Synthesis of doubly 13C-labelled antalarmin isotopomers for pharmacokinetic studies

Article abstract of DOI:10.1002/jlcr.576

Antalarmin (butyl-ethyl-[2,5,6-trimethyl-7-(2,4,6-trimethyl-phenyl)-7H-pyrro-lo[2, 3-d]pyrimidin-4-yl]-amine) was doubly labelled with carbon-13. The synthesized butyl-[¹³C₂]ethyl-[2,5,6-trimethyl-7-(2,4, 6-trimethyl-phenyl)-7H-pyrro

Full text of DOI:10.1002/jlcr.576

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2002; 45: 637–645.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.576
Research Article
13
Synthesis of doubly C-labelled antalarmin
isotopomers for pharmacokinetic studies
Elisabeth Greiner1, Arthur J. Atkinson Jr.2, Alejandro Ayala3,
George P. Chrousos4, Carlo Contoreggi5, William C. Eckelman6,
Philip W. Gold3, Kamal E. Habib3, Arthur E. Jacobson1,
Noel Whittaker7, Elizabeth L. Webster3,4y and Kenner C. Rice1*
1 Laboratory of Medicinal Chemistry, Building 8, Room B1-23, National
Institutes of Diabetes and Digestive and Kidney Diseases, National Institutes of
Health, 8 Center Drive, MSC 0815, Bethesda, MD 20892, USA
2 Office of the Director, Clinical Center, Building 10, Room 1C227, National
Institutes of Health, 10 Center Drive, MSC 1180, Bethesda, MD 20892-1504,
USA
3 Clinical Neuroendocrinology Branch, NIMH, National Institutes of Health,
10 Center Drive, MSC 1284, Bethesda, MD 20892, USA
4 Pediatric Endocrinology Section, PREB, National Institutes of Child Health
and Human Development, National Institutes of Health, 10 Center Drive,
MSC 1583, Bethesda, MD 20892, USA
5 Brain Imaging Branch, NIDA, National Institutes of Health, 5500 Nathan
Stock Drive, Baltimore, MD 21224, USA
6 PET Department, Clinical Center, Building 10, Room 1C495, National
Institutes of Health, 10 Center Drive, MSC 1180, Bethesda, MD 20892, USA
7 Laboratory of Bioorganic Chemistry, Building 8, Room B1-23, National
Institutes of Diabetes and Digestive and Kidney Diseases, National Institute of
Health, 8 Center Drive, MSC 0815, Bethesda, MD 20892 USA
Summary
Antalarmin (butyl-ethyl-[2,5,6-trimethyl-7-(2,4,6-trimethyl-phenyl)-7H-pyrro-
lo[2,3-d]pyrimidin-4-yl]-amine) was doubly labelled with carbon-13. The
*Correspondence to: K.C. Rice, Laboratory of Medicinal Chemistry, Building 8, Room B1-23,
National Institutes of Diabetes and Digestive and Kidney Diseases, National Institutes of Health,
8 Center Drive, MSC 0815, Bethesda, MD 20892, USA. E-mail: kr2lf@nih.gov.
yCurrent address: University of North Carolina, Chapel Hill, NC.
Copyright # 2002 John Wiley & Sons, Ltd.
Received 25 September 2001
Revised 17 January 2002
Accepted 30 January 2002

638
E. GREINER ET AL.
synthesized butyl-[13C2]ethyl-[2,5,6-trimethyl-7-(2,4,6-trimethyl-phenyl)-7H-
pyrrolo[2,3-d]pyrimidin-4-yl]-amine (1) and butyl-ethyl-[2-13C]-[2,5,6-tri-
methyl-7-(2,4,6-trimethyl-phenyl)-7H-pyrrolo[2,3-d]-[2-13C]
pyrimidin-4-yl]-
amine, (2) were prepared for use as substrates for pharmacokinetic studies.
These compounds were obtained in fair overall yield in a 5 and 6 step synthesis
(20–24.5%, respectively) and high isotopic purity (about 99 at% 13C). Copy-
right # 2002 John Wiley & Sons, Ltd.
