Synthesis and biological evaluation of coumarin-1,3,4-oxadiazole hybrids as selective carbonic anhydrase IX and XII inhibitors

Article abstract of DOI:10.1016/j.bioorg.2019.04.004

With an aim to develop novel heterocyclic hybrids as potent anticancer agents, we synthesized a series of coumarin-1,3,4-oxadiazole hybrids (7a-t) and evaluated for their inhibitory activity against the four physiologically relevant human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms CA I, CA II, CA IX and CA XII. The CA inhibition results clearly indicated that the coumarin-1,3,4-oxadiazole derivatives (7a-t) exhibited selective inhibition of the tumor associated isoforms, CA IX and CA XII over CA I and II isoforms. Among all, compound 7b, exhibited significant inhibition in lower micromolar potency against hCA XII, with a K_i of 0.16 μM and compound 7n, exhibited significant inhibition in lower micromolar potency against hCA IX, with a K_i of 2.34 μM respectively. Therefore, compound 7b and 7n could be the potential leads for development of selective anticancer agents by exhibiting a novel mechanism of action through hCA IX and XII inhibition.

Full text of DOI:10.1016/j.bioorg.2019.04.004

Accepted Manuscript
Synthesis and biological evaluation of coumarin-1,3,4-oxadiazole hybrids as
selective carbonic anhydrase IX and XII inhibitors
Nerella Sridhar Goud, S. Mahammad Ghouse, Mohammed Arifuddin, Mallika
Alvala, Andrea Angeli, Claudiu T. Supuran
PII:
S0045-2068(19)30283-4
DOI:
https://doi.org/10.1016/j.bioorg.2019.04.004
YBIOO 2910
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
19 February 2019
22 March 2019
4 April 2019
Please cite this article as: N. Sridhar Goud, S. Mahammad Ghouse, M. Arifuddin, M. Alvala, A. Angeli, C.T.
Supuran, Synthesis and biological evaluation of coumarin-1,3,4-oxadiazole hybrids as selective carbonic anhydrase
IX and XII inhibitors, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.04.004
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Synthesis and biological evaluation of coumarin-1,3,4-oxadiazole hybrids as
selective carbonic anhydrase IX and XII inhibitors
a
a
a
a*
Nerella Sridhar Goud , S. Mahammad Ghouse , Mohammed Arifuddin , Mallika Alvala ,
Andrea Angeli b and Claudiu T. Supuran b*
a
Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and
Research (NIPER), Balanagar, Hyderabad 500037, India
b Università degli Studi di Firenze, Neurofarba Dept., Sezione di Scienze Farmaceutiche e
Nutraceutiche, Via Ugo Schiff 6, 50019 Sesto Fiorentino, Florence, Italy
___________________
Corresponding authors
Mallika Alvala a* mallikaalvala@yahoo.in
Prof. Claudiu T. Supuran b* claudiu.supuran@unifi.it
Abstract
With an aim to develop novel heterocyclic hybrids as potent anticancer agents, we
synthesized a series of coumarin-1,3,4-oxadiazole hybrids (7a-t) and evaluated for their
inhibitory activity against the four physiologically relevant human carbonic anhydrase (hCA,
EC 4.2.1.1) isoforms CA I, CA II, CA IX and CA XII. The CA inhibition results clearly
indicated that the coumarin-1,3,4-oxadiazole derivatives (7a-t) exhibited selective inhibition
of the tumor associated isoforms, CA IX and CA XII over CA I and II isoforms. Among all,
compound 7b, exhibited significant inhibition in lower micromolar potency against hCA XII,
with a Ki of 0.16 µM and compound 7n, exhibited significant inhibition in lower micromolar
potency against hCA IX, with a Ki of 2.34 µM respectively. Therefore, compound 7b and 7n
could be the potential leads for development of selective anticancer agents by exhibiting a
novel mechanism of action through hCA IX and XII inhibition.
Keywords: Coumarin-1,3,4-Oxadiazole hybrids, Cancer, Carbonic anhydrase, hCAs I, II, IX
and XII.
1. Introduction
Cancer is one of most life-threatening diseases in the world and characterized by uncontrolled
growth and metastasis. The dysfunctional oncogenes, microRNA genes, and tumor
suppressor genes are the essential factors to cause different types of cancer [1]. Recent
studies from GLOBOCAN, 2018., showed that about 18.1 million new cases were diagnosed
and 9.6 million cancer death cases were reported [2]. Most of the cancer cells are recognized
by uncontrolled proliferation due to deregulation of key essential enzymes and proteins

