Synthesis of novel pyrazoline-based bis(1,2,3-triazole) scaffolds via click chemistry

A series of novel bis(1,2,3-triazoles) derivatives 7a–m were synthesized by the 1,3-dipolar cycloaddition (click-reaction) of 1-methyl-3,5-bis(2-(prop-2-yn-1-yloxy)phenyl)-4,5-dihydro-1H-pyrazole (5) with various aralkyl azides 6a–m in the presence of sodium ascorbate and copper sulphate with good yields. The required precursor 5 was synthesized by reacting (E)-1,3-bis(2-hydroxyphenyl)prop-2-en-1-one (3) with methylhydrazine hydrate via 2,2′-(1-methyl-4,5-dihydro-1H-pyrazole-3,5-diyl)diphenol 4, followed by reaction with propargyl bromide. The homogeneity of all the newly synthesized compounds was checked by TLC. The IR, NMR, mass spectral data and elemental analysis were in accord with the assigned structure. The title compounds were evaluated for their antibacterial activity against various bacterial strains, i.e., Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus subtilis; compounds 7f–7h and 7j were found to be moderately active against the bacteria, when compared with that of the standard drug. Furthermore, the same library of compounds was evaluated for their antioxidant activity using the nitric oxide radical scavenging activity. The results of the study showed that compounds 7e–7h and 7k–7m showed good radical scavenging activity.


INTRODUCTION
Five-membered heterocyclic compounds occupy a distinctive place in the realm of natural and synthetic organic chemistry.1,2,3-Triazoles have received great attention due to their contribution in pharmaceutical drugs, regardless of their scarcity in nature.In this respect, various approaches for the preparation of these privileged structures with drug-like properties have been developed using various synthetic strategies. 1,2,3-Triazole derivatives are revealed in prominent pharmaceutical drugs, such as, carboxyamidotriazole, cefatrizine and tazobactam.
Pyrazolines are a significant class of heterocyclic compounds comprising two nitrogen atoms in a five-membered ring.Pyrazoline derivatives are the electron rich nitrogen heterocycles that play an essential role in various biological activities.These heterocyclic compounds occur widely in the environment, in the form of alkaloids, vitamins, pigments and as constituents of plant and animal cells.Considerable attention has been focused on pyrazolines and substituted pyrazolines because of their inspiring biological activities.Pyrazolines constitute an interesting class of heterocycles due to their synthetic versatility and effective biological activities, such as anticancer, 19 antioxidant, 20 antibacterial, 21 antifungal, 22 antidepressant, [23][24][25] anti-inflammatory, 26 anticonvulsant, 27 antitumor 28 and analgesic 29 properties.As far as the different pyrazoline isomers are concerned, 2-pyrazoline derivatives became the most frequently studied pyrazoline.Various methods are used for the preparation of 2-pyrazolines.Reaction of α,β-unsaturated ketones with substituted hydrazines seems to be the most popular procedure for the synthesis of 2-pyrazolines.
As mentioned, 2-pyrazolines and 1,2,3-triazole derivatives possess valuable bioactivities, which stimulated the preparation of their various derivatives.Recently, much consideration has been paid toward the synthesis and pharmacological evaluation of triazoles, and bis-triazoles, as potent HIV-1 protease inhibitors 30,31 and size-specific ligands for mRNA hairpin loops, 32 respectively.Some derivatives containing a triazole and pyrazoline moiety were synthesized and investigated for their potential antidepressant activities. 33According to these studies, a system combining two biolabile components, 2-pyrazoline and 1,2,3--triazole, were synthesized and their potential antibacterial and antioxidant effects investigated.Bearing in mind the tremendous biological potency of bis(1,2,3--triazoles) with pyrazoline, the endeavour was the synthesis of pharmacologically active molecules, and then their evaluation for antibacterial and antioxidant activities.In this regard, our research group has focused on the design, synthesis of 1,2,3-triazoles and pyrazolines, thereby contributing to research 34,35 on these biologically important heterocycles.In the current study, the aim was to obtain new compounds containing both pyrazolines and 1,2,3-triazole rings in the same structure, via the click reactions shown in Schemes 1 and 2.

EXPERIMENTAL
Melting points were determined by the open capillary method using an electrical melting point apparatus and are uncorrected.The IR spectra were recorded as KBr pellets on a Shimadzu FT-IR-8400s spectrophotometer.The 1 H-NMR (400 MHz) and 13 C-NMR (100 MHz) spectra were recorded on a Bruker DPX 400 spectrophotometer using tetramethylsilane (TMS) as the internal standard, with DMSO-d 6 and CDCl 3 as solvents.The mass spectra were recorded on a GCMS-QP 1000 EX mass spectrometer.Thin layer chromatography (TLC) was performed to check the purity of the compounds, the spot being located under UV light and iodine vapours.
Analytical and spectral data for the synthesized compounds are given in Supplementary material to this paper. 36,37o a vigorously stirred solution of 2-hydroxy acetophenone (25 g, 184 mmol) and Salicylaldehyde (22.4 g, 184 mmol) in ethanol (200 mL), KOH (30.92 g, 551 mmol) was added in small portions over 1 h at 0 °C.The reaction mixture was stirred at room temperature for 2 h and refluxed for 1 h.After completion of the reaction and cooling to room temperature, the mixture was poured into ice-cold water, neutralized with concentrated HCl and stirred for 1 h._________________________________________________________________________________________________________________________ (CC) 2017 SCS.

