Efficient synthesis of14C-labeled 1H-pyrazolo[3,4-d]pyrimidine and related [4.3.0]-bicyclic pyrimidino systems

Article abstract of DOI:10.1002/hlca.200890102

In support of a research program aimed at discovering purine-related anticancer drug candidates, a method for the ¹⁴C-labeling of pyrazolopyrimidines utilizing the readily available labeled starting material, sodium [¹⁴C]formate, has been developed with a good overall yield. This new method was proven to be general in the preparation of other related [4.3.0]heterocycles containing N, O, and S atoms. A concise synthesis of a model compound, 8-aza-7-deaza-5′-[¹⁴C]noraristeromycin, was achieved utilizing this methodology as a key step.

Full text of DOI:10.1002/hlca.200890102

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Helvetica Chimica Acta – Vol. 91 (2008)
Efficient Synthesis of 14C-Labeled 1H-Pyrazolo[3,4-d]pyrimidine and Related
[4.3.0]-Bicyclic Pyrimidino Systems
by Jonathan Z.Ho* , Kyle R.Van Arsdale , and Matthew P.Braun
Department of DMPK Global Technologies, Merck Research Laboratories, P.O. Box2000,
126 E. Lincoln Avenue, Merck & Co., Inc., Rahway, New Jersey 07065, USA
J. Z. H. dedicates this paper to his postdoctoral research advisor, the late Professor Henry Rapoport,
Ph.D., Department of Chemistry, University of California, Berkeley, California 94720, USA,
on the occasion of his 90th birthday.
In support of a research program aimed at discovering purine-related anticancer drug candidates, a
method for the 14C-labeling of pyrazolopyrimidines utilizing the readily available labeled starting
material, sodium [14C]formate, has been developed with a good overall yield. This new method was
proven to be general in the preparation of other related [4.3.0]heterocycles containing N, O, and S atoms.
A concise synthesis of a model compound, 8-aza-7-deaza-5’-[14C]noraristeromycin, was achieved utilizing
this methodology as a key step.
Introduction. – 9H-Purine (1) and 1H-pyrazolo[3,4-d]pyrimidine (2) are a class of
molecules that have a five-membered ring fused to a six-membered ring. In each ring,
there are two N-atoms, but there is no N-atom at the ring junction (Figure)1). The
biological activity of compounds that contain these ring systems has generated
increased interest in their chemistry [2]. It was reported that many purine nucleosides
and nucleotides are anticancer or antiviral agents [3]. 1H-Pyrazolo[3,4-d]pyrimidine
(2), an isomer of purine, was widely used as a purine replacement in the synthesis of
compounds aimed at improved target activity [4]. An efficient synthetic route to
prepare 14C-labeled 4-hydroxy-1H-pyrazolo[3,4-d]pyrimidine is important to build
radiolabeled tracers used in absorption, distribution, metabolism, and excretion
(ADME) studies, and investigations of activation potentials [5].
Figure. Structures of 9H-purine (1) and 1H-pyrazolo[3,4-d]pyr-
imidine (2)
1
)
For recent reviews on purine and related compounds, see [1a][1b]. For reviews on 1H-pyrazolo[3,4-
d]pyrimidine and related compounds, see [1c].
ꢀ 2008 Verlag Helvetica Chimica Acta AG, Zürich

