Design, synthesis and biological evaluation of coumarin-3-carboxamides as selective carbonic anhydrase IX and XII inhibitors

Article abstract of DOI:10.1016/j.bioorg.2019.02.004

A series of novel 7-hydroxycoumarin-3-carboxamides was synthesized by the reaction of 7-hydroxy-2-oxo-2H-chromene-3-carboxylic acid with various substituted aromatic amines. The newly synthesized compounds were 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 show that the newly synthesized 7-hydroxycoumarin-3-carboxamides (4a-n) exhibited selective inhibition of the tumor associated isoforms, CA IX and CA XII over CA I and II isoforms. The inhibition constants ranged from sub micromolar to low micromolar. Amongst all the compounds tested, compound 4m was the most effective inhibitor exhibiting sub micromolar potency against both hCA IX and hCA XII, with a K_i of 0.2 μM. Therefore, it can be anticipated that compound 4m can serve as a lead for development of anticancer therapy by exhibiting a novel mechanism of action. The binding modes of the most potent compounds within hCA IX and XII catalytic clefts were investigated by docking studies.

Full text of DOI:10.1016/j.bioorg.2019.02.004

Accepted Manuscript
Design, synthesis and biological evaluation of coumarin-3-carboxamides as se-
lective carbonic anhydrase IX and XII inhibitors
Pavitra S. Thacker, Mallika Alvala, Mohammed Arifuddin, Andrea Angeli,
Claudiu T. Supuran
PII:
S0045-2068(18)31426-3
DOI:
https://doi.org/10.1016/j.bioorg.2019.02.004
YBIOO 2779
Reference:
Bioorganic Chemistry
To appear in:
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Revised Date:
Accepted Date:
6 December 2018
29 January 2019
3 February 2019
Please cite this article as: P.S. Thacker, M. Alvala, M. Arifuddin, A. Angeli, C.T. Supuran, Design, synthesis and
biological evaluation of coumarin-3-carboxamides as selective carbonic anhydrase IX and XII inhibitors, Bioorganic
Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.02.004
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Design, synthesis and biological evaluation of coumarin-3-carboxamides as
selective carbonic anhydrase IX and XII inhibitors
Pavitra S. Thackera, Mallika Alvalaa, Mohammed Arifuddina*, Andrea Angeli,b and Claudiu T.
Supuranb *
aDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education and
Research (NIPER), Balanagar, Hyderabad 500037, India
bUniversità degli Studi di Firenze, Neurofarba Dept., Sezione di Scienze Farmaceutiche e
Nutraceutiche, Via Ugo Schiff 6, 50019 Sesto Fiorentino, Florence, Italy
Abstract
A series of novel 7-hydroxycoumarin-3-carboxamides was synthesized by the reaction of 7-
hydroxy-2-oxo-2H-chromene-3-carboxylic acid with various substituted aromatic amines. The
newly synthesized compounds were 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 show that the newly synthesized 7-hydroxycoumarin-
3-carboxamides (4a-n) exhibited selective inhibition of the tumor associated isoforms, CA IX
and CA XII over CA I and II isoforms. The inhibition constants ranged from sub micromolar to
low micromolar. Amongst all the compounds tested, compound 4m was the most effective
inhibitor exhibiting sub micromolar potency against both hCA IX and hCA XII, with a Ki of 0.2
µM. Therefore, it can be anticipated that compound 4m can serve as a lead for development of
anticancer therapy by exhibiting a novel mechanism of action. The binding modes of the most
potent compounds within hCA IX and XII catalytic clefts were investigated by docking studies.
Keywords: Cancer, Carbonic anhydrase, 7-hydroxycoumarin-3-carboxamides, Isoforms IX and
XII, Hypoxic tumors
____________________
Corresponding authors
Mohammed Arifuddina* arif@niperhyd.ac.in Prof. Claudiu T. Supuran claudiu.supuran@unifi.it

1. Introduction
Cancer can be defined as a condition arising due to the uncontrolled growth and malignancy,
which in turn arise due to aberrations in normal tissue progenitor and stem cells [1]. The
mechanisms underlying cancer have been deeply studied till the genetic level, according to
which, cancer can be caused due to dysfunctional oncogenes, tumor suppressor genes or
microRNA genes [2]. According to Hanahan and Weinberg, cancer is characterized by several
signs or “hallmarks” which include sustained proliferative signaling, evasion of growth
suppressors, resistance to cell death, induction of replicative immortality, induction of
angiogenesis and activation of invasion and metastasis [3]. According to GLOBOCAN, as of
2018, about 18.1 million new cases were diagnosed and about 9.6 million people have
succumbed to the deadly disease [4].
Carbonic anhydrases (CAs, EC 4.2.1.1) are a superfamily of ubiquitous metalloenzymes,
which play a catalytic role in the reversible hydration of CO2 to bicarbonate and proton. Their
presence is omnipotent across all biological species, ranging from plants to animals. CAs are
distributed into seven families, which are genetically distinct, on their own. These seven families
are named as: α, β, γ, δ, ζ, η and θ. CAs are considered to be one of the most efficient
physiological catalysts present in the living species, eliciting a wide array of functions [5-7].
The human carbonic anhydrases (hCAs) primarily belong to the α-family of carbonic
anhydrases. This family of CA is, in turn, divided into 16 isoforms, which exhibit distinct
molecular attributes, protein structure, kinetics, localization and catalytic behavior. The isoforms,
with their localizations, can be listed as follows: CA I, CA II, CA III, CA VII and CA XIII are
cytosolic, CA IV, CA IX, CA XII, CA XIV and CA XV are transmembrane bound, CA Va and
Vb mitochondrial and CA VI secreted in body fluids like saliva and colostrum. The remaining
three isoforms, namely CA VIII, CA X and CA XI are catalytically inactive isoforms, also called
as CA-related proteins (CARPs). In recent years, various carbonic anhydrase inhibitors have
been reported [8,9].
A plethora of research is ongoing currently in order to facilitate the treatment of cancer. As
mentioned above, hCAs IX and XII are transmembrane bound, tumor-associated enzymes. In

particular, hCA IX is strongly overexpressed in a variety of solid tumors, mainly hypoxic
tumors, with a limited expression in normal cells. It’s overexpression contributes in the survival
and metastasis of tumor cells due to its property of acidifying the extracellular matrix. In
addition, it is also implied in promoting chemoresistance of particular weakly basic anticancer
drugs. Its presence in hypoxic tumors makes it an attractive marker and drug target for hypoxic
tumors and metastatic hypoxic tumors. CA IX inhibition can mainly be achieved via three ways:
(i) Usage of monoclonal antibodies; (ii) Designing novel small molecule inhibitors and (iii)
formulating nanoparticles derivatized with small molecule inhibitors. Similarly, hCA XII is also
overexpressed primarily in the case of tumors wherein its expression is controlled by hypoxia
and estrogen receptors [10-14].
Coumarins are basically 2H-chromenes, which means they are actually bicyclic oxygen
heterocycles, arising from the fusion of a benzene ring with 5,6 positions of a 2H-pyran ring
system [15]. Natural as well as synthetic coumarins have been implicated in a variety of
biological indications namely, anticancer agents, antimicrobial and antiviral agents, anti-
inflammatory and antioxidant agents, anti-Alzheimer agents, aldose reductase inhibitors,
antithrombotic agents, in various CNS disorders, alpha-amylase inhibitors and thymidine
phosphorylase inhibitors [16]. In the recent years coumarin and its derivatives are reported as
inhibitors of CAs by a non-classical mechanism i.e., by occlusion of the entrance leading to the
enzyme’s active site. This is in contrast to the conventional CA inhibitory mechanism of binding
within the active site metal ion. It is reported that umbelliferone (7-hydoxy coumarin) and it’s
analogues have the capacity of selectively inhibiting hCA(s) IX and/or XII, over CA I and CA II
[9,17-20].
In view of the selective nature of coumarin towards CA IX and XII, it is decided to synthesize
a series of 7-hydroxy coumarin-3- carboxamides (Fig 1) derivatives which have not been
reported earlier as carbonic anhydrase inhibitors. Coumarin-3-carboxamide derivatives have
been previously reported as FXIIa inhibitors and as antiproliferative agents [21] but not for
carbonic anhydrase inhibition. Thus, novel 7-hydroxycoumarin-3-carboxamides (4a-n) were
synthesized. The newly synthesized compounds were evaluated for CA inhibitory activity

