Design, synthesis, characterization of some new 1,2,3-triazolyl chalcone derivatives as potential anti-microbial, anti-oxidant and anti-cancer agents via a Claisen–Schmidt reaction approach

Abstract

The synthesis of a new series of (2E)-1-{1-[2,3-dichloro-6-methyl-5-(trifluoromethyl)phenyl]-5-methyl-1H-1,2,3-triazol-4-yl}-3-aryl prop-2-en-1-one (5a–k) and (2E)-1-{1-[2,3-dichloro-6-methyl-5-(trifluoromethyl)phenyl]-5-methyl-1H-1,2,3-triazol-4-yl}-3-(1,3-diaryl-1H-pyrazol-4-yl)prop-2-en-1-one (6a–e) were carried out via a Claisen–Schmidt condensation of 1-{1-[2,3-dichloro-6-methyl-5-(trifluoromethyl)phenyl]-5-methyl-1H-1,2,3-triazol-4-yl}ethanone (4) with different aryl and 1,3-diaryl-1H-pyrazole-4-carbaldehydes iii(a-e) in the presence of ethanol and aqueous sodium hydroxide mixture respectively. The newly synthesized compounds were characterized by IR, ¹H NMR, ¹³C NMR, mass spectral data and elemental analysis. Further, they were screened for their in vitro anti-microbial, anti-oxidant and anti-cancer activities. Most of the synthesized compounds were displayed broad spectrum of anti-microbial, anti-oxidant activities and some of them exhibits moderate to excellent anti-cancer activities on breast cancer cell lines. Overall, this work has contributed to the development of promising leads for anti-microbial, anti-oxidant and anti-cancer activities.

---

1. Introduction

During the past few decades, there has been a severe increase in the population suffering with infectious diseases due to multi-drug resistance, which often results from the over-expression of multi-drug efflux systems and their wide spread usage.¹ Microbial infections are the most prominent death causing diseases after heart attack, because of their ability to spread rapidly, combined with their toxicity and resistance towards existing anti-biotic drugs.^2,3 These trends have emphasized the urgent need for the development of more effective, potent and broad spectrum antimicrobial novel drugs with good bioavailability, no or fewer side effects to cure microbial infections.⁴ In this context, the drug discovery has acquired a considerable interest in microbial target based synthesis of novel antimicrobial agents.

On the other hand, cancer remains to be the leading cause of death in humans second only to cardiovascular diseases and more than 70% of all cancer deaths occur in developing and under-developed countries.^5,6 Cancers have been reported as one of the Global Burden of Diseases (World Health Organization, 2008) and are estimated to be one of main causes of death in the coming decades.^7,8 Breast cancer is one of the most commonly diagnosed cancers, accounting for ∼20% of all malignancies worldwide and over half a million women develop breast cancer every year. In India, almost 100 000 women are diagnosed with breast cancer every year and a rise to 131 000 cases is predicted by 2020.^9,10 Many current breast cancer chemotherapy treatments are often associated with side effects and the development of drug resistance in cancer cells, whereby majority of the patients succumb to their disease within 2 years of diagnosis. Hence, the design and development of new drugs for different types of breast cancer therapeutics remains to be an important and challenging task for medicinal chemist's worldwide.^11–13

Similarly, numerous studies have demonstrated that in addition to cancer, anti-oxidants plays an important role in the body defense mechanism by regulating the generation and elimination of reactive oxygen species (ROS) like hydroxyl radicals, superoxide radicals etc. which are generated from excessive oxidative stress and normal metabolic activities. The oxidative stress reflects an imbalance between the oxidants and the antioxidant favoring the oxidants implies damage of all essential bio-compounds like proteins, DNA and membrane lipids and can result in cell death.¹⁴ There is increasing evidence showing the involvement of oxidative stress induced by free radicals and reactive oxygen species (ROS) in a variety of diseases and pathophysiological events including inflammation, cancer, myocardial infraction, arthritis and neurodegenerative disorders.¹⁵ Antioxidants can minimize or inhibit the oxidative damage by interrupting the free radical formation or terminating the chain reaction. Antioxidants may slow or possibly prevent the development of the above mentioned diseases.^16,17 On the other hand, many chemotherapeutic agents act by producing free radicals, causing oxidative stress in normal cells.¹⁸ A mono therapy of an anticancer drug with antioxidant properties will probably become more advantageous from the pharmaco-economic point of view. Thus, the discovery and development of novel anti-oxidants attained great importance in organic chemistry.

In recent years, heterocyclic compounds containing nitrogen plays important role in agrochemical and pharmaceuticals. In the family of nitrogen heterocycles, triazoles and their derivatives occupy a central position due to their biologically active nature.¹⁹ Moreover, 1,2,3-triazoles derivatives present in various medicinal agents were capable of forming hydrogen bonds which in turn improves their solubility and ability to interact with bimolecular targets.²⁰ Most of the literature studies revealed that, 1,2,3-triazoles are the key moieties in heterocyclic chemistry and have been used for broad therapeutic applications due to their diverse biological activities.²¹ They are known to possess a wide range of pharmacological activities like anti-microbial,^22,23 anti-convulsant,²⁴ anti-tubercular,²⁵ anti-diabetic,²⁶ anti-malarial,²⁷ anti-oxidant,²⁵ and anti-cancer activities.^28,29 Similarly, chalcones and their derivatives are abundant in edible plants where they are considered to be the precursors of flavonoids or isoflavonoids³⁰ and constitute an important group of natural and synthetic products with wide range of pharmacological activities as its derivatives found to exhibit variety of biological and pharmacological activities, including effects as antimicrobial,^31,32 anti-inflammatory,³³ anti-oxidant,³⁴ anti-malarial,³⁵ anticonvulsant,³⁶ anticancer,^37,38 etc. Fig. 1.

Fig. 1 Examples of biologically active 1,2,3-triazole and chalcone pharmacophores.

In view of the above mentioned findings and in-continuation of our interest in exploration of novel heterocyclic scaffolds for anti-microbial, anti-oxidant and anti-cancer activities we hereby report the synthesis of some novel 1,2,3-triazolyl-chalcones as key nucleus and evaluate their anti-microbial, anti-oxidant and anti-cancer potential with the hope of improving its biological activities. Most of the compounds exhibits good biological activities. The synthetic strategy and representative examples of 1,2,3-triazole-chalcones via a molecular hybridization technique was represented in Fig. 2.

Fig. 2 Representative examples A and B of anticancer, antimicrobial and anti-oxidant active heterocyclic molecules possessing 1,2,3-triazole, chalcone and pyrazole moieties and targeted molecules (5a–k) and (6a–e).

2. Results and discussion

2.1. Chemistry

The synthetic protocol for the title compounds, (2E)-1-{1-[2,3-dichloro-6-methyl-5-(trifluoromethyl)phenyl]-5-methyl-1H-1,2,3-triazol-4-yl}-3-aryl prop-2-en-1-one (5a–k) and (2E)-1-{1-[2,3-dichloro-6-methyl-5-(trifluoromethyl)phenyl]-5-methyl-1H-1,2,3-triazol-4-yl}-3-(1,3-diaryl-1H-pyrazol-4-yl)prop-2-en-1-one (6a–e) was outlined in Scheme 1, and were synthesized by the Claisen–Schmidt condensation of 1-{1-[2,3-dichloro-6-methyl-5-(trifluoromethyl)phenyl]-5-methyl-1H-1,2,3-triazol-4-yl}ethanone (4) with different aryl and 1,3-diaryl-1H-pyrazole-4-carbaldehydes iii(a-e) in presence of ethanol and aqueous sodium hydroxide mixture respectively with good yields (70–80%). The key starting material 1-{1-[2,3-dichloro-6-methyl-5-(trifluoromethyl)phenyl]-5-methyl-1H-1,2,3-triazol-4-yl}ethanone (4) were synthesized by 1,3-dipolar cyclo addition reaction between 3-azido-1,2-dichloro-4-methyl-5-(trifluoromethyl)benzene (3) and acetyl acetone in presence of sodium methoxide in methanol. The intermediate 3-azido-1,2-dichloro-4-methyl-5-(trifluoromethyl)benzene (3) were synthesized from 1,2-dichloro-4-methyl-3-nitro-5-(trifluoromethyl)benzene (1) following literature procedures.³⁹

Scheme 1 Synthetic scheme for the compounds (5a–k) and (6a–e). (A) Zn/HCl/methanol (B) NaNO₂/HCl/0–5 °C and NaN₃/H₂O/0–5 °C (C) NaOCH₃/acetyl acetone/methanol/50 °C (D) ArCHO/NaOH/ethanol/30 °C (E) iii(a-e)/NaOH/ethanol (Claisen–Schmidt reaction). (a) AcOH/NaOH/30 °C (b) DMF/POCl₃/70–75 °C (Vilsmeier–Haack reagent).

Structures of all the synthesized compounds (5a–n) and (6a–e) were established on the basis of their spectral (IR, NMR and mass) and elemental (C, H and N) analysis. Analytical and spectral data of all the synthesized compounds were in full agreement with the proposed structures and also discussed for a representative compound 5g: the IR spectrum of (5g) showed absorption peak at 2936 cm⁻¹ assigned to aromatic C–H stretch. The peak for C=N was observed at 1573 cm⁻¹. The peak at 1593 and 1256 cm⁻¹ were assigned to C=O and C–O stretch respectively. The medium absorption at 1177 and 824 cm⁻¹ was due to the presence of C–F and C–Cl bonds respectively. The ¹H NMR spectrum of (5g) showed three singlet's at δ (ppm) 2.09, 2.50 and 3.87 were assigned to methyl protons of 1,2,3-triazole ring, 2,3-dichloro-6-methyl-5-(trifluoromethyl)phenyl and 4-methoxy phenyl respectively. The two distinct doublet signals at δ 6.94 and 7.69 (J = 8.6 Hz) integrating to four protons were assigned to aromatic protons of 4-methoxy phenyl ring. The singlet signal at δ 8.00 were assigned to aromatic protons of 2,3-dichloro-6-methyl-5-(trifluoromethyl)phenyl moiety. Similarly two doublets at δ 7.92 and 7.98 (J = 15.8 Hz) integrating for two protons were assigned to two olefin (CH=CH) protons. The signals due to carbonyl methyl protons at δ 2.77 were disappeared indicating the formation of the compound (5c). The ¹³C NMR spectrum of (5g) showed signals at δ (ppm) 9.23, 14.29, 55.42 and 184.00 corresponds to three methyl carbons of 1,2,3-triazole, 2,3-dichloro-6-methyl-5-(trifluoromethyl)phenyl, 4-methoxy phenyl ring and carbonyl carbon attached to triazole ring respectively along with other characteristic signals. Similarly for compound (6e), the IR spectrum showed absorption peak at 3066 cm⁻¹ assigned to aromatic C–H stretch. The peak for C=N was observed at 1567 cm⁻¹. The peak at 1668 and 1260 cm⁻¹ were assigned to C=O and C–O stretch respectively. The medium absorption at 1177, 1035 and 824 cm⁻¹ was due to the presence of C–F, C–Br and C–Cl bonds respectively. The ¹H NMR spectrum of (6e) showed two singlet's at δ (ppm) 2.09 and 2.49 were assigned to methyl protons of 1,2,3-triazole ring and 2,3-dichloro-6-methyl-5-(trifluoromethyl)phenyl respectively. The distinct triplet signal at δ 7.34 (J = 7.6 Hz) integrating to two protons were assigned to aromatic protons of phenyl and 3-bromo phenyl ring respectively. The three singlet signals at δ 7.94, 7.99 and 8.53 were assigned to aromatic protons of 3-bromo phenyl, 2,3-dichloro-6-methyl-5-(trifluoromethyl)phenyl and pyrazole moiety respectively. Similarly three doublets at δ 7.51, 7.73 and 7.81 (J = 8.0 Hz) integrating for six protons were assigned to aromatic protons of phenyl and 3-bromo phenyl ring respectively. Similarly two doublets at δ 8.0 and 8.02 (J = 17.0 Hz) integrating for two protons were assigned to two olefin (CH=CH) protons. The signals due to carbonyl methyl protons at δ 2.77 were disappeared indicating the formation of the compound (6e). The ¹³C NMR spectrum of (6e) showed signals at δ (ppm) 9.29, 14.24 and 184.10 corresponds to two methyl carbons of 1,2,3-triazole and 2,3-dichloro-6-methyl-5-(trifluoromethyl)phenyl ring and carbonyl carbon attached to triazole ring respectively along with other characteristic signals. The IR, ¹H NMR and ¹³C NMR spectra of other compounds (5a–k) and (6a–e) has shown similar characteristic properties. The physicochemical data of the compounds (5a–k) and (6a–e) were presented in Table 1.

