Novel Triazole-Piperazine Hybrid Molecules Induce Apoptosis via

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Novel Triazole-Piperazine Hybrid Molecules Induce Apoptosis via Activation of the Mitochondrial Pathway and Exhibit Antitumor Efficacy in Osteosarcoma Xenograft Nude Mice Model Chandra Bhushan Mishra, Raj Kumar Mongre, Shikha Kumari, Dong Kee Jeong, and Manisha Tiwari ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.6b01007 • Publication Date (Web): 13 Jan 2017 Downloaded from http://pubs.acs.org on January 15, 2017

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ACS Chemical Biology

Novel Triazole-Piperazine Hybrid Molecules Induce Apoptosis via Activation of the Mitochondrial Pathway and Exhibit Antitumor Efficacy in Osteosarcoma Xenograft Nude Mice Model Chandra Bhushan Mishra‡1, Raj Kumar Mongre‡2, Shikha Kumari‡1, Dong Kee Jeong2*, Manisha Tiwari2* 1

Bio-organic Chemistry Laboratory, Dr. B.R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi

110007, India 2

Laboratory of Animal Genetic Engineering and Stem Cell Biology, Department of Animal Biotechnology and

Advance Next Generation Convergence, Faculty of Biotechnology, Jeju National University, Jeju-Do, Republic of Korea

*Correspondence Dr. Manisha Tiwari Dr. B. R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi 110007, India E-mail: [email protected]

Professor Dong Kee Jeong Department of Animal Biotechnology and Advance Next Generation Convergence, Faculty of Biotechnology, Jeju National University, Ara-1 Dong, Jeju-city, Jeju-Do 690-756, Republic of Korea E-mail: [email protected]

‡

These authors have equal contribution.

1

ACS Paragon Plus Environment


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Abstract Mitochondria imparts crucial role in the regulation of programmed cell death, reactive oxygen species (ROS) generation besides serving as a primary energy source. Mitochondria appeared as an important target for therapy of cancer due to its significant contribution in cell survival and death. Here, we report the design and synthesis of a novel series of triazole-piperazine hybrids as potent anticancer agents. MCS-5 emerged as an excellent anticancer agent which showed better anticancer activity than standard drug Doxorubicin in-vitro and in-vivo studies. MCS5 displayed an IC50 value of 1.92 µM and induced apoptosis in Cal72 (human osteosarcoma cell line) cells by targeting mitochondrial pathway. This compound arrested G2/M phase of cell cycle, induced ROS production and mitochondrial potential collapse in Cal72 cells. MCS-5 displayed excellent anticancer activity in Cal72 Xenograft nude mice model where, it significantly reduced tumor progression leading to enhanced life span in treated animals compared to control and Doxorubicin treated animals without exerting noticeable toxicity. In addition, 2DG optical probe guided study clearly evokes that MCS-5 remarkably reduced tumor metastasis in Cal72 Xenograft nude mice model. These results indicate that MCS-5 appeared as a novel chemical entity which is endowed with excellent in-vitro as well as in-vivo anticancer activity and may contribute significantly for the management of cancer in the future. Introduction Cancer is a foremost reason of death worldwide and exerts a major health problem globally. 8.2 million deaths due to cancer in 2012 have been reported, which represents 13% of all deaths and 12.7 million new cases per year are being stated worldwide.1 Cancer is a result of uncontrolled growth of abnormal cells and characterized by multiple structural, molecular and behavioral features. Out of all cancers types, liver, lung, bone, stomach, colon and breast cancer are 2


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predominantly responsible for cancer mortality.2 Treatment of cancer relies heavily on conventional therapies such as chemotherapy, radiotherapy, surgical removal as well as immunotherapy.3-4Since long time, widespread efforts have been done to counteract malignancies and in result various anticancer agents have been discovered.5-8 However, clinically running anticancer drugs exert dreadful side effects, including cytotoxicity against normal cells.910

