Carbonic Anhydrase Glycoinhibitors belonging to the Aminoxysulfonamide Series

Article abstract of DOI:10.1021/acsmedchemlett.5b00175

Abstract A general approach for the synthesis of carbonic anhydrases glycoinhibitors belonging to an aminoxysulfonamide series is presented using a Ferrier sulfonamidoglycosylation reaction on glycals. All the compounds showed good in vitro inhibitory activity against four human carbonic anhydrase isoforms, with selectivity against the cytosolic (hCA II) vs the tumor associated (hCA IX and XII) enzymes.

Full text of DOI:10.1021/acsmedchemlett.5b00175

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Letter
Carbonic Anhydrase Glycoinhibitors
belonging to the Aminoxysulfonamide Series
Joanna Ombouma, Daniella Vullo, Pascal Dumy, Claudiu T Supuran, and Jean-Yves Winum
ACS Med. Chem. Lett., Just Accepted Manuscript • Publication Date (Web): 02 Jun 2015
Downloaded from http://pubs.acs.org on June 4, 2015
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Carbonic Anhydrase Glycoinhibitors belong-
ing to the Aminoxysulfonamide Series
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†
‡
†
§
†
Joanna Ombouma, Daniella Vullo, Pascal Dumy, Claudiu T. Supuran, * and JeanꢀYves Winum *
†
Institut des Biomolécules Max Mousseron (IBMM) UMR 5247 CNRSꢀENSCMꢀUniversité de Montpellier, Bâtiment de Recherche Max
Mousseron, Ecole Nationale Supérieure de Chimie de Montpellier, 8 rue de l’Ecole Normale, 34296 Montpellier Cedex, France.
‡
Laboratorio di Chimica Bioinorganica, Polo Scientifico, Università degli Studi di Firenze, Sesto Fiorentino, Florence, Italy.
§
Neurofarba Department, Section of Pharmaceutical and Nutriceutical Sciences, Università degli Studi di Firenze, Via Ugo Schiff 6, 50019
Sesto Fiorentino, Florence, Italy.
KEYWORDS: glycoinhibitor, carbonic anhydrasse, aminoxysulfonamide, zinc binding function, Ferrier rearrangement
ABSTRACT: A general approach for the synthesis of carbonic anhydrases glycoinhibitors belonging to an aminoxysulfonamide
series is presented using a Ferrier sulfonamidoglycosylation reaction on glycals. All the compounds showed good in vitro inhibitoꢀ
ry activity against four human carbonic anhydrase isoforms, with selectivity against the cytosolic (hCA II) vs the tumor associated
(hCA IX and XII) enzymes.
The field of carbonic anhydrase (hCA, EC 4.2.1.1) inhibitors
is still currently a very active area of research taking account
of the involvement of different isoforms of this enzyme (15
known in human) in major pathological processes found in
glaucoma, CNS diseases, obesity and cancer. Nowadays, the
development of new concepts for designing new families of
compounds, capable of blocking the process catalyzed by
our research on the aminoxysulfonamide motif as new zinc
binding function.
In this paper we wish to disclose our latest results towards
the synthesis and activity of 2,3ꢀunsaturated glycosides in
aminoxysulfonamide series using a Ferrier sulfonamidoglyꢀ
1
3
cosylation reaction with NOBF as catalyst.
4
these enzymes (ie reversible hydration of CO into bicarboꢀ
2
nate and a proton) remains a major concern of the research
1
teams working in this field.
The sugar approach is currently considered as one of the
most successful strategy for designing small molecule CAIs
1
ꢀ5
targeting all isoforms known to date.
Throughout the last
years this approach attracted considerable attention especialꢀ
ly in the in the field of tumor associated carbonic anhydrase
isoforms hCA IX and hCA XII. Extensive searches in the
design of selective glycoinhibitors have been accomplished,
some of them having shown interesting inhibition profiles
and promising antitumoral effects in preclinical experiꢀ
Figure 1. 2,3ꢀunsaturated glycosides in hydroxysulfamide series (A)
and aminoxysulfonamide series (B).
