Solid-phase synthesis of α-substituted 3-bisarylthio N-hydroxy propionamides as specific MMP Inhibitors

Article abstract of DOI:10.1016/S0968-0896(01)00311-X

A novel series of potent and specific MMP-2,3,9,13 inhibitors has been obtained by modulation on solid phase by α and aryl substitutions on 3-arylthio-N-hydroxy-propionamides starting from itaconic acid.

Full text of DOI:10.1016/S0968-0896(01)00311-X

Bioorganic & Medicinal Chemistry 10 (2002) 531–544
Solid-phase Synthesis of ꢀ-substituted 3-Bisarylthio N-Hydroxy
Propionamides as Specific MMP Inhibitors
Anne-Marie Chollet,a Thierry Le Diguarher,a Nathalie Kucharczyk,a Armelle Loynel,a
Marc Bertrand,c Gordon Tucker,b Nicolas Guilbaud,b Mike Burbridge,b
Philippe Pastoureau,b Armel Fradin,b Massimo Sabatini,b
Jean-Luc Faucherea and Patrick Casaraa,*
aInstitut de Recherches Servier, 125 chemin de Ronde, 78290 Croissy sur Seine, France
b
´
Institut de Recherches Servier, 3 rue de la Republique, 92150 Suresnes, France
c
´
Technologie Servier, 25-27 rue E. Vignat, 45007 Orleans, France
Received 28 June 2001; accepted 15 August 2001
Abstract—A novel series of potent and specific MMP-2,3,9,13 inhibitors has been obtained by modulation on solid phase by a and
aryl substitutions on 3-arylthio-N-hydroxy-propionamides starting from itaconic acid. # 2002 Elsevier Science Ltd. All rights
reserved.
Introduction
fore the synthesis of the corresponding bisaryl thioether
2 by a methodology applicable to solid phase was envi-
saged to allow an extensive investigation of the P1/P2
mimic part of the molecule (Scheme 1).
Matrix metalloproteinases (MMPs) are a class of zinc
dependent proteolytic enzymes involved in the degrada-
tion and regeneration of the extracellular matrix.1
Upregulation of specific MMPs has been associated
with various pathologies including arthritis (MMP-
1,3,13),2 and metastatic cancer (MMP-2,9),3 and there-
fore inhibition of these enzymes has been recognised as
a valuable therapeutic approach. Among the com-
pounds in clinical development we can distinguish those
active on a large spectrum of MMPs (CGS 27023A4 and
Ro32-3555[Trocade]5 for arthrosis; BB-2516[Marima-
stat],6 AG3340[Prinomastat]7 and BMS-2752918 for
cancer), and those more recently studied selective for
MMP-13 (RS-1308309 for arthrosis), or for MMP-2,9
(ABT-51810 for cancer). The optimal degree of selectiv-
ity of MMPinhibitors as drugs is still debatable but
access to specific inhibitors would help to understand
the relationship between the various enzymes and dif-
ferent pathologies. For this purpose, we recently descri-
bed a series of bisarylether tetrahedral analogues of the
substrate transition-state, and for example 111 is a very
potent and specific inhibitor of MMP-2,3,9,13. There-
Results and Discussion
Chemical synthesis
A retrosynthetic analysis, using classical modifications,
could link 2 to a-substituted 4-hydroxybutyrate 4. Then
the correlation of 4 to itaconic acid 5, based on a
Michael addition of thiophenolate, could give a direct
access to this new class of compounds. This approach is
attractive for a rapid SAR study, since P1 and P01 sub-
stitutions (respectively X and Y), could be exemplified
independently from the common precursor 4. More-
over, this type of synthon 4 could also be adapted to
solid phase synthesis by anchoring the ester function on
a hydroxamate resin12 (Scheme 2).
Investigation on the P01 side. By a Michael addition of 4-
bromophenyl-thiolate on the mono t-butyl ester of ita-
conic acid 5a, followed by reduction of the carboxylic
acid, the synthon 4 could be obtained in fairly good
yield and in large scale from inexpensive starting mate-
rial. In order to validate the chemical sequence, a SAR
*Corresponding author. Tel.: +33ꢀ1-55-72-2361; fax: +33ꢀ1-55-72-
2470; e-mail: patrick.casara@fr.netgrs.com
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00311-X

532
A.-M. Chollet et al. / Bioorg. Med. Chem. 10 (2002) 531–544
investigation on the P01 side by aryl substitution was
carried out. Previous work in our laboratory had shown
11
that a phenyltriazino group was well accepted at P1
imidine 3–6 as an example. The carboxylic acids 3–1 to
3–6 were then converted into their corresponding
hydroxamic acids 2–1 to 2–6 in reasonable yield by
coupling with allylhydroxamate and subsequent depro-
tection (Scheme 3 and Table 1).
and therefore it was used to start this study. The sub-
stitution of the hydroxyl function of 4 was carried out
under Mitsunobu reaction conditions with benzo-
triazine-4-one in very good yield to give 7. As previously
described,13 a bisaryl substituant at P01 improved the
selectivity index versus MMP-1, since crystallographic
data showed a narrow S01 pocket, more shallow in
MMP-1 than in the other MMPs (replacement of Arg
by Leu). A series of aryl and heteroaryl were envisaged
in order to select a bisaryl moiety for the solid phase
procedure. These P01 terminal modifications were per-
formed by substitution of the 4-bromophenyl on 7 by a
Stille reaction14 with various arylstannanes to give bis
arylderivatives and then hydrolysis of carboxylic acids
3–1 to 3–5. As an alternative, 7 could be transformed
into a stannyl derivative and then arylated with 5-bro-
mopyrimidine to gave the corresponding 4-phenylpyr-
Investigation on the P1/P2 sides. Based on the studies of
the active site specificity of MMP-2,9,15 a preference for
much lower hydrophobic residues was observed at the
P1/P2 sites than at P01 (57/29% compared to 100%,
respectively). We decided to take advantage of this to
randomly modify the P1/P2 side, to combine potency,
selectivity and improved pharmacokinetic values. For
this purpose the solid phase methodology described by
Floyd12a to generate hydroxamic acid from its corre-
sponding carboxylic analogue was applied. The use of O-
hydoxylamine Wang-resin allowed the last three steps of
the synthesis described above to be by passed. In order to
cover a larger substituent diversity, we considered either
acyclic or cyclic variations of X1 and X2 in formula 2.
Scheme 1.
Scheme 2. Retrosynthesis.
Scheme 3. Synthesis of compounds 2-1 to 2-6 and 3-1 to 3-6. (i) (a) MeOH, amberlyst-15, rt 80 h, (97%); (b) isobutene, H2SO4, CH2Cl2, rt 12 h
(67.5%); (c) LiOH, H2O/dioxane, 0 ꢁC, 1.5 h, rt, 2 h (75%); (ii) MeONa, MeOH, 4-BrPhSH, rt, 12 h (6, 80%); (iii) (a) ClCO2iPr, NMM, DME, 0 ꢁC,
+12 h, rt; (c) NaBH4, H2O/DME, ꢀ10 ꢁC, 1 h, (4, 74%); (iv) PPh3, DIAD, benzotriazin-4-one, THF, 0 ꢁC–rt (7, 88%); (v) (a) YSnBu3, Pd(PPh3)4,
LiCl, toluene, Á, 12 h; (b) TFA, CH2Cl2, 0 ꢁC–rt, 18 h (3-1 to 3-5, %, Table 1); (vi) (a) (Bu3Sn)2, P d(OAc)2(P(OPhCH3)3)2, toluene, 80 ꢁC, 12 h,
2
(66%); (b) YBr, Pd2(OAc)2(P(OPhCH3)3)2, NMP, 80 ꢁC, 18 h; (c) TFA, CH2Cl2, 0 ꢁC–rt, 18 h (3-6, %, Table 1); (vii) (a) H2NOAllyl, HOBT, EDC,
NEt3, CH2Cl2, rt; 12 h; (b) Bu3SnH, PdCl2(PPh3)2, AcOH, CH2Cl2, rt, 2 h (2-1 to 2-6, %, Table 1).

