Discovery of a thieno[2,3-d]pyrimidine-2,4-dione bearing a p-methoxyureidophenyl moiety at the 6-position: A highly potent and orally bioavailable non-peptide antagonist for the human luteinizing hormone-releasing hormone receptor

Article abstract of DOI:10.1021/jm020180i

We have previously disclosed the first potent and orally effective non-peptide antagonist for the human luteinizing hormone-releasing hormone (LHRH) receptor, a thieno[2,3-b]pyridin-4-one derivative, T-98475 (1). Extensive research on developing non-peptide LHRH antagonists has been carried out by employing a strategy of replacing the thienopyridin-4-one nucleus with other heterocyclic surrogates. We describe herein the design and synthesis of a series of thieno-[2,3-d]pyrimidine-2,4-dione derivatives containing a biaryl moiety, which led to the discovery of a highly potent and orally active non-peptide LHRH antagonist, 5-(N-benzyl-N-methylaminomethyl)-1-(2,6-difluorobenzyl)-6-[4- (3-methoxyureido)phenyl]-3-phenylthieno[2,3-d]pyrimidine-2,4(1H,3H)-dione (9k: TAK-013). Compound 9k showed high binding affinity and potent in vitro antagonistic activity for the human receptor with half-maximal inhibition concentration (IC₅₀) values of 0.1 and 0.06 nM, respectively. Oral administration of 9k caused almost complete suppression of the plasma LH levels in castrated male cynomolgus monkeys at a 30 mg/kg dose with sufficient duration of action (more than 24 h). The results demonstrated that the thienopyrimidine-2,4-dione core is an excellent surrogate for the thienopyridin-4-one and that thienopyrimidine-2,4-diones and thienopyridin-4-ones constitute a new class of potent and orally bioavailable LHRH receptor antagonists. Furthermore, molecular modeling studies indicate that the unique methoxyurea side chain of 9k preferentially forms an intramolecular hydrogen bond between the aniline NH and the methoxy oxygen atom. The hydrogen bond will shield the hydrogen bonding moieties from the solvent and reduce the desolvation energy cost. It is therefore speculated that the intramolecular hydrogen bond resulting from judicious incorporation of an oxygen atom into the terminal alkyl group of the urea may increase the apparent lipophilicity to allow increased membrane permeability and consequently to improve the oral absorption of 9k in monkeys. On the basis of its profile, compound 9k has been selected as a candidate for clinical trials and it is expected that it will provide a new class of potential therapeutic agents for the clinical treatment of a variety of sex-hormone-dependent diseases.

Full text of DOI:10.1021/jm020180i

J . Med. Chem. 2003, 46, 113-124
113
Discover y of a Th ien o[2,3-d ]p yr im id in e-2,4-d ion e Bea r in g a
p-Meth oxyu r eid op h en yl Moiety a t th e 6-P osition : A High ly P oten t a n d Or a lly
Bioa va ila ble Non -P ep tid e An ta gon ist for th e Hu m a n Lu tein izin g
Hor m on e-Relea sin g Hor m on e Recep tor
Satoshi Sasaki,∇,† Nobuo Cho,*,∇,† Yoshi Nara,[,‡ Masataka Harada,∇,§ Satoshi Endo,[,† Nobuhiro Suzuki,[,|
Shuichi Furuya,# and Masahiko Fujino∇,[,#
Pharmaceutical Research Division, Takeda Chemical Industries, Ltd., 10 Wadai, Tsukuba, Ibaraki 300-4293, J apan,
Pharmaceutical Research Division, Takeda Chemical Industries, Ltd., 17-85, J usohonmachi 2-chome, Yodogawa-ku,
Osaka 532-8686, J apan, and Strategic Product Planning Department, Takeda Chemical Industries, Ltd.,
1-1, Doshomachi 4-chome, Chuo-ku, Osaka 540-8645, J apan
Received April 29, 2002
We have previously disclosed the first potent and orally effective non-peptide antagonist for
the human luteinizing hormone-releasing hormone (LHRH) receptor, a thieno[2,3-b]pyridin-
4-one derivative, T-98475 (1). Extensive research on developing non-peptide LHRH antagonists
has been carried out by employing a strategy of replacing the thienopyridin-4-one nucleus with
other heterocyclic surrogates. We describe herein the design and synthesis of a series of thieno-
[2,3-d]pyrimidine-2,4-dione derivatives containing a biaryl moiety, which led to the discovery
of a highly potent and orally active non-peptide LHRH antagonist, 5-(N-benzyl-N-methylami-
nomethyl)-1-(2,6-difluorobenzyl)-6-[4-(3-methoxyureido)phenyl]-3-phenylthieno[2,3-d]pyrimi-
dine-2,4(1H,3H)-dione (9k : TAK-013). Compound 9k showed high binding affinity and potent
in vitro antagonistic activity for the human receptor with half-maximal inhibition concentration
(IC50) values of 0.1 and 0.06 nM, respectively. Oral administration of 9k caused almost complete
suppression of the plasma LH levels in castrated male cynomolgus monkeys at a 30 mg/kg
dose with sufficient duration of action (more than 24 h). The results demonstrated that the
thienopyrimidine-2,4-dione core is an excellent surrogate for the thienopyridin-4-one and that
thienopyrimidine-2,4-diones and thienopyridin-4-ones constitute a new class of potent and orally
bioavailable LHRH receptor antagonists. Furthermore, molecular modeling studies indicate
that the unique methoxyurea side chain of 9k preferentially forms an intramolecular hydrogen
bond between the aniline NH and the methoxy oxygen atom. The hydrogen bond will shield
the hydrogen bonding moieties from the solvent and reduce the desolvation energy cost. It is
therefore speculated that the intramolecular hydrogen bond resulting from judicious incorpora-
tion of an oxygen atom into the terminal alkyl group of the urea may increase the apparent
lipophilicity to allow increased membrane permeability and consequently to improve the oral
absorption of 9k in monkeys. On the basis of its profile, compound 9k has been selected as a
candidate for clinical trials and it is expected that it will provide a new class of potential
therapeutic agents for the clinical treatment of a variety of sex-hormone-dependent diseases.
In tr od u ction
mone (FSH), which regulate gonadal steroidogenesis
and gametogenesis.
Luteinizing hormone-releasing hormone (LHRH, also
known as gonadotropin-releasing hormone (GnRH)),
discovered by Schally et al. in 1971,1 is a decapeptide
amide that plays a pivotal role in modulating reproduc-
tive functions. It is secreted from the hypothalamus in
a pulsatile pattern and binds to the LHRH receptor on
the anterior pituitary. The activation of this G-protein-
coupled receptor causes the biosynthesis and release of
luteinizing hormone (LH) and follicle-stimulating hor-
Several peptidic LHRH agonists,2 which ultimately
suppress gonadal steroid production and achieve a
condition called “biochemical castration”3,4 through a
receptor down-regulation mechanism, have been ap-
proved for the clinical treatment of a variety of endocrine-
based diseases such as prostate cancer, breast cancer,
endometriosis, uterine leiomyoma, and precocious pu-
berty.5,6 One of the major drawbacks of these LHRH
agonists is the initial gonadal hormone surge, the “flare
effect”, which tends to exacerbate the symptoms of the
above conditions. In contrast, LHRH antagonists are
expected to suppress gonadotropins from the onset of
administration.4 In fact, recent clinical studies have
demonstrated that peptidic LHRH antagonists directly
lowered the steroid hormone levels without the adverse
flare effect.4,7 However, peptidic LHRH antagonists still
suffer from insufficient oral bioavailability because of
* To whom correspondence should be addressed. Phone: +81-298-
64-6351. Fax: +81-298-64-6308. E-mail: Cho_Nobuo@takeda.co.jp.
∇ Takeda Chemical Industries, Ltd., 10 Wadai, Tsukuba.
† Medicinal Chemistry Research Laboratories I.
[ Takeda Chemical Industries, Ltd., 17-85, J usohonmachi 2-chome,
Yodogawa-ku.
‡ Medicinal Chemistry Research Laboratories II.
§ Discovery Research Laboratories I.
| Pharmacology Research Laboratories II.
#
Takeda Chemical Industries, Ltd., 1-1, Doshomachi 4-chome,
Chuo-ku.
10.1021/jm020180i CCC: $25.00 © 2003 American Chemical Society
Published on Web 11/27/2002

114 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1
Sasaki et al.
F igu r e 1.
being peptidic in nature, and therefore, potent and
orally effective small-molecule LHRH antagonists could
be superior drugs for clinical applications in the above
conditions.
essentially the same strategy described here, i.e., using
the 6-phenylthieno[2,3-d]pyrimidine-2,4-dione core as a
scaffold for non-peptide ET antagonists. In addition, the
thienopyrimidine-2,4-dione core could possess important
functional groups participating in ligand-receptor in-
teraction at the corresponding positions of the thienopy-
ridin-4-one nucleus. On the basis of the latter strategy,
we therefore used the thienopyrimidine-2,4-dione core
as a scaffold for the LHRH antagonist and conducted
synthetic studies on this surrogate.
To date, four different classes of non-peptide LHRH
antagonists have been reported: 2-phenylthienopyridin-
4-ones8 from our laboratory, more recently, 3-phenylqui-
nolones9 and 2-phenylindoles10 from Merck, and 2-phen-
ylpyrrolopyrimid-7-ones11 from Neurocrine Biosciences.
In our previous paper, we have described the discovery
of the first potent and orally active non-peptide LHRH
receptor antagonist T-98475 (1), a thieno[2,3-b]pyridin-
4-one derivative, and the initial structure-activity
relationship (SAR) of this class8 (Figure 1). Our ap-
proach for developing non-peptide LHRH antagonists
is based on the introduction of crucial functional groups
for receptor binding into a bicyclic scaffold, which
mimics a type II â-turn involving residues 5-8 (Tyr-
Gly-Leu-Arg) of LHRH that has been proposed as the
putative bioactive conformation.6 Although T-98475 (1)
showed excellent in vitro LHRH antagonistic activities,
the in vivo antagonism of 1 when administered orally
was not as effective as we had expected,8 which may be
due to the inadequate oral bioavailability conferred by
the nature of its substituents.
In this article, we describe the synthesis of the thieno-
[2,3-d]pyrimidine-2,4-dione derivatives and their bio-
logical activity. This study revealed that the thienopy-
rimidine-2,4-dione nucleus was an excellent surrogate
for the thienopyridin-4-one and culminated in the
discovery of a highly potent and orally bioavailable non-
peptide LHRH antagonist, a thienopyrimidine-2,4-dione
bearing the unique p-(3-methoxyureido)phenyl group at
the 6-position (9k : TAK-013). Furthermore, molecular
modeling studies on 9k that indicate the beneficial effect
of the methoxyureido group on the oral absorption in
monkeys are also described.
