Synthesis of3H-, 13C3-, and 14C-labeled Sch 727965

Article abstract of DOI:10.1002/jlcr.1845

The preparation of [³H]Sch 727965 from unlabeled compound and tritiated water was base catalyzed. Diethyl [¹³C₃]malonate was used to prepare [¹³C₃]Sch 727965 in five steps in 21.8% overall yield. In a similar manner, [¹⁴C]Sch 727965 was prepared in five steps from diethyl [2-¹⁴C]malonate in 11.1% radiochemical yield. Copyright

Full text of DOI:10.1002/jlcr.1845

Research Article
Received 21 July 2010,
Revised 15 September 2010,
Accepted 30 September 2010
Published online 30 November 2010 in Wiley Online Library
(
wileyonlinelibrary.com) DOI: 10.1002/jlcr.1845
3
Synthesis of H-, C -, and C-labeled
13
14
3
Sch 727965
Ã
C. Flader Lavey, D. Hesk, D. Koharski, V. Truong, and P. McNamara
3 13
The preparation of [ H]Sch 727965 from unlabeled compound and tritiated water was base catalyzed. Diethyl [ C
3
]malonate
1
was used to prepare [ C
3
14
]Sch 727965 in five steps in 21.8% overall yield. In a similar manner, [ C]Sch 727965 was prepared
3
1
in five steps from diethyl [2- C]malonate in 11.1% radiochemical yield.
4
Keywords: carbon-13; carbon-14; Sch 727965; synthesis
Introduction
Bioscan 1000 linear analyzer using the solvent systems
indicated.
The cyclin-dependent kinases (CDKs) are crucial for executing
HPLC analyses were performed on a Waters instrument
1
the correct order of the phases of the cell cycle. This is required equipped with a Waters 2487 dual programmable wavelength
for normal cell growth. Inhibitors of the CDKs can be used to
treat diseases such as cancer, which is characterized as Radiomatic Flo-Scint III liquid scintillation cocktail. [ H] and
detector and a Radiomatic C150TR radioflow detector utilizing
3
2
14
uncontrolled cell proliferation, by causing apoptosis. Sch [ C]Sch 727965 were analyzed by HPLC for radiochemical and
3
27965 (1, Figure 1) is a CDK inhibitor that can potentially be
13
7
3
chemical purity. [ C ]Sch 727965 was analyzed for chemical
used for the treatment of diseases associated with the CDKs, purity by HPLC. The following systems were used for in process
such as cancer. Sch 727965 was labeled with three different and final product chemical and radiochemical (where appro-
isotopes within our group. Initially, the compound was tritiated
for preliminary ADME work. This was followed by the synthesis
of carbon-13-labeled Sch 727965 for use as an internal standard 254 nm, 0.05 M aqueous triethylammonium acetate pH 9:
priate) purity:
System 1: Zorbax Extend C18 column, 3.0 mm I.D. ꢀ 150 mm,
for Bioanalytical support and carbon-14-labeled Sch 727965 for
definitive ADME work. The synthesis of each of these labeled
compounds is described.
acetonitrile (72:28) at 0.5 ml/min for 15 min followed by a step
gradient to acetonitrile for 15 min.
System 2: Agilent SB-C3 column, 3.0 mm ID ꢀ 150 mm,
260 nm, 301C, water:acetonitrile:trifluoroacetic acid (80:20:0.1)
at 0.5 ml/min for 15 min followed by a step gradient to
Experimental
acetonitrile for 15 min.
System 3: Waters Xterra RP-18 column, 4.6 mm ID ꢀ 150 mm,
Low specific activity tritiated water and diethyl [2- C]malonate 260 nm, 301C, 0.05 M aqueous triethylammonium acetate pH
Materials
1
4
1
3
were purchased from GE Healthcare and diethyl [ C ]malonate 10:acetonitrile (75:25) at 1 ml/min for 20 min followed by a step
3
gradient to acetonitrile for 15 min.
was purchased from Isotec. All anhydrous solvents and reagents
not listed above were purchased from commercial suppliers
(
3
Acros Organics, Fisher, or Aldrich Chemical Company) and used Synthesis of [ H]Sch 727965 (2)
as received. Compounds 1, 3, 6, and 8 were obtained from
Sch 727965 (1, 11.8 mg, 0.0298 mmol), sodium hydroxide (4.7 mg,
.118mmol), dioxane (0.10ml), and low specific activity tritiated
water (478 mCi, 50 Ci/ml, 900 mCi/mmol) were added to a 1 ml
either Medicinal or Process Chemistry.
