Synthesis and Biological Assessment of Triazolo-Quinazoline Carbothioamide Derivatives for p38 MAP Kinase Inhibition: In-Silico and In-Vitro Approaches

doi:10.21203/rs.3.rs-5053758/v1

A series of 4-Alkyl-5-oxo-N-(pyridin-3-yl)-4,5-dihydro [1,2,3] triazolo[1,5-a] quinazoline-3-carbothioamide compounds (8a-8k) were synthesized as p38 MAP kinase inhibitors, which could potentially be used as anticancer agents. The synthesized compounds were assessed for their effectiveness in inhibiting cancer using the MCF-7 cancer cell line. The results showed that compound 8a had the highest potency, with an IC₅₀ value of 39.76 ± 0.25 µM. Compound 8f and 8d exhibited noteworthy activity, with IC₅₀ values of 40.43 ± 2.04 µM and 42.15 ± 2.15 µM, respectively. Compound 8a was found to effectively bind with the active site of p38α MAP kinase, with the PDB ID 1W7H. The docking score was found to be -8.8 kcal/mol. The ADME experiments, following Lipinski's rule of five and Ergan's egg graph, showed that all the synthesized compounds had excellent oral bioavailability and acceptable stomach absorption. Compound 8a stood out as the most potent drug in the series, exhibiting considerable docking affinity, ADME profile, and p38 MAP kinase inhibitory action. The findings indicated that compound 8a has promising p38 kinase inhibition and can be a possible therapeutic drug for further investigation.

Triazolo quinazoline carbothioamide derivatives

p38 MAP kinase

MCF-7 cells

ADME

Breast cancer is the second most prevalent kind of cancer. It is characterized by the excessive growth of malignant cells in the tissue lining the mammary glands [1]. Proliferation and metastasis continue to be significant challenges in cancer treatment and research, leading to recurrence, resistance to drugs, and a dismal prognosis. Although the exact cause of breast cancer is still a mystery, researchers suspect that hormonal and genetic variables contribute significantly to the disease's progression [2].

There is a great deal of clinical and genetic diversity within the breast cancer spectrum, which makes the illness extremely diverse [3]. Three receptor phenotypes- estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2 or ErbB2)- categorize 84 subtypes of breast cancer, which include variants of MCF‐7 cells [4]. The hormone receptors that regulate the proliferation of breast cancer cells include ER, PR, and HER2, in addition to the epidermal growth factor receptor (EGFR) [5].

Crucial mitogen-activated protein kinases (MAPKs) that respond to stress signals are p38 [6]. There is some evidence that p38 MAPK activation can prevent breast cancers from developing, slow tumor spread, and serve as an underlying factor in the susceptibility to tumor suppression and cell death [7].

Triazoloquinazoline is a compound that consists of two basic heterocyclic moieties, triazole and quinazoline, fused together. This category of molecules possesses numerous pharmacological applications such as anticancer, antimicrobial [8], anti-inflammatory [9], antiviral [10], anticonvulsant [11], antidiabetic [12], antihypertensive [13], antioxidant etc [14]. Figure 1 depicts a few anticancer drugs based on triazole [15, 16] and quinazoline moiety [17, 18] while Fig. 2 displays triazoloquinazoline compounds that have substantial anticancer properties.

In this research, various substituted 4-Alkyl-5-oxo-N-(pyridin-3-yl)-4,5-dihydro [1, 2, 3] triazolo [1,5-a] quinazoline-3-carbothioamide derivatives are synthesized.

Chemistry

The synthesis of substituted 4-Alkyl-5-oxo-N-(pyridin-3-yl)-4,5-dihydro [1,2,3] triazolo[1,5-a] quinazoline-3-carbothioamide derivatives (8a to 8k) involves 6 steps. The synthesis of substituted methyl 2-aminobenzoate (2) from substituted 2-aminobenzoic acid involves an esterification reaction using diazomethane (CH₃N₂) as the methylating agent which convert into substituted methyl 2-azidobenzoate (3), through diazotization reaction. When the product 3 reacts with substituted O-ethyl cyanoethanethioate (4) in the presence of sodium ethoxide (NaOEt), it produces substituted O-ethyl 5-oxo-4,5-dihydro [1,2,3] triazolo [1,5-a] quinazoline-3-carbothioate (5). The compound 4-alkyl-O-ethyl 5-oxo-4,5-dihydro [1,2,3] triazolo[1,5-a] quinazoline-3-carbothioate (6), produced by dissolving the substituted triazoloquinazoline derivative in dry dimethylformamide (DMF) at low temperature. Sodium hydride (NaH) was added gradually over 10 minutes, and then the chosen alkyl iodide (RI) was added dropwise to the reaction mixture. The mixture was then stirred continuously at room temperature for 3 hours. The substituted triazoloquinazoline thioester (6) was dissolved in dimethylformamide (DMF) in a round-bottom flask equipped with a magnetic stirrer. To maintain an inert environment, the flask was set up with a reflux condenser under a nitrogen atmosphere. Next, substituted pyridin-3-amine (7) and N,N-diisopropylethylamine (DIPEA) were added to the solution and stirring was done at room temperature overnight to yield substituted 4-Alkyl-5-oxo-N-(pyridin-3-yl)-4,5-dihydro [1,2,3] triazolo[1,5-a] quinazoline-3-carbothioamide derivatives (8a to 8k) [Fig 3]. The product was successfully achieved with the yield ranging from 81% to 90%. All the compounds were identified by spectral data like ¹H NMR, ¹³C NMR, and mass spectrometry (MS).

