Synthesis and evaluation of new tripeptide phosphonate inhibitors of MMP-8 and MMP-2

Article abstract of DOI:10.1016/j.ejmech.2004.10.013

The phosphotryptophan derivative l-Pro-l-Leu-l-(P)Trp(OH)₂ (2b) was reported as the first example of left-hand-side¹ phosphonate inhibitor of MMP-8. Its uncommon mode of binding to MMP-8 was mainly ascribed to the presence of the pro

Full text of DOI:10.1016/j.ejmech.2004.10.013

European Journal of Medicinal Chemistry 40 (2005) 271–279
www.elsevier.com/locate/ejmech
Original article
Synthesis and evaluation of new tripeptide phosphonate inhibitors
of MMP-8 and MMP-2
Mariangela Agamennone a, Cristina Campestre a, Serena Preziuso a, Valerio Consalvi b,
Marcello Crucianelli c, Fernando Mazza c, Vincenzo Politi d, Rino Ragno e, Paolo Tortorella f,
Carlo Gallina a,*
a Dipartimento di Scienze del Farmaco, Università G. d’Annunzio, Via dei Vestini 31, 66013 Chieti, Italy
b Dipartimento di Scienze Biochimiche, Università La Sapienza, P. le A. Moro 5, 00185 Rome, Italy
c Dipartimento di Chimica, Ingegneria Chimica e Materiali, Università degli Studi dell’Aquila, Via Vetoio, 67010 L’Aquila, Italy
d Polifarma Research Centre, Via Tor Sapienza 138, 00185 Rome, Italy
e Dipartimento di Scienze del Farmaco, Università La Sapienza, P. le A. Moro 5, 00185 Rome, Italy
f Dipartimento Farmaco-Chimico, Università degli Studi di Bari, Via Orabona 4, 70125 Bari, Italy
Received 13 July 2004; received in revised form 12 October 2004; accepted 28 October 2004
Available online 19 January 2005
Abstract
The phosphotryptophan derivative L-Pro-L-Leu-L-(P)Trp(OH)2 (2b) was reported as the first example of left-hand-side1 phosphonate
inhibitor of MMP-8. Its uncommon mode of binding to MMP-8 was mainly ascribed to the presence of the proline residue in P3. Ten new
analogues of 2b were obtained by replacement of the aminoterminal L-Pro with aminoacid residues bearing small side chains. Most of the new
analogues show an increase of affinity for MMP-2 and MMP-8, and different profiles of selectivity. Computer simulations were performed to
explain the effects of substitutions on the preferred mode of binding. They reveal that most of the new analogues are probably accommodated
in the right, rather than left-hand side of MMP-8 active site.
© 2005 Elsevier SAS. All rights reserved.
Keywords: MMP; Matrix metalloproteinases inhibitors; Tripeptide phosphonates; Right-hand inhibitors; Left-hand inhibitors
1. Introduction
some processes such as ovulation, embryogenic growth,
angiogenesis, differentiation and healing [1,2]. Overexpres-
sion of MMPs activity, however, or inadequate levels of the
natural MMPs tissue inhibitors, can contribute to the patho-
physiology of a variety of disease states and conditions such
as psoriasis [3], multiple sclerosis [4,5] rheumatoid arthritis
[6,7], and osteoporosis [8,9]. In addition, the angiogenetic
process favoured by MMPs [10,11] is essential for vascolari-
sation and growth of tumours beyond the size of approxi-
mately 2 mm in diameter.
Thus, inhibitors of MMPs are actively studied for their
utility in slowing or halting degradation of extracellular matrix
and progression of tumour invasion and metastasis. A great
variety of synthetic, low molecular weight, MMPs inhibitors
have been prepared and tested [12,13]; some of them, such as
marimastat, prinomastat and neovastat [14] are presently in
advanced clinical trials. Their structures include a peptide or
a peptidomimetic chain, which is generally accommodated
Matrix metalloproteinases (MMPs) are a family of zinc
endopeptidases that can degrade virtually all the constituents
of the extracellular matrix. These enzymes are necessary for
normal tissue remodelling and are particularly implicated in
Abbreviations: BSA, bistrimethylsilyl acetamide; DCC, 1,3-dicyclo-
hexylcarbodiimide; HOBt, 1-hydroxybenzotriazole; QF24, McaPro-Leu-
Gly-Leu-Dpa-Ala-Arg-NH2; QF41, McaPro-Cha-Gly-Nva-His-Ala-Dpa-
NH2; TMIS, trimethylsilyl iodide.
* Corresponding author. Tel.: +39-0871-355-5373; fax:
+39-0871-355-5322.
E-mail address: cgallina@unich.it (C. Gallina).
1 Left-hand-side inhibitors: inhibitors that bind in the unprime region of
the enzyme active site, in reference to the convention of drawing the unpri-
med residues of a peptide substrate on the left side. [R.P. Beckett et al., Drug
Discov. Today 1 (1996) 16–26]. The opposite applies to right-hand-side inhi-
bitors.
0223-5234/$ - see front matter © 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2004.10.013

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M. Agamennone et al. / European Journal of Medicinal Chemistry 40 (2005) 271–279
in the S′ region of the active site, and a zinc-binding function,
capable of coordinating the catalytic zinc ion of the enzyme.
Hydroxamate is, by far, the most effective zinc-binding
group. Direct replacement of this function with carboxylate
or phosphonate, for example, causes a 100–2000-fold de-
crease in potency [15,16]. Hydroxamate inhibitors, however,
are generally affected by lack of specificity [17] due to the
overwhelming contribution to binding of the hydroxamic
group relative to that of the peptidomimetic moiety. In addi-
tion, they show, poor pharmacokinetic properties [18] and
may cause toxic effects, in long-term treatments, due to the
release of hydroxylamine, a well known carcinogenic com-
pound [19]. Therefore, MMP inhibitors based on less potent
zinc-binding functions, such as carboxylate, phosphonate and
thiolate, are also currently investigated.
Fig. 1
II and MMPs.
. Natural (1a) and synthetic (1b) carboxylate inhibitors of adamalysin
The pyroglutamate-containing tripeptide 1a (Fig. 1), an
endogenous MMP inhibitor isolated from snake venom [20],
and its synthetic analogue 1b were reported as carboxylate
inhibitors of adamalysin II [21], a snake venom peptidyl-
endopeptidase with an active site structure closely related to
that of MMPs. We have recently studied tripeptide phospho-
nate MMP inhibitors 2a [22] and 2b [23] (Fig. 2), structur-
ally related to the carboxylate inhibitors 1b and 1a.
Fig. 2
. Structure of the phosphonate inhibitors 2a and 2b and their mode of
binding in the active site of adamalysin II and MMP-8, respectively.