controlling cell division and growth [3]. Many of chemotherapeutic drugs act on various
cancer cells but the drug induced toxicity, poor selectivity and poor tolerance are indicative
for the need to develop new anticancer agents with improved cytotoxicity and lower adverse
effects.
The human carbonic anhydrases (hCAs) are belong to α-family of carbonic anhydrases and
this hCAs, in turn have 16 isoforms. CA I, CA II, CA III, CA VII and CA XIII are cytosolic,
CAs, CA IV, CA IX, CA XII, CA XIV and CA XV are transmembrane bound, CA Va and
Vb mitochondrial and CA VI secreted from saliva and colostrum. CA VIII, CA X and CA XI
are catalytically inactive isoforms, named as CA-related proteins (CARPs) [4-5]. The hCA IX
and XII are overexpressed in cancer cells as they are transmembrane bound, tumor-associated
enzymes, mainly hypoxic tumors, with a low expression in normal cells. The overexpression
further contributes for the tumor growth, proliferation, angiogenesis, and metastasis of variety
of cancer cells [6-7]. An anticancer agent, in order to exhibit potent cytotoxicity without
adverse effects, should selectively inhibit tumor-associated hCAs IX and XII over cytosolic
CAs like hCA I and II. Therefore, development of novel anticancer agents through targeting
tumor-associated hCA isozymes (hCA IX and XII) would be the fruitful and effective
strategy in cancer therapy [8].
Coumarin (2H-chromen-2-one) is a fused heterocyclic ring system consisting of benzene and
2-pyrone ring. It belongs to the family of neo flavonoids of plant secondary metabolites.
Coumarins have ability to make noncovalent interactions viz. hydrogen bonds, hydrophobic,
electrostatic, metal coordination, van der Waals force etc. with various active sites of the
proteins and enzymes [9], and thus display a wide range of pharmacological activities like
antimicrobial,
anti-inflammatory,
anti-tuberculosis,
antihyperlipidemic,
antiviral,
antidepressant, antioxidant and anticancer activities etc [10]. Coumarin heterocycle is
present in a number of well-established clinical drugs with diverse therapeutic activities
[11]. Maresca, A & Supuran CT.et al, have reported that coumarins (1) are a new class of
non-zinc mediated carbonic anhydrases inhibitors and suggested that the coumarin
hydrolysis product, a cis-2-hydroxy-cinnamic acid derivative, but not the coumarin
moiety bound within the enzyme active site. The same research group have also reported
umbelliferone (7-hydoxy coumarin, 2) and its derivatives as selective inhibitors of IX and/or
XII, over CA I and CA II [12]. Aikaterini P & Supuran CT.et al, have reported a novel 6-
and 7-substituted coumarins (
3) as selective tumor-associated carbonic anhydrases IX
and XII inhibitors (Figure 1) [13].

On the other hand, 1,3,4-oxadiazoles are thermally stable and neutral heteroaromatic
compounds containing two nitrogens and oxygen atom, affects the pharmacokinetic and
physicochemical properties of the compounds in which it is present. It makes diverse non-
covalent interactions with various active sites of enzymes and receptors in biological systems
and, thus display versatile pharmacological activities like anti-inflammatory, antibacterial,
muscle relaxing, antihypertension, and anticancer activities [14] etc. Giulia Bianco et al, have
reported a series of N-acyl benzene sulfonamide dihydro-1,3,4-oxadiazole hybrids (4)
selective tumor-associated carbonic anhydrases IX and XII inhibitors and demonstrated
that stereochemistry of dihydro-1,3,4-oxadiazole could be considered as key factors to
determine activity and selectivity towards hCA isozymes IX and XII [15a]. Angapelly S et
al., have reported the sulfocoumarin, Coumarin, 4-Sulfamoylphenyl-bearing Indazole-3-
carboxamide hybrids as selective tumor associated CA IX and XII inhibitors[15b]. Adriano
Mollica et al., have reported the new amide derivatives of Probenecid as selective inhibitors
of CA IX and XII[15c]. Fabrizio Carta et al., have reported novel coumarins and 2-thioxo-
coumarins as inhibitors of the tumor-associated carbonic anhydrases IX and XII [15d].
In view of the selective nature of coumarin and 1,3,4-oxadiazole towards CA IX and XII, it is
decided to synthesize a new series of coumarin-1,3,4-oxadiazole hybrids (Figure 1), which
have not been reported earlier. Thus, novel coumarin-1,3,4-oxadiazole hybrids (7a-t) were
synthesized and evaluated for CA inhibitory activity against the four major CA isoforms, CA
I, II, IX and XII, using acetazolamide (AAZ) as standard drug.
Figure 1: Representative examples of coumarin & 1,3,4-oxadiazole derivatives as selective
tumor-associated hCAs IX & XII inhibitors and rationale for designed molecules (7a-t).
2. Result and Discussion
2.1 Chemistry

The target coumarin-1,3,4-oxadiazole hybrids (7a-t) were synthesized (Scheme 1) by
refluxing various aromatic and/or, aliphatic halides with 7-((5-mercapto-1,3,4-oxadiazol-2-yl)
methoxy)-4-methyl-2H-chromen-2-one (6). Briefly, synthesis of 7-hydroxy-4-methyl
coumarin (3) was done by condensation of resorcinol (1) with ethyl acetoacetate (2) in the
presence of concentrated sulphuric acid [16]. In next step, it is converted into ethyl 2-(4-
methyl-2-oxo-2H-chromen-7-yloxy) acetate (4) by reaction with ethyl bromoacetate and
anhydrous potassium carbonate in freshly distilled acetone. The 2-((4-methyl-2-oxo-2H-
chromen-7-yl) oxy) acetohydrazide (5) was synthesized from the compound (4) by reacting
with hydrazine hydrate in THF as solvent [17]. Finally, the Cyclization of compound (5) was
achieved by refluxing with carbon disulfide in ethanol in the basic condition thus, 7-((5-
mercapto-1,3,4-oxadiazol-2-yl) methoxy)-4-methyl-2H-chromen-2-one (6) was obtained
[18].
Scheme 1: Synthesis of coumarin-1,3,4-oxadiazole hybrids (7a-t).
1
All synthesized compounds (7a-t) were characterized by spectral techniques viz. H-NMR,
1
13C NMR and HRMS. The H-NMR spectrum of 7a showed characteristic protons of 4-
methyl group of coumarin ring around δ 2.40 ppm and 7- methyleneoxy group of coumarin
ring around δ 5.20 ppm respectively. All the remaining protons appeared in the range of δ
5.0-8.50 ppm. 13C NMR spectrum of 7a showed the characteristic carbonyl and 4-methyl
group of coumarin ring at δ 166.0 and δ 18.6 ppm respectively, and all the remaining carbons

