- Email: [email protected]
Synthesis and in vitro antitumor activity of novel diaryl urea derivatives
Chinese Chemical Letters 24 (2013) 386–388
Contents lists available at SciVerse ScienceDirect
Chinese Chemical Letters journal homepage: www.elsevier.com/locate/cclet
Original article
Synthesis and in vitro antitumor activity of novel diaryl urea derivatives Yan-Fang Zhao, Zi-Jian Liu, Xin Zhai, Dan-Dan Ge, Qiang Huang, Ping Gong * Key Lab of New Drugs Design and Discovery of Liaoning Province, School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 13 December 2012 Received in revised form 18 December 2012 Accepted 26 December 2012 Available online 14 March 2013
A series of novel diaryl ureas containing 4-[(2-amino-6-trifluromethyl)pyrimidine-4-yl]piperazine-1-yl group were synthesized and evaluated for their cytotoxic activities in a panel of human cancer cell lines. Compared with the reference drug Sorafenib, some compounds showed more potent and a broader spectrum of anti-cancer activities. Among them, compound 2p demonstrated significant inhibitory activities against MDA-MB-231, HT-29 and MCF-7 cell lines with IC50 values of 0.016, 0.63, 0.001 mmol/L, respectively. ß 2013 Ping Gong. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
Keywords: Diaryl ureas Synthesis Antitumor activity in vitro
1. Introduction Kinase inhibitors have been in use as cancer therapeutics for nearly a decade, and their utility in targeting specific malignancies has significantly changed the landscape of current cancer therapy. Among them, some typical structural moieties have emerged and held great potential as novel anticancer agents, such as 4-phenylaminoquinazoline, the diaryl ureas, etc. Diaryl urea derivatives played an important role in anticancer agents because of their good inhibitory activity against receptor tyrosine kinases (RTKs), Raf kinases, protein tyrosine kinases (PTKs), and NADH oxidase, which play critical roles in many aspects of tumor genesis [1]. There are several diaryl urea compounds that have been in clinical use or clinial trials, including Sorafenib [2], ABT-869 [3], KRN-951 [4]. Sorafenib (1, Fig. 1) is a potent inhibitor of Raf-1, which is a member of the RAF/MEK/ERK signaling pathway. It also demonstrates significant inhibitory activity against several receptor tyrosine kinases involved in neovascularization and tumor progression, including vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF) receptor families [5]. It was approved by the U.S. Food and Drug Administration (FDA) for the treatment of patients with advanced renal cell carcinoma (RCC) [6] and unresectable hepatocellular carcinoma (HCC) [7] in December 2005 and November 2007, respectively.
* Corresponding author. E-mail address: [email protected] (P. Gong).
In our continued search for potent and novel RTKIs as potential anticancer agents, we designed and synthesized a series of novel diaryl urea derivatives (2, Fig. 1) containing the 4-[(2-amino-6trifluromethyl)pyrimidine-4-yl]piperazine-1-yl group. 2. Experimental The general synthetic route of target diaryl ureas is depicted in Scheme 1. Ethyl trifluoromethylacetoacetate 3 was condensed with guanidine to give 2-amino-4-hydroxy-6-trifluoromethylpyrimidine 4, which was treated with POCl3 to yield compound 5. Treatment of p-nitrobenzyl bromide 8 with excessive piperazine in ethanol at 0 8C formed 1-(4-nitrobenzyl)piperazine 9. Nucleophilic substitution of 5 with 9 gave intermediate 6, which was reduced with FeCl36H2O and 80% hydrazine hydrate to yield 7. Reaction of 7 with suitable phenyl isocyanates 10, prepared from substituted anilines and triphosgene, and then purified via recrystallization afforded the desired series of diaryl urea derivatives 2a–r. Some of them were acidified with HCl in ether to give hydrochloride salts. The spectral data of all the target compounds are in full agreement with the proposed structures. 2d: mp 206–208 8C. 1H NMR (300 MHz, DMSO-d6): d 2.40 (s, 4H), 3.45 (s, 2H), 3.61 (s, 4H), 6.41 (s, 1H), 6.59 (brs, 2H), 7.01 (d, 1H, J = 6.0 Hz), 7.22–7.30 (m, 4H), 7.42 (d, 2H, J = 9.0 Hz), 7.72 (s, 1H), 8.81 (s, 1H), 8.95 (s, 1H). MS (ESI) m/z: 507.9 (M+H). Anal. Calcd. for C23H23ClF3N7O (%): C, 54.60; H, 4.58; Cl, 7.01; F, 11.27; N, 19.38. Found (%): C, 54.48; H, 4.41; Cl, 7.19; F, 11.33; N, 19.43. 2g: mp 235–237 8C. 1H NMR (300 MHz, DMSO-d6): d 2.40 (s, 4H), 3.45 (s, 2H), 3.61 (s, 4H), 6.41 (s, 1H), 6.59 (brs, 2H),7.25 (d, 2H, J = 9.0 Hz), 7.42 (d, 2H, J = 9.0 Hz), 7.63 (m, 4H, J = 9.0 Hz), 8.79
1001-8417/$ – see front matter ß 2013 Ping Gong. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. http://dx.doi.org/10.1016/j.cclet.2013.02.004
[(Fig._1)TD$IG]
Y.-F. Zhao et al. / Chinese Chemical Letters 24 (2013) 386–388
Cl
CF3
O O
O N H
N
N H
N H
O
R N H
387
N N
N NH2
2
1 (Sorafenib)
CF3
N
N H
Fig. 1. Structures of Sorafenib and target compounds.
