Structure-based design generated novel hydroxamic acid based preferential HDAC6 lead inhibitor with on-target cytotoxic activity against primary choroid plexus carcinoma

Article abstract of DOI:10.1080/14756366.2019.1613987

Histone deacetylase 6 (HDAC6) is an attractive target for cancer therapeutic intervention. Selective HDAC6 inhibitors is important to minimise the side effects of pan inhibition. Thus, new class of hydroxamic acid-based derivatives were designed on struct

Full text of DOI:10.1080/14756366.2019.1613987

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Structure-based design generated novel
hydroxamic acid based preferential HDAC6 lead
inhibitor with on-target cytotoxic activity against
primary choroid plexus carcinoma
Shaymaa E. Kassab, Samar Mowafy, Aya M. Alserw, Joustin A. Seliem,
Shahenda M. El-Naggar, Nesreen N. Omar & Mohamed M. Awad
To cite this article: Shaymaa E. Kassab, Samar Mowafy, Aya M. Alserw, Joustin A. Seliem,
Shahenda M. El-Naggar, Nesreen N. Omar & Mohamed M. Awad (2019) Structure-based design
generated novel hydroxamic acid based preferential HDAC6 lead inhibitor with on-target cytotoxic
activity against primary choroid plexus carcinoma, Journal of Enzyme Inhibition and Medicinal
Chemistry, 34:1, 1062-1077, DOI: 10.1080/14756366.2019.1613987
To link to this article: https://doi.org/10.1080/14756366.2019.1613987
© 2019 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
Group.
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2019, VOL. 34, NO. 1, 1062–1077
https://doi.org/10.1080/14756366.2019.1613987
RESEARCH PAPER
Structure-based design generated novel hydroxamic acid based preferential
HDAC6 lead inhibitor with on-target cytotoxic activity against primary choroid
plexus carcinoma
Shaymaa E. Kassaba, Samar Mowafyb, Aya M. Alserwc, Joustin A. Seliemc, Shahenda M. El-Naggarc,
Nesreen N. Omard and Mohamed M. Awade,f
b
aPharmaceutical Chemistry Department, Faulty of Pharmacy, Damanhour University, Damanhour, Egypt; Pharmaceutical Chemistry Department,
c
Faculty of Pharmacy, Misr International University, Cairo, Egypt; Basic Research Unit, Department of Research, Children’s Cancer Hospital in
d
Egypt, Cairo, Egypt; Biochemistry Department, Faculty of Pharmacy, Modern University for Technology and Information, Cairo, Egypt;
f
eDepartment of Pharmacology and Toxicology, Faculty of Pharmacy, Helwan University, Cairo, Egypt; Canadian Academy of Research and
Development (CARD), Mississauga, ON, Canada
ABSTRACT
ARTICLE HISTORY
Received 26 March 2019
Revised 19 April 2019
Accepted 23 April 2019
Histone deacetylase 6 (HDAC6) is an attractive target for cancer therapeutic intervention. Selective HDAC6
inhibitors is important to minimise the side effects of pan inhibition. Thus, new class of hydroxamic acid-
based derivatives were designed on structural basis to perform preferential activity against HDAC6 target-
ing solid tumours. Interestingly, 1-benzylbenzimidazole-2-thio-N-hydroxybutanamide 10a showed impres-
sive preference with submicromolar potency against HDAC6 (IC50 ¼ 510nM). 10a showed cytotoxic activity
with interesting profile against CCHE-45 at (IC50 ¼ 112.76 mM) when compared to standard inhibitor
Tubacin (IC50 ¼ 20 mM). Western blot analysis of acetylated-a-tubulin verified the HDAC6 inhibiting activity
of 10a. Moreover, the insignificant difference in acetylated-a-tubulin induced by 10a and Tubacin implied
the on-target cytotoxic activity of 10a. Docking of 10a in the binding site of HDAC6 attributed the activity
of 10a to p-p stacking with the amino acids of the hydrophobic channel of HDAC6 and capture of zinc
metal in bidentate fashion. The therapeutic usefulness besides the on-target activity may define 10a as an
interesting safe-lead inhibitor for future development.
KEYWORDS
Preferential HDAC6
inhibitor; acetylated-
a-tubulin; on-target activity;
benzimidazole; acute
promyeloblastic leukemia;
choroid plexus carcinoma;
cytotoxicity
R
N
S
O
N
HN OH
6a-d
R
1. Enzymatic assay
2. Cell-based assay
N
N
N
S
S
N
O
O
10a-d
10a
HN OH
HN OH
Preferential HDAC6 Lead inhibitor
HDAC6 IC50 = 510 nM
R = C6H5CH2, 4-BrC6H4CH2, C6H11CH2, C6H11C2H4
Hydrphobic cap Linker ZBG
Brain cancer cells CCHE-45 IC50 = 112.67µM
CONTACT Shaymaa E. Kassab
shaymaakassab@yahoo.com
Pharmaceutical Chemistry Department, Faulty of Pharmacy, Damanhour University, Damanhour, El-
Buhaira 22516, Egypt
Supplemental data for this article can be accessed here.
ß 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1063
Figure 1. Schematic representation and functional domains of human HDAC6. HDAC6 is the only HDAC with two tandem deacetylase domains (DD1 and DD2) includ-
ing catalytic activity. A nuclear export signal (NES) prevents the accumulation of the protein in the nucleus and the Ser-Glu-containing tetrapeptide (SE14) region
ensures stable anchorage of the enzyme in the cytoplasm. The nuclear localisation signal (NLS) translocates HDAC6 into nucleus. The linker (dynein motor binding,
DMB) between both CATs can bind to dynein and the high affinity ubiquitin-binding zinc finger domain (BUZ). aa, amino acid.
HDAC8 due to the distinct protein structural differences between
these two specific isoforms and other isoforms.
1. Introduction
Mechanisms and signalling pathways that lead to transformation
of normal cell into cancer cell occupy a remarkable space in the
experimental oncology researches. This type of biological studies
succeeded to identify some important genes1,2, proteins3, tran-
scriptional and epigenetic factors4–6 that contribute to the hall-
marks of cancer7. Epigenetic modifications are associated with
changes in gene transcription, and alteration in chromatin struc-
ture8. The main epigenetic modifications include histone methyla-
tion and acetylation8 the reversible addition and removal of acetyl
group is governed by the controlled expression and activity of his-
tone acetyltransferases (HATs) as well as histone deacetylases
(HDACs)9,10. Regularly, chromatin is switched between two states:
the loose state and condensed state. The loose form of the chro-
matin (euchromatin) in which the histone protein is acetylated in
lysine residues by HATs, exposes genes for transcription11. On con-
trary, HDACs are associated with chromatin condensed form12,
this heterochromatin structure intervenes with gene expression.
There are four classes and two families of human HDACs
involving 18 mammalian isoforms with different physiological
functions and distinct cellular compartments; some are either
solely present in the nucleus or the cytosol, while other isoforms
are shuttling between the nucleus and the cytosol13–15. Eleven iso-
forms of deacetylases (HDAC 1–11) which constitute the classical
family, share similar structure, and require Zn2þ for their activity
onset. While the sirtuin family contains 7 isoforms (SIRT 1–7),
which are structurally different from the classical family, and they
are NAD-dependent16,17. HDACs are deregulated in different can-
HDAC6 is found to be overexpressed in several cancer cell
types39–44, it is also implicated in the onset or the progression of
many neurodegenerative diseases45–47 and autoimmune disor-
ders48–50. HDAC6 protein is the only isoform with two active
deacetylase domains that are identical and function independ-
ently51; the linker between the two domains is the dynein motor
binding (DMB) domain, and cytoplasmic retention signal (SE14)
motif that enables the enzyme to reside in the cytoplasm to per-
form its functions regularly (Figure 1)52. Zinc finger ubiquitin bind-
ing domain (BUZ) is located at the C-terminal and is absent in the
other HDAC isoforms14. Due to that unique structure, and cyto-
plasmic localisation, HDAC6 is able to deacetylate non-histone
proteins; such as a-tubulin, heat shock protein 90 (Hsp90), and
cortactin53. The post-translational modification of these non-his-
tone proteins contribute to cancer cell proliferation, migration,
protein homoeostasis, regulation of expression of critical immune
modulators, the stability and activity of transcriptional factors such
as hypoxia inducible factors (HIFs), the activity of estrogenic and
androgenic receptors, and platelet aggregation in coagulation
process14,36,44,53,54
.
Upon surveying the literature on the chemical structures of the
reported selective HDAC6 inhibitors, we have found that all are
with large rigid or non-rigid cap; a common structural feature as
depicted in the representative examples; Tubacin55, Rocillinostat39
and Tubastatin A56 (Figure 2). The linker properties regarding the
length and saturation/unsaturation varied from structure to
another, and ZBG group was conserved with all the reported
structures57. Tubacin that is the first reported as potent selective
HDAC6 inhibitor (HDAC6 IC50 ¼ 4 nM)55,56; however, it has never
been able to be tested in vivo due to its complex structure and
high lipophilicity44. Rocillinostat; although it is the considered the
first selective inhibitor of HDAC6, which is orally available and has
moved to phase I/II of clinical trials for treating multiple myeloma
as well as lymphoma58–60, its potency in solid tumours is very low
as a single agent61 and has not been extensively investigated.
Tubastatin A was reported as selective HDAC6 inhibitor with neu-
roprotective activity56 and no study proved its potential antiproli-
ferative activity against any type of cancer, but it increased the
sensitivity of non-small cell lung cancer (NSCLC) to cisplatin62.
Furthermore, Tubastatin A is of rigid tertahydro-c-carboline hydro-
phobic cap that is not accessible to introduce different variables
to develop but ring extension31. This gap encouraged scientists to
initiate serious attempts to identify new HDAC6 selective inhibi-
tors to combat specific types of cancer. Accordingly, we aimed at
generating a lead compound that initially performs preferential
HDAC6 inhibition, with new chemical entity, and is feasible to be
cer types,8 however, the intricate physiological role of HDAC and
its involvement in normal cell proliferation18, mitosis,
develop-
19
ment, cardiac morphogenesis20, signal transduction pathways21,22
,
and apoptosis19,23–25 made pan inhibition of HDACs leads to
exhibition of toxicity26–30. Researchers worked extensively on pro-
viding structural determinants for the generation of selective or
even preferential inhibitors against one isoform12,31–33 or group of
isoforms13,34,35. Selective HDAC inhibition reduces the toxicity that
results from complete shutdown of normal physiological functions
of HDACs due to pan inhibition. It was reported that the zinc-
dependent HDAC isoforms require an inhibitor with the following
characterisations: hydrophobic cap, metal deter and a linker to
link between the metal deter and the cap36. Only HDAC6 and
HDAC8 that can accommodate large hydrophobic cap due to the
presence of a second tube-like pocket with a different shape and
close to the active hydrophobic pocket37,38 when compared to
the other isoforms, but the difference between HDAC6 and
HDAC8 is the protein surface flexibility that was identified as a
factor of selectivity36. The other isoforms are closely similar and
the elaboration of selective inhibitors against them is yet, very
limited and not feasible to achieve36. Thus, many selective inhibi- synthesised and developed. Enzymatic assay against all human
tors that were discovered where almost against HDAC6 and/or HDAC isoforms are going to be conducted to determine the

