Initial Steps in the Degradation of Methoxychlor by the White Rot Fungus Phanerochaete chrysosporium

The white rot fungus Phanerochaete chrysosporium mineralized [ring-(sup14)C]methoxychlor [1,1,1-trichloro-2,2-bis(4-methoxyphenyl)ethane] and metabolized it to a variety of products. The three most prominent of these were identified as the 1-dechloro derivative 1,1-dichloro-2,2-bis(4-methoxyphenyl)ethane, the 2-hydroxy derivative 2,2,2-trichloro-1,1-bis(4-methoxyphenyl)ethanol, and the 1-dechloro-2-hydroxy derivative 2,2-dichloro-1,1-bis(4-methoxyphenyl)ethanol by comparison of the derivatives with authentic standards in chromatographic and mass spectrometric experiments. In addition, the 1-dechloro-2-hydroxy derivative was identified from its (sup1)H nuclear magnetic resonance spectrum. The 1-dechloro and 2-hydroxy derivatives were both converted to the 1-dechloro-2-hydroxy derivative by the fungus; i.e., there was no requirement that dechlorination precede hydroxylation or vice versa. All three metabolites were mineralized and are therefore likely intermediates in the degradation of methoxychlor by P. chrysosporium.     

Microsomal monooxygenation of the carcinostatic 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea. Synthesis and identification of cis and trans monohydroxylated products.

Liver microsomal hydroxylation of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea was shown to occur on the cyclohexyl ring at positions 3 and 4. Four metabolites were isolated by selective solvent extraction and purifed by high-pressure liquid chromatography. cis-4-, trans-4-, cis-3-, and trans-3-OH derivatives of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea were synthesized and their chromatographic, mass spectral, and nuclear magnetic resonance characteristics matched those of the metabolites. The position of ring hydroxylation and the identity of each geometric isomer were established by nuclear magnetic resonance using a shift reagent in conjunction with spin decoupling techniques. Microsomes from rats pretreated with phenobarbital showed a sixfold increase in hydroxylation rate (19.5 vs. 3.3 nmol per mg per min). The induction was quite selective for cis-4 hydroxylation (19-fold); however, induction of trans-4 (threefold), cis-3 (threefold), and trans-3 (twofold) hydroxylation did occur. Quantitatively the cis-4-hydroxy metabolite was 67of the total product by phenobarbital-induced microsomes and 21% for normal microsomes. Microsomes from animals pretreated wit- 3-methyl-cholanthrene gave about the same rate and product distribution that normal microsomes gave. A mixture of 80% carbon monoxide-20% oxygen inhibited formation of all four hydroxy metabolites with the inhibition ranging from 55 to 78%.     

Regioselective oxygenation of fatty acids, fatty alcohols and other aliphatic compounds by a basidiomycete heme-thiolate peroxidase

Reaction of fatty acids, fatty alcohols, alkanes, sterols, sterol esters and triglycerides with the so-called aromatic peroxygenase from Agrocybe aegerita was investigated using GC-MS. Regioselective hydroxylation of C(12)-C(20) saturated/unsaturated fatty acids was observed at the ω-1 and ω-2 positions (except myristoleic acid only forming the ω-2 derivative). Minor hydroxylation at ω and ω-3 to ω-5 positions was also observed. Further oxidized products were detected, including keto, dihydroxylated, keto-hydroxy and dicarboxylic fatty acids. Fatty alcohols also yielded hydroxy or keto derivatives of the corresponding fatty acid. Finally, alkanes gave, in addition to alcohols at positions 2 or 3, dihydroxylated derivatives at both sides of the molecule; and sterols showed side-chain hydroxylation. No derivatives were found for fatty acids esterified with sterols or forming triglycerides, but methyl esters were ω-1 or ω-2 hydroxylated. Reactions using H(2)(18)O(2) established that peroxide is the source of the oxygen introduced in aliphatic hydroxylations. These studies also indicated that oxidation of alcohols to carbonyl and carboxyl groups is produced by successive hydroxylations combined with one dehydration step. We conclude that the A. aegerita peroxygenase not only oxidizes aromatic compounds but also catalyzes the stepwise oxidation of aliphatic compounds by hydrogen peroxide, with different hydroxylated intermediates.     

