Synthesis, structure-activity relationships, and antitumor studies of 2-benzoxazolyl hydrazones derived from alpha-(N)-acyl heteroaromatics

Article abstract of DOI:10.1021/jm060232u

Recently we have described the antitumor activities of 2- benzoxazolylhydrazones derived from 2-formyl and 2-acetylpyridines. In search of a more efficacious analogue, compounds in which the 2-acetylpyridine moiety has been replaced by 2-acylpyridine and α-(N)-acetyldiazine/quinoline groups have been synthesized. The 2-acylpyridyl hydrazones inhibited in vitro cell proliferation in the nM range, whereas the hydrazones derived from the α-(N)-acetyldiazines/quinolines inhibited cell growth in the μM range. Compounds tested in the NCI-60 cell assay were effective inhibitors of leukemia, colon, and ovarian cancer cells. E-13k [N-benzoxazol-2-yl-N′-(1- isoquinolin-3-yl-ethylidene)-hydrazine] inhibited the proliferation of MCF-7 breast carcinoma cells more efficiently than nontransformed MCF-10A cells. It is not transported by P-glycoprotein and a weak MRP substrate. Increased concentrations of serum or α₁-acid glycoprotein did not reduce the antiproliferative activity of the compound. In the in vivo hollow fiber assay, E-13k achieved a score of 24, with a net cell kill of OVCAR-3 (ovarian) and SF2-95 (CNS) tumor cells.

Full text of DOI:10.1021/jm060232u

J. Med. Chem. 2006, 49, 6343-6350
6343
Synthesis, Structure-Activity Relationships, and Antitumor Studies of 2-Benzoxazolyl
Hydrazones Derived from Alpha-(N)-acyl Heteroaromatics
Johnny Easmon,*,† Gerhard Pu¨rstinger,† Katrin-Sofia Thies,† Gottfried Heinisch,† and Johann Hofmann*,‡
Institute of Pharmacy, Department of Pharmaceutical Chemistry, UniVersity of Innsbruck, Innrain 52a, A-6020 Innsbruck, Austria, and
DiVision of Medical Biochemistry, Medical UniVersity Innsbruck, Fritz-Pregl-Str. 3, A-6020 Innsbruck, Austria
ReceiVed February 28, 2006
Recently we have described the antitumor activities of 2-benzoxazolylhydrazones derived from 2-formyl
and 2-acetylpyridines. In search of a more efficacious analogue, compounds in which the 2-acetylpyridine
moiety has been replaced by 2-acylpyridine and R-(N)-acetyldiazine/quinoline groups have been synthesized.
The 2-acylpyridyl hydrazones inhibited in vitro cell proliferation in the nM range, whereas the hydrazones
derived from the R-(N)-acetyldiazines/quinolines inhibited cell growth in the µM range. Compounds tested
in the NCI-60 cell assay were effective inhibitors of leukemia, colon, and ovarian cancer cells. E-13k
[N-benzoxazol-2-yl-N’-(1-isoquinolin-3-yl-ethylidene)-hydrazine] inhibited the proliferation of MCF-7 breast
carcinoma cells more efficiently than nontransformed MCF-10A cells. It is not transported by P-glycoprotein
and a weak MRP substrate. Increased concentrations of serum or R1-acid glycoprotein did not reduce the
antiproliferative activity of the compound. In the in vivo hollow fiber assay, E-13k achieved a score of 24,
with a net cell kill of OVCAR-3 (ovarian) and SF2-95 (CNS) tumor cells.
Introduction
thereby interfering with the enzyme activity.7 The third class
of RR inhibitors bind with high affinity to the nonheme iron in
the M2 subunit.6 Potent inhibitors interfering with the M2
subunit include the R-(N)-hetrocyclic carboxaldehyde thiosemi-
carbazones (TSCs, compound type A, Figure 1)8 and 2-acylpy-
ridine-R-(N)-hetarylhydrazones9 (compound type B, Figure 1).
From these compounds, we have deduced that an N*-N*-S* or
N*-N*-N* structural motif is essential for RR inhibition. Based
on this hypothesis, we have synthesized 2-benzothiazolylhy-
drazones derived from R-(N)-acetylpyridines, -diazines, and
-(iso)quinolines (e.g., Ia/b, Figure 2) as potential RR inhibitors.
In an in vitro antiproliferative assay, compounds Ia/b were found
to be 80- to 100-fold more active than the TSC analogues.10
Based on the interesting preliminary results we have investigated
the effects of the replacement of the benzothiazole ring by the
isosteric heterocycles benzoxazole and benzimidazole. Com-
pounds IIa/b (Figure 1) turned out to be more active than the
benzothiazole analogues Ia/b and, moreover, were found to
exhibit potent growth inhibitory activity against the proliferation
of a number of cancer cell lines where effective agents are
currently sought.11,12 However, it turned out that all these
compounds do not inhibit RR activity.