0
Methods
screw-cap reaction vial. The reaction mixture was heated at 801C
Proton NMR spectra were recorded on either a 400 MHz Mercury for 16h, then cooled to RT and diluted with methylene chloride
4
00BB spectrometer or a 500 MHz Varian INOVA spectrometer. (5 ml) and 10% sodium bicarbonate (5ml). The organic layer was
removed and the aqueous layer was extracted with methylene
Mass spectra were recorded on either a JEOL MStation double-
focusing magnetic sector mass spectrometer operating in the
FAB ionization mode or a Waters ZQ2000 LC/MS with an APCI
source operating in the positive mode. Quantitation of radio- Merck Research Laboratories, 2015 Galloping Hill Road, Kenilworth, NJ 07033,
USA
activity was performed using
a Packard 2200CA liquid
scintillation analyzer and Scintiverse BD cocktail. Thin layer
chromatography was performed using Whatman LK6CF (silica
*Correspondence to: C. Flader Lavey, Merck Research Laboratories, 2015
Galloping Hill Road, Kenilworth, NJ 07033, USA.
gel 60) 5 ꢀ 20 cm, 0.25 mm plates. The plates were scanned on a E-mail: carolee.lavey@spcorp.com
J. Label Compd. Radiopharm 2011, 54 196–201
Copyright r 2010 John Wiley & Sons, Ltd.

C. F. Lavey et al.
give compound 4 plus salts (4.82 g). This mixture was used
directly in the next reaction.
N
N
1
3
5
,6,7-[ C ]-5,7-Dichloro-3-ethyl-pyrazolo[1,5-a]pyrimidine (5)
3
N
N
To the round-bottomed flask containing a mixture of compound
and salts (4.82 g, assume 0.0141 mol) was added anhydrous
4
OH
HN
acetonitrile (35 ml). The mixture was stirred in an ice bath under
argon while phosphorus oxychloride (13 ml, 0.140 mol) was
added dropwise, followed by N,N-dimethylaniline (5.36 ml,
0
.0423 mol). The resulting brown solution was warmed to RT
+
N
-
and stirred for 10 min, then heated at 851C for 23 h. The thick,
dark red colored reaction was cooled to RT and added to 200 ml
of ice to give compound 5 as a yellow solid. The aqueous
mixture was extracted with methylene chloride (3 ꢀ 100 ml) and
the combined organic extracts were dried (sodium sulfate),
filtered, and evaporated to give crude 5 (4.5 g) as a brown solid.
The compound was purified by column chromatography (110 g
O
Sch 727965 (1)
Figure 1. Structure of Sch 727965.
chloride (2ꢀ 5 ml). The combined organic extracts were washed silica gel; 5:95 ethyl acetate:hexanes) to give 5 as a pale yellow
1
with water (3ml), dried (sodium sulfate), filtered, and evaporated, solid (2.26 g, 73%). H NMR (400 MHz, CDCl
): d 8.10 (s, 1H), 6.92
3
3
and the resulting crude [ H]Sch 727965 (2, 39 mCi, RCP 97.3%, (d, 1H), 2.82 (q, 2H), 1.32 (t, 3H).
HPLC system 1) was dissolved in ethanol (10ml) for storage at
801C. Compound 2 was purified by RP-HPLC [Zorbax Extend 5,6,7-[ C ]-(5-Chloro-3-ethyl-pyrazolo[1,5-a]pyrimidin-7-yl)-(1-oxy-
1
3
ꢁ
3
C18, 9.4 mm I.D. ꢀ 250mm, 254nm, 0.05 M aqueous triethylam- pyridin-3-ylmethyl)-amine (7)
monium acetate pH 9:acetonitrile (72:28) at 6 ml/min]; 37.8mCi of
3
Compound
1
5 (2.10 g, 9.59 mmol), 3-(aminomethyl)pyridine
-oxide hydrochloride (6, 1.76 g, 10.93 mmol), sodium bicarbo-
[
H]Sch 727965 (2) at a specific activity of 1.39 Ci/mmol was
obtained. The radiochemical purity as determined by radio-HPLC
System 1) and radio-TLC plate scanning (5:95 2 M methanolic
ammonia:methylene chloride; R = 0.18) was 98.9 and 99.1%,
respectively. H-NMR (500 MHz, CDCl ): d 8.25 (s), 8.18 (m), 5.22 (m),
.05 (m), 4.50 (m), 1.75 (m).
