New potential anticancer agents based on the anthranilic acid scaffold. Synthesis and evaluation of biological activity

Article abstract of DOI:10.1021/jm050711d

The synthesis and anticancer activity of new compounds designed on the anthranilic acid scaffold are reported. The antiproliferative activity was assayed by the National Cancer Institute in established in vitro and in vivo anticancer experimental models.

Full text of DOI:10.1021/jm050711d

J. Med. Chem. 2005, 48, 8245-8252
8245
New Potential Anticancer Agents Based on the Anthranilic Acid Scaffold.
Synthesis and Evaluation of Biological Activity
Cenzo Congiu,* Maria Teresa Cocco, Valentina Lilliu, and Valentina Onnis
Dipartimento di Tossicologia, Universita` di Cagliari, Via Ospedale 72, Cagliari, I-09124, Italy
Received July 25, 2005
The synthesis and anticancer activity of new compounds designed on the anthranilic acid
scaffold are reported. The antiproliferative activity was assayed by the National Cancer Institute
in established in vitro and in vivo anticancer experimental models. Structural variations based
on the flufenamic acid motif afforded a series of (hetero)aryl esters of N-(2-(trifluoromethyl)-
pyridin-4-yl)anthranilic acid, which showed in vitro growth inhibitory properties against human
tumor cell lines in nanomolar to low micromolar concentrations. The pyridinyl ester 25 exhibited
very potent in vitro antiproliferative efficacy, with a chemosensitive profile showing a number
of GI50 values at concentrations lower than 10-7 M in the full panel of human tumor cell lines.
Compound 25 was also tested in vivo as a potential anticancer agent in the hollow fiber assay
and in human tumor xenografts, showing moderate inhibitory properties. Analysis of biological
activities and the COMPARE procedure was utilized to support putative biochemical mecha-
nisms implicated with the antiproliferative activity.
Introduction
Among the wide variety of synthetic compounds
recognized as potential anticancer drugs, molecules
based on the anthranilic acid scaffold have attracted
great interest in recent years. Experimental and pre-
clinical models demonstrated that a number of these
compounds elicited outstanding anticancer activity
through a range of biological activities implicated with
the development and maintenance of tumor cells. In this
context, several reports describing the antitumor evalu-
ation of anthranilate derivatives appeared in the recent
literature. For example, Tranilast (Figure 1) has been
reported to exhibit antiproliferative activity against
cultured leiomyoma cells, through the suppression of
cyclin-dependent kinase (CDK) 2 activity.1 Also, Yashiro
et al. have described that Tranilast decreases the
production of matrix metallo-proteinase-2 (MMP-2) and
transforms the growth factor-â1 (TGF-â1) from fibro-
blasts, resulting in significant suppression of the inva-
sion ability of gastric cancer cells.2 Farnesyl anthra-
nilate has been shown to reveal tumor growth-sup-
pressive action in experimental murine melanomas
models, as a probable consequence of down regulation
of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA)
reductase activity.3
Antitumor activity of the anthranilamide CI-1040 has
been demonstrated in preclinical models, particularly
for pancreas, colon, and breast cancers.4 The CI-1040
activity has been correlated with its inhibition of mito-
gen-activated protein kinase (MAPk) cascade pathway.
Moreover, the anthranilamide AAL993 has been de-
scribed as a lead compound of a new structural class of
vascular endothelial growth factor (VEGF) kinase in-
hibitors, which possess potent antiangiogenic and an-
titumor properties.5 Flufenamic acid (FA), a nonster-
oidal antiinflammatory drug (NSAID) belonging to the
Figure 1.
structural class of fenamates, has been recently found
to exhibit inhibitory properties against cell proliferation.
Pharmacological activity of NSAIDs is mainly attributed
to inhibition, to different extents, of the two isoforms of
prostaglandin H synthase, cyclooxygenase (COX) 1 and
2, the key enzyme that converts arachidonic acid to
prostaglandin H2, the precursor of a wide group of
biologically active mediators such as prostaglandins,
prostacyclin, and thromboxane A2.6 In recent years, an
important development in oncology is the assessment
that various NSAIDs played an important role as
antiproliferative agents against a broad spectrum of in
vivo and in vitro models of human malignancies, result-
ing in cell cycle arrest, increased apoptosis, and inhibi-
tion of angiogenesis.7 Although it has become widely
accepted that the antiinflammatory and antineoplastic
activities of NSAIDs are interrelated and mediated by
COX-2 inhibition,8 it has been pointed out that both
COX isoforms and/or their products may act in promot-
ing and maintaining the neoplastic state.9 Whether the
NSAIDs block tumor progression only by blocking COX
activity is a theme still being debated. On the other
hand, several research groups have shown that NSAIDs
can produce some of their cancer chemopreventive and
antiproliferative effects via mechanisms that are inde-
pendent of COX inhibition.10 Thus, recent experimental
* To whom correspondence should be addressed. Tel: +39-070-
675 8630. Fax: +39-070-675 8612. E-mail: ccongiu@unica.it.
10.1021/jm050711d CCC: $30.25 © 2005 American Chemical Society
Published on Web 11/15/2005

8246 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 26
Congiu et al.
findings, we planned to explore putative key portions
of the FA molecule, i.e., the carboxylic group, the
anthranilic portion, and the lipophilic trifluorometh-
ylphenyl moiety, by introducing structural modifications
in these regions and evaluating the effects of the
changes on the activity. In this paper, we describe the
synthesis and biological properties as potential antitu-
mor agents of FA aryl esters A, pyridinyl analogs B,
and nicotinic congeners C (Figure 2). Selected com-
pounds were assayed at the National Cancer Institute
(NCI) for their cytotoxicity and antitumor properties in
the in vitro anticancer screening. NCI also evaluated
the most potent compound 25 for its in vivo anticancer
efficacy in the hollow fiber assay and human tumor
xenografts.
Chemistry
Figure 2. Structural variations of the flufenamic acid motif.
The synthesis of the target compounds was conve-
niently undertaken as outlined in Schemes 1 and 2. Aryl
esters A were obtained by coupling FA with an ap-
propriate phenolic compound by the dicyclohexylcarbo-
diimide (DCC) method (vide infra). The first step of the
synthetic approach to compounds B and C was based
on a previously described procedure that allows the
construction of the trifluoromethylpyridine moiety linked
by an amino nitrogen to an aromatic ring.14 With some
modifications, this synthetic pathway is now success-
fully extended to the preparation of intermediates 5a,b
by using anthranilic acid and 2-aminonicotinic acid as
easily available starting materials. Anthranilic acid 1a
was reacted with trifluoroacetylvinyl ether 2 in 1:1.5
molar ratio in refluxing acetonitrile solution to yield the
acid 3a in almost quantitative yield (Scheme 1). Under
the same conditions, 2-aminonicotinic acid failed to react
with 2.
findings have shown FA to elicit antiproliferative activ-
ity by COX inhibition independent pathways. Weiser
et al. have reported that FA exhibits growth inhibition
properties on a mouse fibroblast cell line LM(TK-)
through blockage of a 28-pS nonselective cation chan-
nel.11 Zhu et al. have shown FA to inhibit the androgen
receptor (AR) expression in human prostate cancer
(LNCaP) cells, providing a new possible therapeutic
approach in prostate cancer prevention and/or treat-
ment.12 Because of our ongoing interest in the search
for novel antitumor compounds arising from anthranilic
acid, we started a study aimed to evaluate new deriva-
tives bearing the FA motif. Recently, we have reported
the preliminary results of the noteworthy anticancer
activity demonstrated by some aromatic esters of N-(2-
trifluoromethylpyridin-4-yl)anthranilic acid, which pos-
sess the aza skeleton of FA.13 Encouraged by the above
Scheme 1. Synthesis of Compounds 4-7a
a Reagents and conditions: (i) MeCN, reflux; (ii) 120 °C; (iii) DMF-DMA, 3 equiv, toluene, reflux; (iv) ammonium acetate, 2 equiv,
DMF, reflux; (v) 10% aq NaOH, reflux, then H3O+; (vi) anhyd EtOH, SOCl2, 4 equiv, reflux.

