Carbonic anhydrase inhibitors. Inhibition of the Rv1284 and Rv3273 β-carbonic anhydrases from Mycobacterium tuberculosis with diazenylbenzenesulfonamides

Article abstract of DOI:10.1016/j.bmcl.2009.07.088

A series of diazenylbenzenesulfonamides obtained from sulfanilamide or metanilamide by diazotization followed by coupling with phenols or amines, was tested for the inhibition of the β-carbonic anhydrases (CAs, EC 4.2.1.1) encoded by the genes Rv1284 and

Full text of DOI:10.1016/j.bmcl.2009.07.088

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4929–4932
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Bioorganic & Medicinal Chemistry Letters
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Carbonic anhydrase inhibitors. Inhibition of the Rv1284 and Rv3273
b-carbonic anhydrases from Mycobacterium tuberculosis with
diazenylbenzenesulfonamides
*
Alfonso Maresca, Fabrizio Carta, Daniela Vullo, Andrea Scozzafava, Claudiu T. Supuran
Università degli Studi di Firenze, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, I-50019 Sesto Fiorentino (Firenze), 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 diazenylbenzenesulfonamides obtained from sulfanilamide or metanilamide by diazotization
followed by coupling with phenols or amines, was tested for the inhibition of the b-carbonic anhydrases
(CAs, EC 4.2.1.1) encoded by the genes Rv1284 and Rv3273 of Mycobacterium tuberculosis. Several low
micromolar inhibitors of the two enzymes were detected, with prontosil being the best inhibitor (KIs
of 126–148 nM). Inhibition of pathogenic b-CAs may lead to the development of antiinfectives with a
new mechanism of action, devoid of resistance problems encountered with classical antibiotics.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 24 June 2009
Revised 13 July 2009
Accepted 16 July 2009
Available online 22 July 2009
Keywords:
Carbonic anhydrase
Mycobacterium tuberculosis
Rv1284
Rv3273
Sulfonamide
Sulfonate
Prontosil
Mycobacterium tuberculosis and related Mycobacteria infections
(e.g., Mycobacterium avium) affect a large number of the world pop-
ulation, with an estimated 9.2 million new cases each year, many
of which leading to deaths.1–4 Multi-drug resistant and extensively
multi-drug resistant tuberculosis (TB) worsens even more the sit-
uation, as such strains are now present in more than 50 countries,
posing serious concern to the global healthcare system, as this dis-
ease is largely unresponsible to the presently available drugs.1 In-
deed, the combination therapy used to treat TB is based on agents
developed in the 60–80s, with no new drugs launched for the last
30 years.2–4 There is thus a huge interest for novel anti-TB drugs,
possessing alternative mechanisms of action compared to the clin-
ically used antibiotics. The complete sequencing of M. tuberculosis
genome some years ago5 greatly facilitated the identification of
possible new drug targets which may lead to the development of
compounds possessing a new mechanism of action, and thus re-
solve the drug resistance problem mentioned above. Among the
new such proteins identified after the M. tuberculosis genome
was published, there are also several carbonic anhydrases (CAs,
EC 4.2.1.1) belonging to the b-class.6–9 M. tuberculosis contains
three b-CA genes in its genome, that is, Rv1284 (encoding for a pro-
tein we named mtCA 1), Rv3588c (encoding for mtCA 2), and
Rv3273 (encoding for a third enzyme, mtCA 3).6,7 The catalytic
activity and inhibition studies with a range of sulfonamides and
one sulfamate of two these enzymes, that is, mtCA 1 and mtCA 3
have been recently reported,7–9 whereas Covarrubias et al. re-
ported the X-ray crystal structure of mtCA 1 and mtCA2.6 CAs
belonging to the b-class10 are found in many pathogenic organisms
such as fungi (Candidaalbicans, C. glabrata, and Cryptococcus neofor-
mans among others)10–12 and bacteria (Helicobacter pylori, Arthro-
bacter aurescens, Leptospira borgpetersenii, Legionella pneumophila,
and Haemophilus influenzae)13,14 but they lack from mammals, in
which only
a-CAs (under the form of 16 different isoforms) are
present.10 Thus, inhibition of such b-CAs started to be considered
as a new possible approach for designing antiinfectives (antifungal
or antibacterial agents) possessing a different mechanism of action
compared to the classical pharmacological agents in clinical use for
a long period, for which pathogenic fungi and bacteria developed
various degrees of resistance.15,16 The drug resistance problem
with presently used antifungals and antibiotics represents a seri-
ous medical problem worldwide.17
In previous work from our group7–9 we have investigated the
inhibition of mtCA 1 and mtCA 3 with the classical sulfonamides/
sulfamates in clinical use as well as with simple, aromatic deriva-
tives used as building blocks in the design of CA inhibitors (CAIs)
targeting the mammalian,
a-class enzymes (of which at least 16
isoforms are presently known and well characterized).10,18 Several
of the clinically used sulfonamides, such as acetazolamide AAZ,
methazolamide MZA, dorzolamide DZA or brinzolamide BRZ
* Corresponding author. Tel.: +39 055 457 3005; fax: +39 055 4573385.
