Organic reactions in ionic liquids:A new method for the synthesis of alkyl aryl sulfones by alkylation of sodium arenesulfinates with unactivated alkyl chlorides

A new method is reported for the synthesis of alkyl aryl sulfones by alkylation of sodium arenesulfinates with unactivated alkyl chlorides using ionic liquid based on 1-butyl-3-methylimidazolium tetrafluoroborate (BmimBF 4) mixed with water (2:1) as reaction media. The ionic liquid can be reused and the procedure gives the sulfones in moderate yields.

Organic reactions in ionic liquids: an efficient method for the N-alkylation of benzotriazole

The N-alkylation of benzotriazole with alkyl halides proceeds efficiently in the presence of potassium hydroxide in ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate ([Bmim][BF4]).

Organic reactions in ionic liquids:An efficient method for selective S-alkylation of 2-mercaptobenzothia(xa)zole with alkyl halides

Ionic liquids based on 1-butyl-3-methylimidazolium hexafluorophosphate (BmimPF6) and 1-butyl-3-methylimidazolium tetrafluoroborate (BmimBF4) are used as reusable reaction medium in the selective S-alkylation of 2-mercaptobenzothia(xa)zole with alkyl halides in the presence of potassium carbonate. This procedure is convenient, efficient, and generally gives rise to the S-alkylated product exclusively.

Organic reactions in ionic liquids: N-alkylation of cyclic imides with alkyl halides promoted by potassium fluoride

An ionic liquid based on 1-butyl-3-methylimidazolium hexafluorophosphate is used as an efficient reusable reaction medium in the N-alkylation of cyclic imides with alkyl halides promoted by fluoride ion.

Organic reactions in ionic liquids: N-alkylation of phthalimide and several nitrogen heterocycles

N-Alkylation of heterocyclic compounds bearing an acidic hydrogen atom attached to nitrogen with alkyl halides is accomplished in ionic liquids ([bmim]BF4 = 1-butyl-3-methylimidazolium tetrafluoroborate, [bmim]PF6 = 1-butyl-3-methylimida-zolium hexafluorophosphate, [buPy]BF4 = butylpyridinium tetra­fluoroborate) in the presence of potassium hydroxide as a base. In this manner, phthalimide, indole, benzimidazole, succinimide can be successfully alkylated. The procedure is convenient, efficient, and generally affords the N-alkylated product exclusively.

Organic reactions in ionic liquids:Alkylation of Meldrum's acid

The room temperature ionic liquid N-butylpyridinium tetrafluoroborate, [bpy]BF4 is used as a "green" recyclable alternative to classical molecular solvents for the alkylation of Meldrum's acid.

Synthesis and evaluation of N1-substituted-3-propyl-1,4-benzodiazepine-2-ones as cholecystokinin (CCK2) receptor ligands

A novel synthetic approach towards N1-alkylated 3-propyl-1,4-benzodiazepines was developed in five synthetic steps from 2-amino-4-chlorobenzophenone, in which the N-oxide 4 served as a key intermediate. The structure-activity relationship optimization of this 3-prophyl-1,4-benzodiazepine template was carried out on the N1-position by selective alkylation reactions and resulted in a ligand with an improved affinity on the cholecystokinin (CCK2) receptor. The N-allyl-3-propyl-benzodiazepine 6d displayed an affinity towards the CCK2 (CCK-B) receptor of 170 nM in a radiolabelled receptor-binding assay. The anxiolytic activity of this allyl-3-propyl-1,4-benzodiazepine 6d was subsequently determined in in-vivo psychotropic assays. This novel ligand had ED50 values of 4.7 and 5.2 mg kg-1 in the black and white box test and the x-maze, respectively, and no significant sedation/muscle relaxation was observed.

Synthesis and evaluation of asperlicin analogues as non-peptidal cholecystokinin-antagonists

The SAR of Asperlicin analogues is reported, leading to bioactive 1,4-benzodiazepine-2-ones, which were prepared in a 3 step reaction sequence. The Asperlicin substructure was built up using Tryptophan and readily available 2-amino-acetophenones. This template, containing a 1,4-benzodiazepin-2-one moiety with a 3-indolmethyl side chain, was transformed into mono- and di-substituted 3-indol-3 '-yl-methyl-1,4-benzodi-azepine-2-ones by selective alkylation and acylation reactions. The SAR optimization of the 1,4-benzodiazepine scaffold has included variations at the 5-, 7-, 8-position, at the N1, N-indole nitrogen and the configuration of the C3-position. The most active Asperlicin analogue, having an IC50 of 1.6 microM on the CCKA receptor subtype, was obtained from Tryptophan in only 3 steps in an overall yield of 48%.

Organic reactions in ionic liquids: ionic liquid-accelerated facile synthesis of 3-alkyl-2,4-thiazolidinediones

The room temperature ionic liquid [bmim]PF6 is a new green solvent for the N-alkylation of 2,4-thiazolidinones. Significant rate enhancement and improved yields have been observed.

