The novel pyrimidine and purine derivatives of l-ascorbic acid: synthesis, one- and two-dimensional 1H and 13C NMR study, cytostatic and antiviral evaluation

The syntheses of the novel C-5 substituted pyrimidine derivatives of l-ascorbic acid containing free hydroxy groups at C-2' (6-10) or C-2' and C-3' (11-15) positions of the lactone ring are described. Debenzylation of the 6-chloro- and 6-(N-pyrrolyl)purine derivatives of 2,3-O,O-dibenzyl-l-ascorbic acid (16 and 17) gave the new compounds containing hydroxy groups at C-2' (18) and C-2' and C-3' (19 and 20). Z- and E-configuration of the C4'C5' double bond and position of the lactone ring of the compounds 6-9 were deduced from their one- and two-dimensional (1)H and (13)C NMR spectra and connectivities in NOESY and HMBC spectra. Compounds 15 and 18 showed the best inhibitory activities of all evaluated compounds in the series. The compound 15 containing 5-(trifluoromethyl)uracil showed marked inhibitory activity against all human malignant cell lines (IC(50): 5.6-12.8 microM) except on human T-lymphocytes. Besides, this compound influenced the cell cycle by increasing the cell population in G2/M phase and induced apoptosis in SW 620 and MiaPaCa-2 cells. The compound 18 containing 6-chloropurine ring expressed the most pronounced inhibitory activities against HeLa (IC(50): 6.8 microM) and MiaPaCa-2 cells (IC(50): 6.5 microM). The compound 20 with 6-(N-pyrrolyl)purine moiety showed the best differential inhibitory effect against MCF-7 cells (IC(50): 35.9 microM).     

Studies on 15-hydroxyprostaglandin dehydrogenase with various prostaglandin analogues.

The NAD+-linked 15-hydroxyprostaglandin dehydrogenase (PGDH) of swine lung was purified to a high specific activity by affinity chromatographies on prostaglandin (PG)-and NAD+-Sepharose. The affinities of the enzyme for various synthetic analogues of PGA, E, F, and I and their inhibitory effects on the enzymatic reaction were examined. The modification of the alkyl side chain of PG, particularly at C-15 or C-16, reduced the affinity of the enzyme for these PG analogues. Furthermore, 14-methyl-13,14-dihydro-PGE1 and 16-cyclopentyl-omega-trinor-15-epi-PGE2 were potent inhibitors of PGDH.     

Stereoselective synthesis of some methyl-substituted steroid hormones and their in vitro cytotoxic activity against human gastric cancer cell line MGC-803

A series of 3-, 7-, 15-, and 16-methyl-substituted steroid analogs were synthesized via a highly stereoselective 1,6-conjugate addition. Under the catalysis of CuBr, AlMe₃ reacted with four steroid dienone precursors to afford either the corresponding α-epimer of C-3 and C-7 methyl-substituted steroids as the major products, and the ratio of α/β was up to 10/1. No β-epimer has been detected for methyl addition at C-16. However, under the same reaction conditions, enantioselective methyl addition at C-15 afforded the 15β-epimer as the major product. The preliminary SAR analysis showed that the methyl substituents at C-7α and C-15β positions lead to a dramatical increase in potency against human gastric cancer cell line MGC-803.     

Synthesis of new glycyrrhetinic acid (GA) derivatives and their effects on tyrosinase activity

To synthesize glycyrrhetinic acid (GA) derivatives (3, 4, 5, 10, 13, 14, 15, and 16), we first removed the ketonic group in the C-11 position, and the carboxylic function at the C-30 position was kept intact, reduced to an alcohol, or transformed to an aldehyde corresponding derivatives 10 and 13. Glycyrrhetinic acid (GA) derivatives (3, 4, 5, 15, and 16) were coupled with 4-amino piperpyridine derivatives (12 and 14) and 4-fluorobenzyl bromide at C-30 carboxylic acid position of glycyrrhetinic acid. In subsequent tyrosinase assays, we found that GA derivatives 4, 5, and 16 were not active at early time points, but strongly inhibited tyrosinase activity at late time points. Of the GA derivatives examined, derivative 5 was most active, with an IC₅₀ value of 50 μM after 2 h reaction. IC₅₀ values of derivatives 4 and 16 were 120 and 170 μM, respectively. Further kinetic data indicated that these derivatives are slow-binding inhibitors of tyrosinase. The time-dependent inhibition was reversed when vitamin C or kojic acid was used, that is, both compounds showed active inhibition at early time points. These results suggest that GA derivatives are much more stable than vitamin C or kojic acid, although their intrinsic inhibitory potentials are relatively low. Higher stability and activity suggest that GA derivative 5 might be a useful candidate for skin whitening.     

