Synthesis of 6-tetrazolyl-substituted sulfocoumarins acting as highly potent and selective inhibitors of the tumor-associated carbonic anhydrase isoforms IX and XII

Article abstract of DOI:10.1016/j.bmc.2014.01.043

A series of 6-substituted sulfocoumarins incorporating substituted-1,2,3,4- tetrazol-5-yl moieties were synthesized by reaction of 6-iodo-sulfocoumarin and the corresponding tetrazole via the CH activation reaction. The new sulfocoumarins incorporating alkyl and substituted aryl moieties at the 1-position of the tetrazole, were investigated for the inhibition of four human (h) carbonic anhydrase (hCA, EC 4.2.1.1) isoforms, the cytosolic hCA I and II; and the transmembrane, tumor-associated hCA IX and XII. The tetrazole- substituted sulfocoumarins did not inhibit the ubiquitous, off-target cytosolic isoforms (K_Is >10 μM) but showed effective inhibition against the two transmembrane CAs, with K_Is ranging from 6.5 to 68.6 nM against hCA IX, and between 4.3 and 59.8 nM against hCA XII. As hCA IX and XII are validated anti-tumor targets, such prodrug, isoform-selective inhibitors as the sulfocoumarins reported here, may be useful for identifying suitable drug candidates for clinical trials.

Full text of DOI:10.1016/j.bmc.2014.01.043

Bioorganic & Medicinal Chemistry 22 (2014) 1522–1528
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of 6-tetrazolyl-substituted sulfocoumarins acting as highly
potent and selective inhibitors of the tumor-associated carbonic
anhydrase isoforms IX and XII
b,c,
Aiga Grandane a, Muhammet Tanc b,c, Raivis Zalubovskis a, , Claudiu T. Supuran
⇑
⇑
a Latvian Institute of Organic Synthesis, Aizkraukles 21, LV-1006 Riga, Latvia
b Università degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, 50019 Sesto Fiorentino (Florence), Italy
c Università degli Studi di Firenze, NEUROFARBA Department, Section of Pharmaceutical Chemistry, Via Ugo Schiff 6, 50019 Sesto Fiorentino (Florence), Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 6-substituted sulfocoumarins incorporating substituted-1,2,3,4-tetrazol-5-yl moieties were
synthesized by reaction of 6-iodo-sulfocoumarin and the corresponding tetrazole via the CH activation
reaction. The new sulfocoumarins incorporating alkyl and substituted aryl moieties at the 1-position of
the tetrazole, were investigated for the inhibition of four human (h) carbonic anhydrase (hCA, EC
4.2.1.1) isoforms, the cytosolic hCA I and II; and the transmembrane, tumor-associated hCA IX and XII.
The tetrazole-substituted sulfocoumarins did not inhibit the ubiquitous, off-target cytosolic isoforms
Received 28 December 2013
Revised 21 January 2014
Accepted 23 January 2014
Available online 1 February 2014
Keywords:
Carbonic anhydrase
Inhibitor
Sulfocoumarin
Tumor-associated isoform IX, XII
Tetrazole
(KIs >10 lM) but showed effective inhibition against the two transmembrane CAs, with KIs ranging from
6.5 to 68.6 nM against hCA IX, and between 4.3 and 59.8 nM against hCA XII. As hCA IX and XII are val-
idated anti-tumor targets, such prodrug, isoform-selective inhibitors as the sulfocoumarins reported
here, may be useful for identifying suitable drug candidates for clinical trials.
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
(i.e., their hydrolysis products) bind around 8 Å away from the zinc
ion, at the entrance of the active site cavity, occluding it, and
Our groups recently identified1 the sulfocoumarins (1,2-benz-
oxathiine 2,2-dioxides) as a new class of prodrug-type inhibitors
of the metallo-enzyme carbonic anhydrase (CA, EC 4.2.1.1).2,3 Sim-
ilar to the coumarins,4–8 the compounds used as lead molecules in
their drug design, the sulfocoumarins undergo a hydrolysis medi-
ated by the zinc hydroxide species of the enzyme, with generation
of the inhibitory species. In the case of the sulfocoumarins this
making interactions with amino acid residues in the most variable
region of the active site of the CA isoforms known to date
(15 mammalian CA isoforms are known).2,4–8 On the other hand,
the hydrolyzed sulfocoumarins bind more internally within the
active site, but again in a different binding site compared to the
sulfonamides.1 By means of kinetic studies and X-ray crystallogra-
phy we showed that the 2-hydroxyphenyl-x-ethenylsulfonic acid
species is a 2-hydroxyphenyl-
x
-ethenylsulfonic acid derivative
formed from the original sulfocoumarin is anchored to the
zinc-coordinated water molecule/hydroxide ion by means of a
hydrogen bond. In addition, the scaffold of the inhibitor partici-
pates in other favorable interactions with amino acid residues
and water molecules from the middle of the active site cavity.
(ii) Coumarins and sulfocoumarins are the most isoform-selec-
tive CAIs known to date.1,4–8 This highly desired feature was
explained by considering the inhibition mechanism of these com-
pounds, briefly outlined above. Indeed, the hydroxy-cinnamic
(as demonstrated by means of X-ray crystallography)1a whereas
for the coumarins, 2-hydroxy-cinnamic acid derivatives are formed
through the active-site mediated hydrolysis.4–8
At least three features are remarkable regarding the CA inhibi-
tion by these types of derivatives: (i) The inhibition mechanism
of coumarins/sulfocoumarins is very different compared to that
of the classical CA inhibitors (CAIs) of the sulfonamide/sulfamate/
sulfamide type,2,9 which coordinate to the metal ion within the
enzyme active site, substituting the nucleophile (water molecule
or hydroxide ion) used in the catalytic process. In fact, coumarins
acids/2-hydroxyphenyl-x-ethenylsulfonic acids formed after the
hydrolysis of the coumarin/sulfocoumarin bind in regions of the
CA active site which differ between the 15 CA isoforms known to
date in humans,2 affording the possibility that the inhibitor interacts
in a specific manner with residues which are found in some but not
in other CA isoforms.1–4 For example, we have reported many CA IX/
XII selective coumarin inhibitors,4–8 compounds showing low
⇑
Corresponding authors. Tel.: +371 67014826; fax: +371 67550338 (R.Z.);
tel.: +39 055 457 3005; fax: +39 055 4573385 (C.T.S.).