[(Schem_1)TD$FIG]
Scheme 1. Reagents and conditions: (a) guanidine nitrate, n-BuONa, n-BuOH, reflux, 6 h, 83%; (b) POCl3, Et3N, CH3CN, reflux, 8 h, 78%; (c) 9, Et3N, EtOH, reflux, 7 h, 69%; (d) 80% hydrazine hydrate, FeCl36H2O, activated carbon, DMF, reflux, 9 h, 79%; (e) 10, anhydrous THF, 5 8C, 6 h, 82%; (f) piperazine, K2CO3, EtOH, 0 8C, 1 h, 92%; and (g) triphosgene, 1,4-dioxane, 80 8C, 24 h, 75%–80%.
(s, 1H), 9.08 (s, 1H). MS (ESI) m/z: 540.2 (M+H). Anal. Calcd. for C24H23F6N7O (%): C, 53.43; H, 4.30; F, 21.13; N, 18.17. Found (%): C, 53.19; H, 4.38; F, 21.05; N, 18.31. 2n: mp 180–182 8C. 1H NMR (300 MHz, DMSO-d6): d 2.19 (s, 3H), 2.25 (s, 3H), 2.40 (m, 4H), 3.45 (s, 2H), 3.60–3.62 (m, 4H), 6.41 (s, 1H), 6.59 (s, 2H), 6.76 (d, 1H, J = 6.0 Hz), 7.04 (d, 1H, J = 6.0 Hz), 7.21 (d, 2H, J = 6.0 Hz), 7.43 (d, 2H, J = 6.0 Hz), 7.66 (s, 1H), 7.85 (s, 1H), 9.01 (s, 1H). MS (ESI) m/z: 501.3 (M+H). Anal. Calcd. for C25H28F3N7O (%): C, 60.11; H, 5.65; F, 11.41; N, 19.63. Found (%): C, 60.33; H, 5.80; F, 11.25; N, 19.41. 2p3HCl: mp 190–192 8C. 1H NMR (300 MHz, DMSO-d6): d 2.99–3.03 (m, 2H), 3.33–3.43 (m, 4H), 4.25 (s, 2H), 4.54 (m, 2H), 6.59 (s, 1H), 7.30–7.37 (m, 2H), 7.48 (d, 2H, J = 9.0 Hz), 7.53 (d, 2H, J = 9.0 Hz), 7.79 (dd, 1H J = 6.0 Hz, 2.4 Hz), 9.58 (s, 1H), 9.70 (s, 1H), 11.14 (brs, 1H). MS (ESI) m/z: 523.8 (M+H). Anal. Calcd. for C23H25Cl4F4N7O (%): C, 43.62; H, 3.98; Cl, 22.39; F, 12.00;
N, 15.48. Found (%): C, 43.47; H, 3.83; Cl, 22.58; F, 12.21; N, 15.38. 2r: mp 197–199 8C. 1H NMR (300 MHz, DMSO-d6): d 2.40 (s, 4H), 3.46 (s, 2H), 3.60–3.62 (m, 4H), 6.41 (s, 1H), 6.59 (brs, 2H), 7.27 (d, 2H, J = 9.0 Hz), 7.37 (dd, 1H, J = 9.0 Hz, 3.0 Hz), 7.45 (d, 2H, J = 9.0 Hz), 7.72 (d, 1H, J = 9.0 Hz), 8.60 (s, 1H), 8.65 (s, 1H), 9.57 (s, 1H). MS (ESI) m/z: 575.7 (M+H). Anal. Calcd. for C24H22ClF6N7O (%): C, 50.23; H, 3.86; Cl, 6.18; F, 19.86; N, 17.08. Found (%): C, 50.45; H, 3.60; Cl, 6.30; F, 19.67; N, 17.19. The cytotoxic activity of synthesized diaryl urea analogs 2a– 2r was evaluated against a panel of human cell lines, including human breast cancer MDA-MB-231, human breast adenocarcinoma MCF-7, human colorectal cancer HT-29, human liver cancer SMMC-7721 and human lung cancer NCI-H446 cell lines. The cancer cell lines were cultured in minimum essential medium (MEM) supplement with 10% fetal bovine serum (FBS).
