Design, synthesis, and biological evaluation of matrix metalloproteinase inhibitors derived from a modified proline scaffold

Article abstract of DOI:10.1021/jm9904699

The synthesis and structure-activity relationship (SAR) studies of a series of proline-based matrix metalloproteinase inhibitors are described. The data reveal a remarkable potency enhancement in those compounds that contain an sp² center at the C-4 carbon of the ring relative to similar, saturated compounds. This effect was noted in compounds that contained a functionalized oxime moiety or an exomethylene at C-4, and the potencies were typically < 10 nM for MMP-3 and < 100 nM for MMP- 1. Comparisons were then made against compounds with similar functionality where the C-4 carbon was reduced to sp³ hybridization and the effect was typically an order of magnitude loss in potency. A comparison of compounds 14 and 34 exemplifies this observation. An X-ray structure was obtained for a stromelysin-inhibitor complex which provided insights into the SAR and selectivity trends observed within the series. In vitro intestinal permeability data for many compounds was also accumulated.

Full text of DOI:10.1021/jm9904699

5426
J . Med. Chem. 1999, 42, 5426-5436
Design , Syn th esis, a n d Biologica l Eva lu a tion of Ma tr ix Meta llop r otein a se
In h ibitor s Der ived fr om a Mod ified P r olin e Sca ffold
Menyan Cheng, Biswanath De, Neil G. Almstead, Stanislaw Pikul, Martin E. Dowty, Charles R. Dietsch,
C. Michelle Dunaway, Fei Gu, Lily C. Hsieh, Michael J . J anusz, Yetunde O. Taiwo, and Michael G. Natchus*
Procter and Gamble Pharmaceuticals, 8700 Mason-Montgomery Road, Mason, Ohio 45040
Tomas Hudlicky and Martin Mandel†
Department of Chemistry, University of Florida, Gainesville, Florida 32611
Received September 16, 1999
The synthesis and structure-activity relationship (SAR) studies of a series of proline-based
matrix metalloproteinase inhibitors are described. The data reveal a remarkable potency
enhancement in those compounds that contain an sp2 center at the C-4 carbon of the ring
relative to similar, saturated compounds. This effect was noted in compounds that contained
a functionalized oxime moiety or an exomethylene at C-4, and the potencies were typically
<10 nM for MMP-3 and <100 nM for MMP-1. Comparisons were then made against compounds
with similar functionality where the C-4 carbon was reduced to sp3 hybridization and the effect
was typically an order of magnitude loss in potency. A comparison of compounds 14 and 34
exemplifies this observation. An X-ray structure was obtained for a stromelysin-inhibitor
complex which provided insights into the SAR and selectivity trends observed within the series.
In vitro intestinal permeability data for many compounds was also accumulated.
In tr od u ction
An important subset of proteinase enzymes consists
of the matrix metalloproteinases (MMPs) which are
characterized by the presence of a zinc ion in the active
site. This family of enzymes is composed of 17 known
members, and new members have been identified with
startling regularity in recent years. These endo pro-
teinases are involved primarily in tissue remodeling and
include the collagenases, gelatinases, and stromelysins.
Under physiological conditions, the proteolytic activity
of the MMPs provides a means for the degradation of
the extracellular matrix tissue to allow for normal
remodeling events such as tissue turnover, wound
healing,1 and angiogenesis.2 This activity is regulated
by the tissue inhibitors of matrix metalloproteases
(TIMPS). Several pathological conditions (osteoarthri-
tis,3 rheumatoid arthritis,4 periodontal disease,5 mul-
F igu r e 1.
tiple sclerosis,6 tumor metastasis7) have been linked to
overexpression of the MMPs. The inhibition of the MMP
on succinamide type structures, our recent effort has
enzymes thus represents an attractive target for me-
been directed toward heterocyclic sulfonamide ana-
dicinal intervention in degenerative conditions where
logues. In the course of our studies, we explored scaf-
excessive tissue remodeling plays a key role. This
folds which could be readily prepared from commercially
opportunity has stimulated extensive research in both
available materials, and we observed a striking potency
academic and industrial laboratories toward the devel-
boost in compounds derived from cis-hydroxy-D-proline
opment of small molecule MMP inhibitors, and efforts
where the C-4 center was oxidized to form an sp2 center.
along these lines have been extensively reviewed.8
The account below will focus primarily on the SAR and
Our group and others have been developing MMP
selectivity profiles for compounds which display this
inhibitors for a number of years, and numerous scaffolds
unexpected potency enhancement.
have been reported in the literature including succin-
amides9 (Marimastat), linear sulfonamides10 (CGS-
Ch em istr y
27023A), and heterocyclic sulfonamides11 (AG-3340)
(Figure 1). While much of our earlier work was based
All compounds were derived from cis-4-hydroxy-D-
proline 1 as exemplified in Scheme 1. A routine se-
quence of esterification, sulfonation, and oxidation
provided ketones of type 2 which served as pivotal
* To whom all correspondence should be addressed.
† Current address: Orgachem, Klapkova 96, 182 00 Praha 8, Czech
Republic.
10.1021/jm9904699 CCC: $18.00 © 1999 American Chemical Society
Published on Web 12/09/1999

Synthesis of Proline-Based MMP Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 26 5427
Sch em e 1a
a
Reagents and conditions: (a) MeOH, SOCl2; (b) ClSO2C6H4OnBu, dioxane, H2O, Et3N; (c) J ones oxidation; (d) NH2OH, KOH, MeOH;
(e) MeONH2‚HCl, MeOH, dioxane, NaOAc; (f) NaCNBH3, MeOH, concd HCl; (g) 1N-aminomorpholine, MeOH, dioxane, NaOAc.
Sch em e 2a
intermediates for further elaboration. Treatment of 2
with excess basic hydroxylamine12 gave oxime hydrox-
amates of type 3 directly; alternatively, sequential
treatment with a controlled amount of alkoxy- or
aryloxyamine followed by treatment with hydroxyl-
amine provided functionalized oximes of type 4. The
oximes were isolated as mixtures of E and Z isomers
which could not be easily separated by HPLC. Hydra-
zones of type 6 were similarly prepared from ketone 2.
Treatment of 2 with a disubstituted hydrazine gave
hydrazone 5. Subsequent treatment with hydroxylamine
then provided the desired hydroxamate 6. These hy-
drazones were isolated and purified as variable mixtures
of E/ Z isomers, but the compounds tended to slowly
darken and decompose over time.
A number of conditions were investigated to reduce
the oxime functionality and convert C-4 from an sp2 to
an sp3 center. Both hydrogenation and hydride reduc-
tion conditions were investigated with limited success.
Hydride addition with cyanoborohydride and catalytic
HCl ultimately emerged as the most synthetically
efficient method for the transformation. The methoxyl-
amine 7 was prepared using this methodology and was
obtained as a mixture of diastereomers (2:1, unassigned,
not separated). The mixture was converted to the
corresponding hydroxamic acid with basic hydroxyl-
amine.
Direct analogues of the oxime moiety were sought
which also contain an sp2 carbon at C4, and thus various
olefinic type compounds were prepared as shown in
Scheme 2. The exomethylene moiety in compound 10
was introduced from keto-sulfonamide 8 using standard
Wittig conditions; however, the yield was generally
<25% and highly variable. This is likely due to a
propensity of the sulfonamide moiety to eliminate under
basic conditions since various elimination products were
observed in these reactions. Similar conditions were also
applied to the keto acid 9 to discourage such elimina-
tions, but yields turned out to be even lower. Better
results were achieved with the Boc-protected ketoproline
ester 12. Standard Wittig conditions were successful at
delivering the target olefination, albeit in a very modest
20% yield. Much better results were achieved under
equilibrating conditions via the Dauben-Conia modifica-
a
Reagents and conditions: (a) ClSO2C6H4OMe, dioxane, H2O,
Et3N; (b) J ones oxidation; (c) MeOH, SOCl2; (d) Ph3PCH3Br,
LiHMDS, THF; (e) NH2OH, MeOH; (f) Boc2O, acetone, H2O, Et3N;
(g) Ph3PCH3Br, THF, tert-amylOK, tert-amylOH; (h) TFA, CH2Cl2;
(i) ClSO2C6H4OMe, NEt3, CH2Cl2.
tion of the Wittig reaction.13 These conditions were
successful in delivering the desired olefination product
and worked especially well with stabilized ylides such
as PhCH2PPh3 and CNCH2PPh3; however, only the
exomethylene case was carried forward to properly
functionalized hydroxamates of type 11 for biological
testing. Other nucleophilic olefination methods were
also pursued without satisfactory results.14
Biologica l Resu lts a n d Discu ssion
SAR E ffect s of t h e Oxim e- a n d Hyd r a zon e-
Bea r in g P yr r olid in es. All compounds were assayed
in vitro for the inhibition of truncated collagenase-1
(MMP-1),15 gelatinase-A (MMP-2),15 stromelysin (MMP-
3),16 matrilysin (MMP-7),15 and collagenase-3 (MMP-
13).15 Several structure-activity relationships become
apparent upon inspection of the binding data. Particu-
larly noteworthy is the consistently high potency of the

5428 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 26
Cheng et al.
Ta ble 1. MMP Inhibition and Absorption Data for Oxime, Hydrazone, and Exomethylene Derivatives
MMP IC50 (nM)a
cmpd
Q
X
R
-1
17
119
10
22
13
26
109
3
3
81
39
20
74
26
24
75
32
127
184
293
90
155
329
1548
15
-2
0.2
0.2
<1
-3
-7
nd
307
82
1058
381
89
612
60
-13
% abs
13
3
14
15
16
4
17
18
19
20
21
22
23
24
25
26
27
28
29
6
-OH
-OH
-N
-N
-N
-N
-N
-N
-N
-N
-N
-N
-N
-N
-N
-N
-N
-N
-N
-N
-N
-N
-CH
-CH
-CH
-CH
-CH
-OMe
-OnBu
-OMe
-OEt
5
5
3
16
2
1
15
2
<1
5
0.2
0.3
<0.5
0.7
<1
0.3
<0.4
<0.4
0.8
<0.2
0.5
<0.5
0.4
<0.4
0.2
<0.1
0.4
0.1
29-35
2-5
78-81
23-42
nt
-OMe
-OMe
-OMe
-OMe
-OMe
-OMe
-OMe
-OMe
-OEt
0.3
<0.4
<1
-OnPr
-OnBu
28-36
nt
-OCH2CH2OMe
-OPh
-OC6H4F
-O-4-Pyr
-OnBu
0.5
<0.4
<0.4
0.3
<0.4
0.7
15-18
nt
50-68
10-17
13-19
nd
10-18
insol.c
nt
nd
nt
10-19
1
<24
922
39
2
9
-OtBu
-OtBu
-OtBu
-OtBu
-OiBu
-OPh
-OMe
263
294
785
1032
420
785
490
343
196
12300
2546
1082
>9994
53
-OnBu
0.5
11
5
<1
12
5
17
3
2
14
16
15
69
14
-O-4-Pyr
-OC6H4F
-OnBu
<0.4
<0.4
<0.4
nd
<0.4
nd
nd
nd
2
0.8
-O-4-Pyr
-OCH2Ph
-N-piperazine
-N-morpholine
-OnBu
-OnBu
<1
-OnBu
<1
11
30
31
32
33
-H
-H
-H
-H
-H
-OMe
4
1
46-66
nt
nt
nt
nt
-OnPr
-OnBu
0.7
11
<0.4
-OCH2CH2OMe
-OPh
9
0.4
a
b
See Experimental Section for enzyme assay details. Standard deviations were typically (60% of the mean or less. Absorption values
were predicted from in vitro rat ileum permeability studies (see Experimental Section). c Not soluble in aqueous buffer used in transport
studied. nt, not tested. nd, none detected.
oxime- and hydrazone-bearing compounds against MMPs
-2, -3, and -13 as seen in Table 1. All of these compounds
were single nanomolar or picomolar inhibitors of these
MMPs and demonstrated very little variation in potency
as functional groups were altered. MMPs -1 and -7
showed a broader range of SAR over the series and could
be manipulated somewhat to give compounds with
unique enzymatic profiles. The high potency observed
with this series seemed to be highly dependent on the
presence of a hydroxamic acid. Replacement of the
hydroxamic acid with a carboxylic acid resulted in
complete loss of potency (IC50 for all enzymes > 10 µM).
