Phosphinic derivatives as new dual enkephalin-degrading enzyme inhibitors: Synthesis, biological properties, and antinociceptive activities

Article abstract of DOI:10.1021/jm990483l

The development of dual inhibitors of the two zinc metallopeptidases, neprilysin (neutral endopeptidase) and aminopeptidase N involved in the inactivation of the opioid peptides, enkephalins, represents an attractive physiological approach in the search f

Full text of DOI:10.1021/jm990483l

1398
J . Med. Chem. 2000, 43, 1398-1408
P h osp h in ic Der iva tives a s New Du a l En k ep h a lin -Degr a d in g En zym e In h ibitor s:
Syn th esis, Biologica l P r op er ties, a n d An tin ocicep tive Activities
Huixiong Chen,† Florence Noble,† Aure´lie Mothe´,† Herve´ Meudal,† Pascale Coric,† Sophie Danascimento,†
Bernard P. Roques,*,† Pascal George,‡ and Marie-Claude Fournie´-Zaluski†
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, and De´partement de Recherche
Syste`me Nerveux Central, Sanofi-Synthelabo, B.P. 110, 31, avenue Paul Vaillant Couturier, 92225 Bagneux Cedex, France
Received September 21, 1999
The development of dual inhibitors of the two zinc metallopeptidases, neprilysin (neutral
endopeptidase) and aminopeptidase N involved in the inactivation of the opioid peptides,
enkephalins, represents an attractive physiological approach in the search for new analgesics
devoid of the major drawbacks of morphine. Phosphinic compounds, corresponding to the general
formula H3N+-CH(R1)-P(O)(OH)-CH2-CH(R2)-CONH-CH(R3)-COO-, able to act as transition-
state analogues and to fit the S1, S1′, and S2′ subsites of both enzymes were designed. Selection
of the R1, R2, and R3 residues for optimal recognition of these enzymes led to the first dual
competitive inhibitors with Ki values in the nanomolar range for neprilysin and aminopeptidase
N. These compounds induce potent analgesic responses after intracerebroventricular or
intravenous administrations in mice (hot plate test), and several of them were shown to be, at
least, 10 times more potent than the previously described dual inhibitors.
In tr od u ction
obtained with different classes of enzyme inhibitors,8,9
which have been shown to induce antinociceptive re-
sponses in all animal models of pain where morphine
is active and were demonstrated to be devoid of the
severe drawbacks of opioids (respiratory depression,
tolerance, reduction in intestinal transit, physical or
psychic dependence, etc.) (reviewed in ref 5). However
these compounds are poorly active after oral adminis-
tration, limiting their possible use in human therapy.
Consequently, a new strategy has been developed for
obtaining orally active dual inhibitors.
This could be achieved by taking into account the
particularities of both enzyme active sites, i.e., a car-
boxyl group facing the amino group of the anionic
subsite in APN10 and an arginine side chain interacting
with the C-terminal carboxyl group of enkephalins11
(Figure 1). The main difficulty, in the present case, was
to develop a single molecule capable to efficiently block
the active site of both an endopeptidase (NEP) and an
exopeptidase (APN) with nanomolar affinities.
We have recently synthesized R-aminophosphinic
derivatives of general formula H3N+-CH(R1)-P(O)(OH)-
CH2-CH(R2)-CONH-CH(R3)-COO-, interacting with the
S1, S1′, and S2′ subsites of the enzymes and fulfilling
the requirements for either selective recognition of
APN12 or dual inhibition of both APN and NEP.13 In
these inhibitors, the phosphinic moiety binds the zinc
atom in the catalytic site and mimics the transition-
state intermediate formed during the cleavage of the
substrate amide bond.14
It has been previously shown that a biphenyl group
in the P1′ position increases significantly NEP inhibi-
tion15 and that the presence of several aromatic or
aliphatic bulky side chains in a pseudo-tripeptide
impairs NEP recognition.16 Therefore, a biphenyl group
was introduced in the P1′ position and a methyl group
as the P2′ component in the phosphinic inhibitors. These
The wide distribution of the opioid peptides enkepha-
lins and dynorphin, and of their receptors (µ, δ, or κ),
has suggested their involvement in a variety of physi-
ological functions. Evidence for their implication in the
control of respiratory, cardiovascular, gastrointestinal,
or renal functions as well as in pituitary hormonal
secretion or in immunological responses has been
reported (reviewed in ref 1). However the most studied
function of the opioid peptides Met- and Leu-enkepha-
lins, in the central nervous system, concerns the control
of pain, which is mainly associated with the activation
of the µ opioid receptor.2 This has been recently con-
firmed by disruption of the µ receptor gene leading to
mice insensitive to morphine treatment.3 Moreover, a
clear demonstration of the antinociceptive properties of
the endogenous enkephalins has been addressed by
invalidation of the preproenkephalin gene giving mice
with enhanced sensitivity to painful stimuli.4
These recent results reinforce our early physiological
approach aimed at developing new analgesics by poten-
tiating the antinociceptive properties of the endogenous
enkephalins. This was achieved by inhibition of their
two catabolizing enzymes, i.e., neutral endopeptidase
(neprilysin, NEP, EC 3.4.24.11) and aminopeptidase N
(APN, EC 3.4.11.2), both enzymes belonging to the
family of zinc metallopeptidases (reviewed in ref 5).
Selective inhibition of only one of these peptidases gives
weak antinociceptive effects6 due to the complementary
role of NEP and APN in enkephalin inactivation, and
strong analgesic responses could be obtained only when
the two enzymes were jointly blocked.7 This has been
* To whom correspondence should be addressed. Tel:
(33)-1-43.25.50.45. Fax: (33)-1-43.26.69.18. E-mail: Roques@
pharmacie.univ-paris5.fr.
† INSERM U266 - CNRS UMR 8600.
‡ Sanofi-Synthelabo.
10.1021/jm990483l CCC: $19.00 © 2000 American Chemical Society
Published on Web 03/15/2000

New Dual Enkephalin-Degrading Enzyme Inhibitors
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7 1399
F igu r e 2. Synthetic pathway used to prepare the phosphinic
inhibitors.
four stereoisomers, due to the presence of two unre-
solved asymmetric carbons. With the exception of
compound 23, it was not possible to completely separate
by HPLC, the four diastereoisomers due to the closeness
of their retention times. However, in all cases, two
fractions were obtained from HPLC under semiprepara-
tive conditions. The first eluted fraction contained a
mixture of two isomers (A+B) and the second fraction,
the two other stereoisomers (C+D).
F igu r e 1. Schematic representation of Leu (or Met) enkepha-
lin and phosphinic inhibitors within the active site of APN
(above) and NEP (below).
compounds were the first reported dual competitive
inhibitors with nanomolar affinities for both enzymes.13
The optically pure diastereoisomers needed both for
the determination of their absolute configurations and
for studying the relationships between stereochemistry
and inhibitory potencies were obtained by a slight
modification of the synthetic pathway. First, the 1-(N-
benzyloxycarbonyl)aminoalkylphosphinic acids 1a ,e,h
were resolved by crystallization of their salts obtained
by using the (+)- and (-)-R-methylbenzylamine as
described.17 The optical purity was checked by HPLC
using a chiral column and found to be >99%. The R
isomers, obtained from (+)-R-methylbenzylamine, and
the S isomers, from (-)-R-methylbenzylamine, were
then submitted to the Michael condensation. The new
asymmetric carbon formed, during this step, led to
mixtures of two diastereoisomers in different proportion
(∼75/25). The (R)-R-aminophosphinic acids led to the
isomers B (75%) and D (25%), whereas the (S)-R-
aminophosphinic acids afforded the isomers A (25%) and
C (75%), which were separated either by semiprepara-
tive HPLC or by chromatography on silica gel. Com-
pounds 10, 13, 22, 23, 25, and 28 were obtained by these
procedures and correspond to pure B isomers. Com-
pounds 30-33 were synthesized directly from the
resolved aminophosphinic acids.
On the basis of these preliminary results, structure-
activity studies have been done in this study by using
a large series of dual phosphinic inhibitors. Several of
these compounds exhibit nanomolar affinities for APN
and NEP. Among these compounds, those showing a
poor recognition of related zinc metallopeptidases such
as angiotensin-converting enzyme (ACE) and endothe-
lin-converting enzyme (ECE) were selected for pharma-
cological studies. The synthesis, inhibitory potencies,
selectivities, and antinociceptive properties, after icv or
iv administration of these compounds, are reported in
this paper.
Resu lts
Syn th esis. The synthetic pathway used for obtaining
the various phosphinic inhibitors is depicted in Figure
2. The benzyloxycarbonyl-protected 1-aminoalkylphos-
phinic acids 1a -i were prepared under racemic forms
as previously described.17 The Michael condensation of
these phosphinic acids with various ethyl 2-substituted
acrylates 2a -e in the presence of N,O-bistrimethylsi-
lylacetamide (BSA) led to the key intermediates 3a -o.
In the case of biphenyl-containing derivatives 3p -r , a
two-step reaction consisting in Michael condensation
with the ethyl p-bromobenzylacrylate followed by Su-
zuki arylation using phenylboronic acid was carried out.
After alkaline hydrolysis of esters, a coupling step with
various amino acids was done followed by sequential
cleavage of the protective groups yielding compounds
6-29. These molecules were obtained as mixtures of
Absolu te Con figu r a tion of th e Asym m etr ic Ca r -
1
bon s in th e Design ed In h ibitor s Deter m in ed by H
NMR Sp ectr oscop y. This study has been first per-
formed on compound 23, for which the four stereoiso-
mers have been separated by semipreparative HPLC.
The stereochemistry of the carbon belonging to the
aminophosphinic moiety was deduced from the synthetic
route (chiral amine separation) as reported above.

1400 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7
Chen et al.
Ta ble 1. Correlation between Chemical Shifts of the Leucyl Side Chain Protons and Absolute Configuration of This Residue in the
Four Stereoisomers of the Phosphinic Inhibitor 23 and Stereochemical Dependence of the Inhibitory Protencies toward APN and NEP
δ (ppm)
Ki (nM)a
compd
H(c)
H(d)
H(e)
APNb
NEPc
23A
23B
23C
23D
(S,S,S)
(R,S,S)
(S,R,S)
(R,R,S)
1.36/1.25
1.36/1.18
1.12/1.04
1.17/1.1
1.36
1.37
0.90
0.88
0.78/0.72
0.79/0.72
0.64/0.58
0.64/0.58
32 ( 6
140 ( 10
43 ( 4
4000 ( 200
10000 ( 500
2.3 ( 0.3
1700 ( 300
1200 ( 100
a
b
Values are the mean ( SEM from three independent experiments performed in triplicate. APN activity was measured using Ala-
pNA as substrate. c NEP activity was measured using DGNPA as substrate.
