Synthesis of several isotopically labeled pyrrolo[1,3-d]pyrimidine analogs

Article abstract of DOI:10.1002/jlcr.506

Four members of a novel class of pyrrolo[2,3-d]pyrimidines that show potential for the treatment of asthma and neurodegenerative disorders, have been prepared with radioisotope labels and in one case with multiple stable isotope labels for ADME studies as

Full text of DOI:10.1002/jlcr.506

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Labelled Cpd Radiopharm 2001; 44: 797–810.
DOI: 10.1002/jlcr.506
Research Article
Synthesis of several isotopically labeled
pyrrolo[1,3-d]pyrimidine analogs
John A. Easter* and Wayne T. Stolle
Global Drug Metabolism, Pharmacia Corporation, 301 Henrietta Street,
Kalamazoo, MI 49007, USA
Summary
Four members of a novel class of pyrrolo[2,3-d]pyrimidines that show potential
for the treatment of asthma and neurodegenerative disorders, have been
prepared with radioisotope labels and in one case with multiple stable isotope
labels for ADME studies as part of the drug development process. The
syntheses utilize an isotopically labeled 2,4,6-trisubstituted pyrimidine as a
common building block, readily prepared from isotopically labeled urea.
Cyclizations of the pyrimidine with bromo-ketones generate the ring fused
pyrrolo[2,3-d]-pyrimidines with elegant efficiency as demonstrated by the
preparation of structures 4; 5; 8 and 12. Copyright # 2001 John Wiley &
% % %
Sons, Ltd.
Key Words: radioactive; stable isotopes; pyrrolo[2,3-d]pyrimidines
Introduction
In 1990, Jacobsen et al. reported that the novel pyrrolo[2,3-d]pyrimi-
dine, 4 was a potent antioxidant and inhibitor of lipid peroxidation.1
%
Since then, several hundred analogs have been synthesized at the
Pharmacia Corporation. A number of the analogs have exhibited
potential as orally active antioxidants for the treatment of asthma and
*Correspondence to: J. A. Easter, Global Drug Metabolism, Pharmacia Corporation, 301 Henrietta
Street, Kalamazoo, MI 49007, USA
Copyright # 2001 John Wiley & Sons, Ltd.
Received 9 March 2001
Revised 11 May 2001
Accepted 6 June 2001

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J. A. EASTER AND W. T. STOLLE
neurodegenerative disorders.2 This paper describes the synthesis of four
of these analogs labeled with carbon-14 at the C-2 position of the
pyrimidine ring, and in one case, labeled with multiple stable isotopic
labels, for preclinical absorption, distribution, metabolism, and excre-
tion (ADME) studies, as part of the drug development process.
Discussion and results
The preparation of 2, as the penultimate intermediate for the synthe-
%
sis of the first three pyrrolopyrimidines, 4, 5, and 8 is illustrated in
% %
%
Scheme 1. Following the chemistry presented in an earlier publication,3
carbon-14 labeled urea (obtained from American Radiolabeled Chemi-
cals) was converted to [14C]barbituric acid and chlorodehydroxylated
with phenylphosphonic dichloride to give 2,4,6-trichloro-[2-14C]pyrimi-
dine. Treatment with an excess of pyrrolidine at low temperature gave
the dipyrrolidine-substituted intermediate 2, in excellent yield. Due to
%
the antioxidant nature of this class of compounds, and hence their
sensitivity toward molecular oxygen especially when in solution, all
solvents and solutions used were purged with nitrogen prior to use.
As shown in Scheme 2, a 25 mCi portion of 2 was treated with
%
methylamine in a sealed pressure tube at 1708C for 26 h to give N-
methyl-2,6-di-1-pyrrolidinyl-4-[2-14C]pyrimidinamine, 3 in 87% radio-
%
chemical yield (RCY). This intermediate was used in a novel cyclization/
dehydration reaction for the preparation of 4 and 5.2 In the presence of
%
%
the non-nucleophilic base, diisopropylethylamine, 3 underwent cycliza-
%
tion and dehydration with 2-bromoacetophenone to give 7.8 mCi of
product. The mesylate salt was prepared by treating with one equivalent
of methanesulfonicacid to give a 74% RCY of 4 having a specific activity
%
of 22.2 mCi/mg (9.85mCi/mmol) and radiochemical purity (RCP) of at
least 98% by both TLC and HPLC. In a similar manner, the cyclization/
dehydration reaction of 3 and 2-bromoacetone gave 8.2 mCi of 5 in 71%
%
%
Scheme 1.
