New hydroxypyrimidinone-containing sulfonamides as carbonic anhydrase inhibitors also acting as MMP inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.04.109

A set of benzenesulfonamide (BSA) derivatives bearing a hydroxypyrimidinone (HPM) moiety were synthesized and investigated for their inhibitory activity against several carbonic anhydrase (CA, EC 4.2.1.1) isozymes. They all revealed to be very potent inhibitors (nanomolar order) of the cytosolic CA I and II isozymes, but especially of the transmembrane, tumor-associated CA IX isozyme, a beneficial feature for a potential antitumor effect of these compounds. Further structure optimization aimed at improving the specificity of CA inhibition and enhancing their matrix metalloproteinase (MMP) inhibitory activity may also lead to new compounds with an attractive dual mechanism of action as antitumor agents.

Full text of DOI:10.1016/j.bmcl.2010.04.109

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3623–3627
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
New hydroxypyrimidinone-containing sulfonamides as carbonic anhydrase
inhibitors also acting as MMP inhibitors
M. Alexandra Esteves a, Osvaldo Ortet a, Anabela Capelo a, Claudiu T. Supuran b,
Sérgio M. Marques c, M. Amélia Santos c,
*
a LNEG, Unidade de Pilhas de Combustível e Hidrogénio, Estrada do Paço do Lumiar, 1649-038 Lisboa, Portugal
b Università degli Studi di Firenze, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, I-50019 Sesto Fiorentino (Firenze), Italy
c Instituto Superior Técnico, Centro de Química Estrutural, Av. Rovisco Pais 1, 1049-001 Lisboa, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
A set of benzenesulfonamide (BSA) derivatives bearing a hydroxypyrimidinone (HPM) moiety were syn-
thesized and investigated for their inhibitory activity against several carbonic anhydrase (CA, EC 4.2.1.1)
isozymes. They all revealed to be very potent inhibitors (nanomolar order) of the cytosolic CA I and II iso-
zymes, but especially of the transmembrane, tumor-associated CA IX isozyme, a beneficial feature for a
potential antitumor effect of these compounds. Further structure optimization aimed at improving the
specificity of CA inhibition and enhancing their matrix metalloproteinase (MMP) inhibitory activity
may also lead to new compounds with an attractive dual mechanism of action as antitumor agents.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 30 March 2010
Revised 23 April 2010
Accepted 24 April 2010
Available online 28 April 2010
Keywords:
Hydroxypyrimidinones
Carbonic anhydrase inhibitors
Sulfonamides
MMP inhibitors
Several isoforms of carbonic anhydrases (CAs, EC 4.2.1.1) are
considered important targets for the design of inhibitors with clin-
ical applications, namely for anti-glaucoma, anti-epileptic, and
obesity treatments, or even in oncology for tumor control and
imageology.1–4 Among them, the transmembrane tumor-associ-
ated isozyme IX and the cytosolic isozymes I and II have been
the object of major interest as druggable targets. Examples of po-
tent CA inhibitors are acetazolamide (AAZ) and ethoxzolamide
(EZA), compounds in clinical use, or indisulam (IND), in phase II
clinical trials as anticancer agent (see Fig. 1). All of these CA inhib-
itors contain a terminal primary sulfonamide (RSO2NH2, where R is
generally an aromatic/heteroaromatic moiety), as the zinc-binding
group (ZBG).3 The wide range of pharmaceutical applications of
this class of compounds justifies its continuing research to over-
pass side-effects or even to get further improvements in terms of
bioavailability, specificity, or eventual adjuvant effects.5,6
volved in carcinogenesis and tumor progression processes.7
Although the HA moiety (CONHOH) has been recognized as one
of the strongest ZBGs for the MMP inhibition (MMPi), there are
some drawbacks associated to its metabolic lability.1b Thus, there
is a recent search trend for effective MMP inhibitors with nonhydr-
oxamic ZBGs, including a variety of heterocycles, such as hydroxy-
pyrone/-pyridinones8,9 and pyrimidinetrione compounds.10
We have recently reported a small set of simple hydroxypyri-
midinones (1-hydroxy-2(1H)-pyrimidinones) (HPM), a kind of
endocyclic secondary HA, which proved to be more potent MMPi
(micromolar range)11 than simple primary HA (millimolar
range).12 That feature, together with the remarkable inertness of
these compounds to the in vivo metabolization, make HPM as po-
tential pharmacophore targets as alternatives to HAs for MMPi.
