N-aryl sulfonyl homocysteine hydroxamate inhibitors of matrix metalloproteinases: Further probing of the S1, S1′, and S2′ pockets

Article abstract of DOI:10.1021/jm010097f

A series of N-arylsulfonyl S-alkyl homocysteine hydroxamic acids were synthesized with variations in three subsites corresponding to P₁, P₁′, and P₂′. Enzyme assays with a variety of MMPs revealed activity at the low nanomolar level.

Full text of DOI:10.1021/jm010097f

3066
J . Med. Chem. 2001, 44, 3066-3073
Articles
N-Ar yl Su lfon yl Hom ocystein e Hyd r oxa m a te In h ibitor s of Ma tr ix
Meta llop r otein a ses: F u r th er P r obin g of th e S1, S1′, a n d S2′ P ock ets
Stephen Hanessian,* Nicolas Moitessier, Ce´cile Gauchet, and Martin Viau
Department of Chemistry, Universite´ de Montre´al, C.P. 6128, Succursale Centre-Ville, Montre´al, Que´bec H3C 3J 7, Canada
Received March 3, 2001
A series of N-arylsulfonyl S-alkyl homocysteine hydroxamic acids were synthesized with
variations in three subsites corresponding to P1, P1′, and P2′. Enzyme assays with a variety of
MMPs revealed activity at the low nanomolar level.
In tr od u ction
The matrix metalloproteinases (MMPs) are zinc-
containing proteases including collagenases, stromel-
ysins, and gelatinases. Their key role in the proteolysis
of extracellular matrix has fostered intensive efforts
worldwide in search of effective inhibitors with a
potential for therapeutic applications in a number of
diseases such as arthritis and cancer.1-4 Consequently,
several MMP inhibitors were discovered during the past
decade, some of which having reached an advanced
clinical trial. Derivatives of succinic acid, as exemplified
by Marimastat (1)5 or Ro32-3555 (2),6 and more recently
N-arylsulfonyl R-aminohydroxamic acid derivatives,
such as CGS 27023A (3)7 or AG3340 (4),8 have been
extensively investigated (Figure 1).
In general, hydroxamic acid analogues have been
significantly more effective than the corresponding
carboxylic acids, no doubt due to the presence of a zinc
F igu r e 1. Selected MMP inhibitors.
atom at the active site of these enzymes.9 Extensive
X-ray and NMR studies of inhibitor/MMP complexes
have provided the basis for a better understanding of
important binding interactions between functional groups
present in the inhibitors and their corresponding sub-
sites in the enzymes.10-15 These elegant studies have
paved the way to effectively design new analogues with
potentially more favorable interactions with the en-
zymes.
As part of our studies on the design and synthesis of
inhibitors of MMPs,16,17 we have reported that N-
substituted D,L-homocysteine hydroxamic acids 5 and
6 exhibit potent and broad enzymatic activity (Figure
2).18 In view of this encouraging result, we thought it
F igu r e 2. Leads from the previous study.
necessary to expand the nature of functional groups at
the P1, P1′, and P2′ sites of the inhibitors as functional
probes for S1, S1′, and S2′ pockets of stromelysin and
Resu lts a n d Discu ssion
Molecu la r Mod elin g a n d Design . As a prelude to
the SAR study, we planned to validate the crucial
interactions between 6 and stromelysin-1 (MMP-3)
based on available information in the literature.9 Using
the Goodford program GRID,19-23 we successfully lo-
cated, among others, the highly hydrophobic S1 pocket
surrounded by Tyr-155, His-166, and Tyr-168 (Figure
related MMPs. The goal was to see if chemical modifica-
tions at a given site would enhance or diminish inhibi-
tory activity in general, but more specifically whether
selectivity for a given MMP could be achieved.
* To whom correspondence should be addressed. Fax: (514) 343-
5728. Tel: (514) 343-6738. E-mail: stephen.hanessian@umontreal.ca.
10.1021/jm010097f CCC: $20.00 © 2001 American Chemical Society
Published on Web 08/04/2001

N-Aryl Sulfonyl Homocysteine Hydroxamate Inhibitors of MMPs J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 19 3067
Sch em e 1a
F igu r e 3. Proposed model of 6/MMP-3 from docking simula-
tion.
3). This result combined with reported structures of
MMP complexes with sulfonamide-based inhibitors10
brought out insights into the possible binding mode.
Docking of compound 6 in the binding site of MMP-3
and further refinement of the complex provide the
a
(a) BrCH2CO2t-Bu, DIPEA, MeCN, 58%; (b) PMP-SO2Cl,
pictorial model shown in Figure 3: the S1 pocket is
occupied by the S-benzyl group. In an effort to check
the proposed model, docking with AutoDock,24 which we
proved to be reliable on MMP systems,25 was performed.
The enhanced enzymatic activity of 6 compared to CGS-
27023A,7 possibly due to a more favored P1/S1 interac-
tion, instigated the exploration of other hydrophobic
substituents at this site. Since compound 5 proved to
possess excellent nanomolar to subnanomolar activity
against most of the MMPs,15 we also explored replace-
ments for the diphenylmethyl amide group,26 presum-
ably occupying the shallow S2′ site in MMP-3.10 Finally
the p-methoxyphenyl sulfone moiety occupying the P1′
site was also replaced with other sulfones in an effort
to assess its contribution.
pyridine, 67%; (c) MeONa/MeOH then BnBr, 82%; (d) TFA,
CH2Cl2, 68%; (e) R1NH2, EDC, HOBt, NMM, CH2Cl2, 79-98%; (f)
(for 12d , 12e, 12f, 12i, 12j, 12k ) LiOH, THF/H2O (26-96%) or
(for 12a , 12b, 12c, 12g, 12h ) LiI, pyridine, reflux (79-83%); (g)
TrONH2, EDC, HOBt, NMM, CH2Cl2, 59-95%; (h) TFA 5%,
CH2Cl2.
Sch em e 2a
Syn th esis. We followed a procedure similar to the
one previously reported,18 which allowed the use of a
common precursor to access the three groups of com-
pounds as illustrated in Scheme 1. D,L-Homocysteine
thiolactone was monoalkylated and the product N-
sulfonylated to afford 8. The thiolactone ring was then
opened with sodium methoxide and the resulting thi-
olate was trapped in situ with benzyl bromide (Scheme
1). The tert-butyl ester was converted to the correspond-
ing acid which served as a key intermediate for further
functionalization. Amide formation with a variety of
amines in a parallel array led to the methyl esters 11a -
k . Subsequent saponification of the methyl esters led
to the corresponding acids which were individually
converted to the hydroxamic acid by coupling with
O-trityl hydroxylamine followed by TFA-mediated depro-
tection. In our previous synthesis of 6, we had alluded
to the surprisingly difficult deprotection of an O-tert-
butyl hydroxamate ester with TFA.18,27 In the present
synthesis, we used the O-trityl hydroxamate instead,
which was smoothly converted to the hydroxamic acid
with TFA. A set of N,N-disubstituted amide derivatives,
represented as structures 5a -k , was rapidly assembled.