Ch em istr y
The thieno[2,3-d]pyrimidine-2,4-dione derivatives ex-
amined in this study were synthesized by the general
procedure shown in Scheme 1. The 2-aminothiophenes
3a ,b prepared from the corresponding phenylacetones
using the previously reported Gewald’s procedure14 were
reacted with a variety of isocyanates. The resulting
ureas were cyclized under basic conditions to afford the
thienopyrimidine-2,4-diones 4a -l. Isobutyl isocyanate,
used in the preparation of 4c, was obtained by the
Curtius rearrangement reaction of isovaleric acid with
diphenylphosphoryl azide (DPPA) and was used without
purification. Difluorobenzylation of 4a -l at the 1-posi-
tion, followed by radical bromination of the 5-methyl
group with N-bromosuccinimide (NBS), furnished the
intermediary bromomethyl analogues, which were con-
The observation prompted us to conduct an extensive
research program to identify small-molecule LHRH
antagonists with improved in vivo efficacy. The follow-
ing two strategies were employed: (i) further optimiza-
tion of each substituent of T-98475 (1), e.g., chemical
modification of the ester moiety; (ii) replacement of the
thienopyridin-4-one scaffold with other suitable hetero-
cyclic surrogates. The former strategy of investigating
T-98475-related compounds led to the identification of
highly potent thienopyridin-4-ones bearing the p-hy-
droxyalkylamidophenyl and ketone moieties at the 2-
and 5-position, respectively.12 Simultaneously, we turned
our attention back to the previous work from this
laboratory on the non-peptide endothelin (ET) antago-
nist 2.13 Compound 2 was discovered according to

Thieno[2,3-d]pyrimidine-2,4-dione Derivative
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1 115
Sch em e 1a
a
Reagents: (a) (1) R2NCO, pyridine, (2) NaOMe, MeOH or NaOEt, EtOH; (b) (1) isovaleric acid, DPPA, Et3N, toluene, (2) NaOMe,
i
MeOH; (c) 2,6-difluorobenzyl chloride, K2CO3, KI, DMF; (d) NBS, AIBN, chlorobenzene; (e) N-benzylmethylamine, Pr2NEt, DMF.
Sch em e 2a
a
Reagents: (a) H2, Pd-C, HCl-Et2O, HCO2H (for the preparation of 7a ); (b) H2, Pd-C, HCO2H (for the preparation of 7b and 8); (c)
R1COCl, Et3N, CH2Cl2; (d) (1) CDI, Et3N, CH2Cl2, (2) alkylamine or O-alkylhydroxylamine, Et3N (if necessary), CH2Cl2 or THF; (e) ethyl
isocyanate, pyridine; (f) TFA, CH2Cl2; (g) BH3‚Me2S, THF; (h) EtCOCl, Et3N, CH2Cl2.
verted to compounds 6a -l by introduction of the N-
benzylmethylamino moiety.
alkylureas 9f-i, hydroxyurea 9j, and alkoxyureas 9k -
m . First, compounds 9a -e and 9g were prepared by
reaction of 7a ,b with the appropriate acyl chlorides or
ethyl isocyanate under basic conditions. Second, com-
pounds 9f,h ,i and 9k -m were obtained by a two-step
methodology, i.e., reaction of 7a with 1,1′-carbonyldi-
imidazole (CDI) to give the intermediary imidazolide
and subsequent nucleophilic substitution with the cor-
responding alkylamines or O-alkylhydroxylamines in a
one-pot reaction. The 3-(2,4-dimethoxybenzyloxy)urea
9i was deprotected by treatment with trifluoroacetic
acid (TFA) to afford the hydroxyurea 9j.15 Finally,
compound 8 was reduced with a borane-methyl sulfide
complex and the resulting amine was subsequently
Chemical modification of the para substituent on the
6-phenyl ring was performed according to the synthetic
route depicted in Scheme 2. Catalytic hydrogenation of
the nitro compound 6b in formic acid produced the
requisite aniline 7b together with the undesired formyl-
ated byproduct 8. This problem was overcome by addi-
tion of excess anhydrous hydrogen chloride to trap 7b
as an inactive hydrochloride. Thus, compound 7a was
successfully obtained from 6a by reduction with pal-
ladium-charcoal in the presence of hydrogen chloride.
Compounds 7a ,b were then converted to the amide,
urea, and urea-related derivatives: the amides 9a -e,

116 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1
Sasaki et al.
Ta ble 1. Physicochemical Data and Binding Affinities of Compounds 6c-l
a
compound
R
mp (°C)
recryst solvent
formula
IC50 (µM)
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
iBu
cHex
Bn
Ph
2-MeOPh
3-MeOPh
4-MeOPh
2-ClPh
3-ClPh
4-ClPh
165-167
145-150
123-127
160-162
148-150
160-163
153-157
150-155
152-157
145-146
EtOAc-Et2O
CH2Cl2-Et2O
EtOAc-Et2O
EtOAc-Et2O
EtOAc-Et2O
EtOAc-Et2O
EtOAc-Et2O
EtOAc-Et2O
EtOAc-Et2O
EtOAc-Et2O
C33H33N3O3SF2‚HCl
0.4
0.7
0.4
0.05
0.2
0.02
0.03
0.08
0.2
C35H35N3O3SF2‚HCl‚0.5H2O
C36H31N3O3SF2‚HCl‚0.5H2O
C35H29N3O3SF2‚HCl‚0.5H2O
C36H31N3O4SF2‚HCl‚1.5H2O
C36H31N3O4SF2‚HCl‚0.8H2O
C36H31N3O4SF2‚HCl‚0.5H2O
C35H28N3O3SClF2‚HCl‚0.5H2O
C35H28N3O3SClF2‚HCl‚H2O
C35H28N3O3SClF2‚HCl‚H2O
0.2
a
The binding affinity is reported as an IC50 value, which is the antagonist concentration required to inhibit the specific binding of
125I]leuprorelin to LHRH receptor by 50%. Chinese hamster ovary (CHO) cells expressing human LHRH receptors were used as the
source for LHRH receptors. All data are expressed as the mean of two or three determinations.
[
acylated with propionyl chloride to yield the N-methyl-
anilide 10.
and physicochemical data are shown in Table 2. In the
m-methoxyphenyl series at the 3-position, nitro (6b) was
equipotent with methoxy (6h ); however, reduction of the
nitro group of 6b led to a boost in activity. The aniline
7b exhibited remarkably improved affinity with an IC50
value of 1 nM, which was further enhanced by acylation
of the amino group (9a -e). The amides 9a ,b,d ,e exhib-
ited subnanomolar affinities (IC50 ) 0.2-0.4 nM), which
Resu lts a n d Discu ssion
In Vitr o Recep tor Bin d in g Stu d ies. Our investi-
gation began with an SAR study of thienopyrimidine-
2,4-diones based on data from an in vitro binding assay
using the cloned Chinese hamster ovary (CHO) cells
expressing the human LHRH receptor and [125I]leupro-
relin as a ligand. The preliminary in vitro studies on
T-98475-related compounds indicated that the binding
affinity was dependent on the para substituent of the
2-phenyl group.8 Accordingly, compounds possessing the
N-benzyl-N-methylaminomethyl and 2,6-difluorobenzyl
moieties8,12 important for receptor binding were syn-
thesized and evaluated for LHRH receptor binding
affinity.
were almost comparable to that of T-98475 (1) (IC50
)
0.2 nM). However, the benzamide 9c was about 3-fold
less potent than 9a ,b. In addition, N-methylation of the
amide nitrogen (10) resulted in a marked decrease in
activity. These results suggest that hydrogen donation
from the aniline NH plays an important role for recep-
tor-ligand interaction and that a spatial limitation may
exist for the recognition site interacting with this part
of the molecule.
First, the 3-substituent of the thienopyrimidine-2,4-
dione, which corresponds to the 5-ester group of T-98475
(1), was investigated with compounds bearing a p-
methoxyphenyl group at the 6-position, and the results
and physicochemical data are presented in Table 1. The
alkyl and aralkyl analogues, e.g., isobutyl (6c), cyclo-
hexyl (6d ), and benzyl (6e), were found to exhibit
noticeable submicromolar affinities. In contrast, aro-
matization of the cyclohexyl ring of 6d (6f) induced a
14-fold increase in affinity, which led us to examine next
the substituted phenyl derivatives. Incorporation of a
methoxy group into the 3-phenyl ring (6h ,i) produced
modest enhancement in affinity except at the ortho
position (6g), whereas introduction of a chlorine atom
into the 3-phenyl ring (6j-l) tended to result in a
deleterious effect on the activity. In view of the limited
effect of the substituent on affinity in the substituted
phenyl series, the m-methoxyphenyl and nonsubstituted
phenyl groups were chosen as the 3-substituents for
further investigation.
