Synthesis of new N-(2-(trifluoromethyl)pyridin-4-yl)anthranilic acid derivatives and their evaluation as anticancer agents

Article abstract of DOI:10.1016/j.bmcl.2004.09.045

A series of N-(2-(trifluoromethyl)pyridin-4-yl)anthranilic acid derivatives was prepared and tested for the in vitro cytotoxic activity against human cancer cell lines. Some aryl esters exhibit antiproliferative activity with GI₅₀ values at nan

Full text of DOI:10.1016/j.bmcl.2004.09.045

Bioorganic & Medicinal Chemistry Letters 14 (2004) 5787–5791
Synthesis of new N-(2-(trifluoromethyl)pyridin-4-yl)anthranilic
acid derivatives and their evaluation as anticancer agents
Maria T. Cocco, Cenzo Congiu,* Valentina Lilliu and Valentina Onnis
`
Dipartimento di Tossicologia, Universita degli Studi di Cagliari, Via Ospedale 72, Cagliari I-09124, Italy
Received 6 July 2004;revised 9 September 2004;accepted 17 September 2004
Available online 5 October 2004
Abstract—The N-(2-(trifluoromethyl)pyridin-4-yl)anthranilic acid 6 and a series of its ester and amide derivatives were synthesized
and evaluated for their in vitro cytotoxic activity against human cancer cells. Ester derivatives 13 and 18 exhibited potent growth
inhibitory activity with GI50 values at nanomolar concentrations. Among amide derivatives, N-anthraniloylglycinate 19 shown mod-
erate inhibitory activity in the full panel cancer cell line screening.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
Farnesyl anthranilate2 suppressed the growth of murine
melanomas in in vivo and in vitro models, in part by
arresting cells in the G1/S interface of the cell cycle
and in part by initiating apoptosis. The anthranilamide
PD 184352 (CI-1040), developed by Parke-Davis,3 is
an inhibitor of both Mitogen Activated Extracellular
Kinases MEK1 and MEK2. In vivo, PD 184352 was
shown to inhibit the growth of colon and pancreatic
tumors. Tariquidar (XR9576),4 is a potent and specific
inhibitor of P-gp, commonly associated with the devel-
opment of multidrug resistance (MDR). In in vitro
and in vivo experimental studies, Tariquidar was shown
to restore the antitumor activity of several drugs includ-
ing doxorubicin, paclitaxel, etoposide, and vincristine
Among the wide variety of chemical structures in devel-
opment as new anticancer drugs, molecules designed on
anthranilic acid scaffold have attracted great interest in
recent years. In experimental models, a number of these
compounds exhibited preventive or inhibitory activity
because they can act through different biological mech-
anisms that are involved in the development and main-
tenance of tumoral cells. For example (Fig. 1),
Tranilast,1 also known as anti-allergic drug, inhibited
the proliferation, chemotaxis, and tube formation of hu-
man microvascular endothelial cells in vitro and angio-
genesis in vivo by VEGF-mediated mechanism.
O
N
O
HN
O
O
OH
O
O
O
N
H
NH
O
O
F
NH
F
Cl
NH
O
O
O
O
O
NH2
N
I
Farnesyl anthranilate
Tariquidar (XR9576)
Tranilast
PD 184352 (CI-1040)
Figure 1. Anthranilic acid derivatives as anticancer agents.
Keywords: Anthranilic acid;Anticancer activity;Antitumoral drugs;Pyridine derivatives.
*
Corresponding author. Tel.: +39 070 675 8630;fax: +39 070 675 8612;e-mail: ccongiu@unica.it
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.09.045

5788
M. T. Cocco et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5787–5791
against two highly resistant MDR human tumor cells.5
Other anthranilamides showed activity as VEGF recep-
tor tyrosine kinase6 and MMP inhibitors.7
6
a
b
O
OAr
Prompted by the above findings, in continuation of our
ongoing research on pyridine derivatives endowed with
anticancer activity,8 we became interested in novel com-
pounds containing the anthranilic acid scaffold bearing
the pyridine moiety. In this communication we report
the synthesis and evaluation for in vitro antitumoral effi-
cacy against human cancer cell lines of the N-(2-(tri-
O
NHR
H
H
N
CF3
N
CF3
N
N
19-24
8-18
Scheme 3. Reagents and conditions: (a) ArOH, DCC, CHCl3, rt, 24h;
(b) R–NH2, CDI, THF, rt, 24h.
fluoromethyl)pyridin-4-yl)anthranilic acid
series of its ester and amide derivatives.