We next turned our attention to the preparation of a
set of N-aryl sulfonyl hydroxamic acids based on the
original structure 6. The synthesis started with sequen-
tial reductive amination of D,L-homocysteine thiolactone
and sulfonylation yielding 15 (Scheme 2). Base-cata-
a
(a) i-PrCHO, Et3N, NaBH4, 73%; (b) PMP-SO2Cl, pyridine,
80%; (c) MeONa/MeOH then RBr, 20-99%; (d) i: MeONa/MeOH
then BnBr, 20-99%; ii: H2O2, 75%; (e) NH2OH, KOH, MeOH, 60-
70%; (f) (for d) i: LiOH, H2O/THF, 81%; ii: TrONH2, EDC, HOBt,
THF, 65%; iii: TFA, CH2Cl2, 50%.
lyzed methanolysis of the thiolactone ring in 15 followed
by trapping of the resulting thiolate methyl ester with
different electrophiles led to a series of S-alkylated
esters. Treatment of the methyl esters with the potas-
sium salt of hydroxylamine afforded the corresponding
hydroxamic acids.28 In one case, we proceeded via a
stepwise formation of the O-trityl hydroxamate followed
by cleavage with TFA. In this manner, we were able to
prepare a set of N-arylsulfonyl S-alkyl homocysteine
hydroxamic acids related to 6 in which the P1 site was

3068 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 19
Hanessian et al.
Sch em e 3a
Ta ble 2. Variations in the P1 Site of 6
IC50 (nM)a
compd MMP1 MMP2 MMP3 MMP9 MMP13
R
b
-SCH2C6H5
6a
6b
104
396
297
126
188
19610 1810
384
nt
0.7
4.22 10.3
3.78
6.3
3.2
0.7
<0.1
0.9
3.2
3.0
1.0
149
7
12
11.8
5.3
16.2
5.1
407
a
-SO2CH2C6H5
-SCH2C6H4-3-(OMe) 6c
-SCH2(3-pyridyl)
-SCH2(3-thiophene)
-SCH2(C6F5)
-Hb
(a) R-SO2Cl, pyridine or Et3N, CH2Cl2, 44-71%; (b) MeONa/
5.7
6.2
5.4
705
45
20
MeOH then BnBr, 41-81%; (c) NH2OH, KOH, MeOH, 31-62%.
6d
6e
6f
6g
6h
Ta ble 1. Variation of P2′ in 5
53
30
96
nt
-SCH2C6H4-
0.2
4-(C6H9)b
-CH2C6H4-
6i
nt
49
29
8
20
4-(OCH2C6H5)b
a
b
See Experimental Section for enzyme assay details. See ref
18. nt, not tested.
amide side chain caused only moderate changes in the
potency, the cyclohexylmethyl analogue 5c was more
potent than the corresponding analogue 5a , exhibiting
picomolar inhibition against MMP-9. Adding an extra
cyclohexyl (5c-f) resulted in loss of activity against
MMP-2 and MMP-9, but the dipyridylmethyl amide 5e
was as potent as the monopyridyl analogue 5b. Intro-
ducing a morpholine amide (5c-k ) diminished the
binding activity. The fairly bulky cyclohexylmethyl
amide group in 5c seemed to best accommodate the
binding site of MMP-2 and MMP-9. These results
suggested that the amide substituents in 5 presumed
to occupy the shallow S2′ site in MMP-3,10 allowing
considerable diversity for hydrophobic aryl groups in the
case of MMP-1 and MMP-13 as well. The high selectivity
for analogue 5c toward MMP-9 compared to other
MMPs is noteworthy. Shape complementarity is known
to be a crucial feature in enzyme-inhibitor interac-
tions.30
IC50 (nM)a
R
compd MMP1 MMP2 MMP3 MMP9 MMP13
NHCH2C6H5
NHCH2(3-pyridyl)
NHCH2(cC6H11
NHCH(C6H5)2
NHCH(2-pyridyl)2
NHCH(cC6H11
NHCH2C6H4-
4-(OMe)
NHCH2C6H3-
3,5-(OMe)2
5a
5b
5c
5d
5e
5f
51
20
nt
nt
39
nt
0.7
1.2
0.30
2.3
0.4
1.6
2.2
nt
3.7
1.1
nt
0.2
0.2
0.01
0.5
0.2
0.5
1.1
nt
4.5
0.4
nt
)
)
8.82
0.7
1.88
0.2
2
5g
19
0.9
0.7
5h
5i
36
49
1.6
1.2
1.0
4.7
2.5
1.6
0.6
0.3
0.2
2.3
1.6
0.8
NHCH2C6H2-
2,4,6-(OMe)3
NH(cC6H11
N(CH2CH2)2O
)
5j
5k
16
532
16
195
29
23
a
See Experimental Section for enzyme assay details. nt, not
tested.
The results of the next series (6a -i) consisting of
variations in the P1 arylthioethyl appendage is shown
in Table 2. As previously mentioned, the hydrophobic
region of the S1 pocket is a few ångstro¨ms away and
required solvent exposed groups as spacers on the
inhibitors.10 The depth of this pocket was probed using
S-benzyl (6a ), p-phenylbenzyl (6h ), or benzyloxybenzyl
(6i) groups that clearly shows the spacial requirements
of this pocket. Indeed, the proposed AutoDock model for
MMP-3 showed that the S1 pocket presents roughly a
half-sphere shape filled up by the benzyl group of 6a .
Thus, lengthening the side chain as in 6h and 6i led to
a loss of activity. We then studied the solvent exposed
nature of this subsite by replacing the S-benzyl groups
in 6a by groups capable of interacting with a polar
environment. The sulfone 6b, the thiophene 6e, and the
pyridyl 6d analogue were less active than 6a , while the
pentafluorophenyl analogue 6f was devoid of activity.
Variations at the P1 site were more effective for MMP-9
than for MMP-2, compared to changes at the P2′ site.
Table 3 shows the inhibition data relative to the last
series (6j-l) in which five different aryl groups were
chosen for their shape and their electronic properties.
modified with a variety of substituted S-benzyl append-
ages.
Variations in the N-arylsulfonyl group occupying the
P1′ subsite was the basis of another set of analogues
related to 6 as shown in Scheme 3. In this case, we were
able to convert the esters into the hydroxamic acids
directly by treatment with potassium or sodium salt of
hydroxylamine.29
Biologica l Assa ys. All compounds were tested in
vitro as racemic mixtures for the inhibition of stromel-
ysin 1 (MMP-3), gelatinase-A (MMP-2), gelatinase-B
(MMP-9), collagenase 1 (MMP-1), and collagenase 3
(MMP-13). The data are summarized in Tables 1, 2, and
3.
