Histone deacetylase 3 (HDAC3) inhibitors as anticancer agents: A review

Journal Pre-proof Histone deacetylase 3 (HDAC3) inhibitors as anticancer agents: A review Rajat Sarkar, Suvankar Banerjee, Sk Abdul Amin, Nilanjan Adhikari, Tarun Jha PII:

S0223-5234(20)30138-0

DOI:

https://doi.org/10.1016/j.ejmech.2020.112171

Reference:

EJMECH 112171

To appear in:

European Journal of Medicinal Chemistry

Received Date: 30 November 2019 Revised Date:

18 February 2020

Accepted Date: 19 February 2020

Please cite this article as: R. Sarkar, S. Banerjee, S.A. Amin, N. Adhikari, T. Jha, Histone deacetylase 3 (HDAC3) inhibitors as anticancer agents: A review, European Journal of Medicinal Chemistry, https:// doi.org/10.1016/j.ejmech.2020.112171. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Masson SAS.

Graphical Abstract

Histone deacetylase 3 (HDAC3) inhibitors as anticancer agents: A review Rajat Sarkar, Suvankar Banerjee, Sk Abdul Amin, Nilanjan Adhikari, Tarun Jha* Natural Science Laboratory, Division of Medicinal and Pharmaceutical Chemistry, Department of Pharmaceutical Technology, P. O. Box 17020, Jadavpur University, Kolkata 700032, India Abstract Among different Histone deacetylases (HDACs), histone deacetylase 3 (HDAC3) is an epigenetic drug target which is currently marked as potential therapeutic strategy to combat various cancers. HDAC3 inhibitors are effective ways for the treatment of cancers, different neurodegenerative disorders, diabetes mellitus, cardiac diseases, HIV, inflammatory diseases, rheumatoid arthritis (RA), etc. Inhibition of HDAC3 metalloenzyme is a dynamic approach for drug design and discovery. This approach has gained considerable interest in recent years. The development of an effective therapeutic agent against HDAC3 is still challenging. A lot of work is still in demand. This current communication is a part of our extended work on HDAC3 inhibitors to achieve deep insight of knowledge about the structural information of HDAC3 inhibitors. This article is unique in terms of detailed structure-activity relationships (SARs) analysis. This may help to find out some important clues to design better active HDAC3Is in the future. Keywords: HDAC3; HDAC3 inhibitor; Metalloenzyme; Cancer; SAR; Drug design and discovery.

Corresponding Author * Phone: +9133-2457 2495 (O). +91-9433187443 (M). E-mail: [email protected] 1

1. Introduction Globally the term cancer indicates uncontrollable cell division of abnormal cells. It is the deadliest disease and recently it is found as the second prime reason for death worldwide according to the world health organization (WHO) in 2018. Apart from other major human diseases (like cardiovascular disease, AIDS and diabetes), cancer becomes a common reason for death throughout the earth. In recent years, cancer incidence and mortality are expeditiously growing worldwide (Figure 1). Among 6 deaths, there is a one due to cancer. The death may be due to lung, colon, breast, prostate, stomach, colorectal and skin cancers or hepatocellular carcinoma (Figure 1) [1]. [Figure 1 maybe placed here] The formation of cancer, known as oncogenesis or carcinogenesis, is the transformation of the normal cells into the malignant cells. During the phase of the carcinogenesis, mutation occurs in tumor suppressor genes and oncogenes, subsequently there is a disruption in cell proliferation and cellular differentiation [2]. To reinforce the outgrowth, malignant cancer cells generally decrease the apoptotic cell death [3]. Targeting and inhibiting different proteins related to cancer occurrence and progression is one of the primary methods to deal with several pathophysiological conditions related to cancer. HDACs are the class of zinc-dependent metalloenzymes those take part profoundly in cellular migration and invasion. Modulation of histone proteins and DNA methylation are roots of several epigenetic modifications that alter the chromatin structure and accessibility of DNA [4]. HDAC inhibitors can accelerate the apoptotic cell death or programmed cell death [3]. The overexpression (Figure 2) of histone deacetylases can lead to various types of cancers such as pancreatic, colorectal, breast, lung, colon and liver cancers as well as leukemia, hepatocellular carcinoma and prostate cancer [5-8]. [Figure 2 may be placed here] Histone deacetylases and histone acetyltransferase (HAT) enzymes are two key components that play pivotal roles in the regulation of the deacetylation and acetylation process of histone proteins. These two enzymes are not only responsible for acetylation and deacetylation of histone proteins but also play important roles in cell survival, homeostasis, cell proliferation and gene expression (Figure 3) [9]. HAT enzymes acetylate the nucleosomal histones and lead to 2

hyperacetylation or hypoacetylation which cause transcriptional activation or repression respectively [9]. [Figure 3 may be placed here] Inhibition of HDAC enzymes (specifically HDAC3) is a dynamic approach for diseases related to it and this approach has acquired considerable value in recent years [9]. Development of small molecule HDAC inhibitors for various disease conditions including cancer is an emerging target in recent times [9-10]. HDAC3 has been established as a potential target of cancer [8]. Apart from cancer, HDAC3 inhibitors are also effective ways for the treatment of the different neurodegenerative disorders such as Huntington’s disease (HD), diabetes mellitus, cardiac diseases, HIV and inflammatory diseases, e.g. rheumatoid arthritis (RA) [11]. HDAC3 belongs to the Zn²+ dependant class I HDAC isoforms those erase out the acetyl group from the acetylated lysine residues at the N-terminal region of the core histone proteins (namely H2A, H2B, H3 and H4). HDAC3 also allows histones to enclose the DNA tightly [12-13]. During DNA replication, the total amount of cellular histone is increased. HDACs acts on these freshly synthesized histones. HDAC3 plays crucially in the regulation of gene transcription by modulating STAT 3 (signal transducer and activator of transported 3). This is controlled by the covalent modifications, i.e., acetylation, phosphorylation and methylation to which they are subject. Although HDAC3 has a role in damage control of DNA structure, inhibitors of HDAC3 induce the DNA damage of cancer cells in human body. This will lead to tumor suppressor genes into a silent phase [14]. Therefore, HDAC3 enzyme is an important target of cancer and other diseases. A lot of work is still needed to identify a HDAC3 selective inhibitor. For the last few years, our research team is in continuous endeavors to design better active and selective HDAC3 inhibitors [8-9, 12-14]. This review is a part of our extended work on HDAC3 inhibitors to find important structural information to design better active molecules in future. This article is quite unique in terms of structure-activity relationships (SARs) deliberation that may help to design better active HDAC3 inhibitors. 2. Classification of HDACs In humans, 18 HDAC enzymes have been found till now (Table 1) [5,15]. HDACs are classified into two major groups such as the zinc (Zn²+)-dependent HDACs where zinc metal ion (Zn²+) 3

acts as the co-factor or catalytic activator and another is nicotinamide adenine dinucleotide (NAD+)-dependent HDACs where NAD+ acts as the co-enzyme for their enzymatic activity. The Zn²+-dependent HDACs can be further classified into three different classes such as class I, class II and class IV. However, class I HDACs (such as HDAC 1, 2, 3 and 8) are predominantly located into the nucleus whereas class II HDACs (such as HDAC 4, 5, 6, 7, 9 and 10) are located into both nucleus as well as cytoplasm because of phosphorylation which is done by protein kinase C or protein kinase D [15]. For class IV HDAC, i.e., HDAC 11, it shares the catalytic domain of class I and class II HDACs. Interestingly, class I and class II HDAC isoforms bear structural similarity with the reduced potassium dependency 3 (RPD3) protein and HDAC1 enzyme of yeast respectively [8,16]. The main target of HDAC inhibitors is to suppress the inherent activity of HDACs those are done by occupying the catalytic core of the Zn²+ binding site [15]. In addition, class III HDAC enzymes, NAD-dependent enzymes, are also called sirtuins (SIRTs 1-7) and possess structural similarity to the yeast sir2 silencing protein [13, 17]. [Table 1 may be placed here] 3. Structure of HDAC3 After a thorough watch over the biological role of HDAC3, it is concluded that this repressive chromatin-modifying enzyme forms a stable and reproducible multi-protein complex with nuclear receptor co-repressor (N-CoR) and silencing mediator of retinoic and thyroid receptors (SMRT) [18]. N-CoR is a bridging molecule between HDAC3 and nuclear receptors [19]. In the stable complex of HDAC3/N-CoR/SMRT, HDAC3 is found as the catalytic component that clarifies the mechanism related to the connection of histone deacetylation and transcriptional repression [17]. The activity of histone deacetylation is enhanced due to the recruitment of HDAC3 by repressive transcription factors into the stable co-repressor complex (Figure 4). [Figure 4 may be placed here] Similarly, other two class I HDAC isoforms namely HDAC1 and HDAC2 show maximal enzymatic action after recruited into the nucleosome remodeling and deacetylase (NuRD), Sin3 and REST corepressor 1 (CoREST) multi-subunit co-repressor complex [20]. Deacetylase activating domain (DAD) mediates the activity of SMRT which is generally preserved into the N-CoR through this activation of HDAC3 [21]. DAD includes a tightly spaced SWI3/ADA2/N4

CoR/TFIIIB (SANT) motifs pair (SANT1 and SANT2) in the N-terminus of repressors SMRT and N-CoR. Having a highly conserved sequence, SANT2 motif functions as a part of the HID (Histone-interacting domain) which is responsible for binding and activation of the HDAC3 enzyme [18]. Nevertheless, HDAC3 has a unique C terminal domain which does not obviously match with other class I HDACs [9]. In the interactions between the SMRT:DAD with the amino and carboxyl terminus of the Nterminus region of HDAC3 lead to the formation of four unique helical structures (namely helix H1, helix H2, loop L2 and strand S2) which is due to the structural rearrangement of DAD. The crystal structure of the co-repressor complex HDAC3-SMRT possesses two monovalent cations (MVCs) as potassium with one MVC binding site which is closer to the active site Zn2+ ion. The second MVC binding site is situated ≥ 20 Å from the active site Zn2+ ion. Structural comparison between HDAC8 and HDAC3 shows that there is no need for co-repressor complex such as SMRT-DAD to activate the deacetylase process of HDAC8. This reveals that the structure of HDAC8 differs markedly with HDAC3 in the region where SMRT and DAD are found to interact [20]. In that large conjugated complex besides SMRT, N-CoR and HDAC3 a WD40 repeat-containing protein, presently known as Transducin β-like 1X (TBL1X) is linked. In 2001, Guenther and co-workers [17] purified the complex SMRT-TBL1X- HDAC3 which possesses lysine deacetylase activity. They also explained that without the co-repressor complex HDAC3 alone has no role in the deacetylation process. These co-repressors contain different active domains such as nuclear receptor interacting domains and multiple repressor domains [17]. Like other class I HDACs (such as HDAC2 and HDAC8), HDAC3 also has an eight-stranded parallel β-sheets encompassed by certain α-helices. In loop L5 of HDAC3, a unique solventexposed tyrosine residue (Tyr198) is situated on the surface of the enzyme besides the phenylalanine residue (Phe199). This phenylalanine residue (Phe199) alters the orientation of neighbour tyrosine residue (Tyr198). This tyrosine residue may contribute to the substrate specificity which is just adjacent to the active site tunnel. Except HDAC8, other isoenzymes of class I HDACs (1, 2 & 3) have a similar kind of structural alignment near the substrate-binding site. Loop L1 of HDAC8 is shorter than the corresponding loop of other class I HDACs. There are significant differences in the sequence alignment and length between HDAC8 and HDAC3 [22]. A comparison of structural alignment between HDAC3 with HDAC1 and HDAC2 indicates five major differences. In case of HDAC3, aspartate residue is located in position 92 in 5

place of glutamate which is situated in the outer rim of its cavity. Phenylalanine present in HDAC3 in position 199 instead of tyrosine residue which is found in both HDAC1 and 2. In position 107 of HDAC1 and 2, serine is present but in the case of HDAC3, it is replaced by tyrosine which creates a steric hindrance. Due to that, inhibitors with bulkier functional groups cannot bind to the foot pocket. This a major difference and helps to develop of selective HDAC inhibitors. Besides the above differences, HDAC3 has more structural dissimilarities at positions 13 and 29 those participate in interactions with selective inhibitors [23]. Ins(1,4,5,6)P4 molecule acts as an ‘intermolecular glue’ which makes extensive contact between HDAC3 and SMRT:DAD and helps in the activation of HDAC3 (Figure 5). Both Ins(1,4,5,6)P4 and SMRT-DAD are mutually required for the enzymatic activation of HDAC3. Ins(1,4,5,6)P4 associates with Arg265 which is an important interaction in loop L6. Other than that in L6, Leu266 makes a wall at the active site tunnel. Due to that, loop L6 has a significant role in substrate accessibility towards the active site of HDAC3. These portions of HDAC3 significantly differ with the structure of HDAC8. The absence of these residues may lead to the loss of the deacetylase activity of HDAC3 [20]. [Figure 5 may be placed here] 4. Role of HDAC3 in various diseases HDAC3 is an important zinc-dependent metalloenzyme that can implicate various types of disease conditions in human through epigenetic modulations. The role of HDAC3 is correlated in several life-threatening diseases like cancers, inflammatory diseases, cardiovascular diseases, neurodegenerative disorders, learning and memory dysfunctions, Huntington’s disease (HD), diabetes, etc. 4.1. Inflammation Fouda and co-workers [24] revealed that A1 (arginase 1) shows its anti-inflammatory effect using ornithine decarboxylase (ODC)-mediated suppression of HDAC3 which is a key player in the macrophage inflammatory response. Inhibition of ODC or removal of A1 increases HDAC3 expression in LPS-treated macrophages (MФ) and aggravated the inflammatory responses like iNOS and PRO-IL-1β. Treatment by PEG-A1 weakens the up-regulation of HDAC3 and also reduces the inflammatory responses. The inhibition of HDAC3 ameliorates inflammatory responses by reducing the production of NO and the expression of TNF-α in A1 KO MФ. 6