Key Words: corticotropin-releasing hormone; CRHR1 antagonist; antalarmin;
carbon-13 labelling
1. Introduction
Corticotropin-releasing hormone (CRH) is a 41 amino acid peptide,
which acts as a major regulator of the hypothalamic–pituitary–adrenal
(HPA) axis and is a principal coordinator of stress response including its
neuroendocrine, autonomic, immune, and behavioral components.1–3 In
the central nervous system, CRH activates the sympathetic nervous
system like a neurotransmitter, thus modulating cardiovascular,
metabolic and stress-like behavioral effects associated with drug
withdrawal syndromes.4–9 Overproduction of CRH in the brain has
been connected with mental disorders such as anxiety, addiction, and
depression, for that reason CRH-antagonists are expected to become a
valuable new class of antidepressants.10–13 Competitive binding studies
with 125I-CRH have established that antalarmin and some of its
analogues display high affinity and are specific for type 1 CRH
receptors. In addition, antalarmin has been shown to block the in vivo
and in vitro biological actions of an CRHR1 agonist.14
13C-labelled isotopes have been used for the in vivo pharmacokinetic
evaluation of drug absorption and disposition.15,16 Stable isotopes
incorporating double 13C-labelling of the highly selective CRHR1
antagonist, antalarmin, were synthesized to explore the pharmacoki-
netic properties of this pharmacologically important compound. The
pharmacokinetic investigation of antalarmin would be helpful in several
ways: to determine the pharmacokinetics of the labelled compound in
the presence of endogenous antalarmin, to facilitate identification of
antalarmin metabolites, and to provide information about a preferred
route of administration. Regarding the latter, stable isotope labelled
compounds are particularly well suited for pharmacokinetic studies in
which a labelled isotopomer and the corresponding non-labelled
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 637–645

DOUBLY 13C-LABELLED ANTALARMIN
639
Scheme 1.
compound are administered simultaneously by different routes, e.g.,
intrathecal and i.v. This type of experiment allows examination of rate
and extent of compound transfer between the two sites, in this example
between spinal fluid and plasma. Doubly labelled compounds are more
useful for pharmacokinetic studies than singly labelled compounds in
that they provide better mass spectral separation of the labelled
compound from the non-labelled compound. In order to investigate the
pharmacokinetics of antalarmin, two labelled derivatives of antalarmin
were prepared, and each of these derivatives had their two 13C atoms in
different molecular positions that might be subject to differential
metabolic cleavage. These labels were placed in the molecular positions
seen in 1 (Scheme 1) and 2 (Scheme 2) because the synthetic route to
those compounds appeared to be the most facile and likely to be
achieved using available starting materials.
Results and discussion
Isotopomer 1 was synthesized as outlined in Scheme 1. The coupling
reaction of 317 with n-butylamine was carried out according to Chorvat
et al.18 and afforded compound 4 in 63% yield.
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640
E. GREINER ET AL.
Scheme 2.
The introduction of the doubly labelled N-ethyl group was achieved
in one step by alkylation of the secondary amine with 13C2-labelled ethyl
bromide to give compound 1. Since 13C2-acetyl chloride was less
expensive than 13C2-labelled ethyl bromide, we initially planned to
install the N-ethyl group by acetylation of 4 with 13C2-acetyl chloride
followed by reduction. However, only very low yields of desired product
were obtained from this acetylation reaction. In an effort to use only a
small excess of 13C2-acetyl chloride, the reaction never went to
completion.
In compound 2, two adjacent 13C atoms were incorporated into the
pyrrolo-pyrimidine backbone of the antalarmin molecule by the route
shown in Scheme 2.
Aminopyrrole 6 was synthesized in one step from 2,4,6-trimethylani-
line essentially according to a known reaction sequence.19 The critical
acetylation of 6 was attempted under various conditions, employing
different solvents, bases, and both acetyl chloride and acetic anhy-
dride.19 The best results were obtained using chloroform as a solvent,
pyridine as a base and acetyl chloride as alkylating agent. The doubly
13C-labelled compound 7 was obtained in high yield (95%). Cyclization
of 7 was carried out in 85% phosphoric acid to afford 4-oxopyrrolo-
pyrimidine derivative 8. Subsequent reaction with phosphorous
oxychloride produced compound 9, which in turn was coupled
with ethyl-n-butylamine to give the double 13C-labelled antalarmin
Copyright # 2002 John Wiley & Sons, Ltd.
