Protease inhibitors: Synthesis of bacterial collagenase and matrix metalloproteinase inhibitors incorporating arylsulfonylureido and 5-dibenzo-suberenyl/suberyl moieties

Article abstract of DOI:10.1016/S0968-0896(03)00113-5

Novel matrix metalloproteinase (MMP)/bacterial collagenase inhibitors are reported, considering the sulfonylated amino acid hydroxamates as lead molecules. A series of compounds was prepared by reaction of arylsulfonyl isocyanates with N-(5H-dibenzo[a,d]cyclohepten-5-yl)- and N-(10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5-yl) methyl glycocolate, respectively, followed by the conversion of the COOMe to the carboxylate/hydroxamate moieties. The corresponding derivatives with methylene and ethylene spacers between the polycyclic moiety and the amino acid functionality were also obtained by related synthetic strategies. These new compounds were assayed as inhibitors of MMP-1, MMP-2, MMP-8 and MMP-9, and of the collagenase isolated from Clostridium histolyticum (ChC). Some of the new derivatives reported here proved to be powerful inhibitors of the four MMPs mentioned above and of ChC, with activities in the low nanomolar range for some of the target enzymes, depending on the substitution pattern at the sulfonylureido moiety and on the length of the spacer through which the dibenzosuberenyl/suberyl group is connected with the rest of the molecule. Several of these inhibitors also showed selectivity for the deep pocket enzymes (MMP-2, MMP-8 and MMP-9) over the shallow pocket ones MMP-1 and ChC.

Full text of DOI:10.1016/S0968-0896(03)00113-5

Bioorganic & Medicinal Chemistry 11 (2003) 2227–2239
Protease Inhibitors: Synthesis of Bacterial Collagenase and
Matrix Metalloproteinase Inhibitors Incorporating
Arylsulfonylureido and 5-Dibenzo-suberenyl/suberyl Moieties
Monica Ilies,a,b Mircea D. Banciu,c Andrea Scozzafava,a Marc A. Ilies,b Miron
T. Caproiud and Claudiu T. Supurana,*
a
`
Universita degli Studi, Laboratorio di Chimica Inorganica e Bioinorganica, Via della Lastruccia 3, Rm 188, Polo Scientifico,
50019-Sesto Fiorentino, Firenze, Italy
bUniversity of Agricultural Sciences and Veterinary Medicine, Faculty of Biotechnologies, Department of Chemistry,
B-dul Marasti 59, 71331-Bucharest, Romania
cPolytechnic University, Department of Organic Chemistry, Splaiul Independentei 313, Bucharest, Romania
d‘C.D. Nenitzescu’ Institute of Organic Chemistry, Splaiul Independentei 202B, Bucharest, Romania
Received 1 October 2002; accepted 11 February 2003
Abstract—Novel matrix metalloproteinase (MMP)/bacterial collagenase inhibitors are reported, considering the sulfonylated
amino acid hydroxamates as lead molecules. A series of compounds was prepared by reaction of arylsulfonyl isocyanates
with N-(5H-dibenzo[a,d]cyclohepten-5-yl)- and N-(10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5-yl) methyl glycocolate, respec-
tively, followed by the conversion of the COOMe to the carboxylate/hydroxamate moieties. The corresponding derivatives
with methylene and ethylene spacers between the polycyclic moiety and the amino acid functionality were also obtained
by related synthetic strategies. These new compounds were assayed as inhibitors of MMP-1, MMP-2, MMP-8 and MMP-9, and of
the collagenase isolated from Clostridium histolyticum (ChC). Some of the new derivatives reported here proved to be powerful
inhibitors of the four MMPs mentioned above and of ChC, with activities in the low nanomolar range for some of the target
enzymes, depending on the substitution pattern at the sulfonylureido moiety and on the length of the spacer through which the
dibenzosuberenyl/suberyl group is connected with the rest of the molecule. Several of these inhibitors also showed selectivity for the
deep pocket enzymes (MMP-2, MMP-8 and MMP-9) over the shallow pocket ones MMP-1 and ChC.
# 2003 Elsevier Science Ltd. All rights reserved.
Introduction
species, are responsible for the efficient degradation of
all components of the extracellular matrix (ECM).
ECM turnover is involved in crucial physiological and
physiopathological events, such as embryonic develop-
ment, blastocyst implantation, nerve growth, ovulation,
morphogenesis, angiogenesis, tissue resorption and
remodeling (such as in the case of wound healing), bone
remodeling, apoptosis, cancer invasion and metastasis,
arthritis, atherosclerosis, aneurysm, breakdown of
blood–brain barrier, periodontal disease, skin and cor-
neal ulceration, gastric ulcer, or liver fibrosis in the case
of the vertebrate enzymes mentioned above.1ꢀ7 In bac-
teria, proteases are involved in critical processes such as
colonization and evasion of host immune defenses,
acquisition of nutrients for growth and proliferation,
facilitation of dissemination, or tissue damage during
infection.3
Proteases, such as the matrix metalloproteinases
(MMPs)1,2 or the bacterial proteases (BPs)3 have
recently become interesting targets for the drug design,
in the search of novel types of anticancer, anti-arthritis,
antibacterial or other pharmacological agents useful in
the management of inflammatory processes.1ꢀ5 All these
conditions are generally associated with enhanced
activity of several zinc endopeptidases, of which the
different MMPs actually known (more than 20 such
enzymes were reported for the moment)1ꢀ7 and the large
number of BPs3 isolated in many pathogenic bacterial
*Corresponding author. Tel.: +39-055-457-3005; fax: +39-055-457-
3385; e-mail: claudiu.supuran@unifi.it
0968-0896/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00113-5

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M. Ilies et al. / Bioorg. Med. Chem. 11 (2003) 2227–2239
After the report8 that CGS-27023A 1 acts as a broad
spectrum, potent inhibitor of some MMPs, a large
number of sulfonylated amino acids and their hydrox-
amate derivatives have been investigated in the search of
more potent and selective such agents.9ꢀ15 All such
derivatives target the zinc binding site in the catalytic
domain of the enzyme, and incorporate a strong zinc-
binding function of the hydroxamate (derivatives 1–3)
or carboxylate (derivatives 4–6) type, as well as a scaf-
fold that assures favorable interactions with the primed
and MMP-11 among others), which possess a somehow
0
smaller specificity S1 pocket due to its partial occlusion
by bulkier amino acid residues, such as those in position
193 (MMP-8 numbering), which from Leu in MMP-8
becomes Arg in MMP-1, Tyr in MMP-7, and Gln in
MMP-11.2b,4 The S2 and S3 subsites are also important
for the binding of inhibitors, as well as for the specificity
of such inhibitors towards the different proteases. The
0
0
0
S2 subsite is generally a solvent-exposed cleft with a
0
preference for hydrophobic P2 residues, in both sub-
0
side of the protease active site (generally with the S1 and
0
strates and MMP inhibitors.1ꢀ4 The S3 subsite on the
0
S2 sites).9ꢀ15 The main problem with such protease
other hand is a relatively ill-defined, solvent exposed
region.1ꢀ4
inhibitors is their lack of selectivity towards the different
MMPs, which are generally classified into two main
0
types, depending on their S1 pocket: (i) the deep pocket
enzymes (such as MMP-2, MMP-3, MMP-8, MMP-9
0
and MMP-13), possessing a relatively big S1 pocket,
and (ii) the shallower pocket enzymes (MMP-1, MMP-7
Some progress has been made ultimately in the design of
both bioavailable and more specific MMP/BP
inhibitors.9ꢀ16 A common feature for many of these
compounds, such as the carboxylate/hydroxamate deri-