Anticancer Anthranilates
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 26 8247
Scheme 2. Synthesis of the Aryl Esters 8-31a
Table 1. Selected Growth Inhibitory Properties of Tested
Compounds in the NCI’s Full Cancer Cell Line Panel Screening
and the Average pGI50 Value over All Cell Linesa
pGI50 (M)b
leu- lung colon CNS
kemia NCI- SW- SF-
mela-
noma
renal breast MG_
compd K-562 H23 620 268 UACC-62 SN12C MCF-7 MIDc
10
17
20
21
22
23
24
25
27
31
5.57 4.70 5.09 4.69
6.13 5.69 5.39 5.33
7.29 6.59 6.44 5.65
5.49 5.62 5.36 5.52
5.44 5.55 5.18 5.36
5.52 5.45 5.34 5.51
5.62 5.59 5.34 5.41
7.37 7.44 7.39 6.48
5.34 5.06 5.43 5.00
5.29 5.20 5.26 5.25
4.77
5.51
6.51
5.69
5.58
5.64
5.76
6.54
5.21
5.24
4.63
5.27
6.48
5.53
5.53
5.52
5.51
7.15
5.39
5.19
4.47
5.14
6.55
5.32
5.43
5.44
5.33
7.34
5.44
5.57
4.67
5.39
6.21
5.28
5.08
5.24
5.28
6.38
4.90
4.84
a Data obtained from the NCI’s in vitro disease-oriented human
tumor cells screen. b pGI50 is the -log of the molar concentration
that cause 50% inhibition of net cell growth. c MG_MID ) mean
graph midpoint ) arithmetical mean value for all tested cell lines.
pounds that reduced the growth of any one of the cell
lines to less than 32% were then evaluated in the full
cell line panel. Compounds 10, 17, 20-25, 27, and 31,
which fulfilled this condition, were assayed for their
antiproliferative activity against a panel of almost 60
cell lines derived from leukemia, lung, colon, CNS,
melanoma, ovarian, renal, prostate, and breast human
cancers. In the full cell line panel screening, the test
compounds were evaluated using five concentrations at
10-fold dilutions, ranging from 10-4 to 10-8 M. For each
compound, anticancer activity was deduced from dose-
response curves and expressed by three parameters
(GI50, TGI, LC50) calculated for each cell line. The GI50
value indicates the concentration of the compound
required to cause 50% inhibition of net cell growth. The
TGI value represents the concentration of the compound
resulting in total inhibition of net cell growth. The LC50
value refers to the concentration of the compound
leading to 50% net cell death. Moreover, for each
antitumor activity parameter, the mean graph midpoint
(MG_MID) was calculated giving an averaged activity
parameter over all cell lines. For the calculation of
MG_MID, insensitive cell lines were included with the
highest concentration tested. Selective activity of a
compound against a certain cancer cell line from a
specific organ is characterized by a high deviation of the
particular cell line parameter compared to the MG_MID
value. Table 1 shows the GI50 values of a subset of the
60 cell line panel, as well as the MG_MID values over
all cell lines. As reported in Table 1, tested compounds
exhibit moderate to excellent activity in the full cell line
panel screening, expressed by pGI50 MG_MID values
ranging from 4.67 (compound 10) to 6.38 (compound 25).
Although compounds 10, 27, and 31 possess moderate
activity, all the other tested compounds show antipro-
liferative activity with pGI50 MG_MID values higher
than 5.0. Compounds 20 and 25 demonstrate the best
inhibitory properties with pGI50 MG_MID value of 6.21
and 6.38, respectively, which compares favorably to
standard compounds such as etoposide (6.00), fluoro-
uracil (6.05), and 6-mercaptopurine (5.13). Compounds
20 and 25 also exhibit very potent growth inhibitory
effects at nanomolar concentrations against various cell
lines. The chemosensitive profile across the full cell line
panel reveals that all the tested compounds showed
particular efficacy against colonic SW-620 cell line, with
a Reagents: (i) DCC, CHCl3.
Attempts to favor the reaction by either prolonging
reaction time or heating the reactant mixture without
solvent resulted in poor yields or extensive decomposi-
tion of the starting materials. However, upon heating
the mixture of methyl ester 1b15 and 2 in 1:5 molar ratio
without solvent, nicotinate 3b was successfully obtained
in 82% yield. Upon reacting with an excess of N,N-
dimethylformamide dimethylacetal (DMF-DMA) in re-
fluxing toluene, compounds 3a,b were converted into
1,1,1-trifluorohexadienones 4a,b in 92 and 84% yields,
respectively. Compound 3a was converted in a one-pot
procedure into ester 4a, resulting in an esterification-
condensation reaction sequence. Upon treatment with
ammonium acetate in hot DMF, the key intermediates
4a,b readily underwent pyridine ring closure to give
compounds 5a,b in 86 and 82% yields, respectively.
Hydrolysis of 5a,b in 10% aqueous sodium hydroxide
afforded acids 6a,b. Upon refluxing an ethanolic solu-
tion of 6a,b in the presence of thionyl chloride, the ethyl
esters 7a,b were obtained in 93 and 91% yields, respec-
tively. Finally (Scheme 2), treatment of FA and 6a,b
with appropriate phenol in the presence of DCC in
chloroform solution afforded the ester derivatives 8-31
in moderate to good yields.
Biological Results and Discussion
In Vitro Antitumor Activity. The in vitro growth
inhibition and cytotoxicity assay was performed by the
NCI according to well-established procedures.16-19 First,
selected compounds were evaluated in a one dose
primary anticancer screen at 10-4 M concentration for
their in vitro cytotoxic activity against NCI-H460 (non-
small cell lung), MCF7 (breast), and SF-268 (central
nervous system, CNS) human cancer cell lines. Com-

8248 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 26
Congiu et al.
Table 2. Hollow Fiber Assay for 25
ip score
14
sc score
14
total score
28
cell kill
no
Similarly, with respect to 15 (log P ) 5.70), 25 (log P )
4.74) showed 240-fold enhancement of potency, while
31 (log P ) 4.08) was 35-fold less potent than 25.
Probably, electronic and/or steric effects, due to ester
moiety, although important, remain no longer the main
structural determinant for the observed antiprolifera-
tive effect. However, differences in hydrophilicity due
to the introduction of pyridine ring(s) appear to be more
important in affecting both potency and selectivity in
these classes of compound.
