Structure-activity relationships of 1,3,5-triazine-2,4,6-triones as human gonadotropin-releasing hormone receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2005.05.038

SAR studies of 1,3,5-triazine-2,4,6-triones as human gonadotropin-releasing hormone receptor antagonists resulted in potent compounds. The best compound from the series had a binding affinity of 2 nM.

Full text of DOI:10.1016/j.bmcl.2005.05.038

Bioorganic & Medicinal Chemistry Letters 15 (2005) 3685–3690
Structure–activity relationships of 1,3,5-triazine-2,4,6-triones
as human gonadotropin-releasing hormone receptor antagonists
Zhiqiang Guo,a,* Dongpei Wu,a Yun-Fei Zhu,a Fabio C. Tucci,a Collin F. Regan,a
Martin W. Rowbottom,a R. Scott Struthers,b Qiu Xie,b Shelby Reijmers,b
Susan K. Sullivan,c Yang Said and Chen Chena,*
aDepartment of Medicinal Chemistry, Neurocrine Biosciences, Inc., 12790 El Camino Real, San Diego, CA 92130, USA
bDepartment of Endocrinology, Neurocrine Biosciences, Inc., 12790 El Camino Real, San Diego, CA 92130, USA
cDepartment of Pharmacology, 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 15 February 2005; revised 9 May 2005; accepted 11 May 2005
Available online 13 June 2005
Abstract—SAR studies of 1,3,5-triazine-2,4,6-triones as human gonadotropin-releasing hormone receptor antagonists resulted in
potent compounds. The best compound from the series had a binding affinity of 2 nM.
ꢀ 2005 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 in and secreted by the hypothalamus in a
pulsatile manner, which interacts with specific GnRH
receptors within the anterior pituitary, releasing both
follicle-stimulating hormone (FSH) and luteinizing hor-
mone (LH) from this gland.1,2 FSH and LH, in turn,
stimulate the ovaries and testes to produce gonadal ste-
roids that act on the reproductive organs. Several repro-
ductive disease conditions such as endometriosis, uterine
fibroids and prostate cancer are caused by overstimula-
tion of the reproductive organs by the gonadal steroids,
and thus can be treated by suppression of the pituitary–
gonadal axis. This can be clinically achieved via activa-
tion or inhibition of the GnRH receptor and a number
of peptide agonists and antagonists are currently
approved for treating these disorders as represented by
leuprorelinꢁ and CetrotideTM,1 respectively.3,4
Thus, intensive efforts have been initiated in the develop-
ment of small molecule GnRH antagonists by many
laboratories.5–8 We have previously reported a class of
uracils as potent human gonadotropin-releasing hor-
mone (hGnRH) receptor antagonists exemplified by
(R)-1-(2,6-difluorobenzyl)-5-(3-methoxyphenyl)-3-{2-[N-
methyl-N-(2-pyridyl)methyl]aminopropyl}-6-methylura-
cil 19,10 (Fig. 1). In a recent letter,11 we reported the
development of a novel and convenient synthesis of
1,3,5-triazine-2,4,6-triones, and initial SAR studies
around this monocyclic nucleus as GnRH antagonists.
Here, we report the SAR in other regions of the core,
at the left-hand side represented by N-3 substitution
(see structure 2, Fig. 1) and the ÔsouthernÕ part of the
1,3,5-triazine-2,4,6-trione core (N-1).
The 1,3,5-triazine-2,4,6-trione core structure was synthe-
sized using a one-pot condensation procedure as
However, treatment with peptide agonists or antagonists
requires parenteral administration due to their poor oral
bioavailability. By contrast, small molecule GnRH
antagonists offer the potential of oral administration
and therefore could gain wider acceptance from patients
given the enhanced flexibility that oral dosaging offers.
*
Corresponding authors. Tel.: + 1 858 617 7657; fax: + 1 858 617
7619; e-mail: zguo@neurocrine.com
URL: http://www.neurocrine.com
Figure 1. Structures of small molecule GnRH antagonists.