In a continual development effort, we have instituted a
systematic structural exploration of compound IIb, with the goal
of synthesizing a more efficacious compound or identifying a
new lead for the development of second-generation compounds.
Herein we report the synthesis, antiproliferative activities, and
the inhibition of the in vivo tumor growth of analogues of
compound IIb.
Chemistry. The 2-hydrazinobenzoxazoles 3a13 and 3b14 were
synthesized according to reported procedures, however, avoiding
the use of dioxane as a solvent in the case of compound 3a.
Reaction of 2-chlorobenzoxazole (1) with hydrazine hydrate (2a)
in tetrahydrofuran (THFa) at -5 °C afforded 3a in high yield.
Reaction of 2-acetylpyridine and hydrazine derivative 3b in
methanol containing traces of glacial acetic acid afforded the
hydrazone 4. Compounds 5 and 6 became accessible from the
condensation of 2-hydrazinobenzoxazole (3a) with 3-acetylpy-
ridine or 2-acetylpyridine-N-oxide,15 respectively (Scheme 1).
Despite recent progress in tumor therapy, the majority of solid
tumors are currently not curable by chemotherapy. Half of all
cancer patients fail to respond to chemotherapy or relapse from
the initial response and ultimately die from their metastatic
disease.1 Therefore, there is the urgency to develop highly active
drugs that are ideally orally active. One possible improvement
in tumor chemotherapy resides in continued research designed
to optimize the administration of the currently available
antitumor drugs or to develop derivatives of the known
antitumor drugs, which are better tolerated than current ones.
Another possibility is to discover novel therapeutic products
by exploiting our increased understanding of tumor biology.
Novel agents, for example taxanes and camptothecins that target
tubulin polymerization and topoisomerase 1, respectively, are
showing promising results in ovarian, breast, and colon cancers.1
Another example is the targeting of signal transduction mol-
ecules such as bcr/abl by STI571 (Glivec).2
Recently we became interested in developing novel inhibitors
of ribonucleotide reductase (RR), the enzyme that converts
ribonucleotide diphosphates to deoxynucleotide diphosphates
with the assistance of free radicals.3 The mammalian enzyme
is composed of two dissimilar subunits, M1, the large subunit
containing allosteric sites and M2, the small subunit containing
a binuclear iron center and a tyrosyl free radical that is essential
for reductase activity.4,5 Either unit by itself is inactive. There
are three broad classes of RR inhibitors. The first class includes
nucleoside analogues, which bind to the M1 subunit of the
enzyme.6 The second class is mainly made up of short chain
peptides that bind at the interface binding site of both units,
* To whom correspondence should be addressed. Phone: +43-512-507
5250(5261) (J.E.); +43-512-507 3505 (J.H.). Fax: +43-512-2940 (J.E.);
+43-512-507-2872(2638) (J.H.). E-mail: johnny.easmon@uibk.ac.at (J.E.);
johann.hofmann@uibk.ac.at (J.H.).
† University of Innsbruck.
‡ Medical University Innsbruck.
1 Abbreviations: CH2Cl2, dichloromethane; DIPE, diisopropyl ether; EA,
ethyl acetate; EtOH, ethanol; MeOH, methanol; THF, tetrahydrofuran; and
BZO, benzoxazole.
10.1021/jm060232u CCC: $33.50 © 2006 American Chemical Society
Published on Web 09/22/2006

6344 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 21
Easmon et al.
form, although the signal for the NH proton appears at ∼14
ppm because of the symmetrical nature of the keto moiety.
Results
Antiproliferative Activity. The growth inhibitory activities
of the new compounds were first tested in a panel of human
tumor cell lines in tissue culture. This panel consists of two
types of leukemia (Burkitt’s lymphoma, CCRF-CEM), a cervix
carcinoma (HeLa), and a colon carcinoma cell line (HT-29).
Compounds E-4-6 were synthesized to explore the importance
of the N-N-N system on the antiproliferative activities of the
novel hydrazones. Compared to E-IIb, the results in Table 1
show that transformation of the NH to N-CH3 (E-4) or the
attachment of the hydrazone moiety beta to the hetero aromatic
nitrogen (E-5) results in a total loss of cytotoxic activity.
However, oxidation of the hetero aromatic nitrogen to give
compound E-6, which can be considered as a potential
metabolite of E-IIb, results in a partial loss of activity by a
factor of ∼54. Although it has been shown that E-IIb does not
inhibit RR activity, it can be inferred from the results elaborated
above that the N*-N*-N* structural motif is essential for the
antiproliferative activities of these compounds.
