Design, synthesis, biological evaluation, and NMR studies of a new series of arylsulfones as selective and potent matrix metalloproteinase-12 inhibitors

Article abstract of DOI:10.1021/jm900335a

Overexpression of macrophage elastase (MMP-12), a member of the matrix metalloproteinases family, can be linked to tissue remodeling and degradation in some inflammatory processes, such as chronic obstructive pulmonary disease (COPD), emphysema, rheumatoi

Full text of DOI:10.1021/jm900335a

J. Med. Chem. 2009, 52, 6347–6361 6347
DOI: 10.1021/jm900335a
Design, Synthesis, Biological Evaluation, and NMR Studies of a New Series of Arylsulfones As
Selective and Potent Matrix Metalloproteinase-12 Inhibitors
Elisa Nuti,† Laura Panelli,† Francesca Casalini,† Stanislava I. Avramova,† Elisabetta Orlandini,† Salvatore Santamaria,†
Susanna Nencetti,† Tiziano Tuccinardi,† Adriano Martinelli,† Giovanni Cercignani,‡ Nicola D’Amelio,§ Alessandro Maiocchi,
Fulvio Uggeri, and Armando Rossello*,†
†
‡
Dipartimento di Scienze Farmaceutiche, Universita di Pisa, via Bonanno 6, 56126 Pisa, Italy, Dipartimento di Biologia, Unita di Biochimica,
ꢀ
Universita di Pisa, Via San Zeno 51, 56127 Pisa, Italy, Bracco Imaging;CRB Trieste Area Science Park, edificio Q S.S. 14 Km 163.5, 34012
ꢀ
§
ꢀ
Basovizza Trieste, Italy, and Centro Ricerche Bracco, Bracco Imaging SpA, Via Ribes 5, 10010 Colleretto Giacosa (TO), Italy
Received March 17, 2009
Overexpression of macrophage elastase (MMP-12), a member of the matrix metalloproteinases family,
can be linked to tissue remodeling and degradation in some inflammatory processes, such as chronic
obstructive pulmonary disease (COPD), emphysema, rheumatoid arthritis (RA), and atherosclerosis.
On this basis, MMP-12 can be considered an attractive target for studying selective inhibitors that are
useful in the development of new therapies for COPD and other inflammatory diseases. We report
herein the design, synthesis, and in vitro evaluation of a new series of compounds, possessing an
arylsulfonyl scaffold, for their potential as selective inhibitors of MMP-12. The best compound in the
series showed an IC50 value of 0.2 nM, with good selectivity over MMP-1 and MMP-14. A docking
study was carried out on this compound in order to investigate its binding interactions with MMP-12,
and NMR studies on the complex with the MMP-12 catalytic domain were able to validate the proposed
binding mode.
1. Introduction
inhibitors have been found to have dose-limiting toxicity in
the form of musculoskeletal side effects, including joint stiff-
nessandinflammation.5 Althoughthe human jointside effects
are reversible upon withdrawal of the drug, musculoskeletal
syndrome (MSS) has halted the clinical trials of many non-
selective MMP inhibitors. While the inhibition of specific
MMPs, such as MMP-1 or MMP-14, has been postulated
as the cause of MSS, the exact basis for this pathology is not
yet clear. For this reason, we started a project aimed at finding
selective MMP-12 inhibitors (sparing MMP-1 and MMP-14)
thatmay beuseful inthe developmentof new therapies against
COPD, emphysema, and other relevant inflammatory dis-
eases, such as RA6 and atherosclerosis,7 as has been more
recently observed.
MMP-12 (macrophage elastase) is a member of the matrix
metalloproteinases (MMPs),a a family of endopeptidases able
to degrade all the components of the extracellular matrix
(ECM). In adult tissue, macrophages are the major source of
MMP-12. Several studies have demonstrated that MMP-12 is
involved in inflammatory processes and contributes to tissue
remodeling and tissue degradation.1 Patients suffering from
chronic obstructive pulmonary disease (COPD) showed high-
er levels of MMP-12 in lung tissue and bronchoalveolar
lavage,2 and MMP-12 knockout mice were resistant to
emphysema after chronic cigarette smoke exposure.3
These observations suggest that MMP-12 plays an impor-
tant role in lung tissue destruction in mice, and MMP-12
inhibitors can be potentially useful for the treatment of lung
pathologies. In fact, it was recently reported that a selective
MMP-12 inhibitor, AS1117934 (Figure 1), was able to prevent
inflammation induced by exposure to cigarette smoke in mice.
Unfortunately, only a few MMP inhibitors have entered
clinical trials for cancer or arthritis and none have been
successful thus far. In fact, many broad spectrum MMP
MMP-12 is a zinc-dependent protease secreted as a 54 kDa
pro-form protein that undergoes self-activation to produce
several active forms of the enzyme. It shares common struc-
tural domains with other MMPs. The most closely related
human matrix metalloproteinases are stromelysin-1 (MMP-3)
and interstitial collagenase (MMP-1), each with a 49% simi-
larity to MMP-12.
Recently, the crystal structure of the catalytic domain of
MMP-12 complexed to a hydroxamic acid inhibitor was
reported.1 The S10-specificity pocket is large and extends into
a channel through the protein, which puts MMP-12 into a
class with MMPs -8 and -13, containing large and open
specificity pockets.
*To whom correspondence should be addressed. Phone: þ39 050
2219562. Fax: þ39 050 2219605. E-mail: aros@farm.unipi.it.
a Abbreviations: MMP, matrix metalloproteinase; ECM, extracellu-
lar matrix; COPD, chronic obstructive pulmonary disease; MSS,
musculoskeletal syndrome; RA, rheumatoid arthritis; ZBG, zinc-bind-
ing group; SAR, structure-activity relationship; EDC, N-(3-Dimethyl-
aminopropyl)-N0-ethylcarbodiimide hydrochloride; MSS, musculoske-
letal syndrome; HSQC, heteronuclear single quantum correlation;
NNGH, N-isobutyl-N-[4-methoxyphenylsulfonyl]glycyl hydroxamic
acid; AHA, acetohydroxamic acid; NOE, nuclear Overhauser effect;
APMA, p-aminophenylmercuric acetate.
In another study, Markus et al.8 published the solution
structure of the catalytic domain of MMP-12 complexed with
a carboxylate inhibitor, using the wild-type protein sequence.
The inhibitor binds in the S10 pocket, and the carboxylate
r
2009 American Chemical Society
Published on Web 09/23/2009
pubs.acs.org/jmc

6348 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20
Nuti et al.
functionality coordinates the catalytic zinc ion. The NMR
structure of MMP-12 in the absence of an inhibitor was solved
by Bhaskaran et al.9 using a mutant form of MMP-12 able to
prevent autolysis.
Finally, the crystal structures of the MMP-12 catalytic
domain complexed with nonzinc chelating inhibitors have
been determined.10 This study pointed out the importance of
hydrophobic interactions between the inhibitors and residues
in the S10 pocket in preserving activity and achieving selecti-
vity toward this enzyme.
stability problems, they are still frequently used because of
studies showing the importance of inhibitor structure in
determining the bioavailability and toxicity of compounds.17
As a matter of fact, many hydroxamate-based MMP inhibi-
tors were active in in vivo models and pharmacologically
effective.4,18
Additional support for the use of hydroxamates can be
found in the field of histone deacetylase (HDAC) inhibitors,
where the relative safety of some hydroxamates for anticancer
In spite of the increasing interest in MMP-12,11 only a
modest number of papers regarding the development of
inhibitors have been reported to date. Among them, the most
promising are the phosphinic peptides discovered by Dive
et al.12 and the nonzinc chelating, nonpeptidic inhibitors
published by Dublanchet et al.13
Chart 1
In our previous papers,14,15 we described the synthesis and
biological evaluation of N-i-propoxy-N-biphenylsulfonylami-
nobutyl hydroxamic acids of type A (Figure 1) as selective
inhibitors of MMP-2 for cancer therapy. In particular, com-
pound 1a (Figure 1) was found to be a potent antiangiogenic
agent, able to block the chemoinvasion of HUVEC cells in the
micromolar range.
In the present paper, we report the optimization of template
A geometry between the hydroxamic zinc-binding group
(ZBG) and the P10 sulfonyl group in order to obtain potent
and selective inhibitors of MMP-12. To improve selectivity,
we decided to replace substituents R and N-O-R1 of A with an
aromatic ring that should confer a major degree of rigidity to
the system. Therefore, in a preliminary study of SAR, some
sulfone-based benzoic hydroxamates (compounds of type B)
were synthesized to evaluate the effects of conformational
restriction and the presence of different P10 substituents on
enzyme inhibition (Figure 1).
On the basis of docking studies, a second series of sulfone-
based inhibitors was then obtained by inserting a methylene
between the ZBG and the aromatic ring in order to modify the
geometry of chelation (compounds of type C). Finally, with
the aim of verifying the influence on activity of a small alkyl
group (R2) R to the ZBG, new R-methyl-substituted phenyl-
acetic derivatives were developed (Chart 1). In these studies,
biphenylsulfonyl or phenoxybenzenesulfonyl moieties were
chosen as P10 substituents because they were considered
suitable for fitting well into a channel-like S10 pocket as is
seen in MMP-12. Both hydroxamic and carboxylic acids were
selected as ZBGs, given their different binding modes,16 good
chelating properties, and presence in the vast majority of
reported MMP inhibitors. In fact, despite the debate over
the feasibility of using hydroxamates as ZBGs due to in vivo
Figure 1. Progression of the SAR.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6349
Table 1. In Vitroa Activity (IC50 nM Values) of Benzoic Hydroxamic Acids 2-7
compd
MMP-1
MMP-2
35000 ( 2500 >50000
1200 ( 110 27000 ( 1500
1300 ( 160 22000 ( 2500 1900 ( 100
MMP-3
MMP-8
MMP-9
MMP-12
MMP-13
MMP-14
MMP-16
2
>100000
>10000
>10000
>200000
72000 ( 8500 24000 ( 2200 14000 ( 1000 28000 ( 1800 25000 ( 1000
620 ( 38
31000 ( 2000
1300 ( 100
6000 ( 290
3
690 ( 72
410 ( 45
1200 ( 160
920 ( 80
2700 ( 230
2700 ( 100
1300 ( 200
4000 ( 310
4
5
9900 ( 1100 60000 ( 4600 9200 ( 370 51000 ( 1000 5800 ( 370 20000 ( 2100 120000 ( 10000 110000 ( 10000
6
73000 ( 7100 1300 ( 100 45000 ( 2600 1000 ( 110
7700 ( 600
930 ( 89
6.7 ( 1.6
590 ( 28
70 ( 2
0.20 ( 0.05
1400 ( 150
260 ( 26
4.1 ( 0.2
8300 ( 360
1300 ( 100
9.8 ( 0.2
5100 ( 320
1200 ( 100
51 ( 3
7
1a
28000 ( 2600
490 ( 76
100 ( 10
0.80 ( 0.20
4000 ( 410
50 ( 2
87 ( 5
1.6 ( 0.1
a Assays were run in triplicate. The final values given here are the mean ( SD of three independent experiments.
Scheme 1. Synthesis of Benzoic Hydroxamate 2a
Scheme 2. Preparation of Phenoxyphenyl Benzoic Hydroxa-
mates 3 and 5a
a Reagents and conditions: (a) Cu, KOH, H2O, microwave, 180 W,
140 °C, 12 min; (b) oxone, MeOH, THF, H2O; (c) TBDMSiONH2,
EDC, CH2Cl2; (d) TFA, CH2Cl2, 0 °C.
and antimalaria therapy has been established. Furthermore, a
member of this family of hydroxamate zinc-chelating agents,
Zolinza (Vorinostat), was launched on the market in the USA
last year.19 These results, obtained from different fields of
drug therapy where hydroxamic acid derivatives have been
used, seem to suggest that the nature of the scaffold around
the ZBG can be very important not only for determining the
potency and selectivity among the different metalloproteins
but alsoforconferring hydrolyticand/or metabolic stability to
the compounds, as previously pointed out.
With regards to compounds presented herein, other scientists
in the past years have disclosed some aryl sulfonyl hydroxamates
as selective MMP-2/MMP-9/MMP-13 inhibitors.20 Among
these sulfonylated compounds, the vast majority of which were
arylsulfonamides, a sulfone hydroxamate, somewhat similar to
our compounds, was also described as an inhibitor of the above
cited MMPs. More recently, we independently developed and
patented some thioaryl substituted derivatives as zinc protease
inhibitors having similar structures to compounds B and C.21
a Reagents and conditions: (a) Cu, KOH, H2O, microwave, 180 W,
140 °C, 12 min; (b) Cu(OAc)2, TEA, CH2Cl2, 4 A molecular sieves,
phenylboronic acid; (c) oxone, MeOH, THF, H2O; (d) TBDMSiONH2,
EDC, CH2Cl2; (e) TFA, CH2Cl2, 0 °C; (f) Cu(OAc)2, TEA, CH2Cl2, 4 A
molecular sieves, 4-(methylthio)phenylboronic acid; (g) oxone, MeOH,
THF, H2O.
4-mercaptophenol with 2-iodobenzoic acid to give 22 under
the same conditions used before for compound 20. The use of
the microwave for this type of reaction allowed for a reduction
in the reaction time with respect to the procedure reported in
the literature22 (from 12 h to 12 min), always maintaining high
yields (>80%).
The phenolic intermediate 22 was subsequently converted
to diaryl ethers 23 and 25 by copper(II)-promoted coupling23
with the appropriate arylboronic acids. The reaction was
carried out using Cu(OAc)2 as the source of Cu(II), in the
presence of triethylamine (TEA) and powdered 4 A molecular
sieves at room temperature. To prepare the corresponding
sulfonyl derivatives, 23 and 25 were oxidized with oxone and
finally converted to hydroxamic acids 3 and 5 by the route
previously described. Oxidation of 25 with 6 equiv of oxone
afforded compound 26, with a sulfone and a sulfoxide group,
which was separated from its bis-sulfone analogue by silica gel
column chromatography.
2. Chemistry
The benzoic hydroxamate 2 (Table 1) was synthesized as
described in Scheme 1. Through a microwave-assisted, Cu-
catalyzed Ullmann-type reaction22 between 2-iodobenzoic
acid and 4-methoxythiophenol, it was possible to obtain the
carboxylic acid 20, which was oxidized using oxone in metha-
nol/THF/H2O to the corresponding sulfone 21. This was
converted into the hydroxamic acid 2 by condensation with
O-(tert-butyldimethylsilyl)hydroxylamine in the presence of
N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide
hydro-
chloride (EDC) and subsequent deprotection with trifluora-
cetic acid in dichloromethane.
The phenoxybenzenesulfonyl benzoic derivatives 3 and 5
(Scheme 2) were prepared starting from the reaction of
Synthesis of 4-methoxyphenoxy benzen sulfone derivatives
4, 9, and 15 was carried out as reported in Scheme 3. Phenyl-
acetic compounds (n=1) 9 and 15 were obtained in a similar
way to that reported in Scheme 2 for benzoic derivatives,

