Introduction

Huisgen’s 1,3-dipolar cycloaddition reaction of azides with substituted acetylenes is a well-established route to 1,2,3-triazoles [1], but the reaction requires high temperature, long reaction times, and is not regiospecific. Copper(I)-catalyzed click chemistry discovered by Sharpless and co-workers [2, 3] has put this reaction center stage and since then it has become a reliable method to synthesize 1,2,3-triazoles. The presence of Cu(I) dramatically accelerates the rate and makes the reaction highly regioselective, leading to only 1,4-disubstituted isomers [4]. Moreover, the reaction can be performed at room temperature and under a variety of conditions [5].

1,2,3-Triazoles have attracted the interest of organic chemists for development of new biologically active molecules owing to their stability, resistance to hydrolysis and metabolic degradation, and capability to form hydrogen bonds. 1,2,3-Triazoles have been shown to possess diverse biological activities, e.g., they are antifungal and antibacterial agents, enzyme inhibitors [6–8], and are useful for the treatment of various diseases [9, 10]. In spite of much progress in diverse areas of research [11–13], the agrochemical potential of 1,2,3-triazoles has not been explored fully so far. Plant pathogenic fungi are of concern as they cause a substantial loss of crop. Among various pathogenic fungi, which alone cause nearly 20% reduction in the yield of major food and cash crops, Sclerotium rolfsii, Rhizoctonia bataticola, and Fusarium oxysporum are soil-borne fungi which devastate a wide range of hosts. These fungi infect seeds, seedlings, and mature plants in the field causing collar rot, wilt, damping off, dry root rot, and spoilage [14–16]. To the best of our knowledge, reports of antifungal activity of 1,2,3-triazoles against plant pathogenic strains are scarce. Here, we report synthesis and antifungal activity of a series of highly substituted biaryl-based bis(1,2,3-triazoles) against three soil-borne pathogenic strains.

Results and discussion

The triazoles were synthesized in good yields (85–98%) from three new bisazides obtained from their respective amines. The position of the azide group was confirmed by 2D NMR spectroscopy of the corresponding nitro compound. The reaction conditions were optimized by examining the reaction of bisazide 1 with phenyl acetylene in different solvents (Table 1), the best results being obtained using t-butanol/water (3:1) and ethanol. However, product isolation was found to be easier in the former case. In the presence of acetonitrile several products were formed, whereas the reaction in tetrahydrofuran (THF) was not complete even after 3 days.

Table 1 Effect of solvent on reaction of bisazide 1 and phenyl acetylene
Full size table

A series of compounds were synthesized bearing a variety of substituents such as alkyl, aromatic, and heterocyclic groups (Table 2).

Table 2 Synthesis of highly substituted biaryl-based bis(1,2,3-triazoles)
Full size table

Studies on antifungal activity

The compounds were tested for their potential antifungal activity using the food poison technique. The results obtained in terms of percentage inhibition (mean of three replicates) and ED 50 values are shown in Table 3.

Table 3 Antifungal activity of newly synthesized triazoles
Full size table

The newly synthesized triazoles showed moderate to good antifungal activity. They were found to be more effective against R. bataticola (55–100% inhibition) than S. rolfsii and F. oxysporum at a concentration of 500 μg/cm3. ED 50 values of most of these compounds were in the range of 200–500 μg/cm3 against all three fungi. The ED 50 values of these compounds against R. bataticola and S. rolfsii were in the range of ~55–800 and ~350–875 μg/cm3, respectively. Except for compounds 1a and 2d, the ED 50 values were 110–450 μg/cm3 against F. oxysporum. Carboxin was used as a standard fungicide and its ED 50 values were 8.73, 11.78, and 10.34 μg/cm3 against R. bataticola, S. rolfsii, and F. oxysporum, respectively. Further work on the synthesis and structure–activity relationship is currently under progress.