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DOUBLY 13C-LABELLED ANTALARMIN
641
isotopomer 2. The identity of the two different isotopomers (1 and 2)
was unequivocally established by the methodology used in their
chemical synthesis, and NMR. The chemical shifts of the methyl group
on the pyrrolopyrimidine ring in 2, and the N-ethyl moiety in
isotopomer 1, were identical to the comparable atoms in antalarmin.
Further, the coupling pattern between the 13C atoms and an attached
proton proved the contiguity of the two 13C atoms in both isotopomers.
Mass spectral data indicated that isotopomers 1 and 2 were about
99 at% isotopically pure by examination of the M+-2 peak at m/z 378
(12C peak). The data were acquired by averaging three electric field
scans over a 40 Da range. Triplicate determinations were made for each
compound. The peak intensity at m/z 378 for the labelled compound
was about 0.33 times the peak intensity of a deliberately added sample
with an additional 2% of the 12C analog (which should, theoretically,
now contain 3 at% 12C). Also, it was found to have about 0.2 times the
peak intensity of a deliberately added sample with an additional 4% of
the 12C analogue (which theoretically should have contained about
5 at% of the 12C compound). Thus, we determined that the synthesized
material was of high isotopic purity, ca. 99 at% 13C.
Experimental
TLC analyses were carried out on Analtech silica gel GHLF 0.25 mm
plates with UV light and I2 detection. Melting points were measured in
open glass capillaries on a Thomas–Hoover melting point apparatus
1
and are uncorrected. H NMR spectra were recorded at 300 MHz on a
Varian Gemini spectrometer and chemical shifts are reported in ppm
relative to tetramethylsilane (TMS). The mass spectra for the
determination of the purity of 1 and 2 were obtained with a JEOL
SX-102 MS utilizing a direct exposure probe in the EI mode, using an
ionizing voltage of 70 V and a source temperature of 1508C ( ꢀ 158C);
all other compounds were examined in the CIMS mode, as noted.
Elemental analyses were performed by Atlantic Microlabs Inc.,
Norcross, GA. Acetyl-13C2 chloride and bromoethane-13C2 were
purchased from Aldrich. The purity of both was 99 at% 13C. The MS
of the labelled compounds were compared to the spectra of the
authentic unlabelled material to confirm their identity. The purity of
the labelled antalarmin isotopomers 1 and 2 was also determined
by MS.
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E. GREINER ET AL.
Butyl-[2,5,6-trimethyl-7-(2,4,6-trimethyl-phenyl)-7H-pyrrolo[2,3-d]pyr-
imidin-4-yl]-amine (4)
4-Chloro-2,5,6-trimethyl-7-(2,4,6-trimethyl-phenyl)-7H- pyrrolo[2,3-d]
pyrimidine (3) (5.00 g, 15.9 mmol) and butylamine (7.85 ml, 80.0 mmol)
were dissolved in DMSO (35 ml) and heated at 1358C under argon, until
TLC (eluent: EtOAc/hexane, 2:8) showed no starting material. The
mixture was cooled and extracted between H2O and petroleum ether
(PE). The organic layer was washed with 15% citric acid, dried over
Na2SO4 and evaporated to give 4.62 g of a yellow oil. This was
crystallized from PE to yield 3.49 g (62.5%) of colorless crystals: mp
114–1168C; 1H NMR (300 MHz, CDCl3) d 6.97 (s, 2H), 4.95 (t, J=7 Hz
1H), 3.61 (m, 2H), 2.45 (s, 3H), 2.41 (s, 3H), 2.33 (s, 3H), 1.90 (s, 3H),
1.84 (s, 6H), 1.67 (m, 2H), 1.49 (m, 2H), 1.00 (t, J=7.2 Hz, 3H); CIMS:
m/z 351 (MH+); Anal. (C22H30N4): C, 75.39, H, 8.63, N, 15.98; found:
C, 75.16, H, 8.63, N, 15.83.
[13C2]Ethyl-[2,5,6-trimethyl-7-(2,4,6-trimethyl-phenyl)-7H-pyrro-
lo[2,3-d]pyrimidin-4-yl]-amine (1).
