Discovery of a novel, potent, and orally active nonpeptide antagonist of the human luteinizing hormone-releasing hormone (LHRH) receptor [3]

Full text of DOI:10.1021/jm9803673

4190
J . Med. Chem. 1998, 41, 4190-4195
over, these peptide antagonists must be administered
by daily subcutaneous injection, intranasal spray, or a
suitable sustained delivery system because of their
insufficient oral bioavailability.
Discover y of a Novel, P oten t, a n d Or a lly
Active Non p ep tid e An ta gon ist of th e
Hu m a n Lu tein izin g Hor m on e-Relea sin g
Hor m on e (LHRH) Recep tor
We sought, therefore, to discover and develop a potent
and orally active nonpeptide LHRH antagonist with
therapeutic efficacy in humans. In this paper, we
describe the design, synthesis, and biological properties
of isopropyl 3-(N-benzyl-N-methylaminomethyl)-7-(2,6-
difluorobenzyl)-4,7-dihydro-2-(4-isobutyrylaminophenyl)-
4-oxothieno[2,3-b]pyridine-5-carboxylate hydrochloride
(1, T-98475), a highly potent and orally active nonpep-
tide antagonist of the human LHRH receptor (Chart 1).
Nobuo Cho,† Masataka Harada,† Toshihiro Imaeda,†
Takashi Imada,† Hirokazu Matsumoto,† Yoji Hayase,†
Satoshi Sasaki,† Shuichi Furuya,*,†
Nobuhiro Suzuki,† Shoichi Okubo,† Kazuhiro Ogi,†
Satoshi Endo,‡ Haruo Onda,† and Masahiko Fujino†,‡
Discovery Research Division, Takeda Chemical Industries,
Ltd., 10 Wadai Tsukuba, Ibaraki 300-4293, J apan, and
Pharmaceutical Research Division, Takeda Chemical
Industries, Ltd., 17-85 J usohonmachi 2-chome
Yodogawa-ku, Osaka 532-8686, J apan
In our search for a novel nonpeptide LHRH antago-
nist, we first focused on a type II â-turn5,6 involving
residues 5-8 (Tyr-Gly-Leu-Arg) of LHRH that was
considered to be a dominant structure for its binding to
the receptor. Introduction of crucial functional moieties
for receptor binding into a bicyclic heterocycle “scaffold”,
which mimics the relatively fixed main chain of the
â-turn, would provide a nonpeptide ligand of the LHRH
receptor. In fact, a number of heterocyclic compounds
bearing important functional groups for receptor binding
have been disclosed as synthetic nonpeptide antagonists
of G protein-coupled receptors (GPCRs), such as chole-
cystokinin,8 angiotensin II,9 substance P,10 vasopressin,11
and endothelin.12 Our approach toward the discovery
of a lead molecule included “directed screening”13 of our
chemical library. For this purpose, compounds selected
for their similarity to antagonists of other GPCRs were
screened for their inhibitory effects on the specific
binding of [125I]leuprorelin to the human LHRH recep-
tor14 expressed in Chinese hamster ovary (CHO) cells.15
This screening led to the identification of a thieno[2,3-
b]pyridin-4(7H)-one derivative (2) that demonstrated
significant inhibition of [125I]leuprorelin binding (67%
inhibition at 20 µM).16 Comparison of the structure of
compound 2 with the â-turn portion of LHRH suggested
that the p-methoxyphenyl group and the ethyl ester
group of 2 would correspond to the side chains of the
Tyr and Leu residues, respectively. Furthermore, by
considering the fact that substituting hydrophobic D-
amino acids for the Gly residue of LHRH increases the
activity,6 we hypothesized that the o-methoxybenzyl
moiety of 2 may mimic the side chains of such hydro-
phobic D-amino acids. According to this hypothesis as
illustrated in Chart 1, compound 2 is lacking a func-
tional group corresponding to the Arg residue of LHRH,
and we thus attempted to introduce an appropriate
amino function into the lead compound (2). We found
that introduction of a basic amino moiety into the
3-methyl group of 2 increased the activity, and further
modification resulted in the discovery of T-98475 (1).
Received J une 17, 1998
Luteinizing hormone-releasing hormone (LHRH; pGlu-
His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) is secreted
in pulses from the hypothalamus and stimulates the
anterior pituitary gland to release the gonadotropins
luteinizing hormone (LH) and follicle-stimulating hor-
mone (FSH). Ultimately, both of these hormones elicit
gonadal production of sex steroids and gametogenesis.