Helvetica Chimica Acta – Vol. 91 (2008)
959
Results and Discussion. – The preparation of 1,5-dihydro-4H-pyrazolo[3,4-d]-
pyrimidin-4-one (4) from cyclocondensation of 5-amino-1H-pyrazole-4-carboxamide
(3) with triethyl orthoformate (Table, Entry 1) was previously documented in the
literature, but the yields of the reaction were generally low [6]. Although better yields
can be achieved by replacement of triethyl orthoformate with formamide (Table, Entry
2) [7], 14C-labeled formamide is expensive and not readily available. Given the
frequent appearance of building block 2 in pharmaceutical compounds, we sought to
expand the scope of this potentially useful cyclization method and optimize the
efficiency. In the present work, a simple procedure by which sodium formate (readily
available in 14C-labeled form) was utilized to construct 4 and its analogs was
established.
We have found that heating a mixture of sodium formate and a b-aminocarbox-
amide as intimately mixed solids can directly afford the desired purine or pyrazolopyr-
imidine. A range of related [4.3.0]heterocycles were explored and found to be
obtainable by this new method (Table, Entries 3 – 7). The reactions were conducted in a
solvent-free manner with three equivalents of sodium formate, and the yields were
moderate to good (61 – 89%), based on the utilized b-aminocarboxamide. This method
is generally applicable, because it tolerates a variety of heteroatoms such as N, O, and S.
Table. Formation of Unlabeled [4.3.0]Pyrimidino Heterocycles via Cyclocondensation of b-Amino-
carboxamides
Entry b-Aminocarboxamide
Condition
Solvent
Time [4.3.0]-
[h]
Yield
Heterocycle [%]a)
Compound
A
B
C
1
2
3
4
5
6
7
3
3
3
5
7
9
11
HꢀC
HꢀC
HꢀC
N
N
N
N
HꢀN HC(OEt)3, 808 Ac2O
HꢀN formamide, 1908 formamide
HꢀN HCO2Na, 1908 none
4
1
4
4
4
4
4
4
4
4
6
8
10
12
11 [6]
85 [7]
89
77 [8]
61 [9]
81 [10]
62
HꢀC HꢀN HCO2Na, 1908 none
MeꢀC
N
N
O
HCO2Na, 1908 none
PhꢀN HCO2Na, 1908 none
HCO2Na, 1908 none
HꢀC
MeꢀC PhꢀC
S
a) All yields refer to isolated products.
The 14C-labeled 1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one, 4*, was prepared
by cyclocondensation of 5-amino-1H-pyrazole-4-carboxamide (3) with 14C-labeled
sodium formate (Scheme 1). When one equivalent of sodium [14C]formate was used,
only 35% of 3 were converted to 4*, as indicated by HPLC. Separation of 4* from 3
was very difficult. Alternatively, heating of 3 with three equivalents of [14C]HCO2Na in
a sealed tube at 1908 for 4 h resulted in a 90% conversion of 3. After FC, the desired
product 4* was obtained in 69% yield, based on 3. In radiochemistry, scientists often
report radiochemical yields, which is calculated based on the consumption of the

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Helvetica Chimica Acta – Vol. 91 (2008)
radiochemical starting material (SM) regardless whether it is the limiting reagent or
not. In our case, the radiochemical SM was [14C]HCO2Na, and it was used in threefold
excess with respect to the limiting reagent 3. Therefore, the radiochemical yield of this
reaction, calculated on the basis of the consumption of [14C]HCO2Na, was 23% (69% ꢁ
1/3 ¼ 23%).
Scheme 1
a) 1908, 4 h, 23%. b) POCl3, CH2Cl2, r.t., 16 h, 88%.
Chlorination of 4* generates the chloride 13 (Scheme 1), a very reactive
intermediate. Reaction of chloride 13 with an alcohol, either aliphatic or aromatic,
under refluxin CHCl , would give rise to a pyrimidine ether 14 (Scheme 2). Since a
3
large number of alcohols are available either commercially or synthetically, this method
should easily lead to many 14C-labeled pyrimidine (or purine) tracers for ADME and
other metabolism related studies.
Furthermore, the Cl group in 13 can be transformed into an amino group, and that
would be a crucial step in the synthesis of many nucleoside tracers. Therefore, we
decided to synthesize 14C-labeled 8-aza-7-deaza-5’-noraristeromycin (15), a natural
Scheme 2
a) NH4OH, reflux2 h; 71%. b) BzCl, pyridine, r.t., 4 h; 94%. c) (1R,4S)-4-Hydroxycyclopent-2-en-1-yl
acetate, NaN(TMS)2, (Ph3P)4Pd, Ph3P, DMSO, THF, 608; 44%. d) OsO4, NMO, THF, H2O. e) NH3/
MeOH; 51%.

