Supported by the Belorussian Foundation for Fundamental Lecular Approaches on Biosynthetic Enzymes And/Or Receptors Research (Grant N X01MC-001)

Chem. Listy 97, s233 ñ s318 (2003) Conference on Isoprenoids 2003

Supported by the Belorussian Foundation for Fundamental lecular approaches on biosynthetic enzymes and/or receptors Research (grant N X01MC-001). will perhaps provide some answers in a near future. Phytoecdysteroids have been found in ca. 6 % of the REFERENCES analyzed plant species (Ferns, Gymnosperms, Angiosperms) and in a few species of fungi; ecdysteroid-related molecules 1. Khripach V. A., Zhabinskii V. N., de Groot A.: Brassi- (pinnatasterols) are present in some Algae too7,8. Although nosteroids: A New Class of Plant Hormones. Academic structurally related to brassinosteroids, ecdysteroids have no Press, San Diego 1999. established physiological role in plants, and they represent 2. Khripach V. A., Zhabinskii V. N., Konstantinova O. V., secondary metabolites able to protect plants against phyto- Khripach N. B., Antonchick A. P., Schneider B.: Steroids phagous Insects (and soil Nematodes?) through toxic and/or 67, 597 (2002). deterrent effects8,9. The diversity of phytoecdysteroids is much higher than that of zooecdysteroids, but this could be a simple consequen- ECDYSTEROIDS AND RELATED MOLECULES ce of their high levels in plants (their concentrations may reach IN ANIMALS AND PLANTS 30 g.kgñ1 dry weight), which allows their more convenient isolation. A single plant species contains in fact a complex REN… LAFONT ecdysteroid cocktail10. Their biosynthetic pathway is not better known than that of Insects, but from an evolutionary point of UniversitÈ Pierre et Marie Curie, Institut de Biologie IntÈgra- view it would be of great interest to elucidate it and thus to tive, Laboratoire díEndocrinologie MolÈculaire et Evolution, determine whether both proceed through the same steps. Ec- 7 Quai Saint Bernard, Case 29, 75252 Paris, France dysteroid distribution within plant organs does not follow E-mail: [email protected] a common pattern ñ they accumulate mainly in underground or aerial parts, in stem bark, flowers and/or seeds, Ö ñ and Ecdysteroids are polyhydroxylated steroids firstly isolated within a given specimen this pattern may change during onto- from insects (1954) and later discovered in plants (1966). This geny, which raises fundamental questions concerning the si- family of molecules bears unique structural features (a cis- te(s) of production and the transport systems within the plant. A/B ring junction, a 7-en-6-one and a 14α-OH) and it compri- These questions will be illustrated with the spinach Spinacia ses more than 300 representatives1. oleracea11. Zooecdysteroids are present in all Arthropods and repre- Such a survey would not be complete without describing sent ìmoulting hormonesî which control in fact not only their the pharmacological effects of ecdysteroids on Mammals and growth (i.e. moults and metamorphosis) but also their repro- Humans. Soon after the discovery of phytoecdysteroids, and duction. If 20-hydroxyecdysone is the most common ecdyste- probably in connection with the idea of using them as pestici- roid, some diversity is observed within arthropod ecdysteroids des, ecdysteroid effects were tested on Mammals (in vivo and as concerns their number of carbon atoms (27 to 29) and the in vitro). These studies showed, besides a very low toxici- number and position of hydroxyl groups. The biosynthetic ty (LD50 >6 g.kgñ1 body weight in mice), a wide array of pathway has not yet been fully elucidated, and a ìblack boxî pharmacological actions12, among which we will essentially remains concerning the early steps, but new approaches have comment the anabolic and hypoglycemic ones. Interestingly, recently allowed the full characterization of several biosynthe- several plants used in traditional medicine belong to the ecdy- tic enzymes belonging to the cytochrome P450 (CYP) family steroid-rich species (e.g. Leuzea carhamoides, Pfaffia panicu- thanks to Drosophila developmental (halloween) mutants2,3. lata, Ajuga iva, Ö), and their effects could be explained (at Besides their classical endocrine roles, ecdysteroids may least in part) by their high ecdysteroid content. fulfill allelochemical functions in some Arthropods, and this Finally, what are the practical uses of ecdysteroids? A first can be examplified by the marine Arachnid (Pycnogonum use connected with their physiological role in Insects con- litorale) which accumulates huge amounts of ecdysteroids in cerns the improvement of silk production13 and of honeybee its integument as a defense mechanism against attack by health14. A second use concerns the development of inducible crabs4, and the Chrysomelid beetle Chrysolina carnifex, which gene expression systems, both in vitro with cell cultures (i.e. incorporates large amounts of ecdysteroids in its defensive for fundamental research on gene function) and in vivo (with secretions5. the aim of developing gene therapy systems)12. A third use is Ecdysteroids have also been found in non-Arthropod In- related with the above-described anabolic effects, and indeed vertebrates including primitive Anthozoans but, although their there is a tremendously developing offer on the web of ec- distribution and titer changes can in some instances be corre- dysteroid-containing preparations for sportsmen and body- lated with developmental events, and although exogenously builders12,15. Natural ecdysteroids have no development in applied molecules may have clearcut effects, attempts to de- agriculture at the moment, however synthetic non-steroidal monstrate their endogenous origin have so far been unsucces- ecdysteroid agonists prove efficient in the field16; a better sful. understanding of ecdysteroid biosynthesis and of its regulation Primitive Invertebrates (e.g. Sponges) contain a wide array will possibly allow new strategies for crop auto-protection to of polyhydroxylated steroids, including true ecdysteroids, the be developed in the future. origin of which need to be determined6. Whether some of these polyhydroxylated steroids are biogenetically related to ecdy- REFERENCES steroids (= proto-ecdysteroids?) and how the Arthropod ecdysteroids have appeared is an important open question. Mo- 1. Lafont R., Harmatha J., Marion-Poll F., Dinan L. N.,

s280 Chem. Listy 97, s233 ñ s318 (2003) Conference on Isoprenoids 2003

Wilson I. D.: Ecdybase (2002); http://ecdybase.org, 5/6 tion of liver mitochondrial glycerophosphate dehydrogenase 2003. (GPDH) and cytosolic malic enzyme (ME) when fed or injec- 2. Gilbert L. I., Rybczynski R., Warren J. T.: Annu. Rev. ted into rats and this assay provides a semi-quantitative mea- Entomol. 47, 883 (2002). sure of one activity of these steroids. 3. Warren J. T., Petryk A., MarquÈs G., Jarcho M., Parvy Hydroxylation of DHEA at any position other than 7 J.-P., Dauphin-Villemant C., OíConnor M. B., Gilbert L. resulted in complete loss of activity, but 7α-hydroxy, 7-oxo-, I.: Proc. Natl. Acad. Sci. U.S.A. 99, 11043 (2002). and 7β-hydroxy DHEA were progressively more active than 4. Tomaschko K. H. : Arch. Insect Biochem. Physiol. 41, DHEA. Among inhibitors tested, glycyrrhetinate, an inhibitor 89 (1999). of 11β-hydroxy steroid dehydrogenase, decreased the activity 5. Laurent P., Braekman J. C., Daloze D., Pasteels J. M.: of the 7α-hydroxy and 7-oxo derivatives but enhanced that of Chemoecology (2003), in press. 7β-hydroxyDHEA. This will be discussed below. 6. Costantino V., DellíAversano C., Fattorusso E., Mangoni DHEA incubated with rat liver homogenate fortified with A.: Steroids 65, 138 (2000). ATP, NADPH and malate is converted to some 40 different 7. Lafont R.: Rus. J. Plant Physiol. 45, 276 (1998). steroids. Repeated sampling and analysis of products formed 8. Dinan L.: Phytochemistry 57, 325 (2001). at short time intervals disclosed the conversion of DHEA to 9. Marion-Poll F., Dinan L. N., Lafont R., in: Biopesticides 7α-hydroxyDHEA, to 7-oxoDHEA, to 7β-hydroxyDHEA in díorigine vÈgÈtale (Regnault-Roger C., Philogène B. J. sequence. Half of the DHEA accumulates as androst-5-ene- R., Vincent C., ed.), p. 97. 2002. -3β,17β-diol (Adiol) thus confirming findings of Schneider 10. B·thori M., Girault J.-P., Kalasz H., MathÈ I., Dinan L. and Mason with rabbit liver4. DHEA is hydroxylated at posi- N., Lafont R.: Arch. Insect Biochem. Physiol. 41, 1 (1999). tion 7α by the known P450 7B. 7α-HydroxyDHEA is oxidized 11. Schmelz E. A., Grebenok R. J., Ohnmeiss T. E., Bowers by 11β-hydroxy steroid dehydrogenase (flip orientation) to W. S.: Arch. Insect Biochem. Physiol. 51, 204 (2002). produce 7-oxoDHEA. 7-oxo-DHEA (in reverse orientation) is 12. Lafont R., Dinan L.: J. Insect Science 3:7 (2003); http: subject to reduction by the same enzyme to produce 7β-hy- //www.insectscience.org/3.7, 9/6 2003. droxyDHEA. These steroids are subject to sulfation at position 13. (a) Chandrakala M. V., Maribashetty V. G., Jyothi H. K.: 3β and to reduction at position 17. Sulfation then also occurs Curr. Sci. 74, 341 (1998); (b) Ninagi O., Maruyama M.: at 17β. The ability to induce the formation of rat liver GPDH Jpn. Agric. Res. Quarterly 30, 123 (1996). and cytosolic ME increases in the order of synthesis: DHEA 14. Kholodova Yu. D.: Ukr. Biokhim. Zh. 73, 21 (2001). <7α-hydroxyDHEA < 7-oxoDHEA < 7β-hydroxyDHEA in- 15. B·thori M.: Mini Rev. Med. Chem. 2, 285 (2002). dicating they are on their way to become an active hormone. 16. Dhadialla T. S., Carlson G. R., Le D. P.: Annu. Rev. 7-OxoDHEA is far more effective than DHEA as an enhancer Entomol. 43, 545 (1998). of memory in old mice. While these in vitro conversions are very rapid and involve relatively large quantities of steroids, we must recall that although DHEA is the most abundant METABOLIC CONVERSIONS steroid in human blood plasma, less than 1 % of the total OF DEHYDROEPIANDROSTERONE (DHEA) circulates as the free steroid. And it is the free steroid that TO NEW ACTIVE STEROIDS: undergoes the metabolic transformations. The low concentra- STRUCTURE / ACTIVITY RELATIONS tion in vivo limits all of the conversions to other active molecules. HENRY LARDY, ASHOK MARWAH, Like rat liver, isolated mouse adipocytes (3T3-L1) also PADMA MARWAH, NANCY KNEER convert DHEA to a variety of steroids; the yield of Adiol is 70 % of the added DHEA and it is excreted from the cells to Enzyme Institute, Department of Biochemistry, University of the aqueous medium5. The formation of Adiol is important Wisconsin, Madison, Wisconsin, USA because Adiol possesses androgen activity that is not inhibited by hydroxyflutamide or bicalutamide, two agents that are 6 The C19 steroids are derived from cholesterol via pre- commonly used to treat prostate cancer . gnenolone to a main source, 3β-hydroxyandrost-5-en-17-one, Human prostate cancer usually responds to anti-androgen commonly referred to as DHEA. This molecule was first therapy by undergoing a period of remission of several months identified by Butenandt and Dannenbaum1 who isolated it to a few years. It then renews growth that is not suppressed by from male human urine as the 3-chloro substituted steroid and the traditional drugs. This final period is termed androgen-in- recognized that the halogen had been introduced by treating dependent or hormone refractory. a urine fraction with HCl. The chemical properties of DHEA Thus it is possible that the reason growth of some prostate have been, and continue to be, actively investigated by several cancers becomes resistant to anti-androgen agents is that Adiol eminent Czech scientists. The physiological actions of DHEA is the androgen that is stimulating their growth. Therefore it is were pioneered by Dr. Jeri Sonka of Charles University who important to find agents that inhibit the androgen activity of summarized ten years of his work in a useful monograph2. Adiol as well as of testosterone and dihydrotestosterone. Two Because DHEA has many desirable physiological proper- steroids bearing ethynyl groups at position 17α have some ties but displays these only weakly, we began to search for ability to thwart the androgen activity of Adiol7 and recent metabolites that might be more active than this parent steroid. work has led to even more effective compounds. For example, There were many known metabolites to be examined and we 3β-acetoxyandrost-1,5-diene-17-ethylene ketal (ADEK) is an developed an assay with which to measure their relative acti- effective inhibitor of the androgenic activity of Adiol as well vities3. Like the thyroid hormone, DHEA induces the forma- as that of dihydrotestosterone8. The agonist effect of ADEK is

s281 Chem. Listy 97, s233 ñ s318 (2003) Conference on Isoprenoids 2003 less than that of hydroxyflutamide and therefore is less likely SYNTHESIS OF15β -SUBSTITUTED STEROIDS to induce withdrawal response in prostate cancer patients. The metabolic conversion of steroids to more active struc- RAISSA P. LITVINOVSKAYA, ALEXANDER tures beyond 7β-hydroxyDHEA has not yet been defined. V. BARANOVSKY, VLADIMIR A. KHRIPACH There are several sulfated esters and glucuronides produced but the ones we have tested are not highly active. Hydroxyla- Institute of Bioorganic Chemistry, National Academy of Scien- tion at position 16a is especially prominent in children. 16α- ces of Belarus, Kuprevicha, 5/2, 220141 Minsk, Belarus -HydroxyDHEA and 16-oxoDHEA are completely inactive in E-mail: [email protected] our rat assay but 3β,16α-Dihydroxyandrost-5-ene-7,17-dione and 3β,7β,16α-trihydroxyandrost-5-ene-17-one are as active Many biologically active steroids possess hydroxy groups as 7-oxoDHEA. Likewise, 3β,16α,17β-androstene-triol is in- linked at the D-ring methylene units, e.g. at the C-15 and C-16 active but introduction of an oxo group at position 7 restores positions. In recent years a lot of methods were proposed for activity. Thus it appears that if the active hormone produced D-ring oxy functionalization. Our interest was to devise a me- from DHEA carries oxygen at position 16, that oxygen must thodology to synthesize steroids with hydroxy(alkoxy)alkyl be introduced after 7 is oxygenated. Expanded A or B ring moiety at C-15 position. derivatives of DHEA are inactive but 3β-acetoxy-17a-oxa-an- In this communication we discuss the application of androst-5-ene-7,17-dione is fully active9. There are many pro- drost-15-en 3-ethers such as 1 for the introduction of 15β-sub- ducts formed in liver tissue from DHEA that have not yet been stituent containing oxygene. The title compounds were prepa- completely characterized. They are the basis of our present work. red according to the following scheme. Details of preparation and identification procedures will REFERENCES be discussed. It schould be noted that using of the obtained compounds 1. Butenandt A.: Dannenbaum H.: Z. Physiol. Chem. 229, 9ñ12 in ene reaction1 opens a way to the corresponding 15β- 192 (1934). -substituted derivatives of pregnane and cholestane series. 2. Sonka J.: Acta Univ. Carol. Med. 71, 171 (1976). 3. Hormones, Thermogenesis and Obesity (Lardy H., Strat- REFERENCES man, ed.), pp. 415ñ426. Elsevier 1989. 4. Schneider J. J., Mason H. L.: J. Biol. Chem. 172, 771 (1948). 1. Mikami K., Shimizu M.: Chem. Rev. 92, 1021 (1992). 5. Gomez F. E., Miyazaki M., Kim Y.-C., Marwah P., Lardy H. A., Ntambi J. M., Fox B. G.: Biochemistry 41,5473 (2002); Lardy H. A., et al.: manuscript in preparation. 6. Miyamoto H., Yeh S., Lardy H., Messing E., Chang C.: Proc. Natl. Acad. Sci. U.S.A. 95, 11083 (1998). 7. Chang H.-C., Miyamoto H., Marwah P., Lardy H., Yeh S., Huang K.-E., Chang C.: Proc. Natl. Acad. Sci. U.S.A. 96, 11173 (1999). 8. Miyamoto H., Marwah P., Marwah A., Lardy H., Chang C.: Proc. Natl. Acad. Sci. U.S.A. 100, 4440 (2003). 9. Reich I. L., Lardy H., Wei Y., Marwah P., Kneer N., Powell D. R., Reich H. J.: Steroids 63, 542 (1998).

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A STEREOSELECTIVE APPROACH based onthestereoselectiveconversionofC22-aldehyde I into TO THE BRASSINOLIDE SIDE CHAIN the isoxazoline III possessing three asimmetric centers with via 22-ISOXAZOLINYLSTEROIDS the chirality desired for brassinolide. Aldehyde I treated with organomagnesium bromide (ob- RAISSA P. LITVINOVSKAYA, MARINA tained from cis-1-bromo-1-propene and magnesium in THF A. AVERKOVA, NADEZHDA V. KOVAL, under inert atmosphere) furnished the 22R-hydroxy-(23Z)- ALEXANDER V. BARANOVSKY, ALEXANDER -olefin II in 70 % yield. 1,3-Dipolar cycloaddition of acetoni- S. LYAKHOV, VLADIMIR A. KHRIPACH trile oxide (generated in situ from corresponding acetaldoxi- me, N-chlorosuccinimide and triethylamine in chloroform) to Institute of Bioorganic Chemistry, National Academy of Scien- allyl alcohol II proceeded slowly to give a cycloadduct III ces of Belarus, Kuprevicha, 5/2, 220141 Minsk, Belarus (25 % with 70 % returning of starting material). Others dias- E-mail: [email protected] tereomers and regioisomers have not been detected in the crude mixture by NMR spectroscopy. As a part of our study on the use of nitrile oxide Taking into account that four diastereomeric products methodology in the synthesis of biologically important could be formed in this reaction the stereochemistry of the steroids we now wish to report a new procedure for obtai- isoxazoline III have been determined by X-ray structure analysis. ning of the 22R,23R- -dihydroxy-24S-methyl functionality Some spectral and X-ray data, reaction mechanisms and chemical transformations of the isoxazolinylsteroid III into steroids with open side chain like IV will be discussed.

SYNTHESIS OF ISOXAZOLE ANALOGUES OF ECDYSTEROIDS

IIIRAISSA P. LITVINOVSKAYA, SVETLANA V. DRACH, ELEHA N. MASALOVA, VLADIMIR A. KHRIPACH

Institute of Bioorganic Chemistry, National Academy of Scien- ces of Belarus, Kuprevicha, 5/2, 220141 Minsk, Belarus E-mail: [email protected]

Earlier we have shown that the application of isoxazoli- IV III nylsteroids as key compounds allows effectively to form the

s283 Chem. Listy 97, s233 ñ s318 (2003) Conference on Isoprenoids 2003 side chain of ecdysteroids, in particular ponasterone and pte- O rosterone C (ref.1). In this work we have put the purpose to HO OH investigate an applicability of application 20-hydroxy-20-iso- O O B xazolinylsteroids such as 1 in reactions of formation of a cyclic moiety of ecdysteroid molecule. The proposed methodology is based on realization of the + R fact that the heterocyclic ring is stable in many reactions that O allowed us to obtaine a number of isoxasoline derivatives 2ñ7. Cl It was surprising the aromatization proceeding under action of [Ni] or [Pd]

Lewis acids and resulting in isoxasole 8. K2CO3,100° Evidence for the structures 2ñ9 was obtained by spectral dioxane or toluene methods; details will be discussed.

REFERENCES O O 1. Khripach V. A., Litvinovskaya R. P., Baranovskii A. V.: O Mendeleev Commun. 1992,117.

