Discovery of aliphatic-chain hydroxamates containing indole derivatives with potent class I histone deacetylase inhibitory activities

Accepted Manuscript Discovery of aliphatic-chain hydroxamates containing indole derivatives with potent class I histone deacetylase inhibitory activities Shi-Wei Chao, Liang-Chieh Chen, Chia-Chun Yu, Chang-Yi Liu, Tony Eight Lin, JihHwa Guh, Chen-Yu Wang, Chun-Yung Chen, Kai-Cheng Hsu, Wei-Jan Huang PII:

S0223-5234(17)30995-9

DOI:

10.1016/j.ejmech.2017.11.092

Reference:

EJMECH 9967

To appear in:

European Journal of Medicinal Chemistry

Received Date: 1 August 2017 Revised Date:

11 November 2017

Accepted Date: 28 November 2017

Please cite this article as: S.-W. Chao, L.-C. Chen, C.-C. Yu, C.-Y. Liu, T.E. Lin, J.-H. Guh, C.-Y. Wang, C.-Y. Chen, K.-C. Hsu, W.-J. Huang, Discovery of aliphatic-chain hydroxamates containing indole derivatives with potent class I histone deacetylase inhibitory activities, European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2017.11.092. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.

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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT

Discovery of aliphatic-chain hydroxamates containing indole derivatives with potent class I histone deacetylase inhibitory activities Shi-Wei Chaoa,b, Liang-Chieh Chena,b, Chia-Chun Yuc, Chang-Yi Liud, Tony Eight Lind, Jih-Hwa Guhc, Chen-Yu Wange, Chun-Yung Chenb, Kai-Cheng Hsud*, Wei-Jan Huangb,f,g,h∗ School of Pharmacy, Taipei Medical University, Taipei, Taiwan

b

Graduate Institute of Pharmacognosy, Taipei Medical University, Taipei, Taiwan

c

School of Pharmacy, National Taiwan University, Taipei, Taiwan

d

Graduate Institute of Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan

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a

TaiMed Biologics Inc., Taipei, Taiwan

f

School of Pharmacy, National Defense Medical Center, Taipei, Taiwan

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e

Ph.D. Program for the Clinical Drug Discovery from Botanical Herbs, Taipei Medical

h

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University, Taipei, Taiwan

Ph.D. Program in Biotechnology Research and Development, College of Pharmacy, Taipei Medical University, Taipei, Taiwan

Abstract

Histone deacetylase (HDAC) is a validated drug target for various diseases. This study combined indole recognition cap with SAHA, an FDA-approved HDAC inhibitor used to treat cutaneous T-cell

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lymphoma (CTCL). The structure activity relationship of the resulting compounds that inhibited HDAC was disclosed as well. Some compounds exhibited much stronger inhibitory activities than SAHA. We identified two meta-series compounds 6j and 6k with a two-carbon linker had IC50 values of 3.9 and 4.5 nM for HDAC1, respectively. In contrast, the same oriented compounds with longer

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carbon chain linkers showed weaker inhibition. The result suggests that the linker chain length greatly contributed to enzyme inhibitory potency. In addition, comparison of enzyme-inhibiting activity

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between the compounds and SAHA showed that compounds 6j and 6k displayed higher inhibiting activity for class I (HDAC1, -2, -3 and -8). The molecular docking and structure analysis revealed structural differences with the inhibitor cap and metal-binding regions between the HDAC isozymes that affect interactions with the inhibitors and play a key role for selectivity. Further biological evaluation showed multiple cellular effects associated with compounds 6j- and 6k-induced HDAC inhibitory activity. Keywords: Histone deacetylase; Structure activity relationship; Indole; Isozyme; Molecular docking

*Corresponding authors E-mail: [email protected] (W.-J. Huang) E-mail: [email protected] (K.-C. Hsu) 1

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1. Introduction

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Reversible histone acetylation is balanced by histone deacetylases (HDAC) and histone acetyltransferases (HAT). These epigenetic processes remodel the chromatin

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structure and control gene expression without changing the gene sequence [1]. Acetylation of the lysine tail of nucleosome-related histone neutralizes its positive

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charge, which causes the relaxed chromatin to induce transcriptional expression. In contrast, deacetylation of histone leads to the formation of condensed chromatin,

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which causes transcriptional repression. Deregulation of such epigenetic systems may induce inappropriate gene expression associated with the pathogenesis of certain

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malignancies [2, 3]. These findings suggest HADCs are involved in regulating various cellular events. HDAC inhibitors are known to exhibit anticancer activity in many

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tumor types as well as in-vivo models, and consequently, inhibition of HDACs has been emerged as an attractive strategy in cancer therapeutics [4, 5]. Mammalian HDACs can be classified into four groups according to their sequence homology, sub-cellular distribution, and catalytic activity. Class I (HDAC1, -2, -3, -8), class II (HDAC4, -5, -6, -7, -9, -10), and class IV (HDAC11) enzymes are

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ACCEPTED MANUSCRIPT zinc-dependent HDACs, whereas class III (Sirtuins 1-7) enzymes require NAD+ as a cofactor for activity. Class II enzymes are further subdivided into class IIa (HDAC4,

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-5, -7, -9) and class IIb (HDAC6, -10) [6]. Class I and IV HDACs are ubiquitously expressed in the nucleus. In contrast, class II HDACs are located in some specific

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tissues and shuttle between the nucleus and cytoplasm [7]. Class IIa HDACs are shown to have certain biological functions. For example, HDAC4 performs a

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prominent neurological function and plays a distinct role in synaptic plasticity and memory [8, 9]. HDAC5 reportedly regulates differentiation-specific functions.

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HDAC9, meanwhile, is highly expressed in the brain and skeletal muscle [8]. Study showed that HDAC5- or HDAC9-deficient mice has an increased incidence of the

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cardiac hypertrophy after stress [10]. HDAC7 is highly expressed in the vascular endothelium. HDAC7 deficiency leads to the embryonic lethality in mice due to

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aberrant circulatory development [11]. These experimental facts revealed that inhibiting class IIa HDACs possibly causes some undesirable side effects. In contrast, extensive studies reported that class I enzymes (HDAC1, -2, -3, -8) are involved with cell cycle progression, metastasis, and apoptosis [12]. Moreover, these enzymes are highly expressed in certain cancer cells compared to their corresponding normal cells.

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ACCEPTED MANUSCRIPT Therefore, the anticancer effects of class I HDAC enzymes have been studied intensively.

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Clinical studies have evaluated the use of HDAC inhibitors for treating various solid and hematologic malignancies. These structurally diverse compounds include compounds

such

as

SAHA,

panobinostat,

PCI-24781,

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hydroxamate-based

JNJ-26481585, benzamides such as MS-275 and MGCD0103 and cyclic tetrapeptides

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such as FK228 and apicidin [13]. The hydroxamate-based compounds are considered pan-HDAC inhibitors due to their broad-spectrum inhibition of HDAC isoforms (Fig.

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1). In contrast, the other two groups are classified as class I-selective HDAC inhibitors because they display specific inhibiting effects on HDAC1, -2, -3 enzymes.

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Among these HDAC inhibitors, SAHA and FK228 were approved for the treatment of cutaneous T-cell lymphoma (CTCL) [13, 14] and PXD101 was used to treat refractory

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peripheral T-cell lymphoma (PTCL) [15]. Recently, panobinostat has been approved for use in treating multiple myeloma [16]. However, in the clinical trials, SAHA and PXD101 reportedly cause more side effects for the patients compared to MS275, which suggested targeting class I HDAC may offer wider therapeutic window [14, 17].

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ACCEPTED MANUSCRIPT The chemical features of all HDAC inhibitors can be divided into three groups: a zinc-binding motif (such as a hydroxamic acid), a hydrophobic cavity-binding linker,

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and a surface recognition cap also with hydrophobic properties. Each group interacts with a discrete region of the active site of HDAC (Fig. 1). Studies also indicate that

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modification of the recognition cap moiety may enable recognition of specific HDACs through binding to the rim surface of the enzyme active site, which

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potentially generates selective HDAC inhibitors [18].

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ACCEPTED MANUSCRIPT Figure 1. Examples of pan and class I-selective HDAC inhibitors in clinical trials. Trimethoxyindole exists in various anticancer agents (Fig. 1S). These compounds

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include synthesized combretastatin A-4 derivatives 1-3 [19-21] and a naturally occurring antibiotic, duocarmycin SA [22-24]. Our previous efforts sought to enhance

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the recognition caps that are fused with reported HDAC inhibitor scaffold such as N-hydroxycinnamide and aliphatic hydroxamate [25, 26]. Accordingly, we previously

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developed trimethoxyindole derivative 6d fused with the aliphatic-hydroxamate core of SAHA (Scheme 1). Comparison of the HDAC-inhibiting activities of compound 6d

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and SAHA indicated that compound 6d displayed higher HDAC1 activity [27]. In addition to its potent enzyme inhibition, compound 6d, in human hormone-refractory

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prostate cancer cells, also significantly affected cell cycle regulators as well as apoptotic signaling, which are induced by its anti-HDAC activity. These experimental

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results revealed such modifications can increase HDAC specificity and produces improved HDAC-related cellular effects. Moreover, the trimethoxyindole of compound 6d structurally resembles the indole cap used for HDAC inhibitors LBH-589, PCI-24781, and JNJ-26481585 currently tested in clinical trials (Fig. 1). Despite its strong anti-HDAC effect, the structure activity relationship of compound

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ACCEPTED MANUSCRIPT 6d is still not disclosed. These results further motivated us to extensively investigate the effect of the chemical motif of 6d-derived compounds on HDAC-inhibiting

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activity such as the orientation of indole moiety added to aliphatic hydroxamate, the linker carbon-chain length between the cap and zinc-binding group, and the

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substituent of indole ring.

Using compound 6d as a lead, this study incorporated varied indole moieties into

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the core structure of SAHA via various carbon linker-chain lengths at different positions (Scheme 1). The HDAC inhibitory activities of the resulting compounds

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were evaluated, which leads to identify their structure activity relationship. Several meta-substituted compounds had higher anti-HDAC activity compared to SAHA. Test

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of cytotoxicity revealed their cytotoxicity against human prostate cancer PC-3 cells was comparable to that of SAHA. Notably, two of these compounds, designated

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compounds 6j and 6k, showed much higher potency than SAHA in class I HDAC-1, -2, -3 and -8. Further assays indicated that both compounds had class IIb HDAC6 inhibitory activities compatible to SAHA, but with only slight effects on most class IIa isoforms. Analysis of the binding pocket revealed structural differences between the HDAC isozymes. Further experiments were performed to profile acetylation of

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ACCEPTED MANUSCRIPT histone and α-tubulin, cell cycle progression, and gene expression and revealed the cellular effects that are relevant to the HDAC inhibition of the resulting compounds.

H3CO

H3CO

OCH3 N

O 2O

N H

O 6

N H

OCH3

H3CO

OH

N

H N nO

H N

6

O

OH

O

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H3CO

n=2-4

6d

N

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R

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ortho-, meta-, para-(6a-i)

2O

O N H

O

N H n=4, 6-8 n

6j-r

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Scheme 1. Design of indole-containg aliphatic hydroxamates derived from compound 6d

8

OH

ACCEPTED MANUSCRIPT 2. Chemistry The hydroxamates 6a-6o were synthesized as described in Scheme 2.

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Monomethyl suburate was reacted with oxalyl chloride to produce the activated acid chloride in situ. Treatment with ortho-, meta- and para-aminophenol 1a-1c then gave

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corresponding methyl esters 2a-2c. Reaction of compounds 2a-2c with the appropriate alkyl bromides, such as 2-chloroethyl, 3-chloropropyl and 4-chlorobutyl,

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provided corresponding 3a-3i. Trimethoxyindole was synthesized according to the reaction approach as previously reported [27]. Compounds 3a-3i coupled with various

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indoles yielded compounds 4a-4o, respectively. Saponification of compounds 4a-4o in the presence of LiOH gave corresponding acids 5a-5o in quantitative yields. To

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activate compounds 5a-5o, the corresponding mixed anhydrides were generated by

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using ethyl chloroformate prior to reaction with NH2OH to give hydroxamates 6a-6o.

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ACCEPTED MANUSCRIPT Scheme 2

HO

6

O

H N

a

O O

NH2

O

HO

H N

b

O

6

O

Br

Cl n

Cl

O

nO

H N

O

6

O

H N

d

O

R

O N

O

O

R

N

O

N

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O

N

Br N

O

nO

H N

6

OH

O

6a-o

O

O

H N

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:

e

5a-o

O R

6

O

nO

4a-o

OH

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n=2-4

1a: o-aminophenol 1b: m-aminophenol 1c: p-aminophenol

nO

O

3a-i

2a-c HO

R

c

O

6

N

N

O

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Reagents and conditions: (a) 1) (COCl)2, CH2Cl2, RT; 2) DMF, 60 oC, N2, 42-46%; (b) K2CO3, MeCN, ∆, N2, 79-99%; (c) substituted indole or indole, K2CO3, DMF, RT, N2, 25-95%; (d) LiOH, MeOH, 95-99%; (e) 1) ClCO2Et, Et3N, THF, RT; 2) NH2OH-HCl, KOH, MeOH, 56-99%

The meta-substituted hydroxamates 6p-6r were synthesized as described in

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Scheme 3. Compounds 7a-7c reacted with oxalyl chloride prior to coupling with compound 1b gave 8a-c, respectively. Using the same method to compounds 6a-6o,

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compounds 6p-6r were synthesized starting from compounds 8a-8c. Details on the synthesis, isolation and characterization of compounds 2-5 and 8-9 can be found in the supplementary material. The estimated purity of compounds 6a-6r is at least 97% as determined by HPLC analysis (supplementary material).

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ACCEPTED MANUSCRIPT Scheme 3

O

H N

a

O

n

O

O

7a-c n=4, 7, 8

O

n O

N

Br

H N O

n O

N

n O

O

Cl

O

c

9a-c

d

O

O

N

H N

O

O

n O

OH

e

4p-r

4p-r

O

H N

O

Cl

8a-c

OH

O

b

O

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HO

H N

O

H N

OH

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O

n O

6p-r

3. Results and Discussion

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Reagents and conditions: (a) 1) (COCl)2, CH2Cl2, RT; 2) 1b, DMF, 60 oC, N2, 65-87%; (b) K2CO3, MeCN, ∆, N2, 8897%; (c) 5-Methoxyindole, K2CO3, DMF, RT, N2, 80-97%; (d) LiOH, MeOH, 97-98%; (e) 1) ClCO2Et, Et3N, THF, RT; 2) NH2OH-HCl, KOH, MeOH, 79-89%

3.1 HeLa nuclear HDAC inhibitory activity

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The enzyme inhibitory activities of all synthesized compounds 6a-6m against HeLa nuclear HDACs were tested using SAHA as a reference (Table 1). The activities

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of compounds 6a-6c with ortho-substituted trimethoxyindole moiety were

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approximately 2-fold lower than that of SAHA, which indicates that the ortho substitution weakens the enzyme binding affinity. The activities of para-substituted compounds 6g and 6i were comparable to that of SAHA, suggesting that para-substituted indole slightly enhances enzyme inhibition. In contrast, the activities of meta-substituted compounds 6d-6f were approximately two- to five-fold higher than that of SAHA. We speculate that the positive contribution of meta-substitution 11

ACCEPTED MANUSCRIPT inhibitory activity was due to increased contact between the indole motif and the rim of HDAC enzyme active site. Comparisons of the meta-series 6d-6f indicated that

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compound 6d had the strongest activity, which suggested that a two-carbon linker chain between the benzene and the recognition cap produced optimal anti-HDAC

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effect. Next, compounds 6j-6m were synthesized with different indoles at the meta-position using a two carbon chain length. These four compounds also showed

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either an equivalent or higher potency compared to compound 6d. Notably, the enzyme-inhibiting activity of compound 6k was within a range of single digit

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nanomolar. In general, we observed that compounds that contained at least a two-carbon linker and indoles at the meta-position had better inhibiting activity. Table 1

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IC50 value for the inhibition against HeLa nuclear HDACa (nM) and cytotoxicity

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against cancer cells (µM) by compounds 6a-m H N

H N 6

N

R

nO

O

OH

O

Substitution

Chain

Substitution

position

length (n)

(R)

6a

ortho

2

5,6,7-TriOMe

109.9±1.2

5.6

5.9

6b

ortho

3

5,6,7-TriOMe

111.0±5.2

3.9

5.0

6c

ortho

4

5,6,7-TriOMe

111.4±9.8

2.4

4.8

6d

meta

2

5,6,7-TriOMe

11.5±0.2

1.3

2.4

6e

meta

3

5,6,7-TriOMe

22.5±0.8

2.7

2.7

Compound

12

HDAC

PC-3

A549

ACCEPTED MANUSCRIPT meta

4

5,6,7-TriOMe

17.8±1.9

2.1

2.4

6g

para

2

5,6,7-TriOMe

57.2±3.3

4.4

4.9

6h

para

3

5,6,7-TriOMe

138.6±1.1

6.9

5.9

6i

para

4

5,6,7-TriOMe

46.4±0.6

7.7

9.7

6j

meta

2

5,6-DiOMe

11.2±1.3

1.2

6.4

6k

meta

2

5-OMe

8.6±0.7

1.3

1.7

6l

meta

2

6-OMe

14.9±0.1

1.8

4.2

6m

meta

2

7-OMe

13.5±0.4

1.4

1.9

51.6±3.6

1.5

1.7

SAHA Data was obtained from three independent experiments.

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a

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6f

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We further synthesized meta-substituted compounds 6n-6o to investigate the effect of indole’s substituent on HDAC inhibition. Table 2 shows that compared to compound 6n with unsubstituted indole, compound 6o with bromo-substituted indole

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exhibited lower enzyme inhibition, which suggested that an electron-withdrawing group on indole negatively contributed to the activity. However, compound 6k

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showed greater activity, which suggested that an electron-donating group

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strengthened the activity. Finally, this study synthesized compounds 6p-6r with the linker-chain length of 4, 7, and 8 carbons, respectively, between the amide and hydroxamate group. Comparisons of the enzyme-inhibiting activities between compounds 6k and 6p-6r show that compound 6k had the most potent activity, which suggested that for this series of compounds, the optimal length of linker-chain is six carbons. 13

ACCEPTED MANUSCRIPT Table 2. IC50 value for the inhibition against HeLa nuclear HDACa (nM) by compounds 6n-6r H N

O

N

O

OH

Chain length (n)

Substitution (R)

6n

6

H

6o

6

5-Br

6p

4

5-OMe

185.8±10.3

6q

7

5-OMe

40.0±3.2

6r

8

5-OMe

298.0±14.2

6k

6

5-OMe

8.6±0.7

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Compound

SAHA a

n O

H N

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R

HDAC

23.1±0.3

41.3±0.9

51.6±3.6

Data was obtained from three independent experiments.

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3.2 Cytotoxicities of potential inhibitors

The cell growth inhibiting effects of compounds 6a-6m were compared in human

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prostate cancer PC-3 cells and lung cancer A549 cells. Table 1 showed that, in both cancer cell lines, all compounds except 6c and 6i had cytotoxic effects consistent with

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the HDAC enzyme inhibition. In contrast to its weak anti-HDAC activity, the cytotoxicity of compound 6c was moderate. The experimental result suggested that, in addition to HDAC, compound 6c likely affects other cell death-related cellular events in the signaling pathway. Although the enzyme inhibitory activity of compound 6i was comparable to that of SAHA, compound 6i had much lower cytotoxicity than

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ACCEPTED MANUSCRIPT SAHA. Compound 6j preferentially inhibited PC-3 cells (IC50=1.2 µM) over A549 cells (IC50= 6.4 µM) when compared to SAHA. The HDAC enzyme inhibitory

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activities of compounds 6d and 6k-6m were much higher than that of SAHA; however, their cytotoxicities were only comparable or equivalent to that of SAHA.

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This may be due to their poor cell membrane permeability. 3.3 HDAC isoforms inhibition

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Next, we selected four compounds (6d, 6f, 6j, and 6k) that showed high anti-HeLa nuclear HDAC activity and determined their enzyme inhibiting effects

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against a panel of HDAC isoforms. These isozymes include class I HDACs 1, 2, 3, 8, class IIa HDACs 4, 5, 7, 9, and Class IIb HDAC6. Table 3 shows that the HDAC1

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inhibiting effects of most compounds were superior to that of SAHA. For example, the activities of compounds 6j and 6k were 14- and 16-fold higher than that of

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SAHA[28]. Similarly, they also had stronger anti-HDAC8 effect than did SAHA with four- to eight-fold greater activities. The activities of compounds 6d, 6j and 6k against HDAC2 were approximately two-, twelve-and seven-fold higher than that of SAHA, respectively; however, the activity of compound 6f was five-fold lower. Compared to SAHA, compounds 6d, 6j and 6k had higher or comparable potency

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ACCEPTED MANUSCRIPT against HDAC3, whereas compound 6f had lower potency. The compounds showed weak activity against class IIa HDACs 4, 5, 7, and 9 (Table 3). Moreover, increasing

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concentrations as high as 10 µM reveal low inhibitory effects. In contrast, the HDAC6 inhibitory activities of compounds 6d, 6f, 6j and 6k were comparable to that of

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SAHA (Table 3). These experimental results indicated the methoxy group when added

and 8. Table 3

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to the indole cap can affect the enzyme-inhibiting activity in class I HDACs 1, 2, 3

IC50a (nM) value for the inhibition against class I and class II HDACs by compounds 6d, 6f, 6j and 6k Compound

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Class I HDAC1 HDAC2 HDAC3

HDAC8

Class II

HDAC4 HDAC5 HDAC7 HDAC9 HDAC6

11.1±1.7 111.2±0.2 72.5±0.5 420.9±3.6 >10000

>10000

>10000 >10000 9.6±0.2

6f

29.3±0.9 999.6±5.9 108.2±0.4 752.0±5.2 >10000

>10000

>10000 >10000 8.5±0.7

6j

3.9±0.3 16.6±1.4 29.7±0.7 398.3±2.3 >10000

>10000

>10000 >10000 7.1±1.0

6k

4.5±0.1 33.1±0.5 35.3±0.2 560.9±1.2 >10000

>10000

>10000 >10000 5.9±2.0

>10000

>10000 >10000 9.1±0.3

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SAHA a

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6d

63.5±1.9 214.0±0.1 72.8±1.8 3400.1±6.2 >10000

Each value is obtained by three independent experiments.

A phylogenetic tree based on the sequence homology between HDAC isozymes separates the HDACs by class (Fig. 2S). When the IC50 values of the identified compounds were placed onto the trees, a bias for class I and HDAC6 isozymes was 16

ACCEPTED MANUSCRIPT observed. This suggested a possible discrepancy between the isozyme structures that determines inhibitor potency. Therefore, we identified inhibitors that are specific for

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class I HDACs.

