Discovery of 1-{4-[1-(2,6-difluorobenzyl)-5-[(dimethylamino)methyl]-3-(6- methoxypyridazin-3-yl)-2,4-dioxo-1,2,3,4-tetrahydrothieno[2,3- d ]pyrimidin-6-yl]phenyl}-3-methoxyurea (TAK-385) as a potent, orally active, non-peptide antagonist of the human gonadotropin-releasing hormone receptor

Article abstract of DOI:10.1021/jm200216q

We previously discovered an orally active human gonadotropin-releasing hormone (GnRH) receptor antagonist, thieno[2,3-d]pyrimidine-2,4-dione derivative 1 (sufugolix). To reduce the cytochrome P450 (CYP) inhibitory activity and improve in vivo GnRH antagonistic activity, further optimization of this scaffold was carried out. We focused our synthetic efforts on chemical modification at the 5 and 3 positions of the thieno[2,3-d]pyrimidine-2,4-dione ring based on computational modeling, which resulted in the discovery of 1-{4-[1-(2,6-difluorobenzyl)-5-[(dimethylamino)methyl]-3-(6-methoxypyridazin-3- yl)-2,4-dioxo-1,2,3,4-tetrahydrothieno[2,3-d]pyrimidin-6-yl]phenyl} -3-methoxyurea (16b) as a highly potent and orally active GnRH antagonist. Compound 16b showed potent in vitro GnRH antagonistic activity in the presence of fetal bovine serum (FBS) without CYP inhibition. Oral administration of 16b maintained the suppressive effect of the plasma luteinizing hormone levels in castrated cynomolgus monkeys at a 3 mg/kg dose for more than 24 h. Compound 16b is currently under clinical development with the code name of TAK-385.

Full text of DOI:10.1021/jm200216q

ARTICLE
pubs.acs.org/jmc
Discovery of 1-{4-[1-(2,6-Difluorobenzyl)-5-[(dimethylamino)methyl]-
3-(6-methoxypyridazin-3-yl)-2,4-dioxo-1,2,3,4-tetrahydrothieno[2,3-d]
pyrimidin-6-yl]phenyl}-3-methoxyurea (TAK-385) as a Potent, Orally
Active, Non-Peptide Antagonist of the Human Gonadotropin-Releasing
Hormone Receptor
Kazuhiro Miwa,*,† Takenori Hitaka,‡ Takashi Imada,§ Satoshi Sasaki,§ Mie Yoshimatsu,‡
Masami Kusaka,† Akira Tanaka,§ Daisuke Nakata,§ Shuichi Furuya,§ Satoshi Endo,‡
Kazumasa Hamamura,‡ and Tomoyuki Kitazaki‡
†CMC Center and ‡Pharmaceutical Research Division, Takeda Pharmaceutical Company, Ltd., 17-85, Jusohonmachi 2-chome,
Yodogawa-ku, Osaka 532-8686, Japan
§Pharmaceutical Research Division, Takeda Pharmaceutical Company, Ltd., 10 Wadai, Tsukuba, Ibaraki 300-4293, Japan
S Supporting Information
b
ABSTRACT: We previously discovered an orally active human gonado-
tropin-releasing hormone (GnRH) receptor antagonist, thieno[2,3-d]
pyrimidine-2,4-dione derivative 1 (sufugolix). To reduce the cytochrome
P450 (CYP) inhibitory activity and improve in vivo GnRH antagonistic
activity, further optimization of this scaffold was carried out. We focused
our synthetic efforts on chemical modification at the 5 and 3 positions of
the thieno[2,3-d]pyrimidine-2,4-dione ring based on computational mod-
eling, which resulted in the discovery of 1-{4-[1-(2,6-difluorobenzyl)-5-
[(dimethylamino)methyl]-3-(6-methoxypyridazin-3-yl)-2,4-dioxo-1,2,3,4-tetrahydrothieno[2,3-d]pyrimidin-6-yl]phenyl}-3-
methoxyurea (16b) as a highly potent and orally active GnRH antagonist. Compound 16b showed potent in vitro GnRH
antagonistic activity in the presence of fetal bovine serum (FBS) without CYP inhibition. Oral administration of 16b maintained the
suppressive effect of the plasma luteinizing hormone levels in castrated cynomolgus monkeys at a 3 mg/kg dose for more than 24 h.
Compound 16b is currently under clinical development with the code name of TAK-385.
’ INTRODUCTION
peptidic antagonists directly lower hormone levels without the
concomitant flare effect.8 However, peptidic antagonists show poor
oral bioavailability and usually need to be administered by daily
subcutaneous injection, intranasal spray, or suitable depot form.
Therefore, orally effective non-peptide GnRH antagonists could
be superior therapeutic agents for clinical applications by over-
coming the above problems.9
Gonadotropin-releasing hormone (GnRH), also known as
luteinizing hormone-releasing hormone (LHRH), plays an im-
portant role in the reproductive system.1 It is a linear decapeptide
that is produced in the hypothalamus and released into the pituitary
in a pulsatile manner. In the pituitary gland, GnRH binds to and
activates the GnRH receptor, which belongs to the class A G-
protein-coupled receptor (GRCR).2 This activation of the Gn-
RH receptor triggers the biosynthesis and release of luteinizing
hormone (LH) and follicle-stimulating hormone (FSH), which
regulate gonadal steroidogenesis and gametogenesis in testes
and ovaries.3 Several peptidic GnRH agonists represented by leu-
prorelin4 paradoxically suppress gonadal steroid production
and subsequently achieve a condition called “chemical castra-
tion” through receptor down-regulation and/or desensitization.5
They have been approved for the clinical treatment of a variety of
endocrine-based diseases such as prostate, ovarian, and breast
cancer as well as endometriosis and uterine leiomyoma.6,7 GnRH
agonists occasionally evoke an initial gonadal hormone surge at-
tributed to overstimulation of the receptor, known as the “flare
effect”, leading to symptom exacerbation. In contrast, GnRH
Several non-peptide GnRH antagonists have been reported from
various research groups;10ꢀ19 however, only a few compounds have
reached clinical trials. We have previously found three cores (1ꢀ3),
thieno[2,3-d]pyrimidine-2,4-dione,11b
thieno[2,3-b]pyridin-4-
one,11a,c and imidazo[1,2-a]pyrimidine-5-one,11d as non-peptide
GnRH receptor antagonists (Figure 1). In particular, the thieno-
[2,3-d]pyrimidine-2,4-dione core acting as a bicyclic scaffold could
mimic a type II β-turn involving residues 5ꢀ8 (Tyr-Gly-Leu-Arg)
of the human GnRH receptor, and structureꢀactivity relationship
(SAR) studies led to identification of 1 (sufugolix) bearing the
unique p-(3-methoxyureido)phenyl group at the 6-position of the
Received: February 25, 2011
Published: June 09, 2011
r
2011 American Chemical Society
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Figure 1. Representative potent small molecule GnRH receptor antagonists.
Figure 2. Binding model of sufugolix 1 in the human GnRH receptor. A
three-dimensional model of the human GnRH receptor in complex with
compound 1 is viewed from the extracellular side. Transmembrane helices
(TM1ꢀTM7) and extracellular loops (ECL1ꢀECL3) are depicted as
green and orange, respectively. Compound 1 is drawn with atoms in gray
(carbon), blue (nitrogen), red (oxygen), yellow (sulfur), and light green
(fluorine). The receptor residues participating in the specific interactions
with 1 areshown asball-and-stickrepresentationwith similarcoloringto1.
The putative intermolecular hydrogen bonds are depicted as cyan lines.
This figure was produced using MOLMOL.25
Figure 3. Chemical modification of sufugolix 1.
scaffold, CYP3A4 inhibition of the selected candidate 1 was weak
but stillremained (36% inhibition at10 μM). Additionally, binding
affinityof 1 was reduced significantlyinthepresence of fetalbovine
serum (FBS) (IC50 = 1.6 nM), in comparison with results in the
absence of FBS (IC50 = 0.1 nM). Therefore, our goal was to
discover and developanorallyactive GnRH antagonistwithpotent
activity, even in the presence of FBS, and with no CYP3A4
inhibition.
We judged that our thieno[2,3-d]pyrimidine-2,4-dione deri-
vatives with potent GnRH anatagonistic activities had high log D
values, which resulted in the potent CYP3A4 inhibitory activities
observed. Thus, we speculated that decreasing the log D values
for our compounds could reduce CYP3A4 inhibition. In order to
reduce the lipophilicity of 1 while retaining high GnRH anatago-
nistic activity, we adopted a strategy using the molecular modeling
of 1 interacting with human GnRH receptor (Figure 2). Our
model was constructed based on the crystal structure of rhodopsin,
core ring.11b Compound 1 showed good in vitro and in vivo GnRH
antagonistic activities20 and therefore was selected as a first clinical
candidate. Recently, compound 4 (elagolix, Figure 1), based on a
series of uracil derivatives, demonstrated suppression of LH in
castrated macaques by oral administration13 and entered clinical
studies for the treatment of endometriosis.
Inhibition of the cytochrome P450 (CYP) 3A4 enzyme is well-
known to potentially induce drugꢀdrug interactions. Our early
thieno[2,3-d]pyrimidine-2,4-dione derivatives had a common is-
sue of CYP3A4 inhibition. In a series of derivatives based on this
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Scheme 1a
a Reagents: (a) 10% Pd/C, H2, MeOH, 96%; (b) R1-X, iPr2NEt, KI, DMF.
whichis a member ofthe largest subfamily constituting∼90% of all
GPCRs. According to this model, the terminal methoxyureido
group at the 6-position of thieno[2,3-d]pyrimidine-2,4-dione ring
interacts with both Gln106 and Asn102 of the GnRH receptor,
resulting in high binding affinity.11b Other important interactions
were found as follows: a hydrophobic interaction between the 2,6-
difluorobenzyl group at the 1-position and Tyr283, and a hydro-
gen-bonding interaction between the basic tertiary amine at the
5-position and Asp302. These two interactions of the 2,6-difluoro-
benzyl and N-methylamino groups with GnRH receptor also
contributed to the high binding affinity (Figure 3). In contrast,
the two phenyl rings (R1 and R2) at the 5 and 3 positions of
the thieno[2,3-d]pyrimidine-2,4-dione ringdonotappeartomake
important interactions with the receptor in our model, and we
predicted that these regions had steric space available for the
introduction of various sizes of substituents. Thus, our synthetic
efforts focused on optimization of these phenyl rings at R1 and R2
in 1 to avoid CYP3A4 inhibition and enhance antagonistic activity,
and we examined the introduction of substituents more polar
than phenyl at these positions.
