Synthesis And Anticancer Screening Of Novel Spiro[Chroman-2,4′- Piperidin]-4-One Derivatives With Apoptosis-Inducing Activity

© 2018 Shaimaa A. Abdelatef et al. This is an open access article distributed under the terms of the Creative Commons Attribution License -NonCommercialShareAlikeUnported License (http://creativecommons.org/licenses/by-nc-sa/3.0/). *Corresponding Author Khaled R. A. Abdellatif, Ph.D. Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Beni-Suef University, Beni-Suef 62514, Egypt. E-mail: khaled.ahmed @ pharm.bsu.edu.eg Synthesis And Anticancer Screening Of Novel Spiro[Chroman-2,4′Piperidin]-4-One Derivatives With Apoptosis-Inducing Activity


INTRODUCTION
Cancer is a life-threating disease that is manifested by out-of-control abnormal cell growth with the possibility of invading other tissues. Currently, there are several approaches for cancer treatment including chemotherapy, hormonal therapy, immunotherapy, surgery and radiation. However, chemotherapy remains one of the most prevailing options used for treating cancer (Gerber, 2008). Despite all the treatment approaches mentioned above and the remarkable breakthrough in cancer chemotherapy, the existence of a perfect and outright cure is still a challenging goal (Raguz and Yague, 2008). Accordingly, there is an urgent need to develop more potential anticancer agents. The discovery of apoptosis-inducing agents has become one of the most promising strategies of cancer treatment (Sun et al., 2004). Apoptosis is considered a highly regulated and critically biological programmed cell death mechanism used to get rid of the defective and imperfect cells (McDonald and El-Deiry, 2005). It has an essential role in maintenance of tissue growth and homeostasis of multicellular organisms (Fulda and Debatin, 2006). Consequently, it was believed that the deregulation or impairment of this pivotal process is one of the main causes of incidence of cancer, autoimmune and neurodegeneration diseases (Lowe and Lin, 2000;Ponder, 2001). On the contrary, the induction of apoptosis machinery is feasibly one of the most potent defense strategies against cancer (Zhang et al., 2007). Therefore, the discovery and design of novel apoptotic-inducing agents have received widespread attention as a potential, less toxic and promising approach in cancer chemotherapy (Sharma et al., 2016;Jung et al., 2017). On the other hand, spiro heterocycles have currently attracted a great attention due to their existence as a main skeleton in a wide variety of biologically active natural as well as synthetic compounds in addition to their interesting conformational features. Indeed, these molecules have become an elegant target for medicinal chemists to develop a large number of derivatives and evaluate their pharmacological potentials with emphasis on their medicinal applications. Among this class of compounds, spirochromanone is a well-recognized privileged structure that has been commonly found in a plenty of chemical compounds with diverse biological activities. These compounds exhibit significant biological activities such as anticancer (Atta et al., 2010;El-Desoky et al., 2013), antitubercular (Mujahid et al., 2013;Mujahid et al., 2015), antimicrobials (Feng et al., 2014), histamine-3 antagonists (Becknell et al., 2012), antiarrhythmic (Elliott et al., 1992), acetyl-CoA carboxylase (ACC) inhibitors (Shinde et al., 2009;Huang et al., 2015), stearoyl-CoA desaturase-1 (SCD)-1 inhibitors (Uto et al., 2010), histone deacetylase (HDAC) inhibitors (Varasi et al., 2011;Thaler et al., 2016), antimalarial (Roberts et al., 2016), growth hormone secretagogues (Yang et al., 1998) and δ opioid receptor agonists (Le Bourdonnec et al., 2008). Thaler et al. have reported a series of spirochromanes and evaluated their activity against HDAC as a well-established anticancer target (Thaler et al., 2016). These studies demonstrated that the prepared spirochromane derivatives possessed potent HDAC inhibitory activity that could be successfully developed into potential anticancer agents. Compounds 1 and 2 were the most potent HDAC inhibitors with good oral bioavailability and tumor growth inhibition. Moreover, a series of spiro furochromones was synthesized and screened for their antitumor activity where the selenium-containing compound 3 recorded the most promising activity against breast carcinoma (MCF-7) (Atta et al., 2010), Figure 1. Another set of benzopyranones was reported and tested in vitro and in vivo for their anticancer potential. Compound 4 was highly potent against a panel of cancerous cell lines with IC 50 values between 0.09 and 0.21 μM compared to 5-fluorouracil as a reference drug (El-Desoky et al., 2013).
Furthermore, trimethoxyphenyl and adamantyl moieties are frequently used in design of several anticancer agents. Trimethoxyphenyl has an important role in mitosis, microtubules which represent a crucial target in development of new anticancer agents (Jordan and Wilson, 2004). In this regard, colchicine 5 (Lin et al., 2016), the diarylthiazole derivative 6  and combretastatin A4 7 (Pettit et al., 1989), are examples of this class where their anticancer activity is mediated by binding to tubulin. Meanwhile, adamantyl moiety was found to enhance the various activities of several compounds positively such as anticancer agents. One of such compounds, the diaminophenyladamantane derivative 8 exhibited strong anticancer activity in vitro and in vitro with G 0 /G 1 cell cycle arrest mechanism (Wang et al., 2004). Moreover, the adamantyl-1,2,4-triazole-3(4H)-thione derivative 9 was found to exert moderate in vitro anticancer activity against breast cancer with IC 50 value of 8.15 μM with a suggested inhibition to BRCA2 protein (Genc et al., 2015). The anti-prostate cancer activity was reported for compound 10 with potent inhibitory activity against 17 α -hydroxylase and C 17,20 -lyase activities of human testicular cytochrome P450 17α (Chan et al., 1996). The adamantine-gold complex 11 displayed potent cytotoxic activity in comparison to cisplatin and auranofin and could inhibit the thioredoxin reductase enzyme (Garcia et al., 2016), Figure 2.
As part of our ongoing research work to develop novel anticancer agents, we describe herein the design and synthesis of some new chemical entities based on a spirochromanone framework tethered to known small moieties such as trimethoxyphenyl and adamantyl via various linkers using fragment-based drug design approach. The evaluation of their anticancer activity against a panel of cancer cell lines in addition to their ability to induce apoptosis and cell cycle analysis were also performed. It was conceptualized that these novel spiro hybrids could serve as promising leads for developing potent and safer anticancer agents. Figure 3 shows the suggested pharmacophore of the newly synthesized compounds.

