Synthesis and SAR studies of novel 1,2,4-oxadiazole-sulfonamide based compounds as potential anticancer agents for colorectal cancer therapy

Article abstract of DOI:10.1016/j.bioorg.2020.103754

A diverse series of 1,2,4-oxadiazoles based substituted compounds were designed, synthesized and evaluated as anticancer agents targeting carbonic anhydrase IX (CAIX). Initial structure-activity analysis suggested that the thiazole/thiophene-sulfonamide conjugates of 1,2,4-oxadiazoles exhibited potent anticancer activities with low μM potencies. Compound OX12 exhibited antiproliferative activity (IC₅₀ = 11.1 μM) along with appreciable inhibition potential for tumor-associated CAIX (IC₅₀ = 4.23 μM) isoform. Therefore, OX12 was structurally optimized and its SAR oriented derivatives (OX17-27) were synthesized and evaluated. This iteration resulted in compound OX27 with an almost two-fold increase in antiproliferative effect (IC₅₀ = 6.0 μM) comparable to the clinical drug doxorubicin and significantly higher potency against CAIX (IC₅₀ = 0.74 μM). Additionally, OX27 treatment decreases the expression of CAIX, induces apoptosis and ROS production, inhibited colony formation and migration of colon cancer cells. Our studies provide preclinical rational for the further optimization of identified OX27 as a suitable lead for the possible treatment of CRC.

Full text of DOI:10.1016/j.bioorg.2020.103754

Journal Pre-proofs
Synthesis and SAR studies of novel 1,2,4-oxadiazole-sulfonamide based com-
pounds as potential anticancer agents for colorectal cancer therapy
Farheen Shamsi, Phool Hasan, Aarfa Queen, Afzal Hussain, Parvez Khan,
Bushra Zeya, Hannah M. King, Sandeep Rana, Jered Garrison, Mohamed F.
Alajmi, M Moshahid Alam Rizvi, Muhammad Zahid, Md Imtaiyaz Hassan,
Mohammad Abid
PII:
S0045-2068(19)31689-X
DOI:
https://doi.org/10.1016/j.bioorg.2020.103754
YBIOO 103754
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
9 October 2019
7 February 2020
11 March 2020
Please cite this article as: F. Shamsi, P. Hasan, A. Queen, A. Hussain, P. Khan, B. Zeya, H.M. King, S. Rana, J.
Garrison, M.F. Alajmi, M. Moshahid Alam Rizvi, M. Zahid, M. Imtaiyaz Hassan, M. Abid, Synthesis and SAR
studies of novel 1,2,4-oxadiazole-sulfonamide based compounds as potential anticancer agents for colorectal
cancer therapy, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.103754
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1
,2,4-oxadiazole-sulfonamide-based CAIX inhibitors
Synthesis and SAR studies of novel 1,2,4-oxadiazole-sulfonamide
based compounds as potential anticancer agents for colorectal
cancer therapy
a,b‡
a‡
c
d
c
Farheen Shamsi , Phool Hasan , Aarfa Queen , Afzal Hussain , Parvez Khan Bushra
,
b
e
e
f
c
Zeya , Hannah M. King , Sandeep Rana , Jered Garrison , Mohamed F. Alajmi , M
Moshahid Alam Rizvi , Muhammad Zahid , Md Imtaiyaz Hassan , Mohammad Abid *
b
g
c
a
aMedicinal Chemistry Laboratory, Department of Biosciences, Jamia Millia Islamia, New Delhi-
1
10025, India
bGenome Biology Laboratory, Department of Biosciences, Jamia Millia Islamia, New Delhi-
10025, India
cCentre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi,
10025, India
1
1
dDepartment of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh, 11451,
Kingdom of Saudi Arabia
eEppley Institute for Research in Cancer and Allied Diseases, Fred & Pamela Buffett Cancer
Center, University of Nebraska Medical Center, Omaha, Nebraska 68198-6805, USA
fDepartment of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical
Center, Omaha, Nebraska, 68198-6805, USA
gDepartment of Environmental, Agricultural and Occupational Health, University of Nebraska
Medical Center, Omaha, Nebraska, 68198-6805, USA, Omaha, USA
✉
To whom correspondence should be addressed: (Phone):- +91-8750295095; (Fax): +91-11-2698 0229; email:
mabid@jmi.ac.in (M. Abid).
‡
These authors contributed equally to this work.
1

1
,2,4-oxadiazole-sulfonamide-based CAIX inhibitors
Abstract: A diverse series of 1,2,4-oxadiazoles based substituted compounds were designed,
synthesized and evaluated as anticancer agents targeting carbonic anhydrase IX (CAIX). Initial
structure-activity analysis suggested that the thiazole/thiophene-sulfonamide conjugates of 1,2,4-
oxadiazoles exhibited potent anticancer activities with M potencies. Compound OX12 exhibited
antiproliferative activity (IC =11.1µM) along with appreciable inhibition potential for tumor-
5
0
associated CAIX (IC =4.23µM) isoform. Therefore, OX12 was structurally optimized and its
5
0
SAR oriented derivatives (OX17-27) were synthesized and evaluated. This iteration resulted in
compound OX27 with an almost two-fold increase in antiproliferative effect (IC =6.0µM)
5
0
comparable to the clinical drug doxorubicin and significantly higher potency against CAIX
IC =0.74µM). Additionally, OX27 treatment decreases the expression of CAIX, induces
(
5
0
apoptosis and ROS production, inhibited colony formation and migration of colon cancer cells.
Our studies provide preclinical rational for the further optimization of identified OX27 as a
suitable lead for the possible treatment of CRC.
Keywords: Colorectal cancer, 1,2,4-oxadiazoles, Sulfonamides, Inhibitors, Apoptosis.
1
.
Introduction
2

1
,2,4-oxadiazole-sulfonamide-based CAIX inhibitors
Recent global cancer data (GLOBOCAN 2018) suggests that the global cancer burden has risen to
8.1 million cases and 9.6 million cancer deaths worldwide. Quite evidently, cancer has become
1
an urgent global challenge with the steep rise in the number of cancer cases and deaths each year.
Over 1.8 million new colorectal cancer (CRC) cases and 881,000 deaths are estimated to occur in
2
018, accounting for about 1 in 10 CRC cases and deaths [1]. CRC ranks third in terms of incidence
but second in terms of mortality and is the fifth most common cancer in India [2]. Several
chemotherapeutic agents’ anticancer efficacy is correlated with their apoptosis-inducing ability.
Therefore, the identification of novel apoptosis-inducing agents represents an attractive therapeutic
strategy against CRC. The new agents may overcome tumor resistance to conventional anticancer
agents by inducing apoptosis [3]. Molecularly targeted cancer drug discovery over the last few
decades has resulted in several small molecule inhibitors being successfully introduced in the clinic
for cancer treatment. Moreover, insights into tumor metabolism have provided novel therapeutic
strategies to exploit it, some of which are being evaluated in preclinical models or clinical trials.
One such feature of solid cancers is hypoxia. Metabolic alterations induced by hypoxia can
promote activities associated with aggressive tumor cell behavior, including survival, invasion and
metastasis [4]. One set of proteins important to this system is the family of carbonic anhydrases
(CAs) [5]. CAs are important regulators of a variety of biological processes, including respiration,
acid-base regulation and biosynthetic processes. In the context of tumors, studies provide strong
preclinical evidence for the use of CAIX inhibitors as targeted pharmacologic agents for the
treatment of hypoxic, aggressive solid cancers [6]. CAIX is found to be overexpressed in majority
of hypoxic tumors including CRC and inhibitors of CAIX continuously exploited for the
development of anticancer drug molecules for the treatment of CRC [7-8]. In the context of cancer
therapeutics, development of small molecule inhibitors is the most sought-after approach to
overcome the downsides of traditional cancer drugs. For instance, nitrogen and sulfur-containing
heterocycles have become dominant over other natural/synthetic bioactive small molecules as
therapeutics due to their activity in biological systems [9-10].
Literature survey reveals 1,2,4-oxadiazoles are statistically significant in bioorganic and
pharmaceutical chemistry. They are known to exhibit varied pharmacological properties. Some
examples of 1,2,4-oxadiazole based inhibitors is illustrated in Figure 1. Compounds I, II & III
have been evaluated for their anticancer properties in various cell lines and have yielded excellent
results in colorectal cell lines, such as HCT-116 and CaCo2 cell lines [11-12]. In this study, a
3

1
,2,4-oxadiazole-sulfonamide-based CAIX inhibitors
series of 1,2,4-oxadiazole based substituted compounds were synthesized and evaluated against
HCT-116 cells. Initial study suggested that among the 16 synthesized compounds,
thiazole/thiophene-sulfonamide conjugate of 1,2,4-oxadiazoles exhibited potent anticancer
activities with M potencies. Compound OX12 exhibited good antiproliferative activity against
HCT-116 cells with an IC value of 11.1µM. The carbonic anhydrase (CA) inhibitory potential
5
0
of sulfonamides has been very well established through the years. A few examples are illustrated
in Figure 1, SLC-0111 is a selective, small molecule inhibitor of CAIX which is under
development for the treatment of solid tumors over-expressing CAIX [13]. Compound IV and V
are reported recently to be active against cancer-related, membrane bound isoform hCAIX [14].
Although primary sulfonamides are known to be good inhibitors of carbonic anhydrases in contrast
to secondary sulfonamides, there are reports that evidence the CA inhibitory potential of secondary
sulfonamides as well. The secondary sulfonamide moiety is present in a large variety of clinically
used drugs, among which the sulfa drugs are the best known examples [15]. Examples of secondary
sulfonamides active against CAs are given in Figure 1. For instance, saccharin VII, a cyclic
secondary sulfonamide is reported to possess exceptionally good CA inhibitory potential against
hCAIX/XII. Compound VI is also reported to show selective inhibitory action against the tumor-
associated isoforms hCA IX/XII, without significant inhibition of hCA I and II [15-16]. We tested
the compound OX-12 for CA inhibitory activity only to find its inhibition potential for tumor-
associated CAIX isoform with an IC value of 4.23µM. We carried on this study by structurally
5
0
optimizing OX12; synthesizing its SAR derivatives (OX17-27) as well as evaluating these for
antiproliferative and CA inhibitory potential which led us to our lead compound OX-27.
4

1
,2,4-oxadiazole-sulfonamide-based CAIX inhibitors
Figure 1. Designing strategy: Structures of some CAIX & other anticancer inhibitors highlighting the
main pharmacophoric moieties that formed the basis in designing new 1,2,4-oxadiazole-sulfonamide
compounds
2
. Results and Discussion
2
.1 In silico prediction of physicochemical properties
To develop small molecule inhibitors against CRC, we identified 1,2,4-oxadiazole as our
core structure and designed new derivatives. Before synthesizing the designed compounds, it is
crucial to determine the drug-like properties since most drugs fail in clinical trials due to their poor
physiochemical properties. The predicted ADME parameters were used to determine drug-like
properties for the designed compounds are summarized in Table-S1 (Supplementary Information).
The values for other important physicochemical parameters such as Lipinski’s parameters,
aqueous solubility (logS), lipophilicity (logD), number of rotatable bonds (RB), bioavailability
(BA), oral bioavailability (OBA), topological polar surface area (TPSA), binding to human serum
albumin (LogK (HSA)), brain/blood partition coefficient (logBB) etc. were also calculated and
a
5

