Arch. Pharm. Chem. Life Sci.

2014, 347, 599608

599

Full Paper
Synthesis, Cytotoxic, and Antitumor Activities of
2-Pyridylhydrazones Derived from 3-Benzoylpyridazines
Johnny Easmon1, Gerhard Prstinger1, Gottfried Heinisch1, Hans H. Fiebig2, Thomas Roth2,
and Johann Hofmann3*
1

2
3

Pharmaceutical Chemistry-Institute of Pharmacy, Centrum fr Chemie und Biomedizin, Leopold-FranzensUniversitt, Innsbruck, Austria
Oncotest, Freiburg, Germany
Division of Medical Biochemistry, Centrum fr Chemie und Biomedizin, Medical University Innsbruck,
Innsbruck, Austria

A series of 2-pyridylhydrazones derived from phenyl-pyridazin-3-yl-methanones were prepared in

search for potential novel antitumor agents. The stereochemistry of these compounds was established
by means of NMR spectroscopy. Whereas hydrazones derived from 3-benzoylpyridazines (IC50 0.99
8.74 mM) inhibited the proliferation of the tumor cell lines tested, the non-fully aromatic 3benzoylpyridazinone hydrazones (IC50 > 10 mM) turned out to be inactive. Compounds E-1b
(IC50 0.12 mM) and E-1d (IC50 0.18 mM) exert high cytotoxic activities in clonogenic assays involving
human tumor cells of different tissue origins. In vivo application of compound E-1b (300 mg/kg/day)
resulted in a 66% reduction in tumor burden.
Keywords: Antitumor activity / 3-Benzoylpyridazine 20 -pyridylhydrazones / Colony forming assay / Cytotoxic activity
Received: April 7, 2014; Revised: April 7, 2014; Accepted: April 23, 2014
DOI 10.1002/ardp.201400137

Introduction
An essential enzyme required for the continuous provision of
the building blocks for DNA synthesis is ribonucleotide
reductase (RR). Because of the critical role that RR plays in the
biosynthesis of deoxyribonucleotides, it is considered as an
important metabolic target for the development of agents
that will be suitable for the treatment of cancer [1, 2].
In a structureactivity relationships study concerning
hydroxyurea and congeners, Larson [3] concluded that the
main pharmacophore requirements of compounds capable of
inhibiting the M2 subunit of the enzyme ribonucleotide
reductase are: (i) ability to act as a radical scavenger, (ii) metal
chelating ability, and (iii) planarity. One such compound
described in the literature, which possesses the above
requirements, is the chromogenic reagent 2-benzoylpyridine
2-pyridylhydrazone (A) [4]. We recently synthesized a large
series of novel analogs of compound A (Fig. 1) and studied
Correspondence: Dr. Johnny Easmon, Pharmaceutical ChemistryInstitute of Pharmacy, Centrum fr Chemie und Biomedizin, LeopoldFranzens-Universitt, Innrain 80/82, A-6020 Innsbruck, Austria.
E-mail: johnny.easmon@uibk.ac.at
Fax: 43 512 507 58299

2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

their effects on cell proliferation in vitro [5]. In our test systems,

compound A as well as the congeners B and C exhibited high
cytotoxic activity. Most of the compounds were highly active
against Burkitts lymphoma cells with an IC50 of 0.011
0.035 mM and were also found to be more potent than
hydroxyurea (IC50 140 mM) [5]. In an indirect measure of RR
activity, it was observed that the ratio of the IC50 values for
[14C]cytidine incorporation into DNA to values for cell
proliferation for the hydrazones was >1 but was less than
one for hydroxyurea, a known RR inhibitor [5]. These results
indicated that the novel hydrazones do not exert their
antiproliferative activity by inhibiting RR activity.
In a recent publication, it has been shown that the E-isomer,
an analog of type A compound, is a selective inhibitor of
Jumonji histone demethylase and shows potent in vitro
anticancer activity. However, the Z-isomer is not an inhibitor
of this enzyme and its pharmacological activity is yet to be
elucidated [6].
In previous studies concerning thiosemicarbazones derived
from pyridazine carbaldehydes and alkyl pyridazinyl ketones,


Additional correspondence: Prof. Johann Hofmann
E-mail: johann.hofmann@i-med.ac.at

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Arch. Pharm. Chem. Life Sci. 2014, 347, 599608

R1
N
N

NH

NH

NH

R3

A : R1 = R2 = R3 = H
B: R1 = OH or OCH3
R2 = NO2, Cl or CF3
R3 = NO2, Cl, CF3, CH3 or OCH3

Figure 1. Analogs of compound A.

we observed that the 3-pyridazinyl derivatives were advantageous compared to the 2-pyridyl congeners with regard to
cytotoxicity and solubility in water [7]. In a continuation of
the study of the structureactivity relationships and the
solubility of this class of compounds, we became interested
in compounds of type D characterized by replacement of
the 2-benzoylpyridine substructure by a 3-benzoylpyridazine
moiety. The solubility of these compounds in DMSO-d6 was
acceptable compared to compounds described in [5].
However, for the cell lines tested, compounds of type D
bearing the 1,2-diazine system exerted an activity lower than
that of compounds of types AC and D (1,3- and 1,4-diazines).
These ndings not-withstanding, we synthesized various
analogs of 3-benzoylpyridazine-derived hydrazones in which
(a) the 3-benzoylpyridazine moiety bears a methoxy or
hydroxy function and (b) electron-withdrawing or -donating
groups are attached to the 20 -pyridylhydrazone substructure.
The results of the in vitro antiproliferative activity as well as
the in vivo antitumor studies of selected compounds in a lung
cancer animal model are presented.

