Total synthesis and evaluation of cytostatin, its C10-C11 diastereomers, and additional key analogues: impact on PP2A inhibition

The total synthesis of cytostatin, an antitumor agent belonging to the fostriecin family of natural products, is described in full detail. The convergent approach relied on a key epoxide-opening reaction to join the two stereotriad units and a single-step late-stage stereoselective installation of the sensitive (Z,Z,E)-triene through a beta-chelation-controlled nucleophilic addition. The synthetic route provided rapid access to the C4-C6 stereoisomers of the cytostatin lactone, which were prepared and used to define the C4-C6 relative stereochemistry of the natural product. In addition to the natural product, each of the C10-C11 diastereomers of cytostatin was divergently prepared (11 steps from key convergence step) by this route and used to unequivocally confirm the relative and absolute stereochemistry of cytostatin. Each of the cytostatin diastereomers exhibited a reduced activity toward inhibition of PP2A (>100-fold), demonstrating the importance of the presence and stereochemistry of the C10-methyl and C11-hydroxy groups for potent PP2A inhibition. Extensions of the studies provided dephosphocytostatin, sulfocytostatin (a key analogue related to the natural product sultriecin), 11-deshydroxycytostatin, and an analogue lacking the entire C12-C18 (Z,Z,E)-triene segment, which were used to define the magnitude of the C9-phosphate (>4000-fold), C11-alcohol (250-fold), and triene (220-fold) contribution to PP2A inhibition. A model of cytostatin bound to the active site of PP2A is presented, compared to that of fostriecin, which is also presented in detail for the first time, and used to provide insights into the role of the key substituents. Notably, the alpha,beta unsaturated lactone of cytostatin, like that of fostriecin, is projected to serve as a key electrophile, providing a covalent adduct with Cys269 unique to PP2A, contributing to its potency (> or =200-fold for fostriecin) and accounting for its selectivity.     

A Cascade of Redox Reactions Generates Complexity in the Biosynthesis of the Protein Phosphatase‐2 Inhibitor Rubratoxin A

Redox modifications are key complexity‐generating steps in the biosynthesis of natural products. The unique structure of rubratoxin A ( 1 ), many of which arise through redox modifications, make it a nanomolar inhibitor of protein phosphatase 2A (PP2A). We identified the biosynthetic pathway of 1 and completely mapped the enzymatic sequence of redox reactions starting from the nonadride 5 . Six redox enzymes are involved, including four α‐ketoglutarate‐ and iron(II)‐dependent dioxygenases that hydroxylate four sp³ carbons; one flavin‐dependent dehydrogenase that is involved in formation of the unsaturated lactone; and the ferric‐reductase‐like enzyme RbtH, which regioselectively reduces one of the maleic anhydride moieties in rubratoxin B to the γ‐hydroxybutenolide that is critical for PP2A inhibition. RbtH is proposed to perform sequential single‐electron reductions of the maleic anhydride using electrons derived from NADH and transferred through a ferredoxin and ferredoxin reductase pair.     

Development of an efficient protein phosphatase-based colorimetric test for okadaic acid detection

Okadaic acid (OA), responsible for gastrointestinal problems, inhibits protein phosphatase 2A (PP2A). Therefore, the inhibition exerted by the toxin on PP2A could be used to detect the presence of OA in aqueous solution and in shellfish sample.In this work, two commercial PP2As (from ZEU Immunotec and Millipore) and one produced by molecular engineering (from GTP Technology) were tested. Enzymes were used in solution and also immobilized within a polymeric gel. In solution, best performances were obtained using PP2A purchased from ZEU Immunotec (Spain). OA was detected in aqueous solution in concentration as low as 0.0124 μg L⁻¹ using the enzyme from ZEU Immunotec whereas the detection limits were 0.47 μg L⁻¹ and 0.123 μg L⁻¹ with PP2As from Millipore and GTP Technology, respectively. Considering that the immobilization step contributes to stabilize the PP2A activity, enzymes were entrapped within a photopolymer and an agarose gel. These different polymeric matrices were optimized, tested and compared. Agarose gel seems to be a good alternative to the photopolymer largely used in our group. For instance, the IC₅₀ value obtained with the test based on PP2A from ZEU Immunotec immobilized within an agarose gel was 1.98 μg L⁻¹. This value was 1.8-fold lower than those obtained with the photopolymer test using the same enzyme. The proposed test is sensitive, fast and does not require expensive equipment. To evaluate the efficiency of the assay, PP inhibition tests based on PP2A from ZEU Immunotec in solution or immobilized within a gel were used for OA detection in contaminated shellfish.     