Mechanistic aspects of rearrangement of 16alpha-hydroxy-17-keto steroids to the 17beta-hydroxy-16-keto isomers

The mechanistic aspects of the alkali-catalyzed rearrangement of 16alpha-hydroxy-17-keto steroid 1 to 17beta-hydroxy-16-keto steroid 2 are elucidated by use of (18)O- and deuterium-labeling experiments. The (18)O-labeling experiments refute the gem-hydration-quasi-diaxial dehydration mechanism for the rearrangement previously proposed and support the conventional enolization mechanism. Moreover, equilibrium by gem-hydration-dehydration occurs at the C-17 carbonyl more efficiently than at the C-16 carbonyl. Enolization rate of a carbonyl group at C-16 of 17beta-ketol 2 toward the C-17 position (k(16,17)) was about 8-10 times higher than those of 16alpha-ketol 1 toward the C-16 position (k(17,16)) and ketol 2 toward the C-15 position (k(16,15)). The marked deuterium-isotope effect on each enolization was observed with k(H)/k(D) ranging between 5.4 and 8.8. The present findings reveal that the initial hydration-dehydration equilibration at the C-17 carbonyl of ketol 1 followed by enolization of the carbonyl gives the ene-diol intermediate that isomerizes quantitatively to the 16-keto isomer of which the 16-carbonyl moiety enolizes preferentially toward the C-17 position rather than the C-15 position, yielding the ene-diol. Computational calculations of ground state energies of ketols 1-M and 2-M, trans-cyclohexane/cyclopentane structures, and their activation energies in the rearrangement support the dynamic aspects of the rearrangement as well as the kinetics data of the enolization.     

Novel Etoposide Analogue Modulates Expression of Angiogenesis Associated microRNAs and Regulates Cell Proliferation by Targeting STAT3 in Breast Cancer

Tumor microenvironment play role in angiogenesis and carcinogenesis. Etoposide, a known topoisomerase II inhibitor induces DNA damage resulting in cell cycle arrest. We developed a novel Etoposide analogue, Quinazolino-4β-amidopodophyllotoxin (C-10) that show better efficacy in regulating cell proliferation and angiogenesis. We evaluated its role on expression of microRNAs-15, 16, 17 and 221 and its targets Bcl-2, STAT3 and VEGF that dictate cell proliferation and angiogenesis. Docking studies clearly demonstrated the binding of Etoposide and C-10 to STAT3. We conclude that combination of Etoposide or C-10 with miR-15, 16, 17 and 221 as a new approach to induce apoptosis and control angiogenesis in breast cancer.     

X-ray structure determination of mexiprostil, a new gastroprotective 16-methoxy-16-methyl-PGE1 analogue

Mexiprostil is a new gastroprotective 16-methoxy-16-methyl-PGE1 methyl ester. To assign the absolute configuration at C-15, a crystalline high-melting C-1 ester analog 5 11,15-dihydroxy-16-methoxy-16-methyl-9-oxoprost-13-en-1-oic acid 4-(4-bromobenzamide)phenyl ester (15R, 16R) was prepared and submitted to single crystal X-ray analysis. Since C-8, C-11, C-12 and C-16 are shown to have R configurations, the X-ray diffraction results established that the configuration at C-15 is also R.     

Alpha-glucosidase and tyrosinase inhibitors from fungal hydroxylation of tibolone and hydroxytibolones

Sixteen new and one known metabolites 4-20 were obtained by incubation of tibolone (1) and hydroxytibolones (2 and 3) with various fungi. Their structures were elucidated by means of a homo and heteronuclear 2D NMR and by HREI-MS techniques. The relative stereochemistry was deduced by 2D NOESY experiment. Metabolites of tibolone (1) exhibited significant inhibitory activities against α-glucosidase and tyrosinase enzymes. Hydroxylations at C-6, C-10, C-11, C-15 positions and α,β-unsaturation at C-1/C-2, C-4/C-5 showed potent inhibitory activities against these enzymes.     

Phragmalin-type limonoids with NF-κB inhibition from Chukrasia tabularis var. velutina

Chemical investigation on Chukrasia tabularis var. velutina led to the identification of eight new phragmalin-type limonoids (1–8), as well as 20 known analogues. Compounds 1–4 are a rare class of C-15-acyl phragmalin-type limonoids, and particularly compounds 2–4 also possess a δ-lactone ring formed between C-16 and C-30. All the isolates were evaluated for inhibitory effects on NF-κB production, and four of which showed significant inhibitions.     