Thus, we have decided to explore new molecular entities, incorpo-
rating hydroxypyrimidinone (HPM) and BSA moieties, and to study
their biological potential. In this Letter, we report the synthesis of a
small series of such novel bifunctional derivatives (Fig. 1, com-
pounds 1–4), as well as their inhibitory properties against the cyto-
solic and tumor-associated carbonic anhydrase isozymes I, II and
IX. All these compounds contain the two key functions, BSA and
HPM, separated by different linkers, that mainly differ on the
chain-size (5–9 atom chain) and positioning of the amide linkage
between both functional groups. The rationalization of their CA
inhibitory profiles is aided by comparison with the reference inhib-
itors and a model compound, 5, as well as by modeling studies.
The conjugation of hydroxamic (HA) and benzenesulfonamide
(BSA) moieties in the same molecular entity, for dual inhibition
of matrix metalloproteinases (MMPs) and CAs, has been recently
reported and its potential pharmacologic interest has been out-
lined, since some members of MMP family are also known to be in-
Abbreviations: BSA, benzenesulfonamide; HPM, hydroxypyrimidinone; CA,
carbonic anhydrase; MMP, matrix metalloproteinase; ZBG, zinc-binding group;
HA, hydroxamic acid.
* Corresponding author. Tel.: +351 21 8419000; fax: +351 21 8464455.
E-mail address: masantos@ist.utl.pt (M.A. Santos).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.04.109

3624
M. A. Esteves et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3623–3627
SO2NH2
N
N
NH
H
CH3CONH
SO2NH2
N
Cl
S
S
O
O
AAZ
IND
SO2NH2
O
N
N
H
EtO
S
SO2NH2
H2NO2S
EZA
SPESB
O
O
O
NH2
HO
S
HO
N
N
N
N
O
H
N
( )
OCH3
( )
N
H
( )
n
m
N
H
5
m = 3, n = 0
m = 3, n = 2
m = 5, n = 0
m = 5, n = 2
(1
(2
(3
(4
)
)
)
)
O
O
5
Figure 1. Reference CA inhibitors (AAZ, EZA, IND, and SPESB), and the new compounds (1–5).
The target compounds (1–4) were synthesized by a multi-step
actions or hindrances with these inhibitors than CA I, allowing a
higher differentiation on the stability of their protein–ligand com-
plexes. As compared with the reference inhibitors, our compounds
showed in general slightly lower activities against CA II.
procedure, as depicted in Scheme 1.13 In the first step, N-protected
amino acids 6–7 were coupled14 with the commercially available
sulfonamides 8–9 using 1-hydroxybenzotriazole hydrate (HOBt)
and N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide hydrochlo-
ride (EDCꢀHCl) as coupling reagents to generate compounds 10–
13. Then, the benzyloxycarbonyl (Cbz) protecting group was re-
moved by catalytic hydrogenation to afford intermediates 14–17.
In the next step these compounds were reacted with 1-(benzyl-
oxy)-4-(10,20,40-triazol-10-yl)-2-(1H)-pyrimidinone 18, a hydroxy-
pyrimidinone derivative previously described,11,15 to afford the
O-protected hydroxypyrimidinone-sulfonamide ligands 19–22. Fi-
nally, removal of the benzyl protecting group by catalytic hydroge-
nation afforded the final compounds 1–4.
Concerning the inhibition of CA IX, all our compounds displayed
very strong activities, with KI values ranging from 3.4 to 8.5 nM.
These values are below the ones observed for the reference inhib-
itors, including the one with closest structural similarity, SPESB (KI
value of 18 nM), or even IND, the inhibitor in phase II clinical trials
against cancer (KI value of 24 nM). The CA IX/II selectivities are
determinant features for the interest of these new compounds,
and, unlike the reference compounds, they demonstrated to be
more selective for inhibiting the cancer-associated isozyme CA IX
than the ubiquitous CA II. These selectivities ranged from 1.5 (for
compound 3) to 9.2 (for 1), thus quite convenient for an eventual
cancer therapy.
