Azinyl and diazinyl hydrazones derived from aryl N-heteroaryl ketones: Synthesis and antiproliferative activity

Article abstract of DOI:10.1021/jm970255w

A series of N-heteroaryl hydrazones derived from aryl N-heteroaryl or bis-N-heteroaryl methanones was prepared in search for potential novel antitumor agents. The stereochemistry of these compounds was established by means of NMR spectroscopy. Antiproliferative activity was determined in a panel of human tumor cell lines (CCRF-CEM, Burkitt's lymphoma, HeLa, ZR-75- 1, HT-29, and MEXF 276L) in vitro. Generally, the new compounds were found to be more potent (IC₅₀ = 0.011-0.436 μM) than the ribonucleotide reductase inhibitor hydroxyurea (IC₅₀ = 140 μM). Most of the compounds exhibited the highest activity against Burkitt's lymphoma with an IC₅₀ of 0.011-0.035 μM. [¹⁴C]Cytidine incorporation into DNA was quantitated for selected hydrazones (Z-A, E-1, Z-3, Z-4, E-5, Z-5, E-13, E-18, Z-19, Z-24, and E-26) as a measure of the inhibition of ribonucleotide reductase in Burkitt's lymphoma cells. The E-configurated compounds were found to inhibit [¹⁴C]cytidine incorporation to a greater extent (IC₅₀ = 0.67-5.05 μM) than the Z-isomers (IC₅₀ = 7.20 to > 10 μM). Principal component analysis of the IC₅₀ values obtained for inhibition of cell proliferation revealed that the cell lines tested can be grouped into three main families showing different sensitivities toward the compounds in our series [(i) CCRF-CEM, Burkitt's lymphoma, and Hela; (ii) HT-29; and (iii) MEXF 276 L].

Full text of DOI:10.1021/jm970255w

4420
J . Med. Chem. 1997, 40, 4420-4425
Azin yl a n d Dia zin yl Hyd r a zon es Der ived fr om Ar yl N-Heter oa r yl Keton es:
Syn th esis a n d An tip r olifer a tive Activity†,‡
J ohnny Easmon, Gottfried Heinisch,* Gerhard Pu¨rstinger, Thierry Langer, and J u¨rgen K. O¨ sterreicher
Institute of Pharmaceutical Chemistry, Innrain 52a, University of Innsbruck, A-6020 Innsbruck, Austria
Hans H. Grunicke and J ohann Hofmann*
Institute of Medical Chemistry and Biochemistry, Fritz-Pregl-Strasse 3, University of Innsbruck, A-6020 Innsbruck, Austria
Received April 17, 1997X
A series of N-heteroaryl hydrazones derived from aryl N-heteroaryl or bis-N-heteroaryl
methanones was prepared in search for potential novel antitumor agents. The stereochemistry
of these compounds was established by means of NMR spectroscopy. Antiproliferative activity
was determined in a panel of human tumor cell lines (CCRF-CEM, Burkitt’s lymphoma, HeLa,
ZR-75-1, HT-29, and MEXF 276L) in vitro. Generally, the new compounds were found to be
more potent (IC50 ) 0.011-0.436 µM) than the ribonucleotide reductase inhibitor hydroxyurea
(IC50 ) 140 µM). Most of the compounds exhibited the highest activity against Burkitt’s
lymphoma with an IC50 of 0.011-0.035 µM. [14C]Cytidine incorporation into DNA was
quantitated for selected hydrazones (Z-A, E-1, Z-3, Z-4, E-5, Z-5, E-13, E-18, Z-19, Z-24, and
E-26) as a measure of the inhibition of ribonucleotide reductase in Burkitt’s lymphoma cells.
The E-configurated compounds were found to inhibit [14C]cytidine incorporation to a greater
extent (IC50 ) 0.67-5.05 µM) than the Z-isomers (IC50 ) 7.20 to >10 µM). Principal component
analysis of the IC50 values obtained for inhibition of cell proliferation revealed that the cell
lines tested can be grouped into three main families showing different sensitivities toward the
compounds in our series [(i) CCRF-CEM, Burkitt’s lymphoma, and Hela; (ii) HT-29; and (iii)
MEXF 276 L].
Ch a r t 1
In tr od u ction
The production of deoxyribonucleotides is a tightly
controlled cellular function. The enzyme responsible for
this reaction is ribonucleotide reductase (RR).1 The
mammalian RR consists of two subunits referred to
commonly as M1 and M2. Whereas the activity of RR
is low in resting cells and high in rapidly growing
normal cells, it is further increased in malignant
transformed cells.2 Thus, inhibitors of this enzyme are
considered as potential antitumor agents.3 Inhibitors
directed at the M2 portion of the enzyme have been
studied in detail, and they fall into two categories:
metal ion chelators or radical scavengers.4 Thus, com-
pounds which can act as metal chelators and with the
ability to undergo one-electron oxidation may combine
two molecular features which may be important for RR
inhibitory activity. One such compound previously
described is 2-benzoylpyridine 2′-pyridylhydrazone (A,
Chart 1) which inhibits the proliferation of the hormone
dependent human mammary tumor cell line MDA-
MB231 (IC50 ) 0.097 µM) and the [3H]thymidine
incorporation into DNA (IC50 ) 0.089 µM).5 However,
there are no reports on the stereochemistry of the
sample employed in this study. In our test systems, Z-A
turned out to be a potent cytotoxic agent with a broad
spectrum of activity (IC50 ) 0.022-0.380 µM, Table 1)
against a panel of human tumor cell lines.