PALLADIUM- AND NICKEL- CATALYZED R CROSS-COUPLING ARYLATION IN A SERIES OF 4- AND 6-HALOGEN SUBSTITUTED STEROIDS R = 4-MeO, 4-Me, 4-F, yields 93ñ100 % NIKOLAI V. LUKASHEVa, GENNADY R = 3-Ac-, 4-COOH, yields 8ñ43 % V. LATYSHEVa, GEORGE A. SKRYABINb, IRINA P. BELETSKAYAa ful type of activity. During our research in modification of aThe Department of Chemistry, Moscow State Lomonosov complex organic molecules by Pd-catalyzed cross-coupling University, Vorobievy Gory, 119992, Moscow, Russia, bAm- reactions we found an easy and convenient approach to 4- and biotech Inc., 300 E Lombard St. STE 1400 Baltimore MD 6-arylsubstituted steroids by cross coupling of 4-bromoan- 21202, USA drost-4-ene-3,17-dione,4-bromo-17-hydroxyprogesterone and E-mail: [email protected], [email protected] 6-chloromadinone acetate with arylboronic acids. Usually aryl- and vinyl bromides are convenient substrates for substi- It is known that some of 4-and 6-substituted derivatives of tution of bromine to aryl group in Suzuki reaction. In some androstene and 17-hydroxyprogesterone can act as aromatase cases spatially hindered substrates like steroids are reluctant or 5-α-reductase inhibitors, posses contraceptive or other use- to take part in such reactions. However we have not meet any problems with arylation of above mentioned 4-bromosubsti- tuted steroids under standard conditions giving the respective XY products in high yields. Cross-coupling with spatially hindered vinyl chlorides is a more difficult task. We have studied an influence of cata- OH lysts, solvents and bases on the yields of 6-anisylmadinone B acetate and found the best conditions, providing a series of O R OH some of 6-aryl substituted derivatives in yields from moderate Br to quantitative. 4-Carboxyphenyl substituent was found to be a convenient I, II XY spacer group for binding above mentioned steroid molecules [Pd], K2CO3,100° dioxane:water to protein carriers to prepare immunogens.

R 79–97 % III, IV

I, III: XY = O R = 4-MeO, 4-Me, 4-F, II, IV: X = β-Ac, Y = β-OH 4-COOH and others

s284 Chem. Listy 97, s233 ñ s318 (2003) Conference on Isoprenoids 2003

DETERMINATION OF LINEAR TERPENES REFERENCES ENANTIOMERS PRESENT AT LOW QUANTITITES IN NATURAL 1. (a) Valterov· I., Svatoö A., Hovorka O.: Collect.Czech. ULTI-COMPOUND MIXTURES Chem. Comunn. 61, 1501 (1996); (b) Hovorka O., Urba- nov· K., Valterov· I.: J. Chem. Ecol. 24, 183 (1998). ANNA LUXOV¡a, ANNA-KARIN BORG- 2. Gessner M, Deger W, Mosandl A.: Z. Lebensm.ñUnters. -KARLSONb, KL¡RA URBANOV¡a, Forsch. 186, 417 (1988). IRENA VALTEROV¡a 3. Borg-Karlson A.-K., Lindstrˆm M., Norin T., Persson M., Valterov· I.: Acta. Chem. Scand. 47, 138 (1993). aInstitute of Organic Chemistry and Biochemistry, Academy 4. Bratschat D., Kuntz C., Heil M., Schittrigkeit A., Schu- of Sciences of the Czech Republic, Flemingovo n. 2, 166 10 macher K., Mang M., Mosandl A., Kaiser R.: Phytochem. Prague 6, Czech Republic, bDepartment of Organic Chemi- Anal. 8, 159 (1997). stry, Royal Institute of Technology, S-100 44 Stockholm, Swe- den E-mail: [email protected] STUDIES ON BRASSINOSTEROIDE HORMONE BINDING PROTEINS FROM PLANTS Chiral linear terpenic alcohols such as citronellol, 2,3-dihydrofarnesol and geranylcitronellol play an important role in TOM¡ä MACEKa,MAREKKAMLARa,b, interspecies chemical communication of bumblebees1. These RUDOLF JEéEKa, ANDREA PIäVEJCOV¡b, compounds were also identified in scents of several flowers CSABA KONCZc, LADISLAV KOHOUTa that are pollinated by bumblebee as well. It is important to know the ratio of enantiomers in chiral natural products. In aDepartment of Natural Products, Institute of Organic Che- many cases, interactions between receptor proteins in insect mistry and Biochemistry, Academy of Sciences of the Czech antenna with just one specific enantiomer of pheromone con- Republic, Flemingovo n. 2, 166 10 Prague 6, bDepartment of stituent were proved2. Biochemistry and Microbiology, ICT Prague, Technick· 3, The composition of secretions produced by bumblebeeís 166 28 Prague 6, Czech Republic, cMax-Planck-Institut f¸r labial gland (LG) and the scent of flowers was identified by Zuechtungsforschung, Carl-von-Linne-Weg 15, 50829 Koeln, gas chromatography with mass spectrometry detector. The Germany structure confirmation was done by comparison of mass spec- E-mail: [email protected] tra the NIST library and the standards. For separation of the linear terpene enantiomers from Brassinosteroids (BRs) are a group of plant steroids, of extracts of male LG and from scent of orchids, a two-dimen- which the molecular and biochemical analysis of Arabidopsis sional gas chromatographic (2D-GC) technique3 was used. mutants has furnished conclusive evidence that these com- This technique is crucial e.g. in case of determination the pounds are plant growth hormones1. They are biologically enantiomeric ratio of key monoterpenes in oviposition attrac- active in the various bioassay systems designed for gibberel- tants of the Cameraria ohridella host plant Aesculus hippo- lins, auxins and cytokinins, eliciting remarkable growth res- castanum. This system represents an enantiospecific reaction ponses2. The molecular mechanism of BRs action is uncertain, between antennal receptor proteins of pest moth C. ohridella although one might argue from structural considerations that and kairomone of the horse chestnut. they are likely to work by a mechanism similar to that of Stationary phase in chiral column for separations of linear animal steroid hormones, which generally act via a soluble terpenic alcohols was 60 % of (heptakis(2,3-di-O-acetyl-6-O- receptor-ligand complex that binds to nuclear sites to regulate -TBDMS)-β-cyklodextrin)inPolysiloxanePS-86(ref.4). A good the expression of specific genes. Despite many studies on plant separation of enantiomers of citronellol and 2,3-dihydrofarne- steroids there is no report on successful isolation of a receptor. sol was reached at the temperature around 110 ∞C. Higher Recently published opinion is, that BRs act in plants after pressure of carrier gas was used to reduce extensive retention binding to a sterol binding protein (SBP), which complexes time of 2,3-dihydrofarnesol. with receptor in the membrane, but also binding of BRst to the At these conditions, the retention time of citronellol was membrane receptor alone is not excluded3. 21 minutes and r.t. of 2,3-dihydrofarnesol was 131 minutes. In order to isolate the BRs and oxysterol-binding proteins Pure (ñ)-(S)-citronellol and (ñ)-(S)-2,3-dihydrofarnesol, or receptors we prepared brassinosteroid based bioaffinity respectively, were found in the LG of all investigated bumble- ligands. Affinity chromatography carrier matrices obtained by bees and cuckoo-bumblebees: Bombus terrestris, B. lucorum, oriented immobilisation of BRs ligands bound covalently by B. jonellus, Psithyrus impatiens, P. bohemicus, and P. pyre- a proper spacer arm were compared for performance with plant naes as well as in the volatiles collected from Orchis pauci- extracts. The columns were used to isolate enough proteins flora, O. boryi, and Barlia robertsiana. This finding may from plant extracts for sequencing. Just now the final goal is indicate a narrow relationship between non-rewarding orchids cloning of receptors using methods of reverse genetics.The and their pollinator bumblebees. A hypothesis of flower mi- ligand must be bound by that part of the molecule which least mics of the bumblebee male pheromone for attracting pollina- participates in the biospecific binding. As far as it is not yet tors will be discussed. known exactly, which parts of the BRs molecule are actually necessary for the proper biological activity and which ones This work was supported by the Grant Agency of the Czech for specific binding, oriented immobilisation of different li- Republic (grant No. 203/02/0158). gands to matrix was necessary. Among others newly synthesi-

s285 Chem. Listy 97, s233 ñ s318 (2003) Conference on Isoprenoids 2003 sed brassinosteroid, (20S)-2α,3α-dihydroxy-7-oxa-B-homo- THE SYNTHESIS OF17β -HYDROXY-3- -5α-pregnan-6-one-20-carboxylic acid, was used for immobi- -METHOXY-7α -METHYL-1,3,5(10)-ESTRATRIENE lisation. This compound was obtained in eight steps by general FROM17β -HYDROXY-4-ESTREN-3-ONE synthesis of brassinosteroid skeleton4 from bisnorcholanic acid. Various new BRs derivatives, tested also for other acti- JACEK MARTYNOW, ANDRZEJ KUTNER, vities5, were used to obtain chromatography carriers with BRs HANNA FITAK bound through the ring A, ring B or the side chain. Different carriers were tested to obtain acceptable yield of proteins in amounts sufficient for analysis of their primary structure. Pharmaceutical Research Institute, 8 Rydygiera Str., 01-793 The plant extracts were obtained by grinding frozen plant Warsaw, Poland leaves or callus tissue, salts were removed by gel filtration and E-mail: [email protected] the extract applied to the bioaffinity matrix. The analysis of primary structures by sequencing of proteins obtained is 17β-Hydroxy-3-methoxy-7α-methyl-1,3,5(10)-estratrie- underway. One of the proteins separated from Nicotiana taba- ne 1 is a key intermediate useful in the synthesis of a cum callus extract was identified as osmotin-like protein pre- number of modified steroids, many of which display important cursor. The aminoacid sequence obtained showed a 100 % biological activities1. Compound 1 is not commercially avail- agreement with this pathogen-related (PR) protein, which able in quantity ñ its preparation from more common ste- appears in tobacco under stress conditions6. Our results thus roids has been the subject of a few published synthetic pro- reveal a connection between brassinosteroid and protein in- tocols2, which, however, are lengthy. The most straightfor- volved in stress response. ward synthesis of 1 disclosed to date appears to be the route based on the transformations of β-estradiol to 3-methoxy- The work was sponsored by grant A4055204 of the Grant -17β-(tetrahydropyranyl)oxy-1,3,5(10)-estratrien-6-one 2, Agency of the Academy of Sciences of the Czech Republic and which subsequently is methylated at C(7), followed by a 17- research project Z4 055 905. -O-deprotection and a C(6)-deoxygenation. This procedure affords 17β-hydroxy-3-methoxy-7α-methyl-1,3,5(10)-estra- REFERENCES triene 1 in six steps from estradiol and in yields close to 40 % (ref.3). 1. Szekeres M., Nemeth K., Kon Kalman Z., Mathur J., However, we found that the synthesis of the 6-oxo deriva- Kauschmann A., Altmann T., Redei G. P., Nagy F., Schell tive 2 according to Scheme 1 is very troublesome at the C(6) J., Koncz C.: Cell 85, 171 (1996). hydroxylation stage, due to the formation of boron-derived 2. Kohout L.: Collect. Czech.Chem. Commun.59, 457 (1994). side products which invariably create serious problems during 3. Mussig C., Altmann T.: Trends Endocrinol. Metab. 12, the work-up. Thus, we felt that an improved synthesis of 398 (2001). compound 2 from commercial steroids was necessary for 4. Kohout L., Chodounsk· H., Macek T., Strnad M.: Col- a more practical route to 17β-hydroxy-3-methoxy-7α-methyl- lect. Czech. Chem. Commun. 65, 1754 (2000). -1,3,5(10)-estratriene 1. In this context, we investigated a num- 5. NeönÏrov· P., Svatoö A., Kohout L., Macek T.: Plant ber of chromium(VI)-based oxidation methods4, starting from Physiol. Biochem. 38, S08 (2000). estradiol or its OH-protected analogues. All these attempts in 6. Abad L., DíUrzo M. P., Liu D., Narasimhan M. L., our hands were essentially futile ñ low yields and poor selec- Reuveni M., Zhu J. K., Niu X., Singh N. K., Hasegawa tivities were observed, perhaps with the exception of the P. M., Bressan R. A.: Plant Sci. 118, 11 (1996) PDC/Pyr conditions4d, which required long reaction times and

OH OTHP

1. MeI 2. dihydropyran

3. LIDAKOR, HO MeO B(OMe)3, OH β-Estradiol H2O2

4. [O]

OTHP OH

5. MeI

MeO 6. Et3SiH, MeO O BF3-Et2O Scheme 1 2 1

s286 Chem. Listy 97, s233 ñ s318 (2003) Conference on Isoprenoids 2003

OH OTHP

1. O2, KOAc / DMF then MeI, 72%

O 2. dihydropyran MeO PTSA / PhMe 91% O 2

3. MeI, KOtBu DME, 62%

OTHP OH

4. Et3SiH BF3-Et2O MeO MeO 86% O Scheme 2 1 gave yields below 60 %. Similarly inefficacious proved the 4. (a) PCC/Celite: Mons S., Lebeau L., Mioskowski C.: β approach based on oxidation of 17 -hydroxy-4-estren-3-one Synth. Commun. 28, 213 (1998); (b) CrO3/AcOH: Dean by molecular oxygen in DMSO, in the presence of a base5.In P. D. G., Exley D., Johnson M. W.: Steroids 30,593 light of these results, it was very gratifying for us to find that (1971); (c) CrO3-DMP: Garza G. A., Rao P. N.: Steroids 17β-hydroxy-4-estren-3-one under the action of molecular 42, 3188 (1983); (d) PDC/Pyr: Bastiaansen P. M. F. M., oxygen in hot DMF, in the presence of potassium acetate6 was Kohout L., Posthumus M. A., Wijnberg J. B. P. A., de quite cleanly transformed to 3,17β-dihydroxy-1,3,5(10)-es- Groot A.: J. Org. Chem. 61, 859 (1996). tratrien-6-one. The latter compound, in the same reaction pot, 5. Hofmeister H., Laurent H., Wiechert R.: Chem. Ber. 106, was subsequently selectively methylated at the phenolic OH, 723 (1973). followed by a separate step of tetrahydropyranylation at the 6. Plate R., van Wuijtswinkel R. C. A. L., Jans C. G. J. M., remaining hydroxyl. In our hands, this procedure ñ carried out Groen M. B.: Steroids 66, 117 (2001). on a 20 g scale ñ gave the desired compound 2 in a 65 % yield 7. Rao P. N., Cessac J. W., Kim H. K.: Steroids 59,621 from 17β-hydroxy-4-estren-3-one (Scheme 2). (1994). We completed the synthesis of 17β-hydroxy-3-methoxy- -7α-methyl-1,3,5(10)-estratriene 1 on the way of α-methyla- tion of the 6-oxo derivative 2 (MeI, KOtBu, DME: 62 % yield after chromatography), followed by C(6)-deoxygenation and In vitro ASSESSMENT OF THE NEUROACTIVE 17-O-deprotection (Et3SiH, BF3.Et2O: 86 %). This gave the POTENTIAL OF PREGNANE DERIVATIVES β key compound 1 (crystalline) in 35 % total yield from 17 - ON GABAA RECEPTOR USING PRIMARY -hydroxy-4-estren-3-one, in only four synthetic steps (Sche- CULTURES OF CORTICAL NEURONS 7 me 2). Equivalent conditions (Et3SiH-TFA) for the deoxygenation reaction of similar C(6)-ketones have also been repor- LIBOR MATY¡äb,c, ZOILA BABOTa, ted. The direct synthesis of compound 1 from 17β-hydroxy-4- ALEXANDER KASALb, CRISTINA SU—OLa* -estren-3-one presented here is conceptually simple and, quite possibly, it is the most practical of all the disclosed appro- aDepartment of Neurochemistry, Institut díInvestigacions Bio- aches to 17β-hydroxy-3-methoxy-7α-methyl-1,3,5(10)-estra- mèdiques de Barcelona, Consejo Superior de Investigaciones triene. CientÌficas (CSIC, IDIBAPS), RossellÛ 161, Barcelona, Spain, bInstitute of Organic Chemistry and Biochemistry, Academy REFERENCES of Sciences of the Czech Republic, Flemingovo n. 2, 166 10 Prague 6, cInstitute of Chemical Technology, 166 28 Prague 6, 1. Lednicer D., Mitscher L. A.: The Organic Chemistry of Czech Republic Drug Synthesis, Vol. 2, Chap. 6. Wiley, New York1980. E-mail: [email protected] 2. (a) Wieland P., Anner G.: Helv. Chim. Acta 50,289 (1967); (b) Anner G., Wieland P.: FR 1418540 19651119. The steroids synthesized by the brain and nervous system, 3. (a) Skaddan M. B., W¸st F. R., Katzenellenbogen J. A.: J. named neurosteroids, have a wide variety of diverse functions. Org. Chem. 64, 8108 (1999); (b) Tedesco R., Katzenellen- In general, neurosteroids mediate their actions, not through bogen J. A., Napolitano E.: Tetrahedron Lett. 38,7997 classic steroid hormone nuclear receptors, but through other (1997); (c) Tedesco R., Fiaschi R., Napolitano E.: Syn- mechanisms such as through ion gated neurotransmitter recep- thesis 1995, 1493; (d) Sala A., Lenna R.: IT 2000MI0918 tors. In the adult, neurosteroid stimulation of neurotransmitter A1 20011022. receptors results in behavioral effects, such as decreased an-