Table 1 Characterization data of the compounds (5a–k) and (6a–e)

Comp.	Ar	Mol. formula	Mol. wt	Mp (°C)	Yield (%)	C, H, N analysis (%)
						Found (calculated)
						C	H	N
5a	Phenyl	C20H14Cl2F3N3O	440.2	145–147	71	54.58 (54.56)	3.24 (3.21)	9.58 (9.54)
5b	3-Cl-2F-phenyl	C20H12Cl2F4N3O	492.7	178–180	70	48.78 (48.76)	2.44 (2.45)	8.55 (8.53)
5c	4-OH-phenyl	C20H14Cl2F3N3O2	456.2	148–150	72	52.62 (52.65)	3.08 (3.09)	9.25 (9.21)
5d	3,4,-(CH3)-phenyl	C22H18Cl2F3N3O	468.3	152–154	74	56.40 (56.42)	3.85 (3.87)	8.92 (8.97)
5e	4-F-phenyl	C20H13Cl2F4N3O	458.2	162–164	72	52.42 (52.42)	2.88 (2.86)	9.19 (9.17)
5f	4-Cl-phenyl	C20H13Cl3F3N3O	474.7	167–169	70	50.62 (50.60)	2.78 (2.76)	8.82 (8.85)
5g	4-OCH3-phenyl	C21H16Cl2F3N3O2	470.3	168–170	74	53.56 (53.63)	3.47 (3.43)	8.98 (8.94)
5h	2-F-phenyl	C20H13Cl2F4N3O	458.2	169–171	71	52.43 (52.42)	2.89 (2.86)	9.19 (9.17)
5i	2-Cl-phenyl	C20H13Cl3F3N3O	474.7	172–174	73	50.64 (50.60)	2.79 (2.76)	8.83 (8.85)
5j	3-Pyridyl	C19H13Cl2F3N4O	441.2	179–181	70	51.67 (51.72)	2.99 (2.97)	12.76 (12.70)
5k	4-SCH3-phenyl	C21H16Cl2F3N3OS	486.3	180–182	73	51.90 (51.86)	3.35 (3.32)	8.70 (8.64)

---

Comp.	Ar2	Ar1	Mol. formula	Mol. wt	Mp (°C)	Yield (%)	C, H, N analysis (%)
							Found (calculated)
							C	H	N
6a	Phenyl	Phenyl	C29H20Cl2F3N5O	582.4	214–216	71	59.75 (59.81)	3.43 (3.46)	12.06 (12.02)
6b	4-Cl-phenyl	Phenyl	C29H19Cl3F3N5O	616.8	226–228	73	56.50 (56.47)	3.13 (3.10)	11.32 (11.35)
6c	Phenyl	4-Cl-phenyl	C29H19Cl3F3N5O	616.8	231–233	72	56.51 (56.47)	3.12 (3.10)	11.37 (11.35)
6d	3-Br-phenyl	4-Cl-phenyl	C29H18BrCl3F3N5O	695.7	256–258	72	50.10 (50.06)	2.57 (2.61)	10.11 (10.07)
6e	3-Br-phenyl	Phenyl	C29H19BrCl2F3N5O	661.3	237–239	73	52.65 (52.67)	2.93 (2.90)	10.62 (10.59)

---

2.2. Biological studies

2.2.1. In vitro anti-microbial activity. The anti-microbial activities of the newly synthesized compounds (5a–k) and (6a–e) were tested against four different bacteria and three different funguses. Two Gram-positive strains Staphylococcus aureus (MTCC 1430) and Bacillus subtilis (MTCC 441). Two Gram-negative strains were Escherichia coli (MTCC 1573) and Pseudomonas aeruginosa (MTCC 424). All bacterial strains were maintained on nutrient agar medium at 37 °C. Fungi: Aspergillus flavus, Chrysosporium keratinophilum and Candida albicans (MTCC 227) was used in this study. All fungi strains were maintained on potato dextrose agar (PDA) at 25 °C.

The synthesized compounds was first dissolved in DMSO to a different concentration (1 and 0.5 mg ml⁻¹), and then sterilized by filtration through 0.22 μm Millipore filters. The antibacterial activity of newly synthesized compounds (5a–k) and (6a–e) was determined by well plate method in nutrient agar media.⁴⁰ The compounds were tested against a panel of pathogenic microorganisms, including Escherichia coli (MTCC 1573), Bacillus subtilis (MTCC 441), Staphylococcus aureus (MTCC 1430) and Pseudomonas aeruginosa (MTCC 424). Microorganism strains were maintained on nutrient agar medium at 37 °C. The cultures were inoculated in fresh 10 ml nutrient broth to yield an initial suspension of approximately 10–100 cfu ml⁻¹. All broths were then incubated statically at the aforementioned temperatures for microorganisms, for 18–24 h so that all cells were in the stationary phase. Susceptibility of the test organism to the compounds was determined by employing in the well plate technique. The bacterial suspensions were diluted tenfold in sterilized distilled water, and 0.1 ml from the appropriate dilution was spread plated on nutrient agar in order to give a population approximately 10⁶ cfu per plate. Six millimeter diameter well were then punched carefully using a sterile cork borer and 30 μl of test solutions of different concentrations were added into each labeled well. The same procedure was repeated for different micro-organisms. Each experiment was carried out in triplicate. After the incubation, the inhibition zone was measured and the values for DMSO were subtracted to get the actual values. Ciproflaxin was used as standard drug. MIC (Minimum Inhibitory Concentration) was determined according to the broth micro dilution method described, with suitable modifications.⁴¹ The test compounds were first (1.5 mg ml⁻¹) dissolved in DMSO and then sterilized by 0.5 μm filtration. The final concentration of DMSO in each medium was maintained 1%, so that it does not affect the growth of the test strain. Broth micro dilution methods were used as previously described with slight modifications.⁴¹ Briefly, logarithmic serial two-fold dilutions (serial dilution method) of the test compounds and controls were prepared in DMSO, and 30 μl of each dilution was added to 3 ml of nutrient broth. Thirty microliters of the exponentially growing bacterial cells of Escherichia coli (MTCC 1573), Bacillus subtilis (MTCC 441), Staphylococcus aureus (MTCC 1430) and Pseudomonas aeruginosa (MTCC 424) (final 5.0 × 10⁵ cfu ml⁻¹) were inoculated into the broth. After the cultures were incubated at 37 °C for 24 h, the MIC (Minimum Inhibitory Concentration) value, representing the lowest concentration (highest dilution) that completely inhibited the formation of visible growth, was evaluated after 24 h of incubation at 37 °C. The experiments were performed in triplicates.

Antifungal studies of newly synthesized compounds (5a–k) and (6a–e) were determined by well plate method against Aspergillus flavus, Chrysosporium keratinophilum and Candida albicans (MTCC 227). Normal saline was used to make a suspension of spore of fungal strains for lawning.⁴² A loop full of particular fungal strain was transferred to 3 ml saline to get a suspension of corresponding species. Twenty milliliters of agar media were poured into each Petri dish. Excess of suspension was decanted and plates were dried by placing in an incubator at 37 °C for 1 h. Using sterile cork borer punched carefully, wells were made on these seeded agar plates different concentrations of the test compounds in DMSO were added into each labeled well. A control was also prepared for the plates in the same way using solvent DMSO. The Petri dishes were prepared in triplicate and maintained at 25 °C for 72 h. Antifungal activity was determined by measuring the diameter of inhibition zone. The activity of each compound was compared with fluconazole as standard. The MIC and MFC (Minimum Fungicidal Concentration) was determined by broth micro dilution method. The nutrient broth, which contained logarithmic serially twofold diluted amount of test compounds and controls, was inoculated with approximately 1.6 × 10⁴ to 6 × 10⁴ cfu ml⁻¹ was used. The cultures were incubated for 48 h at 35 °C and the growth was monitored. The lowest concentration (highest dilution) required to arrest the fungus growth was regarded as MIC (Minimum Inhibitory Concentration). To obtain MFC (Minimum Fungicidal Concentration), 0.1 ml volume was taken from each tube and spread on agar plates. The number of cfu was counted after 48 h of incubation at 35 °C. MFC was defined as the lowest drug concentration at which 99.9% of the inoculum was killed. The experiments were performed in triplicates.

The antibacterial activity evaluation of newly synthesized compounds (5a–k) and (6a–e) revealed that, most of the tested compounds exhibited moderate to very good antibacterial activity against all the tested different strains (Tables 2 and 3). Among them, the compounds 5d, 5h, 5i, 5j, 5k, 6b and 6d exhibited very good activities against all four tested bacterial strains namely Escherichia coli (ZOI = 14–19 mm and MIC = 3.12–6.25 μg ml⁻¹), Staphylococcus aureus (ZOI = 13–17 mm and MIC = 3.15–12.5 μg ml⁻¹), Pseudomonas aeruginosa (ZOI = 12–17 mm and MIC = 3.13–12.5 μg ml⁻¹) and Bacillus subtilis (ZOI = 13–16 mm and MIC = 3.12–6.5 μg ml⁻¹) with respect to the standard drug Ciproflaxin (ZOI = 17–24 mm and MIC = 3.10–3.25 μg ml⁻¹ against all strains) at 0.5–1 mg ml⁻¹ concentration. Similarly the derivatives 5b, 5c, 5e, 5f, 6c and 6e shown good activities against tested two Gram positive and two Gram negative bacterial strains namely Escherichia coli (ZOI = 11–17 mm and MIC = 6.12–12.5 μg ml⁻¹), Staphylococcus aureus (ZOI = 6–17 mm and MIC = 7.15–13.2 μg ml⁻¹), Pseudomonas aeruginosa (ZOI = 9–15 mm and MIC = 6.25–12.5 μg ml⁻¹) and Bacillus subtilis (ZOI = 9–16 mm and MIC = 6.25–14.2 μg ml⁻¹) when compared to the standard drug Ciproflaxin. The compounds 5a, 5g and 6a were non-significant with ZOI = 07–15 mm and MIC = 6.25–25 μg ml⁻¹ as it shows moderate inhibitory activity towards the panel of human pathogens.

Table 2 Determination of anti-bacterial activity of the synthesized compounds (5a–k) and (6a–e)

Organic compounds	Escherichia coli		Staphylococcus aureus		Pseudomonas aeruginosa		Bacillus subtilis
Concn. in mg ml^−1	1	0.5	1	0.5	1	0.5	1	0.5
ZOI in mm^a	mm	mm	mm	mm	mm	mm	mm	mm
a The experiment was performed in triplicate and the values are expressed as mean ± SD.
Control	00	00	00	00	00	00	00	00
Ciproflaxin	24 ± 0.2	18 ± 0.1	21 ± 0.2	17 ± 0.2	19 ± 0.2	17 ± 0.2	21 ± 0.4	18 ± 0.3
5a	12 ± 0.3	09 ± 0.4	12 ± 0.3	11 ± 0.4	09 ± 0.1	07 ± 0.3	13 ± 0.2	10 ± 0.3
5b	15 ± 0.2	11 ± 0.2	11 ± 0.3	08 ± 0.2	12 ± 0.2	09 ± 0.3	15 ± 0.2	12 ± 0.3
5c	16 ± 0.2	12 ± 0.3	13 ± 0.3	10 ± 0.2	14 ± 0.2	10 ± 0.4	13 ± 0.3	09 ± 0.3
5d	19 ± 0.3	15 ± 0.4	17 ± 0.1	14 ± 0.4	16 ± 0.3	13 ± 0.6	16 ± 0.5	14 ± 0.2
5e	17 ± 0.3	14 ± 0.1	11 ± 0.2	06 ± 0.3	15 ± 0.1	13 ± 0.2	14 ± 0.2	11 ± 0.2
5f	15 ± 0.1	13 ± 0.2	13 ± 0.2	11 ± 0.3	15 ± 0.3	13 ± 0.4	11 ± 0.3	09 ± 0.1
5g	14 ± 0.3	11 ± 0.2	12 ± 0.3	10 ± 0.2	12 ± 0.3	10 ± 0.2	15 ± 0.3	12 ± 0.2
5h	18 ± 0.4	14 ± 0.3	16 ± 0.3	13 ± 0.2	15 ± 0.2	13 ± 0.2	15 ± 0.2	13 ± 0.3
5i	19 ± 0.4	15 ± 0.3	16 ± 0.3	14 ± 0.2	15 ± 0.2	13 ± 0.2	15 ± 0.2	13 ± 0.3
5j	19 ± 0.2	16 ± 0.1	15 ± 0.5	13 ± 0.3	16 ± 0.4	14 ± 0.2	15 ± 0.3	13 ± 0.5
5k	18 ± 0.4	15 ± 0.3	15 ± 0.3	13 ± 0.2	14 ± 0.2	12 ± 0.2	16 ± 0.2	14 ± 0.3
6a	15 ± 0.4	12 ± 0.3	14 ± 0.3	11 ± 0.2	10 ± 0.2	07 ± 0.2	12 ± 0.2	10 ± 0.3
6b	17 ± 0.1	15 ± 0.2	16 ± 0.2	13 ± 0.3	17 ± 0.3	13 ± 0.3	15 ± 0.2	13 ± 0.2
6c	14 ± 0.4	12 ± 0.3	13 ± 0.5	10 ± 0.3	13 ± 0.4	11 ± 0.2	13 ± 0.3	11 ± 0.5
6d	18 ± 0.2	14 ± 0.3	16 ± 0.2	13 ± 0.3	16 ± 0.3	14 ± 0.2	15 ± 0.3	13 ± 0.2
6e	14 ± 0.3	13 ± 0.4	17 ± 0.1	14 ± 0.2	13 ± 0.3	10 ± 0.2	16 ± 0.3	14 ± 0.2