1064
S. E. KASSAB ET AL.
O
OH
NH
O
O
H
N
O
N
O
O
N
OH
OH
N
N
H
N
H
S
N
N
N
O
Rocilinostat (ACY-1215)
HDAC6 IC50 = 4.7 nM
Tubastatin A
HDAC6 IC50 = 15 nM
HO
N
H
O
Tubacin
HDAC6 IC50 = 4 nM
Hydrophobic cap
Linker
ZBG
Figure 2. Representative examples of most popular selective HDAC6 inhibitors with the corresponding potencies.
Active sites accept variables
R
H
N
N
S
O
SH
N
N
n
HN OH
R = C6H5, 4-BrC6H4, C6H11CH2, C6H11C2H4
n = 1, 3
Site for introduction of variables to build up large hydrophobic cap
Site for introduction of linker variables
Figure 3. Rational design of preferential HDAC6 lead inhibitor.
preferential inhibition activity of the synthesised compounds, spectra were recorded as potassium bromide discs on Schimadzu
1
which will be coupled with the cytotoxic activity against specific FT-IR 440 spectrometer. H NMR spectra were recorded on Bruker
type of children brain cancer; primary choroid plexus carcinoma spectrophotometer at 400 MHz in DMSO-d6; values (d) are given in
(CCHE-45) and cell-based assays for detection of acety-
lated a-tubulin.
parts per million (ppm) downfield from tetramethylsilane (TMS) as
internal reference. 13 C NMR spectra were recorded using the same
1
Thus, it is important to emphasise the critical approaches
toward suggesting a structural design for elaboration of new lead
with preferential HDAC6 inhibiting activity: i) large hydrophobic cap
for possible accommodation in the HDAC6 hydrophobic channel
which reduces the chance of perfect fitting with the other HDAC iso-
forms, ii) different linker lengths were used because no definite report
generally defines the recommended length of linker, iii) zinc binding
group (ZBG) is included as a conservative fragment63,64 that is essen-
tial to capture the zinc metal of the enzyme to stop its catalysis and
enhance the stable residence of inhibitor into the catalytic domain.
Consequently, it was suggested to work on binuclear aromatic
heterocycle with flexible chemical entity to accommodate differ-
ent variables for further modifications that might be useful for
future development of the generated lead inhibitor to improve
both the potency and selectivity. 2-Mercaptobenzimidazole was
chosen as the scaffold of the target inhibitor based on the accessi-
bility of two structural active sites for introduction of different var-
iables which are N1-benzimidazole and 2-mercaptobenzimidazole.
Both sites, are acidic with different pKa values, would play a role
in optimisation of the reaction conditions to introduce the differ-
ent variables at the target site and avoid multiple alkylation or
even getting undesirable regioisomers (Figure 3).
spectrophotometer that used for recording H NMR at 101 MHz in
DMSO-d6. Mass spectra were recorded on ACQUITY UPC2 System
mass spectrometer for ES detection and Schimadzu Triple
Quadrupole GC-MS mass spectrometer for EI detection. The elem-
ental analyses were performed at the Microanalytical Center, Cairo
University, Cairo, Egypt. Reactions were followed up by thin layer
chromatography (TLC) using Merck Silica gel/TLC cards with fluor-
escent indicator UV254 using Hexane:Ethyl acetate (EtOAc) 4:1 and
DCM:MeOH 9.5:0.5 as the eluting systems and the spots were
visualised using Spectroline E series dual wavelength UV lamp
at k ¼ 254 nm.
2.1.1. Synthesis of ethyl (1H-benzo[d]imidazol-2-yl)thioacetate (3)
A mixture of 2-mercaptobenzimidazole 1 (0.75 g, 5 mmol) and
ethyl chloroacetate 2 (0.75 ml, 7.5 mmol) was added to a stirred
solution of absolute ethanol at 60ꢀ C, then anhydrous K2CO3
(1.035 g, 7.5 mmol) was added. The reaction mixture was left stir-
ring overnight. The inorganic salts were filtered off, the solvent
was evaporated under reduced pressure and the white organic
residue was washed with water several times to furnish white
fibrous crystals that was pure enough to be submitted to the next
step of the reactions.
Yield: 34%, m.p.: 60 ꢀC, white microcrystals; 1H NMR (DMSO-
d6, 400 MHz): d 12.60 (s, 1H, NH), 7.48 (d, J ¼ 5.1 Hz, 1H, 8-H), 7.38
(s, 1H, 5-H), 7.11 (dd, J ¼ 5.7, 2.2 Hz, 2H, 6,7-H), 4.20 (s, 2H, S-CH2),
2. Materials and methods
2.1. Chemistry
Melting points were determined on digital Gallen-Kamp MFB-595 4.12 (q, J ¼ 7.1 Hz, 2H, COOCH2CH3), 1.17 (t, J ¼ 7.1 Hz,
instrument using open capillary tubes and are uncorrected. IR 3H, COOCH2CH3).