The relative significance for stem elongation and flowering in Lolium temulentum of 3β-hydroxylation of gibberellins

In previous experiments with many gibberellins (GAs) and GA derivatives applied to Lolium temulentum L., quite different structural requirements were evident for stem elongation on the one hand and for the promotion of flowering on the other. Whereas hydroxylation at carbons 12, 13 and 15 enhanced flowering relative to stem growth, the reverse was the case at carbon 3 (L.T. Evans et al. 1990, Planta 182, 97–106). The significance of hydroxylation at carbon 3 is examined in this paper. The application of inhibitors of 3β-hydroxylation, including C/D-ring-rearranged GAs, reduced stem growth but, in the case of the two acylcyclohexanediones, increased the flowering response when applied on the inductive long day. Later applications of the acylcyclohexanediones, made after floral initiation had occurred, were inhibitory to flowering, suggesting that subsequent inflorescence development requires 3β-hydroxylated GAs. Applications of the 3α-hydroxy epimers of GA₁, GA₃ and GA₄ gave slightly less promotion of flowering in comparison with the 3β-hydroxy GAs, but far less promotion of stem elongation, except in the case of 3-epi-GA₄, which was comparable to GA₄. The 3α-hydroxy epimer of 2,2-dimethyl GA₄ gave less promotion of flowering than its 3β-hydroxy epimer but almost no promotion of stem elongation. The 3α-hydroxy epimers of GA₃ and 2,2-dimethyl GA₄ did not act as competitive inhibitors of the stem elongation elicited by GA₃ and 2,2-dimethyl GA₄, respectively. These results extend the differences in GA structure which favour flowering as opposed to stem elongation, and indicate that 3-hydroxylation and its epimeric configuration are of much greater importance to stem elongation than to flower initiation in Lolium.     

3-Methylene-substituted androgens as novel aromatization inhibitors. Evidence of a requirement for C-3 oxygen in C-19 hydroxylations

Substitution of a methylene group for the C-3 oxygen in androstenedione, testosterone, and the corresponding 19-hydroxy and 19-oxo derivatives results in a new category of inhibitors of estrogen biosynthesis by human placental microsomes. The inhibition is of the competitive type with the most effective inhibitors being the 17-ketonic compounds, 3-methyleneandrost-4-en-17-one, 19-hydroxy-3-methyleneandrost-4-en-17-one, and 3-methylene-19-oxoandrost-4-en-17-one with apparent Ki values of 4.7, 13, and 24 nM, respectively. The 3-methylene derivatives of androstenedione and 19-hydroxyandrostenedione were effective substrates for the placental microsomal 17 beta-hydroxy-steroid oxidoreductase but were only marginally hydroxylated at the C-19 position to the respective 19-hydroxy and 19-oxo derivatives. The 3-methylene analogs are thus competitive inhibitors of aromatization but are not substrates for this enzyme complex. Time-dependent inhibition of aromatization by 10 beta-difluoromethylestr-4-ene-3,17-dione and 10 beta-(2-propynyl)estr-4-ene,3,17-dione was abolished by substitution of a methylene function for the C-3 oxygen, suggesting that the presence of an oxygen at C-3 is required for an oxidative transformation at C-19, an initial step in aromatization. The essential role of the C-19 hydroxylation in aromatization is supported by the observation that the 3-methylene derivatives of 19-hydroxy- and 19-oxoandrostenedione showed time-dependent inhibition, but the corresponding 19-methyl compound did not. The 3-methylene androgens are potent inhibitors of placental aromatization but are themselves only marginal substrates for the enzyme. Their high affinity for and inertness to the placental aromatase complex makes them valuable probes of the aromatization process.     