Thermodynamics of binding of regulatory ligands to tissue transglutaminase

The transamidating activity of tissue transglutaminase is regulated by the ligands calcium and GTP, via conformational changes which facilitate or interfere with interaction with the peptidyl-glutamine substrate. We have analysed binding of these ligands by calorimetric and computational approaches. In the case of GTP we have detected a single high affinity site (K (D) approximately 1 muM), with moderate thermal effects suggestive that binding GTP involves replacement of GDP, normally bound to the protein. On line with this possibility no significant binding was observed during titration with GDP and computational studies support this view. Titration with calcium at a high cation molar excess yielded a complex binding isotherm with a number of "apparent binding sites" in large excess over those detectable by equilibrium dialysis (6 sites). This binding pattern is ascribed to occurrence of additional thermal contributions, beyond those of binding, due to the occurrence of conformational changes and to catalysis itself (with protein self-crosslinking). In contrast only one site for binding calcium with high affinity (K (D) approximately 0.15 muM) is observed with samples of enzyme inactivated by alkylation at the active site (to prevent enzyme crosslinkage and thermal effects of catalysis). These results indicate an intrinsic ability of tissue transglutaminase to bind calcium with high affinity and the necessity of careful reassessment of the enzyme regulatory pattern in relation to the concentrations of ligands in living cells, taking also in account effects of ligands on protein subcellular compartimentation.

Synthesis of triplex-forming oligonucleotide conjugates of the anticancer drug temodal

Covalent attachment of the anticancer drugs temozolomide (Temodal) and mitozolomide to triplex-forming oligonucleotides (TFOs) is a potential way of targeting these alkylating agents to specific gene sequences to maximise site-selectivity. In this work, polypyrimidine TFO conjugates of both drugs were synthesised and targeted to duplex DNA in an attempt to effect site-specific alkylation of guanine residues. Concurrently, in an attempt to enhance the triple helix stability of TFOs at neutral pH, the thermal stabilities of triplexes formed from TFOs containing isoguanine, 2-O-benzyl- and 2-O-allyl-adenine were evaluated. A novel cleavage and deprotection procedure was developed which allowed for the solid phase synthesis of the base-sensitive TFO-drug conjugates using a recently developed silyl-linked controlled pore glass (SLCPG) support. Covalent attachment of either temozolomide or mitozolomide at the 5'-end of TFO conjugates caused no destabilisation of the triplexes studied. The synthesis of a phosphoramidite derivative of mitozolomide enabled direct incorporation of this reagent into a model sequence during DNA synthesis. After cleavage and deprotection of the TFO-drug conjugate, the 5'-end mitozolomide residue was found to have decomposed presumably as a result of ring-opening of the tetrazinone ring. The base-sensitive antibacterial and antitumour agent, metronidazole, was also successfully incorporated at the 5'-end of the oligonucleotide d(T8) using conventional methods. Two C2-substituted derivatives of 2'-deoxyadenosine containing 2-O-benzyl and 2-O-allyl groups were synthesised. Hydrogenolysis of the 2-O-benzyl analogue provided a useful route, amenable to scale-up, for the synthesis of the rare nucleoside 2'-deoxyisoguanosine (isoG). Both the 2-O-allyl and 2-O-benzyl derivatives were incorporated into TFO sequences using phosphoramidite methodology. Thermal melting experiments showed that the 2-O-allyl and 2-O-benzyl groups caused marked destabilisation of the triple helices studied, in contrast to hexose-DNA duplexes, where aralkyl substituents caused significant stabilisation of duplexes. TFOs containing isoG were synthesised by Pd(O)-catalysed deallylation of 2-0-allyl adenine residues. These sequences containing isoG, in its N3- or 02-H tautomeric form, formed triple helices which were equally as stable as those containing adenine.

Combinatorial approach to multi-substituted 1,4-Benzodiazepines as novel non-peptide CCK-antagonists

For the drug discovery process, a library of 168 multisubstituted 1,4-benzodiazepines were prepared by a 5-step solid phase combinatorial approach. Substituents were varied in the 3,5, 7 and 8-position on the benzodiazepine scaffold. The combinatorial library was evaluated in a CCK radiolabelled binding assay and CCKA (alimentary) and CCKB (brain) selective lead structures were discovered. The template of CCKA selective 1,4-benzodiazepin-2-ones bearing the tryptophan moiety was chemically modified by selective alkylation and acylation reactions. These studies provided a series of Asperlicin naturally analogues. The fully optimised Asperlicin related compound possessed a similar CCKA activity as the natural occuring compound. 3-Alkylated 1,4-benzodiazepines with selectivity towards the CCKB receptor subtype were optimised on A) the lipophilic side chain and B) the 2-aminophenyl-ketone moiety, together with some stereochemical changes. A C3 unit in the 3-position of 1,4-benzodiazepines possessed a CCKB activity within the nanomolar range. Further SAR optimisation on the N1-position by selective alkylation resulted in an improved CCKB binding with potentially decreased activity on the GABAA/benzodiazepine receptor complex. The in vivo studies revealed two N1-alkylated compounds containing unsaturated alkyl groups with anxiolytic properties. Alternative chemical approaches have been developed, including a route that is suitable for scale up of the desired target molecule in order to provide sufficient quantities for further in vivo evaluation.

Studies on the mechanism of action on antitumour imidazotetrazinones

The imidazotetrazinones are clinically active antitumour agents, temozolomide currently proving successful in the treatment of melanomas and gliomas. The exact nature of the biological processes underlying response are as yet unclear.This thesis attempts to identify the cellular targets important to the cytotoxicity of imidazotetrazinones, to elucidate the pathways by which this damage leads to cell death, and to identify mechanisms by which tumour cells may circumvent this action. The levels of the DNA repair enzymes O6-alkylguanine-DNA-alkyltransferase (O6-AGAT) and 3-methyladenine-DNA-glycosylase (3MAG) have been examined in a range of murine and human cell lines with differential sensitivity to temozolomide. All the cell lines were proficient in 3MAG despite there being 40-fold difference in sensitivity to temozolomide. This suggests that while 3-methyladenine is a major product of temozolomide alkylation of DNA it is unlikely to be a cytotoxic lesion. In contrast, there was a 20-fold variation in O6-AGAT levels and the concentration of this repair enzyme correