Design, synthesis, and anticancer properties of 4,4′ -dihydroxybenzophenone-2,4-dinitrophenylhydrazone and analogues

Article abstract of DOI:10.1021/jm0301080

4,4′-Dihydroxybenzophenone-2,4-dinitrophenylhydrazone (A-007) has recently completed a phase I clinical trial in advanced cancer with minimal toxicity, and impressive objective responses were noted. A-007 possesses three moieties that appear to have an influence on its anticancer activities: diphenylmethane, hydrazone, and dinitrophenyl. The goals of this study were to modify A-007's chemical moieties with the ultimate goal of maximizing its anticancer activity through increased planarity and introduction of functional groups. Thirty-five phenylhydrazone analogues of A-007 were synthesized and evaluated in vitro in a human primary cancer explant assay. Anticancer activities for selected analogues were also assayed for activity vs established human/murine cell lines. One-hundred-eighty-six fresh human solid tumors were used to screen for anticancer activity. Selected analogues were assayed for therapeutic indices (vs GM-CFC from bone marrow) in preparation for preclinical studies. Several polyaryl phenylhydrazones demonstrated improved cytotoxic activities by factors of 102-103 when compared with A-007. However, the polyaryl quinone moieties of the latter analogues introduced potential toxic properties (cardiac, hematological) that do not exist with A-007.

Full text of DOI:10.1021/jm0301080

4552
J . Med. Chem. 2003, 46, 4552-4563
Design , Syn th esis, a n d An tica n cer P r op er ties of
4,4′-Dih yd r oxyben zop h en on e-2,4-d in itr op h en ylh yd r a zon e a n d An a logu es
Lee Roy Morgan,*,† Kanappan Thangaraj,† Blaise LeBlanc,† Andrew Rodgers,† Lionel T. Wolford,†
Catherine L. Hooper,† Dominic Fan,‡ and Branko S. J ursic§
DEKK-TEC, Inc., 4200 Canal Street, Suite A, New Orleans, Louisiana 70119, Department of Cell Biology, MD Anderson
Cancer Hospital, Houston, Texas 77005, and Department of Chemistry, University of New Orleans, Lakefront, New Orleans,
Louisiana 70148
Received March 5, 2003
4,4′-Dihydroxybenzophenone-2,4-dinitrophenylhydrazone (A-007) has recently completed a
phase I clinical trial in advanced cancer with minimal toxicity, and impressive objective
responses were noted. A-007 possesses three moieties that appear to have an influence on its
anticancer activities: diphenylmethane, hydrazone, and dinitrophenyl. The goals of this study
were to modify A-007’s chemical moieties with the ultimate goal of maximizing its anticancer
activity through increased planarity and introduction of functional groups. Thirty-five
phenylhydrazone analogues of A-007 were synthesized and evaluated in vitro in a human
primary cancer explant assay. Anticancer activities for selected analogues were also assayed
for activity vs established human/murine cell lines. One-hundred-eighty-six fresh human solid
tumors were used to screen for anticancer activity. Selected analogues were assayed for
therapeutic indices (vs GM-CFC from bone marrow) in preparation for preclinical studies.
Several polyaryl phenylhydrazones demonstrated improved cytotoxic activities by factors of
102-103 when compared with A-007. However, the polyaryl quinone moieties of the latter
analogues introduced potential toxic properties (cardiac, hematological) that do not exist with
A-007.
Ch a r t 1. X-ray Crystallographic Characteristics of 13
In tr od u ction
4,4′-Dihydroxybenzophenone-2,4-dinitrophenylhydra-
zone (A-007, 1, Chart 1) has recently completed a phase
I clinical trial, and objective responses were seen in
advanced breast cancer, melanoma, and non-Hodgkin’s
lymphoma (NHL). 1 was applied topically to the sites
of cancer involvement as a 0.25% gel and was well
tolerated, without toxicity.1,2
X-ray crystallography data revealed that 1 exists as
monoclinic crystals (Chart 1).3 Furthermore, 1 exits as
two unique molecules, which differ only in the orienta-
tion within the diphenylmethane moiety, where the
rings are approximately perpendicular to each other
(and rotated approximately 90° from the orientation of
the rings in each rotamer). Both rotamers show strong
intramolecular hydrogen bonding between the -NH of
the -HN-NdC- moiety and an oxygen of the o-nitro
group.3 Thus, there are at least three unique moieties
present in 1 that may contribute to its overall biological
activity: a dihydroxydiphenylmethane, a hydrazone, and
a dinitrophenyl. An impressive characteristic of 1 is that
despite the significant electronegativity that exits within
the structure, it is a relatively stable molecule; e.g., 1
is excreted unchanged in the urine.4,5
Ch a r t 2. Comparative Structures for 1 and Tamoxifen
(under Physiological Conditions)
ize in vivo to an estrogen, as does tamoxifen.6 The
skeletal backbones for 1 and tamoxifen are similar
(Chart 2); thus, it was anticipated that the former might
have similar binding properties to the latter with the
antiestrogen receptor.7 Both the N+-O- group of 1 and
the N+-H group of tamoxifen form strong hydrogen
bonds with complement groups. The latter can form a
pseudoring, similar to the third ring of 1 (Chart 2). The
overall similarities are highlighted for comparison. The
distances between the two electrostatically rich comple-
Ra tion a le
1 was initially designed as an antiestrogen that was
similar isosterically to tamoxifen but unable to isomer-
* To whom correspondence should be addressed. Telephone: (504)
488-5415. Fax: (504)-488-4451. E-mail: lrm1579@aol.com.
† DEKK-TEC, Inc.
‡ MD Anderson Cancer Hospital.
§ University of New Orleans.
10.1021/jm0301080 CCC: $25.00 © 2003 American Chemical Society
Published on Web 09/06/2003

Dinitrophenylhydrazones and Analogues
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21 4553
mentary centers (aryl vs electrophilic) are the same in
both structures. Unfortunately, 1’s antiestrogen activity
was minimal and it did not bind effectively to the
antiestrogen receptor.8 1 inhibits thioredoxin reductase
and blocks [14C]cytidine uptake/incorporation.9 The
exact mechanism(s) for 1’s anticancer activity is still
unknown.10 However, the recent finding that 1 is able
to induce cell membrane modulations through interac-
tions with surface tumor markers and clusters of
differentiations (CD) expands its therapeutic potential
horizons.11
Easmon et al. have reported on 1’s wide spectrum of
activities, including activity vs Burkitt’s lymphoma,
MCF-7 breast cancer, and HeLa cells.9 Easmon’s group
noted improved activity for 1 analogues when both
electron-withdrawing and donating groups (amino vs
methoxy) were substituted into the meta-5-position of
1’s dinitrophenyl ring. A chlorine inserted in the 5-posi-
tion (ortho and para to the 2- and 4-nitro groups,
respectively) of the phenylhydrazone moiety improved
activity by ∼800-fold vs 1.9 The Easmon group has
further demonstrated that replacement of one or both
of the diphenyl rings with a N-heteroaryl moiety (i.e.,
pyridyl) did not improve activity. Acute lymphoblastic
leukemia, Burkitt’s lymphoma, colon, cervical, and
breast adenocarcinomas and melanoma cell lines were
used by the Easmon group to document activities.9
In the present paper, three of the potential pharma-
cophores that are present in 1 have been modified in
an attempt to optimize and understand the basis for 1’s
activity.
fications of ref 13). Analogue 36 was synthesized by
using a similar scheme as described for 35. The last two
heterocyclic analogues are polyaryl hydrazones possess-
ing 1’s diphenyl skeletal moiety.
Biologica l E va lu a t ion s. All of the described new
phenylhydrazone analogues were tested in vitro for
cytotoxicity vs a battery of human cancers removed at
surgery and grown as primary explants in culture.
Selected analogues were also tested vs various estab-
lished human tumor lines, and therapeutic indices were
calculated vs normal FDCP-1 murine GM-CFC cells.
The human primary cancers used were obtained at the
time of surgery and placed fresh into culture. The
CYTOTEC assay, developed by DEKK-TEC, was used
to measure the anticancer activities.14 With use of
DEKK-TEC’S CYTOTEC monolayer tissue culture as-
say, 186 fresh human cancer tissues were grown as
explants in tissue culture and analogues were evaluated
for in vitro activities using a MTT (3-(4,5-dimethylthi-
azol-2-yl)-2,5-diphenyltetrazolium bromide (thiazolyl
blue)) assay to determine IC50 values.15-18 All human
specimens were collected in accordance with an ap-
proved Human Investigational Review Committee pro-
tocol. A total of 186 human cancers were used to obtain
the data reviewed in this paper. Doxorubicin (DOX) and
cisplatinum (cis-DDP) were selected as positive controls,
with concentrations in the 0.1-10 µg/mL range. For IC50
values greater than 5 µg/mL, SD values are not in-
cluded.
Fresh human tumor explants were selected as a
screening system because of the cellular heterogeneity,
i.e., lymphocytes, stromal components, cancer cells, etc.
present in primary cancer tissue, compared to estab-
lished xenograft cell lines.8,14 In addition, visualization
of each CYTOTEC plate allows for individual apprecia-
tion of the effects/impact that the analogues may have
had on the entire ecology of the malignant explants, i.e.,
cancer cells, lymphocytes, fibroblasts, endothelial cells,
etc.8,14
Ch em istr y
Chemical modifications of 1’s moieties are shown in
structures 2-36, which include (a) changes to the
benzophenone moiety (analogues 2-17), (b) coplanar
transformations for the diphenyl rings (analogues 18-
36), and (c) changes to the hydrazine moiety (analogues
23, 35, and 36) (Chart 3). The hydrazones were prepared
via standard procedures, as well as with a new cationic
resin column method. The latter procedure allows purer
hydrazones to be generated with minimal steps.
The influence that functional group substitutions
have on 1’s intra-/intermolecular hydrogen bonding and
electrostatic interactions was studied through the in-
troduction of hydroxyl, amine, methyl, and chlorine
groups (analogues 2-17). The two phenyl rings in 1’s
diphenylmethane moiety are almost perpendicular to
each other, providing a reason to fuse the two rings,
introduce coplanarity, and document the respective
influences. Since polyaryl structures can influence bind-
ing to DNA, anthraquinone, xanthone, and thioxan-
thone analogues were introduced in place of the diphe-
nylmethane moiety (analogues 18-34). Coplanarity was
introduced into 1 by fusing the dN-NH- group to the
benzophenone ring system, resulting in anthrapyridazi-
nones 35 and 36.12 Analogue 33 is an anthrapyrazole
dinitrophenylhydrazone that further challenges the
influences of planarity in the system.
