Design and synthesis of novel HDAC8 inhibitory 2,5-disubstituted-1,3,4-oxadiazoles containing glycine and alanine hybrids with anti cancer activity

Article abstract of DOI:10.1016/j.bmc.2016.09.022

Oxadiazole is a heterocyclic compound containing an oxygen atom and two nitrogen atoms in a five-membered ring. Of the four oxadiazoles known, 1,3,4-oxadiazole has become an important structural motif for the development of new drugs and the compounds containing 1,3,4-oxadiazole cores have a broad spectrum of biological activity. Herein, we describe the design, synthesis and biological evaluation of a series of novel 2,5-disubstituted 1,3,4-oxadiazoles (10a–10j) as class I histone deacetylase (HDAC) inhibitors. The compounds were designed and evaluated for HDAC8 selectivity using in silico docking software (Glide) and the top 10 compounds with high dock score and obeying Lipinski's rule were synthesized organically. Further the biological HDAC inhibitory and selectivity assays and anti-proliferative assays were carried out. In in silico and in vitro studies, all compounds (10a–10j) showed significant HDAC inhibition and exhibited HDAC8 selectivity. Among all tested compounds, 10b showed substantial HDAC8 inhibitory activity and better anticancer activity which is comparable to the positive control, a FDA approved drug, vorinostat (SAHA). Structural activity relation is discussed with various substitutions in the benzene ring connected on 1,3,4-oxadizole and glycine/alanine. The study warranted further investigations to develop HDAC8-selective inhibitory molecule as a drug for neoplastic diseases. Novel 1,3,4-oxadizole substituted with glycine/alanine showed HDAC8 inhibition.

Full text of DOI:10.1016/j.bmc.2016.09.022

Accepted Manuscript
Design and Synthesis of Novel HDAC8 Inhibitory2,5-disubstituted-1,3,4-Ox-
adiazolescontaining Glycine and Alanine hybrids with Anti Cancer Activity
Vijaya Rao Pidugu, Nagendra Sastry Yarla, Srinivasa Rao Pedada, Arunasree
M. Kalle, A. Krishna Satya
PII:
S0968-0896(16)30709-X
http://dx.doi.org/10.1016/j.bmc.2016.09.022
BMC 13275
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
18 August 2016
7 September 2016
9 September 2016
Please cite this article as: Pidugu, V.R., Yarla, N.S., Pedada, S.R., Kalle, A.M., Krishna Satya, A., Design and
Synthesis of Novel HDAC8 Inhibitory2,5-disubstituted-1,3,4-Oxadiazolescontaining Glycine and Alanine hybrids
with Anti Cancer Activity, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.
2016.09.022
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Design and Synthesis of Novel HDAC8 Inhibitory 2,5-disubstituted-
1,3,4-Oxadiazoles containing Glycine and Alanine hybrids with Anti
Cancer Activity
Vijaya Rao Pidugua,b*, Nagendra Sastry Yarlac, Srinivasa Rao Pedadad, Arunasree M
Kallee,1, A Krishna Satyab*
aGVK Biosciences Private Limited, IDA Mallapur, Hyderabad, Telangana, India -500076
bDepartment of Biotechnology, Acharya Nagarjuna University, Guntur, AP, India -
522510
cDepartment of Biochemistry/Bioinformatics, Institute of Science, GITAM University,
Vishakhapatnam, Andhra Pradesh, India-530045
dPRIST University, Thanjavur-613 403, Tamilnadu, India
eDepartment of Animal Biology, School of Life Sciences, University of Hyderabad,
Hyderabad, Telangana, India – 500046
Short title: 2,5-Disubstituted-1,3,4-Oxadiazoles as HDAC8 inhibitors
*Corresponding author:
Vijaya Rao Pidugu: pvijayarao@gmail.com;
Ph: +91-9849634187
A. Krishna Satya: akrishnasatya78@gmail.com; Ph: + 91-863-2346361
1Present address: Department of Environmental Health Sciences, Laboratory of Human
Environmental Epigenomes, Bloomberg School of Public Health, Johns Hopkins
University, 615 N. Wolfe Street, Baltimore, MD, 21205, USA
1

Abstract
Oxadiazole is a heterocyclic compound containing an oxygen atom and two nitrogen
atoms in a five-membered ring. Of the four oxadiazoles known, 1,3,4-oxadiazole has
become an important structural motif for the development of new drugs and the
compounds containing 1,3,4-oxadiazole cores have a broad spectrum of biological
activity. Herein, we describe the design, synthesis and biological evaluation of a series of
novel 2,5-disubstituted 1, 3, 4-oxadiazoles (10a-10j) as class I histone deacetylase
(HDAC) inhibitors. The compounds were designed and evaluated for HDAC8 selectivity
using in silico docking software (Glide) and the top 10 compounds with high dock score
and obeying Lipinski’s rule were synthesized organically. Further the biological HDAC
inhibitory and selectivity assays and anti-proliferative assays were carried out. In in silico
and in vitro studies, all compounds (10a-10j) showed significant HDAC inhibition and
exhibited HDAC8 selectivity. Among all tested compounds, 10b showed substantial
HDAC8 inhibitory activity and better anticancer activity which is comparable to the
positive control, a FDA approved drug, vorinostat (SAHA). Structural activity relation is
discussed with various substitutions in the benzene ring connected on 1,3,4- oxadizole
and glycine/alanine. The study warranted further investigations to develop HDAC8-
selective inhibitory molecule as a drug for neoplastic diseases. Novel 1,3,4- oxadizole
substituted with glycine/alanine showed HDAC8 inhibition.
Key words
1, 3, 4-oxadiazole, Glycine, Alanine, HDAC inhibition activity, class selective, Anti-
proliferative effect.
2

