Long lasting antinociceptive properties of enkephalin degrading enzyme (NEP and APN) inhibitor prodrugs

Article abstract of DOI:10.1021/jm0102248

Prodrugs of phosphinic dual inhibitors of the enkephalin degrading enzymes, neutral endopeptidase (NEP) and aminopeptidase N (APN), corresponding to the formula H₃N⁺CH-(R₁)P(O)(OR)CH₂CH(CH ₂Bip)CONHCH

Full text of DOI:10.1021/jm0102248

J . Med. Chem. 2001, 44, 3523-3530
3523
Lon g La stin g An tin ocicep tive P r op er ties of En k ep h a lin Degr a d in g En zym e
(NEP a n d AP N) In h ibitor P r od r u gs
Huixiong Chen, Florence Noble, Bernard P. Roques,* 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
Received May 21, 2001
Prodrugs of phosphinic dual inhibitors of the enkephalin degrading enzymes, neutral endopep-
tidase (NEP) and aminopeptidase N (APN), corresponding to the formula H3N+CH-
(R1)P(O)(OR)CH2CH(CH2Bip)CONHCH(CH3)COOCH2Ph, with R1 ) CH3 or Ph and R being a
benzyl ester, a S-acyl-2-thioethyl derivative, or an acyloxyalkyl group, were synthesized to
improve the poor central bioavailability of their precursors. As expected, these compounds (50
mg/kg, iv or ip) induced long lasting (∼2 h) antinociceptive responses in the hot plate test in
mice with a ceiling effect varying between 25 and 42% of analgesia. A very rapid hydrolysis of
the carboxylate ester contrasting with a slow deprotection of the phosphinate group (t1/2 ∼ 1 h)
was observed in serum while 80% of free drug was obtained after 1 h incubation with brain
membranes. These results account for the long duration of action observed with these prodrugs.
In tr od u ction
NSAIDs and opioids and thus to alleviate the various
types of nociceptive stimuli, in particular, severe os-
teoarticular, post-operative, or neuropathic pain.
In the central nervous system (CNS), the modulation
of nociceptive stimuli at both the spinal and central
levels depends on the equilibrium between endogenous
counteracting systems. The opioid system involves
several peptides deriving from three independent pre-
cursors designated pro-enkephalin, pro-dynorphin, and
proopiomelanocortin. These peptides induce analgesia
through interaction with one or several of the three
types of opioid receptors µ, δ, and κ, which seem to be
differently involved in the treatment of nociceptive
signals.1
Conversely, anti-opioid systems involve endogenous
peptides able to potentiate pain sensation. This is the
case of calcitonin gene releasing peptide (CGRP), sub-
stance P, and neurokinin A at the spinal cord level
(review in 2) whereas the C-terminal fragment of
cholecystokinin CCK8,3 the neuropeptide FF,4 or the
recently discovered peptide nociceptin5 facilitate spinal
and central nociceptive stimuli.
Pain control by these counteracting systems has
generated numerous studies aimed at developing new
analgesics with reduced side effects as compared to
morphine and surrogates. However, synthetic analogues
of opioid peptides were shown to produce the same type
of drawbacks as morphine,6 and antagonists of the anti-
opioid systems were reported to have a low activity or
to induce important undesirable side effects.7
Other approaches such as development of antagonists
of bradykinin receptors and inhibitors of cyclo-oxyge-
nases have been achieved to try to reduce nociceptive
stimuli at the peripheral nociceptor level. Thus selective
Cox-2 inhibitors behave as interesting new NSAIDs
(nonsteroidal antiinflammatory drugs) since they are
devoid of the gastrointestinal nuisance of most of these
latter compounds.8 However, none of these compounds
resulted in analgesics able to fill the gap between
Numerous experiments have shown that one of the
most promising approaches appears to potentiate the
antinociceptive effects induced by the endogenous opioid
peptides Met- and Leu-enkephalin (review in 9). Indeed,
intracerebroventricularly administered, these morphine-
like peptides interact selectively with µ and δ opioid
receptors to trigger a strong but brief analgesic re-
sponse,10 due to the rapid physiological inactivation of
the peptides by two zinc metallopeptidases, the neutral
endopeptidase (NEP, neprilysin, EC 3.4.24.11),11 and
the neutral aminopeptidase (APN, EC 3.4.11.2).12 J oint
inhibition of both enzymes allows the potent antinoci-
ceptive properties of enkephalins to be strongly potenti-
ated.13 Thus, icv or iv administration in mice and rats
of a dual inhibitor such as kelatorphan, OHNH-CO-CH2-
CH(CH2Ph)-CONH-CH(CH3)-COOH (Ki values 1.7 nM
and 7 µM on NEP and APN, respectively (Scheme 1)14
or the prodrug RB 101, H2N-CH(CH2CH2SCH3)-CH2-
S-S-CH2-CH(CH2Ph)-CONH-CH(CH2Ph)-CO2CH2Ph15
(Scheme 1), produce antinociceptive responses, only
slightly lower than those produced by morphine, in all
animal models of pain used for screening new analge-
sics. In the case of RB 101, the biologically dependent
reduction of the disulfide bond in brain tissue was
shown to release, respectively, two selective thiol-
containing inhibitors which display nanomolar affinities
for NEP and APN, respectively.16
However, due to the short duration of action of these
compounds, a new series of dual inhibitors has been
designed. Among these molecules, R-aminophosphinic
derivatives,17,18 which behave as “transition state”
analogues interacting with the S1, S1′ and S2′ subsites
of both NEP and APN and are endowed with Ki values
in the nanomolar range for the two targeted enzymes,
were shown to be the most interesting.
