The synthesis and tubulin binding activity of thiophene-based analogues of combretastatin A-4

A number of analogues of combretastatin A-4 (1), containing a thiophene ring interposed between the two phenyl groups, have been prepared. The synthesis of these compounds employed a combination of palladium-mediated coupling and iodocyclization techniques. The thiophene compounds 11, 14, 18, and 19 also represent non-benzofused analogues of some recently described tubulin binding benzo[b]thiophenes 3-5. The most active thiophene compounds identified in this study were 11, 14, and 18. Overall they are less active than 1 but exhibit comparable activity to the most active of the benzo[b]thiophenes 3-5. A structure-activity relationship of these compounds is considered.     

Control of thiophene accumulation in calli of two Tagetes species

The relation between root differentiation and accumulation of biocidal thiophenes was studied in stem calli of two Tagetes species. Disorganized tissues of T. erecta were low in thiophene production. A sharp rise in thiophene content coincided with the emergence of roots on the calli. Root regeneration and the amount of thiophenes produced were found to be quantitatively related. Callus tissues of T. minuta did not differentiate into organs. Nevertheless, they accumulated thiophenes throughout the incubation period. Light at 12 W m^- stimulated thiophene production in T. erecta without having an apparent effect on root regeneration. In T. minuta even low irradiance levels (2 W m∼²) strongly inhibited thiophene accumulation. Under favorable conditions thiophene concentrations in calli of both species were comparable to or somewhat lower than the levels in roots on the plants and in excised, cultured roots of T. erecta. We conclude that in calli of T. erecta thiophene accumulation is coupled to root regeneration whereas a different control mechanism allowing for accumulation in disorganized tissues is operative in T. minuta.     

Thiophene production in transformed roots cultures of Tagetes filifolia

Transformed root cultures of Tagetes filifolia were established by infection with Agrobacterium rhizogenes LBA 9402. Several clones were obtained with different growth index, content of thiophenes and pattern. The isolated clones showed considerable variations in total thiophene content (27 to 11764 g/g fresh weight) but all had similar patterns of the different thiophenes. Only one clone excreted thiophenes (about 80% of total amount). © Rapid Science Ltd. 1998     

Postharvest changes in endogenous ABA levels and ABA metabolism in relation to dormancy in potato tubers

In this paper the effects of indole-3-acetic acid (IAA) on growth of Tagetes patula hairy root cultures and secondary product formation are presented. The biosynthesis of thiophenes, sulfurous compounds with nematicidal activity, was inhibited by IAA application, as was evident from a decrease of [³⁵S] sulfur incorporation. The inhibition only occurred after the roots had developed numerous laterals as a result of auxin action. However, in roots cultured in the absence of IAA, there was no significant correlation between branching and thiophene accumulation. Therefore, development of lateral roots is not a sufficient condition for a low capacity to synthesize thiophenes. The highest rate of thiophene accumulation in the roots culture is at its maximum. Hence, growth and the production of thiophenes appear to be compatible in T. Patula hair roots.     

Growth and Secondary Metabolite Production by Hairy Roots of Tagetes Patula in Aqueous Two-Phase Systems

Hairy roots of Tagetes patula have been grown in aqueous two-phase systems. After selecting suitable polymers from single-phase experiments (in which salt phases were unable to support growth in the desired concentrations) several two-phase systems were tested for their influence on cell growth and thiophene production. Cell growth occurred in all aqueous two-phase systems, but the highest growth rate was achieved in normal medium. There was no difference in thiophene production between medium and aqueous two-phase systems. The partition of thiophenes favoured slightly the more hydrophobic top phase in most cases, while the cells were confined to the bottom phase. One aqueous two-phase system (15% polyethyleneglycol 10,000 and 15% Reppal PES 200) was tested in a stirred tank reactor with normal medium as a control. The growth rate in medium was higher than in the aqueous two-phase system, while the thiophene production per unit cell weight was in the same range for both systems. The excretion of thiophenes in the reactor with the aqueous two-phase system was about ten times as high as in the control reactor. The amount excreted was however still not more than 3% of the total production.     

