1804
CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
Synthesis and Biological Assays of 9-(Acylamino)homocamptothecins as DNA Topoisomerase I Inhibitors by Wei Guo a ), Guoqiang Dong a ), Lingjian Zhu a ), Wenfeng Liu a ), Chunlin Zhuang a ), Zizhao Guo a ), Jianzhong Yao a ), Chunquan Sheng a ), Huojun Zhang* b ), Zhenyuan Miao* a ), and Wannian Zhang* a ) a
) School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, P. R. China (e-mail: [email protected], [email protected]) b ) Department of Radiation Oncology, Changhai Hospital, Second Military Medical University, 168 Changhai Road, Shanghai 200433, P. R. China (e-mail: [email protected])
In an effort to improve the stability of homocamptothecin and reduce the toxicity, novel homocamptothecin analogs with acylamino groups at C(9) were designed and synthesized. The cytotoxic activities of all the synthetic compounds against three cancer cell lines were evaluated by the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, and irinotecan was used as reference compound. Compound 7c with a piperidinylacetamido group and 10a with phenylacetamido group at C(9) showed potent activities both in vitro and in vivo. In addition, they also revealed remarkable topoisomerase I inhibitions which were exhibited with well-established bonds with amino acid residues Arg364 and Asp533 in the active pocket. On the basis of the biological activities, 7c and 10a would be potential candidates for further studies.
Introduction. – Camptothecin (CPT; Fig. 1), which was isolated from the Chinese plant Camptotheca acuminate by Wall et al. in 1966, exhibited potent antitumor activity [1]. However, with the development of camptothecin, serious problems appeared, such as its insolubility and toxicity [1 – 5]. The formal studies showed that camptothecin had a unique mechanism of action, i.e., selective inhibition of DNA topoisomerase I (Topo I) [6]. Currently, three important drugs, topotecan, irinotecan, and belotecan, are used in clinical treatment. Topotecan is used as a second-line agent for advanced ovarian cancer or small cell lung cancer, and irinotecan is used against refractory advanced colorectal cancer. Several other camptothecin analogs are in clinical trials [7]. Another camptothecin analog, homocamptothecin (hCPT), was discovered by Lavergne et al. in late 1990s and showed potent antiproliferative activity in cancer cell lines [8]. The hCPT is structurally similar to camptothecin, differing only by changing a six-membered a-hydroxy lactone to a seven-membered b-hydroxy lactone [9]. hCPT can also cleave DNA in the presence of Topo I despite a different sequence selectivity than CPT. The introduction of a CH2 group in ring E of CPT not only led to enhanced lactone stability but also decreased protein binding in human plasma without affecting its capability to poison topoisomerase I-DNA. Two hCPT drugs, diflomotecan (BN80915) and BN80927, were evaluated in Phase II and I trials, respectively [10 – 12]. We have already reported several new classes of 9-substituted hCPTs, such as 9benzylidenamino, 9-(heteroarylmethylidene)amino derivatives of homocampthecins 2013 Verlag Helvetica Chimica Acta AG, Zrich
CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
1805
Fig. 1. Structures of camptothecin analogs
[13 – 20]. These compounds not only have obvious cytotoxic activities in vitro towards several human cancer cell lines but also help to further define the structureactivity relationship of hCPT as Topo I inhibitors. In the present work, we designed and synthesized a series of novel 9-acylhomocaptothecins and investigated whether these modifications would improve the biological properties of hCPT. Results and Discussion. – Chemistry. The preparation of derivatives 7a – 7h was carried out as outlined in Scheme 1 1). Hydrolysis of 1 [13] in refluxing aqueous HCl gave the amino compound 2 [21 – 25]. The intermediate 3 was achieved by acylation of 2 with ClCH2COCl in nearly quantitative yield [26 – 29], which was then reacted with the appropriate secondary amines in the presence of K2CO3 and gave the desired analogs 4b – 4h, catalytic hydrogenation of which afforded the amines 5a – 5h, respectively, in satisfactory yields. The key intermediate 6 was synthesized according to our previously reported procedure, and the process was optimized [13 – 20]. Finally, 7a and the aminoacyl amide hCPTs 7b – 7h were obtained by Friedlnder condensation of 5a – 5h and 6 with Me3SiCl (TMSCl) in moderate yields. Another series of derivatives, 10a – 10h, were prepared by an analogous synthetic approach as depicted in Scheme 2. The amides were obtained from 2 and differently substituted benzoyl chlorides with the appropriate tertiary amine as catalyst. After hydrogenation of the NO2 group, the amino compounds 9a – 9h were obtained, and the 1)
Arbitrary atom numbering; for systematic names, see Exper. Part.
1806
CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
Scheme 1
a) 6n HCl (aq.), 608, 4 h. b) 4-(Dimethylamino)pyridine (DMAP), Et3N, THF, ClCH2COCl, 08. c) K2CO3 , THF, secondary amine, 608, 2 h. d) H2 , 10% Pd/C, EtOH, r.t., overnight. e) Me3SiCl (TMSCl), DMF, 1008, 2 h.
final benzamide derivatives of homocaptothecins 10a – 10h were formed by the condensation reactions of 9a – 9h, respectively, with 6. Cytotoxicity and StructureActivity Relationships. The cytotoxic activities of the synthesized compounds 7a – 7h and 10a – 10h were evaluated against human lung cancer (A549), breast cancer (MDA-MB-435), and colon cancer (HCT-116) cell lines using 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) method. The IC50 values of the samples, compiled in the Table, revealed that most of the tested compounds were active in inhibiting cancer cell lines. For 9-heterocyclic-amidosubstituted hCPT, most of the compounds were sensitive to the breast cancer cell lines, and less active against the lung and colon cancer cell lines. For cell lines MDA-MB-435, eight compounds were more active than the standard drug irinotecan. Among them, compound 7c was the most efficient one with an IC50 value of 0.558 mm, which was 30 times higher than that of irinotecan. For benzamido derivatives, most of the compounds were similarly active as heterocyclic amido-hCPTs, and showed activities against breast cancer cell lines. Six compounds, 10a, 10c, 10d, 10e, 10f, and 10h, were more active than irinotecan. Among these, the activity of 10d, with an IC50 value of 1.76 mm, was ten times higher than that of irinotecan.
CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
1807
Scheme 2
a) DMAP, Et3N, THF, 08. b) H2 , 10% Pd/C, EtOH, r.t., overnight. c) TMSCl, DMF, 1008, 2 h.
Table. Structures and Growth-Inhibitory Activities ( IC50 ) of Target Compounds Compound
7a 7b 7c 7d 7e 7f 7g 7h 10a 10b 10c 10d 10e 10f 10g 10h Irinotecan a
R
Cl Et2N 2-Methylpiperidin-1-yl 3-Methylpiperidin-1-yl 4-Methylpiperidin-1-yl 3,5-Dimethylpiperidin-1-yl (2-Methylpiperidin-1-yl)methyl (4-Methylpiperidin-1-yl)methyl Ph 2-MeC6H4 3-MeC6H4 4-MeC6H4 Bn 3-MeOC6H4 2-ClC6H4 3-ClC6H4 –
IC50 a ) [mm] A549
MDA-MB-435
HCT-116
62.9 26.3 49.6 86.9 96.9 151 > 200 31.9 29.6 133 42.6 > 200 114 > 200 > 200 > 200 48.5
8.32 1.31 0.558 2.34 2.75 7.15 89.2 3.82 7.70 30.1 6.44 1.76 3.60 8.07 23.9 4.40 16.9
33.9 > 200 36.6 109 143 > 200 20.2 29.8 47.4 > 200 124 146 143 158 182 > 200 19.5
) Values were determined by using the MTT method, and the experiments were repeated twice.
Structureactivity relationship study revealed that substitution with a single Me group resulted in a substantial increase in the cytotoxic activity compared to multi Me substitution. For instance, compounds 7c – 7e with a single Me group were more active
1808
CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
than dimethyl-substituted compound 7f. Second, the results suggested that length of the alkyl side chains affected the cytotoxic potencies. The activity decreased with increasing side-chain length. For example, the acetamido compound 7c was more active than the propanamido compound 7g. Antitumor Activities in vivo. Compounds 7c and 10a were chosen for evaluation for 2-week tumor-growth inhibition in vivo assays using nude mice implanted with Lewis lung xenografts (Fig. 2). Treatment with compounds 7c and 10a at 10 mg/kg body weight, tumor growths were suppressed by 32.0 and 45.7%, respectively, as compared with placebo-treated controls. Furthermore, the body weights of all the mice treated with compounds 7c and 10a were increased during the treatment period. Accordingly, compounds 7c and 10a may exert lower toxicities.
Fig. 2. Tumor-growth inhibition by compounds 7c and 10a against A549 xenografts in nude mice administered i.p.
Topoisomerase I-Mediated DNA Cleavage. Compounds 7c and 10a were further evaluated for their possible activities in the inhibition of Topo I (Fig. 3). These compounds showed stable ternary complex activities compared to that of the reference compound camptothecin at the concentrations of 50 and 100 mm. Compound 7c were inactive at concentrations below 10 mm, while 10a exhibited modest activity.
Fig. 3. Effects of compounds 7c and 10a on Topoisomerase I-mediated DNA relaxation. Lane 1, supercoiled DNA pBR322; Lane 2, supercoiled DNA and Topo I; Lanes 3 – 5, 6 – 8, 9 – 11, three samples CPT, 7c, and 10a at 10, 50, and 100 mm, respectively.
CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
1809
Molecular Docking. Considering the in vitro, in vivo, and topoisomerase I-mediated DNA cleavage results, it was worthy to screen for supportive coordination between these results. Topoisomerase I was used as the target enzyme, and automated molecular docking was studied with newly synthesized compound 7c. 9-Amino-hCPT was the reference compounds to determine the binding mode of the compound and enzyme. Using the docking program AutoDock 4.0 furnished the results shown in Figs. 4 and 5.
Fig. 4. Predicted binding mode of 9-aminohomocamptothecin to topoisomerase I active site. The amino acids Arg364 and Asp533, involved in the interactions with the compound, are highlighted.
Fig. 5. Predicted binding mode of compound 7c to topoisomerase I active site. The amino acids Lys425, Arg364, and Asp533, involved in the interactions with compound, are highlighted.
1810
CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
The orientation in the active site cavity was perpendicular to the main axis of the DNA and parallel to the bases. Topo I and hCPTs can interact at the active site. These two homocamptothecin derivatives exhibited well-established bonds with amino acids in the active pocket. At the positions 1 and 20, both of the two compounds had the active pocket consisting of two amino acid residues Arg364 and Asp533. The molecule 7c had also one more site with O-atom which formed another H-bond with amino residue Lys425 as shown in Fig. 5. This study revealed that the molecules 7c showed better binding results towards the target enzyme Topo I than the 9-amino-hCPT. Finally, considering the cytotoxic activites in vitro, in vivo, and molecular-docking results among our synthesized compounds, 7c provided the best results, and it was considered as the best inhibitor of Topo I in this series compounds and could be effective with human cancer cell lines. Conclusions. – Two series of homocamptothecin derivatives with a heterocyclic amido or a benzamido group at C(9) were synthesized and characterized. These compounds were evaluated for in vitro activities against human lung cancer (A549), breast cancer (MDA-MB-435), and colon cancer (HCT-116) cell lines. On basis of the biological activities, the 9-(acylamino)homocaptothecins were found to be potent homocamptothecin derivatives. Among these, 7c with a piperidinylacetamido group and 10a with benzamido group were found to be the most active agents against the breast cell lines MDA-MB-435. They also showed obvious activities in vivo and remarkable Topo I inhibitory activities. Therefore, 9-(acylamino)homocaptothecins could be potential topoisomerase I inhibitors with homocamptothecin skeleton for anticancer agents. This work was supported by the National Natural Science Foundation of China (No. 81171435) and the Shanghai Leading Academic Discipline Project (No. B906).
