Chem Biol Drug Des 2014; 84: 497–504 Research Article
Design, Synthesis and Biological Evaluation of Peptidyl Epoxyketone Proteasome Inhibitors Composed of b-amino Acids Jiankang Zhang1,†, Mengmeng Han2,†, Xiaodong Ma1, Lei Xu2, Jiayi Cao2, Yubo Zhou2, Jia Li2, Tao Liu1,* and Yongzhou Hu1,* 1
ZJU-ENS Joint Laboratory of Medicinal Chemistry, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China 2 National Center for Drug Screening, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China *Corresponding author: Tao Liu, [email protected]; Yongzhou Hu, [email protected] † These authors contributed equally to this work. A series of novel di- and tripeptidyl epoxyketone derivatives composed of b-amino acids were designed, synthesized and evaluated for their proteasome inhibitory activities and anti-proliferation activities against two multiple myeloma cell lines RPMI 8226 and NCI-H929 and normal cells (peripheral blood mononucleated cells). Among these tested compounds, tripeptidyl analogues showed much more potent activities than dipeptides, and four tripeptidyl compounds exhibited proteasome inhibitory activities with IC50 values ranging from 0.97 0.05 to 1.85 0.11 lM. In addition, all the four compounds showed anti-proliferation activities with IC50 values at low micromolar levels against two multiple myeloma cell lines and weak activities against normal cells. Furthermore, Western blot analysis was performed to verify the proteasome inhibition induced by compounds 21d and 21e. All the experimental results validated that the b-amino acid building block has the potential for the development of proteasome inhibitors.
which comprises a 20S proteolytic core and two 19S regulatory particles. The 20S proteasome consists of four stacked rings that form an a7-b7-b7-a7 hollow cylindrical structure (4). Three of the seven b-subunits (b1, b2 and b5) performing unique enzymatic activities are classified as postglutamyl peptidyl hydrolytic-like (PGPH, b1), trypsin-like (T-L, b2) and chymotrypsin-like (CT-L, b5) activity site, respectively (4,5). The 19S regulatory particles bind at both ends of 20S core and control the recognition, unfolding and translocation of ubiquitylated protein substrates before they enter the chamber of the 20S catalytic core (6,7). To date, various proteasome inhibitors have been identified, and most of these compounds sharing a similar peptide skeleton can be mainly classified into four types due to different peptide terminal groups: peptide boronates (bortezomib, CEP18770), peptide epoxyketones (carfilzomib, ONX-0912), peptide aldehydes (MG-132) and peptide vinyl sulphones (ZLVS, NLVS; 8). Some of these compounds have been extensively evaluated in various clinical trials, with bortezomib and carfilzomib (Figure 1) being approved by the US FDA for the treatment of multiple myeloma (MM) in 2003 and 2012, respectively. Although bortezomib is successful in MM therapy, severe side effects and drug resistance are associated with prolonged drug usage (9). A significant advantage of carfilzomib lies in its low rates of peripheral neuropathy in comparison with bortezomib (10); herein, carfilzomib analogues containing epoxyketone fragment may possess enhanced safety profiles compared to boronic acid analogues. In fact, there are some other well-studied epoxyketone peptidyl proteasome inhibitors including ONX-0912 in a phase I clinical trial and ONX-0914 in preclinical development (Figure 1; 11,12).
Key words: anti-cancer, di- and tripeptides, epoxyketone, proteasome inhibitors, b-amino acid
Methods and Materials Received 19 February 2014, revised 3 April 2014 and accepted for publication 9 April 2014
Over the past decade, the proteasome has been considered to be an important anticancer drug target since the understanding of its critical roles in controlling apoptotic and tumour suppressor protein levels (1–3). The most common form of the proteasome is known as the 26S proteasome, ª 2014 John Wiley & Sons A/S. doi: 10.1111/cbdd.12342
Chemistry €chi B-540 apparaMelting points were determined on a Bu €chi Labortechnik, Flawil, Switzerland) and are uncortus (Bu €ker rected. 1H NMR spectra were recorded on a Bru €ker Bioscience, Billerica, MA, 500 MHz spectrometer (Bru USA) with CDCl3 or DMSO-d6 as solvent. Chemical shifts (d) are reported in parts per million (ppm) relative to internal TMS, and coupling constants (J) are reported in Hertz (Hz).
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Figure 1: Structures of some proteasome inhibitors.
Scheme 1: Synthesis of Leu-epoxyketones (7a and 7b). Reagents and conditions: (a) HOBt, EDCI, N, O-dimethylhydroxylamine hydrochloride, diisopropylethylamine, DCM, 0 °C; (b) isopropenylmagnesium bromide, THF, 0 °C; (c) NaBH4, CeCl37H2O, MeOH, THF, 0 °C-rt; (d) mCPBA, DCM, 0 °C; (e) Dess-Martin periodinane, DCM, 0 °C-rt; (f) trifluoroacetic acid, DCM, 0 °C-rt.
Splitting patterns are designated as singlet (s), broad singlet (brs), doublet (d), triplet (t), quartet (q) and multiplet (m). Mass spectral data were obtained using an Esquire-LC€ker Bioscience). Reagents and 00075 spectrometer (Bru solvents were purchased from common commercial suppliers and were used without further purification unless stated otherwise. Column chromatography was 498
performed using silica gel (300–400 mesh). All yields are unoptimized and generally represent the result of a single experiment. Detailed synthetic procedures and characterization data for the synthesized compounds are available in the Supporting Information (Appendix S1) of this manuscript. Chem Biol Drug Des 2014; 84: 497–504
Peptidyl Epoxyketone Proteasome Inhibitors
20S proteasome chymotrypsin-like inhibition assay Chymotrypsin-like enzyme activity assay was carried out in 50 lL volume. One microlitre compound was added into 10 lL purified human proteasome (25 lg/mL), a gift from Dr. Jiang-ping Wu (Notre-Dame Hospital, Montreal, QC, Canada), incubated for 15 min and then combined with 39 lL synthesized substrate Suc-Leu-Leu-Val-Tyr-AMC
(50lM, GL Biochem Ltd., Shanghai, China; 13). The AMC of the probe was detected by monitoring the increase of fluorescence with Envision, at 355 nm excitation and 460 nm emission. The IC50 data were calculated using the software GraphPad Prism, using the equation ‘sigmoidal dose response (variable slope)’ for curve fitting.
