Insights into radicicol biosynthesis via heterologous synthesis of intermediates and analogs

Resorcylic acid lactones are fungal polyketides that display diverse biological activities, with the potent Hsp90 inhibitor radicicol being an important representative member. Two fungal iterative polyketide synthases (IPKSs), Rdc5, the highly reducing IPKS, and Rdc1, the nonreducing IPKS, are required for the biosynthesis of radicicol in Pochonia chlamydosporia. In this study, the complete reconstitution of Rdc5 and Rdc1 activities both in vitro and in Saccharomyces cerevisiae uncovered the earliest resorcylic acid lactone intermediate of the radicicol biosynthetic pathway, (R)-monocillin II. The enzymatic synthesis of (R)-monocillin II confirmed the exquisite timing of the Rdc5 enoyl reductase domain. Using precursor-directed biosynthesis, the chemical modularity of the dual IPKS system was determined. Rdc1 readily accepted an N-acetylcysteamine thioester mimic of the reduced pentaketide product of Rdc5 to synthesize (R)-monocillin II with four additional iterations of polyketide elongation, indicating the C2' ketone group found in (R)-monocillin II is incorporated via the functions of Rdc1 instead of Rdc5. The involvement of the thioesterase domain in Rdc1 in macrolactonization was confirmed through both site-directed mutagenesis and domain deletion. The Rdc1 thioesterase domain was also shown to be tolerant of the opposite stereochemistry of the terminal hydroxyl nucleophile, demonstrated in the precursor-directed synthesis of the enantiomeric (S)-monocillin II. Finally, reconstitution of the halogenase Rdc2 was demonstrated both in vivo and in vitro in the synthesis of pochonin D and a new halogenated analog 6-chloro, 7',8'-dehydrozearalenol.     

Design,synthesis and evaluation against Mycobacterium tuberculosis of azole piperazine derivatives as dicyclotyrosine (cYY) mimics

Three series of azole piperazine derivatives that mimic dicyclotyrosine (cYY), the natural substrate of the essential Mycobacterium tuberculosis cytochrome P450 CYP121A1, were prepared and evaluated for binding affinity and inhibitory activity (MIC) against M. tuberculosis. Series A replaces one phenol group of cYY with a C3-imidazole moiety, series B includes a keto group on the hydrocarbon chain preceding the series A imidazole, whilst series C explores replacing the keto group of the piperidone ring of cYY with a CH₂-imidazole or CH₂-triazole moiety to enhance binding interaction with the heme of CYP121A1. The series displayed moderate to weak type II binding affinity for CYP121A1, with the exception of series B 10a, which displayed mixed type I binding. Of the three series, series C imidazole derivatives showed the best, although modest, inhibitory activity against M. tuberculosis (17d MIC?=?12.5?μg/mL, 17a 50?μg/mL). Crystal structures were determined for CYP121A1 bound to series A compounds 6a and 6b that show the imidazole groups positioned directly above the haem iron with binding between the haem iron and imidazole nitrogen of both compounds at a distance of 2.2?Å. A model generated from a 1.5?Å crystal structure of CYP121A1 in complex with compound 10a showed different binding modes in agreement with the heterogeneous binding observed. Although the crystal structures of 6a and 6b would indicate binding with CYP121A1, the binding assays themselves did not allow confirmation of CYP121A1 as the target.     

Genomic Mining for Novel FADH2-Dependent Halogenases in Marine Sponge-Associated Microbial Consortia

Many marine sponges (Porifera) are known to contain large amounts of phylogenetically diverse microorganisms. Sponges are also known for their large arsenal of natural products, many of which are halogenated. In this study, 36 different FADH₂-dependent halogenase gene fragments were amplified from various Caribbean and Mediterranean sponges using newly designed degenerate PCR primers. Four unique halogenase-positive fosmid clones, all containing the highly conserved amino acid motif “GxGxxG”, were identified in the microbial metagenome of Aplysina aerophoba. Sequence analysis of one halogenase-bearing fosmid revealed notably two open reading frames with high homologies to efflux and multidrug resistance proteins. Single cell genomic analysis allowed for a taxonomic assignment of the halogenase genes to specific symbiotic lineages. Specifically, the halogenase cluster S1 is predicted to be produced by a deltaproteobacterial symbiont and halogenase cluster S2 by a poribacterial sponge symbiont. An additional halogenase gene is possibly produced by an actinobacterial symbiont of marine sponges. The identification of three novel, phylogenetically, and possibly also functionally distinct halogenase gene clusters indicates that the microbial consortia of sponges are a valuable resource for novel enzymes involved in halogenation reactions.     

