Structure-based design and synthesis of potent matrix metalloproteinase inhibitors derived from a 6H-1,3,4-thiadiazine scaffold

Article abstract of DOI:10.1021/jm010887p

We describe a new generation of heterocyclic nonpeptide matrix metalloproteinase (MMP) inhibitors derived from a 6H-1,3,4-thiadiazine scaffold. A screening effort was utilized to identify some chiral 6-methyl-1,3,4-thiadiazines that are weak inhibitors of

Full text of DOI:10.1021/jm010887p

J . Med. Chem. 2001, 44, 3231-3243
3231
Str u ctu r e-Ba sed Design a n d Syn th esis of P oten t Ma tr ix Meta llop r otein a se
In h ibitor s Der ived fr om a 6H-1,3,4-Th ia d ia zin e Sca ffold
J o¨rg Schro¨der,† Andreas Henke,† Herbert Wenzel,† Hans Brandstetter,‡ Hans G. Stammler,§ Anja Stammler,§
Wolf D. Pfeiffer,| and Harald Tschesche*,†
Abt. Biochemie I, Universita¨t Bielefeld, Fakulta¨t fu¨r Chemie, Universita¨tsstr. 25, D-33615 Bielefeld, Germany;
Abt. Strukturforschung, Max-Plank-Institut fu¨r Biochemie, Am Klopferspitz 18a, D-82152 Martinsried, Germany;
Abt. Anorganische Chemie III, Universita¨t Bielefeld, Fakulta¨t fu¨r Chemie, Universita¨tsstr.25, D-33615 Bielefeld, Germany;
Institut fu¨r Chemie und Biochemie, Ernst-Moritz-Arndt Universita¨t Greifswald, Soldmannstr. 16/ 17,
D-17487 Greifswald, Germany
Received March 28, 2001
We describe a new generation of heterocyclic nonpeptide matrix metalloproteinase (MMP)
inhibitors derived from a 6H-1,3,4-thiadiazine scaffold. A screening effort was utilized to identify
some chiral 6-methyl-1,3,4-thiadiazines that are weak inhibitors of the catalytic domain of
human neutrophil collagenase (cdMMP-8). Further optimization of the lead compounds revealed
general design principles that involve the placement of a phenyl or thienyl group at position
5 of the thiadiazine ring, to improve unprimed side affinity; the incorporation of an amino
group at position 2 of the thiadiazine ring as the chelating agent for the catalytic zinc; the
placement of a N-sulfonamide-substituted amino acid residue at the amino group, to improve
primed side affinity; and the attachment of diverse functional groups at position 4 or 5 of the
phenyl or thienyl group at the unprimed side, to improve selectivity. The new compounds were
assayed against eight different matrix metalloproteinases, MMP-1, cdMMP-2, cdMMP-8, MMP-
9, cdMMP-12, cdMMP-13, cdMMP-14, and the ectodomain of MMP-14, respectively. A unique
combination of the above-described modifications produced the selective inhibitor (2R)-N-[5-
(4-bromophenyl)-6H-1,3,4-thiadiazin-2-yl]-2-[(phenylsulfonyl)amino]propanamide with high af-
finity for MMP-9 (Ki ) 40 nM). X-ray crystallographic data obtained for cdMMP-8 cocrystallized
with N-allyl-5-(4-chlorophenyl)-6H-1,3,4-thiadiazin-2-amine hydrobromide gave detailed design
information on binding interactions for thiadiazine-based MMP inhibitors.
In tr od u ction
laboratories working in the area have provided several
classes of MMP inhibitors that have been extensively
reviewed.9 One key issue in the clinical development of
MMP inhibitors relates to whether the development of
broad-spectrum inhibitors, active against a range of
different enzymes, or of selective inhibitors, targeted
against a particular subset of the MMPs, represents the
optimal strategy. Some orally active compounds for the
treatment of cancer and/or arthritis are currently under
clinical investigation. Representative examples include
succinamides10 (Marimastat), linear sulfonamides11 (CGS-
27023A), heterocyclic sulfonamides12 (Prinomastat), and
biphenylbutanoic acid derivatives13 (BAY-129566) (Fig-
ure 1).
Matrix metalloproteinases (MMPs) are a superfamily
of zinc- and calcium-dependent endopeptidases involved
in the degradation and remodeling of connective tissues.
They are secreted as proenzymes that are subsequently
processed by other proteinases to generate the active
forms. The proteolytic activity is directed against most
constituents of the extracellular matrix, like proteogly-
cans, fibronectin, laminin, and interstitial collagens.1
Under normal physiological conditions, the proteolytic
activities are controlled by maintaining a balance
between the synthesis of the active forms and their
inhibition by tissue inhibitors of matrix metalloprotein-
ases (TIMPs) or by nonspecific R2-macroglobulin.2 In
pathological conditions this equilibrium is shifted to-
ward increased MMP activity leading to tissue degrada-
tion,3 which has been linked to several disease states
such as osteoarthritis,4 rheumatoid arthritis,5 periodon-
tal disease,6 multiple sclerosis,7 and tumor metastasis.8
Consequently, there has been significant interest in the
development of drugs to control the aberrent regulation
of MMP production. Recent efforts by a number of
Within three of these compound classes the hydrox-
amate moiety plays a decisive role in achieving inhibitor
potency. Because of the biologically labile nature of
hydroxamates, we undertook additional efforts toward
the discovery of new chelating groups suitable for use
in MMP inhibitor templates. In recent years, interest
in 1,3,4-thiadiazines has increased due to their high
biological activity and the broad-spectrum action of their
derivatives.14 The value of thiadiazines as MMP inhibi-
tors has, however, not hitherto been recognized. As part
of a collaborative effort, a series of chiral 6-methyl-1,3,4-
thiadiazines previously reported15 was assayed utilizing
the catalytic domain of human neutrophil collagenase
(cdMMP-8) as the screening enzyme. From this analysis,
a small number of compounds (1-3) were determined
* To whom correspondence should be adressed. Tel: +49-(0)521-
106-2081. Fax: +49-(0)521-106-6014. E-mail: harald.tschesche@
uni-bielefeld.de.
† Abt. Biochemie I, Universita¨t Bielefeld.
‡ Abt. Strukturforschung, Max-Plank-Institut fu¨r Biochemie.
§ Abt. Anorganische Chemie III, Universita¨t Bielefeld.
| Institut fu¨r Chemie und Biochemie, Ernst-Moritz-Arndt Univer-
sita¨t Greifswald.
10.1021/jm010887p CCC: $20.00 © 2001 American Chemical Society
Published on Web 08/31/2001

3232 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 20
Schro¨der et al.
Sch em e 1. Method Aa
a
Reagents and conditions: (i) thiosemicarbazide, EtOH, 0-20
°C; (ii) EtOH, 48% aq HBr, reflux; (iii) (a) K2CO3/H2O, R4SO2Cl,
reflux, (b) concd HCl; (iv) EDC, HOBt, NMM, DMF, 5 °C.
F igu r e 1. Selected MMP inhibitors.
Ta ble 1. Synthesized 5-Substituted
6H-1,3,4-Thiadiazin-2-amine Hydrohalides (6a -k )
compd
R1
formula
mp (°C)
6a
6b
6c
6d
6e
6f
6g
6h
6i
Ph
4-FPh
4-ClPh
4-BrPh
4-O2NPh
4-NCPh
4-F3CPh
4-H3COPh
4-H3CPh
1-adamantyl
5-Cl-2-thienyl
C9H10ClN3S
206
231
227
218
228
248
224
185
220
251
249
C9H9ClFN3S
C9H9BrClN3S
C9H9Br2N3S
C9H9BrN4O2S
C10H9BrN4S
C10H9BrF3N3S
C10H12BrN3OS
C10H12BrN3S
6j
6k
C
13H20BrN3S
C7H7BrClN3S2
F igu r e 2. Thiadiazine screening leads.
Ta ble 2. Synthesized N-Sulfonylated Amino Acids (8a -g)
to be competitive inhibitors with weak (Ki > 40 µM)
inhibitory activity (Figure 2).
compd
R2
R3
R4
formula
mp (°C)
8a (S)
8b(R) CH3
8c(S)
8d (S)
8e(S)
H
CH3
H
CH3
CH3
CH(CH3)2 Ph
Ph
Ph
C9H11NO4S 123-125
C9H11NO4S 124-126
Further chemical modifications including the lack of
the 6-methyl group resulted in novel 6H-1,3,4-thiadi-
azine derivatives that are potent inhibitors of matrix
metalloproteinases with an exceptional binding mode
to the active site. The account below will focus on the
synthesis, structure-activity relationship (SAR) studies,
and in vitro activity of this series of compounds.
H
H
H
2-thienyl C7H9NO4S2
CH2Ph
85-87
C10H13NO4S 125-127
C11H15NO4S 149
8f(R) CH(CH3)2
8g CH3
H
CH3
Ph
Ph
C11H15NO4S 148-149
C10H13NO4S 146-147
Sch em e 2. Method Ba
Syn th esis
Synthesis of the 6H-1,3,4-thiadiazine-based inhibitors
was accomplished using two methods. In the first
method, a substituted R-bromo-keto compound (4) was
allowed to react with thiosemicarbazide in chilled etha-
nol (Scheme 1). The resulting linear intermediate (5)
was ring-closed by heating this compound in an ethanol/
H2O/HBr mixture to afford the 5-substituted 6H-1,3,4-
thiadiazine-2-amines (6c-i) as their hydrobromide
salts.16 A D- or L-configurated amino acid (7) was heated
with the appropriate sulfonyl chloride in an aqueous
potassium carbonate solution to produce the desired
sulfonamides (8a -g). Acylation of the 5-substituted 6H-
1,3,4-thiadiazine-2-amine hydrohalides (6a -k ) (Table
1) with the carboxylic group of the sulfonamides (8a -
g) (Table 2) was mediated by a mixture of N-ethyl-N′-
(3-dimethylaminopropyl)-carbodiimide hydrochloride,
1-hydroxybenzotriazole, and 4-methylmorpholine in DMF
at 5 °C. The compounds with the general structure 9
were obtained as white or yellow solids that could be
a
Reagents and conditions: (i) thiosemicarbazide hydrohalide;
MeOH; reflux.
crystallized from methanol/acetonitrile mixtures as
described in the Experimental Section.