¹H NMR and ¹³C NMR spectra of compounds 8a–8k were recorded in DMSO, and the peak assignment has been mentioned in the characterization section. The ¹H NMR spectra reveal that the singlet peak of methyl-protons attached to nitrogen of triazoloquinazoline occurred in the range of 3.591 ppm for all the compounds. In compounds 8b, 8c, and 8d, the chemical shift value shifted toward the downfield at 3.764 ppm, respectively, exhibiting a singlet peak as three protons are attached to oxygen, while in compound 8e, the chemical shift value shifted toward the upfield at 2.515 ppm. The ¹³C NMR spectra reveal that methyl-carbon (-CH₃) attached to nitrogen of triazoloquinazoline, in the range near 39 ppm for all the compounds except 8i and 8j, where ethyl group is attached instead of methyl, resulting in two carbon peaks at around 39 (-CH₂CH₃) and 13 ppm (-CH₂CH₃). In compounds 8b, 8c and 8d, methoxy carbon (-OCH₃) was in the range of 55.74 ppm for all. Carbon attached to three fluorine groups (-CF₃) as in 8j exhibited a peak near 124.26 ppm. All compounds (8a-8k) exhibited the corresponding M+1 peaks for their respective molecular formulas. All spectrum data results revealed that the final compounds may be successfully produced.

Anticancer Activity

MTT Assay

The cytotoxicity of the synthesized triazolo-quinazoline carbothioamide derivatives (8a-8k) were assessed using the MTT assay on the MCF-7 cell line. The IC₅₀ values are detailed in Table 1. All derivatives exhibited efficacy comparable to the conventional drug doxorubicin. Notably, compound 8a emerged as the most potent, with an IC₅₀ of 39.76 ± 0.25 µM, which is close to the standard IC₅₀ value of 3.48 ± 0.076 µM. Compounds 8f and 8d, featuring a bromo substitution on the fifth and fourth position of the pyridine ring, demonstrated IC₅₀ values of 40.43 ± 2.04 µM and 42.15 ± 2.15 µM, respectively.

Table 1. IC₅₀ of Compounds 8a-8k against MCF-7cells

Code	IC₅₀ in (mean±SEM) µM
8a	39.76 ± 0.25
8b	56.32 ± 1.02
8c	61.23 ± 1.03
8d	42.15 ± 2.15
8e	83.27 ± 2.27
8f	40.43 ± 2.04
8g	79.14 ± 1.14
8h	76.54 ± 1.54
8i	74.02 ± 2.02
8j	53.74 ± 2.74
8k	90.67 ± 2.64
Doxorubicin	3.48 ± 0.076

SEM=standard error mean; each value is the mean of three measures

Invitro p38 MAP kinase Activity

The in-vitro p38 kinase assay results for the synthesized triazolo-quinazoline carbothioamide derivatives are summarized in Table 2, with IC₅₀ values expressed in nM. Compound 8a demonstrated the highest potency among the tested derivatives, with an IC₅₀ value of 293.42 ± 1.42 nM, indicating potent inhibition of p38 kinase, in comparison to the Adezmapimod, the standard reference compound with an IC₅₀ of 232.13 ± 2.13 nM. This suggests that 8a has activity that is comparable but slightly less potent relative to the standard.

The inhibition efficacy of other compounds varied. Compound 8d and 8f showed a moderate IC₅₀ values of 381.11 ± 2.11 nM and 459.6 ± 2.60, reflecting reasonable inhibition of p38 kinase. Conversely, other compounds had higher IC₅₀ indicating reduced efficacy in p38 kinase inhibition. Overall, while most of the synthesized derivatives demonstrated effective inhibition of p38 kinase, Compound 8a emerged as the most promising candidate in the series, with activity closely approaching that of the standard control, Adezmapimod.

Table 2. In-vitro p38 MAP kinase assay of compounds 8a-8k

Code	IC₅₀ in nM
8a	293.42± 1.42
8b	821.22 ± 1.22
8c	893.54 ± 3.54
8d	381.11 ± 2.11
8e	1544.23 ± 4.23
8f	459.6 ± 2.60
8g	1192.63 ± 2.63
8h	1254.9 ± 2.90
8i	1205.72 ± 2.72
8j	1132.8 ± 2.80
8k	1496.3 ± 3.30
Adezmapimod	232.13 ± 2.13

In-silico Studies

ADME Studies

Multiple approaches have been devised to assess the drug-like properties of bioactive compounds utilizing topological descriptors of their molecular structure or other attributes such as molecular weight, water solubility, cLogP, etc. The characteristics of compounds (8a-8k) are listed in Table 3. All the compounds (8a-8k) synthesized in this study satisfied the criteria for orally active drugs, as per Lipinski's rule of five, which includes characteristics such as polar surface area (<140 Å), rotatable bonds (< 10), lipophilicity (within the suggested range of −2.0 to 6.5), and the BOILED-egg or Ergan's egg approach.

The graph shows that all of the compounds demonstrated satisfactory levels of gastrointestinal absorption (GIA), with WLOGP values less than or equal to 5.88 and TPSA values below 131.6. None of the compounds were found to across the blood-brain barrier (BBB). The blue dot symbolizes molecules that are anticipated to be ejected from the central nervous system via p-glycoprotein as indicated in Table 4 and Fig 4.

Table 3. Physicochemical Properties of compounds 8a-8k.

Code	MW	Log P	Log S	HBA	HBD	RB	MR	Fraction Csp3	Lipinski Violation
8a	336.37	2.3	-3.25	4	1	3	95.39	0.06	0
8b	366.4	2.52	-3.3	5	1	4	101.89	0.12	0
8c	400.84	2.67	-3.89	5	1	4	106.9	0.12	0
8d	445.29	2.81	-4.21	5	1	4	109.59	0.12	0
8e	446.41	2.69	-4.23	8	1	5	110.59	0.16	0
8f	500.37	2.79	-4.21	6	1	6	125.8	0.2	1
8g	435.5	2.95	-3.6	6	1	6	123.06	0.24	0
8h	489.47	2.63	-4.16	9	1	7	123.1	0.24	0
8i	469.95	3.09	-4.13	6	1	7	127.92	0.24	0
8j	435.5	2.95	-3.6	6	1	6	123.06	0.24	0
8k	435.5	2.95	-3.6	6	1	6	123.06	0.24	0

MW: Molecular Weight, Log P: Lipophilicity, Log S: Solubility, HBA: Hydrogen Bond Acceptors, HBD: Hydrogen Bond and Donors Rotatable bonds: RB, MR: Molar Refractivity