Variation of the aromatic sulfonamide substituent (R)
had moderate effects on the potency against MMP-1 and
tended to give compounds with lower potency as the
chain length of the substituent increased (compare 13
vs 3, 14 vs 4, and 22 vs 23). This is consistent with the
finding that this substituent fits into the P1′ pocket
which is much more shallow for MMP-1 and -7 than the
other enzymes of the family.17 Compounds with aryl
groups in this position such as 18 and 19 tended to
increase the potency for all enzymes and produce broad
spectrum inhibitors. Heterocyclic sulfonamides includ-
ing the pyridine in 20 were less potent against MMPs
-1 and -7 than the analogous aryl substituents.
substituents in 29 and 6. A modest decrease in potency
for MMPs -1, -3, and -7 was observed when the oxime
moiety was exchanged for an exomethylene group,
indicating that the heteroatoms do play a role in binding
with the enzyme. Particularly noteworthy is the incre-
mental increase in selectivity of MMP-3 over MMP-1
observed with the exomethylene-containing compounds
as the sulfonamide (R) substituent increased in chain
length. The ratio of MMP-1/MMP-3 increased from 6 to
10 to 21 as the substituents were changed from methyl
to n-propyl to n-butyl in compounds 11, 30, and 31,
respectively.
SAR Effects of a Red u ced sp 2 Cen ter on th e
P yr r olid in e Rin g. A very interesting observation was
made upon reduction of the oxime double bond to a
single bond to give compounds of the type shown in
Table 2. The binding potency observed with these
compounds decreased by roughly 1 order of magnitude
across the entire family of enzymes (compare 4 vs 7).
As with the oxime compounds, extending the chain
length of the S1′ substituent (R) tended to decrease the
potency of MMP-1. Modifications of the hydroxyl sub-
stituent (W) had little effect on the potency of this subset
of molecules as exemplified in the comparison of 34 vs
37. On the basis of these results, the presence of an sp2
center at C-4 of the pyrrolidine ring is apparently
responsible, at least in part, for the extremely potent
nature of the oxime-bearing molecules, and this may be
due to the influence of an sp2 center on the conformation
of the pyrrolidine ring.
The oxime hydroxyl substituents (Q) tended to exert
little influence on potency within the series, and the
enzymes seemed to tolerate hydroxy, alkoxy, and aryl-
oxy groups equally well. There appeared to be a slight
decrease in potency for MMP-1 with the hydrazone

Synthesis of Proline-Based MMP Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 26 5429
Ta ble 2. MMP Inhibition and Absorption Data for Substituted Hydroxylamine Derivatives
MMP IC50 (nM)a
compd
W
R
-1
-2
-3
-7
-13
% absb
34
7
35
36
37
-Me
-Me
-Me
-Me
-tBu
-OMe
212
790
4235
86
5
1
19
21
222
6
3393
2083
6724
1792
5235
3
2
8
12-15
14-17
nt
nt
nt
-OnBu
-OCH2CH2OMe
-OPh
-OMe
11
<0.4
4
<0.4
3
255
20
a
b
See Experimental Section for enzyme assay details. Standard deviations were typically (60% of the mean or less. Absorption values
were predicted from in vitro rat ileum permeability studies (see Experimental Section). nt, not tested.
carbonyl oxygen at 1.95 Å and the N-hydroxyl oxygen
at 1.89 Å. The hydroxamate nitrogen then contributes
a hydrogen bond to Ala-165 at 1.97 Å, and a final
hydrogen bond from Glu-202 anion to the hydroxyl
hydrogen at 2.69 Å completes the binding network.
The sulfonamide moiety also possesses favorable
binding interactions with the enzyme. A dominant
feature of this group is a strong hydrogen bond from
the R-configured sulfonamide oxygen which is within
bonding distance of both Leu-164 at 1.90 Å and Ala-
165 at 2.33 Å.20 The relative orientation of this oxygen
on the sulfonamide moiety allows for proper alignment
of the methoxyphenyl group which fits nicely into the
deep P1′ pocket where it experiences a very compatible
hydrophobic binding environment.
While the binding roles of the sulfonamide and
hydroxamate groups appear to be well explained by the
crystal structure in Figure 2, there seem to be no
obvious interactions which can explain the potency
increase that was observed with molecules possessing
an sp2 center at C-4. The oxime moiety appears to
extend into the S2′ pocket on the surface of the enzyme
which is a very hydrophobic environment. The flat SAR
in this part of the molecule tends to suggest that this
region of the molecule is contributing little to the overall
binding, so we speculate that the sp2 orientation at C-4
alters the conformation of the ring to a more efficiently
bound orientation.
F igu r e 2. Structure of stromelysin-14 complex.
In Vitr o Absor p tion . The relative peroral intestinal
absorption potential (% abs.) shown in Tables 1 and 2
were predicted from in vitro rat ileum transport stud-
ies.21 The results suggested that there was a general
trend toward lower absorption when the substituent on
the molecules became larger. Examples of this phenom-
enon can be seen in series 13 and 3, series 20, 24, and
27, and series 14, 15, 4, and 18. However, compound
20, a pyridyl derivative, showed a greater absorption
potential than would be anticipated based upon size
alone (compare to 18). A recent report has shown that
several pyridyl-substituted compounds have a greater
transcellular permeability than the corresponding phenyl-
substituted molecule by an, as yet, undetermined mech-
anism.22 This phenomenon is also supported when
comparing compounds 22 and 24. The addition of
hydrophilicity in the molecules also reduced the absorp-
tion potential as exemplified by comparing compounds
29 and 6. Two compounds, 23 and 27, showed no
F igu r e 3. Schematic diagram of binding interactions between
14 and truncated stromelysin.
Str u ctu r e of Str om elysin -In h ibitor Com p lex.18
A
crystal structure of the truncated stromelysin-14 com-
plex was obtained from a soaked crystal sample and
solved using the molecular replacement method and is
shown in Figure 2. This structure has provided insight
into the key enzyme-substrate interactions that were
playing a role in binding, and some of these are
represented schematically in Figure 3. Many of the
binding modes observed were consistent with others
which have been described in the literature.19 The
hydroxamic acid plays a dominant role in the enzyme-
substrate interactions, and this is due to a chorus of
closely knit interactions which work in harmony to
anchor the inhibitor. The ensemble is rooted by the
bidentate chelation with the Zn2+ ion to both the

5430 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 26
Cheng et al.
(ppm, δ units). Coupling constants are reported in units of
hertz (Hz). Splitting patterns are designated as s, singlet; d,
doublet; t, triplet; q, quartet; m, multiplet; br, broad. Low-
resolution mass spectra (MS) were recorded on a Micromass
Platform quadrupole mass spectrometer. Mass spectra were
acquired in either the positive or negative ion mode under
electrospray ionization (ESI). Combustion analyses were per-
formed internally.
Human synovial proMMP-3 was obtained from Dr. Hideaki
Nagase, University of Kansas Medical Center, Kansas City,
KS. Human fibroblast proMMP-1, human MMP-9, and human
recombinant MMP-7 catalytic domain were obtained from Dr.
Howard Welgus, J ewish Hospital, St. Louis, MO. Human
recombinant MMP-8 catalytic domain was obtained from Dr.
Harald Tschesche, University Bielefeld, Bielefeld, Germany.
Human recombinant proMMP-2 was purified from CHO cells
as described below. Human recombinant truncated MMP-3
and truncated MMP-1 were purified from Escherichia coli cells
as described below. Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2
was purchased from Bachem Bioscience, King of Prussia, PA.
Phenoxyphenylsulfonyl chloride23 and (pyrid-4-yl)oxyphenyl-
sulfonyl chloride24 were prepared following literature proce-
dures.
detectable transport, which may be attributed, in part,
to the low solubility of these molecules. In fact, signifi-
cant amounts of these two compounds partitioned into
and remained in the intestinal tissue during the experi-
ment. Moreover, there was some evidence that other
compounds from this general class were substrates for
the various concentration-dependent efflux systems
(including P-glycoprotein), encoded by the MDR1 gene,
present in the enterocytes of the intestinal tract.
Further characterization would be necessary to better
understand potential solubility, tissue partitioning be-
havior, and/or efflux mechanisms on overall peroral
absorption.
Con clu sion
In summary, we have described an extremely potent
series of heterocyclic MMP inhibitors which depend on
the presence of an sp2 configuration at the C-4 center
of the proline ring for added potency. The compounds
can be prepared through a brief synthetic sequence
which relies on cis-4-hydroxy-D-proline as a chiral
template. The resulting oxime- and exomethylene-
bearing inhibitors are very potent inhibitors for MMP-3
and -13 and are moderately selective against MMPs -1
and -7. Substitutions on the oxime portion of the
molecule exerted little influence on potency, while
substitutions on the sulfonamide portion of the molecule
produced a more pronounced effect and could be ma-
nipulated somewhat to alter selectivity profiles of
molecules. This is consistent with X-ray data which
shows that the sulfonamide portion of the molecule goes
into the P1′ pocket of the enzymes which is known to
be shallower for MMPs -1 and -7 than other enzymes
in the family. Studies into the relative absorption
potentials of various members of the series helped to
identify characteristics which serve to increase their
absorption potential.
Meth yl 1N-(4-n -Bu toxyp h en ylsu lfon yl-(4R)-h yd r oxy-
p yr r olid in e-(2R)-ca r boxyla te (2a ). cis-4-Hydroxy-D-proline
(14.8 g, 112.95 mmol) was mixed with water:dioxane (1:1, 90
mL), triethylamine (39.3 mL, 282 mmol), and 4-(dimethylami-
no)pyridine (1.3 g, 11.3 mmol). The 4-(n-butoxy)phenylsulfonyl
chloride (29.5 g, 118.6 mmol) was then added, and the mixture
was stirred for 14 h at room temperature. The mixture was
then concentrated and diluted with EtOAc and 1 N HCl. The
layers were separated, and the organic layer was washed twice
with 1 N HCl and once with brine, dried over MgSO4, filtered,
and evaporated to give 37.4 g of solid material which was
dissolved in MeOH (200 mL). Thionyl chloride (20 mL, 272
mmol) was added dropwise, and the resulting mixture was
stirred for 14 h. The mixture was then evaporated to dryness
to give a white solid which was sufficiently pure to carry
forward without purification. 1H NMR (CDCl3): δ 1.01 (t, J )
7.4 Hz, 3H), 1.38-1.52 (m, 2H), 1.66-1.82 (m, 2H), 2.01-2.20
(m, 2H), 2.72-2.94 (m, 1H), 3.24-3,36 (m, 1H), 3.44-3.58 (m,
1H), 3.58 (s, 3H), 3.98-4.08 (m, 2H), 4.12-4.19 (m, 2H), 6.98
(d, J ) 8.7 Hz, 2H), 7.80 (d, J ) 8.7 Hz, 2H). ESI MS: m/z
+
375 [M + NH4] +, 358.3 [M + H]
.
