螺卷毛壳霉代谢产物的抗菌活性研究(英文)

采用管碟法首次对螺卷毛壳霉（Chaetomium cochloides）的含硫次生代谢产物（1—5）进行了体外抗细菌和抗真菌活性测定,并对其构效关系进行了研究。活性测定显示化合物1—5对革兰氏阳性细菌（Staphylococcus aureus）具有较强的抑制活性,其中化合物1的抑菌活性最强。构效关系研究表明位于C-3’和C-6’之间的二硫桥为化合物的抑菌活性中心,C-3与C-11a之间的二硫桥和N-6与O—3’之间的大环结构能够增强活性。     

Metabolism of 1alpha,25-dihydroxyvitamin D(3) in human promyelocytic leukemia (HL-60) cells: in vitro biological activities of the natural metabolites of 1alpha,25-dihydroxyvitamin D(3) produced in HL-60 cells

The secosteroid hormone, 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)], induces differentiation of the human promyelocytic leukemia (HL-60) cells into monocytes/macrophages. At present, the metabolic pathways of 1alpha,25(OH)(2)D(3) and the biologic activity of its various natural intermediary metabolites in HL-60 cells are not fully understood. 1alpha,25(OH)(2)D(3) is metabolized in its target tissues via modifications of both the side chain and the A-ring. The C-24 oxidation pathway, the main side chain modification pathway initiated by hydroxylation at C-24 leads to the formation of the end product, calcitroic acid. The C-23 and C-26 oxidation pathways, the minor side chain modification pathways initiated by hydroxylations at C-23 and C-26 respectively together lead to the formation of the end product, 1alpha,25(OH)(2)D(3)-lactone. The C-3 epimerization pathway, the newly discovered A-ring modification pathway is initiated by epimerization of the hydroxyl group at C-3 to form 1alpha,25-dihydroxy-3-epi-vitamin-D(3). We performed the present study first to examine in detail the metabolism of 1alpha,25(OH)(2)D(3) in HL-60 cells and then to assess the ability of the various natural intermediary metabolites of 1alpha,25(OH)(2)D(3) in inducing differentiation and in inhibiting clonal growth of HL-60 cells. We incubated HL-60 cells with [1beta-(3)H] 1alpha,25(OH)(2)D(3) and demonstrated that these cells metabolize 1alpha,25(OH)(2)D(3) mainly via the C-24 oxidation pathway and to a lesser extent via the C-23 oxidation pathway, but not via the C-3-epimerization pathway. Three of the natural intermediary metabolites of 1alpha,25(OH)(2)D(3) derived via the C-24 oxidation pathway namely, 1alpha,24(R),25-trihydroxyvitamin D(3), 1alpha,25-dihydroxy-24-oxovitamin D(3) and 1alpha,23(S),25-trihydroxy-24-oxovitamin D(3) [1alpha,23(S),25(OH)(3)-24-oxo-D(3)] were almost as potent as 1alpha,25(OH)(2)D(3) in terms of their ability to differentiate HL-60 cells into monocytes/macrophages. We then selected 1alpha,23(S),25(OH)(3)-24-oxo-D(3) which has the least calcemic activity among all the three aforementioned natural intermediary metabolites of 1alpha,25(OH)(2)D(3) to examine further its effects on these cells. Our results indicated that 1alpha,23(S),25(OH)(3)-24-oxo-D(3) was also equipotent to its parent in inhibiting clonal growth of HL-60 cells and in inducing expression of CD11b protein. In summary, we report that 1alpha,25(OH)(2)D(3) is metabolized in HL-60 cells into several intermediary metabolites derived via both the C-24 and C-23 oxidation pathways but not via the C-3 epimerization pathway. Some of the intermediary metabolites derived via the C-24 oxidation pathway are found to be almost equipotent to 1alpha,25(OH)(2)D(3) in modulating growth and differentiation of HL-60 cells. In a previous study, the same metabolites when compared to 1alpha,25(OH)(2)D(3) were found to be less calcemic. Thus, the findings of our study suggest that some of the natural metabolites of 1alpha,25(OH)(2)D(3) may be responsible for the final expression of the noncalcemic actions that are presently being attributed to their parent, 1alpha,25(OH)(2)D(3).     

