Synthesis of [N-methyl- 11C]-3-[(6-dimethylamino)pyridin-3-yl]-2,5-dimethyl-N, N-dipropylpyrazolo[1,5-a]pyrimidine-7-amine: A potential PET ligand for in vivo imaging of CRF1 receptors

Article abstract of DOI:10.1002/jlcr.738

A convenient synthesis of [N-methyl- ¹¹C]-3-[(6-dimethylamino)pyridin-3-yl]-2,5-dimethyl-N, N-dipropylpyrazolo[1,5-a]pyrimidine-7-amine (R121920), a highly selective CRF₁ antagonist has been developed as a potential PET ligand. 3-[(6-methylamino)pyridin-3-yl]-2,5-dimethyl-N,N-dipropylpyrazolo [1,5-a]pyrimidine-7-amine (7), the precursor for radiolabelling was synthesized through a novel palladium catalyzed Suzuki coupling of aryl bromide 5 with heteroaryl boronate ester 4. The requisite boronate ester 4 was synthesized in four steps from 2-amino-4-bromopyridine in 50% overall yield. Although the synthesis of cold R121920 proceeded in 93% yield by sodium hexamethyl-disilazide (NaHMDS) mediated N-methylation of the desmethylamine 7 at -78°C, the attempted radiosynthesis under various conditions using conventional bases were not successful. However, the radiolabeling of [ ¹¹C]R121920 was successfully carried out with [¹¹C]MeOTf in acetone at -20°C in the absence of added basic reagents. The radiotracer was purified by RP-HPLC followed by RP-solid phase extraction. The yield of the reaction was 5% (at EOB) and the specific activity was > 1000 Ci/mmol (at EOB) with a radiochemical purity > 99%. Copyright

Full text of DOI:10.1002/jlcr.738

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2003; 46: 1055–1065.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.738
Research Article
Synthesis of [N-methyl-11C]-3-[(6-
dimethylamino)pyridin-3-yl]-2,5-dimethyl-N,
N-dipropylpyrazolo[1,5-a]pyrimidine-7-amine:
A potential PET ligand for in vivo imaging of
CRF1 receptors
J.S. Dileep Kumar1,*, Vattoly J. Majo1, Jaya Prabhakaran1,
Norman R. Simpson2, Ronald L. Van Heertum2 and
J. John Mann1,2
1 Department of Psychiatry and Division of Neuroscience, Columbia University
and New York State Psychiatric Institute, 1051 Riverside Drive, Box: 42,
New York, NY 10032, USA
2 Department of Radiology, Columbia University, New York, NY 10032, USA
Summary
A convenient synthesis of [N-methyl-11C]-3-[(6-dimethylamino)pyridin-3-yl]-
2,5-dimethyl-N,N-dipropylpyrazolo[1,5-a]pyrimidine-7-amine (R121920),
a
highly selective CRF1 antagonist has been developed as a potential PET
ligand. 3 - [(6 - methylamino)pyridin - 3 - yl]-2,5-dimethyl-N,N-dipropylpyrazolo
[1,5-a]pyrimidine-7-amine (7), the precursor for radiolabelling was synthesized
through a novel palladium catalyzed Suzuki coupling of aryl bromide 5 with
heteroaryl boronate ester 4. The requisite boronate ester 4 was synthesized in
four steps from 2-amino-4-bromopyridine in 50% overall yield. Although the
synthesis of cold R121920 proceeded in 93% yield by sodium hexamethyl-
disilazide (NaHMDS) mediated N-methylation of the desmethylamine 7 at
À788C, the attempted radiosynthesis under various conditions using
*Correspondence to: J. S. Dileep Kumar, Department of Psychiatry and Division of Neuroscience,
Columbia University and New York State Psychiatric Institute, 1051 Riverside Drive, Box: 42,
New York, NY 10032, USA. E-mail: dk2038@columbia.edu
Contract/grant sponsor: National Institutes of Health; contract/grant number: R21MH066620
Copyright # 2003 John Wiley & Sons, Ltd.