On the other hand, during our studies, Betz et al. published
detailed mutagenesis data on the human GnRH receptor.21 Their
data clearly showed the importance of Asp302 and aromatic
residues such as Tyr283/284 in the binding of 1. They also
indicated that a cluster of residues in the transmembrane helix
7 form the binding site of 1. These observations are consistent
with our binding model presented in Figure 2. Although they
argued that the methoxyureido part of 1 interacts with Asp302,
their data did not exclude the possibility of the interaction with
Asn102. We think that the binding site of the methoxyureido part
of 1 remains to be elucidated. In the meantime, our hypothetical
binding model was used to gain insight into the design of non-
peptide GnRH antagonists as described here.
’ CHEMISTRY
Modification at the 5-position of the thieno[2,3-d]pyrimidine-
2,4-dione ring was carried out accordingto Scheme 1 startingfrom
1 in two steps. Thus, reductive debenzylation using palladiumꢀ
carbon under hydrogen atmosphere and subsequent alkylation
with various halides or mesylates provided the corresponding R1-
substituted derivatives 6aꢀh.
The synthesis of R2-substituted compounds 15 and 16 is
outlined in Scheme 2. Ethoxycarbonylation of 2-aminothiophene
7
11b followed by alkylation with 2,6-difluorobenzylchloride gave
N,N-disubstituted amine 9. Bromination of 9 with N-bromosuc-
cinimide (NBS) and 2,20-azobis(isobutyronitrile) (AIBN) pro-
ceeded at the 3-methyl group, and the resulting bromomethyl
derivative 10 was reacted with N-(2-methoxyethyl)methylamine
to give tertiary amine 11. Hydrogenation of nitro compound 11
was then carried out. Aniline 12 was converted to urea 13 by a
two-step methodology. Thus, reaction of 12 with 1,10-carbonyl-
diimidazole (CDI) afforded the intermediary imidazolide, and
subsequent nucleophilic substitution with N-methoxyamine hy-
drochloride gave 13 in a one-pot reaction. Next, selective alkaline
hydrolysis of ethyl ester yielded thiophene-3-carboxylic acid 14.
Condensation reaction of 14 with various amines using diethyl
phosphorocyanidate (DEPC) followed by intramolecular cycli-
zation under basic conditions using sodium methoxide provided
the corresponding thieno[2,3-d]pyrimidine-2,4-dione derivatives
15aꢀj. Finally, the (2-methoxyethyl)(methyl)aminomethyl group
at the 5-position of 15i and 15j was converted to a dimethyl-
aminomethyl group in two steps. Thus, reaction of 15i and 15j
with 1-chloroethylchloroformate afforded the intermediary quar-
ternary ammonium salts, and subsequent nucleophilic substitution
with dimethylamine gave the 5-dimethylaminomethyl derivatives
16a and 16b, respectively.
In this article, we report the discovery of a highly potent and
orally active GnRH antagonist 16b (TAK-385) as a clinical
candidate without CYP3A4 inhibition and with improved in vivo
efficacy.
’ RESULTS AND DISCUSSION
In Vitro Activities and CYP3A4 Inhibition. As a primary
assay for establishing SAR studies, all synthesized compounds
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Scheme 2a
a Reagents: (a) ClCO2Et, toluene, 94%; (b) 2,6-difluorobenzylchloride, K2CO3, KI, DMF, 93%; (c) NBS, AIBN, EtOAc, quant; (d) MeOCH2CH2NHMe,
iPr2NEt, DMF, quant; (e) 10% Pd/C, H2, EtOH, 97%; (f) (1) CDI, iPr2NEt, CH2Cl2, (2) MeONH2 HCl, iPr2NEt, 89%; (g) 2 N NaOH, EtOH, 96%;
3
(h) (1) R2NH2, DEPC, iPr2NEt, DMF, (2) MeONa, MeOH; (i) (1) ClCO2CH(Cl)CH3, CH2Cl2, (2) Me2NH/THF, iPr2NEt, DMF.
were evaluated for their in vitro binding affinities, using cloned
Chinese hamster ovary (CHO) cells expressing the human GnRH
receptor and [125I]leuprorelin as a ligand, expressed as IC50.11a All
compounds were also tested for their ability to inhibit ligand
binding to the CYP3A4 enzyme in vitro assay, expressed as the
percent inhibition at 10 μM. For in vitro antagonistic activity, key
compounds were evaluated for their inhibitory potencies of
GnRH-stimulated arachidonic acid (AA) release from CHO cells
expressing the human GnRH receptor, expressed as IC50.11a
Compound log D values were determined at pH 7.4 by a reported
method.22
First, thieno[2,3-d]pyrimidine-2,4-diones having substituents
(R1) at the 5-(aminomethyl) group were investigated, and the
results are shown in Table 1. As expected, introduction of various
substituents such as pyridyl ring, amide group, or hydrophilic side
chain into the R1 region was tolerated in terms of activity. All com-
pounds (6aꢀh) showed high binding affinities (IC50 e 0.3 nM),
equivalentto sufugolix (1), over a wide range of log D(2.12ꢀ3.15).
Substitution for phenyl with pyridyl brought a decrease of log D;
however, CYP3A4 inhibition was not eliminated (6a, 33%; 6b,
66% inhibition at 10 μM). Extension of the methylene linker in
6a and incorporation of a hydroxymethyl group into the pyridyl
ring of 6a also did not reduce the CYP3A4 inhibitory activity (6c,
64%; 6d, 45% inhibition at 10 μM), although their log D values
decreased. In addition, replacement of the pyridyl ring with a
triazolyl ring or cyclic carboxamide group was not effective for
removal of CYP inhibition. On the other hand, introduction of an
alkylsulfamoyl or methoxyethyl group resulted in the reduction
of CYP3A4 inhibitory activities along with a decrease of log D
(6g, 17%; 6h, 14% inhibition at 10 μM). As a result, compounds
bearing a linear hydrophilic substituent with low log D seem to be
preferable. These results suggest that not only an increase in
hydrophilicity (decreasing log D) but also the size and shape of
the substituent in the R1 position contribute to the reduction of
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Table 1. In Vitro Activities and CYP3A4 Inhibitory Activities of Compounds 1 and 6aꢀh
a Binding affinity is reported as IC50, which is the antagonist concentration required to inhibit the specific binding of [125I]leuprorelin to human
GnRH receptor by 50%. CHO cells expressing human GnRH receptor were used as the source for GnRH receptor. All data are expressed as the mean
of two or three determinations. b Inhibition of GnRH-stimulated AA release from CHO cells expressing human GnRH receptor was measured to
evaluate the GnRH antagonistic activities of the test compounds. The IC50 is the antagonist concentration required to inhibit the GnRH-stimulated
c
AA release from CHO cells by 50%. All data are expressed as the mean of the two or three determinations. Measured at pH 7.4. d ND = not
determined.
CYP3A4 inhibitory activity. Compound 6h also showed potent
antagonistic activity compared with 1 (6h, IC50 = 0.08 nM).
Next, the effect of modifying the 3-substituent (R2) on the
thieno[2,3-d]pyrimidine-2,4-dione ring of 6h was investigated,
and the results are shown in Table 2. Compounds 15aꢀd, bearing
an alkoxy or hydroxyalkyl group at the R2 position, maintained
high binding affinities (IC50 = 0.2ꢀ0.3 nM). CYP3A4 inhibition
for 15aꢀd (8ꢀ30% inhibition at 10 μM) with a linear sub-
stituent was almost comparable to that for 6h, and the lower
log D (1.38) compound 15c showed 23% CYP3A4 inhibition at
10 μM. This result suggests that a linear hydrophilic substituent
at the R2 position does not seem to contribute to a major decrease
in CYP3A4 inhibition, and this encouraged us to introduce more
polar aromatic heterocycles into the 3-position. Among several
five- and six-membered heterocycle derivatives synthesized, pyr-
idine derivatives were preferred for showing high binding affinities.
Substitution of a phenyl group with a pyridyl ring, 15e, provided a
lower log D (1.62) and equally lower CYP3A4 inhibitory activity
(10% inhibition at 10 μM) while maintaining high binding affinity
(15e, IC50 = 0.2 nM). In addition, the introduction of methyl
group into the para-position on the pyridyl ring of 15e resulted
in a slight increase in antagonistic activity while maintaining a
lower CYP3A4 inhibitory activity. For these pyridine derivatives,
when log D is below 2, CYP3A4 inhibitory activity tended to be
less than 10% at 10 μM; however, antagonistic activities were
generally weaker than that of 6h. We found that the introduction
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Table 2. In Vitro Activities and CYP3A4 Inhibitory Activities of Compounds 15aꢀg
a Binding affinity is reported as IC50, which is the antagonist concentration required to inhibit the specific binding of [125I]leuprorelin to human GnRH
receptor by 50%. CHO cells expressing human GnRH receptor were used as the source for GnRH receptor. All data are expressed as the mean of two or
three determinations. b Inhibition of GnRH-stimulated AA release from CHO cells expressing human GnRH receptor was measured to evaluate the
GnRH antagonistic activities of the test compounds. The IC50 is the antagonist concentration required to inhibit the GnRH-stimulated AA release from
CHO cells by 50%. All data are expressed as the mean of the two or three determinations. c Measured at pH 7.4. d ND = not determined.
of a hydrophilic heterocyclic substituent into the 3-position led to
a reduction in CYP3A4 inhibitory activity with a corresponding
decrease in log D.
attributed to their lower lipophilicities (log D = 2.27 for 6h and
log D = 1.62 for 15e). It is known that high lipophilicity is likely to
result in high plasma protein binding; in fact, compound 1 showed
98% plasma protein binding. Therefore, the activity of the less
lipophilic compounds is more effectively translated into in vitro
binding affinity and antagonistic activity in the presence of FBS.