---

Table 3 Minimum inhibitory concentration (MIC) of the synthesized compounds (5a–k) and (6a–e)

Organic compounds in μg ml^−1^a	Escherichia coli	Staphylococcus aureus	Pseudomonas aeruginosa	Bacillus subtilis
a The experiment was performed in triplicate and the values are expressed as mean.
5a	12.5	12.5	25	12.5
5b	6.52	12.5	12.5	6.25
5c	6.25	12.5	12.5	12.5
5d	6.25	3.15	12.5	6.25
5e	6.12	13.2	6.25	12.5
5f	6.25	12.5	6.25	14.2
5g	12.5	12.5	12.5	6.25
5h	6.25	6.25	12.5	6.25
5i	6.25	6.25	6.25	6.25
5j	12.5	6.25	3.13	6.25
5k	3.12	6.25	12.5	3.12
6a	6.25	12.5	25.0	12.5
6b	3.15	12.5	3.15	6.25
6c	12.5	12.5	12.5	6.25
6d	3.15	6.25	12.5	6.50
6e	6.25	7.15	12.5	6.25
Ciproflaxin	3.25	3.15	3.12	3.10

---

Among the newly synthesized compounds (5a–k) and (6a–e) most of the compounds inhibited the growth of most of the human pathogenic fungi tested (Tables 4 and 5). The sensitivity of the fungal strains varies according to the species. Among the tested fungi, Candida albicans was highly sensitive compared to the other fungi species. Aspergillus flavus and Chrysosporium keratinophilum showed some differing responses to each organic compound. The compounds 5i, 5j and 6d showed very good activity against tested fungal strains Aspergillus flavus with ZOI = 9–12 mm and MIC = 7.7–7.9 μg ml⁻¹ with MFC = 16.1–16.8 μg ml⁻¹, Chrysosporium keratinophilum with ZOI = 13–15 mm and MIC = 8.15–8.49 μg ml⁻¹ with MFC = 16.6–17.9 μg ml⁻¹ and Candida albicans with ZOI = 15–18 mm and MIC = 4.95–5.35 μg ml⁻¹ with MFC = 7.38–7.75 μg ml⁻¹ respectively when compared standard drug fluconazole with ZOI = 12–22 mm and MIC = 3.12–6.25 μg ml⁻¹ with MFC = 6.18–12.7 μg ml⁻¹ against all the tested strains at 0.5–1 mg ml⁻¹ concentration. On the other hand, derivatives 5b, 5e, 5f, 5h, 5k, 6b and 6c shown good activity with tested fungal strains Aspergillus flavus (ZOI = 8–12 mm and MIC = 8.35–8.73 μg ml⁻¹ with MFC = 18.3–19.2 μg ml⁻¹), Chrysosporium keratinophilum (ZOI = 11–14 mm and MIC = 8.7–9.15 μg ml⁻¹ with MFC = 18.9–19.9 μg ml⁻¹) and Candida albicans (ZOI = 13–16 mm and MIC = 6.85–7.95 μg ml⁻¹ with MFC = 11.8–12.8 μg ml⁻¹) against the standard drug fluconazole with the tested fungal strains. The rest of the synthesized compounds failed to show good and comparable antifungal activity with ZOI = 5–12 mm and MIC = 10.5–25.8 μg ml⁻¹ with MFC = 50–100 μg ml⁻¹ against the panel of tested fungal strains when compared to the standard drug fluconazole.

Table 4 Determination of anti-fungal activity of the synthesized compounds (5a–k) and (6a–e)

Organic compound	Aspergillus flavus		Chrysosporium keratinophilum		Candida albicans
Concn. in mg ml^−1	1	0.5	1	0.5	1	0.5
ZOI in mm^a	mm	mm	mm	mm	mm	mm
a The experiment was performed in triplicate and the values are expressed as mean ± SD.
Control	00	00	00	00	00	00
Fluconazole	14 ± 0.3	12 ± 0.4	17 ± 0.4	15 ± 0.5	22 ± 0.2	20 ± 0.3
5a	06 ± 0.2	04 ± 0.3	07 ± 0.2	06 ± 0.2	08 ± 0.2	05 ± 0.2
5b	11 ± 0.3	08 ± 0.2	14 ± 0.2	11 ± 0.3	16 ± 0.1	14 ± 0.1
5c	08 ± 0.2	05 ± 0.3	08 ± 0.1	04 ± 0.1	12 ± 0.3	07 ± 0.3
5d	08 ± 0.3	07 ± 0.2	10 ± 0.2	07 ± 0.3	11 ± 0.1	07 ± 0.1
5e	11 ± 0.2	09 ± 0.3	13 ± 0.4	11 ± 0.2	16 ± 0.4	14 ± 0.3
5f	12 ± 0.3	08 ± 0.2	14 ± 0.3	11 ± 0.4	15 ± 0.2	13 ± 0.4
5g	07 ± 0.3	05 ± 0.2	12 ± 0.1	10 ± 0.3	12 ± 0.4	10 ± 0.2
5h	11 ± 0.4	09 ± 0.3	14 ± 0.3	11 ± 0.2	16 ± 0.3	14 ± 0.2
5i	12 ± 0.2	09 ± 0.3	15 ± 0.3	13 ± 0.3	18 ± 0.3	15 ± 0.1
5j	12 ± 0.3	09 ± 0.6	15 ± 0.4	13 ± 0.4	18 ± 0.2	15 ± 0.2
5k	09 ± 0.3	08 ± 0.2	13 ± 0.2	11 ± 0.3	16 ± 0.1	14 ± 0.1
6a	08 ± 0.2	06 ± 0.3	11 ± 0.3	09 ± 0.2	11 ± 0.3	08 ± 0.2
6b	11 ± 0.3	09 ± 0.2	13 ± 0.2	11 ± 0.2	16 ± 0.2	14 ± 0.2
6c	11 ± 0.1	08 ± 0.4	14 ± 0.2	11 ± 0.3	16 ± 0.3	14 ± 0.4
6d	12 ± 0.4	09 ± 0.2	15 ± 0.2	13 ± 0.3	18 ± 0.3	15 ± 0.3
6e	09 ± 0.3	07 ± 0.4	10 ± 0.2	07 ± 0.4	11 ± 0.2	07 ± 0.3

---

Table 5 MIC and MFC of the synthesized compounds (5a–k) and (6a–e)

Organic compounds in μg ml^−1^a	Aspergillus flavus		Chrysosporium keratinophilum		Candida albicans
	MIC	MFC	MIC	MFC	MIC	MFC
a The experiment was performed in triplicate and the values are expressed as mean and values were expressed as μg ml^−1.
5a	15.5	50.5	16.3	62.5	10.5	50
5b	8.52	18.5	8.7	18.9	7.5	12.5
5c	19.5	70.5	20.8	80.5	13.5	75
5d	20.4	74.5	22.8	85.5	14.8	80
5e	8.73	18.8	8.9	18.9	7.8	12.7
5f	8.42	18.4	8.76	19.2	7.95	12.8
5g	22.5	95.5	23.9	95.5	15.6	85
5h	8.44	18.3	8.72	19.2	7.85	12.7
5i	7.8	16.2	8.25	16.8	5.25	7.55
5j	7.9	16.8	8.49	17.9	4.95	7.38
5k	8.68	18.9	8.95	19.8	7.65	12.5
6a	22.8	98	23.9	95.5	15.6	85
6b	8.35	19.2	9.15	19.9	6.95	12.2
6c	8.7	18.8	8.85	19.8	6.85	11.8
6d	7.7	16.1	8.15	16.6	5.35	7.75
6e	23.8	100	25.8	100	15.8	100
Fluconazole	6.25	12.5	6.18	12.7	3.12	6.18

---

Further, the structure–activity relationship of the compounds (5a–k) and (6a–e) revealed that the compounds 5d, 5h, 5i, 5j, 5k, 6b and 6d containing 3,4-dimethyl phenyl, 2-fluoro phenyl, 2-chloro phenyl, 3-pyridyl, 4-thiomethyl phenyl, 1-(phenyl)-3-(4-chloro phenyl)-1H-pyrazole and 1-(4-chloro phenyl)-3-(3-bromo phenyl)-1H-pyrazole substituent's at the 3^rd position of prop-2-en-1-one (chalcone) moiety respectively were found to exhibit enhanced anti-bacterial activity compared to the less or unsubstituted phenyl derivatives (i.e. like 5a, 6a) against the standard drug Ciproflaxin. While the derivatives 5i, 5j and 6d also found to exhibit enhanced anti-fungal activities against tested fungal strains compared to standard drug fluconazole. Similarly the molecules 5b, 5c, 5e, 5f, 5h, 6c and 6e containing 3-chloro-2-fluoro phenyl, 4-hydroxy phenyl, 4-fluoro phenyl, 4-chloro phenyl, 2-fluoro phenyl, 1-(4-chloro phenyl)-3-(phenyl)-1H-pyrazole, 1-(phenyl)-3-(3-bromo phenyl)-1H-pyrazole groups respectively at 3^rd position of chalcone were found to exhibit good antimicrobial activities. Further SAR, reveals that the electron donating groups like methyl, thiomethyl, pyridyl and electron donating groups like chloro, fluoro in the molecules at different positions were generally more beneficial than the unsubstituted ones.

2.2.2. In vitro anti-oxidant activity. In order to investigate the possible biological studies for the synthesized compounds (5a–k) and (6a–e), were also screened for their in vitro antioxidant activity by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging method using ascorbic acid as standard drug.⁴³ The spectrophotometric assay uses the stable radical DPPH as a reagent.

21 mg of DPPH was dissolved with methanol in 100 ml volumetric flask to get DPPH stock solution. Further, 18 ml of above DPPH stock solution was pipette out into a 100 ml volumetric flask and diluted with methanol to obtain 100 μM DPPH working solution.

The sample solution was prepared by mixing 1 ml of various concentrations of the test compounds (5a–n) (62.5 μg ml⁻¹, 31.25 μg ml⁻¹, 15.62 μg ml⁻¹) in DMSO with 5 ml DPPH working solution (100 μM) in each test tube. 1 ml of DMSO was taken in a test tube and then 5 ml of DPPH working solution (100 μM) was added and kept as control sample. 1 ml of DMSO was taken in a test tube and then 5 ml of methanol was added and kept as blank. These test tubes were incubated at 37 °C for 20 min and the absorbance was determined by using UV spectrophotometer at 517 nm. The % inhibition was calculated by using formula,

% inhibition = [(Abs_control − Abs_sample)/Abs_control] × 100

where, Abs_control is the absorbance of the control reaction (containing all reagents, except the test compound) and Abs_sample is the absorbance of the test compound. Tests were carried at in triplicates. The IC₅₀ value was obtained by plotting the graph, taking % inhibition on y-axis and concentration on x-axis.⁴³

The anti-oxidant activity of the compounds (5a–k) and (6a–e) were further evaluated by ABTS (2,2′-azino bis(3-ethyl benzothiazoline-6-sulfonic acid)) antioxidant assay method by bleaching of ABTS derived radical cations. The radical cation derived from ABTS was prepared by reaction of 0.06 ml ABTS with 3 ml MnO₂ (25 mg ml⁻¹) in 5 ml aqueous buffer solution (pH 7). After shaking the solution for a few minutes, it was centrifuged and filtered. The absorbance (A_control) of the resulting green-blue solution (ABTS radical solution) was recorded at λ_max = 734 nm. The absorbance (A_test) was measured upon the addition of 0.02 ml of 1 mg ml⁻¹ solution of the tested sample in spectroscopic grade methanol/buffer (1 : 1 v/v) to the ABTS solution. The inhibition ratio (%) was calculated using the following formula:

% inhibition = [(A_control − A_test)/A_control] × 100

Ascorbic acid (0.02 ml, 2 mm) solution was used as a standard anti-oxidant (positive control). Blank sample was run using solvent without ABTS.