Besides that, this inhibition improves the neuronal cell survival and prevents retinal thinning. It concludes that targeting HDAC3 is a novel strategy for treating neurovascular injury as well as ischemic retinopathy (Figure 6) [24]. [Figure 6 may be placed here] Moreover, macrophages are directly linked with the disease progression of a lipid-driven inflammatory condition like atherosclerosis. Specifically, regulation of the histone acetylation is crucial in inherent immune responses which affects the development of atherosclerosis. 4.2. Cardiovascular Disease Around 31% of global deaths are caused due to the disorders related to the cardiovascular system. Myocardial infarction, cardiac hypertrophy, atherosclerosis, cardiac fibrosis and arrhythmia are the different cardiovascular diseases. These are now the major reason behind numerous deaths worldwide. Histone deacetylase, the epigenetic regulator, has a key role in the pathophysiological conditions of cardiovascular diseases. Schiattarella et al. [25] reported that HDAC inhibitors have a triggering effect in various preclinical models of cardiovascular diseases but it produced some serious cardiac side effects. Class I HDACs specifically HDAC1 and HDAC2 are acting as a mediator of congestive heart failure (CHF) which is characterized by cardiac remodelling, acute myocardial infarction (AMI) and interstitial fibrosis. Nural-Guvener and co-workers [26] demonstrated that there is upregulation of class I HDAC isoforms (HDAC1 and HDAC2) in cardiac fibroblasts. Lkhagva and the group [27] revealed that class I HDACs (HDAC1, 2, 3 & 8) play pivotal roles in the tumor necrosis factor-α (TNF-α)-induced mitochondrial dysfunction in myocardiocytes. Xu and co-workers [28] explained that inhibition of HDAC3 was phenomenally related to the prevention of diabetic myocardiopathy (DCM) in mice model. Janardhan et al. [29] investigated that lymphatic endothelial HDAC3 regulates the lymphatic transport system through the development of both lymphovenous valves and lymphatic valves which maintain blood-lymph separation. Moreover, HDAC3 also regulates oscillatory shear stress (OSS)-mediated activation of the GATA-binding factor 2 (GATA2) intragenic enhancer through the recruitment of E1A-associated protein p300 (EP300). Extracellular superoxide dismutase (EC-SOD3), a chief vascular antioxidant enzyme, has an effective role in idiopathic pulmonary arterial hypertension (IPAH). The expression of SOD3 along with activity is selectively decreased in IPAH which is regulated by HDAC3 by enhancing

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cell proliferation [30]. Hoeksema et al. [31] demonstrated that the upregulation of HDAC3 is related to the macrophage phenotype, plaque phenotype, collagen deposition, and TGFB1 expression. Its enhanced collagen deposition and developed atherosclerosis. Selective inhibition of HDAC3 might help to discover some anti-atherosclerotic drugs [31-33]. Butyrate is a bacterial metabolite that induces antimicrobial activity via its inhibitory action against HDAC3. This gives a driving force to the differentiation process of macrophages leading to the alteration of their metabolism and enhancement of production of antimicrobial peptides in vivo and in vitro [34]. The enzymatic activity of HDAC3 has a pivotal role in the regulation of muscle fuel metabolism and contractile functions which increase the possibility of HDAC inhibitors to enhance or to modulate exercise performance or to manipulate muscle energy metabolism [35]. 4.3. Diabetes Zhao and co-workers [36] suggested that overexpression of HDAC3 has an implication in diabetic stroke. Use of HDAC3 inhibitor or suppression of HDAC3 gives a protective effect against the cerebral H/R and I/R injury in the diabetic state by reducing the cerebral infarct volume, improving cell viability and cytotoxicity, alleviating apoptosis, modulating oxidative stress and enhancement of autophagy. This protective effect might be due to the upregulation of Bmal1 gene [36]. Rosenberg and his group [37] showed that HDAC3 plays a specific role as the positive regulator of myelin gene expression. He et al. [38] described that after inhibition of HDAC3 there is an enhancement of myelin growth and regeneration. 4.4. Memory and learning HDAC3 has an important role in the long-term memory process. Expression of gene related to the memory storage cells is modulated by some HDAC enzymes (specially HDAC3) which take part crucially in memory function by epigenetic remodelling of histones. Recently, Amin and coworkers [9] elaborately discussed the role of HDAC3 in memory and learning. Selective inhibition of HDAC3 ameliorates long term memory function by upregulating hippocampal NR2B mRNA and phosphorylation of cAMP -response element-binding (CREB) at the NR2B gene [9]. 5. Role of HDAC3 in cancer The expression of both HDAC3 and HDAC1 are significantly correlated with the B7-H1 expression in gastric cancer (GC) (Figure 6). Deng et al. [39] revealed that HDACs (namely

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HDAC1-3) might have some potential role in the regulation of B7-H1. Inhibition of HDACs leads to the reduction of interferon gamma (IFN-γ) induced B7-H1 expression in GC (Figure 7). In many cases, overexpression of HDAC3 is directly correlated with the expression of different cancer regulating genes. In colorectal cancer, overexpression of HDAC3 causes a decrease in the expression of p21. Inhibition of HDAC3 induces the acetylation process of p53 and p21 expression that results in G1 phase arrest in triple-negative breast cancer. HDAC3 inhibits mitogen-activated protein kinase (MAPK)-dependent activation of transcription factor-2 (ATF-2) to repress the expression of TNF-α [40]. HDAC3 can repress the metastatic potential of colorectal cancers (Figure 6) by binding to the promoter sequences of Runt-related transcription factor 2 (Runx 2) [41]. [Figure 7 may be placed here] It can decrease the proliferative potential of metastatic breast cancer cells by binding to the CREB protein. Not only that, it also can deteriorate the expression of Hypoxia-inducible factor1α (HIF-1α) by the negative regulation of NF-κB and takes part in the modulation of STAT3 which inhibits beclin 1, autophagy and acts as a prognostic marker in non-small cell lung cancer (NSCLC) cells [42-44]. HDAC3 which is a downstream target of glycogen synthase kinase 3 β (GSK3β) and AKT serine/threonine kinase (Akt) by which it can enhance β-cell apoptosis and causes neuronal death via inflammatory cytokines. Downregulation of HDAC3 enhances the chances of tumor formation of lung cancer cells and also inhibits the growth of cholangiocarcinoma by increasing apoptosis. HDAC3 can also minify the potential of angiogenic growth in melanoma cells. Spurling and co-workers [45] performed immunohistochemical study and showed that HDAC3 has a major role in the regulation of p21 expression as well as takes part in differentiation and proliferation of colon cancer cell lines. According to Narita and co-workers [46], inhibition of HDAC3 may lead to the enhancement of apoptosis of human maxillary cancer. They further demonstrated that specific HDAC3 inhibitors with paclitaxel potentiate the paclitaxel-induced apoptosis in IMC-3 human maxillary cancer cell line [47]. Lucas et al. [48] revealed that resveratrol and piceatannol, two dietary stilbenoids enhance the expression of programmed cell death ligand 1 (PD-L1) in colorectal cancer and breast cancer through HDAC3/p-300-mediated NF-κB signalling. Thus, inhibition of HDAC3 blocks the induction of PD-L1 expression. According to Jiang et al. [49], HDAC3 is required for 9

the proliferation of adult neural stem/progenitor cells (NSPCs). It controls the progression of G2/M phase by post-transitional stabilization of cyclin-dependent kinase 1 (CDK1). Thus, HDAC3 plays a pivotal role in NSPC proliferation which indicates that the use of HDAC3 inhibitors is a good strategy for controlling of cancer growth. Edderkaoui and co-workers [50] revealed that HDAC3 has a specified role in smoking-induced pancreatic adenocarcinoma. It has been seen that due to smoking there was a significant increase in the phosphorylation level of HDAC3 [50]. Thus, it causes translocation of phosphorylated HDAC3 in the nucleus. Inhibition of HDAC3 reduces the level of IL-6 produced by the cancer cells and plays a significant role as a stimulator in the interaction between tumor-promoting macrophages and cancer cells. Therefore, targeting HDAC3 by a novel mechanism can be utilized in cancer therapy. HDAC3 plays key role in the development of acute promyelocytic leukemia (APL) [51]. Harada and co-workers [52] demonstrated that selective inhibition of HDAC3 downregulates DNA methyltransferase 1 (DNMT1) expression in multiple myeloma (MM). HDAC3 is associated with the proliferation of hepatocellular carcinoma (HCC) and acts as the biomarker for fixing the tumor recurrence of hepatitis B mediated hepatocellular carcinoma [53]. Some workers from China reported that the expression of HDAC2 and HDAC3 were upregulated in tectal cells specifically in the ventricular layer of the brain lipid-binding protein (BLBP) positive radial glial cells (RGs). The differentiation or proliferation of radial glial cells is associated with various factors in that HDAC activity is one of the important ones. The specific knockdown of HDAC3 has been found to reduce the BrdU and BLBP-labelled cells which suggests that proliferation of redial glia was selectively mediated by HDAC3 [54]. Zhang and colleagues [55] found that cMyc oncogene decreases the pyruvate level in cancer cells by enhancing the enzymatic action of lactate dehydrogenase A (LDHA) and pyruvate kinase M2 (PKM2) those lead to decrease in inhibition of HDAC3. Hence, slowdown of apoptosis of cancer cells occurs. Reversibly, when the activity of HDAC3 is enhanced, it stabilized the cMyc protein by deacetylating cMyc at Lys323 which reduces the level of pyruvate. This makes a positive feedback loop that elevates the Warburg effect and cell proliferation of cancer. 6. FDA approved HDAC inhibitors Over a long period of time, the outcome of the rigorous studies on several HDAC inhibitors is good for having six approved molecules namely vorinostat, romidepsin, belinostat, panobinostat, chidamide and pracinostat (Figure 8, Table 2) for the treatment of different cancers [14]. 10

Although the number is very less related to prevention of cancers, but it helps continuously to find newer and better molecules in present days. [Figure 8 may be placed here] [Table 2 may be placed here] 6.1. Vorinostat (SAHA/suberoylanilide hydroxamic acid) Vorinostat or SAHA (suberoylanilide hydroxamic acid) (FDA-01) (Figure 8), marketed as Zolinza, is a hydroxamic acid derivative. It was the first FDA approved HDAC inhibitor which has nanomolar activity against various HDACs including HDAC3. It is used for the management of relapsed and refractory cutaneous T- cell lymphoma (CTCL) [13, 56]. It is a linear hydroxamic acid compound having molecular weight of 264.32, empirical formula C14H20N2O3 and pKa approximately 9. In case of hematological cancers, orally administered dose of Vorinostat (200-600 mg per day) is well tolerated. On the other hand, the dose may be increased up to 600 mg for solid tumors. It binds with the zinc metal ion in the catalytic domain of the HDAC enzymes. Entinostat and SAHA increase the expression of thioredoxin binding protein-2 (TBP-2) which inhibits thioredoxin in bladder cancer cell line T24, breast cancer cell line MCF7 and prostate cancer cell LNcap. Vorinostat along with temozolomide which is an alkylating agent and in combination with radiotherapy is now under clinical trial process (NCT00731731) for the treatment of glioblastoma multiforme (GBM) which is the most common as well as a fast-growing malignant brain tumor. It was found that vorinostat exerted an effective apoptotic activity along with antiproliferative action in type I and type II human endometrial cancers by altering the expression of specific genes those were related to the insulin-like growth factor-I (IGF-I) receptor signalling pathway [61]. In type I cell lines, SAHA triggered the IGF-IR phosphorylation and upregulated phosphatase and tensin homolog (PTEN) as well as p21 expression. It also reduced the levels of p53 and cyclin D1 in type I cell lines. However, in type II cell lines, it upregulated IGF-IR and p21 expression and also downregulated AKT, PTEN and cyclin D1 expression. In the case of both type I and type II endometrial cancer cell lines, SAHA hyperacetylated histone H3 [62-63]. In clinical trial, SAHA in combination with front-line anticancer drugs such as doxorubicin, vincristine, cyclophosphamide, and prednisone exhibit poor efficacy in treating untreated peripheral T-cell lymphoma (PTCL) patients [64]. It has potential antineoplastic 11

activity with broad inhibitory action against class I and class II HDACs. In preclinical studies, SAHA also showed its use in human glioblastoma cell line as a potent radiosensitizer [65]. Under hypoxic condition, radiosensitization by SAHA along with capecitabine reduced colonogenicity and also inhibited tumor growth in xenograft models of colorectal carcinoma [66]. Vorinostat shows its transcriptional effects by directly binding with HDAC enzymes or by indirectly acting on numerous transcriptional factors like YY-1, Bcl-6, E2F-1, Smad7, GATA-1 and p53. 6.2. Romidepsin (Istodax) In the year of 2009, romidepsin (FDA-02) got the FDA approval for CTCL treatment (Figure 8) [16]. The maximum tolerated dose (MTD) of romidepsin was found in phase I clinical trial conducted on 37 diseased patients [67]. The approval for the treatment of CTCL was carried out two large phases II studies. The first one was a multi-institutional study performed at the national cancer institute (NCI) in USA with 71 patients and the second one was conducted on 96 patients. In both cases, the response rate was found to be 34%. It also responded strongly for patients having relapsed or refractory PTCL [58]. Along with bortezomib, romidepsin was also found to be a good anticancer agent effective against non-small cell lung cancer (NSCLC). They together inhibit A549 NSCLC cell proliferation [16,68]. Robertson and co-workers [69] revealed that romidepsin alone or with paclitaxel was synergistically effective to eliminate both primary and metastatic tumors formed by SUM 149 1BC cell line. It was also found that romidepsin potentially enhanced the destruction of inflammatory breast cancer (IBC) tumor emboli and lymphatic vascular architecture for the treatment of IBC and advanced breast cancer [69]. Romidepsin is now being evaluated for treating mainly T-cell lymphoma either as a single or in combination with other drugs. 6.3. Belinostat (Beleodaq) Belinostat (FDA-03) (Figure 8) was the third HDAC inhibitor approved by FDA in 2014 for the management of relapsed multiple myeloma with 25.8% objective response rate (ORR). It is a sulphonamide-based hydroxamate derivative. Belinostat (FDA-03) was also investigated in patients having advanced thymic epithelial tumors but no response was found among these patients [58,70].

