Introduction of the 4-(4-bromophenyl)benzenesulfonyl group to hydrazide analogs of Ilomastat leads to potent gelatinase B (MMP-9) inhibitors with improved selectivity

Article abstract of DOI:10.1016/j.bmc.2008.07.041

Hydrazide derivatives of Ilomastat, carrying either aryl groups or distinct alkyl and arylsulfonyl moieties were synthesized and evaluated for their MMP inhibitory activity. Potent and selective MMP-9 inhibition (IC₅₀ = 3 nM) was observed for c

Full text of DOI:10.1016/j.bmc.2008.07.041

Bioorganic & Medicinal Chemistry 16 (2008) 8745–8759
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Introduction of the 4-(4-bromophenyl)benzenesulfonyl group to hydrazide
analogs of Ilomastat leads to potent gelatinase B (MMP-9) inhibitors
with improved selectivity
Gwennaël LeDour a, Gautier Moroy b, Matthieu Rouffet a, Erika Bourguet a, Dominique Guillaume a,
Martine Decarme c, Haquima ElMourabit c, Franck Augé a, Alain J. P. Alix b, Jean-Yves Laronze a,
Georges Bellon c, William Hornebeck c, Janos Sapi a,
*
a Université de Reims-Champagne-Ardenne, CNRS UMR 6229, Institut de Chimie Moléculaire de Reims, Faculté de Pharmacie, IFR53 Biomolécules, 51 rue de Cognacq-Jay,
51096 Reims Cedex, France
b Université de Reims-Champagne-Ardenne, Laboratoire de Spectroscopies et Structures Biomoléculaires, (LSSBM), Faculté des Sciences, IFR53 Biomolécules, BP 1039,
51687 Reims Cedex 2, France
c Université de Reims-Champagne-Ardenne, CNRS UMR 6237, Laboratoire de Biochimie Médicale, Faculté de Médecine, IFR53 Biomolécules, 51 rue de Cognacq-Jay,
51096 Reims Cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 August 2006
Revised 8 July 2008
Accepted 16 July 2008
Available online 20 July 2008
Hydrazide derivatives of Ilomastat, carrying either aryl groups or distinct alkyl and arylsulfonyl moieties
were synthesized and evaluated for their MMP inhibitory activity. Potent and selective MMP-9 inhibition
(IC50 = 3 nM) was observed for compound 3m (arylsulfonyl group: 4-(4-Br–C6H4)–C6H4–SO2–). Interac-
tion with the S2 enzyme subsite is mainly responsible for the inhibitory properties of this derivative as
confirmed by molecular docking computation.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Matrix metalloproteinase
Selective MMP-9 inhibition
Sulfonylhydrazide
Zinc-binding group
Ilomastat
Molecular modeling
1. Introduction
Indeed, besides acting as matrix degrading enzymes, MMPs can
hydrolyze or activate cytokines and growth factors, shed or acti-
Matrix metalloproteinases (MMPs) belong to the M10 clan of
zinc containing endopeptidases that have been shown to play an
important role in a variety of physiological functions.1 Their activ-
ity is controlled by endogenous inhibitors, mainly tissue inhibitors
of metalloproteinases (TIMP). Disruption of the finely tuned MMPs/
TIMPs balance is observed in several pathologies including osteoar-
thritis, atherosclerosis, aneurysm, pulmonary emphysema, neuro-
degenerative diseases, and cancer.
Consequently, the development of synthetic MMP inhibitors
(MMPIs) possessing a high potency and selectivity has attracted
significant efforts aiming to interfere with the progression of these
pathologies.2
vate receptors, and modulate the control of proteinases from other
families through proteolysis of proteinase inhibitors. Important
side effects and lack of selectivity owing to similarities of the active
sites of other metalloproteinases (ACE, ADAM, ADAMT) were also
reported to be responsible for the inefficiency of early MMPIs.4
Finally, clinical trials have also evidenced that some MMPIs may
have disease-promoting or suppressing activities, leading to the
recent classification of MMPs as being either target or antitarget
in cancer progression.5 It needs to be emphasized that gelatinases,
that is, MMP-2 and MMP-9 have been considered to play a pivotal
role in the development of several cancer types and aneurysm
formation.
To date a myriad of broad spectrum MMP inhibitors has been
synthesized but the results of the first clinical trials, notably in can-
cer area, proved to be rather disappointing due to the multifaceted
functions of this endopeptidase family.3
A vast majority of the thousands competitive MMPIs bears an
hydroxamic acid moiety as zinc-binding group (ZBG).6 However,
hydroxamate-type inhibitors display several drawbacks as low
biodisponibility and stability as well as poor pharmacokinetic
properties. In addition, the high potency of hydroxamic acid as zinc
chelator might be somewhat disadvantageous since it can mask
the contribution of other subsites in enzyme inhibition. This lack
* Corresponding author. Tel.: +33 (0) 326918022; fax: +33 (0) 326918029.
E-mail address: janos.sapi@univ-reims.fr (J. Sapi).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.07.041

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G. LeDour et al. / Bioorg. Med. Chem. 16 (2008) 8745–8759
of selectivity has triggered important synthetic efforts to develop
alternative ZBGs such as carboxylate, thiol, phosphonic acid, re-
versed hydroxamate, sulfonamide functions and other heterocy-
cles7 and to better exploit the neighboring subsites (S1 , S2 . . ., S1,
S2. . .).
0
0
To that respect, we recently initiated a research program sup-
ported by molecular modeling to identify MMPIs containing novel
zinc-binding groups.8 For our pharmacomodulation we chose
Ilomastat 1, a potent but non-selective hydroxamic acid containing
inhibitor (Fig. 1).9 From this preliminary investigation a few hydra-
zide-type functions (2) have emerged as novel ZBGs. A second ser-
ies of Ilomastat analogs (3) bearing sulfonylhydrazide-type ZBG
has also been proposed. By incorporating a more electron-with-
drawing arylsulfonyl group on the hydrazide function we envis-
aged to possibly reinforce the interactions between our inhibitors
and the Glu residue in MMP active site (Fig. 2).10
Figure 2. Bindings of hydroxamate and (sulfonyl)-hydrazide type ZBG.
after purification by chromatography. Coupling of acid 5 with
aryl(or methyl)sulfonylhydrazines according to the same path-
way afforded the corresponding sulfonylhydrazides 3a–d in
moderate yield. Sulfonylhydrazines used for direct coupling were
prepared by sulfonylation of N-Boc-hydrazine followed by hydro-
chloric acid mediated deprotection.14 We attributed the low
yield of the direct coupling to the low nucleophilic character
of sulfonylhydrazines.
To overcome such drawbacks, we tried a two-step pathway via
sulfonylation of the corresponding non-substituted hydrazide 2f
which was prepared by coupling 5 with N-Boc-hydrazine and sub-
sequent acid catalyzed deprotection of 2e. Sulfonylation of 2f affor-
ded the targeted arylsulfonylhydrazide Ilomastat analogs 3e–m in
variable yields (11–70%). 2-Aminophenyl substituted analog 3k
was obtained by catalytic reduction (10% Pd/C, H2) of the corre-
sponding nitro derivative 3f. Aliphatic long chain analogs 3g,h
were prepared using dodecansulfonylchloride and hexadecansulf-
onychloride as sulfonating agent, respectively.
Importantly, hydrazide-type ZBG presents the advantage to ex-
tend the molecule to the left-hand side thus allowing to study the
contribution of non-prime MMP subsite (S1, S2) occupancy in inhi-
bition potency and selectivity. Such bis-arylsulfonylamide-type
group containing inhibitors have already been described11 occupy-
0
ing mainly the S1 enzyme subsite.
As an extension of our program, herein we report in full details
the synthesis and biological evaluation of new hydrazide (2) and
sulfonylhydrazide (3) group containing Ilomastat analogs. In addi-
tion, a complementary study has been carried out from the most
potent 4-(4-bromophenyl)benzenesulfonylhydrazide derivative to
0
0
better exploit S1 and S2 enzyme subsites. To this end, different
alkyl(aryl)alkylidene side chains and 2-phenyl substituted tryp-
tophanamide were incorporated into the pseudopeptide backbone
(analogs of type 4). Inhibitory activities were measured on the
main MMPs and the results were analyzed by molecular docking
experiments.
2.2. Synthesis of alkyl(aryl)alkylidene side-chain bearing
Ilomastat analogs with 4-(4-bromophenyl)phenylsulfonyl-
hydrazide as zinc-binding group (4)
2. Chemistry
The synthesis of these Ilomastat analogs was based on a similar
linear approach starting from monoesters 7 or 915,16 and (S)-try-
ptophanmethylamide (6)17 or its 2-phenyl-subtituted counterpart
(8).18 DMTMM-assisted coupling of 6 or 8 with (E)-monoallyl es-
ters 9v,w or with chiral ester-acid 7 smoothly afforded the corre-
sponding succinyltryptophanamide derivatives 10v–y or 11z,
respectively. Deprotection of esters 10v–y (Pd(PPh3)4) or 11z
(TFA) led to the free acids 12v–z.
The final products 4v,w,x,z were obtained by coupling the free
acids 12v,w,x,z with 4-(4-bromophenyl)benzenesulfonylhydrazide
hydrochloride. Product 4y was prepared in three steps from 12y via
the Boc-hydrazide analog 13y. Deprotection of this latter gave 14y
which was sulfonylated with 4-(4-bromophenyl)benzenesulfonyl-
chloride to afford the aryl-alkylidene side-chain containing Ilomas-
tat analog 4y (Scheme 2).
2.1. Synthesis of Ilomastat analogs containing hydrazide (2) or
sulfonylhydrazide functions (3) as ZBG
The synthesis of the derivatives containing hydrazide- or sul-
fonylhydrazide functions as ZBG followed a linear pathway (Scheme
1). Pseudopeptide backbone 5 was assembled by 4-(4,6-dimethoxy-
1,3,5-triazin-2-yl)-methylmorpholinium chloride (DMTMM) assisted
coupling12 between N-methyltryptophanamide 6 and functionalized
chiral succinic ester-acid 713 (Scheme 1).
For the introduction of new ZBGs, two pathways were elabo-
rated. The first one, consisted in the direct amidification of the
carboxylic acid function of 5 with arylhydrazines. In this way,
arylhydrazides 2a–d were obtained in moderate (45–52%) yield
Figure 1. Ilomastat (1), its hydrazide (2), sulfonylhydrazide (3), and various alkylidene side-chain containing analogs (4).

G. LeDour et al. / Bioorg. Med. Chem. 16 (2008) 8745–8759
8747
Scheme 1. Synthesis of hydrazide (2) and sulfonylhydrazide analogs (3) of Ilomastat. Reagents and conditions: (a) DMTMM, MeOH, 69–81%; (b) TFA, CH2Cl2, 57–80%; (c)
R1NHNH2 (or HCl salt), DMTMM, NMM, THF, 45–92%; (d) EtOH/HCl, 73%; (e) R2SO2NHNH2 (or HCl salt), DMTMM, NMM, THF, 11–67%; (f) R2SO2Cl, pyridine (or Et3N, or
NaHCO3), THF (co-solvent DMSO), 0 °C, 11–70%; (g) Pd–C, H2, EtOH, 57%.
Scheme 2. Synthesis of alkyl(aryl)alkylidene side-chain bearing Ilomastat analogs with 4-(4-bromophenyl)phenylsulfonylhydrazide as ZBG. Reagents and conditions: (a)
DMTMM, THF, 53–93%; (b) Pd(PPh3)4, morpholine, THF, 40–74%; (c) TFA, CH2Cl2, 64%; (d) 4-Br–C6H4–C6H4–SO2NHNH3+Clꢀ, DMTMM, NMM, THF, 14–41%; (e) BocNHNH2,
DMTMM, NMM, THF, 92%; (f) HCl, MeOH, 55%; (g) 4-Br–C6H4–C6H4–SO2Cl, Et3N, THF, 54%.
in all series of biological evaluations.13a,19 For the most active mol-
3. Biological evaluation and molecular modeling studies
ecules, the test panel was enlarged to MMP-3 (stromelysin), MMP-
7 (matrilysin), and MMP-9 (gelatinase B).
3.1. Enzyme inhibition experiments
Aryl-substituted hydrazide analogs (2a–d) displayed low inhib-
itory capacity toward MMPs, whatever electron-donating or elec-
tron-withdrawing group bearing aryl-substituted hydrazides was
considered (Table 1).
Among the sulfonylhydrazide analogs, the p-tosyl derivative 3a
was first subjected to biological evaluation toward MMP-1, MMP-
Hydrazide (2a–d) and sulfonylhydrazide analogs (3a–m and
4v–z) were tested for their ability to inhibit MMP-1 (collagenase),
MMP-2 (gelatinase A), and MMP-14 (membrane type-1 matrix
metalloproteinase) using the corresponding fluorimetric substrates
of these enzymes. For comparison, Ilomastat (1) was also included