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1065
2.1.2. General synthesis of ethyl 2-((1-(benzyl/cycloalkyl-1H-benzo[- 2.1.5. 2-((1-benzyl-1H-benzo[d]imidazol-2-yl)thio)-N-hydroxyaceta-
mide (6a)
d]imidazol-2-yl)thio)acetates (5a-d)
Yield: 22.0%, m.p.: 97 ꢀC, golden yellow microcrystals; IR (KBr)
V
Anhydrous K2CO3 (0.21 g, 1.5 mmol) was added to a stirred mix-
ture of ethyl (benzimidadol-2-yl)thioacetate 3 (0.236 g, 1 mmol)
and benzyl chloride 4a (0.12 ml, 1 mmol) or 4-bromobenzyl brom-
ide 4b (0.25 g, 1 mmol) or cyclohexylmethyl bromide 4c (0.14 ml,
1 mmol) or cyclohexylethyl bromide 4d (0.16 ml, 1 mmol) in DMF
and the temperature was raised to 100ꢀ C. The reaction mixture
was judged complete after 1 h upon checking with TLC using
Hexane:EtOAc 4:1 as eluting system. The reaction mixture was
poured onto water and the organic product was extracted by
ethyl acetate, the organic extract was washed with water several
times then dried over anhydrous sodium sulphate. The solvent
was evaporated under reduced pressure to give the product as
dark oil residue. All trials to recrystallise the product from
Methanol or ethanol furnished only Ethyl 2-((1–(4-bromobenzyl)-
1H-benzo[d]imidazol-2-yl)thio)acetate (5b) from methanol as
golden yellow microcrystals but all the other derivatives 5a, 5c,
and 5d were obtained as oil products in yields 45%, 54% and
65% respectively with enough purity to be submitted to the next
step of reactions.
max/cmꢁ1: 3427 (broad band NH/OH), 1717 (CO). 1H NMR
(DMSO-d6, 400 MHz): d 7.57 – 7.51 (m, 1H, Ar-H)), 7.51–7.44 (m,
1H, Ar-H)), 7.36–7.22 (m, 5H, Ar-H), 7.19–7.12 (m, 2H, Ar-H), 5.41 (s,
2H, C6H5CH2), 4.20 (s, 2H, S-CH2), 3.60 (broad s, 2H). 13 C NMR
(DMSO-d6, 101 MHz):d 169.68 (CO), 151.16 (S-C), 142.91, 136.32,
128.82, 127.85, 127.20, 121.92, 121.82, 117.77, 109.92 (Ar-C), 46.82
(C6H5CH2), 34.37 (S-CH2). MS LCMS (ES): (calc) 313.09 (found)
314.1561 (MH)þ. Anal. Calcd for C16H15N3O2S: C, 61.32; H, 4.82; N,
13.41. Found: C, 61.49; H, 5.02; N, 13.25.
2.1.6.
2-((1–(4-bromobenzyl)-1H-benzo[d]imidazol-2-yl)thio)-N-
hydroxyacetamide (6b)
Yield: 19.0%, m.p.: 167 ꢀC, golden yellow microcrystals; IR (KBr)
V
max/cmꢁ1: 3405 (broad band NH/OH), 1713 (CO). 1H NMR
(DMSO-d6, 400 MHz): d 7.52 (d, J ¼ 8.1 Hz, 3H, Ar-H), 7.45 (d,
J ¼ 4.8 Hz, 1H, Ar-H), 7.16 (dd, J ¼ 11.6, 6.1 Hz, 4H, Ar-H), 5.39 (s,
2H, 4-BrC6H4CH2), 4.46 (broad s, 2H), 4.14 (s, 2H, S-CH2). 13 C NMR
(DMSO-d6, 101 MHz): d 169.84 (CO), 151.73 (S-C), 142.97, 136.09,
135.81, 131.65, 129.31, 121.79, 121.72, 120.86, 117.70, and 109.68
(Ar-C), 46.08 (4-BrC6H4CH2), 36.04 (S-CH2). MS LCMS (ES): (calc)
391.00 (found) 392.0124(MH)þ. Anal. Calcd for C16H14BrN3O2S: C,
48.99; H, 3.60; N, 10.71. Found: C,48.71; H, 3.81; N, 10.94.
2.1.3. Ethyl 2-((1–(4-bromobenzyl)-1H-benzo[d]imidazol-2-yl)thio)a-
cetate (5b)
Yield: 40%, m.p.: 107 ꢀC, golden yellow needles; IR (KBr) Vmax
/
cmꢁ1:1741 (CO). 1H NMR (DMSO-d6, 400 MHz): d 7.56–7.52 (m,
3H, Ar-H), 7.50–7.46 (m, 1H, Ar-H), 7.20–7.14 (m, 4H, Ar-H), 5.41 (s,
2H, 4-BrC6H4CH2), 4.26 (s, 2H, S-CH2), 4.11 (q, J ¼ 7.1 Hz, 2H,
COOCH2CH3), 1.16 (t, J ¼ 7.1 Hz, 3H, COOCH2CH3). 13 C NMR
(DMSO-d6, 101 MHz): d 168.35 (CO), 150.63 (S-C), 142.86, 136.22,
2.1.7.
2-((1-(Cyclohexylmethyl)-1H-benzo[d]imidazol-2-yl)thio)-N-
hydroxyacetamide (6c)
Yield: 10.0%, m.p.: 160 ꢀC, golden yellow needle crystals; IR (KBr)
V
max/cmꢁ1: 3429 (broad band NH/OH), 1712 (CO). 1H NMR
(DMSO-d6, 400 MHz): d 7.50 (dd, J ¼ 8.4, 6.8 Hz, 2H, 6,7-H), 7.21 –
135.73, 131.68, 129.29, 122.01, 121.88, 120.94, 117.85, and 109.85 7.09 (m, 2H, 6,7-H), 4.18 (s, 2H, S-CH2), 3.98 (d, J ¼ 7.3 Hz, 2H,
C6H11CH2), 3.45 (broad s, 2H), 1.82–1.86 (m, 1H, C6H11-H),
1.66–1.51 (m, 5H, C6H11-H), 1.13–1.04 (m, 5H, C6H11-H). 13 C NMR
(DMSO-d6, 101 MHz): d 169.68 (CO), 151.06 (S-CH2), 142.64,
136.72, 121.64, 121.50, 117.60, and 110.02 (Ar-C), 49.60 (C6H11CH2),
37.81(S-CH2), 34.29 (C6H11-C-1), 30.23(C6H11-C-2,6), 25.80 (C6H11-C-
3,5), 25.22 (C6H11-C-4). MS LCMS (ES): (calc) 319.14 (found)
320.1853 (MH)þ. Anal. Calcd for C16H21N3O2S: C, 60.16; H, 6.63; N,
13.16. Found: C, 60.37; H, 6.94; N, 13.38.
(Ar-C), 61.27 (COOCH2CH3), 46.11(4-BrC6H4CH2), 33.83(S-CH2), 14.01
(COOCH2CH3). MS LRMS (EI): (calc) 404.02 (found) 404.08 (M)þ.
Anal. Calcd for C18H17BrN2O2S: C, 53.34; H, 4.23; N, 6.91. Found: C,
53.56; H, 4.11; N, 7.19.
2.1.4. General synthesis of 2-((1-benzyl/cycloalkyl-1H-benzo[d]imi-
dazol-2-yl)thio)-N-hydroxyacetamides (6a-d)
Hydroxylamine hydrochloride (10 equivalent) was neutralised in
1 M NaOMe solution in methanol (14 equivalent) via stirring for
10 min and filtered off to discard sodium chloride. Hydroxylamine
solution was added to a stirred solution of the respective ester
(5a-d) (1 equivalent) in methanol (5 ml) at room temperature. The
reaction mixture was left stirring until judged complete upon
observing disappearance of the spot of ester starting material on
TLC after 10 min using DCM:MeOH 9.5:0.5 as eluting system. The
solvent was evaporated under reduced pressure and the crude
residue was quenched with distilled water (15–20 ml). All the
organic dirt of the aqueous reaction mixture was scavenged while
vigorous shaking with EtOAc and the aqueous extract of the prod-
uct was separated free from the organic dirt remained from the
reaction. The aqueous extract of the product was acidified with
drop wise addition of 10% HCl until observing precipitation at
pH ¼ 5.0. The resulting suspension was left in the fridge overnight
for the product to complete its precipitation. The precipitate was
filtered off, washed several times with distilled water and dried
under vacuum to give crystalline product.
2.1.8.
2-((1–(2-cyclohexylethyl)-1H-benzo[d]imidazol-2-yl)thio)-N-
hydroxyacetamide (6d)
Yield: 9.0%, m.p.: 157 ꢀC, light brown fibrous crystals; IR (KBr) Vmax
/
cmꢁ1: 3430 (broad band NH/OH), 1712 (CO). 1H NMR (DMSO-d6,
400 MHz):
d
7.50 (dd, J ¼ 18.6, 6.7 Hz, 2H, 5,8-H), 7.18 (d,
J ¼ 6.3 Hz, 2H, 6,7-H), 4.18 (m, 4H, S-CH2, C6H11CH2CH2), 3.41
(broad s, 2H), 1.78–1.61 (m, 7H, C6H11-H), 1.30–1.18 (m, 4H, C6H11-
H), 0.96 (q, J ¼ 10.9 Hz, 2H, C6H11-H). 13 C NMR (DMSO-d6,
101 MHz): d 169.65 (CO), 150.56 (S-C), 142.83, 136.05, 121.68,
121.53, 117.68, and 109.52 (Ar-C), 41.57 (C6H11CH2CH2), 36.32 (S-
CH2), 34.55 (C6H11CH2CH2), 34.10 (C6H11-C-1), 32.62 (C6H11-C-2,6),
26.01 (C6H11-C-3,5), 25.65 (C6H11-C-4). MS LCMS (ES): (calc) 333.15
(found) 334.2125 (MH)þ. Anal. Calcd for C17H23N3O2S: C, 61.23; H,
6.95; N, 12.60. Found: C, 61.47; H, 7.20; N, 12.91.
2.1.9. Methyl 4-((1H-benzo[d]imidazol-2-yl)thio)butanoate (8)
Potassium hydroxide (0.112 g, 2 mmol) was dissolved in DMF
(5 ml) upon stirring at 90ꢀ C, the resulting solution left to cool to