Results and discussion

Chemistry
Synthesis
Condensation of 2-pyridylhydrazine (7a) with the ketones 5a
e in reuxing methanol-containing traces of glacial acetic
acid afforded the hydrazones 1ae (Scheme 1). The 3benzoylpyridazines 5a, 5b, 5d, and 5e were obtained according
to reported procedures [8]. Treatment of 5b with boron
tribromide solution in dichloromethane gave the ketone 5c
in high yield. The hydrazones 2ag and 3ag became
accessible by reacting equimolar amounts of the ketone 5b
or 5d with the appropriately substituted 2-pyridylhydrazines
6ag (Scheme 1). The reaction of the hydrazines 6f,g
2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

with ketone 5b or 5d were carried out in an inert

atmosphere due to the sensitivity of the hydrazones 2/3f,
g in reuxing methanol and air. Even with these
precautions no hydrazone 2f was formed, isolating only
the starting materials 5b and 6f. The 2-pyridylhydrazines
6a [9], 6b [10], 6c [9], 6d [5], and 6e [11] were prepared
according to the references cited. Hydrazone 4 was
obtained by reacting 3-benzoylpyridazinone 8 [12] with
hydrazine 6 [9].

Spectroscopic investigations
The structures of all novel compounds were conrmed by
elemental analyses (Table 1), IR, and NMR spectroscopy.
Compounds containing the C N substructure can exist
either in the E- or Z-form or as mixtures of E/Z-isomers.
Based on 1H NMR studies, the most remarkable differences
regarding the chemical shifts of the corresponding protons
of the two isomeric forms are the resonance signals
attributable to the NH protons: that is, d(NH) 1415 ppm
for the Z-form and 812 ppm for the E-form. This conclusion is
further supported by homonuclear 1H NOE-difference experiments [5, 13, 14].
For compounds existing in the Z-conguration, irradiation
of the NH resonance leads to a positive NOE on the
pyridazinyl H6 protons, whereas for compounds existing
in the E-conguration, irradiation of the NH resonance
leads to a positive NOE on the phenyl H2/6 and the pyridine
H30 protons.
In analogy to the above-reported studies, E-conguration
was assigned to compounds 1b, 1d, 1e, 2b, 2d, 3a, 3ce, and 4
and Z-conguration to compound 2c. The hydrazones 1a, 2e,
3b, 3f, and 3g were found to be mixtures of E/Z-isomers of
various proportions. After several attempts, only the isomeric
pair 2e could be separated by column chromatography on
silica gel.
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Arch. Pharm. Chem. Life Sci. 2014, 347, 599608

2-Pyridylhydrazones as Antitumor Agents

R1

R
N

R1

H2N-HN

7a

5a e

601

NH
N

5a: R = R1 = H
5b: R = H, R1 = OCH3
5c: R = H, R1 = OH
5d: R = OCH3, R1 = H
5e: R = OCH3, R1 = OCH3

1a
1b
1c
1d
1e

R
H
H
H
OCH3
OCH3

R1
H
OCH3
OH
H
OCH3

1a e
OCH3
N

N
N
NH

R2

2/3a
2/3b
2/3c
2/3d
2/3e
2/3f
2/3g

R3

R3

2ag

NH-NH2

R2

5b

H3CO

5d
6a g

R2

R3

H
H
H
H
H
Cl
CF3

5'-Cl
6'-Cl
5'-NO2
5'-CF3
5'-CH3
5'-CF3
5'-Cl

N
N
NH
R2

R3

3a g
O
HN

Cl

+
N
O

H2N-HN

O
HN

N
N
NH

6a
N

Cl

4
Scheme 1. Synthesis of target hydrazones.

2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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Arch. Pharm. Chem. Life Sci. 2014, 347, 599608

Table 1. Yield and physical characteristics of 3-benzoylpyridazine 2-pyridylhydrazone (1a) and congeners 1be, 2ag, 3ag, and 4.
R1

R
N

OCH 3
N

H 3CO
N

HN

NH

NH

NH
R2

N
NH
R2

R3

R3

Cl

2af

1ae

Compd.
E-/Z-1a
E-1b
E-1c
E-1d
E-1e
E/Z-2a
E-2b
E-2c
E-2d
E-2f
E-3a
E/Z-3b
E-3c
E-3d
E-3e
E/Z-3f
E/Z-3g
E-4

3ag

R1

R2

R3

Yield (%)

M.p. (C)

Solvent

MW

Analysis C, H, N

H
H
H
OCH3
OCH3

H
OCH3
OH
H
OCH3

H
H
H
H
50 -Cl
H
H
H
H
H
Cl
CF3

50 -Cl
60 -Cl
50 -NO2
50 -CF3
CF3
50 -Cl
60 -Cl
50 -NO2
50 -CF3
50 -CH3
50 -CF3
50 -Cl
5-Cl

59
70
60
80
70
55
70
65
54
46
60
60
55
58
55
55
48
60

190192
191194

EtOH
96% EtOH
96% EtOH
96% EtOH
EA/DIPE
EA/PE
EA
EtOH
DIPE
EA/DIPE
EA/DIPE
2-PrOH
2-PrOH
EA/2-PrOH
96% EtOH
EA/PE
EA/PE
2-PrOH

305.34
291.31
305.34
335.37
339.79
339.79
350.34
373.34
407.78
339.79
339.79
350.34
373.34
319.37
407.78
407.78
325.76

C16H13N5
C17H15N5O
C16H13N5O
C17H15N5O
C18H17N5O2
C17H14ClN5
C17H14ClN5
C17H14N6O2
C18H14F3N5
C18H13ClF3N5
C17H14ClN5
C17H14ClN5
C17H14N6O2
C18H14F3N5
C18H17N5
C18H13ClF3N5
C18H13ClF3N5
C16H12ClN5O

193195
184186
165167
186189
190192
158161
160163
184186
207209
238240
198200
184186
155157
164166
255258

Solvent: DIPE, diisopropyl ether; EA, ethylacetate; EtOH, ethanol; PE, petrolether; PrOH, propanol; THF, tetrahydofuran.

