Design, synthesis, and structure-activity relationships of thieno[2,3-b]pyridin-4-one derivatives as a novel class of potent, orally active, non-peptide luteinizing hormone-releasing hormone receptor antagonists

Article abstract of DOI:10.1021/jm0512894

Design, synthesis, and structure-activity relationships of thieno[2,3-b]pyridin-4-one-based non-peptide luteinizing hormone-releasing hormone (LHRH) receptor antagonists are described. Starting with the thienopyridin-4-one derivative 26d (T-98475) an optimization study was performed, which resulted in the identification of a highly potent and orally bioavailable LHRH receptor antagonist, 3-(N-benzyl-N-methylaminomethyl)-7-(2,6- difluorobenzyl)-4,7-dihydro-2-[4-(1-hydroxy-1-cyclopropanecarboxamido)phen-yl] -5-isobutyryl-4-oxothieno[2,3-b]pyridine (33c). Compound 33c displayed subnanomolar in vitro activities for the human receptor and its oral administration caused effective suppression of the plasma LH levels in castrated male cynomolgus monkeys. Furthermore, SAR studies revealed that a hydroxyalkylamido moiety on the 2-phenyl ring is virtually equivalent to an alkylureido moiety, at least in this series of compounds.

Full text of DOI:10.1021/jm0512894

J. Med. Chem. 2006, 49, 3809-3825
3809
Design, Synthesis, and Structure-Activity Relationships of Thieno[2,3-b]pyridin-4-one
Derivatives as a Novel Class of Potent, Orally Active, Non-Peptide Luteinizing
Hormone-Releasing Hormone Receptor Antagonists
Takashi Imada,*,† Nobuo Cho,† Toshihiro Imaeda,† Yoji Hayase,‡ Satoshi Sasaki,‡ Shizuo Kasai,† Masataka Harada,§
Hirokazu Matsumoto,§ Satoshi Endo,| Nobuhiro Suzuki,§ and Shuichi Furuya∧
Medicinal Chemistry Research Laboratories and DiscoVery Research Center, Pharmaceutical Research DiVision,
Takeda Pharmaceutical Company, Ltd., 17-85, Jusohonmachi 2-chome, Yodogawa-ku, Osaka 532-8686, Japan, and
Medicinal Chemistry Research Laboratories, Frontier Research Laboratories, and Pharmacology Research Laboratories II,
Pharmaceutical Research DiVision, Takeda Pharmaceutical Company, Ltd., 10 Wadai, Tsukuba, Ibaraki 300-4293, Japan
ReceiVed December 29, 2005
Design, synthesis, and structure-activity relationships of thieno[2,3-b]pyridin-4-one-based non-peptide
luteinizing hormone-releasing hormone (LHRH) receptor antagonists are described. Starting with the
thienopyridin-4-one derivative 26d (T-98475) an optimization study was performed, which resulted in the
identification of a highly potent and orally bioavailable LHRH receptor antagonist, 3-(N-benzyl-N-
methylaminomethyl)-7-(2,6-difluorobenzyl)-4,7-dihydro-2-[4-(1-hydroxy-1-cyclopropanecarboxamido)phen-
yl]-5-isobutyryl-4-oxothieno[2,3-b]pyridine (33c). Compound 33c displayed subnanomolar in vitro activities
for the human receptor and its oral administration caused effective suppression of the plasma LH levels in
castrated male cynomolgus monkeys. Furthermore, SAR studies revealed that a hydroxyalkylamido moiety
on the 2-phenyl ring is virtually equivalent to an alkylureido moiety, at least in this series of compounds.
Introduction
oral bioavailability, and they are usually administered by daily
subcutaneous injection, intranasal spray, or suitable depot form.
In contrast, non-peptide LHRH antagonists offer the potential
for oral administration, and therefore, could provide desirable
therapeutic agents for clinical applications in the above condi-
tions. These observations have fueled the recent surge of
intensive efforts for developing orally active non-peptide LHRH
antagonists.9 Until recently, several research groups have
engaged in this project and a plethora of small molecule LHRH
antagonists have been reported.10-17
Luteinizing hormone-releasing hormone (LHRH), also known
as gonadotropin-releasing hormone (GnRH), is a hypothalamic
decapeptide hormone that controls the reproductive axiss
gonadal function and ovarian cycles.1 It is synthesized and
released from the hypothalamus in a pulsatile manner and
interacts with the specific G-protein coupled receptor (GPCR)2
on the pituitary gonadotrope cells, where it triggers the
biosynthesis and secretion of both luteinizing hormone (LH)
and follicle-stimulating hormone (FSH).3 These gonadotropins,
in turn, stimulate gonadal steroidogenesis and gametogenesis
in testes and ovaries. Continuous stimulation of the LHRH
receptor by potent LHRH agonists ultimately causes down-
regulation and receptor desensitization, resulting in the blockade
of reproductive hormonal signalings“biochemical castration”.4
Through this peculiar mechanism, several peptidic LHRH
agonists, represented by leuprorelin ([D-Leu6,Pro9-NHEt]L-
HRH),5 paved the way for useful therapeutic application in the
treatment of endocrine-based diseases, such as prostate cancer,
breast cancer, endometriosis, uterine leiomyoma, and precocious
puberty.6,7 However, it is well recognized that these LHRH
agonists occasionally evoke an initial gonadal hormone surge
due to overstimulation of the receptor, known as the “flare
effect”,4 leading to symptom exacerbation.
Lead Generation and Drug Design
In our search for a non-peptide LHRH antagonist, we first
focused on the type II â-turn involving residues 5-8 (Tyr-Gly-
Leu-Arg) of LHRH, which was considered to be the dominant
structure for its binding to the receptor.7 The underlying premise
of our strategy is that the key structural element, the â-turn
structure of LHRH or leuprorelin, could be replaced by a
bicyclic “scaffold”, and introduction of important functional
moieties for receptor binding on to the scaffold would provide
a non-peptide LHRH receptor ligand (Chart 1). To explore a
lead molecule, “directed screening” of our chemical library was
conducted. Screening of compounds selected for their similarity
to antagonists of other GPCRs led to the identification of thieno-
[2,3-b]pyridin-4-one 4a as a weak LHRH receptor binder.
Comparing the structure of the lead compound 4a with the
â-turn portion of LHRH and leuprorelin, we hypothesized that
the 4-methoxyphenyl, 2-methoxybenzyl, and ethyl ester groups
would correspond to the side chains of the Tyr5, D-Leu6, and
Leu7 residues of leuprorelin, respectively.10 From this point of
view, compound 4a is lacking in a basic moiety corresponding
to the Arg8 residue. We thus attempted to introduce an
appropriate amino functionality into 4a and found that introduc-
tion of a basic amino moiety into the 3-position significantly
increased the receptor binding affinity. Encouraged by this
success, we started a medicinal chemistry program to develop
thienopyridin-4-one-based non-peptide LHRH antagonists. Fol-
Currently, several peptidic LHRH antagonists have been
developed and there is compelling evidence indicating their
advantage. Clinical studies have demonstrated that they attenuate
steroid hormone levels from onset and thus alleviate disease
symptoms without the concomitant flare effect.8 One of the
major drawbacks of these peptidic antagonists, however, is poor
* To whom correspondence should be addressed. Tel: +81-6-6300-6120.
Fax: +81-6-6300-6306. E-mail: Imada_Takashi@takeda.co.jp.
† Medicinal Chemistry Research Laboratories, Osaka.
‡ Medicinal Chemistry Research Laboratories, Ibaraki.
§ Frontier Research Laboratories, Ibaraki.
| Discovery Research Center, Osaka.
∧ Pharmacology Research Laboratories II, Ibaraki.
10.1021/jm0512894 CCC: $33.50 © 2006 American Chemical Society
Published on Web 06/03/2006

3810 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13
Imada et al.
Chart 1
Scheme 1a
a Reagents: (a) diethyl ethoxymethylenemalonate; (b) KOH-EtOH, dioxane; (c) PPE; (d) 2-methoxybenzyl chloride, NaH, DMF; (e) NBS, AIBN, CCl4;
i
(f) R2H (amine), Pr2NEt, DMF; (g) N-benzylacetamide, NaH, DMF.
lowing this line, we discovered the first potent and orally active
non-peptide LHRH receptor antagonist, 26d (T-98475), and we
reported the initial structure-activity relationship (SAR) of this
class.10 However, the in vivo antagonism of 26d when admin-
istered orally was not as effective as we had expected, which
was considered to be due to its inadequate oral absorption and/
or insufficient in vitro antagonism. In search for a more potent
LHRH antagonist with improved in vivo efficacy, the following
two strategies were employed: (i) further optimization of each
substituent of 26d and (ii) replacement of the thienopyridin-4-
one scaffold with other heterocyclic surrogates. The latter
strategy of investigating novel classes of LHRH antagonists
resulted in the identification of thieno[2,3-d]pyrimidine-2,4-
diones11 and imidazo[1,2-a]pyrimidin-5-ones.12
In this paper, we wish to report the design, synthesis, and
SARs of thieno[2,3-b]pyridin-4-one derivatives and their bio-
logical activity. The subsequent optimization study culminated
in the identification of a more potent and orally bioavailable
non-peptide LHRH receptor antagonist, a thienopyridin-4-one
bearing a 4-(1-hydroxy-1-cyclopropanecarboxamido)phenyl group
at the 2-position (33c). We also describe how molecular
modeling studies suggested a good correlation between com-
pound 9j and the â-turn portion of LHRH and how this helped
the design of the para-substituent of the 2-phenyl ring.
Chemistry
The thieno[2,3-b]pyridin-4-one derivatives were generally
synthesized according to the previously reported procedure10
shown in Scheme 1. Reaction of the multifunctionalized
aminothiophenes 1a-c,18 prepared from the corresponding
phenylacetones using Gewald’s methodology,19 with diethyl

Non-Peptide LHRH Receptor Antagonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13 3811
Scheme 2a
i
a Reagents: (a) PhCOCl, pyridine; (b) NBS, AIBN, CCl4; (c) N-benzylmethylamine, Pr2NEt, DMF; (d) NaOEt, EtOH, CH2Cl2; (e) R-X, NaH, DMF.
Scheme 3a
a Reagents: (a) diethyl ethoxymethylenemalonate; (b) KOH-EtOH, dioxane; (c) Br2, pyridine, CHCl3; (d) PPE; (e) 2-methoxybenzyl chloride, K2CO3,
i
KI, DMF; (f) 3-methoxyphenylboronic acid, Pd(PPh3)4, 2 M Na2CO3, DME; (g) NBS, AIBN, CCl4; (h) N-benzylmethylamine, Pr2NEt, DMF.
ethoxymethylenemalonate, followed by selective alkaline hy-
drolysis of the 3-ethyl ester, gave the thiophene-3-carboxylic
acids 2a-c. Subsequently, intramolecular cyclization of 2a-c
in polyphosphate ester (PPE) afforded the thieno[2,3-b]pyridines
3a-c. Benzylation of 3a-c predominantly occurred at the
7-position to give the N-benzyl analogues 4a-c contaminated
with a small amount of the O-benzyl byproducts, which were
easily removed by recrystallization. Radical bromination of
4a-c with N-bromosuccinimide (NBS) proceeded selectively
at the 3-methyl group, and the intermediary bromomethyl
compounds were derivatized by reaction with a variety of amines
or N-benzylacetamide under basic conditions to provide the
corresponding amines 5a-h and amide 5i.
Using the general procedure shown in Scheme 1, it would
be cumbersome to prepare various 7-substituted thienopyridin-
4-ones. Hence, we developed the convenient synthetic route
depicted in Scheme 2. Acylation of the thienopyridine 3a
selectively took place at the 7-position, in a manner similar to
that described above for benzylation to give the N-benzoyl
analogue 6. The benzoyl group of 6 was sufficiently stable in
the bromination step, and the resulting 3-bromomethyl com-
pound was treated with 1 equiv of N-benzylmethylamine to
generate the tertiary amine 7. Deprotection of the benzoyl group
of 7 with sodium ethoxide cleanly gave the requisite thienopy-
ridine 8, which was derivatized with various alkyl halides to
afford the target compounds 9a-j.
commercially available 2-aminothiophene 10, was converted to
the 5-bromothiophene-3-carboxylic acid 12 by ionic bromina-
tion. Ring closure of 12 with PPE, followed by benzylation,
produced the 2-bromothienopyridin-4-one 14. Compound 14 was
then subjected to the Suzuki coupling reaction with 3-methox-
yphenylboronic acid in the presence of a zerovalent palladium
catalyst to furnish the coupling product 15, which was converted
to the desired compound 16 by bromination and subsequent
incorporation of an N-benzylmethylamino moiety.
The 7-(2-fluorobenzyl) derivatives 21a-g bearing 4-amido
or 4-ureido moieties on the 2-phenyl ring were prepared by the
route shown in Scheme 4. Nitration of the thienopyridine 3c
gave rise to a mixture of the mono- and dinitrated products,
presumably due to its electron-rich properties. The mixture was
successfully separated by silica gel column chromatography to
afford the desired 4-nitrophenyl derivative 17 in moderate yield.
After benzylation of 17, an N-benzylmethylamino moiety was
introduced into the 3-methyl group of 18 by the usual two-step
protocol to yield the nitrophenyl compound 19. Reduction of
19 with iron powder-hydrochloric acid provided the corre-
sponding aniline 20, which in turn was converted to the target
amides 21a-d and ureas 21e,f by reaction with appropriate acyl
chlorides, acid anhydrides, or isocyanates. In addition, methy-
lation of the methylurea 21e occurred with complete regiose-
lectivity toward the aniline nitrogen to furnish compound 21g.
The structure of 21g was confirmed by the different chemical
1
To develop an efficient route for preparing substituted
2-phenyl analogues, the Suzuki aryl coupling reaction was
employed as a key step and the preparation of the 2-(3-
methoxyphenyl) derivative 16 is outlined in Scheme 3. The
thiophene-3-carboxylic acid 11, readily obtained from the
shifts of the two methyl groups of the urea in its H NMR
spectrum (see the Experimental Section).
The synthetic route to the 2-(4-amidophenyl) and 2-(4-
ureidophenyl) derivatives 26a-d bearing a 2,6-difluorobenzyl
group at the 7-position is presented in Scheme 5. Since handling