E-mail address: claudiu.supuran@unifi.it (C.T. Supuran).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.07.088

4930
A. Maresca et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4929–4932
H3C
constants of 170 nM–2.21 lM. We have thus decided to explore
N
N
N
N
whether compounds with an elongated molecule may have better
mycobacterial CA inhibitory activity, and the diazenylsulfonamides
appeared to us as interesting candidates, also because we have re-
cently investigated two such compounds (4a and 4b) as human (h)
CA II inhibitors, observing that their hCA II inhibitory activity is not
very good21 (which may be a positive feature for a compound that
should target the pathogenic, bacterial b-CAs and not the mamma-
CH3CONH
SO2NH2
S
CH3CON
SO2NH2
S
AAZ
MZA
HN
S
HN
SO2NH2
SO2NH2
N
S
lian, a-class enzymes). It should be noted that azo-derivatives
S
H3C
CH3O(CH2)3
S
O
O
O
O
incorporating sulfonamide moieties are highly important from
the drug design point of view, since the antibacterial sulfa drugs
still widely used in therapy22a were discovered considering pronto-
sil E as a lead molecule. Indeed, prontosil was the first chemother-
apeutic agent to show effective antibacterial effects in animals and
humans with bacterial infections untreatable before the advent of
this drug, being subsequently shown that the active metabolite of
E is sulfanilamide 1.22b–d Although sulfones such as dapsone F and
its methylsulfinate sodium salt sulfoxone sodium G do not possess
a sulfonamide moiety, their mechanism of antibacterial activity is
similar to that of the antibacterial sulfonamides (such as E, 1, etc.),
that is, these clinically used antileprosy agents23 (targeting Myco-
bacterium leprae, a pathogen related to M. tuberculosis) block the
bacterial folate biosynthesis by antagonizing 4-amino-benzoic acid
reaction with pteridine, and acting thus as dihydropteroate syn-
thase inhibitors.22,23 It should be noted that G incorporates al-
kane-sulfinic acid moieties in its molecule, which may be zinc
binders in metalloenzymes such as CAs.18 Thus, we have used com-
pounds A–G as lead molecules in the drug design of antimyobacte-
rial CAs mtCA 1 and mtCA 3 reported here.
We prepared a series of para- and meta-substituted diazenyl-
benzenesulfonamides 4 and 5, starting from sulfanilamide 1 or
metanilamide 2, which have been converted to the corresponding
diazonium salts 3 by treatment with sodium nitrite and hydrochlo-
ric acid at low temperatures (Scheme 1). The diazonium salts 3
were then coupled with phenols or amines, leading to the target
compounds 4 and 5 (Scheme 1), which are obviously similar to
prontosil E as lead molecule.24 The R moieties present in com-
pounds 4 and 5 have been chosen in such a way as to provide
structure–activity relationship (SAR) insights regarding the inhibi-
DZA
BRZ
showed interesting inhibitory activity against mtCA 1 and mtCA 3,
with inhibition constants in the submicromolar ranges (KIs of 481–
839 nM against mtCA 18 and of 104–562 nM against mtCA 37), but
all these compounds were much more potent inhibitors of the host,
human isoforms (such as the ubiquitous CA II).10
Screening analysis for genes specifically required for the myco-
bacterial growth recently showed that some genes encoding b-CAs
(e.g., Rv3588c) are required whereas others (e.g., Rv3273) are not
essential for the bacterial growth in vivo.19 However, such findings
cannot conclusively exclude the biological significance of these en-
zymes in the survival and/or pathogenicity of M. tuberculosis. For
example, an elegant study by Miltner et al.20 identified six inva-
sion-related genes in M. avium. Constitutive expression of proteins
encoded by these genes, among which an orthologue of Rv3273,
showed significantly increased ability of bacterial invasion into
HEp-2 and HT-29 intestinal epithelial cells. These findings indicate
that the mycobacterial CAs plays a crucial role in the infection pro-
cess, which is however poorly understood at the present time. Con-
sidering the small number of inhibitors investigated to date for
their interaction with the mycobacterial CAs,7–9 and the fact that
potent such inhibitors may be relevant for developing novel drugs,
we decided to investigate other classes of sulfonamides as possible
mtCA 1 and mtCA 3 inhibitors. In this Letter we report inhibition
studies of these enzymes with a series of diazenylbenzenesulfona-
mides and two simple sulfonates. Actually sulfonates were not
investigated up to now for their interaction with b-CAs.