Evaluation of antioxidant activity revealed that, most of the compounds tested compounds exhibited moderate to excellent DPPH and ABTS radical scavenging ability compared with the positive control ascorbic acid (Table 6). Among the synthesized compounds, compounds 5a, 5d, 5g, 5j, 5k and 6a bearing phenyl, 3,4-dimethyl phenyl, 4-methoxy phenyl, 3-pyridyl, 4-thiomethyl and 1,3-(biphenyl)-1H-pyrazole substituent's at the 3^rd position of prop-2-en-1-one (chalcone) moiety respectively were found to be more effective and potent DPPH radical scavenging ability with IC₅₀ values 16.36 μM, 15.33 μM, 15.38 μM, 17.48 μM, 16.99 μM and 14.48 μM compared to the standard drug ascorbic acid with IC₅₀ value 12.27 μM at 31.5 μg ml⁻¹ concentration by DPPH radical scavenging activity method. The anti-oxidant activity of the derivatives 5a, 5d, 5g, 5j, 5k and 6a were further illustrated by ABTS assay method with 78.5%, 80.4%, 81.2%, 76.9%, 77.8% and 81.8% inhibition against the positive control ascorbic acid with 88.5% inhibition. Further the derivatives 5f, 5h, 6b, 6c and 6e containing 4-chloro phenyl, 2-fluoro phenyl, 1-(phenyl)-3-(4-chloro phenyl)-1H-pyrazole, 1-(4-chloro phenyl)-3-(phenyl)-1H-pyrazole and 1-(phenyl)-3-(3-bromo phenyl)-1H-pyrazole exhibited moderate DPPH radical scavenging activities with IC₅₀ values 43.76 μM, 45.33 μM, 34.91 μM, 45.48 μM and 39.82 μM respectively with moderate ABTS radical inhibition with 53.1%, 58.6%, 63.5%, 59.7% and 58.6% respectively compared with the positive control ascorbic acid. The remaining compounds non-significant radical scavenging activity with IC₅₀ values in the range of 50.21–255.13 μM and 15.8–43.3% inhibition in DPPH method and ABTS assay methods respectively. Generally according to structure activity relationship (SAR), it was observed that the compounds with less or unsubstituted phenyl at C-1 and C-3 of the pyrazole ring like in 6a, electron donating groups like methyl, methoxy, thiomethyl at C-3 and C-4 position of the phenyl ring as in 5d, 5g, 5k and heterocyclic pyridyl on 3^rd position of the prop-2-en-1-one (chalcone) (i.e. 5j) moiety were found to possess potent radical scavenging ability than the corresponding electron withdrawing substituents in phenyl rings (i.e. 5b, 5c, 6b, 6c etc.).

Table 6 Antioxidant activity of the synthesized compounds (5a–k) and (6a–e) by DPPH method

Compound	Concentration 15.62–62.5 μg ml^−1	IC50 ± SD (μg ml^−1)	% inhibition
	% inhibition^a	DPPH method^a	ABTS method^b
a Data presented is the mean ± SD value of three independent determinations.b Data presented is the mean value of three independent determinations.
5a	37.30–87.12	16.36 ± 1.633	78.5
5b	8.92–71.42	50.21 ± 1.241	35.6
5c	2.57–58.2	255.13 ± 3.697	15.8
5d	38.81–90.83	15.33 ± 1.727	80.4
5e	3.81–76.12	67.87 ± 1.357	25.3
5f	2.7–81.07	43.76 ± 1.898	53.1
5g	39.33–89.24	15.38 ± 1.635	81.2
5h	21.7–73.08	45.53 ± 1.629	58.6
5i	3.4–80.12	62.9 ± 1.286	43.2
5j	34.61–85.89	17.48 ± 1.627	76.9
5k	35.67–83.63	16.99 ± 1.584	77.8
6a	42.7–91.18	14.48 ± 1.629	81.8
6b	32.9–78.82	34.91 ± 1.629	63.5
6c	6.81–71.55	45.48 ± 1.432	59.7
6d	4.87–70.22	62.94 ± 1.267	43.3
6e	28.9–72.23	39.82 ± 1.425	58.6
Ascorbic acid	44.95–95.5	12.7 ± 0.68	88.5
Blank	—	—	—

---

2.2.3. In vitro anti-cancer activity. Cytotoxicity of all the synthesized compounds were determined on the basis of measurement of in vitro growth inhibition of tumor cell lines in 96 well plates by cell-mediated reduction of tetrazolium salt to water insoluble formazan crystals using doxorubicin as a standard. The cytotoxicity was assessed against human breast adenocarcinoma cell lines (MCF-7, MDA-MB-231) in comparison with non-tumorous breast cell line MCF-10A (human mammary epithelial cell line) and non-tumorous VERO cell line (normal monkey kidney epithelial cell line) and using the standard MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) colorimetric assay.^44,45 Estrogen receptor (ER)-positive MCF-7 and ER negative MDA-MB-231 breast cancer cells, non-tumorous MCF-10A and VERO cell lines were procured from National Centre for Cell Science (NCCS), Pune, Maharashtra, India. In detail, exponentially growing cells were harvested and seeded in sterile 96 well flat bottom tissue culture plates at a density of 5000 cells per well and allowed to attach overnight. Test compounds were prepared just prior to the experiment and serially diluted with suitable medium to get different concentration from 25–200 μg. The final concentration of DMSO was not more than 0.05%. After 24 h of incubation, cells were treated with 100 μl of test compounds for 48 h. After 48 h, 50 μl of MTT reagent was added and incubated for 3 h at 37 °C. After 3 h of incubation medium along with MTT was aspirated and 200 μl of 100% DMSO was added to each well to solubilize the formazan crystals. The optical density was measured by a well plate reader at 540 nm using a well plate reader (ELx800, BioTek Instruments Inc., Winooski, VT, USA). The IC₅₀ values (50% inhibitory concentration) were calculated from the plotted absorbance data for the dose–response curves. IC₅₀ values (in μM) were shown as mean ± SD of three independent experiments.

The newly synthesized 1,2,3-triazolyl chalcones (5a–k) and (6a–e) as potential anticancer agents were evaluated for their in vitro cytotoxicity against human breast adenocarcinoma (MCF-7, MDA-MB-231) cell lines using the standard MTT assay. From the data reported in Table 7, it was revealed that the most of compounds were non-toxic against normal non-tumor cell lines MCF-10A (breast) and VERO and some of them exhibits good cytotoxic activities against the breast cancer cell lines MCF-7, MDA-MB-231. The compounds 5b, 5e, 5f, 6c and 6d exhibits good cytotoxic activities against both human breast adenocarcinoma cell lines (MCF-7 and MDA-MB-231) with IC₅₀ values 10.3 μM, 7.8 μM, 8.3 μM, 8.7 μM and 7.9 μM (for MDA-MB-231 cell line) and 3.2 μM, 6.4 μM, 3.1 μM, 3.2 μM and 3.4 μM (for MCF-7 cell line) respectively compared to the positive control doxorubicin with IC₅₀ values 1.1 μM (for MCF-7 cell line) and 0.9 μM (for MDA-MB-231 cell line) respectively. On the other hand, compounds 5g and 5k exhibits moderate cytotoxic activities with IC₅₀ values 16.3 μM, 21.3 μM (for MDA-MB-231 cell line) and 5d with 25.6 μM (for MCF-7 cell line) respectively when compared to the standard drug. However, compounds 5b, 5g and 6e showed equal cytotoxicity on normal non-tumor breast cell line MCF-10A with IC₅₀ values 23.5 μM, 26.7 μM and 36.6 μM respectively. Further the derivatives 5b and 5g exhibited equal cytotoxicity against normal non-tumor VERO cell line with IC₅₀ values 20.9 μM and 25.3 μM respectively. Similarly the rest of the derivatives were non-significant with IC₅₀ values ranging from 30.5 μM to more than 100 μM (for MDA-MB-231 cell line) and 48.3 μM to more than 100 μM (for MCF-7 cell line) respectively and failed to show good and comparable cytotoxic activity when compared to the standard drug doxorubicin.

Table 7 The in vitro cytotoxic activity of (5a–k) and (6a–e) on cancer cells by MTT assay at 48 h of exposure

Compound	IC50^a in μM
	MDA-MB-231	MCF-7	VERO	MCF-10A
a Data presented is the mean ± SD value of three independent determinations.b Positive control.
5a	>100	>100	>100	>100
5b	10.3 ± 0.4	3.2 ± 0.3	20.9 ± 0.3	23.5 ± 0.3
5c	>100	74.8 ± 0.2	>100	>100
5d	>100	25.6 ± 0.2	>100	>100
5e	7.8 ± 0.3	6.4 ± 0.4	>100	>100
5f	8.3 ± 0.2	3.1 ± 0.1	>100	>100
5g	16.3 ± 0.4	10.4 ± 0.2	25.3 ± 0.2	26.7 ± 0.2
5h	67.6 ± 0.3	74.8 ± 0.5	>100	>100
5i	69.9 ± 0.4	78.3 ± 0.4	>100	>100
5j	>100	91.5 ± 0.2	>100	>100
5k	21.3 ± 0.2	>100	>100	>100
6a	>100	>100	>100	>100
6b	57.9 ± 0.4	54.8 ± 0.4	>100	>100
6c	8.7 ± 0.2	3.2 ± 0.2	>100	>100
6d	7.9 ± 0.1	3.4 ± 0.1	>100	>100
6e	30.5 ± 0.4	48.3 ± 0.4	>100	36.6 ± 0.2
Doxorubicin^b	0.9 ± 0.2	1.1 ± 0.1	>100	>100

---

Further, the structure–activity relationship of the compounds (5a–k) and (6a–e) revealed that the compounds 5b, 5e, 5f, 6c and 6d containing 3-chloro-2-fluoro phenyl, 4-fluoro phenyl, 4-chloro phenyl, 1-(4-chloro phenyl)-3-(phenyl)-1H-pyrazole and 1-(4-chloro phenyl)-3-(3-bromo phenyl)-1H-pyrazole substituent's at the 3^rd position of prop-2-en-1-one (chalcone) moiety respectively were found to exhibit enhanced anti-cancer activity compared to the less or unsubstituted phenyl derivatives (i.e. like 5a, 6a). A closer look into the structure activity relationship suggests that the cytotoxic potency was highly dependent, not surprisingly, on the substitution types and patterns on the phenyl rings. The different substituents on the phenyl rings attached at the C-1 and C-3 of the pyrazole ring like in 6c, 6d and C-3 of the chalcone moiety as in 5b, 5e, 5f can slightly alter the cytotoxicity against the cancer cell lines tested compared to non-substituted analogues (5a, 5b). However, replacing the hydrogen with an electron withdrawing group on the phenyl ring attached to the C-3 of the chalcone (as in 5b, 5e, 5f) and C-1 and C-3 position of the pyrazole ring (as in 6c, 6d) resulted in a significant activity increase. Also, the results clearly indicate that the halogenated substitution on the phenyl ring attached to the C-3 of the chalcone and C-1 and C-3 position of the pyrazole ring showed better activity against the MCF-7 and MDA-MB-231 cancer cell lines (Table 7). Hence, the biological response increased by halogenated analogs than their non-halogenated motifs. This is probably due to enhanced lipophilicity, pharmacokinetic properties, physicochemical properties, endurance for metabolic destruction and electronegativity of the molecules due to presence of halo substituents like chloro, fluoro etc. as shown by earlier literature studies on halogen containing molecules.^46,47

