Synthesis and biological evaluation of novel 4,7-disubstituted coumarins as selective tumor-associated carbonic anhydrase IX and XII inhibitors

Article abstract of DOI:10.1016/j.bmcl.2021.127877

With an aim to develop novel potential anti-cancer agents we have designed a series of novel 4,7-disubstituted coumarin hybrids synthesis and evaluated for their inhibitory activity against the human carbonic anhydrase isoforms namely CA I, CA II, CA IX and CA XII. The results of CA inhibition clearly showed that the novel 4, 7-disubstituted coumarin hybrids (7a–i & 8a–j) exhibited selective inhibition towards tumor associated isoforms, CA IX and CA XII without inhibiting CA I and CA II isoforms. Among all the compound 8b showed a significant inhibition against hCA IX with a K_i of 0.58 μM whereas, the compound 7c showed a significant inhibition against hCA XII with a K_i of 0.36 μM respectively. All other compounds have shown a good inhibition against hCA IX and hCA XII over hCA I and hCA II within the range of 0.46 to 9.35 μM. Therefore, compound 8b and 7c would be the potential leads for developing selective cytotoxic agents targeting hCA IX and XII.

Full text of DOI:10.1016/j.bmcl.2021.127877

Bioorg. Med. Chem. Lett. 39 (2021) 127877
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of novel 4,7-disubstituted coumarins as
selective tumor-associated carbonic anhydrase IX and XII inhibitors
,
f
,
f
,
,e,*
K. Muni Chandraa , Nerella Sridhar Gouda , Mohammed Arifuddina d, Mallika Alvalaa
,
Ravi Alvalac, Andrea Angelib, Claudiu T. Supuranb
,
*
a Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research (NIPER), Balanagar, Hyderabad 500037, India
b
`
Universita degli Studi di Firenze, Neurofarba Dept., Sezione di Scienze Farmaceutiche e Nutraceutiche, Via Ugo Schiff 6, 50019 Sesto Fiorentino, Florence, Italy
c G. Pulla Reddy College of Pharmacy, Mehdipatnam, Hyderabad, India
d Department of Chemistry, Anwarul Uloom College, 11-3-918, New Malleypally, Hyderabad 500001, T. S., India
e School of Pharmacy & Technology Management, SVKM’S NMIMS (Deemed to be University), Hyderabad Campus, Plot No. B4, Green Industrial Park, Polepally SEZ,
TSIIC, Jadcherla, Telangana 509 301, India
A R T I C L E I N F O
A B S T R A C T
Keywords:
With an aim to develop novel potential anti-cancer agents we have designed a series of novel 4,7-disubstituted
coumarin hybrids synthesis and evaluated for their inhibitory activity against the human carbonic anhydrase
isoforms namely CA I, CA II, CA IX and CA XII. The results of CA inhibition clearly showed that the novel 4, 7-
disubstituted coumarin hybrids (7a–i & 8a–j) exhibited selective inhibition towards tumor associated isoforms,
CA IX and CA XII without inhibiting CA I and CA II isoforms. Among all the compound 8b showed a significant
inhibition against hCA IX with a Ki of 0.58 µM whereas, the compound 7c showed a significant inhibition against
hCA XII with a Ki of 0.36 µM respectively. All other compounds have shown a good inhibition against hCA IX and
hCA XII over hCA I and hCA II within the range of 0.46 to 9.35 µM. Therefore, compound 8b and 7c would be the
potential leads for developing selective cytotoxic agents targeting hCA IX and XII.
4,7-disubstituted coumarin hybrids
Cancer
Carbonic anhydrase
hCAsI, II, IX and XII
Cancer is the second leading cause of death globally. The most
common effective types include lung cancer (1.76 million deaths),
stomach cancer (8.62 deaths), liver cancer (7.83deaths), colorectal
cancer (7.82 deaths), and breast cancer (6.27 deaths) as per the reports
of the WHO. The estimation of new cancer cases in each year is expected
to increase to 30 millionby 2030. The poor selectivity, drug induced
toxicity and tolerance of currently available anticancer agents shows the
necessity to develop novel potential anticancer agents.1
proliferation, growth, metastasis, and angiogenesis etc.4,5 An ideal
cytotoxic agent should selectively inhibit tumor-associated hCAs IX and
XII without inhibiting cytosolic hCAs I and II in order to show potential
cytotoxicity without adverse effects. Therefore, development of new
cytotoxic agents via targeting tumor-associated hCA IX and XII enzymes
would be the effective, and strategic approach in cancer treatment.6
Coumarin belongs to a family of neo flavonoids which are plant
secondary metabolites. Coumarin nucleus has an ability to make non-
covalent interactions through hydrophobic, electrostatic, hydrogen
bonds and, metal coordination with different active sites present in
various proteins and enzymes, there by elicit a diverse range of phar-
macological activities like anti-tuberculosis, antimicrobial, antiviral,
anti-inflammatory, anti-hyperlipidemic, antioxidant antidepressant,
and anticancer activities etc.7 Recently, in our previous studies, we have
reported the synthesis of novel coumarin-triazole hybrids as selective
carbonic anhydrase IX and XII inhibitors, where the active molecule (1)
has shown a significant inhibition against hCA IX with Ki value of 32.7
A total of 16 human carbonic anhydrase (hCA) isoforms are avail-
able, which are belong to α-family of carbonic anhydrases. Out of 16, the
CA I, II, III, VII and XIII are cytosolic, whereas CA IV, IX, XII, XIV, and XV
are transmembrane bound, the CA VIII, X and XI are inactive isoforms,
called as CA-related proteins (CARPs), the CA Va and Vb mitochondrial,
and CA VI secretes from saliva.2,3 Many reports suggested the over-
expression of hCA IX and XII in various cancer cells as they are tumor-
associated, and transmembrane bound enzymes, mainly in hypoxic tu-
mors. The enzymes over-expression mainly contributes in the tumor
* Corresponding authors at: School of Pharmacy & Technology Management, SVKM’S NMIMS (Deemed to be University), Hyderabad Campus, Plot No. B4, Green
Industrial Park, Polepally SEZ, TSIIC, Jadcherla, Telangana 509 301, India.
E-mail addresses: mallikaalvala@yahoo.in (M. Alvala), claudiu.supuran@unifi.it (C.T. Supuran).
f
Equal contribution to the authors.
https://doi.org/10.1016/j.bmcl.2021.127877
Received 17 December 2020; Received in revised form 8 February 2021; Accepted 10 February 2021
Available online 26 February 2021
0960-894X/© 2021 Elsevier Ltd. All rights reserved.

K.M. Chandra et al.
Bioorganic & Medicinal Chemistry Letters 39 (2021) 127877
Fig. 1. Reported coumarin derivatives as selective tumor-associated hCAs IX & XII inhibitors, and rationale for designed compounds (7a–i & 8a–j).
Scheme 1. Synthesis of 4,7-disubstituted coumarin hybrids (7a–i & 8a–j).
nM, and against hCA XII with Ki value of 623.5 nM over CA I and CA II
isoforms.8 We have also reported coumarin-1,3,4-oxadiazole hybrids as
selective carbonic anhydrase IX and XII inhibitors, the active molecule
(2) exhibited good inhibition against hCA XII, with a Ki of 0.16 µM, and
another active molecule (3) showed a significant inhibition against hCA
IX, with a Ki of 2.34 µM respectively.9 Aikaterini et al., reported a novel
6- and 7-substituted coumarins (4) as selective tumor-associated car-
bonic anhydrases IX and XII inhibitors10 (Fig. 1).
clinical drug development studies of the piperazine compounds in small-
animal models by the US National Cancer Institute (NCI) demonstrated
that these compounds had the ability to suppress experimental tumors.
Hence, the ideology towards design, synthesis of novel hybrids
comprising coumarin and piperazine would be a more fruitful approach
for cancer drug discovery.13 Based on the selectivity of coumarin nu-
cleus towards CA IX and XII inhibition, and importance of piperazine
moiety for potential anticancer activity, we have designed and, syn-
thesized a new series of 4,7-disubstituted coumarin hybrids (Fig. 1) and,
evaluated for CA inhibitory activity against the four physiological hCA I,
II, IX and XII enzymes, with acetazolamide (AAZ) as a standard drug.
The target 4,7-disubstituted coumarin hybrids (7a–i & 8a–j) were
synthesized (Scheme 1) by refluxing various substituted benzyl bro-
mides (6a–j) with piperazin-coumarin intermediates (5a & 5b). In the
Piperazine is a six-membered nitrogen-containing heterocyclic ring.
It has been reported to elicit a broad spectrum of pharmacological ac-
tivities viz antidepressant, anthelmintic, antibacterial, antifungal, anti-
mycobacterial, antimalarial, anticonvulsant and anticancer etc.11 In the
last decade a number of piperazine derivatives have been synthesized
and evaluated for their cytotoxic activity.12 Additional preclinical/
2