We now set out to prepare various analogues of
compound A in which (a) the phenyl ring bears a OCH3
or OH function (1,2), (b) electron-withdrawing or -do-
nating groups are attached to the 2′-pyridylhydrazone
moiety (B), (c) the 2-benzoylpyridine system is replaced
formally by a benzoyldiazine partial structure (C), and
(d) the 2′-pyridylhydrazone moiety is replaced by a
diazinyl hydrazone or a benzoazinyl hydrazone sub-
structure (D) (see Chart 1). Moreover, we investigated
the antiproliferative activity of compounds of types 1,
2, B, C, and D in a panel of human tumor cell lines as
well as the incorporation of cytidine into DNA
* Address correspondence to these authors.
† Taken in part from the Diploma Thesis of J .K.O¨ ., University of
Innsbruck, 1994.
‡ Part of this study was presented at the XVIth European Collo-
quium on Heterocyclic Chemistry (abstract no. P-30), September 1994,
Bled, Slovenia.
X Abstract published in Advance ACS Abstracts, November 15, 1997.
S0022-2623(97)00255-0 CCC: $14.00 © 1997 American Chemical Society

Notes
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 26 4421
Ta ble 1. Antiproliferative Activity of 2-Benzoylpyridine
2′-Pyridylhydrazone and Related Compounds Against Various
Human Tumor Cell Linesa
ride in diethyl ether at -5 to -10 °C. The novel
hydrazones 12-15 then became accessible by reacting
equimolar amounts of the ketones 33a -d and 2-py-
ridylhydrazine (30) in methanol containing traces of
acetic acid. The hydrazone 16 in which the 2-ben-
zoylpyridine system is replaced by a 4-benzoylpyridine
moiety became accessible from the ketone 33e and
hydrazine 30.
Condensation of the ketone 29a with the hydrazines
34a ,17 34b,5 34c, 34d 18 34e,19 34f, 34g,20 34h ,21 34i,22
34j,5 and 34k in methanol containing traces of glacial
acetic acid afforded the hydrazones 17, 18, and 20-28
in high yields. The 4-pyrimidinylhydrazine which was
obtained by treatment of 34b with Pd/C and ammonium
formate in methanolic solution was reacted without
further purification with the ketone 29a to give the
hydrazone 19 in 45% yield (Scheme 2). Reaction of
methyl acetoacetate with formamidine acetate in a
sodium methoxide-methanol solution in analogy to the
procedure reported by Butters23a for the preparation of
6-ethyl-4(3H)-pyrimidinone afforded 6-methyl-4(3H)-
pyrimidinone23b in 72% yield. Thus, 4-chloro-6-meth-
ylpyrimidine then became available in 70% yield by
chlorination of the methylpyrimidinone with phosphorus
oxychloride. It was subsequently reacted with hydra-
zine hydrate (neat) at 40 °C to give 34c23c in 75% yield.
Considering the fact that compounds of type A-D can
exist as the E- or Z-isomer, or as E/ Z-mixtures, a
detailed examination of the stereochemistry at the
-CdN- bond was performed. The configuration of the
isolated compounds was determined by 1H and 13C NMR
spectroscopy and homonuclear NOE-difference experi-
ments in analogy to the reported findings related to
thiosemicarbazones.24 Thus, the E-configuration has to
be assigned to compounds 1, 13, 16, 18, 23, 26, and 28
and the Z-configuration to compounds A, 2-4, 9-11,
19-21, and 24. The hydrazones 5-8, 12, 14, 15, 17,
22, 25, and 27 were found to be mixtures of E/Z-isomers
of various ratios. Due to problems of solubility, only
the E/ Z-isomeric mixtures of compounds 5 and 7 were
separable by careful fractional crystallization from ethyl
acetate.
cytoxicity IC50 (µM)
MEXF
compd config CCRF-CEM Burkitt’s HeLa HT-29 276 L
Z-A
E-1
Z-2
Z-3
Z-4
E-5
Z-5
E/Z-6
E-7
Z-7
E/Z-8
Z-9
Z-10
Z-11
E/Z-12 E/ Z
E-13
E/Z-14 E/ Z
E/Z-15 E/ Z
E-16
E/Z-17 E/ Z
E-18
Z-19
Z-20
Z-21
Z
E
Z
Z
Z
E
Z
E/ Z
E
Z
E/ Z
Z
Z
Z
0.040
0.081
0.269
0.162
0.068
0.286
0.506
3.210
0.082
0.110
0.546
0.140
1.032
1.676
0.586
1.161
0.147
0.212
> 10
0.145
0.112
1.507
1.128
> 10
0.658
1.093
0.877
0.312
0.022
0.068
0.979
0.022
0.011
0.518
0.014
0.010
0.032
0.151
3.245
0.022
0.023
0.105
0.134
0.423
0.591
0.585
0.283
0.032
0.035
> 10
0.073 0.280 0.380
0.048 0.154 0.293
0.440 0.736 0.847
0.054 0.160 0.264
0.046 0.77
0.370
0.113 0.101 0.619
0.515 0.484 8.098
3.541 2.957 4.370
0.041 0.155 0.182
0.060 0.053 0.669
0.301 0.326 3.141
0.228 0.342 1.910
0.793 0.702 1.493
1.246 0.705 2.183
1.614 1.990 3.157
1.209 1.585 2.423
0.273 0.638 1.570
0.142 0.668 1.203
E
E
> 10
> 10
> 10
0.103
0.045
0.347
0.463
0.331 0.855 1.844
0.211 0.372 0.493
0.739 3.078 3.217
0.583 4.645 > 10
E
Z
Z
Z
12.361 > 10
> 10
> 10
E/Z-22 E/ Z
E-23
Z-24
E/Z-25 E/ Z
E-26
E/Z-27 E/ Z
0.338
1.424
0.463
0.257
0.015
0.154
0.266
0.417 2.740 2.295
1.011 1.006 2.563
0.517 0.608 2.614
0.314 1.264 1.820
0.014 0.029 0.244
0.084 0.301 0.774
0.647 0.696 0.969
E
Z
E
E-28
E
a
Data represent the mean of at least three independent
experiments in which duplicate determinations were taken within
each experiment.
as a measure of RR inhibition with several selected
compounds. Finally, we tried to find out whether it is
possible to deduce a 3D-QSAR model in this series.
