Bioorganic & Medicinal Chemistry Letters 24 (2014) 2319–2323
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Design, syntheses, and characterization of piperazine based chemokine receptor CCR5 antagonists as anti prostate cancer agents Christopher K. Arnatt a, Joanna L. Adams a, Zhu Zhang b, Kendra M. Haney a, Guo Li a, Yan Zhang a,c,⇑ a
Department of Medicinal Chemistry, School of Pharmacy, Virginia Commonwealth University, 800 East Leigh Street, Richmond, VA 23298, USA Department of Chemistry, College of Pharmacy, Tianjin Medical University, Tianjin 300070, China c Massey Cancer Center, Virginia Commonwealth University, Richmond, VA 23298, USA b
a r t i c l e
i n f o
Article history: Received 13 January 2014 Revised 19 March 2014 Accepted 24 March 2014 Available online 3 April 2014 Keywords: CCR5 Antagonists Prostate cancer Piperazine ring
a b s t r a c t Chemokine receptor CCR5 plays an important role in the pro-inflammatory environment that aids in the proliferation of prostate cancer cells. Previously, a series of CCR5 antagonists containing a piperidine ring core skeleton were designed based upon the proposed CCR5 antagonist pharmacophore from molecular modeling studies. The developed CCR5 antagonists were able to antagonize CCR5 at a micromolar level and inhibit the proliferation of metastatic prostate cancer cell lines. In order to further explore the structure–activity-relationship of the pharmacophore identified, the molecular scaffold was expanded to contain a piperazine ring as the core. A number of compounds that were synthesized showed promising anti prostate cancer activity and reasonable cytotoxicity profiles based on the biological characterization. Published by Elsevier Ltd.
Besides being a co-receptor for HIV-1 invasion to host cells, chemokine receptor CCR5 has been implicated in several types of cancer development due to its role in the inflammatory network of cells.1–8 Prostate cancer is the most prevalent form of cancer found in American men and is the second only to lung cancer as the most lethal one in men.9 It has been reported that the expression of the inflammatory chemokine RANTES (CCL5) correlates with the growth and survival of prostate cancer cells and mediates these actions through activation of the G protein-coupled receptor (GPCR) chemokine receptor CCR5 (CCR5).10–13 Among several CCR5 antagonists, TAK-779 (1), maraviroc (2), and our piperidine core containing lead compound (3), have been shown to inhibit prostate cancer cell proliferation and/or invasion induced by RANTES.10,14 Previously, the rational design of novel CCR5 antagonists containing the piperidine core skeleton was based on the homology modeling study of the CCR5 and the conformation analysis of several well-known CCR5 antagonists.14 It was found that these well-known CCR5 antagonists all shared a scaffold with a secondary or tertiary amino group at the center, then connected to an amide moiety with an aromatic moiety attached, and on the side of the scaffold a hydrophobic moiety was linked to the secondary or tertiary amine.14 From those observations, a novel molecular skeleton was designed accordingly to fit this proposed pharmacophore. The scaffold included a tri-substituted phenyl ring as the
⇑ Corresponding author. Tel.: +1 804 828 0021; fax: +1 804 828 7625. E-mail address: [email protected] (Y. Zhang). http://dx.doi.org/10.1016/j.bmcl.2014.03.073 0960-894X/Published by Elsevier Ltd.
spacer which connected the amino and amide groups (Fig. 1). Among all the ligands synthesized compound 3 was identified as a CCR5 antagonist and inhibited prostate cancer proliferation in vitro and in vivo.14 However, the therapeutic index of this compound against prostate cancer was hampered by its cytotoxicity. Therefore, another series of compounds was designed as isosteres15,16 of the previously explored ligands, that is, the piperidine ring was replaced with a piperazine ring system (Fig. 2), in order to improve their biological activity profile. It was believed that an additional nitrogen atom in the piperazine ring might allow another possible interaction between the receptor and the ligand to reinforce the binding of the ligand to the receptor. The lead compound 3 had an ethyl group as its bulky group and a phenyl ring as its aromatic group. However, the congruent compound with an isopropyl group as its bulky group and a pyrazinyl ring for its aromatic group had negligible cytotoxicity and still inhibited prostate cancer growth.14 Therefore, both the isopropyl group and pyrazinyl ring were adopted in the new molecular skeleton and the substituents on the phenyl group were varied based on the Topliss-tree scheme (Fig. 2). Scheme 1 showed the 11-step synthetic route applied to prepare the piperazine-based antagonists 13 through 32 (Fig. 3). A Williamson ether synthesis was used to alkylate 4-nitrophenol (4) with 2-bromopropane. This reaction was done in the presence of K2CO3 in dimethylformamide (DMF). Temperature control proved to be critical in this reaction; the reaction was kept constant at 105 °C and after 1 h of reaction, 99% yields of 5 were regularly achieved.
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F F H N O
HN
O N
N
N+
N N
H 2N Maraviroc 1
O
TAK-779 2
O H 2N
O
N H
N
Piperidine based lead 3 Figure 1. Example CCR5 antagonists used as the basis of CCR5 antagonist pharmacophore analysis.
O R
N N
NH O
O
R Bulky
R Aromatic
R
N N
N H
O N N
Figure 2. Molecular modeling based pharmacophore analysis, and designed CCR5 antagonist scaffold.