(3Z)-2-Acetylamino-3-octadecen-1-ol as a potent apoptotic agent against HL-60 cells

(2R,3Z)-, (2R,3E)-, (2S,3Z) and (2S,3E)-2-Acetylamino-3-octadecen-1-ol, and (2R)- and (2S)-2-acetylamino-octadecan-1-ol were prepared using the Wittig olefination of Garner's aldehyde (N-Boc-N,O-isopropylidene-L- or D-serinal) from L- or D-serine. The apoptotic activities of these saturated and unsaturated 2-acetylaminoalcohols were examined in human leukemia HL-60 cells using MTT assay. Among the newly synthesized compounds, the cis-isomers were the most potent. Despite their simple structures, (2R,3Z)- and (2S,3Z)-2-acetylamino-3-octadecen-1-ol showed high and comparable apoptotic activities compared with N-acetyl-D-erythro-sphingosine (D-e-C2-Cer, a well-known inducer of apoptosis). Their apoptotic activities were in the order D-e-C2-Cer approximately L-e-C2-Cer approximately (2R,3Z)- approximately (2S,3Z)->(2R,3E)- approximately (2S,3E)- approximately (2R)- approximately (2S)-derivative. Qualitative analysis of DNA fragmentation caused by these compounds was conducted using agarose gel electrophoresis, and typical DNA fragmentation was found in the cases of (2R,3Z)- and (2S,3Z)-isomers such as C2-Cer, but not trans and saturated isomers. The morphological features of the cells, the proteolytic processing of pro-caspase-3, and the cleavage of PARP as a result of exogenous treatment with (2R,3Z)- and (2S,3Z)-isomers indicated that cell death induced by these compounds was apoptosis. These observations suggest that these newly synthesized compounds, (3Z)-2-Acetylamino-3-octadecen-1-ol, have similar characteristics and apoptosis-inducing activities against HL-60 cells with C2-Cer.     

Two novel engineered bacteria for secretory expression of glutaryl 7-amino-cephalosporanic acid acylase

Two novel engineered bacteria, BL21(DE3)/pETCA1S and TG1/pSuperCA1S, were obtained which can secretory express the gene encoding glutaryl 7-amino-cephalosporanic acid acylase (GL-7ACA acylase) from Pseudomonas sp. 130 with high activity. The growth conditions of transformants for overproduction of GL-7ACA acylase were optimized: in intact cells of BL21(DE3)/pETCA1S and TG1/pSuperCA1S the activity of GL-7ACA acylase was 415 and 600 units g^–1 dry cells, respectively. The highest specific activity of GL-7-ACA acylase is in the intact cell as compared with that of transformants constructed in our laboratory. In fiftieth generation of mutants transferred on agar plates the specific activity of GL-7ACA acylase remained constant.     

Structure-activity relationships of ABA analogs based on their effects on the gas exchange of clonal white spruce (Picea glauca) emblings

ABA analog structure-function relationships were determined by testing an array of 19 different ABA analogs on 1-year-old clonal white spruce ( Picea glauca [Moench.] Voss) raised from somatic embryos. The contribution of specific structural features to analog activity was determined from the relative effect of aeroponically applied analog solutions (10⁻³ M ) on seedling gas exchange. Seedling transpiration rate (E) and carbon assimilation rate (A) were measured continuously during treatment by means of a whole plant cuvette system. The analogs were racemic about the C-1' chiral center and were derived from changes imposed on six regions of the ABA molecule. The activity of optically pure (+)-S-ABA and (−)-R-ABA were also determined. Analog activity was reduced by changing the oxidation level at C-1 from the carboxylic acid. The ring C-2', C-3' double bond was important but not essential to activity. The activity lost through changes in ring structure and C-1 oxidation level was, in many cases, almost fully restored by replacing the C-4, C-5 double bond with a triple bond. Therefore, analogs with a triple bond at C-4 were more active than their equivalents with a dienoic side chain. Fluorination of the C-7' methyl caused a relatively moderate reduction in analog activity. Truncation of C-1 and C-2 from the side chain reduced activity to near zero. The unnatural (−)-ABA enantiomer was inactive.     