In the second method, the thiadiazine ring was
prepared in one step from the substituted R-halogeno-
keto compound and thiosemicarbazide hydrochloride or
thiosemicarbazide hydrobromide16 (Scheme 2). Com-
pounds (10) were slowly heated with the corresponding
thiosemicarbazide hydrohalides in methanol to produce
the 5-substituted 6H-1,3,4-thiadiazine-2-amine hydro-
halides (6a ,b,j,k ) as crystalline solids that could be
purified by recrystallization from an appropriate solvent
as described in the Experimental Section. With this
method the novel compound 5-(5-chloro-2-thienyl)-6H-
1,3,4-thiadiazin-2-amine hydrobromide could be ob-

Potent Matrix Metalloproteinase Inhibitors
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 20 3233
F igu r e 3. Structure of compound 11.
tained in an acceptable yield. The remaining transfor-
mation leading to compounds with the general structure
9 was performed as illustrated in Scheme 1.
F igu r e 4. Proposed 6H-1,3,4-thiadiazine SAR studies.
Resu lts a n d Discu ssion
within the compound series, the inhibition of these
MMPs showed great variability. On the other hand, the
inhibition of MMP-1 and cdMMP-12 was less potent
relative to the other enzymes and demonstrated very
few variations in potency as functional groups were
altered. Surprisingly, the inhibition of cdMMP-13, in
general, occurs in the micromolar to submicromolar
range within the tested 6H-1,3,4-thiadiazine series
(Tables 3-7).
SAR of th e P osition 5 Su bstitu en t. As a first
modification, we prepared analogues substituting the
phenyl ring in position 5 of the 6H-1,3,4-thiadiazine
moiety with various halogens (12a -c), electron-with-
drawing groups (12d -f), and moderately electron-
donating groups (12g,h ) (Tables 3 and 4) and assessed
the effects that these changes have on MMP inhibitory
activity.
As can be seen in Table 3, halogens attached to the
4-position of the phenyl ring increased the potency
against cdMMP-8, MMP-9, and cdMMP-14. This in-
crease had the maximum level with an appended chloro
substituent in the case of MMP-9 and cdMMP-14 and
with an appended bromo substituent in the case of
cdMMP-8. However, electron-withdrawing groups and
moderately electron-donating groups at the 4-position
of the phenyl ring were well-tolerated by the tested
MMPs (Table 4). Surprisingly, the electron-withdrawing
CN group of compound (12e), which can be described
as a pseudohalogen,19 also improved inhibitory activity
against MMP-9 in a manner comparable to the halo-
gens. The moderately electron-donating CH3 group of
compound (12h ) tended to have selective potency against
cdMMP-12. Substitution by electron-withdrawing sub-
stituents produces an electron-deficient phenyl ring,
Str u ctu r e-Activity-Rela tion sh ip (SAR) An a ly-
sis. All compounds were tested in vitro for the inhibition
of PMNL-gelatinase (MMP-9) and the recombinant
catalytic domains of human neutrophil collagenase
(cdMMP-8), human gelatinase A (cdMMP-2), macro-
phage elastase (cdMMP-12), collagenase-3 (cdMMP-13),
and membrane-type-1 MMP (cdMMP-14). Selected com-
pounds have also been tested for the inhibition of
collagenase-1 (MMP-1) and the ectodomain of membrane-
type-1 MMP (MMP-14). The first molecule prepared to
test the concept of 5-substituted-6H-1,3,4-thiadiazine-
2-amide linked MMP inhibitors was the dihydroorotic
acid derivative (11) (Figure 3). This compound showed
a promising potency of Ki ) 1.2 µM against MMP-9. It
was recently discovered that sulfonylated amino acid
hydroxamates and sulfonylated amino acids (carboxyl-
ates) act as efficient MMP inhibitors.17 The most active
compounds from this class of nonpeptide MMP inhibi-
tors possess an arylsulfonyl group, occupying the speci-
ficity S1′ pocket of the enzyme. It was also shown that
the -SO2- moiety of the inhibitors is involved in several
strong hydrogen bonds with amino acid residues from
the active site cleft, which considerably stabilize the
enzyme-inhibitor adduct.18 Assuming that the phenyl
ring of 11 occupies the unprimed side of MMP-9, we
focused on preparing 5-substituted-6H-1,3,4-thiadiaz-
ine-2-amides with a sulfonamide moiety to improve the
primed side affinity. The promising concept of these
novel sulfonamide inhibitors prompted us to establish
SAR considerations that apply to most of the 6H-1,3,4-
thiadiazines described in this paper (Figure 4).
The class in general inhibits cdMMP-2, cdMMP-8,
MMP-9, and cdMMP-14 selectively in the nanomolar
range. Depending on functional group manipulations
Ta ble 3. In Vitro Activity of Halogenated 6H-1,3,4-Thiadiazine Derivatives
MMPs Ki (µM)b
compd
R
formulaa
mp (°C)
method
1c
2
8
9c
12
13
14
14Ed
12a
12b
12c
F
Cl
Br
C18H17FN4O3S2
C18H17ClN4O3S2
166-167
184-185
180-181
B
A
A
0.44
0.65
nt
0.45
0.14
0.27
0.19
0.73
0.11
0.16
0.06
0.16
0.28
0.52
0.44
0.65
0.18
0.37
0.44
0.10
0.34
nte
0.21
nt
C
18H17BrN4O3S2
a
b
Analytical results are within (0.4% of the theoretical values. MMP inhibition in vitro. Assays were run at pH 7 against the catalytic
domains of the enzymes. See the Experimental Section for complete protocols. Standard deviations were typically (15% of the mean or
less. c Full-length version of the enzyme was used. The ectodomain of the enzyme was used. e nt denotes not tested.
d

3234 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 20
Schro¨der et al.
Ta ble 4. In Vitro Activity of 6H-1,3,4-Thiadiazine Derivatives Substituted with Electron-Withdrawing or Electron-Donating
Functionalities
MMPs Ki (µM)b
compd
R
formulaa
mp (°C)
method
1c
2
8
9c
12
13
14
14Ed
12d
12e
12f
12g
12h
NO2
CN
CF3
OCH3
CH3
C18H17N5O5S2
C19H17N5O3S2
C19H17F3N4O3S2
C19H20N4O4S2
C19H20N4O3S2
201-202
186-187
187-188
192-193
184-185
A
A
A
A
A
0.26
0.39
0.43
0.58
nt
0.30
0.24
nt
0.52
0.41
0.17
0.22
0.26
0.18
0.30
0.13
0.08
0.24
0.32
0.16
0.28
0.37
0.30
0.34
0.11
0.26
0.25
0.57
1.33
0.62
0.29
0.24
0.58
0.59
0.38
nte
0.40
0.39
nt
nt
a-e See footnotes in Table 3.
Ta ble 5. In Vitro Activity of 6H-1,3,4-Thiadiazine Derivatives with Different Position 5 Residues
MMPs Ki (µM)b
9c
12
>15 0.41 >15
0.17 0.24
compd
R
formulaa
1-adamantyl C22H28N4O3S2
5-Cl-thienyl
mp (°C)
method
1c
2
8
13
14
14Ed
13a f
13b
183-184
16H15ClN4O3S3 198-199
B
B
nte
0.45
>15
>15
3.07
nt
0.20
C
1.87
0.18
0.47 0.32
a-e See footnotes in Table 3. f At high assay concentrations (>5 µM) the compound showed fluorescence quenching effects.
Ta ble 6. Modifications of the Sulfonamide Residue
MMPs Ki (µM)b
compd
R
formulaa
mp (°C)
method
1c
2
8
9c
12
13
14
14Ed
12b
14a
14b
Ph
CH2Ph
2-thienyl
C18H17ClN4O3S2
C19H19ClN4O3S2
C16H15ClN4O3S3
184-185
169-170
175-176
A
A
A
0.65
0.36
1.04
0.14
0.09
0.55
0.73
0.20
0.06
0.06
0.04
0.80
0.52
0.48
0.36
0.18
0.12
3.81
0.10
0.19
0.25
0.21
0.18
nte
a-e See footnotes in Table 3.
which in turn can improve aryl-aryl stacking interac-
tions with aromatic side chains in the active site. The
reduction in affinity, resulting from a reduced aryl-aryl
stack by phenyl rings para-substituted by moderate
electron-releasing groups (e.g. CH3), appears to be more
than offset by the increase in steric bulk/lipophilic
contacts that these groups provide in the case of
cdMMP-12. Consequently, the replacement of the 4-
methylphenyl ring in compound (12h ) with the bulky
adamantyl residue verified in compound (13a ) resulted
in a 10-1000-fold decrease of inhibitory activity against
the tested MMPs, with the exception of cdMMP-12
(Table 5). This enzyme was inhibited by 13a very
selectively in the submicromolar range. The bioiso-
steric20 replacement of the 4-chlorophenyl residue with
a 5-chlorothienyl moiety represented by compound 13b
demonstrated enzyme selectivity between cdMMP-2 and
MMP-9 by a factor of 10.
broad-spectrum inhibitor. The more flexible benzylsul-
fonamide analogue 14a was also prepared and tested
for in vitro activity. This compound possessed a slight
increase of inhibitory activity with cdMMP-2, cdMMP-
13, and MMP-9, all of which are characterized by a deep
S1′ pocket.21 The results obtained with the thienylsul-
fonamide 14b were remarkable. Replacement of the
phenyl group with a thienyl substituent led to a
significant shift in enzyme selectivity. The compound
was found to be even more selective for cdMMP-8 (Ki )
60 nM) compared with the other tested MMPs.
SAR of Va r ia tion s of th e r-Ca r bon Su bstitu en ts.
The substituents at the R-carbon of the N-sulfonylated
amino acid residue of the molecules were also modified.
To determine if either the R-isomer or the S-isomer
binds tighter to the active site, we synthesized enan-
tiomeric pairs of most of the compounds. The results of
the in vitro tests revealed that the corresponding
R-isomers were, in general, more potent than the parent
S-isomers (Table 7). Replacement of the methyl group
with the bulky isopropyl substituent (15f,g) led to a
decrease of the inhibitory activity against the tested
MMPs. Attachment of a second methyl group at the
SAR of th e Su lfon a m id e Resid u e. The sulfonamide
portion of the molecules and the influence of this group
on enzyme inhibition was also proven (Table 6).
