Synthesis of 3-Trifluoromethyl-5,6-dihydro-[1,2,4]triazolo Pyrazine Derivatives and Their Anti-Cancer Studies

1. Introduction

Cancer is a leading cause of death worldwide, it is therefore urgent for medical researchers to discover efficient drugs for this disease [1]. Pyrazines, an important class of six-membered nitrogen-based heterocycles, are well known for their utility in rational drug design [2]. Ligands encompassing a pyrazine ring have been well studied and their p-donor properties have shown to be promising [3]. Pyrazine has generated great attention due to the diazine rings which form an elegant class of molecular entities, widely occurring in natural and synthetic compounds [4]. This novel unit is found in a wide variety of promising drugs pertaining to diversified therapeutic applications, namely in antidepressant [5], antipsychotic [6], antihistamine [7], anti-fungal [8], anticancer [9], antioxidant [10], and anti-inflammatory [11], etc. drugs. Structural modification with respect to substitution patterns towards the pyrazine moiety, leads to appreciable differences in the bio-active properties towards the target molecules. Therefore, continuous efforts have been undertaken to synthesize a diverse class of pyrazine derivatives which are hoped to be useful in the fight against cancer cells [12]. It is important to mention here that uncontrolled cancer cells can spread and invade different parts of the body through the blood vessels or lymphatic framework. The body is comprised of numerous sorts of cells which are important units of life and these cells develop and isolate in a limited manner to keep the body functioning normally. Tumor growth is a major clinical issue globally and it is one of the primary reasons for mortality. Commonly occurring compounds are routinely used for anticancer medication in human cancer growth [13]. Pyrazine derivatives are known for their significant action against various human tumors. Through tubulin polymerization, the anticancer action of various subordinates of pyrazine has been accounted for in the previous report. Since various derivatives of pyrazine molecules are well reported for their anti-cancer properties, we envisioned that 3-trifluoromethyl-5,6-dihydro-[1,2,4]triazolo pyrazine derivatives (RB1-9) may exhibit interesting bio-active properties. In the literature, we could not find any report for the utilization of pyrazine derivatives (RB1-9) towards anti-cancer studies, which enthralled us to screen them for up-regulation of BAX and down-regulation of Bcl2 in HT-29 colon cancer cell lines.

2. Results and Discussion

2.1. Chemical Section

Though various reports are available in the literature for the synthesis of pyrazine derivatives (RB), there is no method available to access them under mild reaction conditions with an excellent yield, which inspired us to focus our efforts in this direction. In order to execute our idea, we have treated 3-trifluoromethyl-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-α]pyrazine hydrochloride with 1-chloromethyl-4-iso-cyanato benzene under the catalytic influence of Et₃N in DCM at room temperature, over a period of 5 h. This produced the anticipated product in a 92% yield (Scheme 1). The structure of the molecule was confirmed by ¹H-NMR, IR, and LCMS analyses. Encouraged by this result, we treated a wide variety of isocyanates derivatives under the aforementioned condition, smoothly affording the desired compounds RB1 to RB9 in excellent yields. It is important to mention here, that all the target molecules were synthesized under mild reaction conditions with excellent reaction conversions, which is very interesting. The results are summarized in Figure 1.

All reactions were carried out using 1 mmoL of 3-trifluoromethyl-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-α]pyrazine hydrochloride with isocyanates (1.2 mmoL) and triethylamine (1.5 mmoL) in DCM (10 mL) at RT for 5 h.

2.2. General Procedure for the Synthesis of Pyrazine Derivatives (RB1–RB9)

Triethylamine (1.5 mmol) was added to a suspension of 3-trifluoromethyl-5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-α]pyrazine hydrochloride (1 mmol) in DCM (dichloro methane) (10 mL) in RT (retention time), followed by the addition of isocyanates (1.2 mmol). The reaction mixture was stirred at room temperature for 5 h and the progress of the reaction was monitored by TLC (Thin layer chromatography). Upon completion of the reaction, the solvent was removed under reduced pressure and the residue obtained was added to water and extracted with ethyl acetate. The organic layer was washed with a brine solution and dried over anhydrous Na₂SO₄. The solvent was evaporated and the crude product was purified by column chromatography over silica gel using chloroform: methanol (9:1) as an eluent to afford the anticipated products (RB1–RB9) (Supplementary Materials).