In vitro studies
The in vitro antiproliferative activity of compounds 1ae, 2/3
(ag), and 4 was determined in a panel of human tumor
cell lines (see the Experimental section). The results expressed
as IC50 values (i.e., effective dose of compound inhibiting 50%
of cell growth) of these studies are given in Table 2.
Whereas compound E/Z-1a is highly active against CCRFCEM cells (IC50 0.39 mM) compared to the other cell lines, the
introduction of an OCH3 or OH function into the 3benzoylpyridazine moiety of 1a (i.e., compounds E-1b, E-1c,
E-1d, and E-1e; IC50 1.181.88 mM) results in a reduction of
cytotoxic activity by a factor of 5. However, compounds E-1b
and E-1d turned out to be equipotent against CCRF-CEM cells
(IC50 1.50 mM) and the solid tumor cell lines HeLa (IC50
1.77 mM) and MEXF 276L (IC50 1.99 mM). Based on these
results, compounds E-1b and E-1d were selected for further
2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

variations of the 20 -pyridylhydrazone moiety, which resulted

in compounds 2ag and 3ag.
Comparing compounds 2ag to E-1b (Table 2), the following
conclusions could be drawn: (a) The introduction of a CF3
group (compound E-2d) or CH3 group (compounds E-2e and
Z-2e) into the 50 -position results in improved activity against
CCRF-CEM and Burkitts lymphoma cells by a factor of 5 and
(b) compounds bearing a chloro substituent in the 50 - (i.e., E-2a)
or 60 - (E-2b) or a nitro substituent in the 50 - (i.e., Z-2c) position
exhibit reduced cytotoxic activity. The results in the table
also show that E-2e exhibits enhanced cytotoxic activity
(IC50 0.181.36 mM) compared to Z-2e (IC50 0.202.73 mM).
In this series, compound E-2a also turned out to be equipotent
(IC50 2.40 mM) for all the cell lines tested. For the
disubstituted compound E-2g, cytotoxic activity is lowered
compared to E-1b.
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Arch. Pharm. Chem. Life Sci. 2014, 347, 599608

2-Pyridylhydrazones as Antitumor Agents

603

Table 2. Concentrations (mM) inhibiting cell proliferation by 50% (IC50) on human tumor cell lines.
Inhibition of cell growth, IC50 (mM)
Compd.
E-/Z-1a
E-1b
E-1c
E-1d
E-1e
E/Z-2a
E-2b
E-2c
E-2d
E-2e
Z-2e
E-2f
E-3a
E/Z-3b
E-3c
E-3d
E-3e
E/Z-3f
E/Z-3g
E-4

CCRF-CEM

Burkitt0 s

HeLa

HT-29

MEXF 276L

0.39
1.178
1.51
1.50
1.88
2.10
7.18
3.19
0.56
0.27
0.20
2.06
1.77
4.71
1.72
5.00
>10
1.39
1.36
>10

2.27
2.966
6.57
3.29
3.42
2.40
8.74
4.10
1.41
0.18
0.44
3.70
2.23
6.76
1.86
4.91
ND
1.82
1.02
>10

2.43
1.77
4.64
1.88
2.72
2.44
7.76
6.12
2.80
1.39
2.73
10.74
1.63
3.80
2.02
4.53
>10
2.04
2.04
>10

4.21
2.69
2.69
3.38
3.95
2.05
6.73
2.62
7.09
1.36
0.99
11.34
2.18
6.99
2.22
4.55
>10
1.47
1.47
>10

4.36
1.90
ND
2.00
3.04
2.60
8.47
5.19
ND
ND
ND
ND
2.86
13.62
7.02
7.52
ND
4.67
3.63
>10

ND, not determined.

The mean values of at least two independent experiments in which duplicate determinations were taken within each experiment are
indicated. For clarity, standard deviation has been omitted. Standard deviation is below 5%.

A reversal in the trend of cytotoxic activity is obtained for

compounds 3ag compared to 2ag. In contrast, the 50 -Cl
analog (E-3a: IC50 1.632.86 mM) and the disubstituted
analogs E/Z-3f (IC50 1.394.67 mM) and E/Z-3g (IC50 1.02
3.62 mM) turned out to be equipotent to the parent compound
E-1d (IC50 1.503.38 mM). Compared to compounds in this
series, E-3a exhibited a broad cytotoxic activity (IC50 1.63
2.86 mM) against the cell lines tested.
To gain further insight into the importance of the nonlactam heteroaromatic ring in the ketone part of compounds
13 in relation to cytotoxic activity, the 3-benzoylpyridazinone-derived hydrazone 4 was synthesized. As shown in
Table 2, this modication resulted in a total loss of cytotoxic
activity (IC50 >10 mM), thus conrming the fact that a nonlactam heteroaromtic ring is important for maintaining
biological activity.
Compounds E-1b and E-1d were selected for further
evaluation in a clonogenic assay based on the fact that these
compounds inhibited the proliferation of the tumor cells
tested in the same concentration range.

Colony forming assay studies

Colony assays with human tumor xenografts have been found
to show excellent correlation of drug response with treatment
in patients [15, 16], thus underlining the usefulness of
2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

this assay system. The results listed in Table 3 show that

compounds E-1b (mean IC50 0.12 mg/mL) and E-1d (mean
IC50 0.18 mg/mL) exert high inhibitory activity on the colony
formation of all tumor xenografts examined. The IC50 values
for both compounds were generally 10-fold lower than the
corresponding values obtained in the SRB and MTT assay
(Table 2). This is mostly due to the longer drug incubation
times in the clonogenic assay (615 days, depending on the
proliferation rate of the cells). From the mean IC50 and IC70
values obtained for the two compounds, it can be concluded
that E-1b and E-1d are equally active. This is in agreement
with the results observed on the permanent human tumor
cell lines shown in Table 2. Furthermore, both compounds
displayed similar activity proles with the colon carcinoma
CXF 280 being the most sensitive and the renal carcinoma RXF
486 the most resistant xenograft. Based on the in vitro results
E-1b was selected for an initial in vivo study on nude mice
xenografted with the large cell lung carcinoma LXFL 529.

