N-Acylbenzenesulfonamide Dihydro-1,3,4-oxadiazole Hybrids: Seeking Selectivity toward Carbonic Anhydrase Isoforms

Article abstract of DOI:10.1021/acsmedchemlett.7b00205

A series of N-acylbenzenesulfonamide dihydro-1,3,4-oxadiazole hybrids (EMAC8000a-m) was designed and synthesized with the aim to target tumor associated carbonic anhydrase (hCA) isoforms IX and XII. Most of the compounds were selective inhibitors of the tumor associated hCA XII. Moreover, resolution of EMAC8000d racemic mixture led to the isolation of the levorotatory eutomer exhibiting an increase of hCA XII inhibition potency and selectivity with respect to hCA II. Computational studies corroborated these data. Overall our data indicate that both substitution pattern and stereochemistry of dihydro-1,3,4-oxadiazole could be considered as key factors to determine activity and selectivity toward hCA isozymes. These results can provide further indication for the design and optimization of selective hCA inhibitors.

Full text of DOI:10.1021/acsmedchemlett.7b00205

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Letter
N-acylbenzenesulfonamide dihydro-1,3,4-oxadiazole hybrids:
seeking selectivity towards carbonic anhydrase isoforms
Giulia Bianco, Rita Meleddu, Simona Distinto, Filippo Cottiglia, Marco Gaspari, Claudia
Melis, Angela Corona, Rossella Angius, Andrea Angeli, Domenico Taverna, Stefano
Alcaro, Janis Leitans, Andris Kazaks, Kaspars Tars, Claudiu T Supuran, and Elias Maccioni
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.7b00205 • Publication Date (Web): 21 Jun 2017
Downloaded from http://pubs.acs.org on June 25, 2017
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N-acylbenzenesulfonamide dihydro-1,3,4-oxadiazole hybrids: seeking selectiv-
ity towards carbonic anhydrase isoforms
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Giulia Biancoa‡, Rita Meleddua‡, Simona Distintoa*, Filippo Cottigliaa, Marco Gasparib, Claudia Melisa, Angela
Coronaa, Rossella Angiusc, Andrea Angelid, Domenico Tavernab, Stefano Alcaroe, Janis Leitansf, Andris Kazaksf,
Kaspars Tarsf, Claudiu T. Supurand* and Elias Maccionia.
a Department of Life and Environmental Sciences, University of Cagliari, Via Ospedale 72, 09124 Cagliari, Italy
b Department of Experimental and Clinical Medicine, “Magna Græcia” University of Catanzaro, Catanzaro, Italy
c Laboratorio NMR e Tecnologie Bioanalitiche, Sardegna Ricerche, Pula, hCA, Italy
d Dipartimento NEUROFARBA, Sezione di Scienze Farmaceutiche, Università degli Studi di Firenze, Sesto Fiorentino, Florence, Italy
e Dipartimento di Scienze della Salute, Università Magna Graecia di Catanzaro, Campus ‘S. Venuta’, Viale Europa, 88100 Catanzaro,
Italy
f Latvian Biomedical Research and Study Center, Ratsupites 1, Riga, Latvia
KEYWORDS: human Carbonic Anhydrase, hCA, N-acylbenzenesulfonamide, 1,3,4-oxadiazole, hybrids, docking.
ABSTRACT A series of N-acylbenzenesulfonamide dihydro-1,3,4-oxadiazole hybrids (EMAC8000a-m) was designed and synthesized with
the aim to target tumor associated carbonic anhydrase (hCA) isoforms IX and XII. Most of the compounds were selective inhibitors of the
tumor associated hCA XII. Moreover, resolution of EMAC8000d racemic mixture led to the isolation of the levorotatory eutomer exhibiting
an increase of hCA XII inhibition potency and selectivity with respect to hCA II. Computational studies corroborated these data. Overall our
data indicate that both substitution pattern and stereochemistry of dihydro-1,3,4-oxadiazole could be considered as key factors to determine
activity and selectivity towards hCA isozymes. These results can provide further indication for the design and optimization of selective hCA
inhibitors.
