[76Br]BMK-I-152, a non-peptide analogue for PET imaging of corticotropin-releasing hormone type 1 receptor (CRHR1)

Article abstract of DOI:10.1002/jlcr.1616

The study of corticotropin-releasing hormone is of significant interest in mental health. We have developed a radiobromination procedure for the preparation of [⁷⁶Br]BMK-I-152, a high-affinity corticotropin- releasing hormone type 1 receptor an

Full text of DOI:10.1002/jlcr.1616

Research Article
Received 14 January 2009,
Revised 24 April 2009,
Accepted 18 May 2009
Published online 7 July 2009 in Wiley Interscience
(www.interscience.wiley.com) DOI: 10.1002/jlcr.1616
[76Br]BMK-I-152, a non-peptide analogue for
PET imaging of corticotropin-releasing
hormone type 1 receptor (CRHR1)
Ã
L. Lang,a Y. Ma,a B. M. Kim,b E. M. Jagoda,a K. C. Rice,b L. P. Szajek,c
C. Contoreggi,b P. W. Gold,d G. P. Chrousos,e W. C. Eckelman,f and
D. O. Kiesewettera
The study of corticotropin-releasing hormone is of significant interest in mental health. We have developed a
radiobromination procedure for the preparation of [76Br]BMK-I-152, a high-affinity corticotropin-releasing hormone type
1 receptor antagonist. The radiobromination procedure resulted in the formation of two radiobrominated products from
the same trialkyltin precursor. Utilizing the results of several reaction conditions and the chromatographic and mass
spectral data obtained from Waters Acquity and Q-TOF, we determined that both 3-bromo and 4-bromo isomers could be
obtained. The authentic sample of the 3-bromo isomer was prepared to confirm the identity of a previously unknown
radioactive side product; affinity assays revealed that the 4-bromo isomer had ꢀ70 times higher affinity than that of the
3-bromo compound. By manipulation of reaction conditions, the individual products could be selected. Under no-carrier-
added conditions at room temperature in aqueous acetonitrile, the major radioactive product (480%) was identified as the
3-[76Br]bromo-4-tributylstannyl analogue of BMK-I-152. The 4-[76Br]bromo isomer accounted for less than 1% of the total
activity. The 3-[76Br]bromo BMK-I-152 could be obtained by treating this intermediate with trifluoroacetic acid to effect
removal of the trialkyltin. If the radiobromination was conducted after first evaporating the water from the aqueous
ammonium hydroxide solution of [76Br]bromide, the desired 4-[76Br]bromo isomer was obtained with a 58% radiochemical
yield.
Keywords: corticotropin-releasing hormone; radiobromination; bromine-76; CRH; PET
bis-methoxyethyl substituent on the 4-amino of the triazolo-
triazine, was the first high-affinity radioligand that crossed the
Introduction
Considerable effort has been put into the development of an
imaging probe for corticotropin-releasing hormone receptors
(CRHR). Corticotropin-releasing hormone (CRH) is an endogen-
ous 41-amino-acid neuropeptide found in the brain and was first
isolated from the hypothalamus of sheep.1 The CRH system
plays an important role in regulating the brain functions in
response to stress. Abnormal function of CRH receptors has
been implicated in anxiety,2 depression3 and stress-related
syndromes.4–6 Specifically, CRHR1 densities were reduced in the
prefrontal cortex of patients with depression, while CRHR1
densities were increased in the cerebral cortex of Alzheimer’s
patients.7,8 A PET ligand for CRHR1 would be able to monitor
in vivo receptor changes in normal and abnormal states and
evaluate the effect of treatment of disorders involving the CRH
system. BMK-I-152 [8-(4-bromo-2,6-dimethoxyphenyl)-2,7-di-
methylpyrazolo[1,5 - a][1,3,5]triazin -4-yl]-N,N-bis(2-methoxyethyl)
amine is an improved non-peptide high-affinity ligand designed
for imaging CRH type 1 receptor over our previously reported
ligand MJL 1-109-2 labeled with bromine-76 (Figure 1).9
Previous attempts to develop a PET or a SPECT ligand based
on antalarmin or CP-154,526 yielded ligands with very high
lipophilicity and, thus, were unsuitable for imaging the
brain.10,11 [76Br]MJL 1-109-2, a derivative of CP-154,526 with
blood brain barrier due to its lower lipophilicity (C log p = 3.05).
Recently, a C-11-labeled compound C-11 DMP696 with a
chemical structure similar to that of MJL 1-109-2 has also been
evaluated for PET imaging.12 Our newest molecule, BMK-I-152,
contains two extra methoxy groups on the phenyl ring further
aPET Radiochemistry Group, National Institute of Biomedical Imaging and
Bioengineering, National Institutes of Health, Building 10, Room 1C401,
10 Center Drive MSC 1180, Bethesda, MD 20892, USA
bChemical Biology Research Branch, National Institute on Drug Abuse and
National Institute on Alcohol Abuse and Alcoholism, Rockville, MD, USA
cPET Department, Clinical Center, National Institutes of Health, Bethesda,
MD, USA
dMood and Anxiety Research Program, National Institute of Mental Health,
Bethesda, MD, USA
eFirst Department of Pediatrics, University of Athens Medical School, Children’s
Hospital Aghia Sophia, Athens, Greece
fMolecular Tracer, LLC, Bethesda, MD, USA
*Correspondence to: L. Lang, PET Radiochemistry Group, National Institute of
Biomedical Imaging and Bioengineering, National Institutes of Health, Building 10,
Room 1C401, 10 Center Drive MSC 1180, Bethesda, MD 20892, USA.
