Bioorganic & Medicinal Chemistry Letters 23 (2013) 1036–1040 Contents lists available at SciVerse ScienceDirect Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl Identification of N-acyl 4-(3-pyridonyl)phenylalanine derivatives and their orally active prodrug esters as dual acting a4b1 and a4b7 receptor antagonists Jefferson W. Tilley a,⇑, Achyutharao Sidduri a, Jianping Lou a, Gerry Kaplan a, Nadine Tare b, Gary Cavallo b, Karl Frank c, Anjula Pamidimukkala c, Duk Soon Choi d, Louise Gerber d, Aruna Railkar d, Louis Renzetti b a Discovery Chemistry, Pharmaceutical Research and Early Drug Development, Hoffmann-La Roche Inc., 340 Kingsland Street, Nutley, NJ 07110, USA Discovery Inflammation and Respiratory Diseases, Pharmaceutical Research and Early Drug Development, Hoffmann-La Roche Inc., 340 Kingsland Street, Nutley, NJ 07110, USA c Drug Metabolism and Pharmacokinetics, Pharmaceutical Research and Early Drug Development, Hoffmann-La Roche Inc., 340 Kingsland Street, Nutley, NJ 07110, USA d PARD, Pharmaceutical Research and Early Drug Development, Hoffmann-La Roche Inc., 340 Kingsland Street, Nutley, NJ 07110, USA b a r t i c l e i n f o Article history: Received 5 October 2012 Revised 30 November 2012 Accepted 10 December 2012 Available online 20 December 2012 a b s t r a c t From a series of N-acyl 4-(3-pyridonyl)phenylalanine derivatives of 4, the trifluoromethyl derivative 28 was identified as a potent, dual acting alpha4 integrin antagonist with activity in primate models of allergic asthma. Investigation of a series of prodrug esters led to the discovery of the morpholinopropyl derivative 48 that demonstrated good intestinal fluid stability, solubility and permeability. Compound 48 gave high blood levels of 28 when dosed orally in cynomolgus monkeys. Surprisingly, hydrolysis of 48 was rapid in liver microsomes from the pharmacological species, mouse, rat and monkey, but slow in dog and human; in vivo studies also indicated there was prolonged exposure to unchanged prodrug in dogs. Ó 2012 Elsevier Ltd. All rights reserved. Alpha4 integrins are expressed on a variety of leukocytes, including B-cells, T-cells, basophils and eosinophils, and are involved in the recruitment, activation and survival of these cell types. Data supporting a role for alpha4 integrins in a number of inflammatory diseases, including multiple sclerosis, inflammatory bowel diseases, asthma, rheumatoid arthritis, and atherosclerosis, have emerged and are summarized in recent reviews.1 A humanized mouse anti-alpha4 antibody, Natalizumab, has been approved for the treatment of multiple sclerosis2 and Crohn’s disease.3 More recently a small molecule alpha4 integrin antagonist was shown to be effective for the treatment of MS,4 fully validating alpha4 integrins as targets for human disease and increasing interest in the discovery of small molecules with superior activities. We previously reported that the potent, dual-acting antagonist of a4b1 and a4b7, 1, is effective for the treatment of asthma in man5 and sought additional compounds that might have lower clearance and greater selectivity for a4b7 to further assess their potential as alpha4 integrin antagonists. Inspired by reports of the alpha4 integrin antagonist activity of members of the biphenylalanine class such as 2,6 as well as the interesting, selective integrin antagonist activity of members of the pyridizinone family represented by 3,7 we report here on a new series of potent alpha4 integrin antagonists, N-acyl 4-(3-pyridonyl)-phenylalanine derivatives 4 and their orally active prodrug esters. The compounds reported in Table 1 were typically prepared by a palladium-catalyzed coupling reaction of the 4-iodophenylalanine derivative 17 with an in situ generated organozinc intermediate derived from the appropriate 4,6-disubstituted 3-iodo-pyridone intermediate such as 11 and 16 (Scheme 3). Cl OCH3 O Cl HN OCH3 O E-mail address: tilleyjk@optonline.net (J.W. Tilley). 