Thioether benzenesulfonamide inhibitors of carbonic anhydrases II and IV: Structure-based drug design, synthesis, and biological evaluation

Article abstract of DOI:10.1016/j.bmc.2010.03.014

A novel series of potent thioether benzenesulfonamide inhibitors of carbonic anhydrases II and IV was discovered using structure-based drug design. Synthesis, structure-activity relationship, and optimization of physicochemical properties are described. Low nanomolar potency was achieved, and selected compounds with improved thermodynamic solubility showed promising in vitro inhibition of carbonic anhydrase activity in rabbit iris ciliary body homogenate.

Full text of DOI:10.1016/j.bmc.2010.03.014

Bioorganic & Medicinal Chemistry 18 (2010) 3307–3319
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Thioether benzenesulfonamide inhibitors of carbonic anhydrases II and IV:
Structure-based drug design, synthesis, and biological evaluation
*
William Vernier , Wesley Chong, David Rewolinski, Samantha Greasley, Thomas Pauly, Morena Shaw,
Dac Dinh, Rose Ann Ferre, Seiji Nukui, Martha Ornelas, Eric Reyner
Pfizer Global Research and Development, La Jolla Laboratories, 10770 Science Center Drive, San Diego, CA 92121, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of potent thioether benzenesulfonamide inhibitors of carbonic anhydrases II and IV was
discovered using structure-based drug design. Synthesis, structure–activity relationship, and optimiza-
tion of physicochemical properties are described. Low nanomolar potency was achieved, and selected
compounds with improved thermodynamic solubility showed promising in vitro inhibition of carbonic
anhydrase activity in rabbit iris ciliary body homogenate.
Received 23 December 2009
Revised 4 March 2010
Accepted 6 March 2010
Available online 11 March 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Carbonic anhydrase
Thioether benzenesulfonamide
Glaucoma
1. Introduction
the uveoscleral outflow. Prostaglandins, specifically PGF2a, have
proven very effective in inducing the latter pathway16 and a syn-
thetic analog, latanoprost (Xalatan), is widely used for the treat-
ment of glaucoma.
Glaucoma is a disease characterized by increased intraocular
pressure (IOP). This increase in intraocular pressure leads to dam-
age of the optic nerve and is an acknowledged risk factor for vision
loss due to glaucoma.1,2 Elevated IOP can result from either in-
creased production or decreased drainage of aqueous humor, the
fluid that provides structural support, oxygen, and nutrition to
eye tissues.
Carbonic anhydrase (CA) inhibition has been demonstrated to re-
duce fluid flow into the eye and alleviate high IOP.3–8 For over
40 years CA inhibitors (CAIs) were available only as pills, and these
were intended for other therapies. These consisted of acetazolamide,
methazolamide, and dichlorphenamide. Although well tolerated by
many patients, they are also associated with serious side effects such
as weight loss, leucopenia, and metabolic acidosis.9–11 To minimize
the incidence and severity of adverse events, topical ocular dosing of
CAIs was explored as alternative treatment for glaucoma. In 1995
Trusopt, the first eye drop for topical dosing was introduced, using
dorzolamide as the CA inhibitor (CAI). Another CAI, brinzolamide,
followed in 1998. The incidence of side effects was lowered by local
versus systemic dosing, but adverse events still occur in some pa-
tients.12–15
A combination therapy that operates through both mechanisms
could provide greater IOP reduction for the treatment of glau-
coma.17–19 A CAI which is dosed once daily would be ideal for such
combination therapy with other once-daily therapies such as
latanoprost. This report discloses our initial effort toward the
discovery of a novel inhibitor of carbonic anhydrase using struc-
ture-based drug design20 and subsequent optimization through
successive small compound arrays.
2. Chemistry
5-(Aminosulfonyl)-N-alkyl-2-substituted benzamides 3 were
prepared as depicted in Scheme 1. 2-Fluorobenzoic acid (1) was
converted to sulfonamide 2 with a slight modification of previously
described conditions.21 The acid underwent coupling to a variety of
amide-building blocks using HATU.22 The fluoride in the resulting
intermediates was displaced with a variety of nucleophiles such
as thiols, phenols, and alcohols using cesium carbonate in DMSO
to generate analogs of interest 3. In a selected case, thioether 3a
was converted to the corresponding sulfone using hydrogen perox-
ide in acetic acid.
Another approach to reduce IOP is to increase the outflow of
aqueous humor. There are two main known pathways for this alle-
viating drainage: through the trabecular meshwork and through
In some cases the amides obtained from 2 were difficult to iso-
late using silica gel chromatography. To facilitate the isolation of
these intermediates, the sulfonamide was protected as dimethyl-
amino-methylidenes31 as depicted in Scheme 1. The resulting
* Corresponding author. Tel.: +1 858 638 6274; fax: +1 858 526 4127.
E-mail address: william.f.vernier@pfizer.com (W. Vernier).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.03.014

3308
W. Vernier et al. / Bioorg. Med. Chem. 18 (2010) 3307–3319
H
O
N
COOH
COOH
X
Y
F
F
a
b, c
R
H2NO2S
H2NO2S
X= alkyl
Y = O
S
2
1
3
d, b
f
c, e
SO2
R = substituted
Ph or Benzyl
H
N
O
X
F
N
N
S
O
O
2a
O
H
O
O
O
N
O
N
X
S
R
g
c, h
Cl
Cl
N
N
H2NO2S
ClO2S
H2NO2S
X = alkyl
6
4
5
R = substituted
Ph or Benzyl
Scheme 1. Reagents and conditions: (a) HOSO2Cl then NH3 (59%); (b) H2NX, HATU (31–94%); (c) Cs2CO3, DMSO, HOR or HSR (7–100%); (d) SOCl2, DMF (98%); (e) HCl, MeOH
(45%); (f) H2O2, AcOH (63%); (g) NH3(g), CH2Cl2 (91%); (h) Me3Al, H2NX, THF (55–84%).
Table 1
methylidene was converted to amide 2a as previously described.
Aryl fluoride 2a furnished analogs of interest 3 by introducing
Summary of crystallographic data
the thioether followed by removal of the sulfonamide-protecting
group using methanolic HCl.
Compound
6f
6d
Dorzolamide
Resolution (Å)a
20–2.05
(2.12–2.05)
19.1
50–2.00
(2.07–2.00)
21.2
30–1.72
(1.78–1.72)
17.6
The synthesis of pyridine-containing sulfonamides started with
ethyl 2-chloro-5-(chlorosulfonyl)nicotinate (4,23 Scheme 1). Using
ammonia in dichloromethane, the sulfonyl chloride 4 was con-
verted to the sulfonamide 5. This useful building block spawned
small arrays of 6–12 analogs. The chloride was first displaced with
thiols, and the resulting esters were then transformed to amides 6
using trimethyl aluminum under Weinreb’s conditions.24–26 When
chloroester 5 was exposed to amines and trimethyl aluminum, the
desired amides were formed as a mixture with the diamino
adducts.
The synthesis of meta-aminomethyl derivatives 8 is depicted in
Scheme 2. The Cl in 5 was displaced with 3,4-difluorothiophenol
and the ester hydrolyzed to the corresponding acid. The resulting
acid was reduced with borane–methyl sulfide. For the conversion
of alcohol 7 to amines 8, it was first necessary to protect the sulfon-
amide using trimethyl orthoacetate.27,28 The resulting alcohol was
then transformed to the tosylate, which was displaced with the
desired amines. Finally, deprotection of the sulfonamide was
achieved with TFA. Analog 14 (Table 2), without a meta substitu-
ent, was prepared following the chemistry described in Scheme
1, starting with 2-chloropyridine-5-sulfonyl chloride.29
b
Rcryst (%)
c
Rfree (%)
25.2
26.4
20.6
a
Numbers in parentheses refer to the highest resolution shell.
P
P
b
Rcryst = ( h|Fo ꢀ Fc|/ hFo) ꢁ 100, where Fo and Fc are the observed and calcu-
lated structure factors, respectively.
c
Rfree is computed as for Rcryst but with the test set (5%) of reflections only.
chlorosulfonic acid and ammonia. To facilitate the purification of
the crude acid 10, its corresponding methyl ester was made. The
resulting ester was purified by normal phase chromatography, and
pure acid 10 was regenerated by hydrolysis of the methyl ester in
aqueous hydrochloric acid. Acid 10 underwent sequential conver-
sion to the corresponding ethyl amide using HATU and fluoride dis-
placement with thiols to give the desired meta-fluoro analogs 11.
The regiochemistry of sulfonylation and fluoride displacement were
confirmed by careful analysis of the patterns for the aromatic pro-
tons for final product 11b (Fig. 1). The coupling constants observed
were consistent with the reported coupling constants for fluoroben-
zene:30 ortho hydrogen–fluorine J = 7.0 Hz and meta hydrogen–
hydrogen J = 1.1 Hz couplings. The reported para hydrogen–fluorine
coupling (0.2 Hz in fluorobenzene)30 was not observed.
meta-Fluoro analogs 11 (Scheme 2) were prepared by first
converting 2,3-difluorobenzoic acid (9) to sulfonamide 10 using
H
OH
O
N
O
N
X
S
S
F
F
F
F
Cl
a, b, c
g, h, i, j
d, e, f
N
H2N
N
S
O
H2NO2S
H2NO2S
O
8 X = alkyl
5
7
H
N
O
OH
O
O
OH
k, l
F
S
F
R
H2N
H2N
S
F
S
F
F
R = alkyl or
substituted Ph
O O
O O
11
9
10
Scheme 2. Reagents and conditions: (a) Cs2CO3, DMSO, 3,4-difluorothiophenol (99%); (b) LiOH, THF, H2O (100%); (c) BH3–SMe2, THF (28%); (d) trimethyl orthoacetate,
CH3CN; (e) TsCl, Et3N, THF; (f) H2NX then TFA (7–44% for three steps); (g) HOSO2Cl; (h) NH3, CH2Cl2; (i) SOCl2, MeOH; (j) HCl (63% for four steps); (k) HATU, EtNH2, DMF (27%);
(l) Cs2CO3, DMSO, HSR (8–73%).

