Toward an optimal joint recognition of the s1′ subsites of endothelin converting enzyme-1 (ECE-1), angiotensin converting enzyme (ACE), and neutral endopeptidase (NEP)

Article abstract of DOI:10.1021/jm0005454

The formation of vasoconstrictors (e.g., angiotensin II and endothelin) and the inactivation of vasodilators (e.g., bradykinin and atrial natriuretic) by membrane-bound zinc metallopeptidases are key mechanisms in the control of blood pressure and fluid h

Full text of DOI:10.1021/jm0005454

J . Med. Chem. 2002, 45, 1477-1486
1477
Tow a r d a n Op tim a l J oin t Recogn ition of th e S1′ Su bsites of En d oth elin
Con ver tin g En zym e-1 (ECE-1), An gioten sin Con ver tin g En zym e (ACE), a n d
Neu tr a l En d op ep tid a se (NEP )
Nicolas Inguimbert, Pascale Coric, Herve´ Poras, Herve´ Meudal, Franck Teffot,
Marie-Claude Fournie´-Zaluski, and Bernard P. Roques*
De´partement de Pharmacochimie Mole´culaire et Structurale, INSERM U266, CNRS UMR 8600,
UFR des Sciences Pharmaceutiques et biologiques, 4 Avenue de l’observatoire, 75270 Paris Cedex 06, France
Received December 22, 2000
The formation of vasoconstrictors (e.g., angiotensin II and endothelin) and the inactivation of
vasodilators (e.g., bradykinin and atrial natriuretic) by membrane-bound zinc metallopeptidases
are key mechanisms in the control of blood pressure and fluid homeostasis. The way in which
these peptides modulate physiological functions has been intensively studied. With the aim to
develop compounds that can jointly block the three metallopeptidasessneutral endopeptidase
(NEP, neprilysin), angiotensin-converting enzyme (ACE), and endothelin-converting enzyme
(ECE-1)swe studied the common structural specificity of the S1′ subsites of these peptidases.
Various mercaptoacyl amino acids of the general formula HS-CH2-CH(R1′)CO-Trp-OH, pos-
sessing more or less constrained R1′ side chains, were designed. The mercapto-acyl synthons
contain one or two asymmetrical centers. The Ki values of the separated stereoisomers of the
most efficient inhibitors were used to determine the stereochemical preference of each enzyme.
A guideline for the joint inhibition of the three peptidases was obtained with the (2R,3R) isomer
of compound 13b. Its Ki values on NEP, ACE, and ECE were 0.7, 43, and 26 nM, respectively.
In tr od u ction
of a large number of compounds with interesting
therapeutic profiles compared to selective inhibitors.5
However, one possible limitation of this approach could
be an increase in plasma levels of ET-1 since the
degradation of this peptide is achieved by NEP.12 This
problem can be overcome by inhibiting ECE with
compounds that block the enzymatic activity of the three
targeted metallopeptidases. This can be achieved by
synthesizing small molecules containing efficient zinc
ligands.
Four peptidergic systems are involved in the regula-
tion of blood pressure, fluid volume, and electrolyte
homeostasis. Two of them, angiotensin II (AII) and
endothelin I (ET-1), are derived from inactive precursors
and are strong vasoconstrictors. Conversely, bradykinin
(Bk) is a potent vasodilatator, and atrial natriuretic
peptide (ANP) has natriuretic and diuretic activities.1
Consequently, modulating these physiologically coun-
teracting systems could be of great interest for the
treatment of various cardiovascular diseases. One way
to obtain such a result is to decrease the formation of
AII and ET-1 and to increase the amount of Bk and
ANP. Theoretically, this could be achieved by simulta-
neously inhibiting the peptidases involved in the gen-
eration of vasoconstrictor peptides, i.e., angiotensin-
converting enzyme (ACE, EC 3.4.15.1) for AII and
endothelin converting enzyme (ECE-1, EC 3.4.24.71) for
ET-1, and in the inactivation of the diuretic and va-
sorelaxant peptides, i.e., neutral endopeptidase (NEP,
EC 3.4.24.11, neprilysin) for ANP and NEP and ACE
for Bk (reviews in refs 2-5).
Therefore, we tried to design such compounds so that
we could optimize the control of the various cardiovas-
cular functions associated with the four peptidergic
systems. We developed compounds that interact with
the S1′ and S2′ subsites of the three enzymes and that
bear a thiol group as a zinc ligand. All of the molecules
designed have the general formula HS-CH2-CH(R1′)-CO-
TrpOH. The tryptophan residue was selected as P2′
component for all of these molecules because the indole
moiety is well recognized by the S2′ subsite of ECE.13,14
Various R1′ side chains containing aromatic rings were
tested for interaction with the S1′ subsite of the three
targeted enzymes. Mercaptoacyl synthons contain one
or two asymmetrical centers, thus the corresponding
inhibitors are mixtures of two or four diastereoisomers.
We separated each of these isomers and used physico-
chemical methods and X-ray crystallography to deter-
mine and to confirm the absolute configuration of each
isomer. Their Ki values on the three peptidases were
measured, and their stereochemical preferences were
determined so that triple inhibitors with about the same
nanomolar inhibitory potency for the three peptidases
could be obtained.
These three peptidases belong to the same family of
membrane-bound zinc metallopeptidases and have the
same mechanism of action.4 Selective inhibitors have
been reported for each enzyme.6-8 ACE inhibitors are
the most commonly used drugs for the treatment of
hypertension and congestive heart failure.9,10 The con-
cept of dual NEP/ACE inhibition11 led to the generation
* To whom correspondence should be addressed: Professor Bernard
P. Roques, De´partement de Pharmacochimie Mole´culaire et Struc-
turale, U266 INSERM, UMR 8600 CNRS, 4, avenue de l’Observatoire,
75270 PARIS Cedex 06, France. Tel: (33)-1-43.25.50.45. Fax: (33)-1-
43.26.69.18. E-mail: roques@pharmacie.univ-paris5.fr.
10.1021/jm0005454 CCC: $22.00 © 2002 American Chemical Society
Published on Web 02/23/2002

1478 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 7
Sch em e 1. Synthetic Pathway of Inhibitors 5-14a
Inguimbert et al.
m er s of In h ibitor s 9, 10, 13, a n d 14. A convergent
approach was used to assign the absolute configuration
of the asymmetrical carbons in the separated isomers
of inhibitors 9, 10, 13, and 14.
In a previous report15 the four stereoisomers of 4e
were separated by chiral amines and their respective
absolute configurations determined by using stereo-
selective syntheses. These results show that, in our
experimental conditions, the same assignment based on
the HPLC retention times could be proposed: the first
set of enantiomers (4e1) is a mixture of 2R,3R and 2S,3S
isomers, the other set (4e2) contained the other two
stereoisomers 2R,3S and 2S,3R. The first set of enan-
tiomers (4e1) only represented 30% of the initial mix-
ture, whereas the second set (4e2) was the most abun-
dant (70%).
As the relative proportion of each pair 4f1/4f2, 4i1/
4i2, and 4j1/4j2 was identical to the relative proportion
of 4e1/4e2, the same stereochemical assignment was
postulated for the four synthons.
a
(a) (EtO)3P; (b) triethylphosphonoacetate, NaH, THF; (c)
(H2CO)n, K2CO3, THF; (d) NaOH, acetone; (e) thioacetic acid,
CHCl3; (f) EDCl, HOBT, Et3N, HCl, Trp-OR; (g) TFA, NaOH,
MeOH.
NMR has been used to assign the stereochemistry of
a dipeptide or a pseudo dipeptide.16 The general rule is
that, in a dipeptide unit containing one aromatic side
chain, the relative absolute configuration of each residue
can be deduced from the chemical shifts of the protons
of the nonaromatic side chain: the protons were shielded
when the two residues had opposite absolute configu-
ration (R,S or S,R) as compared to those having identi-
cal configurations (R,R or S,S).
All of the inhibitors studied have an S-tryptophan in
the C terminal position. Thus, the relative chemical
shifts of the γ methyl side chain in 9 and 10, or of the
γ methylene in 13 and 14, allow the absolute configura-
tions of C2 to be determined in the four stereoisomers
(Table 1).
The γ methyl in 9 and 10 and the γ methylene in 13
and 14 are more shielded in the “b” and “d” isomers than
in the “a” and “c” isomers indicating that in the former
C2 has an R configuration and that in the latter it has
an S configuration (Table 1).
Taken together these data allow a complete stereo-
chemical assignment of the four stereoisomers of 9, 10,
13, and 14. The absolute configurations of the carbon
C2 and C3 in the mercaptoacyl synthons are 2S,3S in
“a”, 2R,3R in “b”, 2S,3R in “c”, and 2R,3S in “d” (Scheme
2).
Furthermore, the empiric rule used to determine the
absolute configuration of the stereoisomers 9, 10, 13,
and 14 was confirmed by X-ray crystallography on the
protected form of compound 13c. As expected, compound
13c is in the 2S,3R absolute configuration (Figure 1).
It is noteworthy that when HPLC was carried out in
identical conditions the elution order was also related
to the stereochemistry of the compounds, the 2S isomers
“a” and “c” being slightly less retained than the 2R
analogues “b” and “d”. This result further confirms our
hypothesis.