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ISOTOPICALLY LABELED PYRROLO[1,3-D]PYRIMIDINE ANALOGS
799
Scheme 2.
RCY, having a specific activity of 30.8mCi/mg (9.9 mCi/mmol) after salt
formation with dry hydrogen chloride in methanol. The RCP as
determined by HPLC was 97% and by TLC was 98%.
A number of amines could be used to prepare 2,4,6-trisubstitued
pyrimidines as illustrated by the preparation of 8. The chloropyrimidine
%
2 was warmed in a pressure tube with aminoethylmorpholine (neat) for
%
24 h at 1558C. Reaction of the crude displacement product 6 with freshly
prepared 2-bromocyclohexanone in the presence of base, gave 15.3 mCi
of 7 in 50% RCY. Dehydrogenation of 7 with 10% palladium on
%
%
%
carbon as the catalyst in anhydrous decahydronaphthalene at 1608C.
This method was superior to the use of quinoline dehydrogenation
reagents (chloranil and DDQ). After 4.5 h, only a small amount of
starting material remained, no other side products were produced.
Filtration to remove the catalyst, followed by acid/base workup to
remove the decahydronaphthalene and purification by column chro-
matography gave, 4.9 mCi of 8, in 82% RCY, with a specific activity of
%
23.9 mCi/mg (10 mCi/mmol), and a RCP of 99% by both HPLC and
TLC.
Copyright # 2001 John Wiley & Sons, Ltd.
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J. A. EASTER AND W. T. STOLLE
Scheme 3.
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ISOTOPICALLY LABELED PYRROLO[1,3-D]PYRIMIDINE ANALOGS
801
A different approach was needed to prepare isotopically labeled 12 as
shown in Scheme 3. Direct nucleophilic displacement of chloride from 2
%
with glycine pyrrolidine amide was unsuccessful, possibly due to the
poor solubility of the amide in solvents like N,N-dimethylformamide.
A sample of 221 mCi of 1a was warmed with glycine ethylester
hydrochloride in the presence of diisopropylethylamine, to give a
mixture of products from either the displacement of the chloride at the
C-2 or C-4 of the pyrimidine ring in a rough statistical ratio of 1:2 (v/v).
Subsequent column chromatography using 25:75 v/v ethyl acetate : hex-
anes resulted in the isolation of 164 mCi of 9a, in 55% yield. Reaction of
9a with excess pyrrolidine at reflux gave 10a in excellent yield.
Cyclization of 10a with freshly prepared 2-bromocyclohexanone in the
presence of base gave 11a, in 89% RCY. Dehydrogenation of 11a using
10% palladium on carbon in anhydrous decahydronaphthalene at
1968C gave 31 mCi (10.3% overall RCY) of 12a, having a specific
activity of 112 mCi/mg (51 mCi/mmol) after salt formation with dry
hydrogen chloride in methanol. The RCP as determined by HPLC was
99.8% and by TLC was 98.7%.
A stable isotopomer of 12 was prepared using a parallel route shown
in Scheme 3, by substituting [13C, 15N2]urea, and diethyl [1,3-13C2]-
malonate as the unlabeled reagent precursors. Isolation of 379 mg
(0.84 mmol) of 12b was obtained with an isotopicenrichment of 99%
for 13C and >98% for 15N (nominally).
Materials and methods
A. Thin layer chromatography (TLC)
TLC analyses were performed on 2.5 ꢀ 10 cm glass plates precoated
with a 250 mm layer of silica gel GF (Analtech, Newark, DE). The
developed zones were visualized by UV (254 nm) light. TLC radio-
chromatograms were obtained with a Bioscan System 200 Imaging
Scanner. Integrated peak ratios were used to determine radiochemical
purity (RCP).