Table 4. Pharmacokinetic Properties of compounds 8a-8k

Code	TPSA	GI absorption	BBB permeant	Pgp substrate
8a	109.2	High	No	No
8b	118.43	High	No	No
8c	118.43	High	No	No
8d	118.43	High	No	No
8e	126.27	High	No	No
8f	129.51	High	No	No
8g	129.51	High	No	No
8h	129.51	High	No	No
8i	129.51	High	No	No
8j	129.51	High	No	No
8k	129.51	High	No	No

TPSA: Topological Polar Surface Area, GI: Gastrointestinal, BBB: Blood Brain Barrier, Pgp: Permeability Glycoprotein

Docking Analysis

The binding aﬃnity (in kcal/mol) of all the docked ligands is given in Table 5. The binding affinity of the substituted 4-Alkyl-5-oxo-N-(pyridin-3-yl)-4,5-dihydro [1,2,3] triazolo[1,5-a] quinazoline-3-carbothioamide derivatives (8a-8k) to the binding site of the p38α MAP kinase protein was investigated in the docking study. The ligands unveiled binding aﬃnities ranging from −8.8 to −3.0 kcal/mol. The molecular docking studies disclosed that the compounds 8a, 8d, and 8f were the most promising candidates, which is justiﬁed by lowest binding energy with −8.8, −8.3. and −8.0 kcal/mol, respectively. Docking pose and 2D diagrams illustrating protein−ligand interactions of these compounds in Fig 5.

Table 5. Docking scores and Binding Interactions of compounds 8a-8k

Code	Binding Energy (Kcal/mol)	H-Bond interaction and other Interactions (pi-pi, pi-cation, pi-anion, pi-alkyl, pi-)
8a	-8.8	Conventional hydrogen bonding (Thr 106), pi-sigma (Ala 51), pi-alkyl (Ile 84, Leu 75, Val 38, Lys 53, Ala 51)
8b	-6.0	pi-pi stacked (Tyr 35), pi-anion (Asp 168), pi-alkyl (Val 30, Leu 75, Lys 53, Tyr 35)
8c	-6.2	Conventional hydrogen bonding (Met 109, Asp 168), pi-pi stacked (Tyr 35), pi-alkyl (Val 30, Lys 53, Ala 51)
8d	-8.3	Conventional hydrogen bonding (Asp 168), pi-pi stacked (Tyr 35), pi-anion (Asp 168), pi-alkyl (Ile 84, Leu 167, Val 38, Lys 53)
8e	-3.0	Conventional hydrogen bonding (Met 109, Asp 168, Lys 53), halogen (fluorine) (Met 109), pi-pi stacked (Tyr 35), pi-alkyl (Leu 108, Val 38, Ala 51)
8f	-8.0	Conventional hydrogen bonding (Met 109, Tyr 35, Gly 110), pi-sulfur (Tyr 35), pi-alkyl (Leu 167, Val 38)
8g	-4.1	Conventional hydrogen bonding (Glu 71), pi-anion (Asp 168), pi-alkyl (Ile 84, Leu 104, Leu 164, Lys 53)
8h	-4.6	pi-pi T shaped (Tyr 35), pi-anion (Asp 168), pi-alkyl (Ile 84, Leu 167, Val 38, Lys 53), halogen (fluorine) (Val 52, Ala 51)
8i	-4.7	Conventional hydrogen bonding (Met 109, Glu 71), pi-pi T shaped (Tyr 35), pi-anion (Asp 168), pi-alkyl (Ala 51)
8j	-5.9	Conventional hydrogen bonding (Met 109, Thr 106, Glu 71), pi-pi T shaped (Tyr 35), pi-anion (Asp 168), pi-alkyl (Val 38, Lys 53, Ala 51)
8k	-3.6	pi-anion (Asp 168), pi-anion (Asp 168), pi-alkyl (Ala 51)

To summarize, a range of substituted 4-Alkyl-5-oxo-N-(pyridin-3-yl)-4,5-dihydro [1, 2, 3] triazolo[1,5-a] quinazoline-3-carbothioamide derivatives (8a-8k) has been synthesized to develop potent inhibitors of p38 MAP kinase that exhibit considerable anticancer properties. The study examined the effectiveness of the anticancer screening on MCF-7 cancer cell lines. The findings indicated that compound 8a demonstrated the highest potency among the investigated compounds against the MCF-7 cancer cell line, with an IC₅₀ value of 39.76 ± 0.25µM. Compounds 8f and 8d also showed significant anticancer activity against the MCF-7 cancer cell line, with IC₅₀ values of 40.43 ± 2.04 µM and 42.15 ± 2.15 µM, respectively. Compound 8a has been identified as the most powerful compound in the series and has a significant inhibitory effect against p38 MAP kinase. In addition, the molecular docking analysis of compound 8a revealed a favorable positioning within the active binding region of p38 MAP kinase, with a docking score of -8.8 Kcal/mol. According to Lipinski's rule of five and Ergan's egg graph, ADME studies demonstrated that all the synthesized compounds exhibited oral bioavailability as drugs. The compounds also exhibited favorable gastric absorption within the acceptable range.

We can conclude that compound 8a was the most active compound of the series concerning docking score, ADME studies, MTT assay and in vitro p38 MAP kinase inhibitory activity. Hence the compound can further be analysed for its potency.

Material and Methods

Merck Specialties Pvt. Limited, Sigma Aldrich Laboratories Pvt. Limited, and SD Fine Chem Mumbai were employed to procure the requisite chemicals for the synthesis. The column chromatography employed silica gel with a mesh range of 60–120, and the solvents utilized were ethyl acetate and distilled hexane. The melting points were obtained utilizing a DBK software within an unsealed glass capillary tube. The findings of this assessment were not rectified. The ¹H NMR and ¹³C NMR spectra were acquired in DMSO-d6 using spectrometers with frequency of 500 MHz and 125 MHz, respectively, carried out by the Bruker Avance 500 spectrometer with tetramethylsilane as an internal standard. The chemical shift values are quantified in parts per million (ppm), the spin multiplicities are represented by singlet (s), doublet (d), doublet of doublet (dd), triplet (t), and multiplet (m), and the coupling constants are measured in hertz (Hz). The Shimadzu mass spectrometer QSTAR XL GCMS was utilized to measure the mass spectra of the sample.

