Tuning the Dual Inhibition of Carbonic Anhydrase and Cyclooxygenase by Dihydrothiazole Benzensulfonamides

Article abstract of DOI:10.1021/acsmedchemlett.8b00352

A novel series of of 4-[(3-phenyl-4-aryl-2,3-dihydro-1,3-thiazol-2-ylidene)amino]benzene-1-sulfonamides (EMAC10111a-g) was synthesized and assayed toward both human carbonic anhydrase isozymes I, II, IX, and XII and cyclooxygenase isoforms. The majority o

Full text of DOI:10.1021/acsmedchemlett.8b00352

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Letter
Tuning the Dual Inhibition of Carbonic Anhydrase and
Cyclooxygenase by Dihydrothiazole Benzensulfonamides
Rita Meleddu, Simona Distinto, Filippo Cottiglia, Rossella Angius, Marco Gaspari, Domenico Taverna,
Claudia Melis, Andrea Angeli, Giulia Bianco, Serenella Deplano, Benedetta Fois, Sonia Del Prete, Clemente
Capasso, Stefano Alcaro, Francesco Ortuso, Matilde Yáñez, Claudiu T Supuran, and Elias Maccioni
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.8b00352 • Publication Date (Web): 17 Sep 2018
Downloaded from http://pubs.acs.org on September 19, 2018
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Tuning the Dual Inhibition of Carbonic Anhydrase and Cyclooxy-
genase by Dihydrothiazole Benzensulfonamides
a‡
a*‡
a
b
c
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Rita Meleddu , Simona Distinto , Filippo Cottiglia , Rossella Angius , Marco Gaspari , Domenico
c
a
d
a
a
a
Taverna , Claudia Melis , Andrea Angeli , Giulia Bianco , Serenella Deplano , Benedetta Fois , Sonia
e
e
f
f
g
Del Prete , Clemente Capasso , Stefano Alcaro , Francesco Ortuso , Matilde Yanez, Claudiu T.
d*
a
Supuran and Elias Maccioni .
a
Department of Life and Environmental Sciences, University of Cagliari, Via Ospedale 72, 09124 Cagliari, Italy
b
Laboratorio NMR e Tecnologie Bioanalitiche, Sardegna Ricerche, 09010 Pula, Cagliari, Italy
c
Department of Experimental and Clinical Medicine, “Magna Græcia” University of Catanzaro, Campus ‘S. Venuta’, Viale
Europa, 88100 Catanzaro, Italy
d
Dipartimento NEUROFARBA, Sezione di Scienze Farmaceutiche, Università degli Studi di Firenze, Sesto Fiorentino,
Florence, Italy
e
Istituto di Bioscienze e Biorisorse, CNR, Via Pietro Castellino 111, 80131, Napoli, Italy
f
Dipartimento di Scienze della Salute, Università Magna Graecia di Catanzaro, Campus ‘S. Venuta’, Viale Europa, 88100
Catanzaro, Italy
g
Department of Farmacology, Faculty of Pharmacy, University of Santiago de Compostela (USC), Santiago de Compostela,
Spain
KEYWORDS: dual inhibitors, hCA XII, hCA II, hCOX-2, tumor, molecular docking.
ABSTRACT:
A novel series of of 4ꢀ[(3ꢀphenylꢀ4ꢀarylꢀ2,3ꢀdihydroꢀ1,3ꢀthiazolꢀ2ꢀylidene)amino]benzeneꢀ1ꢀsulfonamides
(EMAC10111 aꢀg) was synthesized and assayed towards both human Carbonic Anhydrase isozymes I, II, IX, and XII and Cyꢀ
clooxygenase isoforms. The majority of these derivatives preferentially inhibit hCA isoforms II and XII and hCOXꢀ2 isozyme, inꢀ
dicating that 2, 3, 4ꢀtri substituted 2,3ꢀdihydrothiazoles is a promising scaffold for the inhibition of hCA isozymes and of hCOXꢀ2
enzyme. The nature of the substituent at the dihydrothiazole ring position 4 influenced the activity and selectivity toward both enꢀ
zyme families. EMAC1011g resulted as the best performing compound toward both enzyme families and exhibited preferential acꢀ
tivity toward hCA XII and hCOXꢀ2 isozymes.
