Synthesis, Hemolytic Studies, and In Silico Modeling of Novel Acefylline-1,2,4-Triazole Hybrids as Potential Anti-cancer Agents against MCF-7 and A549.

A series of novel theophylline-7-acetic acid (acefylline)-derived 1,2,4-triazole hybrids with N-phenyl acetamide moieties (11a-j) have been synthesized and tested for their inhibitory (in vitro) potential against two cancer cell lines, A549 (lung) and MCF-7 (breast), using MTT assay. Among these derivatives, 11a, 11c, 11d, 11g, and 11h displayed remarkable activity against both cancer cell lines having cell viability values in the 21.74 ± 1.60-55.37 ± 4.60% range compared to acefylline (86.32 ± 1.75%) using 100 μg/μL concentration of compounds. These compounds were further screened against the A549 cancer cell line (lung) to find their half-maximal inhibitory concentration (IC50) by applying various concentrations of these compounds. Compound 11g (2-(5-((1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)methyl)-4-phenyl-4H-1,2,4-triazol-3-ylthio)-N-p-tolylacetamide) with the least IC50 value (1.25 ± 1.36 μM) was discerned as a strong inhibitor of cancer cell multiplication in both cell lines (A549 and MCF-7). Their hemolytic studies revealed that all of them had very low cytotoxicity. Finally, in silico modeling was carried out to find the mode of binding of the highly active compound (11g), which was according to the results of anti-cancer activity.

Introduction

n class="Disease">Cancer is the second most fatal disease in the world after cardiovascular diseases.[1] Every year, about 7.6 million people die of cancer globally, and this number is expected to reach 13 million by 2030.[2] According to an estimate published by the WHO, the number of new cases is expected to rise by about 70%, that is, from 14 million to 22 million over the next 2 decades. Cancer was responsible for 10 million deaths in 2020. Globally, approximately, 19 million new cases were registered in 2020, and nearly, one out of six deaths is due to cancer and this is projected to increase by 45% (during 2007–2030), killing more people than HIV/AIDS, malaria, and tuberculosis combined. The major organs affected by cancer in men and women include the prostate, breast, lung and bronchus, thyroid, uterine, carpus, colon, and rectum.[3] It is important to note that female breast cancer diagnosis has exceeded with an estimated 2.3 million new cases, that is, 11.7%. It was found to be 11.4% for lung followed by colorectal (10.0%), prostate (7.3%), and stomach (5.6%) cancers. Cancer is currently being treated with chemotherapy, surgery, and radiotherapy. The cancerous cell curability via chemotherapy is attributed only to 11%, while surgery accounts for 49% and radiotherapy accounts for 40%. Various chemotherapeutic medicines are in market at very high price and have adverse side effects and low efficacy. Thus, it is one of the leading interests in drug development and discovery to look for novel anti-cancer drugs.[4,5]

In this regard, n class="Chemical">1,2,4-triazole-derived heterocycles have gained significant attention in the last few years owing to their chemotherapeutic values.[6,7] The literature reveals that 1,2,4-triazole derivatives hold numerous therapeutic features such as analgesic,[8] anti-microbial,[9,10] local anesthetic, anti-inflammatory,[11] anti-malarial,[12] anti-convulsant,[13] anti-viral,[14] anti-neoplastic,[15] and anti-cancer activities.[16−18] It is important to note that 1,2,4-triazole-containing anti-cancer drugs such as letrazole and anastrozole (Figure ) are already in use for the treatment of breast cancer.[19]

Figure 1

Bioactive 1,2,4-triazole anti-cancer drugs letrazole (1A) and anastrozole (1B).

Bioactive n class="Chemical">1,2,4-triazole anti-cancer drugs letrazole (1A) and anastrozole (1B).

Similarly, medicinal plants are the basis for exploring different marketing drugs. One of such prominent moleculn class="Chemical">es is xanthine. Different forms of xanthines (theophylline, theobromine, doxophylline, and caffeine,Figure )[20] are known for their wide applications in pharmaceutical industry as anti-microbial,[21] anti-inflammatory,[22] anti-oxidant, cyclic nucleotide phosphodiesterase inhibition, adenosine receptor antagonist,[23] and anti-tumor activities.[24]

Figure 2

Structures of xanthine (2A) and various xanthine derivatives such as theobromine (2B), theophylline (2C), and doxophylline (2D).

Structurn class="Chemical">es of xanthine (2A) and various xanthine derivatives such as theobromine (2B), theophylline (2C), and doxophylline (2D).

n class="Chemical">Theophylline has gained considerable interest among the widely distributed methylxanthines, as it has been the commonly used dimethylxanthine for treating respiratory diseases such as asthma for over 80 years.[25] Theophylline comprises an important scaffold for modifications in the structure to develop CNS stimulant,[26] analgesic and anti-inflammatory,[27,28] anti-bacterial,[29] hypotensive,[30] hypoglycemic,[31] anti-HIV,[32] and anti-cancer[33] derivatives. Theophylline is an appropriate drug used in cardiology for the treatment of bradyarrhythmias and disorder of atrioventricular conduction.[34] Theophylline-7-acetic acid (acefylline, Figure ), a pharmacologically active derivative of theophylline, is widely used as a bronchodilator, cardiac stimulant, diuretic, and smooth muscle-relaxing agent.[35] Its amide and methyl ester derivatives are active against myeloid leukemia cells,[36] mycobacterium tuberculosis,[37] and cancer cell lines.[38]

Figure 3

Structure of acefylline.

Structure of pan class="Chemical">acefylline.

Our rn class="Chemical">esearch group has previously reported some 1,3,4-oxadiazoles derived from acefylline as anti-cancer agents with least toxicity.[39] Considering the chemotherapeutic importance of acefylline and 1,2,4-triazole derivatives and part of our ongoing studies toward novel biologically relevant molecules,[40,41] it has been decided to synthesize 1,2,4 triazole hybrids of acefylline and investigate their cytotoxicity.