1
,2,4-oxadiazole-sulfonamide-based CAIX inhibitors
found within the range with reference to 95% drugs. These results suggested that the designed
compounds possess drug-like properties.
2
.2 Chemistry
Based on positive ADME predictions viz. drug-like properties, a focused library of 16 new
,2,4-oxadiazole based compounds were synthesized and evaluated for their antiproliferative
1
effects in HCT-116 cells.
2
.2.1 Synthesis of 1,2,4-oxadiazoles
The synthetic route to prepare all the precursors and final compounds is illustrated in Scheme 1-
1. The synthesis of a 1,2,4-oxadiazole core required the formation of a common intermediate, N-
1
hydroxybezamidine (2), which was accomplished by the alkaline treatment of benzonitrile (1) in
refluxing ethanol-water mixture (Scheme1). The amidoxime route to get 1,2,4-oxadiazole core
generally requires activated carboxylic acid, which can be prepared either beforehand or in situ by
several methods [17], e.g., as an acyl chloride or using N, N′-carbonyldiimidazole (CDI). In this
study, all the corresponding acids were used directly or synthesized by multi-step procedures that
were activated by using CDI and amidoxime was O-acylated with the activated acid, which was
cyclized at high temperature in toluene to afford 3,5-disubstituted 1,2,4-oxadiazole derivatives in
moderate to good yield [18]. The acids 3 and 4 were directly converted to oxadiazole derivatives
(OX1 and OX2) following the O-acylation and cyclodehydration protocol (Scheme 2). OX2,
when treated with p-fluorophenylboronic and tetrakis(triphenylphosphine)palladium(0) gave
oxadiazole OX3 (Scheme 3).
a
N
OH
N
NH2
2
1
Scheme 1. General synthesis of N-hydroxybezamidine. Reagents and conditions: NH OH.HCl,
2
EtOH/H O, reflux, 6 hours.
2
6

1
,2,4-oxadiazole-sulfonamide-based CAIX inhibitors
O
N
R
O
N
Ph
N
O
a
b
OH
O
R
+
Ph
N
NH2
2
R
OH
, 4
Ph
NH2
OX 1, 2
3
R =
N
Cl
N
N
OX1
OX2
Scheme 2. Synthesis of OX1 and OX2. Reagents and conditions: (a) CDI in DCM, 30 min; (b)
toluene 120 °C, 4-16 h.
N
O
N
O
HO
B
HO
N
+
N
F
a
N
N
Cl
OX3
OX2
F
Scheme 3. Synthesis of OX3. Reagents and conditions: (a) Pd(PPh ) , 4-fluorophenylboronic acid,
3
4
Na CO , DME and water mixture at 80 °C for 16 h.
2
3
Commercially available esters 5 and 6 were hydrolyzed to corresponding acids 7 and 8 in
the presence of lithium hydroxide in THF/H O. The reaction of 7 and 8 with intermediate 2 yielded
2
oxadiazoles (OX4 and OX5) (Scheme 4). Synthesis of OX6 and OX7 was achieved by the
nucleophilic addition of different amines to ethyl 4,6-dichloronicotinate, 9 resulting in the
formation of esters 10 and 11, followed by basic hydrolysis to yield acids 12 and 13, respectively.
The reaction of corresponding acids 12 and 13 with intermediate 2 yielded OX6 and OX7 (Scheme
5
).
7

1
,2,4-oxadiazole-sulfonamide-based CAIX inhibitors
O
N
R
O
O
O
N
c
Ph
N
b
a
O
R
OH
+
R
O
R
OH
7, 8
Ph
N
NH2
2
5, 6
Ph
NH2
OX4, 5
OX4
OX5
N
N
R =
Cl
Cl
Br
Scheme 4. Synthesis of OX4 and OX5. Reagents and conditions: (a) LiOH, THF:Water (2:1), 16
hours; (b) CDI in DCM, 30 min; (c) toluene 120 °C, 4-16 h.
OH
O
O
Ph
N
b
N
O
R
R NH2
a
N
O
R
OH
+
c
N
O
NH2
2
Cl
N
Cl
N
Cl
Cl
H
H
1
2, 13
10, 11
9
R =
NH2
N
O
N
NH2
d
OX6
O
O
N
N
R
NH
R
HN
OX7
Cl
N
O
H
N
Cl
Scheme 5. Synthesis of OX6 and OX7. Reagents and conditions: (a) DIPEA, DMA, rt, 16 h; (b)
LiOH, THF:Water (2:1), 16 h; (c) CDI in DCM, 30 min; (d) Toluene, 120°C, 4-16 h.
Aryl sulfonyl chlorides 14 and 15 reacted with ethyl piperidine-4-carboxylate in the presence of
pyridine yielded sulfonamide esters 16 and 17 which were subjected to basic hydrolysis to yield
corresponding acids 18 and 19, respectively. The reaction of amidoxime 2 with corresponding
acids (18 and 19) resulted in oxadiazoles OX8 and OX9 respectively (Scheme 6).
8