The phenylsulfonamide 12b, one of the first com-
pounds we examined, was determined to be a potent

Potent Matrix Metalloproteinase Inhibitors
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 20 3235
Ta ble 7. Modifications of the R-Carbon Substituents
MMPs Ki (µM)b
compd
R, R1, R2
H, CH3, F
formulaa
mp (°C)
method
1c
2
8
9c
12
13
14
14Ed
12a (S)
15a (R)
12b(S)
15b(R)
12c(S)
15c(R)
12e(S)
15d (R)
12h (S)
15e(R)
15f(S)f
C18H17FN4O3S2
C18H17FN4O3S2
C18H17ClN4O3S2
C18H17ClN4O3S2
C18H17BrN4O3S2 180-181
C18H17BrN4O3S2 179-180
C19H17N5O3S2
C19H17N5O3S2
C19H20N4O3S2
C19H20N4O3S2
166-167
167-168
184-185
183-184
B
B
A
A
A
A
A
A
A
A
A
A
A
0.44
0.63
0.65
0.21
nt
0.42
0.39
0.40
nt
0.46
nt
0.55
0.30
0.45
0.34
0.14
0.50
0.27
0.15
0.24
0.21
0.41
0.44
0.88
0.85
0.08
0.19
0.35
0.73
0.15
0.11
0.21
0.22
0.23
0.30
0.22
0.78
4.37
0.48
0.16
0.05
0.06
0.08
0.16
0.04
0.08
0.08
0.16
0.21
0.20
0.28
0.35
0.52
0.32
0.44
0.34
0.37
0.36
0.11
0.09
0.38
0.35
0.20
0.65
0.30
0.18
0.14
0.37
0.13
0.25
0.21
0.62
0.32
0.44
0.39
0.10
0.05
0.34
0.20
0.24
0.24
0.38
0.29
0.74
0.59
0.15
nte
CH3, H, F
0.33
0.21
0.21
nt
0.18
0.40
0.40
nt
0.15
nt
0.33
0.11
H, CH3, Cl
CH3, H, Cl
H, CH3, Br
CH3, H, Br
H, CH3, CN
CH3, H, CN
H, CH3, CH3
CH3, H, CH3
186-187
185-186
184-185
183-184
201-202
189-190
204-205
H, (CH3)2CH, Cl C20H21ClN4O3S2
>15
>15
0.18
15g(R)f (CH3)2CH, H, Cl C20H21ClN4O3S2
15h
>15
CH3, CH3, Cl
C19H19ClN4O3S2
0.09
f
a-e See footnotes in Table 3. At high assay concentrations (>5 µM) the compound showed fluorescence quenching effects.
F igu r e 5. The ORTEP-III view of N-allyl-5-(4-chlorophenyl)-
6H-1,3,4-thiadiazin-2-amine hydrobromide showing the two
independent molecules and the atom-numbering scheme.
Displacement ellipsoids are drawn at the 50% probability level
and H atoms are shown as small spheres of arbitrary radii.
The hydrogen positions on all endocyclic nitrogens are calcu-
lated as half-occupied.
F igu r e 6. The electron density at the active site of the
catalytic domain of human neutrophil collagenase (cdMMP-
8) complexed with N-allyl-5-(4-chlorophenyl)-6H-1,3,4-thiadi-
azin-2-amine.
R-carbon was verified in compound 15h . This compound
is the first example of a potent nonchiral 6H-1,3,4-
thiadiazine-based MMP inhibitor with nanomolar af-
finity for cdMMP-2 and MMP-9.
X-r a y Cr yst a llogr a p h y. A crystal structure of
the catalytic domain of human neutrophil collagenase
(cdMMP-8) complexed with N-allyl-5-(4-chlorophenyl)-
6H-1,3,4-thiadiazin-2-amine hydrobromide (16) (Ki )
41 µM) was determined at 2.7 Å resolution and is shown
in Figure 6. This structure provides insights into the
enzyme-inhibitor interactions that play a role in the
exceptional binding of 6H-1,3,4-thiadiazine-based MMP
inhibitors. These interactions are represented schemati-
cally in Figure 7.
Notably, the inhibitor is coordinated to the catalytic
zinc cation via the exocyclic nitrogen of the thiadiazine
moiety. The ring nitrogens are involved in specific
hydrogen bonds with the backbone of cdMMP-8. The
crystal structure of free inhibitor (16) reveals that the
thiadiazine ring deviates from planarity (Figure 5).
F igu r e 7. Schematic diagram of binding interactions be-
tween N-allyl-5-(4-chlorophenyl)-6H-1,3,4-thiadiazin-2-amine
and cdMMP-8. Interatomic distances given in ångstroms are
those observed between protein and inhibitor heteroatoms.
Hydrogen positions are inferred from heavier atom positions.
Zn2+ is the catalytic zinc of cdMMP-8.
Consistent with previous reports,22 the puckering of the
thiadiazine ring may be described as out-of-plane dis-
placements of the vertexes of the symmetric flat polygon

3236 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 20
Schro¨der et al.
using group theory. The method applicable to any real
cyclic compound was introduced by Cremer and Pople
and defines a unique mean plane for the ring atoms.
The displacements of atoms from this plane are treated
as an adequate measure of ring puckering. The calcu-
lated puckering parameters are Q ) 0.651 Å for both
molecules in the asymmetric unit, θ ) 109.9° (molecule
1) and 109.7° (molecule 2) and æ ) 218.8° (molecule 1)
and 218.3° (molecule 2), respectively. The large æ value
for both molecules indicates that the direction of the ring
distortion is toward an inverted screw-boat conforma-
tion.23 The space group P21/c of the uncomplexed 16
crystals implies that there is an equal subset of mol-
ecules with the non-inverted screw-boat conformation
in the crystal structure due to an inversion-center. By
contrast, when complexed with cdMMP-8 the puckering
parameters of the thiadiazine ring are Q ) 0.619 Å,
θ ) 71.3°, and æ ) 36.9°. This corresponds to the non-
inverted screw-boat conformation of the heterocycle and
fits the 4-chloro-substituted phenyl residue into the S3
subsite formed by Phe-164, His-162, and Ser-151 on the
unprimed side. Consequently, stabilization of the non-
inverted screw-boat conformation of the thiadiazine core
structure is expected to result in an inhibitor with
higher affinity. The allyl substituent is directed toward
the S1′ pocket of cdMMP-8. The hydrogen on the
endocyclic nitrogen closest to the exocyclic nitrogen is
prototropically shifted from the exocyclic nitrogen and
stabilized by a hydrogen bond to the carboxylate oxygen
of Glu-198. This glutamate is conserved in all zinc
proteinases, serving as the general base crucial for
catalysis.24 The other double-bound nitrogen accepts a
hydrogen bond from the backbone amide of Ala-163. The
endocyclic sulfur does not appear to interact with any
protein group but makes a specific hydrogen bond to a
structural water molecule. It is further required for
electronic delocalization of charge within the thiadiazine
ring, which is necessary for Zn2+ coordination. The
electron density of the exocyclic nitrogen is directed to
the catalytic zinc and does not interact with the
backbone. The unique hydrogen bonding of the thiadi-
azine core explains why other related thiadiazines were
not identified through the screening effort. Furthermore,
the complex structure rationalizes the selectivity profile
of the compounds described herein toward certain
MMPs. 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. In fact, the potency
of N-allyl-5-(4-chlorophenyl)-6H-1,3,4-thiadiazin-2-amine
hydrobromide is remarkable, given that the inhibitor
lacks a substituent capable of filling the S1′ pocket.
Consequently, it makes sense to attach a sulfonamide
moiety to the thiadiazine core structure to improve
primed-side affinity.
F igu r e 8. View of a model of compound (14b) in the active
site of cdMMP-8. Amino acid residues that form the binding
cleft are labeled, and a solid surface has been added.
protein-ligand complex is estimated in FlexX as the
sum of free energy contributions from hydrogen bonds,
ion-pair interactions, hydrophobic and π-stacking in-
teractions of aromatic groups, and lipophilic interac-
tions. The underlaying assumption for the docking
experiments is that the orientation of the 5-(4-chlo-
rophenyl)-6H-1,3,4-thiadiazin-2-amine moiety in the
binding site of cdMMP-8 is highly conserved. This
assumption appears to be reasonable, since the interac-
tions of this residue (Zn2+ coordination, a.o.) are highly
favorable and specific. The flexible ligand docking
results (highest scoring binding modes) for 14b in
cdMMP-8 were calculated in the form of rmsd values
between the crystallographic binding mode and the
docked binding mode of the core fragment. The rmsd
value calculated for the docked structure of Figure 8
was 3.6 Å with a corresponding free binding energy of
∆G ) -24.2 kJ /mol.
Surprisingly, in this binding prediction the thiophene
ring does not occupy the S1′ pocket but is positioned
above the catalytic zinc. Such electrostatic interactions
between the positive charge of a cation and the negative
partial charge of aromatic π clouds have been proposed
in molecular recognition processes and recently explored
by experimental and theoretical works.26 A physical
model of this interaction has been reported in a review.27
The catalytic zinc ion is positioned on the normal vector
to the aromatic thiophene ring at 5.1 Å distance. A
similar interaction involving the catalytic zinc ion
has already been described for the complex between
cdMMP-8 and batimastat,28 where the thiophene aro-
matic ring of the inhibitor faces the cation at a distance
of 4.5 Å. The contribution of these attractive electro-
static interactions, at a relatively large distance, should
not be neglected in the evaluation of the stabilization
of the complex model and should be taken into account
in the selectivity of compound 14b as well as other
compounds of this series.
Dock in g Exp er im en ts. We examined the interac-
tion of compound 14b with the active site of cdMMP-8
to understand the observed SARs and to rationalize the
specificity profile. Since suitable crystals of a cdMMP-
8/compound (14b) complex could not be obtained for
X-ray studies, we modeled 14b with the help of the
program FlexX25 into the protein by using coordinates
from the above-described cdMMP-8/inhibitor complex
structure as a reference. The free binding energy of a
Con clu sion
A series of potent matrix metalloproteinase inhibitors
based on a 6H-1,3,4-thiadiazine scaffold was discovered
and optimized. In contrast to the established MMP-
inhibitors, which exclusively bind to the primed side,
the compounds described interact with both the primed

Potent Matrix Metalloproteinase Inhibitors
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 20 3237
acetonitrile as the organic modifier and a flow rate of 250 µL/
min. Solvent A ) 0.1% TFA in water. Solvent B ) 0.09% TFA
and 80% CH3CN in water. The following gradient was used:
t ) 0 min (0% B), t ) 5 min (40% B), t ) 25 min (60% B).
Analytical data is reported as retention time, tR, in minutes.
Melting points (mp) were determined with a Bu¨chi 510 melting
point apparatus and are uncorrected. Proton (1H) nuclear
magnetic resonance (NMR) spectra were measured with a
Bruker DRX-500 (500.1 MHz) spectrometer, and 13C NMR
spectra were measured with a Bruker DRX-500 (125.8 MHz)
spectrometer, both with TMS as an external standard. Chemi-
cal shifts are reported in parts per million (ppm, δ units).