Since its discovery in 1971,1 LHRH (synonymous with
gonadotropin-releasing hormone), which has a pivotal
role in the modulation of reproductive functions, and
its synthetic analogues have attracted considerable
scientific interest because of their usefulness in the
treatment of endocrine-based diseases such as prostate
cancer, breast cancer, endometriosis, uterine leiomyoma,
and precocious puberty.2 Several LHRH agonists, rep-
resented by leuprorelin (pGlu-His-Trp-Ser-Tyr-D-Leu-
Leu-Arg-Pro-NHEt),3 are currently used in the treat-
ment of the above conditions.4-6 They bind to the
LHRH receptor in the pituitary gonadotrophs inducing
the synthesis and release of gonadotropins. Chronic
administration of potent LHRH agonists, which are
referred to as 'superagonists', depletes gonadotropins
and subsequently down-regulates the receptor in the
pituitary gonadotrophs, resulting in suppression of
steroidal hormones in mammals. Due to this peculiar
mechanism of action, however, LHRH agonists suppress
gonadotropins and sex steroids only following chronic
administration (2-4 weeks) and occasionally evoke the
release of the gonadal hormones2 in the first days of
administration.
On the other hand, LHRH antagonists are expected
to suppress gonadotropins from the onset and be devoid
of this initial hormone surge. Consequently, intense
research over the past 20 years has focused on the
development of potent and safe antagonists. The rela-
tively low potency and the adverse effects, due to
histamine release, have been the main obstacles to their
acceptance and clinical use. Recently, several peptide
antagonists with low histamine-release properties were
reported,7 but they are still in clinical studies. More-
Ch em istr y. The synthetic route of compound 1 (T-
98475) is shown in Scheme 1. Reaction of ethyl 2-amino-
4-methyl-5-phenylthiophene-3-carboxylate (3)17 with di-
ethyl ethoxymethylenemalonate, followed by alkaline
hydrolysis of the 3-ethyl ester moiety and subsequent
ring closure, produced the thieno[2,3-b]pyridine 4 ac-
cording to the procedure in the literature.18 Benzylation
of 4 occurred predominantly at the 7-position, and the
* To whom correspondence should be addressed.
† Discovery Research Division.
‡ Pharmaceutical Research Division.
10.1021/jm9803673 CCC: $15.00 © 1998 American Chemical Society
Published on Web 10/07/1998

Communications to the Editor
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 22 4191
Ch a r t 1
Sch em e 1a
a
Reagents: (a) diethyl ethoxymethylenemalonate, 120 °C (92%); (b) KOH-EtOH, dioxane, 70 °C (98%); (c) PPE, 120 °C (60%); (d)
2,6-difluorobenzyl chloride, K2CO3, KI, DMF, room temperature (90%); (e) NaNO3, concd H2SO4, 0 °C (71%); (f) NBS, AIBN, CCl4, reflux
(79%); (g) N-benzylmethylamine, N,N-diisopropylethylamine, DMF, room temperature (91%); (h) iron powder, concd HCl, EtOH, 0 °C
(96%); (i) isobutyric anhydride, triethylamine, CH2Cl2, room temperature (quant); (j) Ti(OiPr)4, iPrOH, room temperature (82%); (k) 10 M
HCl-EtOH, 0 °C (quant).
resultant thieno[2,3-b]pyridin-4(7H)-one was selectively
nitrated at the para position of the 2-phenyl ring to
afford 5. Radical bromination of the 3-methyl group of
5, followed by installation of the N-benzylmethylamino
moiety, furnished the nitro compound 6. Reduction of
6 with iron powder-concentrated hydrochloric acid
produced the amine 7, which was converted to the amide
8 by acylation with isobutyric anhydride. The prepara-
tion of 119 was accomplished by transesterification of 8
with titanium(IV) isopropoxide-2-propanol20 and sub-

4192 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 22
Communications to the Editor
Ta ble 1. Structures and Binding Affinities of
Thieno[2,3-b]pyridin-4-ones
IC50 (nM)a
compd
R1
MeO
MeO
MeO
NH2
R2
MeNH
PhCH2N(Me) Et 2-OMe
PhCH2N(Me) Et 2,6-F
PhCH2N(Me) Et 2,6-F
R3
R4
humanb ratc
F igu r e 1. Inhibition of [125I]leuprorelin binding to the human
LHRH receptor by T-98475 (1) (O), leuprorelin (0), and LHRH
(4).
