Acylhydrazone derivatives as potential anticancer agents: Synthesis, bio-evaluation and mechanism of action

Article abstract of DOI:10.1016/j.bmcl.2015.10.069

A series of novel acylhydrazone derivatives were designed, synthesized and evaluated for their potential cytotoxic effects against human cancer cell lines. The preliminary results indicated that some of the obtained compounds (such as 8b, 13c) exhibited good to moderate cytotoxic activities against human HepG2, Huh-7, and BCG-823 cell lines. Especially, compounds 8c and 8e presented obviously selective cytotoxic activities against Huh-7 in vitro (8c, IC₅₀ = 7.74 ± 2.18 μg/mL; 8e, IC₅₀ = 4.46 ± 1.05 μg/mL) compared to 5-FU (IC₅₀ = 10.41 ± 3.41 μg/mL). The highly potential compounds to induce apoptosis in HepG2 cells were analyzed by flow cytometry, and the apoptotic effects of compounds 8b and 13c were further evaluated using Annexin V-FITC/propidium iodide dual staining assay.

Full text of DOI:10.1016/j.bmcl.2015.10.069

Accepted Manuscript
Acylhydrazone Derivatives as Potential Anticancer Agents: Synthesis, Bio-
evaluation and Mechanism of Action
Xifang Yu, Liqiao Shi, Shaoyong Ke
PII:
S0960-894X(15)30178-5
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.10.069
BMCL 23225
Reference:
Bioorganic & Medicinal Chemistry Letters
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Received Date:
Revised Date:
Accepted Date:
21 August 2015
18 September 2015
22 October 2015
Please cite this article as: Yu, X., Shi, L., Ke, S., Acylhydrazone Derivatives as Potential Anticancer Agents:
Synthesis, Bio-evaluation and Mechanism of Action, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://
dx.doi.org/10.1016/j.bmcl.2015.10.069
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Acylhydrazone Derivatives as Potential Anticancer Agents: Synthesis,
Bio-evaluation and Mechanism of Action
Xifang Yu,a Liqiao Shi,b Shaoyong Keb,*
aHuangshi Central Hospital, Hubei Polytechnic University, Huangshi 435000, People’s Republic of
China
bNational Biopesticide Engineering Research Center, Hubei Academy of Agricultural Sciences,
Wuhan 430064, People’s Republic of China
* To whom correspondence should be addressed. E-mail: keshaoyong@163.com
Abstract: A series of novel acylhydrazone derivatives were designed, synthesized and evaluated for
their potential cytotoxic effects against human cancer cell lines. The preliminary results indicated
that some of the obtained compounds (such as 8b, 13c) exhibited good to moderate cytotoxic
activities against human HepG2, Huh-7, and BCG-823 cell lines. Especially, compounds 8c and 8e
presented obviously selective cytotoxic activities against Huh-7 in vitro (8c, IC50 = 7.74 2.18 µg/mL;
8e, IC50 = 4.46 1.05 µg/mL) compared to 5-FU (IC50 = 10.41 3.41 µg/mL). The highly potential
compounds to induce apoptosis in HepG2 cells were analyzed by flow cytometry, and the apoptotic
effects of compounds 8b and 13c were further evaluated using Annexin V-FITC/propidium iodide
dual staining assay.
Keywords: Acylhydrazone, Synthesis, Cytotoxic activity, Apoptosis
1

