Sugar-Based Arylsulfonamide Carboxylates as Selective and Water-Soluble Matrix Metalloproteinase-12 Inhibitors

Article abstract of DOI:10.1002/cmdc.201600235

Matrix metalloproteinase-12 (MMP-12) can be considered an attractive target to study selective inhibitors useful in the development of new therapies for lung and cardiovascular diseases. In this study, a new series of arylsulfonamide carboxylates, with increased hydrophilicity resulting from conjugation with a β-N-acetyl-d-glucosamine moiety, were designed and synthesized as MMP-12 selective inhibitors. Their inhibitory activity was evaluated on human MMPs by using the fluorimetric assay, and a crystallographic analysis was performed to characterize their binding mode. Among these glycoconjugates, a nanomolar MMP-12 inhibitor with improved water solubility, compound 3 [(R)-2-(N-(2-(3-(2-acetamido-2-deoxy-β-d-glucopyranosyl)thioureido)ethyl)biphenyl-4-ylsulfonamido)-3-methylbutanoic acid], was identified.

Full text of DOI:10.1002/cmdc.201600235

DOI: 10.1002/cmdc.201600235
Full Papers
Sugar-Based Arylsulfonamide Carboxylates as Selective
and Water-Soluble Matrix Metalloproteinase-12 Inhibitors
Elisa Nuti,[a] Doretta Cuffaro,[a] Felicia D’Andrea,[a] Lea Rosalia,[a] Livia Tepshi,[b] Marina Fabbi,[c]
Grazia Carbotti,[c] Silvano Ferrini,[c] Salvatore Santamaria,[d] Caterina Camodeca,[e]
Lidia Ciccone,[a] Elisabetta Orlandini,[a] Susanna Nencetti,[a] Enrico A. Stura,[b] Vincent Dive,[b]
and Armando Rossello*[a]
Matrix metalloproteinase-12 (MMP-12) can be considered an
attractive target to study selective inhibitors useful in the de-
velopment of new therapies for lung and cardiovascular dis-
eases. In this study, a new series of arylsulfonamide carboxy-
lates, with increased hydrophilicity resulting from conjugation
with a b-N-acetyl-d-glucosamine moiety, were designed and
synthesized as MMP-12 selective inhibitors. Their inhibitory ac-
tivity was evaluated on human MMPs by using the fluorimetric
assay, and a crystallographic analysis was performed to charac-
terize their binding mode. Among these glycoconjugates,
a nanomolar MMP-12 inhibitor with improved water solubility,
compound 3 [(R)-2-(N-(2-(3-(2-acetamido-2-deoxy-b-d-glucopyr-
anosyl)thioureido)ethyl)biphenyl-4-ylsulfonamido)-3-methylbu-
tanoic acid], was identified.
Introduction
MMP-12, or macrophage metalloelastase, belongs to the
matrix metalloproteinase (MMP) family of enzymes, a well-
known class of endopeptidases able to degrade all the compo-
nents of the extracellular matrix (ECM). MMP-12 is a zinc-de-
pendent enzyme mainly produced by macrophages, and its
principal substrates are elastin, the major constituent of alveo-
lar walls, and other proteins such as type IV collagen, fibronec-
tin, laminin, gelatin, vitronectin, and chondroitin sulfates.[1]
MMP-12 was previously shown to be involved in acute and
chronic pulmonary inflammatory diseases such as chronic ob-
structive pulmonary disease (COPD) and emphysema.[2] More-
over, overexpression of MMP-12 was reported to be associated
with lung cancer. In particular, the clinical importance of MMP-
12 was demonstrated in non-small cell lung cancer (NSCLC) de-
velopment[3] with a critical role played by MMP-12 upregula-
tion in the transition from emphysema to lung cancer.[4] Finally,
MMP-12 was previously shown to be critical in the initiation
and progression of atherosclerosis by stimulating the transfor-
mation of fatty streaks into fibrous plaques.[5] These observa-
tions suggest that MMP-12 selective inhibitors could be poten-
tially useful for the treatment of lung and cardiovascular dis-
eases. Selective MMP-12 inhibitors were recently reported by
Dive et al. for atherosclerosis (RXP470.1),[6] by Li et al. for the
potential treatment of asthma (MMP145),[7] and by Astra
Zeneca for COPD (AZD1236),[8] among others. The principal ob-
stacles that hinder clinical development of MMP inhibitors
(MMPIs) are related to inadequate selectivity for the target
enzyme, poor water solubility with consequent poor oral bio-
availability, and long-term toxicity. Good water solubility of
MMPIs is highly desirable above all for inhibitors used in the
topical treatment of lung pathologies, such as emphysema
and COPD, which require good solubility in aqueous biological
fluids. Compounds with improved hydrophilicity are likely to
have better bioavailability and fewer side effects owing to
cross-inhibition of anti-target zinc proteases (caused by high
dosage).[9] Therefore, to obtain potent and selective MMP-12
inhibitors endowed with increased water solubility, we under-
took the design and synthesis of a series of arylsulfonamide
[a] Dr. E. Nuti, D. Cuffaro, Dr. F. D’Andrea, Dr. L. Rosalia, Dr. L. Ciccone,
Prof. E. Orlandini, S. Nencetti, Prof. A. Rossello
Department of Pharmacy, University of Pisa,
via Bonanno 6, 56126 Pisa (Italy)
E-mail: armando.rossello@farm.unipi.it
[b] L. Tepshi, Dr. E. A. Stura, Dr. V. Dive
CEA-Saclay, Service d’Ingenierie Moleculaire des Proteines,
CEA, iBiTec-S, 91191 Gif sur Yvette (France)
[c] Dr. M. Fabbi, G. Carbotti, Dr. S. Ferrini
Biotherapy Unit, IRCCS AOU San Martino-IST,
Largo R. Benzi 10, 16132 Genoa (Italy)
[d] Dr. S. Santamaria
carboxylates conjugated with
a b-N-acetyl-d-glucosamine
Kennedy Institute of Rheumatology,
University of Oxford, Roosevelt Drive, OX37FY Oxford (UK)
moiety, compounds 2–11 (Figure 1). Our starting hypothesis
was that the insertion of a sugar moiety in the proper position
of the arylsulfonamido scaffold could increase water solubility
without interfering with the potency of the inhibitor.[10] To
prove that, the inhibitory activities of the new glycoconjugates
were tested in vitro by using recombinant MMPs, and X-ray
crystallographic studies were performed to rationalize the re-
[e] Dr. C. Camodeca
Division of Immunology, Transplants and Infectious Diseases,
San Raffaele Scientific Institute, Via Olgettina, 20132 Milan (Italy)
Supporting Information (synthesis and characterization of all intermedi-
ate compounds and detailed crystallographic data) and the ORCID iden-
tification number(s) for the author(s) of this article can be found under
http://dx.doi.org/10.1002/cmdc.201600235.
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Figure 1. Structure of reference compound 1 and glycoconjugated analogues 2–11.
sults. Furthermore, the physicochemical properties of the most
promising derivatives were evaluated.
pounds 2–5, 10, and 11; Tables 1 and 3) or a 1,2,3-triazole
group (compounds 6–9, Table 2). These groups were chosen as
linkers because 1,2,3-triazoles show topological and electronic
similarities with amide bonds and for their inertness in vivo
with respect to oxidation and reduction processes,[14] whereas
the thioureido group is biocompatible and stable in most bio-
logical systems. A n-propyloxy chain was inserted between the
inhibitor and the sugar moiety in both series of derivatives,
compounds 4/5 (Scheme 3) and 8/9 (Scheme 4), to evaluate
the influence of a longer spacer on the affinity for the enzyme.
Finally, sugar-protected analogues, acetylated on the hydroxy
groups (compounds 2/4 and 6/8), were also evaluated.
Results and Discussion
Design of glycoconjugate MMP-12 inhibitors
Previous studies by Attolino et al.[11] proved that the introduc-
tion of hydrophilic chains on the nitrogen atom of a hydroxa-
mate-based sulfonamide scaffold does not substantially
change the affinity of the inhibitors for MMPs. On the basis of
these findings, in the present study we modified the structure
of recently reported carboxylate MMP-12 inhibitor 1[12]
(Figure 1) by introducing a glycosidic residue on its sulfona-
mide nitrogen atom (P2’ substituent) through insertion of
a suitable spacer. This choice was also supported by the X-ray
structure of the 1–MMP-12 complex,[12] which showed that the
benzamidoethyl group does not directly participate in binding.
b-N-Acetyl-d-glucosamine (GlcNAc) was chosen for the prepa-
ration of novel inhibitors 2–11 (Figure 1), because it is a very
common monosaccharide in nature. In fact, GlcNAc is present
in many glycostructures (e.g., chitin and chito-oligomers), is
crucial for protein and lipid glycosylation, and has a relevant
role in the synthesis of the aberrant glycosylated molecules on
cancer cell surfaces (multiantennary structures).[13] The conjuga-
tion between GlcNAc and the MMP inhibitor scaffold was ach-
ieved through the introduction of a thioureido group (com-
Chemistry
The preparation of all sugar moieties used for the coupling
with the MMP inhibitor is shown in Scheme 1. Known oxazo-
line 12[15] and azide 17[16] were obtained in two steps from
commercial d-glucosamine hydrochloride according to pub-
lished procedures. Glycosylation of 12 with 3-O-tosyl-1,3-prop-
andiol [HO(CH2)3OTs][17] was conducted in 1,2-dichloroethane
(DCE) with camphorsulfonic acid (CSA) as the catalyst, which
afforded b-glycoside 13 (J1,2 =8.5 Hz) stereoselectively in good
yield (69%). Known azidopropyl N-acetyl-d-glucosamine 14[18]
was prepared by conversion of 13 into the azide through an
SN2 reaction [NaN3, tetrabutylammonium iodide (TBAI), DMF] in
nearly quantitative yield (98%). Azido compounds 14 and 17[16]
Scheme 1. Synthesis of sugar intermediates 14, 16, 17, and 19. Reagents and conditions: a) CSA, HO(CH2)3OTs, 4 ꢁ molecular sieves, DCE, 808C, 12 h, 69%;
b) NaN3, Bu4NI, DMF, 508C, 1 h, 98%; c) 1. PPh3-resin, THF/H2O (20:1), 24 h; 2. DPT, CH2Cl2, 1.5–3 h, 46–64% (from azide).
