Synthesis and structure-activity relationships of thieno[2,3-d]pyrimidine-2,4-dione derivatives as potent GnRH receptor antagonists

Article abstract of DOI:10.1016/S0960-894X(03)00746-7

The synthesis and SAR studies of thieno[2,3-d]pyrimidine-2,4-diones as human GnRH receptor antagonists to treat reproductive diseases are discussed. It was found that the 2-(2-pyridyl)ethyl group on the 5-aminomethyl functionality of the core structure wa

Full text of DOI:10.1016/S0960-894X(03)00746-7

Bioorganic & Medicinal Chemistry Letters 13 (2003) 3617–3622
Synthesis and Structure–Activity Relationships of
Thieno[2,3-d]pyrimidine-2,4-dione Derivatives as Potent
GnRH Receptor Antagonists
Zhiqiang Guo,a,* Yongsheng Chen,a Dongpei Wu,a Yun-Fei Zhu,a R. Scott Struthers,b
John Saunders,a Qiu Xieb and Chen Chena,*
aDepartment of Medicinal Chemistry, Neurocrine Biosciences, Inc., 10555 Science Center Drive, San Diego, CA92121, USA
bDepartment of Exploratory Discovery, Neurocrine Biosciences, Inc., 10555 Science Center Drive, San Diego, CA92121, USA
Received 28 January 2003; accepted 16 April 2003
Abstract—The synthesis and SAR studies of thieno[2,3-d]pyrimidine-2,4-diones as human GnRH receptor antagonists to treat
reproductive diseases are discussed. It was found that the 2-(2-pyridyl)ethyl group on the 5-aminomethyl functionality of the core
structure was a key feature for good receptor binding activity. SAR study of the 6-(4-aminophenyl) group suggests that hydro-
phobic substituents were preferred. The best compound from this series had binding affinity (Ki) of 0.4 nM to the human GnRH
receptor.
# 2003 Elsevier Ltd. All rights reserved.
Gonadotropin-releasing hormone (GnRH), also known
as luteinizing hormone-releasing hormone (LHRH), is a
decapeptide (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-
Gly-NH2), which is produced and secreted by the
hypothalamus in a pulsatile manner.1,2 It acts on the
pituitary gland to stimulate the secretion of both
luteinizing hormone (LH) and follicle-stimulating hor-
mone (FSH). These gonadotropines, in turn, act on the
reproductive organs, where they participate in the reg-
ulation of gonadal steroid production, spermatogenesis
in males and follicular development in females. Several
reproductive disease conditions such as endometriosis,
uterine fibroids and prostate cancer are caused by over
stimulation of the reproductive organs by the gonadal
steroids, and thus can be treated by suppression of the
pituitary–gonad hormonal axis. Currently depot forms
of peptidic GnRH agonists, represented by leuprorelin1
are used to treat such conditions through a receptor
down-regulation mechanism to suppress gonadal ster-
oid production.3 However, recent clinical evidence has
shown that peptidic GnRH antagonists can act imme-
diately at the receptor to lower steroid levels and there-
fore alleviate disease symptoms without the
concomitant ‘flare effect’, which is exhibited by the
peptide agonists due to their initial overstimulation of
the GnRH receptor.4 In the clinic, it has been shown
that peptidic GnRH antagonists can directly lower
gonadal sex hormone levels to alleviate disease symp-
toms without the concomitant flare effect. Nevertheless,
whether the patient is treated with the peptide agonists
or antagonists, both regimens still require parenteral
administration, typically in depot form due to their
poor oral bioavailability. By contrast, small molecule
GnRH antagonists offer the potential of oral adminis-
tration and therefore could gain wider acceptance from
patients. In response to this need, intensive efforts have
been initiated in the development of small molecule
GnRH antagonists by many laboratories. Compound 1
(T-98475) and its analogues are the first small molecules
reported5 to have high affinity on the human GnRH
receptor (IC50 0.2 nM) albeit with reduced binding affi-
nity for the rat receptor (IC50 60 nM).5 In addition, a
series of papers on quinolones and tryptamines as
potent GnRH antagonists were recently published. For
example, quinolone 2 possesses high binding affinity
(IC50 0.4 nM) for the human GnRH receptor as well as the
rat GnRH receptor (IC50 4 nM).6 Indole derivatives such
as 3 were reported to be potent and orally bioavailable
*Corresponding author. Tel.: +1-858-658-7657; fax: + 1-858-658-
7601; e-mail: zguo@neurocrine.com
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00746-7

