Discovery of 1,1′-Biphenyl-4-sulfonamides as a New Class of Potent and Selective Carbonic Anhydrase XIV Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.5b01144

New 1,1′-biphenylsulfonamides were synthesized and evaluated as inhibitors of the ubiquitous human carbonic anhydrase isoforms I, II, IX, XII, and XIV using acetazolamide (AAZ) as reference compound. The sulfonamides 1-21 inhibited all the isoforms, with K_i values in the nanomolar range of concentration, and were superior to AAZ against all of them. X-ray crystallography and molecular modeling studies on the adducts that compound 20, the most potent hCA XIV inhibitor of the series (K_i = 0.26 nM), formed with the five hCAs, provided insight into the molecular determinants responsible for the high affinity of this molecule toward the target enzymes. The results pave the way to the development of 1.1′-biphenylsulfonamides as a new class of highy potent hCA XIV inhibitors.

Full text of DOI:10.1021/acs.jmedchem.5b01144

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Article
Discovery of 1,1'-Biphenyl-4-sulfonamides as a New Class
of Potent and Selective Carbonic Anhydrase XIV Inhibitors
Giuseppe La Regina, Antonio Coluccia, Valeria Famiglini, Sveva Pelliccia, Ludovica Monti,
Daniela Vullo, Elisa Nuti, Vincenzo Alterio, Giuseppina De Simone, Peiwen Pan, Seppo
Parkkila, Claudiu T Supuran, Armando Rossello, Romano Silvestri, and Simona Maria Monti
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01144 • Publication Date (Web): 25 Oct 2015
Downloaded from http://pubs.acs.org on October 30, 2015
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Discovery of 1,1'-Biphenyl-4-sulfonamides as a New Class of Potent and Selective Car-
bonic Anhydrase XIV Inhibitors
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†
†
†
†
†
Giuseppe La Regina, Antonio Coluccia, Valeria Famiglini, Sveva Pelliccia, Ludovica Monti,
æ
±
≠
≠
≠
Daniela Vullo, Elisa Nuti, Vincenzo Alterio, Giuseppina De Simone, Simona Maria Monti,
§
§
æ
±
†,*
Peiwen Pan, Seppo Parkkila, Claudiu T. Supuran, Armando Rossello, and Romano Silvestri,
†
Istituto Pasteur Italia - Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Far-
maco, Sapienza Università di Roma, Piazzale Aldo Moro 5, I-00185 Roma, Italy
æ
Dipartimento Neurofarba, Sezione di Scienze Farmaceutiche e Nutraceutiche, Università di Firenze,
Via Ugo Schiff 6, I-50019 Sesto Fiorentino, Firenze, Italy
±
Dipartimento di Farmacia, Università di Pisa, Via Bonanno Pisano 6, I-56126 Pisa, Italy
≠
Istituto di Biostrutture e Bioimmmagini – Consiglio Nazionale delle Ricerche, Via Mezzocannone 16,
I-80134-Napoli, Italy
§
School of Medicine, University of Tampere and Tampere University Hospital, Tampere, Finland
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ABSTRACT
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New 1,1'-biphenylsulfonamides were synthesized and evaluated as inhibitors of the ubiquitous human
carbonic anhydrase isoforms I, II, IX, XII and XIV using acetazolamide (AAZ) as reference compound.
The sulfonamides 1-21 inhibited all the isoforms with Ki values in the nanomolar range of concentra-
tion, and were superior to AAZ against all of them. X-ray crystallography and molecular modeling
studies on the adducts that compound 20, the most potent hCA XIV inhibitor of the series (Ki = 0.26
nM), formed with the five hCAs, provided insight into the molecular determinants responsible for the
high affinity of this molecule toward the target enzymes. The results pave the way to the development
of 1.1’-biphenylsulfonamides as a new class of highy potent hCA XIV inhibitors.
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INTRODUCTION
Carbonic anhydrases (CAs, EC 4.2.1.1) are metal containing enzymes which catalyze the production of
monohydrogen carbonate from carbon dioxide and water.1 Currently, there are at least six well de-
scribed genetically distinct CA families, and fifteen human CA (hCA) isoforms belonging to the α-
class.2,3 CAs are involved in key physiological processes, such as pH homeostasis, cell differentiation
and proliferation, and neurotransmission. Consequently, their disregulated expression and/or abnormal
activity may have important pathological consequences. For this reason, in the past years there has
been an ever increasing interest in the design of new CA inhibitors.4-6
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CAs I and II are isoforms ubiquitously spread throughout the human body. CA I is involved in
retinal and cerebral edema, whereas CA II is involved in several pathologies, for example glaucoma,7
epilepsy,8 as well as prevention of acute mountain sickness.9 CA IX and CA XII are well described tar-
gets for the design of anticancer drugs.10-13 Finally, CA XIV is a potential target for the design of
agents against epilepsy6 and some retinopathies.14 Over the past decade several new classes of CA in-
hibitors (CAIs) have been described in search for potent inhibitors of a specific hCA isoform.15 The
strategy to incorporate a linker between a benzensulfonamide moiety and an appropriate tail has been
successfully adopted in some cases.16 However, despite the variety of CAIs developed, effective inhibi-
tors of CA XIV have not been exhaustively explored so far.
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Comparison between the recently solved structure of hCA XIV and other human isoforms iden-
tified the region of the active site containing residues 127-136 as a hot spot to be considered in the de-
sign of selective hCA XIV inhibitors.17 We hypothesized that the unexplored 1,1'-biphenyl-4-
sulfonamide scaffold, extending the chemospace of the benzensulfonamides, could allow for the devel-
opment of inhibitors able to interact with this active site region. On the basis of this hypothesis, the pre-
sent study was designed to investigate new 1,1'-biphenyl-4-sulfonamide derivatives as CA XIV inhibi-
tors. Herein, we describe the synthesis, biological evaluation of 1,1'-biphenyl-4-sulfonamides 1-21 and
the structural characterization, by using X-ray crystallography and molecular modeling techniques, of
the most active compound of the series in complex with the target enzymes.