3
ꢀ8
ments. Moreover, efforts have been made to find different
synthetic methodology to prepare CAIs incorporating sugar
moieties such as for example the use of click reactions and
In order to prepare glycoinhibitor series
B, aminooxysulfoꢀ
namide synthons were synthesized according to scheme 1.
Direct sulfamoylation of the commercially available NꢀCbzꢀ
hydroxylamine with sulfamoyl chloride (prepared in situ by
reaction of chlorosulfonyl isocyanate (CSI) with formic acid)
led to 1a with high yield. Compound 1b was also prepared as
9
ꢀ10
its variant,
or by using Ferrier sulfonamidoglycosylation
1
1
of glycals. Recently, we reported effective synthetic
methodology allowing the access to 2,3ꢀunsaturated glycoꢀ
sides in hydroxysulfamide series
lated glycals platform.(Figure 1) By using the nonꢀmetallic
catalyst nitrosyl tetrafluoroborate we developed
straightforward Ferrier sulfamidoglycosylation allowing us
to access to original and effective CAIs in ꢀglycosylꢀ
In order to develop chemical
diversity using the same carbohydrate scaffolds, we focused
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A starting from a peracetyꢀ
reference compound for inhibitory assay. Reaction of
sulfamoylchloride (prepared in situ by reaction of CSI with
benzyl alcohol) on ꢀBocꢀhydroxylamine followed by acidic
NꢀCbzꢀ
a
N
N
Nꢀ
deprotection with TFA solution in dichloromethane afforded
1b in good yields. (scheme 1)
1
2
hydroxysulfamides series.

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Scheme 1. Synthesis of 1a and 1b
Table 1. Optimized results for the Ferrier sulfonamidoglycosylation
and deprotection steps.
Glycals
[a]
[b]
Step 1
Steps 2+3
2
yield %/ α:β ratio/ reaction time
ꢀ: 70 / 35:65 / 30 min.
MW: 70 / 35:65 / 6 min.
ꢀ: 75 / 43:57 / 30 min.
MW: 75 / 43:57 / 6 min.
ꢀ: 80 / 48:52 / 30 min.
MW: 86 / 48:52 / 6 min.
ꢀ: 87 / 47:53 / 30 min.
MW: 87 / 47:53 / 6 min.
ꢀ: 90 / 47:53 / 30 min.
MW: 93 / 47: 53 / 6 min.
yield %/ α:β ratio
2
a
78 / 35:65
2b
78 / 43:57
78 / 48:52
78 / 47:53
78 / 47:53
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2e
[
a] Reaction conditions: glycal (1) 1 equiv., 1a 1.1 equiv., NOBF
4
0.1
Glycoinhibitors
scheme 2. Starting from peracetylated glycals
the same methodology previously reported by our group on
5
were then synthesized as depicted in
equiv., CH Cl . [b] 10% Pd/C, Et SiH, MeOH then NH /MeOH
2
2
3
3
2
, and using
Compounds 1a-1b, 4a-4e and 5a-5e as well as clinically
used acetazolamide (standard compound) were tested for
their inhibitory activity against the two cytosolic CA
isoforms hCA I and II and the two membrane tumorꢀ
ssociated isoforms hCA IX and XII using a StoppedꢀFlow,
1
2
Ferrier sulfamidoglycosylation, we were able to obtain
compounds by reacting with 1a in the presence of
NOBF catalyst. By optimizing the amount of NOBF , the
3
2
4
4
best results were obtained with 0.1 equivalent of catalyst
leading to yields ranging from 70 to 90%. (Table 1)
16
CO2 Hydration Assay Method. Results are reported in
Table 2.
