Zwitterionic uracil derivatives as potent GnRH receptor antagonists with improved pharmaceutical properties

Article abstract of DOI:10.1016/j.bmcl.2008.07.059

A novel series of potent zwitterionic uracil GnRH antagonists were discovered that showed reduced liability for CYP3A4 enzyme inhibition.

Full text of DOI:10.1016/j.bmcl.2008.07.059

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4503–4507
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Zwitterionic uracil derivatives as potent GnRH receptor antagonists
with improved pharmaceutical properties
Colin F. Regan a, Zhiqiang Guo a, Yongsheng Chen a, Charles Q. Huang a, Mi Chen a, Wanlong Jiang a,
Jaimie K. Rueter a, Timothy Coon a, Chen Chen a, John Saunders a, Michael S. Brown b, Steve F. Betz b,
R. Scott Struthers b, Chun Yang c, Jenny Wen c, Ajay Madan c, Yun-Fei Zhu a,
*
a Department of Medicinal Chemistry, Neurocrine Biosciences, Inc., 12780 El Camino Real, San Diego, CA 92130, USA
b Department of Endocrinology, Neurocrine Biosciences, Inc., 12780 El Camino Real, San Diego, CA 92130, USA
c Department of Preclinical Development, Neurocrine Biosciences, Inc., 12780 El Camino Real, San Diego, CA 92130, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 19 March 2008
Revised 9 July 2008
Accepted 14 July 2008
Available online 17 July 2008
A novel series of potent zwitterionic uracil GnRH antagonists were discovered that showed reduced
liability for CYP3A4 enzyme inhibition.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Gonadotropin-releasing hormone
Antagonist
Non-peptide
Uracil
Zwitterions
CYP3A4
The potential of non-peptide Gonadotropin-releasing hormone
(GnRH) receptor antagonists serving as novel therapeutics for hor-
mone-dependent disease states such as prostate cancer, endome-
triosis, and benign prostate hyperplasia has led to discovery of a
wide range of small molecule antagonists.1,2 We have reported that
uracil-based analogs are potent GnRH receptor antagonists based
on in vitro and in vivo characterizations.3 However, CYP3A4 inhibi-
tion was a common issue for early uracil analogs that contained a
basic amine such as 1 in Figure 1. Since inhibition of CYP3A4 en-
zyme is well known to potentially induce drug–drug interactions,
elimination of such undesired property from this class of molecules
was clearly necessary. Recently, we have shown4 that addition of
an acid can drastically reduce the possibility of this class of mole-
cules to inhibit CYP3A4 enzyme activity regardless of the location
of the acid. However, the GnRH receptor-binding affinity was heav-
ily dependent on the exact location of this acidic functional group.
For example, the acid linked to the phenyl group at the right hand
side of the molecule (2b) diminishes the GnRH receptor-binding
affinity compared to its ester precursor (2a), yet the acid attached
to the amine group through a propylene chain (3b) is highly potent
GnRH receptor binder. As a matter of fact, such combination of the
amino and acid functionalities offered similar potency to its amine
precursor 1, yet without the CYP3A4 liability. Interestingly, such
zwitterionic molecules show good oral bioavailability in cynomol-
gus monkeys, albeit relatively poor exposure in rats.4 To further ex-
pand the SAR on zwitterionic uracils, we report here a novel way of
linking an acid group to the 5-phenyl uracils, which generated a
series of novel and potent zwitterionic molecules without
inhibition of CYP3A4 enzyme.
The initial syntheses of such molecules are outlined in Scheme
1. The starting compounds (4a–d)3,4 were first treated with BBr3 to
remove the methyl group; subsequently, the amino group was pro-
tected using Boc2O to give compounds 5a–d. Alkylation with
Br(CH2)nCO2Et, followed by hydrolysis of the ethyl ester and re-
moval of the Boc protecting group, yielded the desired zwitterionic
compounds 6a, 6b, 7a–c, 8a–c, 8e, and 9a. Compound 8d was pre-
pared alternatively according to Scheme 2 where 5c was first alkyl-
ated with 3-bromopropanol, followed by the oxidation of the
hydroxyl group to the corresponding carboxylic acid functionality
and then removal of the Boc-protecting group. These compounds
were assayed against the human GnRH receptor binding, IP3 func-
tion, and CYP3A4 inhibition.6 The results are summarized in Table
1. Our previous SAR has indicated that polar group cannot be toler-
ant around the 3-methoxylphenyl region at 5-uracil, thus our cam-
paign to introduce the acid functionality on 3-methoxyphenyl
* Corresponding author. Tel.: +1 858 617 7745; fax: +1 858 617 7919.
E-mail address: fzhu@neurocrine.com (Y.-F. Zhu).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.07.059

