Synthesis of two non-peptidyl GnRH receptor antagonists via [ 14C]carbonylation

Article abstract of DOI:10.1002/jlcr.733

In support of a program to develop a new gonadotropin releasing hormone (GnRH) receptor antagonist, two ¹⁴C labelled candidate tracers, ¹⁴C-1 and ¹⁴C-2, were synthesized for utilization in metabolism studies. A slight modi

Full text of DOI:10.1002/jlcr.733

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2003; 46: 993–1000.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.733
Research Article
Synthesis of two non-peptidyl GnRH receptor
antagonists via [14C]carbonylation
Charles S. Elmore1,*, Dennis C. Dean1, Robert J. DeVita2 and
David G. Melillo1
1 Department of Drug Metabolism, Merck Research Laboratories, RY80R-104,
PO Box 2000, Rahway, NJ 07065, USA
2 Department of Basic Chemistry, Merck Research Laboratories, RY121E-22,
PO Box 2000, Rahway, NJ 07065, USA
Summary
In support of a program to develop a new gonadotropin releasing hormone
(GnRH) receptor antagonist, two 14C labelled candidate tracers, 14C-1 and
14C-2, were synthesized for utilization in metabolism studies. A slight
modification of the Medicinal Chemistry route for the synthesis of the
antagonists provided iodide 4. Palladium (0) catalyzed [14C]carbonylation of 4
proceeded in good chemical yield to afford acid 14C-3 which served as a com-
mon precursor to 14C-1 and 14C-2. Copyright # 2003 John Wiley & Sons, Ltd.
Key Words: [14C]carbon monoxide; GnRH receptor antagonist; [14C]carbony-
lation
Introduction
Gonadotropin releasing hormone (GnRH) is a decapeptide secreted by
the hypothalamus which binds to GnRH receptors in gonadotropic cells
of the pituitary gland; this binding activates the receptor and causes a
G-protein coupled cascade that results in the release of follicle
stimulating hormone (FSH) and luteinizing hormone (LH).1 LH
*Correspondence to: C.S. Elmore, Department of Drug Metabolism, Merck Research Labora-
tories, RY80R-104, PO Box 2000, Rahway, NJ 07065, USA. E-mail: charles elmore@merck.com
Copyright # 2003 John Wiley & Sons, Ltd.
Received 28 March 2003
Revised 13 May 2003
Accepted 15 May 2003

994
C.S. ELMORE ET AL.
primarily regulates gonadal steroid biogenesis (estrogen and progester-
one in females and androgen in males) while FSH stimulates
spermatogenesis in males and follicular ripening in females. GnRH
receptor antagonist and agonists have shown clinical efficacy for the
treatment of several disease states including prostate cancer, breast
cancer and endometriosis.2 Chloroquinolones 1 and 2 were identified as
potent and selective antagonists of the human GnRH receptor.3,4 In
order to perform preliminary drug metabolism and distribution studies,
14C labelled tracers of each were required.
NH
NH
S
N
O
O
N
O
O
N
N
N
N
H
H
Cl
N
H
Cl
N
H
O
O
1
2
Results and discussion
DeVita et al. have reported the synthesis of quinolone 1 in 10 steps from
methyl 2-(acetylamino)-4-chlorobenzoate3; however, this synthesis was
not readily amenable to late stage incorporation of 14C into the central
core of the molecule. A simple modification of this route and analogous
to that reported by Walsh5 would provide iodide 4 in three steps from 5
(Scheme 1). A [14C]carbonylation of 4 could be used to access 14C-3, a
common labelled intermediate for synthesis of both 14C-1 and 14C-2.
NH
NBoc
O
*
O
O
I
CO2Me
NHAc
I
X
Cl
Cl
N
H
O
Cl
N
H
O
4
5
H
N
14C-1
X=
N
S
N
H
N
N
14C-2
14C-3
N
* denotes 14C label
HO
Scheme 1. Retrosynthetic analysis of GnRH receptor antagonists 14C-1 and
14C-2
Copyright # 2003 John Wiley & Sons, Ltd.