3812 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13
Imada et al.
Scheme 4a
i
a Reagents: (a) NaNO3, concd H2SO4; (b) 2-fluorobenzyl chloride, K2CO3, KI, DMF; (c) NBS, AIBN, CCl4; (d) N-benzylmethylamine, Pr2NEt, DMF;
(e) Fe, concd HCl, EtOH; (f) R1COCl or (R1CO)2O, Et3N, CH2Cl2; (g) MeNCO or PhNCO, pyridine, THF; (h) MeI, K2CO3, DMF.
Scheme 5a
i
a Reagents: (a) 2,6-difluorobenzyl chloride, K2CO3, KI, DMF; (b) NaNO3, concd H2SO4; (c) NBS, AIBN, CCl4; (d) N-benzylmethylamine, Pr2NEt,
i
i
DMF; (e) Fe, concd HCl, EtOH; (f) EtCOCl or PrCOCl, Et3N, CH2Cl2; (g) MeNCO, pyridine, THF; (h) Ti(OiPr)4, PrOH.
of the 4-nitrophenyl derivative 17 proved to be tedious due to
its low solubility, an alternative route was developed. To
circumvent the intermediate 17, benzylation was conducted prior
to nitration. After difluorobenzylation of 3c, nitration of 22
successfully proceeded at the para-position of the 2-phenyl ring
to generate the 4-nitrophenyl compound 23 in good yield. This
may be attributed to the relative electron-deficiency of the
thienopyridin-4-one nucleus and the 2,6-difluorophenyl ring.
Subsequently, conversion to the aniline 25 from 23 was carried
out in a manner similar to that described above for the
preparation of the 7-(2-fluorobenzyl) derivative 20 shown in
Scheme 4. The aniline 25 was then reacted with acid chlorides
or methyl isocyanate to afford the amides 26a,b and methylurea
26c. Finally, transesterification of 26b with titanium(IV) iso-
propoxide-2-propanol20 produced the isopropyl ester 26d.
Chemical modification of the 5-ester group was performed
by the route depicted in Scheme 6. Facile one-step amidation
of the esters 26a,b with the appropriate amine-aluminum
complex using trimethylaluminum21 worked well to provide the
diamides 27a,b. The Weinreb amide 27b was subsequently
reacted with appropriate Grignard reagents to give the corre-
sponding ketones 28a-c. In addition, the isopropyl ketone 28b
was reduced by sodium borohydride to yield the secondary
alcohol 29.
position of the 2-phenyl ring was conducted by the procedure
shown in Scheme 7. For this purpose, an efficient route that
involved initial ketone formation prior to amide or urea
derivatization was carried out. Protection of the primary amine
of 25 with a trifluoroacetyl group, followed by exposure to N,O-
dimethylhydroxylamine-aluminum complex afforded the Wein-
reb amide 30 in excellent yield. Compound 30 served as a
versatile precursor and was converted to the aniline 31 by
treatment with isopropylmagnesium chloride and subsequent
removal of the trifluoroacetyl group. The preparation of the
target amide and urea derivatives was accomplished as fol-
lows: reaction of 31 with methyl isocyanate to provide the
methylurea 32a, condensation of 31 with Boc-glycine using
(benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluoro-
phosphate (PyBOP reagent) and subsequent deprotection with
trifluoroacetic acid to give the 2-aminoacetamide 32b, conden-
sation of 31 with methoxymethoxyacetic acid using PyBOP
reagent and successive deprotection to furnish the 2-hydroxy-
acetamide 33a, and acylation of 31 with appropriate acetoxyacyl
chlorides and subsequent removal of the acetyl group to afford
the hydroxyalkylamides 33b,c.
Results and Discussion
In Vitro Receptor Binding Studies. The SAR studies of
thienopyridin-4-one derivatives were undertaken on the basis
of data from an in vitro binding assay using cloned Chinese
The synthesis of the ketone derivatives 32a,b and 33a-c
bearing various hydrogen-bonding functional groups at the para-

Non-Peptide LHRH Receptor Antagonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13 3813
Scheme 6a
i
a Reagents: (a) R2NHR3, Me3Al, Pr2NEt (if necessary), CH2Cl2; (b) R4MgX, THF or dioxane; (c) NaBH4, MeOH.
Scheme 7a
a Reagents: (a) (CF3CO)2O, Et3N, CH2Cl2; (b) N,O-dimethylhydroxylamine hydrochloride, iPr2NEt, Me3Al, CH2Cl2; (c) iPrMgCl, dioxane; (d) 2 N NaOH,
i
i
MeOH; (e) MeNCO, pyridine, THF; (f) (1) Boc-Gly-OH, PyBOP, Pr2NEt, CH2Cl2, (2) TFA; (g) (1) MeOCH2OCH2CO2H, PyBOP, Pr2NEt, CH2Cl2, (2)
TFA; (h) (1) AcOC(R2R3)COCl, Et3N, CH2Cl2, (2) 5 N NaOH, EtOH.
Table 1. Physicochemical Data and Binding Affinities of Compounds 4a, 5a, and 5d-i
a
IC50
compd
R
mp (°C)
recryst solvent
formula
(µM)
4a
5d
5e
5f
5g
5a
5h
5i
H
MeNH
PhCH2NH
PhNH
Me2N
PhCH2N(Me)
PhCH2N(Et)
PhCH2N(Ac)
165-167
118-120
118-119
173-174
117-119
147-152
87-91
CH2Cl2-Et2O
Et2O
EtOAc-Et2O
Et2O-hexane
Et2O
EtOAc-Et2O
acetone-Et2O
EtOAc-iPr2O
C26H25NO5S·0.5H2O
10
2
2
C27H28N2O5S·HBr·0.5H2O
C33H32N2O5S·HCl·0.9H2O
C32H30N2O5S·0.2H2O
42%b
0.5
0.07
0.3
7
C28H30N2O5S·HBr·1.75H2O
C34H34N2O5S·HCl·1.8H2O
C35H36N2O5S·(CO2H)2·1.5H2O
C35H34N2O6S·0.5EtOAc
77-82
a The binding affinity is reported as the 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 means of two or three determinations. b Percent inhibition at 20 µM.
hamster ovary (CHO) cells expressing the human LHRH
receptor and [125I]leuprorelin as a ligand.
First, thienopyridin-4-ones possessing various amino func-
tionalities at the 3-position were investigated, and the results
and physicochemical data are shown in Table 1. Introduction
of a basic amino moiety, important for receptor binding, into
the 3-methyl group generally induced a significant increase in
affinity.10 In fact, the methylamino derivative 5d displayed
5-fold higher affinity than the initial lead 4a. The benzylamine
(5e) was equipotent with the methylamine (5d), whereas the
aniline (5f) showed marked decrease in binding affinity. In
contrast, incorporation of secondary amino groups led to a boost

3814 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13
Imada et al.
Table 2. Physicochemical Data and Binding Affinities of Compounds 5a, 8, and 9a-j
a
IC50
compd
R
mp (°C)
recryst solvent
formula
C26H26N2O4S
(µM)
8
H
Me
PhCH2
175-177
217-220
203-208
147-152
109-113
200-204
203-207
221-224
227-230
196-198
214-216
189-192
EtOAc
>20
5
0.2
0.07
0.4
0.3
0.03
0.3
0.4
0.1
0.1
9a
9b
5a
9c
9d
9e
9f
9g
9h
9i
EtOAc-iPr2O
EtOAc-iPr2O
EtOAc-Et2O
Et2O
C27H28N2O4S·HCl·2.5H2O
C33H32N2O4S·HCl·0.2H2O
C34H34N2O5S·HCl·1.8H2O
C34H34N2O5S·HCl·H2O·0.2Et2O
C34H34N2O5S·HCl·0.95H2O
C33H31N2O4SF·HCl·0.6H2O
C33H31N2O4SF·HCl·0.5H2O
C33H31N2O4SF·HCl·0.5H2O
C34H31N3O4S·HCl·0.5H2O
C33H31N3O6S·HCl·0.5H2O
C33H30N2O4SF2·HCl·1.5H2O
(2-OMe)PhCH2
(3-OMe)PhCH2
(4-OMe)PhCH2
(2-F)PhCH2
(3-F)PhCH2
(4-F)PhCH2
(2-CN)PhCH2
(2-NO2)PhCH2
(2,6-F2)PhCH2
EtOAc-iPr2O
EtOAc-iPr2O
EtOAc-iPr2O
EtOAc-iPr2O
EtOAc-iPr2O
EtOAc-iPr2O
EtOAc-iPr2O
9j
0.006
a The binding affinity is reported as the 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 means of two or three determinations.
in activity. The dimethylamino derivative 5g was 4-fold more
potent than 5d,e. Surprisingly, when one methyl group of 5g
was replaced with a benzyl group, the N-benzylmethylamino
derivative 5a showed excellent affinity with a half-maximal
inhibition concentration (IC50) of 70 nM, which was ca. 140-
fold higher than that of 4a. In addition, substitution of an ethyl
(5h) or acetyl (5i) moiety for the methyl group of 5a diminished
the affinity. Accordingly, the N-benzyl-N-methylaminomethyl
group was selected as the 3-substituent for subsequent investiga-
tion.
Second, effects of the 7-substituent on receptor binding
affinity were examined for compounds bearing the N-benzyl-
N-methylaminomethyl group at the 3-position, and the results
and physicochemical data are presented in Table 2. The
nonsubstituted analogue 8 showed greatly reduced activity, but
the methyl derivative 9a was found to exhibit significant affinity,
although it was ca. 70-fold less potent than the 2-methoxybenzyl
derivative 5a. Thus, substituted benzyl derivatives were next
examined. Deletion (9b) or transposition (9c,d) of the 2-methoxy
group of 5a caused a deleterious effect on binding affinity.
Replacement of the methoxy group with a fluorine atom (9e)
led to about a 2-fold increase in activity compared to 5a. As in
the case of the methoxybenzyl compounds, transposition (9f,g)
of the fluorine atom of 9e resulted in more than 10-fold decrease
in activity, which indicates that ortho-substitution is preferable
to meta- or para-substitution of the 7-benzyl moiety. With
respect to the ortho-substituent, fluoro (9e) was more potent
than methoxy (5a), cyano (9h), and nitro (9i). It should be noted
that addition of another fluorine atom into the ortho-position
of 9e produced a further increase in activity, and the affinity of
the 2,6-difluorobenzyl analogue 9j reached a nanomolar level.
The result suggested that 2-fluoro- or 2,6-difluorobenzyl groups
are favorable 7-substituents and the binding site around the
7-substituent is mainly hydrophobic.
is preferable to ortho- or meta-substitution and led us to further
examine the para-substituent. As discussed below in molecular
modeling studies, superimposition of compound 9j on LHRH
suggested that the para-substituent on the 2-phenyl ring could
correspond to the C-terminal region of LHRH. Since the
C-terminal amide of LHRH has been reported to interact with
the hydrogen-bonding amino acid residue (Asn102 of the second
transmembrane helix) of the receptor,22 introduction of hydrogen-
bonding functional groups, such as an amide or urea, were
investigated at this position. In the 7-(2-fluorobenzyl) series,
the amino compound 20 showed almost equal affinity to the
methoxy compound 9e; however, acylation of the amino group
of 20 elicited a notable enhancement in activity, as we had
anticipated. Indeed, the amide derivatives 21a-d exhibited
subnanomolar to nanomolar affinities and the activities increased
in the order of acetamide 21a < benzamide 21d < isobutyry-
lamide 21c < propionylamide 21b. Introduction of a methy-
lureido group led to a further enhancement in activity and the
methylurea 21e displayed extremely high affinity with an IC50
value of 70 pM. Conversely, incorporation of sterically demand-
ing phenylurea (21f) and N-methylation of the aniline nitrogen
(21g) caused more than 30-fold decrease in activity compared
with 21e. Furthermore, additional inclusion of a fluorine atom
into the ortho-position of the 7-benzyl group of 21b,c (26a,b)
increased the activity by more than 3-fold in the amide series.
On the other hand, additional inclusion of a fluorine atom
produced a minimal effect on the affinity in the urea series,
and the difluorobenzyl derivative 26c possessed almost com-
parable affinity to the monofluorobenzyl counterpart 21e. With
respect to the para-substituent on the 2-phenyl ring, it can be
seen that both the hydrogen-donating NH and terminal small
alkyl groups are crucial for high-affinity binding and that a steric
constraint may exist around the recognition site interacting with
this part of the molecule.
After obtaining the successful results above, we began to
probe for effects of the substituent on the 2-phenyl ring, and
the results and physicochemical data are shown in Table 3.
Initially, a methoxy group on the 2-phenyl ring was investigated
for compounds bearing the 2-methoxybenzyl moiety at the
7-position. Omission (5c) as well as transposition (5b, 16) of
the 4-methoxy group resulted in a 3-10-fold decrease in affinity
compared with 5a, which clearly indicated that para-substitution
Table 4 shows the effects of the 5-substituent on binding
affinity, with compounds bearing the 4-propionyl- or 4-isobu-
tyrylamidophenyl groups at the 2-position. Since it is widely
acknowledged that an ester group is metabolically labile in vivo,
a more stable 5-substituent, such as an amide, ketone, or alcohol,
was explored. Although the amide 27a was less potent than the