Acylhydrazone scaffold (-CONHNH=) has attracted considerable attentions for decades due to
their broad applications range from medicinal agents, agrochemicals to functional materials.
Recently, many compounds containing this moiety have been reported, and which demonstrate that
the introduction of this pharmacophore may result in highly potential activities such as antivirus1,2,
antibacterials3,4, antitumor5,6, antioxidation7, antileishmanial agents8 et c. Meanwhile, some
compounds bearing acylhydrazone unit have also been evaluated as herbicides9, insecticides10,
nemacides11. It’s worthy of attention that acylhydrazone derivatives have also been widely used as
ligands to prepare various complexes and chemosensors12-16, and which are always the focus field to
researchers in materials due to its reversible acylhydrazone bond.
Meanwhile, benzoheterocycles always display extensive biological properties, and which are also
extremely versatile structural units serving as important synthons for construction of various
heterocyclic derivatives in the field of drug design and agrochemical industry17-19. Among these
heterocyclic molecules, benzothiazole skeleton has emerged as a most promising class of building
blocks for pharmaceutical drugs20,21. Up to now, many small molecules derived from benzothiazole
have been identified as potential antibacterial22, anticancer23,24, antiviral25 agents and BCL-XL
inhibitors26. In addition, the exploration of new molecular entities based on privileged structures
have been identified as a effective method in drug discovery process, and so the promising bioactive
diversity of this class of benzoheterocycle compounds urges us to synthesize and biologically
evaluate a series of novel acylhydrazone derivatives based on these heterocycles.
Thus, based on the aforementioned description, this work focused on the design, convenient
synthesis, and cytotoxic evaluation of a series of novel acylhydrazone derivatives based on
pharmacophores hybridization. We utilized acylhydrazone scaffold as key prototype structural unit
2

and planed for the integration of the corresponding heterocycle moiety and acylhydrazone
pharmacophore to the core structure as shown in Fig. 1, which might be developed as lead
compounds for highly potential cytotoxic agents. Therefore, a series of novel acylhydrazone
derivatives were designed, synthesized and evaluated for their potential cytotoxic effects against
human cancer cell lines (HepG2, Huh-7, and BCG-823), and the possible mechanism of action for
the highly potential compounds were also evaluated using Annexin V-FITC/propidium iodide dual
staining assay.
Fig. 1. Design strategy of heterocycle-acylhydrazone hybrids.
The general procedures for preparation of target benzothiazol-acylhydrazone hybrids 4a-e and
8a-e are outlined in Scheme 1.
R1
N
S
N
S
a
R2
N
S
S
OEt
O
b
S
HN NH2
O
c
S
HN
O
N
N
S
2
3
4a-e
SH
N
S
N
1
d
e
b
S
OH
O
S
O
S
O
N
S
5
6
R1
R2
N
c
S
HN NH2
S
HN
O
N
S
O
7
8a-e
o
Scheme 1. Reagents and conditions: (a) Ethyl 2-bromoacetate, K2CO3, MeCN, rt to 45 C; (b)
Hydrazine hydrate, EtOH, 45-50 oC; (c) Aldehyde or Ketone, EtOH, rt to 40 oC; (d)
4-(Chloromethyl)benzoic acid, K2CO3, MeCN, rt to 45 oC; (e) Conc. H2SO4, MeOH, 65-70 oC.
The key building block 4-((benzo[d]thiazol-2-ylthio)methyl)benzohydrazide 7 was obtained via
sequence reactions mainly including nucleophilic substitution, esterification, hydrazinolysis reaction,
3

and 2-(benzo[d]thiazol-2-ylthio)acetohydrazide 3 was also conveniently prepared using a similar
method. Then these acylhydrazines were treated with various aldehydes or ketones resulting in the
corresponding benzothiazol-acylhydrazone hybrids 4a-e and 8a-e via classical condensation reaction.
Furthermore, for a few comparative activity measurements of compounds with other heterocycle
moiety, four pyrimidine-acylhydrazone conjugates 13a-d as shown in Scheme 2 have also been
synthesized using similar method.
N
N
a
b
N
N
S
N
S
OH
OMe
N
SH
9
10
11
O
O
c
N
N
d
N
S
R1
N
S
H
N
H
N
N
R2
NH2
O
O
13a-d
12
o
Scheme 2. Reagents and conditions: (a) 4-(Chloromethyl)benzoic acid, K2CO3, MeCN, rt to 45 C;
o
o
(b) Conc. H2SO4, MeOH, 65-70 C; (c) Hydrazine hydrate, EtOH, 50 C; (d) Aldehyde or Ketone,
EtOH, rt to 40 oC.
All obtained acylhydrazone derivatives 4a-e, 8a-e and 13a-d were screened for their in vitro
cytotoxic effects against HepG2 (hepatocellular liver carcinoma), BCG-823 (gastric cancer), and
Huh-7
(hepatoma
cell
line)
cell
lines
by
the
modified
MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay using 5-FU (5-Fluorouracil)
as a positive control27-29. The preliminary screening results were described in Fig. 2.
4