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.05.038

3686
Z. Guo et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3685–3690
described in our previous paper.11 In our effort to opti-
mize this series of compounds, we first utilized readily
available a- and b-amino alcohols to attach substituents
at the 3-position via the Mitsunobu reaction, while
keeping constant the 3-methoxyphenyl or 2-fluoro-3-
methoxyphenyl group at the 5-position and the 2,6-
difluorobenzyl group at the 1-position. Compounds 9
and 10 were synthesized according to the procedure de-
picted in Scheme 1. Thus, 3-methoxyphenyl isocyanate
(3a) was treated with 1 equiv of 2,6-difluorobenzylamine
in dichloromethane to form a urea intermediate, to
which 1.5 equiv of chlorocarbonyl isocyanate was subse-
quently added to generate the disubstituted triazinetri-
one compound 4a in 87% yield. Compound 4b was
prepared in a similar manner from 2-fluoro-3-methoxy-
phenyl isocyanate prepared from 2-fluoro-3-methoxy-
benzoic acid via a Curtis reaction.12 Boc-protected
a-amino alcohols that are unavailable from commercial
sources were prepared from the corresponding a-amino
acids by a lithium aluminum hydride (LAH) reduction,
followed by Boc protection in a one-pot procedure.
b-Amino acids 6 were readily prepared from aldehydes
Scheme 1. Reagents and conditions: (a) (1) 2,6-difluorobenzylamine, dichloromethane, 2 h; (2) ClCONCO, DCM, rt, 12 h; (b) malonic acid,
ammonium acetate, EtOH, reflux, 16 h; (c) LAH/THF, then Boc2O/DCM; (d) PPh3, di-t-butyl-azodicarboxylate, THF, 5 h; (e) TFA/CH2Cl2 (1:1),
1 h.
Scheme 2. Reagents and conditions: (a) MsCl, NEt3, dichloromethane, 2 h; (b) NaN3, DMF, 80 ꢂC, 8 h; (c) LiAlH4, THF, 10 h; (d) (1) 2-fluoro-
3-methoxyphenyl isocyanate, dichloromethane, 2 h; (2) ClCONCO, DCM, room temperature, 12 h; (e) R2OH, PPh3, DEAD, THF, 5 h; (f) alkyl
halide, K2CO3, DMF, 12 h; (g) TFA, dichloromethane, 1 h.

Z. Guo et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3685–3690
3687
Table 1. Binding affinities of compounds 2, 9, and 10 on the hGnRH
receptor15
Table 1 (continued)
Compound
X
H2N–Z–CH(R1)–Y–CH2– Chirality Ki (nM)
10i
H
RS
S
28
24
10j
F
F
Compound
X
H
H2N–Z–CH(R1)–Y–CH2– Chirality Ki (nM)
10k
R
1500
2
R
37
120
9
5, via reaction with malonic acid and ammonium acetate
in refluxing ethanol in 40–80% yields.13 These intermedi-
ates were converted to the b-amino alcohols 7 by LAH
reduction, followed by Boc-protection in THF. These
alcohols were then coupled with 1,3,5-triazine-2,4,6-tri-
ones (4) using Mitsunobu conditions, followed by
deprotection of the Boc-group with TFA in dichloro-
methane (1:1, v/v) to afford the primary amines 9 and
10, respectively, in overall 70–90% isolated yields.
9a
H
F
RS
R
9b
9c
F
RS
RS
RS
RS
RS
RS
RS
RS
RS
RS
20
We were also particularly interested in varying the sub-
stituent at the 1-position of the 1,3,5-triazine-2,4,6-tri-
ones. An alternative synthetic approach (Scheme 2)
was developed to explore the SAR at this position while
keeping the (R)-2-amino-2-phenylethyl group at the 3-
position and the 2-fluoro-3-methoxyphenyl group at
the 5-position constant. Thus, (R)-N-Boc-phenylglycinol
8a was treated with methanesulfonyl chloride in dichlo-
romethane, followed by replacement with sodium azide
in DMF at 80 ꢂC to yield the azido compound 12 in 82%
yield. The azido intermediate was then reduced by
lithium aluminum hydride in THF to give the Boc-
protected diamino compound 13 in 96% yield. Subse-
quent urea formation with 2-fluoro-3-methoxyphenyl
isocyanate, followed by a cyclization with chlorocar-
bonyl isocyanate, gave the triazine-trione 14 in 65%
yield. Alkylation of 14 with alkyl halides in the presence
of potassium carbonate in DMF, followed by Boc-
deprotection, gave the corresponding tri-substituted tri-
azine-triones 15, which were isolated in 30–90% yields.
Alkyl alcohols could also be coupled to this position
under standard Mitusnobu conditions. Over 80 triazine-
triones were prepared using the chemistry described
and a wide variety of substitutions were explored. A
selection of these compounds is listed in Table 2.