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G. LeDour et al. / Bioorg. Med. Chem. 16 (2008) 8745–8759
Table 1
(3m) appeared to be the most potent gelatinase inhibitor with Ki
values of 8.5 and 3.6 nM for gelatinases A and B, respectively,
and only weak inhibition was measured on MMP-3 (415 nM),
MMP-7 (523 nM), and MMP-14 (1900 nM) (Tables 2 and 3).
As 3m proved to be a potent and selective gelatinase inhibitor,
in a second phase of our program, we were interested in studying
the influence of S01 subsite substitution. Formerly, we evidenced
that replacement of the iso-butyl group of Ilomastat by (E)-alkyl-
alkylidene side-chains afforded good MMP-2 selectivity,15 while
the (E)-phenyl-propylidene analog showed significant MMP-9
selectivity.16 In addition, introduction of a phenyl group in position
Inhibition of Ilomastat (1) and some hydrazide (2) and sulfonylhydrazide (3) ZBG
containing analogs (IC50, nM)
Compound
R
MMP-1
MMP-2
MMP-14
1
0.4
330
5600
>10,000
>10,000
0.4
5.2
>10,000
1000
>10,000
>10,000
2a
2b
2c
2d
4-CH3O–C6H4–
3,4-Di-Cl–C6H3–
4-NO2–C6H4–
10,000
10,000
10,000
10,000
2,4-Di-NO2–C6H3–
R1
3a
3b
3c
3d
3e
3f
3g
3h
3k
4-Me–C6H4–
4-Cl–C6H4–
2,4,6-Tri(i-Pr)–C6H2–
CH3–
4-NO2–C6H4–
2-NO2–C6H4–
CH3(CH2)11
CH3(CH2)15
250
290
4000
5600
490
850
980
440
93
24
18
2600
190
2
of indole of Ilomastat increases the MMP-2 inhibition
100
740
310
320
1400
300
92
3100
3700
1500
1100
9700
5100
730
(IC50 = 0.092 nM).22
Combining these diverse structural modifications, we prepared
a series of Ilomastat analogs (4v–z) with 4-(4-bromophenyl)benze-
nesulfonylhydrazide as zinc-binding group focusing on the modifi-
cations of S01 and S02 sites of the pseudopeptide backbone (Table 4).
Generally, 4-(4-bromophenyl)benzenesulfonylhydrazide type
analogs (4v–z) were less potent inhibitors than their hydroxamic
acid containing counterparts. Thus, 4v was nearly 14 times less ac-
tive than corresponding hydroxamic acid analog (IC50 = 123 nM)16
and the MMP-2 selectivity over other MMPs was lost. Similarly
weaker inhibitory activity and no effective selectivity were ob-
served for phenylpropylidene substituted analog (4w).
Introduction of a phenyl group on C-2 of the indole ring in the
heptylidene series (4x vs 4v) had low influence on enzyme inhibi-
tion contrary to the phenylpropylidene side-chain bearing analog
(4y) where better MMP-2 inhibition (IC50 = 65 nM) with slight
selectivity was observed. Comparing enzyme inhibitions of 4z with
3m, it is interesting to note that 2-phenyltryptophan containing
–
–
2-NH2–C6H4–
2, and MMP-14. Its MMP-2 enzyme inhibition (IC50 = 24 nM) was
in good correlation with formerly obtained data (IC50 = 30 nM).20
However, a lower MMP-2/MMP-1 selectivity (10-fold) was found.
Interestingly this compound, contrary to Ilomastat, is a poor
MMP-14 inhibitor.
Comparing hydrazide (2c) and sulfonylhydrazide (3e) ZBG con-
taining analogs, the observed difference of inhibition highlighted
the importance of the acid character of sulfonamide hydrogen in
metal chelation. Replacement of the 4-methyl group by chlorine
(3a vs 3b) allowed to maintain MMP-2 and MMP-1 inhibitory
activity at similar IC50 values but selectivity toward MMP-14 was
partly lost. Other sulfonamides with electron-withdrawing groups
(3e,f,k) showed neither higher inhibition, nor better selectivity.
Incorporation of tri-iso-propylphenyl substitution (3c) afforded a
more selective MMP-2 vs MMP-1 profile.
Table 3
Relative selectivity data of some substituted bis-arylsulfonylhydrazide ZBG contain-
ing derivatives (3j,l,m)
S01 subsite of MMP-2 has been reported to accommodate long al-
kyl chain and fibronectin type II domains of both gelatinases A and
B have been shown to interact with long chain fatty acids.21 Thus,
we explored the possibility that S2 subsite of enzyme could simi-
larly exhibit hydrophobic interactions. To this end, several
alkylsulfonylhydrazide derivatives (3d,g,h) were synthesized and
tested. Unfortunately, these derivatives did not display good inhib-
itory properties (Table 1).
Compound Selectivity
MMP-1/ MMP-2/ MMP-3/ MMP-7/ MMP-14/
MMP-9 MMP-9 MMP-9 MMP-9 MMP-9
(vs MMP-9)
R1
1
3j
3l
3m
0.7
13
4-Succinylimidyl-C6H4– 11
4-(4-Br–C6H4)–C6H4– 10
0.7
3
43
127
108
567
—
559
13
8.5
909
85
2-Naphthalene-
9
3.3
158
5666
On the contrary, bis-arylsulfonyl group substituted analogs dis-
played enhanced MMP-2 and MMP-1 enzyme inhibitory capacities
(Table 2). Thus, biphenyl-ether (3i) or succinimidylphenyl (3l)
substituted analogs presented good MMP-2 and MMP-9 inhibitory
activities, but without real selectivity comparing to other MMPs.
None of these compounds had any major influence on MMP-14
activity.
Naphthyl substitution (3j) enhanced matrilysin (MMP-7) and
MMP-14 selectivity which was maintained by the incorporation
of 4-(4-bromophenyl)phenyl group (3m). Among the sulfonylhyd-
razide analogs, the 4-(4-bromophenyl)benzene substituted one
Table 4
Inhibition of some alkyl(aryl)alkylidene side-chain containing Ilomastat analogs with
4-(4-bromophenyl)phenylsulfonylhydrazide as zinc-binding group (4) (IC50, nM)
Compound R2
R3 MMP-1 MMP-2 MMP-3 MMP-9 MMP-14
4v
4w
4x
4y
4z
@CH–(CH2)5Me
@CH–(CH2)2Ph
H
H
NI
1033
1813
310
1273
65
2538
3863
174
463
53
2563
1246
732
827
18
NI
105
3784
NI
@CH–(CH2)5Me Ph 2043
@CH–(CH2)2Ph Ph 1075
–CH2–CH(CH3)2 Ph 350
247
1237
NI, no inhibition.
Table 2
Inhibition of some substituted aryl- or bis-arylsulfonylhydrazide ZBG containing derivatives (3i,j,l,m) (IC50, nM)
Comound
R1
MMP-1
MMP-2
MMP-3
MMP-7
MMP-9
MMP-14
1
0.4
110
150
91
0.4
45
37
79
9.8
26
—
1400
920
1700
—
—
6150
110
475
0.6
—
11
8.5
3
5.2
3i
3j
3l
3m
4-(C6H5–O)C6H4–
2-Naphthalene-
4-Succinylimidyl-C6H4–
4-(4-Br–C6H4)–C6H4–
2800
10,000
720
30
17,000
3m
4-(4-Br–C6H4)–C6H4–
Ki = 8.5
Ki = 415
Ki = 523
Ki = 3.6
Ki = 1900
—, not tested.

G. LeDour et al. / Bioorg. Med. Chem. 16 (2008) 8745–8759
8749
analog 4z conserved important gelatinase inhibitory ability (MMP-
2 and MMP-9: IC50 = 247 and 18 nM, respectively) associated to a
potent MMP-3 inhibitory activity (IC50 = 53 nM).
3.2. Molecular modeling studies
The docking computations by Autodock23,24 have been carried
out with several hydrazide compounds using MMP-2 and MMP-9
as target proteins. The structural analysis of the lowest energy
model for each MMP will be detailed. The residue numberings em-
ployed for each MMP are those used in the Swissprot database.25
In keeping with enzyme inhibition studies, we initially focused
our efforts on the 4-(4-bromophenyl)benzenesulfonylhydrazide
zinc-binding group containing Ilomastat analog 3m which potently
and selectively inhibited gelatinases (MMP-2 and MMP-9).
The lowest energy model proposed for compound 3m showed a
similar docked position at the surface of both MMP-2 and MMP-9
(Figs. 3 and 4). As expected, the 4-(4-bromophenyl)phenyl group of
3m is localized in the S2 subsite of MMP-2 and presents van der
Waals interactions with residues Asn111, Gly181-Tyr-Pro-Phe184
,
Leu190, and Ala192-His-Ala194. According to our previous reported
model20 the sulfonylhydrazide group occupies the S1 subsite and
chelates the zinc atom. The sulfonylhydrazide group forms further
two H-bonds with backbone of residues Ala192 and Ala194. The iso-
butyl group presents van der Waals interactions with the amino
Figure 4. Lowest energy model for the interactions between 3m and the catalytic
site of MMP-9. (C atoms are colored in magenta, the MMP-9 is in cartoon
representation; and the zinc atom is displayed in grey sphere.)
acids located at the entrance of S01 subsite: Leu191, Ala192, Val400
His403, and Ala404. The indole group is located in the S02 subsite
forming van der Waals contacts with His413, Pro423, and Ile424
,
,
energy model with MMP-2 as target protein, involve the residues
Gly186 and Leu188 in the upper rim, and the amino acids Pro421
and Tyr423 in the lower rim of the catalytic site. The indole group,
located in the S02 subsite, interacts with Pro421 and Met422 of MMP-
9 via van der Waals forces. The measured affinity of 3m is better
for MMP-9 than for MMP-2. In our docking models, the main dif-
ference relies in the van der Waals interaction of the 4-(4-bromo-
phenyl)phenyl group with Phe110 of MMP-9 contrary to the
interaction with Asn111 in MMP-2. In our experimental conditions,
belonging to the lower rim of the catalytic site. The pseudopeptide
backbone of 3m establishes two H-bonds with the amino acids of
the lower rim (Pro423 and Tyr425) and two other H-bonds with
Gly189 and Leu191 (upper rim).
The position and the orientation of the compound 3m docked to
MMP-2 and MMP-9 are similar as confirmed by the root mean
square deviation (RMSD) value of 0.91 Å, calculated between the
atoms of the two inhibitor models. At the MMP-9 surface, the 4-
(4-bromophenyl)phenyl group has van der Waals contacts with
it is most likely that a p-stacking interaction occurs between the 4-
Phe110
,
Tyr179
,
Pro180
,
Leu187
,
and Ala189-His-Ala191 residues
(4-bromophenyl)phenyl group of 3m and Phe110 of MMP-9, thus
explaining the observed affinity differences (Table 2). Moreover,
the S2 pocket in MMP-9 is smaller than in MMP-2. Accordingly,
the 4-(4-bromophenyl)phenyl group can easily insert into the S2
pocket of both gelatinases, but the smaller size of MMP-9 induces
stronger van der Waals interactions favoring its binding.
(MMP-9 numbering). The sulfonylhydrazide group is engaged in
two H-bonds with the amino acids Ala189 and Ala191, respectively.
The iso-butyl group side chain presents van der Waals interactions
with Leu188, Ala189, Val398, Glu402, and Tyr423, forming the entrance
of the S01 subsite. The four H-bonds, already detected in the lowest
Introduction of a phenyl group at R3 afforded a less potent
inhibitor of gelatinases, particularly MMP-2 inhibitor (4z vs 3m).
Accordingly, we attempted to explain differences in MMP-9 inhibi-
tion of 4z as compared to 3m (IC50 = 18 nM and 3 nM, respec-
tively). The lowest energy model of 4z and 3m displayed almost
similar position and conformation within the MMP-9 catalytic site
(Fig. 4). The same interactions are highlighted except for the 2-
phenylindole group. This group interacts with Pro421 and Met422
,
belonging to the lower rim of the MMP-9 active site, as it was ob-
served in the lowest energy model of the compound 3m. Moreover,
phenyl ring of this group has specific van der Waals interactions
with Leu187 and His411 (Fig. 5).
On the contrary, the molecular docking computations have
failed to demonstrate any realistic model concerning the interac-
tion between 4z and MMP-2. In keeping with the relatively high
measured IC50 values, docking results suggested low MMP-2 affin-
ity for 4z. Indeed, incorporation of a phenyl group in 3m (leading to
4z) did not allow to obtain a coherent interaction model by docking
computations with MMP-9. To explain these discrepancies, the
lowest energy model of the 4z/MMP-9 complex was superimposed
with the MMP-2 structure. The superimposed structures of the
MMPs are very close and 4z fits well the catalytic site of the
Figure 3. Lowest energy model for the interactions between 3m and the catalytic
site of MMP-2. (C atoms are colored in magenta, the MMP-2 is in cartoon
representation; and the zinc atom is displayed in grey sphere.)
0
MMP-2, except for the 2-phenylindole group in the S2 subsite that

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G. LeDour et al. / Bioorg. Med. Chem. 16 (2008) 8745–8759
side-chain carrying carbon atom is displaced by 1.5–3 Å toward
outside of the S1 subsite. If this crucial carbon atom is sp3-hybrid-
0
ized, its conformational mobility allows the insertion of the
0
side-chain in the S1 subsite affording higher inhibition activity
(3m and 4z).
4. Conclusion
From our previous investigation, we initially hypothesized that
incorporation of an electron-withdrawing moiety to hydrazide
analog of Ilomastat would amplify their MMP inhibition activity.
Indeed, all synthesized sulfonylhydrazide derivatives appeared
more active than the aryl-substituted analogs, confirming the
importance of sulfonamide hydrogen in metal chelation. Increasing
the electron-withdrawing character of this group did not improve
their MMP inhibitory potential but elongation with diaryl moiety
afforded potent derivatives that inhibit preferentially gelatinases.
Strikingly, the 4-(4-bromophenyl)phenylsulfonylhydrazide analog
(3m) inhibited MMP-9 with an IC50 comparable to that of Ilomastat
(IC50 = 3 nM and 0.6 nM, respectively). Furthermore, this derivative
possesses MMP-9 selectivity over MMP-1, MMP-3, and MMP-14
which are 14-, 13-, and 666-fold greater, respectively, than
Ilomastat.
The importance of S3 and S2 subsites has been highlighted pre-
viously for selective inhibition.26 Computation methods followed
by statistical analysis have provided a ranking of the relative
importance of the MMP subsites for inhibitor selectivity.27 Accord-
ing to this study, the S2 subsite is the second most important sub-
site for selectivity.
The 4-(4-bromophenyl)phenyl group might occupy the S2 sub-
site of MMP-9, a contention which has been supported by molecu-
lar docking studies. The small size, as well as the presence of Phe110
in the S2 pocket of MMP-9 confer to this enzyme a better affinity
for the 4-(4-bromophenyl)phenyl moiety compared to MMP-2
and probably to other MMPs.
Figure 5. Lowest energy model for the interactions between 4z and the catalytic
site of MMP-9. (C atoms are colored in magenta, the MMP-2 is in cartoon
representation; and the zinc atom is displayed in grey sphere.)
exhibits steric hindrance with Ile424 localized in the lower rim of
the MMP-2 active site. Precisely, the ethyl group of the Ile424
side-chain adopts a particular conformation which leads to unfa-
vorable van der Waals-type interaction. The same three-dimen-
sional conformation of Ile424 has also been observed in other
MMPs (PDB codes: 1HOV, 1QIB, 1GXD, and 1EAK). Conversely,
the equivalent amino acid in MMP-9, Met422, has its side-chain di-
rected in opposite direction allowing the binding of 4z with the 2-
0
phenylindole group in its S2 subsite. The 2-phenylindole group
Attempts to further improve the affinity or selectivity of 3m
presents both rigid and voluminous properties and consequently
through pharmacomodulation of R2 or R3 positions of the pseudo-
0
cannot fit properly the S2 cleft of MMP-2. Therefore, the lower
0
0
peptidic backbone targeting the primed subsites (S1 , S2 ) proved to
be unsuccessful mainly due to steric hindrance. Nevertheless, we
have evidenced the contribution of the S2 subsite in the develop-
ment of MMP-9 selective inhibitors and this concept might be
valuable in the design of therapeutic agents in elastic tissue disease
as aneurysms.
affinity of the compound 4z for the MMP-2 is likely due to the
0
Ile424 side-chain localized in the S2 subsite.
Molecular docking computations for compounds 4x and 4y have
failed to deliver realistic positions and plausible conformations for
these ligands in the active site of both MMP-2 and MMP-9, sug-
gesting a low affinity between these inhibitors and both enzymes.
Therefore, a virtual derivative with a methyl group as side-chain
function was built up and tested using the same protocol. Again,
no correct conformation of this compound in the enzyme active
site could be evidenced, indicating that its low affinity was not
due to the bulkiness of the phenyl group or the alkyl chain.
In order to explain why replacement of the hydroxamate moi-
ety by 4-(4-bromophenyl)phenylsulfonylhydrazide as zinc-binding
group led to loss of enzyme inhibition selectivity we calculated the
most probable conformation of the zinc chelated forms of 4x and
4y and docked them into the active site of MMP-2 and MMP-9.
Our data indicated that both 4x and 4y adopted a new conforma-
tion in which sp2-hybridized carbon atom of the alkylidene
5. Experimental
All solvents were of reagent grade and, when necessary, were
purified and dried by standard methods. Reaction and products
were routinely monitored by thin-layer chromatography (TLC) on
silica gel (Kieselgel 60 PF254, Merck). Flash chromatography was
performed on Kieselgel 60 (40–63 lm, Merck). Melting points were
determined on a Reichert Thermovar hot-stage apparatus and are
uncorrected. UV spectra were recorded in methanol solution on a
Unicam 8700 UV/vis apparatus. IR spectra were measured on a Per-
kin-Elmer Spectrum BX FTIR instrument. 1H NMR (300 MHz) and
13C NMR (75 MHz) spectra were recorded on a Bruker AC 300 spec-
trometer using TMS as internal standard, chemical shifts were gi-
ven in ppm (d). Couplings expressed as s, br s, d, t, and m
correspond to singlet, broad-singlet, doublet, triplet, and multiplet,
respectively. Mass spectra were recorded on a MSQ ThermoFinni-
gan apparatus using electrospray ionization (ESI) method or on a
GCT Micromass apparatus using electronimpact (EI) or chemical
ionization (CI) method. Optical rotations were measured on a Per-
kin-Elmer 241 polarimeter.
0
side-chain led to steric hindrance impeding the entrance in S1
subsite. This is particularly true for Pro423 in MMP-2 or Pro421 in
MMP-9. Consequently, although the S2 subsite is occupied no
synergistic effect could be obtained as a consequence of inappro-
priate occupancy of primed subsites.
Finally, we tried to explain why sulfonylhydrazide ZBG contain-
ing inhibitors had good but generally lower enzyme inhibitory
activity than their hydroxamic acid type analogs. Our calculations
pointed out that in the case of sulfonylhydrazide structures the