E-mail: llang@mail.nih.gov
J. Label Compd. Radiopharm 2009, 52 394–400
Copyright r 2009 John Wiley & Sons, Ltd.

L. Lang et al.
reducing the lipophilicity, increasing the brain uptake, and
reducing the non-specific binding. In this article, we describe the
radiochemical challenges in the radiobromination of BMK-I-152.
Results and discussion
Chemistry
The syntheses of BMK-I-152 (13), its 3-bromo analogue (15), and
its tributylstannyl analogue (12b), the precursor for radiolabel-
ing, are outlined in Schemes 1 and 2. A commercially available
aldehyde (1) was protected with a benzyl group on its phenol
moiety and the corresponding benzyl ether 2 was then treated
with toluenesulfonylmethyl isocyanide (TosMIC)13 to provide the
arylacetonitrile derivative 3 in 84% yield. Synthesis from the
cyanide 3 to compound 9 was carried out basically following
the procedure developed by DuPont chemists.14 Deprotection
of the benzyl protecting group (H2, Pd/C) followed by
conversion to triflate yielded compound 11, which was
converted to the trimethyltin precursor 12a or tributyltin
precursor 12b for the radiobromination.
The trimethyltin precursor 12a was treated with N-bromo-
succinimide (NBS) in THF to provide the non-radioactive
4-bromo derivative, 13 (BMK-I-152), almost quantitatively. An
authentic standard of the 3-bromo compound 15 was prepared
by catalytic hydrogenolysis of the 4-bromo substituent using
10% Pd/C, which provided 14 in 92.6% yield. Treatment of 14
with NBS provided the 3-bromo compound 15.
MeO
OMe
MeO
OMe
N
N
N
N
N
N
N
N
N
N
N
N
N
N
OMe
Cl
MeO
Br
Br
BMK-I-152
antalarmin
MJL1-109-2
MeO
OMe
N
N
N
N
N
N
N
Cl
N
Cl
DMP696
CP-154,526
Figure 1. Structures of CRH ligands.
O
CN
OMe
CN
OMe
CHO
CHO
Na, EtOAc
heat
t-BuOK, TosMIC
BnCl, K2CO3
MeO
OMe
MeO
OMe
MeO
MeO
acetone, reflux
1,2-dimethoxyethane
-60 ~ -50 ˚C
OH
OBn
OBn
70-85%
OBn
1
2
3
4
O
N
AcOH
HN
H
H2N
NH
NH.HCl
N
N
HN
N
N
N
N
, AcOH
Na, EtOCOOEt
EtOH
NH2NH2, H2O, AcOH
toluene
OEt
MeO
OMe
MeO
OMe
MeO
OMe
CH3CN
50-60%
quant.
OBn
OBn
OBn
5
6
7
MeO
MeO
OMe
OMe
Cl
N
N
N
N
N
N
N
N
N
N
N
OMe
MeO
H3C
NEt2
N
N
N
N
H
10% Pd/C
MeOH
MeO
OMe
MeO
OMe
MeO
OMe
POCl3, 24h
THF, rt
91%
OBn
OH
OBn
85%
OH
t-Bu
Pd(PPh3)4, LiCl,
R3SnSnR3, dioxane
8
10
9
MeO
MeO
OMe
N
Bu-t
OMe
N
N
N
N
N
N
N
Tf2O
pyr
N
N
MeO
OMe
MeO
OMe
57%
SnR3
OTf
12a (R = Me)
12b (R = n-Bu)
11
Scheme 1. Synthesis of precursors for radiolabeling.
J. Label Compd. Radiopharm 2009, 52 394–400
Copyright r 2009 John Wiley & Sons, Ltd.
www.jlcr.org

L. Lang et al.
MeO
OMe
MeO
OMe
MeO
OMe
MeO
OMe
N
N
N
N
N
N
N
N
NBS
THF
NBS
THF
H2, Pd/C
MeOH
N
N
N
N
N
N
N
N
N
N
N
N
OMe
Br
OMe
OMe
OMe
MeO
MeO
MeO
MeO
SnMe3
Br
12a
13 (BMK-I-152)
14
15
Scheme 2. Synthesis of BMK-I-152 and its 3-bromo analogue.
MeO
OMe
MeO
OMe
MeO
OMe
N
N
N
N
N
N
N
N
N
N
N
N
N
76Br]NH4Br
Peracetic acid
N
N
[
OMe
+
OMe
OMe
MeO
76Br
MeO
MeO
SnBu3
76Br
76Br]13
SnBu3
16
12b
[
MeO
OMe
MeO
OMe
N
N
N
N
N
N
N
N
N
TFA
OMe
76Br
OMe
76Br
N
MeO
MeO
SnBu3
[
76Br]15
16
MeO
OMe
MeO
OMe
MeO
TFA
OMe
N
N
N
N
[
76Br]NH4Br
N
N
N
N
N
N
N
N
N
OMe
OMe
76Br
N
OMe
N
Peracetic acid
MeO
MeO
MeO
SnBu3
[
76Br]15
12b
14
Scheme 3. Radiosynthesis of [76Br]BMK-I-152 and its 3-bromo analogue.
the long-retained peak from this no-carrier-added synthesis was
collected from the HPLC and treated with trifluoroacetic acid to
remove the tributyltin and reaction mixture was re-injected onto
the HPLC. A single radioactive product was obtained with the
same retention time as the BMK-I-152 standard and the long-
retained radioactive component had completely disappeared.