SO2NH2
SO2NH2
O
H
H2N
S
N
(
)n
SO2NH2
O
C: n = 1, Ki(mtCA 1)=612 nM; Ki(mtCA 3)=2210 nM
D: n = 2, Ki(mtCA 1)=853 nM; Ki(mtCA 3)=170 nM
H2N
A
NH2
B
Ki(mtCA 1)=9560 nM
Ki(mtCA 3)=3420 nM
Ki(mtCA 1)=8690 nM
Ki(mtCA 3)=7330 nM
O
O
S
SO2NH2
NH2
H2N
F
OH
N
N
AcNH
HO3S
O
O
S
SO3H
SO2Na
N
NaO2S
N
H
H
E
G
In the initial work7,8 for discovering mtCA 1/3 inhibitors we
observed that the simple aminosulfonamides A and B show rather
modest inhibitory activity against these enzymes, with KIs in the
range of 3.42–9.56 lM, whereas compounds C and D, incorporating
an additional sulfanilyl moiety and thus possessing an elongated
tion of mtCAs with this class of compounds. Thus, both hydroxy-,
amino, methylamino and dimethylamino such moieties were
incorporated in the molecules of sulfonamides 4 and 5 reported
here. Since water solubility is not one of the principal feature of
sulfonamides, we have also prepared the sulfonate derivatives
4e, 4f and 5e, 5f, which incorporate a water solubilizing moiety
molecule, were more effective mycobacterial CAIs, with inhibition

A. Maresca et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4929–4932
4931
Table 1
SO2NH2
SO2NH2
+
SO2NH2
NH2
R
phenol/amine
coupling
Inhibition of CAs of human (hCA II) or mycobacterial origin mtCA 1 and mtCA 3 with
sulfonamides 4 and 5, the sulfonates 6 and 7. Standard sulfonamide inhibitors data
are also provided for comparison26
NaNO2
HCl, 0°C
N
N
SO2NH2
N
N
-
SO2NH2
Cl
1: 4-NH2
2: 3-NH2
3
SO3Na
SO3Na
H
R
N
N
4: para
5: meta
N
N
N
R
N
Scheme 1. Preparation of diazenylbenzenesulfonamides 4 and 5 from sulfanil-
amide 1 or metanilamide 2, by diazotization followed by coupling of the diazonium
salts 3 with phenols/amines.
R
4
5
6
7
(as sodium salts) and confer increased hydrosolubility to these sul-
fonamides, similar to the sulfinate sodium salt moieties present in
the antileprosy agent G mentioned above. The intermediate sulfo-
nates 6 and 7 used to prepare these hydrosoluble sulfonamides (4e,
4f and 5e, 5f) were also tested for their CA inhibitory properties,
since this class of derivatives was scarcely investigated on these
No.
R
KI (l
M)#
mtCA 1b
hCA IIa
mtCA 3b
4a
4b
4c
4d
4e
4f
OH
NH2
NHMe
NMe2
0.665
0.106
0.093
0.638
0.105
0.104
0.106
0.088
0.105
0.107
0.109
58.3
63.6
13
0.012
0.014
0.009
0.003
9.27
7.20
7.69
6.86
6.78
8.71
8.97
7.00
7.54
7.51
63
8.67
7.86
0.126
0.481
0.781
0.744
0.839
12.40
8.78
9.18
30.7
8.90
9.03
9.23
8.68
9.36
9.45
7.40
8.90
9.11
0.148
0.104
0.562
0.137
0.201
enzymes (very few studies for the inhibition of human, a-CAs are
available,25 but no b-CA were investigated up until now for their
NHCH2SO3Na
N(Me)CH2SO3Na
ꢀ
interaction with such compounds, possessing a SO3 instead of a
5a
OH
NH2
NMe2
SO2NHꢀ zinc-binding group—ZBG).