The potential of human Carbonic anhydrases (hCAs) and huꢀ
manCyclooxygenase (hCOX) dual inhibitors for the treatment
of cancer is an attractive yet challenging goal in the field of
medicinal chemistry. CAs are a class of wellꢀstudied metalꢀ
loenzymes that are widely distributed in all living organisms.
COXꢀdependent evasion of immunity has been observed in
31
tumors. Therefore, hCOX inhibitors could be considered as
adjuvant agents for the therapy of different cancers. On the
basis of the above, the identification of dual inhibitors of
hCOX and hCA might lead to highly efficient anticancer
agents, capable of simultaneously interacting with two differꢀ
ent tumor metabolic pathways. Such compounds are intrinsiꢀ
cally advantageous, as they are less prone to induce drug reꢀ
sistance, drugꢀdrug interactions and, not last, they might inꢀ
crease drugꢀcompliance. A key step for the design of dual inꢀ
hibitors is represented by the identification of the common
pharmacophoric features between hCAs inhibitors (hCAIs)
and hCOX inhibitors (hCOXIs), particularly hCOXꢀ2Is.
1-4
These enzymes are encoded by seven different genes famiꢀ
5ꢀ8
lies, αCA, βCA, γCA, δCA, ζCA, ηCA and θCA.
Sixteen
αCA isozymes have been identified in humans so far, each9
differing for cellular localization and tissue distribution.
Thus, cytosolic forms (hCA I–III, VII), membraneꢀbound
(
hCA IV, IX, XII, and XIV), mitochondrial form (hCA V),
9
and secreted (hCA VI) isozymes can be distinguished. The
role of hCAs in the regulation of hypoxicꢀtumors pH has been
extensively reported
idated targets for cancer therapy.
relevant role of hCOX 1 and 2 in different tumors has been
4
, 10ꢀ19
and isoforms II, IX and XII are valꢀ
2
0ꢀ22
On the other hand, the
23ꢀ26
outlined.
Celecoxib, a selective hCOXꢀ2 inhibitor, has
been approved by FDA for adjuvant treatment of patients with
27
familial adenomatous polyposis. Furthermore, the role of
Aspirin as an adjuvant agent in the prevention and treatment of
2
8ꢀ30
several solid tumors has been reported.
Furthermore, a
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Compounds EMAC10111 a-g were characterized by means of
a
O
O
O
O
O
2
O
analytical and spectroscopic methods (Figures S2ꢀ22 and Taꢀ
ble S1ꢀ2) and then submitted for biological evaluation toward
hCA isoforms I, II, IX, and XII and hCOX isozymes 1 and 2
(Table 1). Acetazolamide (AAZ) was chosen as reference
compound for hCA activity while indomethacin, diclofenac,
FR122047, nimesulide, and DuP 697 were selected as referꢀ
ence compounds for hCOX activity.
1
2
3
4
5
6
7
8
9
S
S
S
H
2
N
H N
H
2
N
CH3
O
N
N
N
N
CF
3
CF
3
N
celecoxib
valdecoxib
SCꢀ236
H
3
C
Cl
b
O
S
O
O
S
O
O
O
O
N
O
N
O
O
S
O
S
S
S
S
H
2
N
H
2
N
With respect to the hCA inhibition, the majority of EMAC deꢀ
rivatives exhibited a preferential activity toward the isoforms
hCA II and hCA XII. Interestingly compound EMAC10111b,
bearing a 2,4ꢀdiꢀchlorophenyl moiety in the position 4 of the
dihydrothiazole ring, exhibited the highest activity toward
hCA II with a Ki value equal to 0.053ꢀM. All the other comꢀ
pounds, with the exception of EMAC10111f, resulted as alꢀ
most equipotent in the inhibition of hCA II with Ki values
ranging from 0.28 to 0.86 ꢀM.
