Cyclic secondary sulfonamides: Unusually good inhibitors of cancer-related carbonic anhydrase enzymes

Article abstract of DOI:10.1021/jm500255y

Carbonic anhydrase IX (CA IX) is a target for hypoxic cancer therapies, and the discovery of CA IX selective ligands is imperative for the development of these agents. Primary sulfonamides are broad specificity inhibitors of CA enzymes, while secondary su

Full text of DOI:10.1021/jm500255y

Article
pubs.acs.org/jmc
Cyclic Secondary Sulfonamides: Unusually Good Inhibitors of Cancer-
Related Carbonic Anhydrase Enzymes
Janina Moeker,† Thomas S. Peat,‡ Laurent F. Bornaghi,† Daniela Vullo,§ Claudiu T. Supuran,*,§
and Sally-Ann Poulsen*,†
†Eskitis Institute for Drug Discovery, Griffith University, Nathan, Queensland 4111, Australia
‡CSIRO Biosciences Program, 343 Royal Parade, Parkville, Victoria 3052, Australia
§Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Universita
̀
degli Studi di Firenze, Via della Lastruccia 3, 50019
Sesto Fiorentino, Florence, Italy
S
* Supporting Information
ABSTRACT: Carbonic anhydrase IX (CA IX) is a target for
hypoxic cancer therapies, and the discovery of CA IX selective
ligands is imperative for the development of these agents.
Primary sulfonamides are broad specificity inhibitors of CA
enzymes, while secondary sulfonamides are generally poor CA
inhibitors. However, saccharin, a cyclic secondary sulfonamide,
has unusually good inhibition of CA IX (Ki = 103 nM). In this
study, we demonstrate that the affinity and selectivity of
saccharin for CA IX can be further modulated when linked to
hydrophobic or hydrophilic substituents. The hydrophilic glycoconjugate derivative (12) showed improved inhibition of CA IX
(Ki = 49.5 nM) and extremely poor inhibition of the predominant off-target CAs (Ki > 50 000 nM) compared to saccharin. This
>1000-fold selectivity for CA IX over off-target CAs is unprecedented for classical primary sulfonamide CA inhibitors. Our study
highlights the potential of cyclic secondary sulfonamides to be exploited for the discovery of potent, cancer-selective CA
inhibitors.
the expression is upregulated in a wide selection of hypoxic
tumors as an aid to survival, proliferation, and metastasis in the
otherwise hostile tumor microenvironment that results from
increased metabolism.5 There is substantial interest in assessing
the impact of disrupting cancer cell pH homeostasis as a
therapy, and central to this challenge is the need for selective
and efficacious inhibitors of CA IX.6 It is widely reported that
primary sulfonamides (RSO2NH2) are excellent inhibitors of
CAs, and in the greater than 200 protein X-ray crystal
structures of this compound class bound to CA II, all have
the sulfonamide anion (RSO2NH−) coordinated to the CA
active site zinc cation.7 In contrast, secondary sulfonamides
(RSO2NHR′) are generally very poor CA inhibitors;8 however,
saccharin as a cyclic secondary sulfonamide is the premier
exception to this general finding. To generate CA isozyme
selectivity is challenging with primary sulfonamide compounds,
and the unusual SAR of saccharin, with an impressive selectivity
profile toward CA IX compared to nearly all other CA isozymes
(with the exception of CA VII, Table 1), is of considerable
interest. Although CA VII is strongly inhibited by saccharin, its
localization in the CNS likely limits the capacity for saccharin to
encounter CA VII in vivo, owing to predicted poor blood−
brain barrier permeability of acidic molecules (pKa of saccharin
INTRODUCTION
■
Saccharin (1,2-benzisothiazol-3-one-1,1-dioxide) has been
widely used as calorie-free sugar substitute for more than a
century1 and features as a substructure in a number of bioactive
compounds, including leukocyte elastase inhibitors and 5-HT1A
agonists (Figure 1).2 Our interest in saccharin stems from its
ability to inhibit carbonic anhydrase enzymes (CAs),
specifically CA IX (Table 1).3 CAs catalyze the interconversion
of CO2 and H2O with HCO3 and a H+ to perform a pivotal
−
role in pH regulation.4 While CA IX expression is lacking in
many healthy cells (the stomach and GI tract are exceptions),
Figure 1. Structure of saccharin and bioactive structures comprising an
N-alkylated saccharin substructure.
Received: February 16, 2014
Published: April 1, 2014
© 2014 American Chemical Society
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Table 1. Inhibition and CA IX Selectivity Data of Human CA Isozymes with Saccharin3a
CA isozyme
Ki (nM)
I
II
IV
VA
10 060
97.7
VB
7210
70
VI
935
9.1
VII
10
IX
103
1.0
XII
633
6.1
XIV
773
7.1
18 540
180
5950
57.8
7920
76.9
a
CA IX selectivity
0.1
a
CA IX selectivity is determined by the ratio of Ki values for CA isozyme relative to CA IX.
Figure 2. Target saccharin−triazole analogues for investigation as CA IX inhibitors.
is 1.3).9 The structure of human CA II (hCA II) bound to
saccharin was reported by Klebe and colleagues in 2007 (PDB
entry 2Q38); this structure shows that saccharin utilizes both
the sulfonamide and lactam carbonyl groups to form favorable
interactions with hCA II.3a Like primary sulfonamides,
saccharin is likely deprotonated with its anionic nitrogen
coordinated to the CA active site zinc. The amino acid
sequence alignment of CA isozymes indicates that several
amino acids are likely responsible for the altered CA selectivity
profile of saccharin compared to that for simple primary
sulfonamides.3a We reasoned that improving CA IX binding
and selectivity may result from the addition of substituents to
the benzene ring of saccharin, which is oriented away from the
CA IX active site zinc. In addition to providing insight on SAR,
our findings demonstrate the potential of the cyclic secondary
sulfonamide motif for the development of effective agents
targeting CA IX.
the hydrophobic half or the hydrophilic half of the CA active
site. Our interest in saccharin stems from its unusual ability to
inhibit CA enzymes. Here we report the design, synthesis, and
SAR for a panel of saccharin analogues to assess if it is possible
to exploit the atypical selectivity profile of saccharin to improve
targeting of CA IX over off-target CA isozymes.
RESULTS AND DISCUSSION
■
There are several synthetically distinct methods employed to
obtain the saccharin core, with functionalization at either the
nitrogen or the carbonyl oxygen readily accomplished either
during or post- synthesis, while functionalization elsewhere is
more challenging. As we were looking to synthesize analogues
of saccharin functionalized on the phenyl ring, we hoped to
develop a straightforward synthesis that would allow this.
Cu(I)-catalyzed azide alkyne cycloaddition (CuAAC) has
contributed to a resurgence in the application of azides in the
synthesis of more complex molecules across a range of
chemistry disciplines.9−11 Azidosaccharins are not known
compounds; however, 6-aminosaccharin (3) is a known
compound,12 and we approached the synthesis of 6-azido-
saccharin (4) by employing the common functional group
interconversion of anilines to phenyl azides via a diazonium
intermediate. 6-Aminosaccharin 3 was synthesized from p-
nitrotoluene (1) in three steps using published procedures,12
and 3 was then treated with t-butyl nitrite (1.5 equiv) and
trimethylsilyl azide (1.2 equiv) to synthesize 6-azidosaccharin 4
in 46% yield. The attempted reaction of azide 4 with 4-
ethynylbenzenesulfonamide (g) and six commercially available
phenyl acetylenes (a−f): phenyl acetylene (a), 1-ethynyl-3-
methylbenzene (b), 1-ethynyl-2-(trifluoromethyl)benzene (c),
1-ethynyl-4-methoxybenzene (d), 4-ethynyl-1-fluoro-2-methyl-
benzene (e), 1-ethynyl-4-methoxy-2-methylbenzene (f); under
typical CuAAC conditions (0.2 equiv of CuSO4·5H2O, 0.4
equiv of sodium ascorbate, tBuOH/water 1:1, 40 °C), failed to
give the target 1,4-disubstituted 1,2,3-triazole (Figure 2) in
Saccharin is a rule-of-three compliant fragment,10 that is,
molecular weight <300 Da (=183 Da), cLog P ≤ 3 (=0.45),
hydrogen bond donors ≤3 (=1), hydrogen bond acceptors ≤3
(=3), and rotatable bonds ≤3 (=0). Ligand efficiency (LE) is
the relationship between a compound’s potency and its
molecular size, and there are several definitions that represent
this ratio; here we will use LE = pKi/MW (MW in kDa).11
Optimizing potency while keeping MW low is important as
larger ligands have inherent drawbacks in terms of most
physicochemical properties (e.g., bioavailability, stability, and
solubility) that are relevant to drug discovery. Perola’s early
analysis of 60 lead/drug pairs revealed that 90% of drug leads
had LE > 12.4, while 90% of drugs had LE > 14.7.11b The LE of
saccharin binding to CA IX is 38.2; this far exceeds the drug
lead and drug thresholds, signifying that the saccharin fragment
is a promising starting point for developing CA IX inhibitors.