Exp er im en ta l Section
Meth yl 1N-(4-n -Bu toxyp h en ylsu lfon yl)-4-oxo-p yr r oli-
d in e-(2R)-ca r boxyla te (2). An 8 N solution of J ones reagent
was prepared (Oxidations in Organic Chemistry, p 273). The
alcohol 2a (40 g, 112 mmol) was dissolved in 300 mL of acetone
and cooled to 0 °C. J ones reagent was added (120 mL, 960
mmol) (color changed from orange-red to green), and the
mixture was stirred at room temperature for 14 h. The reaction
mixture was diluted with water and extracted three times with
EtOAc. The organic layers were washed three times with water
and once with brine, dried over magnesium sulfate, and
evaporated. The product was crystallized from EtOAc to give
Gen er a l. All commercial chemicals and solvents are reagent
grade and were used without further purification unless
otherwise specified. The following solvents and reagents have
been abbreviated: tetrahydrofuran (THF), ethyl ether (Et2O),
dimethyl sulfoxide (DMSO), ethyl acetate (EtOAc), dichloro-
methane (DCM), trifluoroacetic acid (TFA), dimethylform-
amide (DMF), methanol (MeOH). All reactions except those
in aqueous media were carried out with the use of standard
techniques for the exclusion of moisture. Reactions were
monitored by thin-layer chromatography on 0.25 mm silica gel
plates (60F-254, E. Merck) and visualized with UV light, iodine
vapors, or 5% phosphomolybdic acid in 95% ethanol. Final
compounds were typically purified either by flash chromatog-
raphy on silica gel (E. Merck, 40-63 mm) or by preparative
reverse-phase high-pressure liquid chromatography (RP-
HPLC) using a Waters model 4000 Delta Prep instrument
equipped with a Waters Symmetry preparative steel column
(C-18, 19 m × 300 mm) as the stationary phase. The mobile
phase employed 0.1% formic acid with acetonitrile as the
organic modifier. Both isocratic and linear gradient methods
were used as appropriate, and the flow rate was 20 mL/min.
Analytical purity was assessed by RP-HPLC using a Waters
600 system equipped with a diode array spectrometer (ı` range
200-400 nm). The stationary phase was a Waters Symmetry
C-18 column (4.6 mm × 200 mm). The mobile phase employed
0.1% formic acid with acetonitrile as the organic modifier and
a flow rate of 1.0 mL/min. Analytical data is reported as
retention time, tR, in minutes (% acetonitrile, time, flow rate).
1H NMR spectra were recorded on a Varian Unity-300
instrument. Chemical shifts are reported in parts per million
1
the desired product as a solid. H NMR (CDCl3): δ 1.01 (t, J
) 7.3 Hz, 3H), 1.43-1.63 (m, 2H), 1.74-1.98 (m, 2H), 2.56 (dd,
J ) 8.1, 3.1 Hz, 1H), 2.80 (dd, J ) 18.2, 9.2 Hz, 1H), 3.68 (s,
3H), 3.78-3.81 (m, 2H), 4.02 (t, J ) 6.6 Hz, 2H), 4.78 (dd, J )
9.1, 3.1 Hz, 1H), 7.01 (d, J ) 9.0 Hz, 2H), 7.79 (d, J ) 9.0 Hz,
2H). ESI MS: m/z 378.3 [M + Na]+, 356.3 [M + H]+.
N-Hyd r oxy 1N-(4-n -Bu toxyp h en yl)su lfon yl-4-(Z,E-N-
h yd r oxyim in o)p yr r olid in e-(2R)-ca r boxa m id e (3). The ke-
toester 2 (0.29 g, 0.8 mmol) was mixed with basic NH2OH
solution (3 mL, 5.1 mmol, 1.7 M in methanol, solution prepared
as described in Fieser and Fieser, Vol. 1, p 478) and stirred
overnight at room temperature. The mixture was then acidified
with 1 N HCl and then extracted three times with EtOAc. The
combined EtOAc layer was dried (MgSO4) and concentrated
under reduced pressure. The crude product was purified by
reverse-phase preparative HPLC (60A40B, A, 95% H2O, 5%
acetonitrile, 0.1% formic acid; B, 80% acetonitrile, 20% H2O;
19 × 300 mm waters SymmetryPrep C18 column) to give 100
mg (35% yield) of a white foaming solid 3:4 mixture of E:Z

Synthesis of Proline-Based MMP Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 26 5431
1
isomers (unassigned). H NMR (DMSO-d6): δ 0.93 (t, J ) 7.8
oxime 4a (0.95 g, 2.47 mmol) in methanol (10 mL) was added
sodium cyanoborohydride (2.2 g, 35 mmol) followed by slow
addition of concentrated HCl (1 mL). The mixture was stirred
overnight at room temperature, and then the pH was adjusted
to 10 with 1 N NaOH. The reaction mixture was extracted
three times with EtOAc. The combined EtOAc layer was
washed with brine, dried over MgSO4, and concentrated under
reduced pressure to give an oil which was purified by column
eluting with EtOAc/hexane (1:1) to give 0.66 g (71% yield) of
the desired product as a 2:1 mixture of R:â epimers (un-
Hz, 3H), 1.37-1.48 (m, 2H), 1.64-1.78 (m, 2H), 2.21-2.60 (m,
3H), 3.52-3.60 (m, 1H), 3.68-3.75 (m, 1H), 3.92-4.12 (m, 3H),
7.16 (d, J ) 9.0 Hz, 2H), 7.79 (d, J ) 9.0 Hz, 2H), 9.02 (br s,
1H), 10.86 (s, 1H). ESI MS: m/z 389 [M + NH4]+, 372 [M +
H]+. HRMS: MH+ calcd for C15H22N3O6S, 372.1229; found,
372.1228.
Meth yl 1N-(4-n -Bu toxyp h en yl)su lfon yl-4-(Z,E-N-m eth -
oxyim in o)p yr r olid in e-(2R)-ca r boxyla te (4a ). To a solution
of ketoester 2 (2.0 g, 5.63 mmol) in dioxane (15 mL), methanol
(10 mL), and water (5 mL) were added methoxylamine
hydrochloride (1.41 g, 16.9 mmol) and sodium acetate (5.23 g,
63.9 mmol). The mixture was stirred overnight at room
temperature and diluted with water. The reaction mixture was
extracted three times with EtOAc. The combined EtOAc layer
was washed with brine, dried over MgSO4, and concentrated
under reduced pressure to give 1.9 g (88%) of the desired
material as a 2:3 mixture of E:Z isomers (unassigned). 1H NMR
(CDCl3): δ 1.02 (t, J ) 7.5 Hz, 3H), 1.44-1.59 (m, 2H), 1.76-
1.85 (m, 2H), 2.72-2.98 (m, 2H), 3.68 (s, 3H, major isomer),
3.71 (s, 3H, minor isomer), 3.84 (s, 3H), 4.05 (t, J ) 6.4 Hz,
2H), 4.12-4.18 (m, 2H), 4.53-4.58 (m, 1H), 6.99 (d, J ) 9.0
Hz, 2H), 7.79 (d, J ) 9.0 Hz, 2H). ESI MS: m/z 402 [M +
NH4]+, 385 [M + H]+.
1
assigned). H NMR (CDCl3): δ 1.00 (td, J ) 7.3, 1.3 Hz, 3H),
1.40-1.54 (m, 2H), 1.73-1.81 (m, 2H), 2.02-2.19 (m, 2H),
3.28-3.37 (m, 2H), 3.41-3.47 (m, 3H), 3.49-3.63 (m, 1H), 3.72
(s, 1H), 3.75 (s, 2H), 4.01 (t, J ) 6.4 Hz, 2H), 4.21-4.33 (m,
1H), 5.17 (d, J ) 7.1 Hz, 1/3H), 5.76 (J ) 9.0 Hz, 2/3H), 6.97
(d, J ) 9.2 Hz, 2H), 7.78 (d, J ) 9.2 Hz, 2H). ESI MS: m/z
387 [M + H]+.
N-Hyd r oxy 1N-(4-n -Bu toxyp h en yl)su lfon yl-4-(N-m eth -
oxya m in o)p yr r olid in e-(2R)-ca r boxa m id e (7). The methyl
ester 7a (0.3 g, 0.78 mmol) was treated with NH2OH (6.4 mL,
10.8 mmol, 1.7 M in methanol, solution prepared as described
in Fieser and Fieser, Vol. 1, p 478) and stirred overnight at
room temperature. The reaction mixture was concentrated
under reduced pressure and purified by column eluting with
CH2Cl2:CH3OH (95:5) to give 0.17 g (56% yield) of a white solid
as a 2:1 mixture of R:â epimers (unassigned). 1H NMR (DMSO-
d6): δ 0.97 (t, J ) 7.2 Hz, 3H), 1.39-1.47 (m, 2H), 1.63-1.79
(m, 3H), 1.81-2.00 (m, 1H), 2.98-3.15 (m, 2H), 3.22-3.42 (m,
3H), 3.81-3.93 (m, 1H), 4.04-4.14 (m, 3H), 6.38 (d, J ) 6.7
Hz, 1/3H), 6.84 (d, J ) 9.0 Hz, 2/3H), 7.08-7.19 (m, 2H), 7.73-
7.81 (m, 2H), 8.97 (s, 1/3H), 9.02 (s, 2/3H), 10.69 (s, 1/3H), 10.79
(s, 2/3H). ESI MS: m/z 388 [M + H]+. HRMS: MH+ calcd for
C16H26N3O6S, 388.1542; found, 388.1559.
N-Hyd r oxy 1N-(4-n -Bu toxyp h en yl)su lfon yl-4-(Z,E-N-
m et h oxyim in o)p yr r olid in e-(2R)-ca r b oxa m id e (4). The
methyl ester 5a (1.0 g, 4.95 mmol) was mixed with NH2OH
(13 mL, 23.4 mmol, 1.7 M in methanol, solution prepared as
described in Fieser and Fieser, Vol. 1, p 478) and stirred
overnight at room temperature. The mixture was then acidified
with 1 N HCl and extracted three times with EtOAc. The
combined EtOAc layer was dried (MgSO4) and concentrated
under reduced pressure. The crude product was purified over
a column of flash silica eluting with CH2Cl2:CH3OH (95:5) to
give 0.69 g (69%) of a white solid as a 2:3 mixture of E:Z
(1N)-4-Meth oxyph en ylsu lfon yl-(2R)-car bom eth oxy-(4R)-
h yd r oxyp yr r olid in e (8a ). cis-Hydroxy-D-proline 1 (50 g, 0.38
mol) was dissolved in water:dioxane (1:1, 300 mL) with
triethylamine (135 mL, 0.96 mol). 4-Methoxyphenylsulfonyl
chloride (87 g, 0.42 mol) was added along with 4-(dimethyl-
amino)pyridine (4.6 g, 0.038 mol), and the mixture was stirred
14 h at room temperature. The mixture was then concentrated
and diluted with EtOAc. Layers were separated, and the
organic layer was washed 2× with 1 N HCl, 1× with brine,
dried over MgSO4, filtered, and evaporated to give 83 g of solid
material which was dissolved in MeOH (500 mL). Thionyl
chloride (50 mL) was added dropwise and the resulting
mixture stirred for 14 h. The mixture was then evaparated to
dryness and triturated with CHCl3 to give 85 g (69%) of white
solid which was sufficiently pure to carry forward without
1
isomers (unassigned). H NMR (DMSO-d6): δ 0.93 (t, J ) 7.3
Hz, 3H), 1.38-1.46 (m, 2H), 1.68-1.80 (m, 2H), 2.32-2.59 (m,
2H), 3.70 (s, 1H), 3.77 (s, 2H), 3.88-4.12 (m, 4H), 4.26-4.38
(m, 1H), 7.16 (d, J ) 9.0 Hz, 2H), 7.78 (dd, J ) 9.0, 3.4 Hz,
2H), 9.02 (s, 1H), 10.86 (s, 1H). ESI MS: m/z 403 [M + NH4]+,
386 [M + H]+. Anal. (C16H23N3O6S) C, H, N.
Meth yl 1N-(4-n -Bu toxyp h en yl)su lfon yl-4-(Z,E-N-m or -
p h olin eim in o)p yr r olid in e-(2R)-ca r boxyla te (5). To the
ketoester 2 (1.5 g, 4.2 mmol) in dioxane (20 mL), methanol (5
mL), and water (5 mL) were added 1-aminomorpholine (0.52
mL, 5.04 mmol) and sodium acetate (3.4 g, 42 mmol). The
mixture was stirred overnight at room temperature and
diluted with water. The reaction mixture was extracted three
times with EtOAc. The combined EtOAc layer was washed
with brine, dried over MgSO4, and concentrated under reduced
pressure to give 1.8 g, (100%) of solid as a 2:3 mixture of E:Z
isomers (unassigned). 1H NMR (CDCl3): δ 1.01 (t, J ) 7.5 Hz,
3H), 1.42-1.58 (m, 2H), 1.78-1.88 (m, 2H), 2.68-2.76 (m, 4H),
2.78-3.01 (m, 2H), 3.69 (s, 3H, major isomer), 3.71 (s, 3H,
minor isomer), 3.78-3.82 (m, 4H), 4.00-4.12 (m, 3H), 4.20 (s,
1H), 4.56-4.60 (m, 1H), 6.98-7.01 (m, 2H), 778-7.81 (m, 2H).