Ta ble 2. Influence of R1 Substituents of Various Phosphinic
Compounds on Their Inhibitory Potencies toward APN and
NEP
The absolute configuration of the second asymmetric
carbon was determined by using a previously described
1H NMR method.18 Thus, it has been demonstrated that
in a dipeptide unit containing one aromatic moiety, the
relative absolute configuration of each amino acid could
be deduced from the chemical shifts of the nonaromatic
side chain protons: a shielding of these protons was
observed when the two amino acids have opposite
absolute configuration (R,S or S,R) as compared to those
having identical configuration (R,R or S,S). Thus, as
shown in Table 1, the isobutyl protons of 23 are more
deshielded in fractions A and B than in fractions C and
D. Accounting for the S configuration of the C-terminal
phenylalanine, the isobutyl side chain has the R con-
figuration in C and D and the S configuration in A and
B. For compounds, such as 6, which contain two benzyl
moieties in positions R1 and R2, the same rule was
shown to be valid and the chemical shift variations
associated with changes in the absolute configuration
of these groups were easily observed on the CH2â
protons of both residues: a shielding of about 0.2 ppm
was observed in the RS isomer as compared to the SS
isomer (data not shown). Moreover, it is interesting to
note that the absolute configuration of the R-amino-
phosphinic moiety has no significant influence on the
proton chemical shifts of the other part of the molecule
(data not shown). This is very likely related to the
flexibility of the R-aminophosphinic moiety, which is
linked to a more rigid dipeptide unit by a methylene
group (Figure 1).
Ki (nM)a
compd
R1
APNb
NEPc
6
7
8
(A+B)
(A+B)
(A+B)
(A+B)
(A+B)
(A+B)
(A+B)
(A+B)
(A+B)
CH3
CH2CH3
2.2 ( 0.2
2.3 ( 0.1
3.2 ( 0.5
6.3 ( 0.3
4.2 ( 0.5
3.1 ( 0.3
130 ( 7
146 ( 2
190 ( 10
790 ( 60
180 ( 10
104 ( 7
CH2CH(CH3)2
CH2CH2SCH3
Ph
Ph(p-Me)
Ph(o,p-diMe)
CH2Ph
9
10
11
12
13
14
160 ( 10
230 ( 8
190 ( 50
540 ( 90
2.9 ( 0.8
2.3 ( 0.4
CH2CH2Ph
a
Values are the mean ( SEM from three independent experi-
b
ments performed in triplicate. APN activity was measured using
Ala-pNA as substrate. c NEP activity was measured using DGNPA
as substrate.
C+D). As illustrated for compound 23 (Table 1), the
fraction A+B is more potent than the fraction C+D by
at least 2 orders of magnitude on both enzymes, a result
which was verified for all compounds (data not shown).
Therefore, only the results corresponding to the A+B
mixture are reported and discussed. Compounds re-
ported in Table 2 contain benzyl moieties in the R2 and
R3 positions for interaction with S1′ and S2′ subsites,
respectively, and differ only by R1 allowing the prefer-
ence of the S1 subsite of each enzyme to be investigated.
All the inhibitors, except 12, are very efficient against
APN with inhibitory potencies in the nanomolar range,
and four compounds, 6, 7, 13, and 14, which have small
linear aliphatic, benzyl, or phenylethyl side chains in
the P1 position, respectively, exhibit Ki values lower
than 3 nM. In contrast, the inhibitory potency of these
compounds on NEP is only in the 10-7 M range (Table
Similar chemical shift scattering was observed for all
described inhibitors leading to the same relationship
between the stereochemistry of each isomer and their
HPLC elution order.
In Vitr o In h ibitor y P oten cy of P h osp h in ic Com -
p ou n d s on NEP , AP N, a n d ACE. The inhibitory
potencies of the four isomers of compound 23 were
determined on both NEP and APN (Table 1) showing
that the isomers A (SSS) and B (RSS) in which the P1′
residues have the S configuration, found in natural
amino acids, are significantly more active than the C
(SRS) and D (RRS) isomers on both enzymes.
In a preliminary screening, the inhibitory potencies
of the phosphinic inhibitors were measured on NEP and
APN, using the mixtures of stereoisomers (A+B and

New Dual Enkephalin-Degrading Enzyme Inhibitors
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7 1401
Ta ble 3. Inhibitory Potencies of Various Phosphinic Compounds on APN and NEP Activities
Ki (nM)a
compd
R1
R2
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph(p-Ph)
CH2Ph(p-Ph)
CH2Ph(p-Ph)
CH2Ph(p-Ph)
CH2CH(CH3)2
CH2Ph
R3
CH2Ph
APNb
NEPc
13
15
16
17
18
19
20
21
22
23
10
24
25
26
6
(A+B)
(A+B)
(A+B)
(A+B)
(A+B)
(A+B)
(A+B)
(A+B)
(A+B)
(A+B)
(A+B)
(A+B)
(A+B)
(A+B)
(A+B)
(A+B)
(A+B)
(A+B)
CH2Ph
2.9 ( 0.8
17.3 ( 0.3
6.4 ( 0.6
12.7 ( 1.3
11.7 ( 1.4
15 ( 2
189 ( 70
185 ( 40
2200 ( 500
560 ( 30
158 ( 10
2.3 ( 0.1
3.3 ( 0.2
290 ( 40
150 ( 30
80 ( 4
CH3
CH2CH(CH3)2
CH2Ph(p-OH)
CH2Ph(p-Ph)
CH3
CH2CH3
CH2CH(CH3)2
CH2Ph
CH2Ph
CH2Ph
Ph
CH3
33 ( 1
6.3 ( 0.7
17 ( 0.3
4.6 ( 0.5
3.8 ( 0.5
10 ( 1
Ph
104 ( 7
CH2Ph
1100 ( 200
2.9 ( 0.1
110 ( 5
CH2Ph(p-Ph)
CH2CH(CH3)2
CH2Ph
CH2Ph(p-Ph)
CH2Ph(p-Ph)
CH2Ph(p-iPr)
14 ( 1
CH2Ph
CH2Ph
CH2Ph
CH3
7.6 ( 0.2
2.2 ( 0.2
3.7 ( 0.3
5.2 ( 0.4
11.4 ( 0.4
CH3
146 ( 2
51 ( 8
1.4 ( 0.2
15.5 ( 1
27
28
29
CH3
a
b
Values are the mean ( SEM from three independent experiments performed in triplicate. APN activity was measured using Ala-
pNA as substrate. c NEP activity was measured using DGNPA as substrate.
2), compounds 6 (R1 ) CH3) and 10 (R1 ) Ph) showing
the best affinities for this peptidase.
B form (RSS isomer) (Table 4) resulting as expected in
an increased affinity by a factor of around 2.
Accounting for these results, three R1 side chains were
selected (methyl, phenyl, and benzyl) for designing new
inhibitors containing various R2 and R3 residues (Table
3). As compared to 13, which contains three benzyl side
chains in the R1, R2, and R3 positions, all the modifica-
tions proposed for R3 (methyl in 15, isobutyl in 16,
p-hydroxybenzyl in 17, and biphenylmethyl in 18) led
to less active compounds on APN with Ki values from 6
to 17 nM, without improvement in NEP recognition. The
same remark could be made when 10 and 24 are
compared. However, a significant increase in NEP
inhibition was obtained by introducing both a biphenyl
methyl moiety in the R2 position and a small residue in
the R3 position (compounds 19, 20, 25, and 28) leading
to Ki values in the nanomolar range. In contrast the
association of the biphenylmethyl moiety and bulky
residues in R3 (compounds 21, 22, and 27) reduced NEP
inhibition.
Regarding APN recognition, the introduction of a
biphenyl moiety in the P1′ position resulted in a
decreased inhibition except for compounds 21, 27, and
28 which have inhibitory potencies lower than 10 nM.
Furthermore, introduction in R2 of an isobutyl side
chain (compounds 23 and 26) or a p-isopropylbenzyl
group (29) was detrimental for both enzymes. From
these data, the best arrangement for dual inhibition of
both NEP and APN in the phosphinic series was to
associate a biphenyl group in R2 and a methyl group in
R3, with a methyl, phenyl, or benzyl group as the R1
moiety (compounds 19, 25, and 28). Finally, the most
active inhibitors, which have been studied as a mixture
of isomers (A+B), were tested under the optically pure
Some additional modifications in the structure of
these very active compounds were investigated: com-
pound 30B, which contains a p-bromobenzyl moiety in
the R2 position, remains very efficient on both enzymes
(4.9 and 1.9 nM on NEP and APN, respectively). Three
others compounds 31B-33B containing more hydro-
philic side chains in the R3 position were also tested
(Table 4). The serine- and threonine-containing inhibi-
tors 31B and 32B are less active on NEP with Ki values
of 11 and 32 nM, respectively, while the glycine in 33B
improves the recognition of this enzyme (Ki ) 0.9 nM).
Regarding APN, the serine (31B) is well-accepted in the
C-terminal position (Ki ) 4.9 nM), but introduction of a
threonine in 32B (Ki ) 10.2 nM) or a glycine in 33B (Ki
) 32.8 nM) reduces the inhibitory potencies. These
compounds were also tested on ACE (Table 4). Com-
pounds 10B, 25B, 31B, and 32B, which possess a
phenyl group in position R1, are very poor ACE inhibi-
tors. Those containing a CH3 in the R1 position are
slightly better with Ki values in the 10-6-10-7 M range
(compounds 28B, 30B, and 33B). In contrast inhibitors
bearing a benzyl moiety in R1 are only 10 times less
active on ACE than NEP and therefore poorly selective.
Finally, compound 28B was also tested on ECE inhibi-
tor, and a Ki value of 3.5 × 10-4 M was measured (data
not shown).
An a lgesic Activity of th e Du a l In h ibitor s. The
antinociceptive properties of the most efficient inhibitors
were investigated under their optically pure RSS iso-
mers, on the mouse hot plate test, following icv admin-
istration 15 min before testing.

1402 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7
Chen et al.