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[
14C]CARBONYLATION LABELLING
995
In order to prepare the iodo compound 4, a four-step sequence shown
in Scheme 2 was used. Methyl 2-(amino)-4-chlorobenzoate was
regioselectively iodinated with silver sulfate and iodine to give aniline
5 which was coupled with 3,4,5-trimethylphenylacetyl chloride in 1,
2-dichloroethane to give amide 6 in 88% yield over 2 steps. Lithium
hexamethyldisilazide initiated cyclization of 6 provided 4-hydroxy-
quinolone 7, and Mitsunobu coupling of 7 and azetidine 8 gave iodide 4
in 54% overall yield from methyl 2-(amino)-4-chlorobenzoate.
Palladium (0) catalyzed [14C]carbonylation of 4 in the presence of
KOAc using ca. 2.7 equivalents of 14CO generated from lithium
[14C]formate6 gave carboxylic acid 14C-3 in 87% chemical yield (32%
radiochemical yield, Scheme 3). An EDC-catalyzed coupling of 14C-3
with either 4-aminopyrimidine or 2-aminothiazole followed by depro-
tection of the Boc group with trifluoroacetic acid provided 14C-1 and
14C-2 respectively. Purification was effected by preparative HPLC to
give 150 mCi of 14C-1 and 190 mCi of 14C-2.
The syntheses of these 14C labelled tracers using a Pd-catalyzed
[14C]carbonylation of an aryl halide precursor as the key label
incorporating step demonstrates the utility of this reaction for late
stage label incorporation.
Experimental
General
Ba14CO3 was obtained from New England Nuclear. [1,10-Bis(di-
phenylphosphino)ferrocene]dichloro-palladium(II) (Pd(dppf)2Cl2) was
O
I
CO2Me
NH
I
CO2Me
NH2
Cl
CO2Me
NH2
Ag2SO4, I2,
EtOH
Cl
Cl
Cl
ClCH2CH2Cl, 80ºC
88%
O
100% (crude)
5
6
Boc
N
Boc
N
OH
8
OH
I
LiN(SiMe3)2,
THF, 0ºC
O
I
PPh3, DEAD, THF
64%
Cl
N
H
O
Cl
N
O
H
96%
7
4
Scheme 2. Synthesis of iodide 4
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996
C.S. ELMORE ET AL.
NBoc
NBoc
Pd(dppf)2Cl2, KOAc,
RNH2, EDC,
O
O
HO214C
Cl
I
14CO, DMSO, 65ºC
87%
DMAP, CH2Cl2
Cl
N
H
O
N
H
O
4
14C-3
NBoc
NH
O
*
O
1. CF3CO2H, Anisole, CH2Cl2
2. Preparative HPLC
O
*
O
RHN
RHN
Cl
N
H
Cl
N
O
O
H
14C-9 (56%)
14C-1 (38%)
R=
R=
N
S
N
N
N
N
N
14C-10 (41%)
S
N
14C-2 (46%)
* denotes 14C label
N
Scheme 3. Synthesis of GnRH receptor antagonist tracer 14C-1 and 14C-2
obtained from Aldrich. Anhydrous solvents were obtained from Aldrich
(
and were dried over 4 A molecular sieves for at least 24 h prior to use
with the exception of THF and CH2Cl2 which were distilled from the
appropriate drying agents. 1H NMR spectra were recorded on a Varian
XL series 400 MHz spectrometer, and 13C NMR spectra were recorded
on a Varian U-400 spectrometer. Low resolution mass spectral analyses
were obtained with a LKB 9000 at an ionizing voltage of 70 eV. High
resolution mass spectral analysis was obtained using a Finnigan New
Star FT/ICR with electrospray ionization. LC/MS analyses were
performed on an HP MSD-100 using a XDB-C8 column, with 5–95%
gradient over 15 min with MeCN-2 mM ammonium formate buffer (pH
3.5) and electrospray ionization. Analytical HPLC was performed using
a Shimadzu HPLC system with LC-10ATVP pumps, a SPD-10AVP UV
detector, a CTO-10ASVP column oven heated to 408C, a SCL-10A
controller and a Packard RadiomaticTM 150TR flow monitor. The
radioactive products were identified by HPLC comparison with
unlabelled reference material using either method A (30–100%
MeCN–0.1% aqueous trifluoroacetic acid over 30 min, Zorbax SB C-
18), method B (23% MeCN-0.1% aqueous trifluoroacetic acid over
30 min, Zorbax SB C-18), method C (40–100% MeCN-0.1% aqueous
HClO4 over 30 min, Zorbax RX C-8), or method D (45% MeOH-0.1%
Copyright # 2003 John Wiley & Sons, Ltd.