against the four major CA isoforms, CA I, II, IX and XII, with the sulphonamide, acetazolamide
(AAZ) being utilized as a drug standard.
Fig 1 General Structure of the designed compounds
2. Result and Discussion
2.1 Chemistry
The current design of experiment (DOE) was aimed at designing molecules which target
tumor-specific carbonic anhydrase isoforms IX and XII. Several coumarin analogues were
shown to be specifically targeting these two isoforms as per previously reported literature.
Hence, we have designed simple coumarin carboxamides for selectively targeting carbonic
anhydrases IX and XII.
The synthesis of the designed 7-hydroxycoumarin-3-carboxamides (4a-n) was performed
according to the general synthetic scheme as illustrated in Scheme 1. The intermediate 7-
hydroxycoumarin-3-carboxylic ester (2) and 7-hydroxycoumarin-3-carboxylic acid (3) were
synthesized according to previously reported literature methods [22,23]. The 7-
hydroxycoumarin-3-carboxylic acid (3) was subjected to acid-amine coupling using various
aromatic amines to afford the corresponding 7-hydroxycoumarin-3-carboxamides (4a-n) (Table
1).

Scheme 1: General synthetic scheme for the synthesis of 7-hydroxy coumarin-3-carboxamides
(4a-n). Reagents and conditions: (i) Diethyl malonate, piperidine, ethanol, reflux, 12-15 hrs,
yield 65% (ii) 2 N NaOH solution, rt, 14 hrs, yield 80% (iii) Substituted aromatic amines,
HATU, DIPEA, anhy. DMF, 0ᵒ-rt, 18-20 hrs, yield 23-90%.
2.2 Carbonic anhydrase inhibition
The newly synthesized novel 7-hydroxycoumarin-3-carboxamides (4a-n) were tested for their
activity against the four physiologically and pharmacologically relevant isoforms, namely, the
cytosolic hCA I and II and the tumor-associated isoforms hCA IX and XII by a stopped-flow
CO2 hydrase assay. The most common clinically used sulphonamide, acetazolamide (AAZ), was
employed as a standard drug. The following structure-activity relationship can be inferred from
the inhibition data of 7-hydroxycoumarin-3-carboxamides (4a-n) as shown in Table 1:
i.
The cytosolic isoforms hCA I and hCA II were not inhibited by the compounds 4a-4n (Ki
> 100 µM).
ii.
The compounds 4a-n showed diverse inhibitory profiles against the transmembrane
tumor-associated isoform hCA IX. All of the compounds elicited inhibitory potencies of
< 10µM with compounds 4b, 4m and 4n eliciting sub micromolar inhibitory potencies
(Kis) of 0.6, 0.2 and 0.3 µM respectively. All the three potent compounds incorporate a
halogen atom in various positions of the aromatic amine ring (variable part) of the 7-
hydroxycoumarin-3-carboxamides.
iii.
iv.
The other transmembrane tumor-associated isoform, hCA XII, was also inhibited in a
variable and diverse manner by the synthesized compounds, 4a-4n, with Kis below 10µM
for all the compounds. Among all the compounds, compound 4m, bearing a chlorine
atom at the para position of the aromatic amine ring (variable part) showed promising
inhibitory activity with a Ki value of 0.2 µM.
All the derivatives exhibited selective inhibition of hCA IX and XII as compared to hCA
I &II in micromolar range.

Table 1. Inhibition of hCA isoforms I, II, IX and XII with compounds 4a-n and acetazolamide
(AAZ) as standard inhibitor.
Compound
4a
Structure
hCA I hCAII hCA IX hCA XII
>100
>100
>100
>100
2.4
7.9
7.7
4b
4c
0.6
>100
>100
7.1
7.5
>100
>100
>100
>100
0.7
8.1
9.0
8.9
4d
4e
>100
>100
>100
>100
9.2
9.1
8.4
8.4
4f
4g

>100
>100
8.4
7.6
4h
>100
>100
>100
>100
9.1
9.4
9.7
8.7
4i
4j
>100
>100
9.1
9.6
4k
>100
>100
>100
>100
9.1
9.1
4l
4m
0.2
0.2
>100
0.25
>100
0.012
7.9
4n
0.3
0.026
0.006
AAZ
* Mean from 3 different assays, by a stopped flow technique (errors were in the range of  5-10
% of the reported values).