Synthetic Procedure.

The synthetic route of target compounds (8a-8k) is outlined in Fig 3.

Synthesis of substituted methyl 2-azidobenzoate (3): To convert substituted methyl 2-aminobenzoate to substituted methyl 2-azidobenzoate, the procedure began by dissolving 10 grams of substituted methyl 2-aminobenzoate in 100 mL of cold aqueous hydrochloric acid (1M) in a 250 mL round-bottom flask. The mixture was cooled in an ice bath to maintain a temperature near 0°C. We then slowly added 6 grams of sodium nitrite (NaNO₂) to the solution over a period of 15 minutes while stirring continuously. This resulted in the generation of nitrous acid (HNO₂) in situ, which reacted with the amine group of substituted methyl 2-aminobenzoate to form a diazonium salt intermediate. Once the addition of sodium nitrite was complete, the reaction mixture was stirred at 0°C for another 30 minutes to ensure complete diazotization. Following this, we added 50 mL of ice-cold sodium azide (NaN₃) solution (1M) dropwise to the diazonium salt solution while maintaining the reaction mixture in the ice bath. The reaction was then allowed to proceed at room temperature for 3 hours under continuous stirring. After completion of the reaction, the reaction mixture was poured into 500 mL of ice water to precipitate the product. The solid was collected by vacuum filtration, washed thoroughly with cold water, and dried under vacuum. The crude product of substituted methyl 2-azidobenzoate was further purified by recrystallization from ethanol, leading to the formation of fine crystals. The purity of the product was confirmed by NMR and IR spectroscopy, and the yield was quantified, confirming an efficient conversion process.

Synthesis of substituted O-ethyl 5-oxo-4,5-dihydro[1,2,3]triazolo[1,5-a]quinazoline-3-carbothioate (5): To synthesize substituted O-ethyl 5-oxo-4,5-dihydro[1,2,3]triazolo[1,5-a]quinazoline-3-carbothioate from substituted methyl 2-azidobenzoate, the procedure began by dissolving 5 grams of substituted methyl 2-azidobenzoate in 50 mL of ethanol (EtOH) in a 250 mL round-bottom flask. We then added 5 grams of substituted O-ethyl cyanoethanethioate to the solution. To this mixture, 10 mL of sodium ethoxide (NaOEt) solution was added dropwise. Sodium ethoxide was prepared by dissolving sodium in ethanol until a concentration of 1M was achieved. The reaction mixture was stirred vigorously at room temperature. After the addition was complete, the mixture was continuously stirred for an additional 4 hours to ensure complete reaction. During this period, the reaction mixture slowly changed from a clear solution to a slightly turbid suspension, indicating the formation of the desired product. Upon completion of the reaction, the mixture was concentrated under reduced pressure to remove the ethanol. The residue was diluted with water and extracted with ethyl acetate to separate the organic layer. The organic extract was washed with brine, dried over anhydrous sodium sulfate, and filtered. The solvent was then removed under reduced pressure. The crude product obtained was purified using column chromatography on silica gel, eluting with a gradient of hexane and ethyl acetate. The purified product was a pale-yellow solid. Its structure was confirmed by ¹H NMR, ¹³C NMR, and mass spectrometry, ensuring the successful synthesis of substituted O-ethyl 5-oxo-4,5-dihydro [1,2,3] triazolo[1,5-a] quinazoline-3-carbothioate. The yield and purity of the product were quantified, indicating a successful synthetic procedure.

Synthesis of 4-alkyl-5-oxo-N-(pyridin-3-yl)-4,5-dihydro[1,2,3]triazolo[1,5-a]quinazoline-3-carbothioamide (6): To convert substituted O-ethyl 5-oxo-4,5-dihydro[1,2,3]triazolo[1,5-a]quinazoline-3-carbothioate to 4-alkyl-O-ethyl 5-oxo-4,5-dihydro[1,2,3]triazolo[1,5-a]quinazoline-3-carbothioate, we initiated the procedure by dissolving 1 gram of the substituted triazoloquinazoline derivative in 40 mL of dry dimethylformamide (DMF) in a 250 mL round-bottom flask equipped with a magnetic stir bar. The flask was then placed in an ice bath to maintain a low reaction temperature during the initial stage. Next, 0.5 grams of sodium hydride (NaH, 60% dispersion in mineral oil) was added slowly to the solution under nitrogen atmosphere to prevent exposure to atmospheric moisture and carbon dioxide. This addition was done gradually over a period of 10 minutes while maintaining vigorous stirring to ensure the complete reaction of NaH with the solvent and substrate. Once the addition of NaH was complete and the mixture had been stirred for an additional 10 minutes while still in the ice bath, the reaction mixture was allowed to warm to room temperature. At this point, 3 equivalents of the chosen alkyl iodide (RI) were added dropwise to the reaction mixture. The alkyl iodide used was selected based on the desired alkyl group to be introduced at the 4-position of the triazoloquinazoline ring. The mixture was then stirred continuously at room temperature for 3 hours. During this period, we monitored the reaction progress through thin-layer chromatography (TLC), observing the gradual disappearance of the starting material and the formation of the product. After confirming the completion of the reaction via TLC, the reaction was quenched by slowly adding water to the reaction mixture. The resulting mixture was extracted with ethyl acetate to separate the organic layer from the aqueous phase. The organic extracts were combined, washed with water, then with brine, and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure using a rotary evaporator, and the crude product was obtained. This crude product was purified using flash column chromatography on silica gel, eluting with a mixture of hexane and ethyl acetate to achieve the desired purity. The purified 4-alkyl-O-ethyl-5-oxo-4,5-dihydro[1,2,3]triazolo[1,5-a] quinazoline-3-carbothioate was obtained as a solid. Final characterization was conducted using ¹H NMR, ¹³C NMR, and mass spectrometry, confirming the structure of the alkylated product and its purity. The overall yield was recorded, completing the synthetic transformation effectively.