The results of introducing diversity at the S2′ pocket
with the set of substituted amides 5a -k are shown in
Table 1. As expected, the hydroxamic acid derivatives
were found to be roughly a 100 times more active than
the corresponding carboxylic acids. All compounds
exhibited broad spectrum inhibition in the nanomolar
range. Although it appeared that the variations of the

N-Aryl Sulfonyl Homocysteine Hydroxamate Inhibitors of MMPs J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 19 3069
Ta ble 3. Variations in the P1′ Site of 6
a solution of 7 (12.0 g, 51.7 mmol) in pyridine (150 mL) was
added p-methoxybenzene sulfonyl chloride (16.0 g, 1.5 equiv)
at 0 °C. The resulting mixture was stirred overnight at room
temperature. The mixture was then concentrated, taken up
in CH2Cl2, washed successively with water, 0.1 N HCl, 0.1 N
NaHCO3, water, and brine, dried over Na2SO4, then concen-
trated in vacuo. The residue afforded, after flash chromatog-
raphy (CH2Cl2/MeOH, 99:1), 8 (13.8 g, 67%): IR (neat/NaCl)
IC50 (nM)a
compd MMP1 MMP2 MMP3 MMP9 MMP13
1745.7, 1705.9, 1596.5, 1579.4 cm-1 1H NMR (300 MHz,
;
CDCl3) δ 7.81 (d, 2H, J ) 8.9 Hz), 6.94 (d, 2H, J ) 8.9 Hz),
4.65 (dd, 1H, J ) 6.8, 13.2 Hz), 4.05 (d, 1H, J ) 18.4 Hz), 3.84
(s, 3H), 3.40 (d, 1H, J ) 18.4 Hz), 3.34-3.16 (m, 2H), 2.64 (m,
1H), 2.20 (ddd, 1H, J ) 5.1, 7.2, 13.0 Hz), 1.45 (s, 9H); 13C
NMR (75 MHz, CDCl3) δ 203.7, 169.2, 163.8, 131.8, 130.5,
114.8, 83.0, 67.7, 56.3, 47.9, 30.4, 28.7, 27.5; LRMS (FAB, NBA,
m/z, %) 402 (5) (M + H+), 373 (6), 346 (100), 318 (25), 300 (15),
171 (64), 146 (35), 130 (63), 114 (43); HRMS calcd for C17H24O6-
NS2 402.10449 (M + H+), found 402.10610. 13C NMR (75 MHz,
CDCl3) δ 203.7, 169.2, 163.8, 131.8, 130.5, 114.8, 83.0, 67.7,
56.3, 47.9, 30.4, 28.7, 27.5; LRMS (FAB, NBA, m/z, %) 402 (5)
(M + H+), 373 (6), 346 (100), 318 (25), 300 (15), 171 (64), 146
(35), 130 (63), 114 (43); HRMS calcd for C17H24O6NS2 402.10449
(M + H+), found 402.10610.
R
-C6H4-4-(OMe)b
-C6H4-4-(Br)
6a
6j
104
164
1450
nt
0.7
16
1.6
4.1
0.7 <0.1
12
14
3.2
3.8
151
4.2
90
4.3
0.5
2.5
-C6H4-4-(OC6H5) 6k
-C6H4-4-(C6H5)b
6l
a
b
See Experimental Section for enzyme assay details. See ref
18. nt, not tested.
The 4-diphenyl analogue 6l18 was less active than the
more flexible 4-phenoxyphenyl analogue 6k . Indeed,
previous work showed that flexible groups fit better in
29,31
the presently probed S1′ pocket.
In conclusion, we have prepared and tested the in
vitro MMP inhibitory activity of a series of acyclic
N-arylsulfonyl homocysteine hydroxamates while prob-
ing the spacial and electronic requirements of different
subsites. Low nanomolar and subnanomolar inhibition
was found in several derivatives, which are being
further evaluated for optimal bioavailablity and related
tests. Variations at the P1, P1′, and P2′ sites revealed
that in each case inhibition of MMP-9 was most effective
among the MMPs tested.
4-Ben zylsu lfan yl-2-[ter t-bu toxycar bon ylm eth yl-(4-m eth -
oxy-ben zen esu lfon yl)-a m in o]-bu tyr ic Acid Meth yl Ester
(9). To a solution of thiolactone 8 (1.67 g, 4.16 mmol) in MeOH
(20 mL) was added sodium methoxide freshly prepared from
Na° (122 mg, 1.28 equiv) in MeOH (13 mL). After stirring for
30 min, benzyl bromide (0.50 mL, 1.0 equiv) was added. The
mixture was stirred for 16 h then concentrated, taken up in
EtOAc, filtrated, and finally concentrated in vacuo. The residue
was chromatographed (CH2Cl2/MeOH, 99:1) to afford 9 (1.78
g, 82%); IR (neat/NaCl) 1746.9, 1596.4 cm-1 1H NMR (300
;
MHz, CDCl3) δ 7.84 (d, 2H, J ) 8.9 Hz), 7.30-7.22 (m, 5H),
6.95 (d, 2H, J ) 8.9 Hz), 4.52 (dd, 1H, J ) 5.5, 8.8 Hz), 4.13
(d, 1H, J ) 18.4 Hz), 3.84 (s, 3H), 3.83 (d, 1H, J ) 18.4 Hz),
3.66 (s, 2H), 3.51 (m, 3H), 2.58 (m, 1H), 2.46 (m, 1H), 1.96 (m,
1H), 1.82 (m, 1H), 1.46 (s, 9H); 13C NMR (100 MHz, CDCl3) δ
171.7, 169.3, 163.7, 138.8, 131.4, 130.7, 129.5, 129.1, 127.6,
114.6, 82.6, 58.6, 56.2, 52.9, 47.5, 36.5, 30.7, 28.6, 28.2; LRMS
(FAB, NBA, m/z, %) 523 (9) (M + H+), 468 (14), 408 (8), 296
(14), 227 (14), 206 (14), 171 (29), 91 (100), 57 (45); HRMS calcd
for C25H33O7NaNS2 546.15961 (M + Na+), found 546.15830.
4-Ben zylsu lfa n yl-2-[ca r boxym eth yl-(4-m eth oxy-ben ze-
n esu lfon yl)-a m in o]-bu tyr ic Acid Meth yl Ester (10). To a
solution of 9 (1.78 g, 3.41 mmol) in CH2Cl2 (10 mL) was added
TFA (10 mL). After being stirred for 4 h, the mixture was
concentrated and chromatographed (CH2Cl2/MeOH, 49:1) to
afford 10 (1.09 g, 68%); IR (neat/NaCl) 3444.7, 1783.7, 1643.7,
1596.1, 1578.6 cm-1; 1H NMR (300 MHz, CDCl3) δ 7.81 (d, 2H,
J ) 8.9 Hz), 7.34-7.24 (m, 5H), 6.97 (d, 2H, J ) 8.9 Hz), 4.58
(dd, 1H, J ) 5.4, 8.8 Hz), 4.20 (d, 1H, J ) 18.8 Hz), 4.00 (d,
1H, J ) 18.8 Hz), 3.87 (s, 3H), 3.67 (s, 2H), 3.55 (s, 3H), 2.51
(m, 1H), 2.41 (m, 1H), 1.99 (m, 1H), 1.83 (m, 1H); 13C NMR
(100 MHz, CDCl3) δ 174.1, 171.4, 163.2, 137.9, 129.9, 128.7,
128.4, 126.9, 114.0, 57.9, 55.5, 52.4, 45.8, 35.8, 29.9, 27.2;
LRMS (FAB, NBA, m/z, %) 468 (20) (M + H+), 307 (22), 154
(100), 137 (64), 136 (67); HRMS calcd for C21H25O7NaNS2
490.09702 (M + Na+), found 490.09650.