2.3 Docking studies
With a view to shed more light on the enzyme inhibition shown by the compounds and to
study their interactions, docking studies of the most active compounds with hCA IX (PDB 3IAI)
and hCA XII (PDB 4WW8) binding site has been performed. Compounds 4b, 4m and 4n which
had shown sub micromolar inhibitory profiles against hCA IX were docked against hCA IX and
compound 4m which had shown sub micromolar inhibitory profile against hCA XII was docked
against hCA XII. Ligand interaction diagram (Fig 2) shows that all the three compounds
exhibited interaction with Zn262 of hCA IX through the ionized hydroxyl group present in the
coumarin moiety. These results are very different from the experimental, X-ray crystallographic
results [17,18], which showed that coumarins bound to other CA isoforms, such as CA II for
example, at the entrance of the cavity by hydrolysis of the lactone ring and conversion to 2-
hydroxy cinnamic acids. Furthermore, there are also differences with the binding of phenols,
which anchor to the zinc-coordinated water molecule. Thus, we consider the binding mode
reported in Fig. 2 as a hypothetical, not yet experimentally confirmed inhibition mechanism
which warrants further studies. Probably the bulky nature of the groups present in position 3 of
the coumarin ring interferes with the hydrolysis of the lactone ring affording thus a novel CA
inhibitory mechanism for these derivatives.
In addition, the coumaryl oxygen of compound 4b showed hydrogen bond interaction with key
active site residue Gln92 and π- π stacking was observed between coumaryl ring system and
His94 in case of compound 4n. The most potent compound 4m showed interactions with Thr199
and Gln 92, in addition to Zn262. Also, other major active site residues like Thr198, Pro200 and
His94 are seen lying in the periphery of the compound. These features allow compound 4m to
exhibit potent sub micromolar inhibition in comparison to other analogues. In case of hCA XII,
compound 4m is shown to interact with Zn301, Thr198 and Asn64, which contributes to its
potency against hCA XII. We stress again, these docking results are not confirmed
experimentally for the moment.

(I)
(II)
(III)
(IV)

Fig 2: - (I) Docking pose of compound 4b with hCA IX (II) Docking pose of compound 4m
with hCA IX (III) Docking pose of compound 4n with hCA IX (IV) Docking pose of compound
4m with hCA XII. The pink arrows in the figure indicate the hydrogen bonding interactions and
the green lines indicate π - π stacking. The compounds coordinate to the Zn ion, although this
binding mode was not confirmed experimentally for other phenols.
3. Conclusion
In conclusion, we have reported the synthesis of a new series of 7-hydroxycoumarin-3-
carboxamide derivatives (4a-n), which have been specifically designed to target the
transmembrane tumor-associated isoforms, hCA IX and XII. The novel 7-hydroxycoumarin-3-
carboxamides (4a-n) were screened against the four physiologically and pharmacologically
relevant 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 exhibited low micromolar to sub
micromolar inhibitory profiles for both the isoforms, particularly for CA IX, with Kis in the
range of 0.2-9.2 µM for CA IX and 0.2-9.7 for CA XII. The newly synthesized 7-
hydroxycoumarin-3-carboxamides (4a-n) proved to be ineffective against both the cytosolic
isoforms, thereby validating our hypothesis of selective inhibition of the two tumor-associated
enzymes. Also, as CA IX has been validated as a target for metastatic hypoxic tumors, the
compound 4m can be considered as a potential lead candidate for the design of novel molecules
which target cancer by a different mechanism.
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
1
uncorrected. H and 13C NMR spectra were recorded using Bruker Avance 500MHz and
125MHz 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 70eV.

