Design of orally active dual inhibitors of neutral endopeptidase and angiotensin-converting enzyme with long duration of action

Article abstract of DOI:10.1021/jm950783c

Mercaptoacyl dipeptides, containing a glycine linked to a C-terminal 5- phenylproline, have been synthesized in order to obtain new highly efficient dual inhibitors of the two zinc metallopeptidases, neutral endopeptidase (NEP) and angiotensin-converting

Full text of DOI:10.1021/jm950783c

2594
J . Med. Chem. 1996, 39, 2594-2608
Design of Or a lly Active Du a l In h ibitor s of Neu tr a l En d op ep tid a se a n d
An gioten sin -Con ver tin g En zym e w ith Lon g Du r a tion of Action
Marie-Claude Fournie-Zaluski,† Pascale Coric,† Vincent Thery,† Walter Gonzalez,‡ Herve´ Meudal,†
Serge Turcaud,† J ean-Baptiste Michel,‡ and Bernard P. Roques*,†
De´partement de Pharmacochimie Mole´culaire et Structurale, U266 INSERM - URA D 1500 CNRS, UFR des Sciences
Pharmaceutiques et Biologiques - Faculte´ de Pharmacie 4, avenue de l’Observatoire, 75270 Paris Cedex 06, France, and
De´partement de Physiologie et Pathologie Expe´rimentale Vasculaires, U 367 INSERM,
17 rue du Fer a` Moulin, 75005 Paris, France
Received October 23, 1995X
Mercaptoacyl dipeptides, containing a glycine linked to a C-terminal 5-phenylproline, have
been synthesized in order to obtain new highly efficient dual inhibitors of the two zinc
metallopeptidases, neutral endopeptidase (NEP) and angiotensin-converting enzyme (ACE),
which are involved in the control of blood pressure and fluid homeostasis. These compounds
have been designed (i) to fit optimally the ACE pharmacophore previously described (Fournie´-
Zaluski, M. C.; et al. J . Med. Chem. 1994, 37, 1070-1083), through interaction with the S1,
S1′, and S2′ subsites of this enzyme, (ii) and to interact with the S1′ and S2′ subsites of NEP
with the 5-phenylproline moiety outside the catalytic domain (Coric, P.; et al. J . Med. Chem.
1996, 39, 1210-1219). Replacement of Gly by Ala in these mercaptoacyl dipeptides induced
an about 100-fold decrease in ACE inhibition. This shows that, in agreement with molecular
modeling studies, a steric constraint as weak as a methyl group hinders optimal ACE active
site recognition. Among these compounds, the dual inhibitor 26 (RB 106) (Ki, ACE ) 0.35 nM;
NEP ) 1.6 nM) showed excellent pharmacokinetic properties with an almost complete in vivo
inhibition of NEP and ACE for more than 4 h after oral administration in mice of a low dose
(2.6 × 10-5 mol/kg) of the inhibitor. Moreover, RB 106 remained active 12 h after oral
administration. In spontaneous hypertensive rats, a chronic treatment of orally administered
RB 106 (25 mg/kg/day) induced a prolonged hypotensive effect (-28 mmHg) still significant 2
days after the end of the treatment. In DOCA salt rats, a hypotensive response and a significant
natriuresis were observed after iv administration. RB 106, which is one of the most potent
dual inhibitors described to date, could have interesting clinical applications in long term
treatment of congestive heart failure and myocardial ischemia.
In tr od u ction
natriuretics properties, without loss of potassium in
various experimental models of hypertension,9-14 but
displayed hypotensive effects only in DOCA salt rats.14-16
Moreover, in patients with heart failure, no significant
reduction in arterial blood pressure and left ventricular
dimensions was observed in spite of the persistent
elevation of plasma ANP levels.17,18 These limited
effects of NEP inhibitors were attributed to the opposite
actions of Ang II and ANP on blood pressure and sodium
excretion, suggesting that coinhibition of NEP and ACE
could be a more efficient approach in the treatment of
cardiovascular diseases.19 Preliminary experiments
have shown that the association of selective inhibitors
of NEP and ACE leads to synergetic beneficial proper-
ties in animal models of hypertension20,21 and congestive
heart failure.22,23 Therefore numerous dual inhibitors
of NEP and ACE have been synthesized, but in most
cases their pharmacological properties have not been
reported.24 The first dual inhibitor described, HS-CH2-
CH(CH2Ph)-CONH-CH[CH2CH-(CH3)2]-COOH,25-27 later
designated SQ-28133,20 elicited hypotensive activity in
spontaneous hypertensive rats (SHR) and deoxycorti-
costerone acetate salt (DOCA salt) hypertensive rats
and potentiated ANP and bradykinin hypotensive re-
sponses in SHR.20 Alatrioprilate, a closely related
derivative of the selective NEP inhibitor thiorphan, HS-
CH2-CH(CH2Ph)-CONH-CH2-COOH,28 was shown to
increase diuresis, natriuresis, and cGMP excretion after
iv administration in anesthetized rats, in agreement
The physiological control of blood pressure and fluid
homeostasis is mainly dependent on regulatory pep-
tides: angiotensin II (Ang II) which induces vasocon-
striction and indirectly sodium retention through al-
dosterone secretion1 and atrial natriuretic peptide (ANP)
which increases diuresis and natriuresis and has a
slight vasodilatatory action. Furthermore, ANP reduces
plasma renin and aldosterone levels.2 The activity of
these two antagonistic systems is regulated essentially
by metabolizing processes involving the zinc metal-
lopeptidases, angiotensin-converting enzyme (ACE, EC
3.4.15.1) and neutral endopeptidase (NEP, EC 3.4.24.11,
neprilysin). ACE is responsible for the maturation of
Ang II from its inactive precursor angiotensin I (Ang
I), and NEP inactivates ANP.3 Moreover, both pepti-
dases are involved in the metabolism of bradykinin,4,5
a vasodilatatory peptide.
Specific inhibitors of ACE6,7 are currently used in the
treatment of hypertension and congestive heart failure
but are generally associated with diuretics which can
be responsible for side effects such as activation of
pressor system and kaliuresis. Selective inhibitors of
NEP8 have been recently shown to have diuretics and
* To whom correspondence should be adressed. Tel: (33) 43-25-
50-45. Fax: (33) 43-26-69-18.
† U266 INSERM - URA D 1500 CNRS.
‡ U 367 INSERM.
X Abstract published in Advance ACS Abstracts, J une 1, 1996.
S0022-2623(95)00783-7 CCC: $12.00 © 1996 American Chemical Society

Orally Active Dual Inhibitors of NEP and ACE
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 13 2595
and short.36 This could result from various factors such
as the production of inactive metabolites, the degrada-
tion of these modified peptides, or their rapid renal
elimination caused by a too high hydrophilicity. It was
therefore necessary to improve the bioavailability of
these dual inhibitors, for instance, by increasing their
hydrophobicity, without decreasing their inhibitory
properties toward ACE and NEP. As ACE was reported
to contain a large and continuous S1′-S2′ subsite,39
a
comparison of the structure of A and B showed that
introduction of the phenyl moiety of A on the proline
ring of B could result in potent inhibitors of ACE with
enhanced lipophilicity, provided that the amino acid
preceding the substituted proline did not hinder the
phenyl ring from fitting the enzyme active site. This
was achieved by introducing a glycine moiety in the new
series of dual inhibitors of type C (Figure 1). Substi-
tuted proline and thiazolidinecarboxylic acid derivatives
have been previously used to obtain selective ACE
inhibitors in both the captopril and enalapril series.6
Given the similarity in their structures, these inhibitors
could be assumed to interact with NEP and ACE as
their parent compounds A and B.36 The synthesis and
biochemical and pharmacological properties of these
new potent and orally active dual inhibitors are reported
in this paper.
F igu r e 1. Schematic representation of the dual NEP-ACE
inhibitors A, [(2S)-2-mercapto-3-phenylpropanoyl]-Ile-Tyr, and
B, [(2S)-2-mercapto-3-phenylpropanoyl]-Ala-Pro,36 which led
to the design of the new inhibitor C, [2-mercapto-3-phenyl-
propanoyl]-Gly-(5-Ph)Pro. The structure of the previously
designed31 dual inhibitor RB 105 is also shown.
Resu lts
Syn th esis. Scheme 1 summarizes the two synthetic
pathways used for the preparation of the inhibitors. The
key step was the condensation of the highly hindered
5-phenylproline derivatives with either N-(acetylthio)-
alkanoyl amino acids (pathway I) or N-protected amino
acids (pathway II). In both cases, the yields were
around 30-50%, whatever the coupling reagents used
(DCC/HOBt, BOP, PyBrop...). Nevertheless, pathway
II using the acyl chloride method of coupling for the
introduction of glycine residue was preferred because
the purification steps were found to be easier. The
various (acetylthio)alkanoic acids 1 were prepared from
the corresponding R-amino acids by the classical two-
step synthesis.40 When optically pure R-amino acids
were used, compounds 1 were obtained with a large
degree (>90%) of enantiomeric enrichment.
The various 5-phenylproline derivatives 2, substituted
or not on the phenyl ring, have been synthesized
(Scheme 2) following the procedures described by Ohta
et al.41 for alkylation of pyroglutamate derivatives and
by Ezquerra et al.42 for the synthesis of the ∆1-pyrroline
derivatives using pyroglutamic esters as starting ma-
terials. These methods have the advantage of giving
the 5-phenylprolines with the same absolute configu-
ration at the R-carbon as the initial pyroglutamates.
Furthermore, in the last step of the synthesis, a catalytic
hydrogenation of the ∆1-pyrroline yielded a cis proline
derivative, while a chemical reduction with sodium
borohydride43 led to a mixture of cis and trans proline
isomers which could be easily separated by crystalliza-
tion43 allowing the trans isomers to be isolated and
studied separately.
with its ability to inhibit renal NEP.29 However, the
relatively weak in vitro inhibitory potency of SQ-28133
and alatrioprilate for ACE limits their in vivo efficien-
cies.30
In contrast, the dual inhibitor RB 105 (or S 21402)
(Figure 1) synthesized in our laboratory displays Ki
values in the nanomolar range for NEP and ACE.30
Intravenous infusion of RB 105 decreased blood pres-
sure and increased natriuresis in three animal models
of hypertension (renovascular, DOCA salt,31 and spon-
taneously hypertensive rats32), and strong dose-depend-
ent hypotensive effects were observed after oral admin-
istration in spontaneously hypertensive rats.31 MDL-
100,240, another orally active thiol-containing dual
inhibitor of NEP and ACE, was reported to have similar
properties to RB 105.33,34
Given their properties, dual inhibitors could be used
in the long term treatment of chronic mild hypertension
or myocardial ischemia.35,64 This requires compounds
with a long duration of action as the second generation
of dual inhibitors. This could be achieved by increasing
the affinities for both NEP and ACE. With this aim,
we have recently synthesized mercaptoacyl dipeptides
such as A and B36 (Figure 1) endowed with nanomolar
and subnanomolar inhibitory potencies toward NEP and
ACE, respectively. These compounds were shown to
interact with the S1, S1′, and S2′ subsites of ACE,
whereas, using both molecular modeling and experi-
ments with mutated Glu102NEP, they have been dem-
onstrated to fit the S1′ and S2′ subsites of NEP with the
C-terminal amino acid located at the surface of the
enzyme.36 This later result agrees with previously
reported data on the localization of various thiol inhibi-
tors within the NEP active site.37,38 However, the in
vivo inhibition of lung ACE induced by oral administra-
tion of A and B, in mice, was found to be relatively weak
In Vitr o In h ibitor y P oten cies. The inhibitory
activities of the newly synthesized compounds were
measured on NEP, purified from rabbit kidney,44 and
ACE, purified from rat testes.45 The IC50 values of the
initial compound 6 and analogues containing a single