Since the studied compounds include two functional groups
that potentially may bind the zinc (the HPM and BSA), we have also
studied a model compound, 5 (containing only the HPM). Thus, by
testing such compound without the BSA (the preferred ZBG in the
case of the CAs), we envisage to get some insight on eventual com-
petition between the HPM and the BSA moieties for the binding to
the catalytic zinc. The low inhibitory activities observed for 5 (with
The model compound 5 was synthesized by the same proce-
dure, starting with the reaction of the hydroxypyrimidinone deriv-
ative 18 with 6-aminohexanoic acid methyl ester hydrochloride16
and potassium carbonate to obtain the O-protected hydroxypyri-
midinone, 4-[5-(methyloxycarbonyl)-pentylamino]-1-(benzyloxy)-
2(1H)-pyrimidinone 23, which was then deprotected by catalytic
hydrogenation to afford ligand 5.
The new HPM–BSA conjugates (1–4) were tested for their inhib-
itory activities towards a set of physico-pathologically relevant hu-
man CAs (the ubiquitous cytosolic CA I and II, and the tumor-
associated isoform CA IX), as previously reported.7 All these hybrid
compounds presented high inhibitory activities (Table 1), with KI
in low nanomolar range (3.4–31 nM), although acting more effi-
ciently against CA IX, a beneficial feature for antitumor effects,
mainly for hypoxic tumors that overexpress this isozyme. Regard-
ing CA I, although this enzyme is usually one of the least inhibited
by the sulfonamides,3,17 in our case, the inhibitory activities for CA
I and II are quite similar. As compared with the reference inhibitors
(i.e., AAZ, IND, EZA, and N-(4-sulfamoylphenylethyl)-4-sulfamoylb-
enzamide (SPESB), with KI values ranging from 250 to 25 nM), in
general, our compounds revealed higher activities, although with
similar values. Regarding CA II, 3 was the most active compound
(KI = 13 nM), while 1 was the less active (KI = 31 nM). For this en-
zyme, the variation in inhibitory activities was higher than for
CA I. This fact suggests that, probably, CA II has more specific inter-
KI 43 and 270 lM for CA I and II, respectively, and higher than 1 lM
for CA IX) indicate that this chelating group hardly interacts with
the metal ion in the active site of the CAs, thus being unable to
block their activity. The inhibitory activity against CA II is compa-
rable but slightly lower than that found for acetohydroxamic acid
(KI = 47 l
M),18 most probably because the monodentate coordina-
tion to the Zn(II) ion by the primary hydroxamate moiety (depro-
tonated at the nitrogen atom) is hampered in the present case,
because the HPM is a secondary HA. Although the inhibition activ-
ity found for this HPM derivative is also in the same range of that
found for phenolic inhibitors, a different metal-binding type
should also be involved. In fact, the phenol–(metal–enzyme) inter-
action involves the OH moiety hydrogen-bonded to the zinc-bound
water/hydroxide ion,19 but the hydroxylic proton is quite more
acidic in HPM than in phenol (pKa ca. 7 and 10, respectively),20,21
thus suggesting that the HPM–(metal–enzyme) interaction should

M. A. Esteves et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3623–3627
3625
O
S
NH2
O
S
O
H
N
O
O
a
Cbz
( )
NH2
( )
Cbz
( )
n
N
H
m
( )
N
H
m
+
n
H2N
OH
O
10
: m = 3, n = 0
11: m = 3, n = 2
8
: n = 0
6
: m = 3
12
: m = 5, n = 0
: m = 5, n = 2
9: n = 2
7: m = 5
13
O
O
b
Bz
N
N
O
S
O
N
N
O
O
S
O
NH2
N
NH2
18
BzO
H
N
N
N
H
N
( )
( )
H2N
m
( )
n
( )
N
m
n
O
H
c
O
14: m = 3, n = 0
15
: m = 3, n = 2
16: m = 5, n = 0
17
19: m = 3, n = 0
b
: m = 5, n = 2
20
21
: m = 3, n = 2
: m = 5, n = 0
22: m = 5, n = 2
O
O
S
O
NH2
HO
N
N
H
N
( )
( )
N
m
n
H
O
1
2
: m = 3, n = 0
: m = 3, n = 2
3: m = 5, n = 0
4
: m = 5, n = 2
Scheme 1. Reagents and conditions: (a) HOBt, EDCꢀHCl, CH2Cl2, DMF, ꢂ10 °C to rt; (b) 10% Pd/C, H2, MeOH, rt; (c) THF, reflux.