6350 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20
Scheme 3. Preparation of compounds 4, 9, and 15a
Nuti et al.
Scheme 4. Synthesis of Compounds 8 and 14a
a Reagents and conditions: (a) Cu, KOH, H2O, microwave, 180 W,
140 °C, 12 min; (b) BnBr, Cs2CO3, DMF, 0 °C-rt; (c) Oxone, MeOH,
THF, H2O; (d) KOH, H2O, 100 °C; (e) TBDMSiONH2, EDC, CH2Cl2;
(f) TFA, CH2Cl2, 0 °C.
Scheme 5. Synthesis of Biphenylic Derivatives 6, 7, 10, 11, 16,
and 17a
a Reagents and conditions: (a) 4-mercaptophenol, Cu, KOH, H2O,
microwave, 180 W, 140 °C, 12 min; (b) Cu(OAc)2, TEA, CH2Cl2, 4 A
molecular sieves, 4-methoxyphenylboronic acid; (c) BnBr, Cs2CO3,
DMF, 0 °C-rt; (d) oxone, MeOH, THF, H2O; (e) KOH, H2O,
100 °C; (f) TBDMSiONH2, EDC, CH2Cl2; (g) TFA, CH2Cl2, 0 °C.
starting from commercially available 2-iodophenylacetic acid.
Before oxidation with oxone, carboxylic acids 28 and 29 were
protected as benzylic ester by reaction with benzyl bromide in
the presence of Cs2CO3 in DMF. This further step was
necessary to improve the following oxidation reaction, which
exhibited poor yields if conducted on carboxylic acids. Hence,
benzylic esters 30 and 31 were oxidized to sulfonyl derivatives
and subsequently hydrolyzed to the corresponding carboxylic
acids 34 and 15 by saponification with KOH in good yields.
Benzoic (4) and phenylacetic (9) hydroxamates were finally
obtained from the corresponding carboxylates as described
for the previous compounds.
a Reagents and conditions: (a) 4-bromobenzenethiol, Cu, KOH,
H2O, microwave, 180 W, 140 °C, 12 min; (b) BnBr, Cs2CO3, DMF,
0 °C-rt; (c) phenylboronic or 4-methoxyphenylboronic acid, Pd(PPh3)4,
K3PO4, dioxane/H2O, 85 °C; (d) oxone, MeOH, THF, H2O; (e) KOH,
H2O, 100 °C; (f) TBDMSiONH2, EDC, CH2Cl2; (g) TFA, CH2Cl2, 0 °C.
A similar sequence of reactions was carried out to synthe-
size 4-methoxybenzen hydroxamate 8 and its carboxylic
analogue 14, as detailed in Scheme 4.
alkylation, racemic esters 52 and 53 were hydrolyzed to the
corresponding carboxylates and then to the hydroxamates 12
and 13, as reported in the previous schemes.
Biphenylsulfonyl derivatives 6, 7, 10, 11, 16, and 17 were
prepared from 4-bromobenzenethiol via a Cu-catalyzed
Ullmann-type reaction (Scheme 5), giving carboxylic acids
38 and 39, which were then protected as benzylic esters.
Palladium-catalyzed cross-coupling (Suzuki conditions24) of
protected arylbromides with phenylboronic or 4-methoxy-
phenylboronic acid afforded biphenyl derivatives 42-45.
Treatment with oxone, followed by basic hydrolysis of the
esters, yielded carboxylates 50, 51, 16, and 17, which were
finally converted to their corresponding hydroxamates by
condensation with O-(tert-butyldimethylsilyl)hydroxylamine
and acid cleavage with TFA.
3. Results and Discussion
MMPs Inhibition. All final hydroxamic acids were tested
in vitro, with a fluorometric assay on purified enzymes, for
their ability to inhibit MMPs. Data obtained for benzoic
hydroxamates 2-7 (type B) are shown in Table 1. Inhibitor
1a was used as a reference compound.
From a brief SAR analysis of the inhibition profile for
these compounds, it becomes apparent that the introduction
of an aromatic ring between the ZBG and the sulfonyl group
causes a drastic decrease in activity for all tested enzymes
with respect to the reference compound 1a. This was a
moderately selective inhibitor, with nanomolar activity on
TopreparetheR-methylsubstituted biphenylderivatives 12
and 13, benzyl esters 48 and 49 were alkylated25 by treatment
with methyl iodide in the presence of sodium hexamethyldi-
silylamide (NaHMDS) in THF at -78 °C (Scheme 6). After

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6351
MMP-3, -8, -9, -13, -14, and -16 and subnanomolar activity
on MMP-2 and MMP-12. Noteworthy was the good selec-
tivity for MMP-2 and -12 over MMP-1, one of the two
enzymes, together with MMP-14, whose inhibition was
postulated to be responsible for musculoskeletal syndrome.
Interestingly, the newly synthesized compounds of type B
show different selectivity profiles based on the kind of aryl-
sulfonyl group (P10) they possess. In fact, the more rigid
biphenylsulfonyl derivatives, 6 and 7, have the best activity
on MMP-12 and good selectivity over MMP-1, -3, and -14,
with 7 being the most active of the two (IC50 =70 nM on
MMP-12) because of its longer P10 group. In contrast, the
more flexible phenoxybenzenesulfonyl derivatives, 3 and 4,
show the best activity on MMP-9 and good selectivity over
MMP-1 and MMP-3.
containing the hydroxamate function as a ZBG suggested
that the hydroxamate interacts with the zinc ion in the same
binding position for all the MMPs. Figure 2B shows the
superimposition of compound 4 and one of the X-ray
structures of the MMP-12/hydroxamate ligand complexes
(PDB code: 1RMZ11). From this picture, it is evident that the
ZBG of compound 4 does not possess the common binding
geometry of the hydroxamate-based ligands because it lacks
interactions with E219. This analysis led to the hypothesis
that the hydroxamic acid could not interact with the typical
binding geometry of hydroxamic acid ligands due to the
direct bond of the ZBG with the benzenesulfonyl group.
Therefore, the distorted interaction of the ZBG with the zinc
could be the cause of the decreased activity.
To improve the activity of these ligands by allowing for a
good interaction of the ZBG, the benzoic hydroxamate func-
tion was modified with different scaffolds and submitted to
docking calculations. From the docking analysis it was appa-
rent that substitution of the benzoic hydroxamate function with
phenylacetic hydroxamate looked very promising.
The presence of a methoxybenzenesulfonyl group in P10,
as in compound 2, caused a reduction in activity for all
MMPs tested, probably due to the small dimensions of this
substituent. A similar drop in activity is observed when a
sulfoxide group is introduced on the terminal ring of the
phenoxybenzenesulfonyl substituent, as in compound 5.
Figure 2A shows the docking results for compound 4 in
MMP-12. The p-methoxyphenoxybenzene group was in-
serted into the S10 pocket and the sulfonyl group was able
to interact with L181 and A182. The ZBG forms an H-bond
with A182 without interacting with E219.
As shown in Figure 3B, the introduction of a methylene
between the ZBG and the aromatic ring allowed for good
coordination of the ZBG with the zinc ion and the interac-
tion with A182 and E219. The docking of compound 9
also showed the formation of H-bonds between the sulfo-
nyl group and L181 and A182, whereas the p-methoxy-
phenoxybenzene group was inserted into the S10 pocket
(Figure 3A).
As already reported,26 the alignment of all the available
experimental structures of MMPs complexed with ligands
On the basis of these docking studies, new phenylacetic
hydroxamates were synthesized and tested for their MMP
inhibitory activities. As shown in Table 2, in agreement with
docking, the introduction of a methylene between the ZBG
and the aromatic ring led to a marked increase in inhibitory
activity for all tested MMPs.
Scheme 6. Preparation of R-Methyl Alkylated Compounds 12
and 13a
This increase is more evident in phenoxybenzenesulfonyl
than in biphenylsulfonyl derivatives. In fact, comparing
compound 9 with its benzoic analogue 4 (Table 1), the first
shows a strong increase in activity ranging from 400 (on
MMP-2) to 4600 (on MMP-12) times that of 4. However,
with biphenylsulfonyl compound 11 and its benzoic analo-
gue 7, activity is increased only 2-15 times due to the
introduction of the methylene spacer.
A strong improvement of inhibitory activity, particularly
marked on MMP-12, was also obtained for compound 8,
bearing a p-methoxybenzenesulfonyl group in P10, relative to
its benzoic analogue 2. In fact, 8 shows an IC50 of 32 nM on
a Reagents and conditions: (a) NaHMDS, MeI, THF, -78 °C; (b)
KOH, H2O, 100 °C; (c) TBDMSiONH2, EDC, CH2Cl2; (d) TFA,
CH2Cl2, 0 °C.
Figure 2. (A) Docking analysis of compound 4 in the MMP-12
binding site. (B) Superimposition of compound 4 (cyan) and the
X-ray structure of one of the MMP-12 hydroxamate-based ligands
(magenta, PDB code: 1RMZ). The H-bond interactions of the
hydroxamic group of compound 4 are also reported.
Figure 3. (A) Docking analysis of compound 9 in the MMP-12
binding site. (B) Superimposition of compound 9 (cyan) and the
X-ray structure of one of the MMP-12 hydroxamate-based ligands
(magenta, PDB code: 1RMZ). The H-bond interactions of the
hydroxamic group of compound 9 are also reported.