s287 Chem. Listy 97, s233 ñ s318 (2003) Conference on Isoprenoids 2003 xiety, sedation, and decreases in seizure activity, these effects being associated with stimulation of neuronal GABAA receptors (for review, see1,2). Thus, one of the primary neurosteroid target receptors considered is the γ-aminobutyric acid type A (GABAA) receptor complex. ñ The GABAA receptor/Cl ionophore complex is an oligo- meric protein that has separate but allosterically interacting binding sites for the endogenous neurotransmitter GABA, for benzodiazepines and for picrotoxinin-like convulsants. Known positive allosteric modulations include the enhanced binding of benzodiazepine agonists by GABA, the enhanced GABA- -induced Clñ flux by benzodiazepines and barbiturates and the different modifications of [35S]tert-butylbicyclophosphoro- thionate binding induced by GABA, benzodiazepines and barbiturates. Certain pregnane steroids produce clear beha- α α vioural effects including, anxiolysis, sedation, analgesia, ane- Fig. 1. A. 3 -OH-5 -pregnan-one B: 3α-fluoro-2β-OH-5α-pregnan-one sthesia and are anti-convulsant. This behavioural profile is C and D: CMO derivatives of 3α-fluoro-5α-pregnan-one characteristic of compounds that act to enhance the actions of GABA acting at the GABAA receptor. It was first shown that the neuroactive steroids 3α,5α-tetrahydrodeoxycorticostero- decreased [3H]flunitrazepam binding by 20ñ25 %. The accom- ne and 3α,5α-tetrahydroprogesterone (3α-hydroxy-5α-preg- panying Fig. 1 shows the effects of these compounds. nan-20-one or allopregnanolone) enhanced the binding of According to the proposed hypothesis, none of the fluorine muscimol and benzodiazepines to GABAA receptors, enhan- synthetized compounds would have a positive allosteric action ñ ced the GABA-elicited Cl current and displaced TBPS bin- in the GABAA receptor similar to that produced by epalon. ding. All these effects were consistent with neuroactive ste- From the compounds tested, compound B may have a positive roids acting as positive modulators of GABAA receptors and, action on GABAA receptor, however its efficacy is lower than hence, modulating neuronal excitability in the nervous system that of epalon, and C and D compounds may directly interact (for review, see3). with the benzodiazepine binding site. Other factors, like bio- Numerous synthetic steroids have been synthesized in an availability and pharmacokinetics among others, should also attempt to therapeutically exploit the behavioural effects of be considered to establish the pharmacological interest of this the pregnane steroids. The conversion of steroid derivatives compound. with better pharmacological profiles has to be considered when evaluating the putative clinical properties of neuroactive Supported by FIS grant 01/1318 and CSIC ñ Academy of Scien- steroids in vivo. Primary cultures of cortical neurons, constitu- ces agreement project 2003CZ0005. Z. Babot and L. Matyas tively expressing GABAA receptors, are a good in vitro system are recipients of a fellowship from the Spanish Ministries of model to study allosteric interactions at the GABAA recep- Education, Culture and Sports and Foreign Offices, respecti- tor4ñ7. The increase of Clñ flux or the increase of [3H]flunitra- vely. zepam binding produced by a compound in this in vitro system may be predictive of the in vivo action of this compound as REFERENCES positive GABAA receptor allosteric modulator. In this work we have determined the effect of several newly synthesised 1. Plassat-Schiess E., Baulieu E. E.: Brain Res. Rev. 37,133 pregnane derivatives on [3H]flunitrazepam binding. In an at- (2001). tempt to increase the stability of the pregnane derivatives, 2. van Broekhoven F., Verkes R.: Psychopharmacology a fluorine atom was introduced in position 3. Primary cul- 165, 97 (2003). tures of cortical neurons were used to assess the effects of 3. Rupprecht R., Holsboer F.: Trends Neurosci. 22,410 these newly synthesized pregnane derivatives on inhibitory (1999). GABAergic neurotransmission. 4. SuÒol C., PomÈs A., RodrÌguez-FarrÈ E., in: European Preparation of primary cultures of cortical neurons from Medicines Research. Perspectives in Pharmacotoxico- 16-day-old mice fetuses and performance of [3H]flunitraze- logy and Pharmacovigilance (Fracchia G. N., ed.), pp. pam binding was performed as described elsewhere6ñ8. 223ñ234. IOS Press, 1994. Allopregananolone (compound A) increased [3H]flunitra- 5. PomÈs A., RodrÌguez-FarrÈ E., SuÒol C.: Dev. Brain Res. zepam binding in a concentration-dependent manner with an 73, 85 (1993). EC50 value of 1.35 µM. Substituting 3α-OH by a F atom led 6. PomÈs A., RodrÌguez-FarrÈ E., SuÒol C.: J. Pharmacol. to a compound that did not increase [3H]flunitrazepam bin- Exp. Ther. 271, 1616 (1994). ding. Further structural modification of this 3-F derivative 7. Vale C., PomÈs A., RodrÌguez-FarrÈ E., SuÒol C.: Eur. J. produced compounds with different effects on [3H]flunitraze- Pharmacol. 319, 343 (1997). pam binding. Introduction of an OH group in position 2 8. Vale C., VilarÛ E., RodrÌguez-FarrÈ E., SuÒol C.: J. (compound B) produced a compound that slightly increased Neurosci. Res. 57, 95 (1999). [3H]flunitrazepam binding, partially recovering the effect of allopregnanolone. However, introducing a longer acidic aliphatic chain produced two compounds (C and D) that even

s288 Chem. Listy 97, s233 ñ s318 (2003) Conference on Isoprenoids 2003

ANALYSIS OF BILE ACID DERIVATIVES Table I BY INHIBITION OF Na+/K+-ATPase Equilibrium and kinetic constants1,4ñ6 of substances in the + + AS TO THEIR POSSIBLE CARDIOACTIVITY Na /K -ATPase (human kidney) inhibition test. kon or koff = velocity constant for formation or decay of the effector-recep- a,b* a RUDOLF MEGGES , RUDOLF SCH÷N , tor complex, respectively.KíD = koff/kon = inhibition constant J‹RGEN WEILANDa, HEINRICH REPKEb, at 37 ∞C and pH 7.4. sl = solubility, r.a.= relative activity KURT R. H. REPKEa (1 = 100). aMax Delbr¸ck Center for Molecular Medicine, D-13092 Ber- b kon koff KíD sl ñ1 ñ1 ñ1 lin-Buch, Germany, German-American Institute for Applied [µM .min ] [min ] [µM] [µM] Biomedical Research GmbH (GAIFAR), D-14 473 Potsdam, Germany E-mail: [email protected] 1 0.0044 0.18 41 15 2a >>20 20 The Na+/K+-ATPase ñ the molecular point of attack of 3 0.011 0.52 48 80 cardioactive steroids ñ has been shown by us to be suited for 4 0.0080 0.13 15.7 50 the analysis of steroids of different types as to their respective 5 0.0047 0.19 40 50 proper-ties1. Cholanic acids (bile acids) have the favourable 6 0.0080 0.13 15.7 50 5β configuration and a 17β substituent with the same number 7 0.0028 0.20 69 50 of carbon atoms as the highly cardioactive bufadienolides. 8 0.014 0.22 15.7 80 Therefore, they are potential and available starting compounds 9 0.0032 0.26 80 100 for the synthesis of cardioactive drugs despite of the less 10 0.0024 0.33 138 >300 favourable 3α-OH and 14α-H (= C/D-trans connection). We have investigated 9 cholanic acid methyl esters (CA) carrying a No inhibition up to solubility limit up to 3 OH groups and one amide with respect to their inhibition of human kidney Na+/K+-ATPase (Table I). Compound 1 has only one equatorial 3-OH group which is common to all investigated CA except 2. Introduction of a second OH group increases [4: 6α- (eq), 6: 7α- (ax), or 8: 12α-OH (ax)] or decreases [7: 7β-OH (eq)] the activity compared to 1, whereas 6β-OH (ax) in 5 has no influence. Thus, improvement of the activity is caused by one OH-group at the α-face of the steroid backbone. A third additional OH group [9: 6α-(eq), 7α-OH (ax)], 10: [7α- (ax), 12α-OH (ax)] did not further increase but decreases the activity. The 3α-O-acetyl- -∆11 derivative of 1 (2) shows a strong decrease of activity. Exchange of -COOCH3 in 1 against -CONHCH2-COOCH3 (3) shows nearly no influence on the activity. This is remarkable, as O → N exchange in the lactone rings of cardenolides2 or 3 R3a = -OH (1,3ñ10); =-OAc; ∆11 = (2) bufadienolides shows a strongly decreased activity of the R24 = -OCH (1,2,4ñ10); -NHCH CO CH (3) lactames compared to the oxygen analogues. Compared to the 3 2 2 3 cardioactive compounds bufalin1 or digitoxigenin1 or canre- 1: 3α-hydroxy-5β-cholan-24-oic acid methyl ester none4, the activity of 1 is about 4 or 3 orders of magnitude (lithocholic acid methyl ester) lower or one order higher, respectively. These differences 2: 3α-acetoxy-5β-chol-11-en-24-oic acid methyl ester reflect the differences in the velocity of the ATPase effector (lithocholenic acid methyl ester 3-acetate) receptor complex formation (k ) whereas the velocities of α β on 3: 3 -hydroxy-5 -cholan-24-oic acid-N-(methoxycarbonyl- complex decay (koff) are similar. methyl) amide Most of other C/D-trans-steroids without 5β configuration 4: 3α,6α-dihydroxy-5β-cholan-24-oic acid methyl ester show a more or less strongly decreased activity1 compared to 1. (hyodeoxycholic acid methyl ester) The solubility of 1 in the measuring buffer solution is about 5: 3α,6β-dihydroxy-5β-cholan-24-oic acid methyl ester 15 µM and increases roughly with an increasing number of (murocholic acid methyl ester) OH-groups. 6: 3α,7α-dihydroxy-5β-cholan-24-oic acid methyl ester (chenodeoxycholic acid methyl ester) REFERENCES 7: 3α,7β-dihydroxy-5β-cholan-24-oic acid methyl ester (ursodeoxycholic acid methyl ester) 1. Repke K. R. H., Megges R., Weiland J., Schˆn R.: An- 8: 3α,12α-dihydroxy-5β-cholan-24-oic acid methyl ester gew. Chem., Int. Ed. Engl. 34, l294 (1995); Angew. (deoxycholic acid methyl ester) Chem. 107, 308 (1995). 9: 3α,6α,7α-trihydroxy-5β-cholan-24-oic acid methyl ester 2. Megges R., Weiland J., Schˆn R., Repke H., Repke K. R. (hyocholic acid methyl ester) H.: Collect. Czech. Chem. Commun. 67, 336 (2002). 10: 3α,7α,12α-trihydroxy-5β-cholan-24-oic acid methyl ester 3. Megges R., Weiland J., Portius H.-J., Glusa E., Messer- (cholic acid methyl ester) schmid A., Repke H., Repke K. R. H.: 23rd IUPAC-2002

s289 Chem. Listy 97, s233 ñ s318 (2003) Conference on Isoprenoids 2003

Int.Symposium on the Chemistry of Natural Products, (Carum carvi L., Anethum graveolens L.). R-(ñ)-carvone is Florence/Italy, 28 Julyñ2 August, 2002, Book of Ab- considered a very promising inhibitor of farnesyltransferase stracts, p. 274. (FTase; IC50= 1,5 mM) ñ an enzyme that attends in post-trans- 4. Repke K. R. H., Schˆn R., Weiland J., Megges R., Nitz lational prenylation of Ras proteins, which are responsible for M.: German Patent (DE-Offenlegungsschrift) 43 21 937 cells proliferation, to function properly. Carvone and dihy- (1995). drocarveol are active against Gramñ and Gram+ bacteria. 5. Repke K. R. H., Sweadner K. J., Weiland J., Megges R., Carvone and its derivatives are also used as starting mate- Schˆn R.: Prog. Drug. Res. 47, 9 (1996). rials for the synthesis of many drugs, e.g. antimalarial and 6. Repke K. R. H., Schˆnfeld W., Weiland J., Megges R., anticancer agents or calcium metabolism regulators. Hache A., in: The Design of Enzyme Inhibitors as Drugs Our interests have been focused on obtaining derivatives (Sandler M., Smith H. J., ed.), pp. 435ñ502. Oxford of 4R-(ñ)- and 4S-(+)-carvone with precisely established ab- University Press, Oxford 1989. solute configuration at C-1 and C-2 stereocentres. These have potential applications as valuable chiral synthons for asym- metric synthesis. The most common pathway for carvone A FACILE SYNTHETIC APPROACH biotransformations described in literature (except for hydro- TO CYCLOPENTACYCLOOCTANE DITERPENOID xylation reactions) comprises a two-step process leading to SKELETON USING RING-CLOSING METATHESIS carveols. The first step is the reduction of the C=C double bond in the cyclohexene ring, followed by the reduction of the MICHA£ MICHALAK, KAROL MICHALAK, carbonyl group1. JERZY WICHA In our studies we used enzymatic systems of celeriac (Apium graveolens L., var. rapaceum) and carrot (Daucus Institute of Organic Chemistry, Polish Academy of Sciences, carota L.) in order to obtain 4R-(ñ)- and 4S-(+)-carvone deri- Kasprzaka 44/52, 01-224 Warsaw, Poland vatives in high optical purity. The biocatalysts we used transformed 4R-(ñ)-carvone to dihydrocarveol also in two steps, The dicyclopenta[a,d]cyclooctene ring system is the struc- but in the opposite order. The carbonyl group was reduced first tural core of numerous diterpenes (such as the fusicoccin1) and to give an allyl alcohol (2S-carveol), and then the reduction of sesterterpenes (such as the ophiobolins and ceroplastin2). The the double bond in the ring occurred, which led to 1R-dihy- wide range of biological activities exhibited by these com- drocarveol. pounds1,2 and their specific structural features have stimulated Transformation of 4S-(+)-carvone proceeded in a different interest in developing synthetic approaches to their core ring manner: the carbonyl group remained intact with 1R-dihydro- system. In this communication we present a new approach to carvone as the only product. synthesis of 5ñ8 ring carbon framework using three consecu- tive reactions: Mukaiyama-Micheal conjugate addition, palla- REFERENCES dium-catalyzed allylation (Tsuji alkylation) and ring closing metathesis (RCM). The reactivity differences in 8-membered 1. Speicher A., Roeser H., Heisel R.: J. Mol. Catal. B: ring closure due to stereochemical factors will be discussed. Enzym. 22, 71 (2003).

REFERENCES FUNGAL CONVERSION 1. Leung P. C., Taylor W. A.,Wang J. H., Tripton C. L.: J. OF DEHYDROEPIANDROSTERONE Biol. Chem. 259, 2742 (1984). 2. Tsipouras A. A., Tkacz J. S., Frazier E. G, Rohrer S. P., ANNA SZPINETER*, TERESA KO£EK, Birzin E., Rosegay A., Zink D. L., Goetz M. A., Singh S. ALINA åWIZDOR, AGNIESZKA MIRONOWICZ B., Schaeffer J. M.: Bioorg. Med. Chem. 4, 53 (1996). Department of Chemistry, Agricultural University of Wro- c≥aw, ul. Norwida 25, 50-375 Wroc≥aw, Poland BIOTRANSFORMATION OF (+)- E-mail: [email protected] AND (ñ)-CARVONES BY CELERIAC AND CARROT ENZYMATIC SYSTEM Dehydroepiandrosterone (DHEA) is a secretory product of adrenal, gonads and the central nervous system. AGNIESZKA MIRONOWICZ, WANDA K. M•CZKA Part of general pule of DHEA is derived from its circula- ting sulfate ester (DHEAS)1. Serum DHEA concentrations Department of Chemistry, Agricultural University, ul. Norwi- decrease with age and this fact leads to speculations about da 25, 50-375 Wroc≥aw, Poland a possible positive role of DHEA hormone in aging. In humans E-mail: [email protected] DHEA is a precursor of sex steroid hormones such as androgens and estrogens. DHEA can influence processes of cogni- S-(+)-carvone, having the scent of caravay, and R-(ñ)-car- tion and memory due to its neuroactive effect. Administration vone, which smells of green pepper, are commonly present in of this hormone to elderly people improves a frame of mind, many plants (Mentha spicata, M. viridis, Cyperus rotundus mood and sexuality2.This compound can play an important root). These compounds are used as fragrants and flavourings, role in enhancing the immune system response and improving and as drugs ñ in therapeutics based on natural plant extracts cell proliferation control3.

s290 Chem. Listy 97, s233 ñ s318 (2003) Conference on Isoprenoids 2003

O while androstenedione was transformated to 6β-, 14α-, 15α- O O -monohydroxy derivatives. In some of the fungi used in present study, DHEA under- went the Baeyer-Villiger oxidation5 to the testolactone (Peni- cillium camembertii, Penicillium lilacinum) and to lactone HO OH HO with 3β-hydroxy-5-ene functions (Penicillium camembertii). 5-Androstene-3β,17β-diol, a known metabolite of nervous i–viii ix, x system tissues, was formed from DHEA in the Botrytis cinerea culture. OH O REFERENCES xi 1. Burger H. G.: Fertil. Steril. 77, Suppl. 4, 3 (2002). 2. Allolio B., Arlt W.: Trends Endocrinol. Metab. 13,228 HO HO (2002). 3. Arlt W., Justl H. G., Callies F., Reincke M.: J. Endocrinol. ix, x Metab. 83, 1928 (1998). 4. Morfin R., Lafaye P., Cotillon A. C., Nato F., Chmielew- O ski V., Pompon D.: Ann. N.Y. Acad. Sci. 917, 971 (2000). O O 5. Roberts S. M., Wan P. W. H.: J. Mol. Catal., B: Enzymatic 4, 111 (1998).

O O NEW BRASSINOSTEROID ANALOGS HAVING Scheme 1. (i) Absidia coerulea; (ii) Fusarium culmorum; (iii) Fusar- NITROGENATED FUNCTIONALITIES AT C3 ium oxysporum; (iv) Nigrospora oryzae; (v) Aspergillus ochraceus; TO PROVIDE MORE INFORMATION ABOUT THE (vi) Mucor hiemalis; (vii) Mucor circinelloides; (viii) Penicillium BRASSINOSTEROID-RECEPTOR INTERACTION frequentans; (ix) Penicillium camembertii; (x) Penicillium lilacium; Botrytis cinerea (xi) MERITXELL MOLIST, MARC AMOR”S, CRISTINA DAVI, XEVI HERNÀNDEZ, LAURA TOBELLA, CARME BROSA* Human and murine species are able to hydroxylate DHEA at 7α-position and the presence of product of this hydroxyla- Institut QuÌmic de Sarrià, C.E.T.S., Universitat Ramon Llull, tion (7α-hydroxy-DHEA) was observed in liver, brain and Via Augusta 390, 08017 Barcelona, Spain other tissues. The 7α-hydroxylated derivative has stronger E-mail: [email protected] biological activity than DHEA and shows more effective activation of immune processes in mouse. It enhances the Brassinosteroids are potent plant growth regulators, which resistance against lethal infections and counteracts the gluco- have an exciting potential use in agriculture for improving the corticosteroid immune supression in peripheral tissues4. yield and quality of crops1. We have studied microbial transformation of DHEA. Three Considering that hydrogen bonding interaction can take types of reactions were observed: hydroxylation, Baeyer-Vil- place in the brassinosteroid-receptor complex2, interesting liger oxidation and reduction of carbonyl group. points of view to be determined are: (1) whether the OH groups 7α-Hydroxy-DHEA was produced from DHEA by the present in an active brassinosteroid act as acceptors or as fungi: Absidia coerulea, Mucor hiemalis, Mucor circinelloi- donors in such hydrogen bonding, (2) the contribution of each des, Penicillium frequentans, Fusarium culmorum, Fusarium OH group presents in the brassinosteroid to develop biological oxysporum, Nigrospora oryzae and Aspergillus ochraceus activity.

