Synthesis of difluoromethyl substituted lysophosphatidic acid analogues

Lysophosphatidic acid (LPA, 1- or 2-acyl-sn-glycerol 3-phosphate) displays an intriguing cell biology that is mediated via interactions both with G-protein coupled seven transmembrane receptors and with nuclear hormone receptor PPARγ. We describe a new and efficient route to enantiomerically homogeneous lysophospholipid analogues from (S)-1,2,4-butanetriol to give two 3-difluoromethyl substituted analogues of 2-acyl-sn-glycerol 3-phosphate. These compounds are migration-blocked analogues of the liable sn-2 LPA species. Preliminary studies were conducted on a nuclear reporter assay in which monocytic cells were transfected with a luciferase construct activated by a PPARγ nuclear receptor response element and have shown that the 3-difluoromethyl substituted analogues are fully active as natural LPA.     

Design,synthesis and evaluation of bifunctional inhibitors of type II dehydroquinase

Inhibitors of type II dehydroquinase were designed to straddle the two distinct binding sites identified for the inhibitor (1S,3R,4R)-1,3,4-trihydroxy-5-cyclohexene-1-carboxylic acid and a glycerol molecule in a crystallographic study of the Streptomyces coelicolor enzyme. A number of compounds were designed to incorporate characteristics of both ligands. These analogues were synthesized from quinic acid, and were assayed against type I (Salmonella typhi) and type II (S. coelicolor) dehydroquinases. None of the analogues showed inhibition for type I dehydroquinase. Six of the analogues were shown to have inhibition constants in the micromolar to low millimolar range against the S. coelicolor type II dehydroquinase, while two showed no inhibition. The binding modes of the analogues in the active site of the S. coelicolor enzyme were studied by molecular docking with GOLD1.2. These studies suggest a binding mode where the ring is in a similar position to (1S,3R,4R)-1,3,4-trihydroxy-5-cyclohexene-1-carboxylic acid in the crystal structure and the side-chain occupies part of the glycerol binding-pocket.     

Fate of benzoate paralytic shellfish poisoning toxins from Gymnodinium catenatum in shellfish and fish detected by pre-column oxidation and liquid chromatography with fluorescence detection

Several cultured strains of Gymnodinium catenatum isolated worldwide have been shown to produce important proportions of the recently discovered benzoate paralytic shellfish poisoning (PSP) toxins GC1 through GC3. These toxins pose a new challenge for the HPLC analysis of shellfish predating during blooms of this microalga because due to their hydrophobicity are retained along the C18 solid-phase extraction step employed to eliminate interferences. The production of GC toxins was confirmed in a clone of G.catenatum isolated from the Portuguese Northwest coast during the winter bloom of 2005, in addition to a clone from 1989 reported previously by other authors. The major peroxide oxidation products of GC1+2 and GC3 were, respectively, dcGTX2+3 and dcSTX. The search of benzoate analogues in bivalves contaminated during the winter 2005 bloom showed these analogues constituted a minor component of the N(1)-H containing toxins, as selectively detected by peroxide oxidation. While in G.catenatum GC1-3 were the major components after C1+2 and B1, in bivalves dcGTX2+3 and dcSTX were the major components after C1+2 and B1. Similar conclusions were later extended to more shellfish species naturally contaminated during the autumn bloom of 2007. In the gut content of sardines GC toxins were present, while in crabs predating upon shellfish, these were absent. A generalised conversion of GC toxins into decarbamoyl analogues was confirmed by in vitro incubations of bivalve's digestive glands with semi-purified GC toxins. This is the first report of widespread carbamoylase activity in shellfish, exclusively targeted at benzoate PSP analogues and that is heat-inactivated. Despite the high proportion of benzoate analogues produced by G.catenatum, analyses of bivalves contaminated with PSP toxins seem to be simplified due to the important conversion of benzoate into decarbamoyl analogues that occurs in bivalves. These last analogues are detected by common HPLC methods used for food protection.     