nate (2.42 g, 28.77 mmol), and anhydrous acetonitrile (48 ml)
were added to a round-bottomed flask. The light yellow mixture
was heated at 701C under argon for 48 h, then cooled to RT.
Water (150 ml) was added and the aqueous mixture was
extracted with methylene chloride (6 ꢀ 100 ml). The combined
organic extracts were washed with brine (150 ml), dried (sodium
sulfate), filtered, and evaporated to give crude 7 (3.2 g) as a
yellow solid. The residue was purified by column chromato-
graphy (96 g silica gel; gradient = 0:100 to 8:92 [methanol:
(
f
3
3
5
1
3
Synthesis of ½ C3ꢂ Sch 727965 (9)
1
3
5
,6,7-½ C3ꢂ-3-Ethyl-4H-pyrazolo[1,5-a]pyrimidine-5,7-dione (4)
methylene chloride]) to give 7 as a light yellow solid (2.55 g,
3
-Amino-4-ethylpyrazole oxalate (3, 2.50g, 0.0124mol) was
1
7%). H NMR (400 MHz, d
8
8
2
6
-DMSO): d 8.95 (dt, 1 H), 8.32 (s, 1 H),
.12 (d, 1 H), 8.06 (s, 1 H), 7.36 (m, 2 H), 6.27 (d, 1 H), 4.60 (t, 2 H),
.63 (q, 2 H), 1.21 (t, 3 H).
added to a separatory funnel containing saturated aqueous
sodium bicarbonate (150ml). The aqueous solution was extracted
with acetone:ethyl acetate (1:1, 4 ꢀ 250 ml) and methanol:ethyl
acetate (1:4, 3 ꢀ 250 ml). The combined organic extracts were
washed with brine (200ml), dried (sodium sulfate), filtered, and
evaporated to give a yellow/orange solid. Ethyl acetate (40 ml)
13
,6,7-[ C ]-2-(1-{3-Ethyl-7-[(1-oxy-pyridin-3-ylmethyl)-amino]-
5
pyrazolo[1,5-a]pyrimidin-5-yl}-piperidin-2-yl)-ethanol (9)
3
was added and the mixture was filtered to remove salts that were Compound 7 (2.40 g, 7.82 mmol), piperidine 8 (1.52 g, 11.73 mmol),
soluble in the (1:4) methanol:ethyl acetate washes. The filtrate sodium bicarbonate (1.97 g, 23.46 mmol), and 1-methyl-2-pyrrolidi-
was evaporated to give 3-amino-4-ethylpyrazole as a yellow/ none (3.4 ml) were combined and heated, under argon, at 1501C
orange solid (1.14 g, 83%). This process was repeated using 1.00g for 48 h. The reaction was cooled to RT. Water (20 ml) and ethyl
of 3-amino-4-ethylpyrazole oxalate to give another batch of acetate (50 ml) were added and the mixture was stirred at 401C for
3-amino-4-ethylpyrazole (0.44 g, 80%). These batches were 10 min. The aqueous layer was removed while the mixture was
combined to give a total of 1.57g (0.0141 mol) of 3-amino-4- warm, 25% aqueous sodium chloride (30 ml) was added, and the
1
ethylpyrazole. Ethanol (16.9 ml) and diethyl [ C
3
3
]malonate mixture was stirred at 401C for 10 min. The aqueous layer was
(
2.38 g, 0.0145mol) were added and the clear, orange solution removed while the mixture was warm and the combined aqueous
was stirred at RT under argon. Sodium ethoxide (21% by weight layers were back-extracted with ethyl acetate (3 ꢀ 30 ml). The
in ethanol, 10.86 ml, 0.0290mol) was added dropwise and the combined organic extracts were dried (sodium sulfate), filtered, and
reaction became cloudy, then thick orange/brown. A reflux evaporated to give crude 9 (6.5 g) as a brown oil. The material was
condenser was attached and the reaction was heated at 781C for purified by column chromatography (110 g silica gel; gradi-
1
8h, then cooled to RT. Ethanol (5 ml) was added and the ent = 0:100 to 20:80 [methanol:ethyl acetate]) to give recovered 7
13
3
reaction was heated at 781C for 24h before being cooled to RT. (0.50 g) and [ C ]Sch 727965 (9, 1.81 g, 58%) as a yellow foam.
