New coumarin/sulfocoumarin linked phenylacrylamides as selective transmembrane carbonic anhydrase inhibitors: Synthesis and in-vitro biological evaluation

Article abstract of DOI:10.1016/j.bmc.2020.115586

Two novel series of phenylacrylamide linked coumarins and sulfocoumarins (6a-p, 8a-i, and 14a-g) were synthesized and evaluated against four physiologically relevant human carbonic anhydrases (hCAs, EC 4.2.1.1), isoforms hCA I, hCA II, hCA IX and hCA XII for their inhibitory action. All new compounds when screened for carbonic anhydrase inhibitory activity have shown selective inhibition towards the tumor associated isoforms hCA IX and XII over CA I and II, with inhibition constants in the submicromolar to low nanomolar range. Compound 6b and 14g exhibited significant inhibition with low nanomolar potency against hCA IX, whereas 6k was effective against hCA XII. Compounds 6b, 14g and 6k may be considered as lead molecules for future development of cancer therapeutics based on a novel mechanism of action.

Full text of DOI:10.1016/j.bmc.2020.115586

Journal Pre-proofs
New Coumarin/Sulfocoumarin linked Phenylacrylamides as Selective Trans‐
membrane Carbonic Anhydrase Inhibitors: Synthesis and In-Vitro Biological
Evaluation
Baijayantimala Swain, Andrea Angeli, Priti Singh, Claudiu T. Supuran,
Mohammed Arifuddin
PII:
DOI:
Reference:
S0968-0896(20)30416-8
https://doi.org/10.1016/j.bmc.2020.115586
BMC 115586
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Bioorganic & Medicinal Chemistry
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Revised Date:
Accepted Date:
9 April 2020
31 May 2020
4 June 2020
Please cite this article as: Swain, B., Angeli, A., Singh, P., Supuran, C.T., Arifuddin, M., New Coumarin/
Sulfocoumarin linked Phenylacrylamides as Selective Transmembrane Carbonic Anhydrase Inhibitors: Synthesis
and In-Vitro Biological Evaluation, Bioorganic & Medicinal Chemistry (2020), doi: https://doi.org/10.1016/
j.bmc.2020.115586
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New Coumarin/Sulfocoumarin linked Phenylacrylamides as Selective Transmembrane
Carbonic Anhydrase Inhibitors: Synthesis and In-Vitro Biological Evaluation
Baijayantimala Swaina, Andrea Angelic, Priti Singha, Claudiu T. Supuranc* and
Mohammed Arifuddina,b*
.
aDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education and
Research (NIPER), Balanagar, Hyderabad 500037, India
bDepartment of Chemistry, Anwarul Uloom College, 11-3-918, New Malleypally,
Hyderabad-500001, T. S., India
cUniversità degli Studi di Firenze, Neurofarba Dept., Sezione di Scienze Farmaceutiche e
Nutraceutiche, Via Ugo Schiff 6, 50019 Sesto Fiorentino, Florence, Italy
Abstract:
Two novel series of phenylacrylamide linked coumarins and sulfocoumarins
(6a-p, 8a-i, and 14a-g) were synthesized and evaluated against four physiologically
relevant human carbonic anhydrases (hCAs, EC 4.2.1.1), isoforms hCA I, hCA II, hCA
IX and hCA XII for their inhibitory action. All new compounds when screened for
carbonic anhydrase inhibitory activity have shown selective inhibition towards the tumor
associated isoforms hCA IX and XII over CA I and II, with inhibition constants in the
submicromolar to low nanomolar range. Compound 6b and 14g exhibited significant
inhibition with low nanomolar potency against hCA IX, whereas 6k was effective against
hCA XII. Compounds 6b, 14g and 6k may be considered as lead molecules for future
development of cancer therapeutics based on a novel mechanism of action.
Keywords:
Carbonic anhydrase, phenylacrylamide, coumarin, sulfocoumarin, hCA IX, XII
isoform, hypoxia tumor.
Corresponding authors: Mohammed Arifuddin arif@niperhyd.ac.in; Prof. Claudiu T. Supuran; claudiu.supuran@unifi.it

Introduction
The coumarin is a privileged structure found in nature and in many
pharmaceutical products because of its various biological activities, as it may intereact
with many pharmacological targets in a selective way. In the last decade, coumarins were
revealed to act as a pro-drug inhibitor of the human tumor-associated zinc metalloenzyme
carbonic anhydrases (CA, EC 4.2.1.1) isoforms hCA IX and hCA XII.1-4 These two
−
extracellular isoforms5 are involved in CO2 metabolism [CO2 + H2O
HCO3 + H+],
acidification of the extracellular microenvironment and protection of the cancer cells
from the acidosis (Eq. 1). CA IX is mostly expressed in hypoxic tissues conditions in
majority of solid tumors. CA IX is typically expressed in the stomach and gut lining.
Indeed, CA IX is a tumor associated isoform.6 The first reported coumarin compound
acting as hCA inhibitor was a natural product (6-(1-S-hydroxy-3-methylbutyl)-
7-methoxy-2H-chromen-2-one) isolated from the Australian plant Leionema elipticum.7
Sulfonamides and their isosteres (sulfamates, sulfamides, etc.), but also phenols and
polyamines inhibit CAs either by complexing with the metal ion within the enzyme
active site or by anchoring to the water molecule coordinated to it.8 However, the
inhibition mechanism of coumarins revealed these compounds to constitute a totally new
class of CA inhibitors (CAIs), as they bind in hydrolyzed form at the entrance of the
enzyme active site.9 (Fig. 1)
OH
(A)
(B)
Hydrophilic part of
active site
O
O
Glu 106
N
O
O
Tail
O
OH
O
CA
Scaffold
AG
O
CA
rt, pH 7.4
Z
H
O
H
COO
OH
OH2
N
Hydrophobic
part of active
site
OH
O
Thr 199
Zn+2
His 94
COO
E
OH
COO
His 119
His 96
OH
Fig. 1. Mechanism of action of coumarins in the CA active site: (A) hydrolysis of the
coumarin in presence of CA, (B) binding of hydrolysed form in CA active cavity

These pro-drugs undergo hydrolysis after binding to CA active site due to esterase
activity of the enzyme (Fig-1A)10 and their hydrolyzed form (2-hydroxy-cinnamic acid
derivatives) blocks the substrate entry and prevent catalysis by binding at the entrance of
the active site (Fig-1B).11 Because of this chemical transformation their inhibitory
properties are time dependent and are known as suicide inhibitors.12 Coumarin based
CAIs exhibit selective inhibition and were usually studied in relation to the tumor
associated isoforms, CA IX and CA XII. Our groups reported that umbelliferone
(7-hydoxy coumarin) (Fig-2a) and its derivatives as selective inhibitors of CA IX and/or
CA XII over CA I and CA II.13-15 Peperidou et al. have reported 6- and 7-substituted
coumarins (Fig-2b) acting as selective tumor-associated carbonic anhydrases IX and XII
inhibitors.16
SO2NH2
F
O
O
O
N
S
O
H
N
N
O
O
HO
O
O
H
H
OCHF2
c
a
b
hCA IX = 1.8 nM
hCA XII = 4.6 nM
hCA IX = 20.2 nM
hCA XII = 6.0 nM
O
X
O
6a-p = 6-NH2, X=CO, R2=H
8a-i = 7-NH2, X=CO, R2= CH3
14a-g = 6-NH2, X= SO2, R2= H
N
R2
R1
H
Present work
Fig. 2. CAIs investigated earlier (a-c) along with the designed molecule 6, 8, 14.
It has been observed that the activity towards specific isoforms improves with
the addition of chemical substituents having various functional groups on the coumarin
scaffold. On the other hand, cinnamic acid and its natural analogues are well known for
their anticancer property over years. This is an α,β-unsaturated carbonyl moiety
consisting of a phenyl ring substituted with an acrylic acid group usually in the
trans-geometry. It has a low toxicity towards human exposure and also acts as a Michael
acceptor because of the α,β-unsaturation, therefore used as an active moiety for the
design of anticancer drugs. Recently, we found benzene sulfonamide linked cinnamic