6352 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20
Nuti et al.
Table 2. In Vitroa Activity (IC50 nM Values) of Phenylacetic Hydroxamic Acids 8-13
compd
MMP-1
MMP-2
MMP-3
MMP-8
MMP-9
MMP-12
MMP-13
MMP-14
MMP-16
8
4100 ( 170
3500 ( 300
29000 ( 3500
8300 ( 360
12000 ( 1300
>10000
230 ( 10
3.5 ( 0.2
38 ( 3
9.7 ( 0.3
73 ( 4
730 ( 32
21 ( 2
280 ( 18
5.1 ( 0.3
370 ( 19
120 ( 10
390 ( 25
54 ( 3
98 ( 7
0.80 ( 0.10
310 ( 28
94 ( 3
340 ( 37
69 ( 2
32 ( 1
84 ( 3
4.1 ( 0.3
62 ( 7
25 ( 2
74 ( 9
12 ( 1
200 ( 8
33 ( 1
130 ( 13
32 ( 1
9
0.20 ( 0.02
6.0 ( 0.4
4.8 ( 0.4
6.0 ( 0.9
2.7 ( 0.2
0.20 ( 0.05
10
11
12b
13b
1a
23000 ( 1600
1600 ( 110
5600 ( 500
1400 ( 100
50 ( 2
940 ( 47
710 ( 60
860 ( 120
360 ( 29
9.8 ( 0.2
220 ( 19
130 ( 10
470 ( 24
150 ( 10
51 ( 3
7.0 ( 0.3
0.80 ( 0.20
490 ( 76
1.6 ( 0.1
6.7 ( 1.6
4.1 ( 0.2
a Assays were run in triplicate. The final values given here are the mean ( SD of three independent experiments. b Racemates.
13) has only a small effect on the inhibitory potency, as can
be seen by comparing compound 10 with 12 and compound
11 with 13.
Hence, the best compound of this series is 9, with an IC50
of 0.2 nM on MMP-12 and a 17500-fold selectivity for
MMP-12 over MMP-1 and 165-fold selectivity over MMP-
14. This compound showed the same activity as reference
compound 1a on MMP-12 but an increased selectivity for
MMP-12 over MMP-1. Moreover, it proved to be a sub-
nanomolar inhibitor of MMP-9 (another MMP implicated
in the pathogenesis of COPD), as well.
Finally, the carboxylic analogues of type C inhibitors
(compounds 14-19) were tested on the principal MMPs in
comparison with 1b, the carboxylate analogue of 1a
(Table 3).
As expected, the carboxylates showed a decrease in activ-
ity (>100-fold) on all the tested MMPs with respect to the
corresponding hydroxamates but they also showed a differ-
ent inhibitory profile. In fact, differently from the hydro-
xamic homologues, the most potent compound of this series
on MMP-12 was the p-methoxybiphenyl derivative 17 (IC50
=139 nM), which also showed good activity on MMP-2, -13,
and -8. This was particularly striking when compared with
1b, which was poorly active on these enzymes. However, the
carboxylic analogue of 9, compound 15, was the most
selective on MMP-12 over MMP-1, -3, and -14.
The results obtained with carboxylates indicate that the
nature of the ZBG, in addition to the structure of the
inhibitors, can strongly influence the inhibitory profile.
NMR Studies. To verify the modeled binding modes of the
inhibitors synthesized, NMR experiments were conducted in
solution. Synergic use of docking and NMR data was
applied successfully in the case of MMPs.28 We selected
compound 9, the most active on MMP-12 (IC50 0.2 nM),
for the study.
Figure 4. Docking analysis of compound 11 in the MMP-12 (A)
and MMP-9 binding site (B). The two nonconserved residues,
V217(A400) and V235(L418), are colored orange.
MMP-12, being about 400 times more active than 2 on this
enzyme.
Moreover, as seen for the previous series of compounds,
derivative 9 bearing a phenoxybenzenesulfonyl group dis-
plays its best activity on MMP-9 (0.8 nM) (besides MMP-
12), while biphenylsulfonyl compounds 10 and 11 have the
best activity on MMP-2 (besides MMP-12). Thus, the pre-
sence of a phenoxybenzenesulfonyl group in P10 causes a
marked increase in potency on MMP-9 in both series of
compounds (except for compound 5). This element can be
important in COPD treatment, considering that expression
of MMP-9 is increased in bronchoalveolar lavage of patients
with COPD.27 The lack of activity of compounds 10 and 11
against MMP-9 was also investigated by means of a docking
analysis. However, comparison of the binding interactions of
these two compounds with MMP-12 and MMP-9 high-
lighted that the compounds interacted with both MMPs
with a very similar disposition. This observation did not
explain the different activity showed against MMP-12 and
MMP-9. As shown in Figure 4, the sulfonyl group of
compound 11 formed a H-bond with L181 (L188 in MMP-
9) and the p-methylbiphenyl group was inserted into the S10
pocket. Analysis of the two S10 pockets revealed that
they were very similar and, regarding the interaction of the
p-methylbiphenyl group, there were two nonconserved re-
sidues, V217 and V235 (A400 and L418 in MMP-9). How-
ever, the different interaction of these two residues with the
p-methylbiphenyl group was not sufficient to explain its
20-fold decrease in activity.
The proton and carbon chemical shifts of 9 (15 mM in
DMSO) are reported in the Experimental Section. The
compound is rather insoluble in water. The 1D spectrum is
quite complex, showing the presence of multiple species for
the methylene moiety. Z and E forms of the hydroxamic
group are easily identified29 and quantified by HN and OH
signals. The E form largely predominates (86.9% compared
to 13.1% of the Z form). The complication regarding the
methylene is probably due to hindered rotation around the
bonds bearing the substituents in the aromatic moiety near
the hydroxamic group.
Because it was not possible to explain the different binding
activities from the binding interaction analysis of com-
pounds 10 and 11 with MMP-12 and MMP-9, we suggest
that the different activities could be due to a differential
ability of the two compounds to enter the S10 pocket of the
two MMPs.
With regards to the selectivity profile, type C compounds
exhibit nanomolar activity on MMP-12 and maintain good
selectivity over MMP-1, -3, and -14. The introduction of a
methyl group R to the hydroxamate (as in compounds 12 and
The 1H,15N-HSQC spectrum of the 15N-labeled wt-MMP-
12 catalytic domain, complexed to the weak inhibitor acet-
ohydroxamic acid (AHA) for stability reasons, is shown in
Figure 5. The dispersion of the peaks clearly indicates that
the protein is well folded. Chemical shift data are in good
agreement with the assignments published elsewhere for its
complex with N-isobutyl-N-[4-methoxyphenylsulphonyl]glycyl

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6353
Table 3. In Vitroa Activity (IC50 nM Values) of Phenylacetic Carboxylic Acids 14-19
compd
MMP-1
MMP-2
MMP-3
MMP-8
MMP-9
MMP-12
MMP-13
MMP-14
MMP-16
14
15
>100000
>10000
58000 ( 2300 >100000
74000 ( 3300 >100000
89000 ( 7100 55000 ( 3700 77000 ( 5900 93000 ( 8800
710 ( 45
510 ( 27
47 ( 3
>10000
74000 ( 1300
7500 ( 750
1200 ( 140
290 ( 12
99 ( 6
420 ( 44
7300 ( 830
730 ( 58
380 ( 25
1500 ( 140
140 ( 22
940 ( 93
260 ( 16
78 ( 10
8100 ( 550
5400 ( 600
740 ( 58
6500 ( 320
4300 ( 380
730 ( 84
16
23000 ( 1000
4400 ( 330
51000 ( 1500 2400 ( 100 >100000
>10000
>100000
17
18b
19b
1b
1900 ( 110
290 ( 12
20000 ( 1200 1900 ( 140
3000 ( 230
1800 ( 190 18000 ( 1700 6300 ( 580
190 ( 17 2100 ( 100 1100 ( 160
7900 ( 390 10000 ( 1600 18000 ( 1000
190 ( 10
8000 ( 570
310 ( 29
410 ( 26
1500 ( 100
54000 ( 4100 2000 ( 100 24000 ( 1800
a Assays were run in triplicate. The final values given here are the mean ( SD of three independent experiments. b Racemates.
Figure 5. (top) 1H,15N-HSQC spectrum of the wt-MMP-12 catalytic domain complexed with acetohydroxamic acid (black) and after addition
of 1 equiv of 9 (red). Magnification of the region around peak G176 at different steps in the inhibitor addition demonstrates the slow exchange
regime on the NMR time scale. (bottom) Combined 15N and 1H chemical shift deviation (calculated as ((ΔδN þ ΔδH2)/2)1/2, where Δδ is the
2
observed chemical shift change) observed after addition of 1 equiv of 9.
hydroxamic acid (NNGH).11 However, as expected, some
peaks display slightly different chemical shifts in the regions
close to the inhibitor.
changes are revealed in the region close to the catalytic zinc
atom (179-183 region), where binding takes place, and in the
S10 pocket (214-217, 236-242 region). It is important to
notice that these changes are not relative to the free form of
the protein (which is not stable) but to its complex with the
weaker acetohydroxamic inhibitor. Accordingly, what we
observe reflects the structural differences of the two inhibitors.
As 9 contains three aromatic rings, it is expected to cause
sizable changes in the shifts of the protein due to the very
well-known anisotropic properties of this chemical moiety.
In addition, the induced shift is highly orientation depen-
dent, causing shielding in the region above and under the
plane of the ring and deshielding in the region along the
plane. Figure 6B shows that the sign of the largest shifts
reflects the orientation of each 9 ring. The figure also shows
how the best results obtained from docking calculations
indeed agree with the observed chemical shift deviations.
For example, residues L181 and K241 display the most
sizable proton deviations (negative, indicating shielding):
-1.09 ppm and -1.70 ppm, respectively. Figure 6B clearly
Addition of 9 caused a substantial change in a large part of
the spectrum. Figure 5 shows how the complex forms in a
slow exchange regime on the NMR time scale, as expected by
the low value of the dissociation constant. Peaks of the “free
form” (complexed to the weaker inhibitor AHA) tend to
reduce in intensity and a new set of peaks, originating from
the form of the enzyme bound to 9, arises. The titration was
performed in steps until the peaks corresponding to the free
form of the protein disappeared.
On the basis of the pattern observed in the 3D-1H,15N-
NOESYHSQC spectrum, the new set of peaks was assigned
and the difference in chemical shift was measured (Figure 5).
Although changes may arise from both structural rear-
rangement of the protein as it harbors the ligand or allosteric
effects, it is reasonable to think that the largest shifts
correspond to regions in close contact with 9.
Differences were graphically mapped onto the structure of
MMP-12 (PDB code: 1RMZ) (Figure 6A). Dramatic

