Synthesis and in vitro cytotoxic evaluation of 2-hydrazinylpyrido[2,3-b] pyrazin-3(4H)-one derivatives

Article abstract of DOI:10.1016/j.cclet.2011.05.031

A series of novel 2-hydrazinylpyrido[2,3-b]pyrazin-3(4H)-one derivatives were synthesized and evaluated for their cytotoxic activities against A549, MDA-MB-231 and HT-29 cell lines in vitro. Pharmacological data indicated that compounds 5b, 5c, 10a and 10

Full text of DOI:10.1016/j.cclet.2011.05.031

Available online at www.sciencedirect.com
Chinese Chemical Letters 22 (2011) 1223–1225
www.elsevier.com/locate/cclet
Synthesis and in vitro cytotoxic evaluation of
2-hydrazinylpyrido[2,3-b]pyrazin-3(4H)-one derivatives
*
Guo Gang Zhang, Ya Jing Liu, Xiao Guang Ma, Hao Dong, Ju Li, Ping Gong
Key Laboratory of Original New Drugs Design and Discovery of Ministry of Education, School of Pharmaceutical Engineering, Shenyang
Pharmaceutical University, Shenyang 110016, China
Received 17 March 2011
Available online 20 July 2011
Abstract
A series of novel 2-hydrazinylpyrido[2,3-b]pyrazin-3(4H)-one derivatives were synthesized and evaluated for their cytotoxic
activities against A549, MDA-MB-231 and HT-29 cell lines in vitro. Pharmacological data indicated that compounds 5b, 5c, 10a
and 10g possessed marked cytotoxicity, especially 10a (with IC50 values of 0.81, 2.56 and 1.63 mmol/L against A549, MDA-MB-
231 and HT29 cell lines, respectively), which had emerged as a lead compound.
# 2011 Ping Gong. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
Keywords: 2-Hydrazinylpyrido[2,3-b]pyrazin-3(4H)-ones; Synthesis; Cytotoxicity
Cancer is a major worldwide health problem. According to the American Cancer Society, 7.6 million people died
from cancer in the world during 2008 [1]. In order to develop more effective and reliable anticancer agents, a large
number of compounds bearing nitrogen-containing fused heterocyclics keletons, such as quinoxalines,
pyrrolopyrimidines, 4-anilinoquinazolines, pyridopyrimidines, and pyrazolopyridazines, have been reported and
many of them exhibited excellent anticancer activity [2–6].
Recently, pyrido[2,3-b]pyrazin-3(4H)-ones have aroused increasing attentions from chemical and biological view
pointssince they were provedto be thepromising anticancer agents with mechanismsof BRAF inhibition [7]. On another
hand, compoundscontainingarylhydrazinewerereportedfortheirgoodcytotoxicity[8,9], which haveinspireduslargely
to develop the related derivatives. With an aim to develop potent pyrido[2,3-b]pyrazin-3(4H)-one derivatives a series of
new molecules containing various arylhydrazones on C-2 position of thescaffold were designed and synthesized. Further
modifications were performed by introducing phenyl or 4-trifluoromethoxyl-phenyl group into N-4 position on the
pyrido[2,3-b]pyrazin-3(4H)-one core. In this paper, we would like to report the synthesis and cytotoxicity of a series of
novel 2-hydrazinylpyrido[2,3-b]pyrazin-3(4H)-ones represented by the generable structures of 5a–5g and 10a–10g.
The title 2-hydrazinylpyrido[2,3-b]pyrazin-3(4H)-one derivatives 5a–5g and 10a–10g were synthesized as shown
in Scheme 1. The commercially available 2-chloro-3-nitropyridine and aniline were treated with N,N-
diisopropylethylamine in isopropanol to give the compound 1. Next, reduction of 1 with zinc powder was carried
out in 95% ethanol at reflux to afford the N2-phenylpyridine-2,3-diamine intermediate, which was converted to 2 by a
* Corresponding author.