3.4 Molecular docking analysis

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We performed an interaction analysis to determine key interacting residues between the strongest (6j) and the weakest (6f) inhibitors for HDAC1 (Table 3). The

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inhibitors were docked into HDAC1 (PDB ID: 4KBX) using the software SYBYL [29] to obtain the binding conformations. The interaction analysis between the enzyme

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residues and inhibitors were determined using the program iGEMDOCK [30]. The modeling of compounds 6j and 6f showed the locations of the indole cap along the

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surface (Fig. 2A-B). Superimposing the two compounds in HDAC1 showed different spatial positions in the surface (Fig. 2C). Within the binding site, the two compounds

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created similar hydrogen bonds with residues H140, H141, D176, H178, D264, and Y303 (Fig. 2D-E). This is due to their hydroxamate moiety. In contrast, compounds 6j and 6f formed different van der Waals interactions with surface residues (Fig. 2C, 2E). Compound 6f contains a four-carbon linker chain. The longer linker allowed the indole of compound 6f to display a higher degree of rotation to form interactions with

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ACCEPTED MANUSCRIPT residues H28 and P29. Compound 6j with a two-carbon linker positioned the indole to form van der Waals interactions with residues H28 and P29 (Fig. 2C). These results

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suggested that the linker length and the interactions between the indole cap and

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surface residues H28 and P29 are important for the potency of the inhibitors.

Figure 2. Molecular docking analysis of inhibitors 6f and 6j in HDAC1. Compound

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6f and 6j docked into HDAC 1 (PDB ID: 4BKX) crystal structure. (A-B) Surface view of compound 6j (yellow) and compound 6f (purple) docked in HDAC 1. (C-D) Compounds 6j (yellow) and compound 6f (purple) docking pose stick view of the HDAC1 (grey) active site reveal interactions with residues. (E) Compounds 6f and 6j were superimposed in HDAC 1 catalytic center. Residues contributed hydrogen bond

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ACCEPTED MANUSCRIPT are colored in green, residues contributed van der Waals interaction are colored in brown. H178 contain hydrogen donor and pi-donor is colored in yellow. (F) Heat map

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of corresponding residue interactions and compounds 6j and 6f. Hydrogen bonds are represented in shades of green and van der Waals in shades of grey.

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3.5 Structural differences between HDAC isozymes

To determine discrepancies between residue interactions and HDAC class

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potency, we compared and analyzed the HDAC isozyme structures. As seen in Table 3, the identified inhibitors show a low IC50 value for class I isozymes, but a high value

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for class IIa isozymes. The identified compounds had a IC50 value comparable to SAHA for HDAC6, a class IIb isozyme (Table 2). HDAC sequences was obtained

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from Uniprot database and a multiple sequence alignment was performed in Jalview [31]. We observed that the histidine residue is relatively conserved across the HDAC

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family; however, the tyrosine and histidine amino acid differs between class I and class IIa HDAC, respectively (Fig. 3A). While residues were relatively conserved, the surface view of the binding site revealed different cavity sizes between the HDAC classes. The crystal structures for HDAC5, 9 and 10 are currently unavailable; as a result, structural analysis and modeling of these structures was not performed. Class I

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ACCEPTED MANUSCRIPT and HDAC6 contain a tyrosine residue, whereas class IIa HDACs contain a histidine residue in the same location [8, 32]. The aromatic ring of the tyrosine residue extends

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into the binding site and creates a narrow pocket for the inhibitor (Fig. 3B). Furthermore, our interaction analysis revealed a hydrogen bond between the tyrosine

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residue and inhibitor 6j (Fig. 2F). The amount of interactions for inhibitor 6j is reduced in class IIa HDACs due to the histidine residue substitution, which produces

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a larger binding site (Fig. 3C). The difference in residues between class IIa and class I and HDAC6 is further amplified when the HDAC isozymes are aligned with inhibitor

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6j (Fig. 3D). The sequence alignment of HDAC8 showed that it contains a leucine and alanine instead of the conserved histidine and proline residues for class I HDAC

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(Fig. 3A). This may explain why the inhibitors and SAHA produced a higher IC50

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value compared to the other class I HDACs (Table 3).

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Figure

3.

Interaction

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ACCEPTED MANUSCRIPT

analysis

of

inhibitors

6f

and

6j.

(A)

Multiple Sequence Alignment (MSA) of HDAC family. Residues were aligned to

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HDAC1. Amino acids labeled using Crustal X color scheme. (B) The docking pose of inhibitor 6j in HDAC1 (yellow) was aligned with class I and class IIb HDAC6 and (C)

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class IIa HDAC4 and HDAC7. (D) All HDAC isozyme structures used previously are

AC C

aligned. Red circle highlights contrasting residues in alignment. HDACs color is labeled as shown.

We next looked at specific indole interactions with surface residues. The HDAC1

and inhibitor 6j complex was aligned to class I isozymes and HDAC6 (Fig. 4A). The inhibitor cap is situated near the HDAC1 residue H28. In contrast, when the 6j-HDAC1 complex is superimposed to class IIa HDACs, the histidine residue is 21

ACCEPTED MANUSCRIPT located on a different plane (Fig. 4B). Alignment of available HDAC structures reveals the histidine residue of class IIa situated away from class I and HDAC6

RI PT

isozymes (Fig. 4C). When the surface is compared, the histidine of HDAC1 forms interactions with the inhibitor cap, whereas HDAC4 contains a non-interacting pocket

SC

due to its histidine residue location (Fig. 4D-E). This observation accounts for the

AC C

EP

TE D

M AN U

increased potency of the compound 6j for class I isozymes and HDAC6.

Figure 4. Surface residues interacts with the inhibitor cap. The surface residues of class I and HDAC6 (A) and class IIa (B) isozymes, as described in Figure 3, are compared. (C) Alignment of all HDAC structures used with Inhibitor 6j docked in HDAC1, were transposed. Red circle denotes contrasting histidine locations between 22

ACCEPTED MANUSCRIPT isozymes. HDACs labeled as shown. The surface model of surface residues of HDAC 1 (D) and HDAC4 (E) reveal non-interacting pocket (red circle).

RI PT

3.6 Hyperacetylation of histone and α-tubulin Compounds 6j and 6k at various concentrations (0.1, 0.3, 1, 3 and 10 µM) were

SC

compared in terms of their enhancement of acetylation of histone and α-tubulin, which are two biomarkers for intracellular HDAC inhibition. Figure 5 shows that, in

M AN U

PC-3 cells, both compounds promoted histone acetylation in a dose-dependent manner. Notably, compounds 6j and 6k at concentrations as low as 1 µM induced higher levels of histone acetylation than did SAHA. In addition to histone acetylation, similar

TE D

concentrations of compounds 6j and 6k enhanced α-tubulin acetylation. The potency

EP

of their enhancing effects was comparable to that of SAHA. These experimental data had strong correlations with the HDAC isoform inhibitory activities of compounds 6j

AC C

and 6k. However, in A-549 cells, compound 6k slightly induced histone acetylation when concentration was increased up to 10 µM (Fig. 3S), which confirmed its preferential cytotoxicity towards PC-3 cells.

23

RI PT

ACCEPTED MANUSCRIPT

Figure 5. Effect of compounds 6j and 6k on induction of histone acetylation and

SC

α-tubulin acetylation in cultured human prostate cancer PC-3 cells. Cells were incubated with the HDAC inhibitors at the indicated concentrations for 16 h. The

M AN U

whole cell lysates were analyzed for histone and α-tubulin acetylation by SDS-PAGE and Western blot for either acetylated histone or α-tubulin.

TE D

3.7 Cell cycle progression

HDACs have been shown to regulate the cell cycle [12]. The effects of

EP

compounds 6j and 6k on cell cycle progression were then investigated by flow cytometry. Different concentrations (1, 3, and 10 µM) of the two compounds were

AC C

tested in PC-3 cells (Fig. 6A). After 24 h incubation, compounds 6j and 6k increased the G2/M population in a dose-dependent manner (Fig. 6B). (A)

24

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

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TE D

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(B)

Figure 6. Effect of compounds 6j and 6k on cell cycle progression in cultured human prostate PC-3 cells. (A) Cells were treated without (ctrl) or with the HDAC inhibitors at the indicated concentrations for 24 h and were analyzed by flow cytometry for cell cycle distribution. (B) Data were presented by mean value of three independent experiments. 25

ACCEPTED MANUSCRIPT

3.8 Gene expression

RI PT

The HDAC inhibitors reportedly affect a spectrum of genes in transformed cells [33]. To compare the effects of compounds 6j, 6k, and SAHA on gene expression

SC

associated with cell proliferation, these three compounds were evaluated for the levels of representative genes in human prostate cancer PC-3 cells and lung cancer A549

M AN U

cells. These genes included p21 (inducing cell cycle arrest) [34], CCND1 (inducing cell cycle progression) [35], RARβ (mediating differentiation and growth arrest) [36],

TE D

TNFα (inducing cell apoptosis) [38], Bcl-2 (inhibiting cell apoptosis) [40], CDH15 [42] (inhibiting migration) as well as TIMP3 [44-46] and CDH1 [47] (supressing

EP

tumor growth). While we observed high RARβ levels induced by compound 6k in PC3 cells, the levels of most genes in PC-3 and A549 cell lines by compounds 6j and

AC C

6k were comparable to those by SAHA (Fig. 4SA). In A549 cells, the TNFα levels induced by compounds 6j and 6k were higher than the TNFα levels induced by SAHA (Fig. 4SB). The levels of compound 6k were the highest. Additionally, compounds 6j and 6k were comparable to SAHA in terms of enhancing effects on TIMP3 expression and CDH1 expression in A549 cells (Fig. 4SC), In contrast, compound 6j, 6k and

26

ACCEPTED MANUSCRIPT SAHA in PC-3 cells showed no significant mediating effects on TNFα, TIMP3 and CDH1 expression (data not shown).

RI PT

4. Conclusion This study synthesized a novel series of aliphatic hydroxamates by incorporating

SC

varied indoles as a recognition cap into SAHA core. The structure activity relationship of the resulting compounds in HDAC-inhibiting activity was extensively disclosed.

M AN U

These findings showed the favored orientation of indole cap, the optimal carbon linker-chain length between the cap and zinc-binding group and the effect of the

TE D

substituent of indole ring in inhibiting HDAC. In class I HDAC enzymes, several of these compounds had much higher enzyme inhibition compared to SAHA. Thus, they

EP

are identified to be potent class I HDAC inhibitors. Notably, two of these compounds, namely 6j and 6k, significantly induced histone and tubulin acetylation.

AC C

Additionally, both compounds arrested cell cycle progression in G2/M phase in a dose-dependent manner. Further mechanism studies associated with the identified inhibitors are being investigated.

27

ACCEPTED MANUSCRIPT 5. Experimental 5.1 General procedures

RI PT

The IR spectra (KBr disk) were recorded using a Jasco Fourier Transform infrared spectrometer (Jasco FT/IR-410). The 1H-NMR spectrum was obtained on a Bruker

SC

AV400 or AV500 spectrometer using standard pulse programs. Melting point was recorded on Fisher-Johns apparatus (uncorrected). The MS data were measured on

M AN U

JEOL JMX-HX110 mass spectrometer (HREIMS and HRFABMS), JMS-SX102A mass spectrometer (EIMS and FABMS), and Finnigan Mat TSQ-7000 mass

TE D

spectrometer (ESIMS). The TLC analyses were performed on silica gel plates (KG60-F254, Merck). The microplate spectrophotometer Victor 2x (Perkin Elmer,

EP

Fremont, CA, USA) was used for fluorometric analysis. Unless otherwise mentioned, all chemicals and materials were used as received from commercial suppliers without

AC C

further purification. Dichloromethane was distilled from calcium hydride under nitrogen. Tetrahydrofuran was distilled from sodium and benzophenone under N2. 5.2 N-Hydroxy-7-(2-(2-(5,6,7-trimethoxy-1H-indol-1-yl)ethoxy)phenylcarbamoyl)heptana mide (6a)

28

ACCEPTED MANUSCRIPT After adding KOH (224 mg, 3.98 mmol) to a solution of NH2OH-HCl (277 mg, 3.98 mmol) in MeOH (5 mL), the resulting solution was stirred in an ice bath for 1 h.

