IL FARMACO 59 (2004) 185–194 www.elsevier.com/locate/farmac Quinoxaline chemistry. Part XVII. Methyl [4-(substituted 2-quinoxalinyloxy) phenyl] acetates and ethyl N-{[4-(substituted 2-quinoxalinyloxy) phenyl] acetyl} glutamates analogs of methotrexate: synthesis and evaluation of in vitro anticancer activity Sandra Piras, Mario Loriga, Giuseppe Paglietti * Dipartimento Farmaco Chimico Tossicologico, Università di Sassari, via Muroni 23A, Sassari 07100, Italy Received 7 January 2003; accepted 13 November 2003 Abstract Fourteen out of 21 quinoxaline derivatives described in the present paper were selected at NCI for evaluation of their in vitro anticancer activity. Preliminary screening showed that some derivatives exhibited a moderate to strong growth inhibition activity on various tumor panel cell lines between 10–5 and 10–4 M concentrations. Interesting selectivities were also recorded between 10–8 and 10–6 M for the compounds 9 and 13. © 2004 Elsevier SAS. All rights reserved. Keywords: Anticancer activity; Quinoxaline derivatives 1. Introduction Quinoxaline derivatives continue to be a standing interest of our research group. So far a great deal of data concerning the anticancer activity of more than 300 compounds constitute a library for the antifolic analogs [1–14]. During our investigation we have taken into account as antifolate models either the classical methotrexate or the non-classical trimetrexate as well as the corresponding dideazafolic derivatives. In this context we have proved that bioisosteric replacement of pteridine ring with 6(7)-trifluoromethylquinoxaline affords a good substrate for the biological activity in the series of the classical antifolate analogs, whereas this was so in a few cases of the series of non-classical ones. Another aspect being considered was also the bioisosteric replacement of 2-NH group with an oxygen that in some cases was of relevance in the anticancer activity [6–8]. Recently, our interest focused on the homologation of quinoxaline analogs of classical type on the side of both amino and carboxamide groups of the aminobenzoylglutamic moiety linked at position 2 [11]. This homologation produced very few examples * Corresponding author. E-mail address: paglietti@ssmain.uniss.it (G. Paglietti). © 2004 Elsevier SAS. All rights reserved. doi:10.1016/j.farmac.2003.11.014 of active compounds in anticancer test at NCI. However, for a development of the knowledge about the pharmacophoric pattern we thought to replace the 2-NH bridge in the abovecited series with oxygen on the ground that very recently American authors have found that quinoxalines bearing a 2-(4-substituted phenoxy) substituent were endowed with potent antitumor activity [15]. Thus, the list of compounds 1–21 (Fig. 1) was prepared and the results of their activity are now presented. 