Received 26 March 2003
Revised 10 April 2003
Accepted 19 May 2003

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J.S. DILEEP KUMAR ET AL.
conventional bases were not successful. However, the radiolabeling of
[11C]R121920 was successfully carried out with [11C]MeOTf in acetone at
À208C in the absence of added basic reagents. The radiotracer was purified by
RP-HPLC followed by RP-solid phase extraction. The yield of the reaction was
5% (at EOB) and the specific activity was >1000 Ci/mmol (at EOB) with a
radiochemical purity >99%. Copyright # 2003 John Wiley & Sons, Ltd.
Key Words: CRF antagonist; positron emission tomography; R121920;
neuropsychiatric diseases
Introduction
Corticotropin releasing factor (CRF or CRH) is a 41 amino acid
neurotransmitter isolated and sequenced by Vale and coworkers.1,2 The
functions of CRF are mainly mediated through two CRF receptor
subtypes (CRF1 and CRF2) and a CRF binding protein (CRF-BP).3
The CRF1 receptors are the most abundant CRF receptor subtype
found in rodent and primate pituitary and are widely distributed in the
brain including cerebral cortex, hippocampus and amygdala. The CRF2
receptors are primarily distributed in lateral septum, choroid plexus,
hypothalamus and sympathetic nuclei.4 The CRF binding protein is
widely distributed in the central nervous system (CNS) including
hippocampus and amygdala and in human, but not in rodent, it is also
found in plasma.4 Abnormal expression of CRF1, mostly over-
expression, comprises part of the pathogenesis of a diverse range of
neuropsychiatric disorders such as anxiety disorders, mood disorders,
obsessive-compulsive disorders, posttraumatic stress disorders and
neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s
disease.5–10 One of the potential therapeutic strategies to block the effect
of CRF elevation is the utilization of selective non-peptidic CRF1
receptor antagonists.11 To be effective, these molecules must have
receptor subtype specificity (a high CRF1/CRF2 ratio for affinity),
aqueous solubility, good oral bioavailability and rapid permeability
across the blood brain barrier (BBB).
Over the years many research laboratories and pharmaceutical
companies have developed a variety of small molecule non-peptide
CRF1 antagonists and some of them have undergone clinical studies
(Figure 1).12 Most of the published preclinical studies using CRF1
receptor antagonists are based on CP-154526 and antalarmin.13
According to these reports CP-154526 binds with high affinity
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 1055–1065

SYNTHESIS OF [11C]CRF1 LIGAND
1057
OMe
N
N
N
N
N
OMe
HN
N
N
N
N
N
N
N
N
N
N
N
Cl
N
Cl
Antalarmin
R121919
CP-154526
DMP-696
Me
O
N
N
N
N
N
N
F
N
N
OMe
N
N
N
X
N
N
N
OMe
NBI-30545
CRA-1000; X = CH3 S
CRA-1001; X = Br
R121920
Figure 1. Highly selective CRF1 receptor antagonists identified recently.
(Ki=2.7 nmol/l) to CRF1 receptor and inhibits intracellular signaling
through g-protein coupled receptor. The intraperitonial administration
of this compound reduces anxiety-like behavior assessed on an elevated
level and shows antidepressant-like effects.14,15 Although the radio-
synthesis of a number of fluorinated and iodinated analogues of CP-
154526 and antalarmin have been developed, the details of the in vivo
validations of these tracers are not reported.16À18
DMP-696, is another potent CRF1 receptor antagonist (Ki=1.6
nmol/l), which reduces stress induced anxiety related behavior without
compromising the HPA system in rodent and primates models.19
Similarly CRA 1000 and CRA 1001 also show potency against CRF-
induced conditioned fear.20
NBI 30545 is a highly selective CRF1 receptor antagonist, which
could not get into clinical trial due to poor BBB permeability and the
lack of its aqueous solubility.21 However, R121919, one of the second-
generation CRF1 receptor antagonists in the NBI series was found to be
a high affinity ligand (Ki=3 nmol/l) with selectivity greater than 1000
times that of CRF2 receptor.22 After successful pre-clinical trials against
anxiety-like behaviors, R121919 was used in a clinical trial for patients
with major depression.23 However, a parallel toxicological study in
normal controls showed significant increases in liver enzymes and
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J.S. DILEEP KUMAR ET AL.
