Synthesis of a series of stromelysin-selective thiadiazole urea matrix metalloproteinase inhibitors

Article abstract of DOI:10.1021/jm9803222

The synthesis and enzyme inhibition data for a series of thiadiazole urea matrix metalloproteinase (MMP) inhibitors are described. A broad screening effort was utilized to identify several thiadiazoles which were weak inhibitors of stromelysin. Optimization of the thiadiazole leads to include an α-amino acid side chain with variable terminal amide substituents provided a series of ureas which were moderately effective stromelysin inhibitors, with K(i)'s between 0.3 and 1.0 μM. The most effective analogues utilized an L-phenylalanine as the amino acid component. In particular, unsubstituted 46 had a K(i) of 710 nM, while the p-fluoro analogue 52 displayed increased potency (100 nM). Stromelysin inhibition was further improved using a pentafluorophenylalanine substituent which resulted in 70, a 14 nM inhibitor. While gelatinase inhibition was generally poor, the use of 1-(2-pyridyl)piperazine as the amide component usually provided for enhanced activity, with 71 inhibiting gelatinase with a K(i) of 770 nM. The combination of this heterocycle with a p-fluorophenylalanine substituent provided the only analogue, 69, with collagenase activity (13 μM). The SAR for analogues described within this series can be rationalized through consideration of the X-ray structure recently attained for 70 complexed to stromelysin. Uniquely, this structure showed the inhibitor to be completely orientated on the left side of the enzyme cleft. These results suggest that thiadiazole urea heterocycles which incorporate a substituted phenylalanine can provide selective inhibitors of stromelysin. Careful selection of the amide substituent can also provide for analogues with modest gelatinase inhibition.

Full text of DOI:10.1021/jm9803222

J . Med. Chem. 1999, 42, 1525-1536
1525
Syn th esis of a Ser ies of Str om elysin -Selective Th ia d ia zole Ur ea Ma tr ix
Meta llop r otein a se In h ibitor s
E. J on J acobsen,* Mark A. Mitchell, Susan K. Hendges, Kenneth L. Belonga, Louis L. Skaletzky,
Lindsay S. Stelzer, Thomas. J . Lindberg, Edward L. Fritzen, Heinrich J . Schostarez, Theresa J . O’Sullivan,
Linda L. Maggiora, Christopher W. Stuchly, Alice L. Laborde, Marc F. Kubicek, Roger A. Poorman,
J oan M. Beck, Henry R. Miller, Gary L. Petzold, Pam S. Scott, Scott E. Truesdell, Tanya L. Wallace,
J ohn W. Wilks, Christopher Fisher, Linda V. Goodman, Paul S. Kaytes, Stephen R. Ledbetter, Elaine A. Powers,
Gabriel Vogeli, J ohn E. Mott, Catherine M. Trepod, Douglas J . Staples, Eric T. Baldwin, and Barry C. Finzel
Departments of Structural, Analytical and Medicinal Chemistry, Protein Science, Genomics, Discovery Technologies, Cell and
Molecular Biology, and Chemical Process Research & Preparations, Pharmacia & Upjohn, Kalamazoo, Michigan 49007
Received May 26, 1998
The synthesis and enzyme inhibition data for a series of thiadiazole urea matrix metallopro-
teinase (MMP) inhibitors are described. A broad screening effort was utilized to identify several
thiadiazoles which were weak inhibitors of stromelysin. Optimization of the thiadiazole leads
to include an R-amino acid side chain with variable terminal amide substituents provided a
series of ureas which were moderately effective stromelysin inhibitors, with Ki’s between 0.3
and 1.0 µM. The most effective analogues utilized an L-phenylalanine as the amino acid
component. In particular, unsubstituted 46 had a Ki of 710 nM, while the p-fluoro analogue 52
displayed increased potency (100 nM). Stromelysin inhibition was further improved using a
pentafluorophenylalanine substituent which resulted in 70, a 14 nM inhibitor. While gelatinase
inhibition was generally poor, the use of 1-(2-pyridyl)piperazine as the amide component usually
provided for enhanced activity, with 71 inhibiting gelatinase with a Ki of 770 nM. The
combination of this heterocycle with a p-fluorophenylalanine substituent provided the only
analogue, 69, with collagenase activity (13 µM). The SAR for analogues described within this
series can be rationalized through consideration of the X-ray structure recently attained for
70 complexed to stromelysin. Uniquely, this structure showed the inhibitor to be completely
orientated on the left side of the enzyme cleft. These results suggest that thiadiazole urea
heterocycles which incorporate a substituted phenylalanine can provide selective inhibitors of
stromelysin. Careful selection of the amide substituent can also provide for analogues with
modest gelatinase inhibition.
In tr od u ction
therapeutic implications for MMP inhibitors (MMPIs)
include periodontal disease,8 atherosclerotic plaque
rupturing,9 and aortic aneurysms.10 Recently several
research groups have shown that related metallopro-
teinases are also involved in the conversion of inactive
tumor necrosis factor-R (TNF-R) into active TNF.11,12
Usually this inflammatory cytokine plays a beneficial
role in physiological defense responses; however, over-
production of TNF-R can lead to systemic toxicity.
Therefore, MMP inhibitors may be indirectly involved
in diseases for which TNF-R has been implicated, such
as Crohn’s disease, MS, cachexia, sepsis, and rheuma-
toid arthritis.13
The matrix metalloproteinases (MMPs) are a family
of zinc endopeptidases which are capable of degrading
the extracellular matrix of connective tissues and base-
ment membranes.1 These proteinases are involved in
tissue remodeling and are generally regulated by selec-
tive endogenous tissue inhibitors of metalloproteinases
(TIMPs).2 Overactivation or increased synthesis of the
MMPs has been implicated in several disease patholo-
gies.3 There are currently at least 18 members of this
family of proteinases which can be roughly associated
into three groups depending on their native substrates.
Thus, the gelatinases are effective proteinases of type
IV collagen, the collagenases degrade interstitial col-
lagen, and the stromelysins are effective as protoglyca-
nases. The discovery and characterization of the MMPs
are summarized in several recent reports.4
MMPs have been implicated in a number of disease
states, including arthritis, cancer progression, and
related connective tissue disorders. For instance, in-
creased levels of MMPs (collagenase, stromelysin) have
been noted in cartilage and synovial fluid in arthritis
with an excellent correlation of the levels of the MMPs
to severity of disease.5 Recent studies with cancer
patients have documented that overexpression of MMPs
is directly correlated with life span.6,7 Other possible
Recent efforts by a number of laboratories have
provided several classes of MMP inhibitors which have
been extensively reviewed.14-16 In general, each class
contains a zinc ligand attached to a small peptide
fragment which is capable of binding to specificity
pockets of the MMP enzymes, usually on the P′ side.
The zinc ligands utilized most frequently are hydrox-
amates, thiols, carboxylic acids, and phosphoric acids.
In general, the use of hydroxamates as the zinc ligand
provides for the most effective MMP inhibitors. How-
ever, hydroxamates are often found to be biologically
labile which has prompted additional efforts toward the
discovery of new chelating groups suitable for use in
10.1021/jm9803222 CCC: $18.00 © 1999 American Chemical Society
Published on Web 04/09/1999

1526 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 9
J acobsen et al.
Sch em e 1. Synthesis of Thiadiazole Aminesa
a
Reagents: (i) H2NNH2, EtOH; (ii) CS2, DMF.
F igu r e 1. Thiadiazole screening leads.
Sch em e 2. Synthesis of Simple Thiadiazolesa
F igu r e 2. Thiadiazole amine and urea targets.
MMPI templates.17 To explore this issue further, a broad
screening effort was undertaken using stromelysin as
the screening enzyme. From this, a number of chemical
classes were identified which were weak (>50 µM)
inhibitors of stromelysin. Of these, several members of
the thiadiazole class (Figure 1, amines and ureas) were
determined to be competitive stromelysin inhibitors.
a
Reagents: (i) THF; (ii) PhCH2NCO, THF.
compounds represented over a 4700-fold improvement
in stromelysin inhibition, as compared to the screening
leads.
Ch em istr y
The general synthesis of the thiadiazole amines 10
following standard procedures22 is illustrated in Scheme
1. Reaction of the desired isothiocyanate 8 with hydra-
zine hydrate provided thiosemicarbazide 9 which was
treated directly with carbon disulfide in DMF at 60 °C
to give aminothiadiazole 10.
In this report we describe the elaboration of this series
to improve enzyme potency, evaluate structure-activity
relationship (SAR), and determine MMP specificity. In
particular, we were interested in the exploration of
compounds in the amine (1, 2) and urea (3, 4) classes.
During this SAR elaboration of the thiadiazole screening
leads, other reports of thiadiazole-based MMP inhibitors
have appeared.18 Additional effort toward the develop-
ment of an amide thiadiazole series is reported sepa-
rately.19 Under the assumption that the exo-cyclic sulfur
chelates to the catalytic zinc, with the side chain of 1-4
generally located toward the S′ side, several analogues
were targeted. By application of known peptidic SAR,14-16
several compounds containing groups capable of inter-
acting with the S2′-S3′ subsites of stromelysin were
designed (see Figure 2). While it was desirable to also
incorporate a P1′ group, the steric bulk of the thiadiazole
heterocycle and linking group (amine, urea) appeared
to effectively prevent this. In addition, exploration of
the thiadiazole core unit was initially of interest.