Table 1 Substituents and cytotoxic activity of target compounds against a panel of human cancer cell lines. Compd.
R
2a3HCl 2b3HCl 2c3HCl 2d 2e 2f3HCl 2g 2h 2i3HCl 2j3HCl 2k3HCl 2l3HCl 2m3HCl 2n 2o3HCl 2p3HCl 2q3HCl 2r Sorafenibb
2-Fluoro 3-Fluoro 4-Chloro 3-Chloro 3-Bromo 3-Trifluoromethyl 4-Trifluoromethyl 4-Trifluoromethoxy 2,6-Difluoro 3,5-Difluoro 3,4-Dichloro 3,5-Dichloro 3,5-Bis(trifluoromethyl) 2,5-Dimethyl 3,4-Dimethyl 3-Chloro-4-fluoro 4-Fluoro-3-trifluoromethyl 2-Chloro-5-trifluoromethyl
a b
IC50 (mmol/L)a MDA-MB-231
HT-29
MCF-7
SMMC-7721
NCI-H446
5.18 0.21 3.84 0.29 2.60 0.30 0.015 0.005 0.15 0.06 2.93 0.34 0.92 0.17 0.61 0.19 6.48 0.42 2.59 0.22 1.85 0.27 2.92 0.19 2.65 0.30 0.43 0.10 0.16 0.04 0.016 0.004 1.35 0.22 0.58 0.12 0.94 0.13
5.01 0.36 5.34 0.20 2.93 0.26 0.38 0.11 4.09 0.42 3.08 0.40 0.77 0.17 4.50 0.38 2.43 0.41 2.59 0.36 1.54 0.20 0.98 0.16 1.95 0.21 2.13 0.35 1.97 0.28 0.63 0.15 1.79 0.24 7.32 0.88 4.30 0.34
1.02 0.15 3.51 0.37 4.88 0.35 5.04 0.62 3.49 0.27 1.11 0.20 1.54 0.33 4.06 0.34 5.19 0.56 1.13 0.21 0.55 0.11 1.46 0.26 1.05 0.23 3.45 0.31 3.40 0.28 0.001 0.001 5.91 0.34 6.15 0.87 36.14 2.45
4.17 0.31 4.78 0.37 4.42 0.34 5.68 0.65 7.43 0.79 4.18 0.29 1.45 0.21 2.56 0.38 2.89 0.35 3.24 0.16 2.57 0.28 2.85 0.32 2.29 0.27 7.55 0.41 23.98 1.69 3.82 0.33 3.29 0.26 2.63 0.40 6.78 0.37
6.85 0.38 10.02 0.57 7.96 0.33 8.28 0.31 9.39 0.16 6.01 0.27 5.55 0.21 7.37 0.36 6.81 0.40 5.99 0.43 6.93 0.28 5.84 0.42 3.77 0.30 8.05 0.67 5.91 0.57 10.42 0.35 11.39 0.89 7.32 0.31 22.13 2.29
IC50 is the concentration of compound required to inhibit the cell growth by 50% compared to an untreated control. Used as a positive control.
388
Y.-F. Zhao et al. / Chinese Chemical Letters 24 (2013) 386–388
Approximately 4 103 cells, suspended in MEM medium, were plated onto each well of a 96-well plate and incubated in 5% CO2 at 37 8C for 24 h. The subject compounds at indicated final concentrations were added to the culture medium and the cell cultures were continued for 72 h. Fresh MTT was added to each well at a terminal concentration of 5 mg/mL and incubated with cells at 37 8C for 4 h. The formazan crystals were dissolved in 100 mL DMSO each well, and the absorbency at 492 nm (for absorbance of MTT formazan) and 630 nm (for the reference wavelength) was measured with the ELISA reader. All of the compounds were tested three times in each of the cell lines. The results expressed as IC50 (inhibitory concentration 50%) were the averages of three determinations and calculated by using the Bacus Laboratories Incorporated Slide Scanner (Bliss) software. The results were illustrated in Table 1 with Sorafenib as the positive control. 3. Results and discussion As shown in Table 1, apparent growth inhibition against MDAMB-231, MCF-7 and SMMC-7721 cell lines was observed for most of the compounds. Among them, compounds 2d, 2g and 2p exhibited more potent anti-tumor activities against MDA-MB-231, HT-29 and MCF-7 cell lines than Sorafenib. Especially, compound 2p, the most promising compound, displayed excellent anti-tumor activity against MDA-MB-231, MCF-7, HT-29 cell lines with IC50 values of 0.016, 0.001, 0.63 mmol/L, respectively. Contrasted to Sorafenib, the tested compounds displayed a broader spectrum of anticancer activity. Some of them showed moderate to strong cytotoxicity against MCF-7 and H446 cell lines. However, Sorafenib exhibited only weak inhibitory activity on these three cell lines. From Table 1, the steric effect of group R appears to have some relationship with the cytotoxicity of the series of compounds. Most of the derivatives with meta-substitution, especially chloro (2d, 2p), bromo (2e), methyl (2n, 2o) group showed significantly higher cytotoxicities than those with fluoro (2b) and trifluoromethyl (2f, 2m) groups or with no substituent. Of them, the chloro group in the meta position appeared to increase