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6.4. Panobinostat (Farydak) Panobinostat (FDA-04, Figure 8), another hydroxamic acid-based pan-HDAC inhibitor, got FDA approval in 2015 for the treatment of multiple myeloma. Objective response rate (ORR) of 27% was observed for panobinostat. It was also effective against hematological cancers and solid tumors in clinical trials. One of the serious side effects of panobinostat (Figure 8) is cardiotoxicity [16]. 6.5 Pracinostat Pracinostat (FDA-05) (Figure 8) is another hydroxamate-based compound approved by FDA for the treatment of Acute myeloid leukemia (AML) and was found to inhibit different classes of HDAC. Besides HDACs, it has no such effect on other zinc-dependent metalloenzymes. It was orally active HDAC inhibitor. After accumulation insight the tumor cells it causes acetylation of histones, remodeling of chromatin and transcription of various tumor suppressor genes [59]. 6.6 Chidamide (Epidaza) Chidamide (FDA-06) (CS055/Tucidinostat/HBI-8000) (Figure 8) was discovered and developed by Chipscreen Biosciences and is a benzamide-based HDAC inhibitor. China Food and Drug Administration (CFDA) approved chidamide in the year 2015 as an HDAC inhibitor for treating relapsed or refractory PTCL and pancreatic cancer. It is orally active having selectivity against HDAC 1, 2, 3 and 10. It acts as a genuine epigenetic modulator that induces apoptosis and arrest the cellular growth [60]. 7. HDAC3 inhibitors A number of academia and industry work are involved in the exploration of HDAC3 inhibitors (Cpds 1-269) over the past decades [71-130]. There are various types of HDAC3 inhibitors such as benzamides, hydroxamates, hydrazides and thiols. Out of these, few compounds show selectivity to some extent. However, this number is very few and still do not pass the clinical trial goals. 7.1. Benzamides as HDAC3 inhibitor Benzamide scaffold mainly targets the zinc ion present in the active site and is a promising zinc binding group (ZBG) for designing effective and potential HDAC3 inhibitors. Many studies have already been done on o-aminoanilide or benzamide-based HDAC inhibitors and still it is going on [8,23]. One of the prime examples of an HDAC inhibitor with benzamide scaffold is entinostat (MS-275) (Cpd 1, Figure 9) which inhibited HDAC3 at a lower concentration (IC50 = 13

0.95 µM). Other than that, it also inhibited HDAC1 and HDAC2 at IC50 values of 0.19 µM and 0.41 µM respectively. Moreover, entinostat was also found to trigger the expression of p21WAF/Cip1 in human Hep3B hepatoma cells [71-73]. [Figure 9 may be placed here] MS-275 also showed greater activity in the prevention of cytokine-induced β-cell apoptosis [74]. A well-known HDAC3 selective inhibitor is RGFP966 (Cpd 2, Figure 9) (IC50 = 0.08 µM) which was used as a molecular tool to study the role of HDAC3 enzyme [75-76]. RG2833/RGFP109 (Cpd 3, Figure 9) is a benzamide-derived inhibitor. It shows better potency for HDAC3 at an IC50 value of 60 nM and Ki value of 5 nM. It blocks the expression of NF-κBdependent transcription process in glioblastoma and used in the treatment of Friedreich’s ataxia [77-78]. Tacedinaline/CI–994 (Cpd 4, Figure 9) was a potent class I inhibitor with promising HDAC3 inhibitory activity (IC50 = 1.2 µM) [79]. It increased the apoptosis in LX- and A-549 cell line [80]. It also potentially inhibited the growth of leukemia BCLO and prostate cancer LNCaP [8182]. BRD3308 (Cpd 5, Figure 9) is a selective HDAC3 inhibitor (IC50 = 0.064 µM) with a minimum 17-fold selectivity over HDAC1 and HDAC2 [83]. Cpd 5 is a modified structure of compound CI-994. There is a fluorine substitution in the para position of the benzamide moiety of BRD3308. It showed better IC50 value than the previous one. It was also used for the therapy of diabetes and HIV infection [84-86]. Mendoza and co-workers [87] synthesized a benzamide-based molecule, RMS-162 (Cpd 6, Figure 9) which has promising HDAC3 inhibitory activity (IC50 = 3.18 µM). It was highly selective over HDAC6 with promising antiangiogenic activity (Luciferase IC50 = 0.72 µM, BRT IC50 = 0.21 µM). Besides that, it also has good antitumor efficacy against breast cancer MCF-7 cell line (IC50 = 1.90) [87]. BG45 (Cpd 7, Figure 9), is a potent HDAC3 inhibitor (IC50 = 289 nM) reported by Minami et al. in 2014 [88]. It was having a minimum of 7-fold selectivity over HDAC1 and HDAC2. It showed dose-dependent inhibition against multiple myeloma cells by inducing acetylation of H2A, H3 and H4 without any toxic effect in peripheral blood mononuclear cells (PBMC). They observed that it acts via caspase-3 and PARP-related apoptotic pathway. BG45 has no effect on α-tubulin which is a biomarker of HDAC6. This showed its

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selectivity towards HDAC3 over HDAC6. Compound 4SC-202 (Cpd 8, Figure 10) potentially reduced the proliferation of urothelial cancer cell lines [89]. [Figure 10 may be placed here] Pinkerneil and co-workers [89] revealed that this compound increases G2/M and sub G1 phase of cell division in VM-CUB1 and UM-UC-3 cells better than SAHA. Another molecule, MI-192 (Cpd 9, Figure 10) showed significant HDAC3 inhibition with an IC50 value of 16 nM. It exhibited significant growth inhibitory activity against a group of leukemia cell lines (namely HL-60, CCRF-CEM, MOLT-4, RPMI-8226 and K562). Along with good growth inhibitory activity, it was also capable to induce apoptosis and differentiation in variety of leukemia cell lines like HL-60, U937 and Kasumi-1. Apart from that, it showed good efficacy against various types of cancers such as non-small cell lung cancer (NSCLC), breast, colon, renal, ovarian and prostate cancers and melanoma. Compared to MS-275, MI-192 exhibited 2 to 3-fold better efficacies in leukemia [90-91]. Chen and co-workers [92] reported that Cpd 10 (Figure 10) was selective against HDAC3 with an IC50 value of 0.12 µM and it did not inhibit other isoforms of HDAC at a concentration up to 30 µM. Suzuki and co-workers [93] screened a series of compounds by using click chemistrybased combinatorial fragment assembly to obtain Cpd 11 (Figure 10) and Cpd 12 (Figure 10) as HDAC3 selective inhibitors. Both compounds have good IC50 value of 0.24 µM and 0.26 µM respectively. During docking study of Cpd 11 with HDAC3 enzyme, it displayed that the amine group (-NH2) and the keto (-CO) group of the benzamide ring bind the zinc atom and form two hydrogen bonds with surrounding amino acids His134 and Gly143. In these compounds, the phenyltriazole group serves as the linker moiety and fits inside the hydrophobic tunnel by hydrophobic interaction. Thiophene ring present in the cap region of the compound interacts with the Pro23 and Phe144 [93]. Cpd 13 (Figure 10) was a potent HDAC3 inhibitor (IC50 = 4.3 µM) synthesized by Hu et al. [94] through SPPS (solid-phase peptide synthesis) process. The specialty of this compound was that it has a flexible linker moiety with amide bonds. More than 19-fold higher concentration was needed for the inhibition of HDAC1 enzyme (IC50 = 83.9 µM). Cpd 14 (Figure 11) was a potent HDAC3 inhibitor (IC50 = 12 nM) having more than 7-fold and 1000-fold selectivity over HDAC2 and HDAC1 respectively [93]. Cpd 14 contained a chiral heterocyclic cap group and

15

produce apoptosis in the Jurkat cell line. Marson et al. [95] revealed that the phenyl ring of the linker moiety entered into the hydrophobic tunnel and produced a hydrophobic interaction between Phe144 and Phe200. The chiral heterocyclic cap region formed hydrophobic interaction with Phe199. Another mocetinostat derivative, Cpd 15 (Figure 11) with an oxazoline cap group synthesized by Marson et al [95]. It showed better selective inhibitory activity against HDAC3NCoR2 complex with an IC50 value of 6 nM [95]. Gao et al. [96] designed and synthesized isatin-cap based series of benzamide derivatives through scaffold hopping strategy. [Figure 11 may be placed here] The antitumor activity of the best active compound (Cpd 15) was found against several cancer cell lines including HEL (IC50 = 1.15 µM), SK-N-BE (2) (IC50= 1.92 µM), MOLT-4 (IC50= 2.46 µM), K562 (IC50 = 2.83 µM) and PC3 (IC50 = 11.35 µM). Hsieh et al. [7] designed Cpd 16 (Figure 11) by replacement of isopropyl group and hydroxamic acid moiety in AR-42 derivatives with dimethyl and N-(2-amino-4-fluorophenyl)amide groups respectively. It showed effective HDAC3 inhibitory activity (IC50 = 0.35 µM) with suppressing the CSCs subpopulation by downregulating the β-catenin in triple-negative breast cancer (TNBC) in vitro. In the in vivo studies in nude mice, this compound showed effective suppression in tumorigenesis via HDAC3 knockdown. Moreover, selectively targeting HDAC3 is required for the treatment of TNBC. Another benzamide derivative Cpd 17 (Figure 11) is a potent HDAC3 inhibitor (IC50 = 0.2 µM) [7]. Cpd 18 (Figure 11), a para-fluoro substituted benzamide derivative, was synthesized by McClure et al. [97] as a selective HDAC3 inhibitor (IC50 = 0.08µM). It was also active against HDAC1 and HDAC2 having an IC50 value of 1.2 µM and 1.5 µM respectively. Cpd 19 (Figure 11) having another fluorine substitution at the meta-position of benzamide ring showed slightly decrease in the inhibitory activity but improved the selectivity towards the HDAC3 [97]. Cpd 20 (Figure 11) was one of the effective inhibitors with an IC50 value of 138 nM. It is also effectively yielded cytotoxicity against a group of cancer cell lines such as K562 (IC50 = 0.98 µM), U937 (IC50 = 1.10 µM), U266 (IC50 = 2.23 µM), HEL (IC50 = 3.02 µM), HCT116 (IC50 = 4.23 µM), ES-2 (IC50 > 20 µM) [98]. Trivedi et al. [14] designed and developed a series of benzamide-based inhibitor which having good selectivity towards HDAC3.

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7.2. Hydroxamates as HDAC3 inhibitor HDAC inhibitors that are currently available possess a small molecular structure with three pharmacophoric subunits. These subunits are composed of a zinc-binding group (ZBG) attached to a hydrophobic cavity binding linker chain which mimics the lysine side chain and terminated by a hydrophobic cap group (surface recognition group). It interacts with the binding site rim at the external surface of the enzyme. The connecting group between the linker motif and the cap group can also be modulated to improve the interactions. This ZBG binds with the zinc ion which is located in the active site of class I HDACs [67,99]. Commonly the strongest ZBGs are hydroxamic acid and benzamides or o-aminoanilides. Vorinostat (Figure 8), belinostat (Figure 8), panobinostat (Figure 8) are some FDA approved synthetic hydroxamic acid-based HDAC inhibitor show high affinity towards the zinc ion. Besides that, there are other hydroxamates which are now in clinical trials such as resminostat (Phase II trial), givinostat (Phase II trial), pracinostat (Phase II trial), abexinostat (Phase I trial), quisinostat (Phase I & II trial), MPT0E028 (Phase I trial), CHR 3996 (Phase I trial), CUDC 101 (Phase I trial) and CUDC 907 (Phase I trial) [100-105]. Trichostatin A (TSA) is a naturally occurring hydroxamic acid HDAC inhibitor which is obtained from Streptomyces hygroscopicus [106]. Compounds with hydroxamate moiety showed the pan-HDAC inhibitory effect with HDAC3 inhibition in nanomolar concentration. Hydroxamic acid has zinc chelating ability which offers a non-selective Zn+dependent enzyme inhibition. It includes histone deacetylases (class I, II, IV), matrix metalloproteinases (MMPs) and aminopeptidase N (APN).Therefore, workers must pay their attention to establish or to design non-hydroxamate moieties which have poor or specific Zn+ chelation effect from hydroxamate derivatives as well as potent inhibitory effect. In case of in vivo studies, the most potent hydroxamate ZBGs display poor absorption as well as rapid metabolism. SAHA has a short half-life (< 2 hr) [56, 107]. Cpd 21 (Figure 12) having a hydroxamic acid group attached with an isoxazole linker moiety showed better and selective HDAC3 inhibitory action with an IC50 value of 30 nM [108]. These compounds contain thiazolylphenyl moiety as a cap group which helps to fit into the hydrophobic tunnel through hydrophobic interaction. [Figure 12 may be placed here]

