Environ-Economic Synthesis and Characterization of Some New 1 , 2 , 4-Triazole Derivatives as Organic Fluorescent Materials and Potent Fungicidal Agents

A multicomponent one-pot clean cyclocondensation reaction of 4-chloro-2-nitro aniline/amino acids and aromatic aldehydes/ indole-2,3-diones with thiosemicarbazide in water yielding triazole/spiro indole-triazole derivatives in high yields and shorter reaction time and displaying excellent florescent property is reported. The developed MCR may provide a valuable practical tool for the synthesis of new drugs containing the title core fragment. All the newly synthesized compounds have been characterized by IR, HNMR, CNMR, and fluorescence study and also been screened for antimicrobial activity.


Introduction
Multicomponent and domino reactions are efficient and effective methods in the sustainable and diversity-oriented synthesis of heterocycles and such reactions have attracted enormous interest in recent years [1].Thiosemicarbazide and its derivatives are an important class of synthetic compounds, having large variety of applications due to their wide spectrum of biological activities [2] including antiviral [3] and antitumoral [4] as well as parasiticidal activity against Plasmodium falciparum, Plasmodium berghei [5], Trypanosoma cruzi [6][7][8] Trypanosoma brucei rhodesiense [9], and Toxoplasma gondii [10].
The 1,2,4-triazoles and their derivatives are found to be associated with various biological activities such as anticonvulsant [11], antifungal [12], anticancer [13], antiinflammatory [14], and antibacterial properties [15].Also several compounds containing 1,2,4-triazole rings are well known as drugs; for example, fluconazole is used as an antimicrobial drug, while vorozole, letrozole, and anastrozole are nonsteroidal drugs used for the treatment of cancer.
The increasing diversity of small molecule libraries is an important source for the discovery of new drug candidates.
The important biological activities of triazole derivatives impelled us to take up the synthesis of these new combinational heterocycles which are likely to have augmented diverse biological activity.The developed MCR may provide a valuable practical tool for the synthesis of novel physiologically active agents containing the title core fragment.Several methods [27][28][29][30] for the synthesis of 1,2,4-triazole derivatives are reported in the literature but all these methods require the presence of organic solvents and also require long reaction time.Further, there are only few reports [31][32][33] available in the literature regarding the synthesis of triazole derivatives under environmentally benign conditions.Earlier we also reported [33] the synthesis of spiro indole-triazoles under microwaves using montmorillonite as an inorganic solid support.
In recent years, organic research is mainly focused on the development of greener and environ-economic methods which involve use of alternative reaction media to replace volatile and hazardous solvents like benzene, toluene, and methanol, commonly used in organic synthesis.Nowadays, many organic transformations have been carried out in water [34][35][36][37][38].It is a unique solvent because it is readily available, inexpensive, nontoxic, safer, and environmentally benign.
Recently, triazole-based fluorescent sensors have been developed for the selective detection of platinum ions in aqueous solutions [38] and synthesis of a pyrenyl-appended triazole-based calyx [4]arene [39] as a fluorescent sensor for Cd +2 and Zn +2 is carried out.Prompted by rapidly expanding applications of organic fluorescent materials for electroluminescence (EL), dyelasers, sensors, probes, and phototherapeutic agents, and in view of the florescent properties associated with triazole derivatives, development of new fluorescent organic compounds with high functionality has been the subject of intense study for more than a decade [40][41][42][43].
In this context, we have synthesized new 1,2,4-triazole derivatives (4/5) using water as a green solvent under the umbrella of green chemistry and synthesized compounds have been evaluated for antibacterial and antifungal activity and also fluorescent properties.
One of the tools used to combine economic aspect of new reactions with environmental aspects is the multicomponent reaction strategy.Compound 4 was synthesized by the onepot multicomponent reaction of 1, 2, and 3 in the presence of lemon juice as a natural acidic catalyst in aqueous medium.There are few reports [49,50] of synthesis of heterocyclic compounds using lemon juice.For the present work, we have used extract of Citrus limonum species of lemon as natural catalyst for the synthesis of triazole derivatives.The main ingredients of lemon juice are moisture (85%), carbohydrates (11.2%), citric acid (5-7%), protein (1%), vitamin-C (0.5%), fat (0.9%), minerals (0.3%), fibers (1.6%), and some other organic acids.As lemon juice is acidic in nature (pH ≈ 2-3) and percentage of citric acid (5-7%) is more than other acids, it works as acid catalyst [49] for synthesis of triazole derivatives.Using this methodology these reactions were completed in shorter reaction times (1-2 hrs) with yields of the product ranging from 78 to 83%.To our satisfaction, we found that the use of 2 mL of lemon juice resulted in quantitative yield of the corresponding triazole derivatives (4) within 1 to 2 hrs.
Mechanism of formation of compound 4 involves acidcatalyzed nucleophilic attack of 4-chloro-2-nitro aniline [1] on carbonyl carbon [7] forming arylimino derivatives preferentially [11].The nucleophilic attack of the amino electrons of thiosemicarbazide at the electrophilic benzylidene carbon [12] accompanied by the migration of a hydrogen atom forms an intermediate thiol derivative [13] which on cyclisation and desulfurization with the loss of H 2 S gives the suggested triazole derivatives (4) (Scheme 2).
On the other hand, synthesis of spiro indole triazole (5) was carried out in the absence of lemon juice by the reaction [11] [13] Scheme 2: Plausible mechanism of formation of compound 4.

