Development of sulfonamides incorporating phenylacrylamido functionalities as carbonic anhydrase isoforms I, II, IX and XII inhibitors

Article abstract of DOI:10.1016/j.bmc.2017.08.047

A series of novel sulfonamides incorporating phenylacrylamido functionalities were synthesized and investigated for the inhibition of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). The physiologically and pharmacologically relevant human (h) isoforms hCA I and II (cytosolic isozymes), as well as the transmembrane tumor-associated hCA IX and XII were included in the study. These compounds showed low nanomolar or sub-nanomolar inhibition constants against hCA II (K_Is in the range of 0.50–50.5 nM), hCA IX (K_Is of 1.8–228.5 nM), and hCA XII (K_Is of 3.5–96.2 nM) being less effective as inhibitors of the off target isoform hCA I. A detailed structure–activity relationship study demonstrates that the nature and position of substituents present on the aromatic part of the scaffold strongly influence the inhibition of CA isoforms. As hCA II, IX and XII are involved in pathologies such as glaucoma and hypoxic, and metastatic tumors, compounds of the type reported in this work may be useful preclinical candidates.

Full text of DOI:10.1016/j.bmc.2017.08.047

Accepted Manuscript
Development of sulfonamides incorporating phenylacrylamido functionalities
as carbonic anhydrase isoforms I, II, IX and XII inhibitors
Srinivas Angapelly, P.V. Sri Ramya, Andrea Angeli, Sonia Del Prete, Clemente
Capasso, Mohammed Arifuddin, Claudiu T. Supuran
PII:
S0968-0896(17)31520-1
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.08.047
BMC 13951
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
25 July 2017
28 August 2017
29 August 2017
Please cite this article as: Angapelly, S., Ramya, P.V.S., Angeli, A., Del Prete, S., Capasso, C., Arifuddin, M.,
Supuran, C.T., Development of sulfonamides incorporating phenylacrylamido functionalities as carbonic anhydrase
isoforms I, II, IX and XII inhibitors, Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/
j.bmc.2017.08.047
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Development of sulfonamides incorporating phenylacrylamido functionalities as
carbonic anhydrase isoforms I, II, IX and XII inhibitors
Srinivas Angapelly,a P.V. Sri Ramya,a Andrea Angelib, Sonia Del Prete,c Clemente Capassoc,
Mohammed Arifuddin,a* 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
cIstituto di Bioscienze e Biorisorse, CNR, via Pietro Castellino 111, 80131 Napoli, Italy
Abstract. A series of novel sulfonamides incorporating phenylacrylamido functionalities
were synthesized and investigated for the inhibition of the metalloenzyme carbonic anhydrase
(CA, EC 4.2.1.1). The physiologically and pharmacologically relevant human (h) isoforms
hCA I and II (cytosolic isozymes), as well as the transmembrane tumor-associated hCA IX
and XII were included in the study. These compounds showed low nanomolar or sub-
nanomolar inhibition constants against hCA II (KIs in the range of 0.50-50.5 nM), hCA IX
(KIs of 1.8-228.5nM), and hCA XII (KIs of 3.5-96.2 nM) being less effective as inhibitors of
the off target isoform hCA I. A detailed structure-activity relationship study demonstrates
that the nature and position of substituents present on the aromatic part of the scaffold
strongly influence the inhibition of CA isoforms. As hCA II, IX and XII are involved in
pathologies such as glaucoma and hypoxic, and metastatic tumors, compounds of the type
reported in this work may be useful preclinical candidates.
Keywords: Phenylacrylamides; Sulfonamide; carbonic anhydrase; isoforms I, II, IX and XII,
Tumors.
_____
* Corresponding authors. E-mail: arif@niperhyd.ac.in (MA); Neurofarba Dept., Sezione di
Scienze Farmaceutiche, E-mail: claudiu.supuran@unifi.it (CTS).