Results and Discussion

Chemistry

n class="Chemical">Acefylline derivatives were synthesized in various steps, as presented in Scheme . Acefylline 3 was esterified with CH3OH using a catalytic amount of sulfuric acid to afford theophylline-7-acetate 4 in 72% yield, followed by the reaction with hydrazine monohydrate to get 7-acetohydrazide of theophylline 5 in 99% yield.[39] The synthesized theophylline-7-acetohydrazide 5 was further treated with phenyl isothiocyanate in ethanol to prepare thiosemicarbazide 6, which was hydrolyzed in basic media to obtain acefylline–triazole hybrid 7 in 70% yield.[42] Various aromatic amines 8a–j were treated with bromo acetyl bromide 9 to obtain 2-bromo-N-substitutedphenyl acetamides 10a–j(43) and were coupled with 7 in dichloromethane to obtain the target compounds in the presence of pyridine 11a–j in 65–82% yield (Scheme , Table ).

Scheme 1

Reaction Conditions and Pathway for the Synthesis of Target Compounds (11a–j)

Reagents and conditions of reactions: (a) methanol, H2SO4, reflux 6 h. (b) Hydrazine monohydrate, RT overnight. (c) Phenyl isothiocyanate, C2H5OH, RT 1 h, reflux 2 h (d) Aq KOH, heat 4 h (e) DCM, pyridine, RT 24–48 h.

Table 1

Anti-cancer and Hemolytic Potential of Thio N-(Substituted-phenyl)acetamide Derivatives of Theophylline-7-acetic Acid (Acefylline) 11a–j

Cell viability, IC50: (Mean ± SD) in triplicate.

Reagents and conditions of reactions: (a) n class="Chemical">methanol, H2SO4, reflux 6 h. (b) Hydrazine monohydrate, RT overnight. (c) Phenyl isothiocyanate, C2H5OH, RT 1 h, reflux 2 h (d) Aq KOH, heat 4 h (e) DCM, pyridine, RT 24–48 h.

Spectral Explanation of the Demonstrative Molecule (11c)

n class="Chemical">Acefylline-derived (compound) 11c was synthesized as a brown amorphous solid, and its structural confirmation was done by IR, 1H-, 13C NMR, and MS-EI spectroscopy (M+) at m/z: 530.1849. To describe various functional groups in Fourier transform infrared spectroscopy (FTIR), different absorption spectra were seen at ν: 3362 (N–H, str), 1643 (CO–amide, str), 1600–1650 (CO–xanthene, str), 1545 (C=N, str), Ph (1447), 1453 (C=C, str), 1473 (CH2, str), 1331 (C–N, str), 801 (C–H), and 650–710 (S–C) cm–1. In the 1H NMR spectrum, methylene and NH of the amide group (signals) are seen at δ 4.11 and δ 10.16, respectively. The most downfield signal was detected at δ 8.00 for N–H of the xanthene heterocyclic ring. 2H-4 of the CH2 group resonated in the upfield region at δ 5.58, whereas 3H-1 and 3H-2 of the purine ring resonated at δ 3.14 and δ 3.41 as a singlet. The presence of two methyl groups at the aromatic ring (CH3-2′ and CH3-4′) was confirmed by two signals at δ 2.15 and δ 2.17, respectively. H-5′ and H-6′ (aromatic protons) reverberated at δ 7.06 (J = 6.4 Hz) as a doublet and at δ 7.25 (J = 7.1 Hz), respectively, while H-3′ reverberated as a singlet at δ 7.31. At δ 7.48; two aromatic protons H-9′ and H-11′ resonated as a multiplet, while H-8′ and H-12′ appeared at δ 7.58 (J = 1.9 Hz) with H-7′ and H-11 as a doublet, respectively. H-10′ of the aromatic ring appeared as a triplet at δ 7.58 and showed ortho couplings with H-9′ and H-11′ (Figure A).

Figure 4

1HNMR (A) and 13C NMR (B) of the compound 11c.

pan class="Chemical">1HNMR (A) and pan class="Chemical">13C NMR (B) of the compound pan class="Chemical">11c.

The n class="Chemical">carbon framework of 11c was also confirmed by 13C NMR. In the spectrum, all of the 26 carbons showed their signals; two resonance signals at δ 37.29 for the methylene group and at δ 165.43 for the carbonyl group confirmed the N-substituted acetamide group. The other two signals belonged to 2 C=O of the purine ring at δ 151.77 and 154.63. Formation of the 1,2,4 triazole ring was confirmed by the downfield signals depicted by quaternary carbons at δ 151.36 and δ 151.70. One signal of methine at δ 143.51 and the two signals of C=C at δ 106.46 and 148.40 exhibited the presence of theophylline ring in the molecule. The signal of the methylene linker between the theophylline and 1,2,4 triazole core appeared at δ 41.33. The 2,4-dimethylphenyl ring attached with the acetamide group showed three methine signals at δ 130.76, δ 120.78, and δ 117.12, while the other three signals of substituted phenyl carbons were seen at δ 136.81 and δ 132.40 for C–CH3 and δ 131.73 for C–N. Two methyl substituents at the phenyl ring were seen in the upfield region of the spectrum at δ 2.15 and δ 2.17. The phenyl ring joined with 1,2,4-triazole was confirmed by one downfield signal of C–N at δ 136.93 and two signals for C-8′ and C-12′ resonated at δ 130.45 and δ 130.06, respectively, while the remaining three carbons of the phenyl ring depicted their signals at δ 127.46 (Figure B). By a similar approach, other synthetic derivatives of the series (11a–j) were also structurally characterized.

Anti-cancer Activity

The cytotoxic prospective of all the target compounds n class="Chemical">11a–j was reviewed against two cancer cell lines, MCF-7 (human breast) and A549 (lung), and found to have lower cell viability values (100 μg/μL) as compared to the reference drug acefylline (86.32 ± 11.75%) using 100 μg/μL concentration of the compound (Table ). In general, compounds 11a, 11c, 11d, 11g, and 11h showed greater activity with both tested cancer cell lines. Compounds 11c (cell viability = 38.74 ± 2.07, 26.14 ± 1.86%) and 11g (cell viability = 31.76 ± 3.16, 21.74 ± 1.60%) for MCF-7 (breast) and A549 (lung) were preferably established more effectively against the lung cancer cell line (A549). The half-maximal inhibitory concentration (IC50) for these compounds was also calculated against the lung cancer cell line A549 applying different concentrations of compounds. All the compounds showed good inhibition potential. 11g with the IC50 value 1.25 ± 1.36 μM was considered a potent anti-cancer derivative among all. Compounds 11e and 11i also exhibited moderate cytotoxic activity with cell viability (54.82 ± 4.88, 52.07 ± 3.66 and 50.82 ± 2.78, 52.477 ± 2.59%), but the compounds 11b, 11f, and 11j with relatively high values of cell viability were considered least active against cancer.

Hemolytic Activity

The n class="Chemical">acefylline-derived analogues (11a–j) were also verified for hemolytic assay. The %age of hemolysis was deliberated, and the data are displayed in Table . Synthesized derivatives revealed low toxicity with RBCs. Least toxicity was detected for molecule 11g (0.39%), which showed minimum binding with the RBC cell membrane as compared to standard ABTS (95.9% hemolysis). The most toxic compound was found to be derivative 11b with hemolysis (15%), whereas all other derivatives, 11a (11.7), 11f (8.6%), 11d (5.5%), 11j (4.6%), 11h (5.9%), and 11i (8.9%), exhibited moderate to low hemolytic activity.

Structure–activity Relationship

n class="Species">SAR (structure–activity relationship) was investigated depending on substituents on the phenyl ring of N-(substituted-phenyl)acetamide to obtain all the comprehensive facts about anti-cancer activities of synthesized molecules. An understanding about the structures and activities of the compounds under examination suggests that incorporating substituents with the electron-donating effect usually increased the anti-cancer activity, for example, compound 11a possessing the unsubstituted phenyl ring of acetanilides exhibited greater activity (34.73 ± 2.49, 59.59 ± 1.36%) against MCF-7 and A549 cancer cell lines as compared to the reference drug acefylline (86.32 ± 11.75%). Substituted phenyl rings with electron-donating groups at different positions exhibited remarkable results. Compound 11g bearing the methyl group on the phenyl ring at the para position showed excellent anti-cancer activity (cell viability = 31.76 ± 3.16, 21.74 ± 1.60%) among all the synthetic derivatives. The activity was slightly decreased in dimethyl-substituted derivatives, such as that compound 11h (cell viability = 33.20 ± 2.77, 55.37 ± 4.60%) was the second most active derivative of the series with two methyl groups adjacently attached on the phenyl ring at meta and para positions. However, the adjustment in the position of methyl groups at ortho and para resulted in a decrease in the activity of compound 11c (cell viability = 38.74 ± 2.07, 26.14 ± 1.86%). This suggests that the presence of the electron-donating substituent in the phenyl ring at para and meta positions compared to that in ortho may be more interactive with the cancer cells and is the reason of its greater potential (Figure ).

Figure 5

SAR of 11a, 11c, 11g, and 11h.

n class="Species">SAR of 11a, 11c, 11g, and 11h.

The activity of compound n class="Chemical">11d (cell viability = 38.14 ± 4.04, 46.21 ± 1.42%) bearing two chloro groups at meta and para positions showed considerable activity, while the activity was decreased when the position of chloro groups was changed to ortho and para in structurally similar hybrids such as 11i (50.82 ± 2.78 ± 52.47 ± 2.59%) and 11b (cell viability = 69.40 ± 7.05, 52.60 ± 5.07%). Compound 11f (cell viability = 72.78 ± 6.35, 55.43 ± 4.11%) having the mono-substituted ortho chloro phenyl ring showed less inhibitory potential toward the MCF-7 (human breast) cancer cell line and found to be least active among all the derivatives (Figure ).

Figure 6

SAR of 11b, 11d, 11f, and 11i.

n class="Species">SAR of 11b, 11d, 11f, and 11i.

Compound n class="Chemical">11e (cell viability = 54.82 ± 4.88, 52.07 ± 4.88%) with the fluoro group at the para position of the phenyl group also showed moderate activity, while the activity of compound n>n class="Chemical">11i (cell viability = 63.72 ± 3.54, 67.54 ± 4.27%) bearing the fluoro substituent at the ortho position was decreased (Figure ). It is obvious from the results that the presence of an electron-donating substituent in the phenyl ring at para and meta positions as compared to that in ortho increased the activity of compounds, while the activity was decreased when an electron-withdrawing substituent is present at the ortho position.

Figure 7

SAR of compounds 11e and 11i.

pan class="Species">SAR of compounds pan class="Chemical">11e and pan class="Chemical">11i.

The same trend was observed in hemolytic activity; compounds having a electron-donating substituent on the phenyl ring—11g, n class="Chemical">11c, and 11h (0.39, 2.7, and 3.9%, respectively)—are less toxic, while compounds bearing an electron-withdrawing substituent on the phenyl ring—11b, 11d, 11f, and 11i (15, 5.5, 8.6, and 7.2%, respectively) and unsubstituted phenyl ring, 11a (17%), with greater hemolytic activity are more toxic.

