Design, synthesis and antitumor activity of new naproxen based 1,2,4-triazole-Schiff base derivatives

In the present work, new Naproxen based 1,2,4-triazole-Schiff base derivatives have been synthesized and screened for in vitro antitumor activity and in silico pharmacokinetic studies. The structure of the newly synthesized compounds (5–12) was elucidated by IR, NMR and mass spectrometry. All the compounds were tested against breast MCF-7, hepatocellular Huh-7 and lung A-549 cancer cell lines using MTT assay. Compound 7 was better in killing A549 cells with IC50 3.71 µM (1.48 fold), compared with Doxorubicin (IC50 5.50 µM). Also, compound 7 was found to be non toxic on MRC-5 normal cells as it depicts IC50 more than 500 μM. Besides, compound 12 also revealed promising activity with IC50 6.94 and 3.33 µM against MCF-7 and Huh-7 respectively. The in silico studies displayed that the synthesized compounds favors the desired pharmacokinetic profile and drug likeness properties. It can be concluded that these new Naproxen based 1,2,4-triazole-Schiff base derivative (7) has the potential to be further investigated as lead molecule in the development of new chemotherapeutic agent.


Introduction
Cancer is the rapid proliferation of abnormal cells that has the tendency to invade other body parts resulting in high mortality rates [1,2].It is the leading cause of death globally accounting for nearly 10 million deaths in 2020 [3] and is expected to rise to 16.4 million by 2040 [4].The most common cancers are breast, lung and colon and 30-50% of cancer mortality can be prevented through early detection, appropriate treatments and care of patients [5].There has been a great advancement in cancer treatment which includes surgery, radiotherapy, chemotherapy and hormonal treatments [6,7].However due to dose toxicity, drug resistance to malignant tumours and selectivity of the current anticancer drugs [8], there is a need to develop effective chemotherapeutics to overcome these obstacles.Naproxen is a COX inhibitor and a potent NSAID.It has now been extensively studied for its anticancer potential as many of its derivatives inhibited proliferation in various cancers [9,10].Urea and propanamide derivatives of Naproxen inhibited cancer proliferation in colon cancer [11], Naproxen-1,3,4-oxaadiazole as EGFR inhibitors [12], Naproxen hydrazide-hydrazones as potent VEGFR-2 inhibitors [13], Naproxen-triazole hybrids as HDAc inhibitors [14] and provided protection in bladed cancer [15].

Chemistry
The intermediates 2-4 were prepared using the reported method [12] with slight change.Naproxen 1 was reacted with methanol in presence of catalytic amount of concentrated sulphuric acid to yield compound 2 in pure form (91% yield).Then compound 2 was refluxed with hydrazine monohydrate in presence of methanol for 4 h to yield compound 3 (86% yield).The hydrazide 3 was dissolved in ethanolic KOH and added carbon disulfide dropwise at 0-5 ℃ followed by stirred at room temperature for 14 h yield potassium thiocarbamate salts, which was filtered.To this crude solid, hydrazine hydrate was added, and reflux for 12 h and then acidification with HCl solution yield main intermediate 4 (76% yield).Compound 4 was reacted with different aromatic aldehydes to yield new compounds 5-10 in 65-85% yield (Scheme 1) and reaction of compound 8 with different alkyl halide afforded compound 11-12 (Scheme 2).All the newly synthesized compounds formation was confirmed by different analytical techniques such as FTIR, 1 H NMR, 13 C NMR and mass spectrometry.The 1 H NMR of compounds 5-7 and 9-10 displayed signals for azomethine protons merged with aromatic protons in the range 6.57-8.55ppm, S-H proton signal in the range 9.93-10.48ppm and aliphatic region showed peaks at 1.79-1.86ppm, 4.43-4.65 ppm and 3.90-3.91ppm ascribed to CH 3 CH-, CH 3 CH-(Ar) 2 , and Ar-O-CH 3 , respectively.These peaks were further supported by 13 C NMR which showed OCH 3 , CH and CH 3 signals in upfield regions at 55. 30