Resu lts a n d Discu ssion
In this paper, the synthesis, characterization, and
evaluation of 36 substituted hydrazones for anticancer
activity in a human cancer explant assay are described.
A total of 186 fresh primary human cancer tissues were
grown as explants, and the analogues were evaluated
for in vitro activities. Selected analogues were assayed
for comparative therapeutic indices vs normal bone
marrow and a battery of human cancer cell lines.
The major objective of this study was to seek correla-
tions between the electronic, planar, hydrophobic, and
steric properties vs anticancer activities for substituted
aryl phenylhydrazones. Early studies with the simple
nonaryl hydrazones demonstrated minimal antiestro-
genic activity.8 However, the introduction of the phe-
nylhydrazone group significantly improved anticancer
activity, with 1 emerging as a novel agent possessing
novel potential for development.10,11 To appreciate struc-
tural reasons for 1’s cytotoxic activity, a wide range of
substitutions/modifications has been made to both the
diphenylmethane (benzophenone) and the phenylhy-
drazone moieties. The data that have been generated
may serve to develop future structure-activity relation-
ships and may suggest directions for the development
of novel anticancer agents with minimal toxicity.
Analogue 35 was synthesized by first converting 1,8-
dichloroanthraquinone into 8-hydroxyanthraquinone-1-
carboxylic acid.11 The latter was then treated with
2-nitrophenylhydrazine followed by thionyl chloride to
obtain the respective anthrapyridazinone 35 (via modi-

4554 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21
Ch a r t 3. Structural Analogues of 1
Morgan et al.
Table 1 represents results from the testing of 1 vs 16
simple biosteric substituted analogues. Functional sub-
stitutions that could influence intra-/intermolecular
hydrogen bonding and electrostatic interactions were
introduced into the basic structure of 1 (Table 1).
Analogue 13 is an acetophenone phenylhydrazone base
structure (in which the diphenylmethane moiety of 1
has been converted to a phenylethane structure in which
a phenyl has been replaced with a methyl group).
However, the other 15 analogues in Table 1 represent
substitutions into the benzophenone phenylhydrazone
ring system of 1. These analogues were designed to
influence electronic and intramolecular hydrogen bond-
ing, i.e., analogues 8 and 11. Analogue 8 is of interest
because it possesses a hydroxyl group juxtapositioned
such that hydrogen bonding occurs with the -NH of the
hydrazone moiety (Chart 4). In contrast, analogue 11
displays intramolecular hydrogen bonding between a
2-nitro group and the -NdNH moiety.
X-ray crystallography supports the structure shown
in Chart 4 with two sets of strong hydrogen bonds in
analogue 8. Analogue 8 has a composite of intramolecu-
lar hydrogen bondings that exist separately in 5 and 7,
i.e., between an o-hydroxyl and the dN-NH-, as well
as between the -NH and 2-NO2 groups.3 Analogue 5 is
a 2,2′-isostere of 1 and possessed anticancer activities
similar to, but with no improvement over, those of 1
(Chart 3). The 5-Cl-substituted analogue (ANG-36) of
Easman et al. was not available for testing at the time
that these studies were conducted.9 Analogues 14-17
contain isosteric substitutions of amines for hydroxyls
in 1. The 4,4′-diamino analogue 14 had improved
activity (∼1 log) vs 1; N-substitutions reduced activity
(analogues 15-17). The activities for the other ana-

Dinitrophenylhydrazones and Analogues
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21 4555
diphenyl rings through an -O- atom, with less planar-
ity (coplanar) than exists in analogue 18. The activities
for 18 and 20 were <0.1 µg/mL, and the IC50 values are
projected values (no SD presented). Blocking the -NH
group in analogue 24 destroyed anticancer activity.
Analogue 20 is a xanthone phenylhydrazone with
significant anticancer activity in at least three tumor
types. These data have been verified, and analogue 20
is being evaluated for anticancer activity in a mouse
model.
Table 3 reviews the biological activities for polyaryl
analogues 25-34 (all possess the skeletal backbone of
1 plus fused/attached diphenyl rings) (Chart 4). The
improved biological properties of the anthraquinone
analogues 25-29 are not surprising. This supports the
concept that the introduction of coplanarity not only
improves 1’s ability to penetrate cell membranes but
also possibly allows the arylhydrazone system to inter-
calate with DNA and other biopolymers.20 Unfortu-
nately, the presence of polycyclic quinone rings would
be expected to introduce new toxic properties (cardiac
and myelosuppression), which 1 does not demon-
strate.5,10 Analogue 33 is an anthrapyrazole derivative
and has no improved activities over other analogues
tested. Analogue 34 is a four-ring polyaromatic system,
which also had some improved activity in two tumor
types. However, the potential toxicities of this ring
system limit the appeal for extended studies.
Table 4 describes activities for two analogues, a
phenylanthracen-1,9-pyridazinone, 35, and a phenylflu-
oren-1,9-pyridazinone, 36. Both analogues were de-
signed and tested because they contained a more rigid
configuration of 1’s basic skeleton. Activity observed
with both analogues was poor. Since the pyridazinone
ring would not be expected to survive more than one
pass through the liver during in vivo testing, interest
in this ring system has been minimal.
F igu r e 1. Inhibition of FDCP-1 and MDA-435 by doxorubin
(adriamycin) and 1.
logues in Table 1 were not significantly different from
the activities of 1, with the possible exception of
analogue 8, which is in preclinical studies to firmly
document anticancer activity. Substitutions on the
phenylhydrazone ring had significant influences on
activity; i.e., absence of or replacement of nitro groups
with methyl groups further reduced activity. Isosteric/
functional substitution in any of the diphenylmethane
rings with 2- and/or 4-chloro, methoxy, methyl, or
acetoxy groups reduced the anticancer activity of 1.
Analogue 14 is a diamine isostere of 1. Analogue 3 is a
simple derivative of 1 that possesses a single 4-hydroxyl
and has improved anticancer activities toward ovarian
cancer cells. Both analogues 3 and 14 deserve more
attention.19
Tables 2 and 3 describe activity for simple polyaryl
analogues 18-24 and 25-34, in which intramolecular
flexibility and hydrogen bonding (which exist in 1’s
diphenylmethane moiety) have been eliminated. In 1,
the last two phenyl groups exist almost perpendicular
to each other (Chart 1, ref 3). The analogues in Table 2
are modified diphenyl ring derivatives of 1, creating a
planar configuration. The fluorene and xanthene ana-
logues 18 and 20, respectively, have significantly im-
proved activity when compared to 1 and the other
simple benzophenone analogues of Table 1. Analogue
18 is a dihydroxyfluorene derivative with fused diphenyl
rings, while analogue 20 possesses fixation of the
Myelotoxicity and therapeutic indices (TI) for 1 vs
doxorubicin (adriamycin) and three analogues 22, 26,
and 27 are available using FDCP-1 murine GM-CFC
cells vs eight tumor cell lines (Table 5 and Figure 1).
The numbers are small (n ) 1-3); however, the data
indicate that 26 and 27 were more toxic than 1 and the
results of the in vitro TI myelotoxicity studies were poor.
This is in contrast to 1, which has minimal toxicity
(Table 5 and Figure 1). In clinical trials, 1 also had
minimal toxicity.1,2 Analogues 26 and 27 demonstrated
excellent responses vs KM12 SM (colon carcinoma) and
SN12 PM6 (renal cell carcinoma), respectively. Analogue
22, on the other hand, demonstrated an excellent TI
against MDA-MB-435 and B16 F10 cell lines. Favorable
hematological responses in vivo would be predicted for
22, compared to 1. Of interest is that preliminary in vivo
studies for 1,8-diacetoxyanthraquinone-10-2,4-DNP, 27,
when administered intraperitoneally (10 mg/(kg‚day))
on days 1 and 5 to C57 mice bearing measurable B-16
F10 melanomas, have been encouraging.8 Responses
noted for analogue 27 vs B-16 mouse melanoma were
75% TGI (tumor growth inhibition) and 175% ILS
(increased life span) vs 35% and 75% for 1, respectively.8
This analogue should also be evaluated in vivo vs
SN12PM6 tumor to document therapeutic indices.19
Table 6 correlates IC50 values for 1 vs cis-DDP and
DOX for melanoma, breast, colon, lung, and ovarian
cancers. The values obtained are in agreement with

4556 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21
Morgan et al.
Ta ble 1. Determination of in Vitro Cytotoxicity of Substituted Benzophenone Phenylhydrazones vs Various Human Tumors
averaage IC50(SD), µg/mL
compd
R1
4-HOPh
Ph
Ph
2,4-(HO)2Ph
2-HOPh
4-HOPh
2-HO-4-MeOPh
Ph
4-AcOPh
4-HOPh
4-HOPh
4-HOPh
Me
R2
HO
R3
X
Y
breast
ovary
colon
lung
melanoma
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
H
H
H
HO
HO
H
HO
HO
H
H
H
H
H
H
H
H
H
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
Me
NO2
H
H
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
Me
c
c
c
c
c
H
>5
5(4)
>10
4.7(3)
8.7(3)
1.5(0.8)
0.5(4)
7b
>10
0.3(1)
0.03b
5b
NAa
0.5(2)
>5
>5
>10
>10
NAa
>5
HO
HO
H
NAa
>5
8(4)
>10
4.5(3)
NAa
>10
NAa
4.7(1)
>5
6(1)
NAa
NAa
NAa
>10
NAa
NAa
>10
NAa
NAa
NAa
NAa
NA
Cl
NAa
>5
NAa
NAa
NAa
>10
NAa
NAa
7b
MeO
HO
AcO
HO
HO
HO
HO
NH2
Me2N
Et2N
AcNH
NAa
>10
NAa
NAa
6.2
10(2)
3.9(3)
>5
H
H
NO2
NO2
NO2
NO2
NO2
5(2)
6b
NAa
8(5)
10
>10
>10
NAa
6b
2-NH2Ph
4-Me2NPh
4-Et2NPh
4-AcNHPh
0.7(2)
4.2(4)
>7
2(2)
NAa
NAa
>10
5b
5b
>10
>10
a
b
NA, not available. One to two samples only. c See Table 6.
Ch a r t 4. Two Possible Intermolecular Hydrogen Bonds
for Analogue 8
probably inducing cell death by a mechanism different
from that for the simple benzophenone analogues. 25-
28 produced death within hours of cell contact; the
analogues in Table 1 seemed to produce their changes
over days.