W. Vernier et al. / Bioorg. Med. Chem. 18 (2010) 3307–3319
3309
Table 2
CAII and CAIV inhibitory activity, kinetic solubility, and docking score in CAIV of analogs 3a–k, 6a–o, 7, 8a–c, lla–b, and 14
X
Y
R
Z
H2NO2S
Analog
X
Y
R
Z
CAII IC50 (nM)
CAIV IC50 (nM)
Kinetic solubility (
lM)
Docking score (kcal/mol)
3a
3b
3c
3d
3e
3f
3g
3h
3i
CONHBn
CONHEt
CONHBn
CONHBn
CONHCH2-2-Pyr
CONHCH2-2-Pyr
CONHCH2-2-Pyr
CONHCH2CH2OMe
CONHiBu
CONHEt
CONHBn
CONHEt
CONHCH2CH2NMe2
CONHCH2CH2OMe
O
S
S
O
S
S
S
S
S
S
S
SO2
S
S
S
Ph
Ph
Ph
Bn
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
12.3
21.3
35.9
10.1
106
84.1
67.6
20.2
21.5
16.5
>5000
20
241
575
<25.1
205
ꢀ7.9
ꢀ6.8
ꢀ6.6
ꢀ7.3
ꢀ6.5
ꢀ7.2
ꢀ7.1
ꢀ7.3
ꢀ5.7
ꢀ6.4
ꢀ6.9
ꢀ6.3
ꢀ6.3
ꢀ6.3
2040
1480
952
>5000
783
1910
>5000
>5000
1450
283
28.5
<24.2
>258
>253
>275
>304
689
628
55.9
118
2-(Hydroxymethyl)Ph
2-Pyr
Ph
CH2CH2Ph
iPr
cPent
Ph
Ph
Ph
Ph
3j
3k
6a
6b
6c
N
N
2710
57.1
>5000
361
>265
35.6
6d
S
Ph
N
48
246
>287
ꢀ6.6
N
O
6e
6f
CONHPr
CONHCH2CH2Ph
S
S
Ph
Ph
N
N
26.7
26.6
229
64
156
<22.2
ꢀ6.4
ꢀ7.7
F
F
6g
CONHCH2-2-Pyr
S
N
23.2
23
<21.2
ꢀ7.2
F
F
6h
6i
CONHEt
CONH-4-MeOBn
O
S
S
3-FPh
3,4-DiFPh
N
N
19.9
2.19
126
46.8
43.2
<19.9
ꢀ6.2
ꢀ7
6j
S
3-FPh
N
N
N
N
25.8
10.6
34.6
18.4
118
23
89.6
214
40.9
211
ꢀ6.2
ꢀ6.6
ꢀ6.4
ꢀ6.7
N
N
O
O
O
F
F
O
6k
F
F
61
S
S
3,4-DiFPh
139
20.9
N
O
F
F
6m
CONHCH2CH2-2-Pyr
F
F
F
F
O
H
N
N
H
6n
6o
S
S
N
N
45.9
11.5
33.1
23
>265
<19
ꢀ4.7
ꢀ6.7
F
F
F
F
CONHCH2CH2-4OMeBn
F
F
7
8a
8b
CH2OH
CH2NH-4-MeOBn
CH2NHEt
S
S
S
3,4-DiFPh
3,4-DiFPh
3,4-DiFPh
N
N
N
79.3
52.8
321
1230
402
2660
>353
449
ꢀ5.4
ꢀ6.2
ꢀ5.1
8c
11a
11b
S
S
S
3,4-DiFPh
iPr
iBu
N
CF
CF
386
3.77
2.79
2190
1890
1250
497
498
553
ꢀ4.3
ꢀ4.7
ꢀ5.1
N
O
CONHEt
CONHEt
F
F
14
H
S
N
8.29
188
558
ꢀ5.1
F
F
3. Results and discussion
identified celecoxib as an inhibitor of CAII.33 For existing assays,
esterase activity for the CAIV isozyme was much weaker than for
the CAII isozyme.34 To improve sensitivity as well as throughput,
a new assay for CAIV was developed.35,36 The best results were
obtained using an acetazolamide-linked fluorescence tracer which
The carbonic anhydrase program started in the legacy Pharma-
cia company with a screening effort of proprietary compounds that
identified pyrazole benzenesulfonamides as inhibitors of CAII.32
There was also a collaboration at Pharmacia with C. Supuran that

3310
W. Vernier et al. / Bioorg. Med. Chem. 18 (2010) 3307–3319
H
N
O
Hb
H2N
S
S
F
1.9 Hz
O O
Ha
11b
Figure 1. Coupling constants for meta-fluoro analog 11b.
9 Hz
showed an improved fluorescence signal.37 Pyrazole benzenesulf-
onamides such as 12 (Fig. 2) were found to have good potency
for the CAII isozyme, but essentially no measurable activity for
the CAIV isozyme in the new assay (CAII IC50 = 46.7 nM and CAIV
IC50 >1000 nM). Since it was thought at the time of this work that
inhibition of both isoforms II and IV of CA was necessary for the de-
sired IOP-lowering effect, we initially focused on improving the
CAIV potency of the lead matter.
Examination of an X-ray crystal structure of the complex of dor-
zolamide in engineered human CAIV (Fig. 3a) obtained in-house38
identified a large lipophilic pocket formed by Lys 91, Glu 123, and
Lys 206 in a rim with Val 121 and Ile 141 at the floor, that could be
used to improve the CAIV potency and maintain CAII potency of the
initial leads. This prompted the design of ligands, which were min-
imized and docked into CAIV and CAII (see Section 5 for computa-
tional methods employed). Modeling of thioethers of the general
structure 13 predicted a good fit in the dorzolamide-CAIV structure
with a good lipophilic interaction in the previously identified pock-
et. Chemistry toward this class of compounds was developed and
the first two analogs made (3a–b, Table 2) showed an improve-
ment in the CAIV potency. The crystal structure of 6d and 6f (
Fig. 3b and c) later obtained in CAIV confirmed the initial docking
experiments. Docking scores (predicted binding free energy, see
Section 5) of the ligands in the CAIV structure associated with 6d
are shown in Table 2. The docking scores generally followed the
CAIV potency trend (3a–c is a good example) but were not predic-
tive for a number of analogs such as 3d, 3f, 3h, 3k, 6n, and 14. As a
whole, the docking was useful for assessing new designs but not
precise enough to rank order the ligands. The sulfonamide coordi-
nation to the zinc for all determined structures displayed identical
geometries to that for dorzolamide with a N–Zn distance of 2.0 Å.
To optimize this series further an empirical approach with small
arrays of analogs (6–12 at a time) and quick assay turnaround
was taken.
Figure 3. Connolly surface of the CAIV-binding pocket obtained from the crystal
structures of CAIV with selected inhibitors. (a) Top view showing H-bonding
network with dorzolamide. (b) Top view showing H-bonding network with 6d. (c)
Top view showing H-bonding network with 6f.
a topical eye drop formulation which requires relatively high aque-
ous solubility, we next focused on improving solubility.
Intermediate fluoro-acid 2 was a key building block with two
diversity points that was used for the rapid generation of small fo-
cused libraries as shown in Scheme 1 with data shown in Table 2.
These libraries were devised to explore the solvent-exposed lipo-
philic pocket as well as the interactions on the other side of the
active site, adjacent to Thr 200 and Asn 62. Initial structure–activ-
ity relationship (SAR) studies showed that a sulfur atom linkage
was more potent than oxygen linkage and that S-aryl substituents
were preferred over S-alkyls (3a–d, Table 2).
Our initial strategy to improve solubility was to introduce
polarity on the thiophenyl and amide substituents. This initial
effort lead to analogs with improved solubility but reduced CAIV
and CAII potency (2-pyridylmethyls 3e–g vs. 3a, Table 2). This
result is consistent with the X-ray data showing the thiophenyl
moiety in the hydrophobic pocket of CAIV described above
(Fig. 3b and c). Oxidizing the sulfur to the sulfone in order to
reduce the lipophilicity of the compounds improved the solubility
only slightly (3k, Table 2), but significantly reduced the CAIV
potency. In another attempt to boost the solubility, the central
phenyl core was replaced with a pyridine core. No improvement
in solubility and a small increase in CAIV potency were observed
Solubility of the analogs made was assessed using a high
throughput kinetic solubility assay (see Section 5). Most of the
compounds initially made had low solubility. Since our goal was
H
O
S
O
N
O
X
S
CF3
N
N
S
H2NO2S
H2NO2S
H2NO2S
Dorzolamide
HN
12
13 X = alkyl
Figure 2. Pyrazole-sulfonamide 12, thioether 13, general structure, and dorzolamide.

W. Vernier et al. / Bioorg. Med. Chem. 18 (2010) 3307–3319
3311
(6a vs 3b, Table 2). The lack of solubility improvement can be
rationalized by the low basicity of the pyridine due to the
electron-withdrawing effects of the sulfonamide and amide sub-
stituents. Introduction of polar substituents on the amide had
mixed results: analogs with a polar group two carbons away from
the amide had reduced CAII and CAIV potency (3g, 6b, Table 2).
However, with greater separation between the polar group and
the amide, the CAII and CAIV potency was maintained (6d, 6m,
6n, Table 2). The methoxy propyl substituent of 6d was particu-
larly noteworthy since it maintained the CAII potency while
improving the CAIV potency and the solubility. A crystal structure
of 3-methoxypropylamide 6d was obtained in CAIV (Fig. 3b) which
revealed a potential hydrogen bond between the side chain amide
oxygen and Asn 62 (2.7–3.7 Å distance) that could account for the
small improvement in CAIV potency. In most cases, however, lipo-
philic amide substituents were required for greater CAII and CAIV
potency (6e, 6f, 6i, Table 2) and these resulted in reduced solubil-
ity. Interestingly, X-ray analysis of 6f in CAIV showed that the
phenethyl substituent pointed inside a pocket of the protein
formed by close interactions with Tyr 7, Asp 62, and His 94 side
chains (Fig. 3c). This may explain the CAIV potency of this analog
and rationalize the preference for lipophilic substituents in this
region of the CAIV-binding pocket.
Introduction of fluorines on the S-aryl substituent resulted in
improved CAIV potency (6h, 6k, 6l, Table 2). By examining the
X-ray structure of 6d in CAIV one can assume that fluorine substit-
uents in the lipophilic pocket interact favorably with the lipophilic
pocket of the protein. This trend is also observed for CAII and has
also been noted by Supuran et al.39,40 The tetrafluorophenyl substi-
tuent of 6k, also identified by Supuran et al.,40 was particularly
intriguing since unlike the other arylfluorides it did not compro-
mise solubility (214 lM kinetic solubility for 6k), and greatly im-
proved CAIV potency. The combination of this substituent with
solubilizing groups on the amide led to analogs with good CAII
and CAIV potency and solubility. For example, amine 6n showed
>265 lM kinetic solubility.
Our next strategy to improve solubility was to design targets
with lower molecular weights.41,42 When replacing the thiophenyl
substituents with smaller thioalkyl substituents, solubility was
greatly improved, but in most cases the CAIV potency was greatly
reduced (3i–j, Table 2). Only branched alkyls such as isopropylthio
11a and isobutylthio 11b retained modest CAIV potency. In a sim-
ilar fashion, the solubility was also improved with the smaller
amide substituents (3b and 6a vs 3a and 6f, Table 2) and the ana-
logs with small non-amide substituents (7, 14, Table 2).
Shepard et al.43 have shown that introducing a fluorine meta to
the sulfonamide resulted in improved CAII potency in their series
of analogs. Similarly, fluorobenzenesulfonamides 11a and 11b inhib-
ited CAII with IC50’s of 3.77 nM and 2.79 nM, respectively, and were
some of the most potent CAII analogs in this series (Table 2).
A selected set of compounds, as shown in Table 3, was selected
for further evaluation. The selection was made based on CAII po-
tency, kinetic solubility, and CAIV potency. Since the kinetic solu-
bility determination was done on a small scale from DMSO liquid
Further examination of the X-ray structure of dorzolamide and
6d and 6f in CAIV showed that the ethylamine group of dorzola-
mide occupied the same region of the protein as the amide substi-
tuent of the thioethers. Analogs with amine substituents at the
meta position of the sulfonamide could potentially maintain po-
tency while increasing the solubility. Unfortunately, these targeted
analogs, while more soluble, had reduced potency for CAII and
CAIV (8a–c, Table 2).
Table 3
Inhibition of carbonic anhydrase in iris ciliary body homogenate (‘pH Stat Spike’) of selected analogs
Analog
Structure
CAII IC50 (nM)
CAIV IC50 (nM)
Thermodynamic
solubility ( M)
pH Stat Spike
(% inh. 0.1 M)
pH Stat Spike
(% inh. 10 M)
l
l
l
F
F
O
O
S
N
H
F
F
6k
10.60
23
124
47.5
80.9
N
NH2
S
O
O
O
F
F
NH2
O
S
N
H
F
F
6n
45.90
33.1
3090
65.4
84.1
N
NH2
S
O
H
O
N
S
F
11b
2.79
8.29
<2
1250
188
43
395
67.6
74.2
68.0
85.5
99.3
86.0
H2N
S
O
O
O
O
S
S
F
F
H2N
N
14
231
F
F
NH
S
NH2
Dorzolamide
>61,600
S
O
O
S
O
O

