Synthesis and structure-activity relationships of uracil derived human GnRH receptor antagonists: (R)-3-[2-(2-amino)phenethyl]-1-(2,6-difluorobenzyl)-6- methyluracils containing a substituted thiophene or thiazole at C-5

Article abstract of DOI:10.1016/j.bmcl.2004.07.022

The design and synthesis of a number of 5-thiazolyl and 5-thienyl substituted uracils is described and results from SAR studies are summarized. The best compound showed K_i = 2 nM. The synthesis of a series of (R)-3-[2-(2-amino)phenethyl]-1-(2,6

Full text of DOI:10.1016/j.bmcl.2004.07.022

Bioorganic & Medicinal Chemistry Letters 14 (2004) 4967–4973
Synthesis and structure–activity relationships of uracil derived
human GnRH receptor antagonists:
(R)-3-[2-(2-amino)phenethyl]-1-(2,6-difluorobenzyl)-6-methyluracils
containing a substituted thiophene or thiazole at C-5
a
a
a
Martin W. Rowbottom,a, Fabio C. Tucci, Patrick J. Connors, Jr., Timothy D. Gross,
Yun-Fei Zhu,a Zhiqiang Guo,a Manisha Moorjani,a Oscar Acevedo,a Lee Carter,b
Susan K. Sullivan,b Qiu Xie,c Andrew Fisher,d R. Scott Struthers,c John Saundersa
*
and Chen Chena,
*
aDepartment of Medicinal Chemistry, Neurocrine Biosciences Inc., 12790 El Camino Real, San Diego, CA 92130, USA
bDepartment of Pharmacology, Neurocrine Biosciences Inc., 12790 El Camino Real, San Diego, CA 92130, USA
cDepartment of Endocrinology, Neurocrine Biosciences Inc., 12790 El Camino Real, San Diego, CA 92130, USA
dDepartment of Preclinical Development, Neurocrine Biosciences Inc., 12790 El Camino Real, San Diego, CA 92130, USA
Received 7 June 2004; accepted 10 July 2004
Available online 31 July 2004
Abstract—The synthesis of a series of (R)-3-[2-(2-amino)phenethyl]-1-(2,6-difluorobenzyl)-6-methyluracils containing a substituted
thiophene or thiazole at C-5 is described. SAR around C-5 of the uracil led to the discovery that a 2-thienyl or (2-phenyl)thiazol-4-yl
group is required for optimal receptor binding. The best compound from the series had a binding affinity of 2nM (Ki) for the human
GnRH receptor. A novel and convenient preparation of N-1-(2,6-difluorobenzyl)-6-methyluracil is also described.
Ó 2004 Elsevier Ltd. All rights reserved.
Gonadotropin-releasing hormone (GnRH) is a decapep-
tide (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2)
produced and secreted by the hypothalamus and which
interacts with specific GnRH receptors located within
the anterior pituitary, releasing both follicle-stimulating
hormone (FSH) and luteinizing hormone (LH) from this
site.1,2 A number of disease states including endometrio-
sis, uterine fibroids, and prostate cancer can be control-
led via the suppression of GnRH stimulated release of
both FSH and LH. This may be clinically achieved via
agonism or antagonism of the GnRH receptor and a
number of peptide agonists and antagonists are cur-
production of both FSH and LH with a concomitant
Ôflare effectÕ, which tends to exacerbate symptoms (in
the short term) in patients. In response to the need for
a more convenient and clinically flexible route of admin-
istration, intensive efforts have been initiated toward the
development of orally bioavailable small-molecule
GnRH antagonists.4–6
Previously we have reported the discovery of a new class
of uracils as orally bioavailable human GnRH [hGnRH]
receptor antagonists, exemplified by 3-(2-aminoethyl)-5-
aryl-6-methyluracils 1 (Fig. 1).5 Subsequent modifica-
tion of the substituent at N-3, led to the discovery of
highly potent (R)-3-[2-(2-amino)phenethyl]-5-aryl-6-
methyluracils 2.6 Furthermore, chemistry was developed
which allowed for facile variation of the N-1 substituent
(of the uracil ring) and we were able to show that the
inclusion of an electron-deficient 2,6-disubstituted ben-
zyl group (represented by 3) was optimal for hGnRH re-
ceptor binding.6 Previously, we have only described