Two different sites of action for platelet activating factor and 1-O-alkyl-2-O-methyl-sn-glycero-3-phosphocholine on platelets and leukemic cells.

2-O-Methyl analogs of platelet activating factor (PAF) are potent anticancer agents. The sites of action and mechanisms of cell toxicity of these agents are as yet unknown. To better understand the mode of action of this class of anticancer agents, we examined the ability of 1-O-hexadecyl-2-acetylglycero-3-phosphocholine with the S or R configuration at C2 ((R)-PAF and (S)-PAF) and 1-O-hexadecyl-2-methoxyglycero-3-phosphocholine with the S or R configuration at C2 ((R)-ET-16-OCH3-GPC and (S)-ET-16-OCH3-GPC) to induce rabbit platelet aggregation and to inhibit [3H]thymidine uptake into WEHI-3B cells, HL-60 cells, and normal blood lymphocytes. The four chiral ether-linked lipids caused aggregation of rabbit platelets with the following order of potency: (R)-PAF greater than (S)-PAF greater than (R)-ET-16-OCH3-GPC greater than (S)-ET-16-OCH3-GPC; the EC50 values were 1 pM, 50 nM, 1 microM, and 50 microM, respectively. The cytotoxic effects of these ether lipids in leukemic cells was in reverse order to that observed for aggregation of platelets. The order of potency for inhibition of [3H]thymidine uptake by WEHI-3B and HL-60 cells was (R)-ET-16-OCH3-GPC = (S)-ET-16-OCH3-GPC greater than (S)-PAF greater than (R)-PAF; the EC50 values were 2, 2, 15, and greater than 40 microM, respectively. PAF antagonists (WEB 2086, CV 3988, triazolam, and SRI 63,441) blocked the action of the four ether lipids on platelets, while SRI 63,441 blocked the antineoplastic activity of the ether lipids on WEHI-3B and HL-60 cells. None of the four lipids was able to kill normal lymphocytes significantly. Scatchard analysis of PAF receptor binding revealed that HL-60 and WEHI-3B cells, which are sensitive to the cytotoxic action of ether-linked lipids, do not possess PAF receptors, whereas both normal lymphocytes and platelets do possess a PAF receptor. The present data indicate that the cytotoxic action of antineoplastic ether-linked lipids does not involve the PAF receptor. The protective role of SRI 63,441 in blocking the proaggregatory activity of the ether lipids in rabbit platelets involves PAF receptor, but cytotoxic activity against WEHI-3B and HL-60 cells does not result from its ability to act as a PAF antagonist.     

Diels-Alder type adducts from Morus cathayana.

Two natural Diels-Alder type adducts, cathayanon A (1) and cathayanon B (2), resembling sanggenon C (4) and O (3), were isolated from the root bark of Morus cathayana. Their structures were elucidated on the basis of spectroscopic evidence. The structure of 1 was confirmed by the results of X-ray crystallographic analysis. The absolute configurations of 1 and 2 were determined as 2S, 3R,14S, 19S, 20R and 2S, 3R, 14R, 19S, 20R, respectively. The absolute configurations at C-2 and C-3 of two other known isomeric Diels-Alder adducts sanggenon O (3) and C (4) were deduced as 2R, 3S. Pharmacological tests indicated that 1 and 2 exhibited potent activities on the inhibition of HL-60 cell adhesion to BAEC at concentrations of 10(-5) mol l(-1), with inhibitory rates of 44.72 and 39.02%, respectively.     