3312
W. Vernier et al. / Bioorg. Med. Chem. 18 (2010) 3307–3319
stocks, more precise thermodynamic solubility44 was determined
with a larger sample in aqueous phosphate buffer. General ranking
of solubility between kinetic and thermodynamic determinations
was consistent, but the latter more relevant for future formulation.
This set of analogs had moderate to good thermodynamic solubil-
ity (although much lower than that of dorzolamide). We next
evaluated this set of compounds with an in vitro experiment where
the inhibition of carbonic anhydrase was measured in rabbit iris
ciliary body homogenate.45,46 Good in vitro carbonic anhydrase
oughly to afford a gray solid, which was added in portions to 28%
aq ammonium hydroxide (700 mL) at 0 °C. The mixture was stirred
at ambient temperature overnight and concentrated to a small
volume under reduced pressure. 0.1 N aq H2SO4 was added slowly
at 0 °C with vigorous stirring until pH 5. The precipitate was fil-
tered, washed with water, and dried under vacuum at 50 °C to give
5-(aminosulfonyl)-2-fluorobenzoic acid (293 g, 58.7%) as a white
solid. 1H NMR (300 MHz, DMSO-d6) d 7.39 (d, J = 1.1 Hz, 1H), 7.43
(br s, 3H), 7.83–7.91 (m, 1H), 8.22 (dd, J = 6.8, 2.5 Hz, 1H). LC/MS
m/z 241.9 [MNa]+. HRMS (TOF) calcd for C7H7FNO4S [MH]+,
220.00743, found 220.00743.
inhibition was achieved at 10 lM and 6n, 11b, and 14 showed
inhibition comparable to dorzolamide at 0.1 lM.
5.3. Method A. General procedure for meta amide preparation
4. Conclusion
To a solution of 2 in DMF (0.23 M) was added HATU (1.5 equiv).
Themixturewasstirredatambienttemperaturefor2 handtheamine
(1.2 equiv) was added. The mixture was further stirred at ambient
temperature for 18 h. The crude material was directly purified by
RPHPLC using acetonitrile in water (5–95%, with 0.1% AcOH or TFA).
A novel series of potent inhibitors of CAII and CAIV has been dis-
covered using a combination of small array synthesis and struc-
ture-based drug design. Each successive small array found more
potent inhibitors and promising leads were co-crystallized to in-
form design of the next iteration. Different strategies to improve
solubility while maintaining CAII and CAIV potency were explored.
Several analogs with improved solubility have been identified and
showed inhibition of CA activity in rabbit iris tissue homogenates
similar to dorzolamide. Such promising activity warrants further
examination, particularly in vivo IOP-lowering studies. Further
studies on this series and related analogs are in progress and will
be reported in due course.
5.3.1. 5-(Aminosulfonyl)-N-benzyl-2-fluorobenzamide (15a)
To a stirred solution of 2 (63.0 g, 0.288 mol) in anhydrous DMF
(1000 mL) at 0 °C were sequentially added HOBt hydrate (62.0 g,
0.461 mol), EDCI (88.0 g, 0.461 mol), N-methyl-morpholine
(126.0 mL, 1.152 mol), and benzylamine (38.0 mL, 0.345 mol).
The mixture was stirred overnight, poured into water, and filtered.
The collected solid was washed with dilute aqueous HCl, dilute
aqueous sodium hydroxide, and water, and dried in vacuo to yield
the title compound (40.0 g, 45.1%) as a white solid. 1H NMR
(300 MHz, DMSO-d6) d 4.51 (d, J = 5.8 Hz, 2H), 7.25–7.32 (m, 3H),
7.35–7.39 (m, 4H), 7.52 (t, J = 9.5 Hz, 1H), 7.91–7.99 (m, 1H), 8.08
(dd, J = 6.5, 2.4 Hz, 1H), 9.11 (s, 1H). LC/MS m/z 309.0 [MH]+. HRMS
(TOF) calcd for C14H14FN2O3S [MH]+, 331.05231; found, 331.05171.
Anal. (C14H13FN2O3Sꢂ0.5HCl) C, H, N.
5. Experimental section
5.1. General methods
All reagents were purchased from commercial sources and were
used without further purification. Solvents were of analytical or
anhydrous grade (Sigma–Aldrich). Reactions were monitored by
HPLC. Analytical HPLC analysis was conducted using an Agilent
1100 HPLC with chromatography performed on a YMC CombiScreen
5.3.2. 5-(Aminosulfonyl)-N-ethyl-2-fluorobenzamide (15b)
Prepared following method A using 2 M ethylamine in THF.
Yield: 87 mg, 31% as a white solid. 1H NMR (300 MHz, DMSO-d6)
d 1.14 (t, J = 7.2 Hz, 3H), 3.22–3.34 (m, 2H), 7.43–7.59 (m, 3H),
7.91–7.98 (m, 1H), 8.06 (dd, J = 6.6, 2.5 Hz, 1H), 8.55 (s, 1H). HPLC
tR = 1.2 min (>95%). LC/MS (ESI) m/z 247.0 [MH]+. HRMS (TOF)
calcd for C9H12FN2O3S [MH]+: 247.05472, found: 247.05745. Anal.
(C9H11FN2O3Sꢂ0.2AcOHꢂ0.5H2O) C, H, N.
ODS-A C18 column (4.6 ꢁ 50 mm, 5
lm) at 25 °C. The mobile phase
was a 4-min binary gradient of acetonitrile (containing 0.1% TFA)
and water (containing 0.1% TFA) (5–95%) with a flow rate of 3 mL/
min. The retention times (tR) are expressed in min with UV detection
at 220 nM, 254 nM, and 280 nM. Reverse-phase preparative HPLC
was performed using a Waters LC 4000 preparative HPLC system
over a 250 ꢁ 50.8 mm Peeke C18 column (10
lm). A gradient of ace-
tonitrile (containing 0.1% TFA or AcOH) and water (containing 0.1%
TFA or AcOH) (5–95%) with a flow rate of 95 mL/min was used. Some
of the partial TFA salt obtained from the RPHPLC purification was
converted to an HCl salt using 3 N HCl in methanol and toluene. 1H
NMR spectra were recorded on an Avance DPX NMR Spectrometer
(Bruker Biospin, Billerica, MA). Chemical shifts (d) are reported in
downfield from TMS. Mass spectra were obtained on an Agilent
G1959A LC/MS with electrospray ionization (ESI) source mass spec-
trometer, and for high resolution, an Agilent G1969A LC/MSD TOF
with electrospray ionization (ESI) source mass spectrometer. Ele-
mental analyses were performed by Atlantic Microlabs, Norcross,
GA, and are within 0.4% of theoretical values.
5.3.3. 5-(Aminosulfonyl)-2-fluoro-N-(pyridin-2-ylmethyl)benz-
amide (15c)
Prepared following method A using 2-(aminomethyl)pyridine.
Yield: 5.47 g, 94%. 1H NMR (400 MHz, DMSO-d6) d 5.02 (d,
J = 5.6 Hz, 2H), 7.78 (s, 2H), 7.83 (dd, J = 10.2, 8.7 Hz, 1H), 8.00 (t,
J = 6.8 Hz, 1H), 8.04 (d, J = 7.8 Hz, 1H), 8.23–8.28 (m, 1H), 8.43
(dd, J = 6.7, 2.4 Hz, 1H), 8.57 (t, J = 7.7 Hz, 1H), 9.02 (d, J = 4.6 Hz,
1H), 9.49–9.52 (m, 1H). LC/MS m/z 310.0 [MH]+. HRMS (TOF) calcd
for C13H13FN3O3S [MH]+, 310.0656; found, 310.0661.
5.3.4. 5-(Aminosulfonyl)-2-fluoro-N-(2-methoxyethyl)benz-
amide (15d)
Prepared following method A using 2-methoxyethylamine. Yield:
0.739 g, 58%. 1H NMR (300 MHz, DMSO-d6) d 3.30 (s, 3H), 3.41–3.53
(m, 4H), 7.55–7.57 (m, 2H), 7.95 (m, 1H), 8.06 (dd, J = 6.5, 2.4 Hz,
1H), 8.35 (dd, J = 6.8, 2.5 Hz, 1H). LC/MS m/z 277.1 [MH]+.
5.2. Chemistry
5.2.1. 5-(Aminosulfonyl)-2-fluorobenzoic acid (2)
2-Fluorobenzoic acid (320 g, 2.28 mol) was added to chlorosul-
fonic acid (750 mL) portionwise at ambient temperature. The
resulting mixture was stirred at ambient temperature for 0.5 h
and then heated at 130–140 °C overnight. The ambient tempera-
ture mixture was slowly poured onto crushed ice. After stirring
for 0.5 h, the precipitate was filtered and washed with water thor-
5.4. Method B. General procedure for the aryl fluoride
displacement
A solution of aryl fluoride, thiophenol (1.5 equiv), and cesium
carbonate (2.5 equiv) in DMSO (0.25 M) was heated at 50–