1
,2,4-oxadiazole-sulfonamide-based CAIX inhibitors
O
O
HO
O
O
O
Ph
N
a
b
OH
NH
N
N
+
O S Ar +
Cl
O
NH2
O S O
Ar
6, 17
O S O
Ar
Ar=
1
4, 15
1
18, 19
c
OX8
N
O
Cl
Cl
O
O
N O
NH2
O
N
N S Ar
O
d
O
N
S
Ar
Ph
OX9
O
N
OX8, 9
Scheme 6. Synthesis of OX8 and OX9. Reagents and conditions: (a) Pyridine, DCM, rt, 16 h; (b)
LiOH, THF:Water (2:1), 16 h; (c) CDI in DCM, 30 min; (c) Toluene, 120°C, 4-16 h.
Substituted aromatic sulfonyl chlorides 22 and 23 reacted with ethyl 2-aminothiazole-4-
carboxylate 20 obtained by the treatment of thiourea with 3-bromo-2-oxo-propionic acid ethyl
ester in refluxing ethanol gave the corresponding thiazole appended sulfonamides esters 25 and 26
[19]. These esters were hydrolyzed (28 and 29) followed by cyclodehydration to yield oxadiazoles
OX10 and OX11. In another set of reaction, sulfonyl chloride 24 reacted with methyl 3-
aminothiophene-2-carboxylate 21 to yield the corresponding thiophenyl sulfonamide ester 27
which on hydrolysis (30) followed by cyclodehydration with 2 led to the formation of oxadiazole
OX12 (Scheme 7).
9

1
,2,4-oxadiazole-sulfonamide-based CAIX inhibitors
Br
O
S
+
O
H N
NH2
2
O
a
S
O
NH2
Ar
N
Ar
O
O
O
S
O
O
O
O
O
b
O
O
S
c
O
20
Het NH
=
Het-NH + O S Ar
Het NH
2
HO
Cl
S
O
2
8, 29, 30
2
2, 23, 24
25, 26, 27
O
NH2
Ph
O
N
d
OH
2
1
NH2
Ar =
Het =
Ar
O
N
S
O
O
S
N
e
HetNH
OX10
N
Het NH
N O
NH2
S
Ph
O
O
Ar
OX10-12
S
N
OX11
OX12
Cl
Cl
S
Scheme 7. Synthesis of OX10, OX11 and OX12. Reagents and conditions: (a) K CO , EtOH,
2
3
reflux; (b) Pyridine, DCM, rt, 16 h; (c) LiOH, THF:Water (2:1), 16 h; (d) CDI in DCM, 30 min;
(e) Toluene, 120 °C, 4-16 h.
The condensation reaction of acetophenone 31 with phenylhydrazine 32 in the presence of
sodium acetate afforded known hydrazone 33 [20] followed by cyclization in the presence of
Vilsmeier reagent gave 1, 3-disubstituted pyrazole-4-carbaldehyde 34 [21-22] followed by
Knoevenagel condensation with malonic acid to yield α, β unsaturated carboxylic acid 35. The
reaction of intermediate 35 with 2 yielded oxadiazole OX13 [23] (Scheme 8).
1
0

1
,2,4-oxadiazole-sulfonamide-based CAIX inhibitors
N
4
Ph
Ph N
Ph
NH2
HN
a
b
H
Ph
N
N
O
Ph
Ph
O
3
31
32
33
c
N
N
Ph
O
OH
Ph
Ph N
N
Ph N
N
Ph
Ph N
e
Ph
NH2
d
O
N
OH
N
O
N
O
H N
Ph
2
Ph
OX13
35
Scheme 8: Synthesis of OX13. Reagents and conditions. (a) NaOAc, EtOAc, reflux, 2 h; (b)
POCl , DMF, 70 °C, 2 h; (c) malonic acid, piperidine: pyridine (1:10), 115 °C, 4 h; (d) CDI in
3
DCM, 30 min; (e) Toluene, 120 °C, 4-16 h.
3
-Amino-1H-pyrazole-4-carboxylic acid, 36 reacted with 2-chloromalonaldehyde in
refluxing ethanol afforded acid, 37, which then coupled with piperidine 4-carboxylate to yield ester
8, followed by hydrolysis and cyclodehydration to yield oxadiazole OX14 (Scheme 9).
3
1
1

1
,2,4-oxadiazole-sulfonamide-based CAIX inhibitors
Cl
O
O
O
N
N
N
Cl
N
NH
N
O
O
N
O
N
N
H
a
HO
H
H
N
Cl
HO
b
NH2
O
O
38
O
36
37
c
Cl
O
N
N
OH
Cl
N
N
N
N
Cl
N
N
O
N
Ph
NH2
e
N
O
N
O
N
d
N
O
OH
N
N
O
N
O
H N
Ph
2
Ph
39
OX14
Scheme 9. Synthesis of OX14. Reagents and conditions: (a) EtOH, reflux, 16 h; (b) HATU,
DIPEA, DCM; (c) LiOH, THF:Water (2:1), 16 h; (d) CDI in DCM, 30 min; (e) Toluene, 120 °C,
4
-16 h.
Commercially available ester 40 reacted with 4-fluorophenylboronic acid via Suzuki cross-
coupling reaction to yield corresponding ester 41. The ester was then hydrolyzed followed by
cyclodehydration yielded oxadiazole OX15 (Scheme 10).
1
2