B. High performance liquid chromatography (HPLC)
HPLC analyses were carried out with a Spectra Physics 8700 solvent
delivery system. Chromatography for 4 and 5 was performed using a
%
%
Supelcosil LC-18 DB, 5 m, 4.6 mm I.D. ꢀ 250 mm column eluted with
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J. A. EASTER AND W. T. STOLLE
85:15 v/v methanol:buffer (buffer=1000 ml water, 5 ml triethylamine,
and 1.4 ml formic acid), pumped isocratically at 1.5 ml/min. Chromato-
graphy for compounds 8 and 12 was performed using a Zorbax Rx-C8,
%
5 m, analytical column (4.6 mm I.D. ꢀ 250 mm), eluted with 60:40 v/v
CH3CN:buffer (buffer=1000 ml water, 5 ml triethylamine, 5 ml formic
acid), pumped isocratically at 1.0 ml/min. All solvents were HPLC
grade. UV detection of the column effluent was performed with a LDC/
Milton Roy Spectromonitor-D variable wavelength detector set at
254 nm. Peak integration used the RadiomaticFlo-One Beta A280.
Integrated peak ratios at the stated wavelength were used to determine
the chemical purity. Radiochemical purity was determined from
integrated radiochromatograms using the Radiomatic Flo-One Beta
A280 radioactivity detector. A 2:1 (v/v) predetection mixing of Ultima
Flo-M1 (Packard Corp) liquid scintillation cocktail and column
effluent was used for the measurement.
C. Radioactivity measurement
Radioactivity determinations were performed with a Pharmacia Wallac
Model 1410 liquid scintillation spectrometer using the external standard
method. Ultima Gold1 (Packard Corp) was used as the liquid
scintillation cocktail. Specific activity determinations were done by
weighing out the compound of interest (ꢁ0.2 mg), dissolving it in
methanol, and counting a known volume of the solution in 10 ml of the
scintillation cocktail.
D. Nuclear magnetic resonance (NMR) spectroscopy
Structural determination was confirmed by proton NMR (1H-NMR)
using a Bruker AM 300 or a Bruker ARX 400 spectrometer at 300 or
400 MHz. The carbon-13 NMR (13C-NMR) was performed on the same
instruments at 75.5 and 100 MHz.
Experimental
N-Methyl-2,6-di-1-pyrrolidinyl-4-[2-14C]pyrimidinamine, 3
%
A mixture of 391.8 mg (1.55 mmol) of unlabeled 2 and 240.6 mg
%
(0.95 mmol, 25 mCi) of carbon-14 labeled 2 was placed in a 15 ml
%
pressure tube equipped with a Teflon magneticstirring bar, and a Teflon
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ISOTOPICALLY LABELED PYRROLO[1,3-D]PYRIMIDINE ANALOGS
803
stopper with a Viton1 O-ring seal. The solids were dissolved in 2 ml of
pyridine and to the solution, 8 ml of 40% methylamine/water was
added quickly under nitrogen atmosphere. A fine precipitate formed.
The pressure tube was stoppered and immersed in an oil bath at
1408C, which dissolved the fine solids. The oil bath temperature was
increased to 1708C and the reaction mixture was stirred for 26 h. The
tube was cooled in an ice bath, carefully opened under nitrogen
atmosphere, and the reaction mixture was mixed with 5 ml of water and
2 ml of ethyl acetate. The mixture was briefly concentrated under
reduced pressure. The aqueous mixture was further diluted with
5 ml of water, and cooled in an ice bath for 30 min. The resulting
solids were collected by filtration, washed with water, and dried
under high vacuum at room temperature to afford 537 mg of 3, in 86%
%
RCY, having a specific activity of 40.2 mCi/mg (9.94 mCi/mmol), and
RCP of 94% by TLC (97:3 v/v, methylene chloride:4 M ammonia
in methanol).
7-Methyl-6-phenyl-2,4-di-1-pyrrolidinyl-7H-pyrrolo[2,3-d,2-14C]pyrimi-
dine monomethanesulfonate, 4
%
A solution of 248 mg (1.0 mmol, 10 mCi) of the freshly prepared 3 in
%
6 ml of acetonitrile was mixed with 1.43 mmol of diisopropylethylamine
and 231 mg (1.16 mmol) of 2-bromoacetophenone. The resulting
solution was stirred at room temperature overnight under nitrogen
atmosphere. Copious solids had precipitated. The mixture was then
refluxed for 1 h and cooled under nitrogen. The resulting solids were
filtered, washed with cold acetonitrile and dried under high vacuum at
room temperature to afford 269 mg of product. The freshly prepared
free base, 265 mg (0.76 mmol), was suspended in 2 ml of methanol at
room temperature, and mixed with 1 ml of methanesulfonicaicd
solution (prepared from 1.83 g of methanesulfonicaicd, brought to
25 ml with methanol). The suspension cleared, and after 10 min, 4 ml
of ethyl acetate was added, and the solution was concentrated under
reduced pressure. The residue was triturated with 4 ml of ethyl
acetate and 8 ml of ether. The solids were filtered, washed with cold
ether, and dried under high vacuum at room temperature to afford
331 mg of 4, in 75% yield, having a specific activity of 22.2 mCi/mg
%
(9.85 mCi/mmol), and a RCP of 99% by HPLC and 98% by TLC
(95:5 v/v methylene chloride:acetone, sample prepared in 4 M ammonia
in methanol).