As for antagonistic activity, the pyridine derivative bearing a methyl
group, 15f, showed about 2-fold improvement in activity ratio
compared with 15e (ratio of 7.2 for 15f and 16 for 15e). In
addition, introduction of a methoxy group into the para position
of the phenyl ring in 6h led to significant improvement in antago-
nistic activity. Thus, the activity ratio for p-methoxyphenyl
derivative 15h was reduced from 17-fold to 2.5-fold and the IC50
of 15h was found to be 0.43 nM in the presence of FBS. On the
basis of these SAR data, we next incorporated a methoxy group
into the para position of a pyridine derivative. Compound 15i
showed potent antagonistic activity (IC50 = 0.49 nM in the
presence of FBS) with negligible CYP3A4 inhibitory activity (7%
inhibition at 10 μM). Considering the improvement found for
the reduction of CYP3A4 inhibition, substitution of a pyridazine
ring for the pyridine ring was performed to reduce logD. The
pyridazine derivative 15j maintained high antagonistic activity
(IC50 = 0.94 nM in the presence of FBS), even though weak
CYP3A4 inhibitory activity remained (9% inhibition at 10 μM).
To further reduce log D and molecular weight, conversion of the
N-2-methoxyethyl group to the smaller N-methyl group at R1 in
15i and 15j was carried out. Dimethylaminomethyl derivatives
Effect of FBS on In Vitro Activities. During the course of our
preclinical development program of GnRH antagonists, in pa-
rallel with the above screening we improved our in vitro assay
methods in order to better mimic our in vivo evaluation system.
Thus, measurements of binding affinity and antagonistic activity
were carried out in the presence of 40% FBS. Unexpectedly, the
binding affinity of 1 was reduced significantly in the presence of
FBS (IC50 = 1.6 nM) in contrast to its strong affinity in the
absence of FBS (IC50 = 0.10 nM) (Table 3). The antagonistic
activity of 1 also decreased 32-fold under the influence of FBS,
suggesting that the apparent antagonistic activity of 1 would not
translate to our in vivo evaluation systems.
We searched for relationships between chemical structure and
the extent of this FBS-induced activity decrease for our 3,5-
disubstituted thieno[2,3-d]pyrimidine-2,4-dione series, and the
results of representative compounds are shown in Table 3. The
ratios of binding affinities and antagonistic activities in the presence
and absence of FBS for 6h (5.0 and 17) were improved 3- and
2-fold compared to compound 1 (ratio of 16 and 32), respec-
tively. Pyridine derivative 15e showed further improvement in
the binding affinity ratio (ratio of 1.5), with high binding affinity
even in the presence of FBS (IC50 = 0.30 nM). These results
suggest thatthe decreasein theinfluence of FBSon GnRH binding
affinities and antagonistic activities for certain compounds can be
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Table 3. Binding to GnRH Receptor and Inhibitory Effects of Compounds 1, 6h, 15e,f,hꢀj, and 16a,b on GnRH-Stimulated
Arachidonic Acid (AA) Release in the Presence or Absence of FBS
a Binding affinity is reported as IC50, which is the antagonist concentration required to inhibit the specific binding of [125I]leuprorelin to human GnRH
receptor by 50%, in the presence or absence of FBS. All data are expressed as the mean of two or three determinations. b Inhibition of GnRH-stimulated
AA release from CHO cells expressing human GnRH receptor was measured to evaluate the GnRH antagonistic activities of the test compounds, in the
presence or absence of FBS. The IC50 is the antagonist concentration required to inhibit the GnRH-stimulated AA release from CHO cells by 50%. All
data are expressed as the mean of two or three determinations. c Measured at pH 7.4. d IC50(40% FBS)/IC50(0% FBS).
16a and 16b showed potent binding affinities and antagonistic
activities comparable to 15i and 15j. Of particular note was pyr-
idazine derivative 16b, which exhibited no CYP3A4 inhibitory
(IC50 = 9800 nM). In contrast, the antagonistic in vitro activity of
16b with respect to the human receptor (IC90 = 18 nM) exceeded
that for the monkey receptor (IC90 = 1700 nM) by 95-fold in the
presence of 40% serum. Thus, compound 16b showed much
more potent activity than 1, giving the expectation that signifi-
cant suppression of LH and testosterone levels may be found in
clinical studies.
As for binding specificity, 16b displayed no significant activity
in 134 different enzyme and radioligand binding assays (<60%
inhibition at 10 μM).
activity (0%) at 10 μM with potent antagonistic activity (IC50
=
0.82 nM) in the presence of FBS. The elimination of CYP3A4
inhibitory activity and improvement in antagonistic activity can
be attributed to its low log D (1.27) and plasma protein binding
(71%). Among the compounds prepared, 16b exhibited the best
overall profile in terms of in vitro GnRH antagonistic activity and
lack of CYP inhibitory activity. Therefore, this compound, 16b,
was selected as a candidate for further investigation.
In Vitro Species Specificity. Species specificities for the
binding affinity and antagonistic activity of 16b were investigated
in a manner similar to that described above. The results are shown
in Table 4. For the monkey receptor, 16b exhibited strong binding
affinity (IC50 = 0.32 nM) comparable to that for the human
receptor while displayinga 30000-fold decrease for the rat receptor
In Vivo Studies. The pharmacokinetic profile of 16b was
evaluated after single oral administration to male cynomolgus
monkeys. As shown in Table 5, Cmax, Tmax, and AUC0ꢀ24h were
16.0 ng/mL, 2.7 h, and 90.1 ng h/mL, respectively, at a dose of
3
1 mg/kg. Although the plasma concentration of 16b at a dose of
3 mg/kg showed an increase over the dose ratio, the value does
not represent a marked variability. In consideration of the in vitro
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Table 4. Species Specificities for Binding to GnRH Receptor
and Inhibitory Effects on GnRH-Stimulated AA Release of
Compounds 1 and 16b
LH levels is illustrated in Figure 4. As expected, a single oral
administration of 16b at doses of 1 and 3 mg/kg exhibited clear
suppressive effects on circulating LH levels in monkeys, and the
suppressive effects lasted for more than 24 h (1 mg/kg) and 48 h
(3 mg/kg). The suppressive effect of 16b at a 1 mg/kg dose was
comparable to that of 1 at a 10 mg/kg dose, and the minimum
effective dose of 16b was 1 mg/kg, which is 10-fold lower than that
of 1.11b It is worth noting that exposure to 16b at a dose as low as
1 mg/kg dose resulted in effective suppression. The potent
antagonistic in vivo activity of 16b may be attributable to its potent
in vitro activity. Consequently, compound 16b dose-dependently
exerted a rapid and prolonged suppressive effect on plasma LH
levels in castrated monkeys. These results clearly demonstrate that
16b is a potent and orally active GnRH antagonist. Moreover, the
large difference between in vitro antagonistic activities of 16b
toward human and monkey receptors implies that a much smaller
dose of 16b should be sufficient to suppress plasma LH levels
in human.
binding affinitya
inhabition of AA releasea
IC50 (nM) (40% FBS)b IC90 (nM) (40% human or monkey serum)c
monkeyd
ratd
human
monkey
1
1.6
1.3
270
18
13694
1700
16b
0.33
0.32
a All data are expressed as the mean of the two or three determinations.
b Binding affinity is reported as IC50, which is the antagonist concentra-
tion required to inhibit the specific binding of [125I]leuprorelin to GnRH
c
receptor by 50%, in the presence of 40% FBS. Inhibition of GnRH-
stimulated AA release from CHO cells expressing human or monkey
GnRH receptor was measured to evaluate the GnRH antagonistic
activities of the test compounds. The IC90 is the antagonist concentra-
tion required to inhibit the GnRH-stimulated AA release from CHO
cells by 90% in the presence of 40% human or monkey serum. d CHO
cells expressing human, monkey, or rat GnRH receptor were used as the
source for the GnRH receptor.
’ CONCLUSION
To develop our nonpeptidic orally active GnRH antagonist,
we focused on optimization at the thieno[2,3-d]pyrimidine-2,4-
dione ring 5 and 3 positions based on computational modeling
results. Introduction of polar substituents gave compounds with
log D of 2 or lower, resulting in decreased CYP3A4 inhibition while
maintaining potent GnRH antagonistic activity. In particular, a
heterocycle at the 3-position was found to be an effective sub-
stituent, giving desirable levels of both reduced CYP3A4 inhibitory
activity and potent GnRH antagonistic activity in the presence of
FBS. Among the compounds synthesized, 16b showed high antag-
onistic activity in the presence of serum without any CYP3A4 inhibi-
tion. Furthermore, 16b exhibited a good pharmacokinetic profile
and obvious suppressive effects of circulating LH levels in monkeys
at a dose of 1 mg/kg. Compound 16b was selected as a candidate for
development and is under clinical study. We strongly believe that
16b represents a novel compound that will be a useful therapeutic
tool for the treatment of sex-hormone-dependent diseases.
Table 5. Oral Absorption of Compound 16b in Cynomolgus
Monkeysa
1 mg/kg (po)
3 mg/kg (po)
Cmax
Tmax
AUC
Cmax
Tmax
AUC
(ng/mL)
(h)
(ng h/mL) (ng/mL)
(h)
(ng h/mL)
3
3
16.0
2.7
90.1
112.8
2.0
533.1
a See Experimental Section.