the antitumor potency. There was no obvious difference between the effects of electrondonating and electron-withdrawing groups on the benzene ring. Compounds 2g and 2h with the trifluoromethyl group and the trifluoromethoxy group at the para-position of the terminal benzene ring, respectively, exhibited much higher cytotoxicity than those with such groups at other positions. This result showed that trifluoromethyl and trifluoromethoxy groups were less effective in the para-position. Moreover, the 3-Cl-4-F analog 2p exhibited comparable cytotoxic activity (IC50 = 0.016 mmol/L) to the 3-Cl analog 2c (IC50 = 0.015 mmol/L) against MDA-MB-231 cell line. Remarkably, compound 2p showed stronger inhibitory activity against MCF-7 cell line than 2c (0.001 mmol/L vs 4.88 mmol/L). The differences resulting from 2q vs 2f and 2p vs
2d also indicated that fluoro at the para-position kept, or improved, the antitumor activity. The effects of fluoro and trifluoromethyl substituents on cytotoxic activities in the ortho-position of benzene ring were also evaluated. Generally, compounds with fluoro or trifluoromethyl group in the ortho-position had weaker cytotoxicity than those with chloro or methyl groups in the ortho-position. It is worth noting that compounds 2a (2-fluoro) and 2i (2,6-difluoro) exhibited weak cytotoxicity, whereas 2r (2-chloro-5-trifluoromethyl) and 2n (2,5-dimethyl) exhibited potent cytotoxicity. The structure-activity relationship (SAR) information indicated that the fluoro group at ortho-postion resulted in less activity. 4. Conclusion In summary, a series of novel diaryl urea derivatives were synthesized and evaluated for their antitumor activities on MDAMB-231, HT-29, MCF-7, SMMC-7721 and NCI-H446 cell lines, with Sorafenib as the reference control. Compounds 2d, 2g, 2l, 2o, 2p and 2q exhibited more potent activity against MDA-MB-231 and HT-29 cell lines as compared with Sorafenib. From preliminary SARs, we may conclude compounds with the chloro substitution in the meta-position are required for optimal potency. Most noteworthy was the steric effect of meta substituents, such as chloro, bromo and methyl, which led to a significant improvement in activity. Moreover, the substitution of the fluoro group at the para-position of the benzene ring further increased cytotoxic activity. This study may provide valuable information for future design and development of antitumor agents with more potent activities. Acknowledgment This work was supported by a grant from the National Natural Science Foundation of China (No. 21002065). References [1] H.Q. Li, P.C. Lv, T. Yan, H.L. Zhu, Urea derivatives as anticancer agents, Anticancer Agents Med. Chem. 10 (2009) 471–480. [2] S. Wilhelm, C. Carter, M. Lynch, et al., Discovery and development of sorafenib: a multikinase inhibitor for treating cancer, Nat. Rev. Drug Discov. 5 (2006) 835–844. [3] Y.J. Dai, K. Hartandi, Z.Q. Ji, et al., Discovery of N-(4-(3-amino-1H-indazol-4yl)phenyl)-N0 -(2-fluoro-5-methylphenyl)urea (ABT-869), a 3-aminoindazolebased orally active multitargeted receptor tyrosine kinase inhibitor, J. Med. Chem. 50 (2007) 1584–1597. [4] K. Nakamura, E. Taguchi, T. Miura, et al., KRN951, a highly potent inhibitor of vascular endothelial growth factor receptor tyrosine kinases, has antitumor activities and affects functional vascular properties, Cancer Res. 66 (2006) 9134–9142. [5] S.M. Wilhelm, C. Carter, L.Y. Tang, et al., BAY 43-9006 exhibits broad spectrum oral antitumor activity and targets the RAF/MEK/ERK pathway and receptor tyrosine kinases involved in tumor, Cancer Res. 64 (2004) 7099–7109. [6] R.C. Kan, A.T. Farrell, H. Saber, et al., Sorafenib for the treatment of advanced renal cell carcinoma, Clin. Cancer Res. 12 (2006) 7271–7278. [7] G.M. Keating, A. Santoro, Sorafenib: a review of its use in advanced hepatocellular carcinoma, Drugs 69 (2009) 223–240.