0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2012.12.019 O O O Cl CH3 1 α4β1 IC50 10 nM α4β7 IC50 43 nM 2 R1 CH3 N N R2 O N O R3 OCH3 OH Cl HN O ⇑ Corresponding author. OH Cl HN OH Cl HN Cl 3 OH Cl HN O O R4 O 4 1037 J. W. Tilley et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1036–1040 Table 1 VCAM/VLA-4 binding inhibition of N-acyl 4-(3-pyridonyl)-L-phenylalanine derivatives R2 R3 N O R4 OH HN R1 No. R1 O O R2 R3 R4 a4b1 Ramos cells/VCAM IC50 nM (Rel Potency)a a4b7 RPMI cells/MadCAM IC50 nM (Rel Potency)a CH3 H H 419 ND CH3 H H 582 (0.03) 687 (0.46) CH3 H CH3 226 (0.23) 363 (0.68) CH3 H CH3 90 (0.37) 179 (1.6) CH3 CH3 CF3 48 (0.47) 154 (3.5) CH3 CH3 CF3 32 (0.81) 42 (6.5) H CH3 CF3 820 (0.08) 810 (0.65) C2H5 CH3 CF3 112 (0.40) 61 (1.9) CH3 CF3 463 (0.03) 1230 (0.34) CH2–C5H6 CH3 CF3 560 (0.12) 600 (0.96) CH3 CH3 CF3 225 (0.41) 767 (0.70) CH3 CH3 CF3 366 (0.36) 1,290 (0.23) CH3 CH3 CF3 140 (0.75) 671 (0.54) CH3 CF3 CH3 269 (0.11) 126 (2.2) CH3 CF3 CH3 267 (0.06) 160 (2.6) H3CO 23 Br Cl 24 CH3 Cl 25 CH3 Cl 26 Cl Cl 27 CH3 Cl 28 Cl Cl 29 Cl Cl 30 Cl Cl 31 Cl Cl 32 Cl CH3 33 C2H 5 CH 3 34 CH(CH3)2 35 CH 3 O Cl 36 Cl Cl 37 CH 3 a Rel potency refers to activity relative to the reference standard RO0270608 run as a positive control in each binding experiment. 1038 J. W. Tilley et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1036–1040 O O OMe O OMe a NH 2 N F F + b H N H 3C CH3 N O O H3C CF 3 7 6 c a, b I CF3 I CF3 8 CH3 N O H3 C CO 2Me F O 5 O OMe BocHN H N H 3C c O d H N H 3C 11 CH 3 O CO2H N H3C e I I CF3 CF 3 9 10 11 H3 C CF3 R OMe ClH.H2N O O F O F F 19 F F F O O c N F3C O OEt CH 3 O O O X = OH or Cl O CH3 O 13 N 14 a, b O CH3 F3C N CH3 O e F 3C N 21 O I CH3 15 CH3 N 16 Scheme 2. Reagents and conditions: (a) pyridine, CH2Cl2, 0 °C to rt, 15 h; (b) NaOEt, EtOH, reflux, 15 h; (c) MeI, K2CO3, DME, reflux, 15 h; (d) LiCl, DMF, 160C, 19 h; (e) NIS, trifluoroacetic anhydride, TFA, 70–85 °C, 2 h. H3C 22 CH 3 N O H 3C c. d CF3 OH Cl HN O Cl The 4,6-disubstituted 3-iodo-pyridone intermediates (11 and 16) were prepared using the process described in Schemes 1 and 2 while others were prepared using similar approach. In both, a 4,6-disubstituted pyridone core was built by a condensation between the mono ester malonamide and the diketone 7 or the related methoxy vinylketone 12 to give 8 and 13, respectively. Treatment of the acid 9 with iodine/KI/NaHCO3 in aqueous methanol at 65 °C led to a nearly quantitative conversion to the iodide 10, which was then alkylated to give 11. Surprisingly, the carboxylic acid derived from 13 did not provide the corresponding iodide under similar conditions. Thus, 13 was converted to the N-methyl intermediate 14 which was then treated with lithium chloride in DMF at 160 °C to effect hydrolysis and decarboxylation followed by treatment with NIS in trifluoroacetic acid in the presence of trifluoroacetic anhydride to give 16 exclusively in 62% yield. The iodide 11 was then reacted with an activated zinc dust in dimethylacetamide at 70 °C to give the corresponding organozinc intermediate.8 The coupling reaction of this organozinc intermediate with 17 proceeded smoothly in the presence of Pd(dba)2 and trifurylphosphine (TFP) in THF at 50 °C to obtain the desired product 18 in 77% isolated yield. The remaining steps, BOC removal, benzoylation, and hydrolysis proceeded in a straightforward manner to obtain the target compounds. In general, the alkyl ester prodrugs were prepared by treatment of the carboxylic acids with the appropriate alkyl iodide or alkyl bromide in the presence of sodium bicarbonate in DMF. For example, the preparation of morpholinopropyl iodide and the corresponding ester prodrug and its HCl salt is shown in Scheme 4. 28 I O O d 20 OEt OEt H 2N O O Scheme 3. Reagents and conditions: (a) Zn dust (activated using 10 mol % dibromoethane and 10 mol % TMSCl in THF), DMAC, 70 °C, 15 h; (b) Pd(dba)2, TFP, THF, 50C, 15 h; (c) 4.0 N HCl in dioxane, dioxane, rt, 5 h; (d) X = OH, HBTU, DIPEA, DMF, rt, 15 h or X = Cl, DIPEA, CH2Cl2, rt, 15 h; (e) 1.0 N NaOH, EtOH, rt, 5 h. CH3 H N F 3C OH HN R1 F 12 b R R1 F O a O CF3 X O O F CH3 N O H 3C CH3 N O d, e Scheme 1. Reagents and conditions: (a) TMSCl, H2O, 0 °C, 15 h; (b) piperidine, EtOH, reflux, 15 h; (c) LiOH, THF, MeOH, reflux, 48 h; (d) NaHCO3, I2, KI, MeOH, H2O, 65–70 °C, 15 h; (e) MeI, K2CO3, DME, rt, 15 h. + 18 17 O CF3 O OEt BocHN O CH3 N O CF3 O O Cl HN O O O N HCl Cl 48 Scheme 4. Reagents and conditions: (a) LAH, THF, rt, 2 