W. Vernier et al. / Bioorg. Med. Chem. 18 (2010) 3307–3319
3313
100 °C for 2 h. The reaction mixture was treated with aq 5 N HCl
(1 mL) and then directly purified by preparative reverse-phase
HPLC using acetonitrile in water (5–95%, with 0.1% acetic acid
or TFA).
HRMS (TOF) calcd for C18H17N4O3S2 [MH]+, 401.0737; found,
401.0738. Anal. (C18H16N4O3S2ꢂ2.0HClꢂ2.1H2O) C, H, N.
5.4.7. 5-(Aminosulfonyl)-2-(phenylthio)-N-(pyridin-2-ylmethyl)
benzamide hydrochloride (3g)
Prepared following method B starting with 15c and thiophe-
nol. Yield: 125 mg (57%). 1H NMR (400 MHz, DMSO-d6) d 4.59
(d, J = 5.6 Hz, 2H), 6.83 (d, J = 8.6 Hz, 1H), 7.23–7.33 (m, 7H),
7.53 (dd, J = 8.6, 2.0 Hz, 1H), 7.60 (t, J = 6.8 Hz, 1H), 7.70 (d,
J = 8.1 Hz, 1H), 7.91 (d, J = 2.0 Hz, 1H), 8.19 (t, J = 7.5 Hz, 1H),
8.60 (d, J = 5.1 Hz, 1H), 9.41 (t, J = 5.7 Hz, 1H). LC/MS m/z
400.0 [MH]+. HRMS (TOF) calcd for C19H18N3O3S2 [MH]+,
400.0784; found, 400.0785. Anal. (C19H17N3O3S2ꢂHClꢂH2O)
C, H, N.
5.4.1. 5-(Aminosulfonyl)-N-benzyl-2-(phenylthio)benzamide
(3a)
Prepared following method B starting with 15a and thiophenol.
Yield: 52 mg (13%). 1H NMR (400 MHz, DMSO-d6) d 4.53 (d, J =
6.1 Hz, 2H), 7.09 (d, J = 8.3 Hz, 1H), 7.28–7.34 (m, 1H), 7.37–7.47
(m, 4H), 7.50–7.58 (m, 5H), 7.75 (dd, J = 8.5, 2.2 Hz, 1H), 7.94 (d,
J = 2.0 Hz, 1H), 9.29 (t, J = 5.9 Hz, 1H). LC/MS m/z 399.0 [MH]+. HRMS
(TOF) calcd for C20H19N2O3S2 [MH]+: 399.0832, found: 399.0836.
Anal. (C20H18N2O3S2ꢂ0.4H2O) C, H, N.
5.4.8. 5-(Aminosulfonyl)-N-(2-methoxyethyl)-2-[(2-phenyl-
ethyl) thio]benzamide (3h)
5.4.2. 5-(Aminosulfonyl)-N-ethyl-2-(phenylthio)benzamide (3b)
Prepared following method B starting with 15b and thiophenol.
Yield: 24 mg (7%). 1H NMR (400 MHz, DMSO-d6)
d 1.00 (t,
Prepared following method B starting 15d and phenethyl mer-
captan. Yield: 116 mg (100%). 1H NMR (300 MHz, DMSO-d6) d
2.89 (t, J = 7.5 Hz, 2H), 3.23–3.32 (m, 5H), 3.37–3.42 (m, 2H),
3.44–3.50 (m, 2H), 7.20–7.35 (m, 5H), 7.40 (s, 2H), 7.65 (d,
J = 8.3 Hz, 1H), 7.75 (d, J = 2.1 Hz, 1H), 7.81 (dd, J = 8.3, 2.1 Hz,
1H), 8.62 (t, J = 5.4 Hz, 1H). LC/MS m/z 395.0 [MH]+. HRMS (TOF)
calcd for C18H23N2O4S2 [MH]+, 395.1094; found, 395.1100. Anal.
(C18H22N2O4S2ꢂ0.1H2O) C, H, N.
J = 7.3 Hz, 3H), 3.09–3.18 (m, 2H), 6.88 (d, J = 8.6 Hz, 1H), 7.25 (s,
2H), 7.31–7.38 (m, 5H), 7.54 (dd, J = 8.5, 2.2 Hz, 1H), 7.69 (d,
J = 2.0 Hz, 1H), 8.54 (t, J = 5.4 Hz, 1H). LC/MS m/z 337.0 [MH]+.
HRMS (TOF) calcd for C15H17N2O3S2 [MH]+: 337.0675, found:
337.0676. Anal. (C15H16N2O3S2ꢂ0.4H2O) C, H, N.
5.4.3. 5-(Aminosulfonyl)-N-benzyl-2-phenoxybenzamide (3c)
Prepared following method B starting with 15a and phenol.
Yield: 405 mg (66%). 1H NMR (400 MHz, DMSO-d6) d 4.47 (d, J =
6.1 Hz, 2H), 6.97 (d, J = 8.8 Hz, 1H), 7.14 (d, J = 7.6 Hz, 2H), 7.21–
7.30 (m, 6H), 7.41 (s, 2H), 7.46 (t, J = 8.0 Hz, 2H), 7.83 (dd, J = 8.8,
2.5 Hz, 1H), 8.09 (d, J = 2.3 Hz, 1H), 8.97 (t, J = 6.1 Hz, 1H). LC/MS
m/z 383.0 [MH]+. HRMS (TOF) calcd for C20H19N2O4S [MH]+:
383.1060, found: 383.1050. Anal. (C20H18N2O4S) C, H, N.
5.4.9. 5-({[(1E)-(Dimethylamino)methylene]amino}sulfonyl)-2-
fluorobenzoic acid (16)
To a solution of 2 (1.0 g, 4.56 mmol) in toluene (10 mL) were
slowly added thionyl chloride (1.66 mL, 22.81 mmol) and DMF
(0.42 mL, 32.1 mmol). The reaction mixture was stirred at 70 °C
for 48 h and then the reaction was carefully quenched with ice
(10 g). The aqueous layer was extracted with ethyl acetate
(3 ꢁ 25 mL) and the combined organic layer was dried (MgSO4)
and concentrated in vacuo to give the title compound (1.23 g,
98%) as a white solid. 1H NMR (300 MHz, DMSO-d6) d 2.93 (s,
3H), 3.17 (s, 3H), 7.51 (t, J = 9.5 Hz, 1H), 7.96–8.08 (m, 1H), 8.20–
8.28 (m, 1H), 13.61 (br. s., 1H). LC/MS m/z 275.2 [MH]+. HRMS
(TOF): calcd for C10H12FN2O4S [MH]+: 275.04963, found:
275.05187. Anal. (C10H11FN2O4Sꢂ0.4HCl) C, H, N.
5.4.4. 5-(Aminosulfonyl)-N-benzyl-2-(benzylthio)benzamide
(3d)
Prepared following method B starting with 15a and benzyl mer-
captan. Yield: 442 mg (67%). 1H NMR (400 MHz, DMSO-d6) d 4.40
(s, 2H), 4.54 (d, J = 6.1 Hz, 2H), 7.32–7.39 (m, 2H), 7.40–7.46 (m,
6H), 7.48–7.55 (m, 4H), 7.77 (d, J = 8.3 Hz, 1H), 7.84–7.91 (m,
2H), 9.22 (t, J = 5.9 Hz, 1H). LC/MS m/z 413.0 [MH]+. HRMS (TOF)
calcd for C21H21N2O3S2 [MH]+: 413.0988, found: 413.0996. Anal.
(C21H20N2O3S2ꢂ0.3H2O) C, H, N.
5.4.10. Methyl 5-({[(1E)-(dimethylamino)methylene]amino}-
sulfonyl) -2-fluorobenzoate (17)
A solution of 16 (160 mg, 0.58 mmol) in methanol was stir-
red at 40 °C for 4 h. The mixture was concentrated under re-
duced pressure and the residue was purified by column
chromatography (silica gel, ethyl acetate–hexane, 50%) to yield
5.4.5. 5-(Aminosulfonyl)-2-{[2-(hydroxymethyl)phenyl]thio}-N-
(pyridin-2-ylmethyl)benzamide hydrochloride (3e)
Prepared following method B starting with 15c and 2-merca-
ptobenzyl alcohol. Yield: 155 mg (33%). 1H NMR (400 MHz, DMSO-
d6) d 4.52 (s, 2H), 4.78 (d, J = 5.8 Hz, 2H), 6.82 (d, J = 8.3 Hz, 1H),
7.38 (t, J = 7.6 Hz, 1H), 7.44 (s, 2H), 7.48 (d, J = 7.8 Hz, 1H), 7.54 (td,
J = 7.5, 1.3 Hz, 1H), 7.65–7.72 (m, 2H), 7.78 (t, J = 6.8 Hz, 1H), 7.88
(d, J = 8.1 Hz, 1H), 8.09 (d, J = 2.0 Hz, 1H), 8.36 (t, J = 7.8 Hz, 1H),
8.80 (d, J = 5.3 Hz, 1H), 9.60 (t, J = 5.7 Hz, 1H). LC/MS m/z 430.1
[MH]+. HRMS (TOF) calcd for C20H20N3O4S2 [MH]+, 430.0890; found,
430.0891. Anal. (C20H19N3O4S2ꢂHClꢂH2O) C, H, N.
the title product as
a
white solid (106 mg, 63%). 1H NMR
(300 MHz, DMSO-d6) d 2.93 (s, 3H) 3.17 (s, 3H) 3.91 (s, 3H)
7.55 (dd, J = 10.6, 8.8 Hz, 1H) 8.01–8.12 (m, 1H) 8.22–8.31 (m,
2H). LC/MS m/z 289.1 [MH]+. HRMS (TOF): calcd for
C11H13FN2O4S [MH]+: 289.06528, found: 289.07424. Anal.
(C11H13FN2O4Sꢂ0.2H2O) C, H, N.
5.5. Method C. Successive fluoride displacement, amide
formation and sulfonamide deprotection
5.4.6. 5-(Aminosulfonyl)-N-(pyridin-2-ylmethyl)-2-(pyridin-2-
ylthio)benzamide dihydrochloride (3f)
To a solution of methyl 17 and Cs2CO3 (1.1 equiv) in DMSO
(0.35 M) was slowly added the thiol (1.1 equiv). The reaction mix-
ture was stirred at 50 °C for 4 h, and diluted in ethyl acetate and
water. The organic layer was washed with water, dried (MgSO4),
and concentrated under reduced pressure to yield the title product
methyl ester intermediate which was used without further purifi-
cation. This material was converted to the corresponding amide
Prepared following method B starting with 15c and 2-mercap-
topyridine. Yield: 127 mg (54%). 1H NMR (400 MHz, DMSO-d6)
d 4.83 (d, J = 5.6 Hz, 2H), 7.27–7.34 (m, 2H), 7.61 (s, 2H), 7.63
(d, J = 8.3 Hz, 1H), 7.78 (td, J = 7.8, 1.9 Hz, 1H), 7.88 (dd, J = 8.3,
2.0 Hz, 1H), 7.94 (t, J = 6.8 Hz, 1H), 7.96 (s, 1H), 8.17 (d, J = 2.0 Hz,
1H), 8.47 (d, J = 4.0 Hz, 1H), 8.51 (td, J = 8.0, 1.5 Hz, 1H), 8.86
(d, J = 5.1 Hz, 1H), 9.66 (t, J = 5.7 Hz, 1H). LC/MS m/z 401.0 [MH]+.

3314
W. Vernier et al. / Bioorg. Med. Chem. 18 (2010) 3307–3319
using method D. The crude product was diluted in 3 N HCl in
methanol (5 mL) and stirred at 60 °C for 3 h before reverse-phase
purification.
the reaction was complete. The reaction mixture was purged with
N2 to remove excess NH3. The suspension was concentrated to give
a light yellow solid, which was filtered, washed with water, and
dried to give the title compound (144 g, 91%) as a white solid. 1H
NMR (300 MHz, DMSO-d6) d 1.37 (t, J = 7.1 Hz, 3H), 4.42 (q,
J = 7.1 Hz, 2H), 7.81 (s, 2H), 8.62 (d, J = 2.5 Hz, 1H), 8.96 (d,
J = 2.5 Hz, 1H). HRMS (TOF) calcd for C8H10ClN2O4S [MH]+,
265.00443; found, 265.00534. Anal. (C8H9ClN2O4S) C, H, N.
5.5.1. 5-(Aminosulfonyl)-N-isobutyl-2-(isopropylthio)benz-
amide (3i)
Prepared via method C starting with 2-propanethiol and isobu-
tylamine. Yield: 129 mg (58%). 1H NMR (400 MHz, DMSO-d6) d 1.00
(d, J = 6.8 Hz, 6H), 1.15 (d, J = 6.6 Hz, 6H), 1.72–1.86 (m, 1H), 2.88
(d, J = 6.8 Hz, 2H), 3.98–4.10 (m, 1H), 7.38 (s, 2H), 7.56 (d,
J = 8.3 Hz, 1H), 7.66 (d, J = 2.3 Hz, 1H), 7.75 (dd, J = 8.3, 2.0 Hz,
1H), 8.39 (d, J = 7.8 Hz, 1H). LC/MS m/z 331.1 [MH]+. HRMS (TOF):
calcd for C14H23N2O3S2 [MH]+: 353.09663, found: 353.09641. Anal.
(C14H22N2O3S2+0.3H2O) C, H, N.
5.6. Method D. General procedure for amide formation from
esters
To a cooled 0 °C solution of the amine (3 equiv) in THF (0.2 M)
was slowly added trimethylaluminum (2 M in toluene, 4 equiv).
The mixture was stirred at ambient temperature for 1 h and the es-
ter (1.0 equiv) was added. The mixture was stirred at 45 °C for 24 h
and then the reaction was carefully quenched with methanol and
1 N HCl. The crude mixture was directly purified by preparative re-
verse-phase HPLC using acetonitrile in water (5–95%, with 0.1%
acetic acid or TFA) to give the desired product.
5.5.2. 5-({[(1E)-(Dimethylamino)methylene]amino}sulfonyl)-N-
ethyl-2-fluorobenzamide (2a)
To a solution of 16 (0.9 g, 3.28 mmol) in DMF (10 mL) were
slowly added triethylamine (0.457 mL, 3.28 mmol) and HATU
(1.25 g, 3.28 mmol). The mixture was stirred at ambient tempera-
ture for 1 h and ethylamine (1.64 mL, 2 M in THF, 3.28 mmol) was
slowly added. The mixture was stirred at ambient temperature for
48 h and then diluted with methylene chloride and water. The or-
ganic layer was washed with water and concentrated in vacuo. The
residue was purified by column chromatography eluting with ethyl
acetate–hexane (30–100%) to give the title compound (455 mg,
51%) as a white solid. 1H NMR (300 MHz, DMSO-d6) d 1.14 (t,
J = 7.3 Hz, 3H), 2.93 (s, 3H), 3.17 (s, 3H), 3.23–3.32 (m, 2H), 7.47
(dd, J = 9.9, 8.8 Hz, 1H), 7.87–7.98 (m, 2H), 8.26 (s, 1H), 8.49–8.59
(m, 1H). LC/MS m/z 302.0 [MH]+. HRMS (TOF): calcd for
C12H17FN3O3S [MH]+: 302.09692, found: 302.09980.
5.6.1. 5-(Aminosulfonyl)-2-chloro-N-ethylnicotinamide hydro-
chloride (18a)
Prepared following method D starting with 5 and ethylamine.
Yield: 580 mg (38%). This material was used without further puri-
fication. HPLC tR = 1.4 min (>98%). LC/MS m/z 264.0 [MH]+.
5-(Aminosulfonyl)-N-ethyl-2-(ethylamino)nicotinamide (365 mg,
30%) was isolated as a byproduct and the TFA salt resulting from the
reverse-phase purification was converted to the HCl salt using 3 N
HCl in MeOH. 1H NMR (300 MHz, DMSO-d6+D2O) d 1.05–1.21 (m,
6H), 3.25 (q, J = 7.3 Hz, 2H), 3.43 (q, J = 7.2 Hz, 2H), 8.20 (d, J = 2.3 Hz,
1H), 8.48 (d, J = 2.3 Hz, 1H). LC/MS m/z 273.1 [MH]+. HRMS (TOF) calcd
for C10H17N4O3S [MH]+, 273.1016; found, 273.1018.
5.5.3. 5-(Aminosulfonyl)-2-(cyclopentylthio)-N-ethylbenzamide
(3j)
Prepared following method B starting with 2a and cyclopentyl
mercaptan. The crude product was diluted in 3 N HCl in MeOH
(3 mL), stirred at 60 °C for 3 h, concentrated, and purified by col-
umn chromatography eluting with ethyl acetate–hexane (10–
100%). Yield: 49 mg (45%). 1H NMR (300 MHz, DMSO-d6) d 1.13
(t, J = 7.2 Hz, 3H), 1.42–1.77 (m, 6H), 2.04–2.20 (m, 2H), 3.17–
3.30 (m, 2H), 3.73–3.86 (m, 1H), 7.38 (s, 2H), 7.62 (d, J = 8.5 Hz,
1H), 7.71 (d, J = 2.1 Hz, 1H), 7.78 (dd, J = 8.5, 2.1 Hz, 1H), 8.49 (t,
J = 5.6 Hz, 1H). LC/MS m/z 329.0 [MH]+. HRMS (TOF) calcd for
C14H21N2O3S2 [MH]+, 329.09881; found, 329.09933. Anal.
(C14H20N2O3S2) C, H, N.
5.6.2. 5-(Aminosulfonyl)-2-chloro-N-(pyridin-2-ylmethyl)-
nicotin-amide (18b)
Prepared following method D starting with 5 and 2-(amino-
methyl)pyridine. Yield: 1.52 g (68%) of an orange solid that con-
tained some of the chloride displacement byproduct. The material
was used without further purification. LC/MS m/z 327.0 [MH]+.
5.7. Method E. General procedure for the 2-chloropyridine
displacement
A solution of 2-chloropyridine, thiophenol (1.2 equiv), and ce-
sium carbonate (2.5 equiv) in DMSO (0.15 M) was heated at 50–
90 °C for 2–18 h. The reaction mixture was treated with aq 5 N HCl
(1 mL), then directly purified by preparative reverse-phase HPLC
using acetonitrile in water (5–95%, with 0.1% acetic acid or TFA).
5.5.4. 5-(Aminosulfonyl)-N-benzyl-2-(phenylsulfonyl)benzamide
(3k)
To a solution of 3a (340 mg, 0.9 mmol) in acetic acid (4 mL) was
added hydrogen peroxide (0.29 mL of 30 wt % in water, 2.56 mmol).
Themixturewasstirredatambienttemperaturefor2 h, thenat75 °C
for 2 h. The crude reaction mixture was directly purified by prepara-
tive reverse-phase HPLC using acetonitrile in water (5–95%, with
0.1% acetic acid or TFA) to give the title compound (272 mg, 63%).
1H NMR (400 MHz, DMSO-d6) d 4.51 (d, J = 5.8 Hz, 2H), 7.29 (t,
J = 7.2 Hz, 1H), 7.38 (t, J = 7.6 Hz, 2H), 7.43–7.47 (m, 2H), 7.62 (t,
J = 7.6 Hz, 2H), 7.67–7.72 (m, 3H), 7.83 (d, J = 1.8 Hz, 1H), 8.01–
8.08 (m, 3H), 8.34 (d, J = 8.3 Hz, 1H), 9.23 (t, J = 5.9 Hz, 1H). LC/MS
m/z 431.0 [MH]+. HRMS (TOF) calcd for C20H19N2O5S2 [MH]+:
431.0730, found: 431.0719. Anal. (C20H18N2O5S2ꢂ0.1H2O) C, H, N.
5.7.1. Ethyl 5-(aminosulfonyl)-2-(phenylthio)nicotinate (19a)
Prepared following method E starting with 5 and thiophenol.
The crude product (1.5 g, 100%) was used without further purifica-
tion. LC/MS m/z 339.0 [MH]+.
5.7.2. Ethyl 5-(aminosulfonyl)-2-[(2,3,5,6-tetrafluorophenyl)th-
io]nicotinate (19b)
Prepared following method E starting with 5 and 2,3,5,6-tetra-
fluorobenzenethiol. The crude product (8.58 g, 100%, white solid)
was used without further purification. 1H NMR (300 MHz, DMSO-
d6) d 1.42 (t, J = 7.1 Hz, 3H), 4.48 (q, J = 7.2 Hz, 2H), 7.71 (s, 2H),
8.10–8.25 (m, 1H), 8.67 (d, J = 2.3 Hz, 1H), 8.81 (d, J = 2.5 Hz, 1H).
LC/MS m/z 411.0 [MH]+. HRMS (TOF): calcd for C14H11F4N2O4S2
[MH]+: 411.00909, found: 411.01768.
5.5.5. Ethyl 5-(aminosulfonyl)-2-chloronicotinate (5)
Ammonia was bubbled through a cooled ꢀ50 °C solution of 4
(170 g, 0.6 mol) in anhydrous CH2Cl2 (1500 mL) for 20–30 min.
TLC (silica gel with EtOAc/petroleum ether = 1:2) showed that

