Bioorganic & Medicinal Chemistry Letters xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and evaluation of novel HDAC inhibitors as potential antitumor agents Jianjun Cheng ⇑, Jihong Qin, Sihua Guo, Hangdeng Qiu, Yun Zhong Shanghai Huilun Life Sciences & Technology Co. Ltd, Shanghai 201108, China
a r t i c l e
i n f o
Article history: Received 9 February 2014 Revised 21 June 2014 Accepted 27 June 2014 Available online xxxx Keywords: Histone deacetylases Hydroxamate Antitumor Vorinostat
a b s t r a c t Phenyl imidazolidin-2-one was introduced as the linker for novel HDAC inhibitors. A focused library of 20 compounds was designed and synthesized, among which eight compounds showed equivalent or higher potencies against HDAC1 as compared to vorinostat. In vitro antitumor activity assays in HCT-116, PC-3 and HL-60 cancer cells revealed six compounds with potent antitumor activities, and compound 1o showed 6- to 9-fold higher potencies compared to vorinostat. In an HCT-116 nude mice xenograft model, compound 1o displayed significant antitumor activity in both continuous and intermittent dosing schedules. Ó 2014 Elsevier Ltd. All rights reserved.
Histone acetylation and deacetylation plays an important role in the regulation of gene expression, influencing the transcription of many genes, including tumor suppressor genes.1 This reversible process is catalyzed by histone acetyl tranferases (HATs) and histone deacetylases (HDACs).2,3 To date, 18 human HDACs have been identified and grouped into four classes: class I (HDAC1, 2, 3 and 8), class II (HDAC4, 5, 6, 7, 9 and 10), class III enzymes (also known as sirtuins, SIRT 1–7) and class IV (HDAC11).4,5 Class I, II and IV HDACs are also referred to as ‘classical’ HDACs and are Zn2+-dependent enzymes whereas sirtuins require NAD+ as a cofactor. Altered HDAC activity has been identified in various types of cancer,6 and developing HDAC inhibitors as novel therapeutics has been validated in both preclinical and clinical studies, in both solid and haematological cancers.7,8 Efforts from the scientific community have led to the discovery of a number of isoform selective HDAC inhibitors,9 which would be very good pharmacological tools to study the different roles each of these HDAC isoforms plays. But there is still a large medical need for pan inhibitors as candidates for cancer therapy, since it has been demonstrated that the various cancer types do not involve the same HDAC isoforms. Most HDAC inhibitors reported so far can be grouped into four chemical families (hydroxamates, benzamides, short-chain fatty acids and macrocyclic peptides),10 and hydroxamates have been proven to be most potent and the major class in clinical trials. Among them, vorinostat was approved by the FDA in 2006 for ⇑ Corresponding author. Tel./fax: +86 21 34304236. E-mail address: [email protected] (J. Cheng).
the therapy of cutaneous T-cell lymphoma.11 The structure of vorinostat represents the common structure of HDAC inhibitors, which constitutes a ‘CAP’ moiety, a linker and the zinc-binding group (ZBG) (Fig. 1). The binding mode of hydroxamates to HDACs was confirmed with the structure of a histone deacetylase homologue bound to vorinostat.12 Several other clinical HDAC inhibitors, such as panobinostat (LBH-589),13 belinostat (PXD-101),14 resminostat (4SC-201), pracinostat (SB-939),15 givinostat (ITF-2357) and quisinostat (JNJ-26481585)16 all contain hydroxamates as their ZBGs and share the same canonical scaffold with vorinostat. In this Letter, we report novel HDAC inhibitors characterized by phenyl imidazolidin-2-one moiety as a new linker and their use as potential antitumor agents (Fig. 1). A focused library was designed based on general structure 1, and a simple and convenient route was applied for the synthesis of compounds 1a–1t (Scheme 1). Commercially available ethyl 4-aminobenzoate was treated with 1-chloro-2-isocyanatoethane to afford the urea intermediate 3, which underwent an intramolecular cyclization to give the phenyl imidazolidin-2-one 4 in good yield.17 Introduction of the R substitution was accomplished via Buchwald coupling of 4 with aryl bromides or alkylation reactions with alkylating agents listed in Scheme 1 (for detailed information on the preparation of intermediates 6a–6t, see Supporting information, Table S1). Most of the intermediates 5a–5t were commercially available or could be prepared according to reported methods,18 and preparations of some of them were depicted in Schemes 2–6. When hydrochloride salts of the reagents were used, the equivalence of base was increased in the alkylating reaction.
http://dx.doi.org/10.1016/j.bmcl.2014.06.080 0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Cheng, J.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.06.080
2
J. Cheng et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
Figure 1. Representative HDAC inhibitors in clinical trials and novel scaffold design.
Scheme 1. Reagents and conditions: (a) 2-chloroethyl isocyanate, toluene, 0–40 °C, 62%; (b) K2CO3, DMF, rt, overnight, 78%; (c) Buchwald coupling with 5a or 5b, or alkylating with 5c–5t; (d) NH2OH, MeOH, 0 °C–rt.
