Efficient Synthesis and Bioactivity of Novel Triazole Derivatives

Triazole pesticides are organic nitrogen-containing heterocyclic compounds, which contain 1,2,3-triazole ring. In order to develop potential glucosamine-6-phosphate synthase (GlmS) inhibitor fungicides, forty compounds of triazole derivatives were synthesized in an efficient way, thirty nine of them were new compounds. The structures of all the compounds were confirmed by high resolution mass spectrometer (HRMS), 1H-NMR and 13C-NMR. The fungicidal activities results based on means of mycelium growth rate method indicated that some of the compounds exhibited good fungicidal activities against P. CapasiciLeonian, Sclerotinia sclerotiorum (Lib.) de Bary, Pyricularia oryzae Cav. and Fusarium oxysporum Schl. F.sp. vasinfectum (Atk.) Snyd. & Hans. at the concentration of 50 µg/mL, especially the inhibitory rates of compounds 1-d and 1-f were over 80%. At the same time, the preliminary studies based on the Elson-Morgan method indicated that the compounds exhibited some inhibitory activity toward glucosamine-6-phosphate synthase (GlmS). These compounds will be further studied as potential antifungal lead compounds. The structure-activity relationships (SAR) were discussed in terms of the effects of the substituents on both the benzene and the sugar ring.


Introduction
Carbohydrates play an important role in the field of pesticide investigation, and many natural carbohydrate products used as pesticides have shown great vitality [1]. Carbohydrate compounds are easy to degrade, have good environmental compatibility [2], and are less resistant to resistance [3]. The new fungicides, which were developed based on carbohydrate compounds, have advantages of safety, high efficiency, low residue and not easy to generate insecticide resistance [4,5], not only ensuring the high yield of vegetables and other agricultural products [6], but also solve the problem of pesticide residues.
Triazole pesticides are the most widely used variety of fungicides in agricultural production, which are mainly divided into fungicides, herbicides, pesticides and plant growth regulators [7]. At this stage, there are more than 30 varieties which have been produced and widely used, such as triazole alcohol [8], triazolone, propiconazole [9], hexaconazole [10] and fluorosilazole [11]. Triazole compounds have been extensively studied in the field of medicine and pesticides, and exhibit a variety of biological activities. In addition to a strong internal fungicidal activity, triazole compounds also have a regulatory role in the growth of plant [12], herbicidal [13], insecticidal [14] and antifungal [15,16]. Because of these multiple effect of the triazole structure, the research of these compounds have been in the ascendant so far. In order to obtain more efficient derivatives, the chemical structure modification of ed better fungicidpromising results, based on the structural characteristics of the substrate fructose-6-phosphate and the current research basis [1, [19][20][21], we introduced the triazole group which possessed various important bioactivities instead of thiadiazole in this article, six series of novel furan glucosyl triazole compounds were designed and synthesized for the first time, their fungicidal activities against P. CapasiciLeonian, Sclerotinia sclerotiorum (Lib.) de Bary, Botrytis cinerea Pers, Pyricularia oryzae Cav., Fusarium oxysporum Schl. F.sp. vasinfectum (Atk.) Snyd. & Hans. and enzyme inhibitory activities against Candida albicans GlmS were evaluated. We would like to report their synthesis (Scheme 1) and bioactivities in much greater details, and also their structure-activity relationship studies. We report herein the preliminary results of the study. Inspired by these promising results, based on the structural characteristics of the substrate fructose-6-phosphate and the current research basis [1, [19][20][21], we introduced the triazole group which possessed various important bioactivities instead of thiadiazole in this article, six series of novel furan glucosyl triazole compounds were designed and synthesized for the first time, their fungicidal activities against P. CapasiciLeonian, Sclerotinia sclerotiorum (Lib.) de Bary, Botrytis cinerea Pers, Pyricularia oryzae Cav., Fusarium oxysporum Schl. F.sp. vasinfectum (Atk.) Snyd. & Hans. and enzyme inhibitory activities against Candida albicans GlmS were evaluated. We would like to report their synthesis (Scheme 1) and bioactivities in much greater details, and also their structure-activity relationship studies. We report herein the preliminary results of the study. chemical structure modification of these compounds as well as the research as medicine and veterinary drugs have been frontier issues both at home and abroad for a long time.