Coupling constants are reported in units of hertz (Hz). All of
the compounds synthesized in the experiments below were
analyzed by NMR, and the spectra were consistent with the
proposed structures in each case. Mass spectral (MS) data were
obtained on a Fisons Autospec VG spectrometer by the DCI/
methane and DEI/isobutane method. MALDI-TOF mass spec-
tral data were obtained on a PerSeptive Biosystems Voyager-
DE spectrometer with DHB as the matrix and PEG 200 as
the calibration standard. Most of the compounds synthesized
in the experiments below were analyzed by mass spectrometry,
and the spectra were consistent with the proposed structures
in each case. Elemental analysis were performed on a Leco
CHNS-932 elemental analyzer. All inhibitors synthesized in
the experiments below were analyzed by elemental analysis,
and the compounds had values within the acceptable range of
(0.4% for C, H, N. 2-Bromo-1-(5-chloro-2-thienyl)ethanone29
was prepared following literature procedures.
and the unprimed side of the MMP. Attachment of a
phenylsulfonamide moiety to the thiadiazine core struc-
ture led to an increase of inhibitory potency against the
tested MMPs. Furthermore, the conformational rigidity
of the series may be advantageous as a means to obtain
the observed selectivity. Using X-ray crystallography,
a novel binding mode of N-allyl-5-(4-chlorophenyl)-6H-
1,3,4-thiadiazin-2-amine in the active site of the cata-
lytic domain of human neutrophil collagenase was
elucidated. This inhibitor binds differently than peptide-
based inhibitors. An extensive network of hydrogen
bonds and chelation of the catalytic zinc stabilize the
latter in the active site. Hydrophobic interactions in the
specificity pockets play a relatively minor role for the
binding energy. In the presence of nonpeptide thiadi-
azine-based inhibitors, loss of binding affinity due to a
reduced number of hydrogen bonds is compensated by
significant hydrophobic interactions and, as suggested
by docking experiments, cation-aromatic interactions.
Thus, the unique combination of the observed SARs
produced the best selective inhibitor, (2R)-N-[5-(4-bro-
mophenyl)-6H-1,3,4-thiadiazin-2-yl]-2-[(phenylsulfonyl)-
amino]propanamide (15c), with high affinity for MMP-9
(Ki ) 40 nM). This compound may be regarded as a
novel, nonpeptidic, and low molecular weight lead for
continued development of MMP inhibitors as drugs.
2-Br om o-1-[4-(tr iflu or om eth yl)p h en yl]eth a n on e. A so-
lution of 4-trifluoromethylacetophenone (1.88 g, 10 mmol) in
40 mL of methylene chloride was heated to reflux. Within 1 h
a solution of bromine (1.6 g, 10 mmol) in 20 mL of methylene
chloride was added dropwise to the boiling solution under rapid
stirring. During reaction the resulting HBr gas was removed
by a positive argon pressure. The orange reaction mixture was
stirred overnight at room temperature within which time it
becomes clear. The mixture was then evaporated under
reduced pressure to give an off-white solid. Recrystallization
from petrolether/n-hexane afforded 2.36 g (88%) of the desired
Exp er im en ta l Section
Gen er a l. All moisture-free reactions were performed in
oven-dried glassware under a positive pressure of argon and
were stirred magnetically. Sensitive liquids and solutions were
transferred via syringe or cannula and were introduced into
reaction vessels through rubber septa. P.a. grade reagents and
solvents were used without further purification, except that
methylene chloride was distilled under argon from calcium
hydride and DMF was distilled under argon on molecular sieve
3 Å. Many of the special organic starting materials and
reagents were obtained from Sigma-Aldrich (Deisenhofen,
Germany) and Lancaster-Synthesis (Mu¨hlheim, Germany).
Solvents were generally obtained from Merck (Darmstadt,
Germany) and Baker (Gross-Gerau, Germany). Abbreviations
that have been used in the descriptions of the schemes and
the descriptions that follow are APMA for p-aminophenyl-
mercuric acetate, DCI for direct chemical ionization, dec for
decomposition, DEI for direct electron impact, DHB for 2,5-
dihydroxybenzoic acid, DMF for dimethylformamide, DMSO
for dimethyl sulfoxide, EDCI for N-ethyl-N′-(3-dimethylami-
nopropyl)-carbodiimide hydrochloride, HEPES for N-[2-hy-
droxyethyl]piperazine-N′-[2-ethanesulfonic acid], HOBt for
1-hydroxy-benzotriazole, MALDI for matrix assisted laser
desorption/ionization, MCA for (7-methoxycoumarin-4-yl)-
acetyl-, MeOH for methanol, MRB for microfluorometric
reaction buffer, NMM for 4-methylmorpholine, PEG 8000 for
poly(ethylene glycol) av mol wt 8000, PMNL for polymorpho-
nuclear neutrophil leucocytes, RP-HPLC for reverse-phase
high-performance liquid chromatography, TFA for trifluoro-
acetic acid, TKI for trypsin-kallikrein-inhibitor, TLC for thin-
layer chromatography, ∆TM for without transmembrane
moiety, TMS for tetramethylsilane, and TOF for time of flight.
Analytical TLC was performed on Alugram Sil G/UV254 pre-
coated aluminum-backed silica gel plates (Macherey-Nagel,
Du¨ren). Visualization of spots was effected by one of the
following techniques: (a) ultraviolet illumination and (b)
immersion of the plate in a 3% solution of ninhydrin in ethanol
followed by heating. Analytical purity was assessed by RP-
HPLC using an Applied Biosystems 130A Separation System.
Compounds were detected at 230 nm. The stationary phase
was an Aquapore OD-300 C18 column (2.1 mm × 220 mm).
The mobile phase employed aqueous trifluoroacetic acid with
1
product as colorless plates. Mp: 53-54 °C. H NMR (CDCl3):
3
3
δ 4.43 (s, 2H), 7.74 (d, J ) 8.3 Hz, 2H), 8.08 (d, J ) 8.2 Hz,
2H). 13C NMR (CDCl3): δ 30.31 (CH2), 123.37 (q, 1J CF ) 272.9
2
Hz, CF3), 125.90, 129.31 (CHarom), 135.05 (q, J CF ) 33.09 Hz,
C-CF3), 136.56 (C-CO), 190.40 (CdO).
Gen er a l P r oced u r e for P r ep a r in g 2-Am in o-6H-1,3,4-
t h ia d ia zin e H yd r oh a lid es (Met h od A). Syn t h esis of
2-Am in o-5-(4-ch lor op h en yl)-6H-1,3,4-th ia d ia zin e Hyd r o-
br om id e (6c). 4-Chlorophenacylbromide (2.34 g, 10 mmol) and
thiosemicarbazide (0.91 g, 10 mmol) were suspended in 30 mL
of ethanol at 0 °C. The mixture was allowed to warm to room
temperature overnight under stirring. The resulting slurry was
cooled to -20 °C and the precipitate was collected by filtration,
washed with cold ethanol, and dried in vacuo. The pale yellow
solid was again suspended in 20 mL of ethanol that contained
1 mL of 48% aqueous hydrobromic acid. The mixture was
heated to reflux for 30 min and was then cooled to room
temperature overnight. The precipitate was filtered, recrystal-
lized from ethanol, and dried in high vacuo at 40 °C over
phosphorus pentoxide. Yield: 1.84 g (60%) of colorless needles.
Mp: 227 °C. 1H NMR (DMSO-d6): δ 4.31 (s, 2H), 7.59 (d, 3J )
3
8.6 Hz, 2H), 7.90 (d, J ) 8.7 Hz, 2H), 9.46 (br s, 1H), 10.11
(br s, 1H), 13.32 (br s, 1H). 13C NMR (DMSO-d6): δ 22.10
(CH2), 128.81, 129.11 (CHarom), 131.86, 136.22, 150.24, 164.17
(C). DEI MS: m/z 225 [M - HBr]+.
2-Am in o-5-(4-br om op h en yl)-6H-1,3,4-th ia d ia zin e Hy-
d r obr om id e (6d ). The compound was prepared starting from
4-bromophenacyl bromide as described for compound 6c.
Yield: 2.33 g (66%) of colorless crystals. Mp: 218 °C. 1H NMR
3
(DMSO-d6): δ 4.30 (s, 2H), 7.73 (d, J ) 8.6 Hz, 2H), 7.83 (d,
3J ) 8.6 Hz, 2H), 9.34 (br s, 1H), 10.09 (br s, 1H), 13.31 (br s,
1H). 13C NMR (DMSO-d6): δ 22.04 (CH2), 125.16 (C), 128.96,
132.03 (CHarom), 132.21, 150.34, 164.16 (C). DEI MS: m/z 270
[M - HBr]+.

3238 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 20
Schro¨der et al.
2-Am in o-5-(4-n itr op h en yl)-6H-1,3,4-th ia d ia zin e Hyd r o-
br om id e (6e). The title compound was prepared starting from
4-nitrophenacyl bromide as described for compound 6c.
was obtained as colorless crystals by evaporation of a hydro-
chloric acid solution of thiosemicarbazide.
A suspension of phenacyl chloride (1.55 g, 10 mmol) and
thiosemicarbazide hydrochloride (1.72 g, 10 mmol) in 25 mL
of methanol was heated to reflux for 15 min. After cooling to
room temperature the white crystalline solid was filtered,
recrystallized from methanol, and dried in high vacuo at 40
°C over phosphorus pentoxide. Yield: 0.99 g (43%) of colorless
needles. Mp: 206 °C. 1H NMR (DMSO-d6): δ 4.29 (s, 2H), 7.50-
7.56 (m, 3H), 7.87-7.89 (m, 2H), 9.63 (br s, 1H), 10.51 (br s,
1H), 13.73 (br s, 1H). 13C NMR (DMSO-d6): δ 21.97 (CH2),
126.97, 129.03, 131.40 (CHarom), 133.06, 150.85, 164.95 (C). DEI
MS: m/z 225 [M - HCl]+.
2-Am in o-5-(4-flu or op h en yl)-6H-1,3,4-th ia d ia zin e Hy-
d r och lor id e (6b). The compound was prepared starting from
4-fluorophenacyl bromide as described for compound 6a .
Yield: 1.79 g (73%) of colorless crystals. Mp: 231 °C. 1H NMR
(DMSO-d6): δ 4.28 (s, 2H), 7.34-7.38 (m, 2H), 7.92-7.96 (m,
2H), 9.59 (br s, 1H), 10.54 (br s, 1H), 13.74 (br s, 1H). 13C NMR
(DMSO-d6): δ 22.02 (CH2), 116.02, 116.19, 129.53 (CHarom),
129.60 (C), 129.64 (CHarom), 149.95, 162.92, 164.94 (C). DEI
MS: m/z 209 [M - HCl]+.
