Cyclocondensation reaction of heterocyclic carbonyl compounds, Part XIII: Synthesis and cytotoxic activity of some 3,7-diaryl-5-(3,4,5-trimethoxyphenyl)pyrazolo[4,3-e][1,2,4]triazines

Article abstract of DOI:10.1016/j.ejmech.2008.05.026

A series of the 3,7-diaryl-5-(3,4,5-trimethoxyphenyl)pyrazolo[4,3-e][1,2,4]triazines (17a-20f) have been synthesized in five steps. The cytotoxic activity of all of the newly synthesized compounds has been tested in vitro against five cancer cell lines. S

Full text of DOI:10.1016/j.ejmech.2008.05.026

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European Journal of Medicinal Chemistry 44 (2009) 891e900
http://www.elsevier.com/locate/ejmech
Original article
Cyclocondensation reaction of heterocyclic carbonyl compounds,
Part XIII: Synthesis and cytotoxic activity of some 3,7-diaryl-
5-(3,4,5-trimethoxyphenyl)pyrazolo[4,3-e][1,2,4]triazines*
a,
a
b
Tomas Gucky , Iveta Frysova , Jan Slouka , Marian Hajduch , Petr Dzubak
b
´ˇ
´
ˇ ´ a
´
´
´
*
ꢀ
a Department of Organic Chemistry, Faculty of Science, Palacky´ University, Tr. Svobody 8, 771 46 Olomouc, Czech Republic
b Laboratory of Experimental Medicine, Departments of Pediatrics and Oncology, Faculty of Medicine,
Palacky University and Faculty Hospital in Olomouc, Czech Republic
Received 1 October 2007; received in revised form 16 May 2008; accepted 23 May 2008
Available online 15 July 2008
Abstract
A series of the 3,7-diaryl-5-(3,4,5-trimethoxyphenyl)pyrazolo[4,3-e][1,2,4]triazines (17ae20f) have been synthesized in five steps. The
cytotoxic activity of all of the newly synthesized compounds has been tested in vitro against five cancer cell lines. Several compounds demon-
strated significant broad cytotoxic activity in low micromolar range, while others were selectively active against lung adenocarcinoma cell line
A549.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: Pyrazolo[4,3-e][1,2,4]triazines; Cytotoxic activity
1. Introduction
[1,2,4]triazines [6e8] and tested their cytotoxic activity which
was, however, very low. For this reason we have decided to
perform another study of synthesis and cytotoxic activity of
novel series of 3,7-diaryl-5-(3,4,5-trimethoxyphenyl)pyra-
zolo[4,3-e][1,2,4]triazines (17ae20f) as well as the study of
cytotoxic activity of the key intermediates, 2-acylamino-
3-(3,4,5-trimethoxyphenyl)acrylic acid hydrazides (5e8),
3-substituted-5-(3,4,5-trimethoxybenzyl)[1,2,4]triazine-6-ones
(9e12) and arylhydrazones of 3-substituted-5-(3,4,5-trime-
thoxybenzoyl)[1,2,4]triazine-6-ones (13ae16f).
The 3,4,5-trimethoxyphenyl group on position 5 of the tri-
azine ring could elevate the cytotoxic activity since a large
number of molecules bearing the 3,4,5-trimethoxyphenyl
group show various biological activity. The vicinal trimethoxy
group is common to Colchicine [9], Demecolcine[10] and Po-
dophyllotoxin [11] which show antimitotic activity as well as
for combrestatins [12] and their analogs [13,14] which simi-
larly to Colchicine and Demecolcine inhibit tubulin polymeri-
sation and also in several synthetic compounds like
Trimethoprim [15], Duocarmine analogs [16e19] and some
The pyrazolo[4,3-e][1,2,4]triazines has been less studied
heterocyclic compounds so far in comparison with other con-
densed heterocycles which bear [1,2,4]triazine ring. There has
been found a few pyrazolo[4,3-e][1,2,4]triazines of the natural
origin. Pseudoiodinine and Normethylpseudoiodinine and
some other related compounds are produced by the microor-
ganisms of the genus Pseudomonas fluorescens var. pseudoidi-
ninum [2e4] and Nostocin A is produced by the cyanobacter
Nostoc spongiaeforme [5]. These natural compounds have pre-
sented slight or moderate antineoplastic and antibacterial
activity.
In our previous works we have described a few series
of 3,5-disubstituted, and 3,5,7-trisubstituted pyrazolo[4,3-e]-
*
See Ref. [1].
* Corresponding author. Tel.: þ420 585634437; fax: þ420 585634405.
´
E-mail address: akrylhydrazid@seznam.cz (T. Gucky).
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.05.026

892
T. Gucky´ et al. / European Journal of Medicinal Chemistry 44 (2009) 891e900
Table 1
Selected NMR data of compounds 9e12
HIV-1 reverse transcriptase inhibitors [20]. The antineoplastic
activity of ansa-titanocene trimethoxy derivatives has been
reported as well [21].
The target of this work was to find the relationship between
structure and cytotoxic activity in the series of studied 3,7-di-
aryl-5-(3,4,5-trimethoxyphenyl)pyrazolo[4,3-e][1,2,4]triazines
and the intermediates.
Compound
d
Rel. int.
(CH2)
d
Rel. int.
(]CH)
Ratio
A/B
(CH2)
(]CH)
9
4.109
4.095
4.114
4.104
2.0
2.0
2.0
2.0
6.349
6.333
6.368
6.351
0.78
0.63
0.49
0.66
1.28
1.58
2.03
1.52
10
11
12
2. Chemistry
3. Pharmacology
The starting (4Z )-2-substituted-4-(3,4,5-trimethoxybenzy-
lidene)-1,3-oxazol-5(4H )-ones (1e4) were prepared by the
The cytotoxic activities were examined under in vitro
conditions using MTT assays towards five tumour cell lines
[CEM e T-lymphoblastic leukemia, CEM DNR Bulk e T-
lymphoblastic leukemia, daunorubicin resistant, K-562 (mye-
loid leukemia), K-562-Tax (myeloid leukemia, paclitaxel
resistant) and A549 (lung adenocarcinoma)]. Those cell lines
we routinely use for characterization of new compounds and
were characterized previously [24]. The presented data repre-
sent mean values from three independent experiments, the
standard deviation did not usually exceed 15% on the mean.
´
modified Erlenmeyers reaction. Compounds 2, 3 and 4 have
not been described in the literature so far. It is probable that
oxazolone derivatives 1e4 form Z isomers only which has
been proven for other similar compounds synthesized under
similar conditions [25].
The hydrazinolysis of oxazolone ring under mild conditions
gave the N-[(Z )-1-(hydrazinocarbonylvinyl)-2-(3,4,5-trime-
thoxyphenyl)]acylamides (5e8). The stereochemistry on the
exocyclic double bond does not change during hydrazinolysis
of oxazolone ring.
4. Results and discussion
The cyclization reaction of the hydrazides 5e8 in boiling
sodium hydroxide solution proceeded smoothly to the
3-substituted-5-(3,4,5-trimethoxybenzyl)[1,2,4]triazin-6(1H )-
ones (9e12). We investigated also the tautomerism of
[1,2,4]triazines (9e12) which can form tautomers A and B.
We have proven that both of the possible forms were present
in dimethylsulfoxide solution. This fact is in agreement with
previously reported findings [22] which were focused on the
study of tautomerism of analogous 3-substituted-5-arylmethy-
lene-[1,2,4]triazin-6(1H )-ones. We have assigned the ratio of
forms A and B by the comparison of relative intensities of
methylene group hydrogens of form A and methine group hy-
drogen of form B (see Scheme 1 and Table 1).
The arylhydrazones of 3-substituted-5-(3,4,5-trimethoxy-
benzoyl)[1,2,4]triazin-6(1H )-ones (13ae16f) were obtained
by the coupling reaction on active methylene group of com-
pounds (9e12) with aryldiazonium salts in pyridine in high
yields and purity. The stereochemistry of the arylhydrazones
13ae16f has not been studied and is difficult to be supposed.
These arylhydrazones were cyclized to the target 3,7-dia-
ryl-5-(3,4,5-trimethoxyphenyl)pyrazolo[4,3-e][1,2,4]triazines
(17ae20f) by the action of phosphorous oxychloride with cat-
alytic amount of pyridine (see Scheme 2).
The hydrazides (5e8) have shown only very slight cyto-
toxic activity against all of the tested cell lines and no struc-
tureeactivity relationship could be found.
The cytotoxic activity of the 3-aryl-5-(3,4,5-trimethoxyben-
zyl)-1,6-dihydro-[1,2,4]triazine-6-ones 9e12 is interesting and
among them 9 showed significant and broad activity against all
tested cell lines.
There has been found significant cytotoxic activity at the
arylhydrazones 13ae13f, 14d, 15a, 15c, 15d, 15f, 16a, 16d
and 16f, however, we were not able to identify any struc-
tureeactivity relationship among these compounds.
The most interesting, 3,7-diaryl-5-(3,4,5-trimethoxyphe-
nyl)pyrazolo[4,3-e][1,2,4]triazines (17b, 17c, 17e, 18b, 19b,
19f, 20b, 20c and 20e) have exhibited significant and relative
selective inhibitory activity against A549 cell line, while they
were generally less active against leukemia cell lines, includ-
ing otherwise highly chemosensitive CEM lymphoblasts.
Those data suggest that this class of compounds could be pre-
dominantly active in epithelial cancers and deserves further
experimental verification.
The results are summarized in Table 2.