4504
C. F. Regan et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4503–4507
O
O
N
R
H
O
N
H2N
O
N
F
F3C
O
2a: R=Et, Ki = 110 nM (hGnRH-R)
IC50=0.35 μM (CYP3A4)
F
O
N
2b: R=H, Ki = 3900 nM (hGnRH-R)
N
IC50= 36 μM (CYP3A4)
H2N
O
F
O
F
O
F3C
N
HN
1: Ki = 0.6 nM (hGnRH-R)
IC50= 0.1 μM (CYP3A4)
O
N
F
O
R
F3C
O
3a: R=Me, Ki = 32 nM (hGnRH-R
IC50= 0.51 μM (CYP3A4)
3b: R=H, Ki = 1.2 nM (hGnRH-R)
IC50= 36 μM (CYP3A4)
Figure 1. Structures and biological activities of previously reported compounds 1–3.
HO
O
OH
F
O
F
( )n
X
O
Y
O
N
O
N
X
O
N
N
a, b
c, d, e
N
R2
O
N
R2 NH
N
O
R1
R2 NH
O
R1
O
R1
O
F
F
F
F
F
F
F
F
F
F
4a–d
5a–d
a: R1=H, R2=H, X=F
b: R1=CH3,R2=H, X=H
c: R1=CH3, R2=H, X=F
d: R1=CH3, R2=CH3, X=F
6a–b, 7a–b
8a–e, 9a
(see Table 1 for
detailed structures)
Scheme 1. Reagents and condition: (a) BBr3, DCM, À78 °C; (b) Boc2O, DCM, Et3N; (c) K2CO3, DMF, Br(CH2)nCO2Et; (d) LiOH, THF/water; (e) TFA/DCM.
activity (Ki = 34 nM); the longer acid 6b did not improve the activ-
ity. Both compounds, as we expected, did not exhibit significant
CYP3A4 inhibition. To search for an improvement on GnRH activ-
ity, we turned our attention to modify on more potent analogs
(4b–d). Indeed, compound 7a, 6-methyluracil analog based on
4b, was much more potent (Ki = 2.1 nM) than that of the non-
methyl analog 6a. However, shortening the chain length (7b and
7c) decreased the potency slightly. Historically, addition of a fluoro
group to the 3-methoxyphenyl ring of 4b further enhances the
GnRH activity. Therefore, compounds 8a–e were prepared accord-
ingly. Enhancement of the potency by fluoro group was not clearly
observed in the binding assay, but was well displayed in the func-
tional assay which measures the ability of a compound to inhibit
GnRH-stimulated [3H] inositol phosphate hydrolysis. Overall, flu-
oro analogs were about 5–10 times more potent than the corre-
sponding non-fluoro analogs (such as 8a and 8b vs 7a and 7b).
Because of their low possibility of CYP3A4 inhibition and potent
GnRH antagonistic activity, pharmacokinetic studies of several
O
O
F
OH
F
O
N
a, b ,c
N
5c
H2N
O
8d
F
F
F
Scheme 2. Reagents and condition: (a) K2CO3, DMF, BrCH2CH2CH2OH, 50 °C;
(b) cat. RuCl3, NaIO4, DCM, MeCN, H2O; (c) TFA/DCM.
position of compound 1 initialized with a long alkyl-acid such as
pentanoic acid (6a), which yielded moderate but encouraging

