The development of a potential single photon emission computed tomography (SPECT) imaging agent for the corticotropin-releasing hormone receptor type 1

Article abstract of DOI:10.1016/S0960-894X(00)00661-2

A high-affinity radioligand for CRHR₁ has been prepared that can serve as a template for the development of SPECT imaging agents. The 5-chloro-N-cyclopropylmethyl-N-(2,6-dichloro-4-iodophenyl)-2-methyl-N-propylp yrimidine-4,6-diamine (6b, K_i = 14 nM), and the corresponding 4-bromophenyl analogue (6a, K_i = 21 nM), were synthesized in four steps from compound 3.

Full text of DOI:10.1016/S0960-894X(00)00661-2

Bioorganic & Medicinal Chemistry Letters 11 (2001) 331±333
The Development of a Potential Single Photon Emission
Computed Tomography (SPECT) Imaging Agent for the
Corticotropin-Releasing Hormone Receptor Type 1
Xinrong Tian,a,y Ling-Wei Hsin,a,{ Elizabeth L. Webster,b,x Carlo Contoreggi,c
George P. Chrousos,d Philip W. Gold,b Kamal Habib,b Alejandro Ayala,b
William C. Eckelman,e Arthur E. Jacobsona and Kenner C. Ricea,*
aLaboratory of Medicinal Chemistry, NIDDK, NIH, Bethesda, MD 20892, USA
bClinical Neuroendocrinology Branch, NIMH, Clinical Center, NIH, Bethesda, MD 20892, USA
cBrain Imaging Unit, NIDA, Baltimore, MD 21224, USA
dPediatric Endocrinology Section, PREB, NICHD, Clinical Center, NIH, Bethesda, MD 20892, USA
ePET Department, Clinical Center, NIH, Bethesda, MD 20892, USA
Received 17 August 2000; accepted 14 November 2000
AbstractÐA high-anity radioligand for CRHR1 has been prepared that can serve as a template for the development of SPECT
imaging agents. The 5-chloro-N-cyclopropylmethyl-N-(2,6-dichloro-4-iodophenyl)-2-methyl-N-propylpyrimidine-4,6-diamine (6b,
Ki=14 nM), and the corresponding 4-bromophenyl analogue (6a, Ki=21 nM), were synthesized in four steps from compound 3.
Published by Elsevier Science Ltd.
Corticotropin-releasing hormone (CRH) is a 41 amino-
acid peptide secreted in response to stress by several
brain nuclei involved in emotions, memory, endocrine
and autonomic regulation. Overstimulation of CRH
type-1 receptor (CRHR1) may be the underlying factor
in the pathogenesis of a variety of mental disorders that
include major depression, anxiety and substance with-
drawal.1 4 Nonpeptide CRHR1 antagonists are there-
fore being developed for their potential of treating these
conditions. Moreover, the advent of a speci®c radio-
ligand to label CRHR1 in vivo would be an invaluable
diagnostic and prognostic tool for several illnesses. We
have previously shown that antalarmin5 (N-butyl-N-
ethyl[2,5,6-trimethyl-7-(2,4,6-trimethylphenyl)pyrrolo-
[2,3-d]pyrimidin-4-yl]-amine, 1), initially patented by
P®zer,6 is a selective high anity CRHR1 antagonist
that exerts anti-anxiety e€ects in rodents and nonhuman
primates. We have also reported the synthesis of poten-
tial PET imaging agents for CRHR1.7 Here we report
the synthesis and binding anity of a potential candi-
date for single photon emission computed tomography
(SPECT). SPECT, like positron emission tomography
(PET), is used for in vivo measurement of hormone
receptors, and has proven to be a non-invasive techni-
que with wide applicability, particularly in neuroscience
(Fig. 1).8 11
*Corresponding author. Tel.: +1-301-496-1856; fax: +1-301-402-
0589; e-mail: kr21f@nih.gov
yCurrent Address: Procter
Mason, OH 45040, USA.
& Gamble Pharmaceuticals, HCRC,
{Current Address: University of Medicine and Dentistry of New Jer-
sey, Robert Wood Johnson Medical School, Department of Pharma-
cology, Piscataway, NJ 08854, USA.
xCurrent Address: University of North Carolina, Department of Psy-
chiatry, Chapel Hill, NC 27599, USA.
Figure 1.
0960-894X/01/$ - see front matter. Published by Elsevier Science Ltd.
PII: S0960-894X(00)00661-2

