Synthesis of Some Novel Fused Pyrimido[4″,5″:5′,6′]-[1,2,4]triazino[3′,4′:3,4] [1,2,4]triazino[5,6-b]indoles with Expected Anticancer Activity

Our current goal is the synthesis of polyheterocyclic compounds starting from 3-amino-[1,2,4]triazino[5,6-b]indole 1 and studying their anticancer activity to determine whether increasing of the size of the molecules increases the anticancer activity or not. 1-Amino[1,2,4]triazino[3′,4′:3,4]-[1,2,4]triazino[5,6-b]indole-2-carbonitrile (4) was prepared by the diazotization of 3-amino[1,2,4]-triazino[5,6-b]indole 1 followed by coupling with malononitrile in basic medium then cyclization under reflux to get 4. Also, new fused pyrimido[4″,5″:5′,6′][1,2,4]triazino-[3′,4′:3,4][1,2,4]triazino[5,6-b]indole derivative 6 was prepared and used to obtain polycyclic heterocyclic systems. Confirmation of the synthesized compounds’ structures was carried out using elemental analyses and spectral data (IR, 1H-NMR and 13C-NMR and mass spectra). The anticancer activity of some of the synthesized compounds was tested against HepG2, HCT-116 and MCF-7 cell lines. The anticancer screening results showed that some derivatives display good activity which was more potent than that of the reference drug used. Molecular docking was used to predict the binding between some of the synthesized compounds and the prostate cancer 2q7k hormone and breast ‎cancer 3hb5 receptors.


Introduction
Interestingly, we are now seeing the emergence of a new generation of drugs with hybrid molecular architectures combining the biological features of two or more small molecules. The expectation is that, in the long term, such molecular conjugates could become a dominant form of targeted infectious diseases. The idea of combining two or more potentially bioactive substructures to make an integrated new molecular framework with a higher anticipated therapeutic power is conceptually simple yet highly successful in the context of infectious diseases that require effective treatment. In previous works, we succeeded in constructing many small 1,2,4-triazine molecules that had a remarkable biological activity on cancer cells [1,2]. In addition, we were able to construct many fused 1,2,4-triazine systems which showed marvelous antimicrobial activities [3][4][5][6][7][8][9][10]. Herein, we aspire to constructed huge fused triazine molecules of increased efficacy and decreased systemic toxicity which are a great challenge for medical science. One of the present goals in heterocyclic compound synthesis is the construction of macromolecules, in particular, by using of sequential reactions [11]. Such molecular explorations, yielding novel architectures, are of particular interest for the investigation of new bioactive agents with possible new modes of action, which could subsequently be elaborated in medicinal chemistry programs [12]. Approaches to the realization of these synthetic goals have often been explored in the context of complex multistep syntheses Molecules 2018, 23, 693; doi:10.3390/molecules23030693 www.mdpi.com/journal/molecules of natural product targets [13]. Among the various clinical applications, heterocyclic compounds have a considerable active role as anti-viral [14], anti-bacterial [15,16], anti-inflammatory [17], anti-fungal [18], and anti-tumor drugs [19][20][21]. Numerous of biologically active macromolecules naturally occurring (e.g., with antimicrobial activity or anticancer) include homofascaplysin [22], fascaplysin (antimicrobial pigment) [23][24][25][26], staurosporine [27], iheyamines [28], and rebeccamycin [29] are shown in Figure 1.
The amazing biological activity of the polycyclic heterocyclic compounds encouraged us to continue our previous work on the synthesis of fused triazine [7,9,10] and their applications, by designing a polycyclic heterocyclic compounds containing five and/or six rings fused with each other in the hope to get a superior biological activity.
The amazing biological activity of the polycyclic heterocyclic compounds encouraged us to continue our previous work on the synthesis of fused triazine [7,9,10] and their applications, by designing a polycyclic heterocyclic compounds containing five and/or six rings fused with each other in the hope to get a superior biological activity.

