Synthesis and antifungal activities of novel 5-amino-6-arylamino-1H- pyrazolo[3,4-d]pyrimidin-4(5H)-one derivatives

Article abstract of DOI:10.1021/jf801359f

A novel approach was developed to regioselectively synthesize new 5-amino-6-arylamino-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one 5 derivatives via a tandem aza-Wittig and annulation reactions of iminophosphorane 2, aromatic isocyanates, and hydrazine in 52-92% isolated yields. The compounds 5 reacted with triethyl orthoformate to give 1,8-2H-pyrazolo[3,4-d][1,2,4]triazolo[1,5-a] pyrimidin-4-ones 6 in good yields (62-94%). Their structures were clearly confirmed by spectroscopy data (IR, ¹H NMR, MS), elemental analysis, or X-ray diffraction crystallography. The results of preliminary bioassay indicated that the compounds 5 and 6 possessed high antifungal activity against Botrytis cinerea Pers and Sclerotinia sclerotiorum. Compounds 5 showed much better antifungal activities when R was Me instead of PhCH₂. Especially, compounds 6c, 6g, and 6i inhibited Sclerotinia by 100% at the concentration of 50 mg/L and by 83, 83, and 82% at the dosage of 10 mg/L, respectively.

Full text of DOI:10.1021/jf801359f

J. Agric. Food Chem. 2008, 56, 7321–7325 7321
Synthesis and Antifungal Activities of Novel
5-Amino-6-arylamino-
1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one Derivatives
HONG-QING WANG,*,† WEI-PIMG ZHOU,§ YU-YUAN WANG,† AND
†
CAN-RONG LIN
College of Chemistry and Chemical Engineering and College of Mathematics and Physics, University
of South China, 28 Changsheng Road Hengyang, Hunan 421001, People’s Republic of China
A novel approach was developed to regioselectively synthesize new 5-amino-6-arylamino-1H-
pyrazolo[3,4-d]pyrimidin-4(5H)-one 5 derivatives via a tandem aza-Wittig and annulation reactions
of iminophosphorane 2, aromatic isocyanates, and hydrazine in 52-92% isolated yields. The
compounds 5 reacted with triethyl orthoformate to give 1,8-2H-pyrazolo[3,4-d][1,2,4]triazolo[1,5-
a]pyrimidin-4-ones 6 in good yields (62-94%). Their structures were clearly confirmed by spectroscopy
data (IR, 1H NMR, MS), elemental analysis, or X-ray diffraction crystallography. The results of
preliminary bioassay indicated that the compounds 5 and 6 possessed high antifungal activity against
Botrytis cinerea Pers and Sclerotinia sclerotiorum. Compounds 5 showed much better antifungal
activities when R was Me instead of PhCH2. Especially, compounds 6c, 6g, and 6i inhibited Sclerotinia
by 100% at the concentration of 50 mg/L and by 83, 83, and 82% at the dosage of 10 mg/L,
respectively.
KEYWORDS: 1H-Pyrazolo[3,4-d]pyrimidin-4(5H)-one; aza-Wittig reaction; synthesis; 1,8-2H-pyrazolo[3,4-
d][1,2,4]triazolo[1,5-a]pyrimidin-4-one; antifungal
INTRODUCTION
of novel 5-amino-6-arylamino-1H-pyrazolo[3,4-d]pyrimidin-
4(5H)-ones 5, having nitrogen atoms from hydrazine, 2H-
pyrazolo[3,4-d][1,2,4]triazolo[1,5-a]pyrimidin-4-one derivatives
(Scheme 1) and a new convenient method of synthesis of
compounds 5 and 6. Their biological activities have been studied
also.
Pyrazolo[3,4-d]pyrimidine derivatives have extremely rich
biological activities because of their structural similarity to
purines (1). They are often employed as antimicrobial and
antifungal agents (2–5), various animal enzyme inhibitors (6–8),
and agrochemicals (9–14). Triazole and its fused heterocycles
have been extensively investigated as biologically active
compounds, such as substituted triazole derivatives (15, 16),
triazolo[1,5-a]pyrimidine derivatives (17), and pyrazolotriaz-
olopyrimidine derivatives (18). Our previous paper (9–11)
reported that a reaction of ethyl 3-alkylthio-1-phenyl-5-triph-
enylphosphoranoimino-1H-pyrazole-4-carboxylate (abbreviation
iminophosphorane) 2 with isocycanates and alklyamine could
give 6-alkylamino-5-aryl-pyrazolo[3,4-d]pyrimidin-4-one de-
rivatives, the nitrogen atoms of which were from isocycanates.