Non-Peptide LHRH Receptor Antagonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13 3815
Table 3. Physicochemical Data and Binding Affinities of Compounds 5a-c, 9e, 16, 20, 21a-g, and 26a-c
a
IC50
compd
R
X
Y
H
mp (°C)
recryst solvent
formula
(nM)
5c
5b
16
5a
9e
20
21a
21b
21c
21d
21e
21f
21g
26a
26b
26c
H
MeO
117-120
119-122
115-120
147-152
203-207
158-160
118-120
221-223
188-192
141-143
216-220
205-207
>300
EtOAc-Et2O
EtOAc-Et2O
EtOAc-Et2O
EtOAc-Et2O
EtOAc-iPr2O
CHCl3-Et2O
EtOH
EtOH-Et2O
EtOH-Et2O
EtOH-Et2O
EtOH-EtOAc
EtOH-EtOAc
EtOH-EtOAc
EtOAc-hexane
EtOH-Et2O
EtOH-EtOAc
C33H32N2O4S·HCl·1.1H2O
C34H34N2O5S·‚HCl·1.5H2O
C34H34N2O5S‚HCl‚1.5H2O
C34H34N2O5S·‚HCl‚1.8H2O
C33H31N2O4S·F‚HCl‚0.6H2O
C32H30N3O3S·F‚0.5H2O
C34H32N3O4S·F‚H2O
C35H34N3O4S·F‚3.0H2O
C36H36N3O4S·F‚2.0H2O
C39H34N3O4S·F‚2.5H2O
C34H33N4O4SF‚2.0H2O
C39H35N4O4SF‚2.5H2O
C35H35N4O4SF‚H2O
200
700
500
70
30
40
2-MeO
3-MeO
4-MeO
4-MeO
4-NH2
F
H
4-MeCONH
2
4-EtCONH
0.4
0.6
1
0.07
30
4-iPrCONH
4-PhCONH
4-MeNHCONH
4-PhNHCONH
4-MeNHCON(Me)
4-EtCONH
2
F
F
100-102
185-186
214-215
C35H33N3O4SF2‚0.75H2O
C36H35N3O4SF2‚HCl‚1.5H2O
C34H32N4O4SF2
0.1
0.2
0.06
4-iPrCONH
4-MeNHCONH
a The binding affinity is reported as the 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 means of two or three determinations.
Table 4. Physicochemical Data and Binding Affinities of Compounds 26a,b,d, 27a, 28a-c, and 29
a
IC50
compd
R1
R2
mp (°C)
recryst solvent
formula
(nM)
26a
26b
26d
27a
28a
28b
28c
29
Et
CO2Et
CO2Et
100-102
185-186
168-170
144-146
250-256
192-197
198-200
232-234
EtOAc-hexane
EtOH-Et2O
EtOH-Et2O
EtOAc-hexane
EtOH-EtOAc
EtOH-EtOAc
EtOAc
C35H33N3O4SF2·0.75H2O
0.1
0.2
0.2
8
0.4
0.2
0.1
0.4
iPr
iPr
Et
C36H35N3O4SF2·HCl‚1.5H2O
C37H37N3O4SF2·HCl‚0.5H2O
C36H36N4O3SF2·3.5H2O
i
CO2 Pr
CONHiPr
COEt
iPr
iPr
iPr
iPr
C36H35N3O3SF2·HCl‚0.5H2O
C37H37N3O3SF2·HCl‚0.5H2O
C40H35N3O3SF2·HCl‚0.5H2O
C37H39N3O3SF2·HCl‚2.0H2O
COiPr
COPh
CH(OH)iPr
EtOAc-Et2O
a The binding affinity is reported as the 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 means of two or three determinations.
esters 26a,d, the ketones 28a-c and alcohol 29 were well-
tolerated and showed low subnanomolar affinities (IC50 < 0.5
nM).
the isopropyl ketone at the 5-position, and the results and
physicochemical data are presented in Table 5. The methylurea
32a exhibited excellent binding affinity with an IC50 value of
0.1 nM, but it was slightly less potent than the ethyl ester
counterpart 26c (see Table 3). It is widely recognized that ureas
tend to show poor oral absorption, resulting from relatively low
solubility and/or poor membrane permeability due to their strong
hydrogen-bonding ability. We thus set out to explore substituents
that could have a similar function to urea and envisioned that
incorporation of a hydrogen-donating group to mimic the
terminal NH of urea (e.g. a hydroxy or amino group), at the
R-carbon of the amido moiety would provide a urea-mimicking
substituent with potent in vitro activity and improved oral
absorption. Incorporation of an amino group into the amide
(32b) reduced the activity, whereas incorporation of a hydroxy
group into the amide did not essentially affect the affinity. The
It is well-known that highly lipophilic compounds tend to be
targets of metabolic enzymes and bind to proteins nonspecifi-
cally. Considering physicochemical properties, the phenyl ketone
28c (log D value of 7.29) is more lipophilic than the isopropyl
ketone 28b (log D value of 6.33) and the affinity of 28b was
acceptable in comparison with that of 28c. Moreover, introduc-
tion of urea or urea-mimicking moieties into the para-position
of the 2-phenyl ring might fill the gap as for the mono- and
difluorobenzyl derivatives shown in Table 3. Hence, the
isopropyl ketone was chosen as the 5-substituent for further
investigation.
Effects of the para-substituent of the 2-phenyl group on
binding affinity were reinvestigated with compounds bearing

3816 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13
Imada et al.
Table 5. Physicochemical Data and Binding Affinities of Compounds 28b, 32a,b, and 33a-c
a
IC50
compd
R
mp (°C)
recryst solvent
formula
(nM)
28b
32a
32b
33a
33b
33c
iPrCONH
192-197
222-226
197-199
197-199
178-180
184-185
EtOH-EtOAc
CHCl3-EtOAc
EtOH
EtOH
CHCl3-Et2O
EtOH
C37H37N3O3SF2·HCl·0.5H2O
C35H34N4O3SF2·HCl·0.5H2O·0.2CHCl3
C35H34N4O3SF2·2.0HCl·1.5H2O
C35H33N3O4SF2·HCl·0.5H2O
C37H37N3O4SF2·0.5H2O
0.2
0.1
9
0.2
0.2
0.1
MeNHCONH
H2NCH2CONH
HOCH2CONH
HOC(Me)2CONH
(1-OH)cPrCONH
C37H35N3O4SF2
a The binding affinity is reported as the 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 means of two or three determinations.
Table 6. Species Specificities for Binding to LHRH Receptors and
displayed 2-2000-fold selectivity for the human receptor over
Inhibitory Effects on LHRH-Stimulated Arachidonic Acid (AA) Release
of Compounds 26d, 32a, and 33c
the monkey and rat receptors. Considering 26d as a “bench-
mark”, incorporation of the additional hydrogen-bonding func-
tionalities into the amide moiety (32a, 33c) significantly
IC50c (nM)
increased the affinities for the human and monkey receptors;
however, it induced no improvement in affinity for the rat
receptor. Thus, it was considered reasonable that further
evaluations of these compounds should be undertaken using
monkey as well as human receptors.
inhibition of
AA releaseb
binding affinitya
monkeyd
compd
LHRH
leuprorelin
26d
32a
33c
humand
rate
7
human
monkey
10
6f
ndg
ndg
0.6
0.07
0.1
ndg
ndg
ndg
ndg
15
0.3
0.2
0.1
0.1
0.8f
2 (4f)
0.2
0.9
0.5
60
200
150
As for in vitro functional antagonism, compounds 32a and
33c had no agonistic effects and potently antagonized LHRH
function on CHO cells expressing the human and monkey
receptors. Moreover, it is noteworthy that the 1-hydroxycyclo-
propanecarboxamide 33c showed almost equal activity to the
methylurea 32a, while the isobutyrylamide 26d was ca. 9-fold
less potent than 32a for the human receptor. Comparing the
binding affinities and in vitro functional antagonism of these
compounds for the human and monkey receptors, compound
33c was essentially equipotent with 32a. This suggests that the
incorporated hydroxy group of 33c might function equivalently
to the terminal NH of 32a. Similar to binding affinity, species
specificities in functional antagonism were again observed. The
in vitro functional antagonism of the human receptor exceeded
that of the monkey receptor by 150-fold.
In Vivo Studies. Finally, the in vivo antagonism of the
1-hydroxycyclopropanecarboxamide 33c was evaluated for the
suppression of plasma LH concentrations in castrated male
cynomolgus monkeys, and the results are shown in Table 7.
As we had expected, oral administration of a 30 mg/kg dose of
compound 33c exhibited almost complete suppression of the
circulating LH levels in monkeys. In addition, exposure to 33c,
even at a 10 mg/kg dose (po), caused effective and sustained
suppression. The suppressive effect of 33c at both 10 and 30
mg/kg doses (po) was unambiguously more potent than that of
26d at a 60 mg/kg dose (po), and compound 33c displayed
longer duration of action than 26d.10 Furthermore, the in vivo
antagonism of 33c was virtually equipotent to that of the
thienopyrimidine-2,4-dione TAK-013 at the same dosages.11 In
these experiments the maximum plasma concentrations of 33c
were 330 nM (reached 8 h after administration) and 56 nM
(reached 4 h after administration) at 30 and 10 mg/kg doses,
respectively, which indicates that compound 33c showed good
oral absorption in monkeys. As a consequence, compound 33c
potently antagonized the elevated LHRH function at the receptor
level that was elicited by castration in male cynomolgus
a The binding affinity is reported as the IC50 value, which is the antagonist
concentration required to inhibit the specific binding of [125I]leuprorelin to
LHRH receptor by 50%. b 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 means 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 (see ref 10). f Monkey anterior pituitaries were
used as the source for LHRH receptors (see ref 10). g nd ) not determined.
hydroxyalkylamides 33a-c proved to be well-tolerated and
displayed comparable, or marginally increased, activities to the
isobutyrylamide 28b. It should be noted that the 1-hydroxycy-
clopropanecarboxamide 33c displayed equal affinity to the
methylurea 32a and was the most potent among the hydroxy-
alkylamide series with an IC50 value of 0.1 nM. Therefore,
compound 33c was selected for further in vitro and in vivo
evaluation.
Species Specificities and In Vitro Functional Antagonism.
The 1-hydroxycyclopropanecarboxamide 33c as well as the
isobutyrylamide 26d and methylurea 32a were evaluated for
their binding affinities to LHRH receptors of particular species
and for in vitro functional antagonism, i.e., inhibitory potencies
of LHRH-stimulated arachidonic acid release from CHO cells
expressing the human and monkey receptors. The results are
summarized in Table 6. Compounds 26d, 32a, and 33c had more
than 50-fold higher affinities than LHRH for the human receptor.
In addition, these three compounds proved to be more potent
than the superagonist leuprorelin for the human receptor. It can
be seen from Table 6 that there are remarkable species
specificities in binding affinities similar to those observed for
the previously reported 26d.10 Compounds 32a and 33c

Non-Peptide LHRH Receptor Antagonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13 3817
Table 7. Time Course of Plasma LH Concentrations in Castrated Male Cynomolgus Monkeys after Oral Administration of Compound 33c 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
dose ) 10 mg/kg
dose ) 30 mg/kg
0.62 ( 0.14
0.48 ( 0.04
0.56 ( 0.08
0.69 ( 0.10
0.38 ( 0.07
0.45 ( 0.05
0.67 ( 0.14
0.27 ( 0.03
0.31 ( 0.05
0.62 ( 0.06
0.13 ( 0.03
0.18 ( 0.06
0.65 ( 0.12
0.11 ( 0.03
0.07 ( 0.03
0.64 ( 0.09
0.56 ( 0.07
0.62 ( 0.07
a Cynomolgus monkeys (male, 4-9 years old) were castrated more than 6 months prior to the examination. Compound 33c (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. Values shown are the mean ( SEM.
para-substituent of the 2-phenyl ring of 9j could correspond to
the C-terminal amide portion of LHRH. Since Asn102 of the
second transmembrane helix was reported as the binding site
for the C-terminal amide of LHRH, based on the mutagenesis
of the human receptor,22 compounds bearing a hydrogen-
bonding functional group at this position would be expected to
interact strongly with the receptor and provide a more potent
antagonist. In fact, incorporation of an amido or ureido moiety
into the para-position of the 2-phenyl ring successfully increased
the activity, as shown in Table 3. The molecular modeling
studies suggested that compound 9j could share a similar
receptor binding site with LHRH, especially in the type II â-turn
portion and C-terminal region. A recent study has revealed that
the binding affinity of a thieno[2,3-d]pyrimidine-based antago-
nist to the human LHRH receptor was affected by mutation of
Ser118, Asp302, and His306.24 Considering that His306 of the
seventh transmembrane helix is located adjacent to Asn102 of
the second transmembrane helix, these data support our revised
Figure 1. Superimposition of the Putative Active Conformation of
hypothesis as well.
LHRH and Compound 9j: Carbon atoms for LHRH and compound 9j
are colored orange and white, respectively. Nitrogen, oxygen, sulfur,
and fluorine atoms are colored cyan, red, yellow, and light green,
Conclusion
respectively.
We have carried out a medicinal chemistry program to
develop thienopyridin-4-one-based non-peptide LHRH receptor
antagonists starting from the lead compound 4a identified by
directed screening. According to the strategy of introducing
important functional groups for receptor binding into a bicyclic
“scaffold”, we first attempted to introduce a basic amino moiety
into 4a that would correspond to the Arg8 residue of LHRH.
Gratifyingly, introduction of a basic moiety into the 3-methyl
group gained a dramatic increase in binding affinity to provide
the N-benzyl-N-methylaminomethyl derivative 5a. The encour-
aging results led us to start a further optimization study of each
substituent surrounding the thienopyridin-4-one scaffold. It was
revealed that the 2-fluorobenzyl or 2,6-difluorobenzyl moieties
were favorable 7-substituents. On the basis of molecular
modeling studies, the 4-amido- or 4-ureidophenyl derivatives
were designed, and introduction of these hydrogen-bonding
moieties into the 2-position caused a remarkable increase in
activity. With respect to the 5-substituent, the ketone was found
to have comparable affinity to the corresponding ester. Finally,
intensive efforts were made toward searching for a urea-
mimicking substituent at the para-position of the 2-phenyl group,
to obtain potent in vitro activity and improved oral absorption.
Compounds with a hydroxy or amino group at the R-carbon of
the amido moiety, e.g. hydroxyalkylamides, were designed,
prepared, and evaluated. Consequently, we have succeeded in
the identification of a highly potent and orally active non-peptide
LHRH antagonist, the 1-hydroxycyclopropanecarboxamide de-
rivative 33c. Compound 33c exhibited high binding affinity and
potent in vitro antagonism for the human receptor, with IC50
values of 0.1 nM for both. Effective and sustained suppression
of the plasma LH levels was accomplished in castrated male
cynomolgus monkeys, with sufficient duration of action (more
than 24 h) after oral administration of 33c at 10 and 30 mg/kg
monkeys, and the antagonism lasted for more than 24 h at both
30 and 10 mg/kg doses (po). These results clearly demonstrate
that compound 33c is a potent and orally active LHRH
antagonist. The excellent in vivo effectiveness of 33c results
from its potent in vitro activities and/or improved oral absorp-
tion.
Molecular Modeling Studies. To rationalize the underlying
premise of our strategy, superimposition of compound 9j on
LHRH was investigated. The putative active conformation of
LHRH was constructed on the basis of the reported dihedral
angles as described by Momany.23 Initially, we hypothesized
that the 4-methoxyphenyl and ethyl ester groups of the lead
compound 4a would correspond to the side chains of the Tyr5
and Leu7 residues of LHRH, respectively. Moreover, we
speculated that the 2-methoxybenzyl group of 4a might mimic
the side chain of the D-Leu6 residue of leuprorelin.10 The SAR
study revealed that incorporation of an amino moiety into the
3-methyl group of 4a led to a significant increase in activity,
as shown in Table 1. Hence, we revised the superimposition of
a thienopyridin-4-one derivative on LHRH, in accordance with
this result. The overlay of the three-dimensional structure of
compound 9j and LHRH is illustrated in Figure 1. In the new
model, good fits were obtained as a whole and certain specific
points of match become apparent. Namely, the thienopyridine
scaffold is superimposed on the type II â-turn portion and the
difluorobenzyl and N-benzyl-N-methylaminomethyl moieties fit
well with the Tyr5 and Arg8 residues, respectively. In addition,
the ethyl ester group extends toward the Gly6 residue, which
corresponds to the D-Leu6 residue of leuprorelin. It should be
noted that a good overlay between the 4-methoxyphenyl group
and the Gly10-NH2 residue is observed. This indicates that the

