Substituted benzene sulfonamides incorporating 1,3,5-triazinyl moieties potently inhibit human carbonic anhydrases II, IX and XII

Article abstract of DOI:10.1016/j.bmcl.2014.01.048

A series of benzene sulfonamides incorporating 1,3,5-triazinyl moieties were synthesized using cyanuric chloride as starting material. Inhibition studies against human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms I, II (cytosolic) and IX, XII (transmembr

Full text of DOI:10.1016/j.bmcl.2014.01.048

Bioorganic & Medicinal Chemistry Letters 24 (2014) 1310–1314
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Substituted benzene sulfonamides incorporating 1,3,5-triazinyl
moieties potently inhibit human carbonic anhydrases II, IX and XII
Amrita K. Saluja a, Meena Tiwari a, , Daniela Vullo b,c, Claudiu T. Supuran b,c,
⇑
⇑
a Shri G.S. Institute of Technology and Science, Department of Pharmacy, 23, Park Road, Indore 452003, Madhya Pradesh, India
b Università degli Studi di Firenze, Dipartimento di Chimica, Lab. Chimica Bioinorganica, Via della Lastruccia 3, 50019 Sesto Fiorentino (Florence), Italy
c Università degli Studi di Firenze, NEUROFARBA Dept., Sezione di Scienze Farmaceutiche e Nutraceutiche, Via Ugo Schiff 6, I-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 benzene sulfonamides incorporating 1,3,5-triazinyl moieties were synthesized using cyanuric
chloride as starting material. Inhibition studies against human carbonic anhydrase (hCA, EC 4.2.1.1) iso-
forms I, II (cytosolic) and IX, XII (transmembrane, tumor-associated) isoforms were performed with the
Received 10 December 2013
Revised 18 January 2014
Accepted 20 January 2014
Available online 27 January 2014
new compounds. hCA I was modestly inhibited (KIs in the range of 87 nM–4.35 lM), hCA II was moder-
ately inhibited by most of the new compounds (KIs in the range of 12.5–130 nM), whereas the tumor
associated isoforms were potently inhibited, with KIs in the range of 1.2–34.1 nM against hCA IX and
of 2.1–33.9 against hCA XII, respectively. Docking studies of some of the new compounds showed an
effective binding mode within the enzyme active site, as demonstrated earlier by X-ray crystallography
for structurally-related sulfonamides incorporating 1,3,5-triazinyl functionalities.
Keywords:
Carbonic anhydrase
1,3,5-Triazine
Benzene sulfonamides
Isoform selectivity
Enzyme inhibition
Molecular docking
Ó 2014 Elsevier Ltd. All rights reserved.
Carbonic anhydrases (CA, E.C.4.2.1.1) are ubiquitous metalloen-
zymes that catalyze a simple reaction, the conversion of CO2 to
bicarbonate ion and a proton.1 They are involved in various phys-
iological and pathological processes, such as gluconeogenesis, lipo-
genesis, ureagenesis and tumorigenicity, serving as an important
target for designing drugs useful in diseases like glaucoma, obesity,
epilepsy and cancer.1–4 CA inhibitors (CAIs) are well established
drugs as diuretics and antiglaucoma agents, but recent research
has shown that several CA isozymes are drug targets for epilepsy,
cancer and infective diseases.2 CA II is responsible for increasing
sodium bicarbonate secretion in the anterior uvea of the eye
leading to visual dysfunctioning and glaucoma.5 Tumour hypoxia,
through hypoxia inducible factor 1 (HIF-1), is involved in the
regulation of expression of several genes involved in pH regulation,
glucose metabolism and angiogenesis, among which the tumor-
associated isoform CA IX. Overexpression of CA IX contributes to
acidification of the extracellular tumor pH, enhancing tumorigene-
sis and metastatic spread.6
action) or CA IX/XII (for the antitumor activity).6,7 We reported ear-
lier that incorporation of a 1,3,5-triazine moiety in 4-aminoben-
zene sulfonamide scaffold leads to compounds with enhanced
efficacy and specificity against the transmembrane isoforms hCA
IX and XII, over the cytosolic forms hCA I and II.10
Considering sulfonamides, which have high binding affinity for
most CA isozymes, we have designed novel compounds to achieve
selectivity and specificity against the transmembranous CA IX iso-
