Pyridyl-2,5-diketopiperazines as potent, selective, and orally bioavailable oxytocin antagonists: Synthesis, pharmacokinetics, and in vivo potency

Article abstract of DOI:10.1021/jm201287w

A six-stage stereoselective synthesis of indanyl-7-(3′-pyridyl)-(3R, 6R,7R)-2,5-diketopiperazines oxytocin antagonists from indene is described. SAR studies involving mono- and disubstitution in the 3′-pyridyl ring and variation of the 3-isobutyl group gave potent compounds (pK_i > 9.0) with good aqueous solubility. Evaluation of the pharmacokinetic profile in the rat, dog, and cynomolgus monkey of those derivatives with low cynomolgus monkey and human intrinsic clearance gave 2′,6′-dimethyl-3′- pyridyl R-sec-butyl morpholine amide Epelsiban (69), a highly potent oxytocin antagonist (pK_i = 9.9) with >31000-fold selectivity over all three human vasopressin receptors hV1aR, hV2R, and hV1bR, with no significant P450 inhibition. Epelsiban has low levels of intrinsic clearance against the microsomes of four species, good bioavailability (55%) and comparable potency to atosiban in the rat, but is 100-fold more potent than the latter in vitro and was negative in the genotoxicity screens with a satisfactory oral safety profile in female rats.

Full text of DOI:10.1021/jm201287w

Article
pubs.acs.org/jmc
Pyridyl-2,5-Diketopiperazines as Potent, Selective, and Orally
Bioavailable Oxytocin Antagonists: Synthesis, Pharmacokinetics, and
In Vivo Potency
Alan D. Borthwick,*,†,▽ John Liddle,*,† Dave E. Davies,† Anne M. Exall,† Christopher Hamlett,†
Deirdre M. Hickey,† Andrew M. Mason,† Ian E. D. Smith,† Fabrizio Nerozzi,† Simon Peace,†
Derek Pollard,† Steve L. Sollis,† Michael J. Allen,§ Patrick M. Woollard,‡ Mark A. Pullen,⊥
Timothy D. Westfall,⊥ and Dinesh J. Stanislaus#
‡
†Departments of Medicinal Chemistry and DMPK, Cardiovascular and Urogenital Centre of Excellence for Drug Discovery,
GlaxoSmithKline Research and Development, Medicines Research Centre, Gunnels Wood Road, Stevenage, Herts SG1 2NY, U.K.
§Department of Assay Development and Compound Profiling, Harlow Research 2, GlaxoSmithKline, New Frontiers, Science Park,
Third Avenue, Harlow, Essex CM19 5AD, U.K.
⊥Department of Urogenital Biology, Cardiovascular and Urogenital Center of Excellence for Drug Discovery, GlaxoSmithKline
Pharmaceuticals, King of Prussia, Pennsylvania, United States
#Safety Assessment, GlaxoSmithKline Pharmaceuticals, King of Prussia, Pennsylvania, United States
S
* Supporting Information
ABSTRACT: A six-stage stereoselective synthesis of indanyl-7-(3′-pyridyl)-(3R,6R,7R)-2,5-
diketopiperazines oxytocin antagonists from indene is described. SAR studies involving
mono- and disubstitution in the 3′-pyridyl ring and variation of the 3-isobutyl group gave
potent compounds (pKi > 9.0) with good aqueous solubility. Evaluation of the
pharmacokinetic profile in the rat, dog, and cynomolgus monkey of those derivatives
with low cynomolgus monkey and human intrinsic clearance gave 2′,6′-dimethyl-3′-pyridyl
R-sec-butyl morpholine amide Epelsiban (69), a highly potent oxytocin antagonist (pKi = 9.9)
with >31000-fold selectivity over all three human vasopressin receptors hV1aR, hV2R, and
hV1bR, with no significant P450 inhibition. Epelsiban has low levels of intrinsic clearance
against the microsomes of four species, good bioavailability (55%) and comparable potency
to atosiban in the rat, but is 100-fold more potent than the latter in vitro and was negative in
the genotoxicity screens with a satisfactory oral safety profile in female rats.
orally active nonpeptide OT antagonists with a higher degree of
selectivity toward the vasopressin receptors (V1a, V1b, V2)
with good oral bioavailability. Although several templates have
been investigated as potential selective OT antagonists, few
have achieved the required selectivity for the OT receptor vs
the vasopressin receptors combined with the bioavailability and
physical chemical properties required for an efficacious oral
drug.4 Therefore our objective was to design a potent, orally
active OT antagonist with high levels of selectivity over the
vasopressin receptor with good oral bioavailability in humans
that would delay labor safely by greater than seven days and
with improved infant outcome, as shown by a reduced com-
bined morbidity score.
INTRODUCTION
■
Preterm labor is a major clinical problem leading to death and
disability in newborns and accounts for 10% of all births and
causes 70% of all infant mortality and morbidity.1 Oxytocin
(OT) is a potent stimulant of uterine contractions and is
responsible for the initiation of labor via the interaction with
the OT receptors in the mammalian uterus. OT antagonists
have been shown to inhibit uterine contractions and delay pre-
term delivery. So there is increasing interest in OT antagonists
because of their potential application in the prevention of
preterm labor. Although several tocolytics have already been
approved in clinical practice, they have harmful maternal or
fetal side effects.2 The first clinically tested OT antagonist
atosiban has a much more tolerable side effect profile and has
recently been approved for use in Europe. However, atosiban is
a peptide and a mixed OT/vasopressin V1a receptor antagonist
that has to be given by iv infusion and is not suitable for long-
term maintenance treatment, as it is not orally bioavailable.3
Hence there has been considerable interest in overcoming the
shortcomings of the peptide OT antagonists by identifying
We recently reported5 the identification of a novel series of
(3R,6R,7R)-2,5-diketopiperazine (2,5-DKP) derivatives with an-
tagonist activity at the human oxytocin receptor (hOTR). The
most potent of these was the 2,4-difluorophenyl dimethylamide 1,
Received: September 27, 2011
Published: January 12, 2012
© 2012 American Chemical Society
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which has good in vitro (pKi = 9.2) and in vivo (IC50 = 227
nM) potency and is 20-fold more potent than atosiban in vitro.
Compound 1 also has good pharmacokinetics with bioavail-
ability >50% in both the rat and the dog. Moreover, it
is >500-fold selective over all three human vasopressin receptors
(hV1aR, hV2R, and hV1bR) and has an acceptable P450 profile.
In addition, it has a satisfactory safety profile in the genotoxicity
screens and in the four day oral toxicity test in rats.
kinetic resolution of a racemic hydantion intermediate with
D-hydantoinase followed by hydrolysis of the resulting urea with
D-carbamoylase. Although this is an efficient synthesis, it is
reasonably long and uses solid-phase enzymes on a plant scale.
A shorter route would have advantages. A very short route (two-
stage process) was developed using the method of Long and
Baldwin7 from commercially inexpensive indene. A 1,3 dipolar
addition of the R-nitrone 28 to indene gave a 75% yield of the
exo and endo cycloadducts 3 and 4 in a ratio of 5:1. The
cycloadducts 3 and 4 were unstable on silica and were purified by
recrystallization. Hydrogenolysis of the cycloadduct mixture of 3
and 4 using Pearlman’s catalyst at 60 psi (∼4 atm) gave
R-indanylglycine 5 in 70% yield, not optimized, which on Cbz-
protection gave N-benzyloxycarbonyl-D-indanylglycine 6 in 95%
yield (Scheme 1).
The synthesis of indane 2,5-diketopiperazine tertiary amides
developed previously5 was used to make a range of amides for
each of the substituted pyridine templates as outlined in
Scheme 2. The four-component Ugi reaction of Z indanyl
R-glycine 6, with the R-aminoesters 8−13, convertible isonitrile 7,
and the pyridylaldehydes 14−19, gave the tripeptide inter-
mediates 20−31, which was followed by hydrogenation to
remove the Z and benzyl groups, enabling cyclization to occur
to give the phenolic 2,5-DKPs 32−43 in good yield.
The cyclic carbamates obtained from carbonyldiimidazole
and phenol’s 32−43 were treated directly with a range of
amines, which gave the required RRR amides 44, 45, 47−59,
and 61−69 in high yield. The 6-OH derivative 46 was obtained
in 91% yield by acid hydrolysis of the 6-MeO derivative 44 and
the N-oxide 60 was prepared in 72% yield by oxidation of 58
with m-chloroperbenzoic acid. The acids 70, 71, and 72 were
prepared by hydrolysis of the cyclic carbamates, obtained from
carbonyldiimidazole and phenols 37, 39, and 43, with aqueous
acetone in high yield. The RRR carboxamides 69, and 73−78
were prepared by treating these with acids 70, 71, and 72 with
carbonyldiimidazole followed by the required amines and
separating the required RRR isomers.
However, 1 had poor aqueous solubility and high intrinsic
clearance in human and cynomolgus monkey liver microsomes,
so a compound was required that retained high antagonist
potency and excellent pharmacokinetics in animal species seen
with 1 but was more soluble and with improved human
intrinsic clearance to decrease the risk of low bioavailability in
humans. Our first approach was to replace the 7-aryl ring with a
five-membered heterocycle, which led to the oxazole Retosiban
(106) as our clinical candidate.6 As a backup to 106, an
alternative replacement of the 7-aryl ring with a six-membered
heterocycle was considered and in this report we describe
how we investigated the modification of the 7-aryl ring to the
7(3′-pyridyl) ring and optimized substitution in this ring as well
as modifying the isobutyl group to obtain good potency, lower
intrinsic clearance in human microsomes, and good pharma-
cokinetics in animal species.