E-mail addresses: raivis@osi.lv (R. Zalubovskis), claudiu.supuran@unifi.it
(C.T. Supuran).
http://dx.doi.org/10.1016/j.bmc.2014.01.043
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

A. Grandane et al. / Bioorg. Med. Chem. 22 (2014) 1522–1528
1523
nanomolar inhibition of these transmembrane, tumor-associated
N
N
N
isoforms CA IX and XII,10,11 but at the same time not inhibiting at
all the cytosolic, off-target isoforms hCA I and II, which are ubiqui-
tous and which are potently inhibited by many sulfonamide CAIs.
This is one of the reasons why the sulfonamide CAIs show a rather
large number of undesired side effects.12,13 Furthermore, several
coumarins showing isoform specificity for the inhibition of hCA XIII,
hCA XIV as well as several other isoforms were also reported.4
(iii) The substitution pattern at the coumarin/sulfocoumarin ring
strongly influences the potency and selectivity against mammalian
CA isoforms of these classes of inhibitors. For example, 7,8-disubsti-
tuted- but not 6,7-disubstituted coumarins were shown to selec-
tively inhibit the transmembrane, tumor-associated isoforms hCA
IX and XII over the cytosolic ones hCA I and II in the low nanomo-
lar/subnanomolar range.5a For the sulfocoumarin derivatives, we
reported that the 6-substituted compounds1a incorporating a range
of simple (halogeno, OH, amino, alkyl, etc) or more complex (substi-
tuted 1,2,3-triazoles) substituents act as low nanomolar hCA IX/XII
R
R
O
S
O
S
O
O
O
O
A
B
R = OH, Br, NO2, NH2
R = Ph, substituted aryl, COOMe, etc.
The rationale of the present study was to replace the 1,2,3-tria-
zole ring present in derivatives B by the isosteric 1,2,3,4-tetrazole
ring, whichaffordstheexplorationof alternative syntheticpathways
as well as a different chemical space (due to the fact that the orien-
tation of the R moiety in compounds B and 5, 7–see later in the
text–are different). Thus we have prepared the key intermediate
6-iodo-sulfocoumarin 3, by the procedure reported earlier for
the synthesis of compounds of type A (Scheme 1).17,18 Reaction of
2-hydroxy-5-iodobenzaldehyde 1 with methanesulfonyl chloride
afforded the methanesulfonate 2 which was cyclized to 3 by the
procedure reported earlier.17,18 The iodo-sulfocoumarin 3 was then
reacted with 1-substituted-1,2,3,4-tetrazoles 419–23 in conditions in
which the CH bond in position 5 of the tetrazole ring is activated (i.e.,
Cs2CO3, CuI, Pd(OAc)2 and tri(2-furyl)phosphine),24 leading to the
desired derivatives 5 with acceptable yields (see Section 4 for
details)–Scheme 1. The R moieties present in derivatives 5 (and orig-
inating from the substituted tetrazoles 4, reported in the
literature19–23) were as variable as possible, that is phenyl, mono-
or di-substituted phenyls (with alkyl, alkoxy and halogens in various
positions of the ring) and 1-naphthyl (Scheme 1), in order to high-
light the structure–activity relationship (SAR) for the interaction of
these compounds with the various CA isoforms (see later in the
paper).
inhibitors, but do not inhibit hCA I and II up to the 50 lM range. On
the contrary, the 7-substituted derivatives1b were shown to act as
low nanomolar hCA II inhibitors, showed some inhibition of hCA
VA, but were not inhibiting hCA I, hCA IX and XII significantly. All
these facts demonstrate that the sulfocoumarins constitute a very
interesting class of CAIs with potential of applications in a range
of disorders in which various CA isoforms are involved, among
which glaucoma,14 obesity15 and cancer.10,11,16
Continuing our interest in the design and investigation of non-
sulfonamide CAIs, we report here the synthesis and inhibition data
against four physiologically relevant isoforms (hCA I, II, IX and XII)
of a series of sulfocoumarins incorporating 1,2,3,4-tetrazolyl moie-
ties which have been prepared by an original method.
2. Results and discussion
2.1. Chemistry
As the procedure employed in Scheme 1 did not work for R = H
or alkyl, we designed an alternative synthesis for obtaining the
unsubstituted tetrazole-sulfocoumarin 7a and the corresponding
In previous work from our laboratories1 we have investigated
6-substituted sulfocoumarins incorporating simple substituents
(of type A) as well as compounds obtained via click-chemistry, of
type B, which possess 1,2,3-triazole moieties in the 6-position of
the heterocyclic ring, observing that the most potent/selective
inhibition of hCA IX and XII was achieved with the last type of
derivatives, B.1a
methyl derivative 7b (Scheme 2). 6-Amino-sulfocoumarin
6
(reported earlier by some of us)17 was reacted with sodium azide,
followed by cyclization of the intermediate with triethyl
orthoformate or triethyl orthoacetate (Scheme 2) leading thus to
derivatives 7 in acceptable yields. The new compounds 5a–5j and
N
N
N
N
R
N
N
4
O
O
N
I
I
I
(i)
(ii)
(iii)
N
O
O
O
Ar
S
S
S
OH
O
O
O
O
O
O
2
1
3
5
Me
R =
;
;
;
;
;
Me
Cl
OMe
d (24%)
CF
3
a (36%)
b (33%)
g (41%)
c (57%)
e (46%)
j (43%)
Br
Cl
Cl
;
;
;
;
.