After establishing the essential structural elements important
for growth inhibitory activity of the hydrazones, we investigated
the effect of replacing the methyl function in E-IIb with various
aliphatic chains and aromatic moieties on cytotoxic activity
(Table 2). Compound E-IIb was highly effective in inhibiting
the proliferation of Burkitt’s lymphoma (IC50 ) 0.005 µM) and
CCRF-CEM (IC50 ) 0.024 µM) cells compared to HeLa (IC50
) 0.77 µM) and HT-29 (IC50 ) 1.43 µM) cell lines. Although
the same trend is observed for compounds where R ) ethyl
(E-11a), n-propyl (E-11b), or isopropyl (E/Z-11c), these
compounds turned out to inhibit the proliferation of HeLa and
HT-29 cells to a greater extent than the lead compound E-IIb.
Furthermore, compounds E-11d and E/Z-11e bearing a highly
branched aliphatic side chain showed a total loss of cytotoxic
activity. Considering what is elaborated above, we can conclude
that the stereochemistry around the -CdN- does not influence
the growth inhibitory activities of compounds 11a-e, therefore,
the loss of cytotoxic activity of E-11d and E/Z-11e can be ex-
plained on the basis that the presence of the tert-butyl and cyclo-
propyl moieties cause a steric hindrance that restricts the proper
binding of these compounds to their molecular target(s).
Of the compounds where R is aromatic (Table 2), (a) the
phenyl analogue E-11g inhibited the proliferation of Burkitt’s
lymphoma, HeLa, and HT-29 cells to the same extent, whereas
the 2-pyridyl derivative 11h was highly active against leukemia
and lymphoma cell proliferation and (b) replacement of the
phenyl with a benzyl moiety (E-11f) results in a compound with
high activity against HeLa-cervix carcinoma cells. Comparison
of the growth inhibitory activities of E-11f, E-11g, and E-11h
gives an indication that these compounds probably induce cell
death through different mechanisms of action.
Figure 1. Proposed pharmacophore model.
Figure 2. Structures of lead compounds.
The alkyl-2-pyridinyl ketones 10a (R ) ethyl),16-19 10b (R
) n-propyl),16,18 10c (R ) isopropyl),18 and 10d (R ) tert-
butyl)16,18 were synthesized by treating 2-cyanopyridine (7) with
the corresponding alkylmagnesium halide, according to reported
procedures (Scheme 1). Several of the reaction procedures were
modified to maximize the yields of the ketones. Cyclopropyl
2-pyridyl ketone (10e)20 was prepared in satisfactory yield by
reacting 2-pyridyllithium (9) and cyclopropyl cyanide. The
synthesis of the lithium compound 9 was accomplished by a
lithium-bromine exchange reaction involving the interaction
of n-butyllithium and 2-bromopyridine (8). The reaction of
benzylmagnesium chloride (which is obtained from benzyl
chloride and magnesium turnings) and 2-cyanopyridine (7)
afforded 2-phenylacetylpyridine (10f)21,22 in a satisfactory yield.
Ketones 12a-k are known compounds and, thus, were synthe-
sized according to reported procedures (see Experimental
Section). The synthesis of the hydrazone derivatives 11a-h and
13a-k (Scheme 1) was accomplished by reacting equimolar
amounts of 2-hydrazinobenzoxazole (3a) with the appropriate
carbonyl compounds 10a-h and 12a-k in methanol containing
traces of glacial acetic acid (Scheme 1).
Spectroscopic Studies. The structures of all novel compounds
were confirmed by IR and NMR spectroscopy, and the purity
was confirmed by elemental analysis. Compounds containing
the -CdN- substructure (e.g., TSCs, hydrazones, and so on)
can exist in the E- or Z-form or as mixtures of E/Z isomers.