9
10
11
7
Et 2-OMe 2000
43%d
34%d
800
70
6
4
0.2
500
60
1 (T-98475) iPrCONH PhCH2N(Me) iPr 2,6-F
Ta ble 2. Species Specificity of T-98475 (1) Binding to the
LHRH Receptors
a
All data are expressed as means of three or more determina-
tions. b Binding to the cloned human receptor; see ref 20. c Binding
IC50 (nM)a
d
to membrane fractions of rat pituitary; see ref 21. Percent
inhibition at 1 µM.
compd
LHRH
leuprorelin
T-98475 (1)
humanb
monkeyc
ratc
sequent treatment with 10 M hydrogen chloride-
ethanol. In addition, compounds 2, 9, 10, and 11 were
prepared using a procedure analogous to that depicted
in Scheme 1.
10
0.3
0.2
6
0.8
4
7
0.5
60
a
All data are expressed as means of three or more determina-
tions. b Binding to the cloned human receptor; see ref 16. c Binding
to membrane fractions of monkey and rat pituitaries; see ref 21.
Resu lts a n d Discu ssion . Compounds 1, 2, 7, 9, 10,
and 11 were evaluated for inhibition of [125I]leuprorelin
binding to the cloned human LHRH receptor and
membrane fractions of the rat anterior pituitary.21 The
results are summarized in Table 1 and are discussed
based on the affinity to the human receptor unless
otherwise mentioned. Introduction of a basic amino
moiety, important for receptor binding, into the 3-posi-
tion of 2 induced a significant increase in affinity. In
fact, the methylaminomethyl derivative (9) had a higher
affinity than the initial lead (2). Optimization of the
amino moiety at the 3-position provided us with the first
breakthrough and led to the N-benzylmethylaminom-
ethyl derivative (10), which was ca. 30-fold more potent
than 9. The structure-activity study of the 7-subsititu-
ent revealed that ortho-substituted benzyl moieties were
favorable. Among them, the 2,6-difluorobenzyl deriva-
tive (11) exhibited excellent affinity with a half-maximal
inhibition concentration (IC50) of 6 nM. Transposition
of the p-methoxy group on the 2-phenyl ring into the
ortho or meta position was detrimental for the binding
affinity. Other para substituents, e.g., amino (7),
maintained affinity almost comparable to methoxy (11).
Acylation of the p-amino group and transesterification,
however, produced another boost in activity. Compound
1 had a 20-fold higher affinity compared to 7, and its
IC50 value was 0.2 nM. From these results, 1 (T-98475)
was selected for further in vitro and in vivo evaluation.
As is shown in Figure 1, 1 (T-98475), leuprorelin, and
LHRH produced a concentration-dependent displace-
ment of the specific binding of [125I]leuprorelin to the
cloned human LHRH receptor. Compound 1 had a 50-
fold higher affinity than LHRH for the human receptor.
In addition, 1 was virtually equipotent to the superago-
nist leuprorelin in this assay (see Table 2). Compound
1 also inhibited LHRH-stimulated arachidonic acid
release from CHO cells expressing the human LHRH
receptor with a pA2 value of 9.4 (data not shown).
Furthermore, 1 had insignificant binding to other
GPCRs of biologically active peptide ligands such as
angiotensin II, cholecystokinin, endothelin, neurotensin,
substance P, thyrotropin-releasing hormone (TRH), and
others. Thus, these results indicate that 1 (T-98475) is
a potent and specific antagonist of the LHRH receptor
in vitro.
To gain insight into the molecular basis for the
interaction of 1 (T-98475) with LHRH receptors, a model
for the human LHRH receptor was constructed (Figure
2, left).22 Figure 2, right, shows the binding mode of 1
to the modeled receptor.23 The bottom part of the
ligand-binding pocket of the receptor is mainly hydro-
phobic and constitutes the binding site of the 2,6-
difluorobenzyl and thieno[2,3-b]pyridin-4-one moieties
of 1. The structure-activity relationship of peptide
LHRH analogues demonstrated the importance of the
positively charged Arg residue of LHRH for high-affinity
binding to the LHRH receptor.6 Mutagenesis studies
of the mouse LHRH receptor revealed a critical role for
Glu301 in recognizing Arg of LHRH.24 In the binding
mode shown in Figure 2, right, the positively charged
amino group of 1 is located in close proximity to Asp302
(equivalent to Glu301 of the mouse receptor) in the
seventh transmembrane domain of the human receptor.
Compound 1 might thus share one of the critical
residues for receptor binding with LHRH.