Contents lists available at SciVerse ScienceDirect
Chinese Chemical Letters journal homepage: www.elsevier.com/locate/cclet
Original article
Synthesis and in vitro antitumor activity of novel diaryl urea derivatives Yan-Fang Zhao, Zi-Jian Liu, Xin Zhai, Dan-Dan Ge, Qiang Huang, Ping Gong * Key Lab of New Drugs Design and Discovery of Liaoning Province, School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 13 December 2012 Received in revised form 18 December 2012 Accepted 26 December 2012 Available online 14 March 2013
A series of novel diaryl ureas containing 4-[(2-amino-6-trifluromethyl)pyrimidine-4-yl]piperazine-1-yl group were synthesized and evaluated for their cytotoxic activities in a panel of human cancer cell lines. Compared with the reference drug Sorafenib, some compounds showed more potent and a broader spectrum of anti-cancer activities. Among them, compound 2p demonstrated significant inhibitory activities against MDA-MB-231, HT-29 and MCF-7 cell lines with IC50 values of 0.016, 0.63, 0.001 mmol/L, respectively. ß 2013 Ping Gong. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
Keywords: Diaryl ureas Synthesis Antitumor activity in vitro
1. Introduction Kinase inhibitors have been in use as cancer therapeutics for nearly a decade, and their utility in targeting specific malignancies has significantly changed the landscape of current cancer therapy. Among them, some typical structural moieties have emerged and held great potential as novel anticancer agents, such as 4-phenylaminoquinazoline, the diaryl ureas, etc. Diaryl urea derivatives played an important role in anticancer agents because of their good inhibitory activity against receptor tyrosine kinases (RTKs), Raf kinases, protein tyrosine kinases (PTKs), and NADH oxidase, which play critical roles in many aspects of tumor genesis [1]. There are several diaryl urea compounds that have been in clinical use or clinial trials, including Sorafenib [2], ABT-869 [3], KRN-951 [4]. Sorafenib (1, Fig. 1) is a potent inhibitor of Raf-1, which is a member of the RAF/MEK/ERK signaling pathway. It also demonstrates significant inhibitory activity against several receptor tyrosine kinases involved in neovascularization and tumor progression, including vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF) receptor families [5]. It was approved by the U.S. Food and Drug Administration (FDA) for the treatment of patients with advanced renal cell carcinoma (RCC) [6] and unresectable hepatocellular carcinoma (HCC) [7] in December 2005 and November 2007, respectively.
* Corresponding author. E-mail address: [email protected] (P. Gong).
In our continued search for potent and novel RTKIs as potential anticancer agents, we designed and synthesized a series of novel diaryl urea derivatives (2, Fig. 1) containing the 4-[(2-amino-6trifluromethyl)pyrimidine-4-yl]piperazine-1-yl group. 2. Experimental The general synthetic route of target diaryl ureas is depicted in Scheme 1. Ethyl trifluoromethylacetoacetate 3 was condensed with guanidine to give 2-amino-4-hydroxy-6-trifluoromethylpyrimidine 4, which was treated with POCl3 to yield compound 5. Treatment of p-nitrobenzyl bromide 8 with excessive piperazine in ethanol at 0 8C formed 1-(4-nitrobenzyl)piperazine 9. Nucleophilic substitution of 5 with 9 gave intermediate 6, which was reduced with FeCl36H2O and 80% hydrazine hydrate to yield 7. Reaction of 7 with suitable phenyl isocyanates 10, prepared from substituted anilines and triphosgene, and then purified via recrystallization afforded the desired series of diaryl urea derivatives 2a–r. Some of them were acidified with HCl in ether to give hydrochloride salts. The spectral data of all the target compounds are in full agreement with the proposed structures. 2d: mp 206–208 8C. 1H NMR (300 MHz, DMSO-d6): d 2.40 (s, 4H), 3.45 (s, 2H), 3.61 (s, 4H), 6.41 (s, 1H), 6.59 (brs, 2H), 7.01 (d, 1H, J = 6.0 Hz), 7.22–7.30 (m, 4H), 7.42 (d, 2H, J = 9.0 Hz), 7.72 (s, 1H), 8.81 (s, 1H), 8.95 (s, 1H). MS (ESI) m/z: 507.9 (M+H). Anal. Calcd. for C23H23ClF3N7O (%): C, 54.60; H, 4.58; Cl, 7.01; F, 11.27; N, 19.38. Found (%): C, 54.48; H, 4.41; Cl, 7.19; F, 11.33; N, 19.43. 2g: mp 235–237 8C. 1H NMR (300 MHz, DMSO-d6): d 2.40 (s, 4H), 3.45 (s, 2H), 3.61 (s, 4H), 6.41 (s, 1H), 6.59 (brs, 2H),7.25 (d, 2H, J = 9.0 Hz), 7.42 (d, 2H, J = 9.0 Hz), 7.63 (m, 4H, J = 9.0 Hz), 8.79
1001-8417/$ – see front matter ß 2013 Ping Gong. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. http://dx.doi.org/10.1016/j.cclet.2013.02.004
[(Fig._1)TD$IG]
Y.-F. Zhao et al. / Chinese Chemical Letters 24 (2013) 386–388
Cl
CF3
O O
O N H
N
N H
N H
O
R N H
387
N N
N NH2
2
1 (Sorafenib)
CF3
N
N H
Fig. 1. Structures of Sorafenib and target compounds.