Experimental Part General. Commercial solvents were used without any pretreatment. TLC: Silica gel plates GF254 (SiO2 ; Qingdao Haiyang Chemical, China). Column chromatography (CC): SiO2 60 G (Qingdao Haiyang Chemical, China). NMR Spectra: Bruker 500 spectrometer in CDCl3 or (D6 )DMSO solns.; d in ppm rel. to Me4Si as internal standard, J in Hz. ESI-MS: API-3000 LC/MS spectrometer; in m/z. Elemental analyses: MOD-1106 instrument; consistent with theoretical values within 0.4%. General Procedure for the Preparation of Compounds 7a – 7h. 9-[(2-Chloroacetyl)amino]-7methylhomocamptothecin ( ¼ 2-Chloro-N-(5-ethyl-4,5,13,15-tetrahydro-5-hydroxy-12-methyl-3,15-dioxo1H,3H-oxepino[ 3’,4’:6,7]indolizino[1,2-b]quinolin-11-yl)acetamide ; 7a) . Compound 2 (600 mg, 3.3 mmol) was dissolved in THF (20 ml) and cooled to 08 prior to the addition of Et3N (0.05 ml, 0.36 mmol) and DMAP ( ¼ 4-(dimethylamino)pyridine; 10 mg, 0.08 mmol). ClCH2COCl (0.27 ml, 3.6 mmol) was added, and the soln. was kept for 4 h at r.t. The mixture was diluted with H2O, extracted with AcOEt, and dried (MgSO4 ). The crude residue was purified by CC (hexane/AcOEt 4 : 1) to give 2[(2-chloroacetyl)amino]-6-nitroacetophenone (3; 800 mg, 94%). Yellow solid. M.p. 135 – 1378. 1H-NMR (CDCl3 ): 2.48 (s, MeCO); 2.85 (s, CH2Cl); 7.63 (t, J ¼ 8.1, HC(4)); 8.00 (d, J ¼ 8.5, HC(5)), 8.53 (d, J ¼ 8.1, HC(3)). ESI-MS: 227.29 ([M H] ). Compound 3 (100 mg, 0.39 mmol) was dissolved in EtOH (20 ml), and 10% Pd/C (100 mg) was added. After stirring overnight at r.t., the mixture was filtered through Celite. The solvent was removed, and the residue was crystallized from hexane to give 2-amino-6-[(2-chloroacetyl)amino]acetophenone (5a; 84 mg, 95%). Yellow solid. 1H-NMR (CDCl3 ): 2.29 (s, MeCO); 2.80 (t, CH2Cl); 6.06 (s, NH2 );
CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
1811
6.51 (d, J ¼ 7.6, HC(5)); 6.58 (t, J ¼ 8.1, HC(3)); 7.10 (d, J ¼ 8.0, HC(4)). ESI-MS: 255.63 ([M H] ). A mixture of 6 (100 mg, 0.36 mmol) and 5a (91 mg, 0.40 mmol) was dissolved in DMF. After dropwise addition of TMSCl (0.1 ml) at r.t., the mixture was heated to reflux under N2 for 2 h at 1008. The solvent was removed, and the crude product was prufied by CC (CH2Cl2/MeOH 100 : 2 – 100 : 5) to give 7a (25 mg, 15%). Yellow solid. M.p. > 3008. 1H-NMR ((D6 )DMSO): 0.87 (t, J ¼ 7.8, Me(18)); 1.86 (q, J ¼ 7.4, CH2(19)); 2.85 (s, MeC(7)); 3.46 (q-like, J ¼ 13.8, CH2(21)); 4.41 (s, CH2Cl); 5.29 (s, CH2(5)); 5.41 (qlike, J ¼ 15.2, CH2(23)); 6.02 (s, HOC(20)); 7.39 (s, HC(14)); 7.51 (d, J ¼ 7.4, HC(12)); 8.12 (t, J ¼ 8.0, HC(11)); 7.38 (d, J ¼ 8.4, HC(10)); 10.41 (s, NHCO). ESI-MS: 466.55 ([M H] ). 9-{[2-(Diethylamino)acetyl]amino}-7-methylhomocamptothecin ( ¼ 2-(Dimethylamino)-N-(5-ethyl4,5,13,15-tetrahydro-5-hydroxy-12-methyl-3,15-dioxo-1H,3H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinolin-11-yl)acetamide; 7b). Compound 3 (100 mg, 0.4 mmol) and Et2NH (0.12 ml, 1.2 mmol) were dissolved in THF, and K2CO3 (100 mg, 0.72 mmol) was added. The mixture was stirred under N2 for 2 h at 608, diluted with H2O, extracted with AcOEt, and then dried (MgSO4 ). The solvent was evaporated under reduced pressure, and 4b (100 mg, 90%) was obtained as a yellow solid. Then, it was dissolved in EtOH (20 ml), and 10% Pd/C (0.1 g) was added. After stirring overnight at r.t., the mixture was filtered through Celite. The solvent was removed, and the residue was crystallized from hexane to give 2-amino-6{[2-(diethylamino)acetyl]amino}acetophenone (5b; 87 mg, 96%), which was dissolved in DMF (20 ml), and 7 (100 mg, 0.18 mmol) was added. After dropwise adding of TMSCl (0.1 ml) at r.t., the mixture was heated to reflux under N2 for 2 h at 1008. The solvent was evaporated, and the residue was purified by CC (CH2Cl2/MeOH 100 : 2 – 100 : 5) to give 7b (130 mg, 71%). Yellow solid. M.p. > 3008. 1H-NMR ((D6 )DMSO): 0.87 (t, J ¼ 7.4, Me(18)); 1.29 – 1.31 (m, 2 MeCH2N); 1.86 (q, J ¼ 7.6, CH2(19)); 2.49 – 2.53 (m, 2 MeCH2N); 2.90 (s, MeC(7)); 3.49 (q-like, J ¼ 13.8, CH2(21)); 4.32 (s, COCH2N); 5.26 (s, CH2(5)); 5.55 (q-like, J ¼ 15.1, CH2(23)); 6.08 (s, HOC(20)); 7.39 (s, HC(14)); 7.59 (d, J ¼ 7.3, HC(12)); 7.86 (t, J ¼ 7.9, HC(11)); 8.15 (d, J ¼ 8.4, HC(10)); 9.85 (s, NHCO). ESI-MS: 503.88 ([M H] ). 7-Methyl-9-{[2-(2-methylpiperidin-1-yl)acetyl]amino}homocamptothecin ( ¼ N-(5-Ethyl-4,5,13,15tetrahydro-5-hydroxy-12-methyl-3,15-dioxo-1H,3H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinolin-11-yl)-2(2-methylpiperidin-1-yl)acetamide; 7c). Yield: 80 mg (42%). Yellow solid. M.p. > 3008. 1H-NMR ((D6 )DMSO): 0.87 (t, J ¼ 7.4, Me(18)); 1.05 (d, J ¼ 7.0, MeC(2’)); 1.06 – 1.17 (m, CH2(4’)); 1.60 – 1.64 (m, CH2(3’,5’)); 1.86 (q, J ¼ 7.4, CH2(19)); 2.92 (s, MeC(7)); 3.04 – 3.07 (m, HC(2’), CH2(6’)); 3.33 (q-like, J ¼ 14.0, CH2(21)); 3.46 (s, COCH2N); 5.29 (s, CH2(5)); 5.52 (q-like, J ¼ 15.1, CH2(23)); 6.03 (s, HOC(20)); 7.38 (s, HC(14)); 7.65 (d, J ¼ 7.2, HC(12)); 7.81 (t, J ¼ 7.9, HC(11)); 8.05 (d, J ¼ 8.2, HC(10)); 10.05 (s, NHCO). ESI-MS: 529.76 ([M H] ). 7-Methyl-9-{[2-(3-methylpiperidin-1-yl)acetyl]amino}homocamptothecin ( ¼ N-(5-Ethyl-4,5,13,15tetrahydro-5-hydroxy-12-methyl-3,15-dioxo-1H,3H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinolin-11-yl)-2(3-methylpiperidin-1-yl)acetamide; 7d). Yield: 90 mg (47%). Yellow solid. M.p. > 3008. 1H-NMR ((D6 )DMSO): 0.87 (t, J ¼ 7.5, Me(18)); 1.05 (t, J ¼ 7.0, MeC(3’)); 1.18 – 1.21 (m, CH2(4’,5’)); 2.91 (s, MeC(7)); 3.03 – 3.09 (m, CH2(2’,6’)); 3.36 – 3.46 (m, 4 H, COCH2N, CH2(21)); 5.29 (s, CH2(5)); 5.52 (qlike, J ¼ 15.1, CH2(23)); 6.08 (s, HOC(20)); 7.39 (s, HC(14)); 7.62 (d, J ¼ 7.3, HC(12)); 7.81 (t, J ¼ 7.9, HC(11)); 8.07 (d, J ¼ 8.5, HC(10)); 8.09 (s, NHCO). ESI-MS: 529.88 ([M H] ). 7-Methyl-9-{[2-(4-methylpiperidin-1-yl)acetyl]amino}homocamptothecin ( ¼ N-(5-Ethyl-4,5,13,15tetrahydro-5-hydroxy-12-methyl-3,15-dioxo-1H,3H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinolin-11-yl)-2(4-methylpiperidin-1-yl)acetamide; 7e). Yield: 70 mg (36%). Yellow solid. M.p. > 3008. 1H-NMR ((D6 )DMSO): 0.87 (t, J ¼ 7.5, Me(18)); 0.92 – 0.93 (m, MeC(4’)); 1.22 – 1.23 (m, HC(4’)); 1.24 – 1.34 (m, CH2(3’,5’)); 1.86 (q, J ¼ 7.7, CH2(19)); 2.90 (s, MeC(7)); 3.04 – 3.07 (m, CH2(2’,6’)); 3.32 (s, COCH2N); 3.48 (q-like, J ¼ 13.7, CH2(21)); 5.27 (s, CH2(5)); 5.52 (q-like, J ¼ 15.2, CH2(23)); 6.04 (s, HOC(20)); 7.38 (s, HC(14)); 7.63 (d, J ¼ 5.6, HC(12)); 7.81 (t, J ¼ 8.2, HC(11)); 8.07 (d, J ¼ 8.4, HC(10)); 9.01 (s, NHCO). ESI-MS: 531.77 ([M H] ). 7-Methyl-9-{[2-(3,5-dimethylpiperidin-1-yl)acetyl]amino}homocamptothecin ( ¼ 2-(3,5-Dimethylpiperidin-1-yl)-N-(5-ethyl-4,5,13,15-tetrahydro-5-hydroxy-12-methyl-3,15-dioxo-1H,3H-oxepino[3’,4’: 6,7]indolizino[1,2-b]quinolin-11-yl)acetamide; 7f). Yield: 60 mg (31%). Yellow solid. M.p. > 3008. 1 H-NMR ((D6 )DMSO): 0.85 – 0.91 (m, Me(18), MeC(3’,5’)); 1.73 (m, CH2(4’)); 1.86 (q, J ¼ 7.5,
1812
CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