Comparison between 9a ,b and 9d ,e indicates that
introduction of the m-methoxy group into the 3-phenyl
ring showed a minimal effect on the binding affinity
when the 6-substituent incorporated is a p-acylami-
nophenyl moiety. Since it is well-known that the meth-
oxyphenyl group tends to be one of the targets for
metabolic enzymes, subsequent investigation into amide-
related substituents of the 6-phenyl group was con-
ducted with compounds bearing a nonsubstituted phen-
yl group at the 3-position. Taking into account the high
affinity of the p-acylaminophenyl derivatives at the
6-position, it is hypothesized that the binding site
accommodating this part of the molecule must be mainly
hydrophilic. Thus, we envisaged that incorporation of
the nitrogen and/or oxygen atoms into the amide moiety
would provide more potent antagonists because of
additional hydrogen bonding interaction with the LHRH
receptor. Incorporation of the NH group into the amide
moiety tended to increase the activity. Indeed, the
alkylureas 9f-h were more potent than the amides
9a ,b. Intriguingly, inclusion of an additional oxygen
atom into the alkylurea moiety of 9f,g had a beneficial
effect on oral absorption compared to the alkylureas,
as mentioned later, without affecting the in vitro
activities. Although the hydroxyurea 9j and isopro-
The preceding SAR studies on the thienopyridin-4-
ones revealed that para substitution on the 2-phenyl
ring was preferable to ortho or meta substitution.8,12 On
the basis of this finding, effects of the para substituent
on the 6-phenyl ring were investigated, and the results

Thieno[2,3-d]pyrimidine-2,4-dione Derivative
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1 117
Ta ble 2. Physicochemical Data and Binding Affinities of Compounds 6b,h , 7b, 9a -h , 9j-m , and 10
a
compound
6h
6b
7b
9d
9e
10
9a
9b
9c
9f
9g
9h
9j
R
X
mp (°C)
recryst solvent
formula
IC50 (nM)
MeO
O2N
H2N
OMe
OMe
OMe
OMe
OMe
OMe
H
160-163
142-144
162-165
170-175
170-173
138-143
197-202
185-190
167-169
175-180
179-182
172-177
180-186
204-205
193-197
201-204
EtOAc-Et2O
CH2Cl2-Et2O
EtOAc-Et2O
EtOAc-Et2O
EtOAc-Et2O
CH2Cl2-Et2O
EtOAc-Et2O
EtOAc-Et2O
EtOAc-Et2O
CH2Cl2-Et2O
CHCl3-Et2O
EtOAc-Et2O
MeOH-Et2O
CHCl3-Et2O
CH2Cl2-Et2O
CH2Cl2-Et2O
C36H31N3O4SF2‚HCl‚0.8H2O
C35H28N4O5SF2‚HCl‚H2O
C35H30N4O3SF2‚2.0HCl‚H2O
C38H34N4O4SF2‚HCl‚1.5H2O
C39H36N4O4SF2‚HCl‚H2O
C39H36N4O4SF2‚HCl‚1.5H2O
C37H32N4O3SF2‚HCl‚H2O
C38H34N4O3SF2‚HCl‚1.5H2O
C41H32N4O3SF2‚HCl‚0.5H2O
C36H31N5O3SF2‚HCl‚H2O
C37H33N5O3SF2‚HCl‚1.5H2O
C38H35N5O3SF2‚HCl‚H2O
C35H29N5O4SF2‚HCl‚H2O
C36H31N5O4SF2
20
20
1
EtCONH
0.2
0.3
20
0.3
0.4
1
0.1
0.1
0.2
0.4
0.1
0.2
0.6
iPrCONH
EtCON(Me)
EtCONH
iPrCONH
H
H
H
H
H
H
H
H
PhCONH
MeNHCONH
EtNHCONH
iPrNHCONH
HONHCONH
MeONHCONH
EtONHCONH
iPrONHCONH
9k (TAK-013)
9l
9m
C37H33N5O4SF2‚HCl‚2.0H2O
C38H35N5O4SF2‚HCl‚0.5H2O
H
a
The binding affinity is reported as an IC50 value, which is the antagonist concentration required to inhibit the specific binding of
125I]leuprorelin to LHRH receptor by 50%. Chinese hamster ovary (CHO) cells expressing human LHRH receptors were used as the
[
source for LHRH receptors. All data are expressed as the mean of two or three determinations.
Ta ble 3. Species Specificities of Compounds 1 and 9a ,g,k
poxyurea 9m were more than 4-fold less potent than
9f,g, the methoxyurea 9k (IC50 ) 0.1 nM) and the
ethoxyurea 9l (IC50 ) 0.2 nM) showed comparable
affinities to 9f,g. With respect to the para substituent
on the 6-phenyl ring, it can be seen that both the
hydrogen-donating NH and terminal small alkyl (C1-
C3) groups are important for high binding affinity. From
the above binding assay data, the methylurea 9f,
ethylurea 9g, and methoxyurea 9k had the optimum
LHRH receptor binding affinities, with IC50 values of
0.1 nM, and compounds 9g,k were selected for further
in vitro and in vivo evaluation.
F u r th er in Vitr o a n d in Vivo Stu d ies. First, the
amide 9a and the ureas 9g,k were evaluated for their
binding affinities to LHRH receptors of the particular
species. It can be seen from Table 3 that there are
marked species specificities in receptor binding affinities
similar to those observed for T-98475 (1).8 Compounds
9a ,g,k exhibited more than 3- and 2000-fold selectivity
for the human receptor over the monkey and rat
receptors, respectively. Hence, it is reasonable that
further in vitro and in vivo evaluations of these com-
pounds are performed utilizing monkey as well as
human receptors.
Binding to the LHRH Receptors and Inhibitory Effects of
Compounds 1 and 9a ,g,k on LHRH-Stimulated Arachidonic
Acid (AA) Release
inhibition of
binding affinitya
IC50 c (nM)
AA releaseb
IC50 c (nM)
compound
humand monkeyd
rate
60
700
200
human monkey
1
0.2
0.3
0.1
0.1
2 (4f)
9
0.3
0.6
0.6
4
ndg
300
9a
9g
0.07
0.06
7
10
9k (TAK-013)
>1000
a
The binding affinity is reported as an IC50 value, which is the
antagonist concentration required to inhibit the specific binding
b
of [125I]leuprorelin to LHRH receptor by 50%. Inhibition of
LHRH-stimulated arachidonic acid (AA) release from CHO cells
expressing human or monkey LHRH receptors was measured to
evaluate the functional LHRH antagonism of the test compounds.
The IC50 value is the antagonist concentration required to inhibit
the LHRH-stimulated AA release from CHO cells by 50%. c All
data are expressed as the mean of two or three determinations.
d
Chinese hamster ovary (CHO) cells expressing human or monkey
LHRH receptors were used as the source for LHRH receptors.e Rat
anterior pituitaries were used as the source for LHRH receptors.
f Monkey anterior pituitaries were used as the source for LHRH
g
receptors (see ref 8). nd ) not determined.
It is widely acknowledged that urea tends to show
poor oral absorption resulting from relatively low solu-
bility and/or poor membrane permeability due to its
strong hydrogen bonding ability. We therefore set out
to probe oral absorption of 9a ,g,k preliminarily using
cynomolgus monkeys, and the results are summarized
in Table 4. As we predicted, the oral absorption of the
amide 9a in monkeys was superior to that of the
ethylurea 9g at a dose of 10 mg/kg. Surprisingly,
judicious incorporation of an oxygen atom into the
terminal alkyl group of 9g provided us with the final
breakthrough. Thus, the methoxyurea 9k exhibits im-
proved oral absorption when compared with 9g at the
Second, for in vitro functional antagonism, assays for
inhibition of LHRH-stimulated arachidonic acid release
from CHO cells expressing the human and monkey
receptors were undertaken. Compounds 9a ,g,k effec-
tively antagonized LHRH function on CHO cells ex-
pressing the human and monkey receptors (Table 3).
Moreover, species specificities in functional antagonism
were observed in a manner similar to that described
above for binding affinity. The in vitro antagonism of
the human receptor (IC50 ) 0.06-4 nM) exceeded that
of the monkey receptor (IC50 ) 7-300 nM), and in
general, the order of the antagonistic effect virtually
paralleled that of the binding affinity.

118 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1
Sasaki et al.
Ta ble 4. Oral Absorption of Compounds 9a ,g,k in Cynomolgus
orally. In fact, when the urea NH was capped with
aliphatic groups, improvements in oral bioavailability
were reported for somatostatin agonists17 and â3-ad-
renergic receptor agonists.18 Ashwood et al. have re-
cently described that the intramolecular hydrogen bond
increased the penetration of the central nervous system
by a neurokinin-1 receptor antagonist.19 Since a urea
moiety can contribute to the stabilization of the drug-
receptor interaction through its strong hydrogen bond-
ing properties, it is often tempting to introduce it into
lead compounds in order to improve their affinities
toward the target receptors or enzymes. As demon-
strated here, we have attained improvement in both the
in vitro activities and oral absorption through judicious
incorporation of the unique methoxyurea moiety at the
para position of the 6-phenyl ring. When the urea NH
participates in the critical hydrogen bonding interaction,
as inferred here, capping of the urea NH may not be
tolerated. Introduction of a properly designed intramo-
lecular hydrogen bond is a versatile strategy for improv-
ing oral absorption without deteriorating the potency
of binding toward the target proteins of lead compounds.
Monkeysa
b
d
compound
9a
Cmax (µM)
Tmax c (h)
AUC0-6 (µM‚h)
0.63
0.063
0.21
3
3
6
2.5
0.27
0.85
9g
9k (TAK-013)e
a
Compounds 9a ,g,k (10 mg/kg) suspended in 0.5% methylcel-
lulose were orally administered to cynomolgus monkeys (female,
b
4-8 years old, n ) 3). Maximum plasma concentration after 10
d
mg/kg oral dosing. c Time to Cmax
.
Area under the concentration-
time curve for 0-6 h after 10 mg/kg oral dosing. e TAK-013
hydrochloride was used (see Experimental Section).
same dosage. These results suggest that the methoxy-
urea 9k is a promising compound for exerting a potent
in vivo efficacy when administered orally.
Finally, the in vivo antagonism of compound 9k was
investigated for the suppression of plasma LH concen-
trations in castrated male cynomolgus monkeys (Table
5). As we had anticipated, oral administration of a 30
mg/kg dose of the methoxyurea 9k caused almost
complete suppression of plasma LH levels (11% of
pretreatment at 24 h) in monkeys. The suppressive
effect of 9k at a 30 mg/kg dose (po) was more potent
than that of T-98475 (1) at a 60 mg/kg dose (po), and
9k exhibited longer duration of action than 1.8 It should
be noted that exposure to 9k even at a 10 mg/kg dose
(po) resulted in effective suppression (approximately
20% of pretreatment from 8 to 24 h). In these experi-
ments the maximum plasma concentrations of 9k were
0.34 µM (reached 6 h after administration) and 0.18 µM
(reached 4 h after administration) at 30 and 10 mg/kg
doses, respectively. As a consequence, the methoxyurea
9k potently antagonized the elevated LHRH function
induced by castration in male cynomolgus monkeys and
the suppressive effects lasted for more than 24 h at both
30 and 10 mg/kg po doses. These data clearly demon-
strate that compound 9k is a potent and orally active
LHRH antagonist. The potent in vivo antagonism of 9k
may result from its potent in vitro activity and/or
excellent pharmacokinetic profiles. Furthermore, the
large difference between in vitro antagonistic activities
of the methoxyurea 9k toward human and monkey
receptors implies that a far smaller amount of 9k would
be sufficient to suppress plasma LH levels in humans.