In Vivo Antitumor Activity. Compound 25, the
most potent of the series, showed interesting in vitro
chemosensitive profile with a number GI50 values at
concentrations lower than 10-7 M in the full panel of
human tumor cell lines. Leukemia (3/4) and colon (5/7)
cancers were the more sensitive, renal (3/8), breast (2/
7), melanoma (2/7), CNS (1/6), ovarian (1/6), and lung
(1/9) cancers were the more resistant tumor types.
Moreover, 25 exhibited noteworthy selectivity, with
inhibitory values in submicromolar range, against cer-
tain colon cell lines at TGI (COLO 205, HCT-116, and
KM12) and LC50 (COLO 205, and KM12) levels. To
evaluate whether the in vitro activity of aryl N-(trifluo-
romethylpyridine)anthranilates can be translated into
anticancer efficacy in human tumor in vivo, compound
25 was assayed by NCI in further in vivo experiments
as described below.
Hollow Fiber Assay. Compound 25, found to have
reproducible activity in the in vitro anticancer drug
screening, was evaluated by NCI in the hollow fiber
assay as the preliminary in vivo experiment, which
provides quantitative indications of drug efficacy.22,23 In
the hollow fiber model, polyvinylidene fluoride fibers
containing various human cancer cell cultures were
implanted intraperitoneally (ip) and subcutaneously (sc)
into athymic nude mice and compounds were adminis-
trated by ip route. The effects of the compounds on
reduction of viable cancer cell mass compared to those
of controls were determined. To simplify evaluation, the
NCI protocol adopts a point system that allows rapid
viewing of the activity of a given compound. For this, a
value of 2 is assigned for each compound dose that
results in a 50% or greater reduction in viable cell mass.
Compounds with a combined ip + sc score g 20, a sc
score g 8, or a net cell kill of one or more cell lines were
considered significantly active. Compound 25 exhibited
a combined ip + sc score of 28, with low cytotoxicity (no
cell kill) (Table 2).
Figure 3. Correlation between the averaged activity (pGI50
MG_MID) parameter and calculated log P values for repre-
a
sentative anthranilates. Extrapolated value from the three-
cell-line prescreen.
GI50 values ranging from nanomolar to low micromolar
concentrations. A similar pattern of activity was also
found against leukemic K-562 cell line. This result
acquires notable significance, since previous studies
indicated that K-562 cells are resistant to the cytotoxic
effect of a variety of different agents, including cytara-
bine and etoposide.20
Among (hetero)aryl ester derivatives of FA, only
2-chlorophenyl ester 10 was endowed with moderate
activity. Although carboxylic acids 6, as well as alkyl
esters 5 and 7, appeared devoid of cytotoxicity, their
conversion into (hetero)aryl esters resulted in genera-
tion of antiproliferative agents. Within the series of the
N-(trifluoromethylpyridine)anthranilates, compounds
17 and 20-25 exhibited good to excellent inhibitory
properties, with 2-chlorophenyl (compound 20) and
pyridin-3-yl (compound 25) esters being the most potent
agents. In contrast, among the nicotinic esters, 2-chlo-
rophenyl and pyridin-3-yl esters, compounds 27 and 31
respectively, revealed moderate to good inhibitory activ-
ity. The 2-chlorine substituent on the phenoxy group
appears to be the most favorable to produce different
extents of antiproliferative efficacy within the three
series of derivatives. Both shifting of the 2-chlorine atom
to other positions of phenyl ring and introducing further
chlorine atoms result in decreasing activity of esters
21-24. In contrast, the same changes on both flufe-
namic and nicotinic esters result in loss of antiprolif-
erative effect. Replacement of the chlorine atom with
an electron-donating substituent, such as the methoxy
group, results in inactive compounds, with the exception
of 3-methoxyphenyl ester 17, which maintains a good
level of activity. Esterification with 3-hydroxypyridine
produces different effects. Flufenamate 15 is devoid of
activity and nicotinate 31 shows moderate cytotoxicity,
while the N-(trifluoromethylpyridine)anthranilate 25
exhibits the highest levels of inhibitory activity among
all the tested compounds.
Moreover, a correlation can be found between the
averaged activity parameter and increasing hydrophi-
licity, expressed by calculated log P values,21 due to
introduction of pyridine ring(s) (Figure 3).
With respect to flufenamic ester 10 (log P ) 7.44), a
single isosteric modification of the phenyl bearing a
trifluoromethyl group with a pyridine ring to give
compound 20 (log P ) 6.48) produced 30-fold enhance-
ment of inhibitory potency. In contrast, the introduction
of a further nitrogen atom in the anthranilic moiety
resulted in reduction of activity. In fact, nicotinic ester
27 (log P ) 5.82) was 20-fold less potent than 20.
Activity against Human Tumor Xenografts. Ac-
cording to the results of the in vitro antiproliferative
activity and in vivo hollow fiber assay, the human
melanoma xenograft LOX IMVI, human nonsmall cell
lung tumor xenograft H522, and human CNS tumor
xenograft U251 were selected by NCI as potentially
sensitive tumor types to evaluate the in vivo efficacy of
compound 25. In the NCI’s protocol, human tumor cells
were implanted subcutaneously into the axillary region
of pathogen-free athymic nude mice and allowed to
establish into a sizable tumor as determined by calliper
measurements. Compound 25 was formulated as a

Anticancer Anthranilates
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 26 8249
Table 3. Antitumor Activity and Toxicity Data in Human CNS
proliferative effect of compounds 10 and 25 and, by
inference, of the other active compounds. Nevertheless,
all the tested compounds also displayed remarkable
inhibitory activity against HCT-15 and HCT-116 colon
cancer cell lines, which, in contrast, have been found
intrinsically deficient in COX-1 and COX-2 expres-
sion.27,28 Taken together, these data indicate that
compounds herein described could exert their antipro-
liferative activity through COX-dependent mechanisms,
and suggest that their cytotoxicity might result in
targeting other proteins or pathways.
COMPARE Analysis. The patterns of activity of
compounds 10, 17, 20, 25, 27, and 31 were also analyzed
by the COMPARE algorithm.29 COMPARE searches the
NCI database of screened agents for those most similar
to the tested compound in their patterns of activity
against the panel of 60 cell lines. Similarity in pattern
often indicates similarity in mechanism of action and
molecular structure. In this analysis, a Pearson cor-
relation coefficient (PCC) > 0.60 is considered signifi-
cant. When tested as seeds against the NCI “Standard
Agents” Database (Table 4), compounds 10, 17, and 27
show, with PCC > 0.60 at GI50 level, a response pattern
that correlated their activity to those of DNA antime-
tabolite agents, including inosine dialdehyde, 6-mer-
captopurine, and 2′-deoxy-6-thioguanosine. COMPARE
analysis also indicates that pyridyl esters 25 and 31
shared a response pattern with topoisomerase II inhibi-
tors, including morpholino-ADR and etoposide. Interest-
ingly, results of the COMPARE procedure indicate that
the tested compounds might exert their antiproliferative
activity through alteration and/or inhibition of processes
crucial for the cell cycle progression.