Gen er a l P r oced u r e for th e P r ep a r a tion of 11a -k . To
a solution of 10 (0.90-1.0 mmol in CH2Cl2 (10 mL) were added
EDC (1.4 equiv), HOBt (1.4 equiv), and NMM (1.8 equiv). After
the mixture was stirred for 20 min, the appropriate amine (2.0
equiv) was added, and the resulting mixture was stirred for
15 h. The mixture was dissolved with CH2Cl2 and successively
washed with water, diluted NaHCO3, diluted HCl water, and
brine, dried over Na2SO4, then concentrated in vacuo. The
residue was purified by flash chromatography to afford 11a -
k . For details, see Supporting Information.
Gen er a l P r oced u r e for th e P r ep a r a tion of 12a -k .
Meth od A. To a solution of 11 (0.4-0.6 mmol) in refluxing
pyridine (6 mL) was added anhydrous lithium iodide (4.0
equiv). The reflux was maintained for 7 h, and then the
solution was cooled to room temperature, concentrated in
Exp er im en ta l Section
Ch em istr y. Solvents were distilled under positive pressure
of dry nitrogen before use and dried by standard methods; THF
and ether, from K/benzophenone; CH2Cl2 and toluene from
CaCl2. All commercially available reagents were used without
further purification. All reactions were performed under
nitrogen atmosphere. NMR (1H, 13C) spectra were recorded on
AMX-300 and ARX-400 spectrometers in CDCl3 or CD3OD
with tetramethylsilane as the internal standard. Low- and
high-resolution mass spectra were recorded on VG Micromass,
AEI-MS 902, or Kratos MS-50 spectrometers using fast atom
bombardement (FAB) or electrospray techniques. Optical
rotations were recorded on a Perkin-Elmer 241 polarimeter
in a 1 dm cell at ambient temperature. Analytical thin-layer
chromatography was performed on Merck 60F254 precoated
silica gel plates. Visualization was performed by UV or by
development using KMnO4 or FeCl3 solutions. Flash column
chromatography was performed using (40-60 µm) silica gel
at increased pressure. Melting points recorded were uncor-
rected. For 13C NMR and other analytical data, see Supporting
Information.
(2-Oxo-tetr ah ydr o-th ioph en -3-ylam in o)-acetic Acid ter t-
Bu tyl Ester (7). To a solution of d,L-homocysteine thiolactone
hydrochloride (12.0 g, 78.1 mmol) in MeCN (150 mL) was
added DIPEA portionwise (28.6 mL, 2.1 equiv) at 0 °C. After
complete dissolution, tert-butyl bromoacetate (15.9 g, 1.1 equiv)
was added dropwise at 0 °C. The resulting mixture was stirred
at room temperature overnight then concentrated. The residue
was taken up in EtOAc, and the precipitate was filtered off.
The organic solution was concentrated in vacuo then chro-
matographed (CH2Cl2/MeOH 99:1) to afford 7 (18.0 g, 58%):
IR (neat/NaCl) 3332.8, 1731.9, 1698.0 cm-1 1H NMR (300
;
MHz, CD3OD) δ 3.51 (dd, 1H, J ) 6.7, 11.5 Hz), 3.40 (m, 2H),
3.30 (m, 2H), 2.57 (m, 1H), 1.95 (m, 1H), 1.46 (s, 9H); 13C NMR
(75 MHz, CD3OD) δ 209.4, 172.3, 82.6, 67.2, 49.9, 33.0, 28.5,
28.3.
[(4-Me t h o x y -b e n ze n e s u lfo n y l)-(2-o x o -t e t r a h y d r o -
th iop h en -3-yl)-a m in o]-a cetic Acid ter t-Bu tyl Ester (8). To

3070 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 19
Hanessian et al.
vacuo, poured into 0.1 N HCl, and extracted with CHCl3. The
resulting organic layer was dried over Na2SO4 and concen-
trated in vacuo. The residue was purified by flash chromatog-
raphy (CH2Cl2/MeOH, 1:0 to 19:1). For details, see Supporting
Information.
Meth od B. To a solution of 11 (0.4-0.6 mmol) in THF (10
mL) was added lithium hydroxide (2.5 equiv) in water (mini-
mum amount). After being stirred for 16 h, the solution was
acidified using diluted HCl and concentrated in vacuo. The
residue was purified by flash chromatography (CH2Cl2/MeOH,
1:0 to 19:1).
(s, 1H), 4.34 (d, 1H, J ) 17.7 Hz), 4.28 (dd, 1H, J ) 7.2, 7.2
Hz), 4.14 (d, 1H, J ) 17.7 Hz), 3.82 (s, 3H), 3.56 (d, 1H, J )
13.0 Hz), 3.51 (d, 1H, J ) 13.0 Hz), 2.28-2.09 (m, 2H), 1.99
(m, 1H), 1.75 (m, 1H).
4-Ben zylsu lfa n yl-2-[[(d icycloh exylm eth yl-ca r ba m oyl)-
m eth yl]-(4-m eth oxy-ben zen esu lfon yl)-am in o]-N-h ydr oxy-
bu tyr a m id e (5f). Yield 66%, colorless oil; Rf ) 0.60 (CH2Cl2/
MeOH, 9:1); IR (neat/NaCl) 3299.7, 1660.5, 1596.1, 1577.3
cm-1; 1H NMR (300 MHz, CDCl3) δ 11.75 (s, 1H). 7.83 (d, 2H,
J ) 8.9 Hz), 7.37-7.21 (m, 5H), 6.96 (d, 2H, J ) 8.9 Hz), 5.71
(d, 1H, J ) 10.1 Hz), 3.97 (dd, 1H, J ) 5.7, 8.1 Hz), 3.84 (s,
3H), 3.83 (d, 1H, J ) 16.8 Hz), 3.70-3.58 (m, 4H), 2.52-2.20
(m, 3H), 1.88-1.48 (m, 11H), 1.48-1.40 (m, 2H), 1.32-0.86
(m, 10H).
Gen er a l P r oced u r e for th e P r ep a r a tion of 13a -k . To
a solution of 12 (0.2-0.4 mmol) in THF (6 mL) was successively
added EDC (1.3 equiv), HOBt (1.3 equiv), and NMM (1.5
equiv). After the mixture was stirred for 20 min, O-trityl
hydroxylamine (1.5 equiv) was added. After stirring for 16 h,
the mixture was dissolved with ether, washed with 0.1 N
NaHCO3, 0.1 N HCl, water, and brine. The solution was then
dried over Na2SO4 and concentrated in vacuo. Purification by
flash chromatography (hexanes/EtOAc, 1:0 then 3:2) afforded
13. For details, see Supporting Information.
Gen er a l P r oced u r e for th e P r ep a r a tion of 5a -k . A
solution of 13 (0.1-0.3 mmol) in CH2Cl2 (2 mL) saturated with
water was added a solution of 10% TFA in CH2Cl2 (1 mL) so
as to maintain a deep yellow color. The solution was stirred
for 1 h, then diluted by CH2Cl2, washed with 0.5 N NaHCO3
and then brine, dried over Na2SO4, and concentrated in vacuo.
The residue was purified by flash chromatography (CH2Cl2/
CH3OH, 1:0 to 19:1) or recrystallization to afford 5. For 13C
NMR and other analytical data see Supporting Information.
2-[(Ben zylcar bam oyl-m eth yl)-(4-m eth oxy-ben zen esu lfo-
n yl)-am in o]-4-ben zylsu lfan yl-N-h ydr oxy-bu tyr am ide (5a).
Yield 65%, colorless oil; Rf ) 0.45 (CH2Cl2/MeOH, 9:1); IR (neat/
NaCl) 3227.4, 1651.8, 1595.6, 1575.9 cm-1; 1H NMR (300 MHz,
CD3OD) δ 7.83 (d, 2H, J ) 8.9 Hz), 7.35-7.17 (m, 10H), 7.02
(d, 2H, J ) 8.9 Hz), 4.41 (s, 2H), 4.25 (dd, 1H, J ) 6.7, 7.2
Hz), 4.16 (d, 1H, J ) 17.5 Hz), 3.97 (d, 1H, J ) 17.5 Hz), 3.85
(s, 3H), 3.60 (d, 1H, J ) 13.0 Hz), 3.45 (d, 1H, J ) 13.0 Hz),
2.31-2.06 (m, 2H), 2.00 (m, 1H), 1.79 (m, 1H).
4-Ben zylsu lfa n yl-2-{(4-m eth oxy-ben zen esu lfon yl)-[(4-
m eth oxy-ben zylca r ba m oyl)-m eth yl]-a m in o}-N-h yd r oxy-
bu tyr a m id e (5g). Yield 63%, colorless oil; Rf ) 0.43 (CH2Cl2/
1
MeOH, 9:1); IR (neat/NaCl) 3365.5, 1651.1, 1595.3 cm-1; H
NMR (300 MHz, CD3OD) δ 7.82 (d, 2H, J ) 8.9 Hz), 7.28-
7.17 (m, 7H), 7.02 (d, 2H, J ) 8.9 Hz), 6.86 (d, 2H, J ) 8.9
Hz), 4.32 (s, 2H), 4.25 (dd, 1H, J ) 6.6, 7.5 Hz), 4.12 (d, 1H, J
) 17.5 Hz), 3.95 (d, 1H, J ) 17.5 Hz), 3.84 (s, 3H), 3.75 (s,
3H), 3.60 (d, 1H, J ) 13.1 Hz), 3.53 (d, 1H, J ) 13.1 Hz), 2.29-
2.12 (m, 2H), 1.99 (m, 1H), 1.77 (m, 1H).
4-Ben zylsu lfan yl-2-[[(3,5-dim eth oxy-ben zylcar bam oyl)-
m eth yl]-(4-m eth oxy-ben zen esu lfon yl)-am in o]-N-h ydr oxy-
bu tyr a m id e (5h ). Yield 82%, colorless oil; Rf ) 0.41 (CH2Cl2/
1
MeOH, 9:1); IR (neat/NaCl) 3182.5, 1654.2, 1597.0 cm-1; H
NMR (300 MHz, CDCl3) δ 7.81 (d, 2H, J ) 8.9 Hz), 7.31-7.18
(m, 5H), 6.92 (d, 2H, J ) 8.9 Hz), 6.47 (d, 2H, J ) 2.2 Hz),
6.34 (dd, 1H, J ) 2.2, 2.2 Hz), 4.45 (dd, 1H, J ) 5.5, 15.0 Hz),
4.32 (m, 2H), 3.90 (d, 1H, J ) 17.1 Hz), 3.81 (s, 3H), 3.73 (s,
6H), 3.71 (d, 1H, J ) 17.1 Hz), 3.60 (d, 1H, J ) 13.1 Hz), 3.53
(d, 1H, J ) 13.1 Hz), 2.38-2.10 (m, 3H), 1.70 (m, 1H).
4-Be n zylsu lfa n yl-2-{(4-m e t h oxy-b e n ze n e su lfon yl)-
[(2,4,6-tr im eth oxy-ben zylca r ba m oyl)-m eth yl]-a m in o}-N-
h yd r oxy-bu tyr a m id e (5i). Yield 49%, colorless oil; Rf ) 0.39
(CH2Cl2/MeOH, 9:1); IR (neat/NaCl) 3341.5, 3196.2, 1651.8,
1
1595.6 cm-1; H NMR (300 MHz, CD3OD) δ 7.80 (d, 2H, J )
8.9 Hz), 7.29-7.17 (m, 5H), 7.02 (d, 2H, J ) 8.9 Hz), 6.20 (s,
2H), 4.49 (d, 1H, J ) 13.5 Hz), 4.29 (d, 1H, J ) 13.5 Hz), 4.27
(dd, 1H, J ) 6.0, 7.1 Hz), 4.12 (d, 1H, J ) 16.8 Hz), 3.88 (d,
1H, J ) 16.8 Hz), 3.85 (s, 3H), 3.81 (s, 6H), 3.78 (s, 3H), 3.56
(d, 1H, J ) 13.0 Hz), 3.50 (d, 1H, J ) 13.0 Hz), 2.25-2.11 (m,
2H), 1.95 (m, 1H), 1.72 (m, 1H).
4-Ben zylsu lfa n yl-2-[cycloh exylca r b a m oylm et h yl-(4-
m et h oxy-b en zen esu lfon yl)-a m in o]-N-h yd r oxy-b u t yr a -
m id e (5j). Yield 65%, white solid; Rf ) 0.50 (CH2Cl2/MeOH,
9:1); IR (neat/NaCl) 3252.0, 1651.8, 1595.6 cm-1; 1H NMR (300
MHz, CD3OD) δ 7.85 (d, 2H, J ) 9.0 Hz), 7.34-7.17 (m, 5H),
7.05 (d, 2H, J ) 9.0 Hz), 4.25 (dd, 1H, J ) 5.4, 7.8 Hz), 4.05
(d, 1H, J ) 17.5 Hz), 3.88 (d, 1H, J ) 17.5 Hz), 3.85 (s, 3H),
3.64 (m, 1H), 3.62 (d, 1H, J ) 13.1 Hz), 3.57 (d, 1H, J ) 13.1
Hz), 2.30-2.11 (m, 2H), 2.01 (m, 1H), 1.92-1.68 (m, 5H), 1.68-
1.56 (m, 1H), 1.42-1.15 (m, 5H).
4-Ben zylsu lfa n yl-2-[(4-m eth oxy-ben zen esu lfon yl)-(2-
m or p h olin -4-yl-2-oxo-et h yl)-a m in o]-N-h yd r oxy-b u t yr a -
m id e (5k ). Yield 51%, colorless oil; Rf ) 0.38 (CH2Cl2/MeOH,
9:1); 1H NMR (300 MHz, CD2Cl2) δ 12.14 (s, 1H), 7.80 (d, 2H,
J ) 9.0 Hz), 7.38-7.20 (m, 5H), 6.98 (d, 2H, J ) 9.0 Hz), 4.28
(m, 1H), 4.25 (d, 1H, J ) 17.3 Hz), 3.85 (s, 3H), 3.76-3.55 (m,
7H), 3.49-3.31 (m, 2H), 2.40-2.27 (m, 3H), 1.78 (m, 1H).