uracils containing a substituted phenyl ring at C-5 (1
rently available as represented by LeuprorelinÒ and
3
Cetrotidee,1 respectively. However, due to the very
low oral bioavailability of these peptides, administration
is normally via injection or depot formulation. In addi-
tion, treatment with agonists initially leads to the over-
*
Corresponding authors. Tel.: +1-858-658-7799; fax: +1-858-658-
7601; e-mail: mrowbottom@neurocrine.com
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.07.022

4968
M. W. Rowbottom et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4967–4973
2
6. Reaction of urea with diketene to give 6-methyluracil
R
N
O
N
O
via a two-step process has previously been reported.7 We
have shown that reaction of (2,6-difluorobenzyl)urea 4
with diketene in pyridine at ambient temperature gave
N-acetoacetylurea 5 (isolated in 60% yield), which upon
refluxing in acetic acid for 1h gave uracil 6 in 95% yield
(Scheme 1).5b,d However, the fact that the preparation of
uracil 6 (from urea 4) still involved two separate steps
and the overall yield was below 60%, encouraged us to
develop a more efficient route. Yamamoto et al. have
reported that reaction of primary amides with diketene
in the presence of trimethylsilyl iodide (generated in situ
from trimethylsilyl chloride and sodium iodide) gave
N-acetoacetylcarboxamides in high yield.8 We reasoned
that in a similar manner the reaction of urea 4 with dike-
tene (in the presence of trimethylsilyl iodide) should give
5. More importantly, 5 should undergo the acid cata-
lyzed cyclodehydration step in situ (mediated by the
hydrogen iodide generated in the reaction) to produce
6 in one step. Gratifyingly, the reaction of urea 4 with
diketene in the presence of trimethylsilyl iodide, gave
uracil 6 as the only product in 93% yield.9,10 Bromina-
tion of 6 (bromine in acetic acid) followed by N-3
3
N
5
3
N
5
1
R
Ar
Ar
6
6
H2N
O
Me
O
N
Me
1
1
3
R
R
2
1
O
N
3
N
5
Ar
6
H2N
O
Me
F
1
F
3
Figure 1. General structures of uracil based GnRH antagonists.
alkylation with (R)-N-boc-2-phenylglycinol (via
Mitsunobu protocol) gave 5-bromouracil 7 in 63% yield
a
(over two steps).
and 2). We also wanted to incorporate substituted
heteroaryl rings at this position, with the hope of discov-
ering further potent analogs with different pharmacoki-
netic and metabolism properties. In this letter we wish
to report the synthesis and hGnRH activity of several
(R)-N-3-[2-(2-amino)phenethyl]-1-(2,6-difluorobenzyl)-6-
methyluracils 3, containing a substituted thiophene or
thiazole ring at C-5.
5-(Thiazol-4-yl)-6-methyluracil derivatives 10 and 11
(Scheme 2 and Tables 1, 2) were prepared directly from
a-bromoketone 9. The reaction of 7 with tri-n-butyl-
(1-ethoxyvinyl)tin via a Stille-coupling11 gave ethylvinyl
ether 8, which upon treatment with NBS in H2O/THF
gave bromoketone 9 in 40% yield (over two steps). Sub-
stituted thiazole rings are easily prepared via the Han-
tzsch thiazole reaction, in which thioureas or
thioamides are condensed with a-bromoketones.12 The
reaction of bromoketone 9 with a variety of thioureas
or thioamides followed by N-Boc deprotection gave 5-
(thiazol-4-yl)-6-methyluracils 10(a, b, d, e, g–m) and
(R)-N-3-[2-(2-Amino)phenethyl]-1-(2,6-difluorobenzyl)-
5-aryl-6-methyluracil analogs 3 were prepared from (R)-
N-3-[2-(2-amino)phenethyl]-5-bromo-1-(2,6-difluorobenz-
yl)-6-methyluracil 7 (Scheme 1), which in turn was
obtained from N-1-(2,6-difluorobenzyl)-6-methyluracil
F
F
O
a
NH2
N
H
NH2
F
F
4
b
d
O
N
F
O
O
O
O
3
Br
5
c
e, f
N
Me
N
N
H
HN
H
6
NH
boc
O
Me
F
O
1 N
Me
F
F
5
F
F
7
6
Scheme 1. Reagents and conditions: (a) urea, water, HCl, reflux, 79%; (b) diketene, pyridine, rt, 24h, 60%; (c) AcOH, reflux, 1h, 95%; (d) diketene,
trimethylsilyl chloride, sodium iodide, MeCN, 5°C to rt, 20h, 93%; (e) Br2, AcOH, rt, 1h, 88%; (f) (R)-N-Boc-2-phenylglycinol, DEAD, PPh3, THF,
rt, 12h, 71%.