1066
S. E. KASSAB ET AL.
room temperature, then benzimidazole-2-thiol (1) (0.15 g, 1 mmol) precipitate was filtered off, washed several times with distilled
and methyl 4-chlorobutyrate (7) (0.145 ml, 1.20 mmol) were added water and dried under vacuum to give powder product. The
to the solution and left stirring overnight at room temperature. resulting products were recrystallised from Hexane:EtOAc 1:1 to
The reaction was found very complete upon checking with TLC
using Hexane:EtOAc3:1. The resulting reaction mixture was poured
onto ice-cold water to give milky solution of oil product. The
product was extracted with EtOAc and the organic extract was
washed several times with water and dried over anhydrous
sodium sulphate. The solvent was evaporated under reduced pres-
sure to give yellowish oil product that upon standing at room
temperature for 10 h furnished off-white needle crystals of
the product.
afford crystalline products.
2.1.12. 4-((1-benzyl-1H-benzo[d]imidazol-2-yl)thio)-N-hydroxybuta-
namide (10a)
Yield: 25.0%, m.p.: 145 ꢀC, off-white microcrystals; IR (KBr) Vmax
/
cmꢁ1: 3426 (broad band NH/OH), 1710 (CO). 1H NMR (DMSO-d6,
400 MHz): d 7.61–7.53 (m, 1H, Ar-H), 7.50–7.43 (m, 1H, Ar-H),
7.36–7.22 (m, 3H, Ar-H), 7.22–7.09 (m, 4H, Ar-H), 5.39 (s, 2H,
C6H5CH2), 3.33–3.37 (m, 2H, S-CH2), 2.37 (t, J ¼ 7.3 Hz, 2H, 2-CH2),
1.95 (m, 2H, 3-CH2). 13C NMR (DMSO-d6, 101 MHz): d 173.98 (CO),
151.43 (S-C), 143.09, 136.43, 136.43, 136.20, 136.20, 128.80, 128.80,
127.75, 127.75, 127.01, 127.01, 121.82, 121.73, 117.76, and 109.85
(Ar-C), 46.69 (C6H5-CH2), 32.42 (S-CH2), 31.25 (2-CH2), 24.56 (3-CH2).
MS LCMS (ES): (calc) 341.12 (found) 342.2353 (MH)þ. Anal. Calcd
for C18H19N3O2S: C, 63.32; H, 5.61; N, 12.31. Found: C, 63.50; H,
5.46; N, 12.57.
Yield: 76.0%, m.p.: 122 ꢀC, off-white needle crystals; IR (KBr)
V
max/cmꢁ1: 3435 (NH), 1752 (CO). 1H NMR (DMSO-d6, 400 MHz):
d 12.54 (s, 1H, NH), 7.43 (s, 2H, 5,8-H), 7.11 (dd, J ¼ 5.9, 3.2 Hz, 2H,
6,7-H), 3.58 (s, 3H, OCH3), 3.29 (t, J ¼ 7.1 Hz, 2H, S-CH2), 2.47 (t,
J ¼ 7.5 Hz, 2H, 2-CH2), 2.01–1.97 (p, J ¼ 7.2, 2H, 3-CH2).13C NMR
(DMSO-d6, 101 MHz): d 172.81 (CO), 149.83 (S-C), 121.35 (Ar-C),
51.37 (COOCH3), 32.00 (S-CH2), 30.43 (2-CH2), 24.79 (3-CH2). MS
LRMS (EI): (calc) 250.08 (found) 250.12 (M)þ. Anal. Calcd for
C12H14N2O2S: C, 57.58; H, 5.64; N, 11.19. Found: C, 57.82; H, 5.31;
N, 11.45.
2.1.13.
4-((1–(4-bromobenzyl)-1H-benzo[d]imidazol-2-yl)thio)-N-
hydroxybutanamide (10b)
Yield: 30.0%, m.p.: 153 ꢀC, off-white macrocrystals; IR (KBr) Vmax
/
2.1.10. General synthesis of methyl 4-((1-benzyl/cycloalkyl-1H-ben-
zo[d]imidazol-2-yl)thio)butanoates (9a-d)
Anhydrous K2CO3 (0.21 g, 1.5 mmol) was added to a stirred mix-
cmꢁ1: 3430 (broad band NH/OH), 1710 (CO). 1H NMR (DMSO-d6,
400 MHz): d 12.19 (s, 1H), 7.62–7.56 (m, 1H, Ar-H), 7.53 (d,
J ¼ 8.3 Hz, 2H), 7.49–7.43 (m, 1H), 7.15 (dt, J ¼ 18.5, 7.4 Hz, 4H),
ture of Methyl 4-((1H-benzo[d]imidazol-2-yl)thio)butanoate
8
(0.25 g, 1 mmol) and benzyl chloride 4a (0.12 ml, 1 mmol) or 4-bro- 5.38 (s, 2H, 4-BrC6H4-CH2), 3.33–3.37 (m, 2H, S-CH2), 2.37 (t,
mobenzyl bromide 4b (0.25 g, 1 mmol) or cyclohexylmethyl brom- J ¼ 7.3 Hz, 2H, 2-CH2), 2.00–1.90 (m, 2H, 3-CH2). 13 C NMR (DMSO-
ide 4c (0.14 ml, 1 mmol) or cyclohexylethyl bromide 4d (0.16 ml, d6, 101 MHz): d 173.90 (CO), 151.33 (S-C), 143.04, 136.08, 135.86,
1 mmol) in DMF and the temperature was raised to100 oC. The 131.67, 129.16, 121.84, 121.76, 120.82, 117.77, and 109.74 (Ar-C),
reaction mixture was judged complete after 2 h upon checking
with TLC using Hexane:EtOAc 4:1 as eluting system. The reaction
mixture was poured onto water and the organic product was
extracted by ethyl acetate, the organic extract was washed with
water several times and dried over anhydrous sodium sulfate. The
solvent was evaporated under reduced pressure to give the prod-
uct as yellowish oil residue. The intermediate ester derivatives
(9a-d) were obtained in yields 85%, 69%, 90%, and 97% respect-
ively with enough purity to be submitted to the next step of reac-
tions without further purification.
45.99 (4-BrC6H4-CH2), 32.36 (S-CH2), 31.22 (2-CH2), 24.49 (3-CH2).
MS LCMS (ES): (calc) 419.03 (found) 420.1528 (MH)þ. Anal. Calcd
for C18H18BrN3O2S: C, 51.44; H, 4.32; N, 10.00. Found: C, 51.69; H,
4.08; N, 9.77.
2.1.14. 4-((1-(Cyclohexylmethyl)-1H-benzo[d]imidazol-2-yl)thio)-N-
hydroxybutanamide (10c)
Yield: 35.0%, m.p.: 149 ꢀC, off-white microcrystals; IR (KBr) Vmax
/
cmꢁ1: 3426 (broad band NH/OH), 1716 (CO). 1H NMR (DMSO-d6,
400 MHz): d 12.18 (s, 1H), 7.54 (d, J ¼ 6.6 Hz, 1H, 8-H), 7.45 (d,
J ¼ 6.3 Hz, 1H, 5-H), 7.22–7.03 (m, 2H, 6,7-H), 3.93 (d, J ¼ 7.0 Hz, 2H,
C6H11-CH2), 3.35 (t, J ¼ 6.8 Hz, 2H, S-CH2), 2.39 (t, J ¼ 7.1 Hz, 2H, 2-
CH2), 2.05–1.87 (m, 2H, 3-CH2), 1.81 (m, 1H, C6H11-H), 1.62–1.48 (m,
5H, C6H11-H), 1.10–1.01 (m, 5H, C6H11-H). 13 C NMR (DMSO-d6,
2.1.11. General synthesis of 4-((1-benzyl/cycloalkyl-1H-benzo[d]imi-
dazol-2-yl)thio)-N-hydroxybutanamides (10a-d)
Hydroxylamine hydrochloride (10 equivalent) was neutralised in
1 M NaOMe solution in methanol (14 equivalent) via stirring for
10 min and filtered off to discard sodium chloride. Hydroxylamine
solution was added to a stirred solution of the respective ester
(5a-d) (1 equivalent) in methanol (5 ml) at room temperature. The
reaction mixture was left stirring until judged complete upon
observing disappearance of the spot of ester starting material on
TLC after 2 h using DCM:MeOH 9.5:0.5 as eluting system. The solv-
ent was evaporated under reduced pressure and the crude resi-
due was quenched with distilled water (15–20 ml). All the organic
dirt of the aqueous reaction mixture was scavenged while vigor-
ous shaking with EtOAc and the aqueous extract of the product
was separated free from the organic dirt that remained from the
101 MHz):
d 173.91(CO), 151.41(S-C), 142.90, 136.56, 121.47,
121.35, 117.58, 109.85 (Ar-C), 49.49 (C6H11-CH2), 37.75 (S-CH2),
32.45 (2-CH2), 31.26 (C6H11-C-1), 30.23 (C6H11-C-2,6), 25.78 C6H11-C-
3,5), 25.20 (C6H11-C-4), 24.58 (3-CH2). MS LCMS (ES): (calc) 347.17
(found) 348.2614 (MH)þ. Anal. Calcd for C18H25N3O2S: C, 62.22; H,
7.25; N, 12.09. Found: C, 62.53; H, 6.98; N, 12.31.
2.1.15. 4-((1–(2-cyclohexylethyl)-1H-benzo[d]imidazol-2-yl)thio)-N-
hydroxybutanamide(10d)
Yield: 31.0%, m.p.: 132 ꢀC, off-white microcrystals; IR (KBr) Vmax
/
cmꢁ1: 3431 (broad band NH/OH), 1715 (CO). 1H NMR (DMSO-d6,
reaction. The aqueous extract of the product was acidified with 400 MHz): d 12.21 (s, 1H), 7.53 (dd, J ¼ 6.7, 2.0 Hz, 1H, 8-H),
drop wise addition of 10% HCl until observing precipitation at pH 7.48–7.41 (m, 1H, 5-H), 7.21–7.08 (m, 2H, 6,7-H), 4.12 (t, J ¼ 7.5 Hz,
¼ 5.0. The resulting suspension was left in the fridge overnight 2H, C6H11-CH2CH2), 3.36–3.33 (m, 2H, S-CH2), 2.38 (t, J ¼ 7.4 Hz, 2H,
for the product to complete its precipitation. The resulting 2-CH2), 1.95 (p, J ¼ 7.3 Hz, 2H, 3-CH2), 1.75–1.54 (m, 7H, C6H11-