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Z. Guo et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3617–3622
GnRH antagonists.7 Most recently, TAK-013 (IC50 0.2
nM) and its analogues were reported as potent and
orally active hGnRH antagonists.8
The thieno[2,3-d]pyrimidine-2,4-dione core was synthe-
sized according to a procedure described in Scheme 1. 4-
Nitrophenylacetone (8) and ethyl cyanoacetate were
treated with sulfur in ethanol to form 2-amino-4-
We have previously described the identification of 7-
phenylpyrrolo[1,2-a]pyrimid-4-ones 5 and 69 (Fig. 1),
and more recently, 2-phenylimidazolo[1,2-a]pyrimidines
710 as potent small molecule GnRH receptor antago-
nists. SAR studies on these series showed that a basic
side-chain is very important for strong receptor-binding
activity and the phenyl ring is a good substitution on the
right side of the molecules. In our efforts to produce
orally active small molecules with good overall PK
profile, we were interested in understanding the SAR on
other bicyclic series as well. In this letter we wish to
report the synthesis and SAR study of thieno[2,3-d]pyr-
imidine-2,4-dione compounds as potent small molecule
GnRH antagonists.
methyl-5-(4-nitrophenyl)thiophene-3-carboxylic
acid
ethyl ester 9.13 Next, 9 and phenylisocyanate were
heated with pyridine in toluene at 100 ꢀC for 4 h to form
a urea intermediate, which was treated with NaOMe/
MeOH in refluxing acetonitrile to give 3-phenyl-thieno-
pyrimidine-2,4-dione 10.14 10 was alkylated at N1 posi-
tion by 2,6-difluorobenzylbromide with K2CO3 in DMF
to give 11.15 The 5-methyl group of 11 was then bromi-
nated with NBS/AIBN in carbon tetrachloride to yield
12. The resulting bromomethyl compound 12 was trea-
ted with N-methylbenzylamine in acetonitrile to give
compounds 13a as the desired products (Scheme 1).
To functionalize the nitro group on the 6-(4-nitrophe-
nyl) moiety for additional modification, 5-(N-benzyl-N-
methylaminomethyl)-1-(2,6-difluorobenzyl)-6-(4-nitro-
phenyl)-3-phenylthieno[2,3-d]pyrimidine-2,4-dione (13a)
was hydrogenated with Pd-C in ethanol to yield the 6-
(4-aminophenyl) analogue 14 (Scheme 2). The aniline
(14) was treated under reductive alkylation conditions
with aldehydes (RCHO) in dichloroethane, followed by
treatment with borane-pyridine, to give the desired
compounds 15. For reductive alkylation with the more
hindered substrate, acetone, NaBH(OAc)3 was used as
the reducing reagent. When R3 was a methyl group, the
second N-methylation was achieved by reductive alkyl-
ation of 15a with another molecule of formaldehyde to
afford the final product 16. Amide formation of 14 was
performed by treatment with acetic anhydride or
Although TAK-013 is a highly potent GnRH antago-
nist, it is highly lipophilic with high molecular weight,
and almost insoluble in water. A quick calculation with
ACD software11 suggests the logD of this compound is
about 6. The measured logD value is >4 due to high
partition in octanol. In this paper we report our results
of screening polar side chain in the efforts to discover
more water-soluble analogues of TAK-013. Based on
our early success on the 2-pyridylethylamine side chain
for several potent classes of GnRH antagonist, we also
like to utilize it to replace the N-benzyl-N-methylamino
side chain. The slightly basic pyridine ring should help
to reduce the lipophilicity therefore increase water solu-
bility (the pKa value of 2-picoline is 5.94).12
Figure 1. Small molecule GnRH antagonists.