Experimental

Chemicals and solvents were purchased from Sigma-Aldrich and E. Merck. Culture media (PDA) were purchased from HiMedia. Reactions were monitored by thin-layer chromatography (TLC) on fluorescent coated plates purchased from E. Merck. Melting points were measured on an electrothermal melting point apparatus. IR spectra were recorded on a Spectrum BX series spectrophotometer using KBr disks. 1H and 13C NMR spectra were recorded on a Bruker 300 MHz spectrometer. TMS was used as the internal standard in all cases. Mass spectra were recorded on a Qstar XL instrument and processed using Analyst QS software.

5,5′-Diazido-2,2′,3,3′-tetramethoxy-1,1′-biphenyl (1, C16H16N6O4)

The diamine (260 mg, 0.73 mmol) was dissolved in 5 cm3 diluted HCl (1:1) and diazotized with 210 mg sodium nitrite (0.03 mmol) below 5 °C. The solution was filtered and added slowly to a freshly prepared solution of 150 mg NaN3 (2.3 mmol) containing excess sodium acetate. The reaction mixture was stirred for 2 h and the resulting solid was filtered, washed with water, and dried. It was recrystallized from petroleum ether to yield 120 mg (40%) 1. M.p.: 143–144 °C; IR: \(\bar{\nu }\) = 2,939, 2,828, 2,111 (N3), 1,583, 1,488, 1,314, 1,246, 1,112, 1,038, 1,002, 954 cm−1; 1H NMR (CDCl3): δ = 3.6 (s, 6H, –OCH3), 3.8 (s, 6H, –OCH3), 6.5 (s, 4H, Ar-H, J = 3 Hz) ppm; 13C NMR (CDCl3): δ = 55.9, 60.8, 103.4, 112.6, 132.8, 135.2, 144.0, 153.7 ppm.

5,5′-Diazido-2,2′,3,3′,6,6′-hexamethoxy-1,1′-biphenyl (2, C18H20N6O6)

The diamino compound (500 mg, 1.3 mmol) was dissolved in 2 cm3 conc. hydrochloric acid and cooled in ice bath. It was diazotized by adding sodium nitrite solution (200 mg in 1 cm3 water) slowly. The diazotized solution was filtered and subsequently added to a stirred solution of 190 mg sodium azide and 10 cm3 sodium acetate in water. The reaction mixture was stirred for 1 h, and the resulting light yellow solid was filtered. Yield 478 mg (83.7%); m.p.: 83–84 °C; IR: \(\bar{\nu }\) = 2,942, 2,834, 2,104 (N3), 1,590, 1,482, 1,246, 1,046, 969, 816 cm−1; 1H NMR (CDCl3): δ = 3.6 (6H, s, –OCH3), 3.7 (6H, s, –OCH3), 3.9 (6H, s, –OCH3), 6.6 (2H, s, Ar-H) ppm; 13C NMR (CDCl3): δ = 56.1, 60.7, 61.5, 104.1, 124.0, 127.9, 149.5 ppm.

Dimethyl 2,2′-diazido-5,5′,6,6′-tetramethoxy-1,1′-biphenyl-3,3′-dicarboxylate (3, C20H20N6O8)

The diamine (140 mg, 3 mmol) was dissolved in 2.5 cm3 conc. HCl and diazotized with 414 mg sodium nitrite (6 mmol) below 5 °C. The solution was filtered and added slowly to a freshly prepared solution of NaN3 (260 mg in 1 cm3 water) containing excess sodium acetate. The reaction mixture was stirred for 2 h and left overnight in the cold. The next day the white solid was filtered, dried, and recrystallized from petroleum ether. Yield 103 mg (92.2%); m.p.: 94–95 °C; IR: \(\bar{\nu }\) = 3,099, 3,003, 2,947, 2,843, 2,125 (N3), 1,719, 1,588, 1,481, 1,427, 1,295, 1,211, 1,161, 1,025, 1,005, 794 cm−1; 1H NMR (CDCl3): δ = 3.7 (s, 6H, –COOCH3), 3.9 (s, 6H, –OCH3), 4.0 (s, 6H, –OCH3), 7.5 (s, 2H, Ar-H) ppm; 13C NMR (CDCl3): δ = 52.4, 56.1, 60.7, 115.1, 119.3, 124.8, 128.8, 133.2, 149.8, 150.9, 165.5 ppm.