The behaviour of phosphonates 2a and 2b was particu-
larly interesting with respect to their binding mode in the
enzyme active site. The phosphonate inhibitor 2a, as well as
its carboxylate analogue 1b, were found to bind in the S′
region of the active site of adamalysin II, adopting a retrob-
inding2 mode [24]. However, when L-Pro was introduced as
the aminoterminal residue in a series of phosphonate ana-
logues, the resulting inhibitor 2b was found to bind in the S
region of the MMP-8 active site, adopting a substrate-like
binding mode [23].
The S3 and S3′ subsites, obviously play an important role
in the accommodation of 2a and 2b phosphonates, and their
orientating effects appear to exceed even that of the S1′ pri-
mary specificity site, in the formation of 2b:MMP-8 com-
plex. Neither structural studies [23], nor molecular dynamic
simulations [25], were able to identify a dominant interac-
tion responsible for this change of binding mode. The unusual
prevalence of the orientating effects of the S3 and S3′ subsites
over those of the S1′ subsite, can be explained on the basis of
a concurrence of circumstances: i) ideal profile of the S3 sub-
site for accommodation of the proline methylenes, ii) addi-
tional cation–p bonding interaction in S3, probably involving
the protonated proline NH, iii) non-bonding interactions that
disfavour the arrangement of the proline residue in S3′, and
iv) non-ideal alignment of the large indolyl group in the S1′
hydrophobic pocket.
Fig. 3. New tripeptide phosphonate inhibitors of MMPs based on L-leucyl-
L-phosphotryptophan.
3b–3f), differing in the aminoterminal residue, was synthe-
sised and tested.
Thus, the aminoterminal L-Pro of 2b was replaced by Ala
and b-Ala residues, as representatives of non-cyclic a and b
aminoacids. Considering that the inhibitor could be accom-
modated either in the substrate-like orientation or in a retrob-
inding mode, the aminoterminal residues have been intro-
duced both in the (S) and in the (R) configurations. The (R)
configuration of the aminoterminal residue, in fact, was
expected to decrease the ligand affinity in the S region, owing
to the inversion of the substrate side chain topology in P3,
and to increase its affinity in the S′ region, due to the topol-
ogy maintenance in P3′.
Both the free aminoterminal (2b–2f) and the N-acetylated
phosphonates (3b–3f) were prepared to evaluate the contri-
bution of possible cation–p interactions in the S3 subsite.
2. Chemistry
In order to further study the behaviour of tripeptide phos-
phonates that may bind both in the S and S′ region of MMPs
active site, a new series (Fig. 3) of analogues (2c–2f and
Inhibitors of the present study were prepared according to
Scheme 1. Cbz-L-leucyl-L-phosphotryptophan diethylester
was obtained by coupling L-phosphotryptophan diethylester
(4) [26], with Cbz-L-leucine, in the presence of DCC and
HOBt [27]. Removal of the Cbz protecting group, by Pd/C
ammonium formate hydrogenolysis, gave L-leucyl-L-phospho-
2 Mode of binding antiparallel to that adopted by the substrate in the enzyme
active site.

M. Agamennone et al. / European Journal of Medicinal Chemistry 40 (2005) 271–279
273
Scheme 1. Synthesis of the tripeptide phosphonate inhibitors 2c–2f and 3b–3f, based on L-leucyl-L-phosphotryptophan. Reaction conditions: (a) Cbz-L-Leu-
OH, DCC, HOBt, THF, 80–90%; (b) 10% Pd/C, HCOONH4, CH3OH, 92%; (c) TMIS, BSA, CH2Cl2, 49–92%; (d) Acetic anhydride, pyridine, 70–90%;
*Phosphonates 6d and 6e required previous hydrogenolysis according to b.
tryptophan diethylester (5), common intermediate for prepa-
ration of the final phosphonates. The required N-protected
aminoacids and the cyclopentane carboxylic acid were
coupled (DCC, HOBt) with 5 to give phosphonate diethylest-
ers 6b–6g. Conversion into phosphonic acids 2b, 2c, 2f and
2g was performed by treatment with TMIS, in the presence
of BSA [28], that also allows effective removal of the Boc
protective group. Under these relatively mild conditions,
required by the presence of the acid sensitive indolyl group,
the Cbz protective group, present in the 6d and 6e intermedi-
ates, was partially retained. These Cbz derivatives were there-
fore deprotected by Pd/C ammonium formate hydrogenoly-
sis, before treatment with TMIS. Free aminoterminal
phosphonates 2b–2f were finally converted into the corre-
sponding N-acetyl derivatives, by acylation with acetic anhy-
dride in pyridine. Internal salts 2b–2f were purified by ion-
exchange and crystallisation. Phosphonic acids 3b–3f and 2g,
after purification by ion-exchange, were crystallised as cyclo-
hexylamine salts.
4. Results and discussion
4.1. Computational studies
All the inhibitors were minimised into the MMP-8 active
site both as left- and right-side inhibitors. The crystallo-
graphic pose of 2b in complex with MMP-8 was used as tem-
plate to simulate the left-side inhibition, while the conforma-
tion of 2a, as found in the crystal complex with adamalysin
II, was employed as template for the right-side inhibition.
The resulting complexes were qualitatively inspected, anal-
ysing how the N-terminal aminoacid replacement influences
the interactions at the active site. Moreover, the inhibitors 2b
and 2c were subjected to conformational search, with the aim
of evaluating the energetic difference between the global mini-
mum geometry of the two inhibitors and the respective poses
found in the complex with MMP-8.
4.2. Discussion
Most of the new analogues, obtained by replacement of
the N-terminal L-Pro in the reference phosphonate 2b and by
acetylation of amino group of the aminoterminal residue, show
increased affinity for MMP-8, although none of them attained
Ki lower than 260 µM.
In order to discuss the effects of the structural variations
on the affinity of the new phosphonates, it would be desirable
to know on which side of the active site they bind.
The mode of binding of the new inhibitors 2c–3f was there-
fore briefly evaluated by means of computer simulations, start-
3. Biochemistry
The ability of the phosphonates 2b–2g and 3b–3f to inhibit
the enzymatic activity of MMP-2 and MMP-8, was mea-
sured by continuous fluorimetric assay, evaluating the residual
enzyme activity in the presence of the fluorescent substrates
QF24 and QF41 [29]. The inhibitory constants were deter-
mined according to the procedure reported in Section 6 and
are expressed as Ki values (Table 1).