Alpha-phosphono lactone analogues of farnesyl pyrophosphate: an asymmetric synthesis via ring-closing metathesis

An alpha-phosphono lactone derivative of farnesol has been prepared, in both racemic and nonracemic forms, to provide a new type of farnesyl pyrophosphate analogue. Attempted preparation of the racemic alpha-phosphono lactone through rearrangement of a vinyl phosphate derived from the parent lactone resulted in both rearrangement and lactone ring opening, revealing that the farnesyl lactone was not stable to the excess of strong base required for the rearrangement. A procedure for C-P bond formation based on generation of the lactone enolate, reaction with a P(III) reagent, and oxidation was successful in providing the racemic alpha-phosphono lactone, in part, because only 1 equiv of strong base was required. The same strategy for phosphonate synthesis then was applied to the nonracemic farnesyl lactone, prepared through a sequence including allylation of farnesal with a nonracemic borane reagent, reaction of the product alcohol with acryloyl chloride, and formation of an unsaturated lactone through ring-closing metathesis. A similar strategy gave the corresponding racemic alpha-phosphono lactam through a six-step sequence from farnesal.     

Studies towards the total synthesis of Phostriecin

A synthetic approach toward the phostriecin, an antitumor natural product is described. The key features of the present synthesis are Wittig reaction, synthesis of homoallylic alcohol using Brown’s protocol (alkoxyallylboration) and RCM for the creation of unsaturated lactone moiety of phostriecin.     

Highly sensitive detection and discrimination of LR and YR microcystins based on protein phosphatases and an artificial neural network

The inhibition characteristics of three different protein phosphatases by three microcystin (MC) variants--LR, YR, and RR--were studied. The corresponding K (I) for each enzyme-MC couple was calculated. The toxicity of MC varies in the following order: MC-LR > MC-YR > MC-RR. The sensitivity of the enzymes increased in the following order: mutant PP2A < mutant PP1 < natural PP2A. The best limit of detection obtained was 21.2 pM MC-LR using the most sensible enzyme. Methanol, ethanol, and acetonitrile up to 2 % (v/v) may be used in inhibition measurements. An artificial neural network (ANN) was used to discriminate two MC variants--LR and YR--using the differences in inhibition percentages measured with mutant PP1 and natural PP2A. The ANN is able to analyze mixtures with concentrations ranging from 8 to 98 pM MC-LR and 31 to 373 pM MC-YR.     

Synthesis of β‐Keto Lactone with Basic Skeleton of Przewalskin B

The synthesis of β‐keto lactone was completed using cyclohexanone as starting material. The α,β‐unsaturated lactone was obtained through Reformatsky reaction and iodolactonization. The aforementioned lactone was converted into target compound 1 (β‐keto lactone) by Nef oxidation and Claisen condensation in six steps with 17.9% overall yield.     

Stereoselective Michael addition of 6-amino-1,3-dimethyl-2,4-pyrimidinedione to the exocyclic methylene of three sesquiterpene lactones. 1H and 13C NMR evidence of a new C-C bond and lactam formation

The Stereoselective addition of the pyrimidine derivative 1 to the exocyclic methylene of the α,β unsaturated dehydrocostus lactone 2, Ivalin acetate 3 and Zaluzanin A diacetate 4, was achieved resulting in a new C-C bond formation. In the cases of compounds 3 and 4, after the addition, the lactone was cleaved followed by reclosure into a lactam ring system.     