Sulfoximine-substituted trifluoromethylpyrimidine analogs as inhibitors of proline-rich tyrosine kinase 2 (PYK2) show reduced hERG activity

The synthesis, in vitro properties, and in vivo pharmacokinetics for a series of sulfoximine-substituted trifluoromethylpyrimidines as inhibitors of proline-rich tyrosine kinase, a target for the possible treatment of osteoporosis, are described. These compounds were prepared as surrogates of the corresponding sulfone compound 1. Sulfone 1 was an attractive PYK2 lead compound; however, subsequent studies determined this compound possessed high dofetilide binding, which is an early indicator of cardiovascular safety. Surprisingly, the corresponding sulfoximine analogs displayed significantly lower dofetilide binding, which, for N-methylsulfoximine (S)-14a, translated to lower activity in a patch clamp hERG K⁺ ion channel screen. In addition, compound (S)-14a shows good oral exposure in a rat pharmacokinetic model.     

Discovery of a novel activator of 5-lipoxygenase from an anacardic acid derived compound collection

Lipoxygenases (LOXs) and cyclooxygenases (COXs) metabolize poly-unsaturated fatty acids into inflammatory signaling molecules. Modulation of the activity of these enzymes may provide new approaches for therapy of inflammatory diseases. In this study, we screened novel anacardic acid derivatives as modulators of human 5-LOX and COX-2 activity. Interestingly, a novel salicylate derivative 23a was identified as a surprisingly potent activator of human 5-LOX. This compound showed both non-competitive activation towards the human 5-LOX activator adenosine triphosphate (ATP) and non-essential mixed type activation against the substrate linoleic acid, while having no effect on the conversion of the substrate arachidonic acid. The kinetic analysis demonstrated a non-essential activation of the linoleic acid conversion with a K_A of 8.65 μM, αK_A of 0.38 μM and a β value of 1.76. It is also of interest that a comparable derivative 23d showed a mixed type inhibition for linoleic acid conversion. These observations indicate the presence of an allosteric binding site in human 5-LOX distinct from the ATP binding site. The activatory and inhibitory behavior of 23a and 23d on the conversion of linoleic compared to arachidonic acid are rationalized by docking studies, which suggest that the activator 23a stabilizes linoleic acid binding, whereas the larger inhibitor 23d blocks the enzyme active site.     

Synthesis and biological evaluation of phenyl-1H-1,2,3-triazole derivatives as anti-inflammatory agents

Rapid and efficient synthesis of a phenyl-1H-1,2,3-triazole library enabled cost-effective biological testing of a range of novel non-steroidal anti-inflammatory drugs with potential for improved drug efficacy and toxicity profiles. Anti-inflammatory activities of the phenyl-1H-1,2,3-triazole analogs synthesized in this report were assessed using the xylene-induced ear edema model in mice. At least four analogs, 2a, 2b, 2c, and 4a, showed more potent effects than the reference anti-inflammatory drug diclofenac at the same dose of 25 mg/kg. To explore relationships between the structural properties of phenyl-1H-1,2,3-triazole analogs and their anti-inflammatory activities in xylene-induced ear edema, comparative molecular field analysis was performed, and pharmacophores showing good anti-inflammatory activities were identified based on an analysis of contour maps obtained from comparative molecular field analysis. The anti-inflammatory effect on the molecular level was tested by the expression of tumor necrosis factor-alpha induced COX-2 using Western blots. Because the addition of the analog 2c caused the expression change of TNF-α induced COX-2, the molecular binding mode between 2c and COX-2 was elucidated using in silico docking.     

Enantioselective resolution of side-chain modified gem-difluorinated alcohols catalysed by Candida antarctica lipase B and monitored by capillary electrophoresis