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were reduced to corresponding amines 15[19] and 18[16]
(Scheme 1) in quantitative yields under Staudinger conditions
(resin-PPh3, THF/H2O). Without further purification, amines 15
and 18 were transformed into corresponding isothiocyanate
derivatives 16 and 19 through treatment with di-2-pyridyl thio-
nocarbonate (DPT). DPT[20] is a commercially available, solid,
nontoxic reagent that can be used in the preparation of iso-
thiocyanates as a safe alternative to thiophosgene. After 5–
24 h, intermediate b-glycosyl isothiocyanates 16 (64% yield
from 14) and 19 (46% yield from 17) were obtained after re-
moval of the solvents and purification by flash chromatogra-
phy. MMP inhibitor precursors 22, 25, and 27 were prepared as
described in Scheme 2. Commercially available biphenyl-4-sul-
fonyl chloride and p-bromobenzenesulfonyl chloride were re-
spectively converted into sulfonamides 20a and 20b by reac-
tion with d-valine in H2O and dioxane in the presence of trie-
thylamine as the base (44–45% yield) according to a previously
reported procedure.[21] Sulfonamides 20a and 20b were then
protected on the carboxylic moiety as tert-butyl esters (R)-21a
and (R)-21b by using N,N-dimethylformamide-di-tert-butyl
acetal at 958C (46–60% yield). Sulfonamide 21a was N-alkylat-
ed by treatment with propargyl bromide in DMF by using po-
tassium carbonate as the base to afford alkyne 22 in 82%
yield. Mitsunobu condensation of sulfonamides 21a and 21b
with commercial tert-butoxycarbonyl (Boc)-protected ethanola-
mine 23 in the presence of diisopropyl azodicarboxylate
(DIAD) and triphenylphosphine gave tert-butyl esters (R)-24a
and (R)-24b (62–85% yield). Pd-catalyzed Suzuki coupling of
commercially available 4-(4’-chlorobenzyloxy)phenylboronic
acid with p-bromo ester 24b in the presence of K3PO4 afforded
(R)-tert-butyl ester 26 in 80.6% yield. Finally, amines 25 and 27
were obtained as trifluoroacetate salts by selective hydrolysis
of the Boc group in the presence of the tert-butyl group upon
treatment of compounds 24a and 26 with trifluoroacetic acid
(TFA) under controlled conditions (2 h at 08C under a argon at-
mosphere, 74–78% yield). Thioureido derivatives 2–5, 10, and
11 were obtained through coupling of MMP inhibitor amino
precursors 25 and 27 with appropriate isothiocyanate b-N-
Scheme 2. Synthesis of MMP inhibitor intermediates 22, 25, and 27. Reagents and conditions: a) biphenyl-4-sulfonyl chloride or p-bromobenzenesulfonyl chlo-
ride, Et3N, H2O, dioxane, RT, 18 h, 44–45%; b) (CH3)2NCH[OC(CH3)3]2, toluene, 958C, 4 h, 46–60%; c) propargyl bromide, K2CO3, DMF, RT, 48 h, 82.6%; d) PPh3,
DIAD, THF, RT, 18 h, 62–85%; e) TFA, CH2Cl2, 08C, 2 h, 74–78%; f) 4-(4’-chlorobenzyloxy)phenylboronic acid, K3PO4, Pd(PPh3)4, dioxane/H2O (1:4.4), 708C, 2 h,
80.6%.
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acetyl-d-glucosamine derivative 16 or 19 (Scheme 3), whereas
copper-catalyzed azide–alkyne cycloaddition (CuAAC)[22] was
used to connect MMP inhibitor alkynyl precursor 22 to azide
b-N-acetyl-d-glucosamine derivatives 14 and 17 (Scheme 4) to
give 1,2,3-triazole derivatives 6–9. Condensation between iso-
thiocyanates 16 and 19 and MMP inhibitor precursors 25 and
27 (Scheme 3) was performed in CH2Cl2/DMF (4:1) in the pres-
ence of Et3N at 608C for 12–24 h. Purification of the crude
product by flash chromatography yielded pure 29 (96%),
whereas thiureido derivatives 28 and 30 were obtained with
an impurity of the corresponding isothiocyanates (ꢀ5%, de-
termined by NMR spectroscopy). Their structures were con-
firmed by the following reactions. b-Glycosyl azides 14 and 17
were conjugated to alkyne 22 (Scheme 4) by CuAAC click
chemistry according to a reported procedure.[22] The reactions
were performed in a mixture DMF/H2O (4:1) with a copper(II)
sulfate and sodium ascorbate catalytic system and were
heated under microwave irradiation at 808C for 30 min. De-
sired 1,2,3-triazole derivatives 31 and 32 were isolated after
purification by flash chromatography with complete regiospe-
cificity in good yields (81 and 94%, respectively).
Analysis of 31 and 32 by NMR spectroscopy (1H, 13C, and 2D
NMR experiments) confirmed their structures. The exclusive
formation of 1,4-disubstituted 1,2,3-triazoles 31 and 32 was
identified by the large D (dC4–dC5) values (ꢀ20 ppm) observed
in the 13C NMR spectra of the cycloadducts. Removal of the
tert-butyl group in 28–32 (Schemes 3 and 4) by treatment with
TFA gave desired carboxylic acids 4, 2, 10, 8, and 6 after tritu-
ration of the crude products with Et2O. Finally, de-O-acetylation
of 4, 2, 10, 8, and 6 by treatment with 3.5n NH3/MeOH afford-
Scheme 3. Synthesis of thioureido derivatives 2–5, 10, and 11. Reagents and conditions: a) Et3N, CH2Cl2/DMF (4:1), 608C, 20–24 h, 96%; b) CF3COOH, CH2Cl2,
RT, 24 h, 92%; c) 7n NH3/MeOH, MeOH, RT, 20–24 h, 79–94%.
Scheme 4. Synthesis of 1,2,3-triazole carboxylates 6–9. Reagents and conditions: a) CuSO4·5H2O, sodium ascorbate, DMF/H2O (4:1), microwave, 808C, 30 min,
81–94%; b) CF3COOH, CH2Cl2, RT, 24 h, 55–81%; c) 7n NH3/MeOH, MeOH, RT, 20–24 h, 67–70%.
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ed deprotected derivatives 5, 3, 11, 9, and 7 in good yields
(67–94%) after trituration of the crude products with Et2O. The
NMR spectra (1H, 13C, and 2D NMR experiments) and elemental
analyses of all the compounds confirmed the structures
shown.
pound. Inhibitory activity against MMP-9 (gelatinase B) was
chosen at first instance to evaluate selectivity for MMP-12.
MMP-9 together with MMP-2 and MMP-8 differ from MMP-12
by having an “intermediate S1’ pocket”, whereas the latter is
classified as a “deep S1’ pocket MMP”.[24] Improved selectivity
for MMP-12 over MMP-9 could be indicative of the ability of
the new derivatives to discriminate among the various MMP
family members. In general, 1) all new derivatives showed
stronger affinity for MMP-12 than for MMP-9 and 2) thioureido
derivatives 2–5 were more active than their triazole analogues,
compounds 6–9, on both enzymes. In particular, among the
thioureido derivatives the best results were obtained for com-
pound 2, an O-acetylated derivative with a short spacer be-
tween the sugar moiety and the biphenyl sulfonamide. The ac-
tivity of compound 2 on MMP-12 (IC50: 18 nm) was similar to
that of reference compound 1 with a fivefold better selectivity
over MMP-9. Noteworthy, in both series of compounds the in-
troduction of an n-propyloxy chain between the linker group
and GlcNAc did not substantially affect activity toward MMP-
12. Only in the thioureido series of compounds did acetylation
of the hydroxy groups on the carbohydrate moiety lead to
a small increase in activity in the compound with a short
spacer (i.e., 2 was twofold more active than 3).
MMP inhibition and crystallographic studies
The first series of carboxylic acids synthesized, that is, thiourei-
do derivatives 2–5 (Table 1) and 1,2,3-triazole derivatives 6–9
(Table 2), were assayed in vitro on human recombinant MMP-
12 by using a fluorogenic peptide[23] as the substrate. Inhibitor
1, devoid of any sugar moiety, was used as the reference com-
Table 1. MMP-12 and MMP-9 inhibitory activities of thioureido derivatives
2–5 and reference compound 1.
To understand the selectivity of compound 2 for MMP-12
over MMP-9, this inhibitor was co-crystallized with the catalytic
domain of both enzymes, and the crystal structure was deter-
mined to discern the details of ligand binding (Figure 2).
Compd
R
n
IC50 [nm][a]
In MMP-12, the acetylated sugar moiety that in compound 2
replaces the phenyl group of compound 1 improves its posi-
tioning to better reflect the change in character from a hydro-
phobic phenyl group to a hydrophilic sugar (Figure 2d). Differ-
ently, in MMP-9 the sugar maintains the same position of the
benzene-dicarboxyamide present in compound 1–1 (a bifunc-
tional analogue of compound 1, the X-ray structure of which
in complex with the catalytic domain of MMP-9 was previously
reported[12]) (Figure 2e). This results in improved MMP-12 in-
hibition and deterioration of MMP-9 inhibition. The sum of the
various effects translates into a substantial increase in the se-
lectivity of 2 for MMP-12 over MMP-9. Comparison of the crys-
tal structures of compounds 2 and 3 with a thioureido linker
with those of compounds 6, 7, and 8 with a triazole linker in
MMP-12 showed that the sugar moieties of the two classes of
compounds do not overlap (Figure 3). As noted for compound
2, the sugar moieties of all sugar derivatives select alternative
positions different from that preferred by compound 1 to opti-
mize their binding (Figure 3a).
MMP-12
MMP-9
2
3
4
5
1
Ac
H
Ac
H
0
0
1
1
18
40
36
28
35
1200
5400
1220
1200
510
[a] Values are the average of three determinations with a standard devia-
tion of <10%.
Table 2. MMP-12 and MMP-9 inhibitory activities of 1,2,3-triazole deriva-
tives 6–9 and reference compound 1.
