N-[2-(Indan-1-yl)-3-mercapto-propionyl] amino acids as highly potent inhibitors of the three vasopeptidases (NEP, ACE, ECE): In vitro and In vivo activities

Article abstract of DOI:10.1016/S0960-894X(02)00248-2

We have previously reported the design of a lead compound 1a for the joint inhibition of neprilysin (NEP, EC 3.4.24.11), angiotensin converting enzyme (ACE, EC 3.4.15.1) and endothelin converting enzyme (ECE-1, EC 3.4.24.71), three metallopeptidases which are implicated in the regulation of fluid homeostasis and vascular tone. We report here the synthesis and biological activities of analogues derived from this lead with inhibitory potencies in the nanomolar range for the three enzymes. Compounds 8b and 15c are the most potent triple inhibitors described to date.

Full text of DOI:10.1016/S0960-894X(02)00248-2

Bioorganic & Medicinal Chemistry Letters 12 (2002) 2001–2005
N-[2-(Indan-1-yl)-3-mercapto-propionyl] Amino Acids as Highly
Potent Inhibitors of the Three Vasopeptidases (NEP, ACE, ECE):
In Vitro and In Vivo Activities
Nicolas Inguimbert,a Herve Poras,a Franck Teffo,a Francoise Beslot,a Mohamed Selkti,b
Alain Tomas,b Elizabeth Scalbert,c Caroline Bennejean,c Pierre Renard,c
Marie-Claude Fournie-Zaluskia and Bernard-Pierre Roquesa,*
a
´
´
Departement de Pharmacochimie Moleculaire et Structurale, U266 INSERM, UMR 8600 CNRS,
UFR des Sciences Pharmaceutiques et Biologiques, 4, avenue de l’observatoire,
75270 ParisCedex 06, France
bLaboratoire de Cristallographie et RMN Biologiques, CNRS UMR 8015, 4, avenue de l’Observatoire,
75270 ParisCedex 06, France
cInstitut de Recherches Internationales Servier, 6, place des Pleiades, 92415 Courbevoie Cedex, France
Received 1 February 2002; revised 9 April 2002; accepted 12 April 2002
Abstract—We have previously reported the design of a lead compound 1a for the joint inhibition of neprilysin (NEP, EC 3.4.24.11),
angiotensin converting enzyme (ACE, EC 3.4.15.1) and endothelin converting enzyme (ECE-1, EC 3.4.24.71), three metallopepti-
dases which are implicated in the regulation of fluid homeostasis and vascular tone. We report here the synthesis and biological
activities of analogues derived from this lead with inhibitory potencies in the nanomolar range for the three enzymes. Compounds
8b and 15c are the most potent triple inhibitors described to date. # 2002 Elsevier Science Ltd. All rights reserved.
Two counteracting peptidergic systems regulate fluid
homeostasis and vascular tone,1 the first one being con-
stituted by endothelin-I (ET-1)2 and angiotensin II (AII)
two potent vasoconstrictive peptides, which are respec-
tively processed by endothelin converting enzyme (ECE-1,
EC 3.4.24.71) and angiotensin converting enzyme
(ACE, EC 3.4.15.1). The second system considered as
physiologically antagonist to the first one, is constituted
by bradykinin (Bk) as vasorelaxant peptide, and the
atrial natriuretic peptide (ANP) which reduces heart
overload by increasing diuresis. The inactivation of the
two latter peptides is essentially controled by ACE and
neutral endopeptidase (neprilysin, NEP, EC 3.4.24.11)
which, like ECE-1, belong to the same family of zinc
metallopeptidases.3 Recent studies have shown that the
effects of dual inhibitors of ACE and NEP appear
superior to those observed with specific ACE inhibitors
like captopril in treatment of severe cardiovascular
diseases.4 However, ET-1 which is the most potent
vasoconstrictive factor is inactivated by NEP, thus
minimizing the vasorelaxant properties of dual ACE/
NEP inhibitors. Therefore, simultaneous inhibition of
NEP, ACE, and ECE by a single inhibitor is expected to
produce a therapeutic efficacy superior to that observed
with dual inhibitors5 with possible use in several dis-
eases including essential hypertension, chronic heart
failure, pulmonary hypertension, vascular remodeling,
renal dysfunction, prostatic hypertrophy and ischemia.6
Up to now, only some compounds acting as inhibitors
of the three enzymes have been reported, most of them
issued from the few pharmaceutical firms involved in
this field.7,8 We have recently designed a lead compound
1 (Table 2), capable of inhibiting NEP, ACE and ECE
with Ki values of 1.8, 20, and 100 nM respectively.9
However, it was necessary to optimize the compound 1
in order to obtain inhibitors nearly equipotent on the
three targeted enzymes. This has been achieved by
introducing various substituents on the indanyl moiety
of the P01 residue, leading to the first inhibitors of NEP,
ACE and ECE with nanomolar inhibitory potencies.
*Corresponding author. Tel.: +33-1-4353-9558; fax: +33-1-4326-
6918; e-mail: roques@pharmacie.univ-paris5.fr
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00248-2