A.-M. Chollet et al. / Bioorg. Med. Chem. 10 (2002) 531–544
Table 1. Structures and yields of compounds 3-1 to 3-6 and 2-1 to 2-6
533
Y
Ph
pCl-Ph
3-thienyl
3-Pyridyl
2-Pyrazinyl
5-Pyrimid
inyl
Compound
iv or v%
Compound
vi %
1-3
60
2-1
40
3-23-3
47
2-2
3-4
3-5
3-6
71
2-3
48
39
2-4
47
59
2-5
16
56
2-6
43
35
To have access to a variety of cyclic amides or imides
(X), we tried using Mitsunobu methodology on solid
phase. Unfortunately, g-hydroxyl hydroxamate lacta-
mized very rapidly during this reaction condition on
solid phase, and an alternative sequential approach had
to be considered. The synthon 4 was first acylated, to
allow the introduction of the 4-chlorophenyl moiety by
a Stille reaction, then transformed to its g-hydroxy acid
and fixed on the Wang resin to give the adduct 8. The
various Mitsunobu reactions were then carried out, and
acidic treatment released the corresponding carboxylic
acids 3–8 to 3–19. They were directly engaged as 3–7 on
the hydroxamate resin to form the adducts 9–7 to 9–19
which were converted into the products 2–7 to 2–19
(Scheme 4 and Table 3).
selectivity versus MMP-1 as was the case in the ether
series,11 but the 4-chloro analogue 2–2 exhibited sig-
nificantly improved inhibition of MMP-2,3,9,13 com-
pared with the non substituted biphenyl analogue 2–1.
In our attempt to replace the chlorophenyl terminal in
2–2 by a heteroaryl, the 3-thiophenyl 2–3 maintained
inhibitory activities for all these MMPs in the same
range as 2–1. However, its replacement by either a 3-
pyridinyl (2–4), or a 5-pyrimidinyl (2–6), increased their
IC50 values by one or two orders of magnitude. The
replacement by a 2-pirazyl (2–5) was the most deleter-
ious, with a loss of activity for all MMPs, illustrating
the lipophilic discriminations of the interactions at P01
(Table 2).
Therefore, the 4-chlorobiphenyl moiety at P01 was
maintained in order to enhance the interactions at the
P1 side. In the first selection, all attempts to replace this
triazino group at P1 by cyclic amide or imide functions
(compounds 2–7 to 2–19 in Table 3) decreased affinities
by factors of 10 to 50, with the exception of 2–9, with a
bulky tricyclic substituent, which showed a more
marked decrease in these affinities. In conclusion, all
these compounds remained potent inhibitors of the
gelatinases (MMP-2,9) with IC50 values in the nM
range. Selectivity indices for the other MMPs remained
the same, except for compound 2–14 which had a
slightly better selectivity versus MMP–13 (Table 3).
For the acyclic series (X2=H), a terminal primary
amine was necessary to achieve N acylation. This pre-
cursor 10 was readily obtained from 4 by introduction
of a phthalimido group, hydrolysis of the ester to give
3–7 and then treatment by hydrazine after the attach-
ment to the resin. Then 8 was coupled with a series of
carboxylic acids, and the final hydroxamates 2–20 to 2–
44 were released by acidic treatment (Scheme 4 and
Table 4).
Biological evaluation
The inhibitory activities of these compounds were
examined against a panel of MMPs and the results were
compared to the various clinical references. Since none
of the carboxylic acids 3–1 to 3–7 displayed significant
activities (IC50 in the mM range; data not shown), car-
boxylic acids were no longer evaluated. In the hydro-
xamate series 2–1 to 2–6, all the compounds with
various aryl or heteroaryl at P01 side showed very good
In the second selection, the random replacement of the
P1-triazine moiety of 2–2 by an aryl/heteroaryl mono or
bicyclic amide significantly reduced the affinities for all
the MMPs (compounds 2–20 to 2–44 in Table 4). How-
ever, three compounds (2–22, 2–28 and 2–34) retained
significant activities for MMP-2,9,13. Interestingly,
there was a 10-fold difference in activity on these
Scheme 4. Synthesis of compounds 2-7 to 2-44. (i) (a) Ac2O, pyridine, CH2Cl2, rt, 18 h, (92%); (b) 4-ClPhSnBu3, Pd(PPh3)4, LiCl, toluene, 80 ꢁC,
12 h, (76%); (c) TFA,CH2Cl2, rt, 48 h, (91.5%); (d) LiOH, H2O/THF, 0 ꢁC; (e) HATU, DIEA, (o-Cl-trityl)-resin; (ii) (a) PPh3, DIAD, XH, THF/
CH2Cl2, rt; (b) 5% TFA, CH2Cl2, 0 ꢁC–rt, 18 h (3-8 to 3-19); (iii) HATU, DIEA, hyroxylamine-resin; (iv) 5% TFA, CH2Cl2 (2-7 to 2-19); (v) (a)
PPh3, DIAD, PhtNH, 0 ꢁC—rt, 12 h, (99%); (b) 4-ClPhSnBu3, Pd(PPh3)4, LiCl, toluene, 80 ꢁC, 18 h, (62%); (c) TFA, CH2Cl2, 0 ꢁC–rt, 18 h, (3-7,
100%); (vi) (a) HATU, DIEA, hyroxylamine-resin; (b) NH2NH2, MeOH; (vii) (a) X1CO2H, HATU, DIEA; (b) 5% TFA, CH2Cl2 (2-20 to 2-44).

534
A.-M. Chollet et al. / Bioorg. Med. Chem. 10 (2002) 531–544
enzymes between the 2- and 3-pyridinylamide, com-
pounds 2–22 and 2–23, respectively. When the two het-
eroatoms were present as in the pyrimidyl derivative 2–
24, the activities were only partially recovered. The
introduction of a phenyl ring close to the nitrogen of the
pyridinyl ring, resulting in the 2-(1-azanaphtyl) 2–34
compound, maintained the activities, but when the
phenyl was in the other side, resulting in the 1-(2-aza-
naphtyl) 2–35 compound, the activities were sig-
nificantly decreased. The synthesis of a new series of
compounds would be planned to investigate the active
site interaction at the S2-S3 region of this hetero-
arylnitrogen atom (Table 4).
As a preliminary evaluation for an oncological applica-
tion, compounds were tested on the experimental B16F10
melanoma metastasis model in mice.16 In this experiment,
compound 2–2 at 100 and 200 mg/kg ip, showed a sig-
nificant reduction of tumour burden (45 and 55%, respec-
tively) and a marked reduction of the size of metastases (50
and 100% reduction of number of the metastases with
diameter over 1 mm, respectively). Some in vitro pharma-
cokinetic parameters were also evaluated using human
hepatic microsomes to predict metabolic stability and first
pass metabolism (MF%) and using Caco2 cell line
monolayers permeability to predict absorption (A%).
Compound 2–2 exhibited a modest metabolic stability
(MF=20%), comparable to most of the reference com-
pounds,5,7 but was well absorbed (A=92%) and is
considered as a good candidate for continued study.
Pharmacological evaluation
Selected compounds with IC50<100 nM on MMP-13
were evaluated in an in vitro model of MMP-dependent
cartilage loss. Except compound 2–29, almost all the
acyclicamides 2–20 to 2–44 showed IC50 values over
1 mM for the inhibition of the degradation of pro-
teoglycan. However, several cyclic derivatives, among
them compounds 2–11, 2–12 and 2–15, showed sig-
nificant activity (IC50<0.5 mM), but relatively lower
than the references compounds. Moreover, these com-
pounds are on the racemate form, and therefore the
synthesis of the active isomer have been planned for
continued study (Table 5).
Conclusion
A synthetic access was developed from itaconic acid to
2-substituted-3-bisarylthiopropionic acid hydroxamates,
a new class of inhibitors of MMPs. Key synthon 4 allowed
0
independent optimisation of Pand P sites adaptable to
solid phase, leading to very potent inhibitors of MMP-2, 9,
13 and selective versus MMP-1. The activities of the selec-
ted compounds were validated in vitro in a cartilage
degradation assay, and in vivo in a metastasis model.
Table 2. Structures and biological evaluation of compounds 2-1 to 2-
6 and references
Experimental
Chemistry general techniques
All reactions were carried out under nitrogen or argon
atmosphere under anhydrous conditions unless other-
wise noted.
IC50 (nM) MMP
Yields refer to chromatographically and spectro-
scopically (1H NMR) homogenous material unless
otherwise stated.
No.
Y
1
2
3
9
13
BB-2516
CGS27023A
Ro32-3555
AG3340
2-1
1.5
1.8
25
14
527
1.1
61
1.6
3.4
12
3.4
1.5
96
(Ki) 7
48
>105
15
154
0.5
10
58
0.2
All reagents were purchased at highest commercial qual-
ity and used without further purification unless otherwise
stated. All reactions were monitored by thin layer chro-
matography carried out on 0.2 mm Merck silicagel plates
using UV light and ethanol phosphomolybdic acid or p-
anisaldehyde and heat as developing agent. Preparative
flash chromatography separations were carried out on
Kiesegel 60 (0.04–0.063 mm) Merck silicagel.
1.2
1.3
0.5
4.7
76
2-2
2-3
2-4
2-5
2-6
>104
>104
>103
>104
>103
0.06
1.3
10
1.2
66
13
Reverse-phase HPLC analysis was performed on an
Agilent 1100 instrument using a Xtera Waters column
with detection at 210 nM using a H2O/CH3CN+0.1%
TFA gradient over 15 min.
11
234
966
164
32
92
>103
20
NMR spectra were recorded on a Brucker PPX 200 or
300 instrument as indicated, calibrated using TMS as an
internal reference.
357
6
375
24
The following abbreviations were used to explain mul-
tiplicities: s, singlet; d, doublet; t, triplet; q, quadruplet;
m, multiplet; b, broad.

A.-M. Chollet et al. / Bioorg. Med. Chem. 10 (2002) 531–544
535
IR spectra were recorded on a Brucker Vector 22 spec-
trophometer.
sealed hermetically and the mixture was warmed to
room temperature overnight. After cooling the flask was
opened and the reaction mixture was carefully con-
centrated to 1 L, washed with diluted NaOH (1 N), and
brine. The organic phase was dried over MgSO4, filtered
and the solvent removed in vacuo to give the title com-
pound (200.8 g, 67.5%). The crude product was used
without further purification. 1H NMR (200 MHz,
DMSO-d6): d (ppm) 6.2–5.5 (2s, 2H, CHCH2), 3.7 (s,
3H, CO2CH3), 3.3 (s, 2H, CH2CO2H), 1.4 (s, 9H, tBu).
Electrospray mass spectra were recorded in a positive
mode on a Finnigan TSQ 7000 spectrophometer, by
infusion at 15 mL/min of a 0.1 mg/mL sample solution in
a mixture of CH3CN/H2O (3/1:v/v).
Preparation of tert-butyl-2-carboxymethyl acrylate (5b).
Step 1: 2-methoxycarbonylmethyl acrylic acid. A het-
erogeneous mixture of itaconic acid (200 g, 1.54 mol)
and Amberlyst 15 H+ resin (240 g, washed to neutral,
rinsed with methanol and dried overnight at 40 ꢁC) in
methanol (4 L) was shaken (120–140 r/min) on an oscil-
lated plate during 3 days at room temperature. The
resin was filtered off, and the mixture was evaporated to
dryness. The residue (214 g, 97%) was used for the next
IR: Vmax 1745, 1714, 1641 cmꢀ1
.
Step 3: Preparation of tert-butyl-2-carboxymethyl acry-
late (5b). To a mixture of the compound obtained in
step 2 (200.8 g, ꢂ1 mol) in dioxane (600 mL) was added
an aqueous solution (600 mL) of LiOH, H2O (62 g,
1.5 mol) at 0 ꢁC. The reaction mixture was stirred 1 h
and 30 min at 0 ꢁC and then 2 h at room temperature.
The residual diester was extracted with ether and the
aqueous phase was acidified to pH 5 with 6 N HCl at
0 ꢁC. The aqueous phase was extracted several times
with ether and the combined organic extracts were
washed with brine. The organic phase was dried over
MgSO4, filtrated and the solvent removed in vacuo to
give the title compound (140.55 g, 75.3%) which was
1
step without further purification. H NMR (200 MHz,
DMSO-d6): d (ppm) 9.5 (m, 1H; OH), 6.2–5.8 (2s, 2H,
CHCH2), 3.65 (s, 3H, CO2CH3), 3.4 (s, 2H, CH2CO2Me).
IR: Vmax 2750–2548, 1732, 1691 cmꢀ1
.
Step 2: tert-butyl-2-methoxy carbonyl methyl acrylate.
To a mixture of the compound obtained in step 1 (214 g)
in CH2Cl2 (3 L) concentrated H2SO4 (15 mL) was added
dropwise at 0 ꢁC. Then a stream of isobutene was bub-
bled on the mixture at 0 ꢁC during 15 min. The flask was
1
used without further purification. H NMR (200 MHz,
DMSO-d6): d (ppm) 9.5 (m, 1H; OH), 6.3–5.7 (2s, 2H,
Table 3. Structures and biological Evaluation of compounds 2-7 to 2-19
No.
X
IC50 (nM) MMPNo.
X
IC
(nM) MMP
3
50
1
2
3
9
13
1
2
7
9
8
13
2–7
2-8
>103
2.8
135
4.5
8.7
2-14
2-15
>104
1100
182
332
>103
3
638
12
84
>103
5
7
53
2-9
>103
63
708
132
42
2
353
16
2-16
2-17
>103
>103
2
2
134
78
6
4
31
23
2-10
311
2
3
2-11
>103
335
4
36
2-18
2-19
>103
>103
1
1
190
225
2
3
55
23
2-12
2-13
>103
>103
5
2
104
652
6
2
25
66