’ EXPERIMENTAL SECTION
Melting points were determined on a Yanagimoto micro melting
point apparatus or a SRS OptiMelt melting point apparatus and are
uncorrected. 1H NMR spectra were measured with a Varian Gemini-200
(200 MHz) or a Varian Mercury-300 (300 MHz) spectrometer. Chemical
shifts are given in δ values (ppm) using tetramethylsilane as the internal
standard, and coupling constants (J) are given in hertz (Hz). Peak multi-
plicities and abbreviations are expressed as follows: s, singlet; d, doublet; t,
triplet; dd, double doublet; dt, double triplet; q, quartet; m, multiplet; br s,
broad singlet. Elemental analyses were carried out by Takeda Analytical
Research Laboratories Ltd., and the results were within (0.4% of the
theoretical values. HPLC analyses were performed on an Agilent HP1100
system. Chromatographic separations were carried out on silica gel
(Kieselgel 60, 63ꢀ200 mesh, Merck) or basic silica gel (Chromatorex
NH-DM1020, 100ꢀ200 mesh, Fuji Silysia Chemical Ltd.) using the
indicated eluents. Yields were not optimized. Purity of compounds (>95%)
was established by elemental analyses and HPLC analyses. The data of
elemental analysis and HPLC analyses are in Supporting Information.
1-(4-{1-(2,6-Difluorobenzyl)-5-[(methylamino)methyl]-2,4-
dioxo-3-phenyl-1,2,3,4-tetrahydrothieno[2,3-d]pyrimidin-
6-yl}phenyl)-3-methoxyurea (5). A mixture of 1-{4-[5-{[benzyl-
(methyl)amino]methyl}-1-(2,6-difluorobenzyl)-2,4-dioxo-3-phenyl-
1,2,3,4-tetrahydrothieno[2,3-d]pyrimidin-6-yl]phenyl}-3-methoxyurea
Figure 4. Time course of plasma LH concentrations in castrated
cynomolgus monkeys after oral administration of compound 16b at
doses of 1 mg/kg (red square) and 3 mg/kg (green triangle). Figures are
percentages of LH concentration at the indicated times, with LH
concentration at 0 h (pretreatment) taken as 100% for each group.
potency of 16b, this PK profile suggests that 16b is a promising
compound for exerting potent in vivo efficacy when administered
orally.
Finally, the in vivo antagonistic activity of 16b was evaluated in
castrated cynomolgus monkeys, and the suppression of plasma
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Journal of Medicinal Chemistry
ARTICLE
(1) (6.68 g, 10.0 mmol), 10%Pd/C(50% wet, 2.23 g), 1 N HCl (11.0 mL,
11.0 mmol), and MeOH (150 mL) was stirred under H2 atmosphere
(1 atm) at room temperature for 2 h. The mixture was filtered through
Celite, and the filtrate was concentrated in vacuo. The residue was diluted
with 1 N NaOH and extracted with EtOAc and THF. The extract was
washed with brine, dried (MgSO4), and concentrated in vacuo. The
residue was washed with Et2O and diisopropyl ether to give 5 (5.52 g,
96%) as a white powder. 1H NMR (CDCl3): δ 2.35 (3H, s), 3.76 (2H, s),
3.82 (3H, s), 5.37 (2H, s), 6.92 (2H, t, J = 8.2 Hz), 7.25ꢀ7.70 (12H, m).
1-{4-[1-(2,6-Difluorobenzyl)-5-{[methyl(pyridin-2-ylme-
thyl)amino]methyl}-2,4-dioxo-3-phenyl-1,2,3,4-tetrahy-
drothieno[2,3-d]pyrimidin-6-yl]phenyl}-3-methoxyurea (6a).
A mixture of 5 (350 mg, 0.61 mmol), 2-chloromethylpyridine hydrochlor-
ide (149 mg, 0.91 mmol), and N,N-diisopropylethylamine (0.32 mL,
1.82 mmol) in DMF (8 mL) was stirred at room temperature for 14 h.
Thereaction mixture was diluted with saturated aqueous NaHCO3 solution
and extracted with EtOAc. The extract was dried (MgSO4) and concen-
trated in vacuo. The residue was purified by basic silica gel column
chromatography (EtOAc) to afford 6a (297 mg, 73%) as colorless crystals,
mp 181ꢀ182 °C. 1H NMR (CDCl3): δ 2.10 (3H, s), 3.71 (2H, s), 3.83
(3H, s), 3.99 (2H, s), 5.36 (2H, s), 6.92 (2H, t, J = 8.2 Hz), 7.00ꢀ7.10 (1H,
m), 7.15 (1H, s), 7.20ꢀ7.35 (4H, m), 7.40ꢀ7.65 (9H, m), 8.40ꢀ8.50 (1H,
m). IR (KBr): 1715, 1667, 1532, 1472, 735 cmꢀ1. LCꢀMS m/z: 669.0
Yield 89%, white crystals, mp 161ꢀ163 °C. 1H NMR (CDCl3): δ 2.14
(3H, s), 2.64 (2H, t, J = 5.9 Hz), 3.27 (3H, s), 3.41 (2H, t, J = 5.9 Hz),
3.83 (5H, s), 5.37 (2H, s), 6.93 (2H, t, J = 8.2 Hz), 7.12ꢀ7.63 (12H, m).
IR (KBr): 1709, 1663, 1560, 1522 cmꢀ1. LCꢀMS m/z: 636.0 [M + H+],
633.9 [M + Hꢀ]. Anal. (C32H31F2N5O5S 0.5H2O) C, H, N.
3
1-{4-[1-(2,6-Difluorobenzyl)-5-({methyl[2-(2-oxopyrroli-
din-1-yl)ethyl]amino}methyl)-2,4-dioxo-3-phenyl-1,2,3,4-
tetrahydrothieno[2,3-d]pyrimidin-6-yl]phenyl}-3-methoxy-
urea (6f). To an ice-cooled solution of 1-(hydroxyethyl)pyrrolidine-2-
one (0.50 g, 3.85 mmol) and triethylamine (0.58 mL, 4.16 mmol) in
THF (5 mL) was added methanesulfonyl chloride (0.27 mL, 3.49 mmol).
After being stirred at room temperature for 1 h, the mixture was diluted
with saturated aqueous NaHCO3 solution and extracted with EtOAc.
The extract was dried (MgSO4) and concentrated in vacuo. The residue
was dissolved in DMF (8 mL), and to this solution were added 5
(404 mg, 0.7 mmol), N,N-diisopropylethylamine (0.24 mL, 1.38 mmol),
and potassium iodide (465 mg, 2.80 mmol). Afterbeing stirredat 50°C for
24 h, the mixture was diluted with water and extracted with EtOAc. The
extract was dried (MgSO4) and concentrated in vacuo. The residue was
purified by basic silica gel column chromatography (EtOAcꢀMeOH,
40:1) to afford 6f (290 mg, 60%) as colorless crystals, mp 134ꢀ136 °C.
1H NMR (CDCl3): δ 1.70ꢀ1.85 (2H, m), 2.14 (3H, s), 2.21 (2H, t,
J = 8.1 Hz), 2.54 (2H, t, J = 6.2 Hz), 3.19 (2H, t, J = 7.0 Hz), 3.29 (2H, t,
J = 6.2 Hz), 3.77 (2H, s), 3.83 (3H, s), 5.38 (2H, s), 6.93 (2H, t, J = 8.1
Hz), 7.18 (1H, s), 7.25ꢀ7.7 (11H, m). IR (KBr): 1715, 1672, 1530,
1470, 1323, 1238, 1032, 735 cmꢀ1. LCꢀMS m/z: 689.1 [M + H+], 687.0
[M + H+], 667.0 [M + Hꢀ]. Anal. (C35H30F2N6O4S 0.5H2O) C, H, N.
3
Compounds 6b,d,e,g,h were prepared by a procedure similar to that
described for 6a, and their physicochemical data are shown below.
1-{4-[1-(2,6-Difluorobenzyl)-5-{[methyl(pyridin-3-ylme-
thyl)amino]methyl}-2,4-dioxo-3-phenyl-1,2,3,4-tetrahy-
drothieno[2,3-d]pyrimidin-6-yl]phenyl}-3-methoxyurea (6b).
Yield 67%, colorless crystals, mp 184ꢀ185 °C. 1H NMR (CDCl3): δ 2.04
(3H, s), 3.58 (2H, s), 3.83 (3H, s), 3.92 (2H, s), 5.37 (2H, s), 6.92 (2H, t,
J = 8.2 Hz), 7.05ꢀ7.70 (14H, m), 8.35ꢀ8.45 (2H, m). IR (KBr): 1713,
1669, 1532, 1464, 1329, 1238, 1032, 787 cmꢀ1. LCꢀMS m/z: 669.1 [M +
[M + Hꢀ]. Anal. (C35H34F2N6O5S 0.5H2O) C, H, N.
3
1-{4-[1-(2,6-Difluorobenzyl)-5-{[methyl(2-pyridin-2-yle-
thyl)amino]methyl}-2,4-dioxo-3-phenyl-1,2,3,4-tetrahy-
drothieno[2,3-d]pyrimidin-6-yl]phenyl}-3-methoxyurea (6c).
Compound 6c was preparedbya procedure similarto that described for 6f
in 56% yield as colorless crystals, mp 159ꢀ160 °C. 1H NMR (CDCl3): δ
2.20 (3H, s), 2.85 (4H, s), 3.82 (5H, s), 5.37 (2H, s), 6.93 (2H, t, J = 8.2
Hz), 6.95ꢀ7.10 (2H, m), 7.14 (1H, s), 7.20ꢀ7.55 (11H, m), 7.60 (1H, s),
8.43 (1H, d, J = 4.0 Hz). IR (KBr): 1717, 1667, 1530, 1470, 1331, 1236,
1030, 735 cmꢀ1. LCꢀMS m/z: 683.1 [M + H+], 681.0 [M + Hꢀ]. Anal.