* Address correspondence to Professor Bernard P. Roques.
Tel: 33-1-43.25.50.45. Fax: 33-1-43.26.69.18. E-mail: roques@
pharmacie.univ-paris5.fr.
Thus, the two dual inhibitors 1 and 2 (Scheme 1)
(compound 1: Ki(NEP) 1.2 nM, Ki(APN) 2.9 nM; com-
10.1021/jm0102248 CCC: $20.00 © 2001 American Chemical Society
Published on Web 09/11/2001

3524 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 21
Chen et al.
Sch em e 1. Structure of the Two Selected Dual NEP/APN Inhibitors 1 and 2 and Their Respective Prodrugs 9a -c
and 10a -f
pound 2: Ki(NEP) 2.0 nM, Ki(APN) 4.8 nM) selected from
structure-activity studies have been shown to induce
strong dose-dependent analgesic responses following icv
administration in mice in the hot plate and the writhing
tests, but high doses were required to obtain these
effects after iv administration. Thus at 100 mg/kg,
compound 1 induced a maximal effect of 25% analgesia,
15 min after administration.18 This was interpreted by
a reduced passage into the brain of these compounds
due to the presence of three highly polar functions. To
increase their brain penetration, polar functions could
be transiently protected by lipophilic groups, able to be
cleaved in vivo by biologically dependent processes.
In this work, we describe the development of such
prodrugs (Scheme 1) differing essentially by the nature
of the phosphinic protecting groups. Their antinocicep-
tive properties and duration of action were investigated
by using the hot plate test in mice after iv or ip
administration.
Resu lts
I. Syn th esis. The ethyl (2R,S)2-biphenylmethyl-3-
[hydroxy[(1′R)1′-(N-benzyloxycarbonylamino)alkyl]phos-
phinyl]propanoate 3 and 4 were synthesized as previ-
ously described18,19 by Michael condensation between
the 2-biphenylmethylacrylate and a (1R)-1-(benzyloxy-
carbonylamino)alkylphosphinic acid20 (Scheme 2). The
Z-protection of the amino function in 3 and 4 was
replaced by a Boc group, and saponification of the ethyl
ester afforded 5 and 6 which are obtained as optically
pure compounds by crystallization in a mixture of
methanol and ethyl acetate, 90/10. These molecules
were coupled with alanine benzyl ester using classical
conditions (EDCI/HOBt or BOP) to give 7 and 8.
Esterification of the phosphinic function was achieved
either by coupling an alcohol ROH using the BOP
method or through alkylation by an alkyl halide in
alkaline conditions as described in Petrillo, J r., et al.21
The final and selective deprotection of the amino group
was performed with mild acid (HCOOH) to prevent
competitive cleavage of the phosphinic ester. After
purification by chromatography on silica gel column,
Several of these dual NEP/APN inhibitor prodrugs
showed, for the first time, potent and long lasting (∼2
h) antinociceptive responses.

Enkephalin Degrading Enzyme Inhibitor Prodrugs
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 21 3525
Sch em e 2. Synthesis of Dual NEP/APN Inhibitor Prodrugsa
a
Reagents: (a) 48% HBr; (b) Boc2O; (c) 1 N LiOH, crystallization; (d) Ala-benzylester/BOP/DIEA (or EDC/HOBT); (e) BOP/DIEA/ROH
or RBr/Et3N; (f) HCO2H.
prodrugs 9a -c and 10a -f were used as trifluoroacetate
or methanesulfonate salts. These prodrugs were mixture
of two diastereoisomers, due to the presence of the chiral
phosphorus atom. This mixture is clearly evidenced by
HPLC.
II. HP LC Stu d y of th e Bioa ctiva tion of Com -
p ou n d 10b in Ra t Ser u m a n d Br a in Hom ogen a te.
Prodrug 10b was incubated at a final concentration of
25 µM with rat serum (3 mg protein/mL) at 37 °C in
Tris HCl buffer pH 7.4. The analysis of the incubation
mixture was performed by HPLC using a C18 kromasil
column with a mixture of 50% CH3CN/50% H2O (0.05%
TFA) as mobile phase.
Prodrug 10b disappeared rapidly in less than 10 min
leading, at nearly 95%, to a compound derived from
benzyl ester hydrolysis (Figure 1). Then, the amount of
this intermediate decreased slowly with concomitant
formation of the completely deprotected and more polar
dual inhibitor. After 1 h, the incubation mixture con-
tains about 40% of the partially protected form and 55%
of the final inhibitor (Figure 1).