6 and a
The key intermediate 4 underwent pyridine ring closure
upon treatment with ammonium acetate in hot DMF to
give 5 in 76% yield (Scheme 2). Hydrolysis of 5 in 10%
aq NaOH solution afforded acid 6. The ethyl ester 7
was obtained in 93% yield by refluxing an ethanolic
solution of 6 in the presence of thionyl chloride.
2. Chemistry
The synthesis of target compounds was conveniently
performed as outlined in Schemes 1–3. By slight modifi-
cations of the procedure previously described by us,9 the
anthranilic acid 1 was reacted with trifluoroacetylvinyl
ether 2 in 1:1.5M ratio, in refluxing acetonitrile to give
the N-alkenylanthranilic acid 3 in almost quantitative
yield (Scheme 1).
Aryl esters and amides were prepared as reported in
Scheme 3. Treatment of 6 with the appropriate phenol
in the presence of DCC gave esters 8–18 in 56–92%
yields. Amide derivatives 19–24 were obtained in 46–
82% yields by reaction of 6 with the appropriate amine
by CDI method.10
Upon reaction with excess of N,N-dimethylformamide
dimethyl acetal (DMF-DMA) in boiling toluene, com-
pound 3 was converted into N-hexadienyl-anthranilic
acid ester 4 in a one-pot procedure, resulting in esterifi-
cation–condensation reaction sequence, in 92% overall
yield.
The stability of ester and amide derivatives was investi-
gated both toward acid (0.1M aq trifluoroacetic acid)
Table 1. Comparison of effects of anthranilic acid derivatives, at
10À4 M concentrations, on the proliferation in a three-cell line pre-
screen, expressed as percentage of proliferation of vehicle treated cells
O
OH
O
CF3
O
OH
NH2
O
O
H
N
O
R
a
+
CF3
H
N
CF3
N
3
2
1
Compd
R
Cell lines
CF3
N
O
OMe O
H
MCF7
(breast)
NCI-H460
(lung)
SF-268
(CNS)
N
b
5
OMe
OH
96
105
98
nta
42
74
nt
100
100
102
nt
13
49
nt
68
3
103
102
103
nt
4
6
7
OEt
OPh-3-Me
Scheme 1. Reagents and conditions: (a) MeCN, reflux, 2h;then rt,
24h;(b) DMF-DMA (3equiv), toluene, reflux, 2h.
8
9
OPh-3-OMe
OPh-4-OMe
OPh-4-OPr
OPh-4-SMe
OPh-2-Cl
43
105
nt
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
O
OMe
O
OH
80
6
81
17
5
H
H
N
CF3
CF3
N
OPh-3-Cl
OPh-4-Cl
12
24
3
3
4
N
N
9
28
9
a
b
5
OPh-2,4-Cl2
OPh-2,4,6-Cl3
O-3-Py
1
6
12
62
60
70
66
56
79
nt
9
55
75
59
97
94
98
96
nt
O
OEt
22
19
105
86
62
99
nt
H
N
NHCH2COOEt
NH(CH2)3OMe
NH-2-Py
CF3
c
N
7
NH-3-Py
NH-4-Py
NHCH2-3-Py
Scheme 2. Reagents and conditions: (a) ammonium acetate (2equiv),
DMF, reflux, 1h;(b) 10% aq NaOH, reflux, 30min, then H +;(c) EtOH
anhyd, SOCl2 (4equiv), reflux, 3h.
a Not tested.