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J. A. EASTER AND W. T. STOLLE
6,7-Dimethyl-2,4-di-1-pyrrolidinyl-7H-pyrrolo[2,3-d,2-14C]pyrimidine
hydrochloride, 5
%
To a stirred solution of 286 mg (1.15 mmol, 11.5 mCi) of 3, in 6 ml of
%
acetonitrile (solvent was purged with nitrogen prior to use) under
nitrogen atmosphere at 08C 260 ml (1.5 mmol) of diisopropylethylamine
and 145 ml (1.73 mmol) of 2-bromoacetone were added. The reaction
mixture was stirred overnight and allowed to reach room temperature as
the ice bath melted. Copious solids resulted. The mixture was refluxed
for 1 h, dissolving all solids. After cooling to room temperature, solids
precipitated. Acetonitrile was removed under reduced pressure to about
12 volume, and the resulting solids were filtered, washed with 2 ml of cold
acetonitrile, and dried under high vacuum at room temperature to
afford 247 mg of product. The material was mixed with 2 ml of methanol
and 300 ml of 9.4 M hydrogen chloride in methanol. The resulting clear
solution was briefly concentrated under reduced pressure to an oil, then
mixed with 2 ml of ether. After stirring for 10 min at room temperature,
solids precipitated. An additional 8 ml of ether was added, and the
solids were filtered, washed with 4 ml of ether and dried under high
vacuum at room temperature to afford 266 mg of 5, in 71% RCY,
%
having a specific activity of 30.8 mCi/mg (9.9 mCi/mmol), with RCP of
97% by HPLC and 98% by TLC (95:5 v/v methylene chloride : acetone,
the sample was prepared in 4 M ammonia in methanol).
N-[2,(4-Morpholinyl)ethyl]2,6-di-1-pyrrolidinyl-4-[2-14C]pyrimidin-
amine, 6
%
A mixture of 101.5 mg (0.40 mmol) of unlabeled 2 and 289.3 mg
%
(1.14 mmol, 26.35 mCi/mmol, 30 mCi) of carbon-14 labeled 2 was
%
placed in a 15 ml pressure tube equipped with a Teflon magnetic stirring
bar, and a Teflon stopper with a Viton1 O-ring seal. Two millilitres of
aminoethylmorpholine was added, the tube was purged with argon and
tightly capped. The solution was placed in an oil bath at 1558C for 24 h.
The pressure tube was cooled in an ice bath and carefully opened under
nitrogen atmosphere. The bright yellow solution was partitioned with
10 ml of water and 20 ml of 1:1 v/v ethyl acetate:hexanes. The aqueous
phase was extracted with an additional 20 ml of 1:1 v/v ethyl
acetate:hexanes. The combined organic extracts were washed with
10 ml of water, 10 ml of saturated brine, dried over anhydrous sodium
sulfate, and concentrated under reduced pressure to afford 594 mg
(28.8 mCi) of 6, RCP of 93% by HPLC and 87% TLC (97:3 v/v
%
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805
methylene chloride:4 M ammonia in methanol). This material was used
without further purification in the next step.
2-Bromocyclohexanone
2-Bromocyclohexanone was prepared in a similar manner to a literature
procedure for 1-bromo-3-methyl-2-butanone.4 Freshly distilled cyclo-
hexanone, 24.5 g (250 mmol) was mixed with 150 ml of methanol and
cooled to ꢂ108C. Bromine, 40 g (250 mmol) was added quickly to the
vigorously stirred solution. There was no noticeable exotherm. After
30 min, the temperature was raised to 08C. Stirring was continued for
8 h during which the reaction mixture came to room temperature. The
colorless solution was quenched with 75 ml of water and stirred
overnight at room temperature. An additional 75 ml of water was
added and the two-phase mixture was concentrated under reduced
pressure to remove the methanol. The remaining mixture was extracted
with 2 ꢀ 100 ml portions of 1:1 v/v ethyl acetate:hexanes. The combined
extracts were washed with 70 ml of water, 30 ml of 1.2 M sodium
bicarbonate, 75 ml of saturated brine, and dried over anhydrous sodium
sulfate. The dry extract was concentrated under reduced pressure and
the residue was distilled under vacuum (B.P. 55–598C/0.9 mm) to afford
34.5 g (78%) of a colorless liquid. A small sample was stored at room
temperature under nitrogen, but within 4 days became a thick black tar.