2596 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 13
Fournie-Zaluski et al.
Sch em e 1. Scheme for the Synthesis of the Dual NEP-ACE Inhibitorsa
Sch em e 2. Scheme for the Synthesis of 5-Phenylproline
subsite is well known to be devoid of stereoselective
preference.8,46 The introduction of a substituent in the
R2 position (compound 8) induced an almost 100-fold
loss of potency for ACE and a 6-fold decrease for NEP.
On the other hand, the replacement of the benzyl side
chain in position R1 of 6 by aliphatic moieties, either
hydrophobic (compound 9) or positively charged (com-
pound 10), was unfavorable for NEP and ACE as shown
by the large enhancement in the respective IC50 values.
Conversely, the presence of substituents, such as OH
(compound 11) or NH2 (compound 12), on the benzyl
moiety of 6 was well accepted by NEP but decreased
slightly the recognition of ACE (factor of 8). Substitu-
tions on the phenyl ring of the phenylproline moiety by
a hydroxyl group in the meta position (compound 14)
or by hydrophobic substituents (compounds 15 and 16)
were slightly detrimental for ACE recognition. In
contrast, 13 (OH group in ortho position) was one of the
most potent compounds of this new series of dual
inhibitors.
Derivativesa
For all the compounds reported in Table 1, the
5-phenylproline moiety has a (2S,5R) configuration. The
influence of this stereochemical parameter on enzyme
inhibition was investigated by using inhibitors with a
5-(ortho-methylphenyl)proline moiety (Table 2). As
compared to 15 (2S,5R), which has a cis configuration,
the inversion of the two asymmetric carbons of the
proline ring in 17 (2R,5S) induces 2- and 10-fold
increases in the IC50 values for NEP and ACE, respec-
tively. For the (2S,5S) (trans isomer) 18, the loss of
activity was more important for ACE with IC50 values
of 48 nM (factor of 16) than for NEP, IC50 ) 17 nM
(factor of 3). The last stereoisomer 19 (2R,5R) was also
less potent than 15 (IC50, NEP ) 25 nM; ACE ) 112
nM).
modification at the level of R1, R2, or R3 are reported in
Table 1. As expected from our initial hypothesis,
compound 6 was efficient in inhibiting both enzymes
with IC50 values in the nanomolar range on NEP and
subnanomolar range for ACE. As expected from previ-
ous studies on selective ACE inhibitors,6 the inversion
of configuration of the mercaptoacyl moiety (compound
7) was responsible for a large decrease in ACE recogni-
tion. The absence of change observed on NEP supports
the localization of the benzyl group of the mercaptoacyl
moiety within the S1′ subsite of NEP37,38 since this
Furthermore, the position of the methyl group on the
5-phenylproline substituent had no significant influence
on the inhibitory potency (compare 15, 16, and 23), and
the presence of a hydroxyl group on the benzyl moiety

Orally Active Dual Inhibitors of NEP and ACE
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 13 2597
Ta ble 1. In Vitro Inhibitory Potencies on NEP and ACE of Various Mercaptoacyl Dipeptides
IC50 (nM)a
no.
R1
abs conf*
R2
R3
NEP
ACE
6
7
8
CH2Ph
CH2Ph
CH2Ph
CH(CH3)CH2CH3
(CH2)4NH3
CH2(p-OH)Ph
CH2(p-NH2)Ph
CH2Ph
CH2Ph
CH2Ph
S
R
S
S
H
H
CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
1.6 ( 0.3
3.2 ( 0.6
10 ( 2
0.55 ( 0.05
25 ( 2
45 ( 5
9
50 ( 7
3.7 ( 0.8
+
10
11
12
13
14
15
16
R + S
500 ( 50
3.4 ( 0.8
1.8 ( 0.4
1 ( 0.5
2.4 ( 0.4
5.3 ( 1.0
2.3 ( 0.6
403 ( 57
S
S
S
S
S
S
4.2 ( 0.4
3.5 ( 0.5
0.5 ( 0.1
1.5 ( 0.5
3.1 ( 0.3
6.2 ( 1.9
o-OH
m-OH
o-CH3
m-CH3
H
H
CH2Ph
a
IC50 values are the mean ( SEM from three independent experiments performed in triplicate.
Ta ble 2. In Vitro Inhibitory Potencies on NEP and ACE of
Various Mercaptoacyl Dipeptides: Influence of 5-Phenylproline
Stereochemistry
Ta ble 3. Influence of R1 and R2 Substituents on the in Vitro
Inhibitory Potencies on NEP and ACE of Various Mercaptoacyl
Dipeptides
IC50 (nM)a
abs conf*
C2 C5
IC50 (nM)a
no.
R1
R3
NEP
ACE
abs
no.
R1
conf*
R3
NEP
ACE
15 CH2Ph
17 CH2Ph
18 CH2Ph
19 CH2Ph
20 CH2(p-OH)Ph
21 CH2(p-OH)Ph
16 CH2Ph
22 CH2(p-OH)Ph
23 CH2Ph
S
R
S
R
S
R
S
S
R
S
S
R
R
S
R
R
o-CH3
o-CH3
o-CH3 17 ( 0.7
o-CH3 25 ( 3
o-CH3
o-CH3
m-CH3
m-CH3
5.3 ( 1.0
8.5 ( 1.6
3.1 ( 0.3
33 ( 7
25 CH2(p-OH)Ph
26 CH2(p-OH)Ph
27 CH2(p-OH)Ph
28 CH2(m-OH)Ph
29 CH2(p-OCH3)Ph
30 CH2(p-OH)Ph
31 CH2(p-OCH3)Ph
32 CH2(p-OH)Ph
33 CH2(p-Cl)Ph
34 CH2(p-F)Ph
S
S
S
o-OH
3.5 ( 0.3
1.6 ( 0.2
0.6 ( 0.2
0.7 ( 0.1
48 ( 5
m-OH
p-OHb
6.36 ( 0.08 0.46 ( 0.02
112 ( 13
5.0 ( 0.3
12.5 ( 1.7
3.1 ( 0.3
3.1 ( 1.6
6.8 ( 1.4
2.5 ( 0.5
2.7 ( 0.5
5.1 ( 1.1
2.3 ( 0.6
1.5 ( 0.3
R,S m-OH
4.3 ( 0.6
2.2 ( 0.5
3.2 ( 0.8
0.42 ( 0.05
4.5 ( 0.1
2.2 ( 0.8
23 ( 3
0.9 ( 0.1
3.8 ( 0.4
5.3 ( 0.7
2.5 ( 0.4
S
S
S
S
S
S
o-OH
o-OCH3 3.4 ( 0.8
o-OCH3 2.5 ( 0.4
cis (racemic) p-CH3 12.5 ( 3.5
b
p-NH2 26 ( 3
24 CH2(p-OH)Ph cis (racemic) p-CH3
5.5 ( 0.8
o-OH
m-OH
1.2 ( 0.2
5.3 ( 0.3
62 ( 1
a
IC50 values are the mean ( SEM from three independent
35 CH2(p-COOH)Ph R,S o-OH
experiments performed in triplicate.
MDL-100,173c
2.2 ( 0.4
a
of the inhibitors (20-22 and 24) is devoid of significant
IC50 values are the mean ( SEM from three independent
b
experiments performed in triplicate. These compounds possess
effect on the recognition of both peptidases recognition.
a racemic (cis) 5-phenylproline. c The Ki values given in ref 50 for
MDL-100,173 are 0.11 and 0.08 nM for ACE and NEP, respec-
tively.
The inhibitory potencies of compounds bearing sub-
stituents of different polarities on the aromatic cycles
are reported in Table 3. It was observed that, when the
benzyl moiety was substituted by a p-OH group (25 to
27), the compounds had a similar activity on the two
enzymes, whatever the position (ortho, meta, or para)
of the hydroxyl group on the phenyl ring of the proline
moiety. Compounds with a para methoxy (29) or a para
chlorobenzyl (33) group also behaved as efficient inhibi-
tors of both enzymes. The introduction of o-OCH3 as
an R3 substituent decreased activity for ACE by 1 order
of magnitude. A large decrease in inhibitory potencies
for both enzymes was observed when R3 ) p-NH2
(compound 32), even taking into account that this
molecule has a racemic phenylproline moiety. Finally,
the presence of a carboxyl group in 35 is particularly
unfavorable for NEP inhibition.
Molecu la r Mod elin g. Molecular modeling studies
were performed with the aim of defining the biologically
active conformations of compound 6 in the active site
of ACE. This was done for ACE by using the previously
described template D30 developed by using the rigid
potent inhibitor (IC50 ) 4 nM) 3-(mercaptomethyl)-
3,4,5,6-tetrahydro-2-oxo-1H-1-benzazocine-1-acetic acid
(MTBA)47 and the proposed biologically active confor-
mation of captopril.48
Two families of conformers characterized by the
presence of the proline peptide bond in cis (16%) and
trans (84%) forms were obtained following molecular
dynamic studies of 6 (as described in materials and
methods), in good agreement with the results of NMR

