Structure-based approach to nanomolar, water soluble matrix metalloproteinases inhibitors (MMPIs)

Article abstract of DOI:10.1016/j.ejmech.2010.09.057

N-Arylsulfonyl-based MMPs inhibitors (MMPIs) are among the most prominent inhibitors possessing nanomolar affinity. However, their poor bioavailability remains critical for the drug development of this family of molecules. The structural analysis of the c

Full text of DOI:10.1016/j.ejmech.2010.09.057

European Journal of Medicinal Chemistry 45 (2010) 5919e5925
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Structure-based approach to nanomolar, water soluble matrix metalloproteinases
inhibitors (MMPIs)
,
,
Emanuele Attolino a, Vito Calderone b, Elisa Dragoni a c, Marco Fragai b c, Barbara Richichi c,
,
,c,
*
Claudio Luchinat b c, Cristina Nativi b
a ProtEra s.r.l. viale delle Idee, 22 I-50019 Sesto F.no (FI), Italy
b CERM, University of Florence, Sesto F.no (FI), Italy
c Department of Chemistry, University of Florence, Sesto F.no (FI), Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
N-Arylsulfonyl-based MMPs inhibitors (MMPIs) are among the most prominent inhibitors possessing
nanomolar affinity. However, their poor bioavailability remains critical for the drug development of this
family of molecules. The structural analysis of the complex of NNGH (the most representative member of
the family) with MMP-12 provided us with the basis to effectively design simple NNGH analogues with
enhanced solubility in water. Following this approach, the sec-butyl residue, not directly involved in the
binding with MMP, has been replaced with hydrophilic residues thus yielding new potent inhibitors
soluble in water.
Received 10 August 2010
Received in revised form
22 September 2010
Accepted 27 September 2010
Available online 20 October 2010
Keywords:
Metalloproteinases
Inhibitors
Ó 2010 Elsevier Masson SAS. All rights reserved.
Structure-based approach
Sulfonamides
Substituent effect
1. Introduction
enzymes. MMPs are involved in many aspects of physiological
cellular processes, as well as in pathologies, such as reumathoid
The decoration of a ligand with hydrophobic groups is often
reported as useful strategy to increase its free-energy of binding to
a target protein. Due to desolvation, the entropy associated to the
binding of a hydrophobic group to the protein surface is usually
large and positive with a significant increase of the overall ligand
affinity [1]. Ultimately, this increase in affinity parallels the
decrease in the solubility of the ligand. However, the increase in
affinity only occurs if the hydrophobic group interacts with the
protein and water molecules are released upon ligand binding,
while if the hydrophobic group sticks-out of the protein surface
into the bulk water, it only worsens the solubility without affecting
the ligand binding. Therefore, a suitable approach to improve both,
affinity and water solubility of a lead compound, lies in the struc-
tural analysis of its complex with the target protein to drive the
decoration of the scaffold with suitable functional groups. Matrix
metalloproteinases (MMPs) are a large class of strictly-related zinc-
dependent enzymes which belong to the family of proteolytic
arthritis, pulmonary emphysema, tumor growth and metastasis.
Since many of these pathologies may benefit from the control of
MMPs activity [2], several MMPs inhibitors (MMPIs) has been
designed and tested in clinical trials [3e8]. However, most of them,
are poorly soluble in water and suffer from a modest oral
bioavailability [9]. A well-balanced hydrophilic/lipophilic character
is crucial for reaching and maintaining high drug levels in plasma.
High lipophilicity negatively affects drug bioavailability, by
preventing the drug from reaching the target site and increasing
the binding to human serum albumin (HSA) [10]. In addition,
a limited drug bioavailability requires the administration of large
doses of drug to achieve and maintain an effective therapeutic
efficacy. In case of MMPI, it means the increasing of adverse effects
due to the modest selectivity which may cause the indiscriminate
inhibition of other zinc-peptidases. Side effects are even worse in
long-term treatments, required in many MMP-related chronic
pathologies [11].
Water soluble inhibitors can be useful in this respect but only
few of the huge number of potentially useful MMPIs reported to
date [12,13] show a good solubility in water preserving a good
bioavailability [14e20]. Water soluble inhibitors are also suitable
candidates for topical treatment of pathologies such as pulmonary
* Corresponding author. Department of Chemistry, University of Florence, via
della Lastruccia, 3,13 Sesto F.no (FI), Italy. Fax: þ 39 0554573540.