acid derivatives (Fig-2c) as selective CAIs against hCA II, IV, IX, XII isoforms
inhibitors.17 In continuation of our interest, herein we report the design, synthesis and
biological evaluation of novel coumarin and sulfocoumarin incorporated
phenylacrylamido functionalities as CA I, CA II, CA IX and CA XII inhibitors.
2. Results and discussion
2.1. Chemistry:
The synthesis of the target compounds (6a-p, 8a-i, and 14a-g) was achieved as outlined
in scheme-1 and the required intermediates were synthesized according to literature
procedure as illustrated in scheme-2. In short the coumarin/sulfocoumarin
phenylacrylamide hybrids (6a-p, 8a-i, and 14a-g) were synthesized from coupling of
cinnamoyl chloride (from phenylacrylic acids with treatment of SOCl2) and amino
coumarin/sulfocoumarin in pyridine at room temperature. (Scheme-1)
The
phenylacrylic acids were synthesized by knoevenagel condensation of benzaldehyde with
malonic acid in the presence of piperidine as base and pyridine as solvent.10 On the
other hand the intermediate 13 was synthesized from 5-nitrosalicylaldehyde 9 which
undergoes mesylation in the presence of MsCl and Et3N to form 10, followed by
intramolecular cyclization in the presence of 1,8- diazabicyclo[5.4.0]undec-7-ene (DBU)
to afford 4-hydroxy-6-nitro-3,4-dihydrobenzo[e][1,2]oxathiine 2,2-dioxide 11 which
undergoes dehydration to form the cyclic scaffold 12 upon treatment with POCl3.24 Then
the reduction of the nitro group of 12 and 4 using iron (Fe0) under acidic conditions
afforded the 6-amino sulfocoumarin 13 and 6-amino coumarin 5 respectively
(Scheme-2).18 All the target molecules were characterized by means of NMR and HRMS.
Almost similar patterns were observed in 1H and 13C NMR spectra of all the synthesized
compounds (6a-p, 8a-i and 14a-g). The fluorine containing compounds were showing
splitting due to J C-F coupling. For example 6b, the value for 1JCF ~245 Hz, ipso-C;
2JFC ~21 Hz, ortho-C; 3JFC ~8 Hz, meta-C; 4JFC ~ 3 were observed (see Experimental
for details).

O
CHO
OH
R1
R1
(i)
2
1
(ii)
O
O
H2N
O
O
O
O
O
H2N
O
5
7
O
Cl
N
R1
O
R1
(iii)
H
N
H
O
O
iii
3
6a-p
R1
8a-i
O
(iii)
S O
H2N
13
O
S O
O
O
N
R1
H
14a-g
Scheme-1. Reagents and conditions: (i) Malonic acid, Piperidine, Pyridine, 90oC, 8-12 h, 75-90% (ii)
SOCl2, DCM, reflux, 2-4 h, (iii) pyridine, rt, 4-8h, 75-80%.
OH
O2N
O2N
CHO
OH
O2N
CHO
OMs
ii
i
O
O
S
O
11
9
10
iii
H2N
O2N
O
O
O
iv
S
S
O
O
O
12
13
O
O
O
O
H2N
iv
O2N
5
4
Scheme-2. Reagents and conditions: (i) MsCl, Et3N, DCM, rt, 18 h, 90%; (ii) DBU, DCM, 0oC; (iii) POCl3,
pyridine, rt, 2 hr, 60%; iv) Fe/AcOH, EtOH-Water, reflux, 1 hr, 90%.
2.2. Carbonic anhydrase inhibition:
Inhibition data for compounds 6a-p, 8a-i and 14a-g reported here with
acetazolamide as standard against four CA isozymes, i.e., the cytosolic hCA I and II and
the transmembrane, tumor-associated hCA IX and XII are listed in Table 1. The
following interesting findings can be evidenced from the data in Table 1 for these
phenylacrylamide linked coumarin/sulfocoumarin derivatives.

Table-1. Inhibition of hCA isoforms I, II, IV and IX with KI (nM) data for compounds
6a-p, 8a-i, 14a-g and acetazolamide (AAZ) as standard inhibitor, by means of the stopped
flow carbon dioxide assays19
Cmp
6a
structure
hCA I
hCA II
>10000
hCA IX
130.7
hCA XII
829.1
O
O
O
>10000
N
H
O
O
O
6b
6c
>10000
>10000
>10000
>10000
40.9
43.0
264.7
410.6
N
H
F
O
O
O
O
N
H
F
O
O
N
H
6d
6e
6f
>10000
>10000
>10000
>10000
>10000
>10000
339.8
91.1
88.0
206.1
235.6
221.0
F
F
O
O
O
N
H
F3C
O
O
O
N
H
CF3
O
O
O
N
H
6g
6h
6i
>10000
>10000
>10000
>10000
>10000
>10000
383.9
472.0
883.7
393.7
136.9
427.4
OCHF2
O
O
O
O
N
H
O
F
O
O
N
H
O
O
O
O
O
O
N
6j
>10000
>10000
>10000
>10000
87.4
445.6
15.5
H
O
O
O
N
H
6k
321.3
O
O

O
O
O
O
O
O
O
N
H
6l
6m
6n
>10000
>10000
>10000
>10000
>10000
>10000
156.7
88.9
149.3
342.8
700.6
O
O
N
H
Cl
O
O
O
O
Cl
N
H
305.4
Cl
O
O
6o
6p
>10000
>10000
>10000
>10000
880.8
7206
760.0
556.7
N
H
NC
O
O
O
N
H
NO2
H
N
O
O
8a
8b
>10000
>10000
>10000
>10000
513.6
3036
648.6
644.3
O
O
H
N
O
O
O
O
O
O
O
H
N
O
O
8c
>10000
>10000
647.0
630.6
O
H
N
O
O
F
F
8d
8e
8f
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
638.8
681.7
772.7
853.6
538.7
604.4
699.4
574.6
O
H
N
O
O
O
O
O
O
F
F
H
N
NC
H
N
O
O
8g
O

H
N
O
O
8h
8i
>10000
>10000
>10000
>10000
488.0
816.7
567.1
847.2
OCHF2
O
O
H
N
O
O
O
F
O
O
O
O
S
O
O
N
H
14a
>10000
>10000
535.5
208.2
O
O
O
O
S
O
14b
14c
>10000
>10000
>10000
>10000
80.4
92.8
398.9
349.3
N
H
O
F
O
O
O
S
O
N
H
OCHF2
O
S O
O
O
14d
14e
>10000
>10000
>10000
>10000
517.3
719.8
505.6
568.4
N
H
F
F
O
S O
O
O
N
H
F
O
O
O
S
O
N
14f
>10000
>10000
65.4
480.0
H
CF3
O
O
O
S
O
14g
>10000
250
>10000
12.1
39.8
25.8
519.9
5.7
N
H
NC
AAZ
* Mean from 3 different assays, by a stopped flow technique (errors were in the range of
5-10 % of the reported values)
 All the synthesized compounds tested here against the cytosolic isoform hCA I
and the most abundant house-keeping isoform hCA II were devoid of any