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[
14C]CARBONYLATION LABELLING
997
aqueous HClO4 over 45 min, Zorbax RX C-8). All HPLC analyses were
conducted with the HPLC column heated to 308C and were concluded
with a 10 min wash of 100% organic. Normal phase column
chromatography was carried out utilizing silica gel 60 (E. Merck).
Methyl 2-amino-4-chloro-5-iodobenzoate (5): To a mixture of 6.8 g
(27 mmol) iodine and 8.4 g (27 mmol) silver (I) sulfate in 270 ml absolute
ethanol was added 5.0 g (27 mmol) methyl 2-amino-4-chlorobenzoate.
The resulting reaction mixture was stirred at room temperature for
45 min. The reaction mixture was filtered through a pad of Celite and the
solvent was removed under vacuum. The residue was dissolved in 400 ml
ethyl acetate and washed with saturated aqueous sodium bicarbonate
(3 ꢀ 50 ml), water (3 ꢀ 50 ml) and once with brine. The organic layer was
dried over magnesium sulfate, filtered and the filtrate concentrated under
vacuum to afford 8.5 g (ꢁ100%) of an off-white solid. 1H NMR
(CDCl3): d 3.85 (s, 3 H), 5.80 (s, 2 H), 6.80 (s, 1 H), 8.24 (s, 1 H). FAB-
MS: calculated for C8H7ClINO2 311; found 312 (M+H, 100).
Methyl 4-chloro-5-iodo-2-N-(3,4,5-trimethylphenyl)acetylaminobenzoate
(6): To a solution of 3.28 g (10.5 mmol) methyl 2-amino-4-chloro-5-
iodobenzoate (5) in 15 ml 1,2-dichloroethane was added 2.08 g
(10.5 mmol) 3,4,5-trimethylphenylacetyl chloride. The resulting reaction
mixture was heated at reflux for 4 hours. The reaction mixture was
cooled to room temperature and the solvent was removed under
vacuum. The resulting off-white solid was triturated in hot methanol.
The mixture was then cooled in an ice bath, solids were filtered, washed
with ice cold methanol and dried to afford 4.36 g (88%) of product as a
white solid. 1H NMR (CDCl3): d 2.14 (s, 3 H), 2.27 (s, 6 H), 3.62 (s, 2 H),
3.85 (s, 3 H), 6.98 (s, 2 H), 8.39 (s, 1 H), 8.94 (s, 1 H).
7-Chloro-4-hydroxy-6-iodo-3- (3,4,5-trimethyl) phenyl-1H-quinolin-2-one
(7): To a solution of 4.36 g (9.2 mmol) amide 6 in 5 ml dry
tetrahydrofuran under nitrogen atmosphere at 08C was added dropwise
via syringe 23.1 ml (23.1 mmol, 2.5 equivalent) of a solution of lithium
bis(trimethylsilyl)amide (1.0 M in THF). The resulting reaction mixture
was stirred at 08C for 1.5 h then acidified with 100 ml 6 N aqueous HCl/
ice (1:1). The resulting solids were stirred vigorously, filtered, washed
with ice cold water followed by ice cold acetonitrile. The resulting off-
white solid was dried in a vacuum oven at 508C for 16 h to afford 3.89 g
(96%) of the product. 1H NMR (DMSO-d6): d 2.14 (s, 3H), 2.24 (s, 6H),
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C.S. ELMORE ET AL.
3.85 (s, 3H), 6.93 (s, 2H), 7.37 (s, 1H), 8.43 (s, 1H). FAB-MS: calculated
for C20H18ClNO4 371; found 372 (M+H, 100).