Cytotoxic and proapoptotic activity of diterpenoids from in vitro cultivated Salvia sclarea roots. Studies on the leukemia cell lines

Four diterpenoids, ferruginol, salvipisone, aethiopinone and 1-oxoaethiopinone, were isolated from transformed roots of Salvia sclarea. Salvipisone and aethiopinone showed relatively high cytotoxicity against HL-60 and NALM-6 leukemia cells (IC50 range 0.6-7.7 microg/ mL which is equal to 2.0-24.7 microM), whereas 1-oxoaethiopinone and ferruginol were less active in this regard. Moreover, we have found that all four diterpenoids of S. sclarea had equal cytotoxic activity against parental HL-60 and multidrug-resistant HL-60 ADR cells, what indicates that they are poor substrates for transport by multidrug resistance-associated protein (MRP1). Caspase-3 activity determinations showed that salvipisone and aethiopinone were able to induce apoptosis in a time- and concentration-dependent manner. The results obtained in this study show that S. sclarea diterpenoids aethiopinone and salvipisone may be useful in the treatment of human cancers, especially in the case of drug resistance.     

Enantioselective synthesis and cytotoxic evaluation of 4,5-dihydro-5-[aryl(hydroxy)methyl]-3-methylidenefuran-2(3H)-ones

A series of enantiomerically enriched 4,5-dihydro-5-[aryl(hydroxy)methyl]-3-methylidenefuran-2(3H)-ones (8) were synthesized by means of asymmetric Sharpless dihydroxylation of the 2-phosphorylated 5-aryl-pent-4-enoic acids 13, followed by Horner-Wadsworth-Emmons reaction of the resulting furanones 15 (Scheme 2). An enantiomeric excess (ee) of 20-95% was achieved for compounds 8, and their absolute configurations were determined by the Mosher ester method. Cytotoxic evaluation against L-1210 and HL-60 leukemia cell lines revealed that the target compounds 8 are active in the micromolar concentration range (Table 2). Thereby, significant differences in activity between the corresponding enantiomers were observed for the HL-60 cell line.     

Epoxide hydrolase activity of Streptomyces strains

The discovery of epoxide hydrolases within a Streptomyces sp. strain collection is described. Screening was performed in 96 well microtiter plates using a modified 4-(p-nitrobenzyl)pyridine assay with styrene oxide, 1,2-epoxy-hexane or 3-phenyl ethylglycidate (3-PEG) as substrates. Out of 120 strains investigated, S. antibioticus Tü4, S. arenae Tü495 and S. fradiae Tü27 exhibited epoxide hydrolase activity. These strains were further investigated by performing laboratory-scale biotransformations utilizing styrene oxide, 1,2-epoxy-hexane and 3-PEG followed by subsequent quantitative analysis employing chiral gas chromatography. The highest conversions were achieved with whole cells from S. antibioticus Tü4 in the presence of 10% (v/v) DMSO. However, enantioselectivity was only satisfying (E = 31) in the presence of 5% (v/v) acetone, which allowed isolation of optically pure non-hydrolyzed (R)-styrene oxide (99% enantiomeric excess (ee)) and (S)-phenyl-1,2-ethandiol (72% ee) at 55% conversion after 24 h. The resolution of 3-PEG proceeded with slightly lower enantioselectivity albeit higher reaction rates. With S. fradiae Tü27 and S. arenae Tü495 enantioselectivity towards styrene oxide was only E = 3-4.     

GL-7ACA酰化酶表达检测系统的建立

戊二酰－7－氨基头孢烷酸（GL－7ACA）酰化酶能够催化GL－7ACA分解生成7－ACA,后者是工业半合成生产头孢类抗菌素所需的重要前体。为了准确地检测GL－7ACA酰化酶及其突变体的表达,本研究通过构建一系列质粒载体,建立了两个简便有效地测定GL－7ACA酰化酶基因acy表达量的系统,从而可对酶的比活力进行定量。我们将两个报告基因,即儿茶酚双加氧酶基因（xylE)和β－半乳糖苷酶基因（lacZ)分别置于acy基因的下游,使之与acy基因共用一个启动子,进行串联表达,各自构成一个多顺反子系统。实验证明,基因融合后的儿茶酚双加氧酶或β－半乳糖苷酶的活力可以间接反映acy的表达量。     