All the compounds had higher affinities for the human
receptor than for the rat receptor (Table 1). Therefore,
species specificity of the binding of 1 (T-98475) to the
LHRH receptors was investigated and compared to
those of peptide ligands, LHRH and leuprorelin. The
binding affinities of 1 to monkey and rat pituitary
membranes21 were 20- and 300-fold less potent than for
the human receptor, whereas LHRH and leuprorelin
had almost the same affinities in these species as in the
human (Table 2). Accordingly, we chose a cynomolgus
monkey and its tissues for evaluation of in vitro and in
vivo functional activities of 1 (T-98475).

Communications to the Editor
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 22 4193
F igu r e 2. Left: Three-dimensional model of the human LHRH receptor viewed from the extracellular side. Right: Detailed
view of the interaction between the human LHRH receptor (thin bonds) and T-98475 (1) (thick bonds).
F igu r e 4. Suppression of plasma LH concentrations after oral
administration of T-98475 (1) in castrated male cynomolgus
monkeys: T-98475 (1) (60 mg/kg) suspended in 0.5% methyl-
cellulose (b) (n ) 4) and 0.5% methylcellulose alone (0) (n )
5). Values shown are the mean ( SEM.
F igu r e 3. Inhibitory effects of T-98475 (1) on LHRH-
stimulated LH release from monkey pituitary cells. Values
shown are the mean ( SEM (n ) 4).
the large difference in the affinities of 1 between human
and monkey receptors, a far smaller amount of 1 should
be enough to suppress plasma LH levels in humans.
Con clu sion . The results of in vitro and in vivo
studies clearly demonstrate that 1 (T-98475) is the first
potent and orally effective nonpeptide antagonist of the
human LHRH receptor. It is expected that 1 (T-98475)
will overcome the shortcomings of the current peptide
antagonists that require daily injection and will provide
a new class of useful therapeutic agents for the treat-
ment of sex hormone-dependent pathologies.
First, the inhibitory effects of 1 on LHRH-stimulated
LH release were examined using cultured cynomolgus
monkey pituitary cells.25 Compound 1 had no agonistic
effect and markedly attenuated the stimulation of LH
release induced by 1 nM LHRH in a concentration-
dependent manner (Figure 3). The concentration of 1
that inhibited 50% of the stimulation of LH release
(IC50) was approximately 100 nM and 25-fold higher
compared to that of receptor binding.
Next, the in vivo pharmacological activity of 1 was
investigated in the suppression of plasma LH concen-
trations in castrated male cynomolgus monkeys.26 Cas-
tration elicited an elevation of the circulating LH levels
in cynomolgus monkeys. After oral administration of
1 (60 mg/kg), suppression of plasma LH levels occurred
in a time-dependent manner. Compound 1 exhibited
more than 70% inhibition of plasma LH levels 8 h after
administration, and this inhibitory effect lasted for more
than 10 h at this dosage (Figure 4). Taking into account
Ack n ow led gm en t. We thank Dr. J . H. Seely (TAP
Pharmaceuticals); Dr. J . Greer, Dr. F. Haviv, and Dr.
E. N. Bush (Abbott Laboratories); and Dr. K. Kato, Dr.
H. Sugihara, and Dr. C. Kitada (Takeda Chemical
Industries) for helpful advice and discussion. We also
acknowledge Mr. A. Horinouchi, Mr. H. Miya, Ms. A.
Asami, and Ms. T. Urushibara (Takeda Chemical In-
dustries) for supporting the biochemical and pharma-
cological experiments.

4194 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 22
Communications to the Editor
a human pituitary λgt 11 cDNA library (Clontech, Palo Alto, CA)
was screened with the [R-32P]dCTP-labeled rat LHRH receptor
cDNA fragment used as a probe. The hybridization of filters was
performed at 55 °C in hybridization buffer (5×SSC, 10×Den-
hardt’s solution, 0.1% SDS, 150 µg/mL heat-denatured salmon
sperm DNA). The filters were washed in 0.2×SSC, 0.1% SDS at
50 °C, dried, and autoradiographed. A 1.1-kb EcoRI fragment
was selected and subcloned into a pUC118 plasmid. The nucle-
otide sequence of the cloned human LHRH receptor cDNA was
identical to that reported in the literature.14 The human LHRH
receptor cDNA was inserted into the pAKKO-111 expression
vector, which had an SR R-promoter and a dihydrofolate reduc-
tase gene (dhfr) as a selection marker. The vector was introduced
into (dhfr-)CHO cells using calcium phosphate-mediated trans-
fection. The transfected CHO cells were cultured in a selection
medium, and a single colony expressing high levels of the
receptor was isolated.
Refer en ces
(1) (a) Schally, A. V.; Arimura, A.; Kastin, A. J .; Matsuo, H.; Baba,
Y.; Redding, T. W.; Nair, R. M. G.; Debeljuk, L.; White, W. F.