The catalytic hydrogenation of 5 to form the primary amine 6 was done with 10% palladium on carbon (Pd/C) under hydrogen gas and 1.3 equiv of concentrated HCl, with yields up to 98% and reaction times ranged from 1 to 2 h for up to 5 g of starting material. The cyclization of 6 to 7 to form the piperazine ring proved to be a very difficult and fastidious reaction. This reaction required temperatures above 130 °C, but below 150 °C in order to get 7 in appreciable yields. Immediately after workup, compound 7 was protected with trifluoroacetic anhydride to form 8 with consistent yields around 94%. Mono-nitration of 8 to introduce the meta-nitro group was not obtainable through normal aromatic nitration. Using several different reaction conditions with acetic acid or acetic anhydride and nitric acid, only di-meta nitration product was obtained. It was speculated that due to the presence of both the 1-propyloxy group and the 4-piperazine group with electron donating capabilities, the aromatic ring was highly activated. This in turn made mono-nitration unlikely with conventional synthetic routes. Therefore, a nitrocyclohexadienone as a mild nitrating reagent was adopted successfully. This reagent has previously been shown to mono-nitrate several activated substrates without leading to the usual oxidative byproducts of normal aromatic nitration.17 Subsequent reduction of the nitro group of 9 to the amine, 10, was done using Pd/C catalyzed hydrogenation with yields around 98% without any complications. The amide-coupling of 10 with pyrazine-2-carboxylic acid to form 11 was done using 1-ethyl3-(3-dimethylaminopropyl) carbodiimide (EDCI) with yields up to 99%. Deprotection of the piperazine moiety was done by refluxing 12 under basic conditions in a methanol/water mixture (1:1). Yields of 12 for this reaction were quantitative. Compounds 13 through 32 all used the key intermediate 12, which allowed for final compounds to be synthesized efficiently. For compounds 13 through 30 the benzyl chloride was coupled with the secondary amine of the piperazine group. This reaction was done in DMF with
K2CO3 and a catalytic amount of KI. The reaction yields ranged from 35% to 94%. Both 31 and 32 had to be prepared via a reductive amination and were synthesized with reasonable yields. This series of newly synthesized compounds were first tested for their CCR5 agonism and antagonism in a calcium mobilization assay using MOLT-4 cells transfected with CCR5 (NIH AIDS Research and Reference Reagent Program).18 Using the calcium sensing dye Fluo-4, compounds 13 through 32 were first tested for their CCR5 agonism and none of them showed any agonism up to 30 lM. The compounds were then tested for their antagonism of RANTESstimulated calcium release. Table 1 showed the results of three independent assays each done in triplicate. All of the compounds antagonized the RANTES stimulated calcium flux on the CCR5 at micromolar levels. These compounds’ antagonism were relatively low compared to Maraviroc and TAK-779,19 but comparable to our original series of designed ligands.14 Some of them actually acted more potently than the lead compound 3. Among them, compound 13 showed an IC50 value of 10.3 ± 4.2 lM. Using this unsubstituted phenyl compound as a reference several observations can be summarized on the structure activity relationship of these newly synthesized ligands. As seen for compounds 13 through 25, there was a general trend that electron withdrawing substituents led to lower IC50 values than electron donating groups with some exceptions, for example, compounds 19 and 20. On another note, the substitution position seemed to play a key role in antagonism as seen for the series of nitro-substituted compounds. para substitution (14) yields the lowest IC50 value of 6.45 ± 1.7 lM, while as ortho substitution (16) is also well tolerated with a slightly higher IC50 value of 16.8 ± 6.2 lM. However, meta and di-meta substituted nitrogroups (15 and 17) did not show any CCR5 antagonism below 50 lM. Overall, CCR5 calcium signaling inhibition was optimized with para substituted electron withdrawing groups. Further exploration of the bulkiness of para-substitutions did not show
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OH
NO2
Cl
O
O
Br
HN
K2CO3 DMF 105 oC
10% Pd/C H2, HCL MeOH
NO2
4
K2CO3 Chlorobenzene Reflux
NH2 . HCl
5
O
Cl
6
N N H 7
Br O
Br
O NO2
THF R.T.
N
NH2 10% Pd/C H2 MeOH
N
F3C
O
N O
F3C
9
8
O 10
Cl
R
O N
N
N
N F3 C
O
Br Br H3C NO2
(CF3CO)2O Py CH2Cl2 M.S. 0 oC to R.T.
O
OH
N
O
H N
N
N O
EDCI, HOBT TEA, DMF, M.S.
O
0 oC to R.T. N F3C
N R N H
O
12
11
DMF R.T.
N O
K2CO3 MeOH/H2O Reflux
N
N H N
13 - 30
KI, K2CO3
O
31, 32
NaBH(OAc) 3 THF, M.S. R.T.
Figure 3. Synthetic route for CCR5 antagonists 13–32.
Table 1 CCR5 antagonism (calcium mobilization) of compounds 14 through 35 using RANTES as the agonist
*
Compound #
Substitution
IC50 (lM)
3 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
N/A H 4-NO2 3-NO2 2-NO2 3,5-NO2 4-Cl 4-CN 4-F 4-Br 4-CO2CH3 4-COOH 4-SO3CH3 4-SO2CH3 4-CH3 4-NH2 4-OCH3 4-SCH3 4-NH2COCH3 4-N(CH3)2 4-N(CH2CH3)2
63.0 ± 26.0 10.3 ± 4.2 6.45 ± 1.7 >50* 16.8 ± 6.2 >50* 6.29 ± 2.4 >50* >50* 23 ± 18 9.8 ± 7.1 41.2 ± 2.9 >50* 45.8 ± 9.1 45.9 ± 26.7 27 ± 14 19.8 ± 2.9 5.1 ± 3.4 25.6 ± 2.1 28.1 ± 4.0 48.0 ± 2.9
(>50) Denotes that the IC50 was above 50 lM and was not tested at higher concentrations due to limited solubility in the specific testing media.