5. Experimental
OMe
OMe
5.1. Apparatus and methods
MeO
OMe
MeO
OMe
Melting points were determined on a Boetius stage and are not
1
corrected. H NMR spectra were measured in DMSO-d6 and
CH2
CH
O
CDCl3 at 300 K on a Bruker Avance 300 spectrometer
(300 MHz) with TMS as an internal standard; chemical shifts
are reported in ppm, and coupling constants in Hz. Mass spectra
wererecordedusinganLCQiontrapmassspectrometer(Finnigan
MAT, San Jose, CA, USA). Elemental analyses were performed
using an EA 1108 Elemental Analyzer (Fison Instruments).
O
N
HN
NH
NH
N
R1
N
R1
9A - 12A
9B-12B
Scheme 1.

T. Gucky´ et al. / European Journal of Medicinal Chemistry 44 (2009) 891e900
893
OMe
MeO
MeO
MeO
MeO
MeO
MeO
MeO
O
O
1. NaOH
2. HCl
N H
2
4
MeO
HN
R1
N
H
NH2
N
O
O
O
N
R1
NH
N
R1
1-4
5-8
9-12
R N
2
2
OMe
MeO
MeO
OMe
MeO
MeO
POCl
3
N
R2
N
N
N
H
O
R2
N
N
N
NH
N
R1
N
13-16
R1
17-20
For compounds 13-20
a) R2 = phenyl
For compounds
1,5,9,13,17
2,6,10,14,18
3,7,11,15,19
4,8,12,16,20
R1= phenyl,
R1= 4-methoxyphenyl
R1= 3,4,5-trimethoxyphenyl,
R1= 4-chlorophenyl
b) R2 = 2-methoxyphenyl
c) R2 = 3-methoxyphenyl
d) R2 = 4-methoxyphenyl
e) R2 = 3,4,5-trimethoxyphenyl
f) R2 = 4-chlorophenyl
Scheme 2.
5.2. Chemistry
(s, 12H, OCH3), 7.29 (s, 1H, ]CH), 7.38 (s, 2H, ArH), 7.79
(s, 2H, ArH), ms: 430.40 (MHþ). Anal. Calcd for
C22H23NO8 (429.43): C, 61.53; H, 5.39; N, 3.26. Found: C,
61.77; H, 5.73; N, 3.30.
5.2.1. (4Z )-2-Substituted-4-(3,4,5-trimethoxybenzyli
dene)-1,3-oxazol-5(4H )-ones 1e4
5.2.1.1. General procedure. The N-acylglycine (56.00 mmol),
3,4,5-trimethoxybenzaldehyde (11.00 g, 56.00 mmol), acetic
anhydride (16.0 ml, 167.5 mmol) and anhydrous pyridine
(4.50 ml, 56.00 mmol) was heated on an oil bath at tempera-
ture 80 ꢀC for 2 h. After cooling to room temperature, the mix-
ture was allowed to stand at 0 ꢀC overnight. The precipitate
was filtered and washed three times with ice-cooled ethanol
(10 ml). The crude product was crystallized from toluene.
5.2.5. (4Z )-2-(4-Chlorophenyl)-4-(3,4,5-trimethoxy
benzylidene)-1,3-oxazol-5(4H )-one 4
Yield: 73%, mp: 178e180 ꢀC, 1H NMR (300 MHz,
DMSO-d6): d 3.78 (s, 3H, OCH3), 3.88 (s, 6H, OCH3), 7.32
(s, 1H, ]CH), 7.70 (d, J ¼ 8.7, 2H, ArH), 7.76 (s, 2H,
ArH), 8.09 (d, J ¼ 8.7, 2H, ArH), ms: 374.90 (MHþ). Anal.
Calcd for C19H16NO5Cl (373.84): C, 61.05; H, 4.31; N,
3.75. Found: C, 60.97; H, 4.54; N, 3.53.
5.2.2. (4Z )-2-Phenyl-4-(3,4,5-trimethoxybenzyli
dene)-1,3-oxazol-5(4H )-one 1
See Ref. [23].
5.2.6. N-[(Z )-1-(Hydrazinocarbonylvinyl)-2-(3,4,5-trime
thoxyphenyl)]acylamides 5e8
5.2.6.1. General procedure. The (4Z )-2-substituted-4-(3,4,5-
trimethoxybenzylidene)-1,3-oxazol-5(4H )-one (14.00 mmol)
was suspended in absolute ethanol (30 ml) and 99% hydrazine
hydrate (0.825 ml, 17.00 mmol) was added. The mixture was
refluxed for 3 min and filtered while hot. The filtrate was
cooled to room temperature and allowed to stand at 0 ꢀC for
2 h. The white precipitate was filtered and washed twice
with ice-cooled ethanol (10 ml). The crude product was crys-
tallized from ethanol.
5.2.3. (4Z )-2-(4-Methoxyphenyl)-4-(3,4,5-trimetho
xybenzylidene)-1,3-oxazol-5(4H )-one 2
Yield: 68%, mp: 166e167 ꢀC, 1H NMR (300 MHz,
DMSO-d6): d 3.77 (s, 3H, OCH3), 3.87 (s, 6H, OCH3), 3.90
(s, 3H, OCH3), 7.18 (d, J ¼ 8.7, 2H, ArH), 7.21 (s, 1H,
]CH), 7.72 (s, 2H, ArH), 8.05 (d, J ¼ 8.7, 2H, ArH), ms:
370.40 (MHþ). Anal. Calcd for C20H19NO6 (369.37): C,
65.03; H, 5.18; N, 3.79. Found: C, 65.23; H, 5.11; N, 3.57.
5.2.4. (4Z )-2-(3,4,5-Trimethoxyphenyl)-4-(3,4,5-trimethoxy
benzylidene)-1,3-oxazol-5(4H )-one 3
Yield: 58%, mp: 194e196 ꢀC, 1H NMR (300 MHz,
DMSO-d6): 3.77 (s, 3H, OCH3), 3.79 (s, 3H, OCH3), 3.89
5.2.7. N-[(Z )-1-(Hydrazinocarbonylvinyl)-2-(3,4,5-trimetho
xyphenyl)]benzamide 5
Yield: 78%, mp: 170e172 ꢀC, 1H NMR (300 MHz,
DMSO-d6): d 3.57 (s, 6H, OCH3), 3.04 (s, 3H, OCH3), 4.40

894
T. Gucky´ et al. / European Journal of Medicinal Chemistry 44 (2009) 891e900
Table 2
Results of MTT cytotoxic activity tests (IC50 in mmol/l)
5.2.8. N-[2-(3,4,5-Trimethoxyphenyl)-1-hydrazino
carbonylvinyl]-(4-methoxy)benzamide 6
Yield: 90%, mp: 184e186 ꢀC, 1H NMR (300 MHz,
DMSO-d6): d 3.58 (s, 6H, OCH3), 3.63 (s, 3H, OCH3), 3.83
(s, 3H, OCH3), 4.38 (br s, 2H, NH2), 6.92 (s, 2H, ArH),
7.02 (d, J ¼ 9.0, 2H, ArH), 7.21 (s, 1H, ]CH), 8.01 (d,
J ¼ 9.0, 2H, ArH), 9.45 (br s, 1H, NH), 9.65 (br s, 1H, NH),
ms: 402.43 (MHþ). Anal. Calcd for C20H23N3O6 (401.42):
C, 59.84; H, 5.77; N, 10.47. Found: C, 60.00; H, 6.01; N,
10.50.
CEM
CEM DNR
Bulk
K562
K562 tax
A549
5
124
144
121
147
126
130
3.21
13.3
129
14.6
18.2
43.2
19.3
9.46
33.2
46.8
39.8
141
132
9.80
47.4
50.1
95.5
203
140
34.6
158
175
37.7
36.6
133
9.16
139
37.7
108
118
99.3
100
129
156
114
119
184
118
117
118
109
125
109
118
201
115
113
35.7
108
139
117
126
162
172
160
166
1.17
3.07
82.1
9.63
11.5
148
12.7
6.27
7.40
12.0
18.4
148
162
2.73
45.5
41.3
44.9
144
112
18.7
94.3
246
17.5
54.1
41.8
7.50
50.1
10.2
169
182
117
84.4
52.6
250
166
178
115
186
230
203
183
182
164
66.2
241
158
220
48.7
192
248
192
190
162
167
153
174
3.88
19.9
138
24.7
19.3
145
13.7
11.0
27.6
38.4
31.8
250
250
8.45
137
238
8.60
186
22.1
8.91
118
128
33.1
250
26.3
8.33
159
12.5
216
94.3
159
150
99.9
173
203
155
174
229
246
232
233
156
141
161
250
196
225
20.7
219
250
210
222
164
134
6
7
123
134
103
161
8
9
10
2.71
3.15
38.4
12.9
10.6
13.7
8.60
4.16
12.2
25.1
9.81
54.6
68.7
6.43
24.2
20.2
5.06
61.9
7.83
5.71
53.8
2.25
10.8
54.5
11.5
9.49
42.1
7.67
109
3.46
9.31
117
11
12
15.8
7.14
3.63
8.30
3.06
3.19
8.55
23.7
250
13a
13b
13c
13d
13e
13f
14a
14b
14c
14d
14e
14f
15a
15b
15c
15d
15e
15f
16a
16b
16c
16d
16e
16f
17a
17b
17c
17d
17e
17f
18a
18b
18c
18d
18e
18f
19a
19b
19c
19d
19e
19f
20a
20b
20c
20d
20e
20f
5.2.9. N-[(Z )-1-(Hydrazinocarbonylvinyl)-2-(3,4,5-trimetho
xyphenyl)](3,4,5-trimethoxy)benzamide 7
Yield: 95%, mp: 188e190 ꢀC, 1H NMR (300 MHz,
DMSO-d6): d 3.62 (s, 6H, OCH3), 3.65 (s, 3H, OCH3), 3.73
(s, 3H, OCH3), 3.83 (s, 6H, OCH3), 4.41 (br s, 2H, NH2),
6.95 (s, 2H, ArH), 7.23 (s, 1H, ]CH), 7.40 (s, 2H, ArH),
9.48 (s, 1H, NH), 9.78 (s, 1H, NH), ms: 462.50 (MHþ).