N-[4-(chloromethyl)phenyl]-3-(trifluromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-α]pyrazine-7(8H)-carboxamide (RB1): (92%, off-white solid), m.p 178.8–183.6 °C ¹H-NMR (CDCL₃, 400 MHz) δ: 4.00 (t, 2H, J = 5.6 Hz), 4.21 (t, 2H, J = 5.2 Hz), 4.46 (d, 2H, J = 5.6 Hz),4.88 (s, 2H), 5.66 (d, 1H, J = 4.8 Hz), 7.28–7.32 (m,4H). LCMS for C₁₄H₁₃ClF₃N₅O (ESI + ion): m/z = 360 [M + H]⁺. IR (KBr, cm⁻¹): 3356.2, 1736, 1621, 1041.

N-[3-methyl-(1,11-biphenyl)-4-yl]-3-(trifluromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-α]pyrazine-7(8H)-carboxamide (RB2): (95%, off-white-solid), 188.6–205.9 °C, ¹H-NMR (CDCl₃, 400 MHz) δ: 4.07 (t, 2H, J = 5.6 Hz), 4.27 (t, 2H, J = 5.6 Hz), 5.02 (s, 2H), 6.90–7.58 (m, 10H). LCMS for C₁₉H₁₆F₃N₅O MS (ESI + ion): m/z = 388 [M + H]⁺. IR (KBr, cm⁻¹): 3429, 3030, 1654, 1283, 1078, 981.

N-(2-fluoro-5-methylphenyl)-3-(trifluromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-α]pyrazine-7(8H)-carboxamide (RB3): (90%, off-white-solid), 196.7–201.7 °C, ¹H-NMR (CDCl₃, 400 MHz) δ: 2.32 (s, 3H), 4.05 (t, 2H, J = 6 Hz), 4.27 (t, 2H, J = 4.8 Hz), 4.99 (s, 2H), 6.78–7.76 (m, 4H) LCMS for C₁₄H₁₃F₄N₅O MS (ESI + ion): m/z = 344 [M + H]⁺. IR (KBr, cm⁻¹): 3240, 2926, 1638, 1498, 1042, 939, 871.

N-(3-methylbenzene)-3-(trifluromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-α]pyrazine-7(8H)-carboxamide (RB4): (87%, white-solid), 178.3, 185.1 °C, ¹H-NMR (CDCl₃, 400 MHz) δ: 2.35 (d, 2H, J = 8 Hz), 3.99 (t, 2H, J = 6.8 Hz), 4.20 (t, 2H, J = 6.8 Hz), 4.43(d, 2H, J = 7.2 Hz), 4.84 (s, 2H), 5.22 (s, 1H), 7.11–7.28 (m, 3H). LCMS for C₁₅H₁₆F₃N₅O MS (ESI + ion): m/z = 341 [M + H]⁺. IR (KBr, cm⁻¹): 3354, 2919, 1629, 1535, 1498, 1138, 1011, 947.

N-(2-methoxyphenyl)-3-(trifluromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-α]pyrazine-7(8H)-carboxamide (RB5): (90%, white-solid), 142.4, 149.9 °C, ¹H-NMR (CDCl₃, 400 MHz) δ: 2.43 (s, 3H), 4.09 (t, 2H, J = 5.6 Hz), 4.29 (t, 2H, J = 5.2 Hz), 5.06 (s, 2H), 7.06–8.11 (m, 5H). LCMS for C₁₄H₁₄F₃N₅O₂ MS (ESI + ion): m/z = 342 [M + H]⁺. IR (KBr, cm⁻¹): 3309, 2962, 1662, 1600, 1389, 1012, 939, 747.