1
,2,4-oxadiazole-sulfonamide-based CAIX inhibitors
B(OH)2
N
N
HO
O
N
O
F
N
2
N
O
O
N
O
b
a
Cl
4
4
1
F
F
4
0
F
OH
OH
N
N
H
N
N
N
Ph
NH2
N
N
O
N
O
c
d
N
F
OX15
Scheme 10. Synthesis of OX15. Reagents and conditions: (a) Na CO , Pd(PPh ) , DME, 16 h; (b)
2
3
3 4
LiOH, THF:Water (2:1), 16 h; (c) CDI in DCM, 30 min; (d) Toluene, 120 °C, 4-16 h.
The reaction of methyl 4-fluoro-3-nitrobenzoate 43 with tetrahydrofuran-3-ol 44 resulted
in ester 45 followed by hydrolysis and cyclodehydration to yield oxadiazole OX16 (Scheme 11).
OH
O
O
O N
O N
2
2
O
O
O N
2
O
O
+ O
O
O
OH
a
O
b
F
46
45
43
44
Ph
N
c
OH
NH2
O
N
O N
2
O
N
O
N
O
H2N
d
O
NO2
O
O
OX16
Scheme 11. Synthesis of OX16. Reagents and conditions: (a) NaH, THF, rt, 16 h; (b) LiOH,
THF:Water (2:1), 16 h (c) CDI in DCM, 30 min; (d) Toluene 120 °C, 4-16 h.
1
3

1
,2,4-oxadiazole-sulfonamide-based CAIX inhibitors
2
.3 X-ray crystallographic analysis
The crystal structures for compounds OX4, OX5, OX7 and OX8 were determined by single crystal
X-ray diffraction analysis. The ORTEP drawings of the compounds and corresponding packing
diagrams are given in Figure 2. The corresponding crystallographic data/parameters are presented
in Table 1. The crystallographic data has been deposited with the Cambridge Crystallographic
Structural Database (CCSD). Complete crystallographic data tables can be found in the
Supplementary Information.
(a)
1
4

1
,2,4-oxadiazole-sulfonamide-based CAIX inhibitors
(b)
Figure 2. (a) ORTEP diagram of compound OX4, OX5, OX7 and OX8 (b) Unit cell diagrams in the crystal
packing of compounds OX4, OX5, OX7, and OX8.
Table 1: Crystal data and structure refinement
Identification code
Empirical formula
OX4
OX5
OX7
C H N O Cl C H N O SCl
2
OX8
C H N OCl
C H N OBr
1
3
7
3
2
13
8
3
17 15
4
2
19 17
3
3
CCDC No.
Formula weight
Temperature/K
1901611
292.12
100
1901612
302.13
100
1901613
342.78
100
1901614
438.31
100
Crystal system
Space group
Monoclinic
monoclinic
monoclinic
orthorhombic
Pbca
P2 /n
P2 /c
P2 /c
1
1
1
a/Å
b/Å
c/Å
α/°
β/°
γ/°
Volume/Å
Z
6.92553(15)
12.9234(3)
13.8420(3)
90
102.574(2)
90
1209.17(5)
4
1.605
6.38104(11)
10.04678(18)
17.8987(3)
90
90.7407(16)
90
1147.37(4)
4
1.749
15.0135(7)
18.2451(6)
12.1729(5)
90
109.713(5)
90
3139.0(2)
8
1.451
8.60462(7)
11.76423(11)
38.5015(4)
90
90
90
3897.38(6)
8
1.494
4.229
1808.0
3
3
ρcalcg/cm
μ/mm
F(000)
-1
4.789
592.0
4.810
600.0
2.314
1424.0
1
5

1
,2,4-oxadiazole-sulfonamide-based CAIX inhibitors
Crystal size/mm3
Radiation
0.5 × 0.1 × 0.05 0.5 × 0.2 × 0.2 0.5 × 0.5 × 0.1 0.6 × 0.5 × 0.2
CuKα (λ =
.54184)
CuKα (λ =
1.54184)
CuKα (λ =
1.54184)
6.254 to
152.988
CuKα (λ =
1.54184)
1
2
Θ range for data collection 9.47 to 151.954 9.884 to
51.048
9.188 to 152.26
1
Index ranges
-7 ≤ h ≤ 8, -15
-7 ≤ h ≤ 8, -12
-18 ≤ h ≤ 17, - -10 ≤ h ≤ 10, -14
≤
k ≤ 13, -14 ≤ ≤ k ≤ 9, -22 ≤ l 22 ≤ k ≤ 18, -
≤ k ≤ 14, -48 ≤ l
≤ 45
l ≤ 17
≤ 20
15 ≤ l ≤ 13
Reflections collected
8100
7487
13528
59071
Independent reflections
2477 [R =
2316 [R =
6295 [R =
4073 [R =
int
int
int
int
0
0
.0420, Rsigma = 0.0487, Rsigma = 0.0557, Rsigma
0.0715, Rsigma
0.0218]
=
.0340]
0.0380]
= 0.0547]
Data/restraints/parameters 2477/0/172
2316/0/163
6295/0/433
4073/0/253
Goodness-of-fit on F2
1.050
1.099
1.071
1.150
Final R indexes [I>=2σ (I)]
R = 0.0368,
1
R = 0.0400,
1
R = 0.0759,
1
R = 0.0441,
1
wR = 0.0946
wR = 0.1059
wR = 0.2114
wR = 0.1158
2
2
2
2
Final R indexes [all data]
Largest diff. peak/hole / e
R = 0.0423,
R = 0.0434,
R = 0.0904,
R = 0.0456,
1
1
1
1
wR = 0.1060
wR = 0.1087
wR = 0.2338
wR = 0.1169
2
2
2
2
0.34/-0.36
0.93/-0.67
0.90/-0.54
0.46/-0.79
-3
Å
2
2
.4 Biological Activity
.4.1 In vitro cell proliferation assay
The 1,2,4-oxadiazole derivatives (OX1 - OX16) were screened for their antiproliferative activity
against HCT-116 cells. Cell-based growth inhibition studies showed better anticancer potential of
OX4, OX11, and OX12 ranging between 11.1-50.6 µM. (Table 2). A preliminary structure-
activity relationship analysis suggested thiazole sulfonamide conjugate of oxadiazole bearing
dichloro substitution (OX11) displayed moderate potency against HCT-116 cells. Replacement of
dichloro substitution by dimethyl increased the antiproliferative potency by more than two-fold
(
IC = 21.4 µM) of OX10. Oxadiazole with dichloro substituted pyridyl ring (OX4) found with
50
equal potency (IC = 23.4 µM) against HCT-116 cells. Interestingly, efficacy on HCT-116 cells
5
0
was enhanced by the replacement of the thiazole ring with a thiophenyl ring in OX12 (IC = 11.1
5
0
µM). Initial screening identified OX12 as lead analog that could be further optimized for improved
potency against CRC or selected CRC model (HCT-116) cells.
1
6

