Discovery of novel tetrahydrobenzo[b]thiophene-3-carbonitriles as histone deacetylase inhibitors

Bioorganic Chemistry 110 (2021) 104801

Contents lists available at ScienceDirect

Bioorganic Chemistry

journal homepage: www.elsevier.com/locate/bioorg

Discovery of novel tetrahydrobenzo[b]thiophene-3-carbonitriles as histone

deacetylase inhibitors

Piyush Gediya^a, Vivek K. Vyas^a, Vincenzo Carafa^b, Nikum Sitwala^a, Laura Della Torre^b,

Angelita Poziello^b, Takashi Kurohara^c, Takayoshi Suzuki^c, Vinod Sanna^d,

,

*

Varalakshmi Raguraman^e, K. Suthindhiran^e, Debarpan Ghosh^f, Dhiraj Bhatia^f, Lucia Altucci^b

,

*

Manjunath D. Ghate^a

^aDepartment of Pharmaceutical Chemistry, Institute of Pharmacy, Nirma University, Ahmedabad 382481, Gujarat, India

b

`

Department of Precision Medicine, Universita degli Studi della Campania “Luigi Vanvitelli”, Via L. De Crecchio 7, 80138 Naples, Italy

^cThe Institute of Scientiﬁc and Industrial Research (ISIR), Osaka University, Mihogaoka, Ibarakishi, Osaka 567-0047, Japan

^dPiramal Pharma Solution, Plot-18 Pharmaceutical Special Economic Zone, Sarkhej-Bawla, NH-8A, Ahmedabad, Gujarat 382213, India

^eSchool of Bio-Sciences and Technology, Vellore Institute of Technology, Vellore, Tamilnadu, India

^fDepartment of Biological Engineering, Indian Institute of Technology, Gandhinagar 382355, Gujarat, India

A R T I C L E I N F O

A B S T R A C T

Keywords:

The discovery and development of isoform-selective histone deacetylase (HDAC) inhibitor is a challenging task

because of the sequence homology among HDAC enzymes. In the present work, novel tetrahydro benzo[b]

thiophene-3-carbonitrile based benzamides were designed, synthesized, and evaluated as HDAC inhibitors.

Pharmacophore modeling was our main design strategy, and two novel series of tetrahydro benzo[b]thiophene-3-

carbonitrile derivatives with piperidine linker (series 1) and piperazine linker (series 2) were identiﬁed as HDAC

inhibitors. Among all the synthesised compounds, 9h with 4-(aminomethyl) piperidine linker and 14n with

piperazine linker demonstrated good activity against human HDAC1 and HDAC6, respectively. Both the com-

pounds also exhibited good antiproliferative activity against several human cancer cell lines. Both these com-

pounds (9h and 14n) also induced cell cycle arrest and apoptosis in U937 and MDA-MB-231 cancer cells. Overall,

for the ﬁrst time, this research discovered potent isoform-selective HDAC inhibitors using cyclic linker instead of

the aliphatic chain and aromatic ring system, which were reported in known HDAC inhibitors.

HDAC inhibitors

Anticancer agents

Cyclic linkers

Benzo[b]thiophene-3-carbonitriles

1. Introduction

transcription causes deregulation of enzyme functions, which leads to

various abrupt effects on the cell cycle. Disruption in the progression of

Histone deacetylases (HDACs) regulate the expression of many pro-

teins, which are studied intensively over the past few years. HDACs are

epigenetically regulated and overexpression play crucial roles in several

biological processes, whose alteration led to the development of many

diseases including cancer [1–3]. Many small molecules are known as

HDAC inhibitors, and they demonstrated signiﬁcant effects against

various types of cancer (Fig. 1) [4]. Histone is a core part of HDAC

enzyme, which plays an important role in epigenetic regulation [5] via

alteration of different chemical reactions i.e. acetylation, deacetylation,

and methylation. Acetylation of histone increases transcription and

deacetylation leads to inhibition of transcription. Inhibition of

the cell cycle causes various types of cancer as well as other diseases.

Inhibition of HDACs leads to an increased level of acetylated histone,

bringing about a variety of dependent cell events like apoptosis, cell

differentiation, cell survival, and inhibition of cell proliferation [6]. In

recent decades, HDACs have emerged as potential targets for the treat-

ment of cancer and neurodegenerative diseases [7]. HDAC enzymes are

classiﬁed into four different classes based on phylogenetic properties:

class I (HDAC1, 2, 3 and 8), class II (class IIa: HDAC4, 5, 7, 9; and class

IIb: HDAC6, 10), class III (sirtuins SIRT1-7), and class IV (HDAC11).

HDAC classes I, II, and IV are zinc-dependent enzymes and class III

HDACs are NAD⁺dependent enzymes. Chemical structures of known

Abbreviations: HDAC, Histone deacetylase; DCM, Dichloromethane; TEA, Triethylamine; TFA, Triﬂuoroacetic acid; TLC, thin-layer chromatography; PI, propidium

iodide.

* Corresponding authors.

E-mail addresses: lucia.altucci@unicampania.it (L. Altucci), manjunath.ghate@nirmauni.ac.in (M.D. Ghate).

https://doi.org/10.1016/j.bioorg.2021.104801

Received 2 January 2021; Received in revised form 27 February 2021; Accepted 1 March 2021

Available online 5 March 2021

P. Gediya et al.

Bioorganic Chemistry 110 (2021) 104801

HDAC inhibitors are comprised of three-components: 1) cap region for

surface recognition; 2) a linker, and; 3) a zinc-binding group (ZBG) [8].

HDAC inhibitors have an electron donor group in their structures, which

is mainly involved in the zinc-binding due to its ability to chelate active

site zinc ions [9]. HDAC1, 2 and 3 inhibitors are considered as the key

targets for the development of anticancer agents. To treat cutaneous T

cell lymphoma (CTCL) and multiple myeloma, USFDA approved few

HDAC inhibitors such as vorinostat (SAHA®) [10], panobinostat (LBH-

589) [11] and belinostat (PXD-101) [12] (Fig. 1). In the year 2015,

panobinostat, a pan-HDAC inhibitor was approved for the treatment of

multiple myeloma.

2. Results and discussion

2.1. Rational design using pharmacophore modeling

The known pharmacophore model of HDAC inhibitors consist of a

cap group, a linker, and a zinc-binding group (ZBG) [19]. Cap moiety

mainly interacts with the important amino acid residues present at the

surface of an enzyme that recognizes the HDAC isoform. A hydrophobic

carbon chain linker joins the cap with ZBG and a zinc-binding group

(ZBG) chelates Zn²⁺ion at the catalytic domain [20]. To design novel

HDAC inhibitors, we performed pharmacophore modeling with the

known HDAC inhibitors (Fig. S1. under Electronic Supplementary In-

formation). Generated pharmacophore models were validated using

receiver operating characteristic (ROC) analysis and Günere-Henry (GH)

scoring method. The best pharmacophore model suggested the presence

of the four features (one acceptor atom, one donor atom, and two hy-

drophobic sites) for the design of novel HDAC inhibitors. For the design

of the inhibitors, we selected a heterocyclic ring system as a core

structure and functional groups as suggested by the generated phar-

macophore model. We have also taken the reported pharmacophore

(cap, linker and ZBG) into the consideration. As suggested by the

pharmacophore model, for the ﬁrst hydrophobic site, which was

recognized as the cap group in the molecules, we selected 2-amino-

4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbonitrile ring. 2-Amino-

4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbonitrile ring also serves the

purpose of the cap region in the molecules [8]. For one acceptor and one

donor atoms identiﬁed in generated pharmacophore model, we selected

an amide linker. This also serves the purpose of connecting unit (CU) as

per the reported HDAC inhibitors pharmacophore model. Piperazine

and piperidin-4-yl methanamine rings were selected as the second hy-

drophobic sites, which are recognized as hydrophobic linkers as per the

Many other HDAC inhibitors are in clinical trials, either as a single

drug or in combination with other agents to treat solid tumors and he-

matological cancers, such as pracinostat (SB939) [13], entinostat

(MS275) [14], and rocilinostat (ACY-1215) [15] (Fig. 1). However, most

of the approved HDAC inhibitors are pan-HDAC or class I inhibitors. Due

to the lack of selectivity or partial selectivity of these inhibitors, some

undesirable responses such as cardiotoxicity was observed. Also, clini-

cally approved HDAC inhibitors are less effective against solid tumors.

Thus, an increasing number of studies concentrate on the discovery and

development of isoform-selective HDAC inhibitors to prevent/reduce

adverse effects and achieve efﬁcacy against solid tumors.

With this aim, herein we described the discovery of novel tetrahy-

drobenzo[b]thiophene-3-carbonitriles as selective HDAC1 and HDAC6

inhibitors as anticancer agents. Our laboratory is mainly involved in the

search of novel anticancer agents [16–18], which are acting on known

biological anticancer targets. Herein, we would like to present novel

HDAC inhibitors as potent anticancer agents.

Fig. 1. Chemical structures of known HDAC inhibitors.

2

P. Gediya et al.

Bioorganic Chemistry 110 (2021) 104801

reported model. Various groups such as methyl, methoxy, halogen, and

amine were introduced as ZBG in the designed molecules. The overall

design strategy is depicted in Fig. 2.

protection was removed using typical acidic condition with triﬂuoro-

acetic acid (TFA) in DCM, which resulted in TFA salt of intermediates

8a-o. The crude intermediates 8a-o were suspended in diethyl ether to

yield pure compounds 8a-o. In the ﬁnal step of Scheme 1, these in-

termediates (8a-o) were reacted with 4 in presence of base TEA in

dioxane (halogen amine coupling) at 60 ^◦C. An excess amount of TEA

was used to neutralize TFA at elevated temperature, which afforded

ﬁnal target compounds 9a-o of series 1. In Scheme 2, one of the com-

pound 9g of series 1 with a nitro group was reduced in the presence of

Zn, NH₄Cl to yield compound 10 with a free amine group. Puriﬁcation of

intermediates and ﬁnal compounds was carried out using column

chromatography. The ﬁnal compounds of series 1 were characterized

using Mass, NMR, and FT-IR spectroscopy. Synthesized compounds (9a-

2.2. Chemistry

A wide variety of synthetic routes were investigated and after ana-

lyses of all possible variations including the use of speciﬁc reaction

conditions, catalysts, temperature range, reagents, etc., target com-

pounds of both the series were synthesized. The general method for the

synthesis of target compounds (9a-o) of series 1 with piperidine linker is

illustrated in Scheme 1. Step 1 involves the Gewald reaction, in which a

tetrahydro benzo[b]thiophene-3-carbonitrile ring was synthesized. This

reaction was carried out in-between readily available cyclohexanone (1)

as a starting material and malononitrile (2), in presence of sulphur (S₈)

and morpholine as a base in absolute ethanol to afford ﬁrst intermediate

2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbonitrile (3). In the

next step, 3 was reacted with chloro acetyl chloride in the presence of

dioxane to obtain 2-chloro-N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thio-

phen-2-yl)acetamide (4). This is a typical amide bond formation reac-

tion using highly reactive acid chloride without any base. After removal

of unreacted acyl chloride through ﬁltration, buff-colored pure inter-

mediate 4 was obtained. In another reaction, commercially available

mono BOC (tert-butyloxycarbonyl) protected aliphatic cyclic diamine

(tert-butyl 4-(aminomethyl)piperidine-1-carboxylate (5) was reacted

with substituted acid chlorides and sulphonyl chlorides (6a-o) to form

amide and sulfonamide bonds, and to obtain compounds 7a-o in the

presence of base triethylamine (TEA) and dichloromethane (DCM). The

crude intermediates 7a-o were dried in rotavapor and suspended in

diethyl ether to get pure compounds 7a-o. To further derivatize, BOC

–

o) of series 1 showed characteristic peaks of carbonyl (C O) stretching

–

around 1600–1500 cm^{ꢀ 1}

,

nitrile (C N) stretching peaks near

–

2210–2200 cm^{ꢀ 1}, aromatic C H stretching peaks near 3045–3010

–

cm^{ꢀ 1}, aliphatic C H stretching peaks near 2970–2930 cm^{ꢀ 1}and amide

–

bond stretching peaks near 3400–3200 cm^{ꢀ 1}in FT-IR spectra. All the

molecules showed M+H stable base peak in the mass analysis (Elec-

tronic Supplementary Information). Signiﬁcant features of all com-

pounds were observed in ¹H NMR spectra. Amide protons of the fused

ring appeared in a range of 6.4 to 8.5 (δ) ppm as a singlet peak based on

the surrounding environment. Protons of the aromatic ring system were

observed as multiplet in a range of 6.7–8.0 (δ) ppm. For para substitution

on the aromatic ring system, we observed doublet of doublet (dd) in the

aromatic regions for all the compounds. The aromatic amide protons

were observed as a broad peak near 10.5 (δ) ppm (Electronic Supple-

mentary Information). Similarly, in ¹³C NMR spectra, the peaks of an

amide carbon atom attached to the aromatic system were observed at

167 (δ) ppm, and carbon atom attached to the aliphatic system was

appeared at 166 ppm for all the compounds along with sets of aromatic

carbon atoms, which were observed in the range of 120–140 (δ) ppm.

Furthermore, the carbon atoms of the cyclic aliphatic ring were observed

in the range of 20–60 (δ) ppm for all the synthesized compounds of series

1 (Electronic Supplementary Information).

To understand the structure–activity relationship (SAR) of linkers,

we synthesized series 2 compounds with piperazine linker group with

decreased chain length as depicted in Scheme 3. Alternatively, free

aliphatic halogen of intermediate 4 was reacted with mono-BOC pro-

tected piperazine (11) through halogen amine coupling reaction using

strong base K₂CO₃at elevated temperature to afford intermediate 12.

BOC protection was removed using typical acidic conditions with tri-

ﬂuoroacetic acid (TFA), which resulted in TFA salt as crude intermediate

13. Compound 13 was evaporated to dryness and suspended in diethyl

ether to give pure intermediate 13. The key intermediate 13 was reacted

with 6a-o (different substituted aliphatic/aromatic acid chlorides and

sulphonyl chlorides) in presence of excess amount of TEA to neutralize

TFA and to obtain the ﬁnal target compounds of series 2 (14a-o).