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Cpd 22 (Figure 12) having the same central structure with modification at the meta-position of the terminal phenyl ring having an amine group led to almost similar HDAC3 inhibitory effect (IC50 = 25 nM). Tapadar and co-workers [108] studied the activity of this compound (Cpd 22) against a group of pancreatic cancer cell lines (such as BxPC-3, HupT3, Mia Paca-2, Panc 04.03 and Su.86.86). Cpd 21 was highly active in all these cell lines. On the other hand, Cpd 22 showed poor cellular permeability [108]. Neelarapu and co-workers [109] reported Cpd 23 and Cpd 24 (Figure 12) having aryloxazole containing hydroxamate function with more than 10-fold better HDAC3 inhibitory activity compared to HDAC8. Cpd 24 showed cytotoxic activity against a variety of cancers (such as hepatocarcinoma, cervical cancer and neuroblastoma). It also displayed some neuroprotective effects [109]. Vaidya et al. [110] reported some novel HDAC inhibitors containing different zinc-binding groups (ZBGs). Cpd 25 (Figure 12) was a hydroxamate-based inhibitor, found to be selectively effective against HDAC3 enzyme with an IC50 value of 44 nM selective over HDAC8 (IC50 = 76 nM). Tashima and co-workers [111] synthesized some hydroxamate-based pan-HDAC inhibitors. Among these compounds, Cpd 26 (Figure 12) displayed potent as well as selective HDAC3 inhibition (HDAC3 IC50 = 28 nM). Li et al. [112] first reported some N-hydroxycinnamide derivatives which are having selectivity preferentially for HDAC1 and 3 over HDAC2 and 6. Cpd 27 (Figure 12) which was a pchlorobenzyl-based compound exhibited promising HDAC3 inhibitory activity with an IC50 value of 3.9 nM. It was also selective towards HDAC3 over other HDAC isoforms. The docking interactions of Cpd 27 with HDAC3 showed that it formed a strong van der Waals interaction due to its flexible methylene group that directs the indole moiety towards the solvent-exposed area. The amide group present in Cpd 27 formed H-bonding interaction with two amino acids (Asp192 and Asp193). This exhibited higher affinity towards HDAC3. Moreover, Cpd 27 displayed the highest U937 cell line inhibition with an IC50 value of 160 nM and decreasd the level of procaspase 3. It also potentially inhibited tumor growth in a U937 xenograft model tested in BALB/c-nu mice. It showed moderate activity against a panel of cancer cell lines including HEL, K562, MDA-MB-231, KG1, HL60, MCF-7, PC3, A-549 and HTC-116. It was less effective against HUVEC cells. In comparison with SAHA, Cpd 27 induced 61.76% cell death at 1 µM concentration and showed a reduced level of histone H3 and H4 acetylation [112]. Zhang et al. [113] reported a series of compounds with greater potency compared to vorinostat (SAHA) towards HeLa nuclear extract. They also evaluated these compounds against different 18

HDAC enzymes (HDAC1, HDAC2, HDAC3, and HDAC6). The SAR data demonstrated that the naphthylcarbonyl/naphthylsulfonyl analogs showed much better affinity towards HDAC3 and HDAC1. Fascinatingly, the o-methoxyphenylaminocarbonyl and m-bromophenylcarbonyl derivatives displayed better activity than the previous ones but nonselectivity was noticed between HDAC3 and HDAC1. These compounds showed potent cytotoxic effect against a list of cancer cell lines. Amongst these compounds, the most potent HDAC3 inhibitor was Cpd 28 (Figure 12) which also showed the greater inhibitory activity against PC3 cell line (IC50 = 1,510 nM). Along with that, it exhibited significant nuclear HDACs inhibitory effect with an increased acetylation level of tubulin. The molecular docking study of Cpd 28 with HDAC3 revealed that the phenyl glycine moiety forms a stable hydrophobic interaction with amino acid residue Phe199. Phe199 was the key residue for HDAC3 selectivity. Cpd 28 also formed unstable interactions with Tyr209 of HDAC2. Due to that, Cpd 28 was more HDAC3 selective over HDAC2 [113]. 7.3. Hydrazides as HDAC3 inhibitor Apart from hydroxamate and benzamide-based inhibitors, there are some hydrazide-based HDAC3 inhibitors with potent activity. Wang et al. [114] identified a series of potent and selective HDAC3 inhibitors having p-substituted benzoylhydrazide moiety (Cpds 29-32, Figure 13). Cpd 29 (IC50 = 70 nM) was the most potent as well as selective HDAC3 inhibitor over other HDAC isoforms. [Figure 13 may be placed here]

Cpd 30 exhibited apoptotic efficacy in HCT116 cell line and arrest the cell cycle at G2/M phase in the MDA-MB-231 cell line. It had no effect on the acetylation of α-tubulin [114]. McClure and co-workers [115] reported some hydrazide-based potent HDAC3 inhibitors (Cpd 33, Figure 13) having efficacy against AML (acute myeloid leukemia). Cpd 33 was a cinnamide derivative containing n-propylhydrazide moiety displayed the highest HDAC3 inhibition with IC50 value of 0.95 nM. These compounds also have selectivity over HDAC1 and 2. Replacement of npropylhydrazide with n-butylhydrazide (Cpd 34, Figure 13) moiety reduced 4-fold HDAC3 inhibitory activity. Cpd 35 and Cpd 36 (Figure 13) having a phenyl group in place of cinnamide moiety reduced inhibitory activity but enhance selectivity towards HDAC3 [115]. Li et al. [116] 19

modified the benzamide moiety with an aryl hydrazide group to design some selective HDAC3 inhibitors having indole moiety as the cap group. 7.4. Thiols as HDAC3 inhibitor Along with benzamides and hydroxamates, some of the thiol-based large molecules displayed potent HDAC3 inhibitory effect. Yao et al. [117] reported a few cyclic depsipeptides as HDAC inhibitors. Among these, Cpd 37 showed better inhibitory activity against HDAC3 with IC50 value of 7.67nM (Figure 14). However, these inhibitors are basically nonselective or pan-HDAC inhibitors [117]. [Figure 14 may be placed here]

In addition, some of the largazole analogs including Cpd 38 were synthesized by Almaliti et al. [118] and was evaluated against colon cancer cell lines (HCT116). These showed promising cytotoxic effects. These compounds also displayed greater potency and higher selectivity towards HDAC3. Cpd 38 exhibited potent HDAC3 inhibitory effect with an IC50 value of 27 nM. On the other hand, Cpd 39 (largazole thiol derivatives) and Cpd 40 (trans-cyclooctane largazole thiol derivative) which are reported by Xu and his group [119] displayed better inhibitory effect towards HDAC3 with IC50 values of 0.7 nM and 1.1 nM respectively. 8. Structure-activity relationship of HDAC3 inhibitors In recent days, HDAC3 inhibitors come up as a useful weapon to fight against numerous diseases. For that reason, development of potent and selective inhibitors is very much in demand. To develop better active HDAC3 inhibitors, it is necessary to understand the structure activity relationship (SAR) of the previously synthesized molecules. Understanding of the SAR analysis is really important as the biological activity is associated with the structural rearrangements of molecules. To improve the biological inhibitory potency of newer molecules, it is essential to interpret the SAR analysis correctly. 8.1. Structure-activity relationship of benzamide based HDAC3 inhibitors Chen and colleagues [92] reported a series of hydroxamate and benzamide derivatives containing phenyl-thiazolyl or triazolyl-phenyl moiety as the cap group. Some of these inhibitors showed potent and selective inhibitory activity against HDAC3. These compounds are more or less similar in cap and linker position. The only difference is in the ZBG of these inhibitors. Before

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going to the SAR of these compounds, we segregate these inhibitors into two major parts on the basis of the ZBG. Firstly, we focused on the benzamide derivatives. The phenyl-thiazole ring and triazolyl-phenyl group show improved inhibitory activity towards HDAC3 as the cap group. Moreover, thiazole derivatives show better inhibitory activity but triazolyl derivatives displayed higher selectivity towards HDAC3 (Cpds 41-42, Cpd 10, Figure 15). [Figure 15 may be placed here] The presence of the phenyl ring in the cap group attached with the linker moiety is not favourable for HDAC3 inhibition (Cpd 41 vs 42). The absence of the phenyl ring improves potency and more suitable to HDAC3 inhibition. Hexamethylene group as the linker moiety is essential for all those HDAC3 inhibitors. Moderately activating groups like acetamido at the meta position of the terminal phenyl ring of phenylthiazole group is effective against HDAC3 inhibition over –NH2, –BocNH substitution (Cpd 41, Cpds 43-44). Interestingly, –NHCOOEt substitution (Cpd 45) shows a slight decrease in the inhibitory activity than Cpd 41. Compound with bis-[(2-aminophenyl)amide] is one as the cap groups and another as the ZBG, shows more or less similar inhibitory potency against HDAC3 (Cpd 46). Moreover, compound containing aminobenzamide group (Cpd 47) as the cap moiety and hydroxamate scaffold as the ZBG shows pan-HDAC inhibition (HDAC1 IC50 = 67.2 nM, HDAC2 IC50 = 101 nM, HDAC3 IC50 = 21.1 nM, HDAC6 IC50 = 9.93 nM, HDAC10 IC50 = 49.3 nM). However, hydroxamate derivatives show the higher activity than benzamide compounds but less selective towards HDAC3. These hydroxamate derivatives with phenyl-thiazole moiety as the cap group show potent inhibitory effect against HDAC3 (Cpd 48 vs Cpd 49). Compound with the –NHCOOEt, –NH2, –NHBoc substitutions at the meta position of the terminal phenyl ring displayed effective inhibitory activity against HDAC3 (Cpds 49-51) [90]. Adhikari et al. [8] also confirmed that the thiazole analog is preferable than triazole or triazolylmethyl analogs. They also explained that hexamethylene linker is favourable instead of the phenyl ring for HDAC3 inhibition [8]. According to them, the acetamido group is better than other substituents. Moreover, the amino group is slightly less effective than the acetamido group for these benzamide derivatives. Benzamide derivatives having the flexible aminobenzyloxy group or acetamide group show promising HDAC3 selectivity over HDAC2. However, HDAC3 selectivity is enhanced by substituting the amino group with the acetyl or ethyl carboxylate function (Figure 16).

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[Figure 16 may be placed here] Marson et al. [95] synthesized a series of 4-substituted analogs of mocetinostat. From the in vitro inhibition assay, it is observed that only a few compounds possess good inhibitory potency against HDAC3. From the SAR study, it is observed that the electron donating group at the 4th position of the aminoanilide group favours the inhibitory activity against HDAC3. Compounds containing the hydroxy (-OH) and methoxy (-OCH3) substituents are highly active against HDAC3 (Cpds 52-53, Table 3). [Table 3 may be placed here] Moreover, compound with the methoxy substituent displayed 6-fold higher selectivity than the hydroxy substituent compound. Compound with the bromo substituent at the 4th position showed less potency than previous two, but it exhibited higher selectivity towards HDAC3 over HDAC2, HDAC6 and HDAC8 (Cpd 54). In addition, when aminoanilide group was replaced with aminonaphthyl moiety it showed the higher selectivity over other HDAC isoforms (Cpd 55) whereas compound with the 4-phenyl group displayed no inhibition on HDAC isoforms (Cpd 56) (Figure 17). [Figure 17 may be placed here] In contrast to all benzamide derivatives, the hydroxamate derivative showed the higher potency against HDAC3 but these were the least selective towards other HDAC isoforms (Cpd 57). Besides this, Marson and colleagues [95] synthesized another series of potent benzamide derivatives with slight modification in the cap region. In this series, compounds having imidazolinone and thiazolidine groups with indolylmethyl, benzyl or phenyl substitutions showed potent inhibitory effects against HDAC3. In addition, compound having the phenyl or benzyl substitution in comparison to the indolylmethyl group displayed the higher selectivity as well as the higher inhibitory activity against HDAC3 (Cpds 58-59 vs Cpd 60). Phenyl and benzyl substitution at the 4th or 5th position of the heterocyclic ring showed more potent and selective compounds. Interestingly, both S and R-configured analogs showed better HDAC3 inhibitory activity with moderate selectivity (Cpd 14, Cpd 61). Compound 14 and 62 have similar kind of structure with a slight difference in the arrangement of the terminal phenyl ring of 22

the cap group where the (S)-phenyl analog is 7 times more potent than the corresponding (R)phenyl analog. For some cases, compound with the thioether group in the linker region displayed potent activity and selectivity towards HDAC3 (Cpds 63-64) (Figure 17) [95]. Marson et al. [120] synthesized a series of N-(2-aminophenyl) benzamide containing aminoimidazoline and oxazoline derivatives as potent and selective chiral HDAC3 inhibitors (Cpd 15, Cpds 65-83, Table 3). Here, N-(2-aminophenyl) benzamide moiety is a common portion for all these inhibitors, served as a zinc binging group. The carbonyl group enters into the active site of HDAC3 and acts as an H-bond acceptor whereas the amide functional group is responsible for zinc chelation. The terminal free amino group (-NH2) serves as H-bond donor which has a key role in the inhibitory activity for all these benzamide derivatives. Hydrophobicity of the terminal phenyl ring may require for settling into the active site of the HDAC3. Inhibitors having aryl linker show potent inhibitory activity than inhibitors containing aliphatic linker group. Aryl linker also imparted steric and hydrophobic characteristics that favoured the inhibition process. The presence of the thioether moiety in the chiral heterocyclic ring plays as a surface recognition group (Cpds 66-69). Five-member semi-saturated heterocyclic rings (Cpd 65 and 15) (such as oxazoline, aminoimidazoline) with different heteroatoms preferred as the cap group over the six-member heterocyclic ring (e.g. MGCD0103/35). Phenyl and benzyl substitution at the 4th or 5th position of the heterocyclic ring showed more potent and selective compounds (Cpd 15, Cpds 65-83). Interestingly, compound having R-configuration shows better inhibitory activity than compound having S-configuration (Cpd 71 vs 70). Moreover, compound with relatively bulky phenyl group at 4th and 5th position of oxazoline ring shows the highest inhibitory activity against HDAC3 with an IC50 value of 6nM (Cpd 15). Electron withdrawing substituent in the cap group improves its selectivity towards HDAC3 (Cpds 73-74) [120] (Figure 18). [Figure 18 may be placed here] Amin et al. [12] demonstrated that the ether linkage in the oxazole ring system has the positive influence over the regulation of HDAC3 inhibition. They conclude that oxazole ring as the cap group is better than an aminoimidazoline moiety. They also explained that aminobenzyl substitution (linker motif) at 2nd position of the oxazole moiety is important for the biological activity. Besides that, the phenyl substitution (cap group) at 5th position enhances the HDAC3

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inhibitory effect. They signified that aryl substituent at ZBG leads to the loss HDAC3 inhibitory activity. Adhikari et al. [8] signified the structural relationship between these compounds quantitatively. They demonstrated that bulky substitution as the cap group may raise unfavourable steric clashes leading to a decrease in the inhibitory activity. On the other hand, the bulky steric substitution in the benzamide moiety decreases in the selectivity towards HDAC3 over HDAC1. Li et al. [98] designed and synthesized a series of potent benzamide derivatives containing indole moiety as the cap group. From the SAR study, it was observed that the 2-thienyl substitution at the R position of benzamide scaffold is detrimental to the inhibitory activity than other analogs (Cpd 84-86, Table 4).