Scheme 2. Synthesis of glycoinhibitors
mide series
4 and 5 in aminoxysulfonaꢀ
Table 2. Inhibitory activity of compounds 4a-4e and 5a-5e against the
four CA isoforms: hCA I, II, IX and XII determined by a Stopped-
16
Flow, CO
2
Hydration Assay Method. Selectivity ratios for the inhibi-
tion of the tumor-associated (hCA IX and XII) over the cytosolic
(hCA II) isozyme are also reported.
a
bK
I
(nM)
Selectivity ratio
b
c
c
hCA I
hCA II
hCA IX
hCA XII
K
I
K
I
hCA
hCA
II/ K
I
II/ K
hCA
IX
I
hCA
XII
AAZ
250
31
300
90
69
37
55
88
552
250
50
12
5
1
3
5
6
4
5
8
25
57
77
90
75
75
86
86
85
61
91
74
61
6
49
556
39
43
43
24
24
58
80
41
29
56
0.48
0.09
0.01
0.03
0.07
0.08
0.05
0.06
0.10
3.55
0.03
0.08
0.06
2
1
a
0.10
0.001
0.08
0.12
0.14
0.17
0.21
0.14
2.71
0.08
0.21
0.07
1b
4a
4
b
Activation of the reaction with microwaves did not allow to
improve the yield of the coupling compared with thermal
activation, but an important reduction of reaction time was
observed by 5 folds.
4
c
4
d
4e
5
a
Compounds
with a slight selectivity for the
selectivity was different of the sulfamidoglycosylation
where the ꢀanomer was mainly obtained suggesting a kiꢀ
3
were obtained as a mixture of
α
and
β
anomer
5b
217
3
6
5
c
β
ꢀanomer. Interestingly, the
12
5d
5e
64
60
4
α
14
a
netic control of the reaction.
Errors in the range of ±5−10% of the reported value from three different
b c
Then removal of the Cbz group was achieved using Pd/C and
determinations. Full length, cytosolic isoform. Catalytic domain, recomꢀ
binant enzyme.
1
5
Et SiH in methanol to yield compounds
4
.
Final deprotecꢀ
tion of acetate groups with methanolic solution of ammonia
led to glycoinhibitors in quantitative yields.
Full characterisation of compounds and
ratio of α/β anomers were unambiguously confirmed using
3
5
Data of Table 2 show that compounds 4 and 5 reported here
do show significant CA inhibitory properties. The cytosolic isoꢀ
form hCA I was inhibited with inhibition constants ranging beꢀ
tween 37 and 552 nM. There were no regularities in the inhibitory
power of the acetylated/deacetylated derivative, and it seems that
the nature of the glycal moiety is the main factor influencing the
CA inhibitory properties of these compounds. hCA II; the physioꢀ
logically dominant isoform, was also the most inhibited one by
3
,
4
5 as well as the
1
13
H, C, 2D COSY pulseprog COSYGPQF (with gradient
quadrature mode; Time domain size, TD = 2k; Relaxation
delay, d1 = 1.5s and scan number = 1), HMQC experiments.
Stereochemical assignment of the major diastereoisomer was
confirmed by NOESY pulseprog NOESYGPPHPP experiꢀ
ments (Time domain size, TD= 2k; the mixing time d8= 0.7s;
the relaxation delay d1=1.5s and Scans Number=16), by
observing a NOE interaction between Hꢀ1 and Hꢀ5 for the βꢀ
anomer (absent for the α anomer). NOE effect between Hꢀ1
and Hꢀ5 allowed to discriminate between both anomers, the
these compounds, which showed K s in the range of 3 – 8 nM
I
(except 5b which was a much weaker inhibitor, with a K of 217
I
nM).