III. In h ibitor y P oten cies of th e Va r iou s In h ibi-
tor s. In a preliminary screening, the synthesized com-
pounds were tested as a mixture of stereoisomers for
their ability to inhibit ECE-1, NEP, and ACE in vitro
(Table 2). The R1′ residues of the first series of com-
pounds (5-7) are bicyclic structures, the spatial orien-
tations of which are constrained by the absence of a
Resu lts
I. Ch em istr y. The various inhibitors were prepared
as outlined in Scheme 1. In brief, compounds 2 were
condensed with formaldehyde by a Wittig-Horner ole-
fination. This was followed by alkaline hydrolysis, which
yielded substituted acrylic acids (3). Racemic mixtures
(4) were obtained by Micha¨el addition of thiolacetic acid
to compounds 3. The inhibitors (5-14) were obtained
by coupling compounds 4 with tryptophan methyl or
tert-butyl ester by use of the EDCI/HOBt method,
followed by alkaline or acidic hydrolysis of the thioester
and ester groups. The 2-substituted phosphonoacetates
2 were obtained by two methods depending on the
nature of the substituent R1′ (Scheme 1). The first
method was an Arbusov reaction between triethyl
phosphite and methyl-1-bromo-1-aryl acetates (1a -c).
The second method involved the reaction between the
triethylphosphonoacetate anion with various bromoalkyl
derivatives (1d -j). Due to the presence of two unre-
solved asymmetrical carbons in synthons 4e-j, the
inhibitors 9-14 were obtained as a mixture of four
stereoisomers. To obtain optically pure species, the most
interesting compounds (9, 10, 13, and 14) were sepa-
rated by a two-step process (Scheme 2). The correspond-
ing synthons (4e, 4f, 4i, and 4j) were separated by
preparative HPLC into two diastereoisomeric sets of
enantiomers, designated 41 and 42 according to their
elution order. The fractions 41 and 42 were present in
30% and 70% of the separated synthons, respectively.
Each pair of enantiomers (41 and 42) was coupled with
the optically pure S-tryptophan tert-butyl ester, leading
to a new mixture of diastereoisomers which were also
separated by preparative HPLC and deprotected (Scheme
2). The different stereoisomers obtained in enantiomeric
pure form (g98% based on analytical HPLC) were
designated “a” and “b” when they were derived from 41
and “c” and “d” when they were derived from 42.
As the HPLC separation was poorly efficient for
compounds 9a -d , the four optically pure stereoisomers
were obtained from the four chiral synthons of 4e,
resolved by chiral amines, as previously described.15
II. Assign m en t of th e Absolu te Con figu r a tion of
th e Asym m etr ica l Ca r bon s in th e Sep a r a ted Iso-

J oint Recognition of ECE-1, ACE, and NEP S1′ Subsites
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 7 1479
Sch em e 2. Separation of the Four Stereoisomers of Compounds 9, 10, 13, and 14a
a
Retention time on a C18 Kromasil column using TFA 0.05%/CH3CN as mobile phase. *Stereochemistry of compound 4e was determined
as previously reported.15
methylene spacer. Compound 5, which has a p-biphenyl
moiety, was the least efficient inhibitor with Ki values
between 10-7 and 10-6 M for the three enzymes. In
contrast, the m-biphenyl residue in 6 significantly
improves ECE recognition, with a Ki value of 0.9 µM
and inhibitory potencies between 10-8 and 10-7 M for
NEP and ACE, respectively. The naphth-1-yl residue
found in 7 is well recognized by NEP (Ki ) 22 nM) but
is less efficient for ECE and ACE.
In the second series of compounds (8 to 12), a
methylene spacer was introduced and was replaced by
a phenyl (compound 8) or methyl group (9 to 12). The
biphenyl methyl moiety of 8 is unfavorable for the three
enzymes with Ki values of about 10-7 M for NEP and
ACE and greater than 10-5 M for ECE. The replacement
of one of the phenyl groups by a methyl in 9 allowed
the recovery of high affinity for NEP and ACE (Ki values
3.0 and 40 nM, respectively) and a significant increase

1480 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 7
Inguimbert et al.
Ta ble 1. Chemical Shift of Some Characteristic Groups of
Ta ble 2. In Vitro Activity (Ki, nM) of the Thiol Inhibitors on
Protons for the Separated Isomers of Compounds 9, 10, 13, and
NEP, ACE, and ECE
14
chemical shift (δ ppm)
pro-
compd
n
R2
H
A
C
D
SH portion config
9a
9b
9c
0
2.6-2.65
2.4-2.65
1.85-2.56 2.65 1.1
1.85-2.40 2.7
2.9
2.85 0.9
1.1
1.75
1.95
1.5
1.85
1.75
1.90
1.50
1.90
15
15
35
35
15
15
35
35
15
15
35
35
10
10
40
40
2S3S
2R3R
2S3R
2R3S
2S3S
2R3R
2S3R
2R3S
2S3S
2R3R
2S3R
2R3S
2S3S
2R3R
2S3R
2R3S
9d
0.8
10a
10b
10c
10d
13a
13b
13c
13d
14a
14b
14c
14d
0
1
2
Ph 2.3-2.70
2.4-2.65
3.2
3.0
1.20
1.05
1.15
0.85
1.86-2.45 2.8
1.9-2.4 2.8
H
H
2.15-2.55 3.35 1.75-1.95 1.65
2.15-2.65 3.2
1.6
2.0
1.60
2.45-2.65 3.05 1.9-2.0
2.45-2.70 3.0
1.50-1.70 2.0
1.90-2.70 3.15 1.5-1.60
1.75
1.9-2.65
2.2-2.6
2.2-2.70
2.9
2.75 1.45-1.75 1.60
2.70 1.1-1.25 2.05
1.05-1.25 1.90
F igu r e 1. Molecular structure of the protected form of 13c,
determined with single-crystal X-ray crystallography.
a
All compounds were tested under mixtures of two or four
stereoisomers.
in ECE recognition (0.3 µM). An additional phenyl group
in 10 was slightly detrimental for NEP and ACE (4.8
and 62 nM, respectively) but increased ECE recognition.
The fusion of the two cycles in a naphth-2-yl (compound
11) and naphth-1-yl (compound 12) moiety was unfavor-
able for ECE in the first case and for the three enzymes
in the latter case.
In the last series of inhibitors (13 and 14), the methyl
group found in 9 was introduced into a cyclic structure,
creating an indanyl group in 13 and a tetrahydronaph-
thyl residue in 14, at the P1′ position. The Ki value
obtained with 14 was not significantly different from
that obtained with 9 but the indanyl moiety in 13
resulted in the most efficient ECE inhibitor described
in this paper with a Ki value of 100 nM.
The four stereoisomers of compound 9 all had the
same effect on NEP activity. However, lengthening the
S1′ side chain of 10 induced a slight stereodependence,
which was attributed to steric hindrance in the “2S”
isomers. Indeed, the affinity of 10a and 10c was
decreased by about 1 order of magnitude, whatever the
configuration at the C3 carbon. For 13 and 14, which
contain a bicyclic moiety in S1′ position, another stereo-
preference was induced. This seemed to be related to
the absolute configuration at the C3 carbon: isomers
13a /13d and 14a /14d were significantly less active than
13b/13c and 14b/14c.
In general, the inhibition of ACE was greatly depend-
ent on the backbone stereochemistry: an S configuration
is required at C2 atom. This was verified for the four
inhibitors. The isomers 9c, 10c, 13c, and 14c, which
have the 2S,3R configuration, were the most active with
Ki values of about 10-8 M. The second most active were
the “a” isomers, which have the 2S,3S configuration. In
On the basis of these preliminary results, we decided
to study compounds 9, 10, 13, and 14 in more detail.
The different stereoisomers were separated so that the
inhibitory potency of each compound could be deter-
mined (Table 3).

J oint Recognition of ECE-1, ACE, and NEP S1′ Subsites
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 7 1481
Ta ble 3. In Vitro Activity (Ki, nM) for the Separated Isomers
of Compounds 9, 10, 13, and 14 on NEP, ACE, and ECE
to compare the S1′ subsites of ECE-1, NEP, and ACE.
We maintained the P2′ moiety so that we could deter-
mine the most favorable spatial disposition of S1′ and
design compounds to inhibit the three peptidases with
equipotent activities.
Compounds 5, 6, and 7, which have P1′ side chains
derived from phenyl glycine moieties, retained a rela-
tively good affinity for NEP and ACE, but their lack of
mobility seemed to preclude an efficient recognition of
the ECE S1′ subsite (Table 2). We also tested â-branched
residues derived from phenylalanine. The second â-
branched aromatic group in 8 was shown to be highly
detrimental compared to the parent compound HSCH2-
(CH2Ph)CO-TrpOH (Ki(ECE) 340 nM),14,22 essentially
for ECE inhibition. However, a â-methyl chain (9) is well
accepted by the active sites of NEP and ACE as
previously demonstrated15 and also by the ECE active
site.
The effect of an additional aromatic ring in the P1′
component of the inhibitor (compounds 10-12) was
highly dependent on its position: the biphenyl moiety
of 10 slightly increased the recognition of ECE, reflect-
ing a better occupancy of this S1′ pocket. Conversely,
the naphthyl moieties of 11 and 12 were significantly
less efficient for both NEP and ECE recognition.
