Synthesis and biological evaluation of some coumarin hybrids as selective carbonic anhydrase IX and XII inhibitors

Article abstract of DOI:10.1016/j.bioorg.2020.104272

Two series, coumarin-linked to thiazolidinone via a pyrazole linker (6a-m, Series 1) and coumarin-linked 1,2,3-triazoles (5a-j, Series 2) were synthesized and the synthesized compounds were subjected for evaluation against the four physiologically and pha

Full text of DOI:10.1016/j.bioorg.2020.104272

BioorganicChemistry104(2020)104272
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis and biological evaluation of some coumarin hybrids as selective
carbonic anhydrase IX and XII inhibitors
Pavitra S. Thackera,1, Nerella Sridhar Gouda,1, Omkar S. Argulwara, Jyothsna Somana,
⁎
⁎
Andrea Angelic, Mallika Alvalaa, Mohammed Arifuddina,b, , Claudiu T. Supuranc,
a Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research (NIPER), Balanagar, Hyderabad 500037, India
b Department of Chemistry, Anwarul Uloom College, 11-3-918, New Malleypally, Hyderabad 500001, T. S., India
c Università degli Studi di Firenze, Neurofarba Dept., Sezione di Scienze Farmaceutiche e Nutraceutiche, Via Ugo Schiff 6, 50019 Sesto Fiorentino, Florence, Italy
A R T I C L E I N F O
A B S T R A C T
Keywords:
Two series, coumarin-linked to thiazolidinone via a pyrazole linker (6a-m, Series 1) and coumarin-linked 1,2,3-
triazoles (5a-j, Series 2) were synthesized and the synthesized compounds were subjected for evaluation against
the four physiologically and pharmacologically relevant hCA isoforms, hCA I, II, IX and XII. The results indicated
selective inhibition of tumor-associated isoforms hCA IX and XII over the off-target isoforms, hCA I and II. The
compounds of series 1 exhibited better hCA IX inhibition compared to hCA XII, with compounds 6i, 6h, 6a and
6k, exhibiting notable Ki values of less than 100 nM. Among all the compounds, compound 6i showed the best
inhibition with a Ki value of 61.5 nM. Among the compounds of series 2, compounds 5a, 5b, 5c, 5d, 5f and 5j
exhibited notable hCA IX inhibition. Compound 5d showed the best inhibition with a Ki value of 32.7 nM. In the
case of hCA XII, compound 5i showed the best inhibition with a Ki value of 84.2 nM. Hence, compound 6i from
Series 1 and 5d from Series 2 could be taken as lead compounds for the further development of selective and
potent hCA IX inhibitors, whereas the compound 5i from Series 2 can be explored further for the design of
selective and potent hCA XII inhibitors.
Carbonic anhydrases
hCA IX & XII isoform
Coumarin
Pyrazole
Thiazolidinone
1, 2, 3-triazole
1. Introduction
present within their active sites. The metal ions which are typically
present are Zn(II), Fe(II), Co(II) and cadmium [1–4].
The living organisms on earth are broadly classified into two major
classes namely prokaryotes and eukaryotes. These two classes, in spite
of being distinct in their own ways, are still related to each other by the
presence of certain common molecules/ions like CO2, bicarbonate and
protons. Carbonic anhydrases (CAs, EC 4.2.1.1) are a superfamily of
enzymes which utilize these simple molecules/ions as substrates and
thereby aid in catalyzing the reversible conversion of CO2 to bicarbo-
nate and proton as depicted in the equation below:-
The carbonic anhydrases are divided into eight genetically distinct
classes namely α-, β-, γ-, δ-, ζ-, η-, θ and ι. All animals, including hu-
mans, mainly contain the α-CAs (denoted as human carbonic anhy-
drases, hCAs, in humans). The α-CA class is in turn comprised of 16
isoforms. These isoforms possess distinct catalytic activities as well as
physiological functions [5,6].
Amongst these 16 isoforms, hCA IX and hCA XII are transmembrane
isoforms which have been characterized as biomarkers for several types
of tumors. The hCA IX is expressed in healthy tissues. However, it is
overexpressed in tumor microenvironment on account of concurrent
conditions like hypoxia and acidity. This in turn promotes the survival
and growth of tumor cells, cancer cell migration, invasion and also
allows the cells to maintain stem-cell like properties. The hCA XII,
originally identified from human renal clear cell carcinoma, also assists
in maintenance of acid-base homoeostasis in normal as well as tumor
cells. The overexpression of both these isoforms in cancer results in the
The expression of these enzymes is widespread across various tis-
sues wherein they play a key role in many physiological processes such
as acid base balance, transport of ions and gases, respiration, ur-
eagenesis, gluconeogenesis and lipogenesis to name a few. These en-
zymes elicit their diverse activities with the help of various metals
⁎ Corresponding authors at: Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research (NIPER), Balanagar, Hyderabad
500037, India (M. Arifuddin).
E-mail addresses: arif@niperhyd.ac.in (M. Arifuddin), claudiu.supuran@unifi.it (C.T. Supuran).
1 Equal contribution by the authors.
https://doi.org/10.1016/j.bioorg.2020.104272
Received 1 May 2020; Received in revised form 30 August 2020; Accepted 7 September 2020
Availableonline08September2020
0045-2068/©2020ElsevierInc.Allrightsreserved.

P.S. Thacker, et al.
BioorganicChemistry104(2020)104272
poor prognosis as well as chemoresistance to weakly basic anti-cancer
drugs [7–9].
reaction afforded the intermediate 5 as per reported literature method
[23]. Finally, the Intermediate 5 was reacted with various anilines and
thioglycollic acid in the presence of silica gel (100–200 mesh) and DCM
as a solvent to afford the final compounds (6a-m) by following the
reported literature method [24].
This warrants the need for discovering selective hCA IX and XII
inhibitors. In recent years, several classes of compounds have been
identified as showing selective and potent inhibition against both of the
aforementioned isoforms. Amongst them, coumarins are a class of
naturally occurring compounds which have been shown to inhibit hCA
IX and XII via a non-classical mechanism, by occlusion of the active-site
entrance [10,11]. Taking a cue from this, several research groups, in-
cluding ours, have reported various derivatives of coumarin as selective
CA IX and XII inhibitors [12–15].
2.1.2. Synthesis of series 1 compounds
The synthesis of coumarin-linked 1,2,3-riazoles (5a-j) was per-
formed as per the general synthetic scheme as illustrated in Scheme 2.
Initially, the intermediate 3 was synthesized by the reaction of re-
sorcinol (1) with ethyl-4-chloroacetoacetate (2) using sulphuric acid as
solvent under cold conditions through Pechmann condensation me-
chanism [25]. Thereafter, the intermediate 4 was synthesized by O-al-
kylation of intermediate 3 with propargyl bromide in the presence of
potassium carbonate as a base and acetone as a solvent. Finally, the
terminal alkyne of intermediate 4 was cyclized with various benzyl
azides furnishing the target compounds 5a-j through copper (I)-cata-
lyzed 1,3-dipolar cycloaddition reaction in moderate to good yields.
Pyrazole is one of the key privileged scaffolds in medicinal chem-
istry which has been shown to possess diverse pharmacological profiles
such as anticancer, anti-inflammatory, antipyretic, analgesic, anti-
psychotic, anti-anxiety and anti-obesity [16]. In addition to that, pyr-
azole has also been shown to possess potent carbonic anhydrase in-
hibition especially against tumor-associated isoforms hCA IX and XII
[17,18]. On the other hand, the 4-thiazolidinone pharmacophore is a
key motif possessing a wide array of pharmacological activities such as
anticancer, anti-diabetic, antimicrobial, cardiovascular, anti-hyperlipi-
demic and anticonvulsant. It’s effectiveness for CA inhibition has also
been explored by various research groups [19,20]. Similarly, the 1,2,3-
triazole scaffold represents another key scaffold which possesses a
broad range of pharmacological activities such as anticancer, anti-
microbial and anti-inflammatory. It also serves as an effective amide
surrogate in bioactive compounds due to its strong dipole moments.
Apart from that, it has been shown to elicit potent CA inhibitory activity
particularly against hCA IX and XII [21,22].
2.2. Carbonic anhydrase inhibition
The compounds synthesized in series 1 (6a-m) and series 2 (5a-j)
were evaluated for their activity against the four physiologically and
pharmacologically relevant isoforms, namely, the cytosolic isoforms,
hCA I and hCA II and the trans-membrane tumor associated isoforms,
hCA IX and hCA XII, by a stopped flow CO2 hydrase assay method.