Anal. Calcd for C22H27N3O8 (461.47): C, 57.26; H, 5.90; N,
9.11. Found: C, 57.38; H, 6.10; N, 8.94.
250
6.20
3.33
18.5
3.30
75.2
3.38
3.39
162
5.2.10. N-[(Z )-1-(Hydrazinocarbonylvinyl)-2-(3,4,5-trimeth
oxyphenyl)](4-chloro)benzamide 8
Yield: 95%, mp: 163e164 ꢀC, 1H NMR (300 MHz,
DMSO-d6): d 3.59 (s, 6H, OCH3), 3.64 (s, 3H, OCH3), 4.41
(br s, 2H, NH2), 6.91 (s, 2H, ArH), 7.23 (s, 1H, ]CH), 7.59
(d, J ¼ 8.7, 2H, ArH), 8.06 (d, J ¼ 8.7, 2H, ArH), 9.54 (s,
1H, NH), 9.90 (s, 1H, NH), ms: 406.93 (MHþ). Anal. Calcd
for C19H20N3O5Cl (405.88): C, 56.23; H, 4.97; N, 10.36.
Found: C, 56.19; H, 4.85; N, 10.16.
1.18
22.1
60.5
8.32
2.34
200
3.81
86.6
5.41
0.61
39.0
2.16
57.3
78.8
6.58
124
5.2.11. 3-Substituted-5-(3,4,5-trimeth
oxybenzyl)[1,2,4]triazin-6(1H )-ones 9e12
109
8.41
117
65.2
150
5.2.11.1. General procedure. The N-[2-(3,4,5-trimethoxyphen
yl)-1-hydrazinocarbonylvinyl]acylamide 5e8 (7.50 mmol)
was refluxed in 1 M sodium hydroxide water solution for
5 min. After cooling to room temperature the solution was
slowly neutralized with acetic acid. The yellow precipitate
was filtered and washed with water (30 ml). The crude product
was crystallized from ethanol.
114
105
54.5
124
113
160
212
115
113
142
126
92.3
15.0
163
5.2.12. 3-Phenyl-5-(3,4,5-trimethoxybenzyl)[1,2,4]tr
iazin-6(1H )-one 9
48.3
36.9
246
75.6
194
Yield: 87%, mp: 169e171 ꢀC, 1H NMR (300 MHz,
DMSO-d6): 9A: 3.639 (s, 3H, OCH3), 3.749 (s, 6H, OCH3),
4.109 (s, 2H, CH2), 6.708 (s, 2H, ArH), 7.456e7.481 (m,
3H, ArH), 8.007e8.040 (m, 2H, ArH), 13.502 (s, 1H, NH),
9B: 3.683 (s, 3H, OCH3), 3.868 (s, 6H, OCH3), 6.349 (s,
1H, ]CH), 7.456e7.481 (m, 3H, ArH), 7.742e7.774 (m,
2H, ArH), 9.823 (s, 1H, NH), 11.139 (s, 1H, NH), 13C
NMR: 55.80, 59.91, 104.53, 105.56, 107.01, 126.01, 126.45,
126.60, 128.26, 128.68, 129.78, 130.03, 130.31, 131.22,
131.79, 133.98, 136.38, 141.84, 146.72, 152.63, 152.81,
154.09, 157.16, 169.42, ms: 354.20 (MHþ). Anal. Calcd for
C19H19N3O4 (353.38): C, 64.58; H, 5.42; N, 11.89. Found:
C, 64.53; H, 5.21; N, 12.01.
82.2
11.6
53.8
54.7
226
8.26
130
3.62
2.86
113
99.9
68.9
15.0
155
(br s, 2H, NH2), 6.92 (s, 2H, ArH), 7.25 (s, 1H, ]CH), 7.49 (t,
J ¼ 7.5, 2H, ArH), 7.58 (t, J ¼ 7.5, 1H, ArH), 8.04 (d, J ¼ 7.5,
2H, ArH), 9.51 (br s, 1H, NH), 9.81 (br s, 1H, NH), ms: 372.38
(MHþ). Anal. Calcd for C19H21N3O5 (371.39): C, 61.45; H,
5.70; N, 11.31. Found: C, 61.15; H, 5.70; N, 5.95.

T. Gucky´ et al. / European Journal of Medicinal Chemistry 44 (2009) 891e900
895
5.2.13. 3-(4-Methoxyphenyl)-5-(3,4,5-trimetho
xybenzyl)[1,2,4]triazin-6(1H )-one 10
and cooled to 0 ꢀC. Then the solution of aryldiazonium salt
was slowly added with stirring so that the temperature was
maintained between 0 and 5 ꢀC. Then the reaction mixture
was allowed to stand at 0 ꢀC overnight. After warming to
room temperature was the solution diluted with water
(25 ml) and the precipitate was filtered off and washed with
water (50 ml). The crude product was used for further reaction
without crystallisation.
Yield: 95%, mp: 192e194 ꢀC, 1H NMR (300 MHz,
DMSO-d6): 12A: 3.639 (s, 3H, OCH3), 3.748 (s, 6H,
OCH3), 4.104 (s, 2H, CH2), 6.698 (s, 2H, ArH), 7.535e
7.564 (m, 2H, ArH), 8.020 (d, J ¼ 8.7, 2H, ArH), 11.178 (s,
1H, NH), 12B: 3.682 (s, 3H, OCH3), 3.860 (s, 6H, OCH3),
6.351 (s, 1H, ]CH), 6.908 (s, 2H, ArH), 7.535e7.564 (m,
2H, ArH), 7.759 (d, J ¼ 8.7, 2H, ArH), 9.890 (s, 1H, NH),
13.567 (s, 1H, NH), ms: 384.45 (MHþ). Anal. Calcd for
C20H21N3O5 (383,4): C, 62.65; H, 5.52; N, 10.96. Found: C,
62.74; H, 5.76; N, 10.84.
5.2.17. 3-Phenyl-5-[(3,4,5-trimethoxyphenyl)
(phenylhydrazono)methyl][1,2,4]-triazin-6(1H )-one 13a
Yield: 87%, mp: 207e209 ꢀC, 1H NMR (300 MHz,
DMSO-d6): d 3.70 (s, 3H, OCH3), 3.77 (s, 6H, OCH3), 6.84
(t, J ¼ 7.2, 1H, ArH), 6.89 (s, 2H, ArH), 7.19 (t, J ¼ 7.5,
2H, ArH), 7.27 (t, J ¼ 7.2, 2H, ArH), 7.40 (t, J ¼ 7.5, 1H,
ArH), 7.51 (d, J ¼ 7.2, 2H, ArH), 8.09 (d, J ¼ 7.5, 2H,
ArH), 10.11 (s, 1H, NH), 13.87 (s, 1H, NH), ms: 458.49
(MHþ). Anal. Calcd for C25H23N5O4 (457.49): C, 65.63; H,
5.07; N, 15.31. Found: C, 65.74; H, 5.07; N, 15.45.
5.2.14. 3-(3,4,5-Trimethoxyphenyl)-5-(3,4,5-trimetho
xybenzyl)[1,2,4]triazin-6(1H )-one 11
Yield: 94%, mp: 157e160 ꢀC, 1H NMR (300 MHz,
DMSO-d6): 10A: 3.642 (s, 3H, OCH3), 3.751 (s, 6H,
OCH3), 3.802 (s, 3H, OCH3), 4.095 (s, 2H, CH2), 6.702 (s,
2H, ArH), 7.037 (d, J ¼ 8.7, 2H, ArH), 7.956 (d, J ¼ 8.7,
2H, ArH), 11.060 (s, 1H, NH), 10B: 3.685 (s, 3H, OCH3),
3.810 (s, 3H, OCH3), 3.868 (s, 6H, OCH3), 6.333 (s, 1H,
]CH), 6.909 (s, 2H, ArH), 7.010 (d, J ¼ 8.7, 2H, ArH),
7.783 (d, J ¼ 8.7, 2H, ArH), 9.726 (s, 1H, NH), 13.381 (s,
1H, NH), ms: 444.13 (MHþ). Anal. Calcd for C22H25N3O7
(443.46): C, 59.59; H, 5.68; N, 9.48. Found: C, 59.47; H,
5.74; N, 9.61.
5.2.18. 3-Phenyl-5-[(3,4,5-trimethoxyphenyl)(2-meth
oxyphenylhydrazono)methyl][1,2,4]-triazin-6(1H )-one 13b
Yield: 86%, mp: 203e205 ꢀC, 1H NMR (300 MHz,
DMSO-d6): d 3.70 (s, 3H, eOCH3), 3.77 (s, 3H, eOCH3),
3.80 (s, 6H, eOCH3), 6.89 (s, 2H, ArH), 6.95e7.04 (m, 3H,
ArH), 7.57e7.65 (m, 4H, ArH), 8.17 (d, J ¼ 8.4, 2H, ArH),
11.89 (br s, 1H, NH), 13.75 (br s, 1H, NH), ms: 488.13
(MHþ). Anal. Calcd for C26H25N5O5 (487.51): C, 64.06; H,
5.17; N, 14.37. Found: C, 64.28; H, 5.31; N, 14.09.
