Targeting the capsid protein of the hepatitis B virus (HBV) has emerged as a promising strategy for developing new antiviral therapies. In this study, we report the discovery of a novel series of pyrrole oxo-carboxamide compounds as HBV capsid assembly modulators (CAMs) that block viral replication. Through a process of focused structure-activity relationship (SAR) optimization, we identified compound 12 (VNRX-9945), which exhibited excellent and broad antiviral activity against multiple HBV genotypes in vitro, along with favorable pharmacokinetic profiles across multiple species. Additionally, 12 demonstrated robust efficacy in the adeno-associated virus mouse model of HBV (AAV-HBV) infection. This compound has advanced into Phase 1 clinical trials to evaluate its safety and pharmacokinetics in healthy volunteers, to enable treatment of chronic HBV infections.

XLS file - 45KB, Sensitivity of a 111 cell line panel to birinapant as a single agent and in combination with TNF or TRAIL.

PDF file - 1384KB, Supplemental Figure 1. Structure of biotinylated birinapant Supplemental Figure 2. Concentration-dependent degradation of GFP-cIAP1/2 by birinapant Supplemental Figure 3. Representative examples of data analysis from 111 cell line panel of birinapant in combination with TRAIL analyzed using the MacSynergy II program. Supplemental Figure 4. Cells which are sensitive to birinapant as a single agent produce TNF in response to birinapant treatment Supplemental Figure 5. Birinapant induces cell death as a single agent in a TNF-dependent manner Supplemental Figure 6. Preferential degradation of TRAF2-associated cIAP1 and cIAP2 by birinapant Supplemental Figure 7. Birinapant inhibits the degradation of IkappaBalpha in cells stimulated with TNF; Supplemental Table 1: Kd values of birinapant and Smac-AVPI peptide for IAP BIR domains.

The inhibitor of apoptosis (IAP) proteins have pivotal roles in cell proliferation and differentiation, and antagonizing IAPs in certain cancer cell lines results in induction of cell death. A variety of IAP antagonist compounds targeting the baculovirus IAP protein repeat 3 (BIR3) domain of cIAP1have advanced into clinical trials. Here we sought to compare and contrast the biochemical activities of selected monovalent and bivalent IAP antagonists with the intent of identifying functional differences between these two classes of IAP antagonist drug candidates. The anti-cellular IAP1 (cIAP1) and pro-apoptotic activities of monovalent IAP antagonists were increased by using a single covalent bond to combine the monovalent moieties at the P4 position. In addition, regardless of drug concentration, treatment with monovalent compounds resulted in consistently higher levels of residual cIAP1 compared with that seen following bivalent compound treatment. We found that the remaining residual cIAP1 following monovalent compound treatment was predominantly tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2)-associated cIAP1. As a consequence, bivalent compounds were more effective at inhibiting TNF-induced activation of p65/NF-κB compared with monovalent compounds. Moreover, extension of the linker chain at the P4 position of bivalent compounds resulted in a decreased ability to degrade TRAF2-associated cIAP1 in a manner similar to monovalent compounds. This result implied that specific bivalent IAP antagonists but not monovalent compounds were capable of inducing formation of a cIAP1 E3 ubiquitin ligase complex with the capacity to effectively degrade TRAF2-associated cIAP1. These results further suggested that only certain bivalent IAP antagonists are preferred for the targeting of TNF-dependent signaling for the treatment of cancer or infectious diseases.

SHP-141 (1) is a hydroxamic acid-based inhibitor of histone deacetylase enzymes which is under development for the treatment of cutaneous T-cell lymphoma. The original synthesis of 1 involved five synthetic steps beginning with suberic acid monomethyl ester. Final deprotection of the O-benzyl hydroxamate moiety using hydrogen and palladium catalyst mandated the use of metal scavengers to reduce palladium levels to within International Council for Harmonisation (ICH) guidance. Owing to the sensitivity of 1 toward self-condensation and the potential for N-O bond cleavage under hydrogenolytic conditions, we developed an alternative route to 1 which avoids Pd-mediated hydrogenation and prolonged metal scavenger treatment. This two-step process employs readily available suberic acid and methyl paraben and has successfully delivered multiple kilograms of 1 for clinical use. Importantly, crude 1 was stabilized for recrystallization in acetonitrile (ACN) solution by the addition of 0.1% citric acid and 4% water. Additionally, the filtration and drying of suitably sized aggregates of 1 with high purity (100 area%) was accomplished via temperature cycling of the 1/ACN solution.