inhibitory activity with
KI >10,000 nM. This lacking was due to the
coumarin/sulfocoumarin moiety.
 The tumor associated hCA IX was well inhibited by all the synthesized
compounds with KI sub nanomolar to sub micromolar range (KI= 39.8 nM to 7.2
µM) with standard AAZ (KI= 25.8 nM). Out of 32 compounds 10 were showing
very good inhibition. Again these molecules were having substitution R1 as 4-F,
3-F, 4-CF3, 3-CF3, 2,4-diOMe, 4-Cl, 2-F-4-OMe, 2-OCHF2, 3-CF3, 4-CN, which
signifies that the electron withdrawing species were showing better inhibition
with compared to the electron donating one like methoxy. Those were 6b (KI of
40.9 nM), 6c (KI of 43.0 nM), 6e (KI of 91.1 nM), 6f (KI of 88.0 nM), 6j (KI of
87.4 nM), 6m (KI of 88.9 nM), 14b (KI of 80.4 nM), 14c (KI of 92.8 nM), 14f (KI
of 65.4 nM), 14g (KI of 39.8 nM).
 Furthermore, it is noteworthy to note that the compounds with 7-NH2-4Me
coumarin moiety (8a-i) were less effective than the molecules with 6-NH2
coumarin (6a-p) and 6-NH2 sulfocoumarin (14a-g) in the case of CA binding part.
 The second tumor associated isoform, hCA XII, was also inhibited by all these
compounds having KI in the range of 15.5 nM-0.8 µM with standard AAZ (KI=
5.7 nM). But all these compounds were less efficient against CA XII as compared
to CA IX. Only one compound 6k was exhibiting good inhibition (KI of 15.5
nM). Therefore, these molecules (6a-p, 8a-i and 14a-g) constitute highly
attractive features for inhibiting CA IX over CA XII.
3. Conclusions
New series of coumarin/sulfocoumarin linked phenylacrylamide hybrids (6a-p,
8a-i and 14a-g) were synthesized and investigated for their CA inhibitory activity against
the physiologically and pharmacologically significant isoforms hCA I, II, IX and XII.
These molecules (6a-p, 8a-i and 14a-g) were ineffective as inhibitors of the
house-keeping, off target isoforms CA I and II, whereas exhibiting quite good inhibition
against the transmembrane tumor-associated isoforms CA IX and XII with a KI value in
the submicromolar to low nanomolar range. It is also noticed that the nature and position
of the groups present on the coumarin ring influenced the CA inhibitory properties,
whereas the 6-NH2 coumarin/sulfocoumarin derivatives were more active than the

7-NH2-4-Me coumarin derivatives. Ten molecules (6b, 6c, 6e, 6f, 6j, 6m, 14b, 14c, 14f,
14g) out of thirty two derivatives were found to be highly potent with a range of KI value
between 39.8 nM-91.1 nM against hCA IX while only one molecule (6k) was found to be
very good against hCA XII with a KI 15.5 nM. From the results obtained in the present
studies it is anticipated that these newly synthesized compounds (6a-p, 8a-i and 14a-g)
will be interesting lead compounds for the design and synthesis of isoform selective
enzyme inhibitors against hCA IX over hCA XII.
4. Experimental Section
4.1. General
Commercially available reagents were used for synthesizing the reported
compounds without further purification. Standard methods were followed to distill and
dry the solvents wherever necessary prior to use. All the air and moisture sensitive
reactions were kept under inert conditions using clean and dried glassware and syringe
technique to transfer solutions. Reactions were monitored by TLC using Merck silica gel
60F-254 plates. Column chromatography, on silica gel (60-120 mesh) using a mixture of
DCM and methanol as eluent, was performed to purify the synthesized molecules.
Melting points were acquired on Stuart digital melting point apparatus / SMP 30 in open
13
capillary tubes and uncorrected. Nuclear Magnetic Resonance (1H NMR and C NMR)
spectra were recorded by using an Avance Bruker 500MHz and 125MHz spectrometer in
DMSO-d6 as solvent and tetramethylsilane (TMS) as internal standard. Chemical shifts
are represented as δ values in parts per million (ppm) and coupling constants (J) in Hz.
Multiplicities are described as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet),
dd (doublet of doublets). HRMS were determined with Agilent QTOF mass spectrometer
6540 series instrument and were performed in the ESI techniques at 70 eV.
4.2. General procedure for the synthesis of compounds (6a-p, 8a-j & 14a-g):
To a stirred solution of amino coumarin/sulfocoumarin (1.0 equiv) in dry pyridine
(5 mL/1 mmol of amine) the appropriate substituted cinnamoyl chlorides 3a-p (1.0 equiv)
prepared from respective cinnamic acids (2a-p) were added. The reaction was carried out
at room temperature under nitrogen atmosphere for 4-8 h and monitored by TLC. The
reaction mixture was quenched with ice and the solid was filtered and washed 2-3 times

with cold water and recrystallized from ethanol to give the products (6a-p, 8a-j & 14a-g)
in good to excellent yields.
Characterization of the compounds:
4.2.1 N-(2-oxo-2H-chromen-6-yl)cinnamamide (6a): White solid, Yield: 93%; mp:
1
236-2380C; H NMR (500 MHz, DMSO-d6) δ 10.44 (s, 1H), 8.22 – 8.08 (m, 2H), 7.79
(dd, J = 8.9, 2.0 Hz, 1H), 7.64 (t, J = 11.2 Hz, 3H), 7.53 – 7.37 (m, 4H), 6.84 (d, J = 15.7
13
Hz, 1H), 6.50 (d, J = 9.5 Hz, 1H); C NMR (125 MHz, DMSO-d6) δ 164.14, 160.44,
149.96, 144.82, 140.95, 136.17, 135.12, 130.34, 129.51, 128.24, 123.89, 122.45, 119.25,
+
118.34, 117.22, 117.04; HRMS (ESI) m/z: [M+H]+ calculated for C18H14NO3 292.0968,
found 292.0968.
4.2.2 (E)-3-(4-fluorophenyl)-N-(2-oxo-2H-chromen-6-yl)acrylamide (6b): White
solid, Yield: 90%; mp: 246-2480C; 1H NMR (500 MHz, DMSO-d6) δ 10.45 (s, 1H), 8.15
(d, J = 2.4 Hz, 1H), 8.12 (d, J = 9.6 Hz, 1H), 7.79 (dd, J = 9.0, 2.5 Hz, 1H), 7.71 (dt, J =
15.9, 8.0 Hz, 2H), 7.62 (d, J = 15.7 Hz, 1H), 7.41 (d, J = 8.9 Hz, 1H), 7.30 (t, J = 8.8 Hz,
2H), 6.80 (t, J = 15.4 Hz, 1H), 6.51 (d, J = 9.5 Hz, 1H); 13C NMR (125 MHz, DMSO) δ
164.38, 164.06, 162.41, 160.47, 149.94, 144.85, 139.75, 136.14, 131.74 (d, J = 3.1 Hz),
130.47 (d, J = 8.6 Hz), 123.85, 122.30, 119.25, 118.30, 117.24, 117.05, 116.60, 116.43.
+
HRMS (ESI) m/z: [M+H]+ calculated for C18H13FNO3 310.0874, found 310.0833.
4.2.3 (E)-3-(3-fluorophenyl)-N-(2-oxo-2H-chromen-6-yl)acrylamide (6c): White solid,
Yield: 84%; mp: 265-2670C; 1H NMR (500 MHz, DMSO-d6) δ 10.45 (s, 1H), 8.14 (d, J
= 2.4 Hz, 1H), 8.11 (d, J = 9.6 Hz, 1H), 7.79 (dd, J = 8.9, 2.5 Hz, 1H), 7.62 (d, J = 15.7
Hz, 1H), 7.54 – 7.46 (m, 3H), 7.41 (d, J = 8.9 Hz, 1H), 7.26 (tdd, J = 7.3, 5.0, 2.6 Hz,
1H), 6.87 (d, J = 15.7 Hz, 1H), 6.50 (d, J = 9.5 Hz, 1H); 13C NMR (125 MHz, DMSO) δ
163.88 (d, J = 10.3 Hz), 161.98, 160.43, 150.03, 144.81, 139.58 (d, J = 2.4 Hz), 137.72
(d, J = 8.0 Hz), 136.04, 131.47 (d, J = 8.5 Hz), 124.34 (d, J = 2.4 Hz), 123.98 (d, J = 17.3
Hz), 119.26, 118.40, 117.24, 117.08, 116.98 (d, J = 21.0 Hz), 114.74, 114.57. HRMS
+
(ESI) m/z: [M+H]+ calculated for C18H13FNO3 310.0874, found 310.0808.
4.2.4 (E)-3-(3,4-difluorophenyl)-N-(2-oxo-2H-chromen-6-yl)acrylamide (6d): White
solid, Yield: 85%; mp: 245-2470C; 1H NMR (500 MHz, DMSO-d6) δ 10.44 (s, 1H), 8.20
– 8.06 (m, 2H), 7.86 – 7.69 (m, 2H), 7.56 (dd, J = 35.6, 9.8 Hz, 3H), 7.40 (d, J = 8.9 Hz,
1H), 6.80 (d, J = 15.7 Hz, 1H), 6.50 (d, J = 9.5 Hz, 1H); 13C NMR (125 MHz, DMSO) δ