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1067
CH2CH2,C6H11-H), 1.29–1.10 (m, 4H, C6H11-H), 0.97–0.89 (m, 2H, and an emission of 460 nm using a Tecan Infinite M1000 micro-
C6H11-H). 13 C NMR (DMSO-d6, 101 MHz): d 173.91(CO), 150.84 (S-
plate reader.
HDAC activity assays were performed in duplicates at each
concentration. The fluorescent intensity data were analysed using
the computer software, Graphpad Prism. In the absence of the
compound, the fluorescent intensity (Ft) in each data set was
defined as 100% activity. In the absence of HDAC, the fluorescent
intensity (Fb) in each data set was defined as 0% activity. The per-
cent activity in the presence of each compound was calculated
according to the following equation: % activity ¼ (F-Fb)/(Ft-Fb),
where F ¼ the fluorescent intensity in the presence of the com-
pound. The values of % activity versus a series of compound con-
centrations were then plotted using non-linear regression analysis
of Sigmoidal dose–response curve generated with the equation
Y ¼ Bþ(T-B)/1 þ 10((LogEC50-X)ꢂHill Slope), where Y ¼ percent activity,
B ¼ minimum percent activity, T ¼ maximum percent activity,
X ¼ logarithm of compound and Hill Slope ¼ slope factor or Hill
coefficient. The IC50 value was determined by the concentration
causing a half-maximal percent activity.
C), 143.04, 135.89, 121.51, 121.39, 117.63, and 109.41(Ar-C), 41.44
(C6H11-CH2CH2), 38.87 (S-CH2), 36.28 (C6H11-CH2CH2), 34.57(2-CH2),
32.58 (C6H11-CH2CH2), 32.39 (C6H11-C-1), 31.03 (C6H11-C-2,6), 25.97
(C6H11-C-3,5), 25.64 (C6H11-C-4), 24.60 (3-CH2). MS LCMS (ES):
(calc) 361.18 (found) 362.2741 (MH)þ. Anal. Calcd for C19H27N3O2S:
C, 63.13; H, 7.53; N, 11.62. Found: C, 63.41; H, 7.18; N, 11.87.
2.2. Biological activity
2.2.1. Histone deacetylase inhibitor assays
All enzymatic assays were conducted in BPS Bioscience Inc. in
6042 Cornerstone Court West, Ste. B, San Diego, California, USA
using the biochemicals, buffers and reagents according to BPS
Bioscience catalog numbers as provided in the supplemen-
tary materials.
2.2.1.1. % inhibition of HDAC enzyme at 10 mM of the test inhibi-
tor. The test compounds were dissolved in DMSO. A series of dilu-
tions for each test compound was prepared with 10% DMSO in
HDAC assay buffer and 5 mL of the final dilution (10 mM) was
added to a 50 mL reaction so that the final concentration of DMSO
is 1% in all of the reactions. The enzymatic reactions for the HDAC
enzymes were conducted in duplicate at 37 ꢀC for 30 min in a
50 mL mixture containing HDAC assay buffer, 5 mg BSA (bovine
serum albumin), HDAC substrate (10 mM), HDAC enzyme (varied in
ng/reaction according to the HDAC subtype as presented in sup-
plementary materials) and a test compound (10 mM). After enzym-
atic reactions, 50 mL of 2 ꢂ HDAC Developer was added to each
well and the plate was incubated at room temperature for an
additional 15 min. Fluorescence intensity was measured at an exci-
tation of 360 nm and an emission of 460 nm using a Tecan Infinite
M1000 microplate reader.
HDAC activity assays were performed in duplicate. The fluores-
cent intensity data were analyzed using the computer software,
GraphPad Prism. In the absence of the compound, the fluorescent
intensity (Ft) in each data set was defined as 100% activity. In the
absence of HDAC, the fluorescent intensity (Fb) in each data set
was defined as 0% activity. The percent activity in the presence of
each compound was calculated according to the following equa-
tion: % activity ¼ (F-Fb)/(Ft-Fb), where F ¼ the fluorescent intensity
in the presence of the compound.
2.2.2. Cytotoxicity assay using xCELLigence system
Established primary Choroid plexus carcinoma (CCHE-45) were cul-
tured as previously described [41], in RPMI-1640 (Lonza, BE-12-
702F) supplemented with 10% Fetal Bovine Serum (FBS, Gibco,
12483–020) and 100 U/ml penicillin and 100 mg/ml streptomycin
(Lonza, DE17-602F). Cells were maintained at 37 ꢀC in a 5% CO2
humidified atmosphere.
Cytotoxicity assay by the xCELLigence system was performed
using RTCA xCELLigence DP system (ACEA Biosciences, Inc., San
Diego, CA, USA) as per manufacturer’s instructions https://www.
aceabio.com/products/rtca-dp/,
https://www.aceabio.com/prod-
ucts/rtca-sp65–68. In brief, after setting up the instruments and
blanking the wells with media, CCHE-45 cells were seeded into
the 96 well E-plate (ACEA Biosciences) with a density of 20,000
cells/well. Attachment and growth of the cells were monitored
every 1 h. Approximately 24 h after seeding, when the cells were
in the log growth phase, the cells were exposed to a range of test
compound 10a concentrations (3.125, 6.25, 12.5, 25, 50, 100, 200,
400) mM for 96 h. Controls received either medium alone, or
medium þ DMSO with a final concentration below of 0.5%. For
each concentration, duplicates were tested. Experiments were con-
ducted in three biological repeats. Results were analyzed using
the RTCA software (Version 2.0). Data was exported and average
IC50 value was analyzed.
2.2.1.2. IC50 of the test inhibitor against HDAC6. All of the com-
pounds are dissolved in DMSO. The serial dilution of the com-
pounds was first performed in 100% DMSO with the highest
concentration at 1 mM. Each intermediate compound dilution (in
100% DMSO) will then get directly diluted 10x fold into assay buf-
fer for an intermediate dilution of 10% DMSO in HDAC assay buf-
fer and 5 mL of the dilution was added to a 50 mL reaction so that
the final concentration of DMSO is 1% in all of reactions. The
enzymatic reactions for the HDAC enzymes were conducted in
duplicate at 37 ꢀC for 30 min in a 50 mL mixture containing HDAC
assay buffer, 5 mg BSA, an HDAC substrate (10 mM), a HDAC
enzyme (10 ng/reaction) and a test compound (10 doses range
from 0.0003 mM to 10 mM upon 3-fold dilution)/standard inhibitor
(10 doses range from 0.00003 mM to 1 mM upon 3-fold dilution).
After enzymatic reactions, 50 mL of 2 x HDAC Developer was
added to each well for the HDAC enzymes and the plate was
incubated at room temperature for an additional 15 min.
2.2.3. Western blot analysis
CCHE-45 cells were treated with 100 mM of test compound for
24 h. HL60 cell line (a kind gift from Professor Azza Kamel from
the National Cancer Institute, Cairo University); and is cultured in
RPMI-1640 (Lonza, BE-12-702F) supplemented with 10% Fetal
Bovine Serum (FBS, Gibco, 12483–020) and 100 U/ml penicillin and
100 mg/ml streptomycin (Lonza, DE17-602F), has also been treated
with different concentrations of 10a in twofold serial dilutions for
24 h. Proteins were extracted from CCHE-45 cells using RIPA Lysis
and Extraction Buffer (ThermoScientific, cat. No: 89901) containing
100x Halt Protease and Phosphate Inhibitor Cocktail
(ThermoScientific, cat. No. 78444). Followed by protein quantifica-
tion using Bradford Reagent (Pierce Coomassie Plus (Bradford)
Assay Reagent) (ThermoScientific, cat. No. 23238). A total of 50 mg
proteins were loaded on a 10% SDS page using 30% Acrylamide/
Bisacrylamide 19:1(Serva, cat. No. 10679.01) then transferred by
Fluorescence intensity was measured at an excitation of 360 nm semidry transfer method using Bio-Rad Trans-Blot Turbo transfer

1068
S. E. KASSAB ET AL.
system on PVDF Transfer Membrane (ThermoScientific, cat. No. chloroacetate (2) in ethanolic suspension of potassium carbonate
88518). The membranes were incubated overnight at 4 ꢀC in the (K2CO3) at 60 ꢀC to afford the corresponding ester (3) in 34% yield
primary antibodies Mouse monoclonal Anti-Beta Actin antibody after stirring for 4 h. The resulting thioacetate ester (3) without
further crystallisation, underwent an alkylation process that
involves alkylation of benzimidazole-NH with 1.0 equivalent of
benzyl chloride (4a), 4-bromobenzyl bromide (4b), cyclohexyl-
methyl bromide (4c) and cyclohexylethyl bromide (4d). The alkyl-
ation reaction was conducted in DMF in the presence of 1.5
equivalent of K2CO3 at 100 ꢀC for 1 h to afford the respective ethyl
1-substituted(benzimidazole-2-yl)thioacetate esters (5a-d) in rea-
sonable yields (40%-65%). The ester products (5a,c,d) were iso-
lated as oil products while 4-bromobenzyl derivative (5b) was
crystallised from methanol as golden yellow needles. The resulting
esters (5a-d) were used for the next step without further chroma-
tographic purification or crystallisation.
Finally, The hydroxylamine hydrochloride (10 equivalent) was
neutralised with 1 M NaOMe (14 equivalent) in methanolic solu-
tion and the produced hydroxylamine was promptly added to the
respective ethyl 1-substituted(benzimidazole-2-yl)thio acetate
esters (5a-d) (1.0 equivalent) dissolved in methanol. The reaction
was considered complete after 10 min of stirring under reflux via
observing disappearance of the ester spot on thin layer chroma-
tography (TLC). The hydroxamic acid products (6b, 6c, 6d) were
obtained in low yields while the benzyl derivative (6a) had the
highest yield of 22%.
(1:100, abcam, ab8224) and Mouse monoclonal Anti-acetylated a
Tubulin (1:200, Santa Cruz Biotechnology, sc-23950). Membranes
were washed 3 times using TBST then incubated for an hour at
room temperature in secondary antibody Anti-Mouse IgG, HRP-
linked Antibody (1:1000, Cell Signaling, cat. No. 7076). Detection
take place by Clarity Western ECL Substrate (Bio-Rad, Cat. No.
170–5061) using ChemiDoc MP imaging system (Bio-Rad).
Densitometry analysis was performed using ImageJ software.
2.3. Docking studies
Molecular docking was performed using Biovia’s Discovery studio
4.0 software using the Dock ligands (CDOCKER) protocol which is
an implementation of the CDOCKER algorithm. The X-ray crystal
structure of the kinase domain of HDAC6 in complex with its pro-
pionic acid derivative inhibitor (PDB entry 5G0H) was recovered
from RSCB protein data bank. The protein structure was prepared
using protein preparation protocol of Biovia’s discovery studio 4.0.
The amino acid residues were ionised using role-based technique
and the missing residues and hydrogen atoms were added and
minimised. The protein structure was typed by CHARMM.
Synthesised compounds were prepared from ligands prepare tool
which fix bad valences, adds hydrogen, and generates a 3 D coor-
dinates using catalyst. Docking was performed using (CDOCKER)
protocol. Top hits were set to 10 and pose cluster radius was set
to 0.5 Å, while other docking parameters were kept as default. The
best docking poses are analyzed according to docking score and
interactions with key amino acids of the receptor using
CDOCKER_ENERGY scoring function.
The structures of N-hydroxyacetamide derivatives (6a-d) were
1
confirmed by IR, H-NMR, 13 C-NMR, Mass spectrometry and micro-
1
analysis. Furthermore, H-NMR confirmed the generation of S-acet-
ate ester regioisomer not N-acetate ester. In 1H-NMR spectrum,
Compound 6b as a representative figure to the whole series (6a-
d) showed a singlet proton peak at chemical shift 4.14 ppm for
methylene proton of S-CH2CONHOH while the reported69 value
for methylene protons of N-CH2CONHOH is 4.98 ppm which verify
the successful synthesis of the target regioisomers (6a-d).
On the other hand, we used different reaction conditions for
preparation of the second series of target HDAC6 inhibitors (ben-
zimidazol-2-yl)thio-N-hydroxybutanamide derivatives (10a-d) start-
3. Results and discussion
3.1. Chemistry
The target product (benzimidazol-2-yl)thio-N-hydroxyacetamide ing from 2-mercaptobenzimidazole (1) as shown in Scheme 2.
derivatives (6a-d) were synthesised as shown in Scheme 1 starting Methyl 4-chlorobutyrate (7) was used to alkylate 2-mercaptobenzi-
from 2-mercaptobenzimidazole (1) to be alkylated with ethyl midazole at position 2, potassium hydroxide was used as strong
H
N
H
N
O
K2CO3/EtOH
60oC/overnight
S
O
O
+
SH
Cl
N
O
N
2
1
3
X
R
4
a
b
c
K2CO3/DMF
100oC/1hr
R
X
R
C6H5CH2
4-BrC6H4CH2
Cl
Br
4
Br C6H11CH2
Br
d
C6H11C2H4
R
N
N
NH2OH.HCl/1M NaOMe
S
O
O
S
O
HN OH
N
N
MeOH/Reflux
10 min
6
5
R
5,6
a
C6H5CH2
b
c
4-BrC6H4CH2
C6H11CH2
d
C6H11C2H4
Scheme 1. Synthesis of (benzimidazol-2-yl)thio-N-hydroxyacetamide derivatives (6).

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1069
H
N
H
N
O
KOH/DMF
r.t./overnight
S
+
SH
Cl
N
O
O
N
O
8
7
1
X
R
4
a
b
c
C6H5CH2
4-BrC6H4CH2
Cl
Br
K2CO3/DMF
100oC/2h
R
4a-d
X
Br C6H11CH2
Br
d
C6H11C2H4
R
R
N
N
NH2OH.HCl/1M NaOMe
S
S
N
O
N
O
O
MeOH/Reflux
2h
HN OH
10
9
R
9,10
a
C6H5CH2
b
c
4-BrC6H4CH2
C6H11CH2
d
C6H11C2H4
Scheme 2. Synthesis of (benzimidazol-2-yl)thio-N-hydroxybutanamide derivatives (10).
base to enhance the formation of thiolate salt in DMF as polar 3.2. Biological activity
aprotic solvent to potentiate the efficacy of alkylation process
3.2.1. Human HDAC inhibition activity
with 4-carbon chain alkylating agent by stirring overnight at room
temperature. The starting ester, methyl 4-((1H-benzo[d]imidazol-2-
yl)thio) butanoate (8) was obtained in 76% yield and in pure nee-
dle crystals. Because the reaction condition was very regioselec-
tive, there were no any by-products require further purification.
Interestingly, the resulting ester (8) gave positive hits on searching
SciFinder for its preparation. But upon searching the references
contents, we did not find the methyl ester of the prepared com-
pound (8) but only the ethyl ester70. The ethyl ester counterpart
of (8) was prepared using triethylamine as a mild base in DMF by
stirring for 12 h at 80 ꢀC and it was purified by column chromatog-
raphy to infer that upon heating for such long period of time,
multiple alkylation might have happened with these reac-
tion conditions.
The prepared ester (8) was heated with 1.0 equivalent of ben-
zyl chloride (4a), 4-bromobenzyl bromide (4b), cyclohexylmethyl
bromide (4c) and cyclohexylethyl bromide (4d) in the presence of
1.5 equivalent of K2CO3 in DMF at 100 ꢀC for 2 h to afford the cor-
responding N-benzyl, N-(4-bromobenzyl), N-(cyclohexylmethyl) and
N-(cyclohexylethyl) esters (9a-d) respectively, in quantita-
tive yields.
Activity of the synthesised benzimidazole-based hydroxamic acid
derivatives (6a-d) and (10a-d) against HDACs isoforms via in vitro
enzymatic assay was investigated. The study revealed that the
derivatives of one-carbon linker (6a-d) performed weak inhibiting
activity against several HDAC isoforms including HDAC6 (Table 1).
Among the investigated derivatives of three-carbon linker (10a-d),
1-benzylbenzimidazolyl-N-hydroxybutanamide 10a showed excel-
lent inhibiting activity at 10 mM (92%) against HDAC6. Moreover,
10a exhibited impressive preferential activity against HDAC6
when compared to the inhibiting activity of the same derivative
against all the other isoforms that represent class I (HDAC1,
HDAC2, HDAC3, HDAC8), class IIa (HDAC4, HDAC5, HDAC7,
HDAC9), class IIb (HDAC10) and class IV (HDAC11) (Table 1). There
was no need to measure the corresponding IC50 values71 of 10a
against other HDAC isoforms due to the weak inhibiting activity
that didn’t reach 75% against any isoform (Table 1). The % inhib-
ition was enough to highlight 10a that showed significant prefer-
ence against HDAC6. Moreover, the other N-hydroxybutanamide
derivatives were with insignificant activity against other HDAC iso-
forms as shown in (Table 1). The % inhibition results promoted
10a among all the synthesised derivatives to measure its HDAC6
IC50 value that specified the corresponding potency at 510 nM. In
The oil products of methyl 4-((1-benzyl/cycloalkyl-1H-benzo[d]i-
midazol-2-yl)thio)butanoates (9a-d) were used to prepare the cor-
responding N-hydroxybutanamide derivatives (10a-d) upon its
treatment with hydroxylamine in methanol at room temperature
after stirring for 2 h as described previously for synthesis of N-
accordance, the nanomolar potency of 10a could primarily define
34,72–74
it as new-preferential HDAC6 lead inhibitor
.
hydroxyacetamide derivatives (6a-b). The target compounds were 3.2.2. Cytotoxic activity assay against choroid plexus carcinoma
obtained in yields (25%–35%).
(CCHE-45) cells
The structures of the final N-hydroxybutanamide derivatives
(10a-d) were confirmed by IR, 1H-NMR, 13 C-NMR, mass spectrom-
etry and microanalysis. In 1HNMR, appearance of triplet proton
peak of methylene group of S-CH2CH2CH2CONHOH for compound
10c at chemical shift 3.35 ppm while the reported value69 for
methylene protons of N-CH2CH2CH2CONHOH is 4.28 ppm, con-
firmed the synthesis of target regioisomers (10a-d).
Choroid plexus carcinoma cell line CCHE-45 was then used to test
the effect of the test HDAC6 inhibitor 10a on cell proliferation.
CCHE-45 cell line is characterised by constitutive formation of
aggresomes41, which are inclusion bodies for highly misfolded
proteins formed by the collapse of the intermediate filament
vimentin. Protein aggregates that are unable to be degraded by
the proteasome are shuttled along the microtubules to the

1070
S. E. KASSAB ET AL.
Table 1. In vitro inhibition activity of test compounds (6a-d) and (10a-d) against human HDACs.
%inhibition of HDACs at 10mM of test compounda
IC50 SEb
HDAC6
Compound
HDAC1
HDAC2
HDAC3
HDAC4
HDAC5
HDAC6
HDAC7
HDAC8
HDAC9
HDAC10
HDAC11
6a
6b
6c
6d
10a
10b
10c
10d
6
18
13
14
36
9
5
6
7
3
19
9
8
8
85
NDc
ND
ND
ND
37
ND
ND
ND
94
ND
ND
ND
ND
1
ND
ND
ND
ND
64
ND
ND
ND
ND
4
ND
ND
ND
ND
64
2
6
2
ND
ND
ND
ND
2
ND
ND
ND
ND
78
14
12
14
1
56
1
12
10
ND
89
ND
ND
ND
ND
5
ND
ND
ND
ND
60
ND
ND
ND
ND
28
ND
ND
ND
97
ND
ND
ND
ND
6
ND
ND
ND
ND
49
ND
ND
ND
ND
2
92
12
14
35
97
100e
510 0.015nM
ND
ND
ND
ND
9
11
98d
ND
SAHA (1mM)
TSA (10mM)
ND
ND
ND
5.0 0.00015 nM
aMean value of two replicates of %inhibition of HDAC at 10 mM of test compound.
bMean value of two replicates of the concentration of test compound required to produce 50% inhibition of HDAC6 in nM standard error.
cNot determined.
dMean value of two replicates of % inhibition of HDAC1 at 3 mM of SAHA.
eMean value of two replicates of % inhibition of HDAC6 at 1 mM of TSA.
Normalized cell index of CCHE-45 cells
1.4
1.2
1
400µM
200µM
100µM
50µM
0.8
0.6
0.4
0.2
0
25µM
12.5µM
6.25µM
3.125µM
0
20
40
60
Time (h)
80
100
120
Figure 4. Real-time cell analysis of the cytotoxic effect of (10a) on CCHE-45. Cells were seeded into the E-plate then 24 h later, treated with a range of concentrations
of (10a) for 96 h. Graph is showing CCHE-45 cell response profiles designated as cell index for the different concentrations of (10a) over 96 h. Graph is a representative
of three independent experiments. Concentrations are 400mM (blue line), 200mM (orange line), 100mM (gray line), 50 mM (yellow line), 25mM (light blue line), 12.5 mM
(green line), 6.25 mM (dark blue line), 3.125 mM (brown Line).
just 5 times lower potency than the standard inhibitor. However,
when compared to their enzymatic activity, where Tubacin has
originally an HDAC6 IC50 ¼ 4 nM55 which is 127.5 times more
potent than the lead inhibitor 10a (HDAC6 IC50 ¼ 510 nM) to infer
that the new lead inhibitor carries remarkable efficacy and inter-
esting cytotoxic profile. Accordingly, it is highly anticipated that
the future structural optimisation of the lead inhibitor 10a might
result in a selective HDAC6 inhibitor with a superior potency
against CCHE-45 cells, compared to Tubacin.
Table 2. In vitro cytotoxic activity of (10a) against CCHE-45.
Compound
CCHE-45 IC50 (mM) SEMa
10a
Tubacin
112.67 11.06
20.00 10.18
aMean value of three replicates of the required concentration of test
compound to produce 50% inhibition of CCHE-45cells standard
error of the mean.
aggresomes assisted by dynein motor proteins and HDAC675. Due
to the essential role of HDAC6 in aggresomes formation, CCHE-45
cell line was chosen to test the cytotoxic activity of HDAC6 inhibi-
tor 10a. xCELLigence system was used to allow real-time monitor-
ing of cell response to the drug; without any need for toxic
labeling. The monitoring of cell proliferation for 96 h (Figure 4)
identified the concentration of 400 mM or 200 mM of 10a led to a
quick and dramatic reduction in cell index (CI), while 100 mM
reduced CI to half the maximum CI after 24 h from addition of the
drug with a mean IC50 of 112.67 11.06 mM (Table 2).
3.2.3. Cell-based assay of acetylated a-tubulin
Western Blot analysis of acetylated a-tubulin was used to detect
and semi-quantify the upregulation of acetylated a-tubulin that
accumulates upon inhibition of HDAC6 by 10a treatment. A sig-
nificant increase in acetylated a-tubulin was observed in CCHE-45
cells at 24 h after treatment with 10a (100 mM), which is statistic-
ally equivalent to the levels of acetylated a-tubulin induced by
Tubacin addition (20 mM) (Figure 5(A)). This increase in acetylated
a-tubulin was consistent through three biological replicates.
Interestingly, the on-target mechanism of antiproliferative activity
When (CCHE-45 IC50 ¼ 112.76 mM) of 10a was compared to
that of Tubacin; a standard HDAC6 selective inhibitor (CCHE-45
IC50 ¼ 20 mM) (Table 2), it showed that the new lead inhibitor has of 10a against brain cancer cells CCHE-45 as HDAC6 inhibitor was

