Optimal recognition of neutral endopeptidase and angiotensin-converting enzyme active sites by mercaptoacyldipeptides as a means to design potent dual inhibitors

Article abstract of DOI:10.1021/jm950590p

An interesting approach for the treatment of congestive heart failure and chronic hypertension could be to avoid the formation of angiotensin II by inhibiting angiotensin converting enzyme (ACE) and to protect atrial natriuretic factor by blocking neutral endopeptidase 24.11 (NEP). This is supported by recent results obtained with potent dual inhibitors of the two zinc metallopeptidases, such as RB 105, HSCH₂CH(CH₃)PhCONHCH(CH₃)COOH (Fournie-Zaluski et al. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 4072-4076), which reduces blood pressure in experimental models of hypertension, independently of the salt and renin angiotensin system status. In order to develop new dual inhibitors with improved affinities, long duration of action, and/or better bioavailabilities, various series of mercaptoacyldipeptides corresponding to the general formula HSCH(R₁)CONHCH(R₁')CON(R)CH(R₂')COOH have been synthesized. The introduction of well-selected β-branched chains in positions R₁ and R₁', associated with a tyrosine or a cyclic amino acid in the C-terminal position, led to potent dual inhibitors of NEP and ACE such as 21 [N-[(2S)-2-mercapto- 3-methylbutanoyl]-Ile-Tyr] and 22 [N-[(2S)-2-mercapte-3-phenylpropanoyl]Ala- Pro] which have IC₅₀ values in the nanomolar range for NEP and subnanomolar range for ACE. These compounds could have different modes of binding to the two peptidases. In NEP, the dual inhibitors seem to interact only with the S₁' and S₂' subsites, whereas additional interactions with the S₁ binding subsite of ACE probably account for their subnanomolar inhibitory potencies for this enzyme. The localization of the Pro residue of 22 outside the NEP active site is supported by biochemical data using (Arg¹⁰²,Glu)NEP and molecular modeling studies with thermolysin used as model of NEP. One hour after oral administration in mice of a single dose (2.7 x 10^-5 mol/kg), 21 inhibited 80% and 36% of kidney NEP and lung ACE, respectively, while 22 inhibited 40% of kidney NEP and 56% of lung ACE.

Full text of DOI:10.1021/jm950590p

1210
J . Med. Chem. 1996, 39, 1210-1219
Op tim a l Recogn ition 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 Active Sites by Mer ca p toa cyld ip ep tid es a s a Mea n s To Design P oten t
Du a l In h ibitor s
Pascale Coric, Serge Turcaud, Herve´ Meudal, Bernard Pierre Roques,* and Marie-Claude Fournie-Zaluski
De´partement de Pharmacochimie Mole´culaire et Structurale, U266 INSERM - URA D 1500 CNRS, UFR des Sciences
Pharmaceutiques et Biologiques, Universite´ Rene´ Descartes-Faculte´ de Pharmacie, 4, avenue de l’Observatoire,
75270 Paris Cedex 06, France
Received December 18, 1995X
An interesting approach for the treatment of congestive heart failure and chronic hypertension
could be to avoid the formation of angiotensin II by inhibiting angiotensin converting enzyme
(ACE) and to protect atrial natriuretic factor by blocking neutral endopeptidase 24.11 (NEP).
This is supported by recent results obtained with potent dual inhibitors of the two zinc
metallopeptidases, such as RB 105, HSCH2CH(CH3)PhCONHCH(CH3)COOH (Fournie´-Zaluski
et al. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 4072-4076), which reduces blood pressure in
experimental models of hypertension, independently of the salt and renin angiotensin system
status. In order to develop new dual inhibitors with improved affinities, long duration of action,
and/or better bioavailabilities, various series of mercaptoacyldipeptides corresponding to the
general formula HSCH(R1)CONHCH(R1′)CON(R)CH(R2′)COOH have been synthesized. The
introduction of well-selected â-branched chains in positions R1 and R1′, associated with a tyrosine
or a cyclic amino acid in the C-terminal position, led to potent dual inhibitors of NEP and ACE
such as 21 [N-[(2S)-2-mercapto-3-methylbutanoyl]-Ile-Tyr] and 22 [N-[(2S)-2-mercapto-3-
phenylpropanoyl]Ala-Pro] which have IC50 values in the nanomolar range for NEP and
subnanomolar range for ACE. These compounds could have different modes of binding to the
two peptidases. In NEP, the dual inhibitors seem to interact only with the S1′ and S2′ subsites,
whereas additional interactions with the S1 binding subsite of ACE probably account for their
subnanomolar inhibitory potencies for this enzyme. The localization of the Pro residue of 22
outside the NEP active site is supported by biochemical data using (Arg102,Glu)NEP and
molecular modeling studies with thermolysin used as model of NEP. One hour after oral
administration in mice of a single dose (2.7 × 10-5 mol/kg), 21 inhibited 80% and 36% of kidney
NEP and lung ACE, respectively, while 22 inhibited 40% of kidney NEP and 56% of lung ACE.
In tr od u ction
Zn2+ metallopeptidases,10,11 and have significant analo-
gies in their active sites, as shown by their capacity to
cleave in vitro identical peptides at the same bond (for
example bradykinin and Leu- or Met-enkephalin12).
Consequently, the same types of molecules are able to
block the active sites of these enzymes, as illustrated
by mercaptoacyl amino acids and carboxyalkyl dipep-
tides (thiorphan, HSCH2CH(CH2Ph)CONHCH2COOH,13
and SCH-32,615, PhCH2CH(COOH)NHCH(CH2Ph)-
CONH(CH2)2COOH,14 for NEP, captopril, HSCH2CH-
(CH3)CO-Pro,15 and enalapril, PhCH2CH2CH(COOH)-
NHCH(CH3)CO-Pro,16 for ACE). The selectivity of these
molecules results from subtle differences in the active
sites of both peptidases.17-19 Efficient NEP inhibitors
preferentially contain aromatic residues in the P1′
position,20 and most ACE inhibitors are characterized
by a small residue (Ala) in the P1′ position and a cyclic
amino acid (Pro) in the P2′ position.21 However, it has
been possible to design dual inhibitors of these pepti-
dases by introducing more hydrophobic residues in the
selective inhibitor of NEP, thiorphan,13 and compounds
such as N-[2-(mercaptomethyl)-3-phenylpropanoyl]leu-
cine17,18 or alatrioprilate22 exhibit relatively good affini-
ties for both enzymes.
The design of dual inhibitors of neutral endopeptidase
(NEP, EC 3.4.24.11 or neprilysin) and angiotensin-
converting enzyme (ACE, EC 3.4.25.1) has become an
important therapeutic challenge in the last few years.1
ACE belongs to the enzymatic cascade of the renin-
angiotensin system and releases the vasoconstrictor
peptide angiotensin II from its inactive precursor,
angiotensin I. NEP inactivates the atrial natriuretic
peptide (ANP)2 which induces diuresis, natriuresis, and
a slight vasodilatation. Furthermore, NEP and ACE are
both involved in the inactivation of bradykinin (Bk), a
vasodilatatory peptide, at their epithelial and endothe-
lial sites, respectively.3,4 However, as shown in various
pharmacological experiments, there is a physiological
antagonism between the renin-angiotensin and ANP
systems, reducing the optimal action expected from the
inhibition of one of these enzymes.5,6 This suggests that
a simultaneous inhibition of ACE and NEP, reducing
angiotensin II formation and protecting ANP and Bk
from inactivation, might have, as proposed some years
ago,1 therapeutic advantages in the treatment of car-
diovascular diseases, as compared to selective inhibition
of a single enzyme.7-9
Recently, more potent dual inhibitors containing
hindered substituents have been designed giving new
information about the structures of the binding domains
of each enzyme. Thus, the development of RB 105
Cloning and sequencing of NEP and ACE have shown
that both enzymes belong to the thermolysin group of
* To whom correspondence should be adressed. Tel: (33) 43-25-50-
45. FAX: (33) 43-26-69-18.
X Abstract published in Advance ACS Abstracts, February 15, 1996.