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Scheme 1. Synthesis of 1,1'-Biphenyl-4-sulfonamides 1-21.a
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5
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7
8
9
O
O
O
O
O
O
S
S
H2N
H2N
S
b
H2N
a
O
B
O
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COOCH3
COOH
22
1
3
O
O
O
O
S
S
H2N
H2N
COOH
COOCH3
a
b
22
22
X
14-16
X
11-13
O
O
O
S
O
O
O
S
S
H2N
H2N
H2N
R1
NH2
c
e
or
X
X
R1
(17, 19)
2, 4-7
20, 21
8-10, 17, 19
d
18
a
.
Reagents and reaction conditions: (a) arylbromide, K3PO4, PdCl2(dppf) CH2Cl2, DMF, 60 °C, 2 h, Ar stream, 22-
.
70%; (b) LiOH H2O, THF/H2O, 25 °C, 12 h, 91-99%; (c) arylbromide, CsCO3, Pd(II) acetate, NMP/H2O, 110 °C, 100
W, 15 min, closed vessel, Pmax = 250 PSI, PowerMAX, 14-77%; (d) NaBH4, THF/H2O, reflux, 2 h, 95%; (e)
SnCl2∙2H2O, AcOEt, reflux, 3 h, 51-58%.
CHEMISTRY
1,1'-Biphenyl-4-sulfonamides 1 and 11-13 were obtained from 4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)benzenesulfonamide (22),18 the appropriate aryl bromide, potassium phosphate tri-
basic and dichloro[1,1'-bis(diphenylphosphino)ferrocene]palladium(II) complex with dichloromethane
(1:1) (PdCl2(dppf)·CH2Cl2) in N,N-dimethylformamide at 60 °C for 2 h under Ar stream. Lithium hy-
droxide hydrolysis of the esters 1, 11-13 in aqueous tetrahydrofuran at 25 °C for 12 h furnished the cor-
responding carboxylic acids 3, 14-16, respectively (Scheme 1). On the other hand, sulfonamides 2, 4-
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10, 17 and 19 were prepared by treatment of 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)benzenesulfonamide18 with the appropriate aryl bromide in the presence of cesium carbonate and
Pd(II) acetate in aqueous 1-methyl-2-pyrrolidinone at 110 °C (100 W) for 15 min. Treatment of 17 with
sodium borohydride in aqueous tetrahydrofuran at reflux temperature for 2 h gave the corresponding
alcohol 18. Amino derivatives 20 and 21 were obtained by reducing the appropriate nitro compounds
17 or 19, respectively, with tin(II) chloride dehydrate in boiling ethyl acetate for 3 h. The synthetic in-
termediates required for the synthesis of 1-21 were obtained as described in Scheme 1S and Scheme 2S
in the Supporting Information.
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RESULTS AND DISCUSSION
Inhibition of hCA I, II, IX, XII and XIV. Sulfonamides 1-21 were assayed for their ability to inhibit
the hCA isoforms I, II, IX, XII and XIV using acetazolamide (AAZ) as reference compound. The Ki
values of sulfonamides 1-21, measured by means of a stopped-flow screening assay19 that monitors the
hydration of carbon dioxide catalyzed by each hCA isoform, are shown in Table 1 along with the hCA
XIV selectivity indexes obtained as ratio between the Kis of the indicated hCA and the corresponding
hCA XIV Kis.
From the results reported in Table 1, it should be noted that each hCA isoform showed a differ-
ent inhibition profile which accounted for distinct structure-activity relationships (SARs). Sulfona-
mides 1-21 inhibited the hCA isoforms I, II, IX, XII and XIV with Ki values in the nanomolar range of
concentration, with the exception of 8 against hCA I. In general, the biphenylsulfonamides 1-21 turned
out to be superior to AAZ against the entire hCAs panel. Most importantly, they were found uniformly
potent inhibitors of the hCA XIV at nano/subnanomolar concentration.
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Table 1. Inhibition of hCA I, II, IX, XII and XIV by 1,1'-Biphenyl-4-sulfonamides 1-21 and Reference Com-
pound AAZ.
6
7
8
9
O
1 1'
O
S
X-R
4'
4
H2N
1-21
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Ki (nM)a/SIb
Compd
1
R
X
hCA I
hCA II
hCA IX
hCA XII
hCA XIV
O
77.0
1145
9.0
13
9.3
14
35.2
53
0.67
—
-
OCH3
O
CH3
78.8
232
42.0
124
71.4
210
44.9
132
0.34
—
2
3
-
CH3
O
CH3
O
340
453
6.2
8.3
3.6
4.8
52.3
70
0.75
—
-
OH
O
67.5
4.9
5.3
0.4
823
60
8.3
0.6
13.8
—
4
-
NH2
O
46.7
68
11.5
17
8.4
12
26.2
40
0.69
—
5
-
HN
OH
62.2
152
11.6
28
9.6
23
7.4
18
0.41
—
NO2
6
-
-
78.5
21
9.1
2.5
605
164
7.7
2.1
3.7
—
NH2
7
1635
1946
4.3
5.1
63.8
76
7.4
8.8
0.84
—
8
C=O
52.7
68
4.9
6.4
8.9
11.6
81.1
105
0.77
—
9
CH(OH)
CH2
75.3
4.6
454
27.9
519
31.8
77.2
4.7
16.3
—
10
11
12
13
14
O
73.8
11
7.8
1.2
42.3
6.3
7.3
1.1
6.7
—
C=O
OCH3
O
6.3
15
6.6
15.7
710
1690
6.7
16
0.42
—
CH(OH)
CH2
OCH3
O
122
27
50.6
11
76.7
17
237
52
4.6
—
OCH3
O
263
283
4.8
5.2
8.5
9.1
45.5
48.9
0.93
—
C=O
OH
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8
O
72.5
18
814
204
450
113
93.9
23
4.0
—
15
16
CH(OH)
CH2
OH
O
236
284
6.9
8.3
31.4
38
90.8
109
0.83
—
OH
9
80.1
23
63.3
18
85.6
24
70.6
20
3.6
—
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17
18
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20
21
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NO2
NO2
NO2
NH2
NH2
17
C=O
8.9
1.0
5.9
0.7
678
80
8.1
0.9
8.5
—
18
CH(OH)
CH2
70.5
19
39.7
11
705
191
9.3
2.5
3.7
—
19
62.0
238
86.2
332
9.2
35
25.4
98
0.26
—
20
C=O
589
879
5.2
7.8
6.0
9
8.1
13
0.67
—
21
CH2
250
6
12
0.3
25
0.7
5.7
0.1
41
—
AAZc
—
—
aSD: Standard deviations went from ±5% to ±10% of the indicated Ki values bSI: hCA XIV selectivity index ob-
tained as ratio between Kis of the indicated hCA and hCA XIV Kis. AAZ: acetazolamide as reference compound.