Results and Discussion
The previously prepared 3-amino-4H- [1,2,4]triazino [5,6-b]indole 1 [30] was used in this work as a starting unit on which the polycyclic compound will built. Compound 1 was subjected to diazotization at 4 °C in conc. HCl with aqueous solution of NaNO2 (Scheme 1). To the stirred solution an aqueous mixture of malononitrile and sodium acetate was added to obtain [4H- [1,2,4]triazino [5,6b]indol-3-yldiazenyl]malononitrile (3). The structure of 3 was elucidated from its IR spectrum, which showed two cyano groups signals at 2210, 2217 cm −1 with disappearance of the band corresponding to the NH2 group of compound 1. Cyclization of compound 3 to 1-amino [1,2,4]triazino[3′,4′:3,4]- [1,2,4]triazino [5,6-b]indole-2-carbonitrile (4) was achieved by refluxing compound 3 in ethanol. The structure elucidation of compound 4 was supported from the disappearance of one cyano group peak in the IR, as only one CN group was detected at 2209 cm −1 , in addition to the disappearance of the triazine NH peak at 3201 cm −1 along with appearance of aNH2 group in the 3351-3313 cm −1 range. The 1 H-, 13 C-NMR data supported the same results, where the disappearance of the NH signal of compound 3 at δ = 11.42 ppm was compensated by the appearance of the NH2 signal of compound 4 at δ = 6.90 ppm. The 13 C-NMRdata, where one of the CN signals disappeared, also advocated the same results. Refluxing of compound 4 with triethyl orthoformate in ethanol resulted in the disappearance of the NH2 signals in both the IR and 1 H-NMR spectra, respectively, due to the formation of the compound ethyl (2-cyano [1,2,4]