4.1.1 Synthesis of ethyl 7-hydroxy-2-oxo-2H-chromene-3-carboxylate (2)
To a stirred solution of 2,4-dihydroxybenzaldehyde (2g, 14.47 mmol) in anhydrous ethanol
(20 ml) was added diethyl malonate (2.3g, 14.47 mmol) and piperidine (1.5 ml). The resulting
solution was allowed to reflux and the progress of the reaction was monitored by TLC. Upon
completion of the reaction on TLC, the reaction solvent was removed under reduced pressure
and the crude residue was subjected to column chromatography using silica gel 60-120 mesh as
stationary phase and EtOAc in Hexane (0 percent to 20 percent) as the mobile phase to afford the
title compound as a yellowish fluffy solid (Yield 65%).
4.1.2 Synthesis of 7-hydroxy-2-oxo-2H-chromene-3-carboxylic acid (3)
Intermediate 2 (2.37g, 10.12 mmol) was dissolved in 2 N NaOH solution (40 ml). The
resulting solution was allowed to stir at rt overnight. On completion of the reaction on TLC, the
solution was acidified using 2 N HCl solution. The resulting precipitates were filtered off,
subsequently washed with water a few times and dried to afford the title compound as a yellow
solid (Yield 90%).
4.1.3 Synthesis of 7-hydroxy-2-oxo-N-phenyl-2H-chromene-3-carboxamides (4a-4n)
To a stirred solution of Intermediate 3 (0.1g, 0.48 mmol) in anhydrous DMF at 0ᵒ C, under
nitrogen, was added HATU (0.276g, 0.72 mmol). The resulting solution was stirred at 0ᵒ C for
half an hour. Thereafter, the corresponding amine (1 eq.) and DIPEA (0.187g, 1.4 mmol) were
added and the resulting solution was allowed to stir at rt for overnight. On completion of the
reaction as checked by TLC, the reaction mixture was dumped in ice water and extracted twice
with ethyl acetate. The ethyl acetate layers were combined, washed with brine, dried over
sodium sulphate and subjected to rotary evaporation under reduced pressure. The crude residue
was subjected to column chromatography using silica gel 60-120 mesh to afford the final
carboxamides 4a-4n.
7-hydroxy-2-oxo-N-phenyl-2H-chromene-3-carboxamide (4a) Yield: 55%; Color: Yellow solid;
mp: 276-278ᵒC; 1H NMR (500 MHz, DMSO-d6) δ 11.14 (s, 1H), δ 10.65 (s, 1H), δ 8.88 (s, 1H),
δ 7.86 (d, J = 8.6 Hz, 1H), 7.71 (d, J = 7.9 Hz, 2H), 7.38 (t, J = 7.8 Hz, 2H), 7.14 (t, J = 7.3 Hz,
1H), 6.91 (dd, J = 8.6, 2.0 Hz, 1H), 6.85 (d, J = 1.7 Hz, 1H); 13C NMR (125 MHz, DMSO-d6) δ
164.44, 161.79, 160.65, 156.87, 148.85, 138.53, 132.62, 129.46, 124.58, 120.33, 115.03, 114.60,
111.72, 102.39; HRMS (ESI): m/z calcd for [M+H]+ C16H12NO4 282.0766; found 282.0733.

N-(4-bromophenyl)-7-hydroxy-2-oxo-2H-chromene-3-carboxamide (4b) Yield: 23%; Color:
Yellow solid; mp: 327-329ᵒC; 1H NMR (500 MHz, DMSO-d6) δ 11.27 (s, 1H), δ 10.70 (s, 1H), δ
8.86 (s, 1H), δ 7.86 (d, J = 8.6 Hz, 1H), 7.70 (d, J = 8.5 Hz, 2H), 7.55 (d, J = 8.5 Hz, 2H), 6.91
13
(d, J = 8.5 Hz, 1H), δ 6.84 (s, 1H); C NMR (125 MHz, DMSO-d6) δ 164.66, 161.65, 160.86,
156.92, 148.94, 137.89, 132.65, 132.22, 122.33, 116.21, 115.10, 114.33, 111.62, 102.41; HRMS
(ESI): m/z calcd for [M+H]+C16H11BrNO4 359.9871; found 359.9826.
7-hydroxy-2-oxo-N-(3,4,5-trimethoxyphenyl)-2H-chromene-3-carboxamide (4c) Yield: 49%;
Color: Yellow solid; mp: 238-240ᵒC; 1H NMR (500 MHz, DMSO-d6) δ 11.17 (s, 1H), δ 10.55 (s,
1H), δ 8.85 (s, 1H), δ 7.83 (d, J = 8.6 Hz, 1H),δ 7.11 (s, 2H), 6.91 (dd, J = 8.6, 2.2 Hz, 1H), 6.84
13
(d, J = 2.0 Hz, 1H), δ 3.79 (s, 6H), δ 3.65 (s, 3H); C NMR (125 MHz, DMSO-d6) δ 164.69,
161.75, 160.53, 156.90, 153.36, 148.80, 134.59, 132.58, 115.12, 114.29, 111.63, 102.42, 98.34,
92.12, 60.59, 56.35; HRMS (ESI): m/z calcd for [M+H]+ C19H18NO7 372.1083; found 372.1050.
N-(4-fluorophenyl)-7-hydroxy-2-oxo-2H-chromene-3-carboxamide (4d) Yield: 90%; Color:
Yellow solid; mp: 293-295ᵒC; 1H NMR (500 MHz, DMSO-d6) δ 11.14 (s, 1H), δ 10.64 (s, 1H), δ
8.86 (s, 1H), δ 7.85 (d, J = 8.6 Hz, 1H), 7.75 (dd, J = 8.8, 5.0 Hz, 2H), 7.21 (t, J = 8.8 Hz, 2H),
13
6.91 (dd, J = 8.5, 2.0 Hz, 1H), 6.84 (d, J = 1.8 Hz, 1H); C NMR (125 MHz, DMSO-d6) δ
164.64, 161.68, 159.92, 158.01, 156.91, 148.75, 134.95, 132.61, 122.30, 115.92, 115.09, 114.37,
111.61, 102.40; HRMS (ESI): m/z calcd for [M+H]+C16H11FNO4 300.0672; found 300.0638.
7-hydroxy-N-(4-methoxyphenyl)-2-oxo-2H-chromene-3-carboxamide (4e) Yield: 56%; Color:
Yellow solid; mp: 271-273ᵒC; 1H NMR (500 MHz, DMSO-d6) ) δ 11.13 (s, 1H), δ 10.52 (s, 1H),
δ 8.86 (s, 1H), δ 7.85 (d, J = 8.6 Hz, 1H), 7.64 (d, J = 8.9 Hz, 2H), 6.95 (d, J = 9.0 Hz, 2H), 6.91
(dd, J = 8.6, 2.1 Hz, 1H), 6.84 (d, J = 1.9 Hz, 1H), 3.76 (s, 3H) ; 13C NMR (125 MHz, DMSO) δ
164.57, 161.83, 160.24, 156.85, 156.33, 156.30, 148.57, 132.53, 131.67, 121.88, 115.09, 114.59,
111.65, 102.39, 55.71.HRMS (ESI): m/z calcd for [M+H]+ C17H14NO5 312.0872; found
312.0872
7-hydroxy-N-(4-hydroxyphenyl)-2-oxo-2H-chromene-3-carboxamide (4f) Yield: 34%; Color:
Yellow solid; mp: 333-335ᵒC; 1H NMR (500 MHz, DMSO-d6) δ 11.02 (s, 1H), δ 10.44 (s, 1H), δ
9.34 (s, 1H), δ 8.85 (s, 1H), δ 7.85 (d, J = 8.6 Hz, 1H), 7.50 (d, J = 8.9 Hz, 2H), 6.91 (dd, J = 8.6,
13
2.2 Hz, 1H), 6.84 (d, J = 2.0 Hz, 1H), 6.78 – 6.75 (m, 2H); C NMR (125 MHz, DMSO-d6) δ