332
X. Tian et al. / Bioorg. Med. Chem. Lett. 11 (2001) 331±333
o
We based our synthesis of a potential SPECT ligand on
a nonpeptide compound (5-chloro-N-cyclopropylmethyl-
2-methyl-N-(2,4,6-trichlorophenyl)-pyrimidine-4,6-di-
amine, 2) known to be a high anity ligand for
CRHR1, and known to have CRH antagonist activity
as shown by a functional assay (inhibition of stimulated
cAMP).12 Our initial work towards the goal of obtain-
ing a template for a CRHR1 SPECT ligand was based
on the synthesis and the determination of the CRHR1
binding anity of compounds 6a and 6b in which a
bromine or iodine atom replaced the chlorine atom in
the C4 position of the substituted phenyl ring of 2. If the
iodo compound was found to have reasonable anity
for the receptor, the further synthesis of a 125I com-
pound based on 6b would be attempted.
propylamine in DMSO at 140 C gave 5a,b, followed by
chlorination, which a€orded the desired 4-halo deriva-
tives (N-(4-bromo-2,6-dichlorophenyl)-5-chloro-N-cyclo-
propylmethyl-2-methyl-N-propylpyrimidine-4,6-diamine
(6a) and 5-chloro-N-cyclopropylmethyl-N-(2,6-dichloro-
4-iodophenyl)-2-methyl-N-propylpyrimidine-4,6-diamine
(6b).
The CRH1 anity of 2, 6a, 6b, and the tributyltin ana-
logue 8 was determined using a previously reported
procedure.7 The binding anity of these compounds in
rat cerebellum against radioligand [125I]Tyr0-sauvagine
is shown in Table 1. Iodo analogue 6b exhibited higher
anity than the formerly known12 chloro analogue 2.
The anity decreased in the order of I>Br>Cl. This
corresponds with the decrease in lipophilicity
(I>Br>Cl). The e€ect of lipophilicity on CRHR1 a-
nity was further demonstrated with compound 8. This
tributyltin analogue, the most lipophilic among the four
synthesized compounds, showed the highest binding
anity.
The synthesis was carried out using a route analogous
to one reported by Chen.12 In addition to 2, bromo (6a)
and iodo (6b) analogues were prepared in four steps from
4,4-dichloro-2-methyl pyrimidine (3), in overall yields of 62
and 47%, respectively (Scheme 1). Coupling of 3 with 4-
bromo and 4-iodo-2,6-dichloroaniline gave the pyr-
imidines 4a and 4b. Amination with N-cyclopropylmethyl-
With the establishment of the high anity of 6b for
CRHR1, we decided to proceed with the synthesis of the
radiolabeled 125I analogue. We envisioned that it could
be derived from the corresponding tributyltin com-
pound 8, which, in turn, should be accessible from the
bromo precursor 6a. As outlined in Scheme 1, conver-
sion of the bromide 6a to the tributyltin compound 8
was not without problems. Initial treatment of 6a with 2
equiv of n-BuLi followed by tributyltin chloride only
gave the debromination product 7. This was probably
due to the bromo-lithium exchange occurring prior to
the deprotonation of the secondary amine; the
generated aromatic anion was immediately quenched by
Table 1. CRH1 binding anity
Compound
Ki (nM)
2
31Æ9.7a
21Æ7.9a
14Æ4.6a
3.0b
6a
6b
8
aThree binding curves conducted in duplicate were generated for each
compound and the Ki values represent the mean of the three experi-
mentsÆSEM.
bThe result of a single experiment.
Scheme 1.