2598 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 13
Fournie-Zaluski et al.
Ta ble 4. Conformers of Compound 6 Fitting the Constrained
ACE Inhibitor Structure D30 Determined by a
Template-Forcing Minimization Procedure
Ta ble 5. In Vivo Inhibition of Lung ACE and Kidney NEP 2 h
after Oral Administration of a Single Dose of the Dual
Inhibitors (c ) 2.6 × 10-5 mol/kg)
rmsd
dev
E(pot)
conformersa
ψ2
φ2
ψ1
ø1
(kcal mol-1
)
b
b
b
b
inhibition (%)
no.
R
R3
lung ACE
kidney NEP
F1
F2
F3
F4
174 -89 -43
174 -92 -41
59 0.326
178 0.323
47 -179 0.325
54 67 0.330
11.9
12.9
11.1
14.1
6
15
23
11
20
24
25
26
27
H
H
H
H
63 ( 3
57 ( 5
42 ( 10
NDa
60 ( 5
33 ( 2
82 ( 7
89 ( 3
ND
75 ( 5
37 ( 5
34 ( 6
27 ( 7
44 ( 3
3 ( 1
69 ( 7
72 ( 11
25 ( 9
-175
-175
87
84
o-CH3
p-CH3
H
o-CH3
p-CH3
o-OH
m-OH
p-OH
a
p-OH
p-OH
p-OH
p-OH
p-OH
p-OH
The atoms taken into account for the rms calculations are
indicated in the formula of D and 6 by dots. The dihedral angles
(in degrees) are defined as follows: ψ2, N6-C7-C8-N9; φ2, C5-
N6-C7-C8; ψ1, S4-C3-C5-N6; ø1, C1-C2-C3-C5.
b
experiments. As the proline peptide bond is trans in
the bioactive conformation of ACE inhibitors,48 only the
trans conformers were submitted to a template-forcing
procedure using the template D, leading to two groups
of molecules composed of 12 and 5 members, respec-
tively, which differ only by the values of the φ2 angle,
-85° ( 10° or 85° ( 10°. All these molecules fit
correctly (rmsd < 0.35) the ACE template D. The
physical characteristics of the four molecules (F1-F4)
having the lowest potential energies are reported in
Table 4.
In the case of 8, the alanine analogue of 6, we did not
find any low-energy conformers able to overlap the D
template with satisfactory values of rmsd. This is very
likely due to the steric hindrance induced by the
5-phenyl substituent of Pro since the removal of this
group led to a compound inhibiting ACE with an IC50
value of 0.3 nM.36 On the other hand, a template-
forcing procedure was achieved with D, compound 6,
and the two recently described highly constrained cyclic
dual inhibitors MDL-100,17350 and BMS-182,657.24b In
both cases, a good overlap (rmsd < 0.4) on the pharma-
cophore and on the conformer (F2) of 6 was observed
(Figure 2). In each case, the orientation of the 2-mer-
capto-3-phenylpropanoyl side chain corresponds to the
proposed biologically active conformation of the enala-
prilate side chain (C6H5CH2CH2CH(COOH))-Ala-Pro.39
Furthermore, it could be observed that the phenyl rings
of the three dual inhibitors partially overlapped. This
defines a large hydrophobic domain which could be filled
with lipophilic moieties interacting with the continuous
S1′-S2′ subsite of ACE.
a
ND: not determined.
The results are summarized in Table 5. The modulation
of the in vivo activity was found more important for
NEP than for ACE, and, as previously noticed, with dual
inhibitors belonging to the RB 105 series,30 there is no
strict relationship between the in vitro IC50 values and
the in vivo efficiencies of these molecules. However,
compounds with IC50 values >100 nM were found to be
inactive (not shown).
The unsubstituted inhibitor 6 exhibited a high in vivo
efficiency on both enzymes since, 2 h after oral admin-
istration, 65% of lung ACE and 75% of kidney NEP were
still blocked. The introduction of hydrophobic groups
in position R3 (compounds 15 and 23) was detrimental
for both enzymes but particularly for NEP. The pres-
ence of only one hydrophilic R group (compound 11)
decreased significantly the in vivo inhibition of NEP,
whereas the conjunction of hydrophilic and hydrophobic
groups in R and R3 positions, respectively (compound
20), restored partially the inhibition of both enzymes
except when the methyl group (R3) was in the para
position (compound 24). Conversely, the introduction
of hydroxyl groups on both R3 and R positions (com-
pounds 25 and 26) yielded inhibitors endowed with good
bioavailabilities, except, once more, when R3 ) p-OH
(27). On the basis of these results, compound 26 which
was, along with 6, the most efficient in vivo dual
inhibitor of this series, was preferred to the latter due
to its better solubility. A time course analysis of the
inhibition of NEP and ACE by 26 and the efficient dual
inhibitor MDL-100,17350 was achieved. As shown in
Figure 3, the two dual inhibitors, orally administered
in mice at the same dose (2.6 × 10-5 mol/kg), have
almost identical in vivo potencies at short times (60 and
120 min), but compound 26 has a longer duration of
action since inhibition of both NEP (Figure 3A) and ACE
(Figure 3B) was still significant 12 h after administra-
tion.
In Vivo In h ib it ion of Kid n ey NE P a n d Lu n g
ACE. The most efficient compounds, selected from their
in vitro inhibitory potencies on NEP and ACE, were
tested to evaluate their in vivo activities by previously
described methods.30 In an initial screening, a single
dose of inhibitor was orally given to mice (2.6 × 10-5
mol/kg) and the inhibition of both enzymes was mea-
sured 2 h after administration. The in vivo efficiency
of the compounds was determined by inhibition of the
binding of a tritiated probe ([3H]trandaloprilate) to lung
ACE and by inhibition of the degradation of the tritiated
substrate [3H]-D-Ala2-Leu-enkephalin by kidney NEP.
P h a r m a cologica l Resp on ses. The antihyperten-
sive activity of the dual inhibitor 26 was tested after
oral administration in SHR. As shown in Figure 4
increasing concentrations of 26, administered daily for

Orally Active Dual Inhibitors of NEP and ACE
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 13 2599
F igu r e 2. (A) Schematic representation of 6, MDL-100,173,50 and BMS-182,657.24b (B) Stable conformers of 6, MDL-100,173,
and BMS-182,657 fitting the previously proposed ACE pharmacophore D (dotted line). (C) Stereoview of the overlapped assumed
biologically active conformation at the ACE active site level of 6 (bold line), MDL-100,173 (dotted line), and BMS-182,657 (medium
line). The superimposition shows the similarity in the spatial disposition of the SH, amide, and carboxyl groups in the three
inhibitors. Moreover, the large hydrophobic domain evidenced by the partial overlap of the phenyl moiety linked to the cyclic
moieties supports the occurrence of a corresponding continuous lipophilic S1′-S2′ subsite in the ACE active site.
5 days, induced a dose-dependent decrease in blood
pressure, statistically significant from 5 mg/kg.
action. This property is crucially dependent on the
affinity of the inhibitors for both peptidases which must
be as high as possible and on their hydrophobic-
hydrophilic balance. This latter parameter is assumed
to control the renal elimination of the compounds with
subsequent inhibition of NEP in the proximal tubule
and the life time in the plasma which must be long
enough to ensure a prolonged blockade of vascular
ACE.31 With this aim, the recently synthesized mer-
captoacyl dipeptides HS-CH(CH2Ph)CO-Ile-Tyr, A, and
HS-CH(CH2Ph)CO-Ala-Pro, B (Figure 1), characterized
by IC50 values in the nanomolar range for NEP and
ACE,36 were used as models. The improvement in
pharmacokinetic properties was obtained by introducing
a phenyl moiety in the proline ring of molecules of type
B in which Ala was replaced by Gly. In addition to
being endowed with a similar lipophilicity but to be
more easily synthesized than the constrained tricyclic
A
maximum reduction of 28 mmHg was observed for 25
mg/kg, and a plateau was observed for higher doses.
Moreover the maximal effect was obtained after 2 days
of treatment (Figure 5). At the end of the experiment,
the recovery of the initial blood pressure required 3
days, its reduction still remaining significant after 48
h (Figure 5).
Compound 26 was also iv administered to DOCA salt
rats at 25 mg/kg-1 + 25 mg/kg-1 h-1 as previously
described.14,32 A decrease in mean blood pressure was
observed (Figure 6A) associated with a significant
increase in natriuresis (Figure 6B) during the time of
the experiment.
Discu ssion
In this work, we report new orally active dual inhibi-
tors of NEP and ACE endowed with a long duration of

2600 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 13
Fournie-Zaluski et al.
F igu r e 5. Variation in blood pressure measured during
chronic oral administration of 25 mg/kg/day RB 106 (compound
26) to SHR.
F igu r e 3. (A) Time course of renal NEP inhibition in mice
after oral administration (2.6 × 10-5 mol/kg) of RB 106
(compound 26) (4) and MDL-100,17350 (9). (B) Time course of
lung ACE inhibition in mice after oral administration (2.6 ×
10-5 mol/kg) of RB 106 (compound 26) (4) and MDL-100,17350
(9).
F igu r e 4. Variation in mean blood pressure observed in
response to increasing doses of daily orally administered RB
106 (compound 26) in SHR; *p < 0.05.
inhibitor MDL-100,17350 or the bicyclic compound BMS-
182,657 reported during this work,24b the mercaptoacyl
dipeptide inhibitors of type C, described here, possess
a residual flexibility which is expected to facilitate their
adaptation in the active site of the two peptidases. The
position of the phenyl substituent on the proline ring
was selected following molecular modeling studies car-
ried out with the recently designed ACE pharmacophore
D in which the bicyclic moiety of MTBA is assumed to
fit the S1′ and S2′ subsites of ACE, which were proposed
to constitute a continuous hydrophobic domain.30,39 The
best overlap with this ACE template was obtained with
the cis isomer of the (2S)-5-phenylproline. The strong
in vitro inhibitory potencies (IC50 ) 1.6 nM for NEP and
0.55 nM for ACE) of 6, which contains the selected
5-phenylproline isomer, confirmed the validity of this
strategy. Moreover, 6 has the advantage of possessing
two aromatic rings which could be easily substituted by
F igu r e 6. (A) Variation in blood pressure measured after iv
administration of 25 mg kg-1 bolus + 25 mg kg-1 h-1 of RB
106 to DOCA salt rats. The arrow represents the begining of
the treatment: b, control animals; O, treated animals. (B)
Variation in natriuresis measured after iv administration of
25 mg kg-1 bolus + 25 mg kg-1 h-1 of RB 106 to DOCA salt
rats. The arrow represents the begining of the treatment:
white stippled bars, control animals; black stippled bars,
treated animals.
hydrophilic or hydrophobic groups in order to modulate
the in vivo targeting of endothelial ACE and epithelial
NEP.30
The conformational analysis of 6 showed that the four
conformers fitting the D template with the lowest
energy differ by the relative disposition of the thiol and
carboxyl group and the spatial orientation of the mer-
captoacyl moiety, defined by ø1 (Table 4). The con-