F
Cl
f (65%)
h (48%)
i (46%)
Scheme 1. Reagents and conditions: (i) MsCl, NEt3, CH2Cl2, rt, 2 h, 99%; (ii) (a) DBU, CH2Cl2, 0 °C, 2 h, (b) POCl3, Py, rt, 3 h, 71%; (iii) Pd(OAc)2, tri(2-furyl)phosphine, CuI,
Cs2CO3, toluene, 40 °C, 20 h.

1524
A. Grandane et al. / Bioorg. Med. Chem. 22 (2014) 1522–1528
N
R
unsubstituted tetrazole 7a and the methyl derivative 7b, together
N
N
with the m-bromophenyl derivative 5f (KIs in the range of 56.8–
68.6 nM). Slightly more effective than these compopunds were
the two sulfocoumarins 5c and 5e, with KIs of 27.1–39.3 nM
(Table 1). They incorporate the m-tolyl and 4-trifluoromethyl-
phenyl moieties as R groups at the tetrazole ring. Thus, the unsub-
stituted tetrazole 7a, the methyl-substituted derivative 7b (R = Me)
and the aryl-substituted ones with meta-methyl and 4-trifluoro-
methyl substituents at the phenyl ring (5c and 5e) were the least
effective hCA IX inhibitors in this series. However, the remaining
derivatives, that is 5a, 5b, 5d, 5g–5i, incorporating phenyl, 1-naph-
thyl, and 4-substituted- or 3,4-disubstituted phenyl groups, were
highly effective, low nanomolar hCA IX inhibitors, with inhibition
constants ranging between 6.5 and 9.1 nM. Thus there are many
substitution patterns leading to highly effective hCA IX inhibitors
in this series of tetrazole-substituted sulfocoumarins. These last
compounds were around 3–4 times more effective hCA IX inhibi-
tors compared to acetazolamide (Table 1).
H N
N
2
(i)
O
O
S
S
O
O
O
O
6
7
R =
H
;
Me.
b (45%)
a (72%)
Scheme 2. Reagents and conditions: (i) NaN3, RC(OEt)3, AcOH, 100 °C, 4 h.
7a,7b were characterized by spectroscopic techniques (IR, 1H and
13C NMR), high resolution mass spectroscopy (HRMS) as well as
elemental analysis, which confirmed their structures (see Section 4
for details).
2.2. CA inhibition
(iii) The second transmembrane isoform, hCA XII, was also highly
inhibited by the new sulfocoumarins reported here, with inhibition
constants ranging between 4.3 and 59.8 nM. Only one compound,
5c with a meta-tolyl substituent, showed a medium potency inhib-
itory power against this isoform, with a KI of 59.8 nM. All the other
compounds were low nanomolar inhibitors with KIs in the range of
4.3–13.5 nM (Table 1). Thus, most substitution patterns present in
these sulfocoumarins, that is both aliphatic and aromatic R groups,
lead to highly effective hCA XII inhibitors. It should be also noted
that acetazolamide is a very effective hCA XII inhibitor, but as men-
tioned earlier, it is a promiscuos CAI, inhibiting significantly most of
the 15 CA isoforms found in mammals.2–4
Sulfocoumarins 5a–5j and 7a,7b were screened for the inhibi-
tion of four human (h) CA isoforms, the cytosolic, widespread
hCA I and II (offtargets in this case) as well as the transmembrane,
tumor-associated hCA IX and XII (anticancer drug targets).4,10,11,16
Table 1 shows inhibition data of sulfocoumarins 5a–5j and 7a,7b
(as well as the sulfonamide acetazolamide AAZ, as standard)
against hCA I, II, IX and XII, after a period of 6 h of incubation of
the enzyme and inhibitor solutions.25 It should be mentioned that
assaying the inhibition with the usual 15 min incubation period (as
for the sulfonamides)9 leads to the measurement of a very weak
inhibition (data not shown). This was also the case with the cou-
marins.4–8 For this reason, a 6 h incubation time has been used
for assaying all sulfocoumarins as CAIs. The following SAR should
be noted regarding the inhibition data of Table 1:
3. Conclusions
(i) The cytosolic isoforms hCA I and II were not inhibited by the
sulfocoumarins 5a–5j and 7a, 7b, which showed inhibition
constants >10 lM. It should be observed that acetazolamide, the
We report here a series of 5-(2,2-dioxido-1,2-benzoxathiin-6-
yl)-1-aryl/alkyl-1H-tetrazoles, obtained by an original synthetic
procedure involving the cyclization of 2-formyl-4-iodophenyl
methanesulfonate to the corresponding 6-iodo-sulfocoumarin,
which was then reacted in conditions of CH activation with
1-substituted 1,2,3,4-tetrazoles, leading to the title compounds.
The new sulfocoumarins incorporating alkyl and substituted aryl
moieties at the 1-position of the tetrazole, were investigated for
the inhibition of four hCA isoforms with medicinal chemistry
applications, the cytosolic hCA I and II, and the transmembrane,
tumor-associated hCA IX and XII. The tetrazole-substituted
sulfocoumarins did not inhibit the ubiquitous, offtarget cytosolic
sulfonamide CAI in clinical use, is a low nanomolar inhibitor of
hCA II, and also significantly inhibits hCA I (KI of 250 nM), Table 1.
The fact that the new sulfocoumarins 5a–5j and 7a, 7b reported
here are not inhibitors of hCA I and II is not surprising, considering
the fact that the structurally related triazole-substituted sul-
focoumarins B reported earlier,1a showed the same behavior.
(ii) The transmembrane, tumor-associated hCA IX was
effectively inhibited by the new sulfocoumarins 5a–5j and 7a, 7b
reported here, with inhibition constants ranging between 6.5 and
68.6 nM. The least effective compounds in the series were the
isoforms hCA I and II (KIs >10 lM) but showed effective inhibition
against the two transmembrane CAs, with KIs ranging from 6.5 to
68.6 nM against hCA IX, and between 4.3 and 59.8 nM against
hCA XII. As hCA IX and XII are validated anti-tumor targets, such
prodrug, isoform-selective inhibitors as the sulfocoumarins re-
ported here, may be useful for identifying suitable drug candidates
for clinical trials.