Chemistry
Chemical reagents and solvents were obtained from commercial sources, Solvents were dried by standard methods when necessary. Elemental analyses were carried out at the microanalytical center in the Faculty of Science, Cairo University. 1 H NMR spectra were recorded with Bruker APX400 spectrometer at 400 MHz in DMSO-d 6 . Chemical shifts were reported on the δ scale. The high-resolution mass spectra (HRMS) were recorded on Agilent 6230 Series Accurate-Mass Time-Of-Flight (TOF) LC/ MS. Thin layer chromatography (TLC) was done by silica gel plates 60 GF254, cellulose plates (20 × 20 cm) from Sigma-Aldrich company for chemicals and ethyl acetate/hexane were used as the eluting system.

Cytotoxicity assay
The cytotoxicity of the five compounds were evaluated by MTT assay, as previously described . The three cell lines were separately cultured in 96-well (3 ×10 3 /well), and incubated at 37°C overnight. Final compound concentrations: 0, 0.05, 0.5, 5, 25, 50 μM (DMSO 0.1%; n = 3). Plates were incubated for 72 h, then MTT was added to each well. Plates were incubated for 3 h, supernatant was aspirated, and DMSO was added to each well. Absorbance was read on multi-plate reader. Optical density of the purple formazan A 550 is proportional to the number of viable cells. Compound concentration causing 50% inhibition (IC 50 ) compared to control cell growth (100%) was determined. GraphPad Prism version 5.00 for Windows, GraphPad Software, San Diego California USA was used for analysis.

Annexin V FITC/PI apoptosis assay
Annexin V FITC/PI assay was used to evaluate possible ability to induce apoptosis. MCF-7 cells were cultured in 6 well plates (1 × 10 5 cells/well) overnight at 37°C. Compound 16 was used to treat cells (0, 5, 10 and 20 µM). After 24 h, supernatant of treated cells was collected in tubes and kept on ice, then cells were trypsinized and incubated at 37°C before being added to the tubes. MCF-7 cells were centrifuged (2000 rpm), washed with PBS (x1), centrifuged again, pellets re-suspended in binding buffer (100 μL, x1) and annexin V FITC (10 μL). Tubes were incubated at room temperature in dark for 20 minutes before adding the binding buffer (400 μL, x1) and 10 μL propidium iodide (PI). Analysis was performed by flow cytometry (NovoCyte Flow Cytometer, Acea Biosciences Inc., California, USA). Different cell populations (early apoptotic, late apoptotic, and necrotic cells) were identified by annexin V and PI staining (Vermes et al., 1995).

Cell cycle analysis
Perturbation of cell cycle was done for MCF-7 cells cultured in 6 well plates (1 × 10 5 cells) overnight at 37°C. Treated cells with compound 3Z (0, 5, 10 and 20 μM) were washed with cold PBS x1 and trypsinized. Collected cells were spinned at 2000 rpm. The resulting pellets were washed in cold PBS x1 again, spinned and fixed overnight in 70% ice cold ethanol. Cells were re-suspended in cold PBS x1 with addition of ribonuclease A (15 min), followed by PI (2 µL/mL). Samples were held on ice and analysed by flow cytometry. Data analysis of DNA contents (PI bound to DNA) of 20000 events was carried out (Bkhaitan et al., 2017).