E-mail address: cristina.nativi@unifi.it (C. Nativi).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.09.057

5920
E. Attolino et al. / European Journal of Medicinal Chemistry 45 (2010) 5919e5925
Table 1
emphysema, and parodontitis where the solubility in aqueous
Enzyme inhibition constants of compounds 12, 17, 22, 23, 24, 28, 30 and 32 (data are
biological fluids is mandatory.
Ki (nM)) towards several MMPs.a
N-Arylsulfonyl
a-aminohydroxamic acid derivatives such as
4 R= H, R' = (CH2)2-Glc, R'' = OMe
NNGH, 1 [21,22] CGS27023, 2 [21] and AG3340, 3 [23] (Fig. 1) are
among the most prominent representatives of a family of nano-
molar inhibitors for some MMPs. Extensive NMR and X-ray studies
to elucidate the binding interactions between functional groups in
the inhibitors and their corresponding subsites in the enzymes
[12,24] have provided the basis to effectively design new analogues
with potentially more favorable interactions and enhanced solu-
bility in water.
R'
O
12
R = H, R' = (CH2)2-OH, R'' = OMe
O
S
HO
N
17
R = H, R' = CH2(CHOH)CH2OH, R'' = OMe
N
H
O
22 R = CH2OH, R' = CH2(CHOH)CH2OH, R'' = OMe
R
23
R = CH2OH, R' = H, R'' = OMe
24
R = CH(CH3)OH, R' = H, R'' = OMe
28 R = H, R' = (CH2)2-OH, R'' = F
30
R = H, R' = (CH2)2-OH, R'' = biphenyl
32
R''
R = H, R' = CH2(CHOH)CH2OH, R'' = biphenyl
X-Ray structures of the NNGH-MMP-12 complex [25] confirmed
that the interaction of inhibitors of this class with the active site of
the enzyme involves the binding of the hydroxamic functional
group to the catalytic Zn ion and the binding of the aromatic group
to the S’1 subsite. On the contrary, the isopropyl group on the
sulfonamide nitrogen atom points away from the shallow S’2 pocket
and does not directly participate in binding. Capitalizing on such
structural insights, we recently reported the synthesis of the water
soluble MMPs inhibitor 4 [16] featuring a glucose residue. As
expected, the correct functionalisation of the sulfonamidic scaffold
allowed to preserve the nanomolar affinity of 4 for several MMPs
while increasing its solubility in water.
Compound MMP-1 MMP-7
MMP-8 MMP-12
9.0
4.3 (1) [16] 3.1 (1) [16] þ1.61
9.1 (8.0) 14.3 (7.0)
7.6 (1.1)
8.0 (1.1)
8.0 (1.2) 7.0 (0.9)
9.0 (1.0) 6.0 (0.7)
MMP-13
C logP
NNGH [25] 174
13000
2000
4 [16]
12
286
1.7 (1.1)
1.0 (0.1)
3.6 (0.8)
1.7 (0.3)
2.2 (0.3)
2.9 (0.3)
46 (6)
ꢀ1.76
ꢀ1.15
ꢀ1.78
ꢀ2.26
ꢀ1.17
ꢀ0.75
ꢀ0.85
þ0.65
þ0.02
128 (18) 1500 (200) 13 (2)
160 (22) 9000 (170) 24 (3)
17
22
32 (5)
344 (65)
23
143 (18) 823 (30)
24
28
287 (33) 984 (110) 10.0(1.8) 11.0 (1.7)
151 (21) 3000 (400) 173 (23) 40 (7)
30
32
67 (9)
1600 (200) 4.6 (0.6) 31 (4.8)
60 (10)
2.7 (0.5)
18 (2)
742 (90) 4000 (900) 48 (7)
a
Values are averaged over triplicate experiments with errors reported in
parentheses.
Relying on this observation, we report here the synthesis and the
binding assessment of a family of new arylsulfonamide derivatives
1a with hydrophilic R’-substituents, replacing isobutyl group, and
differently para-substituted aryl groups. R and R’-substituents with
various degrees of hydrophilicity and R00 substituents with different
size and shape were used, featuring new inhibitors with C logP
[Calculator Plugins, ChemAxon Ltd.] ranging from þ0.65 to ꢀ2.26
(see Table 1).