2. Chemistry The quinoxaline derivatives 1–21 were obtained by a convergent synthesis outlined in Scheme 1. The key intermediate chloroquinoxalines 22a–f, already known as referenced in the experimental section, were reacted with methyl 4-hydroxyphenylacetate (23) to give the esters (1–6) among which 2–6 were converted into the corresponding acids (7–11) on alkaline hydrolysis (see Fig. 1). This process failed in the case of compound 1 that, even under the mild conditions used, alternatively gave the products 24 (10%) 25 (60%), and 26 (25%) instead of the expected acid. This result clearly indicated that a displacement of O-side chain had 186 S. Piras et al. / IL FARMACO 59 (2004) 185–194 and in the presence of TEA and diethylcyanophosphonate, gave the following results. Compounds 7 and 8 yielded the glutamates (12 and 13) while the acids (9 and 10) in an identical run, slightly modifying the reactants ratio, behave differently. In the case of 9 we obtained only the diglutamate (14) in poor yield (14%), whereas in the case of 10 we were able to isolate either the mono- (15) or the diglutamate (16) in 1:1 ratio in 40% yield. This fact seems to confirm our previous observations that when an extra carboxylic group is present on quinoxaline ring, formation of the amide can take place without any selectivity. Alkaline hydrolysis of 12, 13, 15 and 14, 16 proceeded normally and gave the corresponding acids (17–21). The structures of the described compounds have been characterized by the whole of analytical and spectroscopic data. In particular in the case of 6,7-difluoro derivatives the 1 H NMR spectrum shows that 5 and 8 protons do not resonate as singlets but were splitted into doublets of doublets by ortho, meta-coupling with fluorine atoms in accordance with the literature reports [21]. 3. Experimental Fig. 1. List of compounds prepared. occurred in a similar fashion to the case of 6-trifluoromethyl3-phenyl-2-phenoxyquinoxaline derivatives previously observed by us [7] restoring the quinoxalinone (26) which underwent nucleophilic substitution by an ethoxide anion at both positions 7 and 2 to give (25) and to a lesser extent at position 2 to give (24). This displacement of fluorine atom under basic conditions was analogous to that previously observed by us in the case of the preparation of 6,7difluorobenzofuroxane [16] and in more general cases of reactivity of 6,7-difluoroquinolinones with bases [17]. Compound 26 has been reported in two patents [18,19] and mentioned in the preparation of 6,7-difluoro-2chloroquinoxaline (22d) [20], but no physical data were described for both. Now, compounds 26 and 22d are described in detail in this paper. The compounds 24 and 25 were obtained for the first time and fully characterized. Analogously, the alkaline hydrolysis of compound 5 gave along with the expected acid (10) also the compound 27 (10%) thus confirming a partial displacement of the O-side chain. When this reaction was carried out for a time longer than 1.5 h, 27 was the sole compound isolated. Conversion of the acids (7–10) into the corresponding glutamates carrying out the reaction with diethyl L-glutamate hydrochloride in dry DMF Melting points are uncorrected and were recorded on a Kofler or an electrothermal melting point apparatus. UV spectra are qualitative and were recorded in nanometer in ethanol solution with a Perkin-Elmer Lambda 5 spectrophotometer. IR Spectra (Nujol mulls) were recorded with PerkinElmer 781 instrument. 