any significant trend though it seemed that larger moiety seemed to be less favorable for the CCR5 antagonism of these compounds. An anti-proliferation assay using the colorimetric reagent, WST1, was applied to test compounds against two prostate cancer cell lines, PC-3, and M12. They were chosen based upon their high expression of CCR5 and RANTES.20 M12 cells were originally isolated from the prostate gland and selected for more metastatic cells from tumors in mice. PC-3 cells were obtained from a metastatic prostate tumor obtained from a lumbar vertebra.21 Table 2 showed the anti-proliferation data in both prostate cancer cell lines for compounds with reasonable CCR5 antagonism. When tested against PC-3 cells, the unsubstituted compound 13 had an IC50 value of 67 ± 15 lM; further substitution has a drastic effect on the activity of the compounds. First, unlike what is seen for calcium antagonism, there is no significant effect on anti-proliferation activity for substitution position, as seen in the series of nitro group substituted compounds. Second, electron donating groups are generally more favored than electron withdrawing groups, and for example, the diethylamino substituted compound 32 had the lowest IC50 of 6.5 ± 0.7 lM. For M12 cells, 32 also showed the lowest IC50 value of 11.4 ± 0.2 lM. Overall, the same trend of electron donating groups having higher anti-proliferation activity was seen for both cell lines. Compound 32 seemed to carry improved anti-proliferation activity against PC-3 and M12 when
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Table 2 Anti-proliferation assays for PC-3 and M12 prostate cancer cells using WST-1 to measure cell proliferation
Table 3 Basal cytotoxicity assays using NRU and WST-1 to test for exogenous toxicity of compounds 14 through 32 in NIH 3T3 cells
Compound #
Substitution
PC-3 IC50 (lM)
M12 IC50 (lM)
Compound #
Substitution
NIH 3T3 (NRU) TC50 (lM)
TAK-779 (2) 3 13 14 16 18 21 22 23 25 26 27 29 30 31 32
N/A N/A H 4-NO2 2-NO2 4-Cl 4-Br 4-CO2CH3 4-COOH 4-SO2CH3 4-CH3 4-NH2 4-SCH3 4-NH2COCH3 4-N(CH3)2 4-N(CH2CH3)2
37.85 ± 0.99 43.0 ± 2.3 67 ± 15 49.1 ± 6.6 62.9 ± 9.5 56.1 ± 9.6 31.5 ± 1.4 91.5 ± 3.7 74 ± 12 >100* 19.7 ± 1.8 20.1 ± 1.3 78.0 ± 19 24.3 ± 1.6 71.2 ± 1.6 6.5 ± 0.7
20.40 ± 1.10 31.9 ± 0.42 29.9 ± 4.3 78.7 ± 4.0 183 ± 2.5 26.3 ± 0.8 48.8 ± 25 73.7 ± 20 129 ± 19 141 ± 26 15.8 ± 4.4 19.7 ± 5.3 >100* 39 ± 12 65.9 ± 2.9 11.4 ± 0.2
3 13 14 16 18 21 27 30 31 32
N/A H 4-NO2 2-NO2 4-Cl 4-Br 4-NH2 4-NH2COCH3 4-N(CH3)2 4-N(CH2CH3)2
84.0 ± 11.0 46.6 ± 3.2 29.3 ± 2.3 >50* 1.6 ± 0.8 >50* 10.7 ± 1.2 7.8 ± 1.1 >50* 31.9 ± 1.6
*
(>100) Denotes that the IC50 was above 100 lM and was not tested at higher concentrations due to limited solubility in the specific testing media.
*
(>50) Denotes that the IC50 was above 50 lM and was not tested at higher concentrations due to limited solubility in the specific testing media.
31.9 ± 1.6 lM. Further structural modification of this lead will be conducted in order to improve its anti cancer activity profile. Acknowledgments
compared to TAK-779 (2) which was shown to have IC50’s of 20.40 ± 1.1 lM (M12) and 37.85 ± 0.99 lM (PC-3), and to the lead (3) which was shown to have IC50’s of 31.9 ± 0.42 lM (M12) and 43.0 ± 2.3 lM (PC-3), respectively.22 In the view of correlating the ligands’ shown anti prostate cancer activity and their CCR5 antagonism activity we did not observe a significant correlation, probably due to our limited size of compound pool. Another possible explanation is that this could be related to the functional selectivity of our ligands on the target receptor CCR5. To ensure the anti-proliferation results are not due to the toxicity of the compounds, a basal cytotoxicity assay was conducted for the more promising ligands from the anti-proliferation assays. The NIH-3T3 cells used for the assay are mouse fibroblasts that have been used extensively along with neutral red uptake (NRU) to assess basal cytotoxicity levels of small molecules.23,24 In all, several of the compounds (e.g., compound 27) that showed high anti-proliferative activity in both M12 and PC-3 were also cytotoxic in NIH3T3 cells, which indicates their observed anti-proliferative activity may be related to their cytotoxicity (Table 3). On the other hand, compound 32 showed a TC50 of 31.9 ± 1.6 lM which indicated a somehow promising toxicity profile for further exploration though the TC50 value was relatively lower than the lead compound 3. Accumulating evidence has shown the multiple roles that chemokine receptor CCR5 plays to promote the progression of several types of cancer. The mechanism of action of this promotion is thought to involve chronic