M. W. Rowbottom et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4967–4973
4969
O
N
O
N
O
N
OEt
O
3
5
Br
Br
a
b
N
N
N
6
NH
O
NH
O
NH
boc
boc
boc
Me
Me
O
Me
F
1
F
F
F
F
F
7
8
9
e or f
then d
c, d
2
S
S
O
N
O
N
R
1
R
N
N
N
3
N
N
R
H2N
H2N
O
Me
O
Me
F
F
F
F
10
11
Scheme 2. Reagents and conditions: (a) tri-n-butyl(1-ethoxyvinyl)tin, Pd(PPh3)4, 1,4-dioxane, reflux, 12h; (b) NBS, THF/H2O, rt, 4h, 40% (from 7);
(c) R1C(@S)NH2, EtOH, reflux, 2–3h; (d) TFA, DCM, rt, 1h; (e) R2R3NC(@S)NH2, EtOH, reflux, 2–3h; (f) ammonium thiocyanate, R2R3NH,
EtOH, reflux, 13h.
11(a–m), respectively (Scheme 2 and Tables 1, 2).13
Alternatively, treatment of a-bromoketones with thiocy-
anate salts followed by the addition of a primary or sec-
ondary amine, gives 2-aminothiazoles in one pot.14 The
reaction of bromoketone 9 with ammonium thiocyanate
and amines (followed by N-Boc deprotection), gave
5-(thiazol-4-yl)-6-methyluracils 10 (c, f) (Scheme 2 and
Table 1).15 5-(2-Thienyl)-6-methyluracil derivatives 12
may be prepared in two steps from bromouracil 7 via
a Suzuki coupling with 2-thienylboronic acids, followed
by an N-Boc deprotection (Scheme 3 and Table 3).
that we suspected 10d would be metabolically more sta-
ble than the N-methylated and N-benzyl analogs (10e
and 10f, respectively), other compounds containing a
substituted N-phenyl ring were prepared (10g–m). It
was observed that a 4-substituent in the phenyl ring lar-
gely maintained activity whilst 2-substituents were not
acceptable. For example, 2-(2-methoxylphenyl)amino-
thiazole 10j (Ki of 130nM) was less potent than the cor-
responding 4-methoxy derivative 10k (Ki of 42nM) or
the unsubstituted derivative 10d (Ki of 70nM). These
data may suggest that the presence of a ring substituent
ortho to the nitrogen prevents the ring adopting a more
planar orientation (with respect to the amino substitu-
ent), which may be required for optimal binding. The
addition of a second methoxy group at position 3 (10l,
Ki of 33nM) was tolerated though did not improve po-
tency. The replacement of the 4-methyl (10i, Ki of
49nM) or 4-methoxy groups (10k) by a nitro group
(10m) reduced potency approximately 3-fold (10m had
a Ki of 160nM).