In vivo antitumor studies

In a combined dose nding/antitumor study doses of 300, 100,
and 30 mg/kg/day of E-1b were given i.p. to LXFL 529 bearing
nude mice at Days 0, 4, and 8 after randomization. None of the
applied doses resulted in a toxic death of an animal. The most
pronounced antitumor effect was obtained in the group
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Arch. Pharm. Chem. Life Sci. 2014, 347, 599608

Table 3. Inhibition of colony formation of tumor cells derived from human tumor xenografts by componds E-1b and E-1d using the
clonogenic assay.
E-1b

Human tumor
IC50a)

IC70a)

IC50a)

IC70a)

BXF 1301
MX1
MAXF 449
CXF 280
CXF 1103
HT29X

0.31
0.07
0.34
0.002
0.31
0.03

0.57
0.28
5.8
0.02
0.76
0.07

0.15
0.42
2.3
0.01
0.10
0.07

0.34
0.89
10.8
0.09
0.46
0.26

LXFA 289
LXFA 526
LXFL 529
LXFS 538
MEXF 514
MEXF 989
PC3MX
LNCAPX
RXF 468

0.20
0.03
0.16
0.38
0.07
0.10
0.20
0.18
0.74
0.12

0.43
0.06
0.35
0.61
0.27
0.27
0.41
0.80
7.8
0.40

0.24
0.18
0.11
0.26
0.41
0.05
0.19
0.29
0.41
0.18

0.47
0.49
0.29
0.54
0.66
0.10
0.44
0.70
10.0
0.57

Xenograft
Bladder
Breast
Colon

Lung
Non-small cell

Small cell
Melanoma
Prostate
Renal
Mean IC in mg/mL
a)

E-1d

Substance concentration required to reduce tumor cell colony formation to 50% (IC50) or 30% (IC70) in mg/mL.

treated with the highest dose of E-1b resulting in a T/C value of

44%. Both lower doses effected only a marginal tumor growth
inhibition with a T/C value of 64% (Fig. 2a). The maximal body
weight loss of 49% was reached between Days 7 and 10. At the
end of the study (Day 17), the animals had gained weight and
the relative body weights did not differ in comparison to
the control group (Fig. 2b). This indicates that the maximal
tolerable dose (MTD) was not reached.

Conclusions
Despite the high antiproliferative activity of the novel
hydrazones in vitro in the mM range, evaluation of compound
E-1b in vivo resulted only in marginal antitumor activity in the
large cell lung carcinoma LXFL 529 animal model. However,
this is still a promising result, since the MTD of E-1b has not
been reached and the LXFL 529 tumor has been shown to be
only a moderately sensitive model according to the in vitro
data given in Table 3. Using longer therapy schedules, applying
higher doses, and examining more E-1b sensitive tumor
models such as the colon carcinoma CXF 280 for example,
might, therefore, result in a better in vivo antitumor activity.

Experimental
Materials and methods
Infrared spectra (IR) were recorded from KBr pellets on a Mattson
Galaxy Series FTIR 3000 spectrophotometer. NMR spectra were
2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

recorded from DMSO-d6 solutions on a Varian Gemini 200 (1H:

199.98 MHz, 13C: 50 MHz) spectrometer, Ar phenyl, Pydz
pyridazine, Pyri pyridine. The center of the solvent signal
was used as internal standard, which was related to TMS with
(2.49 ppm (1H) and 39.50 (13C)). Melting points were determined
on a Reichert Thermovar hot stage microscope and are
uncorrected. Elemental analyses were performed at the Institut
fr Physikalische Chemie, University of Vienna, Austria and the
data for C, H, and N are within 0.4% of the calculated values.
Reactions were monitored by thin layer chromatography using
Polygram SIL G/UV254 (Macherey-Nagel) plastic backed plates
(0.25 mm layer thickness) and visualized using UV lamp. Column
chromatography was performed using Kieselgel 60 (0.040
0.063 mm).

Synthesis of 4-hydroxyphenyl(3-pyridazinyl)methanone
(5c)
To a well-stirred solution of 4-methoxyphenyl-3-pyridazinyl
ketone (5.20 g, 24.28 mmol) in dry CH2Cl2 (100 mL) under argon
and cooled to 80C, 72.84 mL of a 1 M solution of BBr3 solution
(72.84 mmol) in CH2Cl2 was added dropwise. After the addition,
the mixture was allowed to warm to room temperature and then
stirred overnight. TLC (eluent: EA/CH2Cl2 6:4) evaluation showed
that only about 20% of product had formed. The mixture was then
heated at 40C for 24 h and cooled to room temperature. After
cooling the solution to 10C, it was made basic to pH 6 by the
addition of 10 N NaOH. After separating the organic phase, the
mixture was further diluted with water (100 mL) and extracted
further with CH2Cl2 (2  100 mL). The combined organic phases
were washed with sat. NaCl, dried over anhyd. Na2SO4, and
evaporated to dryness to provide 5c as brown needles (3.65 g,
75%), m.p. 296298C (EtOH). IR (cm1) 3443, 3061, 2957, 2693,
2619, 1649, 1604, 1595, 1319, 1156, 1008, 759, 617. 1H NMR (d,
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Arch. Pharm. Chem. Life Sci. 2014, 347, 599608

2-Pyridylhydrazones as Antitumor Agents

(a)

dryness and the residues obtained were dissolved in hot EA or in a

EA/DIPE mixture and chilled. Then, the crystals that separated
were collected by ltration and recrystallized. Compounds 2g, 3f,
and 3g were synthesized using dry methanol as solvent and under
nitrogen atmosphere.

900

Relative Tumor Volume in %

605

800
700

(4-Methoxyphenyl)(pyridazin-3-yl)-N 0 -(2-pyridyl)methanone hydrazone E-1b

600

IR (cm1): 3434, 3322, 1592, 1562, 1494, 1251, 1139. 1H NMR (d)
3.87 (s, 3H, OCH3), 6.866.91 (m, 1H, Pyri-H5), 7.16 (ABd, J 8.7 Hz,
2H, Ar-H3/5), 7.37 (ABd, J 8.5 Hz, 2H, Ar-H2/6), 7.427.45 (m, 1H,
Pyri-H3), 7.687.82 (m, 2H, Pyri-H4, Pydz-H5), 8.068.18 (m, 1H,
Pydz-H4), 8.348.46 (m, 1H, Pyri-H6), 8.80 (s, 1H, NH), 9.089.25
(m, 1H, Pydz-H6).