1
Reports in the literature show that H NMR spectroscopy can
be used to differentiate between the two isomers. The most
remarkable differences regarding the chemical shifts of the
corresponding protons in the two isomeric forms are the
resonance signals attributable to the NH protons: that is, δ (NH)
) 14-15 ppm for the Z-form and δ (NH) ) 9-12 ppm for the
E-form.23
On the other hand, replacement of the 2-pyridyl moiety of
E-IIb (IC50 ) 0.005-0.024 µM) with a diazine or (iso)quino-
line ring resulted in compounds with decreased activity (IC50
) 0.03-7.04 µM; Table 2). However, compounds E-13a-k
were equipotent in inhibiting the proliferation of the solid tumor
cell lines HeLa and HT-29 compared to the 2-acylpyridyl
hydrazone analogues 11a-h described above. The acetyldiazinyl
hydrazone derivatives (compounds E-13c, 13d, 13e, 13f, and
13h in which Het is a 2-pyrimidine, 4-pyrimidine, or 2-pyrazine
moiety) inhibited the proliferation of Burkitt’s, CCRF-CEM,
HeLa, and HT-29 cells in the submicromolar range. Compara-
This conclusion is further supported by nuclear Overhauser
effect (NOE) difference experiments.24 For compounds existing
in the E-form, irradiation of the NH resonance leads to a positive
NOE on the acetyl-CH3 protons. Thus, considering these
findings, E-configuration was assigned to compounds 4-6,
11a,b,d, 11f,g, and 13a-k. Compounds 11c (30:70) and 11e
(10:90) were mixtures of E- and Z-isomers in the ratios indicated
1
in brackets. In the H NMR spectrum, the signals of the E/Z-
isomers overlap except for the NH proton signals. On the other
hand, compound 11h cannot be assigned to an E/Z-isomeric

Antitumor Studies of 2-Benzoxazolyl Hydrazones
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 21 6345
Scheme 1a,b
a Reagents: (i) (3a) THF, 0-5 °C; (3b) EtOH, reflux; (ii) MeOH, CH3COOH, 60-80 °C; (iii) 2-acetylpyridine; (iv) 3-acetylpyridine; (v) 2-acetylpyridine-
N-oxide; (vi) diethyl ether, -40 to -50 °C; (vii) (a) diethyl ether/THF -15 °C; (b) 2 N HCl; (c) 2 N NaOH. bFor structures of Het for compounds 12a-k,
see Experimental Section, and for compounds 13a-k, see Table 2.
Table 1. Inhibition of Cell Proliferation by Hydrazones IIa/b and
the molecular target(s) might not be known. The GI50 values
obtained along with the mean graph midpoint (MG MID) values
E-4-6a
are summarized in Table 3. The MG MID value is the average
of the GI50 values for all the cell lines tested for drug
concentrations in the range of 10-4-10-8 M.26
As can be seen, all the compounds exhibited high growth
inhibitory activity across the cell lines tested, with an MG MID
of 0.036-10.20 µM. All the compounds tested at the NCI
inhibited the proliferation of colon (mean GI50 ) 0.012-3.9
µM), ovarian (mean GI50 ) 0.018-10.47 µM), and leukemia
(mean GI50 ) 0.012-5.13 µM) cell growth at lower concentra-
tions compared to the MG-MID values given in Table 3. It
should be mentioned that the compounds exhibited striking
sensitivity to these cancers inhibiting six out of seven colon,
five out of six leukemic, and four out of six ovarian cell lines.
On the other hand, breast and prostate cancer cells are insensitive
to the inhibitory activities of the novel hydrazones described.
Only compounds E-11f, E-11h, E-13d/e, and E-13k inhibited
a The mean values of at least two independent experiments in which
the growth of nonsmall cell lung (NSCL) cancer cells at GI50
duplicate determinations were taken within each experiment are indicated.
values equal to or below the MG-MID values. Furthermore,
compounds E-11f, E-11h, E-13c, E-13d, E-13e, E-13f, and
E-13k were also highly effective in renal cancer cells (mean
GI50 ) 0.018-0.199 µM), whereas only E-11h and E-13f
inhibited the growth of melanoma cells above the MG-MID
values.
For clearity, standard deviation has been omitted. Standard deviation is
below 5%, except at low nanomolar IC50s.
tively, the pyridazine derivatives E-13a/b were less active by a
factor of 10-24. Among the acetyl-quinoline derivatives, the
relative potency was in the following order: E-13k > E-13j >
E-13i.
Organ-Specific Tumor Cell Lines. Selected compounds
were submitted for testing in the organ-specific panel of human
tumor cell lines at the National Cancer Institute, Bethesda, MD.
This screen utilizes 60 human tumor cell lines, representing
leukemia, melanoma, and cancers of the lung, colon, brain,
ovary, kidney, prostate, and breast.25 Advantages of the NCI-
60 cell line in vitro assay and microarray data are that the results
can be used to determine the uniqueness of a new lead for which
The results are encouraging because colorectal, ovarian, and
renal cancers are one of the leading causes of cancer mortality
worldwide, and current chemotherapeutic agents administered
alone or in combination, are frequently compromised by the
development of drug resistance or dose-limiting toxicities. In
view of the above-elaborated negatives of cytotoxics in current
use, we conducted further studies to evaluate the potential
usefulness of these agents. Compound E-13k was chosen as a
reference substance for the following studies.

6346 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 21
Easmon et al.
Table 2. Physical Data, Yields and Antiproliferative Activity of
2-benzoxazolylhydrazones 11a-h and 13a-ka
antiproliferative activity in normal cells compared to tumor cells.
A comparison with hydroxyurea implicates a better therapeutic
window for E-13k.
