New orally active dual enkephalinase inhibitors (DENKIs) for central and peripheral pain treatment

Article abstract of DOI:10.1021/jm500602h

Protecting enkephalins, endogenous opioid peptides released in response to nociceptive stimuli, is an innovative approach for acute and neuropathic pain alleviation. This is achieved by inhibition of their enzymatic degradation by two membrane-bound Zn-metallopeptidases, neprilysin (NEP, EC 3.4.24.11) and aminopeptidase N (APN, EC 3.4.11.2). Selective and efficient inhibitors of both enzymes, designated enkephalinases, have been designed that markedly increase extracellular concentrations and half-lives of enkephalins, inducing potent antinociceptive effects. Several chemical families of Dual ENKephalinase Inhibitors (DENKIs) have previously been developed but devoid of oral activity. We report here the design and synthesis of new pro-drugs, derived from co-drugs combining a NEP and an APN inhibitor through a disulfide bond with side chains improving oral bioavailability. Their pharmacological properties were assessed in various animal models of pain targeting central and/or peripheral opioid systems. Considering its efficacy in acute and neuropathic pain, one of these new DENKIs, 19-IIIa, was selected for clinical development.

Full text of DOI:10.1021/jm500602h

Article
pubs.acs.org/jmc
New Orally Active Dual Enkephalinase Inhibitors (DENKIs) for Central
and Peripheral Pain Treatment
Herve Poras,† Elisabeth Bonnard,† Emilie Dange,† Marie-Claude Fournie-Zaluski,†
́
́
́
and Bernard P. Roques*,†,§
†Pharmaleads, Paris BioPark, 11 Rue Watt, 75013 Paris, France
§Universite
́
Paris-Descartes, 4 Avenue de l'Observatoire, 75006 Paris, France
S
* Supporting Information
ABSTRACT: Protecting enkephalins, endogenous opioid peptides released
in response to nociceptive stimuli, is an innovative approach for acute and
neuropathic pain alleviation. This is achieved by inhibition of their enzymatic
degradation by two membrane-bound Zn-metallopeptidases, neprilysin (NEP,
EC 3.4.24.11) and aminopeptidase N (APN, EC 3.4.11.2). Selective and
efficient inhibitors of both enzymes, designated enkephalinases, have been
designed that markedly increase extracellular concentrations and half-lives of enkephalins, inducing potent antinociceptive effects.
Several chemical families of Dual ENKephalinase Inhibitors (DENKIs) have previously been developed but devoid of oral
activity. We report here the design and synthesis of new pro-drugs, derived from co-drugs combining a NEP and an APN
inhibitor through a disulfide bond with side chains improving oral bioavailability. Their pharmacological properties were assessed
in various animal models of pain targeting central and/or peripheral opioid systems. Considering its efficacy in acute and
neuropathic pain, one of these new DENKIs, 19-IIIa, was selected for clinical development.
including those which are not physiologically involved in pain
control but are accountable for MO side effects.11,12
INTRODUCTION
■
In spite of the constant quest for new analgesics devoid of the
severe side effects of morphine (tolerance, dependence,
respiratory depression, sedation, constipation, nausea), few
new painkillers have been developed during the last decades.1
This is particularly true for neuropathic and inflammatory pain,
which are not efficiently treated by morphine or synthetic
opiates.2 However, in the 1970s, the discovery that morphine and
its surrogates bind to specific targets designated opioid
receptors3 and physiologically activated by two peptides, Met-
and Leu-enkephalins (Tyr-Gly-Gly-Phe-Met and Tyr-Gly-Gly-
Phe-Leu),4 has led to a new approach for pain alleviation. These
opioid peptides, expressed as preproenkephalin, are processed
within specific neurons and released through a Ca2+-dependent
mechanism, to interact with two G protein-coupled receptors,
the μ and δ-opioid receptors, to act as physiological regulators of
pain.5 The affinity of enkephalins (ENKs) for μ receptors is
similar to that of morphine (MO) and is 10-fold higher for δ
receptors.6
Consistently, the intracerebroventricular administration of
enkephalins in rodents in animal models of acute pain induced
antinociceptive responses7 similar to morphine. However, this
effect was transient due to the rapid enzymatic degradation of
ENKs.7,8 Consequently, enzyme-resistant ENKs, such as (D-Ala2-
N-Me-Phe4-Met(O)5-OL)enkephalin (FK33−8249), have been
synthesized and were shown to induce potent antinociceptive
responses in clinical trials while keeping all unwanted effects of
morphine.10 This was a consequence of the exogenous
administration of these compounds (MO or modified ENKs)
which interact ubiquitously with all opioid receptors in the body,
We have therefore proposed a novel physiological approach
for pain alleviation based on endogenous ENKs protection from
their enzymatic degradation.13 In vivo, enkephalins are
inactivated by two membrane-bound Zn-metallopeptidases,
neprilysin (NEP, EC 3.4.24.11), which cleaves the Gly3-Phe4
bond, and aminopeptidase N (APN, EC 3.4.11.2), which releases
the N-terminal Tyr1 of these pentapeptides.13
The inhibition of neprilysin by thiorphan,14 or of APN by
bestatin,15 was insufficient for completely protect ENKs16,17 and
therefore unable to show antihyperalgesic or analgesic efficient
effects in rodents18 and in humans,19 respectively. The
combination of both inhibitors was more effective20 in humans,
but dual inhibitors (i.e., one molecule inhibiting both enzymes)
were found more appropriate as shown with the first described
Dual ENKephalinase Inhibitor (DENKI)17 essentially for
pharmacokinetic reasons.13,17,18,21
Accordingly, selective and potent inhibition of both ENK-
metabolizing peptidases largely increases extracellular concen-
trations and half-life of ENKs released in response to a noxious
stimulus.16 This occurs at the three levels of pain control
(peripheral, spinal, and central) without any modification in
ENK secretion, as demonstrated by brain microdialysis and
spinal superfusion experiments in rats16,22,23 without any
saturation of opioid receptors, taken as one of the responsible
effects of exogenous opioids such as MO, as shown by binding
experiments.24
Received: April 17, 2014
Published: June 13, 2014
© 2014 American Chemical Society
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Figure 1. Representation of 1 (RB101) and 2 (RB120) in the subsites of neprilysin (NEP) and aminopeptidase N (APN) (from the nomenclature of
Schechter and Berger43).
a
Scheme 1. Synthesis of APN Inhibitor
a
Reagents and conditions: (a) (i) NMM, iBuOCOCl, DME, (ii) NaBH4, THF; (b) CHCl3, Et3N, CH3SO2Cl; (c) CH3COSK, DMF; (d) NaOH 1N,
MeOH; (e) TFA, CH2Cl2.
a
Scheme 2. Synthesis of NEP Inhibitor
a
Reagents and conditions: (a) CH3COSH, CH2Cl2, overnight reflux; (b) EDCI, HOBt, DIEA, CH2Cl2; (c) semipreparative HPLC; (d) NaOH 1N,
MeOH; (e) isobutylene, H2SO4 cat., tBuOH or (tBu)2CHNMe2, toluene, 80 °C; (f) LDA, −78 °C, paraformaldehyde; (g) MsCl, Et2O, Et3N; (h)
CH3COSK, DMF, 50 °C; (i) TFA, CH2Cl2, RT; (j) EDCI, HOBt, DIEA, CH2Cl2; (k) NaOH 3N, MeOH.
Different chemical families of NEP/APN inhibitors (abbre-
viated in DENKIs)17,18,25−27 were developed in order to improve
their efficacy and druggability. Their efficacy in reducing
nociception17 was unambiguously demonstrated in various
rodent pain models.18,25 It was also shown in vivo that
enkephalins, when fully protected from catabolism by DENKIs,
have an intrinsic efficacy higher than that of morphine, which is
actually a partial agonist.28 Among DENKIs, benzyl N-(3-{[(2S)-
2-amino-4-(methylthio)butyl]dithio}-2-benzylpropanoyl)-L-
phenylalaninate 118 and N-((S)-2-benzyl-3[(S)-2-amino-4-
methylthio)butyldithio-]-1-oxopropyl)-L-alanine benzyl ester
226 (Figure 1), two benzyl ester pro-drugs derived from co-
drugs formed by the combination, through a disulfide bridge, of
an APN and a NEP inhibitor, have been extensively
investigated.18,25,26 The disulfide bond was shown to be reduced
in vivo, after administration of the pro-drug, releasing the two
Zn-metallopeptidase inhibitors endowed with nanomolar
inhibitory potency for their target-peptidase.25
Accounting for its high hydrophobicity, 1 was solubilized in
lipophilic solvent and most of the pharmacological tests were
performed by iv route, where it exhibited high antinociceptive
effects, but this vehicle was not suitable for human use.18,25 The
lack of oral pharmacological activity of 1, solubilized in lipophilic
solvent, is probably related to its precipitation in gut after oral
administration.18 2 had an improved bioavailability and was
slightly active orally but only at very high doses.26
The endogenous enkephalinergic system (made up of μ and δ
receptors, enkephalins, and their degrading enzymes) is not only
present in the medulla and in the brain but also at the peripheral
level29 where noxious stimuli are generated.30 It was therefore
relevant to develop compounds able to target pain through the
peripheral components of the endogenous enkephalinergic
system to avoid central side effects.31,32
The present paper shows how these new disulfide co-drugs
were designed, aiming at improving their oral bioavailability, and
reports the pharmacokinetic properties, making them efficacious
and safe painkillers suitable for oral administration in humans.