Novel vitamin D3 derivatives, 26-homo-delta 22-dehydro-1 alpha,25(S)-dihydroxyvitamin D3 and 26-homo-delta 22-dehydro-1 alpha,25(R)-dihydroxyvitamin D3: preferential activity in c-myc mRNA production and in induction of phenotypic differentiation of HL-60 cells

Novel vitamin D3 derivatives, 26-homo-delta 22-1 alpha,25(S)-dihydroxyvitamin D3 and 26-homo-delta 22-1 alpha,25(R)-dihydroxyvitamin D3 were compared with the native hormone, 1,25-dihydroxyvitamin D3, and with other vitamin D3 derivatives, in inhibition of cell growth, induction of phenotypic differentiation, and c-myc mRNA reduction of HL-60 cells. The degree of inhibition in cell growth caused by 26-homo-delta 22-1 alpha,25(S)-(OH)2D3 was the greatest followed by 26-homo-delta 22-1 alpha,25(R)-(OH)2D3. The ability to reduce NBT was parallel to that to inhibit cellular proliferation. 26-homo-delta 22-1 alpha,25(S)-(OH)2D3, 26-homo-delta 22-1 alpha,25(R)-(OH)2D3, 24-homo-24-F2-1 alpha,25-(OH)2D3, and 1 alpha,24(R)-(OH)2-26-Cl-D3 were more active than 1 alpha,25-(OH)2D3 in the induction of OK-M1+ and OK-Mo-2+ HL-60 cells. Using two color flow cytometric analysis, the percentages of OK-M5(+)- and OK-DR(+)-HL-60 cells were 33% in the treatment with 26-homo-delta 22-1 alpha,25(S)-(OH)2D3 plus interferon-gamma (IFN-gamma) but 14% in the treatment with 1 alpha,25-(OH)2D3 plus IFN-gamma. 26-Homo-delta 22-1 alpha,25(S)-(OH)2D3 has an inhibitory effect on c-myc reduction in treated HL-60 cells. These results suggest that the novel vitamin D3 derivatives, 26-homo-delta 22-1 alpha,25(S)-(OH)2D3 and 26-homo-delta 22-1 alpha,25(R)-(OH)2D3, have preferential activity in inducing phenotypic differentiation and in inducing cell proliferation related c-myc mRNA activity.     

Proton and carbon-13 nuclear magnetic resonance spectroscopy of diastereoisomeric 3- and 17 beta-tetrahydropyranyl ether derivatives of estrone and estradiol

Protection of 3- and 17 beta-hydroxyl groups of estrone and estradiol as tetrahydropyranyl ether derivatives led to mixtures of 2'(R)- and 2'(S)-diastereoisomers which were separated by crystallization (3-tetrahydropyranyl ethers), or by thin-layer chromatography (17-tetrahydropyranyl ethers), and characterized by 1H and 13C nuclear magnetic resonance (NMR). Assignments for NMR signals of estradiol 3,17 beta-ditetrahydropyranyl ether were facilitated by comparison with those of its 15 zeta, 16 zeta-dideuterio analog and by 2D 1H-13C heteroshift correlation experiments. Diastereoisomers of 3-tetrahydropyranyl ether derivatives could be identified through the 13C NMR doublet signals of the anomeric C-2' and the aromatic C-4 carbon atoms in CDCl3. Diastereoisomers of 17-tetrahydropyranyl ether derivatives were recognized from characteristic modifications of 1H NMR signals of H-2', H-6', H-1, H-17, and 18-CH3 protons as well as from the 13C NMR doublet signals corresponding to C-2', C-4', C-6', C-12, C-13, C-16, and C-17 carbon atoms. Low-temperature experiments showed a splitting of the C-2', C-6', and C-17 13C NMR signals of each of the two 17-tetrahydropyranyl ether isomers. The downfield signal (equatorial conformer) of the three resulting doublets was more intense for the 17-tetrahydropyranyl ether 2'(S)-isomer, whereas the upfield signal (axial conformer) was more intense for the 2'(R)-isomer.     