ESI MS: m/z 440 [M + H]+.
N-Hyd r oxy 1N-(4-n -Bu toxyp h en yl)su lfon yl-4-(Z,E-N-
m or p h olin eim in o)p yr r olid in e-(2R)-ca r boxa m id e (6). The
methyl ester (2 g, 4.2 mmol) was mixed with NH2OH (13 mL,
22 mmol, 1.7 M in methanol, solution prepared as described
in Fieser and Fieser, Vol. 1, p 478) and stirred overnight at
room temperature. The mixture was neutralized with 1 N HCl
to pH ∼ 7 and then concentrated under reduced pressure. The
crude product was purified by column eluting with CH2Cl2:
CH3OH (95:5) to give 750 mg (43% yield) of a foaming solid as
1
purification. H NMR (CDCl3): δ 2.07 (ddd, J ) 14.1, 3.8, 1.6
Hz, 1H), 2.16 (ddd, J ) 14.1, 9.5, 4.4 Hz, 1H), 3.31 (dd, J )
10.3, 4.2 Hz, 1H), 3.48-3.54 (m, 2H), 3.76 (s, 3H), 3.89 (s, 3H),
4.28-4.34 (m, 2H), 6.98 (ddd, J ) 9.0, 2.5, 2.5 Hz, 2H), 7.79
(ddd, J ) 9.1, 2.6, 2.6 Hz, 2H). CI+ MS: m/z 316 [M + H]+,
256, 146, 114.
Meth yl 1N-(4-Meth oxyp h en ylsu lfon yl)-4-oxo-p yr r oli-
d in e-(2R)-ca r boxyla te (8). An 8 M batch of J ones reagent
was prepared. The alcohol 8a (10.0 g, 31.7 mmol) is dissolved
in 175 mL of acetone and cooled to 0 °C. J ones reagent was
added until the solution remains an orange color, and the
mixture is stirred at room temperature for 14 h. 2-Propanol
was added to the solution to quench the excess chromium
reagent, and the resulting solid was filtered through Celite.
The filtrate was concentrated under reduced pressure, and the
residue was dissolved in methylene chloride and washed with
water. The resulting solution was dried over magnesium
sulfate and concentrated under reduced pressure. Purification
of the product by chromatography on silica gel using EtOAc:
hexane (1:1) provided the desired ketone. 1H NMR (CDCl3): δ
2.57 (dd, J ) 18.3, 3.3 Hz, 1H), 2.80 (dd, J ) 18.3, 9.2 Hz,
1H), 3.66 (s, 3H), 3.80 (s, 1H), 3.82 (s, 1H), 3.71 (s, 3H), 4.78
(dd, J ) 8.8, 3.3 Hz, 1H), 7.02 (br d, J ) 8.8 Hz, 2H), 7.80 (br
d, J ) 8.8 Hz, 2H). ESI MS: m/z 374 [M+ + H]+.
1
a 2:3 mixture of E:Z isomers (unassigned). H NMR (DMSO-
d6): δ 0.93 (t, J ) 7.5 Hz, 3H), 1.38-1.52 (m, 2H), 1.63-1.79
(m, 2H), 2.39-2.63 (m, 6H), 3.56-3.70 (m, 4H), 3.92-4.12 (m,
4H), 4.20-4.37 (m, 1H), 7.12 (d, J ) 9.0 Hz, 2H), 7.77 (d, J )
9.0 Hz, 2H), 9.01 (s, 1H), 10.83 (s, 1H). ESI MS: m/z 479 [M
+ K]+, 441 [M + H]+. Anal. (C19H28N4O6S‚0.25H2O) C, H, N.
Meth yl 1N-(4-n -Bu toxyp h en yl)su lfon yl-4-(N-m eth oxy-
a m in o)p yr r olid in e-(2R)-ca r boxyla te (7a ). To a solution of
Met h yl 1N-(4-Met h oxyp h en yl)su lfon yl-4-m et h ylen e-
p yr r olid in e-(2R)-ca r boxyla te (10). To a solution of meth-

5432 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 26
Cheng et al.
yltriphenylphosphonium bromide (1.75 g, 4.78 mmol) in 10 mL
of anhydrous THF at 0 °C under argon was added lithium bis-
(trimethylsilyl)amide (5.74 mL, 5.74 mmol, 1.0 M solution in
THF) dropwise and stirred for 15 min. Then, a solution of
ketone 8 (1.5 g, 4.78 mmol) in THF (25 mL) was added slowly.
The mixture was stirred overnight at room temperature and
diluted with ammonium chloride. The reaction mixture was
extracted three times with EtOAc. The combined EtOAc layer
was washed with 1 N HCl, water, aqueous NaHCO3, and brine,
dried over MgSO4, and concentrated under reduced pressure
to an oil which was purified by column chromatography eluting
with EtOAc:hexane (3:7) to give 0.35 g (25%) of the desired
Hz, 2H), 7.79 (d, J ) 9.0 Hz, 2H), 9.02 (s, 1H), 10.86 (s, 1H).
ESI MS: m/z 366 [M + Na]+, 344 [M + H]+. Anal. (C13H17
N3O6S‚0.75H2O ) C, H, N.
-
N-Hydr oxy 1N-(4-Eth oxyph en yl)su lfon yl-4-(Z,E-N-m eth -
oxyim in o)p yr r olid in e-(2R)-ca r boxa m id e (15). The title
compound was prepared as described for compound 4 and gave
a
white foaming solid as a 2:3 mixture of E:Z isomers
(unassigned). 1H NMR (DMSO-d6): δ 1.28-2.01 (m, 3H), 2.24-
2.59 (m, 2H), 3.68-3.75 (m, 3H), 3.93-4.18 (m, 4H), 4.26-
4.35 (m, 1H), 7.08 (d, J ) 9.0 Hz, 2H), 7.74 (d, J ) 9.0 Hz,
2H), 9.02 (s, 1H), 10.86 (s, 1H). ESI MS: m/z 375 [M + NH4]+,
358 [M + H]+. Anal. (C14H19N3O6S‚0.25H2O) C, H, N.
N-H yd r oxy 1N-(4-P r op oxyp h en yl)su lfon yl-4-(Z,E-N-
m eth oxyim in o)p yr r olid in e-(2R)-ca r boxa m id e (16). The
title compound was prepared as described for compound 4 and
gave a white foaming solid as a 2:3 mixture of E:Z isomers
(unassigned). 1H NMR (DMSO-d6): δ 0.99 (t, J ) 7.3 Hz, 3H),
1.64-1.79 (m, 2H), 2.25-2.59 (m, 2H), 3.72 (d, J ) 9.5 Hz,
3H), 3.83-4.08 (m, 4H), 4.26-4.35 (m, 1H), 7.09 (d, J ) 9.0
Hz, 2H), 7.69-7.99 (m, 2H), 9.02 (s, 1H), 10.86 (s, 1H). ESI
MS: m/z 389 [M + NH4]+, 372 [M + H]+. Anal. (C15H21N3O6S‚
0.25H2O) C, H, N.
1
product. H NMR (CDCl3): δ 2.58-2.87 (m, 2H), 3.63 (s, 3H),
3.83 (s, 3H), 4.01-4.15 (m, 2H), 4.43-4.59 (m, 1H), 5.01 (d, J
) 10.3 Hz, 2H), 6.99 (dd, J ) 9.0, 2.0, Hz, 2H), 7.82 (dd, J )
9.0, 2.0 Hz, 2H). ESI MS: m/z 329 [M + NH4]+, 312 [M + H]+.
N-Hyd r oxy 1N-(4-Meth oxyp h en yl)su lfon yl-4-m eth yl-
en e-p yr r olid in e-(2R)-ca r boxa m id e (11). The methyl ester
10 (0.26 g, 0.83 mmol) was mixed with NH2OH (3.7 mL, 6.64
mmol, 1.7 M in methanol, solution prepared as described in
Fieser and Fieser, Vol. 1, p 478) and stirred overnight at room
temperature. Neutralized with 1 N HCl, the mixture was
extracted three times with EtOAc. The combined EtOAc layer
was dried (MgSO4) and concentrated under reduced pressure.
The crude product was purified by column eluting with CH2Cl2:
CH3OH (95:5) to give 0.18 g (70% yield) of the desired product
as a white solid. 1H NMR (DMSO-d6): δ 2.26-2.42 (m, 2H),
3.83-3.90 (m, 1H), 3.87 (s, 3H), 3.95-4.02 (m, 2H), 4.08-4.16
(m, 1H), 4.94 (d, J ) 10.3 Hz, 2H), 7.16 (d, J ) 9.0 Hz, 2H),
7.79 (d, J ) 9.0 Hz, 2H), 10.86 (s, 1H). ESI MS: m/z 330 [M +
NH4]+, 313 [M + H]+. Anal. (C13H16N3O5S‚0.75H2O) C, H, N.
N-Hyd r oxy 1N-(4-(2-Meth oxyeth oxy)p h en yl)su lfon yl-
4-(Z,E-N-m eth oxyim in o)-p yr r olid in e-(2R)-ca r boxa m id e
(17). The title compound was prepared as described for
compound 4 and gave a white solid as a 1:2 mixture of E:Z
1
isomers (unassigned). H NMR (DMSO-d6): δ 2.37-2.60 (m,
2H), 3.63-3.72 (m, 4H), 3.74 (s, 1H), 3.78 (s, 2H), 3.89-4.08
(m, 3H), 4.15-4.23 (m, 2H), 4.28-4.37 (m, 1H), 7.12 (d, J )
9.0 Hz, 2H), 7.78 (dd, J ) 9.0 Hz, 2H), 7.78 (dd, J ) 9.0, 1.7
Hz, 2H), 9.01 (s, 1H), 10.88 (s, 1H). ESI MS: m/z 405 [M +
NH4]+, 388 [M + H]+. HRMS: MH+ calcd for C15H22N3O7S,
388.1178; found, 388.1184.
(1N)-ter t-Bu toxyca r bon yl-(2R)-ca r bom eth oxy-(4R)-h y-
d r oxyp yr r olid in e (12a ). cis-Hydroxy-D-proline methyl ester
hydrochloride (40 g, 220 mmol, the methyl ester was obtained
from methanolic HCl treatment of the relative acid which was
obtained from Aldrich) was taken in 205 mL of acetone:water
(3:2) in the presence of 100 mL of Et3N and 1 g of 4-(dimethyl-
amino)pyridine, and the mixture was treated in portions with
di-tert-butyl dicarbonate (53 g, 243 mmol). After warming
slightly, the mixture was cooled back to room temperature and
N-H yd r oxy 1N-(4-P h en oxyp h en yl)su lfon yl-4-(Z,E-N-
m eth oxyim in o)p yr r olid in e-(2R)-ca r boxa m id e (18). The
title compound was prepared as described for compound 4. The
crude product was purified by reverse-phase preparative
HPLC (55A45B, A, 95% H2O, 5% acetonitrile, 0.1% formic acid;
B, 80% acetonitrile, 20% H2O; 19 × 300 mm waters Sym-
metryPrep C18 column) to give 250 mg (44% yield) of a white
foaming solid as a 1:2 mixture of E:Z isomers (unassigned).