K.M. Chandra et al.
Bioorganic & Medicinal Chemistry Letters 39 (2021) 127877
Table 1
Inhibition of hCA I, II, IX and XII isoforms with 4,7-disubstituted coumarin hybrids (7a–i & 8a–j) and acetazolamide (AAZ) as a standard drug (Ki-µM).*
Compound
Structure
hCA I
hCAII
hCA IX
hCA XII
0.86
7a
>100
>100
5.52
7b
>100
>100
0.89
3.47
3.44
0.36
7c
>100
>100
7d
7e
>100
>100
7.29
3.85
4.58
2.45
>100
>100
7f
>100
>100
5.39
5.25
4.83
7g
7h
>100
>100
>100
>100
2.66
2.55
7.30
(continued on next page)
3

K.M. Chandra et al.
Bioorganic & Medicinal Chemistry Letters 39 (2021) 127877
Table 1 (continued)
Compound
Structure
hCA I
hCAII
hCA IX
hCA XII
7i
>100
>100
4.55
4.22
8a
8b
>100
>100
>100
>100
4.90
0.58
6.34
4.06
8c
8d
>100
>100
>100
>100
0.85
4.60
4.24
5.79
8e
>100
>100
0.91
0.82
0.68
8f
>100
>100
>100
>100
0.93
3.16
8g
0.46
(continued on next page)
4

K.M. Chandra et al.
Bioorganic & Medicinal Chemistry Letters 39 (2021) 127877
Table 1 (continued)
Compound
Structure
hCA I
hCAII
hCA IX
hCA XII
8h
8i
>100
>100
>100
>100
4.93
0.78
5.57
7.07
8j
>100
>100
4.43
5.50
AAZ
–
0.25
0.012
0.025
0.0057
*
Mean from 3 different assays, by a stopped flow technique (errors were in the range of ±5–10% of the reported values).
first step the resorcinol (1) (1 eq) was treated with ethyl-4-
chloroacetoacetate (2) (1.1 eq) in the presence of catalytic amount of
cold sulphuric acid to give 4-chloromethyl-7-hydroxycoumarin (3).14
The chloro methyl group of (3) was converted to methyl piperazine via
N-Alkylation by treating it with various substituted Phenylpiperazins
(1.1 eq) (4a & 4b) in the presence of catalytic amount of mild base
i The newly synthesized compounds 7a–i & 8a–j had shown no
inhibition against the cytosolic isoforms like hCA I & II (Ki > 100
µM).
ii The target compounds 7a–i & 8a–j exhibited a significant inhi-
bition against the tumor-associated transmembrane isoforms like
hCA IX & XII. All the synthesized compounds showed inhibitory
potencies of <10 µM.
◦
K2CO3 (2.5 eq) in Acetone at 80 C to give different Phenylpiperazins
substituted coumarins (5a & 5b).15 In the final step, a series of
Piperazin-Coumarin derivatives (7a–i & 8a–j) were synthesized using
various substituted benzylbromides (6a–j) (1.1 eq) through O-alkylation
at 7-Hydroxy position of (5a & 5b) in the presence of catalytic amount of
K2CO3 in Acetone at 80 ◦C.16
iii The compounds 7b, 8b, 8c and 8i exhibited low micromolar
inhibitory potencies (Kis) of 0.89, 0.58, 0.85 and 0.78 µM
respectively against hCA IX isoform.
iv The potent inhibition order of 4,7-disubstituted coumarin hybrids
(7a–i & 8a–j) for hCA IX inhibition: 4-bromobenzyl (8b) > 3,5-
dimethyl benzyl (8i) > 3-bromobenzyl (8c) > 4-bromobenzyl of
4-methoxy phenyl piperazine series (7b).
All synthesized compounds (7a–i & 8a–j) were characterized by
spectral techniques viz. [1]H, [13]C NMR and HRMS. The 1H NMR
spectrum of 7a clearly showed characteristic methylene group protons
at 4th position of coumarin ring around δ 3.60 ppm and other methylene
group protons of 7th position of coumarin ring around δ 5.30 ppm
respectively. All the remaining protons showed in the range of δ
2.0–8.70 ppm. 13C NMR spectrum of 7a showed the characteristic
carbonyl and 4-methylene group of coumarin ring around δ 162.0 and δ
60 ppm respectively and all the remaining carbons appeared in the range
of δ 50.0–160.0. The similar pattern was observed for the rest of com-
pounds (7a–i & 8a–j).
v The compounds 7c, 8e, 8f and 8g exhibited low micromolar
inhibitory potencies (Kis) of 0.36, 0.82, 0.68 and 0.46 µM
respectively against hCA XII isoform.
vi The potent inhibition order of 4,7-disubstituted coumarin hybrids
(7a–i & 8a–j) for hCA XII inhibition: 3-bromobenzyl of 4-
methoxy phenyl piperazine series (7c) > 2-fluorobenzyl (8e) >
2-nitrobenzyl (8f) > 4-nitrobenzyl (8g).
vii Rest all the 4,7-disubstituted coumarin derivatives have shown
selective inhibition of hCA IX and XII over hCA I &II in micro-
molar range.
The target 4,7-disubstituted coumarin hybrids (7a–i & 8a–j) were
screened for their potential activities against the four physiological hCA
enzymes, which include the cytosolic (hCA I, II) and the tumor-
associated (hCA IX, XII) enzymes by a stopped-flow CO2hydrase assay,
by taking acetazolamide (AAZ), as a standard drug. The below Structure-
Activity Relationship (SAR) can be obtained from the results of enzyme
inhibition of 4,7-disubstituted coumarin hybrids (7a–i & 8a–j) as shown
in Table 1:
The inhibition assay of selected CA isozymes was performed using
SX.18V-R Applied Photophysics (Oxford, UK) stopped flow instru-
ment.17 10 mM Hepes (pH 7.4) as a buffer, with Phenol Red (at a con-
centration of 0.2 mM) as an indicator, 0.1 M Na2SO4 or NaClO4 (To
maintain constant the ionic strength; these anions are not inhibitory in
the used concentration), following the CA-catalyzed CO2 hydration re-
action for a period of 5–10 s. Saturated CO2 solutions in water at 25 ◦C
5

