 5.3 Hz, 2H), 8.34 (d, J = 1 Hz, 1H), 7.75−7.67 (m, 2H), 7.60 (d, J = 1 Hz, 1H), 5.51 (s, 2H), 3.88 (s, 3H), 2.27 (s, 3H); MS (ESI): m/z calcd for C21H16Cl2N4O4S: 490.03. Found: 488.9 [M − H]−. 4.2.6. 2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4(pyridin-4-ylmethoxy)benzo[d]thiazole-6-carboxylic Acid (2): Typical procedure E for Methyl Ester Hydrolysis. A solution of the above methyl ester 6s2 (60 mg, 0.122 mmol) in MeOH (3 mL) and 2 M NaOH (0.3 mL) was stirred at 40 °C for 24 h. NaOH (2 M, 0.3 mL) 1398 https://doi.org/10.1021/acs.jmedchem.2c01597 J. Med. Chem. 2023, 66, 1380−1425 Journal of Medicinal Chemistry pubs.acs.org/jmc Article of THF (4.5 mL) and DMF (3 mL), methyl iodide (19 μL, 0.31 mmol) was added and the reaction mixture was stirred in a pressure tube at 60 °C overnight. The volatiles were removed under reduced pressure, and the residue was triturated with a mixture of DMF (450 μL) and toluene (3 mL) to get the title compound as a gray solid (13.2 mg 68% yield). 1H NMR (400 MHz, DMSO-d6): δ 13.04 (s, 1H), 12.33 (s, 1H), 12.07 (s, 1H), 8.99 (d, J = 6 Hz, 2H), 8.33 (d, J = 1.4 Hz, 1H), 8.20 (d, J = 6 Hz, 2H), 7.95 (s, 1H), 7.60 (d, J = 1.4 Hz, 1H), 5.76 (s, 2H), 4.35 (s, 3H), 2.28 (s, 3H); HRMS (ESI) m/z: [M − I]+ calcd for C21H17Cl2N4O4S 491.0348; found 491.0334; HPLC purity (254 nm): 95%. 4.2.14. 3-(((6-Carboxy-2-(3,4-dichloro-5-methyl-1H-pyrrole-2carboxamido)benzo[d]thiazol-4-yl)oxy)methyl)-1-methylpyri-din1-ium Iodide (6). Prepared according to the typical procedure G from 3 (10 mg, 0.021 mmol); beige solid (7.3 mg, 56% yield). 1H NMR (400 MHz, DMSO-d6): δ 13.08 (s, 1H), 12.31 (s, 1H), 12.02 (s, 1H), 9.22 (s, 1H), 9.02 (d, J = 6.1 Hz, 1H), 8.74 (d, J = 8.1 Hz, 1H), 8.34 (s, 1H), 8.23 (dd, J = 8.1, 6.1 Hz, 1H), 7.68 (d, J = 1.4 Hz, 1H), 5.58 (s, 2H), 4.41 (s, 3H), 2.27 (s, 3H); HRMS (ESI) m/z: [M − I]+ calcd for C21H17Cl2N4O4S 491.0348; found 491.0335; HPLC purity (254 nm): 97%. 4.2.15. Methyl 2-Amino-4-((4-mesylbenzyl)oxy)benzo[d]thiazole-6-carboxylate (5s8). Prepared according to the typical procedure F from 1s (514 mg, 2.29 mmol); bright yellow powder (135 mg, 15% yield). 1H NMR (400 MHz, DMSO-d6): δ 7.99 (d, J = 1.5 Hz, 1H), 7.97 (d, J = 8.4 Hz, 2H), 7.94 (s, 2H), 7.76 (d, J = 8.4 Hz, 2H), 7.46 (d, J = 1.5 Hz, 1H), 5.39 (s, 2H), 3.83 (s, 3H). 13C NMR (101 MHz, DMSO-d6): δ 168.87, 166.04, 147.67, 146.76, 143.08, 140.17, 132.02, 128.15, 127.13, 122.20, 116.24, 110.18, 69.15, 51.99, 43.54. 4.2.16. Methyl 2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4-((4-mesylbenzyl)oxy)benzo[d]thiazole-6-carboxylate (6s8). Prepared according to the typical procedure D from 5s8 (120 mg, 0.303 mmol); gray powder (76 mg, 44% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.28 (s, 1H), 12.18 (s, 1H), 8.33 (s, 1H), 8.00 (d, J = 8 Hz, 2H), 7.81 (d, J = 8 Hz, 2H), 7.63 (s, 1H), 5.48 (s, 2H), 3.89 (s, 3H), 3.25 (s, 3H), 2.27 (s, 3H). 4.2.17. 2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4{(4-mesylphenyl)methoxy}benzo[d]thiazole-6-carboxylic Acid (8). Prepared according to the typical procedure E from 6s8 (70 mg, 0.123 mmol); light brown powder (47 mg, 69% yield). 1H NMR (400 MHz, DMSO-d6): δ 13.03 (s, 1H), 12.27 (s, 1H), 12.15 (s, 1H), 8.28 (s, 1H), 8.00 (d, J = 8.2 Hz, 2H), 7.80 (d, J = 8.2 Hz, 2H), 7.62 (s, 1H), 5.47 (s, 2H), 3.25 (s, 3H), 2.27 (s, 3H); HRMS (ESI) m/z: [M − H]− calcd for C22H16Cl2N3O6S2 551.9858; found 551.9863; HPLC purity (254 nm): 97%. 4.2.18. 2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4((3-fluorobenzyl)oxy)benzo[d]thiazole-6-carboxylic Acid (9). The compound was prepared according to our published procedure.16 4.2.19. Methyl 3-(2-Methoxyethoxy)-4-nitrobenzoate (3s10). The compound was prepared according to a typical procedure H (see below) from 2s (0.70 g, 3.55 mmol) and was used in the next step without further purification. 4.2.20. Methyl 4-Amino-3-(2-methoxyethoxy)benzoate (4s10). Prepared according to the typical procedure I (see below) from crude 3s10 (1.00 g, 3.92 mmol); yellowish oil (640 mg, 80% yield for two steps). 1H NMR (400 MHz, CDCl3): δ 7.56 (dd, J = 8.2, 1.8 Hz, 1H), 7.47 (d, J = 1.8 Hz, 1H), 6.67 (d, J = 8.2 Hz, 1H), 4.31 (s, 2H), 4.24− 4.16 (m, 2H), 3.85 (s, 3H), 3.80−3.73 (m, 2H), 3.44 (s, 3H). 4.2.21. Methyl 2-Amino-4-(2-methoxyethoxy)benzo[d]thiazole6-carboxylate (5s10). Prepared according to the typical procedure C from 4s10 (299 mg, 1.33 mmol); yellow solid (150 mg, 40% yield); 1 H NMR (400 MHz, DMSO-d6): δ 7.96 (d, J = 1.6 Hz, 1H), 7.91 (s, 2H), 7.35 (d, J = 1.6 Hz, 1H), 4.27−4.18 (m, 2H), 3.83 (s, 3H), 3.72−3.66 (m, 2H), 3.32 (s, 3H); 13C NMR (101 MHz, DMSO-d6): δ 168.60, 166.12, 148.17, 146.53, 131.82, 122.22, 115.80, 109.45, 70.44, 67.69, 58.16, 51.96; HRMS (ESI) m/z: [M + H]+ calcd for C12H15N2O4S 283.0753; found 283.0744. 4.2.22. Methyl 2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4-(2-methoxyethoxy)benzo[d]thiazole-6-carboxylate (6s10). Prepared according to the typical procedure D from 5s10 (129 mg, was added and stirred at 40 °C for an additional 24 h and then concentrated. The solid residue was dispersed in water (3 mL), filtered through cotton, and the filtrate was brought to pH = 1 by adding 4 M HCl. The precipitate was collected on a frit, washed with water (0.6 mL), and air-dried. The crude product was triturated with MeOH (2 × 12 mL) to get the title compound as a brown solid (25 mg, 43% yield). 1H NMR (400 MHz, DMSO-d6): δ 13.02 (s, 1H), 12.38 (s, 1H), 12.18 (s, 1H), 8.83−8.76 (m, 2H), 8.30 (d, J = 1 Hz, 1H), 7.83 (d, J = 5.4 Hz, 2H), 7.60 (d, J = 1 Hz, 1H), 5.57 (s, 2H), 2.28 (s, 3H); HRMS (ESI) m/z: [M + H]+ calcd for C20H15Cl2N4O4S 477.0191; found 477.0187; HPLC purity (254 nm): 92%. 4.2.7. Methyl 2-Amino-4-(pyridin-3-ylmethoxy)benzo[d]thiazole6-carboxylate (5s3): Typical Procedure F for Alkylation of 2Aminobenzo[d]thiazol-4-ols. To a solution of methyl 2-amino-4hydroxybenzo[d]thiazole-6-carboxylate (1s) (682 mg, 3.04 mmol) and 3-(chloromethyl)pyridine hydrochloride (598 mg, 3.65 mmol) in DMF (30 mL), K2CO3 (841 mg, 6.09 mmol) and KI (101 mg, 0.61 mmol) were added. The reaction mixture was stirred at 60 °C overnight; then, the volatiles were removed under reduced pressure. The residue was percolated with ethyl acetate, and the crude product was purified by flash column chromatography, using as eluent 1:2 ethyl acetate/hexane and then 1:1 ethyl acetate/hexane to get the title compound as a yellow powder (79 mg, 8% yield). 1H NMR (400 MHz, DMSO-d6): δ 8.71 (d, J = 1.7 Hz, 1H), 8.56 (dd, J = 4.8, 1.7 Hz, 1H), 7.99 (d, J = 1.5 Hz, 1H), 7.96−7.88 (m, 3H), 7.48 (d, J = 1.5 Hz, 1H), 7.47−7.42 (m, 1H), 5.29 (s, 2H), 3.83 (s, 3H); MS (ESI): m/z calcd for C15H13N3O3S: 315.07. Found: 316.1 [M + H]+. 4.2.8. Methyl 2-(3,4-dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4-(pyridin-3-ylmethoxy)benzo[d]thiazole-6-carboxylate (6s3). Prepared according to the typical procedure D from 5s3 (59 mg, 0.252 mmol); gray solid (57 mg 46% yield). 1H NMR (400 MHz, DMSO): δ 12.33 (s, 1H), 12.20 (s, 1H), 8.95−8.92 (m, 1H), 8.77 (dd, J = 5.2, 1.6 Hz, 1H), 8.37−8.31 (m, 2H), 7.83−7.78 (m, 1H), 7.66 (d, J = 1.5 Hz, 1H), 5.48 (s, 2H), 3.90 (s, 3H), 2.26 (s, 3H); MS (ESI): m/z calcd for C21H16Cl2N4O4S: 490.03. 489.2 [M − H]−. 4.2.9. 2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4(pyridin-3-ylmethoxy)benzo[d]thiazole-6-carboxylic Acid (3). Prepared according to the typical procedure E from 6s3 (48 mg, 0.097 mmol); light brown powder (18 mg, 39% yield). 1H NMR (400 MHz, DMSO-d6): δ 13.03 (s, 1H), 12.30 (s, 1H), 12.17 (s, 1H), 8.94−8.88 (m, 1H), 8.75 (dd, J = 5.2, 1.6 Hz, 1H), 8.32−8.25 (m, 2H), 7.76 (dd, J = 7.9, 5.2 Hz, 1H), 7.66 (d, J = 1.5 Hz, 1H), 5.46 (s, 2H), 2.26 (s, 3H); HRMS (ESI) m/z: [M + H]+ calcd for C20H15Cl2N4O4S 477.0191; found 477.0187; HPLC purity (254 nm): 93.0%. 4.2.10. Methyl 2-Amino-4-(pyrimidin-2-ylmethoxy)benzo[d]thiazole-6-carboxylate (5s4). Prepared according to the typical procedure F from 1s (99 mg, 0.441 mmol); yellow crystals (53 mg, 38% yield). 1H NMR (400 MHz, DMSO-d6): δ 8.85 (d, J = 4.9 Hz, 2H), 7.95 (d, J = 1.5 Hz, 1H), 7.89 (s, 2H), 7.48 (t, J = 4.9 Hz, 1H), 7.36 (d, J = 1.5 Hz, 1H), 5.40 (s, 2H), 3.80 (s, 3H); HRMS (ESI) m/ z: [M + H]+ calcd for C14H13N4O3S: 317.0708; found 317.0700. 4.2.11. Methyl 2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4-(pyrimidin-2-ylmethoxy)benzo[d]thiazole-6-carboxylate (6s4). Prepared according to the typical procedure D from 5s4 (52 mg, 0.164); gray solid (59 mg, 73% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.27 (s, 1H), 12.20 (s, 1H), 8.87 (d, J = 4.9 Hz, 2H), 8.29 (s, 1H), 7.59−7.47 (m, 2H), 5.51 (s, 2H), 3.86 (s, 3H), 2.27 (s, 3H). 4.2.12. 2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4(pyrimidin-2-ylmethoxy)benzo[d]thiazole-6-carboxylic Acid (4). Prepared according to the typical procedure E from 6s4 (55 mg, 0.112 mmol); brown solid (31 mg 58% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.95 (s, 1H), 12.27 (s, 1H), 12.15 (s, 1H), 8.87 (d, J = 4.9 Hz, 2H), 8.24 (d, J = 1.4 Hz, 1H), 7.54−7.47 (m, 2H), 5.49 (s, 2H), 2.26 (s, 3H); HRMS (ESI) m/z: [M + H]+ calcd for C19H14Cl2N5O4S 478.0144; found 478.0136; HPLC purity (254 nm): 98%. 4.2.13. 4-(((6-Carboxy-2-(3,4-dichloro-5-methyl-1H-pyrrole-2carboxamido)benzo[d]thiazol-4-yl)oxy)methyl)-1-methylpyri-din1-ium Iodide (5): Typical Procedure G for Quaternization Using Iodomethane. To a solution of 2 (15 mg, 0.031 mmol) in a mixture 1399 https://doi.org/10.1021/acs.jmedchem.2c01597 J. Med. Chem. 2023, 66, 1380−1425 Journal of Medicinal Chemistry pubs.acs.org/jmc Article mg 0.133 mmol); brown solid (25 mg, 40% yield). 1H NMR (400 MHz, DMSO-d6): δ characteristic peaks: 8.12 (s, 1H), 7.49 (s, 1H), 4.25 (m, 1H), 4.13 (m, 1H), 2.24 (s, 3H), 1.14 (d, J = 6.4 Hz, 3H); HRMS (ESI) m/z: [M + H]+ calcd for C19H21Cl2N4O4S 471.0661; found 471.0654; HPLC purity (254 nm): 96%. 4.2.29. Methyl 4-Nitro-3-(2,2,2-trifluoroethoxy)benzoate (3s12). Prepared according to the typical procedure A from 2s (1.25 g, 6.34 mmol), and used in the next step without any further purification. 4.2.30. Methyl 4-Amino-3-(2,2,2-trifluoroethoxy)benzoate (4s12). Prepared according to the typical procedure B from 3s12 (500 mg, 1.79 mmol); white crystals (181 mg 41% yield). 1H NMR (400 MHz, DMSO-d6): δ 7.46 (dd, J = 8.2, 1.8 Hz, 1H), 7.43 (d, J = 1.8 Hz, 1H), 6.71 (d, J = 8.2 Hz, 1H), 5.70 (s, 2H), 4.76 (q, J = 8.9 Hz, 2H), 3.76 (s, 3H); MS (ESI): m/z calcd for C10H10F3NO3: 249.06. Found: 249.9 [M + H]+. 4.2.31. Methyl 2-Amino-4-(2,2,2-trifluoroethoxy)benzo[d]thiazole-6-carboxylate (5s12). Prepared according to the typical procedure C from 4s12 (169 mg, 0.680 mmol); yellow solid (75 mg, 36% yield). 1H NMR (400 MHz, DMSO-d6); δ 8.09−8.02 (m, 3H), 7.45 (d, J = 1.6 Hz, 1H), 4.97−4.88 (m, 2H), 3.84 (s, 3H); MS (ESI): m/z calcd for C11H9F3N2O3S: 306.03. Found: 306.8 [M + H]+. 4.2.32. Methyl 2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4-(2,2,2-trifluoroethoxy)benzo[d]thiazole-6-carboxylate (6s12). Prepared according to the typical procedure D from 5s12 (75 mg, 0.245 mmol); gray solid (102 mg 86% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.32 (s, 1H), 12.29 (s, 1H), 8.40 (d, J = 1.4 Hz, 1H), 7.62 (d, J = 1.5 Hz, 1H), 5.06 (q, J = 8.8 Hz, 2H), 3.90 (s, 3H), 2.28 (s, 3H); MS (ESI): m/z calcd for C17H12Cl2F3N3O4S: 480.99. Found: 480.1 [M − H]−. 4.2.33. 2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4(2,2,2-trifluoroethoxy)benzo[d]thiazole-6-carboxylic Acid (12). Prepared according to the typical procedure E from 6s12 (75 mg, 0.156 mmol); brown solid (60 mg, 82% yield). 1H NMR (400 MHz, DMSO-d6): δ 13.09 (s, 1H), 12.29 (s, 2H), 8.35 (d, J = 1.4 Hz, 1H), 7.61 (d, J = 1.4 Hz, 1H), 5.04 (q, J = 8.8 Hz, 2H), 2.28 (s, 3H); 13C NMR (101 MHz, DMSO-d6); δ 166.87, 160.28, 156.58, 148.44, 141.62, 133.28, 130.06, 126.69, 124.05 (q, J = 277 Hz), 117.72, 116.70, 116.05, 110.11, 109.79, 65.19 (q, J = 34.2 Hz), 11.12; HRMS (ESI) m/z: [M − H]− calcd for C16H9Cl2F3N3O4S: 465.9643; found 465.9646; HPLC purity (254 nm): 95%. 4.2.34. Methyl 3-(Benzyloxy)-4-nitrobenzoate (3s1). Prepared according to the typical procedure A from 2s (500 mg, 2.54 mmol); yellow solid (620 mg, 85% yield). mp 90−93 °C. 1H NMR (400 MHz, CDCl3): δ 7.85 (d, J = 8.3 Hz, 1H), 7.83 (d, J = 1.6 Hz, 1H), 7.70 (dd, J = 8.3, 1.6 Hz, 1H), 7.51−7.43 (m, 2H), 7.45−7.36 (m, 2H), 7.39−7.30 (m, 1H), 5.29 (s, 2H), 3.96 (s, 3H); MS (ESI): m/z calcd for C15H13NO5: 287.08. Found: 310.1 [M + Na]+. 4.2.35. 3-(Benzyloxy)-4-nitrobenzohydrazide (7s14). To a solution of ester 3s1 (4.60 g, 16.0 mmol) in MeOH (100 mL) and THF (100 mL), 80% of hydrazine monohydrate solution in water (7.78 mL, 160 mmol) was added. The reaction mixture was stirred at 65 °C overnight, and the solvent was removed under reduced pressure. The residue was suspended in ethanol and macerated at 5 °C for 1 h. The precipitate was collected and triturated with water to give the first crop (1.44 g) of the title compound. Ethanol from the mother liquor of the preceding filtration was evaporated, and the residue was purified by flash column chromatography (dichloromethane/methanol = 10:1) to give the second crop (2.10 g) of the title compound as beige solid (3.54 g, 77% combined yield); 1H NMR (400 MHz, DMSO-d6): δ 10.06 (s, 1H), 7.98 (d, J = 8.4 Hz, 1H), 7.83 (d, J = 1.6 Hz, 1H), 7.55 (dd, J = 8.4, 1.6 Hz, 1H), 7.50−7.39 (m, 4H), 7.38− 7.33 (m, 1H), 5.37 (s, 2H), 4.63 (s, 2H); MS (ESI): m/z calcd for C14H13N3O4: 287.09. Found: 285.9 [M − H]−. 4.2.36. 5-(3-(Benzyloxy)-4-nitrophenyl)-1,3,4-oxadiazol-2(3H)one (8s14). To a solution of hydrazide 7s14 (3.50 g, 12.2 mmol) in 1,4-dioxane (175 mL), CDI (2.96 g, 18.3 mmol) was added and the reaction mixture was stirred at 100 °C overnight. The solvent was removed under reduced pressure, and the residue was triturated with hot methanol to give 3.22 g of the title compound. The mother liquor was evaporated and purified by flash column chromatography 0.458 mmol); gray solid (143 mg, 68% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.28 (s, 1H), 12.25 (s, 1H), 8.28 (d, J = 1.4 Hz, 1H), 7.49 (d, J = 1.4 Hz, 1H), 4.41−4.27 (m, 2H), 3.88 (s, 3H), 3.79−3.71 (m, 2H), 2.28 (s, 3H). 4.2.23. 2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4(2-methoxyethoxy)benzo[d]thiazole-6-carboxylic Acid (10). Prepared according to the typical procedure E from 6s10 (90 mg, 0.196 mmol); off-white solid (54 mg, 62% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.98 (s, 1H), 12.25 (s, 2H), 8.23 (d, J = 1.4 Hz, 1H), 7.49 (d, J = 1.5 Hz, 1H), 4.47−4.18 (m, 2H), 3.95−3.67 (m, 2H), 3.34 (s, 3H), 2.27 (s, 3H); 13C NMR (101 MHz, DMSO-d6): δ 167.08, 159.50, 156.59, 150.30, 141.65, 132.80, 129.98, 126.76, 116.87, 116.15, 115.66, 109.96, 108.45, 70.33, 67.48, 58.12, 11.08; HRMS (ESI) m/z: [M + H]+ calcd for C17H16Cl2N3O5S 444.0188; found 444.0184; HPLC purity (254 nm): 99%. 4.2.24. Methyl 3-(2-(Dimethylamino)propoxy)-4-nitrobenzoate (3s11): Typical Procedure H for Alkylation of 3-Hydroxy-4nitrobenzoates Using the Mitsunobu Reaction. To a stirred solution of methyl 3-hydroxy-4-nitrobenzoate (2s) (492 mg, 2.49 mmol) and triphenylphosphine (1.31 g, 4.98 mmol) in anhydrous tetrahydrofuran (15 mL) was added 2-(dimethylamino)propan-1-ol (283 mg, 2.74 mmol), and the mixture was stirred at 22 °C for 10 min. DIAD (1.01 g, 4.98 mmol) was added dropwise, and the mixture was stirred at 22 °C for 15 h. The volatiles were removed under reduced pressure, and a mixture of two regioisomers was separated by column chromatography on silica, using hexane/ethyl acetate (2:1) as an eluent to give a colorless oil (176 mg, 25% yield). 1H NMR (400 MHz, CDCl3): δ 7.83 (d, J = 8.3 Hz, 1H), 7.75 (s, 1H), 7.69 (d, J = 8.3 Hz, 1H), 4.24 (dd, J = 9.1, 5.3 Hz, 1H), 4.02 (dd, J = 9.1, 6.2 Hz, 1H), 3.06 (h, J = 6.2 Hz, 1H), 2.36 (s, 6H), 1.18 (d, J = 6.2 Hz, 3H); 13C NMR (101 MHz, CDCl3): δ 165.20, 151.84, 142.59, 134.80, 125.27, 121.41, 115.56, 77.32, 77.00, 76.68, 71.86, 58.01, 52.82, 41.54, 12.38; MS (ESI): m/z calcd for C13H18N2O5: 282.12. Found: 283.13 [M + H]+. 4.2.25. Methyl 4-Amino-3-(2-(dimethylamino)propoxy)benzoate (4s11): Typical Procedure I for Reduction of 4-Nitrobenzoates with Pd/C-Catalyzed Hydrogenation. Methyl 3-(2-(dimethylamino)propoxy)-4-nitrobenzoate (3s11) (1.00 g, 3.54 mmol) was dissolved in methanol/tetrahydrofuran (7:3, 100 mL) and flushed with argon. Pd/C (10%, 16 mg) was added, and the reaction mixture was hydrogenated at 1 atm and 22 °C for 5 h. The catalyst was filtered off, and the solvent was removed in vacuo to get the title compound as a colorless oil (143 mg, quant. yield). 1H NMR (400 MHz, CDCl3): δ 7.55 (d, J = 8.2 Hz, 1H), 7.45 (s, 1H), 6.65 (d, J = 8.2 Hz, 1H), 4.50 (s, 1H), 4.07 (dd, J = 9.8, 6.4 Hz, 1H), 3.94 (dd, J = 9.8, 5.3 Hz, 1H), 3.86 (s, 3H), 3.02 (h, J = 6.5 Hz, 1H), 2.36 (s, 6H), 1.13 (d, J = 6.5 Hz, 3H) ppm; MS (ESI): m/z calcd for C13H20N2O3: 252.15. Found: 253.16 [M + H]+. 4.2.26. Methyl 2-Amino-4-(2-(dimethylamino)propoxy)benzo[d]thiazole-6-carboxylate (5s11). Prepared according to the typical procedure C from aniline 4s11 (157 mg, 0.621 mmol); orange solid (73 mg, 38% yield); 1H NMR (400 MHz, DMSO-d6): δ = 8.02 (s, 1H), 7.94 (s, 2H), 7.45 (s, 1H), 4.30 (m, 1H), 3.83 (s, 3H), 1.23 (s, 3H) (some peaks overlapped by DMSO and water signals); MS (ESI): m/z calcd for C14H19N3O3S: 309.11. Found: 310.1 [M + H]+. 4.2.27. Methyl 2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4-(2-(dimethylamino)propoxy)benzo[d]thiazole-6-carboxylate (6s11): Typical Procedure J for Acylation with 2-Trichloroacetylpyrroles. A mixture of 2-aminobenzothiazole 5s11 (74 mg, 0.240 mmol), 2-trichloroacetyl-3,4-dichloro-5-methyl-1H-pyrrole (71 mg, 0.240 mmol), and Na2CO3 (25 mg, 0.240 mmol) was suspended in dry DMF (5 mL) under Ar and stirred at 60 °C overnight; then, it was concentrated under reduced pressure and triturated sequentially with 10% citric acid, water, EtOAc, and MeOH to get the title compound as a black solid (64 mg, 55% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.36 (s, 1H), 8.33 (s, 1H), 7.62 (s, 1H), 4.50 (s, 3H), 3.89 (s, 3H), 2.27 (s, 3H), 1.39 (s, 3H) (some peaks overlapped by DMSO and water signals); MS (ESI): m/z calcd for C20H22Cl2N4O4S: 484.07. Found: 485.08 [M + H]+. 4.2.28. 2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4(2-(dimethylamino)propoxy)benzo[d]thiazole-6-carboxylic Acid (11). Prepared according to the typical procedure E from 6s11 (65 1400 https://doi.org/10.1021/acs.jmedchem.2c01597 J. Med. Chem. 2023, 66, 1380−1425 Journal of Medicinal Chemistry pubs.acs.org/jmc Article dried (Na2SO4), and concentrated to obtain a light brown solid (1.27 g, 76% yield). 1H NMR (400 MHz, DMSO-d6): δ 8.39 (d, J = 1.5 Hz, 1H), 7.65 (d, J = 1.5 Hz, 1H), 7.57−7.49 (m, 2H), 7.49−7.39 (m, 2H), 7.42−7.33 (m, 1H), 5.38 (s, 2H), 3.90 (s, 3H). 4.2.43. Methyl 4-(Benzyloxy)-2-(methylamino)benzo[d]thiazole6-carboxylate: Typical Procedure L for the Nucleophilic Substitution of 2-Bromobenzothiazoles. The Sandmeyer product 19s18 (1.27 g, 3.36 mmol) was dissolved in THF (100 mL), and methylamine (40% aqueous solution; 3.0 mL, 34 mmol) was added; the mixture was stirred at 22 °C overnight and concentrated under reduced pressure. The residue was partitioned between NH4Cl(aq) and EtOAc. The organic layer was dried (Na2SO4) and concentrated to give the title product (850 mg, 77% yield). 1H NMR (400 MHz, DMSO-d6) δ 8.35 (q, J = 4.7 Hz, 1H), 7.99 (d, J = 1.5 Hz, 1H), 7.53−7.46 (m, 2H), 7.47−7.36 (m, 3H), 7.38−7.29 (m, 1H), 5.28 (s, 2H), 3.82 (s, 3H), 2.96 (d, J = 4.7 Hz, 3H). 4.2.44. 4-(Benzyloxy)-2-(methylamino)benzo[d]thiazole-6-carboxylic Acid. Prepared according to the typical procedure E from methyl 4-(benzyloxy)-2-(methylamino)benzo[d]thiazole-6-carboxylate (850 mg, 2.59 mmol); white solid (750 mg, 92% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.67 (s, 1H), 8.34 (q, J = 4.5 Hz, 1H), 7.94 (d, J = 1.5 Hz, 1H), 7.53−7.45 (m, 2H), 7.46−7.36 (m, 3H), 7.38−7.29 (m, 1H), 5.28 (s, 2H), 2.96 (d, J = 4.5 Hz, 3H). 4.2.45. 4-Methoxybenzyl 4-(Benzyloxy)-2-(methylamino)benzo[d]-thiazole-6-carboxylate (20s18): Typical Procedure M for the Synthesis of 4-Methoxybenzyl Esters. A solution of 4-(benzyloxy)-2(methylamino)benzo[d]thiazole-6-carboxylic acid (750 mg, 2.38 mmol) in DMF (8 mL) was treated with K2CO3 (495 mg, 3.58 mmol) and 4-methoxybenzyl chloride (0.39 mL, 2.85 mmol). The resulting mixture was stirred at 22 °C overnight. After concentration under reduced pressure, the residue was partitioned between EtOAc and water, and the organic layer was dried over Na2SO4 and concentrated. The title compound was obtained after trituration with ether; beige solid (850 mg, 82% yield); 1H NMR (400 MHz, DMSOd6): δ = 8.36 (q, J = 4.7 Hz, 1H), 7.98 (d, J = 1.5 Hz, 1H), 7.52−7.44 (m, 2H), 7.44 (d, J = 1.6 Hz, 1H), 7.44−7.34 (m, 4H), 7.38−7.28 (m, 1H), 5.28 (s, 2H), 5.23 (s, 2H), 3.76 (s, 3H), 2.95 (d, J = 4.6 Hz, 3H) 13 C NMR (101 MHz, DMSO-d6): δ 165.43, 159.16, 148.06, 146.83, 137.08, 131.43, 129.90, 128.41, 128.23, 127.86, 127.83, 122.17, 116.03, 113.86, 110.77, 70.18, 65.79, 55.12, 30.80. 4.2.46. 4-Methoxybenzyl 4-(Benzyloxy)-2-(3,4-dichloro-N,5-dimethyl-1H-pyrrole-2-carboxamido)benzo[d]thiazole-6-carboxylate (21s18). Prepared according to the typical procedure D from 20s18 (850 mg, 1.96 mmol); gray solid (831 mg, 73% yield); 1H NMR (400 MHz, DMSO-d6): δ 12.41 (s, 1H), 8.29 (d, J = 1.5 Hz, 1H), 7.59 (d, J = 1.5 Hz, 1H), 7.56−7.49 (m, 2H), 7.47−7.30 (m, 5H), 6.97 (d, J = 8.7 Hz, 2H), 5.41 (s, 2H), 5.29 (s, 2H), 3.79 (s, 3H), 3.77 (s, 3H), 2.25 (s, 3H). 4.2.47. 4-(Benzyloxy)-2-(3,4-dichloro-N,5-dimethyl-1H-pyrrole-2carboxamido)benzo[d]thiazole-6-carboxylic acid (18): Typical Procedure N for Deprotection of 4-Methoxybenzyl and tert-Butyl Esters. 4-Methoxybenzyl ester 21s18 (120 mg, 0.20 mmol) was dissolved in 1 M HCl in acetic acid and stirred at 22 °C overnight. The volatiles were removed under reduced pressure, and the solid residue was triturated with diethyl ether to give the title compound as a brown solid (20 mg, 20% yield). 1H NMR (400 MHz, DMSO-d6): δ = 13.01 (s, 1H), 12.41 (s, 1H), 8.25 (d, J = 1.4 Hz, 1H), 7.59 (d, J = 1.4 Hz, 1H), 7.57−7.50 (m, 2H), 7.47−7.38 (m, 2H), 7.40−7.30 (m, 1H), 5.40 (s, 2H), 3.80 (s, 3H), 2.26 (s, 3H); HRMS (ESI) m/z: [M − H]− calcd for C22H16Cl2N3O4S 488.0244; found 488.0242; HPLC purity (254 nm): 96%. 4.2.48. 4-Methoxybenzyl 4-(Benzyloxy)-2-(3,4-dichloro-5-(phthalimidomethyl)-N-methyl-1H-pyrrole-2-carboxamido)benzo-[d]thiazole-6-carboxylate (22s19). A suspension of 3,4-dichloro-5(phthalimidomethyl)-1H-pyrrole-2-carboxylic acid36 (63 mg, 0.186 mmol) in SOCl2 (1 mL) was refluxed for 1 h; then, the reaction mixture was concentrated under reduced pressure to get a red tinted white solid. To this, crude acyl chloride 20s18 (81 mg, 0.186 mmol) and toluene (64 mL) were added and the resulting suspension was refluxed overnight. Upon cooling, the precipitate was collected, washed with toluene, and air-dried to get the product as a white (dichloromethane/methanol 30:1) to give a second crop (0.52 g) of the product as a light yellow fine powder (3.74 g, 98% combined yield). 1H NMR (400 MHz, DMSO-d6): δ 12.91 (s, 1H), 8.06 (d, J = 8.4 Hz, 1H), 7.73 (d, J = 1.6 Hz, 1H), 7.54 (dd, J = 8.4, 1.6 Hz, 1H), 7.51−7.43 (m, 2H), 7.47−7.38 (m, 2H), 7.40−7.31 (m, 1H), 5.43 (s, 2H); MS (ESI): m/z calcd for C15H11N3O5: 313.07. Found: 312.0 [M − H]−. 4.2.37. 5-(4-Amino-3-(benzyloxy)phenyl)-1,3,4-oxadiazol-2(3H)one (9s14). Prepared according to the typical procedure B from 8s14 (523 mg, 1.67 mmol); white solid; (269 mg, 57% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.21 (s, 1H), 7.55−7.47 (m, 2H), 7.45− 7.35 (m, 2H), 7.37−7.28 (m, 1H), 7.21 (d, J = 1.9 Hz, 1H), 7.16 (dd, J = 8.2, 1.8 Hz, 1H), 6.73 (d, J = 8.2 Hz, 1H), 5.54 (s, 2H), 5.18 (s, 2H); MS (ESI): m/z calcd for C15H13N3O3: 283.09. Found: 283.9 [M + H]+. 4.2.38. 5-(2-Amino-4-(benzyloxy)benzo[d]thiazol-6-yl)-1,3,4-oxadiazol-2(3H)-one (10s14). Prepared according to the typical procedure C from aniline 9s14 (201 mg, 0.709 mmol); yellow solid (200 mg, 83% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.49 (s, 1H), 7.97 (s, 2H), 7.80 (d, J = 1.5 Hz, 1H), 7.52−7.48 (m, 2H), 7.45−7.38 (m, 2H), 7.38−7.33 (m, 1H), 7.29 (d, J = 1.5 Hz, 1H), 5.27 (s, 2H); MS (ESI): m/z calcd for C16H12N4O3S3: 340.06. Found: 339.1 [M − H]−. 4.2.39. N-(4-(Benzyloxy)-6-(5-oxo-4,5-dihydro-1,3,4-oxadiazol-2yl)benzo[d]thiazol-2-yl)-3,4-dichloro-5-methyl-1H-pyrrole-2-carboxamide (14). Prepared according to the typical procedure D from the 2-aminobenzothiazole 10s14 (146 mg, 430 mmol); brown solid (200 mg, 90% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.61 (s, 1H), 12.22 (s, 1H), 12.14 (s, 1H), 8.17−8.04 (m, 1H), 7.61−7.49 (m, 2H), 7.50−7.32 (m, 4H), 5.34 (s, 2H), 2.26 (s, 3H); 13C NMR (101 MHz, DMSO-d6): δ 158.88, 156.37, 154.45, 153.84, 150.75, 140.91, 136.45, 133.65, 129.96, 128.47, 128.31, 128.16, 119.76, 116.75, 115.63, 112.04, 109.94, 104.98, 70.21, 11.04; HRMS (ESI) m/z: [M + H]+ calcd for C22H16Cl2N5O4S 516.0300; found 516.0296; HPLC purity (254 nm): 98%. 4.2.40. tert-Butyl 2-(4-(Benzyloxy)-2-(3,4-dichloro-5-methyl-1Hpyrrole-2-carboxamido)benzo[d]thiazole-6-carbonyl)hydrazine-1carboxylate (11s15). To a suspension of 1 (400 mg, 0.840 mmol) in DMF (20 mL), TBTU (324 mg, 1.00 mmol) and N-methylmorpholine (0.18 mL, 1.68 mmol) were added. After stirring the reaction mixture for 30 min, tert-butyl carbazate (122 mg, 0.923 mmol) was added. The resulting solution was stirred at 22 °C for 8 h; then, the volatiles were removed under reduced pressure. The residue was triturated with water and ethyl acetate, and the precipitated product was collected (468 mg, 94% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.23 (s, 1H), 12.15 (s, 1H), 10.31−10.22 (m, 1H), 8.97 (s, 1H), 8.13 (s, 1H), 7.96 (s, 1H), 7.62 (s, 1H), 7.55 (d, J = 7.0 Hz, 2H), 7.49−7.34 (m, 3H), 5.32 (s, 2H), 2.26 (s, 3H), 1.45 (s, 9H); MS (ESI): m/z calcd for C26H25Cl2N5O5S: 589.10. Found: 588.1 [M − H]−. 4.2.41. N-(4-(Benzyloxy)-6-(hydrazinecarbonyl)benzo[d]thiazol2-yl)-3,4-dichloro-5-methyl-1H-pyrrole-2-carboxamide Hydrochloride (15). Boc-protected compound 11s15 (400 mg, 0.677 mmol) was suspended in THF (5.0 mL), treated with 4 M HCl in dioxane (2.0 mL), and stirred at 22 °C overnight. The precipitate was collected and washed with THF to obtain the title compound as a white solid (260 mg, 73% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.36 (s, 1H), 12.24 (s, 1H), 11.81 (s, 1H), 10.70 (s, 3H), 8.25 (d, J = 1.5 Hz, 1H), 7.72 (d, J = 1.5 Hz, 1H), 7.60−7.51 (m, 2H), 7.49− 7.40 (m, 2H), 7.43−7.34 (m, 1H), 5.36 (s, 2H), 2.26 (s, 3H); HRMS (ESI) m/z: [M − Cl]− calcd for C21H18Cl2N5O3S 490.0502; found 490.0506; HPLC purity (254 nm): 95%. 4.2.42. Methyl 4-(Benzyloxy)-2-bromobenzo[d]thiazole-6-carboxylate (19s18): Typical Procedure K for the Sandmeyer Reaction of 2-Aminobenzothiazoles. Methyl 2-aminobenzo[d]thiazole-6carboxylate (18s18) (1.39 g, 4.42 mmol) and CuBr2 (1.98 g, 8.85 mmol) were dissolved in acetonitrile (30 mL) and tert-butyl nitrite (1.05 mL, 8.85 mmol) was added dropwise at 0 °C. The mixture was stirred at 22 °C overnight; then, the volatiles were removed under reduced pressure. The residue was partitioned between EtOAc (30 mL) and brine (30 mL), and the organic layer was washed with brine, 1401 https://doi.org/10.1021/acs.jmedchem.2c01597 J. Med. Chem. 2023, 66, 1380−1425 Journal of Medicinal Chemistry pubs.acs.org/jmc crystalline solid (112 mg, 80% yield). 