The compact size of the saccharin fragment within the open CA
active site architecture (PDB entry 2Q38) allows space for
saccharin to be further grown to add interactions with either
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a
Scheme 1
a
Reagents and conditions: (i) (a) 5.0 equiv of ClSO3H acid, 60 °C, 48 h; (b) NH4OH, 50 °C, 2 h, 35%; (ii) 45.0 equiv of CrO3, H2SO4, rt, 24 h,
40%; (iii) H2, 10% Pd/C, MeOH, rt, 4 h, 80%; (iv) 1.5 equiv t-butyl nitrite, 1.2 equiv of trimethylsilyl azide, acetonitrile, 0 °C to rt, 16 h, 46% (4),
88% (8); (v) 1.0 equiv of alkyne (a−g, 10), 0.2 equiv of CuSO4·5H2O, 0.4 equiv of sodium ascorbate, tBuOH/water 1:1, 40 °C, 24 h, no product
(5a−5g), 34−90% (9a−9g), 89% (11); (vi) 1.0 equiv of alkyne (a or b), 1.0 equiv of CuSO4·5H2O, 2.0 equiv of sodium ascorbate, tBuOH/water
1:1, 60 °C, 24 h, 43% (5a), 33% (5b); (vii) (a) 10.0 equiv of ClSO3H, −20 °C, 1 h then rt, 4 h; (b) tBuNH2/Et3N, 0 °C, 2 h, then rt, 6 h, 52%; (viii)
8.0 equiv of H5IO6, 5 mol % of CrO3, 7.0 equiv of Ac2O, acetonitrile, reflux, 5−10 h, 48%; (ix) H2, 10% Pd/C, EtOAc, rt, 4 h, 73%; (x) TFA, rt, 1−6
days, 23−89% (5a−5g), 34% (12).
useful yield (Scheme 1, route A). Metal complexes of saccharin
have been described wherein the NH hydrogen is lost (pKa of
this hydrogen is 1.3),9 and the resulting anion coordinates to
the metal(II) cation.13 Specifically, a crystal structure of a 2:1
saccharin/Cu(II) complex has been reported,14 and related
complexes in solution may account for the poor conversion of
alkynes a−g and azide 4 to triazole products under the standard
CuAAC reaction conditions with catalytic source of Cu(I).
Next, with 1.0 equiv of CuSO4·5H2O, 2.0 equiv of sodium
ascorbate, and a higher reaction temperature of 60 °C, we were
able to demonstrate conversion to triazoles; however, both the
1,4-disubstituted and 1,5-disubstituted 1,2,3-triazoles were
formed. Workup of these reactions required challenging
chromatography to separate regioisomers, and this was further
hampered by the increased amount of CuSO4·5H2O and
sodium ascorbate used for the reaction. Nevertheless, we were
able to prepare 1,4-disubstituted triazoles 5a and 5b using this
method, and isolated yields were 43 and 33%, respectively.
With the intention to improve our synthetic methodology,
we explored the use of a NH-protected 6-azidosaccharin as an
alternate approach to the target compounds 5a−5g. This NH-
protected compound should allow standard CuAAC conditions
to be used to form the triazole link, with subsequent removal of
the NH protecting group from the purified saccharin−triazole
product. The synthesis of the saccharin heterocycle may be
achieved by the direct oxidation of 2-methylbenzene-
sulfonamide using the H5IO6−CrO3 as the oxidation system.
Xu and colleagues investigated these oxidation conditions with
a number of N-alkyl-o-toluenesulfonamides and were able to
show that oxidation of N-t-butyl-2-methyl-5-nitrobenzene-
sulfonamide with H5IO6−CrO3 in acetonitrile gave the
corresponding N-t-butyl saccharin compound (N-t-butyl-6-
nitro-1,2-benzisothiazole-3-one-1,1-dioxide, 6) in 88% yield
(62% yield after recrystallization). This good yield and
straightforward purification encouraged us to utilize the t-
butyl moiety as a protecting group strategy, with removal of the
group under acidic conditions as a final step in the synthesis of
target compounds.15 The synthetic route using this NH-t-butyl-
protected azidosaccharin approach is depicted in Scheme 1
(route B). The synthesis of 5a−5g from compound 6
comprises four steps, while compound 6 is synthesized from
p-nitrotoluene in two steps.16 Compound 6 was reduced with
H2, 10% Pd/C to give the new compound, 6-amino-2-N-t-butyl
saccharin/N-t-butyl-6-amino-1,2-benzisothiazole-3-one-1,1-di-
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Table 2. Inhibition of human CA Isozymes I, II, IX, and XII with saccharin and saccharin analogues 2, 3, 4, 5a−5g and 12 and
selectivity ratio data for cancer-associated CA IX and CA XII
a
b
Ki (nM)
selectivity ratio
compd
cLogP
CA I
CA II
CA IX
CA XII
I/IX
II/IX
I/XII
II/XII
saccharin
+0.45
+0.39
−0.66
+0.76
+2.19
+2.70
+3.06
+2.03
+2.84
+2.54
+0.79
−2.30
18540 62
>50000
5950 24
>50000
103
95.8
5
4
633 24
15600 49
2750 121
665 27
180
>522
>82
57.8
>522
>82
29.3
>3.2
>18.2
7.4
9.4
>3.2
>18.2
12.1
108.1
141.5
>67
2
3
>50000
>50000
610 30
466 18
484 14
4
4930 230
>50000
8058 326
9315 318
8106 119
>50000
10.6
17.3
5a
5b
5c
5d
5e
5f
5g
12
86.2
57.3
3
3
>103
>557
18.1
19.2
>580
>873
10.7
6.1
>50000
89.7
5
90.4
8025 372
5320 212
>50000
443 16
341 16
747 28
866 38
>113
>147
101.9
>522
10.2
>50000
15.6
>58
4870 206
>50000
47.8
95.7
2
5
81.2
201
4
9
>1046
91.4
>616
43.5
37.3
>87
60.0
>249
9.2
8745 314
354 13
>50000
87.3
2
8.6 0.4
49.5
9.5 0.2
572 15
41.2
>50000
1
>1010
>1010
>87
a
b
Errors in the range of 5% of the reported value, from three determinations. Selectivity is determined by the ratio of Ki values for CA isozyme
relative to CA IX and XII.
oxide (7). The amino function of 7 was next converted into the
azido group using t-butyl nitrite and trimethylsilyl azide to give
the new compound N-t-butyl-6-azido-1,2-benzisothiazole-3-
one-1,1-dioxide (8) in 82% yield. The CuAAC reaction of 8
with alkynes a−g under standard conditions (0.2 equiv of
CuSO4·5H2O and 0.4 equiv of sodium ascorbate, tBuOH/water
1:1, 40 °C) gave triazoles 9a−9g in yields ranging from 34 to
90%. The N-t-butyl group of compounds 9a−9g was removed
in refluxing trifluoroacetic acid15 with variable isolated yields
(23−89%). Long reaction times (between 4 and 7 days) were
needed for this final reaction to complete; however, the relative
ease of workup compared to the initial approach (route A)
compensated for the extended reaction times.
exploit the atypical selectivity profile of saccharin to improve
targeting of CA IX over the two major off-target CA isozymes
(I and II). In addition to CA IX expression, CA XII expression
is upregulated in a wide selection of hypoxic tumors, so it is of
general interest to also monitor for inhibition of CA XII to
provide a comprehensive understanding of any individual
compound’s potential as a cancer therapeutic. The CA
inhibition data to block the interconversion of CO2 with
HCO3 and a H+, for the saccharin−triazole analogues 5a−5g
−
and 12, the simple saccharin analogues 2, 3, and 4, as well as
saccharin itself as a reference compound, were measured for CA
I, II, IX, and XII, results are presented in Table 2. The
selectivity ratios for CA IX and CA XII inhibition over the off-
target CA I and CA II are presented in Table 2.
In the past few years, our group has synthesized a large
collection of glycoconjugate CA inhibitors, wherein the
hydrophilic carbohydrate moiety is employed to reduce the
inhibitor’s Log P.17 The calculated Log P (cLog P) parameter
generally provides a good correlation with experimental
permeability data,18 and molecules with cLogP values between
1 and 3 typically have good passive membrane permeability
properties while those with cLogP values of <0 are more likely
to have a low capacity for penetrating cell membranes. Herein,
we have designed and synthesized the saccharin glycoconjugate
12 (Figure 2), in order to compare the CA inhibition profile
with less hydrophilic compounds 5a−5g. The cLogP for the
saccharin glycoconjugate 12 is −2.30, compared to values of
+2.09 to +3.06 for 5a−5f (Table 2). This structure−property
relationship for 12 is consistent with very poor passive
membrane permeability, a useful property for selective targeting
of cell surface CA IX. The synthesis of 12 is shown in Scheme
1. Reaction of the peracetylated O-propynyl glucoside 10 with
the t-butyl-protected 6-azidosaccharin 8 gave the protected
glycoconjugate 11 in 89% yield. The treatment of 11 with
refluxing TFA conveniently removed both the sugar acetate
protecting groups and the NH-t-butyl protecting group to give
the target hydrophilic saccharin analogue 12 in 34% yield from
11. Selected compounds of this study have been submitted to
the Compounds Australia (formerly Queensland Compound
Library) Open Compound Collection (Academic) (see http://
www.griffith.edu.au/science-aviation/queensland-compound-
library).
