Accepted Manuscript

Inhibitors of histone deacetylase as antitumor agents: A critical review

Manal Mohammed, M.J.N. Chandrasekar, Gomathi Priya Jeyapal, M.J. Nanjan

PII: S0045-2068(16)30047-5
DOI: http://dx.doi.org/10.1016/j.bioorg.2016.05.005
Reference: YBIOO 1910

To appear in: Bioorganic Chemistry

Received Date: 16 February 2016

Revised Date: 29 April 2016
Accepted Date: 15 May 2016

Please cite this article as: M. Mohammed, M.J.N. Chandrasekar, G.P. Jeyapal, M.J. Nanjan, Inhibitors of histone
deacetylase as antitumor agents: A critical review, Bioorganic Chemistry (2016), doi: http://dx.doi.org/10.1016/
j.bioorg.2016.05.005

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 INHIBITORS OF HISTONE DEACETYLASE AS ANTITUMOR AGENTS: A CRITICAL REVIEW

a a a b
Manal Mohammed* , Chandrasekar M J N , Gomathi Priya Jeyapal , Nanjan M J .

a
Department of Pharmaceutical Chemistry, JSS College of Pharmacy (A Constituent College of JSS University,
Mysore), Ootacamund 643 001, TamilNadu, India.
b
TIFAC CORE, JSS College of Pharmacy (A Constituent College of JSS University, Mysore), Ootacamund,
TamilNadu, India.
*Corresponding author

Manal Mohammed,
Department of Pharmaceutical Chemistry,
J. S. S. College of Pharmacy,
Rock lands,
Ootacamund,
Tamil Nadu, India - 643 001.
E-mail: manal_mohd@rediffmail.com

KEYWORDS

Histone deacetylase, Anticancer activity, HDAC Inhibitors, Short Chain Fatty Acids, Hydroxamic Acids.

ABSTRACT

Histone Deacetylase (EC 3.5.1.98 - HDAC) is an amidohydrolase involved in deacetylating the histone lysine

residues for chromatin remodeling and thus plays a vital role in the epigenetic regulation of gene expression. Due to

its aberrant activity and over expression in several forms of cancer, HDAC is considered as a potential anticancer

drug target. HDAC inhibitors alter the acetylation status of histone and non-histone proteins to regulate various

cellular events such as cell survival, differentiation and apoptosis in tumor cells and thus exhibit anticancer activity.

Till date, four drugs, namely Vorinostat (SAHA), Romidepsin (FK-228), Belinostat (PXD-101) and Panobinostat

(LBH-589) have been granted FDA approval for cancer and several HDAC inhibitors are currently in various phases

of clinical trials, either as monotherapy and/or in combination with existing/novel anticancer agents. Regardless of

this, today scientific efforts have fortified the quest for newer and novel HDAC inhibitors that show isoform

selectivity. This review focuses on the chemistry of the molecules of two classes of HDAC inhibitors, namely short

chain fatty acids and hydroxamic acids, investigated so far as novel therapeutic agents for cancer.

1
ABBREVIATIONS

ADP, Adenosine diphosphate; Arg, Arginine; Asp, Aspartic acid; Cys, Cysteine; DNA, Deoxyribonucleic acid; Gly,
Glycine; HAT, Histone Acetyl Transferase; HDAC, Histone deacetylase; HDAH, HDAC-like amidohydrolase;
HDLP, Histone Deacetylase Like Protein; His, Histidine; IC50, Half maximal inhibitory concentration; K+,
Potassium ion; Leu, Leucine; Lys, Lysine; µM, Micromolar; nM, Nanomolar; Phe, Phenylalanine; SAHA,
Suberoylanilide hydroxamic acid; SCFA, Short Chain Fatty Acids; Ser, Serine; SIRT, Sirtuins; Trp, Tyrptophan;
Tyr, Tyrosine; ZBD, Zn2+ binding domain; Zn2+, zinc ion.

1. INTRODUCTION

It is well known that the mammalian genome is compressed into chromatin which consists of DNA together with

histone and non-histone proteins. Nucleosome, the basic unit of chromatin, consists of 147 bp of DNA enveloped

around histone, an octamer enclosing two copies each of H2A, H2B, H3 and H4 [1]. Lysine in the histone tails

extend through the DNA strands and post-translation modification of histones occur on the lysines, serines and

arginines present in the tail. These modifications bring out alteration in the structure of nucleosome in the higher

order. Epigenetic regulation of gene expression is explicitly modulated through chromatin remodelling, which

involves nucleosome structural modification. The known post-translational histone modifications include

methylation, sumoylation, ADP-ribosylation, ubiquitination and acetylation of histone lysine in addition to

phosphorylation of histone serine. Among these, acetylation is the well understood process [2]. The regulation in

acetylation and deacetylation of the terminal ε-amino group of specific lysine residues of histone proteins, displays a

pivotal role in the transcription and control of cell survival [3]. The equilibrium of the N-ε-acetylation is controlled

by two enzymes, namely HAT (Histone Acetyl Transferase) and HDAC (Histone Deacetylase) which mediates the

addition and removal, respectively, of the acetyl moieties from the ε-amino group of the lysines. Histone acetylation

(and subsequent DNA unfolding) controls the transcription and replication to activate gene expression. The

deacetylation of histones is known to elevate the positive charge density due to the protonation of the ε-amino group

which in turn strengthens the electrostatic interaction between histones and the negatively charged DNA leading to a

more condensed chromatin and consequent repression of transcription [4, 5] (Fig. 1).

2
 HAT
Lysine (After Deacetylation) Acetylated Lysine
HDAC

Condensed Chromatin Relaxed Chromatin

DNA supercoiling DNA unfolding

Inhibition of gene transcription Activation of gene transcription

Cell proliferation Growth arrest and Apoptosis
Tumor growth and Metastasis Tumour growth inhibition

Fig. 1. Mechanism of histone acetylation and deacetylation.

HDACs are known to act in parallel with tumor growth as they trigger the abnormal transcription of crucial genes

that control essential cell functions, namely proliferation, cell cycle regulation and apoptosis. It also imparts its role

in several genome functions such as DNA repair, chromatin assembly and recombination. The irregularity in HDAC

function thus contributes to the initiation and progress of several tumors. HDAC inhibitors suppress histone

deacetylation to regulate gene expression for increased levels of acetylated histones and induce chromatin relaxation

and alter gene expression [6].

In humans, nearly 18 HDAC isoforms have been identified. They have been categorized into five classes based on

their cellular location, size, number of catalytic pockets and homology to yeast prototypes [7, 8]. Class I (HDACs 1,

2, 3, 8), class IIa (HDACs 4, 5, 7, 9), class IIb (HDACs 6, 10) and class IV (HDAC11) that require a zinc ion (Zn2+)

for their action are referred as the ‘classical family’. Class I and class IV are mainly found in the nucleus whereas

class IIb is found solely in the cytoplasm. As to class IIa, these are found to shuttle between the nucleus and the

cytoplasm. Class III, the Zn2+ independent enzymes, are classified as sirtuins (SIRT 1-7) and thus are not responsive

to compounds that inhibit Zn2+ dependent enzymes. These NAD+ dependent proteases are regulators for gene

expression in cellular redox status [9]. Class III HDACs do not show structural resemblance with Zn2+ dependent

enzymes and are not referred in this review. Sirtuins are known for their therapeutic potential in diseases such as

cancer, cardiovascular and neurodegenerative disorders. A recent publication has discussed on sirtuins as agents for

cancer therapy [10].

3
2. CRYSTAL STRUCTURE OF HDACS

Finnin et al. have explained the structural elements responsible for HDAC inhibition using Trichostatin A (TSA) and

Vorinostat (SAHA) along with the crystal structure of an HDAC related protein namely, histone deacetylase like

protein (HDLP) [11]. The study revealed the presence of a hydrophobic pocket which has a tube-like 11 A° internal

channel that accommodates the inhibitors. The active site Zn2+ center, present at the bottom of the internal channel,

is bound to the hydroxamic acid end of the inhibitors. Another deep internal cavity, constituted by Arg, Tyr and Cys

residues, is located near to this channel.

The crystal structures of Class I HDACs are known to share a common feature, namely an 11 A° deep channel

followed by 14 A° internal cavity in proximity to the active site [12]. A carbonyl of a glycine residue and the side

chain of a tyrosine residue point inside the 11 A° channel of the class I isoforms, namely HDAC1–Gly149 and

Tyr303, HDAC2–Gly154 and Tyr308, HDAC3–Gly143 and Tyr298 and HDAC8–Gly151 and Tyr306 [13]. The

tubular shaped active site with the catalytic Zn2+ ion is situated near the bottom of the pocket. The Zn2+ ion is in

penta-coordination with the side chains of conserved His and Asp residues together with the carbonyl of the

substrate/inhibitor and one molecule of water [14-16]. Though class I isoforms resemble in their structures, the

sequence in HDAC8 is an exception [17]. HDAC8 (Fig. 2) comprises of two molecules with a head-to-head dimer.

Each monomer, with 30% sequence resemblance with HDLP [11], is formed with a single compact α/β domain–

enclosing 11 α-helices and 8 parallel strands of β-sheets. This region represents a remarkable divergence, from that

in HDLP, resulting in better ease for accessing the ~12 A° active site in the isoform. The internal cavity is found to

be dynamic due to variation in the conformation at Ser30–Lys36 loop, thus assisting in the accommodation of the

substrate/inhibitor and progress of the deacetylation reaction by means of additional interactions. The cavity is also

believed to act as a duct to remove the acetate byproduct away from the catalytic site after deacetylation [17]. It has

also been suggested that reduction of the HDAC8 cavity by Trp14 occludes the lower corner of the internal cavity

[18]. Furthermore, two K+ ions are present bound at two different sites. Site 1 is in close proximity to the Zn2+ active

sites as the residues involved in Zn2+ chelation (Asp178 and His180) are also in co-ordination with site 1 K+ ion. The

residues at the site 1 K+ binding region are present in all Zn2+ dependent HDACs, except for Leu200–substituted

with Phe in HDACs 1-3 and Tyr in HDAC10. Similarly, the importance of site 2 K+ binding region (approximately

15 A° away from site 1) for enzyme is not evident but it is supportive in maintaining protein structure integrity.

4
Another striking feature is the existence of a conserved residue Asp101 at the rim of the Zn2+ binding site. The

importance of this residue, critical for positioning the substrate or the inhibitor, has been established by enzyme

activity and also through binding assay with Asp101 HDAC8 mutants [19].

Class IIa HDACs consists of two functionally crucial regions, namely a C-terminal catalytic domain and an N-

terminal extension of about 600 residues, which is responsible for the nucleus-cytoplasm shuttling and also holding

the binding sites for transcriptional regulators [20, 21]. The crystal structure of bacterial class IIb HDAC-like

amidohydrolase (HDAH) has been reported to show structural resemblance to human class II HDACs, thus making

it a good model for the class [22-24]. The structural fold in HDAC4 and HDAC7 is similar to that in class I HDACs.

The catalytic domains of these two isoforms (Fig. 2) have been shown to exhibit a major variation in class IIa

HDACs from class I [25, 26]. A distinct second Zn2+ binding motif is found to define an extended groove adjacent to

the entrance of the active site, thus stabilizing the deep hydrophobic pocket. Here the Zn2+ ion is coordinated with

the Cys and His residues, Cys533, Cys535, Cys618 and His514 in HDAC7 and His665, Cys667, His678 and Cys751

in HDAC4. These residues thus seem to be exclusive for class IIa HDACs. Moreover, the hydroxyl group of Try306

present in class I/IIb HDACs, to stabilize the transition state by hydrogen bond to yield an oxyanion intermediate in

enzyme catalysis (Fig. 3), is substituted with His 976 and His 843 in HDAC4 and HDAC7, respectively. The side

chains of these His residues are rotated away from the catalytic pocket and together with a water molecule, it bridges

as a hydrogen bond with Gly975 and the oxygen atom either in the carbonyl of the substrate or the inhibitor. Since

Tyr306 serves the stability of the oxyanion intermediate, the interaction mediated by class IIa enzymes is believed to

be feeble, enlightening the diminished enzyme activity of this class.

Class IIb HDACs also possesses two catalytic domains [20, 21, 27]. HDAC10 is leucine rich and the C-terminal

portion is found to share significant homology with the N-terminal catalytic domain. This observation implicates the

existence of a partial second catalytic domain [28]. Human HDAC6 contains a specific SE14-repeat domain which

possesses 8 successive tetradecapeptide repeats that are crucial for the stable cytoplasmic retention of the isoforms

[29, 30]. Moreover, with two deacetylase catalytic domains in the isoform, any modification in the linker region

between the two domains or in the spatial arrangement within each domain, drastically hinders the deacetylation

catalysis, both in vitro and in vivo [31].

5
HDAC11 of class IV displays an amino acid sequence frame of 347 residues with a molecular mass of 39 kDa.

Throughout this nuclear protein, the amino acid sequence is only slightly homologous to the other isoforms. But, the

sequence required for deacetylase function is conserved in nine blocks. Further, a catalytic core is presumed to be

present, suggesting its potential for HDAC enzyme activity [32].

Fig. 2. Catalytic domains of HDAC8 in yellow (PDB ID: 1W22), HDAC4 in blue (PDB ID: 2VQM) and HDAC7 in

red (PDB ID: 3C0Z). The violet balls represent the Zn2+ ions and the green balls show the K+ ions.

3. HDAC CATALYTIC MECHANISM AND BIOLOGICAL ACTIONS MEDIATED

The proposed catalytic mechanism intervened by Zn2+ dependent HDACs is believed to share the characteristics

mediated by metallo-proteases and serine proteases [33] (Fig. 3). The bidentate metal ion binds to the carbonyl

oxygen of the acetylated lysine residues of histones to polarize the carbonyl group and activate for elevated

electrophilic carbon. A catalytic water molecule also queues in to bind with the metal ion. As the side chains of two

histidine residues (His142, His143 in HDAC8; His802, His803 in HDAC4; His669, His670 in HDAC7) link through

hydrogen bonds, the water molecule becomes highly nucleophilic and attacks the carbonyl carbon of the acetylated

lysine to form a tetrahedral oxyanion intermediate. As mentioned earlier, the stability of the intermediate is

6
governed by the metal ion as well as the hydroxyl group of tyrosine residue, Tyr306. A proton transfer, therefore,

occurs from the side chain of the ‘outer’ histidine (His143 in HDAC8) to the amino nitrogen of the lysine and the

deacetylation reaction gets completed with acetate and lysine as byproducts.

O
NH Acetylated lysine O
NH Acetylated lysine
Tyr T yr
H H
OH N N
NH His
O His OH NH
N N
H O
N H
O Water N -
N H 2+ O
Zn

-
His H N H 2+
H N His Zn Water H
His His
- - N N
O O - -
O O O
N
Asp O
Asp O H
Asp Asp O

O
NH O
Lysine
NH
Ty r His
Ty r His

OH N NH
NH2 O +
OH HN NH
NH
N HO - OH
2+ HN N O
N H N HN N
His Zn
N H 2+
His His Zn
His
- -
O O
- - Oxyanion intermediate
O O O
Asp Asp O O O
Asp Asp
Products after deacetylation

Fig. 3. Proposed mechanism for histone deacetylase catalysis

Several related works have revealed the aberrant expression of HDACs in different types of tumors. The over

expressed levels of individual isoforms serve as biomarkers in tumors such as breast [34], lung [35], prostate [36],

colorectal [37], liver [38], cervical [39] and gastric cancer [40], implicating initiation and progression of the disease.

Experimental data on genetic knockdown and HDAC knockouts demonstrate strong evidence of tumor suppression

due to the disruption in the upcoming epigenetic regulation [41]. Further, inhibitors of HDAC are also known to

induce and modulate diverse biological effects leading to tumor growth arrest, apoptosis, senescence, autophagy,

7
anti-angiogenesis and immunomodulation (Table 1). The cytotoxic actions of HDAC inhibitors also extends to non-

histone protein targets that include transcription factors and regulators, nuclear import regulators, chaperones,

structural proteins, inflammatory mediators, signaling mediators, DNA repair enzymes and viral proteins [42].

Table 1.

HDAC inhibitor mediated biological effects

Associated Mediators/Molecules/Proteins and their effects References

Cell cycle Arrest at G1/S or G2/M*phases

• Induction of CDKN1A to encode p21waf1/CIP1 promoting pRb hypophosphorylation. 7, 43-48.

• Repression of genes, Cyclin D and Cyclin A, to reduce CDK2 and CDK4 kinase activities
and hypophosphorylation of pRb.
• Induction of p27 which inhibits CDK2- and CDK4- contained complexes.
• Repression of genes for DNA synthesis, CTP synthase and thymidylate synthetase.
• Induction of p15 to induce other CDK inhibitors.
• *
Activation of G2-phase checkpoint through heterochromatin hyperacetylation-resulting
abnormal chromosomal segregation and fragmentation of nucleus.
Oxidative stress and DNA damage**

• Accumulation of ROS in tumor cells to induce mitochondrial disruption. 49-55.

• Down regulation of TrX (scavenger of ROS).
• Induction of TBP2 (negative regulator of TrX).
• **
Indirect DNA damage by increased accumulation of phosphorylated histone variant,
γH2AX (marker of DSB in DNA).
• **
Repression of genes for DNA repair proteins, RAD51, BRAC1, BRCA2, Ku70 and Ku86.
Extrinsic pathway (Death receptor-DR) mediated Apoptosis

• Upregulation of TRAIL, DR5, Fas, Fas-L and TNF α in transformed tumor cells; thus 7,49,56,57.
activation of Casp 8 and 10.
• Repression of genes encoding for DR inhibitors, FLIP and IAP2.
Intrinsic pathway (Mitochondrial) mediated Apoptosis

• Upregulating expression of pro-apoptotic (anti-survival) proteins of BCL2 family, namely 7,50,58-60.

Bid, Bim, Bmf.
• Downregulation of expression of anti-apoptotic (pro-survival) BCL2 family proteins (BCL2,
BCL-XL, BCL-w), MCL-1 and XIAP.

8
 • Gene induction for encoding mediators causing mitochondrial damage (Diablo, Casp 9,
Apaf1, Cyt C and Htr A2).
Autophagy and Senescence#

• Overexpression of BCL-XL thereby inducing autophagy mediated cell death. 42,61-63.

• #
Senescence through Polyploidy and withdrawal of cell cycle.
Anti-angiogenesis

• Repression of pro-angiogenesis factors, HIF-1α, VEGF and CXCR4. 7,64-67.

• Upregulation of tumor suppressor VHL protein and p53.

Immunomodulation

• Augmenting tumor cell antigenicity by upregulating MHC class I and II proteins, 7,68-72.
costimulatory molecules (CD40, CD80 and CD86) and ICAM1.
• Inducing expression of MHC class I chain-related molecules, MICA and MICB.
• Suppression in secretion of pro-inflammatory cytokines, TNF α, IL-1 and IFNγ.
Cell migration and motility

• Hyperacetylation of Cortactin to prevent cell periphery translocation, block F-actin 73-78

association and thus impair cell motility.
• Tubulin hyperacetylation to obstruct fibroblast motility and invasion through decreased
microtubule dynamics and increased focal adhesion accumulation.
• Downregulation of MMP-1, MMP-2, MMP-9 and uPA.
• Upregulation of RECK glycoprotein, an inhibitor of MMP-2, MMP-9 and MT1-MMP.
• Overexpression of TIMP-1 and TIMP-3.
Non-histone substrate mediated

• Hyperacetylation of transcription factors, E2F1, p53, STAT1, STAT3 and NF-κB. 7,42,79-85.

• Hyperacetylation of proteins, Hsp90, α-tubulin, to induce protein degradation, cell growth

inhibition, cell death.
• Disruption of HDAC-PP1 complexes and activation of PP1 to cause irregularity in tumor
cell phosphorylation and acetylation.
• Disruption of aggresome pathway for degradation of misfolded protein aggregates.

Abbreviations: BCL, B-cell Lymphoma; CDK, Cyclin Dependent Kinase; CXCR4, Chemokine (C-X-C motif)
Receptor 4; DSB, Double Strand Breaks; FLIP, Flice Inhibitory Protein; HIF, Hypoxia Inducible Factor; IAP2,
Inhibitor of Apoptosis ; ICAM1, Intercellular Adhesion Molecule 1; IFN γ, Interferon γ; IL-1, Interleukin 1; MCL 1,
Myeloid Cell Leukemia sequence 1; MHC, Major Histocompatibility Complex; MMP, Matrix Metalloproteinase;

9
MT1-MMP, Membrane Type 1- Matrix Metalloproteinase; NF-κB, Nuclear factor - κB;PP1, Protein Phosphatase
1; pRb, Retinoblastoma protein; RECK, Reversion-inducing cysteine-rich protein with Kazal motifs; ROS, Reactive
Oxygen Species; STAT, Signal Transducer and Activator of Transcription; TIMP, Tissue Inhibitors of MMPs; TNF
α, Tumor Necrosis Factor α; TrX, Thioredoxin; TBP2,TrXBinding Protein 2; VEGF, Vascular Endothelial Growth
Factor; VHL, von Hippel-Lindau; uPA, Urokinase Plasminogen Activator; XIAP, X-linked Inhibitor of Apoptosis.

4. AN INSIGHT INTO DEVELOPED HDAC INHIBITORS

HDAC inhibitors have set a paradigm to reverse abnormal epigenetic changes related to cancer. Several HDAC

inhibitors have undergone clinical trials for different types of cancer (Fig. 4). With emphasis on their structural

diversity, the different classes of HDAC inhibitors include hydroxamic acids, benzamides, short chain fatty acids,

macrocyclic peptides and others. These Zn2+ dependent HDAC inhibitors share three pharmacophoric motifs, a cap

group or a surface recognition unit (usually a hydrophobic and aromatic group interacting with the rim of the

binding pocket), a Zn2+ binding domain (ZBD) or chelating group (hydroxamic acid, carboxylic acid or benzamide

groups), that coordinates with Zn2+ ion and a saturated or unsaturated linker with linear or cyclic structure, joining

the cap group to the ZBD (Fig. 4). Alterations in any or all three motifs have variably affected the potency and

selectivity of the HDAC inhibitors. Several reviews have also been reported based on various structural aspects of

HDAC inhibitors [6, 86-91]. n-Butyrate, phenyl butyrate and valproic acid have proved themselves as HDAC

inhibitors, becoming the initial members of short chain fatty acids studied. Hydroxamic acids, however, have turned

out and still continue to be the most studied class of HDAC inhibitors. This comprehensive review aims to

summarize the progress that have been made so far on HDAC inhibitors as anticancer agents with respect to the two

classes, namely short chain fatty acids and hydroxamic acids.

10
 O H O O
H O O
N OH N O
N S H OH
H N NH
O O S HN
Vorinostat (SAHA) O S O
H
Belinostat (Beleodaq) N
HN
O
O Romidepsin (Depsipeptide)

O O
N H O
OH
H N
OH N O N
H
N O S N
O
Abexinostat (PCI-24781) Reminostat

O
H
O OH
N N
H
O N OH
N H N
H
Givinostat O
Panobinostat (LBH589)

HN NH
H
O N N N NH NH2
N OH
O N
N O
CUDC-101 Mocetinostat (MGCD0103)
O
H
N O N NH2 N
H
H NH N
O H2N
O O
Entinostat (MS-275) Tacedinaline (C1994)
O
N OH
N
H
O OH N
O
-
O N

Valproic acid Phenylbutyrate Pracinostat(SB939)

Fig. 4. Approved HDAC inhibitors in clinical trials with their pharmacophores. The cap, linker and the Zn2+ binding

group are represented in blue, green and red, respectively.

4.1. Short Chain Fatty Acids as HDAC inhibitors

Fig. 5 shows the short chain fatty acids (SCFA) that have been investigated so far. The reversible acetylation of

histones and its association to transcription was initially proposed by Allfrey and co-workers [92, 93] and the quest

for the mechanism of histone acetylation continued until it was substantiated using n-butyrate by Riggs and co-

workers [94]. The authors highlighted the use of n-butyrate as a non-competitive inhibitor of HDAC at millimolar

concentration to induce reversible acetylation of hyperacetylated histones. The hyperacetylated state was shown to

trigger a sequence of biological effects such as cell differentiation and cell cycle arrest at G1 and G2 phases [95, 96].

11
Moreover, n-butyric acid is also known to be an endogeneous colonic anaerobic bacterial fermentation product,

which may also be associated with cancer therapy [97]. Despite these properties, n-butyrate displayed its pleotropic

effects on other enzymes, cytoskeleton and cell membranes [98]. Its therapeutic potential for cancer in clinics was

also hindered because of its short half-life, low plasma levels due to first-pass metabolism and doses in grams to

attain therapeutic concentrations [99-102]. In due course, phenyl butyrate and valproic acid turned out to be

successful in vivo HDAC inhibitors and displayed marginal potency against solid tumors and leukemias [103-105].

Besides, a structure-activity relationship study conducted on SCFA revealed that non-branched SCFA with 3-6

carbons (Fig. 5A) show good HDAC inhibitory profile [106]. Decreased enzyme inhibition was observed with

branched SCFA when compared to n-propionate, n-butyrate and n-pentanoate. In the branched chain series, greatest

activity was exhibited by valproic acid followed by 2,2-dimethylbutyric acid and 2-ethylbutyric acid.

Simultaneously, the prodrug approach was exploited to attain better results. Prodrugs of n-butyrate (Fig. 5B), such

as Pivanex (AN-9) [107,108], AN-1[109], AN-7 [110] and tributyrin [102, 111] were successful to overcome the

limitations but turned out as non-viable therapeutic agents. Also, for enhancing cellular uptake regulated by

carbohydrate-specific transporter and cell surface receptors [112, 113], glucose scaffold was attached to n-butyrate

as in Bu4ManNAc [114] and 3,4,6-OBu3GlcNAc [115] (Fig. 5B). Another methodology using acylcarnitine

transport facilitated the development of butyryl-L-carnitines, namely PMX 550B and PMX 550D (Fig. 5B) with

increased potency as HDAC inhibitors than the parent drug, n-butyrate. These prodrugs were acting as mediators for

transporting n-butyrate into cells in vivo [116, 117]. Although short chain fatty acids were extensively utilized as

tools in clinical research, reports on their activity continues to be very limited.

12
(A) Short chain fatty acid as HDAC inhibitors

R1 R2 R3 Fatty acid % Inhibition of HDAC

Me H H Propionic acid 78
O Et H H Butyric acid 82
R1 Et Me Me 2,2-Dimethyl butyric acid 20
OH Et Et H 2-Ethyl butyric acid 24
R2 R3 n-Pr H H Pentanoic acid 78
n-Pr n-Pr H Valproic acid 52
n-Bu H H Hexanoic acid 34
Me(CH2)4 H H Heptanoic acid 40
Me(CH2)5 H H Octanoic acid 53

(B) Butyrated based prodrugs

O O O O
O O
Et Et
O O O O
O O

Pivanex O AN - 1
Tributyrin
O

+ - + - O O
N Me 3Cl N Me 3Br Et P Et
O O O O O
O O O
AN - 7 Et
COOH COOEt
PMX500B PMX500D

O O
O O
O O
H H
N O N O

O O O O
O O O
HO
O O
O
Bu4ManNAc 3,4,6-Bu3GlcNAc

Fig. 5. Short chain fatty acids

4.2. Hydroxamic Acids as HDAC inhibitors

Vorinostat (also known as SAHA-Suberoylanilide hydroxamic acid) was the first clinically approved hydroxamic

acid based HDAC inhibitor for the treatment of cutaneous T-cell lymphoma (CTCL) [118]. Following this,

Belinostat (PXD101, Beleodaq) (Fig. 4) was approved by FDA in 2014 for the management of refractory peripheral

T-cell lymphoma (PTCL) [119] and Panobinostat (LBH589) (Fig. 4) in 2015 for treatment in multiple myeloma in

combination with Bortezomib and Dexamethasone [120]. Givinostat (ITF2357), Abexinostat (PCI-24781),

13
Quisinostat (JNJ-26481585), Resminostat (4SC-201), Practinostat (SB939), CUDC-101, CHR-2845 and CHR-2847

are the other hydroxamic acid based HDAC inhibitors currently in various phases of clinical investigations [6, 86,

121-125]. Tubacin and Tubastatin A (Fig. 6), reported as HDAC6 specific inhibitors, also contain hydroxamic acid

as chelating group [126, 127].

H O
N OH
N O
O H
N OH
N H
O O
S O
N
HO N

Tubacin Tubastatin A

Fig. 6. Specific HDAC6 Inhibitors

Several studies have extensively exploited the various structural elements of hydroxamic acids and thus continues as

a thrust area of research today. In 2001, Lavoie et al. [128] have reported a series of sulfonamide based hydroxamic

acids based on a reported [129] lead compound 1 (Fig. 7) with HDAC inhibition IC50 of 1 µM. With sulfonamide

group as connecting unit, the designed and synthesized molecules 2, exhibited an HDAC1 inhibition with IC50 in

range of 0.075-0.2 µM. When subjected to antiproliferative activity in human colon cancer HCT-116 cells,

compound 2j (HDAC inhibition IC50 = 0.09 µM) demonstrated an EC50 of 0.2 µM. On further in vivo evaluation, in

nude mice with non-small cell lung carcinoma A549 tumors, significant tumor growth inhibition was observed with

no gross toxicity. Marson et al. [130] have studied another set of sulfonamide based analogues, (2E,4E)-4-methyl-5-

arylpenta-2,4-dienoic acid hydroxamides and (2E,4E)-4-methyl-5-arylpenta-2,4-dienoic acid hydroxamides. The

trans,trans-1,3-butadiene linked derivatives 3a and 3b were found to possess a 10-fold potent HDAC inhibition with

higher IC50 of 49 and 74 nM, respectively compared to oxamflatin with IC50 of 2 µM (Fig. 7). The radioactive active

assay carried out was unable to arrive at any specific HDAC isoform inhibition.

14
 H
H
N O
O OH N
S H
S O O
N N
X O OH
R
O
1 R
2
R= 2a: 4-Cl; 2b: 3,4-Cl2; 2c: 2,4-Cl2;
2d: 4-NO2; 2e: SO2NH2; 2f: OCF3;
2g: 4-Me; 2h: 3-Me; 2i: 4-OMe;
O 2j: 3,4-(OMe)2.
OH
O N
S H H
N O
O N
H S H
O N
Oxamflatin OH
R O
3
R = 3a: 4-Cl; 3b: OMe

Fig. 7. Sulfonamide based hydroxamic acids

Macrocyclic and monocyclic succinimide based hydroxamic acids have been reported as HDAC inhibitors [131]

(Fig. 8). The representative compound 4, a monocyclic derivative demonstrated enzyme inhibitory potency of 99

nM in nuclear enzyme assay and significant cytotoxicity (0.67 µM) in human breast carcinoma MDA435 cells. The

report, however, claims to confirm HDAC mediated inhibition by hyperacetylation of nuclear histones and

expression of p21WAF1/CIP1 with no data. Using bicyclic aryl as cappings, Uesato and workers have developed a series

of hydroxamic acid analogues (N-hydroxycarboxamides) as HDAC inhibitors [132]. Using different chemical

approaches, the synthesized derivatives 5 showed significant HDAC inhibition in nanomolar range (IC50 = 39–2000

nM) and antiproliferative activity profile (IC50 = 0.7-9.7 µM) against HCT116 colon cancer cells. Selected

molecules 5a-g were further analyzed against a panel of 39 human cancer cell lines [133] resulting in GI50 of 20.4,

6.5, 3.6, 0.81, 4.1, 2.7 and 20.4 µM, respectively. These results when studied for statistical correlation using

COMPARE analysis yielded an outcome of correlation coefficient (r) less than 0.75 except for 5d.

15
 O
O H
O N
OH R1 N H
N R
N H N
O OH
O H
Ph O
5
4
R = 1,4-cyclohexylene
R1 = 5a: 4-dimethylaminophenyl;
5b: 2-naphthyl; 5c: 1,4-biphenyl;
5d: adamantylamino;
5h: 1-naphthyl;
R = 1,4-phenylene
R1 = 5e: 2-naphthyl; 5f: 1,4-biphenyl;
5g: 6-amino-2-naphthyl;
5i: 1-naphthyl;5j: adamantylamino;

Fig. 8. Succinimide and bicyclic aryl cap based hydroxamic acids

Dai et al. [134] have synthesized a series of hydroxamic acid based HDAC inhibitors through an indole amide

residue at the cap (Fig. 9). A methyl group on the indole nitrogen 6a caused a 3-fold decline in activity (IC50 = 47

nM) and the 3-substituted indole 6b exhibited the same trend (IC50 = 56 nM). However, an aromatic group at the

nitrogen of indole 6c and 6d demonstrated better HDAC inhibitory profile with IC50 value of 3.5 and 12 nM,

respectively. Furthermore, enhanced potency (IC50 = 3.1-9.9 nM) was observed with substituents incorporated on

the indole ring 7. By adding a five membered hetero aromatic ring to the hydrophobic cap, the same authors

designed and synthesized a set of novel hydroxamate based HDAC inhibitors [135]. The molecules 8 and 9 were

considerably more potent than SAHA (IC50=140 nM) with IC50 = 2.9-89 nM. The same trend was observed in

antiproliferative activity in HT1080 fibrosarcoma and MDA435 breast carcinoma cells.

16
 H H H
N N OH N
OH OH
N 4 O N 4 O
4 O
H
N R
6a R 7
6b-d
R = 6b: Me; 6c: Ph; 6d: Bn R = 7a: 4-OMe; 7b: 5-OMe; 7c: 6-OMe;
7d: 5-Br; 7e: 5-Me; 7f: 5-F;
7g: ; 4,6-(OMe)2

H H
O N
OH N N
4 O OH
R N 4 O
R S

8
9
R = 8a: H; 8b: 4-OMe; R = 9a: H; 9b: 4-OMe; 9c: 4-Br; 9d: 4-CF3;
8c: 4-Br; 8d: 4-(4-ClC6H4)
9e: 4-C6H5; 9f: 4-OCF3; 9g: 3-Br;
9h: 2,6-(OMe)2; 9i: 2-naphthyl

Fig. 9. Indole amide and hetero aromatic based hydroxamic acids

Suzuki et al. [136] have synthesized a series of compounds (categorized as acetyl lysine analogues) designed after

SAHA (Fig. 10). In this series, semicarbazide derivative 10 and bromoacetamides 11a-b were found to show potent

HDAC inhibitory activity with an IC50 of 150, 14 and 17 µM, respectively. The binding modes of compound 10,

analyzed by in silico docking, showed that it was slightly removed from the Zn2+ion (CO-Zn 3.1A°; NH2-Zn 2.1

A°). But with SAHA, an optimal bond distance was observed at the hydroxamic acid chelating end (CO-Zn 2.7A°;

NH2-Zn 1.8 A°). A covalent bond of compound 11b with His 132 was shown near to the reactive site of

bromoacetamide. The same authors also have reported another set of SAHA based non-hydroxamates for HDAC

inhibitory activity using SAHA as the standard [137]. The methyl sulfoxide derivative 12 exhibited HDAC

inhibiting profile at 68% at a 100 µM, with IC50 of 48 µM. The compound was also investigated for

hyperacetylation levels using Western blot analysis, treated on HCT116 human colon cancer cells for 8 h at 37°C.