Table 1
compounds. The models were docked into the crystal structures
of CA II and CA IX (respectively, entries 1G54 and 3IAI of the RCSB
Protein Data Bank),23 using the GOLD software,24 following a previ-
ously validated method.25 The docking results showed a major
binding feature, common to all ligands, which is due to the good
accommodation of the benzenesulfonyl moiety into the active site
of the CAs, and the coordination of catalytic zinc ion by the anionic
N-atom of the sulfonamide. Two H-bonds are also formed, one be-
tween its NH group and the hydroxyl group of Thr198 (numeration
according to the hCA II structure of UniProtKB database, entry
P00918),26 and another one between its sulfonyl oxygen and the
backbone NH moiety of the same residue. In Figure 2 are displayed
some of the docking results for CA II, which analysis clearly evi-
dences that all these compounds bind in very similar manners.
The benzene ring of the BSA moiety interacts at the hydrophobic
surface of the enzyme, namely with the residues Leu197, Val142
and Val121. The rest of the molecule is extended outwards the ac-
tive site, but, unexpectedly, it has the spacer bent over, which al-
lows the HPM moiety to interact with the hydrophilic part of the
cavity. The HPM moiety of the different ligands may form at least
one H-bond, namely through the carbonyl O-atom or the OH
groups. For 1 and 3, this H-bond is established between the
HPM-carbonyl and the hydroxyl group of Thr199, while for 2 and
4 that H-bonding involves the HPM-hydroxyl and the side-chain
carbonyl group of Asn67. In the case of 3, an additional H-bond
(which is not seen for the other compounds) is established be-
tween the HPM-NH group and the side-chain carbonyl of Asn67.
This extra H-bond may explain the highest inhibitory activity to-
wards CA II, observed for compound 3. In addition, all the com-
pounds, but 2, form H-bonds between the carbonyl oxygen atom
of the spacer chains (between the HPM and the sulfonamide
groups) and the NH2 group of Gln92.
Inhibitory activities of the target compounds (1–4) towards human CA I, II, and IX,
compared with the model compound 5 and the reference inhibitors
Compounda
KI (nM)
Selectiv.
CA IX/II
CA I
CA II
CA IX
m
n
1
2
3
4
3
3
5
5
0
2
0
2
23
22
27
22
31
28
13
25
3.4
3.5
8.5
4.1
>1000
25
34
24
18
9.2
8.1
1.5
6.2
5
4.3 ꢁ 104
250
25
2.7 ꢁ 105
AAZ
EZA
IND
12
8
15
5
0.48
0.25
0.62
0.28
31
40
SPESB
a
See Figure 1.
involve the anionic O-atom. Thus, for the BSA/HPM conjugates the
interaction with Zn(II) at the active site of the CAs should occur
through the BSA moiety. However, further favorable interactions
between the HPM group and other regions of these enzyme active
site should occur, in order to explain the high inhibitory activities
evidenced by these new compounds towards the tested enzymes.
Moreover, since the mechanism of the new CA inhibitors seems
to be determined by the sulfonamide moieties, they are most prob-
ably reversible noncompetitive inhibitors as well.2,22
The interaction of these conjugates with MMPs was not studied
herein because, based on preliminary studies with a series of sim-
ple HPM model compounds with a set of MMP isoforms (MMP-2,
-7, -9, -14), the inhibitory activities were found to be in the micro-
molar range (see Table S1 of Supplementary data).11 For the pres-
ent set of compounds, no further structural modifications were
made for enhancing their binding to the MMPs, thus identical order
of magnitude is expected for their MMP inhibitory activities.