Tumor Xenograft U251 for 25a
no.
of
opt
%
dose,
%T/C growth max %
drug
mg/kg/day mice route schedule (d)b
delayc wt loss deathsd
100
67
10
10
ip
Q4D × 3, 27 (24)
67
15
no wt loss
no wt loss
0
0
day 5
ip Q4D × 3, 75 (24)
day 5
a Data obtained from the NCI Division of Cancer Treatment and
Diagnosis. b Opt % T/C (d): optimum tumor weight of treated/
control animals in percent (day). c Tumor growth delay according
to the formula [(T - C)/C] × 100. d Number of mice in the
treatment group which were lost as result of test agent related
toxicity.
solution in 10% DMSO in saline/Tween 80 and admin-
istrated by ip route. In the CNS tumor xenograft U251,
25 was administrated in groups of 10 mice. A group of
20 mice served as a vehicle control group. Doses of 100,
67, and 45 mg/kg were administered every 4 days, for a
total of three treatments, with the first treatment given
on day 5 postimplant (Q4D × 3, day 5) (Table 3). All
the ip dosages were very well tolerated and resulted in
no signs of toxicity or body weight loss (maximally
tolerated dose was not achieved). Treatment with 25
resulted in moderate growth inhibition (67% of control
at 100 mg/kg/day). In contrast, the melanoma xenograft
LOX IMVI and human nonsmall cell lung tumor xe-
nograft H522 were resistant toward therapy with
compound 25.
On the basis of structural similarity, the possibility
exists that anthranilate derivatives herein described
share some pharmacological properties with NSAIDs
fenamates, i.e., COX-inhibitory activity, which ac-
counted, at least in part, for the observed anticancer
activity. To investigate whether these compounds are
endowed with NSAIDs-type activity, preliminary ex-
periments (data not shown) were carried out on 2-chlo-
rophenyl ester 10 and pyridyl ester 25. In the acetic
acid-induced writhing test in rats,24 after intraperito-
neal administration, the tested compounds exhibited
inhibition of stretching on the same order of magnitude
as compared to flufenamic acid. In addition, all com-
pounds that were active in the full cell line panel
screening exhibited noteworthy activity against both
SW-620 colon cancer cells, which express a high level
of COX-2 protein,25 and MCF-7 breast cancer cells,
which constitutively express COX-1.26 These results
provide evidence supporting the hypothesis that COX-
inhibitory activity may be involved, at least in part, in
biochemical mechanisms, which resulted in the anti-
Conclusions
An efficient synthesis of new anthranilic acid deriva-
tives led us to identify a series of potential anticancer
agents. The in vitro anticancer screening performed by
the NCI reveals that some esters of N-(2-(trifluoro-
methyl)pyridin-4-yl)anthranilic acid demonstrated in-
teresting inhibitory properties against a wide array of
human tumor cell lines. In particular, compounds 17,
20, and 25 exhibited antiproliferative activity in nano-
molar to low micromolar concentrations against most
of the tested cell lines. On the basis of observed
biological activities and COMPARE analysis, putative
COX-dependent/independent mechanisms responsible
for antitumor activity were proposed.
Table 4. Results of COMPARE Analysis Procedure against the NCI Standard Agents Database, Using Compounds 10, 17, 20, 25, 27,
and 31 as Seeds
no. of
common
cell lines
endpoint
level
compd
standard agent
mechanism of action
correlation
10
17
inosine dialdehyde
vincristine
6-mercaptopurine
flavone acetic acida
2-deoxy-6-thioguanosine
morpholino-ADR
vincristine
DNA antimetabolite
antimitotic agent
GI50
GI50
GI50
GI50
GI50
GI50
TGI
GI50
GI50
0.61
0.68
0.67
0.68
0.58
0.62
0.61
0.66
0.61
41
48
35
32
32
21
31
38
34
DNA antimetabolite
20
25
DNA antimetabolite
topoisomerase II inhibitor
antimitotic agent
DNA antimetabolite
topoisomerase II inhibitor
27
31
macbecin II
etoposide
a NCI’s Standard Agents Database does not report the mechanism of action. Recently, it has been reported that flavone acetic acid
induces a G2/M cell cycle arrest in mammary carcinoma cells.30

8250 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 26
Congiu et al.
FA and 2-methoxyphenol: 0.225 g, yield 58%; mp 43-45 °C
(petroleum ether). 1H NMR (DMSO-d6) δ 3.89 (s, 3H, CH3),
6.87 (m, 1H), 7.13 (m, 2H), 7.30-7.68 (m, 8H), 8.28 (m, 1H),
9.38 (s, 1H, NH); IR (Nujol) 3330 (NH), 1700 (CO), 1630, 1582
cm-1. Anal. (C21H16F3NO3) C, H, N.
Flufenamic Acid 3-Methoxyphenyl Ester (9). Following
the general procedure, the title compound was obtained from
FA and 3-methoxyphenol: 0.256 g, yield 66%; mp 47-48 °C
(petroleum ether); 1H NMR (DMSO-d6) δ 3.88 (s, 3H), 6.46 (m,
1H), 6.97 (m, 3H), 7.12 (m, 1H), 7.46 (m, 3H), 7.64 (m, 3H),
8.26 (m, 1H), 9.38 (s, 1H, NH); IR (Nujol) 3329 (NH), 1699
(CO), 1582 cm-1. Anal. (C21H16F3NO3) C, H, N.
Flufenamic Acid 2-Chlorophenyl Ester (10). Following
the general procedure, the title compound was obtained from
FA and 2-chlorophenol: 0.207 g, yield 53%; mp 64-66 °C (n-
hexane); 1H NMR (DMSO-d6) δ 7.15 (m, 1H), 7.49 (m, 3H),
7.57 (m, 2H), 7.68 (m, 3H), 7.74 (m, 2H), 8.32 (m 1H), 9.30 (s,
1H, NH); IR (Nujol) 3321 (NH), 1702 cm-1. Anal. (C20H13ClF3-
NO2) C, H, N.
Flufenamic Acid 3-Chlorophenyl Ester (11). Following
the general procedure, the title compound was obtained from
FA and 3-chlorophenol: 0.180 g, yield 46%; mp 56-58 °C
(petroleum ether); 1H NMR (DMSO-d6) δ 7.14 (m, 1H), 7.38-
7.70 (m, 10H), 8.26 (m, 1H), 9.31 (s, 1H, NH); IR (Nujol) 3322
(NH), 1700 (CO) cm-1. Anal. (C20H13ClF3NO2) C, H, N.
Experimental Section
Chemistry. Melting points were determined on a Stuart
Scientific Melting point SMP1 and are uncorrected. Infrared
spectra were run on Bruker Vector 22 spectrophotometer.
Absorption band position is given in cm-1 1H NMR spectra
.
were recorded on a Varian Unity 300 spectrometer (300 MHz).
Chemical shifts are expressed in ppm relative to tetrameth-
ylsilane. Silica gel thin-layer chromatography (TLC) sheets
from Fluka (silica gel precoated aluminum sheets with fluo-
rescent indicator at 254 nm) were used for TLC. Developed
plates were visualized by a Spectroline ENF 260C/F UV
apparatus. Concentration and evaporation of the solvent after
reaction or extraction were carried out on a rotary evaporator
(Bu¨chi Rotavapor) operating at reduced pressure. Petroleum
ether refers to the 40-60 °C boiling range fractions. Elemental
analyses were carried out with a Carlo Erba model 1106
elemental analyzer and the values found to be within 0.4% of
the theoretical values.
General Procedure for the Synthesis of 5a,b. To a
solution of 4a or 4b (2 mmol) in dry DMF (5 mL) was added
ammonium acetate (0.308 g, 4 mmol) and the mixture was
gently refluxed for 3 h. The mixture was carefully concentrated
in vacuo and to the residue was added ice-water (15 mL). The
formed solid was filtered off, washed with water, air-dried, and
then crystallized from n-hexane to give 5a or 5b.