The compound (100 µM) did not induce α-tubulin acetylation but was producing Histone H4 hyperacetylation to the

same extent as that of SAHA, thus implicating its inactiveness towards TDAC (α-tubulin deacetylase or HDAC6) in

HCT116 human colon cancer cells.

17
 O O O
R S
NH NH Me
n 6

10 R = NHCONHNH2 n = 5 12

11a R = NHCOCH2Br n = 6

11b R = NHCOCH2Br n = 5

Fig. 10. SAHA based non-hydroxamates as HDAC inhibitors

Shinji C and co-workers [138] have reported an approach to design and synthesize phthalimide based hydroxamic

acid analogues and another series of compounds structurally derived from thalidomide (Fig. 11). The acrolyl

derivative 13, from the first group, exhibited good HDAC inhibiting potency. In the analysis for inhibitory activity

towards HDAC1, HDAC4 and HDAC6, compound 13 was found to be a pan-HDAC inhibitor at nanomolar

concentration of 572, 358 and 709, respectively, which was about 1/10 to 1/30 of that of TSA. The same authors also

have reported the synthesis of hydroxamic acid based HDAC inhibitors carrying a cyclic amide or imide group as

linker as that of thalidomide [139]. The synthesized derivatives were evaluated for HDAC inhibitory potential using

fluorescence activity as well as p21 promoter assay. N-(1,2-diphenylethyl) derivative 14 was found to be a pan

inhibitor [EC50 =190 nM (HDAC1); 150 nM (HDAC4); 250 nM (HDAC6)] with potent p21 promoter activity

(EC1000 = 96 nM) and prominent cytostatic activity (IC50 = 75 nM) against human prostate cancer cells LNCaP than

SAHA. Replaced with isoindolinone group and 4-(3-pyridyl)phenyl group as surface recognition moiety 15 also

provided a pan-HDAC inhibitor [IC50 = 92 nM (HDAC 1); 56 nM (HDAC 4); 280 nM (HDAC 6)] [140]. Compound

15 was found to be promising as antiproliferative candidate against human pancreatic cell lines, PANC-1 and PT-45

with IC50 value of 35 and 200 nM, respectively.

18
 O O
HO O O
N HO
N N
H N
H
O
13
14

O O
HO
N
N
H

15
N

Fig. 11. Phthalimide, thalidomide and indolinonebased hydroxamates

Mai A. and co-workers [141] have designed, synthesized and screened a set of hydroxamates carrying a uracil

moiety for HDAC inhibiting activity (Fig. 12). Compounds 16 and 17 displayed better potency than SAHA in HD2,

HDI-A and HDI-B (maize derived HDACs) isoforms with IC50 ranging from 18-29 and 6-19 nM, respectively. The

hyperacetylation effect caused by the compounds in NIH3T3 cell core histones, analyzed by immunoblotting,

increased considerably compared to the control histones. Moreover, these compounds were able to induce

antiproliferative and cytodifferentiating effects in HL-60 human leukemia cells (viability index for 17 = 78% and

64% at 10 and 15 µM concentrations) when compared to TSA (30 nM) and SAHA (1.2 µM).

O
R N S OH
N
NH 5 H

O
R = 16: benzyl; 17: phenyl

Fig. 12. Uracil based hydroxamic acids

Krennhrubec et al. [142] have reported a set of molecules that were specifically designed for incorporation into the

sub-pocket of HDAC8 active site (Fig. 13). These ‘linkerless’ hydroxamates 18c and 18d displayed more than 100

fold selectivity for HDAC8 with IC50 0.7 and 0.3 µM, respectively, over the other members of the class I HDACs.

The selectivity was further confirmed by hyperacetylation studies and more binding studies. Compound 18a and 18b

19
demonstrated weak HDAC8 inhibition (IC50 = 20.0 and 66.0 µM, respectively). However, as for compound 18b, N-

hydroxy-2,2-diphenylacetamide, class IIa HDAC inhibition was high in nanomolar levels (HDAC4 IC50 = 0.75 µM;

HDAC5 = 0.14 µM; and HDAC7 = 0.39 µM). On this basis, Tessier et al. [143] have optimized compound 18b

through rigidifying the diphenyl system and substitution on the phenyl rings. The ortho position of the phenyl rings

were linked by carbon 18e, oxygen 18f and nitrogen 18g. Compound 18f was found to be more selective than 18e

and 18g towards class IIa HDAC enzymes, especially HDAC 7 (IC50 0.05 µM).

H
H N O
H N O OH
H
N O OH N O
OH OH H
N O
OH R = 18e: CH2; 18f: O;
18a 18b 18c 18d 18g: N.

Fig. 13.‘Linkerless’ and diphenyl methylene hydroxamic acids

Witter et al. [144] have identified three scaffolds, with different substitutions at C-5 and C-6 in benzo[b]thiophene

moiety for the development of hydroxamate based HDAC inhibitors (Fig. 14). Among the synthesized molecules,

compounds 19a-c, the C-6 substituted derivatives, exhibited potent HDAC1 inhibitory profile with IC50 of 10, 50

and 10 nM, respectively. The same trend was observed for 19a-c in cell proliferation studies conducted in murine

erythroleukemia SC-9 cells with IC50 value of 220, 160 and 190 nM, respectively. A structure-activity relationship

study carried out through molecular docking, depicted a 3-atom linker with C-6 substitution, for optimal HDAC1

inhibitory activity.

20
 H
O S N
O S N
O
H N H
OH H N
OH
19a 19b
O
O S N
H
H N
OH
19c

Fig. 14. Benzo[b]thiophene based hydroxamic acids

Price et al. [145] have developed and synthesized a series of 5-(1H-pyrazol-3-yl)-thiophene-2-hydroxamic acid

analogues 20a-d (Fig. 15) by optimizing ADS1003800, a sub-micromolar HDAC inhibitor, identified by virtual

screening. These compounds showed significant HDAC inhibitory potential in addition to proliferation assay (breast

cancer MCF-7 and MDA-MB231 cancer cells). The benzyl-tethered compounds displayed five times better activity

than the lead ADS1003800. Further functionalisation of the pendant phenyl group of compound 20b-c resulted in

compounds with improved enzyme inhibition and antiproliferative activity. Compound 20e, a benzodioxole

analogue, was 4 fold more potent than others in enzyme inhibition and 5-20 times better in cell proliferation assay

against MCF-7, MDA-MB231, HCT116 and PC3 cancer cell lines. The same authors also have reported compounds

21a-e with pyrazole replaced with pyridine that demonstrated enhanced HDAC inhibition (IC50 = 0.004-0.011 µM)

[146].

21
 F F
F O
S
N H ADS100380
N N
HO

O R O
N S S
R N N H N N H
HO 20 21
HO
O H
O N
R = 20a: R = 21a:

O
20b: 21b: O N
N H
O
H

N Cl
20c: 21c:
H
N
H
20d: O Me
21d:
O
N
H
O N F
20e: 21e:
O H
N
H

Fig. 15. Pyrazoyl thiophene and pyridyl thiophene based hydroxamic acids

Charrier and co-workers [147] have designed and synthesized a library of molecules containing hybrids between

indanone, SAHA and MS-275 to restrict conformational mobility (Fig. 16). In the tumor viability studies evaluated

on non-small cell lung cancer cells H661, compound 22 demonstrated an IC50 of 20 µM. The HDAC inhibition

assay, however, was not performed. Instead, the molecule was studied for its molecular dynamics (MM2 method)

and optimized for stable conformers. The study deduced the activity of the molecule 22 based on its aliphatic chain

flexibility and minor interactions due to the methyl group at C-2. The same authors also have reported

benzofuranone based HDAC inhibitors 23. These were evaluated against H661 cancer cells [148]. Unfortunately, the

benzofuranone group was found not suitable as a cap due to lack of favorable orientation.

22
 O O
O
OH
OH
N N
O H
5 H R
O
22 23

Fig. 16. Indanone and benzofuranone based hydroxamic acids

Chen et al. [149] have synthesized a series of triazole linked SAHA like hydroxamates using ‘click chemistry’ (Fig.

17). The incorporation of 1,2,3-triazole ring as surface recognition cap with 5 to 6 methylene spacer to the Zn2+

binding hydroxamate, as in compounds 24a and 24e, markedly enhanced in vitro HDAC inhibition with IC50 of 14.2

and 9.6 nM, respectively. The heteroaromatic caps with fused six-six rings like naphthalene and quinoline (24b-d)

exhibited potent enzyme inhibition. SAR studies revealed compounds with the triazole ring directly attached to the

aromatic group was more potent than when it was spaced with methylene group to the aromatic cap, indicating the

interaction of the triazole ring in the enzyme active site. He et al. [150] also have investigated 1-aryl-1H-[1,2,3]-

triazolylphenyl derivatives for HDAC isoforms selectivity and cytotoxic profile against several pancreatic cell lines

(Fig. 17). Compound 25a was found to show an IC50 value of 20 nM against MiaPaca-2 cancer cells. It was also

shown to reactivate CDK inhibitor proteins expression and effective in in vivo pancreatic tumor growth suppression.

Moreover, compounds 25b and 26 were found to be very effective inhibitors of cancer cell growth in the HupT3

(IC50 = 50 nM) and MiaPaca-2 (IC50 = 40 nM) cancer cell lines, respectively. Suzuki et al. [151] also have

synthesized and reported another series of triazole based hydroxamates with aromatic/heteroaromatic cappings as

selective HDAC8 inhibitors (Fig. 17). Compounds 27a-d displayed better in vitro HDAC8 inhibition with IC50 of

0.35, 0.18, 0.10 and 0.070 μM, respectively. Furthermore, compounds 27b and 27d demonstrated good growth-

inhibitory effects on T-cell lymphoma cells (Jurkat, HH, MT2 and MT4) and neuroblastoma cell lines (NB-1and

LA-N-1), displaying the involvement of HDAC8 in the growth of these cells. Based on these results, compound 27d

was further modified to obtain more derivatives [152]. Among these derivatives, compound 28, a reversed triazole

analogue, demonstrated better selectivity for HDAC8 (IC50 = 0.053 μM) than compound 27d (IC50 = 0.070 μM), but

weak T-cell lymphoma cell growth inhibition.

23
 N N
R O O H
N N
OH N
N N N N OH
3 4 O
H H
24
26
R = 24a: H, 24b: N

H
O N
24c: Me OH
24d: X N
N R1 N N O

27
N N 27a: X = CH2; R1 = Ph;
O 27b: X = CH2CH2; R1 = Ph;
N
OH 27c: X = CH2CH2; R1 = 3-thienyl;
N
4 27d: X = SCH2; R1 = Ph.
H
24e

H
O H N
N OH
N N
N OH S N N O
N
H 4 O
N 28
R1
25

R1 = 25a: C6H5 - ; 25b: 4-F,3-OMe C6H3 -.

Fig. 17. Aryl triazolyl hydroxamic acid derivatives

A series of N-hydroxy-1,2-disubstituted-1H-benzimidazol-5-ylacrylamides have been developed as selective

HDAC1 inhibitors and the effect of an olefinic linker in these analogues was examined [153] (Fig. 18). With the

propanol group at position 1 of benzimidazole, the compounds carrying α-substituted phenyl alkyl chain at C-2 of

benzimidazole (29a-c) were found to be potent HDAC1 inhibitors with IC50 of 0.17, 0.08 and 0.17 µM, respectively,

as well as potent antiproliferative activity in human colon cancer cells COLO205 with IC50 of 3.2, 1.7 and 4.1 µM,

respectively. Again with phenethyl at C-2 of benzimidazole, compound 29d showed significantly improved enzyme

inhibition (IC50 = 0.047 µM) and cellular profiling (IC50 = 0.32µM). Further, with the basic side chains at 1H in

benzimidazole moiety, compounds 29e-g with monoamine basic amine with a two carbon linker showed better

potency in HDAC1 inhibition with IC50 of 0.052, 0.035 and 0.026 µM, respectively. The docking studies of 29f into

24
HDAC1 homology model depicted an electrostatic force of attraction between the basic center of 1H of

benzimidazole and Asp99. An additional hydrogen bond was found between the N-3 of benzimidazole and hydroxyl

of Tyr204, implying that basic centers are crucial for the compound’s potency. Selected compounds, on further in

vitro evaluation in HCT116, A2780 and PC3 cancer cells, demonstrated encouraging results. Compound 29f showed

good efficacy in in vivo xenograft analysis on HCT116 tumor induced in nude mice. Bressi et al. [154] have

investigated a series of N-hydroxy monosubstituted benzimidazolyl and N-hydroxy trisubstituted imidazolyl phenyl

acrylamides as HDAC inhibitors (Fig. 18). Compounds 30a and 30b exhibited HDAC2 inhibition at IC50 of 10 nM.