Ta ble 4. Inhibitory Potencies of the Most Efficient Stereoisomers of R-Aminophosphinic Inhibitors on NEP and APN Activities
Ki (nM)a
compd
R1
R2
CH2Ph
CH2Ph(p-Ph)
CH2CH(CH3)2
CH2Ph
CH2Ph(p-Ph)
CH2Ph(p-Ph)
CH2Ph(p-Br)
CH2Ph(p-Ph)
CH2Ph(p-Ph)
CH2Ph(p-Ph)
R3
CH2Ph
APNb
NEPc
ACEd
13B
22B
23B
10B
25B
28B
30B
31B
32B
33B
CH2Ph
CH2Ph
CH2Ph
Ph
1.5 ( 0.05
5.3 ( 0.7
2.3 ( 0.3
4.2 ( 0.1
4.8 ( 0.7
2.9 ( 0.3
1.9 ( 0.1
4.9 ( 0.3
10.2 ( 1.4
32.8 ( 2.0
190 ( 10
2.2 ( 0.3
43 ( 4
660 ( 70
39 ( 2
440 ( 20
13000 ( 1000
2500 ( 500
120 ( 8
CH3
CH2Ph
CH2Ph
CH3
CH3
CH3
CH2OH
CH(CH3)OH
H
70 ( 8
Ph
2.0 ( 0.5
1.2 ( 0.2
4.9 ( 0.4
11.8 ( 1.2
32.5 ( 3.6
0.94 ( 0.3
CH3
CH3
Ph
Ph
Ph
1400 ( 100
>5000
>20000
7800 ( 600
a
b
Values are the mean ( SEM from three independent experiments performed in triplicate. NEP activity was measured using DGNPA
as substrate. c APN activity was measured using Ala-pNA as substrate. ACE activity was measured using Cbz-Phe-His-Leu as substrate.
d
F igu r e 3. Antinociceptive responses induced by icv admin-
istration of various phosphinic inhibitors in the hot plate test
F igu r e 4. Time course of the antinociceptive responses
induced by iv administration of 100 mg/kg 28B (white col-
umns) and its benzyl ester (black columns) in the hot plate
test in mice (jump response).
in mice (jump response). ED50 values: 9 (28B) 6 µg; 0 (25B)
10.5 µg; b (22B) 11 µg; O (13B) 16 µg; 2 (23B) 13.5 µg; [ (10B)
35 µg (see tables for formulas and inhibitory potencies of the
various compounds).
Discu ssion
The aim of this work was to develop a new series of
As observed in Figure 3, these compounds gave highly
efficient antinociceptive responses on the jump latency
time. The statistical analysis of the curves showed that
they can be considered as parallel, allowing the relative
efficiencies of the studied inhibitors to be compared
using their ED50 values. The order of increasing ef-
ficiency was 10B (35 µg) < 13B (16 µg) < 23B (13.5 µg)
< 22B (11 µg) < 25B (10.5 µg) < 28B (6 µg). The more
efficient inhibitor compound 28B gave 100% analgesia
at 20 µg.
potent dual inhibitors of NEP and APN able to induce
in vivo antinociceptive responses by fully protecting the
endogenous enkephalins from their degrading enzymes.
These two peptidases belong to the thermolysin family
of Zn2+ metallopeptidases (gluzincins) and have similar
mechanisms of action19 indicating that they can be
inhibited by compounds containing a zinc ligand and
residues selected for optimal recognition of their main
binding subsites. In NEP, there is only two important
binding sites, the hydrophobic S1′ and S2′ pockets, and
inhibitors blocking these two domains have Ki values
in the nanomolar range.20 For APN, its exopeptidase
nature requires essentially the occupancy of both the
S1 and the anionic subsites responsible for its N-
terminus specificity,21 but additional interactions with
the S1′ and S2′ subsites have been shown to enhance
the affinity of the inhibitors for this enzyme.12 These
requirements were fulfilled with R-aminophosphinic
compounds giving highly potent APN inhibitors. Struc-
Compound 28B was then studied on the same test
after iv administration. At high doses, 100 mg/kg, 28B
induced significant antinociceptive responses (26% ( 3)
in mice (Figure 4). With the same experimental condi-
tions, the benzyl ester of 28B was iv administered at
the dose of 100 mg/kg in mice (Figure 4), and a delayed
response and a longer duration of action were observed,
with an analgesic peak at 45 min.

New Dual Enkephalin-Degrading Enzyme Inhibitors
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7 1403
Ta ble 5. Physical Data for 2-Alkyl-3-[hydroxy[1′-(N-benzyloxycarbonylamino)-2′-alkyl]phosphinyl]propanoate Derivatives 3 and 4
HPLC tR,
min (B%)
HPLC tR,
min (B%)
MS (ESI)
compd
R1
R2
CH2Ph
CH2Ph(p-Br)
CH2Ph(p-iPr)
CH2Ph
yield (%) mp (°C)
compd yield (%) mp (°C)
(M+ + 1)
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
CH3
CH3
CH3
68.9
90
77
154-155 9.1 (45)
4a
4b
4c
4d
4e
4f
4g
4h
4i
90.2
98
96
175-178 4.2 (65)
406.1
484.1, 486.1
448.2
120-122 6.5 (60)
176-177 4.2 (60)
110
104
9.4, 9.7 (60)
7.1 (50)
158
122
5.4 (60)
5.3 (50)
CH2CH3
CH2CH(CH3)2 CH2Ph
CH2CH2SCH3 CH2Ph
64
97
420.0
74.2
52
55
150-152 9.1, 9.4 (55)
97
65
159-162 6.1 (55)
448.2
466.1
468.0
120-122 8.0, 8.1 (50)
134
164
5.8, 5.9 (48)
Ph
Ph
Ph
CH2Ph
152
6.0 (60)
77
4.5 (60)
CH2Ph(p-Br)
CH2CH(CH3)2
CH2Ph
88
51
191-193 7.5 (60)
147-149 5.8, 7.0 (60)
176-178 22.2 (45)
170-172 7.7 (60)
NPa
96
160
7.2 (45)
434.1
482.0
496.1
482.0
Ph(p-Me)
Ph(o,p-diMe) CH2Ph
51.6
51.2
67
84.1
81.3
85.9
85
4j
4k
90
73
182-184 11.7 (45)
186-188 5.3 (60)
178-179 12.6 (45)
CH2Ph
CH2Ph
CH2Ph
CH2CH2Ph
CH3
CH2Ph
161-163 23.2, 24.2 (45) 4l
100
NPa
99.8
99.7
88.2
85
CH2Ph(p-Br)
CH2CH(CH3)2
CH2Ph
CH2Ph(p-Ph)
CH2Ph(p-Ph)
CH2Ph(p-Ph)
124-125 10.2, 10.7 (60) 4m
143-144 7.7, 8.0 (60)
163-166 35.6 (45)
4n
40
4p
4q
170-172 4.7 (60)
179-180 176, 18.1 (45) 496.1
216-218 4.2 (66)
184 7.1 (60)
180-182 7.1, 8.0 (60)
448.21
198
8.2 (60)
482.0
544.0
558.2
Ph
82
80
163-164 15.2 (60)
164-165 13.9, 14.6 (60) 4r
CH2Ph
97.4
a
NP ) not prepared.
Ta ble 6. Physical Data for 2-Alkyl-3-[hydroxy[1′-(N-benzyloxycarbonylamino)-2′-alkyl]phosphinyl]propanoyl Amino Acids 5
compd
R1
R2
CH2Ph
CH2Ph(p-Br)
CH2Ph(p-Ph)
CH2Ph(p-Ph)
CH2Ph(p-iPr)
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
R3
CH2Ph
CH3
CH2Ph
CH3
yield (%)
mp (°C)
HPLC tR, min (B%)
5a
5b
5c
5d
5e
5f
5g
5h
5i
CH3
CH3
CH3
CH3
CH3
81.3
74
56
58.5
43
92
69.9
47.5
31
175-177
178-180
186-188
162-164
230
200
192-193
152
10.0, 14.3 (45)
4.3, 4.5 (60)
6.1, 6.9 (60)
4.4, 5.1 (60)
7.2, 8.7 (50)
6.8, 8.4 (50)
6.2, 7.5 (60)
5.5, 6.9 (55)
4.6, 6.1 (60)
CH3
CH2CH3
CH2CH(CH3)2
CH2CH2SCH3
Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
Ph
152
220
5j
Ph
81
7.5, 9.2 (60)
5k
5l
Ph
Ph
CH2Ph(p-Ph)
CH2CH(CH3)2
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph(p-Ph)
CH2Ph(p-Ph)
CH2Ph(p-Ph)
CH2Ph(p-Ph)
CH2CH(CH3)2
CH2Ph
CH3
80
79
60
61.5
93
77.6
91
68
56
97.4
97
156
9.8, 11.7 (50)
5.6, 7.1 (60)
7.7, 9.6 (55)
7.0, 8.6 (60)
6.3, 7.4 (50)
CH2Ph
CH2Ph
CH2Ph
CH3
CH2CH(CH3)2
CH2Ph
CH2Ph(p-OH)
CH2Ph(p-Ph)
CH3
CH2CH3
CH2CH(CH3)2
CH2Ph
CH2Ph
CH2Ph
147-149
166-168
184-185
192-195
196-199
203-205
207-209
210-212
180-182
181-182
185-187
196-198
171-173
181-182
5m
5n
5o
5p
5q
5r
Ph(p-Me)
Ph(o,p-diMe)
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2CH2Ph
5.4, 6.9 (60)
8.6, 9.6 (60)
13.9, 14.8, 16.1 (42)
10.5, 13.0, 13.6 (60)
6.2, 7.2, 7.5 (60)
6.6, 8.3, 9.1 (60)
9.5, 11.9, 12.8 (60)
10.0, 12.0, 12.7 (60)
6.0, 7.4, 8.0 (60)
7.1, 8.7 (60)
5s
5t
5u
5V
5w
5x
5y
69
75
58.4
78.9
ture-activity relationships have shown that the pres-
ence of small hydrophobic residues in the P1 position
and hydrophobic aromatic or aliphatic side chains in the
P1′ and P2′ positions are preferred by this peptidase.12
However, accounting for the specificity of NEP active
site discussed above, it is somewhat surprising that
R-aminophosphinic compounds such as those reported
in Table 2 have modest inhibitory potencies on this
enzyme with Ki values in the 10-7 M range. Three
hypotheses could be proposed to explain these apparent
discrepancies: (i) the presence of a free amino group
would be detrimental for NEP inhibition; this argument
can be discarded since we have shown that removing
this function in 6 decreased its Ki value for NEP only
by a factor of 2 (13); (ii) the phosphinic group, in these
molecules, would not be well-oriented for an optimal
interaction with the zinc ion; or (iii) the benzyl moiety
may not fit perfectly the S1′ subsite. The two latter
explanations are probably associated to account for the
present results and for those previously reported in

1404 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7
Chen et al.
other series of phosphorus-containing inhibitors. Indeed
phosphonamidates such as phosphoramidon22 and the
shorter derivative phosphoryl-Leu-Phe23 exhibit nano-
molar affinities for NEP.
However, it is obvious that the development of new
drugs for possible use in human therapy requires orally
active compounds. It was interesting to observe that
compound 28B, administered iv at 100 mg/kg (Figure
4), induces 25% analgesia on the hot plate test. This
indicates that aminophosphinic derivatives could be
active by systemic route even under nonprotected forms.