1H NMR spectra were recorded at 200 MHz with a Varian XL-200 instrument using TMS as internal standard. Elemental analyses were performed at Laboratorio di Microanalisi, Dipartimento di Scienze Farmaceutiche, University of Padua. The analytical results for C, H, and N were within ±0.4% of the theoretical values. 3.1. Chemistry 3.1.1. Intermediates The intermediate chloroquinoxalines necessary for this work were known and prepared according to the data of the literature as follows: 22a [22], 22b [23], 22c [3], 22e [9], and 22f [1]. Compound 22d was previously mentioned in a paper [24] without reporting any data and has been obtained by us from 6,7-difluoroquinoxalinone (26) as described below. 3.1.1.1. 6,7-Difluoroquinoxalin-2(1H)-one (26). A mixture of equimolar amounts (7 mmol) of 4,5-difluoro-1,2-diamino benzene, obtained as described [20], and glyoxylic acid in ethanol (20 ml) was refluxed under stirring for 1 h. On cooling, a precipitate was collected and washed with the same solvent to give 26 (0.70 g, 70% yield), m.p. 277–282 °C (from ethanol). Analysis for C8H4 F2N2O: C, H, N. mmax (nujol) cm–1: 3150, 1680. kmax (EtOH) nm: 342, 273, 223, 205. 1 H NMR (CDCl3–DMSO-d6 3/1) d: 12.6 (1H, br s, NH); 8.14 (1H, s, H-3); 7.67 (1H, dd, JH5,F6 = 10.4 Hz, S. Piras et al. / IL FARMACO 59 (2004) 185–194 187 Scheme 1 JH5,F7 = 8.2 Hz, H-5); 7.22 (1H, dd, JH8,F7 = 10.6 Hz, JH8,F6 = 7.6 Hz, H-8). 3.1.1.2. 2-Chloro-6,7-difluoroquinoxaline (22d). A mixture of 26 (3 g, 15 mmol) and POCl3 (30 ml) was stirred under heating at 120 °C for 2 h. The obtained solution was taken up with water and ice and the formed solid was filtered off and washed with water to give 22d (2.7 g, 90% yield), m.p. 92–94 °C (from EtOH). Analysis for C8H3ClF2N2: C, H, N. kmax (EtOH) nm: 321, 236, 204. 1 H NMR (CDCl3) d: 8.77 (1H, s, H-3); 7.88 (1H, dd, JH5–F6 = 10.0 Hz and JH5–F7 = 8.0 Hz, H-5); 7.78 (1H, dd, JH8–F7 = 10.4 Hz and JH8–F6 = 8.2 Hz, H-8). 3.1.2. General procedure for the preparation of the esters 1–6 A mixture of equimolar amounts (1.6 mmol) of chloroquinoxalines (22a–f) and commercially available (Aldrich) me- thyl 4-hydroxyphenylacetate (23) in the presence of Cs2CO3 in anhydrous dimethylformamide (3.5 ml) was stirred under heating at 70 °C for 13 h. On cooling, the mixture was diluted with water and the precipitates were collected and washed with water to give 1–6 as crude products, which were recrystallized from ethanol. Yields, m.p. values, analytical and spectroscopic (IR; UV; 1 H NMR ) data are reported in Table 1. 3.1.3. General procedure for the preparation of the acids 7–11 and isolation of compound 27 A mixture of the ester 2–6 (2 mmol) in ethanol (20 ml) and 2 M NaOH (14 ml) was stirred at room temperature for 2.5 h (2), 24 h (3 and 4), and 1 h (5 and 6). The reaction mixture was diluted with water and after evaporation of the solvent was made acidic with 2 M HCl. The beige-yellow products (7–11) were collected and washed with water. Yields, m.p. 188 S. Piras et al. / IL FARMACO 59 (2004) 185–194 Table 1 The m.p., yields, analytical and spectroscopic (IR, UV, 1H NMR) data Compounds m.p. (°C) a 1 C17H12F2N2O3 IR (nujol) (mmax cm–1) 1730, 1580 UV (EtOH) (kmax nm) 325, 225, 204 90 C23H18N3O3 1750, 1580 341, 242, 206 159–161 (a) 98 C21H16N2O3S 1740, 1670, 1580 360, 266, 212 