Biological activity
Antibacterial assay.The in vitro antibacterial studies against the test organisms were realised by the agar well diffusion method. 38Nutrient broth (NB) plates were swabbed with 24 h old broth culture (100 mL) of the test bacteria.Using a sterile cork borer, wells (6 mm) were made into each Petri plates.Different concentrations of the test samples dissolved in DMSO were added into the wells by using sterile pipettes.Gentamicin was used as the standard antibiotic for the antibacterial activity.The plates were incubated at 37 °C for 24 h.After the incubation, the diameter of zone of inhibition of each well was measured.Duplicates were maintained and the average values were calculated for eventual antibacterial activity.The broth dilution test was used to determine minimum inhibitory concentration (MIC) of the above-mentioned samples. 39Freshly prepared nutrient broth was used as the diluent.The 24 h old culture of the test bacteria Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus subtilis were diluted 100 fold in nutrient broth (100 μL bacterial cultures in 10 mL NB).Increasing concentrations of the test samples were added to the test tubes containing the bacterial cultures.All the tubes were incubated at 37 °C for 24 h.The tubes were examined for visible turbidity and using NB as a control.The lowest concentration that inhibited visible growth of the tested organisms was recorded as the MIC.

Antioxidant activity
Nitric oxide radical scavenging activity.The scavenging effect on nitric oxide was measured according to the method of Marcocci 40 et al. with a little modification. 41Briefly, 4 mL of a drug solution was added (in a test tube) to 1 mL of sodium nitroprusside (SNP) solution (25 mM) and the tubes incubated at 29 °C for 2 h.A 2 mL aliquot of the incubation solution was diluted with 1.2 mL Griess reagent (1 % sulphanilamide in 5 % H 3 PO 4 and 0.1 % N-1-naphthylethylenediamine dihydrochloride).The absorbance of the chromophore that was formed during diazotization of the nitrite with sulphanilamide and subsequent coupling with N-1-naphthylethylenediamine dihydrochloride was immediately read at 550 nm and the concentration determined from a standard curve (y = mx+c) of sodium nitrite salt treated in the same way with Griess reagent.Inhibition of nitrite formation by the drug or the standard plant antioxidant (vitamin C) was calculated relative to the control: Inhibition, % = 100(A control -A test )/A control where A test is the absorbance of the control reaction mixture excluding the test compound/drug solution and A control is the absorbance of the test compounds/drug solution.

Chemistry
The route for the synthesis of the title compounds 7a-m is outlined in Scheme 1.Initially, the title compound 7a was synthesised as shown in Scheme 2, when conversion of compound 9 (triazole derivative) to the N-substituted dihydro pyrazole derivative 7a was obtained in very low yield making the purification difficult.The bis-propargyl derivative 8 was obtained in 82 % yield from the corresponding chalcone derivative on treatment with NaH and DMF.The bispropargyl derivative 8 was converted to the bis triazole derivatives 9 via coppercatalyzed click reaction with the aralkyl azides in 85 % yield.The bis-triazole derivatives 9 were treated with methylhydrazine hydrate in ethanol at reflux temperature.Finally, the target compound 7a was obtained in very low yield.Due to the low yield obtained following Scheme 2, Scheme 1 was approached, whereby the target compounds were isolated in good yields.The precursor 5 was synthesized by reacting (E)-1,3-bis(2-hydroxyphenyl)prop-2-en-1-one (3) with methylhydrazine hydrate via 2,2′-(1-methyl-4,5-dihydro-1H-pyrazole-3,5-diyl)diphenol (4), followed by reaction with propargyl bromide.The structures of newly synthesized compounds 7a-m were characterized by IR, 1 H-NMR, 13 C-NMR, mass and CHNS spectral analysis.Except the aralkyl azides 6h, 6j, all the other aralkyl azides 6a, 42 6b, 43 6c, 44 6d, 45 6e, 46 6f, 47 6g, 48 6i, 49 6k, 50 6l 51 and 6m 44 are known in the literature.After isolation, the aralkyl azides were treated immediately with compound 5 without further purification to obtain compounds 7a-m.
The IR, 1 H-NMR, 13 C-NMR and mass spectra for the synthesised compounds are given in the Supplementary material to this paper.
The elucidation of the structure of pyrazoline ring protons is usually realised by 1 H-NMR spectroscopy.In the 1 H-NMR spectrum, the signals of the res-pective protons of the pyrazoline ring are verified based on their chemical shifts, multiplicities, and coupling constants.The formation of a pyrazoline ring was confirmed by the presence of an ABX system in the 1 H-NMR due to geminal--vicinal coupling between protons H A , H B and H X .The H A and H B protons appeared as a doublet of doublet due to geminal and vicinal coupling as shown in Scheme 1, bottom inset (compound 7a).These H A and H B differ in their coupling with H X and hence, they are anisogamous.H A , which appeared as a doublet of doublet at δ 2.67 ppm, is the proton cis to H X and geminal to H B (J AB = 16.8Hz, J AX = 14.8 Hz).H B is the proton trans and vicinal to H X and appeared as a doublet of doublet at δ 3.52 ppm (J BA = 16.8Hz, J BX = 9.6 Hz).Moreover, H X appeared as doublet of doublet at δ 4.24 ppm (J XA = 14.8,J XB = 9.6 Hz). 30,52ll the other signals from NMR spectra are in agreement with the proposed structures.

