A new synthesis of 2,8-disubstituted pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidines

Article abstract of DOI:10.1016/j.tetlet.2009.07.113

A practical synthesis of pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidines, which are key intermediates in the preparation of adenosine receptor antagonists, is developed. The method allows introduction of a variety of aryl substituents at position 2 of th

Full text of DOI:10.1016/j.tetlet.2009.07.113

Tetrahedron Letters 50 (2009) 5617–5621
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A new synthesis of 2,8-disubstituted pyrazolo[4,3-e][1,2,4]triazolo
[1,5-c]pyrimidines
*
Anton V. Dolzhenko , Giorgia Pastorin, Anna V. Dolzhenko, Wai Keung Chui
Department of Pharmacy, Faculty of Science, National University of Singapore, 18 Science Drive 4, Singapore 117543, Singapore
a r t i c l e i n f o
a b s t r a c t
Article history:
A practical synthesis of pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidines, which are key intermediates in
the preparation of adenosine receptor antagonists, is developed. The method allows introduction of a
variety of aryl substituents at position 2 of the pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine system
via cyclocondensation of 5-amino-4-iminopyrazolo[3,4-d]pyrimidine with benzaldehydes accompanied
with oxidation by iodobenzene diacetate. Some unexpected reactions are observed and the structures
of the products are confirmed using NMR spectroscopy and X-ray crystallography.
Received 10 June 2009
Revised 7 July 2009
Accepted 17 July 2009
Available online 24 July 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Adenosine is a major modulator of cellular response in different
organs. The effects of adenosine are mediated through four adeno-
sine receptor subtypes, namely A1, A2A, A2B and A3.1 These subtypes
are different in terms of tissue distribution and affinity to adeno-
sine, and have unique pharmacological profiles. Adenosine
receptor antagonists have been widely explored as potential ther-
apeutic agents for treatment of nervous system disorders, cardio-
vascular diseases, and immune and inflammatory disorders.2
The pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine nucleus has
been recognized as a promising template for the development of
new adenosine receptor antagonists.3 Through installation of
appropriate substituents at R1 and R3 in adenosine receptor antag-
onists 4 (Scheme 1), it was found that it was possible to tune the
activity and selectivity of the new agents towards specific adeno-
sine receptor subtypes. However, systematic studies on the effect
of the structure of the R2 substituent on the interaction of 4 with
adenosine receptors are not available.
For the synthesis of adenosine receptor antagonists of general
structure 4, the preparation of intermediate 3 is a critical step that
limits variation of R2 (Scheme 1). The cyclocondensation of hydra-
zides with imidate 2, prepared from aminopyrazoles 1, required
harsh conditions (260 °C) and chromatographic purification of
the products.4 The poor solubility of 3, particularly when R1 = Me
and R3 = Ar, made chromatographic purification a time- and sol-
vent-consuming process.
We hypothesized that the reaction of 5-amino-4-imino-pyraz-
olo[3,4-d]pyrimidine (5) with aromatic aldehydes followed by oxi-
dation might be
a potential approach for the synthesis of
pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidines 3 (Scheme 2). A
variety of commercially available benzaldehydes allow the intro-
duction of chemical diversity at position 2 of the heterocyclic
nucleus.
Imidate 2a5 was prepared from aminopyrazole 1a and triethyl
orthoformate using a known procedure (Scheme 3).4,6 The reaction
of 2a with hydrazine in ethanol on heating was reported7 to pro-
duce 5-amino-4-imino-2-methylpyrazolo[3,4-d]pyrimidine (5).
Surprisingly, our attempt to repeat this synthesis using the condi-
tions described by Baraldi et al.7 resulted in the formation of a dif-
ferent product.
The downfield shift of the amino group signal at 6.65 ppm as well
as the two doublets at 7.47 and 11.83 ppm in the 1H NMR spectrum
could indicate the formation of pyrazolotriazepine 6. However, the
large value of the coupling constant for these doublets
(J = 11.3 Hz) suggested an arrangement of the NH–CH fragment pro-
tons in a trans-configuration; this was also confirmed by a 2D NOESY
experiment. Finally, using X-ray crystallography (Fig. 1),8 the struc-
ture of the product was revealed as a new fused macrocyclic system,
namely 4,12-diamino-2,10-dimethyl-2,8, 10,16-tetrahydrodipyraz-
olo[3,4-e:30,40-l][1,2,4,8,9,11]hexaazacyclotetradecine (7).9
We were able to isolate the desired intermediate 5 when the
reaction of 2a with hydrazine was carried out in ethanol at 0–
20 °C for 5 h (Scheme 3).10 Additionally, pure 7 (6–8%) precipitated
from the filtrate on standing overnight at ambient temperature.
Broadening of the NH and C-4 signals in the NMR spectra of 5
was observed which could be attributed to the dynamic inversion
of the imino group configuration.
Therefore, a new method for the preparation of pyrazolo[4,3-
e][1,2,4]triazolo[1,5-c]pyrimidines 3 via a more practical, chroma-
tography-free process under mild reaction conditions would be
beneficial for further exploration of the chemistry and pharmacol-
ogy of adenosine receptor antagonists.
In the trial reactions of 5 with benzaldehyde (Table 1), we found
that varying amounts of 3 formed after initial condensation with
* Corresponding author. Tel./fax: +65 6779 1554.
E-mail address: phada@nus.edu.sg (A.V. Dolzhenko).
0040-4039/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2009.07.113

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A. V. Dolzhenko et al. / Tetrahedron Letters 50 (2009) 5617–5621
R2
R2
N
N
N
CN
NH2
CN
R2CONHNH2
N
HC(OEt)3
N
N
O
R1 N
R1 N
OEt
1 N
1 N
N
R
R
N
N
N
N
N
H
R3
N
N
1
2
3
4
Scheme 1. General synthetic approach to pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidines—antagonists of adenosine receptors.
Table 1
Ar
A
study of the reaction of 5-amino-4-iminopyrazolo[3,4-d]pyrimidine (5) with
1) ArCHO;
2) [O]
NH
N
benzaldehyde
N
NH2
N
N
Ph
N
Me N
Me N
NH
N
N
N
N
N
PhCHO
NH2
3
5
N
N
Me N
N
Me N
N
Scheme 2.
EtOH, reflux
N
N
3
5
Equiv of
PhCHO
Catalyst
(equiv)
Combined yielda
(%)
Quantity of 3 in the mixtureb
(%)
the aldehyde even without using an oxidant. Improved results
were achieved in the presence of 0.3 equiv of triethylamine.
Increasing the quantity of the aldehyde or triethylamine did not
improve the reaction yield and did not significantly change the
composition of the obtained mixtures.
When this method was applied for the reactions of 5 with
substituted benzaldehydes, the extent of oxidation with aerial oxy-
gen was strongly dependent on the structure of the aldehydes. In
some cases (e.g., Ar = 4-PhC6H4), pure 3 was isolated directly after
1
1
1
1
2
1
—
ꢀ40
ꢀ40
ꢀ65
ꢀ60
ꢀ65
ꢀ35
ꢀ50
ꢀ60
ꢀ70
ꢀ70
ꢀ70
ꢀ10
Et3N (0.1)
Et3N (0.3)
Et3N (1.0)
Et3N (0.3)
Piperidine
(0.3)
a
Approximate values without characterization of non-oxidized intermediates.
Estimated by 1H NMR spectroscopy.
b
H2N
N
Me
N
CN
NH2
CN
N
N
HN
i
iii
OEt
N
N
N
Me
Me
NH
N
N
N
N
N
N
Me
N
NH2
1a
2a
7
H2N
ii
Me N
N
NH
N
N
NH2
H
6
N
Me N
N
N
5
Scheme 3. Reagents and conditions: (i) HC(OEt)3 (5–10 equiv), reflux, 8 h (85%); (ii) N2H4 (4 equiv), EtOH, 0–20 °C, 5 h (88%); (iii) N2H4 (4 equiv), EtOH, reflux, 2 h (62%).