A significant increase in the bioavailability of these
charged inhibitors is illustrated with the benzyl ester
of 28B. Its iv administration in mice (Figure 4) shows:
(i) an increased antinociceptive response as compared
to the free inhibitor and (ii) a delayed and prolonged
effect reflecting an improvement in the pharmacokinetic
properties. This relatively long duration of action is very
different from that observed with RB 101, which is
formed by the association through a disulfide bond, an
inhibitor of NEP and a blocker of APN both exhibiting
nanomolar affinity for their proper enzyme. Thus, the
preliminary results obtained with the benzyl ester of
28B appear very favorable since a more constant
concentration-time profile as compared to RB 101 or
analogues is observed. This interesting pharmacological
result obtained with protection of only one ionizable
group could be improved, for instance, by using protect-
ing groups employed in ACE inhibitors such as fosino-
pril.33 Indeed the ionization state of the inhibitor
phosphinic group (pKa ∼ 1.8-2.0) restricts the bioavail-
ability of this kind of compound.
In contrast, other phosphonamidate peptide ana-
logues24 are relatively poor NEP inhibitors (IC50 g 10-7
M). On the other hand, some N-phosphonomethyl
dipeptides characterized by a free phosphonate group
(-CH2-PO3H2) exhibit nanomolar IC50 values,25 but it can
be noticed that in this series the best compounds contain
a biphenylmethyl moiety in the P1′ position. It has been
previously demonstrated that this large residue is well-
recognized by NEP S1′ subsite.15,26 Only one series of
phosphinic acids ressembling the inhibitors reported in
this study, but without a free amino group, have been
tested on NEP.27,28 None of the compounds contained a
biphenylmethyl group, and the IC50 of the best inhibitor
on NEP is 55 nM.28
In our series, the introduction of the biphenylmethyl
group in the P1′ position, in association with a methyl
side chain in the P2′ position, afforded inhibitors with
nanomolar potencies on NEP (Table 3). Interestingly
these two modifications did not perturb significantly
APN recognition, and as shown in Table 4, three
compounds which differ only by the nature of R1 have
nanomolar Ki values on both enzymes. A decrease in
the size of the R3 residue facilitates NEP recognition
(compound 32B), whereas an increase of R3 enhances
APN inhibition (compound 13B). Another intriguing
result is the fact that these molecules, which could be
assumed to block the S1, S1′, and S2′ subsites of NEP
and act as transition-state analogues, are not better
inhibitors than thiorphan and analogues. This suggests,
as previously discussed,16 that the occupancy of the
assumed S1 subsite of NEP is unable to significantly
increase the affinity for this enzyme.
Exp er im en ta l Section
Ch em istr y. The natural amino acid derivatives were
purchased from Bachem (Bubbendorf, Switzerland). Reagents
were from Aldrich Chimie (Strasbourg, France). The solvents
were from SDS (Peypin, France). TLC experiments were
revealed with UV, iodine vapor, or ninhydrin. The purity of
the compounds was checked by HPLC on a reverse-phase
kromasil C8 (5 µm, 100 Å) column (4.6 × 250 mm) with 0.05%
TFA in H2O (solvent A)/CH3CN (solvent B), as mobile phase,
using isocratic conditions at a flow rate of 1 mL/min on a
Shimadzu apparatus (detector SPD-6AV, pumps LC-9A, re-
corder CR-6A). The eluted peaks were monitored at 210 nm.
The four stereoisomers of the final products were separated
into two parts by semipreparative HPLC using a reverse-phase
kromasil C8 (10 µm, 100 Å) column (20 × 250 mm) with 0.05%
TFA in H2O (solvent A)/CH3CN (solvent B), as mobile phase,
in isocratic conditions at a flow rate of 15 mL/min on a Waters
apparatus (detector Waters 486, pumps Waters 600, recorder
Servogor 120). The eluted peaks were monitored at 210 nm.
Since various phosphinic compounds, such as fosino-
pril, have been described as potent ACE inhibitors,29 it
was important to verify the selectivity of the compounds
reported here. None of them behave as very efficient
ACE inhibitors. However accounting for the specificity
of the S1 subsite of ACE,30 compounds 18-21 which
have a benzyl group in the R1 position and hydrophobic
residues in R2 and R3 have affinities for ACE around
10-8 M. Interestingly, the presence of a phenyl residue
in R1 precludes the recognition of ACE active site
(compounds 10 and 24-26) leading to the most selective
NEP/APN inhibitors of the series.
1
The structure of all compounds was confirmed by H NMR
spectroscopy (Bru¨ker AC 270 MHz) in DMSO-d6 using HMDS
as internal reference (values in δ, ppm). The signals are
described as s (singlet), d (doublet), t (triplet), q (quartet), m
(multiplet), and br (broad). Mass spectral analyses were
performed by Quad Service (Poissy, France), using the elec-
trospray ionization (ESI) technique. Melting points of the
compounds were determined on an Electrothermal apparatus
(Buˆchi Melting Point B-450) and are reported uncorrected.
Satisfactory analyses were obtained (C, H, N) for all final
compounds.
pKa determination of compound 28B was obtained by
classical titration methods (pH meter, Tacussel, Lyon, France).
Abbr evia tion s: AcOH, acetic acid; BOP, 1H-benzotriazol-
1-yloxytris(dimethylamino)phosphonium hexafluorophosphate;
BSA, N,O-bistrimethylsilylacetamide; Chex, cyclohexane; DMF,
dimethylformamide; DMSO, dimethyl sulfoxide; Et2O, ethyl
ether; EtOH, ethanol; EtOAc, ethyl acetate; HMSD, hexa-
methyldisiloxane; Hex, hexane; Hep, heptane; MeOH, metha-
nol; TFA, trifluoroacetic acid; Et3N, triethylamine.
Owing to their high efficiency on both NEP and APN
in vitro, it was important to study the ability of these
new inhibitors to protect the endogenous enkephalins
by in vivo experiments. As expected (Figure 3) these
molecules induced antinociceptive responses at low
doses on the hot plate test in mice after icv administra-
tion. This test represents one of the most severe animal
models of pain used for the selection of new analgesics.31
The percentage of analgesia obtained at doses as low
as 10 or 20 µg/kg reflects the high efficiency of these
molecules and the validity of the concept of a true “dual
inhibitor” for analgesia. It is worth noting that inhibi-
tion of only one enzyme, NEP or APN, was shown to
produce slight antinociceptive responses as compared
to dual inhibitors32 even following icv administration.7
Gen er a l P r oced u r e for th e Syn th esis of 1-(Ben zyloxy-
ca r bon yl)a m in oa lk ylp h osp h on ou s Acid s 1. They were

New Dual Enkephalin-Degrading Enzyme Inhibitors
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7 1405
prepared, under racemic forms, in three steps as described by
Baylis.17 Compounds 1a ,e,h have been resolved by chiral
amine as described.17
alkyl]phosphinyl] propanoate 3a -r was dissolved in ethanol,
and 1 M NaOH (5 equiv) was added. The mixture was stirred
for 6-8 h at room temperature. After acidification with 2 M
HCl, ethanol was evaporated, diluted in water, and extracted
with EtOAc. The organic layer was washed with water and
brine, dried over Na2SO4 and evaporated in vacuo.
Gen er a l P r oced u r e for th e Syn th esis of 2-Alk yl-3-
[h yd r oxy[1′-(N-ben zyloxyca r bon yla m in o)-2′-a lk yl]p h os-
p h in yl]p r op a n oyl Am in o Acid s 5a -y (Ta ble 6). To a
solution of 1 equiv of a 2-alkyl-3-[hydroxy[1′-(N-benzyloxycar-
bonylamino)-2′-alkyl]phosphinyl]propanoic acid 4a -r in DMF
were added the chlorhydrate of a given amino acid ester (1
equiv), Et3N (1 equiv) and BOP (2.5 equiv). After stirring 30
min at room temperature, the mixture was treated with water
and the formed product extracted using EtOAc. The organic
layer was washed with water and brine and dried over Na2-
SO4. After filtration and evaporation of the solvent, the residue
was purified by chromatography on silica gel using CH2Cl2/
MeOH/AcOH, 9:1:0.5, as eluent.
The compound (1 equiv) was dissolved in ethanol and 1 M
NaOH (5 equiv) or 1 M LiOH (5 equiv) was added. The mixture
was stirred for 8 h at room temperature. After acidification
with 2 M HCl, the solution was evaporated, diluted in water.
The precipitate was filtered and dried.
Gen er a l P r oced u r e for th e Syn th esis of 2-Alk yl-3-
[h ydr oxy(1′-am in o-2′-alkyl)ph osph in yl]pr opan oyl Am in o
Acid . The protected inhibitor (1 equiv) was dissolved in CH2-
Cl2 and 1 M BBr3 in CH2Cl2 (5 equiv) was added at -10 °C.
The mixture was stirred at this temperature for 1 h and at
room temperature for 2 h. The organic layer was evaporated.
The aqueous layer was washed with Et2O and evaporated in
vacuo.
Com p ou n d 6: white solid; mp 230 °C dec (91%); HPLC
(30% B) 5.4 and 8.8 min; 1H NMR (DMSO-d6 and TFA) 1.07-
1.2 (dd, 3H, CH3â); 1.45-2.2 (m, 2H, P-CH2); 2.6-3.1 (m, 6H,
2xCH2Ph, CHCO, NCHR); 4.5 (m, 1H, CHCO2); 7.1-7.3 (m,
10H, Ar); 8.0 (s, br, 3H, NH3); 8.38-8.62 (m, 1H, NH); MS
(ESI) (M + 1)+ m/z 419.0. Anal. (C21H28BrN2O5P) C, H, N.
Com p ou n d 7: white solid; mp 204 °C dec (57%); HPLC
(28% B) 6.2 and 10.1 min; 1H NMR (DMSO-d6 and TFA) 1.07-
1.2 (dd, 3H, CH3â); 1.45-2.2 (m, 4H, CH2, P-CH2); 2,6-3,1
(m, 6H, 2xCH2Ph, CHCO, NCHR); 4,5 (m, 1H, CHCO2); 7.1-
7.3 (m, 10H, Ar); 8.0 (s, br, 3H, NH3); 8.38-8.62 (m, 1H, NH);
MS (ESI) (M + 1)+ m/z 433.1. Anal. (C22H30BrN2O5P) C, H, N.
