Synthesis of [3H]-labelled 4-[ethyl[2,5,6-trimethyl-7-(2,4,6- trimethylphenyl)pyrrolo[2,3-d]pyrimidin-4-yl]amino]-2,3-[3H]-butan-1- ol: A high affinity radioligand for the corticotropin-releasing hormone type 1 receptor

Article abstract of DOI:10.1002/jlcr.1082

[³H]-Labelled 4-[ethyl[2,5,6-trimethyl-7-(2,4,6-trimethylphenyl) pyrrolo[2,3-d]pyrimidin-4-yl]amino]-2,3-[³H]-butan-1-ol (3b) was prepared as a novel non-peptidic radiolabelled high affinity antagonist of the corticotropin-releasing

Full text of DOI:10.1002/jlcr.1082

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2006; 49: 635–640.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.1082
Research Article
Synthesis of [3H]-labelled 4-[Ethyl[2,5,6-trimethyl-7-
(2,4,6-trimethylphenyl)pyrrolo[2,3-d]pyrimidin-4-
yl]amino]-2,3-[3H]-butan-1-ol: a high affinity
radioligand for the corticotropin-releasing hormone
type 1 receptor
Anne-Ce
´
cile Hiebel1, John S. Partilla2, Richard B. Rothman2,
Arthur E. Jacobson1 and Kenner C. Rice1,*
1 Laboratory of Medicinal Chemistry, Building 8, Room B1-23, National Institute of
Diabetes and Digestive and Kidney Diseases, National Institutes of Health, DHHS,
8 Center Drive, MSC 0815, Bethesda, MD 20892, USA
2 Behavioral Neuroscience Branch, National Institute for Drug Abuse, Intramural
Research Program, National Institutes of Health, DHHS, 5500 Nathan Shock Dr,
Baltimore, MD 21224, USA
Summary
[3H]-Labelled 4-[ethyl[2,5,6-trimethyl-7-(2,4,6-trimethylphenyl)pyrrolo[2,3-d]pyrimi-
din-4-yl]amino]-2,3-[3H]-butan-1-ol (3b) was prepared as a novel non-peptidic
radiolabelled high affinity antagonist of the corticotropin-releasing hormone type 1
receptor (CRHR1) that could be useful as a more stable and receptor-selective
alternative to the radiolabelled peptides now used to label the CRHR1 receptor for
displacement studies in cell-based binding assays. The precursor (Z)-4-[ethyl[2,5,6-
trimethyl-7-(2,4,6-trimethylphenyl)pyrrolo[2,3-d]pyrimidin-4-yl]amino]but-2-en-1-ol
(2) was reduced with tritium gas using palladium as the catalyst. After HPLC
purification 3b was obtained with a specific activity of 35 Ci/mmol in high
radiochemical purity (>97%). Copyright # 2006 John Wiley & Sons, Ltd.
Received 11 April 2006; Revised 19 April 2006; Accepted 19 April 2006
Key Words: corticotropin-releasing hormone; CRHR1 antagonist; radioligand;
tritium-labelled; antalarmin
*Correspondence to: Kenner C. Rice, Laboratory of Medicinal Chemistry, Building 8, Room B1-23,
National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, DHHS,
8 Center Drive, MSC 0815, Bethesda, MD 20892, USA. E-mail: kr21f@nih.gov
Contract/grant sponsor: National Institute of Diabetes and Digestive and Kidney Diseases
Contract/grant sponsor: National Institute on Drug Abuse
Copyright # 2006 John Wiley & Sons, Ltd.