However, as related non-thiadiazole heterocycles were
ineffective stromelysin inhibitors (also see Discussion
section), this effort was abandoned. Because of the lack
of similarity between the thiadiazoles and known pep-
tide mimetics,14-16 it was extremely difficult to have
confidence in these predictions, despite the appearance
of stromelysin/inhibitor complexes20,21 and modeling
studies, as these leads were so novel. Overall, through
a “loose” (albeit incorrect, vide infra) application of
peptidic hydroxamate SAR, this effort led to a series of
thiadiazole benzenepropanamides, of which the best
The thiadiazole urea analogues were synthesized as
shown in Schemes 2-5. The simple thiadiazole ureas
13 were generally prepared by condensation of 11 with
2-amino-1,3,4-thiadiazole-2-thiol (12a ) as shown in
Scheme 2. In one case, an N-substituted thiadiazole
(12b) was used. The isocyanates 11 utilized were either
commercially available or were generated by Curtius
rearrangement23 of the corresponding carboxylic acid
and used in situ. A desthiocarbonyl analogue (15) was
also prepared within this series. Treatment of 14 with
benzyl isocyanate provided 15. To further evaluate the
role that the thiol tautomer may play in MMP inhibi-
tion, a 3-methyl analogue (18) was prepared as shown
in Scheme 3. Reaction of 1-methylthiosemicarbazide 16,
prepared by J ensen’s procedure,24 with thiocarbonyl-
diimidazole in warm DMF gave 17. Exposure of this
heterocycle to phenethyl isocyanate, as described above
for 12a , did not provide any of the desired product.
However, treatment of 17 with sodium hydride followed
by phenethyl isocyanate afforded 18 in good yield.
The general strategy for the preparation of 13 was
readily adapted toward the synthesis of the more
complex, amino acid-derived thiadiazoles as depicted in
Scheme 4. The isocyanate of the desired amino acid 20
was formed by treating amino acid methyl ester 19 with
phosgene and pyridine, following the procedure recently

Thiadiazole Urea MMP Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 9 1527
Sch em e 3. Synthesis of Urea 18a
a
Reagents: (i) 1,1′-thiocarbonyldiimidazole, THF; (ii) NaH, THF, PhCH2CH2NCO.
Sch em e 4. Synthesis of Amino Acid-Derived Thiadiazole Ureasa
a
Reagents: (i) ClCOCl, CH2Cl2, pyridine; (ii) THF; (iii) R2NH2, EtOH.
Sch em e 5. Synthesis of Amino Acid-Derived Thiadiazolesa
a
Reagents: (i) t-BDMSCl, imidazole, DMF; (ii) ammonium formate, Pd/C, MeOH; (iii) ClCOCl, CH2Cl2, pyridine; (iv) THF; (v) Bu4NF,
THF; (vi) 40% MeNH2.
outlined by Nowick.25 The prerequisite methyl ester
starting materials 19 were either commercially avail-
able or prepared by standard methods. The reaction of
isocyanate 20 with thiadiazole 12a in THF provided
urea 21 in acceptable yield. The ester analogues were
converted to amide 22 by treatment of 21 with the
desired amine, either neat or diluted with ethanol.
Nowick reported25 that no racemization occurred during
the formation of 20. However, some loss of chirality is
possible during the conversion of the isocyanate to 21,
as well as the formation of amide 22 from ester 21.
Chiral HPLC analysis (Welk-O (R,R) column; 30%
2-propanol/hexane) of 46 and rac-46 indicated that no
racemization had occurred. Other analogues were not
evaluated.
Analogues containing either a serine or tyrosine
amino acid derived-thiadiazole were synthesized as
shown in Scheme 5. Protection of N-CBZ-L-serine meth-
yl ester (23a ) or N-CBZ-L-tyrosine methyl ester (23b)
with tert-butyldimethylsilyl chloride provided silyl ethers
24a and 24b. Deprotection of these carbamates under
transfer hydrogenation conditions provided amines 25a
and 25b. Conversion of these amines to isocyanates 26a
and 26b, as detailed above, using phosgene and pyri-

1528 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 9
Ta ble 1. Physical Data for Thiadiazole Amines and Ureas
J acobsen et al.
compd
core structure
R1
R2
mp (°C)
formula
anal.
12b
31
32
33
34
35
36
37
38
39
15a
40
18b
41
42
43
44
10
10
10
10
10
10
13
13
13
13
13
13
13
13
13
13
13
Et
Ph
CH2Ph
(CH2)2Ph
(CH2)4Ph
CH2-2-furyl
H
H
H
H
H
H
H
Et
H
H
H
139-140
213-215
138-141
146-148
117-119
131-133
227-228
235-237
232-233
200-202
235-237
196-198
222-223
153-154
176-178
175-177
198-201
C4H7N3S2
C8H7N3S2
C9H9N3S2
C10H11N3S2
C12H15N3S2
C7H7N3S2
C9H8N4S2O
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
Ph
4-MeO-Ph
4-F-Ph
CH2Ph
CH2Ph
(CH2)2Ph
(CH2)2Ph
(CH2)2Ph
(CH2)3Ph
(CH2)4Ph
CH2-naphth
C10H10N4S2O2
C9H7N4S2OF
C10H10N4S2O
C10H10N4SO
C11H12N4S2O
C12H14N4S2O
C13H16N4OS2
C12H14N4S2O
C13H16N4S2O
C14H12N4S2O
a
b
See Scheme 2. See Scheme 3.
Ta ble 2. Physical Data for Thiadiazole Ureas
compd
R1
R2
mp (°C)
method yield (%)
formula
anal.
45
46
H
NHMe
NHMe
NHMe
244-246
234-236
228
246-247
110-112
240-241
192
195-196
102-105
225-227
198-200
232-233
228-230
114-116
175-177
196-197
206-211
198-200
207-210
185
B
B
B
B
B
B
B
B
A
B
B
B
B
63
51
81
77
41
23
49
47
58
54
73
94
91
97
31
61
77
61
34
85
79
63
50
30
67
56
67
55
39
46
C6H9N5O2S2‚(H2O)1/3
C13H15N5O2S2‚(H2O)1/4
C13H15N5O2S2
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
CH2Ph
en t-46 ent-CH2Ph
47
48
49
29a
50
51
52
29b
53
54
55a
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
CH2cyclohexane NHMe
iBu
Ph
CH2OH
CH2OCH2Ph
CH2Ph
C13H21N5O2S2‚(CH2Cl2)1/8
NHMe
NHMe
NHMe
NHMe
OMe
NHMe
NHMe
NHMe
NHMe
OH
NH2
NMe2
NHiPr
NHCH2Ph
veratrylamine
NHCH2cyclohexane
morpholine
pyrrolidine
thiomorpholine
N-benzylpiperazine
1-(2-pyridyl)piperazine
C10H17N5O2S2‚(H2O)1/4
C12H13N5O2S2
C7H11N5O3S2‚(MeOH)1/2
C14H17N5O3S2‚(H2O)1/3
C13H14N4O3S2‚(C6H14
)
1/8
CH2Ph-p-F
CH2Ph-p-OH
CH2Ph-p-OMe
CH2Ph-p-NO2
CH2Ph
C13H14FN5O2S2‚(C4H8O2)1/8
C13H15N5O3S2‚(H2O)1/2‚(C4H2O2)1/2 C, H, N
C14H17N5O3S2
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C13H14N6O4S2
C12H12N4O3S2‚(H2O)1/4
C12H13N5O2S2‚(CH4O)1/4
C14H17N5O2S2
CH2Ph
B
B
C
C
B
C
C
C
C
C
C
B
B
B
B
B
CH2Ph
CH2Ph
C15H19N5O2S2
CH2Ph
C19H19N5O2S2‚(H2O)1/8
CH2Ph
C21H23N5O4S2
CH2Ph
C19H25N5O2S2‚(H2O)1/6
C16H19N5O3S2‚(H2O)2/5
C16H19N5O2S2‚(H2O)1/3
C16H19N5O2S3
CH2Ph
213-216
185
210-214
210-213
220-222
CH2Ph
CH2Ph
CH2Ph
C23H26N6O2S2‚(H2O)1/4
CH2Ph
C21H23N7O2S2
CH2Ph
1-(2-pyrimidinyl)piperazine 235-237
C20H22N8O2S2
CH2Ph
4-MeO-phenylpiperazine
1-(2-pyridyl)piperazine
NHMe
201-203
240-241.5
229-232
233-235
C23H26N6O3S2
CH2Ph-p-F
CH2C6F5
CH2C6F5
C21H22FN7O2S2‚(H2O)1/8
C13H10F5N5S2O2
1-(2-pyridyl)piperazine
C21H18F5N7O2S2‚(H2O)1/3
dine, proceeded uneventfully. Exposure of each to 12a
provided thiadiazoles 27a and 27b. Each of these
intermediates was deprotected using tetrabutylammo-
nium fluoride to provide the hydroxyl derivatives. The
methylamide analogues 29a and 29b were prepared
under the standard conditions. The physical data for all
analogues synthesized are shown in Tables1 and 2.