HepG2
Huh-7
BCG-823
90
80
70
60
50
40
30
20
10
0
Compd. No.
Fig. 2. Antitumor activities of compounds 4a-e, 8a-e and 13a-d at 40 µg/mL. Abbreviations: HepG2
– Human hepatocellular liver carcinoma cell line; Huh-7 – Human hepatoma cell line; BCG-823 –
Human gastric cancer cell line; 5-FU – 5-Fluorouracil, used as a positive control.
Generally, as shown in Fig. 2, the newly prepared heterocycle-acylhydrazone hybrids displayed
moderate to good inhibitory activities against the three human cancer cell lines. Notably, the
compounds 4a, 4b, 8b, and 13c exhibited significant inhibitory activities against all tested cell lines
at the concentration of 40 µg/mL. Also, it is interesting to note that compound 4c and 8c presented
selective cytotoxicity against a special human hepatoma cell line, and the inhibition rates against
Huh-7 cell were up to 55% and 74% respectively. The results of preliminary cytotoxic assay
indicated this combination of heterocycle moiety and acylhydrazone unit leads to a highly potential
active scaffold, and which can be used as lead compounds for optimization of anticancer agents.
According to the preliminary results, some of the target compounds showed good inhibitory
activities against the tested cell lines, so in order to further explore the potential activities, the IC50
values were further evaluated using the above cell-based method. The inhibitory activities expressed
as IC50 values for the synthesized compounds are presented in Table 1.
Table 1 In vitro cytotoxic activities of the target compounds against human cancer cell lines
Entry
Compd.
No.
Substituents
In vitro cytotoxicity IC50a (µg/mL)
HepG2b BCG-823b Huh-7b
R1
R2
5

1
2
Me
4-Me-3-NO2-Ph
3-Indolin-2-one
1H-Indol-3-yl
3,4,5-(MeO)3-Ph
4-NO2-Ph
32.96 5.34
23.94 4.65
>80
17.82 3.54
17.70 0.74
>80
10.06 0.88
13.13 2.02
32.86 1.85
>80
4a
4b
3
H
H
4c
4
>80
>80
4d
5
Me
Me
>80
35.69 7.77
>80
39.67 2.55
>80
4e
6
4-Me-3-NO2-Ph
3-Indolin-2-one
1H-Indol-3-yl
3,4,5-(MeO)3-Ph
4-NO2-Ph
>80
8a
7
5.71 0.11
>80
11.89 2.33
>80
4.58 0.73
7.74 2.18
>80
8b
8
H
H
8c
9
>80
>80
8d
10
11
12
13
14
15
Me
H
35.73 1.60
33.28 5.15
34.14 7.11
4.85 1.02
>80
31.31 6.35
36.77 3.96
35.53 3.20
3.88 0.32
>80
4.46 1.05
17.42 1.73
23.23 1.12
3.25 0.48
>80
8e
3,4,5-(MeO)3-Ph
1H-Indol-3-yl
4-F-Ph
13a
13b
13c
13d
5-FUc
H
Me
Me
-
4-Me-3-NO2-Ph
-
35.99 5.23
17.41 4.09
10.41 3.41
a IC50 – Compound concentration required to inhibit tumor cell proliferation by 50%.
b
Abbreviations: HepG2 – Human hepatocellular liver carcinoma cell line; BCG-823 – Human
gastric cancer cell line; Huh-7 – Human hepatoma cell line.
c 5-Fluorouracil, used as a positive control.
The screening results also testified that some of the prepared heterocycle-acylhydrazone hybrids
exhibited higher inhibitory activities compared to the positive control 5-FU under the same
conditions. Generally, for series of compounds 4a-e, the ketone-hydrazone derivatives suach as 4a,
4b, 4e exhibited relatively good activities than that of aldehyde-hydrazones 4c and 4d. Meanwhile,
the ketone-hydrazone derivatives 8b and 8e also presented better activities than that of
aldehyde-hydrazone compounds 8c and 8d. Replacement of 3-indolin-2-one substituent of 4b and 8b
with a similar 1H-indol-3-yl group to afford compounds 4c and 8c resulted in an obvious decrease in
activities. For compounds 4b and 8b, containing the different linker, the compound bearing aromatic
ring linker 8b indicated better activities than that of alkyl linker. For series of compounds 13a-d, the
ketone-hydrazone 13c also exhibited better activities than the aldehyde-hydrazones 13a and 13b.
From Table 1, we also can find that the compound bearing pyrimidine core 13c showed the
strongest inhibitory effect on all three cell lines, with IC50 values of 4.85 1.02 (HepG2), 3.88 0.32
6