1. Introduction
Carbonic anhydrases (CA, EC 4.2.1.1) are metalloenzymes that catalyze the reversible
hydration of carbon dioxide to bicarbonate and a proton via a ping-pong mechanism (see Eqs.
1 and 2 below), a pivotal reaction for regulation of many physiologic processes. Some of the
members of the CA superfamily are among the most effective enzymes known to date, with
kcat/KM values close to the limit of diffusion-controlled processes.1 Indeed, the products
formed in the catalyzed reaction are either ions with strong buffering activity (bicarbonate) or
hydrated protons (H+ ions). Seven genetically distinct families of CAs (α-, β-, γ-, δ-, ζ-, η-
and θ-) have been reported, and they possess a highly characteristic active site architecture
with half of the cavity aligned only with hydrophilic amino acid residues whereas the
opposite half is composed of hydrophobic residues (X-ray crystal structures are available for
many enzymes belonging to the α-, β-, γ and ζ-class).2-5
Sulfonamide based compounds are the most well studies class of CA inhibitors (CAIs), and
they bind to the metal ion from the enzyme active site, displacing the metal-bound hydroxide
ion/water which is situated at the bottom of a 15 Å deep active site and also coordinated by
three conserved amino acid residues in all α-CAs, His94, His96 and His119 in a tetrahedral
geometry.6-7 In most CA classes, a Zn(II) ion is crucial for the catalytic activity, but examples
of enzymes working with Cd(II), Fe(II) or Co(II) at the active site are also known.5-7
Up until now, 15 human (h) isozymes, all belonging to the α-class, have been identified,
displaying different activities, subcellular localization and tissue expression profiles. They
play key roles in intracellular and extracellular pH homeostasis, respiration, transport of
CO2/bicarbonate between metabolizing tissues and lungs, and in several biochemical
pathways where either CO2 or bicarbonate is required as substrate, bone resorption, renal
acidification, formation of cerebrospinal fluid and lipogenesis.8-11 An extensive research in
this field suggested the role of hCAs in various pathological processes (e.g. glaucoma
formation, tumorigenicity, obesity, and epilepsy).12 Apart for the mammalian enzymes, the
microorganisms CAs also play a crucial role in growth, pH and CO2 sensing as well as
virulence of various fungal/bacterial/protozoan pathogens.13-15
In the past 5 decades, most of the CAIs have been clinically employed as anti-glaucoma
drugs (targeting hCA II, hCA IV, and hCA XII), anticonvulsants (targeting hCA II, hCA VII,

and hCA XIV) and antiobesity agents (targeting hCA VA and hCA VB).16 Isozyme CA XIV
is a trans membrane isoform predominantly expressed on neuronal bodies and axons in the
human brain,17 implicated in regulation of excitatory synaptic transmission and is an
emerging target for neuroprotective agents.18 Neuronal hCA VII facilitate GABAergic
excitation which is an important target for the development of antiepileptics as well as
neuropathic pain killers.19 Two trans membrane hCA isozymes (hCA IX and hCA XII) are
strongly over expressed in a broad range of tumor types and were shown to be functionally
related to oncogenesis/metastatic spread. Targeting these tumor-specific hCA isozymes with
specific inhibitors thus became a promising strategy for cancer therapy, with a sulfonamide
CAI (SLC-0111) in Phase I/II clinical trials.20,25
CA II is the most widely distributed member of the α-CA gene family, being present in
virtually every human tissue and organ and catalytically one of the most efficient among all
the α-CA isoforms.1 Yoshiura et al.21 demonstrated that CA II is expressed in the tumor
vessel endothelium of melanoma, oesophageal, renal, and lung cancers and later it was also
found in the neo-vessels of malignant astrocytic gliomas.22-24
Some of the CAI drugs and drug candidates possessing sulfonamide moiety are depicted in
Fig-1, among which acetazolamide is in clinical use for more than 60 years,1 whereas SLC-
0111 successfully ended a Phase-I trial (see clinical trials. Gov. NCT02215850 and entered
Phase II recently) for the treatment of solid tumors overexpressing hCA IX and XII, and
DTP-348 a 5-nitroimidazole sulfonamide derivative in preclinical investigations for the
treatment of solid tumors such as neck and head neoplasms (see clinical trials. Gov.
NCT02216669).25-26 Pazopanib is clinically used for several years whereas indisulam and
celecoxib are currently under clinical investigations in various types of tumors (Fig. 1).1,25
Thus, investigation of novel aromatic/heterocyclic scaffolds incorporating sulfonamide
moiety is critical for identifying CAIs with interesting inhibitory and selectivity profiles. In
our previous work, we explored that 4-sulfamoylbenzene-β-lactams, which showed
significant inhibitory effects against several hCA isozymes at subnano/nanomolar
concentrations.27 In continuation of our interest in the design of such pharmacological agents,
herein we describe the synthesis and biological evaluation of novel sulfonamide
incorporating phenylacrylamido functionalities as CA I, CA II, CA IX and CA XII inhibitors.