[(2S,3R)-HSCH2CH(CH(CH3)Phe)CO-L-Ala]23,24
has
0022-2623/96/1839-1210$12.00/0 © 1996 American Chemical Society

Design of Potent Dual Inhibitors
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 6 1211
Sch em e 1a
a
(a) H2NCH(R2)COOtBu, DCC, HOBt in CH2Cl2/THF ) 1/1; (b) TFA, CH2Cl2; (c) HN(R)CH(R3)COOCH3, DCC, HOBt in CH2Cl2/THF
) 1/1; (d) OH- then H+ in H2O/MeOH ) 2/1; (e) HN(R)CH(R3)COOCH3, DCC, HOBt in CH2Cl2/THF ) 1/1; (f) CH3COSCH(R1)COOH,
DCC, HOBt, Et3N in CH2Cl2/THF ) 1/1; (g) R, R3 may constitute the side chain of a cyclic amino acid.
shown that the S1′ subsite of both peptidases is able to
accommodate constrained moieties such as a â-methyl-
benzyl group. However, conformational analysis of RB
105 and its derivatives, to determine thermodynamically
acceptable overlaps between stable conformers of these
inhibitors and the ACE pharmacophore, designed from
rigid selective inhibitors, showed that the presence of
the â-methylbenzyl group in the P1′ position, precludes
the presence of bulky residues in the P2′ position.23
Recently, a great number of molecules containing poly-
cyclic residues at the P2′ position have been described,
showing that NEP seems to possess, like ACE, a large
S2′ domain,25 in agreement with earlier observations.19
Taken together, these data support the idea that the
S1′ and S2′ subsites of NEP and ACE constitute hydro-
phobic domains which could be efficiently occupied by
cyclic or linear structures.20,21 The introduction of cyclic
moieties in the structure of the inhibitors is expected
to reduce the unfavorable entropic factor in enzyme
binding. However, this advantage is often outweighed
by (i) difficulties in synthesizing these compounds,
particularly if they have to be obtained in optically pure
forms; (ii) the problems of manipulating the hydro-
philic-hydrophobic balance to ensure the inhibition of
vascular ACE and kidney NEP, with the same ef-
ficiency.24 In order to improve our knowledge of the
active site of both peptidases and to try to obtain new
potent dual inhibitors of NEP and ACE, we have
designed compounds corresponding to mercaptoacyl-
dipeptides of general formula HSCH(R1)COAA1AA2, in
which linear or cyclic constraints have been introduced.
The synthesis and inhibitory potencies of these com-
pounds on both NEP and ACE are reported in this
paper. The blockade of lung ACE and renal NEP by
the two best inhibitors have been studied after oral
administration. Finally, structure-activity studies in-
cluding the use of a mutated NEP and molecular
modeling studies with thermolysin (TLN) have allowed
a model to be proposed for the recognition of the NEP
and ACE subsites by these new inhibitors.
dure which allowed these compounds to be obtained
with a high degree of optical purity from the corre-
sponding R-amino acids of defined absolute configura-
tion. The R-amino acids led to the corresponding
2-bromoalkanoic acids with retention of configuration
by the Fischer procedure.26 Subsequent SN2 thioacety-
lation of these bromo derivatives15 yielded the expected
(acetylthio)alkanoic acids. The coupling steps were
performed by the DCC/HOBt method, and the depro-
tection of the sulfhydryl and carboxyl groups was
achieved by alkaline hydrolysis (Scheme 1).
2. In h ibitor y Activities on NEP a n d ACE.
A
comparison of the inhibitory potencies of compound 1
on NEP and ACE activities (Table 1), with those of the
corresponding precursors A and B, shows that com-
pound 1 is significantly better than A, which does not
possess a substituted mercaptoalkyl chain (R1 ) H), and
is also more potent than B, which does not contain the
C-terminal alanine (AA2 ) OH), for both enzymes.
Therefore, the inhibitory potencies of compounds con-
taining a N-[2-mercapto-3-phenyl propanoyl]-L-pheny-
lalanyl moiety, and differing only by the nature of the
C-terminal amino acid (compounds 2-7), were com-
pared to those of compound 1. For NEP inhibition, only
one derivative, which contains a C-terminal tyrosine
(compound 7), was as efficient as 1. All of the other
compounds, which have more hydrophobic moieties,
were significantly less active by approximately 1 order
of magnitude. For ACE inhibition, compounds 6 and
7, which contain respectively a tryptophan and a ty-
rosine residue, had activities in the same range as
compound 1. For this latter enzyme, slight increases
in IC50 values were also observed with compounds 2-5.
Taking into account that compound 7 is a relatively good
dual NEP/ACE inhibitor, some modifications were
introduced on the mercaptoalkanyol moiety of this
molecule in order to optimize the recognition of both
enzymes. The lengthening of the alkyl chain (compound
8 versus 7 or 10 versus 9) led to a slight reduction in
the affinity for NEP (factors 7 and 3, respectively) and
a small improvement in ACE recognition. Furthermore,
the shortening of the thiol-containing chain (compound
11 versus 12) had no significant effect on the inhibitory
potencies for both peptidases. The nature of the amino
Resu lts
1. Syn th esis. The various 2-(acetylthio)alkanoic
acids were synthesized by the classical two-step proce-

1212 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 6
Coric et al.
Ta ble 1. Structure-Activity Relationships for Dual Inhibition of NEP and ACE by Various Mercaptoacyl Dipeptides
HS
CO – AA1 – AA2
(S)
R1
*
IC50 (nM)a
compd
R1
AA1
Phe
AA2
NEP
ACE
A
B
C
1
2
3
4
5
6
H
Ala
OH
OH
Ala
14 ( 2
23 ( 3
4 ( 1
2.3 ( 0.2
45 ( 6
50 ( 5
40 ( 3
120 ( 10
50 ( 4
2.9 ( 0.6
20 ( 5
3.6 ( 0.5
10 ( 2
4.5 ( 0.3
3.0 ( 0.3
2.8 ( 0.6
2.2 ( 0.4
5.5 ( 0.6
5.3 ( 0.6
3.5 ( 0.5
32 ( 2
220 ( 20
44 ( 4
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
Phe
Ala
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Val
Val
Ile
40 ( 4
7.1 ( 0.5
1-ACPb
O(Bzl)Ser
S(Bzl)Cys
1-Nalc
Trp
30 ( 4
12 ( 2
14 ( 4
50 ( 5
7.9 ( 0.6
5.6 ( 0.6
1.5 ( 0.3
4.0 ( 1.0
1.6 ( 0.3
3.5 ( 0.3
2.5 ( 0.2
4.0 ( 0.5
3.0 ( 0.3
6.3 ( 0.8
1.9 ( 0.4
10 ( 2
7
8
9
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
(CH2)2Ph
CH2Ph
10
11
12
13
14
15
16
17
18
19
20
21
(CH2)2Ph
Ph
CH2Ph
CH2Ph
CH2Ph
Ile
Nle
Leu
(O-Et)Thr
Val
CH2Ph
CH(CH3)CH2CH3
CH(CH3)CH2CH3
CH(CH3)CH2CH3
CH(CH3)CH2CH3
CH(CH3)OCH2Ph
CH(CH3)2
Phe
â(Me)Phed
(OBzl)Thr
Ile
33 ( 4
34 ( 4
7.1 ( 0.6
0.26 ( 0.02
100 ( 30
4.1 ( 0.6
1.4 ( 0.1
Ile
a
b
IC50 values are the means ( SEM of three independent determinations performed in triplicate. 1-ACP, 1-aminocyclopentanoic acid.
c 1-Nal, 1-naphthylalanine. â(Me)Phe is a mixture of the four stereoisomers.
d
acid “AA1” is not of major importance since the replace-
ment of Phe by amino acids with alkylated side chains
such as Val (compound 9), Ile (compounds 11 and 12),
Nle (compound 13), Leu (compound 14), or (O-Et)Thr
(compound 15) led to nanomolar IC50 values on both
enzymes. In addition, given the selective beneficial role
of a C-terminal â-branched amino acid in the recognition
of the active site of ACE, previously demonstrated in
RB 105 series,23 a similar modification was introduced
in the mercaptoalkanoyl moiety of the inhibitors: three
models of â-branched residues, corresponding respec-
tively to the lateral chains of Ile (compounds 16-19)
(O-benzyl)Thr (compound 20) and Val (compound 21),
were chosen. It is interesting to observe that the best
dual inhibitor of this series contains two short â-branched
chains (compound 21) with IC50 values of 1.4 and 0.26
nM for NEP and ACE, respectively. The presence of
more hindered substituents induced a significant de-
crease in the inhibitory potencies for both peptidases
(compounds 18 and 19).