CO2 hydrase, stopped-flow method, mean from 3 different assays.
c
As inhibitors of hCA I, the ester derivatives were generally more potent than the corresponding
carboxylic acids (compare 1 and 2 with 3, 11 with 14, 12 with 15, and 13 with 16). Esters 1 and 2 were
equipotent to the carboxamides 4 and 5. Nitro derivatives 6 and 17 were at the same level of potency as
the corresponding amino compounds 7 and 20. On the contrary, the amino derivative 21 was 8.3-fold
less potent than its parent nitro derivative 19. Compounds 12 (Ki = 6.3 nM) and 18 (Ki = 8.9 nM), the
most potent inhibitors of hCA I, were 40- and 28-fold, respectively, more potent than the reference
compound AAZ.
Several sulfonamides inhibited hCA II at low nanomolar concentrations. Acid 3 (Ki = 6.2 nM)
and carboxamide 4 (Ki = 5.3 nM), ketones 8, 11 and 14 (Kis’ range = 4.3-7.8 nM), alcohols 9, 12, and
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18 (Kis’ range = 4.9-6.6 nM), and derivative with methylene spacers 16 and 21 (Ki = 6.9 nM and 5.2
nM, respectively) were potent inhibitors of hCA II. Against this isoform, the terminal function of the
ester, acid, nitro or amino type, seemed to be equally beneficial for inducing good inhibitory activity.
Compounds 8 (Ki = 4.3 nM), 9 (Ki = 4.9 nM) and 14 (Ki = 4.8 nM) were the most potent inhibitors of
hCA II within the investigated series.
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Eight compounds, 1, 3, 5, 6, 9, 14, 20 and 21, inhibited hCA IX with Ki <10 nM. High inhibi-
tion of hCA IX was observed for compounds bearing either carboxylic acid or amino group as a termi-
nal function on the biphenylsulfonamide scaffold. The most potent hCA IX inhibition was exhibited by
compound 3 (Ki = 3.6 nM) and 21 (Ki = 6.0 nM).
Compounds 4, 6, 7, 8, 11, 12, 18, 19 and 21 inhibited hCA XII isoform with Ki values within
the range of 6-9 nM. As inhibitors of hCA XII, nitro derivatives 6 and 18 were as potent as the corre-
sponding amino compounds 7 and 21. Alcohol 12 (Ki = 6.7 nM) was found to be the most potent inhib-
itor within the series.
Twelve derivatives, namely 1-3, 5, 6, 8, 9, 12, 14, 16, 20 and 21, proved to be exceptionally po-
tent towards hCA XIV with subnanomolar inhibition constants. Most biphenyl derivatives were highly
potent, although carboxamide 4 and the amino compound 7 were somehow less potent than the other
congeners. Among derivatives bearing the third phenyl ring, ketones 8 and 14 were more potent than
17; alcohol 12 was one order of magnitude superior to 15 and 18. Nitro derivatives 17 and 19 were less
potent than the corresponding amino compounds 20 and 21. Compound 20 was the most potent inhibi-
tor of hCA XIV (Ki = 0.26 nM) reported so far.
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Figure 1. Active-site region of the hCA XIV/20 (A) and hCA II/20 (B) adducts. In both adducts the inhibitor is shown
in association with a σA-weighted |2Fo-Fc| map (contoured at 1.0 σ). Hydrogen bonds, residues involved in van der
Waals interactions (distance <4.0 Å) and the active-site Zn2+ ion coordination are also shown. Conf.1 and Conf.2 indi-
cate the two conformations of Ser132 in hCA XIV/20 adduct.
X-ray Crystallography. In order to understand the molecular basis responsible of the high inhibition
efficiency of 1,1'-biphenyl-4-sulfonamides against hCAs, we solved the crystal structure of the adduct
formed by compound 20, the most potent inhibitor of hCA XIV within the series, with isoforms XIV
and II. Crystals of the adducts were obtained by co-crystallization experiments as previously reported
for other sulfonamide CA inhibitors.17 Data were collected to a resolution of 1.83 Å for hCA XIV/20
and of 1.60 Å for hCA II/20, respectively. The structures were analyzed by difference Fourier tech-
niques and the final refined models had a Rfactor and Rfree values of 19.7% and 21.6% for hCA XIV/20,
and of 17.2% and 19.7% for hCA II/20 (Table S1, Supporting Information). Analysis of the electron
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density maps at various stages of the crystallographic refinement showed the two adducts the binding
of one inhibitor molecule within the enzyme active site. In both cases these maps were very well de-
fined for the 1,1'-biphenyl-4-sulfonamido-4'-carbonyl moiety of 20, but showed poor definition for the
4-aminophenyl tail, suggesting a high flexibility of this group within the two active sites (Figures 1A
and 1B).
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The sulfonamide group of 20 binds to hCA XIV in the typical manner observed in all
CA/sulfonamide complexes20 (Figure 1A). In particular, it coordinates the catalytic zinc ion, and forms
two hydrogen bonds with residue Thr199. The 1,1'-biphenyl scaffold is located in the hydrophobic part
of the active site17 establishing several van der Waals interactions (<4.0 Å) with enzyme residues (Fig-
ures 1A and 2A), while the carbonyl group forms a hydrogen bond with the Ser132OG atom in one of
the two conformations observed for this residue, located in the aforementioned region 127-136. In its
second conformation Ser132 side chain is hydrogen bonded with the Ser130OG atom. Finally, the 4-
aminophenyl moiety of the inhibitor is rather disordered, since it forms only weak binding interactions
with the enzyme.