Table 1
Inhibition constants of phosphonates R-L-Leu-L-(P)Trp(OH)2 against MMP-2 and MMP-8
Phosphonate
R
MMP-2
Ki (mM)a
1.53b
N.I.d
MMP-8
Ki (mM)a
3.94c
N.I.d
Phosphonate
R
MMP-2
Ki (mM)a
1.76
MMP-8
Ki (mM)a
0.34
Number
2b
2c
2d
2e
Number
3b
3c
3d
3e
L-Pro
N-Ac-L-Pro
D-Pro
L-Ala
D-Ala
b-Ala
N-Ac-D-Pro
N-Ac-L-Ala
N-Ac-D-Ala
N-Ac-b-Ala
N.I.d
N.I.d
0.40
0.50
0.55
0.26
N.I.d
0.41
3.77
2.95
2f
N.I.d
0.34
3f
0.56
0.49
2g
C5H9CO
0.40
1.36
a Replicate determinations indicate standard deviations for the kinetic parameters less than 20%.
b Eleven micromolar of IC50 was erroneously reported previously [30].
c 3.2 µM IC50 was erroneously reported previously [30].
d No inhibition observed up to 1.0 mM, upper solubility limit of the inhibitor.

274
M. Agamennone et al. / European Journal of Medicinal Chemistry 40 (2005) 271–279
ing from the experimental evidence that the reference phos-
phonate 2b binds in the S side of MMP-8 active site [23]. As
previously reported [23,25], this orientation of the inhibitor
can be explained by favourable accommodation of the termi-
nal L-Pro pyrrolidine ring into the small hydrophobic pocket
formed by the side chains of His162 and Phe 164. A possible
cation–p interaction, involving the protonated pyrrolidine
nitrogen and the aromatic ring of Phe164, has been also pro-
posed for further stabilisation of the complex. In addition,
the alternative binding of 2b in the S′ side, burying the hydro-
phobic indolyl group of the (P)Trp residue in the large S1′
lipophilic pocket, is disfavoured by serious steric clashes in
S3′, between the proline pyrrolidine ring and Tyr189 (Fig. 4).
Alternative orientations of the Leu and Pro residues would
prevent this unfavourable interaction, but would also increase
the conformational strain of the ligand and/or reduce the
favourable interactions with the protein.
Acetylation of the N-terminal L-Pro of 2b causes an about
10-fold increase of the affinity for MMP-8. The modelling of
the N-acetyl derivative 3b in the MMP-8 active site suggests
that the orientation of the pyrrolidine ring, in the MMP-
8 complex, should not be affected by N-acetylation and that
the cation–p interaction in the 2b:MMP-8 complex could be
replaced by p–p interactions between the phenyl ring of the
Phe164 and the amide group of the N-acetyl-proline of 3b.
The observed increase of binding affinity by L-Pro acetyla-
tion seems to indicate that the cation–p interaction, in the
2b:MMP-8 complex, is not an important contributor to the
overall binding affinity. Probably, the aromatic N-acetylamide
p–p interaction of 3b in S3, due to acetylation of the
N-terminal Pro residue, largely exceeds the consequent loss
of the proposed cation–p interaction.
The slight increase of the binding affinity of 2g, with
respect to 2b, is also in agreement with this hypothesis, assum-
ing that the cyclopentyl ring could be easily accommodated
in the S3 subsite of the enzyme.
Modelling results confirm these observations and suggest
different preferences by replacement of the L-Pro unit and by
acetylation of the N-terminal residue.
Replacement of the N-terminal L-Pro by D-Pro led, instead,
to complete loss of observable inhibition. This result can be
explained with the unfavourable accommodation of the D-Pro
pyrrolidine ring both in S3′ and in S3. Proper alignment of the
D-Pro pyrrolidine ring, to maintain both favourable hydro-
phobic and cation–p interactions in the S3 subsite, could only
be achieved when the D-Pro NH is rotated toward the Ala163
(Fig. 5). In this arrangement, an additional hydrogen bond
between D-Pro NH and Ala163 CO would further improve
the stability of the complex, contrary to the experimental activ-
ity data. However, the internal energy of the two inhibitors
2b and 2c in their bound conformations was, respectively,
26 and 44 kJ mol–1 higher, respectively, than the energy of
the global minimum geometry obtained from the conforma-
tional searches of the two compounds. The larger internal
energy cost paid by 2c to bind to the enzyme, can explain the
decrease of binding affinity of 2c with respect to 2b.
Fig. 4. Representation of the right-side MMP-8 active site complexed with
2b (yellow) in its minimised conformation. Tyr189 and Tyr219 side chains
and the catalytic zinc ion are depicted. Steric bump between proline ring and
Tyr189 is highlighted. Hydrogen atoms on carbon are omitted for sake of
clarity and H-bonds are represented by white dotted lines.
Replacement of the N-terminal L-Pro by L-Ala, D-Ala or
b-Ala leads to molecules that could preferentially be accom-
Fig. 5. Representation of the left-side MMP-8 active site complexed with 2b (A) and 2c (B) in their minimised conformations. Side chains of His162, Ala163,
Phe164 and the catalytic zinc are depicted; p interactions between pyrrolidine ring of the ligands (yellow) and Phe164 are evident. Hydrogen atoms on carbon
are omitted for sake of clarity and H-bonds are represented by white dotted lines.

M. Agamennone et al. / European Journal of Medicinal Chemistry 40 (2005) 271–279
275
modated in the S′ side of the MMP-8 active site. The smaller
size, the lower hydrophobic character and the greater flexibil-
ity of the side chains of these residues, should decrease their
affinity for the MMP-8 S3 subsite and allow easier accommo-
dation in the MMP-8 S3′ subsite [31].
Phosphonates 2d and 3d, containing L-Ala or N-acetyl-L-
Ala as the N-terminal residue, show increased affinity both
for MMP-2 and MMP-8. Introduction of D-Ala or b-Ala in
P3′ causes an analogous increase of affinity for MMP-8, but
strongly decreases MMP-2 inhibition and suggests a way to
achieve selectivity between MMP-2 and MMP-8.
(2 mmol) was added dropwise, under stirring, at 0 °C. The
reaction mixture was further stirred for 1 h at 0 °C and stored
for 20 h at r.t. After removal of N,N′-dicyclohexylurea by fil-
tration, the solution was diluted with EtOAc and sequentially
washed with 2 N HCl, 2 N NaOH and brine. The organic
phase was dried over Na2SO4 and concentrated under reduced
pressure to give the crude product as a slight yellow syrup,
that was purified by silica gel column chromatography
(CHCl3/iPrOH 99:1).According to this procedure the follow-
ing compounds were prepared.