E-mail address: gongpinggp@126.com (P. Gong).
1001-8417/$ – see front matter # 2011 Ping Gong. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
doi:10.1016/j.cclet.2011.05.031

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G.G. Zhang et al. / Chinese Chemical Letters 22 (2011) 1223–1225
Scheme 1. Reagents and conditions: (a) DIPEA/MeOH, r.t., 1 h, 60 8C, 8 h, yield: 53–75%; (b) zinc powder/NH4Cl/EtOH, r.f., 5 h; (c) oxalic acid/
4 mol/L HCl, r.f., 15 h, yield: 33–52%; (d) POCl3, r.f., 3 h, yield: 75–82%; (e) 80% NH2NH2ÁH2O, 60 8C, 1 h, yield: 85–91%; (f) EtOH, 60 8C, 5 h,
yield: 45–65%.
cyclization reaction with oxalic acid. Subsequent treatment of 2 with phosphorus oxychloride and acetonitrile afforded
intermediate 3 [10], which was reacted with an excess of 80% hydrazine hydrate in ethanol to furnish 4. Another
important intermediate 9 was obtained according to the same method as described for compound 4 when aniline was
replaced by 4-(trifluoromethoxy)aniline, respectively. Finally, the target compounds 5a–5g and 10a–10g were
successfully obtained via the reaction of intermediate 4 and 9 with different aromatic aldehydes in the refluxing
ethanol, respectively. The products were purified by silica gel column chromatography, using EtOAc/petroleum ether
1
as eluent and the structures of the target compounds were confirmed by MS, H NMR and 13C NMR [11].
Cytotoxicity of compounds 5a–5g and 10a–10h against A549, MDA-MB-231 and HT29 cell lines were determined
by MTT assay, procaspase activating compound 1 (PAC-1) [9], a well-known hydrazine-contain anticancer agent, as
positive control and the results expressed as IC50 are summarized in Table 1. As shown in Table 1, compounds 5b, 10a
and 10g exhibited good cytotoxicity, in vitro against HT-29 cell lines. Compound 10a was of particular interest
because of its marked activity (IC50 values of 0.81, 1.56 and 2.63 mmol/L against A549, MDA-MB-231 and HT29 cell
lines, respectively) and had emerged as a lead compound.
The data indicated that substituents on N-4 position of pyrido[2,3-b]pyrazin-3(4H)-one scaffold had a very
important effect on antitumor activity, and variation of arylidene on hydrazine at C-2 position would optimize the
Table 1
Cytotoxicity of the tested compounds against A549, MDA-MB-231 and HT-29 cell lines in vitro.
Compd.
X
Ar
IC50 (mmol/L)
A549