The effects in terms of H3 and H4 hyperacetylation and p21induction profile were also found to be promising.

O
OH O
N N
R1 Ph N OH
H N
N
N H
R2
HO 29 Me
O
R2 = 29d:
R1 = 29a: PhCH2CH2- ; Me
O
29b: PhCH(CH)CH2- ;
O
29c: PhCH2OCH2- Me

29e: -CH2CH2N(CH2CH3)2

O 29f:
N N
OH
N N
29g :
H N

N
R
30

R = 30a: -CH3 ; 30b : -CH2CH3

Fig. 18. Benzimidazole based hydroxamic acid derivatives

Tapadar et al. [155] have reported isoxazole based hydroxamic acid derivatives varying the aromatic, heteroaromatic

and alicyclic amino surface caps attached to an isoxazole linker as HDAC inhibitors (Fig. 19). On screening for

class I and II HDAC potency, compound 31 exhibited better HDAC3 inhibition at nanomolar levels with IC50 of 30

± 1 nM and HDAC6 inhibition with IC50 of 68 ± 5 nM. It was equally effective against various pancreatic cancer

cells (BxPC-3, HupT3, Mia Paca-2, panc 04.03 and Su.86.86) with IC50 ranging from 1-4 µM. The authors,

however, conclude that rigidification with isoxazole failed to arrive at an isoform selective HDAC inhibitor.

25
Prompted by these efforts, Conti et al. [156] have carried out investigation to evaluate the role of the isoxazole

moiety inserted near the Zn2+ binding group (Fig. 19). Analogues designed were such that the cap of SAHA (Ph-

NH-CO-) was identical, but vary in the spacer length along with the isoxazole group placed vicinal to the Zn2+

binding group. Substituents mimicking the hydroxamate carbonyl oxygen and nitrogen were also appended at

position 3 of the isoxazole for structure optimization. Unfortunately, the bioisosteric replacement and the isoxazole

insertion did not display better results. However, a thiol analogue 32 exhibited moderate inhibitory profile (22%

decrease at 1 µM and 80% decrease at 10 µM), suggesting a new non-hydroxamate HDAC inhibitor.

H
O N O
N N HS Ph
O N N H N
S O H
HO 3 O

31 32

Fig. 19. Isoxazole based hydroxamic acids

The design, synthesis and preliminary evaluation of N-hydroxy-4-(3-phenylpropanamido)benzamides as HDAC

inhibitors have been reported [157] (Fig. 20). Most of the compounds showed IC50 value below 1 µM but analogues

with alkyl substitutions displayed an IC50 value ranging from 1.6 to 4.0 µM. However, the thiophene derivative 33a

(HDAC inhibition IC50 = 0.3 µM) and the benzo[d][1,3]dioxole analogue 33b (HDAC inhibition IC50 = 0.4 µM)

were promising in cell proliferation assay in HCT116 colon cancer cells and A459 non-small cell lung cancer cells.

These compounds were also found to induce cell cycle arrest at G2 phase, mediating p21-independent apoptosis.

O
H
O N
R OH
N
H 33

O O
Me
R = 33a: 33b:
O O
S

Fig. 20. N-hydroxy-4-(3-phenylpropanamido)benzamide based hydroxamates

Dallavalle et al. [158] have synthesized a series of biphenyl-4-yl-acrylohydroxamic acid derivatives with compound

34a as prototype (Fig. 21). On HDAC2 inhibitory assay, compounds 34b-d with IC50 of 0.59, 0.22 and 0.33 µM,

26
respectively exhibited similar inhibitory profile as 34a (IC50 = 0.82 µM). The presence of double bond between the

cap group and the chelating end (35a-c) was seen to be appreciable in terms of activity. The proximal phenyl system

replaced with cyclohexyl ring (36) and the oxygen atom inserted between the double bond and the biphenyl system

(37) was also tolerable. But the corresponding saturated systems of 35 were detrimental.

O
O
H
N H
OH
OH

R1 35
34 R2

R2 = 35a: H; 35b: OMe; 35c: CN

R1 = 34a: H; 34b: OH;
34c: CH2OH; 34d: CN

O
H H
N O N
OH OH
O

HO
36
37

Fig. 21. Biphenyl-4-yl-acrylohydroxamic acid derivatives

Acylurea (-CONHCONH-) moiety has also been incorporated to form straight chain hydroxamates as HDAC

inhibitors [159] (Fig. 22). This linker, claimed to be more versatile due to the presence of more hydrogen bond

acceptors and donors and also optimal length favourable for interaction, was adjustable. In addition, the cap group

was diversified with varied substituted aromatic and heteroaromatic groups. Compounds 38a-e demonstrated 10-20

fold increase in HDAC1 inhibition compared to SAHA. Reduced HDAC potency was observed with N-alkylated

ureas and acylurea connected straight chain hydroxamates. Compound 38a was further screened against a panel of

human tumor cancer cells, resulting in IC50 values in the range of 0.50 to 1.58 µM. Tazzari et al. [160] have

designed and synthesized a series of aryldithioethione analogues based on SAHA and MS-275 as HDAC inhibitors

(Fig. 22). Majority of the compounds showed enzyme inhibition in varying degrees. Some of the compounds

paralleled the A546 cell proliferation inhibition. Compounds 39a and 39b with IC50 of 0.08 and 0.01 µM,

respectively were more potent than SAHA (IC50 = 0.1µM) in enzyme inhibitory activity. Though a two-fold

27
enhanced H4 hyperacetylation was observed in A549 cells, a 7-8 fold decrease was observed in the antiproliferative

activity in the same cells. The study, however, lacks specificity towards any of the HDAC isoforms known.

O
O O OH
O H
N N
R N N H n O
H H O S H
38 S S 39
39a: n = 4;
R = 38a: C6H5; 38b: 4-Br-C6H4;
39b: n = 6
38c: 4-I-C6H4; 38d: 2-naphthyl;
38e: 3-NH2-4-Cl-C6H3

Fig. 22. Acylurea incorporated and aryldithioethione based hydroxamates

In an attempt to arrive at novel δ-lactams based on KBH-A118 as histone deacetylase inhibitors for improved

metabolic stability, Yoon et al. [161] have synthesized a series of compounds by N-alkylation of various amines

with 4-bromo-1-butene to afford the secondary amines which were then coupled with monoacid (Fig. 23). The

resultant amides were subjected to ring closure metastasis using Grubb’s catalyst (I) and then altering to the desired

compounds with hydroxamic acids as chelating group. The HDAC inhibitory activity of 40 ranged with IC50 0.05 to

0.55 µM. Bulkier substituted compounds 40a-f possessed enhanced activity than the smaller grouped compounds. In

microsomal stability assay, compounds with high lipophilicity illustrated high metabolic clearance whereas those

with more number of rotatable bonds displayed a negative impact. The same authors have also reported a series of

HDAC inhibitors with 3-4 carbon units between the δ-lactam ring and the chelating group [162]. The potency of this

structural composition was also confirmed via docking studies. They have also designed novel δ-lactam analogues

with propionic hydroxamic acid as end group to achieve HDAC inhibitors [163] (Fig. 23). Compounds 41a and 41b

exhibited HDAC inhibition with IC50 of 0.35 and 0.50 µM, respectively in comparison to SAHA (IC50 = 0.11 µM).

Both the compounds showed more antiproliferative effect against human breast cancer cells MDA-MB-231 with a

GI50 of 1.62 and 2.67 µM, respectively (GI50 of SAHA = 0.66 µM). δ-Lactam analogues as HDAC inhibitors have

been reported to give compounds 41c with IC50 of 0.23 µM, 42a-b with IC50 of 37 and 30 nM, respectively. On

cytotoxic evaluation using ACHN (renal cancer), MDA-MB-231 (breast cancer), PC-3 (prostate cancer), NUGC-3

28
(gastric cancer), HCT-115 (colon cancer) and NCI-H23 (lung cancer) cell lines, compounds 41c and 42a-b showed

good GI50 values in the range of 0.28 to 1.18 µM with more sensitivity towards MDA-MB-231 and PC-3 cancer

cells. Compound 42a also exhibited 51% reduction in tumor growth in in vivo MDA-MB-231 breast xenograft

models of nude mice.

O O
OH O O 40a: n=2, R = -4-t-Bu-C6H4;
N N n
OH 40b: n=2, R = -4-Me-C6H4;
H R N N
40c: n=1, R = -3-CN-C6H4;
H
KBH-A118 40d: n=1, R = -4-CN-C6H4;
40e: n=2, R = -4-NO2-C6H4;
40
40f: n=1, R = -2-F-4-Br-C6H3

OH OH
N N N N
R1 H H
n O n
O O O
41 42
41a: n=2, R1 = -H; 42a: n=2
41b: n=1, R1 = - 2,4-(OMe)2; 42b: n=3
41c: n=2, R1 = -4-C6H5

Fig. 23. δ–Lactam based hydroxamates

Prompted by the rationale of the above investigation, another class of γ-lactam derivatives have been designed,

synthesized and reported for their HDAC inhibitory potential [164] (Fig. 24). A good HDAC inhibitory profile with

IC50 in the range of 0.01 to 0.80 µM was obtained with all the synthesized molecules. Compounds 43a-c

demonstrated good enzyme inhibition with IC50 of 0.01, 0.04 and 0.02 µM, respectively. This was justified due to its

compatible sized core favoring good binding to the Zn2+ ion. Additionally, these analogues showed a dose dependent

enhancement in the hyperacetylated levels of H3 and H4. Based on these results, γ-lactams with diversified cap

groups have also been designed and synthesised [165] (Fig. 24). The molecules were varying with 1-3 carbon chain

linkers between the γ-lactam ring and the aromatic cap (with triflouromethyl and methoxy groups at ortho-, meta-

and para- positions). The two carbon chain derivatives were potent in HDAC inhibition (IC50 = 0.07 to 0.19 µM),

prominent ones being 44a-c. Docking studies showed that compound 44c to interact with the enzyme by means of

hydrogen bonds at His145 and His146 as well as π-π interaction between γ-lactam core and aromatic side chain of

29
Phe155 and Phe210. A good correlation was obtained in the in vitro cytotoxic evaluation with that of HDAC

inhibition. However, only a moderate stability was observed in metabolic stability evaluation conducted in mouse

liver microsomes.

O O
O O
N N
R N H N H
R1
HO HO
43
44
R = 43a: 2-naphthyl;
R1 = 44a: -H; 44b: -4-OMe; 44c: -4-CF3
43b: biphenyl;
43c: 1-bromonaphthyl

Fig. 24. γ-Lactam based hydroxamic acid analogues

Belvedere et al. [166] have reported aminosuberoyl hydroxamic acids as a potent novel class of HDAC inhibitors

(Fig. 25). With anilide and aminoquinolide cappings, these novel compounds showed HDAC inhibition (IC50 = 4-42

nM) and murine erythroleukemia SC-90 cell proliferation inhibitory activities at nanomolar concentrations (IC50 =

33-367 nM) as well as tumor growth inhibition in both colon and breast xenograft models. Compounds with 6-

aminoquinolide groups 45 demonstrated good HDAC inhibitory potency (IC50 = 1.3-54 nM), followed by 8-

aminoquinolide and anilide group containing analogues. Lactam based 7-aminosuberoyl hydroxamic acids have

also been further investigated as HDAC inhibitors [167] (Fig. 25). The novel compounds 46-48 containing (S)-7-

aminocarboxy lactams demonstrated HDAC inhibition in nanomolar levels. Compound 45e showed class I HDAC

specificity with IC50 values between 2 and 10 nM. An IC50 of 0.5 µM was also observed against H460 cells in

antiproliferative evaluation.