Incubation of 10b with a homogenate of brain mem-
branes in Tris HCl buffer pH 7.4 showed a HPLC profile
different from that observed in the previous experiment
(Figure 2). The prodrug disappears slowly and requires
about 1 h for a complete conversion. The two metabolites
appear simultaneously, and the amount of the partially
protected inhibitor culminates after 20 min of incuba-
tion at around 40%. Then, this intermediate decreases
slowly down to 20% after 1 h, leading to 80% of
completely deprotected drug (Figure 2).
III. An t in ocicep t ive P r op er t ies of t h e Va r iou s
P r od r u gs a fter iv Ad m in istr a tion . Antinociceptive
properties of the various prodrugs were studied after
mice iv administration by the hot plate test. Two
parameters were measured, the jump and the paw lick
latencies. All compounds were tested at the unique dose
of 50 mg/kg (∼6 × 10-5 mol/kg). In these conditions, the
three prodrugs 9a -c present a significant antinocicep-
tive activity (Figure 3). The response was relatively low
for 9a , affording less than 20% analgesia 30 min after
administration, but this effect was maintained for 30
min. With prodrug 9b, a significant response appeared
40 min after injection with a maximum of 25% analgesia
at 50-60 min, the effect remaining significant for 40
F igu r e 1. Time dependent biotransformation of prodrug 10b
incubated with rat serum (pH 7.4). Values are the mean of
three independent experiments with variations remaining in
the range of ( 5% for each point. 0 R1 ) CH2CH2SCOCH3,
R2 ) OCH2-Ph; b R1 ) CH2CH2SCOCH3, R2 ) OH; 2 R1
H, R2 ) OH.
)
min. The antinociceptive profile of 9c was similar to that
of 9a for the duration of action, but a slightly improved
percentage of analgesia (30% at 30 min) was obtained
each time during the study. Moreover, this latter
prodrug induced a significant response (20% analgesia)
on the paw lick latency 30 and 40 min after administra-
tion (data not shown), whereas the two other prodrugs
were inactive in the same conditions.
For the second series of prodrugs 10a -d , the phos-
phinic acid was protected by a benzyl group (10a ) or
various SATE (S-acyl-2-thioethyl derivatives (10b-d ).
A very long duration of action was observed with the
four derivatives with significant responses obtained
from 20-30 min to 90-100 min after injection (Figure
4A,B). Compound 10b emerges from this study with

3526 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 21
Chen et al.
F igu r e 2. Time dependent biotransformation of prodrug 10b
incubated with rat brain membranes (pH 7.4). Values are the
mean of three independent experiments with variations re-
maining in the range of ( 5% for each point. 0 R1 ) CH2CH2-
SCOCH3, R2 ) OCH2-Ph; b R1 ) CH2CH2SCOCH3, R2 ) OH;
2 R1 ) H, R2 ) OH.
F igu r e 4. Time course of antinociceptive responses induced
after iv administration of 50 mg/kg (6 × 10-5 mol/kg) of
prodrugs 10 a -d in the hot plate test (52 °C) in mice (n )
10). (A) Prodrugs 10a (O) and 10b (b). (B) Prodrugs 10c (9)
and 10d (∆). Results are expressed as percentage of analgesia
( SEM. *P < 0.05, **P < 0.01 as compared to control
(Dunnett’s t test).
F igu r e 3. Time course of the antinociceptive responses
induced after iv administration of 50 mg/kg (6 × 10-5 mol/kg)
of prodrugs 9 a -c in the hot plate test (52 °C) in mice (n )
10). Prodrugs 9a (0), 9b (2), 9c (9). Results are expressed as
percentage of analgesia ( SEM. *P < 0.05, **P < 0.01 as
compared to control (Dunnett’s t test).
more than 40% analgesia at 60 min, and a long-lasting
effect exceeding 100 min.
IV. An tin ocicep tive P r op er ties of th e P r od r u gs
a fter ip Ad m in istr a tion . The antinociceptive proper-
ties of two of the most efficient prodrugs 10c and 10d
were studied after i.p. administration under methane-
sulfonate salts at the unique dose of 50 mg/kg (∼6 ×
10-5 mol/kg). As shown in Figure 5, they induced
antinociceptive responses with a ceiling effect at 30-
35% analgesia, but with a long duration of action since
both prodrugs were still active 100 min after adminis-
tration. Maximum activity was observed in the 70-80
min range. Moreover, a significant response was ob-
served on the paw lick latency (28% analgesia at 60 min)
with compound 10c (data not shown).
F igu r e 5. Time course of antinociceptive responses after i.p.
administration (50 mg/kg) (6 × 10-5 mol/kg) of prodrugs 10
c-d in the hot plate test (52 °C) in mice (n ) 10). Prodrug
10c (b) and 10d (O). Results are expressed as percentage of
analgesia ( SEM. *P < 0.05, **P < 0.01 as compared to control
(Dunnett’s t test).
at 50 min with about 35% analgesia, while 10f exhibited
a very delayed response leading to a significant activity
starting 80 min after administration (data not shown).