N-(2-(Trifluoromethyl)pyridin-4-yl)anthranilic acid
methyl ester (5a): 0.510 g, 86%, mp 87-89 °C. Anal.
(C14H11F3N2O2) C, H, N.
2-(2-(Trifluoromethyl)pyridin-4-ylamino)nicotinic acid
methyl ester (5b): 0.487 g, 82%, mp 110-112 °C. Anal.
(C13H10F3N3O2) C, H, N.
Flufenamic acid 4-Chlorophenyl Ester (12). Following
the general procedure, the title compound was obtained from
FA and 4-chlorophenol: 0.282 g, yield 72%; mp 76-78 °C
(petroleum ether); 1H NMR (DMSO-d6): δ 7.13 (m, 1H), 7.43-
7.70 (m, 10H), 8.26 (m, 1H), 9.32 (s, 1H, NH); IR (Nujol) 3318
(NH), 1693 (CO) cm-1. Anal. (C20H13ClF3NO2) C, H, N.
General Procedure for the Hydrolysis of 5a,b. A
mixture of 5a or 5b (1 mmol) in 10% aqueous sodium hydroxide
(15 mL) was refluxed for 30 min, during which a homogeneous
solution was formed. After cooling, the solution was acidified
with 20% aqueous hydrochloric acid to pH 3-4. The formed
solid was filtered off, washed with water, air-dried, and
crystallized from ethanol to give 6a or 6b.
N-(2-(Trifluoromethyl)pyridin-4-yl)anthranilic acid
(6a): 0.260 g, 92%, mp 222-224 °C. Anal. (C13H9F3N2O2) C,
H, N.
Flufenamic Acid 2,4-Dichlorophenyl Ester (13). Fol-
lowing the general procedure, the title compound was obtained
from FA and 2,4-dichlorophenol: 0.336 g, yield 79%; mp 104-
1
106 °C (isopropyl ether); H NMR (DMSO-d6) δ 7.14 (m, 1H),
7.49 (m, 2H), 7.67 (m, 6H), 7.96 (s, 1H), 8.32 (m, 1H), 9.27 (s,
1H, NH); IR (Nujol) 3326 (NH), 1703 (CO) cm-1. Anal. (C20H12-
Cl2F3NO2) C, H, N.
Flufenamic Acid 2,4,6-Trichlorophenyl Ester (14). Fol-
lowing the general procedure, the title compound was obtained
from FA and 2,4,6-trichlorophenol: 0.290 g, yield 63%; mp
128-130 °C (isopropyl ether); 1H NMR (DMSO-d6) δ 7.15 (m,
1H), 7.48 (m, 2H), 7.74 (m, 4H), 8.03 (s, 1H), 8.36 (m, 1H),
9.23 (s, 1H, NH); IR (Nujol) 3290 (NH), 1716 (CO) cm-1. Anal.
(C20H11Cl3F3NO2) C, H, N.
2-(2-(Trifluoromethyl)pyridin-4-ylamino)nicotinic acid
(6b): 0.252 g, 89%, mp 242-244 °C. Anal. (C12H8F3N3O2) C,
H, N.
General Procedure for the Synthesis of 7a,b. To an ice-
cooled solution of 6a or 6b (1 mmol) in dry ethanol (10 mL)
was added thionyl chloride (0.476 g, 4 mmol) dropwise with
stirring. The ice bath was removed and the mixture was
refluxed for 6 h. The volatile components were carefully
eliminated in vacuo, and to the residue was added aqueous
sodium bicarbonate (15 mL, saturated). The formed solid was
filtered off, washed with water, air-dried, and then crystallized
from n-hexane to give 7a or 7b.
Flufenamic Acid Pyridin-3-yl Ester (15). Following the
general procedure, the title compound was obtained from FA
and 3-hydroxypyridine: 0.262 g, yield 73%; mp 46-48 °C
1
(petroleum ether); H NMR (DMSO-d6) δ 7.14 (m, 1H), 7.47
(m, 2H), 7.65 (m, 5H), 7.91 (m, 1H), 8.30 (m, 1H), 8.67 (m,
2H), 9.30 (s, 1H, NH); IR (Nujol) 3334 (NH), 1702 (CO), 1584
cm-1. Anal. (C19H13F3N2O2) C, H, N.
N-(2-(Trifluoromethyl)pyridin-4-yl)anthranilic Acid
2-Methoxyphenyl Ester (16). Following the general proce-
dure, the title compound was obtained from 6a and 2-meth-
oxyphenol: 0.248 g, yield 64%; mp 64-66 °C (isopropyl ether);
1H NMR (DMSO-d6) δ 7.38 (m, 1H), 7.68 (m, 3H), 8.16 (m,
1H), 8.53 (s, 1H), 8.68 (m, 1H), 8.83 (m, 2H), 10.41 (s, 1H, NH);
IR (Nujol) 3277 (NH), 1702 (CO), 1623, 1587 cm-1. Anal.
(C20H15F3N2O3) C, H, N.
N-(2-(Trifluoromethyl)pyridin-4-yl)anthranilic Acid
3-Methoxyphenyl Ester (17). Following the general proce-
dure, the title compound was obtained from 6a and 3-meth-
oxyphenol: 0.280 g, yield 72%; mp 86-88 °C (isopropyl ether);
1H NMR (DMSO-d6) δ 3.87 (s, 3H, CH3), 6.88-7.87 (m, 9H),
8.28 (m, 1H), 8.49 (m, 1H), 9.54 (s, 1H, NH); IR (Nujol) 3288
(NH), 1703 (CO), 1610, 1588 cm-1. Anal. (C20H15F3N2O3) C,
H, N.
N-(2-(Trifluoromethyl)pyridin-4-yl)anthranilic acid eth-
yl ester (7a): 0.288 g, 93%, mp 82-84 °C. Anal. (C15H13F3N2O2)
C, H, N.
2-(2-(Trifluoromethyl)pyridin-4-ylamino)nicotinic acid
ethyl ester (7b): 0.283 g, 91%, mp 65-67 °C. Anal.
(C14H12F3N3O2) C, H, N.
General Procedure for the Synthesis of 8-31. A mix-
ture of FA, 6a or 6b (1 mmol), and DCC (0.227 g, 1.1 mmol) in
dry chloroform (10 mL) was stirred at room temperature for 1
h and then treated with the appropriate phenol (1.1 mmol).
The mixture was stirred at room temperature for an additional
24 h. The formed precipitate was eliminated by filtration and
the solution evaporated to dryness in vacuo. To the residue
was added aqueous hydrochloric acid (20 mL, 1 M) and the
mixture was extracted with ethyl ether (3 × 10 mL). The
combined organic layers were washed with brine and then
dried over anhydrous magnesium sulfate. Concentration of the
dried extracts yielded a residue which was purified by crystal-
lization to give the ester derivatives 8-31.