inflammation, which creates a microenvironment that enhances tumor survival. Blocking CCR5 function with an antagonist may provide a novel treatment of cancers such as prostate cancer.25–28 Currently, several CCR5 antagonists are available, but all have been optimized for their anti-HIV entry inhibition rather than inhibition in endogenous signaling. Thus, there is need to develop antagonists focused on blocking CCR5 signaling, and inhibiting CCR5 related prostate cancer proliferation. Using a combination of pharmacophore analysis and CCR5 docking studies, a unique CCR5 antagonist skeleton was designed and functionalized at multiple positions to optimize its activity. A combination of calcium inhibition, anti-proliferation, and basal cytotoxicity assays were used to screen for active compounds. In the CCR5 calcium mobilization inhibition assays all of the synthesized compounds acted as antagonists. From the anti-proliferation assays and the basal cytotoxicity assays, compound 32 was identified as a favorable lead compound by carrying an IC50 value of 11.4 ± 0.2 lM and 6.5 ± 0.7 lM in M12 and PC-3 prostate cancer cells, respectively, and a basal cytotoxicity of TC50 of
We are grateful for the partial funding support from US Army Prostate Cancer Research Program PC073739 and A. D. Williams MultiSchool Research Funds at Virginia Commonwealth University. The content of this report is solely the responsibility of the authors and does not necessarily represent the official views of the US Army Prostate Cancer Research Program. We thank the NIH AIDS Research and Reference Reagent Program for providing the MOLT-4/CCR5 cell line. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.03. 073. References and notes 1. Chen, W.; Zhan, P.; DeClercq, E.; Liu, X. Curr. Pharm. Des. 2012, 18, 100. 2. Dorr, P.; Westby, M.; Dobbs, S.; Griffin, P.; Irvine, B.; Macartney, M.; Mori, G.; Rickett, G.; Smith-Burchnell, C.; Napier, C.; Webster, R.; Armour, D.; Price, D.; Stammen, B.; Wood, A.; Perros, M. Antimicrob. Agents Chemother. 2005, 49, 4721. 3. Wood, A.; Armour, D. Prog. Med. Chem. 2005, 43, 239. 4. Tagat, J. R.; McCombie, S. W.; Nazareno, D.; Labroli, M. A.; Xiao, Y.; Steensma, R. W.; Strizki, J. M.; Baroudy, B. M.; Cox, K.; Lachowicz, J.; Varty, G.; Watkins, R. J. Med. Chem. 2004, 47, 2405. 5. Strizki, J. M.; Tremblay, C.; Xu, S.; Wojcik, L.; Wagner, N.; Gonsiorek, W.; Hipkin, R. W.; Chou, C. C.; Pugliese-Sivo, C.; Xiao, Y.; Tagat, J. R.; Cox, K.; Priestley, T.; Sorota, S.; Huang, W.; Hirsch, M.; Reyes, G. R.; Haroudy, B. M. Antimicrob. Agents Chemother. 2005, 49, 4911. 6. Palani, A.; Tagat, J. J. Med. Chem. 2006, 49, 2851. 7. Baba, M.; Nishimura, O.; Kanzaki, N.; Okamoto, M.; Sawada, H.; Iizawa, Y.; Shiraishi, M.; Aramaki, Y.; Okonogi, K.; Ogawa, Y.; Meguro, K.; Fujino, M. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 5698. 8. Maeda, K.; Nakata, H.; Koh, Y.; Miyakawa, T.; Ogata, H.; Takaoka, Y.; Shibayama, S.; Sagawa, K.; Fukushima, D.; Moravek, J.; Koyanagi, Y.; Mitsuya, H. J. J. Virol. 2004, 78, 8654. 9. American Cancer Society. http://www.cancer.org/research/cancerfactsstatistics/ index (accessed Nov 9, 2012). 10. Vaday, G. G.; Peehl, D. M.; Kadam, P. A.; Lawrence, D. M. Prostate 2005, 66, 124. 11. Coussens, L. M.; Werb, Z. Nature 2002, 420, 860. 12. Robinson, S. C.; Scott, K. A.; Wilson, J. L.; Thompson, R. G.; Proudfoot, A. E. I.; Balkwill, F. R. Cancer Res. 2003, 63, 8360. 13. Koenig, J. E.; Senge, T.; Allhoff, E. P.; Koenig, W. Prostate 2004, 58, 121. 14. Arnatt, C. K.; Zaidi, S. A.; Zhang, Z.; Li, G.; Richardson, A. C.; Ware, J. L.; Zhang, Y. Eur. J. Med. Chem. 2013, 69, 647. 15. Langmuir, I. J. Am. Chem. Soc. 1919, 41, 1543. 16. Patani, G. A.; LaVoie, E. J. Chem. Rev. 1996, 96, 3147. 17. Arnatt, C. K.; Zhang, Y. Tetrahedron Lett. 2012, 53, 1592. 18. Baba, M.; Miyake, H.; Okamoto, M.; Iizawa, Y.; Okonogi, K. AIDS Res. Hum. Retroviruses 2000, 16, 935. 19. Yuan, Y.; Arnatt, C. K.; Li, G.; Haney, K. M.; Ding, D.; Jacob, J. C.; Selley, D. E.; Zhang, Y. Org. Biomol. Chem. 2012, 10, 2633.
C. K. Arnatt et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2319–2323 20. Zhang, X.; Haney, K. M.; Richardson, A. C.; Wilson, E.; Gewirtz, D. A.; Ware, J. L.; Zehner, Z. E.; Zhang, Y. Bioorg. Med. Chem. Lett. 2010, 20, 4627. 21. Webber, M. M.; Bello, D.; Quadar, S. Prostate 1997, 30, 58. 22. Haney, K. M.; Zhang, F.; Arnatt, C. K.; Yuan, Y.; Li, G.; Ware, J. L.; Gewirtz, D. A.; Zhang, Y. Bioorg. Med. Chem. Lett. 2011, 21, 5159. 23. Borenfreund, E.; Puemer, J. A. Toxicol. Lett. 1985, 24, 119. 24. National Toxicology Program (NTP) Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM) Test method protocol for the
25. 26. 27. 28.
