Ethyl 7-Methyl-1-(4-nitrophenyl)-5-phenyl-3-(thiophen-2-yl)-1,5-dihydro-[1,2,4]triazolo[4,3-a]pyrimidine-6-carboxylate

Abstract

A novel compound, ethyl 7-methyl-1-(4-nitrophenyl)-5-phenyl-3-(thiophen-2-yl)-1,5-dihydro[1,2,4]triazolo[4,3-a]pyrimidine-6-carboxylate (7) was synthesized by reaction of N-(4-nitrophenyl)thiophene-2-carbohydrazonoyl chloride (1) with ethyl 6-methyl-4-phenyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (2). The mechanism of the studied reaction is discussed and the assigned structure was confirmed by elemental analysis and spectral data. Moreover, the in vitro antitumor activities against human lung (A-549) and human hepatocellular carcinoma (HepG-2) cell lines were determined by the MTT method, and the results indicated a high potency compared with the employed standard antitumor drug (Cisplatin).

1. Introduction

It has been reported in the literature that functionalized thiophene derivatives have received considerable attention over the last two decades because they are endowed with a wide range of anti-tumor activities [1,2,3,4,5]. On the other hand, the synthesis of triazolopyrimidine derivatives has attracted a great deal of attention in view of their potent biological and pharmacological activities, especially their antitumor properties [6,7,8,9,10,11,12,13,14,15,16]. This was exemplified by a series of triazolo[4,3-a]pyrimidin-6-sulfonamide derivatives I that demonstrated potent inhibitory effects on the growth of a wide range of cancer cell lines including leukemia and prostate and lung cancer at low dose levels [15]. Moreover, pyrido[2,3-d][1,2,4]triazolo[4,3-a]pyrimidinone derivative II showed relatively potent antitumor activity against PC-3 and A-549 cell lines [16]. In particular, Compound II was able to cause cell cycle arrest at least partly through enhancing the expression level of the cell cycle inhibitor p21 and induced cancer cell apoptosis via a caspase-3 dependent pathway. Additionally, Compound II exhibited pro-apoptotic activity as evidenced by its ability to induce nuclear fragmentation, in addition to augmenting caspase-3 activation (Figure 1).

Encouraged by the above observations and in continuation of our previous work on the synthesis of antitumor agents [17,18,19,20,21], the synthesis of a 1,2,4-triazolopyrimidine, incorporating the thiophene moiety as a promising antitumor agent, was planned, using N-(4-nitrophenyl)thiophene-2-carbohydrazonoyl chloride as a versatile building block.

2. Results and Discussion

2.1. Chemistry

N-(4-Nitrophenyl)thiophene-2-carbohydrazonoyl chloride (1) [22] was reacted with ethyl 1,2,3,4-tetrahydro-6-methyl-4-phenyl-2-thioxopyrimidine-5-carboxylate (2) [23] in 1,4-dioxane solution in the presence of triethylamine under reflux to give a single product consistent with Structure 7 based on spectroscopic data (IR, ¹H-NMR, and MS) and elemental analyses (Scheme 1). The mass spectrum of the isolated product 7 showed the molecular ion peak at the expected m/z value. Their IR spectrum showed the disappearance of the two NH groups, and revealed a carbonyl band at 1733 cm⁻¹. Additionally, the ¹H-NMR spectrum showed the characteristic signals of the ester protons at δ 1.07 and 4.27 ppm, and one singlet signal at δ = 2.76 ppm assigned to the CH₃ protons, in addition to the expected signals assigned to the aromatic protons. The ¹³C-NMR spectrum provided the expected number and types of carbons. It showed five characteristic signals at δ = 13.8 (CH₃CH₂), 23.2 (pyrimidine-CH₃), 56.3 (pyrimidine-CH), 60.8 (CH₃CH₂), and 167.7 (COCH₂CH₃), in addition to 16 signals due to the aromatic carbons.

As depicted in Scheme 1, the reaction proceeds through S-alkylation to yield the non-isolated S-alkylated intermediate 3 followed by Smiles rearrangement to afford the thiohydrazide intermediate 5, which is consumed in situ via the elimination of hydrogen sulfide gas to yield the final product of 7.