163.79, 160.42, 151.63 (d, J = 12.8 Hz), 151.15 (d, J = 12.9 Hz), 150.02, 149.64 (d, J =
12.6 Hz), 149.19 (d, J = 12.9 Hz), 144.79, 138.7, 136.03, 133.08 (dd, J = 6.3, 3.7 Hz),
125.40, 123.92, 119.25, 118.96 – 118.17 (m), 117.27, 117.27 – 116.75 (m). HRMS (ESI)
+
m/z: [M+H]+ calculated for C18H12F2NO3 328.0780, found 328.0777.
4.2.5 (E)-N-(2-oxo-2H-chromen-6-yl)-3-(4-(trifluoromethyl)phenyl)acrylamide (6e):
1
White solid, Yield 79%; mp: 278-2790C; H NMR (500 MHz, DMSO-d6) δ 10.54 (s,
1H), 8.14 (dd, J = 17.9, 6.0 Hz, 2H), 7.82 (dt, J = 11.4, 5.4 Hz, 5H), 7.69 (d, J = 15.8 Hz,
13
1H), 7.42 (d, J = 8.9 Hz, 1H), 6.96 (d, J = 15.8 Hz, 1H), 6.51 (d, J = 9.5 Hz, 1H); C
NMR (125 MHz, DMSO) δ 163.65, 160.42, 150.07, 144.79, 139.17, 135.98, 129.88,
128.86, 126.35 (dd, J = 7.2, 3.5 Hz), 125.29, 123.92, 123.47, 119.27, 118.43, 117.26,
+
117.08. HRMS (ESI) m/z: [M+H]+ calculated for C19H13F3NO3 360.0842, found
360.0858.
4.2.6 (E)-N-(2-oxo-2H-chromen-6-yl)-3-(3-(trifluoromethyl)phenyl)acrylamide (6f):
1
White solid, Yield: 80%; mp: 236-238 0C; H NMR (500 MHz, DMSO-d6) δ 10.46 (s,
1H), 8.13 (dd, J = 21.1, 6.0 Hz, 2H), 8.04 – 7.90 (m, 2H), 7.85 – 7.73 (m, 2H), 7.70 (dd, J
= 15.5, 6.2 Hz, 2H), 7.41 (d, J = 8.9 Hz, 1H), 6.96 (d, J = 15.8 Hz, 1H), 6.50 (d, J = 9.5
Hz, 1H); 13C NMR (125 MHz, DMSO) δ 163.71, 160.42, 150.04, 144.79, 139.12, 136.34,
136.02, 132.00, 130.64, 130.43, 130.17, 126.54 (d, J = 3.7 Hz), 125.58, 124.94 – 124.05
(m), 123.86, 123.41, 119.27, 118.35, 117.26, 117.07. HRMS (ESI) m/z: [M+H]+
+
calculated for C19H13F3NO3 360.0842, found 360.0853.
4.2.7 (E)-3-(2-(difluoromethoxy)phenyl)-N-(2-oxo-2H-chromen-6-yl)acrylamide (6g):
1
White solid, Yield: 85%; mp: 209-2100C; H NMR (500 MHz, DMSO-d6) δ 10.53 (s,
1H), 8.18 (d, J = 2.3 Hz, 1H), 8.11 (d, J = 9.6 Hz, 1H), 7.86 – 7.74 (m, 3H), 7.55 – 7.49
(m, 1H), 7.36 (ddd, J = 24.0, 20.5, 8.6 Hz, 4H), 6.90 (d, J = 15.8 Hz, 1H), 6.51 (d, J = 9.5
Hz, 1H); 13C NMR (125 MHz, DMSO) δ 163.87, 160.46, 150.01, 149.65 (t, J = 2.9 Hz), 144.84, 136.04,
134.09, 131.84, 128.38, 126.59, 126.32, 124.57, 123.88, 119.62, 119.19 (d, J = 17.4 Hz), 118.42, 117.26,
+
117.08 (d, J = 2.6 Hz).HRMS (ESI) m/z: [M+H]+ calculated for C19H13F2NO4 358.0885,
found 358.0849.
4.2.8 (E)-3-(2-fluoro-4-methoxyphenyl)-N-(2-oxo-2H-chromen-6-yl)acrylamide (6h):
1
White solid, Yield: 79%; mp: 265-2670C; H NMR (500 MHz, DMSO-d6) δ 10.43 (s,
1H), 8.24 – 8.04 (m, 2H), 7.78 (d, J = 7.9 Hz, 1H), 7.63 (dd, J = 23.4, 12.4 Hz, 2H), 7.40

(d, J = 8.6 Hz, 1H), 7.01 – 6.75 (m, 3H), 6.50 (d, J = 9.3 Hz, 1H), 3.83 (s, 3H); 13C NMR
(125 MHz, DMSO) δ 164.28, 163.15, 162.40 (d, J = 11.7 Hz), 161.16, 160.45, 149.93,
144.83, 136.22, 133.52, 130.99 (d, J = 5.0 Hz), 123.85, 122.43 (d, J = 7.0 Hz), 119.23,
118.29, 117.19, 117.02, 115.20 (d, J = 12.0 Hz), 111.89, 102.60, 102.40, 56.39. HRMS
+
(ESI) m/z: [M+H]+ calculated for C19H15FNO4 340.0980, found 340.0983.
4.2.9 (E)-3-(4-methoxyphenyl)-N-(2-oxo-2H-chromen-6-yl)acrylamide (6i): White
solid, Yield: 81%; mp: 242-2440C; 1H NMR (500 MHz, DMSO-d6) δ 10.37 (s, 1H), 8.13
(dd, J = 21.6, 6.0 Hz, 2H), 7.78 (dd, J = 9.0, 2.5 Hz, 1H), 7.58 (t, J = 12.0 Hz, 3H), 7.40
(d, J = 8.9 Hz, 1H), 7.04 (t, J = 14.1 Hz, 2H), 6.69 (d, J = 15.7 Hz, 1H), 6.50 (d, J = 9.5
Hz, 1H), 3.81 (s, 3H); 13C NMR (125 MHz, DMSO-d6) δ 164.47, 161.17, 160.46,
149.85, 144.84, 140.72, 136.32, 129.89, 127.69, 123.81, 119.84, 119.23, 118.22, 117.17,
+
117.00, 114.99, 55.79; HRMS (ESI) m/z: [M+H]+ calculated for C19H16NO4 322.1074,
found 322.1084.
4.2.10 (E)-3-(2,4-dimethoxyphenyl)-N-(2-oxo-2H-chromen-6-yl)acrylamide (6j):
White solid, Yield: 83; mp: 216-2180C; 1H NMR (500 MHz, DMSO-d6) δ 10.32 (s, 1H),
8.21 - 8.06 (m, 2H), 7.77 (t, J = 12.4 Hz, 2H), 7.53 (d, J = 8.4 Hz, 1H), 7.40 (d, J = 8.9
Hz, 1H), 6.74 (t, J = 16.9 Hz, 1H), 6.63 (dd, J = 21.0, 12.4 Hz, 2H), 6.50 (d, J = 9.5 Hz,
1H), 3.85 (dd, J = 37.5, 18.1 Hz, 6H); 13C NMR (125 MHz, DMSO-d6) δ 164.98, 162.63,
160.51, 159.76, 149.76, 144.91, 136.46, 136.21, 130.30, 123.75, 119.94, 119.21, 118.11,
117.16, 116.98, 116.38, 106.54, 99.00, 56.21, 55.93; HRMS (ESI) m/z: [M+H]+
+
calculated for C20H18NO5 352.1179, found 352.1182.
4.2.11 (E)-3-(3,4-dimethoxyphenyl)-N-(2-oxo-2H-chromen-6-yl)acrylamide (6k):
1
White solid, Yield: 77%; mp: 210-2120C; H NMR (500 MHz, DMSO-d6) δ 10.32 (s,
1H), 8.21 - 8.06 (m, 2H), 7.77 (t, J = 12.4 Hz, 2H), 7.53 (d, J = 8.4 Hz, 1H), 7.40 (d, J =
8.9 Hz, 1H), 6.74 (t, J = 16.9 Hz, 1H), 6.63 (dd, J = 21.0, 12.4 Hz, 2H), 6.50 (d, J = 9.5
13
Hz, 1H), 3.85 (dd, J = 37.5, 18.1 Hz, 6H); C NMR (125 MHz, DMSO-d6) δ 164.45,
160.49, 150.96, 149.82, 149.41, 144.89, 141.10, 136.35, 127.83, 123.73, 122.35, 119.94,
119.22, 118.10, 117.20, 117.00, 112.22, 110.51, 56.04, 55.89; HRMS (ESI) m/z: [M+H]+
+
calculated for C20H18NO5 352.1179, found 352.1178.
4.2.12 (E)-N-(2-oxo-2H-chromen-6-yl)-3-(3,4,5-trimethoxyphenyl)acrylamide (6l):
1
White solid, Yield: 72%; mp: 213-2150C; H NMR (500 MHz, DMSO-d6) δ 10.43 (s,