CH2(19)); 2.89 (s, MeC(7)); 2.90 – 2.92 (m, CH2(2’,6’)); 3.04 – 3.49 (m, COCH2N, CH2(21)); 5.29 (s, CH2(5)); 5.52 (q-like, J ¼ 15.1, CH2(23)); 6.04 (s, HOC(20)); 7.38 (s, HC(14)); 7.64 (d, J ¼ 7.4, HC(12)); 7.79 (t, J ¼ 7.9, HC(11)); 8.05 (d, J ¼ 8.4, HC(10)); 10.03 (s, NHCO). ESI-MS: 543.28 ([M H] ). 7-Methyl-9-{[ 3-(2-methylpiperdin-1-yl)propanoyl]amino}homocamptothecin ( ¼ N-(5-Ethyl4,5,13,15-tetrahydro-5-hydroxy-12-methyl-3,15-dioxo-1H,3H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinolin-11-yl)-3-(2-methylpiperidin-1-yl)propanamide; 7g). Yield: 150 mg (76%). Yellow solid. M.p. > 3008. 1H-NMR ((D6 )DMSO): 0.80 – 0.87 (m, Me(18), MeC(2’)); 1.86 (q, J ¼ 6.4, CH2(19)); 1.88 – 1.89 (m, CH2(4’,5’)); 1.91 – 1.92 (m, CH2(3’)); 2.50 (s, MeC(7)), 2.86 – 2.87 (m, NHCOCH2 ); 3.01 – 3.02 (m, HC(2’)); 3.38 – 3.40 (m, CH2(21,6’)); 4.11 – 4.12 (m, CH2N); 5.28 (s, CH2(5)); 5.52 (q-like, J ¼ 14.9, CH2(23)); 6.06 (s, HOC(20)); 7.38 (s, HC(14)); 7.46 (d, J ¼ 7.3, HC(12)); 7.80 (t, J ¼ 7.8, HC(11)); 8.09 (d, J ¼ 8.4, HC(10)); 10.10 (s, NHCO). ESI-MS: 543.27 ([M H] ). 7-Methyl-9-{[ 3-(4-methylpiperdin-1-yl)propanoyl]amino}homocamptothecin ( ¼ N-(5-Ethyl4,5,13,15-tetrahydro-5-hydroxy-12-methyl-3,15-dioxo-1H,3H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinolin-11-yl)-3-(4-methylpiperidin-1-yl)propanamide; 7h). Yield: 20 mg (10%). Yellow solid. M.p. > 3008. 1 H-NMR (D6(DMSO)): 0.80 – 0.87 (m, 6 H, Me(18), MeC(4’)); 1.86 (q, J ¼ 6.4, CH2(19)); 1.88 – 1.89 (m, CH2(2’,3’,5’,6’)); 2.50 (s, MeC(7)); 2.86 – 2.87 (m, NHCOCH2 ); 3.38 – 3.39 (m, CH2(21)); 4.11 – 4.13 (m, CH2N); 5.28 (s, CH2(5)); 5.52 (q-like, J ¼ 14.9, CH2(23)); 6.06 (s, HOC(20)); 7.38 (s, HC(14)); 7.46 (d, J ¼ 7.3, HC(12)); 7.80 (t, J ¼ 7.8, HC(11)); 8.09 (d, J ¼ 8.4, HC(10)); 10.10 (s, NHCO). ESI-MS: 543.21 ([M H] ). General Procedure for the Preparation of Compounds 10a – 10h. 9-(Benzoylamino)-7-methylhomocamptothecin ( ¼ N-(5-Ethyl-4,5,13,15-tetrahydro-5-hydroxy-12-methyl-3,15-dioxo-1H,3H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinolin-11-yl)benzamide; 10a). Compound 2 (100 mg, 0.6 mmol) was dissolved in THF (20 ml) and cooled to 08 prior to the addition of Et3N (0.05 ml, 0.36 mmol) and DMAP (10 mg, 0.08 mmol). PhCOCl (0.07 ml, 0.6 mmol) was added and reacted at r.t. for 4 h. The mixture was diluted with H2O, extraed with AcOEt, and dried (MgSO4 ). Crude residue was purified by CC (SiO2 ; hexane/AcOEt 4 : 1) to give 2-(benzoylamino)-6-nitroacetophenone (8a; 60 mg, 38%) as a yellow solid, which was dissolved in EtOH (20 ml), and 10% Pd/C (0.1 g) was added. After stirring overnight at r.t., the mixture was filtered through Celite. The solvent was evaporated, and the residue was crystallized from hexane to give a yellow solid 9a (51 mg, 96%). A mixture of 6 (100 mg, 0.4 mmol) and 9a (0.05 ml, 0.4 mmol) were dissolved in DMF. After dropwise addition of TMSCl (0.1 ml) at r.t., the mixture was refluxed under N2 for 2 h at 1008. The solvent was removed, and the crude product was purified by CC (SiO2 ; CH2Cl2/MeOH 100 : 2 – 100 : 5) to give 10a (110 mg, 62%). Yellow solid. M.p. > 3008. 1H-NMR ((D6 )DMSO): 0.87 (t, J ¼ 7.3, Me(18)); 1.86 (q, J ¼ 7.3, CH2(19)); 2.81 (s, MeC(7)); 3.46 (q-like, J ¼ 13.8, CH2(21)); 5.27 (s, CH2(5)); 5.51 (q-like, J ¼ 15.2, CH2(23)); 6.05 (s, HOC(20)); 7.04 (s, HC(14)); 7.58 – 7.68 (m, HC(12,3’,4’,5’)); 7.87 (t, J ¼ 7.9, HC(11)); 8.08 – 8.09 (m, HC(2’,6’)); 10.62 (s, NHCO). ESIMS: 494.79 ([M H] ). 7-Methyl-9-[(2-methylbenzoyl)amino]homocamptothecin ( ¼ N-(5-Ethyl-4,5,13,15-tetrahydro-5-hydroxy-12-methyl-3,15-dioxo-1H,3H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinolin-11-yl)-2-methylbenzamide; 10b). Yield: 29 mg (16%). Yellow solid. M.p. > 3008. 1H-NMR ((D6 )DMSO): 0.87 (t, J ¼ 7.4, Me(18)); 1.86 (q, J ¼ 7.3, CH2(19)); 2.51 (s, MeC(2’)); 2.73 (s, MeC(7)); 3.08 (q-like, J ¼ 13.9, CH2(21)); 5.29 (s, CH2(5)); 5.52 (q-like, J ¼ 15.2, CH2(23)); 6.00 (s, HOC(20)); 7.35 – 7.46 (m, HC(14,3’,4’,5’)); 7.64 (d, J ¼ 7.2, HC(12)); 7.68 (d, J ¼ 7.5, HC(6’)); 7.87 (t, J ¼ 7.9, HC(11)); 7.92 (d, J ¼ 8.4, HC(10)); 10.46 (s, NHCO). ESI-MS: 508.66 ([M H] ). 7-Methyl-9-[(3-methylbenzoyl)amino]homocamptothecin ( ¼ N-(5-Ethyl-4,5,13,15-tetrahydro-5-hydroxy-12-methyl-3,15-dioxo-1H,3H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinolin-11-yl)-3-methylbenzamide; 10c). Yield: 29 mg (16%). Yellow solid. M.p. > 3008. 1H-NMR ((D6 )DMSO): 0.86 (t, J ¼ 7.3, Me(18)); 1.86 (q, J ¼ 6.9, CH2(19)); 2.43 (s, MeC(3’)); 2.80 (s, MeC(7)); 3.49 (q-like, J ¼ 13.8, CH2(21)); 5.28 (s, CH2(5)); 5.54 (q-like, J ¼ 15.2, CH2(23)); 6.05 (s, HOC(20)); 7.30 – 7.49 (m, HC(14,4’,5’)); 7.58 (d, J ¼ 7.1, HC(12)); 7.77 – 7.90 (m, HC(11,2’,6’)); 8.16 (d, J ¼ 8.4, HC(10)); 10.57 (s, NHCO). ESI-MS: 508.68 ([M H] ). 7-Methyl-9-[(4-methylbenzoyl)amino]homocamptothecin ( ¼ N-(5-Ethyl-4,5,13,15-tetrahydro-5-hydroxy-12-methyl-3,15-dioxo-1H,3H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinolin-11-yl)-4-methylbenz-
CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
1813
amide; 10d). Yield: 29 mg (16%). Yellow solid. M.p. > 3008. 1H-NMR ((D6 )DMSO): 0.86 (t, J ¼ 7.3, Me(18)); 1.86 (q, J ¼ 7.1, CH2(19)); 2.36 (s, MeC(4’)); 2.79 (s, MeC(7)); 3.49 (q-like, J ¼ 13.8, CH2(21)); 5.28 (s, CH2(5)); 5.54 (q-like, J ¼ 15.1, CH2(23)); 6.06 (s, HOC(20)); 7.38 – 7.40 (m, HC(14,3’,5’)); 7.59 (d, J ¼ 7.3, HC(12)); 7.84 – 7.87 (t, J ¼ 7.9, HC(11)); 7.98 – 8.00 (m, HC(2’,6’)); 8.35 (d, J ¼ 8.4, HC(10)); 10.54 (s, NHCO). ESI-MS: 508.59 ([M H] ). 7-Methyl-9-[(2-phenylacetyl)amino]homocamptothecin ( ¼ N-(5-Ethyl-4,5,13,15-tetrahydro-5-hydroxy-12-methyl-3,15-dioxo-1H,3H-oxepino[ 3’,4’:6,7]indolizino[1,2-b]quinolin-11-yl)-2-phenylacetamide ; 10e) . Yield : 20 mg (11%) . Yellow solid. M.p. > 3008. 1H-NMR ((D6 )DMSO): 0.87 (t, J ¼ 7.4, Me(18)); 1.86 (q, J ¼ 6.4, CH2(19)); 2.71 (s, MeC(7)); 3.38 (q-like, J ¼ 13.9, CH2(21)); 4.17 (s, PhCH2 ); 5.22 (s, CH2(5)); 5.52 (q-like, J ¼ 14.9, CH2(23)); 6.00 (s, HOC(20)); 7.33 – 7.38 (m, HC(14,3’,4’,5’)); 7.44 (d, J ¼ 7.2, HC(12)); 7.50 (dd, J ¼ 2.6, 9.0, HC(11)); 7.73 – 7.79 (m, HC(2’,6’)); 8.04 (d, J ¼ 9.0, HC(10)), 10.34 (s, NHCO). ESI-MS: 499.49 ([M H] ). 9-[(3-Methoxybenzoyl)amino]-7-methylhomocamptothecin ( ¼ N-(5-Ethyl-4,5,13,15-tetrahydro-5hydroxy-12-methyl-3,15-dioxo-1H,3H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinolin-11-yl)-3-methoxybenzamide; 10f). Yield: 72 mg (38%). Yellow solid. M.p. > 3008. 1H-NMR ((D6 )DMSO): 0.87 (t, J ¼ 7.4, Me(18)); 1.86 (q, J ¼ 7.3, CH2(19)); 2.73 (s, MeC(7)); 3.08 (q-like, J ¼ 13.9, CH2(21)); 3.86 (s, MeOC(3’)); 5.29 (s, CH2(5)); 5.52 (q-like, J ¼ 15.2, CH2(23)); 6.00 (s, HOC(20)); 7.35 – 7.46 (m, HC(14,4’,5’,6’)); 7.58 (s, HC(2’)); 7.64 (d, J ¼ 7.2, HC(14)); 7.68 (d, J ¼ 7.5, HC(12)); 7.87 (t, J ¼ 7.9, HC(11)); 8.17 (d, J ¼ 8.4, HC(10)); 10.61 (s, NHCO). ESI-MS: 524.89 ([M H] ). 