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1071
Figure 5. The effect of (10a) on the acetylation of a-tubulin. Treatment of CCHE-45 cells for 24 h with 100 mM of (10a) upregulated acetylated a-tubulin using
Tubacin as standard HDAC6 inhibitor at 20 mM. (A) Representative Western Blot analysis of acetylated a-tubulin protein levels of three replicates showing the effect of
(10a) on acetylation of a-tubulin. (B) Relative mean densitometry measurement value of protein abundance levels using ImageJ software. b-Actin was used as a load-
ing control.
Figure 6. The effect of (10a) on the acetylation of a-tubulin. Treatment of HL60 cells for 24 h with several concentrations of (10a); 6.25, 12.5, 25, 50 and 100 mM upre-
gulated acetylated a-tubulin at concentration starting from 12.5 mM. (A) Representative Western Blot analysis of acetylated a-tubulin protein levels of three replicates
showing the effect of (10a) on acetylation of a-tubulin. (B) Relative mean densitometric value of protein abundance levels using ImageJ software. GAPDH was used as
a loading control.
confirmed via exhibition of insignificant difference between the inhibitor according to Graham Patrick’s definition74 of the new
test inhibitor 10a and Tubacin according to densitometry meas-
urements (Figure 5(B)) at their corresponding CCHE-45 IC50 values,
(Table 2). Moreover, these results excluded the possibility of off-
target mechanism of action of 10a against brain cancer cells
CCHE-45 and the new inhibitor doesn’t have multiple targets by
which it caused cancer cell death. This observation might high-
light the new lead compound 10a as safe inhibitor with controlled
biological interference as antiproliferative agent.
We have extended the work to determine the effect of 10a on
HL60; an acute promyeloblastic leukemia cell line, as HDAC6 has
been observed to be overexpressed in acute myeloid leuke-
mia76,77. Treating HL60 with the test inhibitor 10a was done
through the addition of a range of concentrations over 2-fold ser-
ial dilutions starting from 100 mM and ended at 6.25 mM.
According to the results shown (Figure 6(A)), acetylated a-tubulin
levels were positively correlated with the test compound concen-
tration, with a significant upregulation of acetylated a-tubulin
detected at 25 mM (Figure 6(B)). This suggests that HL60 might be
more sensitive to 10a compared to CCHE-45; as much lower con-
centration was able to induce the acetylation of a-tubulin.
lead compound in drug discovery studies that says “the lead is
the compound that exhibits therapeutic usefulness and the level
of activity is not crucial”. Thus, the newly generated lead 10a is
therapeutically useful lead for future development to improve the
potency besides the selectivity, and eventually elaborate potent
antiproliferative agent against solid tumour of choroid plexus car-
cinoma in addition to acute promyeloblastic leukaemia.
Noteworthy to mention is that the generated lead inhibitor 10a
gained its importance from being the first preferential HDAC6
inhibitor with verified cytotoxic activity against brain cancer CCHE-
45 as type of serious solid tumours having such interesting profile
and verified on-target cytotoxic activity when compared to tuba-
cin standard inhibitor.
3.3. Docking studies
A molecular docking study using discovery studio 4.0 http://www.
3dsbiovia.com/events/webinars/discovery-studio-25/index.html was
performed using the Dock ligands (CDOCKER) protocol78. It is
used to visualise the binding modes and orientation of some rep-
resentative examples of the synthesised compounds into the
Conclusively, the enzymatic, cytotoxic activity and cell-based
assays’ results could define benzimidazole-based hydroxamic acid
derivative 10a of the new class as preferential HDAC6 lead active binding site of HDAC6. The coordinates of the target

1072
S. E. KASSAB ET AL.
Figure 7. (A) Redocking of TSA (was built as solid stick model) into active biding site of HDAC6 (PDB entry 5G0H) in 3 D style and atoms are assigned by colors; (blue
nitrogen, red oxygen and gray carbon). p-p stacking between the benzene ring of TSA and benzene rings of active binding site amino acid residues presented as pink
dotted line. Alkyl-p stacking between alkyl chain of TSA and benzene rings of amino acid residues presented as light pink dotted line. Zinc metal ion-TSA coordinate
bond formation presented as grey-dotted line. (B) Docking solution of TSA into the active binding site of HDAC6 (PDB entry 5G0H) in 2D style (C) Docking solution of
compound (10a) (was built as solid stick model) into the active binding site of HDAC6 (PDB entry 5G0H). Yellow sulphur atom and hydrogen bond formation between
compound (10a) and the amino acid residues represented as green dotted line, the description of the rest of binding interaction representations are the same as A
and B. (D) Docking solution of compound (10a) into the active binding site of HDAC6 (PDB entry 5G0H) in 2D style.
protein structure were obtained from the crystal structure of the unsaturated aliphatic chain of the linker showed Alkyl-p inter-
HDAC6 (PDB entry 5G0H)51 in complex with the preferential action with Phe583 and Phe643 (Figure 7(A,B)).
HDAC6 ligand inhibitor Trichostatin A (TSA) that shows high simi-
The binding mode of the synthesised compounds of benzimi-
larity to the human orthologue79. Validation of docking algorithm dazole-based derivatives was investigated to justify the nanomolar
was achieved by redocking of co-crystallised structure of the activity of compound 10a (IC50 ¼ 510 nM) against HDAC6 among
inhibitor inside the active binding site of HDAC6. The root mean all the other derivatives (5a-d) and (10b-d) that showed remark-
square difference (RMSD) between the top docking pose and ori- ably low %inhibition against HDAC6 compared to 10a (Table 1).
ginal crystallographic geometry was 0.44 Å. This provides sufficient On the other hand, identifying the binding mode of some repre-
confidence in drawing meaningful conclusions from the docking sentative examples of low-active benzimidazole derivatives 6a and
study. In addition, redocking of TSA retrieved the reported bind- 10b will provide us reliable clues for future development and
ing mode of the inhibitor into the X-ray crystal structure of the optimisation of the identified lead inhibitor 10a.
active binding site of HDAC6 (C-docker energy ¼ ꢁ25.49) as
Docking of 1-benzylbenzimidazolyl-N-hydroxybutanamide 10a
depicted in (Figure 7(A,B)), where the carbonyl and hydroxyl oxy- into the catalytic domain of HDAC6 (PDB entry 5G0H) revealed
gens of hydroxamate moiety complexes with the corresponding the (C-docker energy ¼ ꢁ39.12 Kcal/mol) to be superior to that of
Zn2þ metal ion in a bidentate fashion at metal-coordinate bond ligand inhibitor, TSA (C-docker energy ¼ ꢁ25.49 Kcal/mol).
distances 2.25 Å and 2.20 Å, respectively (Figure 7(A,B)). The unsat- Binding interactions of 10a with the active binding site amino
urated aliphatic linker of TSA was nearly planar and sandwiched acid residues showed formation of three hydrogen bonds
between the aromatic side chains of Phe583 and Phe643. Whereas between hydroxamic acid and His573, Tyr745 and Gly582, p-p
the carbonyl group of the hydroxamate was almost coplanar with stacking between 1-benzyl radical and Phe643. Moreover, benzimi-
the unsaturated aliphatic chain and appeared twisted by ꢃ30ꢀ dazole ring interacted with Phe642 via p-p stacking and with
towards the zinc metal ion. The hydroxyl oxygen of hydroxamate Leu712 via alkyl-p stacking (Figure 7(C,D)). Carbonyl and hydroxyl
makes hydrogen bonding with His573. Phenyl radical of docked oxygens of hydroxamate moiety captured zinc metal ion in biden-
ligand inhibitor TSA interacted with Phe643 via p-p stacking while tate manner at respective bond distances 2.21 Å and 2.97 Å