1H NMR (400 MHz, DMSOd6): δ = 12.67 (s, 1H), 8.27 (s, 1H), 7.94−7.89 (m, 2H), 7.89−7.84 (m, 2H), 7.57 (s, 1H), 7.54−7.48 (m, 2H), 7.45−7.37 (m, 4H), 7.37−7.31 (m, 1H), 6.96 (d, J = 8.7 Hz, 2H), 5.39 (s, 2H), 5.28 (s, 2H), 4.81 (s, 2H), 3.76 (s, 3H), 3.74 (s, 3H); 13C NMR (101 MHz, DMSO-d6): δ 167.32, 165.26, 161.51, 159.22, 150.49, 141.77, 136.78, 134.53, 133.92, 131.75, 129.98, 128.50, 128.02, 127.95, 127.71, 127.66, 127.39, 126.12, 123.24, 118.77, 116.36, 113.88, 112.92, 109.90, 108.67, 70.30, 66.18, 55.14, 38.17, 33.04. 4.2.49. (5-((4-(Benzyloxy)-6-carboxybenzo[d]thiazol-2-yl)(methyl)-carbamoyl)-3,4-dichloro-1H-pyrrol-2-yl)methanaminium Chloride (19). To phthalimide 22s19 (100 mg, 0.132 mmol) in dry EtOH (2.6 mL) was added 80% hydrazine hydrate (85 μL, 1.35 mmol) and the suspension was stirred at 50 °C for 40 min. The reaction mixture was concentrated under reduced pressure, suspended in MeOH (5 mL), treated with 37% HCl(aq) (3 drops), and evaporated to get a white solid; 1H NMR (400 MHz, DMSO-d6): δ 12.54 (s, 1H), 11.25 (s, 1H), 10.66−9.74 (br s, 2H), 9.02 (t, J = 5.0 Hz, 1H), 8.30 (d, J = 1.4 Hz, 1H), 7.75−7.70 (m, 1H), 7.65−7.56 (m, 3H), 7.55−7.49 (m, 3H), 7.45−7.32 (m, 6H), 6.97 (d, J = 8.7 Hz, 2H), 5.41 (s, 2H), 5.29 (s, 2H), 4.47 (d, J = 5.0 Hz, 2H), 3.79 (s, 3H), 3.77 (s, 3H). The solid was suspended in 96% EtOH, stirred at 80 °C overnight, concentrated, and triturated with methanol to get 23s19 phthalhydrazide salt as an off-white solid (68 mg, 65%); 1H NMR (400 MHz, DMSO-d6): δ 12.67 (s, 1H), 11.66 (s, 1H), 8.31 (d, J = 1.3 Hz, 1H), 8.24 (m, 2H), 7.89 (m, 2H), 7.61 (d, J = 1.3 Hz, 1H), 7.56−7.49 (m, 2H), 7.47−7.32 (m, 5H), 6.97 (d, J = 8.7 Hz, 2H), 5.41 (s, 2H), 5.30 (s, 2H), 4.07 (s, 2H), 3.81 (s, 3H), 3.77 (s, 3H). This solid was suspended in 1 M solution of HCl in AcOH (5 mL), stirred at 22 °C overnight, and then concentrated under reduced pressure. The crude product in the form of phthalhydrazide salt was triturated with a fresh solution of HCl in acetone (5 drops of 37% HCl(aq) added to 5 mL of acetone) and washed with acetone to get the chloride salt 19 as a white solid (41 mg, 57% yield). 1H NMR (400 MHz, DMSO-d6): δ 13.05 (s, 1H), 12.77 (s, 1H), 8.32 (s, 3H), 8.27 (d, J = 0.7 Hz, 1H), 7.61 (d, J = 0.7 Hz, 1H), 7.54 (d, J = 7.2 Hz, 2H), 7.43 (t, J = 7.4 Hz, 2H), 7.36 (t, J = 7.1 Hz, 1H), 5.41 (s, 2H), 4.08 (s, 2H), 3.82 (s, 3H); HRMS (ESI) m/z: [M + H]+ calcd for C22H19Cl2N4O4S 505.0499; found 505.0488; HPLC purity (254 nm): 98%. 4.2.50. Methyl 4-(1-Phenylethoxy)-2-bromobenzo[d]thiazole-6carboxylate (19s20). Prepared according to the typical procedure K from 18s20 (1.58 g, 4.32 mmol); light brown solid (1.7 g, 90% yield); 1 H NMR (400 MHz, DMSO-d6): δ 8.30 (d, J = 1.5 Hz, 1H), 7.52− 7.42 (m, 3H), 7.36 (t, J = 7.6 Hz, 2H), 7.32−7.22 (m, 1H), 3.83 (s, 3H), 1.66 (d, J = 6.3 Hz, 3H). 4.2.51. 4-Methoxybenzyl 4-(1-Phenylethoxy)-2-(methylamino)benzo[d]thiazole-6-carboxylate (20s20). 4.2.51.1. Methyl 4-(1Phenylethoxy)-2-(methylamino)benzo[d]thiazole-6-carboxylate. Prepared according to the typical procedure L from 19s20 (1.60 g, 4.08 mmol); light yellow solid (852 mg, 61% yield). 1H NMR (400 MHz, DMSO-d6): δ 8.40 (q, J = 4.7 Hz, 1H), 7.92 (d, J = 1.6 Hz, 1H), 7.48−7.40 (m, 2H), 7.38−7.29 (m, 2H), 7.28 (d, J = 1.6 Hz, 1H), 7.29−7.19 (m, 1H), 5.79 (q, J = 6.4 Hz, 1H), 3.76 (s, 3H), 3.00 (d, J = 4.7 Hz, 3H), 1.59 (d, J = 6.4 Hz, 3H). 4.2.51.2. 4-(1-Phenylethoxy)-2-(methylamino)benzo[d]thiazole6-carboxylic Acid. Prepared according to the typical procedure E from methyl 4-(1-phenylethoxy)-2-(methylamino)benzo[d]-thiazole6-carboxylate (798 mg, 2.33 mmol); beige solid (750 mg, 98% yield). 1 H NMR (400 MHz, DMSO-d6): δ 8.45−8.34 (m, 1H), 7.88 (d, J = 1.5 Hz, 1H), 7.48−7.41 (m, 2H), 7.33 (dd, J = 8.3, 6.7 Hz, 2H), 7.27 (d, J = 1.5 Hz, 1H), 7.27−7.22 (m, 1H), 5.77 (q, J = 6.4 Hz, 1H), 3.00 (d, J = 3.9 Hz, 3H), 1.60 (d, J = 6.4 Hz, 3H). 4.2.52. 4-Methoxybenzyl 4-(1-Phenylethoxy)-2-(methylamino)benzo[d]thiazole-6-carboxylate (20s20). Prepared according to the typical procedure M from 4-(1-phenylethoxy)-2-(methylamino)benzo[d]thiazole-6-carboxylic acid (703 mg, 2.14 mmol); white solid (594 mg, 62% yield). 1H NMR (400 MHz, DMSO-d6): δ 8.39 (q, J = 4.7 Hz, 1H), 7.90 (d, J = 1.6 Hz, 1H), 7.45−7.38 (m, 2H), 7.39−7.25 (m, 5H), 7.28−7.19 (m, 1H), 6.95 (d, J = 8.7 Hz, 2H), Article 5.74 (q, J = 6.4 Hz, 1H), 5.20 (d, J = 12.1 Hz, 1H), 5.15 (d, J = 12.1 Hz, 1H), 3.76 (s, 3H), 2.99 (d, J = 4.7 Hz, 3H), 1.59 (d, J = 6.3 Hz, 3H). 4.2.53. 4-Methoxybenzyl 2-(3,4-Dichloro-N,5-dimethyl-1H-pyrrole-2-carboxamido)-4-(1-phenylethoxy)benzo[d]thiazole-6-carboxylate (21s20). Prepared according to the typical procedure D from 20s20 (365 mg, 0.814 mmol); white solid (213 mg, 44% yield). 1 H NMR (400 MHz, DMSO-d6): δ 12.42 (s, 1H), 8.22 (d, J = 1.5 Hz, 1H), 7.50−7.41 (m, 3H), 7.38 (d, J = 8.7 Hz, 2H), 7.38−7.28 (m, 2H), 7.30−7.21 (m, 1H), 6.97 (d, J = 8.7 Hz, 2H), 5.85 (q, J = 6.4 Hz, 1H), 5.25 (d, J = 12.1 Hz, 1H), 5.21 (d, J = 12.1 Hz, 1H), 3.85 (s, 3H), 3.77 (s, 3H), 2.26 (s, 3H), 1.66 (d, J = 6.4 Hz, 3H). 4.2.54. 2-(3,4-Dichloro-N,5-dimethyl-1H-pyrrole-2-carboxamido)-4-hydroxybenzo[d]thiazole-6-carboxylic Acid (20). The above 4-methoxybenzyl ester 21s20 (48 mg, 0.08 mmol) was dissolved in dichloromethane (2 mL), and 1 M solution of SnCl4 in dichloromethane (0.08 mL, 0.08 mmol) was added. The reaction mixture was stirred at 22 °C for 2 h, water was added to the mixture, and the light purple precipitate of product 20 was filtered off (30 mg, 94% yield). 1 H NMR (400 MHz, DMSO-d6): δ 12.85 (s, 1H), 12.40 (s, 1H), 10.27 (s, 1H), 8.05 (d, J = 1.6 Hz, 1H), 7.45 (d, J = 1.7 Hz, 1H), 3.80 (s, 3H), 2.26 (s, 3H); HRMS (ESI) m/z: [M + H]+ calcd for C15H12Cl2N3O4S 399.9926; found 399.9942; HPLC purity (254 nm): 96%. 4.2.55. 4-Methoxybenzyl 2-Aminobenzo[d]thiazole-6-carboxylate (18s21). Prepared according to the typical procedure M from 2-aminobenzo[d]thiazole-6-carboxylic acid (1.00 g, 5.16 mmol); light yellow solid (730 mg 45% yield). 1H NMR (400 MHz, DMSO-d6): δ 8.84−8.81 (m, 1H), 8.11−8.07 (m, 2H), 7.45 (d, J = 8.7 Hz, 2H), 6.97 (d, J = 8.7 Hz, 2H), 5.32 (s, 2H), 3.77 (s, 3H); MS (ESI): m/z calcd for C16H14N2O3S: 314.07. Found: 313.0 [M − H]−. 4.2.56. 4-Methoxybenzyl 2-Bromobenzo[d]thiazole-6-carboxylate (19s21). Prepared according to the typical procedure K from amine 18s21 (232 mg, 0.740 mmol); orange powder (280 mg, 100% yield). 1H NMR (400 MHz, DMSO-d6): δ 8.87−8.79 (m, 1H), 8.14− 8.04 (m, 2H), 7.45 (d, J = 8.6 Hz, 2H), 6.97 (d, J = 8.6 Hz, 2H), 5.32 (s, 2H), 3.77 (s, 3H); MS (ESI): m/z calcd for C16H12BrNO3S: 376.97. Found: 398.8 [M + Na − 2H]−. 4.2.57. 4-Methoxybenzyl 2-((2-Methoxyethyl)amino)benzo[d]thiazole-6-carboxylate (20s21). Prepared according to the typical procedure L from 19s21 (451 mg, 1.19 mmol) and 2-methoxyethylamine; beige solid (244 mg, 55% yield). 1H NMR (400 MHz, DMSOd6): δ 8.54 (t, J = 5.1 Hz, 1H), 8.30 (d, J = 1.7 Hz, 1H), 7.82 (dd, J = 8.4, 1.9 Hz, 1H), 7.45−7.38 (m, 3H), 6.96 (d, J = 8.7 Hz, 1H), 5.25 (s, 2H), 3.76 (s, 3H), 3.61−3.48 (m, 4H), 3.28 (s, 3H); MS (ESI): m/z calcd for C19H20N2O4S: 372.11. Found: 371.0 [M − H]−. 4.2.58. 4-Methoxybenzyl 2-(3,4-Dichloro-N-(2-methoxyethyl)-5methyl-1H-pyrrole-2-carboxamido)benzo[d]thiazole-6-carboxylate (21s21). Prepared according to the typical procedure D from 20s21 (123 mg, 0.331 mmol); pink solid 96 mg (56% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.51 (s, 1H), 8.68 (d, J = 1.7 Hz, 1H), 8.03 (dd, J = 8.6, 1.8 Hz, 1H), 7.90 (d, J = 8.5 Hz, 1H), 7.45 (d, J = 8.7 Hz, 2H), 6.97 (d, J = 8.7 Hz, 2H), 5.31 (s, 2H), 4.54 (t, J = 4.8 Hz, 2H), 3.77 (s, 3H), 3.60 (t, J = 4.8 Hz, 2H), 3.07 (s, 3H), 2.25 (s, 3H); MS (ESI): m/z calcd for C25H23Cl2N3O5S: 547.07. Found: 546.0 [M − H]−. 4.2.59. 2-(3,4-Dichloro-N-(2-methoxyethyl)-5-methyl-1H-pyrrole-2-carboxamido)benzo[d]thiazole-6-carboxylic Acid (21). Prepared according to the typical procedure N from ester 21s21 (56 mg, 0.108 mmol); light pink powder (19 mg, 41% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.98 (s, 1H), 12.50 (s, 1H), 8.63 (d, J = 1.6 Hz, 1H), 8.02 (dd, J = 8.5, 1.6 Hz, 1H), 7.88 (d, J = 8.5 Hz, 1H), 4.55 (t, J = 4.8 Hz, 2H), 3.61 (t, J = 4.8 Hz, 2H), 3.08 (s, 3H), 2.25 (s, 3H); HRMS (ESI) m/z: [M + H]+ calcd for C17H16Cl2N3O4S 428.0239; found: 428.0229; HPLC purity (254 nm): 97%. 4.2.60. tert-Butyl 4-Nitrobenzoate (13s). To a solution of 4nitrobenzoic acid (1.67 g, 10.0 mmol) in pyridine (50 mL), tosyl chloride (3.8 g, 20 mmol) was added and the reaction mixture was cooled to 0 °C. tert-Butanol (1.48 g, 20 mmol) was added, and the mixture was stirred for 2 h at 0 °C and 16 h at 22 °C. The volatiles were removed under reduced pressure, and the mixture was 1402 https://doi.org/10.1021/acs.jmedchem.2c01597 J. Med. Chem. 2023, 66, 1380−1425 Journal of Medicinal Chemistry pubs.acs.org/jmc partitioned between DCM (150 mL) and saturated NaHCO3 solution (150 mL). The organic layer was washed twice with saturated NaHCO3 solution, and the combined NaHCO3 layers were extracted once with DCM (100 mL). The combined organic layers were washed with brine and subsequently with 1 M NaHSO4 (3 × 100 mL), dried over Na2SO4, filtered, and concentrated in vacuo to give a yellow solid (2.0 g, 90% yield). 1H NMR (500 MHz, CDCl3): δ 8.28−8.23 (m, 2H), 8.17−8.12 (m, 2H), 1.62 (s, 9H). 4.2.61. tert-Butyl 4-Aminobenzoate (14s). Prepared according to the typical procedure I from 13s (2.0 g, 9 mmol); off-white solid (1.60 g, 92% yield). 1H NMR (500 MHz, CDCl3): δ 7.80 (d, J = 8.2 Hz, 2H), 6.62 (d, J = 8.1 Hz, 2H), 3.93 (bs, 2H), 1.56 (s, 9H). 4.2.62. tert-Butyl 2-Aminobenzo[d]thiazole-6-carboxylate (15s). Prepared according to the typical procedure C from 14s (1.60 g, 8.28 mmol); dark yellow solid (1.70 g, 82% yield). 1H NMR (500 MHz, DMSO-d6): δ 8.21 (d, J = 1.8 Hz, 1H), 7.87 (s, 2H), 7.75 (dd, J = 8.4 Hz, 1.8 Hz, 1H), 7.34 (d, J = 8.4 Hz, 1H), 1.54 (s, 9H). 13C NMR (126 MHz, DMSO-d6): δ 169.99, 165.39, 157.07, 131.45, 127.44, 123.96, 122.84, 117.42, 80.60, 28.37. MS (ESI): m/z calcd for C12H14N2O2S: 250.08. Found: 251.2 [M + H]+. 4.2.63. tert-Butyl 2-(3,4-Dichloro-5-methyl-1H-pyrrole-2carboxamido)benzo[d]thiazole-6-carboxylate (16s). Prepared according to the typical procedure D from 2-amino-benzothiazole 15s (150 mg, 0.597 mmol); gray solid (145 mg, 57% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.38 (s, 1H), 11.93 (s, 1H), 8.59 (s, 1H), 7.97 (dd, J = 8.5 Hz, 1.8 Hz, 1H), 7.81 (s, 1H), 2.27 (s, 3H), 1.58 (s, 9H). 4.2.64. tert-Butyl 3-Benzyl-2-((3,4-dichloro-5-methyl-1H-pyrrole2-carbonyl)imino)-2,3-dihydrobenzo[d]thiazole-6-carboxylate (17s22): Typical Procedure O for Benzothiazole N-Alkylation. To a suspension of 16s (100 mg, 0.235 mmol), KI (20 mg, 0.12 mmol), and NaHCO3 (24 mg, 0.28 mmol) in DMF (5 mL) was added benzyl bromide (31 μL, 0.26 mmol), and the resulting mixture was stirred at 60 °C overnight. The volatiles were removed under reduced pressure, and the residue was triturated successively with water and methanol to obtain the title compound as a gray solid (98 mg, 81% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.09 (s, 1H), 8.46 (d, J = 1.7 Hz, 1H), 7.95 (dd, J = 8.6 Hz, 1.7 Hz, 1H), 7.64 (d, J = 8.6 Hz, 1H), 7.42−7.37 (m, 2H), 7.35−7.30 (m, 2H), 7.30−7.23 (m, 1H), 5.92 (s, 2H), 2.25 (s, 3H), 1.55 (s, 9H). 4.2.65. 3-Benzyl-2-((3,4-dichloro-5-methyl-1H-pyrrole-2carbonyl)imino)-2,3-dihydrobenzo[d]thiazole-6-carboxylic Acid (22). Prepared according to the typical procedure N from 17s22 (80 mg, 0.155 mmol); gray solid (60 mg, 84% yield). 1H NMR (400 MHz, DMSO-d6): δ 13.0 (s, 1H), 12.08 (s, 1H), 8.50 (s, 1H), 7.99 (d, J = 8.5 Hz, 1H), 7.64 (d, J = 8.5 Hz, 1H), 7.50−7.15 (m, 5H), 5.92 (s, 2H), 2.25 (s, 3H); 13C NMR (101 MHz, DMSO-d6): δ 167.15, 166.51, 165.67, 139.62, 135.71, 129.24, 128.75, 128.40, 127.73, 127.36, 126.32, 126.23, 124.65, 121.93, 114.41, 112.37, 109.68, 48.23, 10.92; 1H−1H NOE observed between benzylic hydrogens and benzothiazole 4-H; HRMS calcd for C21H16Cl2N3O3S: 460.0289. Found: 460.0287 [M + H]+; HPLC purity (254 nm): 99%. 4.2.66. tert-Butyl 2-((3,4-Dichloro-5-methyl-1H-pyrrole-2-carbon-yl)imino)-3-(4-(methylsulfonyl)benzyl)-2,3-dihydroben-zo[d]thiazole-6-carboxylate (17s23). Prepared according to the typical procedure O from 16s (123 mg, 0.288 mmol) and 4-mesylbenzyl bromide (79 mg, 0.317 mmol); gray solid (125 mg, 73% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.10 (s, 1H), 8.49 (d, J = 1.7 Hz, 1H), 7.96 (dd, J = 8.6 Hz, 1.7 Hz, 1H), 7.89 (d, J = 8.5 Hz, 2H), 7.70−7.55 (m, 3H), 6.03 (s, 2H), 3.17 (s, 3H), 2.25 (s, 3H), 1.55 (s, 9H). 4.2.67. 2-((3,4-Dichloro-5-methyl-1H-pyrrole-2-carbonyl)imino)3-(4-(methylsulfonyl)benzyl)-2,3-dihydrobenzo[d]thiazole-6-carboxylic Acid (23). Prepared according to the typical procedure N from tert-butyl ester 17s23 (100 mg, 0.169 mmol); gray solid (70 mg, 77% yield). 1H NMR (400 MHz, DMSO-d6): δ 13.08 (s, 1H), 12.08 (s, 1H), 8.53 (d, J = 1.7 Hz, 1H), 8.00 (dd, J = 8.6, 1.7 Hz, 1H), 7.89 (d, J = 8.5 Hz, 2H), 7.67−7.58 (m, 3H), 6.03 (s, 2H), 3.17 (s, 3H), 2.25 (s, 3H); HRMS (ESI) m/z: [M − H] − calcd for C22H16Cl2N3O5S2 535.9908; found 535.9913; HPLC purity (254 nm): 95%. Article 4.2.68. tert-Butyl 2-((3,4-Dichloro-5-methyl-1H-pyrrole-2-carbon-yl)imino)-3-(2-(4-(methylsulfonyl)phenyl)-2-oxoethyl)-2,3dihydrobenzo[d]thiazole-6-carboxylate (17s24). Prepared according to the typical procedure O from 16s (117 mg, 0.275 mmol) and 4methylsulfonylphenacyl bromide (84 mg, 0.302 mmol); gray solid (120 mg, 70% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.13 (s, 1H), 8.53 (d, J = 1.7 Hz, 1H), 8.40 (d, J = 8.5 Hz, 2H), 8.21 (d, J = 8.5 Hz, 2H), 7.98 (dd, J = 8.7 Hz, 1.7 Hz, 1H), 7.79 (d, J = 8.7 Hz, 1H), 6.33 (s, 2H), 2.20 (s, 3H), 1.58 (s, 9H). 4.2.69. 2-((3,4-Dichloro-5-methyl-1H-pyrrole-2-carbonyl)imino)3-(2-(4-(methylsulfonyl)phenyl)-2-oxoethyl)-2,3-dihydro-benzo[d]thiazole-6-carboxylic Acid (24). Prepared according to the typical procedure N from 17s24 (100 mg, 0.160 mmol); gray solid (48 mg, 53% yield). 1H NMR (400 MHz, DMSO-d6): δ 13.09 (s, 1H), 12.11 (s, 1H), 8.56 (d, J = 1.7 Hz, 1H), 8.40 (d, J = 8.6 Hz, 2H), 8.21 (d, J = 8.6 Hz, 2H), 8.03 (dd, J = 8.6 Hz, 1.7 Hz, 1H), 7.79 (d, J = 8.6 Hz, 1H), 6.33 (s, 2H), 3.36 (s, 3H), 2.19 (s, 3H); HRMS (ESI) m/z: [M + H]+ calcd for C23H18Cl2N3O5S2: 566.0014; found: 566.0007; HPLC purity (254 nm): 96%. 4.2.70. tert-Butyl 2-((3,4-Dichloro-5-methyl-1H-pyrrole-2-carbon-yl)imino)-3-(2-oxo-2-phenylethyl)-2,3-dihydrobenzo[d]thiazole-6-carboxylate (17s25). Prepared according to the typical procedure O from 16s (100 mg, 0.234 mmol) and phenacyl bromide (51 mg, 0.257 mmol); gray solid (80 mg, 63% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.13 (s, 1H), 8.52 (d, J = 1.7 Hz, 1H), 8.20− 8.13 (m, 2H), 7.97 (dd, J = 8.7 Hz, 1.7 Hz, 1H), 7.81−7.73 (m, 2H), 7.70−7.62 (m, 2H), 6.27 (s, 2H), 2.19 (s, 3H), 1.58 (s, 9H). 4.2.71. 2-((3,4-Dichloro-5-methyl-1H-pyrrole-2-carbonyl)imino)3-(2-oxo-2-phenylethyl)-2,3-dihydrobenzo[d]thiazole-6-carboxylic Acid (25). Prepared according to the typical procedure N from tertbutyl ester 17s25 (70 mg, 0.128 mmol); gray solid (10 mg, 16% yield). 1H NMR (400 MHz, DMSO-d6): δ 13.08 (s, 1H), 12.11 (s, 1H), 8.55 (d, J = 1.7 Hz, 1H), 8.16 (d, J = 7.6 Hz, 2H), 8.02 (dd, J = 8.6 Hz, 1.7 Hz, 1H), 7.84−7.72 (m, 2H), 7.70−7.61 (m, 2H), 6.28 (s, 2H), 2.19 (s, 3H); HRMS (ESI) m/z: [M + H]+ calcd for C22H16Cl2N3O4S 488.0239; found 488.0232; HPLC purity (254 nm): 95%. 4.2.72. Ethyl 2-Bromobenzo[d]thiazole-6-carboxylate (19s26). Prepared according to the typical procedure K from ethyl 2aminobenzo[d]thiazole-6-carboxylate (2.95 g, 13.27 mmol); light brown solid (3.80 g, 100% yield). 1H NMR (400 MHz, DMSO-d6): δ 8.82 (dd, J = 1.6 Hz, 0.8 Hz, 1H), 8.14−8.07 (m, 1H), 8.12−8.04 (m, 1H), 4.36 (q, J = 7.1 Hz, 2H), 1.35 (t, J = 7.1 Hz, 3H). 4.2.73. 4-Methoxybenzyl 2-(Methylamino)benzo[d]thiazole-6carboxylate (20s26). 4.2.73.1. Ethyl 2-(Methylamino)benzo[d]thiazole-6-carboxylate. Prepared from the Sandmeyer product 19s26 (1.01 g, 3.53 mmol); beige solid (250 mg, 30% yield). 1H NMR (400 MHz, DMSO-d6): δ 8.35 (q, J = 5 Hz, 1H), 8.30 (d, J = 1.8 Hz, 1H), 7.83 (dd, J = 8.4 Hz, 1.8 Hz, 1H), 7.43 (d, J = 8.4 Hz, 1H), 4.29 (q, J = 7.1 Hz, 2H), 2.97 (d, J = 4.7 Hz, 3H), 1.32 (t, J = 7.1 Hz, 3H). 4.2.73.2. 2-(Methylamino)benzo[d]thiazole-6-carboxylic Acid. Prepared according to the typical procedure E from ethyl 2(methylamino)benzo[d]thiazole-6-carboxylate (603 mg, 2.55 mmol); white solid (340 mg, 64% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.90−12.43 (m, 1H), 8.50−8.05 (m, 2H), 8.10−7.67 (m, 1H), 7.59−7.26 (m, 1H), 3.04−2.86 (m, 3H); MS (ESI): m/z calcd for C9H8N2O2S: 208.03. Found: 208.8 [M + H]+. 4.2.74. 4-Methoxybenzyl 2-(Methylamino)benzo[d]thiazole-6carboxylate (20s26). Prepared according to the typical procedure M from 2-(methylamino)benzo[d]thiazole-6-carboxylic acid (327 mg, 1.57 mmol); off-white solid (155 mg, 30% yield). 1H NMR (400 MHz, DMSO-d6): δ 8.36 (q, J = 4.7 Hz, 1H), 8.31 (d, J = 1.8 Hz, 1H), 7.83 (dd, J = 8.5 Hz, 1.8 Hz, 1H), 7.46−7.38 (m, 3H), 6.96 (d, J = 8.7 Hz, 2H), 5.25 (s, 2H), 3.76 (s, 3H), 2.96 (d, J = 4.7 Hz, 3H). 4.2.75. 4-Methoxybenzyl 2-(3,4-Dichloro-N,5-dimethyl-1H-pyrrole-2-carboxamido)benzo[d]thiazole-6-carboxylate (21s26). Prepared according to the typical procedure D from 20s26 (154 mg, 0.468 mmol); off-white solid (40 mg, 18% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.43 (s, 1H), 8.70 (d, J = 1.7 Hz, 1H), 8.04 (dd, J = 8.5 Hz, 1.7 Hz, 1H), 7.93 (d, J = 8.5 Hz, 1H), 7.45 (d, J = 8.7 Hz, 1403 https://doi.org/10.1021/acs.jmedchem.2c01597 J. Med. Chem. 2023, 66, 1380−1425 Journal of Medicinal Chemistry pubs.acs.org/jmc 2H), 6.97 (d, J = 8.7 Hz, 2H), 5.31 (s, 2H), 3.80 (s, 3H), 3.77 (s, 3H), 2.26 (s, 3H); MS (ESI): m/z calcd for C23H19Cl2N3O3S: 503.1. Found: 501.9 [M − H]−. 4.2.76. 2-(3,4-Dichloro-N,5-dimethyl-1H-pyrrole-2-carboxamido)-benzo[d]thiazole-6-carboxylic Acid (26). Prepared according to the typical procedure N from PMB ester 21s26 (37 mg, 0.078 mmol); light brown solid (20 mg, 66% yield). 1H NMR (400 MHz, DMSO-d6): δ 13.0 (s, 1H), 12.43 (s, 1H), 8.65 (d, J = 1.7 Hz, 1H), 8.02 (dd, J = 8.5 Hz, 1.7 Hz, 1H), 7.91 (d, J = 8.5 Hz, 1H), 3.80 (s, 3H), 2.26 (s, 3H); HRMS (ESI) m/z: [M − H]− calcd for C15H10Cl2N3O3S 381.9820; found 381.9825; HPLC purity (254 nm): 96%. 4.2.77. Methyl 2-Amino-4-(1-phenylethoxy)benzo[d]thiazole-6carboxylate (5s27). Prepared according to the typical procedure F from 1s33 (682 mg, 3.04 mmol) and 1-chloro-1-phenylethan (0.50 mL; 3.65 mmol); white solid (131 mg, 13% yield). 1H NMR (400 MHz, DMSO-d6): δ 7.94 (s, 2H), 7.87 (d, J = 1.5 Hz, 1H), 7.45−7.39 (m, 2H), 7.33 (app t, J = 7.5 Hz, 2H), 7.27−7.23 (m, 2H), 5.73 (q, J = 6.3 Hz, 1H), 3.76 (s, 3H), 1.57 (d, J = 6.4 Hz, 3H). 4.2.78. Methyl 2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4-(1-phenylethoxy)benzo[d]thiazole-6-carboxylate (6s27). Prepared according to the typical procedure D from 24s27 (250 mg, 0.762 mmol); white solid (210 mg, 55% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.32 (s, 1H), 12.28 (s, 1H), 8.19 (s, 1H), 7.46 (d, J = 7.3 Hz, 2H), 7.40−7.33 (m, 3H), 7.29−7.24 (m, 1H), 5.81 (q, J = 6.5 Hz, 1H), 3.81 (s, 3H), 2.29 (s, 3H), 1.65 (d, J = 6.5 Hz, 3H); 13 C NMR (101 MHz, DMSO-d6): δ 166.25, 160.33, 156.97, 149.52, 142.95, 133.39, 130.36, 129.10 (2C), 128.09, 126.03 (2C), 125.59, 117.21, 116.36, 116.33, 110.74, 110.52, 76.08, 52.59, 24.75, 11.55. 4.2.79. 2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4(1-phenylethoxy)benzo[d]thiazole-6-carboxylic Acid (27). Prepared according to the typical procedure E from the methyl ester 6s27 (100 mg, 0.198 mmol); white solid (75 mg, 77% yield). Mp > 300 °C; 1H NMR (400 MHz, DMSO-d6): δ 12.87 (s, 1H), 12.29 (s, 2H), 8.15 (s, 1H), 7.49−7.43 (m, 2H), 7.40−7.32 (m, 3H), 7.26 (t, J = 7.3 Hz, 1H), 5.79 (q, J = 6.3 Hz, 1H), 2.28 (s, 3H), 1.64 (d, J = 6.3 Hz, 3H); 13 C NMR (101 MHz, DMSO-d6): δ 166.88, 159.56, 156.65, 149.00, 142.56, 142.12, 132.81, 129.90, 128.61, 127.56, 126.51, 125.54, 116.86, 115.98, 115.76, 110.59, 109.98, 75.57, 24.31, 11.06; HRMS (ESI) m/z: [M + H]+ calcd for C22H18Cl2N3O4S 490.0395; found 490.0382; HPLC purity (254 nm): 96%. 4.2.80. Methyl (S)-2-Amino-4-(1-phenylethoxy)benzo[d]thiazole6-carboxylate (24sS27). 4.2.80.1. Methyl (S)-4-Nitro-3-(1phenylethoxy)benzoate. Prepared according to the typical procedure H from 2s (2.52 g, 12.77 mmol) and (R)-1-phenylethanol (1.72 g,14.05 mmol); white solid (3.00 g, 78% yield). 1H NMR (400 MHz, CDCl3): δ 7.76 (d, J = 8.3 Hz, 1H), 7.64 (d, J = 1.5 Hz, 1H), 7.60 (dd, J = 8.3 Hz, 1.5 Hz, 1H), 7.44−7.40 (m, 2H), 7.39−7.33 (m, 2H), 7.31−7.26 (m, 1H), 5.54 (q, J = 6.4 Hz, 1H), 3.89 (s, 3H), 1.70 (d, J = 6.4 Hz, 3H). 4.2.80.2. Methyl (S)-4-Amino-3-(1-phenylethoxy)benzoate. Prepared according to the typical procedure B from methyl (S)-4-nitro-3(1-phenylethoxy)benzoate (3.00 g, 9.95 mmol); white solid (2.70 g, 100% yield). 1H NMR (400 MHz, DMSO-d6): δ 7.44 (d, J = 7.2 Hz, 2H), 7.34 (t, J = 7.5 Hz, 2H), 7.30 (dd, J = 8.2 Hz, 1.7 Hz, 1H), 7.25 (t, J = 7.3 Hz, 1H), 7.21 (d, J = 1.7 Hz, 1H), 6.62 (d, J = 8. Hz, 1H), 5.70 (s, 2H), 5.49 (q, J = 6.3 Hz, 1H), 3.68 (s, 3H), 1.56 (d, J = 6.3 Hz, 3H). 4.2.81. Methyl (S)-2-Amino-4-(1-phenylethoxy)benzo[d]thiazole6-carboxylate (24sS27). Prepared according to the typical procedure C from methyl (S)-4-amino-3-(1-phenylethoxy)-benzoate (2.31 g, 8.53 mol); white powder (925 mg, 33% yield). 1H NMR (400 MHz, DMSO-d6): δ 8.02 (s, 2H), 7.88 (d, J = 1.5 Hz, 1H), 7.45−7.40 (m, 2H), 7.34 (t, J = 7.5 Hz, 2H), 7.28−7.22 (m, 2H), 5.73 (q, J = 6.3 Hz, 1H), 3.76 (s, 3H), 1.58 (d, J = 6.3 Hz, 3H); 68% ee determined by chiral HPLC analysis on a Kromasil 3-CelluCoat column (4.6 mm × 150 mm), eluent hexane/MeOH/i-PrOH = 90:5:5; tR = 6.8 min (S enantiomer), 7.4 min (R enantiomer). 4.2.82. Methyl (S)-2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4-(1-phenylethoxy)benzo[d]thiazole-6-carboxylate (25sS27). Prepared according to the typical procedure D from 2- Article aminobenzothiazole 24sS27 (253 mg, 0.77 mmol); white solid (220 mg, 57% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.30 (s, 1H), 12.26 (s, 1H), 8.18 (s, 1H), 7.46 (d, J = 7.4 Hz, 2H), 7.43−7.33 (m, 3H), 7.26 (t, J = 7.3 Hz, 1H), 5.81 (q, J = 6.4 Hz, 1H), 3.81 (s, 3H), 2.28 (s, 3H), 1.65 (d, J = 6.4 Hz, 3H). 4.2.83. (S)-2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)4-(1-phenylethoxy)benzo[d]thiazole-6-carboxylic Acid [(S)-27]. Prepared according to the typical procedure E from methyl ester 25sS27 (150 mg, 0.297 mmol); brown powder (125 mg, 86% yield). 1 H NMR (400 MHz, DMSO-d6): δ = 12.85 (s, 1H), 12.26 (s, 2H), 8.15 (s, 1H), 7.46 (app d, J = 7.2 Hz, 2H), 7.40−7.32 (m, 3H), 7.26 (app t, J = 7.5 Hz, 1H), 5.79 (q, J = 6.3 Hz, 1H), 2.28 (s, 3H), 1.65 (d, J = 6.3 Hz, 3H); MS (ESI): m/z calcd for C22H17Cl2N3O4S: 489.03. Found 488.2 [M − H]−; HPLC purity (254 nm): 96% (sum of enantiomers); 74% ee determined by chiral HPLC analysis on a Kromasil 3-CelluCoat column (4.6 mm × 150 mm), eluent hexane/ MeOH/0.1%TFA in i-PrOH = 90:5:5, flow rate: 1 mL/min; tR = 7.7 min (S enantiomer), 9.5 min (R enantiomer). The absolute configuration of the major product was assigned based on the starting (R)-1-phenylethanol. 4.2.84. Methyl (R)-2-Amino-4-(1-phenylethoxy)benzo[d]thiazole6-carboxylate (24sR27). 4.2.84.1. Methyl (R)-4-Nitro-3-(1phenylethoxy)benzoate. Prepared according to the typical procedure H from 2s (2.62 g, 13.28 mmol) and (S)-1-phenylethanol (1.79 g, 14,62 mmol); white solid (2.08 g, 52% yield). MS (ESI): m/z calcd for C16H15NO5: 301.09. Found: 300.3 [M − H]−. 4.2.84.2. Methyl (R)-4-Amino-3-(1-phenylethoxy)benzoate. Prepared according to the typical procedure B from methyl (R)-4-nitro-3(1-phenylethoxy)benzoate (2.05 g, 6.80 mmol); (1.42g, 77% yield). 1 H NMR (400 MHz, DMSO-d6): δ 7.47−7.39 (m, 2H), 7.36−7.19 (m, 5H), 6.62 (d, J = 8.0 Hz, 1H), 5.70 (s, 2H), 5.48 (d, J = 6 Hz, 1H), 3.68 (s, 3H), 1.56 (d, J = 6 Hz, 3H); MS (ESI): m/z calcd for C16H17NO5: 271.12. Found: 272.2 [M + H]+. 4.2.85. Methyl (R)-2-Amino-4-(1-phenylethoxy)benzo[d]thiazole6-carboxylate (24sR27). Prepared according to the typical procedure C from methyl (R)-4-amino-3-(1-phenylethoxy)-benzoate (1.36 g, 5.00 mmol); (443 mg, 27% yield). 1H NMR (400 MHz, DMSO-d6): δ 7.95 (s, 2H), 7.88 (d, J = 1.5 Hz, 1H), 7.44−7.41 (m, 2H), 7.34 (app t, J = 7.5 Hz, 2H), 7.29−7.20 (m, 2H), 5.73 (q, J = 6.3 Hz, 1H), 3.76 (s, 3H), 1.58 (d, J = 6.3 Hz, 3H); MS (ESI): m/z calcd for C17H16N2O3S: 328.09. Found: 327.3 [M − H]−; 88.6% ee determined by chiral HPLC analysis on a Kromasil 3-CelluCoat column (4.6 mm × 150 mm), eluent hexane/MeOH/i-PrOH = 90:5:5; tR = 6.8 min (S enantiomer), 7.4 min (R enantiomer). 4.2.86. Methyl (R)-2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4-(1-phenylethoxy)benzo[d]thiazole-6-carboxylate (25sR27). Prepared according to the typical procedure D from 2aminobenzothiazole 24sR27 (240 mg, 0.73 mmol); (48 mg, 13% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.30 (s, 1H), 12.26 (s, 1H), 8.19 (s, 1H), 7.46 (app d, J = 7.5 Hz, 2H), 7.40−7.33 (m, 3H), 7.29−7.24 (m, 1H), 5.81 (q, J = 6 Hz, 1H), 3.81 (s, 3H), 2.29 (s, 3H), 1.65 (d, J = 6 Hz, 3H). 4.2.87. (R)-2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4-(1-phenylethoxy)benzo[d]thiazole-6-carboxylic Acid [(R)27]. Prepared according to the typical procedure E from the methyl ester 25sR27 (45 mg, 0.089 mmol); red-brown powder (26 mg, 61% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.86 (s, 1H), 12.26 (s, 2H), 8.14 (d, J = 1.1 Hz, 1H), 7.46 (app d, J = 7.2 Hz, 2H), 7.39− 7.32 (m, 3H)., 7.29−7.23 (m, 1H), 5.79 (q, J = 6.3 Hz, 1H), 2.28 (s, 3H), 1.65 (d, J = 6.3 Hz, 3H); HRMS (ESI) m/z: [M + H]+ calcd for C22H18Cl2N3O4S 490.0395; found 490.0389; HPLC purity (254 nm): 97% (sum of enantiomers); 63% ee determined by chiral HPLC analysis on a Kromasil 3-CelluCoat column (4.6 mm × 150 mm), eluent hexane/MeOH/0.1% TFA in i-PrOH = 90:5:5; tR = 7.6 min (S enantiomer), 9.5 min (R enantiomer). The absolute configuration of the major product was assigned based on the starting (S)-1phenylethanol. 4.2.88. Methyl 4-Amino-3-(2,2,2-trifluoro-1-phenylethoxy)benzoate (4s28). 2,2,2-Trifluoro-1-phenylethanol (0.546 mL, 4.02 mmol) was dissolved in dry DMF (20 mL) on an ice bath and flushed with argon; then, NaH (60% suspension in mineral oil; 177 mg, 4.42 1404 https://doi.org/10.1021/acs.jmedchem.2c01597 J. Med. Chem. 2023, 66, 1380−1425 Journal of Medicinal Chemistry pubs.acs.org/jmc Article °C for 3 h, the volatiles were removed in high vacuum (<10−2 mbar) and the solid residue was washed several times with hot toluene to remove most of the 4-TBDMSO impurity, yielding the title compound; gray solid (30 mg, 13% yield). 1H NMR (400 MHz, DMSO-d6) (characteristic signals): δ 8.35 (s, 1H), 7.71 (s, 1H), 6.06 (s, 1H), 3.92 (s, 3H), 2.26 (s, 3H). 