Butyl-[2,5,6-trimethyl-7-(2,4,6-trimethyl-phenyl)-7H-pyrrolo[2,3-d]pyri-
midin-4-yl]-amine (4) (2.63 g, 7.50 mmol) was dissolved in DMSO
(52.6 ml) and cooled to 08C under argon. NaH (0.54 g, 22.5 mmol, 95%
dispersion) was added slowly, and the mixture stirred for 20 min. Then
double 13C-labelled ethyl bromide (1.00 g, 9.01 mmol) was added and
stirring continued at room temperature for 3 h. The mixture was poured
into water, and extracted with ethyl acetate (3 ꢁ 80 ml). The combined
organic layers were washed with brine (3 ꢁ 50 ml), dried over Na2SO4
and evaporated to give a yellow oil (2.98 g). This was crystallized from
MeOH/H2O to give 2.51 g (88.3%) colorless crystals. mp 80–818C;
1H NMR (300 MHz, CDCl3) d 6.97 (s, 2H), 3.59 (m, 1JH,13C=145.8 Hz,
3
2JH,13C=2.7 Hz, JH,H=7.2 Hz, 2H), 3.56–3.50 (m, 2H), 2.46 (s, 3H),
2.37 (s, 3H), 2.34 (s, 3H), 1.93 (s, 3H), 1.84 (s, 6H), 1.65–1.58 (m, 2H),
1
3
1.20 (m, JH,13C=126.0 Hz, JH,H =3.9 Hz, 3H); CIMS: m/z 379
(M-1+); Anal. (C24H34N4): C, 76.15, H, 9.05, N, 14.08; found: C, 76.07,
H, 9.08, N, 14.67.
N-[3-Cyano-4,5-dimethyl-1-(2,4,6-trimethyl-phenyl)-1H-pyrrol-2-
yl][13C2]-acetamide (7).
2-Amino-4,5-dimethyl-1-(2,4,6-trimethyl-phenyl)-1H- pyrrole-3-carbo-
nitrile (6) (5.73 g, 22.6 mmol) was dissolved in CHCl3 (14 ml). After
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DOUBLY 13C-LABELLED ANTALARMIN
643
addition of dry pyridine (2 ml), the mixture was cooled under argon
atmosphere to 08C. Doubly 13C-labelled acetyl chloride (2.00 g,
24.9 mmol) was slowly added, while allowing the temperature to rise
to 208C. Stirring at rt was continued until TLC showed no more starting
material. The reaction mixture was washed with saturated NaHCO3
(3 ꢁ 50 ml), then 2% HCl (3 ꢁ 30 ml), and H2O (2 ꢁ 30 ml). The organic
layer was dried over Na2SO4 and evaporated to give a yellow oil. This
oil was crystallized from ether to afford 6.35 g (95%) as colorless
1
crystals, m.p. 121–1228C; H NMR (300 MHz, CDCl3) d 6.96 (s, 2H),
2.33 (s, 4.5H), 2.16 (s, 4.5H), 1.90 (s, 6H), 1.78 (s, 3H), 1.57 (s, 3H);
CIMS: m/z 297 (MH+); Anal. (C18H21N3O ꢂ 0.3H2O): calc.: C, 71.87, H,
7.24, N, 13.97; found: C, 71.92, H, 7.06, N, 14.00.
[2-13C]-2,5,6-Trimethyl-7-(2,4,6-trimethyl-phenyl)-3,7-dihydro-pyrro-
lo[2,3-d]-[2-13C]pyrimidin-4-one (8)
N-[3-Cyano-4,5-dimethyl-1-(2,4,6-trimethyl-phenyl)-1H-
pyrrol-2-yl]
[13C2]-acetamide (7) (5.60 g, 18.8 mmol), AcOH (7.36 ml) and 85%
H3PO4 (7.36 ml) were combined and heated at 1208C until TLC (eluent:
CHCl3/MeOH/NH4OH, 90:10:1) showed no more starting material.