Com p ou n d 8: white solid; mp 195 °C dec (86%); HPLC
(35% B) 5.3 and 7.2 min; 1H NMR (DMSO-d6 and TFA) 0.65-
0.9 (m, 6H, 2xCH3); 1.3-2.2 (m, 5H, CH2â-CH, P-CH2); 2.5-
3.2 (m, 6H, 2xCH2Ph, CHCO, NCHR); 4.4 (m, 1H, CHCO2);
7.05-7.3 (m, 10H, Ar); 7.98 (s, br, 3H, NH3); 8.3-8.55 (m, 1H,
NH); MS (ESI) (M + 1)+ m/z 461.0. Anal. (C24H34BrN2O5P) C,
H, N.
1a : R1 ) CH3; white solid; mp 112-3 °C (110 °C lit.) (51%).
20
Isomer R: [R]D -48.3 °C (1% in AcOH).
1b: R1 ) CH2CH3; white solid; mp 106 °C (32%).
1c: R1 ) CH2CH(CH3)2; white solid; mp 148-9 °C (150-2°
lit.) (55%).
1d : R1 ) CH2CH2SCH3; white solid; mp 105 °C (108-9 °C
lit.) (21%).
1e: R1 ) Ph; white solid; mp 220-2 °C (47%). Isomer R:
20
[R]D +14.3 °C (1% in AcOH).
1f: R1 ) Ph(p-Me); white solid; mp 255 °C (52%).
1g: R1 ) Ph(o,p-diMe); white solid; mp 267 °C (49%).
1h : R1 ) CH2Ph; white solid; mp 135-6 °C (137 °C lit.)
20
(50%). Isomer R: [R]D -72.6 °C (1% in AcOH).
1i: R1 ) CH2CH2Ph; white solid; mp 142-3 °C (44%).
Gen er a l P r oced u r e for th e Syn th esis of Acr yla te
Der iva tives 2. Meth od A. To a solution of triethyl phospho-
acetate in DMF, at -10 °C, were added sodium hydride (1.1
equiv) and, after 15 min, the alkyl or aryl bromide (1 equiv).
The mixture was stirred at room temperature for 16 h and
concentrated in vacuo. The residue was dissolved in EtOAc,
washed with brine and dried over Na2SO4. After filtration and
evaporation of the solvent, the crude product was purified by
chromatography on silica gel using EtOAc/Hex, 1:1, as eluent.
The 2-substituted triethyl phosphoacetate (1 equiv), form-
aldeyde (5 equiv) and potassium carbonate (3 equiv) were
treated at 100 °C under stirring for 3 h. Diethyl ether and
water were added. The organic layer was separated, washed
with brine, dried over Na2SO4 and evaporated to yield 2a -d .
2a : R2 ) CH2Ph; oil; Rf (Hep/EtOAc, 3:1) 0.59 (42%).
2b: R2 ) CH2Ph(p-Br); oil; Rf (Hep/EtOAc, 9:1) 0.57 (33%).
2c: R2 ) CH2CH(CH3)2; oil; Rf (Hep/EtOAc, 3.5:6.5) 0.32
(36.7%).
2d : R2 ) CH2Ph(p-CH(CH3)2); oil; Rf (Chex/EtOAc, 10:1) 0.6
(34%).
Meth od B (for 2e). A solution of 4.8 g (16.1 mmol) of ethyl
4-phenylbenzylmalonic acid obtained from the corresponding
diethyl ester34 was treated by 1.68 mL (16.1 mmol) of diethyl-
amine and 1.97 mL (24.2 mmol) of formaldehyde following the
procedure of Mannich and Ritsert.35 The crude product was
purified by chromatography over silica gel using Hep/EtOAc/
AcOH, 9:1:0.2, as eluents to yield 3.52 g (82.1%) of oil; Rf (Hep/
EtOAc/AcOH, 9:1:0.2) 0.55.
Gen er a l P r oced u r e for th e Syn th esis of Eth yl 2-Alk yl-
3-[h ydr oxy[1′-(N-ben zyloxycar bon ylam in o)-2′-alkyl]ph os-
p h in yl]p r op a n oa te Der iva tives 3 (Ta ble 5). Meth od A
(for 3a -o). The solution of various phosphonic acids 1
synthesized as described15 (1 equiv) and the acrylate deriva-
tives (1.2 equiv) in BSA (2-3 equiv) were stirred overnight at
room temperature or at 70 °C. After cooling, the mixtures were
taken off with water and extracted by EtOAc. The organic
layers were washed with water and brine and dried over Na2-
SO4. After filtration and evaporation of the solvent, the solid
residues were triturated in hexane and purified by chroma-
tography on silica gel using CH2Cl2/MeOH/AcOH, 9:1:0.3, as
eluents.
Com p ou n d 9: white solid; mp 175 °C (85%); HPLC (30%
B) 6.5 and 10.4 min; 1H NMR (DMSO-d6 and TFA) 1.4-2.2
(m, 4H, CH2â, P-CH2); 1.96 (s, 3H, SCH3); 2.35-3.08 (m, 7H,
CH2γ, 2xCH2Ph, CHCO); 3.1-3.3 (m, 1H, NCHR); 4.45 (m, 1H,
CHCO2); 6.95-7.2 (m, 10H, Ar); 8.0 (s, br, 3H, NH3); 8.35-
8.6 (m, 1H, NH); MS (ESI) (M + 1)+ m/z 479.1. Anal. (C23H32
BrN2O5PS) C, H, N.
-
Meth od B, Su zu k i Con d en sa tion (for 3p -r ). To a
solution of the ethyl 2-p-bromobenzyl-3-[hydroxy[1′-(N-benzyl-
oxycarbonylamino)alkyl]phosphinyl]propanoate (3b,h ,m ) (10
mmol) and Ph(Ph3)4 (0.3 mmol) in 20 mL of toluene were added
under inert atmosphere 10 mL of 2 M aqueous solution of Na2-
CO3 and 12 mmol of the phenylboronic acid in 5 mL of MeOH.
The vigorously stirred mixture was warmed to 80 °C for 10 h,
then cooled, acidified at pH 3 and extracted with EtOAc. The
organic layer was washed with water and brine and dried over
Na2SO4. After filtration and evaporation, the crude product
was purified by chromatography on silica gel using CH2Cl2/
MeOH/AcOH, 9:1:0.3, as eluents.
Com p ou n d 10: white solid; mp 173 °C dec (85%); HPLC
(30% B) 8.25, 8.7 and 14.0 min; 1H NMR (DMSO-d6 and TFA)
1.4-2.05 (m, 2H, P-CH2); 2.55-3.05 (m, 5H, 2xCH2Ph,
CHCO); 4.35-4.5 (m, 2H, NCHR, CHCO2); 6.9-7.4 (m, 15H,
Ar); 8.2-8.55 (m, 1H, NH), 8.65 (s, br, 3H, NH3); MS (ESI) (M
+ 1)+ m/z 481.1. Anal. (C26H30BrN2O5P) C, H, N.
Com p ou n d 10B: HPLC (30% B) 8.25 min; [R]20 -6.5° (c
D
0.2 in CH3CN/H2O, 50/50).
Com p ou n d 11: white solid; mp 165 °C dec (90%); HPLC
(35% B) 10.1, 10.5 and 12.7 min; 1H NMR (DMSO-d6 and TFA)
1.4-2.02 (m, 2H, P-CH2); 2.2 (s, 3H, CH3Ar); 2.54-3.0 (m,
5H, 2xCH2Ph, CHCO); 4.25-4.52 (m, 2H, NCHR, CHCO2);
6.85-7.38 (m, 14H, Ar); 8.15-8.5 (m, 1H, NH), 8.6 (s, br, 3H,
NH3); MS (ESI) (M + 1)+ m/z 495.0. Anal. (C27H32BrN2O5P)
C, H, N.
Gen er a l P r oced u r e for th e Syn th esis of 2-Alk yl-3-
[h yd r oxy[1′-(N-ben zyloxyca r bon yla m in o)-2′-a lk yl]p h os-
p h in yl]p r op a n oic Acid s 4a -r (Ta ble 5). One equivalent of
an ethyl 2-alkyl-3-[hydroxy[1′-(N-benzyloxycarbonylamino)-

1406 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7
Chen et al.
Com p ou n d 12: white solid; mp 174 °C dec (91%); HPLC
(30% B) 16.7, 28.8 and 31.9 min; 1H NMR (DMSO-d6 and TFA)
1.4-2.0 (m, 2H, P-CH2); 2.18 (d, 6H, 2xCH2); 2.6-3.1 (m, 5H,
2xCH2Ph, CHCO); 4.35-4.6 (m, 2H, NCHR, CHCO2); 6.8-7.4
(m, 13H, Ar); 8.15-8.65 (m, 1H, NH), 8.5 (s, br, 3H, NH3); MS
(ESI) (M + 1)+ m/z 509.0. Anal. (C28H34BrN2O5P) C, H, N.
8.5 (m, 1H, NH); MS (ESI) (M + 1)+ m/z 571.1. Anal. (C33H36
-
BrN2O5P) C, H, N.
Com p ou n d 22B: HPLC (45% B) 8.7 min; [R]20 +1.4° (c
D
0.1 in CH3CN/H2O, 50/50).
Com p ou n d 23: white solid; mp 230 °C dec (86%); HPLC
(30% B) 8.5, 10.1, 14.3 and 15.1 min; 1H NMR (DMSO-d6 and
TFA) 0.6-0.85 (4d, 6H, 2xCH3); 1.15-1.5 (m, 3H, CH2-CH);
1.55-2.0 (m, 2H, P-CH2); 2.65 (m, 1H, CHCO); 2.7-3.1 (m,
4H, CH2Ph, CH2â); 3.45 (m, 1H, NCHR); 4.35 (m, 1H, CHCO2);
7.1-7.3 (m, 10H, Ar); 7.05 (s, br, 3H, NH3); 8.3-8.45 (m, 1H,
NH); MS (ESI) (M + 1)+ m/z 461.0. Anal. (C24H34BrN2O5P) C,
H, N.
Com p ou n d 13: white solid; mp 220 °C dec (89%); HPLC
(30% B) 12.0, 12.8, 14.7 and 22.0 min; 1H NMR (DMSO-d6 and
TFA) 1.4-2.0 (m, 2H, P-CH2); 2.6-3.1 (m, 7H, CH2â, 2xCH2-
Ph, CHCO); 3.4 (m, 1H, NCHR); 4.4 (m, 1H, CHCO2); 7.0-7.3
(m, 15H, Ar); 8.0 (s, br, 3H, NH3); 8.4-8.6 (m, 1H, NH); MS
(ESI) (M + 1)+ m/z 495.0. Anal. (C27H32BrN2O5P) C, H, N.
Com p ou n d 23B: HPLC (30% B) 10.1 min; [R]20 -4.9° (c
Com p ou n d 13B: HPLC (30% B) 12.8 min; [R]20 -3.1° (c
D
D
0.1 in CH3CN/H2O, 50/50).