Resu lts a n d Discu ssion
Three slightly different assays were utilized for
enzyme inhibition studies of the analogues reported
herein. An enzymatic assay using â-casein as the
substrate was utilized in the broad screening effort. This
particular assay was also used for the evaluation of

Thiadiazole Urea MMP Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 9 1529
Ta ble 3. Stromelysin Inhibition for Thiadiazole Amines and Ureas
compd
core structure
R1
R2
IC50 str (µM)a
Ki str (µM)b
12b
31
32
33
34
35
36
37
38
39
44
15 (desthio)
40
18 (4-methyl)
41
42
43
1
2
3
10
10
10
10
10
10
13
13
13
13
13
13
13
13
13
13
13
10
10
13
13
Et
Ph
CH2Ph
(CH2)2Ph
(CH2)4Ph
CH2-2-furyl
H
H
H
H
H
H
H
H
Et
H
H
>2000
190
97
47
318
289
99
Ph
4-MeO-Ph
4-F-Ph
CH2Ph
CH2-naphth
CH2Ph
(CH2)2Ph
(CH2)2Ph
(CH2)2Ph
(CH2)3Ph
(CH2)4Ph
69
16
29
23
>2000
7
694
>1000
16
34
97
173
143
125
C
12H23
2-Cl-6-Me-Ph
H
H
CH2CO2Et
4-Cl-2-Me-Ph
85
119
4
a
b
These analogues were evaluated against stromelysin-1 using the screening assay. IC50 values are provided. These compounds were
evaluated against stromelysin-1 using the continuous fluorescence assay. Ki values are provided.
Ta ble 4. Stromelysin and Gelatinase A Inhibition for
Thiadiazole Urea Methylamides
Ta ble 5. Stromelysin and Gelatinase A Inhibition for
Thiadiazole Ureas
compd
R1
Ki str (µM)a
Ki gel (µM)a
45
46
en t-46
47
48
49
29a
50
H
166
0.71
5.3
4.1
17
4.1
31
2.0
85
>100
31
Ki str
Ki gel
compd
R2
R3
(µM)a (µM)a
CH2Ph
ent-CH2Ph
CH2cyclohexane
iBu
Ph
CH2OH
>200
46
51
52
29b
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
NHMe
OMe
H
H
F
OH
OMe
NO2
H
H
H
H
H
H
H
H
H
H
H
0.71
0.29
0.10
0.40
0.46
0.33
3.2
3.3
2.3
1.3
0.51
0.49
31
17
8.9
21
16
15
67
>200
41
NHMe
NHMe
NHMe
NHMe
OH
>200
CH2OCH2Ph
52
3
>200
>200
226
a
All analogues were evaluated against stromelysin-1 and
NH2
gelatinase A using the PCA assay. Ki values provided.
NMe2
NHiPr
NHCH2Ph
many of the thiadiazole analogues reported in Table 3.
A number of the thiadiazoles listed in Table 3 were
evaluated against stromelysin using a continuous fluo-
rescence inhibition assay. In addition, a particle con-
centration fluorescent assay (PCFA) was developed
based on that reported by Manndetta26 for screening
HIV protease inhibitors. This assay was used for the
evaluation of the analogues reported in Tables 4 and 5.
Most analogues were also evaluated for their ability to
inhibit TNF-R production after an endotoxin challenge.27
Our initial screening efforts focused on the thiadiazole
amines (10) and thiadiazole ureas (13). As shown in
Table 3, little improvement in activity was observed for
the amine analogues as compared to 1 and 2. For
example in comparison to 2, only a 4-fold increase in
MMP inhibition was attained with 33, a 47 µM inhibi-
tor. Substituents on the aryl ring of 2 were not critical,
15
6.2
veratrylamine
NHCH2cyclohexane
morpholine
0.81 >200
0.93
0.66
0.38
0.86
0.52
0.42
1.0
32
20
12
29
1.5
16
6.0
1.8
3.0
0.77
pyrrolidine
thiomorpholine
N-benzylpiperazine
(2-pyridyl)piperazine
2-(pyrimidinyl)piperazine
4-MeO-phenylpiperazine
(2-pyridyl)piperazine
NHMe
H
H
H
F
0.27
pentafluorob 0.018
pentafluorob 0.014
(2-pyridyl)piperazine
a
All analogues were evaluated against stromelysin-1 and
gelatinase A using the PCA assay. Ki values are provided. Ph-
b
R3 ) C6F5.
as 31 was equipotent. Only the benzyl (32) and phen-
ethyl (33) analogues represented a modest improvement
in activity over 2. The N-ethyl analogue, 12b , was

1530 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 9
J acobsen et al.
completely inactive. The inhibition of stromelysin was
slightly improved for compounds in the urea series (13).
By simply varying the chain length and substitution
pattern of 4, roughly a 17-fold improvement in stromel-
ysin inhibition was noted. In this case, 40, with a
2-carbon side chain, was the most effective inhibitor,
with a Ki of 7 µM. As compared to 4, both the benzyl
(39) and phenpropyl (42) analogues also displayed
increased potency. Extending the side chain further
resulted in diminished potency, although bulky substit-
uents (44) were still tolerated. Like 4, phenyl 36 and
the 4-methoxy analogue 37 were weak inhibitors. In-
terestingly, incorporation of a 4-fluoro group (38) did
result in a slight (ca. 3-fold) improvement in stromelysin
inhibition over 4 and 36. The thiocarbonyl group of 40
is essential, as the desthio isomer 15 was devoid of
activity. Additionally, methyl substitution at the aryl
urea nitrogen (41), or the 3-nitrogen on the thiadiazole
ring (18), led to a complete loss of activity. These results
suggest that these hydrogens are necessary for enzyme
interaction. It is also possible that the 100-fold loss in
activity of 18 as compared to 40 is due to the require-
ment of the thio tautomer.
F igu r e 3. Hydrogen bonding between thiadiazole inhibitor
70 and the stromelysin active site. Dashed lines indicate
potential hydrogen bonds between protein and inhibitor het-
eroatoms. Hydrogen positions must be inferred from heavier
atom positions.
the NH and carbonyl group of the amide participate in
hydrogen-bonding interactions.
A number of other secondary and tertiary amides
were evaluated within this series. In general, a rela-
tively flat SAR was observed with the secondary amides
inhibiting stromelysin 0.5-1.3 µM. The tertiary amines
were slightly more effective, having Ki’s of 0.39-1.0 µM.
Interestingly, several compounds were identified which
had moderate activity against gelatinase. Of these, 60,
66, and 68 were the most effective (1.5-6.0 µM).
Thiadiazole 66, which contained a 1-(2-pyridyl)pipera-
zine amide, was unique, inhibiting stromelysin at 0.50
µM and gelatinase at 1.5 µM. Several analogues which
had para-substituents on the aryl ring, while maintain-
ing a methylamide (R2 ) NHMe), were also evaluated.
Of these, the p-fluoro analogue 52 differentiated itself
from the rest, inhibiting stromelysin at 100 nM and
gelatinase at 8.9 µM. Compounds with other para-
substituents displayed activity similar to 46. Combining
a p-fluoro R3 group with a 1-(2-pyridyl)piperazine R2
substituent provided 69, which was the only compound
from this series to have modest collagenase inhibitory
activity (13 µM). To follow up on fluoro 69, and to
support NMR structure elucidation efforts,29 two pen-
tafluorophenyl analogues incorporating either a methyl
or a 1-(2-pyridyl)piperazine amide substituent were
prepared and evaluated. Both of these compounds were
extremely effective stromelysin inhibitors (<20 nM). In
addition, 1-(2-pyridyl)piperazine 71 inhibited gelatinase
with a Ki of 770 nM, while methyl 70 was less effective,
at 3.0 µM. Overall, while the terminal amide substituent
could be widely varied, only the 1-(2-pyridinyl)pipera-
zine group provided unique activity against gelatinase.
In addition, a pentafluoro-substituted aromatic ring (70)
was required for exceptional potency against stromel-
ysin. None of the thiadiazole analogues that were tested
inhibited the proteolytic conversion of the 26-kDa
TNF-R precursor to the 17-kDa soluble cytokine.
When this work was nearly complete, an X-ray
structure of 70 complexed to stromelysin30 was eluci-
dated. Much to our surprise, as shown in Figures 3 and
4, 70 bound to stromelysin completely on the unprimed
side of the enzyme substrate binding cleft. As depicted
in Figure 3, the thiocarbonyl interacts with the catalytic
zinc with the remainder of 70 located toward the S1-
S3 portion of stromelysin. The NH on the thiadiazole
One of our primary interests in the thiadiazole urea
series was to develop a SAR around 3, the most potent
of the screening leads. In particular, we were interested
in the incorporation of a L-amino acid side chain,
effectively providing 7. Compounds of this nature were
roughly designed29 through consideration of the MMP
peptide literature14-16 and modeling efforts, which
indicated that the R1 and R2 groups of 7 could be
accommodated by the S2′ and S3′ pockets, respectively.