Ch em istr y
The hydrazones 1 and 2 were prepared by reaction of
the substituted 2-benzoylpyridines 29b/c6 with 2-py-
ridylhydrazine (30). The synthesis of 3 from bis-2,2′-
pyridylmethanone (29d ) and the hydrazine 30 has been
described previously.7 Compounds 4-11 became acces-
sible by reacting equimolar amounts of 29a and the
appropriately substituted 2-pyridylhydrazines 32a -h .
The 2-pyridylhydrazines 32b,8 32c,9 32d ,10 and 32f 2
were prepared according to the cited references. Reac-
tion of 2-chloro-5-nitropyridine (31a ) or 2-chloro-5-
(trifluoromethyl)pyridine (31e) with hydrazine hydrate
gave the 2-pyridylhydrazine derivatives 32a and 32e
in 90% yield (Scheme 1).
Phenyl 2-pyrazinyl ketone (33a )12 was prepared in a
57% yield from methyl 2-pyrazinecarboxylate13 and
phenyllithium at -70 °C. Reaction of pyrazine-2-
carbonitrile with (p-methoxyphenyl)magnesium bromide
at -30 °C afforded 33b12 in 54% yield. Treatment of
methyl 4-pyrimidinecarboxylate14 with phenyllithium in
a mixture of THF/diethyl ether at -90 °C gave the
ketone 33c15 in 73% yield. In a one-step reaction,
pyrimidine-2-carbonitrile16 was obtained in 49% yield
from 2-chloropyrimidine and sodium cyanide. Thus,
ketone 33d 15 was obtained in 82% yield by reacting the
pyrimidine-2-carbonitrile with phenylmagnesium chlo-
Resu lts a n d Discu ssion
Inhibition of cell proliferation was determined in five
human cell lines (see the Experimental Section). Since
Z-A is more active against leukemia and Burkitt’s
lymphoma compared to colon and melanoma cells by a
factor of 10 (Table 1), we studied the effect of subtle
changes to Z-A on cytotoxic activity. Compounds bear-
ing a p-methoxybenzylidene group (E-1) or the bis-2,2′-
pyridyl moiety (Z-3) showed a marginal improvement
in cytotoxic activity as compared to Z-A. The activity
of the p-OH congener (compound Z-2) is reduced by a
factor of 5-23, depending on the cell line. Further
variations were made in the 2-pyridylhydrazone moiety
of Z-A (i.e. compounds 4-11). Comparing the effect of
monosubstitution on cytotoxic activity, the substituents
in the 5′-position (compounds Z-4, E-5, E-7, and E/Z-8)
generally led to retention of activity with the rank order
of CH3 g NO2 > Cl > CF3. In contrast, a chloro
substituent in the 6′-position (E/Z-6) led to reduced
activity by a factor of 100 (Burkitt’s lymphoma, IC50
)
3.245 µM) compared to the 5′-chloro analogue E-5 (IC50
) 0.032 µM) and Z-A (IC50 ) 0.022 µM). Furthermore,

4422 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 26
Notes
Sch em e 1
Sch em e 2
idine (E/Z-15) moieties (series C) were synthesized and
evaluated for cytotoxic activity. The data given in Table
1 indicate a reduction in activity for all compounds as
compared to Z-A. Furthermore, to study the relevance
of the point of attachment of the benzylidene moiety to
the pyridine ring for cytotoxic activity, the hydrazone
16 which is a derivative of 4-benzoylpyridine was
synthesized. A total loss of activity was observed for
E-16 (IC50 >10 µM). This confirms that like observed
for N-heteroaromatic thiosemicarbazones,25 the point of
attachment of the side chain should be R to the nitrogen
of the N-heteroaromatic ring.
For the series D compounds, replacement of the 2′-
pyridylhydrazone substructure of Z-A by either a 2′-
pyrazinylhydrazone (compound E/Z-17) or a 4′-pyrimi-
dinylhydrazone (compound Z-19) was found to lead to
a reduction in activity by a factor of 8-37. For the
compounds bearing a pyrimidinylhydrazone moiety (E-
18 and Z-19-Z-21), the 6′-Cl derivative (E-18) turned
out to be the most active. Interestingly, the shift of the
hydrazine moiety from the 4-position of the pyrimidine
ring (Z-19, IC50 ) > 0.35-3.22 µM) to the 2-position
(Z-21, IC50 ) > 10 µM) results in a total loss of activity
(Table 1). Also with compounds E/Z-22, E-23, and Z-24,
the activity is lowered compared to Z-A, indicating that
replacement of the 2′-pyridylhydrazone by a pyridazi-
nylhydrazone and the various substituents it bears
offers no advantage. Of the hydrazones (i.e. compounds
25-28) derived from the benzoanulated azinyl- and
diazinylhydrazines (34h -k ), the 1-isoquinoline deriva-
tive E-26 (IC50 ) 0.015-0.244 µM) showed a significant
improvement in cytotoxic activity as compared to Z-A
(IC50 ) 0.022-0.380 µM). In general, all the other
compounds were less active compared to Z-A and E-26,
with the loss of activity being greater with E/Z-25.
we studied the influence of stereochemistry on cytotoxic
activity. Whereas E-5 shows enhanced activity (IC50
)
0.032-0.619 µM) compared to Z-5 (IC50 ) 0.151-8.098
µM), there is only a small difference between the data
obtained for E-7 and Z-7 for the cell lines tested except
for MEXF 276L (E-7, IC50 ) 0.182 µM, and Z-7, IC50
)
0.669 µM). For the disubstituted compounds Z-9-11,
cytotoxic activity is lowered compared to Z-A and the
parent monosubstituted analogues Z-4, E-5, and E/Z-8
(Table 1).