1. INTRODUCTION
Histone deacetylases (HDACs) are family of enzymes involved in removal of
acetyl group of lysine residues on histone proteins leading to gene repression[1]. The 18
known human HDACs have been classified into four classes (Class I, II, III & IV) [2].
Overexpression of HDACs has been observed in many pathological conditions including
cancer and neurodegenerative diseases [3]. Evidences indicate that HDACs are linked
with several malignancies by promoting the repression of tumor suppressor genes. Thus,
inhibition of HDACs has been proven to be a promising way to treat several cancers [4].
Several synthetic HDAC inhibitors such as hydroxamates, cyclic peptides,
aliphatic acids and benzamides with anticancer activities have been developed and
patented by many pharmaceutical industries and academic institutions [2, 5-10]. Some of
them are already at various phases of clinical trials [11].. Trichostatin (TSA) is the first
natural hydroxamate found to inhibit HDACs [12]. Vorinostat, a structural analog of TSA,
is the first FDA approved HDAC inhibitor for the treatment of progressive and persistent
cutaneous T-cell lymphoma (CTCL)[13]. However, due to the overlapping functions of
the HDACs in different classes, many adverse effects are observed. Therefore
development of class selective and isoforms-specific HDAC inhibitors has been the
recent research focus [14].
Oxadiazoles are important class of organic molecules with wide range of
medicinal applications [15]. Valente et al synthesized 1,3,4-oxadiazole-containing
hydroxamates with HDAC1 selective histone deacetylase inhibitory activity [16]. Rajak
et al., designed and synthesized 2,5-Disubstituted-1,3,4-oxadiazoles based HDAC
inhibitors and demonstrated their in vitro and in vivo anticancer activity [17]. The study
3

suggested that 1,3,4 oxadiazole linking enhanced HDAC inhibition activity and raising
hope of 1,3,4-oxadiazole skeleton for the development of novel HDAC inhibitors. In this
point of view, we have designed a series of 2,5-disubstituted-1,3,4-oxadiazoles. Several
substitutions were designed using chemically diverse groups including amino acid
groups. During virtual screening using in silico docking approach, amino acids,
specifically alanine and glycine showed good docking results. The 10 compounds with
high docking score were synthesized with high yields and performed in vitro HDACs
assays. Further, preliminary antiproliferative effects on MDA-MB 231 breast cancer cell
line were also evaluated.
2. MATERIALS AND METHODS
2.1.
In silico analysis
Taking 1,3,4-oxadiazole as a backbone, several compounds were designed by
various substitutions. Crystal structures of HDAC1, 2, 3, 8 were retrieved from protein
data bank and binding of the compounds to the active site was analyzed bioinformatically
by docking studies using Glide software (Schrodinger). The top 10 compounds (10a-10j)
showing good dock score with HDAC8 expressed in Kcal/mol and representing the
binding affinity of ligand to protein were synthesized.
2.1.1. Druggable properties
Druggable properties of the 10(a-j) compounds were calculated according to Lipinski`s
rule of 5 using online software Molinspiration [18].
4

2.2.
Chemistry
2.2.1. Material and methods
All chemicals used for the synthesis were of reagent grade and procured from Sigma-
1
Aldrich, Bangalore, India. H and 13C NMR spectra were recorded on AS 400 MHz
Varian NMR spectrometer using TMS as an internal standard. IR spectra were recorded
by using Perkin-Elmer Spectrum 100 Series FT-IR spectrometer. Mass spectra were
recorded on Agilent 1200 Series LC/MSD VL system. Melting points were determined
by using Buchi melting point B-545 instrument and are uncorrected. All the reactions
were monitored by thin layer chromatography (TLC) using percolated silica 60 F254,
0.25 mm aluminum plates (Merck). The crude compounds were purified by column
chromatography using silica gel (100-200 mesh) and gradient (0-20%) ethyl acetate in
hexane as the eluent system.
2.2.2. General Procedure for the synthesis of 9(a-j):
To the stirring mixture of substituted 1,3,4-oxadizole (1.0 eq) in DMF (10.0 T) was added
HATU (1.2 eq) and DiPEA (2.5 eq) at R.T, stirred for 40.0 min then added Boc-protected
glycine/alanine (1.0 eq). The above reaction mixture was stirred for 1.0 hr and checked
on TLC. The completed reaction mixture was extracted with ethyl acetate. The obtained
ethyl acetate layer was dried over sodium sulphate and distilled under vacuum to obtain
crude 9(a-j), which was further purified by column chromatography using ethyl acetate
and hexane mixture (80:20) and concentrated under vacuum to obtain compound 9(a-j) in
75 % yield.
5