2-Aminothiophene-3-carboxylates and carboxamides as adenosine A1 receptor allosteric enhancers

Three series of 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene and 2-amino-5,6,7,8-tetrahydrocyclohepta[b]thiophenes with 3-carboxylates and carboxamides have been prepared using the Gewald synthesis and evaluated as A(1)AR allosteric enhancers. The structure-activity relationships of these classes of compound are described. A number of compounds, notably 7b, are more potent and efficacious than PD81,723 (1).     

Design and synthesis of 4,5-disubstituted-thiophene-2-amidines as potent urokinase inhibitors

A study of the S1 binding of lead 5-methylthiothiophene amidine 3, an inhibitor of urokinase-type plasminogen activator, was undertaken by the introduction of a variety of substituents at the thiophene 5-position. The 5-alkyl substituted and unsubstituted thiophenes were prepared using organolithium chemistry. Heteroatom substituents were introduced at the 5-position using a novel displacement reaction of 5-methylsulfonylthiophenes and the corresponding oxygen or sulfur anions. Small alkyl group substitution at the 5-position provided inhibitors equipotent with but possessing improved solubility.     

Thieno[3,4-f]isothianaphthene and its N-substitutes: a theoretical insight

Density functional theory is used to study the geometries, electronic structure, aromaticity, the singlet/triplet splitting energies and the vertical ionization energy of tricyclic nonclassical thiophene, Thieno[3,4-f]isothianaphthene, and its five N-substitutes at B3LYP/aug-cc-PVDZ level of theory. Geometry studies show that all of the compounds have a highly symmetric structure with a rigid planar. Calculated results confirm that there exists considerable conjugation and aromaticity over the rings and those tricyclic nonclassical thiophenes may be candidates for conductive polymers. The studies of electronic structure and splitting energies show that those nonclassical thiophenes have more or less a diradical structure.     

Discovery of novel dialkyl substituted thiophene inhibitors of HCV by in silico screening of the NS5B RdRp

A novel 5,4-dialkyl substituted thiophene was discovered by in silico screening of the 3D polymerase crystal structure (1GX6) that demonstrated single digit micromolar HCV inhibition activity in the replicon assay and dose-dependent inhibition in the replicase complex assay. Subsequently, SAR was explored with a small set of dialkyl and tetrahydro-benzo thiophenes. Since these thiophenes inhibit synthesis of both, single- and double-stranded RNAs, their mechanism of action is distinct from other known HCV inhibitors.     

Elicitation of thiophene production by cultured hairy roots of Tagetes patula

Hairy roots of Tagetes patula were initiated by infecting the seedlings with Agrobacterium rhizogenes. The hairy roots were grown in liquid medium MS (Murashige and Skoog 1962) supplemented with sucrose. The roots were treated with three different elicitors obtained from mycelial culture of Aspergillus niger, Rhizopus oligosporus and Pencillium notatum. Accumulation of biomass and the production of thiophenes were studied over a period of six weeks in culture. The HPLC separation profile of the thiophenes indicated the presence of several structurally different thiophenes. α-terthienyl being predominent. Maximum production of thiophene was recorded at the end of the fourth week in culture with a content of 0.138 % (w/w on dry weight basis). Treatment of hairy roots with mycelial extract of Aspergillus niger (1.5 % v/v) elicited an increase in thiophene content by 1.6 folds over the control.     

Synthesis and evaluation of new arylbenzo[b]thiophene and diarylthiophene derivatives as inhibitors of the NorA multidrug transporter of Staphylococcus aureus

The synthesis based on palladium catalytic coupling of 38 new-arylated benzo[b]thiophenes or thiophenes is described in a few steps. We also report the direct arylation of the position 3 of the benzo[b]thiophenic structure, a 'one pot' 2,5-heterodiarylation of thiophenes as well as the synthesis of precursors of amino-acids with a 2-arylated benzo[b]thiophene core. These compounds were evaluated on bacteria strains: most of them did not exhibit any antibiotic activity but were found to selectively inhibit the NorA multidrug transporter of Staphylococcus aureus. As such, they restored the activity of the NorA substrates ciprofloxacin against a resistant S. aureus strain in which this efflux pump is over-expressed.     