Synthesis of the Title Compounds
As shown in Scheme 1, we envisioned that the target compounds triazole derivatives 1, 2, 3, 4, 5 and 6 could be synthesized from the intermediates 7 [22] and 8 [23] which could be prepared using 1,2,5,6-di-isopropylidene-D-glucose as the starting material for five steps according to the known methods [22,23]. Then cyclization of 7 and 8 with alkyne provided the desired triazole derivatives 1 and 2 under the conditions of copper sulfate and sodium ascorbate in high yields, respectively. Then the target compounds 3 or 4 were prepared by treating compounds 1 or 2 with 90% trifluoroacetic acid. Compounds 5 or 6 were obtained by acetylation of compound 3 or 4.
All the derivatives were synthesized according to the procedures described in Scheme 1 in good yields of 60-98%. The structures of all the synthesized compounds were confirmed from its 1 H-NMR, 13 C-NMR spectra and HRMS. The 1 H-NMR experiments of compounds 1/2/5/6 were conducted in CDCl3 as the solvent. Nevertheless, because of the poor solubility of compounds 3/4, so we had to switch the solvent to methanol-d4 or DMSO-d6. The physical data of the target compounds were given in Table 1.

Synthesis of the Title Compounds
As shown in Scheme 1, we envisioned that the target compounds triazole derivatives 1, 2, 3, 4, 5 and 6 could be synthesized from the intermediates 7 [22] and 8 [23] which could be prepared using 1,2,5,6-di-isopropylidene-D-glucose as the starting material for five steps according to the known methods [22,23]. Then cyclization of 7 and 8 with alkyne provided the desired triazole derivatives 1 and 2 under the conditions of copper sulfate and sodium ascorbate in high yields, respectively. Then the target compounds 3 or 4 were prepared by treating compounds 1 or 2 with 90% trifluoroacetic acid. Compounds 5 or 6 were obtained by acetylation of compound 3 or 4.
All the derivatives were synthesized according to the procedures described in Scheme 1 in good yields of 60-98%. The structures of all the synthesized compounds were confirmed from its 1 H-NMR, 13 C-NMR spectra and HRMS. The 1 H-NMR experiments of compounds 1/2/5/6 were conducted in CDCl 3 as the solvent. Nevertheless, because of the poor solubility of compounds 3/4, so we had to switch the solvent to methanol-d 4 or DMSO-d 6 . The physical data of the target compounds were given in Table 1. Fungicidal activities of the target compounds 1, 2, 3, 4, 5, 6 against five fungal species were evaluated as previously reported [25] and compared with the commercial fungicide chlorothalonil. The inhibition rates were given in Table 2. The determination results showed that, most of the tested compounds displayed a certain degree of fungicidal activity against the five species at the concentration of 50 µg/mL.  In general, the following structure-activity relationships (SAR) in compounds 1, 2, 3, 4, 5 and 6 were observed: (1) As a whole, series 1 and 2 exhibited good fungicidal activities against P. CapasiciLeonian, Sclerotinia sclerotiorum (Lib.) de Bary, Pyricularia oryzae Cav. and Fusarium oxysporum Schl. F.sp. vasinfectum (Atk.) Snyd. & Hans. than the other series. (2) For the series 1 and 2, the fungicidal activities were increased by improving the electron-withdrawing ability of substituents on the benzene ring such as compounds 1-d (R 2 = 4-F-C 6 H 4 -), 1-e (R 2 = 4-NO 2 -C 6 H 4 -), 1-f (R 2 = 4-Cl-C 6 H 4 -), 2-d (R 2 = 4-F-C 6 H 4 -), 2-e (R 2 = 4-NO 2 -C 6 H 4 -) and 2-f (R 2 = 4-Cl-C 6 H 4 -); When the substituent group (R 2 ) were substituted phenyl, the fungicidal activities of series compounds were superior to that with substituent alkyl.
(3) Compared series 1 and 2, on an overall level the former (R 1 = Me) displayed a better fungicidal activities than the latter (R 1 = Bn). (4) Series 3 and 4 were obtained by deisopropylidenation of the compounds 1 and 2, they had the better water-solubility, but the fungicidal activities of compounds 3 and 4 against five species were decreased obviously. (5) In order to improve the fat solubility, the compounds 5 and 6 were synthesized. The fungicidal activities of compounds 5 and 6 were better than compounds 3 and 4, but lower than compounds 1 and 2.