Results and Discussion
The previously prepared 3-amino-4H- [1,2,4]triazino [5,6-b]indole 1 [30] was used in this work as a starting unit on which the polycyclic compound will built. Compound 1 was subjected to diazotization at 4 • C in conc. HCl with aqueous solution of NaNO 2 (Scheme 1). To the stirred solution an aqueous mixture of malononitrile and sodium acetate was added to obtain [4H- [1,2,4]triazino [5,6-b]indol-3-yldiazenyl]malononitrile (3). The structure of 3 was elucidated from its IR spectrum, which showed two cyano groups signals at 2210, 2217 cm −1 with disappearance of the band corresponding to the NH 2 group of compound 1. Cyclization of compound 3 to 1-amino [1,2,4]triazino [3 ,4 :3,4]- [1,2,4]triazino [5,6-b]indole-2-carbonitrile (4) was achieved by refluxing compound 3 in ethanol. The structure elucidation of compound 4 was supported from the disappearance of one cyano group peak in the IR, as only one CN group was detected at 2209 cm −1 , in addition to the disappearance of the triazine NH peak at 3201 cm −1 along with appearance of a NH 2 group in the 3351-3313 cm −1 range. The 1 H-, 13 C-NMR data supported the same results, where the disappearance of the NH signal of compound 3 at δ = 11.42 ppm was compensated by the appearance of the NH 2 signal of compound 4 at δ = 6.90 ppm. The 13 C-NMR data, where one of the CN signals disappeared, also advocated the same results. Refluxing of compound 4 with triethyl orthoformate in ethanol resulted in the disappearance of the NH 2 signals in both the IR and 1 H-NMR spectra, respectively, due to the formation of the compound ethyl (2-cyano [1,2,4]triazino [3 ,4 :3,4] [1,2,4]triazino [5,6-b]indol-1-yl)imidoformate (5) with appearance of ethyl group signals in the 1 H-and 13 C-NMR, respectively. Compound 5 was subjected to reaction with hydrazine hydrate in boiling ethanol to get the first fifth cyclic compound 3(4H)-amino-4-iminopyrimido [4",5":5 ,6 ] [1,2,4]triazino [3 ,4 :3,4] [1,2,4]triazino [5,6-b]indole (6), which was considered as a second starting material for the formation of six-ring cyclic compounds (Scheme 1). Conformation of the structure of compound 6 was achieved from it 1 H-NMR, where the signals corresponding to the ethyl group of compound 5 at δ = 1.26 and 3.51 ppm disappeared and new bands due to NH2 and NH at δ = 5.83 and 10.13 ppm appeared, while the 13 C-NMR showed the disappearance of the sp 3 carbons of the ethyl group of compound 5.  [5,6-b]indole-4(3H)-thione (11) were formed by the reactions of 4 with hydroxylamine hydrochloride in glacial acetic acid containing anhydrous sodium acetate as a catalyst and ammonium thiocyanate in glacial acetic acid. The structures of compounds 9, 10 and 11 were confirmed from their spectral Conformation of the structure of compound 6 was achieved from it 1 H-NMR, where the signals corresponding to the ethyl group of compound 5 at δ = 1.26 and 3.51 ppm disappeared and new bands due to NH 2 and NH at δ = 5.83 and 10.13 ppm appeared, while the 13 C-NMR showed the disappearance of the sp 3 carbons of the ethyl group of compound 5. The first starting material, 4, reacted with carbon disulphide in alcoholic KOH under reflux, to get the carbamodithioic acid derivative 7 which was cyclized to give pyrimido[4",5":5 ]triazino indole-2,4(1H,3H)-dithione (8) by refluxing compound 7 in hot ethanolic sodium ethoxide for 8 h. Compound 8 was also prepared by directly refluxing compound 4 with CS 2 in boiling pyridine. The structures of compounds 7 and 8 were confirmed by their spectral analysis, where the 1 H-NMR of compound 7 showed the appearance of a signal at δ = 13.98 ppm due to a SH group, which disappeared in compound 8 and 2 NH signals shown at δ = 11.40 and 12.50 ppm, respectively (Scheme 2).
Reactions of compound 4 with urea and/or thiourea in boiling glacial acetic acid in presence of a catalytic amount of HCl yielded 2,4-diaminopyrimido [4",5 (11) were formed by the reactions of 4 with hydroxylamine hydrochloride in glacial acetic acid containing anhydrous sodium acetate as a catalyst and ammonium thiocyanate in glacial acetic acid. The structures of compounds 9, 10 and 11 were confirmed from their spectral data, where the IR of all compounds showed the disappearance of the cyano group peak with appearance of peaks corresponding to NH 2 , NH in the 3413-3754 cm −1 range, and the IR of compound 11 showed a C=S moiety at 1349 cm −1 . The 1 H-NMR of the three compounds show signals due to NH 2 and in some NH signals in the δ = 5.55-6.93 and 11.12-12.33 ppm ranges, respectively (Scheme 2). The mechanism proposed for the formation of compound 11 is illustrated in Scheme 3. data, where the IR of all compounds showed the disappearance of the cyano group peak with appearance of peaks corresponding to NH2, NH in the 3413-3754 cm −1 range, and the IR of compound 11 showed a C=S moiety at 1349 cm −1 . The 1 H-NMR of the three compounds show signals due to NH2 and in some NH signals in the δ = 5.55-6.93 and 11.12-12.33 ppm ranges, respectively (Scheme 2). The mechanism proposed for the formation of compound 11 is illustrated in Scheme 3.   The product of the reaction of glucose with compound 4 varied depending on the solvent used in the reaction, whereby when the reaction carried out intert-butanol the reaction followed the Maillard reaction pathway and the resulting compound was1-(β-D-glucopyranosyl-methylamino) [ data, where the IR of all compounds showed the disappearance of the cyano group peak with appearance of peaks corresponding to NH2, NH in the 3413-3754 cm −1 range, and the IR of compound 11 showed a C=S moiety at 1349 cm −1 . The 1 H-NMR of the three compounds show signals due to NH2 and in some NH signals in the δ = 5.55-6.93 and 11.12-12.33 ppm ranges, respectively (Scheme 2). The mechanism proposed for the formation of compound 11 is illustrated in Scheme 3.   The product of the reaction of glucose with compound 4 varied depending on the solvent used in the reaction, whereby when the reaction carried out intert-butanol the reaction followed the Maillard reaction pathway and the resulting compound was1-(β-D-glucopyranosyl-methylamino) [  The product of the reaction of glucose with compound 4 varied depending on the solvent used in the reaction, whereby when the reaction carried out in tert-butanol the reaction followed the Maillard reaction pathway and the resulting compound was 1-(β-D-glucopyranosyl-methylamino) -triazino [3 ,4 :3,4][1,2,4]triazino [5,6-b]indole-2-carbonitrile (12), while, when the reaction was performed in hot acetic acid the main product is the Schiff base type product, 1-(2,3,4,5,6pentahydroxyhexylideneamino) [1,2,4]triazino [3 ,4 :3,4][1,2,4]triazino [5,6-b]indole-2-carbonitrile (13). The 1 H-NMR spectra differentiate between the two compounds, whereby compound 12 showed two signals at δ = 3.93 and 5.12 ppm due to CH 2 and NH groups, respectively, whereas the Schiff base compound showed an olefinic proton absorption at δ = 7.34 ppm; the 13 C-NMR also showed a signal at δ = 163.2 ppm in compound 13 corresponding to the HC=N carbon, while, in compound 12 the δ = 52.66 ppm signal is due to a CH 2 group. The mechanisms illustrating the formation of compounds 12 and 13 are shown in Schemes 4 and 5. signals at δ = 3.93 and 5.12 ppm due to CH2 and NH groups, respectively, whereas the Schiff base compound showed an olefinic proton absorption at δ = 7.34 ppm; the 13 C-NMR also showed a signal at δ = 163.2 ppm in compound 13 corresponding to the HC=N carbon, while, in compound 12 the δ = 52.66 ppm signal is due to a CH2 group. The mechanisms illustrating the formation of compounds 12 and 13 are shown in Schemes 4 and 5.   [5,6-b]indole (14) was synthesized by the reaction of compound 4 with formamide, while, the reaction of 4 with malononitrile in basic medium yielded the corresponding 2,4-diaminopyrido[2",3":5 ,6 ][1,2,4]triazino [3 ,4 :3,4] triazino [5,6-b]indole-3-carbonitrile (15). On the other hand, the most amazing compound in this group of products, N,N -bis(2-cyano [1,2,4]triazino [3 ,4 :3,4]- [1,2,4]triazino [5,6-b]indol-1-yl)decanediamide (16) was formed from the reaction of 4 with sebacoyl chloride in basic medium [Scheme 6]. The marvelous feature of compound 16 is the anticancer activity it shows against all types of cell lines, more than the standard drug used. The structures of compounds 14, 15 and 16 are proved using their spectral data (see the Experimental section).
A group of six-ring compounds are synthesized using compound 6 as starting material. The reaction of compound 6 with carbon disulphide in alcoholic KOH under reflux for 9 h gave the cyclic derivative [1,2,4] (17) directly and no evidence for the formation of the dithioic acid derivative, The 1 H-NMR of compound 17 showed no signal due to a SH group with the presence of a signal due to the NH of a triazolo ring at δ = 11.30 ppm. The reaction of compound 17 with hydrazine hydrate in boiling ethanol gave the starting material 6 in poor yield.
The mechanism proposed for this amazing reaction is illustrated in Scheme 7. Methylation of compound 17 with methyl iodide in sodium acetate yielded the S-methyl derivative 18. The structure of compound 18 was confirmed by its IR, 1 H, and 13 C-NMR data. The IR showed the disappearance of the NH peak at 3266 cm −1 and the C=S peak at 1338 cm −1 in compound 17. The 1 H-NMR of compound 18 showed a new signal due to the SCH 3 protons at δ = 3.01 ppm and no signal for a NH group. The 13 C-NMR spectrum also supported the methylation of the S atom by a signal appearing at δ = 13.5 ppm due to the SCH 3 carbon.   Mannich reaction on compound 6 with formaldehyde and piperidine (Scheme 8) afforded 1-(piperidin-1-ylmethyl) [1,2,4]  The IR of compound 21 showed a peak due to a carbonyl group at 1654 cm −1 with disappearance of the NH2 and NH peaks, while compound 22 showed no major functional groups in its IR except the C=N group.