4-(2-(N-t-Butoxycarbonylazetidin-2(S)-yl)ethoxy-7-chloro-6-iodo-3-
(3,4,5-trimethyl)phenyl-1H-quinolin-2-one (4): To a vigorously stir-
red solution of 2.52 g (12.5 mmol) N-t-butoxycarbonylazetidin-2(S)-
ethanol (8) in 50 ml dry tetrahydrofuran under nitrogen was added 6.6 g
(15 mmol) quinolin-2-one 7. To the resulting mixture was added 3.94 g
(15 mmol) of PPh3 then dropwise by syringe 2.43 ml (15 mmol)
diethylazodicarboxylate. The resulting mixture was stirred at room
temperature for 16 h at which time 50 ml silica gel was added to the
reaction mixture. The excess solvent was removed under vacuum to
provide a free flowing powder which was applied to the top of a
prepacked silica gel column. The column was eluted with hexanes/ethyl
acetate (65/35) to provide 4.97 g of the product contaminated with a
minor amount of diethyl N; N0-hydrazinedicarboxylate. 1H NMR
(DMSO-d6): d 1.30 (s, 9H), 1.63 (m, 1H), 1.73 (m, 1H), 2.03 (m, 2H),
2.16 (s, 3H), 2.25 (m, 6H), 3.61 (m, 4H), 4.03 (m, 1H), 7.00 (s, 2H), 7.46
(s, 1H), 8.22 (s, 1H), 11.85 (s, 1H) and 1.16 (t, J=7.1 Hz, 0.7H) and 4.02
(q, J=7.1 Hz, 0.5H) arising from diethyl N,N0-hydrazinedicarboxylate.
FAB-MS: calculated for C28H32ClIN2O4 622; found 623 (M+H, 100).
4-(2-[1-[(tert-butyl)oxycarbonyl]azetidin-2-yl]ethoxy)-7-chloro-2-oxo-3-
(3,4,5-trimethylphenyl)hydroquinoline-6-[14C]carboxylic acid (14C-3):
A three-necked flask containing ca. 20 mCi of LiO124CH (54 mCi/mmol,
0.36 mmol) generated from 74 mg (0.38 mmol, 54 mCi/mmol) of
Ba14CO3, 830 mg (2.99 mmol) of PbCl2, and 0.6 ml (0.6 mmol) of 1 M
LiBEt3H was connected to a vacuum manifold, a septum, and a round
bottom flask via a 908 bent adapter.6 The round bottom flask was
charged with iodide 4 (93 mg, 0.13 mmol), Pd(dppf)2Cl2 (5 mg,
0.007 mmol), KOAc (42 mg, 0.43 mmol), and DMSO (1 ml). Both flasks
were cooled in liquid nitrogen and evacuated to 0.05 mm Hg. The
apparatus was isolated from the vacuum, and the cooling baths
removed. Concentrated H2SO4 (10 ml) was added slowly through
the septum on the three-necked flask to the solid at 08C. The three-
necked flask was heated at 708C for 1 h, and the one-necked flask was
heated at 708C overnight after which the vacuum was released. The
solution was diluted with CH2Cl2 (20 ml) and 1% NaOH (20 ml). The
layers were separated and the organic layer was washed with NaOH
(2 ꢀ 20 ml). The aqueous layer was acidified with 1 M HCl and was
extracted with CH2Cl2 (3 ꢀ 20 ml). The organic layer was dried
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[
14C]CARBONYLATION LABELLING
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(MgSO4) and filtered to give 10.2 mCi which had 64% radiochemical
purity by HPLC (method A) (ca. 87% chemical yield; 32% radio-
chemical yield from Ba14CO3).
1[4-(2-(N-t-butoxycarbonylazetidin-2(S)-ylethoxy)-7-chloro-2-oxo-3-
(3,4,5-trimethylphenyl)(6-hydroquinolyl)]-N-pyrimidin-4-yl-[14C]car-
boxamide (14C-9): To a solution of acid 14C-3 (1.0 mCi, 0.018 mmol,
54 mCi/mmol), 4-aminopyrimidine (18 mg, 0.19 mmol), dimethylamino-
pyridine (5.4 mg, 0.04 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide hydrochloride (20 mg, 0.10 mmol) in CH2Cl2 (1 ml) was
stirred under N2 for 3 days. The solution was diluted with 4 ml of
CH2Cl2 and the organic solution was washed with water (2 ꢀ 2 ml),
dried (MgSO4), and filtered to give a solution containing 0.90 mCi. The
solution was concentrated to afford a yellow solid which was purified by
column chromatography using 80:20 EtOAc:hexane as the eluant to
give 0.56 mCi (95% radiochemical purity by HPLC method A, 56%
yield). LC/MS (M/Z, % abundance): 618 (13), 620 (100, M+1), 621
(36), 622 (38), 623 (12), 624 (2).