3-Isobu tyla m in o-d ih yd r o-th iop h en -2-on e (14). To a so-
lution of D,L-homocysteine hydrochloride (0.201 g, 1.31 mmol)
in MeOH (5 mL) was added freshly distilled isobutyraldehyde
(0.24 mL, 2.62 mmol) at 0 °C. After complete dissolution, Et3N
(0.2 mL, 1.5 mmol) was added. The resulting mixture was
stirred at room temperature for 16 h and then cooled to 0 °C
before adding NaBH3CN (0.172 g, 5.48 mmol) portionwise over
45 min. The mixture was stirred for a further 30 min at 0 °C,
quenched with water, and extracted with ether, and the
organic layer was washed with brine, dried over Na2SO4, and
concentrated in vacuo. The residue was chromatographed
(CH2Cl2/MeOH, 99:1) to afford 14 (0.226 g, 77%), Rf ) 0.60
4-Ben zylsu lfa n yl-2-((4-m et h oxy-b en zen esu lfon yl)-{-
[(p yr id in -3-ylm eth yl)-ca r ba m oyl]-m eth yl}-a m in o)-N-h y-
d r oxy-bu tyr a m id e (5b). Yield 61%, colorless oil; Rf ) 0.28
(CH2Cl2/MeOH, 9:1); IR (neat/NaCl) 3217.0, 1667.4, 1595.1,
1574.2 cm-1; 1H NMR (300 MHz, CD3OD) δ 8.79 (m, 1H), 8.67
(d, 1H, J ) 5.2 Hz), 8.44 (d, 1H, J ) 8.1 Hz), 7.90 (dd, 1H, J
) 5.2, 8.1 Hz), 7.83 (d, 2H, J ) 8.8 Hz), 7.30-7.18 (m, 5H),
7.05 (d, 2H, J ) 8.8 Hz), 4.61 (s, 2H), 4.25 (dd, 1H, J ) 7.4,
7.4 Hz), 4.16 (d, 1H, J ) 17.6 Hz), 4.01 (d, 1H, J ) 17.6 Hz),
3.85 (s, 3H), 3.60 (d, 1H, J ) 13.4 Hz), 3.54 (d, 1H, J ) 13.4
Hz), 2.30-2.10 (m, 2H), 1.96 (m, 1H), 1.73 (m, 1H).
4-Ben zylsu lfa n yl-2-[[(cycloh exylm et h yl-ca r b a m oyl)-
m eth yl]-(4-m eth oxy-ben zen esu lfon yl)-am in o]-N-h ydr oxy-
bu tyr a m id e (5c). Yield 69%, colorless oil; Rf ) 0.51 (CH2Cl2/
1
MeOH, 9:1); H NMR (300 MHz, CDCl3) δ 11.61 (s, 1H), 7.82
(d, 2H, J ) 9.0 Hz), 7.40-7.20 (m, 5H), 6.95 (d, 2H, J ) 9.0
Hz), 6.31 (m, 1H), 4.36 (dd, 1H, J ) 5.9, 7.1 Hz), 3.85 (m, 1H),
3.84 (s, 3H), 3.73-3.58 (m, 3H), 3.20-3.04 (m, 2H), 2.55-2.18
(m, 3H), 1.82-1.60 (m, 6H), 1.58-1.40 (m, 1H), 1.35-1.10 (m,
2H), 1.03-0.80 (m, 3H).
2-[[(Ben zh yd r yl-ca r ba m oyl)-m eth yl]-(4-m eth oxy-ben -
zen esu lfon yl)-a m in o]-4-b en zylsu lfa n yl-N-h yd r oxy-b u -
tyr a m id e (5d ). Yield 64%, white solid; Rf ) 0.55 (CH2Cl2/
1
MeOH, 9:1); H NMR (300 MHz, CDCl3) δ 11.15 (s, 1H), 7.82
(d, 2H, J ) 9.0 Hz), 7.40-7.15 (m, 15H), 7.01 (d, 1H, J ) 8.0
Hz), 6.85 (d, 2H, J ) 9.0 Hz), 6.20 (d, 1H, J ) 8.0 Hz), 4.34
(dd, 1H, J ) 4.0, 8.0 Hz), 3.88 (d, 1H, J ) 17.0 Hz), 3.81 (s,
3H), 3.67 (d, 1H, J ) 17.0 Hz), 3.62 (d, 1H, J ) 13.8 Hz), 3.55
(d, 1H, J ) 13.8 Hz), 2.40-2.12 (m, 3H), 1.70 (m, 1H).
4-Ben zylsu lfa n yl-2-[{[(d i-p yr id in -2-yl-m eth yl)-ca r ba m -
oyl]-m eth yl}-(4-m eth oxy-ben zen esu lfon yl)-a m in o]-N-h y-
d r oxy-bu tyr a m id e (5e). Yield 72%, white solid; Rf ) 0.31
1
(CH2Cl2/MeOH, 9:1); H NMR (300 MHz, CD3OD) δ 8.59 (m,
2H), 8.02 (m, 2H), 7.83 (d, 2H, J ) 9.0 Hz), 7.81-7.71 (m, 2H),
7.50 (m, 2H), 7.30-7.16 (m, 5H), 7.01 (d, 2H, J ) 9.0 Hz), 6.39

N-Aryl Sulfonyl Homocysteine Hydroxamate Inhibitors of MMPs J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 19 3071
(CH2Cl2/MeOH, 1:9); IR (neat/NaCl) 3228.0, 2956.2, 1698.5
cm-1; 1H NMR (300 MHz, CD3OD) δ 3.40 (m,1H), 3.25 (m, 2H),
2.60 (m, 1H), 2.56 (dd, 1H, J ) 6.6, 10.8 Hz), 2.42 (dd, 1H, J
) 6.6, 10.8 Hz), 2.00 (m, 1H), 1.65 (m, 1H), 0.92 (d, 3H, J )
6.6 Hz), 0.88 (d, 3H, J ) 6.6 Hz).
(dd, 1H, J ) 7.9, 12.9 Hz), 2.10 (m, 2H), 1.98 (m, 1H), 1.43 (m,
2H), 0.90 (d, 3H, J ) 6.5 Hz), 0.84 (d, 3H, J ) 6.5 Hz).