Gonadotropin-Releasing Hormone: One Polypeptide Regulates
Secretion of Luteinizing and Follicle-Stimulating Hormones.
Science 1971, 173, 1036-1038. (b) Matsuo, H.; Baba, Y.; Nair,
R. M. G.; Arimura, A.; Schally, A. V. Structure of the Porcine
LH- and FSH-Releasing Hormone. I. The Proposed Amino Acid
Sequence. Biochem. Biophys. Res. Commun. 1971, 43, 1334-
1339. (c) Amoss, M.; Burgus, R.; Blackwell, R.; Vale, W.; Fellows,
R.; Guillemin, R. Purification, Amino Acid Composition and
N-Terminus of the Hypothalamic Luteinizing Hormone Releas-
ing Factor (LRF) of Ovine Origin. Biochem. Biophys. Res.
Commun. 1971, 44, 205-210. (d) Baba, Y.; Matsuo, H.; Schally,
A. V. Structure of the Porcine LH- and FSH-Releasing Hormone.
II. Confirmation of the Proposed Structure by Conventional
Sequential Analyses. Biochem. Biophys. Res. Commun. 1971, 44,
459-463.
(2) Filicori, M.; Flamigni, C. GnRH Agonists and Antagonists:
Current Clinical Status. Drugs 1988, 35, 63-82.
(3) Fujino, M.; Fukuda, T.; Shinagawa, S.; Kobayashi, S.; Yamazaki,
I.; Nakayama, R.; Seely, J . H.; White, W. F.; Rippel, R. H.
Synthetic Analogues of Luteinizing Hormone Releasing Hormone
(LH-RH) Substituted in Position 6 and 10. Biochem. Biophys.
Res. Commun. 1974, 60, 406-413.
(16) The binding data in this text for all the compounds was obtained
using the cloned human receptor expressed in CHO cells15 for
initial screening. The CHO cells expressing the human LHRH
receptor (1 × 109 cells) were suspended in 5 mM EDTA-PBS
and centrifuged. The pellet suspended in 10 mL of homogenate
buffer (10 mM NaHCO3, 5 mM EDTA, pH 7.5) was homogenized
using a Polytron homogenizer. After the resulting homogenate
was centrifuged for 15 min at 400g, the supernatant was
centrifuged for 1 h at 100000g. The pellet, resuspended in 10
mL of assay buffer A [25 mM Tris, 1 mM EDTA, 0.1% bovine
serum albumin (BSA), 0.03% NaN3, 0.25 mM phenylmethane-
sulfonyl fluoride, 1 µg/mL pepstatin A, 20 µg/mL leupeptin, and
100 µg/mL phosphoramidon, pH 7.5], was centrifuged for 1 h at
(4) Garnick, M. B.; Glode, L. M. (The Leuprolide Study Group)
Leuprolide versus Diethylstilbestrol for Metastatic Prostate
Cancer. N. Engl. J . Med. 1984, 311, 1281-1286.
(5) Dutta, A. S. Luteinizing Hormone-Releasing Hormone (LHRH)
Agonists. Drugs Future 1988, 13, 43-57.
100000g. The membrane fraction obtained as
a pellet was
suspended in 20 mL of assay buffer A and was stored at -80
°C. The protocol of the binding experiments was as follows:
Labeled leuprorelin (0.15 nM) and the membrane fractions (0.2
mg/mL) of CHO cells expressing the human LHRH receptor were
incubated at 25 °C for 60 min in 0.2 mL of assay buffer A in the
presence of various concentrations of test compounds. The
reaction was terminated by the addition of 2 mL of ice-cold assay
buffer A, and bound and free ligands were separated by filtration
through a poly(ethylenimine)-coated glass microfiber filter (What-
man, GF/F). The filter was washed twice with 2 mL of assay
buffer A, and radioactivity was measured using a γ-ray counter.
Specific binding was determined by subtraction of the nonspecific
binding, which was measured in the presence of 1 µM unlabeled
leuprorelin. The concentration of a test compound causing 50%
inhibition of the specific binding (IC50 value) was derived by
fitting the data into a pseudo-Hill equation: log[%SPB/(100 -
%SPB)] ) n[log(C) - log(IC50)], where %SPB is specific binding
as a percentage of maximum specific binding, n is a pseudo-Hill
constant, and C is the concentration of a test compound.
(17) Gewald, K.; Schinke, E.; Bo¨ttcher, H. 2-Amino-thiophene aus
Methylenaktiven Nitrilen, Carbonylverbindungen und Schwefel.