The absence of the acetyl groups in compound 3 makes the
site used by 2 less favorable, as the ligand is forced to dock
closer to the MMP-12 surface. The improved activity of the in-
hibitors with the more flexible thioureido linker is likely to be
associated to their ability to select more appropriate positions
to improve their binding. On the basis of these findings, to fur-
ther increase the activity and selectivity for MMP-12 of this
class of inhibitors we decided to replace the biphenyl group
present in the sulfonamide moiety (P1’ group) of 2 with a 4-
(4’-chlorobenzyloxy)biphenyl group. In fact, the introduction of
this substituent in P1’ was previously reported to confer good
Compd
R
n
IC50 [nm][a]
MMP-12
MMP-9
6
7
8
9
1
Ac
H
Ac
H
0
0
1
1
72
53
63
73
35
4400
3100
6800
9400
510
[a] Values are the average of three determinations with a standard devia-
tion of <10%.
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Table 3. MMP-12 and MMP-9 inhibitory activities of 4-(4’-chlorobenzylox-
y)biphenyl thioureido derivatives 10 and 11.
Compd
R
IC50 [nm][a]
MMP-12
MMP-9
10
11
Ac
H
12
42
860
1470
[a] Values are the average of three determinations with a standard devia-
tion of <10%.
Figure 2. a) Molecular surface of MMP-9 with 2 (PDB ID: 5I12) bound show-
ing the positioning of the sugar moiety. b) Molecular surface of MMP-12
with 2 (PDB ID: 5IOL) in the same orientation. The acetylated sugar moiety
fits well in a pocket on the molecular surface. The sugar portion of the com-
pound is in a poor electron density area in both MMP-9 and MMP-12, which
indicates that the interactions it makes with either protein are weak and un-
likely to contribute to the enthalpy substantially. c) Comparison of the bind-
ing of compound 1 to MMP-12 (PDB ID: 4H84)[12] and 1–1 bound to MMP-9
(PDB ID: 4HMA)[12] to show they bind in a similar manner to the two differ-
ent MMPs. d) Comparison of the binding of 1 and 2 to MMP-12 to show the
change in position of the acetylated glucosamine of 2 relative to the phenyl
ring of 1. e) Comparison of the binding of compounds 1–1 and 2 to MMP-9
to show that the sugar portion follows the same “natural” binding tendency
as evidenced by the phenyl group of 1–1.
Also for these last compounds, similar to the other thiourei-
do derivatives endowed with a short spacer (compounds 2
and 3), O-acetylation led to a small increase in activity, and
sugar-deprotected derivative 11 was less potent (IC50 =42 nm)
than its acetylated analogue 10. At this point, the selectivity
profiles of the most promising MMP-12 sugar-based inhibitors,
that is, 2, 3, and 10, were evaluated by testing them on MMP-
1, MMP-2, and MMP-14 and by comparing the results with
those obtained with 1 (Table 4). Compounds 2 and 10 showed
similar inhibitory potency on MMP-12 (IC50 =18 and 12 nm, re-
spectively), slightly higher than that of reference compound
1 (IC50 =35 nm). However, as expected, the introduction of the
4-(4’-chlorobenzyloxy)biphenyl group in P1’ led to a great im-
provement in selectivity over MMP-1 and MMP-14, two MMPs
widely believed to be responsible for some side effects that
have been clinically observed with the use of broad-spectrum
selectivity for MMP-12 and MMP-13 to similar sulfonamido-
based derivatives.[25] 4-(4’-Chlorobenzyloxy)biphenyl thioureido
derivatives 10 and 11 (Table 3) were subsequently synthesized
and tested on MMP-12 and MMP-9. Unsurprisingly, 10 was the
most promising compound, with nanomolar activity against
MMP-12 (IC50 =12 nm) and a 70-fold selectivity over MMP-9.
Figure 3. a) Superimpositions of compounds 1 (yellow), 2 (cyan), and 7 (pink) (PDB ID: 5I4O) showing their relative positions in the catalytic domain of MMP-
12. b) Compounds 2 and 3 (green) (PDB ID: 5I3M) with the more flexible thioureido linker place their sugar moiety in a totally different orientation relative to
those with the triazole linker. c) Compounds 6 (ochre) (PDB ID: 5I2Z) and 7 and 8 (white) (PDB ID: 5I43) with the triazole linker extended along the same
path. The only noticeable difference with increasing linker length is a worse definition of the sugar in the electron density, which implies greater mobility.
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positive effect resulting from the presence of the glycosidic
moiety.
Table 4. Selectivity profiles of compounds 2, 3, and 10 in comparison
with that of 1.
Overall these biological and physicochemical data show that
the introduction of a glycosidic portion in P2’, linked through
a flexible linker (as in compound 3), represents a good com-
promise to maintain nanomolar activity against MMP-12 while
boosting bioavailability.
Compd
IC50 [nm][a]
MMP-9
MMP-1
MMP-2
MMP-12
MMP-14
2
3
10
1
19000
40000
50000
16000
330
320
100
170
1200
5400
860
18
40
12
35
3900
8900
15500
1830
510
[a] Values are the average of three determinations with a standard devia-
tion of <10%.
Conclusions
In the present study, a new series of carboxylate-based MMP-
12 inhibitors were designed and synthesized starting from pre-
viously reported compound 1. To improve the hydrophilicity
and bioavailability of these arylsulfonamides without decreas-
ing their affinity for the target, we linked a b-N-acetyl-d-glucos-
amine (GlcNAc) moiety in P2’. Conjugation between GlcNAc
and the MMP inhibitor scaffold was achieved through the in-
troduction of a thioureido group or a 1,2,3-triazole group as
the linker. The new glycoconjugates were tested on human
MMPs by using a fluorimetric assay. All new derivatives
showed stronger affinity for MMP-12 than for MMP-9, and the
thioureido derivatives were more active than their triazole ana-
logues. In particular, compound 2, an O-acetylated thioureido
biphenyl sulfonamide, was selected for further studies given its
high affinity for MMP-12 (IC50 =18 nm). Crystallographic analy-
sis provided insight into the binding mode of 2 to MMP-12, its
selectivity over MMP-9, and the higher affinity for MMP-12
with respect to that of the triazole analogues. To further opti-
mize its activity and selectivity, the structure of 2 was modified
by introducing a 4-(4’-chlorobenzyloxy)biphenyl group in P1’.
As expected, 4-(4’-chlorobenzyloxy)biphenyl thioureido deriva-
tive 10 showed nanomolar activity against MMP-12 (IC50 =
12 nm) and 70-fold selectivity over MMP-9. At that point, the
physicochemical properties of the most promising derivatives,
that is, 2, 10, and their respective sugar-deprotected deriva-
tives 3 and 11, were evaluated, and the results were compared
with those obtained with starting compound 1. Compound 3
was the one able to conjugate a nanomolar activity for MMP-
12 and good selectivity over MMPs with improved hydrophilici-
ty (water solubility>5 mm and clogP=3.15). These preliminary
results suggest that the introduction of a sugar moiety in P2’,
linked through a flexible linker as in compound 3, not only im-
proves the hydrophilicity of MMP-12 inhibitors but also influ-
ences their biological activity on isolated enzymes (higher se-
lectivity over other MMPs). Of course, to fully appreciate the
effect of the improved bioavailability of 3 with respect to that
of 1, an in vivo assay will be necessary.
MMP inhibitors, such as musculoskeletal syndrome (MSS).[26] In
fact, 4-(4’-chlorobenzyloxy)biphenyl thioureido derivative 10
displayed a 6-fold increase in selectivity for MMP-12 over
MMP-14 and a 41-fold increase in selectivity for MMP-12 over
MMP-1 with respect to the selectivity of biphenyl analogue 2
for MMP-12. Compound 3 showed the same potency as 1 on
MMP-12 and a selectivity profile similar to that of its sugar-ace-
tylated analogue 2. In particular, it had 200-fold selectivity for
MMP-12 over MMP-14 and 1000-fold selectivity for MMP-12
over MMP-1.
The improvement in hydrophilicity caused by the presence
of the glycosidic residue was evaluated by the octanol/water
partition coefficient (logP). The logP values for most active thi-
oureido derivatives 2, 3, 10, and 11 were calculated as clogP
values, and the results were compared with the value calculat-
ed for starting compound 1, devoid of the glycosidic residue
(Table 5). Deprotected glycosidic derivative 3 had a clogP
value of 3.15, which was lower than that of its acetylated ana-
logue 2 and that of reference compound 1, with clogP values
of 4.75 and 5.68, respectively. Also, the water solubility of the
new compounds was determined at pH 7.4 (see Table 5), and
both glycoconjugates 2 and 3 showed increased solubility
(>5 mm) with respect to that of reference compound 1. Re-
garding 4-(4’-chlorobenzyloxy)biphenyl thioureido derivatives
10 and 11, they showed high clogP values (>5) and low solu-
bility (ꢀ200 mm). In this case, the introduction of the 4-(4’-
chlorobenzyloxy)biphenyl portion strongly contributed to im-
proving the lipophilicity of the molecules, which countered the
Table 5. Physicochemical properties of compounds 2, 3, 10, and 11 in
comparison with those of 1.
Compd
clogP[a]
Solubility[b] [mm]
1
2
3
10
11
5.68
4.75
3.15
6.81
5.21
790
>5000
>5000
180
Experimental Section
230
Chemistry
[a] Hydrophobicity was calculated as the partition coefficient between oc-
tanol and water, logP (o/w) by using ACD laboratory software ver-
sion 14.0 (Advanced Chemistry Development, Inc. Toronto, Canada).
[b] Solubility was determined in 50 mm phosphate buffer, pH 7.4, at room
temperature after 24 h of equilibration. Concentrations were assessed for
supernatants of centrifuged samples by UV/Vis spectroscopy at l=
270 nm.
Melting points were determined with a Kofler hot-stage apparatus.
Optical rotations were measured with a PerkinElmer 241 polarime-
1
ter at (20ꢁ2)8C. H NMR spectra were recorded in appropriate sol-
vents with a Bruker Avance II operating at 250.12 MHz or a Bruker
Avance III HD 400 spectrometer operating at 400 MHz. 13C NMR
spectra were recorded with the above spectrometers operating at
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&
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Full Papers
62.9 or 100.57 MHz. The assignments were made, if possible, with
the aid of DEPT, COSY, and HSQC experiments. The first-order
proton chemical shifts, d, were referenced to residual solvents.