Scheme 2. Reagents and conditions: (a) LiAlH4, THF, 0 °C–rt, 1 h, 78%; (b) SOCl2, toluene, rt, 1 h, 98%.
The esters 6a–6t were converted to the corresponding hydroxamic acids by treating with methanol solution of hydroxylamine. Among the various isoforms, overexpression of HDAC1 has been found in gastric, breast, pancreatic, hepatocellular, lung and prostate carcinomas and in most cases HDAC1 up-regulation associates
with poor prognosis.19–21 We screened compounds 1a–1t for their IC50s on HDAC1 and the result was listed in Table 1.22 A direct substitution of the imidazolidin-2-one with pyridine (compound 1a) or quinoline (compound 1b) showed poor HDAC1 inhibitory activities, as compared to vorinostat. However, aryl substitution with a methylene spacer displayed much better potencies, as shown by a benzyl substitution in compound 1c, which showed equivalent potency with vorinostat (IC50 = 17 nM for 1c vs 13 nM for vorinostat). Substitution with a fluorine atom (compound 1d) or methoxy group (compound 1e) on the benzene ring slightly altered the activities, but an electron-withdrawing trifluoromethyl group (compound 1f) reduced the potency significantly. In order to explore heteroaryl substitutions at this position, pyridine was introduced to replace the benzene ring, however both 4-pyridine
Please cite this article in press as: Cheng, J.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.06.080
J. Cheng et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
3
Scheme 3. Reagents and conditions: (a) Me2NH in water (33%, w/w), THF, rt, overnight, quantitative; (b) MeOH, SOCl2, 70 °C, overnight, 94–97%; (c) LiAlH4, THF, 0 °C–rt, 1 h, 64–93%; (d) SOCl2, toluene, rt, 1 h, 75–99%.
Scheme 4. Reagents and conditions: (a) LiAlH4, THF, 0 °C–rt, 1 h, 51%; (b) SOCl2, toluene, rt, 1 h, 98%.
and 3-pyridine substitutions (compounds 1g and 1h) reduced enzyme inhibition slightly. We predicted that an electron-donating substitution might be favored for the benzyl group, and substitutions with amino groups were introduced. Compound 1i with a dimethylamino substitution showed an enhanced activity (IC50 = 9.5 nM), but an introduction of methylene link between dimethylamino group and the benzene reduced the inhibitory activity more than 2-fold (compounds 1j and 1k). Bicyclic substitutions were also introduced to explore the SAR of the CAP group. To our delight, single digit nanomolar potencies were observed for both naphthyl (compounds 1l and 1m) and quinolone (compound 1n) groups. And compound 1o, with a further introduction of diethylamino methylene group to the naphthyl ring (the same CAP group as givinostat), displayed the most potent HDAC1 inhibition in this series, with a 5-fold more potency compared to vorinostat (IC50 = 2.7 nM for 1o vs 13 nM for vorinostat). Elongation of the linker between the aromatic ring and imidazolidin-2-one, from one carbon to two carbons, also displayed potent activities, as represented by compound 1p and 1q, which showed 13 nM and 4.0 nM IC50 values respectively. Alkyl
substitutions without an aromatic ring showed much less activity, and compounds 1r, 1s and 1t displayed poor HDAC1 inhibition. The eight compounds with equivalent or higher potencies compared to vorinostat in Table 1 were elevated for in vitro antitumor activity.23 Growth inhibition assays in HCT-116 cells were run firstly, and most of the compounds showed potent inhibition of cell proliferation, with GI50 values below 1 lM (Table 2). Five most potent compounds 1i, 1m, 1n, 1o and 1q were further evaluated for their inhibition of PC-3 and HL-60 cell proliferation. All these five compounds showed at least 2-fold higher inhibitory potencies on all the 3 cell lines and compound 1o showed 6 to 9-fold potencies compared to vorinostat, with GI50 values around 0.1 lM against all 3 cell lines. As many HDAC inhibitors from diverse structural classes have been reported to be associated with QT prolongation in humans,24,25 we tested compound 1o for hERG inhibition, which would indicate potential influence on K+ ion channel.26 Compound 1o displayed hERG inhibition of 27% at a concentration of 10 lM (n = 3), which predicted a very low risk of inducing QT prolongation. Furthermore, compound 1o showed excellent physiochemical properties, with a molecular weight of 446.5, C log P value of 3.3 (calculated with Chemdraw) and high water solubility (data not shown). Based on its potent in vitro activity and favorable druglike profile, compound 1o was then chosen for further in vivo evaluation.27 The HCT-116 xenograft model in nude mice was selected for preliminary in vivo anticancer activity test of 1o.28 Because most HDAC inhibitors that are currently in the clinical trial can only be given intermittently due to the side effect of fatigue, which is
Scheme 5. Reagents and conditions: (a) DMF, Et2NH, HATU, DIPEA, rt, overnight, 73%; (b) MeOH, SOCl2, 70 °C, overnight, 83%; (c) BH3–SMe2 (2 M in THF), THF, 50 °C, overnight; then 2 M HCl, 70 °C, 1 h, 90%; (d) SOCl2, toluene, rt, 1 h, 87%.