Compound 14g was reduced using Zn and NH₄Cl to obtain compound 15

with a free amine group (Scheme 4). The ﬁnal compounds of series 2

were characterized using Mass, NMR, and FT-IR. Puriﬁcation of in-

termediates and ﬁnal compounds was carried out using column chro-

matography. Series 2 molecules (14a–o), indicated characteristic peaks

of acidic carbonyl (C O) stretching around 1700–1550 cm^{ꢀ 1}, nitrile

–

ꢀ 1

–

H

(C N) stretching peaks around 2210–2200 cm , aromatic C

stretching peaks near 3050–2950 cm^{ꢀ 1}, aliphatic C H stretching peaks

near 2850–2650 cm^{ꢀ 1}and amide stretching peaks near 3500–3300

cm^{ꢀ 1}in FT-IR spectra. In the mass spectrum, all the molecules demon-

strated a stable base peak as M+H (Electronic Supplementary Infor-

mation). Signiﬁcant features of all the synthesized compounds were

observed in ¹H NMR spectra. A peak of amide proton was appeared in a

range of 10.30–10.40 (δ) ppm as singlet based on the surrounding

environment, along with aromatic protons mostly observed as a multi-

plet at 6.7–8.0 (δ) ppm. For, para substitution on the aromatic system,

we observed doublet of doublet (dd) in the aromatic regions for all the

compounds. In ¹³C NMR spectra, the peaks of amide carbon attached to

–

Fig. 2. Design strategy for the design of tetrahydrobenzo[b]thiophene-3-car-

bonitrile using pharmacophore modelling. (A) Generated two-dimensional

pharmacophore hypothesis: AA_1 is acceptor atom; DA_1 is donor atom; and

HY_1 and HY_2 are hydrophobic sites (B) Known pharmacophore model of

HDAC inhibitors. (C) Design of tetrahydrobenzo[b]thiophene-3-carbonitrile

derivatives as per the generated pharmacophore model (A) and correlated with

reported pharmacophore model (B) with distance measured in Å.

3

P. Gediya et al.

Bioorganic Chemistry 110 (2021) 104801

Scheme 1. Synthetic scheme for the synthesis of 2-(4-(aminomethyl)piperidin-1-yl)-N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)acetamide derivatives (9a-

o). Reagents and condition: (a) sulphur S₈, morpholine, ethanol, 0–5 ^◦C to rt, 1 h (b) chloro acetyl chloride, dioxane, rt, 30 min (c) TEA, DCM, rt, 30 min (d) TFA,

DCM, rt, 30 min to 4 h. (e) TEA, dioxane, 60 ^◦C.

Scheme 2. Synthetic scheme for the synthesis of 4-amino-N-((1-(2-((3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)amino)-2-oxoethyl)piperidin-4-yl)methyl)

benzamide (10). Reagents and condition: (a) Zn, NH₄Cl, water, methanol, 45 ^◦C, 3 h.

the aromatic system were observed at 170–171 (δ) ppm and carbon

atoms attached to the aliphatic system appeared at 166–167 (δ) ppm.

Carbon atoms of the cyclic aliphatic ring were observed in the range of

21–61(δ) ppm for all the synthesized molecules along with sets of aro-

matic carbons observed from 126 to 146 (δ) pm (Electronic Supple-

mentary Information). The purity of the ﬁnal compounds of both the

series was assessed by high-performance liquid chromatography (HPLC)

and all the ﬁnal compounds have >95% purity (Electronic Supple-

mentary Information).

ylmethanamine linker) with amide and sulphonamide groups as ZBG

was explored here. Compound 9h consisted of methyl amide, was one of

the most potent compound with an IC₅₀value of 1.7 µM. Compound 10

with a 4-NH₂group (reduction of 4–NO₂of 9g) and 9o with 4-F group

were also found active with IC₅₀values of 2.9 and 2.8 µM, respectively.

To study the role of linkers, we synthesized series 2 with a piperazine

aliphatic ring system, which is one carbon short than piperidin-4-

ylmethanamine analogs. In series 2 compounds, a less crowded sulfon-

–

amides group with CH₃substitution in 14i was observed with an IC₅₀

value of 2.8 µM against HeLa cell nuclear broth. Similarly, compound 15

with 4-NH₂had an IC₅₀value of 3.2 µM. Standard vorinostat was

observed with an IC₅₀value of 3.3 µM. These results indicated that an

2.3. Biology

–

aliphatic CH₃group attached with amide and sulphonamide ZBG,

2.3.1. In vitro inhibition of HeLa cell nuclear broth

instead of an aromatic ring is important for better activity.

We initiated the screening of all the synthesized compounds of both

the series against Hela cell nuclear broth as it is a rich source of HDAC1

and HDAC2 at a concentration of 10 µM. The inhibitory data is presented

in Table S1 and S2 under Electronic Supplementary Information.

Selected compounds with the highest inhibition were tested at different

concentrations and IC₅₀values were determined (Table 1). Structure-

activity relationship (SAR) studies of series 1 compounds (piperidin-4-

2.3.2. In vitro HDAC isoform selectivity

HDAC isoforms selective activity was performed against HDAC1,

HDAC2, HDAC4, and HDAC6 enzymes at 10 µM concentration for the

selected synthesized compounds (Table S3 under Electronic Supple-

mentary Information). HDAC1 and HDAC2 are the member of class I

4

P. Gediya et al.

Bioorganic Chemistry 110 (2021) 104801

Scheme 3. Synthetic scheme for the synthesis of N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-2-(piperazin-1-yl)acetamide derivatives (14a-o). Reagents

and condition: (a) K₂CO₃, DMF, 80 ^◦C. 4 h; (b) DCM, TFA, rt, 3 h; (c) TEA, DMF, rt, 30 min.

Scheme 4. Synthetic scheme for the synthesis of 2-(4-(4-aminobenzoyl)piperazin-1-yl)-N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)acetamide (15). Re-

agents and condition: (a) Zn, NH₄Cl, Water, Methanol, 45 ^◦C, 3 h.

isozymes, however, 14n displayed selective inhibitory activity against

Table 1

HDAC6 with an IC₅₀value of 13.5 µM against HDAC6 with more than

IC₅₀values describing the effect of representative

7.41-fold HDAC6 selectivity over HDAC1 and HDAC4.

compounds on HeLa cell nuclear broth.

Compounds

IC₅₀(µM)

2.3.3. In vitro antiproliferative assay

9h

1.7 ± 0.12

2.8 ± 0.14

2.9 ± 0.13

2.8 ± 0.11

3.2 ± 0.09

3.3 ± 0.09

Most of the synthesized compounds demonstrated good inhibition

against Hela cell nuclear broth. Selected compounds were screened by a

more traditional inhibition of cell proliferation (IC₅₀) assay to determine

that these compounds can inhibit cancer cell growth. In vitro anti-

proliferative activity of these compounds was assessed against the panel

of three different cancer (MDA-MB-231, A549 and HeLa) cell lines.

Compounds were screened at several concentrations to obtain a dose-

response for calculation of the IC₅₀values. Vorinostat was used as a

positive control. Compound 9e displayed potent activity against breast

cancer cell line (MDA-MB- 231) and lung cancer cell line (A549) with an

IC₅₀value of 0.31 and 0.02 µM, respectively (Table 3). Other com-

pounds, 15 and 14k were found active against MDA-MB-231 cell lines,

while 9i, 9o, 14i, and 15 were found active against A549 with an IC₅₀

value of <0.55 µM (Table 3). In addition, synthesized compounds were

also screened against normal human ﬁbroblasts (hTERT RPE-1) to

observe the cytotoxicity of these compounds on normal cells. Synthe-

sized compounds were found non-toxic on hTERT RPE-1 cells and did

not showed signiﬁcant inhibition (at least 50%) of the hTERT RPE-1

cells at 50 µM.

9o

10

14i

15

vorinostat

IC₅₀values were determined as the mean ± SD of

two independent experiments performed.

enzymes, HDAC4 belongs to class IIa and HDAC6 is a member of class IIb

enzymes. Three compounds (9h, 9o, and 14n) were found active and

selective against different HDAC isoforms. These compounds were

selected for the determination of IC₅₀values at different concentrations

(Table 2) along with three reference standards (vorinostat, trichostatin

A, and TMP 269). Compound 9h was found active against HDAC1 and

HDAC6 with an IC₅₀value of 23.2 µM and 33.9 µM, respectively.

Compound 9h displayed 1.46-fold selectivity for HDAC1 over HDAC6

and >4.31-fold selectivity for HDAC1 over HDAC4 (Table 2). Compound

9o displayed no selectivity and showed week inhibition against HDAC

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P. Gediya et al.

Bioorganic Chemistry 110 (2021) 104801

Table 2

IC₅₀values describing the effect of representative compounds on HDAC isoform enzymes.

Compound

HDAC1

HDAC4

HDAC6

Selectivity Index

1/6

IC₅₀(µM)

1/4

6/1

6/4

9h

23.2 ± 2.47

70.8 ± 1.62

45.3 ± 3.43

0.065 ± 0.0027

0.003 ± 0.00017

—

>100

33.9 ± 0.80

84.7 ± 3.87

13.5 ± 0.45

0.006 ± 0.00033

0.001 ± 0.00002

—

1.46

1.20

0.30

0.094

0.37

—

>4.31

>1.41

>2.21

—

0.68

0.84

3.36

10.6

2.73

—

>2.95

>1.18

>7.41

—

9o

>100

14n

>100

vorinostat

trichostatin A

TMP 269

—

0.19 ± 0.0015

—

IC₅₀values were determined as the mean ± SD of three independent experiments performed.

2.3.4. Analysis of cell death regulation in human cancer cell lines

Table 3

To investigate the effects of synthesized compounds on cell death

induction, we performed ﬂuorescence-activated cell sorting (FACS)

analysis, evaluating hypodiploid sub-G1 peak on ﬁxed cells and propi-

dium iodide (PI) incorporation in live cells that reveal DNA fragmen-

tation and dead-cell membrane permeabilization, respectively. The

study was performed on two different human cancer cell lines, pro-

monocytic human myeloid leukemia (U937) cells and triple-negative

breast cancer (MDA-MB-231) cells. The effect was compared with vor-

inostat as a reference compound. Compounds 9d, 9e, 9o, and 15 dis-

played an effect on membrane permeabilization to PI in both the cancer

cell lines (Fig. 3). The comparative study revealed that a higher per-

centage of PI incorporation was observed in U937 cells, which

conﬁrmed that this cell line was more sensible for the induction as

compared to MDA-MB-231 cells. Compound 9d showed 25.75% of PI-

positive cells in U937; and 10.3% in MDA-MB-231, compound 9e dis-

played 11.40% of PI-positive cells in U937 and 9.80% in MDA-MB-231.

Similarly, with compound 9o an effect of 15.1% of PI-positive cells in

U937 and 9.7% in MDA-MB-231 was observed; with compound 15, the

percentage of PI-positive cells observed was 8,75% in U937 cells and

11.5% in MDA-MB-231 cells, respectively (Fig. 3A). A different

IC₅₀values describing the cytotoxic effects of representative compounds on the

panel of three cancer cell lines (MDA- MB-231, A549 and HeLa) and on normal

human retina epithelial cell line (hTERT RPE-1).

Compound

MDA- MB-231

A549

HeLa

hTERT RPE-1

IC₅₀(µM)

9b

NA

22.79

0.02

1.2

NA

>50

9e

0.31

NA

6.65

4.25

9.28

23.4

14.4

13.5

16.6

12.1

20.3

NA

9h

9i

17.28

24.25

NA

0.41

0.15

7.73

8.75

0.55

0.29

NA

9o

10

14e

14i

NA

21.29

4.84

NA

14k

14m

14n

15

20.19

2.72

7.22

0.8

0.15

3.26

14.8

15.7

vorinostat

IC₅₀values were determined as the mean of three independent experiments

performed.

NA = not active at 100 µM.

Fig. 3. Evaluation of induction of cell death after treatment with the synthesized compounds using propidium iodide (PI) incorporation in two different cell lines

U937 (A) and MDA-MB-231 (B). In brief, cells were harvested with PBS, centrifuged at 1200 rpm for 5 min, and resuspended in 500 μL 1X PBS and 0.2 mg/mL PI.

Vorinostat was used as a positive control. Data were acquired using BD Accuri TM C6 ﬂow cytometer system (BD Biosciences). Each experiment was performed in

biological triplicates and values expressed as mean ± SD.

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P. Gediya et al.

Bioorganic Chemistry 110 (2021) 104801

response, in terms of “death induction” was observed in both the cancer

cell lines used, after the treatment with other compounds. Treatment

with compounds 9a, 9k, and 14a in U937 cells, determined a percentage

of PI incorporation of 10.25%, 16.15%, and 14.15%, respectively; this

effect was comparable to that of vorinostat (14.55%) (Fig. 3A). In MDA-

MB-231 breast cancer cells, treatment with 14m, determined an effect

higher compared to that vorinostat; (19.95% vs 12.45%, respectively). A

lower percentage of cell death was revealed after the induction with

compound 9g (13%) (Fig. 3B).

(24.5%) and 9g (23.9%), caused a reduction of cells in G2/M phase.

Concerning the S phase, its increments were observed after the treat-

ment with compounds 9b, 9c, and 9d (31.9%, 33.3% and 30.8%,

respectively, vs 23.7% in control cells) (Fig. 5A) and with compounds

14g, 14h, 14i and 14o (26.5%, 22.2%, 21.3% and 19.5% respectively, vs

12.75% in control cells) (Fig. 5A). In contrast, a strong reduction of the

percentage of cells in the S phase was observed with compounds 15

(7.8% vs 12.75%), and with compounds 10 and 9n (18.9% and 14.7% vs

23.7% in control cells) (Fig. 5A). Concerning the effects in MDA-MB-231

cells, the strongest variations in the percentage of cells in G1 and S

phases were observed after treatment with compounds 9n (G1, 67.4%;

S, 2.3% vs G1, 59.8% and S 7.6% in control cells) and 14c (G1, 59.7%; S,

10.2% vs G1, 50.3% and S 17% in control cells). Speciﬁcally, these

compounds determined an increment of the G1 phase and a reduction of

the S phase, compared to control cells (Fig. 5B). A variation in the G1

phase was observed after treatment with other compounds. Particularly,

an increment of this phase was observed after treatment with com-

pounds 9k, 14a, 14b, 14e, and 14h (59%, 62.4%, 56.5%, 56%, and

56.1%, respectively, vs 50.3% in control cells), and with compounds 9h

and 10 (both 64.9% vs 59.8% in control cells) (Fig. 5B). A reduction of

the percentage of cells in the S phase was observed after treatment with

compounds 14d and 14o (11.2% and 10%, respectively vs 17% in

control cells). Moreover, a slight increment in the S phase was revealed

after the induction with compounds 9c (10.8% vs 7.6% in control cells)

and 9g (11.7% vs 7.6% in control cells) (Fig. 5B). Concerning the G2/M

progression, different effects were observed. Speciﬁcally, an increment

was obtained after treatment with compounds 14l and 14o (35.6% and

35%, respectively, vs 27.6% in control cells) and a reduction of this

cellular phase after stimulation with compounds 9c, 9d, 9f, 9h, 9j, 9m

and 10 (24.5%, 24.1%, 25.7%, 25.7%, 20.2%, 21.9%, and 23.6%,

respectively, vs 30.4% in control cells).