Molecu la r Mod elin g Stu d ies. Incorporation of an
oxygen atom into the methylurea moiety of 9f (9k )
resulted in respectable improvement of the oral absorp-
tion in monkeys. During the conformational analysis of
9k , using high-temperature molecular dynamics calcu-
lation, it was observed that the cis conformer of the
methoxyurea was more populated than the trans con-
former (see Table 6 for the definition of the cis and trans
conformers). Both the molecular mechanics calculations
and ab initio calculations confirmed that the cis con-
former is more stable than the trans conformer by about
6-7 kcal/mol, as shown in Table 6. The stability of the
cis conformer of the methoxyurea can be attributed to
the intramolecular hydrogen bond between the aniline
NH and the methoxy oxygen atom. This intramolecular
hydrogen bond shields the hydrogen bonding moieties
from the solvent and will reduce the desolvation energy
cost during intestinal absorption. It is widely acknowl-
edged that urea is one of the strongest hydrogen bonding
functional groups,16 which renders compounds with the
urea moiety poorly bioavailable when administered
Con clu sion
Starting from T-98475 (1), we have replaced the
thieno[2,3-b]pyridin-4-one core with a thieno[2,3-d]-
pyrimidine-2,4-dione scaffold to attempt to develop a
new non-peptide LHRH antagonist. Prominent improve-
ments in both in vitro activities and oral absorption for
this class have been accomplished through introduction
of the unique methoxyurea moiety into the para position
of the 6-phenyl ring. Consequently, we have succeeded
in the discovery of a highly potent and orally bioavail-
able non-peptide antagonist for the human LHRH
receptor, the methoxyurea 9k (TAK-013). Compound 9k
showed high binding affinity and potent in vitro an-
tagonistic activity for the human receptor, with IC50
values of 0.1 and 0.06 nM, respectively. The methoxy-
urea 9k exhibited improved oral absorption in monkeys
compared to the ethylurea 9g, and oral administration
of 9k produced almost complete suppression of plasma
LH levels in castrated male cynomolgus monkeys at a
30 mg/kg dose, with sufficient duration of action (more
than 24 h). The SAR of compounds described here
demonstrated that the thienopyrimidine-2,4-dione nu-
cleus is an excellent surrogate for the thienopyridin-4-
one and that both the p-acylamino- and p-ureidophenyl
groups at the 6-position and the 3-phenyl ring play
important roles in potent antagonistic activity. Further-
more, molecular modeling studies suggest that 9k forms
an intramolecular hydrogen bond between the aniline
NH and the methoxy oxygen atom, which shields the
hydrogen bonding moieties from the solvent and results
in increasing apparent lipophilicity. On the basis of this
finding, we speculate that the presence of the intramo-
lecular hydrogen bond may increase the membrane
permeability of the alkoxyurea during intestinal absorp-
tion, which ultimately causes the improved oral absorp-
tion of 9k in monkeys.
From these biochemical and pharmacological results,
compound 9k (TAK-013) has been selected as a candi-
date for clinical trials and it is expected that the
methoxyurea 9k will provide a new class of useful
therapeutic agents for the treatment of sex-hormone-

Thieno[2,3-d]pyrimidine-2,4-dione Derivative
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1 119
Ta ble 5. Time Course of Plasma LH Concentrations in Castrated Male Cynomolgus Monkeys after Oral Administration of
Compound 9k at 10 and 30 mg/kg Doses
LH concentrationa (mU/mL)
at 0 h
at 2 h
at 4 h
at 8 h
at 24 h
at 48 h
vehicle
1.13 ( 0.20
(100.0 ( 0.0)
1.01 ( 0.18
(100.0 ( 0.0)
0.91 ( 0.10
(100.0 ( 0.0)
0.99 ( 0.17
(87.5 ( 3.9)
0.74 ( 0.17
(72.9 ( 3.5)
0.55 ( 0.10
(59.5 ( 5.7)
0.89 ( 0.13
(80.0 ( 3.2)
0.37 ( 0.12
(35.7 ( 4.9)
0.25 ( 0.03
(27.7 ( 0.6)
0.97 ( 0.17
(85.5 ( 0.4)
0.19 ( 0.06
(18.4 ( 2.8)
0.13 ( 0.02
(14.5 ( 2.2)
0.93 ( 0.16
(82.2 ( 0.2)
0.18 ( 0.01
(19.7 ( 4.1)
0.10 ( 0.04
(10.8 ( 4.6)
0.85 ( 0.14
(75.8 ( 2.3)
0.76 ( 0.17
(74.7 ( 5.3)
0.71 ( 0.15
(76.7 ( 10.6)
dose ) 10 mg/kg
dose ) 30 mg/kg
a
Cynomolgus monkeys (male, 4-9 years old) were castrated more than 6 months prior to the examination. Compound 9k (10 or 30
mg/kg, 3 mL/kg, n ) 3 for each group) suspended in 0.5% methylcellulose containing 1.2% citric acid, or 0.5% methylcellulose containing
1.2% citric acid alone (3 mL/kg, n ) 3), was administered orally. Figures in parentheses are percentages of LH concentration at the
indicated times, when LH concentration at 0 h (pretreatment) is taken as 100% for each group. Values shown are the mean ( SEM.
Ta ble 6. Energy Differences between the Cis (I) and Trans (II) Conformers of N-Ethyl-N′-phenylurea and N-Methoxy-N′-phenylureaa
Discover (CVFF)
DMol
b
c
d
c
E
(kcal/mol)
∆Ecis-trans
E
(kcal/mol)
∆Ecis-trans
N-ethyl-N′-phenylurea
-13.08
-2970.24
1.71
-0.67
-14.79
-9.50
-2969.57
-2723.95
N-methoxy-N′-phenylurea
-5.75
-7.42
-3.75
-2716.53
a
The cis (I) and trans (II) conformers were constructed using Insight II and subjected to either energy minimization by Discover,
using the CVFF force field, or geometry optimization by DMol. b Internal energy obtained by Discover for each conformer after minimization.
c Energy difference between the two conformers as calculated by subtracting the energy value of the trans conformer from that of the cis
d
conformer. Final binding energy obtained by DMol for each conformer after geometry optimization.
in toluene (100 mL) was heated under reflux for 20 h, while
water was azeotropically removed using Dean-Stark ap-
paratus. After the mixture was cooled to room temperature,
the mixture was concentrated in vacuo. The residue was
diluted with saturated NaHCO3 and extracted with CHCl3.
The extract was washed with brine and dried (MgSO4). After
evaporation of the solvent in vacuo, the residue was purified
by flash column chromatography (CH2Cl2-hexane, 2:4 to 4:1)
to give a red liquid (43.6 g), which was dissolved in EtOH (130
mL). To the solution were added sulfur powder (5.6 g, 0.18
mol) and diethylamine (18 mL, 0.18 mol), and the resulting
mixture was stirred at 65 °C for 2 h. After removal of the
solvent in vacuo, the residue was diluted with brine and
extracted with CHCl3. The extract was washed with brine and
dried (MgSO4). The solution was concentrated in vacuo, and
the residue was recrystallized from CHCl3 to afford 3a (32.9
g, 67%) as dark-orange crystals: mp 168-170 °C. 1H NMR
(CDCl3): δ 1.39 (3H, t, J ) 7.1 Hz), 2.40 (3H, s), 4.34 (2H, q,
J ) 7.1 Hz), 6.27 (2H, br), 7.48 (2H, d, J ) 8.7 Hz), 8.23 (2H,
d, J ) 8.7 Hz). IR (KBr): 3446, 3324, 1667, 1580, 1545, 1506,
1491, 1475, 1410, 1332 cm-1. FAB-MS m/z: 306 (M + H). Anal.
(C14H14N2O4S) C, H, N, S.
dependent pathologies. Further detailed pharmacologi-
cal studies on 9k are in progress, and the results will
be presented elsewhere.
Exp er im en ta l Section
Ch em istr y. Gen er a l P r oced u r es. All melting points were
determined on a Yanagimoto micromelting point apparatus
and are uncorrected. The proton nuclear magnetic resonance
(1H NMR) spectra were recorded on a J EOL J NM-LA300 (300
MHz) spectrometer. Chemical shifts are given in δ values
(ppm) using tetramethylsilane as the internal standard. All J
values are given in hertz. The infrared (IR) spectra were
measured on a J ASCO FTIR spectrometer. FAB mass spectra
were recorded on a J EOL J MS-HX110. Elemental analyses
were within (0.4% of theoretical values and were determined
at Takeda Analytical Research Laboratories, Osaka. Reagents
and solvents were obtained from commercial sources and used
without further purification. Flash chromatography was per-
formed with Merck silica gel 60 (art. 9385; 230-400 mesh),
and reaction progress was determined by thin-layer chroma-
tography (TLC) analysis on silica gel 60 F254 plates (Merck).
Visualization was done with UV light (254 nm) or iodine.
Yields are of purified compounds and were not optimized.
Eth yl 2-Am in o-4-m eth yl-5-(4-n itr op h en yl)th iop h en e-
3-ca r boxyla te (3a ). A mixture of 4-nitrophenylacetone (39.0
g, 0.22 mol), ethyl cyanoacetate (25.1 g, 0.22 mol), ammonium
acetate (3.36 g, 0.04 mmol), and acetic acid (10.0 mL, 0.17 mol)
Eth yl 2-Am in o-5-(4-m eth oxyph en yl)-4-m eth ylth ioph en e-
3-ca r boxyla te (3b). Compound 3b was prepared in 40% yield
from 4-methoxyphenylacetone by a method similar to that
described for 3a , as pale-yellow plates: mp 79-80 °C. 1H NMR
(CDCl3): δ 1.37 (3H, t, J ) 7.1 Hz), 2.28 (3H, s), 3.83 (3H, s),
4.31 (2H, q, J ) 7.1 Hz), 6.05 (2H, br), 6.91 (2H, d, J

120 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1
Sasaki et al.
) 8.8 Hz), 7.27 (2H, d, J ) 8.8 Hz). IR (KBr): 3426, 3328,
1651, 1586, 1550, 1505, 1485 cm-1. FAB-MS m/z: 291 (M +
H). Anal. (C15H17NO3S) C, H, N, S.
J ) 8.7 Hz), 7.25-7.53 (7H, m), 8.92 (1H, s). IR (KBr): 1719,
1659, 1607 cm-1. Anal. (C20H16N2O3S) C, H, N.
3-(2-Met h oxyp h en yl)-6-(4-m et h oxyp h en yl)-5-m et h yl-
th ien o[2,3-d ]p yr im id in e-2,4(1H,3H)-d ion e (4g). Compound
4g was prepared in 81% yield from 3b and 2-methoxyphenyl
isocyanate by a method similar to that described for 4a , as a
5-Meth yl-6-(4-n itr op h en yl)-3-p h en ylth ien o[2,3-d ]p yr i-
m id in e-2,4(1H,3H)-d ion e (4a ). A mixture of 3a (40.0 g, 0.13
mol) and phenyl isocyanate (17.0 mL, 0.16 mol) in pyridine
(240 mL) was stirred at 45 °C for 2 h. The mixture was
concentrated in vacuo, and the residual solid was suspended
in MeOH (400 mL). To this suspension was added sodium
methoxide (28% in MeOH solution, 63.2 g, 0.33 mol). After
being stirred at room temperature for 6 h, the mixture was
acidified with 2 N HCl (200 mL) at 0 °C. After evaporation of
the solvent in vacuo, the yellow precipitate was collected by
filtration, washed with H2O, and dried over P2O5 in vacuo to
give 4a (50.4 g, 100%) as a yellow powder: mp >300 °C. 1H
NMR (DMSO-d6): δ 2.50 (3H, s), 7.30 (2H, d, J ) 6.9 Hz),
7.42-7.51 (3H, m), 7.77 (2H, d, J ) 8.7 Hz), 8.31 (2H, d, J )
1
colorless powder: mp 257-258 °C. H NMR (CDCl3): δ 2.46
(3H, s), 3.82 (3H, s), 3.86 (3H, s), 6.95-7.09 (4H, m), 7.21-
7.42 (4H, m). IR (KBr): 1711, 1655, 1605, 1570, 1531 cm-1
.