Chemistry
The general synthetic pathway used for the preparation of the novel targeted spiro derivatives is shown in Scheme 1. The starting spiro material, tert-butyl 4-oxospiro[chroman-2,4'-piperidine]-1'-carboxylate 12, was prepared using the reported multi-component reaction developed by Kabbe where a thermal condensation reaction of 2-hydroxyacetophenone and N-Boc piperidone in methanol was carried out in presence of pyrrolidine (Kabbe, 1978). The furnished spiro protected compound 12 was subjected to a deprotecting process using TFA in DCM to give the free-boc intermediate 13 in a quantitative yield which in turn was coupled with the appropriate acid chloride or sulfonyl chloride reagents in presence of TEA affording the final targeted compounds (14-18) in good yields. All the final compounds were fully characterized using NMR, mass, CHN spectral analysis.

Cytotoxic activity
The cytotoxic activity of the newly synthesized compounds was assessed in vitro against three cancer cell lines; MCF-7 (human breast carcinoma), A2780 (human ovarian cancer) and HT-29 (human colorectal adenocarcinoma) using MTT assay . The results were summarized in Table 1 and expressed in terms of IC 50 values where doxorubicin was used as a positive control. Generally, it was noticed that the compounds containing a sulfonyl moiety (16-18) exhibited higher activity than compounds (14, 15) containing a carbonyl spacer. Among the tested derivatives, compound 16 was the most potent anticancer agent against the three cancer cell lines with IC 50 values between 0.31 and 5.62 μM. Nevertheless, the trimethoxyphenyl derivative 15 showed the least potency with IC 50 values between 18.77 and 47.05 μM. It was found that the replacement of the phenyl group in compound 16 with a p-tolyl group in compound 17 resulted in a sharp decrease in the potency, especially against HT-29 cell line suggesting a possible significant effect of electron-donating substituents on the activity. However, the replacement with a naphthyl moiety as in compound 18 slightly affected the activity against MCF-7 and HT-29 cell lines compared with compound 16. Of the other substituents, the car-bonyl adamantyl bearing analog 14 displayed moderate activity against HT-29 with IC 50 value of 8.46 μM but with a weak activity against the other used cell lines. These results indicate that the influence of the substitutions on the activity was mainly electronically more than sterically.

Annexin V FITC/PI apoptosis assay
Compound 16, with the highest activity against the three cell lines, was selected for further investigations to explain its mechanism of action. Annexin V FITC/PI assay was used to evaluate whether it can induce apoptosis or not in MCF-7 cells following 24 h treatment. Doses of compound 16 were selected based on its IC 50 (x1, x2 and x4, respectively). Compound 16 increased the early apoptotic MCF-7 cell populations in a dose dependent manner, more than three folds compared to control; combined by increase in late apoptosis at 5 and 10 μM; all at expense of live cells which decreased consequently, Figure 4. Three human cancer cell lines were used; MCF-7 (human breast carcinoma), A2780 (human ovarian cancer) and HT-29 (human colorectal adenocarcinoma). Cells were treated with the test compounds or vehicle for 72 h and cell viability was assessed by MTT assay. Data were reported as mean ± S.D. (n = 6). Doxorubicin was used as a positive control.

Cell cycle analysis
The cell cycle distribution assay of MCF-7 cells treated with compound 16 (24 h), revealed an increase in the sub-G1 pro-apoptotic cells (from 0.30% at 0 μM, to 1.02% and 1.54% at 5 μM and 10 μM, respectively). Compound 16 blocked MCF-7 cells at the G2-M phase in a dose dependent manner ( Figure  5 and Table 2) and that was supported by the annexin V assay results.

CONCLUSION
A new series of spiro[chroman-2,4'-piperidin]-4-one derivatives was synthesized and evaluated as anticancer agents against a panel of human cancer cell lines using MTT assay. Compound 16 with a sulfonyl spacer exhibited the most potent activity with IC 50 values between 0.31 and 5.62 μM. However, the trimethoxyphenyl derivative 15 was the least potent with IC 50 values between 18.77 and 47.05 μM. The most active compound 16 could induce more than three folds early apoptosis in MCF-7 cells treated for 24 h. Moreover, it increased MCF-7 cells in the sub-G1 and G2-M cell cycle phases, following the same treatment duration. These compounds could be considered as good leads that need further structural optimization to be developed into potential anticancer agents.