Synthesis and biological evaluation of 4-oxoquinoline-3-carboxamides derivatives as potent anti-fibrosis agents

Article abstract of DOI:10.1016/j.bmcl.2014.10.071

Thirty-one 4-oxoquinoline-3-carboxamides derivatives were synthesized and evaluated for their anti-fibrotic activities by the inhibition of TGF-β1-induced total collagen accumulation and anti-inflammatory activities by the inhibition of LPS-stimulated TNF-α production. Among them, three compounds (10a, 10l and 11g) exhibited potent inhibitory effects on both TGF-β1-induced total collagen accumulation and LPS-stimulated TNF-α production. Furthermore, oral administrations of 10l at a dose of 20 mg/kg/day for 4 weeks effectively alleviated lung inflammation and injury, and decreased lung collagen accumulation in bleomycin-induced pulmonary fibrosis model. Histopathological evaluation of lung tissue confirmed 10l as a potential, orally active agent for the treatment of pulmonary fibrosis.

Full text of DOI:10.1016/j.bmcl.2014.10.071

Bioorganic & Medicinal Chemistry Letters 24 (2014) 5666–5670
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of 4-oxoquinoline-3-
carboxamides derivatives as potent anti-fibrosis agents
Jun Zhu a, , Lin He a, , Liang Ma a, , Zhe Wei a, Jiqiang He a, Zhuang Yang b, Yuzhi Pu a, Dong Cao a,
Yuzhe Wu b, Mingli Xiang a, Aihua Peng a, Yuquan Wei a, Lijuan Chen a,
⇑
a State Key Laboratory of Biotherapy and Cancer Center/Collaborative Innovation Center for Biotherapy, West China Hospital, West China Medical School, Sichuan University,
Chengdu, Sichuan 610041, PR China
b College of Chemistry, Sichuan University, Chengdu, Sichuan 610064, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Thirty-one 4-oxoquinoline-3-carboxamides derivatives were synthesized and evaluated for their anti-
Received 3 September 2014
Revised 9 October 2014
Accepted 22 October 2014
Available online 29 October 2014
fibrotic activities by the inhibition of TGF-b1-induced total collagen accumulation and anti-inflammatory
activities by the inhibition of LPS-stimulated TNF-
and 11g) exhibited potent inhibitory effects on both TGF-b1-induced total collagen accumulation and
LPS-stimulated TNF- production. Furthermore, oral administrations of 10l at a dose of 20 mg/kg/day
a production. Among them, three compounds (10a, 10l
a
for 4 weeks effectively alleviated lung inflammation and injury, and decreased lung collagen accumula-
tion in bleomycin-induced pulmonary fibrosis model. Histopathological evaluation of lung tissue con-
firmed 10l as a potential, orally active agent for the treatment of pulmonary fibrosis.
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
4-Oxoquinoline-3-carboxamides
Pulmonary fibrosis
TGF-b1
Collagen accumulation
TNF-a
Anti-inflammatory
Pulmonary fibrosis (PF), characterized by the disruption of nor-
mal lung tissue architecture and loss of pulmonary function, is one
of the major clinical problems of cystic fibrosis and chronic
obstructive pulmonary disease.1–4 Its incidence most recently has
been estimated to be between 14 and 42.7 per 100,000 and has
been increasing,5–7 the expected survival time of patients suffered
from PF is estimated to be 2.5–3.5 years.8 However, the pathogen-
esis of PF is still unknown, the excessive deposition of extracellular
matrix (ECM) in lung tissues caused by inflammation has been rec-
ognized as a crucial reason leading to the destruction of the lung
architecture.9,10
sis,16 which is studied for chronic obstructive pulmonary disease
in clinic.17 Treatment with Ivacaftor had significant and sustained
improvement in their lung function in cystic fibrosis.18,19 In our
preliminary studies, Ivacaftor was also observed to reveal in vitro
anti-fibrotic activities by the inhibition of TGF-b1-induced total
collagen accumulation in rat fibroblast cells (NRK-49F), which
has been recognized an effect of good and convenient origin for
anti-fibrotic agent in vitro screening model.20 Therefore, these
pharmacological properties of Ivacaftor provided us the impetus
to develop novel and potent anti-fibrotic agents containing 4-oxo-
quinoline-3-carboxamide moiety by using Ivacaftor as a lead.
Ivacaftor derivatives (9a–9f) were synthesized according to pre-
vious reported procedures (Scheme 1).21–23 The 2,4-di-tert-butyl-
phenol 1 was treated with methyl chloroformate to give 2.