Chem. Ber. 1966, 99, 94-100.
(6) Karten, M. J .; Rivier, J . E. Gonadotropin-Releasing Hormone
Analog Design. Structure-Function Studies toward the Devel-
opment of Agonists and Antagonists: Rationale and Perspective.
Endocr. Rev. 1986, 7, 44-66.
(7) Karten, M. J . An Overview of GnRH Antagonist Development:
Two Decades of Progress. In Modes of Action of GnRH and
GnRH Analogs; Crowley, W. F., J r., Conn, P. M., Eds.; Elsevier:
New York, 1992; pp 277-297.
(8) Freidinger, R. M. Cholecystokinin and Gastrin Antagonists. Med.
Res. Rev. 1989, 9, 271-290.
(9) Duncia, J . V.; Carini, D. J .; Chiu, A. T.; J ohnson, A. L.; Price,
W. A.; Wong, P. C.; Wexler, R. R.; Timmermans, P. B. M. W. M.
The Discovery of DuP 753, a Potent, Orally Active Nonpeptide
Angiotensin II Receptor Antagonist. Med. Res. Rev. 1992, 12,
149-191.
(10) Snider, R. M.; Constantine, J . W.; Lowe, J . A., III; Longo, K. P.;
Lebel, W. S.; Woody, H. A.; Drozda, S. E.; Desai, M. C.; Vinick,
F. J .; Spencer, R. W.; Hess, H.-J . A Potent Nonpeptide Antago-
nist of the Substance P (NK1) Receptor. Science 1991, 251, 435-
437.
(11) Yamamura, Y.; Ogawa, H.; Chihara, T.; Kondo, K.; Onogawa,
T.; Nakamura, S.; Mori, T.; Tominaga, M.; Yabuuchi, Y. OPC-
21268, An Orally Effective, Nonpeptide Vasopressin V1 Receptor
Antagonist. Science 1991, 252, 572-574.
(18) Kuwata, Y.; Meguro, K.; Satoh, Y.; Fugono, T. Synthetic Methods
of Thieno[2,3-b]pyridinecarboxylic Acid Derivatives. J pn. Pat-
ent No. 50-32195 (A), 77394 (A), 1975.
(19) The physicochemical data of T-98475 (1) was as follows: 1H NMR
(free base) (300 MHz, CDCl3) δ 1.28 (6H, d, J ) 6.8 Hz), 1.36
(6H, d, J ) 6.3 Hz), 2.10 (3H, s), 2.53-2.61 (1H, m), 3.65 (2H,
s), 4.16 (2H, s), 5.19-5.27 (1H, m), 5.23 (2H, s), 7.00 (2H, t, J )
8.1 Hz), 7.10-7.26 (5H, m), 7.34-7.42 (1H, m), 7.63 (2H, d, J )
8.3 Hz), 7.78 (2H, d, J ) 8.6 Hz), 8.29 (1H, s). The hydrochloride
salt was obtained by treatment of the free base with 10 M HCl-
EtOH to afford colorless needles (from EtOH-ether): mp 168-
170 °C; IR (KBr) 3400, 2976, 1690, 1603, 1504, 1473 cm-1; FAB-
MS m/e 658 (MH+). Anal. (C37H37N3O4SF2‚HCl‚0.5H2O) C, H,
N.
(12) (a) Clozel, M.; Breu, V.; Burri, K.; Cassal, J .-M.; Fischli, W.; Gray,
G. A.; Hirth, G.; Lo¨ffler, B.-M.; Mu¨ller, M.; Neidhart, W.; Ramuz,
H. Pathophysiological Role of Endothelin Revealed by the First
Orally Active Endothelin Receptor Antagonist. Nature 1993, 365,
759-761. (b) Cho, N.; Nara, Y.; Harada, M.; Sugo, T.; Masuda,
Y.; Abe, A.; Kusumoto, K.; Itoh, Y.; Ohtaki, T.; Watanabe, T.;
Furuya, S. Thieno[2,3-d]pyrimidine-3-acetic Acids, A New Class
of Nonpeptide Endothelin Receptor Antagonists. Chem. Pharm.
Bull., in press.
(13) Elliott, J . D.; Lago, M. A.; Cousins, R. D.; Gao, A.; Leber, J . D.;
Erhard, K. F.; Nambi, P.; Elshourbagy, N. A.; Kumar, C.; Lee,
J . A.; Bean, J . W.; DeBrosse, C. W.; Eggleston, D. S.; Brooks, D.
P.; Feuerstein, G.; Ruffolo, R. R., J r.; Weinstock, J .; Gleason, J .