536
A.-M. Chollet et al. / Bioorg. Med. Chem. 10 (2002) 531–544
CHCH2), 3.4 (s, 2H, CH2CO2H), 1.4 (s, 9H, tBu). IR: Vmax
2500–2400, 1714, 1641 cmꢀ1. Anal. (C9H14O4,0.14H2O):
C,H calcd: 57.29; 7.58. Found: 57.14; 7.51.
methanol (400 mL). Then to this mixture was added
dropwise a solution of compound prepared in step 3
(32 g, 0.17 mol) in methanol (150 mL).
Preparation of tert-butyl-2-carboxy methyl-3-(4-bromo-
phenylthio) propionate (6). To a mixture of NaOMe
(13.8 g, 0.255 mol) in methanol (300 mL) was added a
solution of 4-bromothiophenol (36.2 g, 0.19 mol) in
The reaction mixture was stirred overnight at room
temperature, then concentrated in vacuo. The residue
was diluted with water and washed several times with
ether. The aqueous phase was cooled at 0 ꢁC, acidified
Table 4. Structures and biological evaluation of compounds 2-20 to 2-44
No.
X
IC50 (nM) MMPNo.
X
IC
(nM) MMP
3
50
1
2
3
9
13
36
1
2
9
13
2-20
647
11
126
28
2-33
>103
12
76
10
22
2-21
2-22
2-23
2-24
2-25
2-26
>103
>103
>103
436
94
2
5258
99
284
8
314
12
2-34
2-35
2-36
2-37
2-38
2-39
328
>104
>103
>104
>103
>104
3
47
57
22
29
61
25
179
3
68
7
45
27
19
25
21
407
173
173
161
66
17
32
12
94
352
208
79
324
123
134
826
115
52
424
795
865
52
>103
37
1311
229
2-27
2-28
2-29
>104
>103
545
25
5
141
35
36
6
234
9
2-40
2-41
2-42
>103
>104
>103
23
1715
20
164
>104
384
55
957
193
92
3580
337
24
34
5
60
2-30
>103
102
520
187
293
2-43
2-44
>103
>103
17
18
274
238
60
35
109
91
2-31
2-32
>103
>103
226
3
5250
144
643
24
787
16

A.-M. Chollet et al. / Bioorg. Med. Chem. 10 (2002) 531–544
Table 5. Inhibition of the proteoglycan degradation
537
No.
IC50 (mM)
No.
IC50 (mM)
No.
IC50 (mM)
No.
IC50 (mM)
BB-2516
CGS27023A
AG3340
2-1
2-2
2-3
0.016
0.022
0.0015
1.7
2-6
2-7
2-8
2-10
2-11
2-12
2-13
>10
0.9
0.8
>10
0.32
0.32
>10
2-15
2-20
2-22
2-23
2-25
2-26
2-28
0.33
2.2
1.3
2-29
2-32
2-33
2-34
2-35
2-40
2-44
0.4
1.9
>10
5
5
>10
3
0.64
>10
>10
1.5
1.1
>10
>10
2-4
with 4 N HCl and extracted several times with ether.
The organic phase was dried over MgSO4 washed with
brine and concentrated in vacuo. Flash chromatography
(silica gel, 20% ethyl acetate in petroleum ether) of the
residue gave compound 6. (48 g, 80.2%). 1H NMR
(200 MHz, DMSO-d6): d (ppm) 12.4 (s, 1H; OH), 7.6–
7.4 (2d, 4H, Ph), 3.2 (m, 2H, CH2S), 2.8 (m, 1H,
CHCO), 2.5 (m, 2H, CH2CO2H), 1.4 (s, 9H, tBu). IR:
Vmax 2665–2575, 1716 cmꢀ1. Anal. (C15H19BrO4S): C,
H, S calcd: 48.01; 5.10; 8.54. Found: 48.43; 5.21; 8.83.
C,H,N,Br,S calcd: 53.88; 4.93; 8.57,6.54,16.29. Found:
54.03; 4.92; 8.58,6.29,15.85.
Preparation of N-hydroxy-2-[2-(4-oxo-4H-benzo[d][,2,3]-
triazin-3-ylethyl)]-3-(biphenyl-4-thio)-propionamide (2.1).
Step 1: Preparation of tert-butyl-2-[2-(4-oxo-4H-ben-
zo[d][,2,3]triazin-3-ylethyl)]-3- biphenyl thio propionate.
To a mixture of compound 7 (3.7 g, 8.16 mmol) in tolu-
ene (80 mL) was added sequentially phenyl tri-n-butyltin
(2.66 mL, 12.2 mmol), tetrakis(triphenyl-phosphine)-
palladium(O) (0.47 g, 0.4 mmol) and lithium chloride
(1.04 g, 24.5 mmol) at room temperature. Then the
reaction mixture was heated under reflux for 12 h and
concentrated in vacuo. The residue was diluted in hep-
tane and extracted several times with acetonitrile. The
organic phases were concentrated in vacuo and the
residue purified by flash chromatography (silica gel,
20% ethyl acetate in petroleum ether) to give the title
Preparation of tert-butyl-2-(2-hydroxy-ethyl)-3-(4-bro-
mophenylthio) propionate (4). To a mixture of com-
pound 6 (48 g, 0.128 mol) in DME (200 mL) at 0 ꢁC was
added sequentially N-methylmorpholine (18 mL,
0.16 mol) and isobutylchloroformiate (22 mL, 0,16 mol).
The reaction mixture was stirred overnight at room
temperature and the precipitate removed by filtration.
To the filtrate was added at ꢀ10 ꢁC a solution of
NaBH4 (7.26 g, 0.19 mol) in water (150 mL). After 1 h,
the reaction mixture was diluted with water (200 mL),
acidified with 1 N HCl (100 mL) and extracted with
ethyl acetate. The organic phase was washed with brine,
dried over NaSO4 and concentrated in vacuo. Flash
chromatography (silica gel, gradient of ethyl acetate in
petroleum ether) gave compound 4 (34 g, 74%). 1H
NMR (200 MHz, CDCl3): d (ppm) 7.4–7.25 (2d, 4H,
Ph), 3.7 (q, 2H, OCH2), 3.25–3.0 (2dd, 2H, CH2S),
2.65 (q, 1H, CHCO), 1.9 (m, 2H, OCH2CH2), 1.6
(t, 1H, OH),1.45 (s, 9H, tBu). IR: Vmax 3427,
1726 cmꢀ1. (M+) (C15H19O4S79Br): calcd: 360.0395.
Found: 360.0378.(C15H19O4S81Br): calcd: 362.0375.
Found: 362.0362.
1
compound (2.56 g, 63%). H NMR (200 MHz, CDCl3):
d (ppm) 8.3–8.1 (2d, 2H, o-Phtriazine), 7.9–7.75 (2t, 2H,
m-Phtriazine), 7.6–7.3 (m, 9H, PhPh), 4.5 (t, 2H,
NCH2), 3.3–3.05 (2dd, 2H, CH2S), 2.65 (m, 1H,
CHCO), 2.3 (m, 2H, NCH2CH2), 1.5 (s, 9H, tBu). IR:
Vmax 1720, 1688 cmꢀ1
.
Step 2: Preparation of 2-[2-(4-oxo-4H-benzo[d][,2,3]-
triazin-3-ylethyl)]-3-(biphenyl-4-thio) propionic acid (3–
1). To a solution of compound obtained in step 1 (2.5 g,
5.15 mmol) in CH2Cl2 was added dropwise tri-
fluoroacetic acid (8 mL) at room temperature. After
18 h, the reaction mixture was concentrated in vacuo
and the residue was crystallised in hexane and diisopro-
pylether to give 3.1 (2.1 g, 95%) which was used for the
next step without further purification.
Preparation of tert-butyl-2-[2-(4-oxo-4H-benzo[d][,2,3]-
triazin-3-ylethyl)]-3-(4-bromo-phenylthio)propionate (7).
To a mixture of triphenylphosphine (37 g, 0.14 mol)
and DIAD (28.6 g, 0.14 mol) in anhydrous THF
(400 mL) at 0 ꢁC was added a mixture of compound 4
(34 g, 0.094 mol) and benzotriazine (14 g, 0.095 mol) in
anhydrous THF (400 mL).After 1 h and 30 min, the
reaction mixture was concentrated in vacuo, diluted
with diisopropylether and the precipitate was removed
by filtration. The filtrate was concentrated in vacuo and
compound 7 (40.6 g, 88%) was obtained by flash chro-
matography of the residue (silica gel, gradient of ethyl
acetate in petroleum ether). 1H NMR (200 MHz,
CDCl3): d (ppm) 8.35–8.15 (2d, 2H, o-Phtriazine), 7.95–
7.8 (2t, 2H, m-Phtriazine), 7.35–7.15 (2d, 4H, Ph), 4.5 (t,
2H, NCH2), 3.25–3.0 (2dd, 2H, CH2S), 2.55 (q, 1H,
CHCO), 2.3 (m, 2H, NCH2CH2), 1.5 (s, 9H, tBu). IR:
Step 3: Preparation of N-allyloxy-2-[2-(4-oxo-4H-ben-
zo[d][,2,3]triazin-3-ylethyl)]-3-biphenyl thio-propiona-
mide. To a solution of compound 3.1 (2.1 g, 4.87 mmol)
in CH2Cl2 (100 mL) was added successively EDC (1.1 g,
5.84 mmol), NEt3 (2 mL, 14.6 mmol) and HOBT (0.8 g,
5.84 mmol) at room temperature. After 12 h, the mix-
ture was dissolved in ethyl acetate. The organic phase
was washed with diluted HCl (0.1 N) and brine and
dried over MgSO4. The solvent was removed in vacuo
and the crude residue (2.1 g, ꢂ87%) was used for next
step.
Step 4: Preparation of 2.1. To a mixture of compound
obtained in step 3 (2.1 g, 4.4 mmol) in CH2Cl2 was
added bis(triphenyl phosphine) palladium (II) chloride
(0.124 g, 0.176 mmol), acetic acid (0.6 mL, 11 mmol) and
tri-n-butyltin hydride (2.3 mL, 8.75 mmol) at room tem-
Vmax 1718, 1686 cmꢀ1
.
Anal. (C22H24BrN3O3S):