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BALB/c 3T3 neutral red uptake cytotoxicity test. A. T. f. B. C. F. a. I. V. V. S., phase III; 2003. Dorff, T. B.; Glode, L. M. Curr. Opin. Urol. 2013, 23, 366. Loblaw, D. A.; Walker-Dilks, C.; Winquist, E.; Hotte, S. J.; G.C.D.S.G. of C.C.O.P. in E.-B Clin. Oncol. (R. Coll. Radiol.) 2013, 25, 406. Jiaoyan, H.; Fangqiang, Z.; Xiang, X.; Hong, H.; Wenqiu, H.; Wenhui, C. J. Third Mil. Med. Univ. 2013, 35, 105. Zou, C.; Li, X.; Jiang, R. Chin. J. Androl. 2012, 26, 66.
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Design, syntheses, and characterization of piperazine based chemokine receptor CCR5 antagonists as anti prostate cancer agents Christopher K. Arnatt a, Joanna L. Adams a, Zhu Zhang b, Kendra M. Haney a, Guo Li a, Yan Zhang a,c,⇑ a
Department of Medicinal Chemistry, School of Pharmacy, Virginia Commonwealth University, 800 East Leigh Street, Richmond, VA 23298, USA Department of Chemistry, College of Pharmacy, Tianjin Medical University, Tianjin 300070, China c Massey Cancer Center, Virginia Commonwealth University, Richmond, VA 23298, USA b
a r t i c l e
i n f o
Article history: Received 13 January 2014 Revised 19 March 2014 Accepted 24 March 2014 Available online 3 April 2014 Keywords: CCR5 Antagonists Prostate cancer Piperazine ring
a b s t r a c t Chemokine receptor CCR5 plays an important role in the pro-inflammatory environment that aids in the proliferation of prostate cancer cells. Previously, a series of CCR5 antagonists containing a piperidine ring core skeleton were designed based upon the proposed CCR5 antagonist pharmacophore from molecular modeling studies. The developed CCR5 antagonists were able to antagonize CCR5 at a micromolar level and inhibit the proliferation of metastatic prostate cancer cell lines. In order to further explore the structure–activity-relationship of the pharmacophore identified, the molecular scaffold was expanded to contain a piperazine ring as the core. A number of compounds that were synthesized showed promising anti prostate cancer activity and reasonable cytotoxicity profiles based on the biological characterization. Published by Elsevier Ltd.
Besides being a co-receptor for HIV-1 invasion to host cells, chemokine receptor CCR5 has been implicated in several types of cancer development due to its role in the inflammatory network of cells.1–8 Prostate cancer is the most prevalent form of cancer found in American men and is the second only to lung cancer as the most lethal one in men.9 It has been reported that the expression of the inflammatory chemokine RANTES (CCL5) correlates with the growth and survival of prostate cancer cells and mediates these actions through activation of the G protein-coupled receptor (GPCR) chemokine receptor CCR5 (CCR5).10–13 Among several CCR5 antagonists, TAK-779 (1), maraviroc (2), and our piperidine core containing lead compound (3), have been shown to inhibit prostate cancer cell proliferation and/or invasion induced by RANTES.10,14 Previously, the rational design of novel CCR5 antagonists containing the piperidine core skeleton was based on the homology modeling study of the CCR5 and the conformation analysis of several well-known CCR5 antagonists.14 It was found that these well-known CCR5 antagonists all shared a scaffold with a secondary or tertiary amino group at the center, then connected to an amide moiety with an aromatic moiety attached, and on the side of the scaffold a hydrophobic moiety was linked to the secondary or tertiary amine.14 From those observations, a novel molecular skeleton was designed accordingly to fit this proposed pharmacophore. The scaffold included a tri-substituted phenyl ring as the
⇑ Corresponding author. Tel.: +1 804 828 0021; fax: +1 804 828 7625. E-mail address: [email protected] (Y. Zhang). http://dx.doi.org/10.1016/j.bmcl.2014.03.073 0960-894X/Published by Elsevier Ltd.
spacer which connected the amino and amide groups (Fig. 1). Among all the ligands synthesized compound 3 was identified as a CCR5 antagonist and inhibited prostate cancer proliferation in vitro and in vivo.14 However, the therapeutic index of this compound against prostate cancer was hampered by its cytotoxicity. Therefore, another series of compounds was designed as isosteres15,16 of the previously explored ligands, that is, the piperidine ring was replaced with a piperazine ring system (Fig. 2), in order to improve their biological activity profile. It was believed that an additional nitrogen atom in the piperazine ring might allow another possible interaction between the receptor and the ligand to reinforce the binding of the ligand to the receptor. The lead compound 3 had an ethyl group as its bulky group and a phenyl ring as its aromatic group. However, the congruent compound with an isopropyl group as its bulky group and a pyrazinyl ring for its aromatic group had negligible cytotoxicity and still inhibited prostate cancer growth.14 Therefore, both the isopropyl group and pyrazinyl ring were adopted in the new molecular skeleton and the substituents on the phenyl group were varied based on the Topliss-tree scheme (Fig. 2). Scheme 1 showed the 11-step synthetic route applied to prepare the piperazine-based antagonists 13 through 32 (Fig. 3). A Williamson ether synthesis was used to alkylate 4-nitrophenol (4) with 2-bromopropane. This reaction was done in the presence of K2CO3 in dimethylformamide (DMF). Temperature control proved to be critical in this reaction; the reaction was kept constant at 105 °C and after 1 h of reaction, 99% yields of 5 were regularly achieved.