Antibacterial activity
The newly synthesized compounds 7a-m were screened in vitro for their antibacterial activity against Escherichia coli (ATCC 11229) and Pseudomonas aeruginosa (ATCC 27853), as examples of Gram-negative bacteria, and Staphylococcus aureus (ATCC 6538) and Bacillus subtilis (ATCC 6633), as examples of Gram-positive bacteria.Agar well-diffusion method was used to assay the antibacterial activity against test strains on Mueller-Hinton agar plates.Gentamicin was employed as a standard antibacterial drug.The results obtained as minimum inhibitory concentration (MIC) in µg mL -1 and measurements are presented in Table I.Investigation of the antibacterial efficiency of the synthesized compounds revealed that most of the tested compounds displayed variable inhibitory effects on the growth of the tested Gram-negative and Gram-positive bacterial strains.It is evident from Table I, that compound 7g exhibited the highest antibacterial effect, but was less potent than gentamicin in inhibiting the growth of E. coli, P. aeruginosa, S. aureus and B. subtilis (MIC = 6.25 µg/mL).Next to 7g, compounds 7f and 7h (6.25-12.5 µg mL -1 ) and 7j (6.25-25 µg mL -1 ) were found less potent as compared to the control drug gentamicin.It was envisaged from the analysis of the antibacterial activity results that the presence of methyl-, chloroand triflouromethyl-substituted derivatives had a moderate effect in determining the antibacterial activity and the exhibited antimicrobial potency.

Antioxidant activity
The antioxidant activity of the synthesized compounds 7a-m were evaluated in vitro by the nitric oxide radical scavenging assay. 40,41The results were compared with that of the standard antioxidant ascorbic acid.Most of the compounds tested significantly inhibited nitric oxide radical levels compared to the standard antioxidant used in the study (Table II).As could be seen in Table II, compounds 7e-h and 7k-m exhibited strong scavenging effects on the nitric oxide stable radical, with respective IC 50 values of 2.50±0.65,2.50±0.60,2.50±0.24,2.50±0.76,2.50±0.37,2.43±0.41 and 2.47±0.38 µg mL -1 .
These values were lower than the positive controls in the study (AA with 5.35±0.67µg/mL), indicating that compounds with hydroxyl, nitro, chloro, methyl, fluoro, trifluoromethyl, methoxy and isopropyl substituents were found to be the most potent antioxidant agents towards nitric oxide.The remaining compounds showed moderate activity.In conclusion, a novel series of bis(1,2,3-triazole) derivatives were successfully synthesized through copper-catalyzed Huisgen [3+2] cycloaddition of various aralkyl azides with bis -propargyl pyrazoline 5 in good to excellent yields.Furthermore, this synthesis approach provided a structural framework that could be explored further in the development of new 1,2,3-triazole derivatives from 2-pyrazolines moieties.It is believed that the procedural simplicity, the efficiency, and the easy accessibility of the reaction partners give access to an array of heterocyclic frameworks.In this study, new hybrid molecules consisting of biologically important 1,2,3-triazole derivatives from 2-pyrazolines pharmacophores were synthesized and their antibacterial and antioxidant activities determined.Amongst the synthesized compounds, 7f-h and 7j showed the moderate antibacterial activity against the tested bacterial strains.The radical scavenging activities of the synthesized compounds also showed that the compounds 7e-h and 7k-m exhibited potent IC 50 values.The experimental results of this study will likely provide a new basis for the design of interesting pyrazoline-based bis(1,2,3-triazoles), and further studies, including the design of new analogues of the heterocyclic moiety, are in progress.

SUPPLEMENTARY MATERIAL
Analytical and spectral data for the synthesized compounds are available electronically at the pages of journal website: http://www.shd.org.rs/JSCS/, or from the corresponding authors on request.
Acknowledgements.The authors are thankful to the Head of Department of Chemistry, Osmania University and JNTU-H Hyderabad for their valuable support.

TABLE II .
Antioxidant activity (IC 50 / µg mL -1 ) of the synthesized compounds 7a-m against nitric oxide; values are the means of three replicates±SD.A lower IC 50 value indicates better _________________________________________________________________________________________________________________________(CC) 2017 SCS.Available on line at www.shd.org.rs/JSCS/