1999;9:425C430. order to further study the structure-activity relationships of YCZ series, we report herein the synthesis of new triazole derivatives with different aromatic structure at the position 2 of the 1,3-dioxolane moiety to mimic the partial structure of 4-chlorophenyl moiety found in YCZ-14 (The general structure of target compounds 7aCh is shown in Figure 1). Structure-activity relationships of newly synthesized compounds were discussed. 2. Results and Discussion 2.1. Chemistry Target compounds 7aCh were prepared according to a synthetic route (Scheme 1) as we previously described [22]. Scheme 1 Open in a separate window The chemical synthesis of target compounds. The key transformation of 2aCh with compound 5 consisted of four steps: (1) formation of ethanones 2aCh; (2) tosylation of isopropylideneglycerol 3; (3) deprotection of isopropylidene ketal 4; and (4) ketal formation to generate 6aCh. Compounds 2aCh were prepared by reacting different kinds of commercially available BR synthesis-deficient mutants such as seedlings grown in the dark, and we co-applied BL and GA with the test compounds to determine the reversibility of their effects. With this assay system, we evaluated the biological activities of synthesized compounds. 2.3. Biological Activities of Newly Synthesized Brassinosteroid Biosynthesis Inhibitors The chemical structures of compounds applied for biological studies are shown in Table 1. To identify the aromatic chemical structure at position 2 of 1 1,3-dioxolane ring responsible for the retardation of seedling growth. stem elongation were calculated as described in experiment section. All of the experiments were performed at least in duplicate to establish the repeatability. We used YCZ-14 and Brz as positive controls. A phenyl analogue (compound 7a) was used as a baseline reference for structure-activity relationships discussions. The concentrations of all of the test compounds as well as Brz were assigned to be 0, 0.01, 0.05, 0.1, 0.5, 1 and 10 M, and the IC50 values were calculated accordingly. As shown in Table 1, compound 7a exhibits inhibitory activity on retarding hypocotyls elongation of seedling grown in the dark, with an IC50 value of 0.46 0.04 M, while the IC50 of YCZ-14 was 0.12 0.04 and Brz was 0.73 0.13 M, respectively. This result indicates that the inhibitory potency of YCZ-14 (4-chlorophenyl analogue) is stronger than that of 7a and that a mono substituent at position 4 of the phenyl moiety may promote the inhibitory activity. To verify this possibility, we further introduce 4-methylphenyl, 4-fluorophenyl and 4-trifluoromethylphenyl moieties into the inhibitor (analogues 7bCd) to evaluate their effect on inhibitory activity. We found that analogues with methyl and fluorine atom substituents at position 4 of the phenyl ring (compounds 7b,c) have a positive effect on promoting the inhibitory activity compared with that of 7a, with IC50 values of 0.26 0.05 and 0.21 0.01 M, respectively. Interestingly, introducing a 4-trifluoromethylphenyl moiety (compound 7d) to the position 2 of 1 1,3-dioxolane, however, showed a significant negative effect on promoting inhibitory activity, with an IC50 value approximately 0.73 0.06 M. It is worthwhile to note that compound 7d shares the common 4-trifluoromethylphenyl moiety with Brz220, the most potent inhibitor of BR biosynthesis inhibitor reported by Asami(the Brz series) [25]. Data obtained in this work suggests that the structure requirements for these two synthetic series (Brz and YCZ) on inhibition of BR biosynthesis are different. This observation implies the binding site of YCZ series may be different from Brz series. In order to further determine the structure-activity relationships of YCZ synthetic series, we next introduced a bulky aromatic moiety to the inhibitor. As shown in Table 1, the IC50 value of the biphenyl.Reagents are of the highest grade commercially available. The compound-induced hypocotyls dwarfism were counteracted by the co-application of 10 nM brassinolide, the most potent BR, but not 1 M GA3, which produced seedlings that resembled BR-deficient mutants. In order to further study the structure-activity associations of YCZ series, we statement herein the synthesis of fresh triazole derivatives with different aromatic structure at the position 2 of the 1,3-dioxolane moiety to mimic the partial structure of 4-chlorophenyl moiety found in YCZ-14 (The general structure of target compounds 7aCh is demonstrated in Number 1). Structure-activity associations of newly synthesized compounds were discussed. 