1
Yield: 1.58 g (49%) of orange crystals. Mp: 228 °C. H NMR
3
(DMSO-d6): δ 4.38 (s, 2H), 8.14 (d, J ) 8.9 Hz, 2H), 8.34 (d,
3J ) 8.9 Hz, 2H), 9.61 (br s, 1H), 10.21 (br s, 1H), 13.42 (br s,
1H). 13C NMR (DMSO-d6): δ 22.22 (CH2), 124.07, 128.36
(CHarom), 139.00, 148.76, 149.34, 164.25 (C). DEI MS: m/z 236
[M - HBr]+.
2-Am in o-5-(4-cya n op h en yl)-6H -1,3,4-t h ia d ia zin e H y-
d r obr om id e (6f). The compound was prepared starting from
4-cyanophenacylbromide as described for compound 6c.
Yield: 1.80 g (61%) of yellow needles. Mp: 248 °C. 1H NMR
3
(DMSO-d6): δ 4.35 (s, 2H), 8.00 (d, J ) 8.4 Hz, 2H), 8.05 (d,
3J ) 8.4 Hz, 2H), 10.02 (br s, 1H), 10.19 (br s, 1H), 13.33 (br
s, 1H). 13C NMR (DMSO-d6): δ 22.08 (CH2), 113.41, 118.30
(C), 127.74, 132.92 (CHarom), 137.30, 149.68, 164.22 (C). DEI
MS: m/z 216 [M - HBr]+.
2-Am in o-5-(4-(tr iflu or om eth yl)p h en yl)-6H-1,3,4-th ia d i-
a zin e Hyd r obr om id e (6g). The compound was prepared
starting from 2-bromo-1-[4-(trifluoromethyl)phenyl]ethanone
as described for compound 6c. Yield: 1.54 g (45%) of colorless
crystals. Mp: 224 °C. 1H NMR (DMSO-d6): δ 4.36 (s, 2H), 7.90
Syn th esis of 2-Am in o-5-(2-a d a m a n tyl)-6H-1,3,4-th ia d i-
a zin e Hyd r obr om id e (6j). Th iosem ica r ba zid e Hyd r obr o-
m id e. This compound was obtained as colorless crystals by
evaporation of a 48% hydrobromic acid solution of thiosemi-
carbazide.
3
3
(d, J ) 8.3 Hz, 2H), 8.10 (d, J ) 8.2 Hz, 2H), 9.92 (br s, 1H),
10.12 (br s, 1H), 13.13 (br s, 1H). 13C NMR (DMSO-d6): δ 22.17
(CH2), 123.88 (q, 1J CF ) 272.3 Hz, CF3), 125.92, 127.86 (CHarom),
2
130.97 (q, J CF ) 32.2 Hz, C-CF3), 137.01, 149.92, 164.18 (C).
DEI MS: m/z 259 [M - HBr]+.
A suspension of 1-(2-bromoacetyl)adamantane (2.57 g, 10
mmol) and thiosemicarbazide hydrobromide (1.72 g, 10 mmol)
in 25 mL of methanol was heated to reflux for 15 min. After
cooling to room temperature, the white crystalline solid was
filtered, recrystallized from methanol, and dried in high vacuo
at 40 °C over phosphorus pentoxide. Yield: 1.97 g (60%) of
2-Am in o-5-(4-m eth oxyph en yl)-6H-1,3,4-th ia d ia zin e Hy-
d r obr om id e (6h ). The title compound was prepared starting
from 4-methoxyphenacylbromide as described for compound
1
6c. Yield: 1.65 g (55%) of pale yellow needles. Mp: 185 °C. H
NMR (DMSO-d6): δ 3.81 (s, 3H), 4.27 (s, 2H), 7.06 (d, 3J )
8.9 Hz, 2H), 7.85 (d, 3J ) 8.9 Hz, 2H), 9.26 (br s, 1H), 9.85 (br
s, 1H), 13.10 (br s, 1H). 13C NMR (DMSO-d6): δ 22.07 (CH2),
55.54 (CH3), 114.46 (CHarom), 125.06 (C), 128.82 (CHarom),
151.04, 161.85, 163.99 (C). DEI MS: m/z 221 [M - HBr]+.
1
colorless crystals. Mp: 251 °C. H NMR (DMSO-d6): δ 1.61-
1.68 (m, 6H), 1.75 (s, 6H), 1.98 (s, 3H), 3.79 (s, 2H), 9.11 (br s,
1H), 9.84 (br s, 1H), 12.91 (br s, 1H). 13C NMR (DMSO-d6): δ
20.39 (CH2), 27.21 (CH), 35.82 (CH2), 38.18 (C), 162.87 (C),
164.84 (C). DEI MS: m/z 249 [M - HBr]+.
2-Am in o-5-(4-m eth ylp h en yl)-6H-1,3,4-th ia d ia zin e Hy-
d r obr om id e (6i). The compound was prepared starting from
4-methylphenacylbromide (20 mmol) as described for com-
pound 6c. Yield: 3.13 g (55%) of colorless crystals. Mp: 220
2-Am in o-5-(5-ch lor o-2-th ien yl)-6H-1,3,4-th iadiazin e Hy-
d r obr om id e (6k ). The title compound was prepared starting
from 2-bromo-1-(5-chloro-2-thienyl)ethanone as described for
compound 6j. Yield: 0.35 g (11%); off-white solid. Mp: 249 °C.
1H NMR (DMSO-d6): δ 4.35 (s, 2H), 7.28 (d, 3J ) 4.1 Hz, 1H),
7.68 (d, 3J ) 4.1 Hz, 1H), 9.63 (br s, 1H), 10.03 (br s, 1H),
13.19 (br s, 1H). 13C NMR (DMSO-d6): δ 21.69 (CH2), 128.36,
131.47 (CH.), 133.86, 135.94, 146.13, 163.71 (C). DEI MS: m/z
231 [M - HBr]+.
1
°C. H NMR (DMSO-d6): δ 2.35 (s, 3H), 4.29 (s, 2H), 7.33 (d,
3
3J ) 8.0 Hz, 2H), 7.78 (d, J ) 8.1 Hz, 2H), 9.31 (br s, 1H)),
10.04 (br s, 1H)), 13.23 (br s, 1H). 13C NMR (DMSO-d6): δ
21.01 (CH2), 22.11 (CH3), 126.96, 129.61 (CHarom), 130.13,
141.60, 151.28, 164.19 (C). DEI MS: m/z 205 [M - HBr]+.
N-Allyl-5-(4-ch lor op h en yl)-6H-1,3,4-th ia d ia zin -2-a m in e
Hyd r obr om id e (16). 4-Chlorophenacyl bromide (2.34 g, 10
mmol) and 4-allylthiosemicarbazide (1.31 g, 10 mmol) were
suspended in 30 mL of ethanol at 0 °C. The mixture was
allowed to warm to room temperature overnight with stirring.
The yellow solution was added dropwise to 50 mL of ethyl
acetate at 0 °C, and the precipitate was collected by filtration,
washed with cold ethanol, and dried in vacuo. The yellow solid
was again suspended in 20 mL of ethanol that contained 1
mL of 48% aqueous hydrobromic acid. The mixture was heated
to reflux for 30 min and was then cooled to room temperature
overnight. After addition of ethyl acetate, the precipitate was
filtered, recrystallized from ethanol/ethyl acetate (1:3), and
dried in high vacuo at 40 °C over phosphorus pentoxide.
Yield: 1.49 g (43%) of colorless crystals. Mp: 210 °C. HPLC
tR: 11.97. 1H NMR (DMSO-d6): δ 4.17 (br s, 2H), 4.31 (s, 2H),
5.24-5.31 (m, 2H), 5.8 g (m, 1H), 7.61 (d, 3J ) 8.6 Hz, 2H),
Gen er a l P r oced u r e for P r ep a r in g N-Su lfon yla t ed
Am in o Acid s. Syn th esis of (2S)-2-[(P h en ylsu lfon yl)a m i-
n o]p r op a n oic Acid (8a ). Benzenesulfonyl chloride (1.80 g,
10 mmol) was added to a solution of L-alanine (1.07 g, 12 mmol)
in aqueous potassium carbonate (20 mL, 1.1 M). Under
vigorous stirring, the mixture was carefully heated to 70 °C
for 30 min. The resulting clear solution was cooled in an ice
bath and then acidified to pH 2.5 by the dropwise addition of
concentrated hydrochloric acid under stirring. The precipitate
was collected by filtration and washed with a minimum
amount of ice-cold water. Recrystallization from distilled water
yielded 1.42 g (62%) of (2S)-2-[(phenylsulfonyl)amino]propanoic
acid as a white crystalline solid. Mp: 123-125 °C. 1H NMR
3
(DMSO-d6): δ 1.13 (d, J ) 7.2 Hz, 3H), 3.76 (m, 1H), 7.54-
3
3
7.61 (m, 3H), 7.78 (d, J ) 7.4 Hz, 2H), 8.14 (d, J ) 8.3 Hz,
1H), 12.64 (s, 1H). 13C NMR (DMSO-d6): δ 18.43 (CH3), 51.15
(CH), 126.40, 129.06, 132.35 (CHarom), 141.34 (C), 173.22
(COOH).
7.92 (d, 3J ) 8.6 Hz, 2H), 10.71 (br s, 1H), 12.87 (br s, 1H). 13
C
NMR (DMSO-d6): δ 22.23 (CH2), 46.01 (CH2), 117.95 (dCH2),
128.82, 129.16 (CHarom), 131.53 (dCH), 131.86, 136.28, 151.29
(C). DEI MS: m/z 265 [M - HBr]+. MALDI-TOF-MS: m/z
calcd for [M - Br]+ 266.1, found 266.1. Anal. (C12H13BrClN3S)
C, H, N.
(2R)-2-[(P h en ylsu lfon yl)a m in o]p r op a n oic Acid (8b).
This compound was prepared starting from D-alanine as
described for compound 8a . Yield: 1.65 g (72%) of white solid.
Mp: 124-126 °C. 1H NMR (DMSO-d6): δ 1.13 (d, 3J ) 7.1 Hz,
3H), 3.76 (m, 1H), 7.54-7.62 (m, 3H), 7.78 (d, 3J ) 7.5 Hz,
3
2H), 8.15 (d, J ) 8.3 Hz, 1H), 12.65 (s, 1H).