General procedure for the synthesis of substituted 4-Alkyl-5-oxo-N-(pyridin-3-yl)-4,5-dihydro [1,2,3] triazolo[1,5-a]quinazoline-3-carbothioamide derivatives (8a to 8k): To convert substituted 4-alkyl-O-ethyl 5-oxo-4,5-dihydro[1,2,3] triazolo[1,5-a]quinazoline-3-carbothioate to substituted 4-alkyl-5-oxo-N-(pyridin-3-yl)-4,5-dihydro[1,2,3]triazolo[1,5-a]quinazoline-3-carbothioamide derivatives, we initiated the procedure by dissolving 1 gram of the substituted triazoloquinazoline thioester in 50 mL of dimethylformamide (DMF) in a 250 mL round-bottom flask equipped with a magnetic stirrer. The flask was set up with a reflux condenser under a nitrogen atmosphere to maintain an inert environment. Next, 3 equivalents of pyridin-3-amine and 4 equivalents of N,N-diisopropylethylamine (DIPEA) were added to the solution. DIPEA was used as a base to neutralize the acidic byproducts formed during the amide coupling reaction and to promote the nucleophilic attack of the amine on the thioester carbonyl carbon. The reaction mixture was then stirred at room temperature overnight to ensure complete reaction. During this period, we monitored the progress of the reaction using thin-layer chromatography (TLC), which indicated the gradual formation of the desired amide product. Upon completion of the reaction, as confirmed by TLC showing no starting material, the reaction mixture was poured into water to precipitate the product. The solid was filtered off and washed with cold water to remove any soluble impurities and unreacted starting materials. The filtered solid was then dried under vacuum to obtain a crude product, which was further purified by column chromatography using silica gel, eluting with a gradient of hexane to ethyl acetate. The purification process was guided by TLC, and fractions containing the pure product were combined and concentrated. The final purified substituted 4-alkyl-5-oxo-N-(pyridin-3-yl)-4,5-dihydro [1,2,3] triazolo[1,5-a] quinazoline-3-carbothioamide derivatives were obtained as a solid. The structure and purity of the synthesized compound were confirmed by ¹H NMR, ¹³C NMR, and mass spectrometry.

Characterization of 4-methyl-5-oxo-N-(pyridin-3-yl)-4,5-dihydro-[1,2,3]triazolo[1,5-a]quinazoline-3-carbothioamide (8a)

The title compound was produced from O-ethyl 4-methyl-5-oxo-4,5-dihydro-[1,2,3]triazolo[1,5-a]quinazoline-3-carbothioate and pyridin-3-amine.

White color powder, yield: 86 %, mp: 219 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 3.591 (s, 3H), 7.413 – 7.535 (m, 4H), 8.005 (d, 1H), 8.162 (d, J = 2.813, 2.813, 6.386, 12.738 Hz, 2H), 8.643 (s, 1H), 10.562 (s, 1H); ¹³C NMR (125 MHz, DMSO-d₆) δ 179.79, 162.89, 144.82, 144.50, 140.53, 135.84, 134.90, 132.11, 130.62, 130.00, 126.42, 125.05, 124.50, 118.78, 118.31, 39.57, 33.15; ESI-HRMS (m/z), of C₁₆H₁₂N₆OS, Calcd: 337.0866 [M+H]^+,Found: 337.0892 [M+H]⁺.

Characterization of 9-methoxy-4-methyl-5-oxo-N-(pyridin-3-yl)-4,5-dihydro-[1,2,3]triazolo[1,5-a]quinazoline-3-carbothioamide (8b)

The title compound was produced from O-ethyl 9-methoxy-4-methyl-5-oxo-4,5-dihydro-[1,2,3] triazolo[1,5-a]quinazoline-3-carbothioate and pyridin-3-amine.

Pale colour powder, yield: 88%, mp: 218 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 3.591 (s, 3H), 3.764 (s, 3H), 7.191 (d, J = 1.492, 7.406 Hz, 1H), 7.461 – 7.535 (m, 2H), 7.747 (s, 1H), 8.074 (d, J = 7.479 Hz, 1H), 8.148 (d, J = 2.826, 6.291 Hz, 1H), 8.643 (s, 1H), 10.562 (s, 1H); ¹³C NMR (125 MHz, DMSO-d₆) δ 179.79, 162.32, 158.79, 144.82, 144.50, 140.54, 134.90, 131.47, 130.62, 125.10, 124.50, 119.83, 119.02, 118.50, 114.36, 55.74, 39.53, 39.51, 33.08; ESI-HRMS (m/z), of C₁₇H₁₄N₆O₂S, Calcd: 367.0971 [M+H]⁺, Found: 367.0928 [M+H]⁺.

Characterization of N-(5-chloropyridin-3-yl)-9-methoxy-4-methyl-5-oxo-4,5-dihydro-[1,2,3]triazolo[1,5-a]quinazoline-3-carbothioamide (8c)

The title compound was produced from O-ethyl 9-methoxy-4-methyl-5-oxo-4,5-dihydro-[1,2,3] triazolo[1,5-a]quinazoline-3-carbothioate and 5-chloropyridin-3-amine.

White color powder, yield: 84%, mp: 234 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 3.591 (s, 3H), 3.764 (s, 3H), 7.191 (d, J = 1.466, 7.427 Hz, 1H), 7.596 (s, 1H), 7.747 (s, 1H), 8.074 (d, J = 7.477 Hz, 1H), 8.381 (s, 1H), 8.709 (s, 1H), 10.950 (s, 1H); ¹³C NMR (125 MHz, DMSO-d₆) δ 179.56, 162.32, 158.79, 142.29, 140.54, 139.02, 136.71, 131.47, 130.95, 126.72, 125.09, 119.83, 119.02, 118.50, 114.36, 55.74, 39.56, 33.08; ESI-HRMS (m/z), of C₁₇H₁₃ClN₆O₂S, Calcd: 401.0581 [M+H]^{+ ,}found: 401.0588 [M+H]^+.