4.2.96. 4-(Benzocyclobutan-1-yloxy)-2-(3,4-dichloro-5-methyl1H-pyrrole-2-carboxamido)-benzo[d]thiazole-6-carboxylic Acid (29). Prepared according to the typical procedure E from methyl ester 6s29 (30 mg, 0.060 mmol); brown powder (12 mg, 41% yield). 1 H NMR (400 MHz, DMSO-d6): δ 13.0 (br s, 1H), 12.24 (s, 1H), 12.19 (s, 1H), 8.30 (s, 1H), 7.71 (s, 1H), 7.46−7.35 (m, 1H), 7.34− 7.22 (m, 3H), 6.04 (d, J = 2.5 Hz, 1H), 3.82 (dd, J = 14.2, 4.1 Hz, 1H), 3.27 (d, J = 13.5 Hz, 1H), 2.26 (s, 3H). HRMS (ESI) m/z: [M + H]+ calcd for C22H16Cl2N3O4S 488. 0239; found 488.0226; HPLC purity (220 nm): 91%. 4.2.97. Methyl 3-(2-(Benzoyloxy)-1-phenylethoxy)-4-nitrobenzoate (3s30). Prepared according to the typical procedure H from 2s (203 mg, 1.03 mmol) and 2-hydroxy-2-phenylethyl benzoate (259 mg, 1.13 mmol); white crystals (341 mg, 79% yield). 1H NMR (400 MHz, DMSO-d6): δ 7.97 (d, J = 8.4 Hz, 1H), 7.91−7.85 (m, 1H), 7.82 (d, J = 1.5 Hz, 1H), 7.69−7.59 (m, 2H), 7.60−7.53 (m, 2H), 7.53−7.48 (m, 2H), 7.48−7.40 (m, 2H), 7.40−7.32 (m, 1H), 6.29− 6.18 (m, 1H), 4.69−4.59 (m, 2H), 3.84 (s, 3H). 4.2.98. Methyl 4-Amino-3-(2-(benzoyloxy)-1-phenylethoxy)benzoate (4s30). Prepared according to the typical procedure B from 3s30 (649 mg, 1.54 mmol); yellow oil (414 mg, 69% yield). 1H NMR (400 MHz, DMSO-d6): δ 7.95−7.91 (m, 2H), 7.70−7.61 (m, 1H), 7.62−7.56 (m, 2H), 7.54−7.48 (m, 2H), 7.44−7.38 (m, 2H), 7.36−7.31 (m, 2H), 7.29 (d, J = 1.8 Hz, 1H), 6.64 (d, J = 8.3 Hz, 1H), 5.78 (t, J = 5.3 Hz, 1H), 4.72 (dd, J = 11.5, 7.6 Hz, 1H), 4.56 (dd, J = 11.5, 3.6 Hz, 1H), 3.68 (s, 3H); MS (ESI): m/z calcd for C23H21NO5: 391.14. Found: 392.2 [M + H]+. 4.2.99. Methyl 2-Amino-4-(2-(benzoyloxy)-1-phenylethoxy)benzo-[d]thiazole-6-carboxylate (5s30). Prepared according to the typical procedure C from aniline 4s30 (344 mg, 0.879 mmol); light yellow solid (126 mg, 32% yield). 1H NMR (400 MHz, DMSO-d6): δ 7.96 (s, 2H), 7.91 (d, J = 1.4 Hz, 1H), 7.89−7.84 (m, 2H), 7.64 (t, J = 7.4 Hz, 1H), 7.57 (d, J = 7.3 Hz, 2H), 7.49 (t, J = 7.7 Hz, 2H), 7.43− 7.36 (m, 3H), 7.35−7.28 (m, 1H), 6.25−6.03 (m, 1H), 4.74−4.47 (m, 2H), 3.76 (s, 3H); MS (ESI): m/z calcd for C24H20N2O5S: 448.11. Found: 449.2 [M + H]+. 4.2.100. Methyl 4-(2-(Benzoyloxy)-1-phenylethoxy)-2-(3,4-dichloro-5-methyl-1H-pyrrole-2-carboxamido)benzo[d]thiazole-6carboxylate (6s30). Prepared according to the typical procedure D from 5s30 (87 mg, 0.194 mmol); gray solid (96 mg, 79% yield). 1H NMR (400 MHz, DMSO-d6): δ = 12.29 (s, 1H), 12.25 (s, 1H), 8.23 (s, 1H), 7.92−7.86 (m, 2H), 7.68−7.58 (m, 3H), 7.55−7.46 (m, 3H), 7.46−7.39 (m, 2H), 7.39−7.31 (m, 1H), 6.23−6.14 (m, 1H), 4.79− 4.62 (m, 2H), 3.82 (s, 3H), 2.28 (s, 3H); MS (ESI): m/z calcd for C30H23Cl2N3O6S: 623.07. Found: 424.5 [M + H]+. 4.2.101. 2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4(2-hydroxy-1-phenylethoxy)benzo[d]thiazole-6-carboxylic Acid (30). Prepared according to the typical procedure E from ester 6s30 (82 mg, 0.131 mmol); brown solid (51 mg, 77% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.85 (s, 1H), 12.27 (s, 2H), 8.14 (s, 1H), 7.53−7.20 (m, 6H), 5.66−5.53 (m, 1H), 5.11 (s, 1H), 3.93− 3.80 (m, 1H), 3.79−3.68 (m, 1H), 2.29 (s, 3H); 13C NMR (101 MHz, DMSO-d6): δ 166.86, 159.63, 156.80, 149.28, 141.62, 138.51, 132.72, 129.93, 128.48, 127.85, 126.51, 126.45, 117.15, 116.06, 115.64, 110.34, 109.96, 81.07, 65.88, 11.08. HRMS (ESI) m/z: [M + H]+ calcd for C22H18Cl2N3O5S 506.0344; found 506.0337; HPLC purity (254 nm): 93%. 4.2.102. Methyl 3-(2-Methoxy-1-phenylethoxy)-4-nitrobenzoate (30s31). Prepared according to the typical procedure H from 2s (2.00 g, 10.14 mmol) and 2-methoxy-1-phenylethanol (1.70 g, 11.15 mmol); white crystals (2.85 g, 85% yield). 1H NMR (400 MHz, DMSO-d6): δ 8.00−7.93 (m, 1H), 7.75 (d, J = 1.5 Hz, 1H), 7.60 (dd, J = 8.3, 1.5 Hz, 1H), 7.49−7.44 (m, 2H), 7.42−7.36 (m, 2H), 7.35− 7.29 (m, 1H), 5.94 (dd, J = 7.5, 3.3 Hz, 1H), 3.84 (s, 3H), 3.73 (dd, J = 11.3, 7.6 Hz, 1H), 3.61 (dd, J = 11.3, 3.4 Hz, 1H), 3.32 (s, 3H). mmol) was added and the mixture was stirred on an ice bath for 30 min. Methyl 3-fluoro-4-nitrobenzoate (396 mg, 2.01 mmol) was added, and the reaction mixture was stirred at 22 °C overnight. The reaction was quenched with water (1 mL), and ethyl acetate (50 mL) was added. The organic phase was washed with water (30 mL), saturated aqueous NaHCO3 (30 mL), and brine (30 mL) and then dried over Na2SO4 and filtered. The solvent was removed in vacuo to obtain crude methyl 4-nitro-3-(2,2,2-trifluoro-1-phenylethoxy)benzoate (3s28) as a brown oil that was immediately transformed according to the general procedure B to give 4s28 as a pale yellow solid (360 mg, 55% yield over the two steps). 1H NMR (400 MHz, DMSO-d6): δ 7.72−7.62 (m, 2H), 7.47−7.41 (m, 3H), 7.39−7.35 (m, 2H), 6.69 (d, J = 8.7 Hz, 1H), 6.35 (q, J = 6.7 Hz, 1H), 5.76 (s, 2H), 3.69 (s, 3H); MS (ESI): m/z calcd for C16H14F3NO3: 325.09. Found: 357.7 [M + MeOH + H]+. 4.2.89. Methyl 2-Amino-4-(2,2,2-trifluoro-1-phenylethoxy)benzo-[d]thiazole-6-carboxylate (5s28). Prepared according to the general procedure C from aniline 4s28 (342 mg, 1.05 mmol); yellow solid (80 mg, 20% yield). 1H NMR (400 MHz, DMSO-d6): δ 8.09 (s, 2H), 7.97 (d, J = 1.5 Hz, 1H), 7.65−7.57 (m, 2H), 7.49−7.36 (m, 4H), 6.63 (q, J = 6.7 Hz, 1H), 3.77 (s, 3H). 4.2.90. Methyl 2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4-(2,2,2-trifluoro-1-phenylethoxy)benzo[d]thiazole-6-carboxylate (6s28). Prepared according to the typical procedure D from 2-aminobenzothiazole 5s28 (80 mg, 0.209 mmol); white solid (80 mg, 69% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.33 (s, 2H), 8.30 (d, J = 1.4 Hz, 1H), 7.65 (d, J = 7.0 Hz, 2H), 7.53 (d, J = 1.5 Hz, 1H), 7.51−7.38 (m, 3H), 6.74 (q, J = 6.7 Hz, 1H), 3.82 (s, 3H), 2.30 (s, 3H). 4.2.91. 2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4(2,2,2-trifluoro-1-phenylethoxy)benzo[d]thiazole-6-carboxylic Acid (28). Prepared according to the typical procedure E from methyl ester 6s28 (60 mg, 0.108 mmol); brown solid (30 mg, 51% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.97 (s, 1H), 12.48−12.12 (m, 2H), 8.25 (s, 1H), 7.73−7.59 (m, 2H), 7.57−7.35 (m, 4H), 6.70 (q, J = 6.7, 6.3 Hz, 1H), 2.29 (s, 3H); HRMS (ESI) m/z: [M + H]+ calcd for C22H15Cl2F3N3O4S 544.0112; found 544.0083; HPLC purity (254 nm): >99%. 4.2.92. Methyl 3-(Benzocyclobutan-1-yloxy)-4-nitrobenzoate (3s29). Prepared according to the typical procedure H from 2s (909 mg, 4.61 mmol) and benzocyclobutan-1-ol (607 mg, 5.05 mmol); orange solid (1.27 g, 84% yield). 1H NMR (400 MHz, DMSO-d6): δ 8.06 (d, J = 8.4 Hz, 1H), 7.97 (d, J = 1.6 Hz, 1H), 7.74 (dd, J = 8.4, 1.6 Hz, 1H), 7.41 (td, J = 7.2, 1.5 Hz, 1H), 7.35−7.26 (m, 3H), 6.09 (dd, J = 4.3, 1.8 Hz, 1H), 3.94 (s, 3H), 3.79 (dd, J = 14, 4.3 Hz, 1H), 3.25 (d, J = 14.0 Hz, 1H). 4.2.93. Methyl 4-Amino-3-(benzocyclobutan-1-yloxy)benzoate (4s29). Prepared according to the typical procedure B from 3s29 (1.21 g, 4.04 mmol); white solid (500 mg, 46% yield). 1H NMR (400 MHz, DMSO-d6): δ 7.52 (d, J = 1.8 Hz, 1H), 7.43 (dd, J = 8.2, 1.8 Hz, 1H), 7.37 (app td, J = 7.3, 1.4 Hz, 1H), 7.30−7.23 (m, 4H), 6.67 (d, J = 8.2 Hz, 1H), 5.78−5.74 (m, 1H), 5.69 (s, 2H), 3.78 (s, 3H), 3.72 (dd, J = 14.2, 4.5 Hz, 1H), 3.30 (d, J = 14.2 Hz, 1H). 4.2.94. Methyl 2-Amino-4-(benzocyclobutan-1-yloxy)benzo[d]thiazole-6-carboxylate (5s29). Prepared according to the typical procedure C from aniline 4s29 (415 mg, 1.54 mmol); white powder (161 mg, 32% yield). 1H NMR (400 MHz, DMSO-d6): δ 8.01 (d, J = 1.4 Hz, 1H), 7.94 (s, 2H), 7.55 (d, J = 1.4 Hz, 1H), 7.38 (app td, J = 7.2, 1.5 Hz, 1H), 7.33−7.22 (m, 3H), 5.94 (dd, J = 4.3, 1.7 Hz, 1H), 3.86 (s, 3H), 3.74 (dd, J = 14.2, 4.3 Hz, 1H), 3.22 (d, J = 14.2 Hz, 1H). 4.2.95. Methyl 4-(Benzocyclobutan-1-yloxy)-2-(3,4-dichloro-5methyl-1H-pyrrole-2-carboxamido)benzo[d]thiazole-6-carboxylate (6s29). Prepared according to the typical procedure D from 2aminobenzothiazole 5s29 (150 mg, 0.459 mmol). Upon cooling, the precipitate was collected and washed with toluene and MeOH to get the crude product (47 mg), containing 20 mol % of O-dealkylated impurity (by 1H NMR). The evaporated toluene and MeOH filtrates contained no product, as established by 1H NMR. The crude product was dissolved in pyridine (0.5 mL) and treated with excess tertbutyldimethylsilyl chloride (TBDMSCl) (50 mg). After stirring at 22 1405 https://doi.org/10.1021/acs.jmedchem.2c01597 J. Med. Chem. 2023, 66, 1380−1425 Journal of Medicinal Chemistry pubs.acs.org/jmc Article 7.45−7.31 (m, 4H), 6.01 (dd, J = 10.3, 2.3 Hz, 1H), 4.14 (dd, J = 15.1, 10.2 Hz, 2H), 3.65−3.58 (m, 1H), 3.29 (s, 3H), 2.29 (s, 3H), 1.51 (s, 9H); MS (ESI): m/z calcd for C27H27Cl2N3O6S2: 623.07. Found: 624.1 [M + H]+ 4.2.111. 2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4(3-(methylsulfonyl)-2-phenylpropyl)benzo[d]thiazole-6-carboxylic Acid (32). Trifluoroacetic acid (0.04 mL, 0.48 mmol) was added to a suspension of tert-butyl ester 33s32 (30 mg, 0.048 mmol) in dichloromethane (2 mL). The reaction mixture was stirred at 22 °C overnight, the solvent was removed under reduced pressure, and the residue was triturated with MeOH to give the product as a dark violet solid (15 mg, 56% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.95 (s, 1H), 12.37 (s, 1H), 12.13 (s, 1H), 8.21 (s, 1H), 7.56 (app d, J = 7.3 Hz, 2H), 7.43−7.37 (m, 3H), 7.35−7.30 (m, 1H), 6.08 (dd, J = 10, 2.7 Hz, 1H), 4.10 (dd, J = 15.0, 10 Hz, 1H), 3.64 (d, J = 13 Hz, 1H), 3.27 (s, 3H), 2.29 (s, 3H); 13C NMR (101 MHz, DMSO-d6): δ (characteristic signals) 167.18, 138.47, 130.54, 129.39, 129.03, 127.01, 126.73, 117.31, 110.80, 110.43, 75.47, 60.70, 43.59, 11.54; HRMS (ESI) m/z: [M + H]+ calcd for C23H20Cl2N3O6S2 568.0171; found 568.0166; HPLC purity (254 nm): 97%. 4.2.112. 2-((6-Carboxy-2-(3,4-dichloro-5-methyl-1H-pyrrole-2carboxamido)benzo[d]thiazol-4-yl)oxy)-2-phenylethan-1-aminium Chloride (33). The Boc-protected amine 39 (53 mg, 0.088 mmol) was suspended in 4 M HCl/dioxane (4 mL) and stirred at 22 °C for 24 h. The precipitate was filtered off and washed with dioxane to obtain the title compound as a violet-tinted solid (13 mg, 46% yield). 1H NMR (400 MHz, DMSO-d6): δ 13.06 (s, 1H), 12.64 (s, 1H), 12.30 (s, 1H), 8.39−8.20 (m, 4H), 7.54−7.49 (m, 2H), 7.48−7.39 (m, 3H), 7.35 (t, J = 7.2 Hz, 1H), 5.90 (t, J = 5.8 Hz, 1H), 2.30 (s, 3H) (CH2−NH2 signals overlayed by the water peak); 13C NMR (101 MHz, DMSOd6): δ (characteristic signals) 166.62, 137.10, 132.92, 130.05, 128.89, 128.80, 126.47, 126.42, 118.13, 116.09, 114.08, 110.12, 78.72, 66.34, 44.89, 11.03; HRMS (ESI) m/z: [M − Cl] + calcd for C22H19Cl2N4O4S 505.0499; found 505.0491; HPLC purity (254 nm): 95%. 4.2.113. Methyl 3-(2-(Dimethylamino)-1-phenylethoxy)-4-nitrobenzoate (30s34). Prepared according to the typical procedure H from 2s (2.00 g, 10.14 mmol) and 2-(dimethylamino)-1-phenylethan1-ol (1.84 g, 11.15 mmol); thick oil (1.84 g, 53% yield). 1H NMR (400 MHz, DMSO-d6): δ 7.94 (d, J = 8.3 Hz, 1H), 7.75 (d, J = 1.5 Hz, 1H), 7.58 (dd, J = 8.3, 1.5 Hz, 1H), 7.46−7.41 (m, 2H), 7.40− 7.33 (m, 2H), 7.32−7.25 (m, 1H), 5.83 (dd, J = 7.6, 4.1 Hz, 1H), 3.83 (s, 3H), 2.23 (s, 6H); MS (ESI): m/z calcd for C18H20N2O5: 344.14. Found: 345.4 [M + H]+. 4.2.114. Methyl 4-Amino-3-(2-(dimethylamino)-1-phenylethoxy)-benzoate (31s34). Prepared according to the typical procedure B from 30s34 (1.84 g, 5.34 mmol); white powder (965 mg, 57% yield). 1H NMR (400 MHz, DMSO-d6): δ 7.52−7.36 (m, 4H), 7.36−7.27 (m, 2H), 7.17 (d, J = 1.4 Hz, 1H), 6.61 (d, J = 8.3 Hz, 1H), 6.16 (s, 2H), 5.92 (dd, J = 10.0, 1 Hz, 1H), 3.71−3.58 (m, 4H), 3.39 (dd, J = 13.3, 1 Hz, 1H), 2.88 (s, 6H); MS (ESI): m/z calcd for C18H22N2O3: 314.16. Found: 315.5 [M + H]+. 4.2.115. Methyl 2-Amino-4-(2-(dimethylamino)-1-phenylethoxy)-benzo[d]thiazole-6-carboxylate (32s34). Prepared according to the typical procedure C from aniline 31s34 (940 mg, 2.99 mmol); off-white solid (585 mg, 53% yield). 1H NMR (400 MHz, DMSOd6): δ 8.10 (s, 2H), 8.01 (d, J = 1.5 Hz, 1H), 7.51−7.31 (m, 5H), 7.25 (d, J = 1.5 Hz, 1H), 6.06 (dd, J = 10.8, 1.0 Hz, 1H), 3.83−3.71 (m, 4H), 3.48−3.40 (m, 1H), 3.04 (s, 6H); MS (ESI): m/z calcd for C19H21N3O3S: 371.13. Found: 372.2 [M + H]+. 4.2.116. Methyl 2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4-(2-(dimethylamino)-1-phenylethoxy)benzo[d]thiazole-6carboxylate (33s34). Prepared according to the typical procedure J from 2-aminobenzothiazole 32s34 (200 mg, 0.538 mmol); gray powder (85 mg, 29% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.20 (s, 2H), 8.22 (s, 1H), 7.66−7.29 (m, 6H), 6.18−5.78 (m, 1H), 3.81 (s, 3H), 2.90 (s, 6H), 2.28 (s, 3H) (the peaks of CH2N are overlapped with DMSO and water signal); MS (ESI): m/z calcd for C25H24Cl2N4O4S: 546.09. Found: 547.4 [M + H]+; HPLC purity (254 nm): 98%. 4.2.103. Methyl 4-Amino-3-(2-methoxy-1-phenylethoxy)benzoate (31s31). Prepared according to the typical procedure B from 30s31 (2.56 g, 7.74 mmol); white solid (2.24 g, 96% yield). 1H NMR (400 MHz, DMSO-d6) δ 7.50−7.44 (m, 2H), 7.39−7.25 (m, 4H), 7.19 (d, J = 1.8 Hz, 1H), 6.64 (d, J = 8.3 Hz, 1H), 5.78 (s, 2H), 5.42 (dd, J = 7.8, 3.5 Hz, 1H), 3.79 (dd, J = 10.8, 7.9 Hz, 1H), 3.67 (s, 3H), 3.55 (dd, J = 10.8, 3.5 Hz, 1H), 3.36−3.33 (m, 3H); MS (ESI): m/z calcd for C17H19NO4: 301.13. Found: 324.0 [M + Na]+. 4.2.104. Methyl 2-Amino-4-(2-methoxy-1-phenylethoxy)benzo[d]-thiazole-6-carboxylate (32s31). Prepared according to the general procedure C from aniline 31s31 (2.25 g, 7.48 mmol); light yellow solid (456 mg, 17% yield). 1H NMR (400 MHz, DMSO-d6): δ 7.97 (s, 2H), 7.87 (d, J = 1.5 Hz, 1H), 7.49−7.40 (m, 2H), 7.39−7.30 (m, 2H), 7.30−7.22 (m, 2H), 5.78 (dd, J = 7.3, 3.8 Hz, 1H), 3.80− 3.70 (m, 4H), 3.60 (dd, J = 10.8, 3.8 Hz, 1H), 3.33 (s, 3H); MS (ESI): m/z calcd for C18H18N2O4S: 358.09. Found: 359.0 [M + H]+. 4.2.105. Methyl 2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4-(2-methoxy-1-phenylethoxy)benzo[d]thiazole-6-carboxylate (33s31). Prepared according to the typical procedure D from 32s31 (200 mg, 0.557 mmol); gray solid (247 mg, 83% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.35 (s, 1H), 12.28 (s, 1H), 8.19 (s, 1H), 7.54−7.44 (m, 2H), 7.43−7.32 (m, 3H), 7.32−7.26 (m, 1H), 5.86 (dd, J = 7.5, 3.4 Hz, 1H), 3.87−3.74 (m, 4H), 3.65 (dd, J = 10.8, 3.4 Hz, 1H), 2.29 (s, 3H); MS (ESI): m/z calcd for C24H21Cl2N3O5S: 533.06. Found: 555.9 [M + Na]+. 4.2.106. 2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4(2-methoxy-1-phenylethoxy)benzo[d]thiazole-6-carboxylic Acid (31). Prepared according to the typical procedure E from methyl ester 33s31 (113 mg, 0.211 mmol); off-white solid (78 mg, 71% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.88 (s, 1H), 12.32 (s, 1H), 12.28 (s, 1H), 8.15 (s, 1H), 7.53−7.46 (m, 2H), 7.41−7.32 (m, 3H), 7.28 (ddd, J = 8.5, 2.3, 1.2 Hz, 1H), 5.84 (dd, J = 7.6, 3.5 Hz, 1H), 3.81 (dd, J = 10.8, 7.6 Hz, 1H), 3.64 (dd, J = 10.8, 3.5 Hz, 1H), 2.29 (s, 3H); 13C NMR (101 MHz, DMSO-d6): δ 166.84, 159.65, 156.63, 149.03, 141.99, 138.01, 132.86, 129.92, 128.55, 128.02, 126.50, 126.43, 116.90, 116.13, 115.74, 78.20, 76.00, 58.37, 11.08; HRMS (ESI) m/z: [M − H]− calcd for C23H20Cl2N3O5S 520.0501; found 520.0497; HPLC purity (254 nm): 98%. 4.2.107. tert-Butyl 3-(2-(Methylsulfonyl)-1-phenylethoxy)-4nitro-benzoate (30s32). Prepared according to the typical procedure H from tert-butyl 3-hydroxy-4-nitrobenzoate (2.62 g, 10.96 mmol) and 2-mesyl-1-phenylethanol (2.41 g, 12.06 mmol); white solid (877 mg, 19% yield). 1H NMR (400 MHz, CDCl3): δ 7.81 (d, J = 8.8 Hz, 1H), 7.64−7.58 (m, 2H), 7.45−7.32 (m, 5H), 6.02 (dd, J = 10.2, 2.2 Hz, 1H), 3.89 (dd, J = 15.5, 10.2 Hz, 1H), 3.30 (ddq, J = 15.5, 2.1, 1 Hz, 1H), 3.12 (d, J = 1 Hz, 3H), 1.54 (s, 9H); MS (ESI): m/z calcd for C20H23NO7S: 421.12. Found: 485.1 [M + Na + CH3CN]+. 4.2.108. tert-Butyl 3-(2-(Methylsulfonyl)-1-phenylethoxy)-4amino-benzoate (31s32). Prepared according to the typical procedure B from 30s32 (847 mg, 2.01 mmol); white solid (472 mg, 60% yield). 1H NMR (400 MHz, DMSO-d6) δ 7.54−7.48 (m, 2H), 7.43−7.37 (m, 2H), 7.36−7.30 (m, 1H), 7.25 (dd, J = 8.3, 1.8 Hz, 1H), 7.08 (d, J = 1.7 Hz, 1H), 5.80 (s, 2H), 5.69 (dd, J = 10.1, 2.2 Hz, 1H), 4.07 (dd, J = 15.1, 10.6 Hz, 1H), 3.52 (d, J = 14 Hz, 1H), 3.10 (s, 3H), 1.43 (s, 9H); MS (ESI): m/z calcd for C20H25NO5S: 391.14. Found: 392.2 [M + H]+. 4.2.109. tert-Butyl 2-Amino-4-(2-(methylsulfonyl)-1-phenylethoxy)-benzo[d]thiazole-6-carboxylate (32s32). Prepared according to the typical procedure C from aniline 31s32 (429 mg, 1.10 mmol); light yellow solid (359 mg, 73% yield). 1H NMR (400 MHz, DMSOd6): δ 7.88 (s, 2H), 7.82 (d, J = 1.5 Hz, 1H), 7.56−7.52 (m, 2H), 7.43−7.37 (m, 2H), 7.35−7.30 (m, 1H), 7.17 (d, J = 1.4 Hz, 1H), 5.85 (dd, J = 10.4, 2.7 Hz, 1H), 4.12 (dd, J = 15.4, 10.3 Hz, 1H), 3.52−3.44 (m, 1H), 3.32 (s, 3H), 1.47 (s, 9H); MS (ESI): m/z calcd for C21H24N2O5S2: 448.11. Found: 449.2 [M + H]+. 4.2.110. tert-Butyl 2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4-(2-(methylsulfonyl)-1-phenylethoxy)benzo[d]thiazole-6-carboxylate (33s32). Prepared according to the typical procedure D from 2-aminobenzothiazole 32s32 (320 mg, 713 mmol); violet powder (147 mg, 33% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.41 (s, 1H), 12.11 (s, 1H), 8.16 (s, 1H), 7.58 (d, J = 7.2 Hz, 2H), 1406 https://doi.org/10.1021/acs.jmedchem.2c01597 J. Med. Chem. 2023, 66, 1380−1425 Journal of Medicinal Chemistry pubs.acs.org/jmc Article CDCl3) δ 7.77 (d, J = 8.4 Hz, 1H), 7.68 (d, J = 1.5 Hz, 1H), 7.60 (dd, J = 8.4, 1.5 Hz, 1H), 7.44−7.39 (m, 2H), 7.38−7.33 (m, 2H), 7.32− 7.27 (m, 1H), 5.55 (dd, J = 8.1, 3.2 Hz, 1H), 3.88 (s, 3H), 3.64 (t, J = 4.7 Hz, 4H), 3.04 (dd, J = 14.1, 8.1 Hz, 1H), 2.73 (dd, J = 14.1, 3.2 Hz, 1H), 2.66−2.50 (m, 4H); MS (ESI): m/z calcd for C20H22N2O6: 386.15. Found: 387.2 [M + H]+. 4.2.124. Methyl 4-Amino-3-(2-morpholino-1-phenylethoxy)benzoate (31s36). Prepared according to the typical procedure B from 30s36 (4.68 g, 12.12 mmol); white solid (4.32 g, 100%). 1H NMR (400 MHz, DMSO-d6) δ 7.45 (app d, J = 7.1 Hz, 2H), 7.40− 7.33 (m, 3H), 7.29 (t, J = 7.2 Hz, 1H), 7.12 (d, J = 1.7 Hz, 1H), 6.64 (d, J = 8.3 Hz, 1H), 6.15 (s, 2H), 5.18 (dd, J = 8.9, 2.6 Hz, 1H), 3.65 (s, 3H), 3.58 (t, J = 4.5 Hz, 4H), 2.97 (dd, J = 13.5, 9.0 Hz, 1H), 2.66−2.51 (m, 4H), 2.47 (dd, J = 13.7, 2.9 Hz, 1H); MS (ESI): m/z calcd for C20H24N2O4: 356.17. Found: 357.1 [M + H]+. 4.2.125. Methyl 2-Amino-4-(2-morpholino-1-phenylethoxy)benzo-[d]thiazole-6-carboxylate (32s36). Prepared according to the typical procedure C from aniline 31s36 (1.11 g, 3.13 mmol); light yellow solid (556 mg, 43% yield). 1H NMR (400 MHz, DMSO): δ 7.93 (s, 2H), 7.86 (d, J = 1.5 Hz, 1H), 7.47−7.40 (m, 2H), 7.35−7.29 (m, 3H), 7.27−7.18 (m, 1H), 5.80 (dd, J = 7.8, 3.9 Hz, 1H), 3.76 (s, 3H), 3.52 (app t, J = 4.6 Hz, 4H), 2.92 (dd, J = 13.4, 7.9 Hz, 1H), 2.62 (dd, J = 13.5, 4.0 Hz, 1H), 2.59−2.51 (m, 4H); 19F NMR (376 MHz, DMSO-d6): δ −67.92 (d, J = 9.0 Hz); 13C NMR (101 MHz, DMSO-d6): δ 168.76, 166.01, 147.43, 146.93, 132.26, 126.15 (q, J = 288.0 Hz), 122.24, 116.58, 111.44, 66.79, 64.56, 63.75 (q, J = 25.0 Hz), 51.98, 50.15; MS (ESI): m/z calcd for C21H23N3O4S: 413.14. Found: 414.2 [M + H]+. 4.2.126. Methyl 2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4-(2-morpholino-1-phenylethoxy)benzo[d]-thiazole-6-carboxylate (33s36). Prepared according to the typical procedure D from 2-aminobenzothiazole 32s36 (250 mg, 0.605 mmol); light pink solid (150 mg, 42% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.27 (s, 2H), 8.18 (s, 1H), 7.51−7.46 (m, 3H), 7.35 (app t, J = 7.5 Hz, 2H), 7.26 (t, J = 7.4 Hz, 1H), 5.86 (dd, J = 7.9, 3.8 Hz, 1H), 3.81 (s, 3H), 3.58−3.51 (m, 4H), 3.07−2.95 (m, 1H), 2.65−2.54 (m, 4H), 2.54−2.51 (m, 1H), 2.29 (s, 3H); MS (ESI): m/z calcd for C27H26Cl2N4O5S: 588.10. Found: 589.3 [M + H]+. 4.2.127. 2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4(2-morpholino-1-phenylethoxy)benzo[d]thiazole-6-carboxylic Acid (36). Prepared according to the typical procedure E from methyl ester 33s36 (75 mg, 0.127 mmol); light pink solid (60 mg, 82% yield). 1H NMR (400 MHz, DMSO-d6): δ 13.37−12.65 (m, 2H), 12.32 (s, 1H), 10.70 (s, 1H), 8.24 (s, 1H), 7.55−7.47 (m, 2H), 7.46−7.37 (m, 3H), 7.39−7.30 (m, 1H), 6.40 (d, J = 10.0 Hz, 1H), 4.14−4.02 (m, 2H), 3.95−3.74 (m, 5H), 3.68−3.55 (m, 1H), 2.30 (s, 3H) (2H overlapped with the water peak); 13C NMR (101 MHz, DMSO-d6): δ (representative signals) 166.62, 136.71, 130.10, 128.98, 126.52, 126.42, 117.73, 110.04, 75.72, 63.27, 60.91, 53.10, 52.01, 10.98; HRMS (ESI) m/z: [M + H]+ calcd for C26H25Cl2N4O5S 575.0923; found 575.0917; HPLC purity (254 nm): 97%. 4.2.128. 2-((6-Carboxy-2-(3,4-dichloro-5-methyl-1H-pyrrole-2carboxamido)benzo[d]thiazol-4-yl)oxy)-N,N,N-trimethyl-2-phenylethan-1-aminium Iodide (37). Prepared according to the general procedure G from the tertiary amine 34 (10 mg, 0.019 mmol); gray solid (4.7 mg, 37%) yield. 1H NMR (400 MHz, CD3OD): δ 8.22 (s, 1H), 7.58−7.50 (m, 2H), 7.49 (s, 1H), 7.45−7.32 (m, 3H), 6.38 (d, J = 10 Hz, 1H), 4.25 (dd, J = 14, 10 Hz, 1H), 3.75 (d, J = 14 Hz, 1H), 3.50 (s, 9H), 2.33 (s, 3H); HRMS (ESI) m/z: [M − I]+ calcd for C25H25Cl2N4O4S 547.0968; found 547.0960; HPLC purity (254 nm): 97%. 4.2.129. Methyl 3-(2-Amino-1-phenylethoxy)-4-nitrobenzoate (30s33). Boc-protected derivative 30s39 (1.09 g, 2.62 mmol) was dissolved in 4 M HCl/dioxane (9 mL, 36 mmol) and stirred at 22 °C for 45 min. The volatiles were evaporated, and the residue was neutralized with saturated aqueous NaHCO3 solution. After extraction with EtOAc, the organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated to get 800 mg (97% yield) of a 60:40 mixture of 30s30 (major isomer) and methyl 3-((2hydroxy-2-phenylethyl)amino)-4-nitrobenzoate (rearranged isomer), which was used without separation in the next step. Major isomer 4.2.117. 2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4(2-(dimethylamino)-1-phenylethoxy)benzo[d]thiazole-6-carboxylic Acid (34). Prepared according to the typical procedure E from methyl ester 33s34 (80 mg, 0.147 mmol); brown powder (43 mg, 55%). 1H NMR (400 MHz, DMSO-d6): δ 13.08−11.69 (m, 3H), 8.11 (s, 1H), 7.60−7.23 (m, 6H), 5.79−5.54 (m, 1H), 3.46−2.70 (m, 8H), 2.25 (s, 3H); HRMS (ESI) m/z: [M + H]+ calcd for C24H23Cl2N4O4S 533.0817; found 533.0802; HPLC purity (254 nm): 98%. 4.2.118. Methyl 4-Nitro-3-(1-phenyl-2-(piperidin-1-yl)ethoxy)benzoate (30s35). Prepared according to the typical procedure H from 2s (1.05 g, 5.35 mmol) and 1-phenyl-2-(piperidin-1-yl)ethanol (1.21 g, 5.88 mmol); yellow oil (1.83 g, 89% yield). 1H NMR (400 MHz, CDCl3): δ 7.76 (d, J = 8.4 Hz, 1H), 7.70 (d, J = 1.5 Hz, 1H), 7.59 (dd, J = 8.4, 1.5 Hz, 1H), 7.41 (m, 2H), 7.37−7.27 (m, 3H), 5.56 (dd, J = 8.1, 3.0 Hz, 1H), 3.89 (s, 3H), 3.02 (dd, J = 14.2, 8.1 Hz, 1H), 2.69 (dd, J = 14.2, 3.1 Hz, 1H), 2.59−2.44 (m, 4H), 1.51 (m, 4H), 1.39 (m, 2H); MS (ESI): m/z calcd for C21H24N2O5: 384.17. Found: 385.2 [M + H]+. 4.2.119. Methyl 4-Amino-3-(1-phenyl-2-(piperidin-1-yl)ethoxy)benzoate (31s35). Prepared according to the typical procedure B from 30s35 (1.61 g, 4.18 mmol); light yellow oil (1.29 g, 87% yield). 1 H NMR (400 MHz, DMSO-d6): δ 7.47−7.41 (m, 2H), 7.41−7.27 (m, 4H), 7.06 (d, J = 1.9 Hz, 1H), 6.64 (d, J = 8.3 Hz, 1H), 6.36 (s, 2H), 5.01 (dd, J = 9.2, 2.2 Hz, 1H), 3.64 (s, 3H), 2.91 (dd, J = 13.6, 9.3 Hz, 1H), 2.64−2.56 (m, 2H), 2.49−2.42 (m, 2H), 2.36 (dd, J = 13.6, 2.5 Hz, 1H), 1.58−1.46 (m, 4H), 1.40 (d, J = 4.4 Hz, 2H); MS (ESI): m/z calcd for C21H26N2O3: 354.19. Found: 355.3 [M + H]+. 4.2.120. Methyl 2-Amino-4-(1-phenyl-2-(piperidin-1-yl)ethoxy)benzo[d]thiazole-6-carboxylate (32s35). Prepared according to the typical procedure C from aniline 31s35 (613 mg, 1.73 mmol); light yellow solid (534 mg, 75% yield). 1H NMR (400 MHz, DMSO-d6): δ 8.00 (s, 2H), 7.98 (d, J = 0.8 Hz, 1H), 7.49−7.44 (m, 2H), 7.40 (app t, J = 7.3 Hz, 2H), 7.34 (t, J = 7.1 Hz, 1H), 7.27 (d, J = 1.5 Hz, 1H), 6.24−6.02 (m, 1H), 4.05−3.90 (m, 1H), 3.76 (s, 3H), 3.77−3.63 (m, 2H), 3.21−3.03 (m, 2H), 1.99−1.62 (m, 5H), 1.53−1.34 (m, 1H); MS (ESI): m/z calcd for C22H25N3O3S: 411.16. Found: 412.2 [M + H]+. 4.2.121. Methyl 2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4-(1-phenyl-2-(piperidin-1-yl)ethoxy)benzo[d]thiazole-6carboxylate Monocitrate Salt (33s35). Prepared according to the typical procedure J from 2-aminobenzothiazole 32s35 (108 mg, 0.261 mmol); dark violet solid (62 mg, 31% yield). 1H NMR (characteristic signals) (400 MHz, DMSO-d6): δ 12.45 (s, 1H), 11.94 (s, 1H), 9.49 (s, 1H), 8.28 (s, 1H), 7.52 (d, J = 7.3 Hz, 2H), 7.48 (s, 1H), 7.41 (t, J = 7.4 Hz, 2H), 7.34 (t, J = 7.3 Hz, 1H), 6.30 (d, J = 8.7 Hz, 1H), 3.89−3.76 (m, 5H), 3.70 (dd, J = 15.4, 8.0 Hz, 1H), 3.61−3.50 (m, 1H), 3.23−3.06 (m, 2H), 2.29 (s, 3H), 1.95−1.83 (m, 2H), 1.80− 1.64 (m, 3H), 1.51−1.34 (m, 1H); MS (ESI): m/z calcd for C28H28Cl2N4O4S: 586.12. Found: 587.4 [M + H]+. 4.2.122. 2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4(1-phenyl-2-(piperidin-1-yl)ethoxy)benzo[d]thiazole-6-carboxylate Sodium Salt (35). To a suspension of methyl ester 33s35 (77 mg, 0.100 mmol) in methanol (2 mL), 1 M NaOH (0.400 mL, 0,400 mmol) was added and the reaction mixture was stirred at 40 °C overnight. An additional 1 M of NaOH (0.400 mL, 0.400 mmol) was added and stirred at 40 °C for a further 24 h. The solvent was removed, and the residue was purified by reverse phase column chromatography, eluent 20−80% acetonitrile in water; brown solid (12.5 mg, 21% yield). 1H NMR (400 MHz, DMSO-d6): δ 7.68 (d, J = 1.3 Hz, 1H), 7.47−7.39 (m, 2H), 7.32−7.24 (m, 3H), 7.23−7.14 (m, 1H), 5.76 (dd, J = 7.5, 4.2 Hz, 1H), 2.92 (dd, J = 13.4, 7.5 Hz, 1H), 2.59 (dd, J = 13.4, 4 Hz, 1H), 2.18 (s, 3H), 1.53−1.41 (m, 4H), 1.39−1.30 (m, 2H) (peaks of pyrrole and amide NH are not observed, and peaks of four piperidine protons are covered by the DMSO signal); HRMS (ESI) m/z: [M−Na + 2H]+ calcd for C27H27Cl2N4O4S 573.1130; found: 573.1112; HPLC purity (254 nm): 94%. 4.2.123. Methyl 3-(2-Morpholino-1-phenylethoxy)-4-nitrobenzoate (30s36). Prepared according to the typical procedure H from 2s (2.51 g, 12.73 mmol) and 2-morpholino-1-phenylethanol (2.90 g, 14.00 mmol); yellow solid (4.77 g, 97%). 1H NMR (400 MHz, 1407 https://doi.org/10.1021/acs.jmedchem.2c01597 J. Med. Chem. 2023, 66, 1380−1425 Journal of Medicinal Chemistry pubs.acs.org/jmc 30s33: 1H NMR (400 MHz, DMSO-d6): δ 7.98 (d, J = 8.3 Hz, 1H), 7.64 (s, 1H), 7.60 (d, J = 8.3 Hz, 1H), 7.49−7.25 (m, 5H), 5.56 (dd, J = 7.1 Hz, 4.0 Hz, 1H), 3.83 (s, 3H), 2.97 (dd, J = 13.4 Hz, 7.7 Hz, 1H), 2.88 (dd, J = 13.5 Hz, 3.2 Hz, 1H), 1.68 (s, 2H); for 1H NMR spectrum of the isolated minor isomer, see the next section. 4.2.130. Methyl 3-(2-Acetamido-1-phenylethoxy)-4-nitrobenzoate (30s38). The above mixture of two isomers (768 mg, 2.43 mmol) was dissolved in prop-1-en-2-yl acetate (5.35 mL, 48.6 mmol) and stirred overnight at 22 °C. The excess reagent was removed under reduced pressure, and the residue was purified by column chromatography, eluent EtOAc/hexane = 1:1 → 3:1, to obtain product 30s38 (562 mg, 65% yield) and 224 mg of unreacted methyl 3-((2-hydroxy-2-phenylethyl)amino)-4-nitrobenzoate. 