Remarkable aliphatic hydroxylation by the diiron enzyme toluene 4-monooxygenase in reactions with radical or cation diagnostic probes norcarane, 1,1-dimethylcyclopropane, and 1,1-diethylcyclopropane

Toluene 4-monooxygenase (T4MO) catalyzes the hydroxylation of toluene to yield 96% p-cresol. This diiron enzyme complex was used to oxidize norcarane (bicyclo[4.1.0]heptane), 1,1-dimethylcyclopropane, and 1,1-diethylcyclopropane, substrate analogues that can undergo diagnostic reactions upon the production of transient radical or cationic intermediates. Norcarane closely matches the shape and volume of the natural substrate toluene. Reaction of isoforms of the hydroxylase component of T4MO (T4moH) with different regiospecificities for toluene hydroxylation (k(cat) approximately 1.9-2.3 s(-)(1) and coupling efficiency approximately 81-96%) revealed similar catalytic parameters for norcarane oxidation (k(cat) approximately 0.3-0.5 s(-)(1) and coupling efficiency approximately 72%). The products included variable amounts of the un-rearranged isomeric norcaranols and cyclohex-2-enyl methanol, a product attributed to rearrangement of a radical oxidation intermediate. A ring-expansion product derived from the norcaranyl C-2 cation, cyclohept-3-enol, was not produced by either the natural enzyme or any of the T4moH isoforms tested. Comparative studies of 1,1-dimethylcyclopropane and 1,1-diethylcyclopropane, diagnostic substrates with differences in size and with approximately 50-fold slower k(cat) values, gave products consistent with both radical rearrangement and cation ring expansion. Examination of the isotopic enrichment of the incorporated O-atoms for all products revealed high-fidelity incorporation of an O-atom from O(2) in the un-rearranged and radical-rearranged products, while the O-atom found in the cation ring-expansion products was predominantly obtained by reaction with H(2)O. The results show a divergence of radical and cation pathways for T4moH-mediated hydroxylation that can be dissected by diagnostic substrate probe rearrangements and by changes in the source of oxygen used for substrate oxygenation.     

Identification of rat cytochrome P450 forms involved in the metabolism of clausenamide enantiomers

To identify which cytochrome P450 (CYP) isoform(s) are responsible for the metabolism of clausenamide (CLA) enantiomers in rats, effects of various CYP isoform inducers and inhibitors on the formation of CLA metabolites were investigated in liver microsomes. In incubations with rat liver microsomes, CLA enantiomers were mainly converted to 4-hydroxy, 5-hydroxy, and 7-hydroxy-metabolites. 4-OH-CLA was the major metabolite of (+)-3R, 4S, 5S, 6R-CLA [(+)-CLA], while 7-OH-CLA was the major one of (-)-3S, 4R, 5R, 6S-CLA [(-)-CLA]. In induction studies, enzymatic parameters were used to assess the role of different CYP forms in CLA hydroxylation reactions. A marked increase in the rate of metabolism of CLA enantiomers was observed in microsomes of dexamethasone treated rats, V(max)/K(m) values for 4-OH-(+)-CLA, 7-OH-, 5-OH-, and 4-OH-(-)-CLA were 5.3, 6.5, 3.0, and 5.9 times higher than those in control microsomes, respectively. Rifampicin treatment caused corresponding 1.7-, 2.6-, 3.1-, and 2.8-fold increases. Dex and Rif also increased in the amount of (+)-5- and (+)-7-OH-CLA that were not detectable in the control group. These results suggested that inducible CYP3A1 was involved in the hydroxylation of CLA enantiomers. In inhibition studies, ketoconazone (6.25 microM) completely inhibited the production of main metabolites of (-)-CLA (100%) and (+)-CLA (97%). Triacetyloleandomycin (12.5 microM) strongly inhibited the corresponding metabolites by 34-85%. These findings also indicated that institutive CYP3A2 shared a major role in the hydroxylation of CLA enantiomers with CYP3A1 in untreated rats. Together, the data suggested that CYP3A was the predominant isoform responsible for the metabolism of CLA enantiomers.     