500
400
300
200
100
0

Relative Body Weight in %

(b)

10

15

Days after Randomisation

130
120
110
100

(4-Hydroxyphenyl)(pyridazin-3-yl)-N 0 -(2-pyridyl)methanone hydrazone E-1c

A 1H NMR spectrum showed the raw compound 1c to be a
mixture of E/Z-isomers with the ratio of 10:1. The pure E-isomer
was obtained by recrystallization from ethanol.
IR (cm1): 3426, 3332, 1600, 1500, 1494, 1277, 1139. 1H NMR (d)
6.856.91 (m, 1H, Pyri-H5), 6.97 (ABd, J 8.4 Hz, 2H, Ar-H3/5), 7.23
(ABd, J 8.8 Hz, 2H, Ar-H2/6), 7.48 (d, J 8.4 Hz, 1H, Pydz-H4),
7.687.78 (m, 1H, Pyri-H3), 7.76 (dd, J 4.8 Hz, J 8.8 Hz, 1H, PydzH5), 8.098.11 (m, 1H, Pyri-H4), 8.39 (dd, J 1.4 Hz, J 8.4 Hz, 1H,
Pyri-H6), 8.81 (s, 1H, OH), 9.12 (dd, J 1.4 Hz, J 4.8 Hz, 1H, PydzH6), 9.84 (s, 1H, NH).

(6-Methoxypyridazin-3-yl)(phenyl)-N 0 -(2-pyridyl)methanone hydrazine E-1d

90
80
0

10

15

Days after Randomisation

Figure 2. (a) Growth inhibition of the large cell lung carcinoma LXFL
529 xenografted s.c. in athymic mice by E-1b () 0, (&) 30, (D) 100,
() 300 mg/kg E-1b given i.p. on Day 0, 4, and 8. (b) Change of
animal body weight treated with E-1b () 0, (&) 30, (D) 100, ()
300 mg/kg given i.p. on Day 0, 4, and 8.

ppm) 6.92 (ABd, J 8.8 Hz, 2H, Ar-H3/5), 7.907.97 (m, 1H, PydzH5), 7.94 (ABd, J 8.8 Hz, 2H, Ar-H2/6), 8.09 (dd, J 2 Hz,
J 8.4 Hz, 1H, Pydz-H4), 9.42 (dd, J 1.6 Hz, J 5.0 Hz, 1H, PydzH6), 10.63 (br.s, 1H, OH). 13C NMR (d, ppm) 115.3 (Ar-C2/6), 126.5
(Ar-C4), 127.1 (Pydz-C4), 133.7 (Ar-C3/5), 152.6 (Pydz-C6), 158.6
(Ar-C1), 162.9 (Pydz-C3), 190.9 (C O).

General procedure for the synthesis of hydrazones 1ae,

2/3ag, and 4
A mixture of equimolar amounts of ketone and hydrazine in
methanol containing 510 drops of glacial acetic acid was
reuxed until TLC monitoring indicated no further conversion.
Reaction times varied from 6 to 12 h. The resulting hydrazones
separated upon cooling the solutions to 5C overnight. In the case
of the more soluble hydrazones, the solutions were evaporated to
2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

IR (cm1): 3446, 3332, 1592, 1500, 1465, 1292, 1018. 1H NMR (d)
4.02 (s, 3H, OCH3), 6.846.90 (m, 1H, Pyri-H5), 7.24 (ABd, J 9.3 Hz,
2H, Pydz-H5), 7.387.61 (m, 6H, Ar-H2-6, Pyri-H3), 7.647.77 (m,
1H, Pyri-H4), 8.068.12 (m 1H, Pyri-H6), 8.38 (ABd, J 9.2 Hz, 1H,
Pydz-H6), 8.58 (s, 1H, NH).

[(4-Methoxyphenyl)(6-methoxypyridazin-3-yl)]-N 0 -(2pyridyl)methanone hydrazone E-1e

IR (cm1): 3440, 3332, 1608, 1509, 1429, 1247, 1143. 1H NMR (d)
3.86 (s, 3H, OCH3), 4.03 (s, 3H, OCH3), 6.836.89 (m, 1H, Pyri-H5),
7.14 (ABd, J 8.9 Hz, 2H, Ar-H3/5), 7.23 (d, J 9.3 Hz, 1H, Pydz-H5),
7.34 (ABd, J 8.9 Hz, 2H, Ar-H2/6), 7.427.47 (m, 1H, Pyri-H3),
7.687.76 (m, 1H, Pyri-H4), 8.078.08 (m, 1H, Pyri-H6), 8.34 (d,
J 9.3 Hz, 1H, Pydz-H4), 8.65 (s, 1H, NH).

N 0 -(5-Chloropyridin-2-yl)(4-methoxyphenyl)-(pyridazin-3yl)methanone hydrazine E-2a

IR (cm1): 3430, 3326, 1590, 1502, 1388, 1249, 1137. 1H NMR (d)
3.85 (s, 3H, OCH3), 7.13 (ABd, J 8.8 Hz, 2H, Ar-H3/5), 7.35 (ABd,
J 8.4 Hz, 2H, Ar-H2/6), 7.52 (d, J 9.2 Hz, 1H, Pyri-H3), 7.707.85
(m, 2H, Pyri-H4, Pydz-H5), 8.15 (d, J 2.6 Hz, Pyri-H6), 8.42 (d,
J 8.8 Hz, 1H, Pydz-H4), 9.14 (dd, J 1.0 Hz, J 4.8 Hz, 1H, PydzH6), 9.23 (s, 1H, NH).

N 0 -(6-Chloropyridin-2-yl)(4-methoxyphenyl)-(pyridazin-3yl)methanone hydrazine E-2b

IR (cm1): 3428, 3316, 1590, 1558, 1425, 1247, 1126. 1H NMR (d)
3.86 (s, 3H, OCH3), 6.94 (d, J 7.6 Hz, 1H, Pyri-H3), 7.13 (ABd,
J 8.6 Hz, 2H, Ar-H3/5), 7.35 (ABd, J 9.0 Hz, 1H, Ar-H2/6), 7.45 (d,
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J 8.4 Hz, 1H, Pyri-H5), 7.707.80 (m, 2H, Pyri-H4, Pydz-H5), 8.41
(dd, J 1.6 Hz, J 8.8 Hz, 1H, Pydz-H4), 9.15 (dd, J 1.6 Hz,
J 4.8 Hz, 1H, Pydz-H6), 9.43 (s, 1H, NH).
0

N -(5-Nitro-2-pyridinyl)(4-methoxyphenyl)-(pyridazin-3-yl)methanone hydrazine Z-2c