OH OH OH

N N O H N 3 H 3 H 2 H O O O I: 22R,23R III: 22R,23RV:22R,23R II:22S,23SIV:22S,23SVI:22S,23S

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OH OH OH

HO HO

H N O N N 2 H 3 H 3 H O O O VII:22R,23RIX: 22R,23RXI: 22R,23R VIII:22S,23SX:22S,23S XII:22S,23S

In this sense and focused on the A ring, the substitution of SYNTHESIS AND BIOLOGICAL ACITIVITY the OH function at C3 by another functional group, amine or OF HETEROCYCLIC STEROIDS: TARGETING azide and the activity evaluation of such compounds should INHIBITION OF CYTOCHROME P450 ENZYMES give us more information about the type of interaction that IN BREAST AND PROSTATE CANCERS could take place upon binding in such positions. Moreover, analogs with the presence or not of an adjacent OH at C2 would VINCENT C. O. NJAR, ANGELA M. H. BRODIE be useful to extend the number of compounds with modifications on the A ring and useful to clarify the contribution of Department of Pharmacology and Experimental Therapeu- OH-C2 and OH-C3 in the activity3. tics, University of Maryland School of Medicine, 685 West In this communication, we present the synthetic strategy Baltimore Street, Baltimore, MD 10201-1559, USA and bioactivity evaluation in the rice lamina inclination test (RLIT) towards different brassinosteroid derivatives with an The cytochromes P450 constitutes a super family of heme- amine or azide function at C3 on the A-ring, having or not OH -thiolate enzymes, present in all species1. The P450 enzymes function at C2. involved in steroid hormone biosynthesis represent an important target for drug discovery and development for the treat- This work was supported by a grant from Generalitat de ment of hormone-dependent cancers (reviewed by Van Wau- Catalunya (No. 2003 F1 00930). M. M. is grateful to Genera- we and Janssen2). The most extensively developed are the in- litat de Catalunya for a fellowship. hibitors of the enzyme cytochrome P450 aromatase (CYP19), responsible for the conversion of androgens into estrogens and REFERENCES a target in the treatment of breast cancer. The sequence of reactions catalyzed by CYP19 involves three sequential enzy- 1. Khripach V. A., Zhabinskii V. N., de Groot A. E.: Bras- matic hydroxylations (Scheme 1). The first two take place on sinosteroid: A New Class of Plant Hormones. Academic the C-19 methyl group, whereas the final hydroxylation step Press, California 1999. is still unclear. 2. Brosa C.: Structure-activity Relationship in Brassinoste- Because ~80 % of patients with prostate cancer have roids: Steroidal Plant Hormones (Sakurai A., Yokota T., androgen-dependent diseases, that respond to hormonal abla- Clouse S., ed.) Chap. 9. Springer-Verlag, Tokyo 1999. tion, the inhibition of androgen synthesis is also an important 3. See poster Marc AmorÛs et al. target for the treatment of prostate cancer. The last step in the biosynthesis of androgens involves the two-step conversion of pregnenolone and progesterone, via their corresponding 17α- -hydroxy derivatives, to dehydroepiandrostenedione and androstenedione respectively. Both reactions are catalyzed by the same enzyme; cytochrome P450 17α-hydroxylase/17,20- -lyase (CYP17)3 (Scheme 2). Extensive worldwide efforts have resulted in the identification of structurally diverse series of inhibitors of CYP19 and

O O

aromatase (CYP19) HCOOH 3 NADPH HO O 3O2 androstenedione estrone

Scheme 1. Action of aromatase (CYP19)

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21 O O O CYP19 CYP17 OH H (hydroxylase) (lyase) 17 CH3COOH NADPH NADPH O2 O2 HO

pregnenolone 17α -hydroxy- dehydroepi- pregnenolone androsterone Scheme 2. Action of 17α-hydroxylase-17,20-lyase (CYP17)

HO OAc NOH OTBDMS

6reactions

AcO HO

3reactions 3reactions

H H N O N H H OH

O O 1: 19R 3: 19R 2: 19S 4: 19S

Scheme 3. Synthesis of 10β-aziridinyl steroids of CYP17. Indeed, four CYP19 inhibitors, namely, 4-hydro- tic route used is briefly summarized in Scheme 3. Rigorous xyandrostenedione (formestane), anastrazole, letrozole and establishment of the C-19 configuration in the aziridinyl ste- exemestane are used clinically for the treatment of breast roids was secured by the X-ray crystallographic analysis of cancer (reviewed by Njar and Brodie4). A number of CYP17 compound 3. The compounds were shown to be powerful and inhibitors are currently in development as potential agents for stereoselective inhibitors of human placental microsomal aro- the treatment of prostate cancer5. The present contribution will matase. We also showed that the nitrogen atom of the most focus on our efforts in the rationale design, synthesis and potent aziridine coordinates to the enzymeís heme iron. These evaluation of inhibitors of CYP19 and of CYP17. Specifically, compounds are amongst the most potent inhibitors of this we describe studies of steroidal inhibitors, involving functio- enzyme to date. nalization (N-heterocycle) at either the C-19 (for CYP19 inhi- CYP17 Inhibitors: In our search for potent and selective bitors) or the C-17 (for CYP17 inhibitors) positions in such inhibitors of CYP17, a variety of novel ∆16-17-azolyl steroids, a way as to mimic the natural substrates of the respective that is, pyrazoles, imidazoles, triazoles and tetrazoles have enzymes. The overall strategy is aimed at producing substra- been prepared by a new synthetic route7ñ9. This involved the te-like compounds which are likely to not only interact with nucleophilic vinylic ìaddition-eliminationî substitution reac- the steroid binding site of the enzyme, thus introducing high tion of 3β-acetoxy-17-chloro-16-formylandrosta-5,16-diene specificity, but also to provide a sixth ligand to the enzymeís and azolyl nucleophiles. These series of azolyl steroids are heme iron resulting in tight binding. unlike the heretofore known 17-heteroaryl steroids, as in this Aromatase(CYP19) Inhibitors: The novel (19R)- and (19S)- case, the azole moiety is attached to the steroid nucleus at C-17 -10β-aziridinylestr-4-ene-3,17-diones 1 and 2 and the corres- via nitrogen of the azole. Most of the compounds are potent ponding (19R)- and (19S)-10β-aziridinyl-17β-hydroxyestr-4- inhibitors of both the human and rat CYP17. The most potent -en-3-ones 3 and 4 (Scheme 3) have been prepared from the compound, 3β-hydroxy-17-(1H-imidazole-1-yl)androsta-5,16- 6 19-oximino-19-methyl intermediate . The key reaction was -diene (VN/85-1), with a Ki value of 1.2 nM is 32 times more β the conversion of the 19-oxime into the diastereomeric 10 - potent than ketoconazole (Ki =38nM). The synthetic route -aziridines by lithium aluminium hydride (LAH). The synthe- used to prepare VN/85-1 is briefly summarized in Scheme 4.

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O Cl Cl

CHO i

AcO AcO AcO 5 6

N N N

N N N iii iv CHO

HO AcO AcO

VN/85-1 8 7

Scheme 4. (i) POCl3-DMF, CHCl3, Ar, reflux; (ii) imidazole, K2CO3, DMF, Ar, 80 ∞C; (iii) 10% Pd on activated charcoal, PhCN, reflux; (iv) 10% methanolic KOH, Ar, rt.

Fig. 1. Alignment of common-feature pharmacophore model with training set of CYP17 inhibitors

Spectroscopic studies with a modified form of human CYP17 85-1 suppressed T and DHT to basal levels after two 2 weeks indicate that the inhibition process involves binding of steroi- of daily dosing at 50 mg.kgñ1.dayñ1 (ref.11). Furthermore, re- dal azole nitrogen to the heme iron of the enzyme. In cultures markable antitumor activity was also observed against human of human prostate cancer cell line (LNCaP), VN/85-1 effecti- prostate cancer (LNCaP) xenografted in combined immuno- vely blocked the growth-stimulating effects of testosterone (T) deficient (SCID) mice10,12. Our most potent inhibitors are and dihydrotestosterone (DHT), and was shown to manifest currently in development. anti-androgenic activity10. In Sprague Dawley male rats, VN/ In the absence of detailed structural information on the

s294 Chem. Listy 97, s233 ñ s318 (2003) Conference on Isoprenoids 2003

CYP17 binding site, we have employed a ligand-based com- putational approach, by analyzing a variety of know CYP17 inhibitors, to identify ligand requirements for inhibiting this enzyme. Alignment of common-feature pharmacophore mo- 1 X = O, R1 = R2 = Cl del with training set of CYP17 inhibitors is presented in Fig. 1. 2 X = O, R1 = H, R2 = Cl The study has provided the first insight into hypothetical 3 X = O, R1 = R2 = H binding requirements for steroidal and non-steroidal inhibitors 4 X = NOH, R1 = R2 = H of CYP17 enzyme13. The model may be useful in identification 5 X = NOH, R1 = H, R2 = Cl of new and potent CYP17 inhibitors.

REFERENCES

1. Nelson D. R., Koymans L., Kamataki T., et al.: Pharma- cogenetics 6, 1 (1996). 2. Van Wauwe J. P., Janssen P. A. J.: J. Med. Chem. 32, 2231 (1989). 3. Hall P. F.: J. Steroid Biochem. Mol. Biol. 20, 527 (1981). 4. Njar V. C. O., Brodie A. M. H.: Drugs 58, 233 (1999). 5. Njar V. C. O., Brodie A. M. H.: Curr. Pharm. Design 5, 163 (1999) 6. Njar V. C. O., Safi E., Silverton J. V., Robinson C. H.: J. Chem. Soc., Perkin Trans. 1 1993, 1161. 7. Njar V. C. O., Klus G. T., Brodie A. M. H.: Bioorg. Med. Chem Lett. 6, 2777 (1996). 8. Njar V. C. O., Kato K., Nnane I, P., Grigoryev D. N., Long B.J.,BrodieA.M.H.:J.Med.Chem.41, 902 (1998). 9. Brodie A., Njar V. C. O.: US Patent: 5,994,335 (1999). tene to the appropriate olefin. For example, 5α-cholestan-3- 10. Grigoryev D. N., Long B. J., Nnane I. P., Njar V. C. O., -one was transformed via 3-methylene-5α-cholestane to spi- Lui Y., Brodie A. M. H.: Brit. J. Cancer 81, 622 (1999). ro-cyclobutanone 1. It can be reduced to partially or fully 11. Nnane I. P., Njar V. C. O., Liu Y., Lu Q., Brodie A. M. dehalogenated products 2 and 3, respectively. The unsubstitu- H.: J. Steroid Biochem. Mol. Biol. 71, 145 (1999). ted and α-chloro-cyclobutanones react with hydroxylamine to 12. Handratta D, H., Jelovac D., Long B. J., Kataria R., Nnane form oximes 4 and 5, respectively. Oximes of α,α-dichloro- I.P.,NjarV.C.O.,BrodieA.M.H.:J.Med.Chem.,inpress. cyclobutanones have not been reported and our attempts to 13. Clement O. O., Freeman C. M., Hartmann R. W., Han- prepare oxime of the ketone 1 failed. dratta V. D., Vasaitis T. S., Brodie A. M. H., Njar V. C. The α-unsubstituted oxime 4 rearranged upon treatment O.: J. Med. Chem., in press. with thionyl chloride in benzene to the expected pyrrolidino- nes 6 and 7 in 62 % isolated yield2. However, the course of reaction was different, when cyclobutanone α-monochloro- oximes were subjected to similar reaction conditions. Thus, STEROIDAL CYCLOBUTANONES, for example, reaction of 5 gave two compounds, which were SYNTHESIS AND TRANSFORMATIONS isolated from the reaction mixture and separated by column chromatography. These were: the chloropyrolidinone 8 (11 % KRZYSZTOF B£ASZCZYK, HANNA KOENIG, yield) and the nitrile 9 (30 % yield). This investigation was KATARZYNA MEL, ZDZIS£AW PARYZEK * extended to rearrangements of few other steroidal α-chloro- cyclobutanone oximes. In every reaction studied the Beck- Faculty of Chemistry, Adam Mickiewicz University, Grun- mann fission-displacement occurred and nitrile was found to waldzka 6, 60-780 PoznaÒ, Poland be the more abundant component of the reaction mixture. E-mail: [email protected] The reactions described are the first examples of the Beck- mann rearrangement of alicyclic α-chloroximes resulting in Cyclobutanone oximes rearrange under the Beckmann simultaneous formation of normal and abnormal products. conditions to give γ-lactams1. Analysis of the product formation and distribution permitted In steroids, 5α-spiro[cholestane-3,1í-cyclobutane]-3í-one us to suggest a possible mechanism of the rearrangement. oximes are transformed to spiropyrrolidinones3. Stereospeci- ficity of the process was clearly established2. In the case of Financial support of the work by the State Committee for α α -substituted oximes ( -OH, -OR, -NR2 groups for example), Scientific Research (KBN, project No. 7 T09A 110 21) is when stabilized cations are formed upon C(α) ñ C(sp2)bond gratefully acknowledged. cleavage, abnormal Beckmann rearrangement has been obser- ved3. However, the Beckmann rearrangement of α-chlorocy- REFERENCES clobutanone oximes has not been reported. We prepared steroidal cyclobutanones from ketones in 1. (a) Jeffs P. W., Molina G., Cortese N. A., Hauck P. R., sequence of reactions involving cycloaddition of dichloroke- Wolfram J.: J. Org. Chem. 47, 3876 (1982); (b) Greene

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A. E., Depres J. P., Meana M. C., Crabbe P.: Tetrahedron 2. Bruno M., Piozzi F., Rosselli S.: Nat. Prod. Rep. 19, 357 (2002). Lett. 17, 3755 (1976). 3. Kubo I., Fukuyama Y., Chapya A.: Chem. Lett. 1983, 223. 2. B≥aszczyk K., Paryzek Z.: Liebigs Ann. Chem. 1993, 1105; Nebois P., Greene A. E.: J. Org. Chem. 61,5210 (1996). 3. (a) Smith P. A. S., in: Molecular Rearrangements (de SYNTHESIS OF 7,16-DIHYDROXYDEHYDRO- Mayo P., ed.), Chap. 8 and references cited therein. EPIANDROSTERONE AND DERIVED HAPTENS Interscience Publishers, New York 1963; (b) Hassner A., Nash E. G.: Tetrahedron Lett. 6, 525 (1965); (c) Fr·ter G., VLADIMÕR POUZARa*, IVAN »ERN›a, M¸ller U., G¸nther W.: Tetrahedron Lett. 25,1133(1984). MARIE BI»ÕKOV¡b, RICHARD HAMPLb

aInstitute of Organic Chemistry and Biochemistry, Academy SCUTEPARVIN, A NEW NEOCLERODANE of Sciences of the Czech Republic, Flemingovo n. 2, 166 10 DITERPENOID FROM Scutellaria parvula Prague 6, bInstitute of Endocrinology, N·rodnÌ 8, 116 98 Prague 1, Czech Republic MAURIZIO BRUNOa*, SERGIO ROSSELLIa, E-mail: [email protected] ANTONELLA MAGGIOa, FRANCO PIOZZIa, LEONARDO SCAGLIONIb, The analysis of polyhydroxylated dehydroepiandrostero- ORIETTA SERVETTAZc ne (DHEA) derivatives is important for the improvement of diagnostic methods for the autoimmune diseases and for the aDipartimento di Chimica Organica ìE. Paternòî, Università further extending of our knowledge about the markers of di Palermo, Viale delle Scienze, Parco díOrleans II, 90128, neoplastic processes. Our project is dealing with rare metabo- Palermo, bDipartimento di Scienze Agroalimentari, Univer- lites, isomers of 7,16-dihydroxy-DHEA (1), and correspon- sità di Milano, cDipartimento di Biologia, Università di Mila- ding haptens for radioimmunoassays. no, Italy E-mail: [email protected] O The genus Scutellaria, Lamiaceae (Labiatae) family, oc- 1 curs with some 360 species spread throughout the world .Itis OH rich with neoclerodane diterpenoids, that usually show some heterocyclic functions: epoxides, lactones, hydrofurans HOHO groups. Many of these products have a remarkable antifeedant activity against pest insects. The chemistry of the diterpenoids Synthetic routes for this type of steroids started with from Scutellaria was recently reviewed2. DHEA, which is transformed initially to a corresponding Continuing our research program on the components of isomer of 7-hydroxy-DHEA. Stereoselective introduction of this genus, we investigated Scutellaria parvula Michx., a spe- 16α-hydroxy group was accomplished using two principal cies originating from North America (Florida to Quebec). ways. The first approach used formation of suitably substitu- From its aerial parts we isolated a new neoclerodane ted enolates from 17-ketones, selective epoxidation of 16,17 diterpenoid, scuteparvin (1). Its structure is rather similar to double bond, and subsequent rearrangement into 16α-hydro- those of ajugarin V, isolated by Kubo3 from Ajuga remota, the xy-17-oxo moiety1,2. Second method used bromination of only difference being the occurrence of the trans-cinnamoyl- 17-ketone into 16α-position and then solvolysis in a mixture oxy group instead of an acetoxy on C-6. of N,N-dimethylformamide ñ water into 16α-hydroxy deriva- tive3. Both these procedures were used also for a preparation α O of 16 -hydroxy derivative from 7-oxo-DHEA. O Haptens were prepared by a modification of above methods,the19-(O-carboxymethyl)group beingintroducedmain- ly in the first stages of syntheses. Final CMO derivatives will be coupled with bovine serum albumin and simultaneously H used for a preparation of respective homologous tracers. This after generation of polyclonal antibodies enables completion of kits for radioimmunoassays. H O O Supported by grant No. 203/03/0142 GA CR; a part of research project Z4 055 905 AS CR. OH REFERENCES 1 1. Pouzar V., »ern˝ I., LapËÌk O., Hill M., Hampl R.: REFERENCES Steroids 68, 149 (2003). 2. Reich I. L., Reich H. J., Kneer N., Lardy H.: Steroids 67, 1. Willis J. C.: A Dictionary of the Flowering Plants and 221 (2002). Ferns. Cambridge University Press, Cambridge 1966. 3. Numazawa M., NagaokaM.: J. Org. Chem.47,4024(1982).

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SYNTHESIS AND OXIDATION LAPACHOL, DERIVATIVES AND ANALOGUES: OF TERPENESULFIDES OF BORNANE ADDITIONAL PHARMACOLOGICAL ACTIVITIES STRUCTURE AGAINST TROPICAL DISEASES

SVETLANA A. RUBTSOVA, ANT‘NIO E. G. SANTANAa, PAVEL N. KAZAKOV, ALEXANDER V. KUTCHIN NADJA M. F. LIMAa,CLARIANES.CORREIAa, LUCIANA A. SILVAa, MARÕLIA O. F. GOULARTa, FERNANDO MENDON«Aa, Institute of Chemistry, Komi Science Center, Ural Division of LEONOR L. LEONb the Russian Academy of Sciences, 48 Str. Pervomaiskaya, 167982, Syktyvkar, Russian Federation E-mail: [email protected] aDepartamento de Quimica, CCEN, Universidade Federal de Alagoas, Maceio, Alagoas, 57072-970, bDepartamento de Terpenic sulfides obtained on the base of available natural Imunologia, FIOCRUZ, Rio de Janeiro, 21045-900, Brazil compounds are of significant interest as perspective biologically active compounds. The development of selective ways Leishmaniasis is one of the major infectious diseases of terpenic sulfides oxidation will allow to synthesize chiral affecting the poorest regions of the world. Human infections terpenic sulfoxides, which can be used as complex formers of with Leishmania protozoan parasites, transmitted via the bite different purpose, including chiral ligands, using in enantio- of a sand fly, cause visceral, cutaneous or mucocutaneous selective reactions. We have carried out the synthesis of dibor- leishmaniasis1. The global burden of leishmaniasis has remai- neonyl sulfide and dibornyl sulfide by the following scheme 1. ned stable for some years, causing 2.4 million disability ad- The structure of terpenic sulfides is confirmed by the justed life years lost and 59 000 deaths in 2001 (ref.2). Drug methods of NMR-spectroscopy. treatment today, besides the pentavalent antimonials, is re- stricted to a limited number of clinically useful drugs like pentamidine-isethionate or amphotericin B1. General treatment is unaffordable for many afflicted countries revealing an urgent need for new, safer and cheaper drugs1. Dengue viruses, single stranded RNA viruses of the family Flaviviridae, are the most common cause of arboviral disease in the world. They are found virtually throughout the tropics and cause an estimated 50ñ100 million illnesses annually, including 250 000ñ500 000 cases of dengue haemorrhagic fever ñ a severe manifestation of dengue ñ and 24 000 deaths. More than two fifths of the worldís population (2.5 billion) live in areas potentially at risk for dengue. The incidence, distribution, and clinical severity of dengue has increased dramatically in the last years3. Numerous quinones play vital roles in the biochemistry of living cells and exert relevant biological activities. More es- pecifically, hydroxyquinones had been recognised as potential lead structures against Leishmania4 and natural lapachol isomers, isolated from Calceolaria andina as a new class of Scheme 1 insecticides5. Naphthoquinones were also reported as larvicidal compounds6. One strategy to discover new drugs leads is to investigate Earlier we shown high hemoselectivity of symmetrical and classes of compounds potentially bioactive or old active com- asymmetrical dialkyl-, alkylaryl-, diaryl-, dihalogendiaryl-, pounds for newer uses. Therefore, the aim of the present study dibenzyl sulfides oxidation by chlorine dioxide1. The study of was to further investigate the natural isoprenic hydroxyquino- chlorine dioxide reaction ability in the reactions of synthesized ne lapachol, isomers and derivatives (Fig. 1) toward their terpenic sulfides oxidation is carried out in the present work. leishmanicidal activity against Leishmania brasiliensis and The reaction products are isolated and their structure is establi- L. amazonensis and, against Aedes aegypti. Brine shrimp le- shed by the methods of IR and NMR-spectroscopy. thality assay (Artemia salina) (BSLA) was also employed, to look for a possible correlation on the different screened biolo- This work is realized under financial support of Russian gical activities7. Foundation Basic Research (grant No. 01-03-96404 ´Ural- The bioassays followed the established protocols. Leish- 2001ª). mania amazonensis (MHOM/BR/77/LTB0016 strain) promastigotes were grown at 26 ∞C in Schneiderís Drosophila me- REFERENCES dium supplemented with 10 % (v/v) heat-inactivated fetal calf serum (FCS), with a pH value of 7.2. L. braziliensis (MCAN/ 1. Kutchin A., Rubtsova S., Loginova I.: Russ. Chem. Bull., BR/98/R619) promastigotes were grown in the same medium, Int. Ed. 2001,432. same temperature and pH, but supplemented with 20 % FCS

s297 Chem. Listy 97, s233 ñ s318 (2003) Conference on Isoprenoids 2003

tive against L. braziliensis, with activities even superior to the reference drug, pentamidine isethionate. In relation to Artemia salina, the most active compounds were isolapachol (2) and its derivatives (4 and 5). Concerning lapachol (1), the salt (3) showed a significant decrease on activity in the BSTA. Several salts of lapachol and isolapachol (Na+,Li+ and K+) were assayed against Aedes aegypti. The sodium salt of isolapachol µ ñ1 was sinificantly active, with IC50 = 3.48 g.ml . Despite the absence of the complete set of activities toward Aedes aegypti, this result is very stimulating. In general, in all the performed assays, isolapachol and derivatives have been shown to be the most active. The results so far obtained suggest a continuing search for active compounds within the class of 3-alkyl-2-hydroxynaphthoquinones.