Synthesis and biological activity of 22-iodo- and (E)-20(22)-dehydro analogues of 1alpha,25-dihydroxyvitamin D3

Construction of 25-hydroxy-steroidal side chain substituted with iodine at C-22 was elaborated on a model PTAD-protected steroidal 5,7-diene and applied to a synthesis of (22R)- and (22S)-22-iodo-1alpha,25-dihydroxyvitamin D3. Configuration at C-22 in the iodinated vitamins, obtained by nucleophilic substitution of the corresponding 22S-tosylates with sodium iodide, was determined by comparison of their iodine-displacement processes and cyclizations leading to isomeric five-membered (22,25)-epoxy-1alpha-hydroxyvitamin D3 compounds. Also, 20(22)-dehydrosteroids have been obtained and their structures established by 1H NMR spectroscopy. When compared to the natural hormone, (E)-20(22)-dehydro-1alpha,25-dihydroxyvitamin D3 was found 4 times less potent in binding to the porcine intestinal vitamin D receptor (VDR) and 2 times less effective in differentiation of HL-60 cells. 22-Iodinated vitamin D analogues showed somewhat lower in vitro activity, whereas (22,25)-epoxy analogues were inactive. Interestingly, it was established that (22S)-22-iodo-1alpha,25-dihydroxyvitamin D3 was 3 times more potent than its (22R)-isomer in binding to VDR and four times more effective in HL-60 cell differentiation assay. The restricted mobility of the side chain of both 22-iodinated vitamin D compounds was analyzed by a systematic conformational search indicating different spatial regions occupied by their 25-oxygen atoms. Preliminary data on the in vivo calcemic activity of the synthesized vitamin D analogues indicate that (E)-20(22)-dehydro-1alpha,25-dihydroxyvitamin D3 and 22-iodo-1alpha,25-dihydroxyvitamin D3 isomers were ca. ten times less potent than the natural hormone 1alpha,25-(OH)2D3 both in intestinal calcium transport and bone calcium mobilization.     

Short-chain 3-ketoceramides, strong apoptosis inducers against human leukemia HL-60 cells

Ceramides act as a second messenger of the apoptotic signaling process. The allylic alcohol portion comprising the C-3, C-4, and C-5 carbons is essential for this function. The suggestion has been made that this alcohol moiety is oxidized in mitochondria to a carbonyl moiety, with the generation of reactive oxygen species. However, there is no established precedent for the apoptotic performance of 3-ketoceramides thus presumed. In this work, we have synthesized three different types of short-chain 3-ketoceramides, that is, (2S,4E)-2-acetylamino-3-oxo-4-octadecen-1-ol (A), (2S,4E,6E)-2-acetylamino-3-oxo-4,6-octadecadien-1-ol (B), and (2S,4E)-2-acetylamino-1-methoxy-3-oxo-4-octadecene (C), and demonstrated that these 3-ketoceramides are capable of inducing effective apoptosis in human leukemia HL-60 cells. In particular, the two monoenoic compounds, A and C, are far more powerful than the corresponding alcoholic analogue, N-acetyl-D-erythro-sphingosine. Observations of DNA fragmentation, caspase-3 activation, and cytochrome c release from mitochondria provide substantiated evidence for mitochondrial apoptosis and the effects of exogenous glutathione on these phenomena are also discussed.     