Finally, the best result was obtained with 13, which
can be considered to be a constrained analogue of 10
and which had the best affinity of this series of
compounds for the three enzymes, suggesting that the
three enzymes share strong similarities for the oc-
cupancy of the S1′ subsite.
compd
n
R2
H
config
NEP
ACE
ECE
9
9a
9b
9c
9d
10
10a
10b
10c
10c
13
13a
13b
13c
13d
14
0
racemic
2S3S
2R3R
2S3R
2R3S
3 ( 0.2
2.2 ( 0.3
2.4 ( 0.8
2.5 ( 0.2
5.0 ( 0.4
40 ( 4
29 ( 6
33 ( 1
15 ( 2
290 ( 10
62 ( 4
65 ( 1
130 ( 6
38 ( 1
330 ( 3
20 ( 2
41 ( 3
43 ( 2
10 ( 1
170 ( 1
18 ( 3
26 ( 2
76 ( 6
10 ( 0.6
134 ( 20
300 ( 20
100 ( 10
200 ( 30
780 ( 40
580 ( 40
220 ( 10
110 ( 5
135 ( 10
920 ( 10
190 ( 10
100 ( 8
0
1
2
Ph racemic 4.8 ( 0.1
2S3S
2R3R
2S3R
2R3S
30 ( 1
1.5 ( 0.3
35 ( 5
5.0 ( 0.4
H
H
racemic 1.8 ( 0.2
2S3S
2R3R
2S3R
2R3S
10 ( 1
1140 ( 60
26 ( 3
290 ( 20
680 ( 9
0.7 ( 0.03
2.1 ( 0.1
27 ( 1
racemic 4.5 ( 0.9
300 ( 40
670 ( 50
250 ( 20
190 ( 8
14a
14b
14c
14d
2S3S
2R3R
2S3R
2R3S
8.0 ( 0.6
3.0 ( 0.4
3.0 ( 0.6
43 ( 5
1240 ( 20
all cases, the least efficient compound had the 2R,3S
configuration, with Ki values of about 10-7 M.
Finally, for ECE inhibitors a different stereoprefer-
ence appeared between compounds 9 and 10, containing
the branched synthons, and compounds 13 and 14,
containing a fused bicyclic structure. For compounds 9
and 10, the “a” (Ki 100 and 110 nM, respectively) and
“b” (Ki 200 and 135 nM, respectively) isomers were the
most efficient. For compound 13, the 2R,3R isomer (13b)
had a Ki value of 26 nM. For compound 14, the 2S,3R
isomer (14c) (Ki 190 nM) and the 2R,3R isomer (14b)
(Ki 250 nM) were virtually equipotent.
These results were analyzed in more detail after
separating the various stereoisomers obtained during
this nondiastereoselective synthesis. The first param-
eter that should be highlighted is the asymmetric
induction observed during the Michael addition of
thioacetic acid on the racemic derivatives 3a -j. The 30/
70 ratio reflects a diastereotopic transition state for this
reaction, with differences in the accessibility of the
intermediate enolate double bond.
Discu ssion
However, whatever the mechanism responsible for
this diastereoselectivity, the stereoisomers c and d were
always obtained in greater proportion than a and b, and
this has to be taken into account for the synthesis of
such inhibitors.
The second interesting point concerns the stereo-
chemistry of the most efficient isomers (Table 3), which
are directed both by the different targeted enzymes and
by the structure of a given inhibitor.
Thus, in the case of ACE, the most active inhibitors
were derived from the 2S3R synthons. This has been
previously observed in the mixanpril series,15 in which
the presence of a small C-terminal residue, such as an
alanine, led to Ki values in the nanomolar range. In the
new inhibitors reported here, the bulky C-terminal
tryptophan slightly decreases the affinity for ACE,
leading to Ki values of about 10-8 M.
The S1′ subsite of NEP is relatively large and can
accommodate various side chains without stereochem-
ical preference.18,23 This has been previously shown with
inhibitors such as thiorphan24 and mixanpril15 and was
also the case with compound 9: the four stereoisomers
of which have Ki values between 2 and 5 nM. However,
the lengthening of the P1′ chain in 10 and the constraint
The three enzymes involved in the maturation or
degradation of the major vasoactive peptides (AII, ET-
1, Bk, and ANP) belong to the gluzincin subfamily of
zinc metallopeptidases. Thus, all three enzymes have
virtually identical mechanisms of action and their active
sites are similarly organized4,17-20 as shown by the study
of various dual NEP/ACE inhibitors.15,20 As a result, a
common pharmacophore has been proposed for NEP and
ACE. ECE-1 presents significant primary sequence
homology with NEP (51% in the active site), including
consensus sequences and essential amino acids in the
catalytic site, suggesting a close similarity between NEP
and ECE.21 This was recently confirmed by a three-
dimensional model of ECE based on the X-ray structure
of NEP.18,19 These studies suggest that there are dif-
ferences in the topological organization of the S2′ sub-
sites and that ECE has a deeper S1′ pocket than NEP.
This is consistent with the relative activities of CGS-
26,303 and CGS-34,043, the P1′ residues of which are
of different sizes and which behave, respectively, as a
selective NEP inhibitor (Ki(NEP) 0.9 nM; Ki(ECE) 410
nM) and a selective ECE inhibitor (Ki(NEP) 2300 nM;
Ki(ECE) 22 nM).8 In this work, our main objective was

1482 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 7
Inguimbert et al.
results should allow triple NEP/ACE/ECE inhibitors
with nanomolar affinities for each enzyme to be de-
signed. Moreover, by comparing the activities of 9 and
10 on ECE, it can be concluded that substituting various
groups in suitable positions on the aromatic rings of 13
should improve the recognition of ECE.
Exp er im en ta l Section
1
Ch em istr y. H NMR spectra were measured on a Bru¨ker
AC 270 MHz or AC 400 MHz spectrometer using tetrameth-
ylsilane as internal standard. Electrospray mass spectra (MS-
ES) were recorded on a Esquier-Bru¨ker spectrometer. Flash
columm chromatography was performed using 40-63 µm silica
gel. Reaction progress was determined by either TLC analysis
or monitored using analytical reverse-phase HPLC (Shimadzu,
LC10 AD-vp with a Class-VP5.03 software or Shimadzu, SCL
6B) using a Kromasil C18 column (Touzart-Matignon, France),
with mobile phase consisting of water containing 0.05% TFA
and acetonitrile. HPLC separations were performed using a
reverse-phase C18 Kromasil column (5 µM, porosity 100 Å)
with CH3CN/TFA 0.05% buffer (pH 4.0) as eluent, on a Waters
apparatus (detector 2487, pump 600 controler). The eluted
peaks were monitored at 210 nm. Reagents were obtained from
commercial sources and are used without further purification.
Optical rotation were determined on a J asco P-1030 polarim-
eter.
Gen er a l P r oced u r e for Syn th esis of Com p ou n d s 1a -
c. A mixture constituted of the appropriate phenylacetic
methyl ester (65 mmol), N-bromosuccinimide (130 mmol), and
a catalytic amount of benzoyl peroxyde (0.65 mmol) in dried
CCl4 (50 mL) was refluxed 24 h. After cooling, the precipitate
was removed by filtration, and the reaction adduct was
evaporated in vacuo, yielding the titled compounds as yellow
oils which were used without further purification.
F igu r e 2. Example of triple NEP/ACE/ECE inhibitors.
introduced by the bicyclic moiety in 13 and 14 led to
some restrictions in the optimal recognition of the S1′
NEP subsite. Thus, the 2R3R isomers of 10b, 13b, and
14b were the most efficient compounds, although the
2S3R isomers of 13 and 14 (13c and 14c) also exhibit
good affinities for NEP. Compounds 13c and 14c were
relatively good dual NEP/ACE inhibitors. This is in
contrast with 10, which had opposite stereopreference
for NEP and ACE (Table 3).
Finally, the proposed molecules are not optimized for
ECE inhibition, and their inhibitory potencies have to
be improved. However, given the molecular modeling
study of the active site of ECE,19 this could be achieved
by modifying the P2′ residue. Our results are more
difficult to interpret in terms of stereochemical recogni-
tion. Thus, for the bicyclic structures 13 and 14, only
one stereoisomer (13b) emerges significantly, with a Ki
value of 26 nM on ECE, indicating that, in this spatial
orientation, the residue fits into the S1′ subsite of ECE
relatively well. The indanyl moiety of 13b is smaller
than the P1′ side chain of CGS-35,066 (Ki(ECE) 22 nM
(Figure 2)), which could be an unfavorable parameter,
but has reduced degrees of freedom, probably resulting
in a more favorable entropic contribution during the
recognition of the active site of the enzyme.
1-Br om o-b ip h en -4-yl-a cet ic a cid m et h yl est er (1a ):
18.2 g (92%). TLC (cyclohexane:ethyl acetate, 80:20): Rf ) 0.36.
1
HPLC (CH3CN:H2O, 60:40): Rt ) 18.2 min. H NMR (CDCl3)
δ 3.7 (s, 3H), 5.45 (s, 1H), 7.3 (m, 4H), 7.5 (m, 5H).
1-Br om o-bip h en -3-yl-a cetic a cid m eth yl ester (1b):30
17.1 g (86%). TLC (cyclohexane:ethyl acetate, 80:20): Rf ) 0.38.
1
HPLC (CH3CN:H2O, 70:30): Rt ) 9.1 min. H NMR (CDCl3) δ
3.7 (s, 3H), 5.45 (s, 1H), 7.25 (m, 4H), 7.5 (m, 4H).
1-Br om o-n a p h th -1-yl-a cetic a cid m eth yl ester (1c): 18
g (99%). TLC (cyclohexane:ethyl acetate, 80:20): Rf ) 0.43.
HPLC (CH3CN:H2O, 70:30): Rt ) 8.7 min. 1H NMR (CDCl3) δ
3.7 (s, 3H), 6.1 (s, 1H), 7.4 (m, 3H), 7.65 (d, J ) 7.9 Hz, 1H),
7.8 (dd, J ) 7.9 Hz, 2H), 8.05 (d, J ) 7.9 Hz, 1H).