S
H2
S
N
O
H
2
N
NH
2
NH
C
N
N
H
C
Cl
C
CH
3
3
CH
3
N
CH
3
Cl
O
2
H C
N
O
CH
3
1
1
1
1
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H
acetazolamide
methazolamide
dorzolamide
dichlorphenamide
Figure 1. Structurally representative sulfonamideꢀbased a)
COXꢀ2 selective and b) hCA inhibitors.
In this respect, it could be evident that the sulfonamide moiety,
3
2ꢀ34
although with different specific roles,
cal feature of several inhibitors of hCAs and hCOXꢀ2 (Figure
).
The sulfonamide moiety is widely represented within the
is a common chemiꢀ
Compounds EMAC10111a, b, and d were the most potent for
the inhibition of hCA I isoform. EMAC10111a was the most
active toward hCA IX, but, on the other hand, it demonstrated
to be one of the less selective derivatives toward a specific
hCA isoform, within the studied compounds. With respect to
the hCA XII isoform, compound EMAC10111g, bearing a
thiophene substituent in the position 4 of the dihydrothiazole
ring, was identified as the most potent and selective inhibitor,
with a Ki value of 0.06ꢀM and with a selective index (Ki hCA
II/Ki hCA XII) higher than 10 folds.
1
3
3, 35, 36
hCAIs.
Moreover it is a synthetically accessible and
versatile scaffold, that can be appropriately decorated to
achieve isozyme selectivity.
2
1, 35, 37ꢀ41
In the case of hCOXꢀ2Is
a benzeneꢀsulfonamide or a benzeneꢀsulfonyl methyl moiety is
present in most of the active compounds in order to occupy a
hydrophilic pocket that is made accessible mainly by the subꢀ
stitution of the hCOXꢀ1 Ile523 residue by a smaller valine in
hCOXꢀ2. The substitution of the residues Ile434 and His513 of
COXꢀ1 with valine and arginine in hCOXꢀ2, respectively, furꢀ
ther contributes to enlarge the hydrophilic pocket and to difꢀ
When tested toward the two isoforms of hCOX, none of the
new compounds exhibited activity on the hCOXꢀ1 isozyme up
to the concentration of 25ꢀM. Unfortunately, at higher conꢀ
centrations the compounds precipitated from the test solution.
On the contrary, all compounds, except for EMAC10111d and
EMAC10111f, were active toward the COXꢀ2 at concentraꢀ
tions comparable with those of the reference inhibitors indoꢀ
methacin, diclofenac and nimesulide.
3
2
ferentiate the two isozyme inhibitors sensitivity. Prompted
35,
by these considerations and pursuing our studies on hCAIs
4
2ꢀ44
we have designed and synthesized a small library of 4ꢀ[(3ꢀ
phenylꢀ4ꢀarylꢀ2,3ꢀdihydroꢀ1,3ꢀthiazolꢀ2ꢀ
ylidene)amino]benzeneꢀ1ꢀsulfonamides (EMAC10111 a-g)
and evaluated their activity against the hCA I, II, IX, and XII
isozymes as well as their inhibition activity toward hCOX 1
and 2 isoforms. These compounds represent a new example of
hybrid structures with CA inhibitory activity.
pathway toward compounds EMAC10111 a-g consists of the
reaction of equimolar amounts of 4ꢀamidobenzensulfonamide
with phenylꢀisothiocyanate in refluxing 2ꢀpropanol (Scheme
1). The obtained 1ꢀphenylꢀ3ꢀ(4ꢀsulphamoylphenyl)thiourea
was further reacted with the appropriate alphaꢀhaloketone to
give the desired compounds in good yields.