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a
Scheme 3. Synthesis of Co-drugs and Pro-drugs 19
a
Reagents and conditions: (a) CHCl3, Et3N, RT; (b) EDCI, HOBt, MeOH, DIEA, CH2Cl2; (c) (1R,2S)-ephedrine, Et2O or α-chymotrypsin, pH
7.8, DMSO; (d) (i) NaOH 1N, MeOH, (ii) Zn, 3N HCl, THF; (e) HX, NH2R (25 or 27), BOP, DIEA, DMF, RT; (f) HCOOH; (g) NaIO4 (0.2 M
water solution), EtOH, 4 °C.
The first step was to design less hydrophobic inhibitors by
keeping their high affinity for their target enzymes. In a second
step, pro-drugs endowed with good solubility in aqueous solvents
were tested for the ability to either cross the gastrointestinal or
the blood−brain barrier in relation with a recruitment of the
peripheral or the central enkephalinergic system. In this paper,
we describe the synthesis of various pro-drugs and their
pharmacological properties in various rodent models of pain.
From these results, 19-IIIa (designated PL37),33 a new highly
efficient DENKI pro-drug, was selected for clinical development.
A complete set of 19-IIIa pharmacological and clinical studies
results will be published elsewhere.
The acetylthioalkanoic acids 9 were obtained as racemic
mixtures by addition of thioacetic acid on various acrylic acids 8,
which were synthesized by alkylation of triethylphosphonoace-
tate36 or by Knoevenagel condensation with diethylmalonate.37
Condensation of these different acids 9 with various α-amino
acids methyl esters using the classical EDCI/HOBt coupling
method led to the corresponding N-(mercaptoacyl)amino
methyl esters 10 (Scheme 2), and the diastereoisomers were
separated by HPLC. The ester and the thioacetate groups were
hydrolyzed, leading to the corresponding inhibitors 11 as
optically pure compounds for the determination of their NEP
inhibitory potency.
For compounds containing a cyclopentyl or a cyclohexyl
moiety in P1′ position, the acetylthioalkanoic acids 14 were
obtained by nucleophilic substitution of the corresponding
alcohols 13, prepared from the commercially available acids 12,
as described by Neustadt et al.38 Condensation of 14 with various
α-amino acid methyl esters using the classical EDCI/HOBt
coupling method followed by saponification led to the NEP
inhibitors 15 (Scheme 2).
Synthesis of the Co-drugs: Formation of the Disulfide Bond.
The dissymmetric disulfides 17 were obtained by activation of
the Boc-β-aminothiols 6 with methoxycarbonylsulfenyl chloride,
prepared in situ from chlorocarbonylsulfenyl chloride and
MeOH,39 then this intermediate was coupled with racemic or
optically pure thioalkanoic acids 16, issued from 9 by resolution
RESULTS
■
Chemistry. Synthesis of APN Inhibitor Moieties. The
synthesis of the β-aminothiols was performed as previously
described from their corresponding Boc α-amino acids34 leading
to optically pure APN inhibitors (Scheme 1).
Synthesis of NEP Inhibitor Moieties. For industrial develop-
ment, it could be interesting to remove the chiral center in P1′
position by inhibitors with a cyclopentyl or a cyclohexyl moiety
in P1′ position which were reported to be NEP inhibitors.35
The preparation of the pseudodipeptides NEP inhibitors
included two main steps: (i) the synthesis of the different
acetylthioalkanoic acids 9 or 14 and (ii) the coupling of several α-
amino acids leading to compounds 11 and 15, respectively
(Schemes 2 and 3).
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Table 1. Inhibitory Potency (Ki) of Thiols 11 and 15 on NEP
a
See ref 14 for literature.
with chiral amines25,40 such as (1R,2S) ephedrine or by
enzymatic process (α-chymotrypsin)41 (Scheme 3).
group (Table 1B) in P1′ and various residues in P2′ position were
synthesized.
Synthesis of the Pro-drugs. Several esters pro-drugs 19 were
synthesized by coupling dissymmetric disulfide 17 with several
hydrophilic α-amino acid alkyl esters 25 or 27, including 1-
(ethoxycarbonyloxy) groups obtained from a procedure
described in the literature42 (see Supporting Information for
experimental procedure for the synthesis of the α-amino acid
alkyl esters (25 or 27) and the table with the corresponding pro-
drugs 19 starting from dissymmetric disulfide racemic or
optically active 17 (Supporting Information Scheme SI-12)).
The introduction of β-methionine sulfoxide as APN moiety in
the compound 18-IVa was achieved by oxidation of β-
methionine. Therefore, compound 19-IVa was obtained by
action of NaIO4 on 18-IIIa in EtOH,34 followed by final
deprotection in HCOOH (Scheme 3).
Inhibitory Potencies of the New NEP and APN
Inhibitors. As these co-drugs are a combination of a NEP and
an APN inhibitor, through a disulfide bond, each moiety was
tested under its free form for its NEP- or APN-inhibiting activity.
For the final selection of the NEP inhibitor moiety, two
parameters were taken into account: (a) the inhibitory potency
versus NEP, and (b) the easiest synthesis including the resolution
step to obtain the inhibitor with a (S) configuration,
corresponding to the most active form.18
NEP Inhibitory Potency. Accounting for the hydrophobic
character of the S1′ subsite (as defined by Schechter and
Berger43) of NEP,44,45 various aromatic and cyclic structures
were introduced in this position and the inhibitory potency of the
obtained molecules was measured on NEP (Table 1). Thiophene
being an usual isostere of benzene,46 3-thienyl methyl was tested
instead of benzyl group, found in the P1′ position of the NEP
inhibitor moiety of 1 or 2.
The S2′ subsite of NEP, which is widely open toward the
external medium,44,45 did not present a clear specificity: small
and bulky chains, hydrophilic and hydrophobic residues were
well-accepted. Consequently, compounds 11a−11e with a
benzyl group (Table 1A) or 11f−11h with a 3-thienyl methyl
Compounds 11a-11e (Table 1A) inhibited NEP with Ki values
in the nanomolar range. For compounds containing a 3-thienyl
methyl group in P1′ position, the best Ki value (2.0 0.5 nM)
was obtained for 11f (Table 1B).
The introduction of a symmetric cyclic moiety without chiral
center in P1′ position led to inhibitors 15 (Scheme 2) and their
inhibitory potencies were evaluated toward NEP (Table 1C).
Compounds 15a−15c, with a cyclopentyl moiety in P1′ position,
had Ki values in the 10−8 M range except when the P2′ residue was
the hydrophilic aspartic acid, compound 15c (Ki = 3.4 0.3 nM).
Compound 15d, with a more flexible cyclohexyl moiety, in P1′
position, was found less efficient than its cyclopentyl analogue
15c.
However, the most potent inhibitor 15c, bearing two
equivalent carboxylic functions, requested the esterification of
both groups leading to a too high molecular weight molecule for
a good bioavailability. Therefore, among the inhibitors reported
in Table 1, compounds 11b, 11d, and 11f were selected for the
synthesis of the co-drugs due to their favorable inhibitory
potency.
APN Inhibitory Potency. In a previous report,34 β-aminothiols
containing various aliphatic or aromatic side chains were tested as
potential APN inhibitors such as 7a and 7b; compounds with
aliphatic side chains have IC50 values around 10−20 nM, and
those with aromatic or bulky side chains display greater IC50
values (30−130 nM). Methioninethiol 7a was the most efficient,
with a Ki value of 11 2 nM. New β-aminothiols, derived from
natural or synthetic α-amino acids, with linear aliphatic side
chains, were prepared and tested toward APN (Table 2).
Interestingly, compound 7c, without a heteroatom in its side
chain, has an inhibitory potency (13 2 nM) close to 7a. The
three compounds derived from homoserine (7d−7e) and serine
(7f) were significantly less active.
Moreover, 7a (Table 2) could be easily transformed in a more
soluble sulfoxide 7b, which could be reduced enzymatically in
vivo.47
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administration, at 20 mg/kg in water/mannitol (50 mg/mL) as
vehicle. The percentage of antinociception was reported in
parallel with the calculated average Log P49,50 (Table 3).
These esters (except 19-Ig) elicited significant antinociceptive
effects 10 min after injection.
Table 2. Inhibitory Potency (Ki) of Aminothiols 7a−7f on
APN
Two compounds emerged with an efficacy of 40% analgesia,
19-Ic and 19-Ie, and their average Log P (1.72 and 0.96,
respectively) correspond to a good balance between solubility
and permeability.49,50 The most hydrophilic and consequently
more soluble in aqueous solvent, compounds 19-Ib, 19-If, and
19-Ig (average Log P < 0.4), were significantly less active.
However, due to the complex synthesis of the triglyceride 19-Ie,
the sole acyloxyethyl ester of 19-Ic was selected for the synthesis
of pro-drugs in the benzyl series.