5.2.15. 3-(4-Chlorophenyl)-5-(3,4,5-trimetho
xybenzyl)[1,2,4]triazin-6(1H )-one 12
Yield: 96%, mp: 179e181 ꢀC, 1H NMR (300 MHz,
DMSO-d6): 11A: 3.632 (s, 3H, OCH3), 3.698 (s, 3H,
OCH3), 3.745 (s, 6H, OCH3), 3.772 (s, 6H, OCH3), 4.114 (s,
2H, CH2), 6.692 (s, 2H, ArH), 7.271 (s, 2H, ArH), 11.112
(s, 1H, NH), 11B: 3.677 (s, 3H, OCH3), 3.710 (s, 3H,
OCH3), 3.821 (s, 6H, OCH3), 3.839 (s, 6H, OCH3), 6.368 (s,
1H, ]CH), 6.901 (s, 2H, ArH), 7.082 (s, 2H, ArH), 9.763
(s, 1H, NH), 13.432 (s, 1H, NH), 13C NMR: 55.61, 55.78,
55.87, 56.02, 59.88, 59.98, 60.06, 103.33, 104.52, 106.08,
107.24, 126.48, 127.26, 129.33, 130.23, 131.19, 136.28,
136.66, 139.05, 139.30, 141.62, 146.22, 152.59, 152.81,
152.99, 154.00, 156.9, 169.40, ms: 388.92 (MHþ). Anal.
Calcd for C19H18N3O4Cl (387.87): C, 58.84; H, 4.68; N,
10.83. Found: C, 58.66; H, 4.94; N, 10.56.
5.2.19. 3-Phenyl-5-[(3,4,5-trimethoxyphenyl)(3-metho
xyphenylhydrazono)methyl][1,2,4]-triazin-6(1H )-one 13c
Yield: 83%, mp: 185e187 ꢀC, 1H NMR (300 MHz,
DMSO-d6): d 3.73b (s, 3H, eOCH3), 3.76 (s, 6H, eOCH3),
3.80 (s, 3H, eOCH3), 6.41 (d, J ¼ 8.9, 1H, ArH), 6.75 (d,
J ¼ 9.3, 2H, ArH), 6.88 (s, 2H, ArH), 7.40 (t, J ¼ 8.1, 1H,
ArH), 7.51e7.56 (m, 3H, ArH), 8.08 (d, J ¼ 8.1, 2H, ArH),
10.08 (s, 1H, NH), 13.89 (br s, 1H, NH), ms: 488.13
(MHþ). Anal. Calcd for C26H25N5O5 (487.51): C, 64.06; H,
5.17; N, 14.37. Found: C, 64.19; H, 5.12; N, 14.35.
5.2.20. 3-Phenyl-5-[(3,4,5-trimethoxyphenyl)(4-methoxy
phenylhydrazono)methyl][1,2,4]-triazin-6(1H )-one 13d
Yield: 94%, mp: 175e177 ꢀC, 1H NMR (300 MHz,
DMSO-d6): d 3.69 (s, 3H, OCH3), 3.77 (s, 6H, OCH3), 3.81
(s, 3H, OCH3), 6.83 (t, J ¼ 7.2, 1H, ArH), 6.87 (s, 2H,
ArH), 7.07 (d, J ¼ 9.0, 2H, ArH), 7.18 (d, J ¼ 7.2, 2H,
ArH), 7.26 (t, J ¼ 7.2, 2H, ArH), 8.02 (d, J ¼ 9.0, 2H, ArH),
10.09 (s, 1H, NH), 13.74 (s, 1H, NH), ms: 488.28 (MHþ).
Anal. Calcd for C26H25N5O5 (487.51): C, 64.06; H, 5.17; N,
14.37. Found: C, 63.96; H, 5.31; N, 14.25.
5.2.16. 3-Substituted-5-[(3,4,5-trimethoxyphenyl)
(arylhydrazono)methyl][1,2,4]triazin-6(1H )-ones 13ae16f
5.2.16.1. General procedure. The appropriate aromatic amine
(3.10 mmol) was dissolved in the mixture of water (4.5 ml)
and conc. hydrochloric acid (0.5 ml) and the solution was
cooled to 0 ꢀC. Then a solution of sodium nitrite (0.23 g,
3.30 mmol) in water (3.0 ml) was slowly added with stirring
so that the temperature was maintained between 0 and 5 ꢀC.
The solution of aryldiazonium salt was stirred at 0 ꢀC for fur-
ther 20 min.
5.2.21. 3-Phenyl-5-[(3,4,5-trimethoxyphenyl)(3,4,5-trimetho
xyphenylhydrazono)methyl][1,2,4]-triazin-6(1H )-one 13e
Yield: 79%, mp: 134e136 ꢀC, 1H NMR (300 MHz,
DMSO-d6): d 3.60 (s, 3H, OCH3), 3.69 (s, 3H, OCH3), 3.77
(s, 6H, OCH3), 3.81 (s, 6H, OCH3), 6.50 (s, 2H, ArH), 6.91
The 3-substituted-5-(3,4,5-trimethoxybenzyl)-1H-[1,2,4]tri
azine-6-one (3.00 mmol) was dissolved in pyridine (15 ml)

896
T. Gucky´ et al. / European Journal of Medicinal Chemistry 44 (2009) 891e900
(s, 2H, ArH), 7.40 (t, J ¼ 7.2, 1H, ArH), 7.51 (t, J ¼ 7.2, 2H,
ArH), 8.09 (d, J ¼ 7.2, 2H, ArH), 10.05 (s, 1H, NH), 13.12 (s,
1H, NH), ms: 548.49 (MHþ). Anal. Calcd for C28H29N5O7
(547.57): C, 61.42; H, 5.34; N, 12.79. Found: C, 61.26; H,
5.51; N, 12.55.
C27H27N5O6 (517.55): C, 62.66; H, 5.26; N, 13.53. Found:
C, 62.47; H, 5.31; N, 13.54.
5.2.27. 3-(4-Methoxyphenyl)-5-[(3,4,5-trimethoxyphenyl)
(3,4,5-trimethoxyphenylhydrazono)methyl]
[1,2,4]-triazin-6(1H )-one 14e
5.2.22. 3-Phenyl-5-[(3,4,5-trimethoxyphenyl)(4-chlorop
henylhydrazono)methyl][1,2,4]-triazin-6(1H )-one 13f
Yield: 68%, mp: 127e128 ꢀC, 1H NMR (300 MHz,
DMSO-d6): d 3.70 (s, 3H, OCH3), 3.78 (s, 6H, OCH3), 6.89
(s, 2H, ArH), 7.19 (d, J ¼ 9.0, 2H, ArH), 7.29 (d, J ¼ 9.0,
2H, ArH), 7.38 (t, J ¼ 7.8, 2H, ArH), 7.78 (t, J ¼ 7.8, 1H,
ArH), 8.57 (d, J ¼ 7.8, 2H, ArH), 10.14 (s, 1H, NH), 13.23
(s, 1H, NH), ms: 492.95, 494.02 (MHþ). Anal. Calcd for
C25H22N5O4Cl (491.98): C, 61.04; H, 4.51; N, 14.24. Found:
C, 61.18; H, 4.73; N, 14.30.
1
Yield: 65%, mp: 162e165, H NMR (300 MHz, DMSO-
d6): d 3.59 (s, 3H, OCH3), 3.69 (s, 3H, OCH3), 3.76 (s, 6H,
OCH3), 3.77 (s, 6H, OCH3), 3.82 (s, 3H, OCH3), 6.50 (s,
2H, ArH), 6.89 (s, 2H, ArH), 7.06 (d, J ¼ 8.7, 2H, ArH),
8.02 (d, J ¼ 8.7, 2H, ArH), 10.03 (s, 1H, NH), 13.74 (s, 1H,
NH), ms: 578.61 (MHþ). Anal. Calcd for C29H31N5O8
(577.60): C, 60.31; H, 5.41; N, 12.12. Found: C, 60.19; H,
5.27; N, 11.94.
5.2.28. 3-(4-Methoxyphenyl)-5-[(3,4,5-trimethoxyphenyl)
(4-chlorophenylhydrazono)methyl][1,2,4]-triazin-6
(1H )-one 14f
5.2.23. 3-(4-Methoxyphenyl)-5-[(3,4,5-trimethoxyphenyl)(3-
phenylhydrazono)methyl][1,2,4]-triazin-6(1H )-one 14a
Yield: 71%, mp: 184e186 ꢀC, 1H NMR (300 MHz,
DMSO-d6): d 3.69 (s, 3H, OCH3), 3.77 (s, 6H, OCH3), 3.81
(s, 3H, OCH3), 6.83 (t, J ¼ 7.2, 1H, ArH), 6.87 (s, 2H,
ArH), 7.07 (d, J ¼ 9.0, 2H, ArH), 7.18 (d, J ¼ 7.2, 2H,
ArH), 7.26 (t, J ¼ 7.2, 2H, ArH), 8.02 (d, J ¼ 9.0, 2H, ArH),
10.09 (s, 1H, NH), 13.74 (s, 1H, NH), ms: 488.48 (MHþ).
Anal. Calcd for C26H25N5O5 (487.52): C, 64.06; H, 5.17; N,
14.37. Found: C, 64.31; H, 5.08; N, 14.62.
Yield: 86%, mp: 110e112 ꢀC, 1H NMR (300 MHz,
DMSO-d6): d 3.81 (s, 3H, OCH3), 3.77 (s, 6H, OCH3), 3.69
(s, 3H, OCH3), 6.88 (s, 2H, ArH), 7.06 (d, J ¼ 9.0, 2H,
ArH), 7.18 (d, J ¼ 8.7), 7.29 (d, J ¼ 9.0, 2H, ArH), 8.02 (d,
J ¼ 8.7, 2H, ArH), 10.13 (s, 1H, NH), 13.25 (s, 1H, NH),
ms: 523.00, 524.94 (MHþ). Anal. Calcd for C26H24N5O5Cl
(521.96): C, 59.83; H, 4.63; N, 13.42. Found: C, 59.59; H,
4.42; N, 13.54.