Osteosarcomas, the utmost communal malignancy of bone which affects 900 people per year in the United States.11 It represents 56% of malignant bone tumors in children and nearby 3.4% of all childhood malignancies.12 Currently, most effective chemotherapeutic agents for the treatment of osteosarcoma are Cisplatin, Doxorubicin and Methotrexate.13 In spite of this, numerous new biological approaches to overcome osteosarcoma are being investigated which appear as fruitful strategies to prevent osteosarcoma and its metastasis.14-16 Although several improvements in the management of osteosarcoma to date, quiet seems as a challenge because the overall survival rate of osteosarcoma patients has remained constant for over two decades therefore, new treatment approaches are necessarily required. The mitochondria is the most important organelle concerned with the cellular bioenergetic and biosynthetic changes associated with cancer.17-18 Mitochondrial alterations contribute to the invasive and metastatic properties in most of typical tumors. Therefore, the mitochondria is considered as a valuable target to induce apoptosis in cancer cells and numerous anticancer drugs follows the mitochondrial pathway to kill cancer cells.19-20 The molecular hybridization of biologically active molecules is a powerful tool for drug discovery and appeared as a novel strategy to identify potential drug molecules against various diseases, including cancer.21 Hybrid drug molecules may serve as a combination therapy in single multifunctional agent, which would 3



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be more potent than conventional treatment approach.22 Therefore, by adopting a molecule hybridization drug discovery approach, we aimed to design triazole-piperazine hybrid molecules as potent anticancer agents. 1,2,4-triazole is an important class of heterocycle which has been widely used to develop a large variety of bioactive molecules such as anti-bacterial, anti-fungal, anti-depressant, anti-oxidant, anti-inflammatory, anti-HIV and anti-tubercular agents.23-26 1,2,4-triazoles have also a successful history for the development of potent anticancer agent.27-29 Various derivatives of 1,2,4-triazole derivatives were synthesized and tested for their anticancer potential in various in-vitro as well as in-vivo models (Fig. 1). For example, 4-amino-5-(5-phenylthiene-2-yl)-2,4-dihydro-3H-1,2,4triazole-3-thione 30 (1) displayed excellent cytotoxicity against thymocytes with an IC50 value of 0.012 µM and 4-amino-5-mercapto-3-(2-chlorophenyl)-1,2,4-triazole

31

(2) displayed admirable

antiproliferative activity in Ehrlich Ascites Carcinoma mice. Romagnoli et al. also synthesized a series of 1,5-disubstituted 1,2,4-triazole as potent anticancer agents.32 Compound 3 showed excellent cytotoxicity against HeLa as well as Jurkat cells by inducing apoptosis. On the other hand the piperazine moiety has also been used constantly to develop various potent anticancer agents and several piperazine containing molecules have shown admirable anticancer activities in both pre-clinical and clinical trials (Fig. 1).33-34 The successful anticancer drug Imatinib 35 (4) also contains piperazine moiety and this drug is widely used to treat various types of cancer including chronic myelogenous leukemia. Additionally, a novel piperazine derivative 5 also (LYG-202) displayed potent anti-cancer activity in vitro and in vivo by inducing apoptosis.36 In continuation of our anticancer drug development agenda37-39 and enthused by diverse anticancer properties of the triazole and piperazine moieties, we have designed a novel series of 4


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triazole-piperazine hybrid molecules to appraise their in-vitro and in-vivo anticancer activity. This molecular hybridization might generate potential anticancer agent because both moieties (triazole and piperazine) are well studied to produce anticancer activity. Synthesized derivatives have been evaluated for their anti-proliferative activity against six types of cancer cell lines and their structure activity relationship has been also studied. The most active novel derivative MCS5 showed remarkable cytotoxicity against Cal72 (human osteosarcoma cell line) without displaying cytotoxicity in normal cells. Therefore, MCS-5 was selected for further studies concerning its mechanism of action which governs apoptosis in Cal72 cells. Our studies have shown that MCS-5 activate the mitochondrial pathway to induce apoptosis process. MCS-5 has also exhibited noteworthy in-vivo antitumor activity in a xenograft mice model of human osteosarcoma. Additionally, this novel compound successfully reduced tumor metastasis in 2DG optical probe guided osteosarcoma-xenograft nude mice model. Our efforts have provided a novel chemical entity (MCS-5) with commendable anticancer activity which may help in the management of osteosarcoma in the future. RESULTS AND DISCUSSION Synthesis of triazole-piperazine hybrids. Designed novel hybrid molecules (5-16) have been synthesized in four steps which are depicted in scheme 1 (Fig. 2). In short, commercially available benzhydrazide 1 was reacted with carbon disulfide (CS2) under reflux condition in dimethylformamide (DMF) to afford oxadiazole intermediate 2. After that, compound 2 was reflux with hydrazine hydrate in 1,4-dioxane to yield aminotriazole intermediate 3. Next, the amino group of aminotriazole (3) was reacted with furan-2- carboxyaldehyde in glacial acetic acid under reflux conditions, which led to the formation of furoyl-triazole derivative 4 in high yield. Finally, substituted piperazines were attached to triazole ring with a methylene linker by 5



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performing the Mannich reaction providing the Mannich bases 5-16. Synthesized compounds were fully characterized by 1HNMR, 13C NMR, Mass spectroscopy and elemental analysis.