An enzymatic alternative to the chemical synthesis of chiral gem-difluorinated alcohols has been developed. The method is highly effective and stereoselective, feasible at laboratory temperature, avoiding the use of toxic heavy metal catalysts which is an important benefit in medicinal chemistry including the synthesis of drugs and drug precursors. Candida antarctica lipases A and B were applied for the enantioselective resolution of side-chain modified gem-difluorinated alcohols, (R)- and (S)-3-benzyloxy-1,1-difluoropropan-2-ols (1a and 1b), compounds serving as chiral building blocks in the synthesis of various bioactive molecules bearing a gem-difluorinated grouping. The catalytic activity of these lipases was investigated for the chiral acetylation of 1a and 1b in non-polar solvents using vinyl acetate as an acetyl donor. The dependence of the reaction course on various substrate and enzyme concentrations, reaction time, and temperature was monitored by chiral capillary electrophoresis (CE) using sulfobutyl ether β-cyclodextrin as a stereoselective additive of the aqueous background electrolyte. The application of CE, NMR, and MS methods has proved that the complex enzyme effect of Candida antarctica lipase B leads to the thermodynamically stable (S)-enantiomer 1b instead of the expected acetylated derivatives. In contrast, the enantioselective acetylation of racemic alcohol 1 was observed as a kinetically controlled process, where (R)-enantiomer 1a was formed as the main product. This process was followed by enzymatic hydrolysis and chiral isomerisation. Finally, single pure enantiomers 1a and 1b were isolated and their absolute configurations were assigned from NMR analysis after esterification with Mosher’s acids.     

来源卤化二型聚酮类生物合成基因簇的两种关键功能基因鉴定与表达

为催化卤化产物,挖掘具有生物活性的新卤化物提供新的方法途径,拟构建卤化酶原核表达载体催化天然卤化物的卤化过程.对链霉菌Streptomyces sp.FJS31-2基因组中一个II型PKS类产物zunyimycin生物合成基因簇进行系列分析,针对其中的后修饰酶卤化酶基因和单加氧酶基因进行原核表达纯化.通过分子生物学手段...     

Antiproliferative activities of halogenated thieno[3,2-d]pyrimidines

The in vitro evaluation of thieno[3,2-d]pyrimidines identified halogenated compounds 1 and 2 with antiproliferative activity against three different cancer cell lines. A structure activity relationship study indicated the necessity of the chlorine at the C4-position for biological activity. The two most active compounds 1 and 2 were found to induce apoptosis in the leukemia L1210 cell line. Additionally, the compounds were screened against a variety of other microbial targets and as a result, selective activity against several fungi was also observed. The synthesis and preliminary biological results are reported herein.     

3-(Cyclopropylmethyl)-7-((4-(4-[11C]methoxyphenyl)piperidin-1-yl)methyl)-8-(trifluoromethyl)-[1,2,4]triazolo[4,3-a]pyridine: Synthesis and preliminary evaluation for PET imaging of metabotropic glutamate receptor subtype 2

Selective metabotropic glutamate receptor 2 (mGluR2) inhibitors have been demonstrated to show therapeutic effects by improving alleviating symptoms of schizophrenic patients in clinical studies. Herein we report the synthesis and preliminary evaluation of a ¹¹C-labeled positron emission tomography (PET) tracer originating from a mGluR2 inhibitor, 3-(cyclopropylmethyl)-7-((4-(4-methoxyphenyl)piperidin-1-yl)methyl)-8-(trifluoromethyl)-[1,2,4]triazolo[4,3-a]pyridine (CMTP, 1a). [¹¹C]CMTP ([¹¹C]1a) was synthesized by O-[¹¹C]methylation of desmethyl precursor 1b with [¹¹C]methyl iodide in 19.7 ± 8.9% (n = 10) radiochemical yield (based on [¹¹C]CO₂) with >98% radiochemical purity and >74 GBq/μmol molar activity. Autoradiography study showed that [¹¹C]1a possessed moderate in vitro specific binding to mGluR2 in the rat brain, with a heterogeneous distribution of radioactive accumulation in the mGluR2-rich brain tissue sections, such as the cerebral cortex and striatum. PET study indicated that [¹¹C]1a was able to cross the blood–brain barrier and enter the brain, but had very low specific binding in the rat brain. Further optimization for the chemical structure of 1a is necessary to increase binding affinity to mGluR2 and then improve in vivo specific binding in brain.     