Gen er a l P r oced u r e for Syn th esis of Com p ou n d s 2a -
c. A mixture constituted of the appropriate 1-bromophenyl-
acetic methyl ester 1 (25 mmol) and triethyl phosphite (37.5
mmol) was heated at 120 °C for 24 h. After cooling, evaporation
in vacuo gave a residue which was purified by column
chromatography on silica gel with 30% of ethyl acetate in
cyclohexane, yielding compound 2 as an oil.
Dieth oxyp h osp h or yl-2-(bip h en -4-yl)-a cetic a cid m eth -
yl ester (2a ): 7.8 g (87%). TLC (cyclohexane:ethyl acetate,
70:30): Rf ) 0.17. HPLC (CH3CN:H2O, 70:30): Rt ) 5.3 min.
1H NMR (CDCl3) δ 1.25 (m, 6H), 3.65 (s, 3H), 4.05 (m, 4H),
4.30 (d, J ) 24 Hz, 1H), 7.25 (m, 3H), 7.5 (m, 6H).
Dieth oxylph osp h or yl-2-(biph en -3-yl)-a cetic a cid m eth -
yl ester (2b): 6.0 g (67%). TLC (cyclohexane:ethyl acetate,
70:30): Rf ) 0.15. HPLC (CH3CN:H2O, 70:30): Rt ) 5.0 min.
1H NMR (CDCl3) δ 1.2 (m, 6H), 3.65 (s, 3H), 4.0 (m, 4H), 4.35
(d, J ) 24 Hz, 1H), 7.4 (m, 8H), 7.7 (s, 1H).
Dieth oxyp h osp h or yl-2-(n a p h th -1-yl)-a cetic a cid m eth -
yl ester (2c): 7.8 g (93%). TLC (cyclohexane:ethyl acetate,
70:30): Rf ) 0.14. HPLC (CH3CN:H2O, 70:30): Rt ) 4.2 min.
1H NMR (CDCl3) δ 0.9 (t, J ) 7 Hz, 3H), 1.1 (t, J ) 7 Hz, 3H),
3.65 (s, 3H), 3.9 (m, 4H), 5.05 (d, J ) 24 Hz, 1H), 7.35 (m,
3H), 7.75 (dd, 7 Hz, 2H), 8 (m, 2H).
Gen er a l P r oced u r e for Syn th esis of Com p ou n d s 2d -
j. To a solution of triethylphosphonoacetate (40 mmol) in dry
DMF (50 mL) was added by portions (44 mmol) sodium hydride
In conclusion, a compound must be nearly equipotent
on the three targeted enzymes if it is to be considered
as a triple inhibitor. None of the separated isomers
completely fulfilled this requirement. Compound 10a ,
with Ki values of 30, 65, and 110 nM on NEP, ACE, and
ECE, respectively, could be considered as a triple
inhibitor, because the ratios between these Ki values
are only 2 and 4. However, their affinities are relatively
weak for pharmacological studies. Compound 13b, with
Ki values of 0.7 nM, 40 nM, and 26 nM on NEP, ACE,
and ECE, respectively, could be an interesting candidate
for the triple inhibition of the targeted enzymes. This
compound is comparable with two other triple inhibi-
tors, SCH-54,470 and CGS-26,58225,26 (Figure 2). SCH-
54,470 is a phosphinic inhibitor, which interacts with
the S2-S2′ subsites of the three vasopeptidases (NEP,
ACE, and ECE) with Ki values of 90 nM, 2.5 nM, and
80 nM, respectively. CGS-26,582 is a macrocyclic inhibi-
tor bearing a thiol as a zinc-chelating group with Ki
values of 4 nM, 175 nM, and 600 nM on NEP, ACE,
and ECE, respectively. Thus, CGS-26,582 can be con-
sidered as a selective inhibitor of NEP. Furthermore, a
previous study27 ruled out the possibility of using a thiol
group in ECE inhibitors. However, our work and recent
results28 demonstrate that this efficient zinc coordinat-
ing agent could be an alternative to the phosphonate
group used to optimize CGS-31,447.29 Our promising

J oint Recognition of ECE-1, ACE, and NEP S1′ Subsites
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 7 1483
at 0 °C, and the mixture was stirred at this temperature for
0.5 h. Compound 1 (40 mmol) in dry DMF (20 mL) was added
to this mixture, and the solution was stirred at room temper-
ature overnight. The solvent was removed in vacuo, and the
residue was diluted with ethyl acetate, washed with H2O, and
dried over Na2SO4. After removal of the solvent, the residue
was purified by column chromatography (4:6 ethyl acetate:
cyclohexane) to give 2.
2-(Dieth oxyph osph or yl)-3-diph en yl-pr opion ic acid eth -
yl ester (2d ):31 7.8 g (87%). TLC (cyclohexane:ethyl acetate,
70:30): Rf ) 0.17. HPLC (CH3CN:H2O, 70:30): Rt ) 5.3 min.
1H NMR (CDCl3) δ 1.25 (m, 6H), 3.65 (s, 3H), 4.05 (m, 4H),
4.30 (d, J ) 24 Hz, 1H), 7.25 (m, 3H), 7.5 (m, 6H).
NMR (CDCl3) δ 1.2 (d, 7 Hz, 3H), 4.0 (q, 7 Hz, 1H), 5.6 (s, 1H),
6.1 (s, 1H), 7.2 (m, 3H), 7.3 (m, 2H).
2-[1-(Bip h en yl-4-yl)-eth yl]-a cr ylic a cid (3f): 2.6 g (70%).
TLC (cyclohexane:ethyl acetate:acetic acid, 6:4:0.5): Rf ) 0.61.
1H NMR (DMSO-d6) δ 1.3 (d, 7 Hz, 3H), 3.9 (q, 7 Hz, 1H), 5.6
(s, 1H), 6.1 (s, 1H), 7.2 (m, 3H), 7.3 (m, 2H), 7.5 (m, 4H).
2-[1-(Na p h th a len -2-yl)-eth yl]-a cr ylic a cid (3g): 2.9 g
(85%). TLC (cyclohexane: ethyl acetate:acetic acid, 5:5:0.1):
Rf ) 0.6. 1H NMR (DMSO-d6) δ 1.4 (d, 7 Hz, 3H), 4.1 (q, 7 Hz,
1H), 5.7 (s, 1H), 6.2 (s, 1H), 7.4 (m, 3H), 7.6 (s, 1H), 7.8 (m,
3H).
2-[1-(Na p h t h a len -1-yl)-et h yl]-a cr ylic a cid (3h ): 1.2 g
(35%). TLC (cyclohexane:ethyl acetate, 5:5): Rf ) 0.5. 1H NMR
(CDCl3) δ 1.5 (d, 7 Hz, 3H), 4.7 (q, 7 Hz, 1H), 5.5 (s, 1H), 6.4
(s, 1H), 7.4 (m, 4H), 7.7 (d, J ) 7 Hz, 1H), 7.8 (d, J ) 7 Hz,
1H), 8.0 (d, J ) 7 Hz, 1H).
2-(In d a n -1-yl)-a cr ylic a cid (3i): 1.9 g (69%). HPLC (CH3-
CN:H2O, 50:50): Rt ) 8.9 min. 1H NMR (CDCl3) δ 1.9 (m, 1H),
2.4 (m, 2H), 2.9 (m, 2H), 4.2 (t, J ) 7 Hz, 1H), 5.4 (s, 1H), 6.4
(s, 1H), 7.1 (m, 4H), 7.2 (m, 1H).
2-(1,2,3,4,Tetr a h yd r o-n a p h th -1-yl)-a cr ylic a cid (3j): 1.6
g (52%). HPLC (CH3CN:H2O, 60:40): Rt ) 6.7 min. 1H NMR
(CDCl3) δ 1.7 (t, J ) 7 Hz, 2H), 1.9 (m, 2H), 2.7 (m, 2H), 4.1 (t,
J ) 7 Hz, 1H), 5.1 (s, 1H), 6.4 (s, 1H), 6.9 (m, 1H), 7.1 (m,
3H).
2-(Diet h oxyp h osp h or yl)-3-m et h yl-3-p h en yl-p r op ion -
ic a cid eth yl ester (2e):15 11.9 g (91%). TLC (cyclohexane:
ethyl acetate:acetic acid, 5:5:0.5): Rf ) 0.54. 1H NMR (DMSO-
d6) δ 0.75 (dd, 3H), 0.95 (td, 3H), 1.1 (m, 6H), 3.3 (m, 1H),
3.65 (m, 2H), 4.0 (m, 4H), 7.25 (m, 5H).
2-(Dieth oxyp h osp h or yl)-3-m eth yl-3-(bip h en -4-yl)-p r o-
p ion ic a cid eth yl ester (2f): 14.2 g (88%). TLC (cyclohexane:
ethyl acetate:acetic acid, 5:5:0.5): Rf ) 0.54. HPLC (CH3CN:
1
H2O, 70:30): Rt ) 8.4-8.7 min. H NMR (DMSO-d6) δ 0.8 (td,
3H), 1.2 (m, 6H), 3.4 (m, 2H), 3.7 (s, 2H), 4.05 (m, 4H), 7.25-
7.6 (m, 9H).
2-(Dieth oxyp h osp h or yl)-3-m eth yl-3-(n a p h th a len -2-yl)-
p r op ion ic a cid eth yl ester (2g): 9.3 g (63%). TLC (cyclo-
Gen er a l P r oced u r e for Syn th esis of Com p ou n d s 4a -
j. Thiolacetic acid (12 mmol) was added to a solution of
compound 3 (10 mmol) in CHCl3 (10 mL). The mixture was
refluxed for 12 h. The solvent was removed under reduced
pressure. The residue was purified by column chromatography
on silica gel (ethyl acetate:cyclohexane 4:6) to give 4 as an oil.
