Synthesis of New 3-Heteroarylindoles as Potential Anticancer Agents

2-(3-(1H-Indol-3-yl)-5-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)-4-substituted-5-(substituted diazenyl)thiazoles and 2-(1H-indol-3-yl)-9-substituted-4,7-disubstituted pyrido[3,2-e][1,2,4]triazolo[4,3-a]pyrimidin-5(7H)-ones were synthesized via reaction of hydrazonoyl halides with each of 3-(1H-indol-2-yl)-5-(p-tolyl)-4,5-dihydro-1H-pyrazole-1-carbothioamide and 7-(1H-indol-3-yl)-2- thioxo-5-substituted-2,3-dihydropyrido[2,3-d]pyrimidin-4(1H)-ones, respectively. Also, hydrazonoyl halides were reacted with N’-(1-(1H-indol-3-yl)ethylidene)-2-cyanoacetohydrazide to afford 1,3,4-thiadiazole derivatives. Structures of the new synthesis were elucidated on the basis of elemental analysis, spectral data, and alternative synthetic routes whenever possible. Fifteen of the new compounds have been evaluated for their antitumor activity against the MCF-7 human breast carcinoma cell line. The results indicated that many of the tested compounds showed moderate to high anticancer activity when compared with doxorubicin as a reference drug.

The mechanism in Scheme 3 outlines what seems to be the most plausible pathway for the formation of 19 from the reaction of thione 7 with 8 via two pathways. In the first, 1,3-addition of the thiol tautomer 7 to the nitrilimine 9 would give the thiohydrazonate ester 16 which would undergo nucleophilic cyclization to yield spiro compounds 17. Ring opening to give 18 followed by cyclization Structure 19 was elucidated by elemental analysis, spectral data, and alternative synthetic routes. Thus, treatment of 7-amino-3-substituted-1-phenyl- [1,2,4]triazolo[4,3-a]pyrimidin-5(1H)-ones 20a,e,i [42] with chalcone 3a in boiling acetic acid made products identical in all respects (mp., mixed mp., and spectra) with the corresponding 19a,e,i.
with loss of hydrogen sulfide would then yield 19. In the second pathway, an initial 1,3-cycloaddition of nitrilimine 9 to the C=S double bond of 7 would give 17 directly (Scheme 3). Attempts to isolate the thiohydrazonate ester 16, spiro intermediate 17 and thiohydrazide 18 did not succeed, even under mild conditions as they readily undergo in situ cyclization followed by elimination of hydrogen sulfide to give the final product 19. This structural assignment is also consistent with literature reports, which indicate that reaction of hydrazonoyl halides with 2-thioxo-pyrimidin-4-one yielded regioselectively the corresponding 1,2,4-triazolo[4,3-a]pyrimidin-5-one derivatives [42].  The mechanism in Scheme 3 outlines what seems to be the most plausible pathway for the formation of 19 from the reaction of thione 7 with 8 via two pathways. In the first, 1,3-addition of the thiol tautomer 7 to the nitrilimine 9 would give the thiohydrazonate ester 16 which would undergo nucleophilic cyclization to yield spiro compounds 17. Ring opening to give 18 followed by cyclization with loss of hydrogen sulfide would then yield 19. In the second pathway, an initial 1,3-cycloaddition of nitrilimine 9 to the C=S double bond of 7 would give 17 directly (Scheme 3). Attempts to isolate the thiohydrazonate ester 16, spiro intermediate 17 and thiohydrazide 18 did not succeed, even under mild conditions as they readily undergo in situ cyclization followed by elimination of hydrogen sulfide to give the final product 19. This structural assignment is also consistent with literature reports, which indicate that reaction of hydrazonoyl halides with 2-thioxo-pyrimidin-4-one yielded regioselectively the corresponding 1,2,4-triazolo[4,3-a]pyrimidin-5-one derivatives [42]. Finally