Characterization of N-(4-bromopyridin-3-yl)-9-methoxy-4-methyl-5-oxo-4,5-dihydro-[1,2,3] triazolo [1,5-a]quinazoline-3-carbothioamide (8d)

The title compound was produced from O-ethyl 9-methoxy-4-methyl-5-oxo-4,5-dihydro-[1,2,3] triazolo[1,5-a]quinazoline-3-carbothioate and 4-bromopyridin-3-amine

Brown color powder, yield: 89%, mp: 226 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 3.591 (s, 3H), 3.764 (s, 3H), 7.191 (d, J = 1.467, 7.427 Hz, 1H), 7.452 (d, J = 7.617 Hz, 1H), 7.747 (s, 1H), 8.074 (d, J = 7.475 Hz, 1H), 8.457 (d, J = 7.395 Hz, 1H), 8.513 (s, 1H), 11.191 (s, 1H); ¹³C NMR (125 MHz, DMSO-d₆) δ 180.16, 162.32, 158.79, 146.83, 144.35, 140.54, 134.62, 131.47, 128.00, 125.42, 123.61, 119.83, 119.02, 118.50, 114.36, 55.74, 39.51, 33.08; ESI-HRMS (m/z), of C₁₇H₁₃BrN₆O₂S, Calcd: 445.0076 [M+H]^+,Found: 445.0091 [M+H]⁺.

Characterization of N-(6-acetylpyridin-3-yl)-4-methyl-5-oxo-9-(trifluoromethyl)-4,5-dihydro-[1,2,3]triazolo[1,5-a]quinazoline-3-carbothioamide (8e)

The title compound was produced from O-ethyl 4-methyl-5-oxo-9-(trifluoromethyl)-4,5-dihydro-[1,2,3]triazolo[1,5-a]quinazoline-3-carbothioate and 1-(3-aminopyridin-4-yl)ethan-1-one.

Cream color flakes, yield: 81%, mp: 263 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 2.515 (s, 3H), 3.591 (s, 3H), 7.366 (d, J = 1.619, 7.469 Hz, 1H), 7.743 – 7.818 (m, 2H), 8.050 (d, J = 7.536 Hz, 1H), 8.231 (s, 1H), 8.465 (s, 1H), 10.585 (s, 1H); ¹³C NMR (125 MHz, DMSO-d₆) δ 199.36, 179.76, 162.36, 149.29, 144.02, 140.55, 138.09, 135.09, 130.64, 130.61, 130.57, 130.53, 129.22, 128.97, 128.71, 128.45, 127.87, 127.00, 126.63, 126.60, 126.56, 126.53, 125.04, 124.82, 124.55, 122.65, 120.47, 118.67, 118.64, 118.61, 118.58, 118.55, 118.51, 118.48, 118.45, 39.56, 33.10, 25.10; ESI-HRMS (m/z), of C₁₉H₁₃F₃N₆O₂S, Calcd: 447.0845 [M+H]^+,Found: 447.0821 [M+H]⁺.

Characterization of N-(5-bromopyridin-3-yl)-9-(dimethylglycyl)-4-methyl-5-oxo-4,5-dihydro-[1,2,3]triazolo[1,5-a]quinazoline-3-carbothioamide (8f)

The title compound was produced from O-ethyl 9-(dimethylglycyl)-4-methyl-5-oxo-4,5-dihydro-[1,2,3]triazolo[1,5-a]quinazoline-3-carbothioate and 5-bromopyridin-3-amine

White colour powder, yield: 87%, mp: 231 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 2.303 (s, 6H), 3.591 (s, 3H), 3.813 (s, 2H), 7.689 (s, 1H), 7.964 (d, J = 1.474, 7.394 Hz, 1H), 8.021 (d, J = 7.470 Hz, 1H), 8.301 (s, 1H), 8.354 (s, 1H), 8.782 (s, 1H), 10.706 (s, 1H); ¹³C NMR (125 MHz, DMSO-d₆) δ 195.54, 179.56, 162.86, 147.02, 142.88, 140.55, 137.75, 135.81, 133.84, 132.09, 129.97, 129.13, 125.04, 120.92, 119.61, 119.12, 63.62, 45.02, 39.59, 33.10; ESI-HRMS (m/z), of C₁₉H₁₃F₃N₆O₂S, Calcd: 500.0498 [M+H]^+,Found: 500.0453 [M+H]⁺.

Characterization of 9-(dimethylglycyl)-4-methyl-N-(6-methylpyridin-3-yl)-5-oxo-4,5-dihydro-[1,2,3]triazolo[1,5-a]quinazoline-3-carbothioamide (8g)

The title compound was produced from O-ethyl 9-(dimethylglycyl)-4-methyl-5-oxo-4,5-dihydro-[1,2,3]triazolo[1,5-a]quinazoline-3-carbothioate and 6-methylpyridin-3-amine.

Pale brown color, yield: 88%, mp: 218 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 2.303 (s, 6H), 2.411 (s, 3H), 3.591 (s, 3H), 3.813 (s, 2H), 7.034 (d, J = 7.619 Hz, 1H), 7.937 – 8.047 (m, 3H), 8.301 (s, 1H), 8.435 (s, 1H), 10.743 (s, 1H); ¹³C NMR (125 MHz, DMSO-d₆) δ 195.54, 180.42, 162.86, 145.04, 143.05, 140.55, 137.75, 137.04, 134.14, 133.84, 132.09, 129.97, 125.61, 125.20, 119.61, 119.12, 63.62, 45.04, 45.02, 39.56, 33.10, 17.54; ESI-HRMS (m/z), of C₂₁H₂₁N₇O₂S, Calcd: 436.1550 [M+H]^+,Found: 436.1584 [M+H]⁺.