Shown in Table 4 is a quick survey of thiadiazole amides
derived from a series of L-amino acids. While the simple
methylamide (45) was relatively inactive (Ki 166 µM)
like 3, introduction of a phenylmethyl R1 (“phe”) group
provided a significant improvement in activity, with 46
having a Ki of 0.71 µM. The corresponding enantiomer,
ent-46, was less potent, although with a Ki of 5.0 µM
was not only surprisingly more effective than the
screening leads but also equipotent to 40. Analogues
derived from several other amino acids were at least
3-fold less active, with 48 and serine 29a particularly
ineffective. Several compounds within this series did
display minimal activity against gelatinase, with 46
having a Ki of 31 µM. None of these analogues were
effective collagenase inhibitors.
With the moderate increase in potency for 46 over the
screening leads, a number of analogues were evaluated
which had different substituents at the methylamide
position (R2, see 30). In addition, several functional
groups at the para-position of the aryl ring (R3) were
explored, as shown in Table 5. Both the primary amide
(56) and dimethylamide (57) derivatives were ineffective
as compared to 46, with Ki’s of 3.3 and 2.3 µM,
respectively. Surprisingly, the methyl ester 51 was an
effective MMPI,28 more potent than 46. In comparison
to 40, incorporation of an ester group resulted in a 23-
fold enhancement in stromelysin inhibitory activity. The
corresponding carboxylic acid 55 was also slightly more
active than 40. The potency of 51 as compared to 46 is
contradictory to the MMP peptide literature, where a
secondary amide is generally required at P3′, since both

Thiadiazole Urea MMP Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 9 1531
F igu r e 4. View of 70 bound to stromelysin. Hydrophobic interactions in the P1 and P3 enzyme binding sites are illustrated.
core participates in a bifurcated hydrogen bond to the
carboxylate of glutamate 202. The other thiadiazole
nitrogen (N4) accepts a hydrogen bond from alanine 167.
Furthermore, both urea hydrogens participate in a
hydrogen bond to the carbonyl of alanine 167 on the
stromelysin backbone. The unique hydrogen bonding of
the thiadiazole core, in part, indicates why 15, 18, and
41 were ineffective stromelysin inhibitors, as well as
why other related heterocycles were not identified
through the screening effort. Furthermore, the general
requirement of a phenethyl side chain (i.e., 40, 46) is
obvious from Figure 4, where this group participates in
a key π-π-stacking interaction with tyrosine 155.
Fluoro substitution on the phenyl can enhance this
interaction and also provide a hydrogen bond to the
structural water located near histidine 166. Presumably
both features contribute to the exceptional potency of
the pentafluoro analogues (70, 71). When the phenyl
group is replaced with smaller groups, the π-stacking
interaction is absent, and accordingly, the stromelysin
activity fell off dramatically. From the structural re-
sults, the carbonyl of the methylamide forms a hydrogen
bond with alanine 169 on the stromelysin backbone as
shown. The NH group on the amide does not interact
with the protein, perhaps explaining the surprising
potency of the tertiary amides and ester 71. Also
apparent from the X-ray results is that the amide
substituent is directed toward solvent, supporting the
SAR where a wide variety of substituents could be
incorporated at this site with minimal loss of activity.
Presumably the 1-(2-pyridyl)piperazine substituent of
66, 69, and 71 interacts with gelatinase A at a remote
site; however this has not been confirmed by modeling.
The X-ray results of the 70-stromelysin complex also
point out why compounds from this series are selective
for stromelysin. Significant enzyme differences are
found for the MMPs in the unprimed side of the enzyme
cleft (S1-S3), as compared to the primed side (S1′-S3′)
where most of the known inhibitors bind. For instance
in collagenase, a serine replaces tyrosine 155 of stromel-
ysin, effectively eliminating the π-stacking observed for
70. These results presumably explain why only one
analogue from this series was (only slightly) active
against collagenase. Furthermore, alanine 169 in stromel-
ysin is a glutamine in collagenase, which would also not
be expected to have a positive interaction with the
terminal amide of 46 and analogues. In fact, the
potencies obtained for several of these inhibitors against
stromelysin are quite remarkable given that they do not
possess substituents capable of interacting with the S1′
recognition site. Clearly, the SAR of this series combined
with the X-ray results indicate that the design of a
broad-based thiadiazole urea MMP inhibitor will be a
major challenge.
Con clu sion
The incorporation of an amino acid side chain into a
thiadiazole series based on ureas 3 and 4 provided a
substantial improvement in enzyme inhibition proper-
ties. In general, a substituted phenylalanine provided
for superior enzyme inhibitory activity. Many analogues
had Ki’s of less than 0.5 µM against stromelysin, with
two of the p-fluorophenylalanine-derived thiadiazoles,
52 and 69, effective at 100 and 270 nM, respectively.
The use of a pentafluorophenylalanine substituent
provided a further gain in potency, with both 70 and
71 inhibiting stromelysin at <20 nM. The SAR observed
for this series is consistent with the crystallographic
results of 70 complexed to stromelysin, which, in itself,
is quite unique as the inhibitor is complexed solely on
the unprimed side of the enzyme cleft. Important
features of this structure involve the thiadiazole urea
moiety exquisitely hydrogen-bonded to the stromelysin
â-sheet, with the phenylalanine group undergoing a
critical π-stacking interaction with tyrosine 155. Fluoro
substitution on the aryl ring improved activity consider-
ably by enhancing the π-π-stacking interaction and by
participating in hydrogen bonding with a structural
water located near the para-position of the phenyl ring.
The terminal amide was widely varied with minimal
gain or loss of stromelysin in vitro activity. The X-ray
structure is consistent with these results as the amide
(or ester) substituent is directed toward solvent, with
only the carbonyl group participating in hydrogen
bonding to stromelysin. Most compounds within this
series were selective for stromelysin. However, the
incorporation of a 1-(2-pyridyl)piperazine group at the
terminal amide position often enhanced gelatinase
activity, with 71 having a Ki of 0.77 µM. This hetero-
cyclic substituent, in combination with a p-fluoro group
on the phenylalanine ring, resulted in 69, the only
analogue within this series to inhibit collagenase (13
µM). Nonetheless, a potent, broad-based inhibitor was
not found within this series. The lack of broad-based

1532 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 9
J acobsen et al.
126.69, 45.29, 34.66; MS (EI) m/z 237 (M+), 146, 133, 91, 105.
Anal. (C10H11N3S2).
activity is consistent with the mode of binding as
observed in the stromelysin complex crystal structure
for 70. Overall, the incorporation of a substituted
phenylalanine side chain into a thiadiazole urea tem-
plate provided a novel series of stromelysin-selective
inhibitors with a potency gain of over 6000-fold attained
over the screening leads. In addition, proper choice of
an amide group resulted in modest gains in gelatinase
potency (770 nM). The further chemical and pharma-
cological evaluation of this series will be reported in due
course.
Typ ica l P r oced u r e for th e P r ep a r a tion of Th ia d ia zole
Ur ea s 13. N-(4,5-Dih yd r o-5-th ioxo-1,3,4-th ia d ia zol-2-yl)-
N′-(2-p h en eth yl)u r ea (40). A solution of 5-amino-1,3,4-
thiadiazole-2-thiol (12a ; 2.0 g, 15 mmol) and phenethyl
isocyanate (11, R2 ) (CH2)2Ph; 2.3 g, 16 mmol) in THF (50
mL) was stirred overnight at room temperature. The THF was
removed in vacuo and the residue purified by preparative
reversed-phase chromatography (C18, acetone/water). Solvent
was removed from the purified fractions to yield the product
(2.68 g, 64%) as a white solid (mp 196-198 °C): 1H NMR (300
MHz, DMSO-d6) δ 13.79 (br s, NH), 10.78 (br s, NH), 7.19-
7.34 (m, 5 H), 6.60 (br s, NH), 3.35 (m, 2 H), 2.75 (t, J ) 7.3
Hz, 2 H); 13C NMR (75 MHz, DMSO-d6) δ 183.0, 154.0, 153.3,
138.9, 128.6, 128.3, 126.1, 40.8, 35.2; MS (EI) m/z 280 (M+),
133, 105, 91, 77, 65. Anal. (C11H12N4S2O).
Exp er im en ta l Section
Ch em istr y. Thin-layer and flash chromatography utilized
E. Merck silica gel (230-400 mesh). Melting points were taken
on a Thomas-Hoover capillary melting point apparatus and
are uncorrected. Mass spectra, infrared spectra, and combus-
tion analyses were obtained by the Physical and Analytical
Chemistry Department of Pharmacia & Upjohn. 1H NMR
spectra were recorded at 300 MHz with a Bruker model AM-
300 spectrometer.
N-(1,3,4-Th ia d ia zol-2-yl)-N′-(p h en ylm eth yl)u r ea (15). A
solution of 2-amino-1,3,4-thiadiazole (14; 2.20 g, 21.7 mmol)
and benzyl isocyanate (2.90 g, 21.7 mmol) in dry THF (100
mL) was stirred overnight at room temperature, during which
time the product crystallized. The THF was removed in vacuo,
and the residue was thoroughly triturated with ether to yield
4.77 g (94%) of the product as a white solid (mp 235-257 °C):
IR (mineral oil) 3348, 2953, 2925, 2854, 1700, 1555, 1456, 1228
In cases where synthetic intermediates or products were
isolated by “aqueous workup (organic solvent, drying agent)”,
the procedure was to quench the reaction with H2O, dilute with
the indicated organic solvent, separate the organic layer,
extract the aqueous layer several times with the organic
solvent, dry the combined organic layers with the indicated
drying agent, filter off the drying agent, and remove solvent
using a rotary evaporator at reduced pressure. When “basic
workup (organic solvent, aqueous basic solvent, drying agent)”
is indicated, the procedure was similar to aqueous workup,
except the indicated aqueous base was used instead of H2O.