Acknowledgements to CNPq, RHAE/CNPq, CAPES and FA- PEAL. Fig. 1. Studied hydroxynaphthoquinones and derivatives REFERENCES and 2 % human urine8. Parasites were harvested from the medium on day 4, in which the high percentage of infective 1. Davies C. R., Kaye P., Croft S. L. Sundar S.: Brit. Med. forms (metacyclic promastigotes) was found. After being har- J. 326, 377 (2003). vested from the medium, parasites were counted in Neubauerís 2. World Health Organization. Annex 3.: Burden of disease chamber and adjusted to a concentration of 4◊106 promatigo- in DALYs by cause, sex and mortality stratum in WHO tes/ml using the supernatant of both cultures as diluents. The regions, estimates for 2001. In: The world health report. substances were added, in different concentrations and were Geneva: WHO, 192 (2002). dissolved in small amounts of DMSO. For the larvicidal 3. Gibbons R. V., Vaughn D. W.: Brit. Med. J. 321,1563 bioassay, 4th instar larvae of Aedes aegypti were used and the (2002). test followed the recommendations of WHO9. BSLA was 4. Kayser O., Kiderlen A. F., Laatsch H., Croft S. L.: Acta conducted as recommended10. Trop. 77, 307 (2000). The results of the bioassays are presented in Table I. IC50: 5. Khambay B. P. S., Jewess P.: Crop Protect. 19,597 values indicate the effective concentration of a compound in (2000). mg.mlñ1 necessary to achieve 50 % growth inhibition. 6. Ioset J.R., Marston A., Gupta M. P., Hostettmann K.: Our data showed that among the lapachol analogues as- Phytochemistry 53, 613 (2000). sayed against L. amazonensis, all the compounds showed IC50 7. Zani B., Chaves P. P. G., Queiroz R., De Oliveira A. B., lower than 10 µg.mlñ1 and the most effective was the acetyl- Cardoso J. E., Anjos A. M. G., Grandi T. S. M.: Phyto- µ ñ1 isolapachol (IC50/24 h = 1.6±0.0 g.ml ) (Table I). Isolapa- medicine 2, 47 (1995). chol (2) and acetylisolapachol (5) were also significantly ac- 8. Shamsuzzaman S. M., Furuya M., Korenaga M., Imamu- ra K., Hashiguchi Y.: Ann. Trop. Med. Parasitol.93,613 (1999). Table I 9. World Health Organization. Instructions for determining Antileishmanial activity (IC , µg.mlñ1) against L. amazonen- 50 the susceptibility of resistance of mosquito larvae to sis (MHOM/BR/77/LTB0016) and L. braziliensis (MCAN/ insecticides, WHO/VBC 81-807, 1 (1981). Artemia salina µ ñ1 BR/98/R619 and, toxicity against in g.ml , 10. Vanhaecke P., Persoone G., Claus C., Sorgeloos P.: Eco- of the assayed hydroxyquinones and derivatives toxicol. Environ. Saf. 5, 382 (1981).

Compounds L. amazo-L. brazi- Artemia nensis liensis salina NEW PENTACYCLIC TRITERPENE ESTERS FROM Peltastes peltatus (VELL.) WOODSON Lapachol (1) 5.2±0.7 11.9±6.9 12.75 [6] Isolapachol (2) 4.4±2.9 9.3±2.7 6.62 ANT‘NIO E. G. SANTíANA, Lapachol, K+ salt (3) 7.7±4.1 21.4±2.9 79.76 MARIA CLEONICE B. S. NASCIMENTO, Isolapachol, K+ salt (4) 7.8±0.2 15.8±0.0 0.21 MARGARIDA M. S. HUMBERTO Acetylisolapachol (5) 1.6±0.0 3.4±0.5 1.94 Dihydrolapachol (6) 7.3±0.3 54.0±9.0 23.56 Departamento de QuÌmica, Universidade Federal de Alagoas, Dihydrolapachol, ND ND 33.19 57.072-970 MaceiÛ-AL, Brazil K+ salt (7) E-mail: [email protected] Pentamidine 0.28±0.05 11.6±1.6 ND Isethionate Peltastes peltatus (VELL.) WOODSON (Apocynaceae) is a creeper widely spread over in the southern states of Brazil.

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EIMS (M+, m/z 582). Its IR spectrum showed absorption bands at 1708 and 1242 cmñ1, while itsUV spectrum showed absorption maxima at 247 and 308 nm (log ε = 4.39; 4.48, MeOH), related to an aromatic system conjugated with a diene (Kˆnig and Rimpler, 1985). The 1H NMR spectrum of 1 revealed the δ presence of eight tertiary methyl groups ( H 0.83, 0.87, 0.88, 0.91, 0.93, 0.98, 0.99 and 1.14) and one olefinic hydrogen δ β ( H 5.18, bt, J = 3.4 Hz), typical of -amyrin. This was also supported by the observation of a base ion peak at m/z 218 corresponding to the ion resultant from the reverse Diels-Alder fragmentation characteristic of the derivatives of ∆12-oleane- R1 R2 ne/ursene. 13 1H Me The C NMR spectrum of 1 showed only 39 carbon 2 Me H signals (two sets of carbon signals being superimposed), thirty of them corresponding to the triterpenoid moiety and the remainder compatible with a phenylpenta-2,4-dienoyl moiety comparable to that described in an iridoid from Avicennia marina2. The ester bonding at C-3 was further confirmed in an HMBC experiment. The relative stereochemistry of the diene was assigned on the basis of coupling constants measured in 1H-NMR and on results obtained from a NOESY experiment. Hydrolysis of 1 furnished a triterpenoid alcohol identified as β-amyrin by comparison with reported spectral data3. The acid obtained from the hydrolysate was identified as 5-phenyl- -(2E,4E)-penta-2,4-dienoic acid. 3 Peltastine A (2) was isolated and characterized as a binary mixture in a ratio of 1:2 with b-amyrin 5-phenylpenta-2,4-di- enoate (1). Its structure was determined mainly from comparison of the 1H and 13C NMR spectral data and mass spectrum of mixture with those of compound 1. Alkaline hydrolysis of the mixture (1+2) furnished the 5-phenyl-(2E,4E)- -penta-2,4-dienoic acid (4) along with the mixture of α- and 4 β-amyrins. Peltastine B (3) was isolated as a white amorphous solid, Species belonging to the genus Peltastes are rarely considered m.p. 170ñ172 ∞C (EtOH). The 1Hand13C NMR spectra of 3 in the chemical literature and no report concerning the chem- were closely related to those of compound 1, except for the istry is available. The mutagenic activity of the aqueous extract triterpenoid moiety. Its molecular formula C41H58O2 was esta- of P. peltatus has been recently reported1. The aim of the blished by EIMS (M+ m/z = 582) in combination with 1H and present work was to investigate the chemical composition of 13C NMR, indicating that 3 was isomeric with compounds 1 P. peltatus. Plant material was collected in Alagoas state and 2. The presence of an ester linkage in 3 was indicated by δ (Brazil) and voucher specimen (number 3268) has been de- the carbonyl signal at C 167.1 together with IR absorption at posited in the herbarium of the Department of Botany of the 1709 cmñ1. University of BrasÌlia. The 13C NMR spectrum of 3 showed similar carbon shifts The air-dried and powdered stem (2.7 kg) of P. peltatus as lupeol acetate and lupeol cinnamate for the lupene part of was extracted in a Soxhlet apparatus with EtOH (10 L) to yield the molecule. Alkaline hydrolysis of this compound furnished 185 g of crude extract. After suspension in MeOH/H2O (3:2) 5-phenyl-(2E,4E)-penta-2,4-dienoic acid (4) and the lupeol, solution and extraction with n-hexane, chloroform and ethyl identified by comparison with reported data4. acetate (2.0 L of each solvent) we obtained the hexane, chlo- To our knowledge, peltastines A (2) and B (3), and β- roform, ethyl acetate and aqueous fraction. Part of the hexane amyrin juarezate (1) are the only representatives of triterpene fraction (58.0 g) was chromatographed on silica gel column, esters bearing a conjugated phenyldiene moiety. The 5-phe- eluted with n-hexane, gradually increasing the polarity with nyl-(2E,4E)-penta-2,4-dienoic acid is not usual as natural pro- CHCl3 to yield a solid material (9.5 g), which, on TLC, gave duct, but it has been found in the form of a triterpenoid ester a positive Liebermann-Burchard test and a violet color with in Marsdenia pringle5 and in iridoid esters in Avicennia ma- ceric sulphate, suggesting a triterpenic nature. Rechromato- rina2. graphy on a silica gel column yielded compound 1 as a crystalline solid (3.1 g) a 2:1 mixture of 1 and 2 (3.2 g), and 3 as The authors would like to thank Professor JosÈ Elias de Pau- an amorphous material (0.8 g). The structures of these com- la for collecting and identifying the plant material, CNPq, pounds were established by MS, 1D- and 2D-NMR experi- CAPES and FAPEAL for financial support and Centro Nor- ments as well as by chemical degradation. destino de AplicaÁão e Uso de RMN da Universidade Federal The molecular formula of compound 1 was assigned as do Cear· (CENAUREMN, Fortaleza, CE, Brazil) for NMR 1 13 C41H58O2 based on elemental analysis, H and C NMR and spectra.

s299 Chem. Listy 97, s233 ñ s318 (2003) Conference on Isoprenoids 2003

REFERENCES products of agricultural or medical relevance, as well as fragrance compounds. Preliminary research has also demonstra- 1. Horn R. C., Vargas V. M. F.: Mutagenesis 18, 113 (2003). ted its usefulness as a starting material for the synthesis of 2. Kˆnig G., Rimpler H.: Phytochemistry 24, 1245 (1985). steroids. Two main pathways are therefore under investigation 3. MaratoS.B., Kundu A.P.:Phytochemistry37, 1517 (1994). using carvone either as ring Dor as ring B of (homo)steroid 4. Ahmad V. U., Bano S., Mohammad F. V.: Planta Med. skeletons. 6, 521 (1985). A route toward C,D-cis coupled steroid skeletons, in which 5. DomÌnguez X. A., MarroquÌn J., OlguÌn L. M., Morales the use of carvone leads to an enantiomerically pure D-homo- F., Valdez V.: Phytochemistry 13, 2617 (1974). steroid skeleton, has already been developed and has been accepted for publication1. Mukaiyama reactions play an important role in our che- CARVONE AS A STARTING MATERIAL mistry and we have been able to show that a MichaÎl-Mukai- FOR THE TOTAL SYNTHESIS OF STEROIDS yama domino reaction sequence can lead to tricyclic systems which, using appropriately functionalised starting materials, FLORENCE C. E. SARABÈRa, SVETLANA could be further converted into steroid-like compounds. This V. DRACHb, TATYANA V. CHARNIKHOVAb, chemistry is currently under investigation. ALEXANDER V. BARANOVSKIb, BEN J. M. JANSENa,AEDEDEGROOTa REFERENCES aLaboratory of Organic Chemistry, Wageningen University, 1. Drach S., Charnikhova T., Sarabèr F., Jansen B., de Groot Dreijenplein 8, 6703 HB Wageningen, The Netherlands, bIn- A.: Tetrahedron (2003), in press. stitute of Bioorganic Chemistry, National Academy of Scien- ces of Belarus, Kuprevich Str. 5/2, 220141 Minsk, Belarus E-mail: [email protected], http://www.ftns.wau.nl/oc/ THE PREPARATION AND MIGRATION OF DOUBLE BONDS IN 22(17→ 28)-ABEO Carvone is a natural product which can be isolated from LUPANE DERIVATIVES caraway seeds (S-(+)-carvone) or from mint (R-(ñ)-carvone). In our lab, these compounds have been applied as a starting JAN SEJBAL, V¡CLAV VANÃK material for the synthesis of several more complex natural Department of Organic Chemistry, Charles University, Alber- tov 6, 128 40 Prague 2, Czech Republic E-mail: [email protected]

Triterpenoids, especially lupane derivatives are subject of intensive biological studies in the last decade because of their anti-cancer and anti-HIV activities. Among biologically active triterpenoids are commonly present lupane derivatives

CH2 OH

AcO

s300 Chem. Listy 97, s233 ñ s318 (2003) Conference on Isoprenoids 2003 bearing more oxygen containing functional groups or degra- REFERENCES ded triterpenois. Anhydrobetulines, 22(17→28)abeo lupane derivatives, were 1. Willis J. C.: Dictionary of the Flowering Plants and studied as possible intermediates for the preparation of lupane Ferns,7th Ed., pp. 10ñ38. Cambridge University Press, derivatives with more degraded skeleton. Migration of double Cambridge 1966. bonds in the solution of hydrogen bromide in acetic acid led 2. Senatore F.: Oli Essenziali. Origine, estrazione ed analisi to the mixture of many dienes with double bonds on rings C, chimica. EMSIñRoma, Roma 2000. Dand E and two unexpected products: Compound with ace- 3. Gonzalez A. G., Luis J. G., Ravelo A. G.: Plantas Iberoa- tylated aromatised ring E and spirocyclic compound. Forma- mericanas Fuentes de Moleculas Bioactivas, Vol. 2, pp. tion of these two compounds is discussed. 125ñ183. Ed. Romero, Tenerife 1990. Structures, configuration and conformation of all com- 4. Laeer U., Glombitza K. W., Neugebauer M.: Planta Med. pounds prepared were studied mainly using correlation NMR 62, 81 (1996). techniques. 5. Kirimer N., Tabanca N., Ozek T., Basher K. H. C., Tumen G.: Chem. Nat. Compd. 35, 61 (1999). 6. Galati E. M., Germano M. P., Rossitto A., Tzakou O., CONSTITUENTS AND BIOLOGICAL ACTIVITY Skaltsa H., Roussis V.: J. Essent. Oil Res. 8, 303 (1996). OF THE ESSENTIAL OILS FROM Sideritis italica 7. Gergis V., Spiliotis V., Argyriadou N., Poulos C.: Fla- (MILLER) GREUTER ET BURDET (LAMIACEAE) vour Fragrance J.6, 93 (1991). 8. Ezer N., Abbasoglu U.: Fitoterapia 67, 474 (1996). FELICE SENATOREa, MAURIZIO BRUNOb, 9. Greuter W.: Med-Checklist, Vol. 3, p. 350. Ed. Conserv. FRANCO PIOZZIb Jard. Botan., Genève 1986. 10. Senatore F., Bruno M.: Flavour Fragrance J. 18, 195 (2003). aDipartimento di Chimica delle Sostanze Naturali, Università degli Studi di Napoli ìFederico IIî ñ Via D. Montesano, 49-I 80131 Napoli, bDipartimento di Chimica Organica, Univer- BIOSYNTHESIS OF 2,3-EPOXYBRASSINO- sità degli Studi di Palermo, Viale delle Scienze, Parco díOr- STEROIDS IN RYE SEEDLINGS leans II-I 90128 Palermo, Italy E-mail: [email protected] ANDREY ANTONCHICKa,b, BERND SCHNEIDERa, OLGA V. KONSTANTINOVAb,VLADIMIR The genus Sideritis (Lamiaceae) counts at least 150 spe- N. ZHABINSKIIb,VLADIMIRA.KHRIPACHb cies1 occurring in temperate and tropical regions of the north- ern hemisphere; the countries around the Mediterranean Sea aMax-Planck-Institute for Chemical Ecology, Beutenberg-Cam- are particularly rich. Several species are widely used in the pus, Winzerlaer Str. 10, 07745 Jena, Germany, bInstitute of folk medicine of many countries for their anti-inflammatory, Bioorganic Chemistry, National Academy of Sciences of Be- antispasmodic, carminative, sedative, antitussive, stomachic, larus, Kuprevich Str. 5/2, 220141 Minsk, Belarus anticonvulsant and antifeedant activities. The essential oils are E-mail: [email protected] used for many therapeutic purposes, for instance as pulmonary disinfectants, diuretics, stomachics, neurorelaxing2. The aerial 2,3-Epoxybrassinosteroids such as secasterone and 24- parts of Sideritis taxa have been largely investigated and -epi-secasterone previously have been reported in seeds of diterpenoids were the main metabolites isolated from them3. Secale cereale1 and Lychnis viscaria2, respectively. We ana- On the other hand, many papers on the composition of the lyzed seedlings of two rye varieties (Secale cereale, cv. ìSo- essential oils have been published4ñ6 and antimicrobial and romî and cv. ìPetkaî) for the occurrence of brassinosteroids analgesic activities have been pointed out7,8. The species Si- with special emphasis on 2,3-epoxybrassinosteroids and puta- deritis italica occurs in Sicily and in Southern Italy9 and tive biosynthetic precursors. Secasterone and 2,3-diepi-seca- previous papers reported the occurrence of several ent-kaurane sterone, which is reported in this study for the first time as diterpenoids in the aerial parts. The present is the first paper a naturally occurring compound, were found in rye seedlings. on the chemical composition of the essential oil from leaves In addition, secasterol, the first 2,3-olefinic brassinosteroid and flower heads of this plant. Oils were extracted and exami- has been identified from the same plant source3. Secasterone ned following the usual procedure10. The yields of the essential and 2,3-diepi-secasterone were synthesized as deuterated ana- oils from flower heads and leaves of S. italica were 0.16 % lytical standards4, and deuterated teasterone, typhasterol, and and 0.14 %, respectively. 49 compounds in the flower heads secasterol were synthesized as putative biosynthetic precur- and 31 compounds in the leaves, amounting to 93.7 % and sors. Feeding experiments using deuterated intermediates we- 92.4 % of the essential oil, were identified. In the oil from re carried out in rye seedlings (Secale cereale) in order to flower heads kaur-15-ene and β-cubebene were the main investigate biosynthetic incorporation into 2,3-epoxybrassi- components representing one third of the oil. p-Methoxyace- nosteroids. Biosynthetic products were identified by GC-MS- tophenone was the major component of the oil from leaves. -SIM analysis. The results of the studies on the allelopathic activity of the oils Deuterated teasterone, and typhasterol, upon administra- put in evidence an inhibiting action, in vitro, as on the percen- tion to rye seedlings, were converted to secasterol, secastero- tage of germination of the seeds as on the development imme- ne and 2,3-diepi-secasterone. Additional feeding experiments diately following to the germination of the seed (to the light) showed a high conversion rate of secasterol to both 2,3-epo- of Raphanus sativus. xybrassinosteroids. From these findings, the biosynthetic se-

s301 Chem. Listy 97, s233 ñ s318 (2003) Conference on Isoprenoids 2003

OH OH

OH OH OH

O HO OH H H O O Teasterone Secasterone H O OH OH Secasterol

OH OH

HO H H O O Typhasterol 2,3-diepi-Secasterone quence teasterone/typhasterol to secasterol to secasterone and 2,3-diepi-secasterone has been demonstrated to be operative in Secale cereale3.