Scheme 6. Reagents and conditions: (a) LiAlH4, THF, rt, overnight, 85%; (b) TBSCl, CH2Cl2, imidazole, Et3N, overnight, 78%; (c) DMF, NaH, MeI, rt, 2 h, 83%; (d) THF, TBAF, rt, overnight, 78%; (e) MeCN/Et2O, imidazole, Ph3P, I2, rt, 0.5 h, 87%.
Please cite this article in press as: Cheng, J.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.06.080
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J. Cheng et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
Table 1 HDAC1 inhibitory activities of compounds 1a–1t Compound
R
1a
Table 2 GI50s of selected compounds (‘—’, not tested). IC50 (nM)
Compound
182
1c 1e 1i 1m 1n 1o 1p 1q Voninostat
N 152
1b
N 1c 1d
17
F
HCT-116
PC-3
HL-60
0.85 0.47 0.33 0.21 0.27 0.11 0.65 0.24 0.65
— — 0.43 0.38 0.34 0.10 — 0.21 0.96
— — 0.44 0.36 0.25 0.086 — 0.48 0.75
21
1e
O
12
1f
F3 C
80
1g
N
41
1h
GI50 (lM)
42
N
N 9.5
1i
N 1j
23
N
1k
25 Figure 2. Antitumor activity of compound 1o in HCT-116 xenograft model. 9.5
1l
1m
5.4
1n
5.4
N N
1o
1p
2.7
13
1q
4.0
Acknowledgment
250
We are grateful to Dr. Joel A. Bergman for helpful discussions and valuable suggestions.
N 1r
1s
O
1t Voninostat
—
for 3 weeks resulted in 54% TGI (Fig. 2).29 Doses in both groups were well tolerated and body weight losses of animals were within 15% (data not shown). The intermittent dosing schedule showed a similar antitumor effect with a total drug dosing amount less than the continuous dosing schedule. In summary, a focused library of HDAC inhibitors with phenyl imidazolidin-2-one as the new linker was designed and synthesized. Several compounds displayed potent HDAC1 inhibition and tumor cell growth inhibition in vitro. Compound 1o was evaluated in an HCT-116 xenograft model in vivo and showed significant tumor growth inhibition in both continuous and intermittent dosing schedules. Further evaluation of compound 1o is ongoing and will be reported in due time.
N
125
Supplementary data
N
114
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2014. 06.080. These data include MOL files and InChiKeys of the most important compounds described in this article.
13
considered to be a class effect, we tested the efficacy of 1o using both continuous and intermittent dosing schedules. Daily oral administration of 1o at 50 mg/kg for 3 weeks resulted in 45% tumor growth inhibition (TGI) when compared to the vehicle control; intermittent dosing schedule with 3 days on plus 3 days off
References and notes 1. Johnstone, R. W. Nat. Rev. Drug Disc. 2002, 1, 287. 2. Roth, S. Y.; Denu, J. M.; Allis, C. D. Annu. Rev. Biochem. 2001, 70, 81. 3. Thiagalingam, S.; Cheng, K.-H.; Lee, H. J.; Mineva, N.; Thiagalingam, A.; Ponte, J. F. Ann. N.Y. Acad. Sci. 2003, 983, 84.
Please cite this article in press as: Cheng, J.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.06.080