1
,2,4-oxadiazole-sulfonamide-based CAIX inhibitors
Table 2: Antiproliferative activities of 3,5-disubstituted 1,2,4-oxadiazoles (OX1-16) in HCT-116
cells
Compound
OX1
Chemical Structure
IC (μM)
50
HCT-116 (48 h)
N O
N
>100
N
N
N O
N
OX2
OX3
>100
>100
N
Cl
N
O
N
N
F
N O
N
Cl
OX4
OX5
23.4  6.0
N
N
Cl
N O
N
>100
Br
OX6
OX7
>100
>100
N O HN
N
N
Cl
O
N O
N
N
N
Cl
Cl
OX8
OX9
N O
N
>100
>100
O
N S
O
Cl
O
N O
N
O
N S
O
N
1
7

1
,2,4-oxadiazole-sulfonamide-based CAIX inhibitors
N O
OX10
21.4  4.60
50.6  0.2
N
N
S
O
HN S
O
N O
OX11
N
S
Cl
Cl
N
O
HN S
O
N O
OX12
OX13
11.1  0.9
S
N
HN
S O
O
>100
N
N
O
N
N
OX14
OX15
>100
>100
N
O
N
N
N
N
O
N
Cl
F
N
N
N
O
N
1
8

1
,2,4-oxadiazole-sulfonamide-based CAIX inhibitors
NO2
OX16
>100
O
N
O
N
O
The SAR derivatives (OX17-27) of OX12 were synthesized (scheme 12) using the same protocol
as applied in Scheme 7 for the synthesis of OX12. The SAR optimization is briefly represented in
Figure 3 below followed by the synthetic procedure of all the derivatives, viz., OX17-27 (scheme
1
2).
Figure 3. SAR Optimization of OX12
1
9

1
,2,4-oxadiazole-sulfonamide-based CAIX inhibitors
OH
O
O
S
O
R1 S Cl
O
S
O
O
S
NH
O S O
R1
O
NH
O S O
R1
a
b
NH2
48a-k
47a-k
21
OH
N
c
R2 NH2
N O
O
S
S
O
N
R2
N
HN
S O
R2
NH2 HN
O
O
S
R1
d
O
R1
OX17-27
49a-k
Scheme 12: Synthesis of SAR derivatives (OX17-OX27). Reagents and conditions: (a) Pyridine,
DCM, rt, 16 h; (c) LiOH, THF:Water (2:1), 16 h; (d) CDI in DCM, 30 min; (e) Toluene, 120°C,
4
-16 h.
Various aliphatic, aryl/heteroaryl sulfonyl chlorides were employed to access the core
structure (Scheme 12). Among all the SAR analogues (OX17-27), the activity of OX18 against
HCT-116 was comparatively enhanced (IC = 9.0 µM) by the replacement of the phenyl group at
5
0
the third position of oxadiazole core by the methyl group. However, this replacement cannot be
considered as the sole reason for the increase in potency as in compound OX27. Replacement of
p-methyl substitution in the sulfonamide group by the p-amino group while keeping the whole
molecule intact led to the identification of most potent inhibitor among all which exhibited IC50
value of 6.0 µM on HCT-116 cells comparable to the standard drug doxorubicin with IC50
concentration 5.1 μM (Table 3).
2
0

1
,2,4-oxadiazole-sulfonamide-based CAIX inhibitors
Table 3: SAR of derivatives of OX12
Compound
R
1
R
2
IC50 (μM)
IC50 (μM)
IC50 (μM)
Selectivity
ratio
HCT-116 (48h)
CAIX
CAII
CAII/IX
OX12
OX17
OX18
OX19
OX20
OX21
OX22
11.1±0.9
> 50
4.23±0.13
9.32±0.45
10.24±0.33
5.24±0.54
4.74±0.33
0.92±0.21
4.67±0.23
12.55±0.42
11.34±0.66
4.71±0.24
8.82±0.64
8.64±0.42
2.01±0.26
6.76±0.44
3.0
1.2
0.5
1.7
1.8
2.2
1.4
9.0 ± 2
> 50
> 50
> 50
> 50
OX23
> 50
2.87±0.16
1.98±0.22
0.7
OX24
OX25
> 50
> 50
4.54±0.44
ND
5.98±0.46
ND
1.3
-
OX26
OX27
> 50
3.34±0.21
0.74±0.11
-
1.94±0.33
10.78±0.44
-
0.6
14.6
-
6.0 ± 3
5.1 ± 0.5
Doxorubicin
2
1