REFERENCES

1. Schmidt J., Spengler B., Yokota T., Nakayama M., Taka- tsuto S.,VoigtB.,Adam G.:Phytochemistry38,1095(1995). 2. Friebe A., Volz A., Schmidt J., Voigt B., Adam G., Schnabl H.: Phytochemistry 52, 1607 (1999). H O III 3. Antonchick A., Schneider B., Konstantinova O. V., Zhabin- skii V. N., Khripach V. A.: Phytochemistry (2003), in press. HO 4. Khripach V. A., Zhabinskii V. N., Konstantinova O. V., Antonchick A. P., Schneider B.: Steroids 67, 587 (2002). HO H O IV SYNTHESIS OF A NEW BRASSINOLIDE ANALOG AND ITS BIOASSAY

MIROSLAV äÕäAa, SONIA ESPELTAb, CARME BROSAb, LADISLAV KOHOUTa O aInstitute of Organic Chemistry and Biochemistry, Academy of Scineces of the Czech Republic, Flemingovo n. 2, 166 10 O Prague 6, Czech Republic, bInstitut QuÌmic de Sarria, Univer- H O V sitat Ramon Llull, Via Augusta 390, 08024 Barcelona, Spain E-mail: [email protected], [email protected], brosa@ iqs.url.es O Brassinolide I is known as the most potent compound for plant growth promoting activity1. It was reported2 that brassinolide analog II showed even better activity under the field conditions, although it was completely inactive under the rice O lamina inclination test. In our structure-activity relationships 3 studies of brassinosteroids we prepared brassinolide analog O H VI, which shows better activity already in the rice lamina O inclination test in comparison with brassinosteroid II. VI

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OH lactone 1 was prepared from 5α-pregn-1-ene-3,20-dione by treatment with lead tetraacetate in aqueous acetic acid and the resulting crude secoaldehyde-acid was reduced with sodium OH borohydride1. The starting material for the synthesis of the HO lactone 2 was progesterone: One-step oxidation of progesterone by means of peroxydisulfuric acid in glacial acetic acid was employed; the reagent was prepared from potassium HO O persulfate and concentrated sulfuric acid2. The lactones 1 and H O 3 O 2 were reduced with DIBAL in toluene at ñ78 ∞C (ref. ). I Biological activity of some products will be reported.

This work was supported by grants No. S5011007 and Z4 055 O 905. O H O REFERENCES O II 1. Pappo R., Jung C. J.: Tetrahedron Lett. 1962, 365. 2. Pettit G. R., Kasturi T. R.: J. Org. Chem. 26, 4557 (1961). Synthesis of our brassinosteroids started from (22E)-5α- 3. Suginome H., Yamada S.: Bull. Chem. Soc. Jpn. 60,2453 -ergosta-2,22-dien-6-one (III), which was carefully hydroxy- (1987). lated by osmium tetroxide for 2 hours and the acquired (22E)- -2α,3α-dihydroxy-5α-ergosta-2,22-dien-6-one (IV) was transformed to its acetonide (V). Subsequent epoxidation with ENANTIOSELECTIVE SYNTHESIS 3-chloro-perbenzoic acid gave compound VI. OF (2R,6R)-2,6,10-TRIMETHYLUNDECANE-1-OL ñ CHIRAL PRECURSOR FOR THE PREPARATION This work was supported by grants Nos. 203/01/0083, IBS OF NATURAL VITAMIN E 4055304, A 4055204 and Z 4055905. ANNA YU. SPIVAK, GULíNARA REFERENCES A. EMELíYANOVA, MARINA I. MALLYABAEVA, VICTOR N. ODINOKOV 1. Sasse J.: Physiological Actions of Brassinosteroids, Chap. 7., in Brassinosteroids: Steroidal Plant Hormones (Sa- Institute of Petrochemistry and Catalysis, Bashkortostan Re- kurai A., Yokota T., Clouse S. D., ed.), pp. 137ñ161. public Academy of Sciences and the Ufa Scientific Centre of Springer-Verlag, Tokyo 1999. the Russian Academy of Sciences, 141 prosp. Oktyabrya, 2. Kakiuchi T., Kamuro Y., Takatsuto S., Kobayashi K.: 450075 Ufa, Russian Federation Agric. Biol. Chem. 52, 2381 (1988). E-mail: [email protected] 3. Strnad M., Kohout L.: Plant Growth Regul. 40, 39 (2003). The condensation of (S)-chromanyl ethanal 2 (synthesized previously by us1) and (R,R)-phosphorane 3 is the key step of DIBAL REDUCTION OF SOME one of the approaches to the synthesis of the natural vitamin STEROIDAL LACTONES Eñ(2R,4íR,8íR)-α-tocopherol (1). (R,R)-phosphorane 3 is obtained from (2R,6R)-2,6,10-trimethylundecan-1-ol (11). BARBORA SLAVÕKOV¡, ALEXANDER KASAL We developed the effective enantiospecific synthesis of alcohol 11 based on the dehydration of (3RS,7R,11R)-isophy- Institute of Organic Chemistry and Biochemistry, Fleminovo n. 2, 166 10 Prague 6 E-mail: [email protected] HO In the course of our work on allopregnanolone analogues, we prepared derivatives of 2- and 4-oxa-5α-pregnane-3,20- O -diones 1 and 2 and studied their reduction with DIBAL. The 1

O O

O O Ph3P AcO O + 3 O O O O O H H 1 2 2

s303 Chem. Listy 97, s233 ñ s318 (2003) Conference on Isoprenoids 2003

HC CH2 R 7 6 3 H3C 5 2 4 8 1 N N 9

20 Mg 10 2st a 19 11 7 3 H N N 11

18 16 12 OH CH CH H3C 17 15 3 3 14 5 H 13 H H C OC ()CH 2 3 3 2 O COCH O H 3 O 4: R = Me, CHO

b +

6 7

+ COOH O 8 9 c

10 11

a: TsOH-SiO2/PhH, r.t., 1 h; b:O3/Ba(OH)2/Me2CO, KMnO4/H2SO4/H2O; c: Pb(OAc)4/Cu(OAc)2.H2O/Py, PhH; d: O3/CH2Cl2-MeOH (5:1), NaBH4/MeOH tol (5) obtained via the ozonolysis of chlorophyll (4) followed STABILITY OF 7-OH EPIMERS by the interaction of R,R-phytone formed with vinylmagne- OF3-β HYDROXYANDROST-5-EN-17-ONE (DHEA) 2 sium bromide . It was found, that the reaction of TsOH-SiO2 with isophytol 5 under the mild conditions resulted in the EVA äçASTN¡, VLADIMÕR POUZAR, mixture of phytadiens 6 (a mixture of E/Z-isomers 1:3) and 7 HANA CHODOUNSK¡* in a ratio 6:1 in a quantitative yield. Ozonolysis of the diene mixture 6, 7 and the further oxidation of the ozonolysis Institute of Organic Chemistry and Biochemistry, Academy of products with KMnO4 led to the easily separated mixture Sciences of the Czech Republic, Flemingovo n. 2, 166 10 of (4R,8R)-4,8,12-trimethyltridecane acid 8 and phytone (9). Prague 6, Czech Republic The oxidative decarboxylation of the acid 8 yielded (3R,7R)- E-mail: [email protected] -3,7,11-dodec-1-ene (10), which ozonolysis followed by the β reaction with NaBH4 gave the target alcohol 11. 7-Hydroxylation of 3 -hydroxyandrost-5-ene steroids is a reaction occuring in many mammalian tissues1. Hydroxy- REFERENCES lation of 3β-hydroxyandrost-5-en-17-one (DHEA) with rat liver homogenate at 7α-position and subsequent conversion 1. Odinokov V. N., Spivak A. Yu., Emelyanova G. A., to othre 7-oxygenated steroids in the sequence DHEA → Gamalevich G. D., Serebryakov E. P.: Izv. Akad. Nauk, Ser. Khim. 2001, 2026. 2. Odinokov V. N., Mallyabaeva M. I., Spivak A. Yu., Emelyanova G. A., Dzhemilev U. M.: Dokl. Akad. Nauk 380, 201 (2001).

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7α-hydroxy-DHEA → 7-oxo-DHEA → 7β-hydroxy-DHEA OH was described2. These 7α-OH and 7β-OH isomers have beneficial biolo- HO gical effects3 (e.g. on immune system) but their activity is different. Therefore, it seems to be useful to prove the stability HO O of both isomers under different conditions. O We have found that both 7α-OH and 7β-OH isomers are 4 unstable under acid conditions, they isomerize to each other. 2 OR2 However, in alkaline solution were both isomers stable. O Supported by research project Z4 055 905 and grant No. IAA4055305 of the Grant Agency of Academy of Sciences of R1 1 O O the Czech Republic. O 5–9 REFERENCES

1. Hampl R., Morfin R., St·rka L.: Endocr. Reg.31, 211 (1997). 2. Lardy H., Marwah A., Marwah P.: Lipids 37, 1187 (2002). OR 3. Morfin R., Courchay G.: J. Steroid Biochem. Mol. Biol. 50, 91 (1994). HO

SIMPLE BRASSINOLIDE ANALOGUES HO O EXHIBITING TYPICAL BRASSINOLIDE ACTIVITY O 10, 11 a b MIROSLAV STRNAD , LADISLAV KOHOUT 5R1 = H, R2 = H 9 R1 = CH , R2 = 3-MeBut 1 2 3 a 6R= H, R = 2-MeBut 10 R = 2-MeBut Laboratory of Growth Regulators, Faculty of Sciences of the 7R1 = H, R2 = 3-MeBut 11 R = 3-MeBut Palack˝ University & Institute of Experimental Botany, Aca- 1 2 8R= CH3, R = H demy of Sciences of the Czech Republic, älechtitel˘ 11, 783 71 Olomouc, bDepartment of Steroid Chemistry, Institute of Or- ganic Chemistry and Biochemistry, Academy of Sciences of OR OR the Czech Republic, Flemingovo n. 2, 166 10 Prague 6, Czech Republic HO HO E-mail: [email protected] HO O HO O A new synthetic brassinolide analogue, 2α,3α-dihydroxy- O O -17β-(3-methylbutyryloxy)-7-oxa-7a-homo-5α-androstan-6- -one (11), has been shown to exhibit typical brassinolide 12 R = 2-MeBut 14 R = 2-MeBut activity ñ splitting of the bean second internode. It was prepa- 13 R = 3-MeBut 15 R = 3-MeBut red from the known lactone 2α,3α,17β-trihydroxy-7-oxa-7a- -homo-5α-androstan-6-one (4) which was transformed to an OR α isopropylidenedioxy derivative. After protection of 2 - and HO 3α-hydroxy group it afforded the 2α,3α-isopropylidenedioxy-17β-(3-methyl-butyryloxy)-7-oxa-7a-homo-5β-androstan- HO -6-one (7) on treating with 3-methylbutyryl chloride in pyri- O dine. The analogue with a 2-methylbutyric moiety (10, 2α,3α- β α -dihydroxy-17 -(2-methyl-butyryloxy)-7-oxa-7a-homo-5 - 17 R = 2-MeBut androstan-6-one) in position 17β stimulated only twisting of 18 R = 3-MeBut the bean second internode. However, in the second bean internode bioassay 100 times more 10 or 11 compared to 24-epibrassinolide is required to 2-MeBut = 2-methyl-butyryl 3-MeBut = 3-methyl-butyryl

OH obtain the same effects. Analogues with β-oriented hydroxyl groups at C-2 and C-3 (14, 15), a 6-ketone (17, 18) or 6-oxa- HO -7-oxo-lactone system (12, 13), in ring B lack the typical brassinolide activity. In addition, the active brassinosteroids HO applied to the second internode stimulated a similar, but 30 % O lower elongation of the first internode. From data presented 3 here we conclude that the presence of two hydroxy groups in

s305 Chem. Listy 97, s233 ñ s318 (2003) Conference on Isoprenoids 2003 the positions 22 and 23 of the brassinolide side chain, which skin diseases, toothache, headache, fever, dysentery, high are considered as a key structural requirement, is not absolu- blood pressure, stomachache, malaria, edema, pneumonia tely necessary for a compound to exhibit typical brassinoste- and liver problems1,2. Results of tests for antifungal and anti- roid activity. Nevertheless, these compounds have generally malarial activities have been reported recently3ñ5. Following 2ñ10 times lower activity than that having 22,23-vicinal diol the observation that African armyworms did not attack A. re- in the side chain. mota leaves, clerodane diterpenes (ajugarin I, II, and III) were isolated as moderately strong antifeedants against Spo- doptera exempta (AJG-I and II min conc 100 ppm) and S. lit- IMMUNOANALYTICAL SYSTEM toralis (AJG-I and II min conc 300 ppm)6. Ajugarin IV was FOR DETERMINATION OF BRASSINOSTEROIDS later isolated in trace amount, exhibiting insecticidal acti- IN PLANT TISSUES vity against Bombyx mori (at 500 ppm, LD95) and growth inhibitory activity against pink bollworm Pectinophora gos- a b b 7 J. SWACZYNOV¡ ,M.äÕäA,L.KOHOUT, sypiella (at 500 ppm, ED50) . Ajugarin V, an additional new M. STRNADa diterpene isolated in trace amount, exhibited neither antifeedant nor insecticidal activity8. The isolation of clerodin (no aLaboratory of Growth Regulators, Institute of Experimental data) as an antifeedant was also reported9. The whole plant Botany, Academy of Sciences of the Czech Republic and A. bracteosa (syn. A. remota) afforded recently bracteonin A, Palack˝ University, älechtitel˘ 11, 783 71 Olomouc, bInstitute 14,15-dihydroajugapitin, and 14-hydro-15-hydroxyajugapi- of Organic Chemistry and Biochemistry, Academy of Sciences tin10. of the Czech Republic, Flemingovo n. 2, 166 10 Prague 6, To test the application of HPLC for the effective and rapid Czech Republic determination and quantification of neo-clerodane diterpenes, E-mail: [email protected] an extract from aerial parts of A. remota was prepared and examined (C18 column; UV detection; waterñmethanol gra- Brassinosteroids (BRs) are widely distributed natural pro- dient elution). The method allowed the quantification of aju- ducts that promote growth and passes all properties necessary garins I, II, IV, V in the extract of A. remota leaves, and suggest for classification as a plant hormone. BRs are group of poly- a higher amount of AJG I than reported previously. Peak area hydroxy steroids causing cell elongation, cell expansion, re- ratios are close to isolated weights of other AJG, and thus, the tard leaf abcission, enhance resistance to stress and promote analytical method can be used to quantify AJG-like neo-cle- xylem differentiation. BRs have been detected and isolated rodane diterpenes. from seeds, fruits, leaves, galls and pollen. Furthermore, it was also found that other prospective We have developed polyclonal and monoclonal antibodies neo-clerodane compounds were present. The suitability of against one of brassinosteroids, 24-epicastasterone. Antise- reversed-phase HPLC for the semi-preparative fractionation rum against this substance was produced by immunizing rab- of extracts from A. remota was then explored. The presence bits and mice with 24-epicastasterone, carboxymethyloxime and reversed-phase chromatographic behavior of dihydrocle- conjugated with bovine serum albumin (BSA). The conjugates rodin, ajugapitin, dihydroajugapitin, and the hemiacetalic 14- were prepared by mixed anhydrides method. Polyclonal anti- -hydro-15-hydroxy-ajugapitin and clerodin in A. remota was bodies were purified from rabbit serum by ammonium sulfate also established. The structures of the known AJG I, II, IV, V precipitation or by affinity purification on protein A columns. (rt 21, 28, 40 and 42 min in HPLC semiprep conditions) and The obtained antibodies were tested in enzyme-linked immu- the newly isolated compounds were established from NMR nosorbent assay (ELISA) using 24-epicastasterone-carboxy- data. methyloxime-peroxidase conjugate. The cross-reactivity of Dihydroclerodin (reported as a natural compound in A. par- the antibodies was defined on the bases of competitive studies viflora11), a newly isolated compound from A. remota, was with other brassinosteroids. eluted at 31 min rt. The presence of a hexahydrofurofuran We suppose to use the selected broad-specific antibodies moiety in the molecule was derived from the chemical shifts in immunoaffinity chromatography (IAC). IAC-HPLC-MS or in 1H NMR [δ 4.07 (dd, J = 11.5, 5.5 Hz), 2.83 (m), 3.83 (2H, IAC-HPLC-ELISA system will be used for analysis of endo- m), 5.61 ppm (d, J = 5.0 Hz); assigned to H1-11, H1-13, H2-15 13 δ genous brassinosteroids in plant tissues. and H1-16], as well as in C NMR [ 85.2, 33.4, 42.1, 32.7, 68.3 and 107.7]. Clerodin eluted at rt 36 min, and ajugapitin12 also isolated for the first time from A. remota at 37 min. NEO-CLERODANE DITERPENES Dihydroajugapitin (reported previously in A. chamaepitys13, 14 15 10 FROM Ajuga remota B.ENTH (LABIATAE) A. pseudoiva , A. iva , and recently in A. bracteosa ) was eluted at 34 min rt. JOSEP COLL* , YUDELSY TANDRON In fractions eluting at 23ñ24 min the epimeric mixture of 15-hydroxydihydroclerodin (scutecyprol A) was also isola- Instituto de Investigaciones QuÌmicas y Ambientales de Barce- ted. The corresponding mixture of 15-hydroxydihydroaju- lona ìJosep Pascual Vilaî, Consejo Superior de Investigacio- gapitin10,16was also present but in trace amounts at 24ñ25 min. nes CientÌficas, Jordi Girona 18-26, 08034 Barcelona, Spain 15-Hydroxydihydroclerodanes have been reported mostly from E-mail: [email protected] the genus Scutellaria17.

Ajuga remota is a shrub growing widely in East Africa, Financial support by MCYT grant (AGL2001/2285) and pre- and has been used in traditional medicine for the treatment of doctoral fellowship (to YT) is gratefully acknowledged.