Figure 1. X-ray crystal structure of 7.

A. V. Dolzhenko et al. / Tetrahedron Letters 50 (2009) 5617–5621
5619
Table 2
Synthesis of 2-aryl-8-methylpyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidines (3)
Ar
N
NH
N
1) ArCHO, Et3N; 2) PhI(OAc)2
NH2
N
N
Me N
Me N
N
N
N
N
3
5
3
a
ArCHO
Mp (°C)
Yield (%)
60
248
CHO
b
c
>300
56
53
62
MeO
Me
F
CHO
278–279
278–279
CHO
d
CHO
CHO
e
235
67
Cl
CHO
f
284–285
>300
64
70
92
68
89a
Cl
Cl
g
h
i
CHO
CHO
CHO
CHO
>300
Br
>300
F3C
Ph
j
>300
CHO
k
l
243–244
289–290
288–289
252–253
54
90
90
68
BnO
CHO
Cl
Cl
Cl
CHO
m
n
Cl
MeO
MeO
MeO
CHO
a
Yield after condensation of 5 with the aldehyde, oxidation with PhI(OAc)2 was not required.

5620
A. V. Dolzhenko et al. / Tetrahedron Letters 50 (2009) 5617–5621
NHNH2
NNH2
NH
N
NNH2
N
CN
NH2
CN
N
ΔG298 = 1.5 kJ
i
ii
N
OEt
N
N
N
N
N
N
N
Me
N
N
N
N
N
H
Me
Me
Me
Me
8
9
10
10'
10''
Scheme 4. Reagents and conditions: (i) HC(OEt)3 (5–10 equiv), reflux, 6 h (84%); (ii) N2H4 (4 equiv), EtOH, rt, 8 h (70%) or reflux, 2 h (86%).
Figure 2. X-ray crystal structure of 10.
the reaction of 5 with the aldehyde, making further oxidation
unnecessary.
Acknowledgements
Organic hypervalent iodine derivatives have been used exten-
sively as convenient oxidants.11 The good oxidizing potential,
convenience in handling, mild reaction conditions minimizing
the possibility of side reactions, benign environmental character
and commercial availability are attractive features of hypervalent
iodine compounds. Therefore, iodobenzene diacetate12 was
chosen for the oxidation step. The oxidation reaction was per-
formed in acetic acid on the crude mixture of 3 and non-oxi-
dized intermediates. Oxidation was found to proceed under
mild conditions, almost quantitatively, affording 2-aryl-8-meth-
ylpyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidines (3) with excel-
lent purity.
This work is supported by the National Medical Research Coun-
cil, Singapore (NMRC/NIG/0020/2008) and the National University
of Singapore (R-148-050-091-101/133). The authors thank Tan
Geok Kheng, Koh Lip Lin and Hong Yimian for the X-ray crystallog-
raphy studies.
References and notes
1. For recent reviews on the therapeutic potential of adenosine receptor
modulation, see: Jacobson, K. A.; Gao, Z. G. Nat. Rev. Drug Disc. 2006, 5, 247–
264; Wilson, C. N. Br. J. Pharmacol. 2008, 155, 475–486; Hasko, G.; Linden, J.;
Cronstein, B.; Pacher, P. Nat. Rev. Drug Disc. 2008, 7, 759–770; Vass, G.; Horvath,
I. Curr. Med. Chem. 2008, 15, 917–922; Boison, D. Curr. Opin. Pharmacol. 2008, 8,
2–7; Benarroch, E. E. Neurology 2008, 70, 231–236.
2. For recent reviews on adenosine receptor antagonists, see: Moro, S.; Gao, Z.
G.; Jacobson, K. A.; Spalluto, G. Med. Res. Rev. 2006, 26, 131–159; Press, N. J.;
Gessi, S.; Borea, P. A.; Polosa, R. Exp. Opin. Ther. Pat. 2007, 17, 979–991;
Gonzalez, M. P.; Teran, C.; Teijeira, M. Med. Res. Rev. 2008, 28, 329–371;
Takahashi, R. N.; Pamplona, F. A.; Prediger, R. D. S. Front. Biosci. 2008, 13,
2614–2632; Baraldi, P. G.; Tabrizi, M. A.; Gessi, S.; Borea, P. A. Chem. Rev.