TLC analysis (5:95 7 M methanolic ammonia : methylene chloride; Compound 9 contained silica gel from the chromatography, so it
R [3-amino-4-ethylpyrazole]= 0.45; R [4] = baseline) indicated the was taken up in methylene chloride (100 ml) and filtered through a
f
f
13
reaction was complete. Hydrochloric acid (12M, 2.7 ml) was sintered glass funnel. The filtrate was evaporated to give [ C
3
]Sch
added and the reaction was stirred for 10min and evaporated to 727965 (1.73 g) as a yellow/brown foam. In order to remove 0.7%
J. Label Compd. Radiopharm 2011, 54 196–201
Copyright r 2010 John Wiley & Sons, Ltd.
www.jlcr.org

C. F. Lavey et al.
1
4
of compound 7, the batch was taken up in ethyl acetate (25 ml) 6-[ C]-(5-Chloro-3-ethyl-pyrazolo[1,5-a]pyrimidin-7-yl)-(1-oxy-pyri-
and heated at 801C. The hot suspension was cooled to RT and the din-3-ylmethyl)-amine (12)
mother liquor was removed by filtration to give compound 9 as a
A mixture of compound 11 (68.2 mCi, 1.26 mmol), 3-(amino-
light brown solid (1.34 g, 43%) with a chemical purity of 99.6% by
methyl)pyridine 1-oxide hydrochloride (6, 0.23 g, 1.44 mmol),
an area normalization HPLC assay (System 1). FAB Mass Spectro-
sodium bicarbonate (0.32 g, 3.78 mmol), and anhydrous acet-
metry of unlabelled Sch 727965 (1): m/z 397.236 (M). FAB Mass
1
onitrile (6.8 ml) was heated at 701C under argon for 22.5 h, then
Spectrometry of 9: m/z 400.246 (M). H NMR of 9 (500 MHz,
-DMSO): d 8.30 (s, 1 H), 8.11 (m, 1 H), 7.98 (dt, 1 H), 7.68 (s, 1 H),
.38 (m, 2 H), 5.58 (d, 1 H), 4.73 (t, 1 H), 4.57 (m, 3 H), 4.28 (broad d,
H), 3.34 (m, 2 H), 2.81 (t, 1 H), 2.49 (q, 2 H), 1.76 (m, 1 H), 1.60
cooled to RT. Radio-TLC analysis (9:91 methanol: methylene
chloride; R = 0.91) indicated that the radiochemical purity of 12
d
7
1
6
f
was 93%. Water (8 ml) was added and the aqueous mixture was
extracted with methylene chloride (5 ꢀ 8 ml). The combined
organic extracts were washed with brine (8 ml), dried (sodium
sulfate), filtered, and evaporated to give the crude product as a
yellow solid (61.5 mCi). The residue was purified by column
chromatography (19 g silica gel; gradient = 0:100 to 8:92
(m, 6 H), 1.35 (m, 1 H), 1.18 (t, 3 H).
1
Synthesis of [ C]Sch 727965 (13)
4
1
-[ C]-3-Ethyl-4H-pyrazolo[1,5-a]pyrimidine-5,7-dione (10)
4
[
(
methanol:methylene chloride]) to give 12 as a thin film
61.9 mCi, 90.7% radiochemical yield).