Helvetica Chimica Acta – Vol. 91 (2008)
961
product, to demonstrate the synthetic potential of this newly developed labeling
technique. Treatment of 14C-labeled 13 with NH4OH in THF at 858 yielded amino
compound 16a, which was converted into benzamide 16b by benzoylation. The latter
was successfully transformed to the desired tracer 15 in three steps, in analogy to a
published procedure [4a] (Scheme 2).
In conclusion, a novel method for the preparation of 14C-labeled pyrazolopyrimi-
dines from the readily available inexpensive reagent sodium [14C]formate was
developed. It was demonstrated that this procedure is general and can be used for
the synthesis of other related [4.3.0]heterocycles.
Experimental Part
General. Anh. solvents were obtained from Aldrich and were dried over 4-Š molecular sieves for at
least 24 h prior to use. All of the commercially available reagents and solvents were from either Aldrich
Chemical Co., USA, or from Fisher Scientific Co., USA, except compounds 5, 7, 9, and 11; they were
purchased from Trega Biosciences, Inc., 16140 El Camino Real, Rancho Santa Fe, CA 92067, USA.
Analytical HPLC: Shimadzu HPLC System with LC-10ATVP pumps, SPD-10AVP UV detector, CTO-
10ASVP column oven heated to 308, a SCL-10A controller and a Packard RadiomaticTM 150TR flow
monitor. The reaction products were identified by HPLC comparison with unlabeled material using
Method A (0 to 20% MeCN/0.1% CF3COOH over 30 min). Reversed phase HPLC analyses: Zorbax RX
C18 analytical column (4.6 ꢁ 250), heated to 308 with a 10 min concluding wash with 100% MeCN.
1
Preparative HPLC purifications: Zorbax RX C18 preparative column (22.5 ꢁ 250). H- and 13C-NMR:
Varian U-400 spectrometer. LC/MS: Agilent 1100 series LC/MSD with an Eclipse XCB C18 column
(3.5 mm; 3.0 ꢁ 150 mm; solvent A: 20 nm of ammonium formate in H2O; solvent B: MeCN; 5% B to 95%
B in 10 min, then 5 min, at 100% B, followed by 5 min 5% B); in m/z.
General Procedure for the Synthesis of the [4.3.0]Pyrimidino Heterocycles 4, 6, 8, 10, and 12 via
Cyclization of the b-Aminocarboxamides. A solvent-free solid mixture of b-aminocarboxamide
(0.5 mmol) and dried sodium formate (34 mg, 0.5 mmol) was heated to 1908 under N2 for 2 h, and
then cooled to r.t. After addition of a additional equiv. of sodium formate (34 mg, 0.5 mmol), the mixture
was stirred with a spatula for 5 min, and heated to 1908 under N2 for 1 h. This procedure was repeated
once more so that a total of 3 equiv. of sodium formate (1.5 mmol) were added. Overall heating time was
4 h. HPLC (Method A) and LC/MS assays showed that most of the starting material was consumed and
the desired product was formed. H2O (2 ml) was added, and the crystalline product was collected by
filtration, followed by drying under high vacuum overnight. The yields for the isolated compounds are
listed in the Table.
Synthesis of 1,5-Dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one (4) [7]. 5-Amino-1H-pyrazole-4-
carboxamide hemisulfate (3; 89 mg, 0.5 mmol) and sodium formate (35 mg ꢁ 3 ¼ 105 mg, 1.5 mmol)
gave 4 (53 mg, 89% based on 3). IR (KBr): 1735, 1721, 1632, 1458. 1H-NMR (CDCl3): 5.1 (br. s, 1 H);
7.53 (s, 1 H); 8.13 (s, 1 H); 11.2 (br. s, 1 H). 13C-NMR (CDCl3): 98.8; 133.9; 148.3; 155.6; 157.9. ESI-MS:
137.2 ([M þ H]þ).
Synthesis of 1,9-Dihydro-6H-purin-6-one (6). 5-Amino-1H-imidazole-4-carboxamide (5; 63 mg,
0.5 mmol) and sodium formate (35 mg ꢁ 3 ¼ 105 mg, 1.5 mmol) gave 6 (53 mg, 77% based on 3). IR
(KBr): 1735, 1613, 1450. 1H-NMR (CDCl3): 5.1 (br. s, 1 H); 7.53 (s, 1 H); 8.13 (s, 1 H); 11.2 (br. s, 1 H).
13C-NMR (CDCl3): 99.1; 132.9; 149.4; 155.0; 158.9. ESI-MS: 137.2 ([M þ H]þ).
Synthesis of 3-Methylisoxazolo[5,4-d]pyrimidin-4(5H)-one (8). 5-Amino-3-methylisoxazole-4-car-
boxamide (7; 71 mg, 0.5 mmol) and sodium formate (35 mg ꢁ 3 ¼ 105 mg, 1.5 mmol) gave 8 (46 mg, 61%
based on 7). IR (KBr): 1731, 1668, 1410. 1H-NMR ((D6)DMSO): 2.42 (s, 3 H); 3.35 (br. s, 1 H); 8.21 (s,
1 H). 13C-NMR ((D6)DMSO): 22.4; 104.8; 149.0; 151.5; 160.1; 164.1. ESI-MS: 152.2 ([M þ H]þ).
Synthesis of 1,5-Dihydro-1-phenyl-4H-pyrazolo[3,4-d]pyrimidin-4-one (10). 5-Amino-1-phenyl-1H-
pyrazole-4-carboxamide (9; 101 mg, 0.5 mmol) and sodium formate (35 mg ꢁ 3 ¼ 105 mg, 1.5 mmol) gave
10 (86 mg, 81% based on the starting material 9). IR (KBr): 1729, 1633, 1458. 1H-NMR ((D6)DMSO):

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Helvetica Chimica Acta – Vol. 91 (2008)
3.30 (br. s, 1 H); 7.36 (t, J ¼ 7.6, 1 H); 7.53 (m, 2 H); 7.98 (d, J ¼ 7.6, 2 H). 13C-NMR ((D6)DMSO): 108.3;
122.4; 127.8; 129.9; 136.7; 138.9; 149.5; 152.5; 157.9. ESI-MS: 213.2 ([M þ H]þ).
Synthesis of 5-Methyl-6-phenylthieno[2,3-d]pyrimidin-4(3H)-one (12). 2-Amino-4-methyl-5-phenyl-
thiophene-3-carboxamide (11, 116 mg, 0.5 mmol) and sodium formate (35 mg ꢁ 3 ¼ 105 mg, 1.5 mmol)
gave 12 (75 mg, 62% based on the starting material 11). IR (KBr): 1730, 1613, 1449. 1H-NMR
((D6)DMSO): 2.46 (s, 3 H); 3.28 (br. s, 1 H); 7.38 (m, 2 H); 7.46 (m, 3 H); 8.05 (s, 1 H). 13C-NMR
((D6)DMSO): 15.1; 128.7; 129.6; 133.4; 146.3; 158.8; 158.9; 163.7; 163.7. ESI-MS: 243.2 ([M þ H]þ).
Anal. calc. for C13H10N2OS: C 64.44, H 4.16, N 11.56; found: C 64.88, H 4.45, N 11.87.
Synthesis of 1,5-Dihydro-4H-[6-14C]pyrazolo[3,4-d]pyrimidin-4-one (4*). The reaction was carried
out according to the General Procedure described above. Compound 3 (89 mg, 0.5 mmol) and sodium
[14C]formate (35 mg ꢁ 3 ¼ 105 mg, 1.5 mmol, 80 mCi) gave 4* (48 mg, 0.345 mmol, 18.3 mCi, i.e. 53 mCi/
mmol). The chemical yield was 69% based on the limiting reagent 3, whereas the radiochemical yield was
23% based on three equivalents of radiochemical reagent. Radiochemical purity for 4* was 99% (Method
A). 1H-NMR (CDCl3): 5.1 (br. s, 1 H); 7.53 (s, 1 H); 8.13 (s, 1 H); 11.2 (br. s, 1 H). 13C-NMR (CDCl3):
99.1; 132.9; 155.0; 158.9. ESI-MS: 138.3 ([M þ H]þ).
Synthesis of 4-Chloro-[6-14C]1H-pyrazolo[4,3-d]pyrimidine (13). POCl3 (40 ml, 0.44 mmol) was
added to a soln. of 4* (18 mCi, 0.036 mmol) in CH2Cl2 (1 ml), and the resulting mixture was stirred at r.t.
for 16 h. Pyridine (100 ml) was added, and the mixture was purified by flash chromatography (FC; SiO2,
CH2Cl2) to give 13 (46.3 mg, 15.84 mCi, 88%). 1H-NMR (CD3OD): 7.55 (s, 1 H); 8.40 (s, 1 H). ESI-MS:
157.2 ([M þ H]þ).
Synthesis of N-([6-14C]1H-Pyrazolo[3,4-d]pyrimidin-4-yl)benzamide (16b). A soln. of NH4OH (6m
in H2O, 0.5 ml, 3 mmol), 13 (46.3 mg, 15.84 mCi, 0.3 mmol) and THF (1 ml) was heated at 858 for 2 h.
After cooling to r.t., the aq. layer was extracted with CH2Cl2 (25 ml ꢁ 2). The combined org. layers were
dried (MgSO4), filtered, and concentrated under vacuum to give 16a (¼ [6-14C]1H-pyrazolo[3,4-d]-
pyrimidin-4-amine, 11.25 mCi, 71%). ESI-MS: 138.2 ([M þ H]þ). Benzoyl chloride (0.25 ml, 2.1 mmol)