Orally Active Dual Inhibitors of NEP and ACE
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 13 2601
former F2 could correspond to the biologically active
form of ACE carboxyl inhibitors such as enalapril and
derivatives which have a P1 benzyl moiety as the dual
inhibitors reported here.6
the essential characteristics of the active site were not
respected. For instance, NEP prefers aromatic residues,
uncharged at physiological pH, in the S1′ subsite.25,27
Accordingly, compounds 9, 10, and 35 which contain
aliphatic or charged P1′ residues have IC50 values in the
10-8-10-7 M range.
Moreover, a template-forcing procedure using 6 and
the rigid dual inhibitors MDL-100,17350 and BMS-182,-
65724b showed that these two compounds also have an
extended conformation of F2 type (Figure 2) and could
bind to the ACE active site in the same manner as 6.
Moreover, the superimposition of these assumed biologi-
cally active conformations shows a partial overlap of the
phenyl rings linked to the cyclic moieties supporting the
occurrence of a large and continuous hydrophobic S1′-
S2′ subsite in ACE.39 It is important to observe that
the mutual orientation of the SH and COOH groups in
6 (conformer F2) defined by the value of the dihedral
For ACE, the various inhibitors interact with the
three S1, S1′, and S2′ subsites, and consequently their
activities were more sensitive to changes affecting the
corresponding P1-P2′ side chains. For the R1 group,
which interacts with the S1 subsite, the benzyl chain in
6 was preferred to an aliphatic residue in 9 and the (S)
configuration of the 2-mercaptoacyl moiety was critical
(compare 6 and 7), as previously shown for carboxyalkyl
dipeptides belonging to the enalapril series.6 Moreover,
the replacement of the central glycine by an alanine
(compound 8 versus 6) led to a 100-fold decreased
activity indicating, as confirmed by molecular modeling,
that the simultaneous presence of a 5-phenyl substitu-
ent on Pro and a methyl group on the preceding amino
acid hinders the molecule from reaching conformations
of low energies fitting the ACE template. This is clearly
illustrated by the nanomolar IC50 values found in
corresponding compounds in which the 5-phenyl group
has been removed.36 In accordance with molecular
modeling studies done with the ACE template D, the
optimal interaction with the S1′-S2′ domains of this
peptidase requires a (2S,5R) configuration for the
5-phenylproline moiety. This is illustrated by the IC50
of compound 15 which was more than 10 times better
than those of 17-19 (Table 2).
The inhibitory potency measured ex vivo after oral
administration in mice appeared to be a good reflection
of the capacity of a tested compound to reach the target
enzymes.30 For NEP, which has essentially a kidney
epithelial localization although the enzyme is also
present in the vascular wall52 and heart,35 it was
striking to observe that the best bioavailabilities were
obtained with the initial inhibitor 6 and the two bis-
hydroxylated derivatives 25 and 26 which are more
hydrophilic. The simplest explanation is that 6 is
rapidly metabolized by hydroxylation of the aromatic
rings. All the other compounds tested were significantly
less efficient (Table 5). For ACE, the results were not
very different. Thus, the unsubstituted compound 6 has
a relatively good in vivo activity, and the introduction
of hydrophilic substituents (compounds 25 and 26)
slightly improved the bioavailability. Curiously, this
was not the case with compounds 11 and 27. Com-
pounds 25 and 26 were the most efficient in this
prescreening test with more than 80% of lung ACE
inhibition and around 70% of kidney NEP inhibition 2
h after a single oral administration of 2.6 × 10-5 mol/
kg. Even though these two compounds have the same
in vivo efficiency, the choice to develop 26 (RB 106) for
a complete pharmacological study was influenced by
chemical considerations due to the global yield of its
synthesis which was always found to be significantly
better than that of 25. Compounds 26 and 6 were
shown to behave as competitive inhibitors of NEP and
ACE. The Ki values of 26 (RB 106), calculated from the
Cheng-Prussof equation, were 1.6 and 0.35 nM for NEP
and ACE, respectively.
angle θ between the noncontinuous atoms S-CRPhe
-
CRPro-CCOOH, θ ) 24°, is similar to that found in the
biologically active conformation of ACE inhibitors, θ )
29°.47,48
The position of 6 inside the NEP active site is very
likely identical with that of B which has been estab-
lished recently by using both molecular modeling stud-
ies and the mutated Glu102NEP.36 In these complexes,
the C-terminal proline residue is located at the surface
of the enzyme active site. The absence of important
hydrophobic interactions with the enzyme surface of the
C-terminal amino acid of 6 is clearly illustrated by the
similarity in the IC50 values for NEP of this compound
and its precursor HS-CH(CH2Ph)-CONH-CH2COOH
which are 1.6 and 2.1 nM, respectively. The corre-
sponding IC50’s toward TLN were 1.2 and 2.2 µM,
respectively, emphasizing again the great analogies
between the active sites of TLN and NEP.46,49,51 Fur-
thermore, due to the location of the 5-phenylproline
moiety outside the active site of NEP, there are no great
differences in the inhibitory potencies of the four ste-
reoisomers 15 and 17-19. The slight changes observed
could be due to steric contacts with the enzyme’s
surface.
The computed conformation of 6 inside TLN (ψ2 )
163°, φ2 ) -146°, ø1 ) -45°) and by extension inside
NEP, obtained as previously described,36 is not very
different from that (F2) fitting the ACE template (Table
4). This shows that the molecule is capable of binding
to the NEP or ACE active site without major confor-
mational readjustment. Likewise, conformers of similar
low energy fitting the ACE and NEP templates were
found in MD-100,173. The binding of these two families
of conformers to their adapted enzymes seems more
convincing than the proposed recognition of ACE and
NEP by MD-100,173 with the amide bond of the
molecule under trans or cis conformation, respectively.50
This would require an isomerization process thermo-
dynamically highly unfavorable.
The concept of mixed inhibitors is based on the
occurrence of important similarities in the active sites
of the targeted peptidases.27,8 This is the case for NEP,
ACE, and TLN which are able to cleave in vitro peptides
such as enkephalins or bradykinin at the same peptide
bond.8 Nevertheless, due to subtle differences in the
NEP and ACE active sites, the various changes intro-
duced in 6 had different effects on each enzyme. For
NEP, almost all the compounds synthesized have in-
hibitory potencies in the nanomolar range, except when
When orally administered in mice at 2.6 × 10-5 mol/
kg, RB 106 led to a prolonged inhibition of renal NEP

2602 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 13
Fournie-Zaluski et al.
derivatives was performed following Winkle et al.:54 O-(meth-
oxymethyl)-2-bromophenol, yellow oily product (91%), Rf (B)
0.71; O-(methoxymethyl)-3-bromophenol, yellow oily product
(92%), Rf (C) 0.60; O-(methoxymethyl)-4-bromophenol, yellow
oily product (94%), Rf (C) 0.64. The protection of the phenol
group of Tyr was performed as described by Koga et al.55
The purity of the synthesized compounds was checked by
thin-layer chromatography on silica gel plates (Merck 60F 254)
in the following solvent systems (v/v): A, n-hexane/EtOAc )
9/1; B, n-hexane/EtOAc ) 8/2; C, n-hexane/EtOAc ) 7/3; D,
n-hexane/EtOAc ) 6/4; E, n-hexane/EtOAc ) 5/5; F, n-hexane/
EtOAc/AcOH ) 7/3/0.5; G, n-hexane/EtOAc/AcOH ) 6/4/0.5;
H, CH2Cl2/MeOH ) 9/1; I, CH2Cl2/MeOH ) 8/2; J , CH2Cl2/
MeOH ) 7/3; K, CH2Cl2/MeOH/AcOH ) 9/1/0.5; L, CH2Cl2/
MeOH/AcOH ) 8/2/0.5; M, CH2Cl2/MeOH/AcOH ) 7/3/0.5; N,
CH2Cl2/MeOH/AcOH ) 6/4/0.5; O, n-BuOH/AcOH/H2O ) 4/1/
1. Plates were revealed with UV, iodine vapor, or ninhydrin.
The purity of the final compounds was checked by HPLC on a
reverse phase chromasil C8 (5 µm, 100 Å) column (SFCC) with
0.05% TFA (solvent A)/CH3CN (solvent B), as the mobile phase,
on a Shimadzu apparatus. The eluted peaks were monitored
at 210 nm.
and lung ACE (Figure 3). Thus, 12 h after RB 105
administration, the blockade of both peptidases was still
higher than 50%. As expected the duration of action of
RB 106 was longer than that of a mercaptoacyl dipeptide
analogue, HS-CH(CH2Ph)CO-Ala-Pro, lacking the 5-
phenyl substituent.36 In identical conditions the inhibi-
tion of NEP and ACE was around 50% after 3 h with
the latter compound, while with RB 106, inhibition of
both enzymes was still at maximal levels. The phar-
macokinetic properties of RB 106 appear to be signifi-
cantly better than those of the tricyclic inhibitor MDL-
100,173, particularly regarding ACE inhibition (Figure
3B). This could be related to the in vitro potencies of
both compounds since, in our hands, the IC50 values of
MDL-100,173 on NEP and ACE were 2.5 ( 0.4 and 2.2
( 0.4 nM, respectively, i.e., slightly higher than the
corresponding values (1.6 ( 0.2 and 0.6 ( 0.2 nM) of
RB 106 (Table 3). Nevertheless various factors, includ-
ing chemical stability, bioavailability, and metabolism,
could explain the observed differences in the duration
of action of both compounds.
In agreement with these results, RB 106 was found
to be very efficient in decreasing the blood pressure of
spontaneous hypertensive rats after oral administration
at a dose as low as 5 mg/kg (Figure 4). Even more
interesting, RB 106 exhibited a strong (-28 mmHg) and
prolonged antihypertensive activity in SHR. Thus, at
least 2 days were required so that rats, orally treated
for 5 days with 25 mg/kg/day RB 106, recovered their
initial blood pressure (Figure 5). As expected from its
dual inhibitory potency, RB 106 induced also a signifi-
cant decrease in blood pressure associated with an
increase in diuresis (not shown) and natriuresis in the
DOCA salt model of hypertension (Figure 6).
In conclusion, the presence of a hydrophobic phenyl
group on the 5 position of proline in a series of 2-mer-
capto-3-phenylpropanoyl-Gly-(5-Ph)-Pro compounds led
to very potent orally active dual inhibitors of NEP and
ACE endowed with, as expected, a long duration of
action. One of these compounds (RB 106) has slightly
but significantly better in vitro and in vivo affinities for
NEP and ACE than the dual inhibitors reported to date
including the highly constrained polycyclic compounds
MDL-100,173 and BMS-182,657. In addition, its phar-
macokinetic properties seem also to be more favorable
than those of the latter compounds. This could be partly
due to a higher flexibility of RB 106 allowing a better
adaptation to various targets, including putative intes-
tinal proline transporters, to be obtained. Owing to the
strong and persistent reduction of blood pressure ob-
served after chronic oral administration in SHR, RB 106
warrants an extensive pharmacological evaluation now
in course in our laboratories.
The structure of all the compounds was confirmed by 1H
NMR spectroscopy (Bru¨ker AC 270 MHz) in DMSO-d6 using
HMDS as an internal reference. Melting points of the crystal-
lized compounds were determined on an Electrothermal ap-
paratus and are reported uncorrected. Satisfactory analyses
were obtained (C, H, N) for all final compounds. The optical
purity of the various compounds was verified by HPLC.
Abbr evia tion s: Et2O, ethyl ether; MeOH, methanol; EtOH,
ethanol; BuOH, n-butanol; EtOAc, ethyl acetate; AcOH, acetic
acid; DMF, dimethylformamide; THF, tetrahydrofuran; DCC,
dicyclohexylcarbodiimide; DCU, dicyclohexylurea; HOBt, 1-hy-
droxybenzotriazole; TFA, trifluoroacetic acid; Et3N, triethyla-
nine; DMSO, dimethyl sulfoxide; HMSD, hexamethyldisilox-
ane.
Gen er a l P r oced u r e for th e Syn th esis of 2-(Acetylth io)-
a lk a n oic Acid s CH3COSCH(R1)COOH 1. The various
2-(acetylthio)alkanoic acids were prepared in two steps as
previously described40 from the corresponding R-amino acids.
Briefly, the R-amino acids (1 equiv) were dissolved in a mixture
of HBr 47% (8 equiv) and H2O (2/3, v/v). At 0 °C, a solution of
NaNO2 (6.5 equiv) in H2O was added. After the classical
treatment the 2-bromoalkanoic acids were isolated. The
2-bromoalkanoic acids (1 equiv) were dissolved in DMF, and
a solution of CH3COSK (1 equiv) in DMF was added at 0 °C
under inert atmosphere. The crude products were purified by
chromatography on silica gel.
1a : R1 ) CH2Ph; oily product (98%); Rf (O) 0.70; NMR δ
2.25, CH3CO; 2.88-3.24, CH2â; 4.17, CHR; 7.18, Ar; 12.85,
COOH. 1b: R1 ) CH(CH3)CH2CH3; oily product (78%); Rf (F)
0.64; NMR δ 0.82, 2 × CH3; 1.20-1.30, CH2γ; 1.92, CHâ; 2.30,
CH3CO; 4.10, CHR; 12.30, COOH. 1c: R1 ) CH2(p-OCO2CH2-
Ph)Ph; oily product (60%); Rf (G) 0.45; NMR δ 2.25, CH3CO;
2.90-3.12, CH2â; 4.16, CHR; 5.20, OCH2; 7.10, 7.22, 7.38, Ar;
12.85, COOH. 1d : R1 ) CH2(p-NO2)Ph; oily product (88%);
Rf (A) 0.64; NMR δ 2.30, CH3CO; 3.10-3.33, CH2â; 4.30, CHR;
7.52-8.14, Ar; 12.90, COOH. 1e: R1 ) CH2(p-Cl)Ph; oily
product (90%); Rf (F) 0.31; NMR δ 2.26, CH3CO; 2.90-3.12,
CH2â; 4.18, CHR; 7.18-7.30, Ar; 12.84, COOH. 1f: R1 ) CH2-
(p-F)Ph; oily product (90%); Rf (K) 0.60; NMR δ 2.26, CH3CO;
2.86-3.12, CH2â; 4.15, CHR; 7.04-7.18, Ar; 12.92, COOH.
1g: R1 ) CH2(p-COOCH3)Ph; white solid; mp 220 °C dec (90%);
Rf (N) 0.37; NMR δ 2.25, CH3CO; 2.96-3.24, CH2â; 3.78, OCH3;
4.20, CHR; 7.30-7.80, Ar; 12.88, COOH. 1h : R1 ) CH2(p-
OCH3)Ph; oily product (40%); Rf (G) 0.42; NMR δ 2.28, CH3-
CO; 2.85-3.04, CH2â; 3.68, OCH3; 4.10, CHR; 6.79-7.08, Ar;
12.80, COOH. 1i: R1 ) (CH2)4NH(Boc). 1i was prepared as
Exp er im en ta l Section
Ch em istr y. The natural amino acid derivatives were
purchased from Bachem (Bubbendorf, Switzerland) and the
nonnatural amino acids and reagents from Aldrich-Chemie
(Strasbourg, France). The synthesis of MDL-100,173 was
performed following procedures described,50 and para-car-
bomethoxyphenylalanine was prepared by alkylation of the
diphenylimine of tert-butyl glycinate with methyl 4-(bromom-
ethyl)benzoate as described by O’Donnell et al.53 followed by
acid hydrolysis of the diphenylimine and tert-butyl ester
groups: white solid; mp 220 °C dec; Rf (L) 0.31; NMR δ 3.20,
CH2â; 3.85, OCH3; 4.30, CHR; 7.42-7.93, Ar; 8.30, NH3+. The
protection of 2-, 3-, and 4-bromophenols as methoxymethyl
a
racemic mixture from 6-aminohexanoic acid, and this
compound was brominated on the R-carbon by Br2 in the
presence of PBr3. Thus, after protection of the amino group
by a tert-butyloxycarbonyl group, the thioacetylation was
performed as described: oily product (59%); Rf (F) 0.61; NMR
δ 1.35, Boc; 1.10-1.80, (CH2)3; 2.30, CH3CO; 2.84, CH2(NH);
3.90, CHR; 6.75, NH; 12.86, COOH.
Gen er a l P r oced u r es for th e Syn th esis of th e 5-(Su b-