When “acidic workup (organic solvent, organic solvent, drying
agent)” is indicated, the procedure was to dilute the reaction
mixture with the first indicated solvent, extract the organic
solution several times with 10% HCl, basify the combined
acidic layers with solid KOH, extract the basic solution with
the second indicated organic solvent several times, dry the
organic layers with the indicated drying agent, filter off the
drying agent, and remove solvent using a rotary evaporator
under reduced pressure. Tetrahydrofuran (THF) and ether
were distilled from sodium and benzophenone. Dichloromethane
was distilled from calcium hydride, and DMF was dried over
3-Å molecular sieves. All other solvents were EM Science
HPLC grade, distilled in glass. Phosgene in toluene (CAU-
TION: phosgene is highly toxic and should be used with
extreme care) was purchased from Fluka Chemie AG or
Columbia. All reactions were run under nitrogen or argon.
1
cm-1; H NMR (300 MHz, DMSO-d6) δ 11.02 (br s, 1 H), 8.99
(br s, 1 H), 7.13-7.36 (m, 6 H), 4.38 (d, J ) 5.9 Hz, 2 H); 13C
NMR (75 MHz, DMSO-d6) δ 160.44, 153.64, 147.43, 139.30,
128.32, 127.10, 126.90, 43.11; MS (EI) m/z 234 (M+), 128, 107,
106, 102, 101, 91, 77, 65, 51; HRMS (EI) calcd for C10H10N4-
SO 234.0575, found 234.0573. Anal. (C10H10N4SO).
5-Am in o-3-m eth yl-1,3,4-th ia d ia zole-3H-2-th ion e (17).
1-Methylthiosemicarbazide24 (16; 235 mg, 2.23 mmol), 1,1′-
thiocarbonyldiimidazole (0.660 g, 3.35 mmol), and anhydrous
THF (20 mL) were combined in a dry flask under N2. The
solution was heated at reflux until complete as determined
by TLC (2 h). The solvent was removed in vacuo, the residue
was purified by chromatography (silica, 1% methanol in
chloroform), and the final product was recrystallized from
methanol/chloroform to yield 17 (199 mg, 61%) as a white solid
(mp 192-193 °C): IR (ATR deposited from MeOH) 3140, 1623,
1556, 1380, 1343, 1106, 1012, 668, 625 cm-1 1H NMR (300
;
MHz, DMSO-d6) δ 7.26 (s, 2 H), 3.34 (s, 3 H); MS (EI) m/e 147
(M+), 105, 72, 41. Anal. Calcd for C3H5N3S2: C, 24.48; H, 3.42;
N, 28.54. Found: C, 24.40; H, 3.35; N, 28.78.
N-(4,5-Dih yd r o-4-m et h yl-5-t h ioxo-1,3,4-t h ia d ia zol-2-
yl)-N′-(2-p h en eth yl)u r ea (18). Sodium hydride (60% disper-
sion in mineral oil, 34 mg, 0.85 mmol) and anhydrous THF
(2.0 mL) were combined in a dry flask under N2. The suspen-
sion was cooled to -78 °C and treated with 5-amino-3-methyl-
1,3,4-thiadiazole-3H-2-thione (17; 125 mg, 0.850 mmol) in
anhydrous THF (2.0 mL). After stirring for 15 min at -78 °C,
a solution of phenethyl isocyanate (125 mg, 0.850 mmol) in
anhydrous THF (1.0 mL) was added via syringe. The mixture
was allowed to slowly warm to room temperature and was
stirred overnight. The solution was carefully quenched with
the addition of water and acetic acid. The THF was removed
in vacuo and the residue partitioned between EtOAc (2×) and
water. The EtOAc layers were washed with brine, dried over
Na2SO4, filtered, and concentrated. The residue was recrystal-
lized from ethanol to afford 213 mg (85%) of N-[4,5-dihydro-
4-methyl-5-thioxo-1,3,4-thiadiazol-2-yl)-N′-(2-phenylethyl)-
urea as a white crystalline product (mp 222-223 °C): IR
(mineral oil) 3377, 1686, 1663, 1578, 1555, 1238, 1131, 700
Typ ica l P r oced u r e for th e P r ep a r a tion of Th ia d ia zole
Am in es 10. 5-(2-P h en eth yla m in o)-1,3,4-th ia d ia zole-3H-
2-th ion e (33). Hydrazine hydrate (1.25 mL, diluted with 1.25
mL of water) was added to a solution of phenethyl isothiocy-
anate (8, R1 ) (CH2)Ph; 3.82 g, 23.4 mmol) and ethanol (25
mL) at room temperature. The solution became warm to the
touch and turned slightly yellow. The solution was allowed to
stir for 15 min at which time an ice bath was applied to induce
crystallization. The resulting precipitate was collected by
filtration, washed with ice-cold ethanol, and dried to yield
4-phenethyl-3-thiosemicarbazide (9, R1 ) (CH2)Ph; 3.36 g, 73%)
as a white solid. This material was used without further
purification. Carbon disulfide (1.11 mL, 18.4 mmol) was added
to a solution of 4-(2-phenethyl)-3-thiosemicarbazide (9, R1
)
(CH2)Ph; 1.8 g, 9.2 mmol) and DMF (15 mL). The mixture was
heated at 65 °C with stirring for 2 h. The solution was allowed
to cool slowly and was stirred at room temperature for 48 h.
The solution was then poured into water forming a precipitate,
which was filtered, washed with water, and dried to yield crude
product (1.95 g, 89%). This material was recrystallized from
hot ethanol to give 33 (mp 146-148 °C): 1H NMR (300 MHz,
DMSO-d6) δ 7.64 (br t, 1 H), 7.18-7.33 (m, 5 H), 3.38 (m, 2
H), 3.29 (br s, 1 H), 2.83 (t, J ) 7.2 Hz, 2 H); 13C NMR (75
MHz, DMSO-d6) δ 180.95, 161.18, 139.48, 129.13, 128.83,
1
cm-1; H NMR (300 MHz, DMSO-d6) δ 10.94 (br s, 1 H), 7.28
(m, 5 H), 6.66 (br m, 1 H), 3.68 (s, 3 H) 3.34 (m, 2 H), 2.75 (m,
2 H); MS (EI) m/e 294 (M+), 147, 105, 91, 71, 51, 42. Anal.
(C12H14N4OS2).
Meth od A: Typ ica l P r oced u r e for th e F or m a tion of
Th ia d ia zole Ester 21. N-[[(4,5-Dih yd r o-5-th ioxo-1,3,4-
th ia d ia zol-2-yl)a m in o]ca r bon yl]-L-p h en yla la n in e Meth yl
Ester (51). The isocyanate of L-phenylalanine methyl ester
was synthesized following the general procedure of Nowick.25

Thiadiazole Urea MMP Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 9 1533
Thus, phosgene (7.90 mL, 15.2 mmol, 1.93 M in toluene) was
added to a mixture of L-phenylalanine methyl ester hydro-
chloride (19, R1 ) CH2Ph; 2.57 g, 11.9 mmol), pyridine (3.90
mL, 48.2 mmol), and CH2Cl2 (40 mL) at 0 °C. The mixture
was stirred for 2 h at 0 °C and then diluted with EtOAc (150
mL). The organic layer was washed with cold 5% HCl (2 × 40
mL) and cold brine (50 mL). The organic layers were dried
(MgSO4), filtered, and concentrated to give 2.52 g of isocyanate
20 (R1 ) CH2Ph) as an oil which was carried on crude.
2.95-3.20 (m, 2 H); MS (FAB) m/z 325 (MH+), 324, 129, 71,
57, 43; HRMS (FAB) calcd for C12H12N4O3S2 + H1 325.0429,
found 325.0411.
L-N -(Be n zyloxyca r b on yl)-O-[(1,1-d im e t h yle t h yl)d i-
m eth ylsilyloxy]ser in e Meth yl Ester (24a ). A solution of
L-N-(benzyloxycarbonyl)serine (23a ; 3.73 g, 14.7 mmol) in DMF
(30 mL) was treated with imidazole (1.10 g, 16.2 mmol) and
tert-butyldimethylsilyl chloride (TBDMSCl; 2.44 g, 16.2 mmol).
The resultant solution was stirred for 60 h at room tempera-
ture. The DMF was removed in vacuo to give a white slurry.