General procedure for the synthesis of triazoles [2]

The bisazide (0.4 mmol) and the alkyne (0.4 mmol) were added to a mixture of t-butanol/water (3:1). To the stirred reaction mixture sodium ascorbate (0.04 mmol) and CuSO4 (0.004 mmol) were added. Completion of the reaction was monitored by TLC. After completion of the reaction (14–48 h) the reaction mixture was diluted with water and the resulting solid filtered. TLC showed that all compounds were pure.

1,1′-(5,5′,6,6′-Tetramethoxy-1,1′-biphenyl-3,3′-diyl)bis(4-phenyl-1H-1,2,3-triazole) (1a, C32H28N6O4)

Yield 97.7%; m.p.: 132–134 °C; R f  = 0.45 (CHCl3); IR: \(\bar{\nu }\) = 3,435, 3,132, 2,941, 1,595, 1,498, 1,466, 1,420, 1,276, 1,233, 1,154, 1,054, 844 cm−1; 1H NMR (CDCl3): δ = 3.8 (s, 6H, –OCH3), 4.0 (s, 6H, –OCH3), 7.4, 7.5, 7.9 (6H, Ar-H), 8.2 (triazole-H) ppm; 13C NMR (CDCl3): δ = 56.1, 60.9, 105.0, 113.9, 117.8, 125.6, 128.3, 128.8, 129.9, 131.9, 132.5, 146.8, 148.3, 153.6 ppm.

1,1′-(5,5′,6,6′-Tetramethoxy-1,1′-biphenyl-3,3′-diyl)bis(1H-1,2,3-triazole-4-methanol) (1b, C22H24N6O6)

Yield 88.1%; m.p.: 152–154 °C; R f  = 0.3 (ethyl acetate); IR: \(\bar{\nu }\) = 3,368, 2,939, 1,592, 1,490, 1,465, 1,417, 1,267, 1,238, 1,150, 1,066, 1,035, 872 cm−1; 1H NMR (acetone-d 6): δ = 3.7 (s, 6H, –OCH3), 4.1 (s, 6H, –OCH3), 4.8 (s, 4H, –CH2–), 7.4 (d, Ar-H, J = 2.1 Hz), 7.6 (d, Ar-H, J = 2.1 Hz), 8.5 (triazole-H) ppm; 13C NMR (acetone-d 6): δ = 56.6, 60.9, 105.7, 114.7, 121.3, 133.5, 133.7, 147.6, 154.5, 206.2, 206.5 ppm.

1,1′-(2,2′,5,5′,6,6′-Hexamethoxy-1,1′-biphenyl-3,3′-diyl)bis(4-phenyl-1H-1,2,3-triazole) (2a, C34H32N6O6)

Yield 95.1%; m.p.: 120–122 °C; R f  = 0.40 (CHCl3); IR: \(\bar{\nu }\) = 3,435, 3,144, 2,935, 1,599, 1,478, 1,420, 1,226, 1,147, 1,053, 831 cm−1; 1H NMR (CDCl3): δ = 3.3 (s, 6H, –OCH3), 3.8 (s, 6H, –OCH3), 4.0 (s, 6H, –OCH3), 7.3, 7.4, 7.5, 7.9 (6X Ar-H), 8.4 (triazole-H) ppm; 13C NMR (CDCl3): δ = 56.3, 60.8, 61.2, 109.1, 121.3, 123.4, 125.8, 126.0, 128.3, 128.9, 130.3, 143.8, 147.9, 148.0, 149.5 ppm.