IR (cm1): 3430, 1596, 1506, 1336, 1253, 1132. 1H NMR (d) 3.81
(s, 3H, OCH3), 7.01 (ABd, J 9.0 Hz, 1H, Ar-H3/5), 7.48 (ABd,
J 9.0 Hz, 1H, Ar-H2/6), 7.51 (d, J 9.2 Hz, 1H, Pyri-H3), 7.75 (dd,
J 1.8 Hz, J 8.6 Hz, 1H, Pydz-H3), 7.89 (dd, J 4.8 Hz, J 8.6 Hz,
1H, Pydz-H5), 8.44 (dd, J 2.6 Hz, J 9.2 Hz, 1H, Pyri-H4), 9.00 (d,
J 3.0 Hz, 1H, Pyri-H6), 9.38 (dd, J 1.8 Hz, J 4.8 Hz, 1H, Pyri-H2),
12.09 (s, 1H, NH).
0

(4-Methoxyphenyl)-(pyridazin-3-yl)-N -[5-(triuoromethyl)pyridin-2-yl]methanone hydrazone E-2d

IR (cm1): 3328, 3008, 1612, 1519, 1409, 1251, 1122, 1074. 1H NMR
(d) 3.84 (s, 3H, OCH3), 7.13 (ABd, J 8.2 Hz, Ar-H3/5), 7.36 (ABd,
J 8.2 Hz, 2H, Ar-H2/6), 7.62 (d, J 8.8 Hz, 1H, Pyri-H3), 7.76 (dd,
J 4.6 Hz, J 8.8 Hz, 1H, Pydz-H5), 7.988.05 (m, 1H, Pyri-H4),
8.368.55 (m, 2H, Pyri-H6, Pydz-H4), 8.158.21 (m, 1H, Pydz-H6),
9.65 (s, 1H, NH).

N-(4-Methylphenyl)-N-(4-methoxyphenyl-pyridazin-3-ylmethylene)-hydrazine 2e
As the reaction proceeded, two new spots were observed on the
TLC plate. After cooling the mixture to room temperature, it was
stored in the fridge overnight, and as no precipitate had formed,
the mixture was evaporated to dryness. The two products were
separated by column chromatography on silica gel rst eluting
with EA/CH2Cl2 (3:7) to give a brown solid. The second product
was isolated with EA/CH2Cl2/MeOH (5:3:0.5) to give reddish
amorphous product.
E-2e: Brown solid (0.287 g, 56%), m.p. 184186C (EA/PE). IR
(cm1): 3440, 3332, 1608, 1509, 1429, 1247, 1143, 827. 1H NMR (d)
2.20 (s, 3H, CH3), 3.85 (s, 3H, OCH3), 7.14 (ABd, J 8.6 Hz, 2H,
Ar-H3/5), 7.35 (ABd, J 8.6 Hz, 2H, Ar-H2/6), 7.42 (d, J 8.6 Hz, 1H,
Pyri-H3), 7.58 (dd, J 2 Hz, J 9.0 Hz, 1H, Pydz-H4), 7.71 (dd,
J 4.6 Hz, J 8.6 Hz, 1H, Pydz-H5), 7.93 (m, 1H, Pyri-H4), 8.41 (dd,
J 1.4 Hz, J 8.6 Hz, 1H, Pyri-H6), 8.79 (s, 1H, NH), 9.12 (dd,
J 1.6 Hz, J 4.8 Hz, 1H, Pydz-H6). Anal. C18H17N5O (319.37).
Calcd. (%): C, 67.70; H, 5.37; N, 21.93. Found (%): C, 68.06; H, 5.41;
N, 22.03.
Z-2e: Reddish solid (0.196 g, 38%), m.p. 190192C (DIPE). IR
(cm1): 3432, 3039, 2935, 2840, 1604, 1508, 1243, 1112, 1051. 1H
NMR (d) 2.21 (s, 3H, CH3), 3.80 (s, 3H, OCH3), 7.00 (ABd, J 9.0 Hz,
2H, Ar-H3/5), 7.157.37 (m, 1H, Pyri-H3), 7.47 (ABd, J 9.0 Hz, 2H,
Ar-H2/6), 7.537.59 (m, 1H, Pyri-H4), 7.64 (dd, J 1.8 Hz, J 8.6 Hz,
1H, Pydz-H4), 7.85 (dd, J 5.0 Hz, J 8.6 Hz, 1H, Pydz-H5), 7.90
7.99 (m, 1H, Pyri-H6), 9.33 (dd, J 1.6 Hz, J 5.0 Hz, 1H Pydz-H6),
8.79 (s, 1H, NH). Anal. C18H17N5O. Calcd. (%): C, 67.70; H, 5.37; N,
21.93. Found (%): C, 67.60; H, 5.51; N, 22.20.
0

(4-Methoxyphenyl)(pyridazin-3-yl)-N -[3-chloro-5(triuoromethyl)pyridin-2-yl]methanone hydrazine E-2f

IR (cm1): 3424, 3064, 2937, 2842, 1596, 1448, 1247, 1128, 833. 1H
NMR (d) 3.81 (s, 3H, OCH3), 7.04 (ABd, 2H, J 8.8 Hz, Ar-H3/5), 7.53
(ABd, J 8.8 Hz, 2H, Ar-H2/6), 7.67 (dd, J 1.4 Hz, J 8.4 Hz, 1H,
Pydz-H4), 7.91 (dd, J 5.2 Hz, J 8.8 Hz, Pydz-H5), 8.26 (br.s, 1H,
2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Pyri-H4), 8.59 (br.s, 1H, Pyri-H6), 9.37 (dd, J 1.8 Hz, J 5.2 Hz, 1H,
Pydz-H6), 10.15 (s, 1H, NH).

N 0 -(5-Chloropyridin-2-yl)(6-methoxypyridazin-3-yl)(phenyl)methanone hydrazone E-3a

IR (cm1): 3420, 3332, 1589, 1500, 1463, 1292, 1008, 833. 1H NMR
4.02 (s, 3H, OCH3), 7.23 (d, J 9.4 Hz, 1H, Pydz-H5), 7.377.41 (m,
2H, Ar-H2/6), 7.49 (d, J 8.8 Hz, 1H, Pyri-H3), 7.537.62 (m, 3H, ArH3-5), 7.79 (dd, J 2.8 Hz, J 8.9 Hz, 1H, Pyri-H4), 8.10 (d,
J 2.2 Hz, 1H, Pyri-H6), 10 02 (s, 1H, NH).