Drug Resistance and Drug Inactivation. Resistance to
anticancer therapy is a major problem in the treatment of cancer
patients. Resistance is, among others, frequently caused by the
expression of the mdr1 (multi-drug resistance gene 1, ABCB1)
or MRPs (multi-drug resistance associated proteins). Therefore,
there is the view that new agents that are not transportable by
these drug transporters can go a long way to alleviate some of
the pitfalls in cancer chemotherapy.27,28
The antiproliferative activity of E-13k was examined in
multidrug resistant HeLa-mdr1 cells in which P-glycoprotein
is overexpressed due to transfection of the mdr1 gene.27
Vinblastine was included as a positive control. The antiprolif-
erative activity of vinblastine, a drug transported by the
P-glycoprotein (P-gp), is strongly enhanced by 5 µM verapamil
(Figure 4). However, 5 µM verapamil does not show this effect
when combined with E-13k, indicating that mdr1 does not
transport E-13k. An interesting observation is that some of the
novel hydrazones inhibit the proliferation of mdr1, overexpress-
ing cancer cells at lower IC50 values compared to the corre-
sponding none P-gp overexpressing cells (results not shown).
A comparison of the MRP (ABCC1) expressing HL60/AR
with the parental sensitive HL60 cell line shows a slight
resistance to E-13k (Figure 5). The resistance ratio of the IC50
values in HL60/AR versus HL60 wild-type cells is ap-
proximately 1.7.
The binding of drugs to plasma proteins can have important
implications on the pharmacodynamics/pharmacokenetics of
such agents. The most important drug binding proteins in plasma
include human serum albumin and alpha-1-acid glycoprotein.29,30
Therefore, we investigated whether the antiproliferative
activity of E-13k is reduced if the serum in the medium is
increased. In medium containing 30% fetal calf serum, the
antiproliferative activity of E-13k was not reduced compared
to that of a 10% serum (Figure 6). In a medium containing 0.1%
R-1-acid glycoprotein, no difference to the standard medium
was observed (Figure 7). Antitumor drugs may be applied
intravenously, which may require heat sterilization. As shown
in Figure 8, the same IC50 is obtained for both heat-treated and
nonheat-treated samples of E-13k. This indicates that the novel
hydrazones are stable to heat sterilization.
Hollow Fiber Assay. On the basis of the in vitro cytotoxic
results elaborated in Table 3, compounds E-13c, E-13e, E-13f,
and E-13k were selected for further evaluation as anticancer
agents in an in vivo hollow fiber assay.31 It has been suggested
that this assay may well be suited for the prioritization of
compounds for more advanced stages of in vivo xenograft drug
evaluation.
An arbitrary score of 2 is assigned each time a compound
produced a 50% or greater reduction in viable cell mass
compared to the vehicle-treated controls. A drug is considered
successful if it has a combined IP+SC score of g 20, an SC
score g 8, or a net cell kill of one or more cell lines that can
be observed. The results, listed in Table 4, show that of the
compounds tested, only E-13k (NSC 693638) fulfilled one or
more of the scoring criteria, with a combined score of 24 (IP )
20 and SC ) 4). Furthermore, E-13k showed a net cell kill of
OVCAR-3 (ovarian) as well as SF-295 (CNS) cancer cells.
a The mean values of at least two independent experiments in which
duplicate determinations were taken within each experiment are indicated.
For clearity, standard deviation has been omitted. Standard deviation is
below 5%, except at low nanomolar IC50s.
Inhibition of Tumor versus Nontumor Cells. To explain
whether tumor cells are inhibited more than nontumor cells,
E-13k was tested in MCF-7 breast adenocarcinoma cells versus
the nontumorigenic breast epithelial cell line MCF-10A. As
shown in Figure 3, the proliferation of the normal MCF-10A
cells was inhibited less than that of the transformed MCF-7 cells.
This is an indication that the compound may exhibit less
Discussion
The in vitro result presented here implies that, the novel
hydrazones may be interesting candidates that can find applica-

Antitumor Studies of 2-Benzoxazolyl Hydrazones
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 21 6347
Table 3. Inhibitory Concentration (GI50 in µM) Values and Histological Cancer Types for the Novel Hydrazones E-5, E-11f/h, E-13c-f, and E-13k
compound (mean GI50 µM)
cancer typea
E-6
E-11f
E-11h
E-13c
E-13d
E-13e
E-13f
E-13k
leucemia
NSCL
colon
5.13
12.30
3.39
0.026
0.059
0.020
0.11
0.025
0.049
0.023
0.112
0.028
0.022
0.069
0.060
0.234
0.050
0.012
0.051
0.031
0.166
0.050
0.018
0.032
0.048
0.079
0.042
0.054
0.148
0.087
0.275
0.224
0.107
0.219
1.51
0.023
0.044
0.023
0.112
0.117
0.039
0.022
0.138
0.123
0.069
0.182
0.251
0.129
0.407
0.174
0.199
0.173
0.199
0.447
0.204
0.017
0.026
0.012
0.089
0.037
0.022
0.015
0.144
0.068
0.036
CNS
7.24
melanoma
ovarian
renal
prostrate
breast
16.98
10.47
11.20
12.30
22.90
10.20
0.10
0.020
0.066
0.062
0.148
0.059
0.302
0.170
MG-MID
a Each cancer type represents the arithmetic mean of five to eight cancer cell lines (NSCL ) nonsmall cell lung; CNS ) central nervous system).