Where indicated, the elemental compositions of the compounds
agreed to within 0.4% of the calculated values. All reactions were
followed by TLC on Kieselgel 60 F254 with detection by UV light
and/or with ethanolic 10% phosphomolybdic or sulfuric acid and
heating. Kieselgel 60 (Merck) was used for column and flash chro-
matography (70–230 and 230–400 mesh, respectively). Some flash
chromatography operations were conducted by the automated
system Isolera Four SV (Biotage), equipped with a UV detector with
variable wavelength (l=200–400 nm) or by using prepacked ISO-
LUTE Flash Si II cartridges (Biotage). Microwave-assisted reactions
were run in a CEM Discover LabMate microwave synthesizer.
Unless otherwise noted, solvents and reagents were obtained from
commercial suppliers and were used without further purification.
All reactions involving air- or moisture-sensitive reagents were per-
formed under an argon atmosphere by using anhydrous solvents.
Anhydrous dimethylformamide (DMF), dichloromethane (CH2Cl2),
1,2-dichloethane (DCE), and THF were purchased from Sigma–Al-
drich. Other dried solvents were obtained by distillation according
to standard procedures[27] and were stored over 4 ꢁ molecular
sieves activated for at least 24 h at 2008C. MgSO4 or Na2SO4 was
used as the drying agent for solutions. 2-Methyl-(3,4,6-tri-O-acetyl-
1,2-dideoxy-a-d-glucopyrano)-[2,1-d]-2oxazoline (12),[15] 2-acetami-
do-3,4,6-tri-O-acetyl-2-deoxy-b-d-glucopyranosyl azide (17),[16] and
3-O-tosyl-1,3-propandiol[17] were prepared according to reported
procedures.
washed with saturated aq NaHCO3 (12 mL); the organic phase was
separated, and the aqueous layer was extracted with Et2O (2ꢂ
12 mL). The collected organic extracts were dried (MgSO4·H2O), fil-
tered, and concentrated under reduced pressure. Purification by
flash chromatography (silica gel) gave the pure triazole-linked de-
rivative.
Triazole-linked tert-butyl ester 31: Flash chromatography (silica
gel, n-hexane/EtOAc 3:7+0.01% Et3N) afforded pure 31 (81% yield
calculated from 22) as a clear syrup; Rf =0.64 (EtOAc); ½aꢂ2D3 = +
1
46.2 (c=1.06 in CHCl3); H NMR (250.12 MHz, CD3CN): d=7.92–7.76
(m, 5H; 4Ar-H, CH-triazole), 7.69–7.64 (m, 2H; 2Ar-H), 7.54–7.40 (m,
3H; 3Ar-H), 6.63 (d, J2,NH =9.2 Hz, 1H; NHAc), 5.16 (dd, J2,3
=
10.6 Hz, J3,4 =9.5 Hz, 1H; H-3), 4.95 (dd, J4,5 =9.9 Hz, 1H; H-4), 4.87,
4.69 (AB system, JA,B =16.6 Hz, 2H; CH2NSO2), 4.61 (d, J1,2 =8.5 Hz,
1H; H-1), 4.37 (t, Jvic =7.2 Hz, 2H; CH2N), 4.22 (dd, J5,6b =4.9 Hz,
J
J
J
6a,6b =12.3 Hz, 1H; H-6b), 4.06 (dd, J5,6a =2.2 Hz, 1H; H-6a), 3.93 (d,
vic =10.6 Hz, 1H; CHNSO2), 3.85 (ddd, 1H; H-2), 3.72, 3.40 (2dt,
vic =6.0 Hz, Jgem =10.6 Hz, each 1H; CH2O), 3.70 (m, 1H; H-5), 2.22
(m, 1H; Me2CH), 2.05 (m, 2H; CH2), 2.00, 1.96, 1.94 (3s, each 3H;
3MeCOO), 1.86 (s, 3H; MeCON), 1.23 (s, 9H; CMe3), 0.90 (d, Jvic
=
6.6 Hz, 3H; Me2CH), 0.72 ppm (d, Jvic =6.5 Hz, 3H; Me2CH); 13C NMR
(62.9 MHz, CD3CN): d=171.3, 171.1, 171.0, 170.5, 170.5 (5C=O),
146.3, 146.0 (Ar-C-SO2, C-triazole), 139.9, 139.8, (2Ar-C), 130.1–128.2
(Ar-CH), 125.4 (CH-triazole), 101.7 (C-1), 82.9 (Me3CO), 73.5 (C-3),
72.4 (C-5), 69.7 (C-4), 67.7 (CHN), 66.9 (CH2O), 62.9 (C-6), 54.8 (C-2),
47.5 (CH2N), 41.0 (CH2NSO2), 30.9 (CH2), 29.7 (CHMe2), 27.9 (Me3C),
23.2 (MeCON), 20.9 (3MeCOO), 20.0, 19.4 ppm (Me2CH); elemental
analysis calcd (%) for C41H55N5O13S: C 57.40, H 6.46, N 8.16; found:
C 57.37, H 6.41, N 8.25.
Synthesis
Triazole-linked tert-butyl ester 32: Flash chromatography on silica
tert-Butyl ester 29: Isothiocyanate 19 (0.49 mmol, 1.1 equiv) was
solubilized in dry CH2Cl2/DMF (4:1, 26 mL), and a solution of amine
salt 25 (0.45 mmol, 1 equiv) in dry CH2Cl2/DMF (4:1, 26 mL) con-
taining Et3N (62 mL, 0.45 mmol) was added dropwise. The mixture
was stirred at 608C until TLC analysis (EtOAc) revealed the com-
plete disappearance of amine salt 25 (7 h), and then the mixture
was concentrated under reduced pressure. Purification by flash
chromatography (petroleum ether/EtOAc 2:1) using a Isolute Flash
Si II cartridge afforded pure 29 (353 mg, 96% yield calculated from
25) as a clear syrup; Rf =0.65 (EtOAc); ½aꢂ2D3 = +14.0 (c=1.07 in
gel (n-hexane/EtOAc 3:7+0.01% Et3N) afforded pure 32 (94%
23
yield calculated from 22) as a white foam; Rf =0.53 (EtOAc); ½aꢂD
=
1
+22.0 (c=0.9 in CHCl3); H NMR (250.12 MHz, CD3CN): d=8.05 (s,
1H; CH-triazole), 7.90–7.77 (m, 4H; Ar-H), 7.69–7.64 (m, 2H; Ar-H),
7.54–7.41 (m, 3H; Ar-H), 6.56 (d, J2,NH =9.2 Hz, 1H; NHAc), 5.19 (d,
J
1,2 =10.0 Hz, 1H; H-1), 5.43 (dd, J2,3 =10.4 Hz, J3,4 =9.5 Hz, 1H; H-
3), 5.17 (dd, J4,5 =9.7 Hz, 1H; H-4), 4.56 (m, 1H; H-2), 4.89, 4.68 (AB
system, JAB =16.7 Hz, 2H; CH2N), 4.21 (dd, J5,6b =5.4 Hz, J6a,6b
12.7 Hz, 1H; H-6b), 4.13–4.04 (m, 2H; H-6a, H-5), 3.88 (d, Jvic
=
=
10.5 Hz, 1H; CHN), 2,21 (m, 1H; CHMe2), 2.01 (s, 3H; MeCOO), 1.98
(s, 6H; 2MeCOO), 1.63 (s, 3H; MeCOON), 1.20 (s, 9H; Me3C), 0.89 (d,
1
CHCl3); H NMR (250.12 MHz, CD3CN): d=7.93–7.78 (m, 4H; Ar-H),
7.65 (m, 2H; Ar-H), 7.53–7.40 (m, 3H; Ar-H), 7.30, 7.16 (2brs, each
1H; 2NHCS), 6.70 (d, J2,NH =8.7 Hz, 1H; NHAc), 5.25–5.15 (m, 2H; H-
J
vic =6.6 Hz, 3H; Me2CH), 0,67 ppm (d, Jvic =6.5 Hz, 3H; Me2CH);
13C NMR (62.9 MHz, CD3CN): d=171.2, 170.9, 170.8, 170.5, 170.4
(5C=O), 146.3 (Ar-C-SO2, C-triazole), 140.0, 139.7 (2Ar-C), 130.1–
128.2 (Ar-CH), 124.8 (CH-triazole), 86.1 (C-1), 82.9 (Me3CO), 75.4 (C-
5), 73.2 (C-3), 69.1 (C-4), 67.6 (CHN), 62.7 (C-6), 53.9 (C-2), 40.7
(CH2NSO2), 29.7 (CHMe2), 27.8 (Me3C), 22.8 (MeCON), 20.8
(3MeCOO), 20.0, 19.4 ppm (Me2CH); elemental analysis calcd (%)
for C38H49N5O12S (799.89): C 57.06, H 6.17, N 8.76; found: C 57.02, H
6.14, N 8.74.
1, H-3), 4.97 (dd, J3,4 =9.6 Hz, J4,5 =9.7 Hz, 1H; H-4), 4.23 (dd, J5,6b
=
4.4 Hz, J6a,6b =12.1 Hz, 1H; H-6b), 4.05 (m, 2H; H-6a, H-2), 3.88–3.20
(m, 5H; H-5, CHNSO2, CH2NSO2, CH2NHCS), 3.35 (m, 1H; CH2NSO2),
2.25 (m, 1H; Me2CH), 1.98, 1.97, 1.96 (3 s, each 3H; 3MeCOO), 1.87
(s, 3H; MeCON), 1.18 (s, 9H; Me3C), 1.00 (d, Jvic =6.3 Hz, 3H;
Me2CH), 0.91 ppm (d, Jvic =6.5 Hz, 3H; Me2CH); 13C NMR (62.9 MHz,
CD3CN): d=184.1 (C=S), 172.7, 171.3, 171.2, 170.6, 170.4 (5C=O),
146.4 (Ar-C-SO2), 139.9, 138.9 (2Ar-C), 130–128.2 (Ar-CH), 83.8 (C-1),
82.8 (Me3CO), 73.4 (C-3), 73.2 (C-5), 69.5 (C-4), 67.7 (CHN), 62.8 (C-
6), 53.6 (C-2), 45.9 (CH2NH), 44.3 (CH2NSO2), 29.8 (CHMe2), 27.9
(Me3C), 23.0 (MeCON), 21.1, 20.9, 20.8 (3MeCOO), 20.5, 19.5 ppm
(Me2CH); elemental analysis calcd (%) for C38H52N4O12S2 (820.97): C
55.59, H 6.38, N 6.82; found: C 55.56, H 6.35, N 6.79.