Figure 1. Chemical structures of sulfonamide containing CAIs in clinical use/clinical trials.
2. Results and Discussion
2.1. Chemistry
The rationale for designing the CAIs reported here is based on the tail approach.26a In several
studies,25,26 it has been shown that compounds containing an elongated scaffold possess CA
selectivity for inhibiting various isoforms of interest due to the fact that the tail fragment of
the inhibitor interacts with the entrance of the CA active site, which is the most variable part
among the various CA isoforms.1 This is the reason why compounds with
elaborated/elongated scaffolds such as SLC-0111, pazopanib, indisulam and celecoxib show
some CA-selective inhibition profiles compared to acetazolamide which is a pan-inhibitor
(Fig. 1).1,25 Thus, we decided to incorporate arylacrylamide moieties as tails in the molecule
of benzenesulfonamides, as this type of substitution pattern was scarcely investigated up until
now for the design of sulfonamide CAIs.
The target sulfonamides bearing acryl amide functionalities 4a-p were synthesized according
to the general procedure outlined in the Scheme 1. Cinnamic acids 2a-p were prepared by
employing the Knoevenagel condensation reaction between substituted benzaldehydes 1a-p
and malonic acid (Scheme 1). Subsequently, cinnamic acids 2a-p were converted to the
corresponding cinnamoyl chlorides 3a-p by using SOCl2. The target compounds 4a-p were
obtained by reacting cinnamoyl chlorides 3a-p with sulfanilamide in pyridine at room
temperature, in the excellent yields (72-85%). All the newly synthesized sulfonamides were
1
characterized by spectroscopic techniques such as HRMS, H and 13C NMR spectrometry.
1
The H NMR spectrum of (E)-3-(3,4-dimethoxyphenyl)-N-(4-sulfamoylphenyl)acrylamide,
4k showed a broad singlet of amide (-NH) proton at the δ 10.47 ppm and two singlets of
methoxy protons appearing at δ 3.83 and 3.80 and the rest of all protons appeared in the

aromatic region. In the 13C NMR spectrum of 4k the amide carbon appeared at δ 164.5 ppm
and the aromatic carbons appeared in the range of δ 151.4 - 111.5 ppm, whereas the methoxy
carbons appeared at δ 55.98 ppm (see Experimental for details and Table 1).
Scheme 1. Preparation of sulfonamides 4a-4p based on the sulfanilamide scaffold.
Table 1: Structure of compounds 4a-p
Compound
R1
R2
CF3
H
F
H
R3
H
CF3
H
F
R4
H
H
H
H
Yield (%)
81
H
H
H
H
H
H
4a
4b
4c
4d
4e
4f
82
76
79
77
H
H
Cl
CN
H
H
80
H
H
H
F
Cl
OCH3
OCH3
H
H
H
OCF3
H
F
H
H
H
Cl
H
OCH3
H
H
H
H
78
75
72
74
85
84
81
85
4g
4h
4i
OCHF2
H
H
H
H
OCH3
H
H
4j
OCH3
OCH3
OCH3
OCH3
OCH(CH3)2
OCH3
4k
4l
4m
4n
4o
4p
H
F
80
74
H

2.2. CA inhibitory assay
The newly synthesized sulfonamide incorporating phenylacrylamido functionalities 4a-p
were evaluated as inhibitors of four physiologically relevant CA isoforms, the cytosolic hCA
I and II and the trans membrane tumor-associated hCA IX and XII, using the CO2 hydrase
assay.29 The clinically employed sulfonamides acetazolamide (AAZ) and sulfanilamide (SA)
have been used as standard, and the results are presented in Table 2.
Table 2. Inhibitory potency data against isoforms hCA I, II, IX and XII along with selectivity ratio for
inhibition of isoforms hCA IX and XII over hCA I and II for compounds 4a-p.
Ki (nM)a
hCA I hCA II
Selectivity ratiob
hCA
IX
hCA
XII
50.5
8.0
I/IX
II/IX
I/XII
II/XII
Compound
314.2
768.6
374.4
586.5
922.5
1892.3
630.3
635.9
275.6
4812.1
210.7
4002.6
465.0
919.4
619.6
383.7
250
3.8
34.5
0.63
5.0
11.6
2.4
27.08
320.25
187.2
244.37
73.21
236.53
128.63
353.27
1.82
0.32
14.37
0.31
2.08
0.27
5.4
6.22
320.25
3.89
0.07
4.31
0.006
0.14
0.09
4a
4b
4c
2.0
96.2
35.5
38.4
3.5
2.4
16.52
24.02
4d
4e
3.5
12.6
8.0
43.2
5.1
540.65 12.34
4f
4.9
3.5
1.04
0.46
0.02
0.22
0.053
0.03
1.51
1.40
0.36
0.40
0.46
1.22
180.08
138.23
30.28
1.45
0.18
0.43
0.79
0.056
0.06
1.66
1.12
1.51
0.91
2.12
6.48
4g
4h
4i
0.83
4.0
1.8
4.6
150.7
228.5
9.3
9.1
50.5
0.50
4.0
67.4
8.9
21.05
22.65
31.71
48.43
95.77
83.72
34.25
9.68
71.39
4j
23.67
4k
4l
126.2
9.6
61.6
8.7
64.97
4m
4n
4o
4p
AAZ
SA
14.5
7.4
53.44
5.3
6.6
139.30
126.44
78.30
7.4
20.3
11.2
25.8
294
4.9
4.5
4.9
12.1
240
5.7
433.85
675.66
25000
37
85.01
AAZ: acetazolamide; SA = sulfanilamide (reference compounds)
aMean from 3 different assays, by a stopped flow technique (errors were in the range of 5-10 % of
the reported values).
bSelectivity as determined by the ratio of Kis for hCA isozyme relative to hCA IX and hCA XII.