弱碱K~2CO~3体系中酚酞盐的结构特征

用红外光谱、核磁共振谱鉴定了弱碱K~2Co~3体系和强碱KOH体系中制备的酚酞钾盐, 在弱碱K~2Co~3体系中, 酚酞成盐以完全内酯式结构为特性, 而在强碱KOH体系中,酚酞成盐主要是醌式结构。从而发现成盐反应中所使用的碱本身的性质是决定生成酚酞盐结构的关键因素。     

cis-Delta(2,3)-double bond of phoslactomycins is generated by a post-PKS tailoring enzyme

The antifungal phoslactomycins (PLM A-F), produced by Streptomyces sp. HK803, are structurally unusual in that three of their four double bonds are in the cis form (Delta12,13, Delta14,15, Delta2,3). The PLM polyketide synthase (PKS) has the predicted dehydratase catalytic domain in modules 1, 2, and 5 required for establishing two of these cis double bonds (Delta12,13, Delta14,15), as well as the only trans Delta6,7 double bond. By contrast, the formation of the cis Delta2,3 in the unsaturated lactone moiety of PLMs has presented an enigma because the predicted dehydratase domain in module 7 is absent. Herein, we have demonstrated that the plmT2 gene product, with no homology to PKS dehydratase domains, is required for efficient formation of the cis Delta2,3 alkene. A series of new PLM products in which the C3 hydroxyl group is retained are made in plmT2 deletion mutants. In all of these cases, however, the hydroxyl group is esterified with malonic acid. These malonylated PLM products are converted to the corresponding cis Delta2,3 PLM products and acetic acid by a facile base-catalyzed decarboxylative elimination reaction. Complete or partial restoration of natural PLM production in a plmT2 deletion mutant can be accomplished by plasmid based expression of plmT2 or fos ORF4 (a homologous gene from the fostriecin biosynthetic gene cluster), respectively. The data indicate that dehydratase-independent pathways also function in establishment of unsaturated 6-membered lactone moieties in other PKS pathways and provide the first biosynthetic insights into the possible routes by which unusual malonylated polyketide products are generated.     

A one-step synthesis of 2,3-dihydro-4H-pyran-4-ones from 3-ethoxy alpha,beta-unsaturated lactones

[reaction: see text] Addition of diverse nucleophiles to the unsaturated lactone 2 that results from hetero Diels-Alder reaction of Brassard's diene 1 with aldehydes leads to an efficient and general approach to the synthesis of 2,3-dihydro-4H-pyran-4-ones 3.     

Thuggacins, macrolide antibiotics active against Mycobacterium tuberculosis: isolation from myxobacteria, structure elucidation, conformation analysis and biosynthesis

Two novel antibiotics, thuggacin A (1) and B (2), were isolated from the myxobacterium Sorangium cellulosum. 1 and 2 are unique thiazole-containing macrolides with side chains on both sides of the lactone group. Upon standing in solution, thuggacin A (1) rearranges by acyl migration of the lactone group to give a mixture with thuggacins B (2) and C (3). NOEs and vicinal coupling constants within the lactone ring provided distinct data for the generation of a structure model by PM3 calculations, which allowed an analysis of the conformation in solution and the relative configuration of six asymmetric centres. A minor sorangium metabolite was identified as 13-methyl-thuggacin A (4). Furthermore, two natural thuggacin variants, 5 and 6, were found in another myxobacterium, Chondromyces crocatus. In these variants, one side chain is replaced by a methyl group and a hydroxy group is repositioned to give a primary alcohol at the former methyl site, in an alpha position with respect to the thiazole ring. 1 proved to be active against clinical isolates and reference strains of Mycobacterium tuberculosis. Preliminary studies on the mechanism of action indicate inhibition of the cellular electron-transport chain.     

Four New Diterpenoids from Ballota limbata

Six diterpenoids ( 1 – 6 ) belonging to the clerodane and tetracyclic diterpene types were isolated from the CHCl₃ extract of Ballota limbata. The structures of the new compounds 1 and 4 – 6 (named ballatenolide A and limbatenolides A–C, resp.) were established by their spectral data, and their relative configuration was determined by 2D NMR. Compounds 2 and 3 were identified as known clerodanes. Compound 1 had a unique feature, i.e., an α,β‐unsaturated γ‐lactone moiety at C(4)/C(6), whereas limbatenolides A–C ( 4 – 6 ) belonged to the rare class of tetracyclic diterpenoids, which are known to occur only in the genus Ballota. All of the isolated compounds showed inhibitory potential in a cholinesterase‐inhibition assay.     