Carbonic anhydrase (hCA, EC 4.2.1.1) are widespread enzymes
that catalyze the hydration reaction of carbon dioxide into bicar-
bonate.1 These enzymes are involved in several physiological pro-
cesses, spacing from pH regulation, ion transport, bone resorption
to the secretion of gastric, cerebrospinal fluid and pancreatic juice.2
In mammals the hCA family is composed of 16 different isoforms,
differing for sequence and, more importantly, for tissue localization,
expression and catalytic activity.3 Briefly, hCA I-III and hCA VII are
cytosolic isoforms, while hCA IV, hCA IX, hCA XII and hCA XIV
are membrane-bound isozymes.3 Due to their key role in cell metab-
olism these enzymes have been deeply investigated as drug targets
and, as a result, a number of hCA inhibitors have been designed and
are currently in clinical use.4-6 In particular the trans-membrane hCA
IX and hCA XII isoforms have been associated with tumor progres-
sion and invasion.7-12 However, most of the clinically available agents
inhibit hCA isoforms unselectively and there is a considerable inter-
est of the scientific community for the development of isozyme-se-
lective agents for the treatment of specific pathologies.13-16 Moreo-
ver, serious drug interactions have been reported for several hCA in-
hibitors, and therefore, selectivity is mandatory. In order to identify
new scaffolds for the selective inhibition of tumor-associated hCAs,
pathway was observed, being thiazoline derivatives preferential hCA
XII inhibitors, while thiazolidine based compounds exhibit, at least
in some cases, a preference towards hCA IX isozyme. Furthermore,
saccharin and open derivatives of saccharin, structurally related to
compounds EMAC8000a-m, have been recently reported as selec-
tive inhibitors of the tumor associated hCA isoforms IX and XII,19, 20
suggesting the potential of secondary benzenesulphonamides as iso-
zyme-selective hCA inhibitors. Considering these observations and
with the aim to investigate new scaffolds and molecular geometries
to target hCA IX and hCA XII, we have designed and synthesized a
series of N-acylbenzenesulfonamide dihydro-1,3,4-oxadiazole hy-
brids (EMAC8000a-m) and evaluated their activity towards hCA
isoforms I, II, IX, and XII.
The new compounds were synthesized slightly modifying an al-
ready reported multi-step synthetic procedure (Scheme 1).21,
22
Briefly, 4-sulfamoylbenzoic acid (1) was reacted with methanol in
the presence of sulfuric acid as a catalyst.
we have already synthesized benzenesulfonamide based hybrid mol-
17, 18
ecules
in which the benzenesulfonamide moiety binds either a
N-cyclohexyl-thiazoline or to a N-methyl-thiazolidine core. With re-
spect to the central core of the hybrid molecule, a different selectivity
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Scheme 1. Synthetic pathway to compounds EMAC 8000a-m.
8000i
8000j
8000k
8000l
8000m
AAZ
3-NO2
2-CH3
3-OCH3
2,4-F
> 10000
9376
120
172
571
327
892
12.0
44.3
489
430
373
468
25
6.7
6.3
1
2
3
4
5
6
7
O
S
O
S
O
C
OH
O
O
a
H2N
H2N
C
8952
28.4
6.8
CH3
O
O
1
2
8926
4-CF3
> 10000
250
53.8
5.7
O
S
O
C
c
b
H2N
8
9
*Mean from 3 different assays, by a stopped flow technique (errors
were in the range of  5-10 % of the reported values).
HN NH2
O
3
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O
H
N
S
O
O
O
C
HN
C
H
O
O
All EMAC8000 derivatives displayed a preferential activity to-
wards the isoform hCA XII, except for compounds EMAC8000 d, e,
f, and i that exhibited a moderate activity on the hCA IX isoform.
Surprisingly, although we had previously observed a pseudo-conju-
gative effect between the two aromatic substituents in the position 2
and 5 of the dihydro-oxadiazole,23 no significant difference in the bi-
ological activity of the EMAC8000 compounds could be related to
the electronic nature of the substitution on the phenyl ring in the po-
sition 5 of the heterocycle. On the contrary, the steric effects of the
substituents seemed to be more relevant for the inhibition potency.