Saccharin, the reference compound for SAR, has a Ki of 103
nM for CA IX and is 180-fold selective over CA I and 58-fold
selective over CA II, while for the other cancer-associated CA,
CA XII, the Ki of saccharin is 633 nM, much weaker inhibition
than for CA IX. Saccharin thus has better CA IX selectivity than
CA XII selectivity (29- and 9-fold for CA I and II, respectively).
Noticeably the simple nitro- and aminosaccharin derivatives,
compounds 2 and 3, as well as the hydrophilic glycoconjugate
12 had extremely poor inhibition of CA I and II (>50 000 nM)
while 2 and 12 were equal or better inhibitors of CA IX than
saccharin, with Ki values of 95.8 and 49.5 nM, respectively. This
SAR is ideal as it leads to very high selectivity for CA IX over
both off-target CAs, >522-fold for 2 and >1010-fold for 12.
This selectivity is substantially higher than for the reported
primary benzenesulfonamide class of CA inhibitors, signifying
that compounds 2 and 12 are excellent candidates for follow-up
cell-based studies. For the phenyltriazole-substituted saccharin
compounds 5a−5f, the SAR was related to the substitution on
the phenyl ring, compounds typically displayed very weak
inhibition of off-target CA I and II. For CA I, inhibition was
very poor with Ki > 50 000 nM for 5a, 5b, and 5e, while for CA
II the Ki values were similarly weak (>50 000 nM) for 5c and
5d. For 5a−5e, the inhibition of the other off-target CA,
although greater, was still weak with Ki values ranging from
4870 to 9315 nM. For CA IX, a methyl substituent on the
phenyl ring gave the best inhibition, with Ki values below 100
nM for 5b, 5e, and 5f. Similarly for CA XII, the 3-methyl
substitution of 5b and 5e also gave Ki values below 100 nM.
Compound 5g comprises a primary sulfonamide group in
CA Inhibition. One goal of this study was to generate
compounds that would enable us to assess if it was possible to
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Figure 3. (A) Difference density map with the density set to 2.5σ following the removal of the ligand from the refined model. (B) Standard density
map contoured at 1.2σ. The saccharin moiety is shown in both figures, and the labeled zinc atom is shown as a gray sphere. Density maps were
generated using the FFT algorithm in the CCP4 suite 6.3.20 The figures were prepared using PyMol.21 Coordinates and structure factors for CA II/
5b have been deposited with the PDB (accession code 4cq0).
addition to the secondary sulfonamide group of the saccharin
fragment. It is expected that the primary sulfonamide group of
5g will dominate the recognition interaction with the CAs, and
inhibition data support this assumption as 5g is the most potent
CA inhibitor at all isozymes tested. When comparing
compound 5g with 5a, the difference in structure is a primary
sulfonamide moiety versus a hydrogen atom, respectively; this
compound pair thus allows the impact of the primary
sulfonamide−CA IX interaction to be disconnected in part
from the saccharin−CA IX interaction. Compound 5g (Ki = 8.6
nM at CA IX) has 56-fold greater activity than for 5a (Ki = 484
nM at CA IX). Our findings present the first report that to the
best of our knowledge has investigated the SAR of the saccharin
fragment against cancer-relevant CA IX, with a single earlier
study reporting data only at off-target CA I and CA II.19
Structure of hCA II/Saccharin Ligand Complex. To
obtain insight into ligand−protein interactions at the atomic
level, we next determined the protein X-ray crystal structure of
hCA II in complex with compound 5b. Almost all of the
residues in and around the active site are conserved among
hCA II, IX, and XII; this includes His94, His96, and His119 as
part of the zinc binding triad and Leu197, Thr198, Thr199,
Val121, Leu140, Val142, and Val206. Near the active site
entrance, there are three residues that differ between CA II, CA
IX, and CA XII: Ile91, Phe130, and Asn67. Asn67 is located
away from where the saccharin derivatives are directed, while
Ile91 (Leu in CA IX, Thr in CA XII) and Phe130 (Val in CA
IX, Ala in CA XII) are located where they are likely to form
interactions with the different substituents on the saccharin
derivatives. There was clear density for the compound adjacent
to the zinc atom in the active site after the initial rounds of
refinement. The model refined well against the data, giving a
Rwork of 16.8% and Rfree of 20.2%. The average B factors were
11.4 Å2 for the protein, 21.2 Å2 for the solvent, and 14.9 Å2 for
the compound. The data were 98.5% complete throughout the
resolution range of 70 to 1.45 Å. As can be seen in Figure 3,
both the difference density map (A) and the standard electron
density map (B) show density for the saccharin moiety of 5b,
but little to no density beyond this ring system. As the data are
complete and high resolution, we hypothesize that this is due to
disorder in this latter part of the compound (i.e., multiple
potential conformations beyond the saccharin ring).
CONCLUSION
■
Saccharin as a cyclic secondary sulfonamide is an exception to
the general finding that secondary sulfonamides are poor
inhibitors of CA enzymes. Selective binding to CA IX is an
important undertaking for targeting hypoxic tumors and is
challenging with classical primary sulfonamide CA inhibitors.
Saccharin and some of the saccharin derivatives reported herein
(e.g., 2 and 12) have very impressive selectivity toward CA IX
compared to the major off-target CA isozymes. This finding is
of substantial interest and highlights the potential of cyclic
secondary sulfonamides to be exploited for the discovery of
potent, cancer-selective CA inhibitors.
EXPERIMENTAL SECTION
■
Compound Synthesis. All reagents were purchased from
commercial suppliers. All reactions were monitored by TLC. TLC
plates were visualized with UV fluorescence (λ = 254 nm). Silica gel
flash chromatography was performed using silica gel 60 Å (230−400
mesh). 1H NMR data were acquired at 500 MHz and 13C NMR at 125
MHz at 30 °C. For 1H and 13C NMR acquired in DMSO-d6, chemical
shifts (δ) are reported in parts per million relative to the solvent
residual peak: proton (δ 2.50 ppm) and carbon (δ 39.5 ppm) signals,
1
1
respectively. Assignments for H NMR were confirmed by H−1H
gCOSY, while assignments for 13C NMR were confirmed by H−13C
1
HSQC. Multiplicity is indicated as follows: s (singlet); d (doublet); t
(triplet); m (multiplet); dd (doublet of doublet); b (broad). Coupling
constants are reported in hertz (Hz). Melting points are uncorrected.
High- and low-resolution electrospray ionization mass spectra were
acquired using electrospray as the ionization technique in positive ion
and/or negative ion modes as stated. All MS analysis samples were
prepared as solutions in MeOH. Purity of all compounds was ≥95% as
determined by HPLC with UV detection. Compounds 2, 3, 6, and 10
were synthesized using previously reported methods.12,16,17c,22 1H
NMR data for compounds were in agreement with literature.
Synthesis of 6-(4-Aryl-1H-1,2,3-triazol-1-yl)-1,2-benziso-
thiazole-3-one-1,1-dioxides. General Procedure 1. A mixture of
azide (4, 1.0 equiv) and alkyne (a and b, 1.0 equiv) was suspended in a
tert-butyl alcohol/distilled water mixture (1:1, 0.2−0.5 M final
concentration). A solution of sodium ascorbate (2.0 equiv) in water,
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followed by a solution of CuSO4·5H2O (1.0 equiv) in water, was
successively added. The bright yellow heterogeneous mixture was
stirred vigorously at 60 °C for 16 h. The solvent was removed under
reduced pressure, and the resulting residue was purified by flash
chromatography as described below. The 1,4-regioselectivity of the
products was verified by characteristic 1H and 13C NMR chemical shift
values for the triazole H-4 proton and C-4 carbon of the 1,4-
disubstituted triazole.23
Synthesis of N-t-Butyl-6-(4-aryl-1H-1,2,3-triazol-1-yl)-1,2-
benzisothiazole-3-one-1,1-dioxides. General Procedure 2. A
mixture of azide (8, 1.0 equiv) and alkyne (a−g, 10, 1.0 equiv) was
suspended in THF. A solution of sodium ascorbate (0.4 equiv) in
water, followed by a solution of CuSO4·5H2O (0.2 equiv) in water,
was successively added. The bright yellow heterogeneous mixture was
stirred vigorously at 40 °C until TLC indicated reaction was complete;
typically, 16 h was sufficient. The solvent was removed under reduced
pressure, and the resulting residue was purified by flash chromatog-
raphy (adsorbed on silica from an acetone solution) and eluted with
hexane/EtOAc as described below.