Anal. (C21H18N2O4S‚0.1H2O) C, H, N.
3-(3-Met h oxyp h en yl)-6-(4-m et h oxyp h en yl)-5-m et h yl-
th ien o[2,3-d ]p yr im id in e-2,4(1H,3H)-d ion e (4h ). Compound
4h was prepared in 93% yield from 3b and 3-methoxyphenyl
isocyanate by a method similar to that described for 4a , as a
1
colorless powder: mp >300 °C. H NMR (CDCl3): δ 2.47 (3H,
s), 3.82 (3H, s), 3.86 (3H, s), 6.81-7.01 (5H, m), 7.32-7.45 (3H,
m), 8.54 (1H, s). IR (KBr): 1719, 1659, 1607 cm-1. Anal.
(C21H18N2O4S‚0.2H2O) C, H, N.
8.7 Hz), 12.50 (1H, s). IR (KBr): 1715, 1657, 1593, 1510 cm-1
.
Anal. (C19H13N3O4S‚0.5H2O) C, H, N.
3-(4-Met h oxyp h en yl)-6-(4-m et h oxyp h en yl)-5-m et h yl-
th ien o[2,3-d ]p yr im id in e-2,4(1H,3H)-d ion e (4i). Compound
4i was prepared in 99% yield from 3b and 4-methoxyphenyl
isocyanate by a method similar to that described for 4a , as a
pale-yellow powder: mp >300 °C. 1H NMR (CDCl3): δ 2.47
(3H, s), 3.84 (3H, s), 3.86 (3H, s), 6.95-7.04 (4H, m), 7.19-
7.35 (4H, m), 8.67 (1H, s). IR (KBr): 1717, 1653, 1609, 1516,
1497 cm-1. Anal. (C21H18N2O4S) C, H, N.
3-(3-Meth oxyph en yl)-5-m eth yl-6-(4-n itr oph en yl)th ien o-
[2,3-d ]p yr im id in e-2,4(1H,3H)-d ion e (4b). Compound 4b
was prepared in 77% yield from 3a and 3-methoxyphenyl
isocyanate by a method similar to that described for 4a , as
pale-yellow plates: mp >300 °C. 1H NMR (CDCl3): δ 2.50 (3H,
s), 3.78 (3H, s), 6.87 (1H, d, J ) 8.1 Hz), 6.92 (1H, s), 7.00
(1H, d, J ) 8.1 Hz), 7.38 (1H, t, J ) 8.1 Hz), 7.77 (2H, d, J )
8.7 Hz), 8.31 (2H, d, J ) 8.7 Hz), 12.48 (1H, s). IR (KBr): 1717,
1661, 1593, 1510, 1429 cm-1. Anal. (C20H15N3O5S) C, H, N.
3-(2-Ch lor op h e n yl)-6-(4-m e t h oxyp h e n yl)-5-m e t h yl-
th ien o[2,3-d ]p yr im id in e-2,4(1H,3H)-d ion e (4j). Compound
4j was prepared in 95% yield from 3b and 2-chlorophenyl
isocyanate by a method similar to that described for 4a , as
3-Isobu tyl-6-(4-m eth oxyp h en yl)-5-m eth ylth ien o[2,3-d ]-
p yr im id in e-2,4(1H,3H)-d ion e (4c). A mixture of isovaleric
acid (1.15 mL, 10.0 mmol), diphenylphosphoryl azide (DPPA;
2.83 g, 10.3 mmol), and triethylamine (1.45 mL, 10.0 mmol)
in toluene (15 mL) was heated under reflux for 1.5 h. After
the mixture was cooled to below 40 °C, a solution of 3b (2.0 g,
6.85 mmol) in toluene (5 mL) was added and the resulting
mixture was heated under reflux for another 4 days. The
reaction mixture was diluted with brine and extracted with
EtOAc. The extract was washed with brine and dried (MgSO4).
After evaporation of the solvent in vacuo, the residue was
purified by flash column chromatography (EtOAc-hexane, 1:6)
to give the urea derivative as a white solid. To a solution of
the solid in MeOH (30 mL) was added sodium methoxide (28%
in MeOH solution, 3.93 g, 20.4 mmol). After being stirred at
room temperature for 16 h, the mixture was acidified with 1
N HCl (22 mL) at 0 °C. The resulting mixture was concen-
trated in vacuo, and the precipitate was collected by filtration,
washed with H2O, and dried over P2O5 in vacuo. Recrystalli-
zation from EtOH afforded 4c (1.61 g, 70%) as colorless
1
colorless crystals: mp 285-286 °C. H NMR (CDCl3): δ 2.45
(3H, s), 3.87 (3H, s), 6.98 (2H, d, J ) 8.7 Hz), 7.29-7.40 (5H,
m), 7.54 (1H, d, J ) 8.4 Hz), 10.07 (1H, s). IR (KBr): 1711,
1657, 1607, 1564, 1531 cm-1. Anal. (C20H15N2O3SCl) C, H, N.
3-(3-Ch lor op h e n yl)-6-(4-m e t h oxyp h e n yl)-5-m e t h yl-
th ien o[2,3-d ]p yr im id in e-2,4(1H,3H)-d ion e (4k ). Compound
4k was prepared in 97% yield from 3b and 3-chlorophenyl
isocyanate by a method similar to that described for 4a , as a
1
colorless powder: mp >300 °C. H NMR (CDCl3): δ 2.37 (3H,
s), 3.81 (3H, s), 7.05 (2H, d, J ) 8.7 Hz), 7.29-7.32 (1H, m),
7.39 (2H, d, J ) 8.7 Hz), 7.47-7.52 (3H, m). IR (KBr): 1717,
1657, 1497 cm-1. Anal. (C20H15N2O3SCl) C, H, N.
3-(4-Ch lor op h e n yl)-6-(4-m e t h oxyp h e n yl)-5-m e t h yl-
th ien o[2,3-d ]p yr im id in e-2,4(1H,3H)-d ion e (4l). Compound
4l was prepared in 95% yield from 3b and 4-chlorophenyl
isocyanate by a method similar to that described for 4a , as a
colorless powder: mp >300 °C. H NMR (CDCl3): δ 2.36 (3H,
s), 3.79 (3H, s), 7.03 (2H, d, J ) 8.4 Hz), 7.33 (2H, d, J ) 8.4
Hz), 7.38 (2H, d, J ) 8.4 Hz), 7.53 (2H, d, J ) 8.4 Hz), 12.35
(1H, s). IR (KBr): 1721, 1663, 1609, 1497 cm-1. Anal.
(C20H15N2O3SCI) C, H, N.
1
needles: mp 215-216 °C. H NMR (CDCl3): δ 0.96 (6H, d, J
) 6.8 Hz), 2.13-2.22 (1H, m), 2.50 (3H, s), 3.85-3.87 (5H, m),
6.96 (2H, d, J ) 8.8 Hz), 7.33 (2H, d, J ) 8.8 Hz), 9.50 (1H, s).
IR (KBr): 1711, 1657, 1537, 1499, 1458 cm-1. FAB-MS m/z:
345 (M + H). Anal. (C18H20N2O3S) C, H, N.
1-(2,6-Diflu or ob en zyl)-5-m et h yl-6-(4-n it r op h en yl)-3-
p h en ylth ien o[2,3-d ]p yr im id in e-2,4(1H,3H)-d ion e (5a ). A
mixture of 4a (50.4 g, 0.13 mol), 2,6-difluorobenzyl chloride
(25.9 g, 0.16 mol), K2CO3 (27.5 g, 0.20 mol), and KI (11.0 g,
66.4 mmol) in DMF (800 mL) was stirred at room temperature
for 4 h. The mixture was concentrated in vacuo, and the
residue was partitioned between CHCl3 and H2O. The aqueous
phase was separated and extracted with CHCl3. The combined
extracts were washed with brine and dried (MgSO4). The
solution was concentrated in vacuo, and the residue was
recrystallized from EtOH to afford 5a (60.7 g, 90%) as yellow
crystals: mp 280-282 °C. 1H NMR (CDCl3): δ 2.57 (3H, s),
5.38 (2H, s), 6.94 (2H, t, J ) 8.1 Hz), 7.42-7.58 (8H, m), 8.29
3-Cycloh exyl-6-(4-m eth oxyp h en yl)-5-m eth ylth ien o[2,3-
d ]p yr im id in e-2,4(1H,3H)-d ion e (4d ). Compound 4d was
prepared in 84% yield from 3b and cyclohexyl isocyanate by a
method similar to that described for 4a , as colorless needles:
mp 275-276 °C. 1H NMR (CDCl3): δ 1.23-1.88 (8H, m), 2.41-
2.55 (5H, m), 3.84 (3H, s), 4.83-4.91 (1H, m), 6.96 (2H, d, J )
8.7 Hz), 7.32 (2H, d, J ) 8.7 Hz), 9.94 (1H, s). IR (KBr): 1709,
1640, 1497 cm-1. Anal. (C20H22N2O3S‚0.1H2O) C, H, N.
3-Ben zyl-6-(4-m eth oxyp h en yl)-5-m eth ylth ien o[2,3-d ]-
p yr im id in e-2,4(1H,3H)-d ion e (4e). Compound 4e was pre-
pared in 92% yield from 3b and benzyl isocyanate by a method
similar to that described for 4a , as a colorless powder: mp
241-242 °C. 1H NMR (CDCl3): δ 2.50 (3H, s), 3.85 (3H, s),
5.21 (2H, s), 6.96 (2H, d, J ) 8.7 Hz), 7.25-7.33 (5H, m), 7.52
(2H, d, J ) 8.7 Hz), 10.07 (1H, s). IR (KBr): 1705, 1649, 1497
cm-1. Anal. (C21H18N2O3S) C, H, N.
(2H, d, J ) 8.8 Hz). IR (KBr): 1719, 1669, 1524, 1473 cm-1
.
Anal. (C26H17N3O2SF2) C, H, N.