30
 O
O
H
H N R1 O
NH
N H
N
O OH H HN O O
N
43
45 N H
R2 N
R1 = 45a: Ph-; 45b: pyridin-2-yl-; O OH
45c: quinolin-2-yl-; 45d: PhCH2O -. 46
R2 = 46a: Ph-; 46b: cyclopentyl-;
O
46c: PhCH2CH2 -.
NH

H HN O O O
N H
R3 N
HN
O OH
47
H HN O O
R3 = 47a: - C6H5;
N H
47b: - CH2CH2C6H5; N
47c: - 3-Me C6H5; O OH
47d: - 4-Me C6H5; 48
47e: - 3-CF3C6H5

Fig. 25. Aminosuberoyl based hydroxamic acids

Using 2,5-disubstituted-1,3,4-oxadiazole and thiadiazole as surface recognition motif and a pyrimidine linker, Rajak

et al. [168] have investigated a series of novel HDAC inhibitors (Fig. 26).The compounds with hydroxy and

methoxy groups on the phenyl ring attached to the oxadiazole and thiadiazole (49a-b, 49h-i) were more potent in

HDAC1 inhibition (IC50 = 0.007-0.017 µM) as well as in in vitro antitumor screening against HCT116 cancer cells

(IC50 = 0.25-0.31 µM). Para- substituted analogues were more favorable than the ortho- and meta- substituted

derivatives. However, halogen attached analogues were detrimental to activity. The study implicated the thiadiazole

analogues to be more active than their oxadiazole counterparts. Guan et al. [169] have reported on hydroxamates

based HDAC inhibitors with 1,3,4-thiadiazole as cap group together with 5-6 carbon chain aliphatic linker (Fig. 26).

The phenyl substituted analogues were not significant in enzyme inhibition, whereas the heteroaromatic derivatives

(IC50 = 286-411 nM) displayed similar or better activity compared to SAHA (IC50 = 416 nM). The thiophene variant

50g exerted higher HDAC inhibition with IC50 of 310 nM (SAHA IC50 = 416 nM) and antiproliferative potency

31
against breast cancer cell MDA-MB-231, chronic myelogenous leukemia cell K562 and prostate cancer cell PC3

with IC50 of 1.21, 1.56 and 3.60 µM, respectively.

O N N O O
OH R2 OH
N N N N S N N
H H n H
X N N
R1
H 50
49
n=6
X = O;
R2 = 50a: -4-NMe2-Ph; 50b: -4-Me-Ph; 50c: -2-Cl-Ph;
R1 = 49a: -4-OH; 49b: -4-OMe; 49c: -2-Cl; 49d: -4-Cl;
50d: -4-Cl-Ph; 50e: -4-NO2-Ph; 50f: -4-CF3-Ph;
49e: -4-NO2; 49f: -4-NH2; 49g: -4-NMe2;
50g: -thiophen-2-yl; 50h: -pyridin-3-yl;
X = S; 50i: -pyridin-4-yl;
R1 = 49h: -4-OH; 49i: -4-OMe; 49j: -2-Cl; 49k: -4-Cl; n=5
49l: -4-NO2; 49m: -4-NH2; 49n: -4-NMe2. R2 = 50j: -4-NMe2-Ph; 50k: -4-Me-Ph; 50l: -2-Cl-Ph;
50m: 4-Cl-Ph; 50n: -4-NO2-Ph; 50o: -4-CF3-Ph;
50p: -thiophen-2-yl; 50q: -pyridin-3-yl;
50r: -pyridin-4-yl.

Fig. 26. 1,3,4-Oxadiazole and 1,3,4-thiadiazole based hydroxamates

1,2,4-Oxadiazole based SAHA analogues have been reported as potent HDAC inhibitors and antitumor agents [170]

(Fig. 27). The compounds were specifically analyzed for class I HDAC inhibition. Compound 51h and 51k

displayed better potent HDAC1 inhibition with IC50 value of 70 and 80 nM, respectively than HDAC2 inhibition

with IC50 of 230 and 390 nM, respectively. Compared to HDAC1 and HDAC2, all the compounds were less potent

in HDAC8 inhibition. Most analogues showed higher antiproliferative activities against acute myeloid leukemia

cells U937 than against human lung cancer cells, A549 and NCI-H661. The docking simulations demonstrated

compound 51h to interact with HDAC2 (PDB ID: 4LY1) by hydrogen bonds with His145, His146 and Tyr308

through its hydroxamate end. The linker portion was occupied in the lipophilic tube formed by Gly154, Phe155,

His183, Phe210 and Leu276. The authors also investigated class I HDAC inhibitory activity on 2-aminobenzamide

and trifluoromethyl ketone derivatives. Using scaffold-hopping strategy, Yao et al. [171] have reported a series of

1,3-disubstituted-pyrazole derivatives as class I and IIb histone deacetylase inhibitors (Fig. 27). The compounds

were diversified by means of difference in the length of the linker as well as the position of the groups attached at

the pyrazole ring. Compounds exceeding six methylene spacers demonstrated decreased selectivity between HDAC1

and HDAC7. However, compounds 52 depicted better selectivity on class I and IIb HDAC isoforms than over class

32
IIa; compound 52f with biphenyl moiety represented high selectivity in class I and IIb HDAC isoform inhibition

(HDAC1 IC50 = 0.033 µM; HDAC2 IC50= 0.226 µM; HDAC3 IC50= 0.030 µM; HDAC6 IC50 = 0.034 µM and

HDAC10 IC50 = 0.029 µM). The in vitro antitumor activities of 52f against several cancer cells were also promising

and acetylation of H3 exhibited dose dependency.

O H
N H N
R1 N R2 N OH
N
N O HO OH
51
52
R1 = 52a: -CH2-C6H4-(3-Ph);
R1 = 51a: Ph; 51b: 4-Me-Ph; 51c: 4-OMe-Ph;
52b: -Ph; 52c: -CH2-C6H4-4-(O-Ph);
51d: -4-CF3-Ph; 51e: -4-F-Ph; 51f: -4-Cl-Ph;
52d: -CH2-C6H4-4-(indol-1-yl);
51g: -2-Cl-Ph; 51h: -4-NO2-Ph;
52e: -CH2-C6H4-4-(NH-Ph);
51i: -3-NO2-Ph; 51j: -thiophen-2-yl;
51k: -pyridin-3-yl. 52f: -CH2-C6H4-(4-Ph);
52g: -CH2-naphth-2-yl.

Fig. 27. 1,2,4-Oxadiazole and 1,3-disubstituted-pyrazole based hydroxamic acids

A novel series of hydroxamates featured by tricyclic core (dibenzo-diazepine, -oxazepine and -thiazepine) have been

synthesized and evaluated for HDAC inhibition [172] (Fig. 28). The compounds 53 were characterized by various

functionalities in the central heptameric ring (53a-c = SO2; 53d-f = S; 53g-I = O; 53j-l = NH; and 53m = NMe). The

HDAC inhibitory activity was evaluated based on rank order, RO, taking the lowest concentration of the molecule

into account as significant HDAC inhibitor. In other words, the higher the RO value, the more active is the

compound as a HDAC inhibitor. Compound 53a and 53b demonstrated the highest RO of 4700 and 4400,

respectively, and the least being 53l and 53m with 530 and 240, respectively. This was further supported by

hyperacetylated levels of H3 and H4, evaluated in myeloid leukemia cells NB4. A good correlation was obtained for

the compounds in the antiproliferative activity (against colon cancer cell HCT116 and myeloid leukemia cell NB4)

corresponding to that of HDAC inhibition with compound 53a displaying HCT116 IC50 of 0.04 µM, NB4 IC50 of

0.02 µM and 51b with HCT116 IC50 of 0.03 µM, NB4 IC50 of 0.02 µM. Unfortunately, these two compounds were

not active enough in the in vivo antitumor screening conducted in HCT116 xenograft models. The same group have

also designed and synthesized a series of 5,11-dihydrodibenzo[b,e]azepine-6-one derivatives 54 that were alkylated

on the amide nitrogen with an alkyl chain bearing an hydroxamic acids moiety at the end [173] (Fig. 28). The

33
diphenyl methyl privileged fragment as recognition motif was explored to arrive at a new series of HDAC inhibitors.

The endocyclic ketone 54a was bearable, but not favorable, as it showed a three-fold fall in the IC50 (0.042-0.153

µM).

O H
O H
N
N OH N
N OH
O
X O
X
R

53
54
53a: X = SO2; R = OMe; 53b: X = SO2; R = Cl;
X = 54 a: CO; 54b: CH2; 54c: CHOH;
53c: X = SO2; R = H; 53d: X = S; R = OMe;
54d: CHF; 54e: CF2; 54f: C(OMe)2.
53e: X = S; R = Cl; 53f: X = S; R = H;
53g: X = O; R = OMe; 53h: X = O; R = Cl;
53i: X = O; R = H; 53j: X = NH; R = Cl;
53k: X = NH; R = OMe; 53l: X = NH; R = H;
53m: X = NMe; R = H.

Fig. 28. Tricyclic core based hydroxamic acids

A novel series of spiro[chromane-2,4’-piperidine] and spiro[benzofuran-2,4’-piperidine]hydroxamic acid derivatives

as HDAC inhibitors has been identified by combining the stated structures with a hydroxamic acid moiety as

chelating group [174] (Fig. 29). The compounds 55-57 were analyzed for their ability to inhibit nuclear extract

HDACs. The benzyl and benzoyl derivatives, 55e and 55f, of N-hydroxy-{4-oxospiro[chromane-2,4’-piperidine]-6-

yl}acrylamides 55 demonstrated remarkable potency in HDAC inhibition with IC50 of 0.121 and 0.316 µM,

respectively. Subsequently, in the 7-spiropiperidines 56, the benzoyl analogue 56f (IC50 = 0.366 µM) was equipotent

to 53f in enzyme inhibition assay. All the spirobenzofuran derivatives 57 were less potent, except for 57a and 57b,

which showed activity in low micromolar range with IC50 of 0.221 and 0.538 µM, respectively. On further

evaluation, compound 55e displayed a dose dependent inhibition with IC50 of 0.1 µM in antiproliferative assay

against HCT116 cancer cells. The cell cycle analysis reported the same compound to induce a strong increment of

G0/G1 phase population at the lowest dose as well as a hike in G2M population at 1 and 3 µM. The compound also

inhibited tumor growth in HCT116 xenograft transplants in a dose dependent pattern.

34
 O
R N O
H H O H
N R N
OH N N
R N O OH OH
O O O O O
56 57
55

R = 55a: H ; 55b: Me ; R = 56a: H ; 56b: Me ; R = 57a: H ; 57b: Me ;

55c: Ac ; 55d: COOEt ; 56c: Ac ; 56d: COOEt ; 57c: Ac ; 57d: COOEt ;
55e: Bn ; 55f: Bz. 56e: Bn ; 56f: Bz. 57e: Bn ; 57f: Bz.