Table 1
hCA I, II, IX and XII inhibition data with sulfocoumarins 5 and 7, by a stopped-flow
CO2 hydrase assay25
Compound
KI (nM)*
hCA I
hCA II
hCA IX
hCA XII
7a
7b
5a
5b
5c
5d
5e
5f
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
250
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
12
68.6
65.9
8.5
9.1
27.1
8.7
39.3
56.8
8.8
6.9
8.2
6.4
8.8
7.1
6.1
59.8
6.3
8.1
7.3
4.3
5.4
13.5
5.7
5.7
4. Experimental
4.1. Chemistry
Reagents and starting materials were obtained from commer-
cial sources (Sigma–Aldrich) and used as received. Compound 1
was sythesized according literature procedure.18 The solvents were
purified and dried by standard procedures prior to use; petroleum
ether of boiling range 40–60 °C was used. Flash chromatography
was carried out using Merck silica gel (230–400 mesh). Thin-layer
chromatography was performed on silica gel, spots were visualized
with UV light (254 and 365 nM). Melting points were determined
5g
5h
5i
5j
6.5
25
AAZ*
Acetazolamide (AAZ) was used as a standard inhibitor.
*
Mean from 3 different assays, errors in the range of 10% of the reported values
(data not shown).

A. Grandane et al. / Bioorg. Med. Chem. 22 (2014) 1522–1528
1525
on an OptiMelt automated melting point system. IR spectra were
4.2.1. 5-(2,2-Dioxido-1,2-benzoxathiin-6-yl)-1-phenyl-1H-
tetrazole 5a
measured on a Shimadzu FTIR IR Prestige-21 spectrometer. NMR
spectra were recorded on Varian Mercury (400 MHz) spectrometer
with chemical shifts values (d) in ppm relative to TMS using the
residual DMSO-d6 signal as an internal standard. Elemental
analyses were performed on a Carlo Erba CHNSeO EA-1108
apparatus.
N
N
N
N
O
S
O
O
4.1.1. 2-Formyl-4-iodophenyl methanesulfonate 218
Obtained from 1-phenyl-1,2,3,4-tetrazole 4a19 (0.190 g,
I
1.30 mmol) as yellow crystalline solid (0.076 g, 36%). Mp 189–
O
190 °C. IR (KBr, cmÀ1 max: 1370 (S@O), 1178 (S@O). 1H NMR
) m
O
(400 MHz, DMSO-d6) d: 7.49–7.56 (m, 2H), 7.58–7.68 (m, 6H), 7.78
(d, 1H, J = 10.4 Hz), 8.09–8.12 (m, 1H). 13C NMR (100 MHz, DMSO-
d6) d: 119.2, 119.3, 121.6, 123.6, 126.0, 130.0, 130.8, 131.0, 132.5,
133.8, 135.9, 152.2, 152.4. Anal. Calcd for C15H10N4O3S (326.33): C,
55.21; H, 3.09; N, 17.17. Found: C, 55.25; H, 3.09; N, 17.08.
O
S O
To
a solution of 2-hydroxy-5-iodobenzaldehyde 1 (3.32 g,
17.4 mmol) in dry CH2Cl2 (30 mL) triethylamine (3.63 mL,
26.1 mmol) was added. The mixture was cooled to 0 °C and mesyl
chloride (2.17 mL, 28.0 mmol) was added. The reaction was stir-
red at room temperature for 4 h. The mixture was diluted with
H2O (150 mL) and extracted with EtOAc (3 Â 80 mL). The organic
phase was washed with brine and dried over Na2SO4, and the sol-
vent was evaporated to yield a yellow oil (5.61 g, 99%). IR (neat,
4.2.2. 5-(2,2-Dioxido-1,2-benzoxathiin-6-yl)-1-(4-
methylphenyl)-1H-tetrazole 5b
N
N
N
N
O
S
O
O
cmÀ1
) mmax: 1694 (C@O),1369 (S@O), 1356 (S@O), 1183 (S@O),
1165 (S@O). 1H NMR (400 MHz, DMSO-d6) d: 3.59 (s, 3H), 7.35–
7.38 (m, 1H), 8.13–8.17 (m, 2H), 10.12 (s, 1H). 13C NMR
(100 MHz, DMSO-d6) d: 37.8, 93.3, 126.1, 130.7, 137.4, 144.2,
149.3, 187.6. HRMS (ESI) [M+Na]+: m/z Calcd for (C8H7INaO4S)
348.9007. Found 348.9015.
Obtained from 1-(p-tolyl)-1,2,3,4-tetrazole 4b19,20 (0.208 g,
1.30 mmol) as light brown solid (0.073 g, 33%). Mp 186–187 °C.
IR (neat, cmÀ1 max: 1372 (S@O), 1177 (S@O), 1143 (S@O). 1H
) m
NMR (400 MHz, DMSO-d6) d: 2.40 (s, 3H), 7.38–7.43 (m, 2H),
7.46–7.49 (m, 2H), 7.50–7.56 (m, 2H), 7.65 (d, 1H, J = 10.4 Hz),
7.79 (d, 1H, J = 10.4 Hz), 8.12 (d, 1H, J = 1.9 Hz). 13C NMR
(100 MHz, DMSO-d6) d: 20.8, 119.2, 119.3, 121.7, 123.6, 125.7,
130.4, 131.0, 131.3, 132.4, 135.9, 140.7, 152.1, 152.3. HRMS (ESI)
[M+H]+: m/z Calcd for (C16H13N4O3S) 341.0708. Found 341.0717.
4.1.2. 6-Iodo-1,2-benzoxathiine 2,2-dioxide 3
I
O
S
O
O
A solution of the above formyl-iodophenyl-mesylate 2 (3.00 g,
9.20 mmol) in dry CH2Cl2 (30 mL) was cooled to 0 °C and DBU
(1.38 mL, 9.20 mmol) was added. The reaction was stirred at 0 °C
for 2 h under argon atmosphere and poured into a mixture of ice
and 10% HCl and extracted with Et2O (3 Â 100 mL). The organic
phase was dried over Na2SO4, and the solvent was evaporated.