(C36H32F2N6O4S) C, H, N.
H+], 667.0 [M + Hꢀ]. Anal. (C35H30F2N6O4S 0.3H2O) C, H, N.
3
1-{4-[1-(2,6-Difluorobenzyl)-5-{[{[6-(hydroxymethyl)py-
ridin-2-yl]methyl}(methyl)amino]methyl}-2,4-dioxo-3-ph-
enyl-1,2,3,4-tetrahydrothieno[2,3-d]pyrimidin-6-yl]phenyl}-3-
methoxyurea (6d). Yield 63%, colorless crystals, mp 178ꢀ180 °C. 1H
NMR (CDCl3): δ 2.13 (3H, s), 3.72 (2H, s), 3.83 (3H, s), 3.97 (2H, s),
4.05ꢀ4.10 (1H, m), 4.65ꢀ4.75 (2H, m), 5.37 (2H, s), 6.93 (2H, t, J = 8.2
Hz), 6.90ꢀ7.05 (1H, m), 7.10ꢀ7.20 (2H, m), 7.20ꢀ7.70 (12H, m). IR
(KBr): 1713, 1669, 1534, 1472, 1032, 789, 735 cmꢀ1. LCꢀMS m/z: 699.1
Ethyl 2-[(Ethoxycarbonyl)amino]-4-methyl-5-(4-nitro-
phenyl)thiophene-3-carboxylate (8). To a suspension of ethyl
2-amino-4-methyl-5-(4-nitrophenyl)thiophene-3-carboxylate 711b (50.0 g,
163 mmol) in toluene (1.31 L) was added ethyl chloroformate (31.2 mL,
326 mmol), and the mixture was refluxed for 3 h. Ethyl chloroformate
(31.2 mL, 326 mmol) was again added, and the mixture was heated at
reflux. After 3 h, more ethyl chloroformate (31.2 mL, 326 mmol) was
added and the reaction mixture was heated at reflux for an additional 6 h.
After cooling, the mixture was concentrated in vacuo. Et2O was added to
the residue and the resulting precipitate was collected by filtration,
washed with Et2O, and dried to give 8 (57.8 g, 94%) as a yellow powder.
1H NMR (CDCl3): δ 1.35 (3H, t, J = 7.1 Hz), 1.42 (3H, t, J = 7.2 Hz),
2.42 (3H, s), 4.31 (2H, q, J = 7.1 Hz), 4.39 (2H, q, J = 7.2 Hz), 7.59 (2H,
d, J = 9.0 Hz), 8.27 (2H, d, J = 9.0 Hz), 10.66 (1H, s). IR (KBr): 1740,
[M + H+], 697.0 [M + Hꢀ]. Anal. (C36H32F2N6O5S 1.1H2O) C, H, N.
3
1-{4-[1-(2,6-Difluorobenzyl)-5-({(methyl)[2-(1H-1,2,3-
triazol-1-yl)ethyl]amino}methyl)-2,4-dioxo-3-phenyl-
1,2,3,4-tetrahydrothieno[2,3-d]pyrimidin-6-yl]phenyl}-3-
methoxyurea (6e). Yield 41%, colorless crystals, mp 194ꢀ196 °C. 1H
NMR (CDCl3): δ 2.14 (3H, s), 2.82 (2H, t, J = 6.0 Hz), 3.80 (2H, s), 3.82
(3H, s), 4.39 (2H, t, J = 6.0 Hz), 5.37 (2H, s), 6.92 (2H, t, J = 8.2 Hz),
6.85ꢀ6.95 (1H, m), 7.14 (1H, s), 7.20ꢀ7.55 (11H, m), 7.63 (1H, s). IR
(KBr): 1719, 1672, 1526, 1470, 1236, 1231, 1028, 824, 733 cmꢀ1. LCꢀMS
m/z: 673.3 [M + H+]. Anal. (C33H30F2N8O4S 0.4H2O) C, H, N.
1665, 1597, 1557, 1533, 1516, 1352, 1257 cmꢀ1
.
3
N-{2-[{[1-(2,6-Difluorobenzyl)-6-(4-{[(methoxyamino)-
carbonyl]amino}phenyl)-2,4-dioxo-3-phenyl-1,2,3,4-te-
trahydrothieno[2,3-d]pyrimidin-5-yl]methyl}(methyl)ami-
no]ethyl}-N-methylmethanesulfonamide (6g). Yield 67%, col-
orless crystals, mp 196ꢀ198 °C. 1H NMR (CDCl3): δ 2.12 (3H, s), 2.54
(2H, t, J = 6.3 Hz), 2.70 (6H, s), 3.16 (2H, t, J = 6.3 Hz), 3.82 (2H, s),
3.83 (3H, s), 5.38 (2H, s), 6.93 (2H, t, J = 8.1 Hz), 7.13 (1H, s),
7.25ꢀ7.65 (11H, m). IR (KBr): 1715, 1667, 1534, 1470, 1335, 1144,
1034, 791 cmꢀ1. Anal. (C33H34F2N6O6S2) C, H, N.
Ethyl 2-[(2,6-Difluorobenzyl)(ethoxycarbonyl)amino]-4-
methyl-5-(4-nitrophenyl)thiophene-3-carboxylate (9). To a
solution of 8 (57.3 g, 151 mmol) in DMF (1.82 L) were added KI (27.6 g,
167 mmol) and K2CO3 (27.1 g, 167 mmol). A solution of 2,6-difluor-
obenzyl chloride (27.1 g, 167 mmol) in DMF (100 mL) was then added
to the mixture. After the mixture was stirred at room temperature for
44 h, the resulting precipitate was filtered off and the filtrate was
concentrated in vacuo. The residue was diluted with EtOAc and water,
then extracted with EtOAc. The extract was washed with water and brine,
dried (Na2SO4), and concentrated in vacuo. Et2O was added to the
residue and the resulting precipitate was collected by filtration, washed
with Et2O, and dried to give 9 (71.1 g, 93%) as a pale yellow powder.
1-{4-[1-(2,6-Difluorobenzyl)-5-{[(2-methoxyethyl)(meth-
yl)amino]methyl}-2,4-dioxo-3-phenyl-1,2,3,4-tetrahydro-
thieno[2,3-d]pyrimidin-6-yl]phenyl}-3-methoxyurea (6h).
5006
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Journal of Medicinal Chemistry
ARTICLE
1H NMR (CDCl3): δ 1.15ꢀ1.35 (6H, m), 2.40 (3H, s), 4.15ꢀ4.29 (4H,
m), 4.97 (2H, s), 6.86 (2H, t, J = 7.8 Hz), 7.25ꢀ7.32 (1H, m), 7.51 (2H,
d, J = 8.8 Hz), 8.25 (2H, d, J = 8.8 Hz). IR (KBr): 1717, 1597, 1524,
silica gel column chromatography (EtOAc) to afford 13 (4.89 g, 89%) as a
pale yellow oil. 1H NMR (CDCl3): δ 1.19 (3H, br s), 1.30 (3H, t, J = 6.9
Hz), 2.04 (3H, s), 2.40 (2H, t, J = 6.0 Hz), 3.27 (3H, s), 3.33 (2H, t, J = 6.0
Hz), 3.60 (2H, s), 3.81 (3H, s), 4.13ꢀ4.24 (4H, m), 5.00 (2H, s), 6.84
(2H, t, J = 7.8 Hz), 7.19ꢀ7.29 (2H, m), 7.36 (2H, d, J = 8.7 Hz), 7.50 (2H,
d, J = 8.7 Hz), 7.60 (1H, s). IR (KBr): 1717, 1590, 1528, 1472, 1408,
1475, 1392, 1348 cmꢀ1
.
Ethyl 4-(Bromomethyl)-2-[(2,6-difluorobenzyl)(ethoxy-
carbonyl)amino]-5-(4- nitrophenyl)thiophene-3-carboxy-
late (10). A mixture of 9 (50.5 g, 100 mmol), NBS (22.3 g, 125 mmol),
and AIBN (1.65 g, 10.0 mmol) in EtOAc (600 mL) was refluxed for 2 h.
NBS (3.56 g, 20.0 mmol) and AIBN (165 mg, 1.00 mmol) were added to
the mixture, and the reaction mixture was heated at reflux for an
additional 1.5 h. After cooling to room temperature, the reaction mixture
was diluted with EtOAc and water and extracted with EtOAc. The
extract was washed with water and brine, dried (Na2SO4), and con-
centrated in vacuo to give crude 10 (63.0 g, quant) as an orange oil,
which was used for the next step without purification. 1H NMR
(CDCl3): δ 1.15ꢀ1.39 (6H, m), 4.09ꢀ4.39 (4H, m), 4.71 (2H, s),
4.99 (2H, s), 6.86 (2H, t, J = 7.8 Hz), 7.22ꢀ7.32 (1H, m), 7.72 (2H, d,
J = 8.0 Hz), 8.32 (2H, d, J = 8.0 Hz). IR (KBr): 1725, 1628, 1522, 1475,
1304 cmꢀ1
.
2-[(2,6-Difluorobenzyl)(ethoxycarbonyl)amino]-5-{4-
{[(methoxycarbamoyl)amino]phenyl}-4-[(2-methoxyethyl)-
(methyl)amino]methyl}thiophene-3-carboxylic Acid (14).
2 N NaOH (18.9 mL, 37.8 mmol) was added to a solution of 13
(4.81 g, 7.58 mmol) in EtOH (114 mL). After the mixture was stirred at
60 °C for 5 h, 1 N HCl (37.8 mL, 37.8 mmol) was added to the mixture
at 0 °C and the whole was concentrated in vacuo. The residue was
dissolved in EtOH and toluene, and the whole was concentrated in vacuo.