MDA-MB-231
HT-29
5a
5b
H
H
4-Fluorobenzyl
47.32 Æ 5.28
1.69 Æ 0.15
25.25 Æ 3.61
74.21 Æ 6.84
60.74 Æ 7.13
19.46 Æ 2.03
>100
14.76 Æ 2.33
2.87 Æ 0.32
2.98 Æ 0.45
11.26 Æ 1.32
14.75 Æ 2.31
9.58 Æ 0.87
>100
38.63 Æ 3.92
2-Hydroxynaphthalen-1-yl
4-Hydroxy-3-methoxybenzyl
1H-Pyrrol-2-yl
3.45 Æ 0.41
6.81 Æ 0.49
87.48 Æ 8.74
63.40 Æ 8.10
13.53 Æ 2.16
5c
5d
H
H
5e
H
1H-Indol-3-yl
5f
5g
H
2,3,4-Trimethoxybenzyl
3-Nitrobenzyl
H
>100
10a
10b
10c
10d
10e
10f
10g
PAC-1
–OCF3
–OCF3
–OCF3
–OCF3
–OCF3
–OCF3
–OCF3
2-Hydroxynaphthalen-1-yl
3,4-Difluorobenzyl
0.81 Æ 0.09
>100
1.56 Æ 0.12
34.71 Æ 3.59
18.84 Æ 3.21
>100
2.63 Æ 0.35
22.8 Æ 1.96
15.62 Æ 2.11
2,4-Dimethoxybenzyl
4-(Methylsulfonyl)benzyl
3-Hydroxy-4-methoxybenzyl
Imidazo[1,2-a]pyridin-3-yl
4-Hydroxy-3-methoxybenzyl
14.39 Æ 1.87
80.01 Æ 7.24
18.56 Æ 1.78
22.38 Æ 2.16
14.21 Æ 0.19
0.66 Æ 0.08
>100
11.04 Æ 1.02
5.64 Æ 0.77
1.52 Æ 0.24
6.63 Æ 0.58
13.22 Æ 1.54
81.47 Æ 8.15
2.77 Æ 0.31
1.64 Æ 0.13

G.G. Zhang et al. / Chinese Chemical Letters 22 (2011) 1223–1225
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activity dramatically. Contrast to benzyl group, 4-(triflouromethyl)benzyl group was more potent substituent which
produced the compounds with excellent activity. A case in point is that compound 5c with benzyl group at N-4 position
had lower activity, whereas compound 10g bearing 4-triflouromethylbenzyl group provided about a 2-fold increase in
potency against three tumor cell lines relative to the 5c. On the other hand, introduction of heterocyclidene (e.g. 1H-
pyrrol-2-ylmethylene, 1H-indol-3-ylmethylene, and imidazo[1,2-a]pyridine-3-yl-methylene) exhibited the selectivity
of MDA-MB-231 cell line, while introduction of 2-hydroxynaphthalen-1-yl showed enhanced antitumor activity
against all three cell lines. In addition, electron-donating group on benzylidene such as hydroxy and methoxy groups
had good contributions to the anti-tumor activity. Compounds 10c, 10e and 10g showed more potent cytotoxicity
superior to the corresponding compounds bearing electron-withdraw group on the benzylidene.
Acknowledgment
This work was supported by the grant from National S & T Major Project of China (No. 2009ZX09301-012).
References
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[6] J.F. Blake, N.C. Kallan, D. Xiao, et al. Bioorg. Med. Chem. Lett. 20 (2010) 5607.