The crude mixture was dissolved in pyridine (30 mL), cooled to
0 °C and POCl3 (1.72 mL, 18.4 mmol) was added. The mixture
was stirred at room temperature for 3 h then poured into a mixture
of ice and water. The resulting precipitate was filtered off yielding
4.2.3. 5-(2,2-Dioxido-1,2-benzoxathiin-6-yl)-1-(3-
methylphenyl)-1H-tetrazole 5c
N
N
N
N
O
S
O
O
Obtained from tetrazole 4c20 (0.208 g, 1.30 mmol) as yellow crys-
talline solid (0.126 g, 57%). Mp 153–154 °C. IR (neat, cmÀ1
max: 1372
a yellow solid (2.00 g, 71%). Mp 140–141 °C. IR (neat, cmÀ1
) mmax:
)
m
(S@O), 1350 (S@O), 1179 (S@O), 1143 (S@O). 1H NMR (400 MHz,
DMSO-d6) d: 2.37 (s, 3H), 7.33–7.37 (m, 1H), 7.44–7.49 (m, 3H),
7.50–7.56 (m, 2H), 7.65 (d, 1H, J = 10.4 Hz), 7.79 (d, 1H, J = 10.4 Hz),
8.12–8.14 (m, 1H). 13C NMR (100 MHz, DMSO-d6) d: 20.7, 119.2,
119.3, 121.6, 123.0, 123.6, 126.3, 129.7, 131.0, 131.5, 132.4, 133.7,
135.9, 139.9, 152.2, 152.3. Anal. Calcd for C16H12N4O3S (340.36): C,
56.46; H, 3.55; N, 16.46. Found: C, 56.65; H, 3.53; N, 16.39.
1369 (S@O), 1171 (S@O). 1H NMR (400 MHz, DMSO-d6) d: 7.27
(d, 1H, J = 8.7 Hz), 7.58 (d, 1H, J = 10.4 Hz), 7.65 (d, 1H,
J = 10.4 Hz), 7.91 (dd, 1H, J = 8.7, 2.3 Hz), 8.13 (d, 1H, J = 2.3 Hz).
13C NMR (100 MHz, DMSO-d6) d: 90.5, 120.7, 120.9, 123.5, 135.4,
138.0, 140.7, 150.5. Anal. Calcd for C8H5IO3S (308.09): C, 31.19;
H, 1.64. Found: C, 31.46; H, 1.69.
4.2. General procedure for the synthesis of tetrazoles 5 via C–H
activation
4.2.4. 5-(2,2-Dioxido-1,2-benzoxathiin-6-yl)-1-(4-
methoxyphenyl)-1H-tetrazole 5d
N
N
Compound 3 (0.200 g, 0.649 mmol), the appropriate tetrazole
(2 equiv, 1.30 mmol), Cs2CO3 (0.233 g, 0.714 mmol), CuI (0.124 g,
0.649 mmol), Pd(OAc)2 (0.0146 g, 0.0649 mmol) and tri(2-furyl)
phosphine (0.030 g, 0.130 mmol)were suspended in dry toluene
(3 mL). The mixture was stirred 40 °C under argon for 20 h, then
EtOAc (20 mL) was added and the mixture was filtered through
celite. Celite was washed with EtOAc (50 mL). The filtrate was
evaporated. The mixture was purified by silica gel chromatography
(petroleum ether/EtOAc 2:1) an recrystallized from EtOH.
N
N
O
S
O
O
O
Obtained from tetrazole 4d19,21 (0.229 g, 1.30 mmol) as white
crystalline solid (0.055 g, 24%). Mp 175–176 °C. IR (KBr, cmÀ1
)

1526
A. Grandane et al. / Bioorg. Med. Chem. 22 (2014) 1522–1528
m
max: 1370 (S@O), 1172 (S@O). 1H NMR (400 MHz, DMSO-d6) d:
152.2, 152.5. Anal. Calcd for C15H9ClN4O3S (360.77): C, 49.94; H,
2.51; N, 15.53. Found: C, 49.80; H, 2.38; N, 15.65.
3.84 (s, 3H), 7.11–7.16 (m, 2H), 7.51–5.57 (m, 4H), 7.65 (d, 1H,
J = 10.4 Hz), 7.80 (d, 1H, J = 10.4 Hz), 8.11 (d, 1H, J = 1.9 Hz). 13C
NMR (100 MHz, DMSO-d6) d: 55.6, 115.0, 119.2, 119.3, 121.8,
123.6, 126.4, 127.5, 130.9, 132.4, 135.9, 152.1, 152.4, 160.6. Anal.
Calcd for C16H12N4O4S (356.36): C, 53.93; H, 3.39; N, 15.72. Found:
C, 53.78; H, 3.18; N, 15.83.