General procedure for the transformation of tert-butyl esters 29,
31, and 32 into carboxylic acids 2, 8, and 6: Ester 29, 31, or 32
(0.1 mmol) was dissolved in CH2Cl2 (1.7 mL) and treated with
CF3COOH (0.6 mL), this mixture was stirred at 08C for 1 h and then
at RT. After 24 h, TLC analysis (EtOAc) revealed complete disappear-
ance of the starting material and the formation of a more retained
product. The solution was co-evaporated with toluene (4ꢂ10 mL)
under reduced pressure, and trituration of the crude product with
Et2O afforded the pure carboxylic acid.
General procedure for the synthesis of triazole-linked tert-butyl
esters 31 and 32: Alkyne 22 (0.1 mmol), sugar azide derivative 14
or 17 (0.11 mmol, 1.1 equiv), CuSO4·5H2O (0.1 mmol, 1.5 equiv),
and sodium ascorbate (0.3 mmol, 3 equiv) were dissolved in DMF/
H2O (4:1, 3.2 mL). The solution was heated under microwave irradi-
ation to 808C for 30 min and then diluted with Et2O (12 mL) and
Carboxylic acid 2: Yield: 92%, white solid; Rf =0.41 (EtOAc); mp:
134–1358C; ½aꢂ2D3 = +9.46 (c=1.3 in CHCl3); 1H NMR (250.12 MHz,
&
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Full Papers
CD3OD/D2O): d=7.93–7.90 (m, 2H; Ar-H), 7.76–7.73 (m, 2H; Ar-H),
7.64 (m, 2H; Ar-H), 7.50–7.35 (m, 3H; Ar-H), 5.52 (brs, 1H; H-1),
5.19 (dd, J3,4 =10.3 Hz, J2,3 =9.6 Hz, 1H; H-3), 5.02 (dd, J4,5 =9.9 Hz,
1H; H-4), 4.28 (dd, J5,6b =3.8 Hz, J6a,6b =12.5 Hz, 1H; H-6b), 4.20–
3.75 [m, 7H; H-6a, H-2, H-5, CHNSO2, CH2NSO2 (1H), CH2NHCS], 3.41
(m, 1H; CH2NSO2), 2.25 (m, 1H; Me2CH), 1.95 (s, 9H; 3MeCOO), 1.91
(s, 3H; MeCON), 0.98 ppm (m, 2H; Me2CH); 13C NMR (62.9 MHz,
CD3OD/D2O): d=182.1 (C=S), 171.7–169.2 (5C=O), 145.8 (Ar-C-
SO2), 138.9 (2Ar-C), 128.9–127.2 (Ar-CH), 83.4 (C-1), 72.8, 72.7 (C-3,
C-5), 67.7, 67.6 (C-4, CHN), 61.8 (C-6), 53.8 (C-2), 43.9, 43.8 (CH2NH,
CH2NSO2), 29.6 (CHMe2), 23.1 (MeCON), 20.7–20.6 (3MeCOO), 20.5,
19.6 ppm (Me2CH); elemental analysis calcd (%) for C34H44N4O12S2
(764.86): C 53.39, H 5.80, N 7.33; found: C 53.38, H 5.79, N 7.30.
d=7.95–7.89 (m, 3H; 2Ar-H, NHCS), 7.83–7.77 (m, 2H; 2Ar-H),
7.68–7.63 (m, 2H; 2Ar H), 7.53–7.40 (m, 3H; 3Ar-H), 6.89 (d, J2,NH
9.4 Hz, 1H; NHAc), 6.68 (brt, J=5.1 Hz, 1H; NHCS), 5.14 (dd, J2,3
=
=
10.7 Hz, J3,4 =9.4 Hz, 1H; H-3), 4.95 (dd, J4,5 =9.9 Hz, 1H; H-4), 4.53
(d, J1,2 =8.4 Hz, 1H; H-1), 4.22 (dd, J5,6b =4.8 Hz, J6a,6b =12.3 Hz, 1H;
H-6b), 4.05 (dd, J5,6a =2.6 Hz, 1H; H-6a), 3.87 (d, Jvic =10.4 Hz, 1H;
CHNSO2), 4.10–3.45 [m, 6H; H-2, H-5, CH2NH (1H), CH2NH (1H),
CH2O (1H), CH2NSO2 (1H)], 3.65–3.45 [m, 3H; CH2NH (1H), CH2O
(1H), CH2NSO2 (1H)], 3.38–3.25 [m, 1H, CH2NH (1H)], 2.21 (m, 1H;
Me2CH), 1.75 (m, 2H; CH2), 2.00, 1.96, 1.95 (3s, each 3H; 3MeCOO),
1.90 (s, 3H; MeCON), 1.19 (s, 9H; CMe3), 1.03 (d, Jvic =6.5 Hz, 3H;
Me2CH), 0.92 ppm (d, Jvic =6.6 Hz, 3H; Me2CH); 13C NMR (62.9 MHz,
CD3CN): d=183.4 (C=S), 172.4, 171.3, 171.1, 170.5, 170.3 (5C=O),
146.2 (Ar-C-SO2), 139.9, 139.1, (2Ar-C), 130.1–128.1 (Ar-CH), 101.8
(C-1), 82.7 (Me3CO), 73.4 (C-3), 72.3 (C-5), 69.7 (C-4), 69.7 (CH2O),
67.6 (CHNSO2), 62.9 (C-6), 55.3 (C-2), 45.3, 44.9, 44.8 (2CH2N,
CH2NSO2), 29.4 (CH2), 29.8 (CHMe2), 27.9 (Me3C), 23.3 (MeCON), 20.9
(3MeCOO), 20.5, 19.5 ppm (Me2CH). Crude 28 (125 mg) was dis-
solved in CH2Cl2 (2.3 mL), treated with CF3COOH (0.7 mL), and
stirred at 08C for 1 h and then at RT. After 24 h, TLC analysis
(EtOAc) revealed the complete disappearance of the starting mate-
rial and the formation of a more retained product (Rf =0.26). The
solution was co-evaporated with toluene (4ꢂ10 mL) under re-
duced pressure, and the trituration of the crude product with Et2O
afforded pure carboxylic acid 4 (101 mg, 72% yield calculated from
16) as a white solid; Rf =0.26 (EtOAc); mp: 103–1058C; ½aꢂ2D3 = +
Carboxylic acid 8: Yield: 55%, white solid; Rf =0.24 (CHCl3/MeOH
95:5); mp: 114–1158C; ½aꢂ2D3 = +6.93 (c=1.0 in CHCl3); 1H NMR
(250.12 MHz, CD3CN/CDCl3): d=7.79–7.42 (m, 11H, CH-triazole,
9Ar-H, COOH), 6.82 (d, J2,NH =7.8 Hz, 1H; NHAc), 5.19 (dd, J2,3
=
10.1 Hz, J3,4 =9.6 Hz, 1H; H-3), 4.94 (t, J4,5 =9.6 Hz, 1H; H-4), 4.80–
4.45 (m, 2H; CH2NSO2), 4.60 (d, J1,2 =8.5 Hz, 1H; H-1), 4.30–4.12 (m,
3H; H-6b, CH2N), 4.04 (dd, J5,6a =1.9 Hz, J6a,6b =12.1 Hz, 1H; H-6a),
3.87–3.72 (m, 3H; H-5, H-2, CHNSO2), 3.70, 3.28 (2brs, each 1H;
CH2O), 2.28–2.08 (m, 3H; CH2, Me2CH), 1.98, 1.96, 1.93 (3s, each
3H; 3MeCOO), 1.85 (s, 3H; MeCON), 0.92, 0.83 ppm (2brs, each
3H; Me2CH); 13C NMR (62.9 MHz, CD3CN/CDCl3): d=171.9, 171.3,
171.2, 171.0, 170.5 (5C=O), 145.7, 147.6 (Ar-C-SO2, C-triazole), 139.8,
139.7 (2Ar-C), 129.9–128.0 (Ar-CH), 128.4 (CH-triazole), 101.4 (C-1),
73.2 (C-3), 72.3 (C-5), 69.6 (C-4), 66.5 (CH2O), 66.2 (CHN), 62.8 (C-6),
54.9 (C-2), 47.5 (CH2N), 41.2 (CH2NSO2), 30.8 (CH2), 30.7 (CHMe2),
23.1 (MeCON), 20.9 (3MeCOO), 20.8, 19.8 ppm (Me2CH); elemental
analysis calcd (%) for C37H47N5O13S (801.86): C 55.42, H 5.91, N 8.73;
found: C 55.40, H 5.89, N 8.70.
1
17.1 (c=0.9 in CHCl3); H NMR (250.12 MHz, CD3CN): d=7.93–7.90
(m, 3H; 2Ar-H, OH), 7.81–7.77 (m, 2H; 2Ar-H), 7.73–7.60 (m, 2H;
2Ar-H), 7.58–7.18 (m, 3H; 3Ar-H, NHCS), 6.893–6.78 (m, 2H; NHAc,
NHCS), 5.104 (dd, J2,3 =10.6 Hz, J3,4 =9.4 Hz, 1H; H-3), 4.95 (dd,
J
4,5 =9.8 Hz, 1H; H-4), 4.56 (d, J1,2 =8.6 Hz, 1H; H-1), 4.22 (dd, J5,6b =
5.0 Hz, J6a,6b =12.3 Hz, 1H; H-6b), 4.03 (dd, J5,6a =2.5 Hz, 1H; H-6a),
4.01 (d, Jvic =10.42 Hz, 1H; CHNSO2), 3.97–3.43 [m, 10H; H-2, H-5,
2CH2NH, CH2O, CH2NSO2), 2.18 (m, 1H; Me2CH), 1.74 (m, 2H; CH2),
1.99, 1.96, 1.94 (3s, each 3H; 3MeCOO), 1.88 (s, 3H; MeCON), 0.95
(d, Jvic =6.6 Hz, 3H; Me2CH), 0.92 ppm (d, Jvic =6.5 Hz, 3H; Me2CH);
13C NMR (62.9 MHz, CD3CN): d=182.4 (C=S), 172.8, 171.3, 171.2,
171.1, 170.5 (5C=O), 146.1 (Ar-C-SO2), 139.9, 139.7, (2Ar-C), 130.0–
128.2 (Ar-CH), 101.7 (C-1), 73.1 (C-3), 72.3 (C-5), 69.7 (C-4), 69.5
(CH2O), 66.4 (CHNSO2), 62.8 (C-6), 54.8 (C-2), 44.9, 44.8, 44.7
(2CH2N, CH2NSO2), 29.4 (CH2), 29.2 (CHMe2), 23.2 (MeCON), 20.9
(3MeCOO), 20.4, 19.7 ppm (Me2CH); elemental analysis calcd (%)
for C37H50N4O13S2 (822.94): C 54.00, H 6.12, N 6.81; found: C 54.02,
H 6.10, N 6.79.