Acetazolamide (AAZ) was taken as the standard drug. The following
inferences regarding the structure–activity relationship of the synthe-
sized compounds can be drawn for both the series:-
Hence, taking into consideration the potency of coumarin, pyrazole,
thiazolidinone and 1,2,3-triazole nuclei to inhibit the tumor-associated
isoforms CA IX and XII, it was decided to synthesize two different series
of compounds through amalgamation of the above said nuclei by ap-
plying molecular hybridization strategy. The first series encompassed
the synthesis of coumarin linked to 4-thiazolidinone hybrids via a
pyrazole linker (6a-m), whereas, the second series consisted of cou-
marin-linked 1,2,3-triazole hybrids (5a-j). The highly selective and
potent hCA IX and hCA XII moiety coumarin, was kept common in both
the series (Fig. 1).
2.2.1. Series 1
The inferences derived for the compounds synthesized in series 1
(6a-m) (Table 1) are summarized below:-
i. The two cytosolic isoforms, hCA I and hCA II, were not at all in-
hibited by the synthesized compounds 6a-m (Ki > 10000 nM).
ii. The tumor-associated isoform hCA IX, was inhibited by the com-
pounds, 6a-m, in a low to high nanomolar range, with the Ki values
ranging from 61.5 to 788.4 nM. The best inhibition constant i.e.
61.5 nM was shown by compound 6i, possessing a methyl group at
the meta position of the phenyl ring located at the 3rdposition of 4-
thiazolidinone ring. Apart from this, the other compounds found to
exhibit a low inhibition on this isoform were compounds 6 h
(Ki = 71.1 nM),6a (Ki = 72.1 nM), 6c (Ki = 72.6 nM), and 6 k
(Ki = 72.7 nM). Barring compounds 6b, 6e and 6j, all the remaining
compounds showed Ki values below 100 nM.
2. Result and discussion
2.1. Chemistry
2.1.1. Synthesis of series 1 compounds
The synthesis of coumarin linked to 4-thiazolidinone via a pyrazole
linker (6a-m) was performed as per the general synthetic scheme as
illustrated in Scheme 1. Firstly, the intermediate 3 was synthesized by
the reaction of salicylaldehyde with ethylacetoacetate in the presence
of piperidine as a catalyst. Consequently, the intermediate 3 was further
reacted with phenylhydrazine using glacial acetic acid as a solvent to
afford intermediate 4 which upon subjection to Vilsmeier-Haack
iii. The compounds, 6a-m exhibited a comparatively weaker inhibitory
profile for the tumor-associated isoform hCA XII with Ki values
ranging between 389.2 and 945.4 nM. The best inhibition constant
i.e. 389.2 nM was shown by compound 6 l, possessing a chloro
group at the meta position of the phenyl ring located at the 3rd
Fig. 1. General structure of the designed compounds.
2

P.S. Thacker, et al.
BioorganicChemistry104(2020)104272
Scheme 1. Synthesis of coumarin linked 4-thiazolidinones via pyrazole linker (6a-m)-Reagents and conditions: (i) Catalytic piperidine, neat, 90% (ii)
Phenylhydrazine, Glacial acetic acid, rt, 30 min, 95% (iii) DMF/POCl3, 0 °C-rt, 24 h, 73% (iv) Substituted anilines, thioglycolic acid, silica gel (100–200 mesh), DCM,
rt, 3–12 h, 42–64%.
Scheme 2. Synthesis of coumarin-linked triazoles (5a-j)-Reagents and conditions: (i) H2SO4, ice bath, 15 min, 95% (ii) Propargyl bromide, K2CO3, acetone, rt, 3–4 h
90% (iii) Substituted benzyl azides, CuSO4·5H2O, sodium ascorbate, EtOH:H2O::1:1, rt, overnight, 72–90%.
position of 4-thiazolidinone ring.
3. Conclusion
2.2.2. Series 2
In conclusion, two series, coumarin linked 4-thiazolidinone via a
pyrazole linker (6a-m, Series 1) and coumarin-linked 1,2,3-triazoles
(5a-j, Series 2) were synthesized. The synthesized molecules were
screened against the four physiologically and pharmacologically re-
levant isoforms namely the cytosolic isoforms hCA I and II as well as the
transmembrane tumor-associated isoforms, hCA IX and XII. All the
compounds showed selective inhibition for hCA IX and XII over hCA I
and II. In the case of series 1, for hCA IX, the values of the inhibition
constants (Kis) ranged from 61.5 to 788.4 nM with the best inhibition
constant i.e. 61.5 nM shown by compound 6i. The other compounds of
this series showing notable hCA IX inhibition were compounds 6 h
(Ki = 71.1 nM), 6a (Ki = 72.1 nM), 6c (Ki = 72.6 nM), and 6 k
(Ki = 72.7 nM). None of the compounds of this series were able to elicit
note worthy hCA XII inhibition. In the case of series 2 the inhibtion
data for hCA IX, the values of the inhibition constants (Kis) ranged from
32.7 to 2152 nM, with the best inhibition constant i.e. 32.7 µM shown
by compound 5d. The other compounds of this series showing notable
The inferences derived for the compounds of series 2 (5a-j) are
summarized below (Table 2):-
i. The two cytosolic isoforms, hCA I and hCA II were not at all in-
hibited by the synthesized compounds 5a-j (Ki > 10000 nM).
ii. The tumor-associated isoform hCA IX was inhibited by the com-
pounds 5a-j in a low to high micromolar range with the Ki values
ranging from 32.7 to 2152 nM. The best inhibition constant i.e.
32.7 nM was shown by compound 5d possessing a cyano group at
the para position of the benzyl ring located at the 1stposition (N-1)
of 1,2,3-triazole ring. Apart from this, the other compounds found
to exhibit a low micromolar inhibition on this isoform were com-
pounds 5c (Ki = 54.2 nM), 5j (Ki = 60.7 nM), 5f (Ki = 62.6 nM),
5a (Ki = 75.3 nM) and 5b (Ki = 92.6 nM).
iii. The compounds 5a-j exhibited a comparatively weaker inhibitory
profile for the tumor-associated isoform hCA XII with Ki values
ranging from 84.2 to 717.1 nM. The best inhibition constant i.e.
84.2 nM was shown by compound 5i possessing a methoxy group at
the para position of the benzyl ring located at the 1st position (N-1)
of 1,2,3-triazole ring.
hCA IX inhibition were compounds 5c (Ki
= 54.2 nM),5j
(Ki = 60.7 nM), 5f (Ki = 62.6 nM),5a(Ki = 75.3 nM) and 5b
(Ki = 92.6 nM). For hCA XII, the values of the inhibition constants (Kis)
ranged between 84.2 and 717.1 nM with the best inhibition constant
i.e. 84.2 nM was shown by compound 5i. Hence, compounds 6i from
Series 1 and 5d from Series 2 could be taken as lead compounds for the
further development of selective and potent hCA IX inhibitors, whereas
3

P.S. Thacker, et al.
BioorganicChemistry104(2020)104272
Table 1
Inhibition data of compounds (6a-m) for hCA I, II, IX and XII isoforms and acetazolamide (AAZ) as a standard drug.
KI (nM)* (IC50
)
Compound
6a
Structure
hCA I
hCAII
hCA IX
hCA XII
> 10000
> 10000
72.1
2.1 (84.6)
945.4
863.1
23.4 (599.7)
25.8 (547.6)
6b
6c
> 10000
> 10000
> 10000
> 10000
788.4
72.6
24.5 (915.0)
3.7 (85.2)
891.0
16.9 (565.3)
6d
6e
> 10000
> 10000
> 10000
> 10000
84.8
2.7 (99.3)
906.3
728.0
22.3 (575.0)
27.3 (462.0)
672.7
25.8 (780.9)
6f
> 10000
> 10000
> 10000
> 10000
90.6
94.3
3.1 (106.0)
1.5 (110.3)
850.6
674.8
13.5 (539.7)
18.3 (428.4)
6g
6h
> 10000
> 10000
71.1
1.3 (83.4)
722.3
15.3 (458.4)
(continued on next page)
4

P.S. Thacker, et al.
BioorganicChemistry104(2020)104272
Table 1 (continued)
KI (nM)* (IC50
)
Compound
6i
Structure
hCA I
hCAII
hCA IX
hCA XII
> 10000
> 10000
61.5
2.0 (72.3)
586.8
17.8
6j
> 10000
> 10000
> 10000
> 10000
404.0
72.7
9.5 (469.4)
502.7
7.2 (372.6)
6k
1.1 (85.3)
618.8
11.8 (392.9)
6l
> 10000
> 10000
250
> 10000
> 10000
12.1
91.1
90.9
25.8
3.0 (106.6)
2.7 (106.4)
389.2
623.4
5.7
8.4
6m
AAZ
8.8 (247.5)
* Mean from 3 different assays, by a stopped flow technique (errors were in the range of
5–10% of the reported values).
compound 5i from Series 2 can be explored further for the design of
4.2. General procedures for the synthesis of series 1 compounds
selective and potent hCA XII inhibitors.