General Procedure for the synthesis of 10(a-j):
To a solution of compound 9(a-j) (0.5 g) in DCM (5.0 ml), was added drop wise TFA
(1.5 ml) and stirred for 16h. The obtained solid was collected by filtration and washed
with ethyl acetate, dried under vacuum to obtain compounds 10 (a-j) with good yields.
The total yield and M.P for the compounds obtained is summarized in Table1.
Table 1: Yield and M.P
Compound
10a
Yield %
70
M.P 0C
152 – 154
215 – 219
130 – 135
220-224
60
10b
75
10c
66
10d
65
157 – 159
222 – 225
176-180
10e
53
10f
72
10g
61
210 – 212
210-215
10h
52
10i
45
212-214
10j
2.2.3. Characterization of compounds data
2-amino-N-((5-phenyl-1,3,4-oxadiazol-2-yl)methyl)acetamide(10a):Yield 70%; off
white solid, m.p.152 - 154 0C;1H NMR (400 MHz, DMSO-d6) δ: 3.70 (s, 2H), 4.71 (d, J
= 4.0 Hz, 2H), 7.65(m, 3H, ArH), 7.98(d, 2H, J = 8.0 Hz, ArH), 8.10 (s, 1H,NH2). 9.22
(s, 1H, NH);13C NMR (100 MHz, DMSO-d6) δ: 34.2, 40.6, 123.7, 127.0 (2C), 130.0
(2C), 132.6, 164.2, 164.7, 167.1; IR (KBr) cm-1 : 3254, 3067, 2986,1691,1620, 1536,
6

1414,1199, 1170, 1109, 959, 929, 907, 828, 794; MS (ESI) m/z [M+H]+: 232.9; Anal.
Calcd. for C11H12N4O2 C, 56.89; H, 5.21; N, 24.12. Found C, 57.10; H, 4.92; N,24.05
(R)-2-amino-N-((5-phenyl-1,3,4-oxadiazol-2-yl)methyl)propanamide(10b):Yield60%;
0
light yellow solid, m.p. 215 - 218 C;1H NMR (400 MHz, DMSO-d6) δ: 1.46 (d, 3H),
3.86 (q, 1H), 3.93 (d, J = 8.0 Hz, 2H), 7.58 (m, 3H, ArH), 7.92 (d, 2H, J = 8.0 Hz, ArH),
8.24 (s, 2H,NH2). 8.84 (t, 1H, NH);13C NMR (100 MHz, DMSO-d6) δ: 17.8, 40.9, 48.6,
127.9 (2C), 129.0 (2C), 132.3, 132.9, 166.7, 168.1, 170.3; ; IR (KBr) cm-1 : 3268, 3097,
2839, 1993, 1683, 1567, 1518, 1486, 1411, 1279, 1227, 1131, 1023, 972, 839, 753; MS
(ESI) m/z [M+H]+: 264.9(H2O aduct); Anal. Calcd. for C12H14N4O2; C, 58.53; H, 5.73;
N, 22.75. Found C,58.20; H, 5.20; N,22.70.
2-amino-N-((5-(4-fluorophenyl)-1,3,4-oxadiazol-2-yl)methyl)acetamide(10c):
Yield 75 %; off white solid, m.p.130 - 132 0C;1H NMR (400 MHz, DMSO-d6) δ: 3.66 (s,
2H), 4.70 (d, J = 4.0 Hz, 2H), 7.49 (t, J = 8.0 Hz, ArH), 8.08 (t, 2H, J = 8.0 Hz, ArH),
9.20 (s, 1H,NH; 13C NMR (100 MHz, DMSO-d6) δ: 34.2, 40.5, 117.1, 117.3, 120.3,
129.6, 163.3, 164.0, 164.2, 165.8, 167.2; IR (KBr) cm-1 : 3257, 3166, 2821, 1966, 1649,
1570, 1521, 1426, 1394,1327, 1269, 1158, 1132, 1091, 972, 875, 810; MS (ESI) m/z
[M+H]+: 268.9(H2O aduct); Anal. Calcd. for C11H11FN4O2: C, 52.80; H, 4.43; F, 7.59; N,
22.39. Found C,52.80; H, 4.20; N,22.70.
(R)-2-amino-N-((5-(4-fluorophenyl)-1,3,4-oxadiazol-2-yl)methyl)propanamide(10d):
Yield 66 %; off white solid, m.p.220 - 223 0C;1H NMR (400 MHz, DMSO-d6) δ: 1.36 (d,
J = 4.0 Hz, 2H ), 3.86 (s, 1H), 3.92 (s, 2H), 7.34 (t, 2H, ArH), 7.94 (t, 2H, ArH), 8.74 (s,
7