1,1′-(2,2′,5,5′,6,6′-Hexamethoxy-1,1′-biphenyl-3,3′-diyl)bis(1H-1,2,3-triazole-4-methanol) (2b, C24H28N6O8)

Yield 87%; m.p.: 192–194 °C; R f  = 0.3 (ethyl acetate); IR: \(\bar{\nu }\) = 3,411, 3,158, 2,941, 1,601, 1,492, 1,471, 1,418, 1,220, 1,054, 1,036, 840 cm−1; 1H NMR (DMSO-d 6): δ = 3.3 (s, 6H, –OCH3), 3.8 (s, 6H, –OCH3), 3.9 (s, 6H, –OCH3), 4.9 (s, 4H, –CH2–), 7.4 (s, 2H, Ar-H), 8.1 (triazole-H) ppm; 13C NMR (DMSO-d 6): δ = 54.8, 56.2, 59.7, 60.2, 60.9, 110.3, 122.8, 124.4, 125.6, 144.5, 147.4, 148.3, 148.7, 170.3 ppm.

Tetraethyl 1,1′-(2,2′,5,5′,6,6′-hexamethoxy-1,1′-biphenyl-3,3′-diyl)bis(1H-1,2,3-triazole-4,5-dicarboxylate) (2c, C34H40N6O14)

Yield 85%; m.p.: 140–142 °C; R f  = 0.62 (CHCl3/ethyl acetate 9:1); IR: \(\bar{\nu }\) = 3,104, 2,981, 2,943, 1,747, 1,602, 1,560, 1,489, 1,421, 1,376, 1,350, 1,284, 1,225, 1,085, 1,021, 841 cm−1; 1H NMR (CDCl3): δ = 1.2 (t, 3H, CH3), 1.4 (t, 3H, CH3), 4.2 (q, 4H, –CH2), 4.5 (q, 4H, –CH2), 3.2 (s, 6H, –OCH3), 3.8 (s, 6H, –OCH3), 3.9 (s, 6H, –OCH3), 7.2 (s, 2H, Ar-H) ppm; 13C NMR (CDCl3): δ = 13.7, 14.2, 56.2, 60.8, 61.3, 61.5, 61.8, 62.6, 110.3, 123.0, 124.5, 133.3, 138.7, 145.1, 149.1, 149.5, 158.4, 159.7 ppm.

1,1′-(2,2′,5,5′,6,6′-Hexamethoxy-1,1′-biphenyl-3,3′-diyl)bis(4-hexyl-1H-1,2,3-triazole) (2d, C34H48N6O6)

Yield 89.3%; m.p.: 54–56 °C; R f  = 0.65 (CHCl3/ethyl acetate 9:1); IR: \(\bar{\nu }\) = 2,929, 2,855, 1,601, 1,496, 1,421, 1,221, 1,143, 1,059, 839 cm−1; 1H NMR (CDCl3): δ = 0.8 (s, 6H, CH3), 1.3 (s, 12H, CH2), 1.7 (s, 4H, CH2), 2.8 (s, 4H, CH2), 3.2 (s, 6H, –OCH3), 3.7 (s, 6H, –OCH3), 3.9 (s, 6H, –OCH3), 7.4 (s, 2H, Ar-H), 7.8 (triazole-H) ppm; 13C NMR (CDCl3): δ = 13.9, 22.5, 25.5, 28.7, 29.3, 29.6, 31.4, 56.1, 60.7, 60.8, 109.1, 122.4, 123.3, 126.2, 143.7, 147.5, 148.6, 149.4 ppm.

1,1′-(2,2′,5,5′,6,6′-Hexamethoxy-1,1′-biphenyl-3,3′-diyl)bis[4-(2-pyridinyl)-1H-1,2,3-triazole] (2e, C32H30N8O6)

Yield 93.1%; m.p.: 98–100 °C; R f  = 0.22 (CHCl3); IR: \(\bar{\nu }\) = 3,424, 2,924, 2,837, 1,606, 1,492, 1,469, 1,425, 1,221, 1,149, 1,058, 1,028, 930 cm−1; 1H NMR (CDCl3): δ = 3.3 (s, 6H, –OCH3), 3.8 (s, 6H, –OCH3), 3.9 (s, 6H, –OCH3), 7.4, 7.8, 8.2, 8.6 (Ar-H and pyridine-H), 8.7 (triazole-H) ppm; 13C NMR (CDCl3): δ = 56.2, 60.8, 61.2, 109.0, 115.0, 121.0, 122.8, 123.7, 125.0, 137.0, 144.0, 148.0, 148.1, 149.5, 149.9 ppm.