Z. Guo et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3617–3622
3619
trifluoroacetic anhydride to yield 17a and 17b. Alter-
natively, amides 17c–d were prepared from the corre-
sponding acid chlorides with diisopropylethylamine in
dichloromethane. Urea functionality was introduced
into the molecule by treatment of 14 with ethyl isocya-
nate to yield 18. (Scheme 2) To introduce an extra basic
amino group into the core structure, aniline 14 was
coupled to N-Boc-protected amino acids using HBTU
as the coupling reagent in DMF. Deprotection of the
amino-Boc group with TFA yielded the desired pro-
ducts 19–20 (Scheme 3).
To explore the SARs on the 5-methyl position of the
thieno[2,3-d]pyrimidine-2,4-dione core structure, the
5-methyl group of 11 was then brominated; and the
resulting bromomethyl compound 12 was treated
with a variety of amines (R1R2NH) in acetonitrile to
give compounds 13(b–i) as the desired products
(Scheme 4).
All synthesized compounds were evaluated for their
ability to compete for des-Gly10[125I-Tyr5,DLeu6,NMe-
Leu7,Pro9-NEt]-GnRH radioligand binding to the
Scheme 1. Reagents and conditions: (a) NCCH2COOEt, S, BuNH2, EtOH; (b) PhNCO, pyridine, toluene; (c) NaOMe, MeOH, ACN; (d) 2,6-
difluorobenzyl bromide, K2CO3, DMF; (e) NBS, AIBN, CCl4; (f) PhCH2NHMe, ACN.
Scheme 2. Reagents and conditions: (a) H2, Pd/C, EtOH; (b) RCHO, BH3/pyridine, dichloroethane; (c) acetone, NaBH(OAc)3, dichloroethane; (d)
CH2O, BH3/pyridine, dichloroethane; (e) (RCO)2O, iPr2EtNH, CH2Cl2; (f) RCOCl, iPr2EtNH, CH2Cl2; (g) EtNCO, iPr2EtNH, CH2Cl2.

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Z. Guo et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3617–3622
Scheme 3. Reagents and conditions: (a) BocNH(CH2)nCOOH, n=1–3, HBTU, NEt3, DMF; (b) N-t-Boc-l-alanine or N-t-Boc-l-leucine, HBTU,
NEt3, DMF; (c) TFA/CH2Cl2 (1:1).
Scheme 4. Reagents and conditions: (a) NBS, AIBN, CCl4; (b) R1R2NH, ACN.
cloned human GnRH receptor stably expressed in
HEK293 cells using a 96-well filtration assay format.16
modeling proposed by Takeda scientists, the counter-
part of this hydrogen bonding on the receptor is Asn102
on top of the helix II.4,8 Interestingly there is an aspartic
acid residue Asp98 one-turn below Asn102, which may
interact with any basic group from the small molecule.
Based on this theory we attempted to introduce a basic
function group to improve the physicochemical proper-
ties of these compounds. Thus, coupling of various
amino acids with the aniline 14 resulted in compounds
19a–c and 20a–b. Surprisingly, all these amides were
much less active than those that did not bear a basic
group, although compounds 20a–b still showed modest
activity (43 and 58 nM, respectively).
The impact of the substitutions on the 6-(4-aminophe-
nyl) group was examined and receptor binding data for
these compounds 14–20 are reported in Table 1.
Reduction of the nitro group in 13a to the amino ana-
logue 14 improved the binding potency by 5-fold.
Alkylation of the amino group with small alkyl groups,
such as ethyl 15b slightly increased its affinity. However,
incorporation of dimethyl (16) or isopropyl group (15d)
on the nitrogen decreased the affinity. Interestingly, with
amide bond linkage, 17 showed good potency. Thus,
acetamide 17a was 9 times better than 14, although the
trifluoroacetyl analogue 17b was less active. These data
suggest that the NH might be involved in hydrogen
bonding with the GnRH receptor. Propionyl amide 17c
was the most potent compound in this class. Not sur-
prisingly, the ethyl urea 18 showed the best potency in
this modification. All these results agree with the
hypothesis that this aniline hydrogen plays an impor-
tant role as a hydrogen-bond donor in hydrogen bond-
ing with the GnRH receptor. Base on the receptor
The effect of substituents on the aminomethyl group at
the 5-position of 13 on hGnRH receptor binding affinity
are summarized in Table 2. Although TAK-013 is a
highly potent GnRH antagonist, it may suffer from high
lipophilicity due to low pKa of the benzylamine (calcu-
lated value for logP and pKa were 6.5 and 5.0, respec-
tively). One of our objectives for this study is to improve
physicochemical properties of this series of compounds
by substituting the lipophilic N-benzyl moiety with a