MMP-7 involved in colorectal carcinoma [32] and MMP-1, impli-
cated in skin photo-aging damages [33].
2. Results and discussion
2.1. Synthesis
Following these leading concepts, eight arylsulfonamide deriv-
atives bearing mono- and poly-hydroxy substituents facing the
solvent and different hydrophobic groups targeted to the hydro-
Non-natural, commercially available, GlyOMe 5, D-SerOMe 6,
and D-ThrOMe 7 were reacted with sulfonyl chloride in the pres-
ence of triethylamine and dimethylaminopyridine in dichloro-
methane as solvent to give sulfonamides 8e10 in high yield
(90e97%) (Scheme 1).
The sulfonamide of glycine methylester 8 was reacted with
ethylene oxide and methyliodide [16] to afford compound 11 which
was directly transformed into the corresponding hydroxamic acid
12 by reaction with hydroxylamine hydrochloride and potassium
hydroxide, in methanol as solvent (Scheme 2). Compound 8
was also reacted with enantiopure glycerine dimethylacetal 13
under Mitsunobu conditions, to give methylester 14. Acidic removal
(CH3COOH/H2O 80:20) of acetal protecting group on 14 afforded
a mixture of the desired dihydroxyl derivative 15 and lactone 16;
the latter obtained from 15 by intramolecular trans-esterification.
0
phobic S1 pocket have been synthesized. Low nanomolar affinities
were achieved for these new inhibitors towards some relevant
MMPs such as: MMP-12, implicated in the development of
emphysema [22,26], MMP-13 involved in rheumatoid arthritis
[27,28], MMP-9 overexpressed in diabetic retinopathy [29e31],
R'
O
O
O
O
S O
O
S O
O
S
HO
N
HO
N
HO
N
N
H
N
H
N
H
O
R
R''
OCH3
OCH3
2 CGS 27023A
O
1 NNGH
1a
Cl
S
O
O
O
H
N
O
OH
.
a
+
NH2 HCl
H3CO
S
H3CO
O
N
O
O
HO
O
R
O
HO
R
O
S
OH
OCH3
O
O
O
S O
HO
N
HO
N
N
OCH3
N
H
5 R = H
R = CH2OH
8 R = H
9
H
6
S
R = CH2OH
10
7
R = CH(OH)CH3
R = CH(OH)CH3
3 AG 3340
4
a
OCH3
a) Et3N, DMAP, CH2Cl2, rt, overnight, 90-97%
Fig. 1. Structure of inhibitors 1e4.
Scheme 1.

E. Attolino et al. / European Journal of Medicinal Chemistry 45 (2010) 5919e5925
5921
OH
OH
O
O
O
N
N
b
a
HO
H3CO
S
N
H
8
O
R
R
11
12
R = H
OCH3
O
OCH3
O
O
O
O
S
c
N
8
+
H3CO
O
O
OH
d
13
14
OH
OH
N
OCH3
OH
O
O
O
N
O
H3CO
OH
S
+
O
O
S
O
16
15
e
OH
O
O
OCH3
OCH3
HO
N
N
H
S
O
17
OCH3
a a) see Ref. 22, 64%; b) NH2OH.HCl, KOH, MeOH, rt, 1h + 6h, 38%; c) Ph3P, DIAD,
THF, rt, overnight, 87%; d) CH3COOH/H2O 80:20, 60 °C, 4h, 75% (15 + 16);
e) NH2OH.HCl, KOH, MeOH, rt, 1h + 18h, 45% (after HPLC purification)
Scheme 2.
The non-easily separable mixture of 15 and 16 was treated with
hydroxylamine hydrochloride and potassium hydroxide in meth-
anol to form hydroxamic acid 17 as white solid in 45% yield
(Scheme 2).
The sulfonamide of serine methyester 9 was firstly transformed,
under standard conditions, into the corresponding tertbutyldime-
thylsilyl ether 18 then reacted with acetal 13 as reported above, to
give compound 19 (Scheme 3).