After analyzing the tin precursor, we concluded that in order to
explain this result, the bromine-76 must be attached at a
position ortho to the tributyltin group. We also obtained this
radioactive product by treating the tributyltin precursor first
with TFA in order to remove the tributyltin and then conduct
the direct radiobromination.
In the mean time, we also utilized the newly acquired high-
resolution Waters Acquity Ultra Performance LC (UPLC) system
coupled with highly sensitive Waters Q-TOF MS to analyze the
radioactive products. The observed mass of the long-retained
chromatographic peak was m/z 783/785, which is consistent with
that of tributyltin precursor plus bromine. Most interestingly, the
mass spectral data for the products obtained from two TFA
treatments to remove tributyltin group before and after radio-
bromination as described earlier (m/z 493) were consistent with the
desired BMK-I-152 but the elution time was not co-incident with the
authentic standard prepared by chemical means. The subsequent
Radiochemistry
Bromine-76 was produced by cyclotron irradiation of high-purity
arsenic targets and isolated by chromic acid dissolution and
oxidation followed by simple distillation of [76Br]HBr.15 The
radionuclide was trapped in an aqueous solution of NH4OH. Our
initial radiolabeling attempts followed previously published
procedures.9 An aliquot of aqueous solution containing
[76Br]NH4Br was added to a reaction vial. The trimethylstannyl
substrate in acetonitrile was added, followed by the addition of
a dilute solution of peracetic acid at room temperature (rt)
(Scheme 3). Unlike the previous labeling experience with MJL
1-109-2, only a small amount of radioactive BMK-I-152 was
observed. Most of the bromine-76 radioactivity was highly
retained on the C-18 HPLC column until a 401min elution with
20% methanol and 80% acetonitrile. In order to analyze this
long-retained peak, a carrier-added reaction was performed.
However, when NH4Br was added at equal molar ratio to the tin
substrate, only the desired product was obtained with a low
radiochemical yield and the long-retained radioactive peak was
negligible. Since this long-retained peak had even longer
retention time than the tributyltin precursor, we postulated
that this radioactive peak still had the tributyltin attached to the
molecule (compound 16 in Scheme 3). To test our hypothesis, chemical synthesis and characterization of the 3-bromo isomer (15)
www.jlcr.org
Copyright r 2009 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2009, 52 394–400

L. Lang et al.
proved that the radiochemical product formed was indeed the 3- Varian Gemini spectrometer in CDCl3 with tetramethylsilane (TMS)
bromo isomer. The Acquity UPLC chromatography system proved as the internal standard. Mass spectra (MS) and HRMS were
capable of separating 3-bromo from 4-bromo isomers by about recorded on JEOL SX 102a. Combustion analyses were performed
0.2 min, a separation not obtained on standard HPLC columns.
Since the 3-bromo isomer (15) only differed from the desired
by Atlantic Microlabs, Inc., Norcross, GA, USA.
All UPLC-MS experiments were performed with a Waters
product by a single position change, we decided to investigate Q-TOF Premier (Waters, Miford, MA, USA) coupled with Waters
the biological properties. To our dismay, the affinity of 3-bromo Acquity UPLC system. LC/MS analysis employed an Acquity
isomer (15) is much lower than that of 4-bromo compound (13). BEH Shield RP18 column (150 ꢁ 2.1 mm) interfaced to the
We evaluated the Ki’s of the two non-labeled isomers in monkey Waters Q-TOF MS. The elution profile had the following
frontal cortex via competition with 125I ovine-CRH, a ligand components: initial conditions 95% 25 mM ammonium acetate;
specific for CRHR1. The BMK-I-152 (13) had a Ki of 0.3570.05 nM, 5% CH3CN; gradient 5–95% acetonitrile over 5 min; isocratic
whereas the 3-bromo isomer (15) had a Ki of 24.474.9 nM.
In light of the in vitro affinity data, we required a new synthetic
elution at 95% acetonitrile for an additional 10 min.
approach to allow the high-yield preparation of the 4-[76Br]bromo 4-Benzyloxy-2,6-dimethoxybenzaldehyde (2)
isomer (13). We first tested the effect of temperature on the
To a solution of 2,6-dimethoxy-4-hydroxybenzaldehyde (10.04 g,
labeling yield. By gradually increasing the temperature, the yield
of the desired product showed an upward trend with the yield
reaching about 5% at 1001C. At higher temperatures (1201C in
acetonitrile), the yield of 4-[76Br]bromo compound was increased
to 10% with the 3-[76Br]bromo compound still as the major
product. We then concluded that lower temperature favored the
formation 3-[76Br]bromo isomer, whereas higher temperature
favored the formation of 4-[76Br]bromo isomer. With even higher
temperatures (1501C, DMF as the solvent and preheating before
adding the peracetic acid), the yield increased to 30%; however,
the product also contained the 3-[76Br]bromo isomer (15), which
was not separable from the 4-[76Br]bromo isomer (13) using
regular reversed-phase HPLC. The 3-[76Br]bromo isomer (15) was
probably produced at a high temperature from the 3-[76Br]bromo
tributyltin compound due to the presence of acetic acid.