appeared in the range of δ 30.0-164.0. The similar pattern was observed for the rest of
compounds (7a-t).
2.2 Carbonic anhydrase inhibition
The target coumarin-1,3,4-oxadiazole hybrids (7a-t) were screened for their potentials
against the four hCA isoforms, namely, hCA I, II (the cytosolic) and hCA IX, XII (the tumor-
associated) by a stopped-flow CO2 hydrase assay, using acetazolamide (AAZ), as a standard
drug. The following Structure-Activity Relationship (SAR) can be inferred from the
inhibition data of coumarin-1,3,4-oxadiazole hybrids (7a-t) as shown in Table 1:
i.
The target compounds 7a-t had not shown any inhibition towards cytosolic isoforms
hCA I and hCA II (Ki > 100 µM).
ii.
The compounds 7a-t have shown significant inhibition against the transmembrane
tumor-associated isoform hCA XII. All the target compounds exhibited inhibitory
potencies of < 10µM with compounds 7a, 7b, 7c and 7d eliciting low micromolar
inhibitory potencies (Kis) of 0.28, 0.16, 0.41 and 0.52 µM respectively.
The potent inhibition order of coumarin-1,3,4-oxadiazole hybrids (7a-d) as follows,
hCA XII inhibition: benzoyl (7b) > benzyl (7a) > Phenacyl (7c) > isobutyl group
(7d).
iii.
iv.
The compounds 7a-t have shown variable and diverse inhibition against the
transmembrane tumor-associated isoform hCA IX, with Kis below 10µM. Among all,
compound 7n, bearing a 4-methoxy benzyl moiety on coumarin-1,3,4-oxadiazole
hybrid, showed promising inhibitory activity with a Ki value of 2.3 µM.
All the others derivatives showed selective hCA IX and XII inhibition over hCA I &II
in micromolar range.
v.
Table 1. Inhibition of hCA isoforms I, II, IX and XII with target compounds (4a-t) and
acetazolamide (AAZ) as a standard drug (Ki-µM)*.
Compound
7a
Structure
hCA I hCAII hCA IX hCA XII
>100
>100
7.04
0.28

>100
>100
>100
>100
>100
>100
7.83
8.04
7.70
0.16
0.41
0.52
7b
7c
7d
>100
>100
>100
>100
>100
>100
7.33
8.16
7.03
0.74
0.60
0.60
7e
7f
7g
>100
>100
8.66
0.82
7h

>100
>100
>100
>100
7.04
6.94
7.96
8.41
7i
7j
>100
>100
>100
>100
9.16
7.40
8.34
8.67
7k
7l
>100
>100
8.94
8.33
7m
>100
>100
>100
>100
2.34
7.11
9.67
9.19
7n
7o

>100
>100
6.21
9.72
7p
>100
>100
6.47
7.80
7q
>100
>100
>100
>100
5.90
7.64
8.78
8.68
7r
7s
>100
0.25
>100
0.012
7.72
9.16
7t
0.025
0.0057
AAZ
* Mean from 3 different assays, by a stopped flow technique (errors were in the range of  5-
10 % of the reported values).
3. Conclusion
In conclusion, we have reported the synthesis of coumarin-1,3,4-oxadiazole hybrids (7a-t),
which are designed to target the transmembrane tumor-associated isoforms, hCA IX and XII.
The target compounds (7a-t) were screened against four hCA isoforms i.e. the cytosolic
isoforms hCA I and II as well as the transmembrane tumor-associated isoforms, hCA IX and

XII. All of the compounds have showed significant lower to sub micromolar inhibition for
both the isoforms, particularly for CA IX, with Kis in the range of 2.34-9.16 µM for CA IX
and 0.16-9.72 for CA XII. The coumarin-1,3,4-oxadiazole hybrids (7a-t) proved to be
ineffective against both the cytosolic isoforms I and II, thereby validating our hypothesis of
selective inhibition of the two tumor-associated enzymes. Therefore, the compound 7b and
7n could be the further potential lead candidates for the design of novel small molecules for
cancer therapy by a different mechanism.
4. Experimental Section
4.1. Chemistry
All reagents and solvents were purchased from commercial suppliers and were used without
further purification. Analytical thin layer chromatography (TLC) was performed on MERCK
precoated silica gel 60-F254 (0.5 mm) aluminum plates. Visualization of the spots on TLC
plates was achieved by UV light. 1H and 13C NMR spectra were recorded on bruker 500 MHz
1
spectrometers using tetramethyl silane (TMS) as the internal standard. Chemical shifts for H
and 13C are reported in parts per million (ppm) downfield from tetra methyl silane. Spin
multiplicities are described as s (singlet), d (doublet), dd (double doublet), t (triplet), q
(quartet), and m (multiplet). Coupling constant (J) values are reported in hertz (Hz). HRMS
were determined with Agilent QTOF mass spectrometer 6540 series instrument. Wherever
required, column chromatography was performed using silica gel 60-120.
4.1.1. Synthesis of 7-hydroxy-4-methyl-2H-chromen-2-one (3)
A solution of resorcinol (9.08 mmol) in ethyl acetoacetate (1.3 mL) was added dropwise to an
externally cooled conc. H2SO4 (10 ml) at 10 °C and the reaction mixture was then stirred at
room temperature for 15 min. The mixture was poured into ice water contained in a beaker,
the precipitate product (3) obtained was collected by suction filtration and washed with cold
water and dried. The product was recrystallized from ethanol. Light yellow solid, yield 90%;
mp 180-182 °C; 1H NMR (500 MHz, DMSO) δ 10.49 (s, 1H), 7.53 (d, J = 8.7 Hz, 1H), 6.77
(d, J = 8.7, 2.1 Hz, 1H), 6.67 (s, 1H), 6.08 (s, 1H), 2.32 (s, 3H). 13C NMR (125 MHz,
DMSO) δ 161.15, 160.28, 154.83, 153.44, 126.51, 112.82, 111.99, 110.25, 102.17, 90.71,
22.35, 18.07; ESI-HRMS (m/z) for C10H8O3, calculated 176.0473, observed 177.0542 [M+1]
+
4.1.2. Synthesis of ethyl 2-(4-methyl-2-oxo-2H-chromen-7-yloxy) acetate (4)