M. Ilies et al. / Bioorg. Med. Chem. 11 (2003) 2227–2239
2229
vatives 1–8, is the presence of rather bulky scaffolds in
their molecule, which by interaction with the protease
subsites mentioned above, lead to selectivity towards
some of the target enzymes for which these inhibitors
have been designed.9ꢀ16 Although no absolutely selec-
tive MMP/BP inhibitors are available up to now, this
‘bulky scaffold’ strategy seems to be one of the best
approaches reported for the design of pharmacologi-
cally interesting such enzyme inhibitors. Indeed, a closer
look to the X-ray crystal structure of some sulfonylated
MMP inhibitors such as 12 and 413 bound within the
catalytic domain of MMP-8, afford important informa-
tion for the drug design of this type of pharmacological
agents (Fig. 1). Thus, in such adducts, the hydroxamate/
carboxylate moiety of the inhibitor is coordinated to the
Zn(II) ion as mentioned above, being also hydrogen-
bonded to the carboxylate of Glu 219, similarly to other
types of MMP inhibitors (such as for example the suc-
cinyl hydroxamates, which are among the best studied
inhibitors of these enzymes).1ꢀ4 The importance of the
sulfonamide moiety is stressed by the fact that one of
the oxygens belonging to the SO2 moiety of the inhibitor
molecule participates in two hydrogen bonds with the
main chain amide nitrogens of Leu 181 and Ala 182,
whereas the sulfonyl substituent makes extensive
well as the tetrahydroisoquinolin moiety of 4 are fixed
within the S2 pocket of the enzyme.2,13 These multiple
interactions may explain the unexpectedly high affinity
of the sulfonylated hydroxamates/carboxylates for dif-
ferent enzymes belonging to this family, but also the
0
0
0
capability of both the S1 and S2 subsites to accom-
modate rather bulky moieties. Unfortunately, no X-ray
structures of adducts of MMPs with inhibitors posses-
0
sing bulkier P2 moieties (such as for example 3, 5, 7, or
8) are available at this moment in order to allow a more
detailed analysis for the influence of this moiety to the
binding of inhibitors. Still, the fact that such com-
pounds were reported to act as very potent MMP inhi-
bitors proves that the bulky scaffold’ strategy is worth
being developed for the design of novel types of such
protease inhibitors.
In this paper, we report the preparation of a series of
MMP and Clostridium histolyticum collagenase (ChC,
EC 3.4.24.3) inhibitors incorporating arylsulfonylur-
eido-glycine hydroxamate, as well as bulky 5H-diben-
zo[a,d]cyclohepten-5-yl (=5-dibenzosuberenyl) and
10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5-yl
(=5-
dibenzosuberyl) moieties in their molecule. Some of the
new compounds reported here, assayed for inhibition of
purified collagenases (MMP-1 and MMP-8), gelatinases
(MMP-2and MMP-9) and ChC, showed affinities in the
low nanomolar range for some of these enzymes, as well
as a good degree of selectivity towards the two classes of
proteases mentioned above (deep/shallow pocket
enzymes).
0
hydrophobic contacts with the S1 site, as shown sche-
matically in Fig. 1.2,13 The pyridylmethyl moiety of 1, as
Results
Synthesis
Classical synthetic procedures,17ꢀ23 adapted and opti-
mized in our laboratory, afforded the key intermediates
[5 - dibenzosuberenyl/5 - dibenzosuberyl - (alkyl) - methyl
glycocolates] A1, A2, B1, B2, C1, C2 (Schemes 1–4).
The alcohols 9 and 11 were transformed to the corre-
sponding chloroderivatives 10 and 12 and then to the
glycine derivatives A1 and A2 by routine synthetic pro-
cedures outlined in Scheme 1. Compounds shown in
Scheme 2 were obtained by literature procedures, as
Figure 1. Key protein–ligand interactions for the binding of 1 to
MMP-8.
Scheme 1.

2230
M. Ilies et al. / Bioorg. Med. Chem. 11 (2003) 2227–2239
Scheme 2.
Scheme 3.
follows: 5-hydroxy-10,11-dihydro-5H-dibenzo[a,d]cy-
clohepten-5-yl-carboxylic acid 14 was prepared17 from
aldehydes 28–31, by treating them with pyridinium
chlorochromate, as described in Scheme 4.
10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5-one
13;
5H-dibenzo[a,d]cyclohepten-5-yl-carboxylic acid 15 was
generated18 from the key intermediate 14; 10,11-dihydro-
5H-dibenzo[a,d]cyclohepten-5-carbonitrile 16 and 10,11-
dihydro-5H-dibenzo[a,d]cyclohepten-5-yl-carboxylic acid
17 were obtained19 from 10,11-dihydro-5H-dibenzo[a,d]-
cyclohepten-5-yl-chloride 12; 5H-dibenzo[a,d]cyclo-
hepten-5-yl-acetic acid 18 and 10,11-dihydro-5H-
dibenzo[a,d]cyclohepten-5-yl-acetic acid 19 were pre-
pared by classical procedures adapted from ref 20,
starting from 5H-dibenzo[a,d]cyclohepten-5-ol 9. Car-
boxylic acids 15, 17, 18 and 19 were converted into the
corresponding ethyl esters 20, 22, 24, 26 (Scheme 3) and
these esters were reduced to the corresponding alcohols
21, 23, 25, 27, using nonexceptional synthetic proce-
dures, as outlined in Scheme 3. Alcohols 21, 23, 25, 27
were then selectively oxidized21 to the corresponding
The key intermediates A1, A2, B1, B2, C1, C2 were
subsequently derivatized with arylsulfonyl isocyanates
32 to sulfonylureas D1–D30. These esters were then
converted either to the corresponding carboxylic acids
E1–E30 (by acid hydrolysis of the methyl ester func-
tionality) or to the hydroxamic acids F1–F30 by reac-
tion with hydroxylamine sodium methoxide, affording
the new MMP/ChC inhibitors reported here (Scheme
5).14ꢀ16
The synthetic routes used for the preparation of the
inhibitors reported here were generally related to those
applied by MacPherson et al.,8 Hanessian et al.,10d
Almstead’s and Natchus’ groups,10a,c and our
group,14ꢀ16 for obtaining sulfonylated/sulfonylureido
amino acid hydroxamates.

M. Ilies et al. / Bioorg. Med. Chem. 11 (2003) 2227–2239
2231
Scheme 4.
0
MMP and ChC inhibitory activity. Inhibition data
against four MMP-s (MMP-1, MMP-2, MMP-8 and
MMP-9) and type II ChC with the compounds E1–E30
and F1–F30 reported in the present paper are shown in
Table 1.
(but generally not filling) the specificity S1 pocket of the
enzyme.8ꢀ13 It has been proved that the SO2 moiety of
such an inhibitor is involved in several strong hydrogen
bonds with amino acid residues from the active site
cleft, which strongly stabilize the enzyme-inhibitor
adduct (Fig. 1).8ꢀ13 Recently, we have shown that this
moiety may be successfully replaced by an arylsulfony-
lureido group;14ꢀ16 (ii) a bulky hydrophobic moiety
possibly placed in a to the hydroxamic/carboxylic acid
group (which acts as the zinc-binding function), which is
thought to play a role in increasing the selectivity/spe-
cificity towards some of the different MMPs/BPs.8ꢀ13 It
must be mentioned that some strong and selective
MMP/BP inhibitors do not possess in their molecule
this structural element; (iii) an isobutyl, pyridylmethyl
or benzyl moiety substituting the amino nitrogen atom,
Inhibition data against the above mentioned five pro-
teases with some of the new compounds reported here
and the analogous derivatives incorporating benzyl
0
moieties at P2 instead of 5-dibenzosuberenyl/5-diben-
zosuberyl-methyl are shown in Table 2. Such data are
useful for validating the bulky scaffold strategy pro-
posed in this paper.
Discussion
Chemistry
0
which binds within the S2 solvent-exposed pocket of the
enzyme. As mentioned earlier, it has recently been
proved that this moiety may also be much bulkier,
leading in this way to MMP inhibitors with increased
selectivity.9,10,19a This was the reason why we decided to
investigate compounds incorporating 5-dibenzosuber-
enyl/5-dibenzosuberyl- moieties in this position, toge-
ther with their congeners containing a methylene or
ethylene spacer between the amino acid functionality
Sulfonylated amino acid hydroxamates and sulfonyl-
ated amino acids (carboxylates) were recently dis-
covered to act as efficient MMP/ChC inhibitors.8ꢀ16 The
most active compounds from this class of non-peptide
protease inhibitors possessed the following general
structural features: (i) an arylsulfonyl group, occupying