zyme over the cytosolic, ubiquitous one CA II.3,4 The rationale for
designing the new compounds reported here is based on the earlier
findings (kinetic and X-ray crystallography) that 1,3,5-triazinyl-
containing sulfonamides show potent and selective inhibition of
CA IX/XII over CA I and II.8–10
The nucleophilic aromatic substitution reaction has been
widely used for the synthesis of sulfonamides incorporating
1,3,5-triazine (Scheme 1), pyridine or diazine moieties (Scheme 2),
as reported earlier.11 Here we explored new substitution patterns
at the triazine ring, as by means of X-ray crystallography it has
been shown that the tails present in the scaffold significantly
contribute to the potency and isoform-selectivity of the
benzenesulfonamides incorporating disubstituted-1,3,5-triazinyl
moieties. Reaction of cyanuric chloride (1) with 4-amino benzene
sulfonamide (2) in presence of base afforded the key intermediate,
the dichlorotriazine-benzenesulfonamide derivative reported
earlier (compound 3, Scheme 1). Reaction of (3) with different
nucleophiles in the molar ratio of 1:1 produced monosubstituted
Sulfonamide and sulfamate CAIs were reported to show sub-
stantial anti-glaucoma as well as anti-tumour activity in vitro
and in vivo, thus constituting interesting leads or new therapeutic
approaches, when targeting either CA II (for the antiglaucoma
⇑
Corresponding authors. Tel.: +91 731 2454095x537 (M.T.); tel.: +39 055
4573005; fax: +39 055 4573385 (C.T.S.).
E-mail addresses: mtiwari@sgsits.ac.in (M. Tiwari), claudiu.supuran@unifi.it
(C.T. Supuran).
products
4 (a–e) whereas working at the ratio of 1:2, the
http://dx.doi.org/10.1016/j.bmcl.2014.01.048
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

A. K. Saluja et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1310–1314
1311
H
N
R
N
RH
H2O
N
N
H2N
SO2NH2
Cl
N
Cl
Cl
SO2NH2
Cl
4
N
N
reflux
N
N
2
(a-e)
NH
Cl
N
1
Cl
Acetone
NaOH
0-5oC
H2NO2S
R' H
reflux
H2O
3
reflux
R
2RH H2O
H
N
N
R
R'
N
N
H2NO2S
H
N
N
R
5
(a-b)
N
N
H2NO2S
(a-e)
6
Scheme 1. Synthesis of sulfonamides 4 (a–e), 5 (a–b), 6 (a–e) incorporating triazine moieties by reaction of dichlorotriazine (3) with nucleophiles.
NH2
H
N
THF
K2CO3
N
Cl
N
SO2NH2
Reflux
+
NH2
10
N
7
DMF
K2CO3
NH2
H
N
Cl
N
NH2
H2N
+
+
N
N
100oC
SO2NH2
NH2
8
NH2
11
SO2NH2
2
H
N
O
DMF
DIPEA
N
N
N
O
N
SO2NH2
r.t.
12
Cl
9
Scheme 2. Synthesis of sulfonamides (10–12) incorporating pyridine, pyrimidine, and pyridazine moieties.
disubstituted products 6 (a–e) were obtained.8–10 The monosubsti-
tuted derivatives (4a and 4b) were treated with different nucleo-
philes to replace the second chlorine atom, leading to the
disubstituted derivatives 5 (a–b).
4-amino benzene sulfonamide, ammonia, anilines, morpholine,
tert-butylamine, N-methyl-2-amino ethanol and 1, 3-diaminopro-
pane, in order to explore a chemical space which has not been
investigated in the previous communications.8–10
Isostructural rings to the 1,3,5-triazine ones, such as pyridine,
pyrimidine and pyridazine incorporating a chlorine in position 2-
, 4-, or 6-, also possess a high reactivity towards various nucleo-
philes, and were less investigated for the synthesis of CA inhibitors
until now. The new monosubstituted products (10–12) were thus
obtained when 3-amino-2-chloropyridine (7), 6-chloropyrimi-
dine-2,4-diamine (8) or 3-chloro-6-methoxypyridazine (9) were
reacted with an equivalent of 4-amino benzene sulfonamide (2)
in the presence of anhydrous potassium carbonate or diisopropyl-
ethylamine under an inert nitrogen atmosphere (Scheme 2).12–14
It was observed that replacement of chlorine atoms from the
triazine nucleus requires harsher conditions after the first chlorine
has been replaced, since the accumulation of electron donating
amine substituents gradually decreases the reactivity of the tri-
azine ring. Then, the subsequent replacements occured slower than
the first one. The nucleophiles used for the synthesis included
The synthesized compounds were evaluated15 using a stopped
flow assay against CA isoforms I, II, IX, XII and the inhibition data
are shown in Table 1.