2008, 108, 238–263.
3. For reviews on pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine antagonists of
adenosine receptors, see: Baraldi, P. G.; Cacciari, B.; Borea, P. A.; Varani, K.;
Pastorin, G.; Da Ros, T.; Tabrizi, M. A.; Fruttarolo, F.; Spalluto, G. Curr. Pharm.
Des. 2002, 8, 2299–2332; Baraldi, P. G.; Tabrizi, M. A.; Romagnoli, R.; Fruttarolo,
F.; Merighi, S.; Varani, K.; Gessi, S.; Borea, P. A. Curr. Med. Chem. 2005, 12, 1319–
1329; Baraldi, P. G.; Romagnoli, R.; El-Kashef, H.; Tabrizi, M. A.; Preti, D.;
Pavani, M. G.; Zanella, L.; Fruttarolo, F. Targets Heterocycl. Syst. 2006, 10, 175–
196; Baraldi, P. G.; Tabrizi, M. A.; Romagnoli, R.; El-Kashef, H.; Preti, D.; Bovero,
A.; Fruttarolo, F.; Gordaliza, M.; Borea, P. A. Curr. Org. Chem. 2006, 10, 259–275;
Cacciari, B.; Bolcato, C.; Spalluto, G.; Klotz, K. N.; Bacilieri, M.; Deflorian, F.;
Moro, S. Purinergic Signalling 2007, 3, 183–193; Bolcato, C.; Cusan, C.; Pastorin,
G.; Spalluto, G.; Cacciari, B.; Klotz, K. N.; Morizzo, E.; Moro, S. Purinergic
Signalling 2008, 4, 39–46.
In general, the method was found to be practical. Reasonable
yields of products 3 were obtained and a variety of readily avail-
able aldehydes could be employed (Table 2).13 The procedure
was clean and the products could be used for the synthesis of
new potential adenosine receptor antagonists without any addi-
tional purification.
Interestingly, the regioisomer of 2, imidate 9,14 prepared from
aminopyrazole 8, reacted differently with hydrazine in ethanol,
affording 4-hydrazinopyrazolo[3,4-d]pyrimidine (10)15 as a rear-
rangement product (Scheme 4). The reaction proceeded analogously
in all temperature parameters (0–70 °C) applied.
Hydrazino–hydrazono tautomerism of 10 was observed in
DMSO-d6 solution. The tautomeric equilibrium was shifted to-
wards hydrazino form 10 with
DG298 = 1.5 kJ. Due to broadening
of the signals in the NMR spectra, it was not possible to differenti-
ate between the two hydrazono forms 100 and 1000. They might ex-
ist together in a dynamic equilibrium. In the solid state, the
product was found to exist exclusively in hydrazino form 10:
two almost identical molecules crystallized together as a dimer
(Fig. 2).16
In summary, a new synthesis of pyrazolo[4,3-e][1,2,4]triazo-
lo[1,5-c]pyrimidines, which are important intermediates in the
synthesis of adenosine receptor antagonists, was successfully
developed. The advantages of the method are simple synthetic pro-
cedures and readily available starting materials.
4. Gatta, F.; Del Giudice, M. R.; Borioni, A.; Borea, P. A.; Dionisotti, S.; Ongini, E.
Eur. J. Med. Chem. 1993, 28, 569–576.
5. 4-Cyano-3-[(ethoxymethylene)amino]-1-methylpyrazole (2a).
A mixture of 1a
(2.44 g, 20 mmol) and triethyl orthoformate (30 ml) was heated under reflux
for 8 h. Excess triethyl orthoformate was removed under reduced pressure and
the residue was recrystallized from AcOEt/hexane. Yield 85%, mp 51–52 °C; LC–
MS (APCI) m/z 179 (MH+); Anal. Calcd for C8H10N4O: C, 53.92; H, 5.66; N, 31.44.