CHEMISTRY
■
Our previous synthesis9 of chirally pure R-indanylglycine 5
The synthesis of an array of ortho-substituted 7-phenyl-2,5-
diketopiperazine secondary amides is outlined in Scheme 3.
was a five-step synthesis from 2-iodoindane involving a dynamic
a
Scheme 1
a
Reagents and conditions: (a) CDCl3, 61 °C, 3 h; (b) Pd(OH)2/C, dioxane−TFA 10:1, H2 (60 psi), 55 °C, 24 h; (c) dioxane−H2O 1:1, Et3N,
N-(benzyloxycarbonyloxy)succinimde, room temp, 2 days.
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a
Scheme 2
a
Reagents and conditions: (a) R-amino methyl ester hydrochloride, methanol/trifluoroethanol (1:1), pyridyl aldehyde, Et3N, room temp, 20 h; then
Cbz-IndGly, 2-benzyloxyphenylisocynanide, room temp, 4 days; (b) H2, Pd/C, EtOAc, AcOH, 18 h; (c) CDI, DCM, 16 h; then R2NH, room temp,
16 h; (d) CDI, DCM, 20 h, then Me2CO, H+, H2O, 20 h; (e) CDI, DCM, 18 h; then R2NH, room temp, 18 h; (f) NaI, Me3CCOCl, MeCN, reflux,
4 h, then H2O 10 min; (g) m-chloroperbenzoic acid, CHCl3 room temp, 16 h.
These ortho-substituted phenyl isopropylamides 97−104
were prepared by a four-component Ugi reaction where the
imines formed by the initial reaction of the ortho-substituted
aryl aldehydes 81−88 and amine 8 D-leucine methyl ester
reacted with the isopropyl isonitrile 80 and the resulting
intermediate imminium ion then further reacted with the acid
79 (boc-protected indanyl R-glycine), which rearranged to give
the tripeptides 89−96. In the second-stage deprotection of the
Boc-amine in 89−96 with TFA followed by cyclization in the
presence of Et3N gave a 1:3−2:3 mixture9 of the RRR/RRS
isomers. The RRR isomers 97−104 were isolated in ≤11% yield
(see Supporting Information).
RESULTS AND DISCUSSION
■
The aim was to discover a compound equally potent and more
soluble than the 2,4-difluorophenyl dimethylamide 1 with
acceptable pharmacokinetics in animal species and with
reduced human intrinsic clearance10 to decrease the risk of
low bioavailability in humans. Pyridyl rings are known
bioisosteres of fluorobenzene, so derivatives based on this
ring system would be expected to be similarly active and would
enable the production of more soluble OT antagonists.
However, the ortho pyridyl derivative 105 (Table 1) was
found to readily epimerize11 at the exocyclic position due to the
electron withdrawing properties of the ortho nitrogen atom. We
reasoned that the meta analogues would be less susceptible
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a
Scheme 3
a
Reagents and conditions: (a) Et3N, methanol, room temp, 16 h; (b) TFA, CH2Cl2, 3 h, then Et3N, room temp, 16 h.
Table 1. Inhibition of Oxytocin Binding at the Human Oxytocin Receptors, Solubility and the Pharmacokinetic Profile of
a
6′-Pyridyl substituted (3R,6R,7R)-2,5-Diketopiperazines in the Rat and Dog
a
Displacement of [3H] oxytocin from hOTR by the test compound.12 pKi values are reported as mean values of three or more determinations and all
b
results were obtained in binding assays with a standard deviation in pKi of less than 0.25. Rat PK (n = 4): AUC (ng·h mL−1) at 5 mg/kg, 5%
c
DMSO/95%PEG400 formulation, Cl in mL min−1 kg−1. Dog PK (n = 3): AUC (ng·h mL−1) at 0.6 mg/kg, 5%DMSO/95%PEG400 formulation, Cl
in mL min−1 kg−1. Solubility (mg mL−1) a precipitation/HPLC based measurement.13 An HPLC method based measurement of lipophilicity.14
d
e
f
g
h
% Human serum albumin binding.15 % Rat serum albumin binding.15 Value obtained for 1 when used as internal standard in OT binding assay for
j
6′-pyridyl 2,5-DKP′s, previous value pKi = 9.2, see ref 5. Previous published data see ref 5.
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to epimerization, so the initial target became a series of
6′-substituted-3′-pyridyl derivative where the substituents were
non-electron-withdrawing and the ortho position to the N atom
was blocked to avoid potential metabolic problems (Table 1).
Also, the 6′-substituents in the pyridyl derivatives are equiva-
lent to the 4′-position in the aryl series, which is the optimal
substitution position for potency in the monosubstituted
phenyl derivatives.9
Monosubstituted Pyridyl iso-Butyl Derivatives. We
initially made the dimethylamides (44, 47, 49) and morpholine
amides (45, 48, 50) in the 6′-MeO, 6′-NMe2, and 6′-Me-3-
pyridyl series. All had better solubility, a lower log D, and lower
protein binding to human and rat serum albumin than 1 (Table
1). All had a similar level of potency (pKi 9.6−8.8), however,
the 6-hydroxy derivative 46 was less potent (pKi 8.5) and all
were less potent than 1. Both the 6′-MeO and 6′-Me derivatives
(44, 45) and (49, 50) had better exposure in the rat than the
6′-NMe2 derivatives (47 and 48), and the best bioavailability in
the rat was achieved by 44, 45, and 49. However, 44 had poor
bioavailability in the dog.
low intrinsic clearance in these species, its intrinsic clearance in
cynomolgus monkey and human microsomes is high (Table 2).
A similar pattern was seen with 1. To increase the chance of
good bioavailability in cynomolgus monkey and humans, a
decrease in intrinsic clearance in microsomes of these species
was sought.
Modification of iso-Butyl in 6′-Me-Pyridyl Deriva-
tives. In an attempt to improve potency and intrinsic
clearance in cynomolgus monkey and human microsomes, we
then investigated modification of the 3-isobutyl group (Table 2).
As expected, all maintained the improved solubility compared
to 1. The cyclopropylmethyl dimethylamide 52 and morpholine
derivative 53 were 3 and 10 times less potent than the cor-
responding isobutyl derivative 49 and 50 (Table 1). In contrast,
both the tertiarybutylmethyl derivatives 54 and 55 had a better
potency than 49; however, they had a worst oral exposure in
the rat than 49 although they had a better intrinsic clearance in
human microsomes than 49.
In the natural ligand oxytocin, the 3-Ile is one of the key
amino acids required for agonist potency and we have ration-
alized from extensive SAR6,9 that the 3-isobutyl group in our
2,5-diketopiperazine oxytocin antagonists mimics the 3-Ile
interaction of the natural ligand. However, the alpha group in
the 3-Ile amino acid of oxytocin is an S-sec-butyl while the
3-isobutyl group in 49 is achiral. By changing the 3-isobutyl
group in 49 for a chiral sec-butyl seen in the natural ligand, it
was hoped that a better fit with the receptor could be achieved
The best overall pharmacokinetic profile in rat and dog was
achieved by the 6′-Me-3′-pyridyl dimethylamide 49. Never-
theless, it was 20-fold less potent than 1. Further amide
derivatives of 6′-methyl pyridyl 2,5-DKP template, the morpho-
line 50 and the pyrrolidinyl 51, were inferior in terms of oral
exposure in the rat compared to the dimethylamide 49. Also,
although 49 had good rat and moderate dog bioavailability with
a
Table 2. Pharmacokinetic Profile of 6′Me-3-Pyridyl Substituted (3R,6R,7R)-2,5-Diketopiperazines in the Rat and Dog
a
Displacement of [3H] oxytocin from hOTR by the test compound.12 pKi values are reported as mean values of three or more determinations, and
all results were obtained in binding assays with a standard deviation in pKi of less than 0.25. Microsomal intrinsic clearance (mL min−1 g liver−1).16
b
c
d
Rat PK (n = 4): AUC (ng·h mL−1) at 5 mg/kg, 5%DMSO/95%PEG400 formulation, Cl in mL min−1 kg−1. Dog PK (n = 3): AUC (ng·h mL−1) at
0.6 mg/kg, 5%DMSO/95%PEG400 formulation, Cl in mL min−1 kg−1. Solubility (mg mL−1) a precipitation/HPLC based measurement.13 Value
e
f
g
obtained for 1 when used as internal standard in OT binding assay for 6′-pyridyl 2,5-DKP′s, previous value pKi = 9.2, see ref 5. Previous published
data see ref 5.
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and a subsequent increase in potency. The R-sec-butyl and the
S-sec-butyl derivatives of 49 were prepared. Both the S-sec-butyl
dimethylamide 58 and morpholine amide 59 were more potent
than the corresponding amides 56 and 57 in the R-sec-butyl
series (Table 2) and more potent than the 3-isobutyl dimethy-
lamide 49. These four derivatives also had a better intrinsic
clearance in human microsomes than 49. The N-oxide 60 of
the S-sec-butyl dimethylamide 58 was 5-fold less active than the
parent 58, and it had a much lower oral exposure in the rat than
58. Of these four derivatives, the S-sec-butyl dimethylamide 58
had the better pharmacokinetic profile across both species and
had better bioavailability in the dog than 49 (Table 2). Also, 58
had a better intrinsic clearance in cynomolgus monkey and
human microsomes than the 3-isobutyl dimethylamide 49 and
was better across all four species than 1. Although 58 was greater
in its in vitro potency than 49, it was however still less potent
than 1. The aim was to try and maintain the good in vitro and in
vivo pharmacokinetic profile and solubility shown by 58 and
increase potency further. Two ways forward were envisaged.