therefore further clinical development of R121919 was discontinued.11
More recently neuroprotective effect of the CRF1 receptor antagonist 3-
[(6 - dimethylamino)pyridin - 3 - yl] - 2,5-dimethyl-N,N -dipropylpyrazolo
[1,5-a]pyrimidine-7-amine (R121920) was reported in rat models of
permanent focal ischemia.24 There is increasing evidence to support the
involvement of CRF in the pathogenesis of ischemic brain damage.25
Up-regulation of CRF mRNA has been reported in the cerebral
parenchyma of rats after permanent middle cerebral artery occlusion
(MCAO) and traumatic brain injury.26 R121920 is a water-soluble high
affinity CRF1 receptor selective antagonist (Ki=4 nmol/l for CRF1;
Ki=>10000 nmol/l for CRF2 receptor), which rapidly crosses the BBB
after intravenous administration with a peak brain concentration
approximately 2-fold greater than those in plasma.24 Hence targeting
R121920 as an imaging probe might provide the detailed pathophysiol-
ogy of CRF expression in brain. Herein we report the synthesis of
[11C]R121920, a potential PET imaging probe for CRF1 receptors.
Results and discussion
The key step in the synthesis of 3-[(6-methylamino)pyridin-3-yl]-2,5-
dimethyl-N,N-dipropylpyrazolo[1,5-a]pyrimidine-7-amine (7), the des-
methyl precursor for radiosynthesis was envisaged to be a biaryl
coupling of aryl bromide 5 with arylboronate ester 4. To this end, 3-
bromo-2,5-dimethyl-7-dipropylaminopyrazolo[1,5-a]pyrimidine (5) was
synthesized by the condensation of 3-iminobutyronitrile and 3-amino-4-
bromo-5-methylpyrazole followed by dipropylation with n-propyl
iodide in presence of NaH in N,N-dimethyl formamide.27 [5-(5,5-
Dimethyl-[1,3,2]dioxaborinan-2-yl)pyridin-2-yl]methylcarbamic acid tert-
butyl ester (4), the requisite counterpart for the biaryl coupling was
prepared by the following sequence of reactions as represented in
Scheme 1. 2-Amino-4-bromopyridine (1) was protected as its N-Boc
derivative in 95% yield in the presence of NaHMDS. Subsequent
methylation of 2 was achieved with methyl iodide in the presence of
NaH to afford 3. Initially, we prepared the requisite Boc-protected 6-
methylamino-3-pyridylboronic acid by lithiation of the corresponding
bromide 3 using n-BuLi at –788C followed by boronylation with
B(OiPr)3. However, the difficulty in isolation, purification and handling
of this boronic acid prompted us to use the corresponding neopentyl
boronate ester 4 in the Suzuki coupling, which could be easily prepared
Copyright # 2003 John Wiley & Sons, Ltd.
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SYNTHESIS OF [11C]CRF1 LIGAND
1059
O
O
N
Br
Br
Br
(Boc) O
2
1. n-BuLi, B(OiPr)
THF, -78 ˚C, 2h
3
CH I, NaH
3
NaHMDS
THF
N
N
N
Neopentyl glycol,
2.
THF
THF, -40 ˚C-rt, 12h
HN
NH
N
2
Boc
Boc
N
Boc
4 (65 %)
1
2 (95%)
3 (80 %)
Scheme 1. Synthesis of boronate ester 4
in good yield by the in situ quenching of the boronylation reaction
mixture with neopentyl glycol at À408C.
We then set out to optimize the yield of the Suzuki reaction of
heteroaryl bromide 5 with phenylboronic acid under several conditions
using various palladium catalysts and bases.28 The ideal condition for
the coupling was obtained by using dichlorobis(triphenylphosphine)-
palladium (II) (PdCl2(PPh3)2) as the catalyst in the presence of 3
equivalents of 2 M NaOH in DME-H2O. Under these conditions, the
biaryl coupling of 4 with 5 in the presence of catalytic amount of
PdCl2(PPh3)2 proceeded to give the 3-arylpyrazolo[1,5-a]pyrimidine
derivative 6 in 63% yield (Scheme 2). The deprotection of the Boc group
was then achieved with 50% trifluoroacetic acid in dichloromethane to
yield the desmethyl precursor 7 (Scheme 2).
