Discovery of RG7388, a Potent and Selective p53–MDM2 Inhibitor in

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Discovery of RG7388, a Potent and Selective p53−MDM2 Inhibitor in Clinical Development Qingjie Ding,†,⊥ Zhuming Zhang,*,†,⊥ Jin-Jun Liu,†,⊥ Nan Jiang,† Jing Zhang,† Tina M. Ross,† Xin-Jie Chu,† David Bartkovitz,† Frank Podlaski,‡ Cheryl Janson,‡ Christian Tovar,§ Zoran M. Filipovic,§ Brian Higgins,§ Kelli Glenn,∥ Kathryn Packman,§ Lyubomir T. Vassilev,§ and Bradford Graves*,‡ †

Discovery Chemistry, ‡Discovery Technologies, §Discovery Oncology, ∥Non-Clinical Development, Roche Research Center, Hoffmann-La Roche, Inc., 340 Kingsland Street, Nutley, New Jersey 07110, United States S Supporting Information *

ABSTRACT: Restoration of p53 activity by inhibition of the p53−MDM2 interaction has been considered an attractive approach for cancer treatment. However, the hydrophobic protein−protein interaction surface represents a significant challenge for the development of small-molecule inhibitors with desirable pharmacological profiles. RG7112 was the first small-molecule p53−MDM2 inhibitor in clinical development. Here, we report the discovery and characterization of a second generation clinical MDM2 inhibitor, RG7388, with superior potency and selectivity.

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INTRODUCTION Tumor suppressor p53 is a powerful growth suppressive and pro-apoptotic protein that plays a central role in protection from tumor development.1,2 A potent transcription factor, p53 is activated following cellular stress and regulates multiple downstream genes implicated in cell cycle control, apoptosis, DNA repair, and senescence.3,4 While p53 is inactivated in about 50% of human cancers by mutation or deletion, it remains wild-type in the remaining cases but its function is impaired by other mechanisms.5−8 One such mechanism is the overproduction of MDM2, the primary negative regulator of p53, which effectively disables p53 function.5−8 An E3 ligase, MDM2 binds p53 and regulates p53 protein levels through an autoregulatory feedback loop.9 Stabilization and activation of wild-type p53 by inhibition of MDM2 binding has been explored as a novel approach for cancer therapy.10,11 The crystal structure of the amino-terminal domain of MDM2 bound to a 15-residue transactivation domain peptide from p53 revealed a triad of critical amino acid residues: Phe19, Trp23, and Leu26.12 The cis-imidazoline compounds, termed Nutlins, were the first potent and selective MDM2 inhibitors and stimulated widespread interest in de novo design of smallmolecule p53−MDM2 inhibitors.13,14 However, the large, hydrophobic protein−protein interaction interface on MDM2 presents a major challenge and renders many designed molecules unsuitable for human use due to weak potency, unfavorable physicochemical properties, or poor pharmacokinetic profiles.15,16 An advanced member of the Nutlin family of molecules, RG7112, is undergoing clinical investigation (Figure 1).17 Ding et al. reported a class of high-affinity spiroindoline3,3′-pyrrolidine MDM2 inhibitors represented by MI-219 (Figure 1).18 In the proposed binding mode, oxindole closely mimics Trp23 of p53 and the spiro-pyrrolidine core projects the 3-chlorophenyl and neopentyl groups into the Phe19 and Leu26 pockets, respectively.18a The stereochemical configuration of the pyrrolidine appears to be suboptimal based on the observation by multiple groups that an aromatic substituent is © XXXX American Chemical Society

Figure 1. Chemical structures of RG7112, MI-219, and RG7388. The binding mode of RG7112 is also shown. “Trans” or “Cis” refers to the relative configuration of aryl rings “A” and “B”.

preferred in the Leu26 pocket as well as the expectation that the MI-219 configuration might not be the most stable.19 For MI-63, a close analogue of MI-219, an alternative diastereomer was observed in a crystal structure (PDB 3LBL) that results in a switch of the groups binding into the Leu26 and Phe19 pockets while maintaining similar affinity (Figure 2).19 Guided by de novo design efforts and X-ray crystal structures, we investigated a different stereochemical configuration of pyrrolidine and identified a series of potent MDM2 inhibitors with a novel core scaffold I (Figure 2).20 Here, we report the discovery of a highly potent pyrrolidine compound, RG7388, highlighting the data supporting its progression into clinical development (Figure 1).