19-IIa and 19-IIIa were tested at different doses after iv
administration (Table 4). At 40 mg/kg, threonine-containing
derivative 19-IIa induced significant responses (61% analgesia),
but glycine-containing derivative 19-IIIa, which produced 35%
analgesia at 10 mg/kg and 100% analgesia at 40 mg/kg, was the
most interesting pro-drug in this series.
The responses to 19-IIIa after iv administration were then
evaluated in various vehicles. In water/mannitol, this compound
induced dose-dependent antinociceptive effects (ED50 = 16 mg/
kg) (Figure 3A) with a relatively short time-course (less than 40
min), measured at the dose of 40 mg/kg (Figure 3B). In EtOH/
Tween80/water (1/1/8), strong dose-dependent antinocicep-
tive effects were obtained with low doses of 19-IIIa (ED50 = 4.5
mg/kg) (Figure 3C). The full reversion of 19-IIIa-induced
antinociception by prior subcutaneous injection of naloxone (0.5
mg/kg) (Nlxe) supports the selective involvement of opioid
receptors (Figure 3D).
Then, antinociceptive responses of 19-IIIa and its sulfoxide
analogue 19-IVa were compared at 10 mg/kg in EtOH/
Tween80/water (1/1/8) after iv administration (Figure 4A). A
significant lower efficacy of 19-IVa (30% analgesia) compared to
19-IIIa (78% analgesia) was observed 10 min after admin-
istration.
Accounting for the marked efficacy of 19-IIIa by iv route, this
compound and other acyloxylalkyl ester pro-drugs (19-IIIb−19-
IIIe) were studied after oral administration (150 mg/kg in
EtOH/PEG400/H2O 10/40/50 as vehicle), in HPT in mice
a
diS homodimer reduction was performed with DTT present in the
b
APN buffer. See ref 34 for literature.
According to these in vitro results, new DENKIs pro-drugs
were selected among compounds containing, for APN inhibition,
a methioninethiol or its sulfoxide and for NEP inhibition, a
pseudodipeptide, made of either benzyl or 3-thienyl methyl as P1′
components and α-amino acid (Gly, Ala, or Thr) for S2′
recognition.
On the basis of these criteria, four co-drugs (19-I to 19-IV,
Figure 2) and their pro-drugs, obtained after introduction of
hydrophilic esters, were tested for their antinociceptive proper-
ties (Tables 3−5).
Pharmacological Studies. The Hot Plate Test (HPT) in
Mice, A Model of Acute Central Pain.48 Consistent with their
charged forms well-known to inhibit membrane crossing, the
selected co-drugs 19-I−19-IV (Figure 2) were found inactive in
the hot plate test at a single dose after either iv (10 mg/kg) or oral
administration (150 mg/kg) (data not shown), highlighting the
necessity of the ester pro-drugs to cross the gastrointestinal or
the BBB barrier.
To evaluate the interest of relatively hydrophilic esters in
improving bioavailability of dual inhibitors, a preliminary study
was performed with a series of pro-drugs derived from the co-
drug 19-I, used as a diastereoisomeric mixture (Table 3). These
compounds (19-Ia−19-Ig) were tested in the HPT after iv
Figure 2. 19-I−19-IV co-drug structures.
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Table 3. Percentage of Analgesia after Intravenous Injection of 19-Ia−19-If Pro-drugs Measured on Hot Plate Test in Mice
a
Hot plate test, male mice. Dose: 20 mg/kg, iv administration, 10 min postinjection. Vehicle: water/mannitol (50 mg/mL). ANOVA + Newman
b
Keuls: ***p < 0.001; **p < 0.01; * p < 0.05. ALOGPs 2.1 Program, Virtual Computational Chemistry Laboratory.
Table 4. Percentage of Analgesia after Intravenous Injection of 19-IIa or 19-IIIa Pro-drugs Measured on Hot Plate Test in Mice
a
Hot plate test, male mice. Dose: iv administration, 10 min postinjection. Vehicle: water/mannitol (50 mg/mL). ANOVA + Newman Keuls: *** p <
b
0.001; **p < 0.01; * p < 0.05. ALOGPs 2.1 Program, Virtual Computational Chemistry Laboratory.
(Table 5). The percentage of antinociception, measured 20, 40,
and 60 min after gavage, showed that 19-IIIa remained the most
active pro-drug of this series. The dose−response curve (Figure
4B), established 20 min after administration, gave an ED50 of 133
mg/kg. The duration of action of all these compounds remained
relatively short (less than 1 h).
Tail-Flick Test in Rat (TFT), A Model of Acute Centrospinal
Nociception.51 As shown in Figure 3E, 19-IIIa produced a
significant dose-dependent antinociceptive effects 10 and 20 min
after iv administration at doses of 2.5, 5, 10, and 20 mg/kg in
EtOH/Tween80/H2O (1/1/8). The maximum effect (40%
analgesia) was obtained 10 min after injection of 20 mg/kg. At
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Figure 3. Antinociceptive effects of compound 19-IIIa by intravenous route. (A) Dose−responses of 19-IIIa obtained in a vehicle (mannitol, 50 mg/mL
in water) 10 min after iv injection in the hot plate test (jump latency) in mice, n = 10 per group. (B) Duration of action of 19-IIIa injected by iv route, 40
mg/kg, in a vehicle (mannitol, 50 mg/mL in water), in the hot plate test (jump latency) in mice, n = 9−10 per group. (C) Dose−responses of 19-IIIa
were obtained in ethanol/Tween80/water (1/1/8), 10 min after iv injection in the hot plate test (jump latency) in mice, n = 18−21 per group. (D)
Inhibition of antinociceptive responses induced by 19-IIIa (10 mg/kg, iv) by prior injection of naloxone (0.5 mg/kg, sc) in the hot plate test in mice, n =
7−10 per group. Results are expressed as mean % MPE SEM, or mean jump latency (s) SEM. ★p < 0.05, ★★p < 0.01, ★★★p < 0.001 versus
vehicle, ###p < 0.001 versus drug, ANOVA followed by Bonferroni test. (E) Tail flick in rat. Antinociceptive effects of 19-IIIa injected by iv route at
different doses in the tail-flick test in rat, n = 8−9 per group. Results are expressed as mean % MPE sem. ★p < 0.05, ★★p < 0.01, ★★★p < 0.001
versus vehicle, ###p < 0.001 versus drug, ANOVA followed by Bonferroni test.
this dose, the antinociceptive effects remained significant until 40
min after administration.
pain.54−56 As shown in Figure 6A (left panel), PSNL induced a
dramatic decrease in the threshold for paw withdrawal during
von Frey filament stimulation, significant when compared to the
noninjured contralateral side (Figure 6A, right panel). Oral
administration of 19-IIIa, 25 mg/kg, significantly increased
mechanical threshold values by 51% at 20 min. Likewise, 19-IIIa
(25 mg/kg, po) induced a moderate antihyperalgesic effect
(increase of thermal threshold values to half of those obtained on
contralateral side) (Figure 6B, left panel). These results support
the antiallodynic and antihyperalgesic effects of 19-IIIa at low
oral doses in animal model of neuropathic pain.
Kinetics of Pro-drug Bioactivation. In Vitro Bioactiva-
tion. Compound 19-IIIa was incubated at a final concentration
of 800 μM in rat plasma (66 mg protein/mL) at 37 °C and the
proportion of intact pro-drug and major metabolites, measured
in function of time, was quantified by LC/MS analysis (Figure 7).
After 5 min, all the pro-drug 19-IIIa had disappeared,
transformed into the co-drug 19-III (100%), which decreased
slowly (80% after 1 h) without appearance of new metabolite.
This latter effect was likely due to plasma protein binding.
In Vivo Bioactivation. Compound 19-IIIa (200 mg) was
administered orally to male rats. Iterative blood samples (0.3
At the oral dose of 100 mg/kg, 19-IIIa was poorly active (10%
analgesia 20 min after administration, data not shown).
Antinociceptive Responses in the Formalin Test, A Model of
Persistent Pain.52 Formalin solution injected subcutaneously
into the plantar hindpaw of mice induces a tissue injury
associated with persistent pain and inducing licking of the
affected paw. Compound 19-IIIa and its sulfoxide analogue, 19-
IVa, were compared in the early phase (0−5 min after formalin
injection) of the test at 50 mg/kg po in EtOH/methyl cellulose
0.5% in water (1.5/98.5) as vehicle (Figure 5). At 30 min after
drug administration, 19-IVa was slightly, but not significantly,
more active than 19-IIIa (54% vs 41% analgesia) in reducing
licking. Interestingly, both compounds remained equipotent at
90 min (41% analgesia).
Model of Neuropathic Pain: Partial Sciatic Nerve Ligation
(PSNL) in Mice. The partial sciatic nerve ligation in mice induces
characteristic symptoms as profound and prolonged mechanical
allodynia and thermal hyperalgesia, characterized respectively by
a lowered paw withdrawal threshold to pressure (von Frey test53)
and thermal stimuli (plantar test52), related to neuropathic
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Figure 4. Antinociceptive effects of 19-IIIa and 19-IVa. (A)
Antinociceptive effects of 19-IIIa and 19-IVa (10 mg/kg) by
intravenous injection in ethanol/Tween80/water (1/1/8) in the hot
plate test in mice, 10 min after injection. Results are expressed as mean %
MPE SEM, n = 8 per group. ★p < 0.05, ★★★p < 0.001 versus
vehicle; ##p < 0.01 versus drug, ANOVA followed by Bonferroni test.