4 86–87 (a) 92 C20H18N2O5 1750, 1580 302, 243, 206 5 139–141 (a) 89 C21H17F3N2O5 1730, 1580 332, 243, 206 6 78–80 (a) 60 C20H16F2N2O5 1730 332, 228, 207 7 246–248 69 C22H16N2O3 1700, 1580, 1560 341, 243, 206 8 215–217 95 C20H14N2O3S 1715, 1620 361, 266, 212 9 163–165 97 C17H12N2O5 1720, 1570 325, 242, 206 10 120–121 89 C18H11F3N2O5 3450, 1730, 1570 323, 207 11 161–163 77 C17H10F2N2O5 3500, 1730, 1570 327, 206 12 139–142 (a) 65 C31H31N3O6 3300, 1730, 1650, 1540 341, 243, 206 13 177–180 (a) 39 C29H29N3O6S 3300, 1750, 1650, 1560 361, 266, 211 14 105–108 (a) 14 C35H42N4O11 3300, 1730, 1670 331, 243, 205 Analysis for 114–116 (a) Yields (%) 89 2 132–135 (a) 3 1 H NMRb, dH (J in Hz) [A] 3.69 (2H, s, CH2); 3.74 (3H, s, CH3); 7.22 (2H, d, J = 8.0, H-3′, 5′); 7.38 (2H, d, J = 8.0, H-2′, 6′); 7.60–7.45 (1H, m, H-5); 7.90–7.75 (1H, m, H-8); 8.67 (1H, s, H-3) [A] 3.69 (2H, s, CH2); 3.73 (3H, s, CH3); 7.24 (2H, d, J = 8.4, H-3′, 5′); 7.40 (2H, d, J = 8.4, H-2′, 6′); 7.74–7.50 (6H, m, arom); 8.23–8.10 (2H, m, H-8 + arom) [B] 3.71 (2H, s, CH2); 3.74 (3H, s, CH3); 7.23–7.19 (2H, m, H3″, 4″); 7.32 (2H, d, J = 8.8, H-2′, 6′); 7.68–7.54 (3H, m, arom); 8.05 (1H, m, H-5″); 8.30 (1H, m, H-8) [A] 1.48 (3H, t, CH2CH3); 3.69 (2H, s, CH2); 3.74 (3H, s, COOCH3); 4.57 (2H, q, CH2CH3); 7.26 (2H, d, J = 8.6, H-3′, 5′); 7.40 (2H, d, J = 8.6, H-2′, 6′); 7.80–7.62 (3H, m, arom); 8.20–8.10 (1H, dd, J = 7.6 and 1.2, H-8) [A] 1.50 (3H, t, CH2CH3); 3.70 (2H, s, CH2); 3.74 (3H, s, COOCH3); 4.58 (2H, q, CH2CH3); 7.24 (2H, d, J = 8.6, H-3′, 5′); 7.40 (2H, d, J = 8.6, H-2′, 6′); 7.86–7.80 (1H, dd, J = 8.6 and 1.8, H-6); 8.08 (1H, s, H-8); 8.25 (1H, d, J = 8.8, H-5) [A] 1.48 (3H, t, CH2CH3); 3.69 (2H, s, CH2); 3.74 (3H, s, CH3); 4.56 (2H, q, CH2CH3); 7.23 (2H, d, J = 8.6, H-3′, 5′); 7.38 (2H, d, J = 8.6, H-2′, 6′); 7.60–7.45 (1H, m, H-5); 7.95–785 (1H, m, H-8) [B] 3.66 (2H, s, CH2); 7.24 (2H, d, J = 8.6, H-3′, 5′); 7.40 (2H, d, J = 8.6, H-2′, 6′); 7.82–7.50 (6H, m, arom); 8.24–8.10 (2H, m, arom) [B] 3.66 (2H, s, CH2); 7.26–7.21 (2H, m, H-3″, 4″); 7.30 (2H, d, J = 8.4, H-3′, 5′); 7.41 (2H, d, J = 8.4, H-2′, 6′); 7.70–7.50 (3H, m, arom); 8.00 (1H, m, H-5″); 8.29 (1H, m, H-8) [B] 3.62 (2H, s, CH2); 7.27 (2H, d, J = 8.4, H-3′, 5′); 7.41 (2H, d, J = 8.4, H-2′, 6′); 7.78–7.75 (3H, m, arom); 8.12–8.10 (1H, m, H-8) [B] 3.66 (2H, s, CH2); 7.25 (2H, d, J = 8.6, H-3′, 5′); 7.37 (2H, d, J = 8.6, H-2′, 6′); 7.82–7.74 (1H, m, H-6); 8.07 (1H, s, H-8); 8.25 (1H, d, J = 8.6, H-5) [A] 3.66 (2H, s, CH2); 7.21 (2H, d, J = 8.2, H-3′, 5′); 7.40 (2H, d, J = 8.2, H-2′, 6′); 7.60–7.45 (1H, m, H-5); 8.00–7.80 (1H, m, H-8) [A] 1.35–1.20 (6H, m, 2CH2CH3); 2.45–1.82 (4H, m, CH2CH2); 3.67 (2H, s, CH2Ph); 4.22 –4.09 (4H, m, 2CH2CH3); 4.62–4.75 (1H, m, CH); 6.28 (1H, d, J = 7.4, NH); 7.29 (2H, d, J = 8.4, H-2′, 6′); 7.80–7.55 (6H, m, arom); 8.25–8.10 (2H, m, arom) [A] 1.32–1.21 (6H, m, 2CH2CH3); 2.40–1.90 (4H, m, CH2CH2); 3.68 (2H, s, CH2Ph); 4.30–4.10 (4H, m, 2CH2CH3); 4.59–4.72 (1H, m, CH); 6.31 (1H, d, J = 7.4, NH); 7.25–7.21 (2H, m, H-3″, 4″); 7.35 (2H, d, J = 8.6, H-3′, 5′); 7.39 (2H, d, J = 8.6, H-2′, 6′); 7.70–7.75 (3H, m, arom); 8.05 (1H, m, H-5″); 8.32 (1H, d, J = 8.2, arom) [A] 1.42–1.20 (12H, m, 4CH2CH3); 2.65–1.80 (8H, m, 2CH2CH2); 3.66 (2H, s, CH2Ph); 4.20–4.10 (4H, m, 2CH2CH3); 4.30–4.20 (4H, m, 2CH2CH3); 4.67–4.50 (1H, m, CH); 4.95–4.85 (1H, m, CH); 6.38 (1H, d, J = 7.2, NH); 7.28 (2H, d, J = 7.0, H-3′, 5′); 7.32 (2H, d, J = 7.0, H-2′, 6′); 7.85–7.70 (3H, m, arom); 8.13 (1H, dd, J = 8.6 and 1.4, H-8); 8.39 (1H, d, J = 7.8, NH) (continued on next page) S. Piras et al. / IL FARMACO 59 (2004) 185–194 189 Table 1 (continued) Compounds m.p. (°C) a 15 IR (nujol) (mmax cm–1) 3300, 1730, 1650, 1580 UV (EtOH) (kmax nm) 325, 240, 228, 204 C36H41F3N4O11 3300, 1730, 1660, 1580 331, 242, 204 95 C27H23N3O6 1730, 1700, 1650 342, 242, 207 212–214 (a) 84 C25H21N3O6S 3300, 1720, 1650 360, 266, 211 19 Oil 42 C27H26N4O11 3400, 1660 228, 193 20 105–108 72 C23H18F3N3O8 1730, 1620 324, 240, 228, 205 21 115–119 43 C28H25F3N4O11 1640, 1580 330, 243, 206 Analysis for 132–135 (b) Yields (%) 40 16 109–111 (b) 40 17 187–190 18 a b C27H26F3N3O8 1 H NMRb, dH (J in Hz) [A] 1.32–1.20 (6H, m, 2CH2CH3); 2.50–1.90 (4H, m, CH2CH2); 3.66 (2H, s, CH2Ph); 4.27–4.05 (4H, m, 2CH2CH3); 4.60–4.56 (1H, m, CH); 6.32 (1H, d, J = 6.0, NH); 7.28 (2H, d, J = 8.8, H-3′, 5′); 7.40 (2H, d, J = 8.8, H-2′, 6′); 7.80 (1H, dd, J = 8.6 and 1.8, H-6); 8.08 (1H, s, H-8); 8.18 (1H, d, J = 8.8, H-5) [A] 1.37–1.15 (12H, m, 4CH2CH3); 2.65–1.90 (8H, m, 2CH2CH2); 3.66 (2H, s, CH2Ph); 4.35–3.95 (8H, m, 4CH2CH3); 4.75–4.52 (1H, m, CH); 5.00–4.86 (1H, m, CH); 6.30 (1H, d, J = 6.0, NH); 7.29 (2H, d, J = 7.0, H-3′, 5′); 7.38 (2H, d, J = 7.0, H-2′, 6′); 7.85 (1H, dd, J = 8.6 and 1.6, H-6); 8.08 (1H, s, H-8); 8.25 (1H, d, J = 8.4, H-5); 8.34 (1H, d, J = 7.8, NH) [A] 2.45–1.90 (4H, m, CH2CH2); 3.65 (2H, s, CH2CO); 4.55–4.49 (1H, m, CH); 7.22 (2H, d, J = 8.4, H-3′, 5′); 7.42 (2H, d, J = 8.4, H-2′, 6′); 7.80–7.50 (6H, m, arom); 8.30–8.10 (2H, m, arom) [B] 2.47–1.85 (4H, m, CH2CH2); 3.64 (2H, s, CH2CO); 4.55–4.40 (1H, m, CH); 7.25–7.20 (2H, m, H-3″, 4″); 7.29 (2H, d, J = 8.8, H-3′, 5′); 7.48 (2H, d, J = 8.8, H-2′, 6′); 7.75–7.55 (3H, m, arom); 8.03–8.10 (1H, m, H-5″); 8.31–8.35 (1H, m, arom) [B] 2.44–1.80 (8H, m, 2CH2CH2); 3.50 (2H, s, CH2Ph); 4.54–4.15 (1H, m, CH); 4.75–4.50 (1H, m, CH); 7.25 (2H, d, J = 7.0, H-3′, 5′); 7.40 (2H, d, J = 7.0, H-2′, 6′); 7.90–7.60 (2H, m, arom); 8.11–8.07 (1H, m, arom); 8.37–8.34 (1H, m, arom) [B] 2.50–1.90 (4H, m, 2CH2CH2); 3.65 (2H, s, CH2Ph); 4.60–4.50 (1H, m, CH); 6.90 (1H, d, J = 6.8, NH); 7.25 (2H, d, J = 8.6, H-3′, 5′); 7.42 (2H, d, J = 8.6, H-2′, 6′); 8.90–8.75 (1H, m, H-6); 8.07 (1H, s, H-8); 8.20 (1H, d, J = 8.8, H-5) [B] 2.60–2.10 (8H, m, 2CH2CH2); 3.64 (2H, s, CH2Ph); 4.55–4.40 (1H, m, CH); 4.85–4.70 (1H, m, CH); 7.26 (2H, d, J = 8.4, H-3′, 5′); 7.42 (2H, d, J = 8.4, H-2′, 6′); 8.30–8.05 (2H, m, arom); 8.70–8.60 (1H, m, arom); 8.90–9.01 (1H, m, NH) Purification procedure: (a) crystallized from ethanol and (b) flash chromatography petrol ether (40–60 °C)/ethyl acetate = 1/1. Solvent: [A] = CDCl3; [B] = CDCl3–DMSO-d6. values, analytical and spectroscopic data are reported in Table 1. From hydrolysis of 5, carried out as above, we obtained compound 27 as by-product (10% yield), m.p. 82–84 °C from ethanol. Analysis for C12H9F3N2O3: C, H, N. Table 2 –log GI50,–log TGI,–log LC50 mean graph midpoints (MG-MID) a of in vitro inhibitory activity test for compounds 3, 6, 7, 8, 9, 12, and 13 against human tumor cell lines b Compounds 3 6 7 8 9 12 13 –log GI50 = µM 4.14 = 72.44 4.10 = 79.43 4.22 = 60.25 4.26 = 54.95 4.20 = 63.09 4.13 = 74.13 4.63 = 23.44 –log TGI = µM 4.02 = 95.49 4.01 = 97.72 4.01 = 97.72 4.01 = 97.72 4.00 = 100 4.00 = 100 4.15 = 70.79 –log LC50 = µM 4.00 = 100 4.00 = 100 4.00 = 100 4.00 = 100 4.00 = 100 4.00 = 100 4.02 = 95.49 a MG-MID, mean graph midpoints; the average sensitivity of all cell lines toward the test agent. b From NCI. mmax (nujol) cm–1: 3450, 1720, 1570. kmax (EtOH) nm: 338, 324, 210. 1 H NMR (CDCl3–DMSO-d6) d: 8.20 (1H, d, J = 8.6 Hz, H-5); 8.15 (1H, s, H-8); 7.77 (1H, dd, J = 8.6 and 1.6 Hz, H-8); 5.20 (1H, br s, OH); 4.64 (2H, q, CH2CH3); 1.51 (3H, t, CH2CH3). 3.1.4. Hydrolysis of compound 1 into 2-ethoxy-6,7-difluoroquinoxaline (24), 2,7-diethoxy-6-fluoroquinoxaline (25), and 6,7-difluoroquinoxalin-2(1H)-one (26) Compound 1 (0.25 g, 8.32 mmol), dissolved in a mixture of ethanol (8.3 ml) and 2 M NaOH (5.5 ml) was heated at 40 °C for 5 h. Then, the precipitated product was collected to give crude 24 (10% yield), m.p. 85–87 °C from ethanol. Analysis for C10H8F2N2O: C, H, N. mmax (nujol) cm–1: 1575, 1513. kmax (EtOH) nm: 336, 322, 222, 207. 1 H NMR (CDCl3) d: 8.42 (1H, s, H-3); 7.76 (1H, dd, JH5,F6 = 10.4 Hz, JH5,F7 = 8.2 Hz, H-5); 7.56 (1H, dd, 190 S. Piras et al. / IL FARMACO 59 (2004) 185–194 Table 3 Percentage tumor growth inhibition recorded on subpanel cell lines at 10–4 M of compounds 3, 6, 7, 8, 9, 12, and 13 Panel/cell lines Compounds 3 6 7 8 9 12 13 Leukemia CCRF-CEM HL-60(TB) K-562 MOLT-4 RPMI-8226 SR – – – – – – 77 89 102 159 108 NT 62 63 51 54 57 83 71 70 50 71 90 89 – 71 81 NT 66 – – 58 – – – – – 42 – – – Non-small cell lung cancer A549/ATCC EKVX HOP-62 HOP-92 NCI-H226 NCI-H23 NCI-H322M NCI-H460 NCI-H522 40 72 124 100 – – – – 44 51 – 42 67 55 – – – 66 111 40 76 85 71 80 87 67 65 55 72 64 – – – – – – – – – 48 – 77 NT 108 44 – – 82 111 76 180 130 104 61 87 64 62 Colon cancer COLO 205 HCC-2998 HCT-116 HCT-15 HT29 KM12 SW-620 – – – – – – – – NT 90 – 62 46 40 69 NT 54 62 64 64 62 112 65 69 88 84 78 48 – – – – – – – – 45 – – 47 – – 61 89 – – 40 – CNS cancer SF-268 SF-295 SF-539 SNB-19 SNB-75 U251 69 92 77 69 172 73 – – – – 43 – 75 67 62 49 60 66 60 77 55 54 108 89 – – – – – – 84 61 96 72 89 56 153 71 147 136 NT 103 Melanoma LOX IMVI MALME-3M M14 SK-MEL-2 SK-MEL-28 SK-MEL-5 UACC-257 UACC-62 – – – 65 – – – – – – NT – – 91 – 47 65 62 83 60 52 66 81 47 63 54 66 58 41 63 – – – – – – – – NT – – 41 – 60 – 46 68 68 Ovarian cancer IGROV1 OVCAR-3 OVCAR-4 OVCAR-5 OVCAR-8 SK-OV-3 – – – – 71 103 – 51 63 – 47 – 75 130 42 60 68 64 65 58 – – – – – – – NT 51 63 – NT 69 69 140 102 75 177 NT 87 63 113 40 – 52 50 43 62 (continued on next page) 191 S. Piras et al. / IL FARMACO 59 (2004) 185–194 Table 3 (continued) Panel/cell lines Compounds 3 6 7 8 9 12 13 Renal cancer 786-0 A498 ACHN CAKI-1 RXF 393 SN12C TK-10 UO-31 71 NT 43 – 139 – 51 – 43 NT – – – 47 – – – – 69 69 101 60 51 85 56 106 68 64 124 54 52 80 – – – 50 – – – – 88 – 42 – 93 – 51 67 173 106 143 65 136 76 114 114 Prostate cancer PC–3 DU-145 – – 60 – 67 56 68 55 – – 77 – 52 84 Breast