Figure 1B reports the main protein/inhibitor interactions of the hCA II/20 complex. Many inter-
actions observed in the hCA XIV/20 adduct, such as the coordination of the catalytic zinc ion, the hy-
drogen bonds with residue Thr199 and the interactions of the 1,1'-biphenyl scaffold with residues
which delimit the hydrophobic part of the active site (Figure 2B), are conserved also in this case. On
the contrary, differences are observed in the interaction of the 4-aminobenzoyl tail of the inhibitor with
the border of the active site. Indeed, in the adduct with isoform II the carbonyl group of 20 does not
form any hydrogen bond with the enzyme due to the substitution of Ser132 with a glycine residue; it is
rotated of about 120° with respect to the corresponding pose in the hCA XIV/20 adduct, interacting
with a water molecule present within the active site cavity. This rotation leads to a different orientation
of the aminophenyl tail (Figure 3).
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Figure 2. (A) Solvent accessible surface of hCA XIV (A) and hCA II (B) in their complexes with compound 20. In both
adducts the inhibitor molecule bound within the active site cavity is shown in ball and stick representation, the hy-
drophobic part of the active site is colored in blue, while the hydrophilic one in red.
Figure 3 Superposition of the hCA XIV/20 (green) and hCA II/20 (yellow) complexes. The inhibitor in both adducts
is represented in ball-and-stick. The protein region 127–136 is highlighted in red.
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Molecular modeling
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We speculated that the 1,1'-diphenylsulfonamide scaffold could be used to discriminate the
binding to the different CA isoforms by interacting with the most variable part of the CA active sites17
(residues 127 to 136). This hypothesis has been confirmed by our crystallographic studies. Indeed, in
the hCA II/20 and hCA XIV/20 complexes, the inhibitor established different interactions with this
protein region (Figure 3). To investigate the role of this region in the interaction of compound 20 also
with the other hCA isoforms (namely I, IX, XII) docking studies were performed by using the pro-
grams Plants21 and Autodock.22 High-resolution crystal structures available within PDB were selected
for each studied CA. To validate the method, docking studies were performed for the interaction of
compound 20 with hCA XIV and hCA II and the obtained models compared with the crystallographic
complexes. The RMSDs for the superposition of the crystallographic hCA XIV/20 and hCA II/20 com-
plexes and those obtained by Plants were lower than 1, thus validating the docking procedure (Figures
1S and 2S, Supporting Information).
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Compound 20 was docked on the studied isoforms CA I (2NMX),23 IX (3IAI),24 and XII
(4KP5)25 and obtained models were superimposed to the crystallographic hCA XIV/20 complex to
highlight eventual differences in binding modes.
Superimposition of hCA XIV/20 and hCA I/20 complexes highlighted three important amino
acid substitutions in the active sites, Val121/Ala, Ala91/Phe and Ser132/Ala. In the hCA XIV/20 ad-
duct, Val121 increases the hydrophobic stabilization of 20; its replacement by Ala resulted in a weaker
binding interaction; introduction of the bulkier Phe91 for Ala slightly changed the 20 binding mode,
(Figure 4A).
By comparing hCA XIV/20 with hCA IX/20, only minor differences in binding mode were
found. Worthy of note is the Ser132 “change” to Glu in hCA IX. No contacts between this residue and
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the carbonyl moiety of 20 were observed (Figure 4B).
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Ser132 is conserved in both hCA XIV and hCA XII binding sites. Nevertheless, in the docking
model of hCA XII/20 no contact of this residue was found with the carbonyl group of 20 due to
Leu131/Ala, Ala91/Thr and Tyr204/Asn substitutions from hCA XIV to hCA XII, that markedly af-
fected the inhibitor binding (Figure 4C).
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Altogether these data highlighted the crucial role played by the region 127-136 in discriminat-
ing the binding of 1,1'-diphenylsulfonamides to hCA XIV. Indeed, only hCA XIV formed favourable
interactions between this region and the inhibitor, particularly between Ser132 and the carbonyl group
of the inhibitor. These results are in good agreement with the kinetic data.
Figure 4. Superposition of the binding poses proposed by Plants of 20 in the catalytic site of (A) hCA I (green); (B)
hCA IX (silver) and (C) hCA XII (brown) with hCA XIV (magenta)/20 (cyan) crystallographic complex. hCA XIV resi-
due numbers are shown out of brackets.
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CONCLUSION
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New 1,1'-biphenylsulfonamides 1-21 were synthesized and evaluated as inhibitors of the ubiquitous
human carbonic anhydrase isoforms I, II, IX, XII and XIV using AAZ as reference compound. The sul-
fonamides 1-21 inhibited these hCA isoforms with Ki values in the nanomolar range of concentration,
with the exception of 8 against hCA I, being superior to AAZ against all the isoforms. Compound 12
uniformly inhibited hCAs I, II, XII and XIV, whereas compound 21 was equipontent against hCAs II,
IX XII and XIV. Compound 18 showed high inhibition of isoforms I, II and XII, but was less effective
against the hCA XIV. Compound 20 was the most potent inhibitor of hCA XIV (Ki = 0.26 nM) report-
ed so far. X-ray crystallography and molecular modeling studies on the adducts that this compound
forms with the five hCAs, provided insights into the molecular determinants responsible for its high af-
finity toward the target enzyme. These results pave the way to the development of 1,1'-
biphenylsulfonamides as a new class of interesting hCA XIV inhibitors.
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EXPERIMENTAL SECTION
Chemistry. MW-assisted reactions were performed on a CEM Discover SP single mode reac-
tor, controlling the instrument settings with PC-running CEM Synergy 1.49 software. Closed vessel
experiments were carried out in dedicated capped vials (10 mL) with cylindrical stirring bar (length 8
mm, diameter 3 mm). Stirring, temperature, irradiation power, maximum pressure (Pmax), PowerMAX
(simultaneous cooling-while-heating), ActiVent (simultaneous venting-while-heating), and ramp and
hold times were set as indicated. Temperature of the reaction was monitored by an external fiber optic
temperature sensor. After completion of the reaction, the mixture was cooled to 25 °C via air-jet cool-
ing. Melting points (mp) were determined on a Stuart Scientific SMP1 apparatus and are uncorrected.