Biological Screening (Cytotoxic Activity)
The in vitro growth inhibitory rates (%) and inhibitory growth activity (as measured by IC50) of the newly synthesized compounds were determined against the MCF-7 human breast carcinoma cell line in comparison with the well-known anticancer drug doxorubicin as the standard, using the MTT viability assay. Data generated were used to plot a dose response curve from which the concentration (μM) of test compounds required to kill 50% of cell population (IC50) was determined. Cytotoxic activity was expressed as the mean IC50 of three independent experiments. The difference between inhibitory activities of all compounds with different concentrations was statistically significant p < 0.001.
The results revealed that the tested compounds showed high variation in the inhibitory growth rates and activities against the tested tumor cell lines in a concentration dependent manner compared to the reference drug as shown in Table 1 and Figure 2.
The descending order of activity of the newly synthesized compounds was as follows:

Biological Screening (Cytotoxic Activity)
The in vitro growth inhibitory rates (%) and inhibitory growth activity (as measured by IC 50 ) of the newly synthesized compounds were determined against the MCF-7 human breast carcinoma cell line in comparison with the well-known anticancer drug doxorubicin as the standard, using the MTT viability assay. Data generated were used to plot a dose response curve from which the concentration (µM) of test compounds required to kill 50% of cell population (IC 50 ) was determined. Cytotoxic activity was expressed as the mean IC 50 of three independent experiments. The difference between inhibitory activities of all compounds with different concentrations was statistically significant p < 0.001.
The results revealed that the tested compounds showed high variation in the inhibitory growth rates and activities against the tested tumor cell lines in a concentration dependent manner compared to the reference drug as shown in Table 1 and Figure 2.

Biological Screening (Cytotoxic Activity)
The in vitro growth inhibitory rates (%) and inhibitory growth activity (as measured by IC50) of the newly synthesized compounds were determined against the MCF-7 human breast carcinoma cell line in comparison with the well-known anticancer drug doxorubicin as the standard, using the MTT viability assay. Data generated were used to plot a dose response curve from which the concentration (μM) of test compounds required to kill 50% of cell population (IC50) was determined. Cytotoxic activity was expressed as the mean IC50 of three independent experiments. The difference between inhibitory activities of all compounds with different concentrations was statistically significant p < 0.001.
The results revealed that the tested compounds showed high variation in the inhibitory growth rates and activities against the tested tumor cell lines in a concentration dependent manner compared to the reference drug as shown in Table 1 and Figure 2.
The descending order of activity of the newly synthesized compounds was as follows:


The activities of the synthesized compounds depend on the structural skeleton and electronic environment of the molecules.  Based on our limited study, the 1,3,4-thiadiazole ring as in 27 has in vitro inhibitory activity greater than the 1,3-thiazole ring in 12 and more than the triazolopyridopyrimidine ring in 19.

For the 1,3-Thiazole Ring 12a-c,e
 The in vitro inhibitory activity of the 4-methylthiazole is greater than 4-phenylthiazole (12a > 12e). This may be due to the positive inductive effect (+I effect) of the methyl group (increase activity) or the steric effect caused by phenyl group (decrease activity).  The introduction of electron-donating group (methyl) at C4 of the phenyl group at position 4 in the 1,3-thiazole ring enhances the antitumor activity. In contrast, introduction of an electron-withdrawing group (chlorine) decreases the antitumor activity (12b > 12a > 12c). The descending order of activity of the newly synthesized compounds was as follows:

‚
The activities of the synthesized compounds depend on the structural skeleton and electronic environment of the molecules.
‚ Based on our limited study, the 1,3,4-thiadiazole ring as in 27 has in vitro inhibitory activity greater than the 1,3-thiazole ring in 12 and more than the triazolopyridopyrimidine ring in 19.