Dimethyl 5,5′,6,6′-tetramethoxy-2,2′-bis(4-phenyl-1H-1,2,3-triazol-1-yl)-1,1′-biphenyl-3,3′-dicarboxylate (3a, C36H32N6O8)

Yield 88.3%; m.p.: 128–130 °C; R f  = 0.55 (CHCl3/ethyl acetate 9:1); IR: \(\bar{\nu }\) = 3,444, 2,947, 1,718, 1,589, 1,479, 1,352, 1,216, 1,049, 997, 772 cm−1; 1H NMR (CDCl3): δ = 3.5 (s, 6H, –OCH3), 3.8 (s, 6H, –OCH3), 3.9 (s, 6H, –OCH3), 7.3, 7.4, 7.8 (6X Ar-H), 8.3 (triazole-H) ppm; 13C NMR (CDCl3): δ = 52.54, 56.07, 61.50, 114.95, 123.44, 124.00, 125.78, 125.94, 127.97, 128.75, 129.15, 130.49, 146.63, 149.57, 152.75, 164.77 ppm.

Antifungal assay

Microorganisms and media used

The newly synthesized triazoles were tested for antifungal activity against three soil-borne pathogenic fungi, namely Sclerotium rolfsii (ITCC 5226), Rhizoctonia bataticola (ITCC 0842), and Fusarium oxysporum (ITCC 2042), by the food poison technique. These fungi were collected from the Indian Type Culture, Division of Plant Pathology, Indian Agricultural Research Institute (New Delhi, India) and were maintained on potato dextrose agar (PDA) at 25 °C and were subcultured on PDA petri dishes for 5–6 days at 28 °C prior to use as inoculums.

Food poison technique

The ready-made PDA medium (39 g) was suspended in distilled water (1,000 cm3) and heated to boiling until completely dissolved. The medium and petri dishes were autoclaved at 120 °C for 30 min. The compounds were tested at concentrations of 500, 250, 125, and 62.5 μg/cm3. A stock solution of 1,000 μg/cm3 was prepared, which was further diluted with DMSO to give the required concentrations. DMSO (1 cm3) was used as the control. These solutions were added to the media (65 cm3) contained in conical flasks to obtain the desired concentrations of the test compounds in the media. The medium was poured into a two petri dishes (90 cm in diameter) under aseptic conditions in a laminar flow hood. The plates were kept under UV light in the laminar flow chamber for solidification of the media. After solidification, a 5-mm mycelial plug cut from the actively growing front of a 2-week-old colony of the desired pathogenic fungus was then placed with the inoculum side down in the center of each treatment plate, aseptically. Treated petri dishes were then incubated at 28 °C until the fungal growth was almost complete in the control plates.

Calculation of ED 50 values

The mycelial growth of fungus (cm) in both treated and control petri dishes was measured diametrically. The mean and standard errors were calculated from the three replicates of each treatment, and the percentage inhibition of growth (I) was calculated using the following equation:

$$ I(\% ) = \frac{C - T}{C} \times 100 $$
(1)

where C is the diameter of fungal growth in the control and T is the diameter fungal of growth in the treated plates.

For calculation of ED 50 values (effective dose required for 50% inhibition of growth), the percentage inhibition was converted to corrected inhibition by using Abbott’s formula:

$${\text{Corrected inhibition}}\;\left( \% \right) = \frac{{\% I - {\text{CF}}}} {{100 - {\text{CF}}}} \times 100$$
(2)

where CF is the correction factor [(9 − C)/C]. ED 50 values were calculated (effective dose for 50% inhibition, μg/cm3) for inhibition of growth using the Basic LD50 program, version 1.1 [17].