Cytotoxicity was measured by the microculture CY-
TOTEC assay, as previously described.14 DOX demon-
strated an IC50 from 0.3 to 0.75 µg/mL for breast cancer
and from <0.1 to 0.4 µg/mL for ovarian cancers. cis-DDP
had IC50 of >10 µg/mL for melanoma, colon, lung, and
ovary cancers. The histology of the cancers were as
follows: breast, infiltrating ductal adenocarcinoma (ER/
PgR +/+, -/+, and -/-); ovarian, adenocarcinoma; lung,
non-small-cell carcinoma; colon/rectal, adenocarcinoma;
melanoma, pigmented and nonpigmented. Each value
in Tables 1-4 represents 5-20 individual specimens
from different patients. Breast cancer tissue was the
most sensitive of the human tumor explants tested and
was the most plentiful for assay (Table 6). The presence
or absence of the ER/PgR (estrogen/progesterone recep-
tors) network may have had an influence, at least for
the activity of 1. When the clinical courses for the
patients with breast cancer are reviewed, those patients
whose tumor tissues were sensitive to 1 at <3 µg/mL
also demonstrated the highest values for their tumor
tissue ER+/PgR+.14,22
previously reported sensitivities for many of the can-
cers.20 Drugs such gemcitabine, etc. were not available
as standards when the project was initiated over 15
years ago; only DOX and cis-DDP were used as refer-
ences.
Although 1 appears to be a simple molecule, it
contains at least three active moieties: a dinitrophenyl,
a hydrazone, and a diphenylmethane. At least one
hydroxyl or an amino group appears to be necessary in
the last moiety for cytotoxic activity. The 2-nitro group
may also influence stability.4,5,21 A free NH- group (in
the hydrazone moiety) is needed for activity.4,8,10 Fusion
of the diphenyl rings produced coplanar systems that
improved anticancer activities significantly in breast
cancer [analogues 20, 25, and 27 vs 1]. Fusion of the
dN-NH- moiety in 1 into an anthrapyrazole ring
system (analogue 33) did not improve activity. A single
2-nitro group on the phenylhydrazone ring slightly
improved activity (analogue 11). 2-Hydroxyl or 2,2′-
dihydroxyl groups on 1’s diphenylmethane moiety main-
tained the activity of 1. Other isosteric/functional group
substitutions (Cl, F, CH3, OCH3, COCH3) in the aryl
rings (of the diphenylmethane moiety) reduced activity
to >5 µg/mL. Blocking the -NH, as in analogue 24 (N-
benzylxanathone-2,4-DNP), also reduced activity (IC50
> 5 µg/mL).4
Selected analogues from this series are currently
being evaluated in vivo in human xenograft tumor
models.19
Con clu sion
In summary, we have described modifications to three
functional moieties present in 1 that could influence
biological activity. Analogues of 1 were prepared to
introduce rigidity in the diphenylmethane ring system,
as well as to influence electrostatic properties. Com-
pounds that have evolved from this study demonstrated
that fusing the diphenyl ring system into polyaryl
Since the polyaryl quinines in Tables 2 and 3 possess
potential cardiotoxic moieties, the specific IC50 values
were not determined. Analogues 18, 20, and 25-28 are

Dinitrophenylhydrazones and Analogues
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21 4557
Ta ble 2. Determination of in Vitro Cytotoxicity of Aryl-2,4-dinitrophenylhydrazones vs Various Human Tumors
averaage IC50(SD), µg/mL
compd
R1
R2
R3
R4
R5
X
Z
breast
ovary
colon
lung
melanoma
18
19
20
21
22
23
24
H
H
AcO
H
MeO
H
HO
H
H
H
H
H
HO
H
H
H
HO
H
AcO
H
H
H
H
H
H
H
MeO
H
H
a
a
a
O
O
O
SO
H
H
H
H
0.05b
7.5(10)
>5
0.08b
0.06b
>5
0.7(4)
0.05b
NAc
NAc
>10
>10
NAc
>5
>10
NAc
NAc
>10
4.1
NAc
NAc
0.5
0.9(2)
0.05b
NAc
>10
>10
0.04b
>5
>10
NAc
>10
>10
H
H
H
H
H
Bzd
H
>10
>10
H
H
a
b
d
Is a straight line between the rings, i.e., fluorene ring. Projected value: activities are <0.1 µg/mL.20,23 c NA, not available. Bz )
C6H5CH2.
Ta ble 3. Determination of in Vitro Cytotoxicity of Polyaryl-2,4-dinitrophenylhydrazones vs Various Human Tumors
averaage IC50(SD), µg/mL
compd
R1
R2
HO
H
H
H
H
HO
NH2
NH2
H
R3
R4
R5
X
breast
ovary
colon
lung
melanoma
25
26
27
28
29
30
31
32
33d
34e
HO
HO
AcO
AcO
AcO
H
NH2
H
NH
HO
H
H
H
H
H
H
HO
H
NH2
H
H
H
O
O
O
O
O
0.007f
0.03f
0.04f
0.05f
0.1f
>10
0.25(2)
0.03f
0.16
0.03f
0.08
3.9(5)
5(4)
>10
0.12f
0.1(3)
NAa
0.07
1(2)
NAa
NAa
NAa
0.1f
>5
H
H
H
H
H
H
H
H
HO
AcO
HO
allylOb
H
NAa
NAa
0.07f
0.097
NAa
NAa
NAa
NAa
NAa
NAa
0.3(0.1)
0.4(0.3)
NAa
DNPc
O
2.5(2)
5(1)
NAa
6(7)
H
H
H
H
DNPc
N
>5
>5
>5
>5
>5
>5
benzene ring
O
0.3(1)
NAa
NAa
a
b
d
NA, not available. allylO ) allyloxy (CH2dCH-CH2O). c DNP ) 2,4-dinitrophenylhydrazone moiety [2,4-(NO2)2C6H4NHN]. For
33, R1 and X are connected by a single bond (R1-X ) NH-N). e For 34, R2 and R3 are connected through CHdCH-CHdCH for an
additional benzene ring. f Projected value: activities are <0.1 µg/mL.20,23
Ta ble 4. Determination of in Vitro Cytotoxicity of 35 and 36
vs Various Human Tumors
Ta ble 5. Determination of in Vitro Cytotoxicity of Analogues
1, 22, 26, and 27 vs Various Tumor Cell Lines and Therapeutic
Indices (vs FDCP-1 Murine GM-CFC Cells)
averaage IC50(SD), µg/mL
a
average IC50 (TI vs FDCP-1) (SD), µg/mL
compd
breast ovary colon lung melanoma
cell lines
FDCP-1
1
22
26
27
>5
>5
>10
NAa
NAa
NAa
9.1(1.0) 0.95(1.00) 0.020(1.0) 0.045(1.00)
2.1(3.5) 0.41(2.3)
MDA-MB-435
0.020(1.0) 0.041(1.09)
0.03(0.6)
MDA-MB-435/ NAb
lung met.
NAb
NAb
>5
>5
>5
0.4(1)
KM12 C
NAb
NAb
0.03(0.6)
0.05(0.4)
NAb
KM12 SM
KM12 L4
B16 F10
K1735 M2
SN12 PM6
5.4(1.9) NAb
NAb
NAb
1.57(0.61) 0.02(0.8)
1.0(9.1) 0.44(2.16) NAb
NAb
0.64(1.48) 0.02(1.2)
0.070(0.64)
0.042(1.07)
0.042(1.07)
0.021(2.14)
a
NA, not available.
4.0(1.9) 0.62(1.53) 0.03(0.6)
a
b
IC50 values are derived from MTT assays. NA, not available.
coplanar rings not only improved anticancer activity but
also introduced potential problems from cardiac and
bone marrow toxicities. Substitutions of electron-
withdrawing groups into the dinitrophenyl ring system
influenced activity. 1 and some of the simple analogues
have intramolecular hydrogen bonding between the
dinitrophenyl ring system and the N-NHd of the

4558 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21
Morgan et al.
1139 cm-1
.
1H NMR (DMSO-d6, 300 MHz): δ 11.13 (1H, s,
Ta ble 6. Comparative IC50 for Primary Human Cancer
Culture Sensitivities: 1 vs Standards
NH), 10.05 (1H, broad singlet, OH), 9.99 (1H, broad singlet,
OH), 8.78 (1H, d, J ) 2.7 Hz, 3-H of 2,4-dinitrophenyl ring),
8.36 (1H, dd, J 1 ) 9.6 Hz, J 2 ) 2.7 Hz, 5-H of 2,4-dinitrophenyl
ring), 8.12 (1H, d, J ) 9.6 Hz, 6-H of 2,4-dinitrophenyl ring),
7.47 (2H, d, J ) 8.7 Hz, o-H of benzophenone ring), 7.22 (2H,
d, J ) 8.4 Hz, o-H of benzophenone ring), 7.01 (2H, d, J ) 8.1
Hz, m-H of benzophenone ring), and 6.81 (2H, d, J ) 8.4 Hz,
m-H of benzophenone ring) ppm. 13C NMR (DMSO-d6, 300
MHz): δ 156.19, 155.31, 152.33, 140.47, 133.27, 126.50, 126.35,
126.02, 125.50, 124.07, 119.46, 118.19, 112.97, 112.90, and
111.91 ppm (15 signals for 15 different carbon atoms in 1).
Anal. Calcd for C19H14N4O6: C, 57.87; H, 3.58; N, 14.21.
Found: C, 57.42; H, 3.81; N, 13.88.
averaage IC50(SD), µg/mL
tissue
N
1
cis-DDP
DOX
breast cancer (ER+/PgR+) 28 3.5(2.7)
2.5(0.9) 0.5(1)
>5
3(2)
breast cancer (ER+/PgR-)
breast cancer (ER-/PgR+)
1
1
>10
>10
0.5
0.4(0.1)
0.2(0.3)
>12(5)
4.2(4)
6(5)
breast cancer (ER-/PgR-) 23 12.6(5.5) 5(2)
colon cancer
ovarian cancer
lung cancer
melanoma
11 15.2(6.1) >12(4)
12 12.3(4.5) 3(3)
10 4.9(4)
32 7(4.1)
5(5)
5(4)
>10
hydrazone group, as well as between the hydrazone
moiety and the hydroxyls of the diphenylmethane ring,
all of which had an impact on biological activity. Fresh
human cancer tissue was selected as a test system
because of the natural heterogeneity that exists in a
tumor nodule or mass, i.e., cancer cells, lymphocytes,
fibroblasts, endothelial cells, and natural extracellular
connective tissue matrices.
N-Ben zh ydr yliden e-N′-(2,4-din itr oph en yl)h ydr azin e (2)
was prepared by method A from benzophenone and 2,4-
dinitrophenylhydrazine in 82% yield and purified by crystal-
lization from EtOH or MeOH, mp 232-234 °C. IR: 3295 (N-
H), 2928 (Ar-H), 1612 (CdN), 1590, 1504, 1416, 1334, 1127,
1091, 834, 777, 698, and 608 cm-1 1H NMR (DMSO, 400
.