RI PT

Free NH2OH in MeOH solution was obtained by filtering out the white salt. A solution of 5a (120 mg, 0.24 mmol) in freshly distilled THF (20 mL) was treated with

SC

ethyl chloroformat (0.047 mL, 0.48 mmol) and triethylamine (0.066 mL, 0.48 mmol), and the resulting solution was stirred at RT for 1 h. The prepared free NH2OH

M AN U

solution was then added to the reaction. After 3 h stirring, the reaction mixture was concentrated in vacuo, and the residue was diluted with distd H2O (50 mL), acidified

TE D

with 1N HCl to pH 2~3 and extracted with EtOAc (50 mL x 3). The organic layer was dried over Na2SO4, and the solvent was removed in vacuo. The residue was purified

EP

by silica gel chromatography (CH2Cl2: MeOH =47:3) to give 6a (124 mg, 56%) as a solid. Mp 95-100 oC. 1H NMR (500 MHz, DMSO-d6): δ 10.32 (s, 1H), 8.64 (s, 1H),

AC C

8.48 (s, 1H), 7.93 (d, J = 7.4 Hz, 1H), 7.40 (d, J = 2.7 Hz, 1H), 6.97 (d, J = 3.8 Hz, 2H), 6.86 (t, J = 5.4 Hz, 2H), 6.29 (d, J = 3.0 Hz, 1H,), 4.67 (t, J = 4.7 Hz, 2H), 4.27 (t, J = 4.7 Hz, 2H), 3.94 (s, 3H), 3.76 (s, 3H), 3.75 (s, 3H), 2.32 (t, J = 7.2 Hz, 2H), 1.95 (t, J = 7.2 Hz, 2H), 1.54 (p, J = 7.0 Hz, 2H), 1.51 (p, J = 7.0 Hz, 2H), 1.30 (m, 4H).

13

C NMR (125 MHz, DMSO-d6): δ 171.2, 169.6, 154.4, 148.9, 140.2, 138.6,

29

ACCEPTED MANUSCRIPT 133.3, 130.7, 125.5, 123.2, 121.2, 115.0, 101.5, 98.4, 68.3, 62.0, 61.3, 56.5, 47.5, 36.7, 32.8, 28.9, 25.5. HR-ESI-MS m/z: (M+Na)+ caclcd for C27H35N3O7Na 536.2373,

RI PT

found 536.2377. 5.3

SC

N-Hydroxy-7-(2-(3-(5,6,7-trimethoxy-1H-indol-1-yl)propoxy)phenylcarbamoyl)hepta namide (6b)

M AN U

Following the procedure as described for 6a, reaction of 5b (311 mg, 0.61 mmol) in THF (20 mL) with ethyl chloroformate (0.12 mL, 1.22 mmol) and triethylamine

TE D

(0.17 mL, 1.22 mmol) gave 6b (301 mg, 94%) as a solid. Mp 55-60 oC. 1H NMR (500 MHz, DMSO-d6): δ 10.29 (s, 1H), 8.79 (s, 1H), 8.62 (s, 1H), 7.80 (d, J = 7.7 Hz, 1H),

EP

7.16 (d, J = 3.0 Hz, 1H), 7.01 (t, J = 7.6 Hz, 1H), 6.91 (d, J = 8.0 Hz, 1H), 6.87 (t, J =7.8 Hz, 1H), 6.83 (s, 1H), 6.26 (d, J = 2.9 Hz, 1H), 4.43 (t, J = 6.7 Hz, 2H), 3.92 (s,

AC C

3H), 3.89 (t, J = 6.0 Hz, 2H), 3.76 (s, 3H), 3.72 (s, 3H), 2.36 (t, J = 7.2 Hz, 2H), 2.19 (p, J = 6.2 Hz, 2H), 1.91 (t, J = 7.3 Hz, 2H), 1.57 (p, J = 7.0 Hz, 2H), 1.47 (p, J = 7.4 Hz, 2H), 1.26 (4H, m). 13C NMR (125 MHz, DMSO-d6): δ 171.5, 170.0, 154.8, 148.8, 140.2, 138.5, 133.0, 130.3, 125.5, 123.2, 121.2, 115.0, 101.4, 98.3, 65.3, 62.1, 61.3, 56.5, 46.2, 45.2, 36.7, 32.7, 31.4, 28.9, 25.6. HR-ESI-MS m/z: (M+Na)+ caclcd for

30

ACCEPTED MANUSCRIPT C28H37N3O7Na 550.2529, found 550.2525. 5.4

RI PT

N-Hydroxy-7-(2-(4-(5,6,7-trimethoxy-1H-indol-1-yl)butoxy)phenylcarbamoyl)heptana mide (6c)

SC

Following the procedure as described for 6a, reaction of 5c (395 mg, 0.75 mmol) in THF (20 mL) with ethyl chloroformate (0.15 mL, 1.50 mmol) and triethylamine

M AN U

(0.21 mL, 1.50 mmol) gave 6c (320 mg, 81%) as a solid. Mp 65-68 oC. 1H NMR (500 MHz, DMSO-d6): δ 10.30 (s, 1H), 8.77 (s, 1H), 8.62 (s, 1H), 7.81 (d, J = 7.6 Hz, 1H),

TE D

7.19 (d, J = 3.0 Hz, 1H), 7.02 (t, J = 7.6 Hz, 1H), 6.96 (d, J = 7.7 Hz, 1H), 6.86 (t, J = 7.6 Hz, 1H), 6.83 (s, 1H), 6.27 (d, J = 3.0 Hz, 1H), 4.29 (t, J = 7.0 Hz, 2H), 3.99 (t, J

EP

= 6.1 Hz, 2H), 3.89 (s, 3H), 3.76 (s, 3H), 3.73 (s, 3H), 2.29 (t, J =7.1 Hz, 2H), 1.91 (t, J = 7.4 Hz, 2H), 1.88 (t, J = 7.7 Hz, 2H), 1.71 (p, J = 6.4 Hz, 2H), 1.50 (p, J = 6.6 Hz,

AC C

2H), 1.45 (p, J = 7.0 Hz, 2H), 1.23 (m, 4H). 13C NMR (125 MHz, DMSO-d6): δ 171.3, 169.7, 154.8, 148.8, 140.2, 138.4, 132.9, 130.2, 125.5, 123.2, 121.1, 114.9, 101.2, 98.3, 67.7, 61.9, 61.3, 56.5, 48.0, 36.7, 32.8, 28.9, 28.5, 26.6, 25.6. HR-ESI-MS m/z: (M+Na)+ caclcd for C29H39N3O7Na 564.2686, found 564.2682. 5.5

31

ACCEPTED MANUSCRIPT N-Hydroxy-7-(3-(2-(5,6,7-trimethoxy-1H-indol-1-yl)ethoxy)phenylcarbamoyl)heptana mide (6d)

RI PT

Following the procedure as described for 6a, reaction of 5d (329 mg, 0.66 mmol) in THF (20 mL) with ethyl chloroformate (0.12 mL, 1.32 mmol) and triethylamine

SC

(0.18 mL, 1.32 mmol) gave 6d (277 mg, 82%) as a solid. Mp 105-108 oC. 1H NMR (500 MHz, DMSO-d6): δ 10.30 (s, 1H), 9.77 (s, 1H), 8.62 (t, J = 0.9 Hz, 1H), 7.26 (s,

M AN U

1H), 7.23 (d, J = 3.1 Hz, 1H), 7.13 (t, J = 8.2 Hz, 1H), 7.07 (d, J = 8.2 Hz, 1H), 6.84 (s, 1H), 6.55 (dd, J = 2.1, 8.1 Hz, 1H), 4.61 (t, J = 5.4 Hz, 2H), 4.20 (t, J = 5.4 Hz, 2H), 3.94 (s, 3H), 3.77 (s, 3H), 3.75(s, 3H), 2.24 (t, J = 7.4 Hz, 2H), 1.92 (t, J = 7.4

TE D

Hz, 2H), 1.52 (p, J = 6.8 Hz, 2H), 1.47 (p, J = 7.0 Hz, 2H), 1.25 (m, 4H). 13C NMR

EP

(125 MHz, DMSO-d6): δ 171.8, 169.6, 158.8, 148.9, 141.0, 140.2, 138.6, 130.7, 130.0, 125.5, 123.2, 112.1, 109.2, 106.0, 101.5, 98.4, 68.0, 62.0, 61.3, 56.5, 47.5, 36.9, 32.8,

AC C

28.9, 25.5. HR-ESI-MS m/z: (M+Na)+ caclcd for C27H35N3O7Na 536.2373, found 536.2369. 5.6 N-Hydroxy-7-(3-(3-(5,6,7-trimethoxy-1H-indol-1-yl)propoxy)phenylcarbamoyl)hepta namide (6e)

32

ACCEPTED MANUSCRIPT Following the procedure as described for 6a, reaction of 5e (711 mg, 1.39 mmol) in THF (20 mL) with ethyl chloroformate (0.27 mL, 2.78 mmol) and triethylamine

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(0.39 mL, 2.78 mmol) gave 6e (566 mg, 77%) as a solid. Mp 108-110 oC. 1H NMR (500 MHz, DMSO-d6): δ 10.31 (s, 1H), 9.80 (s, 1H), 8.63 (s, 1H), 7.15 (t, J = 8.2 Hz,

SC

2H), 7.13 (s, 1H), 7.07 (d, J = 8.0 Hz, 1H), 6.83 (s, 1H), 6.56 (dd, J = 3.9, 8.0 Hz, 1H), 6.26 (d, J = 3.0 Hz1H,), 4.39 (t, J = 6.9 Hz, 2H), 3.93 (s, 3H), 3.85 (t, J = 6.0 Hz, 2H),

M AN U

3.76 (s, 3H), 3.74 (s, 3H), 2.26 (t, J = 7.4 Hz, 2H), 2.15 (p, J = 6.2 Hz, 2H), 1.93 (t, J = 7.3 Hz, 2H), 1.53 (p, J = 6.8 Hz, 2H), 1.48 (p, J = 7.1 Hz, 2H), 1.26 (m, 4H). 13C

TE D

NMR (125 MHz, DMSO-d6): δ 171.8, 169.6, 159.2, 148.8, 141.0, 140.2, 138.5, 130.2, 129.9, 125.5, 123.2, 111.9, 109.3, 106.0, 101.4, 98.4, 65.1, 62.0, 61.2, 56.5, 45.3, 36.9,

EP

32.8, 31.4, 28.9, 25.5. HR-ESI-MS m/z: (M+Na)+ caclcd for C28H37N3O7Na 550.2529, found 536.2369.