M. T. Cocco et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5787–5791
Table 2. GI50 values, in lM concentrations, of anthranilic acid derivatives 9, 13–19
5789
Panel/cell line
Compd
9
1
3
1
4
1
5
1
6
1
Leukemia
CCRF-CEM
HL-60(TB)
K-562
nta
0.032
0.47
0.89
13
3.2
4.9
6.4
3.6
3.5
6.6
3.0
nt
0.033
44
2.2
2.1
3.9
2.4
33
0.74
0.69
1.0
0.051
0.028
0.24
0.043
0.039
0.054
0.010
18
16
11
17
MOLT-4
RPMI-8226
SR
0.93
1.8
100
100
100
nt
2.9
4.0
2.4
0.77
nt
0.030
0.44
nt
Nonsmall cell lung cancer
A549/ATCC
EKVX
3.2
6.6
2.8
5.7
8.1
2.0
7.4
4.1
7.6
0.73
8.6
18
15
25
nt
21
2.9
2.8
17
10
4.3
11
19
nt
12
3.4
3.5
14
3.9
0.72
0.39
0.019
0.48
0.51
0.066
2.9
29
31
21
22
25
3.3
2.7
HOP-62
HOP-92
2.7
0.027
0.29
0.25
4.8
8.7
100
0.057
NCI-H226
NCI-H23
2.1
2.4
5.9
2.6
100
22
NCI-H322M
H460
H522
23
11
4.2
20
32
20
0.66
0.51
4.3
4.6
3.4
5.7
0.34
0.41
22
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
2.2
nt
2.2
nt
3.5
nt
13
nt
nt
nt
nt
6.3
0.14
0.36
19
71
21
29
29
27
20
nt
3.2
2.2
nt
1.1
2.8
3.7
2.6
4.4
1.8
1.6
3.7
1.7
2.7
4.6
4.1
4.6
0.036
0.29
0.22
0.39
0.33
0.36
2.1
HT29
KM12
SW-620
nt
nt
0.055
0.31
0.062
9.5
4.4
8.9
6.6
3.7
4.6
CNS cancer
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
4.7
2.5
0.22
0.24
0.58
7.4
3.0
4.4
3.1
3.9
4.2
0.063
0.31
20
71
28
30
nt
18
2.8
nt
63
15
nt
15
nt
3.5
nt
3.8
nt
nt
0.010
0.33
nt
nt
nt
7.1
3.0
0.68
0.27
nt
nt
nt
nt
nt
2.9
0.52
18
Melanoma
LOX IMVI
M14
1.1
1.3
0.026
0.036
nt
nt
nt
nt
nt
nt
2.0
2.1
0.052
0.031
0.66
17
25
23
10
69
17
1.8
nt
2.6
nt
2.0
2.1
1.9
2.8
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
12
24
3.1
12
2.2
5.4
3.1
0.26
nt
17
0.17
4.5
2.6
11
55
0.31
2.3
1.7
0.35
Ovarian cancer
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
1.6
0.22
0.26
0.46
0.24
8.0
3.8
nt
14
3.5
nt
11
11
4.3
0.14
0.46
0.49
0.69
0.25
24
35
26
26
29
nt
nt
nt
nt
nt
3.5
3.6
26
7.9
4.8
4.1
3.0
4.1
nt
nt
nt
Renal cancer
786-0
A498
3.4
nt
0.25
3.9
3.5
2.7
1.9
2.9
0.27
0.52
0.26
0.052
0.34
0.41
0.27
29
64
34
16
19
24
37
nt
nt
nt
nt
21
13
2.1
nt
ACHN
CAKI-1
RXF 393
SN12C
TK-10
3.4
2.7
0.28
0.29
nt
11
2.4
7.1
9.5
3.1
3.4
2.1
nt
3.0
85
2.3
21
5.4
11
nt
0.33
0.65
3.0
3.0
4.1
18
Prostate cancer
PC-3
DU-145
3.2
nt
0.30
4.3
7.4
14
17
19
8.3
15
0.42
0.22
22
27
16
nt
Breast cancer
MCF7
7.6
nt
2.1
0.28
0.32
0.32
4.8
3.7
16
1.8
3.6
3.6
2.2
4.7
3.2
0.16
0.081
0.39
25
51
24
NCI/ADR-RES
MDA-MB-231/ATCC
2.2
1.7
nt
(continued on next page)

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M. T. Cocco et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5787–5791
Table 2 (continued)
Panel/cell line
Compd
9
1
3
1
4
1
5
1
6
HS 578T
MDA-MB-435
BT-549
4.9
1.0
8.1
0.32
0.13
0.34
21
3.0
6.8
30
2.1
15
25
2.9
6.8
2.6
1.6
2.4
0.57
0.064
0.016
46
39
36
a Not tested.
and base (0.1M aq NaOH) hydrolysis. After 48h at
room temperature, the formation of acid 6 was moni-
tored by TLC (chloroform/n-hexane 4:1). In all cases,
examination of TLC plates revealed a single spot with
rf identical to starting compound. Thus, anthranilic es-
ters and amides do not appear to be hydrolytically labile.