Among the new derivatives, EMAC10111g resulted as the
most potent COXꢀ2 inhibitor, with an IC50 equal to 12.61 ꢀM.
Interestingly, EMAC10111g was the most selective inhibitor
of hCA XII indicating that the presence of the thiophene group
in the position 4 of the dihydrothiazole could be optimal for
the interaction with both COXꢀ2 and hCA XII. To achieve a
better understanding on the recognition of EMAC10111g by
both targets, and to obtain useful information to further develꢀ
op such compounds as tumor hCAs and COXꢀ2 dual inhibiꢀ
tors, molecular modeling studies were performed. Due to the
presence of the double bond between the amino benzene sulꢀ
fonamide and the dihydrothiazole moieties, merging known
45, 46
The synthetic
Scheme 1. Synthetic pathway to compounds EMAC10111
a-g.
47, 48
theoretical approaches,
the population of “E” and “Z”
O
isomers was preliminarily investigated. Conformational search
(Supporting information) was carried out on both
EMAC10111g isomers. The internal energy of each generated
conformer was considered in a Boltzmann analysis computed
at 300 K. Interestingly theoretical results indicated the “Z”
isomer population of about 100%, basically discarding the
presence of “E” isomer. As a consequence, further molecular
modeling was carried out taking into account the (Z)-
EMAC10111g configuration only.
O
S
O
S
NH
2
O
O
S
O
NH
2
2
H N
i
ii
N
S
NH
C
S
N
NH
2
NH
Ar
EMAC 10111 a-g
Ar = 4-ClPh, 2,4-ClPh, 4-BrPh, 4-CH
3
Ph, 4-OCH
3
Ph, Ph, Thiophene-2-yl
Docking protocols were first validated with self and crossꢀ
docking experiments. In particular, the validation highlighted
the ability of all settings protocols to reproduce, with acceptaꢀ
ble RMSD, experimental binding mode of most of ligands.
Reagents and conditions: (i) phenylꢀisothiocyanate, 2ꢀ
propanol, reflux; (ii), αꢀhaloketone, RT/50°C, 1−2 h.
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0
Furthermore, all methods clearly confirmed that high selective
hCOXꢀ2Is cannot be docked inside hCOXꢀ1.
both the previously applied 3F8E and the 3K34 crystal (resoꢀ
51
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6
7
8
9
1
1
1
1
1
1
1
1
1
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lution 0.9 Å), where the His64 shows an alternate conforꢀ
mation compared to the previously considered 3F8E. The new
receptor model improved crossꢀdocking RMSD results. Conꢀ
cerning the validation, it was observed that while the docking
Therefore, not surprisingly, we observed that (Z)-
EMAC10111g was not able to recognize hCOXꢀ1 active site,
thus corroborating biological results. On the contrary, the puꢀ
tative binding mode depicted in Figure 2 shows that the comꢀ
pound can be accommodated into hCOXꢀ2 pocket. The theoꢀ
retical complex resulted stabilized by hydrophobic interactions
with several residues such as Val89, Pro86, Leu123, Tyr115,
Ala527, Val116, Tyr355, Leu531, Leu83, Pro84. Furthermore,
the sulfonamide moiety was involved in a hydrogen bond with
Leu82. Finally, the aromatic moieties interacted with Lys83
and Arg120, which acts as channel gate that opens hCOX acꢀ
2
+
program was able to reproduce the binding mode of the Zn
interacting portion, with both the ion and the other catalytic
site residues. On the contrary, different binding conformations,
due to the absence of anchoring residues, were observed when
the solvent accessible ligand portion was docked. Furtherꢀ
more, in our theoretical protocol, we considered the receptor
without waters and other agents, such as glycerol and ethylene
glycol, used for crystallization. Considering that these latter
compounds often occupy the entrance cavity in the crystals,
more space was available to accommodate the cap of docked
hCAIs. The binding mode of most promising compound (Z)-
EMAC10111g in all isoform showed how the compound was
able to reach the catalytic site with the benzene sulfonamide
group. Furthermore, despite the bulky cap exposed to solvent,
the “Z” configuration allowed for a Y shaped geometry of the
three aromatic groups that can, therefore, be accommodated in
hCA II, IX, and XII. However, when docked in hCA II, the
cap aromatic rings are both pushed toward one side (Figure
3c). The reason was mainly addressed to the presence of
Phe131, which is replaced by an Ala by a Val in hCAXII and
hCAIX respectively.