All 5-thiazolyl and 5-thienyl-6-methyluracils described
(Tables 1–3) were assayed for their ability to bind com-
petitively to the cloned hGnRH receptor expressed in
RBL cells, using a 96-well filtration apparatus16,17 and
125I-[His5, D-Tyr6]GnRH as the radiolabeled ligand.18
The functional antagonism of the described compounds
was confirmed by their ability to inhibit GnRH-stimu-
lated Ca2+ flux in the transfected cells in a dose-depend-
ent manner.4g,19 Activity in the 5-(thiazol-4-yl)uracil
series was dependent upon the nature of the thiazole
ring substituent. Thus polar functional groups (10a, c,
Table 1) were less tolerated by the receptor than those
containing more lipophilic substituents. For instance,
2-(ethylamino)thiazole derivative 10b had a Ki of
270nM, which was approximately 10-fold more potent
than the unsubstituted aminothiazole 10a (which had a
Ki of 3000nM) and 5-fold more potent than the 2-(mor-
pholino)thiazole derivative 10c (with a Ki of 1500nM).
Binding affinity was further increased by the incorpora-
tion of either a 2-aminophenyl (10d, Ki of 70nM), 2-
(methylamino)phenyl (10e, Ki of 34nM), or benzyl
group (10f, 35nM). Because of the affinity of 10d, and
Removal of the 2-amino functionality of the thiazole
ring increases the lipophilicity of these compounds (rel-
ative to the amino derivatives), which we reasoned
might improve potency. 5-(2-Methylthiazolyl)-6-methyl-
uracil 11a had a Ki of 120nM (Table 2). Incorporation
of the more lipophilic phenyl (11b) or benzyl group
(11c) improved potency by approximately 5- and 13-fold
to 26 and 9nM, respectively. As with aminothiazole
derivatives 10, 4-substituted phenyl rings (11d, f–h, j–l)
were preferred over those containing a 2- or 3-substitu-
ent. 4-(Trifluoromethyl)phenyl derivative 11d (Ki
of 47nM) was 3-fold more potent than the correspond-
ing 3-substituted analog 11e (Ki of 150nM). Similarly,

4970
M. W. Rowbottom et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4967–4973
Table 1. Binding affinities of 5-(2-aminothiazol-4-yl)-6-methyluracils
10a–m toward the hGnRH receptor
Table 2. Binding affinities of 5-(thiazol-4-yl)-6-methyluracils 11a–m
toward the hGnRH receptor
2
S
S
O
R
O
1
R
N
N
N
3
N
N
R
H2N
H2N
O
N
Me
F
O
N
Me
F
F
F
Compd
NR2R3
Ki (nM)a
Compd
R1
Ki (nM)a
Me
11a
120
NH2
10a
3000
H
N
10b
10c
270
Me
O
11b
11c
11d
26
9
1500
N
H
N
CF
3
10d
10e
10f
10g
70
34
47
Me
N
11e
11f
11g
150
42
CF
3
F
H
N
35
Me
Me
Me
Me
H
N
18
210
F
H
N
11h
49
10h
10i
130
49
Me
Me
H
N
Cl
11i
11j
30
15
OMe
H
N
Cl
O
10j
130
42
H
N
10k
10l
11k
11l
20
7
OMe
OMe
H
N
H
N
OEt
33
O
OMe
H
N
Me
Me
Me
10m
160
11m
500
NO2
a Data are average of three or more independent measurements.17
a See footnote to Table 1.
2-(4-chlorophenyl)thiazole 11j (Ki of 15nM) was 2-fold
more active than the 2-chloro derivative 11i (Ki of
30nM). However, there seems to be a limitation as to
the steric bulk tolerated. The 4-tert-butylphenyl analog
11m had a Ki of 500nM, which was over 20-fold less po-
tent than the 4-methylphenyl derivative 11g, which had
a Ki of 18nM. Surprisingly the N-carbamoyl derivative

M. W. Rowbottom et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4967–4973
4971
O
O
R 4
3
5
Br
a, b
S
N
N
6
NH
H2N
boc
O
N
Me
O
N
Me
1
F
F
F
F
7
12
Scheme 3. Reagents and conditions: (a) thiopheneboronic acid, Pd(PPh3)4, aq Ba(OH)2, DME/PhH/EtOH, 90°C, 15h; (b) TFA, DCM, rt, 1h.
Table 3. Binding affinities of 5-(2-thienyl)-6-methyluracils 12a–c and 5-(4-chlorophenyl)-6-methyluracil 13 toward the hGnRH receptor
Cl
O
N
O
N
4
R
S
N
N
H2N
H2N
O
Me
F
O
Me
F
F
F
12a-c
13
Compd
R4
Ki (nM)a
12a
12b
12c
13
H
10
3
Me
Cl
––
2
14
a See footnote of Table 1.
