sinosteroids by isotopes of hydrogen and carbon

The brassinosteroids (BRs) are a class of native plant growth regulating substances with high biological activity even at very low concentration. These compounds have been rigorously explored and it has been found that they are not only growth regulators in plants but also promising antiviral agents. Recently, it has been reported that natural BRs exhibit relatively interesting anticancer activities. Up to now, the basic anticancer potential of BRs against several normal and human cancer cell lines has been determined. Natural BRs, at micromolar concentrations, impart cell growth-inhibitory responses in several human cancer cell lines without affecting the normal cells. To study the mechanism of action of BRs at the molecular level, the corresponding isotopically labelled compounds are essential. The latter BRs are essential for the investigation of biosynthesis, metabolism, transport and distribution in plants. This venture ultimately led us to explore the labeling of BRs by isotopes of hydrogen and carbon and the related technique to do this. The present review will shed light on the synthetic avenues in this field from the time of the discovery of labelled BRs up until their most recent advances.


Introduction
Brassinosteroids (BRs) represent a class of naturally occurring phytohormones with various physiological activities and ubiquitous distribution in the plant kingdom. 1 The vicinal diol grouping on ring A is typical for BR-plant hormones discovered thirty years ago. 2 In Figure   1, the formulae of two typical BRs-24-epibrassinolide 1 and castasterone 2 -are given. These compounds have been intensively studied and found that they exhibit not only growth regulation Recently, molecular studies directed towards the essential role of BRs in plant growth and development 4 and their chemical synthesis, biological mode of action, and practical application in agriculture and horticulture 5,6 have been greatly intensified. The essentialities of isotopically labelled BRs in the investigation of the biosynthesis, metabolism, transport and distribution of endogenous BRs in plants have been documented. In such studies, BRs labelled with isotopes of hydrogen are most frequently used to explain the biosynthesis, metabolism and mode of action at a molecular level. Therefore, the numerous strategies for the synthesis of BRs labelled with deuterium ( 2 H or D) and tritium ( 3 H or T) in the side chain or in the ring system have been developed. 7 BRs are formed by a biosynthetic network of alternative pathways and sub-pathways. A number of 7 cells. 23 In this review, a variety of labelling procedures of BRs with stable and radioactive isotopes of hydrogen and carbon will be presented. Brassinolide (BL) and related C-28 BRs as castasterone (CS), typhasterol (TY), and teasterone (TE) occur in a wide variety of higher plants. 24,25 The synthesis of [26,28-2 H 6 ] brassinolide 1, [26,28-2 H 6 ]CS 2, [26,28-2 H 6 ]TY 3 and [26,28-2 H 6 ]TE 4 as an internal standards for GC-MS assays of BRs was reported in the literature. 26,27 [26,28-2 H 6 ]crinosterol 28 35,36 The synthesis of precursor of [26-2 H 3 ]-brassinosteroids 34 for biochemical studies from the starting material 25 is depicted in Scheme 3. In the first convergent strategies based on the Claisen rearrangement of a single isomer of allylic alcohol is considered useful in this rearrangement to access certain product. 27,28,37,38 A Claisen rearrangement has been widely used in the preparation of ∆ 22 -steroids containing an alkyl substituent with predictable stereochemistry at C-24. Then the ester compounds 32 were prepared via isomeric acetylenic alcohols 28 and 30 and the allylic alcohol   preparation of the labelled BRs containing six deuterium atoms at C-26 and C-27. 35

Synthesis of [26-2 H 3 ]-epibrassinolide and its precursors
A further study towards the synthesis of labelled BRs containing three deuterium atoms in the terminal part of the side chain starting from commercially available stigmasterol 24 is reported by Khripach et al. 32   Stigmasterol 24 was a starting compound that was converted in five steps to 22-aldehyde 124. Claisen rearrangement was the key step in conversion of 22-aldehyde 124 to the ester 125.
Using the reduction with LiAlD 4 -mesylation-reduction with LiAlD 4 sequence as described above the ester group of 125 was converted to [ 2 H 3 ] methyl group in 128. Rearrangement of 3,5-cyclo derivative 128 to ∆ 2 derivative 129 was achieved by boiling with pyridinium hydrobromide.

Stereospecific labelling of brassinosteroids with hydrogen isotope.
As the deuterium labelled BRs must have at least 4 deuterium atoms in metabolically stable positions to be useful as MS internal standards the deuterium labelling in ring A is not very attractive. On the other hand ring A is good target for labelling with tritum especially if the aim is to prepare suitable precursor for tritiation from the non-labelled target BR. It is the rule to model the tritiation reactions first with deuterium and therefore we developed recently general stereospecific method of introduction of deuterium to ring A of BRs having 2α,3α-dihydroxy group.
In quest for the suitable precursor for tritiation we discovered a stereospecific reaction of α-hydroxy ketone 135, prepared in three steps from 24-epicastasterone 2 in 47% yield, with triphosgene 48 giving in 99% yield 3β-chloro-2α,3α-(carbonyldioxy) derivative 136 (Scheme 17). 45 While catalytic reductive dechlorination is frequently used for the introduction of hydrogen isotopes on aromatic rings 49

Synthesis of 24-[7,7-2 H 2 ]epiBL
The intended transformation of lactone group in epibrassinolide 146 was carried out after protecting both diol functions by isopropylidenation. The key transformation is the preparation of 6, 7-seco diacid 153 from diacetonide 147 in three step and then formation of the cyclic anhydride

Scheme 21
Labelling of biogenetic BRs precursors

Summary
The number of published synthesis of BRs labelled either by stable isotopes or by radioisotopes reflects their importance for the biochemical studies of this interesting group of plant growth regulators. The main effort was devoted to the synthesis of multideuterated BRs in side chain as internal standards for MS. For labelling with tritium the methods developed for multideuterated BRs are not useful. The methods using exchange with tritiated water were described. However, of in this way prepared BRs have specific activities only in order of several mCi/mmol. What remains to be done is to prepare the BRs ligands with the specific activities of order tens of Ci/mmol to enable the search for BRs receptors. Only one example of the synthesis of 14 C-labelled BR reflects the shortage and fast increase of price of Ba 14 CO 3 in recent years.
Notwithstanding, some BRs are considered as potential drugs and if they will pass the preclinical sieve, there will be certainly need for their labelling with 14 C.

Acknowledgements
This work was realized at IOCB, Prague, Czech Republic and supported by Academy of Sciences of the Czech Republic (RVO 61388963), by Grant IAA400550801 of the Grant Agency of the Academy of Sciences of the Czech Republic and by CNMS, Jain University, Bangalore.
The authors wish to express thanks to all for financial support.