The following structure activity relationship (SAR) may be noted regarding the inhibition
data of Table 2:
(i) The ubiquitous cytosolic hCA I was inhibited by all compounds in the high nanomolar to
micromolar range. The interesting case for these molecules was the position of substituent on
the benzene ring. Compounds with one substituent, in position R2, showed a better activity
than those possessing substituents in position R1 or R3. Moreover, monofluorine-containing
scaffolds prove to be more active than other mono substituent scaffolds. An addition of
second substituent in the R2 and R3 position, such as for compound 4i and 4k, led to a
significant enhancement of the inhibitory potency. A further addition of substituents,
however decreased the activity.
(ii) The other cytosolic isoform, hCA II, was effectively inhibited by almost all compounds
here synthesized, in the subnanomolar to low/medium nanomolar range (KI comprised
between 0.5 and 50.5 nM). In analogy to hCA I, the position of the mono substituent in the
scaffold was crucial for modulating the potency of inhibition. Placement in R2 position (4a
and 4c) increased the activity near 10 times than the same scaffold with the substituent in
position R3 (4b and 4d). The second analogy with the other cytosolic isoform was the
presence of fluorine atom in monosubstituted scaffolds, which prove to be essential for
increased potency of inhibition. A difference with hCA I was that a substituent in position R1
improved the hCA II inhibitory activity of these sulfonamides. Compound 4i with two
methoxy groups in R2 and R3 had the best inhibition profile with Ki 0.5 nM.
(iii) The transmembrane, tumor-associated hCA IX was effectively inhibited by all
compounds herein reported, with KI values comprised between 2.0 and 228.5 nM.
Substituents in R2 versus R3 this time did not improve the inhibitory activity. A rather
decreased inhibition potency for compounds 4a compared to 4b-d, of nearly 5 times, was
observed. Furthermore, the R1 position showed the best position for increased the activity. In
the end, scaffolds with two halogen atoms (4i and 4j) or three substituents such as 4l showed
a decreased inhibition potency of up to high nanomolar range (KI 126.2 to 228.5 nM)
compared to 4b-d.
(iv) The second transmembrane isoform here considered, hCA XII, was effectively inhibited
by all compounds in the low to medium nanomolar range (KIs of 3.5 to 96.2 nM). In analogy
with the other transmembrane isoforms, a substituent in R3 position increased the potency

compared to the same substituent in position R2. Moreover, scaffolds with more substituents
did not show an improved inhibition potency for this isoform.
(v) An interesting inhibition profile was observed for compounds 4a and 4b. When CF3
group was in the R2 position, a 45 fold selectivity for inhibiting isoform hCA II over hCA IX
was observed. On the other hand when CF3 was in R3 (4b), the compound showed selectivity
for the transmembrane hCA IX over the cytosolic hCA I and II isoforms. Another interesting
example is furnished by compounds 4i and 4j (incorporating two substituents) and 4l (with
three methoxy groups in its scaffold), which showed a remarkable selectivity against hCA II
over hCA IX and XII. Finally, compound 4f possessing a CN moiety as R3 showed an
interesting selectivity profile for inhibition of the transmembrane isoforms in particular for
hCA XII over hCA I and II (Table 2).
3. Conclusions
A series of novel sulfonamide incorporating phenylacrylamido functionalities have been
synthesized. These molecules were investigated as in vitro inhibitors of physiologically and
pharmacologically relevant isoforms of the human (h) carbonic anhydrases (CAs), the
cytosolic hCA I and II, and the transmembrane tumor associated hCA IX and XII. The hCA
inhibitory activities results showed that these molecules are not very active against hCA I, an
offtarget isoform. Compounds 4c (3-F), 4h (2-OCHF2) and 4k (3,4-dimethoxy) were found to
effectively inhibit hCA II (0.50−0.83 nM) at subnanomolar concentrations, Compounds 4b
(4-CF3, Ki = 2.4 nM) and 4f (4-CN, Ki = 8 nM) were found to selectively inhibit hCA IX,
which is over expressed due to hypoxia, and crucial for tumor cell survival and proliferation.
hCA XII was also well inhibited by some of the new compounds reported here (Kis of 3.5-
96.2 nM) and specifically inhibited by 4f (4-CN, Ki = 3.5 nM).
4. Experimental section
4.1. General
All solvents were purified and dried using standard methods prior to use. Commercially
available reagents were used without further purification. All reactions involving air- or
moisture-sensitive compounds were performed under a nitrogen atmosphere using dried
glassware and syringe techniques to transfer solutions. Analytical thin-layer chromatography
(TLC) was carried out on Merck silica gel 60 F-254 aluminium plates. Melting points were
determined on Stuart digital melting-point apparatus/SMP 30 in open capillary tubes and are

