Synthesis and Pharmacological Studies of Unprecedented Fused Pyridazino[3′,4′:5,6][1,2,4]triazino[3,4-b][1,3,4]thiadiazine Derivatives

A novel fused system with three or four fused rings—pyridazino[3′,4′:5,6][1,2,4]triazino[4,3-b][1,2,4,5]tetrazine and pyridazino[3′,4′:5,6][1,2,4]triazino[3,4-b]pyrimido[4,5-e][1,3,4]thiadiazine was obtained from the starting materials 4(6H)-amino-3-hydrazino-7-(2-thienyl)pyridazino[3,4-e][1,2,4]-triazine 2 and 9-amino-3-(2-thienyl)-2H,8H-pyridazino[3′,4′:5,6][1,2,4]triazino[3,4-b][1,3,4]thiadiazine-8-carbonitrile 12. Each of the starting compounds was subjected to a number of cyclization reactions to obtain a series of new heterocyclic fused systems, 3–10 and 13–23, via bifunctional reagents. Some of the synthesized compounds were screened against three cell lines including HepG2, HCT-116 and MCF-7 to discover their anticancer activity. The synthesized compounds were characterized depending on their elemental analyses and spectral data.


Introduction
As the world's population increases, so too does the number of health problems, and the need to discover new therapeutics becomes even more urgent. Drug design represents the greatest hope for success in the present and future era. Discovering new drugs means saving the lives of many people. Heterocyclic compounds are widely distributed everywhere and area mainstay for life. Now, a vast number of heterocyclic compounds are pharmacologically active and are actually in clinical use [1]. To make scientific progress in the field of drug discovery, our main goal was to discover new drug classes with much better therapeutic profiles. In this paper, we synthesized two novel heterocyclic fused systems containing mainly thiophene as a substituent in pyridazinotriazine, which fused with tetrazine, thiadiazine, pyridine, and pyrimidine, rings in one way or another.