0.1 in CH3CN/H2O, 50/50).
Com p ou n d 24: white solid; mp 190 °C dec (46%); HPLC
Com p ou n d 14: white solid; mp 205 °C dec (89%); HPLC
1
1
(30% B) 5.7 and 9.4 min; H NMR (DMSO-d6 and TFA) 1.4-
(30% B) 5.1, 5.4 and 7.0 min; H NMR (DMSO-d6 and TFA)
2.2 (m, 2H, P-CH2); 2.6-3.2 (m, 3H, CH2Ph, CHCO); 4.3-4.7
(m, 1H, NCHR); 5.3 (m, 1H, CHCO2); 7.0-7.5 (m, 15H, Ar);
8.6 (s, br, 3H, NH3); 8.5-8.8 (m, 1H, NH); MS (ESI) (M + 1)+
m/z 467.0. Anal. (C25H28BrN2O5P) C, H, N.
1.4-2.2 (m, 4H, CH2â, P-CH2); 2.5-3.2 (m, 8H, CH2γ, 2xCH2-
Ph, CHCO, NCHR); 4.38 (m, 1H, CHCO2); 6.9-7.3 (m, 15H,
Ar); 8.1 (s, br, 3H, NH3); 8.35-8.6 (m, 1H, NH); MS (ESI) (M
+ 1)+ m/z 509.0. Anal. (C28H34BrN2O5P) C, H, N.
Com p ou n d 25: white solid; mp 194 °C dec (45%); HPLC
(33% B) 7.5 and 10.1 min; 1H NMR (DMSO-d6 and TFA) 1.0-
1.3 (dd, 3H, CH3); 1.45-2.2 (m, 2H, P-CH2); 2.55-3.05 (m,
3H, CH2Ph, CHCO); 4.15 (m, 1H, CHCO2); 4.4-4.7 (m, 1H,
NCHR), 7.1-7.6 (m, 14H, Ar); 8.1-8.4 (m, 1H, NH); 8.65 (s,
Com p ou n d 15: white solid; mp 210 °C dec (85%); HPLC
1
(30% B) 4.4, 5.5 and 5.9 min; H NMR (DMSO-d6 and TFA)
1.0-1.28 (m, 3H, CH3); 1.4-2.25 (m, 2H, P-CH2); 2.5-2.8 (m,
2H, CH2â); 2.95 (m, 2H, CH2Ph); 3.05 (m, 1H, CHCO); 3.3-
3.52 (m, 1H, NCHR); 4.15 (m, 1H, CHCO2); 7.1-7,3 (m, 10H,
Ar); 8.0 (s, br, 3H, NH3); 8.15-8.48 (m, 1H, NH); MS (ESI) (M
+ 1)+ m/z 419.0. Anal. (C21H28BrN2O5P) C, H, N.
br, 3H, NH3); MS (ESI) (M + 1)+ m/z 481.1. Anal. (C26H30
-
BrN2O5P) C, H, N.
Com p ou n d 25B: HPLC (45% B) 7.5 min; [R]20 -3.4° (c
D
Com p ou n d 16: white solid; mp 230 °C dec (83%); HPLC
0.1 in CH3CN/H2O, 50/50).
1
(30% B) 9.2, 9.6, 15.6 and 17.2 min; H NMR (DMSO-d6 and
Com p ou n d 26: white solid; mp 150 °C dec (56%); HPLC
TFA) 0.6-0.85 (m, 6H, 2xCH3); 1.4-2.25 (m, 5H, P-CH2,
CH2-CH); 2.5-2.8 (m, 2H, CH2â); 2.9-3.15 (m, 3H, CH2Ph,
CHCO); 3.3-3.5 (m, 1H, NCHR); 4.15 (m, 1H, CHCO2); 7.2
(m, 10H, Ar); 8.0 (s, br, 3H, NH3); 8.12-8.42 (m, 1H, NH); MS
(ESI) (M + 1)+ m/z 461.0. Anal. (C24H34BrN2O5P) C, H, N.
1
(30% B) 8.0, 8.7 and 12.5 min; H NMR (DMSO-d6 and TFA)
(DMSO-d6 and TFA) 0.55-0.8 (dd, 6H, 2xCH3); 0.8-1.4 (m,
3H, CH2-CH); 1.5-2.0 (m, 2H, P-CH2); 2.55-3.1 (m, 3H, CH2-
Ph, CHCO); 4.35-4.6 (m, 2H, NCHR, CHCO2); 7.1-7.5 (m,
10H, Ar); 8.1-8.45 (m, 1H, NH); 8.65 (s, br, 3H, NH3); MS
(ESI) (M + 1)+ m/z 447.0. Anal. (C23H32BrN2O5P) C, H, N.
Com p ou n d 27: white solid; mp 215 °C dec (91%); HPLC
Com p ou n d 17: white solid; mp 160 °C dec (44%); HPLC
(25% B) 8.9, 12.0 and 14.3 min; 1H NMR (DMSO-d6 and TFA)
1.3-2.2 (m, 2H, P-CH2); 2.5-3.2 (m, 7H, CHâ, 2xCH2Ph,
CHCO); 3.65 (m, 1H, NCHR); 4.6 (m, 1H, CHCO2); 6.55-6.95
(dd, 4H, Ar); 7.0-7.3 (m, 10, Ar); 8.0 (s, br, 3H, NH3); 8.3-8.5
1
(35% B) 8.5 and 10.9 min; H NMR (DMSO-d6 and TFA) 1.1
(4d, 3H, CH3â); 1.45-2.2 (m, 2H, P-CH2); 2.6-3.4 (m, 6H,
2xCH2Ph, CHCO, NCHR); 4,4 (m, 2H, CHCO2); 7.1-7.65 (m,
14H, Ar); 8.0 (s, br, 3H, NH3); 8.3-8.6 (m, 1H, NH); MS (ESI)
(M + 1)+ m/z 495.0. Anal. (C27H32BrN2O5P) C, H, N.
Com p ou n d 28: white solid; mp 220°-222 °C (92%); HPLC
(30% B) 7.2 and 10.3 min; 1H NMR (DMSO-d6 and TFA) 1.15
(dd, 3H, CH3â); 1.22 (m, 3H, CH3); 1.5-2.2 (m, 2H, P-CH2);
2.65 (m, 1H, CHCO); 2.95 (m, 2H, CH2Ph); 3.2 (m, 1H, NCHR);
4.17 (m, 2H, CHCO2); 7.24-7.6 (m, 9H, Ar); 8.0 (s, br, 3H,
NH3); 8.3-8.5 (m, 1H, NH); MS (ESI) (M + 1)+ m/z 418.9. Anal.
(C21H28BrN2O5P) C, H, N.
(m, 1H, NH); MS (ESI) (M + 1)+ m/z 511.0. Anal. (C27H32
BrN2O6P) C, H, N.
-
Com p ou n d 18: white solid; mp 170 °C dec (50%); HPLC
1
(43% B) 7.1, 8.8 and 9.2 min; H NMR (DMSO-d6 and TFA)
1.45-2.2 (m, 2H, P-CH2); 2.6-3.1 (m, 7H, CH2â, 2xCH2Ph,
CHCO); 3.45 (m, 1H, NCHR); 4.45 (m, 1H, CHCO2); 7.1-7.6
(m, 19H, Ar); 8.0 (s, br, 3H, NH3); 8.4-8.6 (m, 1H, NH); MS
(ESI) (M + 1)+ m/z 571.1. Anal. (C33H36BrN2O5P) C, H, N.
Com p ou n d 19: white solid; mp 210 °C dec (86%); HPLC
(30% B) 15.7, 21.3 and 23.2 min; 1H NMR (DMSO-d6 and TFA)
1.04-1.3 (m, 3H, CH3); 1.5-2.25 (m, 2H, P-CH2); 2.55-3.1
(m, 5H, CH2Ph, CH2â, CHCO); 3.32-3.55 (m, 1H, NCHR); 4.15
(m, 1H, CHCO2); 7.15-7.6 (m, 14H, Ar); 8.02 (s, br, 3H, NH3);
8.2-8.5 (m, 1H, NH); MS (ESI) (M + 1)+ m/z 495.0. Anal.
(C27H32BrN2O5P) C, H, N.
Com p ou n d 20: white solid; mp 205 °C dec (82%); HPLC
(30% B) 10.0, 14.4 and 15.5 min; 1H NMR (DMSO-d6 and TFA)
0.56-0.88 (2t, 2H, CH3); 1.4-2.3 (m, 4H, CH2, P-CH2); 2.6-
3.15 (m, 5H, CH2Ph-Ph, CH2â, CHCO); 3.35-3.55 (m, 1H,
NCHR); 4.15 (m, 1H, CHCO2); 7.15-7.6 (m, 14H, Ar); 8.02 (s,
br, 3H, NH3); 8.3-8.4 (m, 1H, NH); MS (ESI) (M + 1)+ m/z
509.0. Anal. (C28H34BrN2O5P) C, H, N.
Com p ou n d 21: white solid; mp 221 °C dec (83%); HPLC
(30% B) 11.4, 14.0 and 14.9 min; 1H NMR (DMSO-d6 and TFA)
0.7-0.9 (m, 6H, 2xCH3); 1.4-2.3 (m, 5H, CH2-CH, P-CH2);
2.6-3.15 (m, 5H, CH2Ph-Ph, CH2â, CHCO); 3.3-3.55 (m, 1H,
NCHR); 4.2 (m, 1H, CHCO2); 7.15-7.6 (m, 14H, Ar); 8.0 (s,
br, 3H, NH3); 8.3-8.45 (m, 1H, NH); MS (ESI) (M + 1)+ m/z
537.0. Anal. (C30H38BrN2O5P) C, H, N.
Com p ou n d 28B: HPLC (30% B) 7.2 min; [R]20 +1.6° (c
D
0.1 in CH3CN/H2O, 50/50).
Com p ou n d 29: white solid; mp 140 °C dec (38%); HPLC
(27% B) 7.8, and 12.6 min; 1H NMR (DMSO-d6 and TFA) 1.0-
1.25 (m, 12H, 2xCH3, CH(CH3)2); 1.45-2.2 (m, 2H, P-CH2);
2.5-3.05 (m, 4H, CH(CH3)2, CH2Ar, CHCO); 3.65 (m, 1H,
NCHR); 4.1 (m, 1H, CHCO2); 7.0-7.15 (dd, 4H, Ar); 8.0 (s, br,
3H, NH3); 8.15-8.5 (m, 1H, NH); MS (ESI) (M + 1)+ m/z 385.0.
Anal. (C18H30BrN2O5P) C, H, N.