6
3
-Amino-4-ethylpyrazole oxalate (3, 1.50 g, 12.4 mmol) was
added to a separatory funnel containing saturated aqueous
sodium bicarbonate (100 ml). The aqueous solution was
extracted with acetone:ethyl acetate (1:1, 3 ꢀ 150 ml) and
methanol:ethyl acetate (1:4, 3 ꢀ 150 ml). The combined organic
extracts were dried (sodium sulfate), filtered, and evaporated to
give a yellow oil. Ethyl acetate (40 ml) was added and the
mixture was filtered to remove salts that were soluble in the
14
-[ C]-2-(1-{3-Ethyl-7-[(1-oxy-pyridin-3-ylmethyl)-amino]-pyrazo-
6
lo[1,5-a]pyrimidin-5-yl}-piperidin-2-yl)-ethanol (13)
Compound 12 (33.0 mCi, 0.61 mmol), piperidine 8 (119 mg,
0
1
.92 mmol), sodium bicarbonate (154 mg, 1.83 mmol), and
-methyl-2-pyrrolidinone (0.53 ml) were combined and heated,
under argon, at 1501C for 44 h. The clear, dark red reaction
was cooled to RT. Radio-TLC (16:84 methanol:ethyl acetate)
(
1:4) methanol:ethyl acetate. The filtrate was evaporated to
f
indicated that the reaction was 11% product (13; R = 0.11)
give 3-amino-4-ethylpyrazole as a brown solid (0.75 g, 90%).
Ethanol (1.1 ml) was added to 3-amino-4-ethylpyrazole (0.20 g,
and 64% compound 12 (R = 0.20). Water (10 ml) and ethyl
f
14
acetate (10 ml) were added and the mixture was stirred
at 401C for 10 min. The aqueous layer was removed while
the mixture was warm, 25% aqueous sodium chloride (8 ml)
was added, and the mixture was stirred at 401C for 10 min.
The aqueous layer was removed while the mixture was warm
and the combined aqueous layers were back-extracted with
ethyl acetate (3 ꢀ 10 ml). The combined organic extracts were
dried (sodium sulfate), filtered, and evaporated to give the
crude product as a red solid (27.6 mCi). The material was
dried under vacuum overnight, then piperidine 8 (119 mg,
1.80 mmol), followed by diethyl [2- C]malonate (100 mCi, 54 mCi/
mmol, 1.85 mmol). The vial originally containing the diethyl
14
[
2- C]malonate was rinsed with ethanol (3 ꢀ 0.33 ml) and these
washes were added to the reaction. The resulting clear, orange
solution was stirred at RT under argon while sodium ethoxide
(21% by weight in ethanol, 1.38 ml, 3.71 mmol) was added
dropwise. The reaction mixture became cloudy, then thick orange
and it was heated under reflux at 781C for 24 h, then cooled to RT.
The reaction was complete by TLC (5:95 7 M methanolic
ammonia:methylene chloride; R [3-amino-4-ethylpyrazole] = 0.33;
f
0
1
.92 mmol), sodium bicarbonate (154 mg, 1.83 mmol), and
-methyl-2-pyrrolidinone (0.53 ml) were added and the mixture
R
f
[10] = 0.02). Hydrochloric acid (12 M, 0.35 ml) was added and the
reaction was stirred for 10 min, then evaporated to give compound
0 plus salts (0.61 g, assume 99.4 mCi by subtracting activity
was heated, under argon, at 1501C for 95 h. The reaction
was cooled to RT. Radio-TLC (16:84 methanol:ethyl acetate)
indicated that the reaction was 26% product (13). The majority
of 12 was gone and a number of byproducts were present.
Water (10 ml) and ethyl acetate (10 ml) were added and the
mixture was stirred at 401C for 10 min. The aqueous layer was
1
present in rotary evaporator liquid waste). This mixture was used
directly in the next step after further drying under vacuum.