Computational Modeling Studies of the Most Active Compound (11g)

Results

The compounds were computationally modeled to explore their mechanism of action in n class="Disease">cancer. The PASS tool predicted the STAT3 as a potential anti-cancer target with Pa ∼ 0.614. STAT3 is a vital transcript factor that regulates the cell propagation, differentiation, and survival. It has been reported that theophylline modulates the STAT3 signaling, and STAT3 dimerization inhibition is a potential therapeutic modality to control the progression, development, and conservation of malignancies.[44−47] Herein, the inhibitory potential of compound 11g was studied by induced fit docking. The 11g docked at the STAT3 hotspot with a superior conformational energy of −6.2789 Kcal/mol. It was found to significantly exceed the standard’s threshold of conformational energy (i.e., −4.6825 Kcal/mol), which suggested the improved STAT3 inhibitory potential of 11g as compared to acefylline (Table ).

Table 2

Parameters for Induced-Fit Docking of Compounds at the STAT3 Hotspot

compounds	binding score (ΔG)Kcal/mol	binding Residues	interactions type
11g	–6.2789	TRP358. ALA356, ILE354, LEU355, LYS351, LYS352	H-bonding, π–π stacking, π–σ, alkyl,π-alkyl
acefylline	–4.6825	TYR353, LYS351, THR151, ILE354, LEU355, LYS352, VAL350	H-bonding, π–σ, alkyl, π-alkyl

The binding pocket consists of n class="Chemical">THR150, THR151, CYC152, LYS352, TYR353, ILE354, LEU355, and ALA356 at the hotspot of STAT3. Acefylline was found to orient itself at the STAT3 hotspot and stabilized its conformation by contacts with vital residues to disrupt the STAT3 interactions (Figure ),[39] whereas conformational analysis of 11g revealed that it preferably binds with STAT3 to block its complexation at the hotspot. Interestingly, conformation of 11g was able to interact with all STAT3 residues at the binding pocket, which may disrupt the formation of the hotspot during STAT3 complexation.

Figure 8

Conformational analysis of docked compounds at the STAT3-binding pocket. Spatial configuration of the simulated greatest binding approach of A (acefylline) and B (11g) at the 3D space of the interacted place for STAT3.

Conformational analysis of docked compounds at the n class="Gene">STAT3-binding pocket. Spatial configuration of the simulated greatn>n class="Chemical">est binding approach of A (acefylline) and B (11g) at the 3D space of the interacted place for STAT3.

n class="Chemical">Acefylline was found to stabilize its complexation by H-bonding with LYS351, TYR353, ILE354, and LEU355. It also established the alkyl and pi-alkyl interactions with ILE354, VAL350, and LYS352. Moreover, it also interacted with THR151 by a pi-sigma bond at the STAT3-binding site (Figure ). On the other hand, 11g efficiently interacted with conserved residues of acefylline but with higher binding affinity. The compound 11g complexed and inhibited the STAT3 hotspot by strong hydrogen bonds with LEU355 and LYS351. It also formed the Pi-alkyl bonds with LYS352, pi-alkyl, and pi–pi stacked bonds with TRP358 and an alkyl bond with ALA356. Additionally, compound 11g further supported this complexation by pi-sigma bonds with ALA356 and ILE354. It is noteworthy that 11g established the diverse interactions with STAT3 residues and disrupted its complexation at the hotspot, thus corroborating its superior binding energy and inhibitory potential.

Figure 9

Docked compounds interacting with the residues of the STAT3-binding pocket.

Docked compounds interacting with the rpan class="Chemical">esidupan class="Chemical">es of the pan class="Gene">STAT3-binding pocket.

The two-din class="Species">mensional (2D) illustration of acefylline (A) and derivative 11g (B) interacting with STAT3-binding pocket residues represented as colored balls by kind of collaboration is provided.

Discussion

The compound 11g was computationally modeled to invn class="Chemical">estigate its superior anti-cancer potential as compared to acefylline. Herein, the PASS predication highlighted the STAT3 as a potential anti-cancer target for 11g. The superior binding affinity of the compound under examination was investigated by the function of induced fit docking with acefylline as a standard. It is a reliable methodology that accounts for the stretchy receptor’ binding pocket to simulate and predicts the binding mode and complexation of the ligand. Interestingly, compound 11g conformed with higher binding affinity, which may justify its improved anti-cancer potential as compared to acefylline. Moreover, compound 11g efficiently oriented itself toward the STAT3 hotspot and inhibited the STAT3 residues with more diverse interactions, which may completely disrupt the STAT3 potential of complexation. Therefore, these insights may further support the compound 11g as a novel acefylline-based lead candidate in cancer therapeutics.

Conclusions

The targeted compounds n class="Chemical">thio N- phenyl/arylacetamide derivativclass="Chemical">n>n class="Chemical">es of acefylline 11a–j were synthesized in good yield. The anti-cancer activity of all derivatives was screened against cell lines of cancer, MCF-7 (breast) and A549 (lung), and it was revealed that most compounds exhibited better anti-proliferative activity. Among these compounds, 11g with the least IC50 value (1.25 ± 1.60 μM) was recognized to be the most potent agent against both cancer cell lines. Almost all molecules showed low cytotoxicity against human RBCs in the hemolysis assay. The mode of action in the inhibition of cancer cells of the compound 11g was also examined by docking studies, and the results of comprehensive docking analysis of compound 11g are consistent with biological diagnostic findings. Overall, current studies suggest that acefylline-linked triazole hybrids are capable of being established as lead compounds; more modifications on triazole derivatives of acefylline may give rise to advanced anti-cancer agents in cancer therapy.

Materials and Methods

Starting materials, chemicals, and solvents of analytical grade were obtained from local traders from Alfa Aen class="Species">sar, Merck, and Sigma-Aldrich (Germany) and were used without distillation. Thin-layer chromatography (TLC) was performed to monitor the reaction using silica gel plates coated with 60 F254 in a mixture of methanol and dichloromethane. UV light was used to detect the spots on TLC plates. Melting points (mp) were noted using Gallenkamp equipment. FTIR spectra were documented on a Bruker FTIR spectrometer in KBr pellets. The spectra of 1H NMR and 13C NMR at 400 MHz and 100 MHz (δ = ppm), respectively, in DMSO-d6 were documented on a Bruker model AV-400 spectrophotometer.