Orally Active Dual Inhibitors of NEP and ACE
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 13 2603
stitu ted p h en yl)p r olin e Der iva tives 2. (a ) Syn th esis of
(S)(or R)-Eth yl 5-Keto-2-Boc-a m in op en ta n oa te. The vari-
ous (substituted phenyl)magnesium bromides were prepared
by addition of a solution of the substituted phenyl bromides
(1 equiv) to magnesium (1.1 equiv) in dry THF. The mixtures
were kept under reflux until complexion of the reaction (2-3
h) and then were cooled at 0 °C and transferred into a solution
of (S)- or (R)-ethyl N-Boc-pyroglutamate (0.85 equiv) in dry
THF at -40 °C, under argon atmosphere. After 1 h at -40
°C and 1 h at 0 °C, the reaction was quenched with HOAc/
MeOH (1:1) and the mixture diluted with Et2O. The organic
layers were washed with H2O and brine, dried over Na2SO4,
filtered, and evaporated leading to crude products, which were
purified.
m-CH3; oily product (96%); Rf (C) 0.34; NMR δ 1.18, CH3-
(CH2O); 1.50-1.90, CH2âPro; 2.00, CH2γPro; 2.25, CH3(Ar);
3.75-4.05, CHR + ∂Pro; 4.05, CH2O; 6.95-7.13, Ar. 2i: R3 )
p-CH3; white foam; Rf (K) 0.48; NMR δ 1.20, CH3(CH2O); 2.10-
2.30, CH2â + γPro; 2.30, CH3(Ph); 4.08, OCH2; 4.56, CHR +
∂Pro; 7.20-7.42, Ar.
(d ) Syn th esis of (2S)- a n d (2R)-tr a n s-Eth yl 5-(or th o-
Meth ylp h en yl)p r olin a te (2j,k ). The (S) and (R) isomers of
ethyl 5-(o-methylphenyl-∆1-pyrroline-2-carboxylate were sa-
ponified by alkaline hydrolysis, and the corresponding acids
were dissolved in water and treated, at 0 °C, by a solution
(1.02 equiv) of NaBH4 in water. The mixtures were stirred
overnight at 0 °C, acidified at pH 2, and evaporated in vacuo.
A 50/50 mixture of cis- and trans-(2S) or (2R)-5-(o-methyl-
phenyl)proline was obtained (HPLC C8 nucleosil CN, CH3CN/
H2O ) 2/98; (tR ) 5.90 and 7.25 min). The trans isomer was
precipitated as a tosylate salt (HPLC tR ) 7.25 min) as
described43 (70%). The ethyl ester of the (2S) and (2R) trans
isomers was prepared by treatment with EtOH and SOCl2
(98%): Rf (K) 0.56; NMR δ 1.20, (OCH2)CH3; 1.40-1.90, CH2â;
2.20, CH2γ; 2.25, CH3(Phe); 4.05-4.50, CHR + ∂Pro; 4.10,
OCH2; 7.10-7.50, Ar.
R3 ) H: recrystallization in n-hexane/ether ) 6/1; white
solid; mp 85 °C (71%); Rf (E) 0.65. R3 ) o-OCH2OCH3:
chromatography in n-hexane/ethyl acetate ) 9/1; oily product
(42%); Rf (B) 0.22. R3 ) m-OCH2OCH3: chromatography in
n-hexane/ethyl acetate ) 9/1; white solid; mp 62 °C (80%); Rf
(C) 0.23. R3 ) p-OCH2OCH3: chromatography in n-hexane/
ethyl acetate ) 9/1; oily product (78%); Rf (C) 0.25. R3
)
o-OCH3: chromatography in n-hexane/ethyl acetate ) 8.5/1.5;
oily product (76%); Rf (D) 0.51. R3 ) o-CH3: chromatography
in n-hexane/ethyl acetate ) 9/1; oily product (30%); Rf (A) 0.27.
R3 ) m-CH3: chromatography in n-hexane/ethyl acetate ) 8/2;
white solid; mp 88 °C (40%); Rf (B) 0.20. R3 ) o-CH3 ((2R)
configuration): chromatography in n-hexane/ethyl acetate )
9/1; oily product (35%); Rf (A) 0.27. R3 ) p-CH3: chromatog-
raphy in n-hexane/ethyl acetate ) 9/1; oily product (42%); Rf
(B) 0.24. R3 ) NHCOCH3: chromatography in n-hexane/ethyl
acetate ) 8/2; oily product (46%); Rf (C) 0.50.
Gen er a l P r oced u r e for th e Syn th esis of N-2-(Acetyl-
th io)-3-a lk a n oyl Am in o Acid s CH 3COSCH(R1)CONHCH-
(R2)COOH 3. To a solution of the 2-(acetylthio)alkanoic acid
in CHCl3 were added, at 0 °C, a solution of the chlorhydrate
of tert-butyl R-amino ester (1 equiv) and Et3N (1 equiv) in dry
THF, a solution of HOBt (1 equiv) in THF, and DCC (1.1 equiv)
in CHCl3. After stirring overnight at room temperature, DCU
was filtered and the mixture evaporated in vacuo. The residue
was taken up in EtOAc and washed as usual. The crude
compounds were dissolved in CH2Cl2 (0.5 mL/mmol), and 10
equiv of TFA was added at 0 °C. After stirring for 30 min at
0 °C and for 3 h at room temperature, the mixture was
evaporated in vacuo and the residue triturated in Et2O.
3a : R1 ) CH2Ph, R2 ) H; oily product (90%); Rf (A) 0.57;
NMR δ 2.25, CH3CO; 2.80-3.16, CH2â(Ph); 3.63, CH2Gly; 4.30,
CHR(Ph); 7.20, Ar; 8.50, NH; 12.50, COOH. 3b: R1 ) CH2-
Ph, R2 ) CH3; white paste (87%); Rf (A) 0.50; NMR δ 1.20,
CH3(Ala); 2.20, CH3CO; 2.74-3.20, CH2â(Ph); 4.10, CHRAla;
(b) Syn th esis of Eth yl 5-(Su bstitu ted p h en yl)-∆1-p yr -
r olin e-2-ca r boxyla te. The ethyl 5-keto-2-Boc-aminopen-
tanoates were dissolved in CH2Cl2 (1 mL/mmol), and 10 equiv
of TFA was added at 0 °C. The mixture was stirred for 2 h at
the same temperature. After evaporation in vacuo, the residue
was taken off with CH2Cl2, washed (×3) with a 10% solution
of NaHCO3, H2O, and brine, and dried over Na2SO4. After
filtration and evaporation the cyclic compounds were isolated
and used without further purification.
R3 ) H: oily product (94%); Rf (E) 0.54. R3 ) o-OH: oily
product (92%); Rf (C) 0.44. R3 ) m-OH: oily product (90%);
Rf (C) 0.19. R3 ) o-OCH3: oily product (85%); Rf (E) 0.56. R3
) o-CH3: oily product (92%); Rf (E) 0.65. R3 ) m-CH3: oily
product (93%); Rf (C) 0.37. R3 ) o-CH3 ((2R) configuration):
oily product (88%); Rf (C) 0.46. R3 ) p-OH: oily product (91%);
Rf (G) 0.28. R3 ) p-NH2: oily product (92%); Rf (G) 0.50. R3
) p-CH3: oily product (88%); Rf (K) 0.25.
(c) Syn th esis of (2S)- or (2R)-cis-Eth yl 5-(Su bstitu ted
p h en yl)p r olin a te 2. The various ethyl 5-(substituted phen-
yl)-∆1-pyrroline-2-carboxylates were dissolved in EtOH (3 mL/
mmol), and Pt2O (3 mg/mmol) was added. The mixtures were
hydrogenated at room temperature under 30 bars of pressure.
After filtration of the catalyst, the organic layers were evapo-
rated in vacuo and the crude products used without further
purification.
2a : R3 ) H; oily product (99%); Rf (E) 0.41; NMR δ 1.20,
(OCH2)CH3; 1.50-1.86, CH2âPro; 2.02, CH2γPro; 3.80-4.08,
CHR + ∂Pro; 4.08, OCH2; 7.20-7.38, Ar. 2b: R3 ) p-NH2; oily
product; Rf (M) 0.52; NMR δ 1.20, CH3(CH2O); 1.42-1.90,
CH2â + γPro; 3.72-3.85, CHR + ∂Pro; 4.05, OCH2; 4.84, NH2;
6.48-7.00, Ar. 2c: R3 ) o-OH; white solid (92%); Rf (C) 0.44;
mp 140 °C dec; NMR δ 1.20, CH3(CH2O); 1.60-1.85, CH2âPro;
2.00-2.15, CH2γPro; 3.82-4.22, CHR + ∂Pro; 4.05, OCH2;
6.60-7.00, Ar; 9.80, OH. 2d : R3 ) m-OH; white solid (98%);
Rf (K) 0.24; mp 150 °C dec; NMR δ 1.18, CH3(CH2O); 1.45-
1.88, CH2âPro; 2.00, CH2γPro; 3.72-4.00, CHR + ∂Pro; 4.10,
OCH2; 6.50-6.75-7.00, Ar; 9.18, OH. 2e: R3 ) p-OH; oily
product; Rf (N) 0.66; NMR δ 1.20, CH3(CH2O); 2.10-2.30, CH2â
+ γPro; 4.10, CH2O; 4.60, CHR + ∂Pro; 6.80-7.30, Ar; 9.10,
OH. 2f: R3 ) o-OCH3; oily product (99%); Rf (D) 0.28; NMR
δ 1.15, CH3(CH2O); 1.40-1.80, CH2âPro; 2.02, CH2γPro; 3.70,
OCH3; 3.75-4.35, CHR + ∂Pro; 4.04, CH2O; 6.85-7.10-7.55,
Ar. 2g: o-CH3; oily product (97%); Rf (C) 0.41; NMR δ 1.18,
CH3(CH2O); 1.35-1.85, CH2âPro; 2.05, CH2γPro; 2.20, CH3-
(Ar); 3.78-4.28, CHR + ∂Pro; 4.05, CH2O; 7.02-7.60, Ar. 2h :
4.30, CHR(Ph); 7.20, Ar; 8.50, NH; 12.40, COOH. 3c: R1
)
CH(CH3)CH2CH3, R2 ) H; white paste (75%); Rf (A) 0.37; NMR
δ 0.85, 2 × CH3; 1.20-1.50, CH2γ; 1.85, CHâ; 2.30, CH3CO;
3.68, CH2Gly; 4.05, CHR; 8.40, NH; 12.40, COOH.
Gen er a l P r oced u r e for th e Syn th esis of Eth yl(or
m eth yl) N-Glycyl-5-(su bstitu ted p h en yl)p r olin a tes 4. (1)
(Z)-Glycine (1 equiv) dissolved in acetone was treated with
cyanuric chloride (0.5 equiv) in the presence of Et3N (1 equiv)
at room temperature. After stirring for 3 h, the solvent was
evaporated in vacuo and the residue dissolved in CH2Cl2. The
solution of ethyl(or methyl) 5-(substituted phenyl)prolinate (1
equiv) was added, and the mixture was stirred overnight at
room temperature. After evaporation, the residue was taken
up with ethyl acetate, washed with 10% NaHCO3 (×3), H2O,
10% citric acid (×3), H2O, and brine, and dried over Na2SO4.
After filtration and evaporation, the crude products was
purified by chromatography on silica gel using n-hexane/ethyl
acetate (5/5) as eluents.
(2) The protected dipeptides were dissolved in ethanol (or
methanol) and hydrogenated over 10% Pd/C in the presence
of 1.1 equiv of HCl. After completion of the reaction the
catalyst was filtered and the mixture evaporated in vacuo. The
crude product was used for the following step without further
purification.
4a : R3 ) p-CH3, R ) CH3; (1) white foam (65%); Rf (E) 0.29;
(2) white solid; mp 115 °C dec (92%); Rf (L) 0.48. 4b: R3
)
p-OH, R ) CH3; (1) white foam (20%); Rf (E) 0.27; (2) white
solid; mp 105 °C (75%); Rf (L) 0.20. 4c: R3 ) H, R ) CH3; (1)
white foam (33%); Rf (E) 0.13; (2) oily product (94%); Rf (K)
0.23. 4d : R3 ) o-OH, R ) C2H5; (1) oily product (42%); Rf (E)
0.20; (2) white wax (95%); Rf (L) 0.58. 4e: R3 ) m-OH, R )
CH3; (1) white solid; mp 173 °C (33%); Rf (E) 0.13; (2) white
foam (99%); Rf (L) 0.51. 4f: R3 ) o-OCH3, R ) C2H5; (1)
colorless oil (45%); Rf (D) 0.19; (2) white foam (85%); Rf (K)
0.42. 4g: R3 ) o-CH3, R ) C2H5; (1) oily product (42%); Rf (E)
0.21; (2) white solid; mp 209 °C (94%); Rf (K) 0.37. 4h : R3 )