3-Acetylsu lfa n yl-2-(bip h en yl-4-yl)-p r op a n oic a cid (4a ):
1.5 g (48%). TLC (cyclohexane:ethyl acetate:acetic acid, 7:3:
1
hexane:ethyl acetate:acetic acid, 5:5:0.1): Rf ) 0.89. H NMR
(DMSO-d6) δ δ 0.7 (td, 3H), 1.25 (m, 6H), 3.4 (m, 2H), 3.7 (s,
2H), 4.1 (m, 4H), 7.4 (m, 3H), 7.8 (m, 4H).
2-(Dieth oxyp h osp h or yl)-3-m eth yl-3-(n a p h th a len -1-yl)-
p r op ion ic a cid eth yl ester (2h ): 12.6 g (83%). TLC (cyclo-
hexane:ethyl acetate, 50:50): Rf ) 0.17. 1H NMR (CDCl3) δ 0.5-
1.5 (m, 12H), 3.4-3.8 (m, 4H), 4.1 (m, 4H), 4.4 (m, 1H), 7.3
(m, 4H), 7.6 (m, 1H), 7.7 (t, J ) 7 Hz, 1H), 8.1 (t, J ) 7 Hz,
1H).
1
0.5): Rf ) 0.28. HPLC (CH3CN:H2O, 65:35): Rt ) 5.9 min. H
NMR (CDCl3) δ 2.2 (s, 3H), 3.3 (m, 2H), 3.8 (td, J ) 7 Hz, 1H),
7.2 (m, 5H), 7.6 (m, 4H). ES-MS [M+Na]+ 323.
3-Acet ylsu lfa n yl-2-(b ip h en yl-3-yl)-p r op a n oic
Diet h oxyp h osp h or yl-(in d a n -1-yl)-a cet ic a cid m et h yl
ester (2i): 13.2 g (97%). TLC (cyclohexane:ethyl acetate, 60:
a cid
1
40): Rf ) 0.14. H RMN (CDCl3): 1.3 (m, 9H), 2.3 (m, 2H), 2.9
(4b): 2.0 g (67%). TLC (cyclohexane:ethyl acetate, 6:4): Rf )
0.1. HPLC (CH3CN:H2O, 65:35): Rt ) 5.8 min. 1H NMR (CDCl3)
δ 2.2 (s, 3H), 3.3 (m, 2H), 3.8 (td, J ) 7 Hz, 1H), 7.2 (m, 5H),
7.5 (m, 4H). ES-MS [M+Na]+ 323.
3-Acetylsu lfa n yl-2-(n a p h th a len -1-yl)-p r op a n oic a cid
(4c): 0.9 g (35%). TLC (cyclohexane:ethyl acetate, 5:5): Rf )
0.15. HPLC (CH3CN:H2O, 65:35): Rt ) 4.8 min. 1H NMR
(CDCl3) δ 2.2 (s, 3H), 3.4 (m, 2H), 4.6 (td, J ) 7 Hz, 1H), 7.4
(m, 4H), 7.7 (m, 2H), 8.2 (d, J ) 7 Hz, 1H). ES-MS [M+H]+
275.
(m, 2H), 3.3 (m, 1H), 3.9 (m, 1H), 4.2 (m, 6H), 7.30 (m, 4H).
Dieth oxyp h osp h or yl-(1,2,3,4,tetr a h yd r o-n a p h th -1-yl)-
a cetic a cid m eth yl ester (2j): 13 g (95%). HPLC (CH3CN:
H2O, 60:40): Rt ) 9.2-9.5 min. 1H NMR (CDCl3) δ 1.2 (m, 9H),
2.0 (m, 4H), 2.7 (m, 2H), 3.3 (m, 1H), 3.55 (m, 1H), 4.15 (m,
6H), 6.9 (m, 4H).
Gen er a l P r oced u r e for Syn th esis of Com p ou n d s 3a -
j. To a solution of a compound 2 (15 mmol) in dry THF (50
mL) were added paraformaldehyde (30 mmol) and potassium
carbonate (30 mmol). The mixture was stirred for 24 h under
reflux. After warm filtration on Celite, THF was removed
under reduced pressure. The residue was disolved in acetone
(180 mL), and 90 mL of a 1 M sodium hydroxide solution was
added to the solution. The mixture was stirred for 12 h at room
temperature. The solvent was removed in vacuo. The residue
was diluted with H2O (100 mL) and washed with ether (2 ×
70 mL). The aqueous layer was acidified with 1 N HCl (120
mL) and extracted with ether (3 × 100 mL). The combined
organic layers were dried over Na2SO4 and evaporated to
dryness to yield the pure acid 3.
2-Acetylsu lfa n ylm eth yl-3,3-d ip h en yl-p r op a n oic a cid
(4d ): 1.8 g (56%). TLC (cyclohexane:ethyl acetate, 6:4): Rf )
1
0.12. H NMR (CDCl3) δ 2.2 (s, 3H), 2.7 (m, 1H), 3.1 (m, 1H),
3.5 (m, 1H), 4.1 (m, 1H), 7.2 (m, 10H). ES-MS [M+Na]+ 337.
2-Acetylsu lfa n ylm eth yl-3-p h en yl-bu ta n oic a cid (4e):15
2.4 g (95%). TLC (cyclohexane:ethyl acetate:acetic acid, 7:3:
1
0.5): Rf ) 0.5. H NMR (CDCl3) δ 1.2 (s, 3H), 2.2 (s, 3H), 2.5-
3.2 (m, 4H), 6.7-7.3 (m, 5H). ES-MS [M+H]+ 253.
Compound 4e was resolved by chiral amine as previously
described, leading to the four separate stereoisomers: 4e1, 4e′1,
4e2, 4e′2.15
2-(Bip h en yl-4-yl)-a cr ylic a cid (3a ):32 3.15 g (95%). TLC
(cyclohexane:ethyl acetate:acetic acid, 7:3:0.5): Rf ) 0.37.
2-Acetylsu lfan ylm eth yl-3-(biph en yl-4-yl)-bu tan oic acid
(4f): 3.2 g (96%). TLC (cyclohexane:ethyl acetate:acetic acid,
5:5:0.5): Rf ) 0.42. HPLC (CH3CN:H2O, 65:35): Rt ) 7.9 min.
1H NMR (DMSO-d6) δ 1.2 (s, 3H), 2.2 (s, 3H), 2.6-3.1 (m, 4H),
7.1-7.6 (m, 9H). ES-MS [M+NH4]+ 346.
The two diastereoisomers of 4f were separated using
preparative HPLC, leading to 4f1 and 4f2. HPLC (CH3CN:H2O,
50:50): Rt (4f1) 20.2 min, (4f2) 21.1 min.
2-Acetylsu lfa n ylm eth yl-3-(n a p h th a len -2-yl)-bu ta n oic
a cid (4g): 2.2 g (74%). TLC (cyclohexane:ethyl acetate:acetic
acid, 5:5:0.1): Rf ) 0.56. 1H NMR (DMSO-d6) δ 1.3 (d, 7 Hz,
3H), 2.2 (s, 3H), 2.7 (m, 2H), 2.9-3.2 (m, 2H), 7.2 (m, 3H), 7.6
(s, 1H), 7.8 (m, 3H).
1
HPLC (CH3CN:H2O, 65:35): Rt ) 5.5 min. H NMR (CDCl3) δ
6.1 (s, 1H), 6.6 (s, 1H), 7.3 (m, 5H), 7.6 (m, 4H).
2-(Bip h en yl-3-yl)-a cr ylic a cid (3b): 1.6 g (47%). HPLC
1
(CH3CN:H2O, 65:35): Rt ) 5.5 min. H NMR (CDCl3) δ 6.0 (s,
1H), 6.5 (s, 1H), 7.3 (m, 5H), 7.5 (m, 4H).
2-(Na p h th a len -1-yl)-a cr ylic a cid (3c):33 2.0 g (60%). (CH3-
1
CN:H2O, 65:35): Rt ) 4.4 min. H NMR (CDCl3) δ 5.9 (s, 1H),
6.8 (s, 1H), 7.2-7.4 (m, 4H), 7.7-7.9 (m, 3H).
2-(1,1-Dip h en yl-m eth yl)-a cr ylic a cid (3d ):34 3.2 g (89%).
TLC (cyclohexane:ethyl acetate:acetic acid, 6:4:0.5): Rf ) 0.55.
1H NMR (CDCl3) δ 5.2 (s, 1H), 5.3 (s, 1H), 6.5 (s, 1H), 7.0 (m,
4H), 7.2 (m, 6H).
2-Acetylsu lfa n ylm eth yl-3-(n a p h th a len -1-yl)-bu ta n oic
a cid (4h ): 2.5 g (83%). TLC (cyclohexane:ethyl acetate, 5:5):
2-(1-P h en yl-eth yl)-a cr ylic a cid (3e):15 2.2 g (82%). TLC
1
1
(cyclohexane:ethyl acetate:acetic acid, 7:3:0.5): Rf ) 0.67. H
Rf ) 0.24. HPLC (CH3CN:H2O, 65:35): Rt ) 5.7 min. H NMR

1484 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 7
Inguimbert et al.
(CDCl3) δ 1.3 (m, 3H), 2.1 (s, 3H), 2.7-3.1 (m, 3H), 4 (m, 1H),
7.3 (m, 4H), 7.7 (m, 2H), 8.1 (m, 1H). ES-MS [M+Na]+ 325.