The following structure activity relationship can be drawn from
the inhibition data of Table 1:
(i) All the 15 synthesized compounds modestly inhibited
hCA
I with KIs in the range of 87 nM–4.35 lM. The
best activity in the series of the synthesized compounds
was shown by (2,6-diaminopyrmidin-4-ylamino) benzene
sulfonamide (compound 11) and 4a which incorporated a
chloro and an anilino moiety, both of them possessing a KI
value of 87 nM. The diamino derivative 6a (repored earlier)9
was also an effective hCA I inhibitor with a much better KI
(90 nM) compared to that of the standard drug acetazolamide
(AAZ). The weakest activity was observed for (4,6-bis

1312
A. K. Saluja et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1310–1314
Table 1
that compounds with small, compact substituents possess-
ing lower molecular weights, and an increased number of
hydrogen acceptor atoms were more effective against hCA
IX isoforms over hCA II (e.g., 6a, 4a–c, 6d, and 10–12). Again
the 1,3,5-triazine ring could be substituted by the isostruc-
tural pyridine, pyrimidine or diazine rings without loss of
hCA IX inhibitory activity or selectivity for inhibiting the
tumor-associated over the cytosolic isoforms (Table 1).
(iv) hCAXII was also potently inhibited by the new compounds
reported here, with inhibition constants ranging from 2.1
to 33.9 nM. Most of the compounds, such as 6a, 4a–c, 6e,
and 11 showed a very good activity with KIs in the low nano-
molar range, of 2.1–4.1 nM, comparable with AZM (KI of
5.7 nM). It was observed that the disubstituted compounds
with bulkier moieties, such as aniline-morpholine, 3-methyl
aniline- and morpholine, or dimorpholino, were less active
against hCA XII, a situation already encountered at the inhi-
bition of the other CA isozymes discussed here, and probably
due to their too bulky nature and difficulties to effectively
bind within the restricted space of the enzyme active site
cavity. The monosubstituted compounds showed better
hCA IX/hCA XII inhibition activity compared to the disubsti-
tuted derivatives incorporating similar scaffolds.
Inhibition data of human carbonic anhydrase isozyme I, II, IX and XII with synthesized
compounds by a stopped-flow, CO2 hydration assay method15
No
R
R0
KIa (nM)
hCA I hCA II hCA IX hCA XII
4a
4b
4c
4d
4e
Cl
Cl
Cl
Cl
Cl
NHPh
87
18.0
18.3
40.7
67.1
5.9
5.1
1.2
21.4
21.2
4.2
4.3
2.1
8.6
25.4
3-NHC6H4Me
HOC2H4N(Me)
H2N(CH2)3NH
4-F-3-
ClC6H3NH
NHPh
3-NHC6H4Me
NH2
O(CH2CH2)2N
t-BuNH
354
1070
1375
2540 130
5a
5b
6a
6b
6c
6d
6e
10
11
12
AAZ
EZA
O(CH2CH2)2N
O(CH2CH2)2N
NH2
O(CH2CH2)2N
t-BuNH
430
447
90
583
410
2365
4350
421
87
34.1
20.1
10.2
2.4
34.1
19.3
5.9
18.3
10.7
3.9
16.4
15.1
3.9
33.9
18.5
12.3
4.1
11.5
5.4
6.7
21.9
21.2
12.5
54.3
72.6
75.9
24.2
15.6
21.4
12.1
8.0
HOC2H4N(Me) HOC2H4N(Me)
H2N(CH2)3NH
H2N(CH2)3NH
—
—
—
—
—
110
250
25.2
8.5
25.0
34.3
5.7
22.1
a
Mean from three different assay, by a stopped flow technique (errors were in
the range of 5–10% of the reported values).