13
1H,NH2), 8.81 (s, 1H, NH); C NMR (100 MHz, DMSO-d6) δ: 17.8, 40.8, 48.7, 115.8,
116.0, 129.3, 130.5, 130.6, 163.4, 164.9, 168.2, 170.83; IR (KBr) cm-1 :3271, 3149, 2842,
1992, 1684, 1602, 1500, 1401, 1328, 1278,1224, 1131, 1078, 906,813, 759; MS (ESI)
m/z [M+H]+: 282.9(H2O aduct); Anal. Calcd. for C12H13FN4O2 C, 54.54; H, 4.96; F,
7.19; N, 21.20. Found C,54.80; H, 4.82; N,21.43.
2-amino-N-((5-p-tolyl-1,3,4-oxadiazol-2-yl)methyl)acetamide(10e):
Yield 65%; off white solid, m.p.157 – 159 0C;1H NMR (400 MHz, DMSO-d6) δ: 2.43 (s,
3H), 3.65 (s, 2H), 4.67 (d, J = 4.0 Hz, 2H), 7.41 (d, 2H, J = 8.0 Hz, ArH), 7.85 (d, 2H, J
= 8.0 Hz, ArH), 8.01 (s, 2H,NH2). 9.15 (t, 1H, NH);13C NMR (100 MHz, DMSO-d6) δ:
21.5, 34.2, 41.0, 120.9, 126.9 (2C), 130.4 (2C), 142.7, 163.9, 164.7, 167.1; IR (KBr) cm-
1
: 3242, 3073, 2842, 2013, 1708,1679, 1646, 1597, 1567, 1499, 1375, 1267, 1167,
1019,968, 871, 821, 798; MS (ESI) m/z [M+H]+: 246.9; Anal. Calcd. for C12H14N4O2 C,
58.53; H, 5.73; N, 22.75. Found C,58.20; H, 5.62; N,21.93.
(R)-2-amino-N-((5-p-tolyl-1,3,4-oxadiazol-2-yl)methyl)propanamide(10f):
Yield 53%; off white solid, m.p 222 – 225 0C;1H NMR (400 MHz, DMSO-d6) δ: 1.48 (d,
J = 8.0 Hz, 3H ), 3.93 (m, 3H), 4.70 (d, J = 4.0 Hz, 2H), 7.36 (dd, J = 8.0 Hz, 2H, ArH),
7.95 (dd, 2H, J = 4.0 Hz, ArH), 8.20 (s, 2H,NH2), 8.81 (s, 1H, NH);13C NMR (100 MHz,
DMSO-d6) δ: 17.5, 34.2, 48.6, 56.0, 115.4(2C), 116.0, 128.8(2C), 162.5, 163.5, 164.5,
170.6; IR (KBr) cm-1 : 3293, 3193, 2013, 1899, 1679, 1691, 1573, 1497, 1396, 1377,
1176, 1104, 957, 839, 760; MS (ESI) m/z [M+H]+: 278.9; Anal. Calcd. for C13H16N4O2
C, 59.99; H, 6.20; N, 21.52. Found C,59.79; H, 6.32; N,21.82.
8

2-amino-N-((5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-yl)methyl)acetamide(10g):
Yield 72 %; off white solid, m.p 180 – 183 0C;1H NMR (400 MHz, DMSO-d6) δ: 2.43 (s,
3H), 3.65 (s, 2H), 4.67 (d, J = 4.0 Hz, 2H), 7.41 (d, 2H, J = 8.0 Hz, ArH), 7.85 (d, 2H, J
= 8.0 Hz, ArH), 8.01 (s, 2H,NH2). 9.15 (t, 1H, NH);13C NMR (100 MHz, DMSO-d6) δ:
34.1, 41.1, 55.9, 115.3(2C), 115.9, 128.8(2C), 162.4, 163.5, 165.3, 168.2.17.5, 40.9,
48.6,124.1(2C), 129.6(2C), 138.5, 149.8, 164.3, 168.0, 170.3; IR (KBr) cm-1 : 3230, 3134,
2818, 2012, 1686, 1617, 1563, 1489, 1432, 1400, 1304, 1282, 1177, 1114, 1046, 962, 901,
844, 786, 701; MS (ESI) m/z [M+H]+: 262.9; Anal. Calcd. for C12H14N4O3 C, 54.96; H,
5.38; N, 21.36. Found C,54.80; H, 5.12; N,21.23.
(R)-2-amino-N-((5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-yl)methyl)propanamide(10h):
Yield 61 %; off white solid, m.p 210 – 212 0C;1H NMR (400 MHz, DMSO-d6) δ: 3.65 (s,
2H), 3.86 (s, 3H), 4.66 (d, J = 4.0 Hz, 2H), 7.16 (d, J = 8.0 Hz, 2H, ArH), 7.92 (d, J =
8.0 Hz, 2H. ArH), 8.23 (s, 3H,NH2). 9.33 (t, 1H, NH);13C NMR (100 MHz, DMSO-d6)
δ: 17.4, 34.2, 48.5, 55.9, 115.3(2C), 116.0, 128.7(2C), 162.5, 163.6, 164.5,170.4; IR
(KBr) cm-1 : 3361, 3092, 2924, 2026, 1988, 1679, 1645, 1611, 1573, 1497, 1421, 1376,
1255, 1177, 1020, 971, 904, 839, 761; MS (ESI) m/z [M+H]+: 276.9; Anal. Calcd. for
C13H16N4O3C, 56.51; H, 5.84; N, 20.28. Found C,56.83; H, 5.42; N,20.33.
2-amino-N-((5-(4-nitrophenyl)-1,3,4-oxadiazol-2-yl)methyl)acetamide(10i):
Yield 52 %; light brown solid, m.p.210 – 212 0C;1H NMR (400 MHz, DMSO-d6) δ: 3.70
(s, 2H), 4.75 (s, 2H), 8.30 (d, 2H, J = 8.0 Hz, ArH), 8.47 (t, 2H, J = 8.0 Hz, ArH), 9.25 (s,
1H,NH);13C NMR (100 MHz, DMSO-d6) δ: 34.1, 41.0, 125.1(2C),
9