Thus, most of the scaffolds present in these derivatives led to
highly efficient CA II inhibitors (better compared to the standard
1
chemical shifts in H NMR allowing to assign which one is
predominant. (see ESI)
sulfonamide drug acetazolamide AAZ, which had a K of 12 nM
I
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ACS Medicinal Chemistry Letters
against this isoform). The two tumorꢀassociated isoforms, hCA IX
anhydrases: how to design specific drugs targeting 15 different
isoforms? Chem. Rev. 2012, 112, 4421ꢀ4468.
3) Supuran, CT. Structureꢀbased drug discovery of carbonic
anhydrase inhibitors. J Enzyme Inhib. Med. Chem. 2012, 27, 759ꢀ
72.
4)
1
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and XII; were less well inhibited compared to hCA II; and the KIs
ranged between 61 and 91 nM for hCA IX, and 24 – 80 nM
against hCA XII. Thus, the glycoinhibitors reported here were
effective but not highly potent as inhibitors of the transmembrane
isoforms hCA IX and XII. They are also nonꢀselective for the
inhibition of the tumorꢀassociated versus the cytosolic isoform
hCA II; being thus CA IIꢀselective inhibitors (Table 2).
(
7
(
Winum, JꢀY.; Poulsen, SA.; Supuran, CT. Therapeutic apꢀ
plications of glycosidic carbonic anhydrase inhibitors. Med. Res. Rev.,
2009, 29, 419ꢀ435.
(
5)
Winum, JꢀY.; Colinas, PA.; Supuran, CT. Glycosidic carꢀ
Herein we reported a series a carbonic anhydrase glyꢀ
coinhibitors in aminoxysulfonamide series. These comꢀ
pounds were prepared according to a Ferrier sulfonamidoꢀ
bonic anhydrase IX inhibitors: a sweet approach against cancer.
Bioorg. Med. Chem. 2013, 21, 1419ꢀ1426.
(
6)
Touisni, N.; Maresca, A.; McDonald, PC.; Lou, Y.; Scozꢀ
0
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zafava, A.; Dedhar, S.; Winum, JꢀY.; Supuran, CT. Glycosyl coumaꢀ
rin carbonic anhydrase IX and XII inhibitors strongly attenuate the
growth of primary breast tumors. J. Med. Chem. 2011, 54, 8271ꢀ
glycosylation synthetic methodology using NOBF as cataꢀ
4
lyst.
Inhibition assays against relevant carbonic anhydrase
isoforms revealed the nanomolar activity of these inhibitors.
Selectivity and strong inhibitory activity with nanomolar
activity was observed against hCA II showing that aminoxyꢀ
sulfonamide moiety is a very good zinc binding function.
Future Xꢀray crystallography study should allow a better
understanding of the interaction between this new ZBF and
the active site that might be exploited for future investigation
of new potent and selective carbonic anhydrase inhibitors.
8
277.
7)
(
Lou, Y.; McDonald, PC.; Oloumi, A.; Chia, S.; Ostlund,
C.; Ahmadi, A.; Kyle, A.; Auf dem Keller, U.; Leung, S.; Huntsman,
D.; Clarke, B.; Sutherland, BW.; Waterhouse, D.; Bally, M.; Roskelꢀ
ley, C.; Overall, CM.; Minchinton, A.; Pacchiano, F.; Carta, F.; Scozꢀ
zafava, A.; Touisni, N.; Winum, JꢀY.; Supuran, CT.; Dedhar, S.
Targeting tumor hypoxia: suppression of breast tumor growth and
metastasis by novel carbonic anhydrase IX inhibitors. Cancer Res.
2011, 71, 3364ꢀ3376.
(
8)
Lock, FE.; McDonald, PC.; Lou, Y.; Serrano, I.; Chafe,
SC.; Ostlund, C.; Aparicio, S.; Winum, JꢀY.; Supuran, CT.; Dedhar,
S. Targeting carbonic anhydrase IX depletes breast cancer stem cells
within the hypoxic niche. Oncogene 2013, 32, 5210ꢀ5219.