Acidic removal of silylether and acetal protecting groups
afforded a mixture of tri-hydroxyl derivative 20 and lactone 21
which was treated with hydroxylamine hydrochloride and
potassium hydroxide to form the expected hydroxamic acid 22 in
50% yield (see Scheme 3). Treatment of sulfonamide of serine
methylester 9 and of threonine methylester 10, with hydroxyl-
amine and potassium hydroxide in methanol as solvent afforded
the N-disubstituted hydroxamic acids 23 and 24 respectively
(Scheme 4).
Derivatives, 23 and 24, as well as hydroxamic acids 12, 17 and 22
are soluble in water and stable as solid or in solution, at room
temperature for several months under neutral (pH ¼ 7) or basic
conditions (pH ¼ 8.5). The same good stability was observed under
slightly acidic conditions (pH ¼ 4.5) with the only exception of
derivative 22 which tends to degrade forming the corresponding
carboxylic acid.
High-resolution structures of a series of enzymes adducts with
homologous ligands we previously reported [34], provided
precious information for the designing of new ligands. Taking in
consideration these subtle factors, in order to improve inhibitors
selectivity, para-methoxybenzyl residue on scaffold 8 was replaced
by para-fluorobenzyl- and biphenyl-moieties. Therefore, the para-
fluorosulfonamide of glycine methyester 25 and the biphe-
nylsulfonamide of glycine methylester 26 were prepared reacting,
as depicted in Scheme 1, ester 5 with para-fluorosulfonyl chloride
and biphenylsulfonyl chloride respectively (Scheme 5).
Reaction of 25 and 26 with acetylbromoethanol in dimethy-
formamide as solvent in the presence of sodium hydride afforded
sulfonamides 27 and 29 respectively which, after removal of acetyl
protecting group under standard conditions, were transformed
into the corresponding hydroxamic acids 28 (72%) and 30 (48%)
by reaction with hydroxylamine hydrochloride and potassium
hydroxide as base, at room temperature (see Scheme 5). Methyl
ester 26 was also reacted with 13 to give the sulfonamide 31 which
was transformed, as reported for 22, into the hydroxamic acid 32 in
54% yield.

5922
E. Attolino et al. / European Journal of Medicinal Chemistry 45 (2010) 5919e5925
O
O
O
O
O
H
N
O
N
H3CO
SiO
S
a
b
H3CO
SiO
S
O
9
O
OH
OH
N
18
19
OCH3
OCH3
O
OH
O
O
N
O
H3CO
S
O
O
S
+
O
OH
HO
HO
d
OH
O
O
HO
N
N
20
21
OCH3
S
OCH3
O
H
HO
22
OCH3
a a) tBuMe2SiOTf, Et3N, rt, 20h, 97%; b) Ph3P, DIAD, THF, rt, overnight, 85%; c) CH3COOH/
H2O 80:20, 60 °C, 90%, (20 + 21); d) NH2OH.HCl, KOH, MeOH, 1h + 15h, 50% (after HPLC
purification)
Scheme 3.
3. Biological assays
evidence was obtained from the X-ray crystal structure of the
MMP-12e17 complex (Fig. 2, PDB code: 1F15). The protein structure
clearly resembles those of the previously solved MMP-12 structures
[25]. The catalytic and structural zinc atoms as well as the three
calcium ions are well visible (Fig. 2A). The inhibitor shows a very
well defined electron density for all atoms, which allows to identify
the absolute configuration of the non-aminoacidic stereogenic
center as S. As expected for NNGH-like ligands, the catalytic zinc
atom is coordinated by the hydroxamic moiety and the methox-
Compounds 12, 17, 22, 23, 24, 28, 30 and 32 were tested “in
vitro” for the inhibition of a panel of six MMPs: MMP-1, -7, -8, -9,
-12, -13 (see Experimental Section for details on the protocol used).
Assays used the catalytic domain of the proteins. Results are shown
in Table 1 in comparison with those of NNGH and compound 4 [16].
Except for MMP-1 and -7, all compounds reported in Table 1
showed low nanomolar Ki values for the MMPs tested. Given the
active site topology of MMPs, most inhibitors likely share similar
features, i.e., the ability to bind to the metal ion, to the hydrophobic
0
yphenyl ring is nested in the S1 pocket through hydrophobic and
aromatic stacking interactions. The hydroxamic and sulphonate
moieties are then establishing favorable interactions with the
backbone atoms of the protein through several strong hydrogen
bonds. The dihydroxypropyl chain protrudes out of the protein
toward the solvent region, placing the two oxygen atoms at
hydrogen bond distance from three well ordered water molecules.