1
-[ C]-5,7-Dichloro-3-ethyl-pyrazolo[1,5-a]pyrimidine (11)
4
6
To the round-bottomed flask containing a mixture of compound removed while the mixture was warm, 25% aqueous sodium
1
0 and salts (99.4 mCi, 0.61 g, assume 1.80 mmol) was added chloride (8 ml) was added, and the mixture was stirred at 401C
anhydrous acetonitrile (4.5 ml). The mixture was stirred in an ice for 10 min. The aqueous layer was removed while the mixture
bath under argon while phosphorus oxychloride (1.66 ml, was warm and the combined aqueous layers were back-
17.82 mmol) was added dropwise, followed by N,N-dimethylani- extracted with ethyl acetate (3 ꢀ 10 ml). The combined organic
line (0.68 ml, 5.40 mmol). The resulting brown solution was extracts were dried (sodium sulfate), filtered, and evaporated to
warmed to RT and stirred for 10 min, then heated at 851C for give the crude product as a brown oil (24.7 mCi). The material
20.5 h. The reaction was cooled to RT and added to 25 ml of ice. was purified by column chromatography (15 g silica gel;
After the ice melted, the aqueous mixture was extracted with gradient = 0:100 to 20:80 [methanol:ethyl acetate]), then by
methylene chloride (4 ꢀ 10 ml) and the combined organic RP-HPLC [Zorbax SB-C3, 9.4 mm ID ꢀ 250 mm, 5 ml/min, 305 nm,
extracts were dried (sodium sulfate), filtered, and evaporated water:acetonitrile:trifluoroacetic acid (78:22:0.1), column was
1
4
to give a dark brown solid (88.2 mCi) that had a radiochemical cleaned with acetonitrile between injections] to give [ C]Sch
purity (RCP) of 64% by radio-TLC (15:85 ethyl acetate:hexanes; 727965 (13, 5.78 mCi, 17.5% radiochemical yield, RCP 98.0%
f
R = 0.52). The compound was purified by column chromato- by HPLC system 2, RCP 96.8% by HPLC system 3) as a yellow
graphy (60 g silica gel; 5:95 ethyl acetate:hexanes) to give 11 as a solid. APCI Mass Spectrometry of unlabelled Sch 727965 (1):
pale yellow solid (68.2 mCi, RCP 99.8%, 68.2% radiochemical m/z 397.19 (M11). APCI Mass Spectrometry of 13: m/z 399.21
1
yield from diethyl [2- C]malonate).
4
(M11).
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Copyright r 2010 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2011, 54 196–201

C. F. Lavey et al.
distributed throughout the compound as shown in Scheme 1.
The placement of tritium in the 2- and 6-positions of the
Results and discussion
3
[
H]Sch 727965 (2) was prepared, as shown in Scheme 1, pyridine N-oxide is supported by work discussed in a review of
4
by treating the unlabeled compound with four equivalents base-catalyzed exchange on heterocyclic compounds.
of sodium hydroxide and 478 mCi of tritiated water
900 mCi/mmol) in dioxane at 801C. The radiochemical purity a standard in a mass spectrometry based bioanalytical assay.
of the crude product was 97.3% and the compound was easily The synthesis, summarized in Scheme 2, was based on the route
Carbon-13-labeled Sch 727965 was prepared for use as
(
3
3,5,6
7
purified by reversed-phase HPLC to give 37.9 mCi of [ H]Sch that was developed and optimized by Medicinal
and Process
7
27965 (2) at a specific activity of 1.39 Ci/mmol. An analysis of a Chemistry and optimized by chemists in the Organic Preparations
portion of the batch by tritium NMR showed that the tritium was Group. The oxalate salt of 3-amino-4-ethylpyrazole (3) was
8
T
9
%
N
N
N
T
N
2
%
T
N
NaOH, THO, dioxane
0 °C, 16 h
N
T
N
N
T
1
%
HN
42%
T
OH
HN
OH
8
2
3%
N+
N+
O-
O-
2
3%
1
2
3
Scheme 1. Synthesis of [ H]Sch 727965 (2).
1
. NaHCO3
O
O
NH2
H
O
O
N
2
3
.
EtO
OEt
OH
.
N
HO
N
H
N
N
NaOEt/EtOH
. HCl
O
O
3
4
.
HCl
N+
H N
2
6
POCl , CH CN
O-
3
3
Cl
N
N,N-dimethylaniline
N
NaHCO , CH CN, 87%
3 3
N
(
73% from 3)
Cl
5
NH
Cl
N
N
N
8
N
N
N
N
OH
HN
OH
HN
NMP, NaHCO , 43%
3
N+
N+
O-
O-
= 13
C
7
9
13
Scheme 2. Synthesis of [
3
C ]Sch 727965 (9).