6.1.1.1. N-Benzyloxycarbonyl-L-leucyl-L-phosphotryptophan
diethylester. Colourless oil (92%); [␣]D = –45° (c 1; CHCl3);
IR (KBr) mmax cm–1 3286, 2960, 1695, 1450, 1236, 1036, 737,
534; 1H-NMR (CDCl3) d 0.83 (6H, m, two CH3 Leu); 1.10
(1H, m, cCH Leu); 1.27 (6H, m, two CH3 diethylester); 1.48
(2H, m, bCH2 Leu); 3.10 e 3.34 (2H, 2m, bCH2 (P)Trp); 4.10
(4H, m, two CH2 diethylester); 4.78 (2H, m, aCH Leu, aCH
(P)Trp); 5.01 (2H, m, CH2 phenyl); 6.32 (1H, d, J = 9.9 Hz,
NH Leu); 6.99 (1H, d, J = 2.6 Hz, indole); 7.12 (2H, m,
indole); 7.32 (7H, m, phenyl and indole); 7.60 (1H, d,
J = 7.7 Hz, NH (P)Trp); 7.77 (1H, s, NH indole).
5. Conclusions
The present study indicates that modification of the
N-terminal residue in the tripeptide phosphonate 2b can
slightly increase the affinity of the inhibitor for MMP-8 and
MMP-2, and probably affects their mode of binding in the
active site of MMP-8. The majority of the new analogues,
containing L-Ala, D-Ala and b-Ala show increased affinity
mainly for MMP-8 and probably bind in the S′ side of the
MMP-8 active site, in agreement with the general remark [12]
that the most potent MMP inhibitors are right-hand inhibi-
tors. Some selectivity with respect to MMP-2 has also been
attained by introduction of D-Ala or b-Ala in P3′.
6.1.1.2. N-tert-Butoxycarbonyl-L-prolyl-L-leucyl-L-phospho-
tryptophan diethylester (6b). Colourless oil (88%);
[␣]D = –78° (c 1; CHCl3); IR (KBr) mmax cm–1 3294, 2974,
1
1673, 1395, 1232, 1023, 737, 534; H-NMR (CDCl3) d
0.83 and 0.85 (6H, m, two CH3 Leu); 1.63 (24H, m, two CH3
diethylester, cCH Leu, bCH2 Leu; C(CH3)3, three CH2 Pro);
3.11 and 3.36 (3H, two m, bCH2Trp, aCHPro); 4.11 (4H, m,
two CH2 diethylester); 4.32 (1H, m, aCH Leu,); 4.78 (1H, m,
aCH (P)Trp); 6.59 (1H, s, NH Leu); 6.87 (1H, s, NH (P)Trp);
7.31 (5H, m, indole); 8.47 (1H, s, NH indole); 31P-NMR
(CDCl3) d 25.08. Anal. C30H47N4O7P.H2O (C, H, N).
6. Experimental protocols
6.1. Chemistry
Reagent grade materials from Fluka Gmbh or Aldrich
Chemical and Co. were used without further purification.
Silica gel 60 (230–400 mesh) for column chromatography
and TLC silica gel 60 F254 plates were from Merck AG
(Darmstadt, Germany). Ion-exchange resins (Dowex 50W and
Amberlite IRA-68) were from Sigma-Aldrich. Melting points
(Büchi B-540 melting point apparatus) are uncorrected. [␣]D
were determined with a Perkin–Elmer 241 digital polari-
meter. IR spectra were obtained with a Perkin–Elmer
6.1.1.3. N-tert-Butoxycarbonyl-D-prolyl-L-leucyl-L-phospho-
tryptophan diethylester (6c). Colourless oil (92%);
[␣]D = –18° (c 1; CHCl3); IR (KBr) mmax cm–1 3286, 2965,
1673, 1412, 1235, 1023, 737, 534; 1H-NMR (CDCl3) d 0.86
(6H, m, two CH3 Leu); 1.55 (24H, m, two CH3 diethylester,
C(CH3)3, cCH Leu, bCH2 Leu, three CH2 Pro); 3.08 and 3.35
(3H, two m, bCH2 (P)Trp, aCH Pro); 4.11 (4H, m, two CH2
diethylester); 4.37 (1H, m, aCH Leu); 4.73 (1H, m, aCH
(P)Trp); 6.31 and 6.45 (2H, 2s, NH Leu and NH (P)Trp);
7.33 (5H, m, indole); 8.72 (1H, s, NH indole) 31P-NMR
(CDCl3) d 25.11; Anal. C30H47N4O7P (C, H, N).
1
1600 FT-IR spectrometer. H-, 13C- and 31P-NMR spectra
were recorded on a Varian VXR 300 spectrometer. Chemical
shifts (d) for 13C and 1H are given in ppm relative to TMS as
the internal standard and the coupling constants (J) are
reported in Hz. Chemical shifts (d) for 31P are given in ppm
relative to phosphoric acid as the internal standard. Elemen-
tal microanalyses (C, H, N) were within 0.4% of the calcu-
lated values.
6.1.1.4. N-Benzyloxycarbonyl-L-alanyl-L-leucyl-L-phospho-
tryptophan diethylester (6d). Colourless oil (92%); [␣]D = –6°
(c 1; CHCl3); IR(KBr) mmax cm–1 3285, 3062, 2951, 1697,
1
6.1.1. General procedure for compounds 6b–6g
and N-benzyloxycarbonyl-L-leucyl-L-phosphotryptophan
diethylester
To a solution of phosphonate diethylester 4 or 5 and the
appropriate N-protected aminoacid (1 mmol) in anhydrous
THF (7 ml), a solution of DCC (1.2 mmol) and HOBt
1654, 1448, 1234, 1036, 967, 736; H-NMR (CDCl3) d
0.81 and 0.84 (6H, two d, J = 5.3 Hz, two CH3 Leu; 1.25
(11H, m, two CH3 diethylester, bCH2 Leu, CH3 Ala); 1.52
(1H, m, cCH Leu); 3.22 (2H, m, bCH2 (P)Trp), 4.10 (5H, m,
two CH2 diethylester and aCH Leu), 4.41 (1H, m, aCH Ala),
4.77 (1H, m, aCH (P)Trp); 5.1 (2H, two d, J = 11.9 Hz, CH2

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M. Agamennone et al. / European Journal of Medicinal Chemistry 40 (2005) 271–279
Ph); 5.31 (1H, d, J = 7.0 Hz, NHAla); 6.29 (1H, d, J = 7.5 Hz,
NH Leu); 6.7 (1H, d, J = 10.1 Hz, NH (P)Trp); 6.99 and 7.11
(2H, two s, indole); 7.33 (7H, m, phenyl and indole); 7.57
(1H, d, J = 7.0 Hz, indole); 8.34 (1H, s, NH indole). Anal.
C31H43N4O7P.H2O (C, H, N).
2 N Na2CO3 and brine, dried over Na2SO4 and evaporated to
give the expected amine, which was employed without fur-
ther purification. According to this procedure the following
compounds were prepared.