To gain further insight into structure-activity rela-
tionships, compounds characterized by replacement of
the 2-benzoylpyridine substructure of Z-A by a 2-ben-
zoylpyrazine (E/Z-12), the p-OCH3 derivative of 12 (i.e.
E-13), 4-benzoylpyrimidine (E/Z-14) or 2-benzoylpyrim-

Notes
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 26 4423
Ta ble 2. Comparison of Inhibition of Cell Proliferation and
[
14C]Cytidine Incorporation into DNA as a Measure of
Ribonucleotide Reductase Activity of Several Selected
Compounds
IC50 (µM)
cell
[
14C]cytidine
ratio of
compd
proliferationa
incorporationb
IC50 valuesc
hydroxyurea
Z-A
E-1
Z-3
Z-4
140
25.69
7.76
0.67
0.57
7.20
3.84
9.04
3.31
5.05
>10
8.44
2.51
0.18
353
60.9
40.7
720
0.022
0.011
0.014
0.010
0.032
0.150
0.283
0.045
0.347
0.463
0.015
E-5
Z-5
120
60.3
11.7
122
28.8
18.2
167
E-13
E-18
Z-19
Z-24
E-26
a
b
IC50 values taken from Table 1 (Burkitt’s lymphoma). De-
termined in Burkitt’s lymphoma cells. The mean of at least two
independent experiments which duplicate determinations were
taken within each experiment. c The ratio IC50 is IC50 ([14C]cytidine
incorporation)/IC50(cell proliferation).
F igu r e 1. Loadings plot of principal component analysis.
[
14C]Cytid in e Meta bolism in Bu r k itt’s
Lym p h om a Cells
Incorporation of [14C]cytidine into DNA was measured
in intact Burkitt’s lymphoma cells in order to provide
evidence that our novel compounds are capable of
inhibiting RR activity in situ. Table 2 shows that all
compounds inhibited cell proliferation (IC50 ) 0.010-
0.463 µM) and [14C]cytidine incorporation (IC50 ) 0.57
to >10 µM) to a larger extent than hydroxyurea (IC50
) 140 µM and 25.69 µM, respectively). The E-configu-
rated compounds 1, 5, 13, 18, and 26 inhibited [14C]-
cytidine incorporation into DNA to a greater extent (IC50
) 0.67-5.05 µM) than the Z-configurated compounds
A, 4, 5, 19, and 24 (IC50 ) 7.20 to >10 µM). Surpris-
ingly, 3, which can be considered to be in a pseudo-Z-
form, turned out to be the most active compound,
inhibiting the incorporation of [14C]cytidine into DNA
with an IC50 of 0.57 µM. In general the IC50 values for
antiproliferative activity are lower than those of inhibi-
tion of [14C]cytidine incorporation into DNA. Since the
ratios of IC50 values for [14C]cytidine incorporation to
the values for cell proliferation with our compounds is
>1 but is less than one with hydroxyurea (Table 2), we
conclude that the novel compounds do not exert their
antiproliferative activity solely by RR inhibition. In-
vestigations of other possible modes of action, however,
showed that the cell cycle regulators cdc2 kinase, and
cdc25 phosphatase are not affected by our compounds
even at concentrations of >100 µM.26
F igu r e 2. Scores plot of principal component analysis.
performed. The latent variables obtained after PCA of
the data matrix as statistical scores are called principal
properties (PPs) and represent in an appropriate way
the multidimensional system by a few descriptors.
Since PPs are orthogonal to each other, they are
particularly suitable as design variables, permitting a
few representative compounds to be selected out of a
larger set. The results of our calculations are given in
Figures 1 and 2. From the loadings plot (Figure 1) it
becomes evident that there exist three main types of
cell lines exhibiting comparable sensitivity toward the
compounds in our test system (the Burkitt’s lymphoma,
CCRF-CEM, and HeLa cells belonging to one type, the
HT-29 and MEXF-276L cells forming two other types).
From the scores plot (Figure 2), compounds exhibiting
unusual activities in certain cell assays can be detected,
since they appear as outliers (Z-2, Z-4, Z-5, E-7). These
results may serve as a guide for further synthesis and
efficient biological testing of selected compounds toward
representatives of sensitive cell lines.
Sta tistica l Eva lu a tion of Biologica l Da ta
Preliminary studies concerning the finding of QSARs
revealed that no significant correlation exists between
cell growth inhibition and structural properties of the
compounds, using neither lipophilicity values nor 3D
molecular interaction field descriptors calculated within
the CoMFA approach.27 Therefore we focused our
interest in the detection of similarities between the
sensitivity of the various cell lines toward our com-
pounds. For determination of intercorrelation among
biological data resulting from the assays done with the
different cell lines, a principal component analysis
(PCA) of the inhibitory activity of the compounds was
Con clu sion
In general, it turned out that the various structural
changes made to compound Z-A are well tolerated and

4424 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 26
Notes
C-4), 125.0 (q, J ) 268 Hz, CF3), 112.8 (q, J ) 32 Hz, C-5),
105.4 (C-3). Anal. (C6H6F3N3) C, H, N.
that several of the newly synthesized compounds are
even more effective in inhibiting cell proliferation than
Z-A. However, a substituent in the 6′-position of the
2′-pyridylhydrazone moiety (e.g. E/Z-6) and the replace-
ment of the 4-pyrimidinyl group in compound Z-19 by
a 2-pyrimidinyl system (compound Z-21) leads to a
substantial loss of activity. Most sensitive to the new
compounds were Burkitt’s lymphoma, CCRF-CEM, and
HeLa; less sensitive were HT-29 and MEXF 276 L cells.