1H NMR (DMSO-d6): δ 2.41-2.77 (m, 2H), 3.68-3.74 (m, 3H),
3.91-4.12 (m, 2H), 4.30-4.38 (m, 1H), 7.06-7.19 (m, 4H),
7.21-7.34 (m, 1H), 7.43-7.52 (m, 2H), 7.77-8.05 (m, 2H), 9.02
(s, 1H), 10.86 (s, 1H). ESI MS: m/z 423 [M + NH4]+, 406 [M
+ H]+. Anal. (C18H19N3O6S) C, H, N.
N-Hyd r oxy 1N-[4-(4-F lu or op h en oxy)p h en yl]su lfon yl-
4-(Z,E-N-m eth oxyim in o)-p yr r olid in e-(2R)-ca r boxa m id e
(19). The title compound was prepared as described for
compound 4 and gave a white foaming solid as a 2:3 mixture
of E:Z isomers (unassigned). 1H NMR (DMSO-d6): δ 2.41-
2.58 (m, 1H), 2.59-2.74 (m, 1H), 3.69-3.77 (m, 3H), 3.92-
4.10 (m, 2H), 4.22-4.36 (m, 1H), 7.12 (d, J ) 9.0 Hz, 2H),
7.19-7.38 (m, 4H), 7.78-7.84 (m, 2H), 9.02 (s, 1H), 10.87 (s,
1H). ESI MS: m/z 441 [M + NH4]+, 424 [M + H]+. Anal.
(C18H18N3FO6S) C, H, N.
1
stirred for 18 h. The mixture was reduced to ∼ /3 of its volume
and then partitioned between EtOAc and dilute NaH2PO4. The
organic layer was washed 1× with dilute NaH2PO4 and 1×
with brine, dried over MgSO4, filtered, and evaporated to give
68 g of white solid which was carried forward without
purification. 1H NMR (CDCl3): This compound appears in the
1H NMR spectrum as a distinct pair of rotomers. δ [1.44 (s),
1.48 (s)] 9H, 2.06-2.16 (m, 1H), 2.27-2.42 (m, 1H), 3.50-3.75
(m, 2H), [3.80 (s), 3.81 (s)] 1H, 4.29-4.42 (m, 2H). ESI MS:
m/z 262.9 [M + NH4]+, 245.9 [M + H]+, 145.8 [M - Boc + H]+.
Boc-Ketop yr r olid in e Meth yl Ester (12). The starting
alcohol 12a (16.4 g, 66.9 mmol) was oxidized as described for
compound 2. The crude syrup was chromatographed over flash
silica with hexane:Et2O (4:1, 1:2) to give 11.1 g (68%) of white
1
solid. H NMR (CDCl3): The compound appears as a pair of
distinct rotomers. δ 1.46 (br s, 9H), 2.58 (dd, J ) 17.0, 1.9 Hz,
1H), 2.56-3.03 (m, 1H), 3.77 (s, 3H), 3.86-3.93 (m, 2H), [4.26-
4.41 (m), 4.67 (dd, J ) 17.6, 9.6 Hz)] 1H.
N-Hyd r oxy 1N-(4-P yr id yloxyp h en yl)su lfon yl-4-(Z,E-N-
m eth oxyim in o)p yr r olid in e-(2R)-ca r boxa m id e (20). The
title compound was prepared as described for compound 4. The
crude product was purified by reverse-phase preparative
HPLC (90A10B, A, 95% H2O, 5% acetonitrile, 0.1% formic acid;
B, 80% acetonitrile, 20% H2O; 19 × 300 mm waters Sym-
metryPrep C18 column) to give a white foaming solid as a 2:3
N-H yd r oxy 1N-(4-Met h oxyp h en yl)su lfon yl-4-(Z,E-N-
h yd r oxyim in o)p yr r olid in e-(2R)-ca r boxa m id e (13). The
title compound was prepared as described for compound 3 and
gave a white cotton like solid as a 2:3 mixture of E:Z isomers
(unassigned). 1H NMR (DMSO-d6): δ 2.26-2.62 (m, 3H), 3.89
(s, 3H), 3.92-4.13 (m, 2H), 4.24-4.39 (m, 1H), 7.12-7.20 (m,
2H), 7.78-7.86 (m, 2H), 9.02 (s, 1H), 10.87 (s, 1H). ESI MS:
m/z 347 [M + NH4]+, 330 [M + H]+. Anal. (C12H15N3O6S‚
0.5H2O ) C, H, N.
N-H yd r oxy 1N-(4-Met h oxyp h en yl)su lfon yl-4-(Z,E-N-
m eth oxyim in o)p yr r olid in e-(2R)-ca r boxa m id e (14). The
title compound was prepared as described for compound 4 and
gave a white solid as a 2:3 mixture of E:Z isomers (unassigned).
1H NMR (DMSO-d6): δ 2.33-2.62 (m, 2H), 1.20-1.38 (m, 6H),
3.72 (s, 3H, minor isomer), 3.76 (s, 3H, major isomer), 3.86 (s,
3H), 3.88-4.13 (m, 2H), 4.26-4.39 (m, 1H), 7.16 (d, J ) 9.0
1
mixture of E:Z isomers (unassigned). H NMR (DMSO-d6): δ
2.41-2.58 (m, 1H), 2.68-2.79 (m, 1H), 3.61-3.79 (m, 3H),
3.97-4.16 (m, 2H), 4.29-4.39 (m, 1H), 7.01-7.12 (m, 2H),
7.28-7.39 (m, 2H), 7.84-7.79 (m, 2H), 8.26 (s, 1H), 8.50-8.59
(m, 2H), 10.87 (s, 1H). ESI MS: m/z 407 [M + H]+. Anal.
(C17H18N4O6S‚H2O) C, H, N.
N-Hyd r oxy 1N-(4-n -Bu toxyp h en yl)su lfon yl-4-(Z,E-N-
eth oxyim in o)p yr r olid in e-(2R)-ca r boxa m id e (21). The title
compound was prepared as described for compound 4. The
crude product was purified by reverse-phase preparative
HPLC (90A10B, A, 95% H2O, 5% acetonitrile, 0.1% formic acid;

Synthesis of Proline-Based MMP Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 26 5433
B, 80% acetonitrile, 20% H2O; 19 × 300 mm waters Sym-
(unassigned). 1H NMR (DMSO-d6): δ 0.96 (t, J ) 7.3 Hz, 3H),
1.40-1.52 (m, 2H), 1.65-1.80 (m, 2H), 2.43-2.63 (m, 2H),
3.98-4.08 (m, 4H), 4.23-4.36 (m, 1H), 5.00 (d, J ) 8.0 Hz,
2H), 7.08 (d, J ) 9.0 Hz, 2H), 7.09-7.39 (m, 5H), 7.77 (d, J )
9.0 Hz, 2H), 9.04 (s, 1H), 10.89 (s, 1H). ESI MS: m/z 479 [M
+ NH4]+, 462 [M + H]+. HRMS: MH+ calcd for C22H28N3O6S,
462.1699; found, 462.1683.
metryPrep C18 column) to give a white foaming solid as a 1:2
1
mixture of E:Z isomers (unassigned). H NMR (DMSO-d6): δ
0.93 (t, J ) 7.5 Hz, 3H), 1.02-1.18 (m, 3H), 1.39-1.52 (m, 2H),
1.66-1.81 (m, 2H), 2.27-2.62 (m, 3H), 3.87-4.10 (m, 5H),
4.28-4.39 (m, 1H), 7.10 (d, J ) 9.0 Hz, 2H), 7.78 (d, J ) 9.0
Hz, 2H), 9.04 (s, 1H), 10.86 (s, 1H). ESI MS: m/z 417 [M +
Na]+, 400 [M + H]+. Anal. (C17H25N3O6S‚0.25H2O ) C, H, N.
N-H yd r oxy 1N-(4-Met h oxyp h en yl)su lfon yl-4-(Z,E-N-
ter t-bu toxyim in o)pyr r olidin e-(2R)-car boxam ide (22). The
title compound was prepared as described for compound 4. The
crude product was purified by reverse-phase preparative
HPLC (90A10B, A, 95% H2O, 5% acetonitrile, 0.1% formic acid;
B, 80% acetonitrile, 20% H2O; 19 × 300 mm waters Sym-
metryPrep C18 column) to give a white foaming solid as a 1:2
N-Hyd r oxy 1N-(4-n -Bu toxyp h en yl)su lfon yl-4-(Z,E-N-
p ip er id in eim in o)p yr r olid in e-(2R)-ca r boxa m id e (29). The
title compound was prepared as described for compound 7 and
1
give a solid as a 4:3 mixture of E:Z isomers (unassigned). H
NMR (DMSO-d6): δ 0.92 (t, J ) 7.5 Hz, 3H), 1.24-1.60 (m,
9H), 1.62-1.78 (m, 2H), 2.26-2.60 (m, 5H), 3.82-4.10 (m, 4H),
4.18-4.34 (m, 1H), 7.08 (d, J ) 9.0 Hz, 2H), 7.77 (d, J ) 9.0
Hz, 2H), 9.00 (s, 1H), 10.86 (s, 1H). ESI MS: m/z 439 [M +
H]+. Anal. (C20H30N4O5S‚0.5H2O) C, H, N.
1
mixture of E:Z isomers (unassigned). H NMR (DMSO-d6): δ
1.19 (s, 9H), 2.26-2.52 (m, 2H), 3.82 (s, 3H), 3.99-4.07 (m,
2H), 4.32-4.40 (m, 1H), 7.12 (d, J ) 9.0 Hz, 2H), 7.80 (d, J )
9.0 Hz, 2H), 9.02 (s, 1H), 10.86 (s, 1H). ESI MS: m/z 403 [M
+ NH4]+, 386 [M + H]+. Anal. (C16H23N3O6S‚0.75H2O ) C, H,
N.
Meth yl 1N-ter t-Bu toxyca r bon yl-4-exom eth ylen e-p yr -
r olid in e-(2R)-ca r boxyla te (30a ). An oven dried/argon cooled
flask is charged with methyltriphenylphosphonium bromide
(1.29 g, 3.61 mmol) and 20 mL of dry benzene. Over a period
of 30 s, sodium tert-amylate (2.4 mL, 1.65 M in toluene, 3.95
mmol) was added via syringe, and the mixture was allowed to
stir at room temperature under argon for 30 min. A solution
of keto ester 8 (0.6 g, 2.47 mmol) in dry benzene was then
added in one portion via syringe, and the mixture was allowed
to stir for an additional 3 h. The mixture was then quenched
with 15 mL of 10% NH4Cl, and the layers were separated. The
aqueous layer was extracted once with diethyl ether, and the
combined organic layers were dried over MgSO4, filtered, and
evaporated to give 1.29 g of very viscous oil with crystals. The
crude product was dissolved in 5 mL of hot hexane/EtOAc and
filtered through 10% deactivated silica eluting with EtOAc:
hexane (1:3). The filtrate was evaporated to dryness, the
residue was dissolved in 1.5 mL of EtOAc, and 5 mL of hexane
was added. The crystallized triphenylphosphine oxide is
filtered off, and after evaporation, the filtrate furnishes 470
mg (71%) of the desired material in the form of a yellowish oil
containing about 5% of triphenylphosphine oxide impurity. 1H
NMR (CDCl3): The compound appears in the spectrum as two
distinct rotomers in a 2:3 ratio. δ [1.44 (s), 1.49 (s)] 9H, 2.58-
2.69 (m, 1H), 2.89-3.06 (m, 1H), 3.74 (s, 3H), 4.04-4.13 (m,
2H), [4.42 (dd, J ) 9.2, 3.5 Hz), 4.51 (dd, J ) 9.5, 2.8 Hz)] 1H,
4.98-5.06 (m, 2H). ESI MS: m/z 259.0 [M + NH4]+, 242.0 [M
+ H]+, 185.8, 141.