6.1.2.1. L-Leucyl-L-phosphotryptophan diethylester (5). Pale
yellow oil (90%); [␣]D = –12° (c 1; CHCl3); IR (KBr) mmax
6.1.1.5. N-Benzyloxycarbonyl-D-alanyl-L-leucyl-L-phospho-
tryptophan diethylester (6e). White solid (85%); [␣]D = –67°
(c 1; CHCl3); IR (KBr) mmax cm–1 3277, 3058, 2957, 1699,
1
cm–1 3294, 3025, 1657, 1226, 1023, 737, 534; H-NMR
(CDCl3) d 0.77 (6H, m, two CH3 Leu); 1.28 (9H, m, bCH2
Leu, cCH Leu, two CH3 diethylester); 1.58 (2H, s, NH2 Leu);
3.24 (3H, m, aCH Leu, bCH2 (P)Trp), 4.15 (4H, m, two CH2
diethylester); 4.77 (1H, m, aCH (P)Trp), 7.11 (3H, m, indole);
7.33 (1H, d, J = 7.5 Hz, indole); 7.55 (1H, s, NH (P)Trp); 7.6
(1H, d, J = 7.8 Hz, indole); 8.21 (1H, s, NH indole); 31P-
NMR (CDCl3) d 25.72. Anal. C20H32N3O4P (C, H, N).
1
1657, 1446, 1235, 1032, 965, 737; H-NMR (CDCl3) d
0.82 and 0.86 (6H, two d, J = 6.6 Hz, two CH3 Leu); 1.21
(11H, m, bCH2 Leu, two CH3 diethylester, CH3 Ala); 1.54
(1H, m, cCH Leu); 3.06 (2H, m, bCH2 (P)Trp); 4.08 (5H, m,
two CH2 diethylester, aCH Leu); 4.57 (1H, m, aCH Ala);
4.81 (1H, m, aCH (P)Trp); 5.12 (2H, two d, J = 11.9 Hz,
CH2Ph); 6.06 (1H, d, J = 9.2 Hz, NHAla); 6.89 (1H, s, indole);
6.96 (1H, s, NH Leu); 7.09 (2H, m, indole); 7.30 (6H, m,
phenyl and indole); 7.51 (1H, d, indole); 7.73 (1H, s, NH
(P)Trp); 9.05 (1H, s, NH indole); Anal. C31H43N4O7P (C, H,
N).
6.1.2.2. L-Alanyl-L-leucyl-L-phosphotryptophan diethyl-
ester. Pale yellow oil (88%); [␣]D = –61° (c 1; MeOH); IR
(KBr) mmax cm–1 3286, 3058, 2957, 1657, 1530, 1446, 1226,
1032, 965, 737; 1H-NMR (CDCl3) d 0.82 and 0.85 (6H, two
d, J = 6.4 Hz, two CH3 Leu); 1.15 (3H, d, J = 7.0 Hz, CH3
Ala); 1.41 (11H, m, two CH3 diethylester, cCH Leu, bCH2
Leu and NH2 Ala); 3.04 (1H, q, J = 7.0 Hz, aCH Ala); 3.23
(2H, m, bCH2 (P)Trp); 4.04 (4H, m, two CH2 diethylester);
4.29 (1H, m, aCH Leu); 4.80 (1H, m, aCH (P)Trp); 6.67 (1H,
d, J = 10.0 Hz, NH (P)Trp); 7.13 (3H, m, indole); 7.23 (1H,
d, J = 8.2 Hz, NH Leu); 7.31 (1H, d, J = 7.0 Hz, indole); 7.61
(1H, d, J = 7.0 Hz, indole); 8.3 (1H, s, NH indole).
6.1.1.6. N-tert-Butoxycarbonyl-b-alanyl-L-leucyl-L-phospho-
tryptophan diethylester (6f). Colourless oil (80%);
[␣]D = –75° (c 1; CHCl3); IR (KBr) mmax cm–1 3284, 2958,
1640, 1236, 1142, 1041, 737; 1H-NMR (CDCl3) d 0.83 (6H,
m, two CH3 Leu); 1.37 (18H, m, two CH3 diethylester,
(CH3)3C, cCH Leu, bCH2 Leu) 2.19 (2H, t, J = 6.0 Hz, aCH2
b-Ala); 3.11 and 3.35 (4H, two m, bCH2 (P)Trp, bCH2 b-Ala);
4.18 (5H, m, two CH2 diethylester and aCH Leu); 4.75 (1H,
m, aCH (P)Trp); 5.48 (1H, s, NH b-Ala) 5.61 (1H, d,
J = 8.7 Hz, NH Leu); 6.37 (1H, d, J = 10.2 Hz, NH (P)Trp);
7.34 (5H, m, indole); 8.84(1H, s, NH indole). Anal.
C28H45N4O7P (C, H, N).
6.1.2.3. D-Alanyl-L-leucyl-L-phosphotryptophan diethyl-
ester. Pale yellow solid (95%); m.p. 129.9–131.7 °C;
[␣]D = –74° (c 1; MeOH); IR (KBr) mmax cm–1 3269, 3067,
1
2957, 1649, 1547, 1230, 1047, 1026, 966, 741; H-NMR
(DMSO-d6) d 0.79 and 0.83 (6H, two d, J = 6.6 Hz, two CH3
Leu); 1.07 (3H, d, J = 7.0 Hz, CH3 Ala); 1.2 (6H, m, two CH3
diethylester) 1.31 (2H, m, bCH2 Leu); 1.46 (1H, m, cCH Leu)
2.9 and 3.21 (5H, m, bCH2 (P)Trp, cCH Leu, NH2 Ala); 3.99
(4H, m, two CH2 diethylester); 4.34 (2H, m, aCH Leu, aCH
(P)Trp); 7.00 (3H, m, indole); 7.29 (1H, d, J = 8.1 Hz, indole);
7.46 (1H, d, J = 7.7 Hz, indole); 7.87 (1H, d, J = 8.1 Hz, NH
Leu); 8.34 (1H, d, J = 9.5 Hz, NH (P)Trp); 10.83 (1H, s, NH
indole).