(decomp.). 1H NMR (400 MHz, DMSO-d6): δ = 9.48 (s, 1H, Htriazole),
8.26 (d, J = 1.6 Hz, 1H, H-7), 8.23 (dd, J = 8.1, 1.9 Hz, 1H, H-5), 7.82
(d, J = 8.1 Hz, 1H, H-4), 7.79 (bs, 1H, H-2′), 7.74 (d, J = 7.7 Hz, 1H,
H-6′), 7.40 (t, J = 7.6 Hz, 1H, H-5′), 7.21 (d, J = 7.4 Hz, 1H, H-4′),
1
2.40 (s, 3H, CH3), assignments were confirmed by H−1H gCOSY.
13C NMR (125 MHz, DMSO-d6): δ = 166.6 (CO), 147.6 (Ctriazole),
147.1 (C-3a), 138.4 (C-7a), 138.2 (C-3′), 134.3 (C-6), 129.9 (C-1′),
129.0 (C-4′), 128.9 (C-5′), 125.9 (C-2′), 124.2 (C-4), 123.0 (C-5),
122.5 (C-6′), 120.0 (CHtriazole), 110.5 (C-7), 21.1 (CH3), assignments
1
were confirmed by H−13C gHSQC. LRMS (ESI−): m/z 339 [M −
H]−. HRMS (ESI): calcd for C16H11N4O3S− 339.0557, found
339.0569. (2) The title compound 5b was prepared from 9b (50
mg, 0.13 mmol) according to General Procedure 3 in 4 days to give 5b
(89%) as a pale yellow solid. The characterization data for 5b prepared
from 9b are consistent with preparation by General Procedure 1
(above).
N-t-Butyl-6-amino-1,2-benzisothiazole-3-one-1,1-dioxide
(7). The title compound (7) was synthesized by reduction of N-t-
butyl-6-nitro-1,2-benzisothiazole-3-one-1,1-dioxide (6) (500 mg, 1.76
mmol) in MeOH (6 mL) with H2 (325 mg, 1 atm) and Pd−C (10%)
as catalyst, rt, 16 h. The crude reaction mixture was filtered through
Celite, and the solvent was removed under reduced pressure to give 7
as a beige solid in 90% yield. The compound was used in the next step
without further purification. Rf = 0.26 (EtOAc/hexane 1:2). Mp =
Removal of the N-t-Butyl Group of 9a−9g, 11. General
Procedure 3. A solution of N-t-butyl-protected compound (9a−9g,
11) in TFA was refluxed for 1−6 days as needed; the TFA was
removed under reduced pressure, the remaining solid was suspended
in EtOAc, and the insoluble components were removed by filtration.
The solvent was removed under reduced pressure, and the resulting
residue was purified by flash chromatography as described below.
6-Azido-1,2-benzisothiazole-3-one-1,1-dioxide (4). To a
suspension of 6-amino-1,2-benzisothiazole-3-one-1,1-dioxide (3, 3.64
g, 18.4 mmol, 1.0 equiv)12 in anhydrous acetonitrile (35 mL) at 0 °C
were added t-butyl nitrite (3.2 mL, 1.5 equiv) and azidotrimethylsilane
(2.9 mL, 1.2 equiv) by dropwise addition. The reaction mixture was
stirred at 0 °C for 1 h and warmed to room temperature for 16 h after
which TLC (DCM/MeOH, 4:1) showed reaction was complete. The
acetonitrile was removed under reduced pressure, and the remaining
residue was purified by column chromatography (solid addition from
MeOH, DCM/MeOH, 8:1) to give the product as a pale yellow solid
1
224−226 °C. H NMR (500 MHz, DMSO-d6): δ = 7.60 (d, J = 8.4
Hz, 1H, H-4), 6.94−6.88 (m, 2H, H-5, H-7), 6.74 (s, 2H, NH2), 1.63
(s, 9H, 3 × CH3), assignments were confirmed by 1H−1H gCOSY. 13
C
NMR (125 MHz, DMSO-d6): δ = 160.2 (CO), 155.4 (C-3a), 139.8
(C-7a), 126.0 (C-4), 118.1 (C-5), 111.7 (C-6), 102.0 (C-7), 59.4
(C(CH3)3), 27.5 (3 × CH3), assignments were confirmed by 1H−13C
gHSQC. LRMS (ESI+): m/z = 277 [M + Na]+. HRMS (ESI): calcd
for C11H14N2O3SNa+ 277.0617, found 277.0618.
N-t-Butyl-6-azido-1,2-benzisothiazole-3-one-1,1-dioxide (8).
To a suspension of N-t-butyl-6-amino-1,2-benzisothiazole-3-one-1,1-
dioxide (7, 240 mg, 0.94 mmol, 1.0 equiv) in anhydrous acetonitrile
(2.6 mL) at 0 °C were added t-butyl nitrite (168 μL, 1.5 equiv) and
azidotrimethylsilane (150 μL, 1.2 equiv) by dropwise addition. The
reaction mixture was stirred at 0 °C for 1 h and warmed to room
temperature for 16 h after which TLC (EtOAc/hexane, 1:2) showed
reaction was complete. The acetonitrile was removed under reduced
pressure, and the remaining residue was purified by column
chromatography (solid addition from EtOAc, EtOAc/hexane, 1:4) to
give the product as a pale yellow solid (216 mg, 46% yield). Rf = 0.67
(EtOAc/hexane, 1:2). Mp = 173−175 °C. 1H NMR (500 MHz,
DMSO-d6): δ = 8.03 (d, J = 1.9 Hz, 1H, H-7), 7.99 (d, J = 8.3 Hz, 1H,
H-4), 7.60 (dd, J = 8.3, 2.0 Hz, 1H, H-5), 1.68 (s, 9H, 3 × CH3),
assignments were confirmed by 1H−1H gCOSY. 13C NMR (125 MHz,
DMSO-d6): δ = 158.9 (CO), 147.5 (C-3a), 138.8 (C-7a), 126.2 (C-
4), 125.5 (C-6), 122.0 (C-5), 111.6 (C-7), 60.5 (C(CH3)3), 27.3 (3 ×
CH3), assignments were confirmed by 1H−13C gHSQC. LRMS
(ESI+): m/z = 303 [M + Na]+. HRMS (ESI): calcd for
C11H12N4O3SNa+ 303.0522, found 303.0519.
1
(46% yield). Rf = 0.29 (DCM/MeOH, 4:1). H NMR (500 MHz,
DMSO-d6): δ = 7.59 (d, J = 8.0 Hz, 1H, H-4), 7.36 (d, J = 1.8 Hz, 1H,
H-7), 7.27 (dd, J = 8.0, 1.8 Hz, 1H, H-5), assignments were confirmed
by 1H−1H gCOSY. 13C NMR (125 MHz, DMSO-d6): δ = 160.6 (C
O), 147.2 (C-3a), 141.6 (C-6), 126.3 (C-7a), 125.0 (C-4), 123.9 (C-
1
5), 112.0 (C-7), assignments were confirmed by H−13C gHSQC.
LRMS (ESI−): m/z = 223 [M−H]−. HRMS (ESI): calcd for
C7H3N4O3S− 222.9931, found 222.9952.
6-(4-Phenyl-1H-1,2,3-triazol-1-yl)-1,2-benzisothiazole-3-
one-1,1-dioxide (5a). (1) The title compound 5a was prepared from
phenyl acetylene a (27 mg, 0.27 mmol) according to General
Procedure 1. Purification of the crude product by flash chromatog-
raphy (EtOAc/hexane 7:1) afforded 5a (43%) as a pale yellow solid. Rf
= 0.11 (EtOAc/hexane 7:1). Mp = 258−260 °C. 1H NMR (400 MHz,
DMSO-d6): δ = 9.50 (s, 1H, Htriazole), 8.26 (s, 1H, H-7), 8.25−8.21
(m, 1H, H-5), 7.95 (d, J = 8.0 Hz, 2H, H-2′, H-6′), 7.82 (d, J = 8.1 Hz,
1H, H-4), 7.52 (t, J = 7.7 Hz, 2H, H-3′, H-5′), 7.40 (t, J = 7.3 Hz, 1H,
H-4′), assignments were confirmed by 1H−1H gCOSY. 13C NMR
(125 MHz, DMSO-d6): δ = 166.6 (CO), 147.5 (Ctriazole), 147.1 (C-
3a), 138.4 (C-7a), 134.3 (C-6), 130.0 (C-1′), 129.0 (C-3′, C-5′), 128.3
(C-4′), 125.3 (C-2′, C-6′), 124.2 (C-4), 123.1 (C-5), 120.1
(CHtriazole), 110.6 (C-7), assignments were confirmed by 1H−13C
gHSQC. LRMS (ESI−): m/z 325 [M − H]−. HRMS (ESI): calcd for
C15H9N4O3S− 325.0401, found 325.0414. (2) The title compound 5a
was prepared from 9a (80 mg, 0.2 mmol) according to General
Procedure 3 in 24 h to give 5a (56%) as a pale yellow solid. The
characterization data for 5a prepared from 9a are consistent with
preparation by General Procedure 1 (above).