164.34, 161.86, 159.99, 156.78, 154.52, 148.46, 132.50, 130.19, 122.02, 115.83, 115.00, 114.64,
111.71, 102.36; HRMS (ESI): m/z calcd for [M+Na]+ C16H12NO5 320.0535; found 320.0489.
7-hydroxy-2-oxo-N-(p-tolyl)-2H-chromene-3-carboxamide (4g) Yield: 66%; Color: Yellow solid;
mp: 299-301ᵒC; 1H NMR (500 MHz, DMSO-d6) δ 11.10 (s, 1H), δ 10.58 (s, 1H), δ 8.86 (s, 1H),
δ 7.85 (d, J = 8.3 Hz, 1H), 7.59 (d, J = 7.5 Hz, 2H), 7.18 (d, J = 7.4 Hz, 2H), 6.91 (d, J = 8.0 Hz,
13
1H), 6.84 (s,1H); C NMR (125 MHz, DMSO-d6) δ 164.51, 161.84, 160.41, 156.85, 148.71,
136.04, 133.63, 132.58, 129.84, 120.28, 115.05, 114.50, 111.68, 102.38, 20.94; HRMS (ESI):
m/z calcd for [M+H]+ C17H14NO4 296.0923; found 296.0883.
7-hydroxy-2-oxo-N-(4-phenoxyphenyl)-2H-chromene-3-carboxamide (4h) Yield: 76%; Color:
Yellow solid; mp: 253-255ᵒC; 1H NMR (500 MHz, DMSO-d6) δ 11.13 (s, 1H), δ 10.64 (s, 1H), δ
8.87 (s, 1H), δ 7.86 (d, J = 8.6 Hz, 1H), 7.74 (d, J = 9.0 Hz, 2H), 7.42 – 7.37 (m, 2H), 7.13 (t, J =
7.4 Hz, 1H), 7.05 (d, J = 8.9 Hz, 2H), 7.01 (d, J = 7.8 Hz, 2H), 6.91 (dd, J = 8.6, 2.2 Hz, 1H),
13
6.84 (d, J = 2.1 Hz, 1H); C NMR (125 MHz, DMSO-d6) δ 164.57, 161.75, 160.55, 157.59,
156.89, 153.08, 148.74, 134.33, 132.60, 130.47, 123.67, 122.11, 119.90, 118.64, 115.09, 114.47,
111.66, 102.40; HRMS (ESI): m/z calcd for [M+H]+ C22H16NO5 374.1028; found 374.0981.
7-hydroxy-2-oxo-N-(4-phenoxyphenyl)-2H-chromene-3-carboxamide (4i) Yield: 62%; Color:
Yellow solid; mp: 256-258ᵒC; 1H NMR (500 MHz, DMSO-d6)δ 11.13 (s, 1H), δ 10.59 (s, 1H), δ
8.88 (s, 1H), δ 7.86 (d, J = 8.7 Hz, 1H), 7.62 (d, J = 8.5 Hz, 2H), 7.25 (d, J = 8.5 Hz, 2H), 6.92
(dd, J = 8.6, 2.2 Hz, 1H), 6.85 (d, J = 2.1 Hz, 1H), δ 2.88 (septet, 1H), δ 1.21 (d, J = 6.9 Hz, 6H);
13C NMR (125 MHz, DMSO-d6) δ 164.46, 161.84, 160.44, 156.86, 148.77, 144.76, 136.28,
132.60, 127.18, 120.43, 115.04, 114.55, 111.71, 102.39, 33.37, 24.37; HRMS (ESI): m/z calcd
for [M+H]+ C19H18NO4 324.1236; found 324.1177.
7-hydroxy-N-mesityl-2-oxo-2H-chromene-3-carboxamide (4j) Yield: 34%; Color: Yellow solid;
mp: 307-309ᵒC; 1H NMR (500 MHz, DMSO-d6) δ 11.06 (s, 1H), δ 9.88 (s, 1H), δ 8.85 (s, 1H), δ
7.84 (d, J = 8.6 Hz, 1H), 6.94 – 6.91 (m, 2H), 6.90 (d, J = 2.2 Hz, 1H), 6.84 (d, J = 2.0 Hz, 1H),
13
)δ 2.25 (s, 3H), δ 2.15 (s, 6H); C NMR (125 MHz, DMSO-d6) δ 164.26, 161.79, 160.73,
156.88, 148.70, 136.14, 134.95, 132.48, 132.41, 128.82, 114.92, 114.63, 111.63, 102.35, 20.96,
18.59; HRMS (ESI): m/z calcd for [M+H]+ C19H18NO4 324.1236; found 324.1211.