9-[(2-Chlorobenzoyl)amino]-7-methylhomocamptothecin ( ¼ 2-Chloro-N-(5-ethyl-4,5,13,15-tetrahydro-5-hydroxy-12-methyl-3,15-dioxo-1H,3H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinolin-11-yl)benzamide ; 10g) . Yield : 25 mg (13%) . Yellow solid. M.p. > 3008. 1H-NMR ((D6 )DMSO): 0.87 (t, J ¼ 7.4, Me(18)); 1.87 (q, J ¼ 7.2, CH2(19)); 2.73 (s, MeC(7)); 3.08 (q-like, J ¼ 13.7, CH2(21)); 5.38 (s, CH2(5)); 5.53 (q-like, J ¼ 15.1, CH2(23)); 6.00 (s, HOC(20)); 7.27 – 7.39 (m, HC(12,14)); 7.61 – 7.63 (m, HC(3’,4’,5’)); 7.68 (d, J ¼ 7.5, HC(12)); 7.79 (t, J ¼ 7.9, HC(11)); 8.05 (d, J ¼ 7.2, HC(10)); 8.10 (d, J ¼ 7.4, HC(6’)); 10.46 (s, NHCO). ESI-MS: 528.14 ([M H] ). 9-[(3-Chlorobenzoyl)amino]-7-methylhomocamptothecin ( ¼ 3-Chloro-N-(5-ethyl-4,5,13,15-tetrahydro-5-hydroxy-12-methyl-3,15-dioxo-1H,3H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinolin-11-yl)benzamide ; 10h) . Yield : 80 mg (42%) . Yellow solid. M.p. > 3008. 1H-NMR ((D6 )DMSO): 0.87 (t, J ¼ 7.3, Me(18)); 1.87 (q, J ¼ 7.1, CH2(19)); 2.73 (s, MeC(7)); 3.08 (q-like, J ¼ 13.8, CH2(21)); 5.38 (s, CH2(5)); 5.53 (q-like, J ¼ 15.1, CH2(23)); 6.00 (s, HOC(20)); 7.27 – 7.39 (m, HC(2’,14)); 7.57 – 7.62 (m, HC(4’,5’)); 7.68 (d, J ¼ 7.5, HC(12)); 7.79 (t, J ¼ 7.9, HC(11)); 8.05 (d, J ¼ 7.2, HC(10)); 8.09 (d, J ¼ 7.3, HC(2’,6’)); 10.46 (s, NHCO). ESI-MS: 508.66 ([M H] ). Cytotoxicity. Cells (1,200 per well) were plated in 96-well plates. After culturing for 24 h, test compounds with a wide range of concentrations (from 100 to 0.001 mg/ml; ten dilutions) were added onto duplicate wells with different concentrations, and 0.1% DMSO for control. After 3 d of incubation, 20 ml of MTT ( ¼ 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) soln. (5 mg/ml) was added to each well, and, after shaking for 1 min, the plate was incubated for further 4 h. Formazan crystals were dissolved in 100 ml of DMSO. The absorbance (OD) was recorded with microplate spectrophotometer at 570 nm. Wells containing no drugs were used as blanks for the spectrophotometer. The survival of the cells was expressed as percentage of untreated control wells. Antitumor Activities in vivo. The in vivo antitumor activities of compounds 7c and 10a were evaluated. BALB/C Nude male mice (certificate SCXK2003-0003, weighing 18 – 20 g) were obtained from Shanghai Experimental Animal Center, Chinese Academy of Sciences. A549 Lung cancer-ell suspensions were implanted subcutaneously into the right axilla region of mice. Treatment was started when implanted tumors had reached a volume of ca. 100 – 300 mm3 (after 17 d). The animals were randomized into appropriate groups (six animals/treatment, and nine animals for the control group) and administered by ip injection once a day on day 17 and consecutive 4 d. Tumor volumes (TV) were monitored by caliper measurement of the length and width, and calculated using the formula of TV ¼ 1/ 2 a b2, where a is the tumor length and b is the width. Tumor volumes and body weights were monitored every 4 d over the course of treatment. Mice were sacrificed on day 30 after implantation of cells, and tumors were removed and recorded for analysis.
1814
CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
DNA Topoisomerase I Activity Assays. Camptothecin was obtained from the Company of Tianzunzezhong in China. Topo I (calf thymus), buffer, BSA, loading buffer, and supercoilded DNA pBR322 were all from TaKaRa Biotechnology Co., Ltd. All reactions were carried out in 20-ml volumes (16 ml of double dist. H2O, 2 ml of DNA Topo I buffer, 2 ml of 0.1% BSA), including 0.25 mg of supercoiled DNA and 0.5 U Topo I with or without drug. The mixtures were incubated at 378 for 15 min and then stopped by adding SDS (0.5% final concentration). To the mixtures, 6 3.5 ml of loading buffer (0.1 mm EDTA, 7% glycerol, 0.01% xylene cyanol FF, Bromopenol Blue 0.01%) was added. The mixtures were submitted to electrophoresis in 0.8% agarose gel in TAE ( ¼ Tris-acetate-EDTA) buffer for 40 min at 120 V. The gel was stained with ethidium bromide at r.t. and photographed with UV transilluminator.
REFERENCES [1] M. E. Wall, M. C. Wani, C. E. Cook, K. H. Palmer, A. T. McPhail, G. A. Sim, J. Am. Chem. Soc. 1966, 88, 3888. [2] A. G. Schultz, Chem. Rev. 1973, 73, 385. [3] J. A. Gottlieb, A. M. Guarino, J. B. Call, V. T. Oliverio, J. B. Block, Cancer Chemother. Rep. 1970, 54, 461. [4] P. J. Creaven, L. M. Allen, Cancer Chemother. Rep. 1973, 57, 175. [5] C. G. Moertel, A. J. Schutt, R. J. Reitemeier, R. G. Hahn, Cancer Chemother. Rep. 1972, 56, 649. [6] Y. H. Hsiang, R. Hertzberg, S. Hecht, L. F. Liu, J. Biol. Chem. 1985, 260, 14873. [7] S. Basili, S. Moro, Expert Opin. Ther. Pat. 2009, 19, 555. [8] O. Lavergne, L. Lesueur-Ginot, F. P. Rodas, D. C. H. Bigg, Bioorg. Med. Chem. Lett. 1997, 7, 2235. [9] D. P. Curran, C. R. Chim. 2008, 11, 1574. [10] O. Lavergne, D. Demarquay, C. Bailly, C. Lanco, A. Rolland, M. Huchet, H. Coulomb, N. Muller, N. Baroggi, J. Camara, L. C. Breton, E. Manginot, J. B. Cazaux, D. C. Biqq, J. Med. Chem. 2000, 43, 2285. [11] Y. Pommier, Nat. Rev. Cancer 2006, 6, 789. [12] O. Lavergne, L. Lesueur-Ginot, F. P. Rodas, P. G. Kasprzyk, J. Pommier, D. Demarquay, G. Prevost, G. Ulibarri, A. Rolland, A. M. Schiano-Liberatore, J. Harnett, D. Pons, J. Camara, D. C. H. Bigg, J. Med. Chem. 1998, 41, 5410. [13] W. Guo, Z. Y. Miao, C. Q. Sheng, J. Z. Yao, H. Feng, W. N. Zhang, L. J. Zhu, W. F. Liu, P. F. Cheng, J. Zhang, X. Y. Che, W. Y. Wang, C. Luo, Y. L. Xu, Eur. J. Med. Chem. 2010, 45, 2223. [14] L. J. Zhu, Z. Y. Miao, C. Q. Sheng, W. Guo, J. Z. Yao, W. F. Liu, X. Y. Che, W. Y. Wang, P. F. Cheng, W. N. Zhang, Eur. J. Med. Chem. 2010, 45, 2726. [15] W. F. Liu, L. J. Zhu, W. Guo, C. L. Zhuang, Y. Q. Zhang, C. Q. Sheng, P. F. Cheng, J. Z. Yao, W. Y. Wang, G. Q. Dong, S. Z. Wang, Z. Y. Miao, W. N. Zhang, Eur. J. Med. Chem. 2011, 46, 2408. [16] Z. Y. Miao, J. Zhang, L. You, J. Wang, C. Q. Sheng, J. Z. Yao, W. N. Zhang, H. Feng, W. Guo, L. Zhou, W. F. Liu, L. J. Zhu, P. F. Cheng, X. Y. Che, W. Y. Wang, C. Luo, Y. L. Xu, G. Q. Dong, Bioorg. Med. Chem. 2010, 18, 3140. [17] Z. Y. Miao, C. Q. Sheng, W. N. Zhang, H. T. Ji, J. Zhang, L. C. Shao, L. You, M. Zhang, J. Z. Yao, X. Y. Che, Bioorg. Med. Chem. 2008, 16, 1493. [18] W. Guo, Z. Y. Miao, C. Q. Sheng, J. Z. Yao, W. F. Liu, L. J. Zhu, Y. Q. Zhang, P. F. Cheng, G. Q. Dong, C. L. Zhuang, W. N. Zhang, W. Y. Wang, Chem. Biodiversity 2011, 8, 1266. [19] W. Guo, W. F. Liu, L. J. Zhu, Y. Q. Zhang, P. F. Cheng, G. Q. Dong, C. L. Zhuang, W. Y. Wang, J. Z. Yao, C. Q. Sheng, Z. Y. Miao, W. N. Zhang. Chem. Biodiversity 2011, 8, 1539. [20] L. J. Zhu, X. H. Zhang, N. Lei, W. F. Liu, Z. Y. Miao, C. L. Zhuang, C. Q. Sheng, W. Guo, G. Q. Dong, J. Z. Yao, P. F. Cheng, W. N. Zhang. Chem. Biodiversity 2012, 9, 1084. [21] L. J. Dolby, D. L. Booth, J. Am. Chem. Soc. 1966, 88, 1049. [22] M. Mentel, R. Breinbauer, Curr. Org. Chem. 2007, 11, 159. [23] E. J. Alford, K. Schofield, J. Chem. Soc. 1953, 609. [24] K. Schofield, R. S. Theobald, J. Chem. Soc. 1949, 796.
CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
1815
[25] D. W. Ockenden, K. Schofield, J. Chem. Soc. 1953, 612. [26] W. C. Drewe, R. Nanjunda, M. Gunaratnam, M. Beltran, G. N. Parkinson, A. P. Reszka, W. D. Wilson, S. Neidle, J. Med. Chem. 2008, 51, 7751. [27] S. de Castro, R. Chicharro, V. J. Ara´n, J. Chem. Soc., Perkin Trans. 1 2002, 790. [28] Y. L. Leu, S. R. Roffler, J. W. Chern, J. Med. Chem. 1999, 42, 3623. [29] K. J. Yoon, J. J. Qi, J. S. Remack, K. G. Virga, M. J. Hatfield, P. M. Potter, R. E. Lee, M. K. Danks, Mol. Cancer Ther. 2006, 5, 1577. Received September 6, 2012
CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
Synthesis and Biological Assays of 9-(Acylamino)homocamptothecins as DNA Topoisomerase I Inhibitors by Wei Guo a ), Guoqiang Dong a ), Lingjian Zhu a ), Wenfeng Liu a ), Chunlin Zhuang a ), Zizhao Guo a ), Jianzhong Yao a ), Chunquan Sheng a ), Huojun Zhang* b ), Zhenyuan Miao* a ), and Wannian Zhang* a ) a
) School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, P. R. China (e-mail: [email protected], [email protected]) b ) Department of Radiation Oncology, Changhai Hospital, Second Military Medical University, 168 Changhai Road, Shanghai 200433, P. R. China (e-mail: [email protected])
In an effort to improve the stability of homocamptothecin and reduce the toxicity, novel homocamptothecin analogs with acylamino groups at C(9) were designed and synthesized. The cytotoxic activities of all the synthetic compounds against three cancer cell lines were evaluated by the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, and irinotecan was used as reference compound. Compound 7c with a piperidinylacetamido group and 10a with phenylacetamido group at C(9) showed potent activities both in vitro and in vivo. In addition, they also revealed remarkable topoisomerase I inhibitions which were exhibited with well-established bonds with amino acid residues Arg364 and Asp533 in the active pocket. On the basis of the biological activities, 7c and 10a would be potential candidates for further studies.
Introduction. – Camptothecin (CPT; Fig. 1), which was isolated from the Chinese plant Camptotheca acuminate by Wall et al. in 1966, exhibited potent antitumor activity [1]. However, with the development of camptothecin, serious problems appeared, such as its insolubility and toxicity [1 – 5]. The formal studies showed that camptothecin had a unique mechanism of action, i.e., selective inhibition of DNA topoisomerase I (Topo I) [6]. Currently, three important drugs, topotecan, irinotecan, and belotecan, are used in clinical treatment. Topotecan is used as a second-line agent for advanced ovarian cancer or small cell lung cancer, and irinotecan is used against refractory advanced colorectal cancer. Several other camptothecin analogs are in clinical trials [7]. Another camptothecin analog, homocamptothecin (hCPT), was discovered by Lavergne et al. in late 1990s and showed potent antiproliferative activity in cancer cell lines [8]. The hCPT is structurally similar to camptothecin, differing only by changing a six-membered a-hydroxy lactone to a seven-membered b-hydroxy lactone [9]. hCPT can also cleave DNA in the presence of Topo I despite a different sequence selectivity than CPT. The introduction of a CH2 group in ring E of CPT not only led to enhanced lactone stability but also decreased protein binding in human plasma without affecting its capability to poison topoisomerase I-DNA. Two hCPT drugs, diflomotecan (BN80915) and BN80927, were evaluated in Phase II and I trials, respectively [10 – 12]. We have already reported several new classes of 9-substituted hCPTs, such as 9benzylidenamino, 9-(heteroarylmethylidene)amino derivatives of homocampthecins 2013 Verlag Helvetica Chimica Acta AG, Zrich
CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
1805
Fig. 1. Structures of camptothecin analogs
[13 – 20]. These compounds not only have obvious cytotoxic activities in vitro towards several human cancer cell lines but also help to further define the structureactivity relationship of hCPT as Topo I inhibitors. In the present work, we designed and synthesized a series of novel 9-acylhomocaptothecins and investigated whether these modifications would improve the biological properties of hCPT. Results and Discussion. – Chemistry. The preparation of derivatives 7a – 7h was carried out as outlined in Scheme 1 1). Hydrolysis of 1 [13] in refluxing aqueous HCl gave the amino compound 2 [21 – 25]. The intermediate 3 was achieved by acylation of 2 with ClCH2COCl in nearly quantitative yield [26 – 29], which was then reacted with the appropriate secondary amines in the presence of K2CO3 and gave the desired analogs 4b – 4h, catalytic hydrogenation of which afforded the amines 5a – 5h, respectively, in satisfactory yields. The key intermediate 6 was synthesized according to our previously reported procedure, and the process was optimized [13 – 20]. Finally, 7a and the aminoacyl amide hCPTs 7b – 7h were obtained by Friedlnder condensation of 5a – 5h and 6 with Me3SiCl (TMSCl) in moderate yields. Another series of derivatives, 10a – 10h, were prepared by an analogous synthetic approach as depicted in Scheme 2. The amides were obtained from 2 and differently substituted benzoyl chlorides with the appropriate tertiary amine as catalyst. After hydrogenation of the NO2 group, the amino compounds 9a – 9h were obtained, and the 1)
Arbitrary atom numbering; for systematic names, see Exper. Part.
1806
CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
Scheme 1
a) 6n HCl (aq.), 608, 4 h. b) 4-(Dimethylamino)pyridine (DMAP), Et3N, THF, ClCH2COCl, 08. c) K2CO3 , THF, secondary amine, 608, 2 h. d) H2 , 10% Pd/C, EtOH, r.t., overnight. e) Me3SiCl (TMSCl), DMF, 1008, 2 h.
final benzamide derivatives of homocaptothecins 10a – 10h were formed by the condensation reactions of 9a – 9h, respectively, with 6. Cytotoxicity and StructureActivity Relationships. The cytotoxic activities of the synthesized compounds 7a – 7h and 10a – 10h were evaluated against human lung cancer (A549), breast cancer (MDA-MB-435), and colon cancer (HCT-116) cell lines using 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) method. The IC50 values of the samples, compiled in the Table, revealed that most of the tested compounds were active in inhibiting cancer cell lines. For 9-heterocyclic-amidosubstituted hCPT, most of the compounds were sensitive to the breast cancer cell lines, and less active against the lung and colon cancer cell lines. For cell lines MDA-MB-435, eight compounds were more active than the standard drug irinotecan. Among them, compound 7c was the most efficient one with an IC50 value of 0.558 mm, which was 30 times higher than that of irinotecan. For benzamido derivatives, most of the compounds were similarly active as heterocyclic amido-hCPTs, and showed activities against breast cancer cell lines. Six compounds, 10a, 10c, 10d, 10e, 10f, and 10h, were more active than irinotecan. Among these, the activity of 10d, with an IC50 value of 1.76 mm, was ten times higher than that of irinotecan.
CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
1807
Scheme 2
a) DMAP, Et3N, THF, 08. b) H2 , 10% Pd/C, EtOH, r.t., overnight. c) TMSCl, DMF, 1008, 2 h.
Table. Structures and Growth-Inhibitory Activities ( IC50 ) of Target Compounds Compound
7a 7b 7c 7d 7e 7f 7g 7h 10a 10b 10c 10d 10e 10f 10g 10h Irinotecan a
R
Cl Et2N 2-Methylpiperidin-1-yl 3-Methylpiperidin-1-yl 4-Methylpiperidin-1-yl 3,5-Dimethylpiperidin-1-yl (2-Methylpiperidin-1-yl)methyl (4-Methylpiperidin-1-yl)methyl Ph 2-MeC6H4 3-MeC6H4 4-MeC6H4 Bn 3-MeOC6H4 2-ClC6H4 3-ClC6H4 –
IC50 a ) [mm] A549
MDA-MB-435
HCT-116
62.9 26.3 49.6 86.9 96.9 151 > 200 31.9 29.6 133 42.6 > 200 114 > 200 > 200 > 200 48.5
8.32 1.31 0.558 2.34 2.75 7.15 89.2 3.82 7.70 30.1 6.44 1.76 3.60 8.07 23.9 4.40 16.9
33.9 > 200 36.6 109 143 > 200 20.2 29.8 47.4 > 200 124 146 143 158 182 > 200 19.5
) Values were determined by using the MTT method, and the experiments were repeated twice.
Structureactivity relationship study revealed that substitution with a single Me group resulted in a substantial increase in the cytotoxic activity compared to multi Me substitution. For instance, compounds 7c – 7e with a single Me group were more active
1808
CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
than dimethyl-substituted compound 7f. Second, the results suggested that length of the alkyl side chains affected the cytotoxic potencies. The activity decreased with increasing side-chain length. For example, the acetamido compound 7c was more active than the propanamido compound 7g. Antitumor Activities in vivo. Compounds 7c and 10a were chosen for evaluation for 2-week tumor-growth inhibition in vivo assays using nude mice implanted with Lewis lung xenografts (Fig. 2). Treatment with compounds 7c and 10a at 10 mg/kg body weight, tumor growths were suppressed by 32.0 and 45.7%, respectively, as compared with placebo-treated controls. Furthermore, the body weights of all the mice treated with compounds 7c and 10a were increased during the treatment period. Accordingly, compounds 7c and 10a may exert lower toxicities.
Fig. 2. Tumor-growth inhibition by compounds 7c and 10a against A549 xenografts in nude mice administered i.p.
Topoisomerase I-Mediated DNA Cleavage. Compounds 7c and 10a were further evaluated for their possible activities in the inhibition of Topo I (Fig. 3). These compounds showed stable ternary complex activities compared to that of the reference compound camptothecin at the concentrations of 50 and 100 mm. Compound 7c were inactive at concentrations below 10 mm, while 10a exhibited modest activity.
Fig. 3. Effects of compounds 7c and 10a on Topoisomerase I-mediated DNA relaxation. Lane 1, supercoiled DNA pBR322; Lane 2, supercoiled DNA and Topo I; Lanes 3 – 5, 6 – 8, 9 – 11, three samples CPT, 7c, and 10a at 10, 50, and 100 mm, respectively.
CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
1809
Molecular Docking. Considering the in vitro, in vivo, and topoisomerase I-mediated DNA cleavage results, it was worthy to screen for supportive coordination between these results. Topoisomerase I was used as the target enzyme, and automated molecular docking was studied with newly synthesized compound 7c. 9-Amino-hCPT was the reference compounds to determine the binding mode of the compound and enzyme. Using the docking program AutoDock 4.0 furnished the results shown in Figs. 4 and 5.
Fig. 4. Predicted binding mode of 9-aminohomocamptothecin to topoisomerase I active site. The amino acids Arg364 and Asp533, involved in the interactions with the compound, are highlighted.
Fig. 5. Predicted binding mode of compound 7c to topoisomerase I active site. The amino acids Lys425, Arg364, and Asp533, involved in the interactions with compound, are highlighted.
1810
CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
The orientation in the active site cavity was perpendicular to the main axis of the DNA and parallel to the bases. Topo I and hCPTs can interact at the active site. These two homocamptothecin derivatives exhibited well-established bonds with amino acids in the active pocket. At the positions 1 and 20, both of the two compounds had the active pocket consisting of two amino acid residues Arg364 and Asp533. The molecule 7c had also one more site with O-atom which formed another H-bond with amino residue Lys425 as shown in Fig. 5. This study revealed that the molecules 7c showed better binding results towards the target enzyme Topo I than the 9-amino-hCPT. Finally, considering the cytotoxic activites in vitro, in vivo, and molecular-docking results among our synthesized compounds, 7c provided the best results, and it was considered as the best inhibitor of Topo I in this series compounds and could be effective with human cancer cell lines. Conclusions. – Two series of homocamptothecin derivatives with a heterocyclic amido or a benzamido group at C(9) were synthesized and characterized. These compounds were evaluated for in vitro activities against human lung cancer (A549), breast cancer (MDA-MB-435), and colon cancer (HCT-116) cell lines. On basis of the biological activities, the 9-(acylamino)homocaptothecins were found to be potent homocamptothecin derivatives. Among these, 7c with a piperidinylacetamido group and 10a with benzamido group were found to be the most active agents against the breast cell lines MDA-MB-435. They also showed obvious activities in vivo and remarkable Topo I inhibitory activities. Therefore, 9-(acylamino)homocaptothecins could be potential topoisomerase I inhibitors with homocamptothecin skeleton for anticancer agents. This work was supported by the National Natural Science Foundation of China (No. 81171435) and the Shanghai Leading Academic Discipline Project (No. B906).