Those compounds showed satisfactory antifungicidal activities.
Therefore, we have attempted to extend this reaction to
hydrazine to synthesize some new antifungicidally active
heterocycle compounds containing triazole. However, we ob-
tained 5-amino-6-arylamino-pyrazolo[3,4-d]pyrimidin-4(5H)-
one derivatives 5 instead of 6-hydrazino-5-aryl-pyrazolo[3,4-
d]pyrimidin-4-one 7 derivatives. In other words, we got a series
MATERIALS AND METHODS
Instruments. The melting points of the products were determined
on an X-4 binocular microscope (Beijing Tech Instrument Co., Beijing,
China) and are uncorrected. MS was measured on a FinniganTrace Mass
2000 spectrometer at 70 eV. IR was recorded on an Avatar 360
1
spectrometer as KBr pellets with absorption given in cm-1. H NMR
spectra were taken on a Varian Mercury 400 (or 300) spectrometer
with TMS as the internal reference and DMSO-d6 or CDCl3 as the
solvent. Elemental analysis was taken on a Vario EL elemental analysis
instrument. X-ray diffraction was performed with a Brucker Smart
1000.
Synthetic Procedures. All of the solvents and materials were of
reagent grade and purified as required. Ethyl 5-amino-3-alkylthio-1-
phenyl-1H-pyrazole-4-carboxylate 1 and iminophosphorane 2 were
prepared according to the literature (19).
General Synthetic Procedures for 3-Alkylthio-5-amino-6-ary-
lamino-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-ones 5. Aryl
isocyanate (0.27 g, 2 mmol) was added to a solution of iminophos-
phorane 2 (1.07 g, 2 mmol) in dry methylene dichloride (20 mL) under
nitrogen atmosphere at room temperature. After the reaction mixture
had been stirred for 1.5 h, 0.094 g (2.0 mmol, 85%) of hydrazine hydrate
* Corresponding author (telephone + 86 734 8281710; fax +86 734
8282521; e-mail hqwang2001cn@yahoo.com.cn).
† College of Chemistry and Chemical Engineering.
§ College of Mathematics and Physics.
10.1021/jf801359f CCC: $40.75  2008 American Chemical Society
Published on Web 08/02/2008

7322 J. Agric. Food Chem., Vol. 56, No. 16, 2008
Wang et al.
Scheme 1
was added, and the resulting mixture was stirred for an additional 30
min. Then most of the solvent was removed in vacuo, and 25 mL of
anhydrous ethanol and 1.5 mL of sodium ethoxide (3 mol/L) in ethanol
were added to the mixture. After 3 h of stirring at room temperature,
the solution was concentrated under reduced pressure and successively
cooled. The crude product was collected by filtration. After recrystal-
lization from DMF/petroleum ether or column chromatography on a
silica gel, a white crystal was obtained.
) 8.0 Hz, Ar), 7.84 (d, 1H, J ) 12.0 Hz, Ar), 7.25-7.54 (m, 10H,
Ar), 6.92 (t, 1H, J ) 7.6 Hz, Ar), 5.61 (br s, 2H, NH2), 4.48 (s, 2H,
PhCH2); EI-MS (70 eV, m/z, rel intensity) 458 (M+, 21). Anal. Calcd
for C24H19FN6OS: C, 62.87; H, 4.18; N, 18.33. Found: C, 62.96; H,
4.22; N, 18.26.
Data for 5g: white crystal, mp 236.2-238.1 °C; IR (KBr) υ (cm-1
)
3336, 3267, 1692, 1596, 1554, 909, 770, 743, 688; 1H NMR (DMSO-
d6, 400 MHz) δ 9.67 (s, 1H, PhNH), 8.03 (d, 2H, J ) 7.8 Hz, Ph),
7.73 (d, 2H, J ) 7.5 Hz, Ph), 7.46 (d, 2H, J ) 7.6 Hz, Ph), 7.36 (t,
2H, J ) 7.6 Hz, Ph), 7.25 (t, 1H, J ) 7.2 Hz, Ph), 7.11 (t, 1H, J ) 7.2
Hz, Ph), 5.57 (br s, 2H, NH2), 2.60 (s, 3H, SCH3); EI-MS (70 eV, m/z,
rel intensity) 65 (M + 1, 49), 364 (M+, 81). Anal. Calcd for
C18H16N6OS: C, 59.32; H, 4.43; N, 23.06. Found: C, 59.1; H, 4.37; N,
22.96.