N-t-Butyl-6-(4-phenyl-1H-1,2,3-triazol-1-yl)-1,2-benziso-
thiazole-3-one-1,1-dioxide (9a). The title compound 9a was
prepared from phenyl acetylene (a) (20 μL, 0.178 mmol) according
to the General Procedure 2. Purification of the crude product by flash
chromatography (EtOAc/hexane 1:4) afforded 9a (90%) as a pale
1
yellow solid. Rf = 0.58 (EtOAc/hexane, 1:2). Mp = 212−214 °C. H
NMR (500 MHz, DMSO-d6): δ = 9.51 (s, 1H, Htriazole), 8.77 (d, J =
1.5 Hz, 1H, H-7), 8.56 (dd, J = 8.4, 1.9 Hz, 1H, H-5), 8.25 (d, J = 8.4
Hz, 1H, H-4), 8.00−7.84 (m, 2H, H-3′, H-5′), 7.52 (t, J = 7.7 Hz, 2H,
H-2′, H-6′), 7.41 (t, J = 7.4 Hz, 1H, H-4′), 1.71 (s, 9H, 3 × CH3),
assignments were confirmed by 1H−1H gCOSY. 13C NMR (125 MHz,
DMSO-d6): δ = 159.0 (CO), 148.1 (Ctriazole), 141.6 (C-3a), 138.8
(C-7a), 129.8 (C-6), 129.4 (C-3′, H-5′), 128.9 (C-4′), 127.6 (C-4),
126.3 (C-5), 125.63 (C-2′, H-6′), 125.57 (C-1′), 120.4 (CHtriazole),
112.0 (C-7), 61.2 (C(CH3)3), 27.5 (3 × CH3), assignments were
confirmed by 1H−13C gHSQC. LRMS (ESI+): m/z = 383 [M + Na]+.
HRMS (ESI): calcd for C19H19N4O3S+ 383.1172, found 383.1171.
6-(4-[3′-Methylphenyl]-1H-1,2,3-triazol-1-yl)-1,2-benziso-
thiazole-3-one-1,1-dioxide (5b). (1) The title compound 5b was
prepared from 1-ethynyl-3-methylbenzene (b) (35 μL, 0.27 mmol)
according to General Procedure 1. Purification of the crude product by
flash chromatography (EtOAc/hexane 7:1) afforded 5b (33%) as a
pale yellow solid. Rf = 0.39 (DCM/MeOH 4:1). Mp = 265−267 °C
3527
dx.doi.org/10.1021/jm500255y | J. Med. Chem. 2014, 57, 3522−3531

Journal of Medicinal Chemistry
Article
N-t-Butyl-6-(4-[3′-methyl(phenyl)]-1H-1,2,3-triazol-1-yl)-1,2-
benzisothiazole-3-one-1,1-dioxide (9b). The title compound 9b
was prepared from 1-ethynyl-3-methylbenzene (b) (69 μL, 0.54
mmol) according to the General Procedure 2. Purification of the crude
product by flash chromatography (EtOAc/hexane 1:4) afforded 9b
(111 mg, 52%) as a white solid Rf = 0.50 (EtOAc/hexane, 1:2). Mp =
175−177 °C. 1H NMR (500 MHz, DMSO-d6): δ = 9.57 (s, 1H,
Htriazole), 8.80 (d, J = 1.6 Hz, 1H, H-7), 8.58 (dd, J = 8.4, 1.8 Hz, 1H,
H-5), 8.27 (d, J = 8.4 Hz, 1H, H-4), 7.77 (bs, 1H, H-2′), 7.73 (d, J =
7.7 Hz, 1H, H-6′), 7.41 (t, J = 7.6 Hz, 1H, H-5′), 7.23 (d, J = 7.6 Hz,
1H, H-4′), 2.40 (s, 3H, CH3), 1.73 (s, 9H, 3 × CH3 (tBu)),
assignments were confirmed by 1H−1H gCOSY. 13C NMR (125 MHz,
DMSO-d6): δ = 158.7 (CO), 147.9 (Ctriazole), 141.4 (C-3a), 138.6
(C-7a), 138.3 (C-3′), 129.6 (C-6), 129.2 (C-1′), 129.0 (C-4′), 126.8
(C-5′), 125.9 (C-2′), 125.6 (C-4), 125.3 (C-5), 122.5 (C-6′), 120.2
(CHtriazole), 111.7 (C-7), 60.9 (C(CH3)3), 27.3 (3 × CH3 (tBu)),
21.04 (CH3), assignments were confirmed by 1H−13C gHSQC. LRMS
(ESI+): m/z = 397 [M + H]+. HRMS (ESI): calcd for C20H21N4O3S+
397.1329, found 397.1346.
(d, J = 3.4 Hz, C-1′), 125.6 (C-5), 125.3 (C-6), 125.1 (d, J = 17.8 Hz,
C-3′), 124.8 (d, J = 8.4 Hz, C-6′), 120.1 (CHtriazole), 115.8 (d, J = 22.8
Hz, C-5′), 111.7 (C-7), 60.9 (C(CH3)3), 27.3 (3 × CH3(tBu)), 14.2
(d, J = 3.2 Hz, CH3), assignments were confirmed by 1H−13C
gHSQC. LRMS (ESI+): m/z = 415 [M + H]+. HRMS (ESI): calcd for
C20H20FN4O3S+ 415.1235, found 415.1254.
N-t-Butyl-6-(4-[4′-methoxy-2′-methyl(phenyl)]-1H-1,2,3-tria-
zol-1-yl)-1,2-benzisothiazole-3-one-1,1-dioxide (9f). The title
compound 9f was prepared from 1-ethynyl-4-methoxy-2-methylben-
zene (f) (78 μL, 0.54 mmol) according to General Procedure 2.
Purification of the crude product by flash chromatography (EtOAc/
hexane 1:7) afforded 9f (120 mg, 53%) as a pale yellow solid. Rf = 0.27
(EtOAc/hexane, 1:2). Mp = 182−184 °C. 1H NMR (500 MHz,
DMSO-d6): δ = 9.22 (s, 1H, Htriazole), 8.90 (d, J = 1.8 Hz, 1H, H-7),
8.63 (dd, J = 8.4, 1.8 Hz, 1H, H-5), 8.26 (d, J = 8.4 Hz, 1H, H-4), 7.78
(d, J = 8.4 Hz, 1H, H-6′), 7.00−6.86 (m, 2H, H-3′, H-5′), 3.81 (s, 3H,
OCH3), 2.52 (s, 3H, CH3), 1.73 (s, 9H, 3 × CH3(tBu)), assignments
were confirmed by 1H−1H gCOSY. 13C NMR (125 MHz, DMSO-d6):
δ = 159.2 (C-4′), 158.7 (CO), 147.1(Ctriazole), 141.5 (C-3a), 138.6
(C-7a), 137.1 (C-5′), 129.7 (C-4), 126.7 (C-3′), 125.6 (C-5), 125.1
(C-6), 121.5 (C-1′), 120.9 (CHtriazole), 116.2 (C-6′), 111.8 (C-2′),
111.7 (C-7), 60.8 (C(CH3)3), 55.1 (OCH3), 27.3 (3 × CH3(tBu)),
N-t-Butyl-6-(4-[2′-(trifluoromethyl)phenyl]-1H-1,2,3-triazol-
1-yl)-1,2-benzisothiazole-3-one-1,1-dioxide (9c). The title com-
pound 9c was prepared from 1-ethynyl-2-(trifluoromethyl)benzene
(c) (57 μL, 0.41 mmol) according to General Procedure 2. Purification
of the crude product by flash chromatography (EtOAc/hexane 1:6)
afforded 9c (158 mg, 86%) as a white solid. Rf = 0.43 (EtOAc/hexane,
1
21.3 (CH3), assignments were confirmed by H−13C gHSQC. LRMS
(ESI+): m/z = 427 [M + H]+. HRMS (ESI): calcd for C21H23N4O4S+
427.1435, found 427.1442.
