Development of selective HDAC6 inhibitors with in vitro and in vivo anti-multiple myeloma activity

Highlights

• A novel series of HDAC inhibitors were designed, synthesized and evaluated.

• Several compounds showed potent in vitro cytotoxicity against human MM cell lines.

• The in vivo anti-MM potency of 12a1 was demonstrated in a xenograft model.

Abstract

Histone deacetylase 6 (HDAC6) is a promising therapeutic target for the treatment of cancers, neurodegenerative diseases and autoimmune disorders. Herein a novel series of pyrrolo[2,3-d]pyrimidine-based HDAC inhibitors were designed, synthesized and biologically evaluated, among which compounds 7a, 12a1, and 16a1 exhibited potent inhibitory activities and selectivities against HDAC6. Notably, compared with the well-known HDAC6 inhibitor Tubastatin A, our pyrrolo[2,3-d]pyrimidine-based HDAC6 inhibitors showed superior in vitro antiproliferative activity against human multiple myeloma cell lines RPMI 8226, U266 and MM.1S, while maintaining the low cytotoxicity against human breast cancer cell line MDA-MB-231 and two normal cell lines. The HDAC6 selective inhibition of one representative compound 12a1 in RPMI 8226 cells was confirmed by western blot analysis. Although pyrrolo[2,3-d]pyrimidine is a privileged structure in many kinase inhibitors, compound 12a1 showed negligible inhibition against several kinases including JAK family members and Akt1, indicating its acceptable off-target profile. Besides, compound 12a1 exhibited desirable metabolic stability in mouse liver microsome. The in vivo anti-multiple myeloma potency of 12a1, alone and in combination with bortezomib, was demonstrated in a RPMI 8226 xenograft model.

Introduction

Zinc²⁺-dependent histone deacetylases (HDACs), a family of hydrolases involved in epigenetic regulation and post-translational modification by removing the acetyl groups from histones and non-histone proteins, have been validated to be important anticancer targets based on the successful launch of five inhibitors (vorinostat, belinostat, panobinostat, romidepsin, and chidamide) [1], [2]. Amongst, panobinostat was approved in combination with bortezomib and dexamethasone for the treatment of patients with multiple myeloma who have received at least two prior treatment regimens, including bortezomib and an immunomodulatory agent [3]. However, diarrhoea and cardiac toxicities are two substantial toxicities of Panobinostat [4]. Considering that panobinostat is a pan-HDAC inhibitor with little isoform selectivity, it was presumed that isoform selective HDAC inhibitors could exhibit improved tolerability.

Among the Zinc²⁺-dependent HDACs, HDAC6 was suggested to be a potential anti-multiple myeloma target dependent on its important roles in degradation of unfolded and misfolded ubiquitinated proteins [5]. To be specific, HDAC6 contains a zinc finger ubiquitin-binding domain and a dynein motor binding domain, through which polyubiquitinated misfolded protein cargo is recruited by HDAC6 to dynein motors for transport to aggresomes, then degraded by autophagy [6], [7], [8]. It has been demonstrated that HDAC6 inhibition could induce accumulation of polyubiquitinated proteins and inhibit the growth of multiple myelomas, especially when combined with proteasome inhibitor bortezomib [5], [9]. More importantly, genetic ablation or pharmacological inhibition of HDAC6 cause no lethality or toxicity typically associated with pan-HDACs inhibition [10], [11], [12], [13]. Currently, at least three HDAC6 inhibitors (ACY-241, ACY-1215, and CS3003) have entered clinical trials for the treatment of multiple myeloma [14]. The chemical structures and HDAC inhibitory activities of ACY-241 and ACY-1215 are present in Fig. 1.

Pyrrolo[2,3-d]pyrimidine is a privileged structure in drug discovery, especially in the design of protein kinase inhibitors. For example, many JAK inhibitors including the approved drugs ruxolitinib and tofacitinib (Fig. 2) use the pyrrolo[2,3-d]pyrimidine group to form key hydrogen bond interactions with the hinge region of JAK proteins [17]. Besides, the clinical Akt inhibitor AZD5363 (Fig. 2) also contains the pyrrolo[2,3-d]pyrimidine group [18]. Interestingly, two series of pyrrolo[2,3-d]pyrimidine-based hydroxamates exemplified by compounds JMC-45 and BMCL-13b respectively were developed as HDAC and JAK dual inhibitors [19], [20], and one series of pyrrolo[2,3-d]pyrimidine-based benzamides exemplified by compound EJMC-7 demonstrated potent HDAC and tubulin polymerization dual inhibition [21] (Fig. 2). These compounds indicated that pyrrolo[2,3-d]pyrimidine is also a privileged structure in HDAC inhibitor design. In the present research, pyrrolo[2,3-d]pyrimidine group was introduced to the terminal cap group of the clinical HDAC6 inhibitors (ACY-241 and ACY-1215) and the HDAC6 inhibitor pharmacophore to design a novel series of pyrrolo[2,3-d]pyrimidine-based hydroxamates. Structure-activity relationship (SAR) study was focused on the linker part connecting the pyrrolo[2,3-d]pyrimidine group and the hydroxamate group, leading to one selective HDAC6 inhibitor 12a1 with remarkable in vitro and in vivo anti-multiple myeloma potency.