3-Acetylsu lfa n yl-2-(in d a n -1-yl)-p r op a n oic a cid (4i): 2.1
g (85%). TLC (cyclohexane:ethyl acetate, 6:4): Rf ) 0.53. HPLC
HPLC (CH3CN:H2O, 50:50): Rt ) 20.6-21.1-24.5-25.6 min.
1H NMR (DMSO-d6) δ 0.8-1.3 (m, 3H, CH3), 1.9 (m, 1H, SH),
2.6 (m, 3H, CH2S, CHCO), 2.9 (m, 2H, CH2 Trp), 3.1 (m, 1H
CH), 4.2-4.6 (m, 1H, HR Trp), 6.8-7.6 (m, 14H, Ar), 8.1-8.4
(m, 1H, NH), 10.7 (m, 1H, NH, indoyl). ES-MS [M+H]+ 473.
Anal. (C28H28N2O3S) C, H, N.
1
(CH3CN:H2O, 40:60): Rt ) 18.9-20.3 min. H NMR (CDCl3) δ
2-2.3 (m, 5H), 2.8-3.1 (m, 5H), 3.6 (m, 1H), 7.1 (m, 4H).
The two diastereoisomers of 4i were separated using
preparative HPLC, leading to 4i1 and 4i2. HPLC (CH3CN:H2O,
50:50): Rt (4i1) 12.1 min, (4i2) 12.8 min.
(2S)-2-[2-Mer captom eth yl-3-(n aph th alen -2-yl)-bu tan oyl-
a m in o]-3-(1(H)-in d ol-3-yl)-p r op a n oic a cid (11): 0.72 g
1
(65%). HPLC (CH3CN:H2O, 65:35): Rt ) 5.4-5.6-5.8 min. H
NMR (DMSO-d6) δ 0.9-1.2 (m, 3H, CH3), 1.7 (m, 1H, SH), 2.7
(m, 3H, CH2S, CHCO), 3.1 (m, 3H, CH, CH2 Trp), 4.2-4.6 (m,
1H, HR Trp), 6.7-7.7 (m, 12H, Ar), 8.1-8.3 (m, 1H, NH), 10.7
(m, 1H, NH, indoyl). ES-MS [M+H]+ 447. Anal. (C26H26N2O3S)
C, H, N.
3-Acet ylsu lfa n yl-2-(1,2,3,4,t et r a h yd r o-n a p h t h a len -1-
yl)-p r op a n oic a cid (4j): 2.3 g (84%). HPLC (CH3CN:H2O,
1
60:40): Rt ) 6.0-6.3 min. H NMR (CDCl3) δ 1.8 (m, 4H), 2.3
(s, 3H), 2.8 (m, 2H), 3.1 (m, 4H), 7.1 (m, 4H). ES-MS [M+H]+
279
(2S)-2-[2-Mer captom eth yl-3-(n aph th alen -1-yl)-bu tan oyl-
a m in o]-3-(1(H)-in d ol-3-yl)-p r op a n oic a cid (12): 0.55 g
(50%). HPLC (CH3CN:H2O, 65:35): Rt ) 5.2-5.4-5.5-5.7 min.
1H NMR (DMSO-d6) δ 0.9-1.2 (m, 3H, CH3), 1.6-1.7 (m, 1H,
SH), 2.7-3.5 (m, 6H, CH2S, CHCO, CH, CH2 Trp), 4.2-4.5
(m, 1H, HR Trp), 6.5-7.7 (m, 12H, Ar), 8.2 (m, 1H, NH), 10.7
(m, 1H, NH, indoyl). ES-MS [M+Na]+ 447. Anal. (C26H26N2O3S)
C, H, N.
The two diastereoisomers of 4j were separated using
preparative HPLC, leading to 4j1 and 4j2. HPLC (CH3CN:H2O,
50:50): Rt (4j1) 20.2 min, (4j2) 21.1 min.
Gen er a l P r oced u r e for Syn th esis of Com p ou n d s 5-14.
Compound 4 (2.5 mmol), was dissolved in a mixture of THF
and CHCl3 (2.2 mL) containing tryptophan methyl ester (3
mmol), triethylamine (3 mmol), EDC (3 mmol), and HOBt (3
mmol). The reaction mixture was stirred for 14 h at room
temperature, then the solvents were removed in vacuo, and
the residue partitioned between EtOAc and satured NaHCO3.
The EtOAc layer was extracted two times with satured
NaHCO3, three times with 10% aqueous citric acid, and finally
with brine. The organic layer was dried with Na2SO4, the
solvent removed in vacuo, and the residue dissolved in MeOH
(5 mL) under argon with stirring. Degassed 2 M NaOH
solution (6 equiv) was added, and the mixture was stirred for
3 h. The organic solvent was evaporated in vacuo, and the
remaining aqueous solution was acidified with 1 M HCl under
stirring until pH 1. The aqueous solution was dried in vacuo,
and the residue was purified by preparative reverse-phase
HPLC on a C18 column, using TFA 0.05% in H2O and CH3CN
as mobile phases. The eluate was lyophilized to afford the
desired compound as a white solid.
(2S)-2-[2-(In d a n -1-yl)-3-m er ca p to-p r op ion yla m in o]-3-
(1(H)-in d ol-3-yl)-p r op a n oic a cid (13): 0.25 g (25%). HPLC
1
(CH3CN:H2O, 60:40): Rt ) 5.1-5.3 min. H NMR (DMSO-d6)
δ 1.5 (m, 1H, SH), 1.8-2.2 (m, 2H, CH2), 2.4-3.1 (m, 8H), 4.4-
4.6 (m, 1H, HR Trp), 6.8-7.5 (m, 9H, Ar), 8.2 (m, 1H, NH),
10.8 (m, 1H, NH, indoyl). ES-MS [M+H]+ 409. Anal. (C23H24
N2O3S) C, H, N.
-
(2S)-2-[3-Mer ca p to-2-(1,2,3,4,tetr a h yd r o-n a p h th a len -1-
yl)-p r op ion yla m in o]-3-(1(H )-in d ol-3-yl)-p r op a n oic a cid
(14): 0.73 g (69%). HPLC (CH3CN:H2O, 60:40): Rt ) 5.7-6.0-
1
6.2 min. H NMR (CDCl3) δ 1.3 (m, 1H, SH), 1.4-1.7 (m, 4H,
CH2CH2), 2.2 (m, 1H, CHCO), 2.5 (m, CH2 Trp), 2.7-3.3 (m,
5H), 4.8 (m, 1H, HR Trp), 5.8 (m, 1H, NH), 6.8-7.4 (m, 8H,
Ar), 8.1 (m, 1H, NH, indoyl). ES-MS [M+H]+ 423. Anal.
(C24H26N2O3S) C, H, N.
(2S)-[2-(4-Bip h en -4-yl)-3-m er ca p to-p r op a n oyla m in o]-
3-(1(H)-in d ol-3-yl)-p r op a n oic a cid (5): 0.88 g (80%). HPLC
(CH3CN:H2O, 65:35): Rt ) 5.9 min. 1H NMR (CDCl3) δ 1.6 (m,
1H, SH), 2.7 (m, 2H, CH2 Trp), 3.1-3.3 (m, 2H, CH2S), 3.5
(m, 1H, CHCO), 4.8 (m, 1H, HR Trp), 6.0 (m, 1H, NH), 6.7-
7.6 (m, 14H, Ar), 7.9 (NH, indoyl). ES-MS [M+H]+ 445. Anal.
(C26H24N2O3S) C, H, N.
(2S)-2-[2-(3-Biph en -4-yl)-3-m er ca p to-p r op a n oyla m in o]-
3-(1(H)-in d ol-3-yl)-p r op a n oic a cid (6): 0.49 g (44%). HPLC
(CH3CN:H2O, 65:35): Rt ) 5.7 min. 1H NMR (CDCl3) δ 1.4 (m,
1H, SH), 2.7 (m, 2H, CH2 Trp), 3.1-3.3 (m, 2H, CH2S), 3.5
(m, 1H, CHCO), 4.8 (m, 1H, HR Trp), 6.1 (m, 1H, NH), 6.7-
7.5 (m, 14H, Ar), 7.6-7.8 (NH, indoyl). ES-MS [M+H]+ 445.
Anal. (C26H24N2O3S) C, H, N.
(2S)-2-[2-(Na p h th a len -1-yl)-3-m er ca p to-p r op a n oyla m i-
n o]- 3-(1(H)-in d ol-3-yl)-p r op a n oic a cid (7): 0.32 g (31%).
HPLC (CH3CN:H2O, 50:50): Rt ) 11.2 min. 1H NMR (CDCl3-
TFA) δ 3.1 (m, 2H, CH2 Trp), 3.3-3.5 (m, 3H, CH-CH2S), 4.5-
4.9 (m, 1H, HR Trp), 6.5 (m, 1H, NH), 6.8-8 (m, 12H, Ar).
ES-MS [M+H]+ 419. Anal. (C24H22N2O3S) C, H, N.