6.1.1.7. Cyclopentylcarbonyl-L-leucyl-L-phosphotryptophan
diethylester (6g). Pale yellow oil (64%); [␣]D = –53° (c 1;
CHCl3); IR (KBr) mmax cm–1 3282, 3060, 2951, 1647, 1229,
1024, 735; 1H-NMR (CDCl3) d 0.86 (6H, m, two CH3 Leu);
1.27 (8H, m, two CH3 diethylester, bCH2 Leu); 1.57 (9H, m,
cCH Leu, four CH2 cyclopentyl); 2.3 (1H, m, aCH cyclopen-
tyl); 3.13 and 3.35 (2H, two m, bCH2 (P)Trp); 4.08 (4H, m,
two CH2 diethylester); 4.39 (1H, m, aCH Leu); 4.77 (1H, m,
aCH (P)Trp); 5.55 (1H, d, J = 8.4 Hz, NH Leu); 6.50 (1H, d,
J = 9.9 Hz, NH (P)Trp); 7.14 (3H, m, indole); 7.32 (1H, d,
J = 8.3 Hz, indole); 7.61 (1H, d, J = 7.9 Hz, indole); 8.27
(1H, s, NH indole); 31P-NMR (CDCl3) d 25.0; Anal.
C26H40N3O5P·1/2H2O (C,H,N).
6.1.3. General procedure for compounds 2b–2g
A solution of the phosphonate diethylester (1 mmol) in
anhydrous CH2Cl2 (5 ml) and bistrimethylsilylacetamide
(BSA; 12 mmol) was stored under nitrogen, for 1 h at r.t.
After cooling at –20 °C, trimethylsilyl iodide (8 mmol) was
added dropwise, via syringe, under stirring. After 20 min at
0 °C and 1 h at r.t., the solvent was removed under reduced
pressure to give a brown oily residue of the phosphonate tri-
methylsilylester that was hydrolysed by treatment with
7:3 CH3CN/H2O (5 ml). After evaporation of the solvents
under reduced pressure, the residue was dissolved in water
and decolourised by addition of solid Na2SO3 and 2 N HCl.
The solution was passed through a column containing
6.1.2. General procedure for Cbz deprotection of
compounds 6d, 6e and N-benzyloxycarbonyl-L-leucyl-
L-phosphotryptophan diethylester
To a solution of the benzyloxycarbonyl derivative (1 mmol)
in MeOH (10 ml) ammonium formate (4 mmol) and 10%
Pd/C (100 mg) were added. After stirring for 3 h at r.t., the
reaction mixture was filtered and evaporated under reduced
pressure.A solution of the residue in EtOAc was washed with

M. Agamennone et al. / European Journal of Medicinal Chemistry 40 (2005) 271–279
277
10 equiv. of a Dowex 50W sulphonic resin and the phospho-
nic acids recovered by elution with 2 N NH4OH. Evapora-
tion of the ammonia solutions and recrystallisation yielded
phosphonates 2b–2f as analytically pure compounds.Accord-
ing to this procedure the following compounds were pre-
pared.
(2H, m, bCH2 b-Ala); 3.90 (1H, m, aCH Leu); 4.23 (1H, m,
aCH (P)Trp); 7.26 (5H, m, indole);Anal. C19H29N4O5P·2H2O
(C, H, N).
6.1.3.6. Cyclopentylcarbonyl-L-leucyl-L-phosphotryptophan
cyclohexylamine salt (2g). The general procedure was modi-
fied as follows: the residue, recovered from the CH3CN/H2O
solution, was dissolved in EtOAc, and sequentially washed
with a 1:1 mixture of 3% Na2SO3/2 N HCl and brine and
dried over Na2SO4.After removal of the solvent under reduced
pressure the phosphonate was crystallised as cyclohexy-
lamine salt (93%); m.p. 248.1–251.5 °C (MeOH/EtOAc);
[␣]D = –69° (c 1; MeOH); IR (KBr) mmax cm–1 3289, 2937,
6.1.3.1. L-Prolyl-L-leucyl-L-phosphotryptophan (2b). White
solid (77%); m.p. 258.1–259.7 °C (MeOH/Et2O) [␣]D = –114°
(c 1; NH4OH 2 N); IR (KBr) mmax cm–1 3335, 3257, 2955,
1645, 1552, 1133, 1071, 737, 551, 1H-NMR (D2O) d 0.60 and
0.64 (6H, two d, J = 6.4 Hz, two CH3 Leu); 1.17 (3H, m,
bCH2 Leu, cCH Leu); 1.46, 1.75 and 2.05 (4H, three m, b
and c CH2 Pro); 2.77 and 3.17 (4H, two m, bCH2 (P)Trp and
dCH2 Pro); 4.03 (2H, m, aCH Leu, aCH Pro); 4.15 (1H, m,
aCH (P)Trp); 7.25 (5H, m, indole). Anal. C21H31N4O5P (C,
H, N).
1
2858, 1636, 1539, 1451, 1035; H-NMR (DMSO-d6) d
0.73 and 0.77 (6H, two d, J = 6.6 Hz, two CH3 Leu); 1.49
(21H, m, four CH2 cyclopentyl, five CH2 cyclohexylamine,
cCH Leu bCH2 Leu); 2.61 (1H, t, J = 7.5 Hz, aCH cyclo-
hexylamine); 2.72 and 3.21 (3H, two m, bCH2 (P)Trp, aCH
cyclopentyl); 3.97 (1H, m, aCH Leu); 4.21 (1H, m, aCH
(P)Trp); 6.97 (3H, m, indole); 7.25 (1H, d, J = 7.5 Hz, indole);
7.31 (1H, d, J = 8.1 Hz, NH (P)Trp); 7.47 (1H, d, J = 7.5 Hz,
indole); 8.16 (1H, d, J = 8.1 Hz, NH Leu); 10.6 (1H, s, NH
indole); Anal. C28H45N4O5P (C, H, N).
6.1.3.2. D-Prolyl-L-leucyl-L-phosphotryptophan (2c). White
solid (88.5%); m.p. 230.4–235.6 °C (dec.) (MeOH/Et2O);
[␣]D = –82° (c 1; NH3 2 N); IR (KBr) mmax cm–1 3291, 2953,
1
1657, 1544, 1152, 1062, 738, 557; H-NMR (DMSO-d6)
d 0.71 and 0.78 (6H, two d, J = 6.4 Hz, two CH3 Leu); 1.30
(3H, m, bCH2 Leu, cCH Leu); 1.95 and 2.35 (4H, two m, b
and c CH2Pro); 3.37 (4H, m, bCH2 (P)Trp, dCH2 Pro); 4.05
(1H, m, aCH Pro); 4.32 (1H, tl, aCH Leu); 4.48 (1H, m, aCH
(P)Trp); 7.32 (5H, m, indole). Anal. C21H31N4O5P (C, H, N).