4.2.1. Synthesis of 3-acetylcoumarin (3)
To an equimolar mixture of salicylaldehyde (1equiv.) and ethyla-
cetoacetate (1equiv.) were added few (5–6) drops of piperidine. The
mixture was stirred for 30 min at rt. resulting in the formation of a
yellowish solid. On completion of the reaction as indicated by the dis-
appearance of staring materials on TLC, crushed ice was added and the
solid was filtered off. The crude product was purified via column
chromatograpy using EtOAc:Hexane (1:5) as eluent to afford 3-acet-
ylcoumarin (3) as a yellow solid in 90% yield.
4. Experimental section
4.1. General
All the chemicals and solvents were procured and utilized as such
from the suppliers. Wherever necessary, anhydrous solvents were used.
Thin Layer Chromatography (TLC) analysis was done by utilizing Merck
silica gel 60 F254 aluminum plates. Stuart Digital Melting Point
Apparatus (SMP 30) was used in determining the melting points of the
compounds, which are uncorrected. 1H and 13C NMR spectra were re-
corded using Bruker Avance 500 MHz and 125 MHz respectively using
DMSO‑d6 as the solvent. Chemical Shift values are recorded in ppm
using TMS as the internal standard. HRMS were determined by Agilent
QTOF mass spectrometer 6540 series instrument and were performed
using ESI techniques at 70 eV.
4.2.2. Synthesis of 3-(1-(2-phenylhydrazono)ethyl)-2H-chromen-2-one (4)
To a stirred solution of 3-acetylcoumarin 3 (1 equiv.) in acetic acid
was added phenylhydrazine (1 equiv.). The solution was allowed to stir
at rt. for half an hour. Thereafter, the resultant suspension was added in
ice-water and filtered off to afford the crude product 4 in 95% yield.
4.2.3. Synthesis of 1,3-disubstituted-4-formylpyrazole (5)
To a stirred solution of DMF (25 mL) and POCl3 (5 mL) cooled to
5

P.S. Thacker, et al.
BioorganicChemistry104(2020)104272
Table 2
Inhibition data of compounds 5a-j against hCA isoforms I, II, IX and XII and acetazolamide (AAZ) as a standard drug.
KI (nM)*
Compound
5a
Structure
hCAI
hCAII
hCA IX
hCA XII
> 10000
> 10000
75.3
1.6 (88.3)
466.6
10.3 (296.7)
9.3 (379.6)
5b
5c
> 10000
> 10000
> 10000
> 10000
92.6
54.2
2.2 (108.4)
597.8
1.9 (63.8)
2.4 (38.9)
717.1
623.5
14.8 (455.1)
16.3 (395.9)
5d
5e
> 10000
> 10000
> 10000
> 10000
32.7
787.7
19.6 (914.2)
632.5
8.4 (401.6)
5f
> 10000
> 10000
> 10000
> 10000
62.6
1.9 (73.6)
590.9
571.7
6.9 (375.2)
5g
2152
20.0
15.3 (363.0)
(continued on next page)
6

P.S. Thacker, et al.
BioorganicChemistry104(2020)104272
Table 2 (continued)
KI (nM)*
hCAI
Compound
5h
Structure
hCAII
hCA IX
hCA XII
> 10000
> 10000
558.7
13.5 (648.7)
1.5 (312.3)
333.0
7.7 (211.9)
5i
> 10000
> 10000
268.5
84.2
2.0 (54.3)
5j
> 10000
> 10000
60.7
25.8
2.0 (71.4)
437.0
5.7
6.0 (277.8)
AAZ
250.0
12.1
* Mean from 3 different assays, by a stopped flow technique (errors were in the range of 5–10% of the reported values).
0–5 °C was added compound 4 (5 mmol) portion wise. The resulting
solution was allowed to stir at rt. for 24 h and the progress of the re-
action was monitored by TLC. On the completion of the reaction as
indicated by TLC using 100% chloroform as mobile phase, the reaction
mixture was poured onto crushed ice and neutralized with conc. NaOH
to pH 7. The resulting solid was filtered off and dried to afford a crude
yellowish solid which was subjected to column chromatography using
silica gel 60–120 mesh as the stationary phase and 100% chloroform as
the mobile phase to afford a crystalline whitish solid 5 in 73% yield.
480.1392 [M + H]+
.
3-(4-Bromophenyl)-2-(3-(2-oxo-2H-chromen-3-yl)-1-phenyl-1H-pyr-
azol-4-yl)thiazolidin-4-one (6b): Yield: 51%; Color: Off white solid; mp:
188–190 °C; 1H NMR (500 MHz, DMSO‑d6) δ 8.86 (s, 1H), 8.12 (s, 1H),
7.89 (d, J = 7.9 Hz, 2H), 7.83 (d, J = 7.6 Hz, 1H), 7.69 (dd, J = 11.4,
4.2 Hz, 1H), 7.54 – 7.49 (m, 3H), 7.44 – 7.40 (m, 3H), 7.35 (dd,
J = 15.7, 8.1 Hz, 3H), 6.50 (s, 1H), 4.08 (d, J = 15.6 Hz, 1H), 3.81 (d,
J = 15.7 Hz, 1H); 13C NMR (125 MHz, DMSO‑d6) δ 170.5, 160.0, 153.8,
145.6, 143.4, 139.4, 137.4, 132.9, 132.0, 130.0, 129.5, 128.8, 128.4,
127.3, 125.3, 123.9, 120.3, 119.8, 119.4, 118.9, 116.5, 56.6, 32.6; FT-
IR (cm−1): 2924, 1709, 1670, 1342, 1213, 758; HR-MS (ESI-QTOF): m/
4.2.4. Synthesis of 1,3-disubstituted-4-thiazolidinonepyrazole (6)
To a stirred solution of aldehyde 5 (1equiv.) in DCM at rt was added
aniline (1 equiv.) and the mixture was stirred for 5 min. Thioglycolic
acid (2 equiv.) was then added to the above reaction mixture and
stirring was continued for another 5 min followed by addition of silica
gel 100–200 mesh (0.5 g for 1 mmol aldehyde). On completion of the
reaction as indicated by TLC the solvent was removed under reduced
pressure. The obtained crude product were subjected to column chro-
matography using silica gel 60–120 mesh as the stationary phase and
Hexane: EtOAc : 5:1 as mobile phase to afford the final compound 5 as a
white solid.
z
calculated for C27H18BrN3O3S; 546.0310, found 546.0309
[M + 2H]+
.
3-(3,4-Dimethoxyphenyl)-2-(3-(2-oxo-2H-chromen-3-yl)-1-phenyl-1H-
pyrazol-4-yl)thiazolidin-4-one (6c): Yield: 64%; Color: Off white solid,
mp: 232–234 °C; 1H NMR (500 MHz, DMSO‑d6) δ 8.91 (s, 1H), 7.92 (d,
J = 5.4 Hz, 3H), 7.75 (d, J = 7.4 Hz, 1H), 7.68 (t, J = 7.5 Hz, 1H),
7.53 (t, J = 7.8 Hz, 2H), 7.47 (d, J = 8.2 Hz, 1H), 7.40 (t, J = 7.5 Hz,
1H), 7.36 (t, J = 7.3 Hz, 1H), 6.87 (s, 1H), 6.70 (q, J = 8.6 Hz, 2H),
6.42 (s, 1H), 4.05 (d, J = 15.5 Hz, 1H), 3.81 (d, J = 15.5 Hz, 1H), 3.54
(s, 3H), 3.52 (s, 3H); 13C NMR (125 MHz, DMSO‑d6) δ 170.2, 160.0,
153.8, 149.0, 148.0, 146.1, 143.1, 139.5, 132.7, 130.5, 130.0, 129.2,
129.1, 127.3, 125.2, 124.0, 120.1, 119.8, 119.2, 118.9, 116.4, 111.5,
111.3, 56.9, 55.8, 55.6, 32.7; FT-IR (cm−1): 2971, 2935, 1686, 1600,
1506, 1238, 765; HR-MS (ESI-QTOF): m/z calculated for C29H23N3O5S;
2-(3-(2-Oxo-2H-chromen-3-yl)-1-phenyl-1H-pyrazol-4-yl)-3-(p-tolyl)
thiazolidin-4-one (6a): Yield: 64%; Color: Pale brown solid; mp:
190–192 °C ; 1H NMR (500 MHz, DMSO‑d6) δ 8.87 (s, 1H), 7.95 (d,
J = 11.6 Hz, 1H), 7.91 (d, J = 8.2 Hz, 2H), 7.80 (d, J = 7.5 Hz, 1H),
7.69 (t, J = 7.4 Hz, 1H), 7.56 – 7.48 (m, 3H), 7.42 (t, J = 7.5 Hz, 1H),
7.36 (t, J = 7.3 Hz, 1H), 7.12 (d, J = 8.2 Hz, 2H), 6.95 (d, J = 8.2 Hz,
2H), 6.45 (s, 1H), 4.09 (d, J = 12.1 Hz, 1H), 3.80 (d, J = 15.6 Hz, 1H),
2.01 (s, 3H); 13C NMR (125 MHz, DMSO‑d6) δ 170.3, 160.0, 153.7,
145.8, 143.1, 139.4, 136.8, 135.3, 132.8, 130.0, 129.6, 129.3, 129.0,
127.3, 126.8, 125.2, 124.1, 120.2, 119.4, 118.8, 116.5, 56.8, 32.7,
20.8; FT-IR (cm−1): 3098, 3048, 1715, 1668, 1289, 1212, 760; HR-MS
526.1437; found 526.1436 [M + H]+
.