in the order: 13 (2-chloro) ꢀ 14 (3-chloro) > 17 (2,4,6-
trichloro) > 16 (2,4-dichloro) > 15 (4-chloro). This fact
suggests that steric and electronic effects of substitu-
ent(s) on phenyloxy moiety might be implicated in the
activity of these types of compound. Among these, the
ester 13, bearing a 2-chlorophenyl ring, exhibits potent
antiproliferative activity against all tested cell lines, with
GI50 values in the range of 0.026–8.6lM. Compound 13
shows particular selectivity against melanoma LOX-
IMVI (GI50 0.026lM), leukemic MOLT-4 (GI50
0.028lM), CCRF-CEM (GI50 0.032lM), and K-562
(GI50 0.051lM) cell lines.
3. Results and discussion
The anthranilate derivatives examined in this study were
evaluated at National Cancer Institute (NCI) for
cytotoxicity in the NCIÕs disease-oriented antitumor
screening.11–13
On other hand, compound 9, bearing a 3-methoxy-phen-
yl ring, is effective against all the tested cell lines. Com-
pound 9 shows GI50 values between 0.69 and 21lM, the
best result being against leukemic MOLT-4 and K-562
cell lines, with GI50 values of 0.69 and 0.74lM, respec-
tively. Comparison of antiproliferative activity of 9 with
the 4-methoxy isomer 10 shows that shifting of 3-meth-
oxy group to 4-position results in dramatic loss of inhib-
itory activity. Introduction of methylthio group in 4-
position of phenyloxy moiety, also results in inactive
compound 12.
Cytotoxic activities of synthesized compounds were first
evaluated in vitro against NCI-H460 (nonsmall cell
lung), MCF7 (breast), or SF-268 (CNS) human cancer
cell lines in a three-cell line, one dose primary anticancer
assay. Results for each test compound are reported as
the percentage of growth of the treated cells when com-
pared to the untreated control cells and are shown in
Table 1. Compounds which reduced the growth of any
one of the cell lines to less than 32% were passed on
for evaluation in the full panel of 60-cell lines.
Among amide derivatives, only ethyl N-anthraniloyl-
glycinate 19 exhibits moderate inhibitory activity in
the full panel cell lines test;however, this compound
shows selective inhibitory activity against nonsmall cell
lung cancer HOP-92 (GI50 0.057lM) and leukemic
CCRF-CEM (GI50 2.2lM) cell lines.
As shown in Table 1, aryl esters 9 and 13–18, and ethyl
N-anthraniloylglycinate 19 fulfilled this condition. These
compounds were then assayed for cytotoxic activity
against leukemia, lung, colon, CNS, melanoma, ovar-
ian, renal, prostate, and breast human cancer cell lines.
The test compounds were evaluated using five concen-
trations at 10-fold dilutions, in the 10À4–10À8 M range.
The anticancer activity of each compound was deduced
from dose–response curves and is presented in Table 2
according to the data provided by NCI. The response
parameter GI50 refers to the drug concentration that
produced 50% of growth inhibition.
4. Conclusions
In summary, we report the synthesis and anti-prolifera-
tive activity against human cancer cell lines of a series
of N-(2-(trifluoromethyl)pyridin-4-yl)-anthranilic acid
derivatives. The first results confirm the validity of our
approach providing practical access to anhranilate-
based derivatives possessing potent in vitro antiprolifer-
ative activity against human tumor cells. Compounds 13
and 18 were found to have GI50 values at nanomolar
concentrations in most of the cell lines assayed.
Although acid 6 and alkyl esters 5 and 7 are devoid of
cytotoxic activity, their conversion into (hetero)aryl
esters leds to compounds 9 and 13–18 that exhibit anti-
proliferative activity. In this series, growth inhibition is
strongly sensitive to the nature of aryloxycarbonyl moi-
ety and position of substituents on aromatic ring. The 3-
pyridyl ester 18 exhibits potent cytotoxic activity, with
GI50 values in the range of 0.010–0.72lM against al-
most all cancer cell lines. In contrast, HL-60 (leukemia),
NCI-H322M (lung), SK-MEL-2, and UACC-257 (mel-
anoma) cell lines are inhibited by higher concentrations
(44, 2.9, 32, and 55lM, respectively) of 18.