538
A.-M. Chollet et al. / Bioorg. Med. Chem. 10 (2002) 531–544
perature. After 1 h, the reaction mixture was con-
centrated in vacuo, and the residue was crystallised
twice in ethyl acetate and in acetonitrile/H2O to give by
filtration 2.1 (0.38 g, 49%) as an orange solid. IR: Vmax
Preparation of N-hydroxy-2-[2-(4-oxo-4H-benzo[d][,2,3]-
triazin-3-ylethyl)]-3-(4-thien-3-ylphenylthio) propioniona-
mide (2-3). Step 1: Preparation of tert-butyl-2-[2-(4-oxo
-4H-benzo[d][,2,3]triazin-3-ylethyl)]-3-(4-thien-3-yl-
phenylthio) propionate. This compound was prepared
by the same procedure as for 3-1 step 1 using thien-3-yl
tri-n-butyl stannane. Yield (2.85 g, 71%). 1H NMR
(200 MHz, CDCl3): d (ppm) 8.3–8.1 (2d, 2H, o-Phtria-
zine), 7.9–7.75 (2t, 2H, m-Phtriazine), 7.5–7.3 (m, 7H,
ThyPh), 4.5 (t, 2H, NCH2), 3.3–3.05 (2dd, 2H, CH2S),
2.6 (m, 1H, CHCO), 2.3 (m, 2H, NCH2CH2), 1.5 (s, 9H,
3500–3150, 1684, 1631 cmꢀ1
.
1H NMR (200 MHz,
DMSO-d6): d (ppm) 10.6–9 (2s, 2H, OH, NH), 8.3 (2d,
2H, o-Phtriazine), 8.15–7.8 (2t, 2H, m-Phtriazine), 7.65–
7.4 (m, 9H, PhPh), 4.4 (t, 2H, NCH2), 3.2 (d, 2H,
CH2S), 2.65 (m, 1H, CHCO), 2.15 (m, 2H, NCH2CH2).
Anal. (C24H22N4O3S,0.65H2O): C,H,N calcd: 62.85;
5.12; 12.22. Found: 62.60; 4.99; 11.90.
tBu). IR: Vmax 1725, 1685 cmꢀ1
.
(C26H27N3O3S2): calcd: 516.1392. Found: 516.1415.
(M+Na+)
Preparation of N-hydroxy-2-[2-(4-oxo-4H-benzo[d][,2,3]-
triazin-3-ylethyl)]-3-(40-chlorobiphenyl-4-thio) propionio-
namide (2–2). Step 1: Preparation of tert-butyl-2-[2-(4-
oxo - 4H - benzo[d][,2,3]triazin - 3 - ylethyl)] - 3 - (40 - chlor-
obiphenyl-4-thio) propionate. This compound was pre-
pared by the same procedure as for 3-1 step 1 using 4-
Step 2: Preparation of 2-[2-(4-oxo-4H-benzo[d][,2,3]-
triazin-3-ylethyl)]-3-(4-thien-3-ylphenyl thio) propionic
acid (3-3). This compound was prepared by the same
procedure as for 3-1 step 2 using the compound
obtained in step 1 (2.13 g, 4.3 mmol) as the starting
material. Yield (1.75 g, 93%). 1H NMR (200 MHz,
CDCl3): d (ppm) 12.5 (s, 1H, CO2H), 8.3–8.1 (2d, 2H,
o-Phtriazine), 8.05–7.9 (2t, 2H, m-Phtriazine), 7.85–7.65
(m, 2H, 2, 5-Thy), 7.55–7.5 (m, 3H, ThyPh) 7.3 (d, 2H,
Thyo-Ph), 4.4 (t, 2H, NCH2), 3.4–3.05 (2dd, 2H, CH2S),
2.6 (m, 1H, CHCO), 2.2 (m, 2H, NCH2CH2). IR: Vmax
3219, 1788, 1663 cmꢀ1. MH+(438).
1
chlorophenyl tri-n-butyl stannane. Yield (2 g, 47%). H
NMR (200 MHz, CDCl3): d (ppm) 8.4–8.15 (2d, 2H,
oPhtriazine), 7.9–7.75 (2t, 2H, m-Phtriazine), 7.4 (m,
8H, PhPh), 4.5 (t, 2H, NCH2), 3.3–3.05 (2dd, 2H,
CH2S), 2.65 (m, 1H, CHCO), 2.35 (m, 2H, NCH2CH2),
1.5 (s, 9H, tBu). IR: Vmax 1723, 1684 cmꢀ1. (M+Na+)
(C28H28ClN3O3S): calcd: 544.1438. Found: 544.1462.
Step 2: Preparation of 2-[2-(4-oxo-4H-benzo[d][,2,3]-
triazin-3-ylethyl)]-3-(40-chlorobiphenyl-4-thio) propion-
ic acid (3–2). This compound was prepared by the same
procedure as for 3-1 step 2 using the compound
obtained in step 1 as the starting material. Yield (2.1 g,
Step 3: Preparation of N-allyloxy-2-[2-(4-oxo-4H-ben-
zo[d][,2,3]triazin-3-ylethyl)]-3-(4-thien-3-ylphenylthio)
propionamide. This compound was prepared by the
same procedure as for 2-1 step 3 using 3-3 as the starting
material (1 g, 2.3 mmol). Yield (0.97 g, 86.5%). 1H
NMR (300 MHz, DMSO-d6): d (ppm) 11.2 (m, 1H,
NH), 8.3–8.2 (dd, 2H, o-Phtriazine), 8.14–7.95 (m, 2H,
m-Phtriazine), 7.85–7.65 (m, 4H, 2, 5-Thyo-Ph), 7.55
(dd, 1H, 4-Thy), 7.35 (d, 2H, m-Ph), 6.05–5.8 (m, 1H,
CHCH2), 5.4–5.2 (m, 2H, CHCH2), 4.4 (t, 2H, NCH2),
4.3 (d, 2H, OCH2), 3.2 (m, 2H, CH2S), 2.35 (m, 1H,
CHCO), 2.15 (m, 2H, NCH2CH2). IR: Vmax 3215, 1682,
1
87%). H NMR (200 MHz, CDCl3): d (ppm) 8.3–8.15
(2d, 2H, o-Phtriazine), 7.9–7.7 (2t, 2H, m-Phtriazine),
7.6–7.25 (m, 8H, PhPh), 4.6 (t, 2H, NCH2), 3.4–3.1
(2dd, 2H, CH2S), 2.7 (m, 1H, CHCO), 2.4 (m, 2H,
NCH2CH2). IR: Vmax 1735, 1649 cmꢀ1. (M+Na+)
(C24H20ClN3O3S): calcd: 488.0812. Found: 488.0841.
Step 3: Preparation of N-allyloxy-2-[2-(4-oxo-4H-ben-
zo[d][,2,3]triazin-3-ylethyl)]-3-(40-chlorobiphenyl-4-thio)
propionamide. This compound was prepared by the
same procedure as for 2-1 step 3 using 3-2 as the starting
material. Yield (1.7 g, 79%). 1H NMR (300 MHz,
DMSO-d6): d (ppm) 11.15 (m, 1H, NH), 8.25 (dd, 2H,
o-Phtriazine), 8.2–7.9 (m, 2H, m-Phtriazine), 7.8–7.5 (m,
6H, PhPh), 7.45 (d, 2H, PhPh),5.9 (m, 1H, CHCH2), 5.3
(m, 2H, CHCH2), 4.35 (t, 2H, NCH2), 4.3 (d, 2H,
OCH2), 3.25 (m, 2H, CH2S), 2.35 (m, 1H, CHCO), 2.15
1655 cmꢀ1
.
515.1188. Found: 515.1221.
(M+Na+)
(C25H24N4O3S2):
calcd:
Step 4: Preparation of 2-3. This compound was pre-
pared by the same procedure as for 2-1 step 4 using the
compound obtained in step 3 (0.95 g, 1.93 mmol) as the
starting material. Yield (0.52 g, 60%). 1H NMR
(200 MHz, DMSO-d6): d (ppm) 10.6–8.9 (2s, 2H, OH,
NH), 8.25 (2d, 2H, o-Phtriazine), 8.1–7.9 (2t, 2H, m-
Phtriazine), 7.85–7.66 (m, 4H, 2, 5-Thyo-Ph), 7.3 (d, 2H,
m Ph), ꢀ4.35 (t, 2H, NCH2), 3.15 (m, 2H, CH2S), 2.4
(m, 1H, CHCO), 2.15 (m, 2H, NCH2CH2). IR: Vmax
3210, 1681, 1627 cmꢀ1. Anal. (C22H20N4O3S2;0.25H2O):
C, H, N calcd: 57.70, 4.48, 12.25. Found: 57.62, 4.48,
11.65.
(m, 2H, NCH2CH2). IR: Vmax 3217, 1681, 1657 cmꢀ1
.
Anal. (C27H25ClN4O3S): C, H, N calcd: 62.24, 4.84,
10.75. Found: 62.19, 4.86, 10.92.
Step 4: Preparation of 2-2: This compound was pre-
pared by the same procedure as for 2-1 step 4 using the
compound obtained in step 3 as the starting material.
Yield (1.1 g, 50.6%). 1H NMR (200 MHz, DMSO-d6): d
(ppm) 10.6–8.9 (2s, 2H, OH, NH), 8.25 (2d, 2H, o-
Phtriazine), 8.2–7.85 (2t, 2H, m-Phtriazine), 7.7–7.35
(m, 8H, PhPh), 4.4 (t, 2H, NCH2), 3.25 (m, 2H, CH2S),
2.45 (m, 1H, CHCO), 2.15 (m, 2H, NCH2CH2). IR:
Vmax 3236, 1687, 1634 cmꢀ1. Anal. (C24H21ClN4O3S; 0.4
H2O): C, H, N calcd: 58.99, 4.46, 11.47. Found: 59.03,
4.49, 11.12.
Preparation of N-hydroxy-2-[2-(4-oxo-4H-benzo[d][,2,3]-
triazin-3-ylethyl)]-3-(4-pyridin-3-ylphenylthio) propionio-
namide (2-4). Step 1: Preparation of tert-butyl-2-(2-
benzotriazoethyl)-3-(4-pyridin-3-ylphenylthio) propio-
nate. This compound was prepared by the same proce-
dure as for 3-1 step 1 using compound 7 (5 g,
10.2 mmol) and pyridin-3-yl tri-n-butyl stannane. (7.5 g,
20.4 mmol). Yield (1.8 g; 39%). 1H NMR (200 MHz,