2-(3-(2-Oxo-2H-chromen-3-yl)-1-phenyl-1H-pyrazol-4-yl)-3-phe-
nylthiazolidin-4-one (6d): Yield: 50%; Color: Off white solid;
mp:209–211 °C; 1H NMR (500 MHz, DMSO‑d6) δ 8.86 (s, 1H), 8.05 (s,
1H), 7.89 (d, J = 8.6, 1.0 Hz, 2H), 7.80 (d, J = 7.7, 1.4 Hz, 1H), 7.69
(t, J = 8.6, 7.4, 1.6 Hz, 1H), 7.54 – 7.49 (m, 3H), 7.42 (t, J = 7.6,
1.0 Hz, 1H), 7.37 – 7.33 (m, J = 11.1, 3.9 Hz, 3H), 7.22 (t, J = 10.8,
5.1 Hz, 2H), 7.06 (t, 1H), 6.50 (s, 1H), 4.08 (d, 1H), 3.80 (d,
(ESI-QTOF): m/z calculated for
C28H21N3O3S; 480.1382; found
7

P.S. Thacker, et al.
BioorganicChemistry104(2020)104272
J = 15.6 Hz, 1H); 13C NMR (125 MHz, DMSO‑d6) δ 170.4, 160.0, 153.9,
145.6, 143.4, 139.4, 138.1, 132.8, 130.0, 129.4, 129.1, 128.8, 127.3,
127.0, 126.5, 125.2, 124.2, 120.4, 119.4, 118.9, 116.5, 56.8, 32.7; FT-
IR (cm−1): 3132, 3062, 1714, 1675, 1389, 1225, 749, 686; HR-MS (ESI-
QTOF): m/z calculated for C27H19N3O3S; 466.1225; found 466.1223
(cm−1): 1717, 1677, 1225, 752; HR-MS (ESI-QTOF): m/z calculated for
C
28H21N3O3S 480.1382; found 480.1385 [M + H]+
.
2-(3-(2-Oxo-2H-chromen-3-yl)-1-phenyl-1H-pyrazol-4-yl)-3-(3,4,5-tri-
methoxyphenyl) thiazolidin-4-one (6j): Yield: 56%; Color: Off white solid;
mp: 228–230 °C; 1H NMR (500 MHz, DMSO‑d6) δ 9.31 (s, 1H), 8.28 (s,
1H), 7.96 (s, 1H), 7.82 (d, J = 7.8 Hz, 2H), 7.67 – 7.59 (m, 2H), 7.53 (t,
J = 7.9 Hz, 2H), 7.42 – 7.30 (m, J = 18.3, 9.1, 5.7 Hz, 3H), 6.21 (s,
1H), 5.72 (s, 1H), 3.82 (s, 3H), 3.47 (d, J = 1.6 Hz, 6H), 3.23 (d,
J = 12.3 Hz, 1H), 2.74 (d, J = 12.1 Hz, 1H).13C NMR (125 MHz,
DMSO‑d6) δ 169.6, 159.4, 153.6, 153.1, 150.8, 145.6, 142.4, 139.8,
139.5, 134.4, 132.4, 130.0, 129.0, 128.7, 126.8, 125.7, 125.1, 121.5,
119.5, 119.1, 118.8, 116.5, 104.6, 79.6, 61.7, 60.4, 55.8, 32.4; FT-IR
(cm−1): 2926, 1728, 1667, 1101, 749; HR-MS (ESI-QTOF): m/z calcu-
[M + H]+
.
2-(3-(2-Oxo-2H-chromen-3-yl)-1-phenyl-1H-pyrazol-4-yl)-3-(4-phe-
noxyphenyl)thiazolidin-4-one (6e): Yield: 47%; Color: Off white solid;
mp: 168–170 °C; 1H NMR (500 MHz, DMSO‑d6) δ 8.88 (s, 1H), 8.08 (s,
1H), 7.91 (d, J = 7.9 Hz, 2H), 7.82 (d, J = 7.3 Hz, 1H), 7.69 (t,
J = 7.3 Hz, 1H), 7.55 – 7.48 (m, 3H), 7.43 (t, J = 7.4 Hz, 1H), 7.36 (dd,
J = 9.4, 6.2 Hz, 3H), 7.28 (d, J = 8.8 Hz, 2H), 7.14 (t, J = 7.4 Hz, 1H),
6.90 (d, J = 7.9 Hz, 2H), 6.79 (d, J = 8.8 Hz, 2H), 6.44 (s, 1H), 4.08 (d,
J = 15.6 Hz, 1H), 3.80 (d, J = 15.6 Hz, 1H); 13C NMR (125 MHz,
DMSO‑d6) δ 170.4, 160.0, 156.3, 155.8, 153.8, 145.7, 143.4, 139.4,
133.0, 132.8, 130.5, 130.0, 129.4, 128.9, 128.5, 127.3, 125.3, 124.3,
124.1, 120.3, 119.6, 119.3, 118.9, 118.4, 116.5, 56.8, 32.6; FT-IR
(cm−1): 2970, 1720, 1680, 1229, 754; HR-MS (ESI-QTOF): m/z calcu-
lated for C30H25N3O6S 556.1542; found 556.1539 [M + H]+
.
3-(4-Methoxyphenyl)-2-(3-(2-oxo-2H-chromen-3-yl)-1-phenyl-1H-pyr-
azol-4-yl) thiazolidin-4-one (6 k): Yield: 52%; Color: Off white solid; mp:
184–186 °C; 1H NMR (500 MHz, DMSO‑d6) δ 8.89 (s, 1H), 7.95 – 7.90
(m, 3H), 7.77 (d, J = 7.3 Hz, 1H), 7.69 (t, J = 7.5 Hz, 1H), 7.53 (t,
J = 7.5 Hz, 2H), 7.48 (d, J = 8.2 Hz, 1H), 7.41 (t, J = 7.3 Hz, 1H),
7.37 (t, J = 7.2 Hz, 1H), 7.11 (d, J = 8.4 Hz, 2H), 6.68 (d, J = 8.4 Hz,
2H), 6.38 (s, 1H), 4.07 (d, J = 15.5 Hz, 2H), 3.80 (d, J = 15.5 Hz, 1H),
3.49 (s, 3H); 13C NMR (125 MHz, DMSO‑d6) δ 170.3, 160.0, 158.3,
153.7, 145.9, 143.1, 139.4, 132.7, 130.4, 130.0, 129.3, 129.1, 128.6,
127.3, 125.2, 124.0, 120.1, 119.3, 118.9, 116.4, 114.3, 56.9, 55.3,
32.6; FT-IR (cm−1): 2923, 1719, 1683, 1224, 756; HR-MS (ESI-QTOF):
lated for C33H23N3O4S 558.1488; found 558.1488 [M + H]+
.
3-(4-Chlorophenyl)-2-(3-(2-oxo-2H-chromen-3-yl)-1-phenyl-1H-pyr-
azol-4-yl)thiazolidin-4-one (6f): Yield: 58%; Color: Off white solid; mp:
180–182 °C; 1H NMR (500 MHz, DMSO‑d6) δ 8.86 (s, 1H), 8.09 (d,
J = 19.4 Hz, 1H), 7.89 (d, J = 7.8 Hz, 2H), 7.82 (d, J = 7.3 Hz, 1H),
7.69 (t, J = 7.5 Hz, 1H), 7.51 (dd, J = 13.7, 7.8 Hz, 3H), 7.42 (t,
J = 7.5 Hz, 1H), 7.38 (d, J = 8.6 Hz, 2H), 7.34 (d, J = 7.2 Hz, 1H),
7.27 (d, J = 8.6 Hz, 2H), 6.50 (s, 1H), 4.08 (d, J = 15.7 Hz, 1H), 3.81
(d, J = 15.7 Hz, 1H); 13C NMR (125 MHz, DMSO‑d6) δ 170.6, 160.1,
153.8, 145.7, 143.4, 139.4, 137.0, 132.9, 131.4, 130.0, 129.4, 129.1,
128.8, 128.2, 127.3, 125.3, 123.9, 120.3, 119.4, 118.9, 116.5, 56.6,
32.6; FT-IR (cm−1): 3098, 3055, 1710, 1672, 1213, 759, 698; HR-MS
m/z calculated for C28H21N3O4S 496.1331; found 496.1327 [M + H]+
.