3-(4-Meth oxy-ben zen esu lfon yl)-5-m eth yl-2-(2-p en ta flu -
or oph en ylm eth ylsu lfan yl-eth yl)-h exan oic Acid Hydr oxya-
m id e (6f). Method D, yield 65%, Rf ) 0.45 (CH2Cl2/MeOH, 9:1);
N-Isobu tyl-4-m eth oxy-N-(2-oxo-tetr a h yd r o-th iop h en -
3-yl)-ben zen esu lfon a m id e (15). To a solution of 14 (0.4 g,
2.31 mmol) in anhydrous pyridine (5 mL) was added p-
methoxybenzene sulfonyl chloride (0.717 g, 3.47 mmol) at 0
°C. The resulting mixture was stirred for 16 h at room
temperature. The mixture was then concentrated, taken up
in CH2Cl2, washed successively with water, 1 N HCl, saturated
NaHCO3, water, and brine, dried over Na2SO4, then concen-
trated in vacuo. The residue afforded, after flash chromatog-
raphy (CH2Cl2/MeOH, 99:1), 15 (0.722 g, 67%), Rf ) 0.83
(MeOH/CH2Cl2, 1:9); IR (neat/NaCl) 2960.8, 1707.0, 1497.6,
1355.8 cm-1; 1H NMR (300 MHz, CDCl3) δ 7.81 m (d, 2H, J )
9.8 Hz), 6.98 (d, 2H, J ) 9.9 Hz), 4.67 (m, 1H), 3.85 (s, 3H),
3.25 (m, 2H), 3.05 (dd, 1H, J ) 6.8, 14.5 Hz), 2.74 (dd, 1H, J
) 6.8, 14.5 Hz), 2.54 (m, 1H), 2.42 (m,1H), 1.95 (m, 1H), 0.88
(d, 3H, J ) 6.5 Hz), 0.86 (d, 3H, J ) 6.5 Hz).
Gen er a l P r oced u r e for th e P r ep a r a tion of 16a -f. To a
solution of 15 (0.4 mmol) in MeOH (3 mL) was added at 0 °C
sodium methoxide freshly prepared from Na (0.6 mmol) in
MeOH (2 mL). After the mixture was stirred for 30 min, the
appropriate alkyl bromide (0.8 mmol) was added. The mixture
was stirred for 16 h at room temperature then concentrated,
taken up in EtOAc, filtered, and concentrated in vacuo. The
residue was chromatographed to afford 16. For details, see
Supporting Information.
IR (neat/NaCl) 3334.5, 2964.2, 1678.1, 1596.6, 1505.3 cm-1
;
1H NMR (300 MHz, CD3OD) δ 7.77 (d, 2H, J ) 7.2 Hz), 7.25
(d, 2H, J ) 7.2 Hz), 4.38 (bs, 1H), 3.86 (s, 3H), 3.51 (m, 2H),
3.16 (dd, 1H, J ) 7.5, 11.5 Hz), 2.96 (dd, 1H, J ) 7.5, 11.5
Hz), 2.24 (m, 2H), 1.96 (m, 1H), 1.51 (m, 1H), 0.91 (d, 3H, J )
4.7 Hz), 0.87 (d, 3H, J ) 4.7 Hz).
N-Hyd r oxy-2-[isobu tyl-(4-m eth oxy-ben zen esu lfon yl)-
a m in o]-4-(p yr id in -3-ylm eth ylsu lfa n yl)-bu tyr a m id e (6d ).
To a solution of the hydroxamate (60 mg, 0.084 mmol) in CH2-
Cl2 (10 mL) was added TFA (1 mL, 10% in CH2Cl2) dropwise
until a yellow color persists. The mixture was stirred for a
further 2 h, then was extracted with CH2Cl2, washed with 0.1
N NaHCO3 and brine, dried over Na2SO4, and concentrated
in vacuo. Flash chromatography (CH2Cl2/MeOH, 1:0 to 9:1)
afforded 6d (20 mg, 50%), Rf ) 0.27 (CH2Cl2/MeOH, 9:1); IR
(neat/NaCl) 3165.1, 2996.4, 1669.3, 1596.7 cm-1; 1H NMR (300
MHz, CDCl3) δ 9.55 (bs, 1H), 8.48 (m, 3H), 7.68 (d, 2H, J )
8.8 Hz), 7.35 (m, 1H), 6.95 (d, 2H, J ) 8.8 Hz), 4.35 (t, 1H, J
) 7.1 Hz), 3.86 (s, 3H), 3.55 (s, 2H), 3.12 (dd, 1H, J ) 5.3,
13.9 Hz), 2.92 (dd, 1H, J ) 5.3, 13.9 Hz), 2.21 (m, 2H), 1.95
(m, 1H), 0.98 (m, 3H, J ) 6.6 Hz), 0.85 (d, 3H, J ) 6.6 Hz).
N-Hyd r oxy-2-[isobu tyl-(4-m eth oxy-ben zen esu lfon yl)-
am in o]-4-(th ioph en -3-ylm eth ylsu lfan yl)-bu tyr am ide (6e).
Method D, yield 60%, Rf ) 0.45 (CH2Cl2/MeOH 9:1); IR (neat/
NaCl) 3335.6, 2961.4, 1674.2, 1595.2 cm-1; 1H NMR (300 MHz,
CD3OD) δ 9.50 (bs, 1H), 7.78 (d, 2H, J ) 7.8 Hz), 7.28 (m,
2H), 7.00 (d, 2H, J ) 7.8 Hz), 6.98 (m, 1H), 4.32 (t, 3H, J )
7.3 Hz), 3.88 (s, 3H), 3.51 (d, 1H, J ) 5.7 Hz), 3.49 (d, 1H, J
) 5.7 Hz), 3.11 (dd, 1H, J ) 7.2, 11.2 Hz), 2.90 (dd, 1H, J )
7.2, 11.2 Hz), 2.23 (m, 2H), 1.90 (m, 1H), 0.94 (d, 3H, J ) 6.2
Hz), 0.84 (d, 3H, J ) 6.2 Hz).
Gen er a l P r oced u r e for t h e P r ep a r a t ion of 6a -f.
Meth od C. To a solution of 16 (0.1-0.2 mmol) in MeOH (1
mL) were added NH2OH‚HCl (2.0 equiv) and NaOMe, 25% in
MeOH solution (3.0 equiv). After being stirred for 16 h at room
temperature, the solution was acidified with 0.5 N HCl to pH
3, and the organic layer was extracted with EtOAc, dried over
Na2SO4, concentrated in vacuo, and chromatographed (CH2-
Cl2/MeOH, 9:1) to afford 6. For 13C NMR and other analytical
data, see Supporting Information.
4-Ben zylsu lfa n yl-2-[(4-br om o-ben zen esu lfon yl)-isobu -
tyl-a m in o]-N-h yd r oxy-bu tyr a m id e (6j). Yield 62%, color-
less oil; Rf ) 0.37 (CH2Cl2/MeOH, 9:1); IR (neat/NaCl) 3345.4,
2961.3, 1679.6, 1157.0 cm-1; 1H NMR (300 MHz, CDCl3) δ 7.67
(m, 5H), 7.31 (d, 2H, J ) 7.8 Hz), 7.27 (d, 2H, J ) 7.8 Hz),
4.35 (t, 1H, J ) 6.8 Hz), 3.52 (dd, 1H, J ) 5.7, 12.6 Hz), 3.12
(dd, 1H, J ) 5.7, 12.6 Hz), 2.22 (m, 2H), 2.11 (m, 1H), 1.91 (m,
2H), 0.89 (d, 3H, J ) 6.5 Hz), 0.85 (d, 3H, J ) 6.5 Hz).