Scheme 2: Synthesis of dipeptidyl target compounds (13a–r and 16). Reagents and conditions: (a) NH4OAc, malonic acid, EtOH, reflux; (b) (Boc)2O, NaOH, THF, H2O, rt; (c) HOBt, EDCI, diisopropylethylamine, DCM, rt; (d) trifluoroacetic acid, DCM, 0 °C-rt; (e) R1COOH, HOBt, EDCI, diisopropylethylamine, DCM, rt.
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Tumour cell anti-proliferation assay 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2(4-sulfophenyl)-2H-tetrazolium (MTS) was purchased from Promega (Madison, WI, USA). Human MM cell lines NCIH929 and RPMI 8226 were a gift from Professor Jian Hou (Chang Zheng Hospital, Shanghai, China), and were grown in RPMI 1640 supplemented with 10% foetal bovine serum (FBS) and penicillin-streptomycin from Invitrogen (Grand Island, NY, USA) at 37 °C in a 5% CO2 humidified atmosphere. A 100 lL NCI-H929 or RPMI 8226 cells (104/well) were seeded into 96-well plates. After treatment of the cells with test compounds for 72 h, MTS was added at a final concentration of 0.5 mg/mL for 2–4 h. Optical density was determined at 490 nm (background subtraction at 690 nm) by SpectraMax 340 microplate reader (Molecular Devices, Sunnyvale, CA, USA). The growth inhibitory ratio was calculated as follows: growth inhibitory ratio = (AcontrolAsample)/Acontrol. IC50 values were derived from a nonlinear regression model (curve fit) based on a sigmoidal dose–response curve (variable slope) and computed using GRAGHPAD PRISM version 5.02, Graphpad Software.
Western blot analysis Poly ADP-ribose polymerase (PARP) and ubiquitin antibody were purchased from Cell Signaling Technology (Boston, MA, USA); GAPDH antibody was obtained from Sigma. All antibodies were used as recommended by the manufacturers. For experiments, cells were grown in 6-well plates incubated with compounds for 12 h. Cells were rinsed twice with ice-cold PBS and lysed with 1 9 SDS loading buffer. Samples were electrophoresed on 10% SDS–polyacrylamide gels and transferred to PVDF membranes. The membranes were blocked for 1 h with 5% w/v milk, incubated with indicated antibodies for 2 h, washed three times with PBS, incubated with the anti-rabbit or antimouse secondary antibody for 1 h, washed three times with PBS and detected with an ECL Kit (Thermo Scientific, Rockford, IL, USA).
Results and Discussion Design of compounds The epoxyketone group of carfilzomib, which plays a critical role in maintaining its proteasome inhibitory activity, forms a
Scheme 3: Synthesis of tripeptidyl target compounds (21a-e). Reagents and conditions: (a) N-Boc-Leu, HOBt, EDCI, diisopropylethylamine, DCM, rt; (b) trifluoroacetic acid, DCM, 0 °C-rt; (c) Boc-3-amino-3-phenylpropionic acid, HOBt, EDCI, diisopropylethylamine, DCM, rt; (d) trifluoroacetic acid, DCM, 0 °C-rt; (e) R1COOH, HOBt, EDCI, diisopropylethylamine, DCM, rt.
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Chem Biol Drug Des 2014; 84: 497–504
Phenyl 4-fluorophenyl 4-methoxyphenyl Pyrazin-2-yl Pyridin-3-yl Biphenyl-4-yl 1-acetylpiperidine-4-yl Cyclohexyl 4-methoxyanilino Phenyl Cyclohexyl Phenyl Cyclohexyl Phenyl Cyclohexyl Phenyl Phenyl Cyclohexyl Pyrazin-2-yl Pyrazin-2-yl Pyridin-3-yl Phenyl 4-fluorophenyl 4-methoxyphenyl – –
13a 13b 13c 13d 13e 13f 13g 13h 13i 13j 13k 13l 13m 13n 13o 13p 13q 13r 16 21a 21b 21c 21d 21e Carfilzomib Bortezomib H H H H H H H H H 2-Cl 2-Cl 3-Cl 3-Cl 4-Cl 4-Cl 3-F 4-OMe 4-OMe H – – – – – – –
R2 2.97 0.62 4.17 6.07 5.22 24.13 13.01 26.06 27.97 0.51 0.23 11.09 20.45 6.41 4.73 34.98 3.88 12.27 2.96 NT NT NT NT NT – –
0.70 0.44 0.18 0.06 0.64 0.29 0.39 0.43 4.07 0.99 1.19 0.03 0.22 0.10 1.08 0.13 1.40 1.38 0.32
Inhibitory rate at 10 lg/mL (%)a
NT, not tested. a The inhibitory rates and IC50 values are an average of three independent determinations.