AC C

5.7

N-Hydroxy-7-(3-(4-(5,6,7-trimethoxy-1H-indol-1-yl)butoxy)phenylcarbamoyl)heptana mide (6f) Following the procedure as described for 6a, reaction of 5f (587 mg, 1.11 mmol) in THF (20 mL) with ethyl chloroformate (0.22 mL, 2.22 mmol) and triethylamine

33

ACCEPTED MANUSCRIPT (0.31 mL, 2.22 mmol) gave 6f (476 mg, 79%) as a solid. Mp 106-110 oC. 1H NMR (500 MHz, DMSO-d6): δ 10.32 (s, 1H), 9.79 (s, 1H), 8.64 (s, 1H), 7.27 (s, 1H), 7.19 (t,

RI PT

J = 3.0 Hz, 1H), 7.13 (t, J = 8.1 Hz, 1H), 7.05 (d, J = 8.1 Hz, 1H), 6.82 (s, 1H), 6.54 (dd, J = 2.1, 8.0 Hz, 1H), 6.26 (d, J = 3.0 Hz, 1H), 4.28 (t, J = 7.0 Hz, 2H), 3.90 (s,

SC

3H), 3.90 (t, J = 7.4 Hz, 2H), 3.76 (s, 3H), 3.74 (s, 3H), 2.25 (t, J = 7.4 Hz, 2H), 1.92 (t, J = 7.3 Hz, 2H), 1.85 (p, J = 7.1 Hz, 2H), 1.65 (p, J = 6.3 Hz, 2H), 1.53 (p, J = 6.9

M AN U

Hz, 2H), 1.47 (p, J = 7.4 Hz, 2H), 1.25 (m, 4H). 13C NMR (125 MHz, DMSO-d6): δ 171.8, 169.6, 159.3, 148.8, 141.0, 140.2, 138.5, 130.2, 129.9, 125.5, 123.2, 111.8, 109.4, 105.9, 101.2, 98.3, 67.5, 61.9, 61.2, 56.5, 48.0, 36.9, 32.8, 28.9, 28.6, 26.6,

EP

564.2689.

TE D

25.5. HR-ESI-MS m/z: (M+Na)+ caclcd for C29H39N3O7Na 564.2686, found

5.8

AC C

N-Hydroxy-7-(4-(2-(5,6,7-trimethoxy-1H-indol-1-yl)ethoxy)phenylcarbamoyl)heptana mide (6g)

Following the procedure as described for 6a, reaction of 5g (250 mg, 0.50 mmol) in THF (20 mL) with ethyl chloroformate (0.098 mL, 1.00 mmol) and triethylamine (0.14 mL, 1.00 mmol) gave 6g (236 mg, 92%) as a solid. Mp 73-78 oC. 1H NMR (500

34

ACCEPTED MANUSCRIPT MHz, DMSO-d6): δ 10.30 (s, 1H), 9.66 (s, 1H), 8.62 (s, 1H), 7.44 (d, J = 9.0 Hz, 2H), 7.22 (d, J = 3.1Hz, 1H), 6.83 (s, 1H), 6.81 (d, J = 9.0 Hz, 2H), 6.29 (d, J = 3.0 Hz,

RI PT

1H), 4.60 (t, J = 5.5 Hz, 2H), 4.21 (t, J = 5.5 Hz, 2H), 3.93 (s, 3H), 3.77 (s, 3H), 3.75 (s, 3H), 2.22 (t, J = 7.3 Hz, 2H), 1.92 (t, J = 7.3 Hz, 2H), 1.54 (t, J = 7.0 Hz, 2H), 1.47 13

C NMR (125 MHz, DMSO-d6): δ 171.5, 169.6,

SC

(t, J = 7.0 Hz, 2H), 1.25 (m, 4H).

148.9, 148.4, 140.1, 138.6, 131.0, 128.0, 125.6, 124.5, 123.1, 121.7, 121.1, 112.4,

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101.4, 98.5, 68.9, 62.0, 61.3, 56.5, 47.6, 36.8, 32.8, 29.0, 25.6. HR-ESI-MS m/z: (M+Na)+ caclcd for C27H35N3O7Na 536.2373, found 536.2365.

TE D

5.9

N-Hydroxy-7-(4-(3-(5,6,7-trimethoxy-1H-indol-1-yl)propoxy)phenylcarbamoyl)hepta

EP

namide (6h)

Following the procedure as described for 6a, reaction of 5h (342 mg, 0.67 mmol)

AC C

in THF (20 mL) with ethyl chloroformate (0.13 mL, 1.34 mmol) and triethylamine (0.19 mL, 1.34 mmol) gave 6h (295 mg, 84%) as a solid. Mp 73-78 oC. 1H NMR (500 MHz, DMSO-d6): δ 10.31 (s, 1H), 9.69 (s, 1H), 7.46 (d, J = 9.0 Hz, 2H), 7.12 (d, J = 3.9 Hz, 1H), 6.82 (d, J = 9.0 Hz, 1H), 6.83 (s, 1H), 6.25 (d, J = 3.0 Hz, 1H), 4.38 (t, J = 7.0 Hz, 1H), 3.93 (s, 3H), 3.84 (t, J = 6.1 Hz, 2H), 3.76 (s, 3H), 3.73 (s, 3H), 2.33 (t,

35

ACCEPTED MANUSCRIPT J = 7.4 Hz, 2H), 2.13 (p, J = 6.3 Hz, 2H), 1.93 (t, J = 7.3 Hz, 2H), 1.53 (p, J = 6.8 Hz, 2H), 1.47 (p, J = 7.1 Hz, 2H), 1.26 (m, 4H). 13C NMR (125 MHz, DMSO-d6): δ 171.7,

RI PT

169.6, 149.8, 148.8, 140.2, 138.5, 131.1, 128.0, 125.5, 125.0, 123.2, 123.1, 120.7, 115.1, 112.8, 101.2, 98.3, 68.4, 61.9, 61.2, 56.5, 47.9, 36.6, 32.8, 29.0, 26.5, 25.7,

SC

25.6. HR-ESI-MS m/z: (M+Na)+ caclcd for C28H37N3O7Na 550.2529, found 550.2527.

M AN U

5.10

N-Hydroxy-7-(4-(4-(5,6,7-trimethoxy-1H-indol-1-yl)butoxy)phenylcarbamoyl)heptana

TE D

mide (6i)

Following the procedure as described for 6a, reaction of 5i (261 mg, 0.49 mmol)

EP

in THF (20 mL) with ethyl chloroformat (0.10 mL, 0.98 mmol) and triethylamine (0.14 mL, 0.98 mmol) gave 6i (257 mg, 96%) as a solid. Mp 110-115 oC. 1H NMR

AC C

(500 MHz, DMSO-d6): δ 7.37 (d, J = 9.0 Hz, 2H), 7.04 (d, J = 3.0 Hz, 1H), 6.83 (s, 1H), 6.79 (d, J = 9.0 Hz, 2H), 6.28 (d, J = 3.0 Hz, 1H), 4.34 (t, J = 7.0 Hz, 2H), 3.96 (s, 3H), 3.89 (t, J = 6.3 Hz, 2H), 3.83 (s, 3H), 3.82 (s, 3H), 2.32 (t, J = 7.4 Hz, 2H), 2.08 (t, J = 7.4 Hz, 2H), 1.95 (p, J = 7.3 Hz, 2H), 1.70 (p, J = 8.0 Hz, 2H), 1.68 (p, J = 6.7 Hz, 2H), 1.62 (p, J = 7.1 Hz, 2H), 1.38 (m, 4H). 13C NMR (125 MHz, DMSO-d6):

36

ACCEPTED MANUSCRIPT δ 171.9, 169.8, 148.8, 140.2, 138.5, 130.4, 127.6, 125.6, 125.3, 123.4, 123.2, 120.7, 112.4, 101.3, 98.4, 65.7, 62.1, 61.2, 56.5, 45.2, 36.6, 32.7, 31.2, 28.9, 25.7, 25.5.

RI PT

HR-ESI-MS m/z: (M+Na)+ caclcd for C29H39N3O7Na 564.2686, found 564.2682. 5.11

SC

N-Hydroxy-7-(3-(2-(5,6-dimethoxy-1H-indol-1-yl)ethoxy)phenylcarbamoyl)heptanami de (6j)

M AN U

Following the procedure as described for 6a, reaction of 5j (84 mg, 0.18 mmol) in THF (10 mL) with ethyl chloroformate (0.03 mL, 0.36 mmol) and triethylamine

TE D

(0.05 mL, 0.36 mmol) gave 6j (61 mg, 71%) as a solid. Mp 55-62 oC. 1H NMR (500 MHz, DMSO-d6): δ 10.31 (s, 1H), 9.80 (s, 1H), 8.64 (s, 1H), 7.30 (s, 1H), 7.21 (d, J =

EP

3.1 Hz, 1H), 7.14 (t, J = 9.9 Hz, 2H ), 7.04 (d, J = 8.1 Hz, 2H), 6.55 (dd, J = 2.2, 8.0 Hz, 1H), 6.28 (t, J = 3.0 Hz, 1H), 4.48 (t, J = 5.1 Hz, 2H), 4.20 (t, J = 5.2 Hz, 2H),

AC C

3.79 (s, 3H), 3.72 (s, 3H), 2.24 (t, J = 7.4 Hz, 2H), 1.92 (t, J = 7.3 Hz, 2H), 1.52 (p, J = 6.8 Hz, 2H), 1.47 (p, J = 7.0 Hz, 2H), 1.24 (m, 4H).

13

C NMR (125 MHz,

DMSO-d6): δ 171.8, 169.6, 158.8, 146.9, 145.0, 141.0, 131.1, 130.0, 127.6, 121.1, 112.0, 109.1, 106.0, 103.3, 100.9, 94.6, 67.5, 56.4, 56.3, 45.5, 36.9, 32.7, 28.9, 25.5, 25.4. HR-ESI-MS m/z: (M+Na)+ caclcd for C26H33N3O6Na 506.2267, found

37

ACCEPTED MANUSCRIPT 506.2262. 5.12

RI PT

N-Hydroxy-7-(3-(2-(5-methoxy-1H-indol-1-yl)ethoxy)phenylcarbamoyl)heptanamide (6k)

SC

Following the procedure as described for 6a, reaction of 5k (218 mg, 0.50 mmol) in THF (20 mL) with ethyl chloroformat (0.09 mL, 1.00 mmol) and triethylamine

M AN U

(0.14 mL, 1.00 mmol) gave 6k (160 mg, 71%) as a solid. Mp 125-128 oC. 1H NMR (500 MHz, DMSO-d6): δ 10.30 (s, 1H), 9.77 (s, 1H), 8.62 (s, 1H), 7.43 (d, J = 8.9 Hz,

TE D

2H), 7.34 (d, J = 3.0 Hz, 1H), 7.26 (s, 1H), 7.12 (t, J = 8.2 Hz, 1H), 7.05 (d, J = 8.2 Hz, 1H), 7.02 (d, J = 2.3 Hz, 1H), 6.76 (dd, J = 2.3, 8.8 Hz, 1H), 6.53 (dd, J = 1.8, 8.1

EP

Hz, 1H), 6.33 (d, J = 3.0 Hz, 1H), 4.49 (t, J = 5.2 Hz, 2H), 4.19 (t, J = 5.2 Hz, 2H), 3.73 (s, 3H), 2.23 (t, J = 7.4 Hz, 2H), 1.91 (t, J = 7.4 Hz, 2H), 1.53 (p, J = 6.7 Hz, 2H),

AC C

1.46 (p, J = 7.1 Hz, 2H), 1.24 (m, 4H).

13

C NMR (125 MHz, DMSO-d6): δ 171.7,

169.6, 158.8, 153.9, 141.0, 131.7, 129.9, 129.0, 112.1, 111.6, 111.1, 109.2, 105.9, 102.6, 100.9, 67.3, 55.8, 45.6, 36.9, 32.7, 28.9, 25.5, 25.4. HR-ESI-MS m/z: (M+Na)+ caclcd for C25H31N3O5Na 476.2161, found 476.2157. 5.13

38

ACCEPTED MANUSCRIPT N-Hydroxy-7-(3-(2-(6-methoxy-1H-indol-1-yl)ethoxy)phenylcarbamoyl)heptanamide (6l)

RI PT

Following the procedure as described for 6a, reaction of 5l (83 mg, 0.19 mmol) in THF (10 mL) with ethyl chloroformate (0.02 mL, 0.38 mmol) and triethylamine (0.05

SC

mL, 0.38 mmol) gave 6l (86 mg, 99%) as a solid. Mp 130-134 oC. 1H NMR (500 MHz, DMSO-d6): δ 10.31 (s, 1H), 9.80 (s, 1H), 8.64 (s, 1H), 7.38 (d, J=8.6 Hz, 1H),

M AN U

7.30 (s, 1H), 7.26 (d, J = 3.2 Hz, 1H), 7.14 (t, J = 8.1 Hz, 1H), 7.06 (t, J = 8.1 Hz, 2H), 6.65 (dd, J = 2.0, 8.6 Hz, 1H), 6.55 (dd, J = 2.0, 8.2 Hz, 1H), 6.34 (d, J = 3.1 Hz, 1H),

TE D

4.50 (t, J = 5.2 Hz, 2H), 4.21 (t, J = 5.2 Hz, 2H), 3.78 (s, 3H), 2.24 (t, J = 7.4 Hz, 2H), 1.92 (t, J = 7.3 Hz, 2H), 1.52 (p, J = 7.1 Hz, 2H), 1.47 (t, J = 7.1 Hz, 2H), 1.24 (m, 13

C NMR (125 MHz, DMSO-d6): δ 171.8, 169.6, 158.8, 156.1, 141.0, 137.2,

EP

4H).

129.9, 128.3, 122.6, 121.3, 112.1, 109.8, 109.1, 106.0, 101.2, 93.9, 67.3, 55.8, 45.4,

AC C

36.9, 32.7, 28.9, 25.5, 25.4. HR-ESI-MS m/z: (M+Na)+ caclcd for C25H31N3O5Na 476.2161, found 476.2168. 5.14 N-Hydroxy-7-(3-(2-(7-methoxy-1H-indol-1-yl)ethoxy)phenylcarbamoyl)heptanamide (6m)

39

ACCEPTED MANUSCRIPT Following the procedure as described for 6a, reaction of 5m (223 mg, 0.51 mmol) in THF (20 mL) with ethyl chloroformate (0.09 mL, 1.02 mmol) and triethylamine

RI PT

(0.14 mL, 1.02 mmol) gave 6m (231 mg, 84%) as a solid. Mp 105-111 oC. 1H NMR (500 MHz, DMSO-d6): δ 10.31 (s, 1H), 9.79 (s, 1H), 8.64 (s, 1H), 7.29 (d, J = 3.0 Hz,

SC

1H), 7.25 (s, 1H), 7.13 (t, J = 8.0 Hz, 1H), 7.10 (d, J = 8.4 Hz, 1H), 7.07 (d, J = 8.2 Hz, 1H), 6.90 (t, J = 7.9 Hz, 1H), 6.66 (t, J = 7.7 Hz, 1H), 6.55 (dd, J = 2.0, 8.0 Hz,

M AN U

1H), 6.37 (d, J = 3.0 Hz, 1H), 4.71 (t, J = 5.5 Hz, 2H), 4.20 (t, J = 5.5 Hz, 2H), 3.87 (s, 3H), 2.24 (t, J = 7.3 Hz, 2H), 1.92 (t, J = 7.3 Hz, 2H), 1.52 (p, J = 6.9 Hz, 2H), 1.47 13

C NMR (125 MHz, DMSO-d6): δ 171.7, 169.6,

TE D

(p, J = 6.9 Hz, 2H), 1.24 (m, 4H).

158.8, 147.6, 141.0, 131.1, 130.7, 129.9, 125.4, 120.2, 113.9, 112.0, 109.2, 106.0,

EP

103.1, 101.5, 68.3, 56.0, 48.2, 36.9, 32.7, 28.9, 25.5, 25.4. HR-ESI-MS m/z: (M+Na)+ caclcd for C25H31N3O5Na 476.2161, found 476.2159.

AC C

5.15 N-Hydroxy-7-(3-(2-(1H-indol-1-yl)ethoxy)phenylcarbamoyl)heptanamide (6n) Following the procedure as described for 6a, reaction of 5n (150 mg, 0.37 mmol)

in THF (20 mL) with ethyl chloroformate (0.07 mL, 0.74 mmol) and triethylamine (0.10 mL, 0.74 mmol) gave 6n (120 mg, 77%) as a solid. Mp 105-111 oC. 1H NMR (400 MHz, DMSO-d6): δ 10.32 (s, 1H), 9.80 (s, 1H), 8.65 (s, 1H), 7.56 (t, J = 8.5 Hz,

40

ACCEPTED MANUSCRIPT 2H), 7.43 (d, J = 3.0 Hz, 1H), 7.29 (s, 1H), 7.15 (t, J = 7.8 Hz, 2H), 7.08 (d, J = 8.0 Hz, 1H), 7.03 (t, J = 7.5 Hz, 1H), 6.56 (d, J = 6.9 Hz, 1H), 6.45 (d, J = 2.9 Hz, 1H),

RI PT

4.57 (t, J = 5.1 Hz, 2H), 4.24 (t, J = 5.0 Hz, 2H), 2.26 (t, J = 7.2 Hz, 2H), 1.94 (t, J = 7.2 Hz, 2H), 1.57 (t, J = 6.8 Hz, 2H), 1.49 (t, J = 6.8 Hz, 2H), 1.27 (m, 4H). 13C NMR

SC

(100 MHz, DMSO-d6): δ 171.7, 169.6, 158.8, 140.9, 136.4, 129.9, 129.5, 128.6, 121.5, 120.8, 119.5, 112.1, 110.4, 109.2, 105.9, 101.2, 67.3, 45.5, 36.9, 32.7, 28.9, 25.5, 25.4.

M AN U

HR-ESI-MS m/z: (M+Na)+ caclcd for C24H29N3O4Na 446.2056, found 446.2050. 5.16

(6o)

TE D

N-Hydroxy-7-(3-(2-(5-bromo-1H-indol-1-yl)ethoxy)phenylcarbamoyl)heptanamide

EP

Following the procedure as described for 6a, reaction of 5o (100 mg, 0.20 mmol) in THF (10 mL) with ethyl chloroformate (0.04 mL, 0.40 mmol) and triethylamine

AC C

(0.05 mL, 0.40 mmol) gave 6o (92 mg, 87%) as a solid. Mp 141-143 oC. 1H NMR (400 MHz, DMSO-d6): δ 10.32 (s, 1H), 9.79 (s, 1H), 8.65 (s, 1H), 7.73 (s, 1H), 7.57 (d, J = 8.7 Hz, 1H), 7.49 (d, J = 3.0 Hz, 1H), 7.26 (m, 2H), 7.14 (t, J = 8.1 Hz, 1H), 7.08 (d, J = 8.0 Hz, 1H), 6.54 (d, J = 8.1 Hz, 1H), 6.45 (d, J = 2.9 Hz, 1H), 4.57 (t, J = 5.0 Hz, 2H), 4.23 (t, J = 5.0 Hz, 2H), 2.26 (t, J = 7.3 Hz, 2H), 1.94 (t, J = 7.3 Hz, 2H),

41

ACCEPTED MANUSCRIPT 1.56 (p, J = 6.6 Hz, 2H), 1.49 (p, J = 6.6 Hz, 2H), 1.27 (m, 4H). 13C NMR (100 MHz, DMSO-d6): δ171.7, 169.5, 158.7, 140.9, 135.2, 131.0, 130.4, 129.9, 123.9, 122.9,

RI PT

112.5, 112.1, 109.1, 105.9, 100.9, 67.3, 45.7, 36.8, 32.7, 28.8, 25.5, 25.4. HR-ESI-MS m/z: (M+H)+ caclcd for C24H29O4N3Br 502.1336, found 502.1336.

SC

5.17

N-Hydroxy-5-(3-(2-(5-methoxy-1H-indol-1-yl)ethoxy)phenylcarbamoyl)pentanamide

M AN U

(6p)

Following the procedure as described for 6a, reaction of 5p (100 mg, 0.24 mmol) in THF (20 mL) with ethyl chloroformate (0.04 mL, 0.48 mmol) and triethylamine

TE D

(0.07 mL, 0.48 mmol) gave 6p (89 mg, 79%) as a solid. Mp 90-93 oC. 1H NMR (400

EP

MHz, DMSO-d6): δ 10.35 (s, 1H), 9.82 (s, 1H), 8.67 (s, 1H), 7.46 (d, J = 8.9 Hz, 1H), 7.37 (d, J = 2.4 Hz, 1H), 7.28 (s, 1H), 7.15 (t, J = 8.0 Hz, 1H), 7.06 (m, 2H), 6.79 (d,

AC C

J = 8.9 Hz, 1H), 6.55 (d, J = 8.2 Hz, 1H), 6.35 (d, J = 2.3 Hz, 1H), 4.52 (t, J = 5.0 Hz, 2H), 4.21 (t, J = 5.0 Hz, 2H), 3.75 (s, 3H), 2.27 (t, J = 6.6 Hz, 2H), 1.97 (t, J = 6.6 Hz, 2H), 1.53 (m, 4H).

13

C NMR (100 MHz, DMSO-d6): δ 171.6, 169.4, 158.8, 153.9,

140.9, 131.7, 129.9, 128.9, 112.1, 111.6, 111.1, 109.2, 105.9, 102.6, 100.9, 67.4, 55.8, 45.6, 36.7, 32.6, 25.3, 25.2. HR-ESI-MS m/z: (M+H)+ caclcd for C23H28O5N3

42

ACCEPTED MANUSCRIPT 426.2023, found 426.2024. 5.18

RI PT

N-Hydroxy-8-(3-(2-(5-methoxy-1H-indol-1-yl)ethoxy)phenylcarbamoyl)octanamide (6q)

SC

Following the procedure as described for 6a, reaction of 5q (100 mg, 0.22 mmol) in THF (10 mL) with ethyl chloroformate (0.04 mL, 0.44 mmol) and triethylamine

M AN U

(0.06 mL, 0.44 mmol) gave 6q (91 mg, 89%) as a solid. Mp 102-104 oC. 1H NMR (400 MHz, DMSO-d6): δ 10.32 (s, 1H), 9.82 (s, 1H), 8.64 (s, 1H), 7.46 (d, J = 8.9 Hz,

TE D

1H), 7.37 (d, J = 2.8 Hz, 1H), 7.29 (s, 1H), 7.14 (t, J = 8.0 Hz, 1H), 7.07 (m, 2H), 6.79 (d, J = 8.9 Hz, 1H), 6.55 (d, J = 8.1 Hz, 1H), 6.35 (d, J = 2.6 Hz, 1H), 4.52 (t, J =

EP

5.0 Hz, 2H), 4.21 (t, J = 5.0 Hz, 2H), 3.75 (s, 3H), 2.26 (t, J = 7.4 Hz, 2H), 1.94 (t, J = 7.4 Hz, 2H), 1.56 (p, J = 6.9 Hz, 2H), 1.48 (p, J = 6.9 Hz, 2H), 1.26 (m, 6H).