Regioselectivity of hydroxylation of prostaglandins by liver microsomes supported by NADPH versus H2O2 in methylcholanthrene-treated and control rats: formation of novel prostaglandin metabolites

The effects of methylcholanthrene (MC) treatment of male rats on the regioselectivity of hydroxylation of prostaglandins E1 and E2 (PGE1 and PGE2) by liver microsomes, supplemented with NADPH or H2O2, was examined. In the presence of NADPH, control microsomes catalyzed the hydroxylation at omega-1 (C19) and at omega-(C20) sites with minimal formation of novel monohydroxy metabolites of PGE1 and PGE2, referred to as compounds X1 and X2, respectively. Similarly, H2O2 supported the 19-hydroxylation and the formation of compounds X1 and X2, but yielded only minimal amounts of 20-hydroxy products. With NADPH, MC-treated microsomal incubations demonstrated only minor quantitative change in the 19- and 20-hydroxylation as compared with controls, but showed a 7- to 11-fold increase in formation of compound X1 and a 10-fold increase in formation of X2. By contrast with H2O2, MC-treatment increased by about 3-fold the 19- and 20-hydroxylation of PGE1 and by 35- to 46-fold the formation of X1; similarly, there was an approximate 2-fold increase in 19- and 20-hydroxylation of PGE2 and a 10-fold increase in formation of X2. These findings suggest that several monooxygenases are involved in catalyzing the hydroxylation at the various sites of the PGE molecule. Inhibitors of monooxygenases (SKF 525A, alpha-naphthoflavone, and imidazole derivatives) provided further evidence that the hydroxylation at the three sites of PGEs is catalyzed by different P-450 monooxygenases. It is striking that the inhibitors had a much lesser effect on the 20-hydroxylation of PGE1 as compared with other sites of hydroxylation. Structural identification of compounds X1 and X2 was elucidated as follows. Resistance of the PGB derivative of X1 to periodate oxidation and mass fragmentation analysis of the t-butyldimethylsilyl ether methyl ester, placed the hydroxylation at C17 or C18. Finally, mass fragmentation of trimethylsilyl ether methyl ester PGB derivatives of X1 and X2 provided conclusive evidence that X1 and X2 are 18-hydroxy-PGE1 and 18-hydroxy-PGE2, respectively. The above findings indicate that the high regioselectivity of hydroxylation of PGE1 and PGE2, resulting in the formation of 18-hydroxy-PGE1 and 18-hydroxy-PGE2, respectively, is catalyzed by P-450 isozyme(s) which are induced by MC, possibly by P-450c.     

Alkylation and carbamoylation intermediates from the carcinostatic 1-(2-chloroethyl)-3-cyclohexyl -1-nitrosourea (CCNU)

Evidence is presented which shows that 1-(2-chloroethyl) -3-cyclohexyl-1-nitrosourea (CCNU) upon degradation provides a 2-chloroethyl alkylating intermediate, possibly 2-chloroethyl carbonium ion, and 2-chloroethanol. Thiol alkylation occurs $\text{in vivo}$ and a major urinary metabolite of CCNU is thiodiacetic acid. A rapid microsomal hydroxylation of the cyclohexyl ring occurs which yields varying ratios of at least five metabolites: cis or trans 2-hydroxy, trans- 3-hydroxy, cis-3-hydroxy, cis-4-hydroxy and trans-4- hydroxy-CCNU. $\text{In vivo}$ carbamoylation appears to not be due to cyclohexylisocyanate but to the various hydroxy-cyclohexylisocyanates which are formed from hydroxy CCNU metabolites.     