128.3(2C),129.1,149.7,163.3, 165.2, 167.2; IR(KBr)cm-1:3200, 3069, 2921, 2012, 1924,
1805, 1679, 1589, 1469, 1433, 1321, 1276,
1165,1039,900,848,709;MS(ESI)m/z[M+H]+:277.8;Anal.Calcd.for
C11H11N5O4C,47.66;H,4.00;N,25.26. Found C,47.79; H, 3.68; N,25.43
(R)-2-amino-N-((5-(4-nitrophenyl)-1,3,4-oxadiazol-2-yl)methyl)propanamide(10j):
0
Yield 45%; light brown solid, m.p.218 – 220 C;1H NMR (400 MHz, DMSO-d6) δ: 1.37
(d, J = 8.0 Hz, 3H), 3.91 (d, J = 4.0 Hz, 1H), 3.96 (d, J = 4.0 Hz, 1H), 8.10(d, 2H, J = 8.0
Hz, ArH), 8.35(d, 2H, J = 8.0 Hz, ArH), 8.78 (s, 1H,NH). 10.25 (s, 1H, NH);13C NMR
(100 MHz, DMSO-d6) δ: 17.5, 40.9, 48.6,124.1(2C), 129.6(2C), 138.5, 149.8, 164.3,
168.0, 170.3;IR (KBr) cm-1 : 3280, 3043, 2839, 1950, 1687, 1601, 1568,1485, 1394, 1312,
1269, 1172, 1010, 970, 869, 778; MS (ESI) m/z [M+H]+: 291.8; Anal. Calcd. for
C12H13N5O4 C, 49.48; H, 4.50; N, 24.04. Found C,49.80; H, 4.52; N,23.73.
2.3.
In vitro biological studies
2.3.1. Compounds
A 10 mM stock solution of compounds was prepared in 100% Dimethyl sulfoxide
(DMSO) and the final concentration of DMSO used in cell culture assays is 0.01%.
2.3.2. Human HDAC inhibition studies
2.3.2.1. HDAC8 purification
Full length human HDAC8 cDNA was cloned into pGEX4T1 bacterial expression vector
and the recombinant protein was expressed in E. coli DH5α (DE3). The culture was then
10

harvested by centrifugation at 8000 rpm for 15 min. The harvested cell pellet was
resuspended in bacteria lysis buffer [50mM Tris-HCl (pH 8.0), glycerol 10%, glucose
20% and 1x protease inhibitor cocktail (SIGMA ALDRICH)], followed by sonication.
The cell lysate was then separated by centrifugation at 11000 rpm for 10 min at 4 °C and
the soluble fraction was collected. Supernatant was incubated with glutathione sepharose
beads for 90 min at 4 °C, followed by four 20 min washes with PBS. The HDAC8 protein
was then eluted using 20 mM reduced glutathione. The purity of the recombinant
HDAC8 protein was checked on SDS-PAGE.
2.3.2.2. Immunoprecipitation
Breast cancer cells, MDA-MB-231, were grown in DMEM medium supplemented with
10% FBS, and grown at 37°C, 5% CO2 until 80% confluency. For immunoprecipitation,
cells were lysed in lysis buffer [20 mM Tris pH 8.0, 137 mM NaCl, 10% glycerol, 1%
Nonidet P-40, 2 mM EDTA, 1x protease inhibitor cocktail] and immunoprecipitation
with the anti-HDAC antibody was carried out for overnight at 4°C. Using 500 ug of total
protein and 1 ug of antibody, the HDAC1, HDAC2, HDAC3 and HDAC8 proteins were
pull down using agarose A/G beads for 1hr at 4°C. The protein complex bound to the
agarose beads were collected by brief centrifugation after washing in lysis buffer, the
beads were suspended in HDAC assay buffer for activity assay.
2.3.2.3.
HDAC8 activity assay
The deacetylation activity of recombinant protein (GST-HDAC8) and
immunoprecipitated HDACs was measured using the Fluor-de-Lys® HDAC fluorometric
activity assay kit (, ENZO Life Sciences, Cat No. BML-AK500-0001) according to
11