1
1:6). Mp = 184−186 °C. H NMR (500 MHz, DMSO-d6): δ = 9.31
N-t-Butyl-6-(4-[3′-sulfonamido(phenyl)]-1H-1,2,3-triazol-1-
yl)-1,2-benzisothiazole-3-one-1,1-dioxide (9g). The title com-
pound 9g was prepared from 3-ethynyl benzenesulfonamide (g) (97
mg, 0.54 mmol) according to General Procedure 2. Purification of the
crude product by flash chromatography (EtOAc/hexane 1:4) afforded
9g (99 mg, 40%) as a pale yellow solid. Rf = 0.14 (EtOAc/hexane,
(s, 1H, Htriazole), 8.92 (d, J = 1.8 Hz, 1H, H-7), 8.63 (dd, J = 8.4, 1.9
Hz, 1H, H-5), 8.27 (d, J = 8.4 Hz, 1H, H-4), 7.94 (d, J = 7.8 Hz, 1H,
H-3′), 7.88−7.79 (m, 2H, H-6′, H-5′), 7.73 (t, J = 7.4 Hz, 1H, H-4′),
1.73 (s, 9H, 3 × CH3), assignments were confirmed by 1H−1H
gCOSY. 13C NMR (125 MHz, DMSO-d6): δ = 158.7 (CO), 145.3
(Ctriazole), 141.2 (C-3a), 138.6 (C-7a), 132.8 (C-5′), 132.1 (C-6′),
129.6 (C-4′), 128.8 (C-1′), 127.0 (q, J = 31 Hz, CF3), 126.78 (C-4),
126.5 (q, J = 5.5 Hz, C-2′), 126.0 (C-5), 125.5 (C-6), 124.9
(CHtriazole), 122.8 (C-3′), 112.2 (C-7), 60.9 (C(CH3)3), 27.8 (3 ×
CH3), assignments were confirmed by 1H−13C gHSQC. LRMS
(ESI+): m/z = 451 [M + H]+. HRMS (ESI): calcd for
C20H18N4O3F3S+ 451.1046, found 451.1056.
1
1:2). Mp = 200−202 °C. H NMR (500 MHz, DMSO-d6): δ = 9.75
(s, 1H, Htriazole), 8.84 (s, 1H, H-7), 8.61 (d, J = 8.3 Hz, 1H, H-5), 8.43
(s, 1H, H-2′), 8.29 (d, J = 8.3 Hz, 1H, H-4), 8.13 (d, J = 7.8 Hz, 1H,
H-6′), 7.87 (d, J = 7.9 Hz, 1H, H-4′), 7.75 (t, J = 7.7 Hz, 1H, H-5′),
7.48 (s, 2H, SO2NH2), 1.73 (s, 9H, 3 × CH3), assignments were
1
confirmed by H−1H gCOSY. 13C NMR (125 MHz, DMSO-d6): δ =
158.7 (CO), 146.6 (Ctriazole), 145.1 (C-3′), 141.3 (C-3a), 138.6 (C-
7a), 130.4 (C-5′), 130.0 (C-1′), 128.4 (C-6′), 126.8 (C-4), 125.8 (C-
5), 125.6 (C-4′), 125.5 (C-6), 122.3 (C-2′), 121.2 (CHtriazole), 111.9
(C-7), 60.9 (C(CH3)3), 27.3 (3 × CH3), assignments were confirmed
N-t-Butyl-6-(4-[4′-methoxy(phenyl)]-1H-1,2,3-triazol-1-yl)-
1,2-benzisothiazole-3-one-1,1-dioxide (9d). The title compound
9d was prepared from 1-ethynyl-4-methoxybenzene (d) (46 μL, 0.36
mmol) according to General Procedure 2. Purification of the crude
product by flash chromatography (EtOAc/hexane 1:6) afforded 9d
(50 mg, 34%) as a pale yellow solid. Rf = 0.50 (EtOAc/hexane, 1:6).
Mp = 214−216 °C. 1H NMR (500 MHz, DMSO-d6): δ = 9.48 (s, 1H,
Htriazole), 8.79 (d, J = 1.8 Hz, 1H, H-7), 8.57 (dd, J = 8.4, 1.9 Hz, 1H,
H-5), 8.27 (d, J = 8.4 Hz, 1H, H-4), 7.87 (d, J = 8.8 Hz, 2H, H-3′, H-
5′), 7.10 (d, J = 8.8 Hz, 2H, H-2′, H-6′), 3.82 (s, 3H, OCH3), 1.73 (s,
1
by H−13C gHSQC. LRMS (ESI+): m/z = 462 [M + H]+. HRMS
(ESI): calcd for C19H19N5O5S2Na1 484.0720, found 484.0733.
6-(4-[2′-Trifluoromethyl(phenyl)]-1H-1,2,3-triazol-1-yl)-1,2-
benzisothiazole-3-one-1,1-dioxide (5c). The title compound 5c
was prepared from 9c (75 mg, 0.167 mmol) according to General
Procedure 3 in 4 days. Purification of the crude product by flash
chromatography (EtOAc/hexane 1:2) afforded 5c (15 mg, 23%) as a
pale yellow solid. Rf = 0.12 (EtOAc/hexane, 1:2). Mp = 276−278 °C
(decomp.). 1H NMR (500 MHz, DMSO-d6): δ = 9.22 (s, 1H, Htriazole),
8.45 (bs, 1H, H-7), 8.33 (d, J = 8.1 Hz, 1H, H-5), 7.95−7.89 (m, 2H,
H-4, H-6′), 7.86−7.78 (m, 2H, H-4′, H-5′), 7.77−7.66 (m, 1H, H-3′),
assignments were confirmed by 1H−1H gCOSY. 13C NMR (125 MHz,
DMSO-d6): δ = 166.9 (CO), 147.1 (Ctriazole), 145.2 (C-7a), 138.5
(C-3a), 132.9 (C-5′), 132.4 (C-6′), 129.6 (C-4′), 128.9 (C-1′), 127.3
(C-6), 127.1 (CF3), 126.6 (C-2′), 124.7 (C-5), 123.8 (C-4), 123.0
(CHtriazole), 122.7 (C-3′), 111.3 (C-7), assignments were confirmed by
1H−13C gHSQC. LRMS (ESI−): m/z = 393 [M − H]−. HRMS: calcd
1
9H, 3 × CH3), assignments were confirmed by H−1H gCOSY. 13C
NMR (125 MHz, DMSO-d6): δ = 159.6 (C-4′), 158.7 (CO), 147.8
(Ctriazole), 141.4 (C-3a), 138.6 (C-7a), 126.79 (C-4), 126.76 (C-3′, C-
5′), 125.6 (C-5), 125.2 (C-6), 122.1 (C-1′), 119.2 (CHtriazole), 114.6
(C-2′, C-6′), 111.6 (C-7), 60.9 (C(CH3)3), 55.2 (OCH3), 27.3 (3 ×
CH3), assignments were confirmed by 1H−13C gHSQC. LRMS
(ESI+): m/z = 413 [M + H]+. HRMS (ESI): calcd for C20H21N4O4S+
413.1278, found 413.1293.
N-t-Butyl-6-(4-[4′-fluoro-3′-methyl(phenyl)]-1H-1,2,3-triazol-
1-yl)-1,2-benzisothiazole-3-one-1,1-dioxide (9e). The title com-
pound 9e was prepared from 4-ethynyl-1-fluoro-2-methylbenzene (e)
(71 μL, 0.54 mmol) according to General Procedure 2. Purification of
the crude product by flash chromatography (EtOAc/hexane 1:6)
afforded 9e (186 mg, 84%) as a pale yellow solid. Rf = 0.50 (EtOAc/
for C16H10F3N4O3S− 393.0274, found 393.0288.
6-(4-[4′-Methoxy(phenyl)]-1H-1,2,3-triazol-1-yl)-1,2-benziso-
thiazole-3-one-1,1-dioxide (5d). The title compound 5d was
prepared from 9d (42 mg, 0.102 mmol) according to General
Procedure 3 in 1 day. Purification of the crude product by flash
chromatography (DCM/MeOH 19:1 to 15:1) afforded 5d (21 mg,
58%) as a pale yellow solid. Rf = 0.14 (DCM/MeOH 19:1). Mp =
280−282 °C. 1H NMR (500 MHz, DMSO-d6): δ = 9.39 (s, 1H,
Htriazole), 8.24 (d, J = 1.5 Hz, 1H, H-7), 8.21 (dd, J = 8.1, 1.8 Hz, 1H,
H-5), 7.87 (d, J = 8.7 Hz, 2H, H-3′, H-5′), 7.81 (d, J = 8.1 Hz, 1H, H-
4), 7.08 (d, J = 8.8 Hz, 2H, H-2′, H-6′), 3.82 (s, 3H, OCH3),
assignments were confirmed by 1H−1H gCOSY. 13C NMR (125 MHz,
1
hexane, 1:2). Mp = 205−207 °C. H NMR (500 MHz, DMSO-d6): δ
= 9.55 (s, 1H, Htriazole), 8.78 (d, J = 1.7 Hz, 1H, H-7), 8.56 (dd, J = 8.4,
1.5 Hz, 1H, H-5), 8.27 (d, J = 8.4 Hz, 1H, H-4), 7.86 (d, JF,H = 7.3 Hz,
1H, H-2′), 7.81−7.71 (m, 1H, H-6′), 7.32−7.28 (m, 1H, H-5′), 2.33
(bs, 3H, CH3), 1.73 (s, 9H, 3 × CH3 (tBu)), assignments were
1
confirmed by H−1H gCOSY. 13C NMR (125 MHz, DMSO-d6): δ =
160.7 (d, J = 244.8 Hz, C-4′), 158.7 (CO), 147.1 (Ctriazole), 141.3
(C-3a), 138.6 (C-7a), 128.6 (d, J = 5.3 Hz, C-2′), 126.8 (C-4), 125.9
3528
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1
DMSO-d6): δ = 166.6 (CO), 159.4 (C-4′), 147.4 (Ctriazole), 147.1
(C-7a), 138.5 (C-3a), 134.2 (C-6), 126.7 (C-3′, C-5′), 124.1 (C-4),
122.9 (C-5), 122.6 (C-1′), 119.0 (CHtriazole), 114.5 (C-2′, C-6′), 110.4
(C-7), 55.2 (OCH3), assignments were confirmed by 1H−13C
gHSQC. LRMS (ESI−): m/z = 355 [M − H]−. HRMS (ESI): calcd
for C16H11N4O4S− 355.0507, found 355.0504.