1H), 8.18 (d, J = 2.4 Hz, 1H), 8.12 (d, J = 9.6 Hz, 1H), 7.78 (dd, J = 9.0, 2.5 Hz, 1H),
7.57 (d, J = 15.6 Hz, 1H), 7.41 (d, J = 8.9 Hz, 1H), 6.99 (s, 2H), 6.77 (d, J = 15.6 Hz,
1H), 6.51 (d, J = 9.5 Hz, 1H), 3.85 (s, 6H), 3.71 (s, 3H); 13C NMR (125 MHz,
DMSO-d6) δ 164.20, 160.49, 153.59, 149.88, 144.90, 141.15, 139.45, 136.26, 130.66,
123.75, 121.65, 119.24, 118.14, 117.25, 117.03, 105.65, 60.59, 56.37; HRMS (ESI) m/z:
+
[M+H]+ calculated for C21H20NO6 382.1285, found 382.1292.
4.2.13 (E)-3-(4-chlorophenyl)-N-(2-oxo-2H-chromen-6-yl)acrylamide (6m): White
solid, Yield: 75%; mp: 274-2780C; 1H NMR (500 MHz, DMSO-d6) δ 10.48 (s, 1H), 8.14
(dd, J = 15.8, 5.6 Hz, 2H), 7.79 (dd, J = 8.9, 2.0 Hz, 1H), 7.68 (d, J = 8.3 Hz, 2H), 7.62
(d, J = 15.7 Hz, 1H), 7.53 (d, J = 8.3 Hz, 2H), 7.42 (d, J = 8.9 Hz, 1H), 6.84 (d, J = 15.7
13
Hz, 1H), 6.51 (d, J = 9.5 Hz, 1H); C NMR (125 MHz, DMSO-d6) δ 163.92, 160.46,
149.97, 144.85, 139.56, 136.08, 134.78, 134.07, 129.95, 129.56, 123.85, 123.22, 119.26,
+
118.31, 117.26, 117.07; HRMS (ESI) m/z: [M+H]+ calculated for C18H13ClNO3
326.0578, found 326.0584.
4.2.14 (E)-3-(3,5-dichlorophenyl)-N-(2-oxo-2H-chromen-6-yl)acrylamide (6n): White
solid, Yield: 73%; mp: 278-2800C; 1H NMR (500 MHz, DMSO-d6) δ 10.47 (s, 1H), 8.13
(dd, J = 12.3, 6.0 Hz, 2H), 7.78 (dd, J = 8.9, 2.5 Hz, 1H), 7.71 (d, J = 1.8 Hz, 2H), 7.65
(t, J = 1.8 Hz, 1H), 7.57 (d, J = 15.7 Hz, 1H), 7.41 (d, J = 8.9 Hz, 1H), 6.92 (d, J = 15.8
13
Hz, 1H), 6.51 (d, J = 9.6 Hz, 1H); C NMR (125 MHz, DMSO-d6) δ 163.48, 160.44,
150.05, 144.81, 138.93, 137.84, 135.91, 135.12, 129.25, 126.69, 125.76, 123.84, 119.26,
+
118.33, 117.29, 117.09; HRMS (ESI) m/z: [M+H]+ calculated for C18H12Cl2NO3
360.0189, found 360.0202.
4.2.15 (E)-3-(4-cyanophenyl)-N-(2-oxo-2H-chromen-6-yl)acrylamide (6o): White
1
solid, Yield: 71%; mp: 275-2760C; H NMR (500 MHz, DMSO) δ 10.57 (s, 1H), 8.18 –
8.09 (m, 2H), 7.93 (d, J = 8.4 Hz, 2H), 7.86 - 7.76 (m, 3H), 7.68 (d, J = 15.8 Hz, 1H),
13
7.44 (dd, J = 13.2, 9.1 Hz, 1H), 6.97 (d, J = 15.8 Hz, 1H), 6.52 (t, J = 8.6 Hz, 1H); C
NMR (125 MHz, DMSO) δ 163.55, 160.44, 150.08, 144.82, 139.72, 139.04, 135.92,
133.38, 128.93, 125.90, 123.91, 119.28, 119.11, 118.42, 117.30, 117.10, 112.23; HRMS
+
(ESI) m/z: [M+H]+ calculated for C19H13N2O3 317.0921, found 317.0916.
4.2.16 (E)-3-(3-nitrophenyl)-N-(2-oxo-2H-chromen-6-yl)acrylamide (6p): White
solid, Yield: 71%; mp: 290-2920C; 1H NMR (500 MHz, DMSO-d6) δ 10.53 (s, 1H), 8.48