3. Conclusion

In conclusion, a series of new series of novel (2E)-1-{1-[2,3-dichloro-6-methyl-5-(trifluoromethyl)phenyl]-5-methyl-1H-1,2,3-triazol-4-yl}-3-aryl prop-2-en-1-one (5a–k) and (2E)-1-{1-[2,3-dichloro-6-methyl-5-(trifluoromethyl)phenyl]-5-methyl-1H-1,2,3-triazol-4-yl}-3-(1,3-diaryl-1H-pyrazol-4-yl)prop-2-en-1-one (6a–e) were synthesized via a Claisen–Schmidt condensation of 1-{1-[2,3-dichloro-6-methyl-5-(trifluoromethyl)phenyl]-5-methyl-1H-1,2,3-triazol-4-yl}ethanone (4) with different aryl and 1,3-diaryl-1H-pyrazole-4-carbaldehydes iii(a-e) in presence of ethanol and sodium hydroxide mixture in quantitative yields and evaluated for their in vitro antimicrobial, anti-oxidant and anti-cancer studies. Among the series, compounds 5b, 5e, 5f, 5i, 5j, 5k, 6c and 6d were found to be potent and broad spectrum anti-microbial agents comparable with respect to the standard drug Ciproflaxin and fluconazole respectively. While the compounds 5c, 5d, 5h and 6b were found to be good anti-bacterial agents and compounds 6e and 6b were found to be good anti-fungal agents against the standard drugs. Similarly derivatives 5a, 5d, 5g, 5j, 5k and 6a were found to exhibit good anti-oxidant properties against the standard ascorbic acid. Anti-cancer studies revealed that, the compounds 5b, 5e, 5f, 6c and 6d has shown good cytotoxic activity against the human breast adenocarcinoma cell lines (MCF-7 and MDA-MB-231) with respect to the positive control doxorubicin. The significant anti-microbial and anti-cancer activities of the synthesized compounds may be due to the presence of the electron-donating and halo substituted groups on the phenyl rings along with core 1,2,3-triazole, chalcone and pyrazole moiety. Furthermore, the observed anti-microbial and anti-cancer activities may be looked at as key steps for designing more potent new chemical entities with comparable pharmacological profiles to that of the standard drugs.

4. Experimental

4.1. General

All solvents used were of analytical grade and the reagents were used was purchased. All melting points were determined by open capillary method and were uncorrected. IR spectra were obtained in KBr disc on a Shimadzu FT-IR 157 spectrometer. ¹H NMR spectra were recorded either on a Perkin-Elmer EM-390 or on a Bruker WH-200 (400 MHz) in CDCl₃ or DMSO-d₆ as solvent, using tetramethylsilane (TMS) as an internal standard and chemical shifts and coupling constants were expressed as δ (ppm) and J (Hz) respectively. Mass spectrum was determined on a Jeol SX 102/Da-600 mass spectrometer/Data System using argon/xenon (6 kV, 10 mA) as the FAB gas. The accelerating voltage was 10 kV and spectra were recorded at room temperature. The elemental analyses (CHN) were performed on using VARIO EL-III (Elementar Analysensysteme GmBH). The progresses of the reactions were monitored by TLC on pre-coated silica gel G plates. All the spectral data's of newly synthesized compounds were consistent with proposed structure and microanalysis within ±0.4 of the calculated values.

4.2. General procedure for the synthesis of 1-{1-[2,3-dichloro-6-methyl-5-(trifluoromethyl)phenyl]-5-methyl-1H-1,2,3-triazol-4-yl}ethanone (4)

The solution of 3-azido-1,2-dichloro-4-methyl-5-(trifluoromethyl)benzene (3) (0.01 mol) in methanol was added slowly to solution of sodium methoxide (0.015 mol) in methanol at 0–5 °C. To this reaction mass, acetyl acetone (0.012 mol) was added slowly at 0–5 °C. The reaction mass was stirred at 25–30 °C for 12 h. The completion of reaction was monitored by TLC. After completion of the reaction, the reaction mixture was quenched to pre cooled water while pale brown precipitation observed and the precipitated solid was filtered, washed with water, dried and recrystallized from ethanol to get pure compound (4).

IR (KBr) γ/cm⁻¹: 2923 (Ar-H), 1670 (C=O), 1582 (C=N), 1086 (C–F) and 841 (C–Cl). ¹H-NMR: (400 MHz, CDCl₃, δ ppm): 2.05 (s, 3H, CH₃ of triazole), 2.40 (s, 3H, CH₃ of 2,3-dichloro-5-trifluoromethyl-6-methyl phenyl), 2.77 (s, 3H, CH₃ of acetyl), 7.98 (s, 1H, Ar-H of 2,3-dichloro-5-trifluoromethyl-6-methyl phenyl). ¹³C NMR: (100 MHz, CDCl₃, δ ppm): 9.03 (CH₃ of triazole), 14.25 (CH₃ of 2,3-dichloro-5-trifluoromethyl-6-methyl phenyl), 27.82 (CH₃ of acetyl), 121.24, 123.97, 129.9, 132.2, 135.1, 135.6, 136.0, 138.9, 143.3, 194.0 (C=O of acetyl). ¹³C NMR-DEPT-135 (100 MHz, CDCl₃, δ ppm): 9.03 (CH₃ of triazole), 14.25 (CH₃ of 2,3-dichloro-5-trifluoromethyl-6-methyl phenyl), 27.82 (CH₃ of acetyl), 129.9. MS: m/z = 352 (M⁺), 354 (M⁺ + 2). Anal. calcd for C₁₃H₁₀Cl₂F₃N₃O: C (44.34%), H (2.86%), N (11.93%). Found: C (44.28%), H (2.81%), N (11.96%).

4.3. General procedure for the synthesis of (2E)-1-{1-[2,3-dichloro-6-methyl-5-(trifluoromethyl)phenyl]-5-methyl-1H-1,2,3-triazol-4-yl}-3-aryl prop-2-en-1-one (5a–k) and (2E)-1-{1-[2,3-dichloro-6-methyl-5-(trifluoromethyl)phenyl]-5-methyl-1H-1,2,3-triazol-4-yl}-3-(1,3-diaryl-1H-pyrazol-4-yl)prop-2-en-1-one (6a–e)

A mixture of 1-{1-[2,3-dichloro-6-methyl-5-(trifluoromethyl)phenyl]-5-methyl-1H-1,2,3-triazol-4-yl}ethanone (4) (0.01 mol), sodium hydroxide (0.02 mol) solution (25% in water) and aryl aldehydes (0.01 mol)/iii(a-e) in methanol was stirred for 12 h at 25–30 °C. The completion of reaction was monitored by TLC. After completion of the reaction, the reaction mixture was quenched to pre cooled water while precipitation observed and the precipitated solid was filtered, washed with water, dried and recrystallized from ethanol to get pure compound (5a–k) and (6a–e) respectively.

4.3.1. (2E)-1-{1-[2,3-Dichloro-6-methyl-5-(trifluoromethyl)phenyl]-5-methyl-1H-1,2,3-triazol-4-yl}-3-phenyl prop-2-en-1-one (5a). IR (KBr) γ/cm⁻¹: 2930 (Ar-H), 1665 (C=O), 1582 (C=N), 1087 (C–F) and 836 (C–Cl). ¹H-NMR: (400 MHz, CDCl₃, δ ppm): 2.07 (s, 3H, CH₃ of triazole), 2.45 (s, 3H, CH₃ of 2,3-dichloro-5-trifluoromethyl-6-methyl phenyl), 6.94 (d, 2H, J = 8.0 Hz, Ar-H of phenyl), 7.02–7.92 (m, 5H, Ar-H of phenyl and CH = CH), 7.96 (s, 1H, Ar-H of 2,3-dichloro-6-trifluoromethyl phenyl). ¹³C NMR: (100 MHz, CDCl₃, δ ppm): 9.18 (CH₃ of triazole), 14.27 (CH₃ of 2,3-dichloro-5-trifluoromethyl-6-methyl phenyl), 112.2, 120.21, 120.78, 123.3, 125.8, 128.7, 129.9, 131.5, 134.6, 135.8, 138.4, 140.3, 142.1, 144.2, 145.3, 161.2, 184.0 (C=O of chalcone). ¹³C NMR-DEPT-135 (100 MHz, CDCl₃, δ ppm): 9.18 (CH₃ of triazole), 14.27 (CH₃ of 2,3-dichloro-5-trifluoromethyl-6-methyl phenyl), 112.1, 120.2, 128.6, 129.7, 131.4, 142.4. MS: m/z = 440 (M⁺), 442 (M⁺ + 2). Anal. calcd for C₂₀H₁₄Cl₂F₃N₃O: C (54.56%), H (3.21%), N (9.54%). Found: C (54.58%), H (3.24%), N (9.58%).

4.3.2. (2E)-1-{1-[2,3-Dichloro-6-methyl-5-(trifluoromethyl)phenyl]-5-methyl-1H-1,2,3-triazol-4-yl}-3-(3-chloro-2-fluoro phenyl)prop-2-en-1-one (5b). IR (KBr) γ/cm⁻¹: 2935 (Ar-H), 1667 (C=O), 1584 (C=N), 1085 (C–F) and 833 (C–Cl). ¹H-NMR: (400 MHz, CDCl₃, δ ppm): 2.09 (s, 3H, CH₃ of triazole), 2.48 (s, 3H, CH₃ of 2,3-dichloro-5-trifluoromethyl-6-methyl phenyl), 7.42 (d, 1H, J = 8.2 Hz, Ar-H of 3-chloro-2-fluoro phenyl), 7.55 (d, 1H, J = 8.2 Hz, Ar-H of 3-chloro-2-fluoro phenyl), 7.89 (d, 2H, J = 8.0 Hz, CH=CH), 7.95 (s, 1H, Ar-H of 2,3-dichloro-6-trifluoromethyl phenyl). ¹³C NMR: (100 MHz, CDCl₃, δ ppm): 9.17 (CH₃ of triazole), 14.25 (CH₃ of 2,3-dichloro-5-trifluoromethyl-6-methyl phenyl), 112.21, 120.23, 120.77, 123.33, 125.82, 128.72, 129.93, 131.54, 134.6, 135.81, 138.43, 140.31, 142.11, 144.23, 145.32, 161.4, 184.3 (C=O of chalcone). MS: m/z = 490 (M⁺), 492 (M⁺ + 2). Anal. calcd for C₂₀H₁₂Cl₂F₄N₃O: C (48.76%), H (2.45%), N (8.53%). Found: C (48.78%), H (2.44%), N (8.55%).

4.3.3. (2E)-1-{1-[2,3-Dichloro-6-methyl-5-(trifluoromethyl)phenyl]-5-methyl-1H-1,2,3-triazol-4-yl}-3-(4-hydroxy phenyl)prop-2-en-1-one (5c). IR (KBr) γ/cm⁻¹: 3068 (Ar-H), 1663 (C=O), 1566 (C=N), 1168 (C–F) and 713 (C–Cl). ¹H-NMR: (400 MHz, CDCl₃, δ ppm): 2.10 (s, 3H, CH₃ of triazole), 2.52 (s, 3H, CH₃ of 2,3-dichloro-5-trifluoromethyl-6-methyl phenyl), 3.85 (s, 1H, –OH), 6.96 (d, 2H, J = 8.4 Hz, Ar-H of 4-hydroxy phenyl), 7.70 (d, 2H, J = 8.2 Hz, Ar-H of 4-hydroxy phenyl), 7.96 (d, 1H, J = 16 Hz, CH=CH), 7.98 (d, 1H, J = 16 Hz, CH=CH), 8.0 (s, 1H, Ar-H of 2,3-dichloro-6-trifluoromethyl phenyl). ¹³C NMR (100 MHz, CDCl₃, δ ppm): 9.24 (CH₃ of triazole), 14.33 (CH₃ of 2,3-dichloro-5-trifluoromethyl-6-methyl phenyl), 114.42, 120.26, 121.27, 124.03, 126.72, 127.55, 129.8, 130.65, 132.17, 135.74, 136.15, 139.87, 143.84, 144.16, 161.82, 183.20 (C=O of chalcone). MS: m/z = 455.1 (M⁺), 457.1 (M⁺ + 2). Anal. calcd for C₂₀H₁₄Cl₂F₃N₃O₂: calculated: C (52.65%), H (3.09%), N (9.21%); found C (52.62%), H (3.08%), N (9.25%).