Although we had a procedure that would give useable amounts
of the 4-[76Br]bromo isomer, the reaction was variable as to yield
and ratio of the 3-[76Br]bromo to the 4-[76Br]bromo isomer.
We formed the hypothesis that the high-temperature reaction
procedure would result in evaporation of most of the water. The
reaction was done in an open vial with small volumes of aqueous
[76Br]bromide and the precursor was preheated before the addition
of the oxidant. Perhaps the presence of water was detrimental to
the desired formation of the 4-isomer via bromodestannylation. We
changed the procedure to first evaporate the aqueous [76Br]-
bromide to dryness, followed by the addition first of the substrate
in dry DMF and, second, by oxidizing the reagent with a minimal
amount of water. Under these conditions, the 4-[76Br]bromo isomer
was obtained in 50% radiochemical yield contaminated with about
1% of the 3-[76Br]bromo isomer. The radiochemical yield for
4-[76Br]bromo isomer was high even when the temperature was
reduced to 501C. The final radiochemical product was purified by
HPLC and obtained in 57.979.0% (n= 8) radiochemical yield with a
52.35 mmol) in acetone (150 mL) were added K2CO3 (21.71 g,
157.1 mmol) and benzyl bromide (8.10 mL, 68.06 mmol). The
mixture was heated to reflux for 14 h, at which time TLC
indicated no starting material. The mixture was cooled to rt,
filtered through a fritted glass filter, and concentrated under
reduced pressure. The residue was recrystallized from CHCl3/
petroleum ether. The precipitate was filtered and washed with
copious amount of petroleum ether to provide 14.02 g (98%
yield) of tan solid after drying. 1H NMR (CDCl3) d 0.36 (1H, s), 7.43
(4H, m), 6.16 (2H, s), 5.13 (2H, s), 3.86 (6H, s); m.p. 118–1191C; MS
(M11)1 273.2; HRMS calcd. for C16H17O4 273.1127, obsd.
273.1124.
(4-Benzyloxy-2,6-dimethoxyphenyl)acetonitrile (3)
A solution of TosMIC (Aldrichs, 11.80 g, 60.4 mmol) in 50 mL 1,2-
dimethoxyethane was added dropwise to a stirred suspension of
tert-BuOK (13.70 g, 114 mmol) in 50 mL of 1,2-dimethoxyethane
in
a three-necked flask while maintaining the reaction
temperature at ꢂ401C to ꢂ301C. A solution of 4-benzyloxy-
2,6-dimethoxybenzaldehyde (15.0 g, 55.1 mmol) in 1,2-di-
methoxyethane (150 mL) was added to the reaction mixture
dropwise over a period of about 30 min while keeping the
reaction temperature at ꢂ60 to ꢂ501C. Stirring was continued
for 2.5 h at ꢂ60 to ꢂ501C. At this point, TLC (hexane–EtOAc 3:1,
v/v) of the reaction mixture showed no trace of the starting
material. Methanol (120 mL) was added at once and the mixture
was heated to reflux for 15 min. The mixture was cooled to rt,
concentrated under reduced pressure, and partitioned between
0.5 M citric acid (100 mL) and dichloromethane (50 mL) and the
aqueous layer was extracted with more dichloromethane
(50 mL ꢁ 2). Combined organic layer was washed with saturated
aqueous NaHCO3 solution (50 mL), the organic layer dried over
anhydrous MgSO4, and concentrated under reduced pressure.
The residue was dissolved in CHCl3 and filtered through a silica
gel bed (ca 10 cm) and briefly eluted with hexane–EtOAc 3:1
(v/v). The residue was recrystallized from EtOAc–hexane to give
radiochemical purity of 498% and
33.077.8 GBq/mmol (n=4).
a specific activity of
Experimental
1
13.02 g (84% yield) of pale yellow plate crystals. H NMR (CDCl3)
d 7.42 (5H, m), 6.21 (2H, s), 5.06 (2H, s), 3.82 (6H, s), 3.60 (2H, s);
m.p. 96–971C; MS (M11)1 283; HRMS calcd. for C17H18O3N
283.1208, obsd. 283.1203; elemental analysis calcd. for
C17H17O3N C 72.07, H 6.05, N 4.94, found C 71.92, H 6.03, N 4.98.