Concerning the evaluation of DNA fragmentation in U937 cells,

compounds 9k and 14a led to a strong increase of sub-G1 peak 29.8%

and 28.3%, respectively, showing a higher effect than vorinostat

(17.6%). (Fig. 4A). With a lower effect, the other compounds 9n

(12.3%), 14c (12.5%), and 14e (14.1%) were also found to induce DNA

fragmentation (Fig. 4A). A lower DNA fragmentation was observed in

MDA-MB-231 cells, conﬁrming that this cell line was more resistant.

Only after treatment with compound 14o a weak increment of the sub-

G1 population was observed (8.6%) (Fig. 4B).

2.3.5. Regulation of cancer cell cycle progression

The biological effects of newly synthesized compounds were also

studied by their capability to regulate cell cycle progression. To

discriminate these effects, FACS analysis was performed in the U937

(Fig. 5A) and MDA-MB-231 cell lines (Fig. 5B). In U937 cells, compare to

untreated cells (G1, 57.7%; G2/M, 23.9%), a strong reduction of G1 and

G2/M phases was observed after induction with compounds 9k (G1,

42.2%; G2/M, 15.5%), 14a, (G1, 36.3%; G2/M, 16.8%), and 14g, (G1,

49.8%; G2/M, 19.9%) (Fig. 5A). A slight reduction of G1 phase was also

observed with compounds 9o (49.6%), 14c (48.1%), 14d (51.2%), 14e

(51%), 14h (51.5%), 14i (51.2%) and 14m (51%) (Fig. 5A). A higher

percentage of cells in the G1 phase, with respect to control cells (39.2%),

was observed with compounds 9g (46.1%), 9i (45.8%), and 10 (46%).

Treatment with compounds 9b (23.4%), 9c (24.7%), 9d (24%), 9f

Fig. 4. Evaluation of percentage of cell in sub-G1 phase of cell cycle after treatment with synthesized compounds by FACS analysis in U937 (A) and MDA-MB-231 (B)

cell lines. In brief, after stimulation, cell lines were harvested with PBS, centrifuged and resuspended in 500 μL of a hypotonic solution (1X PBS, 0.1% sodium citrate,

0.1% NP-40, freshly added RNAase A, and 50 mg/mL PI). Vorinostat was used as a positive control. Data were acquired using BD Accuri TM C6 ﬂow cytometer

system (BD Biosciences). Each experiment was performed in biological triplicates and values expressed as mean ± SD.

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Bioorganic Chemistry 110 (2021) 104801

Fig. 5. Evaluation of percentage of cells in G1, S and G2/M phases of cell cycle, after treatment with synthesized compounds by FACS analysis in U937 (A) and MDA-

MB-231 (B) cell lines. In brief, after stimulation, cell lines were harvested with PBS, centrifuged and resuspended in 500 μL of a hypotonic solution (1X PBS, 0.1%

sodium citrate, 0.1% NP-40, freshly added RNAase A, and 50 mg/mL PI). Vorinostat were used as a positive control. Data were acquired using BD Accuri TM C6 ﬂow

cytometer system (BD Biosciences). Each experiment was performed in biological triplicates and values expressed as mean ± SD.

2.3.6. Study of biological function at a molecular level

over time with kinetic monitoring. Compounds 9h, 14n, and verapamil

(standard) were incubated with human (HLM) and rat liver microsomes

(RLM) containing cytochrome P450 in the presence of an NADPH

generating system. The concentrations of these compounds in micro-

somal incubations were calculated from their calibration curves. The

remaining concentrations (%) were plotted against incubation times

(Fig. 7). In vitro half-lives (t_1/2) were determined using Graphpad prism.

The in vitro half-lives (t_1/2) were found with HLM in the following

ascending order, verapamil < 14n < 9h, which is in agreement with the

order obtained with RLM (Table 4). However, for 9h, the results sug-

gested slower metabolism with HLM than with RLM. Among these

compounds, 9h clearly showed better metabolic stability in comparison

with that of the reference verapamil (Table 4).

To extend the study at a molecular level, Western blot analysis was

performed. U937 and MDA-MB-231 cells were treated with represen-

tative compounds chosen among those that induced cell death or cell

cycle alteration, in a speciﬁc cell line or in both the cell lines. The bio-

logical properties of synthesized compounds were tested for the acety-

lation levels of histone H3 (lysine K9-14ac) and

α-tubulin. H3 and

α

-tubulin are recognised as molecular targets of class I HDACs and

HDAC6 inhibition, respectively. Slow effects in terms of the acetylation

status of both the molecular targets were observed after Western blot

analysis (Fig. 6A, B). To corroborate the effect on cell cycle progression

observed after treatment with selected compounds, a panel of cell cycle-

regulating elements were examined. To this aim, a comparative study of

cyclin A, D1, and E was performed in U937 (Fig. 6C) and MDA-MB-

231cells (Fig. 6D).

2.4. In silico ADME prediction

A different effect was found between the two cell lines used. In U937,

compounds 9k and 9o led to a decrease of cyclin A expression; com-

pounds 9d, 9n, 9o, and 14a caused a signiﬁcant reduction of cyclin D1

expression, and compounds 9o and 14a caused a signiﬁcant reduction of

cyclin E (Fig. 6C). Treatment with compounds 14e and 15 determined a

strong decrease of cyclin A and D1 protein expression, but no variation

was observed for cyclin E (Fig. 6C). A different regulation was observed

in MDA-MB-231. Particularly, compounds 9k, 9o, 14a, 14e, and 15 led

to an increment of cyclin A, and treatment with compounds 9o and 14a

caused a decrease of cyclin E protein expression (Fig. 6D). All tested

compounds were able to induce a hyperexpression cyclin D1 protein

(Fig. 6D).

ADME properties play a signiﬁcant role in the determination of the

activity and stability of the compounds. We predicted pharmacokinetic

and physicochemical properties such as partition coefﬁcient (log P_o/w),

aqueous solubility (log S), molecular weight (MW), hydrogen bod donor

(HBD), hydrogen bond acceptor (HBA), total polar surface area (TPSA),

molar refractivity (MR), number of rotatable bonds with passive

gastrointestinal absorption (GIA), brain penetration (BBB) and cyto-

chromes P₄₅₀(CYPs) inhibition and compared with vorinostat (Table S4

under Electronic Supplementary Information). All the physico-

chemical properties were found comparable with vorinostat and found

in the acceptable range as per Lipinski’s rule of ﬁve. GI absorption is a

critical parameter for bioavailability, compound 9h and 14n demon-

strated high GI absorption same as vorinostat. No compounds were

predicted with blood brain barrier (BBB) penetration, and some of the

synthesized compounds were predicted with inhibition of isoforms of

CYPs.

2.3.7. In vitro microsomal stability of 9h and 14n

For the estimation and prediction of in vivo metabolism of these

newly synthesized compounds, in vitro microsomal stability was per-

formed using LC-MS/MS [21,22]. Metabolic stability was expressed as

the percentage (%) of the remaining parent-compound concentration

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P. Gediya et al.

Bioorganic Chemistry 110 (2021) 104801

Fig. 6. Effect on synthesized compounds on molecular marker of HDAC (Ac-tubulin and H3 K9-14ac) and cell cycling regulating element (cyclin A, D1 and E) in

U937 and MDA-MB-231 cells using western blot analysis. Numbers on western blot indicated the results of the densitometry analysis, performed using the Image J

Gel Analysis tool.

2.5. Molecular docking

observed with Gly143, His174, Gly303, and Lys272, while π-π in-

teractions were observed with Phe144 and Trp203 and metal ion

interaction with Zn366 in the active site of HDAC6 enzyme (Fig. 8).

Docking results were compared with HDAC inhibitory activity of the

synthesized compounds and found in agreement with in vitro results.

Compounds 9h, 9o, and 14n displayed the highest activity among all the

synthesized compounds and exhibited the highest docking score of

A molecular docking study was carried out to predict the possible

binding orientation of the synthesized compounds with human HDAC6

protein (PDB ID: 5G0G, 1.499 Å). Vorinostat (SAHA®) and trichostatin

A were also docked into human HDAC6 to compare the docking results

with the synthesized compounds. Hydrogen bond interactions were

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P. Gediya et al.

Bioorganic Chemistry 110 (2021) 104801

isoform selectivity assay was performed against HDAC1, HDAC2,

HDAC4 and HDAC6 using selected compounds that demonstrated more

than 74% inhibition in preliminary screening against Hela nuclear

extract. First of all, a preliminary assay was performed at 10 µM con-

centration with these selected synthesized compounds against HDAC

isoforms. Compounds which showed better inhibition against HDAC

isoforms were further selected for the determination of IC₅₀values.

Compound 9h with 4-acetyl group at 4^thposition of linker (piperidin-4-

ylmethanamine) was found active against HDAC1 (IC₅₀= 23.2 ± 2.47

µM) and HDAC6 (IC₅₀= 33.9 ± 0.80 µM) amongst the selected com-

pounds. Compound 9o with 4-ﬂuro-phenyl sulfonamide group at 4^th

position of linker was found moderately active against HDAC1 (IC₅₀

=

70.8 ± 1.62 µM) and HDAC6 (IC₅₀= 84.7 ± 3.87 µM). Compound 14n

with methyl sulfonamide group at 4^thposition of the linker (piperazine)

was found highest active against HDAC1 (IC₅₀= 45.3 ± 3.43 µM) and

HDAC6 (IC₅₀= 13.5 ± 0.45 µM) among all the selected compounds.

Furthermore, based on these results, it was found that increase in

electron-withdrawing characteristics at 4^thposition of linker leads to

decrease in activity viz. 9b with 4-methoxy-benzamide, 14e with 4-

bromo benzamide at 4^thposition of the linker were found less active

against HDAC1 (10.9%), HDAC6 (3.08%) and HDAC1 (4.0%), HDAC6

(4.5%) respectively. Anti-proliferative activity was performed using

MTT assay against three different cancer cell lines (MDA-MB-231, A549,

and Hela) for selected synthesized compounds (active against Hela nu-

clear broth). Compound 9e consisted of 4-bromo-benzamide and com-

pound 14k with 4-methoxy benzenesulfonamide were found active

against all the three cell lines, they demonstrated IC₅₀values of 0.31 µM

and 4.84 µM against MDA-MB-231, IC₅₀values of 0.02 µM and 0.29 µM

against A549 and IC₅₀values of 6.65 µM and 12.1 µM against Hela

cancer cell line, respectively. Compound 9h was found active against

A549 (IC₅₀= 1.2 µM) and Hela (IC₅₀= 4.25 µM) and 9o was found

active against A549 (IC₅₀= 0.15 µM) and moderately active against the

rest of the cell lines. Compound 14n was found highly active against

A549 cells (IC₅₀= 0.8 µM).

Fig. 7. Metabolic-stability proﬁles of 9h, 14n and verapamil in human liver

microsomes (A) and in rat liver microsomes (B).

71.02, 70.40, and 71.97 respectively. Other compounds also showed

good docking scores and formed important interactions with the target

(Table S5 Electronic Supplementary Information). Interactions of

these molecules with crucial amino acid residues like Phe144, His174,

and Trp203 and metal ion Zinc366 are important and found similar with

reference molecules trichostatin A and vorinostat (Fig S2 Electronic

Supplementary Information).

Based on the above results, it is evident that for isoform selectivity,

length of the linker group and bridging moiety play a major role. Finally,

it is concluded that the compounds consisted of piperazine linker with

aliphatic substitution bridged by sulfonamide moiety displayed better

activity against HDAC1 and HDAC6 viz. compound 14n. This suggested

that the linker group cannot tolerate the bulky substitution at the 4^th

position as it may interfere with the entry of molecules in the enzyme

pocket, which is evident from the docking study.

2.6. Structure-activity relationship (SAR)

To understand SAR, we synthesized two series of tetrahydrobenzo[b]

thiophene-3-carbonitriles with 16 compounds in each series. Both the

series comprised of tetrahydrobenzo[b]thiophene-3-carbonitriles as cap

group and different substituted amide and sulfonamide as zinc-binding

group. Series 1 comprised of piperidin-4-yl-methanamine as linker

group and series 2 consisted of piperazine as linker group.

3. Conclusion

To summarize, we discovered novel tetrahydrobenzo[b]thiophene-3-

carbonitrile as histone deacetylase inhibitors. These HDAC inhibitors are

consisted of tetrahydrobenzo[b]thiophene-3-carbonitrile pharmaco-

phore as cap group, various amide and sulfonamides as ZBG. To un-

derstand the SAR, we introduced 4-(aminomethyl) piperidine and

piperazine as linker groups. SAR study revealed that less bulky group

preferred for substitution at 4^thposition of linker compared to a bulkier

hydrophobic moiety. Variation in the linker group is essential for

achieving the desire HDAC selectivity and potency. Compounds 9h and

14n with 4-(aminomethyl) piperidine and piperazine groups,

Initially, SAR study was evaluated based on preliminary screening of

synthesized compounds at 10 µM on Hela nuclear extract, which is a

reach source of HDAC1 and 2. The compounds which showed more than

74% inhibition (9o, 9h, 10, 14i, and 15) were selected for further

determination of IC₅₀values and tested at a deferent concentration of (2,

4, 6, 8, and 10 µM). Compound 9h (IC₅₀= 1.7 ± 0.12 µM), 9o (IC₅₀

=

2.8 ± 0.14 µM), 14i (IC₅₀= 2.8 ± 0.11 µM) and 15 (IC₅₀= 3.2 ± 0.09

µM) displayed good activity against Hela cell nuclear broth. Next,

Table 4

Microsomal stabilities of compounds 9h, 14n and verapamil.

Compound

Human liver microsomes (HLM)

Rat liver microsomes (RLM)

Half Life t_1/2

(min.)

Intrinsic clearance, Clint (µl/min/mg

Clearance

category

protein)

9h

78.50

22.34

9.74

17.66

62.04

142.36

medium

high

21.89

15.95

9.18

63.32

86.90

150.98

medium

high

14n

verapamil

high

Metabolic stability is expressed as the in vitro half-life time.