1-(2,6-Diflu or oben zyl)-3-(3-m eth oxyp h en yl)-5-m eth yl-
6-(4-n itr op h en yl)th ien o[2,3-d ]p yr im id in e-2,4(1H,3H)-d i-
on e (5b). Compound 5b was prepared in 84% yield from 4b
by a method similar to that described for 5a , as a yellow
6-(4-Meth oxyp h en yl)-5-m eth yl-3-p h en ylth ien o[2,3-d ]-
p yr im id in e-2,4(lH,3H)-d ion e (4f). Compound 4f was pre-
pared in 98% yield from 3b and phenyl isocyanate by a method
similar to that described for 4a , as colorless crystals: mp >300
1
solid: mp 231-234 °C. H NMR (CDCl3): δ 2.57 (3H, s), 3.83
1
°C. H NMR (CDCl3): δ 2.47 (3H, s), 3.86 (3H, s), 6.98 (2H, d,
(3H, s), 5.38 (2H, s), 6.80-7.01 (5H, m), 7.29-7.46 (2H, m),

Thieno[2,3-d]pyrimidine-2,4-dione Derivative
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1 121
7.56 (2H, d, J ) 8.8 Hz), 8.29 (2H, d, J ) 8.8 Hz). IR (KBr):
1719, 1678, 1595, 1522, 1491 cm-1. Anal. (C27H19N3O5SF2) C,
H, N.
powder: mp 278-279 °C. 1H NMR (CDCl3): δ 2.46 (3H, s),
3.85 (3H, s), 5.35 (2H, s), 6.89-6.97 (4H, m), 7.17-7.21 (1H,
m), 7.29-7.48 (6H, m). IR (KBr): 1717, 1665, 1531, 1470 cm-1
.
Anal. (C27H19N2O3SClF2) C, H, N.
1-(2,6-Diflu or oben zyl)-3-isobu tyl-6-(4-m eth oxyp h en yl)-
5-m eth ylth ien o[2,3-d ]p yr im id in e-2,4(1H,3H)-d ion e (5c).
Compound 5c was prepared in 73% yield from 4c by a method
similar to that described for 5a , as colorless needles: mp 121-
3-(4-Ch lor oph en yl)-1-(2,6-diflu or oben zyl)-6-(4-m eth oxy-
p h en yl)-5-m eth ylth ien o[2,3-d ]p yr im id in e-2,4(1H,3H)-d i-
on e (5l). Compound 5l was prepared in 91% yield from 4l by
a method similar to that described for 5a , as a colorless
1
122 °C. H NMR (CDCl3): δ 0.96 (6H, d, J ) 6.8 Hz), 2.18-
1
2.24 (1H, m), 2.49 (3H, s), 3.84 (3H, s), 3.91 (2H, d, J ) 6.8
Hz), 5,34 (2H, s), 6.87-6.95 (4H, m), 7.26-7.29 (3H, m). IR
(KBr): 1698, 1659, 1533, 1470 cm-1. Anal. (C25H24N2O3SF2‚
0.1H2O) C, H, N.
powder: mp >300 °C. H NMR (CDCl3): δ 2.46 (3H, s), 3.84
(3H, s), 5.35 (2H, s), 6.89-6.97 (4H, m), 7.21-7.35 (5H, m),
7.48 (2H, d, J ) 9.0 Hz). IR (KBr): 1715, 1665, 1470 cm-1
Anal. (C27H19N2O3SClF2) C, H, N.
.
3-Cycloh exyl-1-(2,6-d iflu or oben zyl)-6-(4-m eth oxyph en -
yl)-5-m eth ylth ien o[2,3-d]pyr im idin e-2,4(1H,3H)-dion e (5d).
Compound 5d was prepared in 73% yield from 4d by a method
similar to that described for 5a , as colorless needles: mp 207-
208 °C. 1H NMR (CDCl3): δ 1.25-1.86 (8H, m), 2.43-2.57 (5H,
m), 3.83 (3H, s), 4.87-4.95 (1H, m), 5.30 (2H, s), 6.86-6.95
(4H, m), 7.26-7.29 (3H, m). IR (KBr): 1705, 1659, 1628, 1609,
1566, 1539 cm-1. Anal. (C27H26N2O3SF2) C, H, N.
3-Ben zyl-1-(2,6-d iflu or oben zyl)-6-(4-m eth oxyp h en yl)-
5-m eth ylth ien o[2,3-d ]p yr im id in e-2,4(1H,3H)-d ion e (5e).
Compound 5e was prepared in 82% yield from 4e by a method
similar to that described for 5a , as a colorless powder: mp
161-163 °C. 1H NMR (CDCl3): δ 2.46 (3H, s), 3.81 (3H, s),
5.26 (2H, s), 5.35 (2H, s), 6.86-6.94 (4H, m), 7.21-7.34 (6H,
m), 7.51 (2H, d, J ) 8.4 Hz). IR (KBr): 1709, 1661, 1535, 1473
cm-1. Anal. (C28H22N2O3SF2) C, H, N.
1-(2,6-Diflu or oben zyl)-6-(4-m eth oxyp h en yl)-5-m eth yl-
3-p h en ylth ien o[2,3-d ]p yr im id in e-2,4(1H,3H)-d ion e (5f).
Compound 5f was prepared in 87% yield from 4f by a method
similar to that described for 5a , as colorless needles: mp 291-
292 °C. 1H NMR (CDCl3): δ 2.47 (3H, s), 3.85 (3H, s), 5.36
(2H, s), 6.88-6.96 (4H, m), 7.29-7.32 (5H, m), 7.43-7.54 (3H,
m). IR (KBr): 1717, 1665, 1626, 1560, 1531, 1508 cm-1. Anal.
(C27H20N2O3SF2‚0.1H2O) C, H, N.
1-(2,6-Diflu or oben zyl)-3-(2-m eth oxyp h en yl)-6-(4-m eth -
oxyp h en yl)-5-m eth ylth ien o[2,3-d ]p yr im id in e-2,4(1H,3H)-
d ion e (5g). Compound 5g was prepared in 86% yield from 4g
by a method similar to that described for 5a , as colorless
crystals: mp 205-208 °C. 1H NMR (CDCl3): δ 2.47 (3H, s),
3.81 (3H, s), 3.84 (3H, s), 5.37 (2H, s), 6.88-6.96 (4H, m), 7.03-
7.09 (2H, m), 7.21-7.32 (4H, m), 7.40 (1H, t, J ) 8.0 Hz). IR
(KBr): 1717, 1680, 1605, 1524, 1506 cm-1. Anal. (C28H22N2O4-
SF2) C, H, N.
5-(N-Ben zyl-N-m eth ylam in om eth yl)-1-(2,6-diflu or oben -
zyl)-6-(4-n itr op h en yl)-3-p h en ylth ien o[2,3-d ]p yr im id in e-
2,4(1H,3H)-d ion e (6a ). A mixture of 5a (30.7 g, 60.7 mmol),
N-bromosuccinimide (NBS; 13.0 g, 72.9 mmol), and 2,2′-
azobisisobutyronitrile (AIBN; 1.20 g, 7.29 mmol) in chloroben-
zene (450 mL) was stirred at 90 °C for 1.5 h. After the mixture
was cooled to room temperature, the mixture was concentrated
in vacuo. The residue was diluted with H2O and extracted with
CH2Cl2. The extract was washed with brine and dried (MgSO4).
The solution was concentrated in vacuo to give the crude
bromomethyl compound (40.1 g) as a yellow solid: 1H NMR
(CDCl3): δ 4.77 (2H, s), 5.38 (2H, s), 6.96 (2H, t, J ) 8.1 Hz),
7.29-7.58 (6H, m), 7.79 (2H, d, J ) 8.5 Hz), 8.35 (2H, d, J )
8.5 Hz). FAB-MS m/z: 584 (M + H).
To a mixture of the bromomethyl compound (40.0 g, 59.1
mmol) and N,N-diisopropylethylamine (9.94 g, 76.8 mmol) in
DMF (300 mL) was added N-benzylmethylamine (8.58 g, 70.9
mmol). After being stirred at room temperature for 2 h, the
mixture was concentrated in vacuo and the residue was diluted
with saturated NaHCO3 and extracted with CHCl3. The extract
was washed with brine and dried (MgSO4). After evaporation
of the solvent in vacuo, the residue was purified by flash
column chromatography (CH2Cl2-EtOAc-hexane, 1:1:4 to 1:1:
3) to give a yellow solid. Recrystallization from EtOAc afforded
1
6a (33.0 g, 88%) as yellow needles: mp 173-174 °C. H NMR
(CDCl3): δ 2.15 (3H, s), 3.60 (2H, s), 3.96 (2H, s), 5.39 (2H, s),
6.95 (2H, t, J ) 8.2 Hz), 7.18-7.55 (11H, m), 8.02 (2H, d, J )
9.0 Hz), 8.26 (2H, d, J ) 9.0 Hz). IR (KBr): 1719, 1678, 1597,
1520 cm-1. Anal. (C34H26N4O4SF2‚0.5H2O) C, H, N.
Compounds 6b-l were prepared by a procedure similar to
that described for 6a , and the physicochemical data are shown
in Table 1 (6c-l) and Table 2 (6b).
6-(4-Am in oph en yl)-5-(N-ben zyl-N-m eth ylam in om eth yl)-
1-(2,6-d iflu or oben zyl)-3-p h en ylth ien o[2,3-d ]p yr im id in e-
2,4(1H,3H)-d ion e (7a ). A solution of 6a (20.0 g, 32.0 mmol)
and 1 M ethereal HCl (96 mL) in formic acid (140 mL) was
hydrogenated over 10% palladium-charcoal (containing 50%
H2O, 2.0 g) under atomospheric pressure at room temperature
for 3.5 h. The mixture was filtered though Celite and the
filtrate was concentrated in vacuo. The residue was diluted
with saturated NaHCO3 and extracted with CH2Cl2. The
extract was washed with brine, dried (MgSO4), and concen-
trated in vacuo. The residue was purified by flash column
chromatography (CH2Cl2-EtOAc-MeOH, 80:20:1 to 20:5:1) to
give a pale-yellow solid. Recrystallization from EtOAc-diethyl
ether afforded 7a (16.5 g, 87%) as pale-yellow crystals: mp
205-207 °C. 1H NMR (CDCl3): δ 2.05 (3H, s), 3.56 (2H, s),
3.83 (2H, s), 3.88 (2H, s), 5.36 (2H, s), 6.70 (2H, d, J ) 8.8
Hz), 6.88-6.94 (2H, m), 7.21-7.31 (8H, m), 7.41-7.53 (5H,
m). IR (KBr): 1715, 1657, 1628, 1537 cm-1. Anal. (C34H28N4O2-
SF2‚H2O) C, H, N.