uncorrected. Nuclear magnetic resonance (1H-NMR, 13C-NMR) spectra were recorded using
an Avance Brucker 500 MHz, 125 MHz spectrometer in DMSO-d6. Chemical shifts are
reported in parts per million (ppm) with TMS as an internal reference, and the coupling
constants (J) are expressed in hertz (Hz). Splitting patterns are denoted as follows: s, singlet;
d, doublet; t, triplet; m, multiplet; dd, double 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 preparation of trans-Cinnamic acids (2a-p): To a stirred
solution of malonic acid (10 mmol) in pyridine (5 mL) and piperidine (3-5 drops) the
corresponding aldehydes 1a-p (8 mmol) were added. The resulting mixture was stirred at
90 oC for 8-12 h. The progress of the reaction was monitored by TLC analyses which
indicated the disappearance of the starting material. After the completion of the reactions the
reaction mixture were cooled to ambient temperature and the reaction mixture was
neutralized with 1N HCl in ice bath resulting a white solid. This soled was filtered and
washed three times with cold water. The recrystallization were done from aqueous ethanol
(1:1) afforded the product 2a-p.
4.3. General procedure for the preparation of trans-Cinnamoyl chlorides (3a-p): To a
solution of appropriate substituted trans-cinnamic acid 2a–p (1 mmol) in dry
dichloromethane the thionyl chloride (2 mmol) and a catalytic amount of DMF was added.
The reaction mixture was refluxed for 2-4 h under anhydrous conditions, the solvent was
evaporated under vacuum to obtain corresponding substituted cinnamoyl chlorides 3a-p
which was further used for the next step without further purification.
4.4. General Procedure for the Synthesis of Compounds (4a-p): To a solution of
sulfanilamide (1.0 equiv) in dry pyridine (5 mL/1 mmol of amine) the appropriate substituted
cinnamoyl chlorides 3a-p (1.1 equiv) were added. The resulting mixture was stirred at room
temperature under nitrogen atmosphere for 4-8 h. After completion of the reaction, the
reaction was quenched with ice which resulted in a solid. The solid were filtered and washed
2-3 times with cold water to obtained crude precipitate which was recrystallized from ethanol
to produces the products (4a-p) in good yields of 72-85%.
4.5. Characterization of compounds 4a-p
4.5.1. (E)-N-(4-sulfamoylphenyl)-3-(3-(trifluoromethyl)phenyl)acrylamide (4a)