Infrared spectra (IR) were run on a Perkin-Elmer SpectrumOne FT-ATR spectrophotometer. Band po-
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sition and absorption ranges are given in cm-1. Proton nuclear magnetic resonance (1H NMR) spectra
were recorded on a Bruker 400 MHz FT spectrometer in the indicated solvent and corresponding fid
files processed by MestreLab Research S.L. MestreReNova 6.2.1-769 software. Chemical shifts are
expressed in δ units (ppm) from tetramethylsilane. Column chromatography was performed on col-
umns packed with silica gel from Macherey-Nagel (70-230 mesh). Silica gel thin layer chromatography
(TLC) cards from Macherey-Nagel (silica gel precoated aluminum cards with fluorescent indicator at
254 nm) were used for TLC. Developed plates were visualized by a Spectroline ENF 260C/FE UV ap-
paratus. Organic solutions were dried over anhydrous Na2SO4. Evaporation of the solvents was carried
out on a Büchi rotavapor R-210 equipped with a Büchi V-850 vacuum controller and a Büchi V-700 or
V-710 vacuum pump. All reagents and solvents are commercially available and were used as pur-
chased, without further purification. Elemental analyses of the compounds were found within ±0.4% of
the theoretical values. The purity of tested compounds was >95%.
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General Procedure for the Preparation of Compounds 1 and 11-13. Example: Methyl 4-
sulfamoyl-1,1'-biphenyl-4'-carboxylate (1). A mixture of 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)benzenesulfonamide18 (22) (0.37 g, 1.3 mmol), methyl ester of the 4-bromobenzoic acid (0.21 g, 1.0
mmol), potassium phosphate tribasic (0.64 g, 3.0 mmol) in N,N-dimethylformamide (5 mL) was de-
gassed for 30 min. PdCl2(dppf)·CH2Cl2 (0.025 g, 0.03 mmol) was added under Ar stream and the reac-
tion mixture was heated at 60 °C for 2 h. After cooling, water and ethyl acetate were added and the re-
sulting mixture was filtered trough a pad of silica gel. The organic layer was washed with brine, dried
and filtered. Removal of the solvent gave a residue that was purified by column chromatography (silica
gel, ethyl acetate:n-hexane = 1:1 as eluent) to furnish 1 (0.17 g, 58%), mp 240-243 °C (from ethanol).
1H NMR (DMSO-d6): δ 3.89 (s, 3H), 7.44 (broad s, 2H, disappeared on treatment with D2O), 7.88-7.96
(m, 6H), 8.07 ppm (d, J = 8.2 Hz, 2H). IR: ν 1701, 3231, 3232 cm-1. Anal. (C14H13NO4S (291.32)) C,
H, N, S.
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Methyl 4-(4-sulfamoyl-1,1'-biphenyl-4'-carbonyl)benzoate (11). Synthesized as 1 starting from
1
methyl 4-(4-bromobenzoyl)benzoate (34). Yield 70%, mp 283-286 °C (from ethanol). H NMR
(DMSO-d6): δ 3.92 (s, 3H), 7.45 (broad s, 2H, disappeared on treatment with D2O), 7.87-7.90 (m, 4H),
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7.94-7.99 (m, 6H), 8.13-8.15 (m, 2H). IR: ν 1644, 1720, 3277, 3375 cm-1. Anal. (C21H17NO5S
(395.43)) C, H, N, S.
Methyl 4-(4-sulfamoyl-1,1'-biphenyl-4'-hydroxymethyl)benzoate (12). Synthesized as 1 starting
from methyl 4-(hydroxy(4-bromophenyl)methyl)benzoate (37). Yield 24%, mp 163-165 °C (from eth-
1
anol). H NMR (DMSO-d6): δ 3.83 (s, 3H), 5.85 (d, J = 3.7 Hz, 1H), 6,16 (d, J = 3.9 Hz, 1H), 7.38
(broad s, 2H, disappeared on treatment with D2O) 7.51 (d, J = 8.2 Hz, 2H), 7.57 (d, J = 8.1 Hz, 2H),
7.68 (d, J = 8.3 Hz, 2H), 7.81-7.88 (m, 4H), 7.92 ppm (d, J = 8.4 Hz, 2H). IR: ν 1721, 3277, 3379,
3506 cm-1. Anal. (C21H19NO5S (397.44)) C, H, N, S.
Methyl 4-(4-sulfamoyl-1,1'-biphenyl-4'-methyl)benzoate (13). Synthesized as 1 starting from
methyl 4-(4-bromobenzyl)benzoate (40). Yield 22%, mp 150-153 °C (from ethanol). 1H NMR (DMSO-
d6): δ 3.82 (s, 3H), 4.08 (s, 2H), 7.36-7.38 (m, 4H), 7.40-7.42 (m, 2H) 7.66 (d, J = 7.9 Hz, 2H), 7.81-
7.91 ppm (m, 6H). IR: ν 1704, 3258, 3353 cm-1. Anal. (C21H19NO4S (381.44)) C, H, N, S.
General Procedure for the Preparation of Compounds 2, 4-10, 17 and 19. Example: 4-
Sulfamoyl-1,1'-biphenyl-4'-amine (7). A mixture of 2218 (0.38 g, 1.3 mmol), 4-bromoaniline (0.17 g,
1.0 mmol), cesium carbonate (0.44 g, 1.3 mmol) and Pd(II) acetate (0.03 g, 0.13 mol) in 1-methyl-2-
pyrrolidinone (2.3 mL) and water (0.2 mL) was placed into the microwave cavity (closed vessel mode,
Pmax = 250 PSI). Starting MW irradiation of 100 W was used, the temperature being ramped from 25
°C to 110 °C. Once 110 °C was reached, taking about 2 min, the reaction mixture was held at the same
temperature for 15 min, while highly stirring and cooling. The mixture was diluted with water and ex-
tracted with ethyl acetate. The organic layer was washed with brine, dried and filtered. Removal of the
solvent gave a residue that was purified by column chromatography (silica gel, ethyl acetate:n-hexane
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= 1:1 as eluent) to furnish 7 (0.11 g, 45%), mp 271-274 °C (from ethanol). Lit.26 266-267 °C. 1H NMR
(DMSO-d6): δ 5.38 (broad s, 2H, disappeared on treatment with D2O), 6.65 (d, J = 8.6 Hz, 2H), 7.29
(broad s, 2H, disappeared on treatment with D2O), 7.44 (d, J = 8.5 Hz, 2H), 7.71 (d, J = 8.7 Hz, 2H),
7.78 ppm (d, J = 8.4 Hz, 2H). IR: ν 1588, 3276, 3434 cm-1. Anal. (C12H12N2O2S (248.30)) C, H, N, S.