cancer MCF-7 MCF-7/ADR-RES MDA-MB-231/ATCC HS578T MDA-MB-435 BT-549 T-47D MDA-N – 53 97 70 – 69 – NT – 109 50 – – – NT NT 65 50 62 77 54 90 48 NT 79 63 – – – – 65 – – NT 52 57 – 81 43 40 100 – 92 133 106 52 74 127 NT 62 52 96 120 56 –, below 40% growth inhibition; NT, not tested at this molar concentration. JH8,F7 = 10.4 Hz, JH8,F6 = 8.2 Hz, H-8); 4.51 (2H, q, CH2CH3); 1.48 (3H, t, CH2CH3). The mother liquors on standing yielded another product constituted by 25 (60% yield), m.p. 57–58 °C from ethanol. Analysis for C12H13 F N2O2: C, H, N. mmax (nujol) cm–1: 1629, 1574. kmax (EtOH) nm: 338, 326, 214. 1 H NMR (CDCl3) d: 8.31 (1H, s, H-3); 7.63 (1H, d, JH–F = 11.4 Hz, H-5); 7.21 (1H, d, JH–F = 8.4 Hz, H-8); 4.49 (2H, q, CH2CH3); 4.25 (2H, q, CH2CH3); 1.55 (3H, t, CH2CH3); 1.47 (3H, t, CH2CH3). The mother liquors on acidification gave crystals of 26 (25% yield), identical with an authentic sample as described above. was shaken with water (35 ml), then with saturated sodium carbonate aqueous solution (42 ml), rewashed with water (35 ml) and, if necessary, with saturated sodium chloride aqueous solution (42 ml) and eventually dried over anhydrous sodium sulfate. On evaporation of the solvent, compounds 12–16 were obtained as crude products, which were recrystallized from ethanol. Compounds 15 and 16 were both obtained in one time from compound 10, and separated by flash chromatography eluting with a mixture of petroleum ether (40–60 °C)/ethyl acetate in a ratio of 1:1. On the contrary from compound 9 only the diglutamate 14 was obtained. Yields, m.p. values, analytical and spectroscopic data are reported in Table 1. 3.1.5. General procedure for the preparation of the esters 12–16 A mixture of equimolar amounts of compounds 7, 8, and 10 (0.42 mmol) in anhydrous DMF (7.2 ml), diethyl L-glutamate hydrochloride and diethylcyanophosphonate in anhydrous DMF (0.6 ml) and in the presence of 2 mol equivalent of TEA was stirred under nitrogen at room temperature for 1.5 h. The above reactants were used in a ratio of 1:2:2 and in the presence of fourfold mole equivalent of TEA in the case of 9 and 10. The resulting solution was poured into a mixture of ethyl acetate and benzene in 3:1 ratio (22 ml). The organic phase 3.1.6. General procedure for the preparation of the acids 17–21 A suspension of the proper ester (12–16) (61 mmol) in a mixture of ethanol (9.3 ml) and 1 M NaOH (2.9 ml) was stirred at room temperature for 3 h. The ethanol was evaporated in vacuo and the residue taken up with water then made acidic with 2 M HCl. A solid (compounds 17–21, respectively) from beige to yelloworange was collected and washed with water. If necessary compounds were recrystallized from ethanol. Yields, m.p. values, analytical and spectroscopic data are reported in Table 1. 