6354 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20
Nuti et al.
Figure 6. (A) Combined chemical shift deviation mapped onto the MMP-12 structure (PDB code: 1RMZ). Combined deviation larger than
2 and 0.3 are displayed in yellow and red, respectively. (B) Positive nitrogen and proton chemical shift deviations larger than 1 and 0.5,
respectively, are displayed in magenta; nitrogen chemical shift variations more negative than -1 (no hydrogen display shifts more negative than
-0.5) are displayed in cyan. (C) Distances smaller than 0.4 nm among MMP-12 amide protons and 9 hydrogen atoms are displayed as red lines.
Amide nitrogens expected to give rise to intermolecular NOE effects are shown as red spheres. Color coding for the protein ribbons are
according to panel A. (D) Expected NOE effect based on distances found in the best docked structure of 9 with the MMP-12 catalytic domain.
In panels A, B, and C, amide protons are displayed as spheres.
shows that these are indeed the only two amide protons
residing on top of two aromatic rings of the inhibitor, where
the shielding effect is the largest. Accordingly, the largest
positive deviation (1.1 ppm, indicating deshielding) is found
for the amide nitrogen of residue H183, which lies in the plane
To further confirm the found structure, we looked for
intermolecular NOE cross peaks. Figure 6C highlights re-
sidues whose amide proton is at least 0.4 nm apart from the
nonexchangeable hydrogen atoms of the inhibitor. The
expected NOEs are reported in panel D.
Figure 7 shows the superimposition of 3D-1H,15N-NOE-
SYHSQC strips for MMP-12 in the absence and in the
presence of 9 in the regions of interest. Indeed, new cross
peaks appear in the presence of the inhibitor. Because slow
exchange is occurring, the signals arising from bound 9 are
likely to be significantly shifted from those found in its free
form. On the other hand, we can still classify the new cross
peaks as aromatic or aliphatic and use this information to
confirm our structure.
of the aromatic ring directly linked to the hydroxamic moiety.
The docked structure suggests hydrogen bonding of one
sulfonyl oxygen of 9 to either the amide of residue L181 or
A182 (or both). To ascertain its presence, we studied the
temperature dependence of amide proton chemical shifts.
We found temperature coefficients of -9.2 ppb/K and
-2.4 ppb/K, respectively, in the absence of 9. In the bound
form these values become -12.3 ppb/K and -2.2 ppb/K.
Because temperature coefficients more positive than -4.5
ppb/K suggest the presence of hydrogen bonds,30 we con-
clude that amide A182 is hydrogen bonded both in the
complex with AHA (our reference) and in that with 9, while
amide L181 appears to be uninvolved in both cases. Inte-
restingly, in the solid state, the X-ray structure of AHA
indicates a hydrogen bond between amide L181 (rather than
A182) and a water molecule (PDB code 1Y9311). Appa-
rently, because the two amides are very close in space, this
H-bond can be established with residue A182 in solution.
However, what matters for the present discussion is that
amide A182 appears to be hydrogen bonded in the complex
with 9, in agreement with docking calculations.
Figure 7 shows how the expected NOE pattern in
Figure 6D is in very good agreement with the experimental
observations. The amide protons L181 and A182 “see” an
extra peak at 8.4 (labeled a, b in panel A of Figure 7),
and HN Y240 and K241 “see” aromatic peaks at around
7 ppm (labeled b, d in panel B of Figure 7). Amide F237
shows a weak extra aromatic peak (labeled a in panel
B of Figure 7), which was not expected but possible because
its distance from the 70 proton is 0.58 nm in the structure.
The extra peak labeled e in panel B of Figure 7 is proba-
bly due to amide Y242 seeing its own aromatic side
chain.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6355
derivatives showed poor activity on MMPs. This activity was
greatly increased by the introduction of a methylene between
the ZBG and the aromatic ring, allowing for a better interac-
tion of the ZBG with the catalytic zinc ion. Five phenylacetic
hydroxamates were identified to have nanomolar IC50 values
on MMP-12. Among them, the most promising inhibitor was
compound 9, with an IC50 value of 0.2 nM, a 17500-fold
selectivity for MMP-12 over MMP-1, and a 165-fold selectiv-
ity over MMP-14. Moreover, this phenoxybenzenesulfonyl
compound was a subnanomolar inhibitor of MMP-9, another
MMP implicated in the pathogenesis of COPD. The intro-
duction of a small alkyl group, such as a methyl, R to the
hydroxamate (as in compounds 12 and 13), had only a small
effect on inhibitory potency. The carboxylic analogues of
phenylacetic hydroxamates were also tested on the principal
MMPs, showing a decrease in activity but also a different
inhibitory profile with respect to the corresponding hydro-
xamates. In fact, in the carboxylic series, the most potent
compound on MMP-12 was the p-methoxybiphenyl deriva-
tive 17 (IC50 = 139 nM). Docking of 9 into the MMP-12
catalytic domain revealed the formation of H-bonds between
the sulfonyl group and L181 and A182, whereas the p-
methoxyphenoxybenzene group was inserted into the S10
pocket. NMR experiments on the 15N-labeled wt-MMP-12
catalytic domain complexed with 9 provided experimental
support for the docking calculations, both through induced
shifts and NOE effects. NOE data also revealed that the
presence of the inhibitor causes some small extra modifica-
tions in the enzyme structure. Further investigations could
lead to optimization of the inhibitory activity of this new class
of arylsulfones toward MMP-12 to be used in treatment of
some inflammatory diseases such as COPD, RA and athero-
sclerosis.
5. Experimental Section
Chemistry. Melting points were determined on a Kofler
hotstage apparatus and are uncorrected. 1H and 13C NMR
spectra were determined with a Varian Gemini 200 MHz
spectrometer. Chemical shifts (δ) are reported in parts per
million downfield from tetramethylsilane and referenced from
solvent references. Coupling constants J are reported in hertz;
13C NMR spectra were fully decoupled. The following abbre-
viations are used: singlet (s), doublet (d), triplet (t), double-
doublet (dd), broad (br), and multiplet (m).Where indicated, the
elemental compositions of the compounds agreed to within
(0.4% of the calculated value. Chromatographic separations
were performed on silica gel columns by flash column chromato-
graphy (Kieselgel 40, 0.040-0.063 mm; Merck). Reverse phase
chromatography were performed using ISOLUTE SPE col-
umns Flash C18 (STEPBIO). Reactions were followed by
thin-layer chromatography (TLC) on Merck aluminum silica
gel (60 F254) sheets that were visualized under a UV lamp, and
hydroxamic acids were visualized with FeCl3 acqueous solution.
Evaporation was performed in vacuo (rotating evaporator).
Sodium sulfate was always used as the drying agent. Micro-
wave-assisted reactions were run in a CEM Discover LabMate
microwave synthesizer. The purity of the final compounds was
determined by reverse-phase HPLC on a Merck Hitachi D-7000
liquid chromatograph. HPLC purity was determined to be
>95% for all final products using a Discovery C18 column
(250 mm ꢀ 4.6 mm, dp=5 μm, Supelco), with a gradient of 40%
water/60% methanol at a flow rate of 1.4 mL/min, with UV
monitoring at the fixed wavelength of 250 nm. See the Support-
ing Information for compound purity analysis data for final
compounds. Commercially available chemicals were purchased
from Sigma-Aldrich.
Figure 7. Superimposition of 3D-1H,15N-NOESYHSQC strips for
MMP-12 in the absence (green) and presence (cyan) of 9 relative to
the regions of interests (negative contours in magenta). (A) Residues
178-183. (B) Residues 237-243. New cross peaks arising in the
presence of the inhibitor are highlighted with circles. Residue
number is displayed for each amide nitrogen of the strips.
Finally, the methyl protons of the methoxy group are seen
by residues Y240 and V243 (labeled c, f in panel B of
Figure 7). As for the cross peaks connecting the methylene
of 9 and amides L181, A182, and H60 with amide T215,
strong overlap does not allow for verification (data not
shown for residue T215).
NOE data also reveal that the presence of the inhibitor
causes some extra modifications in the structure: amide
protons Y240 and Y242 see extra peaks at 0.2 ppm (g, h in
panel B of Figure 7), which are most likely ascribable to the
methyls of L214, indicating that the loop is slightly deformed
to accommodate the inhibitor. In addition, the region from
G178 to A182 comes closer to the methyls of I180 (c, d, e, f, g
in panel A of Figure 7). This indicates that the side chain of
I180, which in the free form of the protein fills a cavity near
the catalytic zinc, moves toward the loop in the region
178-182 in order to make room for 9.
4. Conclusions
A new series of arylsulfones was designed and synthesized
as potential MMP-12 selective inhibitors. The first benzoic