4.2.131. Methyl 3-(2-Acetamido-1-phenylethoxy)-4-nitrobenzoate (30s38). 1H NMR (400 MHz, DMSO-d6): δ 8.15 (t, J = 5.4 Hz, 1H), 7.97 (d, J = 8.3 Hz, 1H), 7.68 (d, J = 1.5 Hz, 1H), 7.61 (dd, J = 8.3 Hz, 1.5 Hz, 1H), 7.42 (m, 4H), 7.36−7.28 (m, 1H), 5.69 (m, 1H), 3.84 (s, 3H), 3.54−3.39 (m, 1H), 1.80 (s, 3H); MS (ESI): m/z calcd for C18H18N2O6: 358.12. Found: 359.1 [M + H]+. 4.2.132. Methyl 3-((2-Hydroxy-2-phenylethyl)amino)-4-nitrobenzoate. 1H NMR (400 MHz, DMSO-d6) δ 8.33 (t, J = 5.3 Hz, 1H), 8.17 (d, J = 8.8 Hz, 1H), 7.55 (d, J = 1.7 Hz, 1H), 7.49−7.43 (m, 2H), 7.39−7.33 (m, 2H), 7.31−7.25 (m, 1H), 7.14 (dd, J = 8.8, 1.7 Hz, 1H), 5.88 (d, J = 4.4 Hz, 1H), 4.91 (dt, J = 8.2, 4.2 Hz, 1H), 3.87 (s, 3H), 3.71−3.58 (m, 1H), 3.51−3.43 (m, 1H). 4.2.133. Methyl 3-(2-Acetamido-1-phenylethoxy)-4-aminobenzoate (31s38). Prepared according to the typical procedure B from 30s38 (500 mg, 1.40 mmol); white solid (312 mg, 68% yield). 1H NMR (400 MHz, DMSO-d6): δ 8.19 (t, J = 5.8 Hz, 1H), 7.40−7.32 (m, 4H), 7.32−7.24 (m, 2H), 7.04 (d, J = 1.7 Hz, 1H), 6.61 (d, J = 8.3 Hz, 1H), 5.86 (s, 2H), 5.28 (dd, J = 7.1 Hz, 4.1 Hz, 1H), 3.64 (s, 3H), 3.57−3.42 (m, 2H), 1.81 (s, 3H); MS (ESI): m/z calcd for C18H20N2O4: 328.14. Found: 329.1 [M + H]+. 4.2.134. Methyl 4-(2-Acetamido-1-phenylethoxy)-2-aminobenzo-[d]thiazole-6-carboxylate (32s38). Prepared according to the typical procedure C from aniline 31s38 (283 mg, 0.862 mmol); yellow solid (216 mg, 65% yield). 1H NMR (400 MHz, DMSO-d6): δ 8.20 (t, J = 5.2 Hz, 1H), 7.95 (s, 2H), 7.89 (d, J = 1.5 Hz, 1H), 7.44− 7.39 (m, 2H), 7.39−7.31 (m, 2H), 7.31−7.24 (m, 2H), 5.62−5.55 (m, 1H), 3.76 (3, 1H), 3.47 (t, J = 5.9 Hz, 2H), 1.82 (s, 3H); MS (ESI): m/z calcd for C19H19N3O4S: 385.11. Found: 386.0 [M + H]+. 4.2.135. Methyl 4-(2-Acetamido-1-phenylethoxy)-2-(3,4-dichloro-5-methyl-1H-pyrrole-2-carboxamido)benzo[d]thiazole-6carboxylate (33s38). Prepared according to the typical procedure D from 2-aminobenzothiazole 32s38 (110 mg, 0.285 mmol); gray solid (90 mg, 56% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.36 (s, 2H), 8.32−8.22 (m, 2H), 7.56−7.50 (m, 2H), 7.49 (s, 1H), 7.47− 7.41 (m, 2H), 7.39−7.34 (m, 1H), 5.78 (dd, J = 7.1 Hz, 5.7 Hz, 1H), 3.89 (s, 3H), 3.72−3.63 (m, 1H), 3.63−3.54 (m, 1H), 2.37 (s, 3H), 1.91 (s, 3H); MS (ESI): m/z calcd for C25H22Cl2N4O5S: 560.07. Found: 559.0 [M − H]−. 4.2.136. 4-(2-Acetamido-1-phenylethoxy)-2-(3,4-dichloro-5methyl-1H-pyrrole-2-carboxamido)benzo[d]thiazole-6-carboxylic Acid (38). Prepared according to the typical procedure E from the methyl ester 33s38 (70 mg, 0.125 mmol); dark gray solid (30 mg, 44% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.90 (s, 1H), 12.28 (s, 2H), 8.19 (t, J = 5.3 Hz, 1H), 8.16 (s, 1H), 7.48−7.42 (m, 2H), 7.41−7.33 (m, 3H), 7.33−7.25 (m, 1H), 5.68 (dd, J = 7.4 Hz, 5.2 Hz, 1H), 3.66−3.45 (m, 2H), 2.29 (s, 3H), 1.83 (s, 3H); 13C NMR (101 MHz, DMSO-d6): δ 169.70, 166.84, 159.88, 156.94, 148.94, 142.09, 138.88, 132.83, 130.00, 128.70 (2C), 128.14, 126.52, 126.22 (2C), 117.03, 116.34, 115.71, 110.49, 110.01, 78.10, 45.75, 22.51, 11.10; HRMS (ESI) m/z: [M + H]+ calcd for C24H21Cl2N4O5S 547.0610; found 547.0602; HPLC purity (254 nm): 93%. 4.2.137. Methyl 3-(2-((tert-Butoxycarbonyl)amino)-1-phenyletho-xy)-4-nitrobenzoate (30s39). Prepared according to the typical procedure H from 2s (2.31 g, 11.72 mmol) and tert-butyl (2-hydroxy2-phenylethyl)carbamate (3.06 g, 12.89 mmol); yellow oil (3.76 g, 77% yield). 1H NMR (400 MHz, DMSO-d6): δ 7.97 (d, J = 8.3 Hz, 1H), 7.68 (s, 1H), 7.60 (dd, J = 8.3 Hz, 1.5 Hz, 1H), 7.47−7.28 (m, 5H), 7.09 (t, J = 5.5 Hz, 1H), 5.73−5.65 (m, 1H), 3.84 (s, 3H), Article 3.47−3.37 (m, 1H), 3.34−3.27 (m, 1H), 1.35 (s, 9H); MS (ESI): m/ z calcd for C21H24N2O7: 416.16. Found: 439.0 [M + Na]+. 4.2.138. Methyl 4-Amino-3-(2-((tert-butoxycarbonyl)amino)-1phenylethoxy)benzoate (31s39). Prepared according to the typical procedure B from 30s39 (3.08 g, 7.39 mmol); white solid (2.17 g, 76% yield). 1H NMR (400 MHz, DMSO-d6): δ 7.38−7.32 (m, 4H), 7.32−7.24 (m, 3H), 7.01 (d, J = 1.6 Hz, 1H), 6.61 (d, J = 8.3 Hz, 1H), 5.92 (s, 2H), 5.27−5.21 (m, 1H), 3.64 (s, 3H), 3.47−3.35 (m, 2H), 1.41−1.20 (m, 9H); MS (ESI): m/z calcd for C21H26N2O5: 386.18. Found: 408.9 [M + Na]+. 4.2.139. Methyl 2-Amino-4-(2-((tert-butoxycarbonyl)amino)-1phenylethoxy)benzo[d]thiazole-6-carboxylate (32s39). Prepared according to the typical procedure C from aniline 31s39 (2.04 g, 5.28 mmol); yellow solid (726 mg, 31% yield). 1H NMR (400 MHz, DMSO-d6): δ 7.96 (s, 2H), 7.88 (s, 1H), 7.44−7.37 (m, 2H), 7.38− 7.30 (m, 2H), 7.30−7.21 (m, 2H), 7.14−7.02 (m, 1H), 5.66−5.50 (m, 1H), 3.76 (s, 3H), 3.44−3.24 (m, 2H), 1.34 (s, 9H); MS (ESI): m/z calcd for C22H25N3O5S: 443.15. Found: 442.0 [M − H]−. 4.2.140. Methyl 4-(2-((tert-Butoxycarbonyl)amino)-1-phenyletho-xy)-2-(3,4-dichloro-5-methyl-1H-pyrrole-2-carboxamido)benzo[d]thiazole-6-carboxylate (33s39). Prepared according to the typical procedure D from 32s39 (256 mg, 0.578 mmol); gray solid (68 mg, 19% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.32 (s, 1H), 12.30 (s, 1H), 8.21 (s, 1H), 7.47−7.25 (m, 6H), 7.14 (s, 1H), 5.68 (t, J = 6.2 Hz, 1H), 3.81 (s, 3H), 3.55−3.40 (m, 2H), 2.29 (s, 3H), 1.34 (s, 9H); MS (ESI): m/z calcd for C28H28Cl2N4O6S: 618.11. Found: 617.0 [M − H]−. 4.2.141. 4-(2-((tert-Butoxycarbonyl)amino)-1-phenylethoxy)-2(3,4-dichloro-5-methyl-1H-pyrrole-2-carboxamido)benzo[d]thiazole-6-carboxylic Acid (39). Prepared according to the typical procedure E from methyl ester 33s39 (56 mg, 0.091 mmol); pinktinted solid (50 mg, 91% yield). 1H NMR (400 MHz, DMSO-d6): δ = 12.98 (s, 1H), 12.36 (s, 2H), 8.24 (s, 1H), 7.54−7.49 (m, 2H), 7.48− 7.42 (m, 3H), 7.36 (t, J = 7.3 Hz, 1H), 7.20 (s, 1H), 5.75 (dd, J = 6.6 Hz, 5.7 Hz, 1H), 3.63−3.53 (m, 2H), 2.37 (s, 3H), 1.42 (s, 9H); HRMS (ESI) m/z: [M + H]+ calcd for C27H27Cl2N4O6S 605.1028; found 605.1017; HPLC purity (254 nm): 93%. 4.2.142. Methyl 4-Nitro-3-(1-phenyl-2-(1H-1,2,4-triazol-1-yl)ethoxy)benzoate (30s40). 4.2.142.1. 1-Phenyl-2-(1H-1,2,4-triazol1-yl)ethan-1-ol (29s40). To a solution of styrene oxide (28s; R = Ph) (3.00 g, 24.97 mmol) and 1,2,4-triazole (1.72 g, 24.97 mmol) in DMF (125 mL), K2CO3 (5.18 g, 37.5 mmol) was added and the reaction mixture was stirred at 90 °C for 20 h. The volatiles were removed under reduced pressure, and the residue was suspended in boiling EtOAc and filtered. The filtrate was concentrated, and the residue was triturated with cold EtOAc to give a white solid (1.91 g, 40% yield). 1 NMR (400 MHz, DMSO-d6): δ 8.36 (s, 1H), 7.95 (s, 1H), 7.38− 7.25 (m, 5H), 5.75 (s, 1H), 4.93 (dd, J = 7.3 Hz, 5.5 Hz, 1H), 4.37− 4.26 (m, 2H); MS (ESI): m/z calcd for C10H11N3O: 189.09. Found: 190.1 [M + H]+. 4.2.143. Methyl 4-Nitro-3-(1-phenyl-2-(1H-1,2,4-triazol-1-yl)ethoxy)benzoate (30s40). Prepared according to the typical procedure H from 2s (2.17 g, 11.03 mmol) and 29s40 (2.30 g, 12.13 mmol); white solid (2.52 g, 62% yield). 1H NMR (400 MHz, DMSO-d6): δ 8.41 (s, 1H), 7.94 (m, 2H), 7.65−7.54 (m, 2H), 7.51− 7.26 (m, 5H), 6.17 (dd, J = 8.1 Hz, 3.5 Hz, 1H), 4.80 (dd, J = 14.5 Hz, 8.3 Hz, 1H), 4.63 (dd, J = 14.4 Hz, 3.6 Hz, 1H), 3.82 (s, 3H). 4.2.144. Methyl 4-Amino-3-(1-phenyl-2-(1H-1,2,4-triazol-1-yl)ethoxy)benzoate (31s40). Prepared according to the typical procedure B from 30s40 (2.50 g, 6.78 mmol); colorless oil (1.56 g, 68% yield). 1H NMR (400 MHz, DMSO-d6): δ 8.64 (s, 1H), 7.97 (s, 1H), 7.47 (m, 2H), 7.33 (m, 3H), 7.27 (dd, J = 8.3 Hz, 1.7 Hz, 1H), 7.06 (d, J = 1.7 Hz, 1H), 6.58 (d, J = 8.3 Hz, 1H), 5.83 (s, 2H), 5.69 (dd, J = 8.8 Hz, 3.3 Hz, 1H), 4.75 (dd, J = 14.2 Hz, 8.8 Hz, 1H), 4.54 (dd, J = 14.1 Hz, 3.3 Hz, 1H), 3.65 (s, 3H); MS (ESI): m/z calcd for C18H18N4O3: 338.14. Found: 339.2 [M + H]+. 4.2.145. Methyl 2-Amino-4-(1-phenyl-2-(1H-1,2,4-triazol-1-yl)ethoxy)benzo[d]thiazole-6-carboxylate (32s40). Prepared according to the typical procedure C from aniline 31s40; (1.39 g, 4.12 mmol); white solid (228 mg, 14% yield). 1H NMR (400 MHz, DMSO-d6): δ 8.53 (s, 1H), 7.96 (s, 2H), 7.94 (s, 1H), 7.88 (d, J = 1.5 Hz, 1H), 1408 https://doi.org/10.1021/acs.jmedchem.2c01597 J. Med. Chem. 2023, 66, 1380−1425 Journal of Medicinal Chemistry pubs.acs.org/jmc Article d6): δ 7.94 (d, J = 8.4 Hz, 1H), 7.85 (d, J = 1.6 Hz, 1H), 7.59 (dd, J = 8.3, 1.6 Hz, 1H), 7.48−7.40 (m, 2H), 7.42−7.33 (m, 2H), 7.34−7.25 (m, 1H), 5.86 (dd, J = 8.2, 3.4 Hz, 1H), 3.85 (s, 3H), 3.01−2.87 (m, 2H), 2.84 (dd, J = 13.8, 8.3 Hz, 1H), 2.59 (dd, J = 13.9, 3.4 Hz, 1H), 2.54−2.51 (m, 1H), 2.15 (s, 6H), 2.12−1.99 (m, 2H), 1.69−1.57 (m, 2H), 1.29−1.11 (m, 2H). MS (ESI): m/z calcd for C23H29N3O5: 427.21. Found: 427.9 [M + H]+. 4.2.151. Methyl 4-Amino-3-(2-(4-(dimethylamino)piperidin-1yl)-1-phenylethoxy)benzoate (31s42). To a solution of 30s42 (350 mg, 0.819 mmol) in ethyl acetate/methanol (2:1, 30 mL), SnCl2 (621 mg, 3.27 mmol) was added and the reaction mixture was stirred at 55 °C overnight. It was concentrated in vacuo, neutralized with saturated NaHCO3 solution to pH = 8, and the water layer was extracted with ethyl acetate (4 × 70 mL). The combined organic layers were dried over Na2SO4, filtered, and the solvent was removed in vacuo; yellow oil (304 mg, 94% yield). 1H NMR (400 MHz, DMSO-d6) δ 7.47− 7.39 (m, 2H), 7.41−7.31 (m, 3H), 7.34−7.24 (m, 1H), 7.09 (d, J = 1.9 Hz, 1H), 6.64 (d, J = 8.3 Hz, 1H), 6.20 (s, 2H), 5.11 (d, J = 8.7 Hz, 1H), 3.16−3.03 (m, 2H), 2.97 (dd, J = 13.6, 9.1 Hz, 1H), 2.49− 2.31 (m, 7H), 2.22 (t, J = 11.5 Hz, 1H), 2.13−2.02 (m, 1H), 1.86− 1.75 (m, 2H), 1.55−1.38 (m, 2H) (1H overlapped by the DMSO peak). MS (ESI): m/z calcd for C23H31N3O3: 397.24. Found: 398.0 [M + H]+. 4.2.152. Methyl 2-Amino-4-(2-(4-(dimethylamino)piperidin-1yl)-1-phenylethoxy)benzo[d]thiazole-6-carboxylate (32s42). Prepared according to the typical procedure C from aniline 31s42 (292 mg, 0.734 mmol); pale yellow oil (107 mg, 32% yield). 1H NMR (400 MHz, DMSO-d6): δ 7.93 (s, 2H), 7.87 (d, J = 1.5 Hz, 1H), 7.49−7.39 (m, 2H), 7.37−7.28 (m, 3H), 7.24 (t, J = 7.2 Hz, 1H), 5.78 (dd, J = 8.9, 3.4 Hz, 1H), 3.76 (s, 3H), 3.24−3.10 (m, 2H), 2.97 (dd, J = 13.5, 8.1 Hz, 2H), 2.75−2.61 (m, 7H), 2.25−2.14 (m, 2H), 1.95−1.81 (m, 2H), 1.62−1.40 (m, 1H). 4.2.153. Methyl 2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4-(2-(4-(dimethylamino)piperidin-1-yl)-1-phenylethoxy)benzo[d]thiazole-6-carboxylate (33s42). Prepared according to the typical procedure J from 2-aminobenzothiazole 32s42 (94 mg, 0.207 mmol); black solid (34 mg, 26% yield). 1H NMR (characteristic signals) (400 MHz, DMSO-d6): δ 8.02 (s, 1H), 7.47 (d, J = 7.5 Hz, 2H), 7.38−7.30 (m, 3H), 7.26 (t, J = 7.3 Hz, 1H), 5.81 (dd, J = 8.5, 3.7 Hz, 1H), 3.78 (s, 3H), 2.34 (s, 6H), 2.24 (s, 3H), 1.85−1.73 (m, 2H), 1.55−1.37 (m, 2H); HRMS (ESI) m/z: [M + H]+ calcd for C30H34Cl2N5O4S 630.1709; found 630.1687. 4.2.154. 2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4(2-(4-(dimethylamino)piperidin-1-yl)-1-phenylethoxy)benzo-[d]thiazole-6-carboxylic Acid (42). Prepared according to the typical procedure E from the methyl ester 33s42 (25 mg, 0.040 mmol); white solid (11 mg, 45% yield). 1H NMR (400 MHz, DMSO-d6): δ 13.60 (s, 1H), 12.93 (s, 1H), 12.64 (s, 1H), 11.21 (s, 1H), 10.92 (s, 1H), 8.23 (s, 1H), 7.52 (d, J = 8.0 Hz, 2H), 7.41 (t, J = 7.4 Hz, 3H), 7.35 (d, J = 8.2 Hz, 2H), 6.46 (d, J = 10.0 Hz, 1H), 2.71 (s, 6H), 2.31 (s, 3H); HRMS (ESI): m/z calcd for C29H30Cl2N5O4S 614.1396; found 614.1403 [M − H]−; HPLC purity (254 nm): 95%. 4.2.154.1. tert-Butyl (1-(2-Hydroxy-2-phenylethyl)piperidin-4-yl)carbamate (29s43). 4-Boc-aminopiperidine (3.00 g, 14.99 mmol) and styrene oxide (1.71 mL, 14.99 mmol) were stirred in a pressure tube at 90 °C for 4 h. The solid crude product was suspended in hexane and filtered off to give a white solid (3.50 g, 73% yield); 1H NMR (400 MHz, DMSO-d6) δ 7.36−7.16 (m, 5H), 6.74 (d, J = 7.9 Hz, 1H), 4.92 (d, J = 3.8 Hz, 1H), 4.65 (dt, J = 7.9, 4.0 Hz, 1H), 3.24−3.11 (m, 1H), 2.92−2.79 (m, 2H), 2.49−2.39 (m, 1H), 2.33 (dd, J = 12.8, 4.5 Hz, 1H), 2.11−1.95 (m, 2H), 1.70−1.56 (m, 2H), 1.39−1.35 (m, 11H); MS (ESI): m/z calcd for C18H28N2O3: 320.21. Found: 321.1 [M + H]+. 4.2.155. Methyl 3-(2-(4-((tert-Butoxycarbonyl)amino)piperidin1-yl)-1-phenylethoxy)-4-nitrobenzoate (30s43). Prepared according to the typical procedure H from 2s (1.79 g, 9.07 mmol) and 29s43 (3.20 g 9.98 mmol); yellow solid (2.81 g, 62%). 1H NMR (400 MHz, DMSO-d6): δ 7.94 (d, J = 8.4 Hz, 1H), 7.80 (d, J = 1.6 Hz, 1H), 7.59 (dd, J = 8.4, 1.5 Hz, 1H), 7.47−7.39 (m, 2H), 7.42−7.33 (m, 2H), 7.34−7.25 (m, 1H), 6.71 (d, J = 7.8 Hz, 1H), 5.85 (dd, J = 7.9, 3.6 Hz, 1H), 3.85 (s, 3H), 3.20−3.08 (m, 1H), 2.95−2.79 (m, 3H), 2.60 7.47−7.39 (m, 2H), 7.39−7.30 (m, 2H), 7.34−7.24 (m, 1H), 7.23 (d, J = 1.6 Hz, 1H), 6.09 (dd, J = 8.0 Hz, 4.4 Hz, 1H), 4.77 (dd, J = 14.2 Hz, 8.1 Hz, 1H), 4.62 (dd, J = 14.2 Hz, 4.4 Hz, 1H), 3.75 (s, 3H); MS (ESI): m/z calcd for C19H17N5O3S: 395.11. Found: 396.2 [M + H]+. 4.2.146. Methyl 2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4-(1-phenyl-2-(1H-1,2,4-triazol-1-yl)ethoxy)benzo[d]-thiazole-6-carboxylate (33s40). Prepared according to the typical procedure D from 32s40 (105 mg, 0.266 mmol); gray solid (140 mg, 92% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.34 (s, 1H), 12.24 (s, 1H), 8.55 (s, 1H), 8.20 (s, 1H), 7.97 (s, 1H), 7.49 (d, J = 7.5 Hz, 2H), 7.43−7.27 (m, 4H), 6.19 (dd, J = 8.1 Hz, 4.4 Hz, 1H), 4.83 (dd, J = 14.2 Hz, 8.2 Hz, 1H), 4.69 (dd, J = 14.2 Hz, 4.5 Hz, 1H), 3.81 (s, 3H), 2.30 (s, 3H); MS (ESI): m/z calcd for C25H20Cl2N6O4S: 570.06. Found: 571.4 [M + H]+. 4.2.147. 2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4(1-phenyl-2-(1H-1,2,4-triazol-1-yl)ethoxy)benzo[d]thiazole-6-carboxylic Acid (40). Prepared according to the typical procedure E from methyl ester 33s40 (51 mg, 0.090 mmol); light brown solid (18 mg, 36% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.90 (s, 1H), 12.34 (s, 1H), 12.21 (s, 1H), 8.54 (s, 1H), 8.17 (s, 1H), 7.96 (s, 1H), 7.48 (d, J = 7.6 Hz, 2H), 7.42−7.26 (m, 4H), 6.27−6.10 (m, 1H), 4.94− 4.76 (m, 1H), 4.75−4.59 (m, 1H), 2.30 (s, 3H); HRMS (ESI) m/z: [M + H]+ calcd for C24H19Cl2N6O4S 557.0566; found 557.0555; HPLC purity (254 nm): 94%. 4.2.148. 4-(2-(4-Aminopiperidin-1-yl)-1-phenylethoxy)-2-(3,4-dichloro-5-methyl-1H-pyrrole-2-carboxamido)benzo[d]thiazole-6carboxylic Acid Hydrochloride (41). Boc-protected compound 43 (30 mg, 0.044 mmol) was dissolved in THF (1 mL), and 4 M of HCl in 1,4-dioxane (2 mL) was added and the reaction mixture was stirred at 22 °C overnight. After the addition of an additional 2 mL of 4 M HCl in 1,4-dioxane, the reaction mixture was stirred for 2 more days and followed with HPLC-MS. The precipitate that formed in the reaction mixture was filtered off, washed with ether, and dried; pale pink solid (25 mg, 92% yield). 1H NMR (400 MHz, DMSO-d6): δ 13.24 (s, 1H), 12.49 (s, 1H), 10.63 (s, 1H), 8.45−8.26 (m, 3H), 8.24 (s, 1H), 7.59−7.47 (m, 2H), 7.46−7.38 (m, 2H), 7.38−7.31 (m, 2H), 6.42 (d, J = 9.9 Hz, 1H), 4.16−3.93 (m, 2H), 3.91−3.69 (m, 2H), 3.40−3.22 (m, 3H), 2.35−2.21 (m, 5H), 2.06−1.89 (m, 2H); HRMS (ESI) m/z: [M − Cl]+ calcd for C27H28Cl2N5O4S 588.1239; found 588.1223; HPLC purity (254 nm): 98%. 4.2.149. Methyl 3-(2-(4-(Dimethylamino)piperidin-1-yl)-1-phenyl-ethoxy)-4-nitrobenzoate (30s42). 4.2.149.1. Methyl 3-(2-(4Aminopiperidin-1-yl)-1-phenylethoxy)-4-nitrobenzoate. To a suspension of methyl 3-(2-(4-((tert-butoxycarbonyl)amino)piperidin-1yl)-1-phenylethoxy)-4-nitrobenzoate (30s43) (1.3 g, 2.6 mmol) in 1,4-dioxane (4 mL), 4 M HCl in 1,4-dioxane (5 mL) was added. The reaction mixture was stirred at 22 °C for 3 days while monitoring with HPLC-MS. The volatiles were evaporated under reduced pressure, and the residue was partitioned between EtOAc and saturated aqueous NaHCO3 solution. The organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated to give the product as a yellow oil (955 mg, 92% yield). 1H NMR (400 MHz, DMSO-d6) δ 7.94 (d, J = 8.3 Hz, 1H), 7.82 (d, J = 1.6 Hz, 1H), 7.59 (dd, J = 8.4, 1.6 Hz, 1H), 7.47−7.39 (m, 2H), 7.41−7.32 (m, 2H), 7.34−7.23 (m, 1H), 5.83 (dd, J = 8.0, 3.7 Hz, 1H), 3.85 (s, 3H), 3.37−2.98 (m, 4H), 2.93−2.76 (m, 4H), 2.59 (dd, J = 13.8, 3.7 Hz, 1H), 2.10 (dtd, J = 23.1, 11.3, 2.5 Hz, 2H), 1.65−1.53 (m, 2H), 1.26−1.03 (m, 2H) (some signals overlayed by the DMSO peak). MS (ESI): m/z calcd for C21H25N3O5: 399.18. Found: 400.0 [M + H]+. 4.2.150. Methyl 3-(2-(4-(Dimethylamino)piperidin-1-yl)-1-phenyl-ethoxy)-4-nitrobenzoate (30s42). To a solution of methyl 3(2-(4-aminopiperidin-1-yl)-1-phenylethoxy)-4-nitrobenzoate (899 mg, 2.25 mmol) in methanol (25 mL), paraformaldehyde (612 mg, 20.38 mmol) was added and the mixture was stirred for 30 min at 0 °C. NaCNBH3 (198 mg, 3.15 mmol) was added, and the reaction mixture was stirred at 22 °C overnight. The solvent was evaporated in vacuo, ethyl acetate was added, and the suspension was washed with water and brine. The organic layer was dried over Na2SO4, filtered, and evaporated to give a crude product that was purified by flash column chromatography (eluent: dichloromethane/methanol 40:1− 9:1); white solid (355 mg, 37% yield). 1H NMR (400 MHz, DMSO1409 https://doi.org/10.1021/acs.jmedchem.2c01597 J. Med. Chem. 2023, 66, 1380−1425 Journal of Medicinal Chemistry pubs.acs.org/jmc (dd, J = 13.8, 3.6 Hz, 1H), 2.19−2.05 (m, 2H), 1.66−1.55 (m, 2H), 1.37 (s, 9H), 1.31−1.21 (m, 2H); MS (ESI): m/z calcd for C26H33N3O7: 499.23. Found: 500.1 [M + H]+. 4.2.156. Methyl 4-Amino-3-(2-(4-((tert-butoxycarbonyl)amino)piperidin-1-yl)-1-phenylethoxy)benzoate (31s43). Prepared according to the typical procedure B from 30s43 (1.90 g, 3.80 mmol); yellow solid (1.57 g, 88% yield). 1H NMR (400 MHz, DMSO-d6): δ 7.47−7.39 (m, 2H), 7.40−7.31 (m, 3H), 7.33−7.24 (m, 1H), 7.10 (d, J = 1.9 Hz, 1H), 6.71 (d, J = 7.6 Hz, 1H), 6.65 (d, J = 8.4 Hz, 1H), 6.12 (s, 2H), 5.11 (dd, J = 8.9, 2.9 Hz, 1H), 3.65 (s, 3H), 3.26−3.14 (m, 1H), 3.03−2.85 (m, 3H), 2.43 (dd, J = 13.5, 3.0 Hz, 1H), 2.28− 2.19 (m, 1H), 2.15−2.05 (m, 1H), 1.75−1.65 (m, 2H), 1.46−1.30 (m, 11H); MS (ESI): m/z calcd for C26H35N3O5: 469.26. Found: 470.2 [M + H]+. 4.2.157. Methyl 2-Amino-4-(2-(4-((tert-butoxycarbonyl)amino)piperidin-1-yl)-1-phenylethoxy)benzo[d]thiazole-6-carboxylate (32s43). Prepared according to the typical procedure C from aniline 31s43 (1.36 g, 2.90 mmol); yellow solid (443 mg. 29% yield). 1H NMR (400 MHz, DMSO-d6) δ 7.94 (s, 2H), 7.85 (d, J = 1.5 Hz, 1H), 7.45−7.38 (m, 2H), 7.36−7.27 (m, 3H), 7.28−7.18 (m, 1H), 6.73 (d, J = 7.9 Hz, 1H), 5.74 (dd, J = 7.6, 4.1 Hz, 1H), 3.76 (s, 3H), 3.23− 3.11 (m, 1H), 3.00 (d, J = 10.4 Hz, 1H), 2.95−2.86 (m, 2H), 2.59 (dd, J = 13.5, 4.0 Hz, 1H), 2.19−2.05 (m, 2H), 1.64 (d, J = 9.4 Hz, 2H), 1.41−1.27 (m, 11H); MS (ESI): m/z calcd for C27H34N4O5S: 526.23. Found: 527.0 [M + H]+. 4.2.158. Methyl 4-(2-(4-((tert-Butoxycarbonyl)amino)piperidin1-yl)-1-phenylethoxy)-2-(3,4-dichloro-5-methyl-1H-pyrrole-2carboxamido)benzo[d]thiazole-6-carboxylate (33s43). Prepared according to the typical procedure J from 2-aminobenzothiazole 32s43 (139 mg, 0.265 mmol); gray solid (93 mg, 50% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.27 (s, 2H), 8.16 (s, 1H), 7.53− 7.21 (m, 6H), 6.74 (s, 1H), 5.79 (s, 1H), 3.80 (s, 3H), 3.06 (d, J = 23.1 Hz, 3H), 2.28 (s, 3H), 1.82−1.60 (m, 2H), 1.43−1.31 (m, 13H) (some piperidine signals are overlapped by water and DMSO peaks); MS (ESI): m/z calcd for C33H37Cl2N5O6S: 701.18. Found: 702.2 [M + H]+. 4.2.159. 4-(2-(4-((tert-Butoxycarbonyl)amino)piperidin-1-yl)-1phenylethoxy)-2-(3,4-dichloro-5-methyl-1H-pyrrole-2carboxamido)benzo[d]thiazole-6-carboxylic Acid (43). Prepared according to the typical procedure E from methyl ester 33s43 (80 mg, 0.113 mmol); off-white solid (43 mg, 55% yield). 1H NMR (400 MHz, DMSO-d6) δ 12.77 (s, 1H), 12.24 (s, 2H), 8.12 (s, 1H), 7.53− 7.22 (m, 6H), 6.73 (d, J = 6.6 Hz, 1H), 5.77 (s, 1H), 3.28−3.19 (m, 1H), 3.13−2.89 (m, 1H), 2.28 (s, 3H), 1.79−1.61 (m, 2H), 1.43− 1.30 (m, 13H) (some piperidine signals are overlapped by water and DMSO peaks); HRMS (ESI) m/z: [M + H] + calcd for C32H36Cl2N5O6S 688.1763; found 688.1747; HPLC purity (254 nm): 97%. 4.2.160. 2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4(2-(4-methylpiperazin-1-yl)-1-phenylethoxy)benzo[d]thiazole-6carboxylic Acid (44). Prepared according to the typical procedure E from methyl ester 33s44 (42 mg, 0.070 mmol) that was synthesized in analogy to 33s36; white crispy foam (7 mg, 17% yield). 1H NMR (500 MHz, CD3OD): δ 8.49 (s, 1H), 8.07 (d, J = 1.3 Hz, 1H), 7.58 (d, J = 1.4 Hz, 1H), 7.53−7.48 (m, 2H), 7.39−7.32 (m, 2H), 7.31− 7.25 (m, 1H), 5.87 (dd, J = 8.8 Hz, 3.1 Hz, 1H), 3.30 (dd, J = 14.2 Hz, 8.9, Hz, 1H), 3.18−3.00 (m, 8H), 2.96 (dd, J = 13.9 Hz, 3.0 Hz, 1H), 2.72 (s, 3H), 2.34 (s, 3H); HRMS (ESI) m/z: [M + H]+ calcd for C27H28Cl2N5O4S 588.1239; found 588.1207; HPLC (254 nm): 97%. 4.2.161. 4-(2-((6-Carboxy-2-(3,4-dichloro-5-methyl-1H-pyrrole-2carboxamido)benzo[d]thiazol-4-yl)oxy)-2-phenylethyl)-1,1-dimethylpiperazin-1-ium Formate (45). Prepared according to the typical procedure E from hydrochloride of methyl ester 33s45 (154 mg, 0.236 mmol; synthesized by methylation of methyl ester 33s44) and purified by reverse phase chromatography to give the title compound as a formic acid salt; white solid (75 mg, 49% yield). 1H NMR (500 MHz, CD3OD): δ 8.33 (s, 1H), 8.08 (d, J = 1.3 Hz, 1H), 7.53 (d, J = 1.5 Hz, 1H), 7.50−7.45 (m, 2H), 7.33 (t, J = 7.6 Hz, 2H), 7.25 (t, J = 7.4 Hz, 1H), 5.77 (dd, J = 8.5 Hz, 3.3 Hz, 1H), 3.40 (t, J = 5.2 Hz, 4H), 3.24 (dd, J = 14.4 Hz, 9.0 Hz, 1H), 3.12 (s, 6H), 3.16−3.07 (m, Article 2H), 3.07−3.00 (m, 2H), 2.93 (dd, J = 14.0 Hz, 3.3 Hz, 1H), 2.31 (s, 3H); 13C NMR (APT) (126 MHz, CD3OD): δ 170.82, 161.14, 158.22, 150.41, 140.62, 134.40, 132.79, 130.99, 129.76, 129.24, 127.51, 118.35, 117.10, 115.28, 113.14, 111.93, 80.59, 64.65, 63.13, 48.05, 40.39, 11.06; HRMS (ESI): m/z calcd for C28H30Cl2N5O4S: 602.1396. Found 602.1364 [M − HCOO−]+; HPLC purity (254 nm): 94%. 4.2.162. tert-Butyl 4-Nitro-3-isopropoxybenzoate (3s46). Prepared according to the typical procedure H from tert-butyl 3-hydroxy4-nitrobenzoate (2.50 g, 10.45 mmol) and 2-propanol (0.88 mL, 11.50 mmol); white solid (2.67 g, 91% yield). 1H NMR (400 MHz, DMSO-d6): δ 7.93 (d, J = 8.3 Hz, 1H), 7.73 (d, J = 1.6 Hz, 1H), 7.57 (dd, J = 8.3 Hz, 1.6 Hz, 1H), 4.90 (hept, J = 6.0 Hz, 1H), 1.57 (s, 9H), 1.31 (d, J = 6.0 Hz, 6H). 4.2.163. tert-Butyl 4-Amino-3-isopropoxybenzoate (4s46). Prepared according to the typical procedure I from 3s46 (2.66 g, 9.44 mmol); white crystals (1.78 g, 75% yield). 1H NMR (400 MHz, DMSO-d6) δ 7.30 (dd, J = 8.2 Hz, 1.8 Hz, 1H), 7.26 (d, J = 1.8 Hz, 1H), 6.62 (d, J = 8.2 Hz, 1H), 5.46 (s, 2H), 4.50 (hept, J = 6.1 Hz, 1H), 1.50 (s, 9H), 1.27 (d, J = 6.1 Hz, 6H). MS (ESI): m/z calcd for C14H21NO3: 251.15. Found: 250.2 [M − H]−, 252.2 [M + H]+. 4.2.164. tert-Butyl 2-Amino-4-isopropoxybenzo[d]thiazole-6carboxylate (5s46). Prepared according to the typical procedure C from aniline 4s46 (1.47 g, 5.84 mmol); light yellow solid (0.99 g, 55% yield). 1H NMR (400 MHz, DMSO-d6): δ 7.87−7.83 (m, 3H), 7.29 (d, J = 1.5 Hz, 1H), 4.81 (hept, J = 6.1 Hz, 1H), 1.54 (s, 9H), 1.28 (d, J = 6.1 Hz, 6H); MS (ESI): m/z calcd for C15H20N2O3S: 308.12. Found: 309.3 [M + H]+. 4.2.165. tert-Butyl 2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4-isopropoxybenzo[d]thiazole-6-carboxylate (6s46). Prepared according to the typical procedure D from 2-aminobenzothiazole 5s46 (254 mg, 0.825 mmol); gray solid (40 mg, 10% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.26 (s, 1H), 12.20 (s, 1H), 8.15 (s, 1H), 7.45 (s, 1H), 4.97−4.83 (m, 1H), 2.27 (s, 3H), 1.58 (s, 9H), 1.36 (d, J = 6.0 Hz, 6H). 4.2.166. 2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4isopropoxybenzo[d]thiazole-6-carboxylic Acid (46). A solution of the tert-Bu ester 6s46 (40 mg, 0.083 mmol) in DCM (3.0 mL) and CF3COOH (0.1 mL) was stirred at room temperature for 9 h (97% conversion by 1H NMR). Additional CF3COOH (0.1 mL) was added and stirred at 22 °C for a further 3.5 h. The reaction mixture was concentrated under reduced pressure, and the solid residue was washed with Et2O (2 × 5 mL) and filtered. The solid residue was washed with MeOH (3 mL) and air-dried. The title compound was obtained as an off-white solid (24 mg, 68% yield). 1H NMR (400 MHz, DMSO-d6) δ 12.99 (s, 1H), 12.26 (s, 1H), 12.19 (s, 1H), 8.20 (s, 1H), 7.49 (s, 1H), 4.96−4.83 (m, 1H), 2.27 (s, 3H), 1.36 (d, J = 6.0 Hz, 6H); HRMS (ESI): m/z calcd for C17H14Cl2N3O4S: 426.0082. Found 426.0089 [M − H]−; HPLC purity (254 nm): 97%. 4.2.167. tert-Butyl 3-((3-Methylbutan-2-yl)oxy)-4-nitrobenzoate (3s47). Prepared according to the typical procedure H from tert-butyl 3-hydroxy-4-nitrobenzoate (2.50 g, 10.50 mmol) and sec-iso-amyl alcohol (1.01 g, 11.50 mmol); white solid (1.68 g, 52% yield). 1H NMR (400 MHz, CDCl3): δ 7.74 (d, J = 8.3 Hz, 1H), 7.68 (d, J = 1.5 Hz, 1H), 7.55 (dd, J = 8.3 Hz, 1.5 Hz, 1H), 4.44−4.36 (m, 1H), 2.06−1.90 (m, 1H), 1.31 (d, J = 6.2 Hz, 3H), 1.01 (d, J = 6.8 Hz, 3H), 0.99 (d, J = 6.8 Hz, 3H). 4.2.168. tert-Butyl 4-Amino-3-((3-methylbutan-2-yl)oxy)benzoate (4s47). Prepared according to the typical procedure I from 3s47 (1.70 g, 5.50 mmol); colorless oil, which solidified upon standing (1.46 g, 95% yield). 1H NMR (400 MHz, CDCl3): δ 7.45 (dd, J = 8.1 Hz, 1.7 Hz, 1H), 7.42 (d, J = 1.7 Hz, 1H), 6.64 (d, J = 8.1 Hz, 1H), 4.26 (p, J = 6.0 Hz, 1H), 4.16 (s, 2H), 2.05−1.88 (m, 1H), 1.25 (d, J = 6.2 Hz, 3H), 1.01 (d, J = 6.8 Hz, 3H), 0.98 (d, J = 6.8 Hz, 3H). 4.2.169. tert-Butyl 2-Amino-4-((3-methylbutan-2-yl)oxy)benzo[d]-thiazole-6-carboxylate (5s47). Prepared according to the typical procedure C from aniline 4s47 (1.46 g, 5.22 mmol); light yellow solid (0.65 g, 37% yield). 1H NMR (400 MHz, DMSO-d6): δ 7.86−7.81 (m, 3H), 7.29 (d, J = 1.4 Hz, 1H), 4.44 (p, J = 6.1 Hz, 1H), 1.91 (dq, 1410 https://doi.org/10.1021/acs.jmedchem.2c01597 J. Med. Chem. 2023, 66, 1380−1425 Journal of Medicinal Chemistry pubs.acs.org/jmc Article C19H18Cl2N3O4S 454.0395; found 454.0380; HPLC purity (254 nm): 94%. 4.2.177. Methyl 4-Nitro-3-((tetrahydrofuran-3-yl)oxy)benzoate (3s49). Prepared following the typical procedure H from 2s (2.11 g, 10.70 mmol) and 3-hydroxytetrahydrofuran (1.04 g, 11.77 mmol); white solid (2.37 g, 83% yield). 1H NMR (400 MHz, DMSO-d6): δ 8.00 (d, J = 8.4 Hz, 1H), 7.76 (d, J = 1.6 Hz, 1H), 7.68 (dd, J = 8.3, 1.6 Hz, 1H), 5.43−5.35 (m, 1H), 3.96−3.87 (m, 4H), 3.87−3.73 (m, 3H), 2.32−2.19 (m, 1H), 2.06−1.94 (m, 1H). 4.2.178. Methyl 4-Amino-3-((tetrahydrofuran-3-yl)oxy)benzoate (4s49). Prepared according to the typical procedure I from 3s49 (2.09 g, 7.81 mmol); white solid (1.00 g, 54% yield). 1H NMR (400 MHz, DMSO-d6): δ 7.38 (dd, J = 8.2 Hz, 1.8 Hz, 1H), 7.24 (d, J = 1.8 Hz, 1H), 6.65 (d, J = 8.2 Hz, 1H), 5.63 (s, 2H), 5.07−4.93 (m, 1H), 3.92−3.75 (m, 4H), 3.75 (s, 3H), 2.25−2.13 (m, 1H), 2.06−1.97 (m, 1H); MS (ESI): m/z calcd for C12H15NO4: 237.10. Found: 238.2 [M + H]+. 4.2.179. Methyl 2-Amino-4-((tetrahydrofuran-3-yl)oxy)benzo[d]thiazole-6-carboxylate (5s49). Prepared according to the typical procedure C from aniline 4s49 (765 mg, 2.60 mmol); light yellow solid (660 mg, 54% yield). 