13

C

AC C

NMR (100 MHz, DMSO-d6): δ171.7, 169.5, 158.7, 153.9, 140.9, 131.6, 129.9, 128.9, 112.1, 111.5, 111.0, 109.1, 105.9, 102.6, 100.8, 67.3, 55.8, 45.6, 36.9, 32.7, 29.0, 28.9, 25.5, 25.4. HR-ESI-MS m/z: (M+H)+ caclcd for C26H34O5N3 468.2493, found 468.2494. 5.19

43

ACCEPTED MANUSCRIPT N-Hydroxy-9-(3-(2-(5-methoxy-1H-indol-1-yl)ethoxy)phenylcarbamoyl)nonanamide (6r)

RI PT

Following the procedure as described for 6a, reaction of 5r (100 mg, 0.21 mmol) in THF (10 mL) with ethyl chloroformate (0.04 mL, 0.42 mmol) and triethylamine

SC

(0.06 mL, 0.42 mmol) gave 6r (84 mg, 83%) as a solid. Mp 135-137 oC. 1H NMR (400 MHz, DMSO-d6): δ 10.31 (s, 1H), 9.79 (s, 1H), 8.64 (s, 1H), 7.46 (d, J = 8.8 Hz,

M AN U

1H), 7.36 (m, 1H), 7.29 (s, 1H), 7.14 (t, J = 8.1 Hz, 1H), 7.07 (m, 2H), 6.79 (d, J = 8.9 Hz, 1H), 6.55 (d, J = 8.1 Hz, 1H), 6.35 (m, 1H), 4.52 (t, J = 5.0 Hz, 2H), 4.21 (t, J

TE D

= 5.0 Hz, 2H), 3.75 (s, 3H), 2.26 (t, J = 7.4 Hz, 2H), 1.93 (t, J = 7.4 Hz, 2H), 1.56 (p, J = 6.4 Hz, 2H), 1.48 (p, J = 6.4 Hz, 2H), 1.26 (m, 8H).

13

C NMR (100 MHz,

EP

DMSO-d6): δ171.7, 169.5, 158.7, 153.9, 140.9, 131.6, 129.9, 128.9, 112.1, 111.6, 111.0, 109.1, 105.9, 102.6, 100.8, 67.3, 55.8, 45.6, 36.9, 32.7, 29.1, 29.0, 25.5.

AC C

HR-ESI-MS m/z: (M+H)+ caclcd for C27H36O5N3 482.2649, found 482.2649. 5.20 Preparation of HDAC4 and HDAC8 Genes encoding HDAC4 (residues 648-1057) and HDAC8 (residues 1-377) flanked with NdeI and EcoRI sites at the 5ʹ- and 3ʹ - ends, respectively, were synthesized by GenScript Corporation (NJ, USA) and subcloned into expression

44

ACCEPTED MANUSCRIPT vectors pET-28a(+) and pET- 24b(+), respectively. Proteins were expressed in BL21(DE3) cells by overnight induction with IPTG (1 mM) at 20-25 ºC and purified

RI PT

from cleared cell lysates by sequential chromatography on Ni-Sepharose 6 fast flow, Mono Q 5/50 GL, and Superdex 75 10/ 300 GL columns (GE Healthcare). Protein

SC

concentrations were quantified with Bradford Reagent (Bio-Rad). 5.21 HDAC activity assay

M AN U

The HDAC activity assay was performed as described previously73. Enzyme, inhibitors and substrate were diluted with HDAC buffer (15 mM TriseHCl pH 8.1,

TE D

0.25 mM EDTA, 250 mM NaCl, 10% v:v glycerol). Briefly, 10 mL diluted HDAC, i.e., HeLa nuclear extract (Enzo), HDAC1 (BPS), HDAC 2 (BPS), HDAC3 (BPS),

EP

HDAC6 (BPS), HDAC4 and HDAC8 and 50 mL test compound solution at varying concentrations were added to each well of a 96-well microtiter plate and

AC C

pre-incubated at 30 ºC for 5 min. The enzymatic reaction was started by adding 40 mL substrate, i.e., Boc-Lys(Ac)-AMC (Bachem) for HeLa nuclear extract and HDAC6, Boc-Lys(TFA)-AMC (Bachem) for HDAC4, -8 and KI 177 (Enzo) for HDAC1, -2 and -3 in HDAC buffer. After incubation at 30 ºC for 30 min, the reaction was stopped by adding 100 mL trypsin solution (10 mg/mL trypsin in 50 mM

45

ACCEPTED MANUSCRIPT Tris-HCl pH 8, 100 mM NaCl, 2 mM SAHA). After incubation at 30 ºC for another 20 min, fluorescence was measured (excitation λ = 355 nm, emission λ = 460 nm). To

RI PT

calculate IC50 values, the fluorescence in wells without test compound (0.1% DMSO, negative control) was set as 100% enzymatic activity, and the fluorescence in wells

were performed in triplicate.

M AN U

5.22 Molecular docking and sequence analysis

SC

with 2µM SAHA (positive control) was set at 0% enzymatic activity. All experiments

The crystal structures of HDAC1 (PDB ID: 4BKX), HDAC2 (PDB ID: 4LXZ),

TE D

HDAC3 (PDB ID: 4A96), HDAC4 (PDB ID: 4CBT), HDAC6 (PDB ID: 5EEI), HDAC7 (PDB ID: 3C10), and HDAC8 (PDB ID: 5FUE) were obtained from RCSB

EP

protein data bank, with binding sites prepared in the co-crystallized ligand. The 3D structures of ligands generated by ACD/ChemSketch were docked into the protein

AC C

binding site of 4BKX and 4CBT. Compounds were docked in SYBYL [29] using default settings. The above HDAC structures were prepared within SYBYL, with the removal of any water molecules. AMBER7 FF99 and Gasteiger-Marsili charges were added to proteins and ligands. The docking mode of Surflex-Dock GeomX (SFXC) was apllied for docking. The charge of Zn ion was assigned with +2. The binding site

46

ACCEPTED MANUSCRIPT was defined by the bound ligands and prepares using SYBYL. Poses that coordinated with the Zn ion were selected for further analysis. Poses and interations were analysed

RI PT

with the Post-Screening Analysis function in iGEMDOCK. Mutliple sequence analysis of human HDACs were performed using Jalview [31] under T-Coffee

SC

parameters. Amino acid sequences were obtained through the Uniprot database [48] . 5.23 Cancer cell lines and cell culture

M AN U

Human prostate cancer PC-3 cells and human lung cancer A549 (American Type Culture Collection, Rockville, MD) were cultured in RPMI 1640 medium with 10%

TE D

FBS (v/v), penicillin (100 U/ml) and streptomycin (100 mg/ml). Cells were maintained in a humidified incubator at 37 ºC in an atmosphere of CO2: air (5: 95).

EP

5.24 The sulforhodamine B (SRB) growth inhibition assay Cells were seeded in 96-well plates in medium containing 5% FBS. After 24 h,

AC C

cells of T0 group were fixed with 10% trichloroacetic acid (TCA) to obtain a representative cell population at the time of hydroxamate addition (T0). After incubation of vehicle (0.1% DMSO) or hydroxamates (all dissolved in media without apparent precipitation) for an additional 48 h, cells were fixed with 10% TCA, and SRB at 0.4% (w/v) in 1% acetic acid was added to the stained cells. Unbound SRB

47

ACCEPTED MANUSCRIPT was washed out by 1% acetic acid, and SRB bound cells were solubilized with 10 mM Trizma base. The absorbance was read at a wavelength of 515 nm. The

RI PT

absorbance measurements obtained at time zero (T0) for each compound concentration level were control growth (C), cell growth in the presence of

SC

hydroxamates (Tx), and percentage growth. Percentage growth inhibition was calculated as [1-(Tx-T0)/(C-T0)] x 100% for concentrations for which Tx ≥ T0.

M AN U

Growth inhibition of 50% (GI50) was defined as the drug concentration that resulted in a 50% reduction in the increase in total protein in control cells during incubation of

TE D

the compound. 5.25 Western blot analysis

EP

After 24 h treatment with compounds, the cells were rapidly washed with PBS then lysed in ice-cold lysis buffer (50 mM TriseHCl, pH 7.4, 1 mM EGTA, 150 mM

AC C

NaCl, 1% Triton X-100, 1 mM PMSF, 5 mg/mL leupeptin, 20 mg/mL aprotinin, 1mM NaF, and 1 mM Na3VO4). The lysates were subjected to SDS-PAGE by using 13% polyacrylamide gels; protein bands were transferred to a nitrocellulose membrane, and immunoblot analysis was performed as described in the literature. Briefly, the membrane was successively incubated with various antibodies at room

48

ACCEPTED MANUSCRIPT temperature for 1 h with 0.1% milk in TTBS. The antibodies included monoclonal antibody specific for β-actin (Santa Cruz biotechnology), polyclonal antibody specific

RI PT

for Ac-histone H3 (Upstate) and monoclonal antibody specific for Ac-α-tubulin (Sigma), and then with horseradish peroxidase-labeled anti-rabbit secondary antibody

SC

for 30 min. After each incubation, the membrane was thoroughly washed with TTBS. Immunoreactive bands were detected by enhanced chemiluminescence (ECL) and

5.26 Cell cycle distribution assay

M AN U

developed with Hyperfilm-ECL (GE Healthcare).

Cell cycle distribution was assayed by flow cytometry. After treatment of

TE D

compounds at indicated concentrations, the cells were harvested by trypsinization, fixed with 70% (v/v) alcohol at 4 ºC for 30 min, and washed with phosphate-buffered

EP

saline (PBS). Cells were centrifuged and incubated in 0.1 M of phosphate-citric acid

AC C

buffer (0.2 M NaHPO4, 0.1 M citric acid, pH 7.8) for 30 mins at room temperature. After another cycle of centrifugation, the cells were resuspended with 0.5 ml PI solution containing Triton X-100 (0.1% v/v), RNase (100 mg/ml), and PI (80 mg/ml). The DNA content was analyzed with the FACScan (Becton Dickinson, Mountain View, CA) and calculated using CellQuest software. Acknowledgements. We gratefully acknowledge the support from the Ministry of 49

ACCEPTED MANUSCRIPT Science

and

Technology

(MOST

105-2311-B-038-001

and

MOST105-2320-B-038-024-MY3) and Health and welfare surcharge of tobacco

partially

supported

by

the

Taiwan

Protein

RI PT

products (MOHW106-TDU-B-212-144001) in Taiwan. This research was also Project

(Grant

No.

SC

MOST105-0210-01-12-01 and Grant No. MOST106-0210-01-15-04). References

389 (1997) 349-352.

M AN U

[1] M. Grunstein, Histone acetylation in chromatin structure and transcription, Nature [2] P.A. Wade, Transcriptional control at regulatory checkpoints by histone deacetylases: molecular connections between cancer and chromatin, Human Mol. Genet. 10 (2001) 693-698.

[3] W.D. Cress, E. Seto, Histone deacetylases, transcriptional control, and cancer, J.

TE D

Cell Physiol. 184 (2000) 1-16.

[4] R.W. Johnstone, Histone-deacetylase inhibitors: novel drugs for the treatment of cancer, Nat. Rev. Drug Discov. 1 (2002) 287-299. [5] M. Jung, Inhibitors of histone deacetylase as new anticancer agents, Curr. Med.

EP

Chem. 8 (2001) 1505-1511.

[6] I.V. Gregoretti, Y.M. Lee, H.V. Goodson, Molecular evolution of the histone

AC C

deacetylase family: functional implications of phylogenetic analysis, J. Mol. Biol. 338 (2004) 17-31.