Gen er a l P r oced u r e for P r ep a r in g 2-Am in o-6H-1,3,4-
t h ia d ia zin e H yd r oh a lid es (Met h od B). Syn t h esis of
2-Am in o-5-p h en yl-6H -1,3,4-t h ia d ia zin e H yd r och lor id e
(6a ). Th iosem ica r ba zid e Hyd r och lor id e. This compound
(2S)-2-[(2-Th ien ylsu lfon yl)a m in o]p r op a n oic Acid (8c).
The title compound was prepared starting from thiophene-2-
sulfonyl chloride as described for compound 8a . Yield: 1.57 g

Potent Matrix Metalloproteinase Inhibitors
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 20 3239
(67%) of white solid. Mp: 85-87 °C. 1H NMR (DMSO-d6): δ
MS: m/z 448 [M + H]+. MALDI-TOF-MS: m/z calcd for [M +
H]+ 448.1, found 448.0; calcd for [M + Na]+ 470.1, found 470.1;
calcd for [M + K]+ 486.0, found 486.0. Anal. (C18H17N5O5S2)
C, H, N.
3
3
1.17 (d, J ) 7.3 Hz, 3H), 7.14 (m, 1H), 7.56 (d, J ) 3.1 Hz,
1H), 7.89 (d, 3J ) 4.8 Hz, 1H), 8.35 (d, 3J ) 8.2 Hz, 1H), 12.71
(s, 1H). 13C NMR (DMSO-d6): δ 27.81 (CH3), 60.83 (CH),
137.09, 141.07, 141.99 (CHarom), 151.69 (C), 182.67 (COOH).
(2S)-2-[(Ben zylsu lfon yl)a m in o]p r op a n oic Acid (8d ).
This compound was prepared starting from benzylsulfonyl
chloride as described for compound 8a . Yield: 1.97 g (81%) of
white solid. Mp: 125-127 °C. 1H NMR (DMSO-d6): 1.23 (d,
(2S)-N-[5-(4-Cya n op h en yl)-6H-1,3,4-th ia d ia zin -2-yl]-2-
[(p h en ylsu lfon yl)a m in o]p r op a n a m id e (12e). Yield: 375
mg (64%) of yellow crystals. Mp: 186-187 °C; HPLC tR: 12.98.
3
1H NMR (DMSO-d6): δ 1.15 (d, J ) 7.1 Hz, 3H), 3.69 (ABq,
2J ) 14.7 Hz, 2H), 4.02 (m, 1H), 7.53-7.61 (m, 3H), 7.78-
2
3J ) 7.3 Hz, 3H), 3.79 (m, 1H), 4.33 (ABq, J ) 13.7 Hz, 2H),
3
3
7.83 (m, 2H), 7.95 (d, J ) 8.4 Hz, 2H), 8.07 (d, J ) 8.3 Hz,
2H), 8.12 (d, 3J ) 7.6 Hz, 1H), 12.11 (br s, 1H). 13C NMR
(DMSO-d6): δ 18.83 (CH3), 20.89 (CH2), 53.03 (CH), 112.48,
118.51 (C), 126.46, 127.69, 129.03, 132.32, 132.74 (CHarom),
138.55, 141.09, 146.96 (C), one C and one CdO were not
detected. DCI MS: m/z 428 [M + H]+. MALDI-TOF-MS: m/z
calcd for [M + H]+ 428.1, found 428.0; calcd for [M + Na]+
450.1, found 450.0; calcd for [M + K]+ 466.0, found 466.0. Anal.
(C19H17N5O3S2) C, H, N.
7.33-7.39 (m, 5H), 7.55 (d, 3J ) 7.9 Hz, 1H), 12.71 (s, 1H).
13C NMR (DMSO-d6): δ 18.67 (CH3), 51.30 (CH), 58.55 (CH2),
128.01, 128.26 (CHarom), 130.40 (C), 130.91 (CHarom), 174.20
(COOH).
(2S)-3-Meth yl-2-[(p h en ylsu lfon yl)a m in o]bu tyr ic Acid
(8e). The compound was prepared starting from L-valine as
described for compound 8a . Yield: 1.75 g (68%) of colorless
1
3
needles. Mp: 149 °C. H NMR (DMSO-d6): δ 0.77 (d, J ) 6.7
3
Hz, 3H), 0.80 (d, J ) 6.7 Hz, 3H), 1.92 (m, 1H), 3.51 (m, 1H),
(2S)-2-[(P h en ylsu lfon yl)a m in o]-N-{5-[4-(tr iflu or om eth -
yl)p h en yl]-6H-1,3,4-th ia d ia zin -2-yl}p r op a n a m id e (12f).
Yield: 396 mg (61%) of colorless needles. Mp: 187-188 °C;
3
3
7.52-7.60 (m, 3H), 7.77 (d, J ) 7.5 Hz, 2H), 8.02 (d, J ) 9.2
Hz, 1H), 12.58 (s, 1H). 13C NMR (DMSO-d6): δ 17.85 (CH3),
19.03 (CH3), 30.41 (CH), 61.26 (CH), 126.52, 128.90, 132.27
(CHarom), 141.19 (C), 172.21 (COOH).
1
3
HPLC tR: 14.17. H NMR (DMSO-d6): δ 1.17 (d, J ) 7.0 Hz,
3H), 3.71 (ABq, 2J ) 14.6 Hz, 2H), 4.03 (m, 1H), 7.54-7.61
3
3
(m, 3H), 7.79 (d, J ) 7.4 Hz, 2H), 7.84 (d, J ) 8.2 Hz, 2H),
8.10-8.12 (m, 3H), 12.10 (br s, 1H). 13C NMR (DMSO-d6): δ
18.85 (CH3), 21.07 (CH2), 53.07 (CH), 122.14 (C), 124.03 (q,
1J CF ) 272.3 Hz, CF3), 125.11, 125.69, 127.76, 129.03 (CHarom),
(2R)-3-Meth yl-2-[(p h en ylsu lfon yl)a m in o]bu tyr ic Acid
(8f). This compound was prepared starting from D-valine as
described for compound 8a . Yield: 1.73 g (67%) of colorless
needles. Mp: 148-149 °C. 1H NMR (DMSO-d6): δ 0.77 (d,
2
3
3J ) 6.7 Hz, 3H), 0.80 (d, J ) 6.7 Hz, 3H), 1.92 (m, 1H), 3.51
130.14 (q, J CF ) 31.9 Hz, C-CF3), 132.32 (CHarom), 138.55,
3
141.09, 146.96 (C), one CdO was not detected. DCI MS: m/z
471 [M + H]+. MALDI-TOF-MS: m/z calcd for [M + H]+ 471.1,
found 471.1; calcd for [M + Na]+ 493.1, found 493.1. Anal.
(C19H17F3N4O3S2) C, H, N.
(m, 1H), 7.52-7.60 (m, 3H), 7.77 (d, J ) 7.5 Hz, 2H), 8.02 (d,
3J ) 9.2 Hz, 1H), 12.58 (s, 1H).
2-Meth yl-N-(p h en ylsu lfon yl)a la n in e (8g). The title com-
pound was prepared starting from 2-aminoisobutyric acid as
described for compound 8a . Yield: 1.84 g (76%) of colorless
crystals. Mp: 146-147 °C. 1H NMR (DMSO-d6): δ 1.25 (s, 6H),
7.52-7.59 (m, 3H), 7.79-7.83 (m, 2H), 7.99 (s, 1H), 12.55 (s,
1H). 13C NMR (DMSO-d6): δ 25.76 (CH3), 57.77 (C), 126.13,
128.92, 131.99 (CHarom), 143.73 (C), 175.32 (COOH).
(2S)-N-[5-(4-Meth oxyp h en yl)-6H-1,3,4-th ia d ia zin -2-yl]-
2-[(p h en ylsu lfon yl)a m in o]p r op a n a m id e (12g). Yield: 345
mg (58%) of colorless plates. Mp: 192-193 °C; HPLC tR: 12.95.
3
1H NMR (DMSO-d6): δ 1.15 (d, J ) 7.0 Hz, 3H), 3.62 (ABq,
3
2J ) 14.5 Hz, 2H), 3.80 (s, 3H), 3.99 (m, 1H), 7.03 (d, J ) 8.6
Hz, 2H), 7.53-7.61 (m, 3H), 7.79 (d, 3J ) 7.4 Hz, 2H), 7.86 (d,
Gen er a l P r oced u r e for P r ep a r in g Su bstitu ted Th ia -
d ia zin e Am id es. Syn th esis of (4S)-2,6-Dioxo-N-(5-p h en yl-
6H-1,3,4-th ia d ia zin -2-yl)h exa h yd r o-4-p yr im id in eca r box-
a m id e (11). To a suspension of 2-amino-5-phenyl-6H-1,3,4-
thiadiazine hydrochloride (6a ) (314 mg, 1.38 mmol), (S)-2,6-
dioxohexahydropyrimidine-4-carboxylic acid (221 mg, 1.40
mmol), and 1-hydroxybenzotriazole (190 mg, 1.40 mmol) in 5
mL of DMF at 0 °C was added 4-methylmorpholine (190 µL,
1.72 mmol) followed by the addition of N-ethyl-N′-(3-dimeth-
ylaminopropyl)-carbodiimide hydrochloride (275 mg, 1.43 mmol).
After 48 h at 5 °C, the mixture was diluted with 20 mL of 0.1
M hydrochloric acid and stirred for 30 min at room tempera-
ture. The precipitate was filtered and washed successively with
water, ice-cold methanol, and diethyl ether. After drying in
vacuo the solid was recystallized from methanol/acetonitrile
(5:1) to yield 225 mg (49%) of the title compound as colorless
crystals. Mp: 198 °C (dec). HPLC tR: 11.30. 1H NMR (DMSO-
d6): δ 2.61 (m, 1H), 2.94 (m, 1H), 3.80 (ABq, 2J ) 14.9 Hz,
2H), 4.18 (m, 1H), 7.48-7.50 (m, 3H), 7.63 (s, 1H), 7.88-7.90
(m, 2H), 10.07 (s, 1H), 12.45 (br s, 1H). 13C NMR (DMSO-d6):
δ 21.63, 33.27 (CH2), 51.64 (CH), 126.95, 128.87, 130.61
(CHarom), 133.97, 148.58, 153.63, 163.32 (C), one C and one
CdO were not detected. DCI MS: m/z 332 [M + H]+. MALDI-
TOF-MS: m/z calcd for [M + H]+ 332.1, found 332.2; calcd for
[M + Na]+ 354.1, found 354.2; calcd for [M + K]+ 370.0, found
370.1. Anal. (C14H13N5O3S) C, H, N.
3
3J ) 8.6 Hz, 2H), 8.06 (d, J ) 7.3 Hz, 1H), 11.99 (br s, 1H).