Organic Chemistry International
Scheme 3: Plausible mechanism of formation of compound 5.
of 1  , 2  , and 3 in the presence of water containing small amount of alcohol as a solvent.The progress of reaction was monitored by TLC.After completion of reaction, mixture was cooled to room temperature and poured on to crushed ice; the product formed was filtered and recrystallized from ethanol.Formation of spiro indole-triazoles (5) (reported by us) [31] took place in the absence of natural acid, formation of which is confirmed by spectral analyses.
The third carbonyl group of isatin being more electrophilic forms 3-aryl imino derivatives preferentially [14].The formation of final compound 5 involves the nucleophilic attack of thiosemicarbazide at the electrophilic indolyl carbon [14] accompanied by the migration of hydrogen atom to form an intermediate thiol derivative [15].This is followed by cyclization and desulphurization with the loss of hydrogen sulphide to give the suggested spiro compound 5 (Scheme 3).
In summary, it can be stated that the present green synthetic protocol is highly efficient as it avoids the use of hazardous solvents at any stage of the reaction.The scope of the method was further studied by reacting differently substituted 1H-indole-2,3diones with amino acids and thiosemicarbazide.The identity of the products (Tables 3  and 4) obtained was confirmed by their IR, 1 H NMR, and 13 C NMR spectral data.

Fluorescence Study
A fluorescence study was carried out with newly synthesized triazole derivatives (4a-j) and (5a-l) in order to relate the fluorescence properties to the nature of donating and acceptor groups attached to these moieties.Fluorescence spectra of 5.7 × 10 −5 M solutions of compounds were measured; excitation and emission maxima are also reported.Emission spectra of compounds (4a-j) and (5a-p) were run in DMSO.The resulting triazoles formed by the reaction of 4-Cl, 2-NO 2 aniline/amino acids and aromatic aldehydes/indole-2,3diones with thiosemicarbazide result in a bathochromic (red) shift of emission maxima (Tables 1 and 2).The combination of 4-chlorophenyl-substituted triazole to 4-chloro-2-nitro aniline in compound 4d results in the shift of em.from 326 to 390.98 nm owing to the electron's withdrawing nature of Cl group.Further, combination of imidazole/indole-substituted triazole in 5j and 5l to 5-chloro-1 H indole-2,3-dione also results in red shift.For amino substituents, the bathochromic shift of the fluorescence as well of the absorption band is due to the large electron releasing ability of the nitrogen in amine group (5a-k) (Figures 1, 2, 3, and 4).

Materials and Methods
4.1.General.Reagents and solvents were obtained from commercial sources and used without further purification.Melting points were determined on a Toshniwal apparatus.The 1 H NMR and 13 C NMR spectra of synthesized compounds have been carried out at SAIF, Punjab University, Chandigarh. 1 H NMR spectra were recorded on Bruker Avance II 400 NMR spectrometer using DMSO-d 6 and CDCl3 as solvent and tetramethylsilane (TMS) as internal reference standard.The purity of compounds was checked on thin layers of silica gel in various nonaqueous solvent systems, for example, benzene : ethyl acetate (8 : 2).IR spectra were recorded in KBr on a Perkin Elmer Infrared L1600300 spectrum Two Li-Ta spectrophotometer and fluorescence studies were carried out on RF-5301 PC spectrofluorophotometer, Shimadzu at FET, MITS, Laxmangarh, Sikar, Rajasthan, India.

General Procedure for the Synthesis of Compounds (4aj).
An equimolar mixture of substituted aromatic aldehyde (0.01 mol), 4-chloro-2-nitro aniline (0.01 mol), and methyl phenyl thiosemicarbazide in water (15 mL containing 1-2 mL of ethanol) in the presence of lemon juice (2 mL) was mixed in round bottom flask and the mixture was refluxed for the time needed to complete the reaction (as monitored by TLC).
After completion of the reaction, mixture was cooled to room temperature and the solid mass was filtered and recrystallized from ethanol.

General Procedure for the Synthesis of Compounds (5ap).
An equimolar mixture of substituted 1H-indole-2,3dione (0.012 mol), substituted amino acids (0.012 mol) and thiosemicarbazide in water (15 mL containing 1-2 mL of ethanol) was mixed in round bottom flask and the mixture was refluxed for the time needed to complete the reaction (as monitored by TLC).The initial syrupy reaction mixture solidifies within 2-3 hours.After completion of the reaction, mixture was cooled to room temperature and the solid mass was poured onto crushed ice, filtered, and recrystallized from ethanol.