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F F H N O
HN
O N
N
N+
N N
H 2N Maraviroc 1
O
TAK-779 2
O H 2N
O
N H
N
Piperidine based lead 3 Figure 1. Example CCR5 antagonists used as the basis of CCR5 antagonist pharmacophore analysis.
O R
N N
NH O
O
R Bulky
R Aromatic
R
N N
N H
O N N
Figure 2. Molecular modeling based pharmacophore analysis, and designed CCR5 antagonist scaffold.
The catalytic hydrogenation of 5 to form the primary amine 6 was done with 10% palladium on carbon (Pd/C) under hydrogen gas and 1.3 equiv of concentrated HCl, with yields up to 98% and reaction times ranged from 1 to 2 h for up to 5 g of starting material. The cyclization of 6 to 7 to form the piperazine ring proved to be a very difficult and fastidious reaction. This reaction required temperatures above 130 °C, but below 150 °C in order to get 7 in appreciable yields. Immediately after workup, compound 7 was protected with trifluoroacetic anhydride to form 8 with consistent yields around 94%. Mono-nitration of 8 to introduce the meta-nitro group was not obtainable through normal aromatic nitration. Using several different reaction conditions with acetic acid or acetic anhydride and nitric acid, only di-meta nitration product was obtained. It was speculated that due to the presence of both the 1-propyloxy group and the 4-piperazine group with electron donating capabilities, the aromatic ring was highly activated. This in turn made mono-nitration unlikely with conventional synthetic routes. Therefore, a nitrocyclohexadienone as a mild nitrating reagent was adopted successfully. This reagent has previously been shown to mono-nitrate several activated substrates without leading to the usual oxidative byproducts of normal aromatic nitration.17 Subsequent reduction of the nitro group of 9 to the amine, 10, was done using Pd/C catalyzed hydrogenation with yields around 98% without any complications. The amide-coupling of 10 with pyrazine-2-carboxylic acid to form 11 was done using 1-ethyl3-(3-dimethylaminopropyl) carbodiimide (EDCI) with yields up to 99%. Deprotection of the piperazine moiety was done by refluxing 12 under basic conditions in a methanol/water mixture (1:1). Yields of 12 for this reaction were quantitative. Compounds 13 through 32 all used the key intermediate 12, which allowed for final compounds to be synthesized efficiently. For compounds 13 through 30 the benzyl chloride was coupled with the secondary amine of the piperazine group. This reaction was done in DMF with
K2CO3 and a catalytic amount of KI. The reaction yields ranged from 35% to 94%. Both 31 and 32 had to be prepared via a reductive amination and were synthesized with reasonable yields. This series of newly synthesized compounds were first tested for their CCR5 agonism and antagonism in a calcium mobilization assay using MOLT-4 cells transfected with CCR5 (NIH AIDS Research and Reference Reagent Program).18 Using the calcium sensing dye Fluo-4, compounds 13 through 32 were first tested for their CCR5 agonism and none of them showed any agonism up to 30 lM. The compounds were then tested for their antagonism of RANTESstimulated calcium release. Table 1 showed the results of three independent assays each done in triplicate. All of the compounds antagonized the RANTES stimulated calcium flux on the CCR5 at micromolar levels. These compounds’ antagonism were relatively low compared to Maraviroc and TAK-779,19 but comparable to our original series of designed ligands.14 Some of them actually acted more potently than the lead compound 3. Among them, compound 13 showed an IC50 value of 10.3 ± 4.2 lM. Using this unsubstituted phenyl compound as a reference several observations can be summarized on the structure activity relationship of these newly synthesized ligands. As seen for compounds 13 through 25, there was a general trend that electron withdrawing substituents led to lower IC50 values than electron donating groups with some exceptions, for example, compounds 19 and 20. On another note, the substitution position seemed to play a key role in antagonism as seen for the series of nitro-substituted compounds. para substitution (14) yields the lowest IC50 value of 6.45 ± 1.7 lM, while as ortho substitution (16) is also well tolerated with a slightly higher IC50 value of 16.8 ± 6.2 lM. However, meta and di-meta substituted nitrogroups (15 and 17) did not show any CCR5 antagonism below 50 lM. Overall, CCR5 calcium signaling inhibition was optimized with para substituted electron withdrawing groups. Further exploration of the bulkiness of para-substitutions did not show
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OH
NO2
Cl
O
O
Br
HN
K2CO3 DMF 105 oC
10% Pd/C H2, HCL MeOH
NO2
4
K2CO3 Chlorobenzene Reflux
NH2 . HCl
5
O
Cl
6
N N H 7
Br O
Br
O NO2
THF R.T.
N
NH2 10% Pd/C H2 MeOH
N
F3C
O
N O
F3C
9
8
O 10
Cl
R
O N
N
N
N F3 C
O
Br Br H3C NO2
(CF3CO)2O Py CH2Cl2 M.S. 0 oC to R.T.
O
OH
N
O
H N
N
N O
EDCI, HOBT TEA, DMF, M.S.
O
0 oC to R.T. N F3C
N R N H
O
12
11
DMF R.T.
N O
K2CO3 MeOH/H2O Reflux
N
N H N
13 - 30
KI, K2CO3
O
31, 32
NaBH(OAc) 3 THF, M.S. R.T.
Figure 3. Synthetic route for CCR5 antagonists 13–32.