11l had a Ki of 7nM, this being the most potent analog
of the thiazole series. Inclusion of a 2-thienyl group at
C-5 of the uracil ring was also investigated (compounds
12a–c, Table 3). The unsubstituted 2-thienyl derivative
12a had a Ki of 10nM and was already equipotent to thi-
azole derivative 11l. Incorporation of a 5-methyl (12b)
or 5-chloro substituent (12c) improved potency further
(with measured KiÕs of 3 and 2nM, respectively). When
compared directly to the corresponding 5-(4-chlorophen-
yl)-6-methyluracil derivative 1320 (Table 3) which had a
Ki of 14nM, compound 12c displayed 7-fold greater
affinity toward the receptor, thus demonstrating that
the 5-phenyl group (of the uracil ring) can be replaced
and potency maintained.
Overall these data suggest that the substituent at C-5 of
the uracil core lies in a relatively lipophilic area of the
binding pocket. Uracils incorporating a 2-thienyl ring
(at C-5) are generally preferred over the more polar thi-
azol-4-yl containing derivatives (compare compounds
11a and 12b).21 Though as previously described, by
increasing the lipophilic nature of the thiazole contain-
ing substituent, potency is much improved (compare
11a with 11b,c). We believe that this increase in lipophi-
licity does not adversely affect metabolic stability, as we
have highlighted examples with an improved in vitro
stability profile (toward HLM), when compared to
potent first generation uracil antagonists. We also
We were also interested in the overall metabolic stability
of compounds described. Incubation with human liver
microsomes (HLM) can be used to help estimate how
much of a target compound will be removed by hepatic
first-pass metabolism in vivo. For instance in an in vitro
HLM assay,5d 5-(thiazol-4-yl)-6-methyluracils 11g and
11j (Table 2) had an intrinsic clearance of 36 and
46mL/min/kg, respectively. When directly compared
to (R)-1-(2,6-difluorobenzyl)-5-(3-methoxyphenyl)-3-(2-
(N-methyl-N-(2-pyridyl)methyl)aminopropyl)-6-methyl-
uracil 145b (Fig. 2) (a potent example from the first gen-
eration of uracil hGnRH antagonists), these compounds
proved to be much more stable. In the same assay, uracil
14 had an intrinsic clearance of 1700mL/min/kg.
OMe
Me
N
O
N
3
N
5
N
6
O
Me
F
1
F
14
Ki (hGnRH) = 5 nM
Figure 2. Structure of uracil derivative 14.

4972
M. W. Rowbottom et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4967–4973
Saunders, C.; Chen, T. K.; Bonneville, A. L. K.; Chen, C.
J. Med. Chem. 2003, 46, 2023; (b) Guo, Z.; Zhu, Y.-F.;
Tucci, F. C.; Gao, Y.; Struthers, R. S.; Saunders, J.;
Gross, T. D.; Xie, Q.; Reinhart, G. J.; Chen, C. Bioorg.
Med. Chem. Lett. 2003, 13, 3311; (c) Tucci, F. C.; Zhu,
Y.-F.; Guo, Z.; Gross, T. D.; Connors, P. J., Jr.; Struthers,
R. S.; Reinhart, G. J.; Saunders, J.; Chen, C. Bioorg. Med.
Chem. Lett. 2003, 13, 3317; (d) Guo, Z.; Zhu, Y.-F.;
Gross, T. D.; Tucci, F. C.; Gao, Y.; Moorjani, M.;
Connors, P. J., Jr.; Rowbottom, M. W.; Yongsheng, C.;
Struthers, R. S.; Xie, Q.; Saunders, J.; Reinhart, G.; Chen,
T. K.; Bonneville, A. L. K.; Chen, C. J. Med. Chem. 2004,
47, 1259; (e) Tucci, F. C.; Zhu, Y.-F.; Zhiqiang, G.; Gross,
T. D.; Connors, P. J., Jr.; Gao, Y.; Rowbottom, M. W.;
Struthers, R. S.; Reinhart, G. J.; Xie, Q.; Chen, T. K.;
Bozigian, H.; Bonneville, A. L. M.; Fisher, A.; Jin, L.;
Saunders, J.; Chen, C. J. Med. Chem. 2004, 47,
3483.