3818 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13
Imada et al.
J ) 7.1 Hz), 6.99-7.16 (2H, m), 7.26-7.30 (1H, m), 7.38-7.47
(1H, m), 8.06 (1H, d, J ) 13.8 Hz), 12.40 (1H, d, J ) 13.8 Hz).
doses. The in vivo efficacy of 33c after oral dosing was
unequivocally more potent than that of the previously reported
26d and almost equipotent to that of the thieno[2,3-d]pyrimidine-
2,4-dione TAK-013. The SAR data obtained here was taken
into consideration during the course of optimizing the thienopy-
rimidine-2,4-dione11 and imidazopyrimidin-5-one12 series. Fur-
thermore, these results indicated that the hydroxyalkylamides
are virtually equivalent to alkylureas with regard to their
hydrogen-bonding ability, at least in the context of in vitro
activity.
Ethyl N-[3-Carboxy-4-methyl-5-phenyl-2-thienyl]aminometh-
ylenemalonate (2c). Compound 2c was prepared in 90% yield from
1c (two steps) by a procedure similar to that described for 2a. Mp:
1
187-190 °C (from EtOH-H2O). H NMR (DMSO-d6): δ 1.24
(3H, t, J ) 7.2 Hz), 1.28 (3H, t, J ) 7.2 Hz), 2.34 (3H, s), 4.15
(2H, q, J ) 7.2 Hz), 4.24 (2H, q, J ) 7.2 Hz), 7.39-7.54 (5H, m),
8.09 (1H, d, J ) 13.3 Hz), 12.42 (1H, d, J ) 13.3 Hz).
Ethyl 4-Hydroxy-2-(4-methoxyphenyl)-3-methylthieno[2,3-b]-
pyridine-5-carboxylate (3a). Compound 2a (6.0 g, 13.8 mmol)
was added portionwise to polyphosphate ester (PPE; 60 g) at 120
°C. The mixture was stirred at 120 °C for 0.5 h. After being cooled,
the reaction mixture was poured into ice-water and extracted with
CHCl3. The extract was washed with brine, dried (MgSO4), and
concentrated in vacuo. The residue was purified by flash column
chromatography (EtOAc-hexane, 1:4). The crystalline product was
recrystallized from EtOH to provide 3a (3.18 g, 67%) as orange
crystals. Mp: 162-163 °C. 1H NMR (CDCl3): δ 1.47 (3H, t, J )
7.1 Hz), 2.63 (3H, s), 3.87 (3H, s), 4.49 (2H, q, J ) 7.1 Hz), 6.99
(2H, d, J ) 8.8 Hz), 7.44 (2H, d, J ) 8.8 Hz), 8.84 (1H, s), 12.11
The medicinal chemistry and biological studies disclosed here
demonstrate that the thieno[2,3-b]pyridin-4-one nucleus is an
excellent scaffold and the thienopyridin-4-one derivatives
constitute the first class of potent non-peptide human LHRH
receptor antagonists.
Experimental Section
Chemistry. General Procedures. 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 either a Varian Gemini-200 (200 MHz)
or a JEOL JNM-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 JACSO FTIR spectrometer. FAB mass spectra
were recorded on a JEOL JMS-HX110. Elemental analyses were
within (0.4% of theoretical values and were determined at Takeda
Analytical Research Laboratories, Osaka, Japan. Reagents and
solvents were obtained from commercial sources and used without
further purification. Flash chromatography was performed with
Merck silica gel 60 (art. 9385; 230-400 mesh), and reaction
progress was determined by thin-layer chromatography (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.
(1H, s). IR (KBr): 3434, 2992, 1692, 1601, 1582, 1535, 1504 cm-1
.
FAB-MS m/z: 344 (M + H). Anal. (C18H17NO4S) C, H, N, S.
Ethyl 4-Hydroxy-2-(2-methoxyphenyl)-3-methylthieno[2,3-b]-
pyridine-5-carboxylate (3b). Compound 3b was prepared in 73%
yield from 2b by a procedure similar to that described for 3a, as
an oil. 1H NMR (CDCl3): δ 1.47 (3H, t, J ) 7.1 Hz), 2.48 (3H, s),
3.85 (3H, s), 4.49 (2H, q, J ) 7.1 Hz), 6.99-7.08 (2H, m), 7.32-
7.45 (2H, m), 8.84 (1H, s), 12.10 (1H, s).
Ethyl 4-Hydroxy-3-methy-2-phenylthieno[2,3-b]pyridine-5-
carboxylate (3c). Compound 3c was prepared in 60% yield from
2c by a procedure similar to that described for 3a. Mp: 155-157
1
°C (from EtOH). H NMR (CDCl3): δ 1.47 (3H, t, J ) 7.1 Hz),
2.66 (3H, s), 4.49 (2H, q, J ) 7.1 Hz), 7.39-7.55 (5H, m), 8.86 (1
H, s), 12.14 (1H, s). Anal. (C17H15NO3S) C, H, N, S.
Ethyl 4,7-Dihydro-7-(2-methoxybenzyl)-2-(4-methoxyphenyl)-
3-methyl-4-oxothieno[2,3-b]pyridine-5-carboxylate (4a). Sodium
hydride (60% in oil; 0.123 g, 3.08 mmol) was added to a mixture
of 3a (1.0 g, 2.91 mmol) in DMF (25 mL) at 0 °C, and the mixture
was then stirred at room temperature for 30 min. Subsequently,
2-methoxybenzyl chloride (0.91 g, 5.81 mmol) was added to the
reaction mixture, and stirring was continued at room temperature
for 23 h and at 70 °C for a further 2 h. After evaporation of the
solvent in vacuo, aqueous NH4Cl was added to the residue and the
resulting mixture was extracted with EtOAc. The extract was
washed with brine and dried (MgSO4). The solution was concen-
trated in vacuo, and the residue was purified by flash column
chromatography (EtOAc-hexane, 4:1, EtOAc, then EtOAc-
MeOH, 9:1) to give 4a (0.95 g, 70%) as a pale-yellow amorphous
powder. Crystallization from CH2Cl2-Et2O afforded yellow col-
Ethyl N-[3-Carboxy-5-(4-methoxyphenyl)-4-methyl-2-thienyl]-
aminomethylenemalonate (2a). A mixture of 1a18 (10 g, 34.3
mmol) and ethyl ethoxymethylenemalonate (7.45 g, 34.5 mmol)
was stirred at 120 °C for 2 h. After being cooled, the mixture was
triturated with Et2O. The yellow precipitate was collected by
filtration, washed with ice-cold Et2O, and dried to give the
corresponding triester compound (14.2 g, 90%) as a yellow
1
crystalline powder. Mp: 122-123 °C. H NMR (CDCl3): δ 1.32
(3H, t, J ) 7.1 Hz), 1.38 (3H, t, J ) 7.2 Hz), 1.41 (3H, t, J ) 7.2
Hz), 2.34 (3H, s), 3.85 (3H, s), 4.25 (2H, q, J ) 7.1 Hz), 4.38 (2H,
q, J ) 7.2 Hz), 4.45 (2H, q, J ) 7.2 Hz), 6.95 (2H, d, J ) 8.8 Hz),
7.31 (2H, d, J ) 8.8 Hz), 8.22 (1H, d, J ) 13.4 Hz), 12.74 (1H, d,
J ) 13.1 Hz); IR (KBr): 2984, 1720, 1707, 1688, 1653, 1599,
1518, 1499 cm-1
.
1
umns. Mp: 165-167 °C. H NMR (CDCl3): δ 1.41 (3H, t, J )
7.1 Hz), 2.65 (3H, s), 3.85 (3H, s), 3.86 (3H, s), 4.39 (2H, q, J )
7.1 Hz), 5.16 (2H, s), 6.92-7.00 (4H, m), 7.21-7.41 (4H, m), 8.41
A solution of KOH (5.0 g, 75.7 mmol) in EtOH (30 mL) was
added to a solution of the triester compound (7.0 g, 15.2 mmol) in
dioxane (60 mL) at 70 °C. After being stirred at 70 °C for 10 min,
the mixture was stirred at room temperature for a further 30 min
and acidified with 2 N HCl (40 mL). The resulting suspension was
concentrated in vacuo (removal of dioxane and EtOH). The
precipitate was collected by filtration and dried to afford 2a (6.1
g, 93%) as a yellow crystalline powder. Mp: 184-187 °C. 1H NMR
(DMSO-d6): δ 1.24 (3H, t, J ) 7.1 Hz), 1.28 (3H, t, J ) 7.2 Hz),
2.30 (3H, s), 3.80 (3H, s), 4.15 (2H, q, J ) 7.1 Hz), 4.24 (2H, q,
J ) 7.2 Hz), 7.03 (2H, d, J ) 8.7 Hz), 7.37 (2H, d, J ) 8.7 Hz),
8.08 (1H, d, J ) 13.6 Hz), 12.41 (1H, d, J ) 13.6 Hz). IR (KBr):
(1H, s). IR (KBr): 2980, 1727, 1684, 1621, 1609, 1589, 1497 cm-1
.
Anal. (C26H25NO5S‚0.5H2O) C, H, N.
Ethyl 4,7-Dihydro-7-(2-methoxybenzyl)-2-(2-methoxyphenyl)-
3-methyl-4-oxothieno[2,3-b]pyridine-5-carboxylate (4b). Com-
pound 4b was prepared in 57% yield from 3b by a procedure similar
to that described for 4a. Mp: 194-196 °C (from EtOAc). 1H NMR
(CDCl3): δ 1.40 (3H, t, J ) 7.1 Hz), 2.51 (3H, s), 3.81 (3H, s),
3.86 (3H, s), 4.39 (2H, q, J ) 7.1 Hz), 5.15 (2H, s), 6.91-7.04
(4H, m), 7.23-7.42 (4H, m), 8.41 (1H, s). Anal. (C26H25NO5S‚
0.1H2O) C, H, N.
3422, 2980, 1719, 1653, 1607, 1551, 1512 cm-1
.
Ethyl 4,7-Dihydro-7-(2-methoxybenzyl)-3-methyl-4-oxo-2-
phenylthieno[2,3-b]pyridine-5-carboxylate (4c). Compound 4c
was prepared in 91% yield from 3c by a procedure similar to that
Ethyl N-[3-Carboxy-5-(2-methoxyphenyl)-4-methyl-2-thienyl]-
aminomethylenemalonate (2b). Compound 2b was prepared in
88% yield from 1b (two steps) by a procedure similar to that
described for 2a. Mp: 167-169 °C (from Et2O-hexane). 1H NMR
(DMSO-d6): δ 1.23 (3H, t, J ) 7.1 Hz), 1.28 (3H, t, J ) 7.1 Hz),
2.13 (3H, s), 3.80 (3H, s), 4.14 (2H, q, J ) 7.1 Hz), 4.24 (2H, q,
1
described for 4a, as an amorphous powder. H NMR (CDCl3): δ
1.41 (3H, t, J ) 7.1 Hz), 2.69 (3H, s), 3.87 (3H, s), 4.40 (2H, q, J
) 7.1 Hz), 5.17 (2H, s), 6.92-7.00 (2H, m), 7.22-7.44 (7H, m),
8.43 (1H, s). Anal. (C25H23NO4S‚0.4H2O) C, H, N.