1H NMR (400 MHz, DMSO-d6): δ 7.97 (d, J = 1.5 Hz, 1H), 7.93 (s, 2H), 7.31 (d, J = 1.5 Hz, 1H), 5.30−5.22 (m, 1H), 3.91−3.84 (m, 3H), 3.83 (s, 3H), 3.81−3.75 (m, 1H), 2.24−2.13 (m, 1H), 2.05−1.97 (m, 1H); MS (ESI): m/z calcd for C13H14N2O4S: 294.07. Found: 295.2 [M + H]+. 4.2.180. Methyl 2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4-((tetrahydrofuran-3-yl)oxy)benzo[d]thiazole-6-carboxylate (6s49). Prepared according to the typical procedure D from 2aminobenzothiazole 5s49 (250 mg, 0.850 mmol); gray powder (296 mg, 74% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.23 (s, 2H), 8.27 (s, 1H), 7.46 (s, 1H), 5.39−5.28 (m, 1H), 3.99−3.91 (m, 2H), 3.88 (s, 3H), 3.85−3.79 (m, 2H), 2.34−2.28 (m, 1H), 2.27 (s, 3H), 2.12−2.03 (m, 1H); MS (ESI): m/z calcd for C19H17Cl2N3O5S: 469.07. Found: 470.3 [M + H]+. 4.2.181. 2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4((tetrahydrofuran-3-yl)oxy)benzo[d]thiazole-6-carboxylic Acid (49). Prepared according to the typical procedure E from methyl ester 6s49 (153 mg, 0.325 mmol); brown solid (89 mg, 60% yield). 1 H NMR (400 MHz, DMSO-d6): δ 12.92 (s, 1H), 12.23 (s, 2H), 8.23 (s, 1H), 7.45 (s, 1H), 5.32 (s, 1H), 4.06−3.69 (m, 4H), 2.27 (s, 4H), 2.16−1.96 (m, 1H). 13C NMR (101 MHz, DMSO-d6): δ = 167.05, 159.69, 156.66, 156.64, 148.78, 133.06, 129.97, 126.70, 116.87, 116.28, 115.79, 110.01, 109.68, 78.06, 72.34, 66.50, 39.52, 32.36, 11.07; HRMS (ESI) m/z: [M + H]+ calcd for C18H16Cl2N3O5S 456.0188; found 456.0182; HPLC purity (254 nm): 97%. 4.2.182. Methyl 2-(4-Chloro-5-methyl-1H-pyrrole-2-carboxamido)-4-(1-phenylethoxy)benzo[d]thiazole-6-carboxylate (38s50). Prepared according to the general procedure J from methyl 2amino-4-(1-phenylethoxy)benzo[d]thiazole-6-carboxylate (5s27) (131 mg, 0.400 mmol) and 2-trichloroacetyl-4-chloro-5-methyl-1Hpyrrole (34s50) (104 mg, 0.400 mmol); white solid (141 mg, 75% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.84 (s, 1H), 12.18 (s, 1H), 8.14 (s, 1H), 7.49−7.42 (m, 3H), 7.40−7.32 (m, 3H), 7.26 (t, J = 7.1 Hz, 1H), 5.81 (q, J = 6.2 Hz, 1H), 3.80 (s, 3H), 2.22 (s, 3H), 1.65 (d, J = 6.2 Hz, 3H); MS (ESI): m/z calcd for C23H20ClN3O4S: 469.09. Found: 470.2 [M + H]+. 4.2.183. 2-(4-Chloro-5-methyl-1H-pyrrole-2-carboxamido)-4-(1ph-enylethoxy)benzo[d]thiazole-6-carboxylic Acid (50). Prepared according to the typical procedure E from methyl ester 38s50 (100 mg, 0.213 mmol); white solid (83 mg, 86% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.82 (s, 2H), 12.20 (s, 1H), 8.12 (d, J = 0.9 Hz, 1H), 7.49−7.42 (m, 3H), 7.40−7.33 (m, 3H), 7.26 (t, J = 7.3 Hz, 1H), 5.79 (q, J = 6.2 Hz, 1H), 2.23 (s, 3H), 1.65 (d, J = 6.2 Hz, 3H); 13 C NMR (101 MHz, DMSO-d6): δ 166.95, 160.15, 158.01, 149.03, 142.58, 142.37, 132.87, 131.67, 128.63, 127.55, 126.25, 125.59, 125.52, 120.73, 115.76, 113.24, 110.23, 109.75, 75.42, 24.40, 10.34; HRMS (ESI) m/z: [M + H]+ calcd for C22H19ClN3O4S 456.0785; found 456.0771; HPLC purity (254 nm): 96%. 4.2.184. Methyl 2-(4-Fluoro-5-methyl-1H-pyrrole-2-carboxamido)-4-(1-phenylethoxy)benzo[d]thiazole-6-carboxylate (39s51). Oxalyl chloride (0.42 mL, 4.88 mmol) was added to a suspension J = 13.5 Hz, 6.7 Hz, 1H), 1.54 (s, 9H), 1.19 (d, J = 6.2 Hz, 3H), 0.96 (dd, J = 9.3 Hz, 6.8 Hz, 6H); MS (ESI): m/z calcd for C17H24N2O3S: 336.15. Found: 335.4 [M − H]−. 4.2.170. tert-Butyl 2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4-((3-methylbutan-2-yl)oxy)benzo[d]thiazole-6-carboxylate (6s47). Prepared according to the typical procedure D from 5s47 (248 mg, 0.736 mmol); gray solid (200 mg, 53% yield). 1H NMR (400 MHz, DMSO-d6) δ 12.28 (s, 1H), 12.18 (s, 1H), 8.14 (s, 1H), 7.44 (s, 1H), 4.52 (p, J = 5.9 Hz, 1H), 2.27 (s, 3H), 1.99 (dq, J = 13.3 Hz, 6.8 Hz, 1H), 1.57 (s, 9H), 1.27 (d, J = 6.1 Hz, 3H), 0.99 (dd, J = 15.0 Hz, 6.8 Hz, 6H); MS (ESI): m/z calcd for C23H27Cl2N3O4S: 511.11. Found: 512.4 [M + H]+. 4.2.171. 2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4((3-methylbutan-2-yl)oxy)benzo[d]thiazole-6-carboxylic Acid (47). A solution of the tert-butyl ester 6s47 (50 mg, 0.098 mmol) and CF3COOH (0.1 mL) in dichloromethane (3 mL) was stirred at 22 °C for 5.5 h (55% conversion by 1H NMR). An additional CF3COOH (0.1 mL) was added, and the mixture was stirred at 22 °C for a further 24 h. The reaction mixture was concentrated under reduced pressure, and the solid residue was dispersed in MeOH (5 mL), filtered, washed with Et2O (3 mL), and air-dried to give an off-white solid (23 mg, 52% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.97 (s, 1H), 12.27 (s, 1H), 12.17 (s, 1H), 8.19 (s, 1H), 7.48 (s, 1H), 4.56−4.47 (m, 1H), 2.27 (s, 3H), 2.02−1.95 (m, 1H), 1.27 (d, J = 6.1 Hz, 3H), 1.01 (J = 6.8 Hz, 3H), 0.98 (J = 6.7 hZ, 3H); 13C NMR (101 MHz, DMSO-d6) (representative signals): δ 167.10, 149.49, 133.04, 129.83, 126.75, 116.87, 115.71, 115.58, 109.95, 109.76, 78.48, 32.38, 18.60, 17.45, 15.89, 11.05; HRMS (ESI) m/z: [M − H]− calcd for C19H20Cl2N3O4S 456.0552; found 456.0536; HPLC purity (254 nm): 99%. 4.2.172. Methyl 3-(1-Cyclopropylethoxy)-4-nitrobenzoate (3s48). Prepared according to the typical procedure H from 2s (1.97 g, 10.00 mmol) and 1-cyclopropylethanol (947 mg, 11.00 mmol); white solid (2.39 g, 90% yield). 1H NMR (400 MHz, CDCl3): δ 7.76 (d, J = 8.4 Hz, 1H), 7.73 (d, J = 1.5 Hz, 1H), 7.66 (dd, J = 8.4 Hz, 1.5 Hz, 1H), 4.13−4.06 (m, 1H), 3.96 (s, 3H), 1.45 (d, J = 6.2 Hz, 3H), 1.21−1.11 (m, 1H), 0.62−0.57 (m, 1H), 0.57−0.52 (m, 1H), 0.43−0.34 (m, 1H), 0.34−0.25 (m, 1H). 4.2.173. Methyl 4-Amino-3-(1-cyclopropylethoxy)benzoate (4s48). Prepared according to the typical procedure I from 3s48 (2.21 g, 8.33 mmol); white solid (1.78 g, 91% yield). 1H NMR (400 MHz, DMSO-d6): δ 7.41−7.33 (m, 1H), 7.34−7.27 (m, 1H), 6.72− 6.60 (m, 1H), 5.54 (s, 2H), 3.95−3.79 (m, 1H), 3.74 (s, 1H), 1.35− 1.22 (m, 3H), 1.18−1.01 (m, 1H), 0.53−0.36 (m, 2H), 0.38−0.21 (m, 2H). 4.2.174. Methyl 2-Amino-4-(1-cyclopropylethoxy)benzo[d]thiazole-6-carboxylate (5s48). Prepared according to the typical procedure C from aniline 4s48 (1.06 g, 4.50 mmol); light yellow powder (724 mg, 55% yield). 1H NMR (400 MHz, DMSO-d6) δ 7.92 (d, J = 1.2 Hz, 1H), 7.86 (s, 2H), 7.33 (d, J = 1.2 Hz, 1H), 4.19−4.10 (m, 1H), 3.82 (s, 3H), 1.30 (d, J = 6.1 Hz, 3H), 1.14−1.04 (m, 1H), 0.52−0.40 (m, 2H), 0.35−0.24 (m, 2H); 13C NMR (101 MHz, DMSO-d6): δ 168.36, 166.13, 147.78, 147.17, 132.15, 122.21, 115.73, 112.75, 78.23, 51.93, 19.53, 16.76, 3.82, 1.60. 4.2.175. Methyl 4-(1-Cyclopropylethoxy)-2-(3,4-dichloro-5-methyl-1H-pyrrole-2-carboxamido)benzo[d]thiazole-6-carboxylate (6s48). Prepared according to the typical procedure D from 2aminobenzothiazole 5s48 (250 mg, 0.854 mmol); white solid (40 mg, 10% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.25 (s, 1H), 12.22 (s, 1H), 8.23 (s, 1H), 7.47 (s, 1H), 4.29−4.15 (m, 1H), 3.87 (s, 3H), 2.27 (s, 3H), 1.38 (d, J = 6.1 Hz, 3H), 1.22−1.11 (m, 1H), 0.58−0.46 (m, 2H), 0.43−0.31 (m, 2H). 4.2.176. 4-(1-Cyclopropylethoxy)-2-(3,4-dichloro-5-methyl-1Hpyrrole-2-carboxamido)benzo[d]thiazole-6-carboxylic Acid (48). Prepared according to the typical procedure E from methyl ester 6s48 (33 mg, 0.070 mmol); brown powder (15 mg, 47% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.94 (s, 1H), 12.25 (s, 1H), 12.19 (s, 1H), 8.18 (s, 1H), 7.47 (s, 1H), 4.33−4.11 (m, 1H), 2.28 (s, 3H), 1.49−1.31 (m, 3H), 1.24−1.07 (m, 1H), 0.63−0.46 (m, 2H), 0.44− 0.26 (m, 2H); HRMS (ESI) m/z: [M + H] + calcd for 1411 https://doi.org/10.1021/acs.jmedchem.2c01597 J. Med. Chem. 2023, 66, 1380−1425 Journal of Medicinal Chemistry pubs.acs.org/jmc of 4-fluoro-5-methyl-1H-pyrrole-2-carboxylic acid (35s51) (70 mg, 0.489 mmol) in anhydrous dichloromethane (5 mL), and the mixture was stirred at 22 °C overnight. The resulting light brown solution was concentrated under reduced pressure, and the reaction vessel was backfilled with argon to obtain the corresponding acyl chloride as a light brown powder. Methyl 2-amino-4-(1-phenylethoxy)benzo[d]thiazole-6-carboxylate (5s27) (161 mg, 0.489 mmol) and normal grade toluene (10.5 mL) were added, and the resulting suspension equipped with a CaCl2 tube was refluxed (oil bath 130 °C) overnight. The reaction mixture was cooled to 22 °C, and the precipitate was collected and washed with toluene. The product was obtained after trituration with cold MeOH as a light brown powder (136 mg, 57% yield). It contained 2−3 mol % (by 1H NMR) of the 4-OH impurity, resulting from acidolytic cleavage of the phenethyl ether. 1H NMR (400 MHz, DMSO-d6): δ 12.74 (s, 1H), 11.85 (s, 1H), 8.15 (d, J = 1.4 Hz, 1H), 7.47−7.42 (m, 2H), 7.38−7.32 (m, 3H), 7.30−7.23 (m, 2H), 5.79 (q, J = 6.3 Hz, 1H), 3.80 (s, 3H), 2.20 (s, 3H), 1.64 (d, J = 6.3 Hz, 3H); 19F NMR (376 MHz, DMSO-d6) δ −166.53; 13C NMR (101 MHz, DMSO-d6): δ 165.90, 160.57, 158.45 (d, J = 3.2 Hz), 149.17, 149.09, 146.82, 133.04, 128.67, 127.62, 125.57, 124.90, 119.14 (d, J = 25.2 Hz), 116.70 (d, J = 7.0 Hz), 115.74, 110.06, 100.84 (d, J = 15.8 Hz), 75.54, 52.16, 24.36, 8.83 (d, J = 2.2 Hz). 4.2.185. 2-(4-Fluoro-5-methyl-1H-pyrrole-2-carboxamido)-4-(1phenylethoxy)benzo[d]thiazole-6-carboxylic Acid (51). Prepared according to the typical procedure E from methyl ester 39s51 (101 mg, 0.222 mmol); light brown solid (80 mg, 82% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.79 (s, 1H), 12.72 (s, 1H), 11.85 (s, 1H), 8.11 (s, 1H), 7.45 (d, J = 7.5 Hz, 2H), 7.40−7.31 (m, 3H), 7.32−7.21 (m, 2H), 5.78 (q, J = 6.3 Hz, 1H), 2.20 (s, 3H), 1.64 (d, J = 6.3 Hz, 3H); 19F NMR (376 MHz, DMSO-d6): δ −166.57; 13C NMR (101 MHz, DMSO-d6): δ 167.45, 160.69, 158.91, 149.49, 147.26, 143.07, 142.89, 133.35, 129.11 (2C), 128.03, 126.68, 126.00 (2C), 119.53 (d, J = 25 Hz), 117.18 (d, J = 7.1 Hz), 116.24, 110.72, 101.25 (d, J = 16 Hz), 75.89, 24.88, 9.29; HRMS (ESI) m/z: [M − H]− calcd for C22H17FN3O4S 438.0924. Found 438.09335; HPLC purity (254 nm): 95%. 4.2.186. Methyl (S)-2-(4-Fluoro-5-methyl-1H-pyrrole-2-carboxamido)-4-(1-phenylethoxy)benzo[d]thiazole-6-carboxylate (39sS51). Prepared from 24sS27 (173 mg, 0.526 mmol) and 35s51 (75 mg, 0.526 mmol) as described above for the synthesis of 39s51; light brown powder (136 mg, 57% yield). 1H NMR and MS data were identical to those of the racemic product. 4.2.187. (S)-2-(4-Fluoro-5-methyl-1H-pyrrole-2-carboxamido)-4(1-phenylethoxy)benzo[d]thiazole-6-carboxylic Acid [(S)-51]. Prepared according to the typical procedure E from methyl ester 39sS51 (100 mg, 0.221 mmol); brown solid (80 mg, 83% yield). 1H NMR and MS data were identical to those of the racemic product; HPLC purity (254 nm): 93% (sum of enantiomers); 82% ee (determined by chiral HPLC analysis on a Kromasil 3-CelluCoat column (4.6 mm × 150 mm)), eluent hexane/MeOH/0.1%TFA in i-PrOH = 90:5:5; tR = 5.4 min (S enantiomer), 6.3 min (R enantiomer). The absolute configuration of the major product was assigned based on the starting (R)-1-phenylethanol. 4.2.188. Methyl (R)-2-(4-Fluoro-5-methyl-1H-pyrrole-2-carboxamido)-4-(1-phenylethoxy)benzo[d]thiazole-6-carboxylate (39sR51). Prepared from 24sR27 (158 mg, 0.481 mmol) and 35s51 (69 mg, 0.481 mmol) as described above for the synthesis of 39s51; white solid (72 mg, 33% yield). 1H NMR and MS data were identical to those of the racemic product. HPLC purity (254 nm): 97.3%. 4.2.189. (R)-2-(4-Fluoro-5-methyl-1H-pyrrole-2-carboxamido)-4(1-phenylethoxy)benzo[d]thiazole-6-carboxylic Acid [(R)-51]. Prepared according to the typical procedure E from methyl ester 39sR51 (60 mg, 0.132 mmol); off-white solid (45 mg, 78% yield). 1H NMR and MS data were identical to those of the racemic product. HPLC purity (254 nm): 96.4% (sum of enantiomers); >95% ee (determined by chiral HPLC analysis on a Kromasil 3-CelluCoat column (4.6 mm × 150 mm)), eluent hexane/MeOH/0.1% TFA in i-PrOH = 90:5:5; tR = 5.4 min (S enantiomer), 6.3 min (R enantiomer). The absolute configuration of the major product was assigned based on the starting (S)-1-phenylethanol. Article 4.2.190. Methyl 2-(4-Cyano-5-methyl-1H-pyrrole-2-carboxamido)-4-(1-phenylethoxy) benzo[d]thiazole-6-carboxylate (39s52). 4Cyano-5-methyl-1H-pyrrole-2-carboxylic acid (35s52) (64 mg, 0.43 mmol) was suspended in SOCl2 (1.55 mL, 21.3 mmol). The mixture was stirred at 22 °C overnight and concentrated under reduced pressure. Compound 5s27 (70 mg, 0.213 mmol) and toluene (4 mL) were added, and the mixture was stirred at 130 °C for 3 h. The precipitate was collected and purified by column chromatography, eluent DCM/THF = 10:1 to get product 39s52 as a beige solid (59 mg, 60% yield). 1H NMR (400 MHz, DMSO-d6): δ 13.08 (s, 1H), 12.76 (s, 1H), 7.50−7.42 (m, 2H), 7.41−7.31 (m, 3H), 7.30−7.23 (m, 1H), 5.80 (q, J = 6 Hz, 1H), 3.80 (s, 3H), 2.40 (s, 3H), 1.65 (d, J = 6 Hz, 3H); MS (ESI): m/z calcd for C24H20N4O4S: 460.12. Found: 459.1 [M − H]−. 4.2.191. 2-(4-Cyano-5-methyl-1H-pyrrole-2-carboxamido)-4-(1phenylethoxy)benzo[d]thiazole-6-carboxylic Acid (52). Prepared according to the typical procedure E from methyl ester 39s52 (44 mg, 0.096 mmol): white solid (30 mg, 70% yield). 1H NMR (400 MHz, DMSO-d6) δ: 13.05 (s, 1H), 12.84 (s, 1H), 12.76 (s, 1H), 8.14 (d, J = 1.4 Hz, 1H), 7.74 (d, J = 2.3 Hz, 1H), 7.48−7.42 (m, 2H), 7.40−7.31 (m, 3H), 7.29−7.22 (m, 1H), 5.79 (q, J = 6.3 Hz, 1H), 2.40 (s, 3H), 1.65 (d, J = 6.3 Hz, 3H); HRMS (ESI) m/z: [M + H]+ calcd for C23H19N4O4S 447.1127; found 447.1133; HPLC purity (254 nm): 97%. 4.2.192. Methyl 2-(5-Methyl-1H-pyrrole-2-carboxamido)-4-(1phenylethoxy)benzo[d]thiazole-6-carboxylate (38s53). Prepared according to the typical procedure J from 5s27 (86 mg, 0.261 mmol) and 2-trichloroacetyl-5-methyl-1H-pyrrole (34s53) (59 mg, 0.261 mmol); white solid (100 mg, 88% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.67 (s, 1H), 11.75 (s, 1H), 8.14 (d, J = 1.2 Hz, 1H), 7.45 (d, J = 7.3 Hz, 2H), 7.41−7.32 (m, 4H), 7.26 (t, J = 7.3 Hz, 1H), 6.00−5.94 (m, 1H), 5.80 (q, J = 6.2 Hz, 1H), 3.80 (s, 3H), 2.26 (s, 3H), 1.65 (d, J = 6.2 Hz, 3H); 13C NMR (101 MHz, DMSO-d6): δ 165.93, 160.80, 158.47, 149.02, 142.84, 142.53, 135.73, 133.04, 128.66, 127.61, 125.57, 124.73, 122.12, 115.72, 115.59, 110.09, 108.82, 75.52, 52.15, 24.36, 12.77; MS (ESI): m/z calcd for C23H21N3O4S: 435.13. Found: 434.5 [M − H]−. 4.2.193. 2-(5-Methyl-1H-pyrrole-2-carboxamido)-4-(1phenylethoxy)benzo[d]thiazole-6-carboxylic Acid (53). Prepared according to the typical procedure E from methyl ester 38s53 (85 mg, 0.195 mmol); beige solid (73 mg, 89% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.79 (s, 1H), 12.64 (s, 1H), 11.75 (s, 1H), 8.10 (s, 1H), 7.55−7.21 (m, 7H), 5.96 (s, 1H), 5.78 (q, J = 6 Hz, 1H), 2.26 (s, 3H), 1.64 (d, J = 6 Hz, 3H); 13C NMR (101 MHz, DMSOd6): δ 167.00, 160.44, 158.47, 148.94, 142.63, 142.51, 135.65, 132.87, 128.63, 127.54, 126.03, 125.53, 122.15, 115.73, 115.53, 110.26, 108.78, 75.41, 24.41, 12.75; HRMS (ESI) m/z: [M + H]+ calcd for C22H20N3O4S 422.1175; found 422.1161; HPLC purity (254 nm): 98%. 4.2.194. Methyl 2-(3-Fluoro-5-methyl-1H-pyrrole-2-carboxamido)-4-(1-phenylethoxy)benzo[d]thiazole-6-carboxylate (39s54). A suspension of 3-fluoro-5-methyl-1H-pyrrole-2-carbonyl chloride (56 mg, 0.347 mmol), prepared from 3-fluoro-1H-pyrrole-2-carboxylic acid (35s54) and oxalyl chloride as described above, and 5s27 (114 mg, 0.347 mmol) in toluene (7 mL) was equipped with a CaCl2 tube and refluxed overnight. After cooling to 22 °C, the precipitate was collected and washed with toluene to obtain the title compound as a light gray powder (91 mg, 57% yield). 1H NMR (400 MHz, DMSOd6): δ 12.23 (s, 1H), 11.37 (s, 1H), 8.17 (d, J = 1.5 Hz, 1H), 7.49− 7.42 (m, 2H), 7.40−7.31 (m, 3H), 7.29−7.25 (m, 1H), 6.91 (app t, J = 4.2 Hz, 1H), 5.80 (q, J = 6.2 Hz, 1H), 3.80 (s, 3H), 1.99 (s, 3H), 1.64 (d, J = 6.2 Hz, 3H); 19F NMR (376 MHz, DMSO-d6): δ −153.97 (d, J = 4 Hz); 13C NMR (101 MHz, DMSO-d6) δ 165.87, 160.07, 156.71, 154.47, 151.93, 149.08, 142.67, 142.50, 133.03, 128.64, 128.54 (d, J = 70.2 Hz), 127.61, 125.58, 125.01, 121.03 (d, J = 6.0 Hz), 115.90, 110.32, 107.78 (d, J = 17.0 Hz), 106.23 (d, J = 13.5 Hz), 75.62, 52.16, 24.28, 7.19; HRMS (ESI) m/z: [M − H]− calcd for C23H19FN3O4S 452.1080; found 452.1092. 4.2.195. 2-(3-Fluoro-5-methyl-1H-pyrrole-2-carboxamido)-4-(1phenylethoxy)benzo[d]thiazole-6-carboxylic Acid (54). A solution 1412 https://doi.org/10.1021/acs.jmedchem.2c01597 J. Med. Chem. 2023, 66, 1380−1425 Journal of Medicinal Chemistry pubs.acs.org/jmc of methyl ester 39s54 (70 mg, 0.154 mmol) and 2 M NaOH (0.40 mL) in MeOH (3.0 mL) was stirred at 40 °C overnight. NaOH (2 M, 0.40 mL) was added, and the mixture was stirred for another night and concentrated. The residue was suspended in water (2 mL), pH was adjusted to 2 by adding 4 M HCl, the precipitate was collected, washed with water, air-dried, and triturated with MeOH to get the title compound as a beige solid (55 mg, 81% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.82 (s, 1H), 12.20 (s, 1H), 11.37 (s, 1H), 8.13 (s, 1H), 7.52−7.40 (m, 2H), 7.40−7.31 (m, 3H), 7.31−7.20 (m, 1H), 6.91 (app t, J = 4.2 Hz, 1H), 5.79 (q, J = 6.3 Hz, 1H), 1.99 (s, 3H), 1.64 (d, J = 6.3 Hz, 3H); 19F NMR (376 MHz, DMSO-d6): δ −154.06 (d, J = 4 Hz); 13C NMR (101 MHz, DMSO-d6): δ 166.92, 159.75, 156.78, 154.36, 151.82, 148.98, 142.57, 142.28, 132.83, 128.61, 127.55, 126.31, 125.53, 120.98 (d, J = 5.3 Hz), 115.90, 110.44, 107.83 (d, J = 15 Hz), 106.18 (d, J = 14 Hz), 75.48, 24.33, 7.17; HRMS (ESI) m/z: [M − H]− calcd for C22H17FN3O4FS 438.0924; found 438.0934; HPLC purity (254 nm): 94%. 4.2.196. Methyl 2-(3,4-Dichloro-5-(phthalimidomethyl)-1H-pyrrole-2-carboxamido)-4-(1-phenylethoxy)benzo[d]thiazole-6-carboxylate (40s55). A suspension of 3,4-dichloro-5-(phthalimidomethyl)-1H-pyrrole-2-carboxylic acid (36s55)36 (155 mg, 0.457 mmol) in SOCl2 (2 mL) was refluxed for 1 h and then concentrated under reduced pressure. To the solid residue were added 5s27 (150 mg, 0.457 mmol) and toluene (9 mL), and the resulting suspension was refluxed overnight. Upon cooling, the precipitate was collected and washed with MeOH to get the crude product (200 mg), containing 10 mol % of an O-dealkylated impurity. After purification with flash chromatography on silica (20 mL), eluent dichloromethane/THF = 20:1, dry-loading with THF, the title compound was obtained as a white solid (126 mg, 42% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.57 (s, 1H), 12.41 (s, 1H), 8.18 (s, 1H), 7.970 7.92 (m, 2H), 7.91− 7.86 (m, 2H), 7.44 (d, J = 7.3 Hz, 2H), 7.39−7.31 (m, 3H), 7.25 (app t, J = 7.3 Hz, 1H), 5.79 (q, J = 6.4 Hz, 1H), 4.84 (s, 2H), 3.80 (s, 3H), 1.62 (d, J = 6.4 Hz, 3H). 4.2.197. (3,4-Dichloro-5-((6-(methoxycarbonyl)-4-(1-phenylethoxy)-benzo[d]thiazol-2-yl)carbamoyl)-1H-pyrrol-2-yl)methanaminium 1,4-Dioxo-3,4-dihydro-1H-phthalazin-2-ide (41s55). Hydrazine hydrate (80%, 0.1 mL, 10 equiv) was added to 40s55 (100 mg, 0.161 mg) in abs. EtOH (3.5 mL), and the suspension was stirred at 40 °C for 40 min. The reaction mixture was concentrated on a water pump, suspended in MeOH, 37% HCl(aq) (3 drops) was added, sonicated to reslurry, and concentrated. The white residue was refluxed in EtOH overnight, then concentrated and triturated with acetone to give 41s55 as an off-white solid (100 mg, 91% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.68 (s, 2H), 11.71 (br s, 1H), 8.37 (s, 3H), 8.21 (d, J = 1 Hz, 1H), 8.16−8.01 (m, 1H), 7.92−7.86 (m, 2H), 7.47 (d, J = 7.3 Hz, 2H), 7.41 (d, J = 1 Hz, 1H), 7.36 (t, J = 7 Hz, 2H), 7.27 (t, J = 7.3 Hz, 1H), 5.82 (q, J = 6.3 Hz, 1H), 4.09 (q, J = 5.3 Hz, 2H), 3.81 (s, 3H), 1.65 (d, J = 6.3 Hz, 3H). 4.2.198. (5-((6-Carboxy-4-(1-phenylethoxy)benzo[d]thiazol-2-yl)carbamoyl)-3,4-dichloro-1H-pyrrol-2-yl)methanaminium Chloride (55). A mixture of methyl ester 41s55 (100 mg, 0.147 mmol), MeOH (3.7 mL), and 2 M NaOH (0.370 mL) was stirred at 50 °C for 24 h. NaOH (2 M, 0.370 mL) was added, and stirring was continued for a further 24 h. The reaction mixture was concentrated, water (1 mL) was added, and the solids were filtered off. The filtrate was acidified to pH 2 by adding 2 M HCl and cooled to 0 °C, and the precipitate was collected, washed with water, and air-dried. The resulting phthalate salt was triturated with a solution of HCl in MeOH (2 × 1 mL) to get 55 as a light yellow powder (23 mg, 29% yield). 1H NMR (400 MHz, DMSO-d6) δ 12.90 (s, 1H), 12.67 (s, 1H), 12.61 (s, 1H), 8.35 (s, 3H), 8.16 (s, 1H), 7.60−7.19 (m, 6H), 5.81 (q, J = 6 Hz, 1H), 4.09 (s, 2H), 1.65 (d, J = 6 Hz, 3H); HRMS (ESI) m/z: [M − Cl]+ calcd for C22H19Cl2N4O4S 505.0499; found 505.0487; HPLC purity (254 nm): 98%. 4.2.199. Methyl 4-Nitro-3-((1,1,1-trifluoro-3-morpholinopropan2-yl)oxy)benzoate (30s57). Prepared according to the typical procedure H from 2s (2.01 g, 10.19 mmol) and 1,1,1-trifluoro-3morpholinopropan-2-ol (2.23 g, 11.21 mmol); light yellow oil solid (810 mg, 21% yield); 1H NMR (400 MHz, CDCl3): δ 7.86 (dd, J = 8.0 Hz, 0.6 Hz, 1H), 7.81−7.73 (m, 2H), 4.46 (dd, J = 10.2 Hz, 8.2 Article Hz, 1H), 4.39 (dd, J = 10.2 Hz, 3.9 Hz, 1H), 3.99 (s, 3H), 3.72−3.57 (m, 4H), 3.61−3.49 (m, 1H), 2.91−2.81 (m, 4H); MS (ESI): m/z calcd for C15H17F3N2O6: 378.10. Found: 379.1 [M + H]+. 4.2.200. Methyl 4-Amino-3-((1,1,1-trifluoro-3-morpholinopropan-2-yl)oxy)benzoate (31s57). Prepared according to the typical procedure I from 30s57 (836 mg, 2.21 mmol); colorless oil (770 mg, 100% yield); 1H NMR (400 MHz, CDCl3): δ 7.59 (dd, J = 8.2 Hz, 1.8 Hz, 1H), 7.49 (d, J = 1.8 Hz, 1H), 6.69 (d, J = 8.3 Hz, 1H), 4.33 (d, J = 6.1 Hz, 2H), 4.26 (s, 2H), 3.87 (s, 3H), 3.76−3.63 (m, 4H), 3.57−3.42 (m, 1H), 2.95−2.81 (m, 4H); 19F NMR (376 MHz, CDCl3): δ −68.37 (d, J = 8.6 Hz). 4.2.201. Methyl 2-Amino-4-((1,1,1-trifluoro-3-morpholinopropan-2-yl)oxy)benzo[d]thiazole-6-carboxylate (32s57). Prepared according to the typical procedure C from aniline 31s57 (781 mg, 2.24 mmol); light orange crispy foam (309 mg, 34% yield). 1H NMR (400 MHz, CDCl3): δ 8.01 (d, J = 1.4 Hz, 1H), 7.56 (d, J = 1.4 Hz, 1H), 5.73 (s, 2H), 4.53 (dd, J = 10.9 Hz, 7.8 Hz, 1H), 4.47 (dd, J = 10.8 Hz, 4.3 Hz, 1H), 3.93 (s, 3H), 3.73−3.58 (m, 5H), 2.94−2.88 (m, 4H); MS (ESI): m/z calcd for C16H18F3N3O4S: 405.09. Found: 406.0 [M + H]+. 4.2.202. Methyl 2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4-((1,1,1-trifluoro-3-morpholinopropan-2-yl)oxy)benzo[d]thiazole-6-carboxylate (33s57). Prepared according to the typical procedure J from 32s57 (152 mg, 0.375 mmol); white solid (48 mg, 22% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.26 (s, 1H), 12.09 (s, 1H), 8.26 (s, 1H), 7.59 (s, 1H), 4.66 (dd, J = 10.9 Hz, 6.8 Hz, 1H), 4.52 (dd, J = 11.0 Hz, 4.6 Hz, 1H), 3.95−3.78 (m, 4H), 3.59− 3.47 (m, 4H), 2.91−2.80 (m, 4H), 2.26 (s, 3H); MS (ESI): m/z calcd for C22H21Cl2F3N4O5S: 580.06. Found: 580.9 [M + H]+. 4.2.203. 2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4((1,1,1-trifluoro-3-morpholinopropan-2-yl)oxy)benzo[d]-thiazole6-carboxylic Acid (57). Prepared according to the typical procedure E from methyl ester 33s57 (46 mg, 0.079 mmol); light brown solid (30 mg, 67% yield). 1H NMR (400 MHz, DMSO-d6): δ 13.01 (s, 1H), 12.34 (s, 1H), 12.04 (s, 1H), 8.29 (s, 1H), 7.63 (s, 1H), 4.73−4.59 (m, 1H), 4.58−4.46 (m, 1H), 3.93−3.80 (m, 1H), 3.53 (s, 4H), 2.86 (s, 4H), 2.28 (s, 3H); 19F NMR (376 MHz, DMSO-d6): δ −67.73 (d, J = 9.4 Hz); HRMS (ESI) m/z: [M − H] − calcd for C21H18Cl2F3N4O5S 565.0327; found 565.0331; HPLC purity (254 nm): 96%. 4.2.204. Methyl 3-(1-Cyclopropyl-2-morpholinoethoxy)-4-nitrobenzoate (30s58). Prepared according to the typical procedure H from 2s (2.10 g, 10.65 mmol) and 1-cyclopropyl-2-morpholinoethan1-ol (2.01 g, 11.72 mmol); yellow oil containing ca. 25 mol % of a DIAD-derived impurity (2.80 g, 75% yield), used as such in the next step. 1H NMR (400 MHz, CDCl3): δ 7.91 (d, J = 1.6 Hz, 1H), 7.76 (d, J = 8.4 Hz, 1H), 7.67 (dd, J = 8.4 Hz, 1.6 Hz, 1H), 4.06 (app td, J = 7.9 Hz, 2.9 Hz, 1H), 3.96 (s, 3H), 3.63−3.48 (m, 4H), 2.83 (dd, J = 13.8 Hz, 8.2 Hz, 1H), 2.66 (dd, J = 13.8 Hz, 2.8 Hz, 1H), 2.51 (ddd, J = 11.5 Hz, 5.8 Hz, 3.5 Hz, 2H), 2.49−2.39 (m, 2H), 1.21−1.06 (m, 1H), 0.65−0.56 (m, 1H), 0.61−0.52 (m, 1H), 0.48−0.34 (m, 1H), 0.36−0.22 (m, 1H). 4.2.205. Methyl 4-Amino-3-(1-cyclopropyl-2-morpholinoethoxy)-benzoate (31s58). Prepared according to the typical procedure I from crude 30s58 (2.80 g); colorless oil containing ca. 0.25 mol % of a DIAD-derived impurity (2.7 g, 100% yield), used as such in the next step. 1H NMR (400 MHz, CDCl3): δ 7.71 (d, J = 1.9 Hz, 1H), 7.58 (dd, J = 8.3 Hz, 1.9 Hz, 1H), 6.63 (d, J = 8.3 Hz, 1H), 4.91 (s, 2H), 3.85 (s, 3H), 3.76−3.61 (m, 5H), 3.42 (app td, J = 8.8 Hz, 2.5 Hz, 1H), 2.83 (dd, J = 13.6 Hz, 9.0 Hz, 1H), 2.65−2.50 (m, 4H), 1.16−1.02 (m, 1H), 0.66−0.58 (m, 1H), 0.57−0.49 (m, 1H), 0.35−0.26 (m, 1H), 0.16−0.08 (m, 1H); MS (ESI): m/z calcd for C17H24N2O4: 320.17. Found: 320.9 [M + H]+. 4.2.206. Methyl 2-Amino-4-(1-cyclopropyl-2-morpholinoethoxy)-benzo[d]thiazole-6-carboxylate (32s58). Prepared according to the typical procedure C from aniline 31s57 (2.67 g, 8.33 mmol); yellow powder (1.1 g, 35% yield). 1H NMR (400 MHz, DMSO-d6): δ 7.91 (s, 1H), 7.84 (s, 2H), 7.48 (s, 1H), 4.28 (td, J = 7.3 Hz, 3.8 Hz, 1H), 3.82 (s, 3H), 3.51−3.44 (m, 3H), 2.73−2.30 (m, 6H), 1.16− 1.03 (m, 1H), 0.51−0.34 (m, 2H), 0.37−0.23 (m, 2H); MS (ESI): m/z calcd for C18H23N3O4S: 377.14. Found: 377.9 [M + H]+. 1413 https://doi.org/10.1021/acs.jmedchem.2c01597 J. Med. Chem. 2023, 66, 1380−1425 Journal of Medicinal Chemistry pubs.acs.org/jmc Article δ 12.91 (s, 1H), 12.65 (s, 1H), 11.91 (s, 1H), 10.57 (s, 1H), 8.20 (s, 1H), 7.57−7.21 (m, 7H), 6.49−6.29 (m, 1H), 4.17−3.33 (m, 10H), 2.21 (s, 3H); 19F NMR (376 MHz, DMSO-d6): δ −166.52; HRMS (ESI) m/z: [M − Cl]+ calcd for C26H26FN4O5S 525.1608; found 525.1599; HPLC purity (254 nm): 95%. 4.2.213. tert-Butyl 2-(4-Fluoro-5-methyl-1H-pyrrole-2-carboxamido)-4-isopropoxybenzo[d]thiazole-6-carboxylate (39s61). To a suspension of 4-fluoro-5-methyl-1H-pyrrole-2-carboxylic acid 35s51 (52 mg, 0.362 mmol) in anhydrous dichloromethane (4 mL), oxalyl chloride (0.31 mL. 3.62 mmol) was added and the reaction mixture was stirred at 22 °C overnight under an argon atmosphere. After the volatiles were removed under reduced pressure, 5s46 (93 mg, 0.302 mmol) and toluene (7 mL) were added to the residue and the reaction mixture was stirred at 130 °C overnight. The gray precipitate was collected and washed with toluene (104 mg, 79% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.66 (s, 1H), 11.85 (s, 1H), 8.13 (d, J = 1 Hz, 1H), 7.44 (d, J = 1 Hz, 1H), 7.26 (d, J = 2.3 Hz, 1H), 4.95− 4.84 (m, 1H), 2.20 (s, 3H), 1.36 (d, J = 6.0 Hz, 6H). MS (ESI): m/z calcd for C21H24FN3O4S: 433.15, found 432.2 [M − H]−. 4.2.214. 2-(4-Fluoro-5-methyl-1H-pyrrole-2-carboxamido)-4-isopropoxybenzo[d]thiazole-6-carboxylic Acid (61). tert-Butyl ester 39s61 (75 mg, 0.173 mmol) was suspended in dichloromethane (3 mL), trifluoroacetic acid (0.13 mL, 1.73 mmol) was added, and the suspension turned into a brown solution. The reaction mixture was stirred at 22 °C overnight, the solvent was removed under reduced pressure, and the residue was triturated with methanol; light brown solid (64 mg, 98% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.92 (s, 1H), 12.65 (s, 1H), 11.84 (s, 1H), 8.17 (d, J = 1.2 Hz, 1H), 7.48 (s, 1H), 7.26 (d, J = 2.7 Hz, 1H), 4.89 (hept, J = 6 Hz, 1H), 2.19 (s, 3H), 1.36 (d, J = 6 Hz, 6H); HRMS (ESI) m/z: [M + H]+ calcd for C17H17FN3O4S 378.0924; found 378.0920; HPLC purity (254 nm): 93%. 