This material was diluted with Et2O (100 mL) and washed
with 0.5 N HCl (2×), dilute NaHCO3, and brine (40 mL). The
organic layer was dried over anhydrous Na2SO4, filtered, and
concentrated under reduced pressure to yield 5.33 g (99%) of
24a as a clear, colorless oil: 1H NMR (300 MHz, CDCl3) δ
7.25-7.45 (m, 5 H), 5.58 (d, J ) 3.8 Hz, 1 H), 5.13 (ABq, J AB
) 12.3 Hz, ∆γ ) 6.6 Hz, 2 H), 4.35-4.45 (m, 1 H), 3.95 (d of
ABq, J d ) 2.8 Hz, J AB ) 10.1 Hz, ∆γ ) 67.3 Hz, 2 H), 3.74 (s,
3 H), 0.85 (s, 9 H), 0.02 (s, 3 H), 0.00 (s, 3 H).
A solution of the isocyanate (11.9 mmol) and THF (35.0 mL)
was cooled to 0 °C. To this was added 5-amino-1,3,4-thiadia-
zole-2-thiol (12a ; 1.60 g, 12.0 mmol). The solution was stirred
at 0 °C for 1 h and at room temperature for 16 h. Aqueous
workup (EtOAc, MgSO4) and purification by flash chromatog-
raphy (5% MeOH/CH2Cl2) gave 2.35 g (58%) of the urea as a
yellow foam (mp 102-105 °C): [R]25D +56° (c 0.94, CHCl3); IR
(mineral oil) 3195, 3088, 3065, 3027, 3002, 1742, 1697, 1575,
1543, 1495, 1321, 1218, 1181, 1067, 1054, 777, 742, 702, 687
cm-1 1H NMR (300 MHz, DMSO-d6) δ 7.10-7.40 (m, 5 H),
;
6.92 (d, J ) 7.7 Hz, 1 H), 4.50-4.65 (m, 1 H), 3.66 (s, 3 H),
2.90-3.15 (m, 2 H); MS (EI) m/ z 338 (M+), 159, 133, 120, 103,
91, 88, 65. Anal. (C13H14N4O3S2).
N-[[(4,5-Dih yd r o-5-th ioxo-1,3,4-th ia d ia zol-2-yl)a m in o]-
ca r b on yl]-O-[(1,1-d im e t h yle t h yl)d im e t h ylsilyloxy]-L-
ser in e Meth yl Ester (27a ). A solution of the protected serine
derivative 24a (6.44 g, 17.6 mmol) in MeOH (125 mL) was
treated with ammonium formate (2.22 g, 35.3 mmol) and 10%
palladium on carbon (644 mg, 10 wt %). After 1 h, the mixture
was filtered through Celite, washing the residual cake with
MeOH (100 mL) and CH2Cl2 (200 mL). The filtrate was
concentrated, reconstituted in CH2Cl2 (200 mL), filtered, dried
over anhydrous Na2SO4, filtered, and concentrated under
reduced pressure to give a quantitative yield of amine 25a as
an opaque oil: 1H NMR (300 MHz, CDCl3) δ 4.45 (br s, 2 H),
3.94 (d of ABq, J d ) 3.7 Hz, J AB ) 10.1 Hz, ∆γ ) 23.0 Hz, 2 H),
3.75 (s, 3 H), 3.65-3.80 (m, 1 H), 0.86 (s, 9 H), 0.09 (s, 3 H),
0.04 (s, 3 H).
Meth od B: Typ ica l P r oced u r e for th e F or m a tion of
Th ia d ia zole Am id e 22. r-[[[(4,5-Dih yd r o-5-th ioxo-1,3,4-
t h ia d ia zol-2-yl)a m in o]ca r b on yl]a m in o]-N,N-d im et h yl-
(S)-ben zen ep r op a n a m id e (57). A solution of 21 (R1
)
CH2Ph; 200 mg, 0.591 mmol), 40% aqueous dimethylamine (1.0
mL), and ethanol (1.0 mL) was stirred for 3 days at room
temperature. The mixture was concentrated and diluted with
EtOAc. The organic layer was washed with 10% HCl (2 × 15
mL) and brine (15 mL), dried (MgSO4), filtered, and concen-
trated to provide 171 mg of crude product. Recrystallization
from hot CH2Cl2/hexane gave 126 mg (61%) of 57 as a white
solid (mp 196-197 °C): [R]25 +24° (c 0.39, DMSO); IR
D
(mineral oil) 3332, 3167, 3129, 3063, 3027, 1693, 1622, 1576,
1547, 1495, 1422, 1405, 1320, 1283, 1231, 1065, 1054, 779, 745,
699, 637 cm-1; 1H NMR (300 MHz, DMSO-d6) δ 13.86 (s, 1 H),
10.76 (s, 1 H), 7.10-7.35 (m, 5 H), 6.90-7.05 (m, 1 H), 4.85-
5.00 (m, 1 H), 2.70-3.00 (m, 2 H), 2.88 (s, 3 H), 2.81 (s, 3 H);
MS (EI) m/z 351 (M+), 306, 159, 120, 103, 101, 91, 72. Anal.
(C14H17N5O2S2).
Following procedure A, crude amine 25a was converted into
2.91 g (47%) of the desired material (27a ) as an off-white,
amorphous solid: [R]25 + 29° (c 1.00, DMSO); IR (mineral
D
oil) 1697, 1668, 1578, 1542, 1490, 1349, 1322, 1259, 1212, 1108,
1
1075, 1058, 836, 779 cm-1; H NMR (300 MHz, DMSO-d6) δ
6.89 (d, J ) 8.3 Hz, 1 H), 4.35-4.50 (m, 1 H), 3.88 (d of ABq,
J d ) 1.9 Hz, J AB ) 11.5 Hz, ∆γ ) 61.4 Hz, 2 H), 3.66 (s, 3 H),
0.82 (s, 9 H), 0.01 (s, 3 H), -0.01 (s, 3 H); MS (FAB) m/z 393
(MH+), 335, 260, 234, 160, 134, 133, 102, 75, 73. Anal.
(C13H24N4O4SiS2‚(AcOH)1/3).
Meth od C: Typ ica l P r oced u r e for th e F or m a tion of
Th ia d ia zole Am id e 22. r-[[[(4,5-Dih yd r o-5-th ioxo-1,3,4-
th iadiazol-2-yl)am in o]car bon yl]am in o]-N-(ph en ylm eth yl)-
(S)-ben zen ep r op a n a m id e (59). A solution of 21 (R1
)
N-[[(4,5-Dih yd r o-5-th ioxo-1,3,4-th ia d ia zol-2-yl)a m in o]-
ca r bon yl]-L-ser in e Meth yl Ester (28a ). A solution of 27a
(1.03 g, 2.63 mmol) in dry THF (25 mL) was treated with
tetrabutylammonium fluoride (1.0 M solution in THF, 6.30 mL,
6.30 mmol). After 1.5 h, the solution was diluted with H2O
(25 mL) and extracted into EtOAc (3 × 50 mL). The extracts
were washed with brine (50 mL), dried over anhydrous Na2-
SO4, filtered, and concentrated to give 1.03 g of a yellow foam.
The foam was purified by silica gel chromatography (2-5%
MeOH/CHCl3) to give 499 mg (68%) of the desired material
as an amorphous, white solid. An analytical sample was
crystallized from hot CH2Cl2/MeOH/hexane to give the desired
CH2Ph; 200 mg, 0.591 mmol) and benzylamine (1.0 mL) was
stirred for 3 days at room temperature. The mixture31 was
diluted with EtOAc and washed several times with 10% HCl
(2 × 20 mL) and brine (20 mL). The organic layer was dried
(MgSO4), filtered, and concentrated to give 214 mg of the crude
amide as a solid. Recrystallization from hot CH2Cl2/hexane
gave 150 mg (61%) of 59 as a white solid (mp 198-200 °C):
[R]25D +13° (c 0.46, DMSO); IR (mineral oil) 3319, 3193, 3109,
3088, 3064, 3028, 1692, 1643, 1604, 1575, 1544, 1497, 1320,
1284, 1230, 1057, 1030, 740, 698, 645 cm-1; 1H NMR (300 MHz,
DMSO-d6) δ 13.83 (s, 1 H), 10.70 (s, 1 H), 8.65-8.80 (m, 1 H),
7.10-7.40 (m, 10 H), 6.80-6.95 (m, 1 H), 4.45-4.60 (m, 1 H),
4.15-4.40 (m, 2 H), 2.85-3.10 (m, 2 H); MS (EI) m/z 413 (M+),
237, 163, 159, 133, 121, 120, 106, 91. Anal. (C19H19N5O2S2‚
(H2O)1/8).
material as white, crystalline needles (mp 180-181 °C): [R]25
D
0° (c 1.01, DMSO); IR (mineral oil) 3387, 3282, 3136, 1726,
1688, 1580, 1544, 1352, 1323, 1287, 1229, 1205, 1070, 1052,
1030 cm-1; 1H NMR (300 MHz, DMSO-d6) δ 13.80 (br s, 1 H),
10.80 (br s, 1 H), 7.03 (d, J ) 8.2 Hz, 1 H), 5.25 (t, J ) 5.2 Hz,
1 H), 4.25-4.35 (m, 1 H), 3.75-3.85 (m, 1 H), 3.55-3.70 (m, 1
H), 3.65 (s, 3 H); MS (EI) m/z 278 (M+), 174, 159, 147, 133, 88,
86, 74, 60, 59, 57. Anal. (C7H10N4O4S2‚(MeOH)1/2‚(H2O)1/2).