(tBu)) assignments were confirmed by H−1H gCOSY. 13C NMR
(125 MHz, DMSO-d6): δ = 170.0, 169.5, 169.2, 169.0 (4 × C
O(Ac)), 158.7 (CO(Sac)), 144.6 (Ctriazole), 141.3 (C-3a), 138.6 (C-
7a), 126.8 (C-5), 125.9 (C-4), 125.4 (C-6), 123.5 (CHtriazole), 112.0
(C-7), 98.5 (C-1′), 72.1 (C-5′), 70.8 (C-3′), 70.7 (C-2′), 68.1 (C-4′),
61.6 (CH2), 61.4 (C-6′), 60.9 (C(CH3)3), 27.3 (3 × CH3(tBu)), 20.5,
20.3, 20.3, 20.2 (4 × OCH3), assignments were confirmed by 1H−13C
gHSQC. LRMS (ESI+): m/z = 667 [M + H]+, 689 [M + Na]+. HRMS
(ESI): calcd for C28H35N4O13S+ 667.1916, found 667.1903.
6-(4-[4′-Fluoro-3′-methyl(phenyl)]-1H-1,2,3-triazol-1-yl)-1,2-
benzisothiazole-3-one-1,1-dioxide (5e). The title compound 5e
was prepared from 9e (50 mg, 0.120 mmol) according to General
Procedure 3 in 4 days. Purification of the crude product by flash
chromatography (DCM/MeOH 15:1) afforded 5e (21 mg, 49%) as a
6-(4-[β-D-Glucopyranoside-O-methylene]-1H-1,2,3-triazol-1-
yl)-1,2-benzisothiazole-3-one-1,1-dioxide (12). The title com-
pound 12 was prepared as a pale white solid (13 mg, 34%) from 11
(50 mg, 0.075 mmol) according to General Procedure 3 in 6 days. Rf =
1
pale yellow solid. Rf = 0.14 (DCM/MeOH 15:1). Mp = >300 °C. H
NMR (500 MHz, DMSO-d6): δ = 9.48 (s, 1H, Htriazole), 8.25 (d, J =
1.8 Hz, 1H, H-7), 8.22 (dd, J = 8.0, 1.8 Hz, 1H, H-5), 7.89 (dd, J = 7.4,
1.5 Hz, 1H, H-2′), 7.82 (d, J = 8.1 Hz, 1H, H-4), 7.81−7.77 (m, 1H,
H-6′), 7.32−7.27 (m, 1H, H-5′), 2.33 (d, J = 1.1 Hz, 3H, CH3),
assignments were confirmed by 1H−1H gCOSY. 13C NMR (125 MHz,
DMSO-d6): δ = 166.6 (C-4′), 159.6 (CO), 147.1 (Ctriazole), 146.7
(C-3a), 138.4 (C-7a), 134.3 (C-6), 128.6 (d, J = 5.2 Hz, C-2′), 126.3
(d, J = 3.2 Hz, C-1′), 124.95 (d, J = 16.9 Hz, C-3′), 124.75 (d, J = 8.2
Hz, C-6′), 124.2 (C-5), 123.0 (C-4), 119.9 (CHtriazole), 115.7 (d, J =
3.2 Hz, C-5′), 110.5 (C-7), 14.2 (d, J = 3.2 Hz, CH3), assignments
were confirmed by 1H−13C gHSQC. LRMS (ESI−): m/z = 357 [M −
H]−. HRMS (ESI): calcd for C16H10FN4O3S− 357.0463, found
357.0454.
1
0.45 (acetonitrile/water 9:1). Mp = 90−92 °C. H NMR (500 MHz,
DMSO-d6, 1% D2O): δ = 9.04 (s, 1H, Htriazole), 8.49 (s, 1H, H-7), 8.34
(d, J = 8.4 Hz, 1H, H-5), 8.02 (d, J = 8.2 Hz, 1H, H-4), 4.95 (d, J =
12.5 Hz, 1H, CH of CH2), 4.77 (d, J = 12.5 Hz, 1H, CH of CH2), 4.34
(d, J = 7.8 Hz, 1H, H-1′), 3.73 (d, J = 10.5 Hz, 1H, H-6′), 3.48 (dd, J =
11.8, 6.1 Hz, 1H, H-6″), 3.21−3.13 (m, 2H, H-2′, H-5′), 3.10−3.05
(m, 1H, H-3′), 3.02 (t, J = 8.4 Hz, 1H, H-4′), assignments were
confirmed by H−1H gCOSY. 13C NMR (125 MHz, DMSO-d6): δ
1
=162.9 (CO), 145.5 (Ctriazole), 143.8 (C-3a), 140.0 (C-7a), 130.1
(C-4), 125.6 (C-5), 124.5 (C-6), 123.1 (CHtriazole), 111.6 (C-7), 102.3
(C-1′), 77.0 (C-3′), 76.6 (C-2′), 73.4 (C-5′), 70.1 (C-4′), 61.3 (CH2),
61.2 (C-6′), assignments were confirmed by H−13C gHSQC. LRMS
1
(ESI−): m/z = 441 [M − H]−. HRMS (ESI): calcd for C16H17N4O9S−
441.0722, found 441.0739.
6-(4-[4′-Methoxy-2′-methyl(phenyl)]-1H-1,2,3-triazol-1-yl)-
1,2-benzisothiazole-3-one-1,1-dioxide (5f). The title compound
5f was prepared as a pale yellow solid (15 mg, 33%) from 9f (53 mg,
0.124 mmol) according to General Procedure 3 in 4 days. Rf = 0.20
(DCM/MeOH 4:1). Mp = 275−277 °C. 1H NMR (500 MHz,
DMSO-d6): δ = 9.17 (s, 1H, Htriazole), 8.64−8.55 (m, 1H, H-7), 8.46−
8.39 (m, 1H, H-5), 8.05−7.98 (m, 1H, H-4), 7.77 (d, J = 8.3 Hz, 1H,
H-5′), 6.95−6.89 (m, 1H, H-3′, H-6′), 3.81 (s, 3H, OCH3), 2.52 (s,
3H, CH3), assignments were confirmed by 1H−1H gCOSY. 13C NMR
(125 MHz, DMSO-d6): δ = 159.2 (C-4′), 158.5 (CO), 146.9
(Ctriazole), 139.8 (C-3a), 137.1 (C-7a), 134.9 (C-4), 129.8 (C-2′),
125.3 (C-5), 124.1 (C-6), 121.72 (C-1′), 121.69 (C-5′), 120.9
(CHtriazole), 116.2 (C-6′), 111.7 (C-3′), 111.5 (C-7), 55.1 (OCH3),
Protein X-ray Crystallography. hCA II was concentrated down
to ∼14 mg mL−1 and set up in SD-2 plates (Molecular Dimensions)
using a Phoenix crystallization robot (ARI) with the following ratio of
protein plus reservoir plus compound: 210 nL + 140 nL + 70 nL. The
compound was dissolved in neat DMSO, and the plate was incubated
at 20 °C. The reservoir condition consisted of 2.9 M ammonium
sulfate with 0.1 M Tris buffer at pH 8.5. Additional dry compound was
added to the crystallization drop after crystals had formed and several
days before data were collected. Then, 360 frames of one degree
oscillation were taken at the MX-2 beamline of the Australian
Synchrotron. The data were indexed using XDS24 and scaled using
SCALA.25 Molecular replacement was done using Phaser26 using
3ML2 as the initial starting model. The model was manually rebuilt
using Coot27 and refined using Refmac.28 The compound was placed
in density using the program Afitt (OpenEye Scientific Software) and
further refined using Refmac.28
Protein Expression and Purification. BL21-DE3 cells were
transformed with 1 μL of the CA II-containing plasmid pACA, and the
cells were plated out onto a YT-Amp plate and left at 37 °C overnight.