2.2. Cytotoxic Activity

The in vitro growth inhibitory activity of the synthesized compound against human lung (A-549) and hepatocellular carcinoma (HepG2) cell lines was investigated in comparison with the well-known anticancer standard drug cisplatin using the MTT assay. Data generated were used to plot a dose–response curve of which the concentration of the test compounds required to kill 50% of cell population (IC₅₀) was determined. The results reveal that the tested compound shows activity that is inhibitory to the tumor cell lines in a concentration-dependent manner. Interestingly, the results represented in

Table 1. The in vitro inhibitory activity of Compound 7 against tumor cell lines expressed as IC₅₀ values (μg/mL) ± standard deviation from three replicates.

Tested Compounds	Tumor Cell Lines
	A-549	HepG2
7	3.46 ± 0.5	5.54 ± 0.7
Cisplatin	19.3 ± 0.9	18.4 ± 1.1

show that Compound 7 exhibits higher activity than the tested cisplatin reference drug under screening conditions. The in vitro growth inhibitory activity of Compound 7 against human lung (A-549) and hepatocellular carcinoma (HepG2) cell lines show promising activities with IC₅₀ equal to 3.46 ± 0.5 and 5.54 ± 0.7 μM, respectively, while the IC₅₀ of cisplatin against A-549 and HepG2 cell lines was found to be 19.3 ± 0.9 and 18.4 ± 1.1 μM, respectively.

3. Experimental

3.1. Chemistry

Melting points were measured on an Electrothermal IA 9000 series digital melting point apparatus (Bibby Sci. Lim. Stone, Staffordshire, UK). IR spectra were recorded in potassium bromide discs on Shimadzu FTIR 8101 PC infrared spectrophotometer (Shimadzu, Tokyo, Japan). ¹H and ¹³C-NMR spectra were recorded in DMSO-d₆ using a Varian Gemini 300 NMR spectrometer (Varian, Inc., Karlsruhe, Germany) (300 MHz for ¹H-NMR and 75 MHz for ¹³C-NMR), and the chemical shifts were related to that of the solvent [24]. Mass spectra were recorded on a Shimadzu GCMS-QP1000 EX mass spectrometer (Tokyo, Japan) at 70 eV. Elemental analysis was 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). The biological evaluation of the products was carried out at the Regional Center for Mycology and Biotechnology at Al-Azhar University, Cairo, Egypt.

3.2. Synthesis of Ethyl 7-Methyl-1-(4-nitrophenyl)-5-phenyl-3-(thiophen-2-yl)-1,5-dihydro-[1,2,4]tri­azolo[4,3-a]pyrimidine-6-carboxylate (7)

Triethylamine (0.15 mL, 1 mmol) was added to a boiling solution containing a mixture of equimolar amounts of N-(4-nitrophenyl)thiophene-2-carbohydrazonoyl chloride (1) (0.281 g, 1 mmol) and ethyl 6-methyl-4-phenyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (2) (0.276 g, 1 mmol) in 1,4-dioxane (15 mL), and reflux continued until all of the starting materials had disappeared (20 h, monitored by TLC). The solvent was evaporated under reduced pressure, and the solid residue was crystallized from acetic acid to yield the pure 1,2,4-triazolo[4,3-a]pyrimidine derivative 7 in 77% yield. Yellow solid, mp 180–182 °C; IR (KBr) ν_max 1036, 1244 (stretch C–O), 1513, 1535, 1552 (stretch C=C), 1586 (C=N), 1733 (C=O), 2927, 3060 (C-H) cm⁻¹; ¹H-NMR (DMSO-d₆) δ 1.07 (t, J = 7.2 Hz, 3H, CH₃CH₂), 2.76 (s, 3H, CH₃), 4.27 (q, J = 7.2 Hz, 2H, CH₃CH₂), 5.06 (s, 1H, pyrimidine-H6), 7.03–7.40 (m, 8H, Ar-H), 7.72 (d, 2H, Ar-H), 8.40 (d, 2H, Ar-H); ¹³C-NMR (DMSO-d₆): δ 13.8, 23.2 (CH₃), 56.3 (CH), 60.8 (CH₂), 114.2, 114.4, 125.6, 127.5, 128.3, 128.4, 129.0, 129.3, 129.6, 138.6, 142.8, 143.4, 147.9, 150.7, 158.0, 159.8 (Ar-C), 167.7 (C=O); MS (70 eV, EI, %), m/z = 487 (M⁺, 19), 461 (51), 329 (18), 272 (22), 180 (28), 113 (25), 87 (36), 59 (100). Anal. Calcd. for C₂₅H₂₁N₅O₄S (487.53): C, 61.59; H, 4.34; N, 14.36. Found: C, 61.74; H, 4.23; N, 14.22%.