9d
F
620
70
10a
10b
10c
10d
10e
10f
10g
10h
H
H
H
H
H
H
H
H
800
66
83
75
180
27
All synthesized compounds were evaluated for their abil-
ity to inhibit des-Gly10[125I-Tyr,5 DLeu,6 NMeLeu,7
Pro10-NEt]-GnRH radioligand binding to the cloned
hGnRH receptor stably expressed in HEK293 cells using
a 96-well filtration assay format as previously reported.14
The binding affinities of 9 and 10 for the hGnRH recep-
tor are summarized in Table 1. A 2-fluoro substituent at
the 3-methoxy phenyl group increased binding affinity 4-
fold, and 9b was the most potent compound in this sub-
series (Ki = 9 nM). We speculate that the 2-fluoro group,
580

3688
Z. Guo et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3685–3690
Table 2. Binding affinities of N-1-alkyl compounds 15 on the hGnRH
receptor15
being slightly larger than a proton, could impede the
free rotation of the 5-phenyl group and thus force the
phenyl ring into a perpendicular conformation with re-
spect to the triazine-trione core, which in turn, may be
the preferred orientation for interaction with the GnRH
receptor. Replacing the phenyl ring of the 3-side chain in
9b with an isobutyl group resulted in significant loss of
affinity as in 9d (Ki = 620 nM). Analogs derived from
b-amino alcohols 10 demonstrated interesting SAR.
The 3-amino-3-phenylpropyl analog 10a (racemic mix-
ture) exhibited similar binding affinity as 2. SAR on
the phenyl ring of the 3-amino-3-phenyl-propyl group
showed that the substituents and position of substitu-
tion had only limited impact on the binding affinity.
As for the heteroaromatic derivatives, the slightly elec-
tron-rich thiophene compound has a Ki of 27 nM
(10g), while the 3-pyridyl analog exhibited poor binding
affinity (Ki = 580 nM). Interestingly, when the phenyl
ring was moved away from the amino group, the corre-
sponding compound 10i was slightly more potent than
10a. The fact that 2, 10a, and 10i had similar binding
affinities indicates that both the amino and phenyl
groups have certain degree of freedom for optimal bind-
ing interaction with the receptor.
Compound
R2
Ki (nM)
9b
9
15a
15b
>10,000
120
15c
15d
15e
29
20
11
Results from the next study clearly indicate that the
ÔsouthernÕ substituent of the triazine-trione core 15 par-
ticipates in a key binding interaction with the hGnRH
receptor. Alkyl substituents on the core 15 as illustrated
by 15a (Table 2) resulted in compounds with low affinity
for the receptor. For compounds containing a substi-
tuted phenyl ring, SAR studies indicate that an elec-
tron-deficient ring was preferred. Compound 15b with
an electron-donating 2-methoxy group was four times
less potent than the 2-methyl analog (15c, Ki = 29 nM).
Compound 15g with the 2-(trifluoromethoxy)benzyl
group was approximately 6-fold more potent than 15b.
Interestingly, the addition of a second substituent at po-
sition 6 of the 2-fluoro-benzyl ring further improved
binding affinity (15h–15j). Compound 15j proved to be
the most potent 1,3,5-triazine-2,4,6-trione from this
study (Ki = 2 nM). Introduction of a methyl group at
the benzylic position (15k, Ki = 630 nM) resulted in sig-
nificant loss of affinity, presumably due to increased ste-
ric hindrance which forces the 2,6-difluorobenzyl ring
out of the optimal binding configuration. Moreover,
bulky 2,6-disubstitutions on the benzyl ring were disfa-
vored, thus, 15l and 15m had decreased binding affinity
than 15d. This result again indicates that the relative po-
sition of the benzyl ring is very important for receptor
binding.