Fig. 29. Spiropiperidine based hydroxamate derivatives

Based on TSA or SAHA, Pabba et al. [175] have reported TSA derivatives containing either an aryl ether or sulfone

group as the connecting unit to the surface recognition motif, a branched chain diene linker and carboxylic or

hydroxamic acid end as chelating group (Fig. 30). A remarkable increase in HDAC inhibitory activity was observed

with derivatives 58 containing the aryloxy capping. The compounds 58a-d were found to be potent in nanomolar

levels with IC50 value of 41, 68, 18 and 20 nM, respectively. Further, the compound 58e, with an extra methyl group

in the diene spacer, was found to be equipotent (IC50 = 55 nM) implying aromatic substitutions at meta-and/or para-

positions are more favorable for enzyme inhibition. Unfortunately, the aryl ethers with carboxylic acid terminal and

the aryl sulfone series with both carboxylic and hydroxamic acid end groups did not show HDAC inhibition (IC50 >

1 µM). The results were confirmed through docking studies where the aryl sulfone series showed unfavorable

interactions with the enzyme, making them unfit for hydrophobic interactions. Zhu et al. [176] have investigated for

a series of diaryl ether hydroxamic acid derivatives as HDAC inhibitors (Fig. 30). The hydroxamic acid end group

lied either in the para- or meta- position of diaryl ether oxygen. The molecules 59 with hydroxamic acid group in the

para- position displayed moderate enzyme inhibition which was dose dependent. The level of inhibition ranged from

50.83 to 69.20% at 1 µM and between 63.18 and 83.56% at 10 µM which was lower compared to SAHA (90.76% at

1 µM and 98.51% at 10 µM). However, these compounds showed no cytotoxicity in in vitro antiproliferation assay

against human acute lymphoblastic leukemia cells (MOLT-4), hepatoma carcinoma cells (SMMC7721), chronic

myeloid leukemia cells (K562) and Burkitt’s lymphoma cells (Raji). The series with hydroxamic acid in meta-

postion of diaryl ether oxygen (60) did not display any significant HDAC inhibition. However, compounds 60c and

60d, demonstrated significant antitumor profile in the cancer cells with IC50 values in micromolar concentrations

(IC50 = 1.1-7.1 µM) suggesting an alternative antitumor mechanism.

35
 O
O OH O
R2 N O H
R1 Me H R N
N N OH
H H O
58
59
R1= H;
R = 59a: -3-pyridinyl;
R2= 58a: -3-biphenyl; 59b: -3-methoxyphenyl;
58b: -4-methoxyphenyl; 59c: -4-methoxyphenyl;
58c: -3-(trifluoromethyl)phenyl; 59d: -4-chlorophenyl;
58d: -7-methoxy-2-naphthyl; 59e: -4-bromophenyl.
R1= Me;
R2= 58e: -3-biphenyl
O
O OH
O N
R H
N N
H H
60

R = 60a: -4-chloro-3-(trifluoromethyl)phenyl;
60b: -3-methoxyphenyl;
60c: -4-methoxyphenyl;
60d: -4-chlorophenyl;
60e: -4-bromophenyl.

Fig. 30. Aryloxy and diaryloxy based hydroxamic acid analogues

Cai et al. [177] have designed and synthesized novel hybrids with indole ring connected to N-hydroxyarylamide

through alkyl substituted triazole (Fig. 31). The synthesized molecules 61 were evaluated for antiproliferative

activities against human colon cancer cells HCT116, Lovo, human leukemia cells K562, breast cancer cells MCF7

and hepatocarcinoma cells Hep G2. Majority of the compounds demonstrated good to moderate antiproliferative

activities. Compound 59 displayed very remarkable antiproliferative activities with IC50 ranging from 3.57-6.21 µM

against the cancer cells that are comparable to/slightly better than SAHA (IC50 = 4.95-7.11 µM). Further, the HDAC

isoforms selectivity profile showed that compounds 61a-f were promising HDAC1, HDAC6 and HDAC8 inhibitors.

Compound 61e exhibited most prominent HDAC1 inhibition with IC50 of 0.078 µM, which is four-fold lower than

that of HDAC6 (IC50 = 0.29 µM) and five-fold lower than that of HDAC8 (IC50 = 0.38 µM). The compound also

demonstrated enhancement in acetylation of H3 and tubulin in a dose dependent manner. A novel set of indole

connected 4-hydroxycinnamide based and 3-hydroxycinnamide hydroxamates as HDAC inhibitors have been

developed wherein the 3-hydroxycinnamide series 62 showed moderate inhibition against the enzymes [178] (Fig.

31). Compound 62d displayed potent inhibition against HDAC4, HDAC8 and HDAC11 with IC50 value of 440, 87

36
and 52 nM, respectively thus establishing them as pan-HDAC inhibitors. But on antiproliferative assay using human

myeloma cells U266, 62d did not show significant activity whereas compounds 62a and 62b with moderate HDAC

inhibition potency demonstrated remarkable activity with IC50 value 0.39 and 0.41 µM, respectively, that was better

than SAHA (IC50 = 1.09 µM). Besides, the in vivo assay in U937 xenograft transplants showed compound 62a as a

potent, orally active HDAC inhibitor. A series of N-hydroxyfurylacrylamide based HDAC inhibitors bearing

branched cap moieties have been reported [179] (Fig. 31). Most of the derivatives were promising in enzyme

inhibition and cancer cytotoxicity assay. Compound 63, on further evaluation for enzyme selectivity profile,

displayed excellent selectivity towards HDAC6 with IC50 in the range of 0.18-0.48 µM.

R1 O
H
OH
N N N
n O m H O OH
N N
N N R O
N H
H R2 H
61 O
62
61a: (para) m = 1, n = 1, R1 = H, R2 = H;
62a: R= -4-methoxyphenyl;
61b: (para) m = 1, n = 1, R1 = H, R2 = OMe; 62b: R = -4-methylphenyl;
61c: (para) m = 1, n = 2, R1 = H, R2 = OMe; 62c: R= -2-chlorophenyl;
61d: (para) m = 1, n = 1, R1 = OMe, R2 = H; 62d: R= -3-methoxyphenyl;
62e: R= -2-methoxyphenyl.
61e: (para) m = 1, n = 1, R1 = OMe, R2 = OMe;
61f: (meta) m = 1, n = 1, R1 = OMe, R2 = H.

R1 O
N O
R2
N OH
H
63
63a: R1 = Benzyl, R2 = Ethyl;
63b: R1 = Benzyl, R2 = Propyl;
63c: R1 = Benzyl, R2 = Butyl;
63d: R1 = Benzyl, R2 = -2-Hydroxy-Ethyl;
63e: R1 = Homopiperonyl, R2 = Ethyl.

Fig. 31. N-hydroxyarylamide, N-hydoxycinnamide and N-hydroxyfurylacrylamide derivatives

Fu et al. [180] have investigated the influence of saccharin in a series of saccharin-based N-hydroxybenzamides as

HDAC inhibitors (Fig. 32). On evaluation for enzyme inhibition, compound 64 displayed good to moderate

37
inhibition profile. The propanamide derivative 64a (IC50 = 0.300 µM) and the butylamide analogue 64c (IC50 =

0.233 µM) exhibited activity similar to SAHA (IC50 = 0.193 µM). Compounds 64d and 64e, with the methylene

linker, also possessed good inhibition with IC50 value of 0.386 and 0.380 µM, respectively. In addition, compounds

64b-c displayed in vitro antiproliferative potential in MDA-MB-231, KG1 and PC-3 cancer cell lines. Compound

64b (IC50 = 3.45 µM) demonstrated similar potential as that of SAHA (IC50 = 4.61 µM) in MDA-MB-231 cells,

confirmed by cell cycle analysis. As an approach to arrive at HDAC inhibitors with better affinity and activity, Lu et

al. [181] have investigated a series of isoferulic acid based hydroxamates and benzamides (Fig. 32). When compared

to SAHA (IC50 = 0.26 µM), the hydroxamate derivatives 65 were more potent than the benzamide analogues.

Compound 63d displayed HDAC inhibitory activity with IC50 of 0.57 µM, whereas compounds 65a-c showed IC50

value of 1.37, 0.94 and 0.73 µM, respectively. Molecular binding modes of 65d with HDAC (PDB ID:3F07)

suggested favorable interactions-two hydrogen bonds of hydroxamates -OH with His142 and His143 at distance of

1.96 A° and 2.41 A°, respectively, one hydrogen bond between N-H and His143 at a distance of 1.79 A° and another

hydrogen bond between carbonyl group with Tyr306 at a distance of 1.75 A°. Further, these compounds also

demonstrated potent antiproliferative activities against MDA-MB-231 and HeLa cell lines with compound 65d

showing an IC50 of 3.91 µM against HeLa cancer cells.

O O
X MeO
S H H
N H
N N
R N O OH
O OH
O O
O
64 R 65

64a: X = -(CH2)2CONH- , R = H; 65a: R = 3,4-Cl2;

64b: X = -(CH2)2CONH- , R = Ph; 65b: R = 4-OMe;
65c: R = 4-Br;
64c: X = -(CH2)3CONH- , R = H;
65d: R = 2-F.
64d: X = -CH2- , R = Ph-CONH-;
64e: X = -CH2- , R = 4-diPh-CONH-.

Fig. 32. Saccharin and isoferulic acid based hydroxamates

Adamantane, homoadamantane and nor-adamantane based potent HDAC inhibitors have been developed as

anticancer [182] (Fig. 33). From the three series, the nor-adamantane based compounds 66a and 66b were shown to

be more potent in enzyme inhibition with IC50 value of 0.02 and 0.03 nM, respectively as well as in antiproliferative

38
assay in non small cell lung carcinomaNCI-H461, colon cancer cells HCT116 and human glioma cancer cells U251

(GI50 = 0.01 - 0.05 µM). With sub-nanomolar HDAC inhibition, compound 66a displayed 45% tumor growth

inhibition at a dose of 5mg/kg, studied in NCI-H460 lung cancer xenograft models. Colchicine based SAHA

hybrids have been reported as a novel class of HDAC inhibitors [183] (Fig. 33). These compounds displayed

synergistic antitumor effect of tubulin inhibitors together with HDAC inhibition. The representative analogue 67c

demonstrated potent HDAC inhibition against HDAC1 (IC50 = 0.72 µM), HDAC3 (IC50 = 0.83 µM) and HDAC6

(IC50 = 0.44 µM). Compound 67c also exhibited potent cytotoxic potential against five cancer cell lines, namely

A431 (IC50 = 0.242 µM), PC-3 (IC50 = 0.813 µM), MCF-7 (IC50 = 0.825 µM), HCT116 (IC50 = 0.903 µM) and A549

(IC50 = 4.672 µM).

O O
O OH
HN N
R MeO n H

N H
H N
OH MeO
O
66 MeO OMe
R = 66a: H; 66b: Me. 67
67a: n = 2; 67b: n = 3; 67c: n = 4;
67d: n = 5; 67e: n = 6.

Fig. 33. Adamantane and colchicine based hydroxamates

Oanh et al. [184] have reported the design and synthesis of a series of benzothiazole analogues based on SAHA (Fig.

34). The synthesized compounds 68 exhibited potent HDAC inhibition and cytotoxicity against five cancer cell

lines, namely colon cancer SW620, breast cancer MCF-7, prostate cancer PC-3, pancreatic cancer AsPC-1and lung

cancer NCI-H460. Compound 68b exhibited cytotoxicity with an IC50 value between 1.59-4.79 µM, comparable to

SAHA (IC50 = 0.49–3.56 µM). The HDAC inhibitory activity of the analogues, analyzed through Western Blot

assay for H3/H4 histone acetylation at 1 µg/mL, was found to be potent. But the compound was less active in the in

vivo profiling due to hepatic oxidation at the methyl group. The same research group also have developed three

compounds, 68g, 68h and 69, as novel HDAC inhibitors [185] (Fig. 34). The compounds were potent to induce

H3/H4 acetylation at 1 µg/mL. Compound 68g was 8-times less potent than SAHA (IC50 = 0.49 µM) against MCF-7

39
cell line (IC50 = 4.10 µM). However, it was more potent than SAHA in prostate cancer PC-3, pancreatic cancer

AsPC-1 and lung cancer NCI-H460 cancer cells. Moreover, the fused benzene ring in the benzothiazole heterocycle

was not vital for cytotoxicity as evidenced by antiproliferative activity profile of 69 in all the cell lines. The in vivo

anticancer studies conducted in PC-3 xenograft models, showed compound 68g to exhibit 41.44 % and 49.00 %

tumor growth inhibition at doses of 10 and 30 mg/kg, respectively (Tumor growth inhibition of SAHA at 30 mg/kg

dose – 48.30 %). The same group have also investigated novel isatin-3’-oxime- and isatin-3’-methoxime-based

hydroxamic acid analogues 70 and 71 as HDAC inhibitors [186] (Fig. 34). Compounds with no substitution (70a

and 71a) or 5’-F, 5’-Cl, 7’-Cl substitution on the isatin ring (70b-d and 71b-d) displayed good HDAC inhibition

against H3 and H4 deacetylation at 1µM. The antiproliferative profile of the compounds against five cancer cells

was found to be potent with IC50 as low as 0.08 µM and compound 70d was more cytotoxic (IC50 = 0.26–0.63 µM).