(s, 1H), 8.25 (d, J = 8.1 Hz, 1H), 8.16 (s, 1H), 8.14 – 8.06 (m, 2H), 7.85 – 7.69 (m, 3H),
7.45 – 7.39 (m, 1H), 7.05 – 6.97 (d, 1H), 6.49 (d, J = 10.1 Hz, 1H); 13C NMR (125 MHz,
DMSO-d6) δ 163.53, 160.42, 150.06, 148.80, 144.80, 138.48, 136.97, 135.97, 134.60,
131.07, 125.37, 124.52, 123.83, 122.19, 119.28, 118.33, 117.29, 117.09; HRMS (ESI)
+
m/z: [M+H]+ calculated for C18H13N2O5 337.0819, found 337.0823.
4.2.17 N-(4-methyl-2-oxo-2H-chromen-7-yl)cinnamamide (8a): White solid, Yield:
1
91%; mp: 320-3210C; H NMR (500 MHz, DMSO-d6) δ 10.66 (s, 1H), 7.91 (d, J = 1.9
Hz, 1H), 7.77 (d, J = 8.6 Hz, 1H), 7.70 – 7.64 (m, 3H), 7.57 (dd, J = 8.7, 2.0 Hz, 1H),
7.51 – 7.42 (m, 3H), 6.86 (d, J = 15.8 Hz, 1H), 6.29 (s, 1H), 2.43 (d, J = 1.1 Hz, 3H); 13C
NMR (125 MHz, DMSO-d6) δ 164.60, 160.49, 154.19, 153.57, 143.04, 141.76, 135.00,
130.54, 129.55, 128.36, 126.48, 122.12, 115.81, 115.64, 112.78, 106.23, 18.45; HRMS
+
(ESI) m/z: [M+Na]+ calculated for C19H15NNaO3 328.0950, found 328.0993.
4.2.18 (E)-3-(3,4-dimethoxyphenyl)-N-(4-methyl-2-oxo-2H-chromen-7-yl)acrylamide
(8b): White solid, Yield: 88%; mp: 316-3180C; 1H NMR (500 MHz, DMSO-d6) δ 10.57
(s, 1H), 7.91 (s, 1H), 7.75 (d, J = 8.5 Hz, 1H), 7.59 (dd, J = 21.9, 12.1 Hz, 2H), 7.27 –
7.18 (m, 2H), 7.04 (d, J = 7.9 Hz, 1H), 6.72 (d, J = 15.6 Hz, 1H), 6.28 (s, 1H), 3.83 (d, J
= 9.3 Hz, 6H), 2.42 (d, J = 1.1 Hz, 3H); 13C NMR (126 MHz, DMSO-d6) δ 164.92,
160.54, 154.20, 151.13, 149.41, 143.23, 141.96, 127.71, 126.49, 122.61, 119.58, 115.69,
115.47, 112.66, 112.24, 110.52, 106.03, 56.07, 55.89, 18.47; HRMS (ESI) m/z: [M+Na]+
+
calculated for C21H19NNaO5 388.1161, found 388.1265.
4.2.19 (E)-N-(4-methyl-2-oxo-2H-chromen-7-yl)-3-(3,4,5-trimethoxyphenyl)
1
acrylamide (8c): White solid, Yield: 81%; mp: 304-3060C; H NMR (500 MHz,
DMSO-d6) δ 10.61 (s, 1H), 7.89 (dd, J = 10.3, 3.3 Hz, 1H), 7.73 (t, J = 14.2 Hz, 1H),
7.66 – 7.48 (m, 2H), 7.00 (s, 2H), 6.81 (dd, J = 33.6, 11.4 Hz, 1H), 6.27 (s, 1H), 3.85 (s,
6H), 3.72 (s, 3H), 2.41 (d, J = 1.2 Hz, 3H); 13C NMR (125 MHz, DMSO-d6) δ 164.67,
160.52, 154.18, 153.60, 143.12, 141.96, 139.61, 130.53, 126.51, 121.31, 115.72, 115.56,
112.73, 106.09, 105.78, 60.60, 56.38, 18.47; HRMS (ESI) m/z: [M+Na]+ calculated for
+
C22H21NNaO6 418.1267, found 418.1285.
4.2.20 (E)-3-(3-fluorophenyl)-N-(4-methyl-2-oxo-2H-chromen-7-yl)acrylamide (8d):
1
White solid, Yield: 77%; mp: 332-3340C; H NMR (500 MHz, DMSO-d6) δ 10.66 (s,
1H), 7.90 (d, J = 2.0 Hz, 1H), 7.77 (d, J = 8.7 Hz, 1H), 7.67 (d, J = 15.7 Hz, 1H), 7.57

(dd, J = 8.7, 2.0 Hz, 1H), 7.53 – 7.47 (m, 3H), 6.88 (d, J = 15.7 Hz, 1H), 6.29 (d, J = 1.2
Hz, 1H), 2.42 (d, J = 1.1 Hz, 3H); 13C NMR (125 MHz, DMSO) δ 164.32, 160.47,
154.18, 153.55, 142.90, 140.37 (d, J = 2.5 Hz), 131.52 (d, J = 8.5 Hz), 126.51, 124.45,
123.72, 117.26, 117.09, 115.79 (d, J = 14.2 Hz), 114.79 (d, J = 22.0 Hz), 112.85, 106.30,
+
18.45. HRMS (ESI) m/z: [M+Na]+ calculated for C19H14FNNaO3 346.0855, found
346.0966.
4.2.21 (E)-3-(4-fluorophenyl)-N-(4-methyl-2-oxo-2H-chromen-7-yl)acrylamide (8e):
1
White solid, Yield: 73%; mp: 318-3200C; H NMR (500 MHz, DMSO-d6) δ 10.53 (s,
1H), 7.81 (d, J = 2.0 Hz, 1H), 7.68 - 7.62 (m, 3H), 7.58 (d, J = 15.7 Hz, 1H), 7.47 (dd, J
= 8.7, 2.0 Hz, 1H), 7.21 (dd, J = 12.3, 5.4 Hz, 2H), 6.70 (d, J = 15.7 Hz, 1H), 6.19 (d, J =
13
1.2 Hz, 1H), 2.33 (d, J = 1.2 Hz, 3H); C NMR (125 MHz, DMSO) δ 164.32, 160.47,
154.18, 153.55, 142.90, 140.37 (d, J = 2.5 Hz), 131.52 (d, J = 8.5 Hz), 126.51, 124.45,
123.72, 117.26, 117.09, 115.79 (d, J = 14.2 Hz), 114.79 (d, J = 22.0 Hz), 112.85, 106.30,
+
18.45. HRMS (ESI) m/z: [M+K]+ calculated for C19H14FKNO3 362.0595, found
362.0638.
4.2.22 (E)-3-(3,4-difluorophenyl)-N-(4-methyl-2-oxo-2H-chromen-7-yl)acrylamide
1
(8f): White solid, Yield: 81%; mp: 330-3320C; H NMR (500 MHz, DMSO-d6) δ 10.66
(s, 1H), 7.90 (d, J = 2.0 Hz, 1H), 7.76 (td, J = 8.0, 3.1 Hz, 2H), 7.67 – 7.63 (m, 1H), 7.58
– 7.52 (m, 3H), 6.81 (d, J = 15.7 Hz, 1H), 6.29 (d, J = 1.2 Hz, 1H), 2.42 (d, J = 1.2 Hz,
3H); 13C NMR (125 MHz, DMSO-d6) δ 164.27, 160.47, 154.17, 153.55, 142.88, 139.54,
126.50, 123.55, 118.75, 118.61, 115.85, 115.73, 112.85, 106.31, 18.44; HRMS (ESI)
+
m/z: [M+K]+ calculated for C19H13F2KNO3 380.0501, found 380.0529.
4.2.23 (E)-3-(4-cyanophenyl)-N-(4-methyl-2-oxo-2H-chromen-7-yl)acrylamide (8g):
1
White solid, Yield: 74%; mp: 331-3330C; H NMR (500 MHz, DMSO-d6) δ 10.64 (s,
1H), 7.84 (d, J = 8.4 Hz, 2H), 7.81 (d, J = 2.0 Hz, 1H), 7.75 (d, J = 8.3 Hz, 2H), 7.68 (d,
J = 8.7 Hz, 1H), 7.63 (d, J = 15.8 Hz, 1H), 7.48 (dd, J = 8.7, 2.0 Hz, 1H), 6.88 (d, J =
15.8 Hz, 1H), 6.20 (d, J = 1.2 Hz, 1H), 2.33 (d, J = 1.2 Hz, 3H);13C NMR (125 MHz,
DMSO-d6) δ 164.04, 154.16, 153.54, 142.77, 139.79, 139.62, 133.39, 129.01, 126.53,
125.60, 119.07, 115.89, 115.84, 112.93, 112.39, 106.37, 18.45; HRMS (ESI) m/z:
+
[M+K]+ calculated for C20H14KN2O3 369.0641, found 369.0699.
4.2.24 (E)-3-(2-(difluoromethoxy)phenyl)-N-(4-methyl-2-oxo-2H-chromen-7-yl)