Synthesis of 7-((5-mercapto-4-phenyl-4H-1,2,4-triazol-3yl)methyl)-1,3-dimethyl-1H-purine-2,6(3H,7H)-dione (7)

To the mixture of n class="Chemical">theophylline acetohydrazide 5 (300 mg, 1.2 mmol) in ethanol (20 mL) was added phenyl isothiocyanate (162 mg, 1.2 mmol) and placed for 1 h; later, the brew was refluxed for 2 h. Precipitates of thiosemicarbazide (intermediate) 6 were formed, which were filtered off and dried. The intermediate was then dissolved in a solution of KOH (0.1 mg, 1.8 mmol) in H2O (5 mL). The mixture was then heated for 4 h in a water bath. It was then cooled and acidified with dilute HCl; precipitates were filtered out and purified by a recrystallization process as a white solid. Yield: 70%; mp 287 °C; IR ν: 3290 (H–Ar), 2500–2600, (S–H), 1605–1652 (2C=O), 1550 (C=C), 1550 (N=C), 1472 (CH2), 600–700 (S–C). 1H NMR, DMSO-d6, 400 MHz (δ/ppm): 1.6 (s, S–H), 3.22, 3.30 (s, 6H, 2N–CH3), 5.79 (s, 2H, N–CH2), 8.20 (s, 1H, CH=N). 13C NMR, DMSO-d6, 100 MHz (δ/ppm): 28 (CH3), 29.6 (CH3), 43, (NCH2), 107, 123, 132, 143, 147, (Ar–C), 153.2, 154.9 (CO–xanthine).

General Synthetic Procedure for the Compounds (11a–j)

To the mixture of n class="Chemical">1,2,4-triazoles analogue 7 (200 mg, 0.54 mmol) and dichloromethane and pyridine (1.89 mmol) were added 2-bromo-N-phenylacetamides 10a–j (2.4 mmol), and the resulting mixture was stirred at room temperature for 24–48 h. Reaction was monitored with the help of TLC. Upon reaction completion, n-hexane was added, and precipitates of N-substituted phenylacetamide analogues of 1,2,4-triazole–acefylline hybrid 11a–j were obtained, which were recrystallized with ethanol.

2-(5-((1,3-Dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)methyl)-4-phenyl-4H-1,2,4-triazol-3-ylthio)-N-phenylacetamide (11a)

Light-yellow powder; yield: 75%; mp 210 °C; IR (KBr) ν: 1600–1650 (2C=O), 3351 (N–H), 1643 (CONH), 1545 (N=C), 1473 (n class="Chemical">CH2), Ph (1476), 1453 (C=C), 1331 (N–C), 600–700 (S–C), 801 (Ar–H). 1H NMR, DMSO-d6, 400 MHz (δ/ppm): 3.15, 3.41 (s, 6H, N–CH3), 4.15 (s, 2H, S-methylene), 5.58 (s, 2H, N-methylene), 7.06 (t, J4′,5/4′,3′ = 5.6 Hz, 1H, H-4′), 7.30 (t, J3′,2 = J5′,6″ = 6 Hz, 2H, H-3′, 5′), 7.48 (d, J8′,9′ = J11′,12′ = 5.8 Hz, 2H, H-8′, 12′), 7.55 (d, J2′,3′ = J5′,6′ = 6.2 Hz, 2H, H-2′, 6′), 7.58 (m, 3H, H-9′, 10′, 11′), 8.0 (s, 1H, HC=N), 10.32 (s, 1H, CO–amide).13C NMR, DMSO-d6, 100 MHz (δ/ppm): 27.93 (methyl), 29.90 (methyl), 37.41 (S-methylene), 41.36, (N-methylene), 106.50, 119.56, 121.09, 123.99, 127.46–132.42 (11C), 139.20, 143.52 (2C), 148.41, 151.37, 151.64 (Ar–C), 151.80, 154.63 (2CO–xanthene), 165.78 (CO–amide). ES+ MS (m/z %): 502.1536 (M+). Anal. Calcd for C24H22N8O3S: C, 56.95; H, 4.48; N, 21.96. Found: C, 57; H, 4.41; N, 22.30.

N-(2,4-Dichlorophenyl)-2-(5-((1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)methyl)-4-phenyl-4H-1,2,4-triazol-3-ylthio)acetamide (11b)

n class="Chemical">Pale-yellow solid; yield: 72%; mp 223 °C; IR (KBr) ν: 1600–1650 (2C=O), 3351 (N–H), 1643 (CONH), 1545 (N=C), 1453 (C=C), 1331 (N–C), 600–700 (S–C), 801 (Ar–H), 1473 (CH2), Ph (1456). 1H NMR, DMSO-d6, 400 MHz (δ/ppm): 3.15, 3.41 (s, 6H, N–CH3), 4.14 (s, 2H, S-methylene), 5.57 (s, 2H, N-methylene), 7.49–7.59 (m, 7H, Ar–H), 7.93 (s, 1H, H-3), 8.0 (s, 1H, HC=N), 10.64 (s, 1H, CO–amide). 13C NMR, DMSO-d6, 100 MHz (δ/ppm): 27.93 (methyl), 29.90 (methyl), 37.19 (S-methylene), 41.31, (N-methylene), 106.50, 119.61, 120.67, 127.41, 130.47–132.35 (7C), 139.27, 143.45 (2C), 148.39, 149.29, 151.43 (Ar–C), 151.85, 154.63 (2CO–xanthene), 166.39 (CO–amide). ES+ MS (m/z %): 570.0756 (M+). Anal. Calcd for C24H20Cl2N8O3S: C, 50.35; H, 3.60; N, 19.53. Found: C, 50.44; H, 4; N, 19.61.