speculate that this area of the binding pocket, which
accommodates the C-5 substituent, is relatively open
and able to tolerate somewhat larger functional groups
(such as 2-phenyl or 2-benzylthiazol-4-yl moieties).22
Acknowledgements
We are indebted to Mr. John Harman for LC–MS sup-
port, Mr. Greg Reinhart for pharmacology support and
Dr. Miklos Feher and Yinghong Gao for computational
advice. This work was partly supported by NIH grants
1-R43-HD38625-01 and 2-R44-HD38625-02.
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M. J. Pharmacol. Exp. Ther. 2003, 305, 688, and references
cited therein; (i) Guo, Z.; Chen, Y.; Wu, D.; Zhu, Y.-F.;
Struthers, R. S.; Saunders, J.; Xie, Q.; Chen, C. Bioorg.
Med. Chem. Lett. 2003, 13, 3617.
9. Initial reaction of urea 4 with diketene via the secondary
nitrogen, would lead to the formation of N-1H-3-(2,6-
difluorobenzyl)-6-methyluracil (a regioisomer of 6). For-
mation of this product was never observed. The identity of
6 was confirmed by NOE NMR experiments, as outlined
in Ref. 5d.
10. Experimental procedure for the preparation of N-1-(2,6-
difluorobenzyl)-6-methyluracil 6. Into a multi-neck 5L
flask fitted with a thermometer, dropping funnel and
mechanical stirrer was placed 2,6-difluorobenzylurea 4
(100g, 537mmol), sodium iodide (121g, 806mmol), and
dry acetonitrile (1L). The reaction mixture was stirred
under nitrogen and cooled to 5°C. Diketene (Aldrich,
stabilized with CuSO4, 62mL, 804mmol) was added
dropwise over 5min, followed by trimethylsilyl chloride
(103mL, 806mmol), added dropwise over 10min (keeping
the temperature below 10°C). After stirring at 5°C for a
further 30min, the reaction mixture was allowed to slowly
warm to rt and stirred for a further 20h. Water (1.5L) was
added and stirring was continued for a further 20h. The
reaction mixture was filtered and the collected solids were
washed with water (500mL), ether (200mL), then dried in
vacuo to give 6 (126g, 93%) as a cream solid; 1H NMR dH
(300MHz; DMSO-d6) 11.23 (1H, s), 7.39 (1H, m), 7.04–
7.13 (2H, m), 5.53 (1H, s), 5.06 (2H, s), 2.22 (3H, s);
HRMS calcd for C12H10F2N2O2 253.0783 (M + H).
Found: 253.0780 (M + H).
11. Chen, C.; Wilcoxen, K.; Huang, C. Q.; Strack, N.;
McCarthy, J. R. J. Flourine Chem. 2000, 101, 285.
12. (a) The Hantzsch reaction proceeds via initial displace-
ment of bromine by the sulfur atom (of the thioamide/
urea), followed by condensation of nitrogen with the
carbonyl group: Babadjamian, A.; Gallo, R.; Metzger, J.
J. Heterocyclic Chem. 1976, 13, 1205; (b) Bredenkamp,
M. W.; Holzapfel, C. W.; Snyman, R. M.; van Zyl, W. J.
Synth. Commun. 1992, 22, 3029.