G. LeDour et al. / Bioorg. Med. Chem. 16 (2008) 8745–8759
8751
5.1. General procedure for coupling of acid 5 with hydrazines
and sulfonylhydrazides
CH2-indole), 4.46 (m, 1H, CH–CH2-indole), 6.68 (dd, 1H, J = 8.7,
2.5 Hz, Haryl), 6.83 (d, 1H, J = 2.5 Hz, Haryl, 6.95 (m, 1H, H6indole),
7.02–7.09 (m, 2H, H2indole, H5indole), 7.32 (m, 2H, Haryl, H7indole),
7.55 (d, 1H, J = 7.7 Hz, H4indole), 7.73 (q, 1H, J = 4.7 Hz, CO–NH–
CH3), 8.00 (d, 1H, J = 8.0 Hz, CONH), 8.13 (br s, 1H, NHNHCO),
9.73 (br s, 1H, NHNHCO), 10.78 (s, 1H, NHindole); 13C NMR
(75 MHz, DMSO-d6): 22.2, 23.1, 25.2, 25.6, 27.7, 36.4, 40.3, 40.9,
53.5, 110.3, 111.2, 112.5, 112.9, 118.6, 118.5, 119.1, 120.8, 123.4,
127.4, 130.3, 131.2, 136.0, 149.5, 170.8, 171.7, 173.7; MS (CI, m/z,
%): 534.77 (M+2, 27), 533.73 (M+1, 22), 531.58 (M0+2, 100), 356
(22), 84 (46), 70 (65); Anal. Calcd for C26H31N5O3Cl2ꢂH2O: C
56.73, H 6.04, N 12.72%. Found: C 56.79, H 5.79, N 12.55%.
A solution of acid 5 and N-methylmorpholine (NMM), in THF
was stirred at room temperature for 30 min. Then dimethoxytri-
azine methylmopholinium chloride (DMTMM) was added to the
reaction mixture and stirring was continued for 0.5–1.5 h. Aryl-
hydrazine, sulfonylhydrazide as base or HCl salt were added to
the reaction mixture, as a solid or THF/DMSO solution in the pres-
ence of NMM. Stirring was continued at room temperature under
nitrogen until the disappearance of the starting material (TLC mon-
itoring). After evaporation of the solvent, the crude residue was
either directly purified by column chromatography, or dissolved
in CH2Cl2. In the latter case, the organic layer was washed succes-
sively with 1 N aqueous HCl and 5% NaHCO3 solution, dried over
Na2SO4, filtered and the solvent was evaporated in vacuo. The res-
idue was purified by column, or preparative thin-layer chromatog-
raphy to afford hydrazides or sulfonylhydrazides.
5.1.3. N0-{3-(R)-[2-(S)-(1H-Indol-3-yl)-1-(methylcarbamoyl)-
ethylcarbamoyl]-5-methylhexanoyl}-N-4-nitrophenylhydrazide
(2c)
Prepared according to the general procedure 5.1: acid
5
(200 mg, 0.54 mmol); NMM (60 L, 0.54 mmol); THF (5 mL); stir-
l
ring at room temperature 0.5 h; DMTMM (180 mg, 0.65 mmol);
time of activation 1.5 h; 4-nitrophenylhydrazine (250 mg,
1.62 mmol); reaction time 20 h. After extraction the product was
crystallized from ethyl acetate and the mother-liquor was purified
by preparative thin-layer chromatography (silica gel, CH2Cl2/EtOH,
98:2). Yield: 2c (123 mg, 45%). Yellow crystals. Mp 230–232 °C;
5.1.1. N0-{3-(R)-[2-(S)-(1H-Indol-3-yl)-1-(methylcarbamoyl)-
ethylcarbamoyl]-5-methylhexanoyl}-N-4-
methoxyphenylhydrazide (2a)
Prepared according to the general procedure 5.1: acid
5
(200 mg, 0.54 mmol); NMM (60 L, 0.54 mmol); THF (5 mL); stir-
l
ring at room temperature 0.5 h; DMTMM (180 mg, 0.65 mmol);
time of activation 1.5 h; 4-methoxyphenylhydrazine HCl salt
(283 mg, 1.62 mmol) dissolved in THF (3 mL) with NMM (180 lL,
½
a 2D1
+3 (c 0.95, DMSO); UV (MeOH, nm): 352, 291, 282, 274, 222,
ꢁ
210, 264; IR (KBr, cmꢀ1): 3286 (NH), 3084 (NH), 2952 (CH), 1665
(CO), 1635 (CO), 1516 (NO2), 1331 (NO2), 1111, 842; 1H NMR
(300 MHz, DMSO-d6): 0.79 and 0.83 (d, 2ꢃ 3H, J = 6.1 Hz,
CH(CH3)2), 1.03–1.44 (m, 3H, CH2–CH(CH3)2), 2.28 (m, 2H,
NH–CO–CH2–), 2.49 (d, 3H, J = 4.5 Hz, CO–NH–CH3), 2.77 (m, 1H,
CH–CH2–CH(CH3)2), 3.02 (m, 2H, CH–CH2-indole), 4.47 (m, 1H,
CH–CH2–indole), 6.76 (d, 2H, J = 9.2 Hz, Haryl), 6.90 (m, 1H, H6in-
dole), 7.05 (m, 1H, H5indole), 7.10 (s, 1H, H2indole), 7.32 (d, 1H,
J = 7.9 Hz, H7indole), 7.56 (d, 1H, J = 7.8 Hz, H4indole), 7.78 (q, 1H,
J = 4.5 Hz, CO–NH–CH3), 8.03 (d, 1H, J = 5.9 Hz, CONH), 8.05 (d,
2H, J = 9.2 Hz, Haryl), 9.05 (br s, 1H, NHNHCO), 10.00 (br s, 1H,
NHNHCO), 10.81 (s, 1H, NHindole); 13C NMR (75 MHz, DMSO-d6):
22.2, 23.0, 25.2, 25.6, 27.8, 36.3, 40.4, 41.1, 53.6, 110.2, 110.5,
111.2, 118.1, 118.4, 120.8, 123.4, 125.8, 127.4, 136.0, 137.9,
1.62 mmol); reaction time 20 h. After extraction the crude product
was purified by flash chromatography (silica gel, CH2Cl2/MeOH,
100:1 to 96:4). Yield: 2a (124 mg, 46%). Yellowish crystals. Mp
200–201 °C; ½a 2D1
ꢀ10 (c 0.65, DMSO); UV (MeOH, nm): 292, 282,
ꢁ
222, 200, 210; IR (KBr, cmꢀ1): 3410 (NH), 3295 (NH), 2943 (CH),
1657 (CO), 1635 (CO), 1507, 1097, 1031, 864, 825; 1H NMR
(300 MHz, DMSO-d6): 0.77–0.84 (m, 6H, CH(CH3)2), 1.09 (m, 1H,
CH2–CH(CH3)2), 1.41 (m, 2H, CH2–CH(CH3)2), 2.17–2.27 (m, 2H,
NH–CO–CH2–), 2.52 (d, 3H, J = 3.7 Hz, CO–NH–CH3), 2.74 (m, 1H,
CH–CH2–CH(CH3)2), 2.99–3.14 (m, 2H, CH–CH2-indole), 3.62
(s, 3H, OCH3), 4.47 (m, 1H, CH–CH2-indole), 6.71 (m, 4H, Haryl),
6.98 (m, 1H, H6indole), 7.07 (m, 1H, H5indole), 7.11 (s, 1H, H2indole),
7.34 (m, 2H, NHNHCO, H7indole), 7.59 (d, 1H, J = 7.7 Hz, H4indole),
7.80 (q, 1H, J = 3.7 Hz, CO–NH–CH3), 8.02 (d, 1H, J = 7.9 Hz, CONH),
9.63 (sl, 1H, NHNH–CO), 10.83 (s, 1H, NHindole); 13C NMR (75 MHz,
DMSO-d6): 22.3, 23.3, 25.4, 25.8, 27.9, 36.9, 40.6, 41.1, 53.7, 55.4,
110.5, 111.4, 113.8, 114.3, 118.3, 118.6, 120.9, 123.6, 127.6,
136.2, 143.4, 152.7, 170.8, 171.9, 174.0; MS (CI, m/z, %): 493.83
(M+., 100), 463 (7), 356 (12), 242 (22); Anal. Calcd for
154.9, 170.8, 171.7, 173.8; MS (CI, m/z, %): 508.88 (M+ , 100), 478
Å
(18), 84 (50), 70 (26); Anal. Calcd for C26H32N6O5: C 61.40, H
6.34, N 16.52%. Found: C 61.12, H 6.16, N 16.30%.
5.1.4. N0-{3-(R)-[2-(S)-(1H-Indol-3-yl)-1-(methylcarbamoyl)-
ethylcarbamoyl]-5-methylhexanoyl}-N-2,4-dinitrophenyl-
hydrazide (2d)
C
26H31N5O3Cl2ꢂ2H2O: C 61.23, H 7.42, N 13.22%. Found: C 60.83,
Prepared according to the general procedure 5.1: acid
5
H 7.02, N 13.60%.
(200 mg, 0.54 mmol); NMM (60 L, 0.54 mmol); THF (5 mL); stir-
l
ring at room temperature 0.5 h; DMTMM (180 mg, 0.65 mmol);
time of activation 1.5 h; 2,4-dinitrophenylhydrazine (320 mg,
1.61 mmol); reaction time 20 h. After extraction the crude product
was purified by flash chromatography (silica gel, CH2Cl2/MeOH,
97:3). Yield: 2d (155 mg, 52%). Orange crystals. Mp 193–195 °C;
5.1.2. N0-{3-(R)-[2-(S)-(1H-Indol-3-yl)-1-(methylcarbamoyl)-
ethylcarbamoyl]-5-methylhexanoyl}-N-3,4-dichlorophenyl-
hydrazide (2b)
Prepared according to the general procedure 5.1: acid
5
(200 mg, 0.54 mmol); NMM (60
lL, 0.54 mmol); THF (5 mL); stir-
½
a 2D1
+2 (c 0.62, DMSO); UV (MeOH, nm): 334, 290, 282, 264, 222,
ꢁ
ring at room temperature 0.5 h; DMTMM (180 mg, 0.65 mmol);
time of activation 1.5 h; 3,4-dichlorophenylhydrazine HCl salt
(345 mg, 1.62 mmol) dissolved in THF (3 mL) with NMM (180 lL,
1.62 mmol); reaction time 20 h. After extraction the crude product
210; IR (KBr, cmꢀ1): 3401 (NH), 3278 (NH), 2952 (CH), 1670
(CO), 1635 (CO), 1520 (NO2), 1335 (NO2), 1097, 921, 829; 1H
NMR (300 MHz, DMSO-d6): 0.81 and 0.86 (d, 2ꢃ 3H, J = 6.0 Hz,
CH(CH3)2), 1.16–1.49 (m, 3H, CH2–CH(CH3)2), 2.35 (m, 2H,
NH–CO–CH2–), 2.51 (d, 3H, J = 4.6 Hz, CO–NH–CH3), 2.82 (m, 1H,
CH–CH2–CH(CH3)2), 3.00 (m, 2H, CH–CH2–indole), 4.48 (m, 1H,
CH–CH2–indole), 6.93 (m, 1H, H6indole), 7.02 (m, 1H, H5indole),
7.07 (s, 1H, H2indole), 7.18 (d, 1H, J = 9.6 Hz, Haryl), 7.26 (d,
J = 8.0 Hz, 1H, H7indole), 7.52 (d, J = 7.8 Hz, 1H, H4indole), 7.79 (q,
1H, J = 4.6 Hz, CO–NH–CH3), 8.10 (d, 1H, J = 8.0 Hz, CONH), 8.18
(dd, 1H, J = 9.6, 2.6 Hz, Haryl), 8.82 (s, 1H, Haryl), 10.05 (br s, 1H,
NHNHCO), 10.41 (br s, 1H, NHNHCO), 10.72 (s, 1H, NHindole); 13C
was crystallized from ethyl acetate. Yield: 2b (130 mg, 45%). Yel-
lowish crystals. Mp 203–205 °C; ½a D21
ꢀ11 (c 1.22, DMSO); UV
ꢁ
(MeOH, nm): 291, 282, 274, 220, 210; IR (KBr, cmꢀ1): 3286 (NH),
3075 (NH), 2951 (CH), 1661 (CO), 1635 (CO), 1093, 851, 811; 1H
NMR (300 MHz, DMSO-d6): 0.77 and 0.81 (d, 2ꢃ 3H, J = 6.2 Hz,
CH(CH3)2), 1.06 (m, 1H, CH2–CH(CH3)2), 1.41 (m, 2H, CH2–
CH(CH3)2), 2.20 (m, 2H, NH–CO–CH2–), 2.50 (d, 3H, J = 4.7 Hz,
CO–NH–CH3), 2.74 (m, 1H, CH–CH2–CH(CH3)2), 3.01 (m, 2H, CH–