2-(5-((1,3-Dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)methyl)-4-phenyl-4H-1,2,4-triazol-3-ylthio)-N-(2,4-dimethylphenyl)acetamide (11c)

Brown solid; yield: 69%; mp 112 °C; IR (KBr) ν: 1600–1650 (2C=O), 3351 (N–H), 1643 (CONH), 1545 (N=C), 1473 (n class="Chemical">CH2), Ph (1476), 1453 (C=C), 1331 (N–C), 600–700 (S–C), 801 (Ar–H). 1H NMR, DMSO-d6, 400 MHz, (δ/ppm): 2.15, 2.17 (6H, Ar–CH3), 3.14, 3.41 (s, 6H, N–CH3), 4.11 (s, 2H, S-methylene), 5.58 (s, 2H, N-methylene), 7.06 (d, J5′,6′ = 6.4 Hz, 1H, H-5′), 7.25 (d, J6′,5′ = 6.4 Hz, 1H, H-6′), 7.31 (s, 1H, H-3′), 7.48 (m, 2H, H-9′, 11′), 7.58 (t, J10′,9′′/J10′,11′′ = 2 Hz, 1H, H-10′), 7.58 (d, J8′,7′′ = J11′,12′′ = 2 Hz, 2H, H-8′, 12′), 8.0 (s, 1H, HC=N), 10.16 (s, 1H, CO–amide). 13C NMR, DMSO-d6, 100 MHz, (δ/ppm): 19.23 (methyl), 20.06 (methyl), 27.93 (methyl), 29.87 (methyl), 37.29 (S-methylene), 41.33, (N-methylene), 106.46, 117.12, 120.78, 127.46 (3C), 130.06, 130.45, 130.76, 131.73, 132.40, 136.81, 136.93, 143.51, 148.40, 151.36, 151.70 (Ar–C), 151.77, 154.63 (2CO–xanthene), 165.43 (CO–amide). ES+ MS (m/z %): 530.1849 (M+). Anal. Calcd for C26H26N8O3S: C, 50.35; H, 3.60; N, 19.53. Found; C, 58.85; H, 4.94; N, 21.12.

N-(3,4-Dichlorophenyl)-2-(5-((1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)methyl)-4-phenyl-4H-1,2,4-triazol-3-ylthio)acetamide (11d)

n class="Chemical">Pale-yellow solid; yield: 71%; mp 222 °C; IR (KBr) ν: 1600–1650 (2C=O), 3351 (N–H), 1643 (CONH), 1545 (N=C), 1473 (CH2), Ph (1476), 1453 (C=C), 1331 (N–C), 600–700 (S–C), 801 (Ar–H). 1H NMR, DMSO-d6, 400 MHz, (δ/ppm): 3.15, 3.41 (s, 6H, N–CH3), 4.14 (s, 2H, S-methylene), 5.57 (s, 2H, N-methylene), 7.44 (t, J9′,10/10′,11′ = 2 Hz, 1H, H-10′), 7.48 (d, J8′,9 = J12′,11′ = 5.6 Hz, 2H, H-8′, 12′), 7.58 (m, 4H, H-5′, 6′, 9′, 11′), 8.0 (s, 1H, HC=N), 10.64 (s, 1H, CO–amide). 13C NMR, DMSO-d6, 100 MHz, (δ/ppm): 27.93 (methyl), 29.90 (methyl), 37.23 (S-methylene), 41.37, (N-methylene), 106.50, 119.64, 120.77, 127.44, 130.47–132.38 (7C), 139.27, 143.52 (2C), 148.41, 151.37, 151.44 (Ar–C), 151.86, 154.63 (CO–xanthene), 166.43 (CO–amide). ES+ MS (m/z %): 570.0756 (M+). Anal. Calcd for C24H20Cl2N8O3S: C, 50.35; H, 3.60; N, 19.53. Found; C, 50.44; H, 3.53; N, 19.61.

2-(5-((1,3-Dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)methyl)-4-phenyl-4H-1,2,4-triazol-3-ylthio)-N-(4-fluorophenyl)acetamide (11e)

Light-yellow powder; yield: 66%; mp 168 °C; IR (KBr) ν: 1600–1650 (2C=O), 3351 (N–H), 1643 (CONH), 1545 (N=C), 1473 (n class="Chemical">CH2), Ph (1476), 1453 (C=C), 1331 (N–C), 600–700 (S–C), 801 (Ar–H). 1H NMR, DMSO-d6, 400 MHz, (δ/ppm): 3.14, 3.41 (s, 6H, N–CH3), 4.13 (s, 2H, S-methylene), 5.58 (s, 2H, N-methylene), 7.14 (d, J3′,2′ = J5′,6′ = 7.2 Hz, 2H, H-3′, 5′),7.48 (d, J2′,3′ = J6′,5′ = 2.8 Hz, 2H, H-2′, 6′), 7.58 (m, 5H, H-8′, 9′, 10′, 11′, 12′), 8.0 (s, 1H, HC=N), 10.40 (s, 1H, CO–amide). 13C NMR, 100 MHz, DMSO-d6, (δ/ppm): 27.93 (methyl), 29.90 (methyl), 37.41 (S-methylene), 41.36, (N-methylene), 106.50, 119.56, 121.09, 123.99, 127.46–132.42 (11C), 139.20, 143.52 (2C), 148.41, 151.37, 151.64 (Ar–C), 151.80, 154.63 (2CO–xanthene), 165.78 (CO–amide). ES+ MS (m/z %): 520.1441 (M+). Anal. Calcd for C24H22FN8O3S: C, 55.35; H, 3.90; N, 21.33. Found; C, 55.38; H, 4.07; N, 21.53.