4.2.8. 1-(3-Chloro-4-fluorophenyl)-5-(2,2-dioxido-1,2-
benzoxathiin-6-yl)-1H-tetrazole 5h
N
N
N
N
O
S
4.2.5. 5-(2,2-Dioxido-1,2-benzoxathiin-6-yl)-1-[4-
(trifluoromethyl)phenyl]-1H-tetrazole 5e
O
O
Cl
F
N
N
N
N
O
Obtained from tetrazole 4h22 (0.258 g, 1.30 mmol) as white
crystalline solid (0.118 g, 48%). Mp 174–175 °C. IR (KBr, cmÀ1
max: 1368 (S@O), 1178 (S@O). 1H NMR (400 MHz, DMSO-d6) d:
S
O
O
)
m
F
F
F
7.56 (d, 1H, J = 8.7 Hz), 7.60 (dd, 1H, J = 8.7, 2.1 Hz), 7.66 (d, 1H,
J = 10.4 Hz), 7.67–7.70 (m, 2H), 7.83 (d, 1H, J = 10.4 Hz), 8.05–8.10
(m, 2H). 13C NMR (100 MHz, DMSO-d6) d: 118.3 (d, J = 23.0 Hz),
119.3, 119.4, 120.9 (d, J = 19.2 Hz), 121.3, 123.6, 127.4 (d,
J = 8.6 Hz), 128.8, 130.7 (d, J = 3.7 Hz), 131.0, 132.6, 135.9, 152.3,
Obtained from tetrazole 4e21 (0.278 g, 1.30 mmol) as yellow
crystalline solid (0.119 g, 46%). Mp 184–185 °C. IR (neat, cmÀ1
max: 1371 (S@O), 1325 (S@O), 1177 (S@O), 1130 (S@O). 1H NMR
)
m
152.6, 158.4 (d, J = 251.7 Hz). Anal. Calcd for
C15H8ClFN4O3S
(400 MHz, DMSO-d6) d: 7.56–7.58 (m, 2H), 7.66 (d, 1H,
J = 10.4 Hz), 7.82 (d, 1H, J = 10.4 Hz), 7.83–7.88 (m, 2H), 7.99–
8.04 (m, 2H), 8.11–8.13 (m, 1H). 13C NMR (100 MHz, DMSO-d6) d:
119.4, 121.4, 123.5 (q, J = 273.0 Hz), 123.6, 126.7, 127.2 (q,
J = 3.6 Hz), 130.7 (q, J = 32.8), 131.2, 132.8, 135.9, 137.0, 152.3,
152.5. Anal. Calcd for C16H9F3N4O3S (394.33): C, 48.73; H, 2.30;
N, 14.21. Found: C, 48.66; H, 2.38; N, 13.98.
(378.77): C, 47.57; H, 2.13; N, 14.79. Found: C, 47.56; H, 2.06; N,
14.59.
4.2.9. 1-(3,4-Dichlorophenyl)-5-(2,2-dioxido-1,2-benzoxathiin-
6-yl)-1H-tetrazole 5i
N
N
N
N
O
4.2.6. 1-(3-Bromophenyl)-5-(2,2-dioxido-1,2-benzoxathiin-6-
yl)-1H-tetrazole 5f
S
O
O
Cl
N
N
Cl
N
N
O
S
Obtained from tetrazole 4i23 (0.280 g, 1.30 mmol) as light yel-
low crystalline solid (0.119 g, 46%). Mp 199–200 °C. IR (KBr,
O
O
Br
cmÀ1 max: 1372 (S@O), 1178 (S@O), 1124 (S@O). 1H NMR
) m
(400 MHz, DMSO-d6) d: 7.55–7.64 (m, 3H), 7.66 (d, 1H,
J = 10.4 Hz), 7.84 (d, 1H, J = 10.4 Hz), 7.90 (d, 1H, J = 8.6 Hz), 8.08
(d, 1H, J = 2.4 Hz), 8.11–8.13 (m, 1H). 13C NMR (100 MHz, DMSO-
d6) d: 119.3, 119.4, 121.3, 123.6, 126.3, 128.1, 131.1, 131.9, 132.4,
132.7, 133.4, 133.9, 135.9, 152.3, 152.6. Anal. Calcd for C15H8Cl2N4-
O3S (395.22): C, 45.58; H, 2.04; N, 14.18. Found: C, 45.46; H, 1.98;
N, 14.12.
Obtained from tetrazole 4f21 (0.293 g, 1.30 mmol) as light yel-
low crystalline solid (0.171 g, 65%). Mp 197–198 °C. IR (KBr,
cmÀ1 max: 1371 (S@O), 1145 (S@O). 1H NMR (400 MHz, DMSO-
) m
d6) d: 7.53–7.59 (m, 3H), 7.60–7.64 (m, 1H), 7.66 (d, 1H,
J = 10.4 Hz), 7.82 (d, 1H, J = 10.4 Hz), 7.84–7.88 (m, 1H), 7.96 (t,
1H, J = 2.0 Hz), 8.11–8.13 (m, 1H). 13C NMR (100 MHz, DMSO-d6)
d: 119.3, 119.4, 121.4, 122.2, 123.6, 125.2, 128.8, 131.1, 131.8,
132.6, 133.8, 135.0, 135.9, 152.2, 152.5. Anal. Calcd for C15H9BrN4-
O3S (405.23): C, 44.46; H, 2.24; N, 13.83. Found: C, 44.49; H, 2.24;
N, 13.73.
4.2.10. 5-(2,2-Dioxido-1,2-benzoxathiin-6-yl)-1-(naphthalen-1-
yl)-1H-tetrazole 5j
N
N
N
4.2.7. 1-(4-Chlorophenyl)-5-(2,2-dioxido-1,2-benzoxathiin-6-
yl)-1H-tetrazole 5g
N
O
S
O
O
N
N
N
N
O
Obtained from tetrazole 4j22 (0.255 g, 1.30 mmol) as light pink
crystalline solid (0.255 g, 43%). Mp 165–166 °C. IR (neat, cmÀ1
max: 1371 (S@O), 1178 (S@O). 1H NMR (400 MHz, DMSO-d6) d:
S
O
O
)
m
Cl
7.23 (d, 1H, J = 8.6 Hz), 7.31 (dd, 1H, J = 8.6, 2.1 Hz), 7.39 (d, 1H,
8.6 Hz), 7.56–7.63 (m, 2H), 7.64–7.70 (m, 1H), 7.71–7.77 (m, 2H),
7.92–7.96 (m, 1H), 8.14–8.20 (m, 2H), 8.30 (d, 1H, J = 8.6 Hz). 13C
NMR (100 MHz, DMSO-d6) d: 119.3, 119.4, 121.2, 121.3, 123.7,
125.7, 126.4, 127.6, 128.2, 128.7, 128.8, 129.7, 130.6, 131.4,
131.8, 133.7, 135.8, 152.2, 153.6. Anal. Calcd for C19H12N4O3S
(376.39): C, 60.63; H, 3.21; N, 14.89. Found: C, 60.49; H, 3.04; N,
14.85.