[Table 4 may be placed here]

In addition, the phenyl or any bulky substitution at this position is unfavourable for HDAC3 inhibition (Cpds 87-88). Moreover, the fluorine substitution at the X position of the benzamide scaffold (Figure 19) is essential for HDAC3 selectivity (Cpd 89). In this series, it was found that phenylcarboxamide function as the linker motif is responsible for less efficacy than pentamethylene carboxamide or benzylcarboxamide functions (Cpd 20, Cpds 90-91) whereas pentamethylene carboxamide function showed improved inhibitory activity against HDAC3 (Cpd 91). S-configuration of the amide group with the benzoyl substitution is important for HDAC3 inhibition (Cpd 20, Cpds 89-91). Adhikari et al. [8] postulated that for the better HDAC3 inhibitory activity, molecules should have lesser steric effect. 2-thienyl substitution produces the higher steric effect at the R position. This is a disadvantage for HDAC3 inhibition (Cpds 84-86). They also suggested that compound having a long chain pentamethylene carboxamide group as the linker moiety is suitable over other analogs (Cpd 91) [98]. [Figure 19 may be placed here] Hsieh et al. [7] synthesized a series of potent and selective benzamide-based HDAC inhibitors. Compound containing heterocyclic group or 5-aminopicolinoyl group as the linker moiety showed improved HDAC3 inhibitory potency over compound containing the phenyl ring as the linker (Cpd 92 vs 93, Figure 20).

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[Figure 20 may be placed here] Compound containing the phenyl ring as the linker displayed high selectivity towards HDAC3 (Cpd 93). 6-aminonicotinoyl moiety as the linker showed a loss of activity against HDAC isoforms (Cpd 94). Interestingly, compounds having ethyl or isopropyl group showed the better activity as well as the better selectivity towards HDAC3 over compounds having the methyl group (Cpd 92 and 95 vs Cpd 96). Compounds with unsubstituted benzyl group as the cap showed improved activity than para-substituted benzyl derivative (Cpd 92 vs Cpd 97). On the other hand, fluorine substitution at ZBG enhances the selectivity of the inhibitors towards HDAC3 (Cpd 17). The presence of the cyclopropyl group between the terminal phenyl ring and the connecting unit may have a role to raise the selectivity towards HDAC3 (Cpd 17). Trifluoro substitution at the para position of the benzyl group reduces the inhibitory activity against HDAC isoforms (Cpd 98). Dimethyl substitution in the cap group may be responsible for the higher selectivity towards HDAC3 over HDAC1 and HDAC2 (Cpd 16). Compound having Sconfiguration of R1 substitution showed the better inhibition than corresponding compound with R-configuration (Cpd 92 vs 99) (Figure 21) [7]. [Figure 21 may be placed here] Yun and co-workers [121] synthesized a series of thioether-based benzamide derivatives as novel HDAC inhibitors where they kept thioether moiety in between the junction of linker and cap groups on the basis of the principle of bioisosterism (Cpd 100-103, Figure 22). [Figure 22 may be placed here] The phenyl ring attached with the thioether moiety is preferred as the cap group over any other bulky substituent. However, compound having the methoxy substitution (Cpd 100) at the para position of the phenyl ring showed the better inhibitory activity against HDAC3. This compound also showed potent inhibitory effect against some cancer cell lines (A549, A375, SMMC7721, HCT116, HeLa). Alkyl substitution at the para position is unfavourable for the inhibitory activity (Cpd 101). The heterocyclic ring as the cap group showed decrease in the activity in comparison to the phenyl ring (Cpd 103 vs 100). Enhancement in the chain length between the thioether moiety and the terminal phenyl ring results in decrease of inhibitory activity (Cpd 102) (Figure 23) [121]. 25

[Figure 23 may be placed here] 8.2. Structure-activity relationship of hydroxamate-based HDAC3 inhibitors Tapadar and co-workers [108] synthesized a series of hydroxamic acid derivatives as HDAC inhibitors in which some of these molecules contain aryl isoxazole moiety as the cap group and other molecules contain isoxazole moiety as the linker function (Cpds 21-22, Cpds 104-111, Figure 24). Isoxazole moiety as the cap group showed the highest inhibitory effect against HDAC3 but these are not selective compared to HDAC6 (Cpds 104-105). However, compound having the isoxazole moiety, which is a more rigid and bulkier linker group attached to the hydroxamic acid moiety showed the better selectivity towards HDAC3 over other HDACs (HDAC6, HDAC2, HDAC1 and HDAC10) (Cpd 21-22). Phenylthiazole moiety as the cap group is essential for the inhibitory activity but compound containing this moiety exhibited the lower HDAC3 inhibitory activity than the compound with isoxazole cap group (Cpds 21-22, Cpds 104-105). Electron donating group, i.e., amino group at the meta position of the terminal phenyl ring improves the HDAC3 inhibitory activity (Cpd 22). The absence of the hydroxamate scaffold diminished the HDAC inhibitory activity (Cpd 106) whereas the carboxylic acid derivative showed 3-fold HDAC3 selectivity over other HDACs (HDAC1, HDAC2, HDAC6 and HDAC10) (Cpd 107). [Figure 24 may be placed here] In addition, carboxamide derivatives exhibited lesser activity than the carboxylic acid derivatives over different HDACs (Cpd 107 vs 108). Tapadar and colleagues [108] also synthesized some new compounds with few modifications in the cap region. They replaced the phenylthiazole group with quinoline-3-yl moiety and performed in vitro inhibitory assay on several HDACs (Cpd 109). Cpd 28 was comparatively less effective than Cpd 23 and Cpd 24 whereas compounds having more bulky groups like biphenyl-4-yl or cycloheptyl moiety as the cap group failed to produce any significant activity on HDAC3 (Cpds 110-111) (Figure 25) [108]. [Figure 25 may be placed here] A series of potent triazol-4-yl phenyl bearing hydroxamates-based HDAC inhibitors were synthesized by He and co-workers [122] with an interesting modification in the cap group. They evaluated these compounds against different HDAC isoforms. Most of these compounds showed 26

pan-HDAC inhibition but some of these showed potent inhibitory activity against HDAC3. From the SAR observation, it may be concluded that any substitution at the terminal phenyl ring of the cap group lost the selectivity against HDAC isoforms although it improves HDAC inhibitory activity (Cpd 112, Figure 26). [Figure 26 may be placed here] As an example, electron withdrawing group at the meta position effectively improved inhibitory activity against HDAC3 but failed to raise selectivity towards HDAC3 (Cpd 112). However, electron donating group at the para position showed activity in nano-molar region for HDAC3 (Cpd 113). Compound containing the iodine substitution at the para position showed improved activity than compound containing the fluorine substitution (Cpd 114 vs 115). However, the trifluoromethyl group at the para position (Cpd 116) showed similar kind of activity with Cpd 14. Replacement of the terminal phenyl ring with the cyclohexyl group resulted in a slight decrease in the inhibitory activity (Cpd 117 vs 118). The presence of the electron withdrawing group (EWG) at the para position along with an electron donating group (EDG) at the meta position showed decreasing inhibitory activity (Cpd 119 vs Cpd 12). Triazol-4-yl phenyl group as the surface recognition moiety is essential for HDAC inhibition. Hydroxamate group as the ZBG showed the higher HDAC3 inhibitory activity but these are nonselective whereas benzamide derivative showed the higher HDAC3 selectivity over other HDACs (HDAC1, HDAC2, HDAC6, HDAC8 & HDAC10) (Cpd 120) (Figure 27). [122]. [Figure 27 may be placed here] Li and co-workers [112] synthesized a series of N-hydroxycinnamamide-based potent HDAC3/HDAC1 inhibitors among those some compounds showed promising HDAC3 and HDAC1 inhibition over HDAC2 and HDAC6 (Cpds 27, 121-126, Table 5). [Table 5 may be placed here] The methylene group present between the ZBG and the aryl linker played an important role in HDAC3 inhibitory activity. The docking study revealed that the flexible methylene group pushes the aryl moiety towards the solvent exposed area which involved better van der Waals interactions. Apart from hydroxamate ZBG, the aryl linker group also favoured the inhibitory

27

activity. The indole cap group is one of the factors for the higher HDAC3 inhibitory activity and it is also preferred over other bulky aryl groups. The presence of an amide group improved the selectivity of these inhibitors towards HDAC3. S-configuration is required to maintain inhibitory activity. The amide group was substituted with the benzoyl moiety which is better than the sulfuryl and acyl groups (Cpds 124, 126-127). The EDG like methoxy or weakly deactivating group e.g. fluorine or chlorine at the para position of the benzoyl moiety improved the potency of these inhibitors (Cpds 27, 123-124). Besides these, the presence of the biphenyl group (Cpd 128) in place of the benzoyl group showed less activity against HeLa extract (IC50 = 1703.4 nM). The docking study of the most potent and selective molecule (Cpd 27) (HDAC1 IC50 = 11.8 nM, HDAC3 IC50 = 3.9 nM) showed that two hydrogen bonds were formed between the cap group and Asp92 as well as Asp93 residues. This may be the more acceptable reason for HDAC3 inhibition. On the other hand, the least active molecule (Cpd 125) did not form a hydrogen bond with Asp92 which may be one of the reasons for its least activity. Apart from that, the most potent molecule showed bidentate interaction with the zinc ion whereas the least potent molecule showed monodentate interaction. Moreover, the methoxy substituted compound is highly potent against HDAC3 with IC50 value 3.2 nM (Cpd 124) (Figure 28). [Figure 28 may be placed here] It also showed 129-fold and 58-fold selectivity towards HDAC3 over HDAC2 and HDAC6 respectively. In addition, the hydroxy substitution at the meta position of the benzoyl group displayed good inhibitory effect on HeLa nuclear extract with an IC50 value of 20.8 nM (Cpd 129). However, the bromo substitution at the para position showed less inhibitory activity than the fluorine substitution against HeLa nuclear extract (Cpds 130 vs 123) [112]. Yang et al. [123] designed and developed a series of potent but nonselective hydroxamate-based compounds by introducing 4-anilinothieno[2,3-d]pyrimidine scaffold as the cap group (Cpds 131-156, Table 6). [Table 6 may be placed here] It was anticipated from the SAR observation that the removal of the 4-anilino fragment from the 4-anilinothieno[2,3-d]pyrimidine scaffold resulted in poor inhibitory activity (Cpd 156). Yang and co-workers [123] also confirmed that the presence of this fragment helped to enhance the 28

hydrophobic interaction with HDACs that may lead to trigger the inhibitory activity against HDAC isoforms. However, the distance between two amide groups is a vital aspect of HDAC inhibition. In some extent, lengthening of the linker chain between two amide groups increased the inhibitory activity (Cpd 134 and 135 vs Cpd 136). The highest inhibitory activity was observed when the chain length was kept between five or six carbon atoms which was similar to SAHA (Cpds 145-146). Moreover, the bis-halogen substitution at the terminal phenyl ring somehow reduced the inhibitory potency against HDAC3 (Cpd 131-141). In addition, the chloro substitution at the meta position showed the better inhibitory activity (Cpd 142) whereas compound with no substitution at the terminal phenyl ring showed the better potency than compound with halogen substitutions (Cpd 144 vs Cpds 131-142). On the other hand, compound having the electron-donating group at the para and/or meta position of the terminal phenyl ring showed better the activity against HDAC3 (Cpd 146, 148). Compound with the pyrrolidine substitution at the para position displayed the lesser inhibitory activity against HDAC3 (Cpds 154-155). In addition, compounds with the dimethylamine or diethylamine substitution at the para position along with the fluoro substitution at the meta position showed the better activity against HDAC3 compared to compound with only diethylamine substitution (Cpd 150 and 151 vs Cpd 149) (Figure 29) [123]. [Figure 29 may be placed here] Zhang and co-workers [113] reported a series of nonselective hydroxamate-based novel histone deacetylase inhibitors with the higher inhibitory potency. These compounds showed significant anti-proliferative activity against several tumor cell lines (such as U937, MCF7, K562, PC3, HL60, and MDA-MB-231). Compounds with carbamido group showed the highest inhibitory potency against HDAC3 with an IC50 value of 5.9 nM (Cpd 28). In addition, compounds with the

bigger

aromatic

groups

like

naphthylcarbonyl,

naphthylsulfonyl

and

dimethylaminonaphthylsulfonyl displayed effective HDAC3 inhibitory activity (Cpds 157-158 and 160, Table 7). [Table 7 may be placed here] From the SAR study, it is anticipated that the sulfonyl group in place of carbamido moiety was not suitable to produce the higher inhibitory potency against HDAC3 (Cpd 158 and 160 vs Cpd

29

28). The S-configuration of these molecules is required to maintain the inhibitory effect. Aryl linker along with connecting unit is essential for HDAC inhibitory effect. From the enzymatic inhibition assay, it was found that compound with the bromo substitution at the meta position of the phenyl ring was better than compound with the chloro substitution at the para position (Cpd 159 vs 161) whereas the o-methoxyphenyl substituent showed the highest inhibitory activity against HDAC3 (Cpd 28). The docking study of the best active compound (Cpd 28) with HDAC3 showed that the terminal phenyl ring interacts with both of amino acid residues Tyr198 and Phe199 present in the opening of the cavity which might play an important role in the selectivity between HDAC3 and HDAC2 whereas the other interactions observed between omethoxyphenyl moiety with Tyr297 and aryl linker with His171. In addition, these interactions are favourable for HDAC3 inhibition (Figure 30) [113]. [Figure 30 may be placed here] Zang

et

al.