N-(2-(Trifluoromethyl)pyridin-4-yl)anthranilic Acid
4-Methoxyphenyl Ester (18). Following the general proce-
dure, the title compound was obtained from 6a and 4-meth-
oxyphenol: 0.299 g, yield 77%; mp 103-105 °C (isopropyl
ether); 1H NMR (DMSO-d6) δ 3.46 (s, 3H, CH3), 7.08-7.86 (m,
Flufenamic Acid 2-Methoxyphenyl Ester (8). Following
the general procedure, the title compound was obtained from

Anticancer Anthranilates
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 26 8251
9H), 8.27 (m, 1H), 8.49 (m, 1H), 9.55 (s, 1H, NH); IR (Nujol)
3323 (NH), 1695 (CO), 1613, 1588 cm-1. Anal. (C20H15F3N2O3)
C, H, N.
3277 (NH), 1702 (CO), 1623, 1587 cm-1. Anal. (C18H11-
ClF3N3O2) C, H, N.
2-(2-(Trifluoromethyl)pyridin-4-ylamino)nicotinic acid
3-Chlorophenyl Ester (28). Following the general procedure,
the title compound was obtained from 6b and 3-chlorophenol:
N-(2-(Trifluoromethyl)pyridin-4-yl)anthranilic Acid
4-Methylthiophenyl Ester (19). Following the general pro-
cedure, the title compound was obtained from 6a and 4-me-
thylthiophenol: 0.328 g, yield 81%; mp 112-114 °C (isopropyl
ether/MeOH 3:1); 1H NMR (DMSO-d6) δ 3.45 (s, 3H, CH3),
7.28-7.88 (m, 9H), 8.29 (m, 1H), 8.50 (m, 1H), 9.54 (s, 1H,
NH); IR (Nujol) 3317 (NH), 1697 (CO), 1612, 1591 cm-1. Anal.
(C20H15F3N2O2S) C, H, N.
N-(2-(Trifluoromethyl)pyridin-4-yl)anthranilic Acid
2-Chlorophenyl Ester (20). Following the general procedure,
the title compound was obtained from 6a and 2-chlorophenol:
0.306 g, yield 78%; mp 132-134 °C (isopropyl ether); 1H NMR
(DMSO-d6) δ 7.30 (m, 1H), 7.51 (m, 4H), 7.56 (m, 1H), 7.72
(m, 2H), 7.87 (m, 1H), 8.35 (m, 1H), 8.48 (m, 1H), 9.49 (s, 1H,
NH); IR (Nujol) 3323 (NH), 1704, 1614, 1596 cm-1. Anal.
(C19H12ClF3N2O2) C, H, N.
1
0.268 g, yield 68%; mp 68-70 °C (isopropyl ether); H NMR
(DMSO-d6) δ 7.34 (m, 1H), 7.69-7.47 (m, 4H), 8.13 (m, 1H),
8.51 (s, 1H), 8.74 (m, 3H), 10.43 (s, 1H, NH); IR (Nujol) 3288
(NH), 1703 (CO), 1610, 1588 cm-1. Anal. (C18H11ClF3N3O2) C,
H, N.
2-(2-(Trifluoromethyl)pyridin-4-ylamino)nicotinic Acid
4-Chlorophenyl Ester (29). Following the general procedure,
the title compound was obtained from 6b and 4-chlorophenol:
1
0.315 g, yield 80%; mp 86-88 °C (isopropyl ether); H NMR
(DMSO-d6) δ 7.35 (m, 1H), 7.53 (d, J ) 8.2 Hz, 2H), 7.69 (d, J
) 8.2 Hz, 2H), 8.10 (m, 1H), 8.52 (s, 1H), 8.79 (m, 3H), 10.45
(s, 1H, NH); IR (Nujol) 3283 (NH), 1695 (CO), 1610, 1584 cm-1
Anal. (C18H11ClF3N3O2) C, H, N.
.
2-(2-(Trifluoromethyl)pyridin-4-ylamino)nicotinic Acid
2,4-Dichlorophenyl Ester (30). Following the general pro-
cedure, the title compound was obtained from 6b and 2,4-
dichlorophenol: 0.368 g, yield 86%; mp 116-118 °C (isopropyl
N-(2-(Trifluoromethyl)pyridin-4-yl)anthranilic Acid
3-Chlorophenyl Ester (21). Following the general procedure,
the title compound was obtained from 6a and 3-chlorophenol:
1
ether); H NMR (DMSO-d6) δ 7.36 (s, 1H), 7.73 (s, 2H), 8.00
1
0.314 g, yield 80%; mp 80-82 °C (isopropyl ether); H NMR
(s, 1H), 8.13 (m, 1H), 8.52 (s, 1H), 8.79 (m, 3H), 10.34 (bs, 1H,
NH); IR (Nujol) 3298 (NH), 1716 (CO), 1610, 1587 cm-1. Anal.
(C18H10Cl2F3N3O2) C, H, N.
(DMSO-d6) δ 7.32 (m, 3H), 7.48 (m, 3H), 7.60 (m, 1H), 7.73
(m, 1H), 7.85 (m, 1H), 8.29 (m, 1H), 8.49 (m, 1H), 9.51 (s, 1H,
NH); IR (Nujol) 3322 (NH), 1700 (CO) cm-1. Anal. (C19H12-
ClF3N2O2) C, H, N.
2-(2-(Trifluoromethyl)pyridin-4-ylamino)nicotinic Acid
Pyridin-3-yl Ester (31). Following the general procedure, the
title compound was obtained from 6b and 3-hydroxypyridine:
N-(2-(Trifluoromethyl)pyridin-4-yl)anthranilic Acid
4-Chlorophenyl Ester (22). Following the general procedure,
the title compound was obtained from 6a and 4-chlorophenol:
1
0.278 g, yield 77%; mp 96-98 °C (isopropyl ether); H NMR
(DMSO-d6) δ 4.05 (s, 3H, CH3), 7.36 (m, 1H), 7.71 (m, 1H),
8.01 (m, 1H), 8.13 (m, 1H), 8.52 (s, 1H), 8.69 (m, 2H), 8.78 (m,
3H), 10.42 (s, 1H, NH); IR (Nujol) 3263 (NH), 1722 (CO), 1597
cm-1. Anal. (C17H11F3N4O2) C, H, N.
1
0.362 g, yield 92%; mp 84-85 °C (isopropyl ether); H NMR
(DMSO-d6) δ 7.30 (m, 1H), 7.40 (m, 2H), 7.46 (d, J ) 8.8 Hz,
2H), 7.64 (d, J ) 8.8 Hz, 2H), 7.73 (m, 1H), 7.85 (m, 1H), 8.29
(m, 1H), 8.49 (m, 1H), 9.51 (s, 1H, NH); IR (Nujol) 3321 (NH),
1697 (CO) 1613, 1592 cm-1. Anal. (C19H12ClF3N2O2) C, H, N.
Acknowledgment. The authors thank the Devel-
opmental Therapeutics Program of the National Cancer
Institute, Bethesda, MD, for providing the in vitro and
in vivo antitumor screening data.
N-(2-(Trifluoromethyl)pyridin-4-yl)anthranilic Acid
2,4-Dichlorophenyl Ester (23). Following the general pro-
cedure, the title compound was obtained from 6a and 2,4-
dichlorophenol: 0.291 g, yield 68%; mp 100-102 °C (isopropyl
1
ether/MeOH 3:1); H NMR (DMSO-d6) δ 7.31 (m, 1H), 7.48-
Supporting Information Available: Experimental de-
tails and structural assignment for compounds 3 and 4.
Elemental analyses, spectral data, and tables of NCI’s in vitro
testing results. This material is available free of charge via
the Internet at http://pubs.acs.org.