Experimental Part General. Commercial solvents were used without any pretreatment. TLC: Silica gel plates GF254 (SiO2 ; Qingdao Haiyang Chemical, China). Column chromatography (CC): SiO2 60 G (Qingdao Haiyang Chemical, China). NMR Spectra: Bruker 500 spectrometer in CDCl3 or (D6 )DMSO solns.; d in ppm rel. to Me4Si as internal standard, J in Hz. ESI-MS: API-3000 LC/MS spectrometer; in m/z. Elemental analyses: MOD-1106 instrument; consistent with theoretical values within 0.4%. General Procedure for the Preparation of Compounds 7a – 7h. 9-[(2-Chloroacetyl)amino]-7methylhomocamptothecin ( ¼ 2-Chloro-N-(5-ethyl-4,5,13,15-tetrahydro-5-hydroxy-12-methyl-3,15-dioxo1H,3H-oxepino[ 3’,4’:6,7]indolizino[1,2-b]quinolin-11-yl)acetamide ; 7a) . Compound 2 (600 mg, 3.3 mmol) was dissolved in THF (20 ml) and cooled to 08 prior to the addition of Et3N (0.05 ml, 0.36 mmol) and DMAP ( ¼ 4-(dimethylamino)pyridine; 10 mg, 0.08 mmol). ClCH2COCl (0.27 ml, 3.6 mmol) was added, and the soln. was kept for 4 h at r.t. The mixture was diluted with H2O, extracted with AcOEt, and dried (MgSO4 ). The crude residue was purified by CC (hexane/AcOEt 4 : 1) to give 2[(2-chloroacetyl)amino]-6-nitroacetophenone (3; 800 mg, 94%). Yellow solid. M.p. 135 – 1378. 1H-NMR (CDCl3 ): 2.48 (s, MeCO); 2.85 (s, CH2Cl); 7.63 (t, J ¼ 8.1, HC(4)); 8.00 (d, J ¼ 8.5, HC(5)), 8.53 (d, J ¼ 8.1, HC(3)). ESI-MS: 227.29 ([M H] ). Compound 3 (100 mg, 0.39 mmol) was dissolved in EtOH (20 ml), and 10% Pd/C (100 mg) was added. After stirring overnight at r.t., the mixture was filtered through Celite. The solvent was removed, and the residue was crystallized from hexane to give 2-amino-6-[(2-chloroacetyl)amino]acetophenone (5a; 84 mg, 95%). Yellow solid. 1H-NMR (CDCl3 ): 2.29 (s, MeCO); 2.80 (t, CH2Cl); 6.06 (s, NH2 );
CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
1811
6.51 (d, J ¼ 7.6, HC(5)); 6.58 (t, J ¼ 8.1, HC(3)); 7.10 (d, J ¼ 8.0, HC(4)). ESI-MS: 255.63 ([M H] ). A mixture of 6 (100 mg, 0.36 mmol) and 5a (91 mg, 0.40 mmol) was dissolved in DMF. After dropwise addition of TMSCl (0.1 ml) at r.t., the mixture was heated to reflux under N2 for 2 h at 1008. The solvent was removed, and the crude product was prufied by CC (CH2Cl2/MeOH 100 : 2 – 100 : 5) to give 7a (25 mg, 15%). Yellow solid. M.p. > 3008. 1H-NMR ((D6 )DMSO): 0.87 (t, J ¼ 7.8, Me(18)); 1.86 (q, J ¼ 7.4, CH2(19)); 2.85 (s, MeC(7)); 3.46 (q-like, J ¼ 13.8, CH2(21)); 4.41 (s, CH2Cl); 5.29 (s, CH2(5)); 5.41 (qlike, J ¼ 15.2, CH2(23)); 6.02 (s, HOC(20)); 7.39 (s, HC(14)); 7.51 (d, J ¼ 7.4, HC(12)); 8.12 (t, J ¼ 8.0, HC(11)); 7.38 (d, J ¼ 8.4, HC(10)); 10.41 (s, NHCO). ESI-MS: 466.55 ([M H] ). 9-{[2-(Diethylamino)acetyl]amino}-7-methylhomocamptothecin ( ¼ 2-(Dimethylamino)-N-(5-ethyl4,5,13,15-tetrahydro-5-hydroxy-12-methyl-3,15-dioxo-1H,3H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinolin-11-yl)acetamide; 7b). Compound 3 (100 mg, 0.4 mmol) and Et2NH (0.12 ml, 1.2 mmol) were dissolved in THF, and K2CO3 (100 mg, 0.72 mmol) was added. The mixture was stirred under N2 for 2 h at 608, diluted with H2O, extracted with AcOEt, and then dried (MgSO4 ). The solvent was evaporated under reduced pressure, and 4b (100 mg, 90%) was obtained as a yellow solid. Then, it was dissolved in EtOH (20 ml), and 10% Pd/C (0.1 g) was added. After stirring overnight at r.t., the mixture was filtered through Celite. The solvent was removed, and the residue was crystallized from hexane to give 2-amino-6{[2-(diethylamino)acetyl]amino}acetophenone (5b; 87 mg, 96%), which was dissolved in DMF (20 ml), and 7 (100 mg, 0.18 mmol) was added. After dropwise adding of TMSCl (0.1 ml) at r.t., the mixture was heated to reflux under N2 for 2 h at 1008. The solvent was evaporated, and the residue was purified by CC (CH2Cl2/MeOH 100 : 2 – 100 : 5) to give 7b (130 mg, 71%). Yellow solid. M.p. > 3008. 1H-NMR ((D6 )DMSO): 0.87 (t, J ¼ 7.4, Me(18)); 1.29 – 1.31 (m, 2 MeCH2N); 1.86 (q, J ¼ 7.6, CH2(19)); 2.49 – 2.53 (m, 2 MeCH2N); 2.90 (s, MeC(7)); 3.49 (q-like, J ¼ 13.8, CH2(21)); 4.32 (s, COCH2N); 5.26 (s, CH2(5)); 5.55 (q-like, J ¼ 15.1, CH2(23)); 6.08 (s, HOC(20)); 7.39 (s, HC(14)); 7.59 (d, J ¼ 7.3, HC(12)); 7.86 (t, J ¼ 7.9, HC(11)); 8.15 (d, J ¼ 8.4, HC(10)); 9.85 (s, NHCO). ESI-MS: 503.88 ([M H] ). 7-Methyl-9-{[2-(2-methylpiperidin-1-yl)acetyl]amino}homocamptothecin ( ¼ N-(5-Ethyl-4,5,13,15tetrahydro-5-hydroxy-12-methyl-3,15-dioxo-1H,3H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinolin-11-yl)-2(2-methylpiperidin-1-yl)acetamide; 7c). Yield: 80 mg (42%). Yellow solid. M.p. > 3008. 1H-NMR ((D6 )DMSO): 0.87 (t, J ¼ 7.4, Me(18)); 1.05 (d, J ¼ 7.0, MeC(2’)); 1.06 – 1.17 (m, CH2(4’)); 1.60 – 1.64 (m, CH2(3’,5’)); 1.86 (q, J ¼ 7.4, CH2(19)); 2.92 (s, MeC(7)); 3.04 – 3.07 (m, HC(2’), CH2(6’)); 3.33 (q-like, J ¼ 14.0, CH2(21)); 3.46 (s, COCH2N); 5.29 (s, CH2(5)); 5.52 (q-like, J ¼ 15.1, CH2(23)); 6.03 (s, HOC(20)); 7.38 (s, HC(14)); 7.65 (d, J ¼ 7.2, HC(12)); 7.81 (t, J ¼ 7.9, HC(11)); 8.05 (d, J ¼ 8.2, HC(10)); 10.05 (s, NHCO). ESI-MS: 529.76 ([M H] ). 7-Methyl-9-{[2-(3-methylpiperidin-1-yl)acetyl]amino}homocamptothecin ( ¼ N-(5-Ethyl-4,5,13,15tetrahydro-5-hydroxy-12-methyl-3,15-dioxo-1H,3H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinolin-11-yl)-2(3-methylpiperidin-1-yl)acetamide; 7d). Yield: 90 mg (47%). Yellow solid. M.p. > 3008. 1H-NMR ((D6 )DMSO): 0.87 (t, J ¼ 7.5, Me(18)); 1.05 (t, J ¼ 7.0, MeC(3’)); 1.18 – 1.21 (m, CH2(4’,5’)); 2.91 (s, MeC(7)); 3.03 – 3.09 (m, CH2(2’,6’)); 3.36 – 3.46 (m, 4 H, COCH2N, CH2(21)); 5.29 (s, CH2(5)); 5.52 (qlike, J ¼ 15.1, CH2(23)); 6.08 (s, HOC(20)); 7.39 (s, HC(14)); 7.62 (d, J ¼ 7.3, HC(12)); 7.81 (t, J ¼ 7.9, HC(11)); 8.07 (d, J ¼ 8.5, HC(10)); 8.09 (s, NHCO). ESI-MS: 529.88 ([M H] ). 7-Methyl-9-{[2-(4-methylpiperidin-1-yl)acetyl]amino}homocamptothecin ( ¼ N-(5-Ethyl-4,5,13,15tetrahydro-5-hydroxy-12-methyl-3,15-dioxo-1H,3H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinolin-11-yl)-2(4-methylpiperidin-1-yl)acetamide; 7e). Yield: 70 mg (36%). Yellow solid. M.p. > 3008. 1H-NMR ((D6 )DMSO): 0.87 (t, J ¼ 7.5, Me(18)); 0.92 – 0.93 (m, MeC(4’)); 1.22 – 1.23 (m, HC(4’)); 1.24 – 1.34 (m, CH2(3’,5’)); 1.86 (q, J ¼ 7.7, CH2(19)); 2.90 (s, MeC(7)); 3.04 – 3.07 (m, CH2(2’,6’)); 3.32 (s, COCH2N); 3.48 (q-like, J ¼ 13.7, CH2(21)); 5.27 (s, CH2(5)); 5.52 (q-like, J ¼ 15.2, CH2(23)); 6.04 (s, HOC(20)); 7.38 (s, HC(14)); 7.63 (d, J ¼ 5.6, HC(12)); 7.81 (t, J ¼ 8.2, HC(11)); 8.07 (d, J ¼ 8.4, HC(10)); 9.01 (s, NHCO). ESI-MS: 531.77 ([M H] ). 7-Methyl-9-{[2-(3,5-dimethylpiperidin-1-yl)acetyl]amino}homocamptothecin ( ¼ 2-(3,5-Dimethylpiperidin-1-yl)-N-(5-ethyl-4,5,13,15-tetrahydro-5-hydroxy-12-methyl-3,15-dioxo-1H,3H-oxepino[3’,4’: 6,7]indolizino[1,2-b]quinolin-11-yl)acetamide; 7f). Yield: 60 mg (31%). Yellow solid. M.p. > 3008. 1 H-NMR ((D6 )DMSO): 0.85 – 0.91 (m, Me(18), MeC(3’,5’)); 1.73 (m, CH2(4’)); 1.86 (q, J ¼ 7.5,
1812
CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