J. Cheng et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx 4. De Ruitjer, A. J. M.; van Gennip, A. H.; Caron, H. N.; Kemp, S.; van Kuilenburg, A. B. P. Biochem. J. 2003, 370, 737. 5. Gao, L.; Cueto, M. A.; Asselbergs, F.; Atadja, P. J. Biol. Chem. 2002, 277, 25748. 6. Weichert, W. Cancer Lett. 2009, 280, 168. 7. Paris, M.; Porcelloni, M.; Binaschi, M.; Fattori, D. J. Med. Chem. 2008, 51, 1505. 8. Venugopal, B.; Evans, T. R. Curr. Med. Chem. 2011, 18, 1658. 9. For examples, see: (a) Balasubramanian, S.; Verner, E.; Buggy, J. J. Cancer Lett. 2009, 280, 211; (b) Gupta, P. K.; Reid, R. C.; Liu, L.; Lucke, A. J.; Broomfield, S. A.; Andrews, M. R.; Sweet, M. J.; Fairlie, D. P. Bioorg. Med. Chem. Lett. 2010, 20, 7067; (c) Huang, W.-J.; Wang, Y.-C.; Chao, S.-W.; Yang, C.-H.; Chen, L.-C.; Lin, M.-H.; Hou, W.-C.; Chen, M.-Y.; Lee, T.-L.; Yang, P.; Chang, C.-I. ChemMedChem 2012, 7, 1815; (d) Marek, L.; Hamacher, A.; Hansen, F. K.; Kuna, K.; Gohlke, H.; Kassack, M. U.; Kurz, T. J. Med. Chem. 2013, 56, 427; (e) Blackburn, C.; Barrett, C.; Chin, J.; Garcia, K.; Gigstad, K.; Gould, A.; Gutierrez, J.; Harrison, S.; Hoar, K.; Lynch, C.; Rowland, R. S.; Tsu, C.; Ringeling, J.; Xu, H. J. Med. Chem. 2013, 56, 7201; (f) Bergman, J. A.; Woan, K.; Perez-Villarroel, P.; Villagra, A.; Sotomayor, E. M.; Kozikowski, A. P. J. Med. Chem. 2012, 55, 9891; (g) Kalin, J. H.; Bergman, J. A. J. Med. Chem. 2013, 56, 6297. 10. Bolden, J. E.; Peart, M. J.; Johnstone, R. W. Nat. Rev. Drug Disc. 2006, 5, 769. 11. Marks, P. A. Oncogene 2007, 26, 1351. 12. Finnin, M. S.; Donigian, J. R.; Cohen, A.; Richon, V. M.; Rifkind, R. A.; Marks, P. A.; Breslow, R.; Pavletich, N. P. Nature 1999, 401, 188. 13. Atadja, P. Cancer Lett. 2009, 280, 233. 14. Plumb, J. A.; Finn, P. W.; Williams, R. J.; Bandara, M. J.; Romero, M. R.; Watkins, C. J.; La Thangue, N. B.; Brown, R. Mol. Cancer Ther. 2003, 2, 721. 15. Wang, H.; Yu, N.; Dizhong Chen, D.; Lee, K. C. L.; Lye, P. L.; Chang, J. W. W.; Deng, W.; Ng, C. Y. M.; Lu, T.; Khoo, M. L.; Poulsen, A.; Sangthongpitag, K.; Wu, X.; Hu, C.; Goh, K. C.; Wang, X.; Fang, L.; Goh, K. L.; Khng, H. H.; Goh, S. K.; Yeo, P.; Liu, X.; Bonday, Z.; Wood, J. M.; Dymock, B. W.; Kantharaj, E.; Sun, E. T. J. Med. Chem. 2011, 54, 4694. 16. Arts, J.; King, P.; Mariën, A.; Floren, W.; Beliën, A.; Janssen, L.; Pilatte, I.; Roux, B.; Decrane, L.; Gilissen, R.; Hickson, I.; Vreys, V.; Cox, E.; Bol, K.; Talloen, W.; Goris, I.; Andries, L.; Du Jardin, M.; Janicot, M.; Page, M.; van Emelen, K.; Angibaud, P. Clin. Cancer Res. 2009, 15, 6841. 17. Saha, A.; Yu, X. Y.; Lobera, M.; Lin, J.; Cheruku, S. R.; Becker, O.M.; Marantz, Y.; Schutz, N. WO2007109330. 18. 5a–5f, 5l, 5m, 5p, 5r, 5s, 5t are commercially available; preparation of some of the chemicals can be found in: 5g, 5h (Ojo, B. Syn. Comm. 2012, 42, 1731.); 5i (Barlin, G. B.; Davies, L. P.; Ireland, S. J.; Ngu, M. M. L. Aust. J. Chem. 1989, 42, 1133); 5q (Liu, C.; Zhang, W.; Dai, L.-X.; You, S.-L. Org. Biomol. Chem. 2012, 10, 7177.). 19. Choi, J. H.; Kwon, H. J.; Yoon, B. I.; Kim, J. H.; Han, S. U.; Joo, H. J.; Kim, D. Y. Jpn. J. Cancer Res. 2001, 92, 1300. 20. Minamiya, Y.; Ono, T.; Saito, H.; Takahashi, N.; Ito, M.; Mitsui, M.; Motoyama, S.; Ogawa, J. Lung Cancer 2011, 74, 300.