The residue was diluted with dry EtOH followed by filtration, and the
filtrate was concentrated in vacuo. Et2O was added to the residue and the
resulting precipitate was collected by filtration, washed with Et2O, and
1
1379, 1348 cmꢀ1
.
dried to give 14 (4.43 g, 96%) as a pale yellow powder. H NMR
(CDCl3): δ 1.17 (3H, br s), 2.45 (3H, s), 2.81 (2H, br s), 3.28 (3H, s),
3.55 (2H, t, J = 4.8 Hz), 3.82 (3H, s), 3.92 (2H, s), 4.10ꢀ4.35 (2H, m),
5.06 (2H, s), 6.82 (2H, t, J = 7.8 Hz), 7.16 (2H, d, J = 8.4 Hz), 7.22ꢀ7.35
(1H, m), 7.60 (2H, d, J = 8.4 Hz), 8.00ꢀ8.50 (2H, br). IR (KBr): 1713,
Ethyl 2-[(2,6-Difluorobenzyl)(ethoxycarbonyl)amino]-
4-{[(2-methoxyethyl)(methyl)amino]methyl}-5-(4-nitro-
phenyl)thiophene-3-carboxylate (11). To a solution of 10 (157 g,
221 mmol) in DMF (1.33 L) was added a mixture of N,N-diisopropyl-
ethylamine (45.7 mL, 265 mmol) and N-(2-methoxylethyl)methylamine
(21.4 g, 240 mmol) in DMF (50 mL). After being stirred at room
temperature for 24 h, the reaction mixture was concentrated in vacuo. The
residue was diluted with EtOAc and water and extracted with EtOAc. The
extract was washed with water and brine, dried (Na2SO4), and concen-
trated in vacuo to give 11 (145 g, quant) as an orange oil, which was used
for the next step without purification. 1H NMR (CDCl3): δ 1.15ꢀ1.30
(3H, m), 1.31 (3H, t, J = 7.4 Hz), 2.11 (3H, s), 2.40ꢀ2.60 (2H, m), 3.28
(2H, s), 3.30ꢀ3.45 (2H, m), 3.68 (2H, s), 4.10ꢀ4.30 (2H, m), 4.23 (2H,
q, J = 7.4 Hz), 5.01 (2H, s), 6.85 (2H, t, J = 7.6 Hz), 7.20ꢀ7.30 (1H, m),
7.69 (2H, d, J = 8.8 Hz), 8.24 (2H, d, J = 8.8 Hz).
1605, 1528, 1472, 1408 cmꢀ1
.
1-{4-[1-(2,6-Difluorobenzyl)-3-(2-hydroxy-2-methylpro-
pyl)-5-{[(2-methoxyethyl)(methyl)amino]methyl}-2,4-di-
oxo-1,2,3,4-tetrahydrothieno[2,3-d]pyrimidin-6-yl]phen-
yl}-3-methoxyurea (15d). Under ice-cooling, to a solution of 14
(303 mg, 0.50 mmol) and 1-amino-2-methylpropan-2-ol (89.0 mg,
1.00 mmol) in DMF (4 mL) were successively added DEPC (0.15 mL,
0.99 mmol) and N,N-diisopropylethylamine (0.19 mL, 1.09 mmol), and
the mixture was stirred at room temperature for 14 h. The reaction mixture
was partitioned between EtOAc and water. The organic layer was
separated, and theaqueous layer was extracted with EtOAc. The combined
extracts were washed with saturated aqueous NaHCO3 solution and brine,
dried (MgSO4), and concentrated in vacuo. The residue was purified by
basic silica gel column chromatography (EtOAcꢀMeOH, 10:1) to afford
the amide intermediate. The amide was dissolved in MeOH (10 mL), and
MeONa (270 mg, 5.00 mmol) was added. After the mixture was stirred at
room temperature for 2 h, 1 N HCl (5.0 mL, 5.0 mmol) was added to the
mixture at 0 °C and the whole was concentrated in vacuo. The residue was
partitioned between EtOAc and water. The organic layer was separated,
and the aqueous layer was extracted with EtOAc. The combined extracts
were washed with saturated aqueous NaHCO3 solution and brine, dried
(MgSO4), and concentrated in vacuo. The residue was purified by basic
silica gel column chromatography (EtOAc) and followed by recrystalliza-
tion from EtOAcꢀEt2O to afford 15d (134 mg, 42%) as colorless crystals,
mp 114ꢀ116 °C. 1H NMR (CDCl3): δ 1.28 (6H, s), 2.30 (3H, s), 2.64
(2H, t, J = 5.8 Hz), 3.30 (3H, s), 3.45 (2H, t, J = 5.8 Hz), 3.82 (5H, s), 3.99
(1H, s), 4.25 (2H, s), 5.36 (2H, s), 6.91 (2H, t, J = 8.2 Hz), 7.12 (1H, s),
7.20ꢀ7.40 (1H, m), 7.50ꢀ7.60 (4H, m), 7.61 (1H, s). LCꢀMS m/z:
Ethyl 5-(4-Aminophenyl)-2-[(2,6-difluorobenzyl)(etho-
xycarbonyl)amino]-4-{[(2-methoxyethyl)(methyl)amino]-
methyl}thiophene-3-carboxylate (12). A solution of 11 (12.4 g,
21.0 mmol) and2 N HCl/Et2O (21.0 mL, 42.0 mmol) inEtOH (315 mL)
was hydrogenated over 10% Pd/C (50% wet, 3.73 g) under atomospheric
pressure at room temperature for 1 h. The mixture was filtered through
Celite, and the filtrate was diluted with saturated aqueous NaHCO3
solution. The mixture was concentrated in vacuo, diluted with EtOAc and
water, and extracted with EtOAc. The extract was washed with brine, dried
(MgSO4), and concentrated in vacuo. The residue was purified by basic
silica gel column chromatography (EtOAcꢀMeOH, 40:1) to afford 12
(11.4 g, 97%) as a yellow oil. 1H NMR (CDCl3): δ 1.12ꢀ1.30 (3H, m),
2.05 (3H, s), 2.39 (2H, t, J = 6.3 Hz), 3.27 (3H, s), 3.32 (3H, t, J = 6.3 Hz),
3.59 (2H, s), 3.78 (2H, s), 4.20 (2H, q, J = 7.1 Hz), 4.10ꢀ4.23 (2H, m),
5.00 (2H, s), 6.66 (2H, d, J = 8.6 Hz), 6.84 (2H, t, J = 8.2 Hz), 7.18 (2H, d,
J = 8.6 Hz), 7.15ꢀ7.30 (1H, m). IR (KBr): 1717, 1626, 1609, 1472, 1406,
1300, 1246 cmꢀ1
.
632.1 [M + H+], 630.0 [M + Hꢀ]. Anal. (C30H35F2N5O6S 0.1H2O)
Ethyl 2-[(2,6-Difluorobenzyl)(ethoxycarbonyl)amino]-5-
{4-[(methoxycarbamoyl)amino]phenyl}-4-{[(2-methoxye-
thyl)(methyl)amino]methyl}thiophene-3-carboxylate (13).
To a solution of 12 (4.89 g, 8.70 mmol) in CH2Cl2 (113 mL) were
addedN,N-diisopropylethylamine (3.06 mL, 17.6mmol) and CDI (2.82 g,
17.4 mmol). After being stirred at room temperature for 67 h, the mixture
was cooled to 0 °C. To the mixture were added O-methylhydroxyamine
hydrochloride (7.26 g, 86.9 mmol) and N,N-diisopropylethylamine
(15.6 mL, 89.6 mmol), and the mixture was stirred at room temperature
for 19 h and then was partitioned between CHCl3 and saturated aqueous
NaHCO3 solution. The organic layer was separated, and the aqueous layer
wasextracted with CHCl3. The combinedextracts were washed with brine,
dried (MgSO4), and concentrated in vacuo. The residue was purified by
3
C, H, N.
Compounds 15aꢀc,eꢀj were prepared by a procedure similar to that
described for 15d, and their physicochemical data are shown below.
1-{4-[1-(2,6-Difluorobenzyl)-3-(2-methoxyethyl)-5-{[(2-
methoxyethyl)(methyl)amino]methyl}-2,4-dioxo-1,2,3,4-
tetrahydrothieno[2,3-d]pyrimidin-6-yl]phenyl}-3-methoxy-
urea (15a). Yield 58%, pale yellow crystals, mp 165ꢀ167 °C. 1H NMR
(CDCl3): δ 2.14 (3H, s), 2.65 (2H, t, J = 5.9 Hz), 3.30 (3H, s), 3.36 (3H,
s), 3.45 (2H, t, J = 5.9 Hz), 3.66 (2H, t, J = 5.9 Hz), 3.81 (3H, s), 3.84
(2H, s), 4.30 (2H, t, J = 5.9 Hz), 5.33 (2H, s), 6.90 (2H, t, J = 8.3 Hz),
7.15 (1H, s), 7.24ꢀ7.34 (1H, m), 7.51 (2H, d, J = 9.0 Hz), 7.56 (2H, d,
J = 9.0 Hz), 7.60 (1H, s). IR (KBr): 2936, 1705, 1663, 1590, 1532,
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1472 cmꢀ1. LCꢀMS m/z: 618.1 [M + H+], 615.9 [M + Hꢀ]. Anal.
(C29H33F2N5O6S) C, H, N.
1H NMR (CDCl3): δ 2.11 (3H, s), 2.64 (2H, t, J = 5.7 Hz), 3.27 (3H, s),
3.42 (2H, t, J = 5.7 Hz), 3.81 (2H, s), 3.83 (3H, s), 3.97 (3H, s), 5.37 (2H,
s), 6.87 (1H, d, J = 8.7 Hz), 6.93 (2H, t, J = 8.1 Hz), 7.19 (1H, s),
7.25ꢀ7.36 (1H, m), 7.50 (1H, dd, J = 2.7, 8.7 Hz), 7.53ꢀ7.60 (4H, m),
7.63 (1H, s), 8.10 (1H, d, J = 2.7 Hz). IR (KBr): 1715, 1672, 1609, 1530,
1470 cmꢀ1. LCꢀMS m/z: 667.1 [M + H+], 665.0 [M + Hꢀ]. Anal.