6356 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20
Nuti et al.
2-(4-Methoxyphenylthio)benzoic Acid (20). A suspension of
2-iodobenzoic acid (1.0 g, 4.03 mmol), 4-methoxythiophenol
(565 mg, 4.03 mmol), KOH (451 mg, 8.1 mmol), and copper
powder (25.6 mg, 0.403 mmol) in deionized water (2 mL) was
submitted to microwave irradiation (two cycles of 6 min each at
the power of 180 W, Tmax=140 °C, 100 psi), cooling the sample
during irradiation with a flow of compressed air. The obtained
suspension was dissolved in a solution of KOH 2N and then
filtered. The filtrate was acidified with HCl 1N, and the formed
precipitate was filtrated and dried under reduced pressure (T=
50 °C). The crude solid was purified by trituration with acetone
to yield 20 (850 mg, 81% yield) as a white solid. 1H NMR
(DMSO-d6) δ: 3.82 (s, 3H), 6.63 (d, J=8.0 Hz, 1H), 7.06-7.20
(m, 3H), 7.30-7.40 (m, 1H), 7.45-7.49 (m, 2H), 7.91 (dd, J1=
7.8 Hz, J2=1.0 Hz, 1H).
to a solution of 23 (200 mg, 0.62 mmol) in THF/MeOH (1:1, 8
mL). The reaction was stirred at room temperature for 24 h, and
the organic solvents were evaporated under reduced pressure.
The obtained suspension was diluted with H2O, and the product
was extracted with EtOAc. The combined organic extracts were
dried over anhydrous Na2SO4, filtered, and evaporated under
reduced pressure to afford 24 as a white solid (190 mg, 86%
yield). H NMR (DMSO-d6) δ: 6.98-7.07 (m, 3H), 7.13-7.23
(m, 2H), 7.37-7.45 (m, 2H), 7.58-7.70 (m, 3H), 7.94-8.02 (m,
2H), 8.30 (d, J=6.8 Hz, 1H).
1
N-Hydroxy-2-(4-phenoxy-benzenesulfonyl)benzamide (3). Com-
pound 3 was synthesized according to the procedure described
above for the preparation of 2, starting from carboxylic acid 24
(180 mg, 0.51 mmol). The O-silylate intermediate was purified by
trituration with n-hexane/CH2Cl2 to afford the protected hydro-
xamate (40 mg) as an oil. 1H NMR (DMSO-d6) δ: 0.24 (s, 6H),
0.97 (s, 9H), 6.98-7.06 (m, 3H), 7.11-7.23 (m, 2H), 7.37-7.45 (m,
2H), 7.57-7.79 (m, 5H), 7.97-8.25 (m, 1H).
After treatment with TFA, the crude product was triturated
with n-hexane to give 3 (15 mg, 12.5% yield) as a white solid. 1H
NMR (DMSO-d6) δ: 7.02-7.12 (m, 3H), 7.15-7.23 (m, 2H),
7.40-7.51 (m, 2H), 7.57-7.79 (m, 5H), 8.01-8.25 (m, 1H), 10.2
(br. s, 1H). Anal. (C19H15NO5S) C, H, N.
2-[4-(4-Methylsulfanyl-phenoxy)-phenylsulfanyl]benzoic Acid
(25). Compound 25 was synthesized according to the procedure
described above for the preparation of 23, starting from phe-
nolic intermediate 22 (400 mg, 1.6 mmol) and 4-(methyl-
thio)phenylboronic acid (539 mg, 3.2 mmol). The obtained
crude was purified by C18 reverse phase chromatography
(MeOH/H2O 3:7, MeOH/H2O 1:1, MeOH/H2O 9:1, MeOH
100%) to yield 25 as solid (225 mg, 38%). 1H NMR (CDCl3) δ:
2.50 (s, 3H), 6.82 (d, J = 8.0 Hz, 1H), 7.02-7.06 (m, 3H),
7.12-7.36 (m, 5H), 7.50-7.54 (m, 2H), 8.11 (d, J=7.8 Hz, 1H).
2-[4-(4-Methanesulfinyl-phenoxy)-benzenesulfonyl]benzoic Acid
(26). A solution of oxone (2 g, 3.2 mmol) in H2O (20 mL) was
added slowly to a solution of 25 (200 mg, 0.54 mmol) in THF/
MeOH (1:1, 6 mL). The reaction was stirred at room temperature
for 24 h, and the organic solvents were evaporated under reduced
pressure. The obtained suspension was diluted with H2O, and the
product was extracted with EtOAc. The combined organic ex-
tracts were driedover anhydrous Na2SO4, filtered, and evaporated
under reduced pressure to afford 26 as a mixture of SO and SO2
derivatives of which the first is the prevalent one (114 mg, 70%
yield). 1H NMR (DMSO-d6) δ: 3.20 (s, 3H), 7.17-7.23 (m, 4H),
7.64-7.71 (m, 3H), 7.89-8.04 (m, 4H), 8.30 (d, J=7.6 Hz, 1H),
13.80 (s, 1H).
2-(4-Methoxy-benzenesulfonyl)benzoic Acid (21). A solution
of oxone (2.2 g, 3.6 mmol) in H2O (15 mL) was added slowly to a
solution of 20 (300 mg, 1.15 mmol) in THF/MeOH (1:1, 12 mL).
The reaction was stirred at room temperature for 24 h, and the
organic solvents were evaporated under reduced pressure. The
obtained suspension was diluted with H2O, and the product was
extracted with EtOAc. The combined organic extracts were
dried over anhydrous Na2SO4, filtered, and evaporated under
reduced pressure to afford 21 as a white solid (202 mg, 60%
yield). 1H NMR (DMSO-d6) δ: 3.83 (s, 3H), 7.11-7.15 (m, 2H),
7.56-7.73 (m, 3H), 7.90-7.94 (m, 2H), 8.07-8.11 (m, 1H).
N-Hydroxy-2-(4-methoxy-benzenesulfonyl)benzamide (2). 1-[3-
(Dimethylamino)propyl]-3-ethyl carbodiimide hydrochloride
(EDC) was added portionwise (65 mg, 0.34 mmol) to a stirred
and cooled solution (0 °C) of the carboxylic acid 21 (100 mg, 0.34
mmol) and O-(tert-butyldimethylsilyl)hydroxylamine (50 mg,
0.34 mmol) in freshly distilled CH2Cl2 (3 mL). After stirring at
room temperature overnight, the mixture was washed with water
and the organic phase was dried and evaporated in vacuo to afford
1
the O-silylate (82 mg, 57% yield) as an oil. H NMR (CHCl3)
δ: 0.25 (s, 6H), 0.97 (s, 9H), 3.82 (s, 3H), 7.10-7.15 (m, 2H),
7.37-7.42 (m, 1H), 7.65-7.70 (m, 2H), 7.95-8.08 (m, 3H), 11.29
(s, 1H).
TFA (0.8 mL, 10.8 mmol) was added dropwise to a stirred and
ice-chilled solution of O-silylate (81 mg, 0.19 mmol) in CH2Cl2
(4 mL). The solution was stirred under these reaction conditions
for 5 h, and the solvent was removed in vacuo to give a solid. The
crude product was triturated with n-hexane/CHCl3 to give
2 (30 mg, 28% yield) as a white solid. 1H NMR (DMSO-d6) δ:
3.82 (s, 3H), 7.08-7.13 (m, 2H), 7.37-7.41 (m, 1H), 7.64-7.69
(m, 2H), 7.96-8.08 (m, 3H), 9.17 (s, 1H), 10.91 (br s, 1H). Anal.
(C14H13NO5S) C, H, N.
2-(4-Hydroxy-phenylsulfanyl)benzoic Acid (22). Compound
22 was synthesized following an analogous procedure to that
described above for the preparation of 20, starting from
2-iodobenzoic acid (1.5 g, 6.05 mmol) and 4-mercaptophenol
(763 mg, 6.05 mmol). The crude solid was purified by trituration
with acetone to yield 22 (1.11 g, 75% yield) as a white solid. 1H
NMR (DMSO-d6) δ: 6.65 (d, J=7.0 Hz, 1H), 6.86-6.91 (m,
2H), 7.09-7.20 (m, 1H), 7.29-7.40 (m, 3H), 7.90 (d, J=7.8 Hz,
1H), 9.98 (br s, 1H).
N-Hydroxy-2-[4-(4-methanesulfinyl-phenoxy)-benzenesulfonyl]-
benzamide (5). Compound 5 was synthesized according to the
procedure described above for the preparation of 2, starting from
carboxylic acid 26 (114 mg, 0.26 mmol). The O-silylate intermedi-
ate was purified by flash chromatography on silica gel (CH2Cl2:
EtOH 95:5, CH2Cl2:EtOH 9:1, CH2Cl2:EtOH 8:2) to afford the
protected SO hydroxamate as an oil. 1H NMR (CDCl3) δ: 0.25 (s,
3H), 0.26 (s, 3H), 1.01 (s, 9H), 3.04 (s, 3H), 7.03-7.21 (m, 4H),
7.50-8.18 (m, 8H), 9.55 (br. s, 1H). After treatment with TFA, the
crude product was triturated with Et2O to give 5 (8.4 mg, 7%
1
2-(4-Phenoxy-phenylsulfanyl)benzoic Acid (23). To a suspen-
sion of 22 (600 mg, 2.43 mmol), phenylboronic acid (592 mg,
4.86 mmol), Cu(OAc)2 (485 mg, 2.43 mmol), and 4 A molecular
sieves in powder (1.0 g) in anhydrous CH2Cl2 (25 mL) was added
triethylamine (1.7 mL, 12.1 mmol). The reaction mixture was
stirred overnight at room temperature and filtered. The filtrated
was treated with HCl 1N and extracted with CH2Cl2. The
organic phases were dried with Na2SO4 and evaporated under
reduced pressure. The obtained crude was purified by tritura-
tion with n-hexane to give 23 (236 mg, 30% yield) as a solid. 1H
NMR (DMSO-d6) δ: 6.73 (d, J=8.0 Hz, 1H), 7.06-7.25 (m,
6H), 7.35-7.56 (m, 5H), 7.91 (d, J=7.8 Hz, 1H).
yield) as a white solid. H NMR (DMSO-d6) δ: 3.20 (s, 3H),
7.17-7.34 (m, 5H), 7.48-8.00 (m, 6H), 8.13 (d, J=7.3 Hz, 1H),
9.4 (br s, 1H), 11.46 (s, 1H). Anal. (C20H17NO6S2) C, H, N.
[2-(4-Hydroxy-phenylsulfanyl)-phenyl]acetic Acid (27). Com-
pound 27 was synthesized following an analogous procedure to
that described above for the preparation of 20, starting from 2-
iodophenylacetic acid (1.0 g, 3.8 mmol) and 4-mercaptophenol
(482 mg, 3.82 mmol). The crude solid was purified by trituration
with acetone to yield 27 (2.5 g, 84% yield) as a white solid. 1H
NMR (DMSO-d6) δ: 3.73 (s, 2H), 6.77-6.81 (m, 2H), 6.92-7.00
(m, 1H), 7.14-7.32 (m, 5H), 9.80 (br s, 1H), 12.38 (br s, 1H).
2-[4-(4-Methoxy-phenoxy)-phenylsulfanyl]benzoic Acid (28).
Compound 28 was synthesized according to the procedure
described above for the preparation of 23, starting from
2-(4-Phenoxy-benzenesulfonyl)benzoic Acid (24). A solution
of oxone (455 mg, 0.74 mmol) in H2O (3 mL) was added slowly