[124]

developed

a

series

of

hydroxamate-based

inhibitor

with

N-

hydroxycinnamamide moiety as the linker motif showing nanomolar activity for HDAC3. The presence of the benzoyl group at R1 position enhanced inhibitory activity against HDAC3 (Cpd 162, Table 8). [Table 8 may be placed here] The benzoyl group was also preferred over acyl or sulfuryl group (Cpd 162 vs 163). Moreover, the methoxy substitution at the para position of the benzoyl group showed the highest inhibitory activity against HDAC3 (Cpd 162). On the other hand, bulky substitution may decrease the activity. Methyl substitution at the para position of the benzoyl moiety improved the inhibitory potency against HeLa nuclear extract with IC50 value of 8.1 nM (Cpd 164). It was observed from the SAR study (Figure 31) that compounds with electron-donating substituents displayed better inhibitory activity than compounds with electron withdrawing substituents (Cpd 162 and 164 vs Cpd 165-166). The terminal indole moiety is essential as the cap group whereas the flexible methylene group of N-hydroxycinnamamide is also important for HDAC3 inhibition. Methoxy substitution at the N-hydroxycinnamamide moiety decreases the inhibitory activity (Cpd 162 vs 167) (Figure 31) [124]. [Figure 31 may be placed here] 30

Abdelkarim and co-workers [125] designed and synthesized a series of potent N-substituted 7aminoheptanoic acid hydroxylamide-based HDAC inhibitors. Compounds with secondary amino groups were found to be more potent than compounds with tertiary amino groups (Cpds 168173, Table 9). [Table 9 may be placed here] The docking study also suggested that the surface binding groups of tertiary amine compounds are unfavourable for binding to the active site whereas the secondary amine with 1H-indol-2ylmethyl group showed the highest inhibitory activity against HDAC3 with an IC50 value of 25 nM (Cpd 168). Compounds with indole moiety as the cap group showed the better inhibitory activity than compounds with the corresponding naphthalene and phenyl ring (Cpd 168 vs 170 and 174). Moreover, electron donating substitution at the meta position or both at the para and meta positions of the phenyl ring exhibited the better inhibitory effect than the unsubstituted phenyl compounds (Cpds 174-176). In addition, the para fluoro substitution (Cpd 177) is good over para-nitro substitution (Cpd 178) whereas bicyclic ring (like phenylthiophene) as the cap group showed improved inhibitory activity with an IC50 value of 38nM (Cpd 169) (Figure 32) [125]. [Figure 32 may be placed here] A series of new hydroxamate derivatives containing naturally occurring β-carbolines was developed by Ling and co-workers [126]. The β-carboline derivatives showed the better activity against HDAC1/HDAC6/HDAC3 and showed significant antiproliferative activity against several cancer cell lines (Cpds 179-183, Table 10).

[Table 10 may be placed here] From the SAR study, it is anticipated that compounds with the hydroxycinnamic acid moiety as the linker group showed good inhibitory activity against HDAC3 (Cpds 179-183) (Figure 33). Moreover, the flexible methylene group is important for the inhibition whereas the methoxy substitution in the linker moieties is slightly tolerable (Cpds 179-180). An increase in the distance between the cap and linker moiety is unfavourable for HDAC3 inhibition (Cpd 179 vs

31

180). The methoxy phenyl substitution in the cap group is important for HDAC3 inhibition (Cpds 179-183) [126]. [Figure 33 may be placed here] Cheng et al. [127] design and developed a group of potent dual inhibitors of HDAC/BRD4 through structure-based design method. Hydroxamate derivative containing indole moiety as the cap group is essential for HDAC3 inhibition (Figure 34). Most of these compounds showed nanomolar activity in nanomolar concentration against HDAC3 (Cpds 184-190, Table 11). [Figure 34 may be placed here] [Table 11 may be placed here] Benzyl substituent at the 3-position of the indole skeleton is important and it can interact with the amino acid residue Leu266 present in the loop 6 of the HDAC3 active site tunnel (Cpds 184185, 187-189). In addition, the presence of a weakly deactivating group, i.e., fluorine improves the selectivity towards HDAC3 and also enhances the inhibitory activity against HDAC3 (Cpds 184, 186, 191). Compounds having the electron donating group at the ortho or meta position slightly decreases the inhibitory activity (Cpds 189, 192-194). Chain length between the carbonyl group and the indole moiety is crucial for the inhibitory activity. The compound having similarity with SAHA in the linker group showed the better inhibitory activity against HDAC3 (Cpds 184-185, 187-190). The docking study (Figure 34) of the highly potent compound exhibited that the indole moiety formed hydrophobic interaction with Phe199 and showed van der Waals interaction with Phe200. In addition, the amido group of the hydroxamic acid forms hydrogen bonds with His134 and His135 those explain the strong inhibitory activity of that compound. Moreover, the flexible aliphatic linker of the compound helps to bind insight into the hydrophobic tunnel of HDAC3 [127]. Taha and co-workers [128-129] design and synthesize a promising series of hydroxamate-based HDAC inhibitors with a simple modification in the cap group (Cpds 195-210, Table 12). [Table 12 may be placed here]

32

They put the tetrahydroisoquinoline group (Cpds 199-210) in the cap region and evaluated against different HDACs. These inhibitors are active against all these class I HDACs and HDAC6. A few compounds showed good inhibitory activity against HDAC3. It was anticipated from the SAR observation (Figure 35) that compound having the hexamethylene linker group showed the better inhibitory activity against HDAC3 (Cpds 195, 199). The shortening of the hexamethylene linker group reduced the inhibitory activity (Cpds 196-197, 200-210). Compound with similar linker chain with SAHA showed the highest inhibitory activity against HDAC3 (Cpd 195 and 199). Apart from hydroxamate as the ZBG, the tetrahydoisoquinoline group (Cpd 199) in the cap region is better than the benzyl moiety (Cpd 195). Compound with secondary amino group (Cpd 195) exhibited slightly better inhibitory effect than compound with tertiary amino moiety (Cpd 198) whereas unsubstituted tetrahydroisoquinoline moiety showed good inhibitory activity against HDAC3 with the IC50 value of 97 nM (Cpd 199). Compound with bulky substitution in the tetrahydroisoquinoline moiety displayed a decrease in the inhibitory activity (Cpds 201-210). [Figure 35 may be placed here] 8.3. Structure-activity relationship of hydrazide-based HDAC3 inhibitors A series of benzoylhydrazide-based potent HDAC3 inhibitors was reported by Wang and colleagues [114]. These hydrazide-based compounds displayed the higher selectivity towards HDAC3 over other class I HDACs and HDAC6. Benzoylhydrazide-based compounds have a unique structure with a central −CO−NH−NH− group (ZBG) bound with a phenyl ring and an aliphatic chain which plays a pivotal role in HDAC3 inhibition (Cpds 29-32, Cpds 211-223, Table 13). The para substitution of the phenyl ring is essential to inhibit the enzymatic activity of HDAC3 (Figure 36). Bulky substitution at the para position is crucial for HDAC inhibition whereas the tert-butyl substitution at the para position showed the highest potency against HDAC3 (Cpd 29). Moreover, weakly deactivating group at the para position although improved selectivity it slightly decreased the inhibitory activity against HDAC3 (Cpds 30, 211, 216-221). In addition, the absence of any substitution at the para position diminished the HDAC3 inhibitory activity (Cpd 213). On the other hand, any change in the aliphatic chain length or structure decreased the HDAC3 inhibitory activity (Cpds 216-223). From the SAR study

33

(Figure 36), it is observed that the n-butyl group is preferred to get better HDAC3 inhibitory potency (Cpds 29-33, 211-215). To some extent the n-propyl group is tolerable (Cpd 216) [114]. [Table 13 may be placed here] [Figure 36 may be placed here] McClure and colleagues [115] synthesized a large number of hydrazide-based inhibitors that showed promising inhibitory activity against different HDAC isoforms (Cpds 33-36, Cpds 224239, Table 13). Here, the hydrazide motif along with carbonyl group may serve as a hydrogen bond donor and weak Zn2+ chelating functionality. From the SAR study, (Figure 37) it is anticipated that the replacement of the phenyl ring attached to the hydrazide motif reduced HDAC3 inhibitory activity. [Figure 37 may be placed here] Moreover, any change in the alkyl chain length or structure attached to the hydrazide motif decreased the inhibitory activity. Compounds displayed that the presence of the heterocyclic ring in the structure reduces the inhibitory activity (Cpd 224-227). In addition, the 4-methoxy substitution (Cpd 228) in the phenyl ring of the cap group showed slightly better inhibitory potency. Moreover, attachment of carbon to the phenyl ring in the form of either aliphatic (Cpd 36) or aromatic (Cpd 229) group displayed an increase in the inhibitory potency. Further, an unsaturated bond between the α- and β- position of carbonyl moiety is 6-fold better than the saturated one (Cpd 230 vs Cpd 231). It is seen that the 2- naphthyl group in place of the phenyl ring dose not favors the inhibitory activity but it showed better activity than 1- naphthyl group (Cpds 232-233). On the other hand, compounds with N-(4-(hydrazide)benzyl)benzamide scaffold displayed better inhibitory activity towards HDAC3. Chain length between 3 and 4 (npropyl and n-butyl) showed the higher inhibitory activity for HDAC3 (Cpd 35). Any change in the chain length or structure decreased the inhibitory activity against HDAC3 (Cpds 234-237) [115]. However, the addition of the cycloalkyl group at the end of the alkyl chain showed lesser inhibition towards HDAC3 (Cpds 238-239). From the SAR observation, it is observed that compound with N-(4-(hydrazide)benzyl)cinnamamide moiety showed selective and potent inhibitory effect towards HDAC3 over HDAC1 and HDAC2 (Cpds 33-34).

34

Li et al. [116] designed and synthesized some potent hydrazide-based HDAC inhibitors having indole moiety as the cap group (Cpds 240-251, Table 14). [Table 14 may be placed here] It was anticipated from the SAR study that substitution at the terminal position with a small linear alkyl group is favored (Cpds 240-243, 247-251) (Figure 38). Any change in the chain length or structure decreased the inhibitory activity against HDAC3 (Cpds 244-246). The compound containing phenylcarboxamide function (Cpd 247) in the linker position showed effective inhibitory potency compared to pentamethylene carboxamide (Cpd 248) or benzylcarboxamide function (Cpds 240-243) containing analogs. In addition, the presence of 4methoxybenzamido group played an important role in the inhibitory effect [116]. [Figure 38 may be placed here] 8.4. Structure-activity relationship of ethyl-ketone-based HDAC3 inhibitors Bresciani and co-workers [130] synthesized a new series of selective HDAC3 inhibitors with an important modification in the zinc-binding group. These compounds are basically nonhydroxamic acid and non-benzamide-based HDAC inhibitors with ethylketone and methylamide as zinc binding group (ZBG) respectively. Compounds with ethylketone as the ZBG showed the higher inhibitory potency against HDAC3 but these are nonselective over HDAC1 and HDAC2 (Cpds 252-259, Table 15). However, non-benzamide derivatives showed improved selectivity against HDAC3 over HDAC1 and HDAC2 (Cpds 260-267). These oxazole-based inhibitors with ethylketone as ZBG (Figure 39) showed nanomolar inhibitory activity against HDAC3 with aryl substitution in the cap group (Cpds 252-259) [130]. [Table 15 may be placed here] [Figure 39 may be placed here] Compound with 2-methoxyquinoline substitution (Cpds 252-255) at the cap region showed the better inhibitory activity than compound with 2-quinolinone group (Cpd 256). Moreover, heterocyclic substitution at the R group is important (Cpds 252-259). N-methylazetidine group (Cpd 252, 255-256) is better than 6-membered heterocyclic rings (Cpds 253, 257). In addition,

35

compound with substituted with heteroalkyl group showed the highest inhibitory activity against HDAC3 (Cpd 254). On the other hand, compound containing β-fluoro substituted N-methylazetidine group displayed less inhibitory effect than unsubstituted N-methylazetidine (Cpd 256 vs 252) whereas the triazole ring-containing compound has similar inhibitory potency like N-methylazetidine containing compound but it has 2-fold selectivity towards HDAC3 (Cpd 259 vs 252). In addition, non-benzamide derivatives (Figure 40) with substitute imidazole ring as the cap group displayed better selectivity towards HDAC3 over HDAC1 (Cpds 260-267, Table 15) [130]. [Figure 40 may be placed here] Compound with N,N-dimethylmethanaminophenyl substitution showed 32-fold selectivity towards HDAC3 over HDAC1 (Cpd 260). However, 2-naphthyl and 4-pyrazolophenyl substituted compounds showed lesser inhibitory activity and lesser selectivity against HDAC3 with respect to the dimethylmethanaminophenyl substituted compound (Cpds 261, 263 vs 260). Compound with 2-methoxyquinoline substitution exhibited the highest inhibitory potency with an IC50 value of 24 nM (Cpd 262). It also exerted 8-fold selectivity towards HDAC3 over HDAC1. Moreover, the amino fragment with a heteroaryl substitution is essential for improving the selectivity of these inhibitors. Compound with indole-5-carbonitrile substitution showed the highest selectivity and better inhibitory potency against HDAC3 compared to the compound containing 3-ethyl-indole and thiazole groups (Cpd 266 vs Cpd 265 and 260) [130].