Cytotoxic Activity
The in vitro growth inhibitory activity of the synthesized compounds was investigated in comparison with a well-known anticancer standard drug (cisplatin) under the same conditions using The IR of compound 21 showed a peak due to a carbonyl group at 1654 cm −1 with disappearance of the NH 2 and NH peaks, while compound 22 showed no major functional groups in its IR except the C=N group.

Cytotoxic Activity
The in vitro growth inhibitory activity of the synthesized compounds was investigated in comparison with a well-known anticancer standard drug (cisplatin) under the same conditions using a colorimetric MTT assay. Data generated were used to plot a dose response curve from which the concentration of test compounds required to kill 50% of cell population (IC 50 ) was determined ( Figure 2). The results revealed that all the tested compounds showed inhibitory activity to the tested tumor cell lines in a concentration dependent manner. Cytotoxic activity was expressed as the mean IC 50 of three independent experiments. Interestingly, the results, presented in Table 1 and Figure 2, show that compound 16 was the most active against the three tested carcinoma cell lines (HepG2, HCT-116 and MCF-7, respectively), giving IC50 values of 3.82, 4.73 and 6.52 μg/mL compared with the cisplatin reference drug.
The order of activity against the liver carcinoma cell line (HepG2) was 16, 4, 7, 8, 12, 15  Interestingly, the results, presented in Table 1 and Figure 2, show that compound 16 was the most active against the three tested carcinoma cell lines (HepG2, HCT-116 and MCF-7, respectively), giving IC 50 values of 3.82, 4.73 and 6.52 µg/mL compared with the cisplatin reference drug.

Molecular Docking Studies
Molecular docking is a key tool in computer drug design [31]. The focus of molecular docking is to simulate the molecular recognition process. Molecular docking aims to achieve an optimized conformation for both protein and drug with relative orientation between them such that the free energy of the overall system is minimized. The results showed a possible arrangement between free compound as a drugs and receptors (2q7k) and (3hb5). The docking study showed a favorable interaction between the compounds and the receptor, and the calculated energy is presented in Tables 2 and 3. According to our results (Figures 3 and 4), HB plots indicate that the drug compounds bind to the 2q7k and 3hb5 proteins with hydrogen bond interactions and there are decomposed interaction energies of synthesized compounds with 2q7k and 3hb5 (Tables 4 and 5). The calculated efficiency is favorable. K i values (estimated using AutoDock) were compared with experimental K i values, when available, and the Gibbs free energy is negative. Also, based on these data, we can propose that an interaction between the 2q7k and 3hb5 receptors and our compounds is possible. The 2D plots of interaction between the compound 3 and compound 4 and prostate cancer receptor (2q7k) and receptor breast cancer enzymes are shown in Figures 5 and 6. From an analysis of the values, it is evident that the binding energy of our compounds decreases. Binding energies are the most widely used mode of measuring the binding affinity of a compound. Thus, a decrease in binding energy due to mutation will increase the binding affinity of the compounds towards the receptor. The characteristic feature of the compounds is represented by the presence of several active sites available for hydrogen bonding. This feature gives them the ability to be good binding inhibitors to the protein and will help to produce augmented inhibitory compounds. The results confirm that the fused triazines as a drug compounds are efficient, preventing prostate cancer 2q7k and breast cancer 3hb5 hormone effectively, which inhibits assembly. This interaction could activate apoptosis in cancer cells for interactions with synthesized compounds. Binding energies are most widely used as a mode of measuring the binding affinity of compounds. Thus, a decrease in binding energy due to mutation will increase the binding affinity of the compounds towards the receptor [25][26][27][28][29]. The characteristic feature of compounds was the presence of several active sites available for hydrogen bonding. This software includes code developed by the Theoretical and Computational Biophysics Group in the Beckman Institute for Advanced Science and Technology at the University of Illinois at Urbana-Champaign [31][32][33][34].  Table 4. Decomposed energy of some synthesized compounds with prostate cancer receptor (2q7k).

General Information
All chemicals were purchased from Sigma-Aldrich (Taufkirchen, Germany). The melting points were measured by a digital Electrothermal IA 9100 Series apparatus Cole-Parmer, Beacon Road, Stone, Staffordshire, ST15 OSA, UK) and were uncorrected. IR spectra were recorded on an ATRAlpha FTIR spectrophotometer (Billerica, MA, USA) from 400 to 4000 cm −1 . 1 H-NMR and 13 C-NMR spectra were recorded on an AC-850 Hz instrument (Bruker, Billerica, Massachusetts). Chemical shifts were expressed as (ppm) relative to TMS as an internal standard, and DMSO-d6 was used as the solvent. Mass spectra were recorded on a GC-MS-QP 1000 EX spectrometer (Shimadzu, Kyoto, Japan). The pharmacological study was carried out at Al-Azhar University, The Regional Center for Mycology & Biotechnology, Elemental analyses were performed at the Micro-analytical Center of Cairo University.   (9). A mixture of 4 (0.26 g, 0.001 mol) and urea and/or thiourea (0.001 mol) was refluxed in glacial AcOH (20 mL) containing HCl (1 mL) for 3 h. After cooling the reaction mixture was poured onto cold water and then neutralized with ammonia solution. The precipitate formed was filtered off and then crystallized from methanol to give orange-red crystals. Yield, 57% from urea and 72% from thiourea, m.p.: over 300 • C. IR: 3413-3761 cm −1 (2NH 2 ), and 1620 cm −1 (C=N). 1 [5,6-b]indole (10). Compound 4 (0.26 g, 0.001 mol) was refluxed for 2 h with hydroxylamine hydrochloride (0.007 g, 0.001 mol) in glacial acetic acid ( 20 mL) containing anhydrous sodium acetate (0.08 g, 0.001 mol) as a catalyst. After cooling the reaction mixture was poured onto cold water to give an orange-yellow precipitate which filtered, dried and crystallized from ethanol. Yield, 81%, m.p.: 297-299 • C. IR: 3392-3754 cm −1 (NH 2 ), and 1621 cm −1 (C=N). 1 (13). A mixture of 4 (0.26 g, 0.001 mol) and glucose (0.18 g, 0.001 mol) in acetic acid/water (15/5 mL) was refluxed for 8 h. After cooling, the resulting solution was poured onto cold water. The red solution was extracted with CH 2 Cl 2 (30 mL). After evaporation of the CH 2 Cl 2 a pale red solid resulted which was filtered off, dried, and crystallized from ethanol. Yield, 52%, m.p.