Nitration of 2 with cooled mixture of HNO3 and H2SO4 (v:v 1:1)
obtained 3. Treatment of 3 with 10% Pd/C, HCOONH4 yielded 4.
The pivotal 4-quinolones intermediates 5a–5f were obtained by
the condensation of appropriate anilines with diethyl ethoxymeth-
ylenemalonate, followed by the cyclization of the intermediate to
give 6a–6f. Compound 6a–6f were hydrolyzed to the correspond-
ing acid 7a–7f, the intermediates 7a–7f were reacted with 4 to
afford 8a–8f, further deprotected to give 9a–9f. Ivacaftor analogues
10a–10o and 11a–11j were outlined in Scheme 2. The intermedi-
ate 7e was treated with anilines to afford the desired products
A number of studies have documented that cytokines such as
TGF-b1, TNF-a
play key roles during the pathogenesis of the PF.11
TGF-b1 is a fibrogenic cytokine involved in pathological fibrosis
of PF by regulating extracellular matrix (ECM) deposition in the
response to lung tissues injury.12–14 TNF-
a, an important pro-
inflammatory cytokine released by macrophages and lymphocytes,
involved in regulating the inflammation respond to lung tissue
injury.15
Ivacaftor (Fig. 1), a 4-oxoquinoline-3-carboxamide compound,
has been approved as a small-molecule drug to treat cystic fibro-
⇑
Corresponding author. Tel.: +86 28 85164063; fax: +86 28 85164060.
E-mail address: chenlijuan125@163.com (L. Chen).
These authors contributed equally.
http://dx.doi.org/10.1016/j.bmcl.2014.10.071
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

J. Zhu et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5666–5670
5667
Figure 1. Ivacaftor and 4-oxoquinoline-3-carboxamides derivatives.
Scheme 1. General synthesis of Ivacaftor derivatives. Reagents and conditions: (a) methyl chloroformate, DMAP, Et3N, DCM, rt, overnight; (b) HNO3/H2SO4 (1:1), 0 °C, rt, 2 h.
(c) 10% Pd/C, HCOONH4, EtOH, reflux, 2 h; (d) EtOH, reflux, 4 h; (e) diphenyl ether, reflux, 1 h; (f) (i) 10% KOH, 100 °C, 3 h; (ii) 1 N HCl, pH = 4–5; (g) HATU, Et3N, DMF, rt,
overnight; (h) KOH, CH3OH, rt, 2 h.
Scheme 2. General synthesis of Ivacaftor analogues. Reagents and conditions: (a) HATU, Et3N, DMF, rt, overnight; (b) Fe powder, NH4Cl, EtOH/H2O (3:1), reflux, 4 h; (c) RX,
Cs2CO3, CH3CN, reflux, overnight.

5668
J. Zhu et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5666–5670
Table 1
10a–10o. Treatment of 10a with various alkyl or benzyl halides
yielded the desired products 11a–11j.24
Inhibitory effects of compounds against TGF-b1-induced total collagen accumulation
in NRK-49F cells at a concentration of 10
l
M
All synthesized Ivacaftor derivatives were evaluated for the
anti-fibrotic activities against TGF-b1-induced total collagen accu-
mulation in NRK-49F cells. Ivacaftor was used as a positive control.
Their initial screening data were depicted in Table 1. As for 9a–9f,
the replacement with different electron-donating group or elec-
tron-withdrawing group on A-ring exhibited comparable inhibi-
tory potency to Ivacaftor (64.7%). Compound 9e exhibited the
most efficacious with the inhibition rate of 73.8%. The introduction
of tert-butyl group (10a–10c) at position C-40 on B-ring led to
increased inhibitory activities. Particularly, compound 10a exhib-
ited the most potent inhibitory activities with the inhibition rate
of 81.0%. However, the replacement of C-40-tert-butyl with elec-
tron-withdrawing group (fluorine, chlorine, trifluoromethyl) or
other electron-donating group (methyl, ethyl, methoxyl) signifi-
cantly reduced the activity. It is surprising that the total of meth-
oxyl group affected activities, and compound 10l with three
methoxyl groups exhibited a 3.3-fold increase in inhibitory effects
(77.1%) compared to 10n with one methoxyl group. Unfortunately,
N1-substitution 10a analogues (11a–11j) were observed much
lower or moderate activities, with the exception being the N1-mor-
pholinoethyl group (11g) which showed approximate inhibitory
ability (71.1%) compared with Ivacaftor.