General
Chemicals were purchased from Aldrichs and used without further
purification unless otherwise noted. TLC analyses were carried out
on Analtech silica gel GHLF 0.25 mm plates with UV and I2
detection. Column chromatography was carried out with silica gel
2-(4-Benzyloxy-2,6-dimethoxyphenyl)-3-oxobutyronitrile (4)
˚
(ICN SiliTech 32-63, 60 A). Melting points (m.p.) were determined in
open glass capillaries on a Thomas-Hoover m.p. apparatus and are
uncorrected. 1H NMR spectra were recorded at 300MHz on a
To a solution of (4-benzyloxy-2,6-dimethoxyphenyl)acetonitrile
(10.77 g, 38.01 mmol) in ethyl acetate (45 mL) were added
J. Label Compd. Radiopharm 2009, 52 394–400
Copyright r 2009 John Wiley & Sons, Ltd.
www.jlcr.org

L. Lang et al.
sodium pellets portionwise at rt. Upon completion of adding the diethyl carbonate (21.3 mL, 0.176 mol). The mixture was heated
sodium, the reaction mixture was heated to reflux. After ca to reflux for 6 h. After cooling to rt, the white precipitate was
50 min, a turbid white precipitate started to appear. The reaction filtered and washed with fresh ethanol (30 mL). The solid was
mixture was allowed to stir at the reflux temperature for another dissolved in 0.1 N aqueous HCl solution and extracted with ethyl
hour. After cooling to rt, more ethyl acetate (90 mL) was added acetate (50 mL ꢁ 3). Combined organic layers were washed with
to the white suspension to facilitate stirring, and the resultant saturated aqueous sodium bicarbonate solution, dried over
mixture was stirred at rt overnight. The mixture was filtered and MgSO4, and filtered. Concentration under reduced pressure
the white solid collected was washed with copious amounts of followed by recrystallization from ethyl acetate provided 6.23 g
1
ethyl ether (500 mL). The solid was collected and suspended in (15.3 mmol, 92% yield) of a white solid. H NMR (CDCl3) d 10.86
water (100 mL) and the solution was acidified to pH = ca 5 using (1H, br s), 7.42 (5H, m), 6.31 (2H, s), 5.10 (2H, s), 3.72 (6H, s), 2.46
0.1 N aqueous HCl solution. It was extracted with ethyl acetate (3H, 2), 2.56 (3H, s); MS (M11) 407.2; HRMS calcd. for C22H23O4N4
(50 mL ꢁ 3) and combined organic layers were dried (MgSO4), 407.1719, obsd. 407.1726.
filtered, and concentrated under reduced pressure. After
pumping at high vacuum, the oily residue converted to a white 8-(4-Benzyloxy-2,6-dimethoxyphenyl)-4-chloro-2,7-dimethylpyrazo-
solid (9.08 g, 73% yield). 1H NMR (CDCl3) d 7.41 (5H, m), 6.24 (2H, s),
lo-[1,5-a][1,3,5]triazine (8)
5.08 (1H, s), 5.07 (2H, s), 3.82 (6H, s), 2.16 (3H, s); m.p. 109–1101C;
A mixture of 8-(4-benzyloxy-2,6-dimethoxyphenyl)-2,7-dimethyl-
MS (M11)1 326.2; HRMS calcd. for C19H20O4N 326.1393, obsd.
3H-pyrazolo[1,5-a][1,3,5]triazin-4-one (7) (4.50 g, 11.1 mmol) and
326.1390; elemental analysis calcd. for C19H19O4N C 70.14, H 5.89, N
N,N-diethyl-p-toluidine (4.33 mL, 24.4 mmol) in POCl3 (50 mL)
4.31, found C 70.00, H 5.93, N 4.43.
was heated to reflux for 24 h. The mixture was cooled to rt and
POCl3 was removed in vacuo. The residue was partitioned
4 - (4 - Benzyloxy-2,6 - dimethoxyphenyl)-5-methyl-2H-pyrazol-3 - yl -
between ice-water (30 mL) and ethyl acetate (50 mL) and the
amine (5)
organic layer was washed with brine, dried over anhydrous
A mixture of 2-(4-benzyloxy-2,6-dimethoxyphenyl)-3-oxobutyro- MgSO4, and concentrated under reduced pressure to a minimal
nitrile (7.29 g, 22.4 mmol), hydrazine monohydrate (1.63 mL, volume. The residue was quickly passed through a silica gel bed
33.6 mmol), and acetic acid (2.14 mL, 37.4 mmol) in toluene (ca 10 cm) and eluted with hexane–EtOAc (1:4, v/v). The
(60 mL) was heated to reflux while removing water using a concentration of the filtrate provided 3.66 g (78% yield) of a
Dean-Stark trap for 5 h. The reaction mixture was cooled to rt brownish gum, which was used for the next reaction without
1
and concentrated using rotary evaporation. To the residue was further purification. H NMR (CDCl3) d 7.42 (5H, m), 6.33 (2H, s),
added 6 N aqueous HCl solution and the resulting suspension 5.12 (2H, s), 3.71 (6H, s), 2.61 (3H, s), 2.36 (3H, s).
was extracted with ether (30 mL ꢁ 2). The aqueous layer was
cooled to 01C and basified with concentrated ammonium [8 - (4-Benzyloxy-2,6-dimethoxyphenyl)-2,7-dimethylpyrazolo[1,5-a][1,3,
5]triazin-4-yl]bis(2-methoxyethyl)amine (9)
hydroxide solution to pH ca 11. The resulting mixture was
extracted with ethyl acetate (100 mL ꢁ 3) and the combined
organic layers were dried over anhydrous MgSO4 and concen-
trated to give a pale yellow foam (7.20 g, 95% yield). 1H NMR
(CDCl3) d 7.41 (5H, m), 6.30 (2H, s), 5.09 (2H, s), 3.75 (6H, s), 2.07
(3H, s); MS (M11)1 340.2; HRMS calcd. for C19H22O3N3 340.1662,
obsd. 340.1655.