The proliferation of Burkitt’s lymphoma cells is inhib-
ited in particular by E-1, Z-3, Z-4, E-7, Z-7, E/Z-14, E/Z-
15, E-18, and E-26 with IC50 in the range of 0.01-0.04
µM. A difference in cytotoxic activity is observed for
E- and Z-isomers (e.g. with compounds 5 and 7), and
this is even more pronounced in the [14C]cytidine
incorporation assay where all of the E-configurated
compounds are more active than the Z-isomers by a
factor of 10-60. In conclusion, our investigations
indicate that inhibition of ribonucleotide reductase
seems not to be the sole mechanism of action of these
compounds.
P h en yl(2-p yr im id in yl)m eth a n on e (33d ). (a) To a stirred
solution of sodium cyanide (12.90 g, 263 mmol) in dry DMF
(200 mL) was added 2-chloropyrimidine (20.00 g, 175 mmol)
portionwise, and the mixture was heated at 60-70 °C for 24
h. After cooling, the mixture was diluted with water (650 mL),
stirred for another 30 min, and extracted with CH2Cl2 (6 ×
250 mL). The combined organic phases were washed with
water, dried over anhydrous Na2SO4, filtered, and evaporated
to dryness. The solid obtained after Kugelrohr distillation in
vacuo was recrystallized from DIPE/PE to give 8.99 g (49%)
of pyrimidine-2-carbonitrile: colorless crystals; mp 39-41 °C
(lit.16 mp 41-42 °C).
(b) To a stirred solution of pyrimidine-2-carbonitrile (5.00
g, 48 mmol) in dry diethyl ether (100 mL) at -5 to -10 °C
was added dropwise a 25% solution of phenylmagnesium
chloride in THF (6.54 g, 48 mmol). The mixture was allowed
to warm to room temperature, and after 4 h of stirring, 6 N
HCl solution (50 mL) was added, and stirring continued for 1
h. The ether layer was separated and extracted with 2 N HCl
solution (3 × 50 mL). The combined acidic aqueous phases
were adjusted to pH 7-8 with solid NaHCO3 and then
extracted several times with diethyl ether. The combined
ether phases were washed with saturated NaCl solution, dried
over anhydrous Na2SO4, filtered, and evaporated to dryness.
The solid remaining was subjected to Kugelrohr distillation
at 180 °C to give 7.15 g (82%) of compound 33d as colorless
Exp er im en ta l Section
Ch em istr y. Infrared spectra (IR) were recorded from KBr
pellets on a Mattson Galaxy Series FTIR 3000 spectropho-
tometer. 1H and 13C NMR spectra were recorded from DMSO-
d6 solutions on a Varian Gemini 200 (1H, 199.98 MHz; 13C,
50.29 MHz) spectrometer with δ 2.49 ppm for 1H and δ 39.50
ppm for 13C as internal standard. Melting points were
determined on a Reichert Thermovar hot stage microscope and
are uncorrected. Elemental analyses were performed at the
“Institut fu¨r Physikalische Chemie”, University of Vienna,
Austria, and the data for C, H, N are within (0.4% of the
calculated values. Reactions were monitored by TLC 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).
2-Ben zoylp yr id in e 4-P yr im id in ylh yd r a zon e (Z-19). To
a solution of 34b5 (1.72 g, 12 mmol) and ammonium formate
(7.05 g, 112 mmol) in methanol (80 mL) was added 10% Pd/C
(0.5 g). The mixture was heated at 50-60 °C for 15 h, cooled
to room temperature, and filtered over Celite. After concen-
tration of the methanolic solution to about 10 mL, 5 drops of
concentrated H2SO4 and 33a (1.00 g, 5.5 mmol) were added,
and the mixture was refluxed for 4 h. After being cooled to
room temperature, the mixture was evaporated to dryness, and
the remaining solid was recrystallized from 95% ethanol to
give 0.69 g (46%) 19 as colorless needles, mp 156-158 °C. Anal.
(C16H13N5) C, H, N.
1
crystals: mp 87-88 °C (lit.15 mp 84-85 °C); H NMR δ 8.94
(d, J ) 5 Hz, 2H, pyrimidine H-4,6), 8.03 (dd, J ) 1.6 Hz, J )
7 Hz, 2H, phenyl H-2,6), 7.66-7.57 (m, 1H, phenyl H-4), 7.52-
7.44 (m, 3H, pyrimidine H-5 and phenyl H-3,5).
Gen er a l P r oced u r e for th e Syn th esis of Hyd r a zon es
1-28. A mixture of equimolar amounts of ketone and hydra-
zine in methanol containing traces of glacial acetic acid was
refluxed until TLC monitoring indicated no further conversion.
Reaction times varied from 6 to 12 h. The resulting hydra-
zones separated when the solutions were cooled to 5 °C
overnight. In the case of the more soluble hydrazones, the
solutions were evaporated to dryness, and the residues ob-
tained were dissolved in hot ethyl acetate or in an ethyl actate/
diisopropyl ether mixture and chilled. Then, the crystals
which separated were collected by filtration and recrystallized.
For compounds of Scheme 1, Z-A: 70% yield; mp 150 °C.