N-(2-Chlorophenyl)-2-(5-((1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)methyl)-4-phenyl-4H-1,2,4-triazol-3-ylthio)acetamide (11f)

Off-white solid; yield: 70%; mp 117 °C; IR (KBr) ν: 1600–1650 (2C=O), 3351 (N–H), 1643 (CONH), 1545 (N=C), 1473 (n class="Chemical">CH2), Ph (1476), 1453 (C=C), 1331 (N–C), 600–700 (S–C), 801 (Ar–H). 1H NMR, DMSO-d6, 400 MHz, (δ/ppm): 3.15, 3.42 (s, 6H, N–CH3), 4.19 (s, 2H, S-methylene), 5.58 (s, 2H, N-methylene), 7.19 (t, J3′,4′/4′,5′ = 5.6 Hz, 1H, H-4′), 7.32 (t, J5′,6′/4′,5′ = 6 Hz, 1H, H-5′), 7.48–7.58 (m, 6H, Ar–H), 7.76 (d, J5′,6′ = 6 Hz, 1H, H-6′), 8.0 (s, 1H, HC=N), 9.89 (s, 1H, CO–amide). 13C NMR, DMSO-d6, 100 MHz, (δ/ppm): 27.93 (methyl), 29.90 (methyl), 37.41 (S-methylene), 41.36, (N-methylene), 106.50, 119.56, 121.09, 123.99, 127.46–132.42 (11C), 139.20, 143.52 (2C), 148.41, 151.37, 151.64 (Ar–C), 151.80, 154.63 (2CO–xanthene), 165.78 (CO–amide). ES+ MS (m/z %): 536.1146 (M+). Anal. Calcd for C24H22ClN8O3S: C, 50.61; H, 3.88; N, 20.69. Found; C, 53.68; H, 3.94; N, 20.87.

2-(5-((1,3-Dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)methyl)-4-phenyl-4H-1,2,4-triazol-3-ylthio)-N-p-tolylacetamide (11g)

Light-brown solid; yield: 73%; mp 141 °C; IR (KBr) ν: 1600–1650 (2C=O), 3351 (N–H), 1643 (CONH), 1545 (N=C), 1473 (n class="Chemical">CH2), Ph (1476), 1453 (C=C), 1331 (N–C), 600–700 (S–C), 801 (Ar–H), 1473 (CH2), Ph (1445).1H NMR, DMSO-d6, 400 MHz, (δ/ppm): 2.23 (s, 3H, Ar–CH3), 3.15, 3.41 (s, 6H, N-methyl), 4.0 (s, 2H, S-methylene), 5.57 (s, 2H, N-methylene), 7.12 (d, J3′,2′ = J5′,6′ = 6.4 Hz, 2H, H-3′, 5′), 7.58 (m, 5H, H-8′, 9′, 10′, 11′, 12′), 7.47 (d, J2′,3′ = J6′,5′ = 3.6 Hz, 2H, H-2′, 6′), 8.0 (s, 1H, HC=N), 10.31 (s, 1H, CO–amide). 13C NMR, DMSO-d6, 100 MHz, (δ/ppm): 20.92 (methyl), 27.92 (methyl), 30.91 (methyl), 37.41 (S-methylene), 41.37, (N-methylene), 106.53–151.78 (Ar–C), 154.65, 162.09 (CO–xanthene), 164.97 (CO–amide). ES+ MS (m/z %): 536.1146 (M+). Anal. Calcd for C24H22ClN8O3S: C, 50.61; H, 3.88; N, 20.69. Found; C, 53.68; H, 3.94; N, 20.87.

2-(5-((1,3-Dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)methyl)-4-phenyl-4H-1,2,4-triazol-3-ylthio)-N-(3,4-dimethylphenyl)acetamide (11h)

Light-gray solid; yield: 67%; mp 148 °C; IR (KBr) ν: 1600–1650 (2C=O), 3351 (N–H), 1643 (CONH), 1545 (N=C), 1473 (n class="Chemical">CH2), Ph (1476), 1453 (C=C), 1331 (N–C), 600–700 (S–C), 801 (Ar–H). 1H NMR, DMSO-d6, 400 MHz, (δ/ppm): 2.17 (6H, Ar–CH3), 3.14, 3.41 (s, 6H, N–CH3), 4.11 (s, 2H, S-methyl), 5.58 (s, 2H, N-methyl), 7.05 (d, J3′,2′ = 6.4 Hz, 1H, H-3′), 7.25 (d, J2′,3′ = 6.4 Hz, 1H, H-2′), 7.31 (s, 1H, H-6′), 7.47–7.58 (m, 5H, Ar–H), 8.0 (s, 1H, HC=N), 10.16 (s, 1H, CO–amide). 13C NMR, DMSO-d6, 100 MHz, (δ/ppm): 19.23 (methyl), 20.06 (methyl), 27.93 (methyl), 29.90 (methyl), 37.39 (S-methylene), 41.33, (N-methylene), 106.46, 117.12, 120.78, 127.46 (3C), 130.06, 130.45, 130.76, 131.73, 132.40, 136.81, 136.93, 143.51, 148.40, 151.33, 151.70 (Ar–C), 151.77, 154.63 (2CO–xanthene), 165.43 (CO–amide). ES+ MS (m/z %): 530.1849 (M+). Anal. Calcd for C26H26N8O3S: C, 50.35; H, 3.60; N, 19.53. Found; C, 58.85; H, 4.94; N, 21.12.

N-(4-Chlorophenyl)-2-(5-((1,3-dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)methyl)-4-phenyl-4H-1,2,4-triazol-3-ylthio)acetamide (11i)

Off-white solid; yield: 73%; mp 136 °C; IR (KBr) ν: 1600–1650 (2C=O), 3351 (N–H), 1643 (CONH), 1545 (N=C), 1473 (n class="Chemical">CH2), Ph (1476), 1453 (C=C), 1331 (N–C), 600–700 (S–C), 801 (Ar–H). 1H NMR, DMSO-d6, 400 MHz, (δ/ppm): 3.13, 3.39 (s, 6H, N–CH3), 4.14 (s, 2H, S-methylene), 5.57 (s, 2H, N-methylene), 7.33–7.56 (m, 9H, Ar–H), 7.99 (s, 1H, HC=N), 10.51 (s, 1H, CO–amide). 13C NMR, DMSO-d6, 100 MHz, (δ/ppm): 27.93 (methyl), 29.90 (methyl), 37.41 (S-methylene), 41.36, (N-methylene), 106.50, 119.56, 121.09, 123.99, 127.46–132.42 (11C), 139.20, 143.52 (2C), 148.41, 151.37, 151.64 (Ar–C), 151.80, 154.66 (2CO–xanthene), 165.75 (CO–amide). ES+ MS (m/z %): 536.1146 (M+). Anal. Calcd for C24H22ClN8O3S: C, 50.61; H, 3.88; N, 20.69. Found; C, 53.68; H, 3.94; N, 20.87.