MHz): δ 11.25 (1 H, s, N-H), 9.07 (1 H, s), 8.37 (1 H, d, J )
8 Hz), 8.22 (1 H, d, J ) 8 Hz), 7.67 (4 H, m, J ) 8 Hz), 7.41 (6
H, m, J ) 8 Hz). Anal. Calcd for C19H14N4O4: C, 62.98; H,
3.86; N, 15.46. Found: C, 63.17; H, 4.06; N, 15.49.
Exp er im en ta l Section
4-H yd r oxyb e n zop h e n on e -2,4-d in it r op h e n ylh yd r a -
zon e (3) was prepared by method B from 4-hydroxybenzophe-
none and 2,4-dihydroxyphenylhydrazine as red needles that
crystallized from EtOH in 80% yield, mp 224-225 °C. IR: 3541
(N-H), 3271 (N-H), 3098 (O-H), 2905 (Ar-H), 1621 (CdN),
Melting points were performed on a Mel-Temp II electro-
thermal melting point apparatus and are reported as uncor-
rected. The infrared spectra were recorded on a Perkin-Elmer
PE 2000 FT-IR spectrometer as Nujol mulls. The UV spectra
were recorded on a Cary 550 Scan UV-vis-NIR spectropho-
tometer in methanol solvent from 800 to 200 nm. The 1H NMR
spectra were recorded on a Varian Unity NMR spectrometer
at 400 MHz or Gemini2000 spectrometer at 300 MHz in
dimethyl sulfoxide (DMSO-d6) solvent or CDCl3 solvent as
indicated. The 13C NMR spectra were recorded at 125 MHz.
As indicated, Me4Si was used as a reference. The mass spectra
1591, 1511, 1418, 1329, 1276, 1130, 1083, 829, and 698 cm-1
.
1H NMR (DMSO, 400 MHz): δ 11.23 (1/2 H, s, N-H), 10.99
(1/2 H, s, N-H), 10.1 (1 H, s, “broad” OH), 8.8 (1 H, s), 8.41 (1
H, d, J ) 8 Hz), 8.19 (1 H, d, J ) 8 Hz), 7.67 (2 H, m, J ) 8
Hz), 7.48 (3 H, m, J ) 8 Hz), 7.28 (1 H, dd, J ) 8 Hz), 7.04 (1
H, dd, J ) 8 Hz), 6.84 (1 H, dd, J ) 8 Hz). Anal. Calcd for
C
19H14N4O5: C, 59.07; H, 3.62. Found: C, 59.71; H, 3.84.
2,2′,4,4′-Tetr a h yd r oxyben zop h en on e-2, 4-d in itr op h e-
(ESI) were obtained from
a Micromass Quatro II triple
n ylh yd r a zon e (4) was prepared by method A from 2,2′,4,4′-
tetrahydroxybenzophenone and 2,4-dinitrophenylhydrazine as
a deep-red crystalline product that crystallized from EtOH in
75% yield, mp 290-291 °C. IR: 3631 (N-H), 3491 (N-H), 3272
(O-H), 2905 (Ar-H), 1614 (CdN), 1518, 1415, 1322, 1206,
quadrapole mass spectrometer with absolute methanol as
solvent. Midwest Micro Lab, Indianapolis, IN, performed the
elemental analyses. The chemicals and tissue culture supplies
were purchased from Aldrich-Sigma-Fluka Chemical Co., St.
Louis, MO.
1. Ch em istr y. The majority of hydrazones were prepared
from corresponding carbonyl compounds (substituted ben-
zophenones, acetophenones, anthraquinones, fluorenones, etc.)
and substituted phenylhydrazones by one of two synthetic
procedures (method A and method B).
Meth od A. MeOH suspensions (300 mL) of substituted
phenylhenylhydrazines (0.148 mol) and concentrated sulfuric
acid (20 mL) were stirred at 50 °C. After the hydrazine
dissolved, a MeOH solution (300 mL) of the corresponding
carbonyl compound (0.1 mol) was added to the hydrazine and
the resulting reaction mixture was stirred at 50 °C for
additional 30 min. The reaction mixture was concentrated to
1/4 of its original volume under vacuum and diluted with water
(500 mL). The precipitates were separated by filtration and
washed with 3% aqueous NaHCO3 (3 × 100 mL) and water (3
× 50 mL). Products were recrystallized from MeOH, EtOH,
or glacial AcOH.
Meth od B. MeOH suspensions (15 mL) of the corresponding
phenylhydrazine (2.7 mmol), ketones (2.1 mmol), and Dowex
50W-50X2-100 cation-exchange resin (0.5 g) [Dow Corp., Baton
Rouge, LA] were refluxed for 1 h. The hot reaction mixture
was separated by filtration and washed with methanol (3 ×
0.5 mL). The filtrate was cooled to room temperature and
diluted with water (20 mL). Resulting precipitates were
filtered, washed with water (3 × 1 mL), and recrystallized from
MeOH, EtOH, or glacial AcOH.
1143, 1107, 974, 864, and 827 cm-1 1H NMR (DMSO, 400
.
MHz): δ 11.59 (1 H, s, O-H), 11.15 (1 H, s, N-H), 10.12 (2 H,
s, O-H), 9.91 (1 H, s, O-H), 8.85 (1 H, s), 8.48 (1 H, d, J ) 8
Hz), 8.19 (1 H, d, J ) 8 Hz), 7.61 (1 H, d, J ) 8 Hz), 6.95 (1 H,
dd, J ) 8 Hz), 6.8 (1 H, dd, J ) 8 Hz), 6.57 (1 H, s), 6.46 (1 H,
dd, J ) 8 Hz), 6.38 (1 H, s), 6.31 (1 H, dd, J ) 8 Hz). Anal.
Calcd for C19H14N4O8: C, 53.52; H, 3.28; N, 13.14. Found: C,
51.79; H, 3.19; N, 13.36.
2,2′-Dih yd r oxyben zop h en on e-2,4-d in itr op h en ylh yd r a -
zon e (5) was prepared by method B from 2,2′-dihydroxyben-
zophenone and 2,4-dinitrophenylhydrazine as deep-red crystals
in 85% yield. The product recrystallized from EtOH, mp 270-
271 °C. IR: 3454 (N-H), 3279 (O-H), 2921 (Ar-H), 1618
(CdN), 1587, 1415, 1330, 1147, 1096, 750, 587, 441, and 415
cm-1 1H NMR (DMSO, 400 MHz): δ 11.13 (1/2 H, s, O-H),
.
10.97 (1/2 H, s, N-H), 10.34 (1 H, s, “broad” O-H), 8.85 (1 H,
s), 8.51 (1 H, d, J ) 8 Hz), 7.79 (1 H, d, J ) 8 Hz), 7.49 (1 H,
t, J ) 8 Hz), 7.35 (1 H, t, J ) 8 Hz), 7.2 (1 H, dd, J ) 8 Hz),
7.15 (1 H, dd, J ) 8 Hz), 7.15 (1 H, dd, J ) 8 Hz), 7.03 (1 H,
m, J ) 8 Hz), 6.87 (1 H, t, J ) 8 Hz). Anal. Calcd for
C
19H14N4O6: C, 57.86; H, 3.55; N, 14.21. Found: C, 57.86; H,
3.54; N, 14.34.
4-Ch lor o-4′-h yd r oxyben zop h en on e-2,4-d in itr op h en yl-
h yd r a zon e (6) was prepared by method B from 4-chloro-4′-
hydroxybenzophenone and 2,4-dinitrophenylhydrazine as deep-
red crystals (87% yield) that crystallized from EtOH, mp 195-
197 °C. IR: 3567 (N-H), 3453 (N-H), 3290 (O-H), 3111
(O-H), 1621 (CdN), 1585, 1500, 1419, 1337, 1306, 1264, 1219,
1134, and 840 cm-1. 1H NMR (DMSO, 400 MHz): δ 11.21 (1/2
H, s, N-H), 10.94 (1/2 H, s, N-H), 10.13 (1 H, d, “broad” O-H),
8.81 (1 H, d, J ) 8 Hz), 8.41 (1 H, t, J ) 8 Hz), 8.18 (1 H, t, J
) 8 Hz), 7.76 (1 H, d, J ) 8 Hz), 7.65 (1 H, d, J ) 8 Hz),
N-(4,4′-Dih ydr oxyben zh ydr yliden e)-N′-(2,4-din itr oph e-
n yl)h yd r a zin e (1) was prepared by method A in 78% yield
from 4,4′-dihydroxybenzophenone and 2,4-dinitrophenylhy-
drazine as deep-red crystals. The product was recrystallized
from MeOH, mp 270-272 °C. IR: 3506 (N-H), 3288 (N-H),
2921 (Ar-H), 1614 (CdN), 1592, 1503, 1417, 1336, 1311, and

Dinitrophenylhydrazones and Analogues
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21 4559
7.5 (4 H, m, J ) 8 Hz), 7.28 (1 H, dd, J ) 8 Hz), 7.05 (1 H, dd,
J ) 8 Hz), 6.84 (1 H, dd, J ) 8 Hz).
4,4′-Dih ydr oxyben zoph en on eph en ylh ydr azon e (12) was
prepared by method A from 4,4′-dihydroxybenzophenone and
phenylhydrazine‚HCl by substituting glacial AcOH for H2SO4.
The yield was 65%, and the product was crystallized from
2,2′-Dih yd r oxy-4,4′-d im eth oxyben zop h en on e-2,4-d in i-
tr op h en ylh yd r a zon e (7) was prepared by method B from
2,2′-dihydroxy-4,4′-dimethoxybenzophenone and 2,4-dinitro-
phenylhydrazine (in 90% yield) as red crystals that crystallized
from EtOH, mp 218-220 °C. IR: 3444 (N-H), 3269 (N-H),
3290 (O-H), 2981 (Ar-H), 1615 (CdN), 1521, 1335, 1209,
1
glacial AcOH as deep-red crystals, mp 185-187 °C. H NMR
(DMSO-d6): δ 9.762 (1H, broad singlet, one of the OH groups),
9.566 (1H, broad singlet, other of two OH groups), 8.440 (1H,
s, NH), 7.299 (2H, d, J ) 8.7 Hz, 2H), 7.159 (4H, d, J ) 4.2
Hz), 7.097 (2H, d, J ) 8.4 Hz), 6.950 (2H, d, J ) 9 Hz), 6.731
(2H, d, J ) 9 Hz), and 6.683 (1H, t, J ) 4.2 Hz) ppm. 13C NMR
(DMSO-d6): δ 154.193, 153.924, 142.078, 140.738, 126.748,
126.668, 125.218, 124.091, 119.853, 115.092, 112.636, 111.482,
and 109.171 ppm (13 signals for 13 different carbons of 12).
Anal. Calcd for C19H16N2O2: C, 74.98; H, 5.26; N, 9.20.
Found: C, 74.52; H, 5.37; N, 9.02.
1
1132, 1607, 843, and 814 cm-1. H NMR (DMSO, 400 MHz):
δ 11.51 (1 H, s, O-H), 11.14 (1 H, s, N-H), 10.38 (1 H, s,
O-H), 8.85 (1 H, s), 8.49 (1 H, dd, J ) 8 Hz), 7.67 (1 H, dd, J
) 8 Hz), 7.1 (1 H, dd, J ) 8 Hz), 6.88 (1 H, dd, J ) 8 Hz), 6.67
(1 H, s), 6.56 (1 H, s), 6.47 (1 H, d, J ) 8 Hz), 3.82 (3 H, s
“methyl”), 3.79 (3 H, s “methyl”). Anal. Calcd for C21H18N4O8:
C, 55.50; H, 3.96; N, 12.33. Found: C, 55.43; H, 3.95; N, 12.28.
4-Hyd r oxya cetop h en on e-4-n itr op h en ylh yd r a zon e (13)
was prepared by method A from 4-hydroxyacetophenone and
4-nitrophenylhydrazine in 82% yield. Deep-red crystals were
crystallized from EtOH, mp 184-185 °C. 1H NMR (DMSO-
d6): δ 10.062 (1H, s, NH), 9.750 (1H, broad singlet, OH), 8.094
(2H, d, J ) 6.6 Hz), 7.678 (2H, d, J ) 6.6 Hz), 7.291 (2H, d, J
) 6.6 Hz), 6.820 (2H, d, J ) 6.6 Hz), and 2.253 (3H, s, CH3)
ppm. 13C NMR (DMSO-d6): δ 154.846, 148.155, 143.186,
134.667, 125.950, 123.910, 122.483, 111.794, 108.312, and
9.896 ppm (10 signals for 10 different carbons of 13). Anal.
Calcd for C14H13N3O3: C, 61.99; H, 4.83; N, 15.49. Found: C,
59.57; H, 5.02; N, 14.73.
4,4′-Dia m in ob en zop h en on e-2,4-d in it r op h en ylh yd r a -
zon e (14) was prepared by method A from 4,4′-diaminoben-
zophenone and 2,4-dinitrophenylhydrazine in MeOH with 10
mL of concentrated H2SO4. The mixture was refluxed for 3 h
and allowed to cool. The MeOH was flash-evaporated under
reduced pressure, resulting in an orange solid. The solid was
added to 100 mL of cold water, and the mixture was stirred
in an ice/water bath. A solution of KOH (15 g in 50 mL of
water) was added until pH ∼3.5 was attained. The fluffy,
purple precipitate was filtered, washed with 100 mL of cold
water, and allowed to air-dry. The yield of 14 was 98%, mp
268-270 °C. 1H NMR (DMSO-d6): δ 11.29 (s, 1H, N-H), 8.81
(s, 1H), 8.35 (d, 1H, J ) 8.8 Hz), 8.12 (d, 1H, J ) 9.6 Hz), 7.36
(dd, 2H, J ) 8.4 Hz), 7.05 (dd, 2H, J ) 8.4 Hz), 6.78 (dd, 2H,
J ) 8.4 Hz), 6.59 (dd, 2H, J ) 8.4 Hz). 13C NMR (DMSO-d6):
δ 153.4, 146.2, 145.2, 139.9, 132.4, 126.0, 125.7, 124.7, 120.9,
119.3, 114.9, 112.6, 111.0, and 109.9 ppm. Anal. Calcd for
2,4-Dih yd r oxyben zop h en on e-2,4-d in itr op h en ylh yd r a -
zon e (8) was prepared by method A in 90% yield from 2,4-
dihydroxybenzophenone and 2,4-dinitrophenylhydrazine as red
crystals that crystallized from MeOH, mp 304-305 °C. IR:
3271 (N-H), 2929 (Ar-H), 2855 (Ar-H), 1619 (CdN), 1518,
1
1421, 1323, 1203, 1112, 868, and 702 cm-1. H NMR (DMSO,
400 MHz): δ 11.14 (1 H, s, N-H), 10.96 (1 H, s, O-H), 10.15
(1 H, s, O-H), 8.80 (1 H, s), 8.48 (1 H, d, J ) 8 Hz), 7.69 (4 H,
m, J ) 8 Hz), 6.74 (1 H, dd, J ) 8 Hz), 6.39 (1 H, s), 6.31 (1 H,
dd, J ) 8 Hz). Anal. Calcd for C19H14N4O6: C, 57.86; H, 3.55;
N, 14.21. Found: C, 57.78; H, 3.51; N, 14.19.
4,4′-Dia cetoxyben zop h en on e-2,4-d in itr op h en ylh yd r a -
zon e (9) was prepared by dissolving 1 (0.01 mol) in 5 mL of 3
M NaOH and 15 g of crushed ice followed by 4.5 g (0.03 mol)
of Ac2O. The mixture was vigorously shaken and allowed to
stand at room temperature. The acetate separated in
a
practically pure state as orange crystals that crystallized from
MeOH; yield 95%, mp 205-207 °C. IR: 3287 (N-H), 3119 (N-
H), 2929 (Ar-H), 1768 (CdO), 1623 (CdN), 1591, 1501, 1263,
1198, 916, and 600 cm-1. 1H NMR (DMSO, 400 MHz): δ 11.07
(1 H, s, N-H), 8.82 (1 H, s), 8.44 (1 H, d, J ) 8 Hz), 8.25 (1 H,
d, J ) 8 Hz), 7.69 (2 H, dd, J ) 8 Hz), 7.58 (2 H, dd, J ) 8 Hz),
7.49 (2 H, dd, J ) 8 Hz), 7.26 (2 H, dd, J ) 8 Hz), 2.35 (3H,s),
2.30 (3H,s). Anal. Calcd for C23H18N4O8: C, 57.70; H, 3.80; N,
11.70. Found: C, 57.76; H, 3.77; N, 11.55.
4,4-Dih yd r oxyb en zop h en on e-2,4-d im et h ylp h en ylh y-
d r a zon e (10) was prepared by method A from 4,4-dihydroxy-
benzophenone and 2,4-dimethylphenylhydrazine as cream-
colored crystals in 75% yield that crystallized from EtOH, mp
140-142 °C. IR (KBr): 3356 (N-H), 2885 (Ar-H), 1663
C
19H16N6O4: C, 58.16; H, 4.11; N, 21.42. Found: C, 58.19; H,
4.17; N, 21.51.
1
(CdN), 1509, 1256, 1228, 1170, 838, and 809 cm-1. H NMR
4,4′-Dim eth yla m in oben zop h en on e-2,4-d in itr op h en yl-
h yd r a zon e (15) was synthesized from the respective hydra-
zine and ketone using the procedure for the synthesis of 14.
(DMSO, 400 MHz): δ 9.87 (1/2 H, s, N-H), 9.6 (1/2 H, s, N-H),
7.36 (4 H, m), 7.16 (2 H, dd, J ) 8 Hz), 6.99 (2 H, dd, J ) 8
Hz), 6.95 (1 H, d, J ) 8 Hz), 6.74 (2 H, dd, J ) 8 Hz), 2.18 (3
H, s), 1.8 (3 H, s). Anal. Calcd for C21H20N2O2: C, 75.90; H,
6.02; N, 8.43. Found: C, 75.74; H, 6.02; N, 8.25.
1
The yield of 15 was 90%, mp 263-264 °C. H NMR (DMSO-
d6): δ 11.29 (s, 1H, N-H), 8.81 (s, 1H), 8.35 (d, 1H, J ) 8.8
Hz), 8.12 (d, 1H, J ) 9.6 Hz), 7.36 (dd, 2H, J ) 8.4 Hz), 7.05
(dd, 2H, J ) 8.4 Hz), 6.78 (dd, 2H, J ) 8.4 Hz), 6.59 (dd, 2H,
J ) 8.4 Hz) ppm. 13C NMR (DMSO-d6): δ 153.4, 146.2, 145.2,
139.9, 132.4, 126, 125.7, 124.7, 120.9, 119.3, 114.9, 112.6,
111.0, and 109.9 ppm. MS (electrospray), m/z: 449 (M + H)+.
Anal. Calcd for C23H24N6O4: C, 61.60; H, 5.39; N, 18.74.
Found: C, 61.65; H, 5.43; N, 18.77.
4,4′-Dih yd r oxyb e n zop h e n on e -2-n it r op h e n ylh yd r a -
zon e (11) was prepared from 4,4′-dihydroxybenzophenone and
2-nitrophenylhydrazine using method A. H3PO4 was substi-
tuted for H2SO4. The yield was 67%, and the product was
recrystallized from aqueous EtOH as red crystals, mp 239-
1
241 °C. H NMR (DMSO-d6): δ 10.806 (1H, NH), 9.930 (2H,
broad, OH), 7.975 (2H, d + d, J 1 ) 6.3, J 2 ) 6.3, two hydrogens
in 3- and 6-position of 2-nitrophenylhydazine moiety), 7.571
(1H, t, J ) 6.0 Hz, hydrogen in 4-position of 2-nitrophenyl-
hyrazine moiety), 7.427 (2H, d, J ) 6.6 Hz, two hydrogens in
o-position of the 4,4′-dihydroxybenzophenone moiety), 7.160
(2H, J ) 6.3 Hz, two hydrogens in o-position of the 4,4′-
dihydroxybenzophenone moiety), 7.005 (2H, J ) 6.6 Hz, two
hydrogens in o-position of the 4,4′-dihydroxybenzophenone
moiety), 6.797 (2H, J ) 6.3 Hz, two hydrogens in m-position
of the 4,4′-dihydroxybenzophenone moiety), and 6.766 (1H, t,
J ) 6.3 Hz, hydrogen in 6-position of the 2-nitrophenylhydra-
zine moiety) ppm. 13C NMR (DMSO-d6): δ 155.529, 155.036,
148.315, 137.906, 133.081, 126.679, 126.352, 125.480, 125.063,
122.150, 119.153, 114.328, 113.107, 112.196, and 111.916 ppm
(15 carbon signals for 15 different carbon atoms in 11). Anal.
Calcd for C19H15N4O6: C, 65.33; H, 4.30; N, 12.03. Found: C,
65.06; H, 4.49; N, 11.92.
4,4′-Dieth yla m in oben zop h en on e-2,4-d in itr op h en ylh y-
d r a zon e (16) was synthesized from the respective hydrazine
and ketone using the procedure for the synthesis of 14. The
yield was 98%, mp 268-270 °C. 1H NMR (DMSO-d6): δ 11.51
(s, 1H, N-H), 9.07 (s, 1H), 8.27 (dd, 1H, J ) 8 Hz), 8.16 (dd,
1H, J ) 8 Hz), 7.58 (dd, 2H, J ) 8.8 Hz), 7.20 (dd, 2H, J ) 8.0
Hz), 6.81 (dd, 2H, J ) 8.4 Hz), 6.65 (dd, 2H, J ) 8.8 Hz), 3.44
(m, 8H, J ) 7.2 Hz), 1.23 (12H, m, J ) 6.8 Hz). 13C NMR
(CDCl3): δ 158.1, 149.4, 149, 144.6, 136.9, 130.3, 130.2, 129.7,
128.7, 124.2, 117.8, 118.8, 111.9, 110.9, 44.6, and 12.8 ppm.
MS (electrospray), m/z: 506.7 (MH)+. Anal. Calcd for
C
27H32N6O4: C, 64.27; H, 6.39; N, 16.66. Found: C, 64.22; H,
6.40; N, 16.59.
4,4′-Dia ceta m id oben zop h en on e-2,4-d in itr op h en ylh y-
d r a zon e (17) was prepared from 14 (0.522 g, 1.33 mmol) and
25 mL of Ac2O. The mixture was refluxed for 1.5 h and cooled

4560 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21
Morgan et al.
to room temperature. The resulting orange crystals were
1256, 1180, 1132, 850, and 740 cm-1. 1H NMR (DMSO-d6, 300
MHz): δ 11.97 (1 H, s, N-H), 8.86 (1 H, s), 8.47 (1 H, d, J )
6), 8.38 (1 H, s), 8.24 (1 H, d, J ) 12 Hz), 8.17 (2 H, m), 8.12
(2 H, m), 7.89 (4 H, m), and 7.75 (3 H, m) ppm. Anal. Calcd
for C21H16N4O7: C, 57.80; H, 3.70; N, 12.84. Found: C, 57.92;
H, 3.68; N, 12.83.
washed with cold water (5 × 50 mL) and allowed to air-dry.
1
The yield was 98%, mp 276-278 °C. H NMR (DMSO-d6): δ
11.12 (s, 1H, N-H), 10.29 (s, 1H, N-H), 10.17 (s, 1H, N-H),
8.77 (s, 1H), 8.37 (d, 1H, J ) 9.6 Hz), 8.16 (d, 1H, J ) 9.6 Hz),
7.87 (dd, 2H, J ) 8 Hz), 7.63 (dd, 2H, J ) 8.4 Hz), 7.55 (dd,
2H, J ) 8.8 Hz), 7.36 (dd, 2H, J ) 8.4 Hz), 2.12 (s, 3H, CH3),
2.05 (s, 3H, CH3) ppm. 13C NMR (DMSO-d6): δ 169.5, 169.2,
155.2, 144.6, 141.8, 141.5, 137.7, 131.4, 130.6, 129.9, 129.6,
128.9, 126.0, 123.4, 120.2, 119.1, 117.1, 24.6, and 24.5 ppm.
MS (electrospray), m/z: 476.8 (MH)+. Anal. Calcd for
N-Ben zyl-N-(2,4-d in itr op h en yl)-N′-xa n th en -9-ylid en e-
h yd r a zon e (23) was synthesized by our reported procedure4
1
with a yield of 50%, mp 219-220 °C. H NMR (CDCl3): δ 4.9
(s, 2H, N-CH2-). Anal. Calcd for C26H18N4O5: C, 66.95; H,
3.89; N, 12.01. Found: C, 67.05; H, 3.87; N, 12.03.
C
23H20N6O6: C, 57.98; H, 4.23; N, 17.64. Found: C, 58.01; H,
9-Su lfoxyxa n th en on e-2,4-din itr op h en ylh ydr a zon e (24)
was synthesized from 9-sulfoxyxanthenone and 2,4-dinitro-
phenylhydrazine in 90% yield using method A. The yellow
crystalline product was crystallized from EtOH, mp 248-250
°C. IR (KBr): 3290 (N-H), 3286 (N-H), 2926 (Ar-H), 2855
(Ar-H), 1615 (CdN), 1590, 1506, 1334, 1101, 1037, 787, and
4.31; N, 17.70.
2,7-Dih yd r oxy-9-flu or en on e-2,4-d in it r op h en ylh yd r a -
zon e (18) was prepared from 2,7-dihydroxy-9-fluorenone and
2,4-dinitrophenylhydrazine using method A. A reddish pow-
dery product was purified by crystallization from glacial AcOH;
yield 78%, mp >325 °C (dec). IR (KBr): 3445 (N-H), 2924
(Ar-H), 2854 (Ar-H), 1615 (CdN), 1585, 1507, 1424, 1335,
551 cm-1 1H NMR (DMSO-d6, 300 MHz): δ 11.97 (1 H, s,
.
N-H), 8.86 (1 H, s), 8.47 (1 H, d, J ) 6 Hz), 8.38 (1 H, s), 8.24
(1 H, d, J ) 12 Hz), 8.17 (2 H, m), 8.12 (2 H, m), 7.89 (4 H, m),
and 7.75 (3 H, m) ppm.4
1308, 1269, 1144, 1117, 1081, 834, and 738 cm-1 1H NMR
.
(DMSO-d6, 400 MHz): δ 11.64 (1 H, s, N-H), 9.87 (1 H, s,
O-H), 9.57 (1 H, s, O-H), 8.77 (1 H, s), 8.36 (1 H, dd, J ) 8
Hz), 8.11 (1 H, dd, J ) 8 Hz), 7.45 (1 H, dd, J ) 8 Hz), 7.40 (1
H, s), 7.35 (1 H, dd, J ) 8 Hz), and 7.12 (1H, s). Anal. Calcd
for C19H12N4O6: C, 58.17; H, 3.08; N, 14.28. Found: C, 56.34;
H, 2.82; N, 13.76.
10-[(2,4-Din itr op h en yl)h yd r a zon o]-1,2-d ih yd r oxy-10H-
a n th r a cen -9-on e (25) was prepared from 1,2-dihydroxyan-
thraquinone and 2,4-dinitrophenylhydrazine by method A,
with the exception that the reaction mixture was refluxed for
48 h. After evaporation of the solvent, the solid residue was
crystallized from dioxine/hexane to afford a pure product in
92% isolated yield, mp 240-242 °C. IR (KBr): 3502 (N-H),
3287 (N-H), 3113 (O-H), 2925 (Ar-H), 2559 (Ar-H), 1618
(CdN), 1595, 1501, 1441, 1335, 1298, 1135, 1091, and 781
1-H y d r o x y -9-flu o r e n o n e -2,4-d in it r o p h e n y lh y d r a -
zon e (19) was prepared from 1-hydroxy-9-fluorenone and 2,4-
dinitrophenylhydrazine using method A. A reddish powdery
product (90% yield) was crystallized from glacial AcOH, mp
>270 °C (dec). IR (KBr): 3338 (N-H), 2927 (Ar-H), 2855 (Ar-
H), 1617 (CdN), 1508, 1427, 1336, 1318, 1142, 1116, 835, and
1
cm-1. H NMR (DMSO-d6, 300 MHz): δ 11.97 (1 H, s, N-H),
8.86 (1 H, s), 8.47 (1 H, d, J ) 6 Hz), 8.38 (1 H, s), 8.24 (1 H,
d, J ) 12 Hz), 8.17 (2 H, m), 8.12 (2 H, m), 7.89 (4 H, m), and
7.75 (3 H, m) ppm. Anal. Calcd for C20H12N4O7: C, 57.15; H,
2.88; N, 13.33. Found: C, 56.58; H, 2.99; N, 12.81.
739 cm-1 1H NMR (DMSO-d6, 400 MHz): δ 11.69 (1 H, s,
.
N-H), 10.1 (1 H, s, O-H), 9.77 (1 H, s, O-H), 8.75 (1 H, s),
8.34 (1 H, dd, J ) 8 Hz), 8.12 (1 H, dd, J ) 8 Hz), 7.92 (1 H,
s), 7.69 (3 H, m), 7.59 (1 H, dd, J ) 8 Hz), 7.52 (1 H, dd, J )
8 Hz), 7.47 (1 H, s), 7.33 (2 H, m), 7.2 (1 H, t, J ) 8 Hz), 7.14
(1 H, s), 6.95 (1 H, dd, J ) 8 Hz), 6.78 (1H, dd, J ) 8 Hz) ppm.
Anal. Calcd for C19H12N4O5: C, 60.64; H, 3.21; N, 14.89.
Found: C, 60.64; H, 3.10; N, 14.64.
10-[(2,4-Din itr op h en yl)h yd r a zon o]-1,8-d ih yd r oxy-10H-
a n th r a cen -9-on e (26) was prepared in 90% yield from 1,8-
dihydroxyanthraquinone (0.5 mmol) and 2,4-dinitrophenylhy-
drazine (0.5 mmol) by method A (using H2S04 as a catalyst).
The orange crystalline product was purified by recrystalliza-
tion from MeOH, mp 284-285 °C (dec). IR (KBr): 3326 (N-
H), 3275 (N-H), 3064 (O-H), 2951 (Ar-H), 2921 (Ar-H), 2852
(Ar-H), 1634 (CdN), 1618 (CdN), 1596, 1504, 1418, 1342,
1320, 1219, 1168, 1102, 985, 830, 741, and 589 cm-1. 1H NMR
(DMSO-d6, 300 MHz): δ 11.93 (1 H, s, N-H), 7.82 (2 H, t, J
) 6 Hz), 7.72 (2 H, d, J ) 6 Hz), and 7.39 (2 H, d, J ) 6 Hz)
ppm. Anal. Calcd for C20H13N3O5: C, 64.00; H, 3.46; N, 11.20.
Found: C, 63.98; H, 3.46; N, 11.13.
2,7-D ia c e t y l-9-flu o r e n o n e -2,4-d in it r o p h e n y lh y r a -
zon e (20) was prepared through acetylation of 18 with Ac2O/
NaOAc in 75% yield. A solid yellow product was recrystallized
from glacial AcOH, mp 295-296 °C. IR (KBr): 3324 (N-H),
3097 (O-H), 2953 (Ar-H), 2825 (Ar-H), 2854 (Ar-H), 1760
(CdO), 1614 (CdN), 1583, 1507, 1422, 1338, 1207, 1123, 914,
834, and 527 cm-1. 1H NMR (DMSO-d6, 300 MHz): δ 11.97 (1
H, s, N-H), 8.86 (1 H, s), 8.47 (1 H, d, J ) 6 Hz), 8.38 (1 H, s),
8.24 (1 H, d, J ) 12 Hz), 8.17 (2 H, m), 8.12 (2 H, m), 7.89 (4
H, m), and 7.75 (3 H, m) ppm. Anal. Calcd for C23H16N4O8: C,
57.99; H, 3.39; N, 11.76. Found: C, 58.02; H, 3.26; N, 11.80.