(BCG-823) and 3.25 0.48 (Huh-7) µg/mL, respectively. In addition, compounds 8c and 8e presented
obviously selective cytotoxic activities against the Huh-7 cell line except HepG2 and BCG-823 cell
lines, and the IC50 values were up to 7.74 2.18 and 4.46 1.05 µg/mL, respectively. These interesting
results might be used to develop novel lead scaffolds for potential anticancer agents.
Furthermore, the dose-response analysis of cell growth inhibitory activities for representation
compounds 4a, 8b, 13c and 5-FU have been displayed in Fig. 3, however, to some extent, these
compounds exhibited cytotoxic effects on HepG2, Huh-7, and BCG-823 cell lines in a
concentration-dependent manner. As we can see form Fig. 3, the inhibitory activities against Huh-7
cell lines for compounds 8b and 13c are not exactly in a concentration-dependent manner, which
may be due to the high cytotoxic activities for these two compounds against the Huh-7 cell lines, and
so the test conditions and concentration gradients are not well suitable.
Huh-7 Cell Line
HepG2 Cell Line
BCG-823 Cell Line
90
80
70
60
50
40
30
20
10
0
80
70
60
50
40
30
20
10
0
100
80
60
40
20
0
4a
4a
4a
8b
8b
8b
13c
5-FU
13c
5-FU
13c
5-FU
1.25
2.5
5
10
20
40
1.25
2.5
5
10
20
40
2.5
5
10
20
40
80
Concentration (µg/mL)
Concentration (µg/mL)
Concentration (µg/mL)
Fig. 3. Dose-response analysis of cell growth inhibitory activity for representative compounds 4a, 8b,
13c and 5-FU (positive control) against HepG2 (left), Huh-7 (middle) and BCG-823 (right) cell
lines.
Potential of the investigated compounds (8b and 13c) to induce apoptosis in HepG2 cells was
analyzed following 24 h of treatment with IC50 or 2×IC50 concentrations, by flow cytometry. The
apoptotic effect of compounds 8b and 13c was evaluated using Annexin V-FITC/PI dual staining
assay, which can examine the occurrence of phosphatidylserine (PS) externalization and also to
understand whether it is due to physiological apoptosis or nonspecific necrosis30. All data obtained
in this study are presented in Fig. 4, as a percentages of an early apoptotic cells, FITC(+)PI(-); late
apoptotic cells, FITC(+)/PI(+); and necrotic cells, FITC(-)/PI(+).
7

Fig. 4. Annexin V-FITC flow cytometry. Annexin V-FITC/PI staining was monitored in HepG2 cells
following 24 h treatment with compounds 8b or 13c at concentrations corresponding to their IC50s or
2×IC50s (B-E). Representative dot plots of three independent experiments are given, presenting intact
cells at lower-left quadrant, FITC(-)/PI(-); early apoptotic cells at lower-right quadrant,
FITC(+)/PI(-); late apoptotic or necrotic cells at upper-right quadrant, FITC(+)/PI(+); necrotic cells
at upper-left quadrant, FITC(-)/PI(+). (F) Apoptotic effect of compounds 8b and 13c was evaluated
after 24 h treatment; bar graphs represent mean SD in at least three independent experiments.
Results indicated that compounds 8b as well as 13c induced apoptotic changes following 24 h
treatment. As shown in Fig. 4F, both compounds initiated apoptosis, in terms of FITC(+)PI(-)
staining, to a larger extent than the control. Compounds 8b and 13c exhibited 9.2% and 11.7% of
apoptosis at IC50 and 16.7% and 18.8% of apoptosis at 2×IC50 concentrations respectively, whereas
1.4% of apoptosis was observed in control (0.1% DMSO). From this experiment it was observed that
these highly potential compounds induced cell death in HepG2 cells at least partly (initially), by
apoptosis. However, the precise mechanisms of cell death induction by tested compounds still
remain to be further explored.
To summary, a series of heterocycle-acylhydrazone hybrids have been synthesized and evaluated
8