Dimethyl-Pyridyl S-sec-Butyl Amides. One was to
further explore the group at the 3-position and the other was
to modify the pyridine ring. Further substitution of the
3′-pyridyl ring to give disubstituted derivatives by maintaining
the 6′methyl group and substituting the 2′, 4′, or 5′ position was
considered. Although the effect of ortho F substitution on the
4′-F-phenyl ring to give 2′,4′-di-F-phenyl ring derivatives (e.g., 1)
was previously shown9 to be an advantage in terms of potency
and rat PK, no systematic investigation of substitution at the
ortho position on potency had ever been carried out. Therefore,
the effect of ortho substitution on the aromatic group was
investigated first with the hope that this would be transferable
to the pyridine system. An array of 7-phenyl substituted
(3R,6R,7R)-2,5-diketopiperazines containing ortho-substituted
phenyl groups were prepared (Table 3).
Table 3. Inhibition of Oxytocin Binding at the Human Oxytocin Receptors by 2′ Phenyl Substituted (3R,6R,7R)-2,5-
Diketopiperazines
a
Functional OT antagonist activity measured by FLIPR assay, see ref 5.
Table 4. Inhibition of Oxytocin Binding at the Human Oxytocin Receptor and the Pharmacokinetic profile of S-sec-Butyl
a
Dimethyl Substituted Pyridyl (3R,6R,7R)-2,5-Diketopiperazines in the Rat and Dog
a
Displacement of [3H] oxytocin from hOTR by the test compound.12 pKi values are reported as mean values of three or more determinations and all
results were obtained in binding assays with a standard deviation in pKi of less than 0.25. Microsomal intrinsic clearance (mL min−1 g liver−1).16
b
c
d
Rat PK (n = 4): AUC (ng·h mL−1) at 5 mg/kg, 5%DMSO/95%PEG400 formulation, Cl in mL min−1 kg−1. Dog PK (n = 3): AUC (ng·h mL−1) at
0.6 mg/kg, 5%DMSO/95%PEG400 formulation, Cl in mL min−1 kg−1.
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These were all in the 3-isobutyl/isopropyl amide series due
to ease of synthesis and availability of starting material. Those
compounds with the highest potency were the chloro 97 and
methyl 98 derivatives (Table 3). Because of the activity of Cl at
the 2-position in a pyridine ring, a series of dimethyl pyridine
templates were prepared with a methyl group in the ortho
position. These were the 2′,6′-dimethyl, the 4′,6′-dimethyl, and
the 2′,5′-dimethyl pyridines (Table 4).
The impact of the ortho methyl group on potency was
achieved with the 2′,6′-dimethyl pyridines (68 and 69) and
4′,6′-dimethyl pyridines (64, 65, and 66) but not with the
2′,5′-dimethyl pyridines (61, 62, and 63), which were less active
than the 6′-methyl pyridines 58 and 59. This confirms that
disubstitution para and ortho is preferred. The increase in
potency seen with the 4′,6′-dimethyl and 2′,6′-dimethyl pyridine
series indicates that the ortho methyl group puts the
6′-methylpyridine ring in a conformation that makes this template
have a more favorable interaction at the oxytocin receptor. We
had previously rationalized from detailed SAR6 that the R aryl
ring in the exocyclic aryl amide restricts the conformational
mobility of the leucine (3-isobutyl) group, which facilitates its
interaction at the oxytocin receptor. Hence the ortho methyl
group in the 6′-methylpyridine ring which increases potency
probably augments this interaction at the receptor.
pyridines. Within the 2′,6′-dimethyl series, the secondary amide
67 and both the tertiary amides 68 and 69 had good phar-
macokinetic profiles in the rat, all with bioavailability >50%.
Both 2′,6′-dimethyl pyridines 68 and 69 were more potent in
vitro than the 6′-methyl pyridine 58, and both have a superior
oral exposure in the rat than 58. The dimethylamide 68 had the
best pharmacokinetic profile in the rat and the dog.
Mono and Di-Me Pyridyl 3-Pentyl Derivatives. The
other way forward to improve potency was to further explore
the group at the 3-position. The S-sec-butyl derivatives (58 and
59) had increased potency over the isobutyl derivative 49, while
the R-sec-butyl derivatives (56 and 57) had a more comparable
potency to the latter (Table 2).
This was taken further by combining the structural elements
of each of these in the more bulky 3-pentyl derivatives (Table 5).
All the 3′-pyridine-2,5-diketopiperazine-3-pentyl tertiary amide
derivatives had increased potency over 68, and all had good oral
exposure in the rat. However, apart from secondary amide 73,
all had an inferior intrinsic clearance in human microsomes and
a lower oral exposure in the rat than 68. The methylamide 73
had the best intrinsic clearance in this series and a good
pharmacokinetic profile in the rat and the dog. However, its
overall profile was slightly inferior to that of the S-sec-butyl-2′,
6′-dimethyl pyridine dimethylamide 68.
Requirements for Further Progression of a Oxytocin
Antagonist Drug. The desirable features that an oral
oxytocin drug should also display are the following:
(1) Sufficient pharmacokinetics in the cynomolgus monkey
to enable preterm labor to be evaluated.
(2) High OT antagonist potency and good selectivity vs
human vasopressin receptors.
(3) High in vivo OT antagonist potency with no significant
P450 inhibition.
The 2′,5′-dimethylpyridine dimethylamide 62 also had an
inferior oral exposure in the rat than the corresponding
dimethylamides 68 and 65 in the other two series. A
comparison of the pharmacokinetic profile of the 4′,6′-dimethyl
and 2′,6′-dimethyl pyridines (Table 4) showed that the
monomethylamide 67 and the dimethylamide 68 in the 2′,6′-
dimethyl series had a better oral exposure in the rat than the
corresponding analogues 64 and 65 in the 4′,6′-dimethyl series.
Also, the intrinsic clearance against the microsomes of all four
species for the 2′,6′-dimethyl pyridines was low (in single
figures) and generally better than that of the 4′,6′-dimethyl
(4) Minimal adverse effects.
(5) Ease of synthesis and formulation.
Table 5. Inhibition of Oxytocin Binding at the Human Oxytocin Receptor and the Pharmacokinetic Profile of 3-Pentyl
a
Substituted Pyridyl (3R,6R,7R)-2,5-Diketopiperazines in the Rat and Dog
a
Displacement of [3H] oxytocin from hOTR by the test compound.12 pKi values are reported as mean values of three or more determinations and all
results were obtained in binding assays with a standard deviation in pKi of less than 0.25. Microsomal intrinsic clearance (mL/min/g liver).16 Rat
b
c
d
PK (n = 4): AUC (ng·h mL−1) at 5 mg/kg, 5%DMSO/95%PEG400 formulation, Cl in mL min−1 kg−1. Dog PK (n = 3): AUC (ng·h mL−1) at
0.6 mg/kg, 5%DMSO/95%PEG400 formulation, Cl in mL min−1 kg−1.−
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Table 6. Inhibition of Oxytocin Binding at the Human Oxytocin Receptors and the Pharmacokinetic Profile of S-sec-Butyl and
a
3-Pentyl Pyridyl (3R,6R,7R)-2,5-Diketopiperazines in the Rat and Cynomolgus Monkey
a
Displacement of [3H] oxytocin from hOTR by the test compound.12 pKi values are reported as mean values of three or more determinations and all
results were obtained in binding assays with a standard deviation in pKi of less than 0.25. Microsomal intrinsic clearance (mL min−1 g liver−1).16
b
c
d
Rat PK (n = 4): AUC (ng·h mL−1) at 5 mg/kg, 5%DMSO/95%PEG400 formulation, Cl in mL min−1 kg−1. Cynomolgus monkey PK (n = 4):
AUC (ng·h mL−1) besylate salt formulated in 0.5% methylcellulose/0.1% Tween 80 in water and administered as a solution at nominal dose of
e
2 mg/kg free base, Cl in mL min−1 kg−1. Previous published data see ref 5.
Table 7. Potency of 2,5-Diketopiperazines 1, 69, and Atosiban at the Human Oxytocin Receptor Compared with the Efficacy
Obtained in the Rat Uterine Contractility Model and Inhibition of OT Binding at the Human OT (hOT) and Vasopressin
a
Binding at the Human (V1a, V1b, and V2) Receptors
a
Displacement of [3H] oxytocin from hOTR or vasopressin from hV1aR, hV1bR, and hV2R by the test compound.12 pKi values are reported as
mean values of three or more determinations and all results were obtained in binding assays with a standard deviation in pKi of less than 0.25, see ref
b
c
d
e
5. Plasma IC50.17 Solubility (mg mL−1) a precipitation/HPLC based measurement.13 An HPLC method based measurement of lipophilicity.14
%
Human serum albumin binding.15 Previous published data, see ref 5. Previous published data, see ref 22.
f
g
Requirement 1: Sufficient Pharmacokinetics in the
Cynomolgus Monkey. Because preterm labor would be
evaluated in the cynomolgus monkey post candidate selection,
pharmacokinetics in this species was considered as a requisite
for the progression of lead compounds. Of the five lead
7-(3′-pyridyl)-2,5-diketopiperazines 58, 68, 69, 73, and 78, the
2′,6′-dimethyl-3′-pyridines morpholine amide 69 had the best
intrinsic clearance in the three animal species and the best
oral exposure and bioavailability in the cynomolgus monkey
(Table 6). Compound 69 was also better than 1 in its oral
exposure and bioavailability in the cynomolgus monkey, with
low microsomal intrinsic clearance in all four species (Table 6),
and a lower log D, lower binding to human serum albumin, and
a better solubility than 1 (Table 7).