The bromoketone when stored in the frozen state at ꢂ108C under
argon, remain colorless for several weeks.
6,7,8,9-Tetrahydro-9-[2-(4-morpholinyl)ethyl]-2,4-di-1-pyrrolidinyl-5-
H-[2-14C]pyrimido[4,5-b]indole, 7
%
The freshly prepared 6 (theory: 1.54 mmol) was transferred to a 15 ml
%
pressure tube with 4 ml of methylene chloride. The reaction mixture was
concentrated under a stream of nitrogen, then under high vacuum to
remove traces of solvent. The remaining yellow glass-like material was
mixed with 700 ml (6.0 mmol) of 2-bromocyclohexanone, 1.1 ml
(6.0 mmol) of diisopropylethylamine, and 1.0 ml of acetonitrile. The
pressure tube was flushed with argon, tightly capped, and placed in an
oil bath at 958C for 34 h. Solids appeared as the reaction mixture cooled
to room temperature. The mixture was partitioned with 10 ml of water
and 20 ml of methylene chloride. The layers were separated and the
aqueous layer was extracted with 20 ml of methylene chloride. The
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J. A. EASTER AND W. T. STOLLE
combined organic extracts were washed with 10 ml of water, 10 ml of
saturated brine, dried over anhydrous sodium sulfate, and concentrated
under reduced pressure. The dark yellow residue was dissolved in
methylene chloride and eluted through a 1 g Whatman Si solid phase
extraction cartridge with 95:5 v/v methylene chloride:methanol to
remove some polar impurities. The eluate was concentrated under
reduced pressure. The residue was mixed with 4 ml of acetonitrile which
on stirring and cooling gave a solid. The solid was filtered, washed with
3 ml of cold acetonitrile, and dried under high vacuum at room
temperature to afford 363 mg (16.6 mCi) of 7. The mother liquor
%
(10.2 mCi) was isotopically diluted with 445 mg of unlabeled 7,
%
crystallized from acetonitrile to afford 408 mg of low specific activity
material. The two crystalline lots of 7 were combined, dissolved in 6 ml
%
of methylene chloride, concentrated under reduced pressure to an oil,
and quickly mixed with 6 ml of acetonitrile at room temperature. The
mixture was cooled in an ice bath for 10min, then the solids were filtered,
washed with 3 ml of cold acetonitrile, and dried under high vacuum at
room temperature to afford 668 mg of 7, having a specific activity of
%
22.9 mCi/mg (9.72 mCi/mmol). The RCP as determined was 99% by
HPLC and 97% by TLC (95:5 v/v methylene chloride:methanol).
9-[2-(4-Morpholinyl)ethyl]2,4-di-1-pyrrolidinyl-9-H-[2-14C]pyrimi-
do[4,5-b]indole, 8
%
A mixture of 262 mg (0.617 mmol, 6.0 mCi) of 7, 50 mg of 10%
%
palladium on carbon catalyst, and 4.5 ml of decahydronaphthalene were
stirred in an oil bath at 1608C for 4.25 h. A long 22 gauge stainless steel
needle was used to sweep nitrogen through the mixture to remove the
hydrogen gas. The reaction mixture was cooled to room temperature
and filtered through a bed of Celite1 and the bed washed with 2 ml of
methylene chloride and 2 ml of hexanes. The filtrate and washings were
partitioned with 10 ml of 0.5 N hydrochloric acid and 20 ml of hexanes.
The aqueous phase was extracted again with 20 ml of hexanes to aid
removal of the decahydronaphthalene. The aqueous phase was basified
with 5 ml of 1.2 M sodium bicarbonate solution, and extracted with
2 ꢀ 20 ml portions of methylene chloride. The combined organic
extracts were washed with 15 ml of saturated brine, dried over
anhydrous sodium sulfate, and concentrated under reduced pressure.