(B) Antinociceptive effects of 19-IIIa after p.o. administration in EtOH/
PEG400/H2O (10/40/50), at different doses in the hot plate test in
OF1 mice, n = 8−9 per group mice. Results are expressed as mean %
MPE sem. ★★p < 0.01, ★★★p < 0.001 versus vehicle, ###p < 0.001
versus drug, ANOVA followed by Bonferroni test.
Figure 5. Antinociceptive effects of oral administration of compounds
19-IIIa and 19-IVa (50 mg/kg) in the formalin test in mice. Formalin
solution was injected sc into the plantar surface of the hindpaw, and
licking behavior (seconds) was recorded during the following 5 min (0−
5 min post injection), corresponding to the early phase of the test.
Compounds 19-IIIa or 19-IVa or vehicle (ethanol/methylcellulose
0.5% in water, 1.5/98.5) were given orally 30 or 90 min before formalin
injection. Results are expressed as mean licking time (s) SEM, n = 5−7
per group. ★★★p < 0.001 versus vehicle, ANOVA followed by
Bonferroni test.
Table 5. Percentage of Analgesia after Oral Administration of 19-IIIa−19-IIIe Pro-drugs Measured on Hot Plate Test in Mice
a
Hot plate test, male mice. Dose: oral administration 150 mg/kg. Vehicle: EtOH/PEG400/H20 (10/40/50). ANOVA + Newman Keuls: *** p <
b)
0.001; **p < 0.01; *p < 0.05. ALOGPs 2.1 Program, Virtual Computational Chemistry Laboratory.
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Figure 6. Antiallodynic and antihyperalgesic effects of 19-IIIa on neuropathic pain in the partial sciatic nerve ligation model in mice. (A) Mechanical
allodynia (von Frey filament) and (B) thermal hyperalgesia (plantar test) induced by partial sciatic nerve ligature in mice were assessed between day 10
and day 20 postsurgery, before (t0) and 20 min after po administration of 19-IIIa (25 mg/kg) or vehicle (ethanol/methylcellulose 0.5% in water, 1.5/
98.5), on both ipsilateral (left panel) and contralateral (right panel) sides and on sham-operated mice. Results are expressed as mean von Frey pressure
(g) sem (allodynia) and mean paw withdrawal latency (s) sem (hyperalgesia), n = 6−9 per group. ★★p < 0.01, ★★★p < 0.001, versus vehicle, #p <
0.05, # #p < 0.01, # # #p < 0.001 versus t0, ANOVA + Bonferroni.
For the NEP moiety, at 30 min in rat plasma, the main metabolite
corresponds to the S-methylation of thiorphan (d).
DISCUSSION
■
The aim of this study was to develop new druggable analgesics
based on the now well-accepted concept of dual enkephalinase
inhibition.12,57,58 However, the poor solubility of early DENKIs
(Figure 1) in aqueous medium and their mediocre bioavailability
have precluded the possibility to develop them.
To overcome this problem, improvements in the chemical
structures of the co-drug and pro-drug moieties were performed.
The oral bioavailability of each compound was estimated by
calculating their average Log P, which is optimal between 1 and
2.49,50
Figure 7. Incubation in vitro of 19-IIIa (800 μM) in male rat plasma (66
mg protein/mL) at 37 °C.
As a first step, we attempted to decrease the lipophilicity of the
NEP thiol inhibitor HS-CH2CH(CH2Ph)CONHCH(CH3)-
CO2H (11a) present in 2. The substitution of the benzyl
group by its more hydrophilic isostere 3-thienyl methyl group in
11f (Table 1) yielded an interesting inhibitor. However, the
difficulty to obtain an optically pure intermediate (S)-9a in large
amounts precluded its use for further development and forced us
to test pro-drugs from 19-I series as a diastereoisomeric mixture
(for details see Supporting Information). Another possibility to
decrease the hydrophobicity of the molecule without loss of
mL) were collected during 8 h after administration. Plasma
samples were centrifuged at 8 °C for analysis by LC/MS/MS.
After 30 min gavage, the pro-drug 19-IIIa disappeared as well as
98% of the co-drug 19-III in favor of the different metabolites
from the two moieties formed by disulfide bridge cleavage
(methioninethiol 7a (20%) and thiorphan 11b (28%), which
were mainly transformed into their S-Me forms (c (20%) and d
(48%), respectively) and various oxidized derivatives such as
sulfoxide from the S-Me derivatives of the latter two (Figure 8).
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Figure 8. Metabolism of 19-IIIa: percentage of metabolites from each moiety, measured in male rat plasma, 30 min after po administration of 19-IIIa.
potency was to change the C-terminal amino acid by substituting
the alanine by a glycyl residue, like in thiorphan.14
were unable to cross the blood−brain and/or the intestinal
barrier.
Therefore, various other pro-drugs were synthesized.
Accounting for the bis-ionic form of the co-drugs, transient
protection of the C-terminal carboxylic or N-terminal groups
could be considered. In this study, the carboxylate protection was
selected. Therefore, esterification of co-drugs with alcohols,
known for their capacity to improve drugs bioavailability, such as
diacetoxy-propyl alcohol, carbethoxyalkyl alcohol, or ethoxycar-
bonyloxyalkyl alcohol,59−61 was performed.
Co-drug 19-I esters were prepared and evaluated for their
efficacy in the HPT. Compounds 19-Ic and 19-Ie were found the
most effective (Table 3). However, due to the intrinsic chemical
instability62 of the triglyceride found in 19-Ie, this compound was
not developed.
The ethoxycarbonyloxyethyl alcohol, present in 19-Ic, was
therefore selected and introduced in co-drugs 19-II, 19-III, and
19-IV, leading to 19-IIa, 19-IIIa (Table 4), and 19-IVa (Figure
4A), respectively. Tested, by iv, in water/mannitol, on the HPT,
19-IIIa was significantly more active than 19-IIa (Table 4) and
19-IVa (Figure 4A), likely in relation with a better brain
penetration due to a higher hydrophobicity.
As a second step, with the aim of removing any chiral center
present in the NEP inhibiting moiety, mercaptomethyl-cyclo-
pentanoyl and -cyclohexanoyl synthons were tested, instead of
the mercaptomethylbenzoyl group. Because of its planar
structure, the cyclopentyl group was relatively well accepted35,38
(see Table 1C) as compared to the cyclohexyl moiety.
Nevertheless, a nanomolar inhibitory potency on NEP was
obtained only with a cyclopentyl group combined with an
aspartic acid as P2′ component (compound 15c) optimizing the
recognition of Arg-102 and Arg-104, present in human NEP
active site S′2 subsite.13,44,45 However, the presence of two
carboxylate groups requires a dual esterification with cascade
ester, of which synthesis is difficult and would lead to a molecule
with a too high molecular weight.
Hydrophobic and linear β-aminothiol side-chains have been
shown to give the best APN inhibition.34 None of the tested
modifications, such as substitution of the sulfur atom by an
oxygen or a CH2 group, increased the inhibitory potency as
compared to the methionine side chain (Table 2).
Four series of co-drugs (Figure 2) were selected and tested for
their antinociceptive properties. As expected, they did not induce
any significant response, showing that, without ester moiety, they
For iv administration of 19-IIIa, another vehicle (ethanol/
Tween 80/H2O: 1/1/8) well-known to facilitate the blood−
brain barrier crossing,63 was used (Figure 3C). As expected, the
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lowest value (ED50 = 4.5 mg/kg) in the HPT was obtained using
this vehicle. The complete reversal of the antinociceptive effects
by naloxone, an antagonist of opioid receptors, demonstrated
that the responses were mediated by the opioidergic system at
the brain and/or spinal levels (Figure 3D).
Considering the results obtained with 19-IIIa on intravenous
administration, various other ethoxycarbonyloxyalkyl esters (19-
IIIc and 19-IIIe) and carbethoxyalkyl esters (19-IIIb and 19-
IIId) were evaluated in the HPT (Table 5) by oral route. The
level of analgesia measured under these conditions reflected the
ability of the molecules to cross both gastrointestinal and blood−
brain barriers. Once more, 19-IIIa, endowed with an average Log
P in the proper range, was found to be the more efficient inhibitor
of the series. In a more severe antinociceptive model, the tail flick
test in rats, 19-IIIa, induced significant effect at relatively low
doses (40% analgesia at 20 mg/kg iv, Figure 3E).
po activity in a central test such as the hot plate assay. Even if the
cleavage of the cascade ester is very rapid, its short lifetime is
sufficient to allow the brain crossing followed by formation of the
two active NEP and APN moieties after cleavage of the disulfide
bridge inside the CNS36 or more likely during the BBB crossing.