To 8-(4-benzyloxy-2,6-dimethoxyphenyl)-4-chloro-2,7-dimethyl-
pyrazolo-[1,5-a][1,3,5]triazine (8) (3.65 g, 8.59 mmol) in dry THF
(30 mL) was added bis(2-methoxyethyl)amine (1.90 mL,
12.9 mmol) and the mixture was stirred for 1 h. The solvent
was removed under reduced pressure and the residue was
purified on a silica gel chromatographic column (elution with
hexane–EtOAc 4:1, v/v) to give 2.89 g (64% yield) of a pale
yellow solid. 1H NMR (CDCl3) d 7.43 (5H, m), 6.31 (2H, s), 5.10
(2H, s), 4.30 (4H, m), 3.76 (4H, t, J = 5.4 Hz), 3.72 (6H, s), 3.38
N-[4-(4-benzyloxy-2,6-dimethoxyphenyl)-5-methyl-2H-pyrazol-3-
yl]acetamidine; acetic acid salt (6)
Glacial acetic acid (1.21 mL, 21.2 mmol) was added to a stirred (6H, s), 2.37 (3H, s), 2.19 (3H, s); m.p. 124–1251C; elemental
mixture of 4-(4-benzyloxy-2,6-dimethoxyphenyl)-5-methyl-2H- analysis calcd. for C28H35N5O5 C 64.47, H 6.76, N 13.43, found C
pyrazol-3-ylamine (7.15 g, 21.7 mmol) and ethyl acetimidate 64.54, H 6.70, N 13.42; MS (M11)1 522; HRMS calcd. for
hydrochloric acid salt (3.93 g, 31.6 mmol) in acetonitrile (90 mL) C28H36O5N5 522.2716, obsd. 522.2715.
and the mixture was stirred for 18 h at rt. A white precipitate was
formed. Ethyl ether (70 mL) was added and the solid was filtered,
washed with ethyl ether (40 mL), and dried under vacuum to
give a white solid (7.33 g, 77% yield), which was used directly for
the next step. H NMR (CDCl3) d 9.66 (1H, br s), 9.00 (1H, br s),
8.59 (1H, br s), 7.43 (5H, m), 6.42 (2H, s), 5.17 (2H, s), 3.69 (6H, s),
2.30 (3H, s), 2.11 (3H, s), 2.00 (3H, s); m.p. 214–2151C.
4 - f-[Bis(2-methoxyethyl)amino]-2,7-dimethyl-pyrazolo[1,5-a][1,3,5]-
triazin-8-ylg -3,5-dimethoxyphenol (10)
1
To a solution/suspension of [8-(4-benzyloxy-2,6-dimethoxyphe-
nyl) - 2,7-dimethylpyrazolo[1,5 - a][1,3,5]triazin - 4 - yl]bis(2 - meth-
oxyethyl)amine (9) (2.80 g, 5.37 mmol) in methanol (75 mL) was
added 10% Pd/C (de Gussa type, Aldrich) and the mixture was
stirred under hydrogen atmosphere for 1 h at rt. After 1 h, when
TLC indicated no starting material, the mixture was diluted with
ethyl acetate (200 mL) and filtered through a bed of Celites
8-(4-Benzyloxy-2,6-dimethoxyphenyl)-2,7-dimethyl-3H-pyrazo-
lo[1,5-a][1,3,5]triazin-4-one (7)
Sodium (5.06 g, 0.22 mol) was added portionwise to absolute (4 cm). The filtrate was concentrated under reduced pressure to
ethanol (200 mL) in a three-necked r.b. flask equipped with a give a white solid (2.28 g, 98% yield). 1H NMR (CDCl3) d 5.87 (2H, s),
condenser. The solution was cooled to rt and N-[4-(4-benzyloxy- 4.37 (4H, m), 3.79 (4H, t, J = 5.4 Hz), 3.60 (6H, s), 3.40 (6H, s), 2.50
2,6 - dimethoxyphenyl) - 5 -methyl - 2H - pyrazol-3-yl]acetamidine; (3H, s), 2.17 (3H, s); m.p. 183–1841C; elemental analysis calcd. for
acetic acid salt (6) (7.33 g, 16.7 mmol) was added followed by C21H29O5N5.0.5H2O C 57.26, H 6.86, N 15.89, found C 57.56, H 6.61,
www.jlcr.org
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J. Label Compd. Radiopharm 2009, 52 394–400

L. Lang et al.
N 15.97; MS (M11)1 432.2; HRMS calcd. for C21H30O5N5 432.2247, hexane, v/v), which provided 284 mg (0.335 mmol, 52% yield) of
1
obsd. 432.2232.
a white solid. H NMR (CDCl3) d6.70 s), 4.27 (4H, m), 3.75 (4H, t,
J = 6.0 Hz), 3.75 (6H, s), 3.37 (6H, s), 2.35 (3H, s), 2.19 (3H, s), 1.58
Trifluoromethanesulfonic acid 4-f4-[bis(2-methoxyethyl)amino]-2,7- di- (6H, m), 1.35 (6H, m), 1.06 (6H, m), 0.90 (9H, t, J = 7.3 Hz);
methylpyrazolo[1,5-a][1,3,5]triazin-8-ylg -3,5-dimethoxyphenyl ester (11)
elemental analysis calcd. for C33H55N5O4Sn C 56.26, H 7.87, N
9.94, found C 56.44, H 7.89, N 9.69.