Com p ou n d 30B: white solid; mp 228 °C dec (57%); HPLC
1
(20% B) 9.8 min; H NMR (DMSO-d6 and TFA) 1.15 (dd, 3H,
CH3â); 1.2 (d, 3H, CH3â); 1,55-2.1 (m, 2H, P-CH2); 2.65-3.2
(m, 6H, 2xCH2Ph, CHCO, NCHR); 4.15 (m, 2H, CHCO2); 7.15-
7.4 (dd, 4H, Ar); 8.0 (s, br, 3H, NH3); 8.45 (d, 1H, NH), [R]20
D
-3.4° (c 0.1 in CH3CN/H2O, 50/50); MS (ESI) (M + 1)+ m/z
497.1, 499.1. Anal. (C21H27Br2N2O5P) C, H, N.
Com p ou n d 31B: white solid; mp 214 °C dec (52%); HPLC
1
(35% B) 7.6 min; H NMR (DMSO-d6 and TFA) 1.5-2.1 (m,
2H, P-CH2); 2.6-3.05 (m, 3H, CHCO, CH2Ph(p-Ph)); 4.2 (m,
2H, CH2O); 4.5 (m, 2H, NCHR, CHCO2); 7.1-7.6 (m, 14H, Ar);
8.38-8.58 (m, 1H, NH), 8.68 (s, br, 3H, NH3); [R]20 -3.2° (c
Com p ou n d 22: white solid; mp 225 °C dec (94%); HPLC
(45% B) 8.7, 10.3 and 10.8 min; 1H NMR (DMSO-d6 and TFA)
1.5-2.2 (m, 2H, P-CH2); 2.6-3.15 (m, 7H, CH2Ph, CH2Ph-
Ph, CH2â, CHCO); 3.35-3.5 (m, 1H, NCHR); 4.36-4.48 (m,
1H, CHCO2); 7.05-7.6 (m, 19H, Ar); 8.0 (s, br, 3H, NH3); 8.38-
D
0.1 in HAc); MS (ESI) (M + 1)+ m/z 467.0. Anal. (C25H28
BrN2O6P) C, H, N.
-
Com p ou n d 32B: white solid; mp 242 °C (55%); HPLC (35%
B) 7.7 min; 1H NMR (DMSO-d6 and TFA) 1.12 (dd, 3H, CH3);

New Dual Enkephalin-Degrading Enzyme Inhibitors
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7 1407
1.44-2.15 (m, 2H, P-CH2); 2.55-3.2 (m, 3H, CHCO, CH2Ph-
(p-Ph)); 4.45 (m, 1H, NCHR); 4.58 (m, 1H, CHCO2); 5.75 (m,
1H, CHO); 7.25-7.6 (m, 14H, Ar); 8.3-8.5 (m, 1H, NH), 8.65
in a volume of 10 µL/mouse according to the method of Haley
and McCormick,42 15 min before the test.
8. System ic In jection s. Inhibitors or vehicle were slowly
injected iv in a volume of 0.1 mL/10 mg in mice. The analgesic
responses were measured at various times after the drug
administration.
(s, br, 3H, NH3); [R]20 -3.4° (c 0.1 in HAc); MS (ESI) (M +
D
1)+ m/z 511.6. Anal. (C27H32BrN2O6P) C, H, N.
Com p ou n d 33B: white solid; mp 230 °C dec (82%); HPLC
1
(20% B) 7.9 min; H NMR (DMSO-d6 and TFA) 1.5-2.25 (m,
9. Hot P la te Test. The test was based on that described
by Eddy and Leimbach.31 A glass cylinder (16 cm high, 16 cm
diameter) was used to keep the mouse on the heated surface
of the plate (53 ( 0.5 °C) using a thermoregulated water-
circulating pump. The latency period until the mouse jumped
was registered by a means of a stopwatch (cutoff time 240 s).
Dose-reponse curves were established by expressing the
data as a percentage of analgesia calculated by the ratio:
% analgesia ) (test latency - control latency)/(cutoff time -
control latency) × 100.
2H, P-CH2); 2.6-3.1 (m, 3H, CH2Ph, CHCO); 3.78 (d, 2H, CH2-
CO2); 4.5 (m, 1H, NCHR); 7.15-7.2 (m, 14H, Ar); 8.5 (t, 1H,
NH); 8.65 (s, br, 3H, NH3), [R]20 +1.6° (c 0.1 in CH3CN/H2O,
D
50/50); MS (ESI) (M + 1)+ m/z 467.0. Anal. (C25H28BrN2O5P)
C, H, N.
Biologica l Tests. 1. En zym es. APN from pig kidney was
purchased from Boehringer Mannheim. NEP was purified to
homogeneity from rabbit kidney.36 ACE was purified from rat
testis.37
2. Su bstr a tes. Inhibitory potencies were determined by
using alanine-â-naphthylamide (AlaâNa) (Km ) 50 µM) for
APN, DGNPA38 (DNS-Gly-(pNO2)Phe-â-Ala) (Km ) 37 µM) for
NEP, and N-CBz-Phe-His-Leu39 (Km ) 50 mM) for ACE as
substrates.
Statistical analysis of data was carried out by analysis of
variance (ANOVA), followed by Dunnett’s test or Newman-
Keulss test. ED50 values and their 95% confidence limits were
calculated by log-probit analysis according to the method of
Litchfield and Wilcoxon.43 The ED50 is defined as the dose
required to elicit 50% analgesia.
3. Assa y for AP N Activity. APN (final concentration 21
ng/mL) was preincubated for 10 min at 37 °C with or without
increasing concentrations of a given inhibitor in a total volume
of 200 µL in 50 mM Tris/HCl buffer, pH 7.4. AlaâNA was added
at a final concentration of 50 µM and the reaction was stopped
after 30 min at 37 °C by adding 10 µM CH3CO2Na 1 M (pH
4.2). The fluorescence of the metabolite was measured at 400
nm (λex ) 340 nm) with a MPF44A Perkin-Elmer spectrofluo-
rimeter. A calibration curve for â-naphthylamide was obtained
by addition of increasing concentrations of âNA into 210 µL
of 50 mM Tris/HCl buffer pH 7.4.
4. Assa y for NEP Activity. NEP (final concentration 250
ng/mL) was preincubated for 15 min at 37 °C with or without
increasing concentrations of a given inhibitor in a total volume
of 225 µL of 50 mM Tris/HCl buffer, pH 7.4. DGNPA (25 µL)
was added to a final concentration of 50 µM, and the reaction
was stopped after 15 min at 37 °C by adding 250 µL of dioxane.
The 500 µL mixture was then transferred into a quartz cell
and the fluorescence measured (λex ) 342 nm, λem ) 525 nm).
Samples, corresponding to 0% hydrolysis, were obtained by
adding the buffer and the substrate only, while samples
corresponding to 100% of relative activity were prepared by
adding all the reagents except the inhibitors. Both solutions
were treated under the same conditions as above. The percent-
age of degradation was evaluated by reference to 100% of
relative activity and the IC50 values of tested inhibitors were
determined accordingly.
5. Assa y for ACE Activity. ACE (final concentration 0.02
pmol/100 µL) 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 follow-
ing the fluorimetric assay described by Cheung et al.40 with a
MPF44A Perkin-Elmer spectrofluorimeter (λex ) 365 nm, λem
) 495 nm). The calibration curve for His-Leu was obtained
by addition of increasing concentrations of His-Leu into 0.1
mL of 50 mM Tris/HCl buffer, pH 8.0, containing the dena-
turated enzyme.
Ack n ow led gm en t. We gratefully acknowledge C.
Dupuis for expert drafting of the manuscript. This work
was supported by grants from CEE (BMH4 CI98 2267).
Refer en ces
(1) Herz, A. Opioids. Handbook Exp. Pharmacol. 1993, 104, I and
II.
(2) Chaillet, P.; Coulaud, A.; Zajac, J . M.; Fournie´-Zaluski, M. C.;
Costentin, J .; Roques, B. P. The mu rather than the delta
subtype of opioid receptors appears to be involved in enkephalin
induced analgesia. Eur. J . Pharmacol. 1984, 101, 83-90.
(3) Matthes, H. W. D.; Maldonado, R.; Simonin, F.; Valverde, O.;
Slowe, S.; Kitchen, I.; Befort, K.; Dierich, A.; Le Meur, M.; Dolle,
P.; Tzavara, E.; Hanoune, J .; Roques, B. P.; Kieffer, B. Loss of
morphine-induced analgesia, reward effect and withdrawal
symptoms in mice lacking the µ-opioid receptor gene. Nature
1996, 383, 819-823.
(4) Ko¨nig, M.; Zimmer, A. M.; Steiner, H.; Holmes, P. V.; Crawley,
J . N.; Brownstein, M. J .; Zimmer, A. Pain responses, anxiety
and agression in mice deficient in pre-proenkephalin. Nature
1996, 383, 535-538.
(5) Roques, B. P.; Noble, F.; Dauge´, V.; Fournie´-Zaluski, M. C.;
Beaumont, A. Neutral endopeptidase 24.11. Structure, inhibi-
tion, and experimental and clinical pharmacology. Pharmacol.
Rev. 1993, 45, 87-146.
(6) Roques, B. P.; Fournie´-Zaluski, M. C.; Soroca, E.; Lecomte, J .
M.; Malfroy, B.; Llorens, C.; Schwartz, J . C. The enkephalinase
inhibitor thiorphan shows antinociceptive activity in mice.
Nature 1980, 288, 286-288.
(7) Fournie´-Zaluski, M. C.; Chaillet, P.; Bouboutou, R.; Coulaud, A.;
Che´rot, P.; Waksman, G.; Costentin, J .; Roques, B. P. Analgesic
effects of kelatorphan, a new highly potent inhibitor of multiple
enkephalin degrading enzymes. Eur. J . Pharmacol. 1984, 102,
525-528.
(8) Schmidt, C.; Peyroux, J .; Noble, F.; Fournie´-Zaluski, M. C.;
Roques, B. P. Analgesic responses elicited by endogenous en-
kephalins (protected by mixed peptidase inhibitors) in a variety
of morphine-sensitive noxious tests. Eur. J . Pharmacol. 1991,
192, 253-262.
(9) Noble, F.; Soleilhac, J . M.; Soroca, E.; Turcaud, S.; Fournie´-
Zaluski, M. C.; Roques, B. P. Inhibition of the enkephalin
metabolizing enzymes by the first systemically active mixed
inhibitor prodrug RB 101 induced potent analgesic responses
in mice and rats. J . Pharmacol. Exp. Ther. 1992, 261, 181-190.