Discu ssion
The aim of this study was to design prodrugs of the
recently reported17,18 dual phosphinic inhibitors of the
two physiological enkephalin degrading enzymes, NEP
and APN, with the purpose of improving the duration
Two other prodrugs 10e and 10f containing an acyl-
oxyalkyl ester as the phosphinic protective group were
also tested. Compound 10e gave a maximum activity

Enkephalin Degrading Enzyme Inhibitor Prodrugs
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 21 3527
analgesic effects suggest the occurrence of differences
during the elimination from the brain.
To investigate the fate of 10b, we have studied its
bioactivation in serum and in homogenates of brain
membranes. A very rapid cleavage of the benzyl ester
occurs in plasma, but hydrolysis of the phosphinic ester
is very slow since after 1 h incubation only 50% of the
final unprotected inhibitor 2 was obtained (Figure 1).
Incubation with a brain membrane homogenate showed
a longer lifetime of the benzyl ester and a more efficient
phosphinic deprotection (Figure 2). Taken together,
these results suggest than an important proportion of
the prodrug is probably rapidly eliminated following
removal of the benzyl ester group, whereas the remain-
ing unchanged prodrug could cross the blood brain
barrier before its slow conversion in brain structures.
Due to the better activity of iv administered prodrugs
issued from inhibitor B, only compounds 10d and 10e
were tested after ip administration, at the same dose
of 50 mg/kg in order to achieve a direct comparison of
their relative efficiencies. Interestingly, both compounds
showed antinociceptive responses not very different
from those obtained after iv administration as il-
lustrated in Figures 4 and 5. This suggests that the two
phosphinic prodrugs are only slowly deprotected before
their entrance in circulation.
In conclusion, iv and ip administration of prodrugs
derived from phosphinic dual inhibitors of NEP and
APN show interesting pharmacological properties. In-
deed, even if their antinociceptive activity is lower than
that observed with RB 101, the best compounds induced
at least 40% analgesia in the hot plate test, considered
as a highly predictive model of severe pain alleviation
in humans. Nevertheless, the most interesting result
is the time course of the pharmacological response which
is much more favorable than that of RB 101 as il-
lustrated by the ceiling antinociceptive effect and the
slow disappearance of this response.
F igu r e 6. Comparison of the antinociceptive responses
induced by iv administration of 10 mg/kg (15 µmol/kg) of RB
101 (2) and 25 mg/kg (30 µmol/kg) of compound 10b (O) in
the hot plate test (52 °C) in mice (n ) 10). RB 101, AUC )
970 (0-30 min); compound 10b, AUC ) 1760 (20-120 min).
Results are expressed as percentage of analgesia ( SEM.
*P < 0.05, **P < 0.01 as compared to control (Dunnett’s t test).
of their antinociceptive properties. For this purpose, the
carboxylate and phosphinate functions of the dual
inhibitors were substituted with hydrophobic groups.
We have previously shown that introduction of a benzyl
ester on the C-terminal carboxyl group of inhibitors
increase the bioavailability but delays the antinocicep-
tive effects of the phosphinic inhibitors.18
Three different protecting groups have been intro-
duced on the phosphinic function: a benzyl group as
described for NEP/ECE phosphonate inhibitors,22 S-acyl-
2-thioethyl (SATE) derivatives developed for the protec-
tion of the phosphate group of nucleotides,23 and acyl-
oxyalkyl esters previously used in the ACE inhibitor
fosinoprilat for the protection of the phosphinic moiety24
(Scheme 1).
Prodrugs 9a -c and 10a -d iv administration induced
intermediate antinociceptive responses, in the hot plate
test, a typical animal model of severe pain, with a
relatively low intensity (20 to 40% analgesia), but with
a long duration of action since some of these compounds
were still active 2 h after administration.
The results obtained in this study confirm, once more,
that complete protection of endogenous enkephalins
could induce antinociceptive responses needed for the
treatment of all types of pain which cannot be treated
with NSAIDs. In addition, as widely demonstrated with
RB 101, dual inhibitors are devoid of the major draw-
backs of opiates including constipation and respiratory
depression.26 Accordingly, it could be now interesting
to investigate the preclinical properties of these com-
pounds and to search for an appropriate form of
administration in humans.
In the two series, the most efficient prodrug contains
an acetylthioethyl group (9c and 10b), the latter being
more active. This is the reverse in the case of the
precursor 1 of prodrugs 9a -c which were found more
efficient after icv administration19 than the precursor
2 of prodrugs 10a -d , in agreement with the better
inhibitory potencies of the latter. This illustrates the
importance of the whole lipophilic content of prodrugs
on their pharmacokinetics properties (Scheme 1).