Molecular modeling studies were performed aimed at getting
some rationalization of the inhibitory activities observed for our
Concerning the docking with CA IX, the results showed that the
binding conformations of the ligands are quite similar to those into
CA II, namely the BSA moiety forming the same interactions with
the enzyme as mentioned for the CA II, and the HPM group binding

3626
M. A. Esteves et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3623–3627
Figure 3. Docking of compounds 1 (green) and 4 (orange) into the CA IX active site.
Black dashed lines represent the ligand coordination to the zinc (gray sphere), and
the full black lines represent the H-bonds formed with the protein.
is with the side-chain carbonyl O-atom of Gln203. Globally, the
docking results seem to have elucidated the high stability of the
complexes formed, and consequently give support the high inhib-
itory activities observed for these compounds. Inhibitor 1 dis-
played the highest number of interactions with the CA IX, thus
resulting in the highest activity observed among this series.
In conclusion, a set of new bifunctional compounds containing
HPM and BSA moieties have been synthesized and evaluated for
their CA inhibitory activities. These compounds demonstrated to
inhibit CA I, II and IX with low nanomolar activity, and showed
preference for inhibiting the tumor-associated isozyme CA IX, over
the ubiquitous cytosolic CA I and II. The high CA inhibitory activi-
ties evidenced for all these compounds are supported by docking
modelling studies which showed that, besides the expected strong
binding between BSA moiety and catalytic zinc ion at the active
site of the CAs, there is an extra interaction between the HPM moi-
ety and the hydrophilic part of these enzymes. Considering the
excellent CA inhibitory activities observed against this set of phys-
iologically relevant CAs, these HPM-containing sulfonamides may
be useful to further design of new CA inhibitors. These might be
endowed with the extra-favorable properties associated with the
HPM (ranging from the MMPi activity, metal chelator, and bio-
availability), and thus reveal interesting for novel applications in
therapy, with eventual dual-drug activity.
Figure 2. Docking of compounds into the CA II active site: 3 (magenta) superim-
posed with 4 (orange) (a), and 1 (green) superimposed with 3 (magenta), with the
protein surface displayed (b). Red surfaces represent the hydrophobic and blue the
hydrophilic regions of the protein; black dashed lines represent the ligand
coordination to the metal (gray sphere), and the full black lines represent the H-
bonds formed with the protein.
Acknowledgments
The authors would like to thank the Portuguese Fundação para
a Ciência e Tecnologia for the post-doc grant SFRH/BPD/29874/
2006 (S. Marques). This research was also financed in part by an
FP7 EU grant (METOXIA) to C.T.S. All molecular modeling figures
and alignments of the enzyme structures were produced using
the UCSF Chimera package, from the Resource for Biocomputing,
Visualization, and Informatics at the University of California, San
Francisco (supported by NIH Grant P41 RR-01081).
with the hydrophilic part of the protein (see Fig. 3). All the ligands
form an H-bond with the side-chain NH2 group of Gln224, through
the carbonyl group of the amide spacer (hCA IX numeration, entry
Q16790 of UniProtKB database). In all cases, the linker chain seems
to establish van der Waals interactions with some residues forming
the hydrophobic part of the active site, namely Val253, Val262 and
Leu272. 1 and 2 are able to form two additional H-bonds with the
protein, through the HPM moiety. In the case of 1, these bonds are
formed between the OH groups of the HPM and Thr333, and be-
tween its carbonyl group and the NH2 of Gln224 (Fig. 3). As for
compound 2, these H-bonds are formed with Gln203, between
the OH of the HPM with the side-chain carbonyl group, and the
carbonyl O-atom of the HPM with the NH2 group, respectively.
Concerning compounds 3 and 4, these form only one extra H-bond
with the enzyme, through the hydroxyl group of the HPM moiety;
while for 3 this interaction is with the OH group of Thr333, for 4 it
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.04.109.
References and notes
1. (a)Drug Design of Zinc–Enzyme Inhibitors; Supuran, C. T., Winum, J.-Y., Eds.; John
Wiley & Sons, Inc.: New Jersey, 2009. pp 13–486, and references cited therein;
(b)Drug Design of Zinc–Enzyme Inhibitors; Supuran, C. T., Winum, J.-Y., Eds.; John
Wiley & Sons, Inc.: New Jersey, 2009. pp 489–671, and references cited therein.