1
White solid, Yield: 81%, m.p 234 – 236 °C; H NMR (500 MHz, DMSO) δ 10.58 (d, J =
21.7 Hz, 1H), 8.01 (s, 1H), 7.96 (d, J = 7.7 Hz, 1H), 7.87 (d, J = 8.8 Hz, 2H), 7.76 (ddd, J =
31.0, 13.4, 8.3 Hz, 5H), 7.29 (s, 2H), 6.99 (d, J = 15.8 Hz, 1H). 13C NMR (126 MHz, DMSO)
δ 164.03, 142.47, 139.66, 139.05, 136.22, 132.07, 130.48 (d, J = 31.8 Hz), 127.29, 126.70,
125.57, 124.63 (d, J = 3.8 Hz), 124.48, 123.40, 119.31. HRMS (ESI) m/z: [M + H]+
calculated for C16H14F3N2O3S 371.0672, found 371.0682.
4.5.2. (E)-N-(4-sulfamoylphenyl)-3-(4-(trifluoromethyl)phenyl)acrylamide (4b)
1
White solid, Yield: 82%, m.p 268.5 – 270 °C; H NMR (500 MHz, DMSO) δ 10.66 (s, 1H),
7.87 (d, J = 8.7 Hz, 4H), 7.82 (t, J = 7.8 Hz, 4H), 7.71 (d, J = 15.8 Hz, 1H), 7.30 (s, 2H), 6.98
(d, J = 15.8 Hz, 1H). 13C NMR (126 MHz, DMSO) δ 163.96, 142.43, 139.69, 139.09, 130.21,
128.93, 127.28, 126.39 (d, J = 3.75 Hz), 125.10, 123.46, 119.37. HRMS (ESI) m/z: [M + H]+
calculated for C16H14F3N2O3S 371.0672, found 371.0679.
4.5.3. (E)-3-(3-fluorophenyl)-N-(4-sulfamoylphenyl)acrylamide (4c)
1
White solid, Yield: 76%, m.p 269 −270 °C; H NMR (500 MHz, DMSO) δ 10.59 (s, 1H),
7.86 (d, J = 8.8 Hz, 2H), 7.80 (d, J = 8.7 Hz, 2H), 7.64 (d, J = 15.7 Hz, 1H), 7.54 – 7.47 (m,
3H), 7.31 – 7.24 (m, 3H), 6.89 (d, J = 15.7 Hz, 1H).13C NMR (126 MHz, DMSO) δ 164.15,
163.90, 161.96, 142.50, 140.11 (d, J = 2.5 Hz), 139.01, 137.61 (d, J = 8.0 Hz), 131.51 (d, J =
8.4 Hz), 127.26, 124.40 (d, J = 2.5 Hz), 123.87, 119.34, 117.12 (d, J = 21.2 Hz), 114.75 (d, J
= 21.9 Hz). HRMS (ESI) m/z: [M + H]+ calculated for C15H14FN3O2S 321.0696, found
321.0641.
4.5.4. (E)-3-(4-fluorophenyl)-N-(4-sulfamoylphenyl)acrylamide (4d)
1
White solid, Yield: 79%, m.p 277 – 278.5 °C; H NMR (500 MHz, DMSO) δ 10.55 (s, 1H),
7.72 (dd, J = 8.5, 5.6 Hz, 2H), 7.64 (d, J = 15.7 Hz, 1H), 7.35 – 7.23 (m, 4H), 6.80 (d, J =
15.7 Hz, 1H). 13C NMR (126 MHz, DMSO) δ 164.38, 162.48, 142.60, 140.29, 138.89,
131.67 (d, J = 3.1 Hz), 130.55 (d, J = 8.6 Hz), 127.25, 122.15, 119.28, 116.54 (d, J = 21.8
Hz). HRMS (ESI) m/z: [M + H]+ calculated for C15H14FN3O2S 321.0696, found 321.0641.
4.5.5. (E)-3-(4-chlorophenyl)-N-(4-sulfamoylphenyl)acrylamide (4e)
1
White solid, Yield: 82%, m.p 278 – 279 °C; H NMR (500 MHz, DMSO) δ 10.58 (s, 1H),
7.86 (d, J = 8.8 Hz, 2H), 7.80 (d, J = 8.8 Hz, 2H), 7.70 – 7.61 (m, 3H), 7.52 (t, J = 9.0 Hz,
2H), 7.28 (s, 2H), 6.86 (d, J = 15.7 Hz, 1H). 13C NMR (126 MHz, DMSO) δ 164.24, 142.54,
140.09, 138.96, 134.91, 133.99, 130.01, 129.58, 127.26, 123.08, 119.31. HRMS (ESI) m/z:
[M + H]+ calculated for C15H14ClN2O3S 337.0408, found 337.0415.