Data for 5a: white crystal, mp 279-281 °C; IR (KBr) υ (cm-1
)
1
3347, 3311, 3207, 1679, 1596, 1563, 1509, 913, 763, 670; H NMR
(DMSO-d6, 300 MHz) δ 9.74 (s, 1H, p-FC6H4NH), 7.99 (d, 2H, J )
8.7 Hz, Ar), 7.70-7.75 (m, 2H, Ar), 7.46 (d, 2H, J ) 7.8 Hz, Ar),
7.18-7.28 (m, 3H, Ar), 5.54 (s, 2H, NH2), 2.59 (s, 3H, SCH3); EI-MS
(70 eV, m/z rel intensity) 384 (M + 2, 16), 383 (M + 1, 47), 382 (M+,
92). Anal. Calcd for C18H15FN6OS: C, 56.53; H, 3.95; N, 21.98. Found:
C, 56.41; H, 3.96; N, 22.85.
Data for 5h: white crystal, mp 226.0-227.8 °C; IR (KBr) υ (cm-1
)
3319, 1684, 1596, 1554, 927, 759, 689, 501; 1H NMR (CDCl3, 300
MHz) δ 9.69 (s, 1H, PhNH), 8.04 (d, 2H, J ) 8.4 Hz), 7.73 (d, 2H, J
) 8.1 Hz), 7.200-7.503 (m, 10H, Ph), 7.12 (t, 1H, J ) 7.2 Hz, Ph),
5.58 (s, 2H, NH2), 4.47 (s, 2H, Ph CH2); EI-MS (70 eV, m/z, rel
intensity) 442 (M + 2, 2), 441 (M+, 7). Anal. Calcd for C24H20N6OS:
C, 65.44; H, 4.58; N, 19.08. Found: C, 65.52; H, 4.43; N, 19.07.
Data for 5b: white crystal, mp 238-239 °C; IR (KBr) υ (cm-1
)
3352, 3314, 3266, 1721, 1621, 1600, 1542 (Ar), 1455, 922, 768; 1H
NMR (DMSO-d6, 400 MHz) δ 9.61(s, 1H, o-FC6H4NH), 7.98 (d, 3H,
J ) 8.4 Hz, Ar), 7.25-7.46 (m, 6H, Ar), 5.65 (s, 2H, NH2), 2.61 (s,
3H, SCH3); EI-MS (70 eV, m/z rel intensity) 384 (M +2, 13), 383 (M
+ 1, 49), 382 (M+, 89). Anal. Calcd for C18H15FN6OS: C, 56.53; H,
3.95; N, 21.98. Found: C, 56.39; H, 3.92; N, 22.03.
Data for 5i: white crystal, mp 245.7-246.4 °C; IR (KBr) υ (cm-1
)
3317, 3268, 3212, 1700, 1633, 1591, 1554, 923, 769; 1H NMR (DMSO-
d6, 300 MHz) δ 9.83 (br s, 1H, NH), 8.16 (s, 1H, Ar), 8.04 (d, 2H, J
) 7.5 Hz, Ar), 7.46-7.56 (m, 3H, Ar), 7.26-7.36 (m, 2H, Ar), 7.11
(d, 1H, J ) 7.8 Hz, Ar), 5.60 (s, 2H, NH2), 2.60 (s, 3H, CH3); EI-MS
(70 eV, m/z, rel intensity) 400 (M + 2, 32), 399 (M + 1, 22), 398
(M+, 95). Anal. Calcd for C18H15ClN6OS: C, 54.20; H, 3.79; N, 21.07.
Found: C, 53.96; H, 3.74; N, 22.27.