4.2.215. tert-Butyl 2-(4-Cyano-5-methyl-1H-pyrrole-2-carboxamido)-4-isopropoxybenzo[d]thiazole-6-carboxylate (39s62). A suspension of 4-cyano-5-methyl-1H-pyrrole-2-carboxylic acid (35s52) (60 mg, 0.400 mmol) in thionyl chloride (1.5 mL) was stirred at 22 °C overnight, and the volatiles were removed under reduced pressure. Compound 5s46 (100 mg, 0.324 mmol) and toluene (4 mL) were added to the residue, and the reaction mixture was stirred at 130 °C overnight. The precipitate was collected, washed with toluene, and triturated with MeOH; white solid (93 mg, 65% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.99 (s, 1H), 12.76 (s, 1H), 8.15 (s, 1H), 7.71 (s, 1H), 7.45 (s, 1H), 4.96−4.83 (m, 1H), 2.39 (s, 3H), 1.57 (s, 9H), 1.36 (d, J = 6 Hz, 6H). MS (ESI): m/z calcd for C22H24FN4O4S: 440.15. Found 439.2 [M − H]−. 60% yield. 4.2.216. 2-(4-Cyano-5-methyl-1H-pyrrole-2-carboxamido)-4isopropoxybenzo[d]thiazole-6-carboxylic Acid (62). To a suspension of tert-butyl ester 39s62 (80 mg, 0.182 mmol) in dichloromethane (3 mL) was added trifluoroacetic acid (0.14 mL, 1.82 mmol). The suspension turned into a light brown solution. The reaction mixture was stirred at 22 °C overnight. The white precipitate was collected and washed with dichloromethane, then triturated with MeOH to get the title compound as a white solid (42 mg, 60% yield). 1 H NMR (400 MHz, DMSO-d6) δ 12.97 (s, 2H), 12.75 (s, 1H), 8.19 (s, 1H), 7.71 (s, 1H), 7.49 (s, 1H), 4.96−4.84 (m, 1H), 2.39 (s, 3H), 1.36 (d, J = 5.3 Hz, 6H). HRMS (ESI) m/z: [M + H]+ calcd for C18H17N4O4S 385.0971; found 385.0962; HPLC purity (254 nm): 98%. 4.2.217. tert-Butyl 2-(4-Chloro-2-methyl-1H-imidazole-5-carboxamido)-4-isopropoxybenzo[d]thiazole-6-carboxylate (42s63). A mixture of 4-chloro-2-methyl-1H-imidazole-5-carboxylic acid (37s63) (90 mg, 0.561 mmol), SOCl2 (2 mL, 28.1 mmol), and DMF (2 drops) was refluxed for 1.5 h. The reaction mixture was concentrated under reduced pressure to obtain the crude acyl chloride as a white solid. Dry toluene (11 mL) and 5s46 (173 mg, 0.561 mmol) were added, and the resulting suspension was stirred at 130 °C overnight. After cooling to 22 °C, the precipitate (starting carboxylic acid) was filtered off and the filtrate was concentrated. To the oily residue was added EtOAc (1 mL), the precipitate (starting 2aminobenzo[d]thiazole) was filtered off, and the filtrate was 4.2.207. Methyl 4-(1-Cyclopropyl-2-morpholinoethoxy)-2-(3,4dichloro-5-methyl-1H-pyrrole-2-carboxamido)benzo[d]thiazole-6carboxylate (33s58). Prepared according to the typical procedure J from 32s57 (200 mg, 0.529 mmol); gray solid (141 mg, 48% yield). 1 H NMR (400 MHz, DMSO-d6): δ 12.25 (br s, 2H), 8.25 (s, 1H), 7.67 (s, 1H), 4.35 (s, 1H), 3.88 (s, 3H), 3.71−3.48 (m, 4H), 3.01− 2.59 (m, 6H), 2.27 (s, 3H), 1.23−1.09 (m, 1H), 0.61−0.22 (m, 4H); MS (ESI): m/z calcd for C24H26Cl2N4O5S: 552.10. Found: 553.2 [M + H]+. 4.2.208. 4-(1-Cyclopropyl-2-morpholinoethoxy)-2-(3,4-dichloro5-methyl-1H-pyrrole-2-carboxamido)benzo[d]thiazole-6-carboxylic Acid (58). Prepared according to the typical procedure E from methyl ester 33s58 (120 mg, 0.216 mmol); brown solid (56 mg, 48% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.90 (s, 1H), 12.20 (s, 2H), 8.18 (s, 1H), 7.64 (s, 1H), 4.43−4.21 (m, 1H), 3.74−3.43 (m, 4H), 2.97−2.65 (m, 6H), 2.27 (s, 3H), 1.27−1.05 (m, 1H), 0.64− 0.22 (m, 4H); 13C NMR (101 MHz, DMSO-d6): δ 167.10, 160.30, 157.41, 149.37, 142.63, 132.87, 129.77, 126.51, 117.43, 116.51, 115.33, 112.94, 109.87, 80.19, 65.46, 61.76, 53.52, 14.10, 11.04, 3.43, 1.88; HRMS (ESI) m/z: [M + H]+ calcd for C23H25Cl2N4O5S 539.0923; found 539.0916; HPLC purity (254 nm): 98%. 4.2.209. Methyl 2-(4-Chloro-5-methyl-1H-pyrrole-2-carboxamido)-4-(2-morpholino-1-phenylethoxy)benzo[d]thiazole-6-carboxylate (33s59). Methyl 2-amino-4-(2-morpholino-1-phenylethoxy)benzo[d]thiazole-6-carboxylate (32s36) (92 mg, 0.222 mmol) was suspended in anhydrous DMF (2.5 mL), and 2,2,2-trichloro-1-(4chloro-5-methyl-1H-pyrrol-2-yl)ethan-1-one (34s50) (70 mg, 0.267 mmol) and Na2CO3 (24 mg, 0.222 mmol) were added, and the reaction mixture was stirred overnight at 60 °C. DMF was removed under reduced pressure, and the residue was triturated successively with 10% aqueous citric acid, 1 M NaOH, and ethyl acetate; light yellow solid (82 mg, 66% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.82 (s, 1H), 12.18 (s, 1H), 8.18−8.13 (m, 1H), 7.52−7.42 (m, 4H), 7.39−7.30 (m, 2H), 7.30−7.21 (m, 1H), 5.85 (dd, J = 7.9, 4.2 Hz, 1H), 3.81 (s, 3H), 3.58−3.50 (m, 4H), 3.01 (dd, J = 13.4, 7.9 Hz, 1H), 2.70−2.62 (m, 1H), 2.56 (dd, J = 9.7, 4.7 Hz, 4H), 2.22 (s, 3H). 4.2.210. 4-(2-((6-Carboxy-2-(4-chloro-5-methyl-1H-pyrrole-2carboxamido)benzo[d]thiazol-4-yl)oxy)-2-phenylethyl)morpholin4-ium Chloride (59). Prepared according to the typical procedure E from methyl ester 33s59 (71 mg, 0.128 mmol); off-white solid (42 mg, 57% yield). 1H NMR (400 MHz, DMSO-d6): δ 13.36−12.59 (m, 2H), 12.34 (s, 1H), 11.40 (s, 1H), 8.22 (s, 1H), 7.70−7.18 (m, 6H), 6.53 (d, J = 8.0 Hz, 1H), 4.20−3.49 (m, 10H), 2.24 (s, 3H); 13C NMR (101 MHz, DMSO-d6): δ 166.72, 160.72, 158.05, 147.47, 142.65, 137.04, 133.22, 131.77, 129.00, 128.85, 126.28, 126.24, 120.69, 117.29, 113.67, 112.23, 109.80, 75.39, 63.22, 61.24, 52.78, 52.29, 10.37. HRMS (ESI) m/z: [M − Cl]+ calcd for C26H26ClN4O5S 541.1312; found 541.1298; HPLC purity (254 nm): 98%. 4.2.211. 4-(2-((2-(4-Fluoro-5-methyl-1H-pyrrole-2-carboxamido)-6-(methoxycarbonyl)benzo[d]thiazol-4-yl)oxy)-2-phenylethyl)morpholin-4-ium Chloride (33s60×HCl). Oxalyl chloride (0.40 mL, 4.76 mmol) was added to a suspension of 4-fluoro-5-methyl-1Hpyrrole-2-carboxylic acid (35s51) (68 mg, 0.476 mmol) in anhydrous dichloromethane (5 mL) and stirred at 22 °C overnight. The resulting light brown solution was concentrated under reduced pressure and filled with argon to obtain the acyl chloride, containing 5 mol % of the starting acid, as a light brown solid. Toluene (9.5 mL) and methyl 2amino-4-(2-morpholino-1-phenylethoxy)benzo[d]-thiazole-6-carboxylate (32s36) (197 mg, 0.476 mmol) were added, and the resulting suspension was refluxed overnight. The reaction mixture was cooled to 22 °C, and the precipitate was collected, washed with toluene, and triturated with methanol to obtain the title compound as a white powder (85 mg, 31% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.67 (s, 1H), 11.92 (s, 1H), 10.50 (s, 1H), 8.25 (s, 1H), 7.56−7.27 (m, 7H), 6.44−6.35 (m, 1H), 4.12−3.96 (m, 2H), 3.87−3.61 (m, 11H), 2.21 (s, 3H). HPLC purity (254 nm): 98.8%. 4.2.212. 4-(2-((6-Carboxy-2-(4-fluoro-5-methyl-1H-pyrrole-2carboxamido)benzo[d]thiazol-4-yl)oxy)-2-phenylethyl)morpholin4-ium Chloride (60×HCl). Prepared according to the typical procedure E from methyl ester 33s60×HCl (81 mg, 0.140 mmol); off-white solid (22 mg, 28% yield). 1H NMR (400 MHz, DMSO-d6): 1414 https://doi.org/10.1021/acs.jmedchem.2c01597 J. Med. Chem. 2023, 66, 1380−1425 Journal of Medicinal Chemistry pubs.acs.org/jmc concentrated. The residue was purified by preparative layer chromatography (20 cm × 20 cm plate, 2 mm silica layer; eluent EtOAc/hexane 2:1) to obtain the crude product (85% pure (by HPLC)). This was dissolved in EtOAc (20 mL) and washed with K2CO3(aq) (2 × 15 mL). The organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated to obtain the title compound as a white solid (59 mg, 23% yield). 1H NMR (400 MHz, DMSO-d6): δ = 12.53 (br s, 1H), 7.95 (s, 1H), 7.31 (s, 1H), 4.97 (hept, J = 6.0 Hz, 1H), 2.28 (s, 3H), 1.56 (s, 9H), 1.30 (d, J = 6.0 Hz, 6H) ppm; MS (ESI): m/z calcd for C20H23ClN4O4S: 450.11. Found: 451.0 [M + H]+; HPLC purity (254 nm): 93.5%. 4.2.218. 2-(4-Chloro-2-methyl-1H-imidazole-5-carboxamido)-4iso-propoxybenzo[d]thiazole-6-carboxylic Acid (63). A suspension of tert-butyl ester 42s63 (59 mg, 0.131 mmol) in dichloromethane (3 mL) was treated with trifluoroacetic acid (0.2 mL), and the resulting solution was stirred at room temperature and monitored by HPLCMS. After complete conversion, the reaction mixture was concentrated under reduced pressure and the solid residue was triturated with water to obtain a beige solid (41 mg, 79% yield). 1H NMR (400 MHz, DMSO-d6) δ 12.9 (br s, 2H), 12.25 (s, 1H), 8.20 (d, J = 1.4 Hz, 1H), 7.49 (d, J = 1.4 Hz, 1H), 4.90 (hept, J = 6.0 Hz, 1H), 2.36 (s, 3H), 1.36 (d, J = 6.0 Hz, 7H); HRMS (ESI) m/z: [M + H]+ calcd for C16H16ClN4O4S 395.0581; found 395.0577; HPLC purity (254 nm): 97%. 4.2.219. 2-Trichloroacetyl-3,4-dichloro-5-methyl-1H-pyrrole (46s). Ethyl 3,4-dichloro-5-methyl-1H-pyrrole-2-carboxylate (44s) (2.00 g, 9.0 mmol) was suspended in water (90 mL), and KOH (2.52 g, 45 mmol) was added. The flask was connected to a Dean− Stark apparatus, evacuated, and backfilled with nitrogen three times. The apparatus was then connected to a nitrogen line with a bubbler. The receiving tube of the Dean−Stark apparatus was prefilled with 1,2-dichloroethane (3 mL) and the rest with water. The reaction mixture was then refluxed (bath temperature between 150 and 190 °C). After 30 min, the starting material was dissolved and the mixture color turned dark brown. The decarboxylated pyrrole started to distill over, and the reaction was finished after 4 h. Refluxing longer sometimes resulted in more pyrrole being collected. The 1,2dichloroethane solution was directly used in the next reaction step. A round bottom flask was charged with Na2SO4 and 1,2-dichloroethane (36 mL) under nitrogen. Under a positive stream of nitrogen, the dichloroethane layer was withdrawn from the Dean−Stark apparatus. To this solution, trichloroacetyl chloride (2.45 g, 13.5 mmol) was added dropwise, and the mixture was stirred at 22 °C overnight (color change from colorless to yellow, pink, dark brown, and black was observed). Saturated aqueous NaHCO3 was added, and the resulting mixture was vigorously stirred (gas evolution). The water layer was separated and extracted three times with dichloromethane; the combined organic layers were washed with water, dried over Na2SO4, and concentrated to afford 46s as a dark green to black oil that solidified upon standing (1.33−0.66 g, 25−50% yield over two steps). 1H NMR (500 MHz CDCl3): δ 9.16 (bs, 1H), 2.39 (s, 3H); 13 C NMR (101 MHz, CDCl3) δ 169.72, 133.56, 123.62, 117.11, 114.70, 94.94, 12.22; MS (ESI): m/z calcd for C7H4Cl5NO: 294.87. Found: 296.2 [M + H]+. 4.2.220. Methyl 2-Amino-4-(1-(4-(methylsulfonyl)phenyl)ethoxy)-benzo[d]thiazole-6-carboxylate (5s64). Prepared according to the typical procedure F from 1s (508 mg, 2.27 mmol) and 1-(1chloroethyl)-4-methylsulfonylbenzene (595 mg, 2.72 mmol); yellow powder (175 mg, 19% yield). 1H NMR (400 MHz, DMSO-d6): δ 7.99 (s, 2H), 7.91 (d, J = 8.4 Hz, 2H), 7.91 (d, J = 1.5 Hz, 1H), 7.71 (d, J = 8.4 Hz, 2H), 7.27 (d, J = 1.5 Hz, 1H), 5.91 (q, J = 6.3 Hz, 1H), 3.76 (s, 3H), 3.20 (s, 3H), 1.59 (d, J = 6.3 Hz, 3H). 4.2.221. Methyl 2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4-(1-(4-(methylsulfonyl)phenyl)ethoxy)benzo[d]thiazole-6carboxylate (6s64). Prepared according to the typical procedure D from 2-aminobenzothiazole 5s64 (155 mg, 0.381 mmol); (191 mg, 86% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.32 (s, 1H), 12.29 (s, 1H), 8.23 (s, 1H), 7.94 (d, J = 8.4 Hz, 2H), 7.74 (d, J = 8.4 Hz, 2H), 7.38 (s, 1H), 6.00 (q, J = 6.3 Hz, 1H), 3.81 (s, 3H), 3.20 (s, 3H), 2.29 (s, 3H), 1.66 (d, J = 6.3 Hz, 3H). Article 4.2.222. 2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4(1-(4-(methylsulfonyl)phenyl)ethoxy)benzo[d]thiazole-6-carboxylic Acid (64). Prepared according to the typical procedure E from methyl ester 6s64 (161 mg, 0.277 mmol); brown solid (148 mg, 94% yield). 1 H NMR (400 MHz, DMSO-d6): δ 12.93 (s, 1H), 12.29 (s, 2H), 8.19 (d, J = 1 Hz, 1H), 7.94 (d, J = 8.5 Hz, 2H), 7.74 (d, J = 8.5 Hz, 2H), 7.37 (d, J = 1 Hz, 1H), 5.98 (q, J = 6.3 Hz, 1H), 3.20 (s, 3H), 2.29 (s, 3H), 1.66 (d, J = 6.3 Hz, 3H); 13C NMR (101 MHz, DMSO-d6): δ 166.86, 159.90, 156.66, 148.66, 148.50, 140.07, 133.01, 130.03, 127.50, 126.57, 126.45, 116.89, 116.45, 115.78, 110.46, 110.03, 74.86, 43.41, 24.10, 11.10; HRMS (ESI) m/z: [M + H]+ calcd for C23H20Cl2N3O6S2 568.0171; found 568.0167; HPLC purity (254 nm): 94%. 4.2.223. 1-(4-(Morpholinomethyl)phenyl)ethan-1-one (47s65). 4′-(Bromomethyl)acetophenone (2.0 g, 9.39 mmol) was dissolved in MeCN (45 mL) followed by the addition of K2CO3 (1.30 g, 9.39 mmol). The mixture was cooled to 0 °C, and morpholine (0.81 mL, 9.39 mmol) was added dropwise. The reaction mixture was stirred overnight at 22 °C. The solvent was removed under reduced pressure, and the residue was dissolved in EtOAc. The organic layer was washed with water and brine and dried with Na2SO4. After filtration, the solvent was removed under reduced pressure; yellow oil (2.02 g, 98% yield). 1H NMR (400 MHz, DMSO-d6): δ 7.92 (d, J = 8.3 Hz, 2H), 7.46 (d, J = 8.3 Hz, 2H), 3.61−3.55 (m, 4H), 3.53 (s, 2H), 2.57 (s, 3H), 2.39−2.29 (m, 4H); MS (ESI): m/z calcd for C13H17NO2: 219.12. Found: 220.0 [M + H]+. 4.2.224. 1-(4-(Morpholinomethyl)phenyl)ethan-1-ol (48s65). To a mixture of ketone 47s65 (1.9 g, 8.66 mmol) and dry EtOH (30 mL), NaBH4 (328 mg, 8.66 mmol) was added in small portions at 0 °C and the reaction mixture was stirred at 22 °C for 1.5 h. The solvent was removed under reduced pressure, and the residue was dissolved in EtOAc. The organic layer was washed with water and brine and dried over Na2SO4. After filtration, the solvent was removed under reduced pressure; yellow oil (1.78 g, 93% yield). 1H NMR (400 MHz, DMSO-d6): δ 7.29 (d, J = 8.1 Hz, 2H), 7.24 (d, J = 8.1 Hz, 2H), 5.10 (d, J = 4.2 Hz, 1H), 4.75−4.65 (m, 1H), 3.61−3.51 (m, 4H), 3.43 (s, 2H), 2.37−2.29 (m, 4H), 1.31 (d, J = 6.4 Hz, 3H); MS (ESI): m/z calcd for C13H19NO2: 221.14. Found: 222.1 [M + H]+. 4.2.225. Methyl 3-(1-(4-(Morpholinomethyl)phenyl)ethoxy)-4nitrobenzoate (49s65). Prepared according to the typical procedure H from 2s (1.17 g, 5.94 mmol) and alcohol 48s65 (1.45 g, 6.53 mmol); yellow oil (1.76 g, 74% yield). 1H NMR (400 MHz, DMSOd6) δ 7.95 (d, J = 8.3 Hz, 1H), 7.71 (d, J = 1.5 Hz, 1H), 7.59 (dd, J = 8.3 Hz, 1.5 Hz, 1H), 7.39 (d, J = 8.1 Hz, 2H), 7.31 (d, J = 8.1 Hz, 2H), 5.86 (q, J = 6.3 Hz, 1H), 3.84 (s, 3H), 3.60−3.49 (m, 4H), 3.42 (s, 2H), 2.39−2.22 (m, 4H), 1.57 (d, J = 6.3 Hz, 3H); MS (ESI): m/z calcd for C21H24N2O6: 400.16. Found: 400.9 [M + H]+. 4.2.226. Methyl 4-Amino-3-(1-(4-(morpholinomethyl)phenyl)ethoxy)benzoate (50s65). Prepared according to the typical procedure B from 49s65 (1.68 g, 4.20 mmol); orange oil (1.23 g, 79% yield). 1H NMR (400 MHz, DMSO-d6): δ 7.40 (d, J = 8.0 Hz, 2H), 7.31 (dd, J = 8.3, 1.7 Hz, 1H), 7.27 (d, J = 8.0 Hz, 2H), 7.21 (d, J = 1.7 Hz, 1H), 6.63 (d, J = 8.3 Hz, 1H), 5.69 (s, 2H), 5.46 (q, J = 6.3 Hz, 1H), 3.68 (s, 3H), 3.60−3.51 (m, 4H), 3.42 (s, 2H), 2.42− 2.21 (m, 4H), 1.56 (d, J = 6.3 Hz, 3H). MS (ESI): m/z calcd for C21H26N2O4: 370.19. Found: 370.2 [M + H]+. 4.2.227. Methyl 2-Amino-4-(1-(4-(morpholinomethyl)phenyl)ethoxy)benzo[d]thiazole-6-carboxylate (51s65). Prepared according to the typical procedure C from aniline 50s65 (1.11 g, 3.00 mmol); yellow solid (654 mg, 51% yield). 1H NMR (400 MHz, DMSO-d6): δ 7.94 (s, 2H), 7.87 (d, J = 1.5 Hz, 1H), 7.38 (d, J = 8.1 Hz, 2H), 7.27 (d, J = 8.1 Hz, 2H), 7.25 (d, J = 1.5 Hz, 1H), 5.71 (q, J = 6.3 Hz, 1H), 3.76 (s, 3H), 3.60−3.50 (m, 4H), 3.41 (s, 2H), 2.39−2.20 (m, 4H), 1.57 (d, J = 6.3 Hz, 3H); MS (ESI): m/z calcd for C22H25N3O4S: 427.16. Found: 428.1 [M + H]+. 4.2.228. Methyl 2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4-(1-(4-(morpholinomethyl)phenyl)ethoxy)benzo[d]thiazole-6-carboxylate (52s65). Prepared according to the typical procedure J from 51s65 (130 mg, 0.304 mmol); gray solid (75 mg, 41% yield). 1H NMR (400 MHz, DMSO-d6) δ 12.29 (br s, 2H), 8.16 1415 https://doi.org/10.1021/acs.jmedchem.2c01597 J. Med. Chem. 2023, 66, 1380−1425 Journal of Medicinal Chemistry pubs.acs.org/jmc Article (376 MHz, DMSO-d6): δ −166.54; HRMS (ESI) m/z: [M − Cl]+ calcd for C27H28FN4O5S 539.1764; found 539.1758; HPLC purity (254 nm): 97%. 4.2.234. 4-(4-(1-((2-(4-Cyano-5-methyl-1H-pyrrole-2-carboxamido)-6-(methoxycarbonyl)benzo[d]thiazol-4-yl)oxy)ethyl)-benzyl)morpholin-4-ium Chloride (53s68×HCl). Prepared according to the typical procedure D from 51s65 (93 mg, 0.218 mmol) and 4-cyano-5methyl-1H-pyrrole-2-carboxylic acid (35s52) (33 mg, 0.218 mmol); white solid (95 mg, 73% yield). 1H NMR (400 MHz, DMSO-d6): δ 13.07 (s, 1H), 12.76 (s, 1H), 8.18 (d, J = 1.1 Hz, 1H), 7.73 (s, 1H), 7.40 (d, J = 8.1 Hz, 2H), 7.38 (d, J = 1.1 Hz, 1H), 7.28 (d, J = 8.1 Hz, 2H), 5.82−5.74 (m, 1H), 3.80 (s, 3H), 3.59−3.50 (m, 4H), 3.41 (s, 2H), 2.33−2.25 (m, 5H), 1.64 (d, J = 6.3 Hz, 3H). 4.2.235. 4-(4-(1-((6-Carboxy-2-(4-cyano-5-methyl-1H-pyrrole-2carboxamido)benzo[d]thiazol-4-yl)oxy)ethyl)benzyl)morpholin-4ium Chloride (68×HCl). Prepared according to the typical procedure E from methyl ester 53s68 hydrochloride (78 mg, 0.131 mmol); white solid (26 mg, 34% yield). 1H NMR (400 MHz, DMSO-d6): δ 13.05 (s, 1H), 12.86 (s, 1H), 12.77 (s, 1H), 10.53−10.23 (m, 1H), 8.16 (s, 1H), 7.75 (s, 1H), 7.64−7.44 (m, 4H), 7.35 (s, 1H), 5.86 (q, J = 6.1 Hz, 1H), 4.41−4.18 (m, 1H), 4.03−3.82 (m, J = 12.1 Hz, 2H), 3.75−3.57 (m, 2H), 3.26−3.15 (m, 2H), 3.14−2.95 (m, 2H), 2.40 (s, 3H), 1.65 (d, J = 6.2 Hz, 3H); HRMS (ESI) m/z: [M − Cl]+ calcd for C28H28N5O5S 546.1811; found 546.1802; HPLC purity (254 nm): 98%. 4.2.236. 2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4(1-(4-(piperazin-1-ylmethyl)phenyl)ethoxy)benzo[d]thiazole-6-carboxylic Acid (69). Hydrochloride of the Boc-protected compound 70 (55 mg, 0.076 mmol) was suspended in 4 M HCl in dioxane (1 mL). The reaction mixture was stirred at 22 °C for 1 h. The solvent was removed under reduced pressure, and to the residue, EtOAc and water were added. The two phases were separated, and to the water phase, 1 M NaOH was added to reach pH 6. The precipitate was collected to obtain the product; brown powder (16 mg, 36% yield). 1 H NMR (400 MHz, DMSO-d6): δ 7.92 (s, 1H), 7.38 (d, J = 7.8 Hz, 2H), 7.24 (d, J = 7.8 Hz, 2H), 7.15 (s, 1H), 5.72 (q, J = 6.2 Hz, 1H), 3.51 (s, 2H), 2.95 (s, 4H), 2.44 (s, 4H), 2.23 (s, 3H), 1.63 (d, J = 6.2 Hz, 3H); HRMS (ESI) m/z: [M + H]+ calcd for C27H28Cl2N5O4S 588.1239; found 588.1227; HPLC purity (254 nm): 93%. 4.2.237. tert-Butyl 4-(4-Acetylbenzyl)piperazine-1-carboxylate (47s70). 4′-(Bromomethyl)acetophenone (2.00 g, 9.39 mmol) and Boc-piperazine (1.75 g, 9.39 mmol) were dissolved in acetonitrile (45 mL) and DMF (7 mL). K2CO3 (1.30 g, 9.39 mmol) was added, and the reaction mixture was stirred vigorously at 60 °C overnight. The solvent was removed under reduced pressure, and the residue was dissolved in EtOAc. The organic layer was washed two times with water, dried over Na2SO4, filtered, and concentrated to get a yellow solid (2.77 g, 93% yield). 1H NMR (400 MHz, DMSO-d6): δ 7.92 (d, J = 8.3 Hz, 2H), 7.45 (d, J = 8.3 Hz, 2H), 3.55 (s, 2H), 3.32 (m, 4H), 2.57 (s, 3H), 2.32 (t, J = 5.1 Hz, 4H), 1.39 (s, 9H); MS (ESI): m/z calcd for C18H26N2O3: 318.19. Found: 319.0 [M + H]+. 4.2.238. tert-Butyl 4-(4-(1-Hydroxyethyl)benzyl)piperazine-1-carboxylate (48s70). Ketone 47s70 (2.65 g, 8.32 mmol) was dissolved in dry EtOH (30 mL) and cooled to 0 °C. NaBH4 (315 mg, 8.32 mmol) was added in small portions, and the reaction mixture was stirred at 22 °C for 2 h. The solvent was removed under reduced pressure, and the residue was dissolved in EtOAc. The organic layer was washed with water and brine, dried over Na2SO4, filtered, and concentrated to get a yellow solid (2.53 g, 95% yield). 1H NMR (400 MHz, DMSO-d6): δ 7.28 (d, J = 8.0 Hz, 2H), 7.22 (d, J = 8.0 Hz, 2H), 5.09 (d, J = 4.2 Hz, 1H), 4.70 (m, 1H), 3.43 (s, 2H), 3.30 (m, 4H), 2.28 (m, 4H), 1.38 (s, 9H), 1.31 (d, J = 6.4 Hz, 3H); MS (ESI): m/z calcd for C18H28N2O3: 320.21. Found: 321.0 [M + H]+. 4.2.239. tert-Butyl 4-(4-(1-(5-(Methoxycarbonyl)-2-nitrophenoxy)-ethyl)benzyl)piperazine-1-carboxylate (49s70). Prepared according to the typical procedure H from 2s (1.11 g, 5.62 mmol) and alcohol 48s70 (1.98 g, 6.19 mmol); brown oil (2.50 g, 89% yield). 1H NMR (400 MHz, DMSO-d6): δ 7.95 (d, J = 8.3 Hz, 1H), 7.71 (d, J = 1.5 Hz, 1H), 7.60 (dd, J = 8.3 Hz, 1.5 Hz, 1H), 7.39 (d, J = 8.2 Hz, 2H), 7.30 (d, J = 8.2 Hz, 2H), 5.86 (q, J = 6.2 Hz, 1H), 3.84 (s, 3H), 3.44 (s, 2H), 3.29 (m, 4H), 2.27 (m, 4H), 1.56 (d, J = 6.2 Hz, 3H), (s, 1H), 7.41 (d, J = 8.1 Hz, 2H), 7.37 (s, 1H), 7.28 (d, J = 8.1 Hz, 2H), 5.79 (q, J = 6.3 Hz, 1H), 3.80 (s, 3H), 3.60−3.49 (m, 4H), 3.41 (s, 2H), 2.36−2.29 (m, 4H), 2.28 (s, 3H), 1.64 (d, J = 6.3 Hz, 3H); MS (ESI): m/z calcd for C28H28Cl2N4O5S: 602.16. Found: 603.2 [M + H]+. 4.2.229. 4-(4-(1-((6-Carboxy-2-((3,4-dichloro-5-methyl-1H-pyrrole-2-carbonyl)oxy)benzo[d]thiazol-4-yl)oxy)ethyl)benzyl)morpholin-4-ium Chloride (65×HCl). Prepared according to the typical procedure E from methyl ester 52s65 (59 mg, 0.097 mmol); brown solid (12 mg, 21% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.88 (s, 1H), 12.32 (s, 1H), 12.25 (s, 1H), 10.29 (s, 1H), 8.17 (s, 1H), 7.90−7.73 (m, 1H), 7.66−7.48 (m, 4H), 7.36 (s, 1H), 5.86 (q, J = 6.3 Hz, 1H), 4.35−4.23 (m, 2H), 4.01−3.86 (m, 2H), 3.74−3.58 (m, 2H), 3.25−3.15 (m, 2H), 3.14−3.00 (m, 2H), 2.29 (s, 3H), 1.65 (d, J = 6.3 Hz, 3H); HRMS (ESI) m/z: [M − Cl]+ calcd for C27H27Cl2N4O5S 589.1079; found 589.1068; HPLC purity (220 nm): 91%. 4.2.230. Methyl 2-(4-Chloro-5-methyl-1H-pyrrole-2-carboxamido)-4-(1-(4-(morpholinomethyl)phenyl)ethoxy)benzo[d]thiazole-6carboxylate Monocitrate (53s66). 2,2,2-Trichloro-1-(4-chloro-5methyl-1H-pyrrol-2-yl)ethan-1-one (34s50) (117 mg, 0.398 mmol) and 51s65 (170 mg, 0.398 mmol) were dissolved in DMF (4 mL), and Na2CO3 (42 mg, 0.398 mmol) was added, and the reaction was stirred at 60 °C overnight. The volatiles were removed under reduced pressure, and the crude residue was triturated with 10% aqueous citric acid and EtOAc to obtain the product as a monocitrate salt; gray solid (209 mg, 69% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.83 (br s, 1H), 12.20 (s, 1H), 8.16 (d, J = 1.1 Hz, 1H), 7.46 (d, J = 2.6 Hz, 1H), 7.43 (d, J = 7.8 Hz, 2H), 7.37 (s, 1H), 7.31 (d, J = 7.8 Hz, 3H), 5.79 (q, J = 6 Hz, 1H), 4.10 (br s, 1H), 3.80 (s, 3H), 3.58 (m, 8H), 3.17 (s, 2H), 2.73 (d, J = 15.3 Hz, 2H), 2.63 (d, J = 15.3 Hz, 2H), 2.44 (s, 3H), 2.22 (s, 3H), 1.64 (d, J = 6 Hz, 3H); MS (ESI): m/z calcd for C28H30ClN4O5S: 569.16. Found: 569.4 [M + H]+. 4.2.231. 4-(4-(1-((6-Carboxy-2-(4-chloro-5-methyl-1H-pyrrole-2carboxamido)benzo[d]thiazol-4-yl)oxy)ethyl)benzyl)morpholin-4ium Chloride (66×HCl). Prepared according to the typical procedure E from methyl ester 53s66 (157 mg, 0.206 mmol); white solid (71 mg, 62% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.81 (s, 2H), 12.20 (d, J = 2.8 Hz, 1H), 10.67 (s, 1H), 8.14 (s, 1H), 7.62−7.51 (m, 4H), 7.46 (d, J = 2.8 Hz, 1H), 7.35 (s, 1H), 5.85 (q, J = 6.2 Hz, 2H), 4.29 (s, 2H), 3.92 (d, J = 12.7 Hz, 2H), 3.70 (t, J = 12.3 Hz, 2H), 3.24−3.13 (m, 2H), 3.13−2.98 (m, 2H), 2.22 (s, 3H), 1.65 (d, J = 6.2 Hz, 4H); HRMS (ESI) m/z: [M − Cl]+ calcd for C27H28ClN4O5S 555.1469; found 555.1457; HPLC purity (254 nm): 98%. 4.2.232. 4-(4-(1-((2-(4-Fluoro-5-methyl-1H-pyrrole-2-carboxamido)-6-(methoxycarbonyl)benzo[d]thiazol-4-yl)oxy)ethyl)-benzyl)morpholin-4-ium Chloride (53s67×HCl). A mixture of 4-fluoro-5methyl-1H-pyrrole-2-carboxylic acid (35s51) (43 mg, 0.300 mmol) and oxalyl chloride (0.26 mL, 3 mmol) in dichloromethane (3 mL) was stirred at 22 °C overnight, and the resulting solution was concentrated under reduced pressure. To the solid residue were added 51s65 (128 mg, 0.300 mmol) and toluene (6 mL), and the reaction mixture was stirred at 130 °C overnight. The precipitate was collected and washed with toluene and EtOAc to get the product; gray solid (49 mg, 27% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.75 (s, 1 H), 11.86 (s, 1 H), 10.28 (br s, 1H), 8.17 (s, 1H), 7.55 (m, 4H), 7.35 (s, 1H), 7.29 (d, J = 2.9 Hz, 1H), 5.87 (q, J = 6.3 Hz, 1H), 4.30 (s, 2H), 3.93 (d, J = 12.6 Hz, 2H), 3.80 (s, 3H), 3.65 (t, J =12.3 Hz, 2H), 3.20 (d, J = 12.6 Hz, 2H), 3.07 (m, 2H), 2.20 (s, 3H), 1.65 (d, J = 6.3 Hz, 3H); 19F NMR (376 MHz, DMSO-d6): δ −166.51; MS (ESI): m/z calcd for C28H30FN4O5S: 553.19. Found: 553.3 [M − Cl]+. 4.2.233. 4-(4-(1-((6-Carboxy-2-(4-fluoro-5-methyl-1H-pyrrole-2carboxamido)benzo[d]thiazol-4-yl)oxy)ethyl)benzyl)morpholin-4ium Chloride (67×HCl). Prepared according to the typical procedure E from methyl ester 53s67 hydrochloride (47 mg, 0.080 mmol); brown solid (10 mg, 22% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.8 (brs, 1H), 12.72 (s, 1H), 11.86 (s, 1H), (brs, 1H), 8.13 (s, 1H), 7.55 (s, 4H), 7.34 (s, 1H), 7.29 (s, 1H), 5.85 (q, J = 6.3 Hz, 1H), 4.29 (s, 2H), 3.92 (d, J = 12.6 Hz, 2H), 3.67 (t, J = 12.2 Hz, 2H), 3.19 (m, 2H), 3.07 (m, 2H), 2.20 (s, 3H), 1.65 (d, J = 6.3 Hz, 3H); 19F NMR 1416 https://doi.org/10.1021/acs.jmedchem.2c01597 J. Med. Chem. 2023, 66, 1380−1425 Journal of Medicinal Chemistry pubs.acs.org/jmc Article 48s71 (532 mg, 2.10 mmol); white solid (322 mg, 39% yield). 1H NMR (400 MHz, CDCl3): δ 7.79 (d, J = 8.4 Hz, 1H), 7.66 (m, 3H), 7.60 (d, J = 1.5 Hz, 1H), 7.54 (d, J = 7.8 Hz, 2H), 5.59 (q, J = 6.4 Hz, 1H), 4.18 (m, 2H), 3.91 (s, 3H) 3.73 (m, 4H), 3.39 (m, 2H), 3.01 (d, J = 14.0 Hz, 2H), 1.70 (d, J = 6.4 Hz, 2H); MS (ESI): m/z calcd for C21H24N2O6S: 432.14. Found: 433.4 [M + H]+. 4.2.247. Methyl 4-Amino-3-(1-(4-((1-oxidothiomorpholino)methyl)-phenyl)ethoxy)benzoate (50s71). Prepared according to the typical procedure B from 49s71 (321 mg, 0.742 mmol); white solid (227 mg, 76% yield). 1H NMR (400 MHz, CDCl3): δ 7.48 (dd, J = 8.2 Hz, 1.8 Hz, 1H), 7.39−7.34 (m, 2H), 7.28 (d, J = 8.2 Hz, 2H), 6.65 (d, J = 8.2 Hz, 1H), 5.42 (q, J = 6.4 Hz, 1H), 4.30 (br s, 2H), 3.79 (s, 3H), 3.55 (s, 2H), 3.05 (m, 2H), 2.85 (m, 4H), 2.68 (m, 2H), 1.66 (d, J = 6.4 Hz, 3H) ppm; MS (ESI): m/z calcd for C21H26N2O4S: 402.16. Found: 402.9 [M + H]+. 4.2.248. Methyl 2-Amino-4-(1-(4-((1-oxidothiomorpholino)methyl)-phenyl)ethoxy)benzo[d]thiazole-6-carboxylate (51s71). Prepared according to the typical procedure C from aniline 50s71 (225 mg, 0.559 mmol); off-white solid (95 mg, 37% yield). 1H NMR (400 MHz, DMSO-d6): δ 7.94 (s, 2H), 7.87 (s, 1H), 7.39 (d, J = 7.9 Hz, 2H), 7.28 (d, J = 7.9 Hz, 2H), 7.25 (s, 1H), 5.71 (q, J = 6.3 Hz, 1H), 3.76 (s, 3H), 3.51 (s, 2H), 2.88−2.77 (m, 4H), 2.70 (m, 2H), 2.62−2.54 (m, 2H), 1.57 (d, J = 6.3 Hz, 2H); MS (ESI): m/z calcd for C22H25N3O4S2: 459.13. Found: 460.0 [M + H]+. 4.2.249. Methyl 2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4-(1-(4-((1-oxidothiomorpholino)methyl)phenyl)ethoxy)benzo[d]thiazole-6-carboxylate (52s71). Prepared according to the typical procedure J from 51s71 (97 mg, 0.211 mmol); black powder (71 mg, 53% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.30 (br s, 1H), 12.27 (br s, 1H), 8.20 (s, 1H), 7.44 (d, J = 8.0 Hz, 2H), 7.40 (s, 1H), 7.31 (d, J = 8.0 Hz, 2H), 5.81 (q, J = 6.2 Hz, 1H), 3.82 (s, 3H), 3.55 (s, 2H), 2.86 (m, 4H), 2.72 (m, 2H), 2.64 (m, 2H), 2.29 (s, 3H), 1.65 (d, J = 6.2 Hz, 3H); MS (ESI): m/z calcd for C28H28Cl2N4O5S2: 634.09. Found: 634.9 [M + H]+. 