Bioassay of Enzyme Inhibitory Activities
Inhibitory activities of all the synthesized compounds towards Candida albicans GlcN-6-P synthase were evaluated using the optimized Elson-Morgan method as previously reported [25]. The absorption value of the solution was measured at 585 nm, and then the concentration was counted by the specification curve which was determined thanks to the relation between the absorption value and the concentration of glucosamine-6-phosphate. The inhibition rates were given in Table 3 at 0.35 mm. As was shown, most of the tested compounds exhibited some enzyme inhibitory activities against glucosamine-6-phosphate synthase at 0.35 mm. On the whole, Although Series 1 and 2 displayed a better fungicidal activities against five species, they exhibited poor enzyme inhibitory activities. Series 3 and 4 without the OH-protection at both 1-and 2-position exhibited better enzyme inhibitory activities than the other series. By and large, the enzyme inhibitory activities of Series 5 and 6 with the OH-acetylation at 1 and 2-position were better than Series 1 and 2 but lower than Series 3 and 4 . Compounds 3-a, 3-d, 3-e and 3-f were more active against glucosamine-6-phosphate synthase than the other compounds.

General Methods
All starting materials and reagents purchased from Sigma-Aldrich (Beijing, China) and Sinopharm Chemical Reagent Beijing Co., Ltd. (Beijing, China). Solvents were purified in the usual way. All reactions were carried out under a nitrogen atmosphere if necessary. All reactions were monitored by thin-layer chromatography (TLC) (the Silica gel thin plate purchased from Yantai Dexin Biological Technology Co., Ltd., Yantai, China) analysis and TLC was performed on silica gel HF with detection by charring with 30% (v/v) H 2 SO 4 in CH 3 OH or by UV detection (254 nm). Column chromatography was conducted by elution of a column (8 × 100, 16 × 240, 18 × 300, 35 × 400 mm) of silica gel (200-300 mesh) with EtOAc-PE (b. p. 60-90 • C) as the eluent. Optical rotations were recorded using a Perkin-Elmer 241 polarimeter (Perkin-Elmer, Waltham, MA, USA). 1 H-NMR (400 MHz) and 13 C-NMR (100 MHz) spectra was recorded in CDCl 3 , Meth-d 4 or DMSO-d 6 with a Bruker DPX400 spectrometer (Brook (Beijing) science and Technology Co., Ltd., Beijing, China), using Tetramethyl silane (TMS) as internal standard; Mass spectra were obtained with Agilent 1100 series LC/MSD mass spectrometer (Agilent Technologies Inc., Beijing, China). High-resolution mass spectra (HRMS) were performed by the Peking University. Melting points were measured on a Yanagimoto melting-point apparatus (Yanagimoto MFG CO, Kyoto, Japan) and are uncorrected. Solutions were concentrated at a temperature <60 • C under diminished pressure.
General procedure for the synthesis of title compounds 3/4. Compound 1 or 2 (0.8 g) was dissolved in 90% aq trifluoroacetic acid (20 mL) and then stirred at 40 • C for 4 h, and TLC (1:1 petroleum ether-EtOAc) indicated that the reaction was complete. The trifluoroacetic acid was evaporated under reduced pressure, then the residue was diluted with CH 2 Cl 2 (50 mL), washed with saturated aq. sodium bicarbonate, and dried over Na 2 SO 4 . The soln was concentrated, and the residue was subjected to column chromatography (2:1 petroleum ether-EtOAc) to give the desired product 3/4.