[(Schem_1)TD$FIG]
Scheme 1. Reagents and conditions: (a) guanidine nitrate, n-BuONa, n-BuOH, reflux, 6 h, 83%; (b) POCl3, Et3N, CH3CN, reflux, 8 h, 78%; (c) 9, Et3N, EtOH, reflux, 7 h, 69%; (d) 80% hydrazine hydrate, FeCl36H2O, activated carbon, DMF, reflux, 9 h, 79%; (e) 10, anhydrous THF, 5 8C, 6 h, 82%; (f) piperazine, K2CO3, EtOH, 0 8C, 1 h, 92%; and (g) triphosgene, 1,4-dioxane, 80 8C, 24 h, 75%–80%.
(s, 1H), 9.08 (s, 1H). MS (ESI) m/z: 540.2 (M+H). Anal. Calcd. for C24H23F6N7O (%): C, 53.43; H, 4.30; F, 21.13; N, 18.17. Found (%): C, 53.19; H, 4.38; F, 21.05; N, 18.31. 2n: mp 180–182 8C. 1H NMR (300 MHz, DMSO-d6): d 2.19 (s, 3H), 2.25 (s, 3H), 2.40 (m, 4H), 3.45 (s, 2H), 3.60–3.62 (m, 4H), 6.41 (s, 1H), 6.59 (s, 2H), 6.76 (d, 1H, J = 6.0 Hz), 7.04 (d, 1H, J = 6.0 Hz), 7.21 (d, 2H, J = 6.0 Hz), 7.43 (d, 2H, J = 6.0 Hz), 7.66 (s, 1H), 7.85 (s, 1H), 9.01 (s, 1H). MS (ESI) m/z: 501.3 (M+H). Anal. Calcd. for C25H28F3N7O (%): C, 60.11; H, 5.65; F, 11.41; N, 19.63. Found (%): C, 60.33; H, 5.80; F, 11.25; N, 19.41. 2p3HCl: mp 190–192 8C. 1H NMR (300 MHz, DMSO-d6): d 2.99–3.03 (m, 2H), 3.33–3.43 (m, 4H), 4.25 (s, 2H), 4.54 (m, 2H), 6.59 (s, 1H), 7.30–7.37 (m, 2H), 7.48 (d, 2H, J = 9.0 Hz), 7.53 (d, 2H, J = 9.0 Hz), 7.79 (dd, 1H J = 6.0 Hz, 2.4 Hz), 9.58 (s, 1H), 9.70 (s, 1H), 11.14 (brs, 1H). MS (ESI) m/z: 523.8 (M+H). Anal. Calcd. for C23H25Cl4F4N7O (%): C, 43.62; H, 3.98; Cl, 22.39; F, 12.00;
N, 15.48. Found (%): C, 43.47; H, 3.83; Cl, 22.58; F, 12.21; N, 15.38. 2r: mp 197–199 8C. 1H NMR (300 MHz, DMSO-d6): d 2.40 (s, 4H), 3.46 (s, 2H), 3.60–3.62 (m, 4H), 6.41 (s, 1H), 6.59 (brs, 2H), 7.27 (d, 2H, J = 9.0 Hz), 7.37 (dd, 1H, J = 9.0 Hz, 3.0 Hz), 7.45 (d, 2H, J = 9.0 Hz), 7.72 (d, 1H, J = 9.0 Hz), 8.60 (s, 1H), 8.65 (s, 1H), 9.57 (s, 1H). MS (ESI) m/z: 575.7 (M+H). Anal. Calcd. for C24H22ClF6N7O (%): C, 50.23; H, 3.86; Cl, 6.18; F, 19.86; N, 17.08. Found (%): C, 50.45; H, 3.60; Cl, 6.30; F, 19.67; N, 17.19. The cytotoxic activity of synthesized diaryl urea analogs 2a– 2r was evaluated against a panel of human cell lines, including human breast cancer MDA-MB-231, human breast adenocarcinoma MCF-7, human colorectal cancer HT-29, human liver cancer SMMC-7721 and human lung cancer NCI-H446 cell lines. The cancer cell lines were cultured in minimum essential medium (MEM) supplement with 10% fetal bovine serum (FBS).