2232
M. Ilies et al. / Bioorg. Med. Chem. 11 (2003) 2227–2239
Scheme 5.
and the polycyclic moiety (it must be mentioned that the
simple 5-dibenzosuberenyl/5-dibenzosuberyl-derivatives
of types 9, 11, 13–15 or 17–19, do not possess MMP/BP
inhibitory properties in the nano-micromolar range—
data not shown). Furthermore, we did not use the sim-
ple sulfonylated amino acids as lead molecules in our
drug design,8ꢀ13 but the structurally related arylsulfo-
nylureido compounds, which, as mentioned above, were
recently shown to possess strong MMP inhibitory
properties.14ꢀ16
approaches were used for preparing these compounds,
which involved the reductive condensation of 5-diben-
zosuberenyl/5 - dibenzosuberyl - methyl/ethyl - carbalde-
hydes 28–31 with methyl glycocolate in the presence of
sodium cyanoborohydride, when the intermediate imi-
nes were converted in situ to the secondary amines B1,
B2, C1, C2 (Scheme 4). Thus, the main problem was to
prepare the four aldehydes mentioned above, 28–31.
For this reason, dibenzosuberone 13 was converted to
the carboxylic acid 15 following literature procedures18
outlined in Scheme 2. The saturated carboxylic acid 17
was on the other hand obtained by a different strategy,
by coupling the chloroderivative 12 mentioned above
with copper(I) cyanide, followed by acidic hydrolysis of
the nitrile 16. Condensation of the unsaturated alcohol
9 with malonic acid led on the other hand to 5-dibenzo-
suberenyl acetic acid 18, which was catalytically (Pd on
charcoal) hydrogenated to the corresponding 5-diben-
zosuberyl derivative 19 (Scheme 2). The carboxylic acids
15, 17–19 were then esterified with ethyl alcohol, and
the corresponding esters 20, 22, 24, 26, reduced with
lithium aluminum hydride to the alcohols 21, 23, 25 and
27, respectively (Scheme 3). Oxidation of the alcohols
21, 23, 25 and 27 with pyridinium chlorochromate
finally afforded the desired aldehydes 28–31 (Scheme 4).
In the last steps, the esters (A,B,C)1,2 were reacted with
The only commercially available compounds possessing
the tricyclic scaffold of interest for us were the alcohols
9 and 11, as well as dibenzosuberone 13. Thus, all the
syntheses were initiated starting from one of them. In
order to obtain the two key intermediates without a
spacer (A1 and A2), the two alcohols mentioned above
(9 and 11) were converted to the corresponding chloro-
derivatives 10 and 12 either with thionyl chloride or
hydrochloric acid in diethyl ether. By reaction with
methyl glycocolate, these chloroderivatives (10 and 12)
afforded the first two key intermediates, A1 and A2,
respectively (Scheme 1). This synthetic strategy did not
work on the other hand for obtaining the remaining
four key intermediates, incorporating a methylene/eth-
ylene spacer, of types B1, B2, C1, C2. Thus, alternative