By oral route, the duration of action was longer than by iv
administration and the metabolism in vivo was very similar to
that obtain in vitro. The important difference was the rapid
transformation of the two active inhibitor entities, thiorphan and
methionine thiol, by S-methylation. In the case of thiorphan, the
metabolite possesses a non-negligible NEP inhibitory potency
(Ki = 46.3 0.3 nM, d in Figure 8), while the S-methylation of
APN blocker methionine thiol leads to an inactive compound (c
in Figure 8), explaining, at least partly, the relatively short
duration of inhibitory potency of the pro-drug 19-IIIa.
CONCLUSION
Since the efficacy of 2 has been previously measured in these
two tests,26 it was interesting to evaluate the increased
bioavailability induced by the ethoxycarbonyloxyethyl ester
present in 19-IIIa in place of the benzyl ester in 2. Thus,
administered by iv route in a lipophilic vehicle containing 10%
EtOH known to facilitate brain crossing, the two pro-drugs gave
similar responses in HPT: 2 (ED50 = 3 mg/kg) in EtOH/
cremophor/H2O and 19-IIIa (ED50 = 4.5 mg/kg) in EtOH/
Tween80/H2O (Figure 3C). In the tail-flick test, 2 induced 35%
analgesia at 25 mg/kg and 19-III 40% analgesia at 20 mg/kg
(Figure 3E). By contrast, antinociceptive responses observed in
HPT after oral administration, are 4-fold better for 19-IIIa (ED50
= 133 mg/kg) than for 2 (ED50 = 410 mg/kg).
Two other nociceptive tests, which are known to involve the
peripheral opioid system,64 were achieved: the formalin test
which produces persistent pain by tissue injury65 and the partial
ligation of sciatic nerve, which is a currently used predictive
model of neuropathic pain.66
The efficiency of 19-IIIa and its sulfoxide analogue 19-IVa at
the same dose (50 mg/kg, per os) was compared in the formalin
test (Figure 5). No difference, neither in the intensity of the
response nor in the duration of action, was observed between
these two compounds. This observation was different from that
obtained in the hot plate test after iv administration, where 19-
IVa was about two times less efficient than 19-IIIa (Figure 4A). It
could be assumed that 19-IVa was rapidly reduced in 19-IIIa
after oral administration, most probably at the level of the
gastrointestinal barrier.59,60
The chronic pain associated with neuropathic syndromes, such
as allodynia and hyperalgesia, results from a wide variety of
pathophysiological mechanisms and the multiplicity of factors
involved in this pathology.67,68 In this study, we used a model of
neuropathic pain based of the lesion of peripheral nerves,53,56 in
which opiates have been demonstrated to be active,69−71
although associated with severe side effects due to their repetitive
administration. In our experiments with 19-IIIa, antiallodynic
and antihyperalgesic responses were obtained at relatively low
doses: 19-IIIa, given orally at 25 mg/kg, reduced both thermal
hyperalgesia and mechanical allodynia by about 50% (Figure 6).
Accounting for the significant increase in the pharmacological
properties observed by modifying the co-drug 19-III into the
pro-drug 19-IIIa, it was interesting to study in details the
metabolism of this latter in vitro and in vivo to understand its
action in both the central nervous system and the periphery.
After in vitro incubation, of 19-IIIa, in plasma, the release of the
ester was very rapid, but the disulfide bridge was not cleaved
(Figure 7). However, the pro-drug ester was essential for an iv or
■
Taken together, these results indicate that the objective of
obtaining a DENKI, active by oral route on various types of pain
was reached. The DENKI 19-IIIa, is active either on acute
centrally controlled pain or on neuropathic pain, in this latter
case essentially by acting at the peripheral level, as shown by
reversion of the analgesia following administration of methyl-
naloxonium, an opioid antagonist unable to enter the brain,72
which has been previously demonstrated by experiments using
DENKis and methyl-naloxonium.73,74 Furthermore, as expected
from the mechanism of action of DENKIs and previous extensive
pharmacological studies with 1,25−28 none of the unwanted
effects of morphine was observed in any of the animal models of
pain tested (not shown), which was confirmed in phase I human
clinical trials with 19-IIIa (to be published). This demonstrates
that DENKIs have the potential to be novel potent analgesics,
effective on various types of pain when administered via
appropriate routes and vehicles. In neuropathic and inflamma-
tory pain, two severe long-lasting painful diseases,75 these
DENKIs, acting at the source of the nocifensive stimulation by
increasing locally the levels and lifetime of endogenous
enkephalins, are devoid of morphine side effects and represent
a novel promising approach of painkillers.
EXPERIMENTAL SECTION
■
Chemistry. All reagents were obtained from Sigma-Aldrich and
solvents from Carlo Erba-SDS (France). TLC plates were Merck TLC
aluminum sheets coated with silica gel 60 F254. Compounds were
purified by flash chromatography (silica gel Si 60, 40−63 μm) or by
semipreparative HPLC purification. 1H NMR spectra were recorded on
200 MHz Bruker instrument. Chemical shift were reported in ppm with
the solvent as internal standard (CDCl3, 7.26 ppm; DMSO-d6, 2.54
ppm). Data are reported as followed: chemical shift, integration,
multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, br = broad, m
= multiplet). Electron spray mass spectroscopy (ESI) was performed
with a LCMS Quad Electrospray Agilent 6120. RP-HPLC separations
were respectively performed on Shimadzu Prominence HPLC at a flow
rate of 1 mL/min for analytic column (Kromasil C18, 100 Å, 5 μm, 250
mm × 4.6 mm, ACE C18, 100 Å, 5 μm, 250 mm × 4.6 mm, or Atlantis
T3, 100 Å, 3 μm, 100 mm × 4.6 mm) and on Waters prep 600 HPLC at a
flow rate of 10 mL/min on the corresponding semipreparative column
(Kromasil C18, 100 Å, 5 μm, 250 mm × 21.2 mm, ACE C18, 100 Å, 5
μm, 250 mm × 21.2 mm, or Atlantis T3, 100 Å, 3 μm, 100 mm × 21.2
mm). Purity (%) was determined from a surface integral of detected
peaks by reversed-phase HPLC using UV detection (210 nm) and all
compounds showed purities greater than 95%. All commercial reagents
and solvents were used without further purification.
General Procedure for the Synthesis of Dissymmetric
Disulfide 2-Alkyl-3-(((S)-2-(tert-butoxycarbonylamino)-4-
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(methylthio)butyl)disulfanyl)propanoic Acid (17). A mixture of
MeOH (23 mL) and THF (23 mL) was cooled to 4 °C, under inert
atmosphere. Then chlorosulfonyl chloride (1.3 mL, 15.25 mmol, 1.09
equiv) was added dropwise. The reaction mixture was stirred for 15 min
at 4 °C to give the methoxycarbonylsulfenyl chloride. Compound 6
(14.86 mmol, 1.06 equiv) in 16 mL of THF/MeOH was added once.
The reaction mixture was warmed to room temperature and stirred for
30 min. The previous solution was added dropwise to a solution of
compound 16 (14.02 mmol, 1 equiv) in 100 mL of degassed CH2Cl2 in
the presence of Et3N (1 equiv). The reaction mixture was stirred 1 h at
room temperature. The solvent was removed under reduced pressure,
and the product was portioned in CH2Cl2 and 10% citric acid solution.
The organic layer was washed with brine and dried over Na2SO4 to give a
crude product, which was purified on silica gel with CHex/AcOEt 8/2
and then CHex/AcOEt 6/4 as eluent to give compound 17.
CH3CN/H2O (0.1% TFA) 40−60: 9.07 and 10.18 min. ESI(+): [M +
H]+ = 581.2.
1-(2-(1-(2-Methanesulfonylethoxycarbonyl)-ethylcarbamoyl)-3-
thiophen-3-ylpropyldisulfanylmethyl)-3-methylsulfanylpropyl-am-
monium Trifluoroacetate (19-Ib) (R2 = CH2−3-Thiophene, R = CH-
(S)-CH3-COOCH2CH2SO2CH3). Solid (yield 54.7%). 1H NMR (DMSO-
d6 + TFA): δ CH31.12 and 1.28 (3H, d), CH2β 1.72−1.92 (2H, m),
SCH3 1.98 (3H, s), CH2γ 2.52 (2H, m), SCH2 + CH + CH2Ar + SCH2
2.60−2.95 (7H, m), SO2CH3 3.0 (3H, s), CHα + CH2SO2 3.35−3.50
(3H, m), CHα 4.17 (1H, m), CH2O 4.20−4.40 (2H, m), Ar 6.92 (1H,
m), Ar 7.09 (1H, m), Ar 7.38 (1H, m), NH3+ 7.87 (3H, br), NH 8.40
(1H, br). HPLC Kromasil C18, 100 Å, 5 μm, 250 mm × 4.6 mm,
CH3CN/H2O (0.1% TFA) 40−60: 5.0 and 5.35 min. ESI(+): [M + H]+
= 529.2.
1-(2-(1-(1-Ethoxycarbonyloxyethoxycarbonyl))-ethylcarbamoyl)-
3-thiophen-3-yl-propyldisulfanylmethyl)-3-methylsulfanylpropyl-
ammonium Trifluoroacetate (19-Ic) (R2 = CH2−3-Thiophene, R =
Because of the poor resolution of 9a (see Supporting Information),
compound 17a was used as a diastereoisomeric mixture.