N-Hyd r oxy 1N-(4-n -Bu toxyp h en yl)su lfon yl-4-(Z,E-N-
ter t-bu toxyim in o)pyr r olidin e-(2R)-car boxam ide (23). The
title compound was prepared as described for compound 4 and
gave a white foaming solid as a 1:1 mixture of E:Z isomers
(unassigned). 1H NMR (DMSO-d6): δ 0.95 (t, J ) 7.5 Hz, 3H),
1.14 (s, 4.5H), 1.19 (s, 4.5H), 1.38-1.52 (m, 2H), 1.68-1.80
(m, 2H), 2.26-2.64 (m, 2H), 3.86-4.12 (m, 4H), 4.28-4.40 (m,
1H), 7.12 (dd, J ) 9.0, 3.6 Hz, 2H), 7.70-7.82 (m, 2H), 9.05 (s,
1H), 10.89 (s, 1H). ESI MS: m/z 450.1 [M + Na]+, 445.1 [M +
NH4]+, 428.1 [M + H]+. Anal. (C19H29N3O6S) C, H, N.
N-Hyd r oxy 1N-(4-P yr id yloxyp h en yl)su lfon yl-4-(Z,E-N-
ter t-bu toxyim in o)-pyr r olidin e-(2R)-car boxam ide (24). The
title compound was prepared as described for compound 4 and
gave a white solid as a 1:2 mixture of E:Z isomers (unassigned).
1H NMR (DMSO-d6): δ 1.23 (s, 9H), 2.42-2.59 (m, 1H), 2.66-
2.81 (m, 1H), 3.98-4.17 (m, 2H), 4.32-4.41 (m, 1H), 6.99-
7.12 (m, 2H), 7.38 (d, J ) 8.4 Hz, 2H), 7.86-8.01 (m, 2H),
8.50-8.61 (m, 2H), 9.06 (s, 1H), 10,86 (s, 1H). ESI MS: m/z
449 [M + H]+. Anal. (C20H24N4O6S‚0.25H2O) C, H, N.
N-Hyd r oxy 1N-[4-(4-F lu or op h en oxy)p h en yl]su lfon yl-
4-(Z,E-N-ter t-bu toxyim in o)-p yr r olid in e-(2R)-ca r boxa m -
id e (25). The title compound was prepared as described for
compound 4 and gave a white foaming solid as a 1:4 mixture
1
of E:Z isomers (unassigned). H NMR (DMSO-d6): δ 1.21 (s,
Meth yl 1N-(4-n -P r op yloxyp h en yl)-su lfon yl-4-exom eth -
ylen e-p yr r olid in e-(2R)-ca r boxyla te (30b). The exometh-
ylene compound 30a (494 mg, 2.04 mmol) was taken in 25 mL
of methylene chloride and treated with 2 mL of trifluoroacetic
acid. The mixture was stirred for 1 h at room temperature,
evaporated to dryness, and triturated once with chloroform.
The residual oil was taken in 25 mL of methylene chloride in
the presence of 3 mL of Et3N and treated with n-propoxyben-
zenesulfonyl chloride. The mixture was stirred for 18 h and
then partitioned between CHCl3 and 1 N HCl. The organic
layer was dried over MgSO4, filtered, and evaporated. The
residue was adsorbed onto silica and then eluted through a
column of flash silica with hexane:EtOAc (8:2 to 5:5) to give
9H), 2.40-2.52 (m, 1H), 2.60-2.72 (m, 1H), 3.88-4.12 (m, 2H),
4.27-4.36 (m, 1H), 7.08 (d, J ) 9.0 Hz, 2H), 7.19-7.25 (m,
2H), 7.28-7.39 (m, 2H), 7.81 (d, J ) 9.0 Hz, 2H), 9.02 (s, 1H),
10.86 (s, 1H). ESI MS: m/z 483 [M + NH4]+, 466 [M + H]+.
Anal. (C21H24N3O6FS‚0.25H2O) C, H, N.
N-Hyd r oxy 1N-(4-n -Bu toxyp h en yl)su lfon yl-4-(Z,E-N-
isobu toxyim in o)p yr r olid in e-(2R)-ca r boxa m id e (26). The
title compound was prepared as described for compound 4 and
gave a white foaming solid as a 1:2 mixture of E:Z isomers
(unassigned). 1H NMR (DMSO-d6): δ 0.76-0.88 (m, 6H), 0.95
(t, J ) 7.4 Hz, 3H), 1.40-1.51 (m, 2H), 1.66-1.87 (m, 3H),
2.30-2.60 (m, 2H), 3.62-3.75 (m, 2H), 3.91-4.10 (m, 4H),
4.28-4.39 (m, 1H), 7.12 (d, J ) 9.0 Hz, 2H), 7.78 (d, J ) 9.0
Hz, 2H), 9.07 (s, 1H), 10.86 (s, 1H). ESI MS: m/z 445 [M +
NH4]+, 428 [M + H]+. Anal. (C19H29N3O6S) C, H, N.
N-Hyd r oxy 1N-(4-P yr id yloxyp h en yl)su lfon yl-4-(Z,E-N-
p h en oxyim in o)p yr r olid in e-(2R)-ca r b oxa m id e (27). The
title compound was prepared as described for compound 4 and
gave a white solid as a 3:4 mixture of E:Z isomers (unassigned).
1H NMR (DMSO-d6): δ 2.68-2.98 (m, 1H), 2.82-3.01 (m, 1H),
3.22-3.40 (m, 2H), 3.79-3.86 (m, 2H), 6.82-6.88 (m, 1H),
6.92-7.03 (m, 2H), 7.19 (d, J ) 8.4 Hz, 1H), 7.27 (d, J ) 8.5
Hz, 1H), 7.36-7.41 (m, 3H), 7.60 (s, 3H), 7.68-7.78 (m, 2H),
10.62 (s, 2H). ESI MS: m/z 469 [M + H]+. HRMS: MH+ calcd
for C22H21N4O6S, 469.1182; found, 469.1177.
1
334 mg (48%) of clear viscous oil. H NMR (CDCl3): δ 1.07 (t,
J ) 7.4 Hz, 3H), 1.79-1.92 (m, 2H), 2.60 (m, 1H), 2.73-2.85
(m, 1H), 3.69 (s, 3H), 4.00 (t, J ) 6.6 Hz, 2H), 4.02 (br s, 2H),
4.46 (dd, J ) 8.8, 3.9 Hz, 1H), 4.97-5.01 (m, 1H), 6.99 (ddd, J
) 9.2, 2.9, 2.2 Hz, 2H), 7.80 (ddd, J ) 9.2, 2.9, 2.2 Hz, 2H).
ESI MS: m/z (rel intensity) 340.0 [M + NH4]+, 357.0 [M +
H]+.
N-H yd r oxyl 1N-(4-n -P r op oxyp h en yl)-su lfon yl-4-exo-
m eth ylen e-p yr r olid in e-(2R)-ca r boxa m id e (30). The com-
pound was prepared from methyl ester 30b (317 mg, 0.93
mmol) as described for compound 11 to give 168 mg of white
1
solid. H NMR (DMSO-d6): δ 1.01 (t, J ) 7.3 Hz, 3H), 1.72-
1.86 (m, 2H), 2.30-2.49 (m, 2H), 3.89 (d, J ) 14.5 Hz, 1H),
4.02 (d, J ) 14.5 Hz, 1H), 4.06 (t, J ) 6.4 Hz, 2H), 4.15 (dd, J
) 8.4, 3.8 Hz, 1H), 4.91 (s, 1H), 4.95 (s, 1H), 7.15 (d, J ) 8.6
Hz, 2H), 7.80 (d, J ) 8.4 Hz, 2H), 8.99 (s, 1H), 10.56 (s, 1H).
N-Hyd r oxy 1N-(4-n -Bu toxyp h en yl)su lfon yl-4-(Z,E-N-
ben zyloxyim in o)p yr r olid in e-(2R)-ca r boxa m id e (28). The
title compound was prepared as described for compound 4 and
gave a white foaming solid as a 2:3 mixture of E:Z isomers

5434 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 26
Cheng et al.
ESI MS: m/z 358.0 [M + NH4]+, 341.0 [M + H]+, 168.9. Anal.
(C15H20N2O5S‚0.1H2O) C, H, N.
Pro249 of proMMP-1 was amplified by polymerase chain
reaction from a commercially available plasmid, p35-1 (ATCC,
Rockville, MD; Templeton, N. S.; et al. (1990) Cancer Res.
1990, 50, 5431-5437) encoding human interstitial MMP-1.
The PCR fragment was ligated into the expression vector, pET-
11a (Novagen, Madison, WI), and expressed in E. coli
BL21(DE3) cells. The protein was solubilized from inclusion
bodies in 6 M urea, 0.15 M NaCl, pH 7.5, refolded in 50 mM
Tris-HCl, pH 7.5, 10 mM CaCl2, 0.1 mM zinc acetate, and then
purified to homogeneity over a hydroxamic acid inhibitor
affinity column as previously described (Moore, W. A.; Spilburg,
C. A. Biochemistry 1986, 25, 5189-5195). N-Terminal se-
quence analysis confirmed the presence of three N-terminal
sequences of Val-Leu-Thr-Glu-Gly-Asn, Met-Val-Leu-Thr-Glu-
Gly-Asn, and Leu-Thr-Glu-Gly-Asn (minor).
N-H yd r oxyl 1N-(4-n -Bu t oxyp h en yl)-su lfon yl-4-exo-
m eth ylen e-p yr r olid in e-(2R)-ca r boxa m id e (31). The com-
pound was prepared as described for compound 30 to give a
white solid. 1H NMR (DMSO-d6): δ 0.2.30-2.49 (m, 2H), 3.35
(s, 3H), 3.68-3.75 (m, 2H), 3.89 (d, J ) 14.7 Hz, 1H), 4.03 (d,
J ) 14.8 Hz, 1H), 4.15 (dd, J ) 8.4, 3.8 Hz, 1H), 4.20-4.27
(m, 2H), 4.92 (s, 1H), 4.95 (s, 1H), 7.16 (d, J ) 8.8 Hz, 2H),
7.82 (d, J ) 8.8 Hz, 2H), 9.00 (s, 1H), 10.77 (s, 1H). ESI MS:
m/z 372.0 [M + NH4]+, 355.0 [M + H]+, 168.9. Anal.
(C16H22N2O5S‚0.3H2O) C, H, N.
N-Hyd r oxy 1N-(4-(2-Meth oxyeth oxy)p h en yl)su lfon yl-
4-exom eth ylen e-p yr r olid in e-(2R)-ca r boxa m id e (32). The
compound was prepared as described for compound 30 to give
a white solid. 1H NMR (DMSO-d6): δ 2.30-2.49 (m, 2H), 3.35
(s, 3H), 3.68-3.75 (m, 2H), 3.89 (d, J ) 14.7 Hz, 1H), 4.03 (d,
J ) 14.8 Hz, 1H), 4.15 (dd, J ) 8.4, 3.8 Hz, 1H), 4.20-4.27
(m, 2H), 4.92 (s, 1H), 4.95 (s, 1H), 7.16 (d, J ) 8.8 Hz, 2H),
7.82 (d, J ) 8.8 Hz, 2H), 9.00 (s, 1H), 10.77 (s, 1H). ESI MS:
m/z 374.0 [M + NH4]+, 357.0 [M + H]+, 168.9. Anal.
(C15H20N2O6S‚0.5H2O) C, H, N.
Exp r ession a n d P u r ifica tion of Hu m a n Recom bin a n t
p r oMMP -2. A partial cDNA clone for MMP-2 known as K-121
(Tryggvason, K. J . Biol. Chem. 1990, 265, 11077-11082) was
obtained from ATCC and subcloned into the pBlueScript SK-
(pBS) plasmid. Sanger dideoxy sequencing revealed that the
first 134 bp of the coding sequence were missing from the 5′
end of K-121. To restore the missing sequence, two overlapping
90+-mer oligonucleotides were designed and synthesized.
These, along with a 3′ antisense oligonucleotide, were used as
primers to the K-121 template in a series of polymerase chain
reaction (PCR) experiments to synthesize a full-length MMP-2
cDNA. The PCR product was then subcloned into pBS. To
express MMP-2 in the mammalian CHO D- cell system, it was
first subcloned into the mammalian expression vector pJ T1
(J . Ting, CRD), which contains the DHFR gene. Recombinant
MMP-2/pJ T1 was Polybrene transfected into CHO D- cells.