N-[[(4,5-Dih yd r o-5-th ioxo-1,3,4-th ia d ia zol-2-yl)a m in o]-
ca r bon yl]-L-p h en yla la n in e (55). A mixture of 51 (528 mg,
1.56 mmol), MeOH (20 mL), H2O (5.0 mL), and KOH (350 mg,
6.24 mmol) was stirred at room temperature for 16 h. The
solution was concentrated (to remove the MeOH), acidified
with 10% HCl, and extracted with EtOAc (3 × 20 mL). The
organic layers were dried (MgSO4), filtered, and concentrated
to give 490 mg (97%) of acid 55. Recrystallization from hot
CH2Cl2/hexane provided an analytical sample as a white solid
r-[[[(4,5-Dih yd r o-5-th ioxo-1,3,4-th ia d ia zol-2-yl)a m in o]-
c a r b o n y l]a m in o ]-3-h y d r o x y -N -m e t h y l-(S )-p r o p a n a -
m id e (29a ). Following general procedure B, 28a (353 mg, 1.20
mmol) was dissolved in a saturated solution of MeNH2 in EtOH
(10 mL) and stirred overnight. After workup (saturating the
aqueous layer with solid NaCl), 162 mg (49%) of the title
compound was recovered as a white, crystalline solid (mp 192
(mp 114-116 °C): [R]25 +15° (c 0.46, DMSO); IR (DRIFT)
D
3341, 3182, 3082, 3062, 3039, 3029, 3005, 2935, 2897, 1709,
1703, 1682, 1564, 1332, 1240 cm-1; 1H NMR (300 MHz, DMSO-
d6) δ 13.83 (s, 1 H), 13.00-13.25 (br s, 1 H), 10.81 (s, 1 H),
7.15-7.35 (m, 5 H), 6.75-6.90 (m, 1 H), 4.40-4.55 (m, 1 H),
°C, dec): [R]25 +10° (c 0.61, DMSO); IR (mineral oil) 3325,
D
3198, 3110, 1706, 1644, 1553, 1490, 1415, 1327, 1261, 1227,
1
1060, 689, 645 cm-1; H NMR (300 MHz, DMSO-d6) δ 13.80

1534 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 9
J acobsen et al.
(br s, 1 H), 10.80 (br s, 1 H), 7.90-8.05 (m, 1 H), 6.93 (d, J )
7.8 Hz, 1 H), 5.03 (t, J ) 5.3 Hz, 1 H), 4.05-4.15 (m, 1 H),
3.45-3.70 (m, 2 H), 2.58 (d, J ) 4.5 Hz, 3 H); MS (FAB) m/z
278 (MH+), 277, 243, 242, 186, 142, 119; HRMS (FAB) calcd
for C7H12N5O3S2 278.0381, found 278.0380. Anal. (Calcd for
C7H11N5O3S2‚(MeOH)1/2).
material was crystallized from MeOH/EtOAc to give 752 mg
(73%) of the desired material as a white, crystalline solid (mp
198-200 °C): [R]25 +72° (c 1.00, DMSO); IR (mineral oil)
D
3371, 3312, 3127, 1705, 1693, 1640, 1578, 1550, 1516, 1491,
1324, 1285, 1260, 1229, 1053 cm-1; 1H NMR (300 MHz, DMSO-
d6) δ 13.75 (br s, 1 H), 10.65 (br s, 1 H), 9.20 (s, 1 H), 7.95-
8.10 (m, 1 H), 6.91 (d, J ) 8.4 Hz, 2 H), 6.77 (d, J ) 8.1 Hz, 1
H), 6.63 (d, J ) 8.4 Hz, 2 H), 4.20-4.35 (m, 1 H), 2.84 (dd, J
) 13.8, 5.4 Hz, 1 H), 2.69 (dd, J ) 13.8, 7.5 Hz, 1 H), 2.55 (d,
J ) 4.5 Hz, 3 H); MS (FAB) m/z 354 (MH+), 196, 195, 105.
Anal. (C13H15N5O3S2‚(H2O)1/2‚(C4H2O2)1/2).
Biologica l Meth od s. A 33-kDa, truncated form of human
stromelysin-1 (stromelysin 1-255), lacking the carboxy ter-
minus, was cloned and expressed using methods analogous to
those previously described.32 The soluble pro-enzyme expressed
in E. coli was pure by SDS-PAGE and amino acid analysis
criteria after ammonium sulfate precipitation and isolated by
anion- and cation-exchange chromatography.
L-N -(Be n zyloxyca r b on yl)-O-[(1,1-d im e t h yle t h yl)d i-
m eth ylsilyloxy]tyr osin e Meth yl Ester (24b). A solution of
L-N-(benzyloxycarbonyl)tyrosine O-methyl ester (23b; 3.93 g,
11.9 mmol) in DMF (25 mL) was treated with imidazole (892
mg, 13.1 mmol) and TBDMSCl (1.98 g, 13.1 mmol). The
resultant solution was stirred for 72 h at room temperature.
The DMF was removed in vacuo to give a white slurry. This
material was diluted with Et2O (100 mL) and washed with
0.5 N HCl (2×), dilute NaHCO3, and brine (50 mL). The
organic layer was dried over anhydrous Na2SO4, filtered, and
concentrated under reduced pressure to give a quantitative
yield of the desired material as a clear, colorless oil: 1H NMR
(300 MHz, CDCl3) δ 7.25-7.45 (m, 5 H), 6.90-7.00 (m, 2 H),
6.65-6.80 (m, 2 H), 5.19 (d, J ) 7.7 Hz, 1 H), 5.10 (ABq, J AB
) 11.5 Hz, ∆γ ) 7.2 Hz, 2 H), 4.55-4.75 (m, 1 H), 3.70 (s, 3
H), 2.95-3.15 (m, 2 H), 0.97 (s, 9 H), 0.18 (s, 6 H).
N-[[(4,5-Dih yd r o-5-th ioxo-1,3,4-th ia d ia zol-2-yl)a m in o]-
ca r bon yl]-O-[(1,1-d im eth yleth yl)d im eth ylsilyloxy]-L-ty-
r osin e Meth yl Ester (27b). A solution of the protected
tyrosine derivative 24b (6.72 g, 15.2 mmol) in MeOH (100 mL)
was treated with ammonium formate (1.92 g, 30.4 mmol) and
10% palladium on carbon (672 mg). After 3 h, the mixture was
filtered through Celite, washing the residual cake with MeOH
(100 mL) and CH2Cl2 (200 mL). The filtrate was concentrated,
reconstituted in CH2Cl2 (200 mL), filtered, dried over anhy-
drous Na2SO4, filtered, and concentrated under reduced pres-
sure to give a quantitative yield of 25b as a cream-colored
solid: 1H NMR (300 MHz, CDCl3) δ 6.95-7.10 (m, 2 H), 6.70-
6.85 (m, 2 H), 4.87 (br s, 2 H), 3.80-4.00 (m, 1 H), 3.71 (s, 3
H), 3.09 (dd, J ) 13.9, 5.6 Hz, 1 H), 2.97 (dd, J ) 13.9, 7.1 Hz,
1 H), 0.97 (s, 9 H), 0.18 (s, 6 H).
En zym a tic Scr een in g Assa y. An enzymatic screening
assay utilized the incubation of activated stromelysin with
radiolabeled â-casein as the substrate, followed by measure-
ment of trichloroacetic acid (TCA)-soluble casein fragments as
a measure of enzyme activity. Stromelysin was activated by
incubation with R-chymotrypsin. â-Casein was radiolabeled by
reductive methylation using NaCNBH3 and [14C]formaldehyde
as previously described.33 Determinations of IC50 values
employed test compounds at concentrations ranging from 0.1
to 2000 µM with 4-6 replicates at each concentration. The
reaction was started in microtiter plates by adding 25 µL of
activated stromelysin (0.01 µg) to test compounds in 25 µL of
assay buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl)
containing labeled substrate (0.1 µCi ) 0.5 µg). Assays were
terminated with ice-cold TCA and centrifugation after an 18-h
incubation at 37 °C. Time course experiments showed that the
activity of stromelysin in this assay did not plateau until after
24 h, indicating that even slow-binding inhibitors would be
detected with this method. An aliquot of the supernatant was
taken for liquid scintillation counting, and data were expressed
as the counts per minute released from 14C-casein, after
subtraction of those counts released due to nonspecific hy-
drolysis (wells containing buffer and substrate only). The
reaction demonstrated first-order kinetics indicating that [14C]-
casein behaves as a single substrate and that its initial
concentration was significantly less than the Km. Actinonin
was used as a reference inhibitor in the 40 assays from which
the data reported here were obtained. The IC50 for actinonin
was 0.63 ( 0.04 µM with a between assay coefficient of
variation of 5.8%. This assay was the first used for this project
and was subsequently replaced by the Ki assays described
below. A total of 44 compounds were evaluated in both the
enzymatic screening assay for determination of IC50 values and
in the Ki assays described below for determination of Ki values.
The IC50 and Ki values were significantly correlated (r ) 0.81),
with Ki values on average being slightly lower (75% of the IC50
value).