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Bioorganic Chemistry 110 (2021) 104801

Fig. 8. Binding modes of 9h and 14n in the catalytic binding domain of HDAC6 (PDB ID: 5G0G, 1.499 Å). (A) 3D view of 9h in HDAC6. HDAC6 is rendered in shaded

ribbon and 9h is in stick model with colour by atoms. (B) 2D view of 9h in HDAC6. Coloured dotted lines represent different binding interactions of 9h with amino

acid residue of HDAC6. 9h is in wire frame with colour by atoms. (C) 3D view of 14n in HDAC6. HDAC6 is rendered in shaded ribbon and 14n is in stick model with

colour by atoms. (B) 2D view of 14n in HDAC6. Coloured dotted lines represent different binding interactions of 14n with amino acid residue of HDAC6. 14n is in

wire frame with colour by atoms.

respectively, selected for various in vitro assays. Both, the compounds

displayed micromolar enzymatic potency against HDACs. Compound 9h

and 14n also exhibited favorable antiproliferative activity in different

cancer cell lines and compared with vorinostat. Compound 9o also

showed an effect on target modulation in cancer cells by Western blot

analysis. Compounds 9h, 9o, and 14n demonstrated their roles in cell

cycle arrest, sub-G1 phase, and also found to induce apoptosis in cancer

cell lines. Overall, these analogs exhibited promising activity against

isoform-selective HDAC with good potency. This study discovered new

molecules as HDAC inhibitors for targeting different cancer via the

HDAC pathway.

4.2. Chemistry

All the chemicals and solvents were purchased from Avra synthesis,

Combi Blocks, Sigma Aldrich, Merck, and Spectrochem and used as

purchased. The progress of reaction were monitored on pre-coated TLC

plate (Merck TLC Silica gel 60 F₂₅₄) using n-hexane/ethyl acetate and

chloroform/methanol as mobile phase. Spots of the reaction mixture on

TLC were visualized under ultraviolet radiation (254 nm) chambers. The

puriﬁcation of various intermediates and ﬁnal compounds was carried

out using column chromatography with Merck silica gel 60

M

(0.015–0.040 mm) as a stationary phase and the mobile phase

mentioned in the speciﬁed corresponding experiment. The melting

points (mp) of intermediates and ﬁnal compounds were determined in

open capillaries on a digital melting-point apparatus and found uncor-

rected. ¹H and ¹³C NMR spectra were recorded on a Bruker biospin

(Switzerland) Avance III 400 (400.13 MHz for ¹H, 100.61 MHz for ¹³C)

using DMSO‑d₆and CDCl₃as solvents. Chemical shifts are given in parts

per million (ppm), (relative to tetramethylsilane). HPLC purity was

determined on JASCO 4000 system (column isocratic eluent: H₂O/

CH₃CN 20:80 (v/v) and H₂O/CH₃CN 30:70 (v/v) ﬂow rate, 1.0 mL/min;

UV wavelength, 254–400 nM; temperature, 25 ^◦C; injection volume, 10

4. Experimental

4.1. Pharmacophore modeling

Eleven molecules (A to K) (Fig. S1 under Electronic Supplemen-

tary Information) were selected from the literature [23–29] to generate

eight pharmacophore models using the DISCOtech module of Sybyl ×

1.2 software. The genetic algorithm similarity program (GASP) was used

to reﬁne the generated models, which resulted in four reﬁned pharma-

cophore models (1–4 models). All the parameters were kept as default,

except population size (125), mutation weight (96), ﬁtness increment

(0.02), and a number of alignment (04). Receiver operating character-

istic (ROC) and Günere-Henry (GH) scoring methods were used to

validate GSAP generated (reﬁned) pharmacophore models 1–4.

μ

L) over 20 min. The purity of the ﬁnal compounds of both the series was

assessed by HPLC and the purity of all ﬁnal compounds was found >95%

or higher. Mass analysis was performed on EI-MS (20 eV): Agilent

Technologies (HP) 5973 mass spectrometer.

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Bioorganic Chemistry 110 (2021) 104801

4.2.1. General procedure for synthesis of 2-amino-4,5,6,7-tetrahydrobenzo

[b]thiophene-3-carbonitrile (3) [30–37]

4.2.5. General procedure for synthesis of substituted 2-(4-(aminomethyl)

piperidin-1-yl)-N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)

acetamide (9a-o) [43]

A clear two-neck round bottom ﬂask (RBF) was charged with

cyclohexanone (1) (10.4 mmol) and ethanol (10.0 mL). The resulting

solution was cooled at 5 ^◦C. To this reaction mixture (RM), malononitrile

(2) (10.4 mmol) was added dropwise using a dropping funnel over a

period of 5 min, and the resulting solution was stirred at 5 ^◦C for 15 min

followed by the addition of elemental sulfur (10.4 mmol) and morpho-

line (10.4 mmol). The resulting reaction mixture was moved to room

temperature. Reaction progress was monitored using TLC. After

completion of the reaction, the reaction mixture was poured in cold

water, which afforded brownish solid crude product. The isolated solid

material was puriﬁed by recrystallization using absolute ethanol to give

the title compound (3) as off-white coloured needle shaped crystal.

A two-neck RBF was charged with a solution of intermediate 4 (0.78

mmol) in dioxane (3 mL) and triethylamine (TEA) (2.35 mmol). The

reaction mixture was stirred at room temperature for 10 min, followed

by the addition of respective 8a-o (0.78 mmol). Then the reaction was

move to 70 ^◦C and stirred until the completion of the reaction. The

progress of the reaction was monitored on TLC, after completion of the

reaction, the temperature was brought to the normal temperature, and

the reaction mixture was poured into ice-cold water. Precipitates were

obtained and collected using vacuum ﬁltration and dried. The crude

products were puriﬁed by column chromatography and/or trituration

using diethyl ether.

4.2.2. General procedure for synthesis of 2-chloro-N-(3-cyano-4,5,6,7-

tetrahydrobenzo[b]thiophen-2-yl) acetamide [38] (4)

4.2.5.1. N-((1-(2-((3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)

amino)-2-oxoethyl)piperidin-4-yl)methyl)-4-methylbenzamide (9a). Com-

pound 9a was synthesized as per general procedure described above as

off-white solid in the yield of 75%, mp 235–237 ^◦C. ¹H NMR (400 MHz,

DMSO‑d₆) : δ 10.91–10.88 (t, J = 11.6, 1H), 8.43 (s, 1H), 7.76–7.74 (d, J

= 8, 2H), 7.26–7.24 (d, J = 7.6, 2H), 3.28 (s, 2H), 3.16–3.13 (t, J = 12,

2H), 2.91–2.88 (d. J = 10.4, 2H), 2.58 (s, 3H), 2.34 (s, 3H), 2.23–2.18 (t,

J = 22, 2H), 1.74–1.68 (m, 7H), 1.58 (s, 1H), 1.30–1.17 (m, 2H); ¹³C

NMR (100 MHz, DMSO‑d₆): δ 167.9, 166.0, 140.7, 131.7, 130.3, 128.7,

127.1, 114.2, 60.1, 54.9, 52.8, 44.5, 40.0, 35.0, 29.7, 23.4, 23.3, 22.5,

21.6, 20.9. MS (ESI) calcd for C₂₅H₃₀N₄O₂S [M⁺] 450.21; found: 451.21

[M+H]. HPLC analysis: retention time = 6.360 min; peak area =

97.63%; eluent A, ACN; eluent B, H₂O; isocratic (80:20) over 20 min

The solution of 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-

carbonitrile) (3) (8.4 mmol) in dioxane (15.0 mL) was added to clean

three-neck RBF and cooled at 5–10 ^◦C. While maintaining the temper-

ature chloroacetyl chloride (12.6 mmol) was added drop-wise over the

period of 10 min by dropping funnel and stirred at 5–10 ^◦C. After

complete addition of chloroacetyl chloride, the reaction mixture was

moved to room temperature and further stirred for 20 min. Reaction

progress was monitored using TLC. After completion of the reaction, the

reaction mixture was poured in ice-cold water to afford intermediate 4

as an off-white coloured solid product. Solid intermediate 4 was ﬁltered

using Büchner funnel by vacuum ﬁltration, dried under IR lamp, and

further puriﬁed by trituration using a mixture of diethyl ether: hexane

(80:20) as a white powder.

with a ﬂow rate of 1 mL min^{ꢀ 1}

.

4.2.5.2. N-((1-(2-((3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)

4.2.3. General procedure for the synthesis of substituted tert-butyl 4-

(methylamino) piperidine-1-carboxylate (7a-o) [39–41]

amino)-2-oxoethyl)piperidin-4-yl)methyl)-4-methoxybenzamide

(9b).

Compound 9b was synthesized as per general procedure described

above as dull white solid in the yield of 70%, mp 190–193 ^◦C. ¹H NMR

(400 MHz, CDCl₃): δ 10.48 (bs, 1H), 7.78–7.76(d, J = 8, 2H), 6.93–6.90

(d, J = 12, 2H), 6.47 (s, 1H), 3.84 (s, 3H), 3.37 (s, 2H), 3.19 (s, 2H),

2.92–2.89 (d, J = 12, 2H), 2.63–2.59 (d, J = 16, 3H), 2.43–2.40 (m,

3H),1.80–1.74 (m, 6H), 1.53–1.44 (m, 3H); ¹³C NMR (100 MHz, CDCl₃):

δ 167.6, 166.2, 162.1, 146.4, 130.7, 128.7, 128.1, 126.8, 114.2, 113.7,

93.8, 60.7, 55.4, 53.8, 45.1, 35.0, 30.2, 24.07, 24.02, 23.1, 22.1. MS

(ESI) calcd for C₂₅H₃₀N₄O₃S [M⁺] 466.20; found: 467.30 [M+H]. HPLC

analysis: retention time = 5.683 min; peak area = 97.46%; eluent A,

ACN; eluent B, H₂O; isocratic (80:20) over 20 min with a ﬂow rate of 1

mL min^{ꢀ 1}. Crude compound was puriﬁed using column chromatography

using n-Hexane and ethyl acetate as an eluent.

In a dry two necked RBF, equipped with nitrogen source, was

charged with solution of commercially available 1-N-BOC-4-(amino-

methyl)piperidine (5) (1.39 mmol) in dichloromethane (DCM) (2.0 mL)

and triethylamine (TEA) (2.09 mmol). The resulting reaction mixture

was cooled at 10 ^◦C. Commercially available different substituted aro-

matic and aliphatic acid chlorides and sulphonyl chlorides (6a-o) (1.39

mmol) were dissolved in DCM (1.0 mL) and added dropwise to the above

solution over the period of 5 min using a glass syringe while maintaining

temperature 5–10 ^◦C. Then, the reaction was moved to room tempera-

ture after the complete addition of 6a-o and monitored on TLC. After

completion of the reaction, the reaction mixture was concentrated under

vacuum to yield semi-solid crude products. Crude products were sus-

pended in a mixture of diethyl ether: hexane (70:30) and cooled at 10 ^◦C.

After cooling, products were fall out as white to off-white precipitates

and ﬁltered using vacuum ﬁltration. This treatment yields high purity of

7a-o, used in the next step as obtained.

4.2.5.3. N-((1-(2-((3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)

amino)-2-oxoethyl)piperidin-4-yl)methyl)benzamide (9c). Compound 9c

was synthesized as per general procedure described above as white solid

in the yield of 73%, mp 194–196 ^◦C. ¹H NMR (400 MHz, CDCl₃): δ 10.76

(bs, 1H), 7.81–7.80 (d, J = 4, 2H), 7.51–7.47 (m, 1H), 7.44–7.40 (m,

2H), 6.65–6.61 (t, J = 16, 1H), 3.40–3.37 (t, J = 12, 3H), 3.19 (s, 2H),

2.92–2.89 (d, J = 12, 2H), 2.63–2.59 (d, J = 16, 4H), 2.36–2.30 (t, J =

24, 2H) 1.85–1.72 (m, 6H), 1.50–1.44 (m, 2H); ¹³C NMR (100 MHz,

CDCl₃): δ 167.7, 167.6, 146.3, 134.5, 131.4, 130.7, 128.5, 128.1, 127.0,

114.2, 93.8, 60.7, 53.8, 45.2, 35.0, 30.2, 29.8, 24.07, 24.01, 23.1, 22.1.

MS (ESI) calcd for C₂₄H₂₈N₄O₂S [M⁺] 436.19; found: 437.20 [M+H].

HPLC analysis: retention time = 5.590 min; peak area = 95.07%; eluent

A, ACN; eluent B, H₂O; isocratic (80:20) over 20 min with a ﬂow rate of

4.2.4. General procedure for the synthesis of substituted methenamine N-

methyl-1-(piperidin-4-yl) (8a–o) [42]

Different substitute aliphatic and aromatic amides and sulphona-

mides (7a-o) were added to clean RBF and dissolved in DCM (5.0 mL),

which was followed by the addition of triﬂuoroacetic acid (TFA) (3.0

eq). The resulting reaction was stirred at room temperature until

completion of reaction and progress was monitored on TLC. After

completion of the reaction, the mixture was concentrated under a vac-

uum to yield crude products in gummy form. The crude products were

puriﬁed by washing with diethyl ether (5 mL × 82). After washing,

products (8a-o) were obtained as white to off-white free-ﬂowing powder

and used in the next step as obtained.

1 mL min^{ꢀ 1}

.

4.2.5.4. 4-Chloro-N-((1-(2-((3-cyano-4,5,6,7-tetrahydrobenzo[b]thio-

phen-2-yl)amino)-2-oxoethyl) piperidin-4-yl)methyl)benzamide (9d).

Compound 9d was synthesized as per general procedure described

above as brownish white solid in the yield of 80%, mp 210–212 ^◦C. ¹H

12

P. Gediya et al.

Bioorganic Chemistry 110 (2021) 104801

NMR (400 MHz, CDCl₃): δ 10.56 (bs, 1H), 7.77–7.74 (d, J = 12, 2H),

7.41–7.39 (d, J = 12, 2H), 6.67 (s, 1H), 3.51–3.47 (m, 1H), 3.41–3.38 (t,

J = 12, 2H), 3.20 (s, 2H), 2.93–2.90 (d, J = 12, 2H), 2.64–2.60 (d, J =

16, 3H), 2.38–2.32 (t, J = 24, 2H) 1.80–1.74 (m, 6H), 1.55–1.46 (m,

2H), 1.29–1.19 (m, 1H); ¹³C NMR (100 MHz, CDCl₃): δ 167.5, 166.8,

146.4, 137.6, 132.9, 130.7, 128.7, 128.5, 128.2, 114.3, 93.7, 65.8, 60.7,

53.7, 45.1, 34.9, 30.1, 29.8, 24.0, 23.1, 22.1, 15.2. MS (ESI) calcd for

1.60–1.43 (m, 2H); ¹³C NMR (100 MHz, CDCl₃): δ 170.6, 167.6, 146.5,

130.7, 128.1, 114.3, 93.7, 60.5, 53.7, 44.5, 34.7, 30.0, 29.4, 24.07,

24.00, 23.2, 23.0, 22.0. MS (ESI) calcd for C₁₉H₂₆N₄O₂S [M⁺] 374.18;

found: 375.50 [M+H]. HPLC analysis: retention time = 5.393 min; peak

area = 95.23%; eluent A, ACN; eluent B, H₂O; isocratic (80:20) over 20

min with a ﬂow rate of 1 mL min^{ꢀ 1}

.