1
acrylamide (8h): White solid, Yield: 79%; mp: 282-2840C; H NMR (500 MHz,
DMSO-d6) δ 10.71 (s, 1H), 7.90 (d, J = 2.0 Hz, 1H), 7.84 (d, J = 15.8 Hz, 1H), 7.80 –
7.75 (m, 2H), 7.59 (dd, J = 8.7, 2.0 Hz, 1H), 7.55 – 7.50 (m, 1H), 7.40 – 7.29 (m, 3H),
13
6.92 (d, J = 15.8 Hz, 1H), 6.29 (s, 1H), 2.43 (d, J = 1.2 Hz, 3H); C NMR (125 MHz,
DMSO) δ 164.34, 160.49, 153.59, 142.90, 134.86, 132.03, 128.52, 126.61 – 126.24 (m),
124.30, 119.62, 117.03, 115.80 (d, J = 13.4 Hz), 106.30, 18.47. HRMS (ESI) m/z:
+
[M+Na]+ calculated for C19H14FNNaO3 394.0867, found 394.0872.
4.2.25 (E)-3-(2-fluoro-4-methoxyphenyl)-N-(4-methyl-2-oxo-2H-chromen-7-yl)
1
acrylamide (8i): White solid, Yield: 74%; mp: 318-3200C; H NMR (500 MHz,
DMSO-d6) δ 10.63 (s, 1H), 7.90 (d, J = 2.0 Hz, 1H), 7.76 (d, J = 8.7 Hz, 1H), 7.68 – 7.62
(m, 2H), 7.57 (dd, J = 8.7, 2.0 Hz, 1H), 6.97 (dd, J = 13.2, 2.5 Hz, 1H), 6.91 (dd, J = 8.7,
2.5 Hz, 1H), 6.82 (d, J = 15.8 Hz, 1H), 6.28 (s, 1H), 3.84 (s, 3H), 2.42 (d, J = 1.1 Hz,
13
3H); C NMR (125 MHz, DMSO) δ 164.75, 163.27, 162.53 (d, J = 11.8 Hz), 161.27,
160.52, 154.16, 153.59 143.08, 134.45, 131.31, 126.46, 122.06 (d, J = 6.9 Hz), 115.75,
115.56, 115.06 (d, J = 11.9 Hz), 112.73, 111.92, 106.14, 102.64, 102.43, 56.41, 18.46.
+
HRMS (ESI) m/z: [M+Na]+ calculated for C20H16FNNaO4 376.0961, found 376.0994.
4.2.26 (E)-N-(2,2-dioxidobenzo[e][1,2]oxathiin-6-yl)-3-(3,4,5-trimethoxyphenyl)
1
acrylamide (14a): White solid, Yield: 73%; mp: 244-2450C; H NMR (500 MHz,
DMSO-d6) δ 10.47 (s, 1H), 8.12 (d, J = 2.5 Hz, 1H), 7.83 – 7.69 (m, 2H), 7.55 (dd, J =
26.2, 13.0 Hz, 2H), 7.44 (d, J = 8.9 Hz, 1H), 6.99 (s, 2H), 6.77 (d, J = 15.6 Hz, 1H), 3.85
(s, 6H), 3.71 (s, 3H); 13C NMR (125 MHz, DMSO-d6) δ 164.33, 153.61, 146.65, 141.41,
139.60, 137.73, 137.21, 130.60, 123.51, 123.40, 121.51, 120.14, 119.51, 119.46, 105.77,
60.60, 56.41; HRMS (ESI) m/z: [M+H]+ calculated for C20H20NO7S+ 418.0955, found
418.1014.
4.2.27
(E)-N-(2,2-dioxidobenzo[e][1,2]oxathiin-6-yl)-3-(2-fluoro-4-methoxyphenyl)acrylami
1
de (14b): White solid, Yield: 73%; mp: 245-2460C; H NMR (500 MHz, DMSO-d6) δ
10.49 (s, 1H), 8.11 (d, J = 2.5 Hz, 1H), 7.79 – 7.73 (m, 2H), 7.63 (dd, J = 22.6, 12.4 Hz,
2H), 7.52 (d, J = 10.3 Hz, 1H), 7.43 (d, J = 8.9 Hz, 1H), 6.96 (dd, J = 13.1, 2.4 Hz, 1H),
13
6.90 (dd, J = 8.7, 2.4 Hz, 1H), 6.80 (d, J = 15.8 Hz, 1H), 3.84 (s, 3H); C NMR (125
MHz, DMSO) δ 164.40, 163.19, 162.45 (d, J = 11.7 Hz), 161.19, 146.67, 137.69, 137.20,

133.83, 131.08 (d, J = 5.0 Hz), 123.47 (d, J = 7.9 Hz), 122.23 (d, J = 6.8 Hz), 120.22,
119.46 (d, J = 8.3 Hz), 115.12 (d, J = 11.8 Hz), 111.91, 102.61, 102.41, 56.40. HRMS
(ESI) m/z: [M+H]+ calculated for C18H15FNO5S+ 376.0649, found 376.0664.
4.2.28 (E)-3-(2-(difluoromethoxy)phenyl)-N-(2,2-dioxidobenzo[e][1,2]oxathiin-6-yl)
1
acrylamide (14c): White solid, Yield: 78%; mp: 230-2320C; H NMR (500 MHz,
DMSO-d6) δ 10.57 (s, 1H), 8.12 (d, J = 2.5 Hz, 1H), 7.84 – 7.73 (m, 4H), 7.57 – 7.42 (m,
13
4H), 7.36 (t, J = 7.6 Hz, 1H), 7.32 (d, J = 1.9 Hz, 1H), 6.89 (d, J = 15.8 Hz, 1H); C
NMR (125 MHz, DMSO) δ 164.01, 146.78, 137.51, 137.17, 134.38, 131.90, 128.44,
126.56, 126.33, 124.42, 123.55, 120.38, 119.74 – 119.37 (m), 119.11, 117.0, 115.00.
HRMS (ESI) m/z: [M+H]+ calculated for C18H14F2NO5S+ 394.0555, found 394.0610.
4.2.29 (E)-N-(2,2-dioxidobenzo[e][1,2]oxathiin-6-yl)-3-(4-fluorophenyl)acrylamide
(14d): White solid, Yield: 77%; mp: 245-2460C; 1H NMR (500 MHz, DMSO-d6) δ 10.49
(s, 1H), 8.10 (d, J = 2.4 Hz, 1H), 7.80 - 7.75 (m, 2H), 7.72 (dd, J = 8.5, 5.6 Hz, 2H), 7.63
(d, J = 15.7 Hz, 1H), 7.53 (d, J = 10.3 Hz, 1H), 7.44 (d, J = 8.9 Hz, 1H), 7.30 (t, J = 8.8
Hz, 2H), 6.78 (d, J = 15.7 Hz, 1H); 13C NMR (125 MHz, DMSO) δ 164.43, 164.19,
162.46, 146.69, 140.03, 137.61, 137.19, 131.69, 130.52 (d, J = 8.5 Hz), 123.50 (d, J = 4.8
Hz), 122.12, 120.25, 119.49 (d, J = 5.8 Hz), 116.61, 116.44. HRMS (ESI) m/z: [M+H]+
calculated for C17H13FNO4S+ 346.0544, found 346.0607.
4.2.30 (E)-3-(3,4-difluorophenyl)-N-(2,2-dioxidobenzo[e][1,2]oxathiin-6-yl)
1
acrylamide (14e): White solid, Yield: 72%; mp: 250-2520C; H NMR (500 MHz,
DMSO-d6) δ 10.52 (s, 1H), 8.09 (d, J = 2.4 Hz, 1H), 7.76 (dt, J = 12.3, 6.7 Hz, 3H), 7.61
(d, J = 15.7 Hz, 1H), 7.52 (dd, J = 12.2, 7.6 Hz, 3H), 7.44 (d, J = 8.9 Hz, 1H), 6.80 (d, J
= 15.7 Hz, 1H); 13C NMR (125 MHz, DMSO) δ 163.91, 151.72, 151.14 (d, J = 13.0 Hz),
149.63, 149.18 (d, J = 13.0 Hz), 146.77, 139.03, 137.50, 137.18, 133.00 (dd, J = 6.3, 3.8
Hz), 125.44 (dd, J = 6.4, 3.2 Hz), 123.61 (d, J = 18.1 Hz), 120.32, 119.51 (d, J = 4.7 Hz),
118.65 (d, J = 17.6 Hz), 117.09 (d, J = 17.4 Hz).HRMS (ESI) m/z: [M+H]+ calculated for
C17H12F2NO4S+ 364.0450, found 364.0491.
4.2.31 (E)-N-(2,2-dioxidobenzo[e][1,2]oxathiin-6-yl)-3-(3-(trifluoromethyl)phenyl)
1
acrylamide (14f): White solid, Yield: 73%; mp: 188-1890C; H NMR (500 MHz,
DMSO-d6) δ 10.54 (s, 1H), 8.11 (s, 1H), 8.01 (s, 1H), 7.96 (d, J = 7.7 Hz, 1H), 7.81 –
7.75 (m, 3H), 7.72 (dd, J = 16.2, 8.9 Hz, 2H), 7.53 (d, J = 10.3 Hz, 1H), 7.45 (d, J = 8.9