2604 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 13
Fournie-Zaluski et al.
m-CH3, R ) C2H5; (1) colorless oil (43%); Rf (E) 0.35; (2) white
solid (98 °C dec) (95%); Rf (L) 0.62. 4l: R3 ) o-CH3, R ) C2H5
(trans isomer); oil (35%); Rf (E) 0.52; (2) hygroscopic solid
(78%); Rf (K) 0.50.
527
:
R1 ) CH2(p-OCOOCH2Ph)Ph, R2 ) H, R3 ) p-NH2;
chromatography in CH2Cl2/MeOH/AcOH ) 9/2/0.1; oily product
(25%); Rf (K) 0.35. 528: R1 ) CH2(p-Cl)Ph, R2 ) H, R3 ) o-OH;
chromatography in n-hexane/ethyl acetate ) 4/6; oily product
(53%); Rf (E) 0.38. 529: R1 ) CH2(p-F)Ph, R2 ) H, R3 ) m-OH;
chromatography in n-hexane/ethyl acetate ) 4/6; white foam
(42%); Rf (E) 0.31. 530: R1 ) CH2(p-COOCH3)Ph, R2 ) H, R3
) o-OH; chromatography in n-hexane/ethyl acetate/acetic acid
) 6/4/0.5; white solid mp 105 °C dec (51%); Rf (E) 0.25.
Gen er a l P r oced u r e for th e P r ep a r a tion of N-[N-(2-
Mer ca p toa lk a n oyl)glycyl]-5-(su bstitu ted p h en yl)p r olin e.
The protected inhibitors (1 equiv) were dissolved in degassed
methanol, and 1 N NaOH (5 equiv) was added at 0 °C under
inert atmosphere. The mixtures were stirred for 2 h at room
temperature. After acidification with 2 N HCl, the methanolic
layers were evaporated, diluted in H2O, and extracted with
degassed EtOAc. The organic layers were washed with H2O
and brine, dried over Na2SO4, and evaporated in vacuo. All
the final compounds were obtained as mixtures of cis (∼20%)
and trans (∼80%) rotamers around the Gly-Pro bond. This
was easily observed on the NMR spectra by the occurrence of
two well-separated peaks for both the CHR and CH∂ protons
of proline.
Com p ou n d 6: oily product (81%); Rf (A) 0.59; HPLC (45%
B) 11.8 min; NMR ∂ (ppm) : 1.78-2.10-2.35, CH2â + γPro;
2.63, SH; 2.70-3.00, CH2âPh; 3.10-3.85, CH2Gly; 3.68, CHRPh;
4.30(t)-4.69(c), CHRPro; 4.90(c)-5.10(t), CH∂Pro; 7.13-7.35-
7.60, Ar; 8.02, NH; 12.40, COOH.
Com p ou n d 7: white solid; mp 92 °C (98%); Rf (K) 0.80;
HPLC (50% B) 7.94 min; NMR δ 1.80-2.12-2.40, CH2â +
γPro; 2.60, SH; 2.68-3.00, CH2âPh; 3.10-3.85, CH2Gly; 3.65,
CHRPh; 4.32(t)-4.70(c), CHRPro; 4.90(c)-5.09(t), CH∂Pro;
7.12-7.35-7.60, Ar; 8.01, NH; 12.50, COOH.
Com p ou n d 8: white solid; mp 98 °C (83%); Rf (G) 0.20;
HPLC (50% B) 8.7 min; NMR δ 1.10, CH3Ala; 2.10-2.35, CH2â
+ γPro; 2.70, SH; 2.80-3.00, CH2âPh; 3.60, CHRPh; 4.28(t)-
4.60(c), CHRPro; 4.80, CHRAla; 4.90(c)-5.02(t), CH∂Pro; 7.20-
7.60, Ar; 8.40, NH; 12.60, COOH.
Com p ou n d 9: white foam (95%); Rf (K) 0.70; HPLC (50%
B) 8.7 min; NMR δ 0.80, 2 × CH3Ile; 1.10-1.35, CH2γIle; 1.62-
1.80-2.16, CHâIle + CH2â + γPro; 2.40, SH; 3.10-3.95, CH2-
Gly; 3.40, CHRIle; 4.35(t)-4.72(c), CHRPro; 4.95(c)-5.12(t),
CH∂Pro; 7.20-7.60, Ar; 8.05, NH; 12.65, COOH.
Com p ou n d 10: white solid (90%); Rf (K) 0.18; HPLC (28%
B) 6.8 min; NMR δ 1.20-1.70, CH2â + γ + ∂, hexanoic chain;
1.80-2.13-2.38, CH2â + γPro; 2.55, SH; 2.68, CH2(NH3+);
3.03-3.85, CH2Gly; 3.75, CHS; 4.31(t)-4.70(c), CHRPro; 4.90-
(c)-5.09(t), CH∂Pro; 7.20-7.31-7.60, Ar; 7.65, NH3+; 7.85, NH;
12.60, COOH.
Com p ou n d 11: white solid (90%); Rf (M) 0.89; HPLC (28%
B) 6.9 min; NMR δ 2.12-2.38, CH2â + γPro; 2.56, SH; 2.70-
2.85, CH2âPh; 3.05-3.85, CH2Gly; 3.52, CHRPh; 4.30(t)-4.70-
(c), CHRPro; 4.90(c)-5.08(t), CH∂Pro; 6.55-7.20-7.30, Ar;
7.80, NH; 9.10, OH; 12.55, COOH.
Syn th esis of Meth yl(or eth yl) N-[N-[2-(Acetylth io)-
a lk a n oyl]glycyl]-5-(su bstitu ted p h en yl)p r olin a tes 5. To
a solution of 2-(acetylthio)alkanoic acids (1 equiv) in THF were
successively added at 0 °C a solution of ethyl(or methyl)
N-glycyl-5-(substituted phenyl)prolinate, hydrochloride (1 equiv),
and Et3N in CHCl3, a solution of HOBt (1 equiv) in THF, and
a solution of DCC (1 equiv) in CHCl3. After stirring for 1 h at
0 °C and overnight at room temperature, DCU was filtered
and the mixture evaporated in vacuo. The residue was taken
up with EtOAc, washed with H2O, 10% NaHCO3, H2O, 10%
citric acid solution, H2O, and brine, and dried over Na2SO4.
After filtration and evaporation in vacuo, the crude product
was purified by chromatography.
51: R1 ) CH2Ph, R2 ) H, R3 ) H ((S) configuration);
chromatography in n-hexane/ethyl acetate ) 4/6; oily product
(46%); Rf (E) 0.33. 52: R1 ) CH2Ph, R2 ) H, R3 ) H ((R)
configuration); chromatography in n-hexane/ethyl acetate/
acetic acid ) 6/4/0.5; oily product (62%); Rf (G) 0.32. 53: R1 )
CH2Ph, R2 ) CH3, R3 ) H; chromatography in n-hexane/ethyl
acetate ) 2/1; oily product (40%); Rf (E) 0.39. 54: R1
)
CH(CH3)CH2CH3, R2 ) H, R3 ) H; chromatography in n-
hexane/ethyl acetate ) 5/5; white foam (42%); Rf (E) 0.37. 55:
R1 ) (CH2)4NHBoc, R2 ) H, R3 ) H; chromatography in
n-hexane/ethyl acetate ) 2/1; oily product (25%); Rf (C) 0.42.
56: R1 ) CH2(p-OCOOCH2Ph)Ph, R2 ) H, R3 ) H; chroma-
tography in n-hexane/ethyl acetate ) 5/5; white solid (32%);
Rf (E) 0.16. 57: R1 ) CH2(p-NO2), R2 ) H, R3 ) H; chroma-
tography in n-hexane/ethyl acetate ) 5/5; oily product (35%);
Rf (E) 0.20. 58: R1 ) CH2(p-NH2), R2 ) H, R3 ) H; obtained
by acid hydrolysis of the preceding compound followed by
catalytic hydrogenation (Pd/C) in the presence of CH3COOH
(95%); Rf (K) 0.33. 59: R1 ) CH2Ph, R2 ) H, R3 ) o-OH;
chromatography in n-hexane/ethyl acetate ) 4/6; white foam
(35%); Rf (E) 0.38. 510: R1 ) CH2Ph, R2 ) H, R3 ) m-OH;
chromatography in n-hexane/ethyl acetate ) 4/6; oily product
(50%); Rf (E) 0.28. 511: R1 ) CH2Ph, R2 ) H, R3 ) o-CH3;
chromatography in n-hexane/ethyl acetate ) 4/6; white solid;
mp 97 °C (58%); Rf (E) 0.40. 512: R1 ) CH2Ph, R2 ) H, R3 )
m-CH3; chromatography in n-hexane/ethyl acetate ) 6/4;
colorless wax (71%); Rf (D) 0.60. 513: R1 ) CH2Ph, R2 ) H, R3
) o-CH3 (cis (2R) configuration); chromatography in n-hexane/
ethyl acetate ) 5/5; oily product (67%); Rf (E) 0.31. 514: R1 )
CH2Ph, R2 ) H, R3 ) o-CH3 (trans (2S) configuration);
chromatography in n-hexane/ethyl acetate ) 5/5; oily product
(47%); Rf (E) 0.40. 515: R1 ) CH2(p-OCOOCH2Ph)Ph, R2
H, R3 ) o-CH3; chromatography in n-hexane/ethyl acetate )
4/6; oily product (56%); Rf (E) 0.28. 516 R1 ) CH2(p-
)
:
OCOOCH2Ph)Ph, R2 ) H, R3 ) o-CH3 (cis (2R) configuration);
chromatography in n-hexane/ethyl acetate ) 5/5; white solid;
mp 70 °C (38%); Rf (E) 0.40. 517: R1 ) CH2(p-OCOOCH2Ph)-
Ph, R2 ) H, R3 ) m-CH3; chromatography in n-hexane/ethyl
acetate ) 5/5; colorless wax (49%); Rf (E) 0.17. 518: R1 ) CH2-
Ph, R2 ) H, R3 ) p-CH3; chromatography in n-hexane/ethyl
acetate ) 6/4; white foam (69%); Rf (D) 0.14. 519: R1 ) CH2(p-
OCOOCH2Ph)Ph, R2 ) H, R3 ) p-CH3; chromatography in
n-hexane/ethyl acetate ) 5/5; white foam (52%); Rf (E) 0.14.
Com p ou n d 12: white foam (89%); Rf (K) 0.30; HPLC (25%
B) 8.57 min; NMR δ 1.80-2.15-2.38, CH2â + γPro; 2.60, SH;
2.76-3.10, CH2âPh; 3.10-3.88, CH2Gly; 3.65, CHRPh; 4.32-
(t)-4.70(c), CHRPro; 4.89(c)-5.05(t), CH∂Pro; 7.20-7.60, Ar;
8.02, NH; 12.46, COOH.
Com p ou n d 13: white foam (93%); Rf (K) 0.53; HPLC (35%
B) 14.5 min; NMR δ 1.75-2.10-2.30, CH2â + γPro; 2.62, SH;
2.70-3.04, CH2âPh; 3.15-3.83, CH2Gly; 3.69, CHRPh;
4.21(t)-4.64(c), CHRPro; 5.15(c)-5.20(t), CH∂Pro; 6.75-7.15-
7.80, Ar; 8.03, NH; 9.52, OH; 12.25, COOH.
Com p ou n d 14: white wax (85%); Rf (G) 0.29; HPLC (30%
B) 30.6 min; NMR δ 1.76-2.10-2.32, CH2â + γPro; 2.62, SH;
2.70-3.05, CH2âPh; 3.15-3.85, CH2Gly; 3.70, CHRPh; 4.30-
(t)-4.60(c), CHRPro; 4.80(c)-4.97(t), CH∂Pro; 6.60-7.10, Ar;
8.00, NH; 9.38, OH; 12.38, COOH.
Com p ou n d 15: white solid; mp 110 °C dec (63%); Rf (E)
0.45; HPLC (45% B) 5.70 min; NMR δ 1.70-2.10-2.35, CH2â
+ γPro; 2.25, CH3(Ar); 2.55, SH; 2.70-2.90, CH2âPhe; 3.08-
3.78, CH2Gly; 3.58, CHRPh; 4.25(t)-4.60(c), CHRPro; 5.02(c)-
5.22(t), CH∂Pro; 7.10, Ar; 8.02, NH; 12.50, COOH.
520
chromatography in n-hexane/ethyl acetate ) 4/6; colorless wax
(45%); Rf (E) 0.29. 521: R1 ) CH2(p-OCOOCH2Ph)Ph, R2
: R1 ) CH2(p-OCOOCH2Ph)Ph, R2 ) H, R3 ) o-OH;
)
H, R3 ) m-OH; chromatography in n-hexane/ethyl acetate )
4/6; oily product (40%); Rf (E) 0.37. 522: R1 ) CH2(p.OCOOCH2-
Ph)Ph, R2 ) H, R3 ) p-OH; chromatography in n-hexane/ethyl
acetate ) 5/5; oily product (50%); Rf (E) 0.26. 523: R1 ) CH2(m-
OCOOCH2Ph)Ph, R2 ) H, R3 ) m-OH; chromatography in
n-hexane/ethyl acetate/acetic acid ) 7/3/0.25; oily product
(19%); Rf (F) 0.19. 524: R1 ) CH2(p-OCH3)Ph, R2 ) H, R3
)
o-OH; chromatography in n-hexane/ethyl acetate ) 4/6; oily
product (50%); Rf (E) 0.28. 525: R1 ) CH2(p-OCOOCH2Ph)-
Ph, R2 ) H, R3 ) o-OCH3; chromatography in n-hexane/ethyl
acetate ) 4/6; oily product (59%); Rf (E) 0.31. 526: R1 ) CH2-
(p-OCH2)Ph, R2 ) H, R3 ) o-OCH3; chromatography in
n-hexane/ethyl acetate ) 4/6; white foam (59%); Rf (E) 0.20.
Com p ou n d 16: white solid; mp ∼105 °C dec (72%); Rf (G)
0.29; HPLC (40% B) 21.3 min; NMR δ 1.80-2.10-2.35, CH2â