For the 1,3-Thiazole Ring 12a-c,e
‚ The in vitro inhibitory activity of the 4-methylthiazole is greater than 4-phenylthiazole (12a > 12e). This may be due to the positive inductive effect (+I effect) of the methyl group (increase activity) or the steric effect caused by phenyl group (decrease activity).

‚
The introduction of electron-donating group (methyl) at C4 of the phenyl group at position 4 in the 1,3-thiazole ring enhances the antitumor activity. In contrast, introduction of an electron-withdrawing group (chlorine) decreases the antitumor activity (12b > 12a > 12c).

Chemistry
All melting points were measured on an Electro thermal IA 9000 series digital melting point apparatus (Bibby Sci. Lim. Stone, Staffordshire, UK). The IR spectra were recorded on potassium bromide discs using a PyeUnicam SP 3300 or Shimadzu FT IR 8101 PC infrared spectrophotometer (Shimadzu, Tokyo, Japan). The NMR Spectra were recorded at 270 MHz on a Varian Mercury VX-300 NMR spectrometer (Varian, Inc., Karlsruhe, Germany). NMR spectra were recorded on a Varian Mercury VX-300 NMR spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany) operating at 300 MHz ( 1 H-NMR) and run in deuterated dimethylsulfoxide (DMSO-d 6 ). Chemical shifts were related to that of the solvent. 13 C-NMR spectra were recorded at 75 MHz. Mass spectra were recorded on a Shimadzu GCMS-QP1000 EX mass spectrometer (Tokyo, Japan) at 70 eV. Elemental analyses and the biological evaluation of the products were carried out at the Microanalytical Centre of Cairo University, Giza, Egypt. All reactions were followed by TLC (silica gel coated aluminum sheets 60 F254, Merck, Merck & Co., Inc., Kenilworth, NJ, USA). Hydrazonoyl halides were prepared as reported in the literature [43][44][45][46]

General Procedure for the Reaction of Hydrazonoyl Halides 8 with Thiones 7a,b
To a solution of thione 7a or 7b (1 mmol) and the appropriate hydrazonoyl halides 8 (1 mmol) in dioxane (20 mL) was added TEA (0.14 mL, 1 mmol). The reaction mixture was refluxed until all of the starting materials had disappeared (8-12 h, monitored by TLC). The solvent was evaporated and the residue was triturated with MeOH. The solid formed was collected and recrystallized from the appropriate solvent to give products 19a-l. The products 19a-l together with their physical constants are listed below.  (15 mL), was refluxed for 10 h then cooled to room temperature. The solid precipitated was collected, washed with water, dried, and recrystallized from DMF to give the corresponding products, 19a,e,i which were identical in all respects (mp, mixed mp and IR spectra) with those obtained from reaction of thione 7a with hydrazonoyl chlorides 8a,e,i but the % yields are 69%, 67%, and 70%, respectively.

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. The cells were maintained at 37˝C in a humidified atmosphere with 5% CO 2 incubator (Shel lab 2406, New York, NY, USA) and were sub-cultured two to three times a week. For antitumor assays, the tumor cell lines were suspended in medium at concentration 5ˆ10 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 (six replicates) to achieve eight concentrations for each compound (started from 200 to 1.56 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. 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 (MTT; Sigma Chemical Co., St. Louis, MO, USA) stock solution (5 mg of MTT in 1 mL of PBS) 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 [1´(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 concentration using Graphpad Prism software (San Diego, CA, USA) [47,48].

Conclusions
3-Acetylindole proved to be a useful precursor for synthesis of various 1,3-thiazoles, 1,2,4-thiadiazoles and pyrido [3,2-e] [1,2,4]triazolo[4,3-a]pyrimidin-5(7H)-one. The structures of the newly synthesized compounds were confirmed by spectral data and elemental analyses. Some of the new compounds were tested in vitro against the MCF-7 human breast carcinoma cell line and compared with doxorubicin as the standard, using the MTT viability assay. Most of the tested compounds were found to have moderate to high anticancer activity.