Table 1 CCR5 antagonism (calcium mobilization) of compounds 14 through 35 using RANTES as the agonist
*
Compound #
Substitution
IC50 (lM)
3 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
N/A H 4-NO2 3-NO2 2-NO2 3,5-NO2 4-Cl 4-CN 4-F 4-Br 4-CO2CH3 4-COOH 4-SO3CH3 4-SO2CH3 4-CH3 4-NH2 4-OCH3 4-SCH3 4-NH2COCH3 4-N(CH3)2 4-N(CH2CH3)2
63.0 ± 26.0 10.3 ± 4.2 6.45 ± 1.7 >50* 16.8 ± 6.2 >50* 6.29 ± 2.4 >50* >50* 23 ± 18 9.8 ± 7.1 41.2 ± 2.9 >50* 45.8 ± 9.1 45.9 ± 26.7 27 ± 14 19.8 ± 2.9 5.1 ± 3.4 25.6 ± 2.1 28.1 ± 4.0 48.0 ± 2.9
(>50) Denotes that the IC50 was above 50 lM and was not tested at higher concentrations due to limited solubility in the specific testing media.
any significant trend though it seemed that larger moiety seemed to be less favorable for the CCR5 antagonism of these compounds. An anti-proliferation assay using the colorimetric reagent, WST1, was applied to test compounds against two prostate cancer cell lines, PC-3, and M12. They were chosen based upon their high expression of CCR5 and RANTES.20 M12 cells were originally isolated from the prostate gland and selected for more metastatic cells from tumors in mice. PC-3 cells were obtained from a metastatic prostate tumor obtained from a lumbar vertebra.21 Table 2 showed the anti-proliferation data in both prostate cancer cell lines for compounds with reasonable CCR5 antagonism. When tested against PC-3 cells, the unsubstituted compound 13 had an IC50 value of 67 ± 15 lM; further substitution has a drastic effect on the activity of the compounds. First, unlike what is seen for calcium antagonism, there is no significant effect on anti-proliferation activity for substitution position, as seen in the series of nitro group substituted compounds. Second, electron donating groups are generally more favored than electron withdrawing groups, and for example, the diethylamino substituted compound 32 had the lowest IC50 of 6.5 ± 0.7 lM. For M12 cells, 32 also showed the lowest IC50 value of 11.4 ± 0.2 lM. Overall, the same trend of electron donating groups having higher anti-proliferation activity was seen for both cell lines. Compound 32 seemed to carry improved anti-proliferation activity against PC-3 and M12 when
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Table 2 Anti-proliferation assays for PC-3 and M12 prostate cancer cells using WST-1 to measure cell proliferation
Table 3 Basal cytotoxicity assays using NRU and WST-1 to test for exogenous toxicity of compounds 14 through 32 in NIH 3T3 cells
Compound #
Substitution
PC-3 IC50 (lM)
M12 IC50 (lM)
Compound #
Substitution
NIH 3T3 (NRU) TC50 (lM)
TAK-779 (2) 3 13 14 16 18 21 22 23 25 26 27 29 30 31 32
N/A N/A H 4-NO2 2-NO2 4-Cl 4-Br 4-CO2CH3 4-COOH 4-SO2CH3 4-CH3 4-NH2 4-SCH3 4-NH2COCH3 4-N(CH3)2 4-N(CH2CH3)2
37.85 ± 0.99 43.0 ± 2.3 67 ± 15 49.1 ± 6.6 62.9 ± 9.5 56.1 ± 9.6 31.5 ± 1.4 91.5 ± 3.7 74 ± 12 >100* 19.7 ± 1.8 20.1 ± 1.3 78.0 ± 19 24.3 ± 1.6 71.2 ± 1.6 6.5 ± 0.7
20.40 ± 1.10 31.9 ± 0.42 29.9 ± 4.3 78.7 ± 4.0 183 ± 2.5 26.3 ± 0.8 48.8 ± 25 73.7 ± 20 129 ± 19 141 ± 26 15.8 ± 4.4 19.7 ± 5.3 >100* 39 ± 12 65.9 ± 2.9 11.4 ± 0.2
3 13 14 16 18 21 27 30 31 32
N/A H 4-NO2 2-NO2 4-Cl 4-Br 4-NH2 4-NH2COCH3 4-N(CH3)2 4-N(CH2CH3)2
84.0 ± 11.0 46.6 ± 3.2 29.3 ± 2.3 >50* 1.6 ± 0.8 >50* 10.7 ± 1.2 7.8 ± 1.1 >50* 31.9 ± 1.6
*
(>100) Denotes that the IC50 was above 100 lM and was not tested at higher concentrations due to limited solubility in the specific testing media.
*
(>50) Denotes that the IC50 was above 50 lM and was not tested at higher concentrations due to limited solubility in the specific testing media.