Z. Guo et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3617–3622
3621
Table 1. Binding affinities of compounds 14–20 on the hGnRH
receptor17
Table 2. Binding affinities of compounds 13a–i on the hGnRH recep-
tor17
R1R2N
Ki (nM)
Compd
14
R3NR4
NH2
Ki (nM)
36
Compd
13a
180
15b
11
13b
13c
640
270
15c
15d
16
17
88
13d
13e
13f
13g
13h
13i
170
76
61
17a
17b
17c
17d
18
4.1
16
460
170
0.6
9
0.8
2.9
0.4
150
360
300
43
19a
19b
19c
20a
20b
By contrast, the phenethyl analogue 13g had similar
activity as 13a.
One explanation for these results is that the substituted
methylamine had interaction with Asp302 on top of
Helix VII of the GnRH receptor.4,8 The extra hetero-
atom O and N offered hydrogen-bonding capability with
this residue, and thus improved the interaction with the
receptor. In addition, the pyridine ring may mimic the
phenyl group, which may interact with the receptor
through p–p stacking. The residue for this interaction
has not been identified from receptor modeling.
58
smaller or more polar group. Replacement of the benzyl
group of 13a with a butyl group made no obvious dif-
ference in affinity while the dimethylamino analogue
was over 3-fold less active. As expected, introduction of
a polar methoxy or diethylamino substituent at the b-
position from the nitrogen (13d and 13e) slightly
improved binding affinity, although the cyclic 1-methyl-
piperidine 13f was less active. When the benzyl moiety
was replaced with a 2-pyridylmethyl group, compound
13i was 20-fold better in binding affinity (Ki=9 nM).
Even more, when a 2-(2-pyridyl)ethyl was used, the
resulting compound 13h showed subnanomolar binding
affinity (Ki=0.6 nM), which is 300-fold better than 13a.
Although the incorporation of a weak basic moiety on
the left side of the molecules resulted in loss of binding
affinity, the replacement of the N-benzyl moiety with the
2-pyridylethyl group did increase 300-fold binding affi-
nity on the GnRH receptor. While the pyridine deriva-
tive 13h (Ki=0.6 nM) exhibited comparable activity
with TAK-013, it was less lipophilic than TAK-013 (the
calculated logD value is 4.81, which is one log unit
lower than TAK-013, and the measured value is 3.3).
This discovery also enables the possibility of further
modification towards more water-soluble analogues of
TAK-013.18

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Z. Guo et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3617–3622
In conclusion, a series of 1-(2,6-difluorobenzyl)-3-phe-
nylthieno[2,3-d]pyrimidine-2,4-dione compounds were
discovered to have excellent binding affinity to the
human GnRH receptor. Hydrophobic substituents on
the aniline nitrogen of the 6-(4-aminophenyl)-
thieno[2,3-d]pyrimidine-2,4-dione core were preferred.
Simple alkyl amides and ureas on the 6-(4-aminophenyl)
moiety were very potent human GnRH receptor
antagonists, whereas extra basic amines were less
favorable. The results of this SAR study suggest that the
2-(2-pyridyl)ethyl group on the 5-aminomethyl func-
tionality of the thieno[2,3-d]pyrimidine-2,4-dione core
was optimal for the human GnRH receptor binding.
This phenomenon may be explained by a predicted
receptor model, in which the pyridyl side chain is in
close proximity to the aspartic acid-302 on helix VII.
The potent compounds discovered from this series may
also have favorable physicochemical properties as
potential orally active drug candidates.
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Acknowledgements
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We are indebted to Dr. Tao Hu and Mrs. Mila Lagman
for assistance in obtaining the logD data.
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17. On each assay plate a standard antagonist of comparable
affinity to those being tested was included as a control for
plate-to-plate variability. Overall, Ki values were highly
reproducible with an average standard deviation of 45% for
replicate Ki determinations. Key compounds were assayed in
3–8 independent experiments.
18. One can envision that replacing the N-benzyl chain of 20a
with the 2-pyridylethyl group will result in compound with
good binding affinity as well as good water solubility.