N-(2-isopropylyphenyl)-3-(trifluromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-α]pyrazine-7(8H)-carboxamide (RB6): (95%, Off-white-solid), 168.1–172.9 °C. ¹H-NMR (CDCl₃, 400 MHz) δ: 1.26 (d, 6H, J = 6.8 Hz), 3.05 (t, 1H, J = 6.8 Hz), 4.02 (t, 2H, J = 5.6 Hz), 4.23 (t, 3H, J = 5.2 Hz), 4.92 (s, 2H), 6.44–7.42 (m, 5H). LCMS for C₁₆H₁₈F₃N₅O MS (ESI + ion): m/z = 355 [M + H]⁺. IR (KBr, cm⁻¹): 3290, 2958, 1636, 1521, 1389, 1246, 1122, 1032, 940.

N-(2-fluoro-4-trifluoromethylphenyl-3-(trifluromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-α]pyrazine-7(8H)-carboxamide (RB7): (88%, off-white-solid), 171.4–176.4 °C, ¹H-NMR (CDCl₃, 400 MHz) δ: 4.10 (t, 2H, J = 7.6 Hz), 4.31 (t, 2H, J = 6.8 Hz), 5.04 (d, 2H, J = 12 Hz), 6.94–8.41 (m, 4H). LCMS for C₁₄H₁₀Cl₂F₇N₅O MS (ESI + ion): m/z = 398 [M + H]⁺. IR (KBr, cm⁻¹): 3342, 646, 1501, 1335, 1277, 1121, 1014, 894.

N-(benzyl)-3-(trifluromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-α]pyrazine7(8H)-carboxamide (RB8): (96%, white-solid), 194.4–199.3 °C. ¹H-NMR (CDCl₃, 400 MHz) δ: 3.99 (t, 2H, J = 5.6 Hz), 4.20 (t, 2H, J = 5.2 Hz), 4.48 (d, 2H, J = 5.6 Hz), 5.39 (s, 1H), 7.28–7.36 (m, 5H). LCMS for C₁₄H₁₄F₃N₅O MS (ESI + ion): m/z = 326 [M + H]⁺. IR (KBr, cm⁻¹): 3325, 2924, 1626, 1543, 1280, 1046, 938.

N-[2-(methylthio)phenyl]-3-(trifluromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-α]pyrazine7(8H)carboxamide (RB9): (94%, off-white-solid), 191.9–197.3 °C. ¹H-NMR (CDCl₃, 400 MHz) δ: 2.41 (d, 3H, J = 10.8 Hz), 4.08 (t, 2H, J = 7.6 Hz), 4.29 (t, 2H, J = 6.8 Hz), 5.04 (s, 2H), 7.05–8.10 (m, 5H) LCMS for C₁₄H₁₄F₃N₅O₅ MS (ESI + ion): m/z = 358 [M + H]⁺. IR (KBr, cm⁻¹): 3299, 2930, 1644, 1398, 1039, 943.

2.3. Biological Studies

The cytotoxic examination and acridine orange and ethidium bromide assay

By MTT assay, resultant compounds were examined for in vitro cytotoxic action in human colon cancer cell lines such as HT-29 and HCT-116. Reduced forms of formazan crystals were obtained from MTT—a tetrazolium salt. The viability of cells was directly measured by the intensity of the formation of formazan. The IC₅₀ estimation of 8.18 µM concentration reveals that the R7 compound shows antitumor activity (

Table 1. IC₅₀ for Pyrazine derivatives.