N 0 -(6-Chloropyridin-2-yl)(6-methoxypyridazin-3-yl)(phenyl)methanone hydrazine E-3b

IR (cm1): 3430, 3332, 1589, 1463, 1292, 1008, 833. 1H NMR (d)
4.03 (s, 3H, OCH3), 7.25 (d, J 9.3 Hz, 1H, Pydz-H5), 7.327.44 (m,
4H, Ar-H2/6, Pyri-H3/5), 7.527.59 (m, 3H, Ar-H3-5), 7.74 (t,
J 8.1 Hz, 1H, pyri-H4), 8.37 (d, J 9.3 Hz, 1H, Pydz-H4), 9.06 (br.s,
1H, NH).

N 0 -(6-Chloropyridin-2-yl)(6-methoxypyridazin-3-yl)(phenyl)methanone hydrazone Z-3b

1
H NMR (d) 4.18 (s, 3H, OCH3), 6.91 (d, J 7.6 Hz, 1H, Pydz-H5),
7.327.44 (m, 4H, Ar-H2/6, Pyri-3/5), 7.527.59 (m, 5H, Ar-H3-5,
Pyri-H4, Pydz-H4), 12.04 (br.s, 1H, NH).

(6-Methoxypyridazin-3-yl)-N 0 -(5-nitropyridin-2-yl)(phenyl)methanone hydrazone E-3c

IR (cm1): 3420, 3320, 1602, 1589, 1463, 1292, 1008, 833. 1H NMR
(d) 4.05 (s, 3H, CH3), 7.30 (d, J 9.4 Hz, 1H, Pydz-H5), 7.397.45 (m,
2H, Ar-H2/6), 7.34 (d, J 7.6 Hz, 1H, Pyri-H3), 7.557.62 (m, 3H, ArH3-5), 8.42 (d, J 9.2 Hz, 1H, Pydz-H4), 8.45 (dd, J 2.6 Hz,
J 7.8 Hz, 1H, Pyri-H4), 8.98 (d, J 3.0 Hz, 1H, Pyri-H6), 9.90 (br.
s, 1H, NH).

(6-Methoxypyridazin-3-yl)-N 0 -[5-(triuoromethylpyridin-2yl](phenyl)methanone hydrazone E-3d

IR (cm1): 3426, 3328, 1614, 1525, 1467, 1328, 1290, 1074, 833. 1H
NMR (d) 4.03 (s, 3H, OCH3), 7.28 (d, J 9.2 Hz, Pydz-H5), 7.397.43
(m, 3H, Ar-H2/6, Pyri-H3), 7.547.63 (m, 3H, Ar-H3-5), 8.02 (dd,
J 2.4 Hz, J 9.0 Hz, 1H, Pyri-H4), 8.40 (d, J 9.4 Hz, Pydz-H4),
8.438.44 (m, 1H, Pyri-H6), 9.25 (8s, 1H, NH).

(6-Methoxypyridazin-3-yl)-N 0 -(5-methylpyridin-2-yl)(phenyl)methanone hydrazone E-3e

IR (cm1): 3430, 3320, 1606, 1508, 1463, 1290, 1010, 835. 1H NMR
(d) 2.20 (s, 3H, CH3), 4.02 (s, 3H, OCH3), 7.24 (d, J 9.6 Hz, 1H, PydzH5), 7.377.41 (m, 3H, Ar-H2/6, Pyri-H3), 7.547.61 (m, 4H, Ar-H3-5,
Pyri-H4), 7.927.93 (m, 1H, Pyri-H6), 8.37 (d, 1H, J 9.6 Hz, PydzH4), 8.49 (s, 1H, NH).

N 0 -[3-Chloro-5-(triuoromethyl)-2-pyridyl](6-methoxypyridazin-3-yl)(phenyl)methanone hydrazone
E-3f

IR (cm1): 3433, 3361, 1598, 1313, 1112, 1045, 700. 1H NMR (d)
4.01 (s, 3H, OCH3), 7.44 (d, J 9.4 Hz, 1H, Pydz-H5), 7.397.65 (m,
5H, Ar-H2-6), 8.128.19 (m, 1H, Pyri-H4), 8.27 (d, J 9.6 Hz, 1H,
Pydz-H4), 8.508.58 (m, 1H, Pyri-H6), 8.70 (br.s, 1H, NH).
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2-Pyridylhydrazones as Antitumor Agents

N 0 -[3-Chloro-5-(triuoromethyl)-2-pyridyl](6-methoxypyridazin-3-yl)(phenyl)methanone
hydrazone Z-3f
H NMR (d) 4.15 (s, 3H, OCH3), 7.31 (d, J 9.2 Hz, 1H, Pydz-H5),
7.397.65 (m, 6H, Ar-H2-6, Pydz-H4), 8.128.19 (m, 1H, Pyri-H4),
8.508.88 (m, 1H, Pyri-H6), 13.83 (s, 1H, NH).
1

N 0 -[5-Chloro-3-(triuoromethyl)-2-pyridyl](6-methoxypyridazin-3-yl)(phenyl)methanone
hydrazone E-3g
1

were added to the cells and exposed continuously for 72 h at

37C in a humidied atmosphere of 95% air and 5% CO2.
The drugs were dissolved in dimethylsulfoxide (DMSO). The
concentration of DMSO was 0.5% and this was not toxic.
Subsequently, the samples were processed and the absorption
was detected by a microplate reader (Model 3550, Bio-Rad).

Colony forming assay

1

IR (cm ): 3430, 3334, 1602, 1465, 1819, 1126, 1091, 705. H NMR
(d) 4.02 (s, 3H, OCH3), 7.44 (d, J 9.4 Hz, 1H, Pydz-H5), 7.487.66
(m, 5H, Ar-H2-6), 8.048.06 (m, 1H, Pyri-H4), 8.32 (d, J 9.4 Hz, 1H,
Pydz-H4), 8.568.65 (m, 1H, Pyri-H6), 9.03 (br.s, 1H, NH).