Requirement 2: High Oxytocin Antagonist Potency and
Selectivity vs Human Vasopressin Receptors. A measure of
the potency of our lead OT antagonist 69 compared to the
previous lead 1 was established by their binding inhibition data
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Table 8. Cyp45018 Inhibition and Microsomal Intrinsic
Clearance16 Profile of 2,5-Diketopiperazines 1 and 69
isozymes. The 2′,6′-dimethyl-3-pyridyl morpholine amide
Epelsiban 69 was therefore chosen as the compound which
fulfilled the selection criteria for further development.
Requirement 4: Minimal Adverse Effects. The 2′,6′-dime-
thyl-3-pyridyl morpholine amide 69 was shown not to be muta-
genic in vitro in both the bacterial (HTFT/mini-Ames assay)20
and mammalian cell (mouse lymphoma assay)21genotoxicity
screens. A seven-day oral safety assessment study was carried
out with 69 in female Sprague−Dawley rats at doses suspended in
0.5% (w/w) methylcellulose/0.1% (w/w) Tween 80 in sterile
water. There was no effect on body weight or food consumption,
no adverse clinical signs, and histological findings did not reveal
any treatment-related effects at doses up to 100 mg/kg/day. The
coverage in toxicity studies based on the minimum effective dose
(MED) allowed the further progression of 69.
Requirement 5: Ease of Synthesis and Formulation. The
6R(indanyl),3R(S-sec-butyl),7R(2′,6′-dimethyl-3′-pyridyl)-2,5-
diketopiperazine morpholine amide 69 can be synthesized in six
high-yield reaction steps from indene. The 2,5-diketopiperazine
69 is a crystalline powder mp 140 °C (2.5% heptane) with
molecular mass of 518.7 Da, and a white crystalline besylate salt
69B mp 179−183 °C with molecular mass of 676.8 Da has
been identified. The compound is ionizable in aqueous solution
(pKa = 5.78) and is moderately lipophilic (log D = 2.55 at pH
7.4). Potential advantages over 106 include greater solubility 33
mg/mL in water at 35 °C. No significant change in terms of assay,
impurity profiles, and appearance was observed when the solid-
state stability of 69B was investigated for up to 3 months at 50 °C
at ambient room humidity. Also, 69B is chemically stable in
aqueous solutions covering the pH range 2−6 and when exposed
to light at room temperature for up to 7 days. The oral bio-
availability of at 5 mg/kg formulated in aqueous solution in the rat
was acceptable (62%), and exposure was approximately propor-
tional when administered in the same formulation at 100 mg/kg
(7-day rat toxicity study). The good aqueous solubility may offer
more solution formulation options, and formulation in solid form
would not be expected to result in any substantial loss of
bioavailability compared to formulations in aqueous solution.
Comparison of Lead Compounds. Although the oxy-
tocin antagonist Epelsiban 69 is comparable in potency to 1
in vitro and in vivo, it is more selective against the human
vasopressin receptors hV1aR and hV2R and has better aqueous
solubility (Table 9). Compared to 1, Epelsiban 69 has a better
P450 profile with no significant inhibition IC50 > 100 μM
against five P450 isozymes and a better microsomal intrinsic
clearance profile, which was low in all four species and
especially in the cynomolgus monkey and human microsomes
(Table 8). Also, it has better pharmacokinetics than 1 in the
cynomolgus monkey (Table 9). In contrast, it is comparable
to Retosiban 1066 in terms of its pharmacokinetic profile in the
rat and cynomolgus monkey, and in its intrinsic clearance in
cynomlogus monkey and human microsomes, but is 5-fold
more potent against the OT receptor, is more selective against
the human vasopressin receptors, especially hV2, and is more
soluble in its salt form (Table 9).
a
b
IC50 μM Cypex.18 Diethoxyfluorescein (DEF) and 7-benzyloxyquino-
c
line (7BQ) are fluorogenic substrates for Cyp3A4. Previous published
data, see ref 6. Microsomal intrinsic clearance (mL min−1 g liver−1).16
d
(pKi) against the isolated recombinant human oxytocin
receptors (Table 7). The morpholine amide 69 has comparable
activity to the previous lead 1 in vitro at the human oxytocin
receptor in the binding assay. Selectivity relative to human
vasopressin receptors for 69 and 1 (Table 7) has been established
by measuring their pKi against hV1aR, hV2R, and hV1bR.
Both OT antagonists, the dimethylamide 1 and the morpholine
amide 69 (Table 7) are >500-fold selective for the hOTR
relative to all three vasopressin receptors hV1aR, hV2R, and
hV1bR. The morpholine amide 69 was more selective than the
dimethylamide 1 against the human vasopressin receptors
hV1aR and hV2R and was also >50000-fold, >63000-fold, and
>31000-fold selective for the human OT receptor relative to the
three vasopressin receptors hV1aR, hV1bR, and hV2R.
Requirement 3: High In Vivo Potency and No Significant
Cyp 450 Inhibition. The in vivo efficacy of the most promising
compound 69 was estimated in an anaesthetized rat model
(see ref 5), where uterine contractions were elicited by iv
administration of oxytocin. The reduction in uterine con-
tractility was measured after subsequent iv administrations of
increasing doses of 69 (0.1, 0.3, and 1 mg/kg iv). The method
used for the measurement of in vivo activity was the plasma
IC50 determination17 [the concentration that causes 50% inhibi-
tion of response to the standard OT dose (0.3 ug/kg iv)]. The
in vivo comparison of the lead 69, and previous lead 1 is
outlined in Table 7. The morpholine amide 69 has comparable
potency (IC50 = 192 nM) to the previous lead 1 and atosiban
(a marketed iv, peptide OT antagonist) in vivo in the rat.
In contrast, 69 is 100-fold more potent than atosiban at the
human receptor in vitro.
CONCLUSION
■
We have shortened the synthesis of indanyl (3R,6R,7R)-2,5-
diketopiperazine OT antagonists to six stages by reducing the
five-stage synthesis of the R-indanylglycine to two stages from
indene. SAR studies have shown that increased aqueous
solubility was possible with a range of 6′-substituted 7-(3′-
pyridyl)-(3R,6R,7R)-2,5-diketopiperazine amides, and the
Cyp 450 Inhibition. Additional predevelopment studies
revealed that 1 inhibited several P450 isozymes, which was
not the case with 69 (Table 8). Also no time-dependent
inhibition19 was observed for 69 against these five P450
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Table 9. Comparison of the Oxytocin Antagonist Potency, Selectivity vs the Human Vasopressin Receptors, Intrinsic Clearance
in Two Species, and Pharmacokinetic Profile in Rat and Cynomolgus Monkey for 1, 106, 69
a
Displacement of [3H] oxytocin from hOTR or vasopressin from hV1aR, hV1bR, and hV2R by the test compound.12 pKi values are reported as
mean values of three or more determinations, and all results were obtained in binding assays with a standard deviation in pKi of less than 0.25, see ref 5.
b
c
d
Plasma IC50 nM in the rat.17 Microsomal intrinsic clearance (mL min −1 g liver−1) (cy = cynomolgus monkey, hu = human).16 Rat PK (n = 4):
e
AUC (ng·h mL−1) at 5 mg/kg, 5%DMSO/95%PEG400 formulation, Cl in mL min −1 kg −1; t1/2 in h. Cynomolgus monkey PK (n = 3): AUC (ng·h
mL−1) administered as a solution at nominal dose of 2.0 mg/kg in 1% DMSO and 6% hydroxypropyl β cyclodextrin (Cavitron) in water (pH 3.5−4).
f
Cynomolgus monkey PK (n = 4): AUC (ng·h mL−1) besylate salt formulated in 0.5% methylcellulose/0.1% Tween 80 in water and administered as
g
kg. Solubility (mg mL −1), a precipitation/HPLC based measurement.13
−1
a solution at nominal dose of 2 mg/kg free base, Cl in mL min
h
i
j
k
Solubility in water for besylate salt. An HPLC method based measurement of lipophilicity.14 % Human serum albumin binding.15 Previous
l
published data, see ref 5. Previous published data, see ref 22.
6′-methyl-3′-pyridyl dimethylamide derivative 49 was the best
in terms of PK in the rat and dog. Also variation of the isobutyl
group showed that the 6-methyl R-sec-butyl derivative 58
maintained good rat and dog pharmacokinetics and had good
intrinsic clearance in cynomolgus monkey and human micro-
somes. Substitution at the ortho position of the 7-phenyl ring of
(3R,6R,7R)-2,5-diketopiperazines with a range of groups has
shown that the chloro and methyl groups were the best at
increasing potency. Substitution at the ortho-position of the
7-(3′-pyridyl)-(3R,6R,7R)-2,5-diketopiperazine gave the 2′,6′,
the 4′,6′, and the 2′,5′-dimethyl-3-pyridyl-2,5-diketopiperazine
templates of which the 2′,6′-dimethyl-3′-pyridyl derivatives 68
and 69 were best in terms of potency and pharmacokinetic
profile in the rat. Although further modification of the S-sec-
butyl group of the 2′,6′-dimethyl-3′-pyridyl 2,5-diketopiperazine
template to the 3-pentyl analogues gave a further increase in
potency, these analogues were worse in terms of pharmacoki-
netics especially in the cynomolgus monkey. The 2′,6′-dimethyl-
3′-pyridyl morpholine amide Epelsiban 69 had low intrinsic
clearance in all four species, a good pharmacokinetic profile in
the rat with a bioavailability of 55%, and the best oral exposure
and bioavailability in the cynomolgus monkey. Evaluation in
vitro has shown that Epelsiban 69 is 100-fold more potent at
the hOTR than atosiban (a marketed iv peptide OT antagonist)
with comparable potency to the latter in vivo in the rat. Also,
69 is >31000-fold selective versus the human vasopressin
receptors (V1aR, V2R, and V1bR) and has a good P450 profile
with no significant inhibition IC50 > 100 μM and had good
aqueous solubility. In addition, it was negative in the
genotoxicity screens and had a satisfactory safety profile in
the seven-day oral toxicity test in female rats.