The resulting oil was purified by chromatography on 35 g of silica gel 60
(70-230 Mesh) packed in and eluted with 70:30 v/v methylene
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ISOTOPICALLY LABELED PYRROLO[1,3-D]PYRIMIDINE ANALOGS
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chloride:acetonitrile. Fractions of 8 were collected, pooled, and
%
concentrated under reduced pressure. The resulting oil was mixed with
1 ml of absolute ethanol at room temperature. Crystals formed quickly
and were further precipitated by dropwise addition of 7 ml of water. The
solids were filtered, washed with 5 ml of water, and dried under high
vacuum at room temperature to afford 205 mg of 8, in 81.7% yield,
%
having a specific activity of 23.9 mCi/mg (10.0 mCi/mmol) and a RCP of
1
99% by HPLC and TLC. H-NMR (CDCl3) d:1.95–1.97 (m, 8H, H-30,
3
40, 300, 400), 2.58 (bs, 4H, H-2-, 5-), 2.74 (t, 2H, J ¼ 6:9 Hz, H-la-),
3.60–3.62 (m, 8H, H-200, 500, 3-, 4-), 3.91 (bs, 4H, H-20, 50), 4.39 (t, 2H,
3J ¼ 6:9 Hz, H-lb-), 7.08 (dd, lH, 3J ¼ 8:0, 3J ¼ 8:0 Hz, H-6), 7.18 (dd,
3
3
3
lH, J ¼ 7:8, J ¼ 7:8 Hz, H-7), 7.28 (d, lH, J ¼ 7:9 Hz, H-8), 7.89
13
(d, lH, J ¼ 7:9 Hz, H-5). C-NMR (CDCl3) d:26.02 (C-30, 40, 300, 400),
38.75 (C-lb-), 46.87 (C-200, 500), 49.97 (C-20, 50), 54.26 (C-2-, 5-), 57.04
(C-la-), 67.36 (C-3-, 4-), 89.97 (C-4a), 108.58 (C-8), 119.98 (C-6),
120.88 (C-5), 121.85 (C-7), 122.45 (C-4b), 137.41 (C-8a), 158.4, 158.8
(C-9a, 4), 159.7 (C-2).
3
[(2,4-Dichloro-4-[2-14C] pyrimidinyl)amino]acetic acid, ethyl ester, 9a,
and stable isotope analog, 9b
A room temperature solution of 802 mg (4.37 mmol, 221 mCi) of 1a in
6 ml of dry tetrahydrofuran (THF) was treated with 628 mg (4.75 mmol)
of glycine ethyl ester hydrochloride. To this mixture under nitrogen
atmosphere, with stirring, 1.6 ml (9 mmol) of diisopropylethylamine
(DIEA) was added dropwise over 10 min at room temperature. After the
addition, the suspension was stirred at room temperature for 30 min,
then refluxed for 5 h. After cooling to room temperature, the reaction
mixture was concentrated under reduced pressure. The residue was
partitioned between 20 ml of saturated sodium bicarbonate and 25 ml of
methylene chloride. The layers were separated and the aqueous layer
was extracted with 2 ꢀ 25 ml portions of methylene chloride. The
combined methylene chloride extracts were dried over anhydrous
sodium sulfate and concentrated under reduced pressure to afford a
crude mixture containing the regioisomers. The crude mixture was
purified by column chromatography on 90 g of silica gel (70-230 Mesh)
packed in and eluted with 25:75 (v/v) ethyl acetate:hexanes. Fractions of
9 were pooled and concentrated under reduced pressure to afford
546 mg, 164 mCi (2.19 mmol, 74% RCY) of 9a, RCP radiochemically
pure by TLC (25:75 (v/v) ethyl acetate:hexanes). In a similar manner,
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J. A. EASTER AND W. T. STOLLE
stable isotope labeled 9b was prepared from 2,4,6-trichloro-[1,3-15N2,
2,4,6-13C3]pyrimidine to give 624 mg (2.49 mmol) of 9b, representing a
1
54% yield, a single zone by TLC (25:75 v/v ethyl acetate:hexanes). H-
3
NMR (CDCl3) d:1.52 (t, 3H, J ¼ 7:14 Hz, CH3CH2ꢂ), 4.19 (bs, 2H,
3
NHCH2ꢂ), 4.28 (q, 2H, J ¼ 7:12 Hz, OCH2), 5.7 (bs, lH, NH), 6.38
(bs, lH, H-5). 13C-NMR (CDCl3) d : 14:50 (CH3CH2ꢂ), 43.42 (OCH2),
62.43 (N-CH2), 159.25 (C-6, enriched), 160.72 (C-4, enriched), 163.8
(C-2, enriched).