(v) All the synthesized compounds showed better inhibition of
the transmembrane tumor associated isoforms hCA IX and
hCA XII over cytosolic isoforms hCA I and hCA II with good
selectivity profile and specificity. For example, 4c showed a
selectivity ratio of 891 for inhibiting hCA IX over hCA I,
and of 33.9 for inhibiting hCA IX over hCA II. The selctivity
ratios for inhibiting hCA XII over hCA I and II were in the
same range like that of hCA IX (selectivity ratio of 509 for
inhibiting hCA XII over hCA I and of 19 over hCAII), making
this compound highly selective for the inhibition of the
tumor-associated over the cytosolic CA isoforms.
(3-amino propyl amino)-1,3,5-triazin-2-ylamino)benzene sul-
fonamide (compound 6e), which had a KI value of 4.35 lM
against this isoform and possesses the rather bulky, long
1,3-propylene diamine arms. The bulkiness present in this or
structurally related compounds (e.g., 6d, 4d and 4e) probably
interferes with the effective binding within the restricted space
of the hCA I active site, leading to a lower activity of these com-
pounds, with inhibition constants in the micromolar range
(Table 1).
(ii) The synthesized compounds showed effective hCA II inhibi-
tory activity with KIs ranging between 12.5 and 130 nM,
having thus a better inhibition profile against this isoform
compared to hCA I (Table 1). Some of the new compounds,
such as 6a, 4a, 4b, 5b, 6b, 10, 11 and 12 showed quite potent
hCA II inhibition, with KIs in the range of 12.5–24.2 nM, com-
parable to the standard drugs AAZ and EZA. They incorpo-
rate both the 1,3,5-triazine or the isostructural rings
(pyridine, pyrimidine or diazine) in their molecules, as well
as amino, methoxy, chloro, anilino and morpholino groups.
Derivatives 5a, 6c, 4c, 6d, 4d, 6e and 4e showed a weaker
hCA II inhibitory activity, with inhibition constants ranging
between 34.1 and 130 nM (Table 1). They seem to incorpo-
rate bulkier moieties compared to the previous ones, of the
tert-butylamino, hydroxyethylamino-methyl or 3-chloro-4-
fluoroanilino. Probably the most important structural
feature interfering with the effective binding is again the
bulkiness of the moieties substituting the 1,3,5-triazine ring,
as the derivatives with compact or less bulky such moieties
showed a better activity compared to those incorporating
one or two bulky functionalities.
(iii) The transmembrane tumor associated isoform hCA IX was
effectively inhibited by the synthesized compounds with
KIs in the range of 1.2–34.1 nM. Compounds 6a, 4a–c, 6d
and 11 showed inhibition constants KI ranging from 1.2 to
5.9 nM, which is better than standard drugs AZM and EZM.
The active compounds incorporated –Cl or –NH2 substitu-
ents at position 2 of the triazine nucleus whereas com-
pounds 5a–b and 6b, having morpholine substitution at
position 2 of triazine ring showed less potent hCA IX inhib-
itory activity. Compound 6b, with the worst activity was
equipotent with EZM and incorporated two morpholine
moieties with the 1,3,5-triazine nucleus. It was observed
In order to have a hint on the binding orientation of these inhib-
itors within the active site of the enzyme, docking of some of the
new compounds was performed using the GOLD suite program.9
The PDB ID 3IAI, of a triazinyl-substituted benzenesulfonamide
bound to hCA II reported earlier by our and McKenna’s group10
was downloaded from RCSB protein data bank and used for the
docking experiments. Protein and ligand files were prepared using
DockPrep tool in CHIMERA software, which included addition of
charges, addition or removal of hydrogens, repairing of residues
by adding missing chains, etc. Binding site was defined at
X = 70.5457, Y = 51.4075, Z = 11.8884 by selecting all atoms within
12 Å, considering the co-crystallized bound ligand. Docking was
carried out using Genetic algorithm and validated using the co-
crystallized bound ligand, acetazolamide. After validation, all the
compounds were docked into the binding pocket keeping the same
value of coordinates and positioned all the ligands in the same ori-
entation as acetazolamide (Fig. 1).
