V. Krysˇtof et al. / European Journal of Medicinal Chemistry 41 (2006) 1405e1411
1409
CDK2, and probably GTP binding with tubulin, because the
structurally similar substance tubulyzine B was recently sug-
gested to localize within the GTP pocket of b-tubulin [17].
As demonstrated previously, combined anticancer chemother-
apies using a CDK inhibitor and MIA could significantly
improve their efficacy [18]. Further studies on the antiprolifer-
ative mechanisms of myoseverin and E2GG are underway.
H0-meta), 6.86 (2H, m, H-meta), 7.27 (2H, m, H0-ortho),
7.29 (2H, m, H-ortho). 13C NMR (100.55 MHz, CDCl3,
0
303 K): 21.5 (C-200), 43.7 (CH2 ), 45.2 (CH2), 49.7 (C-100),
55.0 (2 ꢂ OCH3), 113.8 (C-meta, C0-meta), 128.9 (C-ortho),
129.1 (C0-ortho), 129.9 (C0-ipso), 131.3 (C-ipso), 149.9 (C-4),
154.3 (C-6), 158.6 (C-para), 158.8 (C0-para), 160.6 (C-2).
Anal. (C23H27N7O2) C, H, N.
4. Experimental
4.3.2. 5-Isopropyl-4-nitro-1(2)H-pyrazole-3-
carboxamide (2)
4.1. Chemicals
5-Isopropyl-4-nitro-1(2)H-pyrazole-3-carboxylic acid (1),
12.5 g (62.76 mmol), was refluxed with 18 ml SOCl2 for 2 h.
Excess SOCl2 was then evaporated under reduced pressure.
The residue was dissolved in acetone and aqueous ammonia
was added dropwise (at ꢃ5 ꢀC), with stirring, until the reac-
tion was complete. The mixture was treated with charcoal, fil-
tered and the filtrate was evaporated. The residual material was
crystallized from ethanol: yield 76%; mp 163e170 ꢀC; 1H
NMR (300 MHz, CD3OD): 1.39 (6H, d, J ¼ 7.1 Hz,
(CH3)2eCH), 3.64 (1H, sept, J ¼ 7.1 Hz, CH(CH3)2), 6.85 (2
H, br s). 13C NMR (300 MHz, CD3OD): 21.8, 24.7, 133.9,
134.6, 148.6, 169.8. Anal. (C7H10N4O3) C, H, N.
Colchicine was obtained from MP Biomedicals. Myose-
verin was synthesized and characterized according to a pub-
lished method [19]. Stock solutions for bioassays were
prepared in DMSO and stored at 2 ꢀC. The highest concentra-
tions of DMSO in the polymerization buffer and the culture
medium were 5% and 0.5%, respectively.
4.2. General experimental procedures
NMR spectra were measured using a VarianUNITY Inova-
400 spectrometer or a Varian Gemini-300 at 303 K. The resid-
1
ual solvent signal was used as an internal standard. H NMR,
13C NMR, COSY, TOCSY, HMQC, and HMBC spectra were
4.3.3. 4-Amino-5-isopropyl-1(2)H-pyrazole-3-
carboxamide (3)
1
analysed using the manufacturer’s software (Varian Inc.). H
NMR spectra were zero-filled and exponential multiplication
was applied prior Fourier transformation. Protons were
assigned by COSY and TOCSY and the assignments were
transferred to carbons by HMQC. Chemical shifts are given
in d-scale [ppm] and coupling constants in Hz. Carbon chem-
ical shifts were read out from HMQC (protonated carbons) and
HMBC. Mass spectra were determined using a Waters Micro-
mass ZMD mass spectrometer (direct inlet, ESI, coin voltage
20 V); standard electron ionization EI mass spectra were
recorded using a Jeol D100 double-focusing mass spectrome-
ter (ionization energy 75 eV, chamber temperature 200 ꢀC,
ionizing current 300 mA, accelerating voltage 3 kV). All com-
pounds gave satisfactory elemental analyses (0.4%). Merck
silica gel Kieselgel 60 (230e400 mesh) was used for column
chromatography.