Interaction of 2-deamino- and 2-deamino-2-nitroactinomycin D with calf-thymus DNA and formation of ion-radicals

2-Deaminoactinomycin D (3a) and 2-deamino-2-nitroactinomycin D (2a) were prepared in one step from actinomycin D (1a, AMD) by reaction with nitrous acid. New DNA-binding (calf-thymus) data obtained by difference uv and CD spectra and ΔTm were presented. In vitro cell growth inhibitory activity of CCRF-CEM cells was also reported. The 2-deamino analog, 3a, does not bind to DNA strongly nor by intercalation of its chromophore. However, some binding with DNA was indicated by CD which is attributed only to hydrogen bondings of the peptides with the DNA helix; the affinity for binding is in the order 1a ? 2a > 3a. The 2-nitro analog, 2a, is a more potent agent against CCRF-CEM cells than the 2-deaminoactinomycin D, 3a; the potencies are in the order 1a > 2a ? 3a. Furthermore, the microsomes activate the analogs to free radical states which catalyze the production of superoxide, as indicated by electron paramagnetic resonance studies and oxygen consumption experiments.     

Highly potent activity of isopulegol-derived substituted octahydro-2H-chromen-4-ols against influenza A and B viruses

A set of (?)-isopulegol derived octahydro-2H-chromen-4-ols was synthesized and evaluated in vitro for antiviral activity against panel of reference influenza virus strains differing in subtype, origin (human or avian) and drug resistance. Compound (4R)-11a produced via one-pot synthesis by interaction between (?)-isopulegol and acetone was found to exhibit an outstanding activity against a number of H1N1 and H2N2 influenza virus strains with selectivity index more than 1500. (4R)-11a was shown to be most potent at early stages of viral cycle. Good correlation between anti-viral activity and calculated binding energy to hemagglutinin TBHQ active site was demonstrated.     

Radiosynthesis and in vivo evaluation of a fluorine-18 labeled pyrazine based radioligand for PET imaging of the adenosine A2B receptor

On the basis of a pyrazine core structure, three new adenosine A_2B receptor ligands (7a–c) were synthesized containing a 2-fluoropyridine moiety suitable for ¹⁸F-labeling. Compound 7a was docked into a homology model of the A_2B receptor based on X-ray structures of the related A_2A receptor, and its interactions with the adenosine binding site were rationalized. Binding affinity data were determined at the four human adenosine receptor subtypes. Despite a rather low selectivity regarding the A₁ receptor, 7a was radiolabeled as the most suitable candidate (K_i(A_2B)?=?4.24?nM) in order to perform in vivo studies in mice with the aim to estimate fundamental pharmacokinetic characteristics of the compound class. Organ distribution studies and a single PET study demonstrated brain uptake of [¹⁸F]7a with a standardized uptake value (SUV) of ≈1 at 5?min post injection followed by a fast wash out. Metabolism studies of [¹⁸F]7a in mice revealed the formation of a blood–brain barrier penetrable radiometabolite, which could be structurally identified. The results of this study provide an important basis for the design of new derivatives with improved binding properties and metabolic stability in vivo.     

Discovery of benzo[d]imidazo[5,1-b]thiazole as a new class of phosphodiesterase 10A inhibitors

The design, synthesis and structure activity relationship studies of a series of compounds from benzo[d]imidazo[5,1-b]thiazole scaffold as phosphodiesterase 10A (PDE10A) inhibitors are discussed. Several potent analogs with heteroaromatic substitutions (9a–d) were identified. The anticipated binding mode of these analogs was confirmed by performing the in silico docking experiments. Later, the heteroaromatics were substituted with saturated heteroalkyl groups which provided a tool compound 9e with excellent PDE10A activity, PDE selectivity, CNS penetrability and with favorable pharmacokinetic profile in rats. Furthermore, the compound 9e was shown to be efficacious in the MK-801 induced psychosis model and in the CAR model of psychosis.     

Asymmetric synthesis and biological evaluation of Danshensu derivatives as anti-myocardial ischemia drug candidates