6.1.4. General procedure for compounds 3b–3f
To a stirred solution of tripeptide phosphonate (1 mmol)
in pyridine (10 ml), acetic anhydride (10 ml) was added drop-
wise, at 0 °C, under stirring. After 20 h at r.t., the volatiles
were removed under reduced pressure and the residue taken
up in a 1:1 MeOH/H2O mixture. The solution was passed
through a column containing 10 equiv. of Amberlite IRA-
68 weakly basic resin. The phosphonic acid retained by the
resin was recovered by elution with 2 N NH4OH and evapo-
ration of the ammonia solution. Addition of cyclohexy-
lamine to a solution of the crude residue in MeOH and evapo-
ration of the solvent gave the pure cyclohexylamine
phosphonates 3b–3f, that were recrystallised from MeOH/
EtOAc. According to this procedure the following com-
pounds were prepared.
6.1.3.3. L-Alanyl-L-leucyl-L-phosphotryptophan (2d). White
solid (58%); m.p. 226.5–227.2 °C (H2O/MeOH); [␣]D = –89°
(c 1; NH3 2 N); IR (KBr) mmax cm–1 3316, 3240, 3079, 2957,
1648, 1541, 1150, 913, 745, 538; 1H-NMR (D2O) d 0.59 and
0.64 (6H, two d, J = 6.6 Hz, two CH3 Leu); 1.07 (3H, m, cCH
Leu, bCH2 Leu); 1.19 (3H, d, J = 7.1 Hz, CH3 Ala); 2.99
(2H, m, bCH2 (P)Trp); 3.8 (1H, m, aCH Ala); 4.03 (1H, m,
aCH Leu); 4.2 (1H, m, aCH (P)Trp); 7.0 (3H, m, indole); 7.3
(1H, d, J = 7.9 Hz, indole); 7.5 (1H, d, J = 7.5 Hz, indole);
Anal. C19H29N4O5P·5/2H2O (C, H, N).
6.1.3.4. D-Alanyl-L-leucyl-L-phosphotryptophan (2e). White
solid (54%); m.p. 225.7–228.0 °C (MeOH/EtOAc);
[␣]D = –84° (c 1; NH3 2 N); IR (KBr) mmax cm–1 3395, 3176,
2957, 1648, 1395, 1150, 914, 737, 551, 526; 1H-NMR (D2O)
d 0.54 and 0.6 (6H, two d, J = 6.3 Hz, two CH3 Leu); 0.79
(2H, tl, bCH2 Leu) 1.14 (1H, m, cCH Leu); 1.30 (3H, d,
J = 7.2 Hz, CH3 Ala); 2.83 and 3.22 (2H, two m, bCH2
(P)Trp); 3.92 (2H, m, aCHAla, aCH Leu); 4.24 (1H, m, aCH
(P)Trp); 7.27 (5H, m, indole);Anal. C19H29N4O5P (C, H, N).
6.1.4.1.
N-Acetyl-L-prolyl-L-leucyl-L-phosphotryptophan
cyclohexylamine salt (3b). White solid (41%); m.p. 264.1–
264.7° (dec.) (EtOAc); [␣]D = –33° (c 0.69; MeOH); IR (KBr)
mmax cm–1 3291, 2940, 1637, 1544, 1042, 727, 556; 1H-NMR
(CD3OD) d 0.84 (6H, m, two CH3 Leu); 1.64 (17H, m, cCH
Leu, bCH2 Leu, five CH2 cyclohexylamine, b and cCH2 Pro);
2.05 (3H, s, CH3CO); 3.01 and 3.45 (5H, two m, bCH2 (P)Trp,
CH cyclohexylamine, dCH2 Pro); 4.34 (2H, m, aCH Leu,
aCH Pro); 5.00 (1H, m, aCH (P)Trp); 7.29 (5H, m, indole);
31P-NMR (CD3OD) d 18.85. Anal. C29H46N5O6P (C, H, N).
6.1.3.5. b-Alanyl-L-leucyl-L-phosphotryptophan (2f). White
solid (77%); m.p. 226.2–227.7 °C (dec.) (MeOH/EtOAc);
[␣]D = –103° (c 1; NH3 2 N); IR (KBr) mmax cm–1 3404, 3286,
6.1.4.2. N-Acetyl-D-prolyl-L-leucyl-L-phosphotryptophan
cyclohexylamine salt (3c). White solid (90%); m.p. 239.3–
244.0 °C (dec.) (EtOAc); [␣]D = –16° (c 0.6; MeOH); IR
(KBr) mmax cm–1 3284, 2940, 1637, 1551, 1400, 1042;
1H-NMR (CD3OD) d 0.82 (6H, m, two CH3 Leu); 1.59 (17H,
1
2957, 1640, 1539, 1142, 1041, 737, 551; H-NMR (D2O)
d 0.54 and 0.6 (6H, two d, J = 6.6 Hz, two CH3 Leu); 0.84
(2H, m, bCH2 Leu); 1.13 (1H, m, cCH Leu); 2.46 (2H, m,
aCH2 b-Ala); 2.82 and 3.21 (2H, two m, bCH2 (P)Trp); 3.02

278
M. Agamennone et al. / European Journal of Medicinal Chemistry 40 (2005) 271–279
m, cCH Leu, bCH2 Leu, five CH2 cyclohexylamine, two CH2
Pro); 2.11 (3H, s, CH3CO) 3.02 and 3.51 (5H, two m, bCH2
(P)Trp, CH cyclohexylamine, dCH2 Pro); 4.34 (3H, m, aCH
Leu, aCH Pro, aCH (P)Trp); 7.29 (5H, m, indole). Anal.
C29H46N5O6P (C, H, N).
To simulate the right-hand-side inhibition, the previously
cited crystal structure of the complex 2b:MMP-8 (entry
1i73 in the Protein Data Bank), was considered. As a tem-
plate for the binding geometry to the enzyme, the Fur-L-Leu-
L-(P)Trp(OH)2 (2a) was used as found in the crystallo-
graphic complex with the adamalysin II (entry 4aig in the
Protein Data Bank); to obtain a correct position of the 2a
inhibitor into the MMP-8 active site, the two complexes
2b:MMP-8 and 2a:adamalysin were aligned by superimpos-
ing the zinc ion and the three His residues.As for the left-side
simulation, the furane ring was replaced to obtain the inhibi-
tors tested in this article.
All the crystallographic water molecules were removed
from the MMP-8 crystal structure. Hydrogen atoms were
added on the heteroatoms and on the aromatic rings, as sug-
gested by the MacroModel Batchmin User Manual. Charges
on the zinc ion were corrected as suggested by Guida et al.
[34]. All ligands were used in their deprotonated forms with
negatively charged phosphonates.