Total synthesis and biological evaluation of the protein phosphatase 2A inhibitor cytostatin and analogues

The total synthesis of the natural product cytostatin is described which inhibits protein phosphatase 2A. Cytostatin has anti-metastatic properties and induces apoptosis. On the basis of this synthesis the relative and absolute configuration of cytostatin could be assigned. Key structural elements of cytostatin are an alpha,beta-unsaturated lactone group and a side chain embodying a phosphate and a rather unstable (Z,Z,E)-triene subunit. In addition, the natural product carries six stereocenters. For the construction of the stereocenters reagent-controlled transformations were used in order to ensure maximum stereochemical flexibility. The Evans syn-aldol reaction was chosen to establish the stereochemistry at C-4, C-5, C-9 and C-10; C-6 was introduced by means of the Evans asymmetric alkylation. In all cases the same chiral auxiliary was employed as stereodirecting group. The stereocenter at C-11 was established by an asymmetric reduction using CBS-oxazaborolidine. Temporary protection of the phosphate group was achieved best by using the base-labile 9-fluorenylmethyl group, which could be cleanly cleaved by an excess of triethylamine; this reaction yielded analytically pure phosphates after a simple aqueous work-up. The (Z,Z,E)-triene embodied in cytostatin was synthesized by means of a Stille coupling as key transformation. The synthesis sequence established in this way readily gave access to a series of analogues with simplified structure. Initial biological testing of these analogues proved that the alpha,beta-unsaturated lactone, the C-11-hydroxy group and a fully deprotected phosphate moiety at C-9 are essential for the PP2A-inhibitory activity of cytostatin. The rather unstable triene moiety in the side chain can be replaced by other lipophilic residues with only moderate decrease of biological activity. Other phosphatases, that is, PP1, VHR, PTP1B, CD45, were not inhibited by cytostatin or any of the analogues, demonstrating the high selectivity of this compound. These findings will be useful for the design and synthesis of cytostatin-derived chemical tools for the study of biological processes influenced by PP2A.     

Chelation of vanadium(V) by difluoromethylene bisphosphonate, a structural analogue of pyrophosphate

The structural and functional analogy between difluoromethylene bisphosphonate (CF2PP) and pyrophosphate (PPi) is investigated in a reaction with V(V) in the form of vanadate. The reaction of CF2PP with vanadate was investigated using 1.00 M KCl as supporting electrolyte over the ranges 3 < or = [CF2PP] < or = 60 mM and 2.06 < or = pH < or = 11.80. 51V, 19F, and 31P NMR spectroscopic studies showed that a 1:1 species was formed with an H+-dependent formation constant of 110 M-1 at pH 7.22. Results of solution experiments and ab initio calculations are consistent with CF2PP coordinating V(V) in a bidentate manner, as previously reported for PPi. Below pH 4, a minor complex forms, which is consistent with a 1:2 stoichiometry. This complex was also observed with pyrophosphate. The X-ray crystal structure of the monoprotonated difluoromethylene bisphosphonate anion (H[CF2PP]3-)-toludine complex is presented. The H[CF2PP]3- anion crystallized in the triclinic space group P with a = 12.7629(7) A, b = 13.3992(7) A, c = 17.1002(9) A, and V = 2584.4(2) A3, and Z = 2. Sheets of the layers of anions are connected through a network of H-bonds and separated by a layer of toludine cations. The structural features are investigated, and the CF2PP anion was found to be longer and wider than the corresponding PPi. Given the larger size of this anion compared to PPi, the chelation affinity upon CF2 substitution was found to be 4-5-fold reduced at neutral pH.     

Simple Synthesis of Versatile Coumarin Scaffolds

We describe an approach using ring‐closing metathesis (RCM) to synthesize versatile coumarin derivatives that present appropriate substitutions both at the aromatic and at the α,β‐unsaturated lactone ring. The obtained compounds can be used as molecular scaffolds suitable for further diversifications through a combinatorial approach.     