W. Vernier et al. / Bioorg. Med. Chem. 18 (2010) 3307–3319
3315
5.7.3. Ethyl 5-(aminosulfonyl)-2-[(3,4-difluorophenyl)thio]nic-
otinate (19c)
J = 7.4 Hz, 2H), 3.48–3.58 (m, 2H), 7.29–7.37 (m, 5H), 7.45–7.53 (m,
6H), 7.60 (s, 2H), 8.12 (d, J = 2.3 Hz, 1H), 8.65 (d, J = 2.3 Hz, 1H),
9.02 (t, J = 5.5 Hz, 1H). LC/MS m/z 414.0 [MH]+. HRMS (TOF) calcd
for C20H20N3O3S2 [MH]+, 414.0941; found, 414.0943. Anal.
(C20H19N3O3S2ꢂ0.05HCl) C, H, N.
Prepared following method E starting with 5 and 3,4-difluoro-
thiophenol. The crude material was triturated twice with 50 mL
of 10% ethyl acetate in hexane to give the title compound (14.5 g,
99%) as a white solid, which was used without further purification.
1H NMR (300 MHz, DMSO-d6) d 1.40 (t, J = 7.1 Hz, 3H), 4.44 (q,
J = 7.1 Hz, 2H), 7.39–7.48 (m, 1H), 7.51–7.63 (m, 1H), 7.69 (s, 2H),
7.71–7.77 (m, 1H), 8.59 (d, J = 2.3 Hz, 1H), 8.76 (d, J = 2.3 Hz, 1H).
LC/MS m/z 375.0 [MH]+. HRMS (TOF): calcd for C14H13F2N2O4S2
[MH]+: 375.02793, found: 375.03571. Anal. (C14H12F2N2O4S2) C,
H, N.
5.7.10. 5-(Aminosulfonyl)-N-(pyridin-2-ylmethyl)-2-[(2,3,5,6-
tetrafluorophenyl)thio] nicotinamide hydrochloride (6g)
Prepared following method E starting with 18b and 2,3,5,6-tet-
rafluorobenzenethiol. Yield: 92 mg (42%). 1H NMR (300 MHz,
DMSO-d6) d 4.87 (d, J = 5.3 Hz, 2H), 7.70 (s, 2H), 7.82 (t, J = 6.5 Hz,
1H), 7.93 (d, J = 8.1 Hz, 1H), 8.08–8.21 (m, 1H), 8.39 (td, J = 7.8,
1.1 Hz, 1H), 8.77 (d, J = 2.1 Hz, 1H), 8.80–8.84 (m, 3H), 10.08 (t,
J = 5.5 Hz, 1H). LC/MS m/z 473.0 [MH]+. HRMS (TOF) calcd for
C18H13F4N4O3S2 [MH]+, 473.0360; found, 473.0374. Anal.
(C18H12F4N4O3S2ꢂ1.4HCl) C, H, N.
5.7.4. 5-(Aminosulfonyl)-N-ethyl-2-(phenylthio)nicotinamide (6a)
Prepared following method E starting with 18a and thiophenol.
Yield: 124 mg (100%). 1H NMR (300 MHz, DMSO-d6) d 1.18 (t,
J = 7.3 Hz, 3H), 3.24–3.34 (m, 2H), 7.44–7.54 (m, 5H), 7.59 (s, 2H),
8.15 (d, J = 2.3 Hz, 1H), 8.64 (d, J = 2.3 Hz, 1H), 8.91 (t, J = 5.4 Hz,
1H). LC/MS m/z 338.1 [MH]+. HRMS (TOF) calcd for C14H16N3O3S2
[MH]+, 338.0628; found, 338.0632. Anal. (C14H15N3O3S2ꢂ0.1HCl) C,
H, N.
5.7.11. 5-(Aminosulfonyl)-N-ethyl-2-[(3-fluorophenyl)thio]nic-
otinamide (6h)
Prepared following method E starting with 18a and 3-fluorothi-
ophenol. Yield: 112 mg (92%). 1H NMR (300 MHz, DMSO-d6) d 1.18
(t, J = 7.3 Hz, 3H), 3.27–3.35 (m, 2H), 7.28–7.43 (m, 3H), 7.47–7.56
(m, 1H), 7.60 (s, 2H), 8.21 (d, J = 2.3 Hz, 1H), 8.69 (d, J = 2.3 Hz, 1H),
8.94 (t, J = 5.4 Hz, 1H). LC/MS m/z 356.0 [MH]+. HRMS (TOF) calcd
for C14H15FN3O3S2 [MH]+, 356.0534; found, 356.0538. Anal.
(C14H14FN3O3S2ꢂ0.1HCl) C, H, N.
5.7.5. 5-(Aminosulfonyl)-N-[2-(dimethylamino)ethyl]-2-
(phenyl-thio)nicotinamide dihydrochloride (6b)
Prepared following method D starting with 19a and N,N0-dime-
thylenediamine. Yield: 194 mg (73%). 1H NMR (400 MHz, DMSO-
d6) d 2.94 (s, 6H), 3.92–4.02 (m, J = 4.0 Hz, 4H), 7.50–7.57 (m,
3H), 7.60–7.68 (m, 2H), 7.86 (s, 2H), 8.50 (d, J = 2.3 Hz, 1H), 8.99
(d, J = 2.5 Hz, 1H). LC/MS m/z 381.1 [MH]+. HRMS (TOF) calcd for
C16H21N4O3S2 [MH]+, 381.1050; found, 381.1061. Anal.
(C16H20N4O3S2ꢂ2HClꢂ0.7H2O) C, H, N.
5.7.12. 5-(Aminosulfonyl)-2-[(3,4-difluorophenyl)thio]-N-(4-
methoxybenzyl)nicotinamide hydrochloride (6i)
Prepared following method D starting with 19c and 4-methoxy-
benzylamine. Yield: 111 mg (69%). 1H NMR (300 MHz, DMSO-d6) d
3.76 (s, 3H), 4.44 (d, J = 5.7 Hz, 2H), 6.95 (d, J = 8.7 Hz, 2H), 7.23–
7.30 (m, 1H), 7.33 (d, J = 8.7 Hz, 2H), 7.49–7.57 (m, 1H), 7.60 (s,
2H), 7.63–7.74 (m, 1H), 8.26 (d, J = 2.3 Hz, 1H), 8.69 (d, J = 2.3 Hz,
1H), 9.44 (t, J = 5.8 Hz, 1H), with rotamer peaks. LC/MS m/z 466.0
[MH]+. HRMS (TOF) calcd for C20H18F2N3O4S2 [MH]+, 466.0702;
found, 466.0705. Anal. (C20H17F2N3O4S2) C, H, N.
5.7.6. 5-(Aminosulfonyl)-N-(2-methoxyethyl)-2-(phenylthio)-
nicotinamide (6c)
Prepared following method D starting with 19a and 2-methoxy-
ethylamine. Yield: 164 mg (69%). 1H NMR (400 MHz, DMSO-d6) d
3.21 (s, 3H), 3.33–3.38 (m, 2H), 3.39–3.43 (m, 2H), 7.34–7.38 (m,
3H), 7.39–7.43 (m, 2H), 7.49 (s, 2 H), 8.05 (d, J = 2.3 Hz, 1H), 8.54
(d, J = 2.3 Hz, 1H), 8.91 (t, J = 5.3 Hz, 1H). LC/MS m/z 368.0 [MH]+.
HRMS (TOF) calcd for C15H18N3O4S2 [MH]+, 368.0733; found,
368.0734. Anal. (C15H17N3O4S2ꢂ0.3HCl) C, H, N.
5.7.13. 5-(Aminosulfonyl)-N-(3-methoxypropyl)-2-[(3-
fluorophe-nyl)thio]nicotinamide (6j)
Prepared following methods E starting with 5 and 3-fluoroben-
zenethiol. The crude intermediate ethyl ester was converted to the
title compound using method D and 3-methoxypropylamine.
Yield: 136 mg (67%). 1H NMR (300 MHz, DMSO-d6) d 1.72–1.88
(m, 2H), 3.27 (s, 3H), 3.30–3.38 (m, 2H), 3.44 (t, J = 6.2 Hz, 2H),
7.27–7.44 (m, 3H), 7.46–7.56 (m, 1H), 7.61 (s, 2H), 8.20 (d,
J = 2.3 Hz, 1H), 8.69 (d, J = 2.3 Hz, 1H), 8.95 (t, J = 5.5 Hz, 1H). LC/
MS m/z 400.1 [MH]+. HRMS (TOF) calcd for C16H19FN3O4S2 [MH]+,
400.0796; found, 400.0807. Anal. (C16H18FN3O4S2ꢂ0.2HCl) C, H, N.
5.7.7. 5-(Aminosulfonyl)-N-(3-methoxypropyl)-2-(phenylthio)-
nicotinamide (6d)
Prepared following method D starting with 19a and 3-methoxy-
propylamine. Yield: 103 mg (62%). 1H NMR (300 MHz, DMSO-d6) d
1.74–1.87 (m, 2H), 3.28 (s, 3H), 3.30–3.38 (m, 2H), 3.44 (t,
J = 6.3 Hz, 2H), 7.45–7.54 (m, 5H), 7.59 (s, 2H), 8.15 (d, J = 2.3 Hz,
1H), 8.65 (d, J = 2.3 Hz, 1H), 8.91 (t, J = 5.6 Hz, 1H). LC/MS m/z
382.1 [MH]+. HRMS (TOF) calcd for C16H20N3O4S2 [MH]+,
382.0890; found, 382.0894. Anal. (C16H19N3O4S2ꢂ0.15HCl) C, H, N.
5.7.14. 5-(Aminosulfonyl)-N-(3-methoxypropyl)-2-[(2,3,5,6-
tetrafluorophenyl) thio]nicotinamide (6k)
5.7.8. 5-(Aminosulfonyl)-2-(phenylthio)-N-propylnicotinamide
(6e)
Prepared following method D starting with 19b and 3-methoxy-
propylamine. Yield: 242 mg (65%). 1H NMR (300 MHz, DMSO-d6) d
1.76–1.88 (m, 2H), 3.28 (s, 3H), 3.36–3.38 (m, 2H), 3.44 (t,
J = 6.3 Hz, 2H), 7.62 (s, 2H), 8.05–8.22 (m, 1H), 8.48 (d, J = 2.1 Hz,
1H), 8.72 (d, J = 2.1 Hz, 1H), 9.18 (t, J = 5.4 Hz, 1H). LC/MS m/z 454.0
[MH]+. HRMS (TOF) calcd for C16H16F4N3O4S2 [MH]+, 454.0513;
found, 454.0513. Anal. (C16H15F4N3O4S2ꢂ0.05HCl) C, H, N.
Prepared following method D starting with 19a and propyl-
amine. Yield: 86 mg (55%). 1H NMR (300 MHz, DMSO-d6) d 0.97
(t, J = 7.4 Hz, 3H), 1.54–1.63 (m, 2H), 3.21–3.31 (m, 2H), 7.45–
7.54 (m, 5H), 7.59 (s, 2H), 8.13 (d, J = 2.3 Hz, 1H), 8.65 (d,
J = 2.3 Hz, 1H), 8.90 (t, J = 5.6 Hz, 1H). LC/MS m/z 352.0 [MH]+.
HRMS (TOF) calcd for C15H18N3O3S2 [MH]+, 352.0784; found,
352.0786. Anal. (C15H17N3O3S2ꢂ0.05HCl) C, H, N.
5.7.15. 5-(Aminosulfonyl)-2-[(3,4-difluorophenyl)thio]-N-(3-
methoxypropyl)nicotinamide (6l)
5.7.9. 5-(Aminosulfonyl)-N-(2-phenylethyl)-2-(phenylthio)-
nicotinamide (6f)
Prepared following method D starting with 19c and 3-methoxy-
propylamine. Yield: 81 mg (60%). 1H NMR (300 MHz, DMSO-d6) d
1.73–1.87 (m, 2H), 3.27 (s, 3H), 3.30–3.35 (m, 2H), 3.43 (t,
J = 6.2 Hz, 2H), 7.36–7.44 (m, 1H), 7.49–7.58 (m, 1H), 7.61 (s, 2H),
Prepared following method D starting with 19a and phenethyl-
amine. Yield: 152 mg (84%). 1H NMR (300 MHz, DMSO-d6) d 2.90 (t,