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6357
phenolic intermediate 22 (400 mg, 1.6 mmol) and 4-(methoxy)-
phenylboronic acid (494 mg, 3.2 mmol). The obtained crude
was purified by C18 reverse phase chromatography (MeOH/
H2O 3:7) to yield 28 as solid (190 mg, 33%). 1H NMR (CDCl3)
δ: 3.82 (s, 3H), 6.80 (d, J=8.0 Hz, 1H), 6.90-7.18 (m, 7H),
7.26-7.35 (m, 1H), 7.45-7.52 (m, 2H), 8.12 (dd, J1=7.7 Hz,
J2=1.6 Hz, 1H).
{2-[4-(4-Methoxy-phenoxy)phenylsulfanyl]phenyl}acetic Acid
(29). Compound 29 was synthesized according to the procedure
described above for the preparation of 23, starting from phenolic
intermediate 27 (1.9 g, 7.3 mmol) and 4-(methoxy)phenylboronic
acid (2.2 g, 14.6 mmol). The obtained crude was purified by flash
chromatography on silica gel (CH2Cl2/Et2O 9:1, CH2Cl2/Et2O 8:2,
CH2Cl2/MeOH 9:1) to yield 29 as solid (840 mg, 31%). 1H NMR
(DMSO-d6) δ: 3.75 (s, 5H), 6.88-7.06 (m, 6H), 7.12-7.36 (m, 6H),
12.34 (s, 1H).
3.91 (s, 2H), 6.98-7.13 (m, 6H), 7.41-7.59 (m, 3H), 7.79-7.83
(m, 2H), 8.02 (d, J = 7.4 Hz, 1H), 12.33 (s, 1H). 13C NMR
(DMSO-d6) δ: 37.88, 55.45, 115.28, 116.83, 121.73, 127.93,
128.99, 129.74, 133.47, 134.02, 134.12, 139.17, 147.33, 156.47,
162.24, 171.29.
N-Hydroxy-2-[4-(4-methoxy-phenoxy)benzenesulfonyl]benzamide
(4). Compound 4 was synthesized according to the procedure
described above for the preparation of 2, starting from carboxylic
acid 34 (100 mg, 0.26 mmol). The O-silylate intermediate was
treated with TFA, and the crude product was triturated with
diisopropyl ether to give 4 (12 mg, 12% yield) as a white solid. 1H
NMR (CDCl3) δ: 3.82 (s, 3H), 6.93-7.13 (m, 5H), 7.33-7.51 (m,
5H), 7.85-7.89 (m, 1H), 8.01-8.20 (m, 1H), 12.33 (s, 1H). Anal.
(C20H17NO6S) C, H, N.
N-Hydroxy-2-{2-[4-(4-methoxy-phenoxy)benzenesulfonyl]-
phenyl}acetamide (9). Compound 9 was synthesized according to
the procedure described above for the preparation of 4, starting
from carboxylic acid 15 (500 mg, 1.06 mmol). The O-silylate
intermediate was treated with TFA, and the crude product was
purified by trituration with diisopropyl ether to give 9 (78 mg, 15%
yield) as a white solid; mp: 75 °C; NMR assignment of a 15 mM
solution in DMSO-d6 (atom numbering as in Figure 5D. The
2-[4-(4-Methoxy-phenoxy)phenylsulfanyl]benzoic Acid Benzyl
Ester (30). A solution of carboxylic acid 28 (162 mg, 0.46 mmol)
in anhydrous DMF (1.0 mL) was cooled to 0 °C. Cesium
carbonate (150 mg, 0.46 mmol) was added, and the mixture
was stirred for 40 min at 0 °C. Benzyl bromide (0.05 mL,
0.46 mmol) was added, and the reaction mixture was stirred at
0 °C for 30 min and then at room temperature overnight. The
mixture was poured into H2O and extracted with Et2O. The
combined extracts were washed with H2O and saturated NaCl,
dried over Na2SO4, filtered, and evaporated under reduced
pressure to afford 30 (183 mg, 91% yield) as a yellow solid that
1
assignment reports the shifts of the main form): H: DMSO-d6
(600 MHz) δ 10.55 (1H, HN), 8.83 (1H, OH), 4.05, 3.69 (2H,
CH2), 7.38 (1H, H-3), 7.64 (1H, H-4), 7.53 (1H, H-5), 8.03 (1H, H-
6), 7.88 (2H, H-20), 7.03 (2H, H-30), 7.11 (2H, H-60), 7.00 (2H, H-
70), 3.77 (3H, Me), 9.95 (1H, HN Z form), 9.18 (1H, OH Z form);
13C: DMSO-d6 (600 MHz) δ 165.8 (1C, carbonyl), 35.3 (1C, CH2),
139.4 (1C, C-1), 133.2 (1C, C-2), 132.4 (1C, C-3), 133.0 (1C, C-4),
127.3 (1C, C-5), 128.7 (1C, C-6), 134.0 (1C, C-10), 129.5 (2C, C-20),
116.4 (2C, C-30), 162.2 (1C, C-40), 146.8 (1C, C-50), 121.4 (2C, C-
60), 114.9 (2C, C-70), 156.8 (1C, C-80), 55.0 (1C, Me). Anal.
(C21H19NO6S) C, H, N.
1
was utilized in the next step without further purification. H
NMR (CDCl3) δ: 3.82 (s, 3H), 5.40 (s, 2H), 6.80 (d, J=7.1 Hz,
1H), 6.90-7.13 (m, 7H), 7.21-7.30 (m, 1H), 7.34-7.51 (m, 7H),
8.02 (dd, J1=7.7 Hz, J2=1.6 Hz, 1H).
{2-[4-(4-Methoxy-phenoxy)phenylsulfanyl]phenyl}acetic Acid
Benzyl Ester (31). Compound 29 (1.56 g, 4.27 mmol) was
submitted to the same procedure described above for the pre-
paration of 30, to afford benzylic ester 31 as a yellow solid (1.7 g,
97% yield). 1H NMR (CDCl3) δ: 3.80 (s, 3H), 3.90 (s, 2H), 5.12
(s, 2H), 6.78-7.00 (m, 6H), 7.17-7.32 (m, 11H).
2-[4-(4-Methoxy-phenoxy)benzenesulfonyl]benzoic Acid Bbenzyl
Ester (32). Compound 32 was synthesized according to the
procedure described above for the preparation of 24, starting from
benzylic ester 30 (180 mg, 0.41 mmol) and using 15 equiv of oxone.
After stirring at room temperature for 3 days, the reaction was
treated as described above to give 32 as a white solid (148 mg, 77%
yield). 1H NMR (CDCl3) δ: 3.82 (s, 3H), 5.40 (s, 2H), 6.83-6.99
(m, 6H), 7.32-7.46 (m, 5H), 7.53-7.61 (m, 3H), 7.84-7.89 (m,
2H), 8.05-8.09 (m, 1H).
2-(2-(4-Methoxyphenylthio)phenyl)acetic Acid (35). Com-
pound 35 was synthesized following an analogous procedure
to that described above for the preparation of 20, starting from
2-iodophenylacetic acid (1.0 g, 3.8 mmol) and 4-methoxythio-
phenol (469 μL, 3.8 mmol). The crude solid product so obtained,
consisting of 35 (870 mg, 83% yield), was utilized in the next step
1
without further purification; mp: 86 °C. H NMR (CDCl3) δ:
3.78 (s, 3H), 3.87 (s, 2H), 6.82-6.86 (m, 2H), 7.16-7.30 (m, 6H).
Benzyl 2-(2-(4-Methoxyphenylthio)phenyl)acetate (36). Ester
36 was synthesized following an analogous procedure to that
described above for the preparation of 30, starting from car-
boxylic acid 35 (700 mg, 2.5 mmol). The crude oil product so
obtained, consisting of 36 (691 mg, 74% yield), was utilized in
the next step without further purification. 1H NMR (CDCl3) δ:
3.79 (s, 3H), 3.89 (s, 2H), 5.13 (s, 2 H), 6.81-6.85 (m, 2H),
7.15-7.28 (m, 7H), 7.33 (m, 4H).
{2-[4-(4-Methoxy-phenoxy)benzenesulfonyl]phenyl}acetic Acid
Benzyl Ester (33). Compound 33 was synthesizedaccording to the
procedure described above for the preparation of 32, starting
from benzylic ester 31 (200 mg, 0.44 mmol). The crude product
was triturated with Et2O to give sulfone 33 (206 mg, 96% yield) as
white solid. 1H NMR (CDCl3) δ: 3.81 (s, 3H), 4.08 (s, 2H), 5.03(s,
2H), 6.87-6.99 (m, 6H), 7.30-7.35 (m, 5H), 7.43-7.59 (m, 3H),
7.72-7.78 (m, 2H), 8.13 (dd, J1=7.7 Hz, J2=1.6 Hz, 1H).
2-[4-(4-Methoxy-phenoxy)benzenesulfonyl]benzoic Acid (34).
A suspension of ester 32 (140 mg, 0.3 mmol) and KOH (50 mg,
0.9 mmol) in H2O (1.0 mL) was refluxed for 18 h. After being
cooled to RT, the reaction was treated with HCl 1N and then
extracted with CH2Cl2. The organic phase was dried over
Na2SO4 and concentrated to give a crude product, which was
purified by trituration with Et2O/n-hexane to yield 34 (106 mg,
Benzyl 2-(2-(4-Methoxyphenylsulfonyl)phenyl)acetate (37). A
solution of oxone (9.3 g, 15.2 mmol) in H2O (37 mL) was added
slowly to a solution of 36 (691 mg, 1.9 mmol) in THF/MeOH
(3:1, 36 mL). The reaction was stirred at room temperature for 3
days and the organic solvents were evaporated under reduced
pressure. The obtained suspension was diluted with H2O, and
the product was extracted with EtOAc. The combined organic
extracts were dried over anhydrous Na2SO4, filtered, and eva-
porated under reduced pressure to afford 37 as a yellow oil (682
mg, 91% yield). 1H NMR (CDCl3) δ: 3.80 (s, 3H), 4.07 (s, 2H),
5.01 (s, 2H), 6.86-6.92 (m, 2H), 7.28-7.37 (m, 6H), 7.42-7.57
(m, 2H), 7.74-7.81 (m, 2H), 8.12 (dd, J1=7.5 Hz, J2=1.8 Hz,
1H).
1
94% yield) as yellow oil. H NMR (CDCl3) δ: 3.80 (s, 3H),
6.87-7.00 (m, 5H), 7.31-7.42 (m, 1H), 7.63-7.75 (m, 3H),
2-(2-(4-Methoxyphenylsulfonyl)phenyl)acetic Acid (14). Com-
pound 14 was synthesized according to the procedure described
above for the preparation of 34, starting from benzylic ester 37
(681 mg, 1.7 mmol). The crude product was purified by tritura-
tion with Et2O to yield 14 (355 mg, 89% yield) as white solid; mp
146 °C. 1H NMR (CDCl3) δ: 3.84 (s, 3H), 4.03 (s, 2H),
6.93-6.99 (m, 2H), 7.35 (d, J = 7.5 Hz, 1H), 7.43-7.60 (m,
2H), 7.76-7.83 (m, 2H), 8.10 (dd, J1=7.7 Hz, J2=1.5 Hz, 1H).
7.90-7.95 (m, 2H), 8.16-8.20 (m, 1H).
{2-[4-(4-Methoxy-phenoxy)benzenesulfonyl]phenyl}acetic Acid
(15). Compound 15 was synthesized according to the procedure
described above for the preparation of 34, starting from benzylic
ester 33 (200 mg, 0.41 mmol). The crude product was purified by
trituration with Et2O/n-hexane to yield 15 (133 mg, 81% yield) as
1
white solid; mp: 159 °C. H NMR (DMSO-d6) δ: 3.76 (s, 3H),

6358 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20
Nuti et al.
13C NMR (DMSO-d6) δ: 37.84, 55.76, 114.75, 127.88, 128.88,
129.45, 132.36, 133.32, 133.98, 139.46, 162.95, 171.32.
procedure. The crude oil was purified by flash chromatography
on silica gel (CH2Cl2/n-hexane 6:4) to yield 43 (72% yield) as a
solid. 1H NMR (CDCl3) δ: 3.86 (s, 3H), 5.41 (s, 2H), 6.90 (d, J=
8.0 Hz, 1H), 6.98-7.02 (m, 2H), 7.08-7.15 (m, 1H), 7.22-7.60
(m, 12H), 8.02 (dd, J1=7.7 Hz, J2=1.6 Hz, 1H).
[2-(Biphenyl-4-ylsulfanyl)phenyl]acetic Acid Benzyl Ester (44).
The title compound was prepared from aryl bromide 41 and
phenylboronic acid following the general procedure. The crude
product was purified by flash chromatography on silica gel
(CH2Cl2/n-hexane 4:6) to yield 44 (72% yield) as a yellow oil.
1H NMR (CDCl3) δ: 3.93 (s, 2H), 5.01 (s, 2H), 7.20-7.56 (m,
18H).
[2-(40-Methoxy-biphenyl-4-ylsulfanyl)phenyl]acetic Acid Ben-
zyl Ester (45). The title compound was prepared from aryl
bromide 41 and 4-methoxyphenylboronic acid following the
general procedure. The crude product was purified by flash
chromatography on silica gel (n-hexane/EtOAc 9:1) to yield 45
(68% yield) as a yellow solid. 1H NMR (CDCl3) δ: 3.85 (s, 3H),
3.93 (s, 2H), 5.10 (s, 2H), 6.93-6.98 (m, 2H), 7.19-7.50 (m,
15H).
General Procedure to Synthesize Sulfones 46-49. A solution
of oxone (18 equiv) in H2O (20 mL) was added slowly to a
solution of sulfinyl derivatives 42-45 (1 equiv) in THF/MeOH
(3:1, 20 mL). The reaction was stirred at room temperature for 3
days, and the organic solvents were evaporated under reduced
pressure. The obtained suspension was diluted with H2O, and
the product was extracted with EtOAc. The combined organic
extracts were dried over anhydrous Na2SO4, filtered, and eva-
porated under reduced pressure.
N-Hydroxy-2-(2-(4-methoxyphenylsulfonyl)phenyl)acetamide
(8). Compound 8 was synthesized according to the procedure
described above for the preparation of 2, starting from car-
boxylic acid 14 (150 mg, 0.5 mmol). The O-silylate intermediate
1
was utilized in the next step without further purification. H
NMR (CDCl3) δ: 0.12 (s, 6H), 0.91 (s, 9H), 3.72 (s, 2H), 3.86 (s,
3H), 6.97-7.02 (m, 2H), 7.36-7.44 (m, 1H), 7.50-7.56 (m, 1H),
7.61-7.65 (m, 1H), 7.77-7.81 (m, 2H), 7.94 (d, J=7.7 Hz, 1H),
9.13 (s, 1H). After treatment with TFA, the crude product was
triturated with Et2O/n-hexane to give 8 (96 mg, 91% yield) as a
1
white solid; mp 109 °C. H NMR (DMSO-d6) δ: 3.67 (s, 2H),
3.83 (s, 3H), 7.09-7.13 (m, 2H), 7.35 (d, J = 7.7 Hz, 1H),
7.47-7.66 (m, 2H), 7.82-7.87 (m, 2H), 8.02 (d, J = 7.7 Hz,
1H), 10.58 (s, 1H). 13C NMR (DMSO-d6) δ: 35.66, 55.74,
114.70, 127.52, 128.83, 129.63, 132.36, 132.69, 133.18, 134.65,
139.55, 162.93, 165.77. Anal. (C15H15NO5S) C, H, N.
2-(4-Bromo-phenylsulfanyl)benzoic Acid (38). Compound 38
was synthesized following an analogous procedure to that
described above for the preparation of 20, starting from
2-iodobenzoic acid (3.0 g, 12.1 mmol) and 4-bromobenzenethiol
(2.2 g, 12.1 mmol). The crude solid was purified by trituration
with acetone to yield 38 (3.1 g, 82% yield) as a white solid; mp
219 °C. 1H NMR (DMSO-d6) δ: 6.77 (d, J = 7.3 Hz, 1H),
7.20-7.28 (m, 1H), 7.36-7.49 (m, 3H), 7.65-7.71 (m, 2H), 7.91
(dd, J1=7.7 Hz, J2=1.5 Hz, 1H), 13.26 (br s, 1H).
[2-(4-Bromo-phenylsulfanyl)phenyl]acetic Acid (39). Com-
pound 39 was synthesized following an analogous procedure
to that described above for the preparation of 20, starting from
2-iodophenylacetic acid (5.0 g, 19.1 mmol) and 4-bromoben-
zenthiol (3.6 g, 19.1 mmol). The crude solid product so obtained,
consisting of 39 (4.3 g, 88% yield), was utilized in the next step
Benzyl 2-(Biphenyl-4-ylsulfonyl)benzoate (46). The title com-
pound was prepared from sulfinyl derivative 42 following the
general procedure. The crude product was purified by tritura-
tion with n-hexane/Et2O to yield 46 as a white solid (80% yield);
1
1
without further purification. H NMR (DMSO-d6) δ: 3.74 (s,
mp 124 °C. H NMR (CDCl3) δ: 5.42 (s, 2H), 7.33-7.65 (m,
2H), 7.07 (d, J=8.1 Hz, 2H), 7.30-7.42 (m, 4H), 7.49 (d, J=8.1
Hz, 2H).
15H), 7.98-8.02 (m, 2H), 8.13-8.18 (m, 1H).
Benzyl 2-(40-Methoxybiphenyl-4-ylsulfonyl)benzoate (47). The
title compound was prepared from sulfinyl derivative 43 follow-
ing the general procedure. The crude product was purified by
trituration with n-hexane/Et2O to yield 47 as a white solid (82%
yield); mp 118 °C. 1H NMR (CDCl3) δ: 3.85 (s, 3H), 5.42 (s, 2H),
6.96-7.00 (m, 2H), 7.33-7.64 (m, 12H), 7.94-7.99 (m, 2H),
8.12-8.16 (m, 1H).
2-(4-Bromo-phenylsulfanyl)benzoic Acid Benzyl Ester (40).
Compound 38 (3.0 g, 9.7 mmol) was submitted to the same
procedure described above for the preparation of 30 to afford
benzylic ester 40 as a yellow solid (3.7 g, 94% yield); mp 69 °C.
1H NMR (CDCl3) δ: 5.39 (s, 2H), 6.83 (d, J = 8.0 Hz, 1H),
7.10-7.18 (m, 1H), 7.23-7.58 (m, 10H), 8.01 (dd, J1=7.7 Hz,
J2=1.6 Hz, 1H).
[2-(Biphenyl-4-sulfonyl)phenyl]acetic Acid Benzyl Ester (48).
The title compound was prepared from sulfinyl derivative 44
following the general procedure. The oil so obtained, consisting
of 48 (90% yield), was utilized in the next step without further
[2-(4-Bromo-phenylsulfanyl)phenyl]acetic Acid Benzyl Ester
(41). Compound 39 (3.3 g, 10.2 mmol) was submitted to the
same procedure described above for the preparation of 30, to
1
1
afford benzylic ester 41 as a yellow oil (4.1 g, 97% yield). H
NMR (CDCl3) δ: 3.87 (s, 2H), 5.01 (s, 2H), 6.95-7.00 (m, 2H),
7.27-7.44 (m, 11H).
purification. H NMR (CDCl3) δ: 4.10 (s, 2H), 4.98 (s, 2H),
7.20-7.64 (m, 15H), 7.88-7.92 (m, 2H), 8.20 (dd, J1=7.5 Hz, J2
=1.8 Hz, 1H).
General Procedure to Synthesize Compounds 42-45 by Suzuki
Coupling. A solution of aryl bromide 40 or 41 (1 equiv) in
anhydrous dioxane (16 mL/mmol)/H2O (2.5 mL/mmol) was
sequentially treated under nitrogen with K3PO4 (2.3 equiv), the
appropriate arylboronic acid (1.1 equiv), and Pd(PPh3)4 (5%
equiv). The resulting mixture was stirred for 4 h at 85 °C. After
being cooled to room temperature, the mixture was treated with
NaHCO3 and extracted with EtOAc The combined organic
extracts were dried over anhydrous Na2SO4, filtered, and eva-
porated under reduced pressure.
Benzyl 2-(Biphenyl-4-ylthio)benzoate (42). The title com-
pound was prepared from aryl bromide 40 and phenylboronic
acid following the general procedure. The crude oil was purified
by flash chromatography on silica gel (CH2Cl2/n-hexane 3:7) to
yield 42 (90% yield) as a yellow oil, which solidified on standing .
1H NMR (CDCl3) δ: 5.41 (s, 2H), 6.91 (d, J = 8.0 Hz, 1H),
7.09-7.17 (m, 1H), 7.23-7.51 (m, 9H), 7.59-7.69 (m, 6H), 8.03
(dd, J1=7.7 Hz, J2=1.6 Hz, 1H).
2-(40-Methoxy-biphenyl-4-ylsulfanyl)benzoic Acid Benzyl Es-
ter (43). The title compound was prepared from aryl bromide
40 and 4-methoxyphenylboronic acid following the general
[2-(40-Methoxy-biphenyl-4-sulfonyl)phenyl]acetic Acid Benzyl
Ester (49). The title compound was prepared from sulfinyl
derivative 45 following the general procedure. The solid product
so obtained, consisting of 49 (85% yield), was utilized in the next
step without further purification; mp 86 °C. 1H NMR (CDCl3)
δ: 3.85 (s, 3H), 4.09 (s, 2H), 4.97 (s, 2H), 6.95-7.01 (m, 2H),
7.20-7.35 (m, 6H), 7.45-7.70 (m, 6H), 7.84-7.89 (m; 2H), 8.21
(dd, J1=7.5 Hz, J2=1.8 Hz, 1H).
General Procedure to Synthesize Carboxylic Acids 50, 51 and
16, 17. A suspension of ester 46-49 (1 equiv) and KOH (3 equiv)
in H2O (3.3 mL/mmol) was refluxed for 18 h. After being cooled
to rt, the reaction was treated with HCl 1N and then extracted
with CH2Cl2. The organic phase was dried over Na2SO4 and
concentrated .
2-(Biphenyl-4-ylsulfonyl)benzoic Acid (50). The title com-
pound was prepared from benzylic ester 46 following the general
procedure. The solid product so obtained, consisting of 50 (86%
yield), was utilized in the next step without further purification;
mp 195 °C. 1H NMR (DMSO-d6) δ: 7.43-7.54 (m, 3H),
7.62-7.79 (m, 5H), 7.87-7.91 (m, 2H), 8.02-8.06 (m, 2H),
8.17-8.21 (m, 1H), 13.62 (s, 1H).