Con tin u ou s F lu or escen ce Assa y. The continuous fluo-
rescence Ki assay monitored the stromelysin-1-catalyzed hy-
drolysis of a fluorogenic peptide substrate. The substrate was
a tryptophan-containing peptide, DNP-Pro-Leu-Ala-Leu-Trp-
Ala-Arg-NH2, internally quenched by a dinitrophenol moiety.
Stromelysin was activated by heating at 50 °C for 30 min and
could be stored at 4 °C for up to 1 week without loss of activity.
Two milliliters of buffer (20 mM Tris-HCl, 10 mM CaCl2, 10
µM ZnCl2, 5 mM CHAPS, pH 7.6) was pipetted into a 3-mL
quartz cuvette in a Perkin-Elmer LS-50 fluorescence spectro-
photometer. Inhibitor or DMSO (20 µL) was added, followed
by the addition of 10 µL of stromelysin (1.5 mg/mL). After
equilibration at 25 °C for 5 min, the reaction was started by
the addition of 2 µL of 0.0102 M peptide substrate. Fluores-
cence was measured at an excitation of 280 nM and an
emission of 340 nM.
Following procedure A, crude amine 25b was converted into
3.75 g (53%) of 27b as a yellow, amorphous solid. An analytical
sample was crystallized from CH2Cl2/hexane to give the
desired material as a white crystalline solid (mp 139-142
°C): [R]25 +65° (c 1.01, DMSO); IR (mineral oil) 3382, 1726,
D
1689, 1575, 1542, 1512, 1329, 1285, 1272, 1256, 1215, 1051,
1
923, 839, 780 cm-1; H NMR (300 MHz, DMSO-d6) δ 7.02 (d,
J ) 8.4 Hz, 2 H), 6.87 (d, J ) 7.9 Hz, 1 H), 6.74 (d, J ) 8.4 Hz,
2 H), 4.40-4.55 (m, 1 H), 3.62 (s, 3 H), 2.85-3.05 (m, 2 H),
0.91 (s, 9 H), 0.14 (s, 6 H); MS (EI) m/z 411, 379, 292, 250,
223, 222, 221, 159, 107, 83, 73, 59. Anal. (C19H28N4O4S2Si).
N-[[(4,5-Dih yd r o-5-th ioxo-1,3,4-th ia d ia zol-2-yl)a m in o]-
ca r bon yl]-L-tyr osin e Meth yl Ester (28b). A solution of 27b
(2.42 g, 5.16 mmol) in dry THF (75 mL) was treated with
tetrabutylammonium fluoride (1.0 M solution in THF, 12.4 mL,
12.4 mmol). After 1.5 h, the solution was concentrated to a
golden oil and then diluted with H2O (50 mL). After the
aqueous layer was acidified with 6 N HCl (pH 2), the product
was extracted into EtOAc (3 × 100 mL). The extracts were
washed with 0.5 N HCl (100 mL) and brine (100 mL), dried
over anhydrous Na2SO4, filtered, and concentrated to yield 1.67
g of yellow foam. The foam was purified by silica gel chroma-
tography (25 g, 3 f 4% MeOH/CHCl3) to give 1.52 g (83%) of
an amorphous, yellow solid: IR (mineral oil) 3282, 3018, 1691,
1614, 1575, 1546, 1515, 1484, 1321, 1222, 1180, 1055, 775, 729,
1
685 cm-1; H NMR (300 MHz, DMSO-d6) δ 13.75 (br s, 1 H),
10.75 (br s, 1 H), 9.25 (s, 1 H), 6.92 (d, J ) 8.4 Hz, 2 H), 6.80
(d, J ) 7.7 Hz, 1 H), 6.66 (d, J ) 8.4 Hz, 2 H), 4.40-4.50 (m,
1 H), 3.63 (s, 3 H), 2.80-3.05 (m, 2 H); MS (EI) m/z 216, 178,
159, 136, 133, 108, 107, 88, 77; MS (FAB) m/z 355 (MH+), 354,
196, 186, 134, 88, 77; HRMS (FAB) calcd for C13H15N4O4S2
355.0535, found 355.0533.
r-[[[(4,5-Dih yd r o-5-th ioxo-1,3,4-th ia d ia zol-2-yl)a m in o]-
ca r b on yl]a m in o]-4-h yd r oxy-N-m et h yl-(S)-b en zen ep r o-
p a n a m id e (29b). Following procedure B, 28b (1.03 g, 2.91
mmol) gave 955 mg of 29b as a white, amorphous solid. The
P a r ticle Con cen tr a tion F lu or escen t Assa y. This assay,
based on that described by Mandetta,26 was used to assess
inhibitory properties of compounds against three MMPs:

Thiadiazole Urea MMP Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 9 1535
(11) Gearing, A. J . H.; Beckett, P.; Christodoulou, M.; Churchill, M.;
Clements, J .; Davidson, A. H.; Drummond, A. H.; Galloway, W.
A.; Gilbert, R.; Gordon, J . L.; Leber, T. M.; Mangan, M.; Miller,
K.; Nayee, P.; Owen, K.; Patel, S.; Thomas, W.; Wells, G.; Wood,
L. M.; Woolley, K. Processing of tumor necrosis factor-R precur-
sor by metalloproteinases. Nature 1994, 370, 555-557.
(12) Mohler, K. M.; Sleath, P. R.; Fitzner, J . N.; Cerretti, D. P.;
Alderson, M.; Kerwar, S. S.; Torrance, D. S.; Otten-Evans, C.;
Greenstreet, T.; Weerawarna, K.; Kronheim, S. R.; Petersen, M.;
Gerhart, M.; Kozlosky, C. J .; March, C. J .; Black, R. A. Protection
against a lethal dose of endotoxin by an inhibitor of tumor
necrosis factor processing. Nature 1994, 370, 218-220.
(13) Vilcek, J .; Lee, T. H. Tumor necrosis factor. New insights into
the molecular mechanisms of its multiple actions. J . Biol. Chem.
1991, 266, 7313-7316.
collagenase, gelatinase A, and stromelysin-1. The same polypep-
tide substrate,34 labeled at the amino end with fluorescein and
biotin at the carboxyl end, was used with all three enzymes.
Hydrolysis results in the creation of two substrate fragments:
one fluorescently tagged and the other containing biotin. The
assays were conducted in microtiter plates containing buffer
(50 mM Tris-HCl, 150 mM NaCl, 5-22 mM CaCl2, 5.55 mM
CHAPS, 0-1.11 µM ZnCl2, 0.02% NaN3, pH 7.5), 100 nM
substrate, and stromelysin-1 (60 nM), collagenase (80 nM), or
gelatinase (10 nM) in a final volume of 100 µL. Various
concentrations of inhibitor, ranging from 0.001 to 200 µM, were
assayed in replicate for determination of Ki values. Enzyme
and inhibitor were allowed to preincubate (15 min) at room
temperature prior to the addition of substrate, followed by
incubation at room temperature (stromelysin-1, 1.5 h; colla-
genase, 2.5 h; or gelatinase, 1.25 h). A 50-µL aliquot of the
reaction was transferred to a plate containing avidin-coated
polystyrene beads and then processed in a IDEXX Screen
Machine 2000. Strepavidin-coated beads sequester the intact
fluorescent peptide substrate, and proteolytic activity was
followed by a decrease in fluorescent signal. Data were
analyzed by curve fitting using an appropriate equation, and
the Ki value was calculated.
(14) Schwartz, M. A.; VanWart, H. E. Synthetic inhibitors of bacterial
and mammalian interstitial collagenases. In Progress in Me-
dicinal Chemistry; Ellis, G. P., Luscombe, D. K., Eds.; Elsevier
Science Publishers B.V.: The Netherlands, 1992; Vol. 29.
(15) For recent reviews see: (a) Porter, J . R.; Millican, T. A.; Morphy,
J . R. Recent developments in matrix metalloproteinase inhibi-
tors. Exp. Opin. Ther. Patents 1995, 5, 1287-1296. (b) Brown,
P. D. Matrix metalloproteinase inhibitors: A new class of
anticancer agent. Curr. Opin. Invest. Drugs 1993, 2, 617-626.
(16) Beckett, R. P.; Davidson, A. H.; Drummond, A. H.; Huxley, P.;
Whittaker, M. Recent advances in matrix metalloproteinase
inhibitor research. Drug Discovery Today 1996, 1, 16-26.
(17) Moderate to low bioavailability, depending on species, has been
reported for BB-2516; see: Drummond, A. H.; Beckett, P.; Bone,
E. A.; Brown, P. D.; Davis, M.; Galloway, W. A. BB-2516: An
orally bioavailable matrix metalloproteinase inhibitor with
efficacy in animal cancer models. 86th Annual Meeting of the
American Association for Cancer Research, Toronto, Ontario,
Canada, Mar 18-22, 1995.
Ack n ow led gm en t. We would like to thank J im
Petke, J ohn VanDrie, Boryeu Mau, and J eff Howe for
their helpful discussions and assistance with modeling
of several of the thiadiazole analogues in a model of the
catalytic portion of stromelysin.
(18) Oleksyszyn, J .; J acobsen, A. R. U.S. Patent 5,677282, Oct 14,
1997.
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