8752
G. LeDour et al. / Bioorg. Med. Chem. 16 (2008) 8745–8759
NMR (75 MHz, DMSO-d6): 22.5, 23.1, 25.4, 25.8, 27.9, 36.5, 40.3,
41.4, 53.7, 110.4, 111.4, 115.6, 118.3, 118.5, 120.9, 123.3, 123.5,
127.5, 129.7, 129.9, 136.1, 136.7, 148.6, 170.7, 171.9, 174.0; MS
(CI, m/z, %): 553.64 (M+.,100), 523 (17), 242 (12), 84 (64), 70 (62);
Anal. Calcd for C26H31N7O7: C 56.41, H 5.65, N 17.71%. Found: C
56.50, H 5.65, N 17.34%.
5.1.7. N0-{3-(R)-[2-(S)-(1H-Indol-3-yl)-1-(methylcarbamoyl)-
ethylcarbamoyl]-5-methylhexanoyl}-N-tosylhydrazide (3a)
Prepared according to the general procedure 5.1: acid
5
(150 mg, 0.40 mmol); NMM (50 lL, 0.44 mmol); THF (5 mL); stir-
ring at room temperature 0.5 h; DMTMM (225 mg, 0.80 mmol);
time of activation 1.5 h; p-toluenesulfonylhydrazide (375 mg,
2.01 mmol); reaction time 18 h. Purification of the crude product
by column chromatography (silica gel, CH2Cl2/MeOH, 99:1). Yield:
3a (146 mg 67%). White solid. Mp 192–193 °C; 1H NMR (300 MHz,
DMSO-d6): 0.75 (d, 6H, J = 6.4 Hz, CH(CH3)2), 0.85 (m, 1H, CH2–
CH(CH3)2), 1.25 (m, 2H, CH2–CH(CH3)2), 1.99 (m, 2H, NH–CO–
CH2–), 2.37 (s, 3H, Ar–CH3), 2.55 (d, 3H, J = 4.4 Hz, CO–NH–CH3),
2.64 (m, 1H, CH–CH2–CH(CH3)2), 3.01 (m, 2H, indole-CH2–), 4.45
(m, 1H, CH–CH2-indole), 6.98 (m, 1H, H5indole), 7.05 (s, 1H, H2indole),
7.07 (m, 1H, H6indole), 7.31 (d, 1H, J = 7.1 Hz, H7indole), 7.40 (d, 2H,
J = 8.1 Hz, Htosyl), 7.57 (d, 1H, J = 7.8 Hz, H4indole), 7.75 (d, 2H,
J = 8.1 Hz, Htosyl), 9.80 (br s, 1H, NH), 10.05 (br s, 1H, NH), 10.80
(br s, 1H, NHindole); 13C NMR (75 MHz, DMSO-d6): 21.3, 22.0,
25.3, 25.8, 27.9, 36.5, 39.9, 43.1, 53.5, 110.5, 111.4, 118.3, 118.6,
121.0, 123.5, 127.5, 127.9, 129.5, 136.2, 143.3, 150.1, 169.6,
171.9, 173.7.
5.1.5. N0-{3-(R)-[2-(S)-(1H-Indol-3-yl)-1-(methylcarbamoyl)-
ethylcarbamoyl]-5-methylhexanoyl}-N-tert-butoxycarbonyl-
hydrazide (2e)
A solution of acid 5 (510 mg, 1.37 mmol) and N-methylmor-
pholine (164 lL, 1.50 mmol) in THF (15 mL) was stirred at room
temperature for 30 min and then dimethoxytriazine meth-
ylmorpholinium chloride (DMTMM, 450 mg, 1.63 mmol) was
added to the reaction mixture and stirring was continued for
1.5 h. Then Boc-hydrazine (200 mg, 1.52 mmol) was added to
the reaction mixture and stirring was continued at room temper-
ature under nitrogen overnight. After evaporation of the solvent,
the crude residue was dissolved in CH2Cl2 (30 mL), the organic
layer was washed at 0 °C successively with water (10 mL), 5%
aq NaHCO3 solution, 5% aq citric acid solution, dried over Na2SO4,
filtered, and the solvent was evaporated in vacuo. The residue
was purified by crystallization from ether containing 5–10%
methanol. Yield: 2e (618 mg, 92%). White crystals. Mp 162–
165 °C; UV (MeOH, nm): 290, 282, 274, 229, 207; IR (KBr,
cmꢀ1): 3387 (NH), 3279 (NH), 2946 (CH), 1712 (CO), 1632 (CO),
1542, 1246, 1161 (SO2), 739; 1H NMR (300 MHz, acetone-d6):
0.75 and 0.79 (d, 2ꢃ 3H, J = 6.3 Hz, CH(CH3)2), 1.20 (m, 1H,
CH2–CH(CH3)2), 1.42 (s, 9H, C(CH3)3), 1.45 (m, 2H, CH2–CH(CH3)2),
2.38 (dd, 2H, J = 15.1, 5.6 Hz, NH–CO–CH2–), 2.63 (d, 3H,
J = 4.6 Hz, CO–NH–CH3), 2.80 (m, 1H, CH–CH2–CH(CH3)2), 3.24
(m, 2H, CH–CH2-indole), 4.62 (m, 1H, CH–CH2-indole), 6.99
(t, 1H, J = 7.4 Hz, H6indole), 7.07 (t, 1H, J = 7.4 Hz, H5indole), 7.20
(s, 1H, H2indole), 7.35 (d, 1H, J = 7.4 Hz, H7indole), 7.41–7.44 (d,
2H, J = 8 Hz, NHNHCO), 7.63 (d, 1H, J = 7.4 Hz, H4indole), 7.97 (q,
1H, J = 4.6 Hz, CO–NH–CH3), 9.14 (d, 1H, J = 5.6 Hz, CONH), 10.07
(br s, 1H, NHindole); 13C NMR (75 MHz, acetone-d6): 22.4, 23.3,
26.2, 28.3, 28.4, 37.4, 41.8, 41.9, 55.0, 80.5, 111.7, 112.0, 119.4,
119.5, 121.9, 124.3, 128.6, 137.5, 156.6, 171.9, 172.9, 175.1; Anal.
Calcd for C25H37N5O5: C 61.58, H 7.65, N 14.37%. Found: C 61.77,
H 7.88, N 14.10%.
5.1.8. N0-{3-(R)-[2-(S)-(1H-Indol-3-yl)-1-(methylcarbamoyl)-
ethylcarbamoyl]-5-methylhexanoyl}-N-4-chlorobenzene-
sulfonylhydrazide (3b)
Prepared according to the general procedure 5.1: acid
5
(500 mg, 1.34 mmol); NMM (162 L, 1.47 mmol); THF (30 mL);
l
stirring at room temperature 0.5 h; DMTMM (484 mg, 1.61 mmol);
time of activation 0.5 h; p-chlorophenylsulfonylhydrazide HCl salt
(980 mg, 4.02 mmol) dissolved in THF (15 mL) with NMM (442 lL,
4.02 mmol); reaction time 20 h. After extraction the crude product
was purified by column chromatography (silica gel, heptane/
AcOEt, 70:30). Yield: 3b (82 mg, 11%) after crystallization from
CH2Cl2. White-yellowish solid. Mp 217–219 °C; ½a D21
ꢀ5 (c 2.54,
ꢁ
DMSO); UV (MeOH, nm): 291, 282, 275, 227, 210; IR (KBr, cmꢀ1):
3396 (NH), 3281 (NH), 2947 (CH), 1633 (CO), 1595 (CO), 1538,
1338 (SO2), 1162 (SO2), 1010, 886, 743; 1H NMR (300 MHz,
CD3OD): 0.73 and 0.77 (d, 2ꢃ 3H, J = 6.4 Hz, CH(CH3)2), 0.87 (m,
1H, CH2–CH(CH3)2), 1.31 (m, 2H, CH2–CH(CH3)2), 2.11 (m, 2H,
NH–CO–CH2–), 2.56 (m, 3H, CO–NH–CH3), 2.57 (m, 1H, CH–CH2–
CH(CH3)2), 3.18 (m, 2H, CH–CH2-indole), 4.53 (m, 1H, CH–CH2-
indole), 6.98 (m, 1H, H6indole), 7.05 (s, 1H, H2indole), 7.07 (m, 1H,
H5indole), 7.31 (d, 1H, J = 8.0 Hz, H7indole), 7.52 (d, 2H, J = 8.6 Hz,
5.1.6. N0-{3-(R)-[2-(S)-(1H-Indol-3-yl)-1-(methylcarbamoyl)-
ethylcarbamoyl]-5-methylhexanoyl}-hydrazide (2f)
H
aryl), 7.57 (d, 1H, J = 7.8 Hz, H4indole), 7.85 (d, 2H, J = 8.6 Hz, Haryl);
A solution of Boc protected hydrazide 2e (2.82 g, 5.79 mmol) in
HCl-saturated ethanol (45 mL, C 7.7 mol/L) was stirred at room
temperature for 15–30 min. After evaporation of the solvent, the
crude residue was dissolved in CH2Cl2 (60 mL) and 1 M aqueous
NaOH solution was added at 0 °C till pH 9. The formed solid was
filtered off, washed with water (5 mL), ether (2ꢃ 5 mL), and dried.
Yield: 2f (1.46 g, 73%). White crystals. Mp 187–188 °C; IR (KBr,
cmꢀ1): 3438 (NH), 3297 (NH), 2961 (CH3), 1633 (CO), 1571
(CONH), 1026, 801; 1H NMR (300 MHz, DMSO-d6): 0.77 (m, 6H,
CH(CH3)2), 0.99 (m, 1H, CH2–CH(CH3)2), 1.35 (m, 2H, CH2–
CH(CH3)2), 2.09 (m, 2H, NH–CO–CH2–), 2.56 (d, 3H, J = 4.4 Hz,
CO–NH–CH3), 2.68 (m, 1H, CH–CH2–CH(CH3)2), 3.05 (m, 2H, CH–
CH2-indole), 4.20 (m, 2H, NH2NHCO), 4.43 (m, 1H, CH–CH2-indole),
6.97 (m, 1H, H5indole), 7.06 (m, 1H, H6indole), 7.11 (s, 1H, H2indole),
7.32 (d, 1H, J = 7.9 Hz, H7indole), 7.54 (m, 1H, NH2NHCO), 7.57 (d,
1H, J = 7.7 Hz, H4indole), 7.88 (q, 1H, J = 4.4 Hz, CO–NH–CH3), 7.99
(d, 1H, J = 7.9 Hz, CONH), 10.07 (br s, 1H, NHindole); 13C NMR
(75 MHz, DMSO-d6): 21.8, 22.5, 25.0, 25.2, 27.3, 36.6, 40.6, 40.8,
53.4, 110.3, 110.9, 117.8, 118.0, 120.4, 123.0, 127.5, 136.0, 169.9,
171.4, 173.7; MS (CI, m/z, %): 388.55 (M+1, 100), 359 (11), 357
(11), 256 (9), 219 (13); Anal. Calcd for C20H29N5O: C 61.99, H
7.54, N 18.08%. Found: C 62.27, H 7.31, N 18.44%.
13C NMR (75 MHz, CD3OD): 22.3, 23.4, 26.3, 26.7, 28.7, 37.3, 42.2,
42.3, 55.7, 111.2, 112.3, 119.4, 119.7, 122.4, 124.5, 128.7, 130.3,
131.0, 138.0, 138.8, 140.6, 172.1, 174.4, 176.8; MS (CI, m/z, %):
562.53 (M+1, 100), 531 (13), 375 (24), 84 (37); Anal. Calcd for
C
26H32ClN5O5Sꢂ0.5H2O: C 54.68, H 5.82, N 12.26%. Found: C
54.31, H 5.93, N 11.91%.
5.1.9. N0-{3-(R)-[2-(S)-(1H-Indol-3-yl)-1-(methylcarbamoyl)-
ethylcarbamoyl]-5-methylhexanoyl}-N-2,4,6-tri-isopropyl-
phenylsulfonylhydrazide (3c)
Prepared according to the general procedure 5.1: acid
(147 mg, 0.39 mmol); NMM (40 lL, 0.36 mmol); THF (5 mL); stir-
5
ring at room temperature 0.5 h; DMTMM (135 mg, 0.49 mmol);
time of activation 1.0 h; 2,4,6-tri-i-propylphenylsulfonylhydrazide
(110 mg, 0.37 mmol); reaction time 1.5 h. After extraction the
crude product was purified by preparative thin-layer chromatogra-
phy (silica gel, CH2Cl2/EtOH, 98:2). Yield: 3c (65 mg, 27%). White
solid. Mp 98–100 °C; ½a D21
ꢀ16 (c 0.44, DMSO); UV (MeOH, nm):
ꢁ
291, 281, 272, 220, 210; IR (KBr, cmꢀ1): 3330 (NH), 3260 (NH),
2952 (CH), 1683 (CO), 1639 (CO), 1529, 1459, 1326 (SO2), 1155
(SO2), 1098, 882; 1H NMR (300 MHz, DMSO-d6): 0.70 and 0.74
(d, 2ꢃ 3H, J = 5.8 Hz, CH(CH3)2), 0.87 (m, 1H, CH2–CH(CH3)2),