Data for 5c: white crystal, mp 246-247 °C; IR (KBr) υ (cm-1
)
3314, 3273, 3212, 1702, 1637, 1604, 1560, 1545, 963, 925, 871, 764;
1H NMR (DMSO-d6, 400 MHz) δ 9.82 (s, 1H, m-FC6H4NH), 8.04 (d,
2H, J ) 7.6 Hz, Ar), 7.88 (d, 1H, J ) 12 Hz, Ar), 7.33-7.59 (m, 5H,
Ar), 6.95 (t, 1H, J ) 2.4 Hz, Ar), 5.59 (s, 2H, NH2), 2.61 (s, 3H, SCH3);
EI-MS (70 eV, m/z rel intensity) 384 (M + 2, 21), 382 (M+, 100).
Anal. Calcd for C18H15FN6OS: C, 56.53; H, 3.95; N, 21.98. Found: C,
56.71; H, 4.01; N, 21.85.
Data for 5j: white crystal, mp 227.0-228.0 °C; IR (KBr) υ (cm-1
)
1
3346, 3302, 3186, 1684, 1599, 1543, 1503, 1398, 930, 773; H NMR
(DMSO-d6, 400 MHz) δ 9.61 (s, 1H, C6H4NH), 8.08 (d, 2H, J ) 8.4
Hz, Ar), 7.80 (s, 1H, Ar), 7.50 (t, 2H, J ) 7.8 Hz, Ar), 7.44 (d, 1H, J
) 8.0 Hz, Ar), 7.32 (t, 1H, J ) 8.0 Hz, Ar), 7.26 (t, 1H, J ) 8.0 Hz,
Ar), 6.96 (d, 1H, J ) 7.2 Hz, Ar), 5.59 (s, 2H, NH2), 2.61(s, 3H, SCH3),
2.35 (s, 3H, m-CH3C6H4); EI-MS (70 eV, m/z, rel intensity) 379 (M +
1, 6), 378 (M+, 27). Anal. Calcd for C19H18N6OS: C, 60.30; H, 4.79;
N, 22.21. Found: C, 60.20; H, 4.81; N, 22.42.
Data for 5d: white crystal, mp 208-209 °C; IR (KBr) υ (cm-1
)
3319, 3207, 3025, 2930, 1686,1609, 1595, 1563, 1509, 1390, 1211,
1
¨
924, 771; H NMR (DMSO-d6, 300 MHz) δ A9.76 (br s, 1H,
p-FC6H4NH), 8.00 (d, 2H, J ) 7.8 Hz, Ar), 7.70-7.75 (m, 2H, Ar),
7.44-7.51 (m, 4H, Ar), 7.19-7.32 (m, 6H, Ar), 5.56 (s, 2H, NH2),
4.46 (s, 2H, PhCH2); EI-MS (70 eV, m/z rel intensity) 460 (M + 2,
12), 459 (M + 1, 28), 458 (M+, 50). Anal. Calcd for C24H19FN6OS:
C, 62.87; H, 4.18; N, 18.33. Found: C, 62.56; H, 4.22; N, 18.50.
General Synthetic Procedures for 3-Alkylthio-8-aryl-phenyl-1,8-
dihydropyrazolo[3,4-d][1,2,4]triazolo[1,5-a]pyrimidin-4-one 6. To a
suspension of 5-amino-3-alkylthio-6-arylamino-1-phenyl-1H-pyrazolo[3,4-
d]pyrimidin-4(5H)-one 5 (1.5 mmol) in 25 mL of triethyl orthoformate
was added p-toluenesulfonic acid (p-TsOH) (0.4 g, 2.25 mmol). After
the reaction mixture was refluxed for 15 h and then cooled, lots of
precipitate appeared. The crude product was collected by filtration and
washed with water. After recrystallization from dimethylformamide, a
white crystal was obtained.
Data for 5e: white crystal, mp 188-189 °C; IR (KBr) υ (cm-1
)
3323, 3274, 3212, 3028, 2924, 1685, 1600, 1553, 1491, 1459, 1390,
1228, 1170, 926, 759; 1H NMR (DMSO-d6, 400 MHz) δ 9.61(br s,
1H, o-FC6H4NH), 7.94-7.99 (m, 3H, Ar), 7.26-7.48 (m, 11H, Ar),
5.67 (br s, 2H, NH2), 4.48 (s, 2H, PhCH2); EI-MS (70 eV, m/z, rel
intensity) 458 (M+, 12). Anal. Calcd for C24H19FN6OS: C, 62.87; H,
4.18; N, 18.33. Found: C, 62.95; H, 4.20; N, 18.21.