1
,2,4-oxadiazole-sulfonamide-based CAIX inhibitors
Acetazolamide
-
0.034±0.002
0.013±.001
-
-
-
An in vitro antiproliferative screening of SAR derivatives in HCT-116 cells identified compound
OX27 as a promising candidate (IC ~ 6.0 μM). For further validation, compound OX27 was
5
0
tested in NCI-60 tumor cell lines screening and showed activity in colorectal cancer cells with
2% growth inhibition at 10 μM (Fig. S4.1, Supplementary Information). The cytotoxicity of
6
OX27 was evaluated in HEK293 cells in the concentration range of 0-200 µM, for 48 h. It was
found that the treatment of compound OX27 does not affect the viability of HEK293 in a
significant manner in the studied concentration range (0-200 µM), as more than 75% cells were
viable even at 200 µM concentration of the compound (Fig. S5.1, S5.2, Supplementary
Information). As CAIX is a marker of hypoxia, in order to ascertain the anti-proliferative potential
of most active compound (OX27) in hypoxic conditions, we also performed the cell viability
studies in hypoxic conditions. The cell viability studies suggested that in hypoxic conditions the
IC dose of OX27 decrease to 3.4 μM (from ~ 6.0 μM). These results support our rational of
5
0
evaluating the therapeutic potential of selected compound as CAIX inhibitor. Together, these
results suggested that compound OX27 is a promising candidate and warrants further biological
investigation.
2
2
.4.2 CA Inhibition studies
.4.2.1CA inhibition and expression studies
Since we achieved the considerable activity of OX12 when carried out against CAIX
IC =4.23±0.13μM), all the synthesized sulfonamide derivatives (OX17-27) were then tested for
(
5
0
CA inhibition assay using NPA as a substrate with hCAII and cancer related hCAIX isoforms. The
CA inhibition studies showed that the synthesized compounds possess distinct inhibition profiles
with submicromolar to subnanomolar potency (Table 3). The data revealed that OX21 and OX27
inhibited CAIX with higher potency, whereas, OX27 displayed better potency and selectivity
counter to off-target hCAII. Due to better inhibition potential and selectivity of OX27 against
2
2

1
,2,4-oxadiazole-sulfonamide-based CAIX inhibitors
HCT-116 as well as CAIX, it was evaluated in detail for protein-ligand binding studies and cell-
based functional assays. It was reported that HCT-116 cells have higher expression of CAIX and
this expression further increases in response to hypoxia [24-25]. Thus, we further evaluated the
effect of OX27 on the expression of CAIX using Western blot both in normoxic and in hypoxic
conditions. Expression studies showed that treatment of OX27 decreases the expression of CAIX
in HCT-116 cells (Figure 4A). Interestingly, it was found that the OX27 treatment inhibited the
expression of CAIX more pronouncedly in hypoxic conditions (Figure 4A). Thus, our results of
CAIX inhibition are consistency with previous reports, which suggested that inhibitors of CAIX
have more effective in hypoxic conditions [24]
2
.4.2.2 Docking studies with CAIX
The synthesized sulfonamide derivative showing the most promising activity was subjected
to molecular docking with the purpose to see the binding mode and prediction of interacting
residues. Results of docking showed that the sulfonamide group of OX27 forms an H-bond with
His64, whereas N-atom of oxadiazole forms one H-bond with Gln92. His64, Gln92 and Ser65 are
the important residues of hydrophilic region of CAIX active site. and –NH forms two additional
H-bonds with Thr200 (Figure 4 A&B). Phenyl linker interacted through van der Waals
interactions as well as different hydrophobic interaction like it forms π-π interactions with His94,
π-sigma with Val121, π-sulfur with His96, π-alkyl with Val131 and Leu198. The binding mode
analysis also suggested that OX27 binds deep into the catalytic domain of hCAIX (Figure 4). As
reported by the crystal structure these residues forms hydrophobic part of CAIX active site [26].
In case of CAIX, Zn ion is located at the bottom of active site (as shown by small brown sphere,
Figure 4), and interacted with His94, His96 and His119 (Figure 4C). When compared with the
binding mode of acetazolamide (AZM, reported inhibitor of CA), it was found that the sulfonamide
of AZM interacts with His94 and His96, while oxadiazole and thiophene of OX27 interacts with
His94 and His96. The sulphonamide moiety of OX27 forms two H-bonds, one with His64 and
other with Asn62 (hydrophilic region of active site). These results clearly suggested that OX27
possess a significant binding affinity with CAIX and mode of binding further showed that it binds
to the catalytically important residues [27] of CAIX that could be the reason for its better potency.
2
.4.2.3 Fluorescence binding studies
2
3

1
,2,4-oxadiazole-sulfonamide-based CAIX inhibitors
The initial screening using CA inhibition studies and in silico analysis suggested OX27
has a promising affinity for hCAIX. So, we then evaluate the actual binding affinity of OX27 with
hCAIX. The binding affinity of OX27 was studied using fluorescence based assay by titrating the
recombinant hCAIX with OX27. The emission intensity of hCAIX decreases with
2
4

1
,2,4-oxadiazole-sulfonamide-based CAIX inhibitors
Figure 4. Expression and binding studies of hCAIX with OX27: (A) Expression studies of CAIX in
OX27 treated/untreated cells under normoxic and hypoxic conditions. (B) 3D representation of the
2
5