500-MHz proton NMR studies of the medium-dependent conformational preference of prostaglandin F2 alpha analogues

The complete assignments of the 1H NMR spectra of 2-10 mM D2O solutions of prostaglandin F2 alpha (PGF2 alpha), its C-15 epimer, and analogues bearing a gem-dimethyl group at C-16 or C-17 are presented. PGF2 alpha and its 1,9- and 1,15-lactones were similarly studied in CDCl3 solution. The assignments follow from extensive scalar decoupling and difference NOE spectra and the examination of a specifically deuterated analogue. These studies also define the conformation (including cyclopentane pseudorotational preference) from C-5 through C-16 in each system. The macrolides show little or no conformational freedom at C-4----C-1, but extensive rotational averaging occurs in the terminal portions of both side chains in the monocyclic compounds. The conformational features so determined are contrasted to those seen in crystal structures and those postulated to occur upon binding to PGF2 alpha-recognizing receptors. The NMR data run counter to the DeTitta hypothesis that changes in the orientation of the C-13,14 pi-bond nodal plane relative to the cyclopentane ring and the C-15-O bond are recognition determinants at PGF2 alpha-specific receptors and account for the medium-dependent chiroptical spectral changes previously reported.     

In vitro modulation of human and murine melanoma growth by prostanoid analogues

The inhibitory effect of various prostaglandin analogues on the anchorage independent growth of murine and human melanoma cells was measured. PGA analogues (which were modified at C-16 and C-18) did not demonstrate any major improvement in activity over PGA alone. These included 16, 16-dimethyl PGA₁, 16,16-dimethyl-PGA₂, 16,16-dimethyl-18-oxa-PGA₂ and trans-δ-2-15-α acetoxy-16,16-dimethyl-18-oxa-11-deoxy-PGE₁-methylester. The thromboxane synthetase inhibitor, U51605, demonstrated weak anti-proliferative activity. PGD₂ (with a ketone at C-11 versus C-9 for PGA and PGE) was the most potent prostaglandin tested. Cells from melanoma lines displayed species differences in their sensitivities. PGA₁ and PGE₁ were the most potent inhibitors of the anchorage independent growth of murine melanoma cells. On human melanoma cells PGD₂ was the most active prostaglandin, 2–3 times more potent than PGA₁; PGE₁ was a very weak inhibitor.     

New Nystatin-Related Antifungal Polyene Macrolides with Altered Polyol Region Generated via Biosynthetic Engineering of Streptomyces noursei

Polyene macrolide antibiotics, including nystatin and amphotericin B, possess fungicidal activity and are being used as antifungal agents to treat both superficial and invasive fungal infections. Due to their toxicity, however, their clinical applications are relatively limited, and new-generation polyene macrolides with an improved therapeutic index are highly desirable. We subjected the polyol region of the heptaene nystatin analogue S44HP to biosynthetic engineering designed to remove and introduce hydroxyl groups in the C-9-C-10 region. This modification strategy involved inactivation of the P450 monooxygenase NysL and the dehydratase domain in module 15 (DH15) of the nystatin polyketide synthase. Subsequently, these modifications were combined with replacement of the exocyclic C-16 carboxyl with the methyl group through inactivation of the P450 monooxygenase NysN. Four new polyene macrolides with up to three chemical modifications were generated, produced at relatively high yields (up to 0.51 g/liter), purified, structurally characterized, and subjected to in vitro assays for antifungal and hemolytic activities. Introduction of a C-9 hydroxyl by DH15 inactivation also blocked NysL-catalyzed C-10 hydroxylation, and these modifications caused a drastic decrease in both antifungal and hemolytic activities of the resulting analogues. In contrast, single removal of the C-10 hydroxyl group by NysL inactivation had only a marginal effect on these activities. Results from the extended antifungal assays strongly suggested that the 9-hydroxy-10-deoxy S44HP analogues became fungistatic rather than fungicidal antibiotics.     

Structural requirements for the inhibitory action of 12,13-epoxytrichothecenes on protein synthesis in eukaryotes

1. The inhibitory actions of ten trichothecene antibiotics were investigated, in reticulocyte cell-free systems synthesizing protein in vitro, by studying polyribosome profiles and kinetics of amino acid incorporation in the presence or absence of the drugs. 2. The modes of action observed were critically dependent on the drug concentrations used, but the antibiotics tested could be divided into four distinct groups, each exerting a characteristic inhibitory response. 3. The inhibitory action observed in every case was controlled by the chemical structure of the individual trichothecene and in particular was closely related to the nature of the substituent groups present on C-3, C-4, C-8 and C-15 of the molecule.     