C

₂₄H₂₇ClN₄O₂S [M⁺] 470.15; found: 471.20 [M+H], 473.30 [M+2].

4.2.5.9. N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-2-(4-(phe-

nylsulfonamidomethyl) piperidin-1-yl)acetamide (9i). Compound 9i was

synthesized as per general procedure described above as off white solid

in the yield of 87%, mp 185–187 ^◦C. ¹H NMR (400 MHz, CDCl₃): δ 10.41

(bs, 1H), 7.78–7.86 (d, J = 8, 2H), 7.61–7.57 (m, 1H), 7.55–7.51 (m,

2H), 5.01–4.97 (t, J = 12, 1H), 3.25–3.18 (m, 3H), 2.88–2.85 (m, 5H),

2.64–2.57 (dt, J = 4, 8, 4H), 2.30–2.25 (t, J = 8, 2H), 1.83–1.73 (m, 4H),

1.57–1.50 (m, 1H), 1.39–1.29 (m, 2H); ¹³C NMR (100 MHz, CDCl₃): δ

167.5, 146.3, 139.9, 132.6, 130.7, 129.1, 128.2, 126.9, 114.2, 93.8,

60.6, 53.6, 48.3, 35.4, 29.9, 24.07, 24.01, 23.1, 22.1. MS (ESI) calcd for

HPLC analysis: retention time = 6.763 min; peak area = 95.22%; eluent

A, ACN; eluent B, H₂O; isocratic (80:20) over 20 min with a ﬂow rate of

1 mL min^{ꢀ 1}

.

4.2.5.5. 4-Bromo-N-((1-(2-((3-cyano-4,5,6,7-tetrahydrobenzo[b]thio-

phen-2-yl)amino)-2-oxoethyl) piperidin-4-yl)methyl)benzamide (9e).

Compound 9e was synthesized as per general procedure described above

as off white solid in the yield of 70%, mp 215–217 ^◦C. ¹H NMR (400

MHz, CDCl₃): δ 10.56 (bs, 1H), 7.77–7.74 (d, J = 12, 2H), 7.41–7.39 (d,

J = 12, 2H), 6.67 (s, 1H), 3.51–3.47 (m, 1H), 3.41–3.38 (t, J = 12, 2H),

3.20 (s, 2H), 2.93–2.90 (d, J = 12, 2H), 2.64–2.60 (d, J = 16, 3H),

2.38–2.32 (t, J = 24, 2H) 1.80–1.74 (m, 6H), 1.55–1.46 (m, 2H),

1.29–1.19 (m, 1H); ¹³C NMR (100 MHz, CDCl₃) : δ 167.5, 166.8, 146.4,

137.6, 132.9, 130.7, 128.7, 128.5, 128.2, 114.3, 93.7, 65.8, 60.7, 53.7,

45.1, 34.9, 30.1, 29.8, 24.0, 23.1, 22.1, 15.2. MS (ESI) calcd for

C

₂₃H₂₈N₄O₃S₂[M⁺] 472.16; found: 473.3 [M+H]. HPLC analysis:

retention time = 5.563 min; peak area = 96.14%; eluent A, ACN; eluent

B, H₂O; isocratic (80:20) over 20 min with a ﬂow rate of 1 mL min^{ꢀ 1}

.

4.2.5.10. N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-2-(4-

(((4-methylphenyl)sulfonamido)methyl)piperidin-1-yl)acetamide

(9j).

C

₂₄H₂₇BrN₄O₂S [M⁺] 514.10; found: 515.10 [M+H], 517.10 [M+2].

Compound 9j was synthesized as per general procedure described above

as white solid in the yield of 95%), mp 182–184 ^◦C. ¹H NMR (400 MHz,

CDCl₃): δ 10.25 (bs, 1H), 7.76–7.74 (d, J = 8, 2H), 7.32–7.30 (d, J = 8,

2H), 5.04 (s, 1H), 3.25–3.18 (m, 2H), 2.92–2.82 (m, 4H), 2.64–2.57 (dt,

J = 4, 8, 4H), 2.43 (s, 3H), 2.33–2.24 (td, J = 8, 12, 2H), 1.83–1.73 (m,

6H), 1.58–1.59 (m, 1H), 1.41–1.28 (m, 2H); ¹³C NMR (100 MHz, CDCl₃):

δ 167.5, 146.3, 143.3, 136.9, 130.7, 129.7, 128.2, 127.0, 114.2, 93.7,

60.6, 53.6, 48.3, 35.4, 29.9, 24.07, 24.01, 23.1, 22.1. MS (ESI) calcd for

HPLC analysis: retention time = 11.510 min; peak area = 100%; eluent

A, ACN; eluent B, H₂O; isocratic (70:30) over 20 min with a ﬂow rate of

1 mL min^{ꢀ 1}

.

4.2.5.6. Ethyl 2-(((1-(2-((3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-

yl)amino)-2-oxoethyl) piperidin-4-yl)methyl)amino)-2-oxoacetate (9f).

Compound 9f was synthesized as per general procedure described above

as white solid in the yield of 85%, mp 141–143 ^◦C. ¹H NMR (400 MHz,

CDCl₃): δ 10.45 (bs, 1H), 7.37–7.34 (t, J = 12, 1H), 4.38–4.33 (q, J = 12,

8, 2H), 3.31–3.28 (t, J = 12, 2H), 3.21 (s, 2H), 2.94–2.91 (d, J = 12, 2H),

2.65–2.57 (dt, J = 8, 8 4H), 2.37–2.30 (td, J = 4, 12, 2H) 1.88–1.78 (m,

6H), 1.68 (s, 1H), 1.51–1.44 (m, 2H), 1.40–1.36 (t, J = 16, 3H); ¹³C NMR

(100 MHz, CDCl₃): δ 167.4, 160.7, 156.8, 146.2, 130.7, 128.1, 114.1,

93.8, 63.2, 60.7, 53.6, 45.0, 34.8, 31.6, 30.0, 29.6, 24.0, 23.9, 23.1,

22.1, 13.9. MS (ESI) calcd for C₂₁H₂₈N₄O₄S [M⁺] 432.18; found: 455

[M+Na], 431 [Mꢀ H]. HPLC analysis: retention time = 4.917 min; peak

area = 96.291%; eluent A, ACN; eluent B, H₂O; isocratic (80:20) over 20

C

₂₄H₃₀N₄O₃S₂[M⁺] 486.18; found: 487.3 [M+H]. HPLC analysis:

retention time = 6.077 min; peak area = 95.185%; eluent A, ACN; eluent

B, H₂O; isocratic (80:20) over 20 min with a ﬂow rate of 1 mL min^{ꢀ 1}

.

Crude compound was puriﬁed using column chromatography using n-

Hexane and ethyl acetate as an eluent.

4.2.5.11. N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-2-(4-

(((4-methoxyphenyl) sulfonamido)methyl)piperidin-1-yl)acetamide (9k).

Compound 9k was synthesized as per general procedure described

above as off white solid in the yield of 80%, mp 210–212 ^◦C. ¹H NMR

(400 MHz, CDCl₃): δ 10.43 (bs, 1H), 7.81–7.79 (d, J = 8, 2H), 7.00–6.98

(d, J = 8, 2H), 4.71–4.68 (t, J = 4, 12, 1H), 3.88 (s, 3H), 3.19 (s, 2H),

2.88–2.83 (m, 4H), 2.65–2.58 (m, 4H), 2.32–2.26 (td, J = 8, 12, 2H),

1.83–1.72 (m, 5H), 1.77–1.73 (m, 2H), 1.55–1.52 (m, 1H), 1.39–1.33

(m, 2H); ¹³C NMR (100 MHz, CDCl₃) : δ 167.5, 162.8, 146.3, 131.5,

130.7, 129.1, 128.2, 114.2, 93.7, 60.7, 55.6, 53.6, 48.3, 35.4, 29.9,

24.07, 24.01, 23.1, 22.1. MS (ESI) calcd for C₂₄H₃₀N₄O₄S₂[M⁺] 502.17;

found: 503.3 [M+H]. HPLC analysis: retention time = 5.683 min; peak

area = 95.57%; eluent A, ACN; eluent B, H₂O; isocratic (80:20) over 20

min with a ﬂow rate of 1 mL min^{ꢀ 1}

.

4.2.5.7. N-((1-(2-((3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)

amino)-2-oxoethyl)piperidin-4-yl)methyl)-4-nitrobenzamide (9g). Com-

pound 9g was synthesized as per general procedure described above as

yellowish solid in the yield of 75%, mp 158–160 ^◦C. ¹H NMR (400 MHz,

CDCl₃): δ 10.61 (bs, 1H), 8.30–8.28 (d, J = 8, 2H), 8.01–7.99 (d, J = 8,

2H), 6.69–6.66 (t, J = 12, 1H), 3.48–3.46 (t, J = 8, 2H), 3.22 (s, 2H),

2.96–2.93 (d, J = 12, 2H), 2.65–2.58 (m, 4H), 2.42–2.36 (td, J = 4, 12,

2H) 1.87–1.76 (m, 6H), 1.62–1.52 (m, 2H), 1.30–1.26 (m, 1H); ¹³C NMR

(100 MHz, CDCl₃): δ 167.4, 165.9, 149.5, 146.5, 140.1, 130.7, 128.3,

128.2, 123.7, 114.4, 93.7, 60.6, 53.7, 45.2, 34.9, 31.5, 30.0, 24.09,

24.04, 23.1, 22.6, 22.0, 14.1. MS (ESI) calcd for C₂₄H₂₇N₅O₄S [M⁺]

481.18; found: 482.30 [M+H]. HPLC analysis: retention time = 5.530

min; peak area = 96.812%; eluent A, ACN; eluent B, CH₃OH; isocratic

min with a ﬂow rate of 1 mL min^{ꢀ 1}

.

4.2.5.12. 2-(4-(((4-Chlorophenyl)sulfonamido)methyl)piperidin-1-yl)-N-

(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)acetamide (9l). Com-

pound 9l was synthesized as per general procedure described above as

yellowish white solid in the yield of 75%, mp 162–164 ^◦C. ¹H NMR (400

MHz, CDCl₃): δ 10.42 (bs, 1H), 7.82–7.80 (d, J = 8, 2H), 7.50–7.48 (d, J

= 8, 2H), 5.23 (s, 1H), 3.19 (s, 2H), 2.88–2.86 (m, 4H), 2.63–2.58 (m,

4H), 2.32–2.26 (m, 2H), 1.76–1.73 (m, 6H), 1.54 (s, 1H), 1.40–1.25 (m,

2H); ¹³C NMR (100 MHz, CDCl₃): δ 167.5, 146.3, 139.0, 138.5, 130.7,

129.4, 128.5, 128.2, 114.2, 93.7, 60.6, 53.5, 48.3, 35.4, 29.9, 24.07,

24.01, 23.1, 22.1. MS (ESI) calcd for C₂₃H₂₇ClN₄O₃S₂[M⁺] 506.12;

found: 506.67 [M+], 508.67 [M+2]. HPLC analysis: retention time =

6.493 min; peak area = 97.38%; eluent A, ACN; eluent B, H₂O; isocratic

(80:20) over 20 min with a ﬂow rate of 1 mL min^{ꢀ 1}

.

4.2.5.8. 2-(4-(Acetamidomethyl)piperidin-1-yl)-N-(3-cyano-4,5,6,7-tetra-

hydrobenzo[b]thiophen-2-yl)acetamide (9h). Compound 9h was synthe-

sized as per general procedure described above as white solid in the

yield of 90%, mp 190–192 ^◦C. ¹H NMR (400 MHz, CDCl₃): δ 10.67 (bs,

1H), 5.79–5.76 (t, J = 12, 1H), 3.28–3.20 (m, 4H), 2.92–2.87 (m, 2H),

2.66–2.59 (dt, J = 8, 12, 4H), 2.39–2.33 (td, J = 8, 2H), 2.03–2.01 (s,

3H) 1.87–1.83 (m, 4H), 1.80–1.76 (m, 2H), 1.67–1.61 (m, 1H),

(80:20) over 20 min with a ﬂow rate of 1 mL min^{ꢀ 1}

.

13

P. Gediya et al.

Bioorganic Chemistry 110 (2021) 104801

4.2.5.13. N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-2-(4-

((thiophene-2-sulfonamido) methyl)piperidin-1-yl)acetamide (9m). Com-

pound 9m was synthesized as per general procedure described above as

brown solid in the yield of 65%, mp 120–122 ^◦C. ¹H NMR (400 MHz,

CDCl₃): δ 10.31 (bs, 1H), 7.72–7.54 (m, 2H), 7.11–7.09 (m, 1H), 5.17 (s,

1H), 3.20 (s, 2H), 2.96–2.87 (dd, J = 8, 24, 4H), 2.64–2.57 (dt, J = 4, 8,

4H), 2.33–2.28 (m, 2H), 1.83–1.77 (m, 6H), 1.55 (s, 1H), 1.42–1.29 (m,

2H); ¹³C NMR (100 MHz, CDCl₃): δ 167.5, 146.3, 140.9, 132.0, 131.7,

130.7, 128.2, 127.4, 114.2, 93.8, 60.7, 53.6, 48.6, 35.3, 29.9, 24.07,

24.01, 23.1, 22.1. MS (ESI) calcd for C₂₁H₂₆N₄O₃S₃[M⁺] 478.12; found:

479.3 [M+H]. HPLC analysis: retention time = 5.377 min; peak area =

97.89%; eluent A, ACN; eluent B, H₂O; isocratic (80:20) over 20 min

4H), 2.23–2.17 (td, J = 4, 12, 2H) 1.78–1.73 (m, 4H), 1.70–1.66 (m,

2H), 1.59–1.49 (m, 1H), 1.28–1.18 (m, 2H); ¹³C NMR (100 MHz,

DMSO‑d₆): δ 167.9, 166.2, 151.4, 147.0, 130.3, 128.6, 127.1, 121.3,

114.2, 112.4, 92.9, 60.2, 52.9, 44.4, 40.0, 38.8, 35.1, 29.8, 23.46, 23.40,

22.6, 21.6. MS (ESI) calcd for C₂₄H₂₉N₅O₂S [M⁺] 451.20; found: 452.30

[M+H]. HPLC analysis: retention time = 6.077 min; peak area =

96.13%; eluent A, ACN; eluent B, H₂O; isocratic (80:20) over 20 min

with a ﬂow rate of 1 mL min^{ꢀ 1}

.