Due to the very different time course of nociceptive
responses observed after iv administration of prodrugs
issued from dual phosphinic inhibitors and from those
obtained with RB 101,16 a comparison of their analgesic
profile was performed. This was achieved by measuring
the area under the curve (AUC) (Figure 6), which
reflects the relationship between the time course and
the percentage of analgesia induced by one dose of RB
101 (10 mg/kg ) 15 µmol/kg) and a 2-fold higher dose
of 10b (25 mg/kg ) 30 µmol/kg). The AUC for 10b is
twice as high as that obtained with RB 101 (Figure 6).
This result indicates that both compounds have a quite
similar in vivo potency, which could be related to a
complete protection of endogenous enkephalins.25 How-
ever, the different time courses of RB 101 and 10b
Exp er im en ta l Section
I. Ch em istr y. Natural amino acid derivatives were pur-
chased from Bachem (Bubbendorf, Switzerland). Reagents
were from Aldrich-chimie (Strasbourg, France). Solvents were
from SDS (Peypin, France). TLC were revealed with UV, iodine
vapor, or ninhydrin. The purity of the final compounds was
checked by HPLC on a reverse phase kromasil C8 (5 µm, 100
Å) column (4.6 × 250 mm) with 0.05% TFA in H2O (solvent
A)/CH3CN (solvent B), as the mobile phase, in isocratic
conditions at a flow rate of 1 mL/min on a Shimadzu apparatus
(detector SPD 6AV, pumps LC9A, recorder CR6A). Eluted
peaks were monitored at 210 nm.
1
The structure of all compounds was confirmed by H NMR
spectroscopy (Bru¨ker AC 270 MHz) in DMSO-d6 using HMDS
as an internal reference (values in δ, ppm). Melting points of
the compounds were determined on an Electrothermal ap-

3528 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 21
Chen et al.
paratus (Buˆchi Melting Point B-450) and are reported uncor-
rected. Mass spectra were performed at the Pierre & Marie
Curie University (CNRS EP 103, Paris, France), on a Esquier-
Bru¨ker spectrometer using the electrospray ionization (ESI)
technique. Satisfactory analysis were obtained (C, H, N) for
all final compounds.
Abbr evia tion s: AcOH, acetic acid; Bip, biphenyl; BOP, 1H-
benzotriazol-1-yloxytri(dimethylamino)phosphonium hexafluo-
rophosphate; BSA, N,O-bistrimethylsilylacetamide; Chex, cyclo-
hexane; DIEA, diisopropylethylamine; DMF, dimethylform-
amide; DMSO, dimethyl sulfoxide; EDCI, 1-ethyl-3-(3-dimeth-
ylaminopropyl)carbodiimide methiodide; EtOAc, ethyl acetate;
Et2O, ethyl ether; Hex, hexane; HMSD, hexamethyldisiloxane;
HOBT, 1-hydroxybenzotriazole; MeOH, methanol; SATE, S-acyl-
2-thioethyl; TFA, trifluoroacetic acid; THF, tetrahydrofuran;
Et3N, triethylamine.
Gen er a l P r oced u r e for th e Syn th esis of (2S)-2-Bip h en -
ylm eth yl-3-{h yd r oxy[(1′R)1′-(N-ter t-bu toxyca r bon yla m i-
n o)-a lk yl]p h osp h in yl}p r op a n oic Acid 5 a n d 6. A solution
of ethyl (2R,S)2-biphenylmethyl-3-{hydroxy[1′-(N-benzyloxy-
carbonylamino)alkyl]phosphinyl}propanoate 3 or 4 (1 mM) in
HBr (33%) was stirred for 30 min at room temperature. After
evaporation of the solvent, the bromhydrate of ethyl (2R,S)2-
biphenylmethyl-3-{hydroxy[1′-aminoalkyl]phosphinyl}propan-
oate was obtained without purification.
To a solution of ethyl (2R,S)2-biphenylmethyl-3-{hydroxy-
[1′-aminoalkyl]phosphinyl}propanoate (1 equiv) in DMF were
added (Boc)2O (1 equiv) and Et3N (4 equiv). The mixture was
stirred for 6-8 h at room temperature. The mixture was taken
off with water and extracted with EtOAc. The organic layer
was washed with water and brine and dried over Na2SO4. After
filtration and evaporation of the solvent, the residue was
purified by chromatography on silica gel using CH2Cl2/MeOH/
AcOH (9:1:0.2) as eluents.
1H, CHR); 4.25 (m, 1H, CH); 5.05 (s, 2H, CH2Ph); 7.0-7.7 (m,
15H, Ar, NH); 8.4 (d, 1H, NH).
Com p ou n d 8: white solid; mp 174-176 °C (40%); HPLC
1
(60% B) 19.9 min; H NMR (DMSO-d6 and TFA): 1.2 (d, 3H,
CH3); 1.25 (s, 9H, tBu); 1.4-2.1 (m, 2H, P-CH2); 2.65 (m, 1H,
CHCO2); 2.9 (m, 2H, CH2Ar); 4.3 (m, 1H, CH); 4.8 (m, 1H,
CHR); 5.05 (s, 2H, CH2Ph); 7.1-7.55 (m, 20H, Ar, NH); 8.4 (d,
1H, NH).