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N. Inguimbert et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2001–2005
Scheme 1. Synthesis of the inhibitors 7–17. Method A: (a) Me3SiBr, CHCl3; (b) triethylphosphonoacetate, NaH, DMF; Method B: (a) Me3SiBr,
THF ꢀ78 ꢁC ; (b) triethylphosphonoacetate, NaH, THF ꢀ78 ꢁC; (c) PhB(OH)2, DME, Na2CO3 ; (d) (H2CO)n, THF, K2CO3; (e) NaOH, acetone;
(f) thioacetic acid, CHCl3; (g) BBr3, CH2Cl2; (h) EDCI, HOBt, HAAOt-Bu, Et3N; (i) TFA, CH2Cl2; (j) NaOH, then HCl, MeOH.
The synthesis, biochemical and preliminary pharmaco-
logical properties of these new vasopeptidase inhibitors
(patent Fr No. 00 01937/17 February 2000) are reported
in this paper. The target molecules 7–17 were pre-
pared (Scheme 1) using the synthetic route previously
described9 starting from various substituted 1-indanol
2a–i as precursors (Table 1). Due to the presence of two
unresolved asymmetric carbons in synthon 6, this
method affords the inhibitors as a mixture of four
stereoisomers.
Nevertheless, we have previously shown that the stereo-
chemistry of metallopeptidase inhibitors is important
for optimal recognition of the enzymes.
It was therefore critical to compare the relative activities
of the separate isomers.
The best compounds 8 and 15 were selected for separ-
ation and structure determination. The separation of
the four stereoisomers was done by semi-preparative
HPLC, as previously described,9 and the determination
Table 1. Various substituted 1-indanol precursors
Table 2. In vitro inhibition of NEP,15 ACE16 and ECE,17 activities
by compounds 1 and 7–17
Compd 2 a10
b11
c12
d13
e10
f10
g10
h14 i10
R1
R2
R3
H
H
H
H
H
OMe
H
H
H
H
OMe
H
H
CN Br
OMe SMe NMe2 OEt OMe
OMe
H
H
H
H
H
H
The compounds thus prepared were tested in an initial
screening as a mixture of stereoisomers for their ability
to inhibit NEP, ACE and ECE activities in vitro
according to previously described assays. Table 2 sum-
marizes the results obtained by introduction of various
groups in the phenyl ring of the indanyl moiety of 1.
Methoxy, methylthio or bromide group which have
about the same dimension were introduced in R2 posi-
tion of the indanyl group (8, 11, and 15) leading to a
significant improvement in the recognition of ECE (Ki
22–35 nM) without change on NEP and ACE affinities
(Table 2). Conversely, the introduction of one methoxy
in R1 or R3 position or two methoxy in R2 and R3
positions of the indanyl moiety decreases the inhibitory
potency on ECE (7, 9, and 10). The presence of a basic
substituent on the P01 moiety (12) is also unfavourable
for ECE recognition. The same result was observed
when linear group (17) or bulky substituent (16) is
introduced at the R2 position of the indanyl group. In
the first round of assays, the various inhibitors were
tested as a mixture of four stereoisomers (Table 2).
Ki (nM)a
Compd
R1
R2
R3
NEP
ACE
ECE
1
7
8
9
10
11
12
13
14
15
16
17
H
OMe
H
H
H
H
H
H
H
H
H
OMe
H
OMe
SMe
NMe2
OEt
OH
Br
H
H
H
OMe
OMe
H
H
H
H
H
1.8ꢂ0.2
16ꢂ3
5ꢂ0.2
11ꢂ1
78ꢂ7
6ꢂ0.1
15ꢂ1
8ꢂ2
20ꢂ2
8ꢂ1
100ꢂ10
760ꢂ40
22ꢂ2
11ꢂ1
35ꢂ3
78ꢂ5
14ꢂ1
44ꢂ3
18ꢂ4
7ꢂ0.2
12ꢂ2
27ꢂ7
3ꢂ0.2
79ꢂ3
560ꢂ20
35ꢂ2
370ꢂ10
47ꢂ3
4ꢂ0.1
5ꢂ0.1
14ꢂ1
11ꢂ1
90ꢂ6
H
H
H
31ꢂ3
Ph
CN
H
H
140ꢂ15
210ꢂ10
aValues are means of three experiments performed in triplicate. All
compounds are mixture of four stereoisomers.