M. Ilies et al. / Bioorg. Med. Chem. 11 (2003) 2227–2239
2233
arylsulfonyl isocyanates 32 leading to the arysulfony-
lureas D1–D30, which were either hydrolyzed with
lithium hydroxide/methanol to the corresponding car-
boxylic acids E1–E30, or transformed to hydroxamic
acids F1–F30 by reaction with hydroxylamine hydro-
chloride and sodium methoxide in methanol (Scheme 5).
Table 1. Inhibition data of several MMP-s and ChC with the com-
pounds E1–E30, F1–F30 and 1
Compd
n
X
KIa (nM)
MMP-1b MMP-2b MMP-8b MMP-9b ChCc
E1
E2
E3
E4
E5
E6
E7
E8
0
0
0
0
0
1
1
1
1
1
2
H
4-F
4-Cl
4-Me
2-Me
H
nt
195ꢁ22
ꢁ1.8
nt
125ꢁ7
MMP and ChC inhibitory activity. Inhibition data of
Table 1 show that the entire class of sulfonylureido-
glycine hydroxamate/carboxylate derivatives incorpor-
>250
>250
nt
nt
>250
15ꢁ1.7 26ꢁ3
2 ꢁ21940
ꢁ5212
133
ꢁ11
17ꢁ3
2 ꢁ2395
ꢁ12
ꢁ9
21ꢁ1.3 30ꢁ2nt
29ꢁ3
43ꢁ2.4
nt
150ꢁ13
ating
5-dibenzosuberenyl/5-dibenzosuberyl-moieties
13ꢁ0.8 20ꢁ1.4 24ꢁ2.1 35ꢁ3.4
reported here, of types E1–E30 and F1–F30, act as effi-
cient MMP/ChC inhibitors, but important differences
of activity were detected against the diverse enzymes,
for the different substitution patterns of the sulfony-
lurea moiety, as well as for the length of the spacer
between the polycyclic and amino acid part of these
molecules. The following features should be noted
regarding MMP/ChC inhibition data of Table 1: (i) the
hydroxamates F1–F30 were stronger inhibitors as com-
pared to the corresponding carboxylates E1–E30. This
is probably due to the enhanced Zn(II) coordinating
properties of the CONHOH moiety (bidentate binding
to Zn(II)) as compared to the COOH group (generally
monodentate binding to the zinc ion) and was thor-
oughly documented by our previous work, as well as by
contributions of other groups.8ꢀ16 It must be noted
anyhow that the differences between the hydroxamates
and the corresponding carboxylates are generally not so
high for this class of MMP/ChC inhibitors as compared
to other inhibitors reported in the literature, and this is
a rather positive aspect of derivatives reported here,
since carboxylates generally possess a reduced toxicity
as compared to the hydroxamates;8ꢀ16 (ii) the com-
pounds incorporating 5-dibenzosuberenyl- moieties
were without exception more active than the corre-
sponding derivatives incorporating 5-dibenzosuberyl-
groups; (iii) the spacer connecting the polycyclic moiety
to the amino acid part of the molecule of both carboxy-
late as well as hydroxamate protease inhibitors reported
here was the most important parameter influencing
biologic activity. Thus, the most active compounds were
those containing a methylene spacer (n=1) between the
two structural elements mentioned above, followed by
the corresponding derivatives with an ethylene spacer
(n=2), which in turn were more inhibitory than the
compounds without a spacer (n=0). This is probably
4-F 180ꢁ9
4-Cl 230ꢁ15 12ꢁ1.7 14ꢁ218
4-Me >250
2-Me nt
9ꢁ1
13ꢁ1.6 15ꢁ1.7 21ꢁ2
ꢁ1.1 25ꢁ1.9
E9
15ꢁ1.3 27ꢁ2.3 26ꢁ1.8 38ꢁ3.1
E10
E11
E12
E13
E14
E15
E16
E17
E18
E19
E20
E21
E22
E23
E24
E25
E26
E27
E28
E29
E30
F1
24ꢁ1.7
nt nt
40ꢁ3.9
ꢁ7.6
H
nt
>250
ꢁ108.8 24ꢁ1.9 30ꢁ286
24-F
13ꢁ1
2 ꢁ2.01 21ꢁ1.5 33ꢁ3.4
ꢁ1.6 34ꢁ2.9 26ꢁ1.8
24-Cl 190 ꢁ14 172ꢁ22
24-Me
>250
nt
nt
>250
>250
nt
24ꢁ231
ꢁ1.8
nt
45
nt
40ꢁ4
ꢁ3
2
0
0
0
0
0
1
1
1
1
1
2
2-Me
H
4-F
4-Cl
4-Me
2-Me
H
25ꢁ1.6 37ꢁ2nt
29ꢁ231 ꢁ1.6
24ꢁ1.9 30ꢁ234
nt
ꢁ2.1 163ꢁ13
28ꢁ2.1 35ꢁ1.6 35ꢁ1.9 170ꢁ15
33ꢁ2.9
38ꢁ1.8
nt
nt
nt
nt
165ꢁ14
189ꢁ20
nt
nt
nt
25ꢁ2.6 27ꢁ1.8 38ꢁ3
4-F 200ꢁ18 19ꢁ1.5
nt
35
nt
nt
nt
60ꢁ5
4-Cl
4-Me >250
2-Me nt
nt
21ꢁ2nt
25ꢁ3.1
28ꢁ1.7
ꢁ2.6
nt
nt
42ꢁ5
40ꢁ3.7 43ꢁ2.5
H
nt
>250
nt
nt
nt
ꢁ22.4 7 28ꢁ3
30ꢁ2.5
nt
24-F
21ꢁ1.3
nt
30ꢁ1.9 50ꢁ4
24-Cl
24-Me
2-2Me
0
0
0
0
0
1
1
1
1
1
42
ꢁ1.8 25ꢁ237
ꢁ3.3
nt
62 ꢁ2.3 33ꢁ1.6
nt
55ꢁ4.9
61ꢁ5
nt
33
ꢁ
nt
H
4-F
2 ꢁ52.4
9ꢁ0.6 14ꢁ1.212 ꢁ0.9 16ꢁ0.9
F2
F3
F4
F5
F6
F7
F8
F9
21ꢁ1.6
5ꢁ0.213 ꢁ1.4
9ꢁ1.1 13ꢁ0.6
4-Cl 24ꢁ0.7
7ꢁ0.3 15ꢁ1.211 ꢁ0.8 14ꢁ0.4
4-Me 32ꢁ2.6 15ꢁ1.1 16ꢁ1.8 14ꢁ0.9 18ꢁ1.1
2-Me 36ꢁ2.5 15ꢁ0.8 18ꢁ2.1 18ꢁ2.5 21ꢁ1.3
H
4-F
13ꢁ0.7 1.9ꢁ1.1 2.4ꢁ0.9 3.0ꢁ0.4
11ꢁ1.0 1.5ꢁ0.6 2.0ꢁ0.1 2.3ꢁ0.3
8ꢁ0.5
6ꢁ0.4
7ꢁ0.3
4-Cl 14ꢁ2.1 1.7ꢁ0..232
ꢁ0..282
ꢁ0.4
4-Me 15ꢁ1.3 1.9ꢁ0.4 3.1ꢁ0.5 3.0ꢁ0.3 12ꢁ0.8
2-Me 15ꢁ0.8 1.9ꢁ0.1 3.3ꢁ0.3 3.6ꢁ0.4 15ꢁ1
F10
F11
F12
F13
F14
F15
F16
F17
F18
F19
F20
F21
F22
F23
F24
F25
F26
F27
F28
F29
F30
1
2
H
nt
17 ꢁ23.4
ꢁ1.6
24-Me 42 ꢁ1.6
ꢁ70.5
ꢁ0.9
nt
nt
nt
nt
nt
nt
nt
nt
13ꢁ0.6
10ꢁ0.8
nt
24-F
24-Cl 02
5ꢁ0.4
8ꢁ0.7
9ꢁ1.0
12ꢁ1.5 15ꢁ0.7
2
0
0
0
0
0
1
1
1
1
1
2
2-Me 29ꢁ2.5
13ꢁ1.5
ꢁ1.3
nt
nt
H
4-F
nt
25ꢁ1.5
nt
13ꢁ1.4 17ꢁ216
6ꢁ0.3
nt
14ꢁ1.6 18ꢁ2
4-Cl
4-Me
7ꢁ0.6 19ꢁ1.6 15ꢁ1.4 21ꢁ1.5
nt
180ꢁ22
ꢁ1.5
nt
nt
nt
28ꢁ3
2-Me 39ꢁ3.3 21ꢁ1.7 24ꢁ2.1
0
due to the steric hindrance within the S2 pocket of the
H
4-F
4-Cl
15ꢁ0.6 2.6ꢁ0.3 2.4ꢁ0.5 3.8ꢁ0.3 10ꢁ1
enzyme, where presumably this moiety binds. Probably
the best binding is assured when the bulky tricyclic
moiety is connected to the amino acid part of the mol-
ecule by means of a CH2 linker (or a CH2–CH2 one),
whereas the absence of such a linker increases the steric
bulk and destabilizes the enzyme-inhibitor adduct (it
13ꢁ1.2.22
ꢁ0.4 2.7ꢁ0.6 2.9ꢁ0.3
2.7ꢁ0.3 nt nt
7ꢁ0.5
9ꢁ0.6
nt
4-Me 18ꢁ
3.5ꢁ0.4 3.4ꢁ0.24.0 ꢁ0.3 19ꢁ2
2-Me nt
H
3.9ꢁ0.5 3.3ꢁ0.3
nt
nt
nt
nt
15ꢁ1
nt
nt
ꢁ91
nt
nt
15ꢁ0.9
13ꢁ1
nt
24-F 12
24-Cl 42
ꢁ1.8
5ꢁ0.6
7ꢁ0.3
ꢁ2.5
9ꢁ0.5
24-Me 30 ꢁ2.8 11ꢁ1.4
nt
18ꢁ0.8
0
should be remembered that the S2 is a relatively shallow
2-2Me 38
—
ꢁ3
13ꢁ2nt
nt0 2
ꢁ1.7
and solvent-exposed pocket);1,4 (iv) the substitution
pattern of the arylsulfonylureido part of the molecule
also drastically influenced the MMP/ChC inhibitory
properties of these derivatives. Inhibition power gen-
erally decreased in the following order for the X-Ar-
33ꢁ1.5 20ꢁ1.210 ꢁ1
9ꢁ0.6 41ꢁ3.5
aKIs values were obtained from Easson–Stedman27 plots using a linear
regression program, from at least three different assays. Standard
errors were of 5–10%; nt, not tested due to the limited amount of
inhibitor available.
bWith the thioester substrate Ac-ProLeuGly-S-LeuLeuGlyOEt, spec-
trophotometrically.25,26
cWith FALGPA as substrate, spectrophotometrically.28
SO2NHCONH substituted congeners: 4-F>4-Cl
H>4-Me >2-Me-substituted derivatives; (v) the four
ffi
MMPs and the bacterial collagenase investigated here