5.2.24. 3-(4-Methoxyphenyl)-5-[(3,4,5-trimethoxyphenyl)
(2-methoxyphenylhydrazono)methyl][1,2,4]-triazin-6
(1H )-one 14b
5.2.29. 3-(3,4,5-Trimethoxyphenyl)-5-[(3,4,5-trimetho
xyphenyl)(phenylhydrazono)methyl][1,2,4]-triazin-6
(1H )-one 15a
Yield: 75%, mp: 203e205 ꢀC, ms: 518.36 (MHþ). Anal.
Calcd for C27H27N5O6 (517.55): C, 62.66; H, 5.26; N,
13.53. Found: C, 62.64; H, 5.17; N, 13.34.
Yield: 56%, mp: 128e130 ꢀC, 1H NMR (300 MHz,
DMSO-d6): d 3.69 (s, 6H, OCH3), 3.78 (s, 6H, OCH3), 3.81
(s, 3H, OCH3), 3.89 (s, 3H, OCH3), 7.12 (s, 2H, ArH),
7.21e7.31 (m, 3H, ArH), 7.36 (s, 2H, ArH), 7.56 (d,
J ¼ 7.8, 2H, ArH), 10.15 (s, 1H, NH), 13.25 (s, 1H, NH)
ms: 548.36 (MHþ). Anal. Calcd for C28H29N5O7 (547.57):
C, 61.42; H, 5.34; N, 12.79. Found: C, 61.13; H, 5.11; N,
12.56.
5.2.25. 3-(4-Methoxyphenyl)-5-[(3,4,5-trimethoxyphenyl)
(3-methoxyphenylhydrazono)methyl][1,2,4]-triazin-6
(1H )-one 14c
Yield: 72%, mp: 100e105 ꢀC, 1H NMR (300 MHz,
DMSO-d6): d 3.73 (s, 3H, eOCH3), 3.76 (s, 6H, eOCH3),
3.80 (s, 3H, eOCH3), 6.41 (d, J ¼ 8.9, 1H, ArH), 6.75 (d,
J ¼ 9.3, 2H, ArH), 6.88 (s, 2H, ArH), 7.40 (t, J ¼ 8.1, 1H,
ArH), 7.51e7.56 (m, 3H, ArH), 8.08 (d, J ¼ 8.1, 2H, ArH),
10.08 (s, 1H, NH), 13.89 (br s, 1H, NH), ms: 518.46
(MHþ). Anal. Calcd for C27H27N5O6 (517.55): C, 62.66; H,
5.26; N, 13.53. Found: C, 62.42; H, 5.34; N, 13.62.
5.2.30. 3-(3,4,5-Trimethoxyphenyl)-5-[(3,4,5-trimethoxy
phenyl)(2-methoxyphenylhydrazono)methyl]
[1,2,4]-triazin-6(1H )-one 15b
Yield: 77%, mp: 106e108 ꢀC, ms: 578.56 (MHþ). Anal.
Calcd for C29H31N5O8 (577.59): C, 60.31; H, 5.41; N,
12.12. Found: C, 60.05; H, 5.36; N, 11.95.
5.2.26. 3-(4-Methoxyphenyl)-5-[(3,4,5-trimethoxyphenyl)
(4-methoxyphenylhydrazono)methyl][1,2,4]-triazin-6
(1H )-one 14d
Yield: 87%, mp: 183e185 ꢀC, 1H NMR (300 MHz,
DMSO-d6): d 3.69 (s, 3H, OCH3), 3.71 (s, 3H, OCH3), 3.76
(s, 6H, OCH3), 3.82 (s, 3H, OCH3), 6.85 (s, 2H, ArH), 6.88
(d, J ¼ 9.0, 2H, ArH), 7.07 (d, J ¼ 8.7, 2H, ArH), 7.13 (d,
J ¼ 8.7, 2H, ArH), 8.02 (d, J ¼ 9.0, 2H, ArH), 10.09 (s, 1H,
NH), 13.70 (s, 1H, NH), ms: 518.36 (MHþ). Anal. Calcd for
5.2.31. 3-(3,4,5-Trimethoxyphenyl)-5-[(3,4,5-trimethoxy
phenyl)(3-methoxyphenylhydrazono)methyl]
[1,2,4]-triazin-6(1H )-one 15c
Yield: 59%, mp: 194e196 ꢀC, ms: 578.11 (MHþ). Anal.
Calcd for C29H31N5O8 (577.60): C, 60.31; H, 5.41; N, 12.12
Found: C, 60.52; H, 5.29; N, 12.15.

T. Gucky´ et al. / European Journal of Medicinal Chemistry 44 (2009) 891e900
897
5.2.32. 3-(3,4,5-Trimethoxyphenyl)-5-[(3,4,5-trimethoxy
phenyl)(4-methoxyphenylhydrazono)methyl]
[1,2,4]-triazin-6(1H )-one 15d
Yield: 63%, mp: 152e155 ꢀC, ms: 578.13 (MHþ). Anal.
Calcd for C29H31N5O8 (577.60): C, 60.31; H, 5.41; N, 12.12
Found: C, 60.48; H, 5.19; N, 11.94.
524.07 (MHþ). Anal. Calcd for C26H24N5O5Cl (580): C,
59.83, H, 4.63, N, 13.42. Found: C, 59.92 C, H, 4.58, N, 13.55.
5.2.38. 3-(4-Chlorophenyl)-5-[(3,4,5-trimethoxyphenyl)
(4-methoxyphenylhydrazono)methyl][1,2,4]-triazin-6
(1H )-one 16d
Yield: 92%, mp: 188e190 ꢀC, 1H NMR (300 MHz,
DMSO-d6): d 3.76 (s, 6H, OCH3), 3.78 (s, 3H, OCH3), 7.79
(s, 3H, OCH3), 6.85 (s, 2H, ArH), 6.88 (d, J ¼ 8.4, 2H,
ArH), 7.12 (d, J ¼ 8.4, 2H, ArH), 7.58 (d, J ¼ 8.1, 2H,
ArH), 8.08 (d, J ¼ 8.1, 2H, ArH), 10.08 (s, 1H, NH), 13.30
(br s, 1H, NH), ms: 523.00, 525.05 (MHþ). Anal. Calcd for
C26H24N5O5Cl (522.01): C, 59.83; H, 4.63; N, 13.42. Found:
C, 59.64; H, 4.92; N, 13.20.
5.2.33. 3-(3,4,5-Trimethoxyphenyl)-5-[(3,4,5-trimethoxy
phenyl)(3,4,5-trimethoxyphenylhydrazono)methyl]
[1,2,4]-triazin-6(1H )-one 15e
1
Yield: 52%, mp: 95e98 ꢀC, H NMR (300 MHz, DMSO-
d6): d 3.67 (s, 3H, OCH3), 3.69 (s, 3H, OCH3), 3.73 (s, 6H,
OCH3), 3.75 (s, 3H, OCH3), 3.78 (s, 6H, OCH3), 3.81 (s,
3H, OCH3), 6.71 (s, 2H, ArH), 6.88 (s, 2H, ArH), 7.12 (s,
2H, ArH), 10.13 (s, 1H, NH), 13.16 (s, 1H, NH), ms: 638.47
(MHþ). Anal. Calcd for C31H35N5O10 (637.65): C, 58.39; H,
5.53; N, 10.98. Found: C, 58.46; H, 5.26; N, 11.15.
5.2.39. 3-(4-Chlorophenyl)-5-[(3,4,5-trimethoxyphenyl)
(3,4,5-trimethoxyphenylhydrazono)methyl]
[1,2,4]-triazin-6(1H )-one 16e
Yield: 93%, mp: 168e170 ꢀC, 1H NMR (300 MHz,
DMSO-d6): d 3.59 (s, 3H, OCH3), 3.69 (s, 3H, OCH3), 3.77
(s, 6H, OCH3), 3.79 (s, 6H, OCH3), 6.49 (s, 2H, ArH), 6.91
(s, 2H, ArH), 7.58 (d, J ¼ 8.4, 2H, ArH), 8.09 (d, J ¼ 8.4,
2H, ArH), 10.03 (s, 1H, NH), 13.39 (s, 1H, NH), ms:
582.07, 584.93 (MHþ). Anal. Calcd for C28H28N5O7Cl
(582.06): C, 57.78; H, 4.85; N, 12.03. Found: C, 57.56; H,
4.59; N, 11.78.
5.2.34. 3-(3,4,5-Trimethoxyphenyl)-5-[(3,4,
5-trimethoxyphenyl)(4-chlorophenylhydrazono)
methyl][1,2,4]-triazin-6(1H )-one 15f
Yield: 58%, mp: 207e209 ꢀC, 1H NMR (300 MHz,
DMSO-d6): d 3.67 (s, 3H, OCH3), 3.69 (s, 6H, OCH3), 3.78
(s, 6H, OCH3), 3.81 (s, 3H, OCH3), 6.72 (s, 2H, ArH), 6.88
(s, 2H, ArH), 7.19 (d, J ¼ 9.0, 2H, ArH), 7.30 (d, J ¼ 9.0,
2H, ArH), 10.17 (s, 1H, NH), 13.30 (s, 1H, NH), ms: 583.05
(MHþ). Anal. Calcd for C28H26N5O7Cl (582.02): C, 57.78;
H, 4.85; N, 12.03. Found: C, 57.59; H, 4.67; N, 12.14.
5.2.40. 3-(4-Chlorophenyl)-5-[(3,4,5-trimethoxyphenyl)
(4-chlorophenylhydrazono)methyl]
[1,2,4]-triazin-6(1H )-one 16f
5.2.35. 3-(4-Chlorophenyl)-5-[(3,4,5-trimethoxyphenyl)
phenylhydrazonomethyl][1,2,4]-triazin-6(1H )-one 16a
Yield: 73%, mp: 228e230 ꢀC, ms: 493.00, 494.95 (MHþ).