2. Results and Conversation 2.1. Chemistry Target compounds 7aCh were prepared relating to a synthetic route (Plan 1) once we previously explained [22]. Plan 1 Open in a separate window The chemical synthesis of target compounds. The key transformation of 2aCh with compound 5 consisted of four methods: (1) formation of ethanones 2aCh; (2) tosylation of isopropylideneglycerol 3; (3) deprotection of isopropylidene ketal 4; and (4) ketal formation to generate 6aCh. Compounds 2aCh were prepared by reacting different kinds of commercially available BR synthesis-deficient mutants such as seedlings grown in the dark, and we co-applied BL and GA with the test compounds to determine the reversibility of their effects. With this assay system, we evaluated the biological activities Dolastatin 10 of synthesized compounds. 2.3. Biological Activities of Newly Synthesized Brassinosteroid Biosynthesis Inhibitors The chemical structures of compounds applied for Dolastatin 10 biological studies are demonstrated in Table 1. Dolastatin 10 To identify the aromatic chemical structure at position 2 of 1 1,3-dioxolane ring responsible for the retardation of seedling growth. stem elongation were calculated as explained in experiment section. All the experiments were performed at least in duplicate to establish the repeatability. We used YCZ-14 and Brz as positive settings. A phenyl analogue (compound 7a) was used like a baseline research for structure-activity associations discussions. The concentrations of all of the test compounds as well as Brz were assigned to be 0, 0.01, 0.05, 0.1, 0.5, 1 and 10 M, and the IC50 ideals were determined accordingly. As demonstrated in Table 1, compound 7a exhibits inhibitory activity on retarding hypocotyls elongation of seedling produced in the dark, with an IC50 value of 0.46 0.04 M, while the IC50 of YCZ-14 was 0.12 0.04 and Brz was 0.73 0.13 M, respectively. This result shows the inhibitory potency of YCZ-14 (4-chlorophenyl analogue) is definitely stronger than that of 7a and that a mono substituent at position 4 of the phenyl moiety may promote the inhibitory activity. To verify this probability, we further expose 4-methylphenyl, 4-fluorophenyl and 4-trifluoromethylphenyl moieties into the inhibitor (analogues 7bCd) to evaluate their effect on inhibitory activity. We found that analogues with methyl and fluorine atom substituents at position 4 of the phenyl ring (compounds 7b,c) have a positive effect on advertising the inhibitory activity compared with that of 7a, with IC50 ideals of 0.26 0.05 and 0.21 0.01 M, respectively. Interestingly, introducing a 4-trifluoromethylphenyl moiety (compound 7d) to the position 2 of 1 1,3-dioxolane, however, showed a significant negative effect on advertising inhibitory activity, with an IC50 value approximately 0.73 0.06 M. It is worthwhile to note that compound 7d shares the common 4-trifluoromethylphenyl moiety with Brz220, the most potent inhibitor of BR biosynthesis inhibitor reported by Asami(the Brz series) [25]. Data acquired in this work suggests Dolastatin 10 that the structure requirements for these two synthetic series (Brz and YCZ) on inhibition of BR biosynthesis are different. This observation indicates the binding site of YCZ series may be different from Brz series. In order to further determine the structure-activity associations of YCZ synthetic series, we next introduced a heavy aromatic moiety to the inhibitor. As demonstrated in Table CXCR7 1, the IC50 value of the biphenyl analogue 7e and naphthalene analogue 7f are greater than 10 and 2.63 0.39 M, respectively. This result shows that a bulky aromatic moiety significantly reduces the inhibitory activity. Even though variance of aromatic structure greatly affects the inhibitory activity of this synthetic series, the 4-chlorophenyl analogue (YCZ-14) is the most potent inhibitor. This observation suggests that the substituent(s) of chlorine atom within the phenyl moiety.