tert-Butyl 4-sulfamoyl-1,1'-biphenyl-4'-carboxylate (2). Synthesized as 7 starting from tert-butyl
3
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4-bromobenzoate (26). Yield 14%, mp 178-181 °C (from ethanol). H NMR (DMSO-d6): δ 1.57 (s,
9H), 7.45 (broad s, 2H, disappeared on treatment with D2O), 7.87 (d, J = 7.9 Hz, 2H), 7.90-7.93 (m,
4H), 8.01 ppm (d, J = 7.9 Hz, 2H). IR: ν 1683, 3232, 3281 cm-1. Anal. (C17H19NO4S (333.40)) C, H, N,
S.
4-Sulfamoyl-1,1'-biphenyl-4'-carboxamide (4). Synthesized as
7
starting from 4-
bromobenzamide (27). Yield 31%, mp >280 °C (from ethanol). 1H NMR (DMSO-d6): δ 7.43 (broad s,
2H, disappeared on treatment with D2O), 7.83 (d, J = 8.3 Hz, 2H), 7.90-7.95 (m, 4H), 8.00 (d, J = 8.1
Hz, 2H), 8.07 ppm (broad s, 2H, disappeared on treatment with D2O). IR: ν 1643, 3163, 3367 cm-1.
Anal. (C13H12N2O3S (276.31)) C, H, N, S.
N-(2-Hydroxyethyl) 4-sulfamoyl-1,1'-biphenyl-4'-carboxamide (5). Synthesized as 7 starting
1
from 4-bromo-N-(2-hydroxyethyl)benzamide (28). Yield 35%, mp 187-190 °C (from ethanol). H
NMR (DMSO-d6): δ 3.31-3.38 (m, 2H), 3.51-3.54 (m, 2H), 4.75 (t, J = 5.5 Hz, 1H), 7.43 (broad s, 2H,
disappeared on treatment with D2O), 7.84 (d, J = 8.6 Hz, 2H), 7.90-7.95 (m, 4H), 7.99 (d, J = 8.2 Hz,
2H), 8.53 ppm (broad s, 1H, disappeared on treatment with D2O). IR: ν 1637, 2922, 3068, 3316, 3389
cm-1. Anal. (C15H16N2O4S (320.36)) C, H, N, S.
4-Sulfamoyl-4'-nitro-1,1'-biphenyl (6). Synthesized as 7 starting from 1-bromo-4-nitrobenzene
(24). Yield 40%, mp 143-145 °C (from ethanol). Lit.27 mp 145 °C.
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4-Sulfamoyl-4'-benzoyl-1,1'-biphenyl (8). Synthesized as
7
starting from (4-
1
bromophenyl)(phenyl)methanone (30). Yield 77%, mp 262-265 °C (from ethanol). H NMR (DMSO-
d6): δ 7.44 (broad s, 2H, disappeared on treatment with D2O), 7.57-7.61 (m, 2H), 7.68-7.98 ppm (m,
11H). IR: ν 1649, 3234, 3299 cm-1. Anal. (C19H15NO3S (337.39)) C, H, N, S.
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4-Sulfamoyl-4'-hydroxymethylphenyl-1,1'-biphenyl (9). Synthesized as 7 starting from (4-
1
bromophenyl)(phenyl)methanol (35). Yield 46%, mp 195-197 °C (from ethanol). H NMR (DMSO-
d6): δ 5.76 (d, J = 3.82 Hz, 1H), 5.96 (d, J = 4 Hz, 1H), 7.19-7.23 (m, 1H), 7.29-7.33 (m, 2H), 7.36
(broad s, 2H, disappeared on treatment with D2O), 7.40-7.42 (m, 2H), 7.50 (d, J = 8.1 Hz, 2H), 7.67 (d,
J = 8.2 Hz, 2H), 7.81-7.88 ppm (m, 4H). IR: ν 1595, 2923, 3337 cm-1. Anal. (C19H17NO3S (339.41)) C,
H, N, S.
4-Sulfamoyl-4'-methylphenyl-1,1'-biphenyl (10). Synthesized as 7 starting from 1-benzyl-4-
1
bromobenzene (38). Yield 57%, mp 165-168 °C (from ethanol). H NMR (DMSO-d6): δ 4.00 (s, 2H),
7.19-7.32 (m, 5H; m, 3H after treatment with D2O), 7.35-7.37 (m, 4H), 7.65 (d, J = 8.1 Hz, 2H), 7.81-
7.88 ppm (m, 4H). IR: ν 3271, 3375 cm-1. Anal. (C19H17NO2S (323.41)) C, H, N, S.
4-Sulfamoyl-4'-(4-nitrobenzoyl)-1,1'-biphenyl (17). Synthesized as 7 starting from (4-
1
bromophenyl)(4-nitrophenyl)methanone (31). Yield 58%, mp 222-225 °C (from ethanol). H NMR
(DMSO-d6): δ 7.46 (broad s, 2H, disappeared on treatment with D2O), 7.89-7.94 (m, 3H), 7.95-8.02
(m, 7H), 8.38-8.41 ppm (m, 2H). IR: ν 1641, 3103, 3211, 3380 cm-1. Anal. (C19H14N2O5S (382.39)) C,
H, N, S.
4-Sulfamoyl-4'-methyl(4-nitrophenyl)-1,1'-biphenyl (19). Synthesized as 7 starting from 1-
1
bromo-4-(4-nitrobenzyl)benzene (39). Yield 60%, mp 165-168 °C (from ethanol). H NMR (DMSO-
d6): δ 4.16 (s, 2H), 7.39-7.41 (m, 4H; m, 2H after treatment with D2O), 7.56 (d, J = 8.7 Hz, 2H), 7.68
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(d, J = 8.2 Hz, 2H), 7.82-7.89 (m, 4H), 8.18 (d, J = 8.8 Hz, 2H). IR: ν 3270, 3367 cm-1. Anal.
