Synthesis and in vivo evaluation of N-ethylamino-2-oxo-1,2-dihydro- quinoline-3-carboxamide for inhibition of intestinal tumorigenesis in APC Min/+ mice

Article abstract of DOI:10.1016/j.bmcl.2014.01.042

A selective KGFR tyrosine kinase inhibitor, N-ethylamino-2-oxo-1,2-dihydro- quinoline-3-carboxamide, was synthesized and its possible inhibitory effects on the development of colon polyps and colorectal tumors was examined in APC ^Min/+ mice, a mouse model of human intestinal familial adenomatous polyposis. The present study shows for the first time that a dietary administration of a selective KGFR tyrosine kinase inhibitor lacks the overt-toxicities and significantly reduced the growth of small intestinal polyps in both male and female APC^Min/+ mice. This inhibition of polyp growth appears to occur at a greater extent in female mice.

Full text of DOI:10.1016/j.bmcl.2014.01.042

Bioorganic & Medicinal Chemistry Letters 24 (2014) 1380–1382
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and in vivo evaluation of N-ethylamino-2-oxo-1,
2-dihydro-quinoline-3-carboxamide for inhibition of intestinal
tumorigenesis in APCMin/+ mice
Gopal Pathuri a,b, Qian Li b,c, Altaf Mohammed b,c, Hariprasad Gali a,d, J. Thomas Pento a,d,
,
⇑
Chinthalapally V. Rao b,c
a Department of Pharmaceutical Sciences, College of Pharmacy, The University of Oklahoma Health Sciences Center, Oklahoma City, OK 73117, USA
b Hematology/Oncology Section, Department of Internal Medicine, College of Medicine, The University of Oklahoma Health Sciences Center, Oklahoma City, OK 73117, USA
c Center for Cancer Prevention and Drug Development, The University of Oklahoma Health Sciences Center, Oklahoma City, OK 73117, USA
d Experimental Therapeutics Program, Peggy and Charles Stephenson Oklahoma Cancer Center, The University of Oklahoma Health Sciences Center, Oklahoma City, OK 73117, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A selective KGFR tyrosine kinase inhibitor, N-ethylamino-2-oxo-1,2-dihydro-quinoline-3-carboxamide,
was synthesized and its possible inhibitory effects on the development of colon polyps and colorectal
tumors was examined in APCMin/+ mice, a mouse model of human intestinal familial adenomatous polyp-
osis. The present study shows for the first time that a dietary administration of a selective KGFR tyrosine
kinase inhibitor lacks the overt-toxicities and significantly reduced the growth of small intestinal polyps
in both male and female APCMin/+ mice. This inhibition of polyp growth appears to occur at a greater
extent in female mice.
Received 6 November 2013
Revised 10 January 2014
Accepted 14 January 2014
Available online 27 January 2014
Keywords:
KFGR
Tyrosine kinase inhibitor
Polyps
Ó 2014 Elsevier Ltd. All rights reserved.
Chemoprevention
Colon cancer
Keratinocyte growth factor (KGF, also known as FGF7) is a
member of the fibroblast growth factor family that is produced
in stromal tissue and stimulates DNA synthesis, proliferation and
migration of epithelial cells.1 It is well established that these target
epithelial cells contain high affinity keratinocyte growth factor
receptor (KGFR, also known as FGFR-2IIIb).2 The biological actions
of KGF are involved in normal morphogenesis and tissue repair.3 In
addition, over-expression of KGF and/or KGFR is associated with
the progression of several types of cancers including colorectal
cancers.4–7 KGF/KGFR signaling via the ERK1/2 pathway often
occurs early in the process of cancer cell initiation by enhancing
cell proliferation and motility resulting in progression to a meta-
static phenotype.8 Colorectal cancers are thought to arise due to
a series of histopathologic and molecular changes that transform
normal colonic epithelial cells into colorectal carcinoma, with an
adenomatous polyp as an intermediate step in this process.9 In
addition, recent developments in the cellular and molecular
pathogenesis of colon cancer provide new insights for developing
selective agents with potential chemopreventive properties
against colon carcinogenesis. Chemoprevention is likely to play
a major role in the management or prevention of colorectal
cancer.10,11 Thus, the present study examines the possible
O
O
O
O
O
O
O
O
CHO
NH2
H2NCH2CH2NH2
RT, 15 hr
NH2
H2O
SOCl2
OH
Cl
NH
+
O
75 oC, 8 hr
Reflux, 2 hr
N
H
O
N
H
N
H
1
2
3
Figure 1. Synthetic scheme for N-ethylamino-2-oxo-1,2-dihydro-quinoline-3-carboxamide (3).