4.3.4. (2E)-1-{1-[2,3-Dichloro-6-methyl-5-(trifluoromethyl)phenyl]-5-methyl-1H-1,2,3-triazol-4-yl}-3-(3,4-dimethyl phenyl)prop-2-en-1-one (5d). IR (KBr) γ/cm⁻¹: 3065 (Ar-H), 1667 (C=O), 1568 (C=N), 1165 (C–F) and 711 (C–Cl). ¹H-NMR: (400 MHz, CDCl₃, δ ppm): 2.09 (s, 3H, CH₃ of triazole), 2.33 (s, 3H, CH₃ of 3,4-dimethyl phenyl), 2.40 (s, 3H, CH₃ of 3,4-dimethyl phenyl), 2.59 (s, 3H, CH₃ of 2,3-dichloro-5-trifluoromethyl-6-methyl phenyl), 7.26 (d, 1H, J = 8.4 Hz, Ar-H of 3,4-dimethyl phenyl), 7.39 (d, 1H, J = 8.2 Hz, Ar-H of 3,4-dimethyl phenyl), 7.59–7.65 (d, 1H, J = 23.2 Hz, CH=CH), 7.81 (s, 1H, Ar-H of 3,4-dimethyl phenyl), 7.94–8.00 (d, 1H, J = 23.0 Hz, CH=CH), 8.04 (s, 1H, Ar-H of 2,3-dichloro-6-trifluoromethyl phenyl). ¹³C NMR (100 MHz, CDCl₃, δ ppm): 8.20 (CH₃ of triazole), 13.28, 14.28 (2CH₃ of 3,4-dimethyl phenyl), 20.83 (CH₃ of 2,3-dichloro-5-trifluoromethyl-6-methyl phenyl), 114.42, 120.26, 121.27, 124.03, 126.72, 127.55, 129.8, 130.65, 132.17, 135.74, 136.15, 139.87, 143.84, 144.16, 161.82, 183.4 (C=O of chalcone). MS: m/z = 468.1 (M⁺), 470.1 (M⁺ + 2). Anal. calcd for C₂₂H₁₈Cl₂F₃N₃O: calculated: C (56.42%), H (3.87%), N (8.97%); found C (56.40%), H (3.85%), N (8.92%).

4.3.5. (2E)-1-{1-[2,3-Dichloro-6-methyl-5-(trifluoromethyl)phenyl]-5-methyl-1H-1,2,3-triazol-4-yl}-3-(4-fluoro phenyl)prop-2-en-1-one (5e). IR (KBr) γ/cm⁻¹: 3065 (Ar-H), 1662 (C=O), 1564 (C=N), 1166 (C–F) and 712 (C–Cl). ¹H-NMR: (400 MHz, CDCl₃, δ ppm): 2.09 (s, 3H, CH₃ of triazole), 2.50 (s, 3H, CH₃ of 2,3-dichloro-5-trifluoromethyl-6-methyl phenyl), 7.33–7.36 (dd, 2H, J = 6.4 Hz, 2.4 Hz, Ar-H of 4-fluoro phenyl), 7.45–7.47 (dd, 1H, J = 6.4 Hz, 2.4 Hz, Ar-H of 4-fluoro phenyl), 7.89–7.92 (dd, 1H, J = 6.8 Hz, 2.0 Hz, Ar-H of 4-fluoro phenyl), 8.0 (s, 1H, Ar-H of 2,3-dichloro-6-trifluoromethyl phenyl), 8.08–8.12 (d, 1H, J = 16 Hz, CH=CH), 8.37–8.41 (d, 1H, J = 16 Hz, CH=CH). ¹³C NMR (100 MHz, CDCl₃, δ ppm): 9.25 (CH₃ of triazole), 14.30 (CH₃ of 2,3-dichloro-5-trifluoromethyl-6-methyl phenyl), 114.40, 120.29, 121.30, 124.06, 126.74, 127.55, 129.83, 130.64, 132.15, 135.71, 136.12, 139.82, 143.81, 144.19, 161.85, 183.4 (C=O of chalcone). MS: m/z = 459.3 (M⁺), 461.3 (M⁺ + 2). Anal. calcd for C₂₀H₁₃Cl₂F₄N₃O: calculated: C (52.42%), H (2.86%), N (9.17%); found C (52.42%), H (2.88%), N (9.19%).

4.3.6. (2E)-1-{1-[2,3-Dichloro-6-methyl-5-(trifluoromethyl)phenyl]-5-methyl-1H-1,2,3-triazol-4-yl}-3-(4-chloro phenyl)prop-2-en-1-one (5f). IR (KBr) γ/cm⁻¹: 3067 (Ar-H), 1668 (C=O), 1567 (C=N), 1169 (C–F) and 715 (C–Cl). ¹H-NMR: (400 MHz, CDCl₃, δ ppm): 2.09 (s, 3H, CH₃ of triazole), 2.59 (s, 3H, CH₃ of 2,3-dichloro-5-trifluoromethyl-6-methyl phenyl), 7.26–7.39 (d, 2H, J = 8.2 Hz, Ar-H of 4-chloro phenyl), 7.52–7.65 (d, 2H, J = 8.0 Hz, Ar-H of 4-chloro phenyl), 7.81 (s, 1H, Ar-H of 2,3-dichloro-6-trifluoromethyl phenyl), 7.94–8.00 (d, 1H, J = 23 Hz, CH=CH), 8.00–8.46 (d, 1H, J = 17 Hz, CH=CH). ¹³C NMR (100 MHz, CDCl₃, δ ppm): 8.20 (CH₃ of triazole), 14.28 (CH₃ of 2,3-dichloro-5-trifluoromethyl-6-methyl phenyl), 114.38, 120.17, 121.28, 124.03, 126.72, 127.52, 129.80, 130.62, 132.11, 135.68, 136.10, 139.79, 143.79, 144.18, 161.84, 183.40 (C=O of chalcone). MS: m/z = 473.1 (M⁺), 475.1 (M⁺ + 2). Anal. calcd for C₂₀H₁₃Cl₃F₃N₃O: calculated: C (50.60%), H (2.76%), N (8.85%); found C (50.62%), H (2.78%), N (8.82%).

4.3.7. (2E)-1-{1-[2,3-Dichloro-6-methyl-5-(trifluoromethyl)phenyl]-5-methyl-1H-1,2,3-triazol-4-yl}-3-(4-methoxy phenyl)prop-2-en-1-one (5g). IR (KBr) γ/cm⁻¹: 2932 (ArCH=CH), 2893 (CH=CH), 1668 (C=O), 1089 (C–F) and 713 (C–Cl). ¹H NMR (400 MHz, CDCl₃, δ ppm): 2.09 (s, 3H, of triazole), 2.50 (s, 3H, CH₃ of 2,3-dichloro-5-trifluoromethyl-6-methyl phenyl), 3.86 (s, 3H, OCH₃ of 4-methoxy phenyl), 6.94 (d, 2H, J = 8.6 Hz, ArH of 4-methoxy phenyl), 7.69 (d, 2H, J = 8.2 Hz, ArH of 4-methoxy phenyl), 7.92–7.96 (d, 1H, J = 15.8 Hz, CH=CH), 7.98–8.02 (d, 1H, J = 15.8 Hz, CH=CH), 8.00 (s, 1H, ArH of 2,3-dichloro-5-trifluoromethyl-6-methyl phenyl). ¹³C NMR (100 MHz, CDCl₃, δ ppm): 9.25 (CH₃ of triazole), 14.32 (CH₃ of 2,3-dichloro-5-trifluoromethyl-6-methyl phenyl), 55.42 (OCH₃ of 4-methoxy phenyl), 114.43, 120.28, 121.28, 124.01, 126.74, 127.56, 129.9, 130.67, 132.18, 135.76, 136.13, 139.85, 143.83, 144.17, 161.8, 184.0 (C=O of chalcone). ¹³C NMR-DEPT-135 (100 MHz, CDCl₃, δ ppm): 9.25 (CH₃ of triazole), 14.32 (CH₃ of 2,3-dichloro-5-trifluoromethyl-6-methyl phenyl), 55.44 (OCH₃ of 4-methoxy phenyl), 114.44, 120.29, 129.90, 130.69, 144.19. MS: m/z = 468 (M⁺), 470 (M⁺ + 2). Anal. calcd for C₂₁H₁₆Cl₂F₃N₃O₂: C (53.63%), H (3.43%), N (8.94%). Found: C (53.56%), H (3.47%), N (8.98%).

4.3.8. (2E)-1-{1-[2,3-Dichloro-6-methyl-5-(trifluoromethyl)phenyl]-5-methyl-1H-1,2,3-triazol-4-yl}-3-(2-fluoro phenyl)prop-2-en-1-one (5h). IR (KBr) γ/cm⁻¹: 3080 (Ar-H), 1665 (C=O), 1563 (C=N), 1163 (C–F) and 712 (C–Cl). ¹H-NMR: (400 MHz, CDCl₃, δ ppm): 2.10 (s, 3H, CH₃ of triazole), 2.51 (s, 3H, CH₃ of 2,3-dichloro-5-trifluoromethyl-6-methyl phenyl), 7.34–7.37 (t, 2H, J = 8.2 Hz, Ar-H of 2-fluoro phenyl), 7.46–7.48 (dd, 1H, J = 6.4 Hz, 2.4 Hz, Ar-H of 2-fluoro phenyl), 7.88–7.91 (dd, 1H, J = 6.8 Hz, 2.0 Hz, Ar-H of 2-fluoro phenyl), 8.01 (s, 1H, Ar-H of 2,3-dichloro-6-trifluoromethyl phenyl), 8.07–8.11 (d, 1H, J = 16 Hz, CH=CH), 8.36–8.40 (d, 1H, J = 16 Hz, CH=CH). ¹³C NMR (100 MHz, CDCl₃, δ ppm): 9.24 (CH₃ of triazole), 14.29 (CH₃ of 2,3-dichloro-5-trifluoromethyl-6-methyl phenyl), 114.40, 120.28, 121.34, 124.08, 126.75, 127.53, 129.81, 130.62, 132.13, 135.69, 136.10, 139.83, 143.82, 144.17, 161.84, 184.01 (C=O of chalcone). MS: m/z = 460.2 (M⁺), 462.2 (M⁺ + 2). Anal. calcd for C₂₀H₁₃Cl₂F₄N₃O: calculated: C (52.42%), H (2.86%), N (9.17%); found C (52.43%), H (2.89%), N (9.19%).

4.3.9. (2E)-1-{1-[2,3-Dichloro-6-methyl-5-(trifluoromethyl)phenyl]-5-methyl-1H-1,2,3-triazol-4-yl}-3-(2-chloro phenyl)prop-2-en-1-one (5i). IR (KBr) γ/cm⁻¹: 3077 (Ar-H), 1667 (C=O), 1566 (C=N), 1167 (C–F) and 715 (C–Cl). ¹H-NMR: (400 MHz, CDCl₃, δ ppm): 2.09 (s, 3H, CH₃ of triazole), 2.51 (s, 3H, CH₃ of 2,3-dichloro-5-trifluoromethyl-6-methyl phenyl), 7.33–7.36 (t, 2H, J = 8.0 Hz, Ar-H of 2-chloro phenyl), 7.44–7.47 (d, 1H, J = 8.0 Hz, Ar-H of 2-chloro phenyl), 7.86–7.88 (d, 1H, J = 8.0 Hz, Ar-H of 2-chloro phenyl), 7.99 (s, 1H, Ar-H of 2,3-dichloro-6-trifluoromethyl phenyl), 8.06–8.10 (d, 1H, J = 16 Hz, CH=CH), 8.37–8.41 (d, 1H, J = 16 Hz, CH=CH). ¹³C NMR (100 MHz, CDCl₃, δ ppm): 9.26 (CH₃ of triazole), 14.29 (CH₃ of 2,3-dichloro-5-trifluoromethyl-6-methyl phenyl), 114.37, 120.19, 121.25, 124.01, 126.74, 127.51, 129.78, 130.61, 132.11, 135.66, 136.12, 139.76, 143.76, 144.19, 161.81, 183.20 (C=O of chalcone). MS: m/z = 474.1 (M⁺), 476.1 (M⁺ + 2). Anal. calcd for C₂₀H₁₃Cl₃F₃N₃O: calculated: C (50.60%), H (2.76%), N (8.85%); found C (50.64%), H (2.79%), N (8.83%).