Carboxylic acid 6: Yield: 81%, white solid; Rf =0.57 (EtOAc); mp:
144–1468C; ½aꢂ2D3 = +18.7 (c=0.98 in MeOH); H NMR (250.12 MHz,
1
CDCl3): d=8.06–7.80 (m, 3H; 2Ar-H, CH-triazole), 7.74–7.35 (m, 8H;
7Ar-H, NH), 5.93 (d, J1,2 =8.9 Hz, 1H; H-1), 5.31 (dd, J2,3 =9.3 Hz,
J
3,4 =10.1 Hz, 1H; H-3), 5.06 (brt, J4,5 =10.1 Hz, 1H; H-4),4.81, 4.45
(AB system, JA,B =17.1 Hz, 2H; CH2N), 4.65 (m, 1H; H-2), 4.24 (d,
vic =9.2 Hz, 1H; NCH), 4.10–3.90 (m, 3H; H-5, H-6a, H-6b), 2.25 (m,
1H; Me2CH), 2.04, 2.00, 1.99 (3s, each 3H; 3MeCOO), 1.84 (s, 3H;
MeCOON), 0.88 (d, Jvic =4.9 Hz, 3H; Me2CH), 0.68 ppm (d, Jvic
J
=
5.5 Hz, 3H; Me2CH); 13C NMR (62.9 MHz, CDCl3): d=174.6 (COOH),
171.8, 171.4, 171.3, 169.8 (4C=O), 146.6 (Ar-C-SO2, C-triazole), 139.7,
139.3 (2Ar-C), 129.9–126.0 (Ar-CH), 122.0 (CH-triazole), 86.8 (C-1),
75.5 (C-5), 73.2 (C-3), 68.8 (C-4), 64.6 (CHN), 62.4 (C-6), 53.7 (C-2),
40.4 (CH2NSO2), 26.4 (CHMe2), 23.6 (MeCON), 21.3–21.2 (3MeCOO),
22.1, 19.4 ppm (Me2CH); elemental analysis calcd (%) for
C34H41N5O12S (743.78): C 54.90, H 5.56, N 9.42; found: C 54.88, H
5.54, N 9.39.
Carboxylic acid 10: Isothiocyanate 19 (49.8 mg, 0.133 mmol) was
solubilized in dry CH2Cl2/DMF (4:1, 13.5 mL), and a solution of
amine 27 (89.4 mg, 0.133 mmol) in dry CH2Cl2/DMF (4:1, 13.5 mL)
containing Et3N (37 mL, 2 equiv) was added dropwise. The mixture
was stirred at 608C for 20 h, until TLC analysis (EtOAc) indicated
the formation of a major product (Rf =0.40). The mixture was con-
centrated under reduced pressure, and the crude residue was
taken up in Et2O (10 mL) and washed with H2O (10 mL). The aque-
ous layer was extracted with Et2O (2ꢂ10 mL). The collected organic
extracts were combined and dried (MgSO4·H2O), filtered, and con-
centrated under reduced pressure. The crude product (114 mg)
was purified by flash chromatography (silica gel, n-hexane/EtOAc
2:8) to afford a white solid (80 mg) constituted (NMR) by ester 30
impure of isothiocyanates 19 (5%). Data for 30: 1H NMR
(250.12 MHz, CD3CN): d=7.88–7.84 (m, 2H, Ar-H), 7.77–7.64 (m,
2H, Ar H), 7.61–7.57 (m, 2H, Ar H), 7.45–7.37 (m, 4H, Ar-H), 7.29
(brs, 1H, NHCS), 7.18 (brt, 1H, J=5.3 Hz, NHCS),7.10–7.04 (m, 2H,
Ar-H), 6.67 (d, 1H, J2,NH =8.8 Hz, NHAc), 5.24 (brs, 1H, H-1), 5.19
(dd, 1H, J2,3 =10.4 Hz, J3,4 =9.3 Hz, H-3), 5.12 (s, 2H, ArCH2O), 4.98
Carboxylic acid 4: Isothiocyanate 16 (76 mg, 0.17 mmol) was solu-
bilized in dry CH2Cl2/DMF (4:1, 17.5 mL), and a solution of amine
25 (93 mg, 0.17 mmol) in dry CH2Cl2/DMF (4:1, 17.5 mL) containing
Et3N (50 mL, 2 equiv) was added dropwise. The mixture was stirred
at 608C for 24 h, until TLC analysis (EtOAc) indicated the formation
of a major product (Rf =0.42). The mixture was concentrated under
reduced pressure, and the crude residue was taken up in Et2O
(10 mL) and washed with H2O (10 mL). The aqueous layer was ex-
tracted with Et2O (2ꢂ10 mL). The collected organic extracts were
combined and dried (MgSO4·H2O), filtered, and concentrated under
reduced pressure. The crude residue (136 mg) was submitted to
flash chromatography (silica gel, n-hexane/EtOAc 2:8) to give
a foam solid (125 mg) constituted (NMR) by ester 28 impure of iso-
thiocyanate 16 (5%). Data for 28: 1H NMR (250.12 MHz, CD3CN):
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(dd, 1H, J4,5 =9.8 Hz, H-4), 4.21 (dd, 1H, J5,6b =4.7 Hz, J6a,6b =12.3 Hz,
H-6b), 4.10–4.05 (m, 2H, H-2, H-6a), 3.84 (d, 1H, Jvic =10.5 Hz,
CHNSO2), 3.98–3.71 [m, 4H, H-5, CH2NH (1H), CH2NSO2], 3.35 m,
1H, CH2NH (1H)], 2.22 (m, 1H, Me2CH), 1.99, 1.97, 1.96 (3s, each
3H, 3MeCOO), 1.86 (s, 3H, MeCON), 1.18 (s, 9H, CMe3), 1.00 (d, 3H,
Carboxylic acid 5: Trituration with Et2O afforded pure 5 (88%
yield) as a white solid; Rf =0.07 (CHCl3/MeOH 8:2); mp: 114–1168C;
½aꢂ2D3 =ꢃ4.0 (c=1.08 in CHCl3); H NMR (250.12 MHz, CD3OD/D2O):
1
d=7.99–7.94 (m, 2H; 2Ar-H), 7.78–7.74 (m, 2H; 2Ar-H), 7.68–7.65
(m, 2H; 2Ar-H), 7.49–7.38 (m, 3H; 3Ar-H), 4.39 (d, J1,2 =8.5 Hz, 1H;
H-1), 4.08–3.78 [m, 5H; H-6b, CH2NSO2, CHNSO2, CH2O (1H)], 3.76–
3.59 [m, 4H; H-2, H-6a, CH2O (1H), CH2NH (1H)], 3.58–3.30 [m, 6H;
H-3, H-4, H-5, CH2NH (1H), CH2NH (2H)], 1.98 (s, 3H; MeCOON),
1.75 (m, 2H; CH2), 0.98 (brd, Jvic =6.8 Hz, 3H; Me2CH), 0.90 ppm
(brs, 3H; Me2CH); 13C NMR (62.9 MHz, CD3OD/D2O): d=179.8 (C=
S), 174.2 174.1 (2C=O), 146.4 (Ar-C-SO2), 140.4, 140.2 (2Ar-C),
130.1–128.2 (Ar-CH), 102.5 (C-1), 77.5 (C-5), 75.8 (C-3), 71.9 (C-4),
70.7 (CHNSO2), 67.9 (CH2O), 62.5 (C-6), 57.2 (C-2), 43.5, 43.0, 42.8
(CH2NSO2, 2CH2NH), 30.0 (Me2CH), 29.8 (CH2), 23.2 (MeCON),
20.4 ppm (2Me2CH); elemental analysis calcd (%) for C31H44N4O10S2
(696.83): C 53.43, H 6.36, N 8.04; found: C 53.41, H 6.33, N 8.01.