4.2.7. General procedure for the synthesis of tert-butyl 4-(2-((3-cyano-

4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)amino)-2-oxoethyl)piperazine-1-

carboxylate (12) [43,46]

with a ﬂow rate of 1 mL min^{ꢀ 1}

In a clean and dry three-necked RBF, a solution of commercially

available 1-N-BOC-piperidine (11) (26.8 mmol) in DMF (60.0 mL) was

added and stirred at room temperature. Followed by the addition of

intermediate 4 (26.8 mmol) and K₂CO₃(80 mmol)_.The reaction was

moved to 80 ^◦C and stirred until the completion. The progress of the

reaction was monitored on TLC. After completion of the reaction, the

temperature was brought to room temperature and the reaction mixture

was poured into ice-cold water. The precipitates were collected using

vacuum ﬁltration and dried using IR lamp. The crude product was

washed with a mixture of diethyl ether: hexane (70:30) (10 mL × 3).

This treatment resulted in off-white powdered 12 and collected using a

vacuum ﬁlter. This yields high purity of 12, which was used in the next

step.

.

4.2.5.14. N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-2-(4-

(methylsulfonamidomethyl) piperidin-1-yl)acetamide (9n). Compound 9n

was synthesized as per general procedure described above as white solid

in the yield of 95%, mp 181–183 ^◦C. ¹H NMR (400 MHz, CDCl₃): δ 10.48

(bs, 1H), 4.62–4.59 (t, J = 12, 1H), 3.22 (s, 2H), 3.10–3.07 (t, J = 12,

2H), 2.98 (s, 3H), 2.94–2.91(m, 2H), 2.65–2.58 (dt, J = 4, 8, 4H),

2.39–2.33 (m, 2H), 1.85–1.81 (m, 6H), 1.64–1.59 (m, 1H), 1.51–1.43

(m, 2H); ¹³C NMR (100 MHz, CDCl₃): δ 167.5, 146.4, 130.7, 128.2,

114.3, 93.7, 60.6, 53.6, 48.3, 40.2, 35.7, 29.9, 24.08, 24.01, 23.1, 22.1.

MS (ESI) calcd for C₁₈H₂₆N₄O₃S₂[M⁺] 410.14; found: 411.3 [M+H].

HPLC analysis: retention time = 4.350 min; peak area = 95.20%; eluent

A, ACN; eluent B, H₂O; isocratic (80:20) over 20 min with a ﬂow rate of

1 mL min^{ꢀ 1}

4.2.8. General procedure for the synthesis of N-(3-cyano-4,5,6,7-

tetrahydrobenzo[b]thiophen-2-yl)-2-(piperazin-1-yl)acetamide (13) [42]

A solution of intermediate 12 (23.5 mmol) in DCM (95.0 mL) was

added in dry RBF and stirred at room temperature, which was followed

by the addition of triﬂuoroacetic acid (TFA) (70.5 mmol). The resulting

reaction mixture was stirred at room temperature until completion of

the reaction, and reaction progress was monitored on TLC. After

completion of the reaction, the mixture was concentrated under a vac-

uum to yield crude material in gummy form. Crude 13 was puriﬁed by

washing with diethyl ether (30 mL × 2). After washing 13 was obtained

as off-white powder and used in the next step as obtained.

.

4.2.5.15. N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-2-(4-

(((4-ﬂuorophenyl)sulfonamido) methyl)piperidin-1-yl)acetamide (9o).

Compound 9o was synthesized as per general procedure described

above as off white solid in the yield of 87%, mp 174–176 ^◦C. ¹H NMR

(400 MHz, CDCl₃): δ 10.19 (bs, 1H), 7.91–7.87 (m, 2H), 7.22–7.18 (m,

2H), 5.05 (s, 1H), 3.19 (s, 2H), 2.9–2.87 (m, 4H), 2.64–2.59 (m, 4H),

2.32–2.27 (m, 2H), 1.83–1.82(m, 4H), 1.76–1.72 (m, 2H),1.54 (s, 1H),

1.38–1.35 (m, 2H); ¹³C NMR (100 MHz, CDCl₃): δ 167.5, 166.2, 163.7,

146.4, 136.1, 130.7, 129.79, 129.7, 128.2, 116.4, 116.2, 114.2, 93.7,

60.6, 53.8, 53.6, 48.3, 35.4, 29.9, 24.07, 24.00, 23.0, 22.0. MS (ESI)

calcd for C₂₃H₂₇FN₄O₃S₂[M⁺] 490.15; found: 491.3 [M+H]. HPLC

analysis: retention time = 5.623 min; peak area = 96.52%; eluent A,

ACN; eluent B, H₂O; isocratic (80:20) over 20 min with a ﬂow rate of 1

4.2.9. General procedure for synthesis of substituted N-(3-cyano-4,5,6,7-

tetrahydrobenzo[b]thiophen-2-yl)-2-(piperazin-1-yl)acetamide (14a-o)

[39–41]

mL min^{ꢀ 1}

To a solution of intermediate 13 (0.75 mmol) with DMF (2.0 mL) in

RBF, triethylamine (2.2 mmol) was added, and the reaction mixture was

stirred at room temperature for 10 min. Followed by the addition of a

solution of commercially available 6a-o (different substituted aromatic

and aliphatic acid chlorides and sulphonyl chlorides) (0.75 mmol) with

DMF (1.0 mL) in dropwise manner over the period of 5 min using a glass

syringe with maintaining temperature 10 ^◦C. The reaction was moved to

room temperature after the complete addition of 6a-o and progress was

monitored on TLC. After completion of the reaction, the temperature

was brought to normal temperature, and the reaction mixture was

poured into ice-cold water. The precipitates were collected using vac-

uum ﬁltration and dried. The crude 14a-o were puriﬁed using column

chromatography and/or trituration using diethyl ether.

.

4.2.6. General procedure for the synthesis of 4-amino-N-((1-(2-((3-cyano-

4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)amino)-2-oxoethyl)piperidin-4-

yl)methyl)benzamide (10) [44,45]

In a clean RBF, 9g was added (0.47 mmol), in a mixture of methanol

(4.0 mL) and water (2.0 mL). To the resulting reaction mixture, dry

activated zinc (2.35 mmol) was added, which was followed by the

addition of NH₄Cl (2.35 mmol). The resulting reaction mixture was

stirred at 45 ^◦C until the completion of the reaction. Progress of the

reaction was monitored using TLC. After completion of the reaction, the

reaction mixture was poured into ice-cold water and extracted with

ethyl acetate (10 mL × 3). The combined organic layer was dried over

anhydrous Na₂SO₄and concentrated in vacuo. Crude 10 was puriﬁed by

column chromatography using hexane: ethyl acetate (70:30) as a mobile

phase.

4.2.9.1. N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-2-(4-(4-

methylbenzoyl)piperazin-1-yl)acetamide (14a). Compound 14a was syn-

thesized as per general procedure described above as off white solid in

the yield of 70%, mp 144–146 ^◦C. ¹H NMR (400 MHz, CDCl₃): δ 10.32

(bs, 1H), 7.33–7.31 (d, J = 8, 2H), 7.23–7.21 (d, J = 8, 2H), 3.91 (bs,

1H), 3.60 (bs, 2H), 3.28 (s, 2H), 2.73 (s, 2H), 2.66–2.59 (dt, J = 4, 8,

6H), 2.38 (s, 3H) 1.86–1.81 (m, 3H), 1.67 (s, 2H); ¹³C NMR (100 MHz,

CDCl₃): δ 170.5, 166.5, 146.0, 140.1, 132.3, 130.8, 129.1, 128.5, 127.2,

114.2, 94.1, 60.5, 24.09, 24.01, 23.0, 22.0, 21.4. MS (ESI) calcd for

4.2.6.1. 4-Amino-N-((1-(2-((3-cyano-4,5,6,7-tetrahydrobenzo[b]thio-

phen-2-yl)amino)-2-oxoethyl) piperidin-4-yl)methyl)benzamide (10).

Compound 10 was synthesized as per general procedure described

above as off white solid in the yield of 65%, mp 258–260 ^◦C. ¹H NMR

(400 MHz, DMSO‑d₆): δ 10.82 (bs, 1H), 8.04–8.02 (t, J = 8, 1H),

7.57–7.55 (d, J = 8, 2H), 6.53–6.51 (d, J = 8, 2H), 5.59 (s, 2H),

3.11–3.09 (t, J = 8, 2H), 2.91–2.87 (m, 2H), 2.60–2.57 (m, 2H), 2.49 (s,

C

₂₃H₂₆N₄O₂S [M⁺] 422.18; found: 423.3 [M+H]. HPLC analysis:

14

P. Gediya et al.

Bioorganic Chemistry 110 (2021) 104801

retention time = 7.793 min; peak area = 97.46%; eluent A, ACN; eluent

analysis: retention time = 5.703 min; peak area = 100.0%; eluent A,

B, H₂O; isocratic (70:30) over 20 min with a ﬂow rate of 1 mL min^{ꢀ 1}

.

ACN; eluent B, H₂O; isocratic (70:30) over 20 min with a ﬂow rate of 1

mL min^{ꢀ 1}

.

4.2.9.2. N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-2-(4-(4-

methoxybenzoyl) piperazin-1-yl) acetamide (14b). Compound 14b was

synthesized as per general procedure described above as buff white solid

in the yield of 80%, mp 177–179 ^◦C. ¹H NMR (400 MHz, CDCl₃): δ 10.32

(bs, 1H), 7.41–7.39 (d, J = 8, 2H), 6.93–6.91 (d, J = 8, 2H), 3.84 (s, 3H),

3.66 (bs, 3H), 3.29 (s, 2H), 2.65–2.60 (m, 8H), 1.84–1.83 (d, J = 8, 3H),

1.66 (bs, 2H); ¹³C NMR (100 MHz, CDCl₃): δ 170.3, 166.5, 160.9, 146.0,

130.8, 129.2, 128.5, 127.3, 114.2, 113.7, 94.1, 60.5, 55.3, 53.5, 24.08,

24.01, 23.0, 22.0. MS (ESI) calcd for C₂₃H₂₆N₄O₃S [M⁺] 438.17; found:

439.3 [M+H]. HPLC analysis: retention time = 6.693 min; peak area =

100.0%; eluent A, ACN; eluent B, H₂O; isocratic (70:30) over 20 min

4.2.9.7. N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-2-(4-(4-

nitrobenzoyl)piperazin-1-yl)acetamide (14g). Compound 14g was syn-

thesized as per general procedure described above as yellowish solid in

the yield of 63%, mp 208–209 ^◦C. ¹H NMR (400 MHz, CDCl₃): δ 10.27 (s,

1H), 8.31–8.29 (d, J = 8, 2H), 7.61–7.59 (d, J = 8, 2H), 3.95 (s, 2H),

3.53 (bs, 2H), 3.32 (s, 2H), 2.79 (s, 2H), 2.65–2.59 (m, 6H), 1.85–1.82

(m, 4H); ¹³C NMR (100 MHz, CDCl₃): δ 168.0, 166.1, 148.4, 146.0,

141.4, 130.8, 128.7, 128.1, 124.0, 114.3, 95.6, 94.1, 60.4, 53.5, 53.0,

47.6, 42.2, 24.08, 24.0, 23.0, 22.0. MS (ESI) calcd for C₂₂H₂₃N₅O₄S

[M⁺] 453.15; found: 454.3 [M+H]. HPLC analysis: retention time =

6.320 min; peak area = 100.0%; eluent A, ACN; eluent B, H₂O; isocratic

with a ﬂow rate of 1 mL min^{ꢀ 1}

.

(70:30) over 20 min with a ﬂow rate of 1 mL min^{ꢀ 1}

.

4.2.9.3. 2-(4-Benzoylpiperazin-1-yl)-N-(3-cyano-4,5,6,7-tetrahydrobenzo

[b]thiophen-2-yl)acetamide (14c). Compound 14c was synthesized as

per general procedure described above as white solid in the yield of

72%, mp 192–193 ^◦C. ¹H NMR (400 MHz, CDCl₃): δ 10.31 (bs, 1H),

7.44–7.70 (m, 5H), 3.93 (bs, 2H), 3.58 (bs, 2H), 3.29 (s, 2H), 2.75 (s,

2H), 2.66–2.59 (dt, J = 4, 8, 6H), 1.87–1.83 (m, 4H); ¹³C NMR (100

MHz, CDCl₃): δ 170.3, 166.5, 146.0, 135.3, 130.8, 129.9, 128.5, 127.0,

126.5, 114.2, 94.1, 60.4, 53.7, 53.2, 47.8, 42.2, 24.07, 24.00, 23.0, 22.0.

MS (ESI) calcd for C₂₂H₂₄N₄O₂S [M⁺] 408.16; found: 409.3 [M+H].

HPLC analysis: retention time = 6.630 min; peak area = 100.0%; eluent

A, ACN; eluent B, H₂O; isocratic (70:30) over 20 min with a ﬂow rate of

4.2.9.8. 2-(4-Acetylpiperazin-1-yl)-N-(3-cyano-4,5,6,7-tetrahydrobenzo

[b]thiophen-2-yl)acetamide (14h). Compound 14h was synthesized as

per general procedure described above as white solid in the yield of

94%, mp 146–148 ^◦C. ¹H NMR (400 MHz, CDCl₃): δ 10.30 (s, 1H), 3.75

(bs, 2H), 3.62–3.59 (t, J = 12, 2H), 3.28 (s, 2H), 3.03–3.00 (t, J = 12,

1H), 2.70–2.61 (m, 7H), 2.12 (s, 3H), 1.88–1.80 (m, 4H); ¹³C NMR (100

MHz, CDCl₃): δ 169.0, 167.3, 166.5, 166.3, 160.7, 146.2, 146.0, 130.87,

130.85, 128.6, 128.5, 128.2, 114.2, 94.19, 94.14, 93.8, 61.0, 60.5, 60.4,

54.3, 53.8, 53.4, 53.1, 52.7, 46.3, 46.1, 45.6, 41.4, 39.9, 24.07, 24.00,

23.0, 22.0, 21.3. MS (ESI) calcd for C₁₇H₂₂N₄O₂S [M⁺] 346.15; found:

347.3 [M+H]. HPLC analysis: retention time = 7.52 min; peak area =

98.89%; eluent A, ACN; eluent B, H₂O; isocratic (85:15) over 20 min

1 mL min^{ꢀ 1}

.

with a ﬂow rate of 1 mL min^{ꢀ 1}

4.2.9.4. 2-(4-(4-Chlorobenzoyl)piperazin-1-yl)-N-(3-cyano-4,5,6,7-tetra-

hydrobenzo[b]thiophen-2-yl)acetamide (14d). Compound 14d was syn-

thesized as per general procedure described above as brownish white

solid in the yield of 60%, mp 152–154 ^◦C. ¹H NMR (400 MHz, CDCl₃): δ

10.26 (s, 1H), 8.31–8.29 (d, J = 8, 2H), 7.61–7.59 (d, J = 8, 2H), 3.95

(bs, 2H), 3.53 (bs, 2H), 3.32 (s, 2H), 2.79 (s, 2H), 2.66–2.59 (m, 6H),

1.87–1.80 (m, 4H); ¹³C NMR (100 MHz, CDCl₃): δ 168.0, 166.1, 148.5,

146.0, 141.1, 130.8, 128.7, 128.1, 124.0, 114.3, 94.1, 60.4, 53.5, 53.0,

47.6, 42.2, 24.08, 24.00, 23.0, 22.0. MS (ESI) calcd for C₂₂H₂₃ClN₄O₂S

[M⁺] 442.12; found: 443.5 [M+H], 445.6 [M+2]. HPLC analysis:

retention time = 8.307 min; peak area = 100.0%; eluent A, ACN; eluent

.