Orally Active Dual Inhibitors of NEP and ACE
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 13 2605
+ γPro; 2.26, CH2(Ar); 2.65, SH; 2.70-3.05, CH2âPh; 3.10-
3.86, CH2Gly; 3.68, CHRPh; 4.30(t)-4.70(c), CHRPro; 4.89(c)-
5.02(t), CH∂Pro; 7.13-7.40, Ar; 8.02, NH; 12.50, COOH.
Com p ou n d 17: white solid; mp 102 °C (45%); Rf (K) 0.78;
HPLC (45% B) 9.10 min; NMR δ 1.75-2.16-2.40, CH2â +
γPro; 2.26, CH3(Ar); 2.65, SH; 2.70-2.96, CH2âPh; 3.10-3.89,
CH2Gly; 3.69, CHRPh; 4.30(t)-4.50(c), CHRPro; 5.02(c)-
5.24(t), CH∂Pro; 7.00-7.13, Ar; 8.05, NH; 12.50, COOH.
Com p ou n d 18: white wax (59%); Rf (K) 0.50; HPLC (45%
B) 13.7 min; NMR δ 1.60-1.80-2.02, CH2â + γPro; 2.30,
CH3(Ar); 2.58, SH; 2.68-3.03, CH2âPh; 3.15-3.92, CH2Gly;
3.63, CHRPh; 4.58(t)-4.92(c), CHRPro; 5.20(c)-5.35(t), CH∂Pro;
6.94-7.12, Ar; 8.05, NH; 12.54, COOH.
Com p ou n d 19: white foam (72%); Rf (K) 0.50; HPLC (45%
B) 15.5 min; NMR δ 1.61-1.81-2.0-2.20, CH2â + ∂Pro; 2.30,
CH3(Ar); 2.60, SH; 2.65-3.02, CH2âPh; 3.20-3.89, Gly; 3.65,
CHRPh; 4.60(t)-4.90(c), CHRPro; 5.20(c)-5.37(t), CH∂Pro;
7.00-7.13, Ar; 8.05, NH; 12.50, COOH.
Com p ou n d 20: white solid; mp 130 °C dec; Rf (K) 0.74;
HPLC (45% B) 6.79 min; NMR δ 1.70-2.10, CH2â + γPro; 2.25,
CH3(Ar); 2.55, SH; 2.70-2.92, CH2âPh; 3.08-3.80, CH2Gly;
3.58, CHRPh; 4.28(t)-4.60(c), CHRPro; 5.01(c)-5.20(t), CH∂Pro;
6.55-6.90-7.00-7.10, Ar; 7.95, NH; 9.12, OH; 12.50, COOH.
Com p ou n d 21: oily product; Rf (K) 0.74; HPLC (45% B)
5.73 min; NMR δ 1.86-2.18, CH2â + γPro; 2.31, CH3(Ar); 2.57,
SH; 2.64-2.95, CH2âPh; 3.05-3.95, CH2Gly; 3.60, CHRPh;
4.35(t)-4.75(c), CHRPro; 5.09(c)-5.70(t), CH∂Pro; 6.60-6.90-
7.06-7.20, Ar; 8.06, NH; 9.12, OH; 12.52, COOH.
Com p ou n d 22: white solid; mp 105 °C dec (78%); Rf (K)
0.50; HPLC (50% B) 8.52 min; NMR δ 1.75-2.10-2.32, CH2â
+ γPro; 2.25, CH3(Ar); 2.52, HS; 2.55-2.90, CH2âPh; 3.09-
3.84, CH2Gly; 3.58, CHRPh; 4.30(t)-4.70(c), CHRPro; 4.88(c)-
5.03(t), CH∂Pro; 6.56-6.90-7.03-7.20-7.40, Ar; 8.00, NH;
9.12, OH; 12.60, COOH.
Com p ou n d 23: white foam (89%); Rf (K) 0.59; HPLC (50%
B) 9.50 min; NMR δ 1.71-2.10-2.30, CH2â + γPro; 2.20,
CH3(Ar); 2.60, SH; 2.70-3.00, CH2âPh; 3.10-3.82, CH2Gly;
3.65, CHRPh; 4.30(t)-4.55(c), CHRPro; 4.84(c)-5.02(t), CH∂Pro;
7.10-7.46, Ar; 8.02, NH; 12.40, COOH.
Com p ou n d 24: racemic mixture; white foam (92%); Rf (K)
0.60; HPLC (45% B) 6.17-6.34 min; NMR δ 1.75-2.10-2.32,
CH2â + γPro; 2.23, CH3(Ar); 2.51, SH; 2.58-2.90, CH2âPh;
3.10-3.90, CH2Gly; 3.56, CHRPh; 4.30(t)-4.64(c), CHRPro;
4.85(c)-5.00(t), CH∂Pro; 6.50-6.86-7.10-7.45, Ar; 7.98, NH;
9.10, OH; 12.35, COOH.
Com p ou n d 25: white foam (80%); Rf (K) 0.18; HPLC (25%
B) 19.4 min (C18); NMR δ 1.70-2.10-2.26, CH2â + γPro; 2.52,
SH; 2.55-2.60, CH2âPh; 3.14-3.80, CH2Gly; 3.58, CHRPh;
4.22(t)-4.62(c), CHRPro; 5.12(c)-5.20(t), CH∂Pro; 6.55-6.75-
6.85-7.04-7.79, Ar; 7.98, NH; 9.12-9.67, OH; 12.50, COOH.
Com p ou n d 26: white foam (71%); Rf (K) 0.34; HPLC (30%
B) 9.94 min (C18); NMR δ 1.76-2.10-2.36, CH2â + γPro; 2.55,
SH; 2.60-2.93, CH2âPh; 3.10-3.89, CH2Gly; 3.60, CHRPh;
4.30(t)-4.68(c), CHRPro; 4.80(c)-4.96(t), CH∂Pro; 6.58-7.10,
Ar; 8.00, NH; 9.14-9.39, OH; 12.40, COOH.
Com p ou n d 31: white foam (89%); Rf (G) 0.19; HPLC (45%
B) 9.90 min; NMR δ 1.70-2.10-2.30, CH2â + γPro; 2.60, SH;
2.64-2.96, CH2âPh; 3.18-3.70, CH2Gly; 3.52-3.77, 2 × OCH3;
3.65, CHRPh; 4.21(t)-4.66(c), CHRPro; 5.20(c)-5.25(t), CH∂Pro;
6.70-6.89-7.00-7.20-7.90, Ar; 8.00, NH; 12.60, COOH.
Com p ou n d 32: white foam (62%); Rf (K) 0.30; HPLC (24%
B) 13.36 min; NMR δ 1.65-1.80-2.10, CH2â + γPro; 2.62, SH;
2.70-2.90, CH2âPh; 3.10-3.70, CH2Gly; 3.60, CHRPh;
4.26(t)-4.60(c), CHRPro; 4.90(c)-5.06(t), CH∂Pro; 6.60-7.70,
Ar; 8.10, NH; 9.00, OH; 12.50, COOH.
Com p ou n d 33: white foam (88%); Rf (K) 0.36; HPLC (45%
B) 9.90 min; NMR δ 1.70-2.10-2.30, CH2â + γPro; 2.66, SH;
2.70-3.00, CH2âPh; 3.15-3.84, CH2Gly; 3.67, CHRPh; 4.24-
(t)-4.50(c), CHRPro; 4.65(c)-5.20(t), CH∂Pro; 6.72-7.02-
7.23-7.79, Ar; 8.03, NH; 9.63, OH; 12.56, COOH.
Com p ou n d 34: white foam (97%); Rf (K) 0.28; HPLC (45%
B) 7.63 min; NMR δ 1.75-2.10-2.30, CH2â + γPro; 2.64, SH;
2.70-3.00, CH2âPh; 3.02-3.82, CH2Gly; 3.54, CHRPh;
4.30(t)-4.70(c), CHRPro; 4.80(c)-4.95(t), CH∂Pro; 6.6-7.10,
Ar; 8.00, NH; 9.40, OH; 12.50, COOH.
Com p ou n d 35: white foam (67%); Rf (E) 0.22; HPLC (30%
B) 10.0 and 11.1 min; NMR δ1.74-2.09-2.32, CH2â + γPro;
2.60, SH; 2.88-3.10, CH2âPh; 3.03-3.82, CH2Gly; 3.70, CHRPh;
4.28(t)-4.68(c), CHRPro; 4.80(c)-4.95(t), CH∂Pro; 6.60-7.85,
Ar; 8.03, NH; 9.40, OH; 12.60, COOH.
Molecu la r Mod elin g. Conformational analyses were per-
formed through high-temperature molecular dynamics (MD)
as described.30,56 The calculations were performed using the
Discover Insight software package (Biosym Technologies Inc.,
San Diego, CA) with the CFF91 force field parameters.57,58 The
first-step calculations consisted of equilibrating the system to
1000 K during 10 ps. The trajectory was then continued for
100 ps, a structure being extracted every 1 ps. The resulting
100 structures were then submitted to 10 ps of simulated
annealing at room temperature (300 K) and further minimized
through a steepest descent followed by a conjugate gradient
algorithm. No conformational constraints were taken into
account during the calculations except for the peptide bond
which is assumed to be planar. The dielectric constant was
kept at 78 during all calculation steps. Each structure was
then graphically analyzed using the insight analysis module,
and the conformers were classified into families. Each family
was characterized by a representative conformation and its
relative population defined as its percentage of occurrence
along the 100 ps simulation. The representative conformers
of each family were then compared with the structure of the
pharmacophore by using a template-forcing procedure (Insight
II User guide, version 2.1.0., 1992, Biosym Technologies Inc.,
San Diego, CA). The force constant in this last procedure was
set to 50 kcal M-1, and the atoms taken into account for the
rms calculation are indicated in Table 1. The relative spatial
orientation of the thiol and carboxyl groups in compound 6
was defined by the value of the dihedral angle θ between the
noncontinuous groups of atoms S4-C3 and C10-C11 (Table 4).
Biologica l Tests. [3H]Tyr-D-Ala2-Leu-enkephalin (52 Ci/
mmol) was obtained from Dositek (CEA Saclay, France);
N-Cbz-Phe-His-Leu59 was from Bachem (Buddendorf, Swit-
zerland). Neutral endopeptidase from rabbit kidney44 and
angiotensin converting enzyme from rat testis were purified
to homogeneity as previously described.45 Thermolysin was
purchased from SIGMA (France) and used without purifica-
tion.
Com p ou n d 27: white wax (77%); Rf (K) 0.24; HPLC (25%
B) 16.4 min; NMR δ 1.72-2.10-2.30, CH2â + γPro; 2.55, SH;
2.58-2.92, CH2âPh; 3.10-3.80, CH2Gly; 3.56, CHRPh;
4.28(t)-4.60(c), CHRPro; 4.82(c)-4.93(t), CH∂Pro; 6.55-6.68-
6.88-7.25, Ar; 7.95, NH; 9.10-9.31, OH; 12.60, COOH.
Com p ou n d 28: white wax (87%); Rf (K) 0.20; HPLC (25%
B) 12.38 and 12.80 min; NMR δ 1.75-2.10-2.32, CH2â + γPro;
2.55, SH; 2.60-2.92, CH2âPh; 3.13-3.82, CH2Gly; 3.61, CHRPh;
4.30(t)-4.68(c), CHRPro; 4.80(c)-4.98(t), CH∂Pro; 6.50-6.62-
7.00, Ar; 8.01, NH; 9.12-9.40, OH; 12.60, COOH.
Com p ou n d 29: white foam (83%); Rf (K) 0.42; HPLC (45%
B) 6.48 min; NMR δ1.72-2.12-2.39, CH2â + γPro; 2.55, SH;
2.62-2.95, CH2âPh; 3.15-3.70, CH2Gly; 3.53, OCH3; 3.55,
CHRPh; 4.20(t)-4.54(c), CHRPro; 5.12(c)-5.20(t), CH∂Pro;
6.72-7.00-7.80, Ar; 8.00, NH; 9.70, OH; 12.30, COOH.
Com p ou n d 30: white foam (91%); Rf (K) 0.50; HPLC (45%
B) 5.95 min; NMR δ1.70-2.10-2.30, CH2â + γPro; 2.52, SH;
2.55-2.90, CH2âPh; 3.16-3.82, CH2Gly; 3.60, CHRPh; 3.70,
OCH3; 4.23(t)-4.66(c), CHRPro; 5.18(c)-5.23(t), CH∂Pro; 6.60-
6.90-7.25-7.90, Ar; 9.13, OH; 12.40, COOH.
In Vitr o In h ibition of NEP , ACE, a n d TLN: Assa y for
Neu tr a l En d op ep tid a se Activity. IC50 values were deter-
mined as previously reported.60 NEP (final concentration, 1
pmol/100 µL; specific activity for [3H]-D-Ala2-Leu-enkephalin,
0.3 nmol/mg/min) was preincubated for 15 min at 25 °C with
or without increasing concentrations of inhibitor in a total
volume of 100 µL in 50 mM Tris-HCl buffer, pH 7.4. [3H]-D-
Ala2-Leu-enkephalin (Km ) 30 µM) was added to a final
concentration of 20 nM, and the reaction was stopped after
30 min by adding 10 µL of 0.5 M HCl. The tritiated metabolite
formed, [3H]Tyr-D-Ala-Gly, was separated on polystyrene
beads61 and the radioactivity measured by scintillation count-
ing. Competitive inhibition was demonstrated for 26 from
Dickson representation of inhibition curves (not shown here).