M. A. Esteves et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3623–3627
3627
2. Supuran, C. T. Nat. Rev. Drug Disc. 2008, 7, 168.
3. Supuran, C. T.; Scozzafava, A.; Casini, A. Med. Res. Rev. 2003, 23, 146.
4. Supuran, C. T. Curr. Pharm. Des. 2008, 14, 603.
5. Wilkinson, B. I.; Bornaghi, L. F.; Houston, T. A.; Innocenti, A.; Vullo, D.; Supuran,
C. T.; Poulsen, S. A. J. Med. Chem. 2007, 50, 1651.
6. Leese, M. P.; Leblond, B.; Smith, A.; Newman, S. P.; Di Fiore, A.; De Simone, G.;
Supuran, C. T.; Purohit, A.; Reed, M. J.; Potter, B. J. Med. Chem. 2006, 49, 7683.
7. Marques, S. M.; Nuti, E.; Rossello, A.; Supuran, C. T.; Tuccinardi, T.; Martinelli,
A.; Santos, M. A. J. Med. Chem. 2008, 51, 7968.
temperature, under N2. Then, the solvent was evaporated under reduced
pressure and the resulting residue was purified by CC on silica gel, using a
mixture of methanol/dichloromethane (0.1:1) as eluent to obtain the product,
which was then recrystallized from methanol/diethyl ether mixtures to afford
pure compound 19–22. Compound 23 was synthesized by the same procedure
but using 6-aminohexanoic acid methyl ester hydrochloride instead of an
amide and adding 1 equiv of potassium carbonate to neutralize the
hydrochloride.
4-{3-[(4-Sulfamoyl-phenyl)carbamoyl]propylamino]-1-(benzyloxy)-2(1H)-pyrimidi-
8. Puerta, D. T.; Mongan, J.; Tran, B. L.; Mc Cammon, J. A.; Cohen, S. M. J. Am. Chem.
Soc. 2005, 127, 14148.
9. Yan, Y. L.; Miller, M. T.; Cao, Y.; Cohen, S. M. Bioorg. Med. Chem. Lett. 2009, 19,
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10. Blagg, J. A.; Noe, M. C.; Wolf-Gouveia, L. A.; Reiter, L. A.; Laird, E. R.; Chang, S. P.
Bioorg. Med. Chem. Lett. 2005, 15, 1807.
11. Esteves, M. A.; Cachudo, A.; Ribeiro, C.; Chaves, S.; Santos, M. A.. In Metal Ions in
Biol. & Med; John Libbey Eurotext: Paris, 2006; Vol. 9. pp 35–39.
12. Puerta, D. T.; Lewis, J. A.; Cohen, S. M. J. Am. Chem. Soc. 2004, 126, 8388.
13. Experimental procedures and spectral data for the synthesized compounds.
The full characterization of all compounds can be found in Supplementary
data.
none (19): light yellow crystals, 42 % yield, mp 255-257 °C. FTIR (KBr)
3261, 2958, 2879, 1699, 1633, 1590, 1536, 1504, 1472, 1402, 1320,
1151, 854, 748, 790 cmꢂ1 1H NMR (DMSO-d6) d 1.80 (2H, quin, J = 6.9 Hz,
CH2CH2CH2), 2.39 (2H, t, J = 7.2 Hz, CH2C(O)), 3.25-3.30 (2H, m, NHCH2), 5.05
(2H, s, CH2O), 5.51 (1H, d, J = 9 Hz, NHC@CH), 7.57 (1H, d, J = 9 Hz, NCH@CH),
7.38-7.43 (4H, m, CHCS(O)2 and (O)CHNCCH), 7.74 (5H, s, ArHCH2) ppm. 13C
NMR (DMSO-d6) d 24.5 (CH2CH2CH2), 33.9 (CH2C(O)), 39.2 (NHCH2), 77.3 (CH2O),
93.2 (NCH@CH), 118.6 (2C, (O)CHNCCH), 126.7 (2C, HCCS(O)2), 127.1, 128.0,
129.7, 142.2 (6C, ArCCH2O), 134.4 (CS(O)2), 138.1 ((O)CHNC), 142.9 (NCH@CH),
152.9 (NC(O)N), 162.4 (N@CNH), 171.5 (C(O)NH) ppm.
mmax 3378,
.