manufacturer's specifications. Purified GST-HDAC8 (histone deacetylase 8) or
immunoprecipitated beads with HDACs along with assay buffer (50mM Tris pH 8.0, 137
mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 1mg/ml BSA) was incubated with 25 μM Fluor-
de-Lys substrate for 15 min at 37 °C, in the presence or absence of either 10 μM SAHA
or test compounds. An equal volume of DMSO was used in control well without drug.
After adding 50 μl of developer solution, the plate was incubated for 45 min at room
temperature in dark and fluorescence was measured in a multimode fluorescence reader
(BioTek) at excitation and emission wavelengths of 350 and 440 nm, respectively. The
inhibition of HDAC activity by the test compounds was calculated as percentage of
control.
2.3.3. Anticancer studies
2.3.3.1.
Cell culture
MDA-MB231 breast cancer cells were grown in complete medium (DMEM medium,
10% heat inactivated fetal bovine serum (FBS), 100 IU/ml penicillin, 100 mg/ml
streptomycin and 2mM L-glutamine) at 37 C and 5% CO2. Cell viability was determined
by trypan blue dye and cells were sub-cultured twice a week, seeding at a density of
about 2×104 cells/ml.
2.3.3.2.
MTT Assay
Cytotoxicity was evaluated by MTT assay [19]. Cells were seeded at a density of 5×103
cells/wells in a 96-well plate and incubated for 4 hr. Cells were then treated with
compounds (0.001, 0.01, 0.1, 1, and 10 µM) for 24 h. 10 µl of 5 mg/ml MTT solution
12

was added and incubated for another 3 hr. The formazan crystals formed were dissolved
in 50 µl DMSO and read at 570 nm. The cell viability was measured as % control
(untreated cells) and the % inhibition of growth was plotted as graph.
2.4 Statistical analysis
For the activity assays, the mean and standard error of mean (SEM) was calculated from
the data obtained from 3 independent experiments with triplicates in each experiment.
IC50 was calculated using non-regression dose-response (inhibition) analysis in Graph
Pad Prism software.
3.
Results
3.3. In silico drug designing
Taking, 1,3,4-oxadiazole as backbone, several substitutions were designed using
chemically diverse groups including amino acid groups. All the designed molecules were
first filtered to eliminate non-drug like sub-structures through a careful visual assessment
and application of Lipinsky`s rule. The selected molecules were then assessed for binding
affinity towards HDAC8 crystal protein for its catalytic activity inhibition followed by
docking with other class I enzymes, HDAC1, HDAC2, HDAC3, for selectivity (Table 2).
Surprisingly, 1,3,4 oxadiazole ring lined with glycine/alanine (compounds 10a-10j)
showed high binding affinity and better selectivity towards HDAC8 protein than other
HDACs (1,2&3) proteins. However, compound 10b showed a higher affinity to HDAC8
than other compounds including SAHA and was also showing least dock score to
HDAC1 when compared to other compounds.
13

Table 2: Molecular docking studies of oxadiazole linked with glycine/alanine
Dock score (-Kcal/mol)
HDAC2 HDAC3
(PDB id: 4BKX) (PDB id: 3MAX) (PDB id: 4A69) (PDB id: 1T64)
Compound
HDAC1
HDAC8
6.112
2.61
6.153
6.281
6.227
5.32
5.612
5.656
5.326
5.982
6.39
6.282
5.991
6.482
6.648
6.287
6.553
6.326
6.163
5.94
6.331
6.191
6.12
7.028
7.918
7.233
7.446
7.251
7.557
7.153
6.652
7.131
7.482
6.42
10a
10b
10c
10d
10e
6.031
6.212
6.051
6.173
6.377
6.462
6.563
6.13
10f
10g
10h
10i
10j
SAHA
6.327
6.51
Since class I HDACs are Zinc-dependent enzymes, any molecules targeting Zn
moiety along with amino acids around Zn in catalytic domain are proven to be effective
inhibitors. The binding site residues important for ligand binding in HDAC1 crystal
structure (PDB ID: 4BKX) are Asp 264, Asp 176 and His 178. In the present study the
compound 10b did not show any interaction with any of these residues and therefore the
docking scores were less (-2.61 Kcal/mol). The crystal structure chosen for HDAC2 was
3MAX and the binding site residues in crystal structure are His 145, His 146, Gly 154
and Tyr 308. Compound 10b showed hydrogen bonding only with Gly 154 along with
covalent bonding with Zn and showed a docking score of -5.991 Kcal/mol. HDAC3
binding site residues (PDB ID: 4A69) are Asp 170, Asp 172 and Asp 259. Compound
10b showed interactions with Gly 143 and Phe 144 along with covalent bonding with Zn
and the binding score was -6.191 kcal/mol. HDAC8 binding site residues in crystal
structure (1T64) were Asp 178, His 180, Asp 267, Gly 151, Phe 152 and Tyr 306.
14