Hz, 1H), 6.96 (d, J = 15.8 Hz, 1H); 13C NMR (125 MHz, DMSO) δ 163.85, 146.82,
139.40, 137.48, 137.15, 136.28, 132.03, 130.65, 124.56 (d, J = 15.0 Hz), 123.54 (d, J =
4.1 Hz), 120.29, 119.52 (d, J = 7.1 Hz). HRMS (ESI) m/z: [M+H]+ calculated for
C18H13F3NO4S+ 396.0512, found 396.0542.
4.2.32
(E)-3-(4-cyanophenyl)-N-(2,2-dioxidobenzo[e][1,2]oxathiin-6-yl)acrylamide
(14g): White solid, Yield: 76%; mp: 288-2890C; 1H NMR (500 MHz, DMSO-d6) δ 10.60
(s, 1H), 8.10 (d, J = 2.5 Hz, 1H), 7.93 (d, J = 8.4 Hz, 2H), 7.84 (d, J = 8.3 Hz, 2H), 7.80 –
7.75 (m, 2H), 7.69 (d, J = 15.8 Hz, 1H), 7.54 (d, J = 10.3 Hz, 1H), 7.45 (d, J = 8.9 Hz,
1H), 6.96 (d, J = 15.8 Hz, 1H); 13C NMR (125 MHz, DMSO-d6) δ 163.69, 139.28,
137.40, 137.13, 133.37, 128.95, 125.78, 123.62, 123.56, 120.40, 119.55, 119.50, 112.31;
HRMS (ESI) m/z: [M+H]+ calculated for C18H13N2O4S+ 353.0591, found 353.2723.
Carbonic anhydrase inhibition assay
The assay of the catalytic/inhibition of various CA isozymes has been done by
using an SX.18MV-R Applied Photophysics (Oxford, UK) stopped-flow instrument.20
Phenol Red (at a concentration of 0.2 mM) has been used as indicator, working at the
absorbance maximum of 557 nm, with 10 mM Hepes (pH 7.4) as buffer, 0.1 M 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 5-10 s. The substrate here used was saturated CO2 solutions in water at 25 °C. Stock
solutions of inhibitors were prepared at a concentration of 10 μM (in DMSO-water 1:1,
v/v) and dilutions up to 0.01 nM done with the assay buffer mentioned above. The
inhibition constant has been measured by using a minimum of 7 different inhibitor
concentrations. Inhibitor and enzyme solutions were preincubated 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 throughout the 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,21 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.22-24

Acknowledgements
The authors BS and PS are thankful to Department of Pharmaceuticals, Ministry
of Chemicals and Fertilizers, Govt. of India, New Delhi for the award of NIPER
fellowship.
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New Coumarin/Sulfocoumarin linked Phenylacrylamides as Selective
Transmembrane Carbonic Anhydrase Inhibitors: Synthesis and In-Vitro Biological
Evaluation
Baijayantimala Swaina, Andrea Angelic, Priti Singha, Claudiu T. Supuranc* and
Mohammed Arifuddina,b*
.
aDepartment of Medicinal Chemistry, National Institute of Pharmaceutical
Education and Research (NIPER), Balanagar, Hyderabad 500037, India
bDepartment of Chemistry, Anwarul Uloom College, 11-3-918, New Malleypally,
Hyderabad-500001, T. S., India
cUniversità degli Studi di Firenze, Neurofarba Dept., Sezione di Scienze
Farmaceutiche e Nutraceutiche, Via Ugo Schiff 6, 50019 Sesto Fiorentino, Florence, Italy
Conflict of Interest
The authors don’t have any conflict of interest.
Graphical Abstract

New Coumarin/Sulfocoumarin linked Phenylacrylamides as Selective
Transmembrane Carbonic Anhydrase Inhibitors: Synthesis and In-Vitro Biological
Evaluation
Baijayantimala Swaina, Andrea Angelic, Priti Singha, Claudiu T. Supuranc* and
Mohammed Arifuddina,b*.
aDepartment of Medicinal Chemistry, National Institute of Pharmaceutical
Education and
Research (NIPER), Balanagar, Hyderabad 500037, India
bDepartment of Chemistry, Anwarul Uloom College, 11-3-918, New Malleypally,
Hyderabad-500001, T. S., India
cUniversità degli Studi di Firenze, Neurofarba Dept., Sezione di Scienze
Farmaceutiche e Nutraceutiche, Via Ugo Schiff 6, 50019 Sesto Fiorentino, Florence, Italy
Abstract:
Novel series of phenylacrylamide linked coumarins and sulfocoumarins (6a-p,
8a-i, and 14a-g) were synthesized and evaluated against four physiologically relevant
human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms CA I, CA II, CA IX and CA XII
for their inhibitory action. All new compounds when screened for carbonic anhydrase
inhibitory activity have shown selective inhibition towards the tumor associated isoforms
CA IX and CA XII over CA I and II, with inhibition constant in the subnanomolar to
medium nanomolar range. Compound 6b, 14g exhibited significant inhibition with
subnanomolar potency against hCA IX, whereas 6k against hCA XII. The compounds
6b, 14g and 6k exhibited as the lead molecules for future development of cancer
therapeutics
with
a
novel
mechanism
of
action.
O
O
O
S O
O
O
O
O
O
O
O
O
O
N
H
N
H
N
N
H
H
O
F
NC
6k
6b
hCA IX = 40.9 nM
14g
O
6c
F
hCA IX = 39.8 nM
hCA XII = 15.5 nM
hCA IX = 43.0 nM

New Coumarin/Sulfocoumarin linked Phenylacrylamides as Selective
Transmembrane Carbonic Anhydrase Inhibitors: Synthesis and In-Vitro Biological
Evaluation
Baijayantimala Swaina, Andrea Angelic, Priti Singha, Claudiu T. Supuranc* and
Mohammed Arifuddina,b*
.
aDepartment of Medicinal Chemistry, National Institute of Pharmaceutical
Education and Research (NIPER), Balanagar, Hyderabad 500037, India
bDepartment of Chemistry, Anwarul Uloom College, 11-3-918, New Malleypally,
Hyderabad-500001, T. S., India
cUniversità degli Studi di Firenze, Neurofarba Dept., Sezione di Scienze
Farmaceutiche e Nutraceutiche, Via Ugo Schiff 6, 50019 Sesto Fiorentino, Florence, Italy
Highlights
 A series of new phenylacrylamide incorporated coumarin/sulfocoumarin
derivatives (6a-p, 8a-i, and 14a-g) were synthesized.
 The newly synthesized molecules were screened for their inhibition potency
against four CA isoforms hCA I, II, IX, and XII.
 All the compounds were ineffective against off target isoform hCA I, and II
isoforms.
 6a-p, 8a-i, and 14a-g are active against hCA IX and XII with low nanomolar
range.
 Ten compounds were showing excellent inhibition against hCA IX with KI
39.8-92.8 nM and only one molecule was inhibiting hCA XII with KI 15.5 nM
value.