More importantly, one of the oxygens of the dihydroxypropyl
portion is also engaged in a strong hydrogen bond with one of the
two sulfonamidic oxygens, locking the inhibitor in the suitable
conformation to be buried into the protein cavity. The inhibitor
shares the same binding mode of other inhibitors known in the
0
pocket termed S1 , and to the substrate binding groove. Direct
O
H
O
HO
N
a
N
H
S
9
O
HO
23
OCH3
O
0
literature and all relevant interactions (with the S1 pocket, the
metal, and the substrate binding groove) are in place.
As expected [16], the hydrophilic chain on the sulfonamidic
nitrogen protrudes out of the protein toward the solvent region and
does not negatively affect the binding properties of the inhibitor,
thus confirming the lack of significant participation in binding of
such chains.
O
H
N
HO
a
N
H
S
O
10
HO
Relying on the different shape and on the different aminoacidic
0
24
residues which characterize the S1 binding pockets of MMPs, we
OCH3
prepared compounds 28 (PDB code: 3F18), 30 and 32 with a fluo-
rine residue (28) and a biphenyl residue (30, 32) replacing the
methoxy group with the aim of taking advantage of specific addi-
tive interactions. However, since besides their intrinsic similarity
MMPs exhibit some capability of adapting the binding pocket to the
inhibitor shape [35] no real advantages were observed in term of
a a) NH2OH.HCl, KOH, MeOH, rt, 1h + 20h, 47% (23);
1h +18h, 36% (24).
Scheme 4.

E. Attolino et al. / European Journal of Medicinal Chemistry 45 (2010) 5919e5925
5923
OR
O
O
O
H
N
Cl
O
S
F
b
O
N
a
H3CO
S
5
H3CO
+
O
25
27 R = Ac
c
F
27a
R = H
OH
F
d
O
O
HO
N
N
H
S
O
28
F
OR
O
O
H
N
O
O
Cl
S
b
a
O
S
O
N
H3CO
S
5 +
O
H3CO
O
26
29 R = Ac
c
29a
R = H
d
OH
O
O
HO
N
N
H
S
O
30
O
OH
O
O
OH
O
O
O
f, g
e
26 + 13
HO
N
N
H3CO
N
H
S
S
O
O
32
31
a
25
26
a) Et3N, DMAP, Ch2Cl2, rt, overnight, 89% ( ); 81% ( ); b) AcOCH2CH2Br,
27
29
27a
); 40%
NaH, DMF, 0 °C - rt, 18h, ( ), 36h ( ); c) MeONa, MeOH, 1h, 50% (
.
29a
28
30
(
).; d) NH2OH HCl, KOH, rt, 1h + 18h 72% ( ); 48% ( ); e) Ph3P, DIAD,
THF, rt, overnight, 64%; f) CH3COOH/H2O 80:20, rt than g) NH2OH. HCl, KOH,
2 h, 54%
Scheme 5.

5924
E. Attolino et al. / European Journal of Medicinal Chemistry 45 (2010) 5919e5925
the solubility, and consequently the bioavailability, of candidate
drugs without affecting their affinity for the target. In particular, the
sulfonamidic-based family of MMPIs we reported here represents
a rare example of water soluble, structurally simple inhibitors with
low nanomolar affinity for MMPs. Noteworthy, the rationale fol-
lowed to synthesize this class of large spectrum, potent inhibitors
clearly demonstrated that the introduction of hydrophilic chains on
the nitrogen atom of the NNGH sulfonamidic scaffold does not
negatively impact upon the affinity of the inhibitors for MMPs. This
is particularly evident in the case of inhibitor 17 which presents the
selectivity profile similar to NNGH but a complete solubility in
water. This insight could be successfully exploited to develop di-
topic devices to efficiently target MMPs and a second biologically
relevant target simultaneously.