Nucleic acid analogues: Synthesis and biomimetic reactions

Several nucleic acid analogues as shown in Scheme 1 were synthesized by alternating copolymerization of nucleoside analogues with maleic anhydride or acrylic anhydride. These polymers showed similar physicochemical properties to those of the natural polymers, such as base-pairing and base-stacking. Polymer 3U, for example, formed 1:1 complex with poly(adenylic acid) by base-pairing. Polymer 4A showed 33% of hypochromicity compared with the UV-absorption of adenosine due to the base-stacking. Polymer 5 catalyzed the cleavage of DNA and the hydrolysis of phosphodiester with a rate acceleration of 10³ compared with the uncatalyzed reaction.     

A practical synthesis of kifunensine analogues as inhibitors of endoplasmic reticulum alpha-mannosidase I

[reaction: see text] A practical synthesis of the potent class I alpha-mannosidase inhibitor kifunensine (1) beginning from the inexpensive and readily available starting material L-ascorbic acid (15) is described. The protected amino-alcohol ((2R,3R,4R,5R)-5-amino-2,3:4,6-diisopropylidenedioxyhexanol, 11) served as a key intermediate from which several N-1 substituted kifunensine analogues (including N-methyl, N-cyclohexyl, and N-bis(hydroxymethyl)methyl) and 2-desoxakifunensine analogues (including N-H and N-methyl) were prepared and screened for inhibition of human endoplasmic reticulum alpha-mannosidase I (ER Man I) and mouse Golgi alpha-mannosidase IA (Golgi Man IA). In addition, several pseudodisaccharide kifunensine analogues in which a mannose residue was tethered to N-1 of kifunensine via a two-, three-, or four-carbon linker and an affinity-bound kifunensine analogue were also prepared and evaluated for biological activity. While the synthesized N-1 kifunesine analogues were found to be less potent inhibitors of Class I alpha-mannosidases than kifuensine itself, the bis(hydroxymethyl)methylkifunensine analogue 6 was shown to selectively inhibit ER Man I over Golgi Man IA.     

Synthesis and Antitumor Activity of Tanshinone Analogues

8, 8-Dimethyl-5, 6, 7,8-tetrahydrophenanthrene-3, 4-dione (3) and 8, 8-dimethyl-2- ( 1-hydroxy ethyl) -5,6, 7, 8-tetrahydrophenanthrene-3,4-dione (4), two analogues of the antitumor active tanshinone, were synthesized from anisole. The synthesized compounds 3 and 4 were shown to be highly active against leukemia P-388 cell fine as assayed by in vitro MTT method.     

Studies on steroidal plant-growth regulator: VII. Synthesis of 2α, 3α-dihydroxy-7-oxa-6-oxo-23, 24-dinor-B-homo-5α-cholanic acid and 2α, 3α-dihydroxy-7-oxa-6-oxo-24-nor-B-homo-5α-cholanic acid

Two brassinolide analogues, (2) and (3), have been synthesized from hyodeoxycholic acid (4) through a sequence of reactions as shown in Scheme 1 and 2 of the text.     

Enzymatic synthesis of beta-mannosyl phosphates on solid support

Synthetic bifunctional analogues 4a, b and 14 of dolichol phosphate 1 were attached to solid support and were shown to be substrates for Dol-P-Man synthase.     

The preparation of novel histrionicotoxin analogues and their activity towards the α4β2 and α7 nicotinic acetylcholine receptors

Four histrionicotoxin analogues were prepared in an efficient manner utilizing a nitrone dipolar cycloaddition reaction as the key step in forming tricyclic intermediate 13. The nitrile in intermediate 13 was reduced with DIBAL to an aldehyde which then underwent Z-selective Wittig reactions to produce intermediates containing the Z-alkene side-chain. Hydrogenation of the Z-alkenes produced saturated histrionicotoxin analogues whereas reduction with SmI₂ afforded the unsaturated histrionicotoxin analogues. The histrionicotoxin analogues were shown to be potent non-competitive antagonists of the α4β2 and α7 nAChR's with the most potent analogue 3 displaying IC₅₀'s of 0.10 μM and 0.45 μM against the α4β2 and α7 nAChR's, respectively. The unsaturated analogues 15 and 18 displayed Hill slope (n_H) of approximately 1 whilst the saturated analogues 16 and 3 had a n_H of approximately 0.5, which may indicate that the saturated analogues are binding to more than one binding site.     