Found: C, 53.78; H, 5.75; N, 31.27. 1H NMR (300 MHz, DMSO-d6): d 1.30 (3H, t, J
7.2 Hz, CH2Me), 3.79 (3H, s, NMe), 4.28 (2H, q, J 7.2 Hz, CH2), 8.30 (1H, s, H-5),
8.37 (1H, s, N = CHOEt); 13C NMR (75 MHz, DMSO-d6): d 13.8 (CH2Me), 39.2
(NMe), 62.8 (CH2), 84.0, 113.7, 137.3 (CH), 156.6, 159.2 (CH).

A. V. Dolzhenko et al. / Tetrahedron Letters 50 (2009) 5617–5621
5621
6. Baraldi, P. G.; Manfredini, S.; Simoni, D.; Zappaterra, L.; Zocchi, C.; Dionisotti, S.;
Ongini, E. Bioorg. Med. Chem. Lett. 1994, 4, 2539–2544.
7. Baraldi, P. G.; El-Kashef, H.; Farghaly, A. R.; Vanelle, P.; Fruttarolo, F.
Tetrahedron 2004, 60, 5093–5104.
12. For reviews on iodobenzene diacetate, see: Varvoglis, A. Chem. Soc. Rev. 1981,
10, 377–407; Prakash, O.; Singh, S. P. Aldrichimica Acta 1994, 27, 15–23;
Kirschning, A. J. Prakt. Chem. 1998, 340, 184–186.
13. General method for the preparation of 8-methyl-2-aryl-pyrazolo[4,3-
8. Dolzhenko, A. V.; Pastorin, G.; Dolzhenko, A. V.; Tan, G. K.; Koh, L. L. Acta Cryst.
2009, 65E, o1578–o1579.
e][1,2,4]triazolo[1,5-e]pyrimidines (3). A mixture of 5 (1.64 g, 10 mmol), the
appropriate benzaldehyde (10 mmol) and Et3N (0.42 ml, 3 mmol) in EtOH
(30 ml) was heated under reflux overnight. After cooling, the resulting
precipitate was filtered, washed with cold EtOH and mixed with AcOH (10 ml).
To the resulting mixture was added an equimolar quantity of iodobenzene
diacetate. After stirring at rt for 1 h, the precipitate was filtered and washed with
EtOH. Analytical samples were recrystallized from DMSO. Data for representative
compound 3k: LC–MS (APCI) m/z 357 (MH+); Anal. Calcd for C20H16N6O: C, 67.40;
H, 4.53; N, 23.58. Found: C, 67.33; H, 4.60;N, 23.43. 1H NMR (300 MHz, DMSO-d6):
d 4.20 (3H, s, Me), 5.23 (2H, s, CH2), 7.20 (1H, dd, J 7.9, 1.9 Hz H-40), 7.30–7.56 (6H,
m, H-30 andPh), 7.78–7.86 (2H, m, H-20 and-60), 8.91 (1H, s, H-9), 9.45(1H, s, H-5);
13C NMR (75 MHz, DMSO-d6): d 40.4 (Me), 69.2 (CH2), 101.5, 112.6 (CH), 117.3
(CH), 119.4 (CH), 126.2 (CH), 127.5 (2CH), 127.8 (CH), 128.4 (2CH), 130.2 (CH),
131.1, 136.8, 139.8 (CH), 148.4, 153.7, 158.7, 162.9.
9. 4,12-Diamino-2,10-dimethyl-2,8,10,16-tetrahydrodipyrazolo-[3,4-e:30,40-l][1,2,4,8,9,
11]hexaazacyclotetradecine (7). Hydrazine (2 ml, 40 mmol) was added to a solution
of 2a (1.78 g, 10 mmol) in EtOH (20 ml). The mixture was heated under reflux for
2 h and cooled to rt. The resulting precipitate was filtered and washed with EtOH.