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A.-C. HIEBEL ET AL.
Introduction
Vale et al., isolated corticotropin releasing hormone (CRH) from ovine
hypothalamus extracts and characterized it as a 41 amino acid neuropeptide in
1981.1 CRH has been found to act as a major regulator of the hypothalamic–
pituitary–adrenal (HPA) axis, coordinating neuroendocrine, autonomic,
immune, and behavioral responses to stress.2,3 The CRH system is known
to be composed of saturable, high affinity corticotropin-releasing
hormone type 1 and type 2 receptors (CRHR1 and CRHR2) and their
endogenous ligands. The characterized and cloned4–9 receptors are located in
anatomically well-defined regions of the CNS and periphery.10 Increases in
brain CRH have been associated with anxiety, depression, and substance
abuse.11–14 Excessive chronic activation of the CRH system is postulated to be
involved in the pathogenesis of eating15 and gastrointestinal disorders.16 To
further study this system, we have been engaged in the synthesis and
evaluation of CRH ligands (e.g. antalarmin)17–23 with the goal of finding those
that act with high affinity and specificity to each of the known receptors.
Antalarmin is being considered for clinical trials and is in the final phase of
preclinical toxicology.
There are two radioligands used for the in vitro determination of the affinity
of CRHR1 and CRHR2 ligands. One of these is a commercially available
peptide, [125I]Tyr0-sauvagine.24 In general, peptide radioligands used for
competitive displacement studies in vitro are large, ‘sticky’ (e.g. non-selective),
ligands and may be more unstable than non-peptides. The [125I]Tyr0-
sauvagine, although very useful, presents these drawbacks. More recently,
Zhang et al.,25 synthesized a tritiated non-peptide, ( ꢀ )-N-[2-methyl-4-
methoxyphenyl]-1-(1-(methoxymethyl)propyl)-6-methyl-1H-1,2,3-triazolo[4,5-c]
pyridin-4-amine ([3H]SN003) for these purposes. That compound, however, is
not commercially available. Both non-peptide and peptide radioligands
would be useful tools, since there is evidence that they bind differently at
CRHR1.25
We sought a high affinity non-peptide ligand that would be easily available
and that could be tritiated for use as a radioligand. We now report the
preparation of 4-[ethyl[2,5,6-trimethyl-7-(2,4,6-trimethylphenyl)pyrrolo[2,3-d]
pyrimidin-4-yl]amino]-2,3-[3H]-butan-1-ol] (3b), a novel, high affinity antago-
nist at CRHR1, and a radioligand that could be useful for in vitro studies in
future cell-based binding assays. We previously reported that the unlabelled
pyrrolopyrimidine compound 3a (Scheme 1) was among the highest affinity
ligands for CRHR1 (Ki ¼ 0:68 nM), as determined in displacement assays
using [125I]Tyr0-sauvagine.10,26 In order to provide a non-peptidic and
hopefully more stable and selective radioligand, pyrrolopyrimidine 2 was
tritiated to afford 3b.
Copyright # 2006 John Wiley & Sons, Ltd.
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DOI: 10.1002/jlcr

CRHR1 RADIOLIGAND
637
DMSO
H2, EtOH, 10% Pd/C
or: 3H2, EtOH, 10% Pd/C
3a: R = H
3b: R = 3H
1
2
Scheme 1.
Results and discussion
The unsaturated pyrrolopyrimidine compound 2 (Z)-4-[ethyl[2,5,6-trimethyl-
7-(2,4,6-trimethylphenyl)pyrrolo[2,3-d]pyrimidin-4-yl]amino]but-2-en-1-ol,26
appeared ideal as the precursor since it had a double bond that could be
tritiated (Scheme 1). It was obtained by reaction of the known 4-chloro-3,5,
6-trimethyl-7-(2,4,6-trimethylphenyl)pyrrolo[2,3-d]pyrimidine (1) (in about 5
steps, as reported by Chen),17 and (Z)-4-ethylaminobut-2-en-1-ol. The
synthesis of 2 was improved in our laboratory, for our use in the synthesis
of radiolabelled fluorinated antalarmin.10 In an initial experiment, we found
that 2 was easily reduced with a balloon pressure of hydrogen over 10%
palladium on charcoal. Compound 3b was then prepared by RC TRITEC
Ltd., 9053 Teufen, Switzerland, through tritiation of 2 using 3H2 and 10% Pd/C
in absolute ethanol, and purified by reverse phase high performance liquid
chromatography. Compound 3b was obtained with high radiochemical purity
(>97%) and a specific activity of 35 Ci/mmol. The radioligand was repurified
for pharmacological evaluation, and the pharmacological data will be reported
elsewhere when completed.
Experimental
1H and 13C NMR spectra were recorded using a Varian Gemini XL-300
spectrometer. Chemical shifts are reported in parts per million with
tetramethylsilane (0.0 ppm) used as the internal standard for 1H NMR spectra
and the CDCl3 absorption (77.23 ppm) for 13C NMR. The tritiation was
carried out by RC TRITEC Ltd., 9053 Teufen, Switzerland.
4-[Ethyl[2,5,6-trimethyl-7-(2,4,6-trimethylphenyl)pyrrolo[2,3-d]pyrimidin-
4-yl]amino]butan-1-ol (3a)
The starting oil 210 (10 mg, 0.025 mmol) was placed in a round bottomed flask
purged with argon. Absolute ethanol (0.4 ml) and 10% Pd/C (1 mg) were
added and the mixture was degassed with argon for 20 min. Hydrogen gas was
then introduced in the reaction mixture via a balloon. The reaction was stirred
for 12 h at room temperature. The crude mixture was filtered (syringe filter,
Copyright # 2006 John Wiley & Sons, Ltd.
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DOI: 10.1002/jlcr