15f
13
18
15g
15h
15i
5
3
15j
15k
15l
2
630
36
Functional activity of a few selected compounds for the
hGnRH receptor was determined by inhibition of
GnRH stimulated inositol phosphate accumulation in
RBL cells.16 The results shown in Table 3 indicate that
they are all potent functional antagonists. For example,
compound 15j exhibited a Ki value of 2 nM on hGnRH
receptor binding, and an IC50 value of 33 nM in the ino-
sitol phosphate accumulation assay. These compounds
also displayed species difference in their binding to the
GnRH receptors.9 They exhibited reduced binding affin-
ity on the monkey GnRH receptor and much lower
15m
15n
73
66

Z. Guo et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3685–3690
3689
Table 3. Data from hGnRH receptor functional assay, binding affinities to the rats and monkey GnRH receptors, human liver microsomes (HLM)
stability and CaCo-2 permeability determination for selected compounds 1515
Compound
Ki (nM)
IC50 (nM)
Cl (HLM) (mL/min/kg)
Caco-2 permeability (Papp · 10À6, cm/s)
Human
Monkey
Rat
15f
15g
15j
13
18
2
86
240
24
13,000
9,400
2,300
190
480
33
249
146
158
25.6
6.2
13.9
7. Ashton, W. T.; Sisco, R. M.; Kieczykowski, G. R.; Yang,
Y. T.; Yudkovitz, J. B.; Cui, J.; Mount, G. R.; Ren, R. N.;
Wu, T. J.; Shen, X.; Lyons, K. A.; Mao, A. H.; Carlin, J.
R.; Karanam, B. V.; Vincent, S. H.; Cheng, K.; Goulet,
M. T. Bioorg. Med. Chem. Lett. 2001, 11, 2597, and
references cited therein.
affinity on the rat GnRH receptor. For example, com-
pound 15j exhibited 10- and 1000-fold lower binding
affinity on the monkey and rat GnRH receptors, respec-
tively (Ki = 24 nM and 2.3 lM), when compared to the
hGnRH receptor (Ki = 2 nM).
8. Sasaki, S.; Cho, N.; Nara, Y.; Harada, M.; Endo, S.;
Suzuki, N.; Furuya, S.; Fujino, M. J. Med. Chem. 2003,
46, 113.
Selected compounds from these novel triazine-triones
were also assayed for cell permeability in Caco-2 cells
and metabolic stability in human liver microsomes
(HLM). The apparent permeability (Papp) of 15f, 15g,
and 15j was determined across Caco-2 cell monolayers
in both the apical (AP) to basolateral (BL) and BL to
AP directions. These three compounds demonstrated
medium (15g, Papp a>b 6.2 · 10À6 cm/s) to very good
(15f, Papp a>b 25.6 · 10À6 cm/s) Papp values in this assay.
Incubation of 15g and 15j with HLM resulted in an
intrinsic clearance of 146 and 158 mL/min/kg, respec-
tively (Table 3). From this assay, these compounds
proved to be much more metabolically stable than 1.9
9. Guo, Z.; Zhu, Y.-F.; Gross, T. D.; Tucci, F. C.; Gao, Y.;
Moorjani, M.; Connors, P. J., Jr.; Rowbottom, M. W.;
Chen, Y.; Struthers, R. S.; Xie, Q.; Saunders, J.; Reinhart,
G.; Chen, T. K.; Bonneville, A. L. K.; Chen, C. J. Med.
Chem. 2004, 47, 1259, and references cited therein.
10. (a) Rowbottom, M. W.; Tucci, F. C.; Zhu, Y.-F.; Guo, Z.;
Gross, T. D.; Reinhart, G. J.; Xie, Q.; Struthers, R. S.;
Saunders, J.; Chen, C. Bioorg. Med. Chem. Lett. 2004, 14,
2269; (b) Tucci, F. C.; Zhu, Y.-F.; Guo, Z.; Gross, T. D.;
Connors, P. J., Jr.; Gao, Y.; Rowbottom, M. W.;
Struthers, R. S.; Reinhart, G.; Xie, Q.; Chen, T. K.;
Bozigian, H.; Bonneville, A. L. K.; Fisher, A.; Jin, L.;
Saunders, J.; Chen, C. J. Med. Chem. 2004, 47, 3483.
11. Guo, Z.; Wu, D.; Zhu, Y.-F.; Tucci, F. C.; Pontillo, J.;
Saunders, J.; Xie, Q.; Struthers, R. S.; Chen, C. Bioorg.
Med. Chem. Lett. 2005, 15, 693.
In conclusion, we have prepared a series of novel 1,3,5-
triazine-2,4,6-triones as potent antagonists of the
hGnRH receptor. SAR study at the left-hand side dem-
onstrated that branched primary amines, such as the
substituted 2-aminoethyl- or 3-aminopropyl groups
were a key feature for high binding affinity. SAR study
around the bottom of the 1,3,5-triazine-2,4,6-trione core
led to the discovery of analogs with better binding affin-
ity than the 2,6-difluorobenzyl compounds.
12. Naito, R.; Takeuchi, M.; Morihira, K.; Hayakawa, M.;
Ikeda, K.; Shibanuma, T.; Isomura, Y. Chem. Pharm.
