Synthesis and Biochemical Evaluation of Novel Coumarin Derivatives as Anticancer and Anti-HIV Inhibitors Targeting CDK2

The present investigation dealt with finding new compounds that can act on both cancer and HIV-1 reverse transcriptase targeting CDK2. We constructed a library of new coumarin derivatives and developed a fingerprint pharmacophore model by using known CDK2 inhibitors (in clinical trial phase) and HIV-1 reverse transcriptase crystal structure (3DLG). The proposed library was mapped to the generated pharmacophore models and according to their fit-values; they were selected for docking into CDK2 enzyme. Compounds with high binding energy were selected for chemical preparation. Developing or adapting methods for the preparation of the selected compounds was applied and structures of the target compounds have been established by spectral analysis data. Five compounds 7b, 9d, 7c, 7d and 8c have shown very good anti-HIV activity in MT-4 cells. Similarly, five compounds 7b, 9d, 8b, 7c and 7d have exhibited very significant CDK2 inhibition activity. Compounds 7b and 7d were found to have dual anti-HIV and anticancer activities. Citation: Ibrahim DA, Al-Wahaibi LH, Abu-Melha HM (2017) Synthesis and Biochemical Evaluation of Novel Coumarin Derivatives as Anticancer and Anti-HIV Inhibitors Targeting CDK2. Chemotherapy 6: 244. doi:10.4172/2167-7700.1000244


Introduction
Cancer is a health threat, which show accidental cell growth, invasion, and sometimes spread in the body via lymph or blood (metastasis). The foremost reasons of cancer is the errors in the genetic material of the malformed cells that results from the effects of carcinogens, like tobacco smoke, radiation, chemicals, or viral infections. Other cancer-promoting genetic deviations could be resulted randomly through errors in the replication of DNA, or are inherited. The heritability of cancers could be affected by complex interactions between carcinogens and the host's genome. Tyrosine kinases, DNA methylation, and microRNAs are considered as massively important goals for cancer treatment.
Training set selection and conformational analysis: A set of 12 clinical CDK2 inhibitors were composed from different literature resources and were cautiously chosen to form a training set which was based on the principles of structural diversity and wide coverage of activity range. Structures of the working out set compounds are shown in (Figure 1). All compounds were constructed in 2D/3D Visualizer within CATALYST4.1 and minimized to the closest local minimum by means of the CHARMm-like force field incorporated in the CATALYST program. A series of energetically rational conformational models, which characterize the flexibility of each compound, were produced by CatConf program within CATALYST. Conformational analysis of all molecules was made by using the 'Best conformer generation with 20 kcal/mol as energy cutoff and 250 as maximum number conformers, while all other parameters were set to default. For HIV-1 reverse transcriptase pharmacophore, we generated it from its crystal structure (PDB: 3DLG) throughout the active site and the heavy atoms around the active site was counted as forbidden area.
Common features pharmacophores: Before carrying out pharmacophore modeling for the CDK2 kinase inhibitors, qualitative HIPHOP [17] models were first created based on the six most-active compounds in the training set, where the purpose was to recognize the common chemical features necessary for potent CDK2 kinase inhibitors, as well as to provide some information, which used to develope the quantitative pharmacophore model. In the HIPHOP run, Roscovotin was considered as reference molecule. Structural data from the training set recognized a set of features vital for activity and was considered to represent a pharmacophore hypothesis. Based on our previous experience [18], HypoGen module in Discovery Studios (DS) 2.5 [19] was used to generate our pharmacophore models wherein it evaluates a collection of conformational models for all compounds and maps them to the designated critical features. The top HIPHOP model created contains 4 kinds of chemical features for CDK2, namely, H-bond donors (HBD), H-bond acceptor (HBA) and hydrophobic features, while there are 6 kinds of chemical features for HIV-1 reverse transcriptase namely, one H-bond donor (HBD), one H-bond acceptor (HBA), three hydrophobics and one aromatic feature. The top classified pharmacophore model is expected to recognize the common binding features and the hypothetical orientation of the active compounds interacting with their target (Figure 2).