Clonal cell populations were isolated and screened for produc-
tion of MMP-2 mRNA using specific oligonucleotide primers
and reverse-transcription PCR. Ten clones producing the
highest levels of MMP-2 mRNA were selected, and the DHFR/
MMP-2 construct was amplified by gradually increasing the
media methotrexate (MTX, a DHFR inhibitor) concentration.
Clonal selection was further narrowed after assessing MMP
bioactivity in an MMP fluorescence assay (M. Anastasio, CRD)
and on zymogram gels. Those clones showing activity were
tested for MMP-2 protein production by sequential Edman
degradation protein sequencing (F. Wang, P&GP Cell &
Molecular Biology Core) and Western blot. On the basis of all
results, one clone was expanded for growth in roller bottles.
Approximately 9 L of serum-free conditioned media with an
MMP-2 concentration of ∼ 35 mG/L was generated. ProMMP-2
was purified from conditioned media as previously described
(Crabbe, T. et al. Eur. J . Biochem. 1993, 218, 431-438) with
the following modifications. Conditioned serum free media was
reduced in volume with an Amicon S1Y30 spiral-wound
cartridge prior to chromatography on a gelatin-Sepharose 4B
column equilibrated in 25 mM Tris/HCl, 30 mM NaCl, 10 mM
CaCl2, pH 7.5 (TNC buffer). The column was washed with
equilibration buffer before elution of the bound protein by TNC
buffer containing 1 M NaCl followed by 1 M NaCl and 10%
(by volume) dimethyl sulfoxide (DMSO) in TNC buffer. Pro-
MMP-2 fractions were concentrated in an Amicon ultrafiltra-
tion cell with a YM30 membrane and diafiltered against 25
mM MES/NaOH, 30 mM NaCl, 10 mM CaCl2, pH 6.0 (MNC
buffer). The concentrate was then chromatographed over a
second gelatin-Sepharose 4B column equilibrated in MNC
buffer, washed with equilibration buffer to remove nonbinding
proteins, and eluted with MNC buffer containing 0.3 M NaCl
and 10% (by volume) DMSO. Elution fractions containing
progelatinase A were pooled, diluted, and loaded onto an
S-Sepharose Fast Flow column equilibrated in MNC buffer and
eluted with MNC buffer containing 0.3 M NaCl. The concen-
trated fractions of purified proMMP-2 were combined and
stored in MNC buffer containing 0.3 M NaCl at -70 °C.
N-Terminal sequence, amino acid, and mass spectrophotomet-
ric analysis confirmed the identity of the purified protein with
the expected sequence for progelatinase A.
N-H yd r oxyl 1N-(4-n -P h en oxyp h en yl)-su lfon yl-4-exo-
m eth ylen e-p yr r olid in e-(2R)-ca r boxa m id e (33). The com-
pound was prepared as described for compound 30 to give a
1
white solid. H NMR (DMSO-d6): δ 2.45 (d, J ) 5.68 Hz, 2H),
3.91 (d, J ) 14.5 Hz, 1H), 4.04 (d, J ) 14.5 Hz, 1H), 4.16 (t, J
) 6.3 Hz, 1H), 4.96 (s, 1H), 4.96 (s, 1H), 7.13-7.22 (m, 4H),
7.30 (ddd, J ) 7.3, 7.3, 0.9 Hz, 1H), 7.51 (dd, J ) 7.9 Hz, 2H),
7.87 (br d, J ) 8.8 Hz, 2H), 8.99 (br s, 1H), 10.77 (s, 1H). ESI
MS: m/z 392.0 [M + NH4]+, 375.0 [M + H]+, 168.9. Anal.
(C18H18N2O5S‚0.2H2O) C, H, N.
N-Hyd r oxy 1N-(4-Meth oxyp h en yl)su lfon yl-4-(N-m eth -
oxya m in o)p yr r olid in e-(2R)-ca r b oxa m id e (34). The title
compound was prepared as described for compound 7 to give
a white solid as a 2:1 mixture of R:â epimers (unassigned). 1H
NMR (DMSO-d6): δ 1.64-2.03 (m, 2H), 2.98-3.19 (m, 1H),
3.22-3.39 (m, 1H), 3.38 (s, 3H), 3.40-4.60 (m, 1H), 3.81-3.97
(m, 1H), 3.85 (s, 3H), 6.38-6.40 (m, 1/3H), 6.80-6.87 (d, J )
8.9 Hz, 2/3H), 7.08-7.18 (m, 2H), 7.73-7.81 (m, 2H), 8.93 (s,
1/3H), 9.04 (s, 2/3H), 10.68 (s, 1/3H), 10.79 (s, 2/3H). ESI MS:
m/z 346 [M + H]. Anal. (C13H19N3O6S) C, H, N.
N-Hyd r oxy 1N-(4-(2-Meth oxyeth oxy)p h en yl)su lfon yl-
4-(N-m eth oxya m in o)-p yr r olid in e-(2R)-ca r boxa m id e (35).
The title compound was prepared as described for compound
7 to give a white solid as a 2:1 mixture of R:â epimers
(unassigned). 1H NMR (DMSO-d6): δ 0.99 (t, J ) 7.51 Hz, 3H),
1.40-1.55 (m, 2H), 1.70-1.81 (m, 2H), 2.30-2.49 (m, 2H), 3.89
(d, J ) 14.8 Hz, 1H), 4.03 (d, J ) 14.7 Hz, 1H), 4.07-4.18 (m,
3H), 4.92 (s, 1H), 4.95 (s, 1H), 7.16 (d, J ) 8.8 Hz, 2H), 7.80
(d, J ) 8.8 Hz, 2H), 9.00 (s, 1H), 10.75 (s, 1H). ESI MS: m/z
390 [M + H]+. HRMS: MH+ calcd for C15H25N3O7S, 390.1335;
found, 390.1348.
N-Hyd r oxy 1N-(4-P h en oxyp h en yl)su lfon yl-4-(N-m eth -
oxya m in o)p yr r olid in e-(2R)-ca r b oxa m id e (36). The title
compound was prepared as described for compound 7 to give
a white solid as a 2:1 mixture of R:â epimers (unassigned). 1H
NMR (DMSO-d6): δ 1.63-1.78 (m, 1H), 1.97-2.07 (m, 1H),
3.13-3.21 (m, 1H), 3.27-3.35 (m, 5H), 3.86-3.93 (m, 1H),
6.81-6.92 (m, 1H), 7.12-7.21 (m, 4H), 7.22-7.39 (m, 1H),
7.43-7.53 (m, 2H), 7.82 (d, J ) 9.0 Hz, 2H), 9.08 (s, 1H), 10.76
(s, 1H). ESI MS: m/z 408 [M + H]+. Anal. (C18H21N3O6S) C,
H, N.
N-Hyd r oxy 1N-(4-Meth oxyp h en yl)su lfon yl-4-(N-ter t-
bu toxyam in o)pyr r olidin e-(2R)-car boxam ide (37). The title
compound was prepared as described for compound 7 to give
a white solid as a 3:4 mixture of R:â epimers (unassigned). 1H
NMR (DMSO-d6): δ 1.02 (s, 4H), 1.19 (s, 5H), 1.82-2.27 (m,
3H), 3.08-3.20 (m, 1H), 3.39-3.67 (m, 2H), 3.84 (s, 3H), 4.14-
4.21 (m, 1H), 7.02 (d, J ) 9.0 Hz, 2H), 7,82 (d, J ) 9.0 Hz,
2H), 9.52-9.79 (m, 1H). ESI MS: m/z 388 [M + H]+. Anal.
(C16H25N3O6S) C, H, N.
Exp r ession a n d P u r ifica tion of Hu m a n Recom bin a n t
Tr u n ca ted MMP -1. Briefly, DNA sequence coding for Val82-
Exp r ession a n d P u r ifica tion of Hu m a n Recom bin a n t
Tr u n ca ted MMP -3. Briefly, DNA sequence coding for Ala-1
-

Synthesis of Proline-Based MMP Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 26 5435
Thr255 of proMMP-3 was amplified by polymerase chain
reaction from a recombinant plasmid encoding human synovial
preproMMP-3 kindly provided by Dr. Hideaki Nagase (Uni-
versity of Kansas Medical Center, Kansas City, KS) and Dr.
Markku Kurkinen (Department of Medicine, UMDNJ -Robert
Wood J ohnson Medical School). After DNA sequence verifica-
tion, this DNA fragment was ligated into the expression vector,
pET-3d (Novagen, Madison, WI), and expressed in E. coli BL21
(DE3) cells as previously described (Marcy, A. I. et al.
Biochemistry 1991, 30, 6476-6483). ProMMP-3 was solubi-
lized form inclusion bodies in 8 M guanidine-HCl, refolded in
100 mM Tris-HCl, pH 7.5, 10 mM CaCl2, and 0.5 mM zinc
acetate, and activated overnight at 37 °C with 1.5 mM
p-aminophenylmercuric acetate. Active, truncated MMP-3 was
purified to homogeneity over a hydroxamic acid inhibitor
affinity column as described (Moore, W. A.; Spilburg, C. A.
Biochemistry 1986, 25, 5189-5195). N-Terminal sequence
analysis confirmed the sequence to be consistent with the
catalytic domain, Phe83-Thr255, of proMMP-3.
MMP In h ibition Assa y. Inhibition of all enzymes was
measured according to the representative procedure described
below for MMP-2. ProMMP-1 was activated prior to assay by
treatment with trypsin. ProMMP-2 was activated prior to
assay by treatment with 1 mM p-aminophenyl-mercuric
acetate for 45 min at 37 °C. ProMMP-3 was activated prior to
assay by treatment with p-aminophenylmercuric acetate or
trypsin. ProMMP-9 was activated with MMP-3 (1:20, MMP-
3:MMP-9) and stored at -80 °C until use. MMP-7, 8, and 13
were all supplied as active enzymes and stored frozen until
use. MMP activity was monitored using a fluorescence assay
previously described,15 modified for a microtiter plate format.
In a Dynatech MicroFLUOR plate, active enzyme was incu-
bated with 8 µM Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 in
50 mM Tris-HCl pH 7.5, 10 mM CaCl2, 0.15 M NaCl, 0.05%
Brij for 20-30 min at 37 °C in the presence of varying
concentrations of inhibitor. The reaction was then quenched
with 50 mM EDTA, and the relative fluorescence was moni-
tored on a Perkin-Elmer LS50B spectrofluorometer (λex 328
nm, λem 393 nm) fitted with a microplate reader attachment.
Activity was measured as a percentage of control activity in
the absence of inhibitor. Inhibitor concentrations were run in
triplicate, and IC50 determinations were calculated from a four-
parameter logistic fit of the data within a single experiment.
In Vitr o In testin a l P er m ea bility Stu d ies.21 Briefly, a
portion of rat ileum was freshly excised and mounted in an in
vitro diffusion chamber system (NaviCyte, San Diego, CA). The
amount of drug transported across the tissue with time at
various donor concentrations of drug was determined at 37
°C and pH 7.4 in modified Krebs bicarbonate buffer. Perme-
ability coefficients, kp (cm/min) were calculated at steady state
with the following equation from Fick’s laws of diffusion
Refer en ces
(1) (a) Lafleur, M.; Underwood, J . L.; Rappolee, D. A.; Werb, Z.
Basement Membrane and Repair of Injury to Periferal Nerve;
Defining a Potential Role for Macrophages, Matrix Metallo-
proteinases and Tissue Inhibitor of Metalloproteinases-1. J . Exp.
Med. 1996, 184, 2311-2326. (b) Buisson, A. C.; Gilles, C.; Polette,
M.; Zahm, J . M.; Birembout, P.; Tournier, J . M. Wound Repair-
Induced Expression of Stromelysins is Associated with the
Acquisition of a Mesenchymal Phenotype in Human Respiratory
Epithilial Cells. Lab. Invest. 1996, 74, 658-669. (c) Woessner,
J . F., J r. Metalloproteinases and Their Inhibitors in Connective
Tissue Remodeling. FASEB J . 1991, 5, 2145-2154.