4.2.250. 2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4(1-(4-((1-oxidothiomorpholino)methyl)phenyl)ethoxy)benzo-[d]thiazole-6-carboxylic Acid (71). Prepared according to the typical procedure E from methyl ester 52s71 (70 mg, 0.110 mmol); black powder (38 mg, 56% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.84 (br s, 1H), 12.26 (br s, 2H), 8.14 (s, 1H), 7.42 (d, J = 7.7 Hz, 2H), 7.37 (s, 1H), 7.30 (d, J = 7.7 Hz, 2H), 5.78 (q, J = 6.2 Hz, 1H), 3.52 (s, 2H), 2.88−2.80 (m, 4H), 2.74−2.64 (m, 4H), 2.28 (s, 3H), 1.64 (d, J = 6.2 Hz, 3H); 13C NMR (101 MHz, DMSO-d6): δ 166.96, 159.68, 156.72, 149.02, 142.08, 141.33, 137.20, 132.82, 129.93, 129.09, 126.55, 125.56, 116.95, 116.00, 115.71, 110.56, 109.98, 75.46, 61.16, 45.47, 43.64, 24.21, 11.09; HRMS (ESI) m/z: [M + H]+ calcd for C27H27Cl2N4O5S2 621.0800; found 621.0788; HPLC purity (254 nm): 92%. 4.2.251. 1-(4-((1,1-Dioxidothiomorpholino)methyl)phenyl)ethan-1-one (47s72). 4′-(Bromomethyl)acetophenone (1.16 g, 5.44 mmol) and thiomorpholine 1,1-dioxide (735 mg, 5.44 mmol) were dissolved in acetonitrile (35 mL) and DMF (0.5 mL). K2CO3 (1.51 g, 10.9 mmol) was added, and the reaction mixture was stirred at 60 °C overnight. The volatiles were removed under reduced pressure, the residue was dissolved in EtOAc, and the organic layer was washed with water, dried over Na2SO4, filtered, and evaporated to get the product as yellow crystals (990 mg, 68% yield). 1H NMR (400 MHz, DMSO-d6): δ 7.93 (d, J = 8.3 Hz, 2H), 7.49 (d, J = 8.3 Hz, 2H), 3.75 (s, 2H), 3.20−3.05 (m, 4H), 2.88 2.92−2.84 (m, 4H), 2.57 (s, 3H); MS (ESI): m/z calcd for C13H17NO3S: 267.09. Found: 267.8 [M + H]+. 4.2.252. 4-(4-(1-Hydroxyethyl)benzyl)thiomorpholine 1,1-Dioxide (48s72). A solution of ketone 47s72 (990 mg, 3.70 mmol) in dry ethanol (13 mL) was cooled to 0 °C, followed by the addition of NaBH4 (140 mg, 3.70 mmol) in small portions. The reaction was stirred at 22 °C for 2.5 h, the solvent was removed under reduced pressure, and the residue was dissolved in EtOAc. The organic layer was washed with water and brine, dried over Na2SO4, filtered, and concentrated to get a yellow oil (828 mg, 83% yield). 1H NMR (400 MHz, DMSO-d6): δ 7.30 (d, J = 8.1 Hz, 2H), 7.26 (d, J = 8.1 Hz, 2H), 5.10 (d, J = 4.2 Hz, 1H), 4.70 (dq, J = 4.2 Hz, 6.4 Hz, 1H), 3.63 1.38 (s, 9H); MS (ESI): m/z calcd for C26H33N3O7: 499.23. Found: 500.1 [M + H]+. 4.2.240. tert-Butyl 4-(4-(1-(2-Amino-5-(methoxycarbonyl)phenoxy)-ethyl)benzyl)piperazine-1-carboxylate (50s70). Prepared according to the typical procedure B from 49s70 (2.29 g, 4.57 mmol); orange oil (1.74 g, 81% yield). 1H NMR (400 MHz, DMSO-d6): δ 7.39 (d, J = 8.0 Hz, 2H), 7.30 (dd, J = 8.3 Hz, 1.5 Hz, 1H), 7.26 (d, J = 8.0 Hz, 2H), 7.20 (d, J = 1.5 Hz, 1H), 6.62 (d, J = 8.3 Hz, 1H), 5.69 (s, 2H), 5.46 (q, J = 6.2 Hz, 1H), 3.68 (s, 3H), 3.43 (s, 2H), 3.29 (s, 4H), 2.28 (s, 4H), 1.55 (d, J = 6.2 Hz, 3H), 1.38 (s, 9H); MS (ESI): m/z calcd for C26H35N3O5: 469.26. Found: 470.2 [M + H]+. 4.2.241. Methyl 2-Amino-4-(1-(4-((4-(tert-butoxycarbonyl)piperazin-1-yl)methyl)phenyl)ethoxy)benzo[d]thiazole-6-carboxylate (51s70). Prepared according to the typical procedure C from aniline 50s70 (1.69 g, 3.61 mmol); yellow solid (855 mg, 45% yield). 1 H NMR (400 MHz, DMSO-d6): δ 7.94 (s, 2H), 7.88 (d, J = 1.5 Hz, 1H), 7.38 (d, J = 8.1 Hz, 2H), 7.29−7.23 (m, 3H), 5.76 (s, 1H), 5.70 (q, J = 6.3 Hz, 1H), 3.76 (s, 3H), 3.44 (s, 2H), 3.29 (s, 4H), 2.28 (s, 4H), 1.57 (d, J = 6.3 Hz, 3H), 1.38 (s, 9H); MS (ESI): m/z calcd for C27H34N4O5S: 526.22. Found: 527.2 [M + H]+. 4.2.242. Methyl 4-(1-(4-((4-(tert-Butoxycarbonyl)piperazin-1-yl)methyl)phenyl)ethoxy)-2-(3,4-dichloro-5-methyl-1H-pyrrole-2carboxamido)benzo[d]thiazole-6-carboxylate (52s70). Prepared according to the typical procedure J from 51s70 (203 mg, 0.385 mmol); dark brown solid (100 mg, 37% yield); 1H NMR (400 MHz, DMSO-d6): δ 12.26 (s, 2H), 8.19 (s, 1H), 7.42 (d, J = 7.8 Hz, 2H), 7.40−7.38 (d, J = 1.1 Hz, 1H), 7.29 (d, J = 7.6 Hz, 2H), 5.80 (q, J = 6.4 Hz, 1H), 3.81 (s, 3H), 3.44 (s, 2H), 3.29 (m, 4H), 2.29 (m, 7H), 1.65 (d, J = 6.5 Hz, 3H), 1.38 (s, 9H); MS (ESI): m/z calcd for C33H36Cl2N5O6S: 700.18. Found: 700.2 [M − H]−. 4.2.243. 4-(tert-Butoxycarbonyl)-1-(4-(1-((6-carboxy-2-(3,4-dichloro-5-methyl-1H-pyrrole-2-carboxamido)benzo[d]thiazol-4-yl)oxy)ethyl)benzyl)piperazin-1-ium Chloride (70×HCl). Prepared according to the typical procedure E from methyl ester 52s70 (80 mg, 0.115 mmol); dark brown powder (68 mg, 82% yield). 1H NMR (400 MHz, DMSO-d6) δ 12.87 (s, 1H), 12.33 (s, 1H), 12.26 (s, 1H), 10.22 (s, 1H), 8.17 (s, 1H), 7.57 (d, J = 8.2 Hz, 2H), 7.53 (d, J = 8.2 Hz, 2H), 7.37 (s, 1H), 5.87 (q, J = 6.3 Hz, 1H), 4.30 (s, 2H), 4.01 (d, J = 14.0 Hz, 2H), 3.25 (m, 2H), 3.11 (m, 2H), 2.99 (m, 2H), 2.29 (s, 3H), 1.66 (d, J = 6.3 Hz, 3H), 1.41 (s, 9H); HRMS (ESI) m/z: [M − Cl]+ calcd for C32H36Cl2N5O6S: 688.1763; found 688.1748; HPLC purity (254 nm): 81% (contains 13% of Boc-deprotected compound 69×HCl). 4.2.244. 1-(4-((1-Oxidothiomorpholino)methyl)phenyl)ethanone (47s71). A mixture of 4′-(bromomethyl)acetophenone (639 mg, 3.00 mmol), K2CO3 (1.67 g, 12 mmol), and 4-thiomor-pholin-1-one hydrochloride (467 mg, 3 mmol) in MeCN (9 mL) was stirred at 22 °C overnight and then for 4 h at 40 °C. EtOAc and water were added, and the organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated to get an off-white solid (738 mg, 98% yield). 1H NMR (400 MHz, CDCl3): δ 7.93 (d, J = 8.3 Hz, 2H), 7.43 (d, J = 8.3 Hz, 2H), 3.64 (s, 3H), 3.08 (m, 2H), 2.85 (m, 4H), 2.69 (m, 2H), 2.60 (s, 3H). MS (ESI): m/z calcd for C13H17NO2S: 251.10. Found: 251.9 [M + H]+. 4.2.245. 4-(4-(1-Hydroxyethyl)benzyl)thiomorpholine 1-Oxide (48s71). Ketone 47s71 (738 mg, 2.94 mmol) was dissolved in MeOH (7.3 mL) and cooled to 0 °C and then NaBH4 (111 mg, 2.94 mmol) was added. After stirring for 30 min at 0 °C, the reaction mixture was allowed to warm to 22 °C and stirred for an additional 2.5 h. EtOAc and water were added, and the organic layer was washed with brine. The aqueous phase was additionally washed with dichloromethane; both organic phases were combined, dried over Na2SO4, filtered, and evaporated to give a white solid 488 mg (66% yield). 1H NMR (400 MHz, CDCl3): δ 7.35 (d, J = 8.1 Hz, 2H), 7.30 (d, J = 8.1 Hz, 2H), 7.30 (d, J = 8.1 Hz, 2H), 4.91 (q, J = 6.5 Hz, 1H), 3.57 (s, 2H), 3.06 (m, 1H), 2.94−2.76 (m, 3H), 2.69 (m, Hz, 1H), 1.83 (br s, 1H), 1.50 (d, J = 6.5 Hz, 3H); MS (ESI): m/z calcd for C13H19NO2S: 253.11. Found: 253.9 [M + H]+. 4.2.246. Methyl 4-Nitro-3-(1-(4-((1-oxidothiomorpholino)methyl)-phenyl)ethoxy)benzoate (49s71). Prepared according to the typical procedure H from 2s (376 mg, 1.91 mmol) and alcohol 1417 https://doi.org/10.1021/acs.jmedchem.2c01597 J. Med. Chem. 2023, 66, 1380−1425 Journal of Medicinal Chemistry pubs.acs.org/jmc (s, 2H), 3.09 (m, 4H), 2.84 (m, 4H), 1.31 (d, J = 6.4 Hz, 3H); MS (ESI): m/z calcd for C13H19NO3S: 269.11. Found: 269.9 [M + H]+. 4.2.253. Methyl 3-(1-(4-((1,1-Dioxidothiomorpholino)methyl)phenyl)ethoxy)-4-nitrobenzoate (49s72). Prepared according to the typical procedure H from 2s (442 mg, 2.24 mmol) and alcohol 48s72 (665 mg, 2.47 mmol); orange oil (855 mg, 85% yield). 1H NMR (400 MHz, DMSO-d6): δ 7.95 (d, J = 8.3 Hz, 1H), 7.72 (d, J = 1.5 Hz, 1H), 7.60 (dd, J = 8.3 Hz, 1.5 Hz, 1H), 7.41 (d, J = 8.2 Hz, 2H), 7.34 (d, J = 8.2 Hz, 2H), 5.87 (q, J = 6.2 Hz, 1H), 3.85 (s, 3H), 3.64 (s, 2H), 3.09 (m, 4H), 2.84 (m, 4H), 1.56 (d, J = 6.2 Hz, 3H); MS (ESI): m/z calcd for C21H24N2O7S: 448.13. Found: 449.0 [M + H]+. 4.2.254. Methyl 4-Amino-3-(1-(4-((1,1-dioxidothiomorpholino)methyl)phenyl)ethoxy)benzoate (50s72). Prepared according to the typical procedure B from 49s72 (810 mg, 1.81 mmol); yellow solid (121 mg, 16% yield). 1H NMR (400 MHz, DMSO-d6): δ 7.41 (d, J = 8.0 Hz, 2H), 7.30 (d, J = 8.0 Hz, 3H), 7.21 (d, J = 1.5 Hz, 1H), 6.62 (d, J = 8.2 Hz, 1H), 5.69 (s, 2H), 5.47 (q, J = 6.1 Hz, 1H), 3.68 (s, 3H), 3.63 (s, 2H), 3.13−3.05 (s, 4H), 2.89−2.81 (s, 4H), 1.55 (d, J = 6.1 Hz, 3H); MS (ESI): m/z calcd for C21H26N2O5S: 418.16. Found: 419.0 [M + H]+. 4.2.255. Methyl 2-Amino-4-(1-(4-((1,1-dioxidothiomorpholino)methyl)phenyl)ethoxy)benzo[d]thiazole-6-carboxylate (51s72). Prepared according to the typical procedure C from aniline 50s72 (130 mg, 0.310 mmol); light yellow solid (93 mg, 63% yield). 1H NMR (400 MHz, DMSO-d6): δ 7.95 (s, 2H), 7.88 (s, 1H), 7.40 (d, J = 6.1 Hz, 2H), 7.29 (m, 3H), 5.73 (m, 1H), 3.76 (s, 3H), 3.64 (s, 2H), 3.10 (s, 4H), 2.85 (s, 4H), 1.58 (s, 3H); MS (ESI): m/z calcd for C22H25N3O5S2: 475.12. Found: 476.1 [M + H]+. 4.2.256. Methyl 2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4-(1-(4-((1,1-dioxidothiomorpholino)methyl)phenyl)ethoxy)benzo[d]thiazole-6-carboxylate (52s72). Prepared according to the typical procedure J from 51s72 (50 mg, 0.106 mmol); gray solid (33 mg, 48% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.30 (s, 1H), 12.27 (s, 1H), 8.20 (s, 1H), 7.44 (d, J = 7.9 Hz, 2H), 7.40 (s, 1H), 7.32 (d, J = 7.9 Hz, 2H), 5.81 (q, J = 6.4 Hz, 1H), 3.82 (s, 3H), 3.63 (s, 2H), 3.14−3.04 (m, 4H), 2.91−2.80 (m, 4H), 2.29 (s, 3H), 1.65 (d, J = 6.4 Hz, 3H); MS (ESI): m/z calcd for C28H28Cl2N4O6S2: 650.08. Found: 651.2 [M + H]+. 4.2.257. 4-(4-(1-((6-Carboxy-2-(3,4-dichloro-5-methyl-1H-pyrrole-2-carboxamido)benzo[d]thiazol-4-yl)oxy)ethyl)benzyl)thiomorpholin-4-ium 1,1-Dioxide Chloride (72×HCl). Prepared according to the typical procedure E from methyl ester 52s72 (30 mg, 0.046 mmol); gray solid (19 mg, 61% yield). 1H NMR (400 MHz, DMSOd6): δ 12.87 (s, 1H), 12.29 (s, 1H), 12.26 (s, 1H), 8.16 (s, 1H), 7.58− 7.41 (m, 4H), 7.37 (s, 1H), 5.83 (q, J = 6.4, 5.9 Hz, 1H), 2.29 (s, 3H), 1.65 (d, J = 6.3 Hz, 3H); some peaks overlaid by DMSO and water signals; HRMS (ESI) m/z: [M − Cl] + calcd for C27H27Cl2N4O6S2 637.0749; found 637.0739; HPLC purity (254 nm): 96%. 4.2.258. 4-(4-Acetylbenzyl)morpholin-3-one (47s73). Sodium hydride (60% dispersion in mineral oil, 208 mg, 5.20 mmol) was added to a stirred solution of morpholin-3-one (404 mg, 4.00 mmol) in DMF (12 mL) at 0 °C. After the addition of 4′-(bromomethyl)acetophenone (852 mg, 4.00 mmol), the reaction mixture was stirred at 22 °C overnight. EtOAc was added and the resulting solution was washed with water and brine, dried over Na2SO4, filtered, and concentrated to give a yellow oil (903 mg, 97% yield). 1H NMR (400 MHz, CDCl3): δ 7.94 (d, J = 8.1 Hz, 2H), 7.37 (d, J = 8.1 Hz, 2H), 4.68 (s, 2H), 4.27 (s, 2H), 3.87 (dd, J = 5.8 Hz, 4.5 Hz, 2H), 3.30 (dd, J = 5.8 Hz, 4.5 Hz, 2H), 2.60 (s, 3H); MS (ESI): m/z calcd for C13H15NO3: 233.11. Found: 234.1 [M + H]+. 4.2.259. 4-(4-(1-Hydroxyethyl)benzyl)morpholin-3-one (48s73). Sodium borohydride (7.74 mmol, 293 mg) was added to a solution of ketone 47s73 (903 mg, 3.87 mmol) in methanol (6 mL) and cooled to 0 °C. After 30 min at 0 °C, the reaction mixture was stirred at 22 °C for 3 h. EtOAc was added, and the resulting solution was washed with brine, dried over Na2SO4, filtered, and concentrated to give a colorless oil (877 mg, 96% yield). 1H NMR (400 MHz, CDCl3): δ 7.36 (d, J = 7.8 Hz, 2H), 7.26 (d, J = 7.8 Hz, 2H), 4.91 (dt, J = 11.0, Article 6.4 Hz, 1H), 4.62 (s, 2H), 4.25 (s, 2H), 3.84 (m, 2H), 3.27 (m, 2H), 1.50 (d, J = 6.4 Hz, 3H); MS (ESI): m/z calcd for C13H17NO3: 235.12. Found: MS 236.1 [M + H]+. 4.2.260. Methyl 4-Nitro-3-(1-(4-((3-oxomorpholino)methyl)phenyl)-ethoxy)benzoate (49s73). Prepared according to the typical procedure H from 2s (723 mg, 3.67 mmol) and alcohol 48s73 (949 g, 4.03 mmol); yellow oil (380 mg, 25% yield). 1H NMR (400 MHz, CDCl3): δ 7.76 (d, J = 8.3 Hz, 1H), 7.65−7.59 (m, 2H), 7.40 (d, J = 8.3 Hz, 2H), 7.26 (d, J = 8.1 Hz, 1H), 5.55 (q, J = 6.4 Hz, 2H), 4.63 (d, J = 14.8 Hz, 1H), 4.57 (d, J = 14.8 Hz, 1H), 4.25 (s, 2H), 3.90 (s, 3H), 3.87−3.82 (m, 2H), 3.29−3.25 (m, 2H), 1.68 (d, J = 6.4 Hz, 3H) ppm; MS (ESI): m/z calcd for C21H22N2O7: 414.14. Found: 414.9 [M + H]+. 4.2.261. Methyl 4-Amino-3-(1-(4-((3-oxomorpholino)methyl)phenyl)ethoxy)benzoate (50s73). Prepared according to the typical procedure B from 49s73 (384 mg, 0.928 mmol); yellow solid (296 mg, 83% yield). 1H NMR (400 MHz, CDCl3): δ 7.48 (d, J = 8.2 Hz, 1H), 7.36 (d, J = 8.0 Hz, 4H), 7.24 (d, J = 8.0 Hz, 3H), 6.65 (d, J = 8.2 Hz, 1H), 5.42 (q, J = 6.4 Hz, 1H), 4.63 (d, J = 14.6 Hz, 1H), 4.57 (d, J = 14.6 Hz, 1H), 4.28 (s, 2H), 4.24 (s, 2H), 3.84 (t, J = 5.1 Hz, 2H), 3.80 (s, 3H), 3.27 (t, J = 5.1 Hz, 2H), 1.65 (d, J = 6.4 Hz, 3H); MS (ESI): m/z calcd for C21H24N2O5: 384.17. Found: 384.9 [M + H]+. 4.2.262. Methyl 2-Amino-4-(1-(4-((3-oxomorpholino)methyl)phenyl)ethoxy)benzo[d]thiazole-6-carboxylate (51s73). Prepared according to the typical procedure C from aniline 50s73 (298 mg, 0.775 mmol); orange powder (229 mg, 67% yield). 1H NMR (400 MHz, DMSO-d6): δ 7.94 (s, 2H), 7.87 (d, J = 1.6 Hz, 1H), 7.41 (d, J = 7.9 Hz, 2H), 7.27−7.20 (m, 3H), 5.73 (q, J = 6.4 Hz, 1H), 4.55− 4.44 (m, 2H), 4.09 (s, 2H), 3.79 (t, J = 5.2 Hz, 2H), 3.76 (s, 3H), 3.23 (t, J = 5.2 Hz, 2H), 1.56 (d, J = 6.4 Hz, 3H); MS (ESI): m/z calcd for C22H23N3O5S: 441.14. Found: 441.9 [M + H]+. 4.2.263. Methyl 2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4-(1-(4-((3-oxomorpholino)methyl)phenyl)ethoxy)benzo[d]thiazole-6-carboxylate (52s73). Prepared according to the typical procedure J from 51s73 (210 mg, 0.475 mmol); brown powder (132 mg, 45% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.29 (br s, 1H), 12.26 (br s, 1H), 8.19 (s, 1H), 7.45 (d, J = 7.8 Hz, 2H), 7.40 (s, 1H), 7.24 (d, J = 7.8 Hz, 2H), 5.81 (q, J = 5.7 Hz, 1H), 4.57−4.45 (m, 2H), 4.09 (s, 2H), 3.80 (m, 5H), 3.26−3.22 (m, 2H), 2.28 (s, 3H), 1.64 (d, J = 6.4 Hz, 3H); MS (ESI): m/z calcd for C28H26Cl2N4O6S: 616.10. Found: 617.2 [M + H]+. 4.2.264. 2-(3,4-Dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4(1-(4-((3-oxomorpholino)methyl)phenyl)ethoxy)benzo[d]thiazole6-carboxylic Acid (73). Prepared according to the typical procedure E from methyl ester 52s73 (125 mg, 0.202 mmol); brown solid (15 mg, 12% yield). 1H NMR (400 MHz, DMSO-d6): δ 12.28 (s, 3H), 8.14 (s, 1H), 7.42 (d, J = 7.9 Hz, 2H), 7.37 (s, 1H), 7.30 (d, J = 7.9 Hz, 2H), 5.78 (q, J = 6.5 Hz, 1H), 3.52 (s, 2H), 2.92−2.78 (m, 4H), 2.76−2.65 (m, 2H), 2.28 (s, 3H), 1.64 (d, J = 6.3 Hz, 3H); HRMS (ESI) m/z: [M − H]− calcd for C27H23Cl2N4O6S 601.0715; found 601.0725; HPLC purity (254 nm): 93%. 4.3. X-ray Crystallography. 4.3.1. Protein Expression and Purification. E. coli GyrB24 protein, purified as described previously,41 was concentrated to approximately 11 mg/mL in 50 mM of tris−HCl pH 7.9, 50 mM NaCl, 5 mM DTT. This construct corresponds to residues 1−220 of the full-length wild-type protein (UniProtKB entry P0AES6), with a calculated molecular weight of 24,157 Da, and is referred to as EcGyrB24. The equivalent ATPase subdomain from A. baumannii 1419130 DNA gyrase B, corresponding to residues 28−233 of the full-length wild-type protein (UniProtKB entry A0A009KIJ4), was cloned into a modified pET28 vector and expressed in T7 Express E. coli cells (New England BioLabs) with an N-terminal His-tag. This was purified using a Ni-chelate column, and the tag was cleaved off using TEV protease before further purification on a second Ni-chelate column and a monoQ ion exchange column. The protein was concentrated to approximately 14 mg/mL in 50 mM of tris−HCl pH 7.5, 1 mM EDTA, 1 mM DTT. The resulting protein had a calculated molecular weight of 22,741 Da and is referred to as AbGyrB23. 1418 https://doi.org/10.1021/acs.jmedchem.2c01597 J. Med. Chem. 2023, 66, 1380−1425 Journal of Medicinal Chemistry pubs.acs.org/jmc Article Crystals of the AbGyrB23-novobiocin complex grew from 0.2 M Mg(NO3)2, 20% (w/v) PEG 3350, and were cryoprotected with this solution supplemented with 20% (v/v) ethylene glycol. Data were collected in space group P41212 to a resolution of 1.9 Å, and the structure was solved by molecular replacement using a template derived from another EcGyrB24 structure (PDB accession code 6YD9; the corresponding domains share a 73% sequence identity), giving a single copy of the protein chain in the ASU, with an estimated solvent content of 52%. Crystals of the AbGyrB23-27 complex were obtained from 30% (w/v) PEG 3350, 0.2 M MgCl2, and 10% (v/v) of ethylene glycol was added to this solution for cryoprotection. The complex crystallized in space group C2, and data were taken to a 1.6 Å resolution. The above model of the AbGyrB23-novobiocin complex (PDB accession code 7PQI) was used as the input template for molecular replacement, which gave two copies of the domain per ASU, corresponding to an estimated solvent content of 45%. Crystals of the AbGyrB23-(S)-27 complex were produced using a precipitant comprised of 31% (w/v) PEG 3350, 0.2 M calcium acetate, and cryoprotected using this solution with the addition of 10% (v/v) of ethylene glycol. These crystals were isomorphous with the AbGyrB23-27 complex above, and the structure was solved in the same way using data to a 1.55 Å resolution. Crystals of the PaGyrB24-novobiocin complex grew from 12.5% (v/v) MPD, 12.5% (v/v) PEG 1000, 25% (w/v) PEG 3350, 30 mM MgCl2, 30 mM CaCl2, and 0.1 M imidazole/MES pH 6.5, and these could be flash-cooled directly without additional cryoprotectant. Data were collected in space group P21 to a resolution of 1.32 Å, and the structure was solved by molecular replacement using a template derived from the EcGyrB43 structure (PDB accession code 6XTJ; the corresponding domains share a 74% sequence identity), giving three copies of the protein chain in the ASU, with an estimated solvent content of 44%. Crystals of the PaGyrB24-(S)-27 complex were obtained from 22% (w/v) PEG 3350, 0.1 M HEPES pH 7, and cryoprotected using this solution with the addition of 20% (v/v) of ethylene glycol. This complex also crystallized in space group P21 but was not isomorphous with the PaGyrB24-novobiocin complex. A single protein chain from the latter (PDB accession code 7PTF) was used as the molecular replacement template using data collected to a 2.2 Å resolution. This gave two copies of the domain per ASU corresponding to an estimated solvent content of 37%. 4.4. Microtiter-Plate-Based Assays for Inhibition of E. coli, P. aeruginosa, and A. baumannii Gyrase Supercoiling and E. coli Topo IV Relaxation. Commercially available assay kits from Inspiralis for the determination of IC50 values of the test compounds for the inhibition of DNA gyrase supercoiling and topoisomerase IV relaxation were used, as described in ref.1111 4.5. Gel-Based Assays for Inhibition of E. coli Gyrase Supercoiling, E. coli Gyrase Relaxation, E. coli Gyrase Cleavage, and Inhibition of A. baumannii and P. aeruginosa Topo IV Decatenation. In all experiments, the activity of the enzymes was determined prior to the testing of the compounds and 1 unit (U) was defined as the amount of enzyme required to just fully supercoil, relax, reach the maximum cleavage of the substrate, or fully decatenate the substrate. This amount of enzyme was initially used in the determination of control inhibitor activity. The experiments were performed in duplicate. For all assays, the final DMSO concentration was 1%. Bands were visualized by ethidium staining for 20 min and destaining for 20 min. Gels were scanned using documentation equipment (GeneGenius, Syngene, Cambridge, U.K.), and % inhibition levels were obtained with gel scanning software (GeneTools, Syngene, Cambridge, U.K.). 4.5.1. Inhibition of E. coli Gyrase Supercoiling. DNA gyrase (1 U) was incubated with 0.5 μg of relaxed pBR322 DNA in a 30 μL reaction (containing the test compound in 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1.0, 5.0, 10, 25, 50, and 100 μM final concentrations) at 37 °C for 30 min under the following conditions: 35 mM tris−HCl (pH 7.5), 24 mM KCl, 4 mM MgCl2, 2 mM DTT, 1.8 mM Spermidine, 1 mM ATP, 6.5% (w/v) glycerol, and 0.1 mg/mL BSA. Each reaction The equivalent ATPase subdomain from P. aeruginosa PAO1 DNA gyrase B, corresponding to residues 1−221 of the full-length wild-type protein (UniProtKB entry Q9I7C2), was cloned into a modified pTTQ18 vector and expressed in T7 Express E. coli cells (New England BioLabs) without an affinity tag. This was purified using successive Q-sepharose, monoQ, and phenyl-sepharose columns. The protein was concentrated to approximately 10 mg/mL in 50 mM of tris−HCl pH 7.5, 10% (v/v) glycerol, 1 mM EDTA, 1 mM DTT. The resulting protein had a calculated molecular weight of 24,502 Da and is referred to as PaGyrB24. 4.3.2. Crystallization, X-ray Data Collection, and Structure Solution. Crystals were grown using the vapor diffusion method from proteins at the aforementioned concentrations in the presence of 1 mM ligand. Commercially available (Molecular Dimensions, Qiagen) and in-house crystallization screens were set up in MRC2 96-well crystallization plates (Swissci) with drops comprised of 0.3 μL of precipitant and 0.3 μL of protein solution using an Oryx 8 liquid handling robot (Douglas Instruments) and then equilibrated against 50 μL of reservoir solution at a constant temperature of 19 °C. In most cases, the optimization of initial hits was required to obtain suitable crystals, which were cryoprotected as necessary and mounted in Litholoops (Molecular Dimensions) before flash-cooling by plunging into liquid nitrogen prior to transport to the synchrotron. X-ray data sets were recorded from single crystals on beamline I03, I04, I04-1, or I24 at the Diamond Light Source (Oxfordshire, U.K.) using an Eiger2 XE 16M, a Pilatus 6M, or a Pilatus 6M-F hybrid photon counting detector (Dectris), with crystals maintained at 100 K by a Cryojet cryocooler (Oxford Instruments). X-ray data were integrated and scaled using DIALS42 via the XIA2 expert system43 and merged using AIMLESS44 (data statistics are shown in Tables S12−S14). All successive data processing was carried out using programs in the CCP4 suite via the CCP4i2 graphical user interface.45 All structures were solved via molecular replacement in PHASER.46 Where the template structure was of the same protein as the target structure, the protein component of one GyrB domain was used directly as the input to PHASER, and the resulting models were finalized by successive iterations of model building in COOT47 and restrained refinement in REFMAC548 until no further improvements could be achieved. Where the template structure was from a related protein to the target structure, an input model for PHASER was prepared from one subunit of the template structure by removing nonconserved side chains using SCULPTOR49 with reference to an alignment of the template and target sequences. Furthermore, in these cases, the PHASER solution was automatically rebuilt using BUCCANEER50 prior to finalizing with COOT and REFMAC. Starting coordinates and restraints for the various ligands were generated using AceDRG51 before docking these into a suitable electron density. Final models were validated using MOLPROBITY52 and the PDB-validation server (https://validate-rcsb-2.wwpdb.org). Refinement and validation statistics for all models are summarized in Tables S12−S14. Crystals of the EcGyrB24-1 complex were obtained using a precipitant comprised of 33% (w/v) PEG 4000, 100 mM tris−HCl pH 8.0, 75 mM MgCl2, and these were cryoprotected using the same solution supplemented with 17.5% (v/v) glycerol and ∼1 mM 1. Data were recorded to a 1.16 Å resolution in space group C2. The structure was solved by molecular replacement using a nonisomorphous structure of EcGyrB24 (PDB accession code 1KZN), giving a single copy of the protein chain in the asymmetric unit (ASU) with an estimated solvent content of 47%. Crystals of the EcGyrB24-7 complex were obtained using a precipitant comprised of 34% (w/v) PEG 4000, 100 mM tris−HCl pH 8.0, 128 mM MgCl2. A similar solution was used for the cryoprotectant, although the PEG 4000 concentration was increased to 40% (w/v) and the mixture was supplemented with ∼1 mM 7. This time, the space group was P212121 and data were collected to a 1.65 Å resolution. Again, the structure was solved by molecular replacement using the same EcGyrB24 structure (PDB accession code 1KZN), giving a single copy of the protein chain in the ASU and an estimated solvent content of 44%. 1419 https://doi.org/10.1021/acs.jmedchem.2c01597 J. Med. Chem. 2023, 66, 1380−1425 Journal of Medicinal Chemistry pubs.acs.org/jmc was stopped by the addition of 30 μL of chloroform/iso-amyl alcohol (26:1) and 20 μL of Stop Dye (40% sucrose, 100 mM tris−HCl (pH 7.5), 10 mM EDTA, 0.5 μg/mL bromophenol blue), before being loaded on a 1.0% TAE (tris acetate 0.04 mM, EDTA 0.002 mM) gel run at 80 V for 2 h. 4.5.2. E. coli DNA Relaxation. DNA gyrase (1 U) was incubated with 0.5 μg of supercoiled pBR322 DNA in a 30 μL reaction (containing the test compound in 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1.0, 5.0, 10, 25, 50, and 100 μM final concentrations) at 37 °C for 2 h under the following conditions: 35 mM tris−HCl (pH 7.5), 24 mM KCl, 4 mM MgCl2, 2 mM DTT, 1.8 mM Spermidine, 6.5% (w/v) glycerol, and 0.1 mg/mL BSA. Each reaction was stopped by the addition of 30 μL of chloroform/iso-amyl alcohol (26:1) and 20 μL of Stop Dye (40% sucrose, 100 mM tris−HCl (pH 7.5), 10 mM EDTA, 0.5 μg/mL bromophenol blue), before being loaded on a 1.0% TAE (tris acetate 0.04 mM, EDTA 0.002 mM) gel run at 80 V for 2 h. 4.5.3. E. coli DNA Gyrase Cleavage Assay. Gyrase (1 U) was incubated with 0.5 μg of supercoiled pBR322 in a reaction volume of 30 μL at 37 °C for 1 h in assay buffer (see above) minus the ATP, in the presence of test compound. SDS (0.2%) and 0.1 mg/mL of proteinase K were added before a further incubation at 37 °C for 30 min. Each reaction was stopped by the addition of 30 μL of chloroform/iso-amyl alcohol (26:1) and 20 μL of Stop Dye (40% sucrose, 100 mM tris−HCl (pH 7.5), 10 mM EDTA, 0.5 μg/mL bromophenol blue), before being loaded on a 1.0% TAE (tris acetate 0.04 mM, EDTA 0.002 mM) gel run at 80 V for 2 h. 4.5.4. Inhibition of A. baumannii and P. aeruginosa Topo IV Decatenation. Topo IV (1 U) was incubated with 0.2 μg of kDNA in a 30 μL reaction (containing the test compound in 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1.0, 5.0, 10, 25, 50, and 100 μM final concentrations) at 37 °C for 30 min under the following conditions: 50 mM HEPES− KOH (pH 7.9), 6 mM magnesium acetate, 4 mM DTT, 1 mM ATP, 100 mM potassium glutamate, 2 mM spermidine, and 0.05 mg/mL albumin. Ciprofloxacin and novobiocin at a 5 μM final concentration were used as controls. Each reaction was stopped by the addition of 30 μL chloroform/ iso-amyl alcohol (24:1) and 30 μL Stop Dye (40% sucrose (w/v), 100 mM tris−HCl (pH 7.5), 10 mM EDTA, 0.5 μg/mL bromophenol blue), before being loaded on a 1.0% TAE gel run at 80 V for 2 h. Due to time constraints, all compounds were assayed at the higher test range of 100 μM only, but the data points were fitted up to 10 μM (apart from the controls) to improve the graph fits. 4.6. Microbiology. 