To a solution of 4- f4-[bis(2-methoxyethyl)amino]-2,7-dimethyl-
pyrazolo[1,5-a][1,3,5]triazin-8-ylg -3,5-dimethoxyphenol (10) (300 mg,
0.695 mmol) in dry pyridine (1.0mL) was added trifluorometha-
nesulfonic anhydride (129mL, 0.765 mmol) at 01C. The mixture
[8-(4-Bromo-2,6-dimethoxyphenyl)-2,7-dimethyl-pyrazolo[1,5-
a][1,3,5]triazin-4-yl]bis(2-methoxyethyl)amine (13)
was stirred at 01C for 30min and then at rt overnight. The mixture To a solution of f8-[2,6-dimethoxy-4-(trimethylstannyl)-phenyl]-
was partitioned between water (3mL) and ethyl acetate (3mL). 2,7 - dimethylpyrazolo[1,5 - a][1,3,5]triazin - 4 -ylg - bis(2 - methox-
The aqueous layer was extracted with ethyl acetate (3 mLꢁ 2). yethyl)amine (12a) (27.7 mg, 0.0467 mmol) in dry THF (0.3 mL)
Combined ethyl acetate layers were washed with water (5mL), was added NBS (9.1 mg, 0.0514 mmol) and the mixture was
1 N aqueous HCl solution (3mL), water, and brine, dried (over stirred for 1 h at rt. The solvent was removed in vacuo and the
anhydrous MgSO4), and the concentrated crude product was residue was purified on a silica gel chromatographic column
purified on a silica gel chromatographic column (elution with (eluted with hexane–EtOAc 5:1, v/v) to provide 21.0 mg
1
hexane–EtOAc 4:1, v/v) to provide 224 mg (0.397mmol, 57% (0.0425 mmol, 91% yield) of a pale yellow solid. H NMR (CDCl3)
yield) of a white foam. 1H NMR (CDCl3) d 6.54 (2H, s), 4.31 (4H, m), d 6.79 (2H, s), 4.30 (4H, m), 3.76 (4H, t, J = 5.7 Hz), 3.74 (6H, s), 3.38
3.76 (4H, t, J = 5.6 Hz), 3.76 (6H, s), 3.39 (6H, s), 2.38 (3H, s), 2.17 (3H, s), 2.37 (3H, s), 2.17 (3H, s); m.p. 112–1131C; elemental
(3H, s); MS (M11)1 564.2; HRMS (M1) calcd. for C22H28F3N5O7S analysis calcd. for C21H28BrNO4 ꢃ 0.5 acetone C 51.63, H 5.97, N
564.1740, obsd. 564.1730.
13.37, obsd. C 51.95, H 6.01, N 13.52; MS (M11)1 494; HRMS
calcd. for C21H30BrN5O4 494.1403, obsd. 494.1403.
f8 - [2,6 -Dimethoxy-4-(trimethylstannyl)-phenyl]-2,7-dimethylpyra-
zolo[1,5-a][1,3,5]triazin-4-ylg -bis(2-methoxyethyl)amine (12a)
[8-(2,6-Dimethoxyphenyl)-2,7-dimethyl-pyrazolo[1,5-a][1,3,5]tria-
zin-4-yl]bis(2-methoxyethyl)amine (14)
To a solution of trifluoromethanesulfonic acid 4- f4-[bis(2-
methoxyethyl)amino]-2,7-dimethylpyrazolo[1,5-a][1,3,5]triazin- To compound 13 (107 mg, 0.216 mmol) in methanol (5.0 mL)
8-ylg-3,5-dimethoxyphenyl ester (11) (216mg, 0.383 mmol) in was added 10 wt% Pd/C (40 mg, Aldrich) and the flask was filled
dioxane (2.5 mL) were added lithium chloride (48 mg, 1.13 mmol), with hydrogen gas. The mixture was stirred under atmospheric
tetrakis(triphenylphosphine)palladium (12 mg, 0.0104 mmol), hydrogen (balloon) for 30 min. The mixture was filtered through
one crystal of 2,6-di-tert-butyl-4-methylphenol, and hexamethyl- a Celite pad (ꢀ2 cm) and the filtrate concentrated under
ditin (180 mg, 0.549 mmol) under argon. The mixture was reduced pressure. The residue was purified on a silica gel
heated to 1101C for 3 h and cooled to rt. It was diluted with column (eluted with hexane–EtOAc 5:1–2:1 and 10% methanol/
ethyl acetate (3 mL) and washed with 10% aqueous ammonium CH2Cl2) to give 83.1 mg (0.200 mmol, 92.6% yield) of compound
hydroxide solution (5 mL) and the organic layer was filtered 14 as a white solid. 1H NMR (CDCl3) d 7.28 (1H, t, J = 8.7 Hz), 6.65
through a bed (1 cm) of Celite. The filtrate was washed with (2H, d, J = 8.7 Hz), 4.30 (4H, br), 3.76 (4H, t, J = 5. Hz), 3.76 (6H, s),
water (5 mL) and brine (5 mL), dried (anhydrous MgSO4), and the 3.39 (6H. s), 2.37 (3H, s), 2.20 (3H, s); HRMS (TOF MS ES1) calcd.