A novel series of parenteral cephalosporins exhibiting potent activities against both Pseudomonas aeruginosa and other gram-negative pathogens. Part 2: Synthesis and structure-activity relationships

A novel series of 7beta-[2-(2-amino-5-chloro-thiazol-4-yl)-2(Z)-((S)-1-carboxyethoxyimino)acetamido]cephalosporins bearing various pyridinium groups at the C-3' position were synthesized and their in vitro antibacterial activities against gram-negative pathogens including Pseudomonas aeruginosa and several gram-positive pathogens were evaluated. Among the cephalosporins prepared, we found that a cephalosporin bearing the 2-amino-1-(3-methylamino-propyl)-1H-imidazo[4,5-b]pyridinium group at the C-3' position (8a) showed potent and well-balanced antibacterial activities against P. aeruginosa and other gram-negative pathogens including the strains which produce class C beta-lactamase and extended spectrum beta-lactamase (ESBL). Compound 8a also showed efficacious in vivo activity and high stability against AmpC beta-lactamase. These findings indicate that 2-aminoimidazopyridinium having an aminoalkyl group at the 1-position as a C-3' side chain is suitable for cephalosporins bearing an aminochlorothiazolyl moiety and a carboxyethoxyimino moiety on the C-7 side chain.     

An efficient system for the asymmetric acylation of (R,S)-3-n-butylphthalide catalyzed by novozyme 435

Novozyme 435 could be a highly efficient catalyst in the asymmetric acylation of (R,S)-3-n-butylphthalide in tetrahydrofuran-hexane solvents. The effect of various reaction parameters such as agitation velocity, water content, mixed media, temperature, concentration of Novozyme 435, molar ratio of acetic anhydride to (R,S)-3-n-butylphthalide, reaction time, enantiomeric excess of substrate (ee(S)), enantiomeric excess of product (ee(P)), and enantioselective ratio (E) were studied. Tetrahydrofuran markedly improved (R,S)-3-n-butylphthalide conversion, enantiomeric excess of remaining 3-n-butylphthalide, and enantiomeric ratio. The optimum media were 50% (v/v) tetrahydrofuran and 50% (v/v) hexane. Other ideal reaction conditions were an agitation velocity of 150 rpm, 0.4% (v/v) water content, temperature of 30 °C, 8 mg/mL dosage of Novozyme 435, 8:1 (0.4 mmol: 0.05 mmol) molar ratio of acetic anhydride to (R,S)-3-n-butylphthalide, and a reaction time of 48 hr. Under the optimum conditions, 96.4% ee(S) and 49.3% conversion of (R,S)-3-n-butylphthalide were achieved. In addition, enantiomeric excess of the product was above 98.0%.     

(1'S)-Acetoxychavicol acetate and its enantiomer inhibit tumor cells proliferation via different mechanisms

Elucidation of the mechanisms underlying potential anticancer drugs continues and unraveling these mechanisms would not only provide a conceptual framework for drug design but also promote use of natural products for chemotherapy. The biological effects of (1'S)-acetoxychavicol acetate ((S)-ACA) have been widely investigated. However, in most cases, a natural product or synthetic racemic compound was used in the study. In this study, we prepared (S)-ACA and its enantiomer (R)-ACA by a lipase-catalyzed esterification method and sought to determine the mechanisms of action of (S)-ACA and (R)-ACA in the growth inhibitory effect in Ehrlich ascites tumor cells (EATC). (S)-ACA caused an accumulation of tumor cells in the G1 phase of the cell cycle, which was accompanied by a decrease in phosphorylated retinoblastoma protein (Rb), an increase in Rb and a decrease in the phosphorylation of p27kip1. However, (R)-ACA caused an accumulation of tumor cells in the G2 phase of the cell cycle, an increase in hyperphosphorylated Rb and an increase in the phosphorylation of p27kip1. The results obtained in the present study demonstrate for the first time, to the best of our knowledge, that both (S)-ACA and (R)-ACA caused the inhibition of tumor cells growth but the inhibition was caused via different mechanisms.     