A.-M. Chollet et al. / Bioorg. Med. Chem. 10 (2002) 531–544
539
CDCl3): d (ppm) 8.8 (s, 1H, 2-Pyr), 8.55 (dd, 1H, 6-
Pyr), 8.3–8.1 (2dd, 2H, o-Phtriazine), 8.0–7.7 (m, 3H, 4-
Pyr, m-Phtriazine), 7.4 (m, 5H, 5-Pyr, Ph), 4.55 (t, 2H,
NCH2), 3.3–3.08 (2dd, 2H, CH2S), 2.65 (m, 1H,
CHCO), 2.35 (m, 2H, NCH2CH2), 1.55 (s, 9H, tBu). IR:
Vmax 1727, 1686 cmꢀ1. Anal. (C27H28N4O3S): C, H, N,S
calcd: 66.37, 5.78, 11.47,6.56. Found: 65.9, 5.84, 11.46,
6.53.
1H, 5-Pyr), 8.3–8.1 (2dd, 2H, o-Phtriazine), 7.9–7.75 (m,
4H, m-Ph, m-Phtriazine), 7.45 (m, 2H, o-Ph), 4.55 (t,
2H, NCH2), 3.35–3.1 (2dd, 2H, CH2S), 2.65 (m, 1H,
CHCO), 2.35 (m, 2H, NCH2CH2), 1.5 (s, 9H, tBu). IR:
Vmax 1727, 1686 cmꢀ1. (M+Na+) (C26H27N5O3S):
calcd: 512.1732. Found: 512.1742.
Step 2: Preparation of 2-[2-(4-oxo-4H-benzo[d][,2,3]-
triazin-3-ylethyl)]-3-(4-pyrazin-2-ylphenyl thio) pro-
pionic acid (3-5).
Step 2: Preparation of 2-[2-(4-oxo-4H-benzo[d][2,3]-
triazin-3-ylethyl)]-3-(4-pyridin-3-ylphenyl thio) pro-
pionic acid (3-4).
This compound was prepared by the same procedure as
for 3-1 step 2 using the compound obtained in step 1
(2.15 g, 4.35 mmol) as the starting material. Yield
(1.75 g, 93%). 1H NMR (200 MHz, DMSO-d6): d (ppm)
12.7 (s, 1H, CO2H), 9.2 (d, 1H, 3-Pyr), 8.7–8.6 (m, 2H,
5,6-Pyr), 8.18–8.12 (2dd, 2H, o-Phtriazine), 8.1–7.8 (m,
4H, m-Ph, m-Phtriazine), 7.4 (m, 2H, o-Ph), 4.45 (t, 2H,
NCH2), 3.3 (2dd, 2H, CH2S), 2.55 (q, 1H, CHCO), 2.2
(q, 2H, NCH2CH2). Anal. (C22H19N5O3S; 0.4H2O):
C,H,N calcd: 59.91, 4.49, 15.88. Found: 59.90, 4.52,
15.17. IR: Vmax 3300–2500, 1676 cmꢀ1. MH+(434).
This compound was prepared by the same procedure as
for 3-1 step 2 using the compound obtained in step 1
(1.7 g, 4.3 mmol) as the starting material. Yield (1.8 g,
1
82%). H NMR (200 MHz, CDCl3): d (ppm) 13 (s, 1H,
CO2H), 9.05 (s, 1H, 2-Pyr), 8.75 (d, 1H, 6-Pyr), 8.5 (d,
1H, 4-Pyr), 8.25–8.15 (2dd, 2H, o-Phtriazine), 8.1–7.8
(m, 3H, 5-Pyr, m-Phtriazine), 7.65–7.45 (m, 4H, Ph),
4.45 (t, 2H, NCH2), 3.3 (m, 2H, CH2S), 2.6 (m, 1H,
CHCO),
2.25
(m,
2H,
NCH2CH2).
Anal.
(C23H20N4O3S; CF3CO2H): C, H, N calcd: 54.94, 3.87,
10.09. Found: 54.86, 3.95, 10.09. IR: Vmax 3300–2500,
1742, 1721, 1674 cmꢀ1. MH+(433).
Step 3: Preparation of N-allyloxy-2-[2-(4-oxo-4H-ben-
zo[d][2,3]triazin-3-ylethyl)]-3-(4-pyrazin-2-ylphenylthio)
propionamide.
Step 3: Preparation of N-allyloxy-2-[2-(4-oxo-4H-ben-
zo[d][,2,3]triazin-3-ylethyl)]-3-(4-pyridin-3-ylphenylthio)
propionamide. This compound was prepared by the
same procedure as for 2-1 step 3 using 3-4 as the starting
material (1.4 g, 2.55 mmol). Yield (1.18 g, 95%). 1H
NMR (200 MHz, CDCl3): d (ppm) 9.5 (m, 1H, NH), 8.6
(m, 2H, 2, 6-Pyr), 8.2–8.05 (2dd, 2H, o-Phtriazine), 7.9–
7.55 (m, 3H, 4-Pyr, m-Phtriazine), 7.4–7.1 (m, 5H, 5-
Pyr, Ph), 6.2–5.9 (m, 1H, CHCH2), 5.4–5.3 (m, 2H,
CHCH2), 4.5 (m, 4H, NCH2, OCH2), 3.35–2.9 (2dd,
2H, CH2S), 2.5–2.0 (m, 3H, CHCO, NCH2CH2). Anal.
(C26H25N5O3S;0.5H2O): C, H, N calcd: 62.90, 5.24,
14.10. Found: 62.97, 5.29, 13.96. IR: Vmax 3600–2500,
This compound was prepared by the same procedure as
for 2-1 step 3 using 3-4 as the starting material (2.1 g,
4.8 mmol). Yield (1.84 g, 78.4%). H NMR (200 MHz,
1
CDCl3): d (ppm) 11.2 (m, 1H, NH), 9.2 (d, 1H, 2-Pyr),
8.7–8.6 (dd, 2H, 4, 5-Pyr), 8.25 (2d, 2H, o-Phtriazine),
8.1 (d, 2H, m-Ph), 8.15–7.85 (2d, 2H, m-Phtriazine),
7.45 (d, 2H, o-Ph) 5.95 (m, 1H, CHCH2), 5.3 (m, 2H,
CHCH2), 4.4 (m, 2H, NCH2), 4.3 (d, 2H, OCH2), 3.3
(2dd, 2H, CH2S), 2.4 (m, 1H, CHCO), 2.15 (m, 2H,
NCH2CH2). IR: Vmax 3346, 1693–1682, 1635 cmꢀ1
.
Anal. (C25H24N6O3S): C, H, N,S calcd: 61.46, 4.95,
17.2, 6.56. Found: 61.25, 4.93 17.23, 6.5.
1680, 1657 cmꢀ1
.
Step 4: Preparation of 2-5. This compound was pre-
pared by the same procedure as for 2-1 step 4 using the
compound obtained above in step 3 (1.8 g, 3.7 mmol) as
the starting material. Yield (0.4 g, 24%). 1H NMR
(200 MHz, DMSO-d6): d (ppm) 10.6–9 (2s, 2H, OH,
NH), 9.2 (s, 1H, 3-Pyr), 8.7-8.6 (m, 2H, 5, 6-Pyr), 8.3-
7.9 (m, 4H, o, m-Phtriazine), 8.05 (dd, 2H, m-Ph), 7.45
(dd, 2H, o-Ph), 4.4 (t, 2H, NCH2), 3.25 (m, 2H, CH2S),
2.50 (m, 3H, CHCO), 2.15 (m, 2H, NCH2CH2). IR:
Step 4: Preparation of 2-4. This compound was pre-
pared by the same procedure as for 2-1 step 4 using the
compound obtained in step 3 (0.95 g, 1.93 mmol) as the
starting material. Yield (0.52 g, 60%). 1H NMR
(200 MHz, DMSO-d6): d (ppm) 10.6–9 (2s, 2H, OH,
NH), 8.85–8.55 (d, 2H, 2,6-Pyr), 8.3–8.15 (m, 2H, o-
Phtriazine), 8.1–7.85 (m, 3H, 4-Pyr, m-Phtriazine), 7.65–
7.5 (m, 5H, 5-Pyr, Ph), 4.38 (t, 2H, NCH2), 3.2 (m, 2H,
CH2S), 2.45 (m, 3H, CHCO), 2.15 (m, 1H, NCH2CH2).
IR: Vmax 3251, 1681, 1641 cmꢀ1. Anal. (C23H21N5O3S):
C, H, N calcd: 61.73, 4.73, 15.65. Found: 61.25, 4.82,
15.08. MH+(448).
Vmax
3192–3071,
1681,
1625 cmꢀ1
.
Anal.
(C22H20N6O3S): C, H, N calcd 58.92, 4.49, 18.74.
Found: 58.72, 4.52, 18.28. MH+(448).
Preparation of N-Hydroxy-2-[2-(4-oxo-4H-benzo[d][,2,3]-
triazin-3-ylethyl)]-3-(4-pyrimidin-5-ylphenylthio) propio-
nionamide (2-6). Step 1: Preparation of 4-[2-tert-
butyloxy-carbonyl-4-(4-oxo-4H-benzo[d][,2,3] triazin-3-
yl)-butylthio] phenyltri-n-butyl stananne.
Preparation of N-hydroxy-2-[2-(4-oxo-4H-benzo[d][,2,3]-
triazin-3-ylethyl)]-3-(4-pyrazin-2-ylphenylthio) propionio-
namide (2-5). Step 1: Preparation of tert-butyl-2-[2-(4-
oxo-4H-benzo[d][,2,3]triazin-3-ylethyl)]-3-(4-pyrazin-2-
ylphenylthio) propionate. This compound was prepared
by the same procedure as for 3-1 step 1 using compound
7 (5 g, 10.2 mmol) and pyrazin-2-yl tri-n-butyl stannane.
(5.5 g, 11.2 mmol). Yield (3.25 g, 59%). 1H NMR
(200 MHz, CDCl3): d (ppm) 9.0 (s, 1H, 3-Pyr), 8.6 (m,
To a mixture of compound 7 (4.26 g, 8.7 mmol) in tolu-
ene (100 mL) was added sequentially bis(tri-n-butyltin)
(5 g,
8.7 mmol)
and
bis(tritoluyloxyphosphine)-
palladium diacetate (0.38 g, 0.05 mmol) at room tem-