Results and Discussion
Once in a while, we look for unprecedented fused systems. Our challenge this time is to use the previously prepared compounds 1 and 11 [30,31] as a kernel for constructing a new fused system through subsequent reactions with a bifunctional reagent.
The simple molecule 4-amino-3-mercapto-6-[2-(2-thienyl)vinyl]-1,2,4-triazin-5(4H)-one 1 reacted with hydrazine hydrate in refluxed ethanol to give the starting compound 4(6H)-amino -3-hydrazino-7-(2-thienyl)pyridazino [3,4-e][1,2,4]triazine 2. This compound showed in its 1 H-NMR the disappearance of each SH group and the two olefinic protons of the side chain double bond in compound 1 and its IR supported the observations where the amide carbonyl also disappeared. These observations led to the conclusion that the hydrazine hydrate cyclizes the side chain double bond with the carbonyl group to give the pyridazine ring along with the substitution of the SH group with the hydrazine group (Scheme 1). Compound 2 reacted with carbon disulphide either on alcoholic KOH and/or pyridine to give the new target heterocylic fused system known as pyridazino [3 ,4 :5,6] [1,2,4]triazino [4,3-b] [1,2,4,5]tetrazine-9(10H)-thione derivative 3. The same was obtained when compound 2 reacted with triethyl orthoformate, thiourea, formamide, acetic formic anhydride, and sebacoyl chloride. Reaction of 2 with triethyl orthoformate in boiling DMF gave 9-ethoxy-3-(2-thienyl)-7,8,9,10-tetrahydro-2H-pyridazino [3 ,4 :5,6] [1,2,4]triazino [4,3-b] [1,2,4,5] tetrazine 4. In addition, the reaction of compound 2 with malononitrile in basic medium yielded 8-Amino-3-(2-thienyl)pyrazolo [5,1-c]pyridazino [3,4-e][1,2,4]triazine-7-carbonitrile 5 through the nucleophilic substitution of the hydrazino group followed by cyclization and in situ auto-oxidation for aromatization of the diazole ring; reaction with thiourea in boiling acetic acid yielded 9-amino-3-(2-thienyl)-7,10-dihydro-2H-pyridazino [3 ,4 :5,6] [1,2,4]triazino [4,3-b] [1,2,4,5]tetrazine 6 (Scheme 1). The structure's characterization of the yielded compounds was based on their spectral data and the elemental analysis. Compound 3 in its mass spectra molecular ion peak M + at 304 (22.1) and its IR showed a + signals due to the formation of a cyanodiazole ring. On the other hand, the structure of 9-aminotetrazino derivative 6 was confirmed from its 1 H-NMR where it showed a signal due to NH 2 and 3 NH groups at δ = 5.68, 9.82, 11.37 and 13.05 ppm, respectively.  . The structures of compounds 7 and 8 were confirmed from their 1 H-NMR and IR where the IR of compound 7 showed a band due to an amide carbonyl at 1654 cm 1 and no evidence supported the presence of S atom functional groups such as SH or C=S and this means that the reaction was carried out through the nucleophilic substitution on thioglycolic acid CH2 followed by cyclization to give the triazinone ring. The 1 H-NMR supported this elucidation by the appearance of doublets due to the electronic environment unsymmetrical CH2 signal at δ = 3.65, 3.89 ppm. The 1 H-NMR of compound 8 showed no NH2 group signals and its 13 C-NMR showed an extra signal due to a carbon atom for the formed tetrazine ring. ,5]tetrazine 9 was prepared by the reaction of compound 2 with acetic formic anhydride [32] in boiling ethanol while the dimmer 10 was formed from the reaction of 2 with sebacoyl chloride in basic conditions (Scheme 2). The structures of compounds 9 and 10 confirmed from their spectral date where the two compounds showed the disappearance of the two NH2 groups of compound 2 in both IR and 1 H-NMR. The 13 C-NMR supported this conclusion by showing signals due to sp 3 carbons in the range δ = 23.68-55.29 ppm. . The structures of compounds 7 and 8 were confirmed from their 1 H-NMR and IR where the IR of compound 7 showed a band due to an amide carbonyl at 1654 cm 1 and no evidence supported the presence of S atom functional groups such as SH or C=S and this means that the reaction was carried out through the nucleophilic substitution on thioglycolic acid CH 2 followed by cyclization to give the triazinone ring. The 1 H-NMR supported this elucidation by the appearance of doublets due to the electronic environment unsymmetrical CH 2 signal at δ = 3.65, 3.89 ppm. The 1 H-NMR of compound 8 showed no NH 2 group signals and its 13 C-NMR showed an extra signal due to a carbon atom for the formed tetrazine ring. Compound 12 was prepared by reaction of compound 11 [31] with hydrazine hydrate to give the new hetrocylic system pyridazino[3′,4′:5,6][1,2,4]triazino[3,4-b][1,3,4]-thiadiazine, which is an unprecedented system. Compound 12 acts