3-(3-Chlorophenyl)-2-(3-(2-oxo-2H-chromen-3-yl)-1-phenyl-1H-pyr-
azol-4-yl)thiazolidin-4-one (6 l): Yield: 42%; Color: Off white solid; mp:
152–154 °C; 1H NMR (500 MHz, DMSO‑d6) δ 8.88 (s, 1H), 8.12 (s, 1H),
7.88 (d, J = 8.1 Hz, 2H), 7.83 (d, J = 7.7 Hz, 1H), 7.70 (t, 1H), 7.56 (t,
1H), 7.52 (t, J = 8.1 Hz, 3H), 7.43 (t, J = 7.5 Hz, 1H), 7.38 – 7.32 (m,
J = 13.5, 7.2 Hz, 2H), 7.27 (t, J = 8.0 Hz, 1H), 7.14 (d, J = 7.2 Hz,
1H), 6.54 (s, 1H), 4.07 (d, J = 15.7 Hz, 1H), 3.82 (d, J = 15.7 Hz, 1H);
13C NMR (125 MHz, DMSO‑d6) δ 170.6, 160.1, 153.9, 145.7, 143.5,
139.5, 139.4, 133.3, 132.9, 130.6, 130.0, 129.4, 128.8, 127.3, 126.8,
126.0, 125.3, 124.7, 123.8, 120.4, 119.4, 118.9, 116.6, 56.5, 32.7; FT-
IR (cm−1): 1717, 1682, 753, 683; HR-MS (ESI-QTOF): m/z calculated
(ESI-QTOF): m/z calculated for
C27H18ClN3O3S 500.0836; found
500.0836 [M + H]+
.
3-(4-Isopropylphenyl)-2-(3-(2-oxo-2H-chromen-3-yl)-1-phenyl-1H-pyr-
azol-4-yl) thiazolidin-4-one (6 g): Yield: 53%; Color: Off white solid; mp:
207–209 °C; 1H NMR (500 MHz, DMSO‑d6) δ 8.88 (s, 1H), 8.04 (s, 1H),
7.92 (d, J = 7.8 Hz, 2H), 7.79 (d, J = 7.7, 1.2 Hz, 1H), 7.69 (t, 1H),
7.55 – 7.48 (m, 3H), 7.41 (t, J = 7.5 Hz, 1H), 7.36 (t, J = 7.4 Hz, 1H),
7.21 (d, J = 8.4 Hz, 2H), 7.07 (d, J = 8.5 Hz, 2H), 6.47 (s, 1H), 4.09 (d,
J = 13.4 Hz, 1H), 3.80 (d, J = 15.6 Hz, 1H), 2.71 – 2.61 (m, 1H), 1.07
(d, J = 6.9 Hz, 3H), 1.02 (d, J = 6.9 Hz, 3H); 13C NMR (125 MHz,
DMSO‑d6) δ 170.4, 160.0, 153.8, 147.2, 145.5, 143.3, 139.4, 135.6,
132.8, 130.0, 129.3, 128.8, 127.3, 127.0, 126.5, 125.2, 124.4, 120.4,
119.3, 118.9, 116.5, 56.8, 33.1, 32.6, 23.9, 23.9.; FT-IR (cm−1): 2957,
1688, 1606, 1326, 756; HR-MS (ESI-QTOF): m/z calculated for
for C27H18ClN3O3S 500.0836; found 500.0828 [M + H]+
.
3-(3-Bromophenyl)-2-(3-(2-oxo-2H-chromen-3-yl)-1-phenyl-1H-pyr-
azol-4-yl) thiazolidin-4-one (6 m): Yield: 47%; Color: Off white solid;
mp: 145–147 °C; 1H NMR (500 MHz, DMSO‑d6) δ 8.88 (s, 1H), 8.11 (s,
1H), 7.88 (d, J = 7.9 Hz, 2H), 7.83 (d, J = 7.4 Hz, 1H), 7.72 – 7.67 (m,
2H), 7.52 (t, J = 8.0 Hz, 3H), 7.43 (t, J = 7.5 Hz, 1H), 7.36 (d,
J = 7.6 Hz, 2H), 7.27 (d, J = 8.0 Hz, 1H), 7.20 (t, J = 8.0 Hz, 1H),
C
30H25N3O3S 508.1695; found 508.1692 [M + H]+
.
6.53 (s, 1H), 4.06 (d, J = 15.7 Hz, 1H), 3.82 (d, J = 15.7 Hz, 1H); 13
C
2-(3-(2-Oxo-2H-chromen-3-yl)-1-phenyl-1H-pyrazol-4-yl)-3-(3-(tri-
NMR (125 MHz, DMSO‑d6) δ 170.6, 160.1, 153.8, 145.9, 143.5, 139.6,
139.4, 132.9, 130.9, 130.0, 129.7, 129.4, 128.9, 127.3, 125.3, 125.1,
123.8, 121.6, 120.4, 119.4, 118.9, 116.6, 56.5, 32.7; FT-IR (cm−1):
1717, 1683, 753, 893; HR-MS (ESI-QTOF): m/z calculated for
fluoromethyl)phenyl) thiazolidin-4-one (6 h): Yield: 51%; Color: Off white
solid; mp: 142–144 °C; 1H NMR (500 MHz, DMSO‑d6) δ 8.92 (s, 1H),
8.09 (s, 1H), 7.86 (d, J = 7.4 Hz, 3H), 7.80 (d, J = 7.5 Hz, 1H), 7.73 –
7.64 (m, 2H), 7.54 – 7.47 (m, 4H), 7.43 (dd, J = 14.2, 7.1 Hz, 2H), 7.35
(t, J = 7.3 Hz, 1H), 6.61 (s, 1H), 4.08 (d, J = 15.7 Hz, 1H), 3.85 (d,
J = 15.7 Hz, 1H); 13C NMR (125 MHz, DMSO‑d6) δ 170.8, 160.1, 153.9,
145.7, 143.4, 139.3, 138.9, 132.9, 130.2, 130.1, 130.0, 129.7, 129.4,
129.0, 127.3, 125.3, 123.7, 123.5, 123.0, 123.0, 120.3, 119.3, 118.8,
116.6, 56.5, 32.7; FT-IR (cm−1): 2924, 1709, 1690, 1326, 1116, 750;
HR-MS (ESI-QTOF): m/z calculated for C28H18F3N3O3S 534.1099;
C
27H18BrN3O3S 546.0310; found 546.0349 [M + 2H]+
.
4.3. General procedures for the synthesis of series 2 compounds
4.3.1. Synthesis of 4-(chloromethyl)-7-hydroxy-2H-chromen-2-one (3)
A
solution of resorcinol (1) (9.08 mmol) in ethyl-4-chlor-
oacetoacetate (2) (1.3 mL) was added drop wise to 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 a beaker
containing ice water. The precipitated compound was collected by
suction filtration, washed with cold water and dried. It was re-
crystallized from ethanol. Yield 91%; Color: white solid; mp 186–188
°C; 1H NMR (500 MHz, DMSO‑d6) δ 10.66 (s, 1H), 7.68 (d, J = 7.8 Hz,
1H), 6.90 – 6.71 (m, 2H), 6.42 (s, 1H), 4.96 (s, 2H);13C NMR (125 MHz,
DMSO‑d6) δ 161.94, 160.61, 155.78, 151.45, 127.01, 113.55, 111.54,
109.83, 103.00, 41.83; HR-MS (ESI-QTOF): m/z calcd for
[M + H] + C10H7ClO3, 210.0084; found 211.0157.
found 534.1110 [M + H]+
.
2-(3-(2-Oxo-2H-chromen-3-yl)-1-phenyl-1H-pyrazol-4-yl)-3-(m-tolyl)
thiazolidin-4-one (6i): Yield: 43%; Color: Off white solid; mp:
165–167 °C; 1H NMR (500 MHz, DMSO‑d6) δ 8.86 (s, 1H), 7.91 (d, 2H),
7.80 (d, 1H), 7.70 (t, 1H), 7.52 (dd, J = 6.8 Hz, 3H), 7.40 (dt,
J = 33.3 Hz, 3H), 7.15 – 7.01 (m, 3H), 6.82 (d, 1H), 6.46 (s, 1H), 4.09
(d, J = 14.8 Hz, 1H), 3.81 (d, J = 14.6 Hz, 1H), 2.06 (s, 3H); 13C NMR
(125 MHz, DMSO‑d6) δ 170.3, 160.0, 153.8, 145.8, 143.3, 139.4, 138.5,
137.9, 132.8, 130.0, 129.3, 128.9, 128.9, 127.9, 127.3, 127.0, 125.3,
124.1, 123.8, 120.3, 119.4, 118.9, 116.5, 56.7, 32.8, 21.1; FT-IR
8

P.S. Thacker, et al.