Mapping of the proposed compounds:
We constructed a proposed library of 7-hydroxy coumarin derivatives and all the proposed compounds (110 compounds) were mapped to the top ranked pharmacophores. The planned compounds with high fit-values were cautiously selected for the docking and binding energy calculations (Figures 3 and 4).
Docking and binding energy calculations: All the proposed coumarin derivatives that had the promising fit value were chosen for docking and binding energy calculations. The enzyme structures were obtained from protein data bank (PDB: 2A4L and 3DLG) and      corrected by DS 2.5. The hydrogen atoms and the absent residues were added and the structure was minimized to relax and correct the clashes. Finally, the binding site was defined and the anticipated compounds were docked using the default C-docker protocol. The planned compounds that exhibited a good-C-docker interaction energy values were subjected to binding energy calculations (Table 1). According to the binding energy values, the promising compounds were selected for synthesis ( Figures 5-7).
The reaction of compound 6 with different aromatic aldehydes was studied in refluxing piperidine and the styryl derivatives 7a-7d were isolated in good yield. Compound 7 was hydrolyzed in acid medium to give the amino derivatives 8a-8d as sole product in very good yield. The structural assignment of compound 7 is based on 1 H-NMR that showed a new singlet signal at δ 7.1 ppm corresponding to NH 2 and disappearance of singlet signal at δ 2 ppm due to the disappearance of CH 3 CO.    Finally, 2-(9-acetamido-2,8-dioxo-2H,8H-pyrano[2,3-f] chromen derivative 6 was reacted with triethyl orthoformate and different amines in refluxing DMF to afford amino derivatives 9a-9d, which also hydrolyzed by concentrated sulfuric acid to give compound 10 (Scheme 2). The structures of compounds 9 and 10 were estimated by different spectroscopic techniques. Biology CDK2 and HIV-1 reverse transcriptase assays: Out of the newly synthesized substituted coumarin derivatives, sixteen analogs (7)(8)(9)(10) have been evaluated as CDK2 inhibitors and as antitumor (MCF-7). The inhibitory concentration (IC 50 ) values were gained according to the described methods [21][22][23]. Olomoucine and Dox had been used as standards and the results are itemized in Table 2.
The results from Table 2 reveal that all compounds, which were active as CDK2 were found to be active as antitumor (MCF-7). Compounds 7b and 7d were found to be more active than the reference compound (DOX: IC 50 =1.17µM) with IC 50 values 1.14 µM and 1.16 µM respectively. Compounds 7b, 9d, 8b and 7c, 7d show very significant CDK2 inhibitory activity. Compound 5d was found to be as active as   olomoucine, while compounds 7b, 7d and 8b were more potent than the standard compound. Once again, the same structural features are present in active compounds as is evident in compounds active as anti-HIV-1 reverse transcriptase. Anti-HIV activity in MT-2 cells was compiled in Table 3. Table 3 showed that five compounds (7b, 9d, 7c and 7d) exhibited remarkable HIV-1 reverse transcriptase inhibition activity. Compounds 7b and 9d can be considered as a significant matrix for the design and synthesis of novel candidates with dual anti-HIV reverse transcriptase and anticancer activities. Further examination into the other aspects of structure activity relationship studies of this series of compounds is required to discover the scope and restriction of its biological activities.

Conclusion
A series of novel coumarin derivatives bearing styryl side chain were mapped to 3D pharmacophore of CDK2 and HIV-1 reverse transcriptase, docked into the active site of CDK2 and the binding energy was calculated. According to the fit-value the proposed compounds were selected for docking and binding energy calculations and again according to the binding energy the promising compounds were selected for synthesis and evaluation as inhibitors of CDK2 and HIV-1. The tested coumarin derivatives showed very good inhibition for both CDK2 and HIV-1 reverse transcriptase in µM range. Compounds 7b, 9d, 7c, 7d and 8c have shown very good anti-HIV activity in MT-2 cells. Similarly, five compounds 7b, 9d, 8b, 7c and 7d exhibited a remarkable CDK2 inhibition activity. Pharmacophore mapping and binding energy calculation were proved to be a very useful method for selecting the promising biological candidates. Compounds 7b and 9d can be considered as a significant matrix for the design and synthesis of novel candidates with dual anti-HIV-1 reverse transcriptase and anticancer activities. Further investigation into the other aspects of structure activity relationship studies of this series of compounds is required to explore the scope and limitation of its biological activities.

Chemistry
All melting points were measured on a Gallenkamp melting point apparatus and are uncorrected. The infrared spectra were recorded using potassium bromide disks on a Mattson FTIR infrared spectrophotometer (Mattson, New York, NY, USA). 1 H-NMR spectra were run at 500 MHz, on a Varian Mercury VX-500 NMR spectrometer (Bruker, Rheinstetten, Germany), using TMS as an internal standard in deuterated dimethyl sulphoxide. Chemical shifts δ are quoted in ppm and J in Hz. The mass spectra were recorded on a GCMS-QP-1000EX mass spectrometer (Shimadzu, Kyoto, Japan) at 70 e.V. All the spectral measurements were carried out at the Microanalytical Center of EL-Mansoura University, EL-Mansoura, Egypt and the Main Defense Chemical Laboratory, Cairo, Egypt. The elemental analyses were carried out at the Microanalytical center of Ain Shams University, Cairo, Egypt. All the chemical reactions were monitored by TLC.

Biological Evaluation
Anti-reverse transcriptase Activity: All the new synthesized compounds have been evaluated for their in vitro anti-HIV activity that was performed on T-4 lymphocytes infected and uninfected with HIV-1 using DMSO as solvent. The assay involves the killing of T-4 lymphocytes by HIV. Uninfected cells with the compound serve as a toxicity control, and infected and uninfected cells without the compound serve as basic controls. Cultures are incubated at 37°C in a 5% carbon dioxide atmosphere for 6 days. The tetrazolium salt, XTT, is added to all wells, and cultures are incubated to allow formazan color development by viable cells. Compounds that degenerate or are rapidly metabolized in the culture conditions may not show activity in this screen. Zidovudine (AZT) at 10 µM was used as a control. The viability of the cells was determined spectro-photometrically to quantitate formazan production and in addition is viewed microscopically for detection of viable cells and confirmation of protective activity. Drugtreated virus-infected cells are compared with drug treated noninfected cells and with other appropriate controls (untreated infected and untreated noninfected cells, drug containing wells without cells) on the same plate. CDK2 Inhibition Assay: CDK2-cyclin E kinase was expressed and assayed as previously described [21]. Kinase activity was expressed as a percentage of maximum activity. The concentration of the test compounds required to decrease the CDK activity by 50% was determined from dose-response curves and designated IC 50 .