Compound 10b was interacting with Tyr 306, gly 151, phe 152 along with Zn and the
binding score was -7.918 kcal/mol. Based on these observations, it was hypothesized that
compound 10b might be selective towards HDAC8 (Fig. 1).
Fig. 1: Docking images of compound 10b showing the interactions with the active site
residues and Zn in the active site of class I HDACs .
15

All compounds obeyed Lipinskys druggability rules (Table 3).
Table 3: Drug-like properties of compounds according to Lipinski’s rule
H bond donors
<= 5
H bond acceptors
<= 10
cLOGP
-1.1742
MW
Compound
n-OHNH
n-OH
3
3
3
3
3
3
3
3
6
6
6
6
6
6
7
7
232.24
246.27
250.23
264.26
246.27
260.29
262.26
276.12
277.24
291.26
10a
10b
10c
10d
10e
10f
10g
10h
10i
-0.8652
-1.0015
-0.692
-0.675201
-0.3662
-1.10217
-0.793166
-1.36755
-1.05855
10j
3.4. Organic synthesis
Chemical synthesis of designed oxadiazoles linked with glycine/alanine (10a – 10j):
Using convergent synthesis coupled with 1,3,4-oxadiazolylmethylamine, synthesis of
designed 1,3,4-oxadiazoles linked with glycine/alanine compound was performed. 1,3,4-
oxadiazolylmethylamine react with boc-protected glycine/alanine in presence of HATU
and followed by de-protection by using TFA. The synthesis of 1,3,4-
oxadiazolylmethylamine was started with aromatic acid to react with MeOH and catalytic
amount of H2SO4 converted in to aromatic ester, which were reacted with hydrazine
hydrate solvent as ETOH to give hydrazide, which was further coupled with Boc-
protected glycine/alanine to give desired compounds (10 a-j) (Scheme 1).
16

All the tested compounds (10 a-j) were characterized by 1H, 13C nuclear magnetic
resonance (NMR), infra red (IR) and liquid chromatography mass spectrometry (LC-MS)
analysis.
Scheme 1
O
O
O
NH2
(b)
(c)
(a)
O
N
H
OH
R
R
R
2
3
1
O
O
N
N
NH2
HCl
N
O
O
HN
N
H
N
(e)
(d)
(f)
O
O
N
N
O
H
H
O
R
R
R
5
6(a-e)
4
Compound
10a
R
R1
R1=H
R=H
O
O
H
O
N
H2N
R=H
R1= CH3
R1=H
OH
OH
10b
R1
R1
O
O
R=F
10c
7
8(a-b)
O
,R1= CH3
R1=H
R1= CH3
R1=H
R1= CH3
R1=H
R1= CH3
HN
O
R= F
10d
N
NH2 HCl
N
N
R1
N
NH
O
H
N
(g)
O
O
R= CH3
R= CH3
R= OCH3
R= OCH3
R= NO2
R = NO2
10e
O
OH
O
R
9(a-b)
10f
R
8(a-b)
6(a-e)
(h)
10g
R= H, CH3, OCH3, NO2, F
R1= H, CH3
10h
H2N
O
10i
N
R1
NH
N
O
10j
R
10(a-b)
(a) MeOH, H2SO4 Reflux (b)
NH2NH2.H2O
(c )
DiPEA, rt
MeOH, rt
DCM, rt, EtOAc in HCl
TFA
Bocglycine HATU, DMF
(f)
(e)
TFA
(d )
(g)
PPh3,CCl4 DMF,TEA, rt
Boc anhydride,CHCl3
DiPEA, rt
(h)
DCM, rt
HATU, DMF,
3.3 In vitro HDAC inhibition activity
To further confirm the HDAC8 inhibitory activity of the synthesized Oxadiazoles linked
with glycine/alanine, activity assay using recombinant purified HDAC8 and HDAC
17

fluorophore substrate was carried out. The results clearly demonstrated that all
compounds showed significant HDAC8 inhibitory activities (Table 4). Similar to the in
silico results, 10b showed substantial HDAC8 inhibitory activity with an IC50 of 98 nM
when compared with other compounds including SAHA (Table 4). 10b showed
comparable or more potency and better HDAC8 selectivity than SAHA. The strong
interactions of 10b with active site amino acids of HDAC8 are responsible for its
HDAC8 inhibition activity (Fig. 1).
Table 4: HDAC inhibition activities of oxadiazoles linked with glycine/alanine
Compounds In vitro HDAC8 activity
IC50 ± SEM (nM)
155 ± 9.5
98 ± 6.0
10a
10b
10c
135 ± 8.1
116 ± 9.5
139 ± 4.2
121 ± 2.1
142 ± 5.6
153 ± 11.6
134 ± 3.1
127 ± 4.5
1480 ± 5.6
10d
10e
10f
10g
10h
10i
10j
SAHA
18