5b
5d
5e
5f
6
Inhibition data of the two mycobacterial enzymes mtCA 1 and 3
with compounds 4–7 and prontosil E are reported in Table 1.26
Inhibition data for the host enzyme hCA II (the dominant CA iso-
form in humans)10 are also provided for comparison in Table 1.
The following SAR is to be noted from data of Table 1:
(i) The mycobacterial enzyme mtCA 1 was inhibited by all dia-
zenylbenzenesulfonamides 4 and 5 reported here, with inhibition
NHCH2SO3Na
N(Me)CH2SO3Na
—
—
7
E
AAZ*
MZA*
DZA*
BRZ*
—
—
constants in the range of 6.78–63
weak inhibitor (5f, KI of 63 M) whereas all the other investigated
sulfonamides showed a compact behavior of effective, micromolar
inhibitors (KIs of 6.78–9.27 M). Thus, most of the substitution
lM. Only one compound was a
l
Mean value from at least three different measurements.26 Errors were in the
range of 5% of the obtained value (data not shown).
#
l
a
h = human cloned full length enzyme.
Mycobacterial, recombinant enzyme.
From Refs. 7,8.
patterns present in 4 and 5 lead to efficient mtCA 1 inhibitors.
The only exception is the metanilamide derivative possessing the
rather bulky aminomethylsulfonate moiety found in 5f. It is inter-
esting to note that the corresponding desmethyl derivative 5e is an
effective mtCA 1inhibitor, similarly to the corresponding para-
substituted compound 4f, the two sulfonamides having KIs of
b
*
tent mtCA 1 inhibitors, this compound being a more effective
inhibitor than the clinically used sulfonamides AAZ, MZA, DZA,
and BRZ. In fact prontosil is among the best mtCA 1 inhibitors de-
tected so far.
(ii) mtCA 3 showed an inhibition profile with compounds 4–7
and E rather similar to that of mtCA 1. Thus, all sulfonamides 4
and 5 were medium potency inhibitors, with KIs in the range of
7.40–30.7
whereas all remaining sulfonamides, together with the sulfamates
6 and 7 had KIs of 7.40–12.40 M. SAR is thus rather simple here
too, with the metanilamide derivatives 5 being slightly more effec-
tive mtCA 3 inhibitor compared to the corresponding sulfanilamide
derivatives 4. Again the clinically used compounds were more
effective inhibitors compared to the sulfonamides reported here,
except prontosil E. Indeed, this compound showed very effective
mtCA 3 inhibitory activity, with a KI of 148 nM (Table 1).
7.51–8.71 lM. There were no other notable differences between
the sulfanilamide and metanilamide derivatives, except 5f men-
tioned above. These compounds were on the other hand less effec-
tive mtCA 1 inhibitors compared to the clinically used drugs AAZ–
BRZ or the leads C and D, but were anyhow more active than the
parent compounds A, B or sulfanilamide (data not shown). Thus,
our working hypothesis that compounds with elongated molecule
will act as better inhibitors, was not entirely confirmed by the ob-
tained data, but the new compounds show anyhow an interesting
SAR and probably better activity may be obtained by changing the
R groups present in derivatives 4 and 5. However, a very interest-
ing result emerged regarding the inhibition data with the simple
sulfonates 6 and 7, which showed comparable inhibition data with
lM. The least effective inhibitor was 4d (KI of 30.7 lM)
l
sulfonamides 4 and 5, with KIs of 7.86–8.67 lM (Table 1), and with
prontosil E. Thus, the sulfonate moiety may be an alternative ZBG
or the design of novel b-CA inhibitors. This result is even more sig-
nificant considering that these sulfonates are an order of magni-
tude weaker CA inhibitors against the major human isoform, hCA
(iii) The host, hCA II isoform was more effectively inhibited by
the diazenebenzenesulfonamides 4 and 5, with inhibition con-
stants in the range of 88–665 nM, but were very weakly inhibited
II, which was inhibited with KIs in the range of 58.3–63.6 lM
by the sulfonates 6 and 7 (KIs of 58.3–63.6 lM) (Table 1). Thus, we
(see discussion later in the text too). On the other hand, the bulkier
(then 4 and 5) azo derivative prontosil E was the best mtCA 1
inhibitor detected here, with an inhibition constant of 126 nM. It
is thus obvious that diazenylbenzenesulfonamides incorporating
bulkier moieties to the second aromatic ring (not the one with
the sulfamoyl moiety), such as prontosil E, may lead to quite por-
confirmed the earlier observation that these azo dyes are not very
good hCA II inhibitors (compared to the clinically used compounds
of Table 1, which have KIs in the low nanomolar range, of 3–14 nM)
but they interact better with the a-class enzyme than with the two
b-CA investigated here). Prontosil E was also a very effective hCA II
inhibitor (KI of 13 nM).