540
A.-M. Chollet et al. / Bioorg. Med. Chem. 10 (2002) 531–544
perature. Then the reaction mixture was heated under
reflux for 12 h and concentrated in vacuo. The residue
was purified by flash chromatography (silica gel, gra-
dient ethyl acetate in petroleum ether) to give the title
2H, m-Phtriazine), 7.75 (d, 2H, o-Ph), 7.5 (d, 2H, m-Ph)
6-5.95 (m, 1H, CHCH2), 5.4–5.2 (m, 2H, CHCH2), 4.4
(t, 2H, NCH2), 4.25 (d, 2H, OCH2), 3.2 (m, 2H, CH2S),
2.45 (m, 1H, CHCO), 2.15 (m, 2H, NCH2CH2). IR:
Vmax 3176, 1680 cmꢀ1. Anal. (C25H24N6O3S): C, H, N
cal. 61.46, 4.95, 17.20. Found: 60.64, 4.89, 16.72.
1
compound (1.65 g, 27%). H NMR (200 MHz, CDCl3):
d (ppm) 8.35–8.15 (2dd, 2H, o-Phtriazine), 7.95–7.8 (2t,
2H, m-Phtriazine), 7.3 (m, 4H, Ph), 4.5 (t, 2H, NCH2),
3.25–3.05 (2dd, 2H, CH2S), 2.65 (q, 1H, CHCO), 2.25
(q, 2H, NCH2CH2), 1.65–1.45 (s+m, 15H, tBu, 3CH2),
1.3 (m, 9H, 3CH3), 1.1–0.8 (m, 12H, 6 CH2). IR: Vmax
Step 5: Preparation of 2-6. This compound was pre-
pared by the same procedure as for 2-1 step 4 using the
compound obtained in step 1 above (1.8 g, 3.7 mmol) as
the starting material. Yield (0.4 g, 24%). 1H NMR
(200 MHz, DMSO-d6): d (ppm) 10.7–8.9 (2s, 2H, OH,
NH), 9.2 (s, 1H, 2-Pyr), 9.15 (s, 2H, 3, 5-Pyr), 8.3–
8.1(2d, 2H, o-Phtriazine), 8.1–7.8 (2t, 2H, m-Phtriazine)
7.75 (d, 2H, m-Ph), 7.45 (d, 2H, o-Ph), 4.4 (t, 2H,
NCH2), 3.2 (m, 2H, CH2S), 2.45 (m, 1H, CHCO), 2.15
1728, 1689 cmꢀ1
.
Step 2: Preparation of tert-butyl-2-[2-(4-oxo-4H-ben-
zo[d][,2,3]triazin-3-ylethyl)]-3-(4-pyrimidin-5-ylphenyl-
thio) propionate. To a mixture of the compound
obtained in step 1 above (1.65 g, 2.35 mmol) in N-
methylmorpholine (25 mL) was added sequentially 5-
bromopyrimidine (0.75 g, 4.7 mmol), tetrakis (triphenyl-
phosphine) palladium (O) (0.11 g, 0.12 mmol) and
lithium chloride (0.3 g, 7.5 mmol) at room temperature.
Then the reaction mixture was heated under reflux for
18 h and cooled at room temperature. Then, this mix-
ture was treated with a saturated solution of KF
(1.4 mL) and stirred for 15 min. After filtration, the fil-
trate was diluted with diethylether and washed by dilu-
ted HCl (1 N) and brine. The organic phase was
concentrated in vacuo and the residue was purified by
flash chromatography (silica gel, 20% ethyl acetate in
(m, 2H, NCH2CH2). IR: Vmax 3257, 1683, 1640 cmꢀ1
.
Anal. (C22H20N6O3S): C, H, N calcd: 58.92, 4.49, 18.74.
Found: 58.21, 4.55, 17.45. MH+(449).
Preparation of N-hydroxy-2-(2-X-ethyl)-3-(40-chlorobi-
phenylthio) propionate (2-7 to 2-19). A: Preparation of
2-(2-phthalimidoethyl)-3-(40-chlorobiphenylthio) propio-
nionamide propionic acid (3-7). Step 1: Preparation of
tert-butyl-2-(2-phthalimidoethyl)-3-(4-bromophe-
nylthio) propionate.
To a mixture of triphenylphosphine (25.5 g, 0.097 mol)
and DIAD (19.58 g, 0.097 mol) in anhydrous THF
(250 mL) at 0 ꢁC was added compound 4 (23.4 g,
0.065 mol) in anhydrous THF (150 mL) and then
phthalimide (14.3 g, 0.097 mol). The reaction mixture
was stirred 12 h at room temperature and concentrated
in vacuo. The residue was diluted with diisopropylether
and the precipitate was removed by filtration. The fil-
trate was concentrated in vacuo and the title compound
(29.7 g, 98%) was obtained by flash chromatography of
the residue (silica gel, gradient of ethyl acetate in petro-
1
CH2Cl2) to give the title compound (0.65 g, 56%). H
NMR (200 MHz, CDCl3): d (ppm) 9.2 (s, 1H, 2-Pyr),
8.9 (s, 2H, 4-5Pyr), 8.35–8.1 (2d, 2H, o-Phtriazine), 7.9–
7.75 (2t, 2H, m-Phtriazine), 7.45 (m, 4H, Ph), 4.55 (t,
2H, NCH2), 3.35–3.1 (2dd, 2H, CH2S), 2.65 (q, 1H,
CHCO), 2.3 (m, 2H, NCH2CH2), 1.5 (s, 9H, tBu). IR:
Vmax 1726, 1685 cmꢀ1. Anal. (C26H27N5O3S): C, H, N,S
calcd: 63.78, 5.56, 14.3, 6.55. Found: 63.47, 5.61 13.86,
6.3.
1
Step 3: Preparation of 2-[2-(4-oxo-4H-benzo[d][,2,3]-
triazin-3-ylethyl)]-3-(4-pyrimidin-5-ylphenylthio) pro-
pionic acid (3-6).
leum ether). H NMR (200 MHz, CDCl3): d (ppm) 7.8–
7.7 (m, 4H, Pht), 7.35–7.2 (dd, 4H, Ph), 3.7 (t, 2H,
NCH2), 3.1 (2dd, 2H, CH2S), 2.45 (m, 1H, CHCO), 2.05
(m, 2H, NCH2CH2), 1.45 (s, 9H, tBu). Anal.
(C23H24BrNO4S): C, H, N, S calcd: 56.33, 4.93, 2.86,
6.54. Found: 57.51, 5.01, 3.33, 6.42.
This compound was prepared by the same procedure as
for 3-1 step 2 using the compound obtained in step 2
above (0.61 g, 1.25 mmol) as the starting material. Yield
(0.415 g, 77.5%). 1H NMR (200 MHz, DMSO-d6): d
(ppm) 12.5 (m, 1H, CO2H), 9.2 (d, 1H, 2-Pyr), 9.1 (s,
2H, 5, 6-Pyr), 8.22–8.15 (2dd, 2H, o-Phtriazine), 8.05–
7.85 (2td, 2H, m-Phtriazine), 7.65–7.4 (2d, 4H, o, m-Ph),
4.45 (t, 2H, NCH2), 3.4–3.15 (m, 2H, CH2S), 2.6 (q, 1H,
CHCO), 2.2 (m, 2H, NCH2CH2) Anal. (C22H19N5O3S):
C, H, N calcd: 60.96, 4.42, 16.16. Found: 60.74, 4.59,
15.71. IR: Vmax 3300–2500, 1674 cmꢀ1. MH+(433).
Step 2: Preparation of tert-butyl-2-(2-phthalimi-
doethyl)-3-(40-chlorobiphenylthio) propionate. To
a
mixture of compound obtained in step 1 above (29.4 g,
0.064 mol) in toluene (80 mL) was added sequentially 4-
chlorophenyl tri-n-butyltin (52.48 g, 0.128 mmol), tetra-
kis (triphenylphosphine) palladium(O) (3.7 g, 3.2 mmol)
and lithium chloride (8.15 g, 0.128 mol) at room tem-
perature. Then the reaction mixture was heated under
reflux for 12 h. Then, this mixture was treated with a
saturated solution of KF and stirred for 15 min. After
filtration, the filtrate was washed with brine and dried
over MgSO4. The organic phase was concentrated in
vacuo. The residue was diluted in heptane and extracted
several times with acetonitrile. The organic phases were
concentrated in vacuo and the residue purified by flash
chromatography (silica gel, 10% ethyl acetate in petro-
leum ether) to give the title compound (20.7 g, 62%). 1H
NMR (200 MHz, CDCl3): d (ppm) 7.9–7.6 (m, 4H, Pht),
Step 4: Preparation of N-allyloxy-2-[2-(4-oxo-4H-ben-
zo[d][,2,3]triazin-3-ylethyl)]-3-(4-pyrimidin-5-ylphenyl-
thio) propio-namide.
This compound was prepared by the same procedure as
for 2-1 step 3 using 3-6 as the starting materia (0.415 g,
1
0.96 mmol). Yield (0.4 g, 85.5%). H NMR (200 MHz,
CDCl3): d (ppm) 11. (s, 1H, NH), 9.2 (s, 1H, 2-Pyr), 9.1
(s, 2H, 4,6-Pyr), 8.2 (m, 2H, o-Phtriazine), 8.05–7.9 (2td,