No loss of optical resolution was observed during the following steps
of the synthesis.
1
CH-(S)-CH3-COOCHCH3OCO2CH2CH3). Solid (yield 51.4%). H NMR
(DMSO-d6 + TFA): δ CH3CH 1.12 (3H, m), CH3CH2 1.22 (3H, m),
CH3CH 1.35 (3H, m), CH2β 1.72−1.92 (2H, m), SCH3 1.98 (3H, s),
CH2γ 2.52 (2H, m), SCH2 + CH + CH2Ar + SCH2 2.60−2.95 (7H, m),
CHα 3.42 (1H, m), CH3CH2 4.08 (2H, q), CH3CH 4.28 (1H, m),
CHCH3 6.58 (1H, m), Ar 6.95 (1H, m), Ar 7.12 (1H, m), Ar 7.39 (1H,
m), NH3+ 7.92 (3H, br), NH 8.57 (1H, br). HPLC Kromasil C18, 100 Å,
5 μm, 250 mm × 4.6 mm, CH3CN/H2O (0.1% TFA) 50−50: 3.84 and
4.03 min. ESI(+): [M + H]+ = 539.2.
1-(2-(1-Ethoxycarbonylmethyloxycarbonylethyl carbamoyl)-3-
thiophen-3-yl-propyldisulfanyl methyl)-3-methylsulfanylpropyl-am-
monium Trifluoroacetate (19-Id) (R2 = CH2−3-Thiophene, R = CH-
(S)-CH3-COOCH2CO2CH2CH3). Solid (yield 59.8%). 1H NMR (DMSO-
d6 + TFA): δ CH3 1.10−1.30 (6H, m), CH2β 1.80−2.0 (2H, m), SCH3
1.98 (3H, s), SCH2 + CH + CH2Ar + SCH2 + CH2γ 2.60−3.05 (9H, m),
CHα 3.46 (1H, m), CH3CH2 4.08 (2H, m), CH3CH 4.38 (1H, m),
OCH2CO2 4.52 (2H, m), Ar 6.98 (1H, m), Ar 7.12 (1H, m), Ar 7.38
(1H, m), NH3+ 7.92 (3H, br), NH 8.52 (1H, br). HPLC Kromasil C18,
100 Å, 5 μm, 250 mm × 4.6 mm, CH3CN/H2O (0.1% TFA) 50−50:
4.04 and 4.23 min. ESI(+): [M + H]+ = 509.2.
(S)-3-(((S)-2-(tert-Butoxycarbonylamino)-4-(methylthio)butyl)-
disulfanyl)-2-(thiophen-3-ylmethyl)propanoic Acid (17a) (R2 = CH2−
3-Thiophene). White solid (yield 77.0%). 1H NMR (CDCl3): δ tBu 1.46
(9H, s), CH2β 1.72 (1H, m) and 1.98 (1H, m), SCH3 2.12 (3H, s), CH2γ
2.56 (2H, m), SCH2 + CH + CH2Ar + SCH2 2.70−3.20 (7H, m), CHα
3.95 (1H, m), NH 4.73 (1H, m), Ar 6.93 (1H, m), Ar 7.08 (1H, m), Ar
7.30 (1H, m). HPLC Kromasil C18, 100 Å, 5 μm, 250 mm × 4.6 mm,
CH3CN/H2O (0.1% TFA) 70−30: 7.36 min.
(S)-2-Benzyl-3-(((S)-2-(tert-Butoxycarbonylamino)-4-
(methylthio)butyl)disulfanyl)propanoic Acid (17b) (R2 = CH2Ph).
White solid (yield 65.9%); ee 88%. 1H NMR (CDCl3): δ tBu 1.48 (9H,
s), CH2β 1.68 (1H, m) and 1.97 (1H, m), SCH3 2.11 (3H, s), CH2γ 2.56
(2H, m), SCH2 + CH + CH2Ar + SCH2 2.70−3.20 (7H, m), CHα 3.95
(1H, m), NH 4.72 (1H, d), Ar 7.20−7.40 (5H, m). HPLC Kromasil
C18, 100 Å, 5 μm, 250 mm × 4.6 mm, CH3CN/H2O (0.1% TFA) 70−
30: 8.20 min.
General Synthesis of 2-(3-(((S)-2-Amino-4-(methylthio)-
butyl)disulfanyl)-2-alkylpropanamido)-2-alkylacetic Ester (19).
The amino acid ester was coupled using BOP (1.2 equiv) and DIEA (1.2
equiv) as reagents, and final deprotection gave the crude compound 19
The disulfide 17 (0.54 mmol) was solubilized in DMF (4 mL)
(Scheme 3). BOP (1.2 equiv; 1.0 g) was added, followed by DIEA (284
μL) and the corresponding amino acid ester 25 or 27 (1.3 equiv) (see
Supporting Information for the experimental procedure for synthesis of
the appropriate 25 or 27 leading to the corresponding pro-drug 19,
Supporting Information Scheme SI-12). The reaction mixture was
stirred for 20 min at room temperature, and then DMF was removed
under reduced pressure. The crude mixture was portioned in AcOEt,
and the organic layer was washed with 10% citric acid solution, 10%
NaHCO3 solution, and brine and dried over Na2SO4 to give a crude
product, which was purified on silica gel with CHex/AcOEt 8/2 and
then CHex/AcOEt 6/4 as eluent to give corresponding compound 18.
Compound 18 (0.38 mmol) was solubilized in HCOOH (1 mL). The
reaction mixture was stirred for 1 h at room temperature. The excess
HCOOH was removed under reduced pressure, and the reaction
mixture was coeluted with cyclohexane to give crude which was purified
by reverse-phase semipreparative HPLC to give the appropriate pro-
drug 19.
Because of the poor resolution of 9a, 19-I series compounds
(Supporting Information Scheme SI-11) were synthesized starting from
a diastereoisomeric mixture of 17a and tested as a mixture (Table 3).
1-(2-(1-(2, 3-Diacetoxypropoxycarbonyl)-ethylcarbamoyl)-3-thio-
phen-3-ylpropyldisulfanylmethyl)-3-methylsulfanylpropyl-ammo-
nium Trifluoroacetate (19-Ia) (R2 = CH2−3-Thiophene, R = CH-(S)-
CH3-COOCH2CH(OAc)CH2OAc). Solid (yield 48.4%). 1H NMR
(DMSO-d6 + TFA): δ CH31.12 and 1.28 (3H, d), CH2β 1.72−1.92
(2H, m), COCH3 1.92 (6H, s), SCH3 1.98 (3H, s), CH2γ 2.52 (2H, m),
SCH2 + CH + CH2Ar + SCH2 2.60−2.95 (7H, m), CHα 3.41 (1H, m),
CHα + CH2OCO 4.05−4.30 (5H, m), CHOAc 5.12 (1H, m), Ar 6.93
(1H, m), Ar 7.12 (1H, m), Ar 7.38 (1H, m), NH3+ 7.88 (3H, br), NH
8.45 (1H, br). HPLC Kromasil C18, 100 Å, 5 μm, 250 mm × 4.6 mm,
1-(2-(1-(2-Acetoxy-1-acetoxymethylethoxycarbonyl)-ethylcarba-
moyl)-3-thiophen-3-ylpropyldisulfanylmethyl)-3methylsulfanyl-
propyl-ammonium Trifluoroacetate (19-Ie) (R2 = CH2−3-Thiophene,
1
R = CH-(S)-CH3-COOCH(CH2OAc)2). Solid (yield 27.1%). H NMR
(DMSO-d6 + TFA): δ CH3CH 1.15 (3H, m), CH2β 1.70−2.0 (2H, m),
CH3CO 2.0 (6H, s), SCH3 1.90 (3H, s), CH2γ + SCH2 + CH + CH2Ar +
SCH2 2.60−2.95 (9H, m), CHα 3.42 (1H, m), CH3CH + CH2O 4.0−
4.30 (5H, m), CHO 5.12 (1H, m), Ar 6.95 (1H, m), Ar 7.05 (1H, m), Ar
7.25 (1H, m), NH3+ 7.85 (3H, br), NH 8.40 (1H, br). HPLC Kromasil
C18, 100 Å, 5 μm, 250 mm × 4.6 mm, CH3CN/H2O (0.1% TFA) 40−
60: 7.35 and 8.09 min. ESI(+): [M + H]+ = 581.1.
1-(2-(1-(2-Hydroxy-1-hydroxymethylethoxycarbonyl)-ethylcarba-
moyl)-3-thiophen-3-ylpropyldisulfanylmethyl)-3-methylsulfanyl-
propyl-ammonium Trifluoroacetate (19-If), (R2 = CH2−3-Thiophene,
R = CH-(S)-CH3-COOCH(CH2OH)2). Solid (yield 18.7%). 1H NMR
(DMSO-d6 + TFA): δ CH3CH 1.10−1.30 (3H, m), CH2β 1.70−2.0
(2H, m), SCH3 1.95 (3H, s), CH2γ + SCH2 + CH + CH2Ar + SCH2
2.60−3.0 (9H, m), CHα + CH2O (2×) 3.20−3.60 (5H, m), CH3CH
3.90 (1H, m), CHCH2OH 4.28 (1H, m), Ar 6.90 (1H, m), Ar 7.08 (1H,
+
m), Ar 7.35 (1H, m), NH3 7.85 (3H, br), NH 8.40 (1H, br). HPLC
Kromasil C18, 100 Å, 5 μm, 250 mm × 4.6 mm, CH3CN/H2O (0.1%
TFA) 30−70: 5.22 and 6.90 min. ESI(+): [M + H]+ = 497.1.