⇑
Corresponding author. Address: The University of Oklahoma College of Pharmacy, 1110 N. Stonewall Avenue, Room 325, Oklahoma City, OK 73117, USA. Tel.: +1 (405) 271
6593x47244; fax: +1 (405) 271 7505.
E-mail address: tom-pento@ouhsc.edu (J.T. Pento).
http://dx.doi.org/10.1016/j.bmcl.2014.01.042
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

G. Pathuri et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1380–1382
1381
Age 5 wks
6 wks
18 wks
shown to reduce KGF-mediated proliferation of MCF-7 human
breast cancer cells in vitro.6
The synthesis of 3 was achieved in a three-step synthetic strat-
egy as shown in Figure 1. The experimental details are presented in
References and notes.12,13 In the first step, condensation reaction
between commercially available Meldrum’s acid and 2-amino-
benzaldehyde was carried at 75 °C for 8 h in water using a previ-
ously reported procedure.14 Compound 1 (2-oxo-1,2-dihydro-
quinoline-3-carboxylic acid) was obtained in a yield of 57.7%. In
the second step, the carboxylic group was converted into carboxyl
chloride 2 by refluxing compound 1 in an excess of thionyl chloride
for 2 h. After distilling off the excess thionyl chloride, compound 2
was reacted with an excess of ethylenediamine at room tempera-
ture for 15 h to obtain compound 3. The excess ethylenediamine
was removed by distillation under vacuum and the solid was
washed several times with diethylether and dichloromethane
and dried in vacuum to obtain compound 3 as a white solid in a
yield of 66% with a purity of >98%. Compound 3 is soluble in water
at a concentration of 2 mg/ml. Compound 3 was characterized by
1H and 13C NMR and HRMS.
AIN-76A
diet
Experimentaldiets
Figure 2. Study design for the evaluation of chemoprevention of intestinal
tumorigenesis by compound 3. Animals: APCMin/+ male and female mice (ꢀ8–10/
group). Experimental diets: (a) AIN-76A diet and (b) AIN-76A diet + Compound 3
(150 ppm).
30
20
10
0
25
20
15
10
5
We selected the APCMin/+ mouse model for evaluating the
potential chemopreventive properties of compound 3 against co-
lon carcinogenesis because it is one of the most studied animal
models of intestinal tumorigenesis and chemoprevention stud-
ies.15 It harbors a dominant germline mutation at codon 850 of
the mouse homolog of human adenomatous polyposis coli (APC)
gene, which is similar to the mutation in patients with familial
adenomatous polyposis (FAP). APCMin/+ mice develop multiple
adenomas throughout the whole intestinal tract that are primar-
ily small intestinal polyps (tubular adenomas) and fewer colon
tumors (adenomas and adenocarcinomas).16 The objective of this
study was to determine whether the continuous administration of
compound 3 in the diet (150 ppm) can prevent or reduce the inci-
dence and size of colon polyps and intestinal tumors in the
0
Control Treated
Control Treated
Figure 3. Effects of compound
3 on small intestinal polyp and colon tumor
incidence in APCMin/+ male and female mice. Date represents mean SEM (ꢀ8–10/
group). P values (P < 0.05, statistically significant) were derived using ANOVA Tukey
post test.
chemoprevention effect of a selective KGFR tyrosine kinase inhib-
itor, N-ethylamino-2-oxo-1,2-dihydro-quinoline-3-carboxamide
(3), on the development of colon polyps and colorectal tumors in
an in vivo animal model. In a previous study, compound 3 was
A
B
E
C
10
8
2.0
P = 0.13
60
40
20
0
P < 0.005
1.5
6
1.0
P < 0.025
4
0.5
0.0
2
0
Control Treated
Control Treated
Control Treated
D
F
60
40
20
0
10
P = 0.21
1.0
0.5
0.0
8
6
4
2
0
P = 0.27
P < 0.013
Control Treated
Control Treated
Control Treated
Figure 4. Changes in body weight over time in APCMin/+ mice. Date represents mean SEM (ꢀ8–10/group). P values (P <0.05, statistically significant) were derived using
ANOVA Tukey post test.