The mixture was diluted by rapid and dropwise addition of H2O
(14.9 ml) while cooling to 208C. The mixture was filtered and washed
with 33% AcOH (14.9 ml) to afford 4.55 g (82%) of the desired material
as colorless crystals: mp >2208C (dec.); 1H NMR (300 MHz, CDCl3) d
6.99 (s, 2H), 2.61 (d, J=7.2 Hz, 1.5H), 2.43 (s, 3H), 2.35 (s, 3H), 2.18 (d,
J=7.2 Hz, 1.5H), 1.88 (s, 3H), 1.87 (s, 6H); CIMS: m/z 297 (MH+);
Anal. (C18H21N3O ꢂ 0.25H2O): calc.: C, 72.09, H, 7.22, N, 14.01; found:
C, 72.21, H, 7.06, N, 14.01.
4-Chloro-[2-13C]-2,5,6-trimethyl-7-(2,4,6-trimethyl-phenyl)-7H-pyrro-
lo[2,3-d]-[2-13C]pyrimidine (9)
[2-13C]-2,5,6-Trimethyl-7-(2,4,6-trimethyl-phenyl)-3,7-dihydro- pyrrolo
[2,3-d]-[2-13C]pyrimidin-4-one (8) (4.00 g, 13.5 mmol) and POCl3 (5 ml)
were combined under argon atmosphere and heated to reflux until TLC
(eluent: CHCl3/MeOH/NH4OH, 390:90:1) showed no more starting
material. The mixture was cautiously poured into H2O (30 ml), while the
temperature was allowed to rise to 608C. The product was extracted
with CHCl3 (80 ml), the combined organic layers were neutralized
with saturated NaHCO3. After drying over Na2SO4 the solvent was
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E. GREINER ET AL.
evaporated to give a colorless foam. Crystallization from EtOH
1
afforded 3.88 g (91.3%) of colorless crystals: mp 204–2068C; H NMR
(300 MHz, CDCl3) d 7.02 (s, 2H), 2.82 (d, J=7.2 Hz, 1.5H), 2.47 (s, 3H),
2.39 (d, J=7.2 Hz, 1.5H), 2.36 (s, 3H), 2.00 (s, 3H), 1.82 (s, 6H); CIMS:
m/z 315 (MH+); Anal. (C18H20N3Cl ꢂ 0.1H2O): calc.: C, 68.49, H, 6.45,
N, 13.31, Cl, 11.23; found: C, 68.39, H, 6.44, N, 13.36, Cl, 11.39.
Butyl-ethyl-[2-13C]-[2,5,6-trimethyl-7-(2,4,6-trimethyl-phenyl)-7H-pyr-
rolo[2,3-d]-[2-13C]pyrimidin-4-yl]-amine (2)
4-Chloro-[2-13C]-2,5,6-trimethyl-7-(2,4,6-trimethyl-phenyl)-7H-pyrro-
lo[2,3-d]-[2-13C]pyrimidine (9) (1.90 g, 6.02 mmol) and N-ethylbutyla-
mine (4.15 ml, 30.4 mmol) were dissolved in DMSO (14 ml) and heated
to reflux at 1358C under argon, until TLC (EtOAc/petroleum ether
(PE), 1:7) showed no more starting material. The mixture was cooled
and treated with 5% NaOH (2 ml) and PE (50 ml) to give two phases.
The organic layer was washed with H2O (3 ꢁ 50 ml), dried over Na2SO4
and evaporated to give of a yellow oil (2.15 g). This was crystallized
from MeOH/H2O to give 1.83 g (80%) colorless crystals: m.p. 81–858C;
1H NMR (300 MHz, CDCl3) d 6.97 (s, 2H), 2.45 (m, 4H), 2.36 (s, 3H),
1
2
2.45 (m, JH,13C =126.6 Hz, JH,13C=6.9 Hz, 3H), 2.34 (s, 3H), 1.93 (s,
3H), 1.84 (s, 6H), 1.62 (m, 2H), 1.36 (s, 2H), 1.20 (t, J=6.9 Hz, 3H), 0.91
(t, J=7.2 Hz, 3H); CIMS: m/z 380 (MH+); Anal. (C24H34N4 ꢂ 0.25
H2O): calc.: C, 75.25, H, 9.08, N, 14.62; found: C, 75.05, H, 8.81, N,
14.53.
Conclusion
Efficient synthetic routes to antalarmin isotopomers 1 and 2 were
successfully developed and carried out. These routes provided the
desired compounds in reasonable chemical yields with high isotopic
purity. Pharmacokinetic studies using these non-peptide stable isotope
CRHR1 antagonists will be reported in due course.
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