concentrated crude product was purified on silica gel column for C21H30N5O4 416.2298, obsd. 416.2280.
chromatography (elution with 4:1 hexane–EtOAc, v/v) to provide
194 mg (0.335 mmol, 88% yield) of a white solid. 1H NMR (CDCl3) [8-(3-Bromo-2,6-dimethoxyphenyl)-2,7-dimethyl-pyrazolo[1,5-
a][1,3,5]triazin-4-yl]bis(2-methoxyethyl)amine (15)
d 6.75 (2H, s), 4.32 (4H, m), 3.76 (4H, t, J = 6.0Hz), 3.76 (6H, s), 3.39
(6H, s), 2.36 (3H, s), 2.21 (3H, s), 0.32 (9H, s); m.p. 153–1551C; MS
(M11)1 578, 580; elemental analysis calcd. for C24H37N5O4Sn C
49.85, H 6.45, N 12.11, obsd. C 50.06, H 6.53, N 11.70.
To a magnetically stirred solution of [8-(2,6-dimethoxyphenyl)-2,7-
dimethyl-pyrazolo[1,5 - a][1,3,5]triazin - 4 -yl]bis(2-methoxyethyl)amine
(14) (50.0 mg, 0.120 mmol) in THF (2.0 mL) was added NBS
(29 mg, 0.163 mmol) and the mixture was stirred for 15 min at rt.
The solvent was evaporated under reduced pressure and the
mixture was purified on a silica gel chromatographic column
(eluted with hexane–EtOAc 7:1–4:1) to provide compound 15
(56.0 mg, 95% yield) as a white solid. 1H NMR (CDCl3, 300MHz) d 7.49
(1H, d, J= 8.7 Hz), 6.66 (1H, d, J= 8.7 Hz), 4.32 (4H, br s), 3.77
(4H, t, J= 5.7 Hz), 3.73 (3H, s), 3.54 (3H, s), 3.39 (6H, s), 2.38 (3H, s), 2.19
(3H, s); HRMS (TOF MS ES1) calcd. for C21H29N5O4Br 494.1403, obsd.
494.1405.
f8 - [2,6-Dimethoxy-4-(tributylstannyl)-phenyl]-2,7-dimethylpyrazo-
lo[1,5-a][1,3,5]triazin-4-ylg -bis(2-methoxyethyl)amine (12b)
To a solution of trifluoromethanesulfonic acid 4-f4-[bis(2-
methoxyethyl)amino]-2,7-dimethylpyrazolo[1,5-a][1,3,5]triazin-8-
ylg-3,5-dimethoxyphenyl ester (438 mg, 0.777 mmol) in dioxane
(4.5 mL) were added lithium chloride (99 mg, 2.33 mmol),
tetrakis(triphenylphosphine)palladium (27 mg, 0.0233 mmol),
one crystal of 2,6-di-tert-butyl-4-methylphenol, and hexabutyl-
ditin (506 mL, 1.01 mmol) under argon. The mixture was heated
to 1101C for 22 h and then cooled to rt. It was diluted with ethyl
acetate (20 mL) and washed with 10% aqueous ammonium
hydroxide solution (10 mL) and the organic layer was filtered
through a bed (2 cm) of Celite. The filtrate was washed with
water (20 mL) and brine (20 mL), dried (over anhydrous MgSO4),
Radiolabeling
[8-(4-[76Br]bromo-2,6-dimethoxyphenyl)-2,7-dimethyl-pyrazolo[1,5-
a][1,3,5]triazin-4-yl]bis(2-methoxyethyl)amine
and the concentrated crude product was purified on silica gel An aliquot of the aqueous solution of [76Br] ammonium
Chromatotrons (4 mm plate, elution with 5, 7, and 10% EtOAc in bromide (10–20 mL, 20.0–40.0 MBq) was added to a 1 mL
J. Label Compd. Radiopharm 2009, 52 394–400
Copyright r 2009 John Wiley & Sons, Ltd.
www.jlcr.org

L. Lang et al.
reaction vial and the solvent was evaporated with argon flow.
Tributyltin substrate 12b (200 mg) in 50 mL of acetonitrile was
added to the vial containing the [76Br] radioactivity and was
followed by the addition of 2 mL of 37% peracetic acid in 10 mL of
acetonitrile. The vial was sealed and placed on an 801C heating
block and heated for 30 min. At the end of the reaction, the
reaction mixture was loaded onto a Phenomenex C-18 (2)
column (250 ꢁ 4.6 mm) and eluted with 100 mM ammonium
acetate/acetonitrile (40/60) at a flow rate of 1.2 mL/min. The
radioactivity peak containing the desired product (tR = 15 min)
was collected and analyzed with another HPLC system for
specific activity and radiochemical purity determination. Chemi-
cal purity and m/z identification utilized UPLC-MS.
Acknowledgement
This work was supported by the intramural programs of the
National Institute of Biomedical Imaging and Bioengineering,
the National Institute on Drug Abuse, the National Institute on
Alcohol Abuse and Alcoholism, and the National Institute on
Diabetes and Digestive and Kidney Diseases.
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Copyright r 2009 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2009, 52 394–400