Gen er a l P r oced u r e for th e Syn th esis of (2S)2-Bip h en -
ylm et h yl-3-{a lk yloxy[(1′R)1′-a m in oa lk yl)]p h osp h in yl}-
p r op a n oyl Ala n in e Ben zyl Ester 9 a n d 10. Meth od A: To
a solution of (2S)2-biphenylmethyl-3-{hydroxy[(1′R)1′-(N-tert-
butoxycarbonylamino)alkyl] phosphinyl} propanoyl alanine
benzyl ester 7 or 8 (1 equiv) and ROH (3-5 equiv) in DMF
were added DIEA (3-4 equiv) and BOP (3-4 equiv). Different
alcohols ROH were used, benzyl alcohol or three S-acyl-2-
thioethanols described in Lefebvre et al.22; S-(2-hydroxyethyl)-
thioacetate, S-(2-hydroxyethyl)thiopivaloate, and S-(2-hydroxy-
ethyl)thiobenzoate. The mixture was stirred for 2-3 h at room
temperature, taken off with water, and extracted with EtOAc.
The organic layer was washed with water and brine and dried
over Na2SO4. After filtration and evaporation of the solvent,
the residue was purified by chromatography on silica gel using
Chex/EtOAc (1.5:1) as eluents.
A solution of the previous compound (2-3 mM) in HCO2H
was stirred for 2-4 h at room temperature. After evaporation
of the solvent, the residue was dissolved in ethyl acetate,
washed with 10% NaHCO3 until pH ) 7, washed with water
and brine, and dried over Na2SO4. After filtration and evapo-
ration of the solvent, the residue was purified by chromatog-
raphy on silica gel using CH2Cl2/MeOH (9:1) as eluents.
Then, the pure compounds were dissolved in Et2O, and 1
equiv of CF3COOH or CH3SO3H was added. After the mixture
was stirred for 15 min, the precipitate formed was filtered,
washed with Et2O and a 50/50 mixture of Et2O/Hex, and dried
under vacuo.
Meth od B: This procedure is described in Petrillo, J r., et
al.21 To a solution of 2-biphenylmethyl-3-{hydroxy[(1′R)1′-(N-
tert-butoxycarbonylamino)alkyl]phosphinyl}propanoyl alanine
benzyl ester (1 equiv) and various alkyl halides RX (3-5 equiv)
in CHCl3 were added NaI (1 equiv), (n-Bu)4NHSO4 (0.5 equiv),
and Et3N (3 equiv). The mixture was maintained at reflux for
7-8 h, cooled, washed with water, and dried over Na2SO4.
After filtration and evaporation of the solvent, the residue was
purified by chromatography on silica gel using Chex/EtOAc
(1.5:1) as eluents.
Three alkyl halides RX were used: benzyl bromide and
tert-butylchloroacetate, which were commercially available
(Aldrich, France), and isobutylchlorobenzoate which was pre-
pared from 2-methylpropionaldehyde and benzoyl chloride
(Aldrich, France) as described in ref 27.
To a solution of ethyl (2R,S)2-biphenylmethyl-3-[hydroxy-
[1′-(N-tert-butoxycarbonylamino)alkyl]phosphinyl]propano-
ate (1 equiv) in ethanol was added 5 equiv of 1 N NaOH. The
mixture was stirred for 6-8 h at room temperature. After
acidification with 2 N HCl, the ethanolic layers were evapo-
rated, diluted in water, and extracted with EtOAc. The organic
layers were washed with water and brine, dried over Na2SO4,
and evaporated in vacuo.
The crude compounds were dissolved in
a mixture of
methanol and ethyl acetate 90/10, and the pure (2S)2-biphen-
ylmethyl-3-hydroxy[(1′R)1′-N-tert-butoxycarbonylamino)alkyl]-
phosphinyl propanoic acids 5 and 6 crystallized and were
isolated by filtration.
Com p ou n d 5: white solid; mp 182-184 °C (73%); HPLC
1
(60% B) 5.1 min; H NMR (DMSO-d6 and TFA): 1.1 (m, 3H,
CH3â); 1.25 (s, 9H, tBu); 1.6-2.1 (m, 2H, P-CH2); 2.9 (m, 3H,
CH2Ar, CHCO2); 3.68 (m, 1H, CHR); 7.1-7.6 (m, 10H, Ar, NH).
Com p ou n d 6: white solid; mp 201-202 °C (70%); HPLC
The compounds obtained were then deprotected as described
in method A.
1
(60% B) 6.7 min; H NMR (DMSO-d6 and TFA): 1.35 (s, 9H,
tBu); 1.5-2.1 (m, 2H, P-CH2); 2.85 (m, 3H, CH2Ar, CHCO2);
Com p ou n d 9a (m eth od A or B): white solid; mp 107-
109 °C (56%); HPLC (50% B) 7.9 and 8.7 min; 1H NMR
(DMSO-d6 and TFA): 1.2 (m, 6H, CH3â, CH3); 1.6-2.4 (m, 2H,
P-CH2); 2.6 (m, 1H, CHCO); 2.95 (m, 2H, CH2Ar); 3.5 (m, 1H,
CHR); 4.15-4.3 (m, 1H, CHCO2); 4.95 (d, 2H, POCH2); 5.05
(d, 2H, CH2Ph); 7.15-7.6 (m, 19H, Ar); 8. (s, br, 3H, NH3); 8.