General procedure for the synthesis of compounds 1–5: Compounds 1–5 were
obtained by deprotection of 19–23, using for removal of the Bz protecting group
the procedure already described for the Cbz group.
General procedure for the synthesis of compounds 10–13: A solution of EDCꢀHCl
(1 mmol) in dichloromethane (3.5 mL) was added to a mixture of the N-Cbz-
amino acid (6 or 7) (1 mmol), sulfonamide (8 or 9) (1 mmol) and HOBt
(1 mmol) in DMF (1.8 mL) at -10 °C. The mixture was stirred under a nitrogen
atmosphere at room temperature for 24 h. After this period, the solvent was
removed under reduced pressure and the residue obtained was diluted in
water (10 mL) and extracted with ethyl acetate (3 ꢁ 10 mL). The combined
organic layers were washed with 0.5 M citric acid and brine, dried over
magnesium sulfate and filtered. The filtrate was evaporated in vacuum to
dryness, followed by recrystallization of the residual solid from MeOH/diethyl
ether to afford the pure products 10–13.
4-{3-[(4-Sulfamoyl-phenyl)carbamoyl]propylamino]-1-hydroxy-2(1H)-pyrimidi-
none (1): white solid, 89% yield, mp 140-142 °C. FTIR (KBr) mmax 3272, 2923,
2874, 1636, 1593, 1533, 1403, 1327, 1157, 837, 764 cmꢂ1 1H NMR (CD3OD) d
.
1.91 (2H, quin, J = 6.9 Hz, CH2CH2CH2), 2.43 (2H, t, J = 7.2 Hz, CH2C(O)), 3.38
(2H, t, J = 6.6 Hz, NHCH2), 5.66 (1H, d, J = 7.2 Hz, NCH@CH), 7.56 (1H, d,
J = 7.2 Hz, NCH@CH), 7.74 (4H, q, J = 11.4 Hz, CHCS(O)2 and (O)CHNCCH) ppm.
13C NMR (CD3OD) d 26.3 (CH2CH2CH2), 35.4 (CH2C(O)), 41.2 (NHCH2), 95.4
(NCH@CH), 143.7 (NCH@CH), 120.5 (2C, (O)CHNCCH), 128.1 (2C, CHCS(O)2),
139.5 (CS(O)2), 143.6 (NC(O)N), 164.3 (N@CNH), 174.2 (C(O)NH) ppm. HRMS
(ESI) calcd for C14H17N5O5S+H 368, 10286, found 368, 10349.
14. Ohkanda, J.; Katoh, A. Tetrahedron 1995, 51, 12995.
15. Sung, W. L. J. Chem. Soc., Chem. Commun. 1981, 1089.
16. Jakobsen, C. M.; Denmeade, S. R.; Isaacs, J. T.; Gady, A.; Olsen, C. E.; Christensen,
S. B. J. Med. Chem. 2001, 44, 4696.
17. (a) Cecchi, A.; Hulikova, A.; Pastorek, J.; Pastorekova, S.; Scozzafava, A.; Winum,
J.-Y.; Montero, J.-L.; Supuran, C. T. J. Med. Chem. 2005, 48, 4834; (b) Casey, J. R.;
Morgan, P. E.; Vullo, D.; Scozzafava, A.; Mastrolorenzo, A.; Supuran, C. T. J. Med.
Chem. 2004, 47, 2337.
18. Sconik, L. R.; Clements, A. M.; Liao, J.; Hellberg, M.; May, J.; Dean, D. R.;
Christiason, D. W. J. Am. Chem. Soc. 1997, 119, 850.
19. Innocenti, A.; Vullo, D.; Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. 2008,
1583.
20. Esteves, M. A.; Cachudo, A.; Chaves, S.; Santos, M. A. Eur. J. Inorg. Chem. 2005,
597.
21. Smith, R. M.; Martell, A. E.. In Critical Stability Constants; Plenum Press: New
York, 1977; Vol. 3. p 181.