3-Methylsulfanyl-1-(2-(1-(3,4,5,6-tetrahydroxytetrahydropyran-
2-ylmethoxycarbonyl)-ethylcarbamoyl)-3-thiophen-3-yl-propyldi-
sulfanylmethyl)-propyo-ammonium Trifluoroacetate (19-Ig) (R2 =
CH2−3-Thiophene, R = CH-(S)-CH3-COGlucose). Solid (yield 12.9%).
1H NMR (DMSO-d6 + TFA): δ CH3 1.10−1.30 (3H, m), CH2β 1.75−
1.95 (2H, m), SCH3 1.98 (3H, s), SCH2 + CH + CH2Ar + SCH2 + CH2γ
2.40−3.05 (9H, m), CHα + CHOH 3.10−3.70 (4H, m), CH3CH +
OCH2CHO 3.95−4.40 (5H, m), OCHOH 4.85 (1H, m), Ar 6.94 (1H,
m), Ar 7.12 (1H, m), Ar 7.38 (1H, m), NH3+ 7.90 (3H, br), NH 8.42
(1H, br). HPLC Kromasil C18, 100 Å, 5 μm, 250 mm × 4.6 mm,
CH3CN/H2O (0.1% TFA) 50−50: 2.34 min. ESI(+): [M + H]+ =
585.2.
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1-(2-((1-(1-Ethoxycarbonyloxy-ethoxycarbonyl)-2-hydroxypro-
pylcarbamoyl)-3-phenylpropyldisulfanylmethyl)-3-methylsulfanyl-
propyl-ammonium Trifluoroacetate (19-IIa) (R2 = (S)-CH2−Phenyl, R
= CH-(S)-CH(CH3)OH-COOCHCH3OCO2CH2CH3). Solid (yield 65.2%).
1H NMR (DMSO-d6 + TFA): δ CH3CHOH 1.20 (3H, d), CH3CH2
1.35 (3H, t), CH3CH 1.60 (3H, d), CH2β 2.01−2.18 (2H, m), SCH3
2.19 (3H, s), CH2γ 2.74 (2H, t), SCH2 + CH + CH2Ar + SCH2 2.90−
3.20 (7H, m), CHα 3.76 (1H, m), CH3CH2 + NHCHα + OHCHα
4.10−4.40 (4H, m), CHCH3 6.84 (1H, m), Ar 7.25−7.40 (5H, m),
NH3+ 7.90 (3H, br). HPLC Kromasil C18, 100 Å, 5 μm, 250 mm × 4.6
mm, CH3CN/H2O (0.1% TFA) 45−55: 7.0 min. ESI(+): [M + H]+ =
563.2.
C18, 100 Å, 5 μm, 250 mm × 4.6 mm, CH3CN/H2O (0.1% TFA) 60−
40: 7.72 min. ESI(+): [M + H]+ = 587.2.
Synthesis of 1-(2-(1-Ethoxycarbonyloxy-ethoxycarbonyl-
methyl)-carbamoyl)-3-phenyl-propyldisulfanylmethyl)-3-
methylsulfinylpropyl-ammonium Trifluoroacetate 19-IVa.
Compound 18a was treated by NaIO4 in EtOH34 as described for the
synthesis of 7b. Final deprotection by HCOOH as previously described
led to compound 19-IVa.
1-(2-(1-Ethoxycarbonyloxy-ethoxycarbonylmethyl)-carbamoyl)-
3-phenyl-propyldisulfanylmethyl)-3-methylsulfinylpropyl-ammo-
nium Trifluoroacetate (19-IVa). Solid (yield 64.0%). 1H NMR
(DMSO-d6 + TFA): δ CH3CH2 1.37 (3H, t), CH3CH 1.58 (3H, d),
CH2β 2.07 (2H, m), SOCH3 2.52 (3H, d), CH2γ 2.72 (2H, t), SCH2 +
CH + CH2Ar + SCH2 2.89−3.17 (7H, m), CHα 3.74 (1H, m), NHCH2
3.90−4.10 (2H, m), CH3CH2 4.27 (2H, q), CHCH3 6.84 (1H, m), Ar
7.26−7.40 (5H, m), NH3+ 7.90 (3H, br). HPLC Kromasil C18, 100 Å, 5
μm, 250 mm × 4.6 mm, CH3CN/H2O (0.1% TFA) 30−70: 14.57 min.
ESI(+): [M + H]+ = 535.1.
1-(2-(1-Ethoxycarbonyloxy-ethoxycarbonylmethyl)-carbamoyl)-
3-phenyl-propyldisulfanylmethyl)-3-methylsulfanylpropyl-ammo-
nium Trifluoroacetate (19-IIIa) (R2 = (S)-CH2−Phenyl, R = CH2-
COOCHCH3OCO2CH2CH3). Solid (yield 72.8%); F = 114 °C. IR (KBr):
3298, 2918, 1765, 1651, 1538, 1445, 1375, 1266, 1183, 1078, 983, 932,
20
851, 790, 743, 702, 651, 559, 500 cm−1; [α]D = +0.7° (5% p/v
1
CH3CN/H2O (1/1, v/v)). H NMR (DMSO-d6 + TFA): δ CH3CH2
Biochemistry. Neprilysin (NEP) enzymatic activity was assessed
using Suc-Ala-Ala↓Phe-AMC76 (20 μM, Bachem) and aminopeptidase
N (APN) activity using L-Ala↓β-NA77 (50 μM, Sigma-Aldrich).
Fluorescence was read on a Berthold fluorimeter (λex = 340 nm and
λem = 360 nm for NEP, and λem = 405 nm for APN, lamp energy =
10000). Inhibitory potencies of active inhibitors were determined using
recombinant human (rh) enzymes (50 ng/mL; R&D Systems). After 30
min of preincubation at 37 °C with increasing concentrations of
inhibitor in Tris 50 mM pH 7.4, their corresponding substrates were
added, and the reaction was left to proceed for 30 min at 37 °C. Each
experiment was performed in duplicate, three times independently.
Animals. Experiments were performed in male OF1 mice, 20−32 g
(Charles River Laboratories, France), and male Sprague−Dawley rats,
200−280 g (Janvier, France). Animals were housed for at least 2 days
before experiments in a room with controlled temperature (21 2 °C)
and a 12 h alternating light−dark cycle. Food and water were available at
libitum. Great care was taken to avoid or minimize discomfort of
animals. Animal experiments were carried out with the European
Communities Council directive (89/609/CEE) and in accordance with
the ethical guidelines of the International Association of Pain. The
maximal volume administered was 0.1 mL/100g for intravenous
injection and 0.2 mL/100g orally.
1.37 (3H, t), CH3CH 1.58 (3H, d), CH2β 2.03−2.16 (2H, m), SCH3
2.19 (3H, s), CH2γ 2.72 (2H, t), SCH2 + CH + CH2Ar + SCH2 2.89−
3.17 (7H, m), CHα 3.74 (1H, m), NHCH2 3.90−4.10 (2H, m),
CH3CH2 4.27 (2H, q), CHCH3 6.84 (1H, m), Ar 7.26−7.40 (5H, m),
+
NH3 7.90 (3H, br). 13C NMR (DMSO-d6): δ 14.3, 14.7, 19.6, 19.7,
29.3, 32.2, 37.8, 40.5, 41.9, 47.0, 49.3, 64.5, 91.7, 126.6, 128.6, 129.3,
139.1, 152.7, 168.4, 173.4. HPLC Kromasil C18, 100 Å, 5 μm, 250 mm ×
4.6 mm, CH3CN/H2O (0.1% TFA) 50−50: 5.33 min. ESI(+): [M +
H]+ = 519.1.
1-(2-(1-Ethylcarbonyloxy-isobutoxycarbonylmethyl)-carbamoyl)-
3-phenyl-propyldisulfanylmethyl)-3-methylsulfanylpropyl-ammo-
nium Trifluoroacetate (19-IIIb) (R2 = (S)-CH2−Phenyl, R = CH2-
COOCHCH(CH3)2OCOCH2CH3). Solid (yield 34.0%). 1H NMR
(DMSO-d6 + TFA): δ (CH3)2CH 1.12 (6H, m), CH3CH2 1.20 (3H,
m), CHCH(CH3)2 1.90 (1H, m), CH2β 2.00−2.20 (2H, m), SCH3 2.20
(3H, s), CH3CH2 2.35 (2H, m), CH2γ 2.70 (2H, t), SCH2 + CH +
CH2Ar + SCH2 2.90−3.20 (7H, m), CHα 3.75 (1H, m), NHCH2 3.90−
4.10 (2H, m), CHCH(CH3)2 6.62 (1H, m), Ar 7.26−7.40 (5H, m),
NH3+ 7.90 (3H, br). HPLC Kromasil C18, 100 Å, 5 μm, 250 mm × 4.6
mm, CH3CN/H2O (0.1% TFA) 50−50: 8.82 min. ESI(+): [M + H]+ =
531.1.