(2S)-2-[2-Mer captom eth yl-3-diph en yl-pr opan oylam in o]-
3-(1(H)-in d ol-3-yl)-p r op a n oic a cid (8): 0.58 g (51%). HPLC
(CH3CN:H2O, 65:35): Rt ) 4.8-5.0 min. 1H NMR (CDCl3) δ
1.2 (m, 1H, SH), 2.5 (m, 2H, CH2 Trp), 2.6 (m, 1H, CHCO),
4.0 (m, 1H, PhCHPh), 4.5(m, 1H, HR Trp), 6.2 (m, 1H, NH),
7.0-7.3 (m, 15H, Ar), 8.1 (m, 1H, NH, indoyl). ES-MS [M+H]+
459. Anal. (C27H26N2O3S) C, H, N.
P r ep a r a tion of th e F ou r Ster eoisom er s of In h ibitor s
9, 10, 13, a n d 14. A coupling step between the two separate
diastereoisomers of 4e-f, 4i-j (41 and 42), and tryptophan tert-
butyl ester was performed by the procedure described for
compound 5-14 followed by a separation of the diastereoiso-
mers by preparative HPLC. After final deprotections, the
diastereoisomers were found to have a purity of g98% by
1
analytical HPLC. They were characterized by H NMR using
two-dimensional Cosy and HMQC experiments.
(2S)-2-[2-(2S)-Mer ca p tom eth yl-3-(3S)-p h en yl-bu ta n oyl-
a m in o]-3-(1H-in d ol-3-yl)-p r op a n oic Acid (9a ). Obtained
1
from 4e1: HPLC CH3CN, 50%, 10.23 min. H NMR δ (DMSO)
1.12 (d, 8.5 Hz, CH3), 1.75 (s, SH), 2.3 (t, 7H, 1H), 2.6 (m, 2H),
2.9 (m, 2H), 3.15 (m, 1H), 4.4 (q, CHRTrp), 6.8 (t, 1H), 6.9 (t,
1H), 7 (m, 6H), 7.25 (d, 1H), 7.5 (d, 1H), 8.15 (d, 7Hg, NH),
10.8 (s, 1H, NH indoyl). [R]20 (c ) 0.288, acetone) +37.4.
D
(2S)-2-[2-(2S)-Mer captom eth yl-3-(3R)-ph en yl-bu tan oyl-
a m in o]-3-(1H -in d ol-3-yl)-p r op a n oic Acid (9b ). Obtained
from 4e′1: HPLC CH3CN, 50%, 11.00 min. 1H NMR δ (DMSO)
0.9 (d, 8.5 Hz, CH3), 1.95 (t, SH), 2.4 (m, 1H), 2.65 (m, 3H),
2.9 (m, 2H), 4.3 (m, CHRTrp), 6.8 (s, 1H), 6.9 (t, 1H), 7.0 (t,
1H), 7.1 (m, 5H), 7.25 (d, 2H), 7.4 (d, 2H), 8.15 (d, NH), 10.7
(s, 1H). [R]20 (c ) 0.246, acetone) -11.8.
D
(2S)-2-[2-(2S)-Mer captom eth yl-3-(3R)-ph en yl-bu tan oyl-
a m in o]-3-(1H-in d ol-3-yl)-p r op a n oic Acid (9c). Obtained
1
from 4e2: HPLC CH3CN, 50%, 11.65 min. H NMR δ (DMSO)
1.1 (d, CH3), 1.5 (t, SH), 1.75 (m, 1H), 2.5 (m, 1H), 2.65 (m,
2H), 3.05 (m, 1H), 3.2 (m, 1H), 4.6 (m, CHRTrp), 6.9 (t, 1H),
7.0 (t, 1H), 7.1 (m, 7H), 7.5 (d, 1H), 8.4 (d, 1H), 10.8 (s, 1H).
(2S)-2-[2-Mer ca p tom eth yl-3-p h en yl-bu ta n oyla m in o]-3-
(1(H)-in d ol-3-yl)-p r op a n oic a cid (9): 0.26 g (26%). HPLC
[R]20 (c ) 0.274, acetone) +34.6.
1
(CH3CN:H2O, 70:30): Rt ) 4.1-4.3 min. H NMR (DMSO-d6)
D
δ 0.8-1.1 (m, 3H, CH3), 1.9 (m, 1H, SH), 2.6 (m, 3H, CH2S,
CHCO), 2.9 (m, 2H, CH2 Trp), 3.1 (m, 1H, CH), 4.4 (m, 1H,
HR Trp), 6.9-7.4 (m, 10H, Ar), 8.1-8.3 (m, 1H, NH), 10.8 (m,
1H, NH, indoyl). ES-MS [M+H]+ 397. Anal. (C22H24N2O3S) C,
H, N.
(2S)-2-[2-(2S)-Mer ca p tom eth yl-3-(3S)-p h en yl-bu ta n oyl-
a m in o]-3-(1H-in d ol-3-yl)-p r op a n oic Acid (9d ). Obtained
from 4e′2: HPLC CH3CN, 50%, 12.20 min. 1H NMR δ (DMSO)
0.8 (d, CH3), 1.8 (t, SH), 1.9 (m, 1H), 2.4 (m, 1H), 2.6 (m, 2H),
3.0 (s, 1H), 3.2 (s, 1H), 4.5 (m, CHRTrp, 1H), 6.9-7.0 (m, 2H),
7.1-7.3 (m, 7H), 7.5 (d, 1H), 8.4 (d, NH), 10.8 (s, NH indoyl).
(2S)-2-[2-Mer captom eth yl-3-(biph en -4-yl)-bu tan oylam i-
n o]-3-(1(H)-in d ol-3-yl)-p r op a n oic a cid (10): 0.86 g (73%).
[R]20 (c ) 0.200, acetone) -51.5.
D

J oint Recognition of ECE-1, ACE, and NEP S1′ Subsites
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 7 1485
(2S)-2-[2-(2S)-Mer ca p t om et h yl-3-(3S)-b ip h en -4-ylb u -
ta n oyla m in o]-3-(1H-in d ol-3-yl)-p r op a n oic Acid (10a ). Ob-
tained from 4f1: HPLC CH3CN, 60%, 7.63 min. 1H NMR δ
(DMSO) 1.2 (d, 8.5 Hz, CH3), 1.7 (t, 7 Hz, SH), 2.3 (m, 1H,
CH2S), 2.7 (m, 1H, CH2S), 2.8 (m, 1H, CHCl), 2.95-3.1 (m,
CH2Trp), 3.2 (m, 1H, CHCH3), 4.4 (m, 1H, CHTrp), 6.9 (m,
1H), 7.0 (m, 1H), 7.1 (m, 1H), 7.3 (m, 1H), 7.4 (m, 4H), 7.45
(m, 1H), 7.5 (m, 1H), 7.6 (m, 2H), 7.7 (m, 2H), 8.2 (d, NH),
1.9 (t, SH), 1.95 (m, 1H), 2.6 (m, 2H), 2.65 (m, 1H), 2.9 (m,
2H), 3.1 (m, 2H), 4.55 (q, CH-Trp), 6.9-7.0 (m, 6H), 7.2 (d,
2H), 7.45 (d, 1H), 8.3 (d, NH), 10.7 (s, NH indoyl). [R]20 (c )
D
0.202, acetone) -26.3.
(2S)-2-[3-Mer capto-2-(2S)-((1R),1,2,3,4-tetr ah ydr o-n aph -
th a len -1-yl)-p r op ion yl-a m in o]-3-(1H-in d ol-3-yl)-p r op a n o-
ic Acid (14c). Obtained from 4j2: HPLC CH3CN, 60%, 6.82
min. 1H NMR δ (DMSO) 1.4 (m, 1H), 1.45 (m, 1H), 1.6 (m,
SH), 1.7 (m, 1H), 2.0 (m, 1H), 2.2 (t, 1H), 2.6 (m, 3H), 2.7-
2.85 (m, 2H), 3.0 (m, 1H), 3.15 (m, 1H), 4.6 (q, 1H), 6.9 (t, 1H),
7.0 (m, 5H), 7.1 (s, 1H), 7.25 (d, 1H), 7.5 (d, 1H), 8.3 (d, NH),
10.8 (s, NH indoyl). [R]20 (c ) 0.218, acetone) +53.8.
D
(2S)-2-[2-(2S)-Mer ca p t om et h yl-3-(3R)-b ip h en -4-ylb u -
ta n oyla m in o]-3-(1H-in d ol-3-yl)-p r op a n oic Acid (10b). Ob-
tained from 4f1: HPLC CH3CN, 60%, 7.85 min. 1H NMR δ
(DMSO) 1.05 (d, 8.5 Hz, CH3), 1.9 (t, 7 Hz, SH), 2.4 (m, 1H),
2.55-2.7 (m, 2H), 2.8 (m, 2H), 3.0 (m, 1H), 4.35 (q, CH-Trp),
6.7-7.0 (m, 4H), 7.2 (m, 2H), 7.4 (m, 4H), 7.6 (s, 1H), 7.7 (m,
3H), 8.2 (d, NH), 10.65 (s, NH indoyl). [R]20D (c ) 0.2, acetone)
-23.2.
10.8 (s, NH indoyl). [R]20 (c ) 0.266, acetone) +26.5.