R1
Compound NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT 15.77 55.00 65.76 70.99 72.99 – –
2.99 3.88 2.18 5.27 2.25
Inhibitory rate at 1 lg/mL (%)a
Table 1: 20S proteasome chymotrypsin-like inhibitory activities of di- and tripeptidyl target compounds (13a–r, 16 and 21a–e)
NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT 1.85 1.42 0.97 1.01 8.60 8.96
0.11 0.02 0.05 0.10 1.40 (nM) 0.56 (nM)
IC50 (lM)a
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morpholine ring with the N-terminal threonine residue of the 20S proteasome. The unique binding modes of carfilzomib make it an irreversible proteasome inhibitor. With the aim to keep this critical covalent interaction with proteasome, epoxyketone fragment of carfilzomib was retained in our designed compounds. Additionally, short peptide proteasome inhibitors constructed from a-amino acid are vulnerable to various proteases and peptidases, which leads to low oral bioavailabilities. Introducing a b-amino acid in the peptide skeleton could improve the enzymatic stability of b-peptides significantly against standard proteases and peptidases compared to that of the a-peptides (14,15). To date, only five enzymes have been described that can induce b-peptide hydrolysis (16). Besides, the additional methylene group of the b-peptide skeleton leads to higher conformational variability, which could form very stable secondary structures such as sheets, helices and reverse turns (17). Most importantly, b-amino-acid-containing boronic acid peptidyl analogues have been proved to be potential proteasome inhibitors with comparable activities to that of bortezomib, which validates the value of this peptide building block (18). In this study, di- and tripeptides containing b-amino acid and epoxyketone fragment were synthesized and evaluated for their proteasome inhibitory activities.
subsequently deprotected and coupled with b-amino acid 10a to afford compound 19. The protected tripeptidyl fragment 19 was further deprotected and treated with various acids to afford tripeptidyl derivatives 21a–e.
Synthesis The epoxyketone fragments 7a and 7b were synthesized following the method described in the literature with modifications (11,19), and the synthetic routes are summarized in Scheme 1. Reaction of N-Boc-protected leucine 1 with N, O-dimethylhydroxylamine hydrochloride gave the Weinreb amide 2. Compound 2 was then treated with isopropenylmagnesium bromide in tetrahydrofuran at 0 °C to form the desired a, b-unsaturated ketone 3. Subsequent reduction of 3 with sodium borohydride and cerium chloride afforded allylic alcohol 4. Without further purification, compound 4 was oxidized into epoxide 5 in the presence of mCPBA, then epoxide 5 was oxidized with Dess-Martin reagent to form the epoxyketones 6a and 6b in a ratio of 2.1:1. Afterwards, deprotection of 6a and 6b with trifluoroacetic acid resulted in compounds 7a and 7b, respectively.
a
The synthetic routes for di- and tripeptidyl epoxyketone derivatives are summarized in Schemes 2 and 3. Reaction of corresponding phenylaldehydes 8a–f with malonic acid and ammonium chloride gave racemic b-amino acids 9a–f (20), and these racemic b-amino acids were employed as the peptide building blocks in the target compounds with the aim to shorten the biological screening cycle. Then, the NH2 of 9a–f was protected using (Boc)2O to afford N-Bocprotected b-amino acids 10a–f. Condensation of compounds 10a–f with epoxyketone fragment 7a or 7b yielded dipeptides 11a–f and 14, which were deprotected and treated with various acids to afford dipeptidyl target compounds 13a–r and 16. In addition, the tripeptidyl derivatives were synthesized through similar procedures. Reaction of 7a with N-Boc-protected leucine yielded dipeptide 17, which was 502
Proteasome inhibitory activities The synthesized compounds 13a–r, 16 and 21a–e were evaluated for their 20S proteasome chymotrypsin-like inhibitory activities in vitro with bortezomib and carfilzomib employed as the positive controls. The results are summarized in Table 1. Table 2: In vitro cytotoxic activities of selected compounds (13p and 21a–e) against two multiple myeloma cell lines and peripheral blood mononucleated cells (PBMC) Cytotoxicity (IC50, lM)a Compound
RPMI 8226
13p 21a 21b 21c 21d 21e Bortezomib Carfilzomib
18.57 8.18 4.53 2.51 2.35 2.00 7.96 13.19
0.38 0.27 0.28 0.10 0.08 0.16 0.11 (nM) 0.56 (nM)
NCI-H929 21.87 10.72 9.70 3.62 3.46 2.92 10.85 21.32
0.50 0.10 0.39 0.11 0.05 0.21 0.51 (nM) 0.83 (nM)
PBMC >50 >50 >50 >50 >50 >50 >1 >1
The IC50 values are an average of three independent determinations.
Figure 2: Immunoblotting analysis of polyubiquitin, poly ADPribose polymerase (PARP), and cPARP in RPMI 8226 cells treated with carfilzomib, DMSO (control) or selected b-amino acid derivatives (13p, 21d and 21e).
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As shown in Table 1, compounds (21a–e) with tripeptidyl skeleton exhibited improved activities than that of dipeptide series, and four compounds (21b–e) showed proteasome inhibitory rates of more than 50% at the concentration of 1 lg/mL, with IC50 values of 1.85 0.11 lM, 1.42 0.02 lM, 0.97 0.05 lM and 1.01 0.10 lM, respectively. In the tripeptide series, when R1 was phenyl or substituted phenyl, compounds (21c–e) displayed more potent inhibitory activities than that of compounds with aromatic heterocyclic rings (21a–b). In the dipeptide series, bulky aromatic (13f) and alicyclic (13 g–h) groups were more tolerable than compounds (13d–e) with aromatic heterocycle at the end of carboxamide. In addition, compound 13i (27.97% inhibition at 10 lg/mL) with a urea group exhibited better proteasome inhibitory activity than compound 13c (4.17% inhibition at 10 lg/mL) with amide as the linkage. To evaluate the influences of substituents (R2) on phenyl ring of 3-amino-3-phenylpropanoic acid moiety, a series of compounds (13j–r) were synthesized. However, most of the compounds exhibited decreased activities than that of compound 13 h without substituent on phenyl ring (R2 = H) except compound 13p. Additionally, 1R-isomer (13d) exhibited more potent inhibitory activity than that of 1S-isomer (16), which indicated that the epoxyketone configuration also influenced the activity of this series of compounds.