[(2,6-Di-1-pyrrolidinyl-4[2-14C]-pyrimidinyl)amino]acetyl pyrrolidine,
10a, and stable isotope analog, 10b
The solid 9a (546 mg, 2.19 mmol) was treated with 10 ml of pyrrolidine at
08C. The reaction mixture was placed under nitrogen atmosphere and
stirred while warming to room temperature, and then heated at reflux for
5.5 h. After cooling to room temperature, the mixture was concentrated
under reduced pressure, followed by high vacuum at room temperature
to remove residual traces of pyrrolidine. The resulting residue was
partitioned between 20 ml of saturated sodium bicarbonate and 25 ml of
methylene chloride. The layers were separated, and the aqueous layer
extracted with 2 ꢀ 25 ml portions of methylene chloride. The combined
methylene chloride extracts were dried over anhydrous sodium sulfate
and concentrated under reduced pressure to afford 103 mCi (63% RCY)
of 10a, RCP 99.8% by HPLC and RCP 95.9% by TLC (95:5 (v/v)
methylene chloride:4 M ammonia in methanol). In a similar manner,
stable isotope labeled 10b was prepared from 9b to give 857 mg
(2.49 mmol) of 10b, representing a 100% yield, a single zone by TLC
(95:2 v/v methylene chloride:4 M ammonia in methanol). 1H-NMR
(CDCl3) d : 2:0122:85 (m, 12H, H-30, 300, 3-, 40, 400, 4-), 3.35–3.69
(m, 12H, H-20, 200, 2-, 50, 500, 5-), 4.00 (bs, 2H, H-lb-), 4.91 (bs, lH, H-5).
13C-NMR (CDCl3) d : 24:45, 25.60, 25.70, 26.51 (C-30, 300, 3-, 40, 400, 4-),
46.40, 46.54, 46.84, 46.92, 47.07 (C-20, 200, 2-, 50, 500, 5-), 157.80 (C-6,
enriched), 159.03 (C-4, enriched), 161.27 (C-2, enriched).
(5,6,7,8-Tetrahydro-2,4-di-1-pyrrolidinyl-9H[2-14C]-pyrimido[4,5-b]in-
doyl-9-yl)-acetic acid, pyrrolidine amide, 11a, and stable isotope analog,
11b
The 10a from above was transferred to a 15 ml pressure tube (Ace glass
No. 8648-04) with 5 ml of methylene chloride. The solution was
Copyright # 2001 John Wiley & Sons, Ltd.
J Labelled Cpd Radiopharm 2001; 44: 797–810

ISOTOPICALLY LABELED PYRROLO[1,3-D]PYRIMIDINE ANALOGS
809
concentrated under a stream of nitrogen, then under high vacuum to
remove traces of solvent. The remaining yellow solid was mixed with
1.4 g (7.81 mmol) of 2-bromocyclohexanone, 1.4 ml (7.81 mmol) of
diisopropylethylamine and 4 ml of acetonitrile (purged in nitrogen). The
pressure tube was flushed with nitrogen, tightly closed with a Teflon
lined screw cap, and placed in an oil bath at 928C for 34 h. All solids
went into solution after 4 h of heating. After cooling to room
temperature, the reaction mixture was partitioned between 25 ml of
water and 25 ml of methylene chloride. The layers were separated and
the aqueous layer was extracted with 2 ꢀ 25 ml portions of methylene
chloride. The combined methylene chloride extracts were washed with
25 ml of water, 25 ml of brine, dried over anhydrous sodium sulfate, and
concentrated under reduced pressure at room temperature. The
resulting residue was triturated with 50 ml of hexane to induce
solidification. The solids were filtered, washed with cold hexane and
dried under high vacuum at room temperature to afford 845 mg
(1.99 mmol, 89% RCY) of 11a, RCP 87.4% by HPLC and 73.7% by
TLC (95:5 (v/v) methylene chloride:4 M ammonia in methanol). In a
similar manner, 988 mg (2.33 mmol, 94% yield) of 11b was prepared,
94.6% pure by HPLC at 254 nm.