Anal. (C17H11N4) C, H, N. E-1: 80% yield; mp 143-145 °C
(EtOH). Anal. (C18H15N4O) C, H, N. Z-2: 76% yield; mp 240-
242 °C (EA/DIPE). Anal. (C17H13N4O) C, H, N. Z-4: 67%
yield; mp 199-202 °C (EtOH). Anal. (C17H13N5O2) H, N; C:
calcd, 63.93, found, 64.57. E-5: 35% yield; mp 134-136 °C
(EtOH). Anal. (C17H13ClN4) C, H, N. Z-5: 45% yield; mp
154-156 °C (EtOH). Anal. (C17H13ClN4) C, H, N. E/Z-6: 75%
yield; mp 166-169 °C (EA/DIPE). Anal. (C17H13ClN4) C, H,
N. E-7: 38% yield; mp 148-151 °C (DIPE). Anal. (C18H16N4)
C, H, N. Z-7: 55% yield; mp 110-113 °C (MeOH). Anal.
(C18H16N4) C, H, N. E/Z-8: 85% yield; mp 108-110 and 120-
123 °C (EA). Anal. (C18H13F3N4) C, H, N. Z-9: 60% yield;
mp 237-239 °C (EtOH/EA). Anal. (C17H12N6O4) C, H, N.
P r ep a r a tion of Hyd r a zin es 32a a n d 32e. 2-Chloro-5-
nitropyridine (31a ) (5.50 g, 34.69 mmol) or 2-chloro-5-(trifluo-
romethyl)pyridine (31e) (18.15 g, 0.1 mol) was dissolved in
ethanol (50 mL) with warming, and to this solution was added
6 equiv of 95% hydrazine hydrate.
Z-10: 70% yield; mp 167-169 °C (MeOH). Anal. (C18H12
-
ClF3N4) C, H, N. Z-11: 65% yield; mp 142-144 °C (EA/DIPE).
Anal. (C18H12ClF3N4) C, H, N. E/Z-12: 71% yield; mp 163-
165 °C (EtOH) (lit.7 mp 188 °C). E-13: 58% yield; mp 173-
175 °C (EA/DIPE). Anal. (C17H15N5O) C, H, N. E/Z-14: 77%
yield; mp 152-155 °C (EtOH). Anal. (C16H13N5) C, H, N. E/Z-
15: 73% yield; mp 170-172 °C (EtOH). Anal. (C16H13N5) C,
H, N. E-16: 44% yield; mp 141-144 °C (PrOH/H2O). Anal.
(C17H11N4) C, H, N.
5-Nitr o-2-p yr id ylh yd r a zin e (32a ). The mixture was then
stirred at room temperature overnight. The dark yellow
precipitate formed was filtered off, washed with cold ethanol,
and dried. Compound 32a was obtained as fine yellow needles
after recrystallization from 95% ethanol: yield 4.28 g (80%);
1
mp 213-215 °C; H NMR δ 8.87 (d, J ) 3 Hz, 1H, H-6), 8.16
(dd, J ) 3 Hz, J ) 9 Hz, 2H, H-4/NH), 6.81-6.77 (m, 1H, H-3)
5.13-4.35 (br s, 2H, NH2). Anal. (C5H6N4O2) C, H, N.
For compounds of Scheme 2, E-18: 64% yield; mp 140-144
°C (EtOH/DIPE). Anal. (C16H12ClN5) C, H, N. Z-19: 50%
yield; mp 156-158 °C (EtOH). Anal. (C16H13N5) C, H, N.
Z-20: 78% yield; mp 97-100 °C (EA). Anal. (C17H15N5) C,
H, N; C: calcd, 70.56, found, 71.21. E/Z-22: 87% yield; mp
166-170 °C (EtOH/DIPE). Anal. (C16H12ClN5) C, H, N.
E-23: 45% yield; mp 209-212 °C (EtOH). Anal. (C17H15ClN5)
C, H, N. Z-24: 60% yield; mp 208-210 °C (THF). Anal.
(C17H14N5) C, H, N. E/Z-25: 77% yield; mp 136-139 and 179-
183 °C (EtOH) (lit.29 mp 193 °C). Anal. (C20H16N4) C, H, N.
5-(Tr iflu or om eth yl)-2-p yr id ylh yd r a zin e (32e). The so-
lution was refluxed for 5 h, the mixture was cooled overnight,
and the product was filtered off, washed with cold water, and
recrystallized from a water-ethanol mixture to give 14.17 g
1
(80%) of 36e as colorless needles. mp 65-67 °C; H NMR δ
8.26 (s, 2H, H-6/NH), 7.68 (dd, J ) 3 Hz, J ) 9 Hz, 1H, H-4/
NH), 6.81 (d, J ) 9 Hz, 1H, H-3), 4.33 (s, 2H, NH2); 13C NMR
δ 163.5 (C-2), 145.2 (q, J ) 4 Hz, C-6), 133.6 (q, J ) 3 Hz,

Notes
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 26 4425
E-26: 70% yield; mp 172-174 °C (toluene) (lit.30 mp 172 °C).
E/Z-27: 64% yield; mp 179-183 °C (MeOH/H2O) (lit.7 mp 177-
179 °C). E-28: 55% yield; mp 218-220 °C (2-PrOH) (lit.31 mp
222 °C). Recrystallization solvents: EtOH ) ethanol, EA )
ethyl acetate, DIPE ) diisopropyl ether, MeOH ) methanol,
PrOH ) propanol, 2-PrOH ) 2-propanol, THF ) tetrahydro-
furan.
(11) Plazek, E. Synthesis of 3,5-Dinitropyridine. Recl. Trav. Chim.
Pays-Bas 1953, 72, 569-575; Chem. Abstr. 1955, 49, 1038c.
(12) Schwaiger, W.; Cornelissen, J .; Ward, J . P. A Convenient
Synthesis of Alkyl- and Arylpyrazinyl ketones. Food Chem. 1984,
13, 225-234.