C. F. Regan et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4503–4507
4505
Table 1
SAR of the uracil ziwitterionic molecules
O
O
OH
( )n
O
O
F
OH
F
X
O
N
O
N
N
N
N
O
NH
R2
O
R1
F
F
F
F
F
F
F
9b
6a-9a
b
d
Compound
R1
R2
X
n
Kia (nM)
IC50 (nM)
CYP3A4 IC50 (lM)
6a
6b
7a
7b
7c
8a
8b
8c
8d
8e
9a
9b
H
H
H
H
H
H
H
H
H
H
H
H
CH3
—
F
F
H
H
H
F
F
F
F
F
4
5
5
4
3
5
4
3
2
1
4
—
34
33
2.1; 1.1
4.1
7.5
1.3; 1.2
1.2; 1.0
4.7
10.1
52
n.d.c
n.d.
2.8
26
23
36
25
n.d.
22
10
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
—
0.6
2.6
8.2
28
n.d.
n.d.
n.d.
n.d.
n.d.
21%e
n.d.
16%e
13
F
—
1.2
15
42
a
Ki values in italic were obtained from the binding assay in competition with [125I]Tyr5, -Leu6, NMeLeu7, and Pro-N-Et-GnRH using cloned human GnRH receptor that was
D
transfected on RBL cells, while others were obtained from HEK cells. The data from these two binding assays correlate well.
b
Inhibition of GnRH-stimulated [3H]inositol phosphate hydrolysis from human GnRH receptors stably transfected on RBL whole cells.
n.d., not determined.
Microtiter plate-based fluorimetric assay using recombinant CYP3A4 with 7-benzyloxy-4-(trifluoromethyl)coumarin (BFC)as the substrate.
Percentage of inhibition at 200 lM concentration.
c
d
e
compounds were conducted in rats (Table 2).7 The results indi-
cated that this class of molecules possessed low bioavailability in
rats, which was similar to the first reported class of zwitterionic
uracils such as 3b. But the terminal half-life of each compound
tested in this class was notably improved over 3b. The poor oral
bioavailability could be attributed to the possible low permeabil-
ity, as Caco-2 cell assay demonstrated that these compounds were
poorly permeable (e.g., Papp (a–b) = 0.1 Â 10À6 cm/s for both 7b and
8b). Since reducing hydrogen bond donors could improve perme-
ability, compounds 9a and 9b were designed and synthesized to
aim for maintaining the target potency with reducing hydrogen
bond donors. Synthesis of 9a was described in Scheme 1, while
9b was accomplished by a simple methylation from 9a (Scheme
3). Indeed, with addition of a methyl group on the basic amine,
the permeability improved in Caco-2 data (Papp(a–b) = 1.2 and
17 Â 10À6 cm/s, respectively, for 9a and 9b). Unfortunately, dime-
thylated 9b with better permeability was only moderately potent
(Ki = 15 nM), while mono-methylated 9a maintained the high po-
tency in GnRH binding assay. Thus, 9a was selected for pharmoc-
okinetic study in rats which demonstrated an improved oral
bioavailability (F = 13%) in rats over the primarily amine analog
8b (F = 6.6%).
Metabolism profiling of 9a in human hepatocytes implicated
that it partially produced a b-oxidative metabolite 10 from the
O-alkyl acid side chain as shown in Figure 2, which probably mim-
ics the fatty acid metabolism pathway.8 Since such unusual metab-
olism pathway gives rise to a speculative safety concern, an effort
to block such oxidation was undertaken. Our strategy was to use
heteroatom replacement and introduction of gem-dimethyl group
on one of the methylene group. The syntheses of these analogs
are described in Scheme 4. For nitrogen insertion, 5d reacted with
1,2-bromoethane and 1,3-dibromopropane to yield 11a and 11b,
()4
OH
Table 2
O
F
Pharmacokinetics of the selected zwitterionic uracils in rats
F
O
Pharmacokineticsa
Cl (iv) (ml/min. kg)
O
N
Compound
F (%)
t1/2 (iv) (h)
N
a
N
3b
7a
7b
8a
8b
9a
2.4
4.8
1.1
3.7
6.6
13
64
36
7.5
21
14
50
0.3
4.9
10
2.9
6.6
5.1
9a
O
F
F
F
a
pharmacokinetic studies in male Sprague–Dawley rats at 10 mg/kg (po) and
2.5 mg/kg (iv) except for 3b, where used 10 mg/kg doses were given orally and
intravenously.
9b
Scheme 3. Reagents: (a) formaldehyde in water, NaBH3CN, DCM.

4506
C. F. Regan et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4503–4507
O
O
O
F
OH
O
F
OH
F
F
O
N
O
N
hepatocytes
N
N
NH
NH
O
O
F3C
F3C
9a
10
Figure 2. b-Oxidation metabolism of 9a in human hepatocytes.
( )n
HO
NH
( )n
Br
O
O
F
N
O
F
R3
F
O
F
O
b, c, d
N
N
N
O
N
O
N
O
O
F
F
F
F
F
F
12a-b; 13a-b
(see Table 3 for
detailed structures)
11a, n=2
11b, n=3
a
e
HO
()n
()n
O
O
O
O
F
OH
O
F
F
O
F
O
O
N
N
f, d
N
N
NH
N
O
N
N
O
N
F
O
O
F
O
O
O
F
F
F
F
F
F
F
F
F
11c, n=2
11d, n=3
O
g, c, d
14, n=2
15, n=3
5d
HO
O
F
O
N
N
NH
O
F
F
F
F
16
Scheme 4. Reagents and condition: (a) K2CO3, Br(CH2)nBr, DMF; (b) R3NHCH2CO2Et, Et3N, DCM; (c) LiOH, THF/H2O; (d) 50% TFA in DCM; (e) CH2@CHCH2O(CH2)nOH, Ph3P,
t-BuO2CN@NCO2Bu-t, THF; (f) cat. RuCl3, NaIO4; (g) BrCH2CH2CMe2CH2CO2Et, K2CO3, DMF.
respectively. The bromo groups on 11a and 11b were then re-
placed with amino esters, followed by hydrolysis of the esters
and removal of the Boc protecting group to afford the desired prod-
ucts 12a and 12b, and 13a and 13b. Compound 5d was also alkyl-
ated with 2-allyloxy-ethanol and 3-allyloxy-propan-1-ol under
Mitsunobu condition to afford 11c and 11d. Oxidative cleavage of
the double bonds on 11c and 11d followed by removal of the
Boc-protecting group yielded the final acids 14 and 15. For the
gem-dimethyl analog, 5d was alkylated first with BrCH2
CH2C(CH3)2CH2CO2Et,9 followed by de-protection and hydrolysis
to produce 16. It turned out that all modifications on the alkyl side
chain reduced the binding affinity to the GnRH receptor as the data
summarized in Table 3.
In summary, attachment of an acid functionality to 5-phenyl-
uracil yielded potent zwitterionic GnRH receptor antagonists that
showed drastically reduced CYP3A4 inhibition. An adequate length
between the core and the acid was required only for the targeted
activity, not for blocking CYP3A4 interaction. Optimal length