Antimicrobial Activity
Synthesized compounds 4a-j and 5a-p were evaluated for antibacterial and antifungal activity.

Antibacterial Activity. Synthesized compounds (4a-j)
were screened for their antibacterial activity against Grampositive bacteria Bacillus licheniformis, Staphylococcus aureus, and Micrococcus luteus and Gram-negative bacteria Pseudomonas aeruginosa and Escherichia coli by the agar well diffusion method.5 mL aliquot of nutrient broth was inoculated with the test organism and incubated at 37 ∘ C for 24 hours.Sterile nutrient agar plates were also prepared and holes of 5 mm diameter were cut using a sterile cork borer ensuring proper distribution.The test organisms after 24 hours of incubation were spread onto separate agar plates.The chemical compounds were dissolved in DMSO at a particular concentration or poured into appropriately labelled holes using a pipette in aseptic conditions.A hole containing DMSO served as a control.The plates were left at room temperature for two hours to allow the diffusion of the sample followed by incubation at 37 ∘ C for 24 hours in inverted position.The antimicrobial activity was determined by measuring the diameter of the zone (mm) showing complete inhibition with respect to control (DMSO) and reference compounds streptomycin and erythromycin.It has been observed that all the compounds tested showed good to excellent activity against tested bacteria.
Compound 4g shows good activity against bacteria Staphylococcus aureus at 250 ppm concentration.On the other hand, compound 4j shows excellent activity against bacteria Pseudomonas aeruginosa, Staphylococcus aureus, and Micrococcus luteus at 500 ppm concentration.Compound 4b shows good activity against bacteria Staphylococcus aureus, Micrococcus luteus, and Escherichia coli and compound 4d also shows good activity against bacteria Pseudomonas aeruginosa, Bacillus licheniformis, and Micrococcus luteus at 500 ppm concentration (Table 5, Figure 5).the sterile agar into Petri dishes in aseptic conditions.0.1 mL of each standardized test organism culture was spread onto agar plates.The test compounds (250 and 500 ppm), the standard drug solutions, and the solvent control (DMSO) were placed in the cavity separately.Then the plates were maintained at −4 ∘ C for 1 hour to allow the diffusion of solution into the medium.All the fungal plates were incubated at 28 ∘ C for 42-72 hrs and the zone of inhibition was measured in mm.The antimicrobial activity was determined by measuring the diameter of the zone (mm) showing complete inhibition with respect to control (DMSO) and reference compounds streptomycin and erythromycin.Compounds 4d and 4g show good activity against fungus Fusarium oxysporum at 250 ppm due to presence of -Cl and -OH substituents.Compound 4c shows excellent activity against fungus Chaetomium orium and 4f shows excellent activity against fungus Alternaria brassicicola and Lycopodium sp. at 500 ppm.Compound 4h shows excellent activity against fungus Fusarium oxysporum, Chaetomium orium, and Lycopodium sp. at 500 ppm.Compound 4a shows good activity against fungus Fusarium oxyforum, Alternaria brassicicola, and Lycopodium sp. at 500 ppm concentration due to the presence of -OH substituent (Table 6, Figure 6).

Antibacterial Activity.
Compound 5j shows good activity against bacteria Pseudomonas aeruginosa and 5k shows good activity against bacteria Staphylococcus aureus, respectively at 250 ppm concentration.Compound 5m shows excellent activity against bacteria Staphylococcus aureus at 250 ppm concentration due to presence of -NO 2 substituent.On the other hand, compounds 5b, 5g, and 5n show excellent activity against bacteria Escherichia coli at 500 ppm concentration (Table 7, Figure 7).

Antifungal Activity.
Compound 5d shows good activity against Fusarium oxysporum at 250 ppm concentration.Compounds 5b, 5h, and 5m show good activity against Lycopodium sp. at 500 ppm concentration.Compounds 5d and 5h show good activity against Chaetomium orium at 500 ppm concentration.Compounds 5f and 5p also show good activity against Alternaria brassicicola at 500 ppm concentration; compounds 5e, 5h, and 5j also show good activity against Fusarium oxysporum at 500 ppm concentration (Table 8, Figure 8).

Conclusion
The use of water as a green solvent offers a convenient, nontoxic, and inexpensive reaction medium for the environeconomic synthesis of triazole derivatives.This procedure is simpler, economical, milder, and faster, including cleaner reactions, high yields of products, and a simple experimental and work-up procedure, which makes it a useful and attractive process and is also consistent with the green chemistry theme which affords good yields.Synthesized compounds are found to be excellent fluorescent materials and potent fungicidal agents.