2234
M. Ilies et al. / Bioorg. Med. Chem. 11 (2003) 2227–2239
Table 2. Inhibition data of several MMP-s and ChC with some of the new compounds reported here (F7–F10) and the analogous derivatives
containing benzyl moieties at P20, of type 33a–33d
Compd
X
KIa (nM)
MMP-1b
MMP-2b
MMP-8b
MMP-9b
ChCc
33a
33b
33c
33d
F7
F8
F9
F10
4-F
4-Cl
4-Me
2-Me
4-F
4-Cl
4-Me
2-Me
135
143
170
162
11
14
15
15
9
10
19
18
1.5
1.7
1.9
1.9
13
15
20
25
2.0
2.3
3.1
3.3
1
122
15
21
24
2.3
2.8
3.0
3.6
15
16
17
6
7
12
15
aKIs values were obtained from Easson–Stedman27 plots using a linear regression program, from at least three different assays. Standard errors were
of 5–10%. Data for 33a–33d are from ref 15a.
bWith the thioester substrate Ac-ProLeuGly-S-LeuLeuGlyOEt, spectrophotometrically.25,26
cWith FALGPA as substrate, spectrophotometrically.28
possessed quite different affinities to this class of inhibi-
tors. Thus, carboxylates E1–E30 were rather ineffective
MMP-1 inhibitors and medium potency ChC inhibitors,
whereas they possessed nanomolar affinities against
MMP-2, MMP-8 and MMP-9 (all these three MMPs
are deep pocket enzymes). The order of affinity of such
inhibitors for the five proteases investigated was MMP-
2>MMP-8 ffi MMP-9>ChC > >MMP-1. The same
order of affinity was also shown by the hydroxamate
inhibitors F1–F30, but the inhibition constants, KIs,
were much lower in this case. Thus, affinity for MMP-1
was in the range of 11–40 nM, that for MMP-2in the
range of 1.5–21 nM, for MMP-8 and MMP-9 in the
range of 2–24 nM, whereas for ChC in the range of 6–
28 nM. It may be seen that these inhibitors generally
discriminate between the deep pocket and shallow
pocket enzymes, with ChC showing a somehow inter-
mediate behaviour.
33a–33d. This applies to all five proteases investigated
here and represents the required proof-of-concept of
this bulky scaffold strategy.
Conclusion
We describe here a novel class of strong inhibitors of the
zinc proteases MMP-1, MMP-2, MMP-8, MMP-9 and
ChC (EC 3.4.24.3), a collagenase from C. histolyticum.
The drug design has been done considering the ‘bulky
scaffold’ strategy, which involved the introduction of
bulky, hydrophobic 5-dibenzosuberenyl and 5-dibenzo-
suberyl moieties, in the molecule of arylsulfonylureido
amino acid derivatives. Both carboxylate and hydrox-
amate inhibitors were obtained in this way, with differ-
ent spacers (i.e., no spacer, methylene- and ethylene
spacers, respectively) between the tricyclic and amino
acid part of the molecule. The key intermediates incor-
porating the 5-dibenzosuberenyl and 5-dibenzosuberyl
moieties, were obtained by nonexceptional but rather
laborious synthetic procedures, and were then converted
into arylsulfonylureido-glycine hydroxamates/carbox-
ylates. These last compounds were assayed for their
MMP and ChC inhibitory activity. Some of the new
derivatives reported here proved to be powerful inhibi-
tors of four MMPs with activities in the low nanomo-
lar range for some of the target enzymes. Some of them
also showed selectivity for the deep pocket enzymes
(MMP-2, MMP-8 and MMP-9) over MMP-1 and
ChC.
In order to better evaluate the validity of the bulky
scaffold strategy proposed here for obtaining MMP/BP
inhibitors, we compared the inhibition data of some of
the new compounds described here (such as, e.g., F7–
F10) with the analogous derivatives incorporating a
benzyl moiety instead of the 5-dibenzosuberenyl/
5-dibenzosuberyl-methyl ones, of type 33a–33b, pre-
viously reported by us15a (Table 2). Data of Table 2
unequivocally show the compounds incorporating
0
bulkier P2 moieties, of type F7–F10, to act as much
stronger MMP/BP inhibitors than the corresponding
0
derivatives incorporating benzyl P2 groups, of types

M. Ilies et al. / Bioorg. Med. Chem. 11 (2003) 2227–2239
2235
Experimental
at a molar ratio of carboxylic acid–alcohol of 1:20, in
the presence of one drop of 98% H2SO4 as catalyst. The
obtained homogenous solution was then refluxed under
anhydrous conditions for 10–24 h (reaction monitored
by TLC). After all the acid was converted to the corres-
ponding ester, the mixture was concentrated to half the
volume by evaporating the solvent in vacuo, when an
abundant white precipitate was obtained. Stirring of
this crude product with 35 mL of water for 30 min, at
room temperature, followed by vacuum filtration,
washing with water until a pH of 5.5 was reached, and
recrystallized from methanol, afforded the pure title com-
pounds.17 Overall yields were in the range of 70–80%.
General
Melting points: heating plate microscope (not cor-
rected); IR spectra: KBr pellets, 400–4000 cmꢀ1 Nicolet
Avatar 360 FTIR spectrometer; NMR spectra: Varian
Gemini 300BB apparatus (chemical shifts are expressed
as d values relative to Me4Si as internal standard for
proton spectra and to the solvent resonance for carbon
spectra). Elemental analysis (ꢁ0.4% of the theoretical
values, calculated for the proposed formulas for all the
compounds reported here): Carlo Erba Instrument
CHNS Elemental Analyzer, Model 1106. All reactions
were monitored by thin-layer chromatography (TLC),
using 0.25 mm-thick precoated silica gel plates (E.
Merck) eluted with MeOH–CHCl3 1:4 v/v. Preparative
HPLC was performed on a Dynamax-60A column (25 ꢂ
250 mm), with a Beckman EM-1760 instrument. The
detection wavelength was 254 nm. 5H-Dibenzo[a,d]cyclo-
General procedure for the preparation of compounds 21,
23, 25, 27
A solution was prepared by dissolving 43 mmol of ester
obtained as described above (compound 20, 22, 24, 26,
respectively) in 220 mL anhydrous diethylether. Sepa-
rately, 52mmol (a 20% excess relative to the stoichio-
metric amount) of LiAlH4 were suspended in 60 mL
anhydrous diethylether, under argon. The etheric solu-
tion of the ester was then slowly added to it, drop by
drop, during a period of 2h, under the argon atmo-
sphere and strictly anhydrous conditions. After this
time, the obtained reaction mixture was refluxed for 8–
14 h (TLC control), then cooled to 0 ꢃC, and quenched
with 15% aqueous solution of H2SO4 added dropwise,
when two distinct layers were obtained. The etheric
layer was separated, and the aqueous one extracted
twice with 75 mL of diethylether. The combined organic
layers were washed with water till the neutral pH, and
dried over anhydrous CaCl2. The solvent was evapo-
rated in vacuo yielding a white oil, which cristallyzed by
standing. Recrystallization from hexane afforded the
pure title compounds, as white crystals, with overall
yields between 70 and 80%.17,18
hepten-5-ol
9,
10,11-dihydro-5H-dibenzo[a,d]cyclo-
hepten-5-ol 11, 5H-dibenzo[a,d]cyclohepten-5-one 13,
methyl glycocolate hydrochloride, arylsulfonyl iso-
cyanates 32, trietylamine, carbodiimides, hydroxylamine,
sodium methoxide, 5,50-dithiobis-(2-nitrobenzoic acid),
FALGPA, buffers and other reagents used in the synthesis
were commercially available compounds, from Sigma,
Acros or Aldrich (Milan, Italy). The thioester MMP sub-
strate, AcProLeuGly-S-LeuLeuGlyOEt was from
Bachem. Acetonitrile, methanol, dioxane, ethyl acetate (E.
Merck, Darmstadt, Germany) or other solvents used in the
synthesis were double distilled and kept on molecular
sieves in order to maintain them in anhydrous conditions.
Preparation of 5H-dibenzo[a,d]cyclohepten-5-yl-chloride,
10. One gram (4.8 mmol) of 5H-dibenzo[a,d]cyclo-
hepten-5-ol 9 was dissolved in 20 mL of anhydrous
benzene, under magnetical stirring, at room tempera-
ture. To the obtained solution were added dropwise a
mixture of 3 mL of anhydrous benzene, 1.5 mL of thio-
nyl chloride and one drop of pyridine. The reaction
mixture was stirred for 10 min at room temperature,
then another 10 min at 70 ꢃC (TLC control). The solvent
was evaporated in vacuo and the resulted crude product
was washed with 30 mL petroleum ether and recrys-
tallized from benzene. Yield was around 75%.
General procedure for the preparation of compounds 28–
31 (adapted from literature procedure)21
Pyridinium chlorochromate (4.5 mmol) was suspended
in about 6 mL of methylene chloride, then the corre-
sponding alcohol (21, 23, 25, 27, respectively), dissolved
in the same solvent, was rapidly added under good stir-
ring, working at room temperature, and afforded to
react for 2–3 h (TLC control). When the reaction was
completed, the resulted dark mixture was diluted with
80 mL of anhydrous diethylether. The organic phase
was decanted whereas the inorganic solid (the reduced
reagent) was washed three times with anhydrous ether.
The pooled organic fractions containing the ether solu-
tion of the aldehyde was evaporated in vacuum and the
resulted crude product was used directly for the reduc-
tive coupling with methylglycocolate/sodium boro-
hydride, as described later in the text.
Preparation of 10,11-dihydro-5H-dibenzo[a,d]cyclohep-
ten-5-chloride, 12 (optimized from the literature proce-
dure).22 An amount of 3 g (14.2mmol) of 10,11-
dihydro-5H-dibenzo[a,d]cyclohepten-5-ol 11 was dis-
solved in 40 mL of anhydrous diethylether, with energic
(magnetic) stirring, at room temperature. HCl gas was
then bubbled into the reaction mixture for 30 min, when
12 was obtained in good yield (TLC control). The sol-
vent was evaporated in vacuo and the resulted crude
product was recrystallized from petroleum ether. A
white needle-shaped crystalline solid was obtained with
an yield around 60%.
General procedure for the preparation of compounds A1–
A2
General procedure for the preparation of compounds 20,
22, 24, 26. An amount of 48 mmol of carboxylic acid
15, 17, 18, or 19, respectively, was treated under stirring
at room temperature with anhydrous ethanol, working
Chloroderivative (4.8 mmol) 10 and 12 respectively, and
the stoichiometric amount of methyl glycocolate hydro-