and pyridine (0.20 ml, 0.25 mmol) were added to a soln. of 16a (11.25 mCi, 0.21 mmol) in CH2Cl2 (2 ml).
The mixture was stirred at r.t. for 4 h and then purified by FC (SiO2, 5% AcOEt in CH2Cl2) to give 16b
(48 mg, 10.6 mCi, 94%). 1H-NMR (CD3OD): 7.33 – 7.48 (m, 3 H); 7.60 (s, 1 H); 7.72 – 7.99 (m, 2 H); 8.30
(s, 1 H). ESI-MS: 242.3 ([M þ H]þ).
Synthesis of N-{1-[(1R,4S)-4-Hydroxycyclopent-2-en-1-yl]-[6-14C]1H-pyrazolo[3,4-d]pyrimidin-4-
yl}benzamide (17). A suspension of 16b (48 mg, 10.6 mCi, 0.2 mmol), (Ph3P)4Pd (46.2 mg, 0.04 mmol),
Ph3P (26.2 mg, 0.1 mmol), DMSO (0.1 ml), and THF (1 ml) was stirred under N2, while NaN(TMS)2
(1.0m, 0.2 ml) was added. After being stirred at r.t. for 1 h, (1R,4S)-4-hydroxycyclopent-2-en-1-yl acetate
[7] (40 mg, 0.24 mmol) was added. The mixture was heated to 608, and stirred for 16 h. HCl (6m, 1 ml)
was added, and the mixture was purified by FC (SiO2, 15% AcOEt in CH2Cl2) to give compound 17
1
(29 mg, 4.8 mCi, 44%). H-NMR (CD3OD): 2.39 – 2.44 (m, 1 H); 2.52 – 2.63 (m, 1 H); 3.99 – 4.11 (m,
1 H); 4.39 – 4.51 (m, 1 H); 5.48 – 5.64 (m, 2 H); 7.38 – 7.45 (m, 4 H); 8.01 – 8.09 (m, 2 H); 8.39 (s, 1 H).
ESI-MS: 324.2 ([M þ H]þ).
Preparation of 8-Aza-7-deaza-[2-14C]5’-noraristeromycin (¼(1S,2R,3S,4R)-4-(4-Amino-[6-14C]1H-
pyrazolo[3,4-d]pyrimidin-1-yl)cyclopentane-1,2,3-triol; 15). N-Methylmorpholine N-oxide (NMO,
10.1 mg, 0.1 mmol) and polymer-bound OsO4 (0.3 mmol/g, 5 mg, 0.0015 mmol) were added to a soln.
of 17 (29 mg, 4.8 mCi, 0.09 mmol) in THF (1 ml) and H2O (0.5 ml). The mixture was stirred at r.t. for 2 h.
The solid was filtered off, and the soln. was treated with NH4OH (6m, 1 ml) and MeOH (1 ml). The
progress of the reaction was monitored by HPLC. After 5 h, TFAwas used to adjust to pH 4. The mixture
was concentrated, and then purified by prep. HPLC (Zorbax RX C18, 21.5 ꢁ 250; A, H2O þ 0.1% TFA;
B, MeCN; A/B 77:23 (isocratic)) to give 15 (2.5 mCi, i.e. 53 mCi/mmol, 51% yield, 99.6% radiochemical
purity). This result is in agreement with that reported by Kitade and co-workers [4a]. 1H-NMR
((D6)DMSO): 1.62 – 1.89 (m, 1 H); 2.22 – 2.62 (m, 1 H); 3.75 (br. s, 1 H); 3.90 (br. s, 1 H); 4.32 – 4.49 (m,
1 H); 4.75 (br. s, 1 H); 4.95 (m, 1 H); 5.05 (br. s, 1 H); 7.65 (s, 2 H); 8.12 (s, 2 H); 8.15 (s, 1 H). 13C-NMR
((D6)DMSO): 35.7; 59.6; 73.1; 74.5; 76.8; 100.1; 131.6; 153.1; 155.4; 157.8. ESI-MS: 254.2 ([M þ H]þ).

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Received August 16, 2007