ASSOCIATED CONTENT
Spectroscopic details of compounds 1, 3, 4 and 5 as well as copies
of spectra. This information is available free of charge via the
Internet at http://pubs.acs.org.
(
9)
Lopez, M.; Salmon, AJ.; Supuran, CT.; Poulsen, SA. Carꢀ
bonic anhydrase inhibitors developed through 'click tailing'. Curr.
Pharm. Des. 2010, 16, 3277ꢀ3287.
(10) Saada, MC.; Ombouma, J.; Montero, JL.; Supuran, CT.;
Winum, JꢀY. Thiolꢀene click chemistry for the synthesis of highly
effective glycosyl sulfonamide carbonic anhydrase inhibitors. Chem.
Commun. 2013, 49, 5699ꢀ5701.
AUTHOR INFORMATION
Corresponding Author
(
11) Colinas, PA.; Bravo, RD. Ferrier sulfonamidoglycosylation
of Dꢀglycals. Carbohydr. Res. 2007, 342, 2297ꢀ2302.
*
2
*
Email: jeanꢀyves.winum@univꢀmontp2.fr Phone: +33 467ꢀ147ꢀ
34. Fax: +33ꢀ467ꢀ144ꢀ344.
Email claudiu.supuran@unifi.it Phone: +39ꢀ055ꢀ4573005. Fax:
(12) Ombouma, J.; Vullo, D.; Supuran, CT.; Winum, JY. Ferrier
sulfamidoglycosylation of glycals catalyzed by nitrosonium tetraꢀ
fluoroborate: towards new carbonic anhydrase glycoinhibitors.
Bioorg. Med. Chem. 2014, 22, 6353ꢀ6359.
+39ꢀ055ꢀ4573385.
(
13) Winum, JꢀY.; Supuran, CT. Recent advances in the discovꢀ
Author Contributions
ery of zincꢀbinding motifs for the development of carbonic anhydrase
inhibitors. J. Enzyme Inhib. Med. Chem. 2015, 30, 321ꢀ324.
(14) Alegre, ML.; Diez, RP.; Colinas. PA. Experimental and
theoretical study of the conformational, vibrational and magnetic
The manuscript was written through contributions of all authors. /
All authors have given approval to the final version of the manuꢀ
script.
properties
enopyranosyl ethanesulfonamide. J. Molec. Struct. 2009, 919, 223ꢀ
26.
15) Mandal, P. K.; McMurray, J. S. PdꢀCꢀinduced catalytic
of
4,6ꢀdiꢀOꢀacetylꢀ2,3ꢀdideoxyꢀDꢀthreoꢀhexꢀ2ꢀ
2
Funding Sources
(
The authors would like to thank the Gabonese Ministry of Reꢀ
search for PhꢀD fellowship (to J.O.) and the CNRS/CNR (CoopInꢀ
tEER Program, Grant No. 131999, to J.ꢀY.W.) for financial supꢀ
port.
transfer hydrogenation with triethylsilane. J. Org. Chem. 2007, 17,
6599ꢀ6601.
(16) Khalifah, RG. The carbon dioxide hydration activity of
carbonic anhydrase. I. Stopꢀflow kinetic studies on the native human
isoenzymes B and C. J. Biol. Chem., 1971, 246, 2561ꢀ2573.
ABBREVIATIONS
hCA: human carbonic anhydrase
CAI : carbonic anhydrase inhibitor
DMA : dimethylacetamide
TFA : trifluoroacetic acid
ZBF : zinc binding function
REFERENCES
(
1)
Carbonic Anhydrases as Biocatalysts From Theory to Medꢀ
ical and Industrial Applications, ed. CT. Supuran, G. de Simone,
Elsevier, Amsterdam, 2015, pp 1ꢀ373.
(
2)
Alterio, V.; Di Fiore, A.; D'Ambrosio, K.; Supuran, C.T.;
De Simone, G. Multiple binding modes of inhibitors to carbonic
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