G. LeDour et al. / Bioorg. Med. Chem. 16 (2008) 8745–8759
8753
1.21 (m, 20H, CH2–CH(CH3)2), 3ꢃ (CH(CH3)2aryl), 2.00–2.18 (m, 2H,
NH–CO–CH2–), 2.52 (d, 3H, J = 5.2 Hz, CO–NH–CH3), 2.62 (m, 1H,
CH–CH2–CH(CH3)2), 2.89–3.20 (m, 3H, CH–CH2-indole, CH(CH3)2ar-
yl), 4.07 (m, 2H, CH(CH3)2aryl), 4.38 (m, 1H, CH–CH2-indole),
6.93–7.08 (m, 3H, H6indole, H5indole, H2indole), 7.21 (s, 2H, Haryl),
7.32 (d, 1H, J = 8.0 Hz, H7indole), 7.54 (d, 1H, J = 7.6 Hz, H4indole),
7.71 (q, 1H, J = 5.2 Hz, CO–NH–CH3), 7.97 (d, 1H, J = 8.0 Hz, CONH),
crude product was purified by column chromatography (silica
gel, CH2Cl2/acetone, 50:50). Yield: 3e (45 mg, 11%). Yellow crystals.
Mp 117–119 °C; ½a D21
ꢀ11 (c 0.092, DMSO); UV (MeOH, nm): 291,
ꢁ
281, 272, 220, 210; IR (KBr, cmꢀ1): 3401 (NH), 3330 (NH), 2934
(CH), 1674 (CO), 1652 (CO), 1533, 1344 (SO2), 1164 (SO2), 1089,
851; 1H NMR (300 MHz, DMSO-d6): 0.69–0.81 (m, 7H, CH(CH3)2,
CH2–CH(CH3)2), 1.20–1.33 (m, 2H, CH2–CH(CH3)2, CH2–CH(CH3)2),
2.02 (m, 2H, NH–CO–CH2–), 2.50–2.58 (m, 4H, CO–NH–CH3, CH–
CH2–CH(CH3)2), 2.87–3.10 (m, 2H, CH–CH2–indole), 4.42 (m, 1H,
CH–CH2-indole), 6.93–7.14 (m, 3H, H6indole, H5indole, H2indole),
7.33 (d, 1H, J = 8.1 Hz, H7indole), 7.55 (d, 1H, J = 7.8 Hz, H4indole),
7.73 (q, 1H, J = 4.6 Hz, CO–NH–CH3), 7.96 (d, 1H, J = 8.1 Hz, CONH),
8.08 (d, 2H, J = 8.7 Hz, Haryl), 8.39 (d, 2H, J = 8.7 Hz, Haryl), 10.17 (br
s, 2H, 2NH), 10.80 (br s, 1H, NHindole); 13C NMR (75 MHz, DMSO-
d6): 22.0, 23.3, 25.3, 25.8, 27.9, 36.3, 38.8, 40.6, 53.6, 110.5,
111.4, 118.3, 118.6, 121.0, 123.6, 124.3, 127.5, 129.6, 136.2,
145.3, 150.0, 170.0, 171.9, 173.6; MS (CI, m/z, %): 573.64 (M+1,
100), 543 (12), 160 (3).
9.47 (br s, 1H, NH), 9.99 (br s, 1H, NH), 10.76 (br s, 1H, NHindole); 13
C
NMR (75 MHz, DMSO-d6): 22.2, 23.3, 23.6, 25.0, 25.4, 25.8, 27.7,
29.7, 33.5, 36.3, 39.2, 42.6, 53.6, 110.6, 111.4, 118.3, 118.6, 121.0,
120.9, 121.0, 127.4, 133.0, 136.2, 150.9, 152.4, 170.2, 171.9,
173.9; MS (CI, m/z, %): 675.97 (M+Na, 100), 653.95 (M, 50), 623
(12), 356 (9), 55 (34), 43 (39); Anal. Calcd for C35H51N5O5Sꢂ0.5H2O:
C 63.42, H 7.91, N 10.56%. Found: C 63.28, H 7.64, N 10.51%.
5.1.10. N0-{3-(R)-[2-(S)-(1H-Indol-3-yl)-1-(methylcarbamoyl)-
ethylcarbamoyl]-5-methylhexanoyl}-N-methanesulfonyl-
hydrazide (3d)
Prepared according to the general procedure 5.1: acid
5
(600 mg, 1.61 mmol); NMM (200 L, 1.77 mmol); THF (35 mL);
l
5.2.2. N0-{3-(R)-[2-(S)-(1H-Indol-3-yl)-1-(methylcarbamoyl)-
ethylcarbamoyl]-5-methylhexanoyl}-N-2-nitrobenzene-
sulfonylhydrazide (3f)
Prepared according to the general procedure 5.2: hydrazide 2f
(600 mg, 1.55 mmol); solvents: THF (10 mL) and EtOH (10 mL);
stirring at room temperature 1.5 h; DMTMM (534 mg, 1.93 mmol);
time of activation 0.5 h; methanesulfonylhydrazide HCl salt
(1.18 g, 8.05 mmol) dissolved in DMSO (20 mL) with NMM
(890 lL, 8.09 mmol); reaction time 20 h. Purification of the crude
product by column chromatography (silica gel, CH2Cl2/MeOH,
99.5:0.5). Yield: 3d (231 mg, 31%) as a yellowish oil which was
crystallized from CH2Cl2. White-yellowish solid. Mp 179–181 °C;
base: pyridine (140 lL, 1.71 mmol); 2-nitrobenzenesulfonylchlo-
ride (344 mg, 1.55 mmol); reaction time 18 h. Purified by column
chromatography (silica gel, CH2Cl2/MeOH, 95:5). Yield: 3f
½
a 2D1
ꢁ
ꢀ6 (c 1.21, MeOH); UV (MeOH, nm): 291, 282, 218, 205; IR
(525 mg, 59%). Yellow crystals. Mp 94–95 °C; ½a D21
ꢀ0.3 (c 1.82,
ꢁ
(KBr, cmꢀ1): 3836 (NH), 3281 (NH), 2937 (CH3), 1633 (CO), 1538,
1329 (SO2), 1157 (SO2), 967, 867; 1H NMR (300 MHz, acetone-
d6): 0.77 and 0.82 (d, 2ꢃ 3H, J = 6.1 Hz, CH(CH3)2), 1.06 (m, 1H,
CH2–CH(CH3)2), 1.39 (m, 2H, CH2–CH(CH3)2), 2.19 (m, 2H, NH–
CO–CH2–), 2.55 (d, 3H, J = 4.6 Hz, CH3NHNH–), 2.73 (m, 1H, CH–
CH2–CH(CH3)2), 2.86 (s, 3H, CH3SO2NH), 3.02 (m, 2H, CH–CH2-in-
dole), 4.45 (m, 1H, CH–CH2-indole), 6.98 (m, 1H, H6indole), 7.06
(m, 1H, H5indole), 7.12 (d, 1H, J = 1.7 Hz, H2indole), 7.33 (d, 1H,
J = 7.9 Hz, H7indole), 7.57 (d, 1H, J = 7.7 Hz, H4indole), 7.81 (d, 1H,
J = 4.7 Hz, NH), 8.04 (d, 1H, J = 8.1 Hz, CONH), 9.40 (s, 1H, NH),
10.13 (s, 1H, NHSO2), 10.80 (s, 1H, NHindole); 13C NMR (75 MHz,
acetone-d6): 22.4, 23.1, 25.3, 25.8, 27.9, 36.2, 40.0, 40.3, 41.1,
53.6, 110.5, 111.4, 118.3, 118.6, 121.0, 123.6, 127.6, 136.2, 170.8,
171.9, 173.9; MS (CI, m/z, %): 466.57 (M+1, 100), 436 (18), 375
(16), 357 (8), 84 (27), 80 (17); Anal. Calcd for C21H31N5O5S: C
54.18, H 6.71, N 15.04%. Found: C 54.17, H 6.60, N 14.60%.
DMSO); UV (MeOH, nm): 291, 281, 272, 220, 210; IR (KBr, cmꢀ1):
3286 (NH), 2952 (CH), 1696 (CO), 1639 (CO), 1542, 1353 (SO2),
1168 (SO2), 851, 781; 1H NMR (300 MHz, DMSO-d6): 0.67–0.71
(m, 6H, CH(CH3)2), 0.79 (m, 1H, CH2–CH(CH3)2), 1.22 (m, 2H,
CH2–CH(CH3)2, CH2–CH(CH3)2), 2.05 (m, 2H, NH–CO–CH2–), 2.48
(m, 4H, CO–NH–CH3, CH–CH2–CH(CH3)2), 2.87–3.15 (m, 2H,
CH–CH2-indole), 4.40 (m, 1H, CH–CH2-indole), 6.89–7.09 (m, 3H,
H6indole, H5indole, H2indole), 7.28 (d, 1H, J = 7.8 Hz, H7indole), 7.51
(d, 1H, J = 7.5 Hz, H4indole), 7.70–8.05 (m, 6H, Haryl, CO–NH–CH3,
CONH), 10.17 (br s, 2H, NHNH), 10.76 (br s, 1H, NHindole); 13C
NMR (75 MHz, DMSO-d6): 22.1, 23.3, 25.3, 25.8, 27.8, 36.3, 38.9,
40.5, 53.6, 110.5, 111.4, 118.3, 118.6, 121.0, 123.6, 124.5, 127.5,
131.0, 132.1, 132.6, 134.7, 136.2, 147.9, 170.2, 171.9, 173.7; Anal.
Calcd for C26H32N6O7SꢂH2O: C 52.87, H 5.80, N 14.23%. Found: C
52.86, H 5.37, N 14.12%.
5.2.3. N0-{3-(R)-[2-(S)-(1H-Indol-3-yl)-1-(methylcarbamoyl)-
ethylcarbamoyl]-5-methylhexanoyl}-N-dodecansulfonyl-
hydrazide (3g)
5.2. General procedure for the sulfonylation of hydrazide 2f
Sulfonylchloride (pure or in THF solution) was added at 0 °C to a
solution of hydrazide 2f in THF containing co-solvents (EtOH or
DMF or DMSO) and a base (NaHCO3 or pyridine or Et3N). Stirring
was continued at room temperature until the disappearance of
the starting material (TLC monitoring). After evaporation of the
solvent, the residue was dissolved in CH2Cl2 and the organic layer
was washed with acid (5% aq HCl or 5% aq citric acid) solution, then
with 5% aq NaHCO3, dried over Na2SO4, filtered, and evaporated to
dryness. In some cases final product precipitated during extraction
and was filtered off. Purification of the products was achieved by
crystallization or column chromatography.
Prepared according to the general procedure 5.2: Hydrazide 2f
(100 mg, 0.258 mmol); solvents: THF (3 mL) and DMSO (0.4 mL);
base: pyridine (23 lL, 0.284 mmol); dodecansulfonylchloride
(69 mg, 0.258 mmol) dissolved in THF (2 mL); reaction time 24 h.
After extraction, the crude product was purified by column chro-
matography (silica gel, CH2Cl2/methanol, 95:5). Yield: 3g (55 mg,
34%). White crystals. Mp 171–173 °C; ½a D21
ꢀ8 (c 0.50, DMSO);
ꢁ
UV (MeOH, nm): 290, 283, 274, 222, 206; IR (KBr, cmꢀ1): 3401
(NH), 3339 (NH), 2916 (CH), 1661 (CO), 1635 (CO), 1538, 1454,
1335 (SO2), 1142 (SO2), 741; 1H NMR (300 MHz, acetone-d6,
DMSO-d6): 0.76–0.89 (m, 9H, CH(CH3)2, CH2–CH3), 1.09 (m, 1H,
CH2–CH(CH3)2), 1.24 (m, 22H, –CH2–), 1.78 (m, 2H, CH2–CH(CH3)2),
2.25 (m, 2H, NH–CO–CH2–), 2.58 (d, 3H, J = 4.6 Hz, CO–NH–CH3),
2.75 (m, 1H, CH–CH2–CH(CH3)2), 2.98–3.18 (m, 2H, CH–CH2-in-
dole), 4.52 (m, 1H, CH–CH2-indole), 6.98 (m, 1H, H5indole), 7.06
(m, 1H, H6indole), 7.14 (s, 1H, H2indole), 7.34 (d, 1H, J = 7.9 Hz,
H7indole), 7.60 (d, 1H, J = 7.8 Hz, H4indole), 7.75 (q, 1H, J = 4.6 Hz,
CO–NH–CH3), 7.97 (d, 1H, J = 8.2 Hz, CONH), 9.33 (br s, 1H,
SO2NH–NHCO), 10.11 (br s, 1H, SO2NH–NHCO), 10.78 (br s, 1H,
5.2.1. N0-{3-(R)-[2-(S)-(1H-Indol-3-yl)-1-(methylcarbamoyl)-
ethylcarbamoyl]-5-methylhexanoyl}-N-4-nitrobenzene-
sulfonylhydrazide (3e)
Prepared according to the general procedure 5.2: hydrazide 2f
(300 mg, 0.78 mmol); solvents: THF (7 mL) and EtOH (4 mL); base:
NaHCO3 (655 mg, 7.8 mmol); 4-nitrobenzenesulfonylchloride
(172 mg, 0.78 mmol); reaction time 18 h. After extraction, the

8754
G. LeDour et al. / Bioorg. Med. Chem. 16 (2008) 8745–8759
NHindole); 13C NMR (75 MHz, acetone-d6, DMSO-d6): 14.0, 22.3,
22.4, 23.2, 23.3, 24.8, 25.0, 28.1, 28.2, 29.3, 29.5, 30.0, 30.3, 31.6,
36.3, 40.5, 40.7, 51.6, 53.7, 110.6, 111.4, 118.3, 118.6, 120.9,
123.6, 127.7, 136.4, 170.8, 172.0, 173.8; MS (CI, m/z, %): 620.40
(M+1, 48), 130 (10), 83 (100); Anal. Calcd for C32H53N5O5S: C
62.01, H 8.62, N 11.30%. Found: C 61.65, H 8.57, N 11.06%.
base: triethylamine (87 lL, 0.62 mmol); 2-naphthalenesulfonyl-
chloride (130 mg, 0.573 mmol); reaction time 3 h. During extrac-
tion the product precipitated and the mother-liquor was also
purified by column chromatography (silica gel, CH2Cl2/MeOH,
98:2 to 90:10). Yield: 3j (70 mg, 23%). Yellowish crystals. Mp 87–
89 °C; ½a 2D1
ꢀ12 (c 1.38, DMSO); UV (MeOH, nm): 291, 281, 272,
ꢁ
220, 210; IR (KBr, cmꢀ1): 3401 (NH), 3357 (NH), 2952 (CH), 1683
(CO), 1635 (CO), 1542, 1335 (SO2), 1164 (SO2), 1128, 860, 816;
1H NMR (300 MHz, CD3OD): 0.63 (m, 6H, CH(CH3)2), 0.81 (m, 1H,
CH2–CH(CH3)2), 1.20 (m, 2H, CH2–CH(CH3)2, CH2–CH(CH3)2), 2.07
(m, 2H, NH–CO–CH2–), 2.45 (m, 1H, CH–CH2–CH(CH3)2), 2.50
(d, 3H, J = 4.4 Hz, CO–NH–CH3), 3.00–3.20 (m, 2H, CH–CH2-indole),
4.45 (m, 1H, CH–CH2-indole), 6.96 (m, 1H, H6indole), 7.00 (s, 1H,
H2indole), 7.06 (m, 1H, H5indole), 7.30 (d, 1H, J = 8.1 Hz, H7indole),
7.53 (d, 1H, J = 7.8 Hz, H4indole), 7.57–8.06 (m, 11H, Haryl, NH),
8.46 (br s, 1H, NHindole); 13C NMR (75 MHz, CD3OD): 22.2, 23.3,
26.2, 26.6, 28.6, 37.3, 42.1, 42.2, 55.7, 111.2, 112.2, 119.4, 119.7,
122.4, 124.5, 128.6, 128.7, 129.0, 130.1, 130.2, 130.4, 130.8,
136.6, 136.9, 138.0, 172.1, 174.3, 176.8; MS (CI, m/z, %): 578.62
(M+1, 100), 548 (14), 350 (5); Anal. Calcd for C30H35N5O5Sꢂ0.5H2O:
C 61.42, H 6.18, N 11.94%. Found: C 61.55, H 5.87, N 11.89%.
5.2.4. N0-{3-(R)-[2-(S)-(1H-Indol-3-yl)-1-(methylcarbamoyl)-
ethylcarbamoyl]-5-methylhexanoyl}-N-hexadecansulfonyl-
hydrazide (3h)
Prepared according to the general procedure 5.2: hydrazide 2f
(150 mg, 0.388 mmol); solvents: THF (3 mL) and DMSO (0.2 mL);
base: NaHCO3 (65 mg, 0.776 mmol); hexadecansulfonylchloride
(126 mg, 0.388 mmol) dissolved in THF (2 mL); reaction time 6 h.
After extraction, the crude product was purified by column
chromatography (silica gel, CH2Cl2/methanol, 97:3) and then crys-
tallized from ether. Yield: 3h (65 mg, 25%). White crystals. Mp
149–152 °C; ½a 2D1
ꢀ5 (c 0.90, DMSO); UV (MeOH, nm): 291, 282,
ꢁ
273, 221, 206; IR (KBr, cmꢀ1): 3392 (NH), 3277 (NH), 2916 (CH),
2846, 1657 (CO), 1635 (CO), 1542, 1454, 1335 (SO2), 1146 (SO2),
1098, 877, 741; 1H NMR (300 MHz, DMSO-d6): 0.75–0.89 (m, 9H,
CH(CH3)2, CH2–CH3), 1.01 (m, 1H, CH2–CH(CH3)2), 1.25 (m, 30H,
–CH2–), 1.73 (m, 2H, CH2–CH(CH3)2), 2.10–2.24 (m, 2H, NH–CO–
CH2–), 2.55 (d, 3H, J = 2.5 Hz, CO–NH–CH3), 2.71 (m, 1H, CH–
CH2–CH(CH3)2), 2.92–3.14 (m, 2H, CH–CH2-indole), 4.46 (m, 1H,
CH–CH2-indole), 6.97 (m, 1H, H5indole), 7.05 (m, 1H, H6indole), 7.11
(s, 1H, H2indole), 7.32 (d, 1H, J = 8.0 Hz, H7indole), 7.57 (d, 1H,
J = 7.8 Hz, H4indole), 7.81 (q, 1H, J = 2.5 Hz, CO–NH–CH3), 8.06 (d,
1H, J = 8.3 Hz CONH), 9.40 (br s, 1H, SO2NH–NHCO), 10.09 (br s,
1H, SO2NH–NHCO), 10.81 (br s, 1H, NHindole); 13C NMR (75 MHz,
DMSO-d6): 14.2, 22.3, 22.4, 23.2, 23.3, 25.4, 25.8, 28.0, 28.9, 29.0,
29.1, 29.3, 31.5, 36.4, 40.5, 41.0, 51.5, 53.7, 110.5, 111.4, 118.3,
118.7, 121.0, 123.6, 127.6, 136.2, 170.7, 171.9, 173.8; MS (CI, m/z,
%): 676.50 (M+1, 100), 645 (9), 120 (10); Anal. Calcd for
5.2.7. N0-{3-(R)-[2-(S)-(1H-Indol-3-yl)-1-(methylcarbamoyl)-
ethylcarbamoyl]-5-methylhexanoyl}-N-(4-succinimidyl)-
benzenesulfonylhydrazide (3l)
Prepared according to the general procedure 5.2: hydrazide 2f
(40 mg, 0.10 mmol); solvents: THF (4 mL) and DMSO (0.4 mL);
base: pyridine (10 lL, 0.11 mmol); 4-succinimidylbenzenesulfo-
nylchloride (28 mg, 0.10 mmol) dissolved in THF (3 mL); reaction
time 2 h. After extraction, the crude product was crystallized in
ether containing 10% methanol. Yield: 3l (15 mg, 63%). Pale yellow-
ish crystals. Mp 210–212 °C; ½a D21
ꢀ5.5 (c 1.54, DMSO); UV (MeOH,
ꢁ
nm): 290, 282, 276, 222, 210; IR (KBr, cmꢀ1): 3721 (NH), 3291
(NH), 2947 (CH), 1710 (CO), 1638 (CO), 1533, 1338 (SO2), 1162
(SO2), 1081, 1005, 757; 1H NMR (300 MHz, acetone-d6): 0.75 and
0.80 (d, 2ꢃ 3H, J = 6.1 Hz, CH(CH3)2), 1.06 (m, 1H, CH2–CH(CH3)2),
1.49 (m, 2H, CH2–CH(CH3)2, CH2–CH(CH3)2), 2.19–2.53 (m, 2H,
NH–CO–CH2–), 2.60 (d, 3H, J = 4.6 Hz, CO–NH–CH3), 2.87 (m, 4H,
COCH2–CH2CO), 2.90 (m, 1H, CH–CH2–CH(CH3)2), 3.15 (m, 2H,
CH–CH2-indole), 4.52 (m, 1H, CH–CH2-indole), 6.99–7.19 (m, 4H,
H2indole, H5indole, H6indole, CONH), 7.37 (d, 1H, J = 7.9 Hz, H7indole),
7.51 (d, 2H, J = 8.5 Hz, Haryl), 7.61 (d, 1H, J = 7.7 Hz, H4), 7.99
(d, 2H, J = 8.5 Hz, Haryl), 8.67 (m, 1H, CO–NH–CH3), 9.58 (br s, 1H,
NH), 10.00 (br s, 2H, NH, NHindole); 13C NMR (75 MHz, acetone-
d6): 22.6, 23.2, 26.2, 26.3, 28.3, 29.3, 36.8, 41.6, 42.1, 54.8, 111.8,
112.0, 119.4, 119.5, 122.0, 124.5, 127.7, 128.7, 129.7, 137.5,
138.0, 138.5, 170.9, 172.6, 174.8, 177.2; MS (CI, m/z, %): 625.53
(M+1, 100), 595 (8), 228 (8); Anal. Calcd for C30H36N6O7S: C
57.68, H 5.81, N 13.45%. Found: C 57.02, H 5.75, N 12.87%.
C
36H53N5O5Sꢂ4H2O: C 57.80, H 9.31, N 9.36%. Found: C 57.77, H
8.96, N 9.56%.
5.2.5. N0-{3-(R)-[2-(S)-(1H-Indol-3-yl)-1-(methylcarbamoyl)-
ethylcarbamoyl]-5-methylhexanoyl}-N-4-phenoxybenzene-
sulfonylhydrazide (3i)
Prepared according to the general procedure 5.2: hydrazide 2f
(203 mg, 0.525 mmol); solvents: THF (3 mL) and DMF (3 mL); base:
triethylamine (90 lL, 0.646 mmol); 4-phenoxybenzenesulfonyl-
chloride (163 mg, 0.607 mmol); reaction time 5 h. After extraction,
the crude product was purified by column chromatography (silica
gel, CH2Cl2/MeOH, 99:1 to 93:7). Yield: 3i (70 mg, 18%). Yellowish
crystals. Mp 107–109 °C; ½a D21
ꢀ9 (c 0.24, DMSO); UV (MeOH, nm):
ꢁ
291, 281, 271, 220; IR (KBr, cmꢀ1): 3401 (NH), 3286 (NH), 2943
(CH), 1670 (CO), 1635 (CO), 1582, 1335 (SO2), 1155 (SO2), 1089,
864, 833, 798, 741; 1H NMR (300 MHz, CD3OD): 0.73 and 0.77 (d,
2ꢃ 3H, J = 6.3 Hz, CH(CH3)2), 0.97 (m, 1H, CH2–CH(CH3)2), 1.30 (m,
2H, CH2–CH(CH3)2, CH2–CH(CH3)2), 2.13 (m, 2H, NH–CO–CH2–),
2.59 (m, 4H, CO–NH–CH3, CH–CH2–CH(CH3)2), 3.10–3.30 (m, 2H,
CH–CH2-indole), 4.54 (m, 1H, CH–CH2-indole), 6.90–7.90 (m, 14H,
5.2.8. N0-{3-(R)-[2-(S)-(1H-Indol-3-yl)-1-(methylcarbamoyl)-
ethylcarbamoyl]-(5-methylhexanoyl)}-N-4-(4-bromophenyl)-
benzenesulfonylhydrazide (3m)
Prepared according to the general procedure 5.2: hydrazide 2f
(510 mg, 1.32 mmol); solvents: THF (8 mL) and EtOH (8 mL); base:
H
aryl, Hindole); 13C NMR (75 MHz, CD3OD): 22.4, 23.4, 26.4, 26.7,
28.7, 37.3, 42.0, 42.1, 55.7, 111.2, 112.3, 118.4, 119.4, 119.7, 121.4,
122.4, 124.5, 126.1, 128.8, 131.3, 131.7, 133.3, 138.0, 156.6, 163.5,
172.1, 174.4, 176.8; MS (CI, m/z, %): 620.63 (M+1, 100), 590 (9),
379 (6); Anal. Calcd for C31H37N5O6Sꢂ2H2O: C 58.61, H 6.30, N
10.68%. Found: C 58.55, H 5.85, N 10.59%.
pyridine (117 lL, 1.45 mmol); 4-(4-bromophenyl)benzenesulfo-
nylchloride (437 mg, 1.32 mmol); reaction time 18 h. During
extraction the product precipitated. Yield: 3m (630 mg, 70%). Pale
yellowish crystals. Mp 126–127 °C; ½a D21
ꢀ1.5 (c 0.73, DMSO); UV
ꢁ
(MeOH, nm): 291, 281, 272, 220, 210; IR (KBr, cmꢀ1): 3436 (NH),
3269 (NH), 2908 (CH), 1657 (CO), 1635 (CO), 1542, 1370 (SO2),
1168 (SO2), 1080, 811, 776; 1H NMR (300 MHz, DMSO-d6): 0.68
(m, 6H, CH(CH3)2), 0.79 (m, 1H, CH2–CH(CH3)2), 1.24 (m, 2H,
CH2–CH(CH3)2, CH2–CH(CH3)2), 1.80–2.01 (m, 2H, NH–CO–CH2–),
2.50 (m, 4H, CO–NH–CH3, CH–CH2–CH(CH3)2), 2.90–3.18 (m, 2H,
CH–CH2-indole), 4.40 (m, 1H, CH–CH2-indole), 6.93–7.94 (m, 15H,
5.2.6. N0-{3-(R)-[2-(S)-(1H-Indol-3-yl)-1-(methylcarbamoyl)-
ethylcarbamoyl]-5-methylhexanoyl}-N-2-naphthalenesulfonyl-
hydrazide (3j)
Prepared according to the general procedure 5.2: hydrazide 2f
(200 mg, 0.517 mmol); solvents: THF (5 mL) and DMF (5 mL);