Tumour cell anti-proliferation activities Based on the evaluation of proteasome chymotrypsin-like inhibitory activities, selected compounds (13p and 21a–e) were further tested for their anti-proliferation activities in vitro against two MM cell lines (RPMI 8226 and NCIH929) and normal cells (peripheral blood mononucleated cells, PBMC) by MTS assay. Bortezomib and carfilzomib were employed as the positive controls, and DMSO was used as the negative control. According to the results summarized in Table 2, the dipeptidyl analogue 13p displayed weaker cytotoxic activities against tested cancer cell lines than that of the tripeptides (21a–e), which was consistent with the proteasome inhibitory activities. In addition, tripeptidyl derivatives with phenyl or substituted phenyl moieties (21c–e) as the R1 substituents were more potent than compounds with aromatic heterocyclic rings (21a–b), which was also validated by the proteasome inhibitory evaluation. All the tested compounds exhibited weak anti-proliferation activities against PBMC (IC50 > 50 lM), which indicated that these compounds had selective cytotoxic effects on tumour cell lines against normal cells.
Western blot analysis Subsequently, the proteasome inhibitory effect of selected compounds 13p, 21d and 21e was tested in a cellular system by Western blot analysis. Carfilzomib was used as the positive control. As illustrated in Figure 2, polyubiquitin Chem Biol Drug Des 2014; 84: 497–504
is clearly accumulated in RPMI 8226 cells treated with tripeptidyl derivatives 21d and 21e, which indicated that the two compounds induced proteasomal dysfunction in the cells. Additionally, cleavage of PARP was observed in the cells, suggesting that the apoptotic pathway was activated. On the contrary, compound 13p was unable to induce PARP cleavage, which was mainly attributed to its poor proteasome inhibitory activity.
Conclusions A series of novel di- and tripeptidyl epoxyketone derivatives were synthesized and evaluated for their proteasome inhibitory activities. Four tripeptidyl compounds displayed potent proteasome inhibitory activities with IC50 values lower than 2 lM, which were consistent with the cytotoxic activities against two MM cell lines RPMI 8226 and NCI-H929. On the other hand, the four compounds displayed weak cytotoxic activities against normal cells, which proved that these compounds had selective cytotoxic effects on tumour cells. In addition, the Western blot analysis of compounds 21d and 21e suggested that the inhibitory effects on cellular growth were induced by cell apoptosis. The SARs validated that compounds (21a–e) with tripeptidyl skeleton exhibited much more potent activities than that of dipeptide series, which suggested that the tripeptidyl skeleton is a potential privileged structure in designing peptidyl epoxyketone proteasome inhibitors. Further studies will focus on the detailed SAR studies of the tripeptidyl analogues together with the in vivo, safety and stability evaluations.
Acknowledgments The authors thank the National Natural Science Foundation of China (81125023, 91029716) and the National Science and Technology Major Projects for Major New Drugs Innovation and Development (2012ZX09301-001-004) for financial support.
Conflict of Interest The authors have declared no conflict of interest.
References 1. Groll M., Huber R. (2004) Inhibitors of the eukaryotic 20S proteasome core particle: a structural approach. Biochim Biophys Acta;1695:33–44. 2. Genin E., Reboud-Ravaux M., Vidal J. (2010) Proteasome inhibitors: recent advances and new perspectives in medicinal chemistry. Curr Top Med Chem;10:232– 256. 3. Borissenko L., Groll M. (2007) 20S proteasome and its inhibitors: crystallographic knowledge for drug development. Chem Rev;107:687–717. 503
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4. Groll M., Ditzel L., Lowe J., Stock D., Bochtler M., Bartunik H.D., Huber R. (1997) Structure of 20S proteasome from yeast at 2.4 A resolution. Nature;386:463–471. 5. DeMartino G.N., Slaughter C.A. (1999) The proteasome, a novel protease regulated by multiple mechanisms. J Biol Chem;274:22123–22126. 6. Hough R., Pratt G., Rechsteiner M. (1987) Purification of two high molecular weight proteases from rabbit reticulocyte lysate. J Biol Chem;262:8303–8313. 7. Nandi D., Tahiliani P., Kumar A., Chandu D. (2006) The ubiquitin-proteasome system. J Biosci;31:137–155. 8. Fuchs V., Jaeger K.E., Wilhelm S., Rosenau F. (2011) The BapF protein from Pseudomonas aeruginosa is a b-peptidyl aminopeptidase. World J Microbiol Biotechnol;27:713–718. 9. Cheng R.P., Gellman S.H., DeGrado W.F. (2001) BetaPeptides: from structure to function. Chem Rev;101:3219–3232. 10. Frackenpohl J., Arvidsson P.I., Schreiber J.V., Seebach D. (2001) The outstanding biological stability of beta- and gamma-peptides toward proteolytic enzymes: an in vitro investigation with fifteen peptidases. ChemBioChem;2:445–455. 11. Zhu Y.Q., Zhu X.R., Wu G., Ma Y.H., Li Y.J., Zhao X., Yuan Y.X., Yang J., Yu S., Shao F., Li R.T., Ke Y.R., Lu A.J., Liu Z.M., Zhang L.G. (2010) Synthesis, in vitro and in vivo biological evaluation, docking studies, and structure–activity relationship (SAR) discussion of dipeptidyl boronic acid proteasome inhibitors composed of beta-amino acids. J Med Chem;53:1990–1999. 12. Zhang J., Wu P., Hu Y. (2013) Clinical and marketed proteasome inhibitors for cancer treatment. Curr Med Chem;20:2537–2551. 13. Chen D., Frezza M., Schmitt S., Kanwar J., Dou Q.P. (2011) Bortezomib as the first proteasome inhibitor anticancer drug: current status and future perspectives. Curr Cancer Drug Targets;11:239–253. 14. Kortuem K.M., Stewart A.K. (2012) Carfilzomib. Blood;121:893–897.