1-{4-[1-(2,6-Difluorobenzyl)-3-(2-ethoxyethyl)-5-{[(2-me-
thoxyethyl)(methyl)amino]methyl}-2,4-dioxo-1,2,3,4-tetra-
hydrothieno[2,3-d]pyrimidin-6-yl]phenyl}-3-methoxyurea
1
(15b). Yield 82%, pale yellow crystals, mp 151ꢀ152 °C. H NMR
(C32H32F2N6O6S 0.5H2O) C, H, N.
(CDCl3): δ 1.15 (3H, t, J = 6.9 Hz), 2.14 (3H, s), 2.66 (2H, t, J = 6.0 Hz),
3.30 (3H, s), 3.45 (2H, t, J = 6.0 Hz), 3.54 (2H, q, J = 6.9 Hz), 3.69 (2H, t,
J = 6.0 Hz), 3.81 (3H, s), 3.84 (2H, s), 4.29 (2H, t, J = 6.0 Hz), 5.32 (2H,
s), 6.89 (2H, t, J = 8.1 Hz), 7.17 (1H, s), 7.23ꢀ7.34 (1H, m), 7.52 (2H, d,
J = 8.7 Hz), 7.57 (2H, d, J = 8.7 Hz), 7.60 (1H, s). IR (KBr): 2975, 1705,
1663, 1590, 1532, 1472 cmꢀ1. LCꢀMS m/z: 632.1 [M + H+], 630.0
[M + Hꢀ]. Anal. (C30H35F2N5O6S) C, H, N.
3
1-{4-[1-(2,6-Difluorobenzyl)-5-{[(2-methoxyethyl)(meth-
yl)amino]methyl}-3-(6-methoxypyridazin-3-yl)-2,4-dioxo-
1,2,3,4-tetrahydrothieno[2,3-d]pyrimidin-6-yl]phenyl}-3-
methoxyurea (15j). Yield 69%, pale yellow powder, mp 201ꢀ203 °C.
1H NMR (CDCl3): δ 2.13 (3H, s), 2.62 (2H, t, J = 5.7 Hz), 3.26 (3H, s),
3.41 (2H, t, J = 5.7 Hz), 3.74 (2H, br s), 3.82 (3H, s), 4.18 (3H, s), 5.32
(2H, br s), 6.92 (2H, t, J = 8.3 Hz), 7.12 (1H, d, J = 9.3 Hz), 7.29ꢀ7.35
(1H, m), 7.41 (2H, d, J = 9.3 Hz), 7.54 (2H, d, J = 9.0 Hz), 7.59 (2H, d,
J = 8.7 Hz), 7.66 (1H, s). IR (KBr): 2936, 1717, 1674, 1591, 1530,
1460 cmꢀ1. LCꢀMS m/z: 668.1 [M + H+], 665.9 [M + Hꢀ]. Anal.
(C31H31F2N7O6S) C, H, N.
1-{4-[1-(2,6-Difluorobenzyl)-5-[(dimethylamino)methyl]-
3-(6-methoxypyridin-3-yl)-2,4-dioxo-1,2,3,4-tetrahydroth-
ieno[2,3-d]pyrimidin-6-yl]phenyl}-3-methoxyurea (16a). To
a solution of 15i (58.9 g, 88.9 mmol) in CH2Cl2 (1.2 L) was added
dropwise 1-chloroethyl chlorocarbonate (12.5 mL, 116 mmol) at ꢀ78 °C.
After the mixture was stirred at room temperature for 2 h, saturated
aqueous NaHCO3 solution and brine were added to the mixture, and the
whole was extracted with CH2Cl2. The organic layer was dried (MgSO4)
and concentrated in vacuo. The resulting precipitates were washed with
Et2O to afford the chloromethyl intermediate, which was dissolved in
DMF (400 mL). Then dimethylamine (2 M in THF, 170 mL, 340 mmol)
and N,N-diisopropylethylamine (17.8 mL, 102 mmol) were added
successively to the DMF solution. After the mixture was stirred at room
temperature for 15 h, saturated aqueous NaHCO3 solution was added to
the mixture and the whole was extracted with EtOAc. The extract was
washed with brine, dried (MgSO4), and concentrated in vacuo. The
residue was purified by basic silica gel column chromatography (EtOAc)
followed by recrystallization from EtOAcꢀTHF to afford 16a (13.1 g,
24%) as colorless crystals, mp 234ꢀ236 °C. 1H NMR (CDCl3): δ 2.13
(6H, s), 3.68 (2H, s), 3.83 (3H, s), 3.96 (3H, s), 5.36 (2H, s), 6.8ꢀ7.0
(3H, m), 7.13 (1H, s), 7.2ꢀ7.4 (1H, m), 7.45ꢀ7.65 (6H, m), 8.10 (1H, d,
J = 2.6 Hz). LCꢀMS m/z: 623.0 [M + H+], 621.0 [M + Hꢀ]. Anal.
(C30H28F2N6O5S) C, H, N.
1-{4-[1-(2,6-Difluorobenzyl)-3-(2-hydroxyethyl)-5-{[(2-
methoxyethyl)(methyl)amino]methyl}-2,4-dioxo-1,2,3,4-
tetrahydrothieno[2,3-d]pyrimidin-6-yl]phenyl}-3-methoxy-
urea(15c). Yield 25%, pale yellow amorphous powder. 1HNMR(CDCl3):
δ 2.11 (3H, s), 2.65 (2H, t, J = 5.8 Hz), 3.30 (3H, s), 3.46 (2H, t, J = 5.8 Hz),
3.82 (5H, s), 3.90ꢀ4.00 (2H, m), 4.35 (2H, t, J = 5.2 Hz), 5.34 (2H, s), 6.92
(2H, t, J = 8.0 Hz), 7.14 (1H, s), 7.20ꢀ7.35 (1H, m), 7.50ꢀ7.65 (5H, m).
LCꢀMS m/z: 604.2 [M + H+]. Anal. (C28H31F2N5O6S 1.0H2O) C, H, N.
3
1-{4-[1-(2,6-Difluorobenzyl)-5-{[(2-methoxyethyl)(meth-
yl)amino]methyl}-2,4-dioxo-3-(pyridin-2-yl)-1,2,3,4-tetra-
hydrothieno[2,3-d]pyrimidin-6-yl]phenyl}-3-methoxyurea
(15e). Yield 33%, colorless crystals, mp196ꢀ198 °C. 1H NMR(CDCl3):
δ 2.15 (3H, s), 2.62 (2H, t, J = 5.9 Hz), 3.26 (3H, s), 3.41 (2H, t, J = 5.9
Hz), 3.80 (3H, s), 3.81 (2H, br s), 5.34 (2H, br s), 6.91 (2H, t, J = 8.1 Hz),
7.24ꢀ7.40 (4H, m), 7.53 (2H, d, J = 8.4 Hz), 7.62 (2H, d, J = 8.4 Hz), 7.65
(1H, s), 7.88(1H, dt, J=1.5, 7.8Hz), 8.67ꢀ8.69 (1H, m). IR (KBr): 1717,
1674, 1591, 1530, 1460, 1329 cmꢀ1. LCꢀMS m/z: 637.0 [M + H+], 634.9
[M + Hꢀ]. Anal. (C31H30F2N6O5S) C, H, N.
1-{4-[1-(2,6-Difluorobenzyl)-5-{[(2-methoxyethyl)(meth-
yl)amino]methyl}-3-(5-methylpyridin-2-yl)-2,4-dioxo-1,2,-
3,4-tetrahydrothieno[2,3-d]pyrimidin-6-yl]phenyl}-3-meth-
1
oxyurea (15f). Yield 71%, pale yellow amorphous powder. H NMR
(CDCl3):δ2.14 (3H, s), 2.39 (3H, s), 2.62 (2H, t, J=5.7Hz), 3.26(3H, s),
3.41 (2H, t, J = 5.7 Hz), 3.77 (2H, br s), 3.80 (3H, s), 5.26 (1H, br s), 5.38
(1H, br s), 6.91 (2H, t, J = 8.3 Hz), 7.23ꢀ7.34 (2H, m), 7.42 (1H, s), 7.53
(2H, d, J = 8.7 Hz), 7.62 (2H, d, J = 8.7 Hz), 7.66 (1H, s), 7.66ꢀ7.69 (1H,
m), 8.48 (1H, d, J = 2.4 Hz). IR (KBr): 2938, 1717, 1675, 1586, 1530,
1462 cmꢀ1. LCꢀMS m/z: 651.0 [M + H+], 649.0 [M + Hꢀ]. Anal.
(C32H32F2N6O5S 0.5H2O) C, H, N.
1-{4-[1-(2,6-Difluorobenzyl)-5-[(dimethylamino)methyl]-
3-(6-methoxypyridazin-3-yl)-2,4-dioxo-1,2,3,4-tetrahydro-
thieno[2,3-d]pyrimidin-6-yl]phenyl}-3-methoxyurea (16b).
Compound 16b was prepared in 44% yield from 15j by a procedure
similar to that described for 16a as colorless crystals, mp 228 °C (dec).
1H NMR (CDCl3): δ 2.15 (6H, s), 3.60ꢀ3.80 (2H, m), 3.82 (3H, s),
4.18 (3H, s), 5.35 (2H, s), 6.92 (2H, t, J = 8.2 Hz), 7.12 (1H, d, J = 8.8
Hz), 7.20ꢀ7.65 (7H, m), 7.69 (1H, s). LCꢀMS m/z: 624.0 [M + H+],
621.9 [M + Hꢀ]. Anal. (C29H27F2N7O5S) C, H, N.
In Vitro Binding Assays. Receptor binding assays were carried out
as described previously.11a Briefly, human GnRH receptor cDNA was
cloned from a pituitary cDNA library, and CHO cells stably expressing
high levels of the recombinant human GnRH receptor were isolated.