Meth od D. To a solution of 16 (0.1-0.2 mmol) was added
NH2OK prepared from KOH and NH2OH (8 equiv, 0.87 M in
MeOH). After being stirred for 16 h at room temperature, the
solution was acidified with diluted HCl, and the organic layer
was extracted with EtOAc and brine, dried over Na2SO4, then
concentrated in vacuo. Flash chromatography (CH2Cl2/MeOH
19:1) afforded 6.
4-Ben zylsu lfa n yl-N-h yd r oxy-2-[isob u t yl-(4-p h en oxy-
b en zen esu lfon yl)-a m in o]-bu t yr a m id e (6k ). Yield 80%,
colorless oil; Rf ) 0.42 (CH2Cl2/MeOH, 9:1); IR (neat/NaCl)
4-Ben zylsu lfa n yl-N-h yd r oxy-2-[isob u t yl-(4-m et h oxy-
ben zen esu lfon yl)-am in o]-bu tyr am ide (6a). Method C, yield
50%, Rf ) 0.20 (CH2Cl2/MeOH, 9:1); IR (neat/NaCl) 334.3,
3337.5, 2962.9, 1676.8, 1583.5, 1488.1 cm-1 1H NMR (300
;
MHz, CD3OD) δ 10.92 (bs,1H), 7.74 (d, 2H, J ) 5.8 Hz), 7.39
(t, 2H, J ) 5.5 Hz), 7.26 (m, 5H), 7.03 (t, 3H, J ) 5.0 Hz), 4.34
(t, 1H, J ) 6.6 Hz), 3.49 (dd, 2H, J ) 7.5, 13.6 Hz), 3.15 (dd,
1H, J ) 10.0, 14.2 Hz), 2.88 (dd, 1H, J ) 10.0, 14.2 Hz), 2.22
(m, 1H), 2.1 (m, 2H), 1.8 (m, 1H), 1.4 (m, 1H), 0.90 (d, 3H, J )
6.6 Hz), 0.85 (d, 3H, J ) 6.6 Hz).
1
2962.1, 1677.0 cm-1; H NMR (300 MHz, CDCl3) δ 9.45 (bs,
1H), 7.82 (d, 2H), 7.23 (m, 5H), 6.95 (d, 2H), 4.35 (t, 1H, J )
7.5 Hz), 3.88 (s, 3H), 3.54 (d, 1H, J ) 13.0 Hz), 3.42 (d, 1H, J
) 13,0 Hz), 3.12 (dd, 1H, J ) 9.8, 13.8 Hz), 2.88 (dd, 1H, J )
4.9, 13.8 Hz), 2.07 (m, 1H), 1.88 (m, 2H), 0.89 (d, 3H, J ) 6.6
Hz), 0.85 (d, 3H, J ) 6.6 Hz).
Molecu la r Mod elin g. Molecular modeling studies were
performed using software programs from InsightII (Molecular
Simulations, 1995, San Diego, CA) using a modified AMBER
force field.32,33 The starting MMP-3 crystallographic structure,
retrieved from the Brookhaven Protein Data Bank (code 1HFS
in the PDB), was relaxed according to standard procedures,
and a GRID binding interaction evaluation was performed.
Probes used: Dry, O), O, N1, 2 planes per angstrom (NPLA
) 2), Flexibility of the side chains (MOVE ) 1). This brought
out the key interaction sites among which the highly hydro-
phobic S1 pocket. Compound 6 was manually docked in the
binding site of MMP-3 according to the sites shown up above
and to the reported structures of similar inhibitors cocrystal-
lized with MMPs.10,34,35 The resulting complex was then energy
minimized. AutoDock study was also performed. Grids sur-
rounding the binding site were computed (61 × 61 points, 0.375
Å spacing) with AutoGrid and used for subsequent docking
study with AutoDock using the Lamarckian genetic algorithm
as search protocol. The conformations from AutoDock and
N-Hyd r oxy-2-[isobu tyl-(4-m eth oxy-ben zen esu lfon yl)-
am in o]-4-ph en ylm eth an esu lfon yl-bu tyr am ide (6b). Method
D, yield 60%, Rf ) 0.80 (CH2Cl2/MeOH, 9:1); IR (neat/NaCl)
3321.6, 2926.1, 1680.9, 1591.0, 1492.2 cm-1 1H NMR (300
;
MHz, CD3OD) δ 9.45 (bs, 1H), 7.75 (d, 2H, J ) 8.8 Hz), 7.48
(m, 5H), 6.98 (d, 2H, J ) 8.8 Hz), 4.51 (t, 1H, J ) 7.2 Hz),
4.18 (s, 2H), 3.84 (s, 3H), 3.05 (dd, 1H, J ) 5.6, 14.4 Hz), 2.90
(dd, 1H, J ) 5.6, 14.4 Hz), 2.75 (m, 1H), 2.64 (m, 1H), 2.35 (m,
1H), 1.82 (m, 1H), 0.85 (d, 3H, J ) 6.6 Hz), 0.83 (d, 3H, J )
6.6 Hz).
N-Hyd r oxy-2-[isobu tyl-(4-m eth oxy-ben zen esu lfon yl)-
a m in o]-4-(3-m eth oxy-ben zylsu lfa n yl)-bu tyr a m id e (6c).
Method D, yield 65%, Rf ) 0.72 (CH2Cl2/MeOH, 9:1); IR (neat/
NaCl) 3332.2, 2961.5, 1677.1, 1596.8 cm-1; 1H NMR (300 MHz,
CD3OD) δ 9.54 (br s, 1H), 7.76 (d, 2H, J ) 8.8 Hz), 7.27 (m,
1H), 6.99 (d, 2H, J ) 8.8 Hz), 6.84 (m, 3H), 4.35 (t, 1H, J )
7.0 Hz), 3.86 (s, 3H), 3.81 (s, 3H), 3.48 (d, 1H, J ) 12.9 Hz),
3.38 (d, 1H, J ) 12.9 Hz), 3.12 (dd, 1H, J ) 7.9, 12.9 Hz), 2.90

3072 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 19
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Biologica l Assa y. Human purified MMPs were purchased
or acquired. MMP-2 gelatinase A and MMP-9 gelatinase B
from Boehringer Mannheim (Meylan, France), MMP-3 stromel-
ysin 1 from Valbiotech (Paris, France). All enzymes were
activated by APMA (4-aminophenylmercuric acetate). Inhibi-
tion of MMP-3 was quantified by using the peptidomimetic
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between amino acids Gly and Cys, was used. The fluorescent
cleavage products were measured with a cytofluorometer
(Cytofluor 2350, Millipore, PerSeptive Systems, Voisins le
Bretonneux, France) equipped with a combination of 340 and
440 nm filters for excitation and emission, respectively. The
IC50 values were the average of at least two determinations
with a standard deviation of less than (30%.
Ack n ow led gm en t. We thank the NSERC of Canada
for financial assistance through the Medicinal Chem-
istry Chair Program. We thank Dr. G. Tucker, Labora-
toires Servier, France, for the biological assays and
Servier for financial support.
Su p p or tin g In for m a tion Ava ila ble: Full characteriza-
tion (1H,13C NMR, LRMS, and HRMS) of all the compounds.
This material is available free of charge via the Internet at
http://pubs.acs.org.
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