Physicochemical properties
Besides effectiveness, many molecules could not enter drug development due to poor bioavailability, absorption, water solubility and lipophilicity [6].Therefore, the molecules must have good pharmacokinetic profile along with excellent pharmacological potential.There are certain criteria such as Lipinski rule of five which should be followed by the molecules in order to enter drug innovation [36].The candidate must have molecular weight (M.W.) less than 500, lipophilicity (log P) below 5, hydrogen bond acceptor/donor must be below 10 and 5, respectively for easy transportation, excretion, diffusion and absorption.The synthesized final molecules were screened for in silico ADME predictions to have a look on their physiochemical and pharmacokinetic properties and the results are shown in Table 1.All the newly prepared compounds (5-12) were screened for their in-silico absorption, distribution, metabolism, excretion and toxicity (ADMET) study using swiss ADME and pKCSM data base tools [37].From the results, it was observed that most of the compounds showed promising pharmacokinetics with molecular weight less than 500 except compound 11, % absorption in the range 61.76-79.04,lipophilicity (log P) was found to be less than 5 in the range 3.06-4.90and water solubility (log S) in the good range − 5.27 to 6.99.Also these compounds could not cross blood brain barrier and displayed desired skin permeability (log Kp) in the range − 5.05 to 5.82 for These data suggests that these Naproxen based 1,2,4-triazole-hydrazone derivatives follow Lipinski rule therefore possess desired pharmacokinetic and drug likeness properties as shown in Table 1.

In vitro antitumor activity
All the target molecules were evaluated for their antitumor activity against the three cancer cell lines MCF-7 (breast), Huh-7 (liver) and A-549 (lungs) by MTT method using the method of Mosmann [38].Doxorubicin was used as standard drug which showed IC 50 1.85 µM, 1.40 µM and 5.50 µM against MCF-7, Huh-7 and A-549, respectively.From the results as shown in Figs. 2, 3 and 4, it was observed that the tested compounds showed variations in their activity from excellent to moderate in the range IC 50 4.72-46.80µM, 1.91-28.10µM and 3.71-56.63µM against MCF-7, Huh-7 and A-549, respectively.Among the tested derivatives, compound 7 was the most promising with IC 50 4.72, 1.91 and 3.71 µM, compared to the standard drug, Doxorubicin against MCF-7, Huh-7 and A-549 respectively.It can be seen that compound 7 was better in killing A549 cells with IC 50 3.71 µM (1.48 fold), compared with Doxorubicin (IC 50 5.50 µM).Also, compound 7 was found to be non toxic on MRC-5 normal cells as it depicts IC 50 more than 500 μM.Besides, compound 12 also showed promising activity with IC 50 6.94 and 3.33 µM against MCF-7 and Huh-7 respectively.Compounds which were moderately toxic (IC 50 < 20 µM) to   2.

Chemistry
All the chemicals and reagent used for the synthesis of the target molecules were procured from Sigma Aldrich, Loba Chem and Across.The proposed structure of all the synthesized compounds was confirmed by different analytical techniques such as FT-IR (Thermo Scientific iS50), 1 H & 13 C NMR (Bruker 850 MHz and 213 MHz respectively), Mass spectrometry (Thermo Scientific LCQ FLEET LCF10605), Elemental Analysis (LEECO Elementar Analyzer) while melting points were recorded on Stuart SMP40 machine which were uncorrected.The intermediate compounds 2-4 were prepared according to our previous method [12].Dimethyl sulfoxide (DMSO), MTT and trypan blue dye was purchased from Sigma (St. Louis, Mo., USA).Fetal Bovine serum, RPMI-1640, HEPES buffer solution, L-glutamine, gentamycin and 0.25% Trypsin-EDTA were purchased from Lonza (Belgium).