7.85 (m, 7H), 8.35 (m, 1H), 8.49 (m, 1H), 9.47 (s, 1H, NH); IR
(Nujol) 3325 (NH), 1698 (CO) 1628, 1588 cm-1. Anal. (C19H11-
Cl2F3N2O2) C, H, N.
N-(2-(Trifluoromethyl)pyridin-4-yl)anthranilic Acid
2,4,6-Trichlorophenyl Ester (24). Following the general
procedure, the title compound was obtained from 6a and 2,4,6-
trichlorophenol: 0.259 g, yield 56%; mp 120-122 °C (isopropyl
ether/MeOH 3:1); 1H NMR (DMSO-d6) δ 7.32 (m, 3H), 7.54
(m, 2H), 7.77 (m, 2H), 7.91 (m, 1H), 7.99 (s, 1H), 8.43 (m, 1H),
8.49 (m, 1H), 9.45 (s, 1H, NH); IR (Nujol) 3328 (NH), 1724
(CO) 1613, 1593 cm-1. Anal. (C19H11Cl3F3N2O2) C, H, N.
References
(1) Shime, H.; Kariya, M.; Orii, A.; Momma, C.; Kanamori, T.;
Fukuhara, K.; Kusakari, T.; Tsuruta, Y.; Takakura, K.; Nikaido,
T.; Fujii, S. Tranilast inhibits the proliferation of uterine
leiomyoma cells in vitro through G1 arrest associated with the
induction of p21(waf1) and p53. J. Clin. Endocrinol. Metab. 2002,
87, 5610-5617.
(2) Yashiro, M.; Murahashi, K.; Matsuoka, T.; Nakazawa, K.;
Tanaka, H.; Osaka, H.; Koyama, T.; Ohira, M.; Chung, K. H.
Tranilast (N-3,4-dimethoxycinamoyl anthranilic acid): A novel
inhibitor of invasion-stimulating interaction between gastric
cancer cells and orthotopic fibroblasts. Anticancer Res. 2003, 23,
3899-904.
(3) Mo, H.; Tatman, D.; Jung, M.; Elson, C. E. Farnesyl anthranilate
suppresses the growth, in vitro and in vivo, of murine B16
melanomas. Cancer Lett. 2000, 157, 145-153.
N-(2-(Trifluoromethyl)pyridin-4-yl)anthranilic Acid
Pyridin-3-yl Ester (25). Following the general procedure, the
title compound was obtained from 6a and 3-hydroxypyridine:
1
0.298 g, yield 83%; mp 66-68 °C (isopropyl ether); H NMR
(DMSO-d6) δ 7.31 (m, 1H), 7.47 (m, 2H), 7.65 (m, 1H), 7.74
(m, 1H), 7.86 (m, 2H), 8.33 (m, 1H), 8.49 (m, 1H), 8.63 (s, 1H),
9.52 (s, 1H, NH); IR (Nujol) 3260 (NH), 1713 (CO), 1613, 1597
cm-1. Anal. (C18H12F3N3O2) C, H, N.
(4) (a) Sebolt-Leopold, J. S.; Dudley, D. T.; Herrera, R.; Van
Becelaere, K.; Wiland, A.; Gowan, R. C.; Tecle, H.; Barrett, S.
D.; Bridges, A.; Przybranowski, S.; Leopold, W. R.; Saltiel, A. R.
Blockade of the MAP kinase pathway suppresses growth of colon
tumors in vivo. Nat. Med. 1999, 5, 810-816. (b) Allen, L. F.;
Sebolt-Leopold, J.; Meyer, M. B. CI-1040 (PD184352), a targeted
signal transduction inhibitor of MEK (MAPKK). Semin. Oncol.
2003, 30, 105-116.
(5) (a) Manley, P. W.; Furet, P.; Bold, G.; Bru¨ggen, J.; Mestan, J.;
Meyer, T.; Schnell, C. R.; Wood, J. Anthranilic Acid Amides: A
Novel Class of Antiangiogenic VEGF Receptor Kinase Inhibitors
J. Med. Chem. 2002, 45, 5687-5693. (b) Manley, P. W.; Bold,
G.; Bru¨ggen, J.; Fendrich, G.; Furet, P.; Mestan, J.; Schnell, C.
R.; Stolz, B.; Meyer, T.; Meyhack, B.; Stark, W.; Strauss, A.;
Wood, J. Advances in the structural biology, design and clinical
development of VEGF-R kinase inhibitors for the treatment of
angiogenesis. Biochim. Biophys. Acta 2004, 1697, 17-27.
2-(2-(Trifluoromethyl)pyridin-4-ylamino)nicotinic Acid
3-Methoxyphenyl Ester (26). Following the general proce-
dure, the title compound was obtained from 6b and 3-meth-
oxyphenol: 0.285 g, yield 73%; mp 76-78 °C (isopropyl ether);
1H NMR (DMSO-d6) δ 4.05 (s, 3H, CH3), 7.08 (m, 2H), 8.09
(m, 1H), 8.51 (m, 3H), 8.68 (m, 4H), 10.62 (s, 1H, NH); IR
(Nujol) 3286 (NH), 1700 (CO), 1614, 1586 cm-1. Anal.
(C19H14F3N3O3) C, H, N.
2-(2-(Trifluoromethyl)pyridin-4-ylamino)nicotinic Acid
2-Chlorophenyl Ester (27). Following the general procedure,
the title compound was obtained from 6b and 2-chlorophenol:
1
0.248 g, yield 63%; mp 64-66 °C (isopropyl ether); H NMR
(DMSO-d6) δ 7.38 (m, 1H), 7.68 (m, 3H), 8.16 (m, 1H), 8.53 (s,
1H), 8.68 (m, 1H), 8.83 (m, 2H), 10.41 (s, 1H, NH); IR (Nujol)

8252 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 26
Congiu et al.
(6) Vane, J. R.; Bakhle, Y. S.; Botting, R. M. Cyclooxygenase 1 and
2. Annu. Rev. Pharmacol. Toxicol. 1998, 38, 97-120.
(7) Thun, M. J.; Henley, S. J.; Patrono, C. Nonsteroidal anti-
inflammatory drugs as anticancer agents: Mechanistic, phar-
macologic, and clinical issues. J. Natl. Cancer Inst. 2002, 94,
252-266.
(8) (a) Xu, X. C. Cox-2 inhibitors in cancer treatment and prevention,
a recent development. Anti-Cancer Drugs 2002, 13, 127-137.
(b) Gupta, R. A.; DuBois, R. N. Colorectal cancer prevention and
treatment by inhibition of cyclooxygenase-2. Nat. Rev. Cancer
2001, 1, 11-21. (c) Dannenberg, A. J.; Altori, N. K.; Boyle, J.
O.; Dang, C.; Howe, L. R.; Weksler, B. B.; Subbaramaiah, K.
Cyclo-oxygenase 2: A pharmacological target for the prevention
of cancer. Lancet Oncol. 2001, 2, 544-551. (d) Turini, M. E.;
DuBois, R. N. Cyclooxygenase-2: A therapeutic target. Annu.
Rev. Med. 2002, 53, 35-57. (e) Simmons, D. L.; Botting, R. M.;
Hla, T. Cyclooxygenase isozymes: The biology of prostaglandin
synthesis and inhibition. Pharmacol. Rev. 2004, 56, 387-437.