Ethyl bromoacetate (0.7 mL, 6.8 mmol) and anhydrous K2CO3 (11.36 mmol) were added to a
solution of 7-hydroxy-4-methyl-2H-chromen-2-one (3) in freshly distilled acetone (15 mL)
and the reaction mixture was stirred under reflux for 6h. Removal of solvent by evaporation
left a pale yellow solid residue that was extracted with ethyl acetate (3x20 ml) and dried over
Na2SO4. The combined organic layer was concentrated in vacuo to give a white color solid
1
purified by recrystallization with ethanol. White solid, yield 85%; mp 167-169 °C; H NMR
(500 MHz, CDCl3) δ 7.52 (d, J = 8.0 Hz, 1H), 6.92 (dd, J = 8.8, 2.6 Hz, 1H), 6.78 (d, J = 2.5
Hz, 1H), 6.16 (s 1H), 4.69 (s, 2H), 4.29 (q, J = 7.1 Hz, 2H), 2.40 (s, 3H), 1.32 (t, J = 7.8 Hz,
3H). 13C NMR (125 MHz, CDCl3) δ 167.93, 161.00, 160.60, 155.02, 152.35, 125.74, 114.37,
112.52, 112.46, 101.66, 65.31, 61.68, 18.63, 14.11; ESI-HRMS (m/z) for C14H14O5,
calculated 262.0841, observed 263.0920 [M+1] +
4.1.3 Synthesis of 2-(4-methyl-2-oxo-2H-chromen-7-yloxy) acetohydrazide (5)
Ethyl 2-(4-methyl-2-oxo-2H-chromen-7-yloxy) acetate (3.8 mmol) was dissolved into THF
(10 mL) and to this hydrazine hydrate (0.28 mL, 5.7 mmol) was added dropwise. The
reaction mixture was then stirred under reflux at 65-70 °C for 4h. On cooling at room
temperature 20-30 mL of cold water was added and stirred for 10 min. The white solid
separated out was filtered at pump and dried. The product obtained was recrystallized from
ethanol. White solid, yield 80%; mp 192-194 °C; ESI-HRMS (m/z) for C12H12N2O4,
calculated 248.0797, observed 249.0874 [M+1] +
4.1.4. Synthesis of 7-((5-mercapto-1,3,4-oxadiazol-2-yl) methoxy)-4- methyl-2H-
chromen-2-one (6)
To a solution of NaOH (4.03 mmol) in EtOH (25 ml) were added 3 (2.48 g, 0.01 mol) and
CS2 (0.36 mL, 4.83 mmol), and the mixture was refluxed overnight while stirring. The
solvent was removed in vacuo and the residue was dissolved in water and acidified with dil.
HCI. The precipitate was washed with water and crystallized from EtOH. Pale yellow solid,
1
yield 76%; mp 202-204 °C; H NMR (500 MHz, DMSO) δ 14.43 (s, 1H), 7.62 – 7.58 (m,
1H), 7.02 – 6.96 (m, 2H), 6.16 (s, 1H), 5.21 (s, 2H), 2.44 (s, 3H). 13C NMR (125 MHz,
DMSO) δ 178.46, 160.20, 159.45, 157.54, 154.36, 151.97, 125.56, 114.23, 112.07, 111.87,
101.56, 59.37, 18.13; ESI-HRMS (m/z) for C13H10N2O4S, calculated 290.0361, observed
291.0405 [M+1] +
4.1.5. General procedure for synthesis of coumarin-1,3,4-oxadiazole hybrids (7a-t)