K.M. Chandra et al.
Bioorganic & Medicinal Chemistry Letters 39 (2021) 127877
Fig. 2A. 3D ligand interaction diagram of compound 7c with the ligand binding domain of hCA IX (A) and hCA XII (B) (PDB ID: 4BCW and 1JD0 respectively).
Fig. 2B. 3D ligand interaction diagram of compound 8b with the ligand binding domain of hCA IX (A) and hCA XII (B) (PDB ID: 4BCW and 1JD0 respectively).
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. 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 re-
ported earlier, and represent the mean from at least three different de-
terminations. All CA isozymes used here were recombinant proteins
obtained as reported earlier by our group.18–20
Thr199 with the bromo moiety and other hydrophobic interactions are
observed. In a similar fashion the docking results in the hCA XII also
showed good interactions with the binding site residues as shown in
Figs. 2A and 2B. The 7c molecule showed a metal coordination inter-
–
action between the C O of coumarin with zinc ion complexed by the
–
histidine residues (His 94,96,119) and π-π stacking interactions of the
methoxyphenyl ring with the Trp5 and His64. Likewise, the 8b also
showed the metal coordination interaction along with the aromatic
hydrogen bonding interactions and other hydrophobic interactions with
the binding site residues were observed.22,23
In the present study we have reported the synthesis of novel 4,7-
disubstituted coumarin hybrids (7a–i & 8a–j) targeting the tumor-
associated transmembrane hCA IX and XII enzymes. The synthesized
compounds (7a–i & 8a–j) were screened against four physiological hCA
enzymes include the cytosolic hCAs I & II and the tumor-associated
transmembrane hCAs IX & XII. All the compounds have shown a sig-
nificant lower to sub micromolar inhibition against hCA IX and hCA XII
over hCA I and hCA II with Ki value in the range of 0.46 µM to 9.35 µM.
Among all the compound 8b and 7c would be the potential leads for
developing selective cytotoxic agents by providing hCA IX and XII in-
hibition as a novel mechanism of action.
Molecular docking calculations were performed to further deduce
the interactions of the molecules 7c and 8b with the binding site of the
human carbonic anhydrases IX and XII using the Glide module of
Schrodinger suite.21 The 3D crystal co-ordinates of hCA IX and hCA XII
were obtained from the protein data bank (PDB ID: 4BCW and 1JD0
respectively). The 2D and 3D ligand interaction diagrams of compound
7c and 8b are shown in Figs. 2A an 2B. Compound 7c showed hydrogen
–
bonding interaction between NH of His94 acting as donor and the C
O
–
of the coumarin ring acting as acceptor and various hydrophobic in-
teractions stabilize the docking pose of the 7c in the binding site.
Whereas, the 8b showed a halogen bonding interaction with NH of
6

K.M. Chandra et al.
Bioorganic & Medicinal Chemistry Letters 39 (2021) 127877
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Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
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Acknowledgements
The authors are thankful to the National Institute of Pharmaceutical
Education and Research (NIPER), Balanagar, Hyderabad & Department
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Appendix A. Supplementary data
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