h; (b) NaI, MeSO3H, CH2Cl2, reflux, 15 h; (c) NaHCO3, DMF, rt, 48 h; (d) TMSCl, iPrOH, rt, 2 h. The in vitro potency of the compounds listed in Table 1 was assessed by determining the ability of serial dilutions to inhibit the binding of RAMOS cells (a4b1-specific binding) and RPMI 8866 cells (a4b7-specific binding) with recombinant human VCAM-1 used as the counter ligand for the RAMOS cell assay and human MAdCAM-1 for the RPMI 8866 cell assay. Cells were labeled with Calcein AM, a fluorescent dye, and then activated with a binding buffer containing Mn2+ to achieve maximum activation prior to assay. RO0270608 (1) was used as a positive control on each plate. The data in Table 1 are reported both as the observed IC50 and as fold-potency relative to 1 since the activity of the cell lines varied over time with the number of passages. Comparing the relative potencies of the compounds reported in Table 1 in the cell based assay with the reference compound 1 indicates that 28 is nearly equally potent to 1 against a4b1 and is several-fold more potent as an antagonist of a4b7. The data indicate that R2 is preferably –CH3 and that R4 is preferentially trifluoromethyl group relative to –CH3 or H. These findings are consistent with our previously reported observation with 4-benzoylcarbonylaminophenylalanines related to 1 that an electron deficient distal aromatic ring was favorable for potency9 and esptablished that R1 is preferably a 2,6-dichlorobenzoyl group.10 Since 28 met our criteria for potency and selectivity as an a4b7 antagonist, it was 1039 J. W. Tilley et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1036–1040 selected for further profiling. Intravenous PK experiments in rat (Table 3) indicated that 28 had an acceptable PK properties. Previous experience with related compounds indicated that the free acids in the acylphenylalanine class have very poor bioavailability; thus, a prodrug strategy was pursued from the outset. The ethyl ester 38 is extremely insoluble in water (6 lg/mL) and while bioavailability in rats was a modest 18%, it was only 2% in dogs. Other candidate prodrugs were assessed for Caco-2 permeability, solubility, and stability in simulated intestinal fluid, and promising compounds were further profiled in rat PK (Tables 2 and 3). Most of the prodrugs have good stability and reasonable conversion in plasma to the parent drug. Only the basic prodrugs had good solubility and of these, the morpholinopropyl ester 48 emerged as the best candidate for further profiling in large animal PK and efficacy experiments. PK data for 48 in rat, dog and monkey are shown in Table 3. Although oral bioavailability was only modest in rat and dog, the relatively high bioavailability in monkey, combined with its good solubility and permeability, prompted us to test this compound further in the pivotal monkey lung inflammation model. This model was used to profile 1, which was validated clinically, and thus became our major in vivo hurdle for taking compounds forward. Thus cynomolgus monkeys were challenged with aerosolized Ascaris suum extract 2 h after dosing. Inflammatory cell accumulation in the lungs was assessed by examination of bronchiolar lavage fluid withdrawn 24 h after the antigen challenge. At oral doses of 10 and 30 mg/kg po, 48 was efficacious in blocking influx of eosinophils, lymphocytes, neutrophils and total leukocytes into Table 3 PK properties of compounds 28 and 48 Compd Dose (iv) (mg/kg) AUC (ng h/mL) Cl (mL/min/kg) Vss (mL/kg) Dose (po) (mg/kg) AUC of 28 (ng h/mL) Cmax (mg/mL) F (%) 28 48 Rat Dog Monkey 10 10,860 15.7 320 50 6100 2.5 11 10 10,880 16.5 250 50 6420 2.4 13 15 35,100 3.0 67 30 30,150 5.0 46 their lungs as shown in Figure 1. Based on its in vivo profile, 48 was selected for preclinical safety studies. Surprisingly, although 48 was readily hydrolyzed in mouse, rat and monkey liver microsomes, ester hydrolysis was slow in dog and human. A graphic representation of the microsomal results are shown in Figure 2. The significance of these findings was highlighted by our observation that there was about 10% unchanged prodrug in dog plasma on days 1 and 6 during subchronic safety studies. This gave rise to concerns about tissue distribution