3.3. Biological Evaluation

Antitumor activity assay:

Mammalian cell lines: A-549 cells (human Lung cancer cell line), HepG-2 cells (human Hepatocellular carcinoma) were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA).

Chemicals used: Dimethyl sulfoxide (DMSO), crystal violet, and trypan blue dye were purchased from Sigma (St. Louis, MO, USA). Fetal Bovine serum, DMEM, RPMI-1640, HEPES buffer solution, l-glutamine, gentamycin, and 0.25% Trypsin-EDTA were purchased from Lonza (BioWhittaker^®, Lonza, Belgium).

Crystal violet stain (1%) was composed of 0.5% (w/v) crystal violet and 50% methanol, then made up to volume with ddH₂O and filtered through Whatmann No.1 filter paper.

Cell line propagation: The cells were propagated in Dulbecco′s modified Eagle′s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, 1% l-glutamine, HEPES buffer, and 50 μg/mL gentamycin. All cells were maintained at 37 °C in a humidified atmosphere with 5% CO₂ and were subcultured twice a week.

Cytotoxicity evaluation using a viability assay: For cytotoxicity assay, the cells were seeded in 96-well plate at a cell concentration of 1 × 10⁴ cells per well in 100 μL of growth medium. Fresh medium containing different concentrations of the test sample was added after 24 h of seeding. Serial two-fold dilutions of the tested chemical compound were added to confluent cell monolayers dispensed into 96-well, flat-bottomed microtiter plates (Falcon, NJ, USA) using a multichannel pipette. The microtiter plates were incubated at 37 °C in a humidified incubator with 5% CO₂ for a period of 48 h. Three wells were used for each concentration of the test sample. Control cells were incubated without the test sample and with and without DMSO. The small percentage of DMSO present in the wells (maximally 0.1%) was found not to affect the experiment. After incubation of the cells for 24 h at 37 °C, various concentrations of sample (50, 25, 12.5, 6.25, 3.125 & 1.56 μg) were added, and the incubation was continued for 48 h and viable cell yield was determined by a colorimetric method.

In brief, after the end of the incubation period, media were aspirated, and the crystal violet solution (1%) was added to each well for at least 30 min. The stain was removed and the plates were rinsed using tap water until all excess stain was removed. Glacial acetic acid (30%) was then added to all wells and mixed thoroughly, and the absorbance of the plates were then measured after gentle shaking on a microplate reader (SunRise, TECAN, Inc., San Diego, CA, USA), using a test wavelength of 490 nm. All results were corrected for background absorbance detected in wells without added stain. Treated samples were compared with the cell control in the absence of the tested compounds. All experiments were carried out in triplicate. The cell cytotoxic effect of each tested compound was calculated. The optical density was measured with the microplate reader (SunRise, TECAN, Inc., San Diego, CA, USA) 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 (IC₅₀), 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) [25,26].

4. Conclusion

Ethyl 7-methyl-1-(4-nitrophenyl)-5-phenyl-3-(thiophen-2-yl)-1,5-dihydro-[1,2,4]triazolo[4,3-a]pyri-midine-6-carboxylate was synthesized and tested for its in vitro antitumor activity against two human cancer cell lines (A-549 and HepG-2).