A triazole brassinosteroid biosynthesis inhibitor

The first inhibitor of brassinosteroid biosynthesis is reported. On the basis of the fact that uniconazole inhibits brassinosteroid biosynthesis as its side effect, uniconazole derivatives were synthesized and evaluated as brassinosteroid biosynthesis inhibitor. Among the newly synthesized derivatives, 3-(4-chlorophenyl)-1-phenyl-2-(1,2,4-triazoyl)but-2-en-1-ol (5) was found to inhibit the growth of cress seedlings but not that of rice. Cress, dwarfed by treating this inhibitor, was recovered by treating brassinolide, suggesting that compound 5 primarily inhibits brassinosteroid biosynthesis.

Brassinosteroids have recently been recognized as a new class of phytohormones[1,2] by coupling molecular genetics with studies of biosynthesis. Since the establishment of brassinosteroid chemistry, their homologues have been found to dramatically affect growth responses in plants, including stem elongation, pollen tube growth, leaf bending, leaf unrolling, root inhibition, proton pump activation,[3] promotion of ethylene production,[4] tracheary element differentiation,[5,6] and cell elongation.[7] In addition, extensive studies on their biosynthesis have begun to elucidate their mechanism of action.[8,9] At present, over 40 brassinosteroids have been identified, and it is thought that most of the C28 brassinosteroids are biosynthesized from campesterol, which is a common plant sterol whose side chain has the same carbon skeleton as brassinolide.

In general, specific inhibitors of biosynthesis are useful for determining the physiological functions of endogenous substances, as shown in a study on the mode of action of gibberellin.[10] Therefore, a specific inhibitor of brassinosteroid biosynthesis could provide a new approach to understanding the functions of brassinosteroids. Uniconazole (1 in Fig.

),
[4]

a gibberellin biosynthesis inhibitor, has been reported to inhibit brassinosteroid biosynthesis,[5,6,11] even though its main target is gibberellin biosynthesis[12] rather than brassinosteroid biosynthesis. Various triazole compounds including uniconazole and other gibberellin biosynthesis inhibitors have been shown to inhibit many types of cytochrome P-450, which are found in many oxidative processes in living systems;[13] however, the inhibition of individual enzymes is strictly controlled by the structure of the inhibitor. On the basis of these facts, we have conducted studies on the design and synthesis of brassinosteroid biosynthesis inhibitors among triazole compounds based on their analogy to uniconazole, since many steps in brassinosteroid biosynthesis are thought to involve cytochrome P-450; for example, the production of 6 $\alpha$ -hydroxycampestanol from campestanol, cathasterone from 6-oxocampestanol, teasterone from cathasterone, castasterone from typhasterol, and brassinolide from castasterone (Table

).[1,14]

Compounds 1-5 in Table 1 were synthesized in good yield according to the procedures in Fig.

. In this procedure, bromine is introduced to the $\alpha$ -position of a ketone (a) to yield $\alpha$ -bromoketone. The resulting $\alpha$ -bromoketone (b) is coupled with triazole under basic conditions to yield c, which in turn yields the $\alpha, \beta$ -unsaturated ketone derivative e, followed by condensation with benzaldehyde under basic conditions. Target compound f is obtained by
[4]

Although the final products consisted of four isomers, these compounds were subjected to biological tests without further purification. To determine brassinosteroid biosynthesis inhibitors, we combined some biological assays. First, compounds were assayed using a rice stem elongation test to eliminate gibberellin biosynthesis inhibitors. It is well known that gibberellin biosynthesis inhibitors retard rice stem elongation, and such retardation is supperssed by treatment with gibberellin. Therefore, we considered that this test would be suitable for identifying gibberellin biosynthesis inhibitors. The results are shown in Fig.

. Except for 4 and 5, other
[4]

Fig.3.Rice stem elongation test. Grains of rice (Oryza sativa L. cv. Koshihikari) were germinated in an incubator at 25for 24 h and then transplanted into water containing chemicals in a glass jar. Stem length was measured seven days after transplantation.

chemicals tested here retarded rice stem elongation, and such retardation was suppressed by the addition of gibberellin (GA

) (data not shown), suggesting that such retardation was due to the inhibition of gibberellin biosynthesis. Therefore, 4 and 5 were considered as possible brassinosteroid biosynthesis inhibitors and subjected to the next test. A good biological system for identifying brassinosteroid biosynthesis inhibitors has not yet been found. Therefore, we tested the ability of some plant systems to evaluate the potency of chemicals. As a result, we selected cress (Le-pidium sativum L.) as the test plant because it responded well to the inhibitors, and the inhibited plant recovered well following the addition of brassinolide, a most potent brassinosteroid. In addition, cress has been used previously to investigate the effects of brassinolide[15,16] and therefore it could be useful to compare the present results with those obtained previously. The present results are shown in Fig.

. Compound 5 inhibited the growth of cress hypocotyl, while 4 did not. An important observation is the recovery of cress growth after 5-induced hypocotyl dwarfism by the co-application of brassinolide with 5. On the other hand, the co-application of GA

had less of an effect on the recovery of cress growth after 5-induced dwarfism than that of brassinolide. This implies that the morphological changes in cress treated with 5 are mainly due to a deficiency of brassinosteroids and partly due to a deficiency of gibberellin in cress seedlings. Thus, the main target of 5 appears to be brassinosteroid biosynthesis. The structural difference between 5and 1 is the presence of a phenyl group instead of a t-buylgroup. This difference makes 5 possess an inhibitory activity
[4]

Fig.4.Effect of 1, 4 and 5 on elongation of cress seedling. Cress seeds were sown on 1% agar-solidified medium containing 0.5 $\times$ Murashige and Skoog salts and 1.5% sucrose (w/v) in Agripot (Kirin Brewery Co., Tokyo, Japan) with or without chemicals. Plants were grown in a growth chamber (25) under a 16-h/8-h light (240 $\bsy{\mu}$ E/m

s)/dark cycle. The hypocotyl length was measured seven days after sowing.

against brassinosteroid biosynthesis. Considering that the t-butyl group is bulky while the phenyl group is planar, this result may reflect the difference in structure of the binding sites. The step that 5 blocks in brassinosteroid biosynthesis is not yet known, but a feeding experiment similar to that done by Fujioka et al. with a brassinosteroid-deficient mutant[17] should reveal the target site of 5.

A triazole brassinosteroid biosynthesis inhibitor

References