as a starting material in some cyclization reactions where it reacted with cyanamine in an EtOH/H2O mixture (4:1 by volume) to give the guanidine derivative 13. The idea of cyclization of compound 13 was intuitive from the appearance of two spots in TLC during compound 13 synthesis. Therefore, we thought to repeat the synthesis of compound 13 but with a longer time reflux. This reaction resulted in [7,7- Compound 12 showed in its IR the disappearance of the amide carbonyl to confirm the formation of the pyridazine ring. The structure of compound 13 was recognized from its 1 H-NMR where it showed three NH signals instead of one in compound 12. The structure of compound 14 was proven by the disappearance of the CN group in its IR chart with the appearance of another NH2 in both IR and 1 H-NMR. The mechanism describes the cyclization of compound 13 shown in Scheme 4 [31]. The structure of compound 15 showed an absorption band at 1345 cm −1 due to C=S with other bands at 3376-3212 cm −1 and 2207 cm −1 due to the 2NH2 and CN groups, respectively.
The To confirm the above structures, we scrutinized their IR, 1 H-NMR,and 13 C-NMR.The IR of compound 16 showed a new band at 2613 and 1345 cm −1 due to SH and C=S groups with the disappearance of the NH2 group of compound 12.Additionally, its 1 H-NMR showed the most important signal at δ = 13.78 ppm, corresponding to the SH group. In the case of compound 17, the groups that underpin the structure of compound 16 vanished and new groups characteristic of the compound 17 structure appeared. The 1 H-NMR of 17 showed the demise of the SH group and a third NH group appeared. The IR of compound 18 showed the disappearance of the CN group, which Compound 12 was prepared by reaction of compound 11 [31] with hydrazine hydrate to give the new hetrocylic system pyridazino [3 ,4 :5,6] [1,3,4]-thiadiazine, which is an unprecedented system. Compound 12 acts as a starting material in some cyclization reactions where it reacted with cyanamine in an EtOH/H 2 O mixture (4:1 by volume) to give the guanidine derivative 13. The idea of cyclization of compound 13 was intuitive from the appearance of two spots in TLC during compound 13 synthesis. Therefore, we thought to repeat the synthesis of compound 13 but with a longer time reflux. This reaction resulted in [7,7- . Compound 12 showed in its IR the disappearance of the amide carbonyl to confirm the formation of the pyridazine ring. The structure of compound 13 was recognized from its 1 H-NMR where it showed three NH signals instead of one in compound 12. The structure of compound 14 was proven by the disappearance of the CN group in its IR chart with the appearance of another NH 2 in both IR and 1 H-NMR. The mechanism describes the cyclization of compound 13 shown in Scheme 4 [31]. The structure of compound 15 showed an absorption band at 1345 cm −1 due to C=S with other bands at 3376-3212 cm −1 and 2207 cm −1 due to the 2NH 2 and CN groups, respectively.
The To confirm the above structures, we scrutinized their IR, 1 H-NMR, and 13 C-NMR. The IR of compound 16 showed a new band at 2613 and 1345 cm −1 due to SH and C=S groups with the disappearance of the NH 2 group of compound 12.Additionally, its 1 H-NMR showed the most important signal at δ = 13.78 ppm, corresponding to the SH group. In the case of compound 17, the groups that underpin the structure of compound 16 vanished and new groups characteristic of the compound 17 structure appeared. The 1 H-NMR of 17 showed the demise of the SH group and a third NH group appeared. The IR of compound 18 showed the disappearance of the CN group, which contributes to the cyclization process. The 1 H-NMR of 19 illustrates a doublet and triplet at δ = 1.20 and 3.33 due to CH 3 and CH 2 , respectively. Additionally, 13 C-NMR supported this evidence; it showed signals due to CH 3 and CH 2 at δ = 13.7 and 62.59 ppm, which disappeared in compound 20 1  . The structure of compound 21 was elucidated from 1 H-NMR to 13 C-NMR where the 1 H-NMR showed two amino groups at δ = 6.02 and 6.59 ppm while 13 C-NMR showed an extra three carbons more than compound 12 due to the pyridine ring formed. . The structure of compound 21 was elucidated from 1 H-NMR to 13 C-NMR where the 1 H-NMR showed two amino groups at δ = 6.02 and 6.59 ppm while 13 C-NMR showed an extra three carbons more than compound 12 due to the pyridine ring formed.  . The structure of compound 21 was elucidated from 1 H-NMR to 13 C-NMR where the 1 H-NMR showed two amino groups at δ = 6.02 and 6.59 ppm while 13 C-NMR showed an extra three carbons more than compound 12 due to the pyridine ring formed.