Steroids excreted in urine by neonates with 21-hydroxylase deficiency. 2. Characterization, using GC-MS and GC-MS/MS, of pregnanes and pregnenes with an oxo- group on the A- or B-ring

Urine from neonates with 21-hydroxylase deficiency contains a large range of metabolites of 17-hydroxyprogesterone, 21-deoxycortisol and androgens but few have been previously described. We present the second part of a comprehensive project to characterize and identify these in order to enhance diagnosis and to further elucidate neonatal steroid metabolism. Steroids were analyzed, after extraction and enzymatic conjugate hydrolysis, as methyloxime-trimethylsilyl ether derivatives on gas-chromatographs coupled to quadrupole and ion-trap mass-spectrometers. GC-MS and GC-MS/MS spectra were used together to determine the structure of the A- and B-rings containing an oxo group. Fragmentations indicating presence of 3-, 6-, and 7-oxo groups and also 1β-, 2α-, 4β-, and 6β-hydroxyls are presented and discussed for the first time. Interpretation was aided by comparison with spectra of available relevant standards, of oxidation products of standards and urinary metabolites and of deuterated derivatives. Endogenous 1-enes and 2(3)-ene artifacts of non-hydrolyzed 3α-sulfates are also reported. D-ring and side chain structure was determined according to our previously published criteria. Likely metabolic relationships were also explored. We conclude that GC-MS combined with GC-MS/MS allows identification of the A- and B-ring structure of pregnane and pregnenes in the presence of an oxo group on one of these rings. Major oxygenations are 1β, 15β, 16α and 21-hydroxy and 6- and 7-oxo groups. Minor positions of hydroxylation are those at 2α, 4β and 6β. Three major metabolic streams exist in affected neonates in addition to the classical 3α-hydroxy-5β-pregnane pathway, i.e. these of the 3-oxo-4-enes as well as 3α- and 3β-hydroxy-5α-anes.     

Anti-mutagenic structural modification by fluorine-substitution in highly mutagenic 4-methylquinoline derivatives

We have previously shown that fluorine-substitution at position 3 of quinoline deprived this molecule of mutagenicity, possibly due to interference with the yield of its metabolically activated form, the 1,4-hydrated 2,3-epoxide (enamine epoxide), which is directly responsible for the mutagenic modification of DNA. To further explore the possibility of a method for anti-mutagenic modification of mutagens by fluorine-substitution, 4-methylquinoline (4-MeQ), the most mutagenic form of all the quinoline derivatives examined so far, was used as a target in the present study. Five mono- and di-fluorinated derivatives of 4-MeQ, 2-fluoro-4-methylquinoline (2-F-4-MeQ), 6-F-4-MeQ, 7-F-4-MeQ, 2,6-difluoro-4-methylquinoline (2, 6-diF-4-MeQ), and 2,7-diF-4-MeQ, were subjected to analysis of their structure-mutagenicity relationships. The 2-fluorinated derivatives (2-F-4-MeQ, 2,6-diF-4-MeQ, and 2,7-diF-4-MeQ) were all non-mutagenic in the Ames test. 7-F-4-MeQ was as highly mutagenic as, and 6-F-4-MeQ was less mutagenic than non-fluorinated 4-MeQ. Metabolic studies were also conducted with 4-MeQ, 2-F-4-MeQ, 6-F-4-MeQ, and 7-F-4-MeQ, using a liver microsomal enzyme fraction prepared from the 3-methylcholanthrene-treated rat. The HPLC analytical data showed that, although the metabolic patterns (hydroxylation at 4-methyl group as a main metabolic pathway and 3-hydroxylation as a minor pathway) of these four F-MeQs were similar to one another, only the 3-hydroxy metabolite of 2-F-4-MeQ was not produced under the present experimental conditions employed. These results suggest that fluorine-substitution at position 2 of 4-MeQ inhibited the formation of the enamine epoxide in the pyridine moiety and deprived this molecule of mutagenicity as in the case of quinoline.     

Plant growth retardant activity of 4-homoisotwistane derivatives

A variety of simple derivatives of 3-substituted 4-homoisotwistane derivatives were prepared, and their effect on the growth of cucumber seedlings in complete darkness was investigated. The 3-hydroxy derivative was found to show a strong inhibitory activity at 50 μg/ml, so a series of other hydroxy derivatives of 4-homoisotwistane, endo-2-, exo-2-, and 5-hydroxy- and exo-2,3-dihydroxy-4-homoisotwistane were prepared in order to obtain information on structure-activity relationships. The endo-2-hydroxy derivative inhibited the growth of cucumber and the germination of lettuce seed at 12.5 μg/ml. All the hydroxy derivatives tested increased the number of adventitious roots in hypocotyls of kidney bean at 100 μg/ml, but they inhibited root formation at the lowest part of the cuttings, and the effect was again exhibited most strongly by the endo-2-hydroxy compound. It is suggested that the 2- and 3-hydroxy derivatives possess a potent activity as plant growth retardants.     