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G. Pathuri et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1380–1382
APCMin/+ mouse model without causing gross toxicity as deter-
mined by a reduction of body weight. The study design is shown
in Figure 2. The study was carried out in a similar fashion to the
previously reported studies with other drugs in the same mouse
model.17,18 The mice were recruited for the study at the age of
5 weeks. The mice were initially fed a control diet (AIN-76A) for
one week and then the control groups were fed with the
AIN-76A diet, while the treatment groups were fed the AIN-76A
diet containing 150 ppm of compound 3.
Figure 3 summarizes the chemopreventive effect of compound
3 on tumor multiplicity in the small intestine. Male and female
APCMin/+ mice fed the control diet developed a total of 45.8 3.9
and 51.0 5.7 (mean SEM) intestinal polyps, respectively
(Fig. 3A and C). Whereas male and female APCMin/+ mice fed with
the compound 3 containing diet developed a total of 53.0 5.1
and 45.3 3.7 intestinal polyps, respectively (Fig. 3B and D).
These results indicate that compound 3 did not prevent small
intestinal polyp formation. However, the number of large-sized
polyps (>2 mm) was significantly reduced with compound 3
treatment in both male (6.9 1.6 vs 2.9 0.7 control vs treatment
groups) and female mice (7.0 1.6 vs 2.1 0.9 control vs treat-
ment groups) as shown in Figure 3B and E. The effect is more pro-
found in female mice (Fig. 3E). As shown in Figure 3C and F,
compound 3 did not inhibit colon tumor formation in either the
male or the female mice. However, the body weights of both con-
trol and compound 3 treated mice were essentially unchanged at
the end of study, indicating that compound 3 did not cause any
gross toxicity at the dosage level used in this study (Fig. 4). In
addition, no evidence of gross toxicity was visually observed in
the major organs when the animals were dissected at the conclu-
sion of the study.
References and notes
1. Rubin, J. S.; Osada, H.; Finch, P. W.; Taylor, W. G.; Rudikoff, S.; Aaronson, S. A.
Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 802.
2. Bottaro, D. P.; Rubin, J. S.; Ron, D.; Finch, P. W.; Florio, C.; Aaronson, S. A. J. Biol.
Chem. 1990, 265, 12767.
3. Finch, P. W.; Cunha, G. R.; Rubin, J. S.; Wong, J.; Ron, D. Dev. Dyn. 1995, 203, 223.
4. Yoshino, M.; Ishiwata, T.; Watanabe, M.; Komine, O.; Shibuya, T.; Tokunaga, A.;
Naito, Z. Oncol. Rep. 2005, 13, 247.
5. Otte, J. M.; Schmitz, F.; Banasiewicz, T.; Drews, M.; Folsch, U. R.; Herzig, K. H.
Eur. J. Clin. Invest. 2000, 30, 222.
6. Mehta, M.; Kesinger, J. W.; Zang, X. P.; Lerner, M. L.; Brackett, D. J.;
Brueggemeier, R. W.; Li, P. K.; Pento, J. T. Anticancer Res. 2010, 30, 4883.
7. Zang, X. P.; Lerner, M.; Brackett, D.; Pento, J. T. Anticancer Res. 2009, 29, 3417.
8. Pento, J. T. Drug Future 2009, 34, 915.
9. Cho, K. R.; Vogelstein, B. Cancer 1992, 70, 1727.
10. Oshima, M.; Dinchuk, J. E.; Kargman, S. L.; Oshima, H.; Hancock, B.; Kwong, E.;
Trzaskos, J. M.; Evans, J. F.; Taketo, M. M. Cell 1996, 87, 803.