Cytotoxic Activity
The in vitro growth inhibitory activity of the synthesized compounds was investigated in comparison with the well-known anticancer standard drugs (cisplatin) under the same conditions using colorimetric MTT assay. Data generated were used to plot a dose-response curve of which the concentration of test compounds required to kill 50% of cell population (IC50) was determined (see Figure  1 and Table 1). The results revealed that all the tested compounds showed inhibitory activity with the tumor cell lines in a concentration-dependent manner. Cytotoxic activity was expressed as the mean IC50 of three independent experiments. The order of activity against the breast carcinoma cell line (MCF-7) was 16, 2, 12, 3, 4, 7, 6, 5 and 10, with IC50 values of 29.2 ± 0.7, 29.6 ± 0.9, 43.9 ± 1.2, 54 ± 0.8, 54.5 ± 1.2, 78.2 ± 1.2, 100 ± 0.9, 145 ± 3.4, 168 ± 4.5 and 404 ± 3.4 μg/mL, respectively.

Cytotoxic Activity
The in vitro growth inhibitory activity of the synthesized compounds was investigated in comparison with the well-known anticancer standard drugs (cisplatin) under the same conditions using colorimetric MTT assay. Data generated were used to plot a dose-response curve of which the concentration of test compounds required to kill 50% of cell population (IC 50 ) was determined (see Figure 1 and Table 1). The results revealed that all the tested compounds showed inhibitory activity with the tumor cell lines in a concentration-dependent manner. Cytotoxic activity was expressed as the mean IC 50 of three independent experiments.

Cytotoxic Activity
The in vitro growth inhibitory activity of the synthesized compounds was investigated in comparison with the well-known anticancer standard drugs (cisplatin) under the same conditions using colorimetric MTT assay. Data generated were used to plot a dose-response curve of which the concentration of test compounds required to kill 50% of cell population (IC50) was determined (see Figure  1 and Table 1). The results revealed that all the tested compounds showed inhibitory activity with the tumor cell lines in a concentration-dependent manner. Cytotoxic activity was expressed as the mean IC50 of three independent experiments. The order of activity against the breast carcinoma cell line (MCF-7) was 16, 2, 12, 3, 4, 7, 6, 5 and 10, with IC50 values of 29.2 ± 0.7, 29.6 ± 0.9, 43.9 ± 1.2, 54 ± 0.8, 54.5 ± 1.2, 78.2 ± 1.2, 100 ± 0.9, 145 ± 3.4, 168 ± 4.5 and 404 ± 3.4 μg/mL, respectively.
Moreover, the compounds 5 and 10 were relatively less active against the tested tumor cell line.

General Information
All chemicals were purchased from Sigma (New York, NY, USA). The melting points were measured by a digital Electro thermal IA 9100 Series and were uncorrected. IR spectra were recorded on an ATRAlpha FTIR spectrophotometer (Billerica, MA, USA) from 400 cm −1 to 4000 cm −1 . 1 H-NMR and 13 C-NMR spectra were recorded on a Brüker AC-600 MHz instrument (Bruker, Billerica, Massachusetts).
Chemical shifts were expressed as ppm relative to TMS as an internal standard and DMSO-d 6 was used as the solvent. Mass spectra were recorded on a Shimadzu GC-MS-QP 1000 EX spectrometer (Shimadzu, Kyoto, Japan). The pharmacological study was carried out at Al-Azhar University (Cairo, Egypt), The Regional Center for Mycology and Biotechnology. Elemental analyses were performed at the Micro-analytical Center, Cairo University (Cairo, Egypt).

Methods
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 a 5% CO 2 incubator (Shel lab 2406, Candler, NC, USA).
For anti-tumor assays, the tumor cell lines were suspended in medium at concentration 5 × 10 4 cell/well in Corning ® 96-well tissue culture plates and 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 [33]. 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, Mannedorf, 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 obtain the survival curve of each tumor cell line after treatment with the specified compound. The 50% inhibitory concentration (IC 50 ), which is the concentration required to cause toxic effects in 50% of intact cells, was estimated from graphic plots of the dose-response curve for each concentration using Graphpad Prism software (San Diego, CA, USA) [34].

Conclusions
Novel derivatives of fused 1,2,4-triazines were obtained via subsequent reaction methodology. The new synthesized compounds were screened against three cell lines, HepG2, HCT-116 and MCF-7, to discover their anti-cancer activity. The results revealed that all the tested compounds showed inhibitory activity against the tumor cell lines in a concentration-dependent manner. We plan to evaluate the affinity and selectivity of these and other related synthetic compounds towards adenosine receptor subtypes A 2A and A 3 . The results will be used to optimize these structures for biological activity and our conclusions will be reported in a future publication.