9-Xa n t h en on e-2,4-d in it r op h en ylh yd r a zon e (21) was
prepared from 9-xanthenone and 2,4-dinitrophenylhydrazine
using method A. A deep-yellow product was crystallized from
EtOH (yield 85%), mp 276-277 °C. IR (KBr): 3266 (N-H),
2926 (Ar-H), 2854 (Ar-H), 1619 (CdN), 1505, 1454, 1425,
1334, 1311, 1127, 1082, 907, 772, 752, and 620 cm-1. 1H NMR
(DMSO-d6, 300 MHz): δ 11.51 (1 H, s, N-H), 8.89 (1 H, s),
8.53 (1 H, d, J ) 6 Hz), 8.42 (1 H, d, J ) 6 Hz), 8.29 (2 H, d,
J ) 6 Hz), 8.24 (2 H, d, J ) 6 Hz), 7.72 (2 H, d, J ) 6 Hz), 7.58
(2 H, d, J ) 6 Hz), 7.48 (2 H, d, J ) 6 Hz), and 7.41 (2 H, t, J
) 6 Hz) ppm.4
N-(3,6-Dim et h oxyxa n t h en -9-ylid en e)-N′-(2,4-d in it r o-
p h en yl)h yd r a zon e (22). 3,6-Dimethoxyxanthenone was pre-
pared by refluxing an acetone solution of 3,6-dihyroxy-9-
xanthenone (7.4 mmol), dimethyl sulfate (28 mmol), and
potassium carbonate (1.6 mmol) for 6 h. The mixture was
evaporated to dryness, and the resulting reddish brown powder
of 3,6-dimethoxy-9-xanthenone was crystallized from dioxane.
The 3,6-dimethoxy-9-xanthenone (0.5 mmol) and 2,4-dinitro-
phenylhydrazine (0.5 mmol) in DEP (16 mL) were reacted
using method A with a 78% yield of analogue 22, mp 288-
289 °C. IR (KBr): 3318 (N-H), 3105 (O-H), 2925 (Ar-H),
2853 (Ar-H), 1617 (CdN), 1586, 1503, 1462, 1414, 1345, 1294,
10-[2,4-Din itr op h en yl)h yd r a zon o]-1,8-a cetoxy-10H-a n -
th r a cen -9-on e (27) was prepared from analogue 26 with
Ac2O/NaOAc and recrystallized from EtOH or AcOH as a
yellow-orange powder; yield 85%, mp 278-279 °C. IR (KBr):
3219 (N-H), 3102 (N-H), 2928 (Ar-H), 2860 (Ar-H), 1775
(CdO), 1666 (CdN), 1599, 1501, 1421, 1329, 1201, 1014, 883,
839, 583, and 589 cm-1 1H NMR (DMSO-d6, 300 MHz): δ
.
12.05 (1 H, s, N-H), 8.88 (1 H, s), 8.45 (2 H, q, J ) 6 Hz), 8.31
(1 H, d, J ) 6 Hz), 8.23 (1 H, d, J ) 6 Hz), 7.96 (1 H, t, J ) 6
Hz), 7.82 (1 H, t, J ) 6 Hz), 7.35 (1 H, d, J ) 6 Hz), 2.36 (3 H,
s-CH3), and 2.34 (3 H, s-CH3) ppm. Anal. Calcd for
C
24H16N4O9: C, 57.15; H, 3.20; N, 11.11. Found: C, 57.13; H,
3.09; N, 11.08.
10-[(2,4-Din itr oph en yl)h ydr azon o]-1-acetoxy-8-h ydr oxy-
10H-a n th r a cen -9-on e (28) was synthesized from analogue
27. To a solution of acetobromoglucose, 1.7 g (4.2 mmol) and
TMSOTf (1.8 g, 8 mmol) in 100 mL of CH2Cl2, under a nitrogen
atmosphere at 0 °C, 27 (1 g, 2 mmol) in 50 mL of CH2Cl2 was
slowly added (over 15 min). Stirring continued for another 90
min, and the reaction was quenched with saturated NaHCO3
(14 g and 140 mL of water) at 5 °C. The solid material was
filtered, and the CH2Cl2 layer was separated, washed with a
saturated NaHCO3 solution, and dried. Analogue 28 was
purified by Al2O3 column chromatography with CH2Cl2 as a
solvent. The above reaction does not require acetobromoglu-
cose; however, an improved yield results; 82%, mp 224-225

Dinitrophenylhydrazones and Analogues
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21 4561
°C. 1H NMR (DMSO-d6, 300 MHz): δ 11.93 (1 H, s, N-H),
7.82 (2 H, t, J ) 6 Hz), 7.72(2 H, d, J ) 6 Hz), and 7.39 (2 H,
d, J ) 6 Hz) ppm. Anal. Calcd for C22H14N4O8: C, 57.15; H,
3.05; N, 12.12. Found: C, 57.32; H, 3.18; N, 11.93.
100 °C for 3 h, mp 330-331 °C. IR (KBr): 3341 (N-H), 3260
(N-H), 3110 (N-H), 2926 (Ar-H), 2851 (Ar-H), 1611 (Cd
N), 1582, 1503, 1414, 1332, 1254, 1125, 1060, 864, 767, and
1
667 cm-1. H NMR (DMSO-d6, 400 MHz): δ 13.56 (1H, NH),
12.06 (1H, NH), 8.88 (1H, s), 8.62 (1H, d, J ) 8 Hz), 8.45 (1H,
d, J ) 8 Hz), 8.32 (2H, m), 8.14 (1H, d, J ) 8 Hz), 7.85 (1H, d,
J ) 8 Hz), 7.76 (1H, t, J ) 8 Hz), 7.64 (1H, t, J ) 8 Hz), 7.56
(1H, t, J ) 8 Hz). Anal. Calcd for C20H12N6O4: C, 60.00; H,
3.02; N, 21.00. Found: C, 60.10; H, 2.97; N, 20.99.
10-[(2,4-Din itr oph en yl)h ydr azon o]-1-acetoxy-8-allyloxy-
10H-a n th r a cen -9-on e (29). A solution of CH2Cl2 (50 mL)
containing analogue 28 (0.89 g, 2 mmol), allyl chloride (0.39
g, 2 mmol), and TEA (0.6 g, 6 mmol) was stirred at room
temperature overnight. The solvent was evaporated, and the
solid residue was crystallized from dioxane/hexane to afford
pure 29 in 62% yield, mp 245-247 °C. IR (KBr): 3300 (N-
H), 2924 (Ar-H), 767 (CdO), 1744 (CdO), 1669 (CdN), 1612
(CdN), 1601, 1506, 1410, 1339, 1199, 1138, 966, 833, and 792
12-[2,4-Din itr oph en ylh ydr azon o]-6-h ydr oxy-12H-n aph -
th a cen -5-on e (34). Analogue 34 was synthesized in 71% yield
from 6-hydroxynaphthacene-5,12-dione and 2,4-dinitrophenyl-
hydrazine (1:1 ratio) by a slight modification of method A. The
reaction mixture was refluxed for 48 h, and the red precipitate
was washed with CHCl3, mp 261-262 °C (dec). IR (KBr): 3307
(N-H), 3265 (N-H), 3099 (N-H), 2920 (Ar-H), 2855 (Ar-
H), 1617 (CdN), 1584, 1506, 1420, 1338, 1282, 1137, 1074,
1040, 872, 757, and 601 cm-1. 1H NMR (DMSO-d6, 400 MHz):
δ 8.81 (1H, s), 8.45 (1H, d, J ) 8 Hz), 8.37 (1H, d, J ) 8 Hz),
8.29 (2H, t, J ) 8 Hz), 8.26 (1H, s), 8.13 (1H, s), 8.06 (2H, d,
J ) 8 Hz), 7.98 (2H, t, J ) 8 Hz), 7.86 (4H). Anal. Calcd for
1
cm-1. H NMR (DMSO-d6, 300 MHz): δ 12.05 (1H, s, N-H),
8.88 (1H, s), 8.47 (2H, m, J ) 6 Hz), 8.32 (1H, d, J ) 6 Hz),
8.24 (1H, t, J ) 6 Hz), 7.97 (1H, q, J ) 6 Hz), 7.82 (1H, q, J )
6 Hz), 7.55 (1H, dd, J ) 6 Hz), 7.38 (1H, dd, J ) 6 Hz), 6.5
(3H, m), 6.23 (1H, m), 2.26 (3H, s), and 2.25 (3H, s) ppm. Anal.
Calcd for C25H16N4O9: C, 58.13; H, 3.10; N, 10.85. Found: C,
58.00; H, 3.10; N, 10.91.
2,6-Dih yd r oxy-9,10-[d i-(2,4-d in itr op h en ylh yd r a zon o)]-
9H,10H-a n th r a cen en e (30) was prepared as red crystals
from 2,6-dihydroxyanthraquinone and 2,4-dinitrophenylhy-
drazine (2 equiv) in 75% yield following method A. The product
was purified by crystallization from MeOH, mp >348 °C (dec).
IR (KBr): 3441 (N-H), 3290 (N-H), 2949 (Ar-H), 2922 (Ar-
H), 2858 (Ar-H), 1672 (CdN), 1611 (CdN), 1517, 1422, 1332,
C
24H14N4O6: C, 63.44; H, 3.11; N, 12.33. Found: C, 63.56; H,
3.11; N, 12.14.
11-Acetoxy-2-p h en yl-2H-d iben zo[d e,h ]cin n olin e-3,7-d i-
on e (35). 11-Hydroxy-2-phenyl-2H-dibenzo[de,h]cinnoline-3,7-
dione was prepared from a pyridine solution (10 mL) of
8-hydroxy-9,10-dioxo-9,10-dihydroanthracene-1-carboxylic acid
(1.5 mmol) and phenylhydrazine (1.5 mmol) by was refluxing
for 30 min. The reaction mixture was cooled to room temper-
ature, and the precipitate was separated by filtration and
washed with 50 mL of water and 25 mL of EtOH. The IR
spectral analysis of the solid product indicated the absence of
-CdO vibration at 1705 cm-1 present in starting material.
The yield of the crystalline product was 92%, mp 288-290 °C.