8.5. Structure-activity relationship of thiol-based HDAC3 inhibitors Yao and group [117] reported some novel cyclic depsipeptides-based HDAC inhibitors (Cpd 37, Cpds 268-269) (Figure 41). [Figure 41 may be placed here] These inhibitors showed the higher inhibitory potency and selectivity towards class I HDACs (HDAC1 and HDAC3). From the SAR study, (Figure 42) it is observed that compound having no substitution at the thiol moiety (Cpd 37) showed the better inhibitory activity against HDAC3 than compound with alkaylcarbonyl substitutions (Cpd 268-269). Small substitution at the thiol moiety always favored the inhibitory activity. Hence, compound with methylcarbonyl

36

substitution (Cpd 268) at thiol moiety displayed 23-fold potency towards HDAC3 than compound with heptacarbonyl substitution (Cpd 269). In addition, the presence of the thiazole group is important. [Figure 42 may be placed here] 9. Future Perspective HDACs are responsible for the winding of the DNA strands around the histone proteins. As HDAC3 enzyme is related to the deacetylation process of histone protein and is expressed in various diseases specifically in cancers, potent and selective HDAC3 inhibitors may be the choice of compounds that can fight against cancer. It is observed and explored in various recent studies [8-14] that inhibition of HDAC3 has been shown to impart a remarkable improvement in slowing down or eliminating cancerous cells. HDAC3 has been used as potential drug targets for diverse disease conditions including cancer, neurodegeneration and inflammation [8-14]. Till now, the availability of selective and potent HDAC3 inhibitors in the market is none. Therefore, synthesizing potent as well as selective inhibitors of HDAC3 is always challenging. This current review will help researchers in various ways in the field of drug design and discovery to achieve deep insight into the structural information of HDAC3 inhibitors. From these detailed structure-activity relationship (SAR) analysis one can find out different aspects of inhibitors of HDAC3 related to its therapeutic potency and selectivity (Figure 43). [Figure 43 may be placed here] Interestingly, most of the potent HDAC3 inhibitors contain a crucial structural framework (cap group-linker-ZBG). In that structural arrangement ZBG plays an important role to execute potent inhibitory effect and selectivity towards HDAC3. Besides that, the distance between the cap group and the ZBG is one of the vital aspects of HDAC3 inhibition. It is observed from the overall study that linear aliphatic chain as the linker moiety may be important for good inhibitory activity. In addition, aryl group can be useful as the linker moiety. On the other hand, the interactions between the cap group and the amino acid residues of hydrophobic pocket is important. Thus, the review is important for those interested in epigenetics in general and histone deacetylases in particular.

37

Acknowledgments RS is thankful to the All India Council for Technical Education (AICTE), New Delhi, India for awarding a fellowship. SB is grateful to RUSA 2.0 of UGC, New Delhi to Jadavpur University for awarding a fellowship. SAA is grateful to the Council of Scientific and Industrial Research (CSIR), New Delhi, India for providing Senior Research Fellowship (SRF). NA sincerely acknowledges Council of Scientific and Industrial Research (CSIR), New Delhi, India for providing research associateship (RA). TJ is thankful for the financial support from RUSA 2.0 of UGC New Delhi to Jadavpur University, Kolkata. The authors heartily thank Sandip Kumar Baidya and Saptarshi Sanyal for their kind support. The authors also thank the support from Department of Pharmaceutical Technology, Jadavpur University, Kolkata for providing research facilities. References 1. J. Ferlay, I. Soerjomataram, R. Dikshit, S. Eser, C. Mathers, M. Rebelo, D.M. Parkin, D. Forman, F. Bray, Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012, Int. J. Cancer. 136 (2015) 359-386. 2. C.A. Godman, R. Joshi, B.R. Tierney, E. Greenspan, T.P. Rasmussen, H.W. Wang, D.G. Shin, D.W. Rosenberg, C. Giardina, HDAC3 impacts multiple oncogenic pathways in colon cancer cells with effects on wnt and vitamin D signaling, Cancer. Biol. Ther. 7 (2008) 1570-1580. 3. Y. Shao, Z. Gao, P.A. Marks, X. Jiang, Apoptotic and autophagic cell death induced by histone deacetylase inhibitors, Proc. Natl. Acad. Sci. U.S.A. 101 (2004) 18030–18035. 4. D.E. Handy, R. Castro, J. Loscalzo, Epigenetic modifications: Basic mechanisms and role in cardiovascular disease, Circulation. 123 (2011) 2145-2156. 5. M. Nakagawa, Y. Oda, T. Eguchi, S. Aishima, T. Yao, F. Hosoi, Y. Basaki, M. Ono, M. Kuwano, M. Tanaka, M. Tsuneyoshi, Expression profile of class I histone deacetylases in human cancer tissues, Oncol. Rep. 4 (2007) 769-774. 6. B.M. Muller, L. Jana, A. Kasajima, A. Lehmann, J. Prinzler, J. Budczies, K.J Winzer, M. Dietel, W. Weichert, C. Denkert, Differential expression of histone deacetylases HDAC1, 2 and 3 in human

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51

Figures

Figure 1. Pie chart representations of a number of new cancer cases and the estimated number of deaths in 2018.

Figure 2. Schematic representation of the pathophysiological conditions related to overexpression of HDAC3 enzyme. 52

Figure 3. Acetylation and deacetylation of Histone proteins done by HAT and HDACs.

Figure 4. Crystal structure of HDAC3 (PDB ID: 4A69). 53

Figure 5. Ins(1,4,5,6)P4 binds with HDAC3 active site (PDB ID: 4A69).

Figure 6. Role of HDAC3 in various diseases. 54

Figure 7. Relation between HDAC3 and various cancer.

Figure 8. Structure of different FDA approved HDAC inhibitors. 55

Figure 9. Benzamide-based HDAC3 inhibitor (compounds 1-7).

56

Figure 10. Benzamide-based HDAC3 inhibitor (compounds 8-13).

Figure 11. Benzamide-based HDAC3 inhibitor (Compounds 14-20). 57

Figure 12. Hydroxamate-based HDAC3 inhibitor with their respective IC50 values (compounds 21-28).

58

Figure 13. Hydrazide-based HDAC3 inhibitor with their respective IC50 values (compounds 2936).

59

Figure 14. Thiol-based HDAC3 inhibitor with their respective IC50 values (compounds 37-40).

60

Figure 15. Structures and IC50 values (HDAC1, HDAC2, HDAC3 and HDAC6) of compound 41-51.

61

Figure 16. Structure exploration of benzamide-based HDAC3 inhibitors. (A) Essential structural features required for promising HDAC3 inhibition of phenylthiazole containing benzamides. (B) Structure of best active (Cpd 41) and least active (Cpd 42) with their IC50 values.

62

Figure 17. Structure exploration of benzamide-based HDAC3 inhibitors. (A) Essential structural features required for promising HDAC3 inhibition. (B) Structure of best active (Cpd 14) and least active (Cpd 55) with their IC50 values.

Figure 18. Structure exploration of benzamide-based HDAC3 inhibitors. Essential structural features required for promising HDAC3 inhibition. 63

Figure 19. Structure exploration of benzamide-based HDAC3 inhibitors. (A) Essential structural features required for promising HDAC3 inhibition of indole containing benzamides. (B) Structure of best active (Cpd 91) and least active (Cpd 85) with their IC50 values.

64

Figure 20. Structures and IC50 values (HDAC1, HDAC2 and HDAC3) of compound 92-99.

65

Figure 21. Structure exploration of benzamide-based HDAC3 inhibitors. Essential structural features required for promising HDAC3 inhibition.

Figure 22. Structures and IC50 values (HDAC1, HDAC2, HDAC3, HDAC6 and HDAC8) of

compound 100-103. 66

Figure 23. Structure exploration of benzamide-based HDAC3 inhibitors. Essential structural features required for promising HDAC3 inhibition of thioether containing benzamides.

67

Figure 24. Structures and IC50 values (HDAC1, HDAC2, HDAC3, HDAC6 and HDAC8) of compound 104-111.

68

Figure 25. Structure exploration of hydroxamate-based HDAC3 inhibitors. Essential structural features required for promising HDAC3 inhibition of iso-oxazole containing hydroxamates.

Figure 26. Structures and IC50 values (HDAC1, HDAC2, HDAC3, HDAC6 and HDAC8) of compound 112-120. 69

Figure 27. Structure exploration of hydroxamate-based HDAC3 inhibitors. Essential structural features required for promising HDAC3 inhibition of triazol-4-yl phenyl containing hydroxamates.

70

Figure 28. Structure exploration of hydroxamate-based HDAC3 inhibitors. (A) Essential structural features required for promising HDAC3 inhibition of N-hydroxy cinnamamide containing hydroxamates. (B) and (C) Docking interaction of best active (Cpd27) and least active (Cpd125) compounds respectively with HDAC3 (PDB ID: 4A69).

71

Figure 29. Structure exploration of hydroxamate-based HDAC3 inhibitors. Essential structural features required for promising HDAC3 inhibition of 4-anilinothieno[2,3-d]pyrimidine containing hydroxamates.

Figure 30. Structure exploration of hydroxamate-based HDAC3 inhibitors. (A) Essential structural features required for promising HDAC3 inhibition of aryl linker containing hydroxamates. (B) Docking interaction of best active compound (Cpd 28) with HDAC3 (PDB ID:4A69).

72

Figure 31. Structure exploration of hydroxamate-based HDAC3 inhibitors. Essential structural features required for promising HDAC3 inhibition of indole cap containing hydroxamates.

Figure 32. Structure exploration of hydroxamate-based HDAC3 inhibitors. Essential structural features required for promising HDAC3 inhibition of N-substituted 7-aminoheptanoic containing hydroxamates.

73

Figure 33. Structure exploration of hydroxamate-based HDAC3 inhibitors. Essential structural features required for promising HDAC3 inhibition of β-carbolines containing hydroxamates.

74

Figure 34. Structure exploration of hydroxamate-based HDAC3 inhibitors. (A) Essential structural features required for promising HDAC3 inhibition. (B) Docking interaction of best active compound (Cpd 184) with HDAC3 (PDB ID: 4A69).

75

Figure 35. Structure exploration of hydroxamate-based HDAC3 inhibitors. Essential structural features required for promising HDAC3 inhibition of tetrahydroisoquinoline containing hydroxamates.

Figure 36. Structure exploration of hydrazide-based HDAC3 inhibitors. Essential structural features required for promising HDAC3 inhibition of benzoyl hydrazide-based inhibitors.

76

Figure 37. Structure exploration of hydrazide-based HDAC3 inhibitors. Essential structural features required for promising HDAC3 inhibition of hydrazide-based inhibitors.

Figure 38. Structure exploration of hydrazide-based HDAC3 inhibitors. Essential structural features required for promising HDAC3 inhibition of phenyl carboxamide containing hydrazides.

77

Figure 39. Structure exploration of ethyl-ketone-based HDAC3 inhibitors. Essential structural features required for promising HDAC3 inhibition of ethyl-ketone-based inhibitors. (B) Structure of best active (Cpd 251) and least active (Cpd 254) with their IC50 values.

78

Figure 40. Structure exploration of non-benzamide-based HDAC3 inhibitors. (A) Essential structural features required for promising HDAC3 inhibition of non-benzamide-based inhibitors. (B) Structure of best active (Cpd 259) and least active (Cpd 264) with their IC50 values.

79

Figure 41. Structures and IC50 values (HDAC1, HDAC2, HDAC3, HDAC6 and HDAC8) of compound 268-269.

Figure 42. Structure exploration of thiol-based HDAC3 inhibitors. Essential structural features required for promising HDAC3 inhibition of cyclic depsipeptides-based inhibitors.

80

Figure 43. Summary of important structural requirements of HDAC3 inhibitors for better and selective inhibition.

81

Tables Table 1. HDAC isoforms with their cellular location, chromosomal location, complex, histone/non-histone protein substrate, physiological function and pattern of gene expression. Class

HDAC Isoform

HDAC 1

No. of Amino acids

Cellular Location

483

Chromosomal Location

Complex

1p35 -p35.1 Sin3, NURD

HDAC 2

488

6q21 Nucleus

I

HDAC 3

428

5q31.3

NCoR1 / NCoR2, GPS2 TBL1X

HDAC 8

377

Xq13.1

-

2q37.3

NCoR1 / NCoR2

HDAC 4

1084

Histone/non-histone protein Substrate

Physiological function

SHP, p53, MyOD, E2FI, STAT3, NFκβ, CtIP, AMPK, RBI

Proliferation and survival of cell

GCCR, BC26, STAT3, YY1

Proliferation and survival of cell and insulin resistance

SHP, YY1, GATA1, p65, STAT3, MEF2D

Proliferation and survival of cell

SMC3, Actin

Proliferation of cell

GATA1, HP1

Regulation of cytoskeletal dynamics and cell mobility

Nucleus/ Cytoplasm

HDAC 6

1215

Cytoplasm

Xp11.23

-

HDAC 7

912

Nucleus/ Cytoplasm

12q13.11

Sin3, NCoR2

PLAG1, PLAG2

HDAC 9

1069

7p21

-

-

Helps in endothelial cell function, Gluconeogenesis, Cardiac myocyte growth function Regulation of cytoskeletal dynamics and cell mobility Helps in thymocyte differentiation, homologous recombination, cardiac cell function -

HDAC 10

669

2q13.33

NCoR2

-

-

HDAC 5

1122

17q21.31

-

SMAD7, HP1

II

Cytoplasm

82

Pattern of expression of gene

Ubiquitous expression

Tissue restricted expression

III

SIRT 1

747

Nucleus/ Cytoplasm

SIRT 2

389

Nucleus

SIRT 3

399

10q21.3

-

NF-κβ, p53,

19q13.2

-

PPAR-γ, p53, p300,αtubulin,FOXO

11p15.5

-

PGC-1α, p53, Ku70, Acetyl CoA synthetase. FOXO

Mitochond ria SIRT 4

314

12q24.31

-

SIRT 5

310

6p23

-

SIRT 6

355

19p13.3

-

TNF-α

17q25.3

-

p53, RNA, Polymerase 1

Apoptosis

HDAC6

DNA replication, Immunomodulation

Nucleus SIRT 7

IV

Glutamate dehydrogenase Carbamoyl phosphate synthetase 1, Cyt c

Autoimmunity, aging, redox balance, cell survival survival, migration and invasion of cell Regulating ATP production and metabolism, cell signalling apoptosis, ureacycle Energy metabolism urea cycle, cell signalling Regulates metabolism

HDAC 11

400

347

Nucleus

3p25.1

-

83

Variable expression

Ubiquitous in nature

Table 2. Different FDA approved HDAC inhibitors for the treatment of cancer. Sl. No.