22. Duffel, M. W.; Ing, I. S.; Segarra, T. M.; Dixson, J. A.; Barfknecht, C. F.;
Schoenwald, R. D. J. Med. Chem. 1986, 29, 1488.
3-(4-Sulfamoyl-phenylcarbamoyl)-propylcarbamic acid benzyl ester (10): white
crystals, 53% yield, mp = 172–174 °C. FTIR (KBr) mmax 3333, 3363, 2936, 2958,
1694, 1651, 1588, 1531, 1507, 1402, 1345, 1160, 898 cmꢂ1 1H NMR (CD3OD) d
.
1.86 (2H, quin, J = 6.9 Hz, CH2CH2CH2), 2.42 (2H, t, J = 7.5 Hz, CH2C(O)), 3.19
(2H, t, J = 6.6 Hz, NHCH2), 5.04 (2H, s, CH2O), 7.72 (2H, d, J = 8.7 Hz, CHCS(O)2),
7.81 (2H, d, J = 9 Hz, HNCCH), 7.31 (5H, s, ArHCH2) ppm. 13C NMR (CD3OD) d
26.9 (CH2CH2CH2), 35.2 (CH2C(O)), 41.3 (NHCH2CH2CH2), 67.5 (CH2O), 120.4
(2C, HNCCH), 128.2 (2C, HCCS(O)2), 139.6 (CS(O)2), 143.6 (HNCCH), 127.7,
128.4, 129.5, 138.5 (6C, ArCCH2O), 166.9 (OC(O)), 174.2 (C(O)NHC) ppm.
General procedure for the synthesis of compounds 14–17: A suspension of Pd/C
10% (20 mg) in dry MeOH (10 mL) was pre-hydrogenated with H2 (1 atm) for
30 min. To this suspension, a solution of N-Cbz protected compound 10–13
(0.5 mmol) in dry MeOH (10 mL) was added and the mixture stirred under H2
(1 atm) for 3 h at room temperature. The catalyst was removed by filtration
and the solvent evaporated under reduced pressure to afford products 14–17
as white solids which were used in the following steps without further
purification.
N-(4-Sulfamoyl-phenyl)-4-aminobutanamide (14): Amorphous white solid, 91%
yield. FTIR (KBr) mmax 3362, 3261, 2958, 1668, 1592, 1541, 1400, 1315, 1158,
23. Berman, H. M.; Westbrook, J.; Feng, Z.; Gilliland, G.; Bhat, T. N.; Weissig, H.;
Shindyalov, I. N.; Bourne, P. E. Nucleic Acids Res. 2000, 28, 235.
24. Jones, G.; Willett, P.; Glen, R. C.; Leach, A. R.; Taylor, R. J. Mol. Biol. 1997, 267,
727.
25. Tuccinardi, T.; Nuti, E.; Ortore, G.; Supuran, C. T.; Rossello, A.; Martinelli, A. J.
Chem. Inf. Model. 2007, 47, 515.
839, 748 cmꢂ1 1H NMR (CD3OD) d 1.82 (2H, quin, J = 6.9 Hz, CH2CH2CH2), 2.42
.
(2H, t, J = 7.2 Hz, CH2C(O)), 2.71 (2H, t, J = 6.9 Hz, NH2CH2), 7.69 (2H, d,
J = 8.7 Hz, CHCS(O)2), 7.78 (2H, d, J = 8.7 Hz, HNCCH) ppm. 13C NMR (CD3OD) d
28.7 (CH2CH2CH2), 35.3 (CH2C(O)), 41.7 (NH2CH2), 120.4 (2C, HNCCH), 128.2
(2C, HCCS(O)2), 139.6 (CS(O)2), 143.5 (HNC), 174.2 (C(O)NH) ppm.
General procedure for the synthesis of compounds 19–23: A solution of amide 14–
17 (0.58 mmol) and 1-(benzyloxy)-4-(10,20,40-triazol-10-yl)-2-(1H)-pyrimidi-
none (0.50 mmol) 18 in dry THF (10 mL) was stirred for 24-48 h at reflux
26. The UniProt Consortium. The Universal Protein Resource (UniProt) in 2010,
Nucleic Acids.