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RESULTS AND DISCUSSION In contrast to rigid six-membered rings, five-membered rings, in general, are more flexible due to pseudorotational mobility.21 The conformational propensity of the pyrrolidine ring in proline has been extensively studied and is known to exhibit two predominant puckering modes.22,23 As part of the effort to Received: April 4, 2013

A

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racemic mixtures from which the two chiral enantiomers could be separated by chiral SFC. The prototype compound 1 (Figure 3) shows good potency (IC50 = 196 nM) in the HTRF (homogeneous time-resolved

Figure 2. The pyrrolidine ring puckering and binding modes are shown for MI-63 diastereomer and scaffold I. “Cis−Cis” or “Trans− Trans” refers to the relative orientation between ring A vs ring B and R vs ring A. endo (“down”) and exo (“up”) refers to the position of Cγ in relation to Cα.

systematically determine if a correlation can be established between pyrrolidine ring puckering and binding modes, the “Trans−Trans” configuration of pyrrolidine, shown in scaffold I (Figure 2), was explored. It is noteworthy that the aryl “A” and “B” rings in RG7112, MI-219 (Figure 1), and MI-63 diastereomer (Figure 2) predominantly adopt a “Cis” configuration. Much less is known about the “Trans” configuration. To achieve this configuration between the two aryl rings while maintaining good activity in scaffold I, the presence of the nitrile in the (Z)-α-cyanostilbene (III, Scheme 1) was found to be critical. The synthesis of analogues in scaffold I is outlined in Scheme 1.24−27 The key step is the AgF mediated 1,3-dipolar cycloaddition reaction between (Z)-α-cyanostilbene (III) and the imine (VII) for efficient formation of core pyrrolidine (VIII). The analogues in scaffold I were synthesized initially as

Figure 3. The chemical structures of pyrrolidine analogues in scaffold I.

fluorescence) binding assay (Table 1). Compound 1 was >50fold more potent than its enantiomer (2, IC50 >10000 nM). In cellular 3-(4,5-dimethylthiazol-2-yl)-2,5,-diphenyltetrazolium bromide (MTT) proliferation assays (Table 1), 1 displayed moderate potency (IC50 = 2.8 μM) against wild-type p53 cancer cell lines but only modest selectivity relative to p53 mutant cell lines (IC50 = 22 μM). Absolute stereochemical configuration and ring puckering of the active enantiomer were confirmed by a high resolution crystal structure of 1 bound to MDM2 (Figure 4). The structure clearly shows that the pyrrolidine ring adopts the Cγ-exo conformation, enabling the 3-chloro-phenyl and neopentyl substituents to be in equatorial positions while maintaining optimal binding in the Trp23 pocket by the 4-chloro-phenyl group. A similar observation was made with the MI-63 diastereomer, except that Cγ has to adopt the endo conformation to position the 3-chloro-2-fluoro-phenyl and neopentyl substituents equatorially (Figure 2, PDB 3LBL).19a Systematic study of structure−activity relationships and analysis of crystal structures quickly established that the three hydrophobic groups in compound 3 were optimal for binding to MDM2 (Supporting Information (SI) Figure S2). However, 1 and 3 were found to have high clearance rates and poor oral bioavailability in mouse pharmacokinetic (PK) studies (Table 2). Thus, the focus of the optimization was shifted to improve PK parameters through alterations to the diol side chain. Another reason for focusing on the diol was that preliminary metabolite identification studies indicated that it could be a metabolic hotspot (data not shown). Despite interacting with a relatively flat surface, the R3 side chain in scaffold I was found to play a pivotal role in modulating cellular potency,