2606 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 13
Fournie-Zaluski et al.
Assa y for Th er m olysin Activity. TLN activity was
assayed at 37 °C in a total volume of 100 µL of 50 mM Tris-
HCl buffer (pH 7.4) containing 100 mM NaCl and 5 mM CaCl2,
with 25 nM [3H][Leu5]enkephalin ([3H]Tyr-Gly-Gly-Phe-Leu)
as a substrate.62 Reactions were stopped by the addition of
10 µL of 0.5 M HCl, and the metabolite, [3H]Tyr-Gly-Gly, was
isolated as previously described for the NEP assay. The
radioactivity was quantified by liquid scintillation counting.
6 days, once a day, at 25 mg/kg and blood pressure was
measured during 9 days, the 6 days of the experiment and 3
days after.
P h a r m a cologica l Effects in DOCA Sa lt Ra ts. RB 106
was administered at 25 mg/kg iv + 25 mg/kg/h in conscious
animals as previously described.32 Urine samples were col-
lected via a poly(ethylene) catheter implanted in the bladder.
The blood pressure was measured as in the case of SHR.
Assa y for An gioten sin -Con ver tin g En zym e Activity.
Enzymatic studies on ACE were performed using N-Cbz-Phe-
His-Leu as substrate (Km ) 50 mM). ACE (final concentration,
0.02 pmol/100 µL; specific activity on Cbz-Phe-His-Leu, 13
nmol/mg/min) was preincubated for 15 min at 37 °C with
various concentrations of the inhibitors in 50 mM Tris-HCl
buffer, pH 8.0. N-Cbz-Phe-His-Leu was added to a final
concentration of 0.05 mM. The reaction was stopped after 15
min by adding 400 µL of 2 M NaOH. After dilution with 3
mL of water, the concentration of His-Leu was determining
following the fluorimetric assay described by Cheung et al.63
with a MPF44A Perkin-Elmer spectrofluorimeter (excitation,
365 nm; emission, 495 nm). The calibration curve for His-
Leu was obtained by addition of increasing concentrations of
His-Leu into 0.1 mL of 5.0 M Tris-HCl buffer, pH 8.0,
containing the denaturated enzyme. Competitive inhibition
of 26 and derived Ki value were established by using Dickson
representation (not shown).
Ack n ow led gm en t. The authors would like to thank
C. Dupuis for expert manuscript drafting, Dr. A. Beau-
mont for stylistic revision, and N. Gomez for excellent
technical assistance. The work was supported by funds
from the Institut National de la Sante´ et de la Recherche
Me´dicale, the Centre National de la Recherche Scien-
tifique, and the University Rene´ Descartes.
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