4.5.6. (E)-3-(4-cyanophenyl)-N-(4-sulfamoylphenyl)acrylamide (4f)
White solid, Yield: 80%, m.p 308 – 309 °C; 1H NMR (500 MHz, DMSO) δ 10.72 (d, J = 51.2
Hz, 1H), 7.98 – 7.90 (m, 2H), 7.90 – 7.76 (m, 6H), 7.70 (d, J = 15.8 Hz, 1H), 7.29 (s, 2H),
6.98 (d, J = 15.8 Hz, 1H). 13C NMR (126 MHz, DMSO) δ 163.86, 150.06, 142.38, 139.64,
139.53, 139.14, 133.39, 128.97, 127.28, 125.76, 119.41, 112.33. HRMS (ESI) m/z: [M + H]+
calculated for C16H14N3O3S 328.0750, found 328.0748.
4.5.7. (E)-N-(4-sulfamoylphenyl)-3-(4-(trifluoromethoxy)phenyl)acrylamide (4g)
1
White solid, Yield: 78%, m.p 251 – 252 °C; H NMR (500 MHz, DMSO) δ 10.61 (s, 1H),
7.90 – 7.84 (m, 2H), 7.83 – 7.76 (m, 4H), 7.67 (d, J = 15.7 Hz, 1H), 7.46 (d, J = 8.1 Hz, 2H),
7.29 (s, 2H), 6.87 (d, J = 15.7 Hz, 1H). 13C NMR (126 MHz, DMSO) δ 164.17, 149.60,
142.52, 139.82, 138.99, 134.35, 130.24, 127.26, 123.46, 121.95, 119.32. HRMS (ESI) m/z:
[M + H]+ calculated for C16H14F3N2O4S 387.0621, found 387.0625.
4.5.8. (E)-3-(2-(difluoromethoxy)phenyl)-N-(4-sulfamoylphenyl)acrylamide (4h)
1
White solid, Yield: 75%, m.p 277 – 278 °C; H NMR (500 MHz, DMSO) δ 10.64 (s, 1H),
7.82 (ddd, J = 20.2, 16.7, 7.8 Hz, 6H), 7.55 – 7.47 (m, 1H), 7.41 – 7.14 (m, 5H), 6.92 (d, J =
15.8 Hz, 1H). 13C NMR (126 MHz, DMSO) δ 164.19, 149.67, 142.50, 139.01, 134.59,
131.96, 128.44, 127.26, 126.51, 126.33, 124.45, 119.62, 119.37, 119.09, 117.04, 114.98.
HRMS (ESI) m/z: [M + H]+ calculated for C16H15F2N2O4S 369.0715, found 369.0726.
4.5.9. (E)-3-(3,4-difluorophenyl)-N-(4-sulfamoylphenyl)acrylamide (4i)
White solid, Yield: 72%, m.p 263 – 264 °C; 1H NMR (500 MHz, DMSO) δ 10.59 (s, 1H),
7.86 (d, J = 8.8 Hz, 2H), 7.82 – 7.73 (m, 3H), 7.62 (d, J = 15.7 Hz, 1H), 7.54 (dd, J = 9.2, 4.6
Hz, 2H), 7.29 (s, 2H), 6.82 (d, J = 15.7 Hz, 1H). 13C NMR (126 MHz, DMSO) δ 164.10 (s),
151.65 (s), 151.20 (s), 149.76 (s), 142.48 (s), 139.15 (d, J = 30.0 Hz), 133.04 (s), 127.24 (d, J
= 4.0 Hz), 125.50 (s), 123.74 (s), 119.36 (s), 118.67 (d, J = 17.4 Hz), 117.11 (d, J = 17.7 Hz).
HRMS (ESI) m/z: [M + H]+ calculated for C15H13F2N2O3S 333.0609, found 339.0615.
4.5.10. (E)-3-(3,5-dichlorophenyl)-N-(4-sulfamoylphenyl)acrylamide (4j)
White solid, Yield: 74%, m.p 283.5 – 285 °C; 1H NMR (500 MHz, DMSO) δ 10.58 (s, 1H),
7.85 (d, J = 8.8 Hz, 2H), 7.80 (d, J = 8.8 Hz, 2H), 7.72 (d, J = 1.6 Hz, 2H), 7.66 (d, J = 1.7
Hz, 1H), 7.60 (d, J = 15.7 Hz, 1H), 7.29 (s, 2H), 6.94 (d, J = 15.8 Hz, 1H). 13C NMR (126
MHz, DMSO) δ 163.81, 142.35, 139.13, 138.85, 138.35, 135.14, 129.37, 127.29, 126.75,

125.64, 119.35. HRMS (ESI) m/z: [M + H]+ calculated for C15H13Cl2N2O3S 371.0018, found
371.0022.
4.5.11. (E)-3-(3,4-dimethoxyphenyl)-N-(4-sulfamoylphenyl)acrylamide (4k)
1
White solid, Yield: 85%, m.p 247 – 248 °C; 1H NMR (500 MHz, DMSO) δ H NMR (500
MHz, DMSO) δ 10.47 (s, 1H), 7.86 (d, J = 8.8 Hz, 2H), 7.79 (d, J = 8.8 Hz, 2H), 7.58 (d, J =
15.6 Hz, 1H), 7.24 (dd, J = 18.1, 13.0 Hz, 4H), 7.03 (d, J = 8.3 Hz, 1H), 6.72 (d, J = 15.6 Hz,
1H), 3.82 (d, J = 10.5 Hz, 6H). 13C NMR (126 MHz, DMSO) δ 164.76, 151.07, 149.42,
142.81, 141.66, 138.68, 127.76, 127.23, 122.48, 119.79, 119.13, 112.25, 110.57, 56.06,
55.91. HRMS (ESI) m/z: [M + H]+ calculated for C17H19N2O5S 363.1009, found 363.1099.
4.5.12. (E)-N-(4-sulfamoylphenyl)-3-(3,4,5-trimethoxyphenyl)acrylamide (4l)
1H NMR (500 MHz, DMSO) δ 10.53 (s, 1H),
−273 °C;
White solid, Yield: 84%, m.p 272
7.83 (dd, J = 35.0, 8.0 Hz, 4H), 7.59 (d, J = 15.6 Hz, 1H), 7.28 (s, 2H), 6.99 (s, 2H), 6.79 (d,
J = 15.6 Hz, 1H), 3.85 (s, 6H), 3.71 (s, 3H). 13C NMR (126 MHz, DMSO) δ 164.51, 153.60,
142.71, 141.69, 139.57, 138.80, 130.58, 127.25, 121.50, 119.18, 105.74, 60.60, 56.38. HRMS
(ESI) m/z: [M + H]+ calculated for C18H21N2O6S 393.1109, found 393.1125.
4.5.13. (E)-3-(2,4-dimethoxyphenyl)-N-(4-sulfamoylphenyl)acrylamide (4m)
White solid, Yield: 81%, m.p 252 – 253 °C; 1H NMR (500 MHz, DMSO) δ 10.42 (s, 1H),
7.86 (d, J = 8.8 Hz, 2H), 7.82 – 7.72 (m, 3H), 7.53 (d, J = 8.5 Hz, 1H), 7.26 (s, 2H), 6.78 (d, J
= 15.7 Hz, 1H), 6.68 – 6.59 (m, 2H), 3.90 (s, 3H), 3.83 (s, 3H). 13C NMR (126 MHz, DMSO)
δ 165.29, 162.75, 159.86, 142.93, 138.55, 136.79, 130.44, 127.20, 119.78, 119.11, 116.31,
106.59, 99.01, 56.22, 55.94. HRMS (ESI) m/z: [M + H]+ calculated for C17H18N2O5S
363.1099, found 363.1022.
4.5.14. (E)-3-(4-methoxyphenyl)-N-(4-sulfamoylphenyl)acrylamide (4n)
1
White solid, Yield: 85%, m.p 255 – 256 °C; H NMR (500 MHz, DMSO) δ 10.47 (s, 1H),
7.85 (d, J = 8.8 Hz, 2H), 7.79 (d, J = 8.7 Hz, 2H), 7.60 (dd, J = 12.2, 5.4 Hz, 3H), 7.27 (s,
2H), 7.02 (d, J = 8.7 Hz, 2H), 6.70 (d, J = 15.7 Hz, 1H), 3.81 (s, 3H). 13C NMR (126 MHz,
DMSO) δ 164.76, 161.26, 142.78, 141.29, 138.69, 130.01, 127.58, 127.23, 119.62, 119.16,
115.00, 55.80. HRMS (ESI) m/z: [M + H]+ calculated for C16H17N2O4S 333.0904, found 333.
0913.
4.5.15. (E)-3-(4-isopropoxyphenyl)-N-(4-sulfamoylphenyl)acrylamide (4o)