Finally, a series of pseudotripeptide inhibitors con-
taining a C-terminal cyclic amino acid was synthesized.
Concerning NEP inhibition, the main observation was
that the introduction of C-terminal cyclic amino acids
such as a proline (compounds 22, 23, and 26), a
pipecolinic acid (32), or a perhydroindole carboxylic acid
(33) did not modify the IC50 values significantly, as
compared to their mercaptoalkanoyl amino acid precur-
sors B or C. Conversely, the majority of the inhibitors
containing these cyclic amino acids have IC50 values in
the nanomolar or subnanomolar range for ACE. Chang-
ing the absolute configuration of the residue R1 did not
modify the activity on NEP but was unfavorable for
ACE. This is illustrated by compound 22 (S configu-
ration) which is 75 times more potent than its R isomer
23 on ACE. Furthermore, in this series of compounds,
the lengthening of R1 (compound 24 versus 22) de-
creased the affinity for NEP, while the shorthening of
this chain (compound 29 versus 28) reduced the activity
on both enzymes. In this series, the replacement of the
benzyl group by a branched chain (compounds 34-37)
in position R1 is highly unfavorable for NEP inhibition.
For interaction with the ACE active site, a â-branched
aliphatic residue is well accepted in the R1 position
(compound 34) if the C-terminal residue is not a bicyclic
amino acid (compounds 35 and 36), and the presence of
an R-methyl amino acid in position AA1 (compound 25),
the modification of the proline residue in position AA2
by decarboxylation (compound 30), or hydroxylation
(compound 31) led to a decreased activity on both
enzymes.
Compounds C and 1 were also tested on the mutated
(Arg102,Glu) NEP.27 The IC50 values of C and 1 on this
enzyme were respectively 150 and 18 nM, i.e. 50-fold
and 6-fold less efficient than for the wild-type NEP.
3. Molecu la r Mod elin g. Molecular modeling stud-
ies were performed with the aim of defining the position
of compound 22 in the active site of NEP. This was done
by using the coordinates of the NEP inhibitor thiorphan
co-crystallized inside the active site of TLN as frame-
work.28
The biologically active conformation of 22 in the NEP
active site was investigated using the template-forcing
procedure. The resulting conformation was first docked
inside the active site of TLN and then refined through
a minimization procedure (see the Experimental Sec-
tion). As compared to the thiorphan-TLN complex, the
distance between the thiol group of 22 and the zinc atom
was found to be almost identical. Due to the presence
in thiorphan of an additional CH2 group between the
SH and the R-carbon of the mercaptoacyl moiety, this
resulted in a slight shift of the benzyl group of 22 in

Design of Potent Dual Inhibitors
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 6 1213
F igu r e 1. Stereoview of the complex between thermolysin and the dual inhibitor 22 [HSCH(CH2Ph)CO-Ala-Pro]. Dotted lines
show the hydrogen bonds between 22 and TLN active site amino acids. In this complex the C-terminal proline moiety is outside
the S2′ subsite.
Sch em e 2
the S1′ subsite (0.9 Å calculated from the barycenter of
the phenyl rings in 22 and in thiorphan). The main
hydrogen bonds found in the thiorphan-TLN complex,
such as the interaction between the carbonyl of the
mercaptoalkanoyl moiety and Arg203, were also observed
in the complex 22-TLN. However, in this complex, the
NH of the amide bond of 22 was hydrogen bonded to
the CO of Ala113 and the free carboxylate of the proline
moiety formed a hydrogen bond with the NH2 group of
Asn112 driving the proline moiety at the edge of the
active site cleft (Figure 1).
catalytic site.29 Mercaptoacetyl dipeptides of type II
(Scheme 2) previously described as NEP inhibitors30,31
have been proposed to interact by the R1′ and R2′ groups
with the S1′ and S2′ subsites of the enzyme, respec-
tively.30 On the basis of these results the alkyl group
R1 introduced on the mercaptoacetyl moiety of com-
pound II (Scheme 2) would be expected to bind the S1
subsite (model 1). Alternatively the new inhibitors of
type I could be considered to derive from the coupling
of an amino acid, H2NCH(R2′)COOH, with the free
carboxylate group of a mercaptoacyl amino acid III
(Scheme 2), which interacts with the S1′ and S2′ subsites
of the enzyme.19 In this case, the additional residue,
R2′, would be outside this part of the active site (model
2).
Discu ssion
In order to develop a new series of dual inhibitors of
NEP and ACE, we have synthesized molecules corre-
sponding to the general formula of compound I (Scheme
2) which have two possible binding modes to the
peptidases depending on the position of the R1 substitu-
ent in the S1 or S1′ subsites. In each case, the sulfhydryl
group is coordinated to the zinc atom present in the
In order to choose between these models, the inhibi-
tory potencies of compounds 1-7 were compared with
those of the corresponding precursors A and B.
Compound 1, resulting from the introduction of a
benzyl group in A, has an inhibitory potency for NEP 6

1214 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 6
Coric et al.
times better than its precursor. This could be inter-
preted as resulting from an additional favorable inter-
action of the benzyl side chain with the putative S1
subsite of NEP (model 1). This is not contradicted by
the IC50 values of compounds 2-6, which are slightly
greater than that of 1. However, the decreased activi-
ties of compound 8 versus 7 and 10 versus 9 are difficult
to explain with this model of interaction. Indeed it has
been shown with carboxyalkyl dipeptide inhibitors of
NEP that the S1 subsite of this enzyme is more ef-
ficiently fitted by a phenethyl group than by a benzyl
group. Thus, the compound, [PhCH2CH(COOH)]-Phe-
Ala, IC50 ) 300 nM, is less active than [PhCH2CH2CH-
(COOH)]-Phe-Ala, IC50 ) 90 nM.32 This is the reverse
in the S1′ subsite where the phenethyl group in
HomoPhe has been shown to be detrimental for NEP
ment with the binding of the benzyl group of 1 in the
well-characterized hydrophobic S1 subsite of this pep-
tidase16,21 (model 1). The S1′ and S2′ domains in ACE
could not be optimally fitted by two concomitant volu-
minous residues as illustrated by the decreased activi-
ties of 2 and 5 as compared to 1 or 7. Furthermore the
slight increase in IC50 values for 8 and 10 as compared
to 7 and 9 is also consistent with model 1, since a
phenethyl chain is preferred to a benzyl chain for S1
binding.21
The presence of a valine as the central residue is
favorable for NEP and ACE inhibition (compound 16),
but the introduction of more bulky groups in 18 and 19
decreased the interaction with both peptidases, probably
due to steric hindrance. It is interesting to note that
21, in which the â-branched chains are reversed as
compared to those in 16, is the most active compound
of this series, on both enzymes, with a subnanomolar
activity (IC50 ) 0.26 nM) on ACE. With this compound
all the hydrophobic interactions with S1, S1′, and S2′
subsites of ACE (model 1, Scheme 2) are probably
satisfied, and therefore â-methyl-substituted alkyl chains
represent an interesting “working model” to further
improve dual inhibition.
recognition: PhCH2CH(COOH)-HomoPhe-âAla, IC50
)
8500 nM; PhCH2CH(COOH)-Phe-âAla, IC50 ) 21 nM.32
Consequently, the relative IC50 values of compounds 7/8
and 9/10 for NEP are more satisfactorily explained by
the second model of enzyme recognition.