A.-M. Chollet et al. / Bioorg. Med. Chem. 10 (2002) 531–544
541
7.45 (m, 8H, PhPh), 3.75 (t, 2H, NCH2), 3.3–3.05 (2dd,
2H, CH2S), 2.55 (m, 1H, CHCO), 2.1 (q, 2H,
NCH2CH2), 1.5 (s, 9H, tBu). Anal. (C29H28ClNO4S): C,
H, N, S calcd: 66.72, 5.41, 2.68, 6.14. Found: 66.67,
(2dd, 2H, CH2S), 2.6 (m, 1H, CHCO), 2.0(m, 2H,
OCH2CH2), 2.0 (s, 3H, COCH3) 1.5 (s, 9H, tBu). IR:
Vmax 1737, 1236–1150 cmꢀ1
.
5.46, 2.74, 6.01. IR: Vmax 1773, 1717 cmꢀ1
.
Step 3: Preparation of 2-(2-acetoxyethyl)-3-(40-chlor-
obiphenylthio) propionic acid.
Step 3: Preparation of (3-7). To a solution of compound
obtained in step 2 above (20.71 g, 39.6 mmol) in
CH2Cl2(300 mL) was added dropwise trifluoroacetic
acid (61 mL) at 0 ꢁC. The reaction mixture was stirred
6 h at room temperature and concentrated in vacuo. The
residue was crystallised in hexane and diisopropylether
to give 3–7 (18.5 g, quant). 1H NMR (200 MHz,
CDCl3): d (ppm) 12.5 (m, 1H, CO2H), 7.8 (m, 4H,
3Pht), 7.65–7.35 (2dd, 8H, PhPh), 3.65 (t, 2H, NCH2),
3.2 (m, 2H, CH2S), 2.5 (m, 1H, CHCO), 2.35 (m, 2H,
NCH2CH2). Anal. (C25H20ClNO4S): C, H, N, S calcd:
64.44, 4.33, 3.01, 6.88. Found: 64.12, 4.40, 3.09, 6.91.
IR: Vmax 3274, 1764, 1734, 1696 cmꢀ1. MH+(466).
To a solution of compound obtained in step 2 above
(23.2 g, 0.053 mol) in CH2Cl2 (100 mL) was added
dropwise trifluoroacetic acid (5 mL) at 0 ꢁC. The reac-
tion mixture was stirred 48 h at room temperature, con-
centrated in vacuo and the residue purified by flash
chromatography (silica gel, 5% ethyl acetate, 1% acetic
acid in CH2Cl2) to give the title compound (18.5 g,
1
89%). H NMR (200 MHz, CDCl3): d (ppm) 12.5 (m,
1H, CO2H), 7.5 (m, 8H, PhPh), 4.15 (t, 2H, OCH2CH),
3.3 (dd, 1H, CH2S), 3.05 (dd, 1H, CH2S), 2.75 (m, 1H,
CHCO), 2.1(t, 2H, OCH2CH2), 1.95 (s, 3H, COCH3).
Anal. (C19H19ClO4S): C, H, S, Cl calcd: 60.23, 5.05,
8.46, 9.36. Found: 60.28, 5.19, 8.78, 9.08. IR: Vmax 3400,
3000, 1726, 1696 cmꢀ1
.
B: Preparation of 2-(2-X-ethyl)-3-(40-chlorobiphenylthio)
propionic acid (3-8 to 3-19). B-1: Preparation of the
carboxylic resin 8. Step 1: Preparation of tert-butyl-2-(2-
acetoxyethyl)-3-(4-bromophenylthio) propionate.
Step 4: Preparation of 2-(2-hydroxyethyl)-(40-chlor-
obiphenylthio) propionic acid.
To
a
solution of 2-(2-acetoxyethyl)-3-(40-chlor-
To a mixture of tert-butyl-2-(2-hydroxy-ethyl)-3-(4-bro-
mophenylthio) propionate (4) (27.85 g, 0.077 mol) in
CH2Cl2 (300 mL) was added sequentially pyridine
(12.5 mL, 0.154 mol), and acetic anhydride (8.8 mL,
0.0925 mol) at room temperature. Then the reaction
mixture was stirred 5 days and concentrated in vacuo.
The residue was diluted with ethyl acetate washed by
diluted HCl (1 N), saturated NaHCO3 and brine. The
organic phase was concentrated in vacuo and the resi-
due was purified by flash chromatography (silica gel,
5% ethyl acetate in heptane) to give the title compound
obiphenylthio) propionic acid (7.2 g, 19 mmol) in THF
(150 mL) was added a solution of LiOH, H2O (2.4 g,
57 mmol) in water (40 mL). After 3 h stirring at room
temperature, the reaction mixture was concentrated in
vacuo and acidified with HCl 0.1 N. The alcohol was
extracted with a mixture of ethylacetate/diethylether
and the solution rapidly concentrated in vacuo (in order
to avoid formation of the lactone) to give 2-(2-hydro-
xyethyl)-3-(40-chlorobiphenylthio) propionic acid as a
white wax (6 g, 93%). The instable g-hydroxy acid was
immediately coupled to the resin.
1
(28.7 g, 92%). H NMR (200 MHz, CDCl3): d (ppm)
7.4–7.2 (2d, 4H, Ph), 4.1 (t, 2H, OCH2), 3.2–2.95 (2dd,
2H, CH2S), 2.6 (m, 1H, CHCO), 2.05 (s, 3H, COCH3),
1.5 (m, 2H, OCH2 CH2), 1.45 (s, 9H, tBu). IR: Vmax
Step 5: Preparation of the resin 8. 2-Chlorotritylchloride
resin (Senn Chemicals, 100–200 mesh, 1.4 mmol/g)
(20.35 g, 28.5 mmol) was pre-swollen in CH2Cl2 for 1 h
in a semi-automated Labortec solid-phase synthesizer
(SP4000). After filtration, a solution of the g-hydroxy
acid (6 g, 17.8 mmol) in dichloromethane (250 mL) was
introduced in the reactor. N,N-diisopropylethylamine
(8.3 mL, 47.5 mmol) was added and the suspension sha-
ken for 16 h at room temperature. 10 mL methanol were
added for capping. After 30 min, the resin was filtered
and rinsed alternatively three times with DMF and iso-
propyl alcohol, three times with DCM, twice with Et2O
and dried in vacuo (24.1 g).
1736, 1234, 1150 cmꢀ1
.
Step 2: Preparation of tert-butyl-2-(2-acetoxyethyl)-3-
(40-chlorobiphenylthio) propionate.
To a mixture of compound obtained in step 1 above
(28.5 g, 0.07 mol) in toluene (80 mL) was added sequen-
tially 4-chlorophenyl tri-n-butyltin (49 g, 0.122 mol),
tetrakis (triphenylphosphine) palladium(O) (4.1 g,
3.5 mmol) and lithium chloride (9.1 g, 0.23 mol) at room
temperature. Then the reaction mixture was heated
under reflux for 12 h. Then, this mixture was treated
with a saturated solution of KF and stirred for 15 min.
After filtration, the filtrate was washed with brine and
dried over MgSO4. The organic phase was concentrated
in vacuo. The residue was diluted in heptane and
extracted several times with acetonitrile. The organic
phases were concentrated in vacuo and the residue pur-
ified by flash chromatography (silica gel, 5% ethyl ace-
tate in petroleum ether) to give the title compound
B-2:
Preparation
of
2-(2-X-ethyl)-3-(40-chloro-
biphenylthio) propionic acid (3-8 to 3-19). Step 1: The
ester resin (16 g) was evenly distributed in 15 reactors of
a robotic Zymark solid-phase synthesizer (1.1 g per
reactor, 0.79 mmol theor). The resins were washed three
times with CH2Cl2, filtered and suspended in 3 mL
CH2Cl2. 15 different imides pre-weighted (2.37 mmol) in
individual vials were dissolved in 15 mL of a solution of
triphenylphosphine (622 mg, 2.37 mmol) in THF/
CH2Cl2 50/50. The solutions were introduced via
1
(23.2 g, 76%). H NMR (200 MHz, CDCl3): d (ppm)
7.5–7.3 (m, 8H, PhPh), 4.1 (t, 2H, OCH2CH), 3.25–3.0