[7] M. Paris, M. Porcelloni, M. Binaschi, D. Fattori, Histone deacetylase inhibitors: from bench to clinic, J. Med. Chem. 51 (2008) 1505-1529. [8] A. Clocchiatti, C. Florean, C. Brancolini, Class IIa HDACs: from important roles in differentiation to possible implications in tumourigenesis, J. Cell Mol. Med. 15 (2011) 1833-1846. [9] M.S. Kim, M.W. Akhtar, M. Adachi, M. Mahgoub, R. Bassel-Duby, E.T. Kavalali, E.N. Olson, L.M. Monteggia, An Essential Role for Histone Deacetylase 4 in Synaptic Plasticity and Memory Formation, J. Neurosci. 32 (2012) 10879-10886. [10] R.B. Vega, K. Matsuda, J. Oh, A.C. Barbosa, X.L. Yang, E. Meadows, J. 50

ACCEPTED MANUSCRIPT McAnally, C. Pomajzl, J.M. Shelton, J.A. Richardson, G. Karsenty, E.N. Olson, Histone deacetylase 4 controls chondrocyte hypertrophy during skeletogenesis, Cell 119 (2004) 555-566. [11] S.R. Chang, B.D. Young, S.J. Li, X.X. Qi, J.A. Richardson, E.N. Olson, Histone deacetylase 7 maintains vascular integrity by repressing matrix metalloproteinase 10,

RI PT

Cell 126 (2006) 321-334. [12] E. Telles, E. Seto, Modulation of cell cycle regulators by HDACs, Front. Biosci. 4 (2012) 831-839.

[13] T. Eckschlager, J. Plch, M. Stiborova, J. Hrabeta, Histone Deacetylase Inhibitors as Anticancer Drugs, Int. J. Mol. Sci. 18 (2017) 1414.

SC

[14] K.M. VanderMolen, W. McCulloch, C.J. Pearce, N.H. Oberlies, Romidepsin (Istodax, NSC 630176, FR901228, FK228, depsipeptide): a natural product recently

M AN U

approved for cutaneous T-cell lymphoma, J. Antibiot. 64 (2011) 525-531. [15] H.Z. Lee, V.E. Kwitkowski, P.L. Del Valle, M.S. Ricci, H. Saber, B.A. Habtemariam, J. Bullock, E. Bloomquist, Y. Li Shen, X.H. Chen, J. Brown, N. Mehrotra, S. Dorff, R. Charlab, R.C. Kane, E. Kaminskas, R. Justice, A.T. Farrell, R. Pazdur, FDA Approval: Belinostat for the Treatment of Patients with Relapsed or Refractory Peripheral T-cell Lymphoma, Clin. Cancer Res. 21 (2015) 2666-2670.

TE D

[16] L.A. Raedler, Farydak (Panobinostat): First HDAC Inhibitor Approved for Patients with Relapsed Multiple Myeloma, Am. Health Drug Benefits 9 (2016) 84-87. [17] B.E. Gryder, Targeted cancer therapy: giving histone deacetylase inhibitors all they need to succeed, Future Med. Chem. 4 (2012) 1369-1370.

EP

[18] K.J. Falkenberg, R.W. Johnstone, Histone deacetylases and their inhibitors in cancer, neurological diseases and immune disorders, Nat. Rev. Drug Discov. 13 (2014)

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673-691.

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ACCEPTED MANUSCRIPT

Figure legends

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Figure 1. Examples of pan and class I-selective HDAC inhibitors in clinical trials. Figure 2. Molecular docking analysis of inhibitors 6f and 6j in HDAC1. Compound

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6f and 6j docked into HDAC 1 (PDB ID: 4BKX) crystal structure. (A-B) Surface view of compound 6j (yellow) and compound 6f (purple) docked in HDAC 1. (C-D)

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Compounds 6j (yellow) and compound 6f (purple) docking pose stick view of the HDAC1 (grey) active site reveal interactions with residues. (E) Compounds 6f and 6j

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were superimposed in HDAC 1 catalytic center. Residues contributed hydrogen bond are colored in green, residues contributed van der Waals interaction are colored in

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brown. H178 contain hydrogen donor and pi-donor is colored in yellow. (F) Heat map of corresponding residue interactions and compounds 6j and 6f. Hydrogen bonds are

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represented in shades of green and van der Waals in shades of grey. Figure 3. Interaction analysis of inhibitors 6f and 6j. (A) Multiple Sequence Alignment (MSA) of HDAC family. Residues were aligned to HDAC1. Amino acids labeled using Crustal X color scheme. (B) The docking pose of inhibitor 6j in HDAC1 (yellow) was aligned with class I and class IIb HDAC6 and (C) class IIa

55

ACCEPTED MANUSCRIPT HDAC4 and HDAC7. (D) All HDAC isozyme structures used previously are aligned. Red circle highlights contrasting residues in alignment. HDACs color is labeled as

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shown. Figure 4. Surface residues interacts with the inhibitor cap. The surface residues of

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class I and HDAC6 (A) and class IIa (B) isozymes, as described in Figure 3, are compared. (C) Alignment of all HDAC structures used with Inhibitor 6j docked in

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HDAC1, were transposed. Red circle denotes contrasting histidine locations between isozymes. HDACs labeled as shown. The surface model of surface residues of HDAC

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1 (D) and HDAC4 (E) reveal non-interacting pocket (red circle). Figure 5. Effect of compounds 6j and 6k on induction of histone acetylation and

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α-tubulin acetylation in cultured human prostate cancer PC-3 cells. Cells were incubated with the HDAC inhibitors at the indicated concentrations for 16 h. The

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whole cell lysates were analyzed for histone and α-tubulin acetylation by SDS-PAGE and Western blot for either acetylated histone or α-tubulin. Figure 6. Effect of compounds 6j and 6k on cell cycle progression in cultured human prostate PC-3 cells. (A) Cells were treated without (ctrl) or with the HDAC inhibitors at the indicated concentrations for 24 h and were analyzed by flow cytometry for cell

56

ACCEPTED MANUSCRIPT cycle distribution. (B) Data were presented by mean value of three independent

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experiments.

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ACCEPTED MANUSCRIPT Table 1 IC50 value for the inhibition against HeLa nuclear HDACa (nM) and cytotoxicity against cancer cells (µM) by compounds 6a-m H N 6

N

O

O

OH

Substitution

Chain

Substitution

position

length (n)

(R)

6a

ortho

2

5,6,7-TriOMe

109.9±1.2

5.6

5.9

6b

ortho

3

5,6,7-TriOMe

111.0±5.2

3.9

5.0

6c

ortho

4

5,6,7-TriOMe

111.4±9.8

2.4

4.8

6d

meta

2

5,6,7-TriOMe

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R

nO

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H N

11.5±0.2

1.3

2.4

6e

meta

3

5,6,7-TriOMe

22.5±0.8

2.7

2.7

6f

meta

4

5,6,7-TriOMe

17.8±1.9

2.1

2.4

6g

para

2

5,6,7-TriOMe

57.2±3.3

4.4

4.9

6h

para

3

5,6,7-TriOMe

138.6±1.1

6.9

5.9

6i

para

4

5,6,7-TriOMe

46.4±0.6

7.7

9.7

6j

meta

2

5,6-DiOMe

11.2±1.3

1.2

6.4

6k

meta

2

5-OMe

8.6±0.7

1.3

1.7

6l

meta

2

6-OMe

14.9±0.1

1.8

4.2

6m

meta

2

7-OMe

13.5±0.4

1.4

1.9

51.6±3.6

1.5

1.7

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Data was obtained from three independent experiments.

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HDAC

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SAHA

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PC-3

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ACCEPTED MANUSCRIPT Table 2. IC50 value for the inhibition against HeLa nuclear HDACa (nM) by compounds 6n-6r H N

O

N

O

OH

Chain length (n)

Substitution (R)

6n

6

H

6o

6

5-Br

6p

4

5-OMe

185.8±10.3

6q

7

5-OMe

40.0±3.2

6r

8

5-OMe

298.0±14.2

6k

6

5-OMe

8.6±0.7

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Compound

SAHA

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Data was obtained from three independent experiments.

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a

n O

H N

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R

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HDAC

23.1±0.3

41.3±0.9

51.6±3.6

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Table 3 IC50a (nM) value for the inhibition against class I and class II HDACs by compounds 6d, 6f, 6j and 6k Class I

Class II

Compound HDAC8

HDAC4 HDAC5 HDAC7 HDAC9 HDAC6

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HDAC1 HDAC2 HDAC3

11.1±1.7 111.2±0.2 72.5±0.5 420.9±3.6 >10000

>10000

>10000 >10000 9.6±0.2

6f

29.3±0.9 999.6±5.9 108.2±0.4 752.0±5.2 >10000

>10000

>10000 >10000 8.5±0.7

6j

3.9±0.3 16.6±1.4 29.7±0.7 398.3±2.3 >10000

>10000

>10000 >10000 7.1±1.0

6k

4.5±0.1 33.1±0.5 35.3±0.2 560.9±1.2 >10000

>10000

>10000 >10000 5.9±2.0

SAHA

63.5±1.9 214.0±0.1 72.8±1.8 3400.1±6.2 >10000

>10000

>10000 >10000 9.1±0.3

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Each value is obtained by three independent experiments.

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a

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6d

60

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Figure 1

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Figure 2

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Figure 3

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Figure 4

64

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Figure 5

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(A)

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(B)

Figure 6

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ACCEPTED MANUSCRIPT H3CO H3CO

H3CO

OCH3 N

O 2O

N H

O 6

N H

OCH3

H3CO

OH

N

H N nO

H N

6

O

OH

O

n=2-4

6d

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ortho-, meta-, para-(6a-i)

O

R

N

2O

N H

O

N H n=4, 6-8 n

6j-r

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Scheme 1. Design of indole-containg aliphatic hydroxamates derived from compound 6d

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OH

ACCEPTED MANUSCRIPT Scheme 2

HO

6

O

H N

a

O O

NH2

O

HO

H N

b

O

6

O

Br

Cl n

Cl

O

nO

H N

O

6

O

H N

d

O

R

O N

O

O

R

N

O

N

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O

N

Br N

O

nO

H N

6

OH

O

6a-o

O

O

H N

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:

e

5a-o

O R

6

O

nO

4a-o

OH

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n=2-4

1a: o-aminophenol 1b: m-aminophenol 1c: p-aminophenol

nO

O

3a-i

2a-c HO

R

c

O

6

N

N

O

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Reagents and conditions: (a) 1) (COCl)2, CH2Cl2, RT; 2) DMF, 60 oC, N2, 42-46%; (b) K2CO3, MeCN, ∆, N2, 79-99%; (c) substituted indole or indole, K2CO3, DMF, RT, N2, 25-95%; (d) LiOH, MeOH, 95-99%; (e) 1) ClCO2Et, Et3N, THF, RT; 2) NH2OH-HCl, KOH, MeOH, 56-99%

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ACCEPTED MANUSCRIPT Scheme 3

O

H N

a

O

n

O

O

7a-c n=4, 7, 8

O

n O

N

Br

H N O

n O

N

n O

O

Cl

O

c

9a-c

d

O

O

N

H N

O

O

n O

OH

e

4p-r

4p-r

O

H N

O

Cl

8a-c

OH

O

b

O

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HO

H N

O

H N

OH

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O

n O

6p-r

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Reagents and conditions: (a) 1) (COCl)2, CH2Cl2, RT; 2) 1b, DMF, 60 oC, N2, 65-87%; (b) K2CO3, MeCN, ∆, N2, 8897%; (c) 5-Methoxyindole, K2CO3, DMF, RT, N2, 80-97%; (d) LiOH, MeOH, 97-98%; (e) 1) ClCO2Et, Et3N, THF, RT; 2) NH2OH-HCl, KOH, MeOH, 79-89%

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ACCEPTED MANUSCRIPT >Novel indole-containing aliphatic hydroxamates have been developed. >Several compounds had much greater enzyme-inhibiting than SAHA towards HDACs.

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>Compounds 6j and 6k displayed potent inhibition for class I HDACs.