In order to confirm this hypothesis, the IC50 values
of C and 1 were measured using recombinant rabbit
NEP and its mutated analog (Arg102,Glu)NEP, both
expressed in Cos cells.27 As previously shown, this
mutated NEP can be used as an index of inhibitor
positioning in the enzyme active site.33,34 Indeed Arg102
is thought to be located at the edge of the S2′ subsite
where it probably interacts with the C-terminal carboxyl
group of a P2′ residue, accounting for the dipeptidyl
carboxypeptidase activity of NEP.35-37 When Arg102 is
replaced by Glu, the IC50 of thiorphan for the mutated
enzyme is increased by more than 2 orders of magni-
tude, due to the ionic repulsion between the two
negatively charged groups. Under, the same conditions,
the IC50 of thiorphan amide which has no free carboxy-
late was increased only by a factor 6. The IC50 values
of C and 1 on the mutated enzyme are respectively 150
and 18 nM, while they are 3.2 and 2.8 nM respectively,
on the recombinant wild-type enzyme. The IC50 ratios
of 50 for C and 6 for 1 support the model 2 of NEP
recognition for these compounds in agreement with
previous results obtained with other thiol inhibitors of
NEP.33,34 Additional evidence, for this model, comes
from the tridimensional structure of the complex formed
between thermolysin and the sulfhyldryl-containing
inhibitor BAG [HSCH2CH(CH2Ph)CO-Ala-Gly-NH2].38
The crystallographic data show that the benzyl moiety
and alanyl residue interact respectively with the S1′ and
S2′ subsites of the enzyme whereas the C-terminal
glycinamide emerges at the surface of the enzyme
(model 2).38 This mode of interaction was also observed
with larger C-terminal amino acids.29 The increased
activity of compound 1 as compared to B remains to be
explained. Delaney et al. has proposed the existence of
an S3′ subsite in NEP.39 Although this hypothesis
cannot be excluded, it is difficult to reconcile it with the
existence of an Arg102 residue at the surface of the
enzyme. The increased potency of 1 versus B could
therefore be due to favorable nonspecific interactions
with the enzyme surface. An increase in the size of the
C-terminal amino acid (compounds 2-6) led to a reduc-
tion in NEP affinity probably for steric and/or confor-
mational reasons.
The proposed mode of interaction of these novel dual
inhibitors is reinforced by the results obtained with the
derivatives containing cyclic amino acids with the ACE
active site such as proline or analogues in the C-
terminal position (Table 2). It is known that a proline
residue is well recognized by the S2′ subsite of ACE.15,21
Thus, the introduction of a proline residue in C (com-
pound 22 in Table 2) induced a large increase in affinity
leading to a subnanomolar IC50 value (0.3 nM). This
compound has been previously described by Ondetti et
al.40 as an ACE inhibitor and was recently reevaluated
by Delaney et al.39 on both NEP and ACE, the IC50
values reported (NEP ) 400 nM and ACE ) 30 nM)
were about 100 times greater than those found in the
present work (IC50 on NEP, 3.8 nM; IC50 on ACE ) 0.3
nM). This discrepancy might be due to differences in
the experimental conditions used for the tests. In all
our measurements, thiol inhibitors were preincubated
in the presence of â-mercaptoethanol, in order to prevent
oxidation of the mercaptans. Subnanomolar potencies
were also obtained with compounds 27 and 33 (Table
2) and probably also for 32, taking into account that
racemic pipecolinic acid was used.
In the case of NEP, it has been shown that a proline
ring does not favorably fit the S2′ subsite.18,20 If we
compare compounds 22, 32, and 33 with the precursor
C, or compound 26 with B, there are no significant
differences in IC50 values. This strongly supports the
second model of NEP recognition in which the C-
terminal amino acid is outside the specific binding
domains of the enzyme.29,33,34 Compound 22 has been
docked in TLN, taken as a model of the NEP active site
(Figure 1). This was carried out, as described in the
Experimental Section, by using the coordinates of the
NEP inhibitor thiorphan, which has been cocrystallized
with TLN.28 This showed that the benzyl moiety fits
the S1′ subsite and that the proline residue of 22
emerged outside the active site cleft of this enzyme as
the glycinamide moiety in the TLN-BAG complex.38 The
conformation of 22 inside the TLN active site is not
crucially different from that of thiorphan except for a
For ACE inhibition an increased potency (30-fold) was
obtained for compound 1 as compared to A, in agree-

Design of Potent Dual Inhibitors
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 6 1215
Ta ble 2. Structure-Activity Relationships for Dual Inhibition of NEP and ACE by Various Mercaptoacyl Dipeptides
O
HS
C – AA1 – AA2
*
R1
IC50 (nM)a
compd
R1
stereo (*)
AA1
AA2
OH
OH
Pro
Pro
Pro
Pro
Pro
NEP
ACE
B
C
CH2Ph
CH2Ph
CH2Ph
CH2Ph
S
S
S
R
S
S
S
S
S
S
S
S
S
S
S
S
S
S
Phe
Ala
Ala
Ala
Ala
Aibb
Phe
Val
Ile
23 ( 3
4 ( 1
3.8 ( 0.6
1.3 ( 0.2
20 ( 2
46 ( 10
30 ( 8
24 ( 9
24 ( 5
130 ( 15
22 ( 5
63 ( 8
9.5 ( 0.8
5.3 ( 0.2
190 ( 30
180 ( 15
175 ( 30
180 ( 30
44 ( 4
40 ( 4
0.3 ( 0.1
25 ( 5
0.35 ( 0.05
1.6 ( 0.6
1.1 ( 0.2
0.85 ( 0.08
1.7 ( 0.6
28 ( 5
150 ( 30
100 ( 20
1.6 ( 0.6
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
(CH2)2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
Pro
Pro
Pro
Ile
Ala
Val
Ala
Ala
Val
Ala
Ile
Pyrc
Hypd
(R,S)Pipe
Phif
0.40 ( 0.03
0.58 ( 0.04
3.3 ( 0.2
28 ( 2
110 ( 20
CH(CH3)CH2CH3
CH(CH3)CH2CH3
CH(CH3)2
CH(CH3)Ph
Pro
Indg
Phif
Val
(R,S)Pipe
a
b
IC50 values are the means ( SEM of three independent determinations performed in triplicate. Aib, 2-amino isobutyric acid. c Pyr,
pyrrolidine. Hyp, 4-hydroxyproline. e Pip, pipecolinic acid. f Phi, 1-perhydroindole-2-carboxylic acid. Ind, indoline-2-carboxylic acid.
d
g
slight displacement of the benzyl moiety in the S1′
subsite, which does not seem to modify the van der
Waals interactions responsible for the preference of TLN
and NEP for aromatic moieties as the P1′ component.
The absence of important hydrophobic interactions
between the C-terminal amino acid of 22 and the
enzyme surface is clearly illustrated by the similarity
in the IC50 values for NEP of this compound and its
precursor C which are 4 and 3.8 nM, respectively.
Likewise, the inhibitory potencies of 22 and its precur-
sor for TLN were 3.5 × 10-7 and 2 × 10-7 M respec-
tively, emphasizing again the great analogies between
the active sites of TLN and NEP.28,42,43 Nevertheless,
compounds 16 and 21 (Table 1) which have two branched
chains followed by a tyrosine residue, and which have
nanomolar IC50 values for both peptidases, exhibit a 30-
fold decrease in NEP affinity when a cyclic amino acid
replaces the tyrosine (compounds 34, 36, and 37, in
Table 2). As discussed before for compounds 18 and 19
compared with 16 and 17, one of the simplest explana-
tions could be that the C-terminal cyclic moieties induce
a conformational change unfavorable for NEP recogni-
tion.
In conclusion, highly potent dual inhibitors can be
obtained in the series of mercaptoacyldipeptides by
introduction of linear constraints, such as â-branched
chains, in favorable positions (compound 21). In this
series, the introduction of a C-terminal cyclic amino acid
does not increase the recognition of either enzyme.
Structure-activity relationships and molecular-model-
ing studies indicate that nanomolar inhibition of NEP
can be obtained with compounds optimized for the
interaction with the S1′ and S2′ domains of the enzyme,
reinforcing the assumption that no true S1 subsite exists
in NEP.41 For ACE inhibition, subnanomolar inhibitory
potencies were obtained by improving interactions with
the S1, S1′, and S2′ subsites of this peptidase. This might
be obtained using C-terminal cyclic amino acids such
as Pro and analogues, but also with Tyr as ultimate
amino acid, if residues in the P1 and P1′ positions are
judiciously chosen. The oral activity of 21 and 22 has
been measured in mice. The inhibition of kidney NEP
and lung ACE was measured 1 h after oral administra-
tion of a single dose (2.7 × 10-5 mol/kg) of these
compounds. In these conditions, 21 inhibits 80% and
36% of kidney NEP and lung ACE, respectively, while
22 inhibits 40% of kidney NEP and 56% of lung ACE.
At the same dose, the potencies of orally administered
dual inhibitors belonging to the mixanpril series were
better on pulmonary ACE and renal NEP. However,
using the approach developed in the present work, new
dual inhibitors, with both high inhibitory potency and
increased oral activity and duration of action, could be
designed by appropiate modifications of the hydrophilic
balance of the constituting P1, P1′, and P2′ residues.