192 S. Piras et al. / IL FARMACO 59 (2004) 185–194 Table 4 Percent growth inhibition recorded by compound 13 between 10–8 and 10–5 M concentrations 10–8 Leukemia K-562 Non-small cell lung cancer A549/ATTC HOP-62 Colon cancer HCT-116 CNS cancer SF-539 SNB-19 U251 Melanoma LOX IMVI MALME-3M Ovarian cancer OVCAR-3 OVCAR-4 OVCAR-5 OVCAR-8 Renal cancer 786-0 ACHN RXF 393 SN12C Prostate cancer DU-145 Breast cancer MCF-7/ADR-RES MDA-MB-231/ATCC HS 578T T-47D 31 31 10–7 10–6 10–5 27 36 60 30 30 36 36 94 94 21 58 66 52 47 31 33 20 37 42 40 15 46 22 32 50 29 18 27 48 26 37 65 42 51 86 15 23 28 16 29 21 28 28 37 26 83 85 72 51 27 39 35 52 22 28 17 29 24 17 47 65 39 61 71 41 61 4. Pharmacology Twenty-one compounds of the list (Fig. 1) were submitted for in vitro anticancer evaluation to NCI. Among these 14 were selected by NCI for the in vitro assay. Only seven emerged from a preliminary screening on three-cell panel lines (MCF-7; NCI-H460; SF-288) and their evaluation is referred to the structures of 3, 6, 7, 8, 9, 12, and 13. The protocol for anticancer activity is well documented [25] and it involves a panel of 60 human tumor cell lines. The anticancer activity is derived from dose–response curves and it is presented in three different Tables 2–4. In Table 2 the response parameters (–log GI50), (–log TGI), and (–log LC50) refer to the concentration of the agent in the assay that produced 50% growth inhibition (GI), total growth inhibition (TGI) and 50% cytotoxicity (LC), respectively, and are expressed as mean graph midpoints. In Table 3, we reported the activities of those compounds, which showed a percent GI greater than 40% on subpanel cell lines at 10–4 M concentration. In Table 4, we reported the activity of compound 13 that exhibited a significant percentage GI at the most diluted concentrations (10–8–10–5 M). In Fig. 2, we reported the percent GI of compound 9 against leukemia HL-60 (TB), K-562 and SR cell lines in the range of 10–8–10–4 M concentration. 5. Results of the in vitro pharmacological antitumor assays The data of in vitro anticancer activity reported in Table 2 established that the average sensitivity of all cell lines towards the tested agents, represented as mean graph midpoints, falls in the range of 10–4.63–10–4.00 M concentration and according to this the following decreasing order of activity was recorded 13 >8 >7 >9 >3 >12 >6. The data of Table 3 better indicate that the highest activities were recorded on all the panel cell lines for the compounds 7, 8, 12, and 13, whereas the compounds 3 and 6 exhibited a moderate range of both cell line sensitivity and percent tumor GI. Interestingly compound 9 (Fig. 2) showed to possess both selective and dose-dependent activity against leukemia subpanel cell lines (HL-60 (TB), K-562 and SR) in the range of 10–8–10–4 M, the other cell lines being almost unaffected. S. Piras et al. / IL FARMACO 59 (2004) 185–194 193 Fig. 2. Percent growth inhibition of compounds 9 against leukemia HL-60 (TB), K-562, and SR cell lines between 10–8 and 10–4. According to the data of Table 4 we can observe that compound 13 still exhibited significant percent tumor GI values at the most diluted concentrations (10–8–10–5 M) whereas, in some case its activity seems to be dose dependent. In conclusion we can say that this type of homologation does not present any significative improvement towards the previously encouraging reports [11] but at least two compounds emerged for the selectivity (9) and low toxicity (13). References [1] [2] [3] [4] [5] [6] M. Loriga, M. Fiore, P. Sanna, G. 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