(13) Goodson, P. A.; Oki, A. R.; Glerup, J .; Hodgson, D. J . Design,
Synthesis, and Characterization of Bis(µ-oxo)dimanganese(III,-
III) Complexes. Steric and Electronic Control of Redox Poten-
tials. J . Am. Chem. Soc. 1990, 112, 6248-6254.
(14) Ho¨ffner, S. Beitrage zur homolytischen Substitution am Pyri-
midinsystem. (On the homolytic substitution of the pyrimidine
system.) Diploma Thesis, University of Vienna, 1988.
(15) Heinisch, G.; Holzer, W; Langer, T; Lukavsky, P. Aryl Diazinyl
Ketoximes: Synthesis and Configurational Assignment. Hetero-
cycles 1996, 43, 151-172.
(16) Case, F. H.; Koft, E. The Synthesis of Certain 1,3,5-Triazines
Containing the Ferroin Group. J . Am. Chem. Soc. 1959, 81, 905-
906.
(17) Spingarn, N. E.; Sartorelli, A. C. Synthesis and Evaluation of
the Thiosemicarbazone, Dithiocarbazonate, and 2′-Pyrazinylhy-
drazone of Pyrazinecarboxaldehyde as Agent for the Treatment
of Iron Overload. J . Med. Chem. 1979, 22, 1314-1316.
(18) Graf, H.; Scheutz, F.; Sauter, H.; Ammermann, E.; Pommer,
E-H. Substituierte 5-Amino-Pyrazole und dies enthaltende
Fungizide. (Fungicidal activity of substituted 5-aminopyrazoles.)
EP Pat. Appl. 0293743, 1988; Chem. Abstr. 1989, 110, 192849t
(19) Scho¨nbeck, R.; Kloimstein, E. U¨ ber Pyridazine mit kerngebun-
denem Chlor. (Studies on chloropyridazones.) Monatsh. Chem.
1968, 99, 15-84.
(20) Cregge, R. G.; Coutant, J . E. 6-Hydroxyalkylamino-8-methyl-
1,2,4-triazolo-[4,3-b]pyridazine and Related Compounds. US
Patent 4,578,464, 1986; Chem. Abstr. 1986, 105, 42830n.
(21) Perkin, W. H.; Robinson, R. Harmine and Harmaline. Part II.
The Synthesis of iso-Harman. J . Chem. Soc. 1913, 1973-1985.
(22) Martin, M. J .; Trudell, M. L.; Arau´zo, H. D.; Allen, M. S.;
LaLoggia, A. J .; Deng, L.; Schutz, C. A.; Tan, Y.-C.; Bi, Y.;
Narayanan, K.; Dorn, L. J . Koehler, K. F.; Skolnick, P.; Cook,
J . M. Molecular Yardsticks. Rigid Probes To Define the Spatial
Dimensions of the Benzodiazepine Receptor Binding Site. J .
Med. Chem. 1992, 35, 4105-4117.
(23) (a) Butters, M. Large Scale Synthesis of 4-Ethylpyrimidine, J .
Heterocycl. Chem. 1992, 29, 1369-1370. (b) Foster H. M.; Snyder
H. R. 4-Methyl-6-hydroxypyrimidine. In Organic Syntheses;
Rabjohn, N., Ed.; J ohn Wiley and Sons Publishing Co.: New
York, 1965; Collect. Vol. IV, pp 638-640. (c) Shiho, D.; Taka-
hayashi, N. Hydrazino derivatives of Pyrimidine III. Syntheses
of derivatives of 4-hydrazino-6-methylpyrimidine. J . Pharm. Soc.
J pn. 1955, 75, 773-775; Chem. Abstr. 1956, 50, 4976f.
(24) Easmon, J .; Heinisch, G.; Holzer, W. Pyridazines 47. The
Configuration of Novel Thiosemicarbazone Derivatives of Py-
ridazinecarbaldehydes and Aryl Pyridazinyl Ketones. Hetero-
cycles 1989, 29, 1399-1408.
In Vitr o Cytotoxicity Assa y a n d [14C]Cytid in e In cor -
p or a tion Stu d ies. CCRF-CEM (acute lymphoblastic leu-
kemia, ATCC CCL 119), Burkitt’s lymphoma (CA 46, ATCC
CRL 1648), HeLa (epitheloid cervix carcinoma, ATCC CCL2),
and MEXF 276 L (melanoma) cells were grown in RPMI 1640,
and HT-29 (colon adenocarcinoma, ATCC HTB 38) in McCoy’s
5A medium. The media were supplemented with 10% fetal
calf serum (except Burkitt’s lymphoma with 15%), 2 mM
glutamine, 50 units/mL penicillin, and 50 µg/mL streptomycin.
Inhibition of cell proliferation in HeLa, HT-29, and MEXF-
276 L was detected by the SRB assay,32 that of CCRF-CEM
and Burkitt’s lymphoma cells by the MTT assay33 from
Boehringer Mannheim, Mannheim, Germany.
[14C]Cytidine incorporation into DNA was performed by a
modification of the procedure previously described34 in intact
Burkitt’s lymphoma cells. Three milliliters ((1-2) × 106/mL)
of exponentially growing cells were incubated at 37 °C in
medium with various drug concentrations of each compound.
After 90 min 0.7 µCi [14C]cytidine (2-10 Ci/mmol, ICN
Biomedicals, Meckenheim, Germany) was added to each
sample for 45 min. Subsequently, the cells were washed twice
in ice-cold phosphate-buffered saline, resuspended in 1 mL of
ice-cold trichloroacetic acid, and transferred to Eppendorf
tubes. The samples were then treated as described by No-
centini et al.34
P r in cip a l Com p on en t An a lysis. Statistical evaluation
of the biological data was performed within the Sybyl QSAR
module:35 factor analysis (NIPALS algorithm without rotation)
yielded principal components (cumulative percentage of vari-
ance in the first three components: 86.7, 94.2, and 97.9
respectively).