CH2(19)); 2.89 (s, MeC(7)); 2.90 – 2.92 (m, CH2(2’,6’)); 3.04 – 3.49 (m, COCH2N, CH2(21)); 5.29 (s, CH2(5)); 5.52 (q-like, J ¼ 15.1, CH2(23)); 6.04 (s, HOC(20)); 7.38 (s, HC(14)); 7.64 (d, J ¼ 7.4, HC(12)); 7.79 (t, J ¼ 7.9, HC(11)); 8.05 (d, J ¼ 8.4, HC(10)); 10.03 (s, NHCO). ESI-MS: 543.28 ([M H] ). 7-Methyl-9-{[ 3-(2-methylpiperdin-1-yl)propanoyl]amino}homocamptothecin ( ¼ N-(5-Ethyl4,5,13,15-tetrahydro-5-hydroxy-12-methyl-3,15-dioxo-1H,3H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinolin-11-yl)-3-(2-methylpiperidin-1-yl)propanamide; 7g). Yield: 150 mg (76%). Yellow solid. M.p. > 3008. 1H-NMR ((D6 )DMSO): 0.80 – 0.87 (m, Me(18), MeC(2’)); 1.86 (q, J ¼ 6.4, CH2(19)); 1.88 – 1.89 (m, CH2(4’,5’)); 1.91 – 1.92 (m, CH2(3’)); 2.50 (s, MeC(7)), 2.86 – 2.87 (m, NHCOCH2 ); 3.01 – 3.02 (m, HC(2’)); 3.38 – 3.40 (m, CH2(21,6’)); 4.11 – 4.12 (m, CH2N); 5.28 (s, CH2(5)); 5.52 (q-like, J ¼ 14.9, CH2(23)); 6.06 (s, HOC(20)); 7.38 (s, HC(14)); 7.46 (d, J ¼ 7.3, HC(12)); 7.80 (t, J ¼ 7.8, HC(11)); 8.09 (d, J ¼ 8.4, HC(10)); 10.10 (s, NHCO). ESI-MS: 543.27 ([M H] ). 7-Methyl-9-{[ 3-(4-methylpiperdin-1-yl)propanoyl]amino}homocamptothecin ( ¼ N-(5-Ethyl4,5,13,15-tetrahydro-5-hydroxy-12-methyl-3,15-dioxo-1H,3H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinolin-11-yl)-3-(4-methylpiperidin-1-yl)propanamide; 7h). Yield: 20 mg (10%). Yellow solid. M.p. > 3008. 1 H-NMR (D6(DMSO)): 0.80 – 0.87 (m, 6 H, Me(18), MeC(4’)); 1.86 (q, J ¼ 6.4, CH2(19)); 1.88 – 1.89 (m, CH2(2’,3’,5’,6’)); 2.50 (s, MeC(7)); 2.86 – 2.87 (m, NHCOCH2 ); 3.38 – 3.39 (m, CH2(21)); 4.11 – 4.13 (m, CH2N); 5.28 (s, CH2(5)); 5.52 (q-like, J ¼ 14.9, CH2(23)); 6.06 (s, HOC(20)); 7.38 (s, HC(14)); 7.46 (d, J ¼ 7.3, HC(12)); 7.80 (t, J ¼ 7.8, HC(11)); 8.09 (d, J ¼ 8.4, HC(10)); 10.10 (s, NHCO). ESI-MS: 543.21 ([M H] ). General Procedure for the Preparation of Compounds 10a – 10h. 9-(Benzoylamino)-7-methylhomocamptothecin ( ¼ N-(5-Ethyl-4,5,13,15-tetrahydro-5-hydroxy-12-methyl-3,15-dioxo-1H,3H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinolin-11-yl)benzamide; 10a). Compound 2 (100 mg, 0.6 mmol) was dissolved in THF (20 ml) and cooled to 08 prior to the addition of Et3N (0.05 ml, 0.36 mmol) and DMAP (10 mg, 0.08 mmol). PhCOCl (0.07 ml, 0.6 mmol) was added and reacted at r.t. for 4 h. The mixture was diluted with H2O, extraed with AcOEt, and dried (MgSO4 ). Crude residue was purified by CC (SiO2 ; hexane/AcOEt 4 : 1) to give 2-(benzoylamino)-6-nitroacetophenone (8a; 60 mg, 38%) as a yellow solid, which was dissolved in EtOH (20 ml), and 10% Pd/C (0.1 g) was added. After stirring overnight at r.t., the mixture was filtered through Celite. The solvent was evaporated, and the residue was crystallized from hexane to give a yellow solid 9a (51 mg, 96%). A mixture of 6 (100 mg, 0.4 mmol) and 9a (0.05 ml, 0.4 mmol) were dissolved in DMF. After dropwise addition of TMSCl (0.1 ml) at r.t., the mixture was refluxed under N2 for 2 h at 1008. The solvent was removed, and the crude product was purified by CC (SiO2 ; CH2Cl2/MeOH 100 : 2 – 100 : 5) to give 10a (110 mg, 62%). Yellow solid. M.p. > 3008. 1H-NMR ((D6 )DMSO): 0.87 (t, J ¼ 7.3, Me(18)); 1.86 (q, J ¼ 7.3, CH2(19)); 2.81 (s, MeC(7)); 3.46 (q-like, J ¼ 13.8, CH2(21)); 5.27 (s, CH2(5)); 5.51 (q-like, J ¼ 15.2, CH2(23)); 6.05 (s, HOC(20)); 7.04 (s, HC(14)); 7.58 – 7.68 (m, HC(12,3’,4’,5’)); 7.87 (t, J ¼ 7.9, HC(11)); 8.08 – 8.09 (m, HC(2’,6’)); 10.62 (s, NHCO). ESIMS: 494.79 ([M H] ). 7-Methyl-9-[(2-methylbenzoyl)amino]homocamptothecin ( ¼ N-(5-Ethyl-4,5,13,15-tetrahydro-5-hydroxy-12-methyl-3,15-dioxo-1H,3H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinolin-11-yl)-2-methylbenzamide; 10b). Yield: 29 mg (16%). Yellow solid. M.p. > 3008. 1H-NMR ((D6 )DMSO): 0.87 (t, J ¼ 7.4, Me(18)); 1.86 (q, J ¼ 7.3, CH2(19)); 2.51 (s, MeC(2’)); 2.73 (s, MeC(7)); 3.08 (q-like, J ¼ 13.9, CH2(21)); 5.29 (s, CH2(5)); 5.52 (q-like, J ¼ 15.2, CH2(23)); 6.00 (s, HOC(20)); 7.35 – 7.46 (m, HC(14,3’,4’,5’)); 7.64 (d, J ¼ 7.2, HC(12)); 7.68 (d, J ¼ 7.5, HC(6’)); 7.87 (t, J ¼ 7.9, HC(11)); 7.92 (d, J ¼ 8.4, HC(10)); 10.46 (s, NHCO). ESI-MS: 508.66 ([M H] ). 7-Methyl-9-[(3-methylbenzoyl)amino]homocamptothecin ( ¼ N-(5-Ethyl-4,5,13,15-tetrahydro-5-hydroxy-12-methyl-3,15-dioxo-1H,3H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinolin-11-yl)-3-methylbenzamide; 10c). Yield: 29 mg (16%). Yellow solid. M.p. > 3008. 1H-NMR ((D6 )DMSO): 0.86 (t, J ¼ 7.3, Me(18)); 1.86 (q, J ¼ 6.9, CH2(19)); 2.43 (s, MeC(3’)); 2.80 (s, MeC(7)); 3.49 (q-like, J ¼ 13.8, CH2(21)); 5.28 (s, CH2(5)); 5.54 (q-like, J ¼ 15.2, CH2(23)); 6.05 (s, HOC(20)); 7.30 – 7.49 (m, HC(14,4’,5’)); 7.58 (d, J ¼ 7.1, HC(12)); 7.77 – 7.90 (m, HC(11,2’,6’)); 8.16 (d, J ¼ 8.4, HC(10)); 10.57 (s, NHCO). ESI-MS: 508.68 ([M H] ). 7-Methyl-9-[(4-methylbenzoyl)amino]homocamptothecin ( ¼ N-(5-Ethyl-4,5,13,15-tetrahydro-5-hydroxy-12-methyl-3,15-dioxo-1H,3H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinolin-11-yl)-4-methylbenz-
CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
1813
amide; 10d). Yield: 29 mg (16%). Yellow solid. M.p. > 3008. 1H-NMR ((D6 )DMSO): 0.86 (t, J ¼ 7.3, Me(18)); 1.86 (q, J ¼ 7.1, CH2(19)); 2.36 (s, MeC(4’)); 2.79 (s, MeC(7)); 3.49 (q-like, J ¼ 13.8, CH2(21)); 5.28 (s, CH2(5)); 5.54 (q-like, J ¼ 15.1, CH2(23)); 6.06 (s, HOC(20)); 7.38 – 7.40 (m, HC(14,3’,5’)); 7.59 (d, J ¼ 7.3, HC(12)); 7.84 – 7.87 (t, J ¼ 7.9, HC(11)); 7.98 – 8.00 (m, HC(2’,6’)); 8.35 (d, J ¼ 8.4, HC(10)); 10.54 (s, NHCO). ESI-MS: 508.59 ([M H] ). 7-Methyl-9-[(2-phenylacetyl)amino]homocamptothecin ( ¼ N-(5-Ethyl-4,5,13,15-tetrahydro-5-hydroxy-12-methyl-3,15-dioxo-1H,3H-oxepino[ 3’,4’:6,7]indolizino[1,2-b]quinolin-11-yl)-2-phenylacetamide ; 10e) . Yield : 20 mg (11%) . Yellow solid. M.p. > 3008. 1H-NMR ((D6 )DMSO): 0.87 (t, J ¼ 7.4, Me(18)); 1.86 (q, J ¼ 6.4, CH2(19)); 2.71 (s, MeC(7)); 3.38 (q-like, J ¼ 13.9, CH2(21)); 4.17 (s, PhCH2 ); 5.22 (s, CH2(5)); 5.52 (q-like, J ¼ 14.9, CH2(23)); 6.00 (s, HOC(20)); 7.33 – 7.38 (m, HC(14,3’,4’,5’)); 7.44 (d, J ¼ 7.2, HC(12)); 7.50 (dd, J ¼ 2.6, 9.0, HC(11)); 7.73 – 7.79 (m, HC(2’,6’)); 8.04 (d, J ¼ 9.0, HC(10)), 10.34 (s, NHCO). ESI-MS: 499.49 ([M H] ). 9-[(3-Methoxybenzoyl)amino]-7-methylhomocamptothecin ( ¼ N-(5-Ethyl-4,5,13,15-tetrahydro-5hydroxy-12-methyl-3,15-dioxo-1H,3H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinolin-11-yl)-3-methoxybenzamide; 10f). Yield: 72 mg (38%). Yellow solid. M.p. > 3008. 1H-NMR ((D6 )DMSO): 0.87 (t, J ¼ 7.4, Me(18)); 1.86 (q, J ¼ 7.3, CH2(19)); 2.73 (s, MeC(7)); 3.08 (q-like, J ¼ 13.9, CH2(21)); 3.86 (s, MeOC(3’)); 5.29 (s, CH2(5)); 5.52 (q-like, J ¼ 15.2, CH2(23)); 6.00 (s, HOC(20)); 7.35 – 7.46 (m, HC(14,4’,5’,6’)); 7.58 (s, HC(2’)); 7.64 (d, J ¼ 7.2, HC(14)); 7.68 (d, J ¼ 7.5, HC(12)); 7.87 (t, J ¼ 7.9, HC(11)); 8.17 (d, J ¼ 8.4, HC(10)); 10.61 (s, NHCO). ESI-MS: 524.89 ([M H] ). 9-[(2-Chlorobenzoyl)amino]-7-methylhomocamptothecin ( ¼ 2-Chloro-N-(5-ethyl-4,5,13,15-tetrahydro-5-hydroxy-12-methyl-3,15-dioxo-1H,3H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinolin-11-yl)benzamide ; 10g) . Yield : 25 mg (13%) . Yellow solid. M.p. > 3008. 1H-NMR ((D6 )DMSO): 0.87 (t, J ¼ 7.4, Me(18)); 1.87 (q, J ¼ 7.2, CH2(19)); 2.73 (s, MeC(7)); 3.08 (q-like, J ¼ 13.7, CH2(21)); 5.38 (s, CH2(5)); 5.53 (q-like, J ¼ 15.1, CH2(23)); 6.00 (s, HOC(20)); 7.27 – 7.39 (m, HC(12,14)); 7.61 – 7.63 (m, HC(3’,4’,5’)); 7.68 (d, J ¼ 7.5, HC(12)); 7.79 (t, J ¼ 7.9, HC(11)); 8.05 (d, J ¼ 7.2, HC(10)); 8.10 (d, J ¼ 7.4, HC(6’)); 10.46 (s, NHCO). ESI-MS: 528.14 ([M H] ). 