Data for 5f: white crystal, mp 229-230 °C; IR (KBr) υ (cm-1) 3320,
1
3208, 2919, 1687, 1598, 1556, 1492, 1390, 1171, 945, 774; H NMR
Data for 6a: white crystal, mp 267-269 °C; IR (KBr) υ (cm-1
)
1
(DMSO-d6, 400 MHz) δ 9.88 (br s, 1H, m-FC6H4NH), 8.05 (d, 2H, J
3139, 2924, 1719, 1583, 1560, 1509, 1400, 1162, 902, 768; H NMR

Pyrazolo[3,4-d]pyrimidin-4(5H)-one
Table 1. Yields of Compounds 5 and 6
J. Agric. Food Chem., Vol. 56, No. 16, 2008 7323
compd
R
Ar
yield of 5(%)
yield of 6(%)
5a, 6a
5b, 6b
5c, 6c
5d, 6d
5e, 6e
5f, 6f
5g, 6g
5h, 6h
5i, 6i
Me
Me
Me
PhCH2
PhCH2
PhCH2
Me
PhCH2
Me
Me
p-FC6H4
o-FC6H4
m-FC6H4
p-FC6H4
o-FC6H4
m-FC6H4
C6H5
C6H5
m-ClC6H4
m-MeC6H4
69
92
73
52
81
86
76
72
64
68
70
78
94
62
76
83
72
69
78
79
5j, 6j
(DMSO-d6, 400 MHz) δ 9.30 (s, 1H, triazole-H), 8.05 (d, 2H, J ) 8.0
Hz, Ar), 7.94-7.98 (m, 2H, Ar), 7.49-7.56 (m, 4H, Ar), 7.31 (t, 1H,
J ) 6.8 Hz, Ar), 2.64 (s, 3H, SCH3); EI-MS (70 eV, m/z, rel intensity)
392 (M+, 9). Anal. Calcd for C19H13FN6OS: C, 58.15; H, 3.34; N, 21.42.
Found: C, 58.21; H, 3.32; N, 21.51.
Figure 1. X-ray structure of compound 5a.
Data for 6b: white crystal, m p 224-225 °C; IR (KBr) υ (cm-1
)
PhCH2); EI-MS (70 eV, m/z, rel intensity) 450 (M+, 8). Anal. Calcd
for C25H18N6OS: C, 66.65; H, 4.03; N, 18.65. Found: C, 66.70; H, 4.02;
N, 18.71.
1
3146, 1712, 1607, 1585, 1525, 1509, 1398, 1234, 904, 769, 752; H
NMR (DMSO-d6, 400 MHz) δ 9.23 (s, 1H, triazole-H), 8.00 (d, 2H, J
) 8.0 Hz, Ar), 7.92 (t, 1H, J ) 7.6 Hz, Ar), 7.44-7.69 (m, 5H, Ar),
7.29 (t, 1H, J ) 7.4 Hz, Ar), 2.65 (s, 3H, SCH3); EI-MS (70 eV, m/z,
rel intensity) 393 (M + 1, 31), 392 (M+, 63). Anal. Calcd for
C19H13FN6OS: C, 58.15; H, 3.34; N, 21.42. Found: C, 58.13; H, 3.32;
N, 21.48.
Data for 6i: white crystal, mp 259.6 -261.0 °C; IR (KBr) υ (cm-1
)
1
3112, 2919, 1735, 1610, 1591, 1525, 1509, 1401, 1121, 800, 764; H
NMR (DMSO-d6, 400 MHz) δ 9.41 (s, 1H, triazole-H), 8.22 (s, 1H,
m-ClC6H4), 8.08 (d, 2H, J ) 8.0 Hz, Ph), 7.93 (d, 1H, J ) 8.0 Hz,
Ph), 7.69 (t, 1H, J ) 7.8 Hz, Ar), 7.61 (d, 1H, J ) 8.0 Hz, Ar), 7.50
(t, 2H, J ) 7.6 Hz, Ar), 7.32 (t, 1H, J ) 7.6 Hz, Ar), 2.64 (s, 3H,
SCH3); EI-MS (70 eV, m/z, rel intensity) 408 (M+, 12). Anal. Calcd
for C19H13ClN6OS: C, 55.81; H, 3.20; N, 20.55. Found: C, 55.89; H,
3.17; N, 20.61.