13. Typical experimental procedure for the reaction of
bromoketone 9 with thioamides: Synthesis of (R)-3-[2-(2-
amino)phenethyl]-5-[2-(4-chlorophenyl)thiazol-4-yl]-1-
(2,6-difluorobenzyl)-6-methyluracil trifluoroacetate 11j. A
5. (a) Zhu, Y.-F.; Gross, T. D.; Guo, Z.; Connors, P. J., Jr.;
Gao, Y.; Tucci, F. C.; Struthers, R. S.; Reinhart, G. J.;

M. W. Rowbottom et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4967–4973
4973
stirred solution of bromoketone 9 (27mg, 0.046mmol) and
4-chlorothiobenzamide (10mg, 0.05mmol) in ethanol
(1mL) was heated at 80°C for 2h. The reaction mixture
was cooled and solvent was removed in vacuo. The residue
was redissolved in a mixture of dichloromethane (1mL)
and trifluoroacetic acid (1mL) and stirred at rt for 1h. The
solvent was removed in vacuo and purification via
preparative LC–MS afforded 11j (15mg, 48%) as a
colorless solid; 1H NMR dH (300MHz; CDCl3) 7.77–
7.80 (2H, m), 7.35–7.41 (5H, m), 7.17–7.32 (4H, m), 6.87–
6.93 (2H, m), 5.19 (2H, s), 4.52–4.61 (2H, m), 3.96 (1H,
m), 2.21 (3H, s); HRMS calcd for C29H23ClF2N4O2S
565.1271 (M + H). Found: 565.1269 (M + H).
6.53 (1H, s), 5.34 (1H, d, J = 16.5Hz), 5.27 (1H, d,
J = 16.5Hz), 4.25–4.44 (4H, m), 4.06 (1H, dd, J = 12.9,
4.2Hz), 2.27 (3H, s); HRMS calcd for C30H27F2N5O2S
560.1926 (M + H). Found: 560.1925 (M + H).
16. Perrin, M. H.; Haas, Y.; Rivier, J. E.; Vale, W. W. Mol.
Pharmacol. 1983, 23, 44.
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. All compounds were
assayed in at least three independent experiments.
18. Flanagan, C. A.; Fromme, B. J.; Davidson, J. S.; Millar,
R. P. Endocrinology 1998, 139, 4115.
19. The functional assay, run using a Fluorometric Imaging
Plate Reader (FLIPR), measured the inhibition of GnRH
(5nM) stimulated Ca2+ release. In this assay, compound
12c is a functional antagonist, with a measured IC50 = 116
( 14)nM (n = 3).
14. (a) Dubey, P. K.; Cheedarala, R. K.; Babu, B. Indian J.
Heterocyclic Chem. 2002, 12, 95; (b) Rudolph, J.; Theis,
H.; Hanke, R.; Endermann, R.; Johannsen, L.; Geschke,
F.-U. J. Med. Chem. 2001, 44, 619.
15. Typical experimental procedure for the reaction of
bromoketone 9 with ammonium thiocyanate and amines:
Synthesis of (R)-3-[2-(2-amino)phenethyl]-5-[2-(benzyl-
amino)thiazol-4-yl]-1-(2,6-difluorobenzyl)-6-methyluracil
10f. A stirred solution of bromoketone 9 (35mg, 0.06
mmol) and ammonium thiocyanate (10mg, 0.13mmol) in
ethanol (1mL) was heated at 80°C for 1h. Benzylamine
(200mg, 1.87mmol) was added and heating continued for
further 12h. The reaction mixture was cooled and the
solvent removed in vacuo. The residue was redissolved in a
mixture of dichloromethane (1mL) and trifluoroacetic
acid (1mL) and stirred at rt for 1h. The solvent was
removed in vacuo and purification via preparative TLC
20. Compound 13 was previously prepared by this group.
Unpublished results.
21. The calculated logP values for 2-methylthiophene, 2-
methylthiazol-4-yl and 2-aminothiazol-4-yl are approxi-
mately 2, 1 and 0.5, respectively. These calculated values
were obtained using ACD/Labs LogP database, version
6.0 (2002), Advanced Chemistry Development Inc.,
Toronto, Ontario, Canada (http://www.acdlabs.com).
22. Based on a docking study using a GnRH homology model
obtained from the crystal structure of b-rhodopsin, we
speculate that the C-5 substituent (of the uracil core) sits
close to the extracellular surface of the receptor between
TM domains 4 and 5. Unpublished results.
1
afforded 10f (7mg, 18%) as a colorless solid; H NMR dH
(300MHz; CDCl3) 7.22–7.45 (11H, m), 6.88–6.95 (2H, m),