In fact, the presence of a bulky substituent in the position 4 of the
phenyl ring such as -Cl, -NO2, –OCH3 or -CF3 generally lead to a
decrease of the inhibitory potency towards hCA XII. According to
these observations, we decided to investigate on the role of the ste-
reochemistry at the position 5 of the dihydro-oxadiazole ring.
EMAC8000d was chosen for this preliminary study due to its activ-
ity towards both hCA IX and XII.
Thus, the enantioseparation of compound EMAC8000d was per-
formed by means of semipreparative HPLC using the amylose-
based Chiralpak IA as a chiral stationary phase. Based on the chro-
matographic results of analytical screening, the ethanol-acetoni-
trile/H2O 55-40-5 (v/v/v) mixture was identified as the most suita-
ble eluent for the enantioseparation of EMAC8000d on the 1-cm
I.D. IA column. An amount of ∼2 mg of racemic samples was re-
solved for each chromatographic run, and both enantiomers were
collected with high enantiomeric purity (>99% ee, Figure S27 and
Table S4, SI). The pure enantiomers were then submitted to biolog-
ical evaluation to investigate the role of stereochemistry on both in-
hibition potency and isozyme selectivity. Results are reported in Ta-
ble 2. The activity toward the hCA isoforms was assessed by a previ-
ously reported procedure.24
H2N
S
CH3
d
N C
R
O
N
N
H
R
C
O
4
H3C
EMAC 8000a-m
Reagents and conditions: (a) H2SO4/MeOH; (b) NH2NH2 H2O,
MeOH; (c) substituted benzaldehydes, 2-propanol/CH3COOH; (d)
acetic anhydride/pyridine.
The obtained ester (2) was converted into 4-(hydrazinecar-
bonyl)benzenesulfonamide (3) by treatment with hydrazine hy-
drate in methanol. 4-[N'-(arylmethylidene)hydrazinecarbonyl]ben-
zenesulfonamide (4) was obtained by reaction of 3 with differently
substituted benzaldehydes in 2-propanol and acetic acid as a cata-
lyst. The formation of the dihydro-oxadiazole derivatives
EMAC8000 a-m, was accomplished by refluxing 4 in acetic anhy-
dride, as already described, with the addition of pyridine in catalytic
amounts. After cooling to room temperature, methanol was added
to the reaction mixture. The obtained yellowish solution was then
poured into ice water and vigorously stirred. A precipitate was
formed which was washed with 10% aqueous NaHCO3 solution and
purified by crystallization. Compounds EMAC8000a-m were char-
acterized by means of both analytical and spectroscopic methods
(see Table S1-S3, Figure S1-S26 Supporting Information (SI)) and
submitted to biological evaluation towards hCA isoforms I, II, IX,
and XII (Table 1).
Table 1. Inhibition data towards hCA I, II, IX, and XII of com-
pounds EMAC8000a–m
O
H
S
N
O
C
H
O
O
CH3
R
Table 2. Inhibition data toward hCA I, II, IX, and XII and tumour
associated hCA IX and XII isoforms selectivity (SI) versus hCA II of
EMAC8000d pure enantiomers.
N
N
C
O
H3C
Ki (nM)
Ki (nM) hCA
EMAC
R
hCA I
hCA
II
hCA IX
hCA XII
SI
SI
hCA
hCA
EMAC
8000
I
II
IX
XII
XII/II
IX/II
8000a
8000b
8000c
8000d
8000e
8000f
8000g
8000h
4-Cl
4-CH3
2-OCH3
2,4-Cl
4-F
8404
9410
485
529
450
83.2
277
158
293
239
502
502
378
25.0
43.6
33.1
159
143
11.6
5.2
4.52
1.55
3.33
2.19
4.84
0.48
3280
> 10000
1010.02
250
83.2
59
25.0
26.9
24.2
25
18.4
38.0
7.5
()-d
(+)-d
(-)-d
AAZ
6776
6.2
3280
18.4
6.8
117.2
12.0
15.63
2.10
8245
5.7
4-NO2
2-NO2
3872
47.7
5.0
*Mean from 3 different assays, by a stopped flow technique (errors
were in the range of  5-10 % of the reported values).