4.2.9.9. N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-2-(4-(phe-

nylsulfonyl)piperazin-1-yl)acetamide (14i). Compound 14i was synthe-

sized as per general procedure described above as off white solid in the

yield of 80%, mp 178–180 ^◦C. ¹H NMR (400 MHz, CDCl₃): δ 9.90 (s, 1H),

7.71–7.70 (d, J = 4, 2H), 7.60–7.56 (t, J = 8, 1H), 7.50–7.52 (d, J = 8,

2H), 3.17 (bs, 2H), 3.11(bs, 3H), 2.67–2.65 (t, J = 4, 4H), 2.55–2.45 (dt,

J = 4, 8, 4H), 1.79–1.69 (m, 4H), 1.60 (s, 1H); ¹³C NMR (100 MHz,

CDCl₃): δ 166.2, 145.8, 133.5, 133.2, 130.8, 129.2, 128.6, 127.5, 114.2,

94.1, 60.2, 52.5, 46.1, 24.0, 23.9, 23.0, 22.0. MS (ESI) calcd for

C₂₁H₂₄N₄O₃S₂[M⁺] 444.13; found: 445.4 [M+H]. HPLC analysis:

retention time = 7.837 min; peak area = 97.44%; eluent A, ACN; eluent

B, H₂O; isocratic (70:30) over 20 min with a ﬂow rate of 1 mL min^{ꢀ 1}

.

B, H₂O; isocratic (70:30) over 20 min with a ﬂow rate of 1 mL min^{ꢀ 1}

4.2.9.5. 2-(4-(4-Bromobenzoyl)piperazin-1-yl)-N-(3-cyano-4,5,6,7-tetra-

hydrobenzo[b]thiophen-2-yl)acetamide (14e). Compound 14e was syn-

thesized as per general procedure described above as brownish white

solid in the yield of 69%, mp 162–164 ^◦C. ¹H NMR (400 MHz, CDCl₃): δ

10.68 (s, 1H), 7.58–7.56 (d, J = 8, 2H), 7.32–7.30 (d, J = 8, 2H), 3.98

(bs, 2H), 3.65 (bs, 3H), 0.95 (bs, 2H), 2.63–2.54 (m, 8H), 1.86–1.79 (m,

3H); ¹³C NMR (100 MHz, CDCl₃): δ 169.5, 165.0, 148.5, 145.5, 133.5,

131.9, 131.7, 131.5, 131.1, 128.89, 128.82, 124.6, 114.2, 94.5, 59.3,

52.9, 24.0, 23.9, 23.0, 22.0. MS (ESI) calcd for C₂₂H₂₃BrN₄O₂S [M⁺]

486.07; found: 487.3 [M+H], 489.2 [M+2]. HPLC analysis: retention

time = 8.353 min; peak area = 96.55%; eluent A, ACN; eluent B, H₂O;

.

4.2.9.10. N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-2-(4-

tosylpiperazin-1-yl)acetamide (14j). Compound 14j was synthesized as

per general procedure described above as off white solid in the yield of

76%, mp 230–232 ^◦C. ¹H NMR (400 MHz, CDCl₃): δ 9.86 (s, 1H),

7.59–7.57 (d, J = 8, 2H), 7.30–7.28 (d, J = 8, 2H), 3.63 (s, 1H), 3.17 (s,

2H), 3.09 (bs, 3H), 2.66–2.64 (t, J = 8, 4H), 2.56–2.48 (dt, J = 4, 8, 4H),

2.38 (s, 3H), 1.75–1.70 (m, 4H); ¹³C NMR (100 MHz, CDCl₃): δ 166.3,

145.7, 144.0, 132.5, 130.8, 129.9, 128.6, 127.6, 114.1, 94.1, 67.0, 60.2,

52.6, 46.0, 24.0, 23.9, 23.0, 22.0, 21.6. MS (ESI) calcd for

isocratic (70:30) over 20 min with a ﬂow rate of 1 mL min^{ꢀ 1}

.

C

₂₂H₂₆N₄O₃S₂[M⁺] 458.14; found: 459.3 [M+H]. HPLC analysis:

retention time = 9.227 min; peak area = 100%; eluent A, ACN; eluent B,

H₂O; isocratic (70:30) over 20 min with a ﬂow rate of 1 mL min^{ꢀ 1}

4.2.9.6. Ethyl 2-(4-(2-((3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-

yl)amino)-2-oxoethyl) piperazin-1-yl)-2-oxoacetate (14f). Compound 14f

was synthesized as per general procedure described above as white solid

in the yield of 78%, mp 198–200 ^◦C. ¹H NMR (400 MHz, CDCl₃): δ 10.23

(s, 1H), 4.37–4.32 (q, J = 4, 12, 2H), 3.79 (bs, 2H), 3.61–3.59 (t, J = 8,

2H), 3.31 (s, 2H), 2.73–2.71 (t, J = 8, 4H), 2.66–2.58 (dt, J = 4, 8, 4H),

1.87–1.81 (m, 4H), 1.39–1.36 (t, J = 12, 3H),; ¹³C NMR (100 MHz,

CDCl₃): δ 166.2, 162.3, 160.0, 145.9, 130.8, 128.6, 114.2, 94.2, 62.3,

60.4, 53.2, 52.6, 47.8, 46.0, 41.4, 24.07, 24.00, 23.0, 22.0, 14.0. MS

(ESI) calcd for C₁₉H₂₄N₄O₄S [M⁺] 404.15; found: 405.3 [M+H]. HPLC

.

4.2.9.11. N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-2-(4-((4-

methoxyphenyl)sulfonyl) piperazin-1-yl)acetamide (14k). Compound 14k

was synthesized as per general procedure described above as brownish

white solid in the yield of 80%, mp 236–238 ^◦C. ¹H NMR (400 MHz,

CDCl₃): δ 9.88 (s, 1H), 7.64–7.62 (d, J = 8, 2H), 6.97–6.94 (d, J = 12,

2H), 3.82 (s, 3H), 3.17 (s, 2H), 3.08 (bs, 3H), 2.67–2.64 (t, J = 12, 4H),

2.56–2.46 (dt, J = 8, 12, 4H), 1.78–1.70 (m, 5H); ¹³C NMR (100 MHz,

CDCl₃): δ 166.38, 166.30, 145.7, 130.8, 129.7, 128.6, 126.9, 114.4,

15

P. Gediya et al.

Bioorganic Chemistry 110 (2021) 104801

114.1, 94.1, 60.2, 55.6, 52.6, 46.0, 24.0, 23.9, 23.0, 22.0. MS (ESI) calcd

for C₂₂H₂₆N₄O₄S₂[M⁺] 474.14; found: 475.4 [M+H]. HPLC analysis:

retention time = 8.420 min; peak area = 98.94%; eluent A, ACN; eluent

reaction. Progress of the reaction was monitored on TLC. After

completion of the reaction, the reaction mixture was poured into ice-

cold water and extracted with ethyl acetate (10 mL × 2). The com-

bined organic layer was dried over anhydrous Na₂SO₄and concentrated

in vacuo. Crude 15 was puriﬁed by column chromatography using

hexane: ethyl acetate (60:40) as a mobile phase.

B, H₂O; isocratic (70:30) over 20 min with a ﬂow rate of 1 mL min^{ꢀ 1}

.

4.2.9.12. 2-(4-((4-Chlorophenyl)sulfonyl)piperazin-1-yl)-N-(3-cyano-

4,5,6,7-tetrahydrobenzo[b] thiophen-2-yl)acetamide (14l). Compound

14l was synthesized as per general procedure described above as off

white solid in the yield of 75%, mp 223–225 ^◦C. ¹H NMR (400 MHz,

CDCl₃): δ 9.90 (s, 1H), 7.65–7.63 (d, J = 8, 2H), 7.48–7.46 (d, J = 8, 2H),

3.19 (s, 2H), 3.13 (bs, 3H), 2.68–2.66 (t, J = 8, 4H), 2.56–2.47 (dt, J = 4,

12, 4H), 1.78–1.70 (m, 5H); ¹³C NMR (100 MHz, CDCl₃): δ 166.2, 145.7,

139.8, 134.1, 130.8, 129.6, 129.0, 128.6, 114.2, 94.2, 60.1, 52.5, 46.0,

24.0, 23.9, 23.0, 22.0. MS (ESI) calcd for C₂₁H₂₃ClN₄O₃S₂[M⁺] 478.09;

found: 479.3 [M+H], 481.3 [M+2]. HPLC analysis: retention time =

10.280 min; peak area = 99.28%; eluent A, ACN; eluent B, H₂O; isocratic

4.2.10.1. 2-(4-(4-Aminobenzoyl)piperazin-1-yl)-N-(3-cyano-4,5,6,7-tet-

rahydrobenzo[b]thiophen-2-yl)acetamide (15). Compound 15 was syn-

thesized as per general procedure described above as pale yellowish

white solid in the yield of 60%, mp 136–138 ^◦C. ¹H NMR (400 MHz,

DMSO‑d₆): δ 11.21 (bs, 1H), 7.13–7.11 (d, J = 8, 2H), 6.56–6.54 (d, J =

8, 2H), 5.54 (s, 2H), 3.53 (bs, 4H), 2.60–2.55 (m, 5H), 2.49(bs, 5H), 1.75

(bs, 4H); ¹³C NMR (100 MHz, DMSO‑d₆): δ 169.8, 167.7, 150.5, 146.0,

130.5, 129.2, 127.4, 114.1, 112.6, 93.1, 59.5, 52.4, 23.4, 23.3, 22.5,

21.6. MS (ESI) calcd for C₂₂H₂₅N₅O₂S [M⁺] 423.17; found: 424.3

[M+H]. HPLC analysis: retention time = 5.10 min; peak area = 100.0%;

eluent A, ACN; eluent B, H₂O; isocratic (70:30) over 20 min with a ﬂow

(70:30) over 20 min with a ﬂow rate of 1 mL min^{ꢀ 1}

.

rate of 1 mL min^{ꢀ 1}

.

4.2.9.13. N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-2-(4-(thi-

ophen-2-yl-sulfonyl) piperazin-1-yl)acetamide (14m). Compound 14m

was synthesized as per general procedure described above as brownish

solid in the yield of 60%, mp 195–197 ^◦C. ¹H NMR (400 MHz, CDCl₃): δ

9.96 (s, 1H), 7.62–7.60 (d, J = 8, 1H), 7.51–7.50 (m, 1H), 7.13–7.11 (t,

J = 8, 1H), 3.20 (s, 2H), 3.16 (s, 3H), 2.72–2.69 (t, J = 12, 4H),

2.57–2.47 (dt, J = 4, 12, 4H), 1.78–1.73 (m, 4H), 1.71 (s, 1H); ¹³C NMR

(100 MHz, CDCl₃): δ 166.2, 145.8, 135.4, 132.7, 132.6, 130.8, 128.6,

127.8, 114.2, 94.1, 60.1, 52.4, 46.2, 24.0, 23.9, 23.0, 22.0. MS (ESI)

calcd for C₁₉H₂₂N₄O₃S₃[M⁺] 450.09; found: 451.2 [M+H]. HPLC

analysis: retention time = 7.783 min; peak area = 100%; eluent A, ACN;

eluent B, H₂O; isocratic (70:30) over 20 min with a ﬂow rate of 1 mL

4.3. Biological assay methods

4.3.1. In vitro HeLa cell broth [47]

The HDAC inhibitory activity was determined using a colorimetric-

based EpiQuik HDAC activity/inhibition assay kit (Epigentek, Farm-

ingdale, NY). Brieﬂy, HeLa cells were treated with the synthesised

compounds (10 µmol) for 24 h. The cell pellets were collected and re-

suspended in lysis buffer and incubated under shaking for 30 min at

4 ^◦C to prepare the nuclear fractions. This was centrifuged at 14,000g for

10 min at 4 ^◦C, and the supernatants (nuclear fractions) were stored at

ꢀ 80 ^◦C for further tests. Initially, the biotinylated HDAC substrate was

added to a 96-well strip plate and incubated for 45 min. HDAC assay

min^{ꢀ 1}

.

buffer and prepared nuclear extracts (2 μL) were added to the plate after

4.2.9.14. N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-2-(4-

(methylsulfonyl)piperazin-1-yl)acetamide (14n). Compound 14n was

synthesized as per general procedure described above as white solid in

the yield of 85%, mp 217–219 ^◦C. ¹H NMR (400 MHz, CDCl₃): δ 10.27 (s,

1H), 3.64 (s, 1H), 3.32 (s, 3H), 3.25 (s, 2H), 2.77 (s, 3H), 2.74–2.71 (t, J

= 12, 4H), 2.59–2.51 (dt, J = 4, 8, 4H), 1.81–1.73 (m, 4H); ¹³C NMR

(100 MHz, CDCl₃): δ 166.2, 146.2, 130.7, 128.6, 114.5, 94.0, 67.1, 60.1,

52.6, 46.0, 34.2, 24.09, 24.00, 23.0, 22.0. MS (ESI) calcd for

C₁₆H₂₂N₄O₃S₂[M⁺] 382.11; found: 383.3 [M+H]. HPLC analysis:

retention time = 5.063 min; peak area = 100%; eluent A, ACN; eluent B,

washing and incubated for 1 h. Then, the plates were treated with

antibody capture and incubated for 1 h. After incubation, antibody

detection and developing solution were added. After addition of the stop

solution, the absorbance was measured at 450 nm in an ELISA plate

reader (Bio-Tek Instruments Inc.). Vorinostat was used as a positive

control (Sigma, India). The given formula was used to calculate HDAC

inhibition.

% Inhibition = (1 ꢀ [(control – blank) – (inhibitor sample – blank)]/

[(control – blank) – (no inhibitor sample – blank)] × 100%.

H₂O; isocratic (70:30) over 20 min with a ﬂow rate of 1 mL min^{ꢀ 1}

.