G.; Peishoff, C. E.; Ohlstein, E. H. 1,3-Diarylindan-2-carboxylic
Acids, Potent and Selective Non-Peptide Endothelin Receptor
Antagonists. J . Med. Chem. 1994, 37, 1553-1557.
(14) Kakar, S. S.; Musgrove, L. C.; Devor, D. C.; Sellers, J . C.; Neill,
J . D. Cloning, Sequencing, and Expression of Human Gonado-
tropin Releasing Hormone (GnRH) Receptor. Biochem. Biophys.
Res. Commun. 1992, 189, 289-295.
(15) The human LHRH receptor cDNA was cloned from a pituitary
cDNA library using a rat LHRH receptor cDNA fragment as a
probe. Briefly, a 658-bp rat LHRH receptor cDNA fragment was
cloned from rat testis poly(A)+RNA with reverse transcription
and polymerase chain reaction methods with oligonucleotide
primers (5′-TGAAGCCTGTCCTTGGAGAAATATGGC-3′ and 5′-
AAAGTTGTAGAAGGCCTGATGCCACCA-3′) synthesized based
on the sequence of the mouse LHRH receptor; see: Tsutsumi,
M.; Zhou, W.; Millar, R. P.; Mellon, P. L.; Roberts, J . L.;
Flanagan, C. A.; Dong, K.; Gillo, B.; Sealfon, S. C. Cloning and
(20) Imwinkelried, R.; Schiess, M.; Seebach, D. Diisopropyl (2S,3S)-
2,3-O-isopropylidenetartrate (1,3-Dioxolane-4,5-dicarboxylic acid,
2,2-dimethyl-, bis(1-methylethyl)ester, (4R-trans)-). Org. Synth.
1987, 65, 230-235.
(21) The binding experiments of test compounds to the LHRH
receptors of other species were performed using membranes
prepared from the anterior pituitaries of male Wistar rats and
cynomolgus monkeys. The protocol of the binding experiments
was essentially the same as that using the cloned human
receptor.16 The membrane fractions of rat and monkey pituitar-
ies were diluted with assay buffer A to 0.2 and 0.5 mg/mL,
respectively. In addition, incubation with these membrane
fractions was carried out at 4 °C for 90 min. For example, the
preparation procedure of rat pituitary membranes was as
follows: The anterior pituitaries obtained from 40 Wistar rats
(male, 8 weeks old) were suspended in homogenate buffer (25
mM Tris, 0.3 M sucrose, 1 mM EGTA, 0.03% NaN3, 0.25 mM
phenylmethanesulfonyl fluoride, 10 U/mL aprotinin, 1 µg/mL
pepstatin A, 20 µg/mL leupeptin, and 100 µg/mL phosphorami-
don, pH 7.5) and homogenized using a Polytron homogenizer.
After the resulting homogenate was centrifuged for 15 min at
Functional Expression of
a Mouse Gonadotropin-Releasing
Hormone Receptor. Mol. Endocrinol. 1992, 6, 1163-1169. Then,

Communications to the Editor
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 22 4195
700g, the supernatant was centrifuged for 1 h at 100000g. The
pellet resuspended in 10 mL of assay buffer A was centrifuged
for 1 h at 100000g. The membrane fraction obtained as a pellet
was suspended in 10 mL of assay buffer A and was stored at
-80 °C. The preparation of monkey pituitary membranes was
performed in a similar manner.
(24) Flanagan, C. A.; Becker, I. I.; Davidson, J . S.; Wakefield, I. K.;
Zhou, W.; Sealfon, S. C.; Millar, R. P. Glutamate 301 of the
Mouse Gonadotropin-Releasing Hormone Receptor Confers Speci-
ficity for Arginine 8 of Mammalian Gonadotropin-Releasing
Hormone. J . Biol. Chem. 1994, 269, 22636-22641.