D
(2S)-2-[3-Mer capto-2-(2R)-((1S),1,2,3,4-tetr ah ydr o-n aph -
th a len -1-yl)-p r op ion yl-a m in o]-3-(1H-in d ol-3-yl)-p r op a n o-
ic Acid (14d ). Obtained from 4j2: HPLC CH3CN, 60%, 7.13
min. 1H NMR δ (DMSO) 1.25 (m, 2H), 1.4 (m, 1H), 1.8 (m,
1H), 2.0 (t, SH), 2.2 (t, 1H), 2.55 (m, 2H), 2.7 (m, 3H), 2.95 (m,
1H), 3.15 (m, 1H), 4.5 (m, CHRTrp), 7.0 (m, 6H), 7.1 (s, 1H),
(2S)-2-[2-(2S)-Mer ca p t om et h yl-3-(3R)-b ip h en -4-ylb u -
ta n oyla m in o]-3-(1H-in d ol-3-yl)-p r op a n oic Acid (10c). Ob-
tained from 4f2: HPLC CH3CN, 60%, 5.03 min. 1H NMR δ
(DMSO) 1.15 (d, 7 Hz, CH3), 1.5 (t, SH), 1.9 (t, 1H), 2.4 (m,
1H), 2.75 (m, 1H), 2.8 (m, 1H), 3.1 (m, 1H), 3.2 (m, 1H), 4.6
(m, 1H, CHRTrp), 6.9-7.0 (m, 2H), 7.2 (s, 1H), 7.3 (d, 1H),
7.35-7.45 (m, 4H), 7.55 (d, 1H), 7.65 (s, 1H), 7.8 (m, 4H), 8.5
7.3 (d, 1H), 7.5 (d, 1H), 8.4 (d, NH), 10.7 (s, NH indoyl). [R]20
(c ) 0.285, acetone) -52.6.
D
In Vitr o In h ibition of NEP , ACE, a n d ECE Activities.
1. En zym es. NEP was purified to homogeneity from rabbit
kidney as previously described.35 ACE was purified from rat
testis.36 Recombinant human ECE-1c (hECE-1c) was expressed
in Cos-7 cells.37
(d, NH), 10.8 (s, NH indoyl). [R]20 (c ) 0.280, acetone) +76.1.
D
2. Su bstr a tes. Inhibitory potencies were determined by
using DGPA (Dansyl-Gly-(p-NO2)Phe-â-ala)38 (Km ) 37 µM)
for NEP, N-Cbz-Phe-His-Leu (Km ) 50 µM)39 for ACE, and the
new substrate (Pya21-(p-NO2)Phe22)bigET(19-35) (Km ) 20 µM)
for ECE-1c.37
(2S)-2-[2-(2S)-Mer ca p t om et h yl-3-(3S)-b ip h en -4-ylb u -
ta n oyla m in o]-3-(1H-in d ol-3-yl)-p r op a n oic Acid (10d ). Ob-
tained from 4f2: HPLC CH3CN, 60%, 8.27 min. 1H NMR δ
(DMSO) 0.85 (d, 7 Hz, CH3), 1.85 (m, 1H), 1.9 (m, SH), 2.45
(m, 1H), 2.7 (m, 2H), 3.05 (m, 1H), 3.2 (m, 1H), 4.6 (m,
CHRTrp), 6.9-7.0 (m, 2H), 7.2 (s, 2H), 7.25 (m, 2H), 7.40 (m,
2H), 7.5 (m, 2H), 7.8 (m, 4H), 8.5 (d, NH), 10.8 (s, NH indoyl).
3. Assa y for NEP . NEP (final concentration 250 ng/mL,
specific activity on DGPA, 345 mmol/mg/min) was preincu-
bated for 10 min at 37 °C in black 96-well microplates with or
without increasing concentrations of inhibitors in a total
volume of 100 µL of 50 mM Tris-HCl buffer pH ) 7.4. DGPA
was added to a final concentration of 50 µM, and the reaction
was stopped after 1 h by adding 150 µL of DMSO. The
fluorescence was measured at λex ) 340 nm, λem ) 530 nm.
4. Assa y for ACE. ACE (final concentration 0.02 pmol/100
µL, specific activity on N-Cbz-Phe-His-Leu, 13 nmol/mg/min)
was preincubated for 10 min at 37 °C in black 96-well
microplates with or without various concentrations of inhibi-
tors in 50 mM Tris/HCl/NaCl 1% buffer pH 7.4. N-Cbz-Phe-
His-Leu was added to a final concentration of 50 µM. The
reaction was stopped after 30 min by adding 50 µL of 2 N
NaOH. After dilution with 50 µL of water, the concentration
of His-Leu was determined following the fluorometric assay
previously described.39
[R]20 (c ) 0.202, acetone) -83.8.
D
(2S)-2-[(2S)-2-(1S)-In dan -1-yl-3-m er capto-pr opion ylam i-
n o]-3-(1H-in d ol-3-yl)-p r op a n oic Acid (13a ). Obtained from
4i1: HPLC CH3CN, 60%, 6.44 min. 1H NMR δ (DMSO) 1.65 (t,
7 Hz, SH), 1.75-1.95 (m, 2H, CH2-CH2-CH), 2.15-2.65 (m,
CH2S), 2.65-2.75 (3H, m), 3.0-3.15 (m, CH2Trp), 3.45 (m, 1H),
4.6 (CH-Trp, m), 6.85-7.0 (m, 4H), 7.1-7.2 (m, 3H), 7.3 (d,
1H), 7.5 (d, 1H), 8.35 (d, 1H), 10.8 (s, 1H). [R]20 (c ) 0.235,
D
acetone) +36.3.
(2S)-2-[(2R)-2-(1R)-In d a n -1-yl-3-m er ca p t o-p r op ion yl-
a m in o]-3-(1H-in d ol-3-yl)-p r op a n oic Acid (13b). Obtained
from 4i1: HPLC CH3CN, 60%, 6.72 min. 1H NMR δ (DMSO)
1.6 (m, 2H, CH-CH2-CH2), 2.0 (t, SH), 2.15 (t, 1H), 2.5-2.65
(m, 3H), 2.75 (m, 1H), 2.9 (m, 1H), 3.15 (m, 2H), 4.6 (m, CH-
Trp), 6.7-7.1 (m, 7H), 7.3 (d, 1H), 7.5 (d, 1H), 8.3 (d, NH),
10.8 (s, NH indoyl). [R]20 (c ) 0.233, acetone) -10.3.
D
5. Assa y for ECE. ECE activity was performed in black
96-well microplates in a final volume of 100 µL with recom-
binant human ECE-1c (hECE-1c). hECE-1c at 4 µg total
protein by well was preincubated for 10 min at 37 °C in 50
mM tris-maleate pH 6-8 with or without increasing concen-
trations of inhibitors. The reaction was initiated by addition
of 20 µM of the substrate (Pya21-(p-NO2)Phe22)bigET(19-35),
and the mixture was incubated 1 h at 37 °C. The reaction was
(2S)-2-[(2S)-2-(1R)-In dan -1-yl-3-m er capto-pr opion ylam i-
n o]-3-(1H-in d ol-3-yl)-p r op a n oic Acid (13c). Obtained from
4i2: HPLC CH3CN, 60%, 6.46 min. 1H NMR δ (DMSO) 1.55
(m, SH), 1.9-2.0 (m, 2H, CH-CH2-CH2), 2.4-2.5 (m, 2H), 2.6
(m, 2H), 2.85-3.0 (m, 2H), 3.15 (m, 2H), 4.5 (m, CHRTrp), 6.9
(t, 1H), 7.0-7.3 (m, 7H), 7.5 (d, 1H), 8.4 (d, NH), 10.8 (s, NH
indoyl). [R]20 (c ) 0.264, acetone) +50.31.
D
(2S)-2-[(2R)-2-(1S)-In dan -1-yl-3-m er capto-pr opion ylam i-
n o]-3-(1H-in d ol-3-yl)-p r op a n oic Acid (13d ). Obtained from
4i2: HPLC CH3CN, 60%, 6.71 min. 1H NMR δ (DMSO) 1.5-
1.7 (m, 2H, CH-CH2-CH2), 2.0 (m, 5H), 2.45 (m, 1H), 2.55 (m,
2H), 2.7-2.9 (m, 3H), 3.05 (m, 1H), 3.1 (m, 1H), 4.5 (m,
CHRTrp), 6.8-7.1 (m, 7H), 7.2 (d, 1H), 7.7 (d, 1H), 8.4 (d, NH),
stopped by cooling, and the fluorescence was measured (λex
340 nm, λem ) 400 nm).
)
Ack n ow led gm en t. We thank N. Luciani and E.
Ruffet for their excellent technical assistance and C.
Dupuis for expert manuscript drafting. We acknowledge
C. Oefner and G. E. Dale for their generous gift of the
NEP coordinates, and A. Tomas and M. Selkti for their
help in crystallographic studies. We thank Servier
Laboratories for financial support.
10.7 (d, NH indoyl). [R]20 (c ) 0.222, acetone) -22.8.
D
(2S)-2-[3-Mer capto-2-(2S)-((1S),1,2,3,4-tetr ah ydr o-n aph -
th a len -1-yl)-p r op ion yl-a m in o]-3-(1H-in d ol-3-yl)-p r op a n o-
ic Acid (14a ). Obtained from 4j1: HPLC CH3CN, 60%, 7.15
min. 1H NMR δ (DMSO) 1.4-1.6 (m, 3H), 1.6 (m, 1H), 1.65 (t,
1H, SH), 1.9 (m, 1H), 2.5-2.7 (m, 3H), 3.0-3.2 (m, 4H), 4.55
(q, 1H, CH-Trp), 6.9-7.1 (m, 5H), 7.15 (s, 1H), 7.2 (m, 2H),
Refer en ces
7.5 (m, 1H), 8.4 (d, 1H, NH), 10.8 (s, 1H, NH indoyl). [R]20 (c
D
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) 0.254, acetone) +41.2.
(2S)-2-[3-Mer capto-2-(2R)-((1R),1,2,3,4-tetr ah ydr o-n aph -
th a len -1-yl)-p r op ion yl-a m in o]-3-(1H-in d ol-3-yl)-p r op a n o-
ic Acid (14b). Obta in ed fr om 4j1: HPLC CH3CN, 60%, 7.43
min. 1H NMR δ (DMSO) 1.1 (m, 1H), 1.3 (m, 2H), 1.6 (m, 1H),

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