解淀粉芽孢杆菌Q-426 Bacillomycin D的分离纯化及其抗肿瘤活性

环脂肽化合物因其独特的结构特点和生物活性在医药领域具有广泛的应用前景。本研究旨在从解淀粉芽孢杆菌Q-426发酵液中分离纯化获得高纯度的环脂肽单体,并对分离得到的环脂肽C-15 Bacillomycin D和C-16Bacillomycin D的抗肿瘤活性及机制进行初步研究。首先,联合使用酸沉淀和双树脂层析去除大量杂质,然后通过制备型HPLC分离得到纯度较高的两种环脂肽,经ESI-MS/MS对其进行结构鉴定,分别确定为C-15Bacillomycin D与C-16 Bacillomycin D。其次,将两种环脂肽单体及其1:1混合液(摩尔比)按不同浓度梯度作用于人癌细胞(Hela、MG、Hep-G2、HT-29),结果表明环脂肽对Hela与MG细胞增殖的抑制作用呈浓度依赖性,长链环脂肽的抑制率较短链环脂肽略高。最后,采用细胞划痕实验和PI染色法考察了C-16 Bacillomycin对细胞侵袭与迁移能力、细胞凋亡和周期的影响,结果显示,C-16 Bacillomycin D可有效影响细胞的迁移能力,诱导细胞凋亡呈浓度依赖性,且阻滞细胞G_0G_1期。     

Green tea catechins as a BACE1 (beta-secretase) inhibitor

In the course of searching for BACE1 (beta-secretase) inhibitors from natural products, the ethyl acetate soluble fraction of green tea, which was suspected to be rich in catechin content, showed potent inhibitory activity. (-)-Epigallocatechin gallate, (-)-epicatechin gallate, and (-)-gallocatechin gallate were isolated with IC(50) values of 1.6 x 10(-6), 4.5 x 10(-6), and 1.8 x 10(-6) M, respectively. Seven additional authentic catechins were tested for a fundamental structure-activity relationship. (-)-Catechin gallate, (-)-gallocatechin, and (-)-epigallocatechin significantly inhibited BACE1 activity with IC(50) values of 6.0 x 10(-6), 2.5 x 10(-6), and 2.4 x 10(-6) M, respectively. However, (+)-catechin, (-)-catechin, (+)-epicatechin, and (-)-epicatechin exhibited about ten times less inhibitory activity. The stronger activity seemed to be related to the pyrogallol moiety on C-2 and/or C-3 of catechin skeleton, while the stereochemistry of C-2 and C-3 did not have an effect on the inhibitory activity. The active catechins inhibited BACE1 activity in a non-competitive manner with a substrate in Dixon plots.     

Biotransformation of dehydroabietic acid with resting cell suspensions and calcium alginate-immobilized cells of Mortierella isabellina

Mortierella isabellina ATCC 38063 is a zygomycete capable of hydroxylating fish-toxic resin acids which occur in certain pulp mill effluents to nontoxic metabolites. Addition of dehydroabietic acid (1) (80 mg/liter) to a freshly inoculated culture of M. isabellina in dextrose-yeast extract broth resulted in precursor disappearance in 28 to 30 h. During growth phase, hydroxylation occurred at C-2, whereas hydroxylation at C-15 and C-16 commenced with onset of stationary phase. Alternatively, 1 added to stationary-phase culture (40 mg/liter) disappeared within 2 h and hydroxylation occurred concurrently at C-2, C-15, and C-16. Enzymatic activity of stationary-phase culture was totally cell associated and was present despite the absence of 1 during the preparatory growth phase. Resuspension of mature fungi as free mycelia or immobilized in calcium alginate beads did not diminish the effectiveness of the biotransformation, although two new metabolites, 15-hydroxy-8,9,11,12-tetradehydro-7,8-dihydroabietic acid (5) and 16-hydroxy-8,9,11,12-tetradehydro-7,8-dihydroabietic acid (7) were formed. Immobilized mycelia retained hydroxylase activity for greater than 110 days whether or not they were challenged with fresh 1 on a regular basis. In this respect they are more long-lived than resuspended free mycelia are.     

Biotransformation of dehydroabietic acid with resting cell suspensions and calcium alginate-immobilized cells of Mortierella isabellina.