Birinapant/TL32711 (1) is a bivalent antagonist of the inhibitor of apoptosis (IAP) family of proteins and was designed to mimic AVPI, the N-terminal tetrapeptide of the second mitochondria-derived activator of caspases (Smac/DIABLO). Birinapant bound to the BIR3 domains of cIAP1, cIAP2, and XIAP with K i values of 1, 36, and 45 nM, respectively. Birinapant-mediated activation of cIAP1 resulted in cIAP1 autoubiquitylation and degradation and correlated with inhibition of TNF-mediated NF-κB activation, induction of tumor cell death in vitro, and tumor regression in vivo. Birinapant is being evaluated in Phase 1/2 trials for the treatment of cancer and hepatitis B virus (HBV) infection. After one year at accelerated storage conditions, a formulation of 1 afforded four degradants in >0.1% abundance by HPLC analysis. The primary degradants (2 and 3) were formed via oxidation of the biindole core, while the secondary degradants (5 and 6) arose via [1,2]-rearrangement of 3 and 2, respectively. Forced degradation conditions were developed, which allowed the isolation of 2 and 3 in multigram quantities. Novel deuterated analogues of 1 were prepared to determine the site of oxidation, and NMR experiments confirmed the chemical structures of 5 and 6. The de novo synthesis of 2, 3, 5, and 6 confirmed these experimental findings.

Birinapant/TL32711 (1) is a novel bivalent antagonist of the inhibitor of apoptosis (IAP) family of proteins which is currently in clinical development for the treatment of cancer and hepatitis B virus (HBV) infection. In this report, we present a detailed description of the 1 drug substance synthesis used to support our ongoing clinical studies. Key transformations in this process included the development of a scalable, high-yielding route to acyl indole 14 as well as a two-step dimerization/oxidation of indole 19 that afforded biindole 21 in excellent yield and purity (70% yield, 2 steps; >95 area% purity by HPLC analysis). In addition, partial defluorination of 21 was observed following hydrogen-mediated benzyloxycarbonyl (Cbz) protective group removal which was obviated by the use of HBr/HOAc for this transformation. The use of commercially available amino acid derivatives afforded related impurities which proved difficult to purge in subsequent steps. Thus, defining the impurity specification for these reagents was critical to providing 1 drug substance of >99 area% chemical purity. Using this process, we have successfully prepared 1 drug substance multiple times on >500-g-scale in support of our clinical development program.

Abstract The second mitochondria-derived activator of caspases (SMAC) is thought to exert its pro-apoptotic activity as a homodimeric protein. Both monovalent and bivalent peptidomimetics of the SMAC tetrapeptide are being developed for cancer therapy. Birinapant/TL32711 is a bivalent SMAC-mimetic that targets the inhibitor of apoptosis (IAP) proteins whose gene abnormalities have been implicated in various cancers. Owing to structural differences between bivalent SMAC-mimetics and monovalent IAP-inhibitors, we sought to compare and contrast the biochemical activity of birinapant with several monovalent IAP-inhibitors including a monovalent- version of birinapant/TL32711, MV711. Previous studies have shown that both bivalent and monovalent agents promote auto-ubiquitylation and subsequent degradation of cIAP1 and cIAP2, which triggers tumor necrosis factor receptor (TNFR)-mediated cell death in certain tumor cell lines. However, birinapant showed substantial differences from IAP-inhibitors in degrading TRAF2-associated cIAP1 and cIAP2. Here we show that MV711 was less efficient at degrading cIAP1 by a factor of 7-fold (16 vs. 118 nM, birinapant vs. MV711, respectively) and inhibiting TNF-mediated NF-κB activation by 220-fold (9 vs. 1985 nM, respectively). In addition, a linker-lengthened variant of birinapant was less able to inhibit NF-κB activation by 71-fold (9 vs. 642 nM, respectively). We also studied the effect of birinapant or IAP-inhibitor treatment on SKOV-3, MDA-MB-231 and EVSA-T cancer cell lines in vitro. Comparable cIAP1 BIR3 domain binding constants and IC50 values for the degradation of cIAP1 and cIAP2 (ΔcIAP1/2) were observed for these two classes of agents, and both birinapant and IAP-inhibitors showed dose-dependent induction of cell death. However, despite such comparable potencies, the IAP-inhibitors did not completely kill SKOV-3 or MDA-MB-231 cells even with concentrations >100-times their ΔcIAP1/2 IC50 values. Birinapant revealed the highest suppression of cancer cell growth in the cell lines tested, even after the agent was removed, whereas the cell lines treated with the IAP-inhibitors showed rapid restoration of cell proliferation within 24 h following removal of the agents. These results suggested that monovalent IAP-inhibitors require maintenance of high steady state levels of drug to effectively suppress tumor growth in vivo. In agreement with their inability to induce cell death, IAP-inhibitors were less efficient in stimulating the formation of a RIPK1:caspase-8 complex when compared to birinapant in EVSA-T or SKOV-3 cells. These observations may be partly attributed to the reduced ability of IAP-inhibitors to degrade TRAF2-associated cIAP1 which serves a central role in the activation of NF-κB via TNFR. Citation Format: Yasuhiro Mitsuuchi, Christopher A. Benetatos, Thomas Haimowitz, Yijun Deng, Angeline C. Mufalli, Martin E. Seipel, Jennifer M. Burns, Gurpreet S. Kapoor, C. Glenn Begley, Stephen M. Condon. Birinapant, a bivalent SMAC-mimetic, promotes efficient cellular IAP E3 ligase activity and formation of a pro-apoptotic RIPK1:caspase-8 complex while monovalent IAP inhibitors are less efficient - implications for therapeutic utility. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1806. doi:10.1158/1538-7445.AM2014-1806

Abstract The acquisition of apoptosis resistance is a fundamental event in cancer development. Among the mechanisms used by cancer cells to evade apoptosis is the dysregulation of inhibitor of apoptosis (IAP) proteins. The activity of the IAPs is regulated by endogenous IAP antagonists such as SMAC (also termed DIABLO). Antagonism of IAP proteins by SMAC occurs via binding of the N-terminal tetrapeptide (AVPI) of SMAC to selected BIR domains of the IAPs. Small molecule compounds that mimic the AVPI motif of SMAC have been designed to overcome IAP-mediated apoptosis resistance of cancer cells. Here, we report the preclinical characterization of birinapant (TL32711), a bivalent SMAC-mimetic compound currently in clinical trials for the treatment of cancer. Birinapant bound to the BIR3 domains of cIAP1, cIAP2, XIAP, and the BIR domain of ML-IAP in vitro and induced the autoubiquitylation and proteasomal degradation of cIAP1 and cIAP2 in intact cells, which resulted in formation of a RIPK1:caspase-8 complex, caspase-8 activation, and induction of tumor cell death. Birinapant preferentially targeted the TRAF2-associated cIAP1 and cIAP2 with subsequent inhibition of TNF-induced NF-κB activation. The activity of a variety of chemotherapeutic cancer drugs was potentiated by birinapant both in a TNF-dependent or TNF-independent manner. Tumor growth in multiple primary patient–derived xenotransplant models was inhibited by birinapant at well-tolerated doses. These results support the therapeutic combination of birinapant with multiple chemotherapies, in particular, those therapies that can induce TNF secretion. Mol Cancer Ther; 13(4); 867–79. ©2014 AACR.

The acquisition of apoptosis resistance is a fundamental event in cancer development. Among the mechanisms used by cancer cells to evade apoptosis is the dysregulation of inhibitor of apoptosis (IAP) proteins. The activity of the IAPs is regulated by endogenous IAP antagonists such as SMAC (also termed DIABLO). Antagonism of IAP proteins by SMAC occurs via binding of the N-terminal tetrapeptide (AVPI) of SMAC to selected BIR domains of the IAPs. Small molecule compounds that mimic the AVPI motif of SMAC have been designed to overcome IAP-mediated apoptosis resistance of cancer cells. Here, we report the preclinical characterization of birinapant (TL32711), a bivalent SMAC-mimetic compound currently in clinical trials for the treatment of cancer. Birinapant bound to the BIR3 domains of cIAP1, cIAP2, XIAP, and the BIR domain of ML-IAP in vitro and induced the autoubiquitylation and proteasomal degradation of cIAP1 and cIAP2 in intact cells, which resulted in formation of a RIPK1:caspase-8 complex, caspase-8 activation, and induction of tumor cell death. Birinapant preferentially targeted the TRAF2-associated cIAP1 and cIAP2 with subsequent inhibition of TNF-induced NF-kB activation. The activity of a variety of chemotherapeutic cancer drugswas potentiated by birinapant both in a TNF-dependent or TNF-independentmanner. Tumor growth in multiple primary patient–derived xenotransplant models was inhibited by birinapant at well-tolerated doses. These results support the therapeutic combination of birinapant with multiple chemotherapies, in particular, those therapies that can induce TNF secretion. Mol Cancer Ther; 13(4); 1–13. 2014 AACR.

Birinapant (1) is a second-generation bivalent antagonist of IAP proteins that is currently undergoing clinical development for the treatment of cancer. Using a range of assays that evaluated cIAP1 stability and oligomeric state, we demonstrated that 1 stabilized the cIAP1-BUCR (BIR3-UBA-CARD-RING) dimer and promoted autoubiquitylation of cIAP1 in vitro. Smac-mimetic 1-induced loss of cIAPs correlated with inhibition of TNF-mediated NF-κB activation, caspase activation, and tumor cell killing. Many first-generation Smac-mimetics such as compound A (2) were poorly tolerated. Notably, animals that lack functional cIAP1, cIAP2, and XIAP are not viable, and 2 mimicked features of triple IAP knockout cells in vitro. The improved tolerability of 1 was associated with (i) decreased potency against cIAP2 and affinity for XIAP BIR3 and (ii) decreased ability to inhibit XIAP-dependent signaling pathways. The P2' position of 1 was critical to this differential activity, and this improved tolerability has allowed 1 to proceed into clinical studies.