Ack n ow led gm en t. Financial assistance was gener-
ously provided by the Austrian “Fonds zur Fo¨rderung
der Wissenschaftlichen Forschung”, project no. P 09879-
MED.
Su p p or tin g In for m a tion Ava ila ble: 1H NMR data for
compounds A, 1-28 (5 pages). Ordering information is given
on any current masterhead page.
(25) Agrawal, K. C.; Sartorelli, A. C. The Chemistry and Biological
Activity of R-(N)-Heterocyclic Carbaldehyde Thiosemicarbazones.
Prog. Med. Chem. 1978, 15, 321-356.
(26) Meijer, L. CNRS, Roscoff, personal communication.
(27) Cramer, R. D., III; Patterson, D. E.; Bunce, J . D. Comparative
Molecular Field Analysis (CoMFA). 1. Effect of Shape on Binding
of Steroids to Carrier Proteins. J . Am. Chem. Soc. 1988, 110,
5959-5967.
(28) Schilt, A. A.; Case, F. H. New Chromogens of the Ferroin Type-
X, Synthesis and Chelation Properties of some 2-Thiazolyl- and
2-Pyrimidinylhydrazones. Talanta 1980, 27, 55-58.
(29) Schilt, A. A.; Di Tusa, M. R.; Case, F. H. Synthesis and
Metallochromic Properties of some Hydrazones of 2-, 3-, and
8-Hydrazinoquinoline, Talanta 1982, 29, 338-341.
(30) Case, F. H.; Schilt, A. A. Synthesis of some Isoquinolyl-1-
hydrazones and Spectroscopic Characterization of some of their
Transition-Metal Chelates. J . Chem. Eng. Data 1986, 31, 503-
504.
(31) Case, F. H.; Schilt, A. A.; Simonzadeh, N. Synthesis and
Chelation Properties of Hydrazones Derived from Isoquinoline-
1-carboxaldehyde, 2-Quinoxalinecarboxaldehyde, 4-Isoquinolyl-
hydrazine, and 2-Quinoxalylhydrazine. Anal. Chem. 1984, 56,
2860-2862.
(32) Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J .;
Vistica, D.; Warren, J . T.; Bokesch, H.; Kenney, S.; Boyd, M. R.
New Colorimetric Cytotoxicity Assay for Anticancer-Drug Screen-
ing. J . Natl. Cancer Inst. 1990, 82, 1107-1112.
(33) Mosman, T. Rapid Colorimetric Assay for Cellular Growth and
Survival: Application to Proliferation and Cytotoxicity Assays.
J . Immunol. Methods 1983, 65, 55-63.
(34) Nocentini, G.; Federici, F.; Franchetti, P.; and Barzi, A. 2,2′-
Bipyridyl-6-carbothioamide and its Ferrous Complex: Their in
Vitro Antitumoral Activity Related to the Inhibition of Ribo-
nucleotide Reductase R2 Subunit. Cancer Res. 1993, 53, 19-
26.
Refer en ces
(1) Cory, J . G. Role of Ribonucleotide Reductase in Cell Division.
In Inhibitors of Ribonucleoside Diphosphate Reductase Activity;
Cory, J . G., Cory, A. M., Eds.; Pergamon Press: Oxford, 1989, p
1-16.
(2) Elford, H. L.; Freese, M.; Passamani, E.; Morris, H.-P. Ribo-
nucleotide Reductase and Cell Proliferation 1. Variations of
Ribonucleotide Reductase Activity with Tumor Growth Rate in
a Series of Rat Hepatomas. J . Biol. Chem. 1970, 245, 5228-
5233.
(3) Weber, G. Biochemical Strategy of Cancer Cells and the Design
of Chemotherapy: G. H. A. Clowes Memorial Lecture. Cancer
Res. 1983, 43, 3466-3492.
(4) Larsen, I. K. Inhibition of the Enzyme Ribonucleotide Reductase.
Front. Drug Res.: Alfred Benzon Sympos. 1990, 28, 47-57.
(5) Schaper, K. J .; Seydel, J . K. Substituted 2-Acylpyridin-R-(N)-
Hetarylhydrazones and Medicaments Containing Same. World
Pat. Appl. WO 88/08842, 1988; Chem. Abstr. 1989, 110, 231439f.
(6) Frank, E.; Gearien, J .; Megahy, M.; Pokorny, C. Analgetic and
Anticonvulsant Activity of Some 2- and 4-Pyridyl Ketones. J .
Med. Chem. 1971, 14, 551-553.
(7) Case, F. H. Preparation of New 2-Pyridyl and Pyrazinylhydra-
zones Containing Ferroin Group. J . Chem. Eng. Data 1976, 21,
124-125.
(8) Dusza, J . P.; J oseph, J . P.; Bernstein, S. Heterocyclic substituted-
amino-pyrazolines. EP Pat. Appl. 0070376, 1983; Chem. Abstr.
1983, 99, 5626n.
(9) Bu¨chi, J .; Fabiani, P.; Frey, H. U.; Hofstetter, A.; Schorno, A.
Synthese und pharmakologische Eigenschaften einiger Pyridyl-
pyrazol-5-one. (Synthesis and pharmacological properties of some
pyridyl-pyrazol-5-ones.) Helv. Chim. Acta 1966, 49, 272-280.
(10) Potts, K. T.; Burton. H. R. 1,2,4-Triazoles. XXI. Derivatives of
the s-Triazolo[4,3-a]pyridine Ring System. J . Org. Chem. 1966,
31, 251-260.
(35) Sybyl Version 6.2, Tripos Assoc., St. Louis, MO.
J M970255W