2236
M. Ilies et al. / Bioorg. Med. Chem. 11 (2003) 2227–2239
chloride were suspended in 10 mL of anhydrous aceto-
nitrile, with magnetical stirring. Triethylamine
(9.6 mmol) was added and the obtained mixture was
refluxed for 24–48 h, until a reasonable conversion was
reached (TLC control). The solvent was evaporated in
vacuum, and the obtained precipitate was extracted
with a mixture of 15 mL CH2Cl2–10 mL water. The
separated aqueous layer was reextracted twice with
10 mL of CH2Cl2. The combined organic layers
(25 mL) were treated with 15 mL of saturated aqueous
NaHCO3 solution, then with water until a pH=7 was
reached. The organic layer was dried over anhydrous
magnesium sulfate, filtered, and the solvent evaporated
in vacuo. In order to eliminate traces of the alcohol
used as starting material, the resulted crude solid
product was washed with 50–60 mL of anhydrous
diethylether, then converted into the corresponding
ester hydrochloride by bubbling dried gaseous HCl
(generated in situ from concentrate H2SO4 and solid
KCl). The obtained precipitate was separated by fil-
tration under argon, washed with ether and retaken in
the minimum volume of anhydrous MeOH. For gen-
erating the free amine, the methanolic solution was
treated with solid NaHCO3 and heated at reflux for
15–30 min. The inorganic precipitate was discarded
and the solvent was evaporated in vacuum. The
obtained crude solid was purified by column chroma-
tography (adsorbent: silicagel—60 A, 40–60 mm,
Merck; eluent: CH2Cl2–MeOH 4:1 v/v). Recrystalliza-
tion from absolute ethanol afforded the pure com-
pounds A1, A2. Overall yields were in the range of
60–70%.
General procedure for the preparation of compounds
D1–D30
An amount of 10 mmol of key intermediate A1, A2, B1,
B2, and C1, C2, respectively, and the stoichiometric
amount of arylsulfonyl isocyanate 32 were suspended in
50 mL of anhydrous acetone, and 150 mL (10 mmol) of
triethylamine were added. The reaction mixture was
stirred at room temperature for 2–6 h (TLC control).
The solvent was evaporated in vacuo, and the crude
product was taken up in ethyl acetate (15 mL), poured
into a 5% solution of NaHCO3 (25 mL), and extracted
with ethyl acetate. The combined organic layers were
dried over anhydrous sodium sulfate, filtered, and the
solvent was removed in vacuo. The obtained crude pro-
ducts were recrystallized from ethanol. Yields were
quantitative.
General procedure for the preparation of compounds E1–
E30
An amount of 5 mmol of methyl ester D1–D30 was dis-
solved in water–methanol–THF (1:1:1, v/v/v) and excess
lithium hydroxide (200 mg) was added. The resulting
mixture was stirred overnight at room temperature. The
reaction was acidified with 1 N HCl and the mixture
extracted with methylene chloride. The organic extracts
were dried over sodium sulfate and then concentrated to
an oil under reduced pressure. Preparative HPLC
(Dynamax-60A column (25 ꢂ 250 mm); 90% acetoni-
trile/10% water; flow rate of 30 mL/min) afforded the
pure carboxylic acids E1–E30.
General procedure for the preparation of compounds B1,
B2, C1, C2 (adapted from literature procedure)23
General procedure for the preparation of compounds F1–
F30
A molar ratio of aldehyde–methyl glycocolate hydro-
chloride–NaBH3CN of 1:3:0.6 was used in the reductive
coupling. An amount of 4.27 mmol aldehyde was trea-
ted with 12.81 mM methyl glycocolate hydrochloride,
then a solution of sodium methoxide required for the
neutralization of the acid was added at room tempera-
ture to the reaction mixture, under magnetical stirring.
A 5% excess relative to the stoichiometric amount of
solid anhydrous Na2SO4 was also introduced in this
reaction mixture, for absorbing the water resulted in
reaction. 2.56 mmol of NaBH3CN were then added
into the reaction mixture, and stirring was continued
for 48 h at room temperature (TLC control). The sol-
vent was evaporated in vacuo and 25 mL of water were
added, then a quench was performed by treating the
resulted mixture with concentrated HCl until the pH
was brought to 1–1.5. Solid NaOH was then added till
pH >8.5 was reached. The resulted mixture was
extracted with methylene chloride, for three times,
each time with a volume of 25 mL. The combined
organic layers were treated with 10 mL of aqueous
NaCl solution, dried over anhydrous sodium sulfate,
filtered, and the solvent evaporated in vacuo. The
resulted crude solid was purified following the proce-
dure described above for obtaining the pure com-
pounds A1, A2. Overall yields were in the range of
60–80%.
An amount of 5 mmol of ester D1–D30 was dissolved in
20 mL of methanol. To this solution hydroxylamine
hydrochloride (0.76 g, 11 mmol) was added, followed by
the addition of sodium methoxide (15 mmol) freshly
obtained from sodium (350 mg) dissolved in methanol
(10 mL). The reaction mixture was stirred overnight at
room temperature, and was then worked up by parti-
tioning between dilute hydrochloric acid (pH=3) and
ethyl acetate. The aqueous phase was extracted with
ethyl acetate, the combined organic layers dried
(Na2SO4) and the solvent evaporated. The products
were then purified by HPLC (Dynamax-60A column (25
ꢂ 250 mm); 90% acetonitrile/ 10% methanol; flow rate
of 30 mL/min). Yields were in the range of 65–82%.
1
All the new compounds were characterized by IR, H
and 13C NMR spectroscopy as well as elemental analy-
sis. Representative data are provided below.
N-(5H-Dibenzo[a,d]cyclohepten-5-yl)methyl glycocolate A1.
White crystals, mp 64–66 ꢃC; IR (nujol), cmꢀ1: 894; 972;
1
1070; 1243; 1560; 1673; 3200 br; H NMR (CDCl3, d,
ppm, J, Hz): 3.12(s, 2H, N–CH 2); 3.67 (s, 3H, OMe);
4.87 (s, 1H, H-5); 6.95 (s, 2H, H-10-11); 7.23 (m, 8H, H-
arom); 13C NMR (CDCl3, d ppm): 172.29 (CO); 138.69
(C-q); 133.57 (C-q); 130.86 (CH-10-11); 130.54 (CH-
arom); 130.46 (CH-arom); 130.22 (CH-arom); 130.07