EXPERIMENTAL PROCEDURES
■
General Procedures. Melting points were obtained using an
Electrothermal digital melting point apparatus and are uncorrected.
Purification using silica or basic alumina cartridges refers to
chromatography carried out using a Combiflash Companion with
Redisep cartridges supplied by Presearch. Hydrophobic frits refer to
filtration tubes sold by Whatman. SPE (solid phase extraction) refers
to the use of cartridges sold by International Sorbent Technology Ltd.
All purifications by flash chromatography were performed using
Kieselgel 60, Merck 9385 silica gel. Preparative plate chromatography
were performed using WhatmanPK6F silica gel 60A plates eluting with
ethyl acetate-cyclohexane or 2-propanol−dichloromethane mixtures.
Monitoring of reactions by TLC used Merck 60 F254 silica gel glass
backed plates (5 cm × 10 cm), eluted with mixtures of ethyl acetate
and cyclohexane, and visualized by UV light, followed by heating with
aqueous phosphomolybdic acid. Purity for final compounds was
greater than 95% and was measured using combustion analysis or
analytical HPLC conducted on a Supelcosil LCABZ+PLUS column
(3.3 cm × 4.6 mm ID), eluting with 0.1% HCO2H and 0.01 M
ammonium acetate in water (solvent A) and 0.05% HCO2H 5% water
in acetonitrile (solvent B), using the either elution gradient 1, 0−
0.7 min 0%B, 0.7−4.2 min 0−100%B, 4.2−5.3 min 100%B, 5.3−5.5 min
0%B, or elution gradient 2, 0−0.7 min 0%B, 0.7−4.2 min 0−100%B,
4.2−4.6 min 100%B, 4.6−4.8 min 0%B at a flow rate of 3 mL/min.
The mass spectra (MS) were recorded on a Waters ZQ 2000 mass
spectrometer using electrospray positive [(ES + ve to give MH+ and
M(NH4)+ molecular ions] or electrospray negative [(ES − ve to give
1
(M − H)− molecular ion] modes. H NMR spectra were recorded
using a Bruker DPX 400 MHz spectrometer using tetramethylsilane as
the external standard. CD spectra were recorded in acetonitrile on a
Jasco J-720A spectropolarimeter. Optical rotations were taken with a
Perkin-Elmer model 241 polarimeter. Enantiomeric excess (% ee) was
determined by chiral HPLC analysis using a Chiracel OJ or Chiral Pak
AD464 column with a UV detector λ = 215 nm and with eluents and
flow rates as indicated in each case. Final organic solutions were dried
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over MgSO4 before filtration and evaporation using a Buchi Rotavapor.
Ambient temperature was 20 °C. All solvents used were Fisons
analytical reagents except for Pentane (Aldrich Chemical Co.) and
anhydrous THF (Fluka sureseal). All other reagents were usually
obtained from Aldrich, Fluka, or Lancaster. Elemental microanalyses
were determined by the Microanalytical Laboratory, GlaxoSmithKline
Stevenage.
All animal studies were ethically reviewed and carried out in
accordance with the Animals (Scientific Procedures) Act 1986 and the
GSK Policy on the Care, Welfare, and Treatment of Laboratory
Animals.
phase was back-extracted with ethyl acetate (50 mL), and the combined
organic extracts were washed successively with semisaturated aqueous
solutions of sodium hydrogen carbonate (100 mL), ammonium
chloride (100 mL), and sodium chloride (100 mL), dried (MgSO4),
and evaporated under reduced pressure to give the crude product
(12.01 g). This was purified on a Redisep silica column (330 g) eluted
with 20−50% ethyl acetate in cyclohexane to afford 7.46 g of methyl
N-[(2R)-2-(2,3-dihydro-1H-inden-2-yl)-2-({[(phenylmethyl)oxy]-
carbonyl}amino)acetyl]-N-[1-(2,6-dimethyl-3-pyridinyl)-2-oxo-2-({2-
[(phenylmethyl)oxy]phenyl}amino)ethyl]-D-alloisoleucinate 30 as a
pair of diastereomers. LCMS m/z 797 (MH+) two components,
gradient 2 (tR 3.88 and 3.96 min). This was dissolved in ethanol
(150 mL) and acetic acid (10 mL) and the mixture was hydrogenated at 1
atm of H2 over 10% palladium on carbon (Degussa type) (1.8 g wetted
with water 1:1 w:w) for 18 h. The reaction mixture was evaporated
under reduced pressure, and the residue was partitioned between ethyl
acetate (250 mL) and water (50 mL) with saturated aqueous sodium
hydrogen carbonate added until the aqueous phase was basic (pH 8).
The aqueous phase was extracted with ethyl acetate 2 (50 mL), and
the combined organic extracts were washed with saturated aqueous
sodium hydrogen carbonate/water 3:1 (100 mL) and then with
saturated brine (50 mL) before being dried (MgSO4) and evaporated
under reduced pressure. The crude product was purified on a Redisep
silica column (120 g) eluted with 0−10% methanol in ethyl acetate to
give 42 as a pair of diastereomers (2.94 g, 34%). 1H NMR (CDCl3) δ
8.39, 8.34 (m, 1H), 7.78 (d, 1H, J = 8 Hz), 7.63, 7.53 (m, 1H), 7.45 (d,
1H, J = 8 Hz), 7.22−7.03 (m, 5H), 6.97 (m, 1H), 6.85 (m, 1H), 6.00,
5.55 (s, 1H), 4.18, 4.10 (m, 1H), 4.00−3.91 (m, 1H), 3.22−2.75 (m,
5H), 2.63, 2.56, 2.55, 2.51 (s, 6H), 1.72 (m, 1H), 1.58 (m, 1H), 1.14−
0.88 (m, 4H), 0.84, 0.71 (t, 3H, J = 7 Hz). LCMS m/z 541 (MH+) two
components, gradient 2 (tR 2.75 and 2.81 min). HRMS calcd for
C32H36N4O4 (MH+) 541.28093, found 541.28103. HPLC: two
components 35% and 58% (tR 10.93 and 11.37 min).
The compounds 32−41 and 43 were similarly prepared; see
Supporting Information for experimental and spectroscopic details.
(2R)-2-{(3R,6R)-3-(2,3-Dihydro-1H-inden-2-yl)-6-[(1S)-1-
methylpropyl]-2,5-dioxo-1-piperazinyl}-2-(2,6-dimethyl-3-pyr-
idinyl)-N-methylethanamide (67). The phenol (42) (0.400 g, 0.74
mmol) and carbonyldiimidazole (0.192 g, 1.18 mmol) in dry dichloro-
methane (10 mL) were stirred at room temperature overnight. The
mixture was treated with a 2 M solution of methylamine in tetra-
hydrofuran (1.849 mL, 3.70 mmol) and left to stand overnight at room
temperature. The solvents were blown down, and the residue was
purified on a Redisep silica column (35 g) eluted with 0−10%
methanol in ethyl acetate followed by further purification on a Kromasil
KR100−10-C18 reverse-phase column eluted with aqueous acetoni-
trile (20−45% MeCN) containing 0.1% formic acid. This gave after
freeze-drying from 1,4-dioxane the amide 67 (102 mg, 30%) as a white
lyophilizate. 1H NMR (CDCl3) δ 7.63 (d, J = 8.1 Hz, 1H, pyridyl-4H),
7.25−7.14 (m, 4H, indanyl-arylH), 7.05 (d, J = 8.1 Hz, 1H, pyridyl-
5H), 6.56 (d, J = 2.8 Hz, lactam-NH), 5.95 (q, J = 4.4 Hz, 1H,
CONHMe), 5.35 (s, 1H, NCHpyridyl), 4.07 (dd, J = 9.9 Hz, 3.3 Hz,
1H, NCHindanyl), 3.89 (d, J = 4.3 Hz, 1H, NCHsec-butyl), 3.19−2.71
(m, 8H, indanyl-3H,-1H,-2H, CONHMe), 2.57−2.53 (m, 6H, pyridyl-
2Me, -6Me), 1.82−1.67 (m, 2H, CHHMe, CHMeCH2), 1.20−1.05 (m,
1H, CHHMe), 0.99 (d, J = 7.1 Hz, 3H, CHMe), 0.90 (t, J = 7.3 Hz,
3H, CH2Me). LCMS m/z 463 (MH+) single component, gradient 2
(tR 2.44 min). HRMS calcd for C27H34N4O3 (MH+) 463.27037, found
463.27036. HPLC: 100% (tR 9.793 min).
(4R,6aR,11aS,11bR)-4-Phenyl-3,4,6a,11,11a,11b-hexahydro-
1H-indeno[2′,1′:4,5]isoxazolo[3,2-c][1,4]oxazin-1-one (3). The
nitrone (5R)-5-phenyl-5,6-dihydro-2H-1,4-oxazin-2-one 4-oxide7
2
(0.500 g, 2.615 mmol) and indene (2 mL) were dissolved in dry
chloroform (20 mL), and the mixture was heated at reflux under
nitrogen for 3 h. The mixture was then evaporated under reduced
pressure, and the gummy solid residue was stirred with cyclohexane to
remove excess indene. The crude solid contained the major
cycloadduct 3 and the minor cycloadduct 4 in a ratio of 5:1, LCMS
m/z 308 (MH+) two components, gradient 2 (major component tR
3.19 min, minor component tR 3.27 min). The crude solid was
recrystallized (×2) from ethyl acetate/cyclohexane to give the major
exo cycloadduct 3 as colorless needles (0.302 g, 38%); mp 220−
222 °C (dec). 1H NMR (CDCl3) δ 7.52−7.21 (m, 9H, ArH), 5.75 (d,
J = 7.6 Hz, 1H, ArCHO), 4.40−4.18 (m, 3H, PhCHCH2O), 3.91 (d,
J = 9.1 Hz, 1H, NCHCOO), 3.85−3.77 (m, 1H, ArCH2CH), 3.41−
3.31 (m, 2H, ArCH2CH). LCMS m/z 308 (MH+) single component,
gradient 2 (tR 3.20 min).