> 10000
4-OCH3 > 10000
34.0
2
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Noteworthy, an increase in the inhibitory potency was observed
1
only towards the hCA XII isozyme in the case of the (-)-
EMAC8000d enantiomer. On the contrary, the (+)-EMAC8000d
enantiomer resulted as the most active toward the II isozyme, mainly
due to the loss of activity of the (-)-EMAC8000d enantiomer hCA
II. All together this behavior lead to a tenfold increase of the selec-
tivity of the (-)-EMAC8000d enantiomer toward hCA XII with re-
spect to the II isozyme. To a lower extent, a similar behavior was ob-
served for the hCA IX isozyme. However, the gain in selectivity to-
ward the hCA IX isoform, with respect to the hCA II isozyme could
only be related to the reduction of the potency of the (-)-
EMAC8000d enantiomer toward the hCA II isozyme, being the two
enantiomers almost equally potent toward the hCA IX. The absolute
configuration of (-)-EMAC8000d and (+)-EMAC8000d enantio-
mers was not assigned. However, the two enantiomers R and S were
considered in the computational investigation. In the light of the bi-
ological results exhibited by the pure enantiomers and to rationalize
the major activity toward hCA XII of the (-)-EMAC8000d enantio-
mers both EMAC8000d enantiomers were subjected to molecular
docking applying the Quantum-mechanics polarized Docking
(QMPL).25, 26 Prior to perform the docking study on unknown com-
pounds, the protocol has been validated through simulations of self-
and cross- docking of known hCA XII inhibitors: the 4-propylthio-
benzenesulfonamide (VD9) and acetazolamide (AAZ) into the
crystal structure with pdb code 4WW8 (resolution 1.42 Å).27 Both
compounds were successfully docked with the QMPL protocol, dis-
playing root-main-square deviation (RMSD) values lower than 2.0
Å (Figure S28 and Table S5 SI). Both oxadiazolidine derivatives (S)-
EMAC8000d and (R)-EMAC8000d have been docked into the
above-mentioned crystal structure and the best scored poses were
subjected to a post-docking protocol based on energy minimization
and binding-free energy calculation, due to lack of accuracy of dock-
ing score in the overall energy evaluation (Table S6 SI). The total
energies of interactions were obtained applying molecular mechan-
ics and continuum solvation models using the molecular mechanics
generalized Born/surface area method (MM-GBSA) (Table S7
SI).28 After these data were collected and analyzed, we were then
able to establish which enantiomer was mainly stabilized within the
hCA XII active site.
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Figure 1. a, c) 3D representation of the putative binding mode obtained
by docking experiment of (R)-EMAC8000d (in red) and S-
EMAC8000d (in pink) into hCA XII (pdb code 4WW8) and b, d) 2D
representation of the complexes stabilizing interactions with the binding
site residues.
The putative binding mode confirmed the importance of the sub-
stituent in the position 5 of the dihydro-oxadiazole ring and the role
of stereochemistry in the affinity for this isoform. In addition, the se-
lectivity toward the hCA XII isoform has been demonstrated
through the analysis of the binding site of all the tested isoforms. In
particular, the substitutions in hCA XII: of Ala126 into Phe131 in
hCA II, of Thr88 into Phe 91 in hCA I, and of Asn203 into Tyr204
in hCA I, lead to relevant differences between the four isoforms.