G. LeDour et al. / Bioorg. Med. Chem. 16 (2008) 8745–8759
8755
Haryl, Hindole, CONH, (CONHCH3), 9.91 (br s, 1H, SO2NHNHCO), 10.03
(s, 1H, SO2NHNH), 10.77 (s, 1H, NHindole); 13C NMR (75 MHz,
DMSO-d6): 22.0, 23.4, 25.3, 25.8, 27.9, 36.5, 39.7, 40.5, 53.5,
110.5, 111.4, 118.3, 118.6, 121.0, 122.4, 123.5, 127.5, 126.2,
126.5, 127.0, 126.6, 129.0, 129.3, 132.0, 132.2, 136.2, 137.8,
143.1, 148.0, 170.0, 171.9, 173.7; MS (CI, m/z, %): 682.54 (M+.,
100), 389 (12), 289 (9), 248 (9); Anal. Calcd for
(m, 2H, –CH2–CH@), 2.41 (d, 3H, J = 4.7 Hz, CO–NH–CH3), 3.15–
3.50 (m, 4H, NH–CO–CH2, CH–CH2–indole), 4.46–4.48 (m, 2H,
CH2–O), 4.62–4.67 (m, 1H, CH–CH2-indole), 5.13–5.25 (m, 2H,
CH2@CH–CH2O), 5.76–5.95 (m, 3H, –CH@C, CH2@CH–CH2O, CO–
NH–CH3), 6.64 (d, 1H, J = 7.3 Hz, CO–NH), 7.03–7.75 (m, 9H, Haryl,
Hindole), 9.15 (s, 1H, NHindole); 13C NMR (75 MHz, CDCl3): 13.9,
22.4, 26.1, 28.1, 28.2, 28.9, 29.3, 31.5, 32.0, 54.3, 65.2, 107.2,
110.9, 118.9, 119.1, 119.7, 122.3, 127.9, 128.0, 128.7, 128.8,
131.6, 132.3, 135.8, 138.3, 168.1, 170.2, 171.3; MS (ESI, m/z, %):
530.3 (M+1, 100), 499 (14).
C
32H36BrN5O5SꢂH2O: C 54.86, H 5.47, N 9.99%. Found: C 54.96, H
5.71, N 9.59%.
5.3. N0-{3-(R)-[2-(S)-(1H-Indol-3-yl)-1-(methylcarbamoyl)-
ethylcarbamoyl]-5-methylhexanoyl}-N-2-aminobenzenesulfonyl-
hydrazide (3k)
5.4.2. (2E)-3-(Allyloxycarbonyl)-2-(3-phenylpropylidene)-
propionyl-(S)-2-phenyltryptophan-N-methylamide (10y)
Prepared according to the general procedure 5.4: acid 9w
A solution of 3f (470 mg, 0.82 mmol) in ethanol (10 mL) was
hydrogenated at atmospheric pressure in the presence of 10% pal-
ladium on charcoal (45 mg) for 2–3 h. After filtration of the cata-
lyst, the solvent was evaporated and the residue was crystallized
from methanol giving 3k (252 mg, 57%), as white crystals. Mp
(162 mg, 0.59 mmol); base: NMM (62
THF (5 mL); DMTMM (233 mg, 0.84 mmol); time of activation
0.5 h; (S)-2-phenyltryptophan-N-methylamide (165 mg,
lL, 0.56 mmol); solvent:
8
0.56 mmol); reaction time 18 h. After extraction the crude product
was purified by column chromatography (silica gel, CH2Cl2/MeOH,
98:2). Yield: 10y (300 mg, 93%). Yellow amorphous solid. IR (film,
cmꢀ1): 3303 (NH), 2916 (CH), 1727 (CO), 1652 (CO), 1599, 1533,
1454, 1366, 1304, 1176, 1132, 987, 930, 740; 1H NMR (300 MHz,
CDCl3): 2.32–2.37 (m, 2H, CH2–CH2–Ph), 2.51 (d, 3H, J = 4.8 Hz,
CO–NH–CH3), 2.57–2.63 (m, 2H, CH2–CH2–Ph), 3.12–3.51 (m, 4H,
CO–CH2, CH–CH2-indole), 4.48–4.50 (m, 2H, CH2–O), 4.69–4.71
(m, 1H, CH–CH2-indole), 5.15–5.29 (m, 2H, CH2@CH–CH2O),
5.78–5.84 (m, 1H, CH2@CH–CH2O), 5.95 (t, 1H, J = 7.3 Hz, –CH@C),
6.57 (d, 1H, J = 7.3 Hz, NH), 7.09–7.57 (m, 14H, Haryl, Hindole), 7.83
(d, 1H, J = 7.6 Hz, NH), 8.41 (s, 1H, NHindole); 13C NMR (75 MHz,
CDCl3): 26.2, 27.2, 30.3, 32.5, 34.6, 55.8, 65.6, 107.8, 110.8, 118.4,
119.5, 120.1, 122.7, 126.1, 127.9, 128.0, 128.2, 128.4, 128.9,
129.1, 130.1, 131.6, 132.4, 135.7, 136.7, 140.7, 168.3, 170.7,
171.4; MS (ESI, m/z, %): 550 (M+1, 90), 519 (83), 287 (15), 274
(25), 257 (100), 239 (25), 218 (63), 206 (82).
217–220 °C; ½a 2D1
ꢁ
ꢀ6 (c 0.68, DMSO); IR (KBr, cmꢀ1): 3358 (NH),
2933 (CH), 1689 (CO), 1637 (CO), 1522, 1323 (SO2), 1178 (SO2);
1H NMR (300 MHz, DMSO-d6): 0.73–0.77 (m, 6H, CH(CH3)2), 0.81
(m, 1H, CH2–CH(CH3)2), 1.27 (m, 2H, CH2–CH(CH3)2, CH2–
CH(CH3)2), 2.03 (m, 2H, NH–CO–CH2–), 2.52 (m, 4H, CO–NH–CH3,
CH–CH2–CH(CH3)2), 2.93 (m, 2H, CH–CH2-indole), 4.42 (m, 1H,
CH–CH2-indole), 6.06 (br s, 2H, Ph–NH2), 6.55 (m, 1H, Haryl), 6.96
(m, 1H, H6indole), 7.06 (m, 2H, H5indole, H2indole), 7.25 (m, 1H, Haryl),
7.33 (d, 1H, J = 7.8 Hz, H7indole), 7.46 (d, 1H, J = 7.8 Hz, Haryl), 7.56
(d, 1H, J = 7.7 Hz, H4indole), 7.75 (d, 1H, J = 4.0 Hz, CO–NH–CH3),
7.93 (d, 1H, J = 8.0 Hz, CONH), 9.51 (br s, 1H, NH), 9.94 (br s, 1H,
NH), 10.78 (br s, 1H, NHindole); 13C NMR (75 MHz, DMSO-d6):
22.1, 23.4, 25.4, 25.8, 27.9, 36.5, 40.3, 40.5, 53.6, 110.5, 111.4,
114.8, 117.1, 117.8, 118.4, 118.6, 121.0, 123.5, 127.5, 130.2,
134.3, 136.2, 147.7, 169.9, 172.0, 173.8; MS (CI, m/z, %): 542.6
(M+., 100), 512 (50), 356 (18), 242 (12), 218 (7); Anal. Calcd for
C
26H34N6O5SꢂH2O: C 55.70, H 6.47, N 14.99%. Found: C 55.65, H
5.4.3. 3-(R)-[2-(S)-(2-Phenyl-1H-indol-3-yl)-methylcarbamoyl)-
ethylcarbamoyl]-5-methyl tert-butylhexanoate (11z)
6.14, N 14.89%.
Prepared according to the general procedure 5.4: acid
(399 mg, 1.73 mmol); base: NMM (205 L, 1.87 mmol); solvent:
THF (20 mL); DMTMM (570 mg, 2.01 mmol); time of activation
0.5 h; (S)-2-phenyltryptophan-N-methylamide (500 mg,
7
5.4. General procedure for the condensation of carboxylic acids
7, 9v,w with (S)-tryptophan-N-methylamide (6) or (S)-2-phenyl-
tryptophan-N-methylamide (8)
l
8
1.70 mmol); reaction time 3 h. After extraction, the crude product
was purified by column chromatography (silica gel, CH2Cl2/MeOH,
A mixture of acid 7 or 9v or 9w, N-methylmorpholine (NMM), and
4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chlo-
ride) (DMTMM) in dry THF was stirred at room temperature for
30 min and then (S)-tryptophan-N-methylamide (6) or (S)-2-phe-
nyltryptophan-N-methylamide (8) was added to the reaction mix-
ture and stirring was continued overnight at room temperature.
After evaporation, the residue was dissolved in CH2Cl2 (10–30 mL),
washed successively with 5% aq citric acid, water, and 5% aq NaHCO3
solution. The organic phase was dried over MgSO4, filtered and con-
centrated in vacuo. The residue was purified by column chromatog-
raphy to give the corresponding derivatives 10 or 11.
98:2). Yield: 11z (620 mg, 72%). Yellowish amorphous solid. ½a D21
ꢁ
+21 (c 0.6, DMSO); IR (KBr, cmꢀ1): 3383 (NH), 3286 (NH), 2925
(CH), 1727 (CO), 1639 (CO), 1547, 1525, 1450, 1366, 1300, 1252,
1150, 838, 767; 1H NMR (300 MHz, DMSO-d6): 0.79 (d, 3H,
J = 6.2 Hz, CH(CH3)2), 0.85 (d, 3H, J = 6.3 Hz, CH(CH3)2), 1.07 (m,
1H, CH2–CH(CH3)2), 1.38 (m, 11H, C(CH3)3, CH2–CH(CH3)2, CH2–
CH(CH3)2), 2.10–2.27 (m, 2H, O–CO–CH2), 2.38 (d, 3H, J = 4.6 Hz,
CONHCH3), 2.70 (m, 1H, CH–CH2–CH(CH3)2), 2.99–3.32 (m, 2H,
CH–CH2-indole), 4.59 (m, 1H, CH–CH2-indole), 7.01 (m, 1H,
H6indole), 7.07 (m, 1H, H5indole), 7.37 (m, 2H, Haryl), 7.50 (m, 2H,
H
aryl, H7indole), 7.62 (d, 1H, J = 4.6 Hz, CO–NH–CH3), 7.67 (d, 1H,
5.4.1. (2E)-3-(Allyloxycarbonyl)-2-heptylidenepropionyl-(S)-2-
phenyltryptophan-N-methylamide (10x)
J = 7.8 Hz, H4indole), 7.73 (m, 2H, Haryl), 11.20 (s, 1H, NHindole); 13C
NMR (75 MHz, DMSO-d6): 22.6, 23.2, 25.4, 25.9, 27.9, 28.8, 38.2,
40.0, 41.3, 54.3, 79.9, 107.9, 111.2, 118.8, 119.5, 121.7, 127.5,
128.3, 128.9, 129.3, 133.0, 135.4, 136.1, 171.1, 171.7, 173.7; MS
(CI, m/z, %): 506.28 (M+1, 100), 450 (29).
Prepared according to the general procedure 5.4: acid 9v
(195 mg, 0.76 mmol); base: NMM (56
THF (5 mL); DMTMM (212 mg, 0.76 mmol); time of activation
0.5 h; (S)-2-phenyltryptophan-N-methylamide (150 mg,
lL, 0.51 mmol); solvent:
8
0.51 mmol); reaction time 18 h. After extraction, the crude product
was purified by column chromatography (silica gel, CH2Cl2/MeOH,
99:1). Yield: 10x (192 mg, 71%). Yellow amorphous solid. IR (film,
cmꢀ1): 3295 (NH), 2916 (CH), 1731 (CO), 1652 (CO), 1502, 1454,
1361, 1308, 1172, 987, 921, 767; 1H NMR (300 MHz, CDCl3): 0.88
(t, 3H, J = 6.5 Hz, –CH3), 1.14–1.26 (m, 8H, CH2), 1.95–2.02
5.5. General procedure for the preparation of carboxylic acids
12v–y
To a solution of allyl ester 10v-y in THF tetrakis(triphenylphos-
phine)palladium (Pd(PPh3)4) and morpholine were added and the
reaction mixture was stirred for 30 min under nitrogen atmo-