(10) Luciani, N.; Marie-Claire, C.; Ruffet, E.; Beaumont, A.; Roques,
B. P.; Fournie´-Zaluski, M. C. Characterization of Glu350 as a
critical residue involved in the N-terminal amine binding site
of aminopeptidase N (EC 3.4.11.2): Insights into its mechanism
of action. Biochemistry 1998, 37, 686-692.
(11) Bateman, R. C.; J ackson, D.; Slaughter, C. A.; Unnithan, S.;
Chai, Y. G.; Moomaw, C.; Hersh, L. B. Identification of the active
site arginine in rat neutral endopeptidase 24.11 (enkephalinase)
as arginine 102 and analysis of a glutamine 102 mutant. J . Biol.
Chem. 1989, 264, 6151-6157.
Ki values were calculated from IC50 statistical values using
the Cheng-Prussoff relationship.41
6. P h a r m a cologica l Assa ys. The inhibitors were dissolved
in water and the pH of the solutions was adjusted to 7.0. Drugs
and vehicle (controls) were administered to male Swiss mice
(20-22 g; Depre´, France). Mice were housed and used strictly
in accordance with European Community guidelines for the
care and use of laboratory animals and after approval of the
proposed experiments by the ethical committee of the faculty.
7. In tr a cer ebr oven tr icu la r In jection s. Inhibitors or
vehicle were slowly (15 s) injected free hand into the left lateral
ventricle of mice using a modified Hamilton microliter syringe
(12) Chen, H.; Roques, B. P.; Fournie´-Zaluski, M. C. Design of the
first highly potent and selective aminopeptidase N (EC 3.4.11.2)
inhibitor. Bioorg. Med. Chem. Lett. 1999, 9, 1511-1516.

1408 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7
Chen et al.
(13) Chen, H.; Noble, F.; Coric, P.; Fournie´-Zaluski, M. C.; Roques,
B. P. Aminophosphinic inhibitors as transition state analogues
of enkephalin-degrading enzymes: A class of central analgesics.
Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 12028-12033.
(14) Matthews, B. W. Structural basis of the action of thermolysin
and related zinc peptidases. Acc. Chem. Res. 1988, 21, 333-340.
(15) Berger, J. A novel approach to analgesia: Synthesis and structure-
activity relationships of some potent and selective inhibitors of
enkephalinase A. 19th National Medicinal Chemistry Sympo-
sium, Tucson, AZ, 1984.
(29) Krapcho, J .; Turk, C.; Cushman, D. W.; Powell, J . R.; DeForest,
J . M.; Spitzmiller, E. R.; Karanewsky, D. S.; Duggan, M.;
Rovnyak, G.; Schwartz, J .; Natarajan, S.; Godfrey, J . D.; Ryono,
D. E.; Neubeck, R.; Atwal, K. S.; Petrillo, E. W., J r. Angiotensin-
converting enzyme inhibitors. Mercaptan, carboxyalkyl dipep-
tide, and phosphinic acid inhibitors incorporating 4-substituted
prolines. J . Med. Chem. 1988, 31, 1148-1160.
(30) Patchett, A. A.; Harris, E.; Tristram, E. W.; Wyvratt, M. J .; Wu,
M. T.; Taub, D.; Peterson, E. R.; Ikeler, T. J .; Broeke, J . T.;
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. A new class of angiotensin-converting enzyme
inhibitors. Nature 1980, 288, 280-283.
(16) Gomez-Monterrey, I.; Turcaud, S.; Lucas, E.; Bruetschy, L.;
Roques, B. P.; Fournie´-Zaluski, M. C. Exploration of neutral
endopeptidase active site by a series of new thiol-containing
inhibitors. J . Med. Chem. 1993, 36, 87-94.
(17) Baylis, E. K.; Campbell, C. D.; Dingwall, J . G. 1-aminoalky-
lphosphonous acids. Part 1. Isosteres of the protein amino acids.
J . Chem. Soc., Perkin Trans. 1 1984, 2845-2853.
(31) Eddy, N. B.; Leimbach, D. Synthetic analgesia (II) diethylbutenyl
and diethylbutylamines. J . Pharmacol. Exp. Ther. 1953, 107,
385-389.
(18) Fournie´-Zaluski, M. C.; Lucas, E.; Devin, J .; Roques, B. P. 1H
NMR configuration correlation for retro-inverso dipeptides:
Application to the determination of the absolute configuration
of “enkephalinase” inhibitors. Relationships between stereo-
chemistry and enzyme recognition. J . Med. Chem. 1986, 29,
751-757.
(32) Noble, F.; Soleilhac, J . M.; Soroca-Lucas, E.; Turcaud, S.;
Fournie´-Zaluski, M. C.; Roques, B. P. Inhibition of the enkepha-
lin metabolizing enzymes by the first systemically active mixed
inhibitor prodrug RB 101 induces potent analgesic responses in
mice and rats. J . Pharmacol. Exp. Ther. 1992, 261, 181-190.
(33) De Forrest, J . M.; Waldron, T. L.; Harvey, C.; Scalese, B.; Rubin,
B.; Powell, J . R.; Petrillo, E. W.; Cushman, D. W. Fosinopril, a
phosphinic acid inhibitor of angiotensin I converting enzyme:
In vitro and preclinical in vivo pharmacology. J . Cardiovasc.
Pharmacol. 1989, 14, 730-736.
(34) Curtius, T.; Sieber, W. Rearrangment of alkylmalonic acids in
R-amino acids (Part II). Synthesis of â-phenyl-R-alanine and
R-amino-n-butyric acid. Chem. Ber. 1922, 55B, 1543-1558.
(35) Mannich, C.; Ritsert, K. About the condensation of malonic ester,
diethylamine and formaldehyde. Chem. Ber. 1924, 57, 1116-
1118.
(36) Aubry, M.; Bertheloot, A.; Beaumont, A.; Roques, B. P.; Crine,
P. The use of a monoclonal antibody for the high yield purifica-
tion of kidney enkephalinase solubilized in â-octylglucoside.
Biochem. Cell Biol. 1987, 65, 398-404.
(37) Pantaliano, M. W.; Holmquist, B.; Riordan, J . F. Affinity
chromatographic purification of angiotensin converting enzyme.
Biochemistry 1984, 23, 1037-1042.
(38) Goudreau, N.; Guis, C.; Soleilhac, J . M.; Roques, B. P. DNS-
Gly-(pNO2)Phe-âAla, a specific fluorogenic substrate for neutral
endopeptidase 24.11. Anal. Biochem. 1994, 219, 87-95.
(39) Piquilloud, Y.; Reinharz, A.; Roth, M. Studies of the angiotensin
converting enzyme with different substrates. Biochim. Biophys.
Acta 1970, 206, 136-142.
(40) Cheung, H. S.; Cushman, D. W. Inhibition of homogeneous
angiotensin converting enzyme of rabbit lung by synthetic venom
peptides of both rops jararaca. Biochim. Biophys. Acta 1973, 293,
451-457.
(41) Cheng, Y. C.; Prussof, W. H. Relationship between the inhibition
constant (Ki) and the concentration of inhibitor which causes
50% inhibition (I50) of an enzyme reaction. Biochem. Pharmacol.
1973, 22, 3099-3108.
(42) Haley, T. J .; McCormick, W. G. Pharmacological effects produced
by intracerebroventricular injections of drugs in conscious
mouse. Br. J . Pharmacol. 1957, 12, 12-16.
(19) Roques, B. P. Zinc metallopeptidases: Active site structure and
design of selective and mixed inhibitors - new approaches in
the search for analgesics and antihypertensives. Biochemical
Society Transactions; Portland Press: London, 1993; Vol. 21, pp
678-685.
(20) Fournie´-Zaluski, M. C.; Lucas, E.; Waksman, G.; Roques, B. P.
Differences in the structural requirements for selective interac-
tion with neutral metalloendopeptidase (enkephalinase) or an-
giotensin converting enzyme. Molecular investigation by use of
new thiol inhibitors. Eur. J . Biochem. 1984, 139, 267-274.
(21) Fournie´-Zaluski, M. C.; Coric, P.; Turcaud, S.; Bruetschy, L.;
Lucas, E.; Noble, F.; Roques, B. P. Potent and systemically active
aminopeptidase N inhibitors designed from active-site investiga-
tion. J . Med. Chem. 1992, 35, 1259-1266.
(22) Rupreht, J .; Ukponmwan, O. E.; Admiral, P. V.; Dzolzic, M. R.
Effect of phosphoramidon - a selective enkephalinase inhibitor
on nociception and behaviour. Neurosci. Lett. 1983, 41, 331-
335.
(23) Altstein, M.; Blumberg, S.; Vogel, Z. Phosphoryl-Leu-Phe: A
potent inhibitor of the degradation of enkephalin by enkepha-
linase. Eur. J . Pharmacol. 1981, 76, 299-300.
(24) Elliot, R. L.; Marks, N.; Berg, M. J .; Portoghese, P. S. Synthesis
and biological evaluation of phosphoramidate peptide inhibitors
of enkephalinase and angiotensin-converting enzyme. J . Med.
Chem. 1985, 28, 1208-1216.
(25) De Lombaert, S.; Erion, M. D.; Tan, J .; Blanchard, L.; El-
Chehabi, L.; Ghai, R. D.; Sakane, Y.; Berry, C.; Trapani, A. J .
N-Phosphonomethyl dipeptides and their phosphonate pro-
drugs, a new generation of neutral endopeptidase (NEP, EC
3.4.24.11) inhibitors. J . Med. Chem. 1994, 37, 498-511.
(26) Tiraboschi, G.; J ullian, N.; The´ry, V.; Antonczak, S.; Fournie´-
Zaluski, M. C.; Roques, B. P. A Three-dimensional construction
of the active site (region 507-749) of human neutral endopep-
tidase (EC 3.4.24.11). Protein Eng. 1999, 12, 141-149.
(27) Chackalamannil, S.; Chung, S.; Stamford, A. W.; McKittrick, B.
A.; Wang, Y.; Tsai, H.; Cleven, R.; Fawzi, A.; Czarniecki, M.
Highly potent and selective inhibitors of endothelin converting
enzyme. Bioorg. Med. Chem. Lett. 1996, 6, 1257-1260.
(28) McKittrick, B. A.; Stamford, A. W.; Weng, X.; Ma, K.; Chack-
alamannil, S.; Czarniecki, M.; Cleven, R. M.; Fawzi, A. B. Design
and synthesis of phosphinic acids that triply inhibit endothelin
converting enzyme, angiotensin converting enzyme and neutral
endopeptidase 24.11. Bioorg. Med. Chem. Lett. 1996, 6, 1629-
1634.
(43) Litchfield, J . T.; Wilcoxon, F. A simplified method of evaluating
dose-effect experiments. J . Pharmacol. Exp. Ther. 1949, 96,
99-113.
J M990483L