N
N
N
N
O
O
N
N
N
N
N
N
N
N
PdCl2(PPh3 )2
CF3 COOH
CH2 Cl2
N
+
N
2M NaOH-DME
100 ˚C, 5h
N
N
N
Boc
Br
NH
Boc
5
4
6 (63 %)
7 (91 %)
Scheme 2. Synthesis of the precursor amine 7
The synthesis of cold R121920 was achieved in 93% yield by sodium
hexamethyldisilazide mediated N-methylation of 7 using methyl iodide at
À788C (Scheme 3). Various bases such as NaH, NaOH, K2CO3 and
KOtBu were ineffective for the desired N-methylation of 7, and instead,
formation of more polar products were observed in these reactions.
However, our labeling experiments of 7 with [11C]MeI or [11C]MeOTf in the
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J.S. DILEEP KUMAR ET AL.
N
N
N
N
N
N
N
N
CH3OTf, acetone
THF, -20 ˚C
N
N
NaHMDS, THF
N
N
N
-78 °C, CH3I
N
N
N
N
NH
8 (7 %)
11 C ]8 (5 %)
8 (93 %)
7
[
Scheme 3. Synthesis of R121920 and [11C]R121920.
presence of NaHMDS did not afford [11C]R121920. This may be due
to the interaction of hexamethyldisilazide with the trace amount of [11C]MeI
or [11C]MeOTf, which is the limiting reagent under hot experimental
conditions. The use of other conventional bases such as NaOH and KOtBu
were also not effective for the [11C]N-methylation reaction due to the
predominant formation of polar radiolabelled products. The radiolabeling
was finally achieved by the use of [11C]MeOTf in acetone at –208C without
using any base (Scheme 3). The crude product was purified by reverse-phase
HPLC to afford [11C]8 in 5% (EOB) yield. Under identical conditions,
reaction of 7 with methyl triflate at À208C in acetone provided 8 in 7%
yield. The chemical identity of [11C]8 was confirmed by co-injection with the
authentic sample of 8 on analytical reverse-phase HPLC (Figure 2). The
specific activity was calculated based on a standard mass curve using HPLC
technique. The chemical and radiochemical purity of [11C]8 was found to be
>99% with a specific activity >1000 Ci/mmol (EOB).
Conclusions
A convenient method for the synthesis of [N-methyl-11C]-3-[(6-
dimethylamino)pyridin-3-yl]-2,5-dimethyl-N,N-dipropylpyrazolo[1,5-
a]pyrimidine-7-amine ([11C]R121920) and its desmethyl precursor 7 is
achieved. The total time required for the synthesis of [11C]R121920 was
30 min from EOB by using [11C]-methyl triflate in acetone at À208C.
The radiochemical purity was >99% with a specific activity >1000 Ci/
mmol. Thus the synthesis of [11C]R121920, a highly selective CRF1
nonpeptide antagonist has been developed as a potential radioligand for
the in vivo quantification of CRF1 receptors using PET.
Copyright # 2003 John Wiley & Sons, Ltd.
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SYNTHESIS OF [11C]CRF1 LIGAND
1061
Figure 2. (a) Radiosynthesis of [11C]R121920 and (b) co-injection with R121920.
Experimental
Melting points were determined on a Fisher Melting point apparatus
and are uncorrected. 1H NMR spectra were recorded on a Bruker PPX
300 and 400 MHz spectrometer. Spectra are recorded in CDCl3 and
chemical shifts are reported in ppm relative to TMS as internal
standards. The mass spectra were recorded on JKS-HX 11UHF/HX110
HF Tandem Mass Spectrometer in the FAB+ mode. The HPLC
analyses were performed using Waters 1525 HPLC system (column:
Phenomenex, Prodigy ODS 4.6 Â 250 mm, 5 m). Flash column chroma-
tography was performed on silica gel (Fisher 200–400 mesh) using the
solvent system indicated. [11C]-Methyl triflate was synthesized in the
radioligand laboratory of Columbia University by transfering [11C]-
methyl iodide through a glass column containing AgOTf at 2008C.29
The radiochemical and chemical purities were analyzed by RP-HPLC
with PDA and NaI detectors.