Yield 62%, mp 269–270 °C (MeOH); LC–MS (APCI) m/z 329 (MH+); Anal. Calcd for
C12H16N12: C, 43.90; H, 4.91; N, 51.19. Found: C, 43.82; H, 5.02; N, 51.02. 1H NMR
(300 MHz, DMSO-d6): d 3.73 (6H, s, 2Me), 6.65 (4H, s, 2NH2), 7.47 (2H, d, J 11.3 Hz,
H-7 and -15), 8.02 (2H, s, H-3 and -11), 11.83 (2H, d, J 11.3 Hz, H-8 and -16); 13C
NMR(75 MHz,DMSO-d6):d38.8(2Me), 100.4(2C), 130.6(2CH),138.1(2CH),147.6
(2C), 152.2 (2C).
10. 5-Amino-4-imino-2-methylpyrazolo[3,4-d]pyrimidine (5). Hydrazine (1.2 ml,
24 mmol) was added to a cold (0–5 °C) solution of 2a (1.08 g, 6 mmol) in
EtOH (20 ml). The mixture was stirred at rt for 5 h. The resulting precipitate
was filtered and washed with EtOH to provide 5. Yield 88%, mp 180 °C (EtOH).
LC–MS (APCI) m/z 165 (MH+); Anal. Calcd for C6H8N6: C, 43.90; H, 4.91; N,
51.19. Found: C, 43.73; H, 5.08; N, 51.05. 1H NMR (300 MHz, DMSO-d6): d 3.92
(3H, s, Me), 5.41 (2H, s, NH2), 7.58 (1H, br s, NH), 7.87 (1H, s, CH), 8.26 (1H, s,
CH); 13C NMR (75 MHz, DMSO-d6): d 39.5 (Me), 105.4, 128.3 (CH), 149.9 (CH),
151.7 (br s), 155.0. Additionally, 7 (6–8%) precipitated from the filtrate on
standing overnight.
11. For recent reviews on organic hypervalent iodine reagents, see: Koser, G. F. Adv.
Heterocycl. Chem. 2004, 86, 225–292; Wirth, T. Angew. Chem., Int. Ed. 2005, 44,
3656–3665; Richardson, R. D.; Wirth, T. Angew. Chem., Int. Ed. 2006, 45, 4402–
4404; Matveeva, E. D.; Proskurnina, M. V.; Zefirov, N. S. Heteroat. Chem. 2006,
17, 595–617; Zhdankin, V. V. Sci. Synth. 2007, 31a, 161–233; Zhdankin, V. V.;
Stang, P. J. Chem. Rev. 2008, 108, 5299–5358.
14. Taylor, E. C., Jr.; Loeffler, P. K. J. Am. Chem. Soc. 1960, 82, 3147–3151; Baraldi, P.
G.; Cacciari, B.; Spalluto, G.; Pineda de Villatoro, M. J.; Zocchi, C.; Dionisotti, S.;
Ongini, E. J. Med. Chem. 1996, 39, 1164–1171.
15. 4-Hydrazino-1-methyl-1H-pyrazolo[3,4-d]pyrimidine (10). Mp 243 °C (EtOH);
LC–MS (APCI) m/z 165 (MH+); Anal. Calcd for C6H8N6: C, 43.90; H, 4.91; N,
51.19. Found: C, 43.82; H, 5.02; N, 51.12. 1H NMR (300 MHz, DMSO-d6): d 3.89
(3H, s, Me), 4.67* and 4.87 (2H, two br s, NH2), 8.03* and 8.11 (1H, two br s, H-
3), 8.30 (1H, br s, H-6), 9.13 and 9.45* (1H, two br s, NH); 13C NMR (75 MHz,
DMSO-d6): d 33.2 and 33.3* (Me), 98.5, 130.8* and 134.6 (two br s, CH), 152.2*
and 153.6 (two br s), 155.0 and 155.5* (two br s, CH), 156.8* and 160.5 (two br
s). *-signals of minor hydrazone tautomer.
16. Dolzhenko, A. V.; Pastorin, G.; Dolzhenko, A. V.; Tan, G. K.; Koh, L. L. Acta Cryst.
2009, 65E, o1720–o1721.