Non-Peptide LHRH Receptor Antagonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13 3819
Ethyl 3-(N-Benzyl-N-methylaminomethyl)-4,7-dihydro-7-(2-
methoxybenzyl)-2-(4-methoxyphenyl)-4-oxothieno[2,3-b]pyridine-
5-carboxylate Hydrochloride (5a). A mixture of 4a (0.35 g, 0.76
mmol), N-bromosuccinimide (NBS; 0.135 g, 0.76 mmol), and 2,2′-
azobis(isobutyronitrile) (AIBN; 13 mg, 0.079 mmol) in CCl4 (5
mL) was heated under reflux for 2 h. After being cooled, an
insoluble material was removed by filtration and the residue was
washed with a small portion of CHCl3. The filtrate was diluted
with CHCl3 and washed with brine. The aqueous phase was re-
extracted with CHCl3. The extract was washed with brine and dried
(MgSO4). After removal of the solvent in vacuo, the residue was
purified by flash column chromatography (CHCl3-MeOH, 99:1)
to provide the corresponding bromomethyl compound (0.37 g, 90%)
as a pale-yellow solid. Recrystallization from EtOAc gave colorless
needles. Mp: 200-201 °C. 1H NMR (CDCl3): δ 1.40 (3H, t, J )
7.1 Hz), 3.86 (6H, s), 4.40 (2H, q, J ) 7.1 Hz), 5.05 (2H, s), 5.16
(2H, s), 6.92-7.04 (4H, m), 7.23-7.28 (1H, m), 7.34-7.43 (1H,
m), 7.57 (2H, d, J ) 8.9 Hz), 8.46 (1H, s). IR (KBr): 2980, 1725,
powder. Crystallization from EtOAc afforded 6 (6.41 g, 98%) as
white crystals. Mp: 110-112 °C. 1H NMR (CDCl3): δ 1.14 (3H,
t, J ) 7.7 Hz), 2.42 (3H, s), 3.85 (3H, s), 4.26 (2H, q, J ) 7.2 Hz),
6.98 (2H, d, J ) 6.7 Hz), 7.40 (2H, d, J ) 8.9 Hz), 7.57 (2H, t, J
) 7.6 Hz), 7.70 (1H, t, J ) 5.9 Hz), 8.27 (2H, d, J ) 7.0 Hz), 9.14
(1H, s). IR (KBr): 2972, 1717, 1607, 1580, 1522, 1502, 1454 cm-1
Anal. (C25H21NO5S) C, H, N.
.
Ethyl 3-(N-Benzyl-N-methylaminomethyl)-4-hydroxy-2-(4-
methoxyphenyl)thieno[2,3-b]pyridine-5-carboxylate (8). A mix-
ture of 6 (6.39 g, 14.3 mmol), NBS (2.67 g, 15.0 mmol), and AIBN
(0.47 g, 2.86 mmol) in CCl4 (100 mL) was heated under reflux for
1 h. After being cooled, the mixture was poured into water and
extracted with CHCl3. The extract was washed with brine and dried
(MgSO4). The solution was concentrated in vacuo to provide the
corresponding bromomethyl compound as a crystalline solid, which
was dissolved in DMF (100 mL). Then, N,N-diisopropylethylamine
(2.52 mL, 14.7 mmol) and N-benzylmethylamine (1.9 mL, 14.7
mmol) were added successively to the DMF solution. After being
stirred at room temperature for 40 min, the mixture was concentrated
in vacuo. Water was added to the residue and the resulting mixture
was extracted with CH2Cl2. The extract was washed with brine and
dried (MgSO4). Evaporation of the solvent in vacuo afforded the
crude 7 as a brown oil, which was dissolved in CH2Cl2 (100 mL)
and EtOH (50 mL). A solution of sodium ethoxide (0.69 g, 10.1
mmol) in EtOH (50 mL) was added to the solution at 0 °C. After
being stirred at room temperature for 4 h, the reaction mixture was
neutralized with AcOH. After removal of the solvent in vacuo, water
was added to the residue and the resulting mixture was extracted
with CHCl3. The extract was washed with brine, dried (MgSO4),
and concentrated in vacuo. The residue was purified by flash column
chromatography (CH2Cl2-MeOH, 95:5) to provide a white amor-
phous powder. Crystallization from EtOAc gave 8 (8.72 g, 61%
1607, 1588, 1497 cm-1
.
N-Benzylmethylamine (1.05 mL, 8.14 mmol) was added to a
mixture of the bromomethyl compound (4.0 g, 7.37 mmol) and
N,N-diisopropylethylamine (1.54 mL, 8.84 mmol) in DMF (70 mL).
After being stirred at room temperature for 1 h, the mixture was
concentrated in vacuo and saturated NaHCO3 was added to the
residue. The resulting mixture was extracted with EtOAc, and the
extract was washed with brine and dried (MgSO4). The solution
was concentrated in vacuo, and the residue was purified by flash
column chromatography (CH2Cl2-MeOH, 99:1 to 4:1) to afford
the free amine of 5a (3.30 g, 77%) as a white amorphous powder.
1H NMR (free amine in CDCl3): δ 1.39 (3H, t, J ) 7.1 Hz), 2.19
(3H, brs), 3.73 (2H, brs), 3.86 (3H, s), 3.87 (3H, s), 4.20 (2H, s),
4.39 (2H, q, J ) 7.1 Hz), 5.18 (2H, s), 6.93-7.01 (4H, m), 7.10-
7.32 (5H, m), 7.37 (1H, t, J ) 3.9 Hz), 7.74 (2H, d, J ) 4.4 Hz),
8.41 (1H, s). The free amine was treated with 1 M ethereal HCl to
afford 5a as a white crystalline powder (from EtOAc-Et2O). Mp:
147-152 °C. IR (KBr): 1719, 1605, 1502, 1253 cm-1. FAB-MS
m/z: 583 (M + H). Anal. (C34H34N2O5S‚HCl‚1.8H2O) C, H, N.
Compounds 5b-h were prepared by a procedure similar to that
described for 5a, and the physicochemical data are shown in Table
1 (5d-h) and Table 3 (5b,c).
1
from 6) as white crystals. Mp: 175-177 °C. H NMR (CDCl3):
δ 1.45 (3H, t, J ) 7.2 Hz), 2.35 (3H, s), 3.75 (2H, s), 3.89 (3H, s),
3.92 (2H, s), 4.44 (2H, q, J ) 7.2 Hz), 7.01 (2H, d, J ) 6.7 Hz),
7.21-7.37 (7H, m), 8.87 (1H, s). IR (KBr): 3424, 3000, 1686,
1607, 1504 cm-1. Anal. (C26H26N2O4S) C, H, N.
Ethyl 3-(N-Benzyl-N-methylaminomethyl)-7-(2,6-difluoroben-
zyl)-4,7-dihydro-2-(4-methoxyphenyl)-4-oxothieno[2,3-b]pyridine-
5-carboxylate Hydrochloride (9j). Compound 9j was prepared
in 66% yield (free amine) from 8 by a procedure similar to that
described for 4a. Mp: 189-192 °C (from EtOAc-iPr2O, HCl salt).
1H NMR (free amine in CDCl3): δ 1.42 (3H, t, J ) 6.9 Hz), 2.91
(3H, s), 3.88 (3H, s), 4.37-4.70 (6H, m), 5.45 (2H, s), 6.95-7.07
(4H, m), 7.28-7.32 (3H, m), 7.42-7.52 (8H, m), 8.64 (1H, m).
Ethyl 3-(N-Acetyl-N-benzylaminomethyl)-4,7-dihydro-7-(2-
methoxybenzyl)-2-(4-methoxyphenyl)-4-oxothieno[2,3-b]pyridine-
5-carboxylate (5i). Sodium hydride (60% in oil; 27 mg, 0.675
mmol) was added to a solution of N-benzylacetamide (99 mg, 0.66
mmol) in DMF (3 mL) at 0 °C under a nitrogen atmosphere. After
being stirred at 0 °C for 30 min, a solution of the intermediary
bromomethyl compound (0.30 g, 0.55 mmol) obtained in the
preparation of 5a in DMF (8 mL) was added to the mixture. Then,
the resulting mixture was stirred at room temperature for 4 h. After
removal of the solvent in vacuo, aqueous NH4Cl was added to the
residue and the resulting mixture was extracted with EtOAc. The
extract was washed with brine, dried (MgSO4), and concentrated
in vacuo. The residue was purified by flash column chromatography
(CHCl3-MeOH, 99:1 to 97:3) to provide 5i (56 mg, 17%) as a
pale-yellow oil. Crystallization from EtOAc-iPr2O afforded pale-
IR (KBr): 3574, 1725, 1605, 1504, 1473, 1259, 1180, 1035 cm-1
.
FAB-MS m/z: 589 (M + H). Anal. (C33H30N2O4SF2‚HCl‚1.5H2O)
C, H, N.
Compounds 9a-i were prepared by a procedure similar to that
described for 4a, and the physicochemical data are shown in Table
2.
Ethyl N-[3-Carboxy-4-methyl-2-thienyl]aminomethylenemal-
onate (11). Compound 11 was prepared in 86% yield from 10 (two
steps) by a procedure similar to that described for 2a. Mp: 187-
1
189 °C (from EtOH-H2O). H NMR (DMSO-d6): δ 1.24 (3H, t,
1
yellow crystals. Mp: 77-82 °C. H NMR (CHCl3): δ 1.38, 1.41
J ) 7.1 Hz), 1.27 (3H, t, J ) 7.1 Hz), 2.30 (3H, s), 4.14 (2H, q,
J ) 7.1 Hz), 4.23 (2H, q, J ) 7.1 Hz), 6.68 (1H, s), 8.08 (1H, d,
J ) 13.4 Hz), 12.54 (1H, d, J ) 13.4 Hz).
(3H, each t, J ) 7.1 Hz), 1.64, 2.16 (3H, each s), 3.84 (3H, s),
3.86, 3.88 (3H, each s), 4.27 (2H, s), 4.37, 4.40 (2H, each q, J )
7.1 Hz), 5.05, 5.37 (2H, each s), 5.11, 5.13 (2H, each s), 6.81-
7.02 (8H, m), 7.13-7.21 (3H, m), 7.30-7.45 (2H, m), 8.38, 8.40
(1H, each s). Anal. (C35H34N2O6S‚0.5EtOAc) C, H, N.
Ethyl N-(5-Bromo-3-carboxy-4-methyl-2-thienyl)aminometh-
ylenemalonate (12). A solution of bromine (5.8 mL, 0.113 mol)
in CHCl3 (50 mL) was added dropwise to a mixture of 11 (30.3 g,
92.6 mmol) and pyridine (38 mL, 0.47 mol) in CHCl3 (200 mL).
After being stirred at room temperature for 40 min, the mixture
was concentrated in vacuo and the residue was treated with dilute
HCl. The precipitate was collected by filtration, washed with water
and a small portion of Et2O, and dried to afford 12 (34.1 g, 91%)
as a brown crystalline powder, which was used in the following
Ethyl 7-Benzoyl-4,7-dihydro-2-(4-methoxyphenyl)-3-methyl-
4-oxothieno[2,3-b]pyridine-5-carboxylate (6). Benzoyl chloride
(1.78 mL, 15.3 mmol) was added dropwise to a mixture of 3a (5.0
g, 14.6 mmol) in pyridine (100 mL) at 0 °C. After being stirred at
room temperature for 2.5 h, the reaction was quenched by adding
EtOH (1 mL). After evaporation of the solvent in vacuo, water
was added to the residue and the resulting mixture was extracted
with CH2Cl2. The extract was washed with brine, dried (MgSO4),
and concentrated in vacuo. The residue was purified by flash column
chromatography (CH2Cl2-MeOH, 95:5) to give a white amorphous
1
reaction without further purification. Mp: 182-185 °C (dec). H
NMR (DMSO-d6): δ 1.25 (3H, t, J ) 7.1 Hz), 1.26 (3H, t, J )
7.1 Hz), 2.27 (3H, s), 4.15 (2H, q, J ) 7.1 Hz), 4.23 (2H, q, J )
7.1 Hz), 7.93 (1H, d, J ) 13.4 Hz), 12.38 (1H, d, J ) 13.2 Hz). IR