Degradation of substituted thiophenes by bacteria isolated from activated sludge

Actinomycetes were isolated from activated sludge acclimated to thiophene-2-carboxylic acid (T2C) or 5-methyl-thiophene-2-carboxylic acid (T5M2C). These isolates were apparently identical and were identified as strains ofRhodococcus. The strains could grow on T2C, T5M2C, or thiophene-2-acetic acid as sole sources of carbon and energy, but could not use thiophene, methyl thiophenes, several other substituted thiophenes, dibenzothiophene, dimethyl sulfide, or pyrrole-2-carboxylic acid. T2C was degraded quantitatively to sulfate, and its carbon was converted almost entirely to cell biomass and carbon dioxide. Growth yields indicated about 25% conversion of T2C-carbon to cell-carbon. Growth was not supported by thiosulfate or methionine, nor were these compounds oxidized.Rhodococcus strain TTD-1 grown on T2C oxidized both T2C and T5M2C with an apparent K_m of 1.3×10^–5 M. Sulfide was also oxidized by T2C-grown organisms. This is the first demonstration of an actinomycete capable of the complete degradation of thiophene derivatives and of their use by it as sole substrates for growth.     

Production of thiophenes from callus cultures of Tagetes patula L. and its mosquito larvicidal activity

Calli were initiated from leaf tissues of T. patula, on Murashige and Skoog's medium supplemented with 2-4 dichlorophenoxacetic acid (2,4-D) and kinetin (Kn). The maximum accumulation of biomass was recorded on 6th week at 1.11 g. dry wt/culture. Maximum thiophene content was recorded on 4th week at 0.008% on dry wt basis in callus cultures of T. patula subjected to nutrient stress. Nitrogen stress induced 3-fold increase in thiophene production level in six weeks (0.024% on dry weight basis). The best hormonal supplementation required for thiophenes production was found to be 2,4-D (2.0 mg L(-1)) and kinetin (2.0 mg L(-1)). The thiophenes produced in callus cultures of T. patula showed larvicidal effect against mosquito larvae.     

GLC and GLC-mS analysis of thiophene derivatives in plants and in in vitro cultures of Tagetes patula L. (Asteraceae)

The occurrence of thiophenic compounds in diverse plant organs and in in vitro root-, callus- and cell suspension cultures of Tagetes patula cv. Carmen was investigated using capillary GLC and GLC-MS. The separation of thiophenes by capillary GLC and the group specific MS fragmentation with the typical sulfur isotope peaks allowed the unequivocal assignment of individual thiophenes in complex mixtures, even when occurring in traces and in the presence of different geometrical isomers. The extracts of Tagetes patula cv. Carmen contained the following 8 thiophene compounds: 5-(3-buten-1-ynyl)-2,2'-bithienyl (BBT), 5'-methyl-5-(3-buten-1-ynyl)-2,2'-bithienyl (MeBBT), 5-(1-pentynyl)-2,2'-bithienyl (PBT), 5-(4-hydroxy-1-butynyl)-2,2'-bithienyl (BBTOH), 2,2',5,2"-terthienyl (alpha-T), 5-(4-acetoxy-1-butynyl)-2,2'-bithienyl (BBTOAc), 5-methylaceto-5'-(3-buten-1-ynyl)-2,2'-bithienyl (AcOCH2BBT), and 5-(3,4-diacetoxy-1-butynyl)-2,2'-bithienyl (BBT(OAc)2). The most complex thiophene profile, including the less common PBT was detected in aerial parts of freshly harvested plant material. Under in vitro conditions only the root cultures, but not callus or cell suspension cultures produced substantial amounts of irregular thiophenes confirming that roots are the main site of thiophene biosynthesis.     

A DFT study of the effects of donor-to-acceptor ratio on the electronic properties of copolymers based on tricyclic non-classical thiophene

The geometries and electronic properties of the 1D polymers composed of thieno[3,2-b]thiophene (TT), thiophene (T), pyrrole (P), furan (F) and tricyclic non-classical thiophenes ([1,2,5]thiadiazolo[3,4-b]thieno-[3,4-e]pyrazine, TTP) are investigated systematically by the density functional theory method at the B3LYP level with 6-31G(d) basis set. The theoretical study suggests that the ratio of donor-to-acceptor (D-A ratio) plays a crucial role in the geometric and electronic properties for the alternating donor–acceptor polymers. The increase in the D-A ratio leads to an increase in the bridge bond length and an inverse change in the bond-length alternation. Furthermore, the increase in the portion of donor units can lead to an obvious reduction in band gap for these studied polymers. The TT-containing polymer possessing the D-A ratio of 2:1 (p-BTTTTP) that is predicted to have a small band gap of 0.25 eV and a relatively small effective mass of carriers is a good candidate for an intrinsic conducting polymer. Therefore, the tricyclic non-classical thiophenes (TTP) and TT are good building blocks that can lead to small band gap polymers.     