[5-(5,5-Dimethyl-[1,3,2]dioxaborinan-2-yl)pyridin-2-yl]methylcarbamic-
acid tert-butyl ester (4)
A solution of the aryl bromide 3 (630 mg, 2.195 mmol) in THF (10 ml)
under argon was treated with n-BuLi (1.0 ml, 2.415 mmol, 2.5 M)
dropwise at –788C and allowed to stir for 20 min. Triisopropyl borate
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J.S. DILEEP KUMAR ET AL.
(1.0 ml, 4.349 mmol) was rapidly added to this solution and the reaction
mixture was further stirred for 2 h; maintaining the temperature at
–788C. The temperature was then gradually allowed to reach –408C and
finely powdered neopentyl glycol (250 mg, 2.195 mmol) was added. The
solution was warmed to r.t. and stirred for further 12 h. The reaction
mixture was then quenched with water, the organic layer was removed
and the aqueous layer was extracted with CH2Cl2 (3 Â 20 ml). The
combined organic layer was concentrated and column chromato-
graphed (70:30 hexane: EtOAc) to give 500 mg (65%) of the boronate
1
ester 4 as a viscous solid. H NMR (400 MHz, CDCl3): d 8.70 (d, 1 H,
J=1.8 Hz, Ar-H), 7.95 (dd, 1 H, J=1.9, 8.4 Hz, Ar-H), 7.63 (d, 1 H,
J=8.3 Hz, Ar-H), 3.76 (s, 4 H, 2 Â OCH2), 3.41 (s, 3 H, NCH3), 1.51 (s,
9 H, NBoc), 1.02 (s, 6 H, 2 Â CH3); HRMS calculated for C16H26BN2O4
(MH+) : 321.1986, found: 321.2010.
[5-(7 - Dipropylamino - 2,5-dimethyl-1H-pyrazolo[1,5-a]pyrimidin-3-yl)-
pyridin-2-yl]methyl carbamic acid tert-butyl ester (6)
A suspension of the aryl bromide 5 (174 mg, 0.535 mmol), aryl boronate
ester 4 (150 mg, 0.643 mmol) and PdCl2(PPh3)2 (37 mg, 10 mol%) in 1,2-
dimethoxyethane (2 ml) was thoroughly deaerated and stirred under
argon. Deionised water (200 ml) and aqueous NaOH (550 ml, 2 M) were
added to the reaction mixture and heated at 1008C for 5 h. The reaction
mixture was diluted with EtOAc, dried over MgSO4, passed through a
short pad of celite, concentrated and column chromatographed (90: 10
Hexane: EtOAc) to give the desired product as a pale yellow viscous
solid in 150 mg (63%) yield. 1H NMR (400 MHz, CDCl3) d 8.77 (d, 1 H,
J=2.3 Hz, Ar-H), 8.05 (dd, 1 H, J=2.4, 8.6 Hz, Ar-H), 7.65 (d, 1 H,
J=8.5 Hz, Ar-H), 5.82 (s, 1 H, 6C-H), 3.71 (t, 4 H, J=7.8 Hz,
2 Â NCH2), 3.43 (s, 3 H, NCH3, 2.56 (s, 3 H, Ar-CH3), 2.46 (s, 3 H,
Ar-CH3), 1.72 (m, 4 H, 2 Â CH2), 1.53 (s, 9 H, NBoc), 0.95 (t, 6 H,
J=7.4 Hz, 2 Â CH3); HRMS calculated for C25H37N6O2 (MH+):
453.2978, found: 453.2989.
3-[(6 - methylamino)pyridin-3 - yl] - 2,5 - dimethyl - N,N-dipropylpyrazolo
[1,5-a]pyrimidine-7-amine (7)
A solution of the Boc-amine 6 (68 mg, 0.1504 mmol) in CH2Cl2 (1 ml)
was treated dropwise with CF3COOH (1 ml) and was stirred at r.t. for
3 h. The reaction mixture was then diluted with CH2Cl2 and neutralized
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SYNTHESIS OF [11C]CRF1 LIGAND
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with saturated NaHCO3 solution. The aqueous layer was extracted with
EtOAc, the combined organic layer was washed with H2O, brine and
concentrated and column chromatographed (4% CH2Cl2 in CH3OH) to
yield the product as pale yellow solid in 91% (48 mg) yield. m.p.