Obtained from tetrazole 4g19,20 (0.235 g, 1.30 mmol) as white
crystalline solid (0.096 g, 41%). Mp 193.5–194.5 °C. IR (KBr, cmÀ1
max: 1373 (S@O), 1184 (S@O). 1H NMR (400 MHz, DMSO-d6) d:
7.55–7.57 (m, 2H), 7.63–7.67 (m, 3H), 7.68–7.72 (m, 2H), 7.82 (d,
1H, J = 10.4 Hz), 8.09–8.11 (m, 1H). 13C NMR (100 MHz, DMSO-
d6) d: 119.3, 121.5, 123.6, 127.8, 130.1, 131.1, 132.6, 135.4, 135.9,
)
m

A. Grandane et al. / Bioorg. Med. Chem. 22 (2014) 1522–1528
1527
4.3. General procedure for the synthesis of tetrazoles 7
were obtained after 6 h incubation of enzyme and inhibitor, as
for the sulfocoumarins and coumarins reported earlier.1,4 The inhi-
bition constants were obtained by non-linear least-squares meth-
ods using PRISM 3, as reported earlier,2 and represent the mean
from at least three different determinations. All CA isofoms were
recombinant ones obtained in-house as reported earlier.1,4–8
To the appropriate aminoderivative 617 (1.0 equiv), NaN3
(1,2 equiv) and triethyl orthoformate or triethyl orthoacetate
(1.5 equiv) AcOH (3 mL per g amine) was added. The mixture
was stirred 100 °C for 4 h, then cooled to room temperature and
poured into ice-water. The precipitate was filtered off and dried
under vacuum.
Acknowledgments
4.3.1. 1-(2,2-Dioxido-1,2-benzoxathiin-6-yl)-1H-tetrazole 7a
This work was financed by two FP7 EU Grants (METOXIA and
DYNANO).
N
N
N
N
O
S
References and notes
O
O
1. (a) Tars, K.; Vullo, D.; Kazaks, A.; Leitans, J.; Lends, A.; Grandane, A.;
Zalubovskis, R.; Scozzafava, A.; Supuran, C. T. J. Med. Chem. 2013, 56, 293; (b)
Tanc, M.; Carta, F.; Bozdag, M.; Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem.
2013, 21, 4502.
2. (a) Alterio, V.; Di Fiore, A.; D’Ambrosio, K.; Supuran, C. T.; De Simone, G. Chem.
Rev. 2012, 112, 4421; (b) Supuran, C. T. Nat. Rev. Drug Disc. 2008, 7, 168; (c)
Neri, D.; Supuran, C. T. Nat. Rev. Drug Disc. 2011, 10, 767; (d) Aggarwal, M.;
Kondeti, B.; McKenna, R. Bioorg. Med. Chem. 2013, 21, 1526.
3. (a) Aggarwal, M.; McKenna, R. Expert Opin. Ther. Pat. 2012, 22, 903; (b) Supuran,
C. T. J. Enzyme Inhib. Med. Chem. 2012, 27, 759; (c) Supuran, C. T.; Scozzafava, A.;
Casini, A. Med. Res. Rev. 2003, 23, 146; (d) Pastorekova, S.; Parkkila, S.; Pastorek,
J.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2004, 19, 199.
4. (a) Maresca, A.; Temperini, C.; Vu, H.; Pham, N. B.; Poulsen, S. A.; Scozzafava, A.;
Quinn, R. J.; Supuran, C. T. J. Am. Chem. Soc. 2009, 131, 3057; (b) Maresca, A.;
Temperini, C.; Pochet, L.; Masereel, B.; Scozzafava, A.; Supuran, C. T. J. Med.
Chem. 2010, 53, 335.
Obtained from compound 617 (2.31 g, 11.7 mmol), NaN3
(0.914 g; 14.1 mmol) and triethyl orthoformate (2.92 mL, 17.6
mmol). The crude was recrystallized from EtOH to yield 7a as yellow
crystalline solid (2.11 g, 72%). Mp 173.5–174.5 °C. IR (KBr, cmÀ1
)
m
max: 1369 (S@O), 1358 (S@O), 1171 (S@O), 1148 (S@O). 1H NMR
(400 MHz, DMSO-d6) d: 7.72 (d, 1H, J = 10.4 Hz), 7.78 (d, 1H,
J = 9.0 Hz), 7.83 (d, 1H, J = 10.4 Hz), 8.13 (dd, 1H, J = 9.0, 2.6 Hz),
8.35 (d, 1H, J = 2.6 Hz), 10.11 (s, 1H). 13C NMR (100 MHz, DMSO-
d6) d: 119.9, 120.3, 122.7, 124.2, 125.1, 131.4, 135.6, 142.6, 150.7.
HRMS (ESI) [M+H]+: m/z Calcd for (C9H7N4O3S) 251.0239. Found
251.0247.
5. (a) Maresca, A.; Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2010, 20,
7255; (b) Touisni, N.; Maresca, A.; McDonald, P. C.; Lou, Y.; Scozzafava, A.;
Dedhar, S.; Winum, J. Y.; Supuran, C. T. J. Med. Chem. 2011, 54, 8271; (c)
Bonneau, A.; Maresca, A.; Winum, J. Y.; Supuran, C. T. J. Enzyme Inhib. Med.
Chem. 2013, 28, 397.
6. Carta, F.; Maresca, A.; Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. Lett.
2012, 22, 267.
7. Carta, F.; Vullo, D.; Maresca, A.; Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem.
Lett. 2012, 22, 2182.
4.3.2. 1-(2,2-Dioxido-1,2-benzoxathiin-6-yl)-1H-tetrazole 7b
N
N
N
N
O
S
O
O
8. (a) Carta, F.; Maresca, A.; Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. 2012,
20, 2266; (b) Davis, R. A.; Vullo, D.; Maresca, A.; Supuran, C. T.; Poulsen, S. A.
Bioorg. Med. Chem. 2013, 21, 1539.