5
21. Zhang, Z.; Yamashita, H.; Toyama, T.; Sugiura, H.; Ando, Y.; Mita, K.; Hamaguchi, M.; Hara, Y.; Kobayashi, S.; Iwase, H. Breast Cancer Res. Treat. 2005, 94, 11. 22. Compounds were screened against HDAC1 (purchased from BPS Bioscience) with Boc-Lys(Ac)-AMC substrate concentration at Km.Tests were run on 384-well plates and compounds were diluted in 10 concentrations and run in duplicate.The reaction contained 6.25 nM HDAC1, assay buffer (50 mM HEPES, pH 7.4, 100 mM KCl, 0.001% Tween-20, 0.05% BSA, 20 lM TCEP). 23. All cell lines were obtained from the American Type Culture Collection and cultivated according to the supplier’s instructions; Seeding density of HCT-116, PC-3 and HL-60 was 2000/well, 2000/well and 10000/well respectively (cells was counted with Vi-Cell XR) and incubation time was 72 h; all tests were done CellTiter Glo assay kit (Promega). 24. Molife, R.; Fong, P.; Scurr, M.; Judson, I.; Kaye, S.; de Bono, J. Clin. Cancer Res. 2007, 13, 1068. 25. Subramanian, S.; Bates, S.; Wright, J.; Espinoza-Delgado, I.; Piekarz, R. Pharmaceuticals 2010, 3, 2751. 26. Recanatini, M.; Poluzzi, E.; Masetti, M.; Cavalli, A.; De, P. F. Med. Res. Rev. 2005, 25, 133. 27. Selected characterization data for compound 1o: MS (ESI+): m/z 447 [M+H]; 1H NMR (300 MHz, DMSO-d6) d 11.13 (br, 1H), 8.91 (br, 1H), 8.13 (s, 1H), 7.97 (d, J = 6.3 Hz, 1H), 7.91 (d, J = 6.6 Hz, 1H), 7.86 (s, 1H), 7.82 (d, J = 6.3 Hz, 1H), 7.75 (d, J = 6.3 Hz, 2H), 7.65 (d, J = 6.6 Hz, 2H), 7.49 (d, J = 6.3 Hz, 1H), 4.57 (s, 2H), 4.40 (s, 2H), 3.84 (t, J = 6.0 Hz, 2H), 3.40 (t, J = 6.0 Hz, 2H), 3.03 (q, J = 5.4 Hz, 4H), 1.25 (t, J = 5.4 Hz, 6H); 13C NMR (75 MHz, DMSO-d6) d 164.4, 157.3, 143.5, 136.4, 133.3, 132.3, 131.0, 129.0, 128.8, 128.7, 128.4, 128.0, 127.2, 126.6, 125.9, 116.4, 55.2, 47.7, 46.2, 42.3, 41.3, 8.7; HRMS (ESI): calcd for C26H31N4O3 (M+H): 447.2396, found: 447.2391. 28. Evaluation of vorinostat in this model has been reported: Novotny-Diermayr, V.; Sangthongpitag, K.; Hu, C. Y.; Wu, X.; Sausgruber, N.; Yeo, P.; Greicius, G.; Pettersson, S.; Liang, A. L.; Loh, Y. K.; Bonday, Z.; Goh, K. C.; Hentze, H.; Hart, S.; Wang, H.; Ethirajulu, K.; Wood, J. M. Mol. Cancer Ther. 2010, 9, 642– 52.Vorinostat at a dose of 200 mg/kg, qd (formulated in 50% PEG400 in water (V/V)) was used as the positive control group for the in vivo test of compound 1o, but it displayed a significant toxicity and this group was terminated after 10 days dosing, with about 50% TGI at that time point. 29. Compound 1o was formulated in 50% PEG400 in water (V/V), vehicle control group was dosed with 50% PEG400 in water, and dosing volume was 10 mL/kg; Tumor volume (mm3) = (w2 l)/2, in which w is the width and l is the length of the tumor in mm; TGI was calculated using the following formula: %TGI = (Tday 21 Tday 0)/(Cday 21 Cday 0) 100 [in which Tday 21 is the median tumor volume for treatment group on day 21; Tday 0 is the median tumor volume for treatment group on day 0; Cday 21 is the median tumor volume for control group (vehicle) on day 21; Cday 0 is the median tumor volume for control group (vehicle) on day 0].
Please cite this article in press as: Cheng, J.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.06.080
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and evaluation of novel HDAC inhibitors as potential antitumor agents Jianjun Cheng ⇑, Jihong Qin, Sihua Guo, Hangdeng Qiu, Yun Zhong Shanghai Huilun Life Sciences & Technology Co. Ltd, Shanghai 201108, China
a r t i c l e
i n f o
Article history: Received 9 February 2014 Revised 21 June 2014 Accepted 27 June 2014 Available online xxxx Keywords: Histone deacetylases Hydroxamate Antitumor Vorinostat
a b s t r a c t Phenyl imidazolidin-2-one was introduced as the linker for novel HDAC inhibitors. A focused library of 20 compounds was designed and synthesized, among which eight compounds showed equivalent or higher potencies against HDAC1 as compared to vorinostat. In vitro antitumor activity assays in HCT-116, PC-3 and HL-60 cancer cells revealed six compounds with potent antitumor activities, and compound 1o showed 6- to 9-fold higher potencies compared to vorinostat. In an HCT-116 nude mice xenograft model, compound 1o displayed significant antitumor activity in both continuous and intermittent dosing schedules. Ó 2014 Elsevier Ltd. All rights reserved.
Histone acetylation and deacetylation plays an important role in the regulation of gene expression, influencing the transcription of many genes, including tumor suppressor genes.1 This reversible process is catalyzed by histone acetyl tranferases (HATs) and histone deacetylases (HDACs).2,3 To date, 18 human HDACs have been identified and grouped into four classes: class I (HDAC1, 2, 3 and 8), class II (HDAC4, 5, 6, 7, 9 and 10), class III enzymes (also known as sirtuins, SIRT 1–7) and class IV (HDAC11).4,5 Class I, II and IV HDACs are also referred to as ‘classical’ HDACs and are Zn2+-dependent enzymes whereas sirtuins require NAD+ as a cofactor. Altered HDAC activity has been identified in various types of cancer,6 and developing HDAC inhibitors as novel therapeutics has been validated in both preclinical and clinical studies, in both solid and haematological cancers.7,8 Efforts from the scientific community have led to the discovery of a number of isoform selective HDAC inhibitors,9 which would be very good pharmacological tools to study the different roles each of these HDAC isoforms plays. But there is still a large medical need for pan inhibitors as candidates for cancer therapy, since it has been demonstrated that the various cancer types do not involve the same HDAC isoforms. Most HDAC inhibitors reported so far can be grouped into four chemical families (hydroxamates, benzamides, short-chain fatty acids and macrocyclic peptides),10 and hydroxamates have been proven to be most potent and the major class in clinical trials. Among them, vorinostat was approved by the FDA in 2006 for ⇑ Corresponding author. Tel./fax: +86 21 34304236. E-mail address: [email protected] (J. Cheng).