J
vic =6.4 Hz, Me2CH), 0.91 ppm (d, 3H, Jvic =6.6 Hz, Me2CH); 13C NMR
(62.9 MHz, CD3CN): d=184.3 (C=S), 172.8, 171.3, 171.2, 170.5,
170.4 (5C=O), 160.0 (Ar-C-O), 145.8 (Ar-C-SO2), 138.2, 137.0, 134.1,
132.5 (4Ar-C), 130.2–128.0 (Ar-CH), 116.3 (2Ar-CH), 83.9 (C-1), 82.8
(Me3CO), 73.4 (C-5), 73.2 (C-3), 69.5 (C-4), 69.8 (ArCH2O), 67.6
(CHNSO2), 62.8 (C-6), 55.6 (C-2), 45.9 CH2NSO2), 44.2 (CH2NH), 29.8
(CHMe2), 27.9 (Me3C), 23.0 (MeCON), 20.9 (3MeCOO), 20.5, 19.5 ppm
(Me2CH). Crude ester 30 (80 mg) was dissolved in CH2Cl2 (1.4 mL)
and treated with CF3COOH (0.54 mL), and the mixture was stirred
at 08C for 1 h and then at RT. After 24 h, TLC analysis (EtOAc) re-
vealed the complete disappearance of the starting material and
the formation of a more retained product (Rf =0.33). The solution
was co-evaporated with toluene (4ꢂ10 mL) under reduced pres-
sure, and trituration of the crude product with Et2O afforded pure
carboxylic acid 10 (52 mg, 43% yield calculated from 19) as
a white solid; Rf =0.33 (EtOAc); mp: 119–1208C; ½aꢂ2D3 = +12.6 (c=
Carboxylic acid 7: Crystallization (MeOH/EtOAc) afforded pure 7
(67% yield) as a white solid; Rf =0.13 (CHCl3/MeOH 8:2); mp: 115–
117 8C; ½aꢂ2D3 =ꢃ10.2 (c=1.2 in MeOH); 1H NMR (250.12 MHz,
CD3OD/D2O): d=7.87–7.27 (m, 10H; 9Ar-H, CH-triazole), 5.87 (brs,
1H; H-1), 4.35–3.15 (m, 9H; H-2, H-3, H-4, H-5, H-6a, H-6b, CH2N,
CHN,), 2.08 (m, 1H; Me2CH), 1.82 (s, 3H; MeCOON), 0.90–0.78 ppm
(m, 6H; Me2CH); 13C NMR (62.9 MHz, CD3OD/D2O): d=176.6,173.6
(2C=O), 146.4 (Ar-C-SO2, C-triazole), 140.4, 139.7 (2Ar-C), 130.4–
128.5 (Ar-CH), 121.8 (CH-triazole), 87.8 (C-1), 80.8 (C-5), 75.4 (C-3),
70.9 (C-4), 62.0 (CHN), 61.9 (C-6), 56.7 (C-2), 40.4 (CH2NSO2), 25.4
(CHMe2), 23.1 (MeCON), 21.0, 20.0 ppm (Me2CH); elemental analysis
calcd (%) for C28H35N5O9S (617.67): C 54.45, H 5.71, N 11.34; found:
C 54.43, H 5.70, N 11.32.
1
0.9 in CH3OH); H NMR (250.12 MHz, CD3CN): d=7.88–7.71 (m, 2H;
2Ar-H), 7.67–7.60 (m, 2H; 2Ar-H), 7.58–7.52 (m, 2H; 2Ar-H), 7.47–
7.38 (m, 5H; 4Ar-H, OH), 7.34–7.17 (m, 2H; 2NHCS), 7.14–7.08 (m,
2H; Ar-H), 6.79 (d, J2,NH =9.0 Hz, 1H; NHAc), 5.41 (brs, 1H; H-1),
5.19 (dd, J2,3 =10.1 Hz, J3,4 =9.4 Hz, 1H; H-3), 5.11 (s, 2H; ArCH2O),
4.97 (dd, J4,5 =10.0 Hz, 1H; H-4), 4.28 (dd, J5,6b =4.4 Hz, J6a,6b
=
12.4 Hz, 1H; H-6b), 3.98 (d, Jvic =10.2 Hz, 1H; CHNSO2), 4.10–3.60
(m, 5H; H-2, H-6a, H-5, CH2NSO2), 3.35 [m, 2H; CH2NH), 2.17 (m,
1H; Me2CH), 1.97, 1.96, 1.94 (3s, each 3H; 3MeCOO), 1.84 (s, 3H;
MeCON), 0.92 (d, Jvic =6.6 Hz, 3H; Me2CH), 0.89 ppm (d, Jvic =6.7 Hz,
3H; Me2CH); 13C NMR (62.9 MHz, CD3CN): d=182.9 (C=S), 173.7,
171.3, 171.2, 170.6, 170.5 (5C=O), 160.0 (Ar-C-O), 145.7 (Ar-C-SO2),
137.9, 137.0, 134.1, 132.5 (4Ar-C), 130.2–127.8 (Ar-CH), 116.3 (2Ar-
CH), 83.2 (C-1), 73.4 (C-5), 73.2 (C-3), 69.5 (C-4), 69.8 (ArCH2O), 66.6
(CHNSO2), 62.8 (C-6), 53.7 (C-2), 45.2 CH2NSO2), 44.4 (CH2NH), 29.1
(CHMe2), 23.0 (MeCON), 20.9 (3MeCOO), 20.3, 19.6 ppm (Me2CH); el-
emental analysis calcd (%) for C41H49ClN4O13S2 (905.43): C 54.39, H
5.45, N 6.19; found: C 54.37, H 5.43, N 6.17.
Carboxylic acid 9: Trituration with Et2O afforded pure 9 (70%
yield) as a white solid; Rf =0.17 (CHCl3/MeOH 8:2); mp: 156–1578C;
½aꢂ2D3 = +12.3 (c=1.08 in MeOH); 1H NMR (250.12 MHz, CD3OD/
D2O): d=7.87–7.27 (m, 10H; 9Ar-H, CH-triazole), 4.32 (d, J1,2
=
8.3 Hz, 1H; H-1), 4.25–3.10 (m, 13H; H-2, H-3, H-4, H-5, H-6a, H-6b,
CH2NSO2, CHN, CH2N, CH2O), 2.18–203. (m, 3H; CH2, Me2CH), 1.89 (s,
3H; MeCOON), 1.02–0.81 ppm (m, 6H; Me2CH);13C NMR (62.9 MHz,
CD3OD/D2O): d=175.3 170.5 (2C=O), 146.3 (Ar-C-SO2, C-triazole),
140.4 (2Ar-C), 130.3–129.1 (Ar-CH), 128.3 (CH-triazole), 102.7 (C-1),
77.9 (C-5), 75.9 (C-3), 72.0 (C-4), 66.9 (CH2O), 66.7 (CHN), 62.7 (C-6),
57.3 (C-2), 47.9 (CH2N), 41.8 (CH2NSO2), 30.7 (CH2), 30.6 (CHMe2),
23.4–22.0 ppm (MeCON, Me2CH); elemental analysis calcd (%) for
C31H41N5O10S (675.75): C 55.10, H 6.12, N 10.36; found: C 55.08, H
6.10, N 10.34.
General procedure for the synthesis of deprotected carboxylic
acids 3, 5, 7, 9, and 11: A solution of protected carboxylic acid 2,
4, 6, 8, or 10 (0.1 mmol) in MeOH (1 mL) was treated with 7n NH3/
MeOH (1 mL), and the solution was stirred at RT until the starting
compound was completely reacted (TLC, CHCl3/MeOH 8:2, 20–
24 h). The solution was co-evaporated with toluene (4ꢂ10 mL)
under reduced pressure, and crystallization (MeOH/EtOAc) or tritu-
ration of the crude product with Et2O afforded the pure carboxylic
acid.
Carboxylic acid 11: Trituration with Et2O afforded pure 11 (79%
yield) as a white solid; Rf =0.10 (CHCl3/MeOH 8:2); mp: 134–1368C;
½aꢂ2D3 = +27.7 (c=0.85 in CHCl3); 1H NMR (250.12 MHz, CD3OD/
D2O): d=7.98–7.82 (m, 2H; 2Ar-H), 7.77–7.73 (m, 2H; 2Ar-H), 7.66–
7.62 (m, 2H; 2Ar-H), 7.45–7.38 (m, 4H; 4Ar-H), 7.12–7.08 (m, 2H;
2Ar-H), 5.12 (s, 2H; ArCH2O), 4.05–3.31 (m, 12H; H-1, H-2, H-3, H-4,
H-5, H-6a, H-6b, CHNSO2, CH2NSO2, CH2NHCS), 2.18 (m, 1H, Me2CH),
1.97 (s, 3H; MeCON), 1.03–0.97 ppm (m, 6H; Me2CH); 13C NMR
(62.9 MHz, CD3OD/D2O): d=181.7 (C=S), 174.8, 173.0 (2C=O),
160.4 (Ar-C-O), 146.2 (Ar-C-SO2), 138.4, 137.3, 134.5, 133.1 (4Ar-C),
129.6–127.8 (Ar-CH), 116.5 (2Ar-CH), 84.5 (C-1), 78.9 (C-5), 75.8 (C-
3), 71.8, (C-4), 70.2 (CHNSO2), 68.7 (ArCH2O), 62.6 (C-6), 56.1 (C-2),
45.9, 44.8 (CH2NH, CH2NSO2), 29.6 (Me2CH), 22.8 (MeCON), 20.7,
19.9 ppm (Me2CH); elemental analysis calcd (%) for C35H43ClN4O10S2
(779.32): C 53.94, H 5.56, N 7.19; found: C 53.91, H 5.53, N 7.17.
Carboxylic acid 3: Trituration with Et2O afforded pure 3 (94%
yield) as a white solid; Rf =0.25 (CHCl3/MeOH 8:2); mp: 131–1338C;
½aꢂ2D3 = +20.3 (c=1.01 in MeOH); 1H NMR (250.12 MHz, CD3OD/
D2O): d=7.96–7.92 (m, 2H; 2Ar-H), 7.81–7.75 (m, 2H; 2Ar-H), 7.64
(m, 2H; 2Ar-H), 7.49–7.39 (m, 3H; 3Ar-H), 4.20–3.25 (m, 12H; H-1,
H-2, H-3, H-4, H-5, H-6a, H-6b, CHNSO2, CH2NSO2, CH2NHCS), 2.19
(m, 1H; Me2CH), 1.93 (s, 3H; MeCON), 0.96 ppm (m, 6H; Me2CH);
13C NMR (62.9 MHz, CD3OD/D2O): d=183.1 (C=S), 174.9,174.8 (2C=
O), 146.7 (Ar-C-SO2), 140.3, 139.0 (2Ar-C), 129.9–128.1 (Ar-CH), 84.3
(C-1), 79.4 (C-5), 75.7 (C-3), 71.7 (C-4), 66.8 (CHN), 62.5 (C-6), 55.9
(C-2), 46.1, 44.4 (CH2NH, CH2NSO2), 29.5 (CHMe2), 22.8 (MeCON),
20.6, 19.9 ppm (Me2CH); elemental analysis calcd (%) for
C28H38N4O9S2 (638.75): C 52.65, H 6.00, N 8.77; found: C 52.63, H
6.01, N 8.76.