6-(4-Am in oph en yl)-5-(N-ben zyl-N-m eth ylam in om eth yl)-
1-(2,6-d iflu or oben zyl)-3-(3-m eth oxyp h en yl)th ien o[2,3-d ]-
p yr im id in e-2,4(1H,3H)-d ion e (7b) a n d 5-(N-Ben zyl-N-
m eth yla m in om eth yl)-1-(2,6-d iflu or oben zyl)-6-(4-for m yl-
a m in o p h e n y l)-3-(3-m e t h o x y p h e n y l)t h ie n o [2,3-d ]p y -
r im id in e-2,4(1H,3H)-d ion e (8). A solution of 6b (2.40 g, 3.66
mmol) in formic acid (30 mL) was hydrogenated over 10%
palladium-charcoal (containing 50% H2O, 240 mg) under
atomospheric pressure at room temperature for 2 h. The
residue obtained after a similar workup as described above
was purified by flash column chromatography (CHCl3-EtOAc,
1-(2,6-Diflu or oben zyl)-3-(3-m eth oxyp h en yl)-6-(4-m eth -
oxyp h en yl)-5-m eth ylth ien o[2,3-d ]p yr im id in e-2,4(1H,3H)-
d ion e (5h ). Compound 5h was prepared in 85% yield from
4h by a method similar to that described for 5a , as a colorless
powder: mp 253-255 °C. 1H NMR (CDCl3): δ 2.47 (3H, s),
3.83 (3H, s), 3.85 (3H, s), 5.36 (2H, s), 6.81-7.00 (7H, m), 7.29-
7.44 (4H, m). IR (KBr): 1719, 1671, 1609, 1535 cm-1. Anal.
(C28H22N2O4SF2) C, H, N.
1-(2,6-Diflu or oben zyl)-3-(4-m eth oxyp h en yl)-6-(4-m eth -
oxyp h en yl)-5-m eth ylth ien o[2,3-d ]p yr im id in e-2,4(1H,3H)-
d ion e (5i). Compound 5i was prepared in 83% yield from 4i
by a method similar to that described for 5a , as colorless
1
needles: mp >300 °C. H NMR (CDCl3): δ 2.47 (3H, s), 3.84
(6H, s), 5.35 (2H, s), 6.88-6.96 (4H, m), 7.02 (2H, d, J ) 9.0
Hz), 7.19 (2H, d, J ) 9.0 Hz), 7.26-7.32 (3H, m). IR (KBr):
1715, 1665, 1473 cm-1. Anal. (C28H22N2O4SF2) C, H, N.
3-(2-Ch lor oph en yl)-1-(2,6-diflu or oben zyl)-6-(4-m eth oxy-
p h en yl)-5-m eth ylth ien o[2,3-d ]p yr im id in e-2,4(1H,3H)-d i-
on e (5j). Compound 5j was prepared in 83% yield from 4j by
a method similar to that described for 5a , as a colorless
powder: mp 178-182 °C. 1H NMR (CDCl3): δ 2.47 (3H, s),
3.85 (3H, s), 5.32 (1H, d, J ) 15.9 Hz), 5.46 (1H, d, J ) 15.9
Hz), 6.89-6.97 (4H, m), 7.29-7.42 (6H, m), 7.56-7.58 (1H,
mn). IR (KBr): 1717, 1673, 1628, 1605, 1560, 1528 cm-1. Anal.
(C27H19N2O3SClF2) C, H, N.
3-(3-Ch lor oph en yl)-1-(2,6-diflu or oben zyl)-6-(4-m eth oxy-
p h en yl)-5-m eth ylth ien o[2,3-d ]p yr im id in e-2,4(1H,3H)-d i-
on e (5k ). Compound 5k was prepared in 93% yield from 4k
by a method similar to that described for 5a , as a colorless

122 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1
Sasaki et al.
1:1) to give 7b (1.37 g, 60%) as a colorless amorphous solid
and 8 (0.26 g, 11%) as a white solid.
7b. H NMR (CDCl3): δ 2.04 (3H, s), 3.56 (2H, s), 3.83 (3H,
ethereal HCl to afford the hydrochloride (TAK-013 hydrochlo-
ride) as a white powder (from MeOH-diethyl ether): mp 182-
185 °C. IR (KBr): 3440, 3042, 1713, 1665, 1628, 1593, 1539,
1473 cm-1. FAB-MS m/z: 668 (M + H). Anal. (C36H31N5O4-
SF2‚HCl‚0.5H2O) C, H, N.
Compounds 9f,h ,l,m were synthesized by a procedure
similar to that described for 9k , and the physicochemical data
are shown in Table 2.
1
s), 3.88 (2H, s), 5.35 (2H, s), 6.71 (2H, d, J ) 8.8 Hz), 6.82-
7.00 (5H, m), 7.16-7.52 (9H, m). The free amine was treated
with 1 M ethereal HCl to afford the hydrochloride as a white
powder (from EtOAc-diethyl ether): mp 162-165 °C. IR
(KBr): 1715, 1659, 1537, 1473 cm-1. Anal. (C35H30N4O3SF2‚
2.0HCl‚H2O) C, H, N.
5-(N-Ben zyl-N-m eth ylam in om eth yl)-1-(2,6-diflu or oben -
zyl)-6-[4-[3-(2,4-d im et h oxyb en zyloxy)u r eid o]p h en yl]-3-
ph en ylth ien o[2,3-d]pyr im idin e-2,4(1H,3H)-dion e (9i). Com-
pound 9i was prepared from 7a and O-(2,4-dimethoxybenzyl)-
hydroxylamine15 by a method similar to that described for 9k ,
as a pale-brown amorphous powder. 1H NMR (CDCl3): δ 2.06
(3H, s), 3.57 (2H, s), 3.80 (3H, s), 3.82 (3H, s), 3.91 (2H, s),
4.87 (2H, s), 5.37 (2H, s), 6.43-6.60 (2H, m), 6.92 (2H, t, J )
8.2 Hz), 7.20-7.31 (13H, m), 7.44-7.53 (4H, m), 8.12 (1H, s).
8. Mp 213-215 °C. 1H NMR (CDCl3): δ 2.06 (3H, s), 3.58
(2H, s), 3.83 (3H, s), 3.90 (2H, s), 5.36 (2H, s), 6.82-7.76 (17H,
m), 8.43-8.75 (1H, m). IR (KBr): 1717, 1669, 1609, 1537, 1464
cm-1. Anal. (C36H30N4O4SF2‚1.2H2O) C, H, N.
5-(N-Ben zyl-N-m eth ylam in om eth yl)-1-(2,6-diflu or oben -
zyl)-3-p h en yl-6-(4-p r op ion yla m in op h en yl)th ien o[2,3-d ]-
p yr im id in e-2,4(1H,3H)-d ion e (9a ). To an ice-cooled solution
of 7a (10.0 g, 15.0 mmol) and triethylamine (8.35 mL, 59.9
mmol) in CH2Cl2 (200 mL) was added propionyl chloride (1.82
mL, 21.0 mmol). After being stirred at room temperature for
4 h, the mixture was diluted with saturated NaHCO3 and
extracted with CHCl3. The extract was washed with brine and
dried (MgSO4). The solution was concentrated in vacuo, and
the residue was purified by flash column chromatography
(CHCl3-EtOAc-MeOH, 80:20:1 to 60:40:5) to give 9a (7.50 g,
77%) as a colorless amorphous powder. 1H NMR (CDCl3): δ
1.25 (3H, t, J ) 7.2 Hz), 2.05 (3H, s), 2.39 (2H, q, J ) 7.2 Hz),
3.56 (2H, s), 3.90 (2H, s), 5.36 (2H, s), 6.89-6.94 (2H, m), 7.15-
7.59 (13H, m), 7.69 (2H, d, J ) 8.8 Hz). The free amine was
treated with 1 M ethereal HCl to afford the hydrochloride as
a white powder (from EtOAc-diethyl ether): mp 197-202 °C.
IR (KBr): 1717, 1655, 1543, 1475 cm-1. Anal. (C37H32N4O3-
SF2‚HCl‚H2O) C, H, N.
5-(N-Ben zyl-N-m eth ylam in om eth yl)-1-(2,6-diflu or oben -
zyl)-6-[4-(3-h yd r oxyu r eid o)p h en yl]-3-p h en ylt h ien o[2,3-
d ]p yr im id in e-2,4(1H,3H)-d ion e (9j). To a solution of 9i (2.70
g, 3.36 mmol) in CH2Cl2 (50 mL) was added trifluoroacetic acid
(TFA; 5 mL). After being stirred at room temperature for 20
min, the mixture was diluted with saturated NaHCO3 and
extracted with CHCl3. The extract was washed with brine and
dried (MgSO4). After evaporation of the solvent in vacuo, the
residue was purified by flash column chromatography (CHCl3-
MeOH, 10:1) to give a colorless amorphous powder. Recrys-
tallization from CHCl3-diethyl ether afforded 9j (2.23 g, 100%)
1
as white crystals; mp 164-165 °C. H NMR (CDCl3): δ 2.05
(3H, s), 3.46 (2H, s), 3.92 (2H, s), 5.35 (2H, s), 6.65 (1H, br),
6.90 (2H, t, J ) 8.0 Hz), 7.28-7.43 (15H, m), 8.04 (1H, s), 9.73
(1H, br). The free amine was treated with 1 M ethereal HCl
to afford the hydrochloride as a white powder (from MeOH-
diethyl ether): mp 180-186 °C. IR (KBr): 3388, 3066, 1713,
1663, 1628, 1593, 1537, 1473 cm-1. FAB-MS m/z: 654 (M +
H). Anal. (C35H29N5O4SF2‚HCl‚H2O) C, H, N.
Compounds 9b-e were synthesized by a procedure similar
to that described for 9a , and the physicochemical data are
shown in Table 2.
5-(N-Ben zyl-N-m eth ylam in om eth yl)-1-(2,6-diflu or oben -
zyl)-6-[4-(3-eth ylu r eid o)p h en yl]-3-p h en ylth ien o[2,3-d ]p y-
r im id in e-2,4(1H,3H)-d ion e (9g). To a solution of 7a (0.70 g,
1.18 mmol) in pyridine (10 mL) was added ethyl isocyanate
(0.11 mL, 1.42 mmol). After being stirred at room temperature
for 18 h, the mixture was concentrated in vacuo. The residue
was diluted with H2O and extracted with CH2Cl2. The extract
was washed with brine and dried (MgSO4). After evaporation
of the solvent in vacuo, the residue was purified by flash
column chromatography (CHCl3-EtOAc-MeOH, 80:20:1) to
give 9g (0.78 g, 100%) as a pale-yellow amorphous powder.
1H NMR (CDCl3): δ 1.19 (3H, t, J ) 7.2 Hz), 2.06 (3H, s), 3.28-
3.37 (2H, m), 3.56 (2H, s), 3.90 (2H, s), 4.62 (1H, t, J ) 5.3
Hz), 5.36 (2H, s), 6.28 (1H, s), 6.92 (2H, t, J ) 8.0 Hz), 7.18-
7.55 (13H, m), 7.69 (2H, d, J ) 8.6 Hz). The free amine was
treated with 1 M ethereal HCl to afford the hydrochloride as
a white powder (from CHCl3-diethyl ether): mp 179-182 °C.