Design, synthesis and evaluation of furanocoumarin monomers as inhibitors of CYP3A4

A number of furanocoumarins isolated from grapefruit juice have been found to inhibit CYP3A4 activity in vitro. In this study, we have designed and synthesised a range of analogues based on bergamottin to investigate the relationship between chemical structure and inhibition of CYP3A4 activity. Studies were performed using human liver microsomes and human intestinal S9 fraction, with testosterone as the marker substrate. With the exception of the coumarin and phenolic furanocoumarin derivatives, which were inactive, the alkyloxy-furanocoumarin analogues were found to inhibit CYP3A4 activity in a dose dependent manner, with observed IC50 values ranging from 0.13 +/- 0.03 to 49.3 +/- 1.9 microM. The unsaturated furan derivatives were found to exhibit time-dependent inhibition, showing a 2-, 4- and 14-fold increase in potency for 6',7'-epoxybergamottin, 6',7'-dihydroxybergamottin and bergamottin, respectively after a preincubation period of ten minutes. Reduction of the furan moiety resulted in an 11-fold decrease in inhibitory potency, suggesting that this functional group is key to the interaction between these compounds and CYP3A4.     

In vivo metabolite profiles of isoimperatorin and phellopterin in rats analyzed using HPLC coupled with diode array detector and electrospray ionization ion trap time‐of‐flight mass spectrometry technique

Isoimperatorin (IP) and phellopterin (PP) are two furocoumarins existing in Angelicae Dahuricae Radix. There is an isopentenyloxyl substituted at C‐5 in IP, and an isopentenyloxyl and a methoxyl substituted at C‐8 and C‐5, respectively, in PP. To elucidate the in vivo metabolic characteristics of PP and IP, HPLC coupled with diode array detector and electrospray ionization ion trap time‐of‐flight mass spectrometry technique was used. In total, 111 metabolites, including 53 new ones, were identified from the urine and plasma samples of rats after oral administration of IP and PP, respectively. The metabolites were formed through eight reactions on IP and PP: oxidation, hydroxylation–hydrogenation, carboxylation on the isopentenyloxyl, O‐dealkylation, hydroxylation on the furocoumarin nucleus, ring‐opening reaction on the furan ring and reduction or ring‐opening reaction on the lactone ring. Among these, hydroxylation on the furocoumarin nucleus was found for the first time for in vivo metabolites of PP and IP, and the ring‐opening reaction on the furan ring or lactone ring was found for the first time for in vivo metabolites of isopentenyloxyl furocoumarins. The research gave us a new insight into the in vivo metabolic profiles of IP and PP, which could help us better understand their important roles as two active constituents of Angelicae Dahuricae Radix.     

NHC‐Catalyzed Chemoselective Reactions of Enals and Aminobenzaldehydes for Access to Chiral Dihydroquinolines

An N‐heterocyclic carbene (NHC)‐catalyzed reaction between α‐bromoenals and 2‐aminoaldehydes has been developed. Key steps include chemoselective reaction of the NHC catalyst with one of the aldehyde substrates (the bromoenal) to eventually generate an α,β‐unsaturated acylazolium intermediate. Addition of the nitrogen atom of aminoaldehyde to the unsaturated azolium ester intermediate followed by intramolecular aldol reaction, β‐lactone formation, and decarboxylation leads to chiral dihydroquinolines with high optical purity. The dihydroquinoline products, which are quickly prepared by using this method, can be readily transformed into a diverse set of functional molecules such as pyridines and chiral piperidines.     

Photocycloaddition of benzophenone to α-angelicalactone and thermal rearrangement of the product

α-Angelicalactone dissolved in thiophene in the presence of benzophenone was irradiated with uv light in order to find out if the unsaturated lactone behaves either as an olefin capable of yielding a [2 + 2] adduct with the heterocycle or if it rather reacts as a substrate for the excited benzophenone. The isolation of two isomeric oxetanes indicates that the latter is the preferred reaction pathway. One of the oxetanes undergoes an interesting thermal rearrangement.