To a solution of 7-((5-mercapto-1,3,4-oxadiazol-2-yl) methoxy)-4- methyl-2H-chromen-2-
one (6) in freshly distilled acetone (20 mL), added K2CO3 as base and different alkyl halides
or aryl halides (3.4 mmol) dropwise and stirred under reflux for 2-3h. After confirmation was
done by TLC, the solvent was evaporated completely and the obtained residue was extracted
with ethyl acetate (3x20 ml) and dried over Na2SO4. The combined organic layer was
concentrated in vacuo and the residues were purified by column chromatography
on silica gel.
7-((5-(benzylthio)-1,3,4-oxadiazol-2-yl) methoxy)-4-methyl-2H-chromen-2-one (7a)
Yellow solid, yield 81%; mp 282-284 °C;1H NMR (500 MHz, CDCl3) δ 7.54 (d, J = 7.0 Hz,
1H), 7.45 – 7.37 (m, 2H), 7.35 – 7.25 (m, 3H), 7.06 – 6.81 (m, 2H), 6.18 (s, 1H), 5.27 (s, 2H),
13
4.49 (s, 2H), 2.41 (s, 3H). C NMR (125 MHz, CDCl3) δ 166.02, 162.51, 160.88, 160.12,
155.04, 152.26, 135.18, 129.14, 128.85, 128.22, 125.97, 114.85, 112.86, 112.16, 102.36,
59.90, 36.78, 18.68; HRMS (ESI): m/z calc. for C20H16N2O4S 380.0831; found 381.0905 [M
+ H] +.
S-(5-(((4-methyl-2-oxo-2H-chromen-7-yl)oxy) methyl)-1,3,4-oxadiazol-2-yl) benzothioate
(7b)
1
Light yellow solid, yield 72%; mp 303-305 °C; H NMR (500 MHz, DMSO) δ 7.95 (d, J =
8.3, 1.3 Hz, 2H), 7.73 (d, J = 8.6 Hz, 1H), 7.65 (t, J = 7.4 Hz, 1H), 7.51 (t, J = 7.7 Hz, 2H),
13
7.19 – 7.04 (m, 2H), 6.25 (s, 1H), 5.43 (s, 2H), 2.40 (s, 3H). C NMR (125 MHz, DMSO) δ
167.78, 160.42, 160.39, 154.95, 153.73, 133.33, 131.21, 129.72, 129.03, 127.20, 114.66,
112.88, 112.32, 102.43, 60.41, 18.60; HRMS (ESI): m/z calc. for C20H14N2O5S 394.0623;
found 395.0685 [M + H] +.
4-methyl-7-((5-((2-oxo-2-phenylethyl) thio)-1,3,4-oxadiazol-2-yl) methoxy)-2H-chromen-
2-one (7c)
White solid, yield 80%; mp 321-323 °C; 1H NMR (500 MHz, CDCl3) δ 8.03 (d, J = 7.5 Hz,
2H), 7.65 (t, J = 7.4 Hz, 1H), 7.56 – 7.50 (m, 3H), 7.08 – 6.79 (m, 2H), 6.18 (s, 1H), 5.29 (s,
2H), 4.95 (s, 2H), 2.41 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 191.74, 165.89, 162.68,
160.87, 160.09, 155.05, 152.24, 134.79, 134.35, 129.01, 128.52, 125.97, 114.87, 112.88,
112.24, 102.32, 59.87, 41.65, 18.68; HRMS (ESI): m/z calc. for C21H16N2O5S 408.0780;
found 409.0850 [M + H] +.

7-((5-(isobutylthio)-1,3,4-oxadiazol-2-yl) methoxy)-4-methyl-2H-chromen-2-one (7d)
Pale yellow solid, yield 71%; mp 231-233 °C; 1H NMR (500 MHz, CDCl3) δ 7.54 (d, J = 8.8
Hz, 1H), 7.11 – 6.85 (m, 2H), 6.18 (s, 1H), 5.28 (s, 2H), 3.18 (s, 2H), 2.41 (d, J = 1.1 Hz,
13
3H), 2.18 – 1.96 (m, 1H), 1.06 (d, J = 6.7 Hz, 6H). C NMR (126 MHz, CDCl3) δ 166.98,
162.28, 160.87, 160.16, 155.04, 152.25, 125.95, 114.82, 112.84, 112.17, 102.37, 59.92,
41.01, 28.39, 21.63, 18.68; HRMS (ESI): m/z calc. for C17H18N2O4S 346.0987; found
347.1064 [M + H] +.
7-((5-((4-chlorobutyl) thio)-1,3,4-oxadiazol-2-yl) methoxy)-4-methyl-2H-chromen-2-one
(7e)
Pale yellow solid, yield 74%; mp 246-248 °C; 1H NMR (500 MHz, CDCl3) δ 7.55 (d, J = 7.2
Hz, 1H), 7.06 – 6.89 (m, 2H), 6.18 (d, J = 1.0 Hz, 1H), 5.29 (s, 2H), 3.64 – 3.52 (t, J = 7.1
13
Hz, 2H), 3.31 (t, J = 7.0 Hz, 2H), 2.41 (d, J = 1.1 Hz, 3H), 2.08 – 1.88 (m, 4H). C NMR
(126 MHz, CDCl3) δ 166.32, 162.47, 160.89, 160.12, 155.04, 152.27, 125.98, 114.85,
112.86, 112.20, 102.32, 59.89, 44.07, 31.78, 31.20, 26.54, 18.69; HRMS (ESI): m/z calc. for
C17H17ClN2O4S 380.0598; found 381.0667 [M + H] +.
ethyl4-(((5-(((4-methyl-2-oxo-2H-chromen-7-yl)oxy)methyl)-1,3,4-oxadiazol-2-yl)thio)
methyl) benzoate (7f)
1
Bright yellow solid, yield 79%; mp 319-321 °C; H NMR (500 MHz, CDCl3) δ 8.07 – 7.96
(d, 2H), 7.56 (d, J = 8.7 Hz, 1H), 7.52 (d, J = 8.3 Hz, 2H), 7.00 – 6.92 (m, 2H), 6.20 (d, J =
1.2 Hz, 1H), 5.29 (s, 2H), 4.53 (s, 2H), 4.39 (q, J = 7.1 Hz, 2H), 2.43 (d, J = 1.2 Hz, 3H), 1.40
13
(t, J = 7.1 Hz, 3H). C NMR (125 MHz, CDCl3) δ 166.07, 165.56, 162.69, 160.88, 160.08,
155.03, 152.27, 140.34, 130.33, 130.03, 129.11, 125.98, 114.87, 112.87, 112.17, 102.31,
61.12, 59.87, 36.24, 18.68, 14.32; HRMS (ESI): m/z calc. for C23H20N2O6S 452.1042; found
453.1119 [M + H] +.
ethyl2-((5-(((4-methyl-2-oxo-2H-chromen-7-yl)oxy)methyl)-1,3,4-oxadiazol-2-yl)thio)
acetate (7g)
White solid, yield 88%; mp 277-279 °C; 1H NMR (500 MHz, CDCl3) δ 7.56 (d, J = 8.7 Hz,
1H), 7.04 – 6.87 (m, 2H), 6.20 (d, J = 1.1 Hz, 1H), 5.31 (s, 2H), 4.27 (q, J = 7.1 Hz, 2H), 4.10
(s, 2H), 2.43 (d, J = 1.1 Hz, 3H), 1.31 (t, J = 7.1 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ
167.19, 165.11, 162.80, 160.86, 160.07, 155.04, 152.25, 125.99, 114.88, 112.87, 112.17,

102.34, 62.52, 59.85, 34.33, 18.68, 14.06; HRMS (ESI): m/z calc. for C17H16N2O6S
376.0729; found 376.0813 [M + H] +.
4-methyl-7-((5-((4-nitrobenzyl) thio)-1,3,4-oxadiazol-2-yl) methoxy)-2H-chromen-2-one
(7h)
Yellow solid, yield 91%; mp 296-298 °C; 1H NMR (500 MHz, CDCl3) δ 7.98 (d, J = 9.5 Hz,
2H), 7.54 (d, J = 8.7 Hz, 1H), 7.50 (d, J = 8.6 Hz, 2H), 7.02 – 6.91 (m, 2H), 6.17 (d, J = 13.3,
4.2 Hz, 1H), 5.29 (s, 2H), 4.98 (s, 2H), 2.41 (d, J = 1.1 Hz, 3H). 13C NMR (125 MHz,
CDCl3) δ 190.65, 165.72, 162.76, 160.92, 160.07, 155.04, 152.27, 141.00, 133.13, 129.93,
129.40, 125.99, 114.89, 112.90, 112.29, 102.28, 59.86, 41.34, 18.71; HRMS (ESI): m/z calc.
for C20H15N3O6S 425.0682; found 426.0742 [M + H] +.
7-((5-((2-(4-chlorophenyl)-2-oxoethyl)thio)-1,3,4-oxadiazol-2-yl)methoxy)-4-methyl-2H-
chromen-2-one (7i)
1
Yellow solid, yield 78%; mp 341-343 °C; H NMR (500 MHz, CDCl3) δ 8.51 – 8.29 (m,
2H), 7.84 (d, J = 8.7 Hz, 2H), 7.75 (d, J = 8.7 Hz, 1H), 7.15 (t, J = 5.6, 2.5 Hz, 2H), 6.40 (d, J
= 0.9 Hz, 1H), 5.48 (s, 2H), 4.75 (s, 2H), 2.61 (t, J = 9.3 Hz, 3H). 13C NMR (125 MHz,
CDCl3) δ 190.65, 165.72, 162.76, 160.92, 160.07, 155.04, 152.27, 141.00, 133.13, 129.93,
129.40, 125.99, 114.89, 112.90, 112.29, 102.28, 59.86, 41.34, 18.71; HRMS (ESI): m/z calc.
for C21H15ClN2O5S 442.0390; found 443.0435 [M + H] +.
7-((5-(allylthio)-1,3,4-oxadiazol-2-yl)methoxy)-4-methyl-2H-chromen-2-one (7j)
Pale yellow solid, yield 85%; mp 252-254 °C; 1H NMR (500 MHz, CDCl3) δ 7.56 (d, J = 8.8
Hz, 1H), 7.03 – 6.89 (m, 2H), 6.20 (d, J = 1.2 Hz, 1H), 5.99 (m, J = 17.0, 10.0, 7.0 Hz, 1H),
5.39 (dd, J = 16.9, 2.4, 1.2 Hz, 1H), 5.30 (s, 2H), 5.23 (dd, J = 10.0, 0.9 Hz, 1H), 3.99 – 3.82
(m, 2H), 2.42 (d, J = 1.2 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 165.86, 162.57, 160.88,
160.12, 155.03, 152.28, 131.35, 125.98, 120.06, 114.84, 112.84, 112.16, 102.34, 59.91,
35.15, 18.69; HRMS (ESI): m/z calc. for C16H14N2O4S 330.0674; found 331.0757 [M + H] +.
4-methyl-7-((5-(prop-2-yn-1-ylthio)-1,3,4-oxadiazol-2-yl)methoxy)-2H-chromen-2-one
(7k)
Yellow solid, yield 87%; mp 260-262 °C; 1H NMR (500 MHz, CDCl3) δ 7.56 (d, J = 8.8 Hz,
1H), 7.07 – 6.86 (m, 2H), 6.20 (d, J = 1.0 Hz, 1H), 5.35 (dd, J = 25.1, 17.3 Hz, 2H), 5.19 (t, J
= 9.6 Hz, 1H), 4.05 (d, J = 2.6 Hz, 1H), 2.43 (d, J = 0.8 Hz, 3H), 2.35 (t, J = 2.6 Hz, 1H). 13C