(25) The anterior pituitaries obtained from three male cynomolgus
monkeys were diced and incubated at 37 °C for 1 h in a buffer
(0.7 mM Na2HPO4, 137 mM NaCl, 5 mM KCl, 25 mM HEPES,
and 50 µg/mL gentamycin sulfate) containing 0.4% collagenase
(Boehringer Mannheim, Mannheim, Germany) and 10 µg/mL
deoxyribonuclease (Sigma, St. Louis, MO); then they were
treated with 0.25% pancreatin (Sigma) at 37 °C for 8 min. The
pituitary cells were washed twice with Dulbecco’s modified
Eagle’s medium containing 20 mM HEPES (DMEM-H), then
seeded in plates (24 wells, 2 × 105/well), and cultured for 3 days
in DMEM-H supplemented with 10% FCS. To evaluate T-98475
(1), the cells were pretreated with DMEM-H containing 0.2%
BSA at 37 °C for 1 h. After the medium was removed, fresh
DMEM-H containing 0.2% BSA and various concentrations of
T-98475 (1) was added and the mixture incubated for 1 h. Then,
LHRH (1 nM) was added and the mixture incubated for 3 more
hours. The medium was harvested, and LH concentration was
measured with bioassays using mouse testicular cells. The
bioassay to determine LH concentration was performed as
follows: Testicular cells were obtained from male BALB/c mice
(8-9 weeks old). An assay buffer, DMEM-H containing 0.2%
BSA, was used in all the steps in this experiment. The cells were
washed, incubated at 37 °C for 1 h, and passed through a nylon
mesh (70 µm). The cells (8 × 105 cells) were washed twice and
then incubated in 0.4 mL of the assay buffer with samples
(conditioned medium of monkey pituitary cells or monkey
plasma) to be tested at 37 °C for 2 h. Equine LH (Sigma) was
used as a standard. Then, the medium was harvested, and
testosterone concentration was measured with radioimmuno-
assay (CIS Diagnostics, Ltd., Sakura, Chiba, J apan).
(22) The LHRH receptor belongs to the G protein-coupled receptor
family and consists of seven transmembrane segments, presum-
ably adopting an R-helical conformation. Modeling procedures
for the human LHRH receptor were similar to those already
described; see: Yamamoto, Y.; Kamiya, K.; Terao, S. Modeling
of Human Thromboxane A2 Receptor and Analysis of the
Receptor-Ligand Interaction. J . Med. Chem. 1993, 36, 820-
825. First, a model for the human â2-adrenergic receptor was
constructed using its amino acid sequence and the known helix
arrangement of bacteriorhodopsin; see: Henderson, R.; Baldwin,
J . M.; Ceska, T. A.; Zemlin, F.; Beckmann, E.; Downing, K. H.
Model for the Structure of Bacteriorhodopsin Based on High-
Resolution Electron Cryo-Microscopy. J . Mol. Biol. 1990, 213,
899-929. Then, a model of the human LHRH receptor was built
upon the scaffold of the â2-adrenergic receptor. Figure 2, left,
shows a three-dimensional model of the human LHRH receptor
viewed from the extracellular side. Transmembrane helices are
depicted as green ribbons, and residues constituting the putative
ligand-binding pocket are shown with CPK models. Colors used
for CPK models are red for acidic residues, cyan for basic
residues, magenta for other hydrophilic residues, and yellow for
hydrophobic residues.
(23) The binding mode of T-98475 (1) to the modeled receptor was
investigated with the automated docking program DOCK 3.5;
see: (a) Shoichet, B. K.; Bodian, D. L.; Kuntz, I. D. Molecular
Docking Using Shape Descriptors. J . Comput. Chem. 1992, 13,
380-397. (b) Meng, E. C.; Shoichet, B. K.; Kuntz, I. D. Auto-
mated Docking with Grid-Based Energy Evaluation. J . Comput.
Chem. 1992, 13, 505-524. (c) Meng, E. C.; Gschwend, D. A.;
Blaney, J . M.; Kuntz, I. D. Orientational Sampling and Rigid-
Body Minimization in Molecular Docking. Proteins 1993, 17,
266-278. Flexibility of the compound was taken into account
by searching the conformational database prepared for T-98475
(1). Figure 2, right (the highest scoring mode), shows a detailed
view of the interaction between the human LHRH receptor (thin
bonds) and T-98475 (1) (thick bonds). T-98475 (1) is drawn with
atoms in light green (carbon), cyan (nitrogen), red (oxygen),
sulfur (yellow), and dark green (fluorine). A solvent-accessible
surface of the receptor molecule is shown as purple dots. Asp302
in the seventh transmembrane domain is drawn as a ball-and-
stick model.
(26) Cynomolgus monkeys (male, 4-8 years old) were castrated more
than 11 weeks prior to the examination. The monkeys were
trained to sit in a primate-restraining chair for administration
of the compounds. T-98475 (1) (60 mg/kg) suspended in 0.5%
methylcellulose or 0.5% methylcellulose alone was orally admin-
istered to the monkeys (volume of the administration: 3 mL/
kg). Blood samples (heparin-plasma) were collected from a fem-
oral vein at 24 h before administration and at 0, 2, 4, 8, 10, 24,
and 48 h after administration. LH concentrations in the plasma
were measured with bioassays using mouse testicular cells.25
J M9803673