13C NMR (DMSO-d6): δ 18.98 (CH3), 21.22 (CH2), 53.22 (CH),
55.38 (CH3), 114.22, 126.47, 128.69, 129.02 (CHarom), 129.70
(C), 132.30 (CHarom), 141.15, 148.03, 161.11 (C), one C and one
CdO were not detected. DCI MS: m/z 433 [M + H]+. MALDI-
TOF-MS: m/z calcd for [M + H]+ 433.1, found 433.2; calcd for
[M + Na]+ 455.1, found 455.2. Anal. (C19H20N4O4S2) C, H, N.
(2S)-N-[5-(1-Adamantyl)-6H-1,3,4-thiadiazin-2-yl]-2-[(phen-
ylsu lfon yl)a m in o]p r op a n a m id e (13a ). Yield: 420 mg (66%)
of colorless prisms. Mp: 183-184 °C. HPLC tR: 14.70. 1H NMR
(DMSO-d6): δ 1.11 (d, 3J ) 7.1 Hz, 3H), 1.64-1.70 (m, 6H),
1.77 (s, 6H), 1.99 (s, 3H), 3.17 (ABq, 2J ) 14.5 Hz, 2H), 3.91
3
(m, 1H), 7.51-7.59 (m, 3H), 7.77 (d, J ) 7.2 Hz, 2H), 7.95 (d,
3J ) 7.5 Hz, 1H), 11.89 (br s, 1H). 13C NMR (DMSO-d6): δ
18.99 (CH3), 20.07 (CH2), 27.32 (CH), 35.95 (CH2), 38.55 (C),
53.59 (CH), 126.38, 128.91, 132.14 (CHarom), 141.19, 159.80 (C),
one C and one CdO were not detected. DCI MS: m/z 461
[M + H]+. MALDI-TOF-MS: m/z calcd for [M + H]+ 461.2,
found 461.0; calcd for [M + Na]+ 483.2, found 483.1. Anal. (C22
-
H
28N4O3S2) C, H, N.
(2S)-N-[5-(5-Ch lor o-2-th ien yl)-6H-1,3,4-th ia d ia zin -2-yl]-
2-[(p h en ylsu lfon yl)a m in o]p r op a n a m id e (13b). Yield: 216
mg (35%) of colorless needles. Mp: 198-199 °C; HPLC tR:
1
3
13.67. H NMR (DMSO-d6): δ 1.13 (d, J ) 6.9 Hz, 3H), 3.65
(ABq, 2J ) 14.7 Hz, 2H), 4.03 (m, 1H), 7.22 (d, 3J ) 3.7 Hz,
3
1H), 7.53-7.61 (m, 4H), 7.77-7.79 (m, 2H), 8.13 (d, J ) 7.5
The following compounds were synthesized following the
general procedure as described for compound 11.
Hz, 1H), 11.85 (br s, 1H). 13C NMR (DMSO-d6): δ 18.80 (CH3),
20.27 (CH2), 52.54 (CH), 127.30, 127.97, 129.04, 129.83, 132.35
(CHarom), 132.49, 138.18, 141.03, 143.49 (C), one C and one
CdO were not detected. DCI MS: m/z 443 [M + H]+. MALDI-
TOF-MS: m/z calcd for [M + H]+ 443.0, found 443.0; calcd
for [M + Na]+ 465.0, found 465.0. Anal. (C16H15ClN4O3S3) C,
H, N.
(2S)-N-[5-(4-Nit r op h en yl)-6H -1,3,4-t h ia d ia zin -2-yl]-2-
[(p h en ylsu lfon yl)a m in o]p r op a n a m id e (12d ). Yield: 278
mg (45%) of yellow crystals. Mp: 201-202 °C; HPLC tR: 13.30.
3
1H NMR (DMSO-d6): δ 1.15 (d, J ) 7.0 Hz, 3H), 3.73 (ABq,
2J ) 14.7 Hz, 2H), 4.03 (m, 1H), 7.53-7.61 (m, 3H), 7.79 (d,
3J ) 7.4 Hz, 2H), 8.15 (m, 3H), 8.31 (m, 2H), 12.15 (br s, 1H).
13C NMR (DMSO-d6): δ 18.82 (CH3), 20.99 (CH2), 53.04 (CH),
123.93, 126.47, 128.19, 129.04, 132.33 (CHarom), 140.32, 141.09,
146.65, 148.17 (C), one C and one CdO were not detected. DCI
(2S)-2-[(Ben zylsu lfon yl)a m in o]-N-[5-(4-ch lor op h en yl)-
6H-1,3,4-th ia d ia zin -2-yl]p r op a n a m id e (14a ). Yield: 288
mg (46%) of colorless needles. Mp: 169-170 °C. HPLC tR:

3240 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 20
Schro¨der et al.
1
3
14.03. H NMR (DMSO-d6): δ 1.26 (d, J ) 7.1 Hz, 3H), 3.75
(ABq, 2J ) 13.8 Hz, 2H), 4.00 (m, 1H), 4.33 (ABq, 2J ) 14.0
Hz, 2H), 7.33-7.39 (m, 5H), 7.50 (d, 3J ) 6.2 Hz, 1H), 7.56 (d,
detected. DCI MS: m/z 417 [M + H]+. MALDI-TOF-MS: m/z
calcd for [M + H]+ 417.1, found 417.1; calcd for [M + Na]+
439.1, found 439.1; calcd for [M + K]+ 455.1, found 455.1. Anal.
(C19H20N4O3S2) C, H, N.
3J ) 8.5 Hz, 2H), 7.93 (d, J ) 8.4 Hz, 2H), 12.12 (br s, 1H).
3
13C NMR (DMSO-d6): δ 19.18 (CH3), 21.19 (CH2), 53.12 (CH),
58.64 (CH2), 127.98, 128.24, 128.74, 128.90 (CHarom), 130.32
(C), 130.86 (CHarom), 133.10, 135.20, 147.41 (C), one C and one
CdO were not detected. DCI MS: m/z 451 [M + H]+. MALDI-
TOF-MS: m/z calcd for [M + H]+ 451.1, found 451.3; calcd for
[M + Na]+ 473.1, found 473.3; calcd for [M + K]+ 489.0, found
489.3. Anal. (C19H19ClN4O3S2) C, H, N.
(2S)-N-[5-(4-Ch lor op h en yl)-6H-1,3,4-th ia d ia zin -2-yl]-3-
m eth yl-2-[(ph en ylsu lfon yl)am in o]bu tan am ide (15f). Yield:
324 mg (51%) of colorless needles. Mp: 201-202 °C. HPLC tR:
14.42. 1H NMR (DMSO-d6): 0.77 (d, 3J ) 6.8 Hz, 3H), 0.80 (d,
2
3J ) 6.7 Hz, 3H), 1.93 (m, 1H), 3.59 (ABq, J ) 14.6 Hz, 2H),
3.76 (m, 1H), 7.49-7.57 (m, 5H), 7.76 (d, 3J ) 7.2 Hz, 2H),
7.91-7.93 (m, 3H), 11.95 (br s, 1H). 13C NMR (DMSO-d6):
17.95 (CH3), 19.11 (CH3), 21.00 (CH2), 30.90 (CH), 50.66 (CH),
126.55, 128.82, 132.18 (CHarom), 133.18, 135.15, 140.99, 147.31
(C), one C and one CdO were not detected. DCI MS: m/z 465
[M + H]+. MALDI-TOF-MS: m/z calcd for [M + H]+ 465.1,
found 465.0; calcd for [M + Na]+ 487.1, found 487.0. Anal.
(C20H21ClN4O3S2) C, H, N.
(2S)-N-[5-(4-Ch lor op h en yl)-6H-1,3,4-th ia d ia zin -2-yl]-2-
[(2-th ien ylsu lfon yl)a m in o]p r op a n a m id e (14b). Yield: 301
mg (49%) of colorless plates. Mp: 175-176 °C. HPLC tR: 13.62.
1H NMR (DMSO-d6): δ 1.19 (d, J ) 7.1 Hz, 3H), 3.67 (ABq,
3
2J ) 14.6 Hz, 2H), 4.08 (m, 1H), 7.14 (m, 1H), 7.55-7.56 (m,
3
3
3H), 7.89 (d, J ) 4.9 Hz, 1H), 7.90 (d, J ) 8.5 Hz, 2H), 8.30
(d, J ) 6.9 Hz, 1H), 12.07 (br s, 1H). 13C NMR (DMSO-d6): δ
3
N-[5-(4-Ch lor oph en yl)-6H-1,3,4-th iadiazin -2-yl]-2-m eth -
yl-2-[(p h en ylsu lfon yl)a m in o]p r op a n a m id e (15h ). Yield:
239 mg (38%) of colorless needles. Mp: 204-205 °C. HPLC tR:
14.17. 1H NMR (DMSO-d6): 1.31 (s, 3H), 1.78 (s, 3H), 3.57
(m, 1H), 3.75 (m, 1H), 7.42 (m, 1H), 7.54-7.57 (m, 3H), 7.67
(m, 1H), 7.74 (m, 1H), 7.82 (m, 1H), 7.90 (m, 1H), 8.05-8.10
(m, 2H), 12.11 (br s, 1H). 13C NMR (DMSO-d6): 21.63 (CH2),
24.13 (CH3), 66.85 (C), 126.30, 127.77, 128.15, 128.88, 129.04,
129.36 (CHarom), 133.82 (C), 134.01 (CHarom), 134.52, 135.16,
139.09, 149.85, 160.63, 173.79 (C). DCI MS: m/z 451 [M +
H]+. MALDI-TOF-MS: m/z calcd for [M + H]+ 451.1, found
18.81 (CH3), 21.11 (CH2), 53.12 (CH), 127.54, 128.75, 128.88,
131.62, 132.51 (CHarom), 133.11, 135.18, 142.02, 147.37 (C), one
C and one CdO were not detected. DCI MS: m/z 443 [M +
H]+. MALDI-TOF-MS: m/z calcd for [M + H]+ 443.0, found
443.0; calcd for [M + Na]+ 465.0, found 465.0. Anal. (C16H15
-
ClN4O3S3) C, H, N.