Inhibitors

Class (structure)

Trade name

Company name

FDA approved Indications

Defects

[FDA-01]

Vorinostat (SAHA)

Hydroxamate

Zolinza

Merck

Cutaneous T-cell lymphoma

Thrombocytopenia, anaemia, diarrhoea, fatigue, nausea

2006

[56]

[FDA-02]

Romidepsin

Cyclicpeptide

Istodax

Celgene

Cutaneous T-cell lymphoma

Nausea, vomiting, thrombocytopenia, pyrexia, anorexia

2009

[16, 57]

[FDA-03]

Belinostat (PXD101)

Hydroxamate

Beleidaq

Spectrum

Relapsed multiple myeloma

Nausea, vomiting, fatigue, diarrhoea

2014

[57, 58]

[FDA-04]

Panobinostat (LBH-589)

Hydroxamate

Farydak

Novartis

Relapsed multiple myeloma

Thrombocytopenia, neutropenia, asthenia, fatigue, diarrhea

2015

[16]

[FDA-05]

Pracinostat (SB939)

Hydroxamate

-

Helsinn Group and MEI Pharma

Acute myeloid leukemia

Fatigue, diarrhea peripheral edema

2016

[57, 59]

2015

[60]

[FDA-06]

Chidamide (HBI-8000)

Benzamide

Epidaza

Relapsed or refractory Shenzhenperipheral Thrombocytopenia, core T-cell neutropenia, biotechnology lymphoma fatigue, anemia limited and pancreatic cancer

84

Year of Ref. approval

Table 3. Structures and IC50 values (HDAC1, HDAC2, HDAC3, HDAC6 and HDAC8) of compound 52-83.

IC50 Value (nM) Cpd

R2

R1 HDAC3

HDAC1

HDAC2

HDAC6

HDAC8

52

-OH

719

-

1,430

>20,000

>20,000

53

OCH3

111

-

248

>20,000

>20,000

54

-Br

4,720

-

>20,000

>20,000

>20,000

56

-C6H5

>20,000

-

16,700

>20,000

>20,000

85

58

H

14

350

560

>20,000

80,00

59

H

30

-

66

>20,000

13,000

60

H

104

190

78

>20,000

>20,000

61

H

36

410

82

>20,000

12,700

H

79

370

56

>20,000

10,700

H

63

-

440

>20,000

5,160

64

H

509

-

580

>20,000

>20,000

65

H

15

240

270

-

5,500

66

H

38

380

180

-

14,300

H

62

S N

H

63

N H

S N

S

86

67

H

66

1,200

360

-

9,500

68

H

56

-

610

-

1,12,000

69

H

71

700

250

-

88,000

70

H

40

530

180

-

14,700

71

H

33

82

180

-

80,700

72

H

34

190

150

-

33,900

73

H

24

240

240

-

52,200

74

H

34

-

340

-

82,100

H

18

290

230

-

83,300

O N

75 HO

N H

87

76

H

31

260

430

-

11,1000

77

H

41

200

130

-

63,000

78

H

11

76

192

-

1,73,000

79

H

35

390

94

-

8,100

80

H

55

-

400

-

53,500

81

H

120

-

280

-

29,200

82

H

21

78

160

-

1,79,000

83

H

18

130

290

-

1,13,000

88

Table 4. Structures and IC50 values (HDAC1, HDAC2 and HDAC3) of compound 84-91.

Cpd

R1

R2

R3

X

IC50 Value (nM) HDAC 1 HDAC2 HDAC3

84

H

48

338

3,569

85

H

36.5

314

62,540

86

H

58.8

320

8,007

87

H

55.5

490

5,626

88

H

58.4

311

30,850

89

89

H

F

511

909

111

90

H

H

49.7

441

227

91

H

H

58.7

296

42.9

Table 5. Structures and IC50 values (HDAC1, HDAC2, HDAC3, HDAC6 and HeLa extract) of compound 121-130. O OH N H O R

HN

IC50 Value (nM) Cpd

R

HDAC1

HDAC2

HDAC3

HDAC6

HeLa extract

121

10.3

535.5

14.1

142.2

10.5

122

13.2

432.1

143.0

143.7

23.2

90

123

16.7

457.0

5.5

101.7

6.7

124

6.0

413.6

3.2

185.6

4.8

125

329.9

498.5

403.2

164.4

389.0

126

355.4

791.2

47.0

293.6

157.8

127

-

-

-

-

636.9

128

-

-

-

-

1703.4

129

-

-

-

-

20.8

130

-

-

-

-

20.4

91

Table 6. Structures and IC50 values (HDAC1, HDAC3 and HDAC6) of compound 131-156.

Cpd

R1

R2

R3

n

131

H

Cl

F

132

H

Cl

133

H

134

IC50 value (nM) HDAC1

HDAC3

HDAC6

4

131.30

126.56

568.76

F

5

35.89

37.67

23.99

Cl

F

6

13.36

17.91

21.96

H

CF3

Cl

4

218.10

607.03

-

135

H

CF3

Cl

5

40.84

48.26

30.00

136

H

CF3

Cl

6

21.67

23.53

11.49

137

F

H

F

6

10.07

16.85

7.88

138

F

H

Cl

6

12.50

14.47

5.58

139

F

H

Br

6

13.54

11.88

8.87

140

F

H

I

6

6.24

13.52

6.55

141

F

Cl

H

6

7.78

14.63

8.46

142

H

Cl

H

6

3.24

6.46

8.58

143

H

H

H

5

11.77

20.77

26.99

144

H

H

H

6

1.90

3.47

12.04

145

H

H

CH3

5

14.01

9.33

19.68

146

H

H

CH3

6

2.01

2.92

5.37

147

H

CH3

CH3

5

29.82

14.74

16.87

148

H

CH3

CH3

6

1.14

3.56

11.43

149

H

H

N(Et)2

6

32.87

39.72

43.16

150

H

F

N(Et)2

6

4.57

3.52

3.44

151

H

F

N(CH3)2

6

4.51

3.22

2.69

152

H

Cl

6

2.97

3.58

1.90

92

153

H

F

6

5.39

8.19

3.42

154

H

F

6

14.49

14.05

9.28

155

H

H

6

13.85

14.10

7.19

156

H

H

6

25.53

26.53

15.10

H

Table 7. Structures and IC50 values (Enzymatic inhibitory assay, HDAC1, HDAC2, HDAC3 and HDAC6) of compound 157-161. O OH O

N H N H

R

IC50 Value (nM) Cpd

157

158

R

Enzymatic inhibitory assay 14.3

15.6

93

HDAC1

HDAC2

HDAC3

HDAC6

28.7

243.5

16.8

375.8

30.7

170.6

23.1

294.5

159

13.7

11.8

89.2

8.5

195.8

160

9.7

24.7

106.7

24.4

365.9

161

348.6

-

-

-

-

Table 8. Structures and IC50 values (HeLa extract, HDAC1, HDAC2, HDAC3 and HDAC6) of compound 162167.

IC50 Value of (nM) Cpd

162

R1

R2

HeLa HDAC1 HDAC2 HDAC3 HDAC6 extract

OCH3

9.6

94

6.0

413.6

3.2

185.6

163

H

-

180.4

321.6

7.5

376.7

164

OCH3

8.1

-

-

-

-

165

OCH3

60.0

-

-

-

-

166

OCH3

23.3

167

H

-

6.0

413.6

3.2

185.6

Table 9. Structures and IC50 values (HDAC1, HDAC2, HDAC3 and HDAC8) of compound 168-178.

Cpd

168

X

Y

n

HDAC 1

H

5

61

95

IC50 Value (nM) HDAC HDAC 2 3 260

25

HDAC 8 620

169

H

5

48

690

38

550

170

H

5

39

320

68

320

171

5

340

3,700

1,300

4,600

172

5

840

3,000

1,100

4,100

173

5

1,100

4,200

950

5,400

174

H

5

340

3,100

430

1,800

175

H

5

140

790

190

2,800

176

H

5

210

1,800

180

1,800

177

H

5

220

1,500

240

1,700

178

H

5

220

2,900

1,000

2,100

96

Table 10. Structures and IC50 values (HDAC1, HDAC3 and HDAC6) of compound 179-183.

Cpd

n

179

X

IC50 Value (nM)

Y

R

1 OCH3

O

CH3

32

125

17

180

2 OCH3

O

CH3

27

148

13

181

2

NH

OH

57

-

-

182

3 OCH3

O

CH3

61

-

-

183

1

NH

OH

75

-

-

H

H

HDAC1 HDAC3 HDAC6

97

Table 11. Structures and IC50 values (HDAC1, HDAC2 and HDAC3) of compound 184-194.

R CH3 O

O

N

N

OH

n

N H

CH3

Cpd

R

n

IC50 Value (nM) HDAC1 HDAC2 HDAC3

184

6

181

298

5

185

6

31

79

13

186

5

259

946

17

187

6

431

438

18

188

6

179

118

23

98

189

6

253

127

24

190

6

240

139

28

191

5

341

517

39

192

5

314

>1,000

62

193

6

220

>1,000

50

194

5

222

568

62

99

Table 12. Structures and IC50 values (HDAC1, HDAC2, HDAC3 and HDAC8) of compound 195-210.

IC50 Value (nM) HDAC2 HDAC3

Cpd

R

m

195

H

6

340

3,100

430

1,800

196

H

4

14,000

58,000

22,000

10,000

197

H

2

50,000

>1,00,000

72,000

>1,00,000

6

1,100

2,700

1,100

4,000

198

HDAC1

HDAC8

199

H

6

98

330

97

1,900

200

H

2

57,000

>1,00,000

98,000

19,000

201

4

770

2,700

810

44

202

2

13,000

69,000

34,000

110

100

203

2

27,000

>1,00,000

>1,00,000

82

204

2

24,000

99,000

>1,00,000

210

205

2

37,000

>1,00,000

>1,00,000

340

206

2

18,000

>1,00,000

27,000

190

207

2

>1,00,000

>1,00,000

75,000

6,700

208

2

18,000

29,000

29,000

480

209

2

2,400

13,000

3,400

390

101

210

2

7,300

47,000

38,000

55

Table 13. Structures and IC50 values (HDAC1, HDAC2 and HDAC3) of compound 211-239.

Cpd

Ar

R

IC50 Value (nM) HDAC1 HDAC2 HDAC3

211

n-butyl

13,200

15,100

1,780

212

n-butyl

8,860

9,090

1,350

213

n-butyl

>50,000

>50,000

>50,000

214

n-butyl

1,910

2,520

430

215

n-butyl

3,000

3,800

2,500

102

216 217 218 219 220 221 222

n-propyl n-pentyl n-hexyl CH2CH2Bn CH2CH2(c-C5H10) CH2(c-C5H10) CH2CH2CH2(cC5H10)

1,700 1,870 10,270 >50,000 7,400 8,810

3880 1,920 16,110 >50,000 7,650 28,450

220 920 20,040 24,180 1,080 6,980

>50,000

>50,000

>50,000

223

CH2(c-C5H10)

1,760

3,250

1,670

224

n-butyl

-

-

1,362

225

n-butyl

-

-

1,547

226

n-butyl

-

-

903.9

227

n-butyl

-

-

5,001

228

n-butyl

-

-

156.7

229

n-butyl

-

-

68.85

230

n-butyl

-

-

294.5

231

n-butyl

-

-

1,892

103

232

n-butyl

-

-

892

233

n-butyl

-

-

>10,000

234

isopropane

-

-

>10,000

235

n-decane

-

-

>10,000

236 237 238 239

n-hexane 2-butene methylcyclopropane methylcyclobutane

-

-

-

-

184.1 1,533 39.93 84.44

104

Table 14. Structures and IC50 values (HDAC1, HDAC2, HDAC3 and HDAC6) of compound 240-251.

Cpd

R1

R2

HDAC1

IC50 value (nM) HDAC2 HDAC3

HDAC6

240

C2H5

95.46

88.77

34.90

392.7

241

C3H7

63.28

287.1

8.50

>1,00,00 0

242

C4H9

53.68

193.5

16.54

>1,00,000

243

C5H11

29.78

332.1

10.87

244

cyclopentyl

>5,000

>5,000

>5,000

245

C6H5

>5,000

>5,000

>5,000

105

>1,00,00 0 >1,00,00 0 >1,00,00 0

247

C3H7

9.54

28.04

1.41

248

C3H7

80.19

209.0

12.44

249

C3H7

91.59

191.3

5.205

250

C3H7

52.77

199.3

12.09

106

>1,00,00 0 >1,00,00 0 >1,00,00 0 >1,00,00 0

Table 15. Structures and IC50 values HDAC3 of compound 252-267.

Y

Q

HDAC3 IC50 Value (nM)

252

O

C

0.9

253

O

C

1.2

254

O

C

0.6

255

O

C

1.1

256

O

C

6.0

257

O

C

7.0

258

O

C

0.9

Cpd

R1

R2

107

259

O

C

0.9

260

NH NH

188

261

NH NH

235

262

NH NH

24

263

NH NH

223

264

NH NH

42

265

NH NH

35

266

NH NH

26

267

O

108

NH

3,360

Highlights


HDAC3 is a crucial validated target of cancer.
HDAC3 inhibitors may be effective strategy to combat various cancers.
The current study deals with the detail SAR study of HDAC3 inhibitors.
This study may be effective in designing HDAC3 inhibitors in future.

Declaration of Interest Statement The authors have no conflict of interests.