Data for 6c: white crystal, mp 247-249 °C; IR (KBr) υ (cm-1
)
3115, 3064, 2924, 1733, 1608, 1586, 1526, 1510, 1402, 1232, 1159,
905, 850, 746; 1H NMR (DMSO-d6, 400 MHz) δ 9.39 (s, 1H, triazole-
H), 8.07 (d, 2H, J ) 8.0 Hz, Ar), 7.93 (d, 1H, J ) 10.0 Hz, Ar), 7.84
(d, 1H, J ) 8.4 Hz, Ar), 7.72 (q, 1H, J ) 7.5 Hz, Ar), 7.52 (t, 2H, J
) 7.8 Hz, Ar), 7.41 (t, 1H, J ) 8.4 Hz, Ar), 7.33 (t, 1H, J ) 7.2 Hz,
Ar), 2.64 (s, 3H, SCH3); EI-MS (70 eV, m/z, rel intensity) 392 (M+,
5). Anal. Calcd for C19H13FN6OS: C, 58.15; H, 3.34; N, 21.42. Found:
C, 58.22; H, 3.30; N, 21.26.
Data for 6j: white crystal, mp 257-259 °C; IR (KBr) υ (cm-1) 3140,
3084, 2930, 1718, 1615, 1586, 1562, 1529, 1510, 1387, 1164, 904,
1
768; H NMR (DMSO-d6, 400 MHz) δ 9.34 (s, 1H, triazole-H), 8.09
(d, 2H, J ) 7.6 Hz, Ar), 7.85 (s, 1H, Ar), 7.70 (d, 1H, J ) 8.0 Hz,
Ar), 7.47-7.54 (m, 3H, Ar), 7.30-7.36 (m, 2H, Ar), 2.63 (s, 3H, SCH3),
2.44 (s, 3H, m-CH3Ph); EI-MS (70 eV, m/z, rel intensity) 389 (M + 1,
42), 388 (M+, 76). Anal. Calcd for C20H16N6OS: C, 61.84; H, 4.15; N,
21.63. Found: C, 61.96; H, 4.12; N, 21.68.
Data for 6d: white crystal, mp 267-268 °C; IR (KBr) υ (cm-1
)
3031, 1723, 1616, 1589, 1528, 1508, 1456, 1234, 1128, 901, 843, 766;
1H NMR (DMSO-d6, 400 MHz) δ 9.30 (s, 1H, triazole-H), 8.06 (d,
2H, J ) 8.8 Hz, Ar), 7.94-7.97 (m, 2H, Ar), 7.26-7.56 (m, 10H,
Ar), 4.51 (s, 2H, PhCH2); EI-MS (70 eV, m/z, rel intensity) 468 (M+,
3). Anal. Calcd for C25H17FN6OS: C, 64.09; H, 3.66; N, 17.94. Found:
C, 64.20; H, 3.68; N, 17.86.
Crystal Structure Determination. The crystal structure of the
compound 5a was determined, and X-ray intensity data were recorded
on a Bruker SMART 1000 CCD diffraction meter using graphite
monochromated Mo KR radiation (λ ) 0.71073 Å). A total of 9306
reflections were measured, of which 3444 were unique (Rint ) 0.0272)
in the range of 2.46 < θ < 26.37° (h, -12 to 17; k, -9 to 8; l, -20
to 20), and 2579 observed reflections with I > 2σ(I) were used in the
refinement on F2. The structure was solved by direct methods with the
SHELXS-97 program. All of the non-H atoms were refined anisotro-
pically by full-matrix least-squares to give the final R ) 0.0362 and
wR ) 0.0878 (w ) 1/[σ2(Fo2) + (0.0473P)2 + 0.3686P], where P )
(Fo2 + 2Fc2)/3) with (∆/σ)max ) 0.001 and S ) 1.043 by using the
SHELXL program. The hydrogen atoms were located from a difference
Fourier map and refined isotropically.
Biological Tests: In Vitro Antifungal Activity. The fungi were
obtained from the College of Plant Protect, Central China Agriculture
University, China. The antifungal activities of compounds 5 and 6
against Botrytis cinerea Pers, Pyricularia oryzae, Gibberella zeae, and
Sclerotinia sclerotiorum were investigated at dosages of 50 and 10 mg/L
using the procedure (20).