4.3.2. In vitro HDACs isoform selectivity assay

HDACs isoform selectivity inhibition assays were conducted based

on the previously reported ﬂuorescent assay system [48]. FLUOR DE

LYS® deacetylase substrate based on residues 379–382 of p53 (Arg-His-

Lys-Lys(Ac), BML-KI 177, Enzo) was used for HDAC1, 2, and 6 (Sig-

nalchem). A synthesized ﬂuorogenic HDAC class II substrate Boc-Lys

(TFA)-AMC was used as a substrate for HDAC4 (Signalchem) [49].

HDAC assay buffer (BPS), developer*2 (BPS), and DMSO (nacalai) were

also used in the assays. Experiments were conducted in 96-well black

plates (Corning Incorporated, 3694) according to the following protocol.

Synthesized compounds (5% DMSO in HDAC assay buffer, 10 µL/well)

and HDACs solution (1.0 ng/µL in HDAC assay buffer, 20 µL/well) were

incubated for 10 min at 25 ^◦C. Then, the reaction was started by the

addition of substrate (12.5 µM in HDAC assay buffer, 20 µL/well) and

incubated for 3 h at 25 ^◦C. Reaction was stopped by addition devel-

oper*2 (doubling dilution with HDAC assay buffer, 50 µL/well) and

further incubated for 30 min at 25 ^◦C. The ﬂuorescence was measured by

Ensight® readers (PerkinElmer Ltd.) with excitation at 380 nm and

emission at 460 nm. The IC₅₀values were calculated from ﬁve data point

dose response curve (n = 3) and determined by regression analysis of the

concentration/inhibition data by GraFit 7.

4.2.9.15. N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-2-(4-((4-

ﬂuorophenyl)sulfonyl) piperazin-1-yl)acetamide (14o). Compound 14o

was synthesized as per general procedure described above as white solid

in the yield of 85%, mp 210–212 ^◦C. ¹H NMR (400 MHz, CDCl₃): δ 9.93

(s, 1H), 7.74–7.69 (m, 2H), 7.21–7.15 (m, 2H), 3.19 (s, 2H), 3.11 (s, 3H),

2.69–2.66 (t, J = 12, 4H), 2.56–2.46 (dt, J = 4, 8, 4H), 2.38 (s, 4H),

1.75–1.70 (m, 1H), 1.60 (s, 1H); ¹³C NMR (100 MHz, CDCl₃): δ 166.7,

166.2, 164.1, 145.8, 131.5, 131.4, 130.8, 130.3, 130.2, 128.6, 116.6,

116.5, 114.1, 94.2, 60.1, 52.5, 46.1, 24.0, 23.9, 23.0, 22.0. MS (ESI)

calcd for C₂₁H₂₃FN₄O₃S₂[M⁺] 462.12; found: 463.3 [M+H]. HPLC

analysis: retention time = 8.447 min; peak area = 98.29%; eluent A,

ACN; eluent B, H₂O; isocratic (70:30) over 20 min with a ﬂow rate of 1

mL min^{ꢀ 1}

.

4.2.10. General procedure for the synthesis of 2-(4-(4-aminobenzoyl)

piperazin-1-yl)-N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)

acetamide (15) [44,45]

In a clean RBF, 14g (2.2 mmol) was added in a mixture of methanol

(1.0 mL) and water (0.5 mL). To this reaction mixture, dry activated zinc

(11 mmol) was added and followed by the addition of NH₄Cl (11 mmol).

The resulting mixture was stirred at 45 ^◦C until completion of the

16

P. Gediya et al.

Bioorganic Chemistry 110 (2021) 104801

4.3.3. In vitro antiproliferative assay [50]

10–15% polyacrylamide gels and 5 µg of histone extract into 15%, and

then transferred to a nitrocellulose membrane using a transfer apparatus

according to the manufacturer’s protocols (Bio-Rad). After blocking step

in 5% non-fat milk in TBST (10 mM Tris pH 8.0, 150 mM NaCl, 0.5%

TWEEN 20) for 60 min, the membrane was washed in TBST and incu-

bated with antibodies. Detection was performed with an ECL system

(Amersham Biosciences) according to the manufacturer’s protocol.

In vitro antiproliferative assay was performed using Cell Quanti-MTT

cell viability assay kit (Bioassay Systems) using MDA-MB-231, A549,

HeLa and hTERT RPE-1 cell lines (2 × 10⁵cells per well). MDA-MB-231

(human breast cancer), A549 (lung cancer) HeLa (human cervical car-

cinoma), and hTERT-RPE1 (normal retina epithelial cells immortalized

with hTERT) cells were obtained from NCCS (Pune, India) and main-

tained in medium DMEM and DMEM F12 (Gibco, Bangalore, India)

supplemented by 10 percent FBS (V/V), streptomycin (100 g/L) and

penicillin (100 IU/mL) (Himedia) to prevent microbial contamination.

In 96-well ﬂat bottom titer plate, the MDA-MB-231, A549, HeLa and

hTERT RPE-1 cells were seeded at a density of 2 × 10⁵cells per well and

incubated at 37 ^◦C for 24 h in an atmosphere containing 5% CO₂. The

media was extracted and replaced by a fresh medium containing syn-

thesized compounds at different concentrations and incubated at 37 ^◦C

for 24 h. After incubation, the culture media was drained and washed

thrice with phosphate buffer saline (PBS) and incubated at 37 ^◦C for 4 h

4.3.7. In vitro microsomal stability

A 10 mM stock solution of 9h, 14n and verapamil was prepared in

DMSO. From the intermediate stock solution of 2 mM, a working solu-

tion of 0.5 mM was prepared by diluting the compounds in acetonitrile:

water (50:50). The compounds (1.8 µL of working solution) were spiked

in 0.1 M potassium phosphate buffer (260.7 µL) at pH 7.4 and a con-

centration of 3 µM (0.1% DMSO). Following this, human and rat liver

microsomes (Invitrogen) (7.5 µL, ﬁnal protein concentration was 0.5

mg/mL) were added. The aforementioned sample was incubated at

37 ^◦C for 5 min. Subsequently, 30 µL of 10 mM NADPH prepared in 0.1

M potassium phosphate buffer was added (as a co-factor) to initiate the

reaction. The samples were then incubated at 37 ^◦C for desired time

points.

with 100

μL of MTT (5 mg/mL in PBS). Then the MTT was substituted

μ

L DMSO and blended well to remove the insoluble formazan.

Further, the absorbance was measured at 570 nm using a microplate

reader (Lark, India). The percentage of cell viability was calculated

using the formula:

At each time point (0, 5, 15, 30, 60, and 120 min), 40 µL of the

samples were withdrawn and reactions were stopped using 360 µL

chilled acetonitrile or methanol containing suitable internal standard

(carbamazepine). The samples were centrifuged and the supernatants

were analyzed in duplicate by LC-MS/MS. The percent compound

remaining at each time point was calculated with respect to that of the 0

min sample. The data were then analyzed in duplicate to calculate half-

life and intrinsic clearance (CLint). Control samples were run without

NADPH for initial and ﬁnal time point and blank samples were prepared

using DMSO (without the test compounds).

Cell viability (%) = (absorbance of sample/absorbance of control) ×

100, and further IC₅₀was determined using GraphPad prism 8.0.

4.3.4. Cell death analysis (FACS) and cell cycle analysis

4.3.4.1. Cell lines. U937 and MDA-MB-231cells were purchased from

DSMZ and grown following standard protocols at 37 ^◦C with 5% CO₂, in

RPMI (U937 cells) and DMEM (MDA-MB-231) supplemented with 10%

fetal bovine serum (FBS; Gibco), 2 mM ^L-glutamine (Euroclone), and

antibiotics (100 U/mL penicillin, 100 µg/mL streptomycin, and 250 ng/

mL amphotericin-B; Euroclone). Cells were mycoplasma free, tested by

EZ-PCR Mycoplasma Test Kit (Biological Industries). Cells were used for

experiments between passages 10 to 20 and then discarded.

4.4. In silico ADME evaluation

Pharmacokinetic and physicochemical properties such partition co-

efﬁcient (log P_o/w), aqueous solubility (log S), molecular weight (MW),

hydrogen bod donor (HBD), hydrogen bond acceptor (HBA), total polar

surface area (TPSA), molar refractivity (MR), number of rotatable bonds

with passive gastrointestinal absorption (GIA), brain penetration (BBB)

and cytochromes P₄₅₀(CYPs) inhibition were predicted for the synthe-

sized compounds of both the series and comapared with vorinostat using

online workstation SwissADME [52]. SwissADME is freely available at

http://www.swissadme.ch/index.php#.

4.3.4.2. Antibodies. Cyclin A and actin were purchased from Santa

Cruz. Acetyl tubulin was purchased from from Sigma. Cyclin D, cyclin E,

and histone H4 were purchased from Abcam. GAPDH from Cell

Signaling, and histone H3 K9/14ac was purchased from Diagenode.

4.3.5. Cell cycle and cell death analysis [51]

For cell cycle analysis and sub-G1 evaluation, U937 and MDA-MB-

231, after stimulation were harvested with PBS, centrifuged, and re-

4.5. Molecular docking

suspended in 500 μL of a hypotonic solution (1X PBS, 0.1% sodium

citrate, 0.1% NP-40, RNAase A, and 50 mg/mL PI). For cell death

evaluation, cells were harvested with PBS, centrifuged at 1200 rpm for 5

Docking study was performed using Gold 5.2.2 software on Intel

Chipset with Intel Core i3 second generation processor, 4 GB DDR3

RAM, and a clock speed of 3.3 GHZ [17]. We docked synthesized com-

pounds in HDAC6 protein (PDB ID: 5G0G, 1.499 Å) co-crystallized with

trichostatin A. HDAC6 co-crystallized structure was downloaded and

imported in the GOLD wizard. Hydrogen atoms were added and water

molecules were deleted. Crystallized trichostatin A was extracted and a

docking site was created. Synthesized compounds were imported with

reference standard ligand. The gold score was used as a ﬁtness function

to evaluate the score of compounds.

min, and re-suspended in 500 μL 1X PBS and 0.2 mg/mL PI. Data was

acquired using the BD Accuri TM C6 ﬂow cytometer system (BD Bio-

sciences). Each experiment was performed in biological triplicates and

values expressed as mean ± SD.

4.3.6. Western blot analysis

After treatment, cells were washed and lysed in two speciﬁc buffers,

one for protein total extract and one for histone proteins extract. For

total protein extract, lysis reaction was carried out for 15 min at 4 ^◦C in a

speciﬁc buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 10

mM NaF, 1 mM PMSF and protease inhibitors). Histone proteins were

obtained as suspended cellular pellet into triton extraction buffer (TEB)

(PBS containing 0.5% Triton X 100 (v/v), 2 mM PMSF, 0.02% (w/v)

NaN₃). The lysis was performed for 10 min at 4 ^◦C. After centrifugation

pellets were washed in TEB (half volume) and then resuspended in 0.2 N

HCl. Acid histone extraction was carried out overnight at 4 ^◦C. Proteins

concentration was quantiﬁed by Bradford assay (Bio-Rad).

Author contributions

The P.G. and M.D.G. designed the study. P.G. synthesized all the

molecules. P.G., and N.S. characterized all the synthesized molecules. P.

G. and V.K.V. designed and performed all the computational studies. V.

R. and K.S. performed a HeLa broth inhibition assay. V.C., L.D.T., A.P

performed cell cycle analysis, cell death evaluation, and western blot

analysis. L.A. evaluated the biomedical analyses and anticancer data. T.

K., and T.S, performed an HDAC enzyme inhibition assay. D.G., and D.B

Western blot was performed by lodging 50 µg total extract into

17

P. Gediya et al.

Bioorganic Chemistry 110 (2021) 104801

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performed an MTT assay. V.S. performed in vitro microsomal stability

assay. The manuscript was written by P.G., V.K.V., M.D.G., and also

through the contributions of all the co-authors. All co-authors have

approved the ﬁnal version of this manuscript.

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C. McCoy, A. Schott, C. Scholz, S.G. Eckhardt, A Phase I and pharmacokinetic study

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[16] V.K. Vyas, B. Variya, M.D. Ghate, Design, synthesis and pharmacological

evaluation of novel substituted quinoline-2-carboxamide derivatives as human

dihydroorotate dehydrogenase (hDHODH) inhibitors and anticancer agents, Eur. J.

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Declaration of Competing Interest

The authors declare that they have no known competing ﬁnancial

interests or personal relationships that could have appeared to inﬂuence

the work reported in this paper.

Acknowledgment

[17] N.D. Sitwala, V.K. Vyas, B.C. Variya, S.S. Patel, C.C. Mehta, D.N. Rana, M.D. Ghate,

Liquid phase combinatorial synthesis of 1,2,5-trisubstituted benzimidazole

derivatives as human DHODH inhibitors, Bioorg. Chem. 75 (2017) 118 –126.

[18] P.K. Parikh, M.D. Ghate, Recent advances in the discovery of small molecule c-Met

Kinase inhibitors, Eur. J. Med. Chem. 143 (2018) 1103 –1138 .

All of the authors are grateful to DST INSPIRE-Department of Science

and Technology, INDIA for ﬁnancial assistance under Project number

DST INSPIRE/IF160300. Vanvitelli per la Ricerca: “CAMPANIA” (ID

342) and “AdipCare” (ID 263); Campania Regional Government Tech-

nology Platform Lotta alle Patologie Oncologiche: iCURE

(B21C17000030007); Campania Regional Government FASE2: IDEAL

(B63D18000560007). MIUR, Proof of Concept POC01_00043; AP holds

a PhD fellowship coded ’B25D18000010006’ PON-2017; LDT holds a

PhD fellowship coded ’B27D18001030006’ PON-2018. PG is thankful to

all the collaborators for their kind support in this research. This research

work is a part of the Ph.D. thesis of Piyush Gediya (PG), Institute of

Pharmacy, Nirma University, Ahmedabad, India.

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[21] K.J. Coe, T. Koudriakova, Metabolic stability assessed by liver microsomes and

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Appendix A. Supplementary material

[25] C. Cheng, F. Yun, J. He, S. Ullah, Q. Yuan, Design, synthesis and biological

evaluation of novel thioquinazolinone-based 2-aminobenzamide derivatives as

potent histone deacetylase (HDAC) inhibitors, Eur. J. Med. Chem. 173 (2019)

185 –202 .

Supplementary data to this article can be found online at https://doi.

org/10.1016/j.bioorg.2021.104801.

[26] T. Abdizadeh, M.R. Kalani, K. Abnous, Z. Tayarani-Najaran, B.Z. Khashyarmanesh,

R. Abdizadeh, R. Ghodsi, F. Hadizadeh, Design, synthesis and biological evaluation

of novel coumarin-based benzamides as potent histone deacetylase inhibitors and

anticancer agents, Eur. J. Med. Chem. 132 (2017) 42 –62 .

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19

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Discovery of novel tetrahydrobenzo[b]thiophene-3-carbonitriles as histone deacetylase inhibitors

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