Compds
Inhibition
(%) SD
Compds
Inhibition
(%) SD
Ivacaftor
9a
9b
9c
9d
64.7 2.1b
49.9 8.6
55.9 5.5b
53.1 4.7b
51.6 7.3
73.8 3.4a
48.7 9.3
81.0 1.2b
75.7 1.6b
67.9 5.6b
54.3 5.5
44.8 4.0a
28.9 17.6
19.4 7.2
23.5 5.2
35.3 6.2
10j
10k
10l
27.2 8.3
30.5 5.9
77.1 1.7b
32.6 4.8a
23.5 7.0
47.9 11.8
34.3 8.5
16.9 7.7
9.1 12.3
35.8 6.5
30.1 13.5
44.8 3.8a
71.1 6.3
46.6 5.9
52.1 5.6a
30.2 4.0
10m
10n
10o
11a
11b
11c
11d
11e
11f
9e
9f
10a
10b
10c
10d
10e
10f
10g
10h
10i
11g
11h
11i
11j
The results are the means SD of at least three independent experiments. aP <0.05,
bP <0.01 versus the TGF-b1-treated group.
Table 2
Inhibitory effects of compounds against the production of TNF-
264.7 cells at a concentration of 10
a in LPS-induced RAW
l
M
Compounds 9e, 10a–10c, 10l and 11g were chosen to investi-
gate inhibitory effects of TNF-
a production in LPS-induced RAW
Compds
TNF-
(%) SD
a
inhibition
Compds
TNF-a inhibition
(%) SD
264.7 cells.25,26 The results were summarized in Table 2. Four com-
pounds 10a, 10c, 10l and 11g were chosen to further investigate
the inhibitory activity against TGF-b1-induced total collagen accu-
mulation in NRK-49F cells. As shown in Table 3, they possessed
Ivacaftor
9e
10a
63.2 1.5b
45.7 0.6b
61.6 9.0
47.7 7.7
10c
10l
11g
71.3 7.0a
70.8 3.6b
69.5 6.7a
10b
potent inhibitory activity with IC50 of 2.0
2.3 M, respectively.
The visible mode of extracellular collagen deposition is estab-
lM, 4.1 lM, 2.5 lM and
The results are the means SD of at least three independent experiments. aP <0.05,
bP <0.01 versus the LPS-treated group.
l
lished upon Picro-Sirius red (PSR) staining to text inhibitory activ-
ity of 10a, 10c, 10l and 11g against TGF-b1-induced total collagen
accumulation. As depicted in Figure 2, treatment with 10a, 10c, 10l
Table 3
IC50 values against TGF-b1-induced total collagen accumulation in NRK-49F cells
and 11g at a concentration of 3 lM, the total collagen accumula-
tion was alleviated remarkably (Fig. 2A4–E4). It is interesting to
Compds
IC50
(
lM)
Compds
IC50 (lM)
note that the total collagen accumulation was hardly visualized
in the presence of 10l and 11g at a concentration of 5 lM
(Fig. 2D6 and E6).
Ivacaftor
10a
10c
4.0
2.0
4.1
10l
11g
2.5
2.3
Figure 2. Picro-Sirius red (PSR) staining for the change of TGF-b1-induced total collagen accumulation in NRK-49F cells.

J. Zhu et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5666–5670
5669
Figure 3. Treatment of bleomycin-induced lung fibrosis, effects on accumulative survival rate (A) and variation of body weight (B), effects on differential cells count in BALF
(C–E) and hydroxyproline analysis in right lung tissue (F). Mice were treated with Ivacaftor, 10a, 10l, 11g at a dose of 20 mg/kg/day. The results were expressed as the
means SEM, ⁄P <0.05, ⁄⁄P <0.01, ⁄⁄⁄P <0.001 versus vehicle.
Pulmonary fibrosis is a chronic, progressive, and irreversible
lung disease.27,28 The severity of pulmonary fibrosis was affected
by individual differences and the fibrotic progression. To evaluate
the in vivo anti-fibrotic potency of 10a, 10l and 11g, bleomycin-
induced lung fibrosis model was employed.29 Compounds 10a,
10l, 11g and Ivacaftor were administered (ip) at a dose of 20 mg/
kg/day prior to the bleomycin injection. Results exhibited that
the cumulative proportion surviving of 10a (83.3%), 10l (72.2%)
were higher than others on day 28 (Fig. 3A). The body weight loss
was reduced more slowly during the treatment with 10a and 10l
than others (Fig. 3B). Inflammatory cells such as leukocytes, lym-
phocytes and neutrophils were documented to play a key role dur-
ing lung fibrotic reactions.30 The amount of inflammatory cells in
bronchoalveolar lavage fluid (BALF) are an important parameter
of acute inflammatory caused by bleomycin for evaluating com-
pounds with potential anti-fibrotic activities. As depicted in Fig-
ure 3C–E, Compound 10l significantly reduced the inflammatory
cells (54.8% for leukocytes, 15.8% for lymphocytes, 57.7% for neu-
trophils). Compound 10a exhibited less effect (40.3% for leuko-
cytes, 33.6% for lymphocytes, 39.7% for neutrophils).