4.3.10. (2E)-1-{1-[2,3-Dichloro-6-methyl-5-(trifluoromethyl)phenyl]-5-methyl-1H-1,2,3-triazol-4-yl}-3-(3-pyridyl)prop-2-en-1-one (5j). IR (KBr) γ/cm⁻¹: 3067 (Ar-H), 1668 (C=O), 1567 (C=N), 1169 (C–F) and 715 (C–Cl). ¹H-NMR: (400 MHz, CDCl₃, δ ppm): 2.10 (s, 3H, CH₃ of triazole), 2.50 (s, 3H, CH₃ of 2,3-dichloro-5-trifluoromethyl-6-methyl phenyl), 7.33–7.35 (d, 2H, J = 8.2 Hz, Ar-H of 3-pyridyl), 7.46–7.49 (t, 1H, J = 8.0 Hz, Ar-H of pyridyl), 7.94 (s, 1H, Ar-H of pyridyl), 8.01 (s, 1H, Ar-H of 2,3-dichloro-6-trifluoromethyl phenyl), 8.07–8.11 (d, 1H, J = 16 Hz, CH=CH), 8.38–8.42 (d, 1H, J = 16 Hz, CH=CH). ¹³C NMR (100 MHz, CDCl₃, δ ppm): 9.24 (CH₃ of triazole), 14.30 (CH₃ of 2,3-dichloro-5-trifluoromethyl-6-methyl phenyl), 114.37, 120.18, 121.29, 124.07, 126.75, 127.51, 129.79, 130.61, 132.10, 135.69, 139.77, 143.78, 144.17, 161.81, 183.15 (C=O of chalcone). MS: m/z = 440.1 (M⁺), 442.1 (M⁺ + 2). Anal. calcd for C₁₉H₁₃Cl₂F₃N₄O: calculated: C (51.72%), H (2.97%), N (12.70%); found C (51.67%), H (2.99%), N (12.76%).

4.3.11. (2E)-1-{1-[2,3-Dichloro-6-methyl-5-(trifluoromethyl)phenyl]-5-methyl-1H-1,2,3-triazol-4-yl}-3-(4-thiomethyl phenyl)prop-2-en-1-one (5k). IR (KBr) γ/cm⁻¹: 3070 (Ar-H), 1667 (C=O), 1568 (C=N), 1171 (C–F) and 710 (C–Cl). ¹H-NMR: (400 MHz, CDCl₃, δ ppm): 2.02 (s, 3H, CH₃ of triazole), 2.43 (s, 3H, CH₃ of 2,3-dichloro-5-trifluoromethyl-6-methyl phenyl), 3.43 (s, 3H, SCH₃ of 4-thiomethyl phenyl) 7.20–7.47 (d, 2H, J = 8.0 Hz, Ar-H of 4-thiomethyl phenyl), 7.58–7.60 (d, 2H, J = 8.2 Hz, Ar-H of 4-thiomethyl phenyl), 7.84–7.88 (d, 1H, J = 15 Hz, CH=CH), 7.93 (s, 1H, Ar-H of 2,3-dichloro-5-trifluoromethyl-6-methyl phenyl), 7.97–8.01 (d, 1H, J = 16 Hz, CH=CH). ¹³C NMR: (100 MHz, CDCl₃, δ ppm): 9.25 (CH₃ of triazole), 14.32 (CH₃ of 2,3-dichloro-5-trifluoromethyl-6-methyl phenyl), 55.42 (SCH₃ of 4-thiomethyl phenyl), 114.34, 120.18, 121.23, 124.05, 126.75, 127.52, 129.75, 130.65, 132.14, 135.62, 136.10, 139.73, 143.71, 144.17, 161.80, 183.9 (C=O of chalcone). MS: m/z = 484.1 (M⁺), 486.0 (M⁺ + 2). Anal. calcd for C₂₁H₁₆Cl₂F₃N₃OS: calculated: C (51.86%), H (3.32%), N (8.64%); found C (51.90%), H (3.35%), N (8.70%).

4.3.12. (2E)-1-{1-[2,3-Dichloro-6-methyl-5-(trifluoromethyl)phenyl]-5-methyl-1H-1,2,3-triazol-4-yl}-3-(1,3-diphenyl-1H-pyrazol-4-yl)prop-2-en-1-one (6a). IR (KBr) γ/cm⁻¹: 3068 (Ar-H), 1664 (C=O), 1565 (C=N), 1164 (C–F) and 712 (C–Cl). ¹H-NMR: (400 MHz, CDCl₃, δ ppm): 2.09 (s, 3H, CH₃ of 2,3-dichloro-5-trifluoromethyl-6-methyl phenyl), 2.49 (s, 3H, CH₃ of triazole), 7.34–7.38 (t, 1H, J = 7.6 Hz, Ar-H of phenyl), 7.45–7.53 (m, 5H, Ar-H of phenyl), 7.73–7.76 (dd, 2H, J = 8.2 Hz, 3.2 Hz, Ar-H of phenyl), 7.82–7.84 (d, 2H, J = 8.0 Hz, Ar-H of phenyl), 7.96–8.00 (d, 1H, J = 16.2 Hz, CH=CH), 8.01 (s, 1H, Ar-H of 2,3-dichloro-5-trifluoromethyl-6-methyl phenyl), 8.04–8.08 (d, 1H, J = 16.0 Hz, CH=CH), 8.53 (s, 1H, Ar-H of pyrazole). ¹³C NMR: (400 MHz, CDCl₃, δ ppm): 9.23 (CH₃ of triazole), 14.31 (CH₃ of 2,3-dichloro-5-trifluoromethyl-6-methyl phenyl), 118.34, 119.42, 121.88, 126.69, 127.33, 128.72, 128.83, 129.63, 129.95, 130.00, 132.15, 132.24, 135.05, 135.23, 136.12, 139.46, 143.72, 154.36, 183.73 (C=O of chalcone). MS: m/z = 582.1 (M⁺), 583.2 (M⁺ + 1). Anal. calcd for C₂₉H₂₀Cl₂F₃N₅O: calculated: C (59.81%), H (3.46%), N (12.02%); found C (59.75%), H (3.43%), N (12.06%).

4.3.13. (2E)-1-{1-[2,3-Dichloro-6-methyl-5-(trifluoromethyl)phenyl]-5-methyl-1H-1,2,3-triazol-4-yl}-3-(1-(phenyl)-3-(4-chloro phenyl)-1H-pyrazol-4-yl)prop-2-en-1-one (6b). IR (KBr) γ/cm⁻¹: 3069 (Ar-H), 1665 (C=O), 1567 (C=N), 1162 (C–F) and 711 (C–Cl). ¹H-NMR: (400 MHz, CDCl₃, δ ppm): 2.09 (s, 3H, CH₃ of 2,3-dichloro-5-trifluoromethyl-6-methyl phenyl), 2.49 (s, 3H, CH₃ of triazole), 7.35–7.39 (t, 1H, J = 7.6 Hz, Ar-H of phenyl), 7.47–7.54 (q, 4H, J = 8.2 Hz, 7.6 Hz, Ar-H of phenyl), 7.68–7.70 (d, 2H, J = 8.4 Hz, Ar-H of 4-chloro phenyl), 7.80–7.82 (d, 2H, J = 8.0 Hz, Ar-H of 4-chloro phenyl), 7.94–7.98 (d, 1H, J = 16.0 Hz, CH=CH), 7.99 (s, 1H, Ar-H of 2,3-dichloro-5-trifluoromethyl-6-methyl phenyl), 8.00–8.04 (d, 1H, J = 16.0 Hz, CH=CH), 8.51 (s, 1H, Ar-H of pyrazole). ¹³C NMR: (400 MHz, CDCl₃, δ ppm): 9.22 (CH₃ of triazole), 14.32 (CH₃ of 2,3-dichloro-5-trifluoromethyl-6-methyl phenyl), 118.33, 119.41, 121.86, 126.68, 127.34, 128.73, 128.81, 129.61, 129.93, 130.02, 132.13, 132.22, 135.03, 135.21, 136.11, 139.44, 143.70, 154.34, 183.72 (C=O of chalcone). MS: m/z = 615.2 (M⁺), 616.2 (M⁺ + 1). Anal. calcd for C₂₉H₁₉Cl₃F₃N₅O: calculated: C (56.47%), H (3.10%), N (11.35%); found C (56.50%), H (3.13%), N (11.32%).

4.3.14. (2E)-1-{1-[2,3-Dichloro-6-methyl-5-(trifluoromethyl)phenyl]-5-methyl-1H-1,2,3-triazol-4-yl}-3-(1-(4-chloro phenyl)-3-(phenyl)-1H-pyrazol-4-yl)prop-2-en-1-one (6c). IR (KBr) γ/cm⁻¹: 3067 (Ar-H), 1663 (C=O), 1565 (C=N), 1163 (C–F) and 713 (C–Cl). ¹H-NMR: (400 MHz, CDCl₃, δ ppm): 2.10 (s, 3H, CH₃ of 2,3-dichloro-5-trifluoromethyl-6-methyl phenyl), 2.50 (s, 3H, CH₃ of triazole), 7.34–7.38 (t, 1H, J = 7.6 Hz, Ar-H of phenyl), 7.46–7.53 (q, 4H, J = 8.0 Hz, 7.6 Hz, Ar-H of phenyl), 7.67–7.69 (d, 2H, J = 8.0 Hz, Ar-H of 4-chloro phenyl), 7.80–7.82 (d, 2H, J = 8.0 Hz, Ar-H of 4-chloro phenyl), 7.93–7.97 (d, 1H, J = 16.0 Hz, CH=CH), 7.98 (s, 1H, Ar-H of 2,3-dichloro-5-trifluoromethyl-6-methyl phenyl), 7.99–8.03 (d, 1H, J = 16.0 Hz, CH=CH), 8.50 (s, 1H, Ar-H of pyrazole). ¹³C NMR: (400 MHz, CDCl₃, δ ppm): 9.23 (CH₃ of triazole), 14.34 (CH₃ of 2,3-dichloro-5-trifluoromethyl-6-methyl phenyl), 118.35, 119.40, 121.85, 126.66, 127.35, 128.75, 128.80, 129.63, 129.91, 130.00, 132.14, 132.21, 135.01, 135.23, 136.10, 139.42, 143.72, 154.31, 183.70 (C=O of chalcone). MS: m/z = 615.4 (M⁺), 616.2 (M⁺ + 1). Anal. calcd for C₂₉H₁₉Cl₃F₃N₅O: calculated: C (56.47%), H (3.10%), N (11.35%); found C (56.51%), H (3.12%), N (11.37%).

4.3.15. (2E)-1-{1-[2,3-Dichloro-6-methyl-5-(trifluoromethyl)phenyl]-5-methyl-1H-1,2,3-triazol-4-yl}-3-(1-(4-chloro phenyl)-3-(3-bromo phenyl)-1H-pyrazol-4-yl)prop-2-en-1-one (6d). IR (KBr) γ/cm⁻¹: 3067 (Ar-H), 1663 (C=O), 1565 (C=N), 1163 (C–F) and 713 (C–Cl). ¹H-NMR: (400 MHz, CDCl₃, δ ppm): 2.093 (d, 3H, J = 0.8 Hz, CH₃ of 2,3-dichloro-5-trifluoromethyl-6-methyl phenyl), 2.49 (s, 3H, CH₃ of triazole), 7.356–7.393 (t, 1H, J = 7.6 Hz, 7.2 Hz, Ar-H of 3-bromo phenyl), 7.477–7.540 (q, 4H, J = 8.4, 7.6 Hz, Ar-H of 4-chloro phenyl), 7.683–7.704 (d, 2H, J = 8.4 Hz, Ar-H of 4-chloro phenyl and 3-bromo phenyl), 7.801–7.822 (d, 2H, J = 8.4 Hz, Ar-H of 3-bromo phenyl and Ar-H of 2,3-dichloro-5-trifluoromethyl-6-methyl phenyl), 7.995–8.006 (d, 2H, J = 4.4 Hz, CH=CH), 8.523 (s, 1H, Ar-H of pyrazole). ¹³C NMR: (400 MHz, CDCl₃, δ ppm): 9.23 (CH₃ of triazole), 14.34 (CH₃ of 2,3-dichloro-5-trifluoromethyl-6-methyl phenyl), 118.35, 119.40, 121.85, 126.66, 127.35, 128.75, 128.80, 129.63, 129.91, 130.00, 132.14, 132.21, 135.01, 135.23, 136.10, 139.42, 143.72, 154.31, 183.70 (C=O of chalcone). MS: m/z = 693.2 (M⁺), 694.2 (M⁺ + 1). Anal. calcd for C₂₉H₁₈BrCl₃F₃N₅O: calculated: C (50.06%), H (2.61%), N (10.07%); found C (50.10%), H (2.57%), N (10.11%).