(2R)-N-[5-(4-F lu or op h en yl)-6H-1,3,4-th ia d ia zin -2-yl]-2-
[(p h en ylsu lfon yl)a m in o]p r op a n a m id e (15a ). Yield: 225
mg (39%) of colorless needles. Mp: 167-168 °C. HPLC tR:
1
3
13.17. H NMR (DMSO-d6): δ 1.15 (d, J ) 7.1 Hz, 3H), 3.65
(ABq, 2J ) 14.6 Hz, 2H), 4.02 (m, 1H), 7.30-7.34 (m, 2H), 7.53-
7.61 (m, 3H), 7.78-7.79 (m, 2H), 7.94-7.97 (m, 2H), 8.09 (d,
3J ) 8.3 Hz, 1H), 12.02 (br s, 1H). 13C NMR (DMSO-d6): δ
18.91 (CH3), 21.22 (CH2), 53.10 (CH), 115.74, 115.91, 126.46,
129.03, 129.39, 129.46 (CHarom), 130.80 (C), 132.31 (CHarom),
141.15, 147.44, 162.40, 164.38 (C), one CdO was not detected.
DCI MS: m/z 421 [M + H]+. MALDI-TOF-MS: m/z calcd for
[M + H]+ 421.1, found 421.0; calcd for [M + Na]+ 443.1, found
443.0. Anal. (C18H17FN4O3S2) C, H, N.
451.7; calcd for [M + Na]+ 473.1, found 473.7. Anal. (C19H19
ClN4O3S2) C, H, N.
-
The stereoisomers 12a -c, 12h , 15d , and 15g were synthe-
sized in a similar manner. Analytical data were consistent with
the proposed structures in each case.
En zym e P r ep a r a tion s. MMP-1 from human rheumatoid
synovial fibroblasts was purchased from Calbiochem-Nova-
biochem Corp. (La J olla, CA). Preparation of the recombinant
catalytic domain of human gelatinase A (cdMMP-2): The
catalytic domain of pro-gelatinase A was prepared using an
Escherichia coli expression system.30 The proenzyme was
activated with 0.5 mM APMA at 37 °C prior to use in the
assay. Preparation of the recombinant catalytic domain of
human neutrophil collagenase (cdMMP-8): The enzyme was
expressed in E. coli as an active variant.31 Preparation of
PMNL-gelatinase (MMP-9): Latent PMNL-pro-gelatinase was
prepared from human plasma buffy coat as described by
Tschesche et al.32 PMNL-pro-gelatinase was activated prior
to use by incubation with trypsin at 37 °C for 10 min.
Inactivation of trypsin was accomplished with TKI. Prepara-
tion of the recombinant catalytic domain of human macrophage
elastase (cdMMP-12): The catalytic domain of MMP-12 was
expressed in E. coli. The overexpressed protein was isolated
as inclusion bodies, and the renatured protein was purified
by affinity chromatography with a hydroxamate inhibitor
coupled column.33 Preparation of the recombinant catalytic
domain of human collagenase-3 (cdMMP-13): Human pro-
cdMMP-13 was prepared using an E. coli expression system.34
The isolated pro-cdMMP-13 was activated prior to use by
incubation with 5 mM HgCl2 for 2 h at 37 °C. Preparation of
the recombinant catalytic domain of membrane-type-1 MMP
(cdMMP-14): The catalytic domain of MMP-14 was expressed
in E. coli and was activated by autocatalysis.35 Preparation of
the recombinant human ectodomain of membrane-type-1 MMP
(∆TM-MMP-14): The ectodomain of MMP-14 was expressed
in Pichia pastoris by the method of Roderfeld et al. and
activated by yeast proteinases (furin-like-proteinases) during
maturation.36
(2R)-N-[5-(4-Ch lor op h en yl)-6H-1,3,4-th ia d ia zin -2-yl]-2-
[(p h en ylsu lfon yl)a m in o]p r op a n a m id e (15b). Yield: 308
mg (51%) of colorless needles. Mp: 183-184 °C. HPLC tR:
1
3
13.18. H NMR (DMSO-d6): δ 1.15 (d, J ) 7.1 Hz, 3H), 3.65
(ABq, 2J ) 14.6 Hz, 2H), 4.01 (m, 1H), 7.53-7.61 (m, 5H), 7.78-
3
3
7.80 (m, 2H), 7.92 (d, J ) 8.6 Hz, 2H), 8.09 (d, J ) 8.2 Hz,
1H), 12.04 (br s, 1H). 13C NMR (DMSO-d6): δ 18.88 (CH3),
21.04 (CH2), 53.07 (CH), 126.46, 128.76, 128.87, 129.02, 132.30
(CHarom), 133.12, 135.18, 141.12, 147.34 (C), one C and one
CdO were not detected. DCI MS: m/z 437 [M + H]+. MALDI-
TOF-MS: m/z calcd for [M + H]+ 437.1, found 437.1; calcd
for [M + Na]+ 459.0, found 459.1. Anal. (C18H17ClN4O3S2) C,
H, N.
(2R)-N-[5-(4-Br om op h en yl)-6H-1,3,4-th ia d ia zin -2-yl]-2-
[(p h en ylsu lfon yl)a m in o]p r op a n a m id e (15c). Yield: 365
mg (55%) of colorless crystals from methanol/acetonitrile (10:
1
1). Mp: 179-180 °C. HPLC tR: 13.90. H NMR (DMSO-d6): δ
3
2
1.15 (d, J ) 7.1 Hz, 3H), 3.65 (ABq, J ) 14.6 Hz, 2H), 4.00
3
(m, 1H), 7.53-7.61 (m, 3H), 7.69 (d, J ) 8.4 Hz, 2H), 7.78 (d,
3J ) 7.3 Hz, 2H), 7.84 (d, J ) 8.4 Hz, 2H), 8.10 (d, J ) 8.0
Hz, 1H), 12.05 (br s, 1H). 13C NMR (DMSO-d6): δ 18.89 (CH3),
21.01 (CH2), 53.07 (CH), 124.05 (C), 126.46, 128.98, 129.03,
131.80, 132.31 (CHarom), 133.48, 141.11, 147.45 (C), one C and
one CdO were not detected. DCI MS: m/z 481 [M + H]+.
MALDI-TOF-MS: m/z calcd for [M + H]+ 481.0, found 481.0;
calcd for [M + Na]+ 503.0, found 503.0; calcd for [M + K]+
519.0, found 519.0. Anal. (C18H17BrN4O3S2) C, H, N.
3
3
(2R)-N-[5-(4-Meth ylp h en yl)-6H-1,3,4-th ia d ia zin -2-yl]-2-
[(p h en ylsu lfon yl)a m in o]p r op a n a m id e (15e). Yield: 308
mg (54%) of colorless needles. Mp: 183-184 °C. HPLC tR:
Cr yst a llogr a p h y. Crystals of N-allyl-5-(4-chlorophenyl)-
6H-1,3,4-thiadiazin-2-amine hydrobromide suitable for dif-
fraction analysis were obtained by slow crystallization from
an ethanol/ethyl acetate (1:3) solution. The crystals grew in
space group P21/c [a ) 21.3350(6) Å, b ) 9.3150(2) Å, c )
14.4910(4) Å, â ) 102.9950(19)°] and contain two molecules
per asymmetric unit. Diffraction data were collected under
cryogenic conditions (100 K) on the Nonius Kappa CCD station
1
3
13.42. H NMR (DMSO-d6): δ 1.15 (d, J ) 7.0 Hz, 3H), 2.34
(s, 3H), 3.63 (ABq, 2J ) 14.5 Hz, 2H), 4.00 (m, 1H), 7.29 (d,
3J ) 7.9 Hz, 2H), 7.53-7.61 (m, 3H), 7.78-7.80 (m, 4H), 8.06
(d, J ) 7.9 Hz, 1H), 12.02 (br s, 1H). 13C NMR (DMSO-d6): δ
3
18.94 (CH3), 20.94 (CH3), 21.23 (CH2), 53.22 (CH), 126.46,
126.94, 129.01, 129.40 (CHarom), 131.41 (C), 132.29 (CHarom),
140.37, 141.15, 148.30 (C), one C and one CdO were not

Potent Matrix Metalloproteinase Inhibitors
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 20 3241
to 0.71 Å resolution. All calculations were performed using
maXus.37 Data reduction was done with Denzo and Scalepak.38
SHELXS-9739 was used to solve the structure, SHELXL-9740
was used to refine the structure, and ORTEP-III41 was used
to generate the molecular graphics. The catalytic domain of
human neutrophil collagenase (cdMMP-8) was concentrated
to 8 mg/mL and then mixed with a 3-fold molar excess of an
aqueous solution of N-allyl-5-(4-chlorophenyl)-6H-1,3,4-thia-
diazin-2-amine hydrobromide for a final cdMMP-8 concentra-
tion of 6 mg/mL. Three microliters of protein/inhibitor complex
was mixed with 2 µL of precipitant solution containing 100
mM cacodylate (pH 5.5-6.5), 10 mM CaCl2, 100 mM NaCl,
and 10% PEG 6000. The hanging drop was equilibrated by
vapor diffusion at room temperature against a reservoir
containing 1.0-1.5 M phosphate buffer. Data were collected
on an imaging plate detector (MAR Research) to 2.7 Å
resolution and processed using CCP4 data reduction software.
The space group of the crystal was determined as P212121 with
unit cell dimensions a ) 33.67 Å, b ) 69.64 Å, and c ) 73.40
Å. The orientation and translation of the molecule within the
crystallographic unit cell was determined with Patterson
search techniques42 using the program AMoRe.43 Electron
density calculation and model building proceeded using the
program MAIN.44 The structure was refined using the program
X-PLOR.45 Ring puckering parameters were calculated using
the program RICON.46 Atomic coordinates of the complex have
been deposited in the Protein Data Bank under accession code
1J H1.
for MMP-1, cdMMP-2, cdMMP-8, MMP-9, cdMMP-12, cdMMP-
13, cdMMP-14, and ∆TM-MMP-14 were 40.3, 9.1, 5.9, 1.8, 27.3,
7.5, 6.8, and 7.5 µM, respectively. Ki values were determined
using the method proposed by Horovitz and Levitski.48 This
approach explicitly takes into account the effect of the sub-
strate, enzyme, and inhibitor concentrations and applies to the
situation of both standard and tight-binding inhibition.
Ack n ow led gm en t. We wish to thank the Deutsche
Forschungsgemeinschaft (DFG) for supporting this
project through grant No. Ts 8/37 (H.T.).
Su p p or tin g In for m a tion Ava ila ble: X-ray crystallo-
graphic data for 16 and analysis data for 11, 12a -h , 13a , 13b,
14a , 14b, 15a -h , and 16. This material is available free of
charge via the Internet at http://pubs.acs.org.
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