The mother liquors from the recrystallization were evaporated
under reduced pressure and a small portion was purified by reverse-
phase HPLC to give an impure sample of the minor endo cycloadduct
4 (contaminated with the exo cycloadduct et al.) as a white solid
(estimated yield of endo cycloadduct from nitrone 8%). LCMS m/z
308 (MH+), gradient 2 (tR 3.27 min). The cycloadducts showed some
instability on silica.
(2R)-Amino(2,3-dihydro-1H-inden-2-yl)ethanoic Acid (5). The
cycloadduct 3 (100 mg, 0.325 mmol) and 20% palladium hydroxide on
carbon (250 mg) were hydrogenated in 1,4-dioxan/trifluoroacetic acid
(10:1 v:v) (11 mL) at 60 psi over atmospheric pressure and 50 °C
using a Parr hydrogenator and a hydrogen generator for 2 h. The crude
reaction mixture was filtered with suction (using a glass microfibre
filter), and the residue was blown down under nitrogen and triturated
under diethyl ether to give 5 as an off-white solid (26 mg, 41%).
LCMS m/z 192 (MH+) single component, gradient 2 (tR 1.32 min).
1H NMR (DMSO-d6) δ 8.42−8.32 (broad s, 3H, exchangeables),
7.26−7.12 (m, 4H, ArH), 4.12−4.06 (m, 1H, NCHCO2), 3.06−
2.80 (m, 5H, Ar(CH2)2CH); only one enantiomer was present as
shown by chiral HPLC comparison with authentic (R)-2-indanylgly-
cine9 and the racemic material (Chirobiotic T column, 25 cm, eluted
with 20% acetonitrile in water containing 0.1% tetraethylammonium acetate
(pH 4.1), single component (tR 5.41 min) (<0.5% (S) enantiomer
present).
(2R)-2,3-Dihydro-1H-inden-2-yl({[(phenylmethyl)oxy]-
carbonyl}amino)ethanoic Acid (6). This was prepared from 5 as
described previously.5
2-{(3R,6R)-3-(2,3-Dihydro-1H-inden-2-yl)-6-[(1S)-1-methyl-
propyl]-2,5-dioxo-1-piperazinyl}-2-(2,6-dimethyl-3-pyridinyl)-
N-(2-hydroxyphenyl)acetamide (42). 2,6-Dimethylpyridine-3-car-
boxaldehyde 19 (2.00 g, 16.1 mmol)) and D-alloisoleucine methyl
ester hydrochloride 11 (2.93 g, 16.1 mmol)) in methanol (50 mL) and
2,2,2-trifluoroethanol (50 mL) were treated with triethylamine
(2.24 mL, 16.1 mmol), and the mixture was stirred under nitrogen
at room temperature for 20 h. (2R)-[(Benzyloxycarbonyl)amino](2,3-
dihydro-1H-inden-2-yl)ethanoic acid 6 (5.24 g, 16.1 mmol) and
2-benzyloxyphenylisonitrile 7 (3.37 g, 16.1 mmol) were added, and the
mixture was stirred at room temperature under nitrogen for 4 days.
The mixture was concentrated under reduced pressure and then
partitioned between ethyl acetate (150 mL) and water (150 mL) plus
saturated aqueous sodium hydrogen carbonate (6 mL). The aqueous
The following compounds 49, 58, and 69 were similarly prepared.
(2R)-2-[(3R,6R)-3-(2,3-Dihydro-1H-inden-2-yl)-6-(2-methyl-
propyl)-2,5-dioxo-1-piperazinyl]-N,N-dimethyl-2-(6-methyl-3-
pyridinyl)ethanamide (49). Similarly prepared as 67, using
dimethylamine and phenol 34, the amide 49 was obtained as a
1
white solid (50%). H NMR (CDCl3) δ 8.58 (d, J = 2.2 Hz, 1H,
pyridyl-2H), 7.66 (dd, J = 8.0 Hz, 2.2 Hz, 1H, pyridyl-4H), 7.26−7.14
(m, 5H, pyridyl-5H, indanyl-arylH), 6.72 (d, J = 4.0 Hz, 1H, lactam-
NH), 6.44 (s, 1H, NCHpyridyl), 4.07 (dd, J = 11.8 Hz, 2.5 Hz, 1H,
NCHisobutyl), 4.00 (dd, J = 10.3 Hz, 4.5 Hz, 1H, NCHindanyl),
3.20−3.03 (m, 3H, indanyl-3H, -1H), 2.99 (s, 3H, CONMeMe),
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2.93−2.74 (m, 4H, CONMeMe, indanyl-2H, indanyl-1H), 2.61 (s, 3H,
pyridyl-6Me), 1.63−1.44 (m, 2H, CHHCHMe2, CH2CHMe2), 0.72−
0.61 (m, 4H, CHHCHMe2, CH2CHMeMe), 0.36 (d, J = 6.5 Hz, 3H,
CH2CHMeMe). LCMS m/z 463 (MH+) single component, gradient 2
(tR 2.92 min). HRMS calcd for C27H34N4O3 (MH+) 463.27037, found
463.27010. HPLC: 100% (tR 10.294 min).
the 2-methyl pyridine 58 (0.232 g, 0.5 mmol) in chloroform (10 mL)
was added to m-chloroperbenzoic acid (∼50%, 0.345 g, ∼1 mmol) and
the reaction mixture stirred at room temperature for 4 h. Methanol
was added and the mixture applied to an aminopropyl SPA column
(10 g), which was eluted with chloroform/methanol (1: 1) to give
after evaporation of the required fractions a white solid (0.300 g). This
was purified on a Redisep silica column (12 g) eluted with ethyl
acetate/methanol (9:1 to 4:1) to give the N-oxide 60 as white solid
(0.172 g, 72%). 1H NMR (CDCl3) δ 8.47 (s, 1H, pyridyl-2H), 7.39 (d,
J = 8.0 Hz, 1H, pyridyl-4H), 7.32 (d, J = 8.0 Hz, 1H, pyridyl-5H),
7.26−7.14 (m, 5H, indanyl-arylH), 6.07 (s, 1H, NCHpyridyl), 4.10−
4.03 (m, 3H, NCHindanyl and NCHsec-butyl), 3.19−3.10 (m, 3H,
indanyl-3H,-1H), 2.98 and 2.96 (2s, 6H, CONMe2), 2.91−2.80 (m,
2H, indanyl-2H, indanyl-1H), 2.55 (s, 3H, pyridyl-6Me), 1.60 (m, 1H,
CHHMe), 1.39 (m, 1H, CHMeCH2), 0.99 (m, 1H, CHHMe), 0.79−
0.73 (m, 6H, CH2Me and CHMe). LCMS m/z 479.4 (MH+) single
component, gradient 2 (tR 2.59 min). HRMS calcd for C27H35N4O4
(MH+) 479.2658, found 479.2656. HPLC: 95% (tR 9.906 min).
{(3R,6R)-3-(2,3-Dihydro-1H-inden-2-yl)-6-[(1S)-1-methyl-
propyl]-2,5-dioxo-1-piperazinyl}(2,6-dimethyl-3-pyridinyl)-
acetic Acid Hydrochloride (70). The phenol 42 (24.25 g,
45 mmol) and carbonyldiimidazole (11.7 g, 72 mmol) were dissolved in
dry dichloromethane (200 mL) and left to stand under nitrogen for
20 h. The solvent was removed in vacuo, and the residue was dissolved
in acetone (200 mL) and 2N hydrochloric acid (20 mL). After stirring
for 20 h, the solvent was removed in vacuo and the residue was
dissolved in methanol (50 mL). The solution was applied to an
aminopropyl cartridge (2 × 70 g) and eluted with methanol (250 mL)
and then 10% acetic acid in methanol (250 mL). The required
fractions were combined and evaporated in vacuo. The residue was
treated with 2N hydrochloric acid and the resulting solution
evaporated in vacuo to give the acid hydrochloride 70 as a tan solid
(2R)-2-{(3R,6R)-3-(2,3-Dihydro-1H-inden-2-yl)-6-[(1S)-1-
methylpropyl]-2,5-dioxo-1-piperazinyl}-N,N-dimethyl-2-(6-
methyl-3-pyridinyl)ethanamide (58). Similarly prepared as 67,
using dimethylamine and phenol 37, the amide 58 was obtained, as a
1
white solid (25%). H NMR (CD3OD) δ 8.57 (d, J = 2.2 Hz, 1H,
pyridyl-2H), 7.83 (dd, J = 8.3 Hz, 2.5 Hz, 1H, pyridyl-4H), 7.43 (d, J =
8.0 Hz, 1H, pyridyl-5H), 7.21−7.08 (m, 4H, indanyl-arylH), 6.31 (s,
1H, NCHpyridyl), 3.95 (d, J = 10.0 Hz, 1H, NCHindanyl), 3.90 (d, J =
5.8 Hz, 1H, NCHsec-butyl), 3.10−3.00 (m, 3H, indanyl-3H, -1H), 2.94
and 2.93 (2s, 6H, CONMe2), 2.87−2.73 (m, 2H, indanyl-2H, indanyl-
1H), 2.58 (s, 3H, pyridyl-6Me), 1.64−1.54 (m, 1H, CHHMe), 1.39−
1.27 (m, 1H, CHMeCH2), 0.92−0.80 (m, 1H, CHHMe), 0.70 (t, J =
7.3 Hz, 3H, CH2Me), 0.58 (d, J = 7.0 Hz, 3H, CHMe). LCMS m/z 463
(MH+) single component, gradient 2 (tR 2.82 min). HRMS calcd for
C27H34N4O3 (MH+) 463.27037, found 463.27018. HPLC: 100% (tR
10.547 min).