Data for 6e: white crystal, mp 231-232 °C; IR (KBr) υ (cm-1
)
3133, 1705, 1587, 1558, 1525, 1507, 1398, 1240, 1068, 901, 767, 754;
1H NMR (DMSO-d6, 400 MHz) δ 9.23 (s, 1H, triazole-H), 8.00 (d,
2H, J ) 8.4 Hz, Ar), 7.92 (t, 1H, J ) 8.0 Hz, Ar), 7.25-7.68 (m, 11H,
Ar), 4.50 (s, 2H, PhCH2); EI-MS (70 eV, m/z, rel intensity) 468 (M+,
1). Anal. Calcd for C25H17FN6OS: C, 64.09; H, 3.66; N, 17.94. Found:
C, 64.15; H, 3.67; N, 17.96.
Data for 6f: white crystal, mp 217-219 °C; IR (KBr) υ (cm-1) 3134,
1718, 1607, 1582, 1524, 1508, 1400, 1228, 1158, 765; 1H NMR
(DMSO-d6, 400 MHz) δ 9.37 (s, 1H, triazole-H), 8.07 (d, 2H, J ) 7.6
Hz, Ar), 7.93 (d, 1H, J ) 8.0 Hz, Ar), 7.84 (d, 1H, J ) 7.2 Hz, Ar),
7.69-7.73 (m, 1H, Ar), 7.26-7.55 (m, 9H, Ar), 4.51 (s, 2H, PhCH2);
EI-MS (70 eV, m/z, rel intensity) 468 (M+, 2). Anal. Calcd for
C25H17FN6OS: C, 64.09; H, 3.66; N, 17.94. Found: C, 64.20; H, 3.65;
N, 17.98.
Data for 6g: white crystal, mp 253-255 °C; IR (KBr) υ (cm-1
)
3144, 3081, 2925, 1718, 1605, 1578, 1560, 1527, 1388, 1265, 1165,
1
768, 685; H NMR (DMSO-d6, 400 MHz) δ 9.34 (s, 1H, triazole-H),
RESULTS AND DISCUSSION
8.08 (d, 2H, J ) 8.4 Hz, Ph), 7.93 (d, 2H, J ) 8.0 Hz, Ph), 7.67 (t,
2H, J ) 8.2 Hz, Ph), 7.49-7.57 (m, 3H, Ph), 7.31 (t, 1H, J ) 6.8 Hz,
Ph), 2.65 (s, 3H, SCH3); EI-MS (70 eV, m/z, rel intensity) 375 (M +
1, 13), 374 (M+, 36). Anal. Calcd for C19H14N6OS: C, 60.95; H, 3.77;
N, 22.45. Found: C, 61.02; H, 3.75; N, 22.53.
Iminophosphorane 2, which was prepared by the reaction of
ethyl 5-amino- 3-alkylthio-1-phenyl-1H-pyrazole-4-carboxylate
1 with triphenylphosphorane and bromine, reacted with isocy-
anates to give the key intermediates carbodiimide 3. Treatment
of 3 with hydrazine hydrate gave the guanidine 4 (9), which, in
the presence of EtONa, proceeded to afford the crude target
compounds at room temperature. The crude products were
collected by filtration. After recrystallization from DMF/
Data for 6h: white crystal, mp 224-226 °C; IR (KBr) υ (cm-1
)
1
3118, 3064, 2924, 1718, 1608, 1580, 1528, 1389, 1163, 757, 685; H
NMR (DMSO-d6, 400 MHz) δ 9.34 (s, 1H, triazole-H), 8.08 (d, 2H, J
) 7.6 Hz, Ph), 7.92 (d, 2H, J ) 7.6 Hz, Ph), 7.50-7.67 (m, 7H, Ph),
7.34 (t, 3H, J ) 7.4 Hz, Ph), 7.26 (t, 1H, J ) 7.2 Hz, Ph), 4.51 (s, 2H,

7324 J. Agric. Food Chem., Vol. 56, No. 16, 2008
Wang et al.