(9) Shiff, S. J.; Rigas, B. The Role of Cyclooxygenase inhibition in
the antineoplastic effects of nonsteroidal anti-inflammatory
drugs (NSAIDs). J. Exp. Med. 1999, 190, 445-450.
(10) Tegeder, I.; Pfeilschifter, J.; Geisslinger, G. Cyclooxygenase-
independent actions of cyclooxygenase inhibitors. FASEB J.
2001, 15, 2057-2072.
(11) (a) Weiser, T.; Wienrich, M. Investigations on the mechanism
of action of the antiproliferant and ion channel antagonist
flufenamic acid. Naunyn Schmiedebergs Arch. Pharmacol. 1996,
353, 452-460. (b) Jung, F.; Selvaraj, S.; Gargus, J. J. Blockers
of platelet-derived growth factor-activated nonselective cation
channel inhibit cell proliferation. Am. J. Physiol. 1992, 262,
C1464-C1470.
(12) Zhu, W.; Smith, A.; Young, C. Y. A nonsteroidal antiinflamma-
tory drug, flufenamic acid, inhibits the expression of the
androgen receptor in LNCaP cells. Endocrinology 1999, 140,
5451-5454).
(13) Cocco, M. T.; Congiu, C.; Onnis, V.; Lilliu, V. Synthesis of new
N-(2-(trifluoromethyl)-pyridine-4-yl)anthranilic acid derivatives
and their evaluation as anticancer agents. Bioorg. Med. Chem.
Lett. 2004, 14, 5787-5791.
(18) Weinstein, J. N.; Myers, T. G.; O’Connor, P. M.; Friend, S. H.;
Fornace, A. J. Jr.; Kohn, K. W.; Fojo, T.; Bates, S. E.; Rubinstein,
L. V.; Anderson, N. L.; Buolamwini, J. K.; van Osdol, W. W.;
Monks, A. P.; Scudiero, D. A.; Sausville, E. A.; Zaharevitz, D.
W.; Bunow, B.; Viswanadhan, V. N.; Johnson, G. S.; Wittes, R.
E.; Paull, K. D. An information-intensive approach to the
molecular pharmacology of cancer. Science 1997, 275, 343-349.
(19) Data concerning the NCI screening methods in detail are
accessible from the NCI via the Internet from following ad-
dresses: http://dtp.nci.nih.gov/branches/btb/ivclsp.html; http://
dtp.nci.nih.gov/branches/btb/hfa.html.
(20) McGahon, A.; Bissonnette, R.; Schmitt, M.; Cotter, K. M.; Green,
D. R.; Cotter, T. G. BCR-ABL maintains resistance of chronic
myelogenous leukemia cells to apoptotic cell death. Blood 1994,
83, 1179-1187.
(21) Calculated log P values were obtained by using the facility
supplied by Molinspiration cheminformatics accessible at the
Internet from following address: http://www.molinspiration.com/
services/logp.html.
(22) Plowman, J.; Dykes, D. J.; Hollingshead, M.; Simpson-Herren,
L.; Alley, M. C. In Anticancer Drug Development Guide: Pre-
clinical Screening, Clinical Trials, and Approval; Teicher, B.,
Ed.; Humana Press: Totowa, NY, 1997; pp 101-125.
(23) Hollingshead, M. G.; Alley, M. C.; Camalier, R. F.; Abbott, B.
J.; Mayo, J. G.; Malspeis, L.; Grever, M. R. In vivo cultivation
of tumor cells in hollow fibers. Life Sci. 1995, 57, 131-141.
(24) (a) Ballou, L. R.; Botting, R. M.; Goorha, S.; Zhang, J.; Vane, J.
R. Nociception in cyclooxygenase isozyme-deficient mice. Proc.
Natl. Acad. Sci. 2000, 97, 10272-10276. (b) Deraedt, R.; Jou-
quey, S.; Delevalle´e, F.; Flahaut, M. Release of prostaglandins
E and F in an algogenic reaction and its inhibition Eur. J.
Pharmacol. 1980, 61, 17-24.
(25) Li, M.; Wu, X.; Xu, X. Induction of apoptosis in colon cancer cells
by cyclooxygenase-2 inhibitor NS398 through a cytochrome
c-dependent pathway. Clin. Cancer Res. 2001, 7, 1010-1016.
(26) Liu, X. H.; Rose, D. P. Differential expression and regulation of
cyclooxygenase-1 and -2 in two human breast cancer cell lines.
Cancer Res. 1996, 56, 5125-5127.
(27) Hanif, R.; Pittas, A.; Feng, Y.; Koutsos, M. I.; Qiao, L.; Staiano-
Coico, L.; Shiff, S. I.; Rigas, B. Effects of nonsteroidal anti-
inflammatory drugs on proliferation and on induction of apop-
tosis in colon cancer cells by a prostaglandin-independent
pathway. Biochem. Pharmacol. 1996, 52, 237-245.
(14) Cocco, M. T.; Congiu, C.; Onnis, V. Annulation of functionalized
hexadienones as an efficient regioselective approach to N-aryl-
2-(trifluoromethyl)-4-pyridinamines. Tetrahedron Lett. 1999, 40,
4407-4410.
(15) Okawa, T.; Osakada, N.; Eguchi, S.; Kakehi, A. One-pot syn-
thesis of novel (2-oxo-1,2-dihydropyridin-3-yl)-1,3,5-triazine de-
rivatives from methyl 2-(N-triphenyl-phosphoranylidene)-ami-
nonicotinate, aryl isocyanate and primary amines: Sequential
aza-Wittig/cycloaddition/ring-transformation reactions. Tetra-
hedron 1997, 53, 16061-16082.
(16) Monks, A.; Scudiero, D.; Skehan, P.; Shoemaker, R.; Paull, K.;
Vistica, D.; Hose, C.; Langley, J.; Cronise, P.; Vaigro-Wolff, A.;
Gray-Goodrich, M.; Campbell, H.; Mayo, J.; Boyd, M. Feasibility
of a high-flux anticancer drug screen using a diverse panel of
cultured human tumor cell lines. J. Natl. Cancer Inst. 1991, 83,
757-766.
(17) Boyd, M. R.; Paull, K. D. Some practical considerations and
applications of the National Cancer Institute in vitro anticancer
drug discovery screen. Drug Dev. Res. 1995, 34, 91-109.
(28) Sheng, H.; Shao, J.; Kirkland, S. C.; Isakson, P.; Coffey, R. J.;
Morrow, J.; Beauchamp, R. D.; DuBois, R. N. Inhibition of
human colon cancer cell growth by selective inhibition of
cyclooxygenase-2. J. Clin. Invest. 1997, 99, 2254-2259.
(29) Paull, K. D.; Shoemaker, R. H.; Hodes, L.; Monks, A.; Scudiero,
D. A.; Rubinstein, L.; Plowman, J.; Boyd, M. R. Display and
analysis of patterns of differential activity of drugs against
human tumor cell lines: Development of mean graph and
COMPARE algorithm. J. Natl. Cancer Inst. 1989, 81, 1088-
1092.
(30) Panaro, N. J.; Popescu, N. C.; Harris, S. R.; Thorgeirsson, U. P.
Flavone acetic acid induces a G2/M cell cycle arrest in mammary
carcinoma cells. Br. J. Cancer 1999, 80, 1905-1911.
JM050711D