(dd, 1H, NH), MS (ESI) (M + 1)+ m/z ) 599.1. Anal.
(C35H39N2O5P) C, H, N.
Com p ou n d 9b (m eth od B): white solid; mp 104-106 °C
(47%); HPLC (50% B) 7.1 and 8.3 min; 1H NMR (DMSO-d6
and TFA): 0.8 (m, 6H, 2xCH3); 1.1-1.35 (m, 7H, CH3â, CH3,
CH); 1.5-2.0 (m, 2H, P-CH2); 2.55 (m, 1H, CHCO); 2.95 (m,
2H, CH2Ar); 3.45 (m, 1H, CHR); 4.35 (m, 1H, CHCO2); 5.05 (s,
2H, CH2Ph); 6.0-6.2 (m, 1H, OCHO); 7.2-7.6 (m, 14H, Ar);
8.15 (s, br, 3H, NH3); 8.7 (m, 1H, NH), MS (ESI) (M + 1)+
m/z ) 623.1. Anal. (C34H43N2O7P) C, H, N.
4.85 (m, 1H, CHR); 7.1-7.7 (m, 15H, Ar, NH).
Gen er a l P r oced u r e for th e Syn th esis of (2S)2-Bip h en -
ylm eth yl-3-{h yd r oxy[(1′R)1′-(N-ter t-bu toxyca r bon yla m i-
n o)a lk yl]p h osp h in yl}p r op a n oyl Ala n in e Ben zyl Ester 7
a n d 8. To a solution of (2S)2-biphenylmethyl-3-{hydroxy[(1′R)-
1′-(N-tert-butoxycarbonylamino)alkyl] phosphinyl}propanoic
acid (1 equiv) and DIEA (1 equiv) in THF was added a solution
of the chlorhydrate of alanine benzyl ester (1 equiv) and DIEA
(1 equiv) in THF, a solution of HOBT (1 equiv) in CH2Cl2,
EDCI (4 equiv), and DIEA (1 equiv). After being stirred for 30
min at room temperature and evaporation of the solvent, the
mixture was taken off with water and extracted with EtOAc.
The organic layer was washed with water and brine and dried
over Na2SO4. After filtration and evaporation of the solvent,
the residue was purified by chromatography on silica gel using
CH2Cl2/MeOH (9:1) as eluents.
Com p ou n d 9c (m eth od A): white solid; mp 113-114 °C
1
Com p ou n d 7: white solid; mp 138-139 °C (47%); HPLC
(66.4%); HPLC (50% B) 6.4 and 6.9 min; H NMR (DMSO-d6
1
(60% B) 11.2 min; H NMR (DMSO-d6 and TFA): 1.1 (m, 3H,
and TFA): 1.0-1.25 (m, 6H, CH3â); 1.3 (m, 3H, CH3); 1.65-
2.4 (m, 2H, P-CH2); 2.2 (d, 3H, COCH3); 2.6 (m, 1H, CHCO);
2.95 (m, 4H, CH2S, CH2Ar); 3.45 (m, 1H, CHR); 3.95 (m, 2H,
CH3â); 1.25 (d, 3H, CH3); 1.3 (s, 9H, tBu); 2.65-3.05 (m, 2H,
P-CH2); 2.7 (m, 1H, CHCO2); 2.95 (m, 2H, CH2Ar); 3.65 (m,

Enkephalin Degrading Enzyme Inhibitor Prodrugs
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 21 3529
chiral phosphorus atom and gives two peaks in HPLC (tR
POCH2); 4.3 (m, 1H, CHCO2); 5.05 (s, 2H, CH2Ph); 7.15-7.6
(m, 14H, Ar); 8.12 (s, br, 3H, NH3); 8.4 (dd, 1H, NH), MS (ESI)
(M + 1)+ m/z ) 611.4. Anal. (C32H39N2O6SP) C, H, N.
Com p ou n d 10a (m eth od A or B): white solid; mp 102-
104 °C (67%); HPLC (50% B) 12.4 and 13.9 min; 1H NMR
(DMSO-d6 and TFA): 1.15 (m, 3H, CH3); 1.4-2.4 (m, 2H,
P-CH2); 2.5-3.05 (m, 3H, CHCO, CH2Ar); 4.15 (m, 1H,
CHCO2); 4.3-5.0 (m, 2H, POCH2); 4.8 (m, 1H, CHR); 5.05 (d,
2H, CH2Ph); 7.0-7.6 (m, 24H, Ar); 8.5 (d, 1H, NH); 8.8 (s, br,
3H, NH3), MS (ESI) (M + 1)+ m/z ) 661.4. Anal. (C40H41N2O5P)
C, H, N.