Compound

R-Group

Compound

Number

Number

5

11

6

12

7

13

8

14

9

NO2

R-Group

15

6


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10

16

Scheme 1: Reagents and conditions: A. CS2, DMF, reflux, 3-4 h; B. NH2NH2, 1,4 dioxane, reflux, 12-14 h; C. Furan-2- carboxyaldehyde, AcOH, reflux, 12-14 h; D. Substituted piperazine, formaldehyde, MeOH (reflux, 12-14 h)/ DMF(RT, 12-15 h).

In-vitro cytotoxicity studies and structure activity relationship (SAR): In-vitro cytotoxic potential of synthesized hybrids 5-16 were assessed against six types of human cancer cell lines; Cal72, HepG-2, SaOS-2, A549, MCF-7 and Hela by using MTT assay. The results are presented in terms of IC50 values as given in table 1. The structure activity relationship (SAR) has been explicated on the basis of observed results in MTT assay. It was observed that phenyl substitution (compound 5) of this core was completely ineffective against all six cell lines. In electronegative element substituted phenyl derivatives, chloro substituted phenyl derivative (compound 7) appeared more effective as compared to fluoro substituted derivative (compound 6). The chlorophenyl substituted derivative (compound 7) was effective in producing cytotoxicity against Cal72 and MCF cell line even as fluorophenyl substituted derivative only showed satisfactory cytotoxicity towards the Hela cell line as compared to Doxorubicin. Ethanone group substitution on the phenyl ring (compound 8) also did not exert satisfactory cytotoxicity against tested cell lines. However, this compound showed acceptable cytotoxicity against SaOS-2 cell line with an IC50 value of 15.76 µM which was almost similar to Doxorubicin. The introduction of NO2 (strong electron withdrawing group) on the phenyl ring (compound 9) displayed venerable cytotoxicity against the Cal72 cell line, while it did not show promising cytotoxicity towards other cell lines as compared to Doxorubicin. Benzoyl group 7



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substitution (compound 10, MCS-5) on this core made it a successful derivative and this compound showed excellent cytotoxicity against all tested six cancer cell lines. This compound displayed prominent cytotoxicity against Cal72, HepG-2, SaoS-2, MCF-7 as well as Hela cell line, where this compound showed much better IC50 value as compared to standard drug Doxorubicin. Electron donor methyl substitution on the phenyl ring (compound 11) was unable to produce significant cytotoxicity against all tested six cancer cell lines. However, methoxy substituted phenyl derivative (compound 12) exhibited promising cytotoxicity towards MCF-7 and Hela cell line as compared to Doxorubicin. Pyrimidine containing derivative (compound 13) also did not produce a satisfactory cytotoxic effect against tested cancer cell lines. However, flexible benzyl moiety bearing derivative 14 showed promising cytotoxic effect against Cal72, HepG-2, SaOS-2 and Hela cell line as compared to Doxorubicin. The insertion of one more phenyl ring to the benzyl derivative led to diphenyl derivative 15 which showed significant cytotoxicity towards Cal72, SaoS-2, MCF-7 and Hela cell lines, where its efficacy was almost similar to Doxorubicin. Fluoro substitution at p-position of the diphenyl ring (compound 16) also produced a remarkable cytotoxic effect against Cal72, HepG-2, MCF-7 and Hela cell line, where it appeared more potent than Doxorubicin. In-vitro cytotoxicity of synthesized derivatives revealed that most of the compounds displayed a notable anticancer effect against tested cancer cell lines. Phase contrast microscopy images have been taken to elucidate morphological changes associated with programmed cell death (Fig. 2a). However, MCS-5 emerged as a most effective derivative in whole series and it showed admirable cytotoxicity against Cal72 with an IC50 value 1.97 µM in comparison with standard drug Doxorubicin which possesses an IC50 value 16.43 µM (Fig. 2b). Moreover, MCS-5 did not show significant cytotoxicity against normal human bone marrow (hBM) cell line and it also 8