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1073
Figure 8. Docking solution of (10b) (was built as solid stick model) into catalytic domain of HDAC6 (PDB entry 5G0H) in 3D style in the left side and 2D style in the
right side. Atoms are assigned by colours; (blue nitrogen, red oxygen, grey carbon, yellow sulphur and maroon bromine). Alkyl-p stacking presented as light pink dot-
ted line. Zinc metal cation-hydroxamate coordinate bond formation presented as grey-dotted line. Hydrogen bond formation between compound (10b) and the amino
acid residues represented as green dotted line.
(Figure 7(C,D)) to imply the formation of metal-coordinate bond
with lower strength due to the longer bond length80–82 when
compared to TSA (Figure 7(A,B)). It is worthy to emphasise that
the generated lead inhibitor 10a succeeded to interact with 3
amino acids; Phe643, Gly582, Leu712 out of 5 amino acids [51]
that constitute the narrow hydrophobic channel of HDAC6 at
which the acetylated lysine is set into the catalytic domain for
deacetylation while the ligand inhibitor TSA interacted with only
Phe643. In addition, 10a formed hydrogen bond as an extra type
of interaction with Tyr745 that TSA in its bioactive conformer was
not able to form (Figure 7(B–D)). Tyr745 is located next to zinc
metal cation and thought to stabilise the transition state of inter-
mediate to eventually release the product with deacetylated lysine
residues51.
Compound 10b; the1–(4-bromobenzyl) counterpart of the lead
inhibitor 10a that with 4-atoms-linker, was chosen to represent
the derivatives of low inhibition activity (12%) against HDAC6
(Table 1). Docking solution of 10b into the catalytic domain of
HDAC6 (PDB entry 5G0H) showed (C-docker energy
¼ ꢁ36.34 Kcal/mol) to excel the binding affinity of ligand inhibitor
TSA (C-docker energy ¼ ꢁ25.49 Kcal/mol). Upon investigating the
real reason(s) that lied behind the discrepancy in activity between
10b and 1-benzyl derivative 10a against HDAC6, it was found that
the orientation of the docked compound 10b flipped around the
S-alkyl chain axis (Figure 8) when compared to 10a (Figure 7(C)).
This flip oriented the 4-bromobenzyl fragment upward in the
hydrophobic channel to interact with Leu712 via alkyl-p stacking
and be away from Phe643. Benzimidazole ring was then oriented
downward to interact with Phe642 via p-p stacking. This might be
attributed to the para substitution of 1-benzyl ring with bromine
that led to steric clash with Phe643 forced the 4-bromobenzyl to
flip and stay closer to Phe642. 4-Bromobenzyl exhibited p-stacking
with Phe642 and Leu712 via bromine substituent and benzyl ring
respectively (Figure 8) and lost the ability to interact with the
hydrophobic channel amino acid Phe643. The hydroxamate moi-
ety captured the metal cation in bidentate manner and formed
two hydrogen bonds with His573 and Tyr745 (Figure 8). Thus, sig-
nificant weaker inhibition activity of 10b might be attributed to
the loss of p-p stacking interaction with Phe643.
Finally, we docked the 1-benzylbenzimidazole-N-hydroxyacta-
mide derivative 6a; the one-carbon linker counter part of 10a that
showed insignificant inhibiting activity (2%) against HDAC6 (Table
1) compared to 10a (92%). The docking solution of 6a into the
active binding site of HDAC6 (PDB entry 5G0H) showed compar-
able binding affinity (C-docker energy ¼ -28.63 Kcal/mol) to ligand
inhibitor TSA (C-docker energy ¼ ꢁ25.49 Kcal/mol) but lower than
that of 10a (-39.12 Kcal/mol) and 10b (ꢁ36.34 Kcal/mol) to reflect
significant alteration in the binding interaction forces with the
hydrophobic channel amino acid residues of the catalytic domain.
The orientation of the docked derivative 6a left an impression
that the shortness of the linker made the compound struggle to
take the pose that fulfills all the required binding interactions for
successful inhibition to the enzyme. This could be justified by the
resulting best pose of 6a that flipped on the S-alkyl chain axis to
let benzimidazole ring catch the opportunity to interact with
Phe643 via p- p stacking and it gives the hydroxamate moiety
that built on short alkyl chain, the chance to face the zinc metal
ion (Figure 9). The docked compound 6a also swung a little to
get the hydroxamate residue closer to zinc metal ion to capture it
via formation of metal-coordinate bond by the hydroxyl oxygen in
a monodentate fashion (Figure 9). Thus, test compound’s flipping
led the1-benzyl group to interact with Phe642 via p- p stacking
and lost the interaction with Leu712 as one of the hydrophobic
channel amino acid residues when compared to N-hydroxybutana-
mide 10b case. Two further hydrogen bonds are formed between
the hydroxamate moiety and His574 and Tyr745 amino acid resi-
dues (Figure 9). Conclusively, the insignificant activity of 1-benzyl-
benzimidazole-N-hydroxyactamide 6a is definitely attributed to
shortness of the linker between the 1-benzylbenzimidazole hydro-
phobic cap and the hydroxamate moiety.
Based on the results of the above investigation that involved
docking the compound 10a of the most inhibition activity (92%)
against HDAC6 (IC50 ¼ 510 nM) and two representative examples
of insignificant inhibiting activity 10b (12%) and 6a (2%), it was
obvious that the length of the linker, interaction of the 1-(4-unsub-
stituted)benzyl hydrophobic cap of benzimidazole ring with the nar-
row hydrophobic channel amino acid residues; Phe643 and Leu712
via p-stacking and bidentate capture of the hydroxamate moiety to
zinc metal ion by the aid of carbonyl and hydroxyl oxygens are

1074
S. E. KASSAB ET AL.
Figure 9. Docking solution of (6a) (was built as solid stick model) into catalytic domain of HDAC6 (PDB entry 5G0H) in 3 D style in the left side and 2D style in the
right side. Atoms are assigned by colours; (blue nitrogen, red oxygen, yellow sulphur and grey carbon). p-p stacking presented as pink dotted line. Zinc metal cation-
hydroxamate coordinate bond formation presented as grey-dotted line. Hydrogen bond formation between compound (6a) and the amino acid residues represented
as green dotted line.
critical determinants that verified the inhibiting activity pattern of
the test inhibitors generated from such benzimidazole skeleton.
This also justifies the weak inhibiting activity of 1-cyclohexyl deriv-
atives 6c,d and 10c,d that lack the interaction with the hydropho-
bic channel amino acids of HDAC6 via p-p stacking which is
considered a critical type of interaction for successful inhibition
of HDAC6.
After exhibiting the binding mode of the generated lead inhibi-
tor; 1-benzylbenzimidazole-N-hydroxybutanamide 10a into the
catalytic domain of HDAC6 and comparing it to that of the ligand
inhibitor TSA, it was shown that the test inhibitor 10a was with
superior binding affinity due to more interaction types with the
narrow hydrophobic channel amino acid residues.
This raises the question: why the lead inhibitor 10a that showed
better binding mode and affinity to the ligand inhibitor TSA was
much less potent in biological evaluation. The lead inhibitor 10a
carries hydrophobic cap that is large enough to maintain the pref-
erential and inhibiting activity against HDAC6 compared to
HDAC1-5 and HDAC7-11 (Table 1) but is not enough to excel the
selectivity. Moreover, the two critical factors that determine the
activity and selectivity of the HDAC inhibitors are the hydrophobic
cap and the linker64,83,84. Those are the two fragments that exten-
sively varied in most of the designed HDAC inhibitors to either
generate new inhibitor or to optimise the discovered lead inhibi-
tor for improvement of the relative potency12,13,31. Since then, it
might be that the three-carbon length of the linker of 10a was
not enough to capture the zinc metal ion in HDAC6 catalytic
domain as strong as TSA due to formation of metal-coordinate
bond at longer distance (Figure 7(B,D)).
Definitely, the interesting results of the docking study could
provide us with an excellent framework for setting up the future
directions towards optimisation of the identified lead inhibitor
10a. The suggested directions involve: (i) longer carbon linker
from five-carbon to seven-carbon to enhance the potency against
the enzyme, (ii) bigger size of 1-arylmethyl residue via trying with
binuclear arylmethyl residues instead of benzyl residue that is
expected to change the profile of 10a from preferential to select-
ive inhibitor and (iii) pyridylmethyl and quinolinyl methyl via try-
ing with heterocycle substitute to benzyl residue that might
enhance the affinity and the corresponding potency by nitro-
gen heteroatom.
Conclusion
The present study introduced molecular-, structural-based design
and identification of new class of benzimidazole-based hydroxa-
mic acid that involves a lead inhibitor with HDAC6 preferential
inhibiting activity (HDAC6 IC50 ¼ 510 nM) and on-target cytotoxic
mechanism of action against CCHE-45 children brain cancer cells
at (CCHE-45 IC50 ¼ 112.76 mM). The generated lead inhibitor
though low potency, it showed moderated cytotoxic activity
against the CCHE-45 cells with interesting profile when com-
pared to Tubacin as standard inhibitor that verified the better
efficacy and the superior activity is anticipated upon enhancing
the potency. The preferential inhibitor gave better activity
against acute leukaemia cells HL60 at 25 mM according to
Western analysis. The new class is feasible to synthesise with
laboratory friendly reaction conditions and accessible to develop
and modify to enhance both the potency and selectivity.
Docking studies gave clues for the appropriate modifications to
develop the structural features of the lead in order to change
the profile from preferential to selective inhibitor. Referring to
docking results, it has been recommended that introducing big-
ger size of arylmethyl and heterocycle substituents at position 1
of the lead scaffold will be in the favor of improving the select-
ivity and the longer carbons of the linker at position 2 might
play a key role in enhancing the potency. For future study, these
recommendations aim to enhance the level of activity and
selectivity without compromising the therapeutic usefulness
exhibited by the original lead inhibitor and ensure the on-target
activity of the same inhibitor that may define it as safe
lead inhibitor.
Disclosure statement
No potential conflict of interest was reported by the authors.
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