Hydroxylation of DHEA and its analogues by Absidia coerulea AM93. Can an inducible microbial hydroxylase catalyze 7α- and 7β-hydroxylation of 5-ene and 5α-dihydro C19-steroids?

In this paper we focus on the course of 7-hydroxylation of DHEA, androstenediol, epiandrosterone, and 5α-androstan-3,17-dione by Absidia coerulea AM93. Apart from that, we present a tentative analysis of the hydroxylation of steroids in A. coerulea AM93. DHEA and androstenediol were transformed to the mixture of allyl 7-hydroxy derivatives, while EpiA and 5α-androstan-3,17-dione were converted mainly to 7α- and 7β-alcohols accompanied by 9α- and 11α-hydroxy derivatives. On the basis of (i) time course analysis of hydroxylation of the abovementioned substrates, (ii) biotransformation with resting cells at different pH, (iii) enzyme inhibition analysis together with (iv) geometrical relationship between the C–H bond of the substrate undergoing hydroxylation and the cofactor-bound activated oxygen atom, it is postulated that the same enzyme can catalyze the oxidation of C₇-H_α as well as C₇-H_β bonds in 5-ene and 5α-dihydro C₁₉-steroids. Correlations observed between the structure of the substrate and the regioselectivity of hydroxylation suggest that 7β-hydroxylation may occur in the normal binding enzyme-substrate complex, while 7α-hydroxylation—in the reverse inverted binding complex.     

Synthesis of 2-bromomethyl-3-hydroxy-2-hydroxymethyl-propyl pyrimidine and theophylline nucleosides under microwave irradiation. Evaluation of their activity against hepatitis B virus

Alkylation of 2-methylthiopyrimidin-4(1H)-one (1a) and its 5(6)-alkyl derivatives 1b-d as well as theophylline (7) with 2,2-bis(bromomethyl)-1,3-diacetoxypropane (2) under microwave irradiation gave the corresponding acyclonucleosides 1-[(3-acetoxy-2-acetoxymethyl-2-bromomethyl)prop-1-yl]-2-methyl-thio pyrmidin-4(1H)-ones 3a-d and 7-[(3-acetoxy-2-acetoxymethyl-2-bromomethyl)prop-1-yl]theophylline (8), which upon further irradiation gave the double-headed acyclonucleosides 1,1 '-[(2,2-diacetoxymethyl)-1,3-propylidene]-bis[(2-(methylthio)-pyrimidin-4(1H)-ones] 4a-c, and 7,7 '-[(2,2-diacetoxymethyl)-1,3-propylidene]-bis(theophylline) (9). The deacetylated derivatives were obtained by the action of sodium methoxide. The activity of deacetylated nucleosides against Hepatitis B virus was evaluated. Compound 5b showed moderate inhibition activity against HBV with mild cytotoxicity.     

Open-chain carbocyclic analogs of adenosine with dihalovinyl unit as potential inhibitors of S-adenosyl-L-homocysteine hydrolase

Vinylogously extended deoxyeritadenine derivatives were synthesized as acyclic/ carbocyclic analogues of the 6'-halo(homovinyl)adenosines, which are known to be potent inhibitors of S-adenosyl-L-homocysteine hydrolase. Swern oxidation of 9-[3-(t-butyldimethylsilyloxy)-4-hydroxybutyl]adenine (4) followed by Wittig olefination and desilylation gave access to ethyl 6-(adenin-9-yl)-4-hydroxy-2(E)-hexenoate (7) and 5-(adenin-9-yl)-1,1-dibromo-1-penten-3-ol (9). No inhibition of AdoHcy Hydrolase was observed with 7 and 9.     