Anal. Calcd for C25H22N5O4Cl (491.98): C, 61.04; H, 4.51; N,
14.24. Found: C, 61.15; H, 4.36; N, 14.51.
Yield: 82%, mp: 210e212 ꢀC, 1H NMR (300 MHz,
DMSO-d6): d 3.69 (s, 3H, OCH3), 3.77 (s, 6H, OCH3), 6.89
(s, 2H, ArH), 7.18 (d, J ¼ 8.7, 2H, ArH), 7.29 (d, J ¼ 8.7,
2H, ArH), 7.58 (d, J ¼ 8.7, 2H, ArH), 8.08 (d, J ¼ 8.7, 2H,
ArH), 10.12 (s, 1H, NH), 13.95 (s, 1H, NH), ms: 523.13
(MHþ). Anal. Calcd for C26H24N5O5Cl (522.01): C, 59.83;
H, 4.63; N, 13.42. Found: C, 59.64; H, 4.92; N, 13.20.
5.2.36. 3-(4-Chlorophenyl)-5-[(3,4,5-trimethoxyphenyl)
(2-methoxyphenylhydrazono)methyl][1,2,4]-triazin-6
(1H )-one 16b
5.2.41. 3,7-Disubstituted-5-(3,4,5-trimethoxyphenyl)
Yield: 96%, mp: 215e217 ꢀC, 1H NMR (300 MHz,
DMSO-d6): d 3.70 (s, 3H, OCH3), 3.78 (s, 6H, OCH3), 3.82
(s, 3H, OCH3), 6.89 (s, 2H, ArH), 6.94e6.99 (m, 2H, ArH),
7.61 (d, J ¼ 8.7, 1H, ArH), 7.66 (d, J ¼ 7.8, 2H, ArH), 7.85
(d, J ¼ 8.7, 1H, ArH), 8.15 (d, J ¼ 7.8, 2H, ArH), 11.81 (s,
1H, NH), 13.47 (s, 1H, NH), ms: 583.05 (MHþ). Anal. Calcd
for C28H26N5O7Cl (580.04): C, 57.78; H, 4.85; N, 12.03.
Found: C, 57.59; H, 4.67; N, 12.14.
pyrazolo[4,3-e][1,2,4]triazines 17ae20f
5.2.41.1. General procedure. The mixture of 3-substituted-
5-[(3,4,5-trimethoxyphenyl)(arylhydrazono)methyl][1,2,4]-tri-
azin-6(1H )-one (1.00 mmol), phosphorous oxychloride
(2.50 ml) and anhydrous pyridine (100 mL) was heated on an
oil bath at 115 ꢀC for 2 h. After cooling to room temperature
the solution was poured over crushed ice (30 g) and left to
stand at room temperature for 30 min. The brown precipitate
was filtered off and washed with water (100 ml). The crude
product was crystallized from an appropriate solvent, resp.
mixture of solvents (see text).
5.2.37. 3-(4-Chlorophenyl)-5-[(3,4,5-trimethoxyphenyl)
(3-methoxyphenylhydrazono)methyl]
[1,2,4]-triazin-6(1H )-one 16c
Yield: 92%, mp: 198e200 ꢀC, 1H NMR (300 MHz,
DMSO-d6): d 3.69 (s, 3H, OCH3), 3.74 (s, 3H, OCH3), 3.77
(s, 6H, OCH3), 6.89 (s, 2H, ArH), 7.13 (t, J ¼ 8.7, 1H,
ArH), 7.48 (d, J ¼ 8.7, 1H, ArH), 7.58 (d, J ¼ 8.5, 2H,
ArH), 7.83 (d, J ¼ 8.7, 1H, ArH), 8.09 (d, J ¼ 8.5, 1H,
ArH), 10.07 (s, 1H, NH), 13.92 (s, 1H, NH), ms: 522.13,
5.2.42. 3,7-Diphenyl-5-(3,4,5-trimethoxyphenyl)
pyrazolo[4,3-e][1,2,4]triazine 17a
1
Yield: 92%, mp: 197e198 ꢀC (ethanoletoluene (2:1)), H
NMR (300 MHz, CDCl3): d 3.99 (s, 3H, OCH3), 4.04 (s,
6H, OCH3), 7.40 (t, J ¼ 7.2, 1H, ArH), 7.56e7.63 (m, 5H,

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T. Gucky´ et al. / European Journal of Medicinal Chemistry 44 (2009) 891e900
ArH), 7.92 (s, 2H, ArH), 8.49 (d, J ¼ 8.1, 2H, ArH), 8.62e
8.64 (m, 2H, ArH), ms: 440.50 (MHþ). Anal. Calcd for
C25H21N5O3 (439.47): C, 68.32; H, 4.82; N, 15.94. Found:
C, 68.15; H, 4.76; N, 15.78.
5.2.48. 3-(4-Methoxyphenyl)-7-phenyl-5-(3,4,5-trime
thoxyphenyl)pyrazolo[4,3-e][1,2,4]triazine 18a
Yield: 95%, mp: 218e219 ꢀC, 1H NMR (300 MHz,
CDCl3): d 3.92 (s, 3H, OCH3), 3.98 (s, 3H, OCH3), 4.06 (s,
6H, OCH3), 7.05 (d, J ¼ 7.5, 2H, ArH), 7.40 (t, J ¼ 7.5, 1H,
ArH), 7.60 (t, J ¼ 7.5, 2H, ArH), 7.93 (s, 2H, ArH), 8.49 (d,
J ¼ 8.7, 2H, ArH), 8.57 (d, J ¼ 8.7, 2H, ArH), ms: 470.50
(MHþ). Anal. Calcd for C26H23N5O4 (469.50): C, 66.51; H,
4.94; N, 14.92. Found: C, 66.73; H, 4.81; N, 14.78.
5.2.43. 7-(2-Methoxyphenyl)-3-phenyl-5-(3,4,5-trimethoxy
phenyl)pyrazolo[4,3-e][1,2,4]triazine 17b
1
Yield: 92%, mp: 186e187 ꢀC (ethanoletoluene (3:1)), H
NMR (300 MHz, CDCl3): d 3.78 (s, 3H, eOCH3), 3.79 (s,
3H, eOCH3), 3.95 (s, 6H, eOCH3), 7.26 (t, J ¼ 7.5, 1H,
ArH), 7.41 (d, J ¼ 7.8, 1H, ArH), 7.63e7.68 (m, 3H, ArH),
7.73 (d, J ¼ 7.8, 1H, ArH), 7.85 (s, 2H, ArH), 8.58 (d,
J ¼ 7.8, 2H, ArH), ms: 470.13 (MHþ). Anal. Calcd for
C26H23N5O4 (469.5): C, 66.51; H, 4.94; N, 14.92. Found: C,
66.78; H, 4.99; N, 14.85.
5.2.49. 3-(4-Methoxyphenyl)-7-(2-methoxyphenyl)-5-(3,4,5-
trimethoxyphenyl)pyrazolo[4,3-e][1,2,4]triazine 18b
Yield: 98%, mp: 176e177 ꢀC, 1H NMR (300 MHz,
CDCl3): d 3.94 (s, 3H, OCH3), 3.97 (s, 3H, OCH3), 3.98 (s,
3H, OCH3), 4.08 (s, 6H, OCH3), 6.97 (m, 2H, ArH), 7.09
(d, J ¼ 8.7, 2H, ArH), 7.51 (t, J ¼ 8.1, 1H, ArH), 7.98 (s,
2H, ArH), 8.12 (d, J ¼ 8.1, 1H, ArH), 8.61 (d, J ¼ 8.7, 2H,
ArH), ms: 500.33 (MHþ). Anal. Calcd for C27H25N5O5
(469.50): C, 64.92; H, 5.04; N, 14.02. Found: C, 64.90; H,
4.89; N, 13.85.
5.2.44. 7-(3-Methoxyphenyl)-3-phenyl-5-(3,4,5-trimethoxy
phenyl)pyrazolo[4,3-e][1,2,4]triazine 17c
1
Yield: 80%, mp: 171e172 ꢀC (ethanoletoluene (2:1)), H
NMR (300 MHz, CDCl3): d 3.79 (s, 3H, eOCH3), 3.92 (s,
3H, eOCH3), 3.99 (s, 6H, eOCH3), 7.26 (t, J ¼ 7.5, 1H,
ArH), 7.41 (d, J ¼ 7.8, 1H,ArH), 7.63e7.68 (m, 3H, ArH),
7.73 (d, J ¼ 7.8, 1H, ArH), 7.85 (s, 2H, ArH), 8.58 (d,
J ¼ 7.8, 2H, ArH), ms: 470.12 (MHþ). Anal. Calcd for
C26H23N5O4 (469.50): C, 66.51; H, 4.94; N, 14.92. Found:
C, 66.39; H, 4.69; N, 14.71.
5.2.50. 3-(4-Methoxyphenyl)-7-(3-methoxyphenyl)-5-(3,4,
5-trimethoxyphenyl)pyrazolo[4,3-e][1,2,4]triazine 18c
Yield: 96%, mp: 142e145 ꢀC, 1H NMR (300 MHz,
CDCl3): d 3.94 (s, 3H, OCH3), 3.96 (s, 3H, OCH3) 3.99 (s,
3H, OCH3), 4.08 (s, 6H, OCH3), 7.10 (d, J ¼ 8.7, 2H, ArH),
7.57 (d, J ¼ 8.7, 2H, ArH), 7.95 (s, 2H, ArH), 8.50 (d,
J ¼ 8.7, 2H, ArH), 8.60 (d, J ¼ 8.7, 2H, ArH), ms: 500.42
(MHþ). Anal. Calcd for C27H25N5O5 (469.50): C, 64.92; H,
5.04; N, 14.02. Found: C, 64.77; H, 5.16; N, 13.96.