These residues are located in the entrance of the binding site which
results bigger in the hCA XII compared to the isoform hCA II and
hCA I (Figure S29-S30). Hence this could explain why our com-
pounds are generally more selective toward the hCA XII isoform and
could give a rationale for the higher activity of one of the two enan-
tiomers. To corroborate our results, we have docked the compounds
EMAC8000f and EMAC8000m into hCA XII. These compounds
are the less active toward the hCA XII isoform and are characterized
by the presence of a NO2 and a CF3 group in the position 4 of the
phenyl ring, respectively. The steric hindrance of this group does not
allow a fully efficient accommodation in the binding site. This is fur-
ther confirmed by their lower value of G (Table S5 and Figure
S31). In summary, a small library of N-acylbenzenesulfonamide di-
hydro-1,3,4-oxadiazole hybrids has been synthesized and character-
ized. Our preliminary data indicate that the hybridization of the di-
hydro-oxadiazole ring with the N-acylbenzenesulfonamide moiety
leads to preferential inhibition of the hCA XII isoform. In this re-
spect both the substitution pattern and the stereochemistry at the
position 5 of the oxadiazole ring appeare to be as key features to
modulate the isoform selectivity. With these data in our hands we
aim to further develop these derivatives to achieve more effective tu-
mor associated hCA inhibitors.
The R enantiomer showed a value of binding free energy of -28.36
Kcal/mol while the S enantiomer a value of -25.75 Kcal/mol. This
difference could partially explain the better activity of the enantio-
mer in the biological test. In order to confirm this hypothesis, a de-
tailed analysis of the crucial interactions is required. In fact, the R
configuration allows the formation of a hydrogen bond between
Asn64 and the amidic oxygen of the dihydro-oxadiazole ring, while
the different spatial distribution of the atoms in the S enantiomer
does not permit this important interaction. It must be pointed out
that the hydrogen bond with Thr198 and His117 and the interaction
with the Zn ion is well conserved with both configurations (Figure
1), confirming the same binding mode of this class of compounds.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Pub-
lications website at DOI:
3
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(13)
D'Ascenzio, M.; Carradori, S.; De Monte, C.; Secci, D.; Ceruso, M.;
Experimental procedures, compounds characterization, tables and
schemes are reported as (PDF).
Supuran, C. T. Design, synthesis and evaluation of N-substituted saccharin
derivatives as selective inhibitors of tumor-associated carbonic anhydrase XII.
Bioorg. Med. Chem. 2014, 22, 1821-1831.
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a therapeutic strategy. Nat. Rev. Drug Discovery 2011, 10, 767-777.
(15) Pacchiano, F.; Carta, F.; McDonald, P. C.; Lou, Y.; Vullo, D.;
Scozzafava, A.; Dedhar, S.; Supuran, C. T. Ureido-substituted
benzenesulfonamides potently inhibit carbonic anhydrase IX and show
antimetastatic activity in a model of breast cancer metastasis. J. Med. Chem.
2011, 54, 1896-1902.
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AUTHOR INFORMATION
Neri, D.; Supuran, C. T. Interfering with pH regulation in tumours as
Corresponding Author
claudiu.supuran@unifi.it, s.distinto@unica.it.
Author Contributions
The manuscript was written through contributions of all authors. All
authors have given approval to the final version of the manuscript.
‡These authors contributed equally.
9
(16)
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.
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Meleddu, R.; Maccioni, E.; Distinto, S.; Bianco, G.; Melis, C.; Alcaro,
ABBREVIATIONS
S.; Cottiglia, F.; Ceruso, M.; Supuran, C. T. New 4-[(3-cyclohexyl-4-aryl-2,3-
dihydro-1,3-thiazol-2-ylidene)amino]benzene-1-sulfonamides, synthesis and
inhibitory activity toward carbonic anhydrase I, II, IX, XII. Bioorg. Med. Chem.
Lett. 2015, 25, 3281-3284.
hCA, Carbonic anhydrase; QMPL, Quantum-mechanics polarized
Docking; VD9, 4-propylthiobenzenesulfonamide; AAZ, acetazolamide;
MM-GBSA, molecular mechanics generalized Born/surface area
method.
(18)
Melis, C.; Meleddu, R.; Angeli, A.; Distinto, S.; Bianco, G.; Capasso,
C.; Cottiglia, F.; Angius, R.; Supuran, C. T.; Maccioni, E. Isatin: a privileged
scaffold for the design of carbonic anhydrase inhibitors. J. Enzyme Inhib. Med.
Chem. 2017, 32, 68-73.
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