31.9 ± 1.6 lM. Further structural modification of this lead will be conducted in order to improve its anti cancer activity profile. Acknowledgments
compared to TAK-779 (2) which was shown to have IC50’s of 20.40 ± 1.1 lM (M12) and 37.85 ± 0.99 lM (PC-3), and to the lead (3) which was shown to have IC50’s of 31.9 ± 0.42 lM (M12) and 43.0 ± 2.3 lM (PC-3), respectively.22 In the view of correlating the ligands’ shown anti prostate cancer activity and their CCR5 antagonism activity we did not observe a significant correlation, probably due to our limited size of compound pool. Another possible explanation is that this could be related to the functional selectivity of our ligands on the target receptor CCR5. To ensure the anti-proliferation results are not due to the toxicity of the compounds, a basal cytotoxicity assay was conducted for the more promising ligands from the anti-proliferation assays. The NIH-3T3 cells used for the assay are mouse fibroblasts that have been used extensively along with neutral red uptake (NRU) to assess basal cytotoxicity levels of small molecules.23,24 In all, several of the compounds (e.g., compound 27) that showed high anti-proliferative activity in both M12 and PC-3 were also cytotoxic in NIH3T3 cells, which indicates their observed anti-proliferative activity may be related to their cytotoxicity (Table 3). On the other hand, compound 32 showed a TC50 of 31.9 ± 1.6 lM which indicated a somehow promising toxicity profile for further exploration though the TC50 value was relatively lower than the lead compound 3. Accumulating evidence has shown the multiple roles that chemokine receptor CCR5 plays to promote the progression of several types of cancer. The mechanism of action of this promotion is thought to involve chronic inflammation, which creates a microenvironment that enhances tumor survival. Blocking CCR5 function with an antagonist may provide a novel treatment of cancers such as prostate cancer.25–28 Currently, several CCR5 antagonists are available, but all have been optimized for their anti-HIV entry inhibition rather than inhibition in endogenous signaling. Thus, there is need to develop antagonists focused on blocking CCR5 signaling, and inhibiting CCR5 related prostate cancer proliferation. Using a combination of pharmacophore analysis and CCR5 docking studies, a unique CCR5 antagonist skeleton was designed and functionalized at multiple positions to optimize its activity. A combination of calcium inhibition, anti-proliferation, and basal cytotoxicity assays were used to screen for active compounds. In the CCR5 calcium mobilization inhibition assays all of the synthesized compounds acted as antagonists. From the anti-proliferation assays and the basal cytotoxicity assays, compound 32 was identified as a favorable lead compound by carrying an IC50 value of 11.4 ± 0.2 lM and 6.5 ± 0.7 lM in M12 and PC-3 prostate cancer cells, respectively, and a basal cytotoxicity of TC50 of
We are grateful for the partial funding support from US Army Prostate Cancer Research Program PC073739 and A. D. Williams MultiSchool Research Funds at Virginia Commonwealth University. The content of this report is solely the responsibility of the authors and does not necessarily represent the official views of the US Army Prostate Cancer Research Program. We thank the NIH AIDS Research and Reference Reagent Program for providing the MOLT-4/CCR5 cell line. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.03. 073. References and notes 1. Chen, W.; Zhan, P.; DeClercq, E.; Liu, X. Curr. Pharm. Des. 2012, 18, 100. 2. Dorr, P.; Westby, M.; Dobbs, S.; Griffin, P.; Irvine, B.; Macartney, M.; Mori, G.; Rickett, G.; Smith-Burchnell, C.; Napier, C.; Webster, R.; Armour, D.; Price, D.; Stammen, B.; Wood, A.; Perros, M. Antimicrob. Agents Chemother. 2005, 49, 4721. 3. Wood, A.; Armour, D. Prog. Med. Chem. 2005, 43, 239. 4. Tagat, J. R.; McCombie, S. W.; Nazareno, D.; Labroli, M. A.; Xiao, Y.; Steensma, R. W.; Strizki, J. M.; Baroudy, B. M.; Cox, K.; Lachowicz, J.; Varty, G.; Watkins, R. J. Med. Chem. 2004, 47, 2405. 5. Strizki, J. M.; Tremblay, C.; Xu, S.; Wojcik, L.; Wagner, N.; Gonsiorek, W.; Hipkin, R. W.; Chou, C. C.; Pugliese-Sivo, C.; Xiao, Y.; Tagat, J. R.; Cox, K.; Priestley, T.; Sorota, S.; Huang, W.; Hirsch, M.; Reyes, G. R.; Haroudy, B. M. Antimicrob. Agents Chemother. 2005, 49, 4911. 6. Palani, A.; Tagat, J. J. Med. Chem. 2006, 49, 2851. 7. Baba, M.; Nishimura, O.; Kanzaki, N.; Okamoto, M.; Sawada, H.; Iizawa, Y.; Shiraishi, M.; Aramaki, Y.; Okonogi, K.; Ogawa, Y.; Meguro, K.; Fujino, M. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 5698. 8. Maeda, K.; Nakata, H.; Koh, Y.; Miyakawa, T.; Ogata, H.; Takaoka, Y.; Shibayama, S.; Sagawa, K.; Fukushima, D.; Moravek, J.; Koyanagi, Y.; Mitsuya, H. J. J. Virol. 2004, 78, 8654. 9. American Cancer Society. http://www.cancer.org/research/cancerfactsstatistics/ index (accessed Nov 9, 2012). 10. Vaday, G. G.; Peehl, D. M.; Kadam, P. A.; Lawrence, D. M. Prostate 2005, 66, 124. 11. Coussens, L. M.; Werb, Z. Nature 2002, 420, 860. 12. Robinson, S. C.; Scott, K. A.; Wilson, J. L.; Thompson, R. G.; Proudfoot, A. E. I.; Balkwill, F. R. Cancer Res. 2003, 63, 8360. 13. Koenig, J. E.; Senge, T.; Allhoff, E. P.; Koenig, W. Prostate 2004, 58, 121. 14. Arnatt, C. K.; Zaidi, S. A.; Zhang, Z.; Li, G.; Richardson, A. C.; Ware, J. L.; Zhang, Y. Eur. J. Med. Chem. 2013, 69, 647. 15. Langmuir, I. J. Am. Chem. Soc. 1919, 41, 1543. 16. Patani, G. A.; LaVoie, E. J. Chem. Rev. 1996, 96, 3147. 17. Arnatt, C. K.; Zhang, Y. Tetrahedron Lett. 2012, 53, 1592. 18. Baba, M.; Miyake, H.; Okamoto, M.; Iizawa, Y.; Okonogi, K. AIDS Res. Hum. Retroviruses 2000, 16, 935. 19. Yuan, Y.; Arnatt, C. K.; Li, G.; Haney, K. M.; Ding, D.; Jacob, J. C.; Selley, D. E.; Zhang, Y. Org. Biomol. Chem. 2012, 10, 2633.
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