Table 2. Antifungal Activity of Compounds 5 and 6 (50 mg/L, Inhibitory
a,b
Rate Percent)
Table 3. Antifungal Activity of Compounds 5 and 6 (10 mg/L, Inhibitory
Rate Percent)a,b
relative inhibition (%)
relative inhibition (%)
Botrytis Sclerotinia
Botrytis
Pyricularia
Gibberella
Sclerotinia
compd
5a, 6a
5b, 6b
5c, 6c
5d, 6d
5e, 6e
5f, 6f
5g, 6g
5h, 6h
5i, 6i
5j, 6j
thiabendazole
5
6
5
6
5
6
5
6
compd
5
6
5
6
45
93
87
55
64
69
13
60
99
71
71
60
91
67
60
29
99
73
99
97
43
62
43
29
14
14
36
50
96
14
9
20
51
43
34
40
46
14
29
57
46
49
26
74
37
37
34
66
60
74
50
94
97
96
83
71
82
84
72
98
86
93
50
100
75
96
29
100
93
5a, 6a
5b, 6b
5c, 6c
5d, 6d
5e, 6e
5f, 6f
5g, 6g
5h, 6h
5i, 6i
9
45
31
70
30
27
5
70
49
71
74
72
73
75
52
33
40
51
31
76
52
70
19
83
32
75
5
83
67
82
73
5
29
43
14
0
80
29
57
70
69
56
12
18
20
3
12
73
36
100
98
5j, 6j
100
87
100
100
a Mean value for relative inhibition calculated from at least three determinations.
b S (standard deviations) are 1-4.
a Mean value for relative inhibition calculated from at least three determinations.
b S (standard deviations) are 1-3.
inhibition against Botrytis at 50 mg/L. For example, the
inhibitory rates of compounds 5b, 5c, 5i, 6c, 6g, 6i, and 6j were
93, 87, 99, 91, 99, 99, and 97%, respectively.
petroleum ether or column chromatography on silica gel, a white
crystal were obtained in 52-92% yield (Scheme 1; Table 1).
The spectral data proved the white crystal was 3-alkylthio-5-
amino-6-arylamino-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-
ones 5 instead of the isomer 3-alkylthio-6-hydrazine-5-aryl-1-
phenyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-ones 7. This did not
agree with our previous findings (9–11) showing the reaction
of carbodiimide 3 with alkyl amine gave only an annulation
compound, the nitrogen atom of which came from the ArNH
group. On refluxing compounds 5 with triethyl orthoformate,
compounds 6 were obtained in 62-94% yields in the presence
of p-TsOH.
In summary, we have developed a novel approach to reg-
ioselectively synthesize 3-alkylthio-5-amino-6-arylamino-1-
phenyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one and 3-alkylthio-
8-aryl-1-phenyl-2H-pyrazolo[3,4-d][1,2,4]triazolo[1,5-a]pyrimidin-
4 -one derivatives via a tandem aza-Wittig and annulations in
good yields. The antifungal tests indicated that most of
compounds 5 and 6 possessed excellent antifungal activities and
could be further developed as fungicides.
LITERATURE CITED
The structures of compounds 5 and 6 were deduced from
their spectral data (IR, 1H NMR, EI-MS, and elementary
analysis). 1H NMR spectra of compounds 5 showed the signal
of NH in the ArNH group at 9.61-9.88 ppm, which agreed
with the one in PhNH (δ > 7.0) (21), and the single signal of
NH2 at 5.54-5.67 ppm. Thus, a combination of chemical shift
and couplings allowed the complete and unambiguous assign-
ment of all signals and demonstrated that the major products
correspond to structure 5. The EI-MS spectra of 5 showed the
molecular ion peak (M+, 7-100%). All of the fragmentation
ions were consistent with their structures and could be clearly
assigned. In addition, the structures of compounds 6 and X-ray
single-crystal diffraction of 5a, which was reported elsewhere
(22), further verified the proposed structure of compound 5
(Figure 1) (23).
The antifungal activities of all compounds 5, 6, and 2-(4′-
thiazoly)benzimidazole (commercial name thiabendazole), a
commercially available fungicide, were discovered, as shown
in Tables 2 and 3 by contrasting to distilled water. It is well-
known that the incorporation of a fluorine instead of a hydrogen
atom can alter biological activity (24, 25). By comparing the
activities of compounds 5a-5f with those of 5g-5j and the
activities of compounds 6a-6f with those of 6g-6j, we came
to the conclusion that introducing a fluorine atom into com-
pounds 5 and 6 could not favorably improve their antifungal
activities. It was also worthy of note that the compounds 5
showed higher antifungal activities in general when R was
substituted for methyl instead of benzyl. Most compounds 5
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