0
1
2
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0
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0
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3
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0
49
tive site.
Figure 2. 3D representation of the putative binding mode obꢀ
tained by docking experiment of (Z)-EMAC10111g into
hCOXꢀ2 and 2D representation of the complex stabilizing inꢀ
teractions with the binding site residues.
Our data indicated that 4ꢀ[(3ꢀphenylꢀ4ꢀarylꢀ2,3ꢀdihydroꢀ1,3ꢀ
thiazolꢀ2ꢀylidene)amino]benzeneꢀ1ꢀsulfonamide derivatives
could be considered as a promising scaffold for the dual inhiꢀ
bition of hCA and hCOXꢀ2 enzymes. The information on the
putative binding modes in both targets and relative isoforms of
the newly synthetized inhibitors, encourage us to further inꢀ
vestigate and optimize these derivatives in order to improve
their activity and selectivity.
The hCA isoforms docking simulations were performed with a
4
3, 44
previously applied protocol.
We improved cross docking
validation and compared the previously utilized crystal strucꢀ
tures with the newest and with better resolution pdb entries.
While in the case of hCA XII and hCA IX we did not change
the receptor, when hCA II was investigated, we considered
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0
Figure 3. 3D representation of the putative binding mode obtained by docking experiment of (Z)-EMAC1011g into a) hCA XII, b)
hCA IX and c) hCA II. Below each is depicted the relative 2D representation of the complexes stabilizing interactions with the resꢀ
idues of the binding site.
Table 1. Inhibition data towards hCA I, II, IX, XII, and hCOX 1 and 2 isozymes of compounds EMAC10111 a-g.
O
N
O
N
N
N
S
S
O
O
S
S
S
NH
2
NH
2
R
EMAC 10111 (a-f)
EMAC 10111g
Ki(µM)
hCA II
0.28
IC50 (µM)
Compound EMAC
R
hCA I
0.49
0.75
3.46
0.96
3.37
8.02
9.07
hCA IX
1.25
hCAXII
0.65
hCOXꢀ1
hCOXꢀ2
16.21
18.32
19.73
*
1
1
1
1
0111a
0111b
0111c
0111d
4ꢀCl
2,4ꢀCl
4ꢀBr
4ꢀCH3
*
*
*
*
*
*
*
0.053
0.32
2.78
0.34
2.44
0.80
0.26
2.26
0.78
10111le
10111f
4ꢀOCH3
0.28
2.25
0.84
21.78
*
H
//
3.10
3.54
0.30
1
0111g
0.86
3.43
0.06
12.61
Reference compounds
AAZ
/
/
/
/
/
/
0.25
0.01
0.02
0.006
/
/
Indomethacin
Diclofenac
FR122047
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
12.16
18.23
0.09
***
35.20
23.62
**
Nimesulide
DuP 697
23.14
0.12
22.61
*
Inactive at 25 µM (highest concentration tested). At higher concentrations, the compounds precipitate.
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*
* Inactive at 100 µM (highest concentration tested). At higher concentrations, the compound precipitates.
** Inactive at 500 µM (highest concentration tested). At higher concentrations, the compound precipitates.
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*
Anhydrase Isozymes. Bioorg. Med. Chem. 2013, 21 (6), 1570ꢀ
582.
8)
ASSOCIATED CONTENT
Supporting Information
1
(
Supuran, C. T. Carbonic Anhydrases: Novel
Therapeutic Applications for Inhibitors and Activators. Nat. Rev.