The freebase 58 was treated with 2N hydrochloric acid, and the
resulting solution evaporated in vacuo to give the hydrochloride 58B
1
as a white solid. H NMR (DMSO-d6) δ 8.76 (broad s, 1H, pyridyl-
2H), 8.51 (d, J = 3.5 Hz, 1H, lactam-NH), 8.33 (broad d, J = 8.1 Hz,
1H, pyridyl-4H), 7.86 (d, J = 8.5 Hz, 1H, pyridyl-5H), 7.23−7.09 (m,
4H, indanyl-arylH), 6.11 (s, 1H, NCHpyridyl), 3.99 (d, J = 2.5 Hz, 1H,
NCHsec-butyl), 3.81 (dd, J = 8.6 Hz, 3.3 Hz, 1H, NCHindanyl), 3.00−
2.73 (m, 14H, indanyl-3H, -1H, CONMe2, indanyl-2H, indanyl-1H,
pyridyl-6Me), 1.74−1.63 (m, 1H, CHHMe), 1.56−1.46 (m, 1H,
CHMeCH2), 0.98−0.85 (m, 4H, CHHMe), 0.73 (t, J = 7.3 Hz, 3H,
CH2Me). LCMS m/z 463 (MH+) single component, gradient 2 (tR
2.79 min). HPLC: 100% (tR 10.40 min). HRMS calcd for C27H34N4O3
(MH+) 463.27037, found 463.27018; circular dichroism (CH3CN)
λmax227.8 nm, dE −5.31, E5795; λmax255.6 nm, dE 0.77, E3881;
λmax280.0 nm, dE −0.97, E3756.
(3R,6R)-3-(2,3-Dihydro-1H-inden-2-yl)-1-[(1R)-1-(2,6-dimeth-
yl-3-pyridinyl)-2-(4-morpholinyl)-2-oxoethyl]-6-[(1S)-1-methyl-
propyl]-2,5-piperazinedione (69). Similarly prepared as 67, using
morpholine and phenol 42, the amide 69 was obtained, after freeze-
drying from 1,4-dioxane as a white solid (23%). Identical in all respects
with that obtained from acid hydrochloride 70.
1
(12.21 g, 56%). H NMR (CDCl3) δ 8.66, 8.60 (d, 1H, J = 3.8 Hz),
8.19, 7.96 (d, 1H, J = 8 Hz), 7.74, 7.68 (d, 1H, J = 8 Hz), 7.22 (m,
2H), 7.13 (m, 2H), 5.53, 5.24 (s, 1H), 4.24, 3.81 (m, 1H), 4.08−4.00
(m, 1H), 3.13−2.84 (m, 4H), 2.81−2.67 (m, 7H), 2.14, 1.82 (m, 1H),
1.46, 1.38 (m, 1H), 1.12, 0.92 (d, 3H, J = 7 Hz), 1.07 (m, 1H), 0.88,
0.58 (t, 3H, J = 7 Hz). LCMS m/z 450.3 (MH+) single components,
gradient 2 (tR 2.41 min). HRMS calcd for C26H31N3O4 (MH+)
450.23873, found 450.23854. HPLC: two components 48% and 48%
(tR 9.104 and 9.172 min).
The compounds 71 and 72 were similarly prepared; see Supporting
Information for experimental and spectroscopic details.
The compounds 44, 45,47, 48, 50−57, 59, and 61−68 were
similarly prepared; see Supporting Information for experimental and
spectroscopic details.
(3R,6R)-3-(2,3-Dihydro-1H-inden-2-yl)-1-[(1R)-1-(2,6-dimeth-
yl-3-pyridinyl)-2-(4-morpholinyl)-2-oxoethyl]-6-[(1S)-1-methyl-
propyl]-2,5-piperazinedione (69). A suspension of the acid
hydrochloride 70 (5.0 g, 10.3 mmol) in dry dichloromethane
(50 mL) was treated with carbonyldiimidazole (2.6 g, 16 mmol), and
the reaction mixture was stirred under nitrogen for 18 h. Morpholine
(4.8 mL, 55 mmol) was added, and the resultant solution was left
to stand under nitrogen for 18 h. The solvent was removed in vacuo,
and the residue was separated between ethyl acetate and water. The
organic phase was washed with brine and dried over anhydrous
magnesium sulfate. The solvent was removed in vacuo, and the residue
was dissolved in dichloromethane. This was applied to a basic alumina
cartridge (240 g) and eluted using a gradient of 0−7.5% methanol in
diethyl ether (9CV), 7.5−10% methanol in diethyl ether (1CV), and
10% methanol in diethyl ether (1CV). The required fractions were
combined and evaporated in vacuo to give 69 as a white solid (2.4 g,
45%). Recystallisation from ethyl acetate/hexane (1:3) gave colorless
(2R)-2-[(3R,6R)-3-(2,3-Dihydro-1H-inden-2-yl)-6-(2-methyl-
propyl)-2,5-dioxo-1-piperazinyl]-N,N-dimethyl-2-(6-oxo-1,6-di-
hydro-3-pyridinyl)ethanamide (46). A solution of the 2-methox-
ypyridyl derivative 44 (76 mg, 0.165 mmol), sodium iodide (30 mg,
0.2 mmol), and trimethylacetyl chloride (22.4 μL, 0.182 mmol) in dry
acetonitrile (7 mL) was refluxed for 4 h and then water (700 μL) was
added and the mixture stirred for a further 10 min. The mixture was
diluted with dichloromethane (15 mL), washed with 2N HCl (5 mL),
a saturated aqueous solution of sodium hydrogen carbonate (5 mL)
and brine (5 mL), dried (MgSO4), and evaporated to give after freeze-
1
drying from 1,4-dioxane 46 as a white solid (70 mgs, 91%). H NMR
(CDCl3) δ 12.36 (broad s, 1H, pyridone NH), 7.53−7.45 (m, 2H,
pyridone-2H, -4H), 7.38 (broad d, J = 3.0 Hz, lactam-NH), 7.25−7.14
(m, 4H, indanyl-arylH), 6.67 (d, J = 9.5 Hz, pyridone-5H), 6.27 (s, 1H,
NCHpyridone), 4.20 (dd, J = 12.1 Hz, 3.5 Hz, 1H, NCHisobutyl),
3.99 (dd, J = 10.31 Hz, 4.5 Hz, 1H, NCHindanyl), 3.20−2.77 (m, 11H,
indanyl-3H, -1H,-2H, CONMe2), 1.74−1.54 (m, 2H, CHHCHMe2,
CH2CHMe2), 1.01−0.92 (m, 1H, CHHCHMe2), 0.74 (d, J = 6.5 Hz,
3H, CH2CHMeMe), 0.64 (d, J = 6.5 Hz, 3H, CH2CHMeMe). LCMS
m/z 465 (MH+) single component, gradient 2 (tR 2.68 min). HRMS
calcd for C26H32N4O4 (MH+) 465.24963, found 465.24938. HPLC:
100% (tR 9.839 min).
1
needles (75%) mp 140 °C. H NMR (CDCl3) δ 7.49 (d, J = 7.8 Hz,
1H, pyridyl-4H), 7.26−7.15 (m, 4H, indanyl-arylH), 7.10 (d, J =
8.1 Hz, 1H, pyridyl-5H), 6.68 (s, 1H, NCHpyridyl), 6.49 (d, J = 2.8 Hz,
1H, lactam-NH), 4.10 (dd, J = 10.1 Hz, 4.0 Hz, 1H, NCHindanyl),
4.01 (d, J = 4.5 Hz, NCHsec-butyl), 3.75−2.71 (m, 13H, 8×
morpholinyl-H, indanyl-3H, -1H, -2H), 2.62 and 2.58 (2s, 6H, pyridyl-
2Me,-6Me), 1.64−1.52 (m, 1H, CHHMe), 0.98−0.79 (m, 2H,
CHHMe, CHMeCH2), 0.70 (t, J = 7.1 Hz, 3H, CH2Me), 0.45 (d,
J = 6.8 Hz, 3H, CHMe). LCMS m/z 519 (MH+) single component,
(2R)-2-{(3R,6R)-3-(2,3-Dihydro-1H-inden-2-yl)-6-[(1S)-1-
methylpropyl]-2,5-dioxo-1-piperazinyl}-N,N-dimethyl-2-(6-
methyl-1-oxido-3-pyridinyl)ethanamide (60). To a solution of
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Journal of Medicinal Chemistry
Article
gradient 2 (tR 2.70 min). HRMS calcd for C30H38N4O4 (MH+)
519.29658, found 519.29667. HPLC: 100% (tR 10.388 min).