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250 mm NaCl, 100 mm Tris-HCl, pH 10.0 used in the crystallization
of other MMP-12 ligand complexes but with insufficient precipitat-
ing power for the MMP-12 glycoconjugate complexes. These pre-
cipitants were supplemented with 5–20% dioxane (Table S1, see
the Supporting Information). Crystals remained small and crystal
growth was so slow that to avoid excessive protein degradation
the E219Q MMP-12 mutant was used instead of the more active
wild-type enzyme for several MMP-12 complexes. Crystals for the
trigonal polymorph of MMP-9 in complex with compound 2 were
obtained from 40% monomethyl PEG 5000 (MPEG-5 K), 100 mm
bicine, pH 7.25 (Table S1), conditions similar to those used for co-
crystals with the hydroxamate-based inhibitor ARP101.[29]
Biological methods
MMP inhibition assays: Recombinant human MMP-14 catalytic
domain was a kind gift from Prof. Gillian Murphy (Department of
Oncology, University of Cambridge, UK). Pro-MMP-1, pro-MMP-2,
and pro-MMP-9 were purchased from Calbiochem (Merck-Milli-
pore). Pro-MMP-12 was purchased from R&D Systems. Proenzymes
were activated immediately prior to use with p-aminophenylmer-
curic acetate (2 mm APMA for 1 h at 378C for MMP-2, 2 mm APMA
for 2 h at 378C for MMP-1, and 1 mm APMA for 1 h at 378C for
MMP-9). Pro-MMP-12 was autoactivated by incubating in fluorimet-
ric assay buffer (FAB: Tris 50 mm, pH 7.5, NaCl 150 mm, CaCl2
10 mm, Brij-35 0.05%, and DMSO 1%) for 30 h at 378C. For assay
measurements, the inhibitor stock solutions (DMSO, 10 mm) were
further diluted in FAB. Activated enzyme (final concentrations of
0.56 nm for MMP-2, 1.3 nm for MMP-9, 1.0 nm for MMP-14 cd,
2.0 nm for MMP-1, 2.3 nm for MMP-12) and inhibitor solutions were
incubated in the assay buffer for 3 h at 258C. After the addition of
200 mm solution of the fluorogenic substrate Mca-Lys-Pro-Leu-Gly-
Leu-Dap(Dnp)-Ala-Arg-NH2 (Bachem) for all the enzymes in DMSO
(final concentration of 2 mm for all enzymes), the hydrolysis was
monitored every 10 s for 15 min, recording the increase in fluores-
cence (lex =325 nm, lem =400 nm) with a Molecular Devices Spec-
traMax Gemini XPS plate reader. The assays were performed in du-
plicate in a total volume of 200 mL per well in 96-well microtiter
plates (Corning black, NBS). Control wells lacked inhibitor. The
MMP inhibition activity was expressed in relative fluorescent units
(RFU). Percent of inhibition was calculated from control reactions
without the inhibitor. IC50 values were determined by using Equa-
tion (1):
Cryoprotection and data collection: The crystals used for data collec-
tion were picked up with a litho-loop from the crystallization tray
and were transferred to cryoprotectant solutions, which in the case
of MMP-9 also contained 1 mm compound 2 (Table S1) to prevent
the loss of the ligand during the cryosoak phase of the experiment.
The cryoprotectant solutions were prepared with mixed compo-
nents either from 40% v/v CryoSol or CryoProtX (Molecular Dimen-
sions, UK) solutions, 50% v/v precipitant, and 10% v/v buffer as
previously described.[30] Crystals were soaked in these solutions
(Table S1) for a few seconds, then picked up into a loop, and finally
flash-cooled in liquid nitrogen by using magnetic SPINE compatible
cryovials for data collection at the ESRF synchrotron facility or
transferred to Unipucks (MiTeGen, USA) for robotic mounting. Data
were collected either on beamlines Proxima 1 or Proxima 2A[31] at
the Soleil synchrotron Facility (St. Aubin, France) or at The ESRF
synchrotron facility (Grenoble, France) on the user-operated beam-
line ID23–2 or dispatched to the fully automatic ID30A beamline.
In all cases, data were collected from a single crystal at 100 K with
MxCube[32] and were reduced by using XDS[33] and XSCALE by
using the script “xdsme” (https://github.com/legrandp/xdsme).
vi
v0
1
¼
ð1Þ
ð1 þ ½Iꢂ=IC50Þ
Crystallographic structure determination: The structures were solved
by molecular replacement by using phaser[34] or MOLREP[35] and
were refined by using REFMAC5[36] and Phenix.[37] The MMP-9 crys-
tal belongs to the space group P3221 diffracting to 1.59 ꢁ resolu-
tion.
in which vi is the initial velocity of substrate cleavage in the pres-
ence of the inhibitor at concentration [I] and v0 is the initial veloci-
ty in the absence of the inhibitor. Results were analyzed by using
SoftMax Pro software and Prism Software version 5.0 (GraphPad
Software, Inc., La Jolla, CA).
Protein preparation: The expression and purification for the catalyt-
ic domains used in the crystallographic studies, MMP-9 (MMP9wt)
and MMP12 (hMMP12wt) and the active-site glutamate to gluta-
mine mutant hMMP12 (E219Q), were described elsewhere.[28] In
brief, the synthetic gene used for the catalytic domain of human
MMP-9 comprised residues Gly106–Gly215 and Gln391–Gly444,
without the fibronectin domains, those for MMP-12. Plasmids were
propagated in the Escherichia coli strain XL1-Blue, and the recombi-
nant catalytic domains expressed in E. coli cells BL21 (DE3 star).
After induction, the cells were harvested by centrifugation, the pel-
lets were resuspended, and the cells were suspended, disrupted,
centrifuged, and redissolved in 8m urea. The protein was then re-
folded, purified and dialyzed as previously described.[28] Acetohy-
droxamic acid (AHA) in the range 10–120 mm was added to pre-
vent self-degradation during concentration.
Model building and electron density interpretation
The ligands were built with the CCP4 ligand sketcher[38] and fitted
into the difference electron density (weighted F0ꢃFc) calculated
and displayed by using COOT.[39] The electronic density for the in-
hibitor portion of the compounds was of excellent quality, whereas
the sugar moiety was poorly defined for most of the copies of the
ligand in the asymmetric unit. The presence of the sugar moiety
on the inhibitor shifted the preference for the P21212 space group
with a single molecule in the asymmetric unit to a monoclinic P21
cell with two molecules in the asymmetric unit, a=39.1 ꢁ b=
62.9 ꢁ c=63.7 ꢁ, b=102.58 for the MMP-12·2 complex or four
molecules in a larger cell a=63.7 ꢁ b=63.1 ꢁ c=78.9 ꢁ, b=103.18
for the MMP-12·3 complex and with similar cell parameters for the
other complexes. The packing of the MMP-12 catalytic domain in
the larger cell was related to that in the smaller one. A strong
pseudo-translational noncrystallographic (NCS) operation related
the two cells with a Patterson off-origin peak that was 38.6% of
the origin peak. The sugar moiety of the inhibitor was positioned
at a crystal contact and was the likely cause of the larger cell.
Crystallization: The crystallization trials were performed by sitting
vapor diffusion with 1 mL equivolumetric drops of protein–sub-
strate and reservoir solution using CrysChem plates stored in
a cooled incubator at 208C. Initial screening for the MMP-12 glyco-
conjugate complexes was performed with five precipitant solu-
tions: 27% polyethylene glycol 10000 (PEG-10 K), 0.2m imidazole
malate, pH 8.5; 45% (PEG-4 K), 0.2m imidazole piperidine, pH 8.5;
17% PEG-20 K, 0.2m imidazole malate, pH 8.5, 250 mm NaCl; 27%
PEG-10 K, 150 mm imidazole piperidine, pH 8.5 and 17% PEG-20 K,
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[17] I. A. Moussa, S. D. Banister, C. Beinat, N. Giboureau, A. J. Reynolds, M.
Kassiou, J. Med. Chem. 2010, 53, 6228–6239.
Acknowledgements
[18] a) I. Choudhury (Mukherjee), N. Minoura, H. Uzawa, Carbohydr. Res.
2003, 338, 1265–1270; b) A. S. Couto, L. L. Soprano, M. Landoni, M.
Pourcelot, D. M. Acosta, L. Bultel, J. Parente, M. R. Ferrero, M. Barbier, C.
Dussouy, M. I. Esteva, J. Kovensky, V. G. Duschak, FEBS J. 2012, 279,
3665–3679.
Allocation of synchrotron beamtime is acknowledged with grati-
tude. We are also grateful to Dr. Didier Nurizzo on beamline
ID23-2 and Dr. Matthew Bowler on MASSIF-1 at the ESRF syn-
chrotron facility and to Dr. Tatiana Isabet on beamline Proxima-
1 and Dr. William Shepard on the Proxima-2A beamline at the
SOLEIL synchrotron for help on the beamlines and with data col-
lection and processing. This study was supported by the Italian
Ministry of Education, University and Research (MIUR) within the
frame of the COFIN national project anno 2010–2011—prot.
2010JMAZML_008 and the Italian Ministry of Health (5ꢀ1000
Funds 2012).
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Received: May 6, 2016
Revised: June 13, 2016
Published online on && &&, 0000
&
ChemMedChem 2016, 11, 1 – 13
www.chemmedchem.org
12
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

FULL PAPERS
Sugar power: Ten glycoconjugate aryl-
sulfonamide carboxylates were de-
signed and synthesized as matrix metal-
loproteinase-12 (MMP-12)-selective in-
hibitors with improved hydrophilicity.
They were tested on recombinant
MMPs by fluorimetric assays, and X-ray
crystallographic studies helped rational-
ize the results. Introduction of b-N-
acetyl-d-glucosamine at the P2’ position
was found to maintain nanomolar activ-
ity against MMP-12 and to boost bio-
availability.
E. Nuti, D. Cuffaro, F. D’Andrea, L. Rosalia,
L. Tepshi, M. Fabbi, G. Carbotti, S. Ferrini,
S. Santamaria, C. Camodeca, L. Ciccone,
E. Orlandini, S. Nencetti, E. A. Stura,
V. Dive, A. Rossello*
&& – &&
Sugar-Based Arylsulfonamide
Carboxylates as Selective and Water-
Soluble Matrix Metalloproteinase-12
Inhibitors
ChemMedChem 2016, 11, 1 – 13
www.chemmedchem.org
13
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