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15. Zhou H.J., Aujay M.A., Bennett M.K., Dajee M., Demo S.D., Fang Y., Ho M.N. et al. (2009) Design and synthesis of an orally bioavailable and selective peptide epoxyketone proteasome inhibitor (PR-047). J Med Chem;52:3028–3038. 16. Verbrugge S.E., Emal D., Al M., Lougheed S., Chan E., Kirk C., Dijkmans B., Scheper R., de Gruijl T. (2012) Targeting the immunoproteasome with the second generation proteasome inhibitor ONX 0914: effects on IFN-alpha-induced monocyte-derived dendritic cells. J Immunol;188:119.4. 17. Adams J., Palombella V.J., Sausville E.A., Johnson J., Destree A., Lazarus D.D., Maas J., Pien C.S., Prakash S., Elliott P.J. (1999) Proteasome inhibitors: a novel class of potent and effective antitumor agents. Cancer Res;59:2615–2622. 18. Seebach D., Abele S., Schreiber J.V., Martinoni B., Nussbaum A.K., Schild H., Schulz H., Henneke H., Woessner R., Bitsch F. (1998) Biological and pharmacokinetic studies with beta-peptides. Chimia;52:734– 739. 19. Spaltenstein A., Leban J.J., Huang J.J., Reinhardt K.R., Humberto Viveros O., Sigafoos J., Crouch R. (1996) Design and synthesis of novel protease inhibitors. Tripeptide a0 , b0 -epoxyketones as nanomolar inactivators of the proteasome. Tetrahedron Lett;37:1343– 1346. 20. Tan C.Y.K., Weaver D.F. (2002) A one-pot synthesis of 3-amino-3-arylpropionic acids. Tetrahedron;58:7449– 7461.
Supporting Information Additional Supporting Information may be found in the online version of this article: Appendix S1. Experimental details and characterization for the synthesized compounds.
Chem Biol Drug Des 2014; 84: 497–504
Design, Synthesis and Biological Evaluation of Peptidyl Epoxyketone Proteasome Inhibitors Composed of b-amino Acids Jiankang Zhang1,†, Mengmeng Han2,†, Xiaodong Ma1, Lei Xu2, Jiayi Cao2, Yubo Zhou2, Jia Li2, Tao Liu1,* and Yongzhou Hu1,* 1
ZJU-ENS Joint Laboratory of Medicinal Chemistry, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China 2 National Center for Drug Screening, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China *Corresponding author: Tao Liu, [email protected]; Yongzhou Hu, [email protected] † These authors contributed equally to this work. A series of novel di- and tripeptidyl epoxyketone derivatives composed of b-amino acids were designed, synthesized and evaluated for their proteasome inhibitory activities and anti-proliferation activities against two multiple myeloma cell lines RPMI 8226 and NCI-H929 and normal cells (peripheral blood mononucleated cells). Among these tested compounds, tripeptidyl analogues showed much more potent activities than dipeptides, and four tripeptidyl compounds exhibited proteasome inhibitory activities with IC50 values ranging from 0.97 0.05 to 1.85 0.11 lM. In addition, all the four compounds showed anti-proliferation activities with IC50 values at low micromolar levels against two multiple myeloma cell lines and weak activities against normal cells. Furthermore, Western blot analysis was performed to verify the proteasome inhibition induced by compounds 21d and 21e. All the experimental results validated that the b-amino acid building block has the potential for the development of proteasome inhibitors.
which comprises a 20S proteolytic core and two 19S regulatory particles. The 20S proteasome consists of four stacked rings that form an a7-b7-b7-a7 hollow cylindrical structure (4). Three of the seven b-subunits (b1, b2 and b5) performing unique enzymatic activities are classified as postglutamyl peptidyl hydrolytic-like (PGPH, b1), trypsin-like (T-L, b2) and chymotrypsin-like (CT-L, b5) activity site, respectively (4,5). The 19S regulatory particles bind at both ends of 20S core and control the recognition, unfolding and translocation of ubiquitylated protein substrates before they enter the chamber of the 20S catalytic core (6,7). To date, various proteasome inhibitors have been identified, and most of these compounds sharing a similar peptide skeleton can be mainly classified into four types due to different peptide terminal groups: peptide boronates (bortezomib, CEP18770), peptide epoxyketones (carfilzomib, ONX-0912), peptide aldehydes (MG-132) and peptide vinyl sulphones (ZLVS, NLVS; 8). Some of these compounds have been extensively evaluated in various clinical trials, with bortezomib and carfilzomib (Figure 1) being approved by the US FDA for the treatment of multiple myeloma (MM) in 2003 and 2012, respectively. Although bortezomib is successful in MM therapy, severe side effects and drug resistance are associated with prolonged drug usage (9). A significant advantage of carfilzomib lies in its low rates of peripheral neuropathy in comparison with bortezomib (10); herein, carfilzomib analogues containing epoxyketone fragment may possess enhanced safety profiles compared to boronic acid analogues. In fact, there are some other well-studied epoxyketone peptidyl proteasome inhibitors including ONX-0912 in a phase I clinical trial and ONX-0914 in preclinical development (Figure 1; 11,12).