5. Experimental
All commercially available reagents used for synthesis were
employed without further purification; solvents used were of
analytical grade and were employed without further purification
unless otherwise stated. The following structures: 3F15 (MMP-12-
17), 3F16 (MMP-12-23), 3F18 (MMP-12-28), 3B2U (MMP-12-4),
3N2 V (MMP-12-30) have been deposited to the PDB.
5.1. Synthesis of compound 14
To a solution of 8 (1.04 g, 4.00 mmol), PPh3 (1.37 g, 5.20 mmol) and
13 (0.69 g, 5.20 mmol) in dry THF (20 mL) was slowly added DIAD
(1.02 mL, 5.20 mmol). The reaction mixture was stirred overnight at
room temperature then concentrated to dryness. 25 mL of a mixture
cyclohexane/EtOAc 6:1 were added and the suspension was stirred
for 30 min at room temperature. The white solid was filtered off and
the filtrate was concentrated to dryness. The crude product was
purified by flash column chromatography on silica gel (CHCl3/EtOAc
7:1) to afford 14 (1.30 g, 87%) as an oil. [
a
]
2D5: þ4.39(c 0.41, CHCl3).
HRMS for C16H23NO7S þ H calcd: 374.1273. Found: 374.1268. 1H
Fig. 2. RX structure of MMP12-17 complex: A) global view; B) inhibitor’s dihydroxyl
NMR (200 MHz, CDCl3): d 7.77e7.74 (AA’ part of an AA’MM’ system,
chain protruding outside the protein.
JAM ¼ 9.2 Hz, 2H), 6.97e6.95 (MM’ part of an AA’MM’ system,
JAM ¼ 9.2 Hz, 2H), 4.34e4.22 (m, 3H, H-2a, H-20, H-2b), 4.09e4.02 (A
part of an ABX system, JAx ¼ 6.4 Hz, JAB ¼ 8.4 Hz, 1H, H-30a), 3.87 (s,
3H, OCH3), 3.68e3.64 (B part of an ABX system, JBx ¼ 6.8 Hz,
JAB ¼ 8.4 Hz, 1H, H-30b), 3.62 (s, 3H, COOCH3), 3.62e3.52 (A part of
an ABX system, JAx ¼ 4.0 Hz, JAB ¼ 14.8 Hz, 1H, H-10a), 3.25e3.14 (B
part of an ABX system, JBx ¼ 7.2 Hz, JAB ¼ 14.8 Hz, 1H, H-10b), 1.35
(s, 3H, C(CH3)2), 1.30 (s, 3H, C(CH3)2). 13C NMR (50 MHz, CDCl3):
selectivity. Indeed, the presence of the bulky biphenyl group did
not improve substantially the affinity vs the enzyme with a larger
0
S1 pocket, like MMP-13, with respect to MMP-12 or MMP-8.
The low affinity of all the inhibitor0s here reported for MMP-7,
characterized by a narrow and short S1 pocket, was not surprising,
while the disappointing micromolar Ki value measured in the case of
0
28 vs MMP-1, featuring at the bottom of the S1 pocket a positively
d
169.7, 162.9, 131.0, 129.4, 114.1, 109.5, 75.4, 67.0, 55.5, 51.9, 50.4,
charged residue, can probably be ascribed to the lack of the expected
favorable interactions between the Arg214 and the fluorine atom.
Positive C logP values reported in Table 1 for 30 and 32, featuring
one and two hydroxyl residues respectively, clearly indicated
a sensible decreasing in hydrophilicity for these inhibitors caused
by the replacement of the para-substituted benzene portion with
a biphenyl residue. In fact, the presence of at least one hydroxyl
group guarantees a global hydrophilic nature for all the others
inhibitors, from a minimum of ꢀ0.75 for 24 (one hydroxyl group on
the aminoacidic skeleton) to a maximum of ꢀ2.26 for 22 (three
hydroxyls, one on the aminoacidic skeleton and two on the side
chain not involved in the binding). Ultimately, the replacement of
the methoxy residue with phenyl ring did not increase selectivity vs
MMPs, conversely it made inhibitors’ worse.
49.2, 26.6, 25.2.