M. Ilies et al. / Bioorg. Med. Chem. 11 (2003) 2227–2239
2237
(CH-arom); 68.88 (C-5); 51.57 (OMe); 48.30 (N–CH2).
Anal., found: C, 77.36; H, 6.16; N, 4.95. C18H17NO2
requires: C, 77.42; H, 6.09; N, 5.02.
the tricyclic ring, 7.4); 3.07 and 3.31 (m, 4H, H-10-11,
syst. A2B2); 3.35 (s, 2H, N–CH2); 3.70 (s, 3H, OMe);
4.74 (t, 1H, H-5, 7.4); 5.65 (bs, 1H, NH, deuterable);
7.16 (m, 8H, H-arom); 13C NMR (CDCl3, d, ppm):
32.90 (C-a to the tricyclic ring); 33.03 (C-10-11); 47.74
(C-b to the tricyclic ring); 49.73 (N–CH2); 51.78 (OMe);
51.82 (C-5); 125.98 (CH-arom); 126.51 (CH-arom); 129.77
(CH-arom); 130.09 (CH-arom); 139.24 (C-q); 141.05 (C-
q); 171.81 (CO). Anal., found: C, 77.52; H, 7.62; N, 4.43.
C20H23NO2 requires: C, 77.67; H, 7.44; N, 4.53.
N-(10,11-Dihydro-5H-dibenzo[a,d]cyclohepten-5-yl)me-
thyl glycocolate A2. White crystals, mp 85–87 ꢃC; IR
(nujol), cmꢀ1: 867; 954; 1082; 1255; 1560; 1674; 3200 br;
1H NMR 2.88 (m, 4H, H-10-11, syst. A2B2); 3.28 (s, N–
CH2); 3.70 (s, 3H, OMe); 4.78 (s, 1H, H-5); 7.18 (m, 8H,
H-1-4 and 6-9); 13C NMR (CDCl3, d, ppm): 32.51 (C-
10-11) 48.72(N-CH ); 51.77 (OMe); 68.93 (C-5); 125.97
2
(CH-arom); 127.81 (CH-arom); 129.92 (CH-arom);
130.55 (CH-arom); 139.22 (C-q); 140.55 (C-q); 173.24
(CO). Anal., found: C, 76.79; H, 6.71; N, 4.88.
C18H19NO2 requires: C, 76.87; H, 6.76; N, 4.98.
N-4-Fluorophenylsulfonylureido-N-(5H-dibenzo[a,d]cy-
clohepten-5-yl)-glycine E2. Colorless crystals, mp 136–
137 ꢃC; IR (KBr), cmꢀ1: 1153 (SOs2ym), 1287 (amide III),
1379 (SOa2s), 1584 (amide II), 1712(amide I), 1755
(COOH); 3060 and 3300 (NH, OH); 1H NMR (DMSO-
d6, d, ppm, J, Hz): 3.62(s, 2H, C H2), 5.73 (s, 1H, HC5),
7.17 (s, 2H, H10,11), 7.55 (m, 8H, HPh), 7.69 (d, 2H,
N - [(5H - Dibenzo[a,d]cyclohepten - 5 - yl)methylen]methyl
glycocolate B1. Amorphous colorless solid; IR (nujol),
cmꢀ1: 895; 966; 1059; 1248; 1560; 1671; 3200 br; 1H
NMR (CDCl3, d, ppm, J, Hz): 2.99 (d, 2H, H-51, 7.9);
3.30 (s, 2H, N–CH2); 3.63 (s, 3H, OMe); 4.32(t, 1H, H-
5, 7.9); 6.88 (s, 2H, H-10-11); 7.22 (m, 8H, H-arom); 13C
NMR (CDCl3, d, ppm): 47.53(CH2NHCH2); 49.31 (N–
CH2); 51.87 (OMe); 54.04 (C-5); 126.98 (CH-arom);
129.89 (CH-arom); 128.99 (CH-arom); 130.21 (CH-
arom); 130.72(CH-10-11); 134.16 (C-q); 138.03 (C-q);
171.46 (CO). Anal., found: C, 77.75; H, 6.39; N, 4.81.
C19H19NO2 requires: C, 77.81; H, 6.48; N, 4.78.
3
Hortho of 4-FC6H4), 7.95 (d, JHH=8.1, 2H, Hmeta of 4-
FC6H4); 8.12(bs, H2, N HCONH); 11.47 (bs, 1H,
COOH); 13C NMR (DMSO-d6), d, ppm: 45.39 (CH2),
68.54 (CH), 128.73 (enlarged), 129.61 (Cq), 130.03 (Ph),
130.10 (Cmeta of FC6H4), 130.26 (Ph), 130.45 (Ph),
130.58 (Ph), 130.85 (Ph), 132.63 (NHCONH), 133.40
(Cq), 135.13 (Cortho of FC6H4), 148.92( Cipso of FC6H4),
149.82( Cpara of FC6H4), 170.31 (COOH). Anal.
(C24H19FN2SO5) C, H, N.
N-4-Toluenesulfonylureido-N-[(5H-dibenzo[a,d]cyclohep-
ten-5-yl)methylen]glycine E9. White crystals, mp 133–
135 ꢃC; IR (KBr), cmꢀ1: 1164 (SOs2ym), 1288 (amide III),
1377 (SOa2s), 1580 (amide II), 1715 (amide I), 1751
(COOH); 3060 and 3300 (NH, OH); 1H NMR (DMSO-
d6), d, ppm: 2.51 (s, 3H, CH3C6H4), 2.93 (d, 2H,
3JHH=7.9, CH–CH2), 3.26 (s, 2H, CH2COO), 4.23 (t,
N-[(10,11-Dihydro-(5H-dibenzo[a,d]cyclohepten-5-yl)me-
thylene]methyl glycocolate B2. Amorphous colorless
solid; IR (nujol), cmꢀ1: 872; 969; 1081; 1267; 1563; 1667;
1
3200 br; H NMR (CDCl3, d, ppm, J, Hz): 3.03 and
3.35 (m, 4H, H-10-11, syst. A2B2); 3.41 (s, 2H, N–CH2);
3.67(d, 2H, H-51, 7.9); 3.68 (s, 3H, OMe); 4.26 (t, 1H,
H-5, 7.9); 7.17 (m, 8H, H-arom); 13C NMR (CDCl3, d,
ppm): 33.29 (C-10-11); 50.53 (CH2–N); 50.53 (N–CH2);
51.71 (OMe); 55.13 (C-5); 126.18 (CH-arom); 126.90
(CH-arom); 130.38 (CH-arom); 130.41 (CH-arom);
139.21 (C-q); 139.85 (C-q); 172.55 (CO). Anal., found:
C, 77.21; H, 7.19; N, 4.69. C19H21NO2 requires: C,
77.29; H, 7.12; N, 4.74.
3
1H, JHH=7.9, HC5), 6.87 (s, 2H, H10,11), 7.30 (m, 8H,
3
HPh)7.68 (d, JHH=8.2, 2H, Hortho of CH3C6H4), 7.99
3
(d, JHH=8.2, 2H, Hmeta of CH3C6H4); 8.25 (bs, 2H,
NHCONH); 11.73 (bs, 1H, COOH); 13C NMR
(DMSO-d6), d, ppm: 26.1 (CH3C6H4), 45.61
(CH2COO), 50.49 (CHCH2), 55.10 (CH), 126.77,
128.49, 129.83, 130.11, 130.63 (all from Ph), 130.54
(Cmeta of CH3C6H4), 132.71 (NHCONH), 134.17 and
138.70 (Cq), 135.62( Cortho of CH3C6H4), 145.80 (Cipso
of CH3C6H4), 148.14 (Cpara of CH3C6H4), 175.91
(COOH). Anal. (C26H24N2SO5) C, H, N.
N - [(5H - Dibenzo[a,d]cyclohepten - 5 - yl)ethylene]methyl
glycocolate C1. Amorphous colorless solid; IR (nujol),
cmꢀ1: 874; 975; 1049; 1241; 1574; 1668; 3200 br; 1H
NMR (CDCl3, d, ppm, J, Hz): 1.91 (q, 2H, CH2-a to
the tricyclic ring, 7.3); 2.26 (t, 2H, CH2-b to the tricyclic
ring, 7.3); 3.24 (s, 2H, N–CH2); 3.65 (s, 3H, OMe); 4.09
(t, 1H, H-5, 7.3); 6.87 (s, 2H, H-10-11); 7.21 (m, 8H, H-
arom); 13C NMR (CDCl3, d, ppm): 30.11 (C-a to the
tricyclic ring); 47.62(C- b to the tricyclic ring); 50.55 (N–
CH2); 51.56 (OMe); 52.28 (C-5); 126.25 (CH-arom);
128.58 (CH-arom); 129.43 (CH-arom); 129.74 (CH-
arom); 130.77 (CH-10-11); 133.94 (C-q); 140.73 (C-q);
172.76 (CO). Anal., found: C, 78.08; H, 6.79; N, 4.51;
C20H21NO2 requires: C, 78.17; H, 6.84; N, 4.56.
N-4-Chlorophenylsulfonylureido-N-(5H-dibenzo[a,d]cy-
clohepten-5-yl)-glycine hydroxamate F3. Colorless crys-
tals, mp 157–158 ꢃC; IR (KBr), cmꢀ1: 1162(SO sym),
2
1281 (amide III), 1385 (SOa2s), 1574 (amide II), 1715
1
(amide I), 1757 (COOH); 3060 and 3300 (NH, OH); H
NMR (DMSO-d6, d, ppm, J, Hz): 3.60 (s, 2H, CH2),
5.77 (s, 1H, HC5), 7.19 (s, 2H, H10,11), 7.55 (m, 8H,
HPh), 7.66 (d, 2H, Hortho of 4-ClC6H4), 7.94 (d,
3JHH=8.1, 2H, Hmeta of 4-ClC6H4); 8.15 (bs, 2H,
NHCONH); 11.41 (bs, 1H, COOH); 13C NMR (DMSO-
d6), d, ppm: 45.2 6 C( H2), 68.37 (CH), 128.79 (enlarged),
129.54 (Cq), 130.12(Ph), 130.15 ( Cmeta of ClC6H4), 130.26
(Ph), 130.44 (Ph), 130.61 (Ph), 130.82(Ph), 13.269
(NHCONH), 133.74 (Cq), 135.10 (Cortho of ClC6H4),
149.14 (Cipso of ClC6H4), 149.87 (Cpara of ClC6H4), 170.20
(COOH). Anal. (C24H20ClN3SO5) C, H, N.
N-[(10,11-Dihydro-(5H-dibenzo[a,d]cyclohepten-5-yl)e-
thylene]methyl glycocolate C2. Amorphous colorless
solid; IR (nujol), cmꢀ1: 863; 975; 1078; 1267; 1565; 1671;
1
3200 br; H NMR (CDCl3, d, ppm, J, Hz): 2.28 (q, 2H,
CH2-a to the tricyclic ring, 7.4); 2.56 (t, 2H, CH2-b to