3316
W. Vernier et al. / Bioorg. Med. Chem. 18 (2010) 3307–3319
7.64–7.72 (m, 1H), 8.22 (d, J = 2.3 Hz, 1H), 8.69 (d, J = 2.3 Hz, 1H),
8.96 (t, J = 5.5 Hz, 1H). LC/MS m/z 418.2 [MH]+. HRMS (TOF) calcd
for C16H18F2N3O4S2 [MH]+, 418.0702; found, 418.0709. Anal.
(C16H17F2N3O4S2ꢂ0.1HCl) C, H, N.
J = 10.3, 8.0, 2.1 Hz, 1H), 8.20 (d, J = 2.3 Hz, 1H), 8.57 (d, J = 2.3 Hz,
1H). LC/MS m/z 333.0 [MH]+. HRMS (TOF) calcd for C12H11F2N2O3S2
[MH]+, 333.0174; found, 333.0166. Anal. (C12H10F2N2O3S2ꢂ0.6HCl)
C, H, N.
5.7.16. 5-(Aminosulfonyl)-N-(2-pyridin-2-ylethyl)-2-[(2,3,5,6-
tetrafluorophenyl)thio] nicotinamide hydrochloride (6m)
Prepared following method D starting with 19b and 2-(2-ami-
noethyl)pyridine. Yield: 90 mg (68%). 1H NMR (300 MHz, DMSO-
d6) d 3.32 (t, J = 6.1 Hz, 2H), 3.73–3.88 (m, 2H), 7.65 (s, 2H), 7.85
(t, J = 6.8 Hz, 1H), 7.94 (d, J = 7.9 Hz, 1H), 8.05–8.24 (m, 1H), 8.41
(t, J = 7.7 Hz, 1H), 8.56 (d, J = 1.7 Hz, 1H), 8.71 (d, J = 1.9 Hz, 1H),
8.81 (d, J = 5.1 Hz, 1H), 9.41 (t, J = 5.4 Hz, 1H). LC/MS m/z 487.0
[MH]+. HRMS (TOF) calcd for C19H15F4N4O3S2 [MH]+, 487.0516;
found, 487.0502. Anal. (C19H14F4N4O3S2ꢂ2.3HClꢂ2H2Oꢂ0.2toluene)
C, H, N.
5.7.21. Methyl (1E)-N-({6-[(3,4-difluorophenyl)thio]-5-(hydroxy-
methyl)pyridine-3-yl}sulfonyl)ethanimidoate (21)
To a solution of 7 (0.286 g, 0.810 mmol) in acetonitrile (4 mL)
was added trimethyl orthoacetate (0.256 mL, 2.01 mmol). The
reaction mixture was stirred at 85 °C for 18 h. The reaction mixture
was concentrated under reduced pressure to give the title product
(367 mg, 100%), which was used without further purification. LC/
MS m/z 389.0 [MH]+.
5.8. Method F. General procedure for the conversion of alcohol
to amine
5.7.17. N-(3-Aminopropyl)-5-(aminosulfonyl)-2-[(2,3,5,6-
tetrafluorophenyl)thio]nicotinamide hydrochloride (6n)
Prepared following method D starting with 19b and 1,3-diami-
nopropane. Yield: 100 mg (83%). 1H NMR (400 MHz, DMSO-d6) d
1.83–1.94 (m, 2H), 2.86–2.96 (m, 2H), 3.40 (q, J = 6.6 Hz, 2H),
7.66 (s, 2H), 7.96 (br s, 3H), 8.07–8.20 (m, 1H), 8.64 (d, J = 2.0 Hz,
1H), 8.72 (d, J = 2.3 Hz, 1H), 9.36 (t, J = 5.6 Hz, 1H). LC/MS m/z
439.0 [MH]+. HRMS (TOF) calcd for C15H15F4N4O3S2 [MH]+,
439.0516; found, 439.0521. Anal. (C15H14F4N4O3S2ꢂ1.2HClꢂ0.5H2O)
C, H, N.
To a cooled 5 °C solution of 21 in THF (0.18 M) was added trieth-
ylamine (2.2 equiv) and p-toluenesulfonyl chloride (2.0 equiv). The
mixture was stirred at 5 °C to ambient temperature over 3 h. The
mixture was cooled back to 5 °C and the amine (30 equiv) was
added. The mixture was stirred at ambient temperature overnight.
The crude mixture was diluted with water (1 mL) and treated with
TFA (0.65 mL) and then directly purified by RPHPLC using acetoni-
trile in water (5–95%, with TFA). The resulting TFA salt was con-
verted to the HCl salt using 3 N HCl in MeOH to give the product.
5.8.1. 6-[(3,4-Difluorophenyl)thio]-5-{[(4-methoxybenzyl)-
amino] methyl}pyridine-3-sulfonamide hydrochloride (8a)
Prepared following method F using 4-methoxybenzylamine.
Yield: 12 mg (7%). 1H NMR (300 MHz, MeOH) d 3.86 (s, 3H), 4.41
(s, 2H), 4.45 (s, 2H), 7.02–7.11 (m, 2H), 7.37–7.43 (m, 2H), 7.50–
7.59 (m, 3H), 8.31 (d, J = 2.3 Hz, 1H), 8.77 (d, J = 2.1 Hz, 1H). LC/
MS m/z 452.1 [MH]+. HRMS (TOF) calcd for C20H19F2N3O3S2
[MH]+, 452.0909; found, 452.0915. HPLC tR = 2.2 min (98%).
5.7.18. 5-(Aminosulfonyl)-N-[2-(4-methoxyphenyl)ethyl]-2-
[(2,3,5,6-tetrafluorophenyl)thio]nicotinamide (6o)
Prepared following method D starting with 19b and 4-methoxy-
phenethylamine. Yield: 107 mg (68%). 1H NMR (300 MHz, DMSO-
d6) d 2.85 (t, J = 7.4 Hz, 2H), 3.45–3.57 (m, 2H), 3.74 (s, 3H), 6.89
(d, J = 8.7 Hz, 1H), 7.22 (d, J = 8.5 Hz, 1H), 7.69 (s, 2H), 8.03–8.22
(m, 1H), 8.55 (d, J = 2.1 Hz, 1H), 8.69 (d, J = 2.1 Hz, 1H), 9.45 (s,
1H). LC/MS m/z 516.0 [MH]+. HRMS (TOF): calcd for
C21H18F4N3O4S2 [MH]+: 516.0670, found: 516.0670. Anal.
(C21H17F4N3O4S2ꢂ0.2HCl) C, H, N.
5.8.2. 6-[(3,4-Difluorophenyl)thio]-5-[(ethylamino)methyl]-
pyridine-3-sulfonamide hydrochloride (8b)
Prepared following method F using ethylamine. Yield: 58 mg
(44%). 1H NMR (300 MHz, DMSO-d6) d 1.32 (t, J = 7.2 Hz, 3H), 3.08–
3.22 (m, 2H), 4.34 (t, J = 5.8 Hz, 2H), 7.44–7.52 (m, 1H), 7.54–7.65
(m, 1H), 7.70 (s, 2H), 7.75–7.83 (m, 1H), 8.43 (d, J = 2.3 Hz, 1H),
8.73 (d, J = 2.1 Hz, 1H), 9.58 (s, 2H). LC/MS m/z 360.0 [MH]+. HRMS
(TOF) calcd for C14H16F2N3O2S2 [MH]+, 360.0647; found, 360.0646.
Anal. (C14H15F2N3O2S2ꢂ1.5HClꢂH2O) C, H, N.
5.7.19. 5-(Aminosulfonyl)-2-[(3,4-difluorophenyl)thio]nicotinic
acid (20)
To a solution of 19c (12.0 g, 32.1 mmol) in THF (60 mL) was
added lithium hydroxide (2.30 g, 96.2 mmol) in water (60 mL).
The reaction mixture was stirred at ambient temperature for
2 h. 5 N aq HCl (21 ml, 106 mmol) was slowly added and the
THF was evaporated. The resulting solid was collected by filtra-
tion and dried overnight under vacuum to give the title
compound (11.1 g, 99%) as a white solid. 1H NMR (400 MHz,
DMSO-d6) d 7.42 (s, 1H), 7.50–7.59 (m, 1H), 7.63 (s, 2H), 7.67–
7.74 (m, 1H), 8.58 (s, 1H), 8.72 (d, J = 2.3 Hz, 1H), 14.19 (br s,
1H). LC/MS m/z 345.0 [MꢀH]ꢀ. HRMS (TOF) calcd for
C12H8F2N2O4S2 [MH]+, 346.9967; found, 346.9972. Anal.
(C12H8F2N2O4S2ꢂ0.5H2O) C, H, N.
5.8.3. 6-[(3,4-Difluorophenyl)thio]-5-{[(3-methoxypropyl)-
amino] methyl}pyridine-3-sulfonamide hydrochloride (8c)
Prepared following method F using 3-methoxypropylamine.
Yield: 49 mg (42%). 1H NMR (300 MHz, DMSO-d6) d 1.92–2.06 (m,
2H), 3.14–3.17 (m, 2H), 3.28 (s, 3H), 3.46 (t, J = 5.9 Hz, 2H), 4.32–
4.41 (m, 2H), 7.44–7.52 (m, 1H), 7.54–7.65 (m, 1H), 7.70 (s, 2H),
7.74–7.82 (m, 1H), 8.43 (d, J = 2.1 Hz, 1 H), 8.74 (d, J = 2.3 Hz,
1H), 9.53 (br s, 2H). LC/MS m/z 404.0 [MH]+. HRMS (TOF) calcd
for C16H20F2N3O3S2 [MH]+, 404.0909; found, 404.0902. Anal.
(C16H19F2N3O3S2ꢂ1.2HCl) C, H, N.
5.7.20. 6-[(3,4-Difluorophenyl)thio]-5-(hydroxymethyl)-
pyridine-3-sulfonamide hydrochloride (7)
To a cooled 0 °C solution of 20 (1.0 g, 2.9 mmol) in dry THF
(5 mL) was slowly added borane–methyl sulfide complex 2 M in
THF (2.9 mL, 5.8 mmol) over 5 min. The mixture was stirred at
0 °C to ambient temperature over 3 h and then the reaction was
carefully quenched with methanol and water. The crude mixture
was directly purified by RPHPLC using acetonitrile in water (5–
95%, with 0.1% acetic acid or TFA). The resulting TFA salt was con-
verted to the HCl salt using 3 N HCl in MeOH to give the title prod-
uct (286 mg, 28%). 1H NMR (300 MHz, DMSO-d6) d 4.61 (s, 2H),
5.87 (br. s., 1H), 7.38–7.45 (m, 1H), 7.50–7.63 (m, 3H), 7.72 (td,
5.8.4. 5-(Aminosulfonyl)-2,3-difluorobenzoic acid (10)
2,3-Difluorobenzoic acid (9; 200 g, 1.26 mol) was added drop-
wise to chlorosulfonic acid (660 mL, 9.6 mol) in an ice-cold water
bath. After the addition, the mixture was stirred at 115 °C for 48 h.
The mixture was slowly poured into ice. The resulting mixture was
stirred rapidly at ambient temperature for 2–3 h and filtered to give
a yellow solid that was dried in vacuum to give 5-(chlorosulfonyl)-
2,3-difluorobenzoic acid (215 g, 66.4%), which was used directly
in the next step without purification. To a solution of CH2Cl2