(4) Borthwick, A. D. Oral Oxytocin Antagonists. J. Med. Chem. 2010,
53, 6525−6538.
To a warm solution of 69 (2.66 g, 5.1 mmol) in acetone (40 mL)
was added a solution of benzene sulfonic acid (0.81 g, 5.1 mmol) in
acetone (40 mL), and the resulting solution was heated to boiling and
allowed to cool to room temperature during 48 h. The resulting
crystals were filtered off, air-dried on the filter pad to give the besylate
(5) Borthwick, A. D.; Davies, D. E.; Exall, A. M.; Hatley, R. J. D.;
Hughes, J. A.; Irving, W. R.; Livermore, D. G.; Sollis, S. L.; Nerozzi, F.;
Valko, K. L.; Allen, M. J.; Perren, M.; Shabbir, S. S.; Woollard, P. M.;
Price, M. A. 2,5-Diketopiperazines as Potent, Selective and Orally
Bioavailable Oxytocin Antagonists 3: Synthesis, Pharmacokinetics and
in Vivo Potency. J. Med. Chem. 2006, 49, 4159−4970.
(6) Borthwick, A. D.; Liddle, J. The design of orally bioavailable 2,5
diketopiperazine oxytocin antagonists: from concept to clinical
candidate for premature labour. Med. Res. Rev. 2011, 31, 576−604.
(7) Long, A.; Baldwin, S. W. Enantioselective syntheses of
homophenylalanine derivatives via nitrone 1,3-dipolar cycloaddition
reactions with styrenes. Tetrahedron Lett. 2001, 42, 5343−5345.
(8) Tamura, O.; Gotanda, K.; Yoshino, J.; Morita, Y.; Terashima, R.;
Kikuchi, M.; Miyawaki, T.; Mita, N.; Yamashita, M.; Ishibashi, H.;
Sakamoto, M. Design, Synthesis, and 1,3-Dipolar Cycloaddition of
(5R)-[and (5S)]-5,6-Dihydro-5-phenyl-2H-1,4-oxazin-2-one N-Oxides
as Chiral (E)-Geometry-Fixed α-Alkoxycarbonylnitrones. J. Org. Chem.
2000, 65, 8544−8551.
(9) Borthwick, A. D.; Davies, D. E.; Exall, A. M.; Livermore, D. G.;
Sollis, S. L.; Nerozzi, F.; Allen, M. J.; Perren, M.; Shabbir, S. S.;
Woollard, P. M.; Wyatt. 2,5-Diketopiperazines as Potent, Selective and
Orally Bioavailable Oxytocin Antagonists 2: Synthesis, Chirality and
Pharmacokinetics. J. Med. Chem. 2005, 48, 6956−6969.
(10) Obach, R. S. The prediction of human clearance from hepatic
microsomal metabolism data. Curr. Opin. Drug Discovery Dev. 2001, 4,
36−44.
1
(3.214 g, 92.6%) as white crystals of 69B mp 179−183 °C. H NMR
(CD3OD) δ 8.30 (d, 1H, J = 8.1 Hz, pyridyl-4H), 7.84−7.80 (m, 2H,
PhSO3− 2× ortho-H), 7.78 (d, J = 8.3 Hz, 1H, pyridyl-5H), 7.45−7.38
−
(m, 3H, PhSO3 2× meta-H, para-H), 7.23−7.09 (m, 4H, indanyl-
arylH), 6.08 (broad s, 1H, NCHpyridyl), 4.00 (d, J = 4.6 Hz, 1H,
NCHsec-butyl), 3.92 (d, J = 9.9 Hz, 1H, NCHindanyl), 3.78−3.39 and
3.14−2.80 (m, 13H, 8× morpholinyl-H, indanyl-3H, -1H, -2H)), 2.79
and 2.78 (2s, 6H, pyridyl-2Me, -6Me), 1.85−1.74 (m, 1H, CHHMe),
1.59−1.48 (m, 1H, CHHMe), 1.15−1.01 (m, 1H, CHMeCH2), 0.92
(d, J = 6.3 Hz, 3H, CHMe), 0.85 (t, J = 7.3 Hz, 3H, CH2Me). LCMS
m/z 519 MH+ single components, tR 2.72 min; circular dichroism
(CH3CN) λmax 225.4 nm, dE −15.70, E15086; λmax 276 nm, dE 3.82,
E5172. HRMS calcd for C30H38N4O4 (MH+) 519.2971, found
519.2972. Anal. (C30H38N4O4·C6H6O3S·3.0H2O) C, H, N, S.
The compounds 73−78 were similarly prepared; see Supporting
Information for experimental and spectroscopic details.
ASSOCIATED CONTENT
■
S
* Supporting Information
C, H, N, analysis for compounds 59B, 68, and 69B.
Experimental and spectroscopic details for similarly prepared
compounds 32−41, 43, 44, 45, 47, 48, 50−57, 59, 61−68, 71,
72, and 73−78. Array chemistry for compounds 95−102. This
material is available free of charge via the Internet at http://
pubs.acs.org.
(11) Wyatt, P. G.; Allen, M. J.; Borthwick, A. D.; Davies, D. E.; Exall,
A. M.; Hatley, R. J. D.; Irving, W. R.; Livermore, D. G.; Miller, N. D.;
Nerozzi, F.; Sollis, S. L.; Szardenings, A. K. 2,5-Diketopiperazines as
Potent and Selective Oxytocin Antagonists 1: Identification,
Stereochemistry and Initial SAR. Bioorg. Med. Chem. Lett. 2005, 15,
2579−2582.
(12) McCafferty, G. P.; Pullen, M. A.; Wu, C.; Edwards, R. M.; Allen,
M. J.; Woollard, P. M.; Borthwick, A. D.; Liddle, J.; Hickey, D. M.;
Brooks, D. P.; Westfall, T. D. Use of a novel and highly selective
oxytocin receptor antagonist to characterize uterine contractions in the
rat. Am. J. Physiol. 2007, 293, R299−R305.
(13) Valko, K. Separation Methods in Drug Synthesis and
Purification. In Handbook of Analytical Separations; Valko, K., Ed.;
Elsevier: Amsterdam, 2000; Vol. 1, Chapter 12 , pp 535−583.
(14) Chromatographic hydrophobicity index (CHI) utilizes the
gradient retention time from rapid gradient reverse-phase elution to
measure lipophilicity. See the following: Valko, K.; Du, C. M.; Bevan,
C.; Reynolds, D. P.; Abrahams, M. H. Rapid method for the estimation
of octanol/water partition coefficient (log Poct) from gradient RP-
HPLC retention and a hydrogen bond acidity term (Σα2H). Curr.
Med. Chem. 2001, 8, 1137−1146.
(15) HSA and RSA binding methods using Chromtech Immobilized
HSA and RSA HPLC columns see: Valko, K.; Nunhuck, S.; Bevan, C.;
Abraham, M. H.; Reynolds, D. P. Fast gradient HPLC method to
determine compounds binding to human serum albumin.
Relationships with octanol/water and immobilized artificial
membrane lipophilicity. J. Pharm. Sci. 2003, 92, 2236−2248.
(16) Intrinsic clearance protocol: NADP regeneration buffer for use
in incubations was prepared fresh on the assay day. It contained 7.8 mg
glucose-6-phosphate (monosodium salt), 1.7 mg NADP and 6 units
glucose-6-phosphate dehydrogenase per 1 mL of 2% sodium
bicarbonate. Microsomes (human, female; cynomolgus monkey,
female; dog, female; rat, female) were prepared in pH 7.4 phosphate
buffer. The final protein concentration in the incubation mixture was
0.5 mg/mL. The microsomal solution containing the compound was
transferred to a microplate and was prewarmed at 37 °C prior to
initiation of incubations. All incubations were initiated by addition of
the NADP regeneration system to the prewarmed microsomes and the
mixtures were incubated at 37 °C. Following 0, 3, 6, 12, and 30 min
incubation, aliquots were taken and added to acetonitrile containing an
internal standard. For determination of the rate of metabolism,
AUTHOR INFORMATION
■
Corresponding Author
*For A.D.B.: phone, +44(0)7772112742; E-mail, alan.d.
borthwick@drugmoldesign.com. For J. L.: phone, +44(0)1438
763643; E-mail, John.2.Liddle@GSK.com.
Present Address
▽DrugMolDesign, 15 Temple Grove, London NW11 7UA, U.K.
ABBREVIATIONS USED
■
Cyno, cynomolgus; CHILog D, chromatographic hydrophobic-
ity index logarithm of the distribution coefficient; 2,5-DKP,
2,5-diketopiperazine; FLIPR, fluorometric imaging plate reader;
hOTR, human oxytocin receptor; hV1aR, human arginine
vasopressin receptor 1A; hV1bR, human arginine vasopressin
receptor 1B; hV2R human arginine vasopressin receptor 2;
HSA, human serum albumin; HTFT, high throughput fluctuation
test; MED, minimum effective dose; OT, oxytocin; P450, human
cytochrome P450 enzyme; PK, pharmacokinetics; pKi, −log 10Ki;
SAR, structure−activity relationships
REFERENCES
■
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