Anal. (C37H33N5O3SF2‚HCl‚1.5H2O) C, H, N.
5-(N-Ben zyl-N-m eth ylam in om eth yl)-1-(2,6-diflu or oben -
zyl)-3-(3-m eth oxyph en yl)-6-[4-(N-m eth yl-N-pr opion ylam i-
n o)ph en yl]th ien o[2,3-d]pyr im idin e-2,4(1H,3H)-dion e (10).
To a solution of 8 (0.73 g, 1.12 mmol) in THF (20 mL) was
added dropwise a solution of borane-methyl sulfide complex
(0.28 mL, 2.8 mmol) in THF (10 mL) at 0 °C. The reaction
mixture was stirred at room temperature for 1 h and then
heated under reflux for 2 h. After the mixture was cooled,
MeOH (5 mL) was added at 0 °C and the mixture was stirred
for 30 min. The resulting mixture was treated with 1 M
ethereal HCl to attain pH < 2 and heated at 80 °C for 1 h.
After evaporation of the solvent in vacuo, the concentrate was
diluted with saturated NaHCO3 and extracted with CH2Cl2.
The extract was washed with brine and dried (MgSO4). The
solution was concentrated in vacuo, and the residue was
purified by flash column chromatography (CHCl3-EtOAc, 10:1
to 4:1) to give the N-methylaniline (0.61 g, 85%) as a white
powder. To an ice-cooled solution of the powder (0.20 g, 0.31
mmol) and triethylamine (43 µL, 0.31 mmol) in CH2Cl2 (10
mL) was added dropwise propionyl chloride (27 µL, 0.31 mmol).
After being stirred at room temperature for 4 h, the mixture
was diluted with saturated NaHCO3 and extracted with CH2-
Cl2. The extract was washed with brine and dried (MgSO4).
After removal of the solvent in vacuo, the residue was purified
by flash column chromatography (CH2Cl2-EtOAc, 10:1 to 4:1)
to afford 10 (0.18 g, 84% for two steps from 8) as a colorless
5-(N-Ben zyl-N-m eth ylam in om eth yl)-1-(2,6-diflu or oben -
zyl)-6-[4-(3-m eth oxyu r eid o)p h en yl]-3-p h en ylth ien o[2,3-
d ]p yr im id in e-2,4(1H,3H)-d ion e (9k ). To an ice-cooled so-
lution of 7a (20.6 g, 34.7 mmol) and triethylamine (9.67 mL,
69.4 mmol) in CH2Cl2 (480 mL) was added 1,1′-carbonyldiimi-
dazole (CDI; 11.3 g, 69.4 mmol). After being stirred at room
temperature for 24 h, O-methylhydroxylammonium chloride
(24.7 g, 296 mmol) and triethylamine (41.2 mL, 296 mmol) at
0 °C were added to the mixture. After being stirred at room
temperature for 3 h, the reaction mixture was washed suc-
cessively with saturated NaHCO3, H2O, and brine. The organic
layer was dried (MgSO4) and concentrated in vacuo. The
residue was purified by flash column chromatography (CHCl3-
EtOAc-MeOH, 60:40:0 to 12:8:1) to give 9k (16.5 g, 71%) as
a colorless solid. Recrystallization from CHCl3-diethyl ether
1
amorphous powder. H NMR (CDCl3): δ 1.09 (3H, t, J ) 7.3
Hz), 2.10-2.20 (5H, m), 3.30 (3H, s), 3.59 (2H, s), 3.84 (3H, s),
3.94 (2H, s), 5.38 (2H, s), 6.82-7.01 (5H, m), 7.15-7.47 (9H,
m), 7.83 (2H, d, J ) 8.6 Hz). The free amine was treated with
1 M ethereal HCl to afford the hydrochloride as a white powder
(from CH2Cl2-diethyl ether): mp 138-143 °C. IR (KBr): 1715,
1665, 1605, 1473 cm-1. FAB-MS m/z: 695 (M + H). Anal.
(C39H36N4O4SF2‚HCl‚1.5H2O) C, H, N.
1
afforded white needles: mp 204-205 °C. H NMR (CDCl3): δ
2.05 (3H, s), 3.57 (2H, s), 3.82 (3H, s), 3.90 (2H, s), 5.37 (2H,
s), 6.92 (2H, t, J ) 8.2 Hz), 7.16-7.31 (9H, m), 7.42-7.57 (5H,
m), 7.63 (1H, s), 7.73 (2H, d, J ) 8.8 Hz). IR (KBr): 3338, 3064,
1717, 1669, 1628, 1591, 1531, 1470 cm-1. Anal. (C36H31N5O4-
SF2) C, H, N. The free amine (TAK-013) was treated with 1 M
In Vitr o Bin d in g Assa ys. Receptor binding assays were
carried out as described previously.8 Briefly, human LHRH
receptor cDNA was cloned from a pituitary cDNA library, and

Thieno[2,3-d]pyrimidine-2,4-dione Derivative
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1 123
CHO cells stably expressing high levels of the recombinant
human LHRH receptor were isolated. [125I][Tyr5]leuprorelin
(0.12-0.15 nM) and the membrane fractions of the CHO cells
(0.2 mg/mL) were incubated at 25 °C for 60 min in 0.2 mL of
assay buffer A [25 mM Tris, 1 mM EDTA, 0.1% bovine serum
albumin (BSA), 0.03% NaN3, 0.25 mM phenylmethanesulfonyl
fluoride, 1 µg/mL pepstatin A, 20 µg/mL leupeptin, and 100
µg/mL phosphoramidon, pH 7.5] containing various concentra-
tions of the test compounds. The reaction was terminated by
adding 2 mL of ice-cold assay buffer A, and the bound and
free ligands were immediately separated by filtration through
a poly(ethylenimine)-coated glass microfiber filter (Whatman,
GF/F). The filter was washed twice with 2 mL of assay buffer
A, and radioactivity was measured using an X-ray counter.
Specific binding was determined by subtracting the nonspecific
binding, which was measured in the presence of 1 µM
unlabeled leuprorelin, from the total binding. The concentra-
tion of each test compound that produced 50% inhibition of
the specific binding (IC50 value) was derived by fitting the data
into a pseudo-Hill equation:
1.2% citric acid, or 0.5% methylcellulose containing 1.2% citric
acid alone (3 mL/kg, n ) 3), were administered orally. Blood
samples (heparin-plasma) were collected from a femoral vein
24 h before administration and 0, 2, 4, 8, 24, and 48 h after
administration. LH concentrations in the plasma were mea-
sured by bioassays using mouse testicular cells.8
Molecu la r Mod elin g Stu d ies. All calculations were per-
formed on a Silicon Graphics O2 R10000 workstation. Molec-
ular modeling was carried out using the Insight II software
package (Accelrys Inc., San Diego, CA). Molecular mechanics
and ab initio calculations were performed to evaluate the
energy difference between the cis and trans conformers
concerning the methoxyurea part of compound 9k . To compare
the intrinsic propensities of the conformation of the urea part,
only the partial structures, N-ethyl-N′-phenylurea and N-
methoxy-N′-phenylurea, were considered. The cis and trans
conformers were constructed using Insight II and subjected
to either energy minimization by Discover using the CVFF
force field or geometry optimization by DMol.
Ack n ow led gm en t . We acknowledge Dr. Haruo
Onda, Dr. Koichi Kato, Dr. Hirosada Sugihara, Dr.
Hideo Shirafuji, Dr. Akio Miyake, Dr. Hidekazu Sawa-
da, Dr. Chieko Kitada, and Dr. Shuji Hinuma for
encouragement and helpful discussions throughout this
work. We also thank Dr. Toshihiro Imaeda for support-
ing the synthetic work, and Dr. Hirokazu Matsumoto,
Ms. Asano Asami, Ms. Tomoko Urushibara, Mr. Shiro
Takekawa, Mr. Hiroyuki Miya, and Mr. Akira Horin-
ouchi for supporting the biochemical and pharmacologi-
cal experiments.
% SPB
100 - % SPB
log
) n[log(C) - log(IC50)]
(
)
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 compound. Similarly,
the binding experiments to the LHRH receptor of other species
were performed as follows. The monkey LHRH receptor cDNA
was cloned from a pituitary cDNA library of cynomolgus
monkeys, and CHO cells stably expressing high levels of the
recombinant monkey LHRH receptor were isolated as de-
scribed previously.8 The binding assays were carried out by
incubating [125I][Tyr5]leuprorelin (0.12-0.13 nM) and mem-
branes prepared from the CHO cells (0.25 mg/mL) at 25 °C
for 60 min, in the presence or absence of compounds. For the
binding experiments to the rat LHRH receptor, [125I][Tyr5]-
leuprorelin (0.15 nM) and membranes (0.2 mg/mL) from the
anterior pituitary of male Wistar rats were incubated with or
without compounds at 4 °C for 90 min.8
In Vitr o F u n ction a l Assa ys. LHRH-stimulated arachi-
donic acid release from CHO cells expressing human or
monkey LHRH receptors was measured according to the
previously reported protocol.20 The receptor-expressing CHO
cells were seeded into 24-well plates at a density of 4 × 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,
NEN Lifescience Products) for 1 day and washed with Dul-
becco’s modified Eagle’s medium (DMEM) supplemented with
20 mM HEPES and 0.2% BSA. The cells were then preincu-
bated with the compounds at 37 °C for 60 min and the reaction
was started by addition of LHRH (1 nM). After incubation at
37 °C for 40 min, radioactivity in the medium was measured
with a liquid scintillation counter.
Or a l Absor p tion in Cyn om olgu s Mon k eys. The hydro-
chloride of 9k (TAK-013 hydrochloride) was used in order to
unify the experimental conditions. Compounds 9a ,g and 9k
(10 mg/kg) suspended in 0.5% methylcellulose were orally
administered to cynomolgus monkeys (female, 4-8 years old,
n ) 3). Blood samples (heparin-plasma) were collected from a
forearm vein 1, 3, and 6 h after administration. To the plasma
(0.3 mL) the same volume of 5% acetonitrile containing 0.05%
TFA was added, and the precipitated plasma proteins were
removed by centrifugation. The compound concentrations in
the supernatant were measured by reverse-phase HPLC using
a TSK ODS-80TM column (4 mm × 250 mm, TOSO) with a
linear gradient of acetonitrile (40-80%) containing 0.05% TFA
(flow rate, 1.0 mL/min; detection, 320 nm; column tempera-
ture, 35 °C).
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