N 0 -[5-Chloro-3-(triuoromethyl)-2-pyridyl](6-methoxypyridazin-3-yl)(phenyl)methanone
hydrazone Z-3g
1
H NMR (d) 4.15 (s, 3H, OCH3), 7.33 (d, J 9.2 Hz, 1H, Pydz-H5),
7.487.66 (m, 5H, Ar-H2-6), 8.008.03 (m, 1H, Pyri-H4), 8.14 (d,
J 9.4 Hz, Pydz-H4), 8.208.24 (m, 1H, Pyri-H6), 14.03 (br.s, 1H,
NH).

6-[[N 0 -(5-Chloropyridin-2-yl)hydrazone]phenylmethyl]pyradazin-3(2H)-one E-4

IR (cm1): 3340, 3333, 3014, 2940, 2862, 1675, 1659, 1568, 1296,
1132, 1009, 780. 1H NMR (d) 6.846.91 (m, 1H, Pyri-H5), 6.96 (d,
J 10.0 Hz, 1H, Pydz-H5), 7.357.40 (m, 2H, Ar-H2/6), 7.47 (d,
J 8.6 Hz, 1H, Pyri-H3), 7.517.59 (m, 3H, Ar-H3-5), 7.697.78 (m,
1H, Pyri-H4), 8.068.09 (m, 1H, Pyri-H6), 8.31 (d, J 10.0 Hz, 1H,
Pydz-H4), 8.57 (s, 1H, NH), 12.88 (br.s, 1H, NH).

Biological methods
Cell proliferation experiments
ZR-75-1 (breast carcinoma, ATCC CRL 1500), Burkitts
lymphoma (CA 46, ATCC CRL 1648), CCRF-CEM (acute
lymphoblastic leukemia, ATCC CCL 119), HeLa (epitheloid
cervix carcinoma, ATCC CCL 2), and HT-29 (colon adenocarcinoma, ATCC HTB 38) cells were obtained from the American
Type Culture Collection, Rockville, MD. MEXF 276 L (melanoma) cells were kindly provided by Dr. H. H. Fiebig, Freiburg,
Germany. HeLa, MEXF, ZR-75, Burkitts lymphoma, and CCRFCEM were grown in RPMI 1640, HT-29 in McCoys 5A medium
supplemented with 10% fetal calf serum (except Burkitts
lymphoma with 15%), 2 mM glutamine, 50 units/mL penicillin, and 50 mg/mL streptomycin.
Inhibition of cell proliferation of ZR-75-1, HeLa, HT-29, and
MEXF-276 L was detected by the SRB-assay [17]. In this assay,
300010,000 cells in 200 mL of medium were seeded per well
into 96-well plates. Doseresponse curves for CCRF-CEM and
Burkitts lymphoma cells were detected by an MTT-assay [18]
from Boehringer Mannheim (Mannheim, Germany). Approximately, 10,000 cells per 100 mL were seeded in 96-well plates.
After an initial incubation of 4 h, various drug concentrations
2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

607

Solid human tumor xenografts were removed, mechanically

disaggregated, and subsequently incubated with an enzyme
cocktail consisting of collagenase (1.21.8 U/mL), DNase
(375 U/mL), and hyaluronidase (29 U/mL) in RPMI 1640
medium at 37C for 30 min. The cell mixture was passed
through sieves of 200 and 50 mm mesh size and washed
thereafter twice with phosphate buffered saline (PBS). The
percentage of viable cells was determined in a Neubauer
counting chamber using Trypan blue exclusion.
The clonogenic assay was performed according to a modied
two-layer soft agar assay introduced by Hamburger and
Salmon [19]. The bottom layer consisted of 0.2 mL of Iscovess
modied Dulbeccos medium with 20% fetal calf serum and
0.75% agar. 8  103 to 1.6  104 cells were added to 0.2 mL of
the same culture medium and 0.4% agar and plated in
24-multiwell dishes onto the base layer. Drugs were applied one
day after cell seeding (continuous exposure) in 0.2 mL medium.
Every dish included six control wells containing the vehicle
and drug treated groups in triplicate at six concentrations.
Cultures were incubated at 37C and 7% CO2 in a humidied
atmosphere for 715 days depending on the doubling time of
the tumor stem cells. At the time of maximum formation
of tumor colonies with a diameter of 50 mm, counts were
performed with an automatic image analysis system. Twentyfour hours prior to evaluation, vital colonies were stained with
a sterile aqueous solution of 2-(4-iodophenyl)-3-(4-nitrophenyl)5-phenyltetrazolium chloride (1 mg/mL, 100mL/well). Colony
growth inhibition was expressed as T/C values in % and used
for the determination of IC50 and IC70 values. Mean IC50 and
IC70 values were calculated according to the formula:
Pn
logIC50;70 x
Mean IC50;70 10 x1
n
with x specic tumor xenograft and n total number of
xenograft studied.

In vivo test procedures

Tumor fragments harvested from the subcutaneously growing human large cell lung carcinoma LXFL 529 in nude mice
hosts were implanted subcutaneously into both anks of 6- to
8-week-old female athymic nude mice (Balb/c). When tumors
were approximately 57 mm in diameter, mice were randomly assigned to treatment groups and untreated controls. The
control group consisted of four mice bearing ve evaluable
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J. Easmon et al.

tumors, and drug-treated groups of two to three mice bearing

three to four evaluable tumors. E-1b was applied i.p. as a
suspension in saline containing 5% DMSO on Days 0, 4, and 8
at doses of 300, 100, and 30 mg/kg/day. Mice were weighed and
tumors were measured using calipers twice weekly. Tumor
volume was calculated according to the formula: Tumor
volume (mm3) Width (mm2)  Length (mm)/2.
Data evaluation was performed using software developed in
our laboratory. Relative tumor volume (RTV) values were
calculated for each single tumor by dividing the tumor
volume Day X by the tumor volume Day 0 at the time of
randomization [RTV (TVX  100)/TV0)]. Median RTV values
were used for further evaluation. Furthermore, T/C values
were calculated by comparing the relative tumor size of
treated groups and controls.
The authors wish to thank the FWF (project No. P-09879-Med) for
nancial support and Mrs. J. Fink for her excellent technical support.
The authors have declared no conict of interest.

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128, Pergamon Press, New York 1989, pp. 245264.

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