Hydroxylation at C4' or C6 is essential for apoptosis-inducing activity of flavanone through activation of the caspase-3 cascade and production of reactive oxygen species

Previous studies demonstrated that hydroxyl groups play important roles in the antioxidative activities of flavonoids; however, the importance of structurally related hydroxylation in their apoptosis-inducing activities is still undefined. In the present study, flavanone with hydroxylation at C4' and C6 had a significant cytotoxic effect in human leukemia HL-60 cells accompanied by the occurrence of DNA ladders, apoptotic bodies, and hypodiploid cells, characteristics of apoptosis. The replacement of a hydroxyl group (OH) by a methoxyl (OCH3) group at C4' or C6 attenuated the apoptotic effect in cells, and there was no significant cytotocity of flavanone or flavanone with OH or OCH3 in C7-treated HL-60 cells. Induction of enzyme activity of caspase-3 and -9, but not caspase-1 and -8, accompanied by release of cytocrome C from mitochondria to cytosol and the appearance of cleaved of PARP (85 kDa), D4-GDI (23 kDa), and caspase-3 (p17/p15) fragments, was identified in 4'-OH- or 6-OH- flavanone-treated HL-60 cells. Caspase-3 and -9 inhibitors Ac-DEVD-FMK and Ac-LEHD-FMK, but not caspase-1 and -8 inhibitors Ac-YVAD-FMK and Ac-LETD-FMK, attenuated 4'-OH- or 6-OH-flavanone-induced cell death. And, inhibition of capsase-9 activity by Ac-LEHD-FMK suppresses caspase-3 protein procession induced by 4'-OH- and 6-OH-flavanone, indicative of caspase-9 activation locating upstream of caspase-3. A decrease in the antiapoptotic protein Mcl-1 and increases in the pro-apoptotic proteins Bax and Bad were found in 4'-OH- or 6-OH-flavanone-treated HL-60 cells. Induction of endogenous ROS production was detected in 4'-OH- or 6-OH-flavanone-treated HL-60 cells by the DCHF-DA assay. Antioxidants such as N-acetylcysteine (NAC), catalase (CAT), superoxide dismutase (SOD), and allopurinol (ALL), but not pyrrolidine dithiocarbamate (PDTC) or diphenylene iodonium (DPI), significantly inhibited 4'-OH- or 6-OH-flavanone-induced ROS production, with blocking of the apoptosis induced by 4'-OH- or 6-OH-flavanone. The apoptosis-inducing activity of 4'-OH- or 6-OH-flavanone was also observed in another leukemia cell line (Jurkat), but was not found in mature monocytic cells (THP-1) and normal human polymorphonuclear neutrophils (PMNs). This suggests that hydroxylation at C4' or C6 is important to the apoptosis-inducing activities of flavanone through ROS production, and that activation of the caspase-3 cascade, downstream of caspase-9 activation, is involved.     

Induction of monocytic differentiation of HL-60 cells by 1,25-dihydroxyvitamin D analogs

1,25-Dihydroxyvitamin D3, the hormonal form of vitamin D, induces differentiation of HL-60 human promyelocytes into monocyte-like cells in vitro. We assessed the relative activity of 30 analogs of 1,25-dihydroxyvitamin D3 in inducing development of monocytic markers in HL-60 cells. The three differentiation markers assayed were nonspecific acid esterase activity, nitro blue tetrazolium reducing activity, and phagocytic capacity. Of the known metabolites of vitamin D, 1,25-dihydroxyvitamin D3 is the most active; 50% of the cells exhibit the mature phenotype following a 4-day treatment with 10(-8) M 1,25-dihydroxyvitamin D3. Removal of either the C-1 or C-25-hydroxyl group reduces activity by 2 orders of magnitude, while epimerization of the 1 alpha- to 1 beta-hydroxyl group virtually abolishes activity. Elongation of the steroidal side chain of 1,25-dihydroxyvitamin D3 by addition of one carbon at C-24 or C-26 improves the potency by an order of magnitude. Truncation of the steroidal side chain leads to a 10-fold reduction in activity for each carbon removed. Elimination of the C-26 and C-27 methyl groups reduces activity 100-fold. Analogs with short aliphatic side chains as 1 alpha-hydroxyhomo- and bishomopregnacholecalciferol have surprisingly high activity, being only 20-fold less potent than the natural hormone. The activity of most analogs in the HL-60 system parallels their known relative affinities for the well characterized 1,25-dihydroxyvitamin D3 receptor in chick intestine, providing further evidence that this function of 1,25-dihydroxyvitamin D3 is receptor mediated.