BioorganicChemistry104(2020)104272
4.3.2. 4-(chloromethyl)-7-(prop-2-yn-1-yloxy)–2H-chromen-2-one (4)
To the solution of compound 3 (1 mmol) in freshly distilled acetone
(10 mL), was added anhydrous K2CO3 (2.5 mmol) as a base and pro-
pargyl bromide (1 mmol) drop wise. The resulting reaction mixture was
stirred under reflux for 2–3 hrs. On completion of the reaction as in-
dicated by TLC, the solvent was evaporated completely and the ob-
tained residue was extracted with ethyl acetate (3x20 mL) and dried
over Na2SO4. The combined organic layer was concentrated in vacuo
and the residue was purified by column chromatography on silica gel.
Yield 85%; Color: Pale yellow solid; mp 123–125 °C; 1H NMR
(500 MHz, CDCl3) δ 7.78 (d, J = 8.8 Hz, 1H), 6.99–6.87 (m, 2H), 6.40
(s, 1H), 4.77 (d, J = 2.3 Hz, 2H), 3.60 (s, 2H), 2.58 (t, J = 2.3 Hz, 1H),
2.55;13C NMR (125 MHz, CDCl3) δ 161.19, 160.34, 155.42, 151.53,
125.97, 113.05, 112.74, 112.47, 102.12, 78.42, 76.53, 59.51, 54.21;
HR-MS (ESI-QTOF): m/z calcd for [M + H] + C13H9ClO3; 248.0204,
found 249.0324
green solid; mp: 119–138 °C; 1H NMR (500 MHz, DMSO‑d6) δ 8.26 (s,
1H), 7.70 (t, J = 11.2 Hz, 1H), 7.50 (d, J = 8.2 Hz, 2H), 7.20 (d,
J = 8.1 Hz, 2H), 7.12 (t, J = 10.2 Hz, 1H), 7.02 – 6.95 (m, 1H), 6.44 (d,
J = 7.7 Hz, 1H), 5.53 (s, 2H), 5.20 (s, 2H), 4.96 – 4.87 (s, 2H); 13C NMR
(125 MHz, DMSO‑d6) δ 161.9, 155.8, 151.5, 148.0, 144.0, 130.0, 129.8,
127.1, 126.2, 124.6, 124.5, 113.6, 112.9, 111.4, 102.7, 62.4, 52.7,
42.0; HR-MS (ESI-QTOF): m/z calc. for (M + 2)+ C20H15BrClN3O
460.0064; found 462.0046.
4-(Chloromethyl)-7-((1-(4-Methylbenzyl)-1H-1,2,3-Triazol-4-yl)
Methoxy)–2H-Chromen −2-one (5f): Yield: 77.3%; Color: Creamy green
solid; mp: 124–130 °C; 1H NMR (500 MHz, DMSO‑d6) δ 8.31 (d,
J = 5.6 Hz, 1H), 7.78 (t, J = 7.9 Hz, 1H), 7.20 (dd, J = 13.2, 10.5 Hz,
5H), 7.11 – 7.05 (m, 1H), 6.53 (d, J = 9.1 Hz, 1H), 5.57 (s, 2H), 5.28 (s,
2H), 5.00 (s, 2H), 2.29 (s, 3H); 13C NMR (125 MHz, DMSO‑d6) δ 161.7,
160.4, 155.5, 151.2, 138.0, 133.4, 129.7, 128.5, 126.9, 125.4, 113.3,
112.6, 111.1, 102.4, 62.2, 53.1, 41.8, 21.1; HR-MS (ESI-QTOF): m/z
calc. for (M + H)+ C21H18ClN3O3 396.1115;found 396.1124.
4-(Chloromethyl)-7-((1-(3-Methoxybenzyl)-1H-1,2,3-Triazol-4-yl)
Methoxy)–2H-Chromen-2-one (5 g): Yield: 74.8%; Color: Yellowish
white solid; mp: 89–117 °C; 1H NMR (500 MHz, DMSO‑d6) δ 8.29 (s,
1H), 7.79 (dd, J = 16.2, 8.9 Hz, 1H), 7.31 (d, J = 8.6 Hz, 2H), 7.10 –
7.06 (m, 2H), 6.93 (d, J = 8.7 Hz, 2H), 6.53 (d, J = 9.6 Hz, 1H), 5.54
4.3.3. General procedure for the preparation of the compounds (5a-j)
A 50 mL round bottom flask was charged with compound 4
(1 mmol), various benzyl azides (0.9 mmol), sodium ascorbate
(0.3 mmol), 2 mL of ethanol and 2 mL of distilled water. Then,
CuSO4·5H2O (0.15 mmol) was added. The reaction mixture was stirred
at rt for overnight. After completion of the reaction by TLC analysis, the
mixture was filtered and extracted with ethyl acetate (3x20 mL) and
dried over Na2SO4. The combined organic layer was concentrated in
vacuo and the residue was purified by column chromatography on silica
gel to afford the final target compounds 5a-j in moderate to good yields.
7-((1-Benzyl-1H-1,2,3-Triazol-4-yl)Methoxy)-4-(Chloromethyl)–2H-
Chromen-2-one (5a): Yield: 66.2%; Color: Creamy white solid; mp:
96–124 °C; 1H NMR (500 MHz, DMSO‑d6) δ 8.35 (s, 1H), 7.79 (dd,
J = 14.2, 8.9 Hz, 1H), 7.41 – 7.31 (m, 5H), 7.18 (d, J = 18.3 Hz, 1H),
7.09 (d, J = 11.6 Hz, 1H), 6.53 (d, J = 8.7 Hz, 1H), 5.63 (s, 2H), 5.29
(s, 2H), 5.00 (d, J = 8.6 Hz, 2H); 13C NMR (125 MHz, DMSO‑d6) δ
161.6, 160.4, 155.5, 151.2, 133.8, 131.8, 129.6, 127.2, 126.8, 126.6,
125.7, 123.7, 113.3, 111.1, 102.4, 62.1, 51.3, 41.7; HR-MS (ESI-QTOF):
m/z calc. for (M + H)+ C20H16ClN3O3,382.0958; found 382.0969.
4-(Chloromethyl)-7-((1-(4-Nitrobenzyl)-1H-1,2,3-Triazol-4-yl)
(s, 2H), 5.27 (s, 2H), 5.00 – 4.96 (s, 2H), 3.75 (d, J = 4.3 Hz, 3H); 13
C
NMR (125 MHz, DMSO‑d6) δ 161.6, 160.8, 160.4, 155.5, 151.2, 141.8,
133.2, 129.2, 126.9, 125.9, 118.9, 113.3, 112.6, 111.4, 111.2, 102.6,
102.4, 79.4, 62.1, 52.7, 41.7; HR-MS (ESI-QTOF): m/z calc. for
(M + H)+ C21H18ClN3O4 412.1064; found 412.1066.
4-(Chloromethyl)-7-((1-(Naphthalen-2-yl-Methyl)-1H-1,2,3-Triazol-4-
yl)Methoxy)–2H-Chromen-2-one (5 h): Yield: 86.8%; Color: Creamy pink
solid; mp: 153–185 °C; 1H NMR (500 MHz, DMSO‑d6) δ 8.31 – 8.18 (m,
2H), 8.01 – 7.95 (m, 2H), 7.76 (d, J = 8.9 Hz, 1H), 7.62 – 7.53 (m, 3H),
7.46 (d, J = 6.8 Hz, 1H), 7.17 (d, J = 1.9 Hz, 1H), 7.06 (d, J = 8.8 Hz,
1H), 6.51 (s, 1H), 6.13 (s, 2H), 5.26 (s, 2H), 4.98 (d, J = 13.1 Hz,
2H);13C NMR (125 MHz, DMSO‑d6) δ 160.1, 159.0, 154.0, 149.8, 132.3,
130.3, 128.1, 125.8, 125.3, 125.2, 124.2, 122.2, 111.8, 109.7, 100.9,
60.6, 49.8, 40.3; HR-MS (ESI-QTOF): m/z calc. for (M + H)+
C24H18ClN3O3 432.1115; found 432.1358.
Methoxy)–2H-Chromen-2-one (5b): 60.2%. Yield; Color: Greenish white
solid; mp: 98–117 °C; 1H NMR (500 MHz, DMSO‑d6) δ 8.42 (s, 1H), 8.25
(d, J = 8.6 Hz, 2H), 7.78 (d, J = 8.9 Hz, 1H), 7.55 (d, J = 8.6 Hz, 2H),
7.21 (d, J = 2.3 Hz, 1H), 7.09 (dd, J = 8.9, 2.4 Hz, 1H), 6.53 (s, 1H),
5.82 (s, 2H), 5.32 (s, 2H), 5.01 (s, 2H); 13C NMR (125 MHz, DMSO‑d6) δ
161.7, 160.4, 155.5, 151.2, 138.0, 133.3, 129.7, 128.5, 126.8, 125.4,
113.3, 112.6, 111.1, 102.4, 102.1, 62.1, 53.1, 41.7; HR-MS (ESI-QTOF):
m/z calc. for (M + H)+ C20H15ClN4O5 427.0809; found 427.0816.