3
1-{4-[1-(2,6-Difluorobenzyl)-3-(5-fluoropyridin-2-yl)-5-
{[(2-methoxyethyl)(methyl)amino]methyl}-2,4-dioxo-1,2,-
3,4-tetrahydrothieno[2,3-d]pyrimidin-6-yl]phenyl}-3-meth-
oxyurea (15g). Yield 46%, pale yellow amorphous powder. 1H NMR
(CDCl3): δ 2.13(3H, s), 2.62 (2H, t, J = 5.9 Hz), 3.26(3H, s), 3.41 (2H, t,
J = 5.9 Hz), 3.80 (2H, br s), 3.82 (3H, s), 5.33 (2H, br s), 6.92 (2H, t, J =
8.3 Hz), 7.19 (1H, s), 7.28ꢀ7.38 (2H, m), 7.52ꢀ7.63 (6H, m), 8.51 (1H,
d, J = 3.0 Hz). IR (KBr): 1715, 1674, 1586, 1530, 1462 cmꢀ1. LCꢀMS m/
z: 655.0 [M + H+], 653.0 [M + Hꢀ]. Anal. (C31H29F3N6O5S) C, H, N.
1-{4-[1-(2,6-Difluorobenzyl)-5-{[(2-methoxyethyl)(meth-
yl)amino]methyl}-3-(4-methoxyphenyl)-2,4-dioxo-1,2,3,4-
tetrahydrothieno[2,3-d]pyrimidin-6-yl]phenyl}-3-methoxy-
1
[
125I][Tyr5]leuprorelin (2.0ꢀ2.5 nM) and the membrane fraction of the
urea (15h). Yield 62%, pale brown solid. H NMR (CDCl3): δ 2.13
(3H, s), 2.64 (2H, t, J = 5.7 Hz), 3.27 (3H, s), 3.41 (2H, t, J = 5.6 Hz),
3.82ꢀ3.84 (8H, m), 5.36 (2H, s), 6.92 (2H, t, J = 8.1 Hz), 7.01 (2H, d, J =
8.7 Hz), 7.11 (1H, s), 7.19 (2H, d, J = 8.7 Hz), 7.26ꢀ7.33 (1H, m),
CHO cells (20 μg/well) were incubated at 25 °C for 60 min with shaking
in 0.2 mL of assay buffer A [25 mM Tris, 1 mM EDTA, 0.1% bovine
serum albumin (BSA), 0.25 mM phenylmethanesulfonyl fluoride, 1 μg/
mL pepstatin A, 20 μg/mL leupeptin, and 10 μg/mL phosphoramidon,
pH 7.5] containing various concentrations of the test compounds. The
bound and free ligands were immediately separated by filtration through
a poly(ethylenimine)-coated glass microfiber filter (Whatman, GF/C).
The filter was washed three times with 2 mL of assay buffer A, and
radioactivity was measured using an X-ray counter. Specific binding was
7.53ꢀ7.70 (5H, m). IR (KBr): 1716, 1661, 1625, 1588, 1500, 1456 cmꢀ1
.
LCꢀMS m/z: 666.3 [M + H+]. Anal. (C33H33F2N5O6S 0.5H2O) C, H, N.
3
1-{4-[1-(2,6-Difluorobenzyl)-5-{[(2-methoxyethyl)(meth-
yl)amino]methyl}-3-(6-methoxypyridin-3-yl)-2,4-dioxo-
1,2,3,4-tetrahydrothieno[2,3-d]pyrimidin-6-yl]phenyl}-
3-methoxyurea (15i). Yield 71%, colorless crystals, mp 167ꢀ169 °C.
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Journal of Medicinal Chemistry
ARTICLE
determined bysubtractingthenonspecificbinding, which was measured in
the presence of 1 μM unlabeled leuprorelin, from the total binding. The
concentration of each test compound that produced 50% inhibition of
the specific binding (IC50) was derived by fitting the data into a pseudo-
Hill equation:
The urea part of 1 seems to participate in the hydrogen bonding
interactions with the side chain of Asn102 (transmembrane 2, TM2) and
the main chain amides of Gln106 and Trp107 (extracellular loop 1, ECL1)
as depicted in Figure 2. All potential hydrogen bonding functionalities of
the urea moiety are utilized for intermolecular hydrogen bonding. Since
Asn102 of TM2 was reported as the binding site for the C-terminal
amide of LHRH based on mutagenesis of the human receptor,24 the urea
part of 1 might be a mimic of the C-terminal part of GnRH. The carbonyl
group at the opposite position of the difluorobenzyl moiety in the six-
membered ring is located within hydrogen bonding distance of the Ser194
side chain of ECL2. Considering the importance of the carbonyl group at
this site, hydrogen bonding interaction with Ser194 might be critical for
the tight binding of 1 with the human LHRH receptor.
log½% SPB=ð100 ꢀ % SPBÞꢁ ¼ n½logðCÞ ꢀ logðIC50Þꢁ
where % SPB is the specific binding expressed as a percentage of the
maximum specific binding, n is the pseudo-Hill constant, and C is the
concentration of the test compounds. Similarly, the binding experiments
to the GnRH receptor of monkey and rat were performed.
In Vitro Functional Assays. GnRH-stimulated arachidonic acid
release from CHO cells expressing human or monkey GnRH receptors
was measured according to the previously reported protocol.23 The
receptor-expressing CHO cellswere seededinto24-well plates ata density
of 4 ꢂ 104 cells/well and cultured for 1 day. The cells were then
incubated with [5,6,8,9,11,12,14,15-3H]arachidonic acid (11 kBq/well,
PerkinElmer Inc.) overnight and washed with Dulbecco’s modified
Eagle’s medium (DMEM) supplemented with 20 mM HEPES and
0.2% BSA. The cells were then preincubated with the compounds at
37 °C for 15 min, and the reaction was started by addition of GnRH
(1 nM). After incubation at 37 °C for 45 min, radioactivity in the medium
was measured with a liquid scintillation counter. IC50 and IC90 inhibitory
concentrations of each compound were calculated by logistic regression
analysis.
’ ASSOCIATED CONTENT
S
Supporting Information. Elemental analysis data and
b
HPLC results for compounds 6aꢀh, 15aꢀj, and 16a,b; enzyme
and radioligand binding assays. This material is available free of
charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: +81-6-6300-6283. Fax: +81-6-6300-6312. E-mail:
Miwa_Kazuhiro@takeda.co.jp.
Measurement of CYP3A4 Inhibitory Activity. Inhibitory
activity of test compounds of CYP3A4 was evaluated by incubating 100
μmol/L testosterone with 10 nmol/L CYP3A4 derived from CYP3A4-
expressing human B-lymphoblastoid cells (BD Biosciences) in the
presence of 10 μmol/L test compound. The incubation mixture was allowed
to stand for 30 min at 37 °C. The concentration of 6β-hydroxytestosterone
was measured by HPLC system equipped with a UV detector.
Oral Absorption in Cynomolgus Monkeys. Compound 16b
(1 or 3 mg) suspended in 0.5% methylcellulose (containing 0.6% citric
acid) was orally administered to fasted cynomolgus monkeys (male, 5ꢀ7
years old, n = 3). Blood samples (heparin plasma) were collected from
a forearm vein 0.25, 0.5, 1, 2, 4, 8, and 24 h after administration. The
plasma samples were deproteinized with acetonitrile. After centrifugation,
the obtained supernatant was diluted with internal standard solution
(0.01 mol/L ammonium formate/acetonitrile = 4/1) and centrifuged
again. The compound concentration in the supernatant was measured
by LC/MS/MS.
The mass spectrometer was equipped with an electrospray ion source
and operated in positive ion mode. The HPLC conditions were as
follows: column, L-column ODS (2.1 mm ꢂ 150 mm); mobile phase,
0.01 mol/L ammonium formate (containing 0.2% formic acid)/
acetonitrile = 6/4; flow rate, 0.2 mL/min; column temperature, 40 °C.
In Vivo Efficacy in Cynomolgus Monkeys. Compound 16b
(1 or 3 mg/kg) suspended in 0.5% methylcellulose containing 1.2%
citric acid, or 0.5% methylcellulose containing 1.2% citric acid alone,
was orally administered to fasted adult castrated cynomolgus monkeys
(6ꢀ11 years old, n = 3). Blood samples (heparin plasma) were collected
from a femoral vein without anesthesia at 0, 1, 2, 4, 8, 24, and 48 h after
administration. LH concentrations in the plasma were measured by
bioassays using mouse testicular cells.11a
’ ACKNOWLEDGMENT
We thank Dr. Keiji Kamiyama, Dr. Akihiro Tasaka, Dr. Nobuhiro
Suzuki, and Tatsuya Watanabe for encouragement and helpful
discussions throughout this work. We also acknowledge Tomohiro
Kaku and Dr. Kenichi Miyata for supporting the synthetic work, and
Dr. Shiro Takekawa, Dr. Masataka Harada, Yumiko Akinaga,
Tomoko Kaisho, Mari Asada, Tomoko Urushibara, Koichi Iida,
Shinichi Niwa, Dr. Yukihiro Ikeda, Dr. Takayasu Ito, and Junzo
Takahashi for supporting the biochemical and pharmacological
experiments.
’ ABBREVIATIONS USED
GnRH, gonadotropin-releasing hormone; LHRH, luteinizing
hormone-releasing hormone; FBS, fetal bovine serum; CYP,
cytochrome P450; GPCR, G-protein-coupled receptor; AA,
arachidonic acid; CHO, Chinese hamster ovary; LH, luteinizing
hormone; FSH, follicle-stimulating hormone; SAR, structureꢀ
activity relationship; NBS, N-bromosuccinimide; AIBN, 2,20-
azobis(isobutyronitrile); CDI, 1,10-carbonyldiimidazole; DEPC,
diethyl phosphorocyanidate
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