Article
2-(40-Methoxybiphenyl-4-ylsulfonyl)benzoic Acid (51). The
title compound was prepared from benzylic ester 47 following
the general procedure. The solid product so obtained, consisting
of 51 (80% yield), was utilized in the next step without further
purification; mp 203 °C. 1H NMR (DMSO-d6) δ: 3.80 (s, 3H),
7.03-7.07 (m, 2H), 7.61-7.78 (m, 5H), 7.83-7.87 (m, 2H),
7.98-8.02 (m, 2H), 8.15-8.20 (m, 1H).
[2-(Biphenyl-4-sulfonyl)phenyl]acetic Acid (16). The title com-
pound was prepared from benzylic ester 48 following the general
procedure. The crude product was purified by trituration with
Et2O to yield 16 as a white solid (88% yield); mp: 182-183 °C.
1H NMR (DMSO-d6) δ: 3.95 (s, 2H), 7.42-7.77 (m, 8H),
7.86-7.96 (m, 4H), 8.13 (d, J=7.7 Hz, 1H), 12.38 (br s, 1H).
13C NMR (DMSO-d6) δ: 38.02, 127.10, 127.72, 127.99, 128.64,
129.04, 129.26, 133.69, 134.09, 134.45, 138.08, 138.70, 139.64,
144.96, 171.34.
[2-(40-Methoxy-biphenyl-4-sulfonyl)phenyl]acetic Acid (17).
The title compound was prepared from benzylic ester 49 follow-
ing the general procedure. The crude product was purified by
trituration with n-hexane/Et2O to yield 17 as a white solid (94%
yield); mp 189 °C. 1H NMR (DMSO-d6) δ: 3.80 (s, 3H), 3.94 (s,
2H), 7.03-7.07 (m, 2H), 7.44 (d, J=7.3 Hz, 1H), 7.57-7.71 (m,
4H), 7.86 (m, 4H), 8.10 (d, J=7.5 Hz, 1H). 13C NMR (DMSO-
d6) δ: 37.95, 55.27, 114.51, 126.97, 127.73, 128.03, 128.35,
129.21, 130.25, 133.63, 134.09, 134.31, 138.77, 138.86, 144.59,
159.82, 171.32.
General Procedure to Synthesize Hydroxamic Acids 6, 7 and
10, 11. 1-[3-(Dimethylamino)propyl]-3-ethyl carbodiimide hy-
drochloride (EDC) was added portionwise (1.5 equiv) to a
stirred solution of the carboxylic acid 50, 51 or 16, 17 (1 equiv),
and O-(tert-butyldimethylsilyl)hydroxylamine (1.5 equiv) in
freshly distilled CH2Cl2 (25 mL/mmol). After stirring at room
temperature overnight, the mixture was washed with water and
the organic phase was dried and evaporated in vacuo to afford
the O-silylate intermediate. TFA (57 equiv) was added dropwise
to a stirred and ice-chilled solution of O-silylate (1 equiv) in
CH2Cl2 (8 mL/mmol). The solution was stirred under these
reaction conditions for 5 h, and the solvent was removed in
vacuo.
2-(Biphenyl-4-ylsulfonyl)N-hydroxybenzamide (6). The title
compound was prepared from carboxylic acid 50 following the
general procedure. The crude product was purified by tritura-
tion with n-hexane/Et2O to yield 6 as a white solid (90% yield);
mp 132 °C. 1H NMR (DMSO-d6) δ: 7.43-7.53 (m, 4H),
7.71-7.74 (m, 4H), 7.86-7.93 (m, 2H), 8.03-8.18 (m, 3H),
10.96 (s, 1H). 13C NMR (DMSO-d6) δ: 127.11, 127.24, 128.66,
129.04, 129.68, 129.96, 130.48, 133.69, 134.98, 138.29, 138.46,
140.01, 144.92, 164.00. Anal. (C19H15NO4S) C, H, N.
N-Hydroxy-2-(40-methoxybiphenyl-4-ylsulfonyl)benzamide (7).
The title compound was prepared from carboxylic acid 51
following the general procedure. The crude product was purified
by trituration with n-hexane/Et2O to yield 7 as a white solid (97%
yield); mp 144 °C. 1H NMR (DMSO-d6) δ: 3.80 (s, 3H),
7.03-7.07 (m, 2H), 7.40-7.45 (m, 1H), 7.67-7.71 (m, 4H),
7.81-7.88 (m, 2H), 8.05-8.16 (m, 3H), 10.95 (s, 1H). 13C
NMR (DMSO-d6) δ: 55.25, 114.50, 126.50, 128.33, 128.66,
129.65, 129.86, 130.45, 133.60, 134.93, 138.60, 139.13, 144.54,
159.74, 164.02. Anal. (C20H16O5S) C, H, N.
2-[2-(Biphenyl-4-sulfonyl)phenyl]N-hydroxy-acetamide (10).
The title compound was prepared from carboxylic acid 16
following the general procedure. The crude product was purified
by trituration with CH2Cl2/Et2O to yield 10 as a white solid
(55% yield); mp: 174 °C. 1H NMR (DMSO-d6) δ: 3.72 (s, 2H),
7.34-7.75 (m, 8H), 7.87-8.02 (m, 4H), 8.12 (d, J=7.5 Hz, 1H),
8.87 (s, 1H), 10.62 (s, 1H). 13C NMR (DMSO-d6) δ: 35.73,
127.11, 127.68, 127.93, 128.64, 129.04, 129.26, 132.94, 133.58,
135.00, 138.13, 138.53, 139.64, 144.96, 165.72. Anal.
(C20H17NO4S) C, H, N.
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6359
17 following the general procedure. The crude product was
purified by trituration with CH2Cl2/Et2O to yield 11 as a white
solid (57% yield); mp 158 °C. 1H NMR (DMSO-d6) δ: 3.71 (s,
2H), 3.80 (s, 3H), 7.04-7.07 (m, 2H), 7.37-8.13 (m, 10H), 10.61
(s, 1H). 13C NMR (DMSO-d6) δ: 35.71, 55.23, 114.50, 126.91,
127.66, 127.90, 128.33, 129.17, 130.26, 132.85, 133.47, 134.94,
138.77, 138.93, 144.58, 159.78, 165.73. Anal. (C21H19NO5S)
C, H, N.
General Procedure to Synthesize Compounds 52, 53. A solu-
tion of benzylic ester 48, 49 (1 equiv) in dry THF (6 mL/mmol)
was cooled to -78 °C under argon and treated dropwise with a 1
M solution of NaHMDS in THF (1.1 equiv). After stirring at
-78 °C for 1 h, MeI (5 equiv) was added. The mixture was stirred
in these conditions for 1 h and then allowed to warm up to
-50 °C for 2 h. The reaction mixture was quenched with AcOH
(3 equiv) in Et2O (4 mL/mmol) and filtered over celite. The
filtrate was concentrated and the crude product was purified by
flash chromatography.
2-[2-(Biphenyl-4-sulfonyl)phenyl]propionic Acid Benzyl Ester
(52). The title compound was prepared from benzylic ester 48
following the general procedure. The crude product was purified
by flash chromatography on silica gel (n-hexane/EtOAc 15:85)
to yield 52 (68% yield) as a yellow oil. 1H NMR (CDCl3) δ: 1.41
(d, J=7.14 Hz, 3H), 4.81 (q, J=7.14 Hz, 1H), 4. 87 (s, 2H),
6.94-7.01 (m, 2H), 7.11-7.19 (m, 3H), 7.39-7.62 (m, 10H),
7.86-7.91 (m, 2H), 8.26 (dd, J1=7.8 Hz, J2=1.5 Hz, 1H).
2-[2-(40-Methoxy-biphenyl-4-sulfonyl)phenyl]propionic Acid
Benzyl Ester (53). The title compound was prepared from
benzylic ester 49 following the general procedure. Yellow solid
(70% yield). 1H NMR (CDCl3) δ: 1.40 (d, J=7.14 Hz, 3H), 3.86
(s, 3H), 4.82 (q, J=7.14 Hz, 1H), 4.87 (s, 2H), 6.95-7.00 (m,
4H), 7.15-7.20 (m, 3H), 7.40-7.61 (m, 7H), 7.83-7.87 (m, 2H),
8.25 (d, J=7.7 Hz, 1H).
2-[2-(Biphenyl-4-sulfonyl)phenyl]propionic Acid (18). Com-
pound 18 was synthesized according to the procedure de-
scribed above for the preparation of 34, starting from
benzylic ester 52 (330 mg, 0.72 mmol). The crude product
was purified by trituration with toluene to afford 18 (214 mg,
81% yield) as white solid; mp: 209 °C. 1H NMR (DMSO-d6) δ:
1.18 (d, J=7.08 Hz, 3H), 4.55 (q, J=7.08 Hz, 1H), 7.40-7.62
(m, 5H), 7.69-7.78 (m, 3H), 7.90-7.96 (m, 4H), 8.17 (d, J=7.8
Hz, 1H), 12.34 (br s, 1H). 13C NMR (DMSO-d6) δ: 18.98,
127.10, 127.68, 127.79, 128.70, 129.08, 129.88, 134.25, 138.02,
139.73, 140.95, 144.90, 174.07.
2-[2-(40-Methoxy-biphenyl-4-sulfonyl)phenyl]propionic Acid
(19). Compound 19 was synthesized according to the procedure
described above for the preparation of 34, starting from benzylic
ester 53 (360 mg, 0.74 mmol). The crude product was purified by
trituration with Et2O to afford 19 (210 mg, 72% yield) as white
solid; mp: 153 °C. 1H NMR (DMSO-d6) δ: 1.17 (d, J=7.08 Hz,
3H), 3.80 (s, 3H), 4.56 (q, J=7.08 Hz, 1H), 7.03-7.08 (m, 2H),
7.46-7.75 (m, 5H), 7.82-7.87 (m, 4H), 8.15 (d, J=7.8 Hz, 1H),
12.32 (br s, 1H). 13C NMR (DMSO-d6) δ: 18.98, 55.25, 114.53,
126.90, 127.77, 128.32, 128.95, 129.85, 130.17, 134.16, 138.22,
138.84, 140.90, 144.52, 159.82, 174.09.
2-[2-(Biphenyl-4-sulfonyl)phenyl]N-hydroxy-propionamide (12).
Hydroxamic acid 12 was synthesized according to the procedure
described above for the preparation of 2, starting from carboxylic
acid 18 (140 mg, 0.44 mmol). The O-silylate intermediate was
utilized in the next step without further purification. After treat-
ment with TFA, the crude product was triturated with toluene to
1
afford 12 (58 mg, 41%) as white solid; mp: 177 °C. H NMR
(DMSO-d6) δ: 1.06 (d, J=7.14 Hz, 3H), 4.39 (q, J=7.14 Hz, 1H),
7.44-7.59 (m, 4H), 7.66-8.13 (m, 9H), 8.82 (s, 1H), 10.35 (s, 1H).
13C NMR (DMSO-d6) δ: 20.01, 37.31, 127.10, 127.46, 127.66,
127.95, 128.66, 129.03, 130.70, 133.69, 138.04, 139.75, 140.66,
144.96, 169.19. Anal. (C21H19NO4S) C, H, N.
N-Hydroxy-2-[2-(40-methoxy-biphenyl-4-sulfonyl)phenyl]pro-
pionamide (13). Hydroxamic acid 13 was synthesized accord-
ing to the procedure described above for the preparation of 2,
N-Hydroxy-2-[2-(40-methoxy-biphenyl-4-sulfonyl)phenyl]aceta-
mide (11). The title compound was prepared from carboxylic acid