NMR (125 MHz, CDCl3) δ 164.78, 162.94, 160.87, 160.07, 155.03, 152.26, 125.99, 114.89,
112.89, 112.19, 102.35, 80.89, 79.77, 59.89, 21.07, 18.69; HRMS (ESI): m/z calc. for
C16H12N2O4S 328.0518; found 329.0594 [M + H] +.
7-((5-((bromomethyl)thio)-1,3,4-oxadiazol-2-yl)methoxy)-4-methyl-2H-chromen-2-one
(7l)
White solid, yield 72%; mp 279-281 °C; 1H NMR (500 MHz, CDCl3) δ 7.54 (d, J = 21.0 Hz,
1H), 6.99 (dd, J = 24.2, 17.9, 13.0 Hz, 2H), 6.20 (d, J = 1.0 Hz, 1H), 5.34 (s, J = 12.3 Hz,
2H), 5.09 (s, J = 11.9 Hz, 1H), 2.43 (d, J = 0.9 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ
164.85, 163.38, 160.88, 160.01, 155.03, 152.29, 126.03, 114.93, 112.90, 112.23, 102.29,
59.86, 29.70, 18.70; HRMS (ESI): m/z calc. for C14H11BrN2O4S 381.9623; found 380.1758
[M - H] +.
7-((5-((2-(4-methoxyphenyl)-2-oxoethyl)thio)-1,3,4-oxadiazol-2-yl)methoxy)-4-methyl-
2H-chromen-2-one (7m)
yellow solid, yield 83%; mp 316-318 °C; 1H NMR (500 MHz, CDCl3) δ 8.18 – 7.73 (d, 2H),
7.52 (d, J = 19.7 Hz, 1H), 7.13 – 6.83 (m, 4H), 6.18 (d, J = 1.2 Hz, 1H), 5.29 (s, 2H), 4.92 (s,
13
2H), 3.90 (s, 3H), 2.41 (d, J = 1.2 Hz, 3H). C NMR (125 MHz, CDCl3) δ 190.19, 166.15,
164.49, 162.59, 160.90, 160.10, 155.05, 152.25, 130.94, 127.78, 125.97, 114.87, 114.19,
112.88, 112.26, 102.32, 59.86, 55.63, 41.57, 18.69; HRMS (ESI): m/z calc. for C22H18N2O6S
438.0886; found 439.0962 [M + H] +.
7-((5-((4-methoxybenzyl)thio)-1,3,4-oxadiazol-2-yl)methoxy)-4-methyl-2H-chromen-2-
one (7n)
Pale yellow solid, yield 86%; mp 278-208 °C; 1H NMR (500 MHz, CDCl3) δ 7.56 – 7.51 (d,
1H), 7.37 – 7.30 (d, 2H), 6.95 (m, J = 8.3, 4.2 Hz, 2H), 6.89 – 6.81 (d, 2H), 6.18 (d, J = 1.2
Hz, 1H), 5.27 (s, 2H), 4.45 (s, 2H), 3.79 (s, 3H), 2.41 (d, J = 1.2 Hz, 3H). 13C NMR (125
MHz, CDCl3) δ 166.15, 162.44, 160.88, 160.13, 159.51, 155.05, 152.25, 130.43, 127.04,
125.97, 114.85, 114.22, 112.86, 112.16, 102.36, 59.91, 55.31, 36.47, 18.69; HRMS (ESI):
m/z calc. for C21H18N2O5S 410.0936; found 411.1012 [M + H] +.
7-((5-((4-chlorobenzyl)thio)-1,3,4-oxadiazol-2-yl)methoxy)-4-methyl-2H-chromen-2-one
(7o)

1
Pale yellow solid, yield 88%; mp 292-294 °C; H NMR (500 MHz, CDCl3) δ 7.52 (d, J =
19.6 Hz, 1H), 7.40 – 7.34 (d, 2H), 7.32 – 7.27 (d, 2H), 6.95 (m, J = 6.8, 2.5 Hz, 2H), 6.19 (d,
13
J = 1.1 Hz, 1H), 5.27 (s, 2H), 4.44 (s, 2H), 2.41 (d, J = 1.1 Hz, 3H). C NMR (126 MHz,
CDCl3) δ 165.59, 162.67, 160.86, 160.08, 155.05, 152.23, 134.14, 133.90, 130.51, 128.99,
125.98, 114.88, 112.90, 112.16, 102.32, 59.88, 35.97, 18.69; HRMS (ESI): m/z calc. for
C20H15ClN2O4S 414.0441; found 415.0519 [M + H] +.
4-methyl-7-((5-((2-oxo-2-(p-tolyl)ethyl)thio)-1,3,4-oxadiazol-2-yl)methoxy)-2H-chromen-
2-one (7p)
White solid, yield 72%; mp 332-334 °C; 1H NMR (500 MHz, CDCl3) δ 7.93 (d, J = 8.3 Hz,
2H), 7.54 (d, J = 8.7 Hz, 1H), 7.30 (d, J = 11.9 Hz, 2H), 7.08 – 6.86 (m, 2H), 6.18 (d, J = 1.2
Hz, 1H), 5.29 (s, 2H), 4.94 (s, 2H), 2.43 (s,3H), 2.41 (d, J = 1.2 Hz, 3H). 13C NMR (125
MHz, CDCl3) δ 191.34, 166.04, 162.62, 160.89, 160.10, 155.05, 152.24, 145.51, 132.30,
129.69, 128.64, 125.97, 114.87, 112.88, 112.24, 102.33, 59.86, 41.71, 21.81, 18.69; HRMS
(ESI): m/z calc. for C22H18N2O5S 422.0936; found 423.1001 [M + H] +.
7-((5-((2-(4-bromophenyl)-2-oxoethyl)thio)-1,3,4-oxadiazol-2-yl)methoxy)-4-methyl-2H-
chromen-2-one (7q)
Yellow solid, yield 75%; mp 357-359 °C;1H NMR (500 MHz, CDCl3) δ 8.00 – 7.82 (d, 2H),
7.76 – 7.61 (d, 2H), 7.60 – 7.50 (d, 1H), 6.98 (m, J = 21.5, 9.8, 6.4 Hz, 2H), 6.19 (d, J = 1.2
13
Hz, 1H), 5.29 (s, 2H), 4.89 (s, 2H), 2.41 (d, J = 1.2 Hz, 3H). C NMR (125 MHz, CDCl3) δ
190.86, 165.69, 165.04, 162.76, 160.07, 155.06, 152.22, 133.54, 132.39, 129.96, 129.79,
125.98, 114.90, 112.92, 112.26, 102.29, 59.84, 41.27, 18.69; HRMS (ESI): m/z calc. for
C21H15BrN2O5S 485.9885; found 486.9966 [M + H] +.
4-methyl-7-((5-((3,4,5-trimethoxybenzyl)thio)-1,3,4-oxadiazol-2-yl)methoxy)-2H-
chromen-2-one (7r)
1
Pale yellow solid, yield 80%; mp 310-312 °C; H NMR (500 MHz, CDCl3) δ 7.52 (d, J =
19.4 Hz, 1H), 7.08 – 6.84 (m, 2H), 6.65 (s, 2H), 6.18 (s, 1H), 5.28 (s, 2H), 4.44 (s, 2H), 3.96
13
– 3.74 (m, 9H), 2.41 (s, 3H). C NMR (125 MHz, CDCl3) δ 166.06, 162.57, 160.89, 160.10,
155.03, 153.39, 152.30, 137.90, 130.68, 125.99, 114.87, 112.86, 112.23, 106.22, 102.29,
60.85, 59.93, 56.19, 37.30, 18.68; HRMS (ESI): m/z calc. for C23H22N2O7S 470.1148; found
471.1221 [M + H] +.