Evaluation in in vivo models of these compounds is
underway at NCI and the results will be disclosed in
due course.
Acknowledgements
We thank the National Cancer Institute, Maryland,
USA for the in vitro anticancer assay in human cancer
cell lines.
Esters bearing chlorine atom(s) on the phenyloxy moiety
show antiproliferative activity decreasing approximately

M. T. Cocco et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5787–5791
5791
8. (a) Cocco, M. T.;Congiu, C.;Onnis, V.
Eur. J. Med.
References and notes
Chem. 2000, 35, 545–552;(b) Cocco, M. T.;Congiu, C.;
Onnis, V. Eur. J. Med. Chem. 2003, 38, 37–47.
9. (a) Cocco, M. T.;Congiu, C.;Onnis, V. Tetrahedron Lett.
1999, 40, 4407–4410;(b) Cocco, M. T.;Congiu, C.;Onnis,
V. Chem. Pharm. Bull. 2001, 49, 703–706.
1. Isaji, M.;Miyata, H.;Ajisawa, Y.;Takehana, Y.;
Yoshimura, N. Br. J. Pharmacol. 1997, 122, 1061–1066.
2. Mo, H.;Tatman, D.;Jung, M.;Elson, C. E. Cancer Lett.
2000, 157, 145–153.
10. All compounds gave correct analytical and spectral data.
For example compound 7; 1HNMR (DMSO-d6): d 1.34 (t,
J = 6.9Hz, 3H, CH3), 4.36 (q, J = 6.9Hz, 2H, CH2), 7.24
(d, J = 5.4Hz, H-5 Py), 7.37 (t, J = 7.7Hz, 1H, Ph), 7.45
(s, H-3, Py), 7.64 (m, 1H, Ph), 7.75 (m, 1H, Ph), 8.05 (d,
J = 7.7Hz, 1H, Ph), 8.47 (d, J = 5.4Hz, H-6, Py), 9.52 (s,
1H, NH). Melting point = 82–84°C (from n-hexane). Infra
3. 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. Nat. Med. 1999, 5, 810–816.
4. Roe, M.;Folkes, A.;Ashworth, P.;Brumwell, J.;Chima,
L.;Hunjan, S.;Pretswell, I.;Dangerfield, W.;Ryder, H.;
Charlton, P. Bioorg. Med. Chem. Lett. 1999, 9, 595–
600.
red (Nujol mull), m = 3252 (NH), 1677 (C@O) cmÀ1
.
11. (a) Grever, M. R.;Sherpartz, S. A.;Chabner, B. A. Semin.
Oncol. 1992, 19, 622–638.
5. Mistry, P.;Stewart, A. J.;Dangerfield, W.;Okiji, S.;
Liddle, C.;Bootle, D.;Plumb, J. A.;Templeton, D.;
Charlton, P. Cancer Res. 2001, 61, 749–758.
6. Manley, P. W.;Furet, P.;Bold, G.;Bruggen, J.;Mestan,
J.;Meyer, T.;Schnell, C. R.;Wood, J.;Haberey, M.;
Huth, A.;Kruger, M.;Menrad, A.;Ottow, E.;Seidel-
mann, D.;Siemeister, G.;Thierauch, K. H. J. Med. Chem.
2002, 45, 5687–5693.
7. Levin, J. I.;Du, M. T.;DiJoseph, J. F.;Killar, L. M.;
Sung, A.;Walter, T.;Sharr, M. A.;Roth, C. E.;Moy, F.
J.;Powers, R.;Jin, G.;Cowling, R.;Skotnicki, J. S.
Bioorg. Med. Chem. Lett. 2001, 11, 235–238.
12. Monks, A.;Scudiero, D. A.;Skehan, P.;Shoemaker, R.
H.;Paull, K. D.;Vistica, D. T.;Hose, C.;Langley, J.;
Cronice, P.;Vaigro-Wolf, M.;Gray-Goodrich, M.;Camp-
bell, H.;Mayo, M. R. J. Natl. Cancer Inst. 1991, 83, 757–
766.
13. Boyd, M. R.;Paull, K. D.;Rubinstein, L. R. In Cytotoxic
Anticancer Drugs: Models and Concepts for Drug Discov-
ery and Development;Vleriote, F. A., Corbett, T. H.,
Baker, L. H., Eds.;Kluwer Academic: Hingham, MA,
1992;pp 11–34.