Key words: anti-cancer, di- and tripeptides, epoxyketone, proteasome inhibitors, b-amino acid
Methods and Materials Received 19 February 2014, revised 3 April 2014 and accepted for publication 9 April 2014
Over the past decade, the proteasome has been considered to be an important anticancer drug target since the understanding of its critical roles in controlling apoptotic and tumour suppressor protein levels (1–3). The most common form of the proteasome is known as the 26S proteasome, ª 2014 John Wiley & Sons A/S. doi: 10.1111/cbdd.12342
Chemistry €chi B-540 apparaMelting points were determined on a Bu €chi Labortechnik, Flawil, Switzerland) and are uncortus (Bu €ker rected. 1H NMR spectra were recorded on a Bru €ker Bioscience, Billerica, MA, 500 MHz spectrometer (Bru USA) with CDCl3 or DMSO-d6 as solvent. Chemical shifts (d) are reported in parts per million (ppm) relative to internal TMS, and coupling constants (J) are reported in Hertz (Hz).
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Figure 1: Structures of some proteasome inhibitors.
Scheme 1: Synthesis of Leu-epoxyketones (7a and 7b). Reagents and conditions: (a) HOBt, EDCI, N, O-dimethylhydroxylamine hydrochloride, diisopropylethylamine, DCM, 0 °C; (b) isopropenylmagnesium bromide, THF, 0 °C; (c) NaBH4, CeCl37H2O, MeOH, THF, 0 °C-rt; (d) mCPBA, DCM, 0 °C; (e) Dess-Martin periodinane, DCM, 0 °C-rt; (f) trifluoroacetic acid, DCM, 0 °C-rt.
Splitting patterns are designated as singlet (s), broad singlet (brs), doublet (d), triplet (t), quartet (q) and multiplet (m). Mass spectral data were obtained using an Esquire-LC€ker Bioscience). Reagents and 00075 spectrometer (Bru solvents were purchased from common commercial suppliers and were used without further purification unless stated otherwise. Column chromatography was 498
performed using silica gel (300–400 mesh). All yields are unoptimized and generally represent the result of a single experiment. Detailed synthetic procedures and characterization data for the synthesized compounds are available in the Supporting Information (Appendix S1) of this manuscript. Chem Biol Drug Des 2014; 84: 497–504
Peptidyl Epoxyketone Proteasome Inhibitors
20S proteasome chymotrypsin-like inhibition assay Chymotrypsin-like enzyme activity assay was carried out in 50 lL volume. One microlitre compound was added into 10 lL purified human proteasome (25 lg/mL), a gift from Dr. Jiang-ping Wu (Notre-Dame Hospital, Montreal, QC, Canada), incubated for 15 min and then combined with 39 lL synthesized substrate Suc-Leu-Leu-Val-Tyr-AMC
(50lM, GL Biochem Ltd., Shanghai, China; 13). The AMC of the probe was detected by monitoring the increase of fluorescence with Envision, at 355 nm excitation and 460 nm emission. The IC50 data were calculated using the software GraphPad Prism, using the equation ‘sigmoidal dose response (variable slope)’ for curve fitting.
Scheme 2: Synthesis of dipeptidyl target compounds (13a–r and 16). Reagents and conditions: (a) NH4OAc, malonic acid, EtOH, reflux; (b) (Boc)2O, NaOH, THF, H2O, rt; (c) HOBt, EDCI, diisopropylethylamine, DCM, rt; (d) trifluoroacetic acid, DCM, 0 °C-rt; (e) R1COOH, HOBt, EDCI, diisopropylethylamine, DCM, rt.
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Tumour cell anti-proliferation assay 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2(4-sulfophenyl)-2H-tetrazolium (MTS) was purchased from Promega (Madison, WI, USA). Human MM cell lines NCIH929 and RPMI 8226 were a gift from Professor Jian Hou (Chang Zheng Hospital, Shanghai, China), and were grown in RPMI 1640 supplemented with 10% foetal bovine serum (FBS) and penicillin-streptomycin from Invitrogen (Grand Island, NY, USA) at 37 °C in a 5% CO2 humidified atmosphere. A 100 lL NCI-H929 or RPMI 8226 cells (104/well) were seeded into 96-well plates. After treatment of the cells with test compounds for 72 h, MTS was added at a final concentration of 0.5 mg/mL for 2–4 h. Optical density was determined at 490 nm (background subtraction at 690 nm) by SpectraMax 340 microplate reader (Molecular Devices, Sunnyvale, CA, USA). The growth inhibitory ratio was calculated as follows: growth inhibitory ratio = (AcontrolAsample)/Acontrol. IC50 values were derived from a nonlinear regression model (curve fit) based on a sigmoidal dose–response curve (variable slope) and computed using GRAGHPAD PRISM version 5.02, Graphpad Software.
Western blot analysis Poly ADP-ribose polymerase (PARP) and ubiquitin antibody were purchased from Cell Signaling Technology (Boston, MA, USA); GAPDH antibody was obtained from Sigma. All antibodies were used as recommended by the manufacturers. For experiments, cells were grown in 6-well plates incubated with compounds for 12 h. Cells were rinsed twice with ice-cold PBS and lysed with 1 9 SDS loading buffer. Samples were electrophoresed on 10% SDS–polyacrylamide gels and transferred to PVDF membranes. The membranes were blocked for 1 h with 5% w/v milk, incubated with indicated antibodies for 2 h, washed three times with PBS, incubated with the anti-rabbit or antimouse secondary antibody for 1 h, washed three times with PBS and detected with an ECL Kit (Thermo Scientific, Rockford, IL, USA).