W. Vernier et al. / Bioorg. Med. Chem. 18 (2010) 3307–3319
3317
(500 mL) saturated with NH3 was added dropwise a solution of 5-
(chlorosulfonyl)-2,3-difluorobenzoic acid (200 g, 0.78 mol) in
CH2Cl2 (500 mL) at 0 °C. After the addition, the suspension was stir-
red at ambient temperature overnight. The mixture was filtered, and
the solid was washed with water and dried under vacuum to give
(1:3 dilutions, 11 points) in duplicate. Highest final concentration
of compound in this CA-II Fluorimetric assay is 1 M. Assays were
conducted in a final volume of 100 L in 50 mM Tris/HCl (pH 7.6),
100 mM Na2SO4, and 0.005% Tween-20 in a 96-well black assay
plate. Fluorescein diacetate was used as the substrate. Enzyme inhi-
l
l
crude 5-(aminosulfonyl)-2,3-difluorobenzoic acid (210 g) as
a
bition was determined by pipetting 8
Sigma–Aldrich, product # C6165) into assay plate that contained
L of compound and 2 L of substrate (10 M) in 88 L of assay
buffer. The rate of the hydrolysis of fluorescein diacetate was mea-
sured spectrophometrically at 488 nm (excitation), 538 nm (emis-
sion), and 530 nm (cutoff) using a Molecular Devices SpectraMax
M2fluorescencereaderat25 °C. TheIC50, theinhibitorconcentration
resulting in 50% inhibition of the enzyme activity, was calculated
using GraphPad Prism or similar in-house software with the IC50
curve fitting using the four parameter logistic equation.
lL of human CA-II (5 nM, from
yellow solid. To facilitate purification, the material was transiently
converted to the methyl ester. Thionyl chloride (250 mL, 3.43 mol)
was added to MeOH (500 mL). The solution was stirred at ambient
temperature for 1 h, and the crude 5-(aminosulfonyl)-2,3-difluoro-
benzoic acid (150 g, 0.63 mol) was added portionwise at 0 °C. The
reaction mixture was heated at 60 °C overnight. The mixture was
concentrated under vacuum and the crude material was purified
by column chromatography(MeOH/CH2Cl2; 1:20) to give the methyl
esterof5-(aminosulfonyl)-2,3-difluorobenzoicacid(100 g) asalight
yellow solid. The solid was added to 3 N aq hydrochloric acid (1.5 L)
portionwise. The mixture was heated at 70 °C overnight and then
evaporated under vacuum to give the title compound (85 g, 63%)
as a white solid. 1H NMR (300 MHz, DMSO-d6) d 7.67 (s, 2H) 8.03–
8.11 (m, 1H) 8.13–8.19 (m, J = 5.6 Hz, 1H) 14.06 (br s, 1H). LC/MS
m/z 235.9 [MꢀH]ꢀ. HRMS (TOF) calcd for C7H6F2NO4S [MH]+,
237.9980; found, 237.9969. Anal. (C7H5F2NO4S) C, H, N.
2
l
l
l
l
5.10. Carbonic anhydrase-IV (CA-IV) fluorescence polarization
assay—IC50 determination
Human CAIV was amplified from a human kidney cDNA library
(Clonetech) using primers: 50-ggaattccatatggcagagtcacactggtgctac-
gag and 50-ccgctcgagttactaggactttatcaccgtgcgctgccc, with KOD Poly-
merase (Novagen). The PCR amplified product was cloned into a
NdeI/ XhoI cut pET-43.1a(+) (Novagen) and transformed into Esche-
richia coli BL21 (DE3) (Invitrogen) cells. These cells were grown in
5.8.5. 5-(Aminosulfonyl)-N-ethyl-2,3-difluorobenzamide (22)
Prepared following method A starting with 10 and ethylamine.
Yield: 76 mg (27%) as a white solid. 1H NMR (300 MHz, DMSO-d6) d
1.14 (t, J = 7.2 Hz, 3H), 3.27–3.34 (m, 2H), 7.63 (s, 2H), 7.83–7.88
(m, 1H), 7.91–8.00 (m, 1H), 8.70 (s, 1H). HPLC tR = 1.4 min
(>95%). LC/MS (ESI) m/z 265.1 [MH]+. HRMS (TOF): calcd for
C9H11F2N2O3S [MH]+: 265.0453, found: 265.0415. Anal.
(C9H10F2N2O3S) C, H, N.
Luria broth (LB) media (Biomyx) supplemented with 800
at 37 °C until an O.D.600 of 0.7, at which point the cells were induced
with 100 M isopropyl-beta- -thiogalactopyranoside (IPTG) for
20 h at 20 °C. The frozen pellet was resuspended in 50 mM 2-mor-
pholinoethanesulfonic acid (MES) at pH 6.0, 100 mM NaCl, 800
lM ZnCl2
l
D
lM
ZnCl2, and EDTA-Free protease inhibitors (Roche). The cells were
lysed with a microfluidizer, the lysate was spun at 40,000 rpm for
45 min at 4 °C, and the soluble fraction was dialyzed overnight at
4 °C in 50 mM MES, pH 6.0, and 100 mM NaCl. The soluble fraction
was then put over a 100 mL SP-Sepharose High Performance (GE
Healthcare) column and eluted with a 50 mM MES, pH 6.0,
750 mM NaCl gradient. The peak fractions were then concentrated,
via an Amicon Ultra-4 10,000 MWCO (Millipore) spin column to
2.0 mL and loaded onto a Sephacryl S-100 High Resolution (GE
Healthcare) column in 25 mM tris(hydroxymethyl)aminomethane
hydrochloride (Tris–HCl), pH 7.0, and 100 mM NaCl. The peak frac-
tions were then concentrated, via an Amicon Ultra-4 10,000 MWCO
(Millipore) spin column, to 7.0 mg/mL and left exposed at ambient
temperature overnight. Fully oxidized protein was characterized
by performing an Ellman’s Assay, utilizing Pierce reagents, and
non-reducing SDS–PAGE. The specific activity of the human CA-IV
enzyme was confirmed with the literature inhibitors; IC50 for aceta-
zolamide = 120 nM, ethoxzolamide = 88 nM, dorzolamide = 43 nM,
and brinzolamide = 45 nM. These IC50 were generated using the FP
assay described below and are comparable to the Ki/IC50 values in
the literature.47,48
5.8.6. 5-(Aminosulfonyl)-N-ethyl-3-fluoro-2-(isopropylthio)-
benzamide (11a)
Prepared following method B starting with 22 and 2-propane-
thiol. Yield: 10 mg (8%). 1H NMR (300 MHz, DMSO-d6) d 1.09–1.22
(m, 9H), 3.20–3.31 (m, 2H), 3.44–3.58 (m, 1H), 7.58 (d, J = 1.5 Hz,
1H), 7.62 (s, 2H), 7.69 (dd, J = 8.9, 1.9 Hz, 1H), 8.54 (t, J = 5.5 Hz,
1H). LC/MS m/z 321.0 [MH]+. HRMS (TOF) calcd for C12H17FN2O3S2
[MH]+, 321.0738; found, 321.0735. HPLC tR = 2.1 min (92%).
5.8.7. 5-(Aminosulfonyl)-N-ethyl-3-fluoro-2-(isobutylthio)-
benzamide (11b)
Prepared following method B starting with 22 and 2-methyl-1-
propanethiol. Yield: 244 mg (73%). 1H NMR (300 MHz, DMSO-d6) d
0.94 (d, J = 6.6 Hz, 6H), 1.15 (t, J = 7.3 Hz, 3H), 1.53–1.71 (m, 1H),
2.81 (d, J = 7.0 Hz, 2H), 3.22–3.32 (m, 2H), 7.56 (d, J = 1.9 Hz, 1H),
7.60 (s, 2H), 7.67 (dd, J = 9.0, 1.9 Hz, 1H), 8.57 (t, J = 5.5 Hz, 1H).
LC/MS m/z 335.0 [MH]+. HRMS (TOF) calcd for C13H20FN2O3S2
[MH]+, 335.0894; found, 335.0889. Anal. (C13H19FN2O3S2) C, H, N.
5.8.8. 6-[(2,3,5,6-Tetrafluorophenyl)thio]pyridine-3-sulfon-
amide (14)
Compounds were dissolved in DMSO at the concentration of
1 mM, then to 250
for further dilutions (1:3 dilutions, 11 points) in duplicate. Final
compound highest concentration in CA-IV FP assay is 5 M. Assays
were conducted in a final volume of 100 L in 50 mM Tris/HCl (pH
lM in DMSO and transferred to a 96-well plate
Prepared following method E starting with 2-chloropyridine-5-
sulfonamide and 2,3,5,6-tetrafluorobenzenethiol. Yield: 1625 mg
(90%). 1H NMR (300 MHz, DMSO-d6) d 7.56–7.70 (m, 3H), 8.11 (dd,
J = 8.5, 2.5 Hz, 1H), 8.15–8.29 (m, 1H), 8.74 (d, J = 1.9 Hz, 1H). LC/
MS m/z 338.9 [MH]+. HRMS (TOF): calcd for C11H7F4N2O2S2 [MH]+:
338.9878, found: 338.9880. Anal. (C11H6F4N2O2S2ꢂ0.4HClꢂ0.7H2O)
C, H, N.
l
l
7.6), 100 mM Na2SO4, and 0.005% Tween-20 in a 96-well black as-
say plate. BODIPYÒ558/568-Acetazolamide (Cat # B-12270 from
invitrogen-molecular, discontinued item) was used as the tracer.
Binding inhibition was determined by pipetting 8
CA-IV (25 nM) into assay plate contained 2 L of compound and
L of tracer (2 nM) in 88 L of assay buffer. The assay plate
lL of human
l
5.9. Carbonic anhydrase-II (CA-II) fluorimetric assay—IC50
determination
2
l
l
was incubated at ambient temperature for 30 min and read in
the fluorescence polarization reader (Molecular Devices, Analyst)
at 524/45 nm (excitation), 595/60 nm (emission), and 561 nm
(beam splitter). IC50, the inhibitor concentration resulting in 50%
Compounds were dissolved in DMSO at a concentration of 1 mM,
then 50 lM, and transferred to a 96-well plate for further dilutions

3318
W. Vernier et al. / Bioorg. Med. Chem. 18 (2010) 3307–3319
inhibition of the enzyme activity, was calculated using GraphPad
Prism or similar in-house software with the IC50 curve fitting using
the four parameter logistic equation.
5.14. Crystallographic methods
Co-crystals of the soluble portion of CA IV (see section on IC50
determination) with compounds (6f, 6d, or dorzolamide) were ob-
tained from 20% to 25% polyethylene glycol (PEG) 3350, 0.1 M so-
dium acetate (pH 4.5), 0.2–0.3 M ammonium sulfate, 5 mM DTT,
glucose (3%) by the method of vapor diffusion in hanging drops.
Crystals grew at 13 °C over a period of 14 days. Data were collected
at ꢀ180 °C after crystals were mounted in a loop (Hampton Re-
search, Laguna Hills, CA) and dragged through a cryo-protecting
solution of mother liquor containing 20% DMSO before flash cool-
ing in liquid nitrogen. Data were collected either in-house or at the
Advanced Light Source (ALS) at Berkeley and processed and scaled
with HKL200059 (see Table 1 for data statistics). The structures
were determined by molecular replacement using the protein
chain only of the CA IV pdb structure 1ZNC60 as the search model.
Molecular replacement was carried out with MOLREP61 through
CCP4.62 Refinement was carried out in REFMAC5,63 with initial
phase improvement using ARP/wARP.64 Statistics for the final re-
fined structures are presented in Table 1. Coordinates for the X-
ray structure of Figures 3a–c have been deposited at the PDB under
the file names 3FW3 (dorzolamide), 3F7U (6d), and 3F7B (6f).
5.11. Kinetic solubility assay
The samples were predissolved as a 30 mM DMSO stock solution
in VWR 96-well plates (200 lL, polypropylene PCR). The plates were
allowed to thaw at room temperature and then placed in a 40 °C so-
nic water bath for 10 min to facilitate resuspension. An automated
liquid handler was used to prepare 50-fold dilutions of stocks by
combining 6 lL aliquots of stock solutions with 294 lL of 50 mM so-
dium phosphate buffer (pH 6.5) directly into a Millipore solubility
filterplate. ThefinalDMSOconcentrationwas2%, andthetheoretical
maximum concentration was 0.6 mM. The plates were heat sealed,
and incubation was carried out at ambient room temperature (ca.
22–25 °C) for 24 h with shaking at 200 rpm. The plates were vacuum
filtered and the filtrates were injected into the nitrogen detector
using an automated liquid handler. The concentrations of filtrates
were determined by chemiluminescent nitrogen detection.49
5.12. Thermodynamic solubility assay
A standard sample was prepared by dissolving 1–3 mg of sam-
ple in 5 mL of methanol. The test sample was prepared by mixing
2–5 mg of sample with 1.0 mL of 100 mM sodium phosphate buffer
at pH 6.5. The mixture was stirred for 4 h at ambient temperature
and centrifuged. The standard and saturated solutions were ana-
lyzed by HPLC. The solubility was calculated using the integrated
areas for analyzed peaks.44
5.15. Inhibition of carbonic anhydrase in iris ciliary body
homogenate (‘pH stat spike’, similar to published procedure by
Ponticello, et al)46
Dutch Belted rabbits (N = 3 per drug concentration) where
euthanized with Beuthenasia and eyes where enucleated. Iris Cili-
ary Body (ICB) was collected via a wet dissection and snap frozen in
liquid nitrogen. Samples were calibrated by using ICB wet weight.
5.13. Computational methods
Compounds were formulated at specified concentrations (10 lM
and 100 nM) in chilled 0.02 M Tris Buffer, pH 8.4, before homoge-
nizing ICB samples (15 mg) in this buffer on the day of the titration
assay. Each sample was assessed for carbonic anhydrase activity
using a pH Stat titration machine (TitraLab). Samples containing
15 mg of homogenized ICB tissue were titrated with 0.025 N NaOH
over 6 minutes while bubbling CO2 (100 mls/min) into the ICB
homogenate. The pH of the sample was held constant at pH 8.3
for at least 3 min. The titration assay was run N = 3 for each sample
and the average was used to calculate the slope of the curve to
determine enzymatic activity. Blank values were subtracted and
the slope of the curve (mls/min NaOH added) was used to calculate
percent inhibition of compound versus placebo-treated ICB.
Reasonable conformers of computationally AMBER-derived candi-
date ligands were docked with the aid of AGDOCK according to a
prior protocol50 into the experimentally observed protein struc-
tures of CAII and CAIV described herein. The sulfonamide nitro-
gen-zinc distance was constrained to that experimentally
observed and AGDOCK employed iteratively to a suitable ligand–
protein complex geometry for further computational analysis. Sub-
sequently, protein–ligand minimization for energetic analysis was
performed using in-house software based on the MacroModel
implementation of the AMBER force field (version 7),51–53 with some
added parameters to support specific chemical moieties found in
Pfizer compounds and solved crystallographic structures (e.g., tet-
razoles and some metal ions).
Acknowledgments
The minimization itself was done using a limited-memory
Truncated-Newton minimizer54,55 with partial preconditioning.
Associated surface areas were computed using the LCPO (Linear
Combination of Pairwise Overlaps) method, a fast method within
the AMBER package that allows rapid and reasonably accurate sur-
face area calculations with derivatives. Partial charges are assigned
from the AMBER force field unless reasonable charges are provided in
the input file, in which case the input charges are used. Bound li-
gand models were compared by calculated binding energies and
visual inspection to try to predict favorable binding modes and at-
tempt to prioritize which targets to synthesize earlier.
For the docking scores given in Table 2, the sulfonamide nitrogen
of the ligands was constrained to that experimentally observed and
the ligands docked using the docking program AGDOCK. This pro-
gram uses an evolutionary algorithm (specifically, Evolutionary Pro-
graming)toexplorethetranslational, rotational, andtorsionalspaces
of the ligand within the binding site.56,57 The docked structures were
scored using the High Throughput Screening program developed at
Agouron. This program uses an empirical energy function that has
been parameterized using experimental data.58
We would like to thank all our colleagues who have contributed
to the work presented here; in particular, Jennifer Lafontaine, Mi-
chael Plewe, and David Anderson for the helpful review of the
manuscript, Mike Niesman for his support, and Patrick Wheeler
and Kristin Magnuson for analytical support.
Supplementary data
Supplementary data (combustion analysis data, summary of
crystallographic data and selected 1H NMR spectra) associated
with this article can be found, in the online version, at
doi:10.1016/j.bmc.2010.03.014.
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