General procedure for synthesis of compound 5-10
Compound 4 (0.001 mol) was taken in 100 mL round bottom flask and added 30 mL absolute ethanol, different aromatic aldehydes (0.001 mol) followed by addition of 3-5 drops of glacial acetic acid.The reactions mixture was stirred at 50-60 ℃ for 6-14 h.After completion of the reaction, the reactions mixture were concentrated to around 10 mL and poured onto the crushed ice, stirred to get solid precipitate.The products were filtered, washed with water and dried.The crude products were recrystallized from ethanol to get pure compounds with 68-86% yield.

Antitumor activity
The antitumor activity of the newly synthesized compounds was tested against Breast MCF-7, Hepatocellular Huh-7 and lung A549 carcinomas using MTT protocol.The cell lines were obtained from the American Type Culture Collection (ATCC, Rockville, MD) and the activity was performed at Regional Center of Microbiology and Biotechnology, Al-Azhar University, Egypt.The positive reference drug used was Doxorubicin and the assay was performed according to published work [39].The optical density was measured at 590 nm with the microplate reader (SunRise, TECAN, Inc, USA) to determine the number of viable cells and the percentage of viability was calculated as [(ODt/ ODc)] × 100% where ODt is the mean optical density of wells treated with the tested sample and ODc is the mean optical density of untreated cells.The graph between surviving cells and drug concentration were plotted to get the survival curve of each tumor cell line after treatment with the specified compound.The IC 50 for each compound was calculated from the dose response curve for each conc.using Graphpad Prism software (San Diego, CA.USA) [38].

Conclusion
In the present work, new Naproxen based 1,2,4-triazole-Schiff base derivatives have been synthesized and screened for in vitro antitumor activity and in silico pharmacokinetic studies.Compound 7 was found to be the most potent with IC 50 4.72, 1.91 and 3.71 µM, against MCF-7, Huh-7 and A-549, respectively and was better in killing A549 cells than doxorubicin with 1.48 fold activity.The in silico studies displayed that the synthesized compounds favors the desired pharmacokinetic profile and drug likeness properties.It can be concluded that these new Naproxen based 1,2,4-triazole-Schiff base derivative (7) could be further investigated as lead molecule in the development of new chemotherapeutic agents.

Scheme 1 Scheme 2
Scheme 1 Synthesis of naproxen based 1,2,4-triazole bearing Schiff base (5-10) the cancer cells were found to be 10 and 14 towards MCF-7; 9 and 10 against Huh-7; and compound 12 towards A-549 cells.Other compounds shown mild cytotoxicity with IC 50 < 50 µM towards the tested cell lines except compound 9 which was less active against A-549 cells.The result of antitumor activity is shown in Table

Fig. 4
Fig. 4 Cell viability (%) of final compounds (5-12) against A-549 cells (8),4-triazole-hydrazone derivative(8)displayed one additional signal at 6.08-6.10ppmforAr-O-CH 2 in 1 H NMR and at 102.13 ppm in 13 C NMR in addition to similar signals observed for compound 7. Compound 10 showed a downfield signal in13C NMR spectra at 176.02 ppm for C = O confirming the presence of cromonyl group.Compounds 11 and 12 showed similar signals to compound 8 but both of them showed disappearance of -SH proton.Also, compound 11 exhibited appearance of additional S-CH 2 and five aromatic protons at 4.46 ppm and 7.21-7.38ppm,occurreddue to S-benzylation whereas compound 12 displayed a triplet at 1.42 ppm and broad singlet at 3.35 ppm integrating for three and two protons, respectively and signals at 14.62 ppm and 27.85 ppm correspond to -S-CH 2 -CH 3 carbons in1H & 13 C NMR, confirming alkylation at sulphur.Lastly, all the final compounds were confirmed by mass spectrometry displaying molecular ion peaks in the positive/negative mode.

Table 1
ADMET studies of the target molecules 5-12 P octanol-water partition coefficient, HBA hydrogen bond acceptor, HDB hydrogen bond donor, Log S water solubility, Log Kp skin permeability, %abs absorption, BBB blood brain barrier