4-(Chloromethyl)-7-((1-(2,5-Dichlorobenzyl)-1H-1,2,3-Triazol-4-yl)
Methoxy)–2H-Chromen-2-one (5c): Yield: 65.2%; Color: Pale yellow
solid; mp:138–148 °C; 1H NMR (500 MHz, DMSO‑d6) δ 8.33 (s, 1H),
7.83 –7.78 (m, 1H), 7.71 (s, 1H), 7.49 (d, J = 8.1 Hz, 1H), 7.31 (d,
J = 6.3 Hz, 1H), 7.20 (s, 1H), 7.10 – 7.07 (m, 1H), 6.53 (d, J = 7.1 Hz,
1H), 5.73 (s, 2H), 5.30 (s, 2H), 4.97 (s, 2H); 13C NMR (125 MHz,
DMSO‑d6) δ 161.6, 160.4, 155.5, 151.2, 147.7, 143.7, 129.7, 129.5,
126.9, 125.9, 124.3, 124.2, 113.3, 112.6, 111.2, 102.4, 79.4, 62.1,
4-(Chloromethyl)-7-((1-(4-Methoxybenzyl)-1H-1,2,3-Triazol-4-yl)
Methoxy)–2H-Chromen-2-one (5i): Yield: 79.9%; Color: Greenish white
solid; mp: 151–157 °C; 1H NMR (500 MHz, CDCl3) δ 8.33 (s, 1H), 7.86
(d, J = 8.9 Hz, 1H), 7.43 – 7.37 (m, 2H), 7.23 – 7.13 (m, 3H), 7.01 (d,
J = 8.8 Hz, 1H), 6.33 (s, 1H), 5.61 (s, 2H), 5.26 (s, 2H), 3.65 (s, 2H),
3.58 (s, 3H); 13C NMR (125 MHz, DMSO‑d6) δ 160.80, 159.93, 159.59,
154.71, 150.41, 140.97, 132.35, 128.34, 126.04, 125.07, 118.11,
112.48, 111.83, 110.59, 101.80, 78.56, 61.27, 51.86, 40.92; HR-MS
(ESI-QTOF): m/z calc. for (M + 2)+ C21H18ClN3O4 412.1064; found
413.2665.
7-((1-(3-Chlorobenzyl)-1H-1,2,3-Triazol-4-yl)Methoxy)-4-
(Chloromethyl) –2H-Chromen-2-one (5j): Yield:84.4%; Color: Pale pink
solid; mp: 97–142 °C;1H NMR (500 MHz, DMSO‑d6) δ 8.32 (d,
J = 8.7 Hz, 1H), 7.70 (d, J = 8.9 Hz, 1H), 7.34 (dd, J = 6.8, 3.4 Hz,
3H), 7.21 (t, J = 2.9 Hz, 1H), 7.12 (d, J = 2.4 Hz, 1H), 7.01 (dd,
J = 8.9, 2.5 Hz, 1H), 6.44 (s, 1H), 5.58 (s, 2H), 5.22 (d, J = 6.6 Hz,
2H), 4.92 (s, 2H);13C NMR (125 MHz, DMSO‑d6) δ 160.7, 159.5, 154.6,
150.3, 137.8, 132.8, 130.2, 127.7, 126.2, 126.1, 126.0, 125.9, 124.8,
112.4, 110.2, 101.4, 61.2, 51.6, 40.8; HR-MS (ESI-QTOF): m/z calc. for
(M + H)+ C20H15Cl2N3O3 416.0569; found 417.0564.
52.4, 41.7; HR-MS (ESI-QTOF): m/z calc. for(M
+
H)+
C20H14Cl3N3O3450.0179; found 451.0653.
4-((4-(((4-(Chloromethyl)-2-Oxo-2H-Chromen-7-yl) Oxy) Methyl)-
1H-1,2,3-Triazol-1-yl) Methyl)Benzonitrile (5d): Yield: 63.7%; Color:
Pale yellow solid; mp: 113–140 °C; 1H NMR (500 MHz, DMSO‑d6) δ
8.41 (s, 1H), 7.87 (d, J = 7.2 Hz, 2H), 7.48 (d, J = 6.9 Hz, 2H), 7.22 –
7.07 (m, 3H), 6.53 (d, J = 5.8 Hz, 1H), 5.76 (s, 2H), 5.31 (s, 2H), 4.99
(d, J = 20.1 Hz, 2H); 13C NMR (125 MHz, DMSO‑d6) δ 161.6, 160.4,
155.5, 151.2, 138.7, 133.7, 131.2, 128.6, 127.1, 125.7, 113.3, 111.1,
102.4, 62.1, 52.5, 41.8, 40.5, 40.3; HR-MS (ESI-QTOF): m/z calc. for
(M + H)+ C21H15ClN4O3407.0911; found 407.0923.
4.4. CA inhibition
An SX.18 V-R Applied Photophysics (Oxford, UK) stopped flow in-
strument has been used to assay the CA catalyzed CO2 hydration ac-
tivity. [26]. Phenol Red (at a concentration of 0.2 mM) has been used as
an indicator, working at an absorbance maximum of 557 nm, with
10 mM Hepes (pH 7.4) as a buffer, 0.1 M Na2SO4 or NaClO4 (for
maintaining constant the ionic strength; these anions are not inhibitory
7-((1-(4-Bromobenzyl)-1H-1,2,3-Triazol-4-yl)Methoxy)-4-
(Chloromethyl)–2H-Chromen-2-one (5e): Yield: 84.5%; Color: Creamy
9

P.S. Thacker, et al.
BioorganicChemistry104(2020)104272
in the used concentration), following the CA-catalyzed CO2 hydration
reaction for a period of 5–10 s. Saturated CO2 solutions in water at 25°C
were used as substrate. Stock solutions of inhibitors were prepared at a
concentration of 10 mM (in DMSO-water 1:1, v/v) and dilutions up to
0.01 nM done with the assay buffer mentioned above. The enzyme
solution was prepared at a concentration of 0.1 µM. At least 7 different
inhibitor concentrations have been used for measuring the inhibition
constant. Inhibitor and enzyme solutions were pre-incubated together
for 6 hr at 24 °C prior to assay, in order to allow for the formation of the
E-I complex. In the case of coumarins the incubation is done for 6 h to
allow the hydrolysis to occur. Triplicate experiments were done for
each inhibitor concentration and the values reported throughout the
paper are the mean of such results. The inhibition constants were ob-
tained 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 [27–30].
103739.
[13] P.S. Thacker, M. Alvala, M. Arifuddin, A. Angeli, C.T. Supuran, Design, synthesis
and biological evaluation of coumarin-3-carboxamides as selective carbonic anhy-
drase IX and XII inhibitors, Bioorg. Chem. 86 (2019) 386–392.
[14] S.G. Narella, M.G. Shaik, A. Mohammed, M. Alvala, A. Angeli, C.T. Supuran,
Synthesis and biological evaluation of coumarin-1,3,4-oxadiazole hybrids as se-
lective carbonic anhydrase IX and XII inhibitors, Bioorg. Chem. 87 (2019) 765–772.
[15] L. De Luca, F. Mancuso, S. Ferro, M.R. Buemi, A. Angeli, S. Del Prete, C. Capasso,
C.T. Supuran, R. Gitto, Inhibitory effects and structural insights for a novel series of
coumarin-based compounds that selectively target human CA IX and CA XII car-
bonic anhydrases, Eur. J. Med. Chem. 143 (2018) 276–282.
[16] M.F. Khan, M.M. Alam, G. Verma, W. Akhtar, M. Akhter, M. Shaquiq-uz-zaman, The
therapeutic voyage of pyrazole and its analogs: a review, Eur. J. Med. Chem. 120
(2016) 170–201.
[17] (a) H.S. Ibrahim, S.M. Abou-Seri, M. Tanc, M.M. Elaasser, H.A. Abdel-Aziz,
C.T. Supuran, Isatin-pyrazolebenzenesulfonamide hybrids potently inhibit tumor-
associated carbonic anhydrase isoforms IX and XII, Eur. J. Med. Chem. 103 (2015)
583–593;
(b) I.N. Cvijetic, M. Tanç, I.O. Juranic, T.Z. Verbic, C.T. Supuran, B.J. Drakulic, 5-
Aryl-1H-pyrazole-3-carboxylic acids as selective inhibitors of human carbonic an-
hydrases IX and XII, Bioorg. Med. Chem. 23 (2015) 4649–4659.
[18] (a) P. Khloya, G. Celik, SitaRam, D. Vullo, C.T. Supuran, P.K. Sharma, 4-
Functionalized 1,3-diarylpyrazoles bearing benzenesulfonamide moiety as selective
potent inhibitors of the tumor associated carbonic anhydrase isoforms IX and XII,
Eur. J. Med. Chem. 76 (2014) 284–290;
Declaration of Competing Interest
(b) C. Yamali, H. I. Gul, A. Ece, S. Bua S, A. Angeli A, H. Sakagami H, E. Sahin, C. T.
Supuran, Synthesis, biological evaluation and in silico modelling studies of 1,3,5-
trisubstituted pyrazoles carrying benzenesulfonamide as potential anticancer agents
and selective cancer-associated hCA IX isoenzyme inhibitors, Bioorg. Chem. 92
(2019) 103222.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
[19] A. C. Tripathi, S. J. Gupta, G. N. Fatima P. K. Sonar, A. Verma, S. K. Saraf, 4-
Thiazolidinones: the advances continue, Eur. J. Med. Chem. 72 (2014) 52–77.
[20] (a) S.U. Mahmood, A. Saeed, S. Bua, A. Nocentini, P. Gratteri, C.T. Supuran,
Synthesis, biological evaluation and computational studies of novel iminothiazoli-
dinonebenzenesulfonamides as potent carbonic anhydrase II and IX inhibitors,
Bioorg. Chem. 77 (2018) 381–386;
Acknowledgements
PST, NSG, OSA and JS would like to thank the Department of
Pharmaceuticals, Ministry of Chemicals and Fertilizers, Govt. of India,
New Delhi for awarding NIPER Fellowship.
(b) O. Guzel-Akdemir, A. Angeli, K. Demir, C.T. Supuran, A. Akdemir, Novel
thiazolidinone-containing compounds, without the well-known sulphonamide zinc-
binding group acting as human carbonic anhydrase IX inhibitors, J. Enzyme. Inhib.
Med. Chem. 33 (2018) 1299–1308;
Declaration of Competing Interest
The authors declare no conflict of interest.
Appendix A. Supplementary material
(c) Z. P. Zhang, Z. F. Yin, J. Y. Li, Z. P. Wang, Q. J. Wu, J. Wang, Y. Liu Y, M. S.
Cheng, Synthesis, Molecular Docking Analysis, and Carbonic Anhydrase Inhibitory
Evaluations of Benzenesulfonamide Derivatives Containing Thiazolidinone,
Molecules.24 (2019) 2418.
[21] D. Dheer, V. Singh, R. Shankar, Medicinal attributes of 1,2,3-triazoles: Current
developments, Bioorg. Chem. 71 (2017) 30–54.
[22] (a) P. Singh, B. Swain, P. S. Thacker, D. K. Sigalapalli, P. Y. Purnachander, A.
Angeli A, C. T. Supuran, M. Arifuddin, Synthesis and carbonic anhydrase inhibition
studies of sulfonamide based indole-1,2,3-triazole chalcone hybrids, Bioorg. Chem.
99 (2020)103839.
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2020.104272.
(b) A. Grandane, M. Tanc, R. Zalubovskis, C.T. Supuran, 6-Triazolyl-substituted
sulfocoumarins are potent, selective inhibitors of the tumor-associated carbonic
anhydrases IX and XII, Bioorg. Med. Chem. Lett. 24 (2014) 1256–1260;
(c) J. Leitans, A. Sprudza, M. Tanc, I. Vozny, R. Zalubovskis, K. Tars, C.T. Supuran,
5-Substituted-(1,2,3-triazol-4-yl)thiophene-2-sulfonamides strongly inhibit human
carbonic anhydrases I, II, IX and XII: solution and X-ray crystallographic studies,
Bioorg. Med. Chem. 21 (2013) 5130–5138
References
[1] C. Capasso, C.T. Supuran, An overview of the alpha-, beta- and gamma-carbonic
anhydrases from Bacteria: can bacterial carbonic anhydrases shed new light on
evolution of bacteria? J. Enzyme. Inhib. Med. Chem. 30 (2015) 325–332.
[2] D.M. Monti, G. De Simone, E. Langella, C.T. Supuran, A. Di Fiore, S.M. Monti,
Insights into the role of reactive sulfhydryl groups of Carbonic Anhydrase III and VII
during oxidative damage, J. Enzyme. Inhib. Med. Chem. 32 (2017) 5–12.
[3] C.T. Supuran, Carbon-versus sulphur-based zinc binding groups for carbonic an-
hydrase inhibitors? J. Enzyme. Inhib. Med. Chem. 33 (2018) 485–495.
[4] V. Alterio, A. Di Fiore, K. D'Ambrosio, C.T. Supuran, G. De Simone, Multiple binding
modes of inhibitors to carbonic anhydrases: how to design specific drugs targeting
15 different isoforms? Chem. Rev. 112 (2012) 4421–4468.
[23] D. Srikrishna P. K. Dubey, Synthesis of novel substituted 3-(4-((1H-benzo[d]imi-
dazol-2-ylthio)methyl)-1-phenyl-1H-pyrazol-3-yl)-2H-chromen-2-ones: various ap-
proaches, Res. Chem. Intermed. 44 (2018) 4455–4468.
[24] M.P. Thakare, P. Kumar, N. Kumar, S.P. Pandey, Silica gel promoted environment
friendly synthesis of 2,3-disubstituted 4-thiazolidinones, Tetrahydron. Lett. 55
(2014) 2463–2466.
[25] N.S. Goud, V.K. Kanth Makani, J. Pranay, R. Alvala, I.A. Qureshi, P. Kumar,
R.D. Bharath, C. Nagaraj, S. Yerramsetty, M. Pal-Bhadra, M. Alvala, Synthesis, 18F-
radiolabeling and apoptosis inducing studies of novel 4, 7-disubstituted coumarins,
Bioorg. Chem. 97 (2020) 103663.
[5] C.T. Supuran, Exploring the multiple binding modes of inhibitors to carbonic an-
hydrases for novel drug discovery [published online ahead of print, 2020 Mar 25],
Expert. Opin. Drug. Discov. (2020) 1–16.
[26] R. G. Khalifah, The carbon dioxide hydration activity of carbonic anhydrase. I. Stop-
flow kinetic studies on the native human isoenzymes B and C, J. Biol. Chem. 246
(1971) 2561-2573.
[6] C.T. Supuran, An update on drug interaction considerations in the therapeutic use
of carbonic anhydrase inhibitors, Expert. Opin. Drug. Metab. Toxicol. 16 (2020)
297–307.
[27] P.S. Thacker, P. Shaikh, A. Angeli, M. Arifuddin, C.T. Supuran, Synthesis and bio-
logical evaluation of novel 8-substituted quinoline-2-carboxamides as carbonic
anhydrase inhibitors, J. Enzyme. Inhib. Med. Chem. 34 (2019) 1172–1177.
[28] W.M. Eldehna, G.H. Al-Ansary, S. Bua, A. Nocentini, P. Gratteri, A. Altoukhy,
H. Ghabbour, H.Y. Ahmed, C.T. Supuran, Novel indolin-2-one-based sulfonamides
as carbonic anhydrase inhibitors: Synthesis, in vitro biological evaluation against
carbonic anhydrases isoforms I, II, IV and VII and molecular docking studies, Eur. J.
Med. Chem. 127 (2017) 521–530.
[7] E. Andreucci, J. Ruzzolini, S. Peppicelli, F. Bianchini, A. Laurenzana, F. Carta,
C.T. Supuran, L. Calorini, The carbonic anhydrase IX inhibitor SLC-0111 sensitises
cancer cells to conventional chemotherapy, J. Enzyme. Inhib. Med. Chem. 34
(2019) 117–123.
[8] O. OzensoyGuler, C.T. Supuran, C. Capasso, Carbonic anhydrase IX as a novel
candidate in liquid biopsy, J. Enzyme. Inhib. Med. Chem. 35 (2020) 255–260.
[9] A. Waheed, W.S. Sly, Carbonic anhydrase XII functions in health and disease, Gene
623 (2017) 33–40.
[29] A. Nocentini, M. Ferraroni, F. Carta, M. Ceruso, P. Gratteri, C. Lanzi, E. Masini,
C.T. Supuran, Benzenesulfonamides incorporating flexible triazole moieties are
highly effective carbonic anhydrase inhibitors: synthesis and kinetic, crystal-
lographic, computational, and intraocular pressure lowering investigations, J. Med.
Chem. 59 (2016) 10692–10704.
[10] C.L. Lomelino, C.T. Supuran, R. McKenna, Non-classical inhibition of carbonic an-
hydrase, Int. J. Mol. Sci. 17 (2016) 1150.
[11] C.T. Supuran, How many carbonic anhydrase inhibition mechanisms exist? J.
Enzyme. Inhib. Med. Chem. 31 (2016) 345–360.
[30] A.G. Uslu, T. GurMaz, A. Nocentini, E. Banoglu, C.T. Supuran, B. Calıskan,
Benzimidazole derivatives as potent and isoform selective tumor-associated car-
bonic anhydrase IX/XII inhibitors, Bioorg. Chem. 95 (2020) 103544.
[12] P.S. Thacker, A. Angeli, O.S. Argulwar, P.L. Tiwari, M. Arifuddin, C.T. Supuran,
Design, synthesis and biological evaluation of coumarin linked 1,2,4-oxadiazoles as
selective carbonic anhydrase IX and XII inhibitors, Bioorg. Chem. 98 (2020)
10