5.2.45. 7-(4-Methoxyphenyl)-3-phenyl-5-(3,4,5-trimethoxy
phenyl)pyrazolo[4,3-e][1,2,4]triazine 17d
1
Yield: 94%, mp: 183e184 ꢀC (ethanoletoluene (2:1)), H
5.2.51. 3-(4-Methoxyphenyl)-7-(4-methoxyphenyl)-5-(3,4,
5-trimethoxyphenyl)pyrazolo[4,3-e][1,2,4]triazine 18d
Yield: 97%, mp: 194e196 ꢀC, 1H NMR (300 MHz,
CDCl3): d 3.92 (s, 3H, OCH3), 3.93 (s, 3H, OCH3), 3.98 (s,
3H, OCH3), 4.07 (s, 6H, OCH3), 7.07 (d, J ¼ 8.4, 2H, ArH),
7.12 (d, J ¼ 8.4, 2H, ArH), 7.95 (s, 2H, ArH), 8.35 (d,
J ¼ 9.0, 2H, ArH), 8.59 (d, J ¼ 9.0, 2H, ArH), ms: 500.50
(MHþ). Anal. Calcd for C27H25N5O5 (469.50): C, 64.92; H,
5.04; N, 14.02. Found: C, 65.08; H, 4.87; N, 13.93.
NMR (300 MHz, CDCl3): d 3.78 (s, 3H, OCH3), 3.88 (s,
3H, OCH3), 3.98 (s, 6H, OCH3), 7.26 (d, J ¼ 8.7, 2H, ArH),
7.66 (d, J ¼ 7.2, 2H, ArH), 7.92 (s, 2H, ArH), 8.28 (d,
J ¼ 8.7, 2H, ArH), 8.61 (d, J ¼ 7.2, 2H, ArH), ms: 470.13
(MHþ). Anal. Calcd for C26H23N5O4 (469.5): C, 66.51; H,
4.94; N, 14.92. Found: C, 66.73; H, 4.88; N, 14.66.
5.2.46. 3-Phenyl-5-(3,4,5-trimethoxyphenyl)-7-(3,4,5-trime
thoxyphenyl)pyrazolo[4,3-e][1,2,4]triazine 17e
1
Yield: 62%, mp: 173e174 ꢀC (ethanoletoluene (1:1)), H
5.2.52. 3-(4-Methoxyphenyl)-7-(3,4,5-trimethoxyphenyl)-5-
(3,4,5-trimethoxyphenyl)pyrazolo[4,3-e][1,2,4]triazine 18e
Yield: 95%, mp: 192e193 ꢀC, 1H NMR (300 MHz,
CDCl3): d 3.93 (s, 3H, OCH3), 3.95 (s, 3H, OCH3), 3.98 (s,
3H, OCH3), 4.04 (s, 6H, OCH3), 4.06 (s, 6H, OCH3), 7.07
(d, J ¼ 9.0, 2H, ArH), 7.76 (s, 2H, ArH), 7.92 (s, 2H, ArH),
8.58 (d, J ¼ 9.0, 2H, ArH), ms: 500.46 (MHþ). Anal. Calcd
for C29H29N5O7 (559.58): C, 62.25; H, 5.22; N, 12.52. Found:
C, 62.38; H, 4.07; N, 12.31.
NMR (300 MHz, CDCl3): d 3.96 (s, 3H, eOCH3), 3.99 (s,
3H, eOCH3), 4.06 (s, 6H, eOCH3), 4.08 (s, 6H, eOCH3),
7.59e7.62 (m, 3H, ArH), 7.78 (s, 1H, ArH), 7.96 (s, 1H,
ArH), 8.65e8.69 (m, 2H, ArH), ms: 530.32 (MHþ). Anal.
Calcd for C28H27N5O6 (529.55): C, 63.51; H, 5.14; N,
13.22. Found: C, 63.50; H, 4.97; N, 13.45.
5.2.47. 7-(4-Chlorophenyl)-3-phenyl-5-(trimethoxyphenyl)
pyrazolo[4,3-e][1,2,4]triazine 17f
Yield: 83%, mp: 241e242 ꢀC, 1H NMR (300 MHz,
CDCl3): d 3.99 (s, 3H, eOCH3), 4.07 (s, 6H, eOCH3),
7.56e7.60 (m, 5H, ArH), 7.95 (s, 2H, ArH), 8.50 (d,
J ¼ 9.0, 2H, ArH), 8.64e8.67 (m, 2H, ArH), ms: 459
(MHþ). Anal. Calcd for C25H20N5O3Cl (473.96): C, 63.36;
H, 4.25; N, 14.78. Found: C, 63.26; H, 3.97; N, 14.55.
5.2.53. 3-(4-Methoxyphenyl)-7-(4-chlorophenyl)-5-(3,4,5-tri
methoxyphenyl)pyrazolo[4,3-e][1,2,4]triazine 18f
Yield: 93%, mp: 255e256 ꢀC, 1H NMR (300 MHz,
CDCl3): d 3.99 (s, 3H, OCH3), 4.08 (s, 6H, OCH3), 7.44 (t,
J ¼ 7.5, 1H, ArH), 7.58 (d, J ¼ 8.7, 2H, ArH), 7.64 (t,
J ¼ 7.5, 2H, ArH), 7.97 (s, 2H, ArH), 8.52 (d, J ¼ 7.5, 2H,

T. Gucky´ et al. / European Journal of Medicinal Chemistry 44 (2009) 891e900
899
ArH), 8.62 (d, J ¼ 8.7, 2H, ArH), ms: 505.00, 505.94 (MHþ).
5.2.59. 7-(4-Chlorophenyl)-3,5-di-(3,4,5-trimethoxy
phenyl)pyrazolo[4,3-e][1,2,4]triazine 19f
Anal. Calcd for C26H22N5O4Cl (503.95): C, 61.97; H, 4.40; N,
13.90. Found: C, 61.90; H, 4.56; N, 13.76.
Yield: 92%, mp: 266e267 ꢀC, 1H NMR (300 MHz,
CDCl3): d 3.98 (s, 3H, eOCH3), 3.97e3.99 (m, 12H,
eOCH3), 4.06 (s, 3H, eOCH3), 7.56 (d, J ¼ 9.0, 2H, ArH),
7.93 (s, 2H, ArH), 7.95 (s, 2H, ArH), 8.49 (d, J ¼ 9.0, 2H,
ArH), ms: 564.73 (MHþ). Anal. Calcd for C28H26N5O6Cl
(564.00): C, 60.09; H, 5.37; N, 11.30. Found: C, 60.01; H,
5.28; N, 11.15.
5.2.54. 7-Phenyl-3-(3,4,5-trimethoxyphenyl)-5-(3,4,5-trime
thoxyphenyl)pyrazolo[4,3-e][1,2,4]triazine 19a
Yield: 94%, mp: 209e211 ꢀC, 1H NMR (300 MHz,
CDCl3): d 3.96 (s, 6H, OCH3), 3.98 (s, 3H, OCH3), 3.99 (s,
3H, OCH3), 4.03 (s, 6H, OCH3), 7.40 (t, J ¼ 7.2, 1H, ArH),
7.60 (t, J ¼ 7.2, 2H, ArH), 7.91 (s, 2H, ArH), 7.93 (s, 2H,
ArH), 8.48 (d, J ¼ 7.2, 2H, ArH), ms: (MHþ). Anal. Calcd
for C27H28N5O6: C, 63.51; H, 5.14; N, 13.22; Found: C,
63.74; H, 5.19; N, 13.11.
5.2.60. 3-(4-Chlorophenyl)-7-(phenyl)-5-(3,4,5-trimethoxy
phenyl)pyrazolo[4,3-e][1,2,4]triazine 20a
Yield: 59%, mp: 233e235 ꢀC, 1H NMR (300 MHz,
CDCl3): d 3.99 (s, 3H, OCH3), 4.08 (s, 6H, OCH3), 7.44 (t,
J ¼ 7.5, 1H, ArH), 7.57 (d, J ¼ 8.7, 2H, ArH), 7.64 (t,
J ¼ 7.5, 2H, ArH), 7.97 (s, 2H, ArH), 8.52 (d, J ¼ 7.5, 2H,
ArH), 8.62 (d, J ¼ 8.7, 2H, ArH), ms: 475.13(MHþ). Anal.
Calcd for C25H20N5O3Cl (473.96): C, 63.36; H, 4.25; N,
14.78. Found: C, 63.59; H, 4.28; N, 14.96.
5.2.55. 7-(2-Methoxyphenyl)-(3,4,5-trimethoxyphenyl)-5-
(3,4,5-trimethoxyphenyl)pyrazolo[4,3-e][1,2,4]triazine 19b
Yield: 95%, mp: 278e279 ꢀC, 1H NMR (300 MHz,
CDCl3): d 3.85 (s, 3H, eOCH3), 3.97 (s, 3H, eOCH3), 3.98
(s, 3H, eOCH3), 4.01 (s, 6H, eOCH3), 4.05 (s, 6H,
eOCH3), 7.19e7.24 (m, 2H, ArH), 7.55e7.61 (d, J ¼ 6.0,
1H, ArH), 7.68 (d, J ¼ 6.6, 1H, ArH), 7.98 (s, 2H, ArH),
8.02 (s, 2H, ArH), ms: 560.57(MHþ). Anal. Calcd for
C29H29N5O7 (559.58): C, 62.25; H, 5.22; N, 12.52. Found:
C, 62.55; H, 5.11; N, 12.74.