[4-(2-azetidin-2(S)-ylethoxy)-7-chloro-2-oxo-3-(3,4,5-trimethylphenyl)
(6-hydroquinolyl)]-N-pyrimidin-4-yl-[14C]carboxamide (14C-1): A so-
lution of 0.40 mCi of amide 14C-9 and 20 mL of anisole in 2 ml of
CH2Cl2 and 1 ml of CF3CO2H was stirred at rt under N2 for 1 h. HPLC
analysis (method A) showed the reaction to be complete; therefore, the
volatiles were removed by passing N2 gas over the solution. The residue
was taken up in 5 ml of EtOH, and the solution was determined to
contain 400 mCi (75% radiochemical purity by HPLC (method B)). The
solution was concentrated to dryness under a N2 stream, and the solid
was taken up in 500 mL of EtOH, purified by preparative HPLC
(21.2 ꢀ 250 mm Zorbax SB C-18, 23% MeCN-0.1% TFA, 20 ml/min),
and isolated by elution from an OasisTM HLB SepPak with MeOH to
afford 150 mCi. HPLC (method B) radiochemical purity 99.0%. LC/MS
(M/Z, % abundance): 518 (13), 520 (M+H, 100), 521 (32), 522 (34), 523
1
(11), 524 (2). H NMR (CD3OD): d 2.07 (m, 2H), 2.20 (m, 2H), 2.25
(s, 3H), 2.34 (s, 6H), 3.74 (m, 1H), 3.78 (m, 2H), 3.98 (q, 1H, J=
9.4 Hz), 4.42 (quintet, 1H, J=7.7 Hz), 7.08 (s, 2H), 7.49 (s, 1H), 8.11 (s,
1H), 8.34 (d, 1H, J=5.8 Hz), 8.71 (m, 1H), 8.89 (s, 1H). 13C NMR
(CD3OD): d 15.4, 20.7, 25.8, 34.7, 44.4, 60.1, 70.2, 111.9, 117.3, 117.5,
122.7, 125.9, 130.3, 130.8, 134.8, 136.6, 137.6, 140.8, 159.1, 159.4, 159.6,
161.0, 165.9.
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[4-(2-(N-t-butoxycarbonylazetidin-2(S)-ylethoxy)-7-chloro-2-oxo-3-(3,
4,5-tri-methylphenyl)(6-hydroquinolyl)]-N-(1,2,5thiadiazol-3-yl)[14C]car-
boxamide (14C-10): The procedure reported for 14C-9 was followed
using 1.0 mCi of acid [14C]-3 to give 0.62 mCi of 14C-10 which assayed at
66% radiochemical purity by HPLC (method C).
[4-(2-azetidin-2-ylethoxy)-7-chloro-2-oxo-3-(3,4,5-trimethylphenyl) (6-hydro-
quinolyl)]-N-(1,2,5thiadiazol-3-yl)[14C]carboxamide (14C-2): The procedure
reported for the synthesis of 14C-1 was followed using 0.62 mCi (66%
radiochemical purity) of 14C-10. Purification by preparative HPLC
(21.2 ꢀ 250 mm Zorbax RX C-8, 28% MeCN-0.1% HClO4, 20ml/min)
afforded 0.19 mCi of antagonist 14C-2 which was diluted with 3.6 mg of
unlabeled 2. HPLC analysis (method D) showed a 98.2% radiochemical
purity, and the specific activity of 24 mCi/mmol was calculated from liquid
scintillation counting and UV spectroscopic determination of concentration
at three different wavelengths.
Acknowledgements
We would like to thank Herb Jenkins and Dr Allen Jones for analytical
support on antagonist 14C-2.
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