Iso-migrastatin congeners from Streptomyces platensis and generation of a glutarimide polyketide library featuring the dorrigocin, lactimidomycin, migrastatin, and NK30424 scaffolds

Iso-Migrastatin (10) has been shown to be the main natural product of Streptomyces platensis, which undergoes a facile, H2O-mediated rearrangement into dorrigocin A (2), 13-epi-dorrigocin A (11), dorrigocin B (3), and migrastatin (1). Eight new congeners (12-19) of 10 were characterized. They can undergo the same H2O-mediated rearrangement into the corresponding 1, 2, 3, and 11 analogues (20-43) or 1,4-Michael addition with cysteine to afford the corresponding analogues (44-51) of NK30424 A and B (5, 6). This study generated a 47-member library of glutarimide polyketides, setting the stage to investigate the SAR for this family of natural products. These results also established the absolute stereochemistry of 5 and 6 and shed new light into the post-polyketide synthase steps for 10 biosynthesis.     

Investigation of substituent effects on the 1H and 13C NMR spectra of ferrocene analogues of chalcones

The transmission of electronic effects across the ferrocene analogues of chalcones [3-aryl-1-ferrocenyl-2-propene-1-ones (series 1) and 1-aryl-3-ferrocenyl-2-propene-1-ones (series 2)], as well as the conformations of both types of ferrocene analogues have been studied. The ferrocene analogues of chalcones of series 1 were found to be in a non-planar conformation. Their H-α and C-α chemical shifts are more sensitive to the resonance than to the inductive effects of substituents. The C-α chemical shifts of the ferrocene analogues of chalcones of series 2 are more sensitive to the inductive than to the resonance effects of substituents. The transmission of the substituent effects to the ferrocene moiety is also briefly discussed.     

Investigation of the surfactant type and concentration effect on the retention factors of glutathione and its analogues by micellar electrokinetic chromatography

In the present study, a micellar electrokinetic chromatographic method was used to determine the retention factors of hydrophilic monomeric and homodimeric forms of glutathione analogues. Ionic‐liquid‐based surfactant, 1‐tetradecyl‐3‐methylimidazolium chloride, as well as cetyltrimethylammonium bromide and phosphate buffer (pH 7.4) were employed in the experiments. Since the studied peptides possess a negative charge under physiological conditions, it is expected that the peptides interact with the oppositely charged 1‐tetradecyl‐3‐methylimidazolium chloride and cetyltrimethylammonium bromide micelles via hydrophobically assisted electrostatic forces. The dependence of the retention factor on the micellar concentration of 1‐tetradecyl‐3‐methylimidazolium chloride and cetyltrimethylammonium bromide is nonlinear and the obtained curves converge to a limiting value. The retention factor values of GSH analogues were in the range of 0.36–2.22 for glutathione analogues and –1.21 to 0.37 for glutathione when 1‐tetradecyl‐3‐methylimidazolium chloride was used. When cetyltrimethylammonium bromide was employed, the retention factor values were in the range of 0.27–2.17 for glutathione analogues and –1.22 to 0.06 for glutathione. If sodium dodecyl sulfate was used, the retention factor values of glutathione analogues with carnosine moiety were in the range of –1.54 to 0.38.     

Synthesis of natural flutimide and analogous fully substituted pyrazine-2,6-diones, endonuclease inhibitors of influenza virus

Flutimide, a fully substituted 1-hydroxy-3H-pyrazine-2,6-dione, is a fungal metabolite isolated from a new species of Delitschia cofertaspora. It has been shown to selectively inhibit cap-dependent endonuclease activity of influenza virus A. The inhibition of this activity is a target for the potential development of a therapeutic agent to treat influenza infections. A convergent total synthesis of flutimide starting from L-leucine has been described. The synthetic methodology has been extended to include the synthesis of specifically designed aromatic analogues of flutimide, some of which exhibited greater than 7-fold improvement in activity. The most potent compounds were those with p-fluorobenzylidene or p-methoxybenzylidene substitutions at C-5 of 3H-pyrazine-2,6-dione and showed IC(50) values of 0.9 and 0.8 microM, respectively. The details of the rationale for the synthetic design, syntheses, and biological activities of these analogues are described.     