This work is now in progress in our laboratory.
Exp er im en ta l Section
I. Biologica l Tests. [3H]Tyr-D-Ala2-Leu-enkephalin (52 Ci/
mmol) was obtained from Dositek (CEA Saclay, France),
N-Cbz-Phe-His-Leu44 was obtained from Bachem (Buddendorf,
Switzerland). Neutral endopeptidase from rabbit kidney45 and
angiotensin converting enzyme from rat testis403 were purified
to homogeneity as previously described. (Arg102,Glu)NEP was
cloned and expressed as described.27 Thermolysin was pur-
chased from Sigma (France) and used without further purifi-
cation.
Assa y for Neu tr a l En d op ep tid a se Activity. IC50 values
were determined as previously reported.47 NEP (final con-
centration 1 pmol/100 µL, specific activity for [3H]Tyr-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]Tyr-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 metabolites formed were separated on polystyrene
beads.48 The assay for the mutated-Glu102 NEP was identical
to that described for wild-type NEP.
Assa y for An gioten sin -Con ver tin g En zym e Activity.
Enzymatic studies on ACE were performed using N-Cbz-Phe-

1216 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 6
Coric et al.
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 per 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 determined
following the fluorimetric assay described by Cheung et al.49
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.
Assa y for Th er m olysin . IC50 values were determined as
previously reported42 using [3H]Leu-enkephalin as substrate.
II. Molecu la r Mod elin g. The docking of 22 in the TLN
active site was done by a template-forcing procedure (Insight
II User guide, Version 2.1.0. copyright 1992, Biosym Technolo-
gies Inc., San Diego, CA) using the coordinates of thiorphan
cocrystallized with TLN28 followed by minimization. For this
purpose, the heavy atoms of the (2-mercapto-3-phenylpro-
panoyl)glycyl moiety of 22 and all the corresponding atoms of
thiorphan were used. The Gly-Pro bond of 22 was fixed in a
trans conformation.
Two minimization steps were performed: first, compound
22 was minimized inside the TLN active site, whose structure
was maintained fixed, and second, minimization of the complex
TLN/22 without any constraints was carried out. A zinc force
field derived from ab initio calculations, parametrized in the
Amber framework, was used in this procedure to take into
account metal-protein interactions.
III. Ch em istr y. The natural amino acid derivatives and
â-naphthyl-L-alanine were purchased from Bachem (Bubben-
dorf, Switzerland). The non-natural amino acids, pipecolinic
acid, homophenylalanine, indoline-2-carboxylic acid, and the
reagents were purchased from Aldrich-Chemie (Strasbourg,
France). The synthesis of â-methylphenylalanine, (âMe)Phe,50
and perhydroindolecarboxylic acid,51 (Phi) were performed
following procedures previously described.
) CH(CH3)2 (70%), Rf (C) 0.52; R1 ) CH(CH3)CH2CH3 (87%),
Rf (C) 0.70; R1 ) CH(CH3)OCH2Ph (98%), Rf (A) 0.56.
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 , R1CH(SCOCH3)COOH. A solution of so-
dium thioacetate (1.5 equiv) in DMF was added at 0 °C to a
solution of 2-bromoalkanoic acid (1 equiv) in DMF. After
stirring overnight at room temperature, the solvent was
evaporated in vacuo. The residue was taken up with water,
acidified with 1 N HCl, and extracted with EtOAc. The organic
layer was washed with water and saturated solution of NaCl,
dried over Na2SO4, filtered, and evaporated in vacuo. An oily
product was obtained.
R1 ) Ph (62%): Rf (F) 0.37; 1H NMR ∂ (ppm) 2.28 (CH3CO),
5.14 (CHR), 7.30 (Ar), 12.60 (COOH). R1 ) CH2Ph (98%): Rf
1
(E) 0.70; H NMR ∂ (ppm) 2.25 (CH3CO), 2.88-3.24 (CH2â);
4.17 (CHR), 7.18 (Ar), 12.85 (COOH). R1 ) (CH2)2Ph (79%):
Rf (D) 0.29; 1H NMR ∂ (ppm) 1.85-2.05 (CH2â), 2.28 (CH3-
CO), 2.55 (CH2γ), 3.90 (CHR), 7.15 (Ar), 12.80 (COOH). R1 )
CH(CH3)Ph (96%): Rf (A) 0.60; 1H NMR ∂ (ppm) 1.18-1.23
(CH3â), 2.15-2.30 (CH3CO), 3.14 (CHâ), 4.18 (CHR), 7.20 (Ar),
12.70 (COOH). R1 ) CH(CH3)2 (94%): Rf (C) 0.40; 1H NMR ∂
(ppm) 0.90 (2CH3), 2.10 (CHâ), 2.30 (CH3CO), 3.85 (CHR),
12.40 (COOH). R1 ) CH(CH3)CH2CH3 (78%): Rf (A) 0.44; 1H
NMR ∂ (ppm) 0.82 (2CH3), 1.20-1.30 (CH2γ), 1.92 (CHâ), 2.30
(CH3CO), 4.10 (CHR), 12.30 (COOH). R1 ) CH(CH3)OCH2Ph
1
(64%): Rf (A) 0.46; H NMR ∂ (ppm) 1.10 (CH3â), 2.34 (CH3-
CO), 3.90 (CHâ), 4.20 (CHR), 4.45 (OCH2), 7.25 (Ar), 12.90
(COOH).
Gen er a l P r oced u r e for th e Cou p lin g Step s. A solution
of the R-amino ester or dipeptide ester (1 equiv) and triethy-
lamine (1.1 equiv) in CHCl3, a solution of HOBt (1 equiv) in
THF, and a solution of DCC (1.5 equiv) in CHCl3 were
successively added to a solution of the compound containing
a free carboxylic group (Boc-amino acid or 2-(acetylthio)-
alkanoic acid) in THF at 0 °C. After stirring overnight, the
mixture was filtered and evaporated in vacuo. The residue
was dissolved in EtOAc, washed, and dried as classically
reported.
Gen er a l P r oced u r es for th e Dep r otection of th e F u n c-
tion a l Gr ou p s. The N-Boc protection was cleaved by treat-
ment with 10 equiv of TFA in CH2Cl2 (50/50, v/v). The ester
and the acetyl protection of thiol group were cleaved by
alkaline hydrolysis with degassed reagents in order to prevent
the oxidation of the free thiol group.
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, CH2Cl2/MeOH ) 9/1;
B, CH2Cl2/MeOH/AcOH ) 9/1/0.5; C, CH2Cl2/MeOH/AcOH )
9/0.5/0.5; D, CH2Cl2/MeOH/AcOH ) 9/0.3/0.1; E, BuOH/AcOH/
H2O ) 4/1/1; F, c-hexane/EtOAc/AcOH ) 7/3/0.5; G, CH2Cl2/
MeOH/AcOH/H2O ) 5/5/0.5/1. Plates were analyzed with UV,
iodine vapor, or ninhydrin. The purity of the final compounds
was checked by HPLC on a reverse phase kromasil C8 (5 µm,
100 Å) column (SFCC) with TFA 0.05% (solvent A)/CH3CN
(solvent B) as the mobile phase on a Shimadzu apparatus. The
eluted peaks were monitored at 210 nm.
Com p ou n d 1: white solid; mp 145 °C; Rf (F) 0.22; HPLC
1
tR (40% B) 13.5 min; H NMR ∂ (ppm) 1.22, (CH3(Ala)), 2.40
(SH), 2.50 and 3.08 (2CH2â), 3.60 (CHSH), 4.12 (CHR(Ala)),
4.48 (CHR(Phe)), 7.17 (Ar), 8.12 and 8.27 (NH), 12.50 (COOH).
Com p ou n d 2: white solid; mp 88-91°C dec; Rf (C) 0.60;
HPLC tR (55% B) 7.9 min; 1H NMR ∂ (ppm) 1.52 (4H), 1.76
(2H), 1.95 (2H, cyclopentyl), 2.55 (HS), 2.70, 2.90, and 3.02
(2CH2â), 3.65 (CHSH), 4.50 (CHR(Phe)), 7.20 (Ar), 8.15 (NH),
12.12 (COOH).
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.
Com p ou n d 3: white solid; mp 125 °C; Rf (C) 0.43; HPLC
1
tR (55% B) 11.2 min; H NMR ∂ (ppm) 2.30 (SH), 2.70-3.00
(2CH2â), 3.45-3.70 (CH2O + CHSH), 4.49 (CHR(Phe) + OCH2-
Ph), 4.60 (CHR(Ser)), 7.10-7.20 (Ar), 8.18-8.30 (NH), 12.10
(COOH).
The following abbreviations were used: Et2O, ethyl ether;
MeOH, methanol; BuOH, 1-butanol; EtOAc, ethyl acetate;
AcOH, acetic acid; DMF, dimethylformamide; THF, tetrahy-
drofuran; DCC, dicyclohexylcarbodiimide; DCU, dicyclohexy-
lurea; HOBt, 1-hydroxybenzotriazole; TFA, trifluoroacetic acid;
DMSO, dimethyl sulfoxide; HMSD, hexamethyldisiloxane.
Gen er a l P r oced u r e for th e Syn th esis of 2-Br om oa l-
k a n oic Acid , R1CH(Br )COOH. The R-amino acid (1 equiv)
was dissolved in a mixture of HBr 48% (8 equiv) and water
(2/3, v/v) at 0 °C. An aqueous solution of NaNO2 (3.2 equiv)
was added slowly, and the mixture was stirred for 2 h at the
same temperature. The mixture was degassed in vacuo and
then extracted by Et2O. The organic layer was washed with
water and saturated NaCl solution, dried over Na2SO4, filtered,
and evaporated in vacuo. The 2-bromoalkanoic acids were
obtained as oily products which crystallized slowly: R1 ) Ph
(96%), Rf (B) 0.63; R1 ) CH2Ph (88%), Rf (A)0.60; R1 ) (CH2)2-
Ph (98%), Rf (B) 0.64; R1 ) CH(CH3)Ph (84%), Rf (E) 0.90; R1
Com p ou n d 4: white solid; mp 135 °C; Rf (C) 0.51; HPLC
tR (55% B) 12.45 min; H NMR ∂ (ppm) 2.30 (HS), 2.75-3.75
1
(3 × CH2â), 3.4 (CHSH), 3.68 (SCH2Ph), 4.36 (CHR(Phe)), 4.57
(CHR(Cys)), 7.12 (Ar), 8.20-8.32 (NH), 12.60 (COOH).
Com p ou n d 5: white solid; mp 145 °C; Rf (A) 0.28; HPLC
1
tR (37% B) 6.2 min; H NMR ∂ (ppm) 2.40 (SH), 2.55-3.05 (3
× CH2â), 3.50 (CHSH), 4.50 (CHR(Phe) + CHR(Nal)), 7.1 (Ar-
(Phe)), 7.3-8.05 (Ar(Nal)), 8.05-8.45 (NH), 12.40 (COOH).
Com p ou n d 6: white solid; mp 156 °C; Rf (C) 0.42; HPLC
tR (55% B) 9.85 min; 1H NMR ∂ (ppm) 2.30 (SH), 2.70-3.05 (3
× CH2â), 3.60 (CHSH), 4.44 (CHR(Trp)), 4.51 (CHR(Phe)),
6.80-7.40 (Ar), 8.10-8.13 (NH), 10.8 (NH(Ind)), 12.60 (COOH).
Com p ou n d 7: white solid; mp 189 °C; Rf (B) 0.56; HPLC
tR (55% B) 6.45 min; 1H NMR ∂ (ppm) 2.38 (HS), 2.60-3.10 (3
× CH2â), 3.60 (CHSH), 4.30 (CHR(Tyr)), 4.50 (CHR(Phe)),
6.40-6.98 (Ar(Tyr)), 7.14 (Ar(Phe)), 8.15-8.20 (NH), 9.18 (OH-
(Tyr)), 12.60 (COOH).

Design of Potent Dual Inhibitors
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 6 1217
Com p ou n d 8: white solid; mp 138 °C; Rf (B) 0.42; HPLC
tR (55% B) 7.50 min; 1H NMR ∂ (ppm) 1.70-1.90 (CH2(CHSH)),
2.50 (HS), 2.65-3.00 (2 × CH2â + CH2γ), 3.70 (CHSH), 4.30
(CHR(Tyr)), 4.50 (CHR(Phe)), 6.60-6.95 (Ar(Tyr)), 7.20 (Ar-
(Phe)), 8.20 (2 × NH), 9.20 (OH(Tyr)), 12.70 (COOH).
Com p ou n d 9: white solid; mp 182 °C; Rf (C) 0.24; HPLC
tR (45% B) 8.00 min; 1H NMR ∂ (ppm) 1.30 (CH3(Val)), 1.86
(CHâ(Val)), 2.64 (SH), 2.70-3.10 (2 × CH2â), 3.70 (CHSH),
4.20 (2 × CHR), 6.60-6.95 (Ar(Tyr)), 7.12 (Ar(Phe)), 7.96-8.18
(NH), 9.12 (OH(Tyr)), 12.45 (COOH).
Com p ou n d 10: white solid; mp 190 °C; Rf (B) 0.44; HPLC
tR (45% B) 9.80-10.35 min; 1H NMR ∂ (ppm) 0.80 (CH3(Val)),
1.75-1.90 (CH2-CH-SH), 1.90 (CHâ(Val)), 2.50 (HS), 2.65-2.95
(CH2â + CH2γ), 3.45 (CHSH), 4.20-4.28 (2 × CHR), 6.00-
6.95 (Ar(Tyr)), 7.15 (Ar(Phe)), 8.00-8.15 (NH), 9.14 (OH(Tyr)),
12.50 (COOH).
Com p ou n d 21: white solid; mp 199 °C; Rf (B) 0.42; HPLC
tR (50% B) 5.84 min; 1H NMR ∂ (ppm) 0.72-0.85 (2 × CH3-
(Val) + 2 × CH3(Ile)), 1.00 (CHâ(Ile)), 1.38-1.65 (CH2γ(Ile)),
1.80 (CHâ(Val)), 2.30 (SH), 2.75-2.98 (CH2â(Tyr)), 3.15 (CHSH),
4.18-4.32 (CHR(Ile + Tyr)), 6.55-6.90 (Ar(Tyr)), 7.90-8.08
(2 × NH), 9.15 (OH(Tyr)), 12.50 (COOH).
Com p ou n d 22: white solid; mp 74 °C; Rf (B) 0.59; HPLC
tR (37% B) 6.96 min; 1H NMR ∂ (ppm) 1.12 (CH3(Ala)), 1.78
(CH2γ(Pro)), 1.81-2.00 (CH2â(Pro)), 2.70 (SH), 2.75-3.08
(CH2â(Ph)), 3.25-3.33 (CH2∂(Pro)), 3.62 (CHSH), 4.10 (CHR-
(Pro)), 4.45 (CHR(Ala)), 7.15 (Ar(Ph)), 8.25 (NH), 12.35 (COOH).
Com p ou n d 23: white solid; mp 157 °C; Rf (B) 0.61; HPLC
tR (37% B) 7.05 min; 1H NMR ∂ (ppm) 0.92 (CH3(Ala)), 1.78
(CH2γ(Pro)), 1.75-2.08 (CH2â(Pro)), 2.69 (SH), 2.88-3.02 (CH2-
(Ph)), 3.45-3.55 (CH2∂(Pro)), 3.65 (CHSH), 4.15 (CHR(Pro)),
4.45 (CHR(Ala)), 7.12 (Ar(Ph)), 8.18 (NH), 12.40 (COOH).
Com p ou n d 24: white paste; Rf (C) 0.60; HPLC tR (45% B)
Com p ou n d 11: white solid; mp >250 °C dec; Rf (B) 0.42;
1
1
HPLC tR (50% B) 6.88 min; H NMR ∂ (ppm) 0.80 (2 × CH3-
6.42 min; H NMR ∂ (ppm) 1.14 (CH3(Ala)), from 1.80 to 2.10
(Ile)), 1.08-1.40-1.68 (CHâ + CH2γ(Ile)), 2.58 (SH), 2.60-2.90
(CH2â(Tyr)), 4.20-4.25 (CHR(Ile + Tyr)), 4.85 (CH(Ph)), 6.55-
6.95 (Ar(Tyr)), 7.30 (Ar(Ph)), 8.15 (2NH), 9.10 (OH(Tyr)), 12.45
(COOH).