Mortierella isabellina ATCC 38063 is a zygomycete capable of hydroxylating fish-toxic resin acids which occur in certain pulp mill effluents to nontoxic metabolites. Addition of dehydroabietic acid (1) (80 mg/liter) to a freshly inoculated culture of M. isabellina in dextrose-yeast extract broth resulted in precursor disappearance in 28 to 30 h. During growth phase, hydroxylation occurred at C-2, whereas hydroxylation at C-15 and C-16 commenced with onset of stationary phase. Alternatively, 1 added to stationary-phase culture (40 mg/liter) disappeared within 2 h and hydroxylation occurred concurrently at C-2, C-15, and C-16. Enzymatic activity of stationary-phase culture was totally cell associated and was present despite the absence of 1 during the preparatory growth phase. Resuspension of mature fungi as free mycelia or immobilized in calcium alginate beads did not diminish the effectiveness of the biotransformation, although two new metabolites, 15-hydroxy-8,9,11,12-tetradehydro-7,8-dihydroabietic acid (5) and 16-hydroxy-8,9,11,12-tetradehydro-7,8-dihydroabietic acid (7) were formed. Immobilized mycelia retained hydroxylase activity for greater than 110 days whether or not they were challenged with fresh 1 on a regular basis. In this respect they are more long-lived than resuspended free mycelia are.     

Microbial transformation of glycyrrhetinic acid derivatives by Bacillus subtilis ATCC 6633 and Bacillus megaterium CGMCC 1.1741

Glycyrrhetinic acid (GA), the major bioactive pentacyclic triterpene aglycone of licorice root, was known to play a vital role in anti-ulcer, anti-depressant, anti-inflammatory, and anti-allergic. In this study, we semi-synthesized five GA derivatives by a series of chemical reactions. They were selected as substrates for the biotransformation and yielded thirteen metabolites by Bacillus subtilis ATCC 6633 and Bacillus megaterium CGMCC 1.1741. Their structures were identified on the basis of extensive spectroscopic methods and nine of them were found for the first time. Two main types of reactions, regio- and stereo-selective hydroxylation and glycosylation, especially in the unactivated C-H bonds including C-11, C-19 and C-27, were observed in the biotransformation process, which greatly expand the chemical diversities of GA derivatives. All compounds were tested for their inhibitory effects on nitric oxide (NO) generation in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Among them, olean-12-ene-3β,7β,15α,19α,30-pentol (16) and olean-12-ene-3β,7β,15α,27,30-pentol (17) showed significant inhibitory effect with IC₅₀ values of 0.64 and 0.07 μM, respectively.     

A peptide based on the sequence of the CDR3 of a murine anti-DNA mAb is a better modulator of experimental SLE than its single amino acid-substituted analogs

A peptide based on the complementarity determining region (CDR) 3 of a pathogenic anti-DNA 16/6 Id(+) monoclonal antibody was previously shown to be a dominant T-cell epitope in experimental SLE, and to be capable of inhibiting SLE-associated proliferative responses. Single amino acid-substituted analogs of pCDR3 were designed and analyzed for their ability to stimulate or inhibit the proliferation of a pCDR3-specific T-cell line. Alterations in positions 9 and 10 neutralized the proliferative potential of pCDR3, whereas alterations in positions 6-8 and 11-15 retained the proliferative potential of the peptides. Similar to pCDR3, its analogs Ala11 and Nle13 inhibited efficiently the in vivo priming of lymph node cells either to pCDR3 or to the human monoclonal anti-DNA 16/6 Id(+) antibody. Substituting both positions 11 (Tyr --> Ala) and 13 (Met --> Nle) reduced this inhibitory capacity compared to the single substituted analogs. Also, truncation of pCDR3 at the C- and/or N-terminus obliterated the inhibitory activities of the peptide. Analogs Ala11 and Nle13 immunomodulated serological and clinical smanifestations of experimental SLE. Nevertheless, the original pCDR3 was a more efficient modulator of the disease.     

Inhibitors of rabbit plasma prostaglandin A isomerase

1. The potent inhibitory activities of three groups of prostaglandin analogues on the prostaglandin A isomerase of rabbit plasma were demonstrated. 2. Six of the compounds were prepared by NaBH(4) reduction of the C-9 oxo groups of prostaglandin A(2) and prostaglandin C(2) and their C-15 epimers. The remaining four were racemates and were synthesized in another laboratory. Unknown configurations at C-9 and at C-15 were assigned. 3. All the compounds were found to be competitive inhibitors of the isomerase in vitro. K(m)/K(i) ratios were determined and it was found that both the 15(S) and 15(R) epimers have potent inhibitory activity. 4. One of the inhibitors was used to study the reversibility of the isomerase. 5. It is suggested that these compounds may be useful for determining the biological significance of prostaglandin A isomerase. In view of its weak biological activity and possibly extended half-life in vivo, the reduction product of 15-epiprostaglandin C(2) may be the most suitable agent for this purpose.