As for all other hCA II–sulfonamide adducts investigated so far,
it was observed that the nitrogen atom of the sulfonamide 6b (a
potent hCA II inhibitor, KI of 12.5 nM) coordinates to Zn(II) ion
and participates in a network of hydrogen bonds involving residues
Thr199 and Glu106. An oxygen function of sulfonamide group also
hydrogen bonds to the –NH moiety of Thr199. Additionally, the
polar amino acids Gln67, Asn62, Ser65 were found to be in close
contact with the inhibitor 6b (Fig. 1a), thus stabilizing the
enzyme–inhibitor adduct. In the docking analysis of compound
4e (the least potent against hCA II in the series reported here, KI
of 130 nM, Fig. 1b), it was observed that due to presence of bulky,
fluorophenyl functionality at the 1,3,5-triazine ring, the orientation
and alignment of the inhibitor was not perfectly fitted within the
hCA II active site. The scaffold of the inhibitor was found

A. K. Saluja et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1310–1314
1313
diazinyl-substituted moieties. The new derivatives selectively
and specifically inhibited the transmembrane, tumor associated
isoforms hCA IX and hCA XII in low nanomolar range over cytosolic
isoforms hCA I and II, which were moderately inhibited. Docking
studies of the synthesized compounds within the hCA II active site
revealed similar orientation and interaction at the active site
as acetazolamide and other triazinyl-substituted sulfonamides
investigated earlier by means of X-ray crystallography.
Acknowledgments
We thankfully acknowledge All Indian Council for Technical
Education (8023/BOR/RID/RPS-125/2009-2010), MHRD-AICTE and
EU FP7 project (Metoxia) for financial assistance.
Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.
01.048.
References and notes
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Figure 1. Orientation and interaction with the metal ion and amino acid residues
from the hCA II active site of some of the synthesized compounds: (a) 6b (brown)
superposed on the acetazolamide (AAZ)—purple adduct, and (b) compound 4e
(brown) superposed to the acetazolamide adduct (purple), obtained by docking 6b
and 4e in the 3IAI PDB file.10
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15. Khalifah, R. G. J. Biol. Chem. 1971, 246, 2561. An Applied Photophysics stopped-
flow instrument has been used for assaying the CA catalyzed CO2 hydration
activity. 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 NaClO4 (for maintaining constant the ionic strength),
following the initial rates of the CA-catalyzed 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
inhibitor at least six traces of the initial 5–10% of the reaction have been used
surrounded by hydrophobic amino acid residues, such as Val121,
Val131, Trp5, Pro201, and Pro202 (Fig. 1b). Thus, the two sulfona-
mides 6b (strong hCA II inhibitor) and 4e (much weaker hCA II
inhibitor), bind with different orienations and make diverse con-
tacts with amino acid residues from the hCA II active site (Fig. 1a
and b), which explains their different inhibition profile against
the enzyme.
In conclusion, we report here a new series of 15 benzene-
sulfonamides incorporating triazinyl- pyridinyl, pyrimidinyl or

1314
A. K. Saluja et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1310–1314
for determining the initial velocity. The uncatalyzed rates were determined in
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Prete, S.; Vullo, D.; Scozzafava, A.; Capasso, C.; Supuran, C. T. Bioorg. Med. Chem.
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the same manner and subtracted from the total observed rates. Stock solutions
of inhibitor (10–50 mM) were prepared in distilled-deionized water and
dilutions up to 0.1 nM were done thereafter with the assay buffer. Inhibitor
and enzyme solutions were preincubated together for 15 min at room
temperature prior to assay, in order to allow for the formation of the E–I
complex or for the eventual active site mediated hydrolysis of the inhibitor.
The inhibition constants were obtained by non-linear least-squares methods
using PRISM 3 and the Cheng–Prusoff equation, as reported earlier,16 and
represent the mean from at least three different determinations. All CA
isoforms were recombinant ones obtained in-house as reported earlier.16–18
hCA I, II, IX and XII were recombinant proteins obtained in-house as described
earlier.16–18