Antitumor Activity Assay
The tested human carcinoma cell lines were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). The cells were grown on RPMI-1640 medium supplemented with 10% heat inactivated fetal calf serum, 1% L-glutamine, and 50 µg/mL gentamycin at 37 • C in a humidified atmosphere with 5% CO 2 incubator (Shel lab 2406, Candler, NC, USA).
For antitumor assays, the tumor cell lines were suspended in medium at concentration 5 × 10 4 cell/well in Corning ® 96-well tissue culture plates, then incubated for 24 h. The tested compounds were then added into 96-well plates (three replicates) to achieve ten concentrations for each compound (started from 500 to 1 µg/mL). Six vehicle controls with media or 0.1% DMSO were run for each 96 well plate as a control. After incubating for 24 h, the numbers of viable cells were determined by the MTT assay [35]. Briefly, the media was removed from the 96 well plate and replaced with 100 µL of fresh culture RPMI 1640 medium without phenol red then 10 µL of the 12 mM MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (Sigma, Taufkirchen, Germany) was added to each well including the untreated controls. The 96 well plates were then incubated at 37 • C and 5% CO 2 for 4 h. An 85 µL aliquot of the media was removed from the wells, and 50 µL of DMSO was added to each well and mixed thoroughly with the pipette and incubated at 37 • C for 10 min. Then, the optical density was measured at 590 nm with the microplate reader (SunRise, TECAN, Inc., Männedorf, Switzerland) to determine the number of viable cells and the percentage of viability was calculated as [(ODt/ODc)] × 100% where ODt is the mean optical density of wells treated with the tested sample and ODc is the mean optical density of untreated cells. The relation between surviving cells and drug concentration is plotted to get the survival curve of each tumor cell line after treatment with the specified compound. The 50% inhibitory concentration (IC 50 ), the concentration required to cause toxic effects in 50% of intact cells, was estimated from graphic plots of the dose response curve for each conc. using Graphpad Prism software (San Diego, CA, USA) [36].

Theoretical Molecular Docking Techniques
The docking calculations were carried out using Docking Server [32]. The MMFF94 force field [31,32] was used for energy minimization of compound molecules and protein models using Docking Server. Gasteiger partial charges were added to the compound atoms. Non-polar hydrogen atoms were merged, and rotatable bonds were defined. Docking calculations were carried out on 2q7k and 3hb5 protein models. Essential hydrogen atoms, Kollman united atom type charges and solvation parameters were added with the aid of AutoDock tools [27]. Affinity (grid) maps of 20 µg/mL × 20 µg/mL × 20 A • grid points and 0.375 A • spacing were generated using the Autogrid program [34]. AutoDock parameter set-and distance-dependent dielectric functions were used in the calculation of van der Waals and electrostatic terms, respectively. Docking simulations were performed using the Lamarckian genetic algorithm and the Solis and Wets local search method [37][38][39][40]. Initial position, orientation and torsions of the compound molecules were set randomly. Each docking experiment was derived from 10 different runs that were set to terminate after a maximum of 250,000 energy evaluations. The population size was set to 150. During the search, a translation step of 0.2 A • and quaternion and torsion steps of 5 were applied.

Conclusions
In conclusion, the anticancer screening showed not all the tested compounds exhibited a good result, except compound 16 which exhibited good cytotoxic activities against the three tested carcinoma cell lines (HepG2, MCF-7, and HCT-116) compared with the reference drug cisplatin. This result confirms that increasing of the compound volume is not necessarily effective in producing more active anticancer compounds. The effectiveness of compound 16 may be due to the octa-CH 2 chain separating two bulky five-ring compounds. Molecular docking modeling was performed on these studied fused triazino compounds against the receptor of prostate cancer 2q7k and breast cancer 3hb5 to get a chance to compare between theoretical and practical results.