Bull. 1998, 46, 1286.
13. Cardillo, G.; Gentilucci, L.; Tolomelli, A.; Tomasini, C.
J. Org. Chem. 1998, 63, 2351.
14. Perrin, M. H.; Haas, Y.; Rivier, J. E.; Vale, W. W. Mol.
Pharmacol. 1983, 23, 44.
15. 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 three to eight independent experiments.
16. Functional activity of the compounds for the hGnRH
receptor was determined by inhibition of GnRH stimu-
lated inositol phosphate accumulation. RBL cells stably
expressing the full-length hGnRH receptor were plated
onto 96 well plates (Corning) at 100,000 cells per well in
100 lL of inositol free medium (inositol free DMEM
supplemented with 10 % dialyzed fetal bovine serum,
2 mM L-glutamine, 10 mM HEPES, 50 lg/mL penicillin/
streptomycin, and 0.5 mg/ml G418) containing 1 lCi/mL
of [3H]myo-inositol and grown overnight. The following
day, cells were washed twice with buffer I (140 mM NaCl,
4 mM KCl, 20 mM HEPES, 0.1% BSA, 8.3 mM D-
glucose, 1 mM MgCl2, and 1 mM CaCl2, pH 7.4). The
appropriate dilutions of antagonists were prepared in
4 nM GnRH diluted in buffer I containing 10 mM LiCl.
Simultaneous agonist/antagonist stimulation was carried
out in the presence of LiCl for 1 h at 37 ꢂC. After
stimulation, the medium was removed and 200 lL of
10 mM formic acid was added to each well and incubated
at 4 ꢂC for 30 min to extract the inositol phosphates from
References and notes
1. Huirne, J. A. F.; Lambalk, C. B. Lancet 2001, 358, 1793.
2. (a) Matsuo, H.; Baba, Y.; Nair, R. M. G.; Arimura, A.;
Schally, A. V. Biochem. Biophys. Res. Commun. 1971, 43,
1334; (b) Amoss, M.; Burgus, R.; Blackwell, R.; Vale, W.;
Fellows, R.; Guillemin, R. Biochem. Biophys. Res. Com-
mun. 1971, 44, 205.
3. Fujino, M.; Fukuda, T.; Shinagawa, S.; Kobayashi, S.;
Yamazaki, I.; Nakayama, R.; Seely, J. H.; White, W. F.;
Rippel, R. H. Biochem. Biophys. Res. Commun. 1974, 60,
406.
4. Goulet, M. T. Gonadotropin Releasing Hormone
Antagonists. In Bristol, J. A., Ed.; Annual Reports in
Medicinal Chemistry; Academic: New York, 1995; Vol.
30, p 169.
5. Cho, N.; Harada, M.; Toshihiro, I.; Imada, T.; Matsu-
moto, H.; Hayase, Y.; Sasaki, S.; Furuya, S.; Suzuki,
N.; Okubo, S.; Ogi, K.; Endo, S.; Onda, H.; Fujino, M.
J. Med. Chem. 1998, 41, 4190.
6. Jiang, J.; DeVita, R. J.; Goulet, M. T.; Wyvratt, M. J.; Lo,
J.-L.; Ren, N.; Yudkovitz, J. B.; Cui, J.; Yang, Y. T.;
Cheng, K.; Rohrer, S. P. Bioorg. Med. Chem. Lett. 2004,
14, 1795, and references cited therein.

3690
Z. Guo et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3685–3690
the cells. The samples were transferred to 96-well What-
ammonium formate and 0.1 M formic acid into solid
scintillant coated, 96-well microplates (Perkin-Elmer) and
after drying the plates were counted in a TopCount NXT
(Perkin-Elmer). IC50 values for the inhibition of GnRH
stimulated inositol phosphate accumulation were calcu-
lated using the Prism software package (GraphPad Soft-
ware) with a Ôsigmoidal dose–response (variable slope)Õ
option for curve fitting.
man GF/C Unifilter Microplates (Whatman) packed with
1:10 w/v AG-1X8 resin in dH2O (Bio-Rad Laboratories).
The plates were centrifuged at a speed of 1500 rpm for
3 min and the flow through was removed. The resins were
washed once with 200 lL dH2O and then once with
200 lL of 60 mM ammonium formate and 5 mM sodium
tetraborate. IP, IP2, and IP3 were eluted with 50 lL of 1 M