1
White solid, Yield: 80%, m.p 238.5 – 240 °C; H NMR (500 MHz, DMSO) δ 10.46 (s, 2H),
7.85 (d, J = 8.6 Hz, 4H), 7.79 (d, J = 8.5 Hz, 4H), 7.62 – 7.52 (m, 6H), 7.27 (s, 4H), 6.99 (d, J
= 8.5 Hz, 4H), 6.69 (d, J = 15.7 Hz, 2H), 4.69 (dt, J = 11.9, 5.9 Hz, 2H), 1.29 (d, J = 5.9 Hz,
13H). 13C NMR (126 MHz, DMSO) δ 164.79, 159.55, 142.79, 141.34, 138.68, 130.07,
127.22, 119.43, 119.15, 116.35, 69.86, 22.24. HRMS (ESI) m/z: [M + H]+ calculated for
C18H21N2O4S 361.1217, found 361.1229.
4.5.16. (E)-3-(2-fluoro-4-methoxyphenyl)-N-(4-sulfamoylphenyl)acrylamide (4p)
White solid, Yield: 74%, m.p 281 – 282 °C; 1H NMR (500 MHz, DMSO) δ 10.55 (s, 1H),
7.86 (d, J = 8.8 Hz, 2H), 7.79 (d, J = 8.8 Hz, 2H), 7.64 (dd, J = 19.5, 12.3 Hz, 2H), 7.28 (s,
2H), 6.96 (dd, J = 13.1, 2.4 Hz, 1H), 6.90 (dd, J = 8.7, 2.4 Hz, 1H), 6.83 (d, J = 15.8 Hz, 1H),
3.83 (s, 3H). 13C NMR (126 MHz, DMSO) δ 164.59 (s), 163.22 (s), 162.49 (d, J = 11.8 Hz),
161.22 (s), 142.68 (s), 138.82 (s), 134.11 (s), 131.15 (d, J = 5.1 Hz), 127.23 (s), 122.23 (d, J
= 7.0 Hz), 119.24 (s), 115.09 (d, J = 11.9 Hz), 111.91 (s), 102.51 (d, J = 25 Hz), 56.40 (s).
HRMS (ESI) m/z: [M + H]+ calculated for C16H16FN2O4S 351.0809, found 351.0820.
4.6. Carbonic anhydrase inhibition
An SX.18MV-R Applied Photophysics (Oxford, UK) stopped-flow instrument has been used
to assay the catalytic/inhibition of various CA isozymes.28 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),1 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. At least 7 different inhibitor concentrations have been used for measuring
the inhibition constant. 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,29 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.29-31

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Development of sulfonamides incorporating phenylacrylamido functionalities as
carbonic anhydrase isoforms I, II, IX and XII inhibitors
Srinivas Angapelly, P. V. Sri Ramya, Andrea Angeli, Sonia Del Prete, Clemente Capasso,
Mohammed Arifuddin,* Claudiu T. Supuran*