J. Label Compd. Radiopharm 2011, 54 196–201
Copyright r 2010 John Wiley & Sons, Ltd.
www.jlcr.org

C. F. Lavey et al.
1. NaHCO3
O
O
NH2
. HO
H
N
O
O
2.
EtO
OEt
OH
N
N
N
H
NaOEt/EtOH
. HCl
N
O
3
O
3
10
.
HCl
H2N
6
N+
POCl , CH CN
3
3
Cl
N
-
O
N
NaHCO , CH CN, 91%
3 3
N,N-dimethylaniline
N
14
(68% from [ C]diethyl
Cl
malonate)
11
NH
Cl
N
N
N
N
8
N
N
OH
N
HN
OH
HN
NMP, NaHCO , 18%
3
N+
N+
O-
O-
= 14C
12
13
14
Scheme 3. Synthesis of [ C]Sch 727965 (13).
neutralized using sodium bicarbonate, and the resulting free 11% conversion to 13. Compound 12 was still present in 64%,
1
3
base was condensed with diethyl [ C
was reacted with phosphorus oxychloride and N,N-dimethyl- reaction was subjected to an aqueous work-up to see if a side-
aniline in acetonitrile at 851C for 23 h to give a 73% yield of product interfering in the reaction progression could be
3
]malonate to give 4. Crude but a number of by-products had formed. As it had stalled, the
4
1
3
[
3
C ]pyrazolopyrimidine 5 after column chromatography. Com- removed. The crude organic mixture from the work-up was
pound 7 was formed in 87% yield by heating 5, pyridine N-oxide 6, dried under vacuum overnight. Fresh reagents were added and
and sodium bicarbonate in acetonitrile at 701C for 48h. the reaction was continued at 1501C for an additional 95 h to
1
3
Subsequently, [ C
compound 7 and piperidine 8 in 1-methyl-2-pyrrolidinone at material 12 remaining and numerous by-products generated.
501C for 48 h. The crude material was purified by column It is assumed that the poorer stability of the carbon-14-labeled
3
]Sch 727965 (9) was generated by heating give product at a radiochemical purity of 26%, with no starting
1
chromatography to give a 55% yield of 9 that contained 0.7% of 7 compound at elevated temperatures, relative to the unlabeled
by HPLC. This impurity was removed by adding ethyl acetate, compound, led to the lower yield of this reaction compared with
heating the mixture to 801C, cooling it to room temperature and the corresponding one carried out with carbon-13-labeled
filtering, resulting in a 77% recovery (43% yield) of purified material. Crude 13 was purified by column chromatography
1
3
[
ization HPLC assay.
3
C ]Sch 727965 (9), which was 99.6% pure by an area normal- followed by reversed-phase prep-HPLC to give a 20% radio-
1
chemical yield of [ C]Sch 727965 (13). A batch with a specific
4
1
4
[
to 9 (Scheme 3). The free base of the 3-amino-4-ethylpyrazole chemical yield of 11.1%.
4
oxalate salt (3) was condensed with diethyl [2- C]malonate to
C]Sch 727965 (13) was synthesized in an analogous manner activity of 56.0 mCi/mmol was synthesized in an overall radio-
1
give 10 after acidic work-up. Crude compound 10 was reacted Acknowledgement
with phosphorus oxychloride and N,N-dimethylaniline at 851C to
give [ C]pyrazolopyrimidine 11 in 68% yield following column The authors thank the Molecular Spectroscopy group for
1
4
1
3
3
chromatography. Subsequently, 12 was synthesized in 91% analysis of [ C ]Sch 727965 and Dr T. M. Chan from this group
yield from 11 and pyridine N-oxide 6. The initial attempt to for tritium NMR work. Thanks are also due to Process and
make 13 from compound 12 and piperidine 8 by heating at Medicinal Chemistry Researchers for intermediates and unlabeled
1501C in 1-methyl-2-pyrrolidinone for 44 h resulted in only an Sch 727965.
www.jlcr.org
Copyright r 2010 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2011, 54 196–201

C. F. Lavey et al.
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J. Label Compd. Radiopharm 2011, 54 196–201
Copyright r 2010 John Wiley & Sons, Ltd.
www.jlcr.org