the therapy of cutaneous T-cell lymphoma.11 The structure of vorinostat represents the common structure of HDAC inhibitors, which constitutes a ‘CAP’ moiety, a linker and the zinc-binding group (ZBG) (Fig. 1). The binding mode of hydroxamates to HDACs was confirmed with the structure of a histone deacetylase homologue bound to vorinostat.12 Several other clinical HDAC inhibitors, such as panobinostat (LBH-589),13 belinostat (PXD-101),14 resminostat (4SC-201), pracinostat (SB-939),15 givinostat (ITF-2357) and quisinostat (JNJ-26481585)16 all contain hydroxamates as their ZBGs and share the same canonical scaffold with vorinostat. In this Letter, we report novel HDAC inhibitors characterized by phenyl imidazolidin-2-one moiety as a new linker and their use as potential antitumor agents (Fig. 1). A focused library was designed based on general structure 1, and a simple and convenient route was applied for the synthesis of compounds 1a–1t (Scheme 1). Commercially available ethyl 4-aminobenzoate was treated with 1-chloro-2-isocyanatoethane to afford the urea intermediate 3, which underwent an intramolecular cyclization to give the phenyl imidazolidin-2-one 4 in good yield.17 Introduction of the R substitution was accomplished via Buchwald coupling of 4 with aryl bromides or alkylation reactions with alkylating agents listed in Scheme 1 (for detailed information on the preparation of intermediates 6a–6t, see Supporting information, Table S1). Most of the intermediates 5a–5t were commercially available or could be prepared according to reported methods,18 and preparations of some of them were depicted in Schemes 2–6. When hydrochloride salts of the reagents were used, the equivalence of base was increased in the alkylating reaction.
http://dx.doi.org/10.1016/j.bmcl.2014.06.080 0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Cheng, J.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.06.080
2
J. Cheng et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
Figure 1. Representative HDAC inhibitors in clinical trials and novel scaffold design.
Scheme 1. Reagents and conditions: (a) 2-chloroethyl isocyanate, toluene, 0–40 °C, 62%; (b) K2CO3, DMF, rt, overnight, 78%; (c) Buchwald coupling with 5a or 5b, or alkylating with 5c–5t; (d) NH2OH, MeOH, 0 °C–rt.
Scheme 2. Reagents and conditions: (a) LiAlH4, THF, 0 °C–rt, 1 h, 78%; (b) SOCl2, toluene, rt, 1 h, 98%.
The esters 6a–6t were converted to the corresponding hydroxamic acids by treating with methanol solution of hydroxylamine. Among the various isoforms, overexpression of HDAC1 has been found in gastric, breast, pancreatic, hepatocellular, lung and prostate carcinomas and in most cases HDAC1 up-regulation associates
with poor prognosis.19–21 We screened compounds 1a–1t for their IC50s on HDAC1 and the result was listed in Table 1.22 A direct substitution of the imidazolidin-2-one with pyridine (compound 1a) or quinoline (compound 1b) showed poor HDAC1 inhibitory activities, as compared to vorinostat. However, aryl substitution with a methylene spacer displayed much better potencies, as shown by a benzyl substitution in compound 1c, which showed equivalent potency with vorinostat (IC50 = 17 nM for 1c vs 13 nM for vorinostat). Substitution with a fluorine atom (compound 1d) or methoxy group (compound 1e) on the benzene ring slightly altered the activities, but an electron-withdrawing trifluoromethyl group (compound 1f) reduced the potency significantly. In order to explore heteroaryl substitutions at this position, pyridine was introduced to replace the benzene ring, however both 4-pyridine
Please cite this article in press as: Cheng, J.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.06.080
J. Cheng et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
3
Scheme 3. Reagents and conditions: (a) Me2NH in water (33%, w/w), THF, rt, overnight, quantitative; (b) MeOH, SOCl2, 70 °C, overnight, 94–97%; (c) LiAlH4, THF, 0 °C–rt, 1 h, 64–93%; (d) SOCl2, toluene, rt, 1 h, 75–99%.