Table 1 Substituents and cytotoxic activity of target compounds against a panel of human cancer cell lines. Compd.
R
2a3HCl 2b3HCl 2c3HCl 2d 2e 2f3HCl 2g 2h 2i3HCl 2j3HCl 2k3HCl 2l3HCl 2m3HCl 2n 2o3HCl 2p3HCl 2q3HCl 2r Sorafenibb
2-Fluoro 3-Fluoro 4-Chloro 3-Chloro 3-Bromo 3-Trifluoromethyl 4-Trifluoromethyl 4-Trifluoromethoxy 2,6-Difluoro 3,5-Difluoro 3,4-Dichloro 3,5-Dichloro 3,5-Bis(trifluoromethyl) 2,5-Dimethyl 3,4-Dimethyl 3-Chloro-4-fluoro 4-Fluoro-3-trifluoromethyl 2-Chloro-5-trifluoromethyl
a b
IC50 (mmol/L)a MDA-MB-231
HT-29
MCF-7
SMMC-7721
NCI-H446
5.18 0.21 3.84 0.29 2.60 0.30 0.015 0.005 0.15 0.06 2.93 0.34 0.92 0.17 0.61 0.19 6.48 0.42 2.59 0.22 1.85 0.27 2.92 0.19 2.65 0.30 0.43 0.10 0.16 0.04 0.016 0.004 1.35 0.22 0.58 0.12 0.94 0.13
5.01 0.36 5.34 0.20 2.93 0.26 0.38 0.11 4.09 0.42 3.08 0.40 0.77 0.17 4.50 0.38 2.43 0.41 2.59 0.36 1.54 0.20 0.98 0.16 1.95 0.21 2.13 0.35 1.97 0.28 0.63 0.15 1.79 0.24 7.32 0.88 4.30 0.34
1.02 0.15 3.51 0.37 4.88 0.35 5.04 0.62 3.49 0.27 1.11 0.20 1.54 0.33 4.06 0.34 5.19 0.56 1.13 0.21 0.55 0.11 1.46 0.26 1.05 0.23 3.45 0.31 3.40 0.28 0.001 0.001 5.91 0.34 6.15 0.87 36.14 2.45
4.17 0.31 4.78 0.37 4.42 0.34 5.68 0.65 7.43 0.79 4.18 0.29 1.45 0.21 2.56 0.38 2.89 0.35 3.24 0.16 2.57 0.28 2.85 0.32 2.29 0.27 7.55 0.41 23.98 1.69 3.82 0.33 3.29 0.26 2.63 0.40 6.78 0.37
6.85 0.38 10.02 0.57 7.96 0.33 8.28 0.31 9.39 0.16 6.01 0.27 5.55 0.21 7.37 0.36 6.81 0.40 5.99 0.43 6.93 0.28 5.84 0.42 3.77 0.30 8.05 0.67 5.91 0.57 10.42 0.35 11.39 0.89 7.32 0.31 22.13 2.29
IC50 is the concentration of compound required to inhibit the cell growth by 50% compared to an untreated control. Used as a positive control.
388
Y.-F. Zhao et al. / Chinese Chemical Letters 24 (2013) 386–388
Approximately 4 103 cells, suspended in MEM medium, were plated onto each well of a 96-well plate and incubated in 5% CO2 at 37 8C for 24 h. The subject compounds at indicated final concentrations were added to the culture medium and the cell cultures were continued for 72 h. Fresh MTT was added to each well at a terminal concentration of 5 mg/mL and incubated with cells at 37 8C for 4 h. The formazan crystals were dissolved in 100 mL DMSO each well, and the absorbency at 492 nm (for absorbance of MTT formazan) and 630 nm (for the reference wavelength) was measured with the ELISA reader. All of the compounds were tested three times in each of the cell lines. The results expressed as IC50 (inhibitory concentration 50%) were the averages of three determinations and calculated by using the Bacus Laboratories Incorporated Slide Scanner (Bliss) software. The results were illustrated in Table 1 with Sorafenib as the positive control. 3. Results and discussion As shown in Table 1, apparent growth inhibition against MDAMB-231, MCF-7 and SMMC-7721 cell lines was observed for most of the compounds. Among them, compounds 2d, 2g and 2p exhibited more potent anti-tumor activities against MDA-MB-231, HT-29 and MCF-7 cell lines than Sorafenib. Especially, compound 2p, the most promising compound, displayed excellent anti-tumor activity against MDA-MB-231, MCF-7, HT-29 cell lines with IC50 values of 0.016, 0.001, 0.63 mmol/L, respectively. Contrasted to Sorafenib, the tested compounds displayed a broader spectrum of anticancer activity. Some of them showed moderate to strong cytotoxicity against MCF-7 and H446 cell lines. However, Sorafenib exhibited only weak inhibitory activity on these three cell lines. From Table 1, the steric effect of group R appears to have some relationship with the cytotoxicity of the series of compounds. Most of the derivatives with meta-substitution, especially chloro (2d, 2p), bromo (2e), methyl (2n, 2o) group showed significantly higher cytotoxicities than those with fluoro (2b) and trifluoromethyl (2f, 2m) groups or with no substituent. Of them, the chloro group in the meta position appeared to increase