(C19H16N2O4S (368.41)) C, H, N, S.
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General Procedure for the Preparation of Compounds 3 and 14-16. Example: 4-Sulfamoyl-
1,1'-biphenyl-4'-carboxylic acid (3). A mixture of 1 (0.1 g, 0.34 mmol) and lithium hydroxide hydrate
(0.043 g, 1.0 mmol) in tetrahydrofuran (3 mL) and water (3 mL) was stirred at 25 °C overnight. The re-
action mixture was made acidic (pH = 2) with 1 N HCl and extracted with ethyl acetate. The organic
layer was washed with brine, dried and filtered. Removal of the solvent gave 3 (0.09 g, 95%), mp >
280 °C (from ethanol). 1H NMR (DMSO-d6): δ 7.43 (broad s, 2H, disappeared on treatment with D2O),
7.86 (d, J = 8.2 Hz, 2H), 7.93 (m, 4H), 8.05 (d, J = 8.3 Hz, 2H), 12.75 ppm (broad s, 1H, disappeared
on treatment with D2O). IR: ν 1670, 3258, 3374 cm-1. Anal. (C13H11NO4S (277.30)) C, H, N, S.
4-(4-Sulfamoyl-1,1'-biphenyl-4'-carbonyl)benzoic acid (14). Synthesized as 3 starting from 11.
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Yield 91%, mp > 280 °C (from ethanol). Lit.28 351-352 °C. 1H NMR (DMSO-d6): δ 7.45 (broad s, 2H,
disappeared on treatment with D2O), 7.86-7.90 (m, 4H), 7.93-7.99 (m, 6H) 8.11-8.13 (m, 2H), 12.85
ppm (broad s, 1H, disappeared on treatment with D2O). IR: ν 1687, 2845, 3273, 3370 cm-1. Anal.
(C20H15NO5S (381.40)) C, H, N, S.
4-(4-Sulfamoyl-1,1'-biphenyl-4'-hydroxymethyl)benzoic acid (15). Synthesized as 3 starting
from methyl 12. Yield 93%, mp >280 °C (from ethanol). 1H NMR (DMSO-d6): δ 5.79 (d, J = Hz, 1H),
6.03 (d, J = Hz, 1H), 7.39-7.42 (m, 4H; m, 2H after treatment with D2O)), 7.50 (d, J = 8.2 Hz, 2H),
7.67 (d, J = 8.0 Hz, 2H), 7.81-7.89 ppm (m, 6H), 12.83 ppm (broad s, 1H, disappeared on treatment
with D2O). IR: ν 1685, 2845, 3360, 3365, 3568 cm-1. Anal. (C20H17NO5S (383.42)) C, H, N, S.
4-(4-Sulfamoyl-1,1'-biphenyl-4'-methyl)benzoic acid (16). Synthesized as 7 starting from 13.
Yield 99%, mp >280 °C (from ethanol). 1H NMR (DMSO-d6): δ 4.08 (s, 2H), 7.31-7.43 (m, 6H; m, 4H
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after treatment with D2O), 7.67 (d, J = 8.2 Hz, 2H), 7.82-7.89 (m, 6H), 12.79 ppm (broad s, 1H, disap-
peared on treatment with D2O). IR: ν 3346, 3258, 1681 cm-1. Anal. (C20H17NO4S (367.42)) C, H, N, S.
General Procedure for the Preparation of Compounds 20 and 21. Example: 4-Sulfamoyl-4'-
(4-aminobenzoyl)-1,1'-biphenyl (20). A mixture of 17 (0.15 g, 0.39 mmol) and tin(II) chloride dihy-
drate (0.44 g, 1.95 mol) in ethyl acetate (5 mL) was refluxed for 3 h. After cooling, the reaction mixture
was made basic (pH = 10) with a saturated aqueous solution of sodium hydrogen carbonate. The organ-
ic layer was dried and filtered. Removal of the solvent gave 20 (0.08 g, 58%), mp 140-145 °C (from
ethanol). ). 1H NMR (DMSO-d6): δ 6.20 (broad s, 2H, disappeared on treatment with D2O), 6.62 (d, J =
8.7 Hz, 2H), 7.44 (broad s, 2H, disappeared on treatment with D2O), 7.57 (d, J = 8.7 Hz, 2H), 7.72 (d, J
= 7.9 Hz, 2H), 7.87 (d, J = 7.9 Hz, 2H), 7.86-7.96 ppm (m, 4H). IR: ν 1638, 3264, 3340 cm-1. Anal.
(C19H16N2O3S (352.41)) C, H, N, S.
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4-Sulfamoyl-4'-methyl(4-aminophenyl)-1,1'-biphenyl (21). Synthesized as 20 starting from 19.
1
Yield 51%, mp 235 °C with decomposition (from ethanol). H NMR (DMSO-d6): δ 3.80 (s, 2H), 4.87
(broad s, 2H, disappeared on treatment with D2O), 6.50 (d, J = 8.2 Hz, 2H), 6.90 (d, J = 8.2 Hz, 2H),
7.30 (d, J = 8.1 Hz, 2H), 7.36 (broad s, 2H, disappeared on treatment with D2O), 7.63 (d, J = 8.1 Hz,
2H), 7.81-7.88 ppm (m, 4H). IR: ν 3310, 3410 cm-1. Anal. (C19H18N2O2S (338.42)) C, H, N, S.