Drug Discov. 2008, 7 (2), 168ꢀ181.
Experimental procedures and compounds characterization are reꢀ
portꢀed (PDF). The Supporting Information is available free of
charge on the ACS Publications website at DOI:
(9)
Supuran, C. T. In Carbonic Anhydrases as Drug
Targets: General Presentation, John Wiley & Sons, Inc.: 2009;
pp 15ꢀ38.
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AUTHOR INFORMATION
Corresponding Author
(10)
Abbate, F. Carbonic Anhydrase Inhibitors: E7070, A
Sulfonamide Anticancer Agent, Potently Inhibits Cytosolic
Isozymes I and II, and Transmembrane, TumorꢀAssociated
Isozyme IX. Bioorg. Med. Chem. Lett. 2004, 14, 217ꢀ223.
*
*
Eꢀmail: claudiu.supuran@unifi.it.
Eꢀmail: s.distinto@unica.it.
(11)
Dorai, T. Role of Carbonic Anhydrases in the
Progression of Renal Cell Carcinoma Subtypes: Proposal of a
Unified Hypothesis. Cancer Invest. 2006, 24, 754ꢀ779.
Author Contributions
These authors contributed equally. The manuscript was written
through contributions of all authors. All authors have given apꢀ
proval to the final version of the manuscript.
‡
(12)
Garaj, V. Carbonic Anhydrase Inhibitors: Synthesis
And Inhibition of Cytosolic/TumorꢀAssociated Carbonic
Anhydrase Isozymes I, II, and IX with Sulfonamides
Incorporating 1,2,4ꢀTriazine Moieties. Bioorg. Med. Chem. Lett.
2004, 14, 5427ꢀ5433.
ACKNOWLEDGMENT
(13)
Ilies, M. A. Carbonic Anhydrase Inhibitors. Inhibition
The authors wish to acknowledge the “Ufficio Valorizzazione dei
Risultati della Ricerca” of Sardegna Ricerche Technological Park,
Pula (CA) – Italy. The authors also thank the COST action
CA15135 (Multitarget Paradigm for Innovative Ligand Identificaꢀ
tion in the Drug Discovery Process MuTaLig) for support.
of TumorꢀAssociated Isozyme IX by Halogenosulfanilamide and
Halogenophenylaminobenzolamide Derivatives. J. Med. Chem.
2003, 46, 2187ꢀ2196.
(14)
Zhou, Y.; Mokhtari, R. B.; Pan, J.; Cutz, E.; Yeger, H.
Carbonic Anhydrase II Mediates Malignant Behavior of
Pulmonary Neuroendocrine Tumors. Am. J. Respir. Cell Mol.
Biol. 2015, 52 (2), 183ꢀ92.
(15)
PerezꢀSayans, M.; GarciaꢀGarcia, A.; Scozzafava, A.;
Supuran, C. T. Inhibition of VꢀATPase and Carbonic Anhydrases
as Interference Strategy with Tumor Acidification Processes.
Curr. Pharm. Des. 2012, 18 (10), 1407ꢀ1413.
ABBREVIATIONS
hCOX, human cyclooxygenase; hCA, human carbonic anhydrase;
hCOXIs, human cyclooxygenase inhibitors; hCAIs, human carꢀ
bonic anhydrase inhibitors.
(16)
Swietach, P.; Hulikova, A.; VaughanꢀJones, R. D.;
Harris, A. L. New Insights into the Physiological Role of
Carbonic Anhydrase IX in Tumor pH Regulation. Oncogene
2
010, 29 (50), 6509ꢀ6521.
(17) Supuran, C. T.; Winum, J.ꢀY. Carbonic Anhydrase IX
Inhibitors in Cancer Therapy: an Update. Future Med. Chem.
015, 7 (11), 1407ꢀ1414.
18) Svastova, E. Hypoxia Activates the Capacity of Tumorꢀ
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