6.1.4.3. N-Acetyl-L-alanyl-L-leucyl-L-phosphotryptophan
cyclohexylamine salt (3d). White solid (98%); m.p. 246.3–
247.2 °C (MeOH/EtOAc); [␣]D = –78° (c 1; MeOH); IR (KBr)
mmax cm–1 3286, 2931, 1640, 1539, 1142, 1041, 914, 737,
551; 1H-NMR (D2O) d 0.62 (6H, two d, J = 6.6 Hz, two CH3
Leu); 1.31 (16H, m, five CH2 cyclohexylamine, CH3 Ala, cCH
Leu, bCH2 Leu), 1.8 (3H, s, CH3CO); 2.80 (1H, m, aCH
cyclohexylamine); 2.97 and 3.22 (2H, two m, bCH2 (P)Trp);
4.00 (2H, m, aCHAla, aCH Leu); 4.18 (1H, m, aCH (P)Trp);
7.02 (3H, m, indole); 7.29 (1H, d, J = 7.9 Hz, indole); 7.56
(1H, d, J = 7.5 Hz, indole); Anal. C27H44N5O6P·2H2O (C, H,
N).
6.1.4.4. N-Acetyl-D-alanyl-L-leucyl-L-phosphotryptophan
cyclohexylamine salt (3e). White solid (82%); m.p. 210–
216.3 °C (dec.) (EtOAc); [␣]D = –25° (c 1; MeOH); IR (KBr)
mmax cm–1 3277, 2940, 1632, 1539, 1146, 1040, 720; 1H-NMR
(CD3OD) d 0.78 and 0.83 (6H, two d, J = 6.6 Hz, two CH3
Leu); 1.54 (16H, m, cCH Leu, bCH2 Leu, CH3 Ala, five CH2
cyclohexylamine); 2.00 (3H, s, CH3CO); 3.05 (3H, m, bCH2
(P)Trp, aCH cyclohexylamine); 3.4 (1H, m, aCH Ala); 4.24
(1H, m, aCH Leu); 4.37 (1H, m, aCH (P)Trp); 7.00 (3H, m,
indole); 7.27 (1H, d, J = 7.5 Hz, indole); 7.6 (1H, d, J = 7.2 Hz,
The enzyme structure was relaxed without inhibitors before
complexes evaluations, with a 10,000 iterations of Polak–
Ribière Conjugate Gradient minimisations until the deriva-
tive convergence was 0.01 kcal Å–1 mol. An energy con-
straint of 400 kJ Å–2 mol was applied to the main chain to
avoid large structural modifications.
The phosphonate function was kept in the crystallographi-
cally observed position, by fixing the distances between the
phosphonate oxygen atoms and the zinc ion and applying an
energy constraint of 500 kcal Å–2 mol, to avoid rearrange-
ment of the zinc coordination.
Minimisations were performed using the truncated New-
ton conjugate gradient (TNCG) algorithm until the derivative
convergence was 0.001 kJ Å–1 mol, the extended non-
bonded treatment and the GB/SA water solvation model
implemented in MacroModel. The inhibitors were free to
minimise, while all the residues in a 10 Å range from the
inhibitor were fixed in the 3D space with an energy restraint
of 200 kcal Å–2 mol, even if their non-bonded interactions
with all the relaxing atoms were calculated. The same proto-
col was applied for all the simulations.
The Batchmin Monte Carlo multiple minimum (MCMM)
methodology was chosen to carry out conformational searches
on 2b and 2c. The conformational space of the two structures
was explored by random changes in the torsional angles of
all the rotatables bonds, as given by the TORS command.
All the conformations obtained were subsequently mini-
mised with the TNCG algorithm to a derivative convergence
of 0.01 kJ Å–1 mol.
indole); 31P-NMR (CD3OD)
C27H44N5O6P·3H2O (C, H, N).
d
18.49; Anal.
6.1.4.5. N-Acetyl-b-alanyl-L-leucyl-L-phosphotryptophan
cyclohexylamine salt (3f). White solid (81%); m.p. 238.2–
244.2 °C (dec.) (MeOH/EtOAc); [␣]D = –53° (c 1; MeOH);
IR (KBr) mmax cm–1 3285, 2939, 1631, 1547, 1040, 720, 534;
1H-NMR (CD3OD) d 0.79 and 0.83 (6H, two d, J = 6.6 Hz,
two CH3 Leu); 1.60 (16H, m, five CH2 cyclohexylamine, cCH
Leu, bCH2 Leu, CH3CO) 2.37 (2H, t, J = 6.4 Hz, aCH2
b-Ala); 3.01 and 3.33 (5H, two m, bCH2 (P)Trp, bCH2 b-Ala,
CH cyclohexylamine); 4.22 (1H, m, aCH Leu); 4.41 (1H, m,
aCH (P)Trp); 7.28 (5H, m, indole);Anal. C27H44N5O6P·H2O
(C, H, N).
6.2. Molecular modelling
All molecular modelling calculations were performed using
the software packages MacroModel 7.1 [32], running on Sili-
con Graphics Octane R12000 workstation. The Amber* [33]
united atom force field was used for all the calculations.
Models of MMP-8 complexed with the compounds 2c–3f
as left-hand inhibitors were constructed based on the crystal
structure of the related complex 2b:MMP-8 (entry 1i73 in
the Protein Data Bank). The L-Pro aminoacid of the crystal-
lographic inhibitor was replaced by the corresponding resi-
dues of the synthesised analogues.
6.3. Enzyme assay and determination of the inhibition
constants
Inhibition of MMP-2 and MMP-8 has been determined at
25 °C in Tris–HCl 50 mM pH 7.5, CaCl2 5 mM, Brij 0.05%,
NaN3 0.02% continuously monitoring the hydrolysis of the
fluorogenic substrates QF24 (0.20 µM) and QF41 (5.00 µM)

M. Agamennone et al. / European Journal of Medicinal Chemistry 40 (2005) 271–279
279
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[19] by MMP-2 and MMP-8, respectively, after 5 min incu-
bation in the presence of the inhibitors. Reactions were started
by addition of the substrates in the cuvette, under continuous
stirring. The hydrolysis was followed by measuring the
increase in relative fluorescence at 393 nm (excitation at
328 nm) due to the formation of product. The highest enzyme
concentration used in the assays was always lower than 3.8 ×
10–10 M. Under these conditions, low enzyme concentration
and substrate concentration lower than the Km values, the sub-
strate consumption is negligible but well measurable, and the
reaction proceeds linearly for at least 1500 s. The rate of enzy-
matic substrate hydrolysis was determined in the 200–1400 s
time interval. Data analysis has been performed assuming a
reversible inhibition with rapid binding of the enzyme to the
inhibitor.
[11] C.M. Overall, V. Lopez-Otin, Nat. Rev. Cancer 2 (2002) 657–672.
[12] J.W. Skiles, N.C. Gonnella, A.Y. Jeng, Curr. Med. Chem. 8 (2001)
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R.A. Kramer, P.A. Trail, Cancer Res. 61 (2001) 8480–8485.
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