1
,2,4-oxadiazole-sulfonamide-based CAIX inhibitors
best binding mode of docked OX27 (maroon), into the active site residues of hCAIX (stick model), H-bond
donor and acceptor residues of the protein are shown in the respective color. Zn atom is shown in brown
ball model. 2D representation of residues of hCAIX with (C) OX27 and (D) acetazolamide involved in
different interactions like van der Waals interactions, hydrogen bonding, π-π, π-sulfur, metal acceptor or π-
sigma interactions (each type of interaction is represented by respective color, see inset). (E) Fluorescence
emission spectra of hCAIX (20 µM) with the increasing concentration of OX27 (excitation wavelength is
2
80 nm and emission range is 300-400 nm). (F) Modified Stern-Volmer plot of fluorescence quenching
results with OX27 for the analysis of binding affinity (Ka).
the increasing concentrations of OX27, this was further analyzed using the modified Stern-Volmer
7
-1
equation. The estimated value of binding constant (K ) is 4.02× 10 M (Figure 4 C&D)
a
Interestingly, these results are in consistency of activity and docking results and suggested that
OX27 strappingly binds with CAIX and this binding could also be the possible reason for higher
potency.
2
.4.3 Colony formation assay
The colony formation assay is a technique widely used to evaluate cellular transformation in vitro.
This is an in vitro cell survival assay based on the ability of a single cell to grow into a colony.
The assay essentially tests every cell in the population for its ability to undergo ‘unlimited’ division
[28]. The results demonstrated inhibition of colony formation by OX27 in a dose-dependent
manner. OX27 remarkably reduces the colony formation potential of HCT-116 cells as less
number of colonies were observed with increasing concentration of OX27 (Figure 4a).
2
.4.4 Migration of HCT-116 cells
The migration potential of HCT-116 cells was analyzed after the IC dose treatment of compound
5
0
OX27. The treatment was given for 48 h, and the width of the wound was measured pre- and post-
treatment. The results of the wound healing assay are shown in Figure 4b. Results showed that
the untreated control cells migrated to fill the area of scratch more rapidly as compared to the
OX27 treated cells. These results suggested that OX27 inhibits the migration of HCT-116 cells.
2
6

1
,2,4-oxadiazole-sulfonamide-based CAIX inhibitors
Figure 4. Colony formation and cell migration of HCT-116 cells: Treatment of OX27 showed inhibition of (a)
colony formation, and (b) cell migration of HCT-116 cells.
2
.4.5 DAPI (4′,6-diamidino-2-phenylindole) staining
Apoptosis plays an important role in development, immunological competence, and homeostasis.
It is characterized by marked changes in cellular morphology, including chromatin condensation,
membrane blebbing, nuclear breakdown, etc. [29]. To assert whether the inhibition in HCT-116
cell growth by OX27 is associated with apoptosis, the effect of compound OX27 in HCT-116 cells
2
7

1
,2,4-oxadiazole-sulfonamide-based CAIX inhibitors
was studied by DAPI (4′,6-diamidino-2-phenylindole) staining assay that distinguishes live cells
from apoptotic cells based on nuclear morphology. DAPI is a fluorescent dye that firmly binds
with A-T rich region in DNA and identifies the nuclear damage or condensation of chromatin.
DAPI cannot pass through the membrane of live cells effectively. Due to the condensed nucleus,
the apoptotic cells appeared as bright-coloured on staining with DAPI which is a distinctive
characteristic of apoptosis [30]. As observed from Figure 5a, i.e., DAPI staining assay, nuclear
morphology of control (DMSO) cells remain intact while nuclear blebbing, condensation and
fragmentation can be easily observed in the OX27 treated cells. These results suggest that OX27
induces apoptotic death in HCT-116 cells.
2
.4.6 DNA fragmentation assay
The laddering phenomenon is a characteristic feature of apoptosis in which the genomic DNA is
cleaved into fragments by the endogenous endonucleases [31]. To further delineate the mechanism
of apoptotic induction by OX27, DNA ladder assay by gel electrophoresis was performed that
detects apoptotic DNA fragments. DNA fragments were observed as a ladder pattern in HCT-116
cells exposed to 6.0 μM concentration of OX27 for 48 hours Figure 5b. The result suggests that
OX27 can induce apoptosis in HCT-116 cells at the IC concentration of 6.0 μM.
5
0
2
.4.7 Flow cytometric analysis
To get insights into the cell cycle phases, flow cytometric analysis for cell cycle arrest using
propidium iodide (PI) staining was performed. This technique reveals the distribution of cells in
three major phases of the cycle (G vs. S vs. G /M) and makes it possible to detect apoptotic cells
1
2
with fractional DNA content. For flow cytometric analysis, control and treated cells were stained
with PI, and the apoptotic ratio was determined. Compound OX27 treatment caused depletion of
HCT-116 cells in the G0/G1 (P2 65.21%) phase with an increase in the accumulation of cells in
G2/M (P4 18.95%) phase, while an increase was also observed in the apoptotic fraction, S-phase
(P3 12.76%) compared to control HCT-116 cells with 74.29%, 15.14%, and 9.50% population,
respectively (Figure 5c and d).
2
.4.8 poly(ADP-ribose) polymerase (PARP) cleavage
2
8

1
,2,4-oxadiazole-sulfonamide-based CAIX inhibitors
Poly(ADP-ribosyl)ation is an important mechanism for the maintenance of genomic integrity in
response to DNA damage. During apoptotic cell death, PARP plays two opposite roles: its
stimulation leads to poly(ADP-ribose) synthesis, whereas caspase activation causes PARP
cleavage and inactivation. PARP-1 cleavage has been used as a prominent biochemical hallmark
of apoptosis [32]. To explore the mechanism underlying apoptosis in HCT-116 cells by compound
OX27, we observed the cleavage of poly(ADP-ribose) polymerase (PARP) upon treatment with
compound OX27 (Figure 5e), OX27 induced apoptosis in a dose-dependent manner.
2
9