Analysis and quantitation of free ceramide containing nonhydroxy and 2-hydroxy fatty acids, and phytosphingosine by high-performance liquid chromatography.

Reaction of ceramides containing nonhydroxy fatty acids with benzoyl chloride in pyridine at 70 degrees C for 1 hr resulted in N-benzoylation to form N,N-acyl,benzoyl derivatives; O-benzoylation also occurred. However with ceramides containing 2-hydroxy fatty acids and phytosphingosine only O-benzoylation occurred even on prolonged treatment. Only O-benzoylation occurred on reaction with benzoic an hydride. However, the benzoylation of ceramides with phytosphingosine could not be achieved with benzoic anhydride and this benzoylation was performed by reaction with benzoyl chloride at 70 degrees C for 4 hr. Because N,N-acyl,benzoyl derivatives of ceramides containing nonhydroxy fatty acids produced by treatment with benzoyl chloride overlap methyl benzoate on high-performance liquid chromatography, benzoic anhydride was preferable for benzoylation of ceramides with nonhydroxy and 2-hydroxy fatty acids. On the other hand, the reaction with benzoyl chloride at 70 degrees C for 4 hr was used for quantitation of benzoylated ceramides containing 2-hydroxy fatty acids and phytosphingosine. 3-(p-Phenylbenzoyl)estrone was used as an internal standard for both reactions and values for ceramides containing 2-hydroxy fatty acids obtained by the two reactions were in good agreement. This procedure was applied to measurement of the ceramide levels in the brain, liver, and kidney of rats during development. The levels of ceramides containing nonhydroxy and 2-hydroxy fatty acids in the brain, liver, and kidney increased to the adult levels and then remained unchanged. Ceramide with phytosphingosine was detected in the liver and kidney, where its concentration gradually increased with age, but it was not found in the brain. The composition of nonhydroxy fatty acids were also analyzed.     

Regioselective ring opening of exo- and endo-3,4-benzylidene acetals of arabinopyranoside derivatives with Lewis acids and reducing agents

Dioxolane type 3,4-benzylidene acetals of benzyl β-l-arabinose either as a mixture or pure exo- and endo-isomers cleavaged with BF₃·OEt₂/Et₃SiH in dichloromethane or acetonitrile regioselectively, provided the 4-O-benzyl-3-hydroxy derivative. The reaction with TiCl₄/Et₃SiH or Cu(OTf)₂/Et₃SiH provided a mixture of 3- and 4-O-benzyl derivatives whereas with Cu(OTf)₂/BH₃·THF gave only hydrolyzed product. The regioselectivity of the reaction was proved to be directed by the acetyl substitution at C-2. Benzyl substitution provided a mixture of 3- and 4-O-benzyl derivatives in 1:1 ratio whereas non-substitution yielded the same mixture in 2:1 ratio.     

Design and synthesis of 4-HPR derivatives for rhabdoid tumors

Rhabdoid tumors (RTs) are aggressive pediatric malignancies with poor prognosis that arise due to loss of the hSNF5/INI1 tumor suppressor. Molecular studies indicate that cyclin D1, a downstream effector of INI1 is up regulated in RT, and is essential for this tumor formation. Previously we demonstrated that 4-HPR, a synthetic retinoid that targets Cyclin D1, is a potential chemotherapeutic agent for RT. To facilitate further chemical development of this retinoid, and to determine its active moiety, we synthesized small chemical libraries of 4-HPR and tested their cytotoxic effect on RT cells. We synthesized 4-HPR (1) and the derivatives (5a-5n) starting from retinoic acid. First, retinoic acid was converted to acid chloride derivatives, then in the presence of DMF, base, and aniline derivatives, we synthesized the corresponding 4-hydroxy phenyl amine derivatives (5a-5n). This procedure gave 70-90% yield. Then, the 4-HPR derivatives were tested for their ability to inhibit RT cells using an in vitro cell survival assay. We found that the 4-hydroxy group at para-position is essential for cytotoxic activity against RT cells. Furthermore, we identified a few derivatives of 4-HPR with higher cytotoxic potencies than 4-HPR. In addition, we demonstrate that either chloro, fluoro or iodo derivatives at meta-position of phenyl ring retain the cytotoxic activity. Interestingly, substitution of iodo-moiety at meta-position (5j) substantially increased the efficacy (IC(50) approximately 3muM, Fig. 1D). These results indicate that chemical modification of 4-HPR may result in derivatives with increased therapeutic potential for RTs and that halogen substituted 4-HPR that retain the activity can be synthesized for further therapeutic and diagnostic use.     

Structure–Activity Studies of Brassinosteroids and the Search for Novel Analogues and Mimetics with Improved Bioactivity

A number of novel brassinosteroid analogues were synthesized and subjected to the rice leaf lamina inclination bioassay. Modified B-ring analogues included lactam, thiolactone, cyclic ether, ketone, hydroxyl, and exocyclic methylene derivatives of brassinolide. Those derivatives containing polar functional groups retained considerable bioactivity, whereas the exocyclic methylene compounds were devoid of activity. Analogues containing normal alkyl and cycloalkyl substituents at C-24 (in place of the isopropyl group of brassinolide) showed an inverse relationship between activity and chain length or ring size, respectively. The corresponding cyclopropyl and cyclobutyl derivatives were significantly more active than brassinolide and appear to be the most potent brassinosteroids reported to date. When synergized with the auxin indole-3-acetic acid (IAA), their bioactivity can be further enhanced by 1–2 orders of magnitude. The cyclopropyl derivative, when coapplied with the auxin naphthaleneacetic acid, gave a significant increase in yield of wheat in a field trial. Certain 25- and 26-hydroxy derivatives are known metabolites of brassinosteroids. All of the C-25 stereoisomers of 25-hydroxy, 26-hydroxy, and 25,26-dihydroxy derivatives of brassinolide were prepared and shown to be much less active than brassinolide. This indicates that they are likely metabolic deactivation products of the parent phytohormone. A series of methyl ethers of brassinolide was synthesized to block deactivation by glucosylation of the free hydroxyl groups. The most significant finding was that the compound where three of the four hydroxyl groups (at C-3, C-22, and C-23) had been converted to methyl ethers retained substantial bioactivity. This type of modification could, in theory, allow brassinolide or 24-epibrassinolide to resist deactivation and thus offer greater persistence in field applications. A series of nonsteroidal mimetics of brassinolide was designed and synthesized. Two of the mimetics showed significant bioactivity and one had bioactivity comparable to brassinolide, but only when formulated and coapplied with IAA. They thus represent the first nonsteroidal analogues possessing brassinosteroid activity.     

Studies on platelet lipoxygenase specificity towards icosapolyenoic and docosapolyenoic acids

Two docosapolyenoic acids (22:5(n-3) and 22:5(n-6)) were isolated from the liver of normal and 18:3(n-3)-deficient trout, respectively. They were prepared by combined thin-layer chromatography (TLC) and reversed-phase high performance liquid chromatography (HPLC). Their purity, checked by capillary gas liquid chromatography, was greater than 95%. Each fatty acid was oxygenated into monohydroxy derivatives by human platelets. The hydroxy compounds were purified by TLC and HPLC and then derivatized for gas chromatography-mass spectrometry analysis. Whereas 22:5(n-6) was only converted into 14-OH-22:5, three hydroxy derivatives (11, 13 and 14) were obtained from 22:5(n-3). However, 13-hydroxy was not formed in the presence of aspirin, indicating that platelet lipoxygenase catalyses the formation of both 11- and 14-hydroxy derivatives from 22:5(n-3), as described previously, from 22:6(n-3). Further studies showed that 22:4(n-6) and 20:5(n-3) were only converted into 14- and 12-hydroxy derivatives. We conclude then that, besides the well-known n-9 oxygenation, lipoxygenase of human platelets is able to catalyse an n-12 oxygenation on docosapolyenoic acids of the n-3 family.