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REFERENCES pria, S. grossa, and S. rubicunda) show the highest antifeedant index against Spodoptera littoralis2. From Ajuga pseudoiva 1. Kokwaro J. O.: Medicinal Plants of East Africa. East leaves, 14,15-dihydroajugapitin displayed the highest activity Africa Lit Bureau/Kenya Lit. Bureau, Nairobi, Kenya. (AI > 90 even at 1 ppm dose) amongst several highly active 106/118 (1976/1993 (2nd ed)); cf2ñ4. compounds isolated3. 2. Odek-Ogunde M., Rajab M. S., Migwi G. J., Ndegwa J. The genus Teucrium (family Labiatae) is one of the richest M.: Planta Med. 59, 573 (1993). sources of clerodane diterpenes, and the new natural products 3. Kariba R. M.: Fitoterapia 72, 177 (2001). are conveniently reviewed periodically4. Most of the reported 4. Kuria K. A. M., De Coster S., Muriuki G., Masengo W., structures display a furan ring system in the side-chain, with Kibwage I., Hoogmartens J., Laekeman G. M.: J. Ethno- an oxygen bearing C-12. The isolates from T. fruticans5,6 pharmacol. 74 141 (2001). fruticolone, isofruticolone and 8β-hydroxyfruticolone are ex- 5. Kuria K. A. M., Chepkwony H., Govaerts C., Roets E., ceptional for the absence of susbstitution at C-11 and C-12. Busson R., De Witte P., Zupko I., Hoornaert G., Quirynen Only a few other exceptions amongst more than 200 com- L., Maes L., Janssens L., Hoogmartens J., Laekeman G.: pounds are known4. J. Nat. Prod. 65, 789 (2002). Since T. fruticans is a widely distributed species and 6. Kubo I., Lee Y.-W., Balogh-Nair V., Nakanishi K., Cha- readily available, it was chosen to evaluate the HPLC behavior pya A.: J. Chem. Soc., Chem. Commun. 1976, 949. of furo-clerodanes. The diterpene fraction on HPLC analysis 7. Kubo I., Klocke J. A., Miura I., Fukuyama Y.: J. Chem. appeared more complex thananticipated. Thus, the isolation, Soc., Chem. Commun. 1982, 618. structural elucidation and antifeedant activity of previously 8. Kubo I., Fukuyama Y., Chapya A.: Chem. Lett. 1983, unreported neo-clerodanes, with different substitution on the 223. decalin portion and in the side chain is here reported. 9. Kubo I., Kido M., Fukuyama Y.: J. Chem. Soc., Chem. In previous studies5ñ7 the structures of neo-clerodane di- Commun. 1980,897. terpenoids isolated from Teucrium fruticans have been repor- 10. Verma V. H., Mahmood U., Singh B.: Nat. Prod. Lett. 16, ted. A DCM extract of the aerial parts of this plant has now 255 (2002). allowed the isolation of a new family of clerodanes present in 11. Beauchamp P. S., Bottini A. T., Caselles M. C., Dev V., the plant. Hope H., Larter M., Pant A. K., Raffel R. J., Sharma V. The wavelength of furo-diterpenes chromophore for UV K., Wyatt D.: Phytochemistry 43, 827 (1996). detection is close to 210 nm, and since low sensitivity results, 12. Coll J.: Phytochem. Anal. 13, 372 (2002). the analysis may be seriously interfered by other compounds 13. Hern·ndez A., Pascual C., Sanz J., RodrÌguez B.: Phyto- with high UV absorption. Both the specificity of the method chemistry 21, 2909 (1982). and detection sensitivity can be improved by adopting evapo- 14. Camps F., Coll J., Dargallo, O.: Phytochemistry 23,387 rative light scattering detector (ELSD) for compounds with (1984). low (or not at all) UV absorption. 15. BondÌ M. L., Al-Hillo M. R. Y., Lamara K., Ladjel S., HPLC coupled on-line to an ELSDprovided data about Bruno M., Piozzi F., Simmonds M. S. J.: Biochem. Syst. the order of elution of the various furo-clerodanes present in Ecol. 28, 1023 (2000). T. fruticans extracts and a preliminary quantification. Semi- 16. Camps F., Coll J., Dargallo O.: Phytochemistry 23,2577 -prep-HPLC proved to be a convenient purification procedure, (1984). occasionally being followed by TLC. 17. Bruno M., Piozzi F., Rosselli S.: Nat. Prod. Rep. 19,357 In addition to three of the reported neo-clerodanes found (2002). in this plant (fruticolone, isofruticolone, 8β-hydroxyfruticolone), 6-acetyl-teujaponin B (previously reported from Teu- crium scordium and Teucrium grisebachii)8,9 was also isolated from T. fruticans. The structures of new neo-clerodanes isola- NEO-CLERODANE DITERPENES ted in the present work were: 7β-hydroxyfruticolone, 11-hy- FROM Teucrium fruticans L. (LABIATAE) droxyfruticolone and 10-hydroxy-6-acetyl-teujaponin B. Unam- biguous structural elucidation was based on extensive spectral JOSEP COLL* , YUDELSY TANDRON (one and two-dimensional NMR experiments) studies. The new compounds were assayed for their antifeedant activity Instituto de Investigaciones QuÌmicas y Ambientales de Bar- against Spodoptera littoralis. celona ìJosep Pascual Vilaî, Consejo Superior de Investiga- ciones CientÌficas, Jordi Girona 18-26, 08034 Barcelona, Financial support by MCYT grant (AGL2001/2285) and pre- Spain doctoral fellowship (to YT) is gratefully acknowledged. E-mail: [email protected] REFERENCES The insect antifeedant property of clerodane diterpenes is the most extensively studied bioactivity of these compounds1. 1. Klein Gebbinck E. A., Jansen B. J. M., de Groot A.: Various genera of the plant family Labiatae, namely Scutella- Phytochemistry 61, 737 (2002). ria and Ajuga are reported to produce some of the most potent 2. Bruno M., Piozzi F., Rosselli S.: Nat. Prod. Rep. 19,357 clerodane antifeedants. In Scutellaria, jodrellin B (occurring (2002). in S. albida, S. galericulata, S. grossa, S. polyodon, and 3. Ben Jannet H., Harzallah-Skhiri F., Mighri Z., Simmonds S. woronowii) and scutecyprol B (found in S. columnae, S. cy- M. S. J., Blaney W. M.: Fitoterapia 71, 105 (2000).

s307 Chem. Listy 97, s233 ñ s318 (2003) Conference on Isoprenoids 2003

4. Piozzi F., Bruno M., Rosselli S.: Heterocycles 48,2185 O (1998) and references cited therein. OH 5. Savona G., Passannanti S., Paternostro M. P., Piozzi F., OH HO O O OH Hanson J. R., Hitchcock P. B., Siverns M.: J. Chem. Soc., HO OH Perkin Trans. 1 1978,356. HO 6. Savona G., Passannanti S., Paternostro M. P., Piozzi F., Hanson J. R., Siverns M.: Phytochemistry 17, 320(1978). O 13 HO 7. Bruno M., Alc·zar R., de la Torre M. C., Piozzi F., [U- C]Glucose O Cnicin RodrÌguez B., Savona G., Perales A., Arnold N. A.: Phytochemistry 31, 3531 (1992). 8. Jakupovic J., Baruah R. N., Bohlmann F., Quack W.: = labelled C2-blok Planta Med. 51, 341 (1985). (originally from [U-13C6]Glucose) 9. Tonn C., Caltabiano J. A., Gianello J. C., Giordano O. S.: = labelled C-atom Rev. Latinoamer. Quim. 21, 52 (1990). Fig. 2. Incorporation of [U-13C]glucose and labelling pattern of cnicin

BIOSYNTHESIS OF THE SESQUITERPENE LACTONE ESTER CNICIN IN Cnicus benedictus L. O O OH * O OH DIRK UMLAUF , HANS BECKER, OH KLAUS PETER ADAM * NH2 Universit‰t des Saarlandes, Pharmakognosie und Analytische O Cnicin Phytochemie, F.R. 8.7, Geb‰ude 32, 66041 Saarbr¸cken [U-13C]Isoleucine HO O E-mail: [email protected], [email protected] = labelled C-block Isoprenoids are the largest group of secondary metabolites. They share the same five carbon building block isoprene 13 which is derived from isopentenyl diphosphate (IPP). This Fig. 3. Incorporation of [U- C]isoleucine and labelling pattern of common precursor can be biosynthesized either via the meva- cnicin lonic acid pathway (MVA) or via the methylerythritol phos- phate pathway (MEP)1,2. Both MEP and MVA pathways are involved in the formation of isoprenoids of higher and lower The [1-13C]glucose experiment demonstrated that the se- plants. squiterpene unit is biosynthesized exclusively via the MVA In the course of our studies on the origin of the isoprene pathway (Fig. 1). building blocks of various plant isoprenoids3,4, we investigated The analysis of the labelling patterns after [U-13C]glucose the sesquiterpene lactone cnicin in axenic cultures of the incorporation indicated the cyclization of farnesyl diphospha- Astereaceae Cnicus benedictus. Cnicin is an ester of a germa- te to germacrene A as a hypothetical intermediate of the crene type sesquiterpene lactone unit with an isoprene like sesquiterpene moiety (Fig. 2). hydroxyethacrylic acid side chain. The [U-13C]isoleucine experiment showed that, despite its The biosynthetic origin of these two moieties has been isoprene like skeleton, the hydroxyethacrylic acid side chain elucidated by incorporation of [1-13C]glucose, [U-13C]glucose is derived from the amino acid isoleucine (Fig. 3). However, and [U-13C]isoleucine into cnicin and subsequent quantitative qualitative and quantitative labelling patterns of the side chain 13C-NMR spectroscopic analysis of the labelled compounds. after [1-13C]glucose and [U-13C]glucose incorporations suggest a formation of isoleucine via a non typical biosynthetic pathway5. O OH REFERENCES OH HO O O OH HO OH HO 1. Qureshi N., Porter J. W.: Biosynthesis of Isoprenoid Compounds (Porter J. W., Spurgeon S. L., ed.), Vol. 1, O pp. 47ñ94. Wiley, New York 1981. 13 HO 2. Rhomer M., Knani K., Simonin P., Sutter B., Sahm H.: [1- C]Glucose O Cnicin Biochem. J. 295, 517 (1993). 3. Adam K. P., Thiel R. Zapp J., Becker H.: Arch. Biochem. < < Biophys. 354, 181 (1998). = labelled C-atom 4. Adam K. P., Zapp J.: Phytochemistry 48, 953 (1998). 5. Dunstan R. H., Whatley F. R., Greenaway F. R. S., Fig. 1. Incorporation of [1-13C]glucose and resulting labelling pattern Greenaway W.: Proc. R. Soc. London, Ser. B 231,349 of cnicin (1987).

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2-(1-NAPHTYL)-2-PHENYLACETIC ACID structure of (ñ)-menthyl ester of 1-NPA was obtained showing IN DETERMINATION OF ABSOLUTE phenyl group facing the alcohol moiety rather than the naph- CONFIGURATION OF CHIRAL SECONDARY thyl unlike the expectations. This may explain why smaller ALCOHOLS BY1H NMR values of ∆δ than presumed were observed, although they exceed those of MTPA. JAN V¡VRAa, LUDVÕK STREINZb*, On the other hand, the anisotropic effect in distant atoms MILOä BUDÃäÕNSK›b, BOHUMÕR KOUTEKb was also observed, as citronellol and similar terpenoids were esterified with 1-NPA. The more detailed conformation ana- aDepartment of Organic Chemistry, Charles University, lysis will be given. 120 00 Prague 2, bInstitute of Organic Chemistry and Bioche- mistry, Academy of Sciences of the Czech Republic, Flemin- Supported by Research Project Z 4055 905 and the Grant govo n. 2, 166 10 Prague 6, Czech Republic Agency of the Czech Republic, grant No. 203/01/0116.

Mosherís acid (MTPA) has been one of the most favourite chiral derivatizing agents based on acetic acid pattern whose power lies in the anisotropic effect of an aromate. It has already BRASSINOSTEROIDS: TRYING TO FIND proven its usefulness in the determination of the absolute A BETTER WAY TO SELECT THE ACTIVE configuration of chiral alcohols and amines. A new compound CONFORMATION TO ESTABLISH A QSAR exploiting the aromatic anisotropic effect, 2-(1-naphthyl)-2- -phenylacetic acid (1-NPA), was designed. A strong enhance- MARC VILAPLANA-POLO, ISMAEL ZAMORA, ment in anisochrony ∆δ (the difference of chemical shifts of CARME BROSA* diastereomeric protons) caused by the present naphthyl ring was expected. Institut QuÌmic de Sarrià, C.E.T.S., Universitat Ramon Llull, Enantiomerically pure acid was obtained from the racemic Via Augusta 390, 08017 Barcelona, Spain one by resolution of diastereomeric mixture of (R)- and (S)-1- E-mail: [email protected] -NPA (ñ)-menthyl esters and subsequent hydrolysis. A set of 11 chiral alcohols was prepared with both racemic and enatio- In the field of brassinosteroids (BRs), we have developed merically pure acid. The ∆δ values were tabulated and their a quantitative structure-activity relationship (QSAR) study absolute values were compared with those of MTPA. After- that provide defined information about the minimum structu- wards, a correlation of the ∆δ values with the structure of the ral requirements necessary to elicit activity as plant growth alcohol moiety revealed that the spectral behaviour followed promoters1. systematic rules reflecting their steric arrangement. X-Ray Since the structure of the receptor is not known, indirect methodologies for QSAR studies are required. These methods assume that all information needed to explain the activity is on the structure of BRs. Even BRs can adopt different confor- mations, due to the presence of flexible points, the active BRs 2 should have a single defined three-dimensional (3D) ìactive R2 O 1 conformationî able to bind to the receptor. On this ìactive R1 conformationî, atoms involved in binding with receptor ought O H to have the same spatial situation in all active molecules. Thus, the more complementary is the ìactive conformationî of a defined BR to the 3Dstructure of receptor, the most active it should be. One should take into account that the so-called ìactive O conformationî is not necessarily the preferred conformation adopted in the steroid-receptor complex. Nevertheless, and O H 1,2 due to the fact that the structure of the receptor is unknown at the moment, the information gained by comparing the ìactive conformationî of all the active BRs will be useful in defining a good QSAR. Moreover, one can assume that the energy needed for the ìactive conformationî of various BRs to adopt the preferred conformation will not differ too much for one active BR to another. In our QSAR studies the ìactive conformationî of BRs was selected based on the methods of active analog approach (AAA) using geometrical descriptors as a selection criteria1. This approach consists on the selection of the ìactive confor- mationî for a reference compound (brassinolide) by comparing all its possible conformers with all the possible conformers of each of the other active BRs. Thus the ìactive confor- mationî of brassinolide will be that which results in highest

s309 Chem. Listy 97, s233 ñ s318 (2003) Conference on Isoprenoids 2003 similarity. Once defined the ìactive conformationî for brassi- were exposed to drought and flooding stresses. These plants nolide, this is taken as reference for further comparison with were treated with new synthetic brassinosteroid analogues and all conformers of a defined BR. The ìactive conformationî of their influence on the suppression of water stress was studied. each BR will be the most similar to the ìactive conformationî The first variant was treated with brassinosteroids 7 days of brassinolide. before the beginning of stress as foliar sprays, the second According to these geometries very good QSAR models variant was sprayed 5 days after the beginning of stress. Two were obtained. Based on GRIDmethodology2 we have found variants were used as controles: the first one without stress and a good correlation between the activity and the areas with high without any treatment, the second one with stress without any probability of H-bonding with receptor1. The results obtained treatment. The influence of stress in all variants was determi- until now, suggested that the region near to 23R-OH was more ned by dry means of weight changes, ethylene production, important for eliciting activity than those near to 22R-OH. fluctuation of abscisic acid and chlorophyll a and b content. Recently, the activity of some of the new compounds The dry weight were increased in both type stresses of obtained in our group donít fully agree with the contribution plants (drought and flooding) treated with 24-epibrassinolide on activity, above suggested, for the hydroxyls at C22 and C23 as well as with brassinosteroid analogues. The production of (ref.3). Considering that the ìactive conformationî for 22S, ethylene was not influenced significantly after treatment of 23S-BRs differs considerably from those of 22R,23R-BRs we neither analogues nor 24-epibrassinolide. suggested a re-evaluation of the methodology used to select the ìactive conformationî. In this communication we will This research was supported by the project of Czech Ministry present another way to select the ìactive conformationî based of Education ñ FRVS 1190 and by projects Nos. 203/01/0083, on activity prediction criteria which will allow us to consider A 4055204 and Z 4055905. the side chain 3Dgeometry from another point of view. Both methodologies will be compared and discussed. REFERENCES

REFERENCES 1. Khripach V. A., Zhabinskii V. N., de Groot A. E.: Physio- logical Mode of Action of Brassinosteroids, in: Brassi- 1. Brosa C.: Structure-activity Relationship,in:Brassino- nosteroids: A New Class of Plant Hormones (Khripach stroids: Steroidal Plant Hormones (Sakurai A., Yokota V. A., Zhabinskii V. N., de Groot A. E., ed.), pp. 220ñ301. T., Clouse S., ed.) Chap. 9. Springer-Verlag, Tokyo 1999. Academic Press, San Diego 1999. 2. Wade R. C., Clark K. J., Goodford P. J.: J. Med. Chem. 2. Mach·Ëkov· I., in: Regul·tory rostlinnÈho r˘stu (Pro- 36, 140 (1993). ch·zka S., äeb·nek J., Mach·Ëkov· I., Krekule J., KamÌ- 3. See poster Iban JovÈ et al. nek M., Borkovec V., HradilÌk J., Havel L., Ond¯ej M., Psota V., Luxov· M., Rauscherov· L., Sladk˝ Z., Viz·- rov· G., »Ìûkov· R., KlÌËov· ä., Rozkoöov· V., ed.), pp. 96ñ99, 350ñ353. Academia, Prague 1997. BRASSINOSTEROIDS AND WATER STRESS IN RAPE (Brassica napus L. )

EVA VLAä¡NKOV¡a, LADISLAV KOHOUTb, THE CHIRAL DERIVATIZATION JAN HRADILÕKa WITH MOSHERíS ACID ACYLISOCYANATE aDepartment of Botany and Plant Physiology, Faculty of Ag- PETR VODI»KA, LUDVÕK STREINZ, ronomy, Mendel University of Agriculture and Forestry, Ze- MILOä BUDÃäÕNSK›, BOHUMÕR KOUTEK mÏdÏlsk· 1, 613 00 Brno, bInstitute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Institute of Organic Chemistry and Biochemistry, Academy of Flemingovo n. 2, 166 10 Prague 6, Czech Republic Sciences of the Czech Republic, Flemingovo n. 2, 166 10 E-mail: [email protected] Prague 6, Czech Republic E-mail: [email protected] One of the most serious environmental stresses is drought. Not only survival during drought stress but also recovery of Trichloroacetyl isocyanate (TAI) has alredy been pro- stressed plant is crucial for the next plant growth. Synthetic ven as a valuable derivatization tool for NMR investigation1. analogues of the brassinosteroids represent new plant growth Its chiral derivative, (S)-2-chloro-2-fluoroethanoyl isocyanate regulators with the activity to increase plant growth but also besides conserving good reactivity of TAI, brings additional to enhance resistence of plants to stress conditions. It was de- informations with respect to the chirality of compound inves- scribed1 that 24-epibrassinolide increases root activity, plant tigated, thus demonstrating the possible role of chiral acyliso- growth and weight of the roots and shoots if applied as foliar cyanates in the analysis of optically pure compounds2. For spray to plants grown after water stress. obtaining good results with those types of substances, how- One of the first reactions of plants to stress is the ethylene ever, the presence of some functional groups is essential. production. The production of ethylene is increase by flooding, Namely, halogen atom(s) as well as aromatic ring in the mo- high temperature, toxic materials etc. Another phytohormone lecule significantly accelerates the reaction and positively af- increase in water stress is abscisic acid in the leaves and roots2. fects the spectral properties, respectively. Since the Mosherís In our experiments the plants of rape were used. The plants acid3 contains both the trifluoro- and phenyl groups, it seemed

s310 Chem. Listy 97, s233 ñ s318 (2003) Conference on Isoprenoids 2003

methyl)benzyl isocyanate4 as the chiral derivatization rea- (1) DMF + (COCl) gents. OMe 2 OMe NH OH (2) aq. NH 2 3 F3C Supported by Research Project Z 4055 905 and the Grant F3C O O Agency of the Czech Republic, grant No. 203/01/0116. 2 (COCl)2 REFERENCES

1. Goodlett V. W.: Anal. Chem. 37, 431 (1965). OMe 2. VodiËka P., Streinz L., Koutek B., BudÏöÌnsk˝ M., On- N C O dr·Ëek J., CÌsa¯ov· I.: Chirality 15, 472 (2003). F3C 3. Dale J.A., Mosher H. S.: J. Am. Chem. Soc. 90,3732 (1968). (R) O 4. Nabeya A., Endo T.: J. Org. Chem. 53, 3358 (1988). 1

R1 (R+S) SYNTHESIS OF STEROID CONTAINING HO R H 2 MACROCYCLES VIA MULTI COMPONENT REACTIONS

BRUNHILDE VOIGT, ANGELA SCHAKS, OMe 1 OMe 2 H R H R MONIKA B÷GEL, WOLFGANG BRANDT, N O 2 N O R + R1 * F3C F3C LUDGER A. WESSJOHANN O O H O O H R R R S Institute of Plant Biochemistry, Weinberg 3, 06120 Halle/ ( , ) ( , ) Saale, Germany E-mail: [email protected] Scheme1. Example for (R)-MTPA The synthesis of molecules which can recognize and bind to be a good starting material for preparing thus derived others or catalyze transformations of the bound molecules like acylisocyanate. an arteficial enzyme is one of the most exciting fields in The preparation of 3,3,3-trifluoro-2-methoxy-2-phenyl- synthetic chemistry. Our aim are molecules with well-defined propanoyl isocyanate (1) follows the easy two step reaction geometries in which conformational freedom is kept under leading from (R)- or (S)-α-methoxy-α-(trifluoromethyl)-phe- close control. This criterion can be met by designs based on nylacetic acid (MTPA) to 3,3,3-trifluoro-2-methoxy-2-phe- rigid frameworks combined with elements of controlled flexi- nylpropanamide (2). Finally, amide 2 with oxalyl chloride bility. At the same time, certain dimensions are required and under the controled conditions affords acylisocyanate 1 pure have to be build up fast and efficiently. enough for in situ reaction with chiral alcohols. The reaction With this background we started a program for the synthesis with alcohols is usually very fast (without any catalysis) giving of macrocycles with steroid containing rigid parts using the mixture of diastereomeric carbamates esters detectable by Ugi-multi component reaction (U-MCR1). Bile acids2 are the means of NMR. The analysis of NMR data of database of most valuable group of steroids due to their relatively rigid prepared diastereomers revealed that 1H, 19F and 13C-NMR moiety, umbrella shape, chemically different hydroxyl groups, shifts well correlating with the compundís structure, so the enantiomeric purity, availability, and low cost. derivatization may be used only for the optical purity determi- First step for the synthesis of cyclopeptide steroids is the nation, but also for estimation of absolute and/or relative bifunctionalization of bile acids as starting compounds. Good configuration. results were obtained with the U-4CR-condensation of the Last but not least, results in the form of the characteristic diamine3 and diisocyanide synthesized from lithocholic acid, chemical shifts have been compared with results of previous together with isobutyraldehyde and acetic acid (Scheme). works where was used MTPA3 and α-methoxy-α-(trifluoro- After chromatography and preparative HPLC-separation eight

NH 2 NH NH O NH NH HN 2 O + O O N O or O O O N O N N O OH

NC NC

s311 Chem. Listy 97, s233 ñ s318 (2003) Conference on Isoprenoids 2003 products were obtained having the molecular ion mass of 969 ne effects in human sperm, neurosteroid action on neuronal in the ESI-MS which showes the successful cyclization. These structures and vascular effects of estrogens. Mechanisms of are the four possible diastereomers of the head-head and action are being studied with regard to signal perception and head-tail regioisomers. When paraformaldehyde was used for transduction involved, and for various steroids including al- the same U-4CR only two main products were formed. Mole- dosterone a patchy sketch of a membrane receptor/second cular modelling investigations showed that the heats of forma- messenger cascade shows up in the mist being not essentially tion of the head to head and head to tail macrocycles is nearly dissimilar to cascades involved in catecholamine or peptide the same, the probability of the formation of both macrocycles hormone action. Aside nonclassical membrane receptors with is close to equal. The cavities of synthesized macrocycles are a high affinity for a particular steroid, these effects appear to large enough to encapsulate small organic substrates, and variably involve phospholipase C, phosphoinositide turnover, more detailed studies in this respect are under way. intracellular pH and calcium, protein kinase C and tyrosine kinases. The physiological and pathophysiological relevance REFERENCES of these effects is not yet clear, but more and more studies indicate that rapid steroid effects on cardiovascular, central 1. Dˆmling A., Ugi I.: Angew. Chem. 112, 3300 (2000). nervous and reproductive functions occur in vivo and seemin- 2. Tamminen J., Kolehmainen E.: Molecules 6, 21 (2001). gly transmit physiological and pathophysiological responses. 3. Lawless L. J., Blackburn A. G., Ayling A. J., Perez-Payan Future research will have to target the cloning of the first M. N., Davis A. P.: J. Chem. Soc., Perkin Trans. 1 2001, membrane receptor for steroids which should be achieved in 1329. near future, and the evaluation of the physiological and clinical relevance of these rapid steroid effects.

REFERENCES NONGENOMIC STEROID ACTION: FROM MEMBRANES TO HUMAN PHYSIOLOGY 1. Wehling M., Christ M., Gerzer R.: Trends Pharmacol. Sci. 14, 1 (1993). M. WEHLING 2. Wehling M.: Annu. Rev. Physiol. 59, 365 (1997). 3. http://steroid-rapid-responses.ucr.edu, 20/5 2003. Institute of Clinical Pharmacology, Faculty for Clinical Me- dicine Mannheim, University of Heidelberg, Germany

According to the traditional model steroid hormones bind ALKYL GLYCOSIDES WITH BIOLOGICAL to intracellular receptors and subsequently modulate transcrip- ACTIVITY tion and protein synthesis, thus triggering genomic events finally responsible for delayed effects. Based upon similarities ZDENÃK WIMMERa, PAVLA SOCHŸRKOV¡a, in molecular structure, specific receptors for steroids, vitamin LAURA SILE b, DAVID äAMANa, a a D3 derivatives, thyroid hormone, retinoids and a vanity of JELENA KULDOV¡ , IVAN HRD› orphan receptors are considered to represent a superfamily of steroid receptors. In addition, very rapid effects of steroids aInstitute of Organic Chemistry and Biochemistry, Academy mainly affecting intracellular signaling have been widely re- of Sciences of the Czech Republic, Flemingovo n. 2, 166 10 cognized which are clearly incompatible with the genomic Prague 6, Czech Republic, bLatvian Institute of Organic Syn- model. These rapid, nongenomic steroid actions are likely to thesis, Aizkraukles 21, 1006 Riga, Latvia be transmitted via specific membrane receptors. Evidences for nongenomic steroid effects and distinct receptors involved are This research has been based on juvenoids (insect juvenile now presented for all steroid groups including related com- hormone mimics), which have been developed in past years. pounds like vitamin D3 and thyroid hormones. The physiolo- All juvenoids studied belong to the 2-(4-hydroxybenzyl)-cy- gical and clinical relevance of these rapid effects is still largely clohexan-1-ol series. Their glycosides belong among hormo- unclear, but their existence in vivo has been clearly shown in nogenic substances (juvenogens) capable of liberating the various settings including human studies. Drugs that specifi- biologically active juvenoids under the effect of biotic factors cally affect nongenomic steroid action may find applications (enzymes) or abiotic factors (environmental conditions ñ pH, in various clinical areas such as cardiovascular and central UV, humidity or oxygen effect etc.)1,2. nervous disorders, electrolyte homeostasis and infertility. In The present study reflects our effort in investigating physi- addition to a short description of genomic steroid action, this co-chemical and biological properties of 2-(4-alkoxybenzyl)- review pays particular attention to the current knowledge and cyclohex-1-yl-β-D-glycopyranosides. In principle, the com- important results on the mechanisms of nongenomic steroid pounds are accessible by several most often used methods of action. The modes of action are discussed in relation to their synthesis of alkyl glycosides: Fischer-Helferich method, Koe- potential physiological or pathophysiological relevance and nigs-Knorr method or a method through auxiliary trichloro- with regard to a cross-talk between genomic and non-genomic acetimidate formation3. We have found that the protected responses. derivatives, 2-(4-alkoxybenzyl)cyclohex-1-yl-2í,3í,4í,6í-tetra- Prominent examples of nongenomic steroid action are -O-acetyl-β-D-glycopyranosides, are available by the Koe- rapid aldosterone effects in lymphocytes and vascular smooth nigs-Knorr synthesis, using 2,3,4,6-tetra-O-acetyl-β-D-glyco- muscle cells, vitamin D3 effects in epithelial cells, progestero- pyranylbromides as glycosyl donors and heavy metal promo-

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OR ROLES OF BRASSINOSTEROID BIOSYNTHESIS IN PEA SEED DEVELOPMENT AND GERMINATION

OH TAKAO YOKOTA, TAKAHITO NOMURA *

OR Department of Biosciences, Teikyo University, Utsunomiya 320-8551, Japan (*Present address: Plant Science Center, AcO RIKEN, Yokohama, 230-0045, Japan) OO OAc Brassinosteroid (BR) is a steroidal plant growth hormone AcO involved in cell elongation/enlargement, cell division and cell OAc differentiation. Major BRs are C28 steroids that are synthesized from campesterol (Fig. 1). Campesterol is hydrogenated to campestanol and then converted to castasterone through OR either the early or late C-6 oxidation pathway. Castasterone is finally converted to brassinolide. The available evidence sug- HO gests that brassinolide and castasterone are both biologically- -active. OO OH To investigate the roles of brassinosteroids in seed development and germination, we quantified the endogenous HO BRs and the transcripts of BR synthesis (LKB, LK, PsDWF4, OH PsCPD1, PsCPD2, DDWF1, PsD), metabolism (PsBAS1) and receptor (LKA) genes in seeds and seedlings of pea (Pisum Scheme 1 sativum L.). As pea seeds rapidly grow, the levels of brassinolide and castasterone were increased but, in fully-expanded seeds, were ters to perform the reactions (Scheme 1). Using cadmium decreased drastically, indicating brassinolide and castasterone carbonate as promoter has resulted in highest yields among are important for seed growth. In support of this, the PsD a series of screened promoters. Subsequent deprotection of the expression was increased in conjugation with the increase of sugar unit may be performed by different methods, depending castasterone and brassinolide. 6-Deoxocastasterone was accu- on functionalities present in the aliphatic chain R (Scheme 1). mulated high in fully expanded seeds but rapidly decreased Rather convenient method of removing acetyl groups consists through desiccation presumably by the action of the PsBAS1 in using alkali metal salts in absolute methanol. If another ester enzyme. The levels of upstream precursors, 6-deoxocathaste- functionality is present in the molecule, in the aliphatic chain rone, 6-deoxoteasterone, 3-dehydro-6-deoxoteasterone and 6- R, deacetylation by zinc acetate in absolute ethanol usually -deoxotyphasterol were not changed much from immature to represents a convenient method. Our investigation has invol- mature stages. 6-Deoxocathasterone was the major brassino- ved synthesis of selected glucosides and galactosides. Selected steroid in mature seeds and is likely to be an important storage biological activity data of several juvenogen example substan- form. Through seed growth, the LK transcript levels remained ces on non-related insect pest species will be presented. constant but those of other genes were fluctuated. In mature seeds, the PsCPD1 gene level increased markedly while the This research was supported by the grant GA CR 203/02/0166. levels of LKB, PsCPD2, DDWF1, PsBAS1 and LKA were International collaboration was supported by the Czech-Lat- considerably decreased although still detectable, suggesting vian bilateral contract and has been a part of the COST that the mRNAs of these genes may be utilized to generate D29/0002/03 network. The authors thank Mrs. M. Wimmerov· brassinosteroids when seeds germinate. for skillful technical assistance. In imbibed seeds, neither castasterone nor brassinolide were detected but the transcripts of PsD, DDWF1, PsDWF4, REFERENCES PsBAS1 and LKA were increased. One and three days after the imbibition, castasterone was detected in germinating seeds. In 1. Sl·ma K., RomaÚuk M.: Insect Biochem. 6, 579 (1976). these plantlets, the level of castasterone as well as the PsD 2. Wimmer Z., Rejzek M., Zarev˙cka M., Kuldov· J., Hrd˝ expression was high in shoots and roots but low in seeds, I., NÏmec V., RomaÚuk M.: J. Chem. Ecol. 23,605 indicating that the PsDprotein seems to be a key enzyme for (1997). seed germination. 3. Schmidt R. R., Jung K.-H., in: Preparative Carbohydrate Chemistry (Hanessian S., ed.), p. 283. Dekker 1997.

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LK LKB

O HO HO H (24R)-5α-ergostan-3-one campesterol 24-methylenecholesterol

HO HO H H O campestanol 6-oxocampestanol PsDWF4 OH PsDWF4 OH

HO HO H H cathasterone 6-deoxocathasterone O PsCPD OH PsCPD OH

OH OH

HO H HO H teasterone 6-deoxoteasterone O OH OH

Early C-6 oxidation pathway Late C-6 oxidation pathway OH OH

O O H H 3-dehydro-6-deoxoteasterone O 3-dehydroteasterone OH OH

OH OH

HO typhasterol H HO H 6-deoxotyphasterol O OH DDWF4 OH DDWF4 OH OH OH OH HO PsD HO HO HO HO H HO O H O H O 6-deoxocastasterone castasterone brassinolide

Fig. 1. Brassinosteroid biosynthesis and related genes in Pisum sativum (Bold arrows indicate major biosynthetic pathways)

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SYNTHESIS OF 5,10- AND 13,14-SECOSTEROIDS Radical oxidation of the tertiary alcohol 1 with lead tetraacetate in the presence of iodine gave a set of intermediates VLADIMIR N. ZHABINSKIIa,GALINA 4ñ7, which are suitable for the preparation of 5,10-seco ste- P. FANDOa, ALLA A. GOVOROVAa, roids. The reaction proceeds via the radicals 2 and 3. The latter MARINUS B. GROENb, AEDE DE GROOTc, undergoes either hydrogen abstraction at C-1 to afford the VLADIMIR A. KHRIPACHa*, ANNA ∆1(10)-olefins 4 and 5, or a transannular hydrogen abstraction I. KOTYATKINAa, JAAP VAN DER LOUWb, at C-4. Reaction of this new radical with iodine then leads to ALEXANDER S. LYAKHOVa, the iodides 6 and 7. YULIYA Y. ZHIBURTOVICHa Compounds 4ñ7 were transformed further into steroids with functional groups characteristic for androgens (e.g. 8). aInstitute of Bioorganic Chemistry, National Academy of Scien- Some fragmentation or intramolecular cyclization products ces of Belarus, Kuprevich Str., 5/2, 220141 Minsk, Belarus, like 9,10 were obtained also. bDepartment of Medicinal Chemistry, N.V. Organon, P.O. An alternative approach to 5,10-seco steroids was found Box 20, 5340 BH Oss, cWageningen University, Laboratory of by ozonolysis of the ∆5(10)-olefin 11, followed by synthetic Organic Chemistry, Dreijenplein 8, 6703 HB Wageningen, transformations of diketone 12. Seco steroids containing two The Netherlands double bonds in the AB-cyclic part (e.g. 13) became available in this way. A distinctive feature of steroids is the presence of rigid Key steps in the preparation of 13,14-seco steroids 17ñ19 tetracyclic skeleton. The greater conformational flexibility in were again found in radical oxidation of the 14α-hydroxy these molecules, which can be obtained by cleavage of an derivative 15, followed by the removal of iodine in 16. internal C-C bond to seco steroids, may lead to new biological A second route to 13,14-seco steroids was based on the properties. Results on the synthesis of 5,10- and 13,14-seco Grob fragmentation of hydroxy tosylate 20. Modifications of steroids using different approaches (radical oxidation, Grob the resulting unsaturated ketone included hydride reduction of fragmentation, oxidative cleavage) will be presented. the 14-carbonyl group and hydroboration-oxidation of ∆13,17-

OAc

LTA, I 2 • AcO AcO AcO OH OH• O 1 2 3 H

+ H + + AcO AcO AcO AcO O O O O 4 5 I 6 I 7

O OAc OH O O

H O AcO H H 10 8 OAc 9

OAc OAc OAc

O O3

AcO AcO AcO O 11 12 13

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O O O

1. TsCl I 2. MeOH, Py LTA, I 2 OH OH O HO OMe OMe 14 15 16

O OH O

OAc

O O O 17 18 19

OTs OAc

HO AcO

OH MeO MeO MeO

20 21 22

-double bond, which both proceeded stereoselectively, to pro- Thea sinensis L.; secasterone, from Secale cereale L.), either vide compound 22 as the only isomer. obtained by addition of adequate prefix(es) to the name of The structural elucidation of new types of seco steroids a previously known brassinosteroid (e.g. 28-homobrassino- and reaction mechanisms will also be discussed. lide, 28-norcastasterone, 25-methyldolichosterone, 3-epi-2- -deoxy-25-methyldolichosterone, etc.). More than 50 known brassinosteroids are 3-oxygenated (22R,23R)-5α-cholestane- SEMISYSTEMATIC NOMENCLATURE -22,23-diols, of plant origin, bearing alkyl or oxy substituents, OF BRASSINOSTEROIDS conjugated or not to sugars or fatty acids1. This general structural feature allows the proposition of semisystematic names MARCO ANT”NIO TEIXEIRA ZULLOa, to the brassinosteroids, in which (22R,23R)-2α,3α,22,23-te- LADISLAV KOHOUTb trahydroxy-5α-campestane, of trivial name 6-deoxocastasterone, is considered the functional parent compound and is aInstituto AgronÙmico (IAC), P.O. Box 28, 13001-970 Cam- named brassinostane or brassinane. The closely related com- pinas, SP, Brazil, bInstitute of Organic Chemistry and Bioche- pounds of trivial names, 6α-hydroxycastasterone, castastero- mistry, Academy of Sciences of the Czech Republic, Flemin- ne and brassinolide, would then be named 6α-brassinosta- govo n. 2, 166 10 Prague 6, Czech Republic nol, brassinostanone and brassinostanolactone, respectively, E-mail: [email protected] or 6α-brassinol, brassinone and brassinolide. The semisystematic names of the other members of the brassinosteroid The assignment of a trivial name to a natural product has family shall be given according to the established rules for the advantage of concentrating all the structural, including naming natural products2. The use of these semisystematic stereochemical, information in a single, or at most a few, names would avoid some mistakes in assigning trivial names simple word(s), since it is unique to this compound, but to the brassinosteroids and the unpractical constant usage of without knowing the compound one cannot advance these their systematic names. information. On the other hand, its systematic name carries all This work was supported by the grant No. A4055204 (GA CR) structural and stereochemical information, but is usually too and research project Z4 055 905. long for being frequently used. The trivial name of a brassinosteroid is either derived from the plant source it was first REFERENCES isolated or detected (brassinolide, from Brassica napus L.; castasterone, from Castanea crenata Sieb. et Zuck; dolicholi- 1. Zullo M. A. T., Kohout L., De Azevedo M. B. M.: Plant de and dolichosterone, from Dolichos lablab Adans.; typha- Growth Regul. 39, 1 (2003). sterol, from Typha latifolia G. F. W. Mey.; teasterone, from 2. IUPAC: Pure Appl. Chem. 61, 1783 (1999).

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