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[10] B.D. Louis, O.D. Rhys, P. Christopher, et al., World Patent 2007020521 (C.A. 2007, 146:274402).
[11] Physical and spectral data for target compounds. 5a: mp 168–170 8C; ESI-MS (m/z, %): 360.1 (M+H)+; 1H NMR (300 MHz, DMSO-d6): d 8.70
(s, 1H), 8.11 (dd, 1H, J = 4.7, 1.5 Hz); 8.03 (dd, 1H, J = 7.9, 1.5 Hz), 7.90 (dd, 2H, J = 8.6, 5.7 Hz), 7.51 (t, 2H, J = 7.3 Hz), 7.44 (t, 1H,
J = 7.2 Hz), 7.39–7.30 (m, 4H), 7.24 (dd, 1H, J = 7.7, 4.7 Hz). 5b: mp 173–175 8C; ESI-MS (m/z, %): 408.1 (M+H)+; 1H NMR (300 MHz,
DMSO-d6): d 13.16 (s, 1H), 11.93 (s, 1H), 9.76 (s, 1H), 8.15 (m, 2H), 7.93 (m, 3H), 7.61 (t, 1H, J = 7.5 Hz), 7.51 (t, 2H, J = 7.4 Hz), 7.44–7.40
(m, 2H), 7.35 (d, 2H, J = 7.2 Hz), 7.27–7.22 (m, 2H). 5c: mp 181–183 8C; ESI-MS (m/z, %): 388.1 (M+H)+; 1H NMR (300 MHz, DMSO-d6): d
11.28 (s, 1H), 9.60 (s, 1H), 8.52 (s, 1H), 8.02 (s, 1H), 7.94 (d, 1H, J = 7.6 Hz), 7.56 (t, 2H, J = 7.4 Hz), 7.48 (t, 1H, J = 7.3 Hz), 7.39 (d, 3H,
J = 7.4 Hz), 7.25 (s, 1H), 7.17 (s, 1H), 6.87 (d, 1H, J = 8.1 Hz), 3.86 (s, 3H); 13C NMR (75 MHz, DMSO-d6): d 153.57, 151.74, 149.93, 145.80,
143.53, 142.76, 138.82, 133.22, 129.62, 129.08, 128.91, 128.68, 128.40, 126.89, 126.22, 120.19, 117.36, 54.87. 5d: mp 160–162 8C; ESI-MS
(m/z, %): 331.1 (M+H)+; 1H NMR (300 MHz, DMSO-d6): d 11.56 (s, 1H), 8.45 (s, 1H), 8.00 (s, 1H), 7.84 (s, 1H), 7.56 (t, 2H, J = 7.3 Hz), 7.48
(t, 2H, J = 7.2 Hz), 7.38 (d, 1H, J = 7.1 Hz), 7.25 (dd, 1H, J = 7.7, 4.7 Hz), 7.02 (s, 1H), 6.54 (s, 1H), 6.19 (s, 1H). 5e: mp 154–156 8C; ESI-MS
(m/z, %): 381.1 (M+H)+; 1H NMR (300 MHz, DMSO-d6): d 11.64 (s, 1H), 8.80 (s, 1H), 8.47 (d, 1H, J = 5.9 Hz), 7.99 (m, 2H), 7.82 (m, 1H),
7.52 (m, 4H), 7.40 (d, 2H, J = 7.2 Hz), 7.34 (d, 1H, J = 7.1 Hz), 7.25 (m, 3H). 5f: mp 156–158 8C; ESI-MS (m/z, %): 432.1 (M+H)+; 1H NMR
(300 MHz, DMSO-d6): d 11.41 (s, 1H), 8.53 (s, 1H), 8.06 (dd, 1H, J = 4.6, 1.6 Hz), 7.95 (dd, 1H, J = 7.9, 1.6 Hz), 7.53 (d, 2H, J = 7.7 Hz), 7.48
(t, 1H, J = 7.4 Hz), 7.37 (d, 2H, J = 7.1 Hz), 7.26 (dd, 1H, J = 7.9, 4.7 Hz), 7.00 (s, 2H), 3.84 (s, 6H), 3.69 (s, 3H). 5g: mp: 169–171 8C; ESI-MS
(m/z, %): 387.1 (M+H)+; 1H NMR (300 MHz, DMSO-d6): d 11.49 (s, 1H), 8.79 (s, 1H), 8.61 (s, 1H), 8.29 (d, 1H, J = 8.2 Hz), 8.21 (d, 1H,
J = 7.8 Hz), 8.12 (d, 1H, J = 3.2 Hz), 8.06 (d, 1H, J = 7.7 Hz), 7.79 (t, 1H, J = 8.0 Hz), 7.58 (t, 2H, J = 7.4 Hz), 7.51 (t, 1H, J = 7.2 Hz), 7.41 (d,