Synthesis and Anticholinesterase Activity of (-)-Physostigmine Analogues with Modifications at C3a and C5

A new series of physostigmine analogues 3a―3j with modifications at the C3a and C5 positions was designed and synthesized. Bioassay of the synthetic analogues 3a―3j, along with the previous synthesized C3a-ethyl-C5-triazole physostigmine analogues 1a―1g and 2a―2j was performed, which indicates that the replacement of the carbamoyl moiety of C3a-ethyl-C5-triazole analogues 1 and 2 with a triazole moiety decreased acetylcholinesterase(AchE) inhibitory activity, whereas the introduction of heterocycles into the triazole ring increased both AChE and butyrylcholinesterase(BchE) inhibitory activities. Structure-activity relationship(SAR) studies of C3a-methyl-C5-triazole analogues 3 reveal the C3a-methyl substituent is important for AChE and BChE inhibition and the introduction of a second ionizable N center improved the binding of the synthetic analogues to both AChE and BChE.     

Synthesis of disparlure analogues,using resolution on microcrystalline cellulose triacetate-I

The gypsy moth, Lymantria dispar, uses a chiral epoxide, (+)-(7R,8S)-2-methyl-7,8-epoxyoctadecane, (+)-disparlure, as its main sex attractant. The moths can detect both enantiomers of disparlure and respond differently to each one. In an effort to understand the structure–activity relationships of the gypsy moth olfactory system, we prepared the analogues of (+)- and (−)-disparlure. The key intermediate in route to the analogues was 2-epoxytridecan-1-ol. Herein we report the resolution of 2-epoxytridecan-1-yl esters on microcrystalline cellulose triacetate and the synthesis of 5-oxa and (5Z)-ene analogues of (+)- and (−)-disparlure. An effort to make 5-aza analogues resulted in the formation of anti-5-(1-hydroxy-1-undecyl)-3-(3-methylbutyl)oxazolidin-2-one. The analogues were tested for their electroantennogram responses and for their ability to bind to pheromone-binding protein 1 (PBP1). We found that the 5-oxa analogues gave strong responses and that the antenna and the PBP1 no longer distinguish the enantiomers of the 5-oxa analogues. The analogues all bound the PBP1 with similar affinity to (−)-disparlure.     

Photoinduced Bergman cycloaromatization of imidazole-fused enediynes

A series of 4,5-bis-(alkyn-1-yl)imidazoles—‘imidazole-fused’ enediynes—were synthesized and their reactivities in photoinduced Bergman cycloaromatization reactions were determined. The more conformationally rigid analogues gave cycloaromatized products in good yields upon irradiation (450 W low-pressure mercury lamp, ambient temperature). A bicyclic analogue (3) was shown to cleave supercoiled plasmid DNA.     

Synthesis of analogues of Eunicea gamma-cembranolides containing cyclic ethers via saponification.

A method for the synthesis of derivatives of the lead structures euniolide (1), 12,13-bisepieupalmerin (2), and eupalmerin acetate (3) containing tetrahydrofuran and tetrahydropyran ring systems was developed on the basis of alkali-induced intramolecular oxacyclizations. Representatives of the new analogues were submitted to the in vitro antitumor cell-line-screening program of the National Cancer Institute (NCI). While it was shown that a variety of structural modifications are possible, these transformations led typically to nontoxic synthetic cembranoids.     

酚酞分子印迹聚合物的制备及特异吸附性能

以泻药酚酞为模板分子,4-乙烯基吡啶为功能单体制备了模板分子和功能单体不同比例的一系列酚酞分子印迹聚合物。利用扫描电镜对聚合物进行了表面形态分析,采用静态平衡实验法研究了聚合物对模板分子及其类似物的吸附行为和选择性识别能力。实验结果表明,所制备的分子印迹聚合物,吸附 3 h 后基本接近最大吸附量,其中模板分子、4-乙烯基吡啶和交联剂的摩尔比为 1∶6∶20的MIP2的印迹因子为 2.30,效果最佳。Scatchard 分析表明, 在所研究的浓度范围内,吸附过程存在两类结合位点,一类高亲和力结合位点的离解常数为Kd1= 0.63 mmol/L,最大表观结合量 Qmax1 = 25.4 umol/g,另一类低亲和力结合位点的离解常数为 Kd2 =3.5 mmol/L,最大表观结合量 Qmax2 = 61.9 umol/g,通过与酚酞类似物质在酚酞分子印迹聚合物上的吸附行为比较,表明对酚酞具有很好的选择性吸附。     