Com p ou n d 10b (m eth od A): white solid; mp 106-107 °C
(72%); HPLC (50% B) 10.1 and 11.2 min; 1H NMR (DMSO-d6
and TFA): 1.28 (m, 3H, CH3); 1.35-2.15 (m, 2H, P-CH2); 2.25
(d, 3H, COCH3); 2.6-3.1 (m, 5H, CH2S, CH2Ar, CHCO); 3.3-
4.0 (m, 2H, POCH2); 4.35 (m, 1H, CHCO2); 4.78 (m, 1H, CHR);
5.05 (s, 2H, CH2Ph); 7.05-7.6 (m, 19H, Ar); 8.55 (dd, 1H, NH);
8.85 (s, br, 3H, NH3), MS (ESI) (M + 1)+ m/z ) 673.4. Anal.
(C37H41N2O6SP) C, H, N.
Com p ou n d 10c (m eth od A): white solid; mp 117-118 °C
(63%); HPLC (60% B) 8.0 and 8.9 min; 1H NMR (DMSO-d6
and TFA): 1.05 (d, 9H, tBu); 1.25 (m, 3H, CH3); 1.4-2.3 (m,
2H, P-CH2); 2.6-2.95 (m, 3H, CH2Ar, CHCO); 3.0-4.0 (m, 4H,
POCH2CH2); 4.3 (m, 1H, CHCO2); 4.75 (m, 1H, CHR); 5.05 (s,
2H, CH2Ph); 7.05-7.6 (m, 19H, Ar); 8.55 (dd, 1H, NH); 8.8 (s,
br, 3H, NH3), MS (ESI) (M + 1)+ m/z ) 715.4. Anal.
(C40H47N2O6SP) C, H, N.
)
4.8 and 5.5 min in 50% CH3CN); tR ) 8.3 min (in 35% CH3-
CN) for the deprotected drug 2.18
III. P h a r m a cologica l Assa ys. An im a ls. Male Swiss al-
bino mice (20-22 g) (Charles River, France) were used.
Animals were housed in groups of 50 mice for at least 2 days
before the experiments, and food and water were available ad
libitum. Each animal was used only once. All antinociceptive
measurements were recorded between 9:00 a.m. and 7:00 p.m.
Hot P la te Test. The test was based on that described by
Eddy and Leimbach.28 A glass cylinder (16 cm high, 16 cm
diameter) was used to keep the mouse on the heated surface
of the plate which was kept at a temperature of 52 ( 0.5 °C
using a thermoregulated water circulating pump. The latency
period until the mouse licked its paws or jumped was regis-
tered by a stop watch (cut-off time, 240 s). The data are
expressed in percent of analgesia using the following equa-
tion: percent analgesia (test latency - control latency)/(cut-
off time - control latency) × 100. Statistical analysis was
carried out by ANOVA followed by the Dunnett’s t test.
System ic In jection s. Prodrugs or vehicle were slowly (15
s) iv or ip injected in mice in a volume of 0.1 mL/10 g in mice
as previously described.15,18 Prodrugs were solubilized in a
mixture of cremophore/ethanol/water ) 10/10/80.
Ack n ow led gm en t. We thank C. Dupuis for expert
manuscript drafting and M. Mas-Nieto, F. Beslot, and
N. Raux for excellent technical assistance. This work
was supported by grants from EEC (BMH4 CI98 2267).
Com p ou n d 10d (m eth od A): white solid; mp 113-114 °C
(77%); HPLC (55% B) 10.9 and 11.8 min; 1H NMR (DMSO-d6
and TFA): 1.25 (m, 3H, CH3); 1.4-2.4 (m, 2H, P-CH2); 2.5-
3.1 (m, 3H, CH2Ar, CHCO); 3.0-4.15 (m, 4H, POCH2CH2); 4.35
(m, 1H, CHCO2); 4.8 (m, 1H, CHR); 5.05 (s, 2H, CH2Ph); 7.05-
7.9 (m, 24H, Ar); 8.57 (dd, 1H, NH); 8.85 (s, br, 3H, NH3), MS
(ESI) (M + 1)+ m/z ) 735.4. Anal. (C42H43N2O6SP) C, H, N.
Com p ou n d 10e (m eth od B): white solid; mp 97-99 °C
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(31%); HPLC (60% B) 9.7 and 10.3 min; H NMR (DMSO-d6
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Com p ou n d 10f (m eth od B): white solid; mp 100-101 °C
(49%); HPLC (60% B) 17.4, 21.7, and 22.5 min; 1H NMR
(DMSO-d6 and TFA): 0.85 (m, 6H, 2 × CH3); 1.3 (m, 3H, CH3);
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HCl buffer pH 7.4. The metabolic process was stopped by
addition of 50 µL of 4 M HClO4. The preparation was cooled
at 0 °C for 10 min, and 200 µL of CH3CN was added. After
being stirred vigorously, the mixture was centrifuged for 5 min
at 100000g. Controls were performed in the same conditions
with proteins inactivated by prior addition of 4 M HClO4. The
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