(CH2â,γ(Pro) + CH2â(Ph)), 2.52 (CH2γ(Ph)), 2.65 (SH), 3.37
(CHSH), 3.46-3.60 (CH2∂(Pro)), 4.18 (CHR(Pro)), 4.67 (CHR-
(Ala)), 7.15 (Ar(Ph)), 8.25 (NH), 12.35 (COOH).
Com p ou n d 25: Oily product; Rf (F) 0.37; HPLC tR (40%
B) 6.6-6.8 min; 1H NMR ∂ (ppm) 1.25 (2 × CH3), 1.70-2.2
(CH2â + γ(Pro)), 2.6 (SH), 2.7-3.1 (CH2â(Phe)), 3.2-3.55
(CHSH + CH2∂(Pro)), 4.1 (CHR(Pro)), 7.18 (Ar(Ph)), 8.12 (NH),
12.1 (COOH).
Com p ou n d 12: white solid; mp 185 °C; Rf (B) 0.28; HPLC
tR (45% B) 9.23 min; 1H NMR ∂ (ppm) 0.80 (2CH3(Ile)), 1.03-
1.35-1.68 (CHâ + CH2γ(Ile)), 2.65 (SH), 2.75-3.20 (CH2â(Ph
+ Tyr)), 3.68 (CHSH), 4.10 (CHR(Ile)), 4.30 (CHR(Tyr)), 6.65-
7.00 (Ar(Tyr)), 7.18 (Ar(Ph)), 8.05-8.20 (NH), 9.10 (OH(Tyr)),
12.55 (COOH).
Com p ou n d 26: white solid; mp 69 °C; Rf (C) 0.60; HPLC
1
(50% B) 8.10 min; H NMR ∂ (ppm) 1.85 (CH2γ(Pro)), 1.85-
Com p ou n d 13: white solid; mp 130-140 °C dec; Rf (D)
2.05 (CH2â(Pro)), 2.50 (SH), 2.72-3.00 (CH2â(Phe + Ph)), 3.61
(CHSH), 4.10 (CHR(Pro)), 4.60 (CHR(Phe)), 7.12 (Ar(Ph +
Phe)), 8.35 (NH), 12.40 (COOH)).
1
0.38; HPLC tR (46% B) 9.4 min; H NMR ∂ (ppm) 0.80 (ꢀCH3-
(Nle)), 1.25 (CH2γ,∂(Nle)), 1.55 (CH2â(Nle)), 2.60 (SH), 2.80-
3.20 (CH2â(Ph + Tyr)), 3.65 (CHSH), 4.00-4.12 (CHR(Nle +
Tyr)), 6.60-6.95 (Ar(Tyr)), 7.24 (Ar(Ph)), 8.05-8.20 (NH), 9.55
(OH(Tyr)), 12.48 (COOH).
Com p ou n d 27: white solid; mp 155 °C; Rf (A) 0.26; HPLC
tR (50% B) 6.25 min; 1H NMR ∂ (ppm) 0.80 (2 × CH3(Val)),
1.78-2.04 (CHâ(Val) + (CH2â, CH2γ(Pro)), 2.65 (SH), 2.78-
3.05 (CH2â(Phe)), 3.48 (CH2∂(Pro)), 3.70 (CHSH), 4.10 (CR-
(Pro)), 4.25 (CHR(Val)), 7.14 (Ar(Ph)), 8.15 (NH), 12.33 (COOH).
Com p ou n d 28: white solid; mp 158-162 °C dec; Rf (D)
Com p ou n d 14: white solid; mp 134 °C; Rf (B) 0.45; HPLC
1
tR (50% B) 7.25 min; H NMR ∂ (ppm) 0.75 (CH3(Leu)), 1.30
(CH2â(Leu)), 1.48 (CHγ(Leu)), 2.60 (SH); 2.70-3.10 (CH2â(Ph
+ Tyr)), 3.54 (CHSH), 4.22 (CHR(Leu + Tyr)), 6.60-6.95 (Ar-
(Tyr)), 7.15 (Ar(Ph)), 7.92-8.10 (NH), 9.15 (OH(Tyr)), 12.50
(COOH).
1
0.22; HPLC tR (45% B) 9.22 min; H NMR ∂ (ppm) 0.80 (2 ×
CH3), 1.04 (CHâ(Ile)), 1.50-1.78 (CH2γ(Ile)), 1.78-2.05 (CH2â
+ γ(Pro)), 2.70 (SH), 2.80-3.05 (CH2â(Ph)), 3.50-3.70 (CH2∂-
(Pro) + CHSH), 4.08 (CHR(Pro)), 4.38 (CHR(Ile)), 7.12 (Ar-
(Ph)), 8.20 (NH), 12.35 (COOH).
Com p ou n d 15: Oily compound; Rf (B) 0.44; HPLC tR (45%
B) 9.00 min; 1H NMR ∂ (ppm) 1.00 (2 × CH3), 2.60 (SH), 2.70-
3.15 (CH2â(Ph + Tyr)), 3.40 (OCH2), 3.70 (CHâ(Thr)), 3.85
(CHSH), 4.34 (CHR(Tyr + Thr)), 6.60-6.95 (Ar(Tyr)), 7.18 (Ar-
(Ph)), 7.80-8.00 (NH), 9.20 (OH(Tyr)), 12.4 (COOH).
Com p ou n d 16: white product; mp 150 °C; Rf (D) 0.20;
HPLC tR (50% B) 5.76 min; 1H NMR ∂ (ppm) 0.75 (CH3(Val +
Ile)), 1.00-1.25 (CH2γ(Ile)), 1.52 (CHâ(Ile)), 1.90 (CHâ(Val)),
2.40 (SH), 2.75-2.88 (CH2â(Tyr)), 3.28 (CHSH), 4.16-4.30
(CHR(Val + Tyr)), 6.58-6.95 (Ar(Tyr)), 7.90-8.12 (NH), 9.12
(OH(Tyr)), 12.56 (COOH).
Com p ou n d 29: Oily compound; Rf (C) 0.33; HPLC tR (50%
B) 7.94 min; 1H NMR ∂ (ppm) 0.80 (2 × CH3), 1.15 (CHâ(Ile)),
1.50-1.80 (CH2â(Ile)), 1.80-2.10 (CH2â + γ(Pro)), 2.45 (SH),
3.25-3.78 (CH2∂(Pro)), 4.10 (CHR(Pro)), 4.32 (CHR(Ile)), 4.80
(CH(Ph)), 7.20-7.40 (Ar(Ph)), 8.40 (NH), 12.36 (COOH).
Com p ou n d 30: Oily product; Rf (A) 0.40; HPLC tR (45%
B) 6.80 min; 1H NMR ∂ (ppm) 1.10 (CH3), 1.79 (CH2â,â′(Pyr)),
2.68 (SH), 2.80-3.08 (CH2â(Ph)), 3.2 (CH2R,R′(Pyr)), 3.64
(CHSH), 4.45 (CHR(Ala)), 7.15 (Ar(Ph)), 8.20 (NH), 12.36
(COOH).
Com p ou n d 17: white product; mp 120 °C; Rf (B) 0.40;
1
HPLC tR (50% B) 8.10 min; H NMR ∂ (ppm) 0.72 (2 × CH3-
Com p ou n d 31: white solid; mp 100 °C dec; Rf (C) 0.30;
1
(Ile)), 0.95-1.20 (CH2γ(Ile)), 1.60 (CHâ(Ile)), 2.15 (SH), 2.60-
3.00 (CH2â(Phe + Tyr)), 3.20 (CHSH), 4.32 (CHR(Tyr)), 4.52
(CHR(Phe)), 6.60-6.95 (Ar(Tyr)), 7.20 (Ar(Phe)), 9.12 (OH-
(Tyr)), 12.62 (COOH).
HPLC tR (50% B) 7.6 min; H NMR ∂ (ppm) 0.50-0.85 (2 ×
CH3(Ile)), 1.05-1.50-1.65 (CHâ, CH2γ(Ile)), 1.90-2.05 (CH2â-
(Hyp)), 2.65 (SH), 2.70-3.10 (CH2â(Ph)), 3.60 (CHR(Ph) +
CH2∂(Hyp)), 4.25 (CHR(Ile) + (CHR + CHγ(Hyp)), 5.10 (OH-
(Hyp)), 7.20 (Ph)), 8.15 (NH), 12.37 (COOH).