Further, to assess the selectivity of 10b, class I HDAC enzymes were immunoprecipitated
from MDA-MB-231 cell lysates and activity assay was carried out using the
immunoprecipitated proteins as enzyme source. The results clearly showed that 10b was
moderately selective in inhibiting HDAC8 (53% inhibition) than HDAC1 (30%),
HDAC2 (19%) and HDAC3 (0%) at 100 nM concentration. A selectivity ratio was
expressed as the ratio of IC50 for HDAC8 to that for HDAC1, 2, or 3 (Table 5) that
showed compound 10b was moderately selective to HDAC8 than other class I HDACs.
Table 5: Selectivity ratio of Compound 10b for class I HDACs.
HDAC IC50 (nM) Selectivity ratio
(IC50 HDAC8/IC50 HDAC)
HDAC1 260
HDAC2 135
HDAC3 >10,000
HDAC8 100
0.38
0.74
ND
1
3.4
In vitro anti-cancer activity
Anticancer activity of oxadiazoles linked with glycine/alanine (10 a-j) was evaluated
against breast cancer MDA-MB-231 cells by MTT assay. All compounds showed
significant anticancer activity. Among the tested compounds, compound 10b has
emerged as most active compound against MDA-MB-231 cancer cells (IC50 = 230 nM)
(Table.6).
19

Table 6: Anti-proliferative effects of compounds
In vitro anticancer activity against MDA-MB231breast
cancer cells
Compounds
IC50 ± SEM (nM)
283 ± 12.3
230 ± 11.6
254 ± 16.9
247 ± 12.6
260 ± 9.8
10a
10b
10c
10d
10e
254 ± 13.4
276 ± 14.8
295 ± 12.4
276 ± 22.4
261 ± 19.4
6000 ± 10.8
10f
10g
10h
10i
10j
SAHA
Discussion
Development of class-selective and isoform-specific inhibitor of HDACs is currently
a major focus of HDAC inhibitor design [20]. Selective HDAC inhibitors, for instance
tubacin, HDAC6 selective inhibitor, not only can elucidate the function of individual
HDAC but also provide candidates for the treatment of cancer or other diseases with less
side effects [21]. Weiping et al., (2011) designed and discovered HDAC8-selective
inhibitors from a novel collection of compounds [22]. However, only limited progress
towards the discovery of HDAC8 selective inhibitors has been made to date.
Oxadiazole is an important pharmacophore, which has been reported to possess
various bioactivities including HDAC inhibition [16]. In the present study, we have
designed a number of molecules on a basic oxadizole core moiety. Surprisingly, glycine
20

and alanine linking to oxadizole moiety showed better selectivity towards HDAC8 than
other isoforms of class I HDACs in both in silico and in vitro studies. Similar selectivity
of oxadiazole-containing hydoxamates towards HDAC6, a class II isoform, has been
reported recently [23].
The structure activity relationship studies showed that, glycine/alanine compounds,
particularly L isomer, exhibited selective HDAC8 inhibition among other class I HDACs.
Methyl substitution in R1 group and H substitution in R group of compound 10b caused
good HDAC8 inhibition and better selectivity. 10b exhibited more HDAC8 inhibition
and moderate selectivity than SAHA (a FDA approved drug). Further anticancer studies
were performed on HDAC inhibitory compounds since HDACs are involved in
pathophysiology of cancer. Among all compounds, HDAC8 inhibitory 10b exhibited
anti-proliferative potential against MDA-MB-231 breast cancer cells. Further detailed
investigations are under progress to demonstrate the molecular mechanism involved in its
anti-proliferative activity.
4.
Conclusions
A series of HDAC8-selective oxadiazoles linked with glycine/alanine (10 a-j) were
designed using in silico tools and chemically synthesized with high yields. In vitro
HDAC and anticancer studies demonstrated that oxadiazoles linked with glycine/alanine
(10 a-j) showed significant class I HDAC inhibition and anticancer activities. Among all
compounds, 10b showed moderate selectivity towards HDAC8 as well as better
anticancer activity than SAHA (positive control). The results of the present study
21

warranted further studies on 10b to develop as a potential candidate drug for the
treatment of HDAC8-overexpressed cancers.
Funding
Financial assistance from DBT-BCIL (Grant # BT/327/NE/TBP/2012) to AMK is
acknowledged.
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Highlights
 Synthesis of 2,5-disubstituted 1, 3, 4-oxadiazoles - glycine/alanine hybrids (10a-
10j).
 In silico and in vitro evaluation of (10a-10j) as class I HDAC inhibitors.
 All compounds showed cytotoxic effects on breast cancer cell line, MDA-MB-
231.
 Compound 10b showed high binding affinity and moderate selectivity to HDAC8.
 Compound 10b showed potent anti-proliferative effects on MDA-MB-231 cells.
26

Graphical Abstract
27