as potential cytotoxic agents. Bio-evaluation indicated that some of the newly synthesized
compounds presented good cytotoxic activities. Especially, the most potent compounds 8b and 13c
exhibited higher cytotoxic activities against the tested cell lines as compared with 5-FU in vitro, and
these interesting results might be used to develop novel lead scaffold for potential anticancer agents.
The further annexin V-FITC assay indicated these highly potential compounds induced cell death in
HepG2 cells at least partly by apoptosis.
Acknowledgment
This work was financially supported by the Supportive Projects of Hubei Academy of Agricultural
Sciences (2014fcxjh17) and Key Laboratory of Integrated Pest Management in Crops in Central
China, Ministry of Agriculture and Key Laboratory for Crop Diseases, Insect Pests and Weeds
Control in Hubei Province (2015ZTSJJ9). The authors also gratefully acknowledge the partial
support from Hubei Agricultural Science Innovation Centre (2012-620-008-002, 2015-620-003-001).
References
1. Barman, S.; You, L.; Chen, R.; Codrea, V.; Kago, G.; Edupuganti, R.; Robertus, J.; Krug, R. M.;
Anslyn, E. V. Eur. J. Med. Chem. 2014, 71, 81-90.
2. Burgeson, J. R.; Gharaibeh, D. N.; Moore, A. L.; Larson, R. A.; Amberg, S. M.; Bolken, T. C.;
Hruby, D. E.; Dai, D. Bioorg. Med. Chem. Lett. 2013, 23, 5840-5843.
3. Chen, C.; Dolla, N. K.; Casadei, G.; Bremner, J. B.; Lewis, K.; Kelso, M. J. Bioorg. Med. Chem.
Lett. 2014, 24, 595-600.
4. Pieczonka, A. M.; Strzelczyk, A.; Sadowska, B.; Mlostoń, G.; Staczek, P. Eur. J. Med. Chem.
2013, 64, 389-395.
9

5. Misra, S.; Ghatak, S.; Patil, N.; Dandawate, P.; Ambike, V.; Adsule, S.; Unni, D.; Swamy, K. V.;
Padhye, S. Bioorg. Med. Chem. 2013, 21, 2551-2559.
6. Liu, T.; Sun, C.; Xing, X.; Jing, L.; Tan, R.; Luo, Y.; Huang, W.; Song, H.; Li, Z.; Zhao, Y.
Bioorg. Med. Chem. Lett. 2012, 22, 3122-3125.
7. Hadjipavlou-Litina, D.; Samadi, A.; Unzeta, M.; Marco-Contelles, J. Eur. J. Med. Chem. 2013,
63, 670-674.
8. Taha, M.; Baharudin, M. S.; Ismail, N. H.; Khan, K. M.; Jaafar, F. M.; Siddiqui, S.; Choudhary,
M. I. Bioorg. Med. Chem. Lett. 2013, 23, 3463-3466.
9. Wang, J.; Tan, H.; Li, Y.; Ma, Y.; Li, Z.; Guddat, L. W. J. Agric. Food Chem. 2011, 59,
9892-9900.
10. Li, W.; Zhang, Z.; Nan, X.; Liu, Y.; Hu, G.; Yu, H.; Zhao, X.; Wu, D.; Yan, L. Pest Manag. Sci.
2014, 70, 667-673.
11. Che, Z.; Zhang, S.; Shao, Y.; Fan, L.; Xu, H.; Yu, X.; Zhi, X.; Yao, X.; Zhang, R. J. Agric. Food
Chem. 2013, 61, 5696-5705.
12. Al-Ne’aimi, M. M.; Al-Khuder, M. M. Spectrochim. Acta A 2013, 105, 365-373.
13. Yan, Z.; Wang, S.; Wang, H.; Wu, S. Mater. Sci. Eng. C 2013, 33, 2562-2568.
14. Bao, X.; Zheng, P.; Liu, Y.; Tan, Z.; Zhou, Y.; Song, B. Supramolecular Chem. 2013, 25,
246-253.
15. Ye, X.; Zhu, T.; Wu, W.; Ma, T.; Xu, J.; Zhang, Z.; Wang, Y.; Jia, L. Inorg. Chem. Commun.
2014, 47, 60-62.
16. Lin, Q.; Zhu, X.; Fu, Y.; Zhang, Y.; Wei, T. Dyes Pigments 2015, 112, 280-282.
17. Gaba, M.; Singh, S.; Mohan, C. Eur. J. Med. Chem. 2014, 76, 494-505.
10