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demonstrated comparatively higher cell viability than Doxorubicin after 24 hours incubation (Fig. 2c). Additionally, time course studies of MCS-5 have been also performed against Cal72 cell line where, it gradually reduced cell viability up to 48 hours (Fig. 2d). Inspired with these results, we have chosen MCS-5 to proceed further for advance anticancer studies to confirm its potency as effective anticancer agent. Table 1: In-vitro cytotoxic effect of novel synthesized compounds (5-16) a IC50 (µM)b Test

Cal72

HepG-2

SaOS-2

A549

MCF-7

Hela

27.623±

23.20±

41.07±

compound 5

6

7

8

44.73

± 38.64

1.902

0.816

5.908

2.109

1.225

3.645

17.20±

19.95±

39.32±

22.36±

42.13±

13.25±

0.130

0.022

0.725

1.556

1.902

0.380

16.04±

56.23±

51.87±

32.15±

19.25±

32.78±

0.227

0.492

0.537

0.529

0.596

0.759

18.75±

52.25±

15.76±

25.468±

34.28±

38.25±

0.435

0.115

0.602

0.378

0.642

3.93±

19.95±

16.65±

30.29±

35.39±

22.64±

0.169

0.663

0.733

0.943

0.482

0.466

0.183 9

± 32.31±

9



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

10 (MCS-5)

7.64±

9.99±

13.58±

6.34±

10.24±

0.509

0.681

0.779

0.465

0.442

23.56±

19.02±

16.52±

26.81±

27.51±

0.345

0.350

0.328

0.623

0.104

81.86±

19.91±

13.42±

10.21±

11.26±

36.234

0.405

0.170

0.520

1.234

75.80±

39.94±

40.25±

52.36±

29.37±

0.600

0.723

1.418

0.378

0.352

9.784±

8.678±

12.54±

16.25±

12.52±

0.285

0.372

0.755

0.111

0.200

19.65±

15.07±

21.45±

14.27±

15.67±

0.226

0.755

1.211

1.139

0.236

10.75±

24.78±

18.39±

12.73±

9.43±

0.837

0.959

0.459

1.100

0.429

16.43±

17.31±

15.23±

8.27±

21.90±

14.52±

0.352

0.136

0.220

0.164

0.432

0.654

1.97 ± 0.090 27.31±

11

0.160 18.14±

12

0.252 22.52±

13

0.240 16.18±

14

0.334 16.39±

15

0.352 4.702±

16

0.432 Doxorubicinc

a

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The data denoted the mean of three experiments in triplicate and were value represented as mean. b

The IC50 denotes the concentration at which 50% of cells survived. 10


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C

Used as positive control

MCS-5 induces cell apoptosis. The generation of apoptosis has been regarded as a praiseworthy strategy for therapy of cancer and nearly all anticancer drugs kill tumor cells by inducing apoptotic processes.40-41 Therefore, apoptotic effect of MCS-5 was assessed by Hoechst 33342 staining and propidium iodide (PI)/Annexin-V FITC double staining method in Cal72 cells. Phase contrast microscopy images have been taken to elucidate morphological alterations in the apoptotic cells by both staining methods as shown in Fig 3a. In Hoechst 33342 staining, MCS-5 treated cells showed distinctive apoptotic characteristics, including chromatin condensation, apoptotic body and nuclear fragmentation as compared to control and Doxorubicin (10 µM). Whereas, untreated cells displayed uniformly dispersed chromatin and stained meager homogeneous blue. Further, apoptosis induced by MCS-5 was also examined by microscopic study and flow cytometer using PI/Annexin-V FITC double staining method. Phase contrast microscopy of PI/Annexin-V FITC double staining MCS-5 treated cells displayed significant early and late apoptosis as compared to untreated cells (Fig. 3b). Flow cytometry studies markedly indicate that MCS-5 showed superior apoptosis against Cal72 cells as compared to Doxorubicin (Fig. 3c). The result of this study indicates that MCS-5 showed significant (P

Novel Triazole-Piperazine Hybrid Molecules Induce Apoptosis via

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