5.2.61. 3-(4-Chlorophenyl)-7-(2-methoxyphenyl)-5-(3,
4,5-trimethoxyphenyl)pyrazolo[4,3-e][1,2,4]triazine 20b
Yield: 96%, mp: 213e215 ꢀC, 1H NMR (300 MHz,
CDCl3): d 3.77 (s, 3H, eOCH3), 3.78 (s, 3H, eOCH3), 3.95
(s, 6H, eOCH3), 7.25 (t, J ¼ 7.9, 1H, ArH), 7.39 (t, J ¼ 7.9,
1H, ArH), 7.65e7.74 (m, 4H, ArH), 7.84 (s, 2H, ArH), 8.58
(d, J ¼ 8.1, 2H, ArH), ms: 504.07(MHþ). Anal. Calcd for
C26H22N5O4Cl (503.99): C, 61.97; H, 4.40; N, 13.90. Found:
C, 61.83; H, 4.51; N, 13.85.
5.2.56. 7-(3-Methoxyphenyl)-(3,4,5-trimethoxyphenyl)-5-(3,
4,5-trimethoxyphenyl)pyrazolo[4,3-e][1,2,4]triazine 19c
Yield: 86%, mp: 216e218 ꢀC, 1H NMR (300 MHz,
CDCl3): d 3.85 (s, 3H, OCH3), 3.94 (s, 3H, OCH3), 3.97 (s,
6H, OCH3), 4.01 (s, 6H, OCH3), 4.05 (s, 3H, OCH3), 7.19e
7.28 (m, 2H, ArH), 7.58 (t, J ¼ 6.3, 1H, ArH), 7.68 (d,
J ¼ 7.2, 1H, ArH), 7.98 (s, 2H, ArH), 8.02 (s, 2H, ArH), ms:
560.07(MHþ). Anal. Calcd for C29H29N5O7 (559.58): C,
62.25; H, 5.22; N, 12.52. Found: C, 62.48; H, 5.13; N 12.34.
5.2.62. 3-(4-Chlorophenyl)-7-(3-methoxyphenyl)-5-(3,
4,5-trimethoxyphenyl)pyrazolo[4,3-e][1,2,4]triazine 20c
Yield: 99%, mp: 182e184 ꢀC, 1H NMR (300 MHz,
CDCl3): d 3.77 (s, 3H, eOCH3), 3.78 (s, 3H, eOCH3), 3.95
(s, 6H, eOCH3), 7.25 (t, J ¼ 7.9, 1H, ArH), 7.39 (t, J ¼ 7.9,
1H, ArH), 7.65e7.74 (m, 4H, ArH), 7.84 (s, 2H, ArH), 8.58
(d, J ¼ 8.1, 2H, ArH), ms: 504.13, 506.05 (MHþ). Anal. Calcd
for C26H22N5O4Cl (503.99): C, 61.97; H, 4.40; N, 13.90.
Found: C, 61.73; H, 4.28; N, 13.97.
5.2.57. 7-(4-Methoxyphenyl)-(3,4,5-trimethoxyphenyl)-5-(3,
4,5-trimethoxyphenyl)pyrazolo[4,3-e][1,2,4]triazine 19d
Yield: 94%, mp: 251e252 ꢀC, 1H NMR (300 MHz,
DMSO-d6): d 3.98 (s, 3H, eOCH3), 3.97e3.99 (m, 12H,
eOCH3), 4.06 (s, 3H, eOCH3), 7.56 (d, J ¼ 9.0, 2H, ArH),
7.93 (s, 2H, ArH), 7.95 (s, 2H, ArH), 8.49 (d, J ¼ 9.0, 2H,
ArH), ms: 470(MHþ). Anal. Calcd for C29H29N5O7
(559.58): C, 62.25; H, 5.22; N, 12.52. Found: C, 62.28; H,
5.51; N 12.43.
5.2.63. 3-(4-Chlorophenyl)-7-(4-methoxyphenyl)-5-
(3,4,5-trimethoxyphenyl)pyrazolo[4,3-e][1,2,4]triazine 20d
Yield: 98%, mp: 233e235 ꢀC, 1H NMR (300 MHz,
CDCl3): d 3.92 (s, 3H, eOCH3), 3.98 (s, 3H, eOCH3), 4.07
(s, 6H, eOCH3), 7.12 (d, J ¼ 9.0, 2H, ArH), 7.55 (d,
J ¼ 8.4, 2H, ArH), 7.92 (s, 2H, ArH), 8.33 (d, J ¼ 9.0 2H,
ArH), 8.58 (d, J ¼ 8.4, 2H, ArH), ms: 504.13, 506.05
(MHþ). Anal. Calcd for C26H22N5O4Cl (503.99): C, 61.97;
H, 4.40; N, 13.90. Found: C, 62.05; H, 4.58; N, 13.72.
5.2.58. 3,5,7-Tri-(3,4,5-trimethoxyphenyl)pyrazolo[4,3-e]
[1,2,4]triazine 19e
Yield: 95%, mp: 223e225 ꢀC, 1H NMR (300 MHz,
CDCl3): d 3.96 (s, 3H, eOCH3), 3.98 (s, 3H, eOCH3), 3.99
(s, 3H, eOCH3), 4.02 (s, 6H, eOCH3), 4.06 (s, 6H,
eOCH3), 4.07 (s, 6H, eOCH3), 7.76 (s, 1H, ArH), 7.95 (s,
2H, ArH), 7.98 (s, 2H, ArH), ms: 620.13(MHþ). Anal. Calcd
for C31H33N5O9 (619.63): C, 60.09; H, 5.37; N, 11.30. Found:
C, 60.01; H, 5.28; N, 11.15.
5.2.64. 3-(4-Chlorophenyl)-5-(3,4,5-trimethoxyphenyl)-7-(3,
4,5-trimethoxyphenyl)pyrazolo[4,3-e][1,2,4]triazine 20e
Yield: 96%, mp: 168e170 ꢀC, 1H NMR (300 MHz,
CDCl3): d 3.77 (s, 3H, eOCH3), 3.79 (s, 3H, eOCH3), 3.94
(s, 6H, eOCH3), 3.98 (s, 6H, OCH3), 7.71 (s, 2H, ArH),
7.73 (d, J ¼ 7.5, 2H, ArH), 7.89 (s, 2H, ArH), 8.59 (d,
J ¼ 7.5, 2H, ArH), ms: 565.06, 567.05 (MHþ). Anal. Calcd

900
T. Gucky´ et al. / European Journal of Medicinal Chemistry 44 (2009) 891e900
for C28H26N5O6Cl (564.04): C, 59.63; H, 4.65; N, 12.42.
Found: C, 59.81; H, 4.47; N, 12.23.
incubation at 37 ꢀC overnight. The optical density (OD) was
measured at 540 nm with a Labsystem iEMS Reader MF. Tu-
mour cell inhibitory concentration (IC) was calculated using
the following equation: IC ¼ (ODdrug-exposed well/mean ODcon-
5.2.65. 3-(4-Chlorophenyl)-7-(4-chlorophenyl)-5-(3,4,5-tri
methoxyphenyl)pyrazolo[4,3-e][1,2,4]triazine 20f
value, the drug concentration lethal
trol wells) ꢁ 100%. The IC50
Yield: 96%, mp: 303e304 ꢀC, 1H NMR (300 MHz,
CDCl3): d 3.99 (s, 3H, eOCH3), 4.07 (s, 6H, eOCH3),
7.55e7.60 (m, 4H, ArH), 7.95 (s, 2H, ArH), 8.49 (d,
J ¼ 9.0, 2H, ArH), 8.65 (d, J ¼ 9.0, 2H, ArH), ms: 509.13,
511.05 (MHþ). Anal. Calcd for C25H19N5O3Cl2 (508.46): C,
59.07; H, 3.77; N, 13.78. Found: C, 59.26; H, 3.49; N, 13.51.
to 50% of the tumour cells, was calculated from appropriate
doseeresponse curves.
Acknowledgement
The study was supported from the research concept of the
Ministry of Education, Youth and Sport of the Czech Republic
(MSM6198959216).
5.3. Biological activity
5.3.1. Cell lines
References
All cells were purchased from the American Tissue Culture
Collection (ATCC), unless otherwise indicated. The daunoru-
bicin resistant subline of CEM cells (CEM DNR Bulk) and
paclitaxel resistant subline K-562-tax were selected in our lab-
oratory by the cultivation of maternal cell lines in increasing
concentrations of daunorubicin or paclitaxel, respectively
[24]. The cells were maintained in Nunc/Corning 80 cm2 plas-
tic tissue culture flasks and cultured in cell culture medium
(DMEM/RPMI 1640 with 5 g/L glucose, 2 mM glutamine,
100 U/mL penicillin, 100 mg/mL streptomycin, 10% fetal
calf serum, and NaHCO3).
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Cell suspensions were prepared and diluted according to
the particular cell type and the expected target cell density
(2500e30,000 cells/well based on cell growth characteristics).
Cells were added by pipette (80 mL) into 96-well microtiter
plates. Inoculates were allowed a pre-incubation period of
24 h at 37 ꢀC and 5% CO2 for stabilisation. Fourfold dilutions,
in 20-mL aliquots, of the intended test concentration were
added to the microtiter plate wells at time zero. All test com-
pound concentrations were examined in duplicate. Incubation
of the cells with the test compounds lasted for 72 h at 37 ꢀC, in
a 5% CO2 atmosphere at 100% humidity. At the end of the in-
cubation period, the cells were assayed using MTT. Aliquots
(10 mL) of the MTT stock solution were pipetted into each
well and incubated for further 4 h. After this incubation period
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100 mL/well of 10% aq SDS (pH ¼ 5.5), followed by a further
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