Results and Discussion Design of compounds The epoxyketone group of carfilzomib, which plays a critical role in maintaining its proteasome inhibitory activity, forms a
Scheme 3: Synthesis of tripeptidyl target compounds (21a-e). Reagents and conditions: (a) N-Boc-Leu, HOBt, EDCI, diisopropylethylamine, DCM, rt; (b) trifluoroacetic acid, DCM, 0 °C-rt; (c) Boc-3-amino-3-phenylpropionic acid, HOBt, EDCI, diisopropylethylamine, DCM, rt; (d) trifluoroacetic acid, DCM, 0 °C-rt; (e) R1COOH, HOBt, EDCI, diisopropylethylamine, DCM, rt.
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Phenyl 4-fluorophenyl 4-methoxyphenyl Pyrazin-2-yl Pyridin-3-yl Biphenyl-4-yl 1-acetylpiperidine-4-yl Cyclohexyl 4-methoxyanilino Phenyl Cyclohexyl Phenyl Cyclohexyl Phenyl Cyclohexyl Phenyl Phenyl Cyclohexyl Pyrazin-2-yl Pyrazin-2-yl Pyridin-3-yl Phenyl 4-fluorophenyl 4-methoxyphenyl – –
13a 13b 13c 13d 13e 13f 13g 13h 13i 13j 13k 13l 13m 13n 13o 13p 13q 13r 16 21a 21b 21c 21d 21e Carfilzomib Bortezomib H H H H H H H H H 2-Cl 2-Cl 3-Cl 3-Cl 4-Cl 4-Cl 3-F 4-OMe 4-OMe H – – – – – – –
R2 2.97 0.62 4.17 6.07 5.22 24.13 13.01 26.06 27.97 0.51 0.23 11.09 20.45 6.41 4.73 34.98 3.88 12.27 2.96 NT NT NT NT NT – –
0.70 0.44 0.18 0.06 0.64 0.29 0.39 0.43 4.07 0.99 1.19 0.03 0.22 0.10 1.08 0.13 1.40 1.38 0.32
Inhibitory rate at 10 lg/mL (%)a
NT, not tested. a The inhibitory rates and IC50 values are an average of three independent determinations.
R1
Compound NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT 15.77 55.00 65.76 70.99 72.99 – –
2.99 3.88 2.18 5.27 2.25
Inhibitory rate at 1 lg/mL (%)a
Table 1: 20S proteasome chymotrypsin-like inhibitory activities of di- and tripeptidyl target compounds (13a–r, 16 and 21a–e)
NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT NT 1.85 1.42 0.97 1.01 8.60 8.96
0.11 0.02 0.05 0.10 1.40 (nM) 0.56 (nM)
IC50 (lM)a
Peptidyl Epoxyketone Proteasome Inhibitors
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morpholine ring with the N-terminal threonine residue of the 20S proteasome. The unique binding modes of carfilzomib make it an irreversible proteasome inhibitor. With the aim to keep this critical covalent interaction with proteasome, epoxyketone fragment of carfilzomib was retained in our designed compounds. Additionally, short peptide proteasome inhibitors constructed from a-amino acid are vulnerable to various proteases and peptidases, which leads to low oral bioavailabilities. Introducing a b-amino acid in the peptide skeleton could improve the enzymatic stability of b-peptides significantly against standard proteases and peptidases compared to that of the a-peptides (14,15). To date, only five enzymes have been described that can induce b-peptide hydrolysis (16). Besides, the additional methylene group of the b-peptide skeleton leads to higher conformational variability, which could form very stable secondary structures such as sheets, helices and reverse turns (17). Most importantly, b-amino-acid-containing boronic acid peptidyl analogues have been proved to be potential proteasome inhibitors with comparable activities to that of bortezomib, which validates the value of this peptide building block (18). In this study, di- and tripeptides containing b-amino acid and epoxyketone fragment were synthesized and evaluated for their proteasome inhibitory activities.
subsequently deprotected and coupled with b-amino acid 10a to afford compound 19. The protected tripeptidyl fragment 19 was further deprotected and treated with various acids to afford tripeptidyl derivatives 21a–e.
Synthesis The epoxyketone fragments 7a and 7b were synthesized following the method described in the literature with modifications (11,19), and the synthetic routes are summarized in Scheme 1. Reaction of N-Boc-protected leucine 1 with N, O-dimethylhydroxylamine hydrochloride gave the Weinreb amide 2. Compound 2 was then treated with isopropenylmagnesium bromide in tetrahydrofuran at 0 °C to form the desired a, b-unsaturated ketone 3. Subsequent reduction of 3 with sodium borohydride and cerium chloride afforded allylic alcohol 4. Without further purification, compound 4 was oxidized into epoxide 5 in the presence of mCPBA, then epoxide 5 was oxidized with Dess-Martin reagent to form the epoxyketones 6a and 6b in a ratio of 2.1:1. Afterwards, deprotection of 6a and 6b with trifluoroacetic acid resulted in compounds 7a and 7b, respectively.
a
The synthetic routes for di- and tripeptidyl epoxyketone derivatives are summarized in Schemes 2 and 3. Reaction of corresponding phenylaldehydes 8a–f with malonic acid and ammonium chloride gave racemic b-amino acids 9a–f (20), and these racemic b-amino acids were employed as the peptide building blocks in the target compounds with the aim to shorten the biological screening cycle. Then, the NH2 of 9a–f was protected using (Boc)2O to afford N-Bocprotected b-amino acids 10a–f. Condensation of compounds 10a–f with epoxyketone fragment 7a or 7b yielded dipeptides 11a–f and 14, which were deprotected and treated with various acids to afford dipeptidyl target compounds 13a–r and 16. In addition, the tripeptidyl derivatives were synthesized through similar procedures. Reaction of 7a with N-Boc-protected leucine yielded dipeptide 17, which was 502
Proteasome inhibitory activities The synthesized compounds 13a–r, 16 and 21a–e were evaluated for their 20S proteasome chymotrypsin-like inhibitory activities in vitro with bortezomib and carfilzomib employed as the positive controls. The results are summarized in Table 1. Table 2: In vitro cytotoxic activities of selected compounds (13p and 21a–e) against two multiple myeloma cell lines and peripheral blood mononucleated cells (PBMC) Cytotoxicity (IC50, lM)a Compound
RPMI 8226
13p 21a 21b 21c 21d 21e Bortezomib Carfilzomib
18.57 8.18 4.53 2.51 2.35 2.00 7.96 13.19
0.38 0.27 0.28 0.10 0.08 0.16 0.11 (nM) 0.56 (nM)
NCI-H929 21.87 10.72 9.70 3.62 3.46 2.92 10.85 21.32
0.50 0.10 0.39 0.11 0.05 0.21 0.51 (nM) 0.83 (nM)
PBMC >50 >50 >50 >50 >50 >50 >1 >1
The IC50 values are an average of three independent determinations.
Figure 2: Immunoblotting analysis of polyubiquitin, poly ADPribose polymerase (PARP), and cPARP in RPMI 8226 cells treated with carfilzomib, DMSO (control) or selected b-amino acid derivatives (13p, 21d and 21e).
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Peptidyl Epoxyketone Proteasome Inhibitors
As shown in Table 1, compounds (21a–e) with tripeptidyl skeleton exhibited improved activities than that of dipeptide series, and four compounds (21b–e) showed proteasome inhibitory rates of more than 50% at the concentration of 1 lg/mL, with IC50 values of 1.85 0.11 lM, 1.42 0.02 lM, 0.97 0.05 lM and 1.01 0.10 lM, respectively. In the tripeptide series, when R1 was phenyl or substituted phenyl, compounds (21c–e) displayed more potent inhibitory activities than that of compounds with aromatic heterocyclic rings (21a–b). In the dipeptide series, bulky aromatic (13f) and alicyclic (13 g–h) groups were more tolerable than compounds (13d–e) with aromatic heterocycle at the end of carboxamide. In addition, compound 13i (27.97% inhibition at 10 lg/mL) with a urea group exhibited better proteasome inhibitory activity than compound 13c (4.17% inhibition at 10 lg/mL) with amide as the linkage. To evaluate the influences of substituents (R2) on phenyl ring of 3-amino-3-phenylpropanoic acid moiety, a series of compounds (13j–r) were synthesized. However, most of the compounds exhibited decreased activities than that of compound 13 h without substituent on phenyl ring (R2 = H) except compound 13p. Additionally, 1R-isomer (13d) exhibited more potent inhibitory activity than that of 1S-isomer (16), which indicated that the epoxyketone configuration also influenced the activity of this series of compounds.
Tumour cell anti-proliferation activities Based on the evaluation of proteasome chymotrypsin-like inhibitory activities, selected compounds (13p and 21a–e) were further tested for their anti-proliferation activities in vitro against two MM cell lines (RPMI 8226 and NCIH929) and normal cells (peripheral blood mononucleated cells, PBMC) by MTS assay. Bortezomib and carfilzomib were employed as the positive controls, and DMSO was used as the negative control. According to the results summarized in Table 2, the dipeptidyl analogue 13p displayed weaker cytotoxic activities against tested cancer cell lines than that of the tripeptides (21a–e), which was consistent with the proteasome inhibitory activities. In addition, tripeptidyl derivatives with phenyl or substituted phenyl moieties (21c–e) as the R1 substituents were more potent than compounds with aromatic heterocyclic rings (21a–b), which was also validated by the proteasome inhibitory evaluation. All the tested compounds exhibited weak anti-proliferation activities against PBMC (IC50 > 50 lM), which indicated that these compounds had selective cytotoxic effects on tumour cell lines against normal cells.
Western blot analysis Subsequently, the proteasome inhibitory effect of selected compounds 13p, 21d and 21e was tested in a cellular system by Western blot analysis. Carfilzomib was used as the positive control. As illustrated in Figure 2, polyubiquitin Chem Biol Drug Des 2014; 84: 497–504
is clearly accumulated in RPMI 8226 cells treated with tripeptidyl derivatives 21d and 21e, which indicated that the two compounds induced proteasomal dysfunction in the cells. Additionally, cleavage of PARP was observed in the cells, suggesting that the apoptotic pathway was activated. On the contrary, compound 13p was unable to induce PARP cleavage, which was mainly attributed to its poor proteasome inhibitory activity.
Conclusions A series of novel di- and tripeptidyl epoxyketone derivatives were synthesized and evaluated for their proteasome inhibitory activities. Four tripeptidyl compounds displayed potent proteasome inhibitory activities with IC50 values lower than 2 lM, which were consistent with the cytotoxic activities against two MM cell lines RPMI 8226 and NCI-H929. On the other hand, the four compounds displayed weak cytotoxic activities against normal cells, which proved that these compounds had selective cytotoxic effects on tumour cells. In addition, the Western blot analysis of compounds 21d and 21e suggested that the inhibitory effects on cellular growth were induced by cell apoptosis. The SARs validated that compounds (21a–e) with tripeptidyl skeleton exhibited much more potent activities than that of dipeptide series, which suggested that the tripeptidyl skeleton is a potential privileged structure in designing peptidyl epoxyketone proteasome inhibitors. Further studies will focus on the detailed SAR studies of the tripeptidyl analogues together with the in vivo, safety and stability evaluations.
Acknowledgments The authors thank the National Natural Science Foundation of China (81125023, 91029716) and the National Science and Technology Major Projects for Major New Drugs Innovation and Development (2012ZX09301-001-004) for financial support.
Conflict of Interest The authors have declared no conflict of interest.
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Supporting Information Additional Supporting Information may be found in the online version of this article: Appendix S1. Experimental details and characterization for the synthesized compounds.
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