X. Tian et al. / Bioorg. Med. Chem. Lett. 11 (2001) 331±333
333
an acidic anilino proton. The preferred formation of an
aromatic anion was con®rmed by the use of 1 equiv of
n-BuLi which also gave the reduction product 7. We
circumvented this problem by a stepwise deprotonation
and lithiation sequence. Compound 6a was ®rst treated
with 1 equiv of sodium hydride to deprotonate the ani-
line followed by 1 equiv of n-BuLi for bromo-lithium
exchange. The resulting dianionic species was quenched
with tributyltin chloride to a€ord the desired tin
compound 813 in 51% yield.
Institute of Diabetes and Digestive and Kidney Dis-
eases, for the mass spectral data.
References and Notes
1. Bremner, J. D.; Licinio, J.; Darnell, A.; Krystal, J. H.;
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Science 1984, 226, 1342.
3. Zhou, Y.; Spangler, R.; LaForge, K. S.; Maggos, C. E.;
Ho, A.; Kreek, M. J. J. Pharmacol. Exp. Ther. 1996, 279, 351.
4. Iredale, P. A.; Alvaro, J. D.; Lee, Y.; Terwilliger, R.; Chen,
Y. L.; Duman, R. S. J. Neurochem. 2000, 74, 199.
5. Webster, E. L.; Lewis, D. B.; Torpy, D. J.; Zackman, E. K.;
Rice, K. C.; Chrousos, G. P. Endocrinology 1996, 137, 5747.
6. Chen, Y. L. Pyrrolopyrimidines as CRF Antagonists.
International Publication Number WO 94/13676, June 23, 1994.
7. Hsin, L.-W.; Webster, E. L.; Chrousos, G. P.; Gold, P. W.;
Eckelman, W. C.; Contoreggi, C.; Rice, K. C. Bioorg. Med.
Chem. Lett. 2000, 10, 707.
8. Diksic, M.; Reba, R. C. Radiopharmaceuticals and Brain
Pathology Studies with PET and SPECT; CRC: Boca Raton,
1991.
Compound 8 was converted to the targeted 125I com-
pound 9 using the following procedure. To a solution of
carrier-free [125I]NaI (5.34 mCi, Amersham-Pharmacia
Biotech, Chicago, IL 60611) were added tributyltin pre-
cursor (150 mL, 0.33 mg/mL) in ethanol, H3PO4 (40 mL,
0.5 M), and peracetic acid (50 mL, 0.2 M). After stand-
ing at room temperature for 30 min, sodium bisul®te
was added to quench the reaction, and 1 mL of satu-
rated sodium bicarbonate solution was added to the
mixture. The [125I] product was extracted with EtOAc (3
 1 mL) and then passed through a short anhydrous
Na2SO4 column. The ®ltrate was evaporated to dryness
under a stream of nitrogen. The residue was dissolved in
50 mL of MeOH and 100 mL of mobile phase (MeOH/
H2O/triethylamine, 90:10:0.3), and puri®ed by HPLC
on a reverse phase column (C-18 Applied Biosystems
Spheri-5 ODS 5 m, 4.6Â250 mm column). The corre-
sponding fraction was collected and evaporated to dry-
ness under a stream of nitrogen to a€ord pure [125I]
product 9 (4.96 mCi) with a radiochemical purity
>99%. Since 8 is eluted after 9 using reversed phase
HPLC, and proton-destannylation has not been
observed under these conditions, we expect the e€ective
speci®c activity of 9 to be 2200 Ci/mmol.
9. Frost, J. J.; Wagner, H. N. Quantitative Imaging. Neuror-
eceptors, Neurotransmitters and Enzymes; Raven: New York,
1990.
10. Andreasen, N. C. Science 1988, 239, 1381.
11. de Costa, B. R.; Iadarola, M. J.; Rothman, R. B.; Berman,
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12. Chen, C.; Dagnino, J. R.; De Souza, E. B.; Grigoriadis,
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13. The procedure used for the preparation of the tributyltin
analogue 8 from bromide 6a is as follows: To a solution of the
bromide 6a (311 mg, 0.65 mmol) in THF (2 mL) was added
sodium hydride (16 mg, 60% in mineral oil) at 0 ꢀC. After
being stirred at 0 ꢀC for 15 min, the reaction mixture was
cooled to 78 ꢀC and n-BuLi (1.6 M in hexane, 0.25 mL) was
added. The mixture was stirred for 20 min and treated with
tributyltin chloride (288 mg, 0.88 mmol). It was then slowly
warmed to room temperature with stirring over 2 h, quenched
with aqueous NH4Cl solution and extracted with EtOAc. The
extract was dried over Na2SO4 and concentrated. The residue
was chromatographed (silica gel, hexanes/EtOAc, 20:1 to 7:1)
to give the tributyltin derivative 8 (230 mg, 51% yield) as a
In summary, we have developed the synthesis of the ®rst
nonpeptide potential SPECT ligand for CRHR1. The
signi®cant binding anity exhibited by iodo analogue
6b makes its 125I analogue 9 an intriguing template for
further development of the SPECT imaging agent for
the CRHR1. It also holds great potential as a selective
nonpeptide radioligand for the CRHR1 binding assay,
replacing the currently used peptide radioligands. A
study along these lines is in progress and the results will
be reported in due course.
1
clear oil; H NMR (300 MHz, CDCl3) 7.39 (s, 2H), 6.71 (s,
Acknowledgements
1H), 3.58 (t, 2H, J=7.8 Hz), 3.42 (d, 2H, J=6.9 Hz), 2.26 (s,
3H), 1.53 (m, 6H), 1.06±1.20 (m, 6H), 0.90 (m, 12H), 0.51 (m,
2H), 0.24 (q, J=4.8 Hz); MS (CI+) m/z 689; HRMS calcd for
C30H47N4Cl3Sn 688.1888, found 688.1892.
The authors thank Noel Whittaker and Wesley White of
the Laboratory of Bioorganic Chemistry, National