Scheme 1. The Racemic Synthesis of Designed Pyrrolidine I Analoguesa

a

Reagents and conditions: (a) MeONa, MeOH, rt; (b) DCM, rt; (c) AgF, NEt3, DCM, rt; (d) DBU, tBuOH, reflux; (e) TFA, DCM, rt; (f) NH2R3, HATU, iPr2NEt, DCM, rt; (g) chiral SFC separation B

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Table 1. In Vitro Activity of Pyrrolidine Scaffold I Analogues and RG7112 in HTRF Binding Assays and MTT Proliferation Assays with Human Cancer Cell Linesa HTRF IC50 (nM) MTT IC50 (μM)b selectivityc

1

3

4

5

6

7

8

9

10

11

12

RG7112

196 2.8 8

74 2.1 8

56 1.4 5

42 0.71 18

22 0.11 109

23 0.18 69

22 0.15 71

20 0.07 176

21 0.21 65

25 0.27 47

6 0.03 344

18 0.4 20

a

IC50 was determined by one experiment performed in duplicate. bAverage IC50 of three wt-p53 cancer cell lines (SJSA1, RKO, HCT116). cRatio of average IC50 of two mutant p53 cell lines (SW480, MDA-MB435) and average IC50 of three wild-type p53 cell lines (as above). Results for the individual cell lines can be found in SI Table S2.

apoptosis in cancer cells expressing wild-type p53, consistent with a nongenotoxic p53 activation mechanism.13 It also achieved impressive in vivo efficacy against established human SJSA1 osterosarcoma xenografts in nude mice (Figure 5) at significantly lower doses and exposures compared to RG7112 (Table 3).28

Figure 4. Crystal structure of 1 bound to MDM2. The 4-chlorophenyl ring is buried in the Trp23 pocket. The 3-chloro-phenyl occupies the Leu26 pocket, forming a π−π stacking interaction with the His96 residue on MDM2. Neopentyl binds to the Phe19 pocket. The pyrrolidine Cα carbonyl forms a hydrogen bond with NH of His96 (PDB 4JRG).

physicochemical properties, and PK profiles, as exemplified by compounds 4−12 (Tables 1 and 2). A large number of analogues with diverse side chains at R3 was synthesized and evaluated in a panel of assays for in vitro potency and human liver microsomal stability. Selected compounds were evaluated in vivo with mouse PK studies (Table 2). Compound 6, with the para-benzoic acid side chain, proved to be critical, as it markedly improved the PK parameters and the cellular potency and selectivity. Further optimization of compound 6 evaluated a range of electron donating and withdrawing groups at different positions. This led to the identification of compound 12, known as RG7388. Compared to 6, compound 12 displayed improved in vitro binding as well as cellular potency/selectivity (Table 1). Thus, compound 12 was chosen for further studies. In cell-based mechanistic studies (SI part S-10), compound 12 induced dose-dependent p53 stabilization, cell cycle arrest, and

Figure 5. Oral in vivo efficacy profile of 12 (RG7388) in nude mice implanted sc with SJSA1 osteosarcoma tumor cells.

Table 3. In Vivo Efficacy Data for RG7388 compared to RG7112 Based on the SJSA1 Tumor Xenograft Model RG7112 dose (mg/kg) tumor growth inhib (%) AUC (μg·h/mL)

50 74 200

RG7388

100 >100 380

12.5 88 23

25 >100 29

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CONCLUSION RG7388 showed all the characteristics expected of an MDM2 inhibitor (see Biology SI for additional details) in terms of specific binding to the target, mechanistic outcomes resulting

Table 2. The Human Liver Microsomal Stability and Mean PK Parameters of Compounds 1, 3, 4, and 6,a 7−12, and RG7112a in C57 Male Mice by Single Oral and IV Dosing b

HLM_CL (mL/min/kg) PO dose (mg/kg) PO AUC/PO dose (μg·h/mL/mg/kg) PO Cmax (μg/mL) IV dose (mg/kg) CL (mL/min/kg) T1/2 (h) F (%)c a

1

3

4

6a

7

8

9

10

11

12

RG7112a

18.7 20 0.02 0.5 5 52.9 0.44 6.2

19.9 20 0.01 0.3 5 51.7 0.52 3.6

4.2 25 0.01 0.01 3.6 19.8 1.48 35. cOral bioavailability. C

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(4) Harris, S. L.; Levine, A. J. The p53 pathway: positive and negative feedback loops. Oncogene. 2005, 24 (17), 2899−2908. (5) Hainaut, P.; Hollstein, M. p53 and human cancer: the first ten thousand mutations. Adv. Cancer Res. 2000, 77, 81−137. (6) Vousden, K. H.; Lane, D. P. p53 in health and disease. Nature Rev. Mol. Cell Biol. 2007, 8, 275−283. (7) Bond, G. L.; Hu, W.; Levine, A. J. MDM2 is a central node in the p53 pathway: 12 years and counting. Curr. Cancer Drug Targets 2005, 5, 3−8. (8) Wade, M.; Wang, Y, V.; Wahl, G. M. The p53 orchestra: MDM2 and MDMX set the tone. Trends Cell Biol. 2010, 20 (5), 299−309. (9) Wu, X.; Bayle, J. H.; Olson, D.; Levine, A. J. The p53−MDM2 autoregulatory feedback loop. Genes Dev. 1993, 7, 1126−1132. (10) Poyurovsky, M. V.; Prives, C. Unleashing the power of p53: lessons from mice and men. Genes Dev. 2006, 20, 125−131. (11) (a) Brown, C. J.; Lain, S.; Verma, C. S.; Fersht, A. R.; Lane, D. P. Awakening guardian angels: drugging the p53 pathway. Nature Rev. Cancer 2009, 9, 862−873. (b) Cheok, C. F.; Verma, C. S.; Baselga, J.; Lane, D. P. Translating p53 into the clinic. Nature Rev. Clin. Oncol. 2011, 8, 25−37. (12) Kussie, P. H.; Gorina, S.; Marechal, V.; Elenbaas, B.; Moreau, J.; Levine, A. J; Pavletich, N. P. Structure of the MDM2 oncoprotein bound to the p53 tumor suppressor transactivation domain. Science 1996, 274, 948−953. (13) Vassilev, L. T.; Vu, B. T.; Graves, B.; Carvajal, D.; Podlaski, F.; Filipovic, Z.; Kong, N.; Kammlott, U.; Lukacs, C.; Klein, C.; Fotouhi, N.; Liu, E. A. In vivo activation of the p53 pathway by small-molecule antagonists of MDM2. Science 2004, 303, 844−848. (14) Wang, S.; Zhao, Y.; Bernard, D.; Aguilar, A.; Kumar, S. Targeting the MDM2−p53 Protein−Protein Interaction for New Cancer Therapeutics. Top. Med. Chem. 2012, 8, 57−80. (15) Popowicz, G. M.; Dömling, A.; Holak, T. A. The StructureBased Design of MDM2/MDMX−p53 Inhibitors Gets Serious. Angew. Chem., Int. Ed. 2011, 50, 2680−2688. (16) Vu, B. T.; Vassilev, L. T. Small-molecule inhibitors of the p53− MDM2 interaction. Curr. Top. Microbiol. Immunol. 2011, 348, 151− 172. (17) Ray-Coquard, I.; Blay, J. Y.; Italiano, A.; Le Cesne, A.; Penel, N.; Zhi, J.; Heil, F.; Rueger, R.; Graves, B.; Ding, M.; Geho, D.; Middleton, S. A.; Vassilev, L. T.; Nichols, G. L.; Bui, B. N. Effect of the MDM2 antagonist RG7112 on the P53 pathway in patients with MDM2amplified, well-differentiated or dedifferentiated liposarcoma: an exploratory proof-of-mechanism study. Lancet Oncol. 2012, 13 (11), 1133−1140. (18) (a) Ding, K.; Lu, Y.; Nikolovska-Coleska, Z.; Qiu, S.; Ding, Y.; Gao, W.; Stuckey, J.; Krajewski, K.; Roller, P. P.; Tomita, Y.; Parrish, D. A.; Deschamps, J. R.; Wang, S. Structure-based design of potent non-peptide MDM2 inhibitors. J. Am. Chem. Soc. 2005, 127 (29), 10130−10131. (b) Yu, S.; Qin, D.; Shangary, S.; Chen, J.; Wang, G.; Ding, K.; McEachern, D.; Qiu, S.; Nikolovska-Coleska, Z.; Miller, R.; Kang, S.; Yang, D.; Wang, S. Potent and Orally Active Small-Molecule Inhibitors of the MDM2−p53 Interaction. J. Med. Chem. 2009, 52 (24), 7970−7973. (19) (a) Popowicz, G. M.; Czarna, A.; Wolf, S.; Wang, K.; Wang, W.; Dömling, A.; Holak, T. A. Structures of low molecular weight inhibitors bound to MDMX and MDM2 reveal new approaches for p53-MDMX/MDM2 antagonist drug discovery. Cell Cycle 2010, 9 (5), 1104−1111. (b) Shu, L.; Li, Z.; Gu, C.; Fishlock, D. Synthesis of a Spiroindolinone Pyrrolidinecarboxamide MDM2 Antagonist. Org. Process Res. Dev. 2013, 17 (2), 247−256. (c) Zhao, Y.; Liu, L.; Sun, W.; Lu, J.; McEachern, D.; Li, X.; Yu, S.; Bernard, D.; Ochsenbein, P.; Ferey, V.; Carry, J.-C.; Deschamps, J. R.; Sun, D.; Wang, S. Diastereomeric Spirooxindoles as Highly Potent and Efficacious MDM2 Inhibitors. J. Am. Chem. Soc. 2013, 135 (19), 7223−7234. (d) Carry, J.-C.; Garcia-Echeverria, C. Inhibitors of the p53/HDM2 protein−protein interaction-path to the clinic. Bioorg. Med. Chem. Lett. 2013, 23, 2480−2485. (20) Bartkovitz, D. J.; Chu, X.-J.; Ding, Q.; Jiang, N.; Liu, J.-J.; Ross, T. M.; Zhang, J.; Zhang, Z. Preparation of substituted pyrrolidine-2-