Complex profiles of hydrophobic paralytic shellfish poisoning compounds in Gymnodinium catenatum identified by liquid chromatography with fluorescence detection and mass spectrometry

The presence of hydrophobic analogues of paralytic shellfish poisoning toxins (PSTs) was studied in a Portuguese strain of Gymnodinium catenatum by conventional pre-column oxidation HPLC after a prolonged acetonitrile gradient coupled with fluorescence detection. Prior separation of hydrophobic PSTs analogues from hydrophilic analogues was done by solid-phase extraction (SPE) partitioning on a C18 cartridge. Several unknown oxidation products, with emission spectra similar to known PSTs, appeared after periodate or hydrogen peroxide oxidation. The compounds producing these oxidation products could be grouped into three major sub-groups according to SPE partitioning. The first one eluting with 10 and 20% MeOH, produced the first set of oxidation products observed after the saxitoxin oxidation product. The second one eluting with 30-100% MeOH produced the second set of oxidation products. The third one eluted with acidified 90% MeOH produced the third and last set of oxidation products. Additionally, the oxidation products corresponding to decarbamoyl gonyautoxins and decarbamoyl saxitoxins were also abundant, resulting from ester cleavage of the benzoate side chain of these compounds during the oxidation. Analysis of these fractions by LC-MS demonstrated the second sub-group was constituted by analogues of the 11-hydroxysulfated GC1/GC2, while the third sub-group was constituted by analogues of GC3, which lack the 11-hydroxysulfate. In addition to GC1/GC2 and GC3, novel analogues differing by 16u could be related, respectively, to the N1-hydroxyl analogues of GC1-GC3, designated GC4-GC6. A novel family of GC analogues, differing, by 16u from GC1-GC6, were hypothesized to possess an extra hydroxyl in the benzoate side chain, existing in both N1-hydroxylated and non-N1-hydroxylated variants, and tentatively designated GC1a-GC6a. The first sub-group was hypothesized to constitute an additional novel family of GC analogues with a hydroxysulfate group instead of the hydroxyl group in the benzoate side chain, tentatively designated GC1b-GC6b.     

Synthesis of oxytocin analogues with replacement of sulfur by carbon gives potent antagonists with increased stability

[Chemical reaction: See text] The neuropeptide oxytocin 1 controls mammary and uterine smooth muscle contraction. Atosiban 2, an oxytocin antagonist, is used for prevention of preterm labor and premature birth. However, the metabolic lifetimes of such peptide drugs are short because of in vivo degradation. Facile production of oxytocin analogues with varying ring sizes wherein sulfur is replaced by carbon (methylene or methine) could be achieved by standard solid-phase peptide synthesis using olefin-bearing amino acids followed by on-resin ring-closing metathesis (RCM). These were tested for agonistic and antagonistic uteronic activity using myometrial strips taken from nonpregnant female rats. Peptide 8 showed agonistic activity in vitro (EC50= 1.4 x 10(3) +/- 4.4 x 10(2) nM) as compared to 1 (EC50= 7.0 +/- 2.1 nM). Atosiban analogues 17 (pA2= 7.8 +/- 0.1) and 18 (pA2= 8.0 +/- 0.1) showed substantial activity compared to the parent oxytocin antagonist 2 (pA2= 9.9 +/- 0.3). Carba analogue 35 (pA2= 6.1 +/- 0.1) had an agonistic activity over 2 orders of magnitude less than its parent 3 (8.8 +/- 0.5). A comparison of biological stabilities of 1,6-carba analogues of both an agonist 8 and antagonist 18 versus parent peptides 1 and 2 was conducted. The half-lives of peptides 8 and 18 in rat placental tissue were shown (Table 2) to be greatly improved versus their parents oxytocin 1 and atosiban 2, respectively. These results suggest that peptides 8 and 18 and analogues thereof may be important leads into the development of a long-lasting, commercially available therapeutic for initiation of parturition and treatment of preterm labor.