the antitumor potency. There was no obvious difference between the effects of electrondonating and electron-withdrawing groups on the benzene ring. Compounds 2g and 2h with the trifluoromethyl group and the trifluoromethoxy group at the para-position of the terminal benzene ring, respectively, exhibited much higher cytotoxicity than those with such groups at other positions. This result showed that trifluoromethyl and trifluoromethoxy groups were less effective in the para-position. Moreover, the 3-Cl-4-F analog 2p exhibited comparable cytotoxic activity (IC50 = 0.016 mmol/L) to the 3-Cl analog 2c (IC50 = 0.015 mmol/L) against MDA-MB-231 cell line. Remarkably, compound 2p showed stronger inhibitory activity against MCF-7 cell line than 2c (0.001 mmol/L vs 4.88 mmol/L). The differences resulting from 2q vs 2f and 2p vs
2d also indicated that fluoro at the para-position kept, or improved, the antitumor activity. The effects of fluoro and trifluoromethyl substituents on cytotoxic activities in the ortho-position of benzene ring were also evaluated. Generally, compounds with fluoro or trifluoromethyl group in the ortho-position had weaker cytotoxicity than those with chloro or methyl groups in the ortho-position. It is worth noting that compounds 2a (2-fluoro) and 2i (2,6-difluoro) exhibited weak cytotoxicity, whereas 2r (2-chloro-5-trifluoromethyl) and 2n (2,5-dimethyl) exhibited potent cytotoxicity. The structure-activity relationship (SAR) information indicated that the fluoro group at ortho-postion resulted in less activity. 4. Conclusion In summary, a series of novel diaryl urea derivatives were synthesized and evaluated for their antitumor activities on MDAMB-231, HT-29, MCF-7, SMMC-7721 and NCI-H446 cell lines, with Sorafenib as the reference control. Compounds 2d, 2g, 2l, 2o, 2p and 2q exhibited more potent activity against MDA-MB-231 and HT-29 cell lines as compared with Sorafenib. From preliminary SARs, we may conclude compounds with the chloro substitution in the meta-position are required for optimal potency. Most noteworthy was the steric effect of meta substituents, such as chloro, bromo and methyl, which led to a significant improvement in activity. Moreover, the substitution of the fluoro group at the para-position of the benzene ring further increased cytotoxic activity. This study may provide valuable information for future design and development of antitumor agents with more potent activities. Acknowledgment This work was supported by a grant from the National Natural Science Foundation of China (No. 21002065). References [1] H.Q. Li, P.C. Lv, T. Yan, H.L. Zhu, Urea derivatives as anticancer agents, Anticancer Agents Med. Chem. 10 (2009) 471–480. [2] S. Wilhelm, C. Carter, M. Lynch, et al., Discovery and development of sorafenib: a multikinase inhibitor for treating cancer, Nat. Rev. Drug Discov. 5 (2006) 835–844. [3] Y.J. Dai, K. Hartandi, Z.Q. Ji, et al., Discovery of N-(4-(3-amino-1H-indazol-4yl)phenyl)-N0 -(2-fluoro-5-methylphenyl)urea (ABT-869), a 3-aminoindazolebased orally active multitargeted receptor tyrosine kinase inhibitor, J. Med. Chem. 50 (2007) 1584–1597. [4] K. Nakamura, E. Taguchi, T. Miura, et al., KRN951, a highly potent inhibitor of vascular endothelial growth factor receptor tyrosine kinases, has antitumor activities and affects functional vascular properties, Cancer Res. 66 (2006) 9134–9142. [5] S.M. Wilhelm, C. Carter, L.Y. Tang, et al., BAY 43-9006 exhibits broad spectrum oral antitumor activity and targets the RAF/MEK/ERK pathway and receptor tyrosine kinases involved in tumor, Cancer Res. 64 (2004) 7099–7109. [6] R.C. Kan, A.T. Farrell, H. Saber, et al., Sorafenib for the treatment of advanced renal cell carcinoma, Clin. Cancer Res. 12 (2006) 7271–7278. [7] G.M. Keating, A. Santoro, Sorafenib: a review of its use in advanced hepatocellular carcinoma, Drugs 69 (2009) 223–240.