General Procedure for the Preparation of Compounds 18 and 35-37 (Supporting Infor-
mation). Example: 4-Sulfamoyl-4'-hydroxymethyl(4-nitrophenyl)-1,1'-biphenyl (18). A mixture of 17
(0.19 g, 0.49 mmol) and sodium borohydride (0.018 g, 0.49 mmol) in tetrahydrofuran (2.4 mL) and wa-
ter (0.14 mL) was heated at reflux for 2 h. After cooling, cold water and ethyl acetate were added and
layers were separated. The organic one was washed with brine, dried and filtered. Evaporation of the
solvent gave a residue that was purified by column chromatography (silica gel, ethyl acetate:n-hexane
1
= 1:1 as eluent) to furnish 18 (0.07 g, 37%), mp 169-172 °C (from ethanol). H NMR (DMSO-d6): δ
5.94 (d, J = 2.5 Hz, 1H), 6.31 (d, J = 3.5 Hz, 1H), 7.37 (broad s, 2H, disappeared on treatment with
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D2O), 7.53 (d, J = 8.2 Hz, 2H), 7.68-7.72 (m, 4H), 7.82-7.89 (m, 4H), 8.19-8.21 ppm (m, 2H). IR: ν
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3258, 3362, 3571 cm-1. Anal. (C19H16N2O5S (384.41)) C, H, N, S.
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X-ray crystallography. Co-crystallization. hCA XIV enzyme was prepared and purified as previously
described.20 The hCA XIV/20 adduct was prepared by adding a 50-fold excess of the inhibitor to a 0.1
mg/mL enzyme solution in 20 mM Tris-HCl pH 7.5. After incubation overnight at 4 °C the complex
concentrated to 10 mg/mL. Crystals were obtained at 20 °C using the hanging drop vapor diffusion
technique by equilibrating drops containing 1 µL of complex solution and an equal volume of precipi-
tant solution consisting of 1.9 M ammonium sulfate, 0.1 M TRIS-HCl, pH 8.5, over a reservoirs con-
taining 1 mL of precipitant solution. Crystals appeared after 3 days. Crystals of the hCA II/20 adduct
were obtained as previously described.29
Diffraction data and collection. X-ray diffraction data for both hCA XIV/20 and hCA II/20
adducts were collected at 100 K, using a copper rotating anode generator developed by Rigaku and
equipped with Rigaku Saturn CCD detector. Prior to cryogenic freezing, the crystals were transferred to
the respective precipitant solution with the addition of 20 % (v/v) glycerol. Data were integrated,
merged and scaled using HKL2000.30 Crystal parameters and data collection for both adducts are re-
ported in Table 1S of Supporting Information.
Structure determination. Phasing of the complexes was carried out with CNS31 using as start-
ing model the previously solved hCA XIV (PDB ID: 4LU3)17 and hCA II (PDB ID:1CA2).32 The
graphic program O33 was used to view the electron density maps, and the structure was adjusted based
on the calculated electron density. Composite simulated-annealing omit maps were used regularly dur-
ing the building process to verify and correct the models.31 Topology files of the inhibitor was generat-
ed using the PRODRG2 server.34 The geometric restraints of the final models were analyzed using
PROCHECK.35 The refinement statistics of the final models are summarized in Table 1S, Supporting
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Information. Coordinates and structure factors were deposited in the Protein Data Bank (accession code
5CJF and 5E2R).
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Molecular modeling. All molecular modeling studies were performed on a MacPro dual 2.66
GHz Xeon running Ubuntu 14.04 LTS. The CA structures were downloaded from the PDB (http://
www.rcsb.org/). Hydrogen atoms were added to the protein, using Molecular Operating Environment
(MOE) 2010 (Molecular Operating Environment (MOE 2010.10), Chemical Computing Group, Inc.,
Montreal, Quebec, Canada, http://www.chemcomp.com) and minimized, keeping all the heavy atoms
fixed until a rmsd gradient of 0.05 kcal mol-1 Å-1 was reached. Ligand structures were built with MOE
and minimized using the MM-FF94x forcefield until a rmsd gradient of 0.05 kcal mol-1 Å-1 was
reached. The docking simulations were performed using PLANTS21 and Autodock 4.0.22 The images
presented here were created with Pymol.36
CA Inhibition Screening Assay. An Applied Photophysics stopped-flow instrument has been used for
assaying the CA-catalyzed CO2 hydration activity.19 Phenol red (at a concentration of 0.2 mM) has
been used as an indicator, working at the maximum absorbance of 557 nm, with 20 mM Hepes (pH
7.5) as buffer, and 20 mM Na2SO4 (for maintaining constant the ionic strength). The initial rates of the
CA-catalyzed CO2 hydration reaction were followed for a period of 10−100 s. The CO2 concentrations
ranged from 1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants. For
each inhibitor, at least six traces of the initial 5−10% of the reaction have been used for determining the
initial velocity. The uncatalyzed rates were determined in the same manner and subtracted from the to-
tal observed rates. Stock solutions of inhibitor (0.1 mM) were prepared in distilled−deionized water,
and dilutions up to 0.01 nM were done thereafter with distilled−deionized water. Inhibitor and enzyme
solutions were preincubated together for 15 min at room temperature prior to assay, to allow for the
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formation of the E−I complex. The inhibition constants were obtained by nonlinear least squares meth-
ods using the Cheng-Prusoff equation and represent the mean from at least three different determina-
tions. Errors were in the range of ± 5-10 % of the reported KI values. CA isoforms were recombinant
enzymes obtained in house as reported earlier.37-39 The enzyme concentrations in the assay system
were: hCA I: 13.2 nM; hCA II: 8.4 nM; hCA IX: 7.9 nM; hCA XII: 15.2 nM; hCA XIV: 10.7 nM.
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ASSOCIATED CONTENT
Additional chemical and biological material is available free of charge via the internet at
http//pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: +39 06 4991 3800. Fax: +39 06 4991 3993. E-mail: romano.silvestri@uniroma1.it.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
This research was supported by PRIN 2009 grant No. 20098SJX4F and Progetti di Ricerca di Universi-
tà, Sapienza Universitàꢀdi Roma (grant No. C26H135FL5). CTS is grateful to an FP7 EU research
grant (Dynano).
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ABBREVIATIONS
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CA, carbonic anhydrase; hCA, human carbonic anhydrase; CAI, CA inhibitor; SAR, structure-activity
relationship; AAZ, acetazolamide, DMF, N,N-dimethylformamide, THF, tetrahydrofuran, NMP, 1-
methyl-2-pyrrolidinone; BOP reagent, (benzotriazol-1-yloxy)tris (dimethylamino)phosphonium hex-
afluorophosphate; MCPBA, m-chloroperbenzoic acid; CDI, 1,1′-carbonyldiimidazole.
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