7-hydroxy-2-oxo-N-(3-(trifluoromethyl)phenyl)-2H-chromene-3-carboxamide (4k) Yield: 35%;
Color: Yellow solid; mp: 294-296ᵒC; 1H NMR (500 MHz, DMSO-d6) δ 11.18 (s, 1H), δ 10.84 (s,
1H), δ 8.87 (s, 1H), δ 8.25 (s, 1H), δ 7.90 (d, J = 8.2 Hz, 1H), 7.86 (d, J = 8.6 Hz, 1H), 7.61 (t, J
= 8.0 Hz, 1H), 7.49 (d, J = 7.8 Hz, 1H), 6.92 (dd, J = 8.6, 2.2 Hz, 1H), 6.85 (d, J = 2.1 Hz, 1H);
13C NMR (125 MHz, DMSO-d6) δ 164.66, 161.46, 161.34, 156.98, 149.08, 139.31, 132.71,
130.61, 130.00, 124.18, 123.44, 120.93, 116.63, 115.09, 114.36, 111.61, 102.43; HRMS (ESI):
m/z calcd for [M+H]+ C17H11F3NO4 350.0640; found 350.0624.
N-(2,4-dimethylphenyl)-7-hydroxy-2-oxo-2H-chromene-3-carboxamide (4l) Yield: 75%; Color:
Yellow solid; mp: 311-313ᵒC; 1H NMR (500 MHz, DMSO-d6) δ 11.15 (s, 1H), δ 10.57 (s, 1H), δ
8.93 (s, 1H), δ 8.05 (d, J = 8.2 Hz, 1H), 7.88 (d, J = 8.6 Hz, 1H), δ 7.07 (s, 1H), 7.03 (d, J = 8.1
Hz, 1H), 6.92 (dd, J = 8.5, 1.8 Hz, 1H), δ 6.85 (s, 1H), δ 2.29 (s, 3H), δ 2.26 (s, 3H); 13C NMR
(125 MHz, DMSO-d6) δ 164.52, 162.38, 160.10, 156.84, 149.11, 134.35, 133.74, 132.68, 131.37,
129.53, 127.28, 121.62, 115.09, 114.14, 111.78, 102.35, 20.91, 17.96; HRMS (ESI): m/z calcd
for [M+H]+ C18H16NO4 310.1079; found 310.1060.
N-(4-chlorophenyl)-7-hydroxy-2-oxo-2H-chromene-3-carboxamide (4m) Yield: 25%; Color:
Yellow solid; mp: 323-325ᵒC; 1H NMR (500 MHz, DMSO-d6) δ 11.16 (s, 1H), δ 10.71 (s, 1H), δ
8.86 (s, 1H), δ 7.86 (d, J = 8.3 Hz, 1H), 7.76 (d, J = 8.0 Hz, 2H), 7.43 (d, J = 8.0 Hz, 2H), 6.91
13
(d, J = 8.0 Hz, 1H), δ 6.84 (s, 1H); C NMR (125 MHz, DMSO-d6) δ 164.63, 161.63, 160.88,
156.93, 148.91, 137.49, 132.66, 129.32, 128.18, 121.99, 115.10, 114.40, 111.64, 102.41; HRMS
(ESI): m/z calcd for [M+H]+C16H11ClNO4 316.0377; found 316.0346.
N-(3-fluorophenyl)-7-hydroxy-2-oxo-2H-chromene-3-carboxamide (4n) Yield: 78%; Color:
Yellow solid; mp: 304-306ᵒC; 1H NMR (500 MHz, DMSO-d6) δ 11.18 (s, 1H), δ 10.77 (s, 1H), δ
8.88 (s, 1H),δ 7.87 (d, J = 8.7 Hz, 1H), 7.78 (d, J = 12.6 Hz, 1H),δ 7.43-7.40 (m, 2H), 6.97 (m,
1H), 6.92 (dd, J = 8.6, 2.2 Hz, 1H), 6.85 (d, J = 2.0 Hz, 1H); 13C NMR (125 MHz, DMSO-d6) δ
164.65, 163.64, 161.60, 161.09, 156.96, 149.04, 132.70, 131.03, 124.01, 116.25, 115.11, 114.36,
111.65, 107.37, 107.16, 102.43; ; HRMS (ESI): m/z calcd for [M+H]+C16H11FNO4 300.0672;
found 300.0650.