Com p ou n d 18 (mixture of diastereoisomers): white prod-
uct; mp 125 °C; Rf (B) 0.50-0.60; HPLC tR (50% B) 9.13 min;
1H NMR ∂ (ppm) 0.50-0.90 (3 × CH3), 0.95-1.20-1.50 (CHâ
+ CH2γ(Ile)), 2.12 (SH), 2.65-3.20 (CH2â(Tyr) + CHâ(Phe)),
3.30 (CHSH), 4.30-4.60 (CHR(Phe + Tyr)), 6.60-6.95 (Ar-
(Tyr)), 7.18 (Ar(Phe)), 7.82-8.50 (NH), 9.10 (OH(Tyr)), 12.52
(COOH).
Com p ou n d 32 (mixture of stereoisomers): oily product; Rf
(C) 0.48; HPLC tR (50% B) 5.75 min; 1H NMR ∂ (ppm) 1.10
(CH3(Ala)), 1.25-1.55-2.04 (CH2â,γ,∂(Pip)), 2.70 (HS), 2.75-
3.08 (CH2â(Ph)), 3.10-3.55 (CH2ꢀ(Pip)), 3.65 (CHR(Ph)), 4.70
(CHR(Ala)), 4.95 (CHR(Pip)), 7.18 (Ar), 8.20 (NH), 12.05
(COOH).
Com p ou n d 19: Oily product; Rf (C) 0.30; HPLC tR (45%
B) 8.72 min; 1H NMR ∂ (ppm) 0.75-1.05 (2 × CH3(Ile) + CH3-
(Thr)), 1.35-1.60-1.70 (CHâ + CH2γ(Ile)), 2.45 (SH), 2.80-
2.90 (CH2â(Tyr)), 3.45 (CHSH), 3.85 (CHR(Tyr)), 4.40 (OCH2
+ CHR + CHâ(Thr)), 6.60-6.85 (Ar(Tyr)), 7.25 (Ar(Ph)), 7.92-
8.08 (NH), 9.15 (OH(Tyr)), 12.70 (COOH).
Com p ou n d 20: white solid; mp 82 °C; Rf (B) 0.45; HPLC
tR (50% B) 8.00 min; 1H NMR ∂ (ppm) 0.75 (2 × CH3(Ile)), 1.15
(CH3(Thr)), 1.00-1.35-1.75 (CHâ + CH2γ(Ile)), 2.55 (SH),
2.68-2.80 (CH2â(Tyr)), 3.62 (CHSH), 3.68 (CHâ(Thr)), 4.28
(CHR(Ile + Tyr)), 4.40 (CH2O), 6.55-6.90 (Ar(Tyr)), 7.18 (Ar-
(Ph)), 8.00-8.20 (2 × NH), 9.12 (OH(Tyr)), 12.43 (COOH).
Com p ou n d 33: white solid; mp 148 °C; Rf (G) 0.79; HPLC
tR (50% B) 7.67 min; H NMR ∂ (ppm) 1.10 (CH3), 1.00-2.30
(CH2(Phi)), 2.65 (SH), 2.75-3.10 (CH2â(Ph)), 3.65 (CHSH), 4.04
(CHR(Phi)), 4.46 (CHR(Ala)), 7.12 (Ar(Ph)), 8.30 (NH), 12.28
(COOH).
1
Com p ou n d 34: oily product; Rf (B) 0.59; HPLC tR (50% B)
1
6.86 min; H NMR ∂ (ppm) 0.90 (4 × CH3), 1.00-2.12 (CHâ-
(Ile) + CH2γ(Ile) + CHâ(Val) + (CH2â + γ(Pro)), 2.40 (SH),
3.54 (CHSH), 3.30-3.76 (CH2∂(Pro)), 4.17 (CHR(Pro)), 4.30
(CHR(Val)), 8.16 (NH), 12.36 (COOH).
Com p ou n d 35: white solid; mp >200 °C dec; Rf (A) 0.61;
1
HPLC tR (50% B) 9.32 min; H NMR ∂ (ppm) 0.80 (2 × CH3-

1218 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 6
Coric et al.
(16) Patchett, A. A.; Harris, E.; Tristram, E. W.; Wyvratt, M. J .; Wu,
H. T.; Taub, D.; Peterson, E. R.; Ikeler, T. J .; ten Brocke, J .;
Payne, L. G.; Ondeyka, D. L.; Thorsett, E. D.; Greenlee, W. J .;
Lohr, N. S.; Hoffsommer, R. D.; J oshua, H.; Ruyle, W. V.;
Rothrock, J . W.; Aster, S. D.; Maycock, A. L.; Robinson, F.M.;
Hirschmann, R.; Sweet, C. S.; Ulm, E. H.; Gross, D. M.; Vassil,
T. C.; Stone, C. A. A new class of angiotensin converting enzyme
inhibitors. Nature (London) 1980, 288, 280-283.
(17) Fournie´-Zaluski, M. C.; Llorens, C.; Gacel, G.; Malfroy, B.;
Swertz, J . P.; Lecomte, J . M.; Schwartz, J . C.; Roques, B. P.
Synthesis and biological properties of highly potent enkephali-
nase inhibitors. Peptides 1980. In Proceedings of the sixteeth
European Peptide Symposium; Brunfeldt, K., Ed.; Scriptor:
Copenhagen 1981; pp 476-481.
(18) Gordon, E. M.; Cushman, D. W.; Tung, R.; Cheung, H. S.; Wang,
F. L.; Delaney, N. G. Rat brain enkephalinase: characterization
of the active site using mercaptopropanoyl amino acids inhibitors
and comparison with angiotensin converting-enzyme. Life Sci.
1983, 33 (Suppl. 1), 113-116.
(19) Fournie´-Zaluski, M. C.; Lucas, E.; Waksman, G.; Roques, B. P.
Differences in the structural requirements for selective interac-
tion with neutral endopeptidase (enkephalinase) or angiotensin
converting enzyme: molecular investigation by use of new thiol
inhibitors. Eur. J . Biochem. 1984, 139, 267-274.
(Ile)), 1.04 (CHâ(Ile)), 1.25 (CH3(Ala)), 1.30-1.50 (CH2γ(Ile)),
1.65 (CH2â(Ind)), 2.38 (SH), 3.60 (CHSH), 4.50 (CHR(Ala)),
5.10 (CHR(Ind)), 7.00-7.18 (Ar(Ind)), 8.10 (NH), 12.36 (COOH).
Com p ou n d 36: white solid mp >250 °C dec; Rf (C) 0.54;
HPLC tR (50% B) 9.03 min; 1H NMR ∂ (ppm) 0.80-0.90 (CH3-
(Val + Ile)), 1.00-2.25 (CHâ(Ile) + CH2γ(Ile) + CHâ(Val) +
CH2(Phi)), 2.40 (SH), 3.58 (CHSH), 4.12 (CHR(Phi)), 4.25
(CHR(Ile)), 8.30 (NH), 12.36 (COOH).
Com p ou n d 37 (mixture of stereoisomers): oily product; Rf
(B) 0.48; HPLC tR (50% B) 9.3, 9.8, 10.3, and 10.6 min; 1H NMR
∂ (ppm) from 0.70 to 2.20 (CH2â,γ,∂(Pip) + CH3(Val) + CH3â-
(Ph) + CHâ(Val)), 2.60 (SH), 2.95 (CHâ(Ph)), 3.60 (CHSH),
3.70-3.95 (CH2∂(Pip)), 4.30 (CHR(Val)), 5.05 (CHR(Pip)), 7.20
(CH(Ar)), 8.20 (NH), 12.60 (COOH).
Ack n ow led gm en t. The authors would like to thank
C. Dupuis for expert manuscript drafting and Dr. A.
Beaumont for stylistic revision. Particular acknowl-
edgements are due to V. Thery and S. Antonczak for
their help in computer-modeling studies. The work was
supported by grants from the “Institut de Recherches
Servier”.
(20) Roques, B. P.; Noble, F.; Dauge´, V.; Fournie´-Zaluski, M. C.;
Beaumont, A. Neutral endopeptidase 24.11; structure, inhibition,
and experimental and clinical pharmacology. Pharmacol. Rev.
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