Characterization of 9-(dimethylglycyl)-4-methyl-5-oxo-N-(5-(trifluoromethyl)pyridin-3-yl)-4,5-dihydro-[1,2,3]triazolo[1,5-a]quinazoline-3-carbothioamide (8h)

The title compound was produced from O-ethyl 9-(dimethylglycyl)-4-methyl-5-oxo-4,5-dihydro-[1,2,3]triazolo[1,5-a]quinazoline-3-carbothioate and 5-(trifluoromethyl)pyridin-3-amine.

Cream color powder, yield: 80%, mp: 221 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 2.303 (s, 6H), 3.591 (s, 3H), 3.813 (s, 2H), 7.648 (s, 1H), 7.964 (d, J = 1.470, 7.401 Hz, 1H), 8.021 (d, J = 7.474 Hz, 1H), 8.301 (s, 1H), 8.432 (s, 1H), 8.660 (s, 1H), 10.747 (s, 1H); ¹³C NMR (125 MHz, DMSO-d₆) δ 195.54, 179.53, 162.86, 144.96, 142.19, 142.15, 142.10, 142.06, 140.55, 137.75, 135.65, 135.62, 135.60, 135.57, 133.84, 132.09, 129.97, 126.43, 126.16, 125.90, 125.65, 125.39, 125.04, 124.26, 122.38, 122.35, 122.32, 122.28, 122.09, 119.91, 119.61, 119.12, 63.62, 45.02, 39.53, 33.10; ESI-HRMS (m/z), of C₂₁H₁₈F₃N₇O₂S, Calcd: 490.1267 [M+H]^+,Found: 490.1226 [M+H]⁺.

Characterization of 9-acetyl-N-(5-chloropyridin-3-yl)-4-ethyl-5-oxo-4,5-dihydro-[1,2,3]triazolo[1,5-a]quinazoline-3-carbothioamide (8i)

The title compound was produced from O-ethyl 9-acetyl-4-ethyl-5-oxo-4,5-dihydro-[1,2,3]triazolo[1,5-a]quinazoline-3-carbothioate and 5-chloropyridin-3-amine.

White color powder, yield: 91%, mp: 213 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 1.342 (t, J = 8.013, 8.013 Hz, 3H), 2.303 (s, 6H), 3.813 (s, 2H), 4.312 (q, J = 7.963, 7.963, 7.982 Hz, 2H), 7.596 (s, 1H), 7.964 (d, J = 1.509, 7.419 Hz, 1H), 8.021 (d, J = 7.411 Hz, 1H), 8.292 (s, 1H), 8.381 (s, 1H), 8.708 (s, 1H), 10.950 (s, 1H); ¹³C NMR (125 MHz, DMSO-d₆) δ 195.54, 179.61, 162.50, 142.29, 140.58, 139.02, 138.12, 136.71, 133.94, 131.95, 130.95, 130.17, 126.72, 125.61, 119.62, 119.35, 63.62, 45.04, 45.02, 42.40, 39.58, 13.51; ESI-HRMS (m/z), of C₂₁H₂₀ClN₇O₂S, Calcd: 470.1160 [M+H]^+,Found: 470.1125 [M+H]⁺.

Characterization of 9-(dimethylglycyl)-4-ethyl-5-oxo-N-(5-(trifluoromethyl)pyridin-3-yl)-4,5-dihydro-[1,2,3]triazolo[1,5-a]quinazoline-3-carbothioamide (8j)

The title compound was produced from O-ethyl 9-(dimethylglycyl)-4-ethyl-5-oxo-4,5-dihydro-[1,2,3]triazolo[1,5-a]quinazoline-3-carbothioate and 5-(trifluoromethyl)pyridin-3-amine.

Brown flakes, yield: 85 %, mp: 256 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 1.342 (t, J = 8.013, 8.013 Hz, 3H), 2.657 (s, 3H), 4.312 (q, J = 7.990, 7.990, 8.039 Hz, 2H), 7.648 (s, 1H), 8.036 (d, J = 2.688 Hz, 2H), 8.369 (s, 1H), 8.432 (s, 1H), 8.660 (s, 1H), 10.747 (s, 1H); ¹³C NMR (125 MHz, DMSO-d₆) δ 196.37, 179.63, 162.52, 144.96, 142.19, 142.15, 142.10, 142.06, 140.58, 138.08, 135.65, 135.62, 135.60, 135.57, 133.98, 133.47, 130.19, 126.43, 126.16, 125.90, 125.65, 125.61, 125.39, 124.26, 122.38, 122.35, 122.32, 122.28, 122.09, 119.91, 119.31, 118.85, 42.40, 39.53, 26.37, 13.51; ESI-HRMS (m/z), of C₂₀H₁₅F₃N₆O₂S, Calcd: 461.1002 [M+H]^+,Found: 461.1087 [M+H]⁺.

Characterization of 9-(dimethylglycyl)-4-methyl-N-(5-nitropyridin-3-yl)-5-oxo-4,5-dihydro-[1,2,3]triazolo[1,5-a]quinazoline-3-carbothioamide (8k)

The title compound was produced from O-ethyl 9-acetyl-4-ethyl-5-oxo-4,5-dihydro-[1,2,3]triazolo [1,5-a]quinazoline-3-carbothioate and 5-nitropyridin-3-amine.

White color powder, yield: 89 %, mp: 222 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 2.303 (s, 6H), 3.591 (s, 3H), 3.813 (s, 2H), 7.964 (d, J = 1.475, 7.410 Hz, 1H), 8.021 (d, J = 7.471 Hz, 1H), 8.301 (s, 1H), 8.415 (s, 1H), 8.907 – 8.953 (m, 2H), 11.245 (s, 1H); ¹³C NMR (125 MHz, DMSO-d₆) δ 195.54, 179.49, 162.86, 149.45, 146.60, 140.68, 140.55, 137.75, 135.76, 133.84, 132.09, 129.97, 125.07, 120.84, 119.61, 119.12, 63.62, 45.02, 39.59, 33.10; ESI-HRMS (m/z), of C₂₀H₁₈N₈O₄S, Calcd: 467.1244 [M+H]^+,Found: 467.1209 [M+H]⁺.