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A. Maresca et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4929–4932
14. Nishimori, I.; Minakuchi, T.; Kohsaki, T.; Onishi, S.; Takeuchi, H.; Vullo, D.;
(iv) Selectivity issues: except for the sulfonates 6 and 7 which
are much better b-CA than
ited much better the host (
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a
-CA inhibitors, all sulfonamides inhib-
a-class) over the pathogenic (b-class en-
zyme). Thus, the main finding of this study, except for the medium-
weak mtCA 1/3 inhibitors belonging to the sulfonamide class that
we evidenced, is that sulfonates may be better binding and also
selectively inhibit the pathogenic over the host CAs. Another impor-
tant aspect is related to the good inhibitory activity of prontosil
against mtCA 1 and 3, although this compound is also not a selec-
tive CAI for the pathogenic over the host enzymes. Work is war-
ranted to confirm these finding on different classes of sulfonates
and to design compounds based on the prontosil scaffold as lead.
In conclusion, we report here a series of diazenylbenzenesulf-
onamides which have been obtained from sulfanilamide or meta-
nilamide by diazotization followed by coupling with phenols or
amines. The compounds were tested for the inhibition of the b-
CAs encoded by the genes Rv1284 and Rv3273 of M. tuberculosis.
Several low micromolar inhibitors of the two enzymes were de-
tected with the best inhibitor being prontosil (KIs of 126–
148 nM). Some sulfonates tested for the same interaction showed
excellent mtCA inhibitory activity and selectivity for the inhibition
of the pathogenic over host enzymes. Inhibition of pathogenic b-
CAs may thus lead to the development of antiinfectives with a
new mechanism of action, devoid of resistance problems encoun-
tered with classical antibiotics
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Acknowledgements
24. Aminonenzenesulfonamide (sulfanilamide
1 or metanilamide 2) (0.20 g,
1.0 equiv) was dissolved in a freshly prepared 40% solution of concentrated
hydrochloric acid in deionised water (3.0 ml) and then cooled down to ꢀ5 °C. A
2.3 M aqueous solution of NaNO2 (1.2 equiv) was added dropwise and the
mixture was kept stirring at the same temperature until a persistent pale
yellow solution, for sulfanilamide, or pale-orange solution, for metanilamide,
was formed (5–10 min). The solution was used freshly prepared for the
This research was financed in part by a grant of the 6th Frame-
work Programme (FP) of the European Union (DeZnIT project), and
by a grant of the 7th FP of EU (Metoxia project).
References and notes
coupling reactions. For example,
a
solution of diazotized sulfanilamide/
solution of N,N-
metanilamide was added dropwise to
3
a
dimethylaminobenzene (0.14 g, 0.15 ml, 1.0 equiv) in a saturated aqueous
solution of AcONa (3.0 ml) at ꢀ5 °C. The solution turned bright orange
immediately and a precipitate was formed. The mixture was stirred at the
same temperature for 15 min, then warmed at room temperature and the pH
adjusted to 7. The solid was collected by filtration, washed with a minimum
amount of H2O, dried under vacuo and purified by silica gel column
chromatography eluting with 5% MeOH in dichloromethane to give 4d as an
orange solid in 79% yield. 4-(40-Dimethylaminophenyl)diazenylbenzene-
sulfonamide 4d: mp260–261 °C silica gel TLC Rf 0.38 (MeOH/DCM 5%); mmax
(KBr) cmꢀ1, 1604 (Aromatic), 1520 (N@N), 1370 (SO2–N); dH (400 MHz, DMSO-
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145.0 (ipso), 144.0 (ipso), 128.5 (C-2), 127.0 (C-3), 123.6, 113.2, 30.8 (2 ꢁ CH3).
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