Scheme 4. Reagents and conditions: (a) LiAlH4, THF, 0 °C–rt, 1 h, 51%; (b) SOCl2, toluene, rt, 1 h, 98%.
and 3-pyridine substitutions (compounds 1g and 1h) reduced enzyme inhibition slightly. We predicted that an electron-donating substitution might be favored for the benzyl group, and substitutions with amino groups were introduced. Compound 1i with a dimethylamino substitution showed an enhanced activity (IC50 = 9.5 nM), but an introduction of methylene link between dimethylamino group and the benzene reduced the inhibitory activity more than 2-fold (compounds 1j and 1k). Bicyclic substitutions were also introduced to explore the SAR of the CAP group. To our delight, single digit nanomolar potencies were observed for both naphthyl (compounds 1l and 1m) and quinolone (compound 1n) groups. And compound 1o, with a further introduction of diethylamino methylene group to the naphthyl ring (the same CAP group as givinostat), displayed the most potent HDAC1 inhibition in this series, with a 5-fold more potency compared to vorinostat (IC50 = 2.7 nM for 1o vs 13 nM for vorinostat). Elongation of the linker between the aromatic ring and imidazolidin-2-one, from one carbon to two carbons, also displayed potent activities, as represented by compound 1p and 1q, which showed 13 nM and 4.0 nM IC50 values respectively. Alkyl
substitutions without an aromatic ring showed much less activity, and compounds 1r, 1s and 1t displayed poor HDAC1 inhibition. The eight compounds with equivalent or higher potencies compared to vorinostat in Table 1 were elevated for in vitro antitumor activity.23 Growth inhibition assays in HCT-116 cells were run firstly, and most of the compounds showed potent inhibition of cell proliferation, with GI50 values below 1 lM (Table 2). Five most potent compounds 1i, 1m, 1n, 1o and 1q were further evaluated for their inhibition of PC-3 and HL-60 cell proliferation. All these five compounds showed at least 2-fold higher inhibitory potencies on all the 3 cell lines and compound 1o showed 6 to 9-fold potencies compared to vorinostat, with GI50 values around 0.1 lM against all 3 cell lines. As many HDAC inhibitors from diverse structural classes have been reported to be associated with QT prolongation in humans,24,25 we tested compound 1o for hERG inhibition, which would indicate potential influence on K+ ion channel.26 Compound 1o displayed hERG inhibition of 27% at a concentration of 10 lM (n = 3), which predicted a very low risk of inducing QT prolongation. Furthermore, compound 1o showed excellent physiochemical properties, with a molecular weight of 446.5, C log P value of 3.3 (calculated with Chemdraw) and high water solubility (data not shown). Based on its potent in vitro activity and favorable druglike profile, compound 1o was then chosen for further in vivo evaluation.27 The HCT-116 xenograft model in nude mice was selected for preliminary in vivo anticancer activity test of 1o.28 Because most HDAC inhibitors that are currently in the clinical trial can only be given intermittently due to the side effect of fatigue, which is
Scheme 5. Reagents and conditions: (a) DMF, Et2NH, HATU, DIPEA, rt, overnight, 73%; (b) MeOH, SOCl2, 70 °C, overnight, 83%; (c) BH3–SMe2 (2 M in THF), THF, 50 °C, overnight; then 2 M HCl, 70 °C, 1 h, 90%; (d) SOCl2, toluene, rt, 1 h, 87%.
Scheme 6. Reagents and conditions: (a) LiAlH4, THF, rt, overnight, 85%; (b) TBSCl, CH2Cl2, imidazole, Et3N, overnight, 78%; (c) DMF, NaH, MeI, rt, 2 h, 83%; (d) THF, TBAF, rt, overnight, 78%; (e) MeCN/Et2O, imidazole, Ph3P, I2, rt, 0.5 h, 87%.
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J. Cheng et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
Table 1 HDAC1 inhibitory activities of compounds 1a–1t Compound
R
1a
Table 2 GI50s of selected compounds (‘—’, not tested). IC50 (nM)
Compound
182
1c 1e 1i 1m 1n 1o 1p 1q Voninostat
N 152
1b
N 1c 1d
17
F
HCT-116
PC-3
HL-60
0.85 0.47 0.33 0.21 0.27 0.11 0.65 0.24 0.65
— — 0.43 0.38 0.34 0.10 — 0.21 0.96
— — 0.44 0.36 0.25 0.086 — 0.48 0.75
21
1e
O
12
1f
F3 C
80
1g
N
41
1h
GI50 (lM)
42
N
N 9.5
1i
N 1j
23
N
1k
25 Figure 2. Antitumor activity of compound 1o in HCT-116 xenograft model. 9.5
1l
1m
5.4
1n
5.4
N N
1o
1p
2.7
13
1q
4.0
Acknowledgment
250
We are grateful to Dr. Joel A. Bergman for helpful discussions and valuable suggestions.
N 1r
1s
O
1t Voninostat
—
for 3 weeks resulted in 54% TGI (Fig. 2).29 Doses in both groups were well tolerated and body weight losses of animals were within 15% (data not shown). The intermittent dosing schedule showed a similar antitumor effect with a total drug dosing amount less than the continuous dosing schedule. In summary, a focused library of HDAC inhibitors with phenyl imidazolidin-2-one as the new linker was designed and synthesized. Several compounds displayed potent HDAC1 inhibition and tumor cell growth inhibition in vitro. Compound 1o was evaluated in an HCT-116 xenograft model in vivo and showed significant tumor growth inhibition in both continuous and intermittent dosing schedules. Further evaluation of compound 1o is ongoing and will be reported in due time.