Growth and Secondary Metabolite Production by Hairy Roots of Tagetes Patula in Aqueous Two-Phase Systems

Hairy roots of Tagetes patula have been grown in aqueous two-phase systems. After selecting suitable polymers from single-phase experiments (in which salt phases were unable to support growth in the desired concentrations) several two-phase systems were tested for their influence on cell growth and thiophene production. Cell growth occurred in all aqueous two-phase systems, but the highest growth rate was achieved in normal medium. There was no difference in thiophene production between medium and aqueous two-phase systems. The partition of thiophenes favoured slightly the more hydrophobic top phase in most cases, while the cells were confined to the bottom phase. One aqueous two-phase system (15% polyethyleneglycol 10,000 and 15% Reppal PES 200) was tested in a stirred tank reactor with normal medium as a control. The growth rate in medium was higher than in the aqueous two-phase system, while the thiophene production per unit cell weight was in the same range for both systems. The excretion of thiophenes in the reactor with the aqueous two-phase system was about ten times as high as in the control reactor. The amount excreted was however still not more than 3% of the total production.     

gamma-Glutamyl dipeptides in Petiveria alliacea

Three gamma-glutamyl dipeptides have been isolated from Petiveria alliacea L. roots. These dipeptides include (S(C2)R(C7))-gamma-glutamyl-S-benzylcysteine together with two diastereomeric sulfoxides, namely (S(C2)R(C7)R(S))- and (S(C2)R(C7)R(S))-gamma-glutamyl-S-benzylcysteine S-oxides (gamma-glutamyl-petiveriins A and B, respectively). Their structures and absolute configurations have been determined by NMR, MALDI-HRMS, IR and CD spectroscopy, and confirmed by comparison with authentic compounds obtained by synthesis.     

Thiophene accumulation in hairy roots ofTagetes patula in response to fungal elicitors

Summary Hairy roots ofTagetes patula were treated with fungal extracts from 3 different fungi. Treated roots were found to accumulate thiophenes at a higher level than untreated control cultures. The kinetics of thiophene formation varied with the fungus from which the elicitor was prepared, elicitor concentration and duration of exposure.     

Molecular cloning and sequence of the thdF gene, which is involved in thiophene and furan oxidation by Escherichia coli.

Our previous work resulted in the isolation of mutant strains of Escherichia coli K-12 which were able to oxidize furans and thiophenes as a result of mutations in several novel genes. Some of the genes involved in thiophene oxidation were cloned into the multicopy vector pUC19. The plasmid pKA10 carries a 3.8-kb chromosomal fragment which encodes a previously undiscovered gene involved in thiophene oxidation. Three proteins with approximate molecular sizes of 48, 30, and 26 kDa were overproduced by cells carrying pKA10. Maxicell experiments and DNA sequence analysis indicated that the 48- and 26-kDa proteins are encoded by pKA10, whereas the 30-kDa protein is apparently chromosomally derived. A cassette specifying kanamycin resistance was inserted into various sites on pKA10. An insertion which abolished the 48-kDa protein also abolished thiophene oxidation. Chromosomal integration of pKA10::Kan allowed us to locate the chromosomal insert of pKA10 at 84 min on the E. coli genetic map by transduction. Since no previously identified genes involved in thiophene metabolism are located in this region, we designated the gene for the 48-kDa protein as thdF. Sequencing of the 3.8-kb insert revealed an overlap of several hundred bases with the regulatory and structural regions of the tnaA gene, which is also located at 84 min. The 26-kDa protein is probably truncated tnaA protein. An open reading frame corresponding to the 48-kDa thdF protein was located next to the tnaA gene, which encodes tryptophanase, but was transcribed in the opposite sense.