Figure 4. H&E staining and Masson staining for the histological changes of left lung tissues.

5670
J. Zhu et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5666–5670
2. Ziobro, R.; Henry, B.; Edwards, M. J.; Lentsch, A. B.; Gulbins, E. Biochem. Biophys.
Anti-fibrotic effects of compounds can also be evaluated by
Res. Commun. 2013, 434, 705.
measuring the content of hydroxyproline, an amino acid that is
specifically found in collagen.31 As displayed in Figure 3F, the con-
tent of hydroxyproline was significantly reduced by the value of
52.4% in the presence of 10l at a dose of 20 mg/kg/day. However,
10a showed slight effect.
3. Wilson, M. S.; Wynn, T. A. Mucosal Immunol. 2009, 2, 103.
4. Ma, Z.; Pan, Y.; Huang, W.; Yang, Y.; Wang, Z.; Li, Q.; Zhao, Y.; Zhang, X.; Shen, Z.
Bioorg. Med. Chem. Lett. 2014, 24, 220.
5. Raghu, G.; Collard, H. R.; Egan, J. J.; Martinez, F. J.; Behr, J.; Brown, K. K.; Colby, T.
V.; Cordier, J. F.; Flaherty, K. R.; Lasky, J. A.; Lynch, D. A.; Ryu, J. H.; Swigris, J. J.;
Wells, A. U.; Ancochea, J.; Bouros, D.; Carvalho, C.; Costabel, U.; Ebina, M.;
Hansell, D. M.; Johkoh, T.; Kim, D. S.; King, T. E. J.; Kondoh, Y.; Myers, J.; Muller,
N. L.; Nicholson, A. G.; Richeldi, L.; Selman, M.; Dudden, R. F.; Griss, B. S.;
Protzko, S. L.; Schunemann, H. J. Am. J. Respir. Crit. Care Med. 2011, 183, 788.
6. Gribbin, J.; Hubbard, R. B.; Le Jeune, I.; Smith, C. J.; West, J.; Tata, L. J. Thorax
2006, 61, 980.
7. Raghu, G.; Weycker, D.; Edelsberg, J.; Bradford, W. Z.; Oster, G. Am. J. Respir. Crit.
Care Med. 2006, 174, 810.
8. Ley, B.; Collard, H. R.; King, T. E., Jr. Am. J. Respir. Crit. Care Med. 2011, 183, 431.
9. Pohlers, D.; Brenmoehl, J.; Loffler, I.; Muller, C. K.; Leipner, C.; Schultze-Mosgau,
S.; Wolf, G. Biochim. Biophys. Acta 2009, 1792, 746.
From the results of H&E staining, massive inflammatory cells
infiltrated into the alveoli and interstitium in bleomycin-induced
group, the walls of the air sacs and the small blood vessels were
destroyed because of inflammation (Fig. 4B) while administrations
of 10l at a dose of 20 mg/kg/day was observed with few signs of
inflammatory infiltrate and a strong amelioration of bleomycin-
induced injury (Fig. 4E). The effects of Ivacaftor and 10a were not
obvious at the same doses (Fig. 4C and D). As shown by Masson
staining, excessive collagen was visualized to accumulate in lung
tissue and the normal air sacs architecture was destroyed in bleo-
mycin-induced group, treatment with 10l resulted in significant
suppression of bleomycin-induced pulmonary fibrosis, character-
ized by a reduction of lung collagen deposition (Fig. 4J). Ivacaftor
and 10a had slight effects (Fig. 4H and I).
10. Chen, J.; Lu, M.; Liu, B.; Chen, Z.; Li, Q.; Tao, L.; Hu, G. Bioorg. Med. Chem. Lett.
2012, 22, 2300.
11. Wynn, T. A.; Ramalingam, T. R. Nat. Med. 2012, 18, 1028.
12. Bartram, U.; Speer, C. P. Chest 2004, 125, 754.
13. Strieter, R. M.; Mehrad, B. Chest 2009, 136, 1364.
14. Arribillaga, L.; Dotor, J.; Basagoiti, M.; Riezu-Boj, J. I.; Borras-Cuesta, F.; Lasarte,
J. J.; Sarobe, P.; Cornet, M. E.; Feijoo, E. Cytokine 2011, 53, 327.
15. Khasnis, A. A.; Calabrese, L. H. Semin. Arthritis Rheum. 2010, 40, 147.
16. Davis, P. B.; Yasothan, U.; Kirkpatrick, P. Nat. Rev. Drug Discovery 2012, 11, 349.
17. http://www.clinicaltrials.gov/ct2/show/NCT02135432?term=VX-
770&rank=13.