4.1.4 CA inhibition
An SX.18V-R Applied Photophysics (Oxford, UK) stopped flow instrument has been used to
assay the catalytic inhibition of various CA isozymes [24]. 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, 0.1M Na2SO4 or NaClO4 (for maintaining 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 25ᵒC 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 [25-27].
4.2 Computational Studies
The 3D crystal coordinates of hCA IX (3IAI) [28] and hCA XII (4WW8) [29] were
downloaded from the PDB and were prepared according to protocols specified in Protein
Preparation Wizard [30] of Schrödinger suite 2017. Briefly, hydrogens were added, bond orders
were assigned, water molecules were removed within 5 Å distance and minimization done using
OPLS-2005 force field. Compounds 4b,4n,4m were drawn and converted to 3D using LigPrep
[31], module of Schrödinger suite 2017. Further Grid has been generated with 10 Å around co-
crystal and flexible ligand docking protocol has been performed by Glide [32], module of
Schrödinger suite 2017. Ten conformations were generated for each compound and based on
docking score and interactions, the final poses were selected. Docking protocol was optimized by
calculating the RMSD of crystal and docked pose of co-crystal ligand with <1 Å.

Acknowledgements
PST will like to thank the Department of Pharmaceuticals, Ministry of Chemicals and
Fertilizers, Govt. of India, New Delhi for awarding NIPER Fellowship.
Conflict of Interest
The authors declare no conflict of interest.
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Highlights


A series of novel 7-hydroxycoumarin-3-carboxamides was synthesized.
Thenovel 7-hydroxycoumarin-3-carboxamides were investigated for inhibition against
four isoforms hCA I, II, IX and XII.


The compounds exhibited low micromolar to sub micromolar inhibitory profiles for both
the isoforms, particularly for CA IX, with Kis in the range of 0.2-9.2 µM for CA IX and
0.2-9.7 for CA XII.
The compounds showed exclusive selectivity for hCA IX and XII over hCA I and II.

Graphical abstract