6360 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20
Nuti et al.
starting from carboxylic acid 19 (80 mg, 0.20 mmol). The
O-silylate intermediate was purified by flash chromatography
on silica gel (n-hexane/EtOAc 6:4) to afford the protected
convergence value of 0.05 kcal/(A mol) was reached, using the
3
same force field and parameters used for the CS. We subjected
the ligands to this protocol in order to achieve reasonable
starting structures.
1
hydroxamate (101 mg) as an oil. H NMR (CDCl3) δ: 0.06 (s,
3H), 0.08 (s, 3H), 0.89 (s, 9H), 1.16 (d, J=6.95 Hz, 3H), 3.86 (s,
3H), 4.35 (q, J=6.95 Hz, 1H), 6.96-7.02 (m, 2H), 7.38-7.47 (m,
1H), 7.51-7.63 (m, 3H), 7.69-7.74 (m, 3H), 7.86-7.90 (m, 2H),
8.03-8.07 (m, 1H), 8.84 (br s, 1H). After treatment with TFA,
the crude product was purified by flash chromatography on
silica gel (CH2Cl2/MeOH 96:4) to give 13 (24 mg, 32% yield) as a
white solid; mp: 153 °C. 1H NMR (DMSO-d6) δ: 1.06 (d, J=6.78
Hz, 3H), 3.81 (s, 3H), 4.40 (q, J=6.78 Hz, 1H), 7.04-7.08 (m,
Automated docking was carried out by means of the Gold
program,37 version 3.0.1. The region of interest used by Gold
was defined in order to contain the residues within 10 A from the
original position of the ligand in the X-ray structures; the zinc
ion was set as possessing a trigonal-bipyramidal coordination.
The “allow early termination” option was deactivated while the
possibility for the ligand to flip ring corners was activated. The
remaining Gold default parameters were used, and the ligands
were submitted to 100 genetic algorithm runs by applying the
ChemScore fitness function. The best docked conformation was
taken into account.
2H), 7.50-8.11 (m, 10H), 8.81 (br s, 1H), 10.33 (br s, 1H). 13
C
NMR (DMSO-d6) δ: 20.01, 37.29, 55.25, 114.50, 126.90, 127.42,
127.95, 128.35, 128.55, 130.19, 130.65, 133.62, 138.20, 138.88,
140.63, 144.58, 159.82, 169.21. Anal. (C22H21NO5S) C, H, N.
MMP Inhibition Assays.31 Recombinant human progelati-
nase A (pro-MMP-2) and B (pro-MMP-9), MMP-16 and
MMP-14 catalytic domains were a kind gift of Prof. Gillian
Murphy (Department of Oncology, University of Cambridge,
UK). Pro-MMP-1, pro-MMP-8, pro-MMP-3, and pro-MMP-
13 were purchased from Calbiochem. Pro-MMP-12 was pur-
chased by R&D Systems.
Proenzymes were activated immediately prior to use with
p-aminophenylmercuric acetate (APMA 2 mM for 1 h at 37 °C
for MMP-2, MMP-1, and MMP-8, 1 mM for 1 h at 37 °C for
MMP-9 and MMP-13). Pro-MMP-3 was activated with trypsin
(5 μg/mL) for 30 min at 37 °C followed by soybean trypsin
inhibitor (SBTI) (62 μg/mL). Pro-MMP-12 was autoactivated
by incubating in FAB for 30 h at 37 °C.
For assay measurements, the inhibitor stock solutions
(DMSO, 10 mM) were further diluted, at 7 different concentra-
tions (0.01 nM-300 μM) for each MMP in the fluorometric assay
buffer (FAB: Tris 50 mM, pH=7.5, NaCl 150 mM, CaCl2 10
mM, Brij 35 0.05% and DMSO 1%). Activated enzyme (final
concentration 2.9 nM for MMP-2, 2.7 nM for MMP-9, 1.5 nM
for MMP-8, 0.3 nM for MMP-13, 5 nM for MMP-3, 1 nM for
MMP-14 cd, 15 nM for MMP-16 cd, 2.0 nM for MMP-1, and 1
nM for MMP-12) and inhibitor solutions were incubated in the
assay buffer for 4 h at 25 °C.32 After the addition of a 200 μM
solution of the fluorogenic substrate Mca-Arg-Pro-
Lys-Pro-Val-Glu-Nva-Trp-Arg-Lys(Dnp)-NH2 (Sigma) for
MMP-3 and Mca-Lys-Pro-Leu-Gly-Leu-Dap(Dnp)-Ala-Arg-
NH2 (Bachem) for all the other enzymes in DMSO (final
concentration 2 μM), the hydrolysis was monitored every 15 s
for 20 min, recording the increase in fluorescence (λex=325 nm,
NMR Studies. All NMR measurements were made at 300 K
on a Bruker Avance III Ultra Shield Plus 600 MHz spectrometer
provided with a two channel BBI probe. Hydrogen and carbon
assignment of 9 15 mM in DMSO was accomplished by a
series of 2D spectra, namely 2D-NOESY, 2D-COSY,
2D-TOCSY,
1H,13C-HSQC,
2D-1H,1H-TOCSY-1H,13C
HSQC, and 2D-1H,13C-HMBC.
The 15N-labeled catalytic domain of wt-MMP-12 was pur-
chased by PROTERA srl (cloned from human cDNA, expressed
in Escherichia coli) and provided at a concentration of 0.3 mM in
a buffer containing 20 mM Tris (pH 7.2), 10 mM CaCl2, 0.1 mM
ZnCl2, 0.3 M NaCl, 0.2 M AHA. The enzyme consists of the
catalytic domain of human MMP-12 (residues 106-263 Swis-
sprot accession P39900).
The NMR backbone assignment of MMP-12 (Bio-
MagResBank entry 6444) was further adjusted in our experi-
mental condition by 3D-1H,15N-NOESYHSQC using a mixing
time of 100 ms to avoid spin diffusion. Compound 9 was added
stepwise using few microliters of a concentrated solution in
DMSO. The experiments were repeated after concentrating the
protein to 1 mM in order to improve the signal/noise ratio and
better ascertain the presence of intermolecular NOEs.
Acknowledgment. Molecular graphics images were pro-
duced 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). We thank Bruker Biospin srl for
providing a 600 MHz Bruker Avance III Ultra Shield
Plus spectrometer, and the CBM (Consorzio per il Centro
di Biomedicina Molecolare S.c.r.l., AREA Science Park,
Basovizza, Trieste, Italy) for hosting the instrumentation.
λem=395 nm) using a Molecular Devices SpectraMax Gemini
XS plate reader. The assays were performed in triplicate in a
total volume of 200 μL per well in 96-well microtiter plates
(Corning, black, NBS). Control wells lack inhibitor. The MMP
inhibition activity was expressed in relative fluorescent units
(RFU). Percent inhibition was calculated from control reactions
without the inhibitor. IC50 was determined using the formula:
Vi/Vo = 1/(1 þ [I]/ IC50), where Vi is the initial velocity of
substrate cleavage in the presence of the inhibitor at concentra-
tion [I] and Vo is the initial velocity in the absence of the
inhibitor. Results were analyzed using SoftMax Pro software33
and GraFit software.34
Supporting Information Available: Table reporting the HPLC
purity analysis and the combustion analysis data of the final
products. Additional experiments and dose-response curves
for proving the dependence of inhibitory activity on preincuba-
tion time. This material is available free of charge via the
Internet at http://pubs.acs.org.
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