Compounds	HCT-116	HT-29	Chang Liver (Normal Cells)
RB1	23.25 ± 0.8	28.32 ± 0.5	>100
RB2	32.21 ± 0.6	29.23 ± 0.6	>50
RB3	28.32 ± 1.2	33.17 ± 1.2	>100
RB4	45.23 ± 0.23	38.26 ± 0.45	>100
RB5	52.32 ± 0.7	32.16 ± 0.78	>50
RB6	22.36 ± 0.65	25.23 ± 0.95	>100
RB7	15.23 ± 0.9	8.18 ± 1.2	>100
RB8	25.23 ± 1.4	19.36 ± 1.23	>50
RB9	22.56 ± 1.12	18.65 ± 0.89	>100
Cisplatin	6.15 ± 1.4	5.23 ± 0.91	>50

IC₅₀ values were expressed in triplicate experiments from 0.7 to 200 micro molar and IC₅₀ values were calculated using prism pad 5.0.

and Figure 2a,b). Acridine orange is a vital dye that recolors live, as well as dead cells. A given sample treated with cells in various concentrations showed a remarkable growth in apoptosis. The report revealed that samples with cell membrane injury were caused due to early apoptosis at 8.18 µM treatment (Figure 2c).

2.4. Up-Regulation of BAX and Down-Regulation of Bcl2 in HT-29 Cell Lines

The apoptosis process is a quick and irreversible procedure to effectively dispense with inadequate cells. The evasion of apoptosis by threatening cells is a sign of malignant growth. For malignant growth advancement, apoptosis can be viewed as a significant obstruction, maintaining a strategic distance from apoptosis is essential to tumor improvement and protection from treatment. The inborn apoptotic pathway is controlled and managed by the Bcl2 family proteins [14]. The Bcl2 family proteins are known to be significant watchmen of apoptotic reactions. The commencement of apoptosis was observed to rely on the harmony between a professional apoptotic and hostility to an apoptotic protein that intercedes cell death and development.

In numerous malignant growth types, the anti-apoptotic protein Bcl2 may assume an important role. The over articulation of Bcl2 in numerous malignant growths may restrain the prop-apoptotic signals which permit cancer cells to undergo death. The counter apoptotic Bcl2 forestalls apoptosis either by forestalling the arrival of mitochondrial apoptosome factors, for example, cytochrome c and Apaf1 into the cytoplasm or by sequestering preforms of death-driving cysteine proteases called caspases. Cells become delicate to apoptosis when the degree of apoptotic protein BAX is up-regulated. An elevated level of master apoptosis protein Bax sharpens the cells for apoptosis. The pro-apoptotic protein Bax triggers the initiation of caspase 3 by inciting the arrival of mitochondrial cytochrome C and AIF into the cytoplasm by means of changes in the pores, subsequently prompting caspase 3 activation [15]. In the current investigation, d1 inhibited the activation of Bc1-2 mRNA articulation and expanded the up-regulation of Bax. After the successful synthesis of pyrazine derivatives (RB1–9), we utilized them for anti-cancer studies. Results revealed that d1 expanded Bax transcripts and hindered Bcl2 transcripts in HT-29 cells (Figure 3) and revealed a logical increment in caspase 3 inception when contrasted with controls (Figure 3), proposing that d1 causes cell death through a mitochondrial apoptotic pathway.

The apoptosis process is a quick and irreversible procedure to effectively dispense with inadequate cells. The evasion of apoptosis by threatening cells is a sign of malignant growth. For malignant growth advancement, apoptosis can be viewed as a significant obstruction, and maintaining a strategic distance from apoptosis is essential to tumor improvement and protection from treatment. The inborn apoptotic pathway is controlled and managed by the Bc1-2 family proteins [14]. The Bc1-2 family proteins are known to be significant watchmen of the apoptotic reaction. It was observed that the commencement of apoptosis relies upon the harmony between a professional apoptotic and hostility to the apoptotic protein that intercedes cell death and development.