5.2. Synthesis of compound 15
A solution of 14 (1.20 g, 3.21 mmol) in 18 mL of a mixture AcOH/
H2O 80:20 was left to stir at 60 ꢁC for 4 h then toluene (20 mL) was
added. Concentration of the solvent under vacuum gave a crude
product which was purified by a flash column chromatography on
silica gel (EtOAc) to afford a mixture of 15 and 16 (total yield 75%) as
an oil. An analytical amount of compound 15 has been purified for
characterization. [
a
]
2D5: ꢀ7.00(c 0.50, CH3OH). Anal. for C13H19NO7S
calcd: C, 46.84; H, 5.74; N, 4.20. Found: C, 47.07; H, 5.01; N, 4.66. 1H
NMR (200 MHz, CDCl3): d 7.78e7.73 (AA’ part of an AA’MM’ system,
JAM ¼ 9.2 Hz, 2H), 7.00e6.96 (MM’ part of an AA’MM’ system,
JAM ¼ 9.2 Hz, 2H), 4.12e4.08 (A part of an AB system, JAB ¼ 18.4 Hz,
4. Conclusion
1H), 4.02e3.97 (B part of an AB system, JAB
¼
18.4 Hz,
1H), 3.92e3.85 (m, 1H), 3.87 (s, 3H), 3.76e3.56 (m, 1H), 3.72 (s, 3H),
3.39e3.28 (A part of an ABX system, JAx ¼ 7.6 Hz, JAB ¼ 14.6 Hz, 1H),
3.24e3.31 (B part of an ABX system, JBx ¼ 4.0 Hz, JAB ¼ 14.6 Hz, 1H),
In conclusion, the present analysis shows that the structure-
based approach to drug design can be easily exploited to modulate

E. Attolino et al. / European Journal of Medicinal Chemistry 45 (2010) 5919e5925
5925
2.42 (bs, 1H). 13C NMR (50 MHz, CDCl3):
114.3, 70.1, 63.4, 55.5, 52.4, 52.1, 50.2.
d 170.6, 163.1, 130.1, 129.4,
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5.3. General procedure for the synthesis of hydroxamic acids.
Synthesis of compound 17
A suspension of KOH (168 mg, 3.00 mmol) and NH2OH$HCl
(167 mg, 2.40 mmol) in CH3OH (2 mL) was stirred at room
temperature for 1 h, then a solution of 15 þ 16 (0.60 mmol) in
CH3OH (2 mL) was added. The reaction mixture was stirred until no
trace of starting material was revealed by TLC analysis (18 h), then
concentrated to dryness. The crude product was dissolved in EtOAc,
washed with a mixture AcOH/H2O 1:1 and dried over Na2SO4.
Concentration of the solvent under vacuum affords a crude product
which was purified by HPLC (C8 column Ultrasphere Beckman
Coulter, AcCN/H2O 10:90, AcCN/H2O 30:70) to afford 17 (90.0 mg,
45%) as a pale yellow glassy solid. [
a
]
2D5: ꢀ22.3(c 0.48, CH3OH).
HRMS for C12H19N2O7S þ H calcd: 335,0913. Found: 335.0909. ESI-
MS [M þ H]þ calcd: 335.1. Found: 335.1. [M þ Na]þ calcd: 357.1.
Found: 357.1. 1H NMR (400 MHz, CD3OD):
d 7.73e7.70 (AA’ part of
an AA’MM’ system, JAM ¼ 8.8 Hz, 2H), 7.05e7.03 (MM’ part of
a AA’MM’ system, JAM ¼ 8.8 Hz, 2H), 3.90e3.78 (m, 3H, CH2-2, H-20),
3.88 (s, 3H, OCH3), 3.52e3.51 (m, 2H, CH-30), 3.35e3.30 (m, 1H, H-
10a), 3.17e3.11 (B part of an ABX system, JBx ¼ 8.8 Hz, JAB ¼ 14.4 Hz,
1H, H-10b). 13C NMR (50 MHz, CD3OD):
114.0, 70.5, 63.6, 54.9, 53.4, 50.0.
d 167.3, 163.3, 129.7, 129.3,
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Author thank Dr. Stefano Roelens for fruitful discussions. This
work was supported by EC (Projects: SFMET nꢁ 201640, SPINE2-
COMPLEXES nꢁ 031220), by MIUR (Prot. RBLA032ZM7, Prot.
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Appendix. Supplementary material
Supplementary data related to this article can be found online at
doi:10.1016/j.ejmech.2010.09.057.
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