4.6.1. Susceptibility Testing. MICs for susceptibility testing and MIC90 assays were determined according to CLSI standards for liquid MIC (M07) using the direct colony suspension method for inoculum preparation. Appropriate inoculation suspension density was assessed using a Sensititre Nephelometer (Thermo Fisher Scientific) with a Sensititre 0.5 McFarland Standard. MBC and time-kill assays were conducted according to CLSI guidelines (M26). MIC in the presence of serum was done using a 50% (final concentration) pooled, heat-inactivated (56 °C, 1 h), sterile-filtered (Filtropur S plus 0.2, Sarstedt) human serum. 4.6.2. Frequency of Resistance and Whole Genome Sequencing. Frequency of resistance was determined by plating dilutions of freshly grown cultures onto freshly poured MH-II plates containing concentrations of the test compounds corresponding to 4- or 8times the MIC. Serial dilutions of the starting cultures were plated nonselectively on MH-II plates to determine the initial culture density. Colonies were counted on selection plates after 24 and 48 h, and the number of colonies after 48 h was divided by the calculated number of cells plated to yield the frequency of resistance. Colonies that grew were restreaked on plates with identical concentrations of the test compounds that they were selected on. Reduced susceptibility of the clones was confirmed by MIC testing. Genomic DNA was extracted from resistant isolates using an Epicentre Masterpure Complete DNA & RNA Purification kit (Lucigen). Genomic DNA was used to prepare genomic libraries for whole genome sequencing using a Nextera XT Library Preparation Kit (Illumina) and Nextera Indexes according to the manufacturer’s protocols. Libraries were sequenced using a Miseq device (Illumina), Article and the resulting sequences were analyzed using CLC Genomics Workbench V11 (Qiagen). 4.6.3. Hemolysis. Hemolysis was tested as previously described by DeRosa et al.53 4.7. Cytotoxicity. 4.7.1. Lactate Dehydrogenase Assay and MTS Assay. Lactate dehydrogenase (LDH) assay15 and MTS assay18 were performed as described. In vitro fluorometric microculture cytotoxicity (FMC) assay was performed according to the published procedure.19 4.8. Genotoxicity and Mutagenicity. 4.8.1. In Vitro Cell Micronucleus Test. The protocol followed the recommendations of the Test Guideline 487 of the OECD guideline for the testing of chemicals.54 The test was performed on rodent CHO cells (ECACC ref: 85050302). The cells were seeded at a density of 2000/well in a black 96-well plate with a clear bottom and were incubated in a humidified atmosphere at 37 °C with 5% CO2. To estimate the micronuclei frequency, the cells scored must have completed one mitosis during the treatment or the post-treatment incubation period. Compounds were prepared at 10 mM in 100% DMSO and were assayed at 100, 50, 25, 12.5, and 6.25 μM for 24 h in six replicates. DMSO did not exceed 1% according to TG-487. Mitomycin C (MitC, Sigma-Aldrich), a known inducer of micronuclei formation, was the positive control used to demonstrate the sensitivity of the test, and cells untreated were used as a negative control. After treatment, cytochalasin B (cytoB) was used as a cytokinesis-blocker of cultures for 28 h. Cells were then fixed with 3.7% formaldehyde and 1% Triton X-100, and nuclei were stained with bisbenzimide (Hoechst dye no. 33258) for 30 min at 22 °C. Imaging acquisition was performed by using the Operetta CLS High-Content Analysis System (Perkin Elmer). Analysis was performed using Harmony software of Perkin Elmer and the Fundacin MEDINA in-house App NucleusFinder, based on an open source processing image program, ImageJ. NucleusFinder identified the regularly shaped mononuclear, binuclear, and multinuclear cells, excluded irregular, small, and isolated nuclei (odd nuclei), and detected valid micronuclei following very conservative conditions as cytoplasmic location without connection with the main nuclei and proper size. NucleusFinder chose the best analysis algorithm for each image capture from six implemented filters, using a smart fit choice calculation. It allowed us to perform classification and a counting of the different elements. The cytokinesis-block proliferation index (CBPI), which indicates the average number of cell cycles per cell during the period of exposure to cytoB, was used to estimate the cytostatic activity of a treatment by comparing values in the treated and control cultures. Cytostasis percentage did not exceed 60% because higher levels may induce micronuclei as a secondary effect of cytotoxicity and was calculated as follows %cytostasis = 100 100{(CBPI T 1)/(CBPIC 1)} where T is the test compound treatment culture, C is the vehicle control culture, and CBPI = {(No. mononucleate cells) + (2 × No. binucleate cells) + (3 × No. multinucleate cells)}/(total number of cells). 4.8.2. AMES Test. A commercial test kit, AMES MPFTM 98/100 Microplate Format Mutagenicity Assay, from Xenometrix AG (Allschwil, Switzerland), was used to evaluate the mutagenicity of compounds with and without metabolic activation S9 using amino acid requiring S. typhimurium strains TA100 and TA98. 4.9. Mitochondrial Toxicity. The protocol adopted by Swiss et al.33 and Marroquin et al.55 was applied for in vitro testing using HepG2 cells cultured in either glucose or galactose. HepG2 cells (50,000) were seeded in each well of two 96-well plates, and 100 μL of growth medium (glucose media) was added. Cells were allowed to adhere overnight in a cell incubator at 37 °C. The next day, the medium was removed, cells were washed with PBS, and 100 μL of medium (with glucose) containing positive control (Rotenone) was added, before the addition of the test compounds. The same procedure was repeated for plate 2 with medium replaced with galactose. Cells were again incubated at 37 °C for 24 h. CellTiter-Glo 2.0 Cell Viability (100 μL) (Promega) was then added to each well to 1420 https://doi.org/10.1021/acs.jmedchem.2c01597 J. Med. Chem. 2023, 66, 1380−1425 Journal of Medicinal Chemistry pubs.acs.org/jmc measure the amount of ATP. The plate was placed in a shaker for 2 min and incubated for 15 min in the dark at 22 °C. Luminescence was read in a luminometer Tecan Infinite 200 pro. Luminescence from each well treated with the compound was compared to the negative control (medium only). 4.10. Ion Channel Screening with Manual Patch-Clamp Method. Stably transfected hERG, Nav1.5 cells (CHO), and inducible Cav1.2 (HEK-293) used in this study were obtained from Dr. Brian T. Donovan (hERG; GSK) and B’SYS GmbH (Nav1.5 and Cav1.2; Witterswil, Switzerland). The cells were maintained as previously described56 or according to the provided datasheets. All culture medium components and chemicals used for patch-clamp solutions were purchased from Thermo Fisher Scientific and SigmaAldrich (Germany), respectively. Tetracycline (2.5 μg/mL) was added to the culture media 48 h prior to recordings to induce the expression of Cav1.2. The extracellular solution for hERG, Nav1.5, and Cav1.2 contained hERG/Nav1.5�137 mM NaCl, 4 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 10 mM D-glucose, 10 mM HEPES, pH 7.4 adjusted with NaOH; Cav1.2�100 mM NaCl, 4 mM KCl, 40 mM NMDG, 5 mM CaCl2, 1 mM MgCl2, 5 mM D-glucose, 10 mM HEPES, and 5 mM sorbitol, pH 7.4 adjusted with HCl; osmolarity 290−300 mOsm. The intracellular solutions for hERG, Nav1.5, and Cav1.2 contained hERG�130 mM KCl, 1 mM MgCl2, 5 mM EGTA, 5 mM Mg-ATP, 10 mM HEPES; Nav1.5�120 mM KCl, 6 mM MgCl2, 5 mM EGTA, 10 mM NaCl, 10 mM HEPES, pH 7.2 adjusted with KOH; Cav1.2� 132 mM CsCl, 1 mM KCl, 0.1 mM CaCl2, 4 mM Mg-ATP, 0.4 mM NaGTP, 10 mM EGTA, 10 mM HEPES, pH 7.2 adjusted with CsOH; osmolarity 280−290 mOsm. The test compounds were first dissolved in DMSO to make a stock solution (10 mM) and then diluted in the extracellular solution to 10 μM. The final DMSO concentration was 0.1%. All recordings (five or six for each assay) were performed at 22 °C. Currents were measured using the whole-cell voltage-clamp method with an Axopatch 200B patch-clamp amplifier (sampling frequency, hERG�2.5 kHz, Nav1.5�20 kHz, Cav1.2�5 kHz; low-pass filter frequency, hERG/Cav1.2�1 kHz, Nav1.5�2 kHz), digitized with a Digidata 1440A/1550A interface under the control of the pCLAMP 10 software (Molecular Devices). Glass pipettes were pulled from borosilicate glass (Harvard Apparatus) by a horizontal puller (DMS universal puller, Germany) and had a resistance of 4−8 MΩ when filled with intracellular solutions. After the rupture of the cell membrane, the cell was allowed to stabilize for 3−5 min before recordings. Currents were induced using multiple sweeps of a voltage waveform at a holding potential of −80 mV for 200 ms, stepping to −50 mV for 200 ms, stepping to +20 mV for 5 s, stepping to −50 mV for 5 s, and returning to −80 mV (delivered once every 15 s for hERG); a holding potential of −120 mV for 20 ms, stepping to −30 mV for 50 ms, and returning to −120 mV (delivered once every 2 s for Nav1.5); a holding potential of −80 mV for 50 ms, stepping to −50 mV for 100 ms, stepping to 0 mV for 200 ms, and returning to −80 mV (delivered once every 10 s for Cav1.2). The access resistance was continuously monitored. A negative control, consisting of extracellular solutions with 0.1% DMSO, was applied until a stable current amplitude was achieved, followed by the application of test compounds. The compound was given 5 min to reach a steady-state block, followed by wash-out with the second negative control. A positive control consisting of quinidine (hERG�50 μM, Nav1.5�1 mM) or verapamil (Cav1.2�1 mM) was further applied after the wash-out. The peak hERG tail current amplitude was measured as the peak positive current at the second −50 mV step minus the initial −50 mV step and further normalized to the peak tail current amplitude in the first negative control to evaluate the level of hERG inhibition. The Nav1.5 current amplitude was measured as the difference between the maximum inward current during the first 10 ms of the −30 mV step minus the mean current during the last 5 ms of the same voltage step and further normalized to the Nav1.5 current amplitude in the negative control to determine the level of Nav1.5 inhibition. The Cav1.2 peak current amplitude was measured as the difference between the maximum inward current of the 0 mV Article depolarizing step and the current of the −50 mV step and further normalized to the Cav1.2 current amplitude in the negative control to evaluate the level of Cav1.2 inhibition The normalized hERG/ Nav1.5/Cav1.2 currents were presented as mean ± standard error of the mean (SEM). 4.11. ADME Assays. 4.11.1. Kinetic Solubility. Kinetic solubility, utilizing a test compound from 10 mM DMSO stock solution, was measured at a final compound concentration of 100 μM and 1% DMSO. The test compound was added to 100 mM potassium phosphate buffer and incubated at 37 °C for at least 20 h in a heatershaker. After incubation, the samples were centrifuged at 3000g at 37 °C for 30 min to pellet the insoluble material, and an aliquot of the supernatant was taken for analysis. After dilution of the sample, the concentration of the dissolved compound was quantified by liquid chromatography coupled to triple quadrupole mass spectrometry (LC-MS/MS). 4.11.2. Thermodynamic Solubility. Thermodynamic solubility assay utilized the solid form of a test compound. The solid test compound (2−3 mg) was weighed in a glass HPLC vial, and 100 mM K3PO4 buffer, pH 7.4 was added to give a theoretical max. concentration of ∼5−6 mg/mL. The vial was incubated in a rotational shaker at 900 rpm, 37 °C for 24 h. After the incubation, an aliquot (200 μL) was transferred to a glass insert and centrifuged at 10,000g, 37 °C for 20 min to separate any solid material from the solution. The supernatant was transferred to a new HPLC vial and analyzed by liquid chromatography coupled to triple quadrupole mass spectrometry (LC-MS/MS). 4.11.3. Chemical Stability. A test compound was pipetted (0.5 μL) into HPLC vials from 10 mM DMSO stocks to yield 5 μM final concentration (1000 μL inc. volume). To the reaction, start buffer/ H2O (1:1) or buffer/isopropanol (1:1) was added. The following buffers were used: pH 2 (H3PO4/KH2PO4 10 mM), pH 4 (ammonium formate 50 mM, isotonic), pH 7.4 (KH2PO4/K2HPO4 10 mM), and pH 10 (glycine/NaOH 10 mM). Immediately (<1 min) after buffer or buffer/IPA addition, a 50 μL aliquot was added to a separate plate containing 150 μL of acetonitrile supplemented with Warfarin as the internal standard (IS), sealed, and stored at −80 °C. This was repeated at 15 and 45 min and after 1, 2, 4, and 24 h. The samples were analyzed by liquid chromatography coupled to triple quadrupole mass spectrometry (LC-MS/MS). 4.11.4. pKa Determination. The pKa measurements were performed on a Sirius T3 automated instrument from Sirius Analytical Ltd. (East Sussex, U.K.) equipped with a D-PAS (dip probe absorption spectroscopy) lamp for spectrophotometric titrations and electrode for potentiometric titrations. The spectrophotometric titrations were performed using 2−5 μL of 10 mM DMSO compound stock. During the titration, the instrument added a predetermined volume of ionic-strength-adjusted (ISA) water or a combination of ISA and ISA containing 80% methanol in the potentiometric titrations. A titration from high-to-low or low-to-high was performed between pH 2 and 12. During the titration, the instrument collected a UV−vis spectrum by using the D-PAS technique to establish a titration curve. In the potentiometric method, the instrument instead bases the titration on the amount of acid (HCl) and base (KOH) that was added. The electrode was calibrated using a blank titration from pH 1.8 to pH 12.0 before every individual determination. The measurements were performed under argon to minimize the effect of dissolved CO2. Precipitation was continuously monitored at 500 nm. The temperature was controlled throughout the experiment at 25 ± 1 °C. 4.11.5. log D Determination. A miniaturized shake-flask method in HPLC vials was applied. Solutions used were potassium phosphate 0.05 M pH 7.4 (KP) saturated with octanol and octanol saturated with KP. The phase ratios used were 1:1 and 1:3, i.e., 0.8/0.4 mL of octanol + 0.8/1.2 mL of KP. Compound (1.6 μL) from 10 mM DMSO stock was pipetted into a HPLC vial. Octanol was added followed by KP. The vial was sealed and vortexed, and phase separation was set for 48 h at an ambient temperature (ca 23 °C) in the dark. Next, the octanol phase was carefully separated with a pipette. Both phases were then analyzed against a separate standard 1421 https://doi.org/10.1021/acs.jmedchem.2c01597 J. Med. Chem. 2023, 66, 1380−1425 Journal of Medicinal Chemistry pubs.acs.org/jmc ■ curve by liquid chromatography coupled to triple quadrupole mass spectrometry (LC-MS/MS). 4.11.6. Metabolic Stability in the Presence of Human and Animal Liver Microsomes. Metabolic stability was determined in 0.5 mg/mL of human or animal liver microsomes at a compound concentration of 1 μM in 100 mM of K3PO4 buffer pH 7.4 in a total incubation volume of 500 μL. The reaction was initiated by the addition of 1 mM NADPH. At various incubation times, i.e., at 0, 5, 10, 20, 40, and 60 min, a sample was withdrawn from the incubation and the reaction was terminated by the addition of cold acetonitrile with warfarin as an internal standard. The amount of parent compound remaining was analyzed by liquid chromatography coupled to triple quadrupole mass spectrometry (LC-MS/MS). 4.11.7. Plasma Protein Binding and Stability in Human and Animal Plasma. Pooled human plasma was provided by Uppsala Academic Hospital and was collected from two donors (nonsmoking) (citric acid). In brief, 0.2 mL of the plasma (50% plasma, 50% isotonic buffer) test solution (typically 10 μM of the final compound concentration) was transferred to the membrane tube in the RED insert (Thermo Fisher Scientific). Isotonic phosphate buffer (0.35 mL, pH 7.4) was added to the other side of the membrane. The 96well base plate was then sealed with an adhesive plastic film (Scotch Pad) to prevent evaporation. The sample was incubated with rapid rotation (>900 rpm) on a Kisker rotational incubator at 37 °C for 4 h to achieve equilibrium. A stability test of the test solution was prepared (to allow the detection of drug degradation), and >100 μL of the plasma test solution (in a plastic vial or on a sealed plate) was incubated at 37 °C for 4 h (or as long as the dialysis time). The plasma test solution was frozen directly after the administration to prevent any degradation. Prior to LC-MS/MS analysis, the plasma and buffer sample were treated with the addition of methanol (1:3) containing warfarin as the internal standard to precipitate proteins. The standard curve was created using the plasma standard. The plate was then sealed and centrifuged, and the supernatant was analyzed by liquid chromatography coupled to triple quadrupole mass spectrometry (LC-MS/MS). 4.11.8. Caco-2 Cell Permeability Assay. Caco-2 cell monolayers (passages 94−105) were grown on a permeable filter support and used for transport study on day 21 after seeding. Prior to the experiment, a 10 μM drug solution was prepared and warmed to 37 °C. The Caco-2 filters were washed with prewarmed HBSS prior to the experiment, and thereafter the experiment was started by applying the donor solution on the apical or basolateral side. The transport experiments were carried out at pH 7.4 in both the apical and basolateral chambers. The experiments were performed at 37 °C and with a stirring rate of 500 rpm. The receiver compartment was sampled at 15, 30, and 60 min, and at 60 min also a final sample from the donor chamber was taken to calculate the mass balance of the compound. The samples (100 μL) were transferred to a 96-well plate containing 100 μL of methanol and warfarin as IS and was sealed until liquid chromatography coupled to triple quadrupole mass spectrometry (LC-MS/MS). The borders for low-moderate-high permeability (Papp) in the assay setup is low: The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.2c01597. IC50 and MIC values, solubility and free fraction data, Xray crystallography details, ADMET data, NMR spectra, and HPLC traces (PDF) Molecular formula strings (CSV) Accession Codes PDB ID Codes complex of E. coli GyrB24 with 1: 7P2M; complex of E. coli GyrB24 with 7: 7P2W; complex of A. baumannii GyrB23 with 27: 7PQL; complex of A. baumannii GyrB23 with (S)-27: 7PQM; complex of A. baumannii GyrB23 with novobiocin: 7PQI; complex of P. aeruginosa GyrB24 with (S)-27: 7PTG; complex of P. aeruginosa GyrB24 with novobiocin: 7PTF. ■ : 1.6 ± 0.2 ( × 10 6cm/s) AUTHOR INFORMATION Corresponding Author Danijel Kikelj − Faculty of Pharmacy, University of Ljubljana, 1000 Ljubljana, Slovenia; Phone: (+386)1476-9500; Email: danijel.kikelj@ffa.uni-lj.si; Fax: (+386)1425-8031 Authors Andrej Emanuel Cotman − Faculty of Pharmacy, University of Ljubljana, 1000 Ljubljana, Slovenia; orcid.org/00000003-2528-396X Martina Durcik − Faculty of Pharmacy, University of Ljubljana, 1000 Ljubljana, Slovenia; orcid.org/00000002-9218-1771 Davide Benedetto Tiz − Faculty of Pharmacy, University of Ljubljana, 1000 Ljubljana, Slovenia; Present Address: University of Perugia, Department of Pharmaceutical Sciences, Via del Liceo 1, 06100 Perugia, Italy Federica Fulgheri − Faculty of Pharmacy, University of Ljubljana, 1000 Ljubljana, Slovenia; Present Address: University of Cagliari, Department of Life and Environmental Sciences - Drug section, via Ospedale 72, 09124 Cagliari, Italy.; orcid.org/0000-0002-7858-1605 Daniela Secci − Faculty of Pharmacy, University of Ljubljana, 1000 Ljubljana, Slovenia; Present Address: University of Cagliari, Department of Life and Environmental Sciences Drug section, via Ospedale 72, 09124 Cagliari, Italy. Maša Sterle − Faculty of Pharmacy, University of Ljubljana, 1000 Ljubljana, Slovenia; orcid.org/0000-0003-38985194 Š tefan Možina − Faculty of Pharmacy, University of Ljubljana, 1000 Ljubljana, Slovenia; Present Address: Lek Pharmaceuticals, Verovškova ulica 57, 1000 Ljubljana, Slovenia.; orcid.org/0000-0002-7416-6981 Ž iga Skok − Faculty of Pharmacy, University of Ljubljana, 1000 Ljubljana, Slovenia; Present Address: Lek Pharmaceuticals, Verovškova ulica 57, 1000 Ljubljana, Slovenia. Nace Zidar − Faculty of Pharmacy, University of Ljubljana, 1000 Ljubljana, Slovenia; orcid.org/0000-0003-19050158 Anamarija Zega − Faculty of Pharmacy, University of Ljubljana, 1000 Ljubljana, Slovenia; orcid.org/00000003-4065-0019 1.6 ± 0.2 ( × 10 6cm/s), high 0.4 ± 0.2 ASSOCIATED CONTENT sı Supporting Information * 0.4 ± 0.2 ( × 10 6cm/s), moderate : Article 4.11.9. Liquid Chromatography Coupled to Triple Quadrupole Mass Spectrometry (LC-MS/MS). The test compounds were optimized on a Waters Acquity UPLC XEVO TQ-S micro system (Waters Corp.) operating in multiple reaction monitoring (MRM) mode with positive or negative electrospray ionization using the QuanOptimize software (Waters Corp.). For chromatographic separation, a C18 BEH 1.7 μm column was used, with a general gradient of 1−90% of mobile phase B over a total running time of 2 min. Mobile phase A consisted of 5% acetonitrile and 0.1% formic acid in purified water, and mobile phase B consisted of 0.1% formic acid in 100% acetonitrile. The flow rate was set to 0.5 mL/min, and 5 μL of the sample was injected. 1422 https://doi.org/10.1021/acs.jmedchem.2c01597 J. Med. Chem. 2023, 66, 1380−1425 Journal of Medicinal Chemistry pubs.acs.org/jmc Janez Ilaš − Faculty of Pharmacy, University of Ljubljana, 1000 Ljubljana, Slovenia; orcid.org/0000-0002-01240474 Lucija Peterlin Mašič − Faculty of Pharmacy, University of Ljubljana, 1000 Ljubljana, Slovenia Tihomir Tomašič − Faculty of Pharmacy, University of Ljubljana, 1000 Ljubljana, Slovenia; orcid.org/00000001-5534-209X Diarmaid Hughes − Department of Medical Biochemistry and Microbiology, Uppsala University, 75123 Uppsala, Sweden Douglas L. Huseby − Department of Medical Biochemistry and Microbiology, Uppsala University, 75123 Uppsala, Sweden; orcid.org/0000-0001-9974-578X Sha Cao − Department of Medical Biochemistry and Microbiology, Uppsala University, 75123 Uppsala, Sweden Linnéa Garoff − Department of Medical Biochemistry and Microbiology, Uppsala University, 75123 Uppsala, Sweden Talía Berruga Fernández − Department of Medical Biochemistry and Microbiology, Uppsala University, 75123 Uppsala, Sweden; orcid.org/0000-0001-6459-1397 Paraskevi Giachou − Department of Medical Biochemistry and Microbiology, Uppsala University, 75123 Uppsala, Sweden Lisa Crone − Department of Medical Biochemistry and Microbiology, Uppsala University, 75123 Uppsala, Sweden Ivailo Simoff − Drug Optimization and Pharmaceutical Profiling Platform (UDOPP), Department of Pharmacy, Uppsala University, 75123 Uppsala, Sweden; orcid.org/ 0000-0001-6522-7191 Richard Svensson − Drug Optimization and Pharmaceutical Profiling Platform (UDOPP), Department of Pharmacy, Uppsala University, 75123 Uppsala, Sweden Bryndis Birnir − Department of Medical Cell Biology, Uppsala University, 75123 Uppsala, Sweden Sergiy V. Korol − Department of Medical Cell Biology, Uppsala University, 75123 Uppsala, Sweden; orcid.org/ 0000-0001-8279-2790 Zhe Jin − Department of Medical Cell Biology, Uppsala University, 75123 Uppsala, Sweden Francisca Vicente − Fundación MEDINA, 18016 Granada, Spain Maria C. Ramos − Fundación MEDINA, 18016 Granada, Spain; orcid.org/0000-0002-3674-615X Mercedes de la Cruz − Fundación MEDINA, 18016 Granada, Spain Björn Glinghammar − Department Chemical Process and Pharmaceutical Development, Unit Chemical and Pharmaceutical Safety, RISE Research Institutes of Sweden, 15136 Södertälje, Sweden Lena Lenhammar − Department of Medical Sciences, Uppsala University Hospital, 75185 Uppsala, Sweden Sara R. Henderson − Department of Biochemistry and Metabolism, John Innes Centre, Norwich NR4 7UH, U.K; Present Address: Institute of Microbiology and Infection, College of Medical and Dental Sciences, University of Birmingham, Birmingham, U.K.; orcid.org/0000-00032078-639X Julia E. A. Mundy − Department of Biochemistry and Metabolism, John Innes Centre, Norwich NR4 7UH, U.K Anthony Maxwell − Department of Biochemistry and Metabolism, John Innes Centre, Norwich NR4 7UH, U.K; orcid.org/0000-0002-5756-6430 Article Clare E. M. Stevenson − Department of Biochemistry and Metabolism, John Innes Centre, Norwich NR4 7UH, U.K David M. Lawson − Department of Biochemistry and Metabolism, John Innes Centre, Norwich NR4 7UH, U.K Guido V. Janssen − Medicinal Chemistry Division, Vrije Universiteit Amsterdam, 1081 HZ Amsterdam, The Netherlands Geert Jan Sterk − Medicinal Chemistry Division, Vrije Universiteit Amsterdam, 1081 HZ Amsterdam, The Netherlands Complete contact information is available at: https://pubs.acs.org/10.1021/acs.jmedchem.2c01597 Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. The authors will release the atomic coordinates and experimental data upon article publication. ■ ACKNOWLEDGMENTS The research presented in this paper was conducted as part of the ND4BB ENABLE Consortium and has received support from the Innovative Medicines Initiative Joint Undertaking under grant no. 115583, resources of which are comprised of financial contributions from the European Union’s seventh framework program (FP7/2007-2013) and EFPIA companies’ in-kind contribution. The views expressed in this article are the views of the authors, and neither IMI nor the European Union or EFPIA is responsible for any use that may be made of the information contained herein. Financial support from the Slovenian Research Agency (grant nos. P1-0208, J1-3030, J13031) was gratefully acknowledged. S.R.H. was supported by an iCASE studentship funded by BBSRC and Redx Pharma Plc (BB/J014524/1). Work in A.M.’s lab was supported by an Investigator Award from the Wellcome Trust (110072/Z/15/ Z) and by a BBSRC Institute Strategic Programme Grant (BB/ P012523/1). The authors thank Diamond Light Source for access to beamlines I03, I04, I04-1, and I24 under proposals MX18565 and MX25108. ■ ABBREVIATIONS USED ■ REFERENCES CDI, 1,1′-carbonyldiimidazole; CFU, colony-forming unit; DCM, dichloromethane; DIAD, diisopropyl azodicarboxylate; DIPEA, N,N-diisopropylethylamine; DMF, N,N-dimethylformamide; FMCA, fluorometric microculture cytotoxicity assay; hERG, human Ether-à-go-go-Related Gene; HPLC, highperformance liquid chromatography; LDH, lactate dehydrogenase; MBC, minimal bactericidal concentration; MDR, multidrug-resistant; MIC, minimum inhibitory concentration; MNT, in vitro cell micronucleus test; MTS, (3-[4,5,dimethylthiazol-2-yl]-5-[3-carboxymethoxy phenyl]-2-[4-sulfophenyl]-2H-tetrazolium); PMB, 4-methoxybenzyl; THF, tetrahydrofurane; TLC, thin-layer chromatography; TBTU, 2-(1Hbenzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate; TPSA, total polar surface area (1) Prioritization of pathogens to guide discovery, research and development of new antibiotics for drug-resistant bacterial infections, 1423 https://doi.org/10.1021/acs.jmedchem.2c01597 J. 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