18. Facchinetti, V.; Reis, R. D. R.; Gomes, C. R. B.; Vasconcelos, T. R. A. Mini-Rev. Org. Chem.
2012, 9, 44-53.
19. Xiao, L.; Gao, H.; Kong, J.; Liu, G.; Peng, X.; Wang, S. Chin. J. Org. Chem. 2014, 34,
1048-1060.
20. Keri, R. S.; Patil, M. R.; Patil, S. A.; Budagupi, S. Eur. J. Med. Chem. 2015, 89, 207-251.
21. Sharma, P. C.; Sinhmar, A.; Sharma, A.; Rajak, H.; Pathak, D. P. J. Enzyme Inhibit. Med. Chem.
2013, 28, 240-266.
22. Hwang, J. Y.; Smithson, D. C.; Holbrook, G.; Zhu, F.; Connelly, M. C.; Kaiser, M.; Brun, R.;
Guy, R. K. Bioorg. Med. Chem. Lett. 2013, 23, 4127-4131.
23. Kamal, A.; Faazil, S.; Ramaiah, M. J.; Ashraf, M.; Balakrishna, M.; Pushpavalli, S. N. C. V. L.;
Patel, N.; Pal-Bhadra, M. Bioorg. Med. Chem. Lett. 2013, 23, 5733-5739.
24. Xie, X.; Yan, Y.; Zhu, N.; Liu, G. Eur. J. Med. Chem. 2014, 76, 67-78.
25. Ke, S.; Wei, Y.; Yang, Z.; Wang, K.; Liang, Y.; Shi, L. Bioorg. Med. Chem. Lett. 2013, 23,
5131-5134.
26. Sleebs, B. E.; Kersten, W. J. A.; Kulasegaram, S.; Nikolakopoulos, G.; Hatzis, E.; Moss, R. M.;
Parisot, J. P.; Yang, H.; Czabotar, P. E.; Fairlie, W. D.; Lee, E. F.; Adams, J. M.; Chen, L.; van
Delft, M. F.; Lowes, K. N.; Wei, A.; Huang, D. C. S.; Colman, P. M.; Street, I. P.; Baell, J. B.;
Watson, K.; Lessene, G. J. Med. Chem. 2013, 56, 5514-5540.
27. Ke, S.; Shi, L.; Cao, X.; Yang, Q.; Liang, Y.; Yang, Z. Eur. J. Med. Chem. 2012, 54, 248-254.
28. Shi, L.; Hu, R.; Wei, Y.; Liang, Y.; Yang, Z.; Ke S. Eur. J. Med. Chem. 2012, 54, 549-556.
29. Ke, S.; Wei, Y.; Shi, L.; Yang, Q.; Yang, Z. Anti-Cancer Agents Med. Chem. 2013, 13,
1291-1298.
11

30. Browne, L. J.; Gude, C.; Rodriguez, H.; Steele, R. E.; Bhatnager, A. J. Med. Chem. 1991, 34,
725-736.
12

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