9-[(3-Chlorobenzoyl)amino]-7-methylhomocamptothecin ( ¼ 3-Chloro-N-(5-ethyl-4,5,13,15-tetrahydro-5-hydroxy-12-methyl-3,15-dioxo-1H,3H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinolin-11-yl)benzamide ; 10h) . Yield : 80 mg (42%) . Yellow solid. M.p. > 3008. 1H-NMR ((D6 )DMSO): 0.87 (t, J ¼ 7.3, Me(18)); 1.87 (q, J ¼ 7.1, CH2(19)); 2.73 (s, MeC(7)); 3.08 (q-like, J ¼ 13.8, CH2(21)); 5.38 (s, CH2(5)); 5.53 (q-like, J ¼ 15.1, CH2(23)); 6.00 (s, HOC(20)); 7.27 – 7.39 (m, HC(2’,14)); 7.57 – 7.62 (m, HC(4’,5’)); 7.68 (d, J ¼ 7.5, HC(12)); 7.79 (t, J ¼ 7.9, HC(11)); 8.05 (d, J ¼ 7.2, HC(10)); 8.09 (d, J ¼ 7.3, HC(2’,6’)); 10.46 (s, NHCO). ESI-MS: 508.66 ([M H] ). Cytotoxicity. Cells (1,200 per well) were plated in 96-well plates. After culturing for 24 h, test compounds with a wide range of concentrations (from 100 to 0.001 mg/ml; ten dilutions) were added onto duplicate wells with different concentrations, and 0.1% DMSO for control. After 3 d of incubation, 20 ml of MTT ( ¼ 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) soln. (5 mg/ml) was added to each well, and, after shaking for 1 min, the plate was incubated for further 4 h. Formazan crystals were dissolved in 100 ml of DMSO. The absorbance (OD) was recorded with microplate spectrophotometer at 570 nm. Wells containing no drugs were used as blanks for the spectrophotometer. The survival of the cells was expressed as percentage of untreated control wells. Antitumor Activities in vivo. The in vivo antitumor activities of compounds 7c and 10a were evaluated. BALB/C Nude male mice (certificate SCXK2003-0003, weighing 18 – 20 g) were obtained from Shanghai Experimental Animal Center, Chinese Academy of Sciences. A549 Lung cancer-ell suspensions were implanted subcutaneously into the right axilla region of mice. Treatment was started when implanted tumors had reached a volume of ca. 100 – 300 mm3 (after 17 d). The animals were randomized into appropriate groups (six animals/treatment, and nine animals for the control group) and administered by ip injection once a day on day 17 and consecutive 4 d. Tumor volumes (TV) were monitored by caliper measurement of the length and width, and calculated using the formula of TV ¼ 1/ 2 a b2, where a is the tumor length and b is the width. Tumor volumes and body weights were monitored every 4 d over the course of treatment. Mice were sacrificed on day 30 after implantation of cells, and tumors were removed and recorded for analysis.
1814
CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
DNA Topoisomerase I Activity Assays. Camptothecin was obtained from the Company of Tianzunzezhong in China. Topo I (calf thymus), buffer, BSA, loading buffer, and supercoilded DNA pBR322 were all from TaKaRa Biotechnology Co., Ltd. All reactions were carried out in 20-ml volumes (16 ml of double dist. H2O, 2 ml of DNA Topo I buffer, 2 ml of 0.1% BSA), including 0.25 mg of supercoiled DNA and 0.5 U Topo I with or without drug. The mixtures were incubated at 378 for 15 min and then stopped by adding SDS (0.5% final concentration). To the mixtures, 6 3.5 ml of loading buffer (0.1 mm EDTA, 7% glycerol, 0.01% xylene cyanol FF, Bromopenol Blue 0.01%) was added. The mixtures were submitted to electrophoresis in 0.8% agarose gel in TAE ( ¼ Tris-acetate-EDTA) buffer for 40 min at 120 V. The gel was stained with ethidium bromide at r.t. and photographed with UV transilluminator.
REFERENCES [1] M. E. Wall, M. C. Wani, C. E. Cook, K. H. Palmer, A. T. McPhail, G. A. Sim, J. Am. Chem. Soc. 1966, 88, 3888. [2] A. G. Schultz, Chem. Rev. 1973, 73, 385. [3] J. A. Gottlieb, A. M. Guarino, J. B. Call, V. T. Oliverio, J. B. Block, Cancer Chemother. Rep. 1970, 54, 461. [4] P. J. Creaven, L. M. Allen, Cancer Chemother. Rep. 1973, 57, 175. [5] C. G. Moertel, A. J. Schutt, R. J. Reitemeier, R. G. Hahn, Cancer Chemother. Rep. 1972, 56, 649. [6] Y. H. Hsiang, R. Hertzberg, S. Hecht, L. F. Liu, J. Biol. Chem. 1985, 260, 14873. [7] S. Basili, S. Moro, Expert Opin. Ther. Pat. 2009, 19, 555. [8] O. Lavergne, L. Lesueur-Ginot, F. P. Rodas, D. C. H. Bigg, Bioorg. Med. Chem. Lett. 1997, 7, 2235. [9] D. P. Curran, C. R. Chim. 2008, 11, 1574. [10] O. Lavergne, D. Demarquay, C. Bailly, C. Lanco, A. Rolland, M. Huchet, H. Coulomb, N. Muller, N. Baroggi, J. Camara, L. C. Breton, E. Manginot, J. B. Cazaux, D. C. Biqq, J. Med. Chem. 2000, 43, 2285. [11] Y. Pommier, Nat. Rev. Cancer 2006, 6, 789. [12] O. Lavergne, L. Lesueur-Ginot, F. P. Rodas, P. G. Kasprzyk, J. Pommier, D. Demarquay, G. Prevost, G. Ulibarri, A. Rolland, A. M. Schiano-Liberatore, J. Harnett, D. Pons, J. Camara, D. C. H. Bigg, J. Med. Chem. 1998, 41, 5410. [13] W. Guo, Z. Y. Miao, C. Q. Sheng, J. Z. Yao, H. Feng, W. N. Zhang, L. J. Zhu, W. F. Liu, P. F. Cheng, J. Zhang, X. Y. Che, W. Y. Wang, C. Luo, Y. L. Xu, Eur. J. Med. Chem. 2010, 45, 2223. [14] L. J. Zhu, Z. Y. Miao, C. Q. Sheng, W. Guo, J. Z. Yao, W. F. Liu, X. Y. Che, W. Y. Wang, P. F. Cheng, W. N. Zhang, Eur. J. Med. Chem. 2010, 45, 2726. [15] W. F. Liu, L. J. Zhu, W. Guo, C. L. Zhuang, Y. Q. Zhang, C. Q. Sheng, P. F. Cheng, J. Z. Yao, W. Y. Wang, G. Q. Dong, S. Z. Wang, Z. Y. Miao, W. N. Zhang, Eur. J. Med. Chem. 2011, 46, 2408. [16] Z. Y. Miao, J. Zhang, L. You, J. Wang, C. Q. Sheng, J. Z. Yao, W. N. Zhang, H. Feng, W. Guo, L. Zhou, W. F. Liu, L. J. Zhu, P. F. Cheng, X. Y. Che, W. Y. Wang, C. Luo, Y. L. Xu, G. Q. Dong, Bioorg. Med. Chem. 2010, 18, 3140. [17] Z. Y. Miao, C. Q. Sheng, W. N. Zhang, H. T. Ji, J. Zhang, L. C. Shao, L. You, M. Zhang, J. Z. Yao, X. Y. Che, Bioorg. Med. Chem. 2008, 16, 1493. [18] W. Guo, Z. Y. Miao, C. Q. Sheng, J. Z. Yao, W. F. Liu, L. J. Zhu, Y. Q. Zhang, P. F. Cheng, G. Q. Dong, C. L. Zhuang, W. N. Zhang, W. Y. Wang, Chem. Biodiversity 2011, 8, 1266. [19] W. Guo, W. F. Liu, L. J. Zhu, Y. Q. Zhang, P. F. Cheng, G. Q. Dong, C. L. Zhuang, W. Y. Wang, J. Z. Yao, C. Q. Sheng, Z. Y. Miao, W. N. Zhang. Chem. Biodiversity 2011, 8, 1539. [20] L. J. Zhu, X. H. Zhang, N. Lei, W. F. Liu, Z. Y. Miao, C. L. Zhuang, C. Q. Sheng, W. Guo, G. Q. Dong, J. Z. Yao, P. F. Cheng, W. N. Zhang. Chem. Biodiversity 2012, 9, 1084. [21] L. J. Dolby, D. L. Booth, J. Am. Chem. Soc. 1966, 88, 1049. [22] M. Mentel, R. Breinbauer, Curr. Org. Chem. 2007, 11, 159. [23] E. J. Alford, K. Schofield, J. Chem. Soc. 1953, 609. [24] K. Schofield, R. S. Theobald, J. Chem. Soc. 1949, 796.
CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
1815
[25] D. W. Ockenden, K. Schofield, J. Chem. Soc. 1953, 612. [26] W. C. Drewe, R. Nanjunda, M. Gunaratnam, M. Beltran, G. N. Parkinson, A. P. Reszka, W. D. Wilson, S. Neidle, J. Med. Chem. 2008, 51, 7751. [27] S. de Castro, R. Chicharro, V. J. Ara´n, J. Chem. Soc., Perkin Trans. 1 2002, 790. [28] Y. L. Leu, S. R. Roffler, J. W. Chern, J. Med. Chem. 1999, 42, 3623. [29] K. J. Yoon, J. J. Qi, J. S. Remack, K. G. Virga, M. J. Hatfield, P. M. Potter, R. E. Lee, M. K. Danks, Mol. Cancer Ther. 2006, 5, 1577. Received September 6, 2012