In numerous malignant growth types, the anti-apoptotic protein Bcl2 may assume an important role. The over articulation of Bcl2 in numerous malignant growths may restrain the pro-apoptotic signals which permit cancer cells to undergo death. The counter apoptotic Bcl2 forestalls apoptosis, either by forestalling the arrival of mitochondrial apoptosome factors, for example, cytochrome c and Apaf1 into the cytoplasm or by sequestering preforms of death-driving cysteine proteases called caspases. Cells become delicate to apoptosis when the degree of genius apoptotic protein BAX is up-regulated. An elevated level of master apoptosis protein Bax sharpens the cells for apoptosis. The pro-apoptotic protein Bax triggers the initiation of caspase 3 by inciting the arrival of mitochondrial cytochrome C and AIF into the cytoplasm by means of changes in the pores, subsequently prompting caspase 3 activation [15]. In the current investigation, d1 inhibits the activation of Bc12 mRNA articulation and expanded the up-regulation of Bax. After the successful synthesis of pyrazine derivatives (RB1-9), we utilized them for anti-cancer studies. The results revealed that d1 expanded Bax transcripts and hindered Bcl2 transcripts in HT-29 cells (Figure 3) and revealed a logical increment in caspase 3 inception when contrasted with the control (Figure 3), proposing that d1 causes cell death through a mitochondrial apoptotic pathway.

3. Materials and Methods

3.1. Chemicals and Cell Lines

We purchased MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] from Sigma Chemicals. We obtained human HT-29 development cell lines—HT-29 and HCT-116 from the National Centre for Cell Science, Pune, India. Cells developed in an ideal media magnified with 10% warmth inactivated fetal bovine serum (FBS), 100 μg of streptomycin/mL, 100 U/mL of penicillin, and were incubated in humidified environment with 5% CO₂ at 37 °C.

3.2. Statistical Analysis

Statistical analysis was accomplished by one-way and one-way ANOVA conveyed by Bonferroni’s multiple comparison test. This was used to examine the correlation p < 0.05 and was used to signify statistical significance when computing the IC₅₀ of all the compounds. Data were analyzed utilizing Graph Pad Prism 5.0 software.

3.3. MTT Cell Viability Assay

Cytotoxic activity of chalcones was examined by applying the MTT assay [16]. In 100 µL DMEM 1 × 10⁴ cells/well had seeded. A total of 10% FBS (fetal bovine serum) was magnified in all wells of a 96-well micro culture plate and incubated at 37 °C in CO₂, followed by 48 h of incubation. A total of 10 µL MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (5 mg mL⁻¹) was added to all wells, and the plates further incubated for 4 h. From the supernatant of each well in 100 µL of DMSO, the crystals of formazan were taken up and absorbance was recorded at 540 nm.

3.4. Acridine Orange/Ethidium Bromide Staining

Through the AO/EB (acridine orange and etidium bromide) double staining method, nuclear recoloring was executed [16]. HT-29 cells were collected from the control and d1 treated category. In a fixative arrangement (methanol: acidic corrosive (3:1)) cells were fixed and spread on clean glass slides, the slides were hydrated with PBS, and recolored by acridine orange/ethidium bromide (1:1). The cells were washed thoroughly by using PBS and detected through fluorescent instruments at 400–500 nm.

3.5. RT-PCR

RT-PCR was performed to analyze β-Actin RNA articulation, BAX, Bcl2, and caspase 3 by the Techno Prime framework. As per the manufacturer’s instruction, cDNA was synthesized from 2 µg of RNA utilizing a verso cDNA synthesis kit, accompanied by an oligo dT primer. The reaction volume was set to 20 μL and synthesis of cDNA was carried out for 1 h at 42 °C, followed by RT inactivation for 5 min at 85 °C. Samples of the resultant products were examined in 1.5% agarose gel [16].

4. Conclusions

We have successfully developed an efficient protocol for the synthesis of a wide variety of 3-trifluoromethyl-5,6-dihydro-[1,2,4]triazolo pyrazine derivatives in excellent yields, under mild reaction conditions. All the synthesized compounds were thoroughly characterized using ¹H-NMR, IR, and LC-MS analyses. All the synthesized compounds were then utilized for anti-cancer studies. The up-regulation of Bax has expansive applications for the enlistment of apoptosis in disease treatment. Overexpression of the Bax protein prompts the arrival of cytochrome C, initiation of the caspase pathway in compound RB7, and actuates apoptosis in HT-29 cells, ultimately leading to activation of the mitochondrial apoptotic pathway and cell death.