8756
G. LeDour et al. / Bioorg. Med. Chem. 16 (2008) 8745–8759
sphere. After evaporation of the solvent, the residue was dissolved
in CH2Cl2 (15 mL) and extracted with 5% aq NaHCO3 (15 mL). The
aqueous phase was acidified (pH 1) with 5% aq citric acid solution,
extracted with CH2Cl2. The organic phase was dried (MgSO4), fil-
tered and the filtrate was concentrated in vacuo to give acids
12v–y.
5.6. General procedure for condensation of carboxylic acids
12v,w,x,z and 4-(4-bromophenyl)-benzenesulfonylhydrazide
HCl salt
A mixture of acid 12 N-methylmorpholine (NMM), and 4-(4,6-
dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium
chloride)
(DMTMM) in dry THF was stirred at room temperature for
30 min. 4-(4-Bromophenyl)benzenesulfonylhydrazide HCl salt in
THF with NMM were added to the mixture. The reaction mixture
was stirred overnight at room temperature. The organic phase
was washed successively with 5% aq citric acid, water, 5% aq NaH-
CO3 and dried over MgSO4. The organic phase was filtered, evapo-
rated in vacuo and residue was purified by column
chromatography to give 4.
5.5.1. (2E)-3-(Hydroxycarbonyl)-2-heptylidenepropionyl-(S)-2-
phenyltryptophan-N-methylamide (12x)
Prepared according to the general procedure 5.5: allyl ester 10x
(190 mg, 0.36 mmol); solvent: THF (5 mL); Pd(PPh3)4 (42 mg,
0.036 mmol); base: morpholine (98 lL, 1.11 mmol); Yield: 12x
(69 mg, 40%). Viscous oil. IR (film, cmꢀ1): 3295 (NH), 2916 (CH),
1709 (CO), 1647 (CO), 1528, 1458, 1304, 1264, 1181, 1009, 745;
1H NMR (300 MHz, CDCl3): 0.87 (t, 3H, J = 6.3 Hz, –CH3), 1.14–
1.43 (m, 8H, CH2), 2.02–2.09 (m, 2H, –CH2–CH@), 2.37 (d, 3H,
J = 4.4 Hz, CO–NH–CH3), 3.08–3.46 (m, 4H, NH–CO–CH2, CH–CH2-
indole), 4.62–4.69 (m, 1H, CH–CH2-indole), 5.86 (q, 1H, J = 4.4 Hz,
CO–NH–CH3), 6.04 (t, 1H, J = 7.3 Hz, CH@), 7.05–7.69 (m, 10H,
NH, Haryl, Hindole), 8.80 (s, 1H, NHindole), 10.50 (br s, 1H, OH); 13C
NMR (75 MHz, CDCl3): 13.9, 22.4, 26.2, 27.6, 28.3, 28.4, 28.8,
31.4, 34.2, 54.1, 106.8, 111.0, 118.9, 119.9, 122.5, 127.5, 127.8,
128.4, 128.6, 128.7, 128.9, 131.8, 132.4, 135.7, 135.9, 140.4,
170.4, 171.4, 172.7; MS (ESI, m/z, %) 490 (M+1, 56), 459 (15), 279
(30), 79 (100).
5.6.1. N0-{3-(E)-[2-(S)-(1H-Indol-3-yl)-1-(methylcarbamoyl)-
ethylcarbamoyl]-decenoyl}-N-4-(4-bromophenyl)benzene-
sulfonylhydrazide (4v)
Prepared according to the general procedure 5.6: acid 12v
(50 mg, 0.12 mmol); base: NMM (20 lL, 0.18 mmol); solvent: THF
(3 mL); DMTMM (50 mg, 0.18 mmol); time of activation 0.5 h;
4-(4-bromophenyl)benzenesulfonylhydrazide HCl salt (132 mg,
0.36 mmol); solvent: THF (3 mL); base: NMM (40 lL, 0.36 mmol);
reaction time 6 h. After extraction the crude product was purified
by column chromatography (silica gel, CH2Cl2/MeOH, 98:2 then
95:5). Yield: 4v (23 mg, 26%). Amorphous solid. IR (film, cmꢀ1):
3295 (NH), 2916 (CH), 1647 (CO), 1537, 1339 (SO2), 1167 (SO2),
1093, 811, 771, 745; 1H NMR (300 MHz, CDCl3): 0.83
(t, 3H, J = 6.6 Hz, –CH3), 1.11–1.25 (m, 8H, CH2), 1.84–1.96 (m, 2H,
–CH2–CH@), 2.66 (d, 3H, J = 4.6 Hz, CO–NH–CH3), 3.02–3.34 (m,
4H, NH–CO–CH2, CH–CH2-indole), 4.71–4.73 (m, 1H, CH–CH2-in-
dole), 5.93 (q, 1H, J = 4.6 Hz, CO–NH–CH3), 6.07 (t, 1H, J = 7.0 Hz,
5.5.2. (2E)-3-(Hydroxycarbonyl)-2-(3-phenylpropylidene)-
propionyl-(S)-2-phenyltryptophan-N-methylamide (12y)
Prepared according to the general procedure 5.5: allyl ester 10y
(281 mg, 0.51 mmol); solvent: THF (7 mL); Pd(PPh3)4 (59 mg,
0.051 mmol); base: morpholine (138 lL, 1.58 mmol); Yield: 12y
(169 mg, 65%). Viscous oil. IR (film, cmꢀ1): 3312 (OH), 3048
(NH), 2925 (CH), 1713 (CO), 1643 (CO), 1528, 1454, 1260, 1185,
908, 740; 1H NMR (300 MHz, CDCl3): 2.41–2.50 (m, 5H, CH2–
CH2–Ph, CO–NH–CH3), 2.57–2.67 (m, 2H, CH2–CH2–Ph), 3.10–3.50
(m, 4H, CO–CH2, CH–CH2-indole), 4.63–4.70 (m, 1H, CH–CH2–in-
dole), 5.44 (q, 1H, J = 4.8 Hz, CO–NH–CH3), 6.11 (t, 1H, J = 7.3 Hz,
CH@), 7.03–7.54 (m, 14H, Haryl, Hindole), 7.77 (d, 1H, J = 7.1 Hz,
NH), 8.35 (s, 1H, NHindole); 13C NMR (75 MHz, CDCl3): 26.3, 27.9,
30.2, 34.5, 34.6, 54.1, 107.1, 111.0, 119.1, 120.2, 122.7, 126.2,
127.9, 128.1, 128.2, 128.5, 128.7, 128.8, 129.1, 132.1, 132.3,
135.7, 135.9, 139.1, 140.4, 170.5, 171.1, 171.8; MS (ESI, m/z, %)
510 (M+1, 100), 479 (40).
CH@), 6.70 (d, 1H, J = 7.7 Hz, CO–NH), 7.09–7.86 (m, 14H, NH, Haryl
H
,
indole), 8.35 (s, 1H, NHindole), 9.51 (d, 1H, J = 5.6 Hz, NH); 13C NMR
(75 MHz, CDCl3): 14.0, 22.4, 26.3, 28.4, 28.6, 29.0, 31.5, 33.3, 54.4,
110.4, 111.4, 118.5, 119.8, 122.3, 123.1, 127.0, 127.3, 128.1, 128.7,
129.1, 132.1, 135.5, 136.0, 137.7, 140.6, 144.9, 168.9, 169.3, 171.7;
HRMS Calcd for C35H40N5O5NaSBr: 744.1831. Found: 744.1843;
Anal. Calcd for C35H40N5O5SBrꢂH2O: C 56.81, H 5.41, N 9.46. Found:
C 56.25, H 5.77, N 9.40.
5.6.2. N0-{3-(E)-[2-(S)-(1H-Indol-3-yl)-1-(methylcarbamoyl)-
ethylcarbamoyl]-6-phenylhexenoyl}-N-4-(4-bromophenyl)-
benzenesulfonylhydrazide (4w)
5.5.3. 3-(R)-[2-(S)-(2-Phenyl-1H-indol-3-yl)-methylcarbamoyl)-
ethylcarbamoyl]-5-methyl hexanoic acid (12z)
Prepared according to the general procedure 5.6: acid 12w
(75 mg, 0.17 mmol); base: NMM (28 lL, 0.26 mmol); solvent:
To a solution of ester 11z (570 mg, 1.13 mmol) in CH2Cl2
(11 mL), were added TFA (2.8 mL) and dithiothreitol (10 mg), and
the mixture was stirred at room temperature for 3.5 h. After evap-
oration, a white solid was precipitated in ether. Yield: 12z (325 mg,
THF (5 mL); DMTMM (72 mg, 0.26 mmol); time of activation
0.5 h; 4-(4-bromophenyl)benzenesulfonylhydrazide HCl salt
(157 mg, 0.43 mmol); solvent: THF (5 mL); base: NMM (48 lL,
0.43 mmol); reaction time 18 h. After extraction the crude product
was purified by column chromatography (silica gel, CH2Cl2/MeOH,
98:2 then 95:5). Yield: 4w (33 mg, 26%). Amorphous solid. IR (film,
cmꢀ1): 3330 (NH), 2916 (CH), 1727 (CO), 1458, 1374 (SO2), 1260,
1163 (SO2), 1093, 1018, 798, 745; 1H NMR (300 MHz, CDCl3):
2.18–2.25 (m, 2H, CH2–CH2–Ph), 2.46 (t, 2H, J = 7.6 Hz, CH2–CH2–Ph),
2.65 (d, 3H, J = 4.8 Hz, CO–NH–CH3), 2.97 (s, 2H, NH–CO–CH2),
3.11–3.32 (m, 2H, CH–CH2-indole), 4.69–4.74 (m, 1H,
CH–CH2-indole), 5.97 (q, 1H, J = 4.8 Hz, CO–NH–CH3), 6.09 (t, 1H,
J = 7.1 Hz, CH@), 6.81 (d, 1H, J = 7.6 Hz, CO–NH), 6.99–8.31 (m,
64%). White crystals. Mp 197–198 °C; ½a D21
ꢁ
+36.5 (c 1.17, DMSO); IR
(KBr, cmꢀ1): 3568 (OH), 3286 (NH), 3057 (NH), 2952 (CH), 1701
(CO), 1635 (CO), 1538, 1454, 1247, 917, 763; 1H NMR (300 MHz,
DMSO-d6): 0.78 (d, 3H, J = 5.8 Hz, CH(CH3)2), 0.85 (d, 3H,
J = 5.8 Hz, CH(CH3)2), 1.11 (m, 1H, CH2–CH(CH3)2), 1.37 (m, 2H,
CH2–CH(CH3)2, CH2–CH(CH3)2), 2.23 (m, 2H, O–CO–CH2), 2.39 (d,
3H, J = 4.1 Hz, CO–NH–CH3), 2.71 (m, 1H, CH–CH2–CH(CH3)2),
3.18 (m, 2H, CH–CH2-indole), 4.59 (m, 1H, CH–CH2–indole), 7.00
(m, 1H, H6indole), 7.10 (m, 1H, H5indole), 7.37 (m, 2H, Haryl), 7.50
(m, 2H, Haryl, H7indole), 7.61 (d, 1H, J = 4.1 Hz, CO–NH–CH3), 7.68
(d, 1H, J = 7.8 Hz, H4indole), 7.74 (m, 2H, Haryl), 8.05 (d, 1H,
J = 8.0 Hz, CO–NH), 11.20 (s, 1H, NHindole), 12.14 (m, 1H, OH); 13C
NMR (75 MHz, DMSO-d6): 22.5, 23.1, 25.4, 25.9, 28.6, 37.1, 39.9,
41.3, 54.3, 108.0, 111.2, 118.8, 119.5, 121.7, 127.5, 128.2, 128.8,
129.3, 132.9, 135.3, 136.1, 171.6, 173.4, 174.0; MS (CI, m/z, %)
450.26 (M+1, 100), 419 (25), 218 (15).
19H, NH, Haryl, Hindole), 8.31 (s, 1H, NHindole), 9.45 (d, 1H,
J = 4.6 Hz, NH); 13C NMR (75 MHz, CDCl3): 26.3, 28.3, 30.3, 33.2,
34.3, 54.3, 110.4, 111.4, 118.5, 119.7, 122.3, 123.0, 126.2, 127.0,
127.3, 128.1, 128.3, 128.5, 128.7, 128.8, 128.9, 129.0, 132.1,
132.1, 135.5, 136.1, 137.7, 139.0, 140.3, 144.9, 168.8, 169.2,
171.6; HRMS Calcd for
764.1522.
C37H36N5O5NaSBr: 764.1518. Found:

G. LeDour et al. / Bioorg. Med. Chem. 16 (2008) 8745–8759
8757
5.6.3. N0-{3-(E)-[2-(S)-(2-Phenyl-1H-indol-3-yl)-1-(methyl-
carbamoyl)-ethylcarbamoyl]-decenoyl}-N-4-(4-bromophenyl)-
benzenesulfonylhydrazide (4x)
Prepared according to the general procedure 5.6: acid 12x
(50 mg, 0.1 mmol); base: NMM (17 lL, 0.15 mmol); solvent: THF
(3 mL); DMTMM (42 mg, 0.15 mmol); time of activation 0.5 h; 4-
(4-bromophenyl)benzenesulfonylhydrazide HCl salt (111 mg,
0.3 mmol); solvent: THF (3 mL); base: NMM (34 lL, 0.3 mmol);
reaction time 7 h. After extraction the crude product was purified
by column chromatography (silica gel, CH2Cl2/MeOH, 99:1 then
98:2). Yield: 4x (24 mg, 30%). Amorphous solid. IR (film, cmꢀ1):
3259 (NH), 2925 (CH), 1661 (CO), 1647 (CO), 1506, 1344 (SO2),
1167 (SO2), 1115, 1093, 811, 771, 740; 1H NMR (300 MHz, CDCl3):
0.84 (t, 3H, J = 6.6 Hz, –CH3), 1.12–1.25 (m, 8H, CH2), 1.77–1.93 (m,
2H, –CH2–CH@), 2.49 (d, 3H, J = 4.7 Hz, CO–NH–CH3), 2.96 (s, 2H,
NH–CO–CH2), 3.29–3.47 (m, 2H, CH–CH2–indole), 4.66–4.70 (m,
1H, CH–CH2-indole), 5.53 (q, 1H, J = 4.7 Hz, CO–NH–CH3), 5.93 (t,
1H, J = 7.1 Hz, CH@), 6.78 (d, 1H, J = 7.3 Hz, CO–NH), 7.26–7.87
(m, 18H, NH, Haryl, Hindole), 8.44 (s, 1H, NHindole), 9.62 (d, 1H,
J = 5.8 Hz, NH); 13C NMR (75 MHz, CDCl3): 14.0, 22.4, 26.3, 28.2,
28.4, 28.6, 29.0, 31.5, 33.5, 54.0, 107.3, 111.0, 119.1, 120.3, 122.7,
123.0, 127.0, 127.9, 128.0, 128.1, 128.4, 128.5, 128.7, 128.9,
129.1, 131.9, 132.1, 132.4, 135.5, 135.7, 136.0, 137.8, 140.2,
144.8, 168.8, 169.2, 171.2; HRMS Calcd for C41H44N5O5NaSBr:
820.2127. Found: 820.2144.
(MgSO4), filtered and evaporated. Yield: 14y (78 mg, 55%). Amor-
phous solid. IR (film, cmꢀ1): 3277 (NH), 2916 (CH), 1647 (CO),
1612 (CO), 1528, 1445, 1405, 1150, 740; 1H NMR (300 MHz,
CD3OD): 2.23–2.31 (m, 2H, CH2–CH2–Ph), 2.41–2.56 (m, 5H, CH2–
CH2–Ph, CO–NH–CH3), 2.76–2.96 (m, 2H, CO–CH2), 3.34–3.49 (m,
2H, CH–CH2–indole), 4.51–4.55 (m, 1H, CH–CH2-indole), 5.86
(t, 1H, J = 7.3 Hz, CH@), 6.98–7.59 (m, 14H, Haryl, Hindole); 13C
NMR (75 MHz, CD3OD): 25.4, 26.7, 29.9, 31.8, 34.0, 54.0, 106.0,
110.7, 118.2, 118.9, 121.6, 125.6, 127.2, 127.6, 127.8, 127.9,
128.3, 128.4, 128.6, 129.2, 132.5, 135.7, 137.9, 140.4, 169.1,
170.5, 172.1; MS (ESI, m/z, %) 524 (M+1, 100), 492 (15).
5.9. N0-{3-(E)-[2-(S)-(2-Phenyl-1H-indol-3-yl)-1-(methyl-
carbamoyl)-ethylcarbamoyl]-6-phenylhexenoyl}-N-4-(4-
bromophenyl)benzenesulfonylhydrazide (4y)
Prepared according to the general procedure 5.2: hydrazide 14y
(52 mg, 0.099 mmol); solvent: THF (5 mL); base: triethylamine
(14 lL, 0.099 mmol); 4-(4-bromophenyl) benzenesulfonylchloride
(36 mg, 0.1 mmol); reaction time 18 h. After extraction, the crude
product was purified by column chromatography (silica gel,
CH2Cl2/MeOH, 98:2). Yield: 4y (44 mg, 54%). Amorphous solid. IR
(film, cmꢀ1): 3392 (NH), 3277 (NH), 2916 (CH), 1661 (CO), 1643
(CO), 1511, 1454, 1339 (SO2), 1255, 1159 (SO2), 1093, 811, 740;
1H NMR (300 MHz, CDCl3): 2.15–2.26 (m, 2H, CH2–CH2–Ph),
2.43–2.55 (m, 5H, CH2–CH2–Ph, CO–NH–CH3), 2.91–2.97 (m, 2H,
NH–CO–CH2), 3.29–3.46 (m, 2H, CH–CH2-indole), 4.61–4.69
(m, 1H, CH–CH2-indole), 5.51 (q, 1H, J = 4.6 Hz, CO–NH–CH3),
5.95 (t, 1H, J = 7.2 Hz, CH@), 6.75 (d, 1H, J = 7.4 Hz, CO–NH),
6.99–7.86 (m, 23H, NH, Haryl, Hindole), 8.33 (s, 1H, NHindole), 9.52
(d, 1H, J = 7.4 Hz, NH); 13C NMR (75 MHz, CDCl3): 26.3, 28.2, 30.4,
33.4, 34.4, 54.0, 107.3, 111.1, 119.1, 120.3, 122.7, 122.9, 126.2,
127.0, 127.9, 128.1, 128.5, 128.7, 128.9, 128.9, 129.1, 129.1,
132.0, 132.4, 135.6, 135.7, 135.9, 137.7, 138.7, 140.4, 144.8,
168.7, 169.1, 171.2; HRMS Calcd for C43H40N5O5NaSBr: 840.1846.
Found: 840.1831.
0
5.7. N-{3-(E)-[2-(S)-(2-Phenyl-1H-indol-3-yl)-1-(methylcarbamoyl)-
ethylcarbamoyl]-6-phenylhexenoyl}-N-tert-butoxycarbonyl-
hydrazide (13y)
A mixture of acid 12y (160 mg, 0.3 mmol), N-methylmorpholine
(NMM) (35
lL, 0.3 mmol) and 4-(4,6-dimethoxy-1,3,5-triazin-2-
yl)-4-methylmorpholinium
chloride) (DMTMM) (130 mg,
0.4 mmol) in dry THF (7 mL) was stirred at room temperature for
30 min. Boc-hydrazine (46 mg, 0.3 mmol) was added to the prece-
dent mixture and stirring was continued for 1.5 h. After evapora-
tion of the solvent, the residue was dissolved in CH2Cl2 (10 mL)
and washed successively with 5% aq citric acid solution, water
and 5% aq NaHCO3 solution. The organic layer was dried (MgSO4),
filtered and the filtrate was evaporated in vacuo. The crude product
was purified by column chromatography (silica gel, elution:
CH2Cl2/MeOH, 98:2 then 96:4) to give 13y as a pale-yellow foam.
5.10. N0-{3-(R)-[2-(S)-(2-Phenyl-1H-indol-3-yl)-1-
(methylcarbamoyl)-ethylcarbamoyl]-5-methylhexanoyl}-N-4-
(4-bromophenyl)benzenesulfonylhydrazide (4z)
Prepared according to the general procedure 5.6: acid 12z
(42 mg, 0.09 mmol); base: NMM (25 lL, 0.13 mmol); solvent:
THF (3 mL); DMTMM (38 mg, 0.14 mmol); time of activation
0.5 h; 4-(4-bromophenyl)benzenesulfonylhydrazide HCl salt
(84 mg, 0.23 mmol); solvent: THF (3 mL); base: NMM (15 lL,
0.14 mmol); reaction time 5 h. After extraction, the crude product
was purified by column chromatography (silica gel, CH2Cl2/MeOH,
Yield: 13y (176.6 mg, 92%). ½a D21
ꢁ
ꢀ20.2 (c 0.5, CHCl3); IR (film,
cmꢀ1): 3295 (NH), 2925 (CH), 1735 (CO), 1652 (CO), 1511, 1458,
1370, 1238, 1159, 740; 1H NMR (300 MHz, CDCl3): 1.43 (s, 9H,
C(CH3)3), 2.39–2.63 (m, 7H, CH2–CH2–Ph, CO–NH–CH3), 2.97–3.11
(m, 2H, CO–CH2), 3.41–3.49 (m, 2H, CH–CH2–indole), 4.64–4.67
(m, 1H, CH–CH2-indole), 5.83 (br s, 1H, NH), 5.91 (t, 1H,
J = 6.6 Hz, CH@), 6.63 (d, 1H, J = 6.9 Hz, NH), 7.12–7.56 (m, 14H,
99:1 then 98:2). Yield: 4z (24 mg, 35%). Amorphous solid. ½a D21
ꢁ
+8
H
aryl, Hindole), 7.76 (d, 1H, J = 7.7 Hz, NH), 8.42 (s, 1H, NHindole),
(c 0.25, MeOH); IR (film, cmꢀ1): 3256 (NH), 3057 (NH), 2919
(CH), 1633 (CO), 1508, 1340 (SO2), 1161 (SO2) 1094, 806, 700; 1H
NMR (300 MHz, CD3OD): 0.68–0.74 (m, 6H, CH(CH3)2), 0.79 (m,
1H, CH2–CH(CH3)2), 1.25–1.28 (m, 2H, CH2–CH(CH3)2), 2.06–2.10
(m, 2H, NH–CO–CH2), 2.38 (s, 3H, CO–NH–CH3), 2.42–2.50 (m,
1H, CH–CH2–CH(CH3)2), 3.16–3.18 (m, 2H, CH–CH2–indole), 4.52–
4.53 (m, 1H, CH–CH2-indole), 6.99–7.92 (m, 17H, Haryl, Hindole);
13C NMR (75 MHz, CDCl3): 22.4, 23.3, 26.4, 26.7, 28.9, 37.1, 42.0,
42.2, 55.7, 108.2, 112.0, 120.0, 120.1, 122.9, 128.2, 128.5, 129.3,
129.9, 130.0, 130.6, 133.2, 133.3, 134.5, 137.1, 137.7, 168.4,
173.9, 176.6; HRMS Calcd for C38H40N5O5NaSBr: 780.1819. Found:
780.1831.
8.53 (s, 1H, NH); 13C NMR (75 MHz, CDCl3): 26.3, 27.5, 28.0, 30.5,
33.5, 34.5, 54.1, 81.4, 107.5, 110.9, 119.3, 120.2, 122.7, 126.1,
127.9, 128.0, 128.3, 128.4, 128.9, 129.1, 129.6, 132.4, 135.7,
138.2, 140.8, 155.2, 169.4, 169.7, 171.3; MS (ESI, m/z, %) 624
(M+1, 100), 568 (48), 537 (20), 524 (15), 492 (40).
5.8. N0-{3-(E)-[2-(S)-(2-Phenyl-1H-indol-3-yl)-1-(methyl-
carbamoyl)-ethylcarbamoyl]-6-phenylhexenoyl}-hydrazide
(14y)
A solution of Boc protected hydrazide 13y (166 mg, 0.26 mmol)
in methanol saturated with HCl gas (1.4 mL, C 11.3 mol/L) was stir-
red at room temperature for 45 min. After evaporation of the sol-
vent, the crude residue was dissolved in CH2Cl2 and made
alkaline (pH 9) at 0 °C by addition of an 1 N aq NaOH solution.
The precipitate was extracted with CH2Cl2 (3ꢃ 5 mL), dried
5.11. Materials and methods for biological evaluation
Quenched fluorogenic substrates DNP-Pro-Cha-Gly-Cys(Me)-
His-Ala-Lys(N-Me-Abz)-NH2 [where DNP: 2,4-dinitrophenyl; Cha:

8758
G. LeDour et al. / Bioorg. Med. Chem. 16 (2008) 8745–8759
b-cyclohexylalanyl; Abz: 2-aminobenzoyl(anthraniloyl)] were pur-
chased from Calbiochem (VWR, Strasbourg, France). Mca-Pro-Leu-
Gly-Leu-Dpa-Ala-Arg-NH2 (where Mca: (7-methoxycoumarin-4-
for the calculations of the electrostatic grid maps. Random starting
positions on the entire protein surface, random orientations and
torsions were used for all ligands. The AutoDock program, version
4.0 was used for docking computations, with a Lamarckian genetic
algorithm.23,24 Each docking experiment was performed with two
runs constituted of a series of 250 simulations. Each docking sim-
ulation was carried out with an initial population of 250 individu-
als, a maximum number of 5,000,000 energy evaluations and a
maximum number of 50,000 generations. The pseudo-Solis and
Wets modification methods were used with default parameters.
Docked conformations of the ligands were clustered with a root
mean square deviation (RMSD) cut-off of 0.5 Å. The molecular
graphics of the three-dimensional structures and their display have
been done using PyMOL program (Warren L. DeLano ‘The PyMOL
Molecular Graphics System.’ DeLano Scientific LLC, San Carlos,
CA, USA. http://www.pymol.org).
yl); Dpa: [N-3-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl] and
NBD- -aminocaproyl-Arg-Pro-Lys-Pro-Leu-Ala-Nva-Trp-Lys(DMAC-
e
A)-NH2 (where NBD: 6-(7-nitro-benzo[1,2,5]oxodiazol-4-yl);
DMACA: 7-dimethylaminocoumarin-4-yl) were obtained from
Bachem (Weil am Rhein, Germany). Human recombinant pro-MMP-
2, pro-MMP-9, pro-MMP-1, pro-MMP-7, and catalytic domains of
MT1-MMP or of MMP-3 were obtained from Calbiochem. The
pro-enzymes were freshly activated with 1
lM (pro-MMP-2) or
2
lM (pro-MMP-9, pro-MMP-7, and pro-MMP-1) p-aminophenyl-
mercuric acetate (Sigma–Aldrich, Saint Quentin Fallavier, France)
at 37 °C in water bath for 2 h.
Enzyme inhibitions were carried out in 50 mM Tris–HCl buffer
(pH 7.5), 150 mM NaCl, 5 mM CaCl2 at 25 °C except for MMP-3
where 50 mM Tris–HCl (pH 7.5), 5 mM CaCl2, 1 mM ZnCl2 was in-
stead used, according to manufacturer instructions. All assays were
performed in black 96-well plates (non-binding surface plates
3650; CorningCostar), in conditions of initial rate of substrate
hydrolysis. Progress curves were monitored by following the in-
crease of fluorescence at (i) 465 nm (kex = 326 nm) induced by
the cleavage of MCA-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 (Bachem)
by MMP-2 or MMP-7; (ii) 465 nm (kex = 360 nm) induced by the
cleavage of Dup-Pro-Cha-Gly-Cys(Me)-His-Ala-Lys (N-MeAbz)-
NH2 (Calbiochem) by either MMP-1 or MMP-9; (iii) 465 nm
Acknowledgments
The authors thank the French Ministry of Education, Research
and Technology (MENRT) for Ph.D. fellowship of G. LeDour and
the ‘Conseil Régional de Champagne-Ardenne’ for Ph.D. fellowship
of M. Rouffet and for postdoctoral fellowship of H. ElMourabit.
Financial support of CNRS, University of Reims-Champagne-Ard-
enne and ‘Cancéropôle Grand-Est’ is gratefully acknowledged. We
thank C. Petermann (NMR), D. Harakat (HRMS) Ph. Sigaut (MS)
and D. Patigny (MS) for spectroscopic measurements.
(kex = 360 nm) induced by the cleavage of NBD-e-aminocaproyl-
Arg-Pro-Lys-Pro-Leu-Ala-Nva-Trp-Lys(DMACA)-NH2 by MMP-3.
The fluorescence was monitored with a Perkin-Elmer HT Soft
7000 Plus spectrofluorimeter (Perkin-Elmer, Courtaboeuf, France)
equipped with a temperature device control and a plate shaker.
References and notes
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Typical experimental conditions were 200
lL of buffer, 1–2 nM
of MMP and 1–10 M of substrate. Inhibitors at various concentra-
l
tions were preincubated for 30 min at 25 °C with enzymes before
additions of substrate. Percentage inhibition was determined in
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5.12.1. MMPs and ligands initial data
Structures of Protein Data Bank (PDB) entries 1CK7 for MMP-228
and 1GKC for MMP-929 were used for docking simulations. While
MMP atoms and the zinc ion in its catalytic site were retained,
all the other atoms were removed. The positions of polar hydro-
gens and charges were assigned using the Kollman algorithm.30
Solvation parameters were added using the ADDSOL utility of
AutoDock 4.0. MMP side chains were kept fixed for all the docking
computations.
Ligands were built using the module BIOPOLYMER of Insight98
software package (MSI, Inc., San Diego, CA, USA). Ligands structure
was optimized and their atoms charges were computed from quan-
tum calculations with the Gaussian 03 program,31 using a 6-31G*
basis set. The AutoDock module AutoTors was used to define li-
gands torsion angles. All the flexible torsions, except the amide
bonds, were allowed to rotate during the docking stage.
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5.12.2. Molecular docking simulations and calculations
Affinity grid maps were calculated for each atom type constitut-
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the MMP catalytic site, with 124 ꢃ 124 ꢃ 124 grid points and a
spacing of 0.214 Å between the grid points. The distance-depen-
dent dielectric permittivity of Mehler and Solmajer32 was used
21. Berton, A.; Rigot, V.; Huet, E.; Decarme, M.; Eeckhout, Y.; Patthy, L.; Godeau, G.;
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G. LeDour et al. / Bioorg. Med. Chem. 16 (2008) 8745–8759
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22. LeDour, G. Ph.D. Thesis, University of Reims-Champagne-Ardenne, November
25, 2005.
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23. Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.; Hart, W. E.; Belew, R. K.;
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