1
136–1378C, H NMR (400 MHz, CDCl3): d 8.39 (d, 1 H, J=9.0 Hz,
Ar-H), 8.14 (s, 1 H, Ar-H), 6.79 (d, 1 H, J=9.2 Hz, Ar-H), 5.82 (s, 1 H,
6C-H), 3.71 (t, 4 H, J=7.3 Hz, 2 Â NCH2), 3.00 (s, 3 H, N-CH3), 2.52 (s,
3 H, Ar-CH3), 2.46 (s, 3 H, Ar-CH3), 1.71 (m, 4 H, 2 Â CH2), 0.95 (t,
6 H, J=7.2 Hz, 2 Â CH3); HRMS calculated for C20H29N6 (MH+):
353.2431, found: 353.2454.
3 - [(6 - dimethylamino)pyridin - 3 -yl]-2,5-dimethyl-N,N-dipropylpyrazolo
[1,5-a]pyrimidine-7-amine (8)
A stirred solution of the amine 7 (30 mg, 0.085 mmol) in THF (1.0 ml)
was treated dropwise at –788C with NaHMDS (1.0 M in THF,
0.128 mmol, 128 ml). A solution of methyl iodide (10% v/v in THF,
159 ml, 0.256 mmol) was added to the reaction mixture and stirred
further for 20 min, maintaining the temperature at –788C. The reaction
was quenched by the addition of water. The reaction mixture was
diluted with CH2Cl2, dried over MgSO4, concentrated and column
chromatographed (4% methanol in CH2Cl2) to give the product as a
pale yellow solid in 93% (28 mg) yield. m.p. 1028C, 1H NMR (300 MHz,
CDCl3): d 8.48 (d, 1 H, J=2.3 Hz, Ar-H), 7.91 (dd, 1 H, J=2.3, 8.8 Hz,
6
Ar-H), 6.65 (d, 1 H, J=8.8 Hz, Ar-H), 5.79 (s, 1 H, C-H), 3.70 (t, 4 H,
J=7.7 Hz, 2 Â NCH2), 3.12 (s, 6 H, N(CH3)2), 2.54 (s, 3 H, Ar-CH3),
2.45 (s, 3 H, Ar-CH3), 1.70 (m, 4 H, 2 Â CH2), 0.94 (t, 6 H, J=7.4 Hz,
2 Â CH3); HRMS calculated for C21H31N6 (MH+): 367.2610, found:
367.2599.
Radiosynthesis of [N-methyl-11C]-3-[(6-dimethylamino)pyridin-3-yl]-
2,5-dimethyl-N,N-dipropylpyrazolo[1,5-a]pyrimidine-7-amine ([11C]8)
The desmethylamine 7 (1.5 mg) was dissolved in 400 ml of anhydrous
acetone in a capped 5 ml V-vial and was cooled to À208C. [11C]-Methyl
triflate was transported by a stream of argon (20–30 ml/min) into the
vial for 5 min at À208C. At the end of the trapping, the reaction mixture
was directly injected into a semi preparative RP-HPLC (Phenomenex
C18, 10 Â 250 mm, 10 m) and eluted with a solvent system (flow rate:
5 ml/min) comprising of acetonitrile: water: triethylamine (85: 15: 0.3).
Copyright # 2003 John Wiley & Sons, Ltd.
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The precursor 7 appeared at 5–6 min during the HPLC analyses. The
product fraction with a retention time of 9–10 min based on g-detector
was collected, diluted with 100 ml of deionized water, and passed
through a classic C-18 Sep-Pak catridge. The Sep-Pak was further
washed with deionized water (2 Â 50 ml) to ensure the complete removal
of triethylamine. Reconstruction of the product in 1 ml of absolute
ethanol afforded [11C]8 (5% yield based on EOB). A portion of the
ethanol solution was analyzed by analytical HPLC (Phenomenex C18)
to determine the specific activity and radiochemical purity. Then the
final solution of the [11C]8 in 10% ethanol-saline was analyzed to
confirm the chemical and radiochemical purities.
Acknowledgements
This work was supported by a research grant from the National
Institutes of Health (R21MH066620). The authors also thank Ms Julie
Arcement for her assistance in the radiolabeling studies.
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