7-((5-((2,4-dichlorobenzyl)thio)-1,3,4-oxadiazol-2-yl)methoxy)-4-methyl-2H-chromen-2-
one (7s)
1
White solid, yield 77%; mp 317-319 °C; H NMR (500 MHz, CDCl3) δ 7.55 (t, J = 8.2 Hz,
2H), 7.42 (d, J = 7.5 Hz, 1H), 7.23 – 7.15 (dd, 1H), 6.95 (m, J = 6.4, 2.5 Hz, 2H), 6.18 (d, J =
1.1 Hz, 1H), 5.27 (s, 2H), 4.54 (s, 2H), 2.41 (d, J = 1.1 Hz, 3H). 13C NMR (125 MHz, CDCl3)
δ 165.76, 162.76, 160.90, 160.07, 155.04, 152.26, 135.07, 134.96, 132.34, 132.06, 129.66,
127.40, 125.98, 114.87, 112.89, 112.19, 102.27, 59.85, 33.91, 18.70; HRMS (ESI): m/z calc.
for C20H14Cl2N2O4S 448.0051; found 449.0174 [M + H] +.
4-(((5-(((4-methyl-2-oxo-2H-chromen-7-yl)oxy)methyl)-1,3,4-oxadiazol-2-yl)thio)
methyl) benzonitrile (7t)
Yellow solid, yield 83%; mp 286-288 °C; 1H NMR (500 MHz, CDCl3) δ 7.77 – 7.46 (m, 5H),
7.03 – 6.83 (m, 2H), 6.19 (d, J = 1.1 Hz, 1H), 5.27 (s, 2H), 4.50 (s, 2H), 2.40 (t, J = 4.7 Hz,
3H). 13C NMR (125 MHz, CDCl3) δ 165.18, 162.90, 160.87, 160.04, 155.02, 152.31, 141.04,
132.53, 129.93, 126.03, 118.38, 114.89, 112.89, 112.24, 112.04, 102.23, 59.88, 35.94, 18.70;
HRMS (ESI): m/z calc. for C21H14ClN3O4S 405.0783; found 406.0858 [M + H] +.
4.2 CA inhibition
The inhibition assay of selected CA isozymes was performed using SX.18V-R Applied
Photophysics (Oxford, UK) stopped flow instrument [19]. 10 mM Hepes (pH 7.4) as a buffer,
with Phenol Red (at a concentration of 0.2 mM) as an indicator, 0.1M Na2SO4 or NaClO4 (To
maintain constant the ionic strength; these anions are not inhibitory in the used
concentration), following the CA-catalyzed CO2 hydration reaction for a period of 5-10s.
Saturated CO2 solutions in water at 25 ᵒC were used as substrate. Stock solutions of inhibitors
were prepared at a concentration of 10mM (in DMSO-water 1:1, v/v) and dilutions up to
0.01nM done with the assay buffer mentioned above. At least 7 different inhibitor
concentrations have been used for measuring the inhibition constant. Inhibitor and enzyme
solutions were pre-incubated together for 10 min at room temperature prior to assay, in order
to allow for the formation of the E-I complex. Triplicate experiments were done for each
inhibitor concentration, and the values reported in this paper are the mean of such results. The
inhibition constants were obtained by non-linear least squares methods using the Cheng-
Prusoff equation, as reported earlier, and represent the mean from at least three different
determinations. All CA isozymes used here were recombinant proteins obtained as reported
earlier by our group [20-22].

Conflicts of interest
The authors declare no conflicts of interest.
Acknowledgements
Thankful to National Institute of Pharmaceutical Education and Research (NIPER),
Balanagar, Hyderabad & Department of Pharmaceuticals, Ministry of Chemicals and
Fertilizers, Govt. of India.
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Synthesis and biological evaluation of coumarin-1,3,4-oxadiazole hybrids as
selective carbonic anhydrase IX and XII inhibitors
a
a
a
a*
Nerella Sridhar Goud , S. Mahammad Ghouse , Mohammed Arifuddin , Mallika Alvala ,
Andrea Angeli b and Claudiu T. Supuran b*
a) Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and
Research (NIPER), Balanagar, Hyderabad 500037, India;
b) Università degli Studi di Firenze, Neurofarba Dept., Sezione di Scienze Farmaceutiche e
Nutraceutiche, Via Ugo Schiff 6, 50019 Sesto Fiorentino, Florence, Italy

Highlights
1. Reporting synthesis of novel coumarin-oxadiazole hybrids (7a-t) as specific hCA IX/XII
inhibition compare to hCAI and II.
2. Compound 7b is specifically inhibiting hCA XII with Ki of 0.16 µM.