Anticancer Activity

MTT Assay

The MCF-7 cells were cultivated in a 96-well tissue culture plate with a transparent bottom, with each well containing 100 L of cells. Following a 24-hour period of seeding, triplicate test samples (8a-8k) were introduced to the cells at concentrations ranging from 5 to 500 µM (5, 10, 25, 50, 100, 250, 500). The cells were then cultured for an additional 24 hours to conclude the treatment period. The cultivation of all samples took place in a collective volume of 20 L of culture media. We disposed of the culture medium and rinsed the cells twice in PBS (Phosphate buffered saline). The MTT reagent was diluted to a final concentration of 0.5 mg/mL in PBS medium and added at a volume of 15 µL per well. The reagent volume will need to be adjusted based on the quantity of cell culture. Cells were incubated at a temperature of 37°C for a duration of 3 hours, during which they developed purple formazan crystals within their own cellular structures. These crystals were subsequently examined using a microscope. Following the removal of any remaining MTT reagent by washing with PBS, 100 L of DMSO was added to each well and gently agitated on an orbital shaker for 1 hour at room temperature. The quantity of DMSO utilized will differ based on the overall volume of the cell culture. We utilized an absorbance plate reader to evaluate the concentration of each sample at a wavelength of 570 nanometers (nm) [19].

Invitro p38 MAP kinase Activity:

This study employed a nonradioactive immunosorbent test for p38 kinase activity, which is a useful tool for systematically screening small-molecule p38 kinase inhibitors. Phosphorylation was carried out using an ATF-2 substrate, which exhibited linearity between 5 and 30 ng/well. This investigation showed that the ideal concentration and incubation time were 15 ng/well for 1.5 hours. ATF-2, the p38 kinase substrate (10 μg/mL in TBS), was applied to microtiter plates and incubated for 1.5 hours at 37°C. After three washes with distilled water, the remaining open binding sites were blocked with blocking buffer (BB; 0.05% Tween 20, 0.25% BSA, 0.02% NaN₃ in TBS) for 30 minutes at room temperature. Following another wash, the plates were incubated for one hour at 37°C. The kinase buffer (50 mM Tris, pH 7.5, 10 mM MgCl₂, 10 mM β-glycerophosphate, 100 μg/mL BSA, 1 mM dithiothreitol, 0.1 mM Na₃VO₄, and 100 μg/mL rATP) was diluted with or without test substance (ranging from 0.01 to 1.0 μM) for test solutions like 8a-8k that contained 15 ng/well p38 MAP kinase. Plates were blocked with BB for 15 minutes and then cleaned four times after that. 50 μL of the particular anti-bis-(Thr69/71)-phospho-ATF-2 (AB, 1:500 in BB) was added to each well. The wells were then washed and then incubated for another hour at 37°C with 50 μL of the secondary antibody [AB (alkaline phosphatase-conjugated), 1:1400 in BB]. After a final washing step, 100 μL of 4-NPP was pipetted into each well, and 1.5−2 hours later, color development was assessed using an enzyme-linked immunosorbent assay reader (Tecan, Sunrise, USA) at 405 nm. Using the metallin software 4.21, percent enzyme activity and IC₅₀ were computed based on the kinase assay values [20].

In-silico Studies

ADME Studies

Using Swiss ADME software, the ADME properties of substances were evaluated. Lipinski's rule of five states that substances with a molecular weight less than 500 have good oral bioavailability. All compounds observed the rule. The Swiss ADME program was also used to conduct the gastrointestinal safety profile. The desired compounds' characteristics were generated after uploading the SMILES list to these web services 2 [21]

Docking Studies

Molecular docking studies were carried out in AutoDock 4.2. The docking study was conducted to find out how well the 4-Alkyl-5-oxo-N-(pyridin-3-yl)-4,5-dihydro[1,2,3]triazolo[1,5-a]quinazoline-3-carbothioamide derivatives (8a to 8k) binds to the p38α MAP kinase.

The PDB format of the p38α MAP kinase protein 3D crystal structure (PDB: 1W7H) was obtained, and prior to docking analysis, the 3D protein structure underwent refinement and energy minimization. To improve the protein, missing atoms, polar hydrogens, and Kollman charges were added; on the other hand, foreign ligands, crystallographic water molecules, and superfluous ions were removed. Both a hard protein and a flexible ligand were used in the docking process. Using ACD Lab Chemsketch, the suggested ligands' 3D structure was created and stored in mol 2 molecular format. Using MGL tools 1.5.7, these mol 2 structures were transformed into pdbqt format. Docking studies were conducted using AutoDock 4.2 and the Lamarckian genetic method. Flexible docking was employed for a p38α MAP kinase protein and a flexible ligand. We generated a grid with 60 points in x, y, and z directions. The energy map was calculated using Autogrid Grid 4 with a 0.375 Å grid spacing and a distance-dependent dielectric constant function. The default settings were applied to all other parameters. After docking, the ligand with the top binding free energy was identified. Each molecule was docked using AutoDock 4.2, with parameters ga_num_evals and ga_run set to 25 000 000 and 50, respectively, as recommended. DS 4.0 visualizer provides molecular interaction graphs. All calculations were done on Linux-based PCs [22].

Conflict of Interest

The authors declare no conflict of interest

Author Contribution

Design and synthesis was done by Keerthi, in-vitro P38 kinase , MTT assay and Manuscript writing by Dr. Divya Pingili and Dr. Archana Awasthi, Molecular docking by Prasanth, Ramesh and Kantlam supervision of the work

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No competing interests reported.

SupplementarydataKeerthi.docx

Synthesis and Biological Assessment of Triazolo-Quinazoline Carbothioamide Derivatives for p38 MAP Kinase Inhibition: In-Silico and In-Vitro Approaches

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Abstract

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Introduction

Results and Discussion

Conclusion

Experimental Section

Declarations

Conflict of Interest

Author Contribution

References

Additional Declarations

Supplementary Files

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