18. Polenakovik, H. M.; Sanville, B. J. Cyst. Fibros. 2013, 12, 530.
19. Kotha, K.; Clancy, J. P. Ther. Adv. Respir. Dis. 2013, 7, 288.
20. Hu, Q.; Noor, M.; Wong, Y. F.; Hylands, P. J.; Simmonds, M. S.; Xu, Q.; Jiang, D.;
Hendry, B. M.; Xu, Q. Nephrol. Dial. Transplant. 2009, 24, 3033.
21. Niedermeier, S.; Singethan, K.; Rohrer, S. G.; Matz, M.; Kossner, M.; Diederich,
S.; Maisner, A.; Schmitz, J.; Hiltensperger, G.; Baumann, K.; Holzgrabe, U.;
Schneider-Schaulies, J. J. Med. Chem. 2009, 52, 4257.
In conclusion, thirty-one 4-oxoquinoline-3-carboxamides
derivatives were synthesized to evaluate for their anti-fibrotic
activities. Three derivatives (10a, 10l, 11g) exhibited potent inhib-
itory activity on both TGF-b1-induced total collagen accumulation
and TNF-a production. Treatment with 10l at a dose of 20 mg/kg/
day clearly reduced lung inflammation and fibrosis. These observa-
tions suggest that 10l might be an effective agent for the treatment
of pulmonary fibrosis.
22. Ding, H. X.; Leverett, C. A.; Kyne, R. E., Jr.; Liu, K. K.; Sakya, S. M.; Flick, A. C.;
O’Donnell, C. J. Bioorg. Med. Chem. 2014, 22, 2005.
23. Sheth, U.; Fanning, L. T. D.; Numa, M. M. D.; Binch, H.; Hurley, D.; Zhou, J.; Ruah,
S. S. H.; Hazlewood, A.; Silina, A.; Vairagoundar, R.; Goor, F.; Grootenhuis, P. D.
J.; Botfield, M. U.S. Patent 20100184739A1, 2010.
24. Wang, G.; Li, C.; He, L.; Lei, K.; Wang, F.; Pu, Y.; Yang, Z.; Cao, D.; Ma, L.; Chen, J.;
Sang, Y.; Liang, X.; Xiang, M.; Peng, A.; Wei, Y.; Chen, L. Bioorg. Med. Chem. 2014,
22, 2060.
Acknowledgments
We greatly appreciated the financial support from China Post-
doctoral Science Foundation (No. 2014M552373) and National
Key R&D Programs of China (2012ZX09103101-017).
25. Wei, Z.; Yang, Y.; Xie, C.; Li, C.; Wang, G.; Ma, L.; Xiang, M.; Sun, J.; Wei, Y.;
Chen, L. Fitoterapia 2014, 97, 172.
26. Wang, F.; Sun, J.; Huang, M.; Wang, H.; Sun, P.; Lin, J.; Chen, W. Eur. J. Med.
Chem. 2014, 72, 35.
Supplementary data
27. Fabre, A.; Marchal-Somme, J.; Marchand-Adam, S.; Quesnel, C.; Borie, R.;
Dehoux, M.; Ruffle, C.; Callebert, J.; Launay, J.-M.; Henin, D.; Soler, P.; Crestani,
B. Eur. Respir. J. 2008, 32, 426.
28. Nagano, J.; Iyonaga, K.; Kawamura, K.; Yamashita, A.; Ichiyasu, H.; Okamoto, T.;
Suga, M.; Sasaki, Y.; Kohrogi, H. Eur. Respir. J. 2006, 27, 460.
29. Moeller, A.; Ask, K.; Warburton, D.; Gauldie, J.; Kolb, M. Int. J. Biochem. Cell Biol.
2008, 40, 362.
30. Shimabukuro, D. W.; Sawa, T.; Gropper, M. A. Crit. Care Med. 2003, 31, S524.
31. Pini, A.; Shemesh, R.; Samuel, C. S.; Bathgate, R. A. D.; Zauberman, A.; Hermesh,
C.; Wool, A.; Bani, D.; Rotman, G. J. Pharmacol. Exp. Ther. 2010, 335, 589.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.
10.071.
References and notes
1. Selman, M.; Thannickal, V. J.; Pardo, A.; Zisman, D. A.; Martinez, F. J.; Lynch, J. P.
Drugs 2004, 64, 405.