N. Inguimbert et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2001–2005
2003
of their absolute configuration was achieved by NMR
spectroscopy.9 The chemical shift of the indanyl CH2
protons in the vicinity of the substituted asymmetric
carbon (C3, Fig. 1) are dependent on the configuration
of the C2, C3 carbons as shown in Table 3. This has
been clearly established previously with dipeptides18 or
thiol-derived inhibitors.19 X-ray crystallography was
used to confirm the absolute configuration proposed
from NMR in the case of compound 15c. For this
purpose, replacement of the tryptophan at the P02
position by alanine was achieved in order to facilitate
structure assignment of synthon 6i1 obtained in
enantiomeric pure form by resolution with R-(+)-a-
methylbenzylamine (Scheme 2). Coupling of 6i1 with
alanine gives compound 18c (Scheme 2). Suitable
crystals were obtained by crystallization in acetonitrile.
X-ray crystallography confirms the 2S,3R absolute
configuration of 18c and thus of 6i1 (Fig. 1).
Scheme 2. Synthesis of the inhibitors 15c and 18c.
recognition with Ki values in the 10ꢀ8 molar range (8b,
15b). These values are of the same order than the Ki
values obtained for CGS 31447.21 This inhibitor has
been synthesized in our laboratory with the aim to
investigate its inhibitory potency towards ACE which
was not reported. As shown in Table 4, CGS 31447 has
a low affinity for this zinc metallopeptidase. Further-
more, it can be noticed that in this series of inhibitors
the stereochemical preferences for ACE and ECE are
different. Thus the 2S,3R stereoisomers are the best
ACE inhibitors whereas the most favourable configur-
ation is 2R,3R for optimal ECE inhibition (Table 4).
NEP displays no clear stereochemical preference
although the best inhibitors belong to the 2R,3R ser-
ies.22 This is consistent with the close similarity of NEP
and ECE active sites.23,24 Due to their efficient inhibitory
potencies in vitro on NEP, ACE, and ECE compound
15c was used to measure its in vivo activity. Furthermore,
Figure 1. Ortep20 view of compound 18c.
As observed in a previous study with compound 1,9 the
2S,3R configuration leads to good NEP/ACE inhibition
with Ki values in the nanomolar range for the tested
compounds 8 and 15 (Table 4). The 2R,3R configur-
ations of 8 and 15 are the most efficient regarding ECE
Table 4. In vitro inhibition of NEP, ACE and ECE, for the pure
stereoisomers of compounds 1, 8 and 15
Table 3. 1H NMR data for compounds 8(a–d), 15(a–d) and 18c
Ki (nM)a
Compd
Stereochemistry
NEP
ACE
ECE
Compd
Stereo
Chemical shifts DMSO-d6
of X protons (d ppm)
[a]2Á3
SCH 244707
CGS 3144714
1a
8a
8b
8c
8d
15a
15b
15c
15d
90b
2.5b
>10000
43ꢂ2
80b
11ꢂ1
2.2ꢂ0.4
0.7ꢂ0.03
30ꢂ2
8a
8b
8c
8d
15a
15b
15c
15d
18c
2S,3S
2R,3R
2S,3R
2R,3S
2S,3S
2R,3R
2S,3R
2R,3S
2S,3R
1.8, 2.0a
1.65
+36.2
ꢀ16.1
+25.2
ꢀ48.2
+11.1
ꢀ5.8
2R,3R
2S,3S
2R,3R
2S,3R
2R,3S
2S,3S
2R,3R
2S,3R
2R,3S
26ꢂ3
70ꢂ2
1460ꢂ240
10ꢂ1
1.3ꢂ0.1
2.7ꢂ0.3
15ꢂ2
24ꢂ3
1.95
1.5, 1.70a
1.8, 2.0a
1.65
4.6ꢂ0.5
30ꢂ4
100ꢂ8
180ꢂ7
110ꢂ10
18ꢂ3
9.4ꢂ0.3
1.8ꢂ0.3
3.8ꢂ0.3
13ꢂ0.7
58ꢂ6
55ꢂ4
1.95
1.55, 1.70a
2.1
+38.0
ꢀ25.9
ꢀ18.7
4.1ꢂ0.3
28ꢂ2
66ꢂ5
90ꢂ10
aValues are means of three experiments performed in triplicate.
bIC50
Optical rotations were recorded in acetone c=1.
aThe values correspond to chemical shifts of non equivalent protons.
.