N
125
Supplementary data
N
114
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2014. 06.080. These data include MOL files and InChiKeys of the most important compounds described in this article.
13
considered to be a class effect, we tested the efficacy of 1o using both continuous and intermittent dosing schedules. Daily oral administration of 1o at 50 mg/kg for 3 weeks resulted in 45% tumor growth inhibition (TGI) when compared to the vehicle control; intermittent dosing schedule with 3 days on plus 3 days off
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21. Zhang, Z.; Yamashita, H.; Toyama, T.; Sugiura, H.; Ando, Y.; Mita, K.; Hamaguchi, M.; Hara, Y.; Kobayashi, S.; Iwase, H. Breast Cancer Res. Treat. 2005, 94, 11. 22. Compounds were screened against HDAC1 (purchased from BPS Bioscience) with Boc-Lys(Ac)-AMC substrate concentration at Km.Tests were run on 384-well plates and compounds were diluted in 10 concentrations and run in duplicate.The reaction contained 6.25 nM HDAC1, assay buffer (50 mM HEPES, pH 7.4, 100 mM KCl, 0.001% Tween-20, 0.05% BSA, 20 lM TCEP). 23. All cell lines were obtained from the American Type Culture Collection and cultivated according to the supplier’s instructions; Seeding density of HCT-116, PC-3 and HL-60 was 2000/well, 2000/well and 10000/well respectively (cells was counted with Vi-Cell XR) and incubation time was 72 h; all tests were done CellTiter Glo assay kit (Promega). 24. Molife, R.; Fong, P.; Scurr, M.; Judson, I.; Kaye, S.; de Bono, J. Clin. Cancer Res. 2007, 13, 1068. 25. Subramanian, S.; Bates, S.; Wright, J.; Espinoza-Delgado, I.; Piekarz, R. Pharmaceuticals 2010, 3, 2751. 26. Recanatini, M.; Poluzzi, E.; Masetti, M.; Cavalli, A.; De, P. F. Med. Res. Rev. 2005, 25, 133. 27. Selected characterization data for compound 1o: MS (ESI+): m/z 447 [M+H]; 1H NMR (300 MHz, DMSO-d6) d 11.13 (br, 1H), 8.91 (br, 1H), 8.13 (s, 1H), 7.97 (d, J = 6.3 Hz, 1H), 7.91 (d, J = 6.6 Hz, 1H), 7.86 (s, 1H), 7.82 (d, J = 6.3 Hz, 1H), 7.75 (d, J = 6.3 Hz, 2H), 7.65 (d, J = 6.6 Hz, 2H), 7.49 (d, J = 6.3 Hz, 1H), 4.57 (s, 2H), 4.40 (s, 2H), 3.84 (t, J = 6.0 Hz, 2H), 3.40 (t, J = 6.0 Hz, 2H), 3.03 (q, J = 5.4 Hz, 4H), 1.25 (t, J = 5.4 Hz, 6H); 13C NMR (75 MHz, DMSO-d6) d 164.4, 157.3, 143.5, 136.4, 133.3, 132.3, 131.0, 129.0, 128.8, 128.7, 128.4, 128.0, 127.2, 126.6, 125.9, 116.4, 55.2, 47.7, 46.2, 42.3, 41.3, 8.7; HRMS (ESI): calcd for C26H31N4O3 (M+H): 447.2396, found: 447.2391. 28. Evaluation of vorinostat in this model has been reported: Novotny-Diermayr, V.; Sangthongpitag, K.; Hu, C. Y.; Wu, X.; Sausgruber, N.; Yeo, P.; Greicius, G.; Pettersson, S.; Liang, A. L.; Loh, Y. K.; Bonday, Z.; Goh, K. C.; Hentze, H.; Hart, S.; Wang, H.; Ethirajulu, K.; Wood, J. M. Mol. Cancer Ther. 2010, 9, 642– 52.Vorinostat at a dose of 200 mg/kg, qd (formulated in 50% PEG400 in water (V/V)) was used as the positive control group for the in vivo test of compound 1o, but it displayed a significant toxicity and this group was terminated after 10 days dosing, with about 50% TGI at that time point. 29. Compound 1o was formulated in 50% PEG400 in water (V/V), vehicle control group was dosed with 50% PEG400 in water, and dosing volume was 10 mL/kg; Tumor volume (mm3) = (w2 l)/2, in which w is the width and l is the length of the tumor in mm; TGI was calculated using the following formula: %TGI = (Tday 21 Tday 0)/(Cday 21 Cday 0) 100 [in which Tday 21 is the median tumor volume for treatment group on day 21; Tday 0 is the median tumor volume for treatment group on day 0; Cday 21 is the median tumor volume for control group (vehicle) on day 21; Cday 0 is the median tumor volume for control group (vehicle) on day 0].
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