3820 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13
Imada et al.
(KBr): 3120, 2984, 1688, 1634, 1574, 1547, 1499 cm-1. FAB-
MS m/z: 406 (M + H; 79Br), 408 (M + H; 81Br).
Ethyl 4,7-Dihydro-7-(2-fluorobenzyl)-3-methyl-2-(4-nitrophen-
yl)-4-oxothieno[2,3-b]pyridine-5-carboxylate (18). Compound 18
was prepared in 60% yield from 17 by a procedure similar to that
described for 14. Mp: 184-186 °C (from EtOH). 1H NMR
(CDCl3): δ 1.41 (3H, t, J ) 7.1 Hz), 2.74 (3H, s), 4.41 (2H, q, J
) 7.1 Hz), 5.26 (2H, s), 7.12-7.28 (3H, m), 7.35-7.48 (1H, m),
7.58 (2H, d, J ) 8.6 Hz), 8.28 (2H, d, J ) 8.6 Hz), 8.39 (1H, s).
Anal. (C24H19N2O5SF) C, H, N.
Ethyl 2-Bromo-4-hydroxy-3-methythieno[2,3-b]pyridine-5-
carboxylate (13). Compound 13 was prepared in 61% yield from
12 by a procedure similar to that described for 3a. Mp: 214-216
1
°C (from EtOH). H NMR (CDCl3-CD3OD): δ 1.47 (3H, t, J )
7.1 Hz), 2.60 (3H, s), 4.50 (2H, q, J ) 7.1 Hz), 8.82 (1H, s). Anal.
(C11H10NO3SBr) C, H, N, S.
Ethyl 3-(N-Benzyl-N-methylaminomethyl)-4,7-dihydro-7-(2-
fluorobenzyl)-2-(4-nitrophenyl)-4-oxothieno[2,3-b]pyridine-5-
carboxylate Hydrochloride (19). Compound 19 was prepared in
41% yield from 18 (two steps) by a procedure similar to that
described for 5a. Mp: 140-144 °C (from CH2Cl2-Et2O, HCl salt).
1H NMR (free amine in CDCl3): δ 1.40 (3H, t, J ) 7.1 Hz), 2.20
(3H, s), 3.67 (2H, s), 4.22 (2H, s), 4.41 (2H, q, J ) 7.1 Hz), 5.27
(2H, s), 7.10-7.28 (8H, m), 8.07 (2H, d, J ) 8.9 Hz), 8.07 (2H, d,
J ) 8.9 Hz), 8.36 (1H, m). Anal. (C32H28N3O5SF‚HCl‚0.85H2O‚
0.1CH2Cl2) C, H, N.
Ethyl 2-Bromo-4,7-dihydro-7-(2-methoxybenzyl)-3-methyl-4-
oxothieno[2,3-b]pyridine-5-carboxylate (14) 2-Methoxybenzyl
chloride (1.60 g, 10.2 mmol) was added to a mixture of 13 (2.15
g, 6.80 mmol), K2CO3 (1.41 g, 10.2 mmol), and KI (0.56 g, 3.37
mmol) in DMF (25 mL). The resulting mixture was stirred at room
temperature for 30 min and at 50 °C for a further 2 h. After being
cooled, the mixture was concentrated in vacuo. Water was added
to the residue and the resulting mixture was extracted with EtOAc-
THF (4:1). 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, 99:1 to 98:2)
to give 14 (2.70 g, 91%) as a pale-yellow solid. Recrystallization
from EtOAc-Et2O afforded white crystals. Mp: 161-163 °C. 1H
NMR (CDCl3): δ 1.40 (3H, t, J ) 7.1 Hz), 2.60 (3H, s), 3.85 (3H,
s), 4.38 (2H, q, J ) 7.1 Hz), 5.09 (2H, s), 6.92-7.02 (2H, m),
7.19-7.24 (1H, m), 7.35-7.44 (1H, m), 8.39 (1H, s). IR (KBr):
2926, 1727, 1690, 1609, 1495, 1439 cm-1. FAB-MS m/z: 436
(M + H; 79Br), 438 (M + H; 81Br). Anal. (C19H18NO4SBr) C, H,
N.
Ethyl 4,7-Dihydro-7-(2-methoxybenzyl)-2-(3-methoxyphenyl)-
3-methyl-4-oxothieno[2,3-b]pyridine-5-carboxylate (15). Tetrakis-
(triphenylphosphine)palladium(0) (0.163 g, 0.141 mmol) was added
to a mixture of 14 (0.615 g, 1.41 mmol), 3-methoxyphenylboronic
acid (0.535 g, 2.11 mmol), and 2 M Na2CO3 (3.53 mL, 7.06 mmol)
in 1,2-dimethoxyethane (30 mL) under an argon atmosphere. The
resulting mixture was refluxed with stirring under an argon
atmosphere for 2 h. After being cooled, the mixture was diluted
with EtOAc and an insoluble material was removed by filtration
through Celite. The filtrate was diluted with water 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 7:3 to 9:1,
EtOAc, and then EtOAc-MeOH 99:5 to 9:1) to afford 15 (0.446
g, 68%) as a white amorphous powder. 1H NMR (CDCl3): δ 1.41
(3H, t, J ) 7.1 Hz), 2.69 (3H, s), 3.84 (3H, s), 3.87 (3H, s), 4.39
(2H, q, J ) 7.1 Hz), 5.16 (2H, s), 6.87-7.02 (5H, m), 7.22-7.42
(3H, m), 8.42 (1H, s). IR (KBr): 3440, 2938, 1727, 1688, 1607,
1493, 1465 cm-1. Anal. (C26H25NO5S‚0.5H2O) C, H, N.
Ethyl 3-(N-Benzyl-N-methylaminomethyl)-4,7-dihydro-7-(2-
methoxybenzyl)-2-(3-methoxyphenyl)-4-oxothieno[2,3-b]pyridine-
5-carboxylate Hydrochloride (16). Compound 16 was prepared
in 65% yield from 15 (two steps) by a procedure similar to that
described for 5a. Mp: 115-120 °C (from EtOAc-Et2O, HCl salt).
1H NMR (free amine in CDCl3): δ 1.39 (3H, t, J ) 7.1 Hz), 2.16
(3H, brs), 3.69 (2H, brs), 3.80 (3H, s), 3.87 (3H, s), 4.23 (2H, s),
4.39 (2H, q, J ) 7.1 Hz), 5.18 (2H, s), 6.90-7.00 (3H, m), 7.12-
7.42 (9H, m), 7.68 (1H, brs), 8.40 (1H, s). Anal. (C34H34N2O5S‚
HCl‚1.5H2O) C, H, N.
Ethyl 4-Hydroxy-3-methyl-2-(4-nitrophenyl)thieno[2,3-b]pyr-
idine-5-carboxylate (17). A solution of sodium nitrate (1.87 g, 22.0
mmol) in concentrated H2SO4 (2 mL) was added dropwise to a
solution of 3c (6.27 g, 20.0 mmol) in concentrated H2SO4 (20 mL)
at 0 °C. After being stirred at 0 °C for 30 min, the mixture was
poured into ice-water and extracted with CHCl3. The extract was
washed with brine, dried (MgSO4), and concentrated in vacuo. The
residue was purified by flash column chromatography (EtOAc-
hexane, 1:4) to give 17 (4.20 g, 55%) as a yellow solid.
Recrystallization from EtOAc-hexane provided yellow crystals.
Mp: 260-261 °C. 1H NMR (CDCl3): δ 1.49 (3H, t, J ) 7.2 Hz),
2.70 (3H, s), 4.51 (2H, q, J ) 7.2 Hz), 7.71 (2H, d, J ) 8.9 Hz),
8.34 (2H, d, J ) 8.9 Hz), 8.89 (1H, s). IR (KBr): 3002, 1692,
1605, 1514, 1350, 1290 cm-1. FAB-MS m/z: 359 (M + H). Anal.
(C17H14N2O5S) C, H, N, S.
Ethyl 2-(4-Aminophenyl)-3-(N-benzyl-N-methylaminomethyl)-
4,7-dihydro-7-(2-fluorobenzyl)-4-oxothieno[2,3-b]pyridine-5-car-
boxylate (20). Concentrated HCl (10 mL) was added dropwise to
a mixture of 19 (3.29 g, 5.0 mmol) and iron powder (1.12 g, 20.0
mmol) in EtOH (10 mL) at 0 °C. After being stirred at 0 °C for 1
h, the mixture was poured into ice-water and treated with 5 N
NaOH. The resulting suspension was diluted with CHCl3 and an
insoluble material was removed by filtration through Celite. The
aqueous phase was separated and extracted with CHCl3. The CHCl3
layer was washed with brine and dried (MgSO4). After removal of
the solvent in vacuo, the residue was purified by flash column
chromatography (CHCl3-MeOH, 9:1) to give 20 (2.18 g, 79%) as
a yellow solid. Recrystallization from CHCl3-Et2O afforded a pale-
1
yellow crystalline powder. Mp: 158-160 °C. H NMR (CDCl3):
δ 1.35 (3H, t, J ) 7.2 Hz), 2.04 (3H, s), 3.65 (2H, s), 4.17 (2H, s),
4.35 (2H, q, J ) 7.2 Hz), 5.47 (2H, s), 6.74 (2H, d, J ) 8.4 Hz),
7.07-7.28 (7H, m), 7.30-7.50 (4H, m), 8.64 (1H, s). IR (KBr):
3330, 1723, 1607, 1495, 1446, 1377, 1311 cm-1. FAB-MS m/z:
556 (M + H). Anal. (C32H30N3O3SF‚0.5H2O) C, H, N.
Ethyl 3-(N-Benzyl-N-methylaminomethyl)-4,7-dihydro-7-(2-
fluorobenzyl)-4-oxo-2-(4-propionylamidophenyl)thieno[2,3-b]-
pyridine-5-carboxylate (21b). A solution of propionic anhydride
(0.26 mL, 2.0 mmol) in CH2Cl2 (1 mL) was added dropwise to a
solution of 20 (0.11 g, 0.20 mmol) and triethylamine (0.56 mL,
4.0 mmol) in CH2Cl2 (2 mL) at 0 °C. After being stirred at room
temperature for 6 h, the mixture was poured into ice-water 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-MeOH, 9:1)
to afford 21b (0.12 g, quant.) as a pale-yellow solid. Recrystalli-
zation from EtOH-Et2O gave white needles. Mp: 221-223 °C.
1H NMR (CDCl3): δ 1.22 (3H, t, J ) 7.5 Hz), 1.41 (3H, t, J ) 7.0
Hz), 2.44 (2H, q, 7.5 Hz), 2.56 (3H, s), 4.16 (2H, s), 4.40 (2H, q,
J ) 7.0 Hz), 4.58 (2H, s), 5.61 (2H, s), 7.20-7.40 (7H, m), 7.40-
7.55 (4H, m), 7.75 (2H, d, J ) 8.7 Hz), 8.89 (1H, s). IR (KBr):
2970, 1692, 1605, 1502, 1446, 1385, 1321, 1270, 1183 cm-1. FAB-
MS m/z: 612 (M + H). Anal. (C35H34N3O4SF‚3.0H2O) C, H, N.
Compounds 21a,c,d were prepared by a procedure similar to that
described for 21b, and the physicochemical data are shown in Table
3.
Ethyl 3-(N-Benzyl-N-methylaminomethyl)-4,7-dihydro-7-(2-
fluorobenzyl)-2-[4-(3-methylureido)phenyl]-4-oxothieno[2,3-b]-
pyridine-5-carboxylate (21e). Methyl isocyanate (0.32 mL, 5.4
mmol) was added dropwise to a solution of 20 (0.57 g, 1.0 mmol)
and pyridine (1 mL) in THF (10 mL) at 0 °C. After stirring at
room temperature for 10 h, the mixture was concentrated in vacuo.
Water was added to the residue and the resulting mixture was
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-MeOH, 95:
5) to give 21e (0.46 g, 73%) as a pale-yellow amorphous powder.
Crystallzation from EtOH-EtOAc provided a pale-yellow powder.
Mp: 216-220 °C. 1H NMR (CDCl3): δ 1.44 (3H, t, J ) 7.3 Hz),

Non-Peptide LHRH Receptor Antagonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13 3821
2.25 (3H, s), 2.84 (3H, s), 4.35 (2H, s), 4.43 (2H, q, J ) 7.2 Hz),
4.90 (2H, s), 5.61 (2H, s), 7.25-7.65 (13H, m), 8.85 (1H, s). Anal.
(C34H33N4O4SF‚2.0H2O) C, H, N.
Compounds 21f were prepared by a procedure similar to that
described for 21e, and the physicochemical data are shown in Table
3.
Ethyl 3-(N-Benzyl-N-methylaminomethyl)-7-(2,6-difluoroben-
zyl)-4,7-dihydro-2-(4-isobutyrylamidophenyl)-4-oxothieno[2,3-
b]pyridine-5-carboxylate Hydrochloride (26b). The free amine
of compound 26b was prepared quantitatively from 25 by a
procedure similar to that described for 21b, and compound 26b
was obtained from the free amine in the usual manner. Mp: 185-
1
186 °C (from EtOH-Et2O, HCl salt). H NMR (free amine in
Ethyl 3-(N-Benzyl-N-methylaminomethyl)-4,7-dihydro-2-[4-
(1,3-dimethylureido)phenyl]-7-(2-fluorobenzyl)-4-oxothieno[2,3-
b]pyridine-5-carboxylate (21g). Iodomethane (14.2 mg, 0.10
mmol) was added dropwise to a mixture of 21e (30.6 mg, 0.05
mmol) and K2CO3 (6.90 mg, 0.05 mmol) in DMF (1 mL) at 0 °C.
After being stirred at room temperature for 14 h, the mixture was
poured into ice-water and extracted with CHCl3. The extract was
washed with brine, dried (MgSO4), and concentrated in vacuo. The
residue was purified by flash column chromatography (CHCl3-
MeOH, 9:1) to give 21g (10 mg, 32%) as a pale-yellow solid.
Recrystallization from EtOH-EtOAc afforded a pale-yellow crys-
CDCl3): δ 1.28 (6H, d, J ) 6.8 Hz), 1.39 (3H, t, J ) 7.1 Hz), 2.12
(3H, s), 2.44 (2H, q, J ) 7.2 Hz), 2.50-2.60 (1H, m), 3.65 (2H,
s), 4.16 (2H, s), 4.40 (2H, q, J ) 7.1 Hz), 5.26 (2H, s), 7.01 (2H,
t, J ) 8.1 Hz), 7.10-7.30 (5H, m), 7.35-7.47 (5H, m), 7.62 (2H,
d, J ) 8.5 Hz), 7.81 (2H, d, J ) 8.5 Hz), 8.35 (1H, s). Anal.
(C36H35N3O4SF2‚HCl‚1.5H2O) C, H, N.
Ethyl 3-(N-Benzyl-N-methylaminomethyl)-7-(2,6-difluoroben-
zyl)-4,7-dihydro-2-[4-(3-methylureido)phenyl]-4-oxothieno[2,3-
b]pyridine-5-carboxylate (26c). Compound 26c was prepared in
79% yield from 25 by a procedure similar to that described for
21e. Mp: 214-215 °C (from EtOH-EtOAc, free amine). 1H NMR
(CDCl3): δ 1.37 (3H, t, J ) 7.2 Hz), 2.00 (3H, s), 2.85 (3H, d, J
) 4.5 Hz), 3.52 (2H, s), 4.07 (2H, s), 4.37 (2H, q, J ) 7.2 Hz),
5.25 (2H, s), 5.80 (1H, br), 6.99 (2H, t, J ) 8.4 Hz), 7.02-7.12
(5H, m), 7.36-7.47 (1H, m), 7.45 (2H, d, J ) 8.7 Hz), 7.64 (2H,
d, J ) 8.7 Hz), 7.72 (1H, br), 8.38 (1H, s). IR (KBr): 3362, 1607,
1499, 1473, 1317, 1238, 1183 cm-1. FAB-MS m/z: 631 (M + H).
Anal. (C34H32N4O4SF2) C, H, N.
1
talline powder. Mp: >300 °C. H NMR (CD3OD): δ 1.29 (3H,
s), 1.41 (3H, t, J ) 7.2 Hz), 2.70 (3H, s), 2.80 (3H, s), 3.62 (2H,
s), 4.43 (2H, q, J ) 7.2 Hz), 4.52 (2H, s), 5.70 (2H, s), 7.17-7.36
(4H, m), 7.39-7.55 (9H, m), 8.96 (1H, s). IR (KBr): 3394, 1690,
1603, 1543, 1499, 1386, 1319 cm-1. FAB-MS m/z: 627 (M + H).
Anal. (C35H35N4O4SF‚H2O) C, H, N.
Ethyl 7-(2,6-Difluorobenzyl)-4,7-dihydro-3-methyl-4-oxo-2-
phenylthieno[2,3-b]pyridine-5-carboxylate (22). Compound 22
was prepared in 90% yield from 3c by a procedure similar to that
described for 14. Mp: 171-173 °C (from EtOAc-Et2O). 1H NMR
(CDCl3): δ 1.41 (3H, t, J ) 6.9 Hz), 2.68 (3H, s), 4.40 (2H, q, J
) 6.9 Hz), 5.26 (2H, s), 7.00 (2H, t, J ) 10.8 Hz), 7.34-7.48
Isopropyl 3-(N-Benzyl-N-methylaminomethyl)-7-(2,6-difluo-
robenzyl)-4,7-dihydro-2-(4-isobutyrylamidophenyl)-4-oxothieno-
[2,3-b]pyridine-5-carboxylate Hydrochloride (26d). Titanium(IV)
isopropoxide (0.30 mL, 1.0 mmol) was added dropwise to a solution
of 26b (1.29 g, 2.0 mmol) in 2-propanol (50 mL) at 0 °C. After
being stirred at room temperature for 12 h, the reaction mixture
was poured into a mixture of CHCl3 and water with vigorous
stirring. An insoluble material was removed by filtration through
Celite. The filtrate was diluted with brine and extracted with CHCl3.
The extract was washed with brine and dried (Na2SO4). After
removal of the solvent in vacuo, the residue was purified by flash
column chromatography (CHCl3-MeOH, 5:1). The crude product
was recrystallized from EtOAc to provide the free amine of 26d
(1.08 g, 82%) as a white crystalline powder. 1H NMR (free amine
in 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 free amine was treated with 10
M ethanolic HCl to give 26d as colorless needles (from EtOH-
Et2O). Mp: 168-170 °C. IR (KBr): 3400, 2976, 1690, 1603, 1504,
1473 cm-1. FAB-MS m/z: 658 (M + H). Anal. (C37H37N3O4SF2‚
HCl‚0.5H2O) C, H, N.
3-(N-Benzyl-N-methylaminomethyl)-7-(2,6-difluorobenzyl)-
4,7-dihydro-N-isopropyl-4-oxo-2-(4-propionylamidophenyl)-
thieno[2,3-b]pyridine-5-carboxamide (27a). Compound 27a was
prepared in 56% yield from 26a by a procedure similar to that
described for 27b (see below). Mp: 144-146 °C (from EtOAc-
hexane, free amine). 1H NMR (CDCl3): δ 1.25-1.31 (9H, m), 2.04
(3H, s), 2.43 (2H, q, J ) 7.2 Hz), 3.63 (2H, s), 4.13 (2H, s), 4.24-
4.30 (1H, m), 5.28 (2H, s), 6.95-7.72 (12H, m), 8.67 (1H, s), 10.20
(2H, d, J ) 7.2 Hz). Anal. (C36H36N4O3SF2‚3.5H2O) C, H, N.
3-(N-Benzyl-N-methylaminomethyl)-7-(2,6-difluorobenzyl)-
4,7-dihydro-2-(4-isobutyrylamidophenyl)-N-methoxy-N-methyl-
4-oxothieno[2,3-b]pyridine-5-carboxamide (27b). Trimethylalu-
minum (15% solution in hexane; 9.64 mL, 20.0 mmol) was added
dropwise to a mixture of N,O-dimethylhydroxylamine hydrochloride
(1.95 g, 20.0 mmol) and N,N-diisopropylethylamine (3.48 mL, 20.0
mmol) in CH2Cl2 (50 mL) at 0 °C. After the mixture was stirred at
0 °C for 1 h, a solution of 26b (5.14 g, 7.98 mmol) in CH2Cl2 (20
mL) was added dropwise to the mixture. Then, the reaction mixture
was stirred at 0 °C for a further 1 h. The mixture was poured into
ice-water and extracted with CHCl3. The extract was washed with
brine, dried (Na2SO4), and concentrated in vacuo. The residue was
crystallized from Et2O-petroleum ether to afford 27b (4.54 g, 86%)
as a yellow crystalline powder. Mp: 152-154 °C. 1H NMR
(6H, m), 8.39 (1H, s). IR (KBr): 1680, 1624, 1497, 1466 cm-1
.
FAB-MS m/z: 440 (M + H). Anal. (C24H19NO3SF2) C, H, N.
Ethyl 7-(2,6-Difluorobenzyl)-4,7-dihydro-3-methyl-2-(4-nitro-
phenyl)-4-oxothieno[2,3-b]pyridine-5-carboxylate (23). Com-
pound 23 was prepared in 71% yield from 22 by a procedure similar
to that described for 17. Mp: 215-217 °C (from EtOAc-CHCl3-
Et2O). 1H NMR (CDCl3): δ 1.42 (3H, t, J ) 7.2 Hz), 2.73 (3H, s),
4.41 (2H, q, J ) 7.2 Hz), 5.27 (2H, s), 7.02 (2H, t, J ) 8.1 Hz),
7.38-7.49 (1H, m), 7.60 (2H, d, J ) 8.7 Hz), 8.30 (2H, d, J ) 8.7
Hz), 8.41 (1H, s). IR (KBr): 1688, 1628, 1518, 1493, 1470, 1348
cm-1. FAB-MS m/z: 485 (M + H). Anal. (C24H18N2O5SF2‚0.5H2O)
C, H, N.
Ethyl 3-(N-Benzyl-N-methylaminomethyl)-7-(2,6-difluoroben-
zyl)-4,7-dihydro-2-(4-nitrophenyl)-4-oxothieno[2,3-b]pyridine-
5-carboxylate (24). Compound 24 was prepared in 72% yield from
23 (two steps) by a procedure similar to that described for 5a. Mp:
145-147 °C (from CH2Cl2-Et2O, free amine). 1H NMR (CDCl3):
δ 1.40 (3H, t, J ) 7.2 Hz), 2.19 (3H, s), 3.67 (2H, s), 4.21 (2H, s),
4.41 (2H, q, J ) 7.2 Hz), 5.28 (2H, s), 7.03 (2H, t, J ) 8.1 Hz),
7.12-7.23 (5H, m), 7.39-7.50 (1H, m), 8.10 (2H, d, J ) 9.0 Hz),
8.26 (2H, d, J ) 9.0 Hz), 8.39 (1H, s). Anal. (C32H27N3O5SF2‚
0.1H2O) C, H, N.
Ethyl 2-(4-Aminophenyl)-3-(N-benzyl-N-methylaminomethyl)-
7-(2,6-difluorobenzyl)-4,7-dihydro-4-oxothieno[2,3-b]pyridine-
5-carboxylate (25). Compound 25 was prepared in 96% yield from
24 by a procedure similar to that described for 20. Mp: 195-196
1
°C (from EtOAc). H NMR (CDCl3): δ 1.25 (3H, t, J ) 7.2 Hz),
2.11 (3H, s), 3.65 (2H, s), 4.14 (2H, s), 4.40 (2H, q, J ) 7.2 Hz),
5.25 (2H, s), 6.73 (2H, d, J ) 8.7 Hz), 7.00 (2H, t, J ) 7.8 Hz),
7.11-7.27 (5H, m), 7.35-7.46 (1H, m), 7.64 (2H, d, J ) 8.7 Hz),
8.33 (1H, s). IR (KBr): 3352, 1682, 1607, 1495, 1473 cm-1. FAB-
MS m/z: 574 (M + H). Anal. (C32H29N3O3SF2‚0.25H2O) C, H, N.
Ethyl 3-(N-Benzyl-N-methylaminomethyl)-7-(2,6-difluoroben-
zyl)-4,7-dihydro-4-oxo-2-(4-propionylamidophenyl)thieno[2,3-
b]pyridine-5-carboxylate (26a). Compound 26a was prepared
quantitatively from 25 by a procedure similar to that described for
1
21b. Mp: 100-102 °C (from EtOAc-hexane, free amine). H
NMR (CDCl3): δ 0.88 (3H, t, J ) 6.6 Hz), 1.37 (3H, t, J ) 7.2
Hz), 2.11 (3H, s), 2.42 (2H, q, J ) 6.6 Hz), 3.64 (2H, s), 4.37 (2H,
q, J ) 7.2 Hz), 4.14 (2H, s), 5.26 (2H, s), 6.97-7.75 (12H, m),
8.00 (1H, s), 8.36 (1H, s). Anal. (C35H33N3O4SF2‚0.75H2O) C, H,
N.