4.3.16. (2E)-1-{1-[2,3-Dichloro-6-methyl-5-(trifluoromethyl)phenyl]-5-methyl-1H-1,2,3-triazol-4-yl}-3-(1-(phenyl)-3-(3-bromo phenyl)-1H-pyrazol-4-yl)prop-2-en-1-one (6e). IR (KBr) γ/cm⁻¹: 3066 (Ar-H), 1668 (C=O), 1567 (C=N), 1164 (C–F) and 711 (C–Cl). ¹H-NMR: (400 MHz, CDCl₃, δ ppm): 2.092 (d, 3H, J = 0.8 Hz, CH₃ of 2,3-dichloro-5-trifluoromethyl-6-methyl phenyl), 2.491 (s, 3H, CH₃ of triazole), 7.348–7.388 (t, 1H, J = 7.6 Hz, 7.6 Hz, Ar-H of 3-bromo phenyl), 7.453–7.537 (m, 5H, Ar-H of phenyl), 7.739–7.760 (dd, 1H, J = 5.2 Hz, 2.0 Hz, Ar-H of 3-bromo phenyl), 7.820–7.840 (d, 2H, J = 8.0 Hz, Ar-H of 3-bromo phenyl), 7.960–8.001 (d, 1H, J = 16.4 Hz, CH=CH), 8.048–8.088 (d, 1H, 16 Hz, CH=CH), 8.287 (s, 1H, Ar-H of 2,3-dichloro-5-trifluoromethyl-6-methyl phenyl), 8.53 (s, 1H, Ar-H of pyrazole). ¹³C NMR: (400 MHz, CDCl₃, δ ppm): 9.29 (CH₃ of triazole), 14.24 (CH₃ of 2,3-dichloro-5-trifluoromethyl-6-methyl phenyl), 118.35, 119.40, 121.85, 126.66, 127.35, 128.75, 128.80, 129.63, 129.91, 130.00, 132.14, 132.21, 135.01, 135.23, 136.10, 139.42, 143.72, 154.31, 184.10 (C=O of chalcone). MS: m/z = 659.3 (M⁺), 660.3 (M⁺ + 1). Anal. calcd for C₂₉H₁₉BrCl₂F₃N₅O: calculated: C (52.67%), H (2.90%), N (10.59%); found C (52.65%), H (2.93%), N (10.62%).

Acknowledgements

One of the authors, Manjunatha Bhat, is grateful to the management of SeQuent Scientific Ltd., New Mangalore, India for encouraging research work. Manjunatha Bhat is also thankful to Late Prof. A. Srikrishna, Dept. of Organic Chemistry, IISc, Bangalore and MIT, Manipal for providing ¹H NMR and ¹³C NMR spectral facilities. We are also thankful to Dept. of Biochemistry, Jnanasahyadri, Kuvempu University, Shankarghatta, Karnataka and Dept. of Pharmacology, Manipal College of Pharmaceutical Sciences, Manipal, Karnataka for providing sincere help for the biological activity studies. Authors are also thankful to Chairman, Dept. of Chemistry, Mangalore University.

Notes and references

1. (a) H. Okusu, D. Ma and H. Nikaido, J. Bacteriol., 1996, 178, 306; (b) R. E. Isturiz, Int. J. Antimicrob. Agents, 2010, 36, S19.
2. M. S. Butler, M. A. Blaskovich and M. A. Cooper, J. Antibiot., 2013, 66(10), 571–591,  DOI:10.1038/ja.2013.86.
3. R. He, Y. F. Chen, Y. H. Chen, A. V. Ougolkov, J. S. Zhang, D. N. Savoy, D. D. Billadeau and A. P. Kozikowski, J. Med. Chem., 2010, 53(3), 1347–1356,  DOI:10.1021/jm901667k.
4. J. Ziemska, A. Rajnisz and J. Solecka, Cent. Eur. J. Biol., 2013, 8, 943–957.
5. O. O. Fadeyi, S. T. Adamson, E. L. Myles and C. O. Okoro, Bioorg. Med. Chem. Lett., 2008, 18, 4172–4176.
6. N. S. H. N. Moorthy, C. Karthikeyan and P. Trivedi, Med. Chem., 2009, 5, 549–557.
7. C. D. Mathers and D. Loncar, PLoS Med., 2006, 3(11), 442,  DOI:10.1371/journal.pmed.0030442.
8. J. Ferlay, I. Soerjomataram, R. Dikshit, S. Eser, C. Mathers, M. Rebelo, D. M. Parkin, D. Forman and F. Bray, Int. J. Cancer, 2015, 136(5), 359–386,  DOI:10.1002/ijc.29210.
9. G. A. Elmegeed, W. K. B. Khalil, R. M. Mohareb, H. H. Ahmed, M. M. Abd-Elhalim and G. H. Elsayed, Bioorg. Med. Chem., 2011, 19, 6860–6872.
10. M. I. Ansari, M. K. Hussain, A. Arun, B. Chakravarti, R. Konwar and K. Hajela, Eur. J. Med. Chem., 2015, 99, 113–124.
11. N. Magne, C. Chargari, D. MacDermed, R. Conforti, L. Vedrine, J. P. Spano and D. Khayat, Crit. Rev. Oncol. Hematol., 2010, 76, 186–195.
12. V. C. Jorden and W. J. Gradishar, Mol. Aspects Med., 1997, 18, 187–247.
13. M. Baum, A. U. Budzar, J. Cuzick, J. Forbes, J. H. Houghton, J. G. Klijn and T. Sahmoud, Lancet, 2002, 359, 2131–2139.
14. V. R. Tandon, S. Sharma, A. Mahajan and G. H. Bardi, JK Science, 2005, 7, 1–3.
15. Dinesha, S. Viveka, P. Naik and G. K. Nagaraja, Med. Chem. Res., 2014, 23, 4189–4197.
16. S. Viveka, Dinesha, L. N. Madhu and G. K. Nagaraja, Monatsh. Chem., 2015, 146, 1547–1555.
17. T. Taj, R. R. Kamble, T. M. Gireesh, R. K. Hunnur and S. B. Margankop, Eur. J. Med. Chem., 2011, 46, 4366–4373.
18. A. M. Pisoschi and A. Pop, Eur. J. Med. Chem., 2015, 97, 55–74.
19. B. Zhang and A. Studer, Chem. Soc. Rev., 2015, 44(11), 3505–3521,  10.1039/c5cs00083a.
20. N. S. Vatmurge, B. G. Hazra, V. S. Pore, F. Shirazi, P. S. Chavan and M. V. Deshpande, Bioorg. Med. Chem. Lett., 2008, 18, 2043–2047.
21. S. G. Agalave, S. R. Maujan and V. S. Pore, Asian J. Chem., 2011, 6, 2696–2718,  DOI:10.1002/asia.201100432.
22. K. Rama, K. Dharmendra, A. Drishti, D. G. Rinkoo, T. Ragini, K. A. Satish and A. Alka, Eur. J. Med. Chem., 2016, 113, 34–49.
23. B. Manjunatha, G. K. Nagaraja, K. Reshma, S. K. Peethamber and M. Shafeeullah, RSC Adv., 2016, 6, 59375–59388,  10.1039/c6ra06093e.
24. E. M. Nassar, F. M. Abdelrazek, R. R. Ayyad and A. F. El-Faragy, Mini-Rev. Med. Chem., 2016, 16(11), 926–936.
25. M. H. Shaikh, D. D. Subhedar, M. Arkile, V. M. Khedkar, N. Jadhav, D. Sarkar and B. B. Shingate, Bioorg. Med. Chem. Lett., 2016, 26(2), 561–569,  DOI:10.1016/j.bmcl.2015.11.071.
26. C. Yakaiah, T. Sneha, T. Shalini, C. Srinivas, K. D. Anand, K. A. Niranjana, K. V. N. S. Srinivas, A. Sarfaraz, K. J. Kotesh, K. Feroz, T. Ashok and G. Paramjit, Eur. J. Med. Chem., 2015, 93, 564–573.
27. R. Pingaew, A. Sakee, P. Mandi, C. Nantasenamat, S. Prachayasittikul, S. Ruchirawat and V. Prachayasittikul, Eur. J. Med. Chem., 2014, 85, 65–76.
28. S. Y. Zhang, D. J. Fu, X. X. Yue, Y. C. Liu, J. Song, H. H. Sun, H. M. Liu and Y. B. Zhang, Molecules, 2016, 21(5), 653,  DOI:10.3390/molecules21050653.
29. S. M. Hussaini, P. Yedla, K. S. Babu, T. B. Shaik, G. K. Chityal and A. Kamal, Chem. Biol. Drug Des., 2016, 88(1), 97–109,  DOI:10. 1111/cbdd.12738.
30. B. B. Chavan, A. S. Gadekar, P. P. Mehta, P. K. Vawhal, A. K. Kolsure and A. R. Chabukswar, Asian J. Biomed. Pharm. Sci., 2016, 6(56), 01–07.
31. B. T. Yin, C. Y. Yan, X. M. Peng, S. L. Zhang, S. Rasheed, R. X. Geng and C. H. Zhou, Eur. J. Med. Chem., 2014, 71, 148–159.
32. R. Suresh, S. Muthusubramanian, N. Paul, N. Kalidhasan and V. Shanmugaiah, Med. Chem. Res., 2016, 23(10), 4367–4375.
33. T. L. B. Ventura, S. D. Calixto, B. A. Abraham-Vieira, A. M. T. Souza, M. V. P. Mello, C. R. Rodrigues, L. S. M. Miranda, R. O. M. A. Souza, I. C. R. Leal, E. B. Lalunskaia and M. F. Muzitano, Molecules, 2015, 20, 8072–8093,  DOI:10.3390/molecules20058072.
34. M. H. Shaikh, D. D. Subhedar, V. M. Khedkar, P. C. Jha, F. A. K. Khan, J. N. Sangshetti and B. B. Shingate, Chin. Chem. Lett., 2016, 27(7), 1058–1063.
35. T. Mahalakshmi, A. Anand and R. Sudha, J. Theor. Biol., 2016, 403, 110–128.
36. C. S. Sharma, K. S. Shekhawat, C. S. Chauhan and N. Kumar, J. Chem. Pharm. Res., 2013, 5(10), 450–454.
37. V. V. Truong, T. D. Nam, T. N. Hung, N. T. Nga, P. M. Quan, L. V. Chinh and S. H. Jung, Bioorg. Med. Chem. Lett., 2015, 25(22), 5182–5185,  DOI:10.1016/j.bmcl.2015.09.069.
38. S. Gurjaspreet, A. Aanchal, S. M. Satinderpal, R. Sunita, K. Hargobinder, G. Kapil, S. Rakesh, M. K. Indresh, T. Rupinder, L. C. Duane, S. Subash and K. Navneet, Eur. J. Med. Chem., 2016, 108, 287–300.
39. B. S. Holla, M. Mahalinga, M. S. Karthikeyan, B. Poojary, P. M. Akberali and N. S. Kumari, Eur. J. Med. Chem., 2005, 40(11), 1173–1178.
40. B. A. Arthington-Skaggs, D. W. Warnock and C. J. Morrison, J. Antimicrob. Chemother., 2000, 44(8), 2081–2085.
41. I. Kubo, K. I. Fujita, A. Kubo, K. I. Nihel and T. Ogura, Journal of Agriculture and Food Chemistry, 2004, 52, 3329–3332.
42. D. J. M. Lowry, M. J. Jaqua and S. T. Selepak, Appl. Microbiol., 1970, 20, 46–53.
43. T. Mosmann, J. Immunol. Methods, 1983, 65, 55–63.
44. (a) A. Prakash, J. Agric. Food Chem., 2001, 44, 701–705; (b) J. A. Vinson, Y. Hao, X. Su and L. Zubik, J. Agric. Food Chem., 1998, 46, 3630–3634.
45. I. Dhamija, N. Kumar, S. N. Manjula, V. Parihar, M. M. Setty and K. S. R. Pai, Exp. Toxicol. Pathol., 2013, 65, 235–242.
46. S. G. Agalave, S. R. Maujan and V. S. Pore, Chem.–Asian J., 2011, 6, 2696–2718,  DOI:10.1002/asia.201100432.
47. M. L. Quan, P. Y. S. Lam, Q. Han, D. J. P. Pinto, M. Y. He, R. Li, C. D. Ellis, C. G. Clark, C. A. Teleha, J. H. Sun, R. S. Alexander, S. Bai, J. M. Luettgen, R. M. Knabb, P. C. Wong and R. R. Wexler, J. Med. Chem., 2005, 48(6), 1729–1744,  DOI:10.1021/jm0497949.

---

Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra22705h

---