2004
N. Inguimbert et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2001–2005
injection of AI in vehicle at 1 nmol/kg iv increased MAP
by 15ꢂ2 mm Hg. In comparison, the sodium salt of 15
administered iv at 30 mg/kg (59 mmol/kg) 5 min prior to
the AI injection completely blocked the pressor
response induced by AI.
The in vivo inhibition of lung NEP in mice was carried
out as previously described.27 A70% blockade of the
enzyme is observed for compound 15c injected at 25mg/
kg iv.
In conclusion, the new potent triple inhibitor of NEP,
ACE and ECE, 15c generated after optimization of the
lead compound 1 fulfils the requirements of potency for
investigations on animal models of cardiovascular dis-
eases. This compound is the first reported inhibitor able
to jointly block in vivo the three enzymes involved in
the regulation of blood arterial pressure. Further phar-
macological studies are necessary to shed more light on
the advantages of this first described triple inhibitor
over dual NEP/ACE inhibitors in order to validate the
concept of triple inhibition.
Figure 2. Inhibition of Big ET-1 (1 nmol/kg, iv) pressor response
by compound 15c iv administered in anesthetized rats. *p<0.05,
**p<0.01 versus control group (Dunnett’s test).
Supplementary material
Crystal data for 18c: formula C21H28BrNO4S mono-
clinic, [a]2Á3=ꢀ18.7 space group P21; a=8.532(3),
b=9.667(4), c=14.571(8) A, b=105.56(2), V=1158(1)
A3, Z=2, 7294 independent reflections, 3920 with I>2
s(I), R1=0.0735, wR2=0.2120. The structure was
solved by SHELXS-9728 and refined using SHELXL-
97.29 Crystallographic data (excluding structure factors)
have been deposited to the Cambridge Crystallographic
Data Center as supplementary publication no. CCDC
177861. Copies of the data can be obtained, free of
charge, on application to CCDC, 12 Union Road,
Cambridge, CB2 1EZ, UK (fax: +44-1223-336033;
e-mail: deposit@ccdc.cam.ac.uk).
Figure 3. Inhibition of angiotensin I (1 nmol/kg, iv) pressor response
by compound 15 iv administered in anaesthetized rats. *p<0.05 versus
control group (Dunnett’s test).
15c was choosen because it can be obtained in larger
amounts than 18c following the synthetic pathway
described in Scheme 2.
Compound 15c was evaluated for its ability to inhibit
the pressor response produced by big ET-1 in anesthe-
tized (pentobarbital sodium 60 mg/kg ip), ganglion-
blocked (chlorisondamine 2 mg/kg iv) male Wistar rats
(Fig. 2).25 Intravenous injection of 1 nmol/kg big ET-1
in rats treated with vehicle at 1 mL/kg iv (150 mL EtOH,
150 mL Chremophor, 10 mL DMSO, 1350 mL saline)
resulted in an 57.2ꢂ4.6 mmHg increase in mean arterial
pressure (MAP).
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