Figure 5. Inhibition of cell proliferation in sensitive HL60 and MRP
(ABCC1) expressing HL60/AR cells. The mean ((SD) of two
independent experiments in which four samples were taken within each
experiment is indicated.
Figure 3. Effects of E-13k on the antiproliferative activity of
nontransformed MCF-10A and transformed MCF-7 breast carcinoma
cells. The cells were treated with the indicated concentrations of the
drugs for 72 h. The mean ((SD) of two independent experiments, in
which two samples were taken within each experiment, is indicated.
Figure 6. Influence of serum on the inhibition of cell proliferation by
E-13k. HT-29 colon carcinoma cells were treated for 72 h with E-13k
in medium containing the indicated concentrations of fetal calf serum.
The mean ((SD) of two independent experiments in which two samples
were taken within each experiment is indicated.
Compounds showing tissue specific profiles in the NCI
disease oriented in vitro antiproliferative assay were advanced
to the in vivo hollow fiber assay. Of the compounds tested in
the in vivo hollow fiber assay, E-13k was found to be effective
Figure 4. Combination of E-13k with verapamil. Multidrug resistant
HeLa-mdr1 cells were treated with the indicated drugs for 72 h.
and also induced a net cell kill in cancer cells. This is important
because it indicates that E-13k is not only cytostatic, but it really
Inhibition of cell proliferation compared to untreated controls was
can kill cancer cells.
evaluated by the SRB assay. The mean ((SD) of two independent
experiments in which two samples were taken within each experiment
An important issue in cancer treatment is antitumor drug
resistance. Verapamil does not increase the activity of E-13k
in HeLa-mdr1 cells (Figure 4), and there is only a small
difference between HL60 and HL60/AR cells (Figure 5). These
are indications that the expression of mdr1 or MRP does not
significantly influence the activity of these compounds.
To date, the putative mechanism(s) by which the novel
hydrazones induce cell death is lacking. Patterns of drug activity
is indicated.
tion, especially for the treatment of colon, ovarian, and renal
cancers. Moreover, it was found that the compounds exhibited
preferential activity also against transformed cells over normal
cells and were active under conditions that otherwise render a
standard class of antitumor agents ineffective.

6348 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 21
Easmon et al.
efficacy compared to compound E-IIb or to obtain a suitable
lead compound(s) for further chemical manipulation, has largely
been met.
Experimental Section
Chemistry. Infrared spectra (IR) were recorded from KBr pellets
on a Mattson Galaxy Series FTIR 3000 spectrophotometer. 1H NMR
spectra were recorded from DMSO-d6 solutions on a Varian Gemini
2000 (1H, 199.98 MHz; 13C, 49.95 MHz) spectrometer. The center
of the solvent signal was used as internal standard, which was
related to TMS with δ 2.49 ppm (1H) and 39.50 (13C). Assignments
are based on chemical shift considerations as well as homonuclear
NOE difference experiments. 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 a UV lamp. Column chromatography was per-
formed using Kieselgel 60 (0.040-0.063 mm).
Figure 7. Inhibition of cell proliferation of Burkitt’s lymphoma cells
in normal medium (control) and in medium containing 0.1% R1-acid
glycoprotein. The mean ((SD) of two independent experiments in
which two samples were taken within each experiment is indicated.
The following ketones utilized as starting materials were obtained
according to the literature cited: methyl 3-pyridazinyl ketone
(12a),34 methyl 3-(6-methylpyridazinyl) ketone (12b),35 methyl
2-pyrimidinyl ketone (12c),36 methyl 4-pyrimidinyl ketone (12d),35
methyl 4-(6-methylpyrimidinyl) ketone (12e),37 methyl 2-(5-me-
thylpyrazinyl) ketone (12h),38 2-acetylquinoline (12i),39 1-acetyl-
isoquinoline (12j),10 and 3-acetylisoquinoline (12k).40 Methyl
2-pyrazinyl ketone (12f) and methyl 2-(3-methylpyrazinyl) ketone
(12g) were purchased from Maybridge Co. 2-Benzoylpyridine (10g)
and di-(pyridin-2-yl)-methanone (10h) were purchased from Sigma-
Aldrich Co.
E-N-Benzoxazolyl-2-yl-N-methyl-N′-[1-pyridin-2-yl-ethylidine]-
hydrazine (4). A mixture of 2-acetylpyridine (0.67 g, 5.58 mmol)
and 3 (0.92 g, 5.58 mmol) in MeOH (25 mL) containing 5 to 10
drops glacial acetic acid was heated at 60 °C, and the reaction was
followed by TLC (CH2Cl2/EA 7:3). After completion (18 h), the
reaction mixture was evaporated to dryness to give a dark brown
oil. The compound was purified by column chromatography, eluting
Figure 8. Inhibition of cell proliferation of Burkitt’s lymphoma cells
by normal (control) and autoclaved (15 min, 121 °C) E-13k. The mean
((SD) of two independent experiments in which two samples were
taken within each experiment is indicated.
1
with CH2Cl2/EA (7:3) to give 0.85 g (53%) of a yellow oil. H
NMR (δ ppm) 2.51 (s, 3H, CH3), 3.49 (s, 3H, N-CH3), 7.08-7.28
(m, 2H, BZO-H5/6), 7.44-7.58 (m, 3H, BZO-H4/7, pyri-H5), 7.59
(dt, J ) 1.8 Hz, J ) 7.6 Hz, 1H, pyri-H4), 8.15 (d, J ) 8.0 Hz,
1H, pyri-H3), 8.72 (dd, J ) 1.2 Hz, J ) 4.8 Hz, 1H, pyri-H6).
Anal. (C15H14N4O) C, H, N.
Table 4. Anticancer Activities of the Hydrazones E-13c, E-13e, E-13f,
and E-13k in the in Vivo Hollow Fiber Assay
E-N-Benzoxazolyl-2-yl-N′-[1-pyridin-3-yl-ethylidine]hydra-
zine (5). A mixture of 3-acetylpyridine (0.50 g, 4.13 mmol) and
2-hydrazinobenzoxazole (2; 0.62 g, 4.13 mmol) in MeOH (25 mL)
containing 5 to 10 drops glacial acetic acid was heated at 70 °C.
The reaction was complete after 18 h (TLC, CH2Cl2/EA, 7:3). After
cooling, the mixture was evaporated to dryness, and the residue
was recrystallized from EA/EtOH to give light yellow needles
(75%): mp 197-199 °C; IR νmax 3126, 2750, 1640, 1578, 1457,
1237, 990, 737 cm-1; 1H NMR (δ ppm) 2.37 (s, 3H, CH3), 7.03-
7.25 (m, 3H, BZO-H5/6, pyri-H5), 7.40-7.47 (m, 2H, BZO-H4/
7), 8.22 (dt, J ) 2.2 Hz, J ) 8.0 Hz, 1H, pyri-H4), 8.57 (dd, J )
1.8 Hz, J ) 4.8 Hz, 1H, pyri-H6), 9.10 (s, 1H, pyri-H2). Anal.
(C14H12N4O) C, H, N.
compound no.
IP score SC score total score cell kill
E-13c
E-13e
E-13f
NSC 693639
NSC 693637
NSC 703100
8
2
2
6
2
4
10
8
N
N
N
Y
10
20
12
24
E-13k NSC 693638
across the NCI-60 human cancer cell lines have been shown to
contain detailed information that can be used to delineate the
mechanism of action of new antitumor agents.32,33 Antitumor
agents with identical mechanisms of action possess identical
or nearly identical cytotoxicity patterns. A Pearson correlation
coefficient (PCC) with the COMPARE algorithm of >0.5
between an unknown and a known compound shows a similar
mechanism of action. Using E-13k (NSC 693638) as a seed, a
COMPARE analysis showed a positive correlation coefficient
of >0.6 with a number of hydrazones of this class of compounds
and a negative correlation with antitumor agents of known
mechanism of action. This indicates that the hydrazones
described represent a novel class of antitumor agents with a
novel mechanism(s) of action. Currently, an intense study is
underway to elucidate the biochemical mode of action of these
compounds, and the results will be published soon.
E-2-[1-(Benzoxazol-2-yl-hydrazono)-ethyl]-pyridin-1-oxide (6).
A mixture of 2-acetylpyridine-N-oxide (0.76 g, 5.58 mmol) and
2-hydrazinobenzoxazole (2; 0.83 g, 5.58 mmol) in MeOH (25 mL)
containing 5 to 10 drops glacial acetic acid was heated at 60 °C.
The reaction was followed by TLC (CH2Cl2/EA 7:3), and a
voluminous light precipitate formed after 3 h. The mixture was
cooled overnight, and the precipitate was collected by filtration and
recrystallized from EtOH to give light yellow crystals (70%): mp
248-252 °C; IR νmax 3006, 1657, 1586, 1458, 1238, 849, 727 cm-1
;
1H NMR (δ ppm) 2.28 (s, 3H, CH3), 7.04-7.27 (m, 3H, BZO-
H5/6, pyri-H5), 7.40-7.50 (m, 3H, BZO-H4/7, pyri-H3), 7.57 (dd,
J ) 2.6 Hz, J ) 6.6 Hz, 1H, pyri-H4), 8.28 (dd, J ) 1.2 Hz, J )
5.8 Hz, 1H, pyri-H6). Anal. (C14H12N4O2) C, H, N.
In summary, one of the main goals of the present research,
which was to design and synthesize compounds that show high

Antitumor Studies of 2-Benzoxazolyl Hydrazones
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 21 6349
(2) Mauro, M. J.; O’Dwyer, M.; Heinrich, M. C.; Druker, B. J. STI571:
a paradigm of new agents for cancer therapeutics. J. Clin. Oncol.