2238
M. Ilies et al. / Bioorg. Med. Chem. 11 (2003) 2227–2239
N-4-Toluenesulfonylureido-N-[(10,11-dihydro-(5H-diben-
zo[a,d]cyclohepten-5-yl)ethylene] glycine hydroxamate
F29. Colorless crystals, mp 108–110 ꢃC; IR (KBr),
cmꢀ1: 1159 (SO2sym), 1285 (amide III), 1381 (SOa2s), 1575
(amide II), 1710 (amide I), 1754 (COOH); 3060 and
3300 (NH, OH); 1H NMR (DMSO-d6), d, ppm: 2.25 (q,
CaCl2, pH 7.5. The rate of hydrolysis was determined
from the change in absorbance at 324 nm using an
extinction coefficient for FALGPA e305=24.700 Mꢀ1
cmꢀ1 in the above-mentioned reaction buffer.28 Mea-
surements were made using a Cary 3 spectrophotometer
interfaced with a PC. Initial velocities were thus esti-
mated using the direct linear plot-based procedure
reported by van Wart and Steinbrink.28 KIs were then
determined according to Easson–Stedman23 plots and a
linear regression program.
3
3
2H, JHH=7.3, CHCH2CH2), 2.60 (t, 2H, JHH=7.3,
CHCH2CH2), 2.66 (s, 3H, CH3C6H4), 3.05 (m, 2H,
H11A, H11B), 3.33 (m, 2H, H10A, H10B), 3.37 (s, 2H,
3
CH2COO), 4.10 (t, 1H, JHH=7.4, HC5), 7.00–7.32(m,
3
8H, HPh), 7.60 (d, JHH=8.3 Hz, 2H, Hortho of
3
CH3C6H4), 7.95 (d, JHH=8.3 Hz, 2H, Hmeta of
CH3C6H4); 8.26 (bs, 2H, NHCONH); 8.79 (bs, 1H,
NHOH); 10.66 (brs, 1H, NHOH); 13C NMR (DMSO-
d6), d, ppm: 26.71 (s, CH3C6H4), 33.12( C10+C11), 47.65
(CH2COO), 49.83 (CHCH2CH2), 51.78 (CH), 125.76,
126.54, 130.19, 130.34 (all from Ph), 130.53 (Cmeta of
CH3C6H4), 132.86 (NHCONH), 135.62( Cortho of
CH3C6H4), 139.24 and 141.05 (Cq), 145.35 (Cipso of
CH3C6H4), 148.97 (Cpara of CH3C6H4), 172.53 (s,
CONHOH). Anal. (C27H29N3SO5) C, H, N.
Acknowledgements
This research was financed by a grant from the Italian
CNR—target project Biotechnology, by the Roumanian
grant- CNFIS 141 and by CSGI, Florence, Italy.
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