3822 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13
Imada et al.
(CDCl3): δ 1.28 (6H, d, J ) 7.0 Hz), 2.13 (3H, s), 2.53-2.62
(1H, m), 3.34 (3H, s), 3.62 (2H, s), 3.73 (3H, s), 4.16 (2H, s), 5.21
(2H, s), 6.99 (2H, t, J ) 8.1 Hz), 7.15-7.30 (5H, m), 7.34-7.44
(1H, m), 7.62 (2H, d, J ) 8.6 Hz), 7.72 (1H, s), 7.83 (2H, d, J )
8.5 Hz). IR (KBr): 1620, 1497, 1473 cm-1. FAB-MS m/z: 659
(M + H). Anal. (C36H36N4O4SF2) C, H, N.
as a white crystalline powder. Mp: 198 °C (from Et2O). 1H NMR
(CDCl3): δ 2.15 (3H, s), 3.35 (3H, s), 3.62 (2H, s), 3.73 (3H, s),
4.15 (2H, s), 5.20 (2H, s), 7.00 (2H, t, J ) 7.8 Hz), 7.10-7.30
(5H, m), 7.38-7.48 (1H, m), 7.64 (2H, d, J ) 8.4 Hz), 7.71 (1H,
s), 7.90 (2H, t, J ) 8.4 Hz). IR (KBr): 3029, 2938, 1723, 1622,
1539, 1497, 1471 cm-1. FAB-MS m/z: 685 (M + H). Anal.
(C34H29N4O4SF5) C, H, N.
3-(N-Benzyl-N-methylaminomethyl)-7-(2,6-difluorobenzyl)-
4,7-dihydro-2-(4-isobutyrylamidophenyl)-4-oxo-5-propionyl-
thieno[2,3-b]pyridine Hydrochloride (28a). Ethylmagnesium chlo-
ride (2.0 M solution in THF; 0.92 mL, 1.84 mmol) was added
dropwise to a suspension of 27b (0.30 g, 0.46 mmol) in dry THF
(10 mL) at 0 °C under an argon atmosphere. The reaction mixture
was stirred at 0 °C under an argon atmosphere for 30 min. Then,
the mixture was poured into ice-water 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-MeOH, 10:1) to afford the
free amine of 28a (0.20 g, 69%) as a pale-yellow amorphous
2-(4-Aminophenyl)-3-(N-benzyl-N-methylaminomethyl)-7-
(2,6-difluorobenzyl)-4,7-dihydro-5-isobutyryl-4-oxothieno[2,3-
b]pyridine (31). The precursor of compound 31 [3-(N-benzyl-N-
methylaminomethyl)-7-(2,6-difluorobenzyl)-4,7-dihydro-5-isobutyryl-
2-(4-trifluoroacetamidophenyl)-4-oxothieno[2,3-b]pyridine] was
prepared in 75% yield from 30 by a procedure similar to that
described for 28a as a white crystalline powder. Mp: 160 °C (from
1
Et2O). H NMR (CDCl3): δ 1.18 (6H, d, J ) 6.6 Hz), 2.12 (3H,
s), 3.64 (2H, s), 4.11-4.19 (1H, m), 4.18 (2H, s), 5.27 (2H, s),
7.00 (2H, t, J ) 8.4 Hz), 7.10-7.22 (5H, m), 7.36-7.46 (1H, m),
7.66 (2H, d, J ) 6.6 Hz), 7.90 (2H, d, J ) 6.9 Hz), 8.29 (1H, s).
FAB-MS m/z: 668 (M + H).
1
powder. H NMR (free amine in CDCl3): δ 1.17 (3H, t, J ) 7.1
Hz), 1.29 (6H, d, J ) 6.8 Hz), 2.10 (3H, s), 2.51-2.58 (1H, m),
3.29 (2H, q, J ) 7.1 Hz), 3.64 (2H, s), 4.16 (2H, s), 5.27 (2H, s),
7.00 (2H, t, J ) 8.2 Hz), 7.16-7.26 (5H, m), 7.36-7.46 (1H, m),
7.62 (2H, d, J ) 8.5 Hz), 7.78 (2H, d, J ) 8.5 Hz), 8.34 (1H, s).
The free amine was treated with 1 M ethereal HCl to give 28a as
a pale-yellow crystalline powder (from EtOH-EtOAc). Mp: 250-
256 °C. IR (KBr): 3422, 3230, 2976, 1694, 1671, 1628, 1595, 1526,
1502, 1475 cm-1. Anal. (C36H35N3O3SF2‚HCl‚0.5H2O) C, H, N.
Compounds 28b,c were prepared by a procedure similar to that
described for 28a, and the physicochemical data are shown in Table
4.
NaOH (2 N, 2 mL, 4.0 mmol) was added dropwise to a solution
of the above trifluoroacetamide compound (0.67 g, 1.0 mmol) in
MeOH (10 mL). After being stirred at 60 °C for 1 h, the reaction
mixture was poured into water and extracted with EtOAc. The
extract was washed with brine, dried (MgSO4), and concentrated
in vacuo. The residue was purified by flash column chromatography
(EtOAc-hexane, 1:1) and the purified compound was then crystal-
lized from Et2O to afford 31 (0.45 g, 79%) as a pale-yellow
1
crystalline powder. Mp: 149 °C. H NMR (CDCl3): δ 1.18 (6H,
d, J ) 6.9 Hz), 2.10 (3H, s), 3.64 (2H, s), 3.83 (2H, s), 4.13-4.23
(1H, m), 4.16 (2H, brs), 5.25 (2H, s), 6.73 (2H, d, J ) 9.0 Hz),
6.99 (2H, t, J ) 8.1 Hz), 7.10-7.25 (5H, m), 7.25-7.42 (1H, m),
7.59 (2H, d, J ) 8.7 Hz), 8.27 (1H, s). IR (KBr): 3445, 3358,
3223, 2971, 2932, 1667, 1595, 1575, 1495, 1472 cm-1. FAB-MS
m/z: 572 (M + H). Anal. (C33H31N3O2SF2) C, H, N.
3-(N-Benzyl-N-methylaminomethyl)-7-(2,6-difluorobenzyl)-
4,7-dihydro-5-(1-hydroxy-2-methylpropyl)-2-(4-isobutyrylami-
dophenyl)-4-oxothieno[2,3-b]pyridine Hydrochloride (29). So-
dium borohydride (0.20 g, 5.4 mmol) was added in one portion to
a solution of 28b (1.0 g, 1.5 mmol) in MeOH (30 mL) at 0 °C.
After being stirred at 0 °C for 1 h, the mixture was diluted with
saturated NaHCO3 and extracted with CH2Cl2. The extract was
washed with brine and dried (Na2SO4). The solution was concen-
trated in vacuo, and the residue was purified by flash column
chromatography (EtOAc-MeOH, 10:1 to 3:1) to give the free
amine of 29 (0.66 g, 68%) as a brown crystalline powder. The free
amine was treated with 1 M ethereal HCl to provide 29 as yellow
3-(N-Benzyl-N-methylaminomethyl)-7-(2,6-difluorobenzyl)-
4,7-dihydro-5-isobutyryl-2-[4-(3-methylureido)phenyl]-4-
oxothieno[2,3-b]pyridine Hydrochloride (32a). Compound 32a
was prepared quantitatively from 31 by a procedure similar to that
described for 21e. Mp: 222-226 °C (from CHCl3-EtOAc, HCl
salt). 1H NMR (free amine in CDCl3): δ 1.19 (6H, d, J ) 6.6 Hz),
2.44 (3H, br), 2.76 (3H, d, J ) 4.8 Hz), 3.95-4.04 (3H, m), 4.34
(2H, s), 5.34 (2H, s), 6.29 (1H, br), 7.00 (2H, t, J ) 8.0 Hz), 7.25
(7H, br), 7.37-7.48 (1H, m), 7.62 (2H, d, J ) 8.4 Hz), 8.35 (1H,
s), 9.03 (1H, br). Anal. (C35H34N4O3SF2‚HCl‚0.5H2O‚0.2CHCl3)
C, H, N.
2-[4-(2-Aminoacetamido)phenyl]-3-(N-benzyl-N-methylami-
nomethyl)-7-(2,6-difluorobenzyl)-4,7-dihydro-5-isobutyryl-4-
oxothieno[2,3-b]pyridine Dihydrochloride (32b). PyBOP reagent
(0.39 g, 0.75 mmol) was added portionwise to a solution of 31
(0.29 g, 0.50 mmol), N-Boc-glycine (0.13 g, 0.75 mmol), and N,N-
diisopropylethylamine (0.13 g, 1.0 mmol) in CH2Cl2 (5 mL) at 0
°C. After being stirred at room temperature for 18 h, the reaction
mixture was poured into water and extracted with CHCl3. The
extract was washed with brine, dried (MgSO4), and concentrated
in vacuo. The residue was purified by flash column chromatography
(CHCl3-MeOH, 19:1), and the purified compound was recrystal-
lized from EtOH to afford the corresponding N-Boc-glycinamide
compound (0.21 g, 58%) as a white crystalline powder. Mp: 135-
137 °C. 1H NMR (CDCl3): δ 1.18 (6H, d, J ) 6.8 Hz), 1.48 (9H,
s), 2.09 (3H, s), 3.63 (2H, s), 4.01 (2H, t, J ) 5.6 Hz), 4.11-4.21
(1H, m), 4.16 (2H, s), 5.24 (2H, s), 5.66 (1H, t, J ) 5.9 Hz), 6.97
(2H, t, J ) 8.1 Hz), 7.10-7.21 (5H, m), 7.33-7.43 (1H, m), 7.61
(2H, d, J ) 8.5 Hz), 7.74 (2H, d, J ) 8.5 Hz), 8.29 (1H, s), 8.82
(1H, brs). FAB-MS m/z: 729 (M + H).
1
crystals (from EtOAc-Et2O). Mp: 232-234 °C. H NMR (HCl
salt in DMSO-d6): δ 0.76-1.05 (6H, m), 1.12 (6H, d, J ) 6.6
Hz), 2.06 (1H, brs), 2.51 (1H, m), 2.68 (3H, s), 4.14-4.79 (4H,
m), 5.32 (1H, brs), 5.65 (2H, d, J ) 12.6 Hz), 5.78 (2H, d, J )
12.6 Hz), 7.24-7.28 (7H, m), 7.39 (2H, d, J ) 8.7 Hz), 7.35-
7.45 (1H, m), 7.84 (2H, d, J ) 8.7 Hz), 8.28 (1H, s), 10.27 (1H,
s). IR (KBr): 3412, 2972, 2362, 1686, 1611, 1522, 1473, 1408
cm-1. FAB-MS m/z: 644 (M + H). Anal. (C37H39N3O3SF2‚HCl‚
2.0H2O) C, H, N.
3-(N-Benzyl-N-methylaminomethyl)-7-(2,6-difluorobenzyl)-
4,7-dihydro-N-methoxy-N-methyl-4-oxo-2-(4-trifluoroacetami-
dophenyl)thieno[2,3-b]pyridine-5-carboxamide (30). A solution
of trifluoroacetic anhydride (0.65 mL, 5.0 mmol) in CH2Cl2 (5 mL)
was added dropwise to a solution of 25 (1.15 g, 2.0 mmol) and
triethylamine (0.56 mL, 4.0 mmol) in CH2Cl2 (20 mL) at 0 °C.
After being stirred at room temperature for 1 h, the mixture was
poured into ice-water and extracted with CH2Cl2. The extract was
washed with brine, dried (MgSO4), and concentrated in vacuo. The
residue was purified by flash column chromatography (EtOAc-
MeOH, 9:1) and the purified compound was then triturated with
Et2O to give the corresponding trifluoroacetamide compound (1.31
g, 98%) as a pale-yellow amorphous powder. Mp: 114 °C. 1H NMR
(CDCl3): δ 1.36 (3H, t, J ) 7.2 Hz), 2.13 (3H, s), 3.64 (2H, s),
4.16 (2H, s), 4.37 (2H, q, J ) 7.2 Hz), 5.27 (2H, s), 7.01 (2H, t, J
) 8.1 Hz), 7.05-7.25 (5H, m), 7.35-7.48 (1H, m), 7.66 (2H, d, J
) 8.7 Hz), 7.92 (2H, t, J ) 8.7 Hz), 8.36 (1H, s). FAB-MS m/z:
670 (M + H).
Trifluoroacetic acid (1 mL) was added to the solution of the
above amide compound (0.15 g, 0.2 mmol) in CH2Cl2 (1 mL) at 0
°C. After being stirred at room temperature for 18 h, the reaction
mixture was concentrated in vacuo, and the residue was dissolved
in CHCl3 and washed with saturated NaHCO3. The organic layer
was dried (MgSO4) and concentrated in vacuo. The residue was
purified by flash column chromatography (CHCl3-MeOH, 9:1),
Compound 30 was then prepared in 94% yield from the above
amide compound by a procedure similar to that described for 27b