Docking studies revealed 70a and 71a to show high affinities to HDAC2 and HDAC8, respectively.

R N O O S O O
OH OH
S N N N N N
6
H 6 H H H
68 69
R = 68a: H; 68b: Me; 68c: OMe;
68d: OEt; 68e: SO2CH3;
68f: NO2; 66g: Cl; 68h: CF3.

O O O
O
HO MeO OH
OH N N N
N N N
H H

R R
70 71
R = 70a: H; 70b: 5-F; 70c: 5-Cl; R = 71a: H; 71b: 5-F; 71c: 5-Cl;
70d: 7-Cl; 70e: 5-NO2; 71d: 7-Cl; 71e: 5-NO2;
70f: 5-Me; 70g: 5-Br. 71f: 5-Me; 71g: 5-Br.

Fig. 34. Benzothiazole, thiazole and isatin based hydroxamic acid analogues

40
A series of 1,3,5-triazine based hydroxamates 72 have been reported as potent inhibitors of HDAC [187] (Fig. 35).

Compounds 72b, 72g and 72i exhibited good HDAC inhibition with IC50 value of 0.72, 0.57 and 0.31 µM,

respectively. On cytotoxic evaluation in three cancer cells, namely HCT116, MCF-7 and HeLa, these compounds

displayed consistent activity profile with the enzyme inhibition profile. Compound 72i also induced apoptosis and

cell cycle arrest at G2/M phase in HCT116 in a dose dependent pattern. Jin et al. [188] have designed a series of

novel indoline-2,3-dione derivatives 73 as HDAC inhibitors (Fig. 35). Most of the derivatives displayed potent

HDAC inhibition in nanomolar concentration with compound 73e showing an IC50 of 10.13 nM. On further

evaluation for cytotoxicity in several cancer cell lines, compound 73e showed the strongest cytotoxicity (IC50 = 0.22

- 5.33 µM). This derivative also demonstrated isoform selectivity against HDAC1 (IC50 = 201.16 nM), HDAC3

(IC50 = 25.01 nM) and HDAC6 (IC50 = 126.48 nM) and potent efficacy in H7402 xenograft mice models.

O O

N N
N N N N
O
N N OH N N O O
O N
O n O OH
H N N
72 H n H

72a: (para) n = 2 ; 72b: (para) n = 3; 72i: n = 6

72c: (para) n = 4; 72d: (para) n = 5;
72e: (meta) n = 2 ; 72f: (meta) n = 3;
72g: (meta) n = 4; 72h: (meta) n = 5.
n
O
O O
O OH
O N
N H
N
R m H

73
73a: R = Cl, m = 1, n = 1 ; 73b: R = Cl, m = 1, n = 2 ;
73c: R = Br, m = 1, n = 1 ; 73d: R = Br, m = 1, n = 2;
73e: R = Br, m = 2, n = 1 ; 73f: R = Br, m = 3, n = 1.

Fig. 35. Triazine and indoline based hydroxamates

41
Tan et al. [189] have designed and synthesized thieno[3,2-d]pyrimidine analogues 74 bearing hydroxamic acid end

group as novel inhibitors of HDAC (Fig. 36). Compounds 74d-f demonstrated potent HDAC inhibition with IC50

value of 0.38, 0.49 and 0.61 µM, respectively. Compound 74d was found to induce apoptosis and cell cycle arrest at

G2/M phase in HCT116 cell lines. Another series of 4-anilinothieno[2,3-d]pyrimidine derivatives 75 have also been

investigated for HDAC inhibitors [190] (Fig. 36). Compound 75b displayed inhibition against HDAC1 (IC50 = 1.14

nM), HDAC3 (IC50 = 3.56 nM) and HDAC6 (IC50 = 11.43 nM). Most of the analogues were also good antitumor

agents against RPM18226 and HCT116 cells, with compound 75b displaying an IC50 value of 2.39 and 1.41 µM,

respectively. The compound also upregulated levels H3 acetylation in comparison to SAHA at the same

concentration. Using benzofuroxan as the surface recognition moiety, Duan et al. [191] have designed and

synthesized a series of hybrids 76 and 77 that served a dual purpose of nitric oxide donor and HDAC inhibition (Fig.

36). Compounds with six carbon chain, 76d, 77c and 77d, were potent in enzyme inhibition in nanomolar levels

with IC50 value of 18.4, 17.0 and 12.8 nM, respectively. The same trend was observed with in vitro antitumor

activity evaluation in various cancer cell lines. The compounds 76d and 77d were found to inhibit the growth of

HCT116 cancer cells and the inhibitory activity was partially diminished by the NO scavenger haemoglobin,

confirming the synergism of HDAC inhibition and NO releasing effect.

42
 O
N
N N S O O
S OH
N NH N N
O 4
R H H
N OH
O N
n H 75
74
75a: R = 3-Cl;
74a: (para) n = 4; 74b: (meta) n = 4; 75b: R = 3,4-Me2;
74c: (para) n = 5; 74d: (meta) n = 5;
74e: (para) n = 6; 74f: (meta) n = 6; 75c: R = 3-F, 4-N(Et)2;
74g: (para) n = 7; 74h: (meta) n = 7. 75d: R = 3-F, 4-N(Me)2;
75e: R = 3-F, 4-
N

O H O N O H
n N n
N N N
O OH O OH
O
N O O

76 77
77a: n = 3; 77b: n = 4;
76a: n = 3; 76b: n = 4;
77c: n = 5; 77d: n = 6;
76c: n = 5; 76d: n = 6;
77e: n = 7.
76e: n = 7.

Fig. 36. Thienopyrimidine and benzofuroxan based hydroxamic acid derivatives

Quinoline based hydroxamates have been developed and screened for HDAC inhibition [192] (Fig. 37). The

compounds were diversified with substituents at position 4 and 6 of the quinoline moiety along with varying number

of methylene as the linker between the cap and the hydroxamic acid end group. Compound 78 with five carbon

chain, halogen substitution at position 6 and hydrogen at position 4 of the quinoline ring, displayed significant

HDAC inhibitory profile (IC50 between 82–155 nM). The chloro-, bromo- and iodo-substituted quinoline analogues

78b-d, on cytotoxic evaluation against MDA-MB-231, PC-3, K562 and A549 cancer cells, were significantly potent.

Besides, these were more cytotoxic against MDA-MB-231 cells (IC50 = 0.90–1.84 µM) when compared to SAHA

(IC50 = 2.02 µM). A series of isoquinoline based HDAC inhibitors have also been designed and synthesised [193]

(Fig. 37). Compound 79 demonstrated good inhibitory profile against HDAC1, 3 and 6. The IC50 values of

derivative 79c against HDAC1, HDAC3 AND HDAC6 were 4.17, 4.00 and 3.77 nM, respectively. The

43
antiproliferative activity of the compounds was assessed against multiple myeloma cells RPMI 8226, HCT116 and

Hep G2 cells. The IC50 value of all the derivatives against RPMI 8226 was below 1µM, more than SAHA (IC50 =

2.39 µM). In addition, on evaluation for in vitro drug safety in terms of cardiac toxicity, no inhibitory effect against

hERG channel was observed. This suggested that the compounds could be non-cardiotoxic compounds for

developing novel anticancer agents.

R2 R
R1 HN H H
H H
5
N N N N
N OH N OH
O 4 O
O O
78 79
78a: R1 = F, R2 = H; 78b: R1 = Cl, R2 = H; 79a: R = 2-F, 4-Br; 79b: R = 2-F, 3-Cl;
78c: R1 = Br, R2 = H; 78d: R1 = I, R2 = H; 79c: R = 4-Me; 79d: R = 3,4 - Ph2;
78e: R1 = Ph, R2 = H. 79e: R = 2-Br, 4-Me; 79f: R = 2-F.

Fig. 37. Quinoline and Isoquinoline based hydroxamic acid analogues

Several other studies reported on hydroxamates as HDAC inhibitors have also led to an advance understanding of

the different facets of medicinal chemistry [194-203].

5. CONCLUSION AND FUTURE PROSPECTS

It is well established that histone deacetylase (HDAC) is a potential target for cancer treatment. Other than cancer,

the therapeutic use of HDAC inhibitors extends to diseases such as microbial diseases, inflammatory diseases,

neurodegenerative, metabolic and autoimmune disorders. Four anticancer agents namely, SAHA, Romidepsin,

Belinostat and Panobinostat have been approved by FDA for the treatment of refractory cutaneous/peripheral T-cell

lymphoma and several candidates are presently in clinical development. In addition, combination therapy of HDAC

inhibitors with different chemotherapeutic drugs and radiation therapy have displayed very good synergism.

Nevertheless, the need for more potent, isoforms specific and tumor specific HDAC inhibitors is mounting due to

the problems of side effects. These toxic effects may be related to the inhibitors acting in multiple pathways or due

to lack of isoform-selective molecules. Moreover, both pan- and class-selective inhibitors have presented almost

similar side effects. In other words, inhibitors with better therapeutic effects and toxicity in lower magnitude are yet

to be realized. Perhaps, a promising strategy would be the effective and continued use of in silico computational

44
techniques that have provided an insight to the crucial interactions between the inhibitors and the target enzyme.

Numerous applications such as molecular docking, pharmacophore modeling, QSAR analysis, lead based or

fragment based design would be of assistance to optimize and fine tune the existing compounds for better lead

molecules and drugs. Together with the knowledge of the essential pharmacophoric assembly, the catalytic

mechanism, the amino acid residues and the size of the internal catalytic zone, several manipulations can be applied

to achieve novel HDAC inhibitors which may be class and/or isoform selective or precise for each tumor. With

further understanding of HDAC inhibitors acting on non-histone or multiple targets, another hopeful approach lies in

exploring the specific mode of action of these inhibitors. Ongoing research with computational tools and

experimental techniques should no doubt facilitate the discovery and development of novel HDAC inhibitors as

anticancer agents in the near future.

CONFLICT OF INTEREST

The authors confirm that this article content has no conflicts of interest.

ACKNOWLEDGEMENT

M.M. would like to thank Department of Science and Technology (DST), Government of India, for the financial

support under DST Women Scientist Scheme, WOS-A (File No: SR/WOS-A/CS-1059/2014).

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 INHIBITORS OF HISTONE DEACETYLASE AS ANTITUMOR AGENTS: A CRITICAL REVIEW

a a a b
Manal Mohammed* , Chandrasekar M J N , Gomathi Priya Jeyapal , Nanjan M J .

a
Department of Pharmaceutical Chemistry, JSS College of Pharmacy (A Constituent College of JSS University,
Mysore), Ootacamund 643 001, TamilNadu, India.
b
TIFAC CORE, JSS College of Pharmacy (A Constituent College of JSS University, Mysore), Ootacamund,
TamilNadu, India.
*Corresponding author

Manal Mohammed,
Department of Pharmaceutical Chemistry,
J. S. S. College of Pharmacy,
Rock lands,
Ootacamund,
Tamil Nadu, India - 643 001.
E-mail: manal_mohd@rediffmail.com

HIGHLIGHTS

• Histone deacetylase (HDAC) is an attractive target of interest for several types of cancer.
• The crystal structure of HDACs and the biological effects mediated by the enzyme are described.
• The chemistry of the molecules of two classes of HDAC inhibitors, short chain fatty acid and hydroxamic
acid, for cancer treatment are covered in detail till date.

GRAPHICAL ABSTRACT

58