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A.-C. HIEBEL ET AL.
Titan HPLC, pore size 0.5 mm, membrane type: PTFE) and the crude product
was purified by Radial PLC (gradient 2% MeOH/CH2Cl2 to 5% MeOH/
CH2Cl2). The product 3a was isolated as a clear oil (8 mg, 80%). The spectral
data was identical to that reported in the literature.26
Radiosynthesis of 4-[Ethyl[2,5,6-trimethyl-7-(2,4,6-trimethylphenyl)pyrrolo
[2,3-d]pyrimidin-4-yl]amino]-2,3-[3H]-butan-1-ol (3b)
The synthesis, HPLC purification and determination of specific activity of 3b
were accomplished by RC TRITEC Ltd., 9053 Teufen, Switzerland as follows:
80 mg of the oily unsaturated starting material10 2 were dissolved in 3.0 ml
absolute ethanol. From this stock solution, 0.113 ml (3.0 mg of 2, 7.6 mmol)
was added to 1.73 mg of the catalyst (10% Pd/C) and diluted with 0.5 ml of
absolute ethanol. The suspension was degassed four times at the stainless steel
vacuum manifold (RC TRITEC Ltd.) and stirred under an atmosphere of
tritium gas (12.4 Ci) for 3.5 h. The solvent was removed in vacuo, and labile
tritium was exchanged by adding 1 ml methanol, stirring the solution, and
removing the solvent again in vacuo. This process was repeated three times.
Finally, the well-dried solid was extracted with 5 ml ethanol and the
suspension filtered through a nylon membrane (0.2 mm), obtaining a clear
and colorless solution with an activity of 513 mCi. The crude product
(radiochemical purity 88%) was purified on a Waters XTerra RP8 (5 mm,
7.8 ꢁ 150 mm) column under isocratic conditions (A: water, 0.1% TFA; B:
acetonitrile 0.1% TFA; 40% B). The compound decomposes slowly under
acidic conditions, thus the compound was isolated by solid phase extraction
(Waters Oasis1, HLB (6 cm3, 200 mg)). The HPLC solvent mixture was first
diluted with water containing 1% ammonia to adjust the acetonitrile content
to 20%. After loading on the cartridge and an additional washing step with
water (containing 1% ammonia), the product was eluted with ethanol. Finally,
10 mCi of purified product 3b with a radiochemical purity of >97% (HPLC)
were obtained. The specific activity of approximately 35 Ci/mmol was
calculated from the activity concentration (determined by LSC) and the
chemical concentration (determined by UV peak area method) of the active
sample.
Conclusion
4-[Ethyl[2,5,6-trimethyl-7-(2,4,6-trimethylphenyl)pyrrolo[2,3-d]pyrimidin-4-yl]
amino]-2,3-[3H]-butan-1-ol (3b) was prepared as a novel non-peptidic
radiolabelled potent antagonist of the corticotropin-releasing hormone type
1 receptor (CRHR1). This compound will be examined for its stability
and its ability to label CRHR1 for displacement studies in cell-based binding
studies.
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm. 2006; 49: 635–640
DOI: 10.1002/jlcr

CRHR1 RADIOLIGAND
639
Acknowledgements
This research was supported by the NIH Intramural Research Program of the
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
and the National Institute on Drug Abuse (NIDA). Anne-Cecile Hiebel
´
thanks NIDDK for an IRTA fellowship. The authors (LMC, NIDDK) also
thank NIDA for partial support of this work.
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