11. Kelloff, G. J. Adv. Cancer Res. 2000, 78, 199.
12. 2-Oxo-1,2-dihydro-quinoline-3-carboxylic acid (1). 2-Aminobenzaldehyde
(1.0 g, 8.26 mmol) and Meldrum’s acid (1.19 g, 8.26 mmol) were combined in
H2O (20 ml). The reaction mixture was stirred at 75 °C for 8 h. The progress of
the reaction was monitored by TLC. After completion of reaction, the reaction
mixture was cooled to room temperature, the precipitate was filtered on
sintered funnel and dried at suction to obtain the 2-oxo-1,2-dihydro-
quinoline-3-carboxylic acid as
300 MHz): d (ppm) = 7.35–7.43 (m, 1H), 7.45–7.52 (m, 1H), 7.72–7.78 (m, 1H),
8.02 (d, J = 8.2 Hz, 1H), 8.96 (s, 1H).
a
solid (0.9 g, 57.7%). 1H NMR (DMSO-d6,
13. N-ethylamino-2-oxo-1,2-dihydro-quinoline-3-carboxamide (3). To a sample of
1 (0.9 g, 4.76 mmol) was added freshly distilled thionyl chloride (10 ml) at
room temperature. The reaction mixture was heated under reflux for 1 h. The
excess thionyl chloride was removed by distillation and co distilled with
dichloromethane (2 Â 10 ml) to obtain
2 in a quantitative yield. This
compound was used immediately for next step without further purification.
To execess of ethylene diamine (10 ml) was added a solution of 2 (0.83 g,
4 mmol) in 10 ml of acetone to at 0 °C. The reaction mixture was stirred at
room temperature for overnight. The ethylene diamine was evaporated on
rotary evaporator and residue was washed several times with ether and
dichloromethane and dried to obtain the L-27 compound as a solid (0.61 g,
66%). 1H NMR (DMSO-d6, 300 MHz): d (ppm) = 2.96–3.02 (m, 2H), 3.55–3.63
(m, 2H) 7.26–7. 32 (m, 1H), 7.42–7.47 (m, 1H), 7.62–7.70 (m, 1H), 7.94 (d,
J = 7.6 Hz, 1H), 8.84 (s, 1H), 9.85–9.92 (m, 1H). 13C NMR (DMSO-d6, 75 MHz): d
(ppm) = 36.75, 37.30, 115.20, 118.40, 121.43, 122.68, 129.61, 132.61, 139.38,
143.73, 161.62, 163.60. Electrospray MS calcd m/z for C12H13N3O2: 231.1;
found: 232.1 ([M+H]+).
In summary, the present study demonstrates for the first time
that dietary administration of a selective KGFR tyrosine kinase
inhibitor may stabilize the growth of small intestinal polyps in
both male and female APCMin/+ mice. Also compound 3 appear to
cause a reduction of tumor development to a greater extent in
the female mice when compared to the male response.
14. Wells, J.; Renslo, A. R.; Wolan, D.; Zorn, J. PCT Patent Application 2009, PCT/
US2009/030680.
15. Rosenberg, D. W.; Giardina, C.; Tanaka, T. Carcinogenesis 2009, 30, 183.
16. Su, L. K.; Kinzler, K. W.; Vogelstein, B.; Preisinger, A. C.; Moser, A. R.; Luongo, C.;
Gould, K. A.; Dove, W. F. Science 1992, 256, 668.
Acknowledgments
17. Swamy, M. V.; Patlolla, J. M.; Steele, V. E.; Kopelovich, L.; Reddy, B. S.; Rao, C. V.
Cancer Res. 2006, 66, 7370.
18. Mohammed, A.; Janakiram, N. B.; Li, Q.; Choi, C. I.; Zhang, Y.; Steele, V. E.; Rao,
C. V. Cancer Prev. Res. 2011, 4, 2015.
We acknowledge funding from the NIH grant NCI-R01-
CA094962 and Kerley-Cade Chair Endowment.