Obtained from 617 (0.400 g, 2.03 mmol), NaN3 (0.159 g;
2.44 mmol) and triethyl orthoacetate (0.56 mL, 3.05 mmol). The
crude was recrystallized from EtOH to yield 7b as yellow crystalline
9. (a) Supuran, C. T.; Scozzafava, A.; Casini, A. Development of Sulfonamide
Carbonic Anhydrase Inhibitors (CAIs). In Carbonic Anhydrase – Its Inhibitors and
Activators; Supuran, C. T., Scozzafava, A., Conway, J., Eds.; CRC Press: Boca Raton
(FL), 2004; p 67; (b) Supuran, C. T. Bioorg. Med. Chem. Lett. 2010, 20, 3467; (c)
Supuran, C. T. Future Med. Chem. 2011, 3, 1165; (d) Aggarwal, M.; Boone, C. D.;
Kondeti, B.; McKenna, R. J. Enzyme Inhib. Med. Chem. 2013, 28, 267; (e) Dubois,
L.; Lieuwes, N. G.; Maresca, A.; Thiry, A.; Supuran, C. T.; Scozzafava, A.; Wouters,
B. G.; Lambin, P. Radiother. Oncol. 2009, 92, 423; (f) Wilkinson, B. L.; Bornaghi, L.
F.; Houston, T. A.; Innocenti, A.; Supuran, C. T.; Poulsen, S. A. J. Med. Chem. 2006,
49, 6539.
10. (a) Svastova, E.; Hulíkova, A.; Rafajova, M.; Zatovicova, M.; Gibadulinova, A.;
Casini, A.; Cecchi, A.; Scozzafava, A.; Supuran, C. T.; Pastorek, J.; Pastorekova, S.
FEBS Lett. 2004, 577, 439; (b) Thiry, A.; Dogné, J. M.; Masereel, B.; Supuran, C. T.
Trends Pharmacol. Sci. 2006, 27, 566; (c) Alterio, V.; Hilvo, M.; Di Fiore, A.;
Supuran, C. T.; Pan, P.; Parkkila, S.; Scaloni, A.; Pastorek, J.; Pastorekova, S.;
Pedone, C.; Scozzafava, A.; Monti, S. M.; De Simone, G. Proc. Natl. Acad. Sci. U.S.A.
2009, 106, 16233; (d) Ahlskog, J. K.; Schliemann, C.; Mårlind, J.; Qureshi, U.;
Ammar, A.; Pedleym, R. B.; Neri, D. Bioorg. Med. Chem. Lett. 2009, 19, 4851.
11. (a) Stander, B. A.; Joubert, F.; Tu, C.; Sippel, K. H.; McKenna, R.; Joubert, A. M.
PLoS One 2013, 8, e53853; (b) Pacchiano, F.; Carta, F.; McDonald, P. C.; Lou, Y.;
Vullo, D.; Scozzafava, A.; Dedhar, S.; Supuran, C. T. J. Med. Chem. 2011, 54, 1896;
(c) Lou, Y.; McDonald, P. C.; Oloumi, A.; Chia, S. K.; Ostlund, C.; Ahmadi, A.;
Kyle, A.; Auf dem Keller, U.; Leung, S.; Huntsman, D. G.; Clarke, B.; Sutherland,
B. W.; Waterhouse, D.; Bally, M. B.; Roskelley, C. D.; Overall, C. M.; Minchinton,
A.; Pacchiano, F.; Carta, F.; Scozzafava, A.; Touisni, N.; Winum, J. Y.; Supuran, C.
T.; Dedhar, S. Cancer Res. 2011, 71, 3364; (d) Pacchiano, F.; Aggarwal, M.;
Avvaru, B. S.; Robbins, A. H.; Scozzafava, A.; McKenna, R.; Supuran, C. T. Chem.
Commun. 2010, 8371.
solid (0.239 g, 45%). Mp 209.5–210.5 °C. IR (KBr, cmÀ1
) mmax: 1363
(S@O), 1175 (S@O). 1H NMR (400 MHz, DMSO-d6) d: 2.60 (s, 3H),
7.72 (d, 1H, J = 10.4 Hz), 7.76 (d, 1H, J = 8.7 Hz), 7.80 (d, 1H,
J = 10.4 Hz), 7.95 (dd, 1H, J = 8.7, 2.6 Hz), 8.13 (d, 1H, J = 2.6 Hz). 13
C
NMR (100 MHz, DMSO-d6) d: 9.3, 119.8, 120.2, 124.0, 126.4, 128.7,
131.2, 135.6, 151.2, 152.5. Anal. Calcd for C10H8N4O3S (264.26): C,
45.45; H, 3.05; N, 21.20. Found: C, 45.40; H, 2.99; N, 20.94.
4.4. CA inhibition
An applied photophysics stopped-flow instrument has been
used for assaying the CA catalysed CO2 hydration activity.25 Phenol
red (at a concentration of 0.2 mM) has been used as indicator,
working at the absorbance maximum of 557 nm, with 20 mM
Hepes (pH 7.5) as buffer, and 20 mM Na2SO4 (for maintaining con-
stant the ionic strength), following the initial rates of the CA-cata-
lyzed CO2 hydration reaction for a period of 10–100 s. The CO2
concentrations ranged from 1.7 to 17 mM for the determination
of the kinetic parameters and inhibition constants. For each inhib-
itor at least six traces of the initial 5–10% of the reaction have been
used for determining the initial velocity. The uncatalyzed rates
were determined in the same manner and subtracted from the
total observed rates. Stock solutions of inhibitor (0.1 mM) were
prepared in distilled–deionized water and dilutions up to
0.01 nM were done thereafter with the assay buffer. Inhibitor
and enzyme solutions were preincubated together for 15 min–6 h
at room temperature (15 min) or 4 °C (6 h) prior to assay, in order
to allow for the formation of the E–I complex. Data from Table 1
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T.; Onishi, S.; Takeuchi, H.; Vullo, D.; Scozzafava, A.; Supuran, C. T. Bioorg. Med.
Chem. Lett. 2007, 17, 3585.
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Med. Chem. 2013, 21, 1377; (c) Briganti, F.; Pierattelli, R.; Scozzafava, A.;
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