(2) Folkman, J .; Shing, Y. Angiogenesis J . Biol. Chem. 1992, 267,
10931-10934.
(3) Cawston, T. Metalloproteinase Inhibitors and the Prevention of
Connective Tissue Breakdown. Pharmacol. Ther. 1996, 70, 163-
182.
(4) (a) Ahrens, D.; Koch, A. E.; Pope, R. M.; Steinpicarella, M.;
Niedbala, M. J . Expression of Matrix Metalloproteinase 9 (96-
kd Gelatinase B) in Human Rheumatoid Arthritis. Arthritis
Rheum. 1996, 39, 1576-1587. (b) Blaser, J ,; Triebel, S.; Maa-
josthusmann, U.; Romisch, J .; Krahlmateblowski, U.; Freuden-
berg, W.; Fricke, R.; Tschesche, H. Determination of Metallo-
proteinases, Plasminogen-Activators and their Inhibitors in the
Synovial Fluids of Patients with Rheumatiod Arthritis During
Chemical Synoviorthesis. Clin. Chim. Acta 1996, 244, 17-33.
(5) Overall, C. M.; Wiebkin, O. W.; Thonard, J . C. Demonstration
of tissue Collagenase Activity in vivo an its Relationship to
Inflammation Severity in Human Gingiva. J . Periodontal Res.
1987, 22, 81-88.
(6) (a) Yong, V. W. The potential Use of MMP Inhibitors to Treat
CNS Diseases. Expert Opin. Invest. Drugs 1999, 94, 2177-2182.
(b) Maeda, A.; Sobel, R. A. Matrix Metalloproteinases in the
Normal Human Nervous System, Microglial Nodules, and
Multiple Sclerosis Lesions. J . Neuropathol. Exp. Neurol. 1996,
55, 300-309. (c) Chandler, S.; Coates, R.; Gearing, A.; Lury, J .;
Wells, G.; Bone, E.; Matrix Metalloproteinases Degrade Myelin
Basic Protein. Neurosci. Lett. 1995, 201, 223-226. (d) Hewson,
A. K.; Smith, T.; Leonard, J . P.; Cuzner, M. L. Suppression of
Experimental Allergic Encephalomyelitis in the Lewis Rat by
the Matrix Metalloproteinase Inhibitor Ro31-9790. Inflamm.
Res. 1995, 44, 345-349.
(7) (a) Brown, P. D. Matrix Metalloproteinases in Gastrointestinal
Cancer. Gut 1998, 43, 161-163. (b) Chambers, A. F.; Matrisan,
L. M. Changing Views of the Role of Matrix Metalloproteinases
in Metastasis. J . Natl. Cancer Inst. 1997, 89, 1260-1270. (c)
Yu, A. E.; Hewitt; R. E.; Connor, E. W.; Stetler-Stevenson, W.
G. Matrix Metalloproteinases: Novel Targets for Directed
Cancer Therapy. Drugs Aging 1997, 11, 229-244. (d) Zuker, S.;
Lystik, R. M.; Zarrabi, H. M.; Moll, U.; Tickle, S. P.; Stetler-
Stevenson, W.; Baker, T. S.; Docherty, A. J . P. Plasma Assay of
Matrix Metalloproteinases (MMP’s) and MMP-Inhibitor Com-
plexes in Cancer. Ann. N.Y. Acad. Sci. 1994, 732, 248-262.
(8) (a) Beckett, R. P.; Whittaker, M. Matrix Metalloproteinase
Inhibitors 1998. Expert Opin. Ther. Patents 1998, 8, 259-282.
(b) White, A. D. Bocan, T. M. A.; Boxer, P. A.; Peterson, J . T.;
Schrier, D. Emerging Therapeutic Advances for the Development
of Second Generation Matrix Metalloproteinase Inhibitors. Curr.
Pharm. Des. 1997, 3, 45-58. (c) Zask, A.; Levin, J . I.; Killar, L.
M.; Skotnicki, J . S. Inhibition of Matrix Metalloproteinases:
Structure Based Design. Curr. Pharm. Des. 1996, 2, 624-661.
(9) (a) Cherney, R. J .; Wang, L.; Meyer, D. T.; Xue, C.-B.; Arner, E.
C.; Copeland, R. A.; Covington, M. B.; Hardman, K. D.; Wasser-
man, Z. R.; J affee, B. D.; Decicco, C. P. Macrocyclic Hydroxamate
Inhibitors of Matrix Metalloproteinases and TNF-R Production.
Biorg. Med. Chem. Lett. 1999, 9, 1279-1284. (b) Natchus, M.
G.; Cheng, M.; Wahl, C. T.; Pikul, S.; Almstead, N. G.; Bradley,
R. S.; Taiwo, Y. O.; Mieling, G. E.; Dunaway, C. M.; Snider, C.
E.; McIver, J . M.; Barnett, B. L.; McPhail, S. J .; Anastasio, M.
B.; De, B. Design and Synthesis of Conformationally-Constrained
MMP Inhibitors. Bioorg. Med. Chem. Lett. 1998, 8, 2077.
(10) (a) MacPherson, L. J .; Parker, D. T. New Aryl-sulfonyl-amino-
kp ) [S]/[ACo]
where S is the slope of the linear (steady state) portion of the
cumulative amount transported through the membrane versus
time plot in µg/min, A is the cross-sectional area of the tissue
surface exposed to solute transport in cm2 (0.636 cm2), and Co
is the initial concentration of solute in the donor chamber in
µg/mL (approximately 25-65 µg/mL for compounds tested). A
predicted percent absorption, Abs, is obtained from the ratio
of the kp value for the test compound divided by the kp of the
mannitol internal standard (kp ratio) from the following
equation:
hydroxamic Acid Derivatived
- Useful as Metalloprotease
Abs ) [100]/[1 + exp{-7.42(kp ratio - 0.812)}]
Inhibitors for Treating Arthritis, Ulcers, Periodontal Disease,
Tumor Metastasis, HIV Infections, etc. European Patent Ap-
plication. EP606046A, 1993. (b) MacPherson, L. J .; Bayburt, E.
K.; Capparelli, M. P.; Carroll, B. J .; Goldstein, R. L.; Doughty,
J . R.; Spirito, S.; Blancuzzi, V.; Wilson, D.; O’Byrne, E. M.; Ganu,
V. S.; Parker, D. T. Discovery of CGS 27023A, a Non-Peptidic,
Potent, and Orally Active Stromelysin Inhibitor That Blocks
Cartilage Degradation in Rabbits. J . Med. Chem. 1997, 40,
2525-2532.
Ack n ow led gm en t. We are grateful to Dr. Fred C.
Wireko and Michael R. Mootz for obtaining X-ray crystal
data which proved the structure shown in ref 14. We
are also grateful to Glen Mieling for formatting the
graphic which was used for Figure 2.

5436 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 26
Cheng et al.
(11) (a) Pikul, S.; McDow-Dunaway, K. L.; Almstead, N. G.; De, B.;
Natchus, M. G.; McPhail, M. V.; Snider, C. E.; Taiwo, Y. O.;
Rydel, T.; Dunaway, C. M.; Gu, F.; Mieling, G. E. Discovery of
Potent, Achiral Matrix Metalloproteinase Inhibitors. J . Med.
Chem. 1998, 41, 3568. (b) Almstead, N. A.; Bradley, R. S.; Pikul,
S.; De, B.; Natchus, M. G.; Taiwo, Y. O.; Gu, F.; Williams, L. E.;
Hynd, B. A.; J anusz, M. J .; Dunaway, C. M.; Mieling, G. E.
Design, Synthesis and Biological Evaluation of Potent Thiazine
and Thiazapine Based MMP Inhibitors. 1999, in press.
(12) (a) Fieser, L. F.; Fieser, M. Reagents for Organic Synthesis; J ohn
Wiley and Sons, Inc.: New York, 1967; Vol. 1, p 478. (b) Hauser,
C. R.; Renfrow, W. B., J r. Org. Synth. Coll. Vol. 1943, 2, 67.
(13) (a) Hudlicky, T.; Zingde, G. S.; Natchus, M. G.; Ranu, B. C.;
Papadopoulos, P. J . System Oriented Design of Triquinanes:
Stereocontrolled Synthesis of Pentallenic Acid and Pentalenene.
Tetrahedron 1987, 43, 5685. (b) Conia, J . M., Limasset, J . C. J .
Bull. Soc. Chim. Fr. 1967, 6, 1936.
(17) There is general knowledge that sulfonyl substituents go into
the P1′ pocket which is much more shallow for MMP-1 and -7
than the other enzymes of the family. Bode, W.; Fernandez-
Catalan, C.; Grams, F.; Gomis-Ruth, F.-Z.; Nagase, H.; Tscheche,
H.; Maskos, K. Insights into MMP-TIMP Interactions. Ann. N.Y.
Acad. Sci. 1999, 878, 73-91.
(18) Protein Data Bank file name: 1D7X.
(19) (a) See ref 12. (b) Babine, R. E.; Bender, S. L. Molecular
Recognition of Protein-Ligand Complexes: Applications to Drug
Design. Chem. Rev. 1997, 97, 1359-1472. (c) Gonella, N. C.; Li,
Y.-C.; Zhang, X.; Paris, C. G. Bioactive Conformation of a Potent
Stromelysin Inhibitor Determined by X-nucleus Filtered and
Multidimensional NMR Spectroscopy. Bioorg. Med. Chem. 1997,
5, 2193-2201.
(20) It has been demonstrated in the literature that the R-oriented
oxygen plays a more significant role in binding than does the
S-oriented oxygen, and this is believed to be due to its closer
relative distance to the enzyme: Pikul, S.; McDow-Dunham, K.;
Almstead, N. G.; De, B.; Natchus, M. G.; Anastasio, M. V.;
McPhail, S. J .; Snider, C. E.; Taiwo, Y. O.; Chen, L.; Dunaway,
C. M.; Gu, F.; Mieling, G. E. Design and Synthesis of Phosphin-
amide-Based Hydroxamic Acids as Inhibitors of Matrix Metallo-
proteinases. J . Med. Chem. 1999, 42, 87-94.
(14) In the course of our olefination experimentation we observed a
curious electrophilic preference of the dithiane anion below for
an ester moiety over a ketone moiety. The structure of the
product was confirmed by X-ray crystallography. For reference,
see: (a) Muzard, M.; Portella, C. Synthesis 1992, 965. (b) Carey,
F. A.; Court, A. S. J . Org. Chem. 1972, 37, 1926.
(21) Dowty, M. E.; Dietsch, C. R. Improved Prediction of In-Vivo
Peroral Absorption from In-Vitro Intestinal Permeability Using
an Internal Standard to Control for Intra- and Inter-Rat
Variability. Pharm. Res. 1997, 14, 1792-1797
(22) Desai, S. R.; Liu, D. Z.; Thakker, D. R. The Unusual Effect of
Pyridyl Group on the Transcellular Transport of Compounds
Across the Caco-2 Cell Monolayers. Pharm. Res. 1997, 14, S-21.
(23) Cremlyn, R. J .; Cronje, T.; Goulding, K. Some Phenoxybenzene-
sulfonyl Chlorides and Related Compounds. Aust. J . Chem. 1979,
32, 445-452.
(15) Knight, C. G., Willenbrock, F., Murphy, G. A. A Novel Coumarin-
labeled Peptide for Sensitive Continuous Assays of the Matrix
Metalloproteinases. FEBS Lett. 1992, 296, 263-266.
(16) Okada, Y.; Nagase, H.; Harris, E. D. A Metalloproteinase from
Human Rheumatiod Synovial Fibroblasts that Digests Connec-
tive Tissue Matrix Components. Purification and Characteriza-
tion. J . Biol. Chem. 1986, 261, 14245-14255.
(24) Schraufstatter, E.; Schmidt-Kastner, G.; Wirth, W. Ger. Patent
1,078,129.
J M9904699