and possible safety issues associated with significant exposure to the unchanged prodrug, particularly as it could be anticipated to occur in humans as well. These concerns were validated when, metabolism studies of radiolabeled 28 and 48 in liver microsomes from rat, dog, monkey and human revealed that significant covalent protein binding occurred with an unknown metabolite derived from the unchanged Table 2 Prodrug esters of the lead compound 28 CH3 H3C N O CF3 O R Cl HN O O Cl Compd 28 38 39 40 41 42 43 44 45 R –H (Parent) –CH2CH3 –CH2CH2CH3 –CH2CH(CH3)2 –CH2CH2OCH3 –(CH2)3OCH3 O N(CH 3)2 CH 2 O CH 2 OH OH N 46 Caco-2 Papp 10 7 cm/s Sol. SGFa lg/ mL Sol. SIFb lg/ mL Stability SIFb t1/2 min Bioavail rat F (%) Comment lM 1.4 271 526 325 499 228 — BQL BQL BQL BQL nd — BQL BQL BQL BQL nd — Stable Stable Stable Stable nd — 2.7 nd nd nd nd — 18 nd nd nd 2 — Dog F = 2% 63 0.8 0.7 Stable nd nd 764 0.03 0.03 Stable nd nd 18 190 140 Stable nd 6 Unchg prodrug in plasma 162 >13,000 200 240 11 15 Unchg prodrug in plasma 96 >5000 Decomp 30 11.7 12 165 3900 330 Stable 2.9 11 4 Stable hERG IC20 O N 47 O 48 49 N O CH3 275 O a b SGF is simulated gastric fluid, pH 2.0. SIF is simulated intestinal fluid, pH 7.5. 17 Mixture of diastereomers 1040 J. W. Tilley et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1036–1040 Allergen-induced Inflammatory Cell Influx in the Atopic Primate 35 Vehicle 10 mg/kg 30 mg/kg 30 Cells X 104/ml 25 20 15 10 5 0 MAC NEU EOS LYM Total Cells Cell Type Figure 1. Accumulation of inflammatory cells in atopic non-human primate BAL fluid 24 h after challenge with Acaris suum 2 h after oral treatment with 48. 100 90 % of 24 Remaining 80 the inhibition of a4b7. Compound 28 stood out among those investigated and a series of prodrugs was investigated to find a suitable derivative for oral delivery of the otherwise poorly absorbed carboxylic acid. Most compounds investigated were rejected due to poor solubility, permeability or oral bioavailability and attention was focused on the morpholinopropyl ester 48 as having the best combination of in vitro properties and reasonable bioavailability, particularly in monkeys. The unanticipated species selective hydrolysis of 48 led to the abandonment of this compound as a lead candidate and raises a significant cautionary flag for others considering a prodrug approach to solving drug delivery issues. Scientists interested in pursuing prodrug strategies should verify that any prodrugs they contemplate undergo sufficiently rapid conversion in all species used in pharmacological and safety studies. A similar incidence of species-selective prodrug ester hydrolysis was observed in a series of structurally similar amino acid derived beta2 integrin antagonists.11 Even though many esters undergo rapid hydrolysis and are rarely employed as drugs per se for this reason, their consistent hydrolysis cannot be taken as a given. It would be interesting to investigate more systematically, how common similar species effects occur. One could, for example examine the time course of hydrolysis of a series of known prodrug esters in liver microsomes and plasma from several species seeking structural patterns. In a forthcoming paper, we will describe a successful prodrug strategy with a series of closely realted pyrimidine-diones in which this phenomena did not occur.12 70 References and notes 60 50 40 30 20 10 0 0 10 30 90 Time (min) Control Mouse Rat Dog Monkey Human Figure 2. Percent of ester hydrolysis of 48 versus time in liver microsomal preparations from various species. Control refers to incubation of 48 in buffer with no microsomes added. prodrug, but not the active pyridone acid 28 in dog and human, but not rat or monkey. It is likely that exposure to 48 in the latter was too transient to observe this metabolite or that the metabolic pathway simply was not operable in these species. In the work presented in this manuscript, a series of pyridones was prepared seeking alpha4 integrin antagonists with a bias for 1. (a) Tilley, J. W. Expert Opin. Ther. Patents 2008, 18, 841; (b) Davenport, R. J.; Munday, J. R. Drug Discovery Today 2007, 12, 569; (c) Bosserhoff, A.-K. Expert Opin. 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