Non-Peptide LHRH Receptor Antagonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13 3823
and the crude product was crystallized from Et2O to provide the
free amine of 32b (0.12 g, 95%) as a white amorphous powder: 1H
NMR (free amine in CDCl3): δ 1.13 (6H, d, J ) 6.6 Hz), 2.76
(3H, s), 3.83-3.87 (1H, m), 4.29 (2H, s), 4.46 (2H, s), 4.65 (2H,
s), 5.35 (2H, s), 6.94 (2H, t, J ) 8.3 Hz), 7.10-7.45 (8H, m), 7.76
(2H, d, J ) 8.6 Hz), 8.32 (1H, s), 11.05 (1H, brs). The free amine
was treated with 5 M ethanolic HCl to afford 32b as a white
crystalline powder. Mp: 197-199 °C (from EtOH). FAB-MS
m/z: 629 (M + H). IR (KBr): 2936, 1682, 1626, 1593, 1539, 1504,
1471, cm-1. Anal. (C35H34N4O3SF2‚2.0HCl‚1.5H2O) C, H, N.
3-(N-Benzyl-N-methylaminomethyl)-7-(2,6-difluorobenzyl)-
4,7-dihydro-2-[4-(2-hydroxyacetamido)phenyl]-5-isobutyryl-4-
oxothieno[2,3-b]pyridine Hydrochloride (33a). Compound 33a
was prepared in 98% yield (two steps) from 31 by a procedure
similar to that described for 32b. Mp: 197-199 °C (from EtOH,
HCl salt). 1H NMR (free amine in CDCl3): δ 1.18 (6H, d, J ) 6.8
Hz), 2.09 (3H, s), 3.61 (2H, s), 4.07-4.19 (1H, m), 4.16 (2H, s),
4.21 (2H, s), 5.24 (2H, s), 6.99 (2H, t, J ) 8.3 Hz), 7.10-7.25
(5H, m), 7.32-7.48 (1H, m), 7.63 (2H, d, J ) 8.6 Hz), 7.72 (2H,
d, J ) 8.6 Hz), 8.25 (1H, s), 8.67 (1H, brs). FAB-MS m/z: 630
(M + H). Anal. (C35H33N3O4SF2‚HCl‚0.5H2O) C, H, N.
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 concentrations 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 (What-
man, 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 concentration 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:
log[%SPB/(100 - %SPB)] ) 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.
3-(N-Benzyl-N-methylaminomethyl)-7-(2,6-difluorobenzyl)-
4,7-dihydro-2-(4-(2-hydroxy-2-methylpropionylamido)phenyl)-
5-isobutyryl-4-oxothieno[2,3-b]pyridine (33b). Compound 33b
was prepared in 70% yield from 31 (two steps) by a procedure
similar to that described for 33c (see below). Mp: 178-180 °C
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 described
previously.10 The binding assays were carried out by incubating
1
(from CHCl3-Et2O). H NMR (CDCl3): δ 1.18 (6H, d, J ) 6.8
Hz), 1.58 (6H, s), 2.10 (3H, s), 3.08 (1H, brs), 3.64 (2H, s), 4.13-
4.21 (1H, m), 4.16 (2H, s), 5.23 (2H, s), 6.99 (2H, t, J ) 8.2 Hz),
7.10-7.25 (5H, m), 7.30-7.44 (1H, m), 7.64 (2H, d, J ) 8.5 Hz),
7.75 (2H, d, J ) 8.5 Hz), 8.25 (1H, s), 8.86 (1H, brs). FAB-MS
m/z: 667 (M + H). Anal. (C37H37N3O4SF2‚0.5H2O) C, H, N.
3-(N-Benzyl-N-methylaminomethyl)-7-(2,6-difluorobenzyl)-
4,7-dihydro-2-[4-(1-hydroxy-1-cyclopropanecarboxamido)-
phenyl]-5-isobutyryl-4-oxothieno[2,3-b]pyridine (33c). 1-Acetoxy-
1-cyclopropanecarbonyl chloride (3.58 g, 22.0 mmol) was added
dropwise to a solution of 31 (11.4 g, 20.0 mmol) and triethylamine
(2.43 g, 24.0 mmol) in CH2Cl2 (40 mL) at 0 °C. After being stirred
at room temperature for 6 h, the reaction mixture was poured into
water 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-
MeOH, 19:1) to afford the corresponding 1-acetoxy-1-cyclopro-
panecarboxamide compound (11.2 g, 80%) as a pale-yellow solid.
1H NMR (CD3OD): δ 1.18 (6H, d, J ) 6.8 Hz), 2.11 (3H, s), 3.47
(3H, s), 3.65 (2H, s), 4.10-4.24 (1H, m), 4.18 (2H, s), 4.26 (2H,
s), 4,65 (2H, s), 5.27 (2H, s), 7.00 (2H, t, J ) 8.3 Hz), 7.10-7.25
(5H, m), 7.33-7.49 (1H, m), 7.67 (2H, d, J ) 8.6 Hz), 7.81 (2H,
d, J ) 8.6 Hz), 8.28 (1H, s), 8.51 (1H, brs). FAB-MS m/z: 674
(M + H).
[
125I][Tyr5]leuprorelin (0.12-0.13 nM) and membranes 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.10
In Vitro Functional Assays. LHRH-stimulated arachidonic acid
release from CHO cells expressing human or monkey LHRH
receptors was measured according to the previously reported
protocol.25 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 [5,6,8,9,11,12,14,15-3H8]-
arachidonic acid (11 kBq/well, NEN Lifescience Products) for 1
day and washed with Dulbecco’s modified Eagle’s medium
(DMEM) supplemented with 20 mM HEPES and 0.2% BSA. The
cells were then preincubated with the compounds at 37 °C for 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.
In Vivo Efficacy in Cynomolgus Monkeys. Cynomolgus
monkeys (male, 4-9 years old) were castrated more than 6 months
prior to the examination. The monkeys were trained to sit in a
primate-restraining chair during administration of the compounds.
Compound 33c (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. Blood samples (heparin-plasma)
were collected from a femoral vein 0, 2, 4, 8, 24, and 48 h after
administration. LH concentrations in the plasma were measured
by bioassays using mouse testicular cells.10
Molecular Modeling Studies. All calculations were performed
on a Silicon Graphics O2 R10000 workstation. Molecular modeling
was carried out using the Insight II software package (Accelrys
Inc., San Diego, CA). Conformer CC of LHRH as described by
Momany23 was reproduced with the reported dihedral angles and
used as the putative active conformation of LHRH.
NaOH (5 N, 10 mL, 50 mmol) was added to a solution of the
above amide compound (5.40 g, 7.74 mmol) in EtOH (40 mL) at
0 °C. After being stirred at 0 °C for 3 h, the mixture was poured
into water and extracted with CHCl3. The extract was washed with
brine, dried (MgSO4), and concentrated in vacuo. The residue was
purified by flash column chromatography (CHCl3-MeOH, 9:1) and
the purified compound was recrystallized from EtOH to give 33c
(4.53 g, 89%) as a white crystalline powder. Mp: 184-185 °C.
1H NMR (CDCl3): δ 1.18 (6H, d, J ) 6.8 Hz), 1.16-1.20 (2H,
m), 1.48-1.51 (2H, m), 2.09 (3H, s), 3.64 (2H, s), 3.95 (1H, brs),
4.10-4.20 (1H, m), 4.14 (2H, s), 5.20 (2H, s), 6.99 (2H, t, J ) 8.0
Hz), 7.10-7.25 (5H, m), 7.34-7.44 (1H, m), 7.56 (2H, d, J ) 8.5
Hz), 7.70 (2H, d, J ) 8.5 Hz), 8.21 (1H, s), 8.82 (1H, brs). IR
(KBr): 3362, 3065, 2976, 2955, 2872, 1688, 1672, 1590, 1497,
1473 cm-1. FAB-MS m/z: 656 (M + H). Anal. (C37H35N3O4SF2)
C, H, N.
Acknowledgment. We thank Dr. Masahiko Fujino (de-
ceased), Dr. Haruo Onda, Dr. Koichi Kato, Dr. Hirosada
Sugihara (deceased), Dr. Hideo Shirafuji, Dr. Akio Miyake, Dr.
Hidekazu Sawada, Dr. Chieko Kitada, and Dr. Shuji Hinuma
for encouragement and helpful discussions throughout this work.
In Vitro Binding Assays. Receptor binding assays were carried
out as described previously.10 Briefly, human LHRH receptor cDNA
was cloned from a pituitary cDNA library, and CHO cells stably
expressing high levels of the recombinant human LHRH receptor
were isolated. [125I][Tyr5]leuprorelin (0.12-0.15 nM) and the

3824 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13
Imada et al.
(12) Sasaki, S.; Imaeda, T.; Hayase, Y.; Shimizu, Y.; Kasai, S.; Cho, N.;
Harada, M.; Suzuki, N.; Furuya, S.; Fujino, M. A New Class of Potent
Nonpeptide Luteinizing Hormone-Releasing Hormone (LHRH) An-
tagonists: Design and Synthesis of 2-Phenylimidazo[1,2-a]pyrimidin-
5-ones. Bioorg. Med. Chem. Lett. 2002, 12, 2073-2077.
We also acknowledge Dr. Shoichi Okubo, Dr. Kazuhiro Ogi,
Dr. Asano Asami, Ms. Tomoko Urushibara, Dr. Shiro Takeka-
wa, Mr. Hiroyuki Miya, and Dr. Akira Horinouchi for supporting
the biochemical and pharmacological experiments.
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Supporting Information Available: Elemental analysis data
of new compounds. This material is available free of charge via
the Internet at http://pubs.acs.org.
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