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General Procedure for the Preparation of the Target Hy-
drazones 11a-h and 13a-k. A mixture of 3.35 mmol of the
appropriate carbonyl compound (10a-h and 12a-k) and 2-hy-
drazinobenzoxazole (2; 3.3 mmol) in methanol (25 mL) containing
5 to 10 drops glacial acetic acid was heated at 80 °C, and the
reaction was followed by TLC (CH2Cl2/EA 7:3). After completion,
the reaction mixture was placed in a fridge overnight. The
precipitates that separated out were filtered. Where there was no
precipitate formed, the reaction mixture was evaporated to dryness,
the residue was treated with DIPE, and the solid product that
separated out was filtered. The compounds were recrystallized from
the appropriate solvents.
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Biological Methods. (a) Cytotoxicity Assays. Burkitt’s lym-
phoma (CA 46, ATCC CRL 1648), CCRF-CEM (acute lympho-
blastic leukemia, ATCC CCL 119), HeLa (epitheloid cervix
carcinoma, ATCC CCL 2), multidrug resistant HeLa-mdr1 (d
HeLa cells transfected with a wild-type multi-drug resistance gene
1, mdr1 ) ABCB141), HL60, and HL60/AR cells (expressing MRP
) ABCC142) were grown in RPMI 1640 medium. To HL60/AR
cells was added 100 nM daunomycin and to HeLa-mdr1 cells was
added 100 nM vinblastine every other week. HT-29 (colon
adenocarcinoma, ATCC HTB 38) were grown in McCoy’s 5A
medium. MCF-7 (ATTC HTB-22) and MCF-10A (ATTC CRL-
10317) were grown in Eagle’s minimum essential medium, Earle’s
BSS adjusted to contain 1.5 g/L sodium bicarbonate, 0.1 mM
nonessential amino acids, 1 mM sodium pyruvate, and 0.01 mg/
mL insulin. The media were supplemented with 10% fetal calf
serum (except Burkitt’s lymphoma with 15% and HL60 and HL60/
AR with 20%), 2 mM glutamine, and 50 µg gentamycin/mL.
Detection of Cellular Proliferation. Inhibition of cell prolifera-
tion of HeLa, HeLa-mdr1, HT-29, KB-3-1, KB-C1, KB-HU, MCF-
7, and MCF-10A cells was detected by the SRB assay.43 Dose-
response curves for Burkitt’s lymphoma, CCRF-CEM, HL60, and
HL60/AR cells were detected by an MTT assay44 (Roche, Vienna,
Austria). Approximately 10 000 cells per well were seeded in 96-
well plates. After an initial incubation of 4 h, various drug
concentrations were added to the cells and exposed continuously
at 37° C in a humidified atmosphere of 95% air and 5% CO2 for
72 h. The drugs were dissolved in dimethyl sulfoxide (DMSO).
The concentration of DMSO was 0.5% and was also added to
controls and not toxic. Subsequently, the samples were processed,
and the absorption was detected by a microplate reader (Model
3550, Bio-Rad, Hercules, CA). Experiments with different con-
centrations of serum or addition of 0.1% R1-acid glycoprotein and
controls were counted with an electronic cell counter (Casy1,
Schaerfe System, Reutlingen, Germany).
NCI Growth Inhibitory Determination. Cell culture and drug
application procedures have been described previously.25 Briefly,
cell lines are inoculated into a series of 96-well microtiter plates,
with varied seeding densities, depending on the growth character-
istics of each cell line. Following a 24-h drug-free incubation, test
agents were added at five 10-fold dilutions, with a maximum
concentration of 100 µM. Cellular protein levels were determined
after 48 h of drug exposure by sulforhodamine B colorimetry.
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Acknowledgment. This work was supported by the Austrian
Science Fund (FWF), Grant Nos. P09879-MED and P12384-
MOB. Special thanks go to the developmental therapeutics
program (DTP) of the NCI for part of the test results discussed.
Supporting Information Available: Experimental details on
1
the preparation and H NMR data of 2-acylpyridines 10a-5 and
the novel hydrazones 11a-h and 13a-k and the microanalytical
data (C, H, N) for compounds 4-6, 11a-h, and 13a-k. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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