2-(5-((1,3-Dimethyl-2,6-dioxo-2,3-dihydro-1H-purin-7(6H)-yl)methyl)-4-phenyl-4H-1,2,4-triazol-3-ylthio)-N-(2-fluorophenyl)acetamide (11j)

Light-brown solid; yield: 68%; mp 127 °C; IR (KBr) ν: 1600–1650 (2C=O), 3351 (N–H), 1643 (CONH), 1545 (N=C), 1473 (n class="Chemical">CH2), Ph (1476), 1453 (C=C), 1331 (N–C), 600–700 (S–C), 801 (Ar–H). 1H NMR, DMSO-d6, 400 MHz, (δ/ppm): 3.15, 3.42 (s, 6H, N–CH3), 4.19 (s, 2H, S-methylene), 5.58 (s, 2H, N-methylene), 7.19 (t, J3′,4′/4′,5′ = 5.6 Hz, 1H, H-4′), 7.32 (t, J5′,6′/4′,5′ = 6 Hz, 1H, H-5′), 7.48–7.58 (m, 6H, Ar–H), 7.77 (d, J5′,6′ = 6 Hz, 1H, H-6′), 8.0 (s, 1H, HC=N), 9.89 (s, 1H, CO–amide). 13C NMR, DMSO-d6, 100 MHz (δ/ppm): 27.93 (methyl), 29.90 (methyl), 37.41 (S-methylene), 41.36, (N-methylene), 106.50, 119.56, 121.09, 123.99, 127.46–132.42 (11C), 139.20, 143.52 (2C), 148.41, 151.37, 151.64 (Ar–C), 151.80, 154.66 (2CO–xanthene), 165.75 (CO–amide). ES+ MS (m/z %): 520.1441 (M+). Anal. Calcd for C24H22FN8O3S: C, 55.35; H, 3.90; N, 21.33. Found; C, 55.38; H, 4.07; N, 21.53.

MTT Assay

Cell Culture

The n class="Species">human MCF-7 breast and A549 lung cancer cell lines were cultured in Dulbecco’s modified Eagle medium containing 10% fetal bovine serum and 1% streptomycin–penicillin (100 units/mL and 1% 100 μg/mL) at 37 °C having 5% carbon dioxide in a moistened atmosphere. Cell lines were treated with compounds in dimethyl sulfoxide (DMSO) (less than 1% final concentration).

Determination of Cell Viability

The cytotoxic prospective was evaluated by n class="Chemical">MTT assay. MCF-7 and A549 cells were sown in 96-well plates (microculture), and with different dilutions of compounds, cultured cells were supplemented for 48 h, following further incubation with 20 μL of MTT mixture (5 mg/mL) at 37 °C for 240 min. Later, formazan crystals were mixed in 150 μL of control (DMSO), and absorbance was quantified in a microplate reader at a wavelength of 490 nm and percentage cell viability was calculated.[48]

Hemolytic Assay

All the synthn class="Chemical">esized derivatives were examined following the literature[49] to find out their hemolytic potential. Blood samples (bovine) collected (3 mL) in EDTA were centrifuged at 1000 × g for 10 min. After the isolation of erythrocytes, it was washed three times with 5 mL of cold sterilized solution of PBS at 7.4 pH. The blood suspension (180 μL) was mixed with 20 μL of sample solution (10 mg/mL in negative control, i.e., DMSO) and incubated for 30 min at 37 °C. ABTS and DMSO were used as positive and negative controls, respectively. At 576 nm absorbance of the sample, it was perceived to the % hemolysis was calculated.

Computational Modeling Method

The in silico studin class="Chemical">es were executed to further delineate the mechanism of action of test compounds with higher pharmacological potential. The PASS prediction tool was utilized to predict the therapeutic target with 95% probability, and anti-cancer targets with probability of activity (Pa) > 50% were selected.[50] In the Molecular Operating Environment 2015.10, by the application of induced fit docking, the synthetic derivative was in silico docked against these targets. The compound was sketched and energy-minimized using the CHARMm force field with the MMFF9x partial charge in DS Visualizer 17.2. The conformer (3D) obtained with acefylline was (PubChem CID: 69550) retrieved from the database (PubChem). From PDB (Protein Data Bank) RSCB (http://www.rscb.org), the 3D X-ray structure (crystallized) of STAT3 (5AX3, 2.984 Å resolution) was retrieved. These structures were prepared by the Quickprep function of MOE to correct the structural problems such as missing residues, alternates, and terminus capping. The structures were protonated to resist the modification (molecular) of binding pose. The molecular system was energy-minimized with Amber10: EHT force field. The Site Finder application was used to identify and isolate the potential binding pocket at the hotspot of STAT3. The Dock function was used to place the compounds in the binding pocket with the triangle placement method (matcher) and recorded with London dG. Redocking was done and ranked with a scoring function (GBVI/WSA dG). The pose with the highest conformational energy (ΔG) was utilized to simulate the protein–ligand interactions in DS Visualizer 17.2. Acefylline served as a standard, and its binding score was used as a standard’s threshold.

Statistical Data

All the measurepan class="Species">ments were carried out in triplicate, and statistical analysis was performed using Prism. The rpan class="Chemical">esults are prpan class="Chemical">esented as mean ± SD.