1
2H, J = 7.4 Hz), 7.31 (dd, 1H, J = 7.9, 4.7 Hz). 10a: mp 179–181 8C; ESI-MS (m/z, %): 492.2 (M+H)+; H NMR (300 MHz, DMSO-d6): d
13.19 (s, 1H), 11.95 (s, 1H), 9.77 (s, 1H), 8.14 (d, 2H, J = 8.9 Hz), 7.95 (d, 2H, J = 9.0 Hz), 7.91 (d, 1H, J = 8.1 Hz), 7.62 (m, 5H), 7.42 (t, 1H,
J = 7.4 Hz), 7.34 (dd, 1H, J = 7.6, 4.6 Hz), 7.27 (d, 1H, J = 8.9 Hz); 13C NMR (75 MHz, DMSO): d 160.57, 151.79, 149.37, 147.98, 145.63,
143.04, 137.10, 134.74, 132.98, 131.52, 131.16, 129.60, 129.00, 124.11, 121.68, 120.27, 113.46, 110.74, 107.66, 102.72. 10b: mp 169–161 8C;
ESI-MS (m/z, %): 462.1 (M+H)+; 1H NMR (300 MHz, DMSO-d6): d 11.62 (s, 1H), 8.61 (s, 1H), 8.11 (d, 1H, J = 4.5 Hz), 8.01 (d, 1H,
J = 7.8 Hz), 7.80–7.73 (m, 2H), 7.58 (s, 4H), 7.53 (d, 1H, J = 4.7 Hz), 7.31 (dd, 1H, J = 7.8, 4.7 Hz); 13C NMR (75 MHz, DMSO): d 167.32,
151.85, 147.98, 146.08, 145.42, 143.41, 142.59, 134.75, 133.25, 131.62, 131.14, 128.84, 128.65, 124.26, 121.68, 120.26, 118.79, 118.19,
118.02, 115.16. 10c: mp 187–190 8C; ESI-MS (m/z, %): 486.1 (M+H)+; 1H NMR (300 MHz, DMSO-d6): d 11.40 (s, 1H), 8.87 (s, 1H), 8.07 (s,
1H), 7.95 (m, 3H), 7.57 (m, 4H), 7.28 (s, 1H), 6.67 (d, 2H, J = 10.3 Hz), 3.87 (s, 3H), 3.84 (s, 3H). 10d: mp 150–152 8C; ESI-MS (m/z, %):
504.1 (M+H)+; 1H NMR (300 MHz, DMSO-d6): d 8.74 (s, 1H), 8.12 (dd, 1H, J = 4.7, 1.5 Hz), 8.05 (d, 1H, J = 7.9 Hz), 8.03 (s, 4H), 7.59 (s,
4H), 7.32 (dd, 1H, J = 7.9, 4.7 Hz), 3.27 (s, 3H). 10e: mp 161–163 8C ESI-MS (m/z, %): 472.1 (M+H)+; 1H NMR (300 MHz, DMSO-d6): d
11.29 (s, 1H), 9.35 (s, 1H), 8.49 (s, 1H), 8.08 (d, 1H, J = 4.2 Hz), 7.95 (d, 1H, J = 7.6 Hz), 7.57 (s, 4H), 7.33 (s, 1H), 7.29 (dd, 1H, J = 7.8,
4.7 Hz), 7.04 (d, 1H, J = 7.7 Hz), 6.98 (d, 1H, J = 8.3 Hz), 3.81 (s, 3H). 10f: mp 160–162 8C; ESI-MS (m/z, %): 466.1 (M+H)+; 1H NMR
(300 MHz, DMSO-d6): d 9.93 (d, 1H, J = 6.8 Hz), 8.95 (s, 1H), 8.22 (s, 1H), 8.09 (d, 1H, J = 4.3 Hz), 8.03 (d, 1H, J = 8.0 Hz), 7.86 (d, 1H,
J = 9.0 Hz), 7.68 (t, 1H, J = 6.9 Hz), 7.59–7.56 (m, 5H), 7.43 (t, 1H, J = 6.8 Hz), 7.32 (dd, 1H, J = 7.8, 4.8 Hz). 10g: mp 160–162 8C; ESI-MS
(m/z, %): 472.1 (M+H)+; 1H NMR (300 MHz, DMSO-d6): d 11.29 (s, 1H), 9.58 (s, 1H), 8.52 (d, 1H, J = 7.2 Hz), 8.09 (s, 1H), 7.97 (d,
J = 7.7 Hz, 1H), 7.58 (s, 4H), 7.32 (m, 2H), 7.15 (d, 1H, J = 17.1 Hz), 6.86 (d, 1H, J = 7.8 Hz), 3.87 (s, 3H).