C. F. Regan et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4503–4507
4507
Table 3
b-oxidation metabolism pathway led to the reduction of potency
for the molecules to bind to the GnRH receptor.
SAR of the zwitterionic uracils with modification on acid side chain
OH
( )
O
nY
Acknowledgments
F
O
O
N
Authors greatly appreciate Mr. Brock Brown and Ms. Qiu Xia for
their assistances in performing the assays for the receptor binding
and functional activity; Mrs. Teresa Dong, Mr. Zhi Yang, and
Mr. Michael Jones for performing assays related to CYP enzyme
inhibitions, metabolism profiling, and Caco-2 permeability, and
Mrs. Aixia Sun and Mrs. Hui Tang for the bio-analytic assays for
rat plasma samples.
N
NH
O
F
F
F
F
b
Kia (nM)
CYP3A4 IC50 (lM)
References and notes
Compound
n
Y
12a
12b
13a
13b
14
2
2
3
3
2
3
2
NH
N–CH3
NH
N–CH3
O
O
27
98
155
35
9.3
38
29
n.d.c
n.d.
33
n.d.
10
1. Chengavala, M. V.; Pelletier, J. C.; Kopf, G. S. Curr. Med. Chem.- Anti-cancer Agents
2003, 3, 399.
2. Betz, S. F.; Zhu, Y. F.; Chen, C.; Struthers, R. S. J. Med. Chem. 2008, 51, 3331.
3. Tucci, F. C.; Zhu, Y. F.; Struthers, R. S.; Guo, Z.; Gross, T. D.; Rowbottom, M. W.;
Acevedo, O.; Gao, Y.; Saunders, J.; Xie, Q.; Reinhart, G. J.; Liu, X. J.; Ling, N.;
Bonneville, A. K.; Chen, T.; Bozigian, H.; Chen, C. J. Med. Chem. 2005, 48, 1169.
4. Chen, Y.; Pontillo, J.; Guo, Z.; Wu, D.; Madan, A.; Chen, T.; Huang, C. Q.; Tucci, F.
C.; Rowbottom, M. W.; Zhu, Y. F.; Wade, W.; Saunders, J.; Struthers, R. S.;
Bozigian, B.; Chen, C. Bioorg. Med. Chem. Lett. 2008, 18, 3301.
5. Guo, Z.; Chen, Y.; Huang, C. Q.; Gross, T. D.; Pontillo, J.; Rowbottom, M. W.;
Saunders, J.; Struthers, S.; Tucci, F. C.; Xie, Q.; Wade, W.; Zhu, Y. F.; Wu, D.; Chen,
C. Bioorg. Med. Chem. Lett. 2005, 15, 2519.
15
16
22
22
C(CH3)2
a
Ki values were obtained from the binding assay in competition with [125I]Tyr5,
D
-Leu6, NMeLeu7, Pro-N-Et-GnRH using cloned human GnRH receptor transfected
on RBL cell line.
b
Microtiter plate-based fluorimetric assay using recombinant CYP3A4 with
6. Crespi, C. L.; Miller, V. L.; Penman, B. W. Anal. Biochem. 1997, 248, 188.
7. Pharmacokinetics and pharmacodynamics of 7a, also known as NBI-70631,
were further evaluated in monkeys and humans. The corresponding data will be
reported elsewhere.
7-benzyloxy-4-(trifluoromethyl)coumarin (BFC) as the substrate.
c
n.d., not determined.
8. Kim, J.-J.; Miura, R. Eur. J. Biochem. 2004, 271, 483.
9. The reagent was prepared according to the following scheme:
between the 5-phenyl on the core and the acid group is –O(CH2)4–
and –O(CH2)5–. Oral bioavailability of such zwitterion molecules
was improved through a simple mono-methylation on the basic
amine moiety. Despite the flexible nature of the O-alkyl acid side
chain, modifications aiming for elimination of the unusual
O
O
a, b
Br
O
O
a) BH3, THF; b) HBr
.