542
A.-M. Chollet et al. / Bioorg. Med. Chem. 10 (2002) 531–544
cannula to the 15 reactors and diisopropyl azodicarbox-
ylate was manually added in four portions (479 mg,
2.37 mmol) at 5 min time intervals. The suspension was
mixed by intermittent nitrogen bubbling for 2 h. After
filtration, a second coupling was performed. The resins
were automatically washed alternatively three times
with DMF and isopropyl alcohol, three times with
CH2Cl2.
0.71 mmol/g, 25 g, 17.75 mmol) was pre-swollen in
CH2Cl2 for 1 h on a semi-automated Labortec solid-
phase synthesizer. After 2 washings with CH2Cl2 and
DMF, the Fmoc protecting group was cleaved with a
solution of 20% piperidine in DMF (2ꢃ20 min). The
resin was rinsed three times with DMF, three times with
CH2Cl2. A solution of 3-7 (12.4 g, 26.62 mmol) and
HATU (10.2 g, 26.62 mmol) in 250 mL DMF was added
to the resin, followed by N,N-diisopropylethylamine
(7.75 mL, 44.38 mmol). The suspension was shaken for
16 h, filtered and washed alternatively three times with
DMF and isopropyl alcohol, three times with CH2Cl2.
Step 2: Preparation of 3-8 to 3-19. The reactors were
transferred to a semi-automated cleavage station and
treated 30 min with 20 mL of 5% trifluoroacetic acid/
CH2Cl2 solution. The filtrates were collected in vials and
concentrated in vacuo in a Christ B-RVC parallel eva-
porator to give the carboxylic acids 3-8 to 3-19 which
were used for next step without further purification.
Step 2: The resin obtained in step 1, was treated with a
10% solution of hydrazine hydrate in methanol for 2 h.
After filtration, a second cleavage was performed for
16 h. The resin was washed five times with DMF, alter-
natively three times with DMF and isopropyl alcohol,
and three times with CH2Cl2.
C: Preparation of 2-7 to 2-19. Step 1: Fmoc-hydro-
xylamine-o-chlorotrityl resin (Novabiochem, 200–400
mesh, 0.76 mmol/g, 15 g) was distributed evenly in 15
reactors of the Zymark solid-phase synthesizer17 (1 g per
reactor, 0.76 mmol). After three washings with CH2Cl2,
20 mL of a 20% piperidine/DMF solution was dis-
pensed in each reactor to cleave the Fmoc protecting
group. The resin was mixed by intermittent nitrogen
bubbling for 20 min, filtered, and a second cleavage was
performed for 20 min. The resin was washed three times
with DMF, three times with CH2Cl2 and was suspended
in 10 mL of a diisopropylethylamine/DMF solution
(270 mL, 1.52 mmol).
B: Preparation of 2-20 to 2-44. Step 1: The resin 10
was introduced in the mixing chamber of the robotic
Zymark synthesizer, evenly distributed in the 25 reac-
tors (0.71 mmol per reactor) and washed three times
with CH2Cl2. Then 25 different acids pre-weighted in 25
vials (2.13 mmol) were dissolved in 7 mL of HATU/
DMF solutions (810 mg per reactor, 2.13 mmol). After
addition of 10 mL of a N,N-diisopropylethylamine/
DMF solution (366 mg per reactor, 2.84 mmol) to the
resins, the acid solutions were introduced via cannula to
their corresponding reactor. The suspensions were
mixed by nitrogen bubbling for 10 h, filtered and rinsed
alternatively three times with DMF and isopropyl alco-
hol, three times with CH2Cl2.
The compounds 3-7 to 3-19 (0.76 mmol theor) prepared
above were introduced in 13 individual vials, dissolved
in a solution of HATU (289 mg per reactor, 0.76 mmol)
in 7 mL DMF. The 13 solutions were introduced via
cannula to the 13 reactors and the suspensions mixed by
nitrogen bubbling for 10 h. After filtration, the resin was
washed alternatively three times with DMF and isopro-
pyl alcohol, three times with CH2Cl2.
Step 2: The reactors were transfered to a semi-auto-
mated cleavage station and treated for 30 min with
15 mL of 5% trifluoroacetic acid/CH2Cl2 solution. The
filtrates were collected in vials and concentrated in
vacuo in a Christ B-RVC parallel evaporator. After
crystallisation in diisopropylether and filtration, the
precipitates were lyophilised in dioxane. Yields and
analytical data of compounds 2-20 to 2-44 were sum-
marized in Table 7.
Step 2: preparation of 2-7 to 2-19: The reactors were
transferred to a cleavage station and treated for 30 min
with 20 mL of 5% trifluoroacetic acid/CH2Cl2 solution.
The filtrates were collected in vials and concentrated in
vacuo in a Christ B-RVC parallel evaporator. After
crystallisation in acetonitrile and filtration, the pre-
cipitates were lyophilised in dioxane. Yields and analy-
tical data of compounds 2-7 to 2-19 were summarized in
Table 6.
Enzyme assays
Human pro-MMPs were dissolved in Novex developing
buffer (Cat. No LC2671) at the following concentra-
tions: MMP-1 (Calbiotech) at 1.25 mg/ml; MMP-2 and
MMP-9 (Boehringer) at 300 and 200 mU/mL respec-
tively; MMP-3 (AbCys) at 1 mg/mL and MMP-13 (Pr G.
Murphy, Univ. East Anglia) at 2 mg/mL. Pro-enzymes
were activated by 2 mM p-aminophenilmercuric acetate
Preparation of N-hydroxy-2-(2-X1-aminocarbonylethyl)-
3-(40-chloro-biphenylthio) propionates 2-20 to 2-44. A:
Preparation of resin 10. Step 1: Fmoc-hydroxylamine-o-
chlorotrityl resin (Novabiochem, 200–400 mesh,
Table 6. Yields and analytical data of compounds 2-7 to 2-19
2-7
2-8
2-9
2-10
2-11
2-12
2-13
2-14
2-15
2-16
2-17
2-18
2-19
Yield %
MH+
Purity %
68
481
95
15
482
91
9
531
87
24
549
90
16
485
79
18
473
93
3
480
94
7
497
92
6
512
83
16
480
88
14
447
97
19
523
88
23
524
88

A.-M. Chollet et al. / Bioorg. Med. Chem. 10 (2002) 531–544
Table 7. Yields and analytical data of compounds 2-20 to 2-44
2-20 2-21 2-22 2-23 2-24 2-25 2-26 2-27 2-28 2-29 2-30 2-31 2-32 2-33 2-34 2-35 2-36 2-37 2-38 2-39 2-40 2-41 2-42 2-43 2-44
543
Yield %
MH+
90
473 491 456 456 457 539 518 513 527 499 620 511 523 495 506 506 508 522 509 509 494 565 539
89 91 84 95 76 85 81 88 96 73 91 70 92 84 93 84 78 82 98 94 94
52
37
23
14
56
22
68
45
64
81
53
93
20
18
35
79
53
92
41
29
55
79
44 39
553 507
Purity % 82
77 43:55 84
(APMA, Sigma) at 37 ꢁC for 30 min (MMP-1, -2, -9) or
1 h (MMP-3,-13). Activation was stopped by transfer-
ring the samples to ice. Inhibitors were dissolved in
dimethyl sulfoxide (DMSO) at 10ꢀ2 M, then serially
diluted (1/10) in developing buffer at concentrations
from 10ꢀ4 to 10ꢀ13 M. Fluorogenic substrates were
purchased from Bachem. Substrate for MMP-3 was (7-
methoxycoumarine-4-yl)-Arg-Pro-Lys-Pro-Tyr-Ala-Nva-
Trp-Met-Lys(Dnp)-NH2.18 Substrate for MMP-1, -2, -
9, -13 was Dnp-Pro-Cha-Gly-Cys(ME)-His-Ala-Lys
(Nma)-NH2.19 They were dissolved in DMSO at 10ꢀ2
and 2ꢃ10ꢀ3 M, respectively, then diluted to 2ꢃ10ꢀ4 M
in water. Assays were performed in 96-well plates by
adding to each well 70 mL of developing buffer, 10 mL of
inhibitor (or buffer for the control) and 10 mL of enzyme
(or buffer for the blank). Each point was run in dupli-
cate and each inhibitor was assayed at least twice. After
a 30 min preincubation at 37 ꢁC, 10 mL of substrate was
added and the plates incubated for 6 h at 37 ꢁC. Reading
was then performed by a Spectrofluor Plus fluorimeter
(Tecan), set at excitation and emission wavelengths of
340 and 440 nm, respectively. Substrate degradation in
the presence of inhibitor at a given concentration was
calculated as % fluorescence of control wells. IC50 of
each product on each enzyme was calculated by EXCEL
software using three points in the central linear range of
fluorescence inhibition.
culture media and in the tissue digest was measured by
liquid scintillation using a b-counter (Beckman). Pro-
teoglycan degradation in each fragment was expressed
as the percentage of released radioactivity. The effect of
MMPinhibitors was calculated from the mean values of
proteoglycan degradation in each group by the formula:
% inhibition=(control-treatment)/(control-basal)ꢃ100.
IC50 of each product on proteoglycan degradation was
calculated by a SAS-based software using at least three
concentration points in the linear range of % inhibition.
Acknowledgements
The authors wish to thank Sophie Sciberras, Christine
Fouache, Sandra Haumont, Marie Thomas, Christophe
Lesur, Virginie Malivet and Frederic Mauppin for their
skilful technical assistance and Solange Huet for the
preparation of the manuscript. We would like also to
acknowledge the analytical department at the IdRS for
performing all the spectral analysis.
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