1-(2-(1-Ethoxycarbonyloxy-isobutoxycarbonylmethyl)-carbamo-
yl)-3-phenyl-propyldisulfanylmethyl)-3-methylsulfanylpropyl-am-
monium Trifluoroacetate (19-IIIc) (R2 = (S)-CH2−Phenyl, R = CH2-
COOCHCH(CH3)2OCO2CH2CH3). Solid (yield 38.5%). 1H NMR
(DMSO-d6 + TFA): δ (CH3)2CH 1.12 (6H, m), CH3CH2 1.30 (3H,
t), CHCH(CH3)2 1.90 (1H, m), CH2β 2.00−2.20 (2H, m), SCH3 2.20
(3H, s), CH2γ 2.70 (2H, t), SCH2 + CH + CH2Ar + SCH2 2.90−3.20
(7H, m), CHα 3.75 (1H, m), NHCH2 3.90−4.10 (2H, m), CH3CH2
4.30 (2H, q), CHCH(CH3)2 6.62 (1H, m), Ar 7.26−7.40 (5H, m),
NH3+ 7.90 (3H, br). HPLC Kromasil C18, 100 Å, 5 μm, 250 mm × 4.6
mm, CH3CN/H2O (0.1% TFA) 50−50: 9.33 min. ESI(+): [M + H]+ =
547.1.
Pharmacological Assays. Hot Plate Test. A glass cylinder (16 cm
× 16 cm) was used to keep the mouse on the heated surface of the plate,
which was maintained at a temperature of 52
1 °C using a
thermoregulated water-circulating pump. Jump latency was registered
by a stop-watch, and cutoff time was set to 240 s. Results are expressed as
percentage of maximum possible effect: % MPE = (drug latency −
control latency)/(cutoff time − control latency) × 100.
Tail-Flick Test. Tail-flick latency was measured automatically using a
tail-flick analgesimeter (Bioseb, France). For each rat, three to four
determinations were performed once a day until response values were
stable. Then at least two determinations were carried out before (t0) and
after drug injection at each time point. A cutoff time of 15 s was selected
to minimize tail damage. Responses are expressed as percentage of
maximal possible effect (% MPE).
Formalin Test. Mice received subcutaneous (plantar surface of the
hindpaw) injection of 5% formalin solution (20 μL, in saline) and were
placed back for nociceptive behavior observation (licking) for the initial
early phase (0−5 min). Vehicle (ethanol/PEG 400/water, 1/4/5) or
compounds were administered by gavage 30 or 90 min before formalin
injection. Results are expressed as licking time (in seconds).
Mechanical Allodynia: von Frey Test. Mechanical allodynia was
quantified by measuring the hindpaw withdrawal in response to von
Frey filament stimulation (bending force ranging from 0.02 to 2 g). First,
the filament of 0.4 g was used. Then the weight of the next filament was
decreased if the mouse responded or increased if the mouse did not
respond. This up−down procedure was stopped four measures after the
first change in animal responding. The threshold of response was
calculated by using the up−down Excel program kindly provided by the
A. Basbaum’s laboratory (UCSF, San Francisco, USA). Clear paw
1-(2-(1-Ethylcarbonyloxy-cyclohexylmethoxycarbonylmethyl)-
carbamoyl)-3-phenyl-propyldisulfanylmethyl)-3-methylsulfanyl-
propyl-ammonium Trifluoroacetate (19-IIId) (R2 = (S)-CH21−Phenyl,
R = CH2-COOCH(C6H11)OCOCH2CH3). Solid (yield 44.0%). H NMR
(DMSO-d6 + TFA): δ CH3CH2 1.20 (3H, m), cyclohexyl 1.10−1.80
(11H, m), CH2β 2.00−2.20 (2H, m), SCH3 2.20 (3H, s), CH3CH2 2.35
(2H, m), CH2γ 2.70 (2H, t), SCH2 + CH + CH2Ar + SCH2 2.90−3.20
(7H, m), CHα 3.75 (1H, m), NHCH2 3.90−4.10 (2H, m), CHC6H12
+
6.63 (1H, m), Ar 7.26−7.40 (5H, m), NH3 7.90 (3H, br). HPLC
Kromasil C18, 100 Å, 5 μm, 250 mm × 4.6 mm, CH3CN/H2O (0.1%
TFA) 50−50: 17.50 min. ESI(+): [M + H]+ = 571.2.
1-(2-(1-Ethoxycarbonyloxy-cyclohexylmethoxycarbonylmethyl)-
carbamoyl)-3-phenyl-propyldisulfanylmethyl)-3-methylsulfanyl-
propyl-ammonium Trifluoroacetate (19-IIIe) (R2 = (S)-CH2−Phenyl, R
1
= CH2-COOCH(C6H11)OCO2CH2CH3). Solid (yield 50.0%). H NMR
(DMSO-d6 + TFA): δ cyclohexyl 1.10−1.80 (11H, m), CH3CH2 1.30
(3H, t), CH2β 2.10 (2H, m), SCH3 2.20 (3H, s), CH2γ 2.70 (2H, t),
SCH2 + CH + CH2Ar + SCH2 2.90−3.20 (7H, m), CHα 3.75 (1H, m),
NHCH2 3.90−4.10 (2H, m), CH3CH2 4.30 (2H, q), CHC6H11 6.65
(1H, m), Ar 7.20−7.50 (5H, m), NH3+ 7.90 (3H, br). HPLC Kromasil
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withdrawal, shaking, or licking was considered as nociceptive-like
response. Both ipsilateral and contralateral side were tested.
neprilysin; Nlxe, naloxone; OR, opioid receptor; PEG, poly-
(ethylene glycol); po, per os; PSNL, partial sciatic nerve ligation;
TFA, trifluoroacetic acid; TFT, tail flick test
Hyperalgesia to Noxious Thermal Stimuli: Plantar Test. Briefly,
paw withdrawal latency in response to radiant heat stimulus was
measured using a plantar test apparatus (Bioseb, France). The intensity
of the noxious stimulus was calibrated to obtain paw withdrawal
latencies in a range of 8−10 s in mice. A cutoff time of 20 s was used to
prevent tissue damage. Mean paw withdrawal latencies for ipsilateral and
contralateral side were determined from the average of three separate
trials, taken at 5 min intervals to prevent thermal sensitization and
behavioral disturbances. Both ipsilateral and contralateral side were
tested.
Seltzer Model of Neuropathic Pain. A partial ligation of the sciatic
nerve at midthigh level was used to induce neuropathic pain. Mice were
anaesthetized by isoflurane (TEM, France; induction 4%, surgery,
1.5%), and the common sciatic nerve was exposed at the level of the
midthigh of the right hindpaw. At about 1 cm proximally to the nerve
trifurcation, a tight ligation was created around 33−50% of the sciatic
nerve using 8−0 18 in. nonabsorbable virgin silk suture (Alcon France),
leaving the rest of the nerve uninjured. The muscle was then stitched,
and the incision was closed with wound clips. Control animals (sham-
operated mice) underwent the same surgical procedure except that the
sciatic nerve was not ligated.
Habituation periods were conducted for two consecutive days on
mice. Then baseline thresholds to pressure (von Frey test) and heat
(plantar test) stimulation were assessed during two consecutive days.
Sciatic nerve surgery was performed the day after. Hypersensitivity to
pain was assessed on mice using von Frey test (mechanical allodynia)
followed by the plantar test (thermal hyperalgesia) between day 3 and
day 20 after surgery (baseline). Compound was tested between day 10
and day 20 after surgery. Mechanical and thermal hypersensitivity were
assessed before (t0) and 20 min after vehicle (ethanol/PEG400/water,
1/4/5) or 19-IIIa (50 mg/kg) oral administration.
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ASSOCIATED CONTENT
* Supporting Information
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AUTHOR INFORMATION
Corresponding Author
*Phone: + 33144067000. E-mail: bernard.roques@pharmaleads.
com.
■
Notes
́
(13) Roques, B. P.; Noble, F.; Dauge, V.; Fournie-Zaluski, M. C.;
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ABBREVIATIONS USED
■
AMC, 7-amino-4-methylcoumarin; APN, aminopeptidase N;
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DIEA, diisopropylethylamine; DMSO, dimethylsulfoxide; EDCI,
1-[3-(dimethylamino)propyl]-3-ethyl-carbodiimide methiodide;
ED50, median effective dose; ee, enantiomeric excess; ENK,
enkephalin; ESI, electrospray ionization; HCOOH, formic acid;
HOBt, hydroxybenzotriazole; HPLC, high performance liquid
chromatography; HPT, hot plate test; iv, intravenous; LC/MS,
liquid chromatography mass spectroscopy; MO, morphine;
MPE, maximal possible effect; β-NA, β-naphthylamine; NEP,
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