The hydroxycinnolinedione was dissolved in a mixture of Ac2O
(4 mL) and NaOAc (0.16 g) and stirred at room temperature
for several hours. The solvent was evaporated at reduced
pressure, and the solid residue was washed with water, 10%
NaHCO3, water, and EtOH to afford analogue 35 as a dark
powder in 55% yield, mp 254-255 °C. IR (KBr): 3073 (N-H),
2951 (Ar-H), 2923 (Ar-H), 2855 (Ar-H), 1744 (CdO), 1677
(CdN), 1667 (CdN), 1590, 1460, 1333, 1227, 1133, 783, and
1128, 828, and 575 cm-1 1H NMR (DMSO-d6, 400 MHz): δ
.
11.99 (1H, s, N-H), 11.94 (1H, s), 10.81 (1H, s-OH), 10.58 (2H,
s), 0.22 (1H, s-OH), 8.88 (2H, s), 8.47 (2H, d, J ) 8 Hz), 8.18
(2H, d, J ) 8 Hz), 7.62 (2H, d, J ) 8 Hz), 7.06 (2H, d, J ) 8
Hz), 2.89 (3H, s), and 2.73 (3H, s) ppm. Anal. Calcd for
C
26H16N8O10: C, 52.00; H, 2.66; N, 18.66. Found: C, 51.25; H,
2.59; N, 18.05.
1,2-Dia m in o-10-[(2,4-d in it r op h en yl)h yd r a zon o]-10H -
a n th r a cen -9-on e (31) was prepared in 80% yield from 1,2-
diaminoanthraquinone (2 equiv) and 1 equiv of 2,4-dinitro-
phenylhydrazine by a slight modification of method A. EtOH,
instead of MeOH, was the solvent, and the catalyst used was
phosphoric acid instead of sulfuric acid. The reaction mixture
was refluxed for 10 h. Red crystals formed from EtOH, mp
>250 °C. IR (KBr): 3479 (N-H), 3389 (N-H), 3298 (N-H),
3104 (N-H), 2957 (Ar-H), 2930 (Ar-H), 2858 (Ar-H), 1615
1
699 cm-1. H NMR (DMSO-d6, 400 MHz): δ 8.73 (2H, q, J )
(CdN), 1585, 1503, 1421, 1333, 1312, 1128, 826, and 570 cm-1
.
8 Hz), 8.33 (1H, d, J ) 8 Hz), 8.19 (2H, t, J ) 8 Hz), 7.79 (2H,
t, J ) 8 Hz), 7.62 (1H, m), and 1.6 (3H, s) ppm. Anal. Calcd
for C23H14N2O4: C, 72.25; H, 3.69; N, 7.33. Found: C, 72.30;
H, 3.61; N, 7.34.
1H NMR (DMSO-d6, 300 MHz): δ 11.93 (2H, s), 8.87 (2H, s),
8.43 (2H, d, J ) 9 Hz), 8.13 (2H, d, J ) 9 Hz), 7.98 (2H, d, J
) 9 Hz), 7.39 (1H, s), 6.79 (2H, d, J ) 9 Hz), and 6.14 (1H, s)
ppm. Anal. Calcd for C20H14N6O5: C, 57.41; H, 3.34; N, 20.09.
Found: C, 57.51; H, 3.29; N, 19.79.
2-(2-Nit r op h e n yl)-2H -in d e n o[1,2,3-d e]p h t h a la zin -3-
on e (36) was prepared by a modified procedure of Campbell
et al.12 9-[(2-Nitrophenyl)hydrazono]-9H-fluorene-1-carboxylic
acid was prepared from a pyridine solution (20 mL) of 9-oxo-
9H-fluorene-1-carboxylic acid (0.02 mol) and 2-nitrophenylhy-
drazine (0.02 mol) by refluxing for 4 h. The reaction mixture
was poured into ice/water, and the precipitate was filtered,
washed with water (50 mL), and EtOH (50 mL). 9-[(2-
Nitrophenyl)hydrazono]-9H-fluorene-1-carboxylic acid was ob-
tained in 85% yield, mp >220 °C (dec). Thionyl chloride (0.13
mmol) and the carboxylic acid (0.01 mol) were refluxed for 1.5
h. Excess thionyl chloride was removed at reduced pressure,
and the oily residue was mixed with ice/water (50 mL). A
precipitate formed which was collected by filtration, washed
with water (50 mL) and 20% NaHCO3 (10 mL), and dried to
afford analogue 36 in 85% yield, mp 220-222 °C. IR (KBr):
3071 (N-H), 3041 (N-H), 2919 (Ar-H), 2886 (Ar-H), 1689
(CdN), 1645 (CdN), 1509, 1359, 1094, 974, 852, 777, and 716
2,6-Dia m in o-9,10-[d i-(2,4-d in it r op h en yl)h yd r a zon o)]-
9H,10H-a n th r a cen e (32) was prepared in 70% yield from 2,6-
diaminoanthraquinone (1 equiv) and 2,4-dinitrophenylhydra-
zine (2 equiv) by slight modification of method A. EtOH was
used as solvent, and phosphoric acid was used as an acid
catalyst. The reaction mixture was refluxed for 24 h. The
resulting product upon cooling was purified by crystallization
from EtOH, mp 328-330 °C. IR (KBr): 3479 (N-H), 3389 (N-
H), 3298 (N-H), 3104 (N-H), 2957 (Ar-H), 2930 (Ar-H), 2858
(Ar-H), 1615 (CdN), 1585, 1503, 1421, 1333, 1312, 1128, 826,
and 570 cm-1 1H NMR (DMSO-d6, 300 MHz): δ 11.93 (2H,
.
s), 8.87 (2H, s), 8.43 (2H, d, J ) 9 Hz), 8.13 (2H, d, J ) 9 Hz),
7.98 (2H, d, J ) 9 Hz), 7.39 (1H, s), 6.79 (2H, d, J ) 9 Hz),
6.14 (1H, s) ppm. Anal. Calcd for C26H18N10O8: C, 52.17; H,
3.01; N, 23.40. Found: C, 51.88; H, 3.03; N, 22.66.
N-(2H-Diben zo[cd ,g]in d a zol-6-ylid en e)-N′-(2,4-d in itr o-
p h en yl)h yd r a zon e (33). 2H-Dibenzo[cd,g]indazol-6-one was
prepared from a pyridine (150 mL) solution of 1-chloroan-
thraquinone (50 mmol) and 1 equiv of hydrazine monohydrate
by refluxing for 6 h. The solution was poured into water, and
the yellow product was filtered and crystallized from chlo-
robenzene, mp 281-282 °C. Analogue 33 was prepared from
2H-dibenzo[cd,g]indazol-6-one and 2,4-dinitrophenylhydrazine
in 62% yield by a slight modification of method A. The solvent
used was diethyl phosphite, and the reactants were heated at
cm-1 1H NMR (DMSO-d6, 400 MHz): δ 8.32 (1H, d, J ) 8
.
Hz), 8.20 (1H, d, J ) 8 Hz), 8.05 (1H, d, J ) 8 Hz), 7.99 (2H,
m, J ) 4 Hz), 7.95 (1H, d, J ) 8 Hz), 7.92 (3H, m), 7.86 (1H,
d, J ) 8 Hz), 7.78 (1H, t, J ) 12 Hz), 7.57 (1H, t, J ) 8 Hz),
7.47 (1H, t, J ) 8 Hz). Anal. Calcd for C20H11N3O3: C, 70.58;
H, 2.94; N, 12.35. Found: C, 70.58; H, 3.10; N, 12.43.
2. Biologica l Stu d ies. In Vitr o Th er a p eu tic In d ex
Da ta . Cytotoxicity for the synthesized hydrazones was evalu-
ated in a human explant system (CYTOTAC).14 Selected

4562 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21
Morgan et al.
analogues were also evaluated in human and murine xenograft
cell lines: FDCP-1, MDA-MB-435, MDA-MB-435/lung met,
KM12 C, KM12 SM, KM12 L4, SN12 PM6, and B16 F10
(ATCC, Manassas, VA).
a . P er m a n en t Mu r in e H em a t op oiet ic P r ogen it or
(P MHP ) Cell Lin es. The FDCP-1 cell line was available in
the laboratory of one of the authors (Dr. Dominic Fan). IL-3,
necessary for cell growth, was obtained from conditioned
medium of murine WEHI-3 myelomonocytic leukemia cell
lines.16,18
slides were air-dried for 10 min and sprayed with a cytofixative
(Pro-Fixx, Lerner Labs). The slides were examined and
counted. The results were interpreted in the following fashion.
(i) Scan high-power fields and count. For controls, 5-10 cells
or clumps of cells per HPF in 5 HPF is considered as 100%
growth. (ii) Average the growth results from duplicate flasks,
and (iii) tabulate the percent growth vs test agents. (iii)
Determine the IC50 (and SD) for A-007 and other agents vs
control cancer cultures as per USP (111) or equivalent
algorithm.22
b. Cell Lin es a n d Cu ltu r e Con d ition s. The following cell
lines were purchased from ATCC (American Tissue Culture
Collection, Gathersburg, MD) or made available by one of the
authors (D.F.): MDA-MB-435 human breast carcinoma (also
lung metastasis variant), SN12 M6 renal cell cancer, KM12
SM/L4 colon cancers, K1735 M2 human melanoma, and B16
F10 murine melanoma. They were maintained in serial culture
in RPMI-1640 media (BioCell) supplemented with 10% fetal
calf serum, 100 mcg/mL streptomycin, and 100 units/mL
penicillin at 36 °C and 10% CO2 (moist).
Ack n ow led gm en t. We thank the NCI/SBIR and
NCI/FLAIR programs for financial support (Grants
CA16672, CA49310, and CA89772) and the Louisiana
Board of Reagents for their financial support (Grant
LEQSF (2001-04)-RD-B-12) for this work. Thanks are
given to Dr. Robert F. Struck, Southern Research
Institute, Birmingham, AL, for reviewing the manu-
script.
c. Assa y for in Vitr o An tip r olifer a tive Effects in
Hu m a n Tu m or Cell Lin es. The cancer cell lines were
suspended in medium and seeded at 2000-3000 cells/well in
96-well tissue culture plates. The cells were fed with fresh
medium (controls) or with medium containing different con-
centrations of the hydrazones after 18 h. After an additional
36 h, antiproliferative activity was determined by monitoring
the number of viable cells. This was accomplished using the
colorimetric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-
razolium bromide (thiazolyl blue)) assay.15-17 Growth inhibi-
tion was calculated from the formula
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A
B
cytostasis (%) ) 1 -
× 100
(
)
where A is the absorbance at 570 nm of treated cells and B is
the absorbance at 570 nm of control cells.
d . Assa y for in Vitr o Myelotoxicity.5,15 FDCP-1 cells were
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cisplatinum (cis-DDP)). DOX and cis-DDP dose ranges were
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Dinitrophenylhydrazones and Analogues
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