1-[(2,4-D-1-pyrrolidinyl-9H-[2-14C]-pyrimido-[4,5-b]indol-9-yl)-acet-
yl]pyrrolidine monohydrochloride, 12a, and stable isotope analog 12b
A mixture of 845 mg (1.99 mmol) of 11a and 400 mg of 10% palladium
on carbon catalyst and 10 ml of anhydrous decahydronaphthalene
under nitrogen atmosphere was stirred in an oil bath at 1968C for 6 h.
The reaction mixture was cooled to room temperature and filtered
through a bed of Celite1, and the bed was washed with 10 ml of
methylene chloride. The combined filtrate and wash were concentrated
under reduced pressure. The residue was partitioned between 10 ml of
1 N HCl and 50 ml of hexanes. The layers were separated and the
aqueous layer was extracted with 25 ml of hexanes to aid the removal of
the decahydronaphthalene. The aqueous phase was basified with
saturated sodium bicarbonate and extracted with 3 ꢀ 25 ml of methylene
chloride. The combined methylene chloride extracts were washed with
25 ml of brine, dried over anhydrous sodium sulfate, and concentrated
under reduced pressure. TLC analysis of the residue showed the
reaction to be incomplete. A mixture of the residue, 400 mg of fresh
10% palladium on carbon and 10 ml of decahydronaphthalene under
Copyright # 2001 John Wiley & Sons, Ltd.
J Labelled Cpd Radiopharm 2001; 44: 797–810

810
J. A. EASTER AND W. T. STOLLE
nitrogen atmosphere were stirred in an oil bath at 1968C for 6 h. The
reaction mixture was worked up the same as above to remove the
decahydronaphthalene to afford the crude free base. The crude mixture
was purified by column chromatography on 90 g of silica gel
(70-230 Mesh) packed in and eluted with 85:15 (v/v) methylene
chloride:acetonitrile. Fractions of 12a were pooled and concentrated
under reduced pressure to afford 277 mg, 32.1 mCi (0.66 mmol) of 12a,
RCP 99.1% by HPLC. The purified free base was dissolved in 0.5 ml of
a 1.2 M HCl in methanol solution. While cooling the methanolic
solution, ether was added dropwise with stirring to a volume of 10 ml to
precipitate the salt. After cooling at ꢂ188C for 2 h, the white solids were
filtered, washed with cold ether and dried under high vacuum at room
temperature to afford 277 mg (0.61 mmol, 31 mCi, 14% overall yield) of
12a, sp. act. 112 mCi/mg (51 mCi/mmol), RCP 99.8% by HPLC and
RCP 98.7% by TLC. In a similar manner, 379 mg (0.84 mmol, 28%
overall yield) of 12b, 99% pure by HPLC at 254 nm. 1H-NMR (CDCl3)
d : 1:821:9, 2.0–2.1 (m, 4H, H-3-, 4-), 2.03–2.09 (m, 8H, H-30, 300, 40, 400)
3.48 (t, 2H, H-2-, 5-), 3.87 (t, 2H, H-2-, 5-), 4.00 (m, 8H, H-20, 50, 200,
500), 6.08 (s, 2H, H-lb-), 7.22–7.33 (m, 3H, H-5, 6, 7), 7.89 (d, lH, H-8).
References
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KL, Braughler JM, Hall ED, Houser DJ, Krook MA, Runge TA. J Med
Chem 1990; 33: 1145.
2. Bundy GL, Ayer DL, Banitt LS, Belonga KL, Miszak SA, Palmer JL,
Tustin JM, Chin JE, Hall ED, Linseman KL, Richards IM, Scherch HM,
Sun FF, Yonkers PA, Larson PG, Lin JM, Padbury GE, Aaron CS, Mayo
JK. J Med Chem 1995; 38: 4161.
3. Stolle WT, Easter JA, Chew EH, McGrath JP, Palmer JR, Hsi RSP.
J Labelled Cpd Radiopharm 1994; 34: 1187.
4. Gaudry M, Marquet A. OrganicSynthesis Collective, Vol. VI. Wiley: New
York, 1988; 193–195.
Copyright # 2001 John Wiley & Sons, Ltd.
J Labelled Cpd Radiopharm 2001; 44: 797–810