from activation of the p53 pathway, and in vivo efficacy. Although the cellular mechanism of action of RG7388 is identical to that of RG7112, it is much more potent and selective. RG7388 activates p53 (Figure 6 and SI) at a

Figure 6. Western blot analysis of the levels of p53 and select target proteins resulting from the treatment of SJSA cells for 20 h with Nutlin-3a, RG7112, or RG7388.

concentration that is an order of magnitude lower than RG7112. Importantly, this trend extends to the in vivo setting, where the exposures required for the same levels of efficacy are much lower for RG7388 (Table 3). In summary, our studies of this pyrrolidine scaffold led to the identification of a highly potent and selective MDM2 antagonist, RG7388. The data reported here show that RG7388 blocks p53−MDM2 binding and effectively activates the p53 pathway, leading to cell cycle arrest and/or apoptosis in cell lines expressing wild-type p53 and tumor growth inhibition or regression of osteosarcoma xenografts in nude mice. RG7388 is undergoing clinical investigation in solid and hematological tumors.

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ASSOCIATED CONTENT

S Supporting Information *

Detailed experimental section. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*For Z.Z.: phone, 908-240-4582; E-mail, zzzhang_3000@ yahoo.com. For B.G.: E-mail, [email protected]. Author Contributions ⊥

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS SFC purification by Theodore Lambros, detailed 1H and NOE NMR analysis for determination of stereochemical configuration by Gino Sasso, high resolution mass spectrometry by George Perkins, and insightful discussion with Binh Vu are gratefully acknowledged.

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