5-Isopropyl-4-nitro-1(2)H-pyrazole-3-carboxamide (3 g,
15.138 mmol) (2) was hydrogenated at atmospheric pressure
and ambient temperature on 1 g of Raney nickel (W5 activ-
ity) in 20 ml MeOH and 5 ml H2O for 9 h. The product was
crystallized from EtOAc. The rest of the product was ob-
tained from mother liquor by chromatography on silica in
CHCl3eMeOH (93:7); overall yield 96%; mp 177e179 ꢀC;
1H NMR (400 MHz; CD3OD): 1.31 (6H, d, J ¼ 7.5 Hz,
(CH3)2eCH), 3.06 (1H, sept, J ¼ 7.5 Hz, CH(CH3)2), 6.98
(2H, br s), 7.12 (2H, br s). 13C NMR (400 MHz, CD3OD):
21.5, 24.0, 128.5, 137.1, 165.6, 172.3. Anal. (C7H12N4O)
C, H, N.
4.3.4. 3-Isopropyl-1(2)H-pyrazolo[4,3-d]-
pyrimidin-5,7-diol (4)
4.3. Prepared compounds
A mixture of 4-amino-5-isopropyl-1(2)H-pyrazole-3-car-
boxamide (3) (770 mg, 4.58 mmol) and urea (1.4 g,
4.58 mmol) was fused at 180 ꢀC for 30 min. After cooling,
the solid residue was dissolved in 2.3 ml of aqueous 2 M
NaOH. The boiling solution was acidified with glacial acetic
acid and the warm solution was filtered. This solution was
cooled to 5 ꢀC and the product started to precipitate after
a few minutes. The product (1.3 g) was recrystallized twice
from hot water; yield 78.5%; mp 295e298 ꢀC; 1H NMR
(300 MHz, DMSO-d6): 1.21 (6H, d, J ¼ 6.6 Hz, CH3CHe),
3.17 (1H, sept, J ¼ 6.6 Hz, CH3CHe). 13C NMR (300 MHz,
DMSO-d6, 303 K): 21.6 (C-200), 25.5 (C-10), 124.9 (7a),
137.6 (7b), 145.4 (3), 150.4 (7), 154.7 (5). EI MS: 194 (94;
Mþ); 179 (100; C7H7N4O2); 162 (51); 136 (15); 123 (7); 95
(8); 81 (13); 54 (26); 43 (23). Anal. (C8H10N4O2) C, H, N.
4.3.1. 3-Isopropyl-5,7-bis[(4-methoxybenzyl)amino]-1,2,3-
triazolo[4,5-d]pyrimidine (8-azamyoseverin)
A reaction mixture containing 5,7-dichloro-3-isopropyl-
1,2,3-triazolo[4,5-d]pyrimidine (1.68 mmol) in 7 ml 1-butanol
with 4-methoxybenzylamine (11.5 mmol) was stirred (110 ꢀC,
17 h) according to a previously described procedure [11].
Crystals of the product were filtered; recrystallization from
CHCl3 afforded the product; yield 50%; mp 162e165 ꢀC,
ESIþMS: 434.3 (100, M þ Hþ); 1H NMR (400 MHz,
CDCl3): 1.65 (6H, d, J ¼ 6.8 Hz, H-200), 3.79 (3H, s,
CH3O0), 3.80 (3H, s, CH3O), 4.60 (2H, d, J ¼ 5.7 Hz, CH2),
0
4.71 (2H, m, CH2 ), 4.95 (1H, septet, J ¼ 6.8 Hz, H-100), 5.52
(1H, br s, 2-NH), 6.53 (1H, br s, 6-NH0), 6.85 (2H, m,