The next day a single colony was selected and grown up in 5 mL of
2xYT overnight to form a starter culture. One liter of 2xYT was started
with 2 mL of overnight culture, and the cells were induced with 1 mM
IPTG at OD595 = 0.68. The cells were allowed to grow for another
3.5 h, and then the cells were spun down, washed with 20 mM Tris pH
7.5 and 100 mM NaCl, and spun again. The cell pellet was frozen at
−80 °C. The cell pellet was brought up in 30 mL (of 50 mL total)
buffer A that had one protease inhibitor tablet (Roche), 20 μL of 50
mg mL−1 lysozyme, and 20 μL benzoase added and used to lyse the
cells with a sonicator. The sample was on ice; the sonicator was used
in 15 s bursts with 15 s of rest between bursts, and the setting was at
80 to 100% for a total of 11 min of sonication. The 20 mL “extra”
buffer A was added when extra cooling was needed throughout the
procedure. When done, the sample was centrifuged at 4000 rpm for 25
min at 4 °C to remove cell debris. Next, 45 mL of the sample was
loaded onto a Sepharose S 5 mL column and washed with buffer A for
10 column volumes. The sample was then eluted from the column
with buffer B in a linear gradient; fractions were collected and analyzed
by SDS-PAGE gel. Fractions containing the majority of the protein of
interest were combined (∼4 mL total). Next, a preparative gel
filtration column, GE Sepharose16/60, was used, and the sample was
run in 2 × 2 mL lots onto this column. Collected fractions were
analyzed by SDS-PAGE. The pure hCA II fractions were combined,
concentrated using an Amicon Ultra 10 kDa concentration device to
1
21.3 (CH3), assignments were confirmed by H−13C gHSQC. LRMS
(ESI−): m/z = 369 [M − H]−. HRMS (ESI): calcd for C17H13N4O4S−
369.0663, found 369.0674.
6-(4-[3′-Sulfonamido(phenyl)]-1H-1,2,3-triazol-1-yl)-1,2-
benzisothiazole-3-one-1,1-dioxide (5g). The title compound 5g
was prepared as a pale yellow solid (35 mg, 79%) from 9g (50 mg,
0.108 mmol) according to General Procedure 3 in 1 day. Rf = 0.10
1
(DCM/MeOH 4:1). Mp = >300 °C. H NMR (500 MHz, DMSO-
d6): δ = 9.69 (s, 1H, Htriazole), 8.51 (d, J = 1.9 Hz, 1H, H-7), 8.44 (d, J
= 1.8 Hz, 1H, H-2′), 8.39 (dd, J = 8.2, 1.9 Hz, 1H, H-5), 8.18−8.09
(m, 1H, H-4′), 8.01 (d, J = 8.2 Hz, 1H, H-4), 7.89−7.83 (m, 1H, H-
6′), 7.74 (t, J = 7.7 Hz, 1H, H-5′), 7.46 (s, 2H, SO2NH2), assignments
were confirmed by 1H−1H gCOSY. 13C NMR (125 MHz, DMSO-d6):
δ = 146.4 (C-3′), 145.1 (Ctriazole), 144.6 (C-7a), 139.6 (C-3a), 130.6
(C-6), 129.9 (C-2′), 128.4 (C-4′), 125.5 (C-6′), 125.4 (C-5′), 124.2
(C-4), 122.3 (C-5), 121.0 (C-1′), 111.5 (CHtriazole), 109.5 (C-7),
1
assignments were confirmed by H−13C gHSQC. LRMS (ESI−): m/z
−
= 404 [M − H]−. HRMS (ESI): calcd for C15H10N5O5S2 404.0129,
found 404.0148.
N-t-Butyl-6-(4-[2′,3′,4′,6′-tetra-O-acetyl-β-D-glucopyrano-
side-O-methylene]-1H-1,2,3-triazol-1-yl)-1,2-benzisothiazole-
3-one-1,1-dioxide (11). The title compound 11 was prepared from
10 (138 mg, 0.36 mmol) according to General Procedure 2.
Purification of the crude product by flash chromatography (hexane/
EtOAc 2:1 to 1:1) afforded 11 (211 mg, 89%) as a pale yellow solid. Rf
1
= 0.45 (hexane/EtOAc 1:2). Mp = 90−92 °C. H NMR (500 MHz,
DMSO-d6): δ = 9.06 (s, 1H, Htriazole), 8.80 (d, J = 1.8 Hz, 1H, H-7),
8.54 (dd, J = 8.4, 1.9 Hz, 1H, H-5), 8.25 (d, J = 8.4 Hz, 1H, H-4), 5.27
(t, J = 9.6 Hz, 1H, H-3′), 4.99−4.88 (m, 3H, H-1′, H-2′, CH of CH2),
4.83−4.76 (m, 2H, H-4′, CH of CH2), 4.21 (dd, J = 12.4, 4.8 Hz, 1H,
H-6′), 4.08 (d, J = 12.1, 2.3 Hz, 1H, H-6″), 4.06−4.02 (m, 1H, H-5′),
2.02, 1.99, 1.94, 1.93 (4 × s, 4 × 3H, 4 × OCH3, 1.72 (s, 9H, 3 × CH3
3529
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14 mg mL−1 (as measured on a NanoDrop spectrophotometer) in GF
buffer, and subsequently flash frozen in 100 μL aliquots in liquid N2
and transferred to −80 °C. Buffers: (buffer A) 20 mM MES pH 6.5, 10
mM NaCl, 0.1 mM ZnSO4, 0.4 mM DTT; (buffer B) 100 mM Tris
pH 8.0, 10 mM NaCl, 0.1 mM ZnSO4, 0.4 mM DTT; (GF buffer) 30
mM Tris pH 8.0, 20 mM NaCl, 0.1 mM ZnSO4, 1 mM DTT.
CA Inhibition Assay. An Applied Photophysics stopped-flow
instrument was used for assaying the CA-catalyzed CO2 hydration
activity.29 IC50 values were obtained from dose−response curves
working at seven different concentrations of test compound, by fitting
the curves using PRISM (www.graphpad.com) and nonlinear least-
squares methods; values represent the mean of at least three different
determinations as described by us previously.30 The inhibition
constants (Ki) were then derived by using the Cheng-Prusoff
equation31 as follows: Ki = IC50/(1 + [S]/Km), where [S] represents
the CO2 concentration at which the measurement was carried out, and
Km is the concentration of substrate at which the enzyme activity is at
half-maximal. All enzymes used were recombinant, produced in
Escherichia coli as reported earlier.32 The concentrations of enzymes
used in the assay were as follows: hCA I, 10.4 nM; hCA II, 8.3 nM;
hCA IX, 8.0 nM; and hCA XII, 12.4 nM.
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ASSOCIATED CONTENT
■
S
* Supporting Information
1H and 13C NMR spectra of compounds 4, 7, 8, 5a−5g, 9a−9g,
11, and 12. Data collection and structure refinement statistics
of 5b/hCA II crystal structure. This material is available free of
charge via the Internet at http://pubs.acs.org.
(7) Alterio, V.; Di Fiore, A.; D’Ambrosio, K.; Supuran, C. T.; De
Simone, G. Multiple binding modes of inhibitors to carbonic
anhydrases: how to design specific drugs targeting 15 different
isoforms? Chem. Rev. 2012, 112, 4421−4468.
(8) Di Fiore, A.; Maresca, A.; Alterio, V.; Supuran, C. T.; De Simone,
G. Carbonic anhydrase inhibitors: X-ray crystallographic studies for
the binding of N-substituted benzenesulfonamides to human isoform
II. Chem. Commun. 2011, 47, 11636−11638.
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chlorine potentials of N-chlorinated organic molecules. J. Chem. Soc. B
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drugs and their leads in successful drug discoveryprograms. J. Med.
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saccharin and cyclamate derivatives bearing polymerizable vinyl group.
Bull. Chem. Soc. Jpn. 1982, 55, 3824−3827.
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AUTHOR INFORMATION
Corresponding Authors
*Telephone: +39 055 457 3729. E-mail: claudiu.supuran@unifi.
■
it.
*Telephone: +61 7 3735 7825. E-mail: s.poulsen@griffith.edu.
au.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was financed by the Australian Research Council
(Grant Nos. DP110100071 and FT10100185 to S-A.P.) and
two EU grants of the seventh framework program (Metoxia and
Dynano projects to C.T.S.). cDNA for hCA II was kindly
provided by C. Fierke. We thank OpenEye Scientific Software
for a license to use Afitt, the CSIRO Collaborative
Crystallization Centre (www.csiro.au/c3) for crystallization,
and the Australian Synchrotron and the beamline scientists at
the MX-2 beamline for their support in data collection.
ABBREVIATIONS USED
■
1,3-DCR, 1,3-dipolar cycloaddition reaction; CuAAC, copper-
catalyzed azide alkyne cycloaddition; CA, carbonic anhydrase;
Ki, inhibition constant
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