The synthesis and bioactivities of Danshensu derivatives (R)-methyl 2-acetoxy-3-(3,4-diacetoxyphenyl)propanoate (1a), (R)-methyl 2-acetoxy-3-(3,4-methylenedioxyphenyl)propanoate (1b) and their racemates 7 and 10 were reported in this paper. These derivatives were designed to improve their chemical stability and liposolubility by protecting Danshensu’s phenolic hydroxyl groups with acetyl or methylene which could be readily hydrolyzed to release bioactive Danshensu. The asymmetric synthesis of 1a and 1b were achieved by catalytic hydrogenation of (Z)-methyl 2-acetoxy-3-(3,4-diacetoxyphenyl)-2-propenoate (6a) and (Z)-methyl 2-acetoxy-3-(3,4-methylenedioxyphenyl)-2-propenoate (6b) in excellent enantiomeric excesses (92% ee and 98% ee, respectively) and good yields (>89%). An unexpected intermediate product, (Z)-2-acetoxy-3-(3,4-dihydroxyphenyl)acrylic acid (4c) was obtained with high chemoselectivity in 86% yield by keeping the reaction temperature at 60 °C and its structure was identified by X-ray single crystal diffraction analysis. 1a, 1b and their racemates 7, 10 as well as 4c exhibited potent protective activities against hypoxia-induced cellular damage. The in vitro test showed that all these compounds could increase cell viability, and inhibit lipid hyperoxidation. Furthermore, 1a and 4c could inhibit apoptosis by regulating the expression of apoptosis-related molecule in gene and protein levels, up-regulating the expression of bcl-2 and down-regulating bax and caspase-3. The in vivo test indicated that 4c exhibited anti-myocardial ischemic effects featured by reducing infarction size and increasing the level of the intracellular enzymes detectable in serum. Therefore, these Danshensu derivatives may be good drug candidates for anti-myocardial ischemia therapy and merit further investigation.     

1,1-Dioxo-5,6-dihydro-[4,1,2]oxathiazines,a novel class of 11ß-HSD1 inhibitors for the treatment of diabetes

Racemic cis-1,1-dioxo-5,6-dihydro-[4,1,2]oxathiazine derivative 4a was isolated as an impurity in a sample of a hit from a HTS campaign on 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1). After separation by chiral chromatography the 4a-S, 8a-R enantiomer of compound 4a was identified as the true, potent enzyme inhibitor. The cocrystal structure of 4a with human and murine 11ß-HSD1 revealed the unique binding mode of the oxathiazine series. SAR elucidation and optimization in regard to metabolic stability led to monocyclic tetramethyloxathiazines as exemplified by compound 21g.     

pH-Dependent mismatch discrimination of oligonucleotide duplexes containing 2'-deoxytubercidin and 2- or 7-substituted derivatives: protonated base pairs formed between 7-deazapurines and cytosine

Oligonucleotides incorporating 2′-deoxytubercidin (1a), its 2-amino derivative 2a and related 2-, or 7-substituted analogs (1d, 2b–d, 3 and 4) are synthesized. For this purpose, a series of novel phosphoramidites are prepared and employed in solid-phase synthesis. Hybridization experiments performed with 12mer duplexes indicate that 7-halogenated nucleosides enhance the duplex stability both in antiparallel and parallel DNA, whereas 2-fluorinated 7-deaza-2′-deoxyadenosine residues destabilize the duplex structure. The 7-deazaadenine nucleosides 1a, 1d and their 2-amino derivatives 2a–d form stable base pairs with dT but also with dC and dG. The mispairing with dC is pH-dependent. Ambiguous base pairing is observed at pH 7 or under acid conditions, whereas base discrimination occurs in alkaline medium (pH 8.0). This results from protonated base pairs formed between 1a or 2a and dC under neutral or acid condition, which are destroyed in alkaline medium. It is underlined by the increased basicity of the pyrrolo[2,3-d]pyrimidine nucleosides over that of the parent purine compounds (pK_a values: 1a = 5.30; 2a = 5.71; dA = 3.50).     

Application of tandem biotransformation for biosynthesis of new pentacyclic triterpenoid derivatives with neuroprotective effect

Tandem whole-cell biotransformation was applied successfully to deliver novel pentacyclic triterpenoid derivatives for the first time. In this process, the starting substrate oleanolic acid (1) was biotransformed into a hydroxylated metabolite 1a by Rhizopus chinensis CICC 40335 and then was further glycosylated to 1b by Bacillus subtilis ATCC 6633. Moreover, metabolite 1a was furtherly oxidized by Streptomyces griseus ATCC 13273 and generated two new derivatives as 1c and 1d. To validate the feasibility, tandem biotransformation of 18β-glycyrrhetinic acid (2) by R. chinensis and B. subtilis was also conducted and offered a glycosylated derivative (2c). Finally, the neuroprotective effects of the derivatives were assessed on neural injury PC12 cell model induced by cobalt chloride.     

Discovery of imidazo[1,2-a]pyrazine-based Aurora kinase inhibitors

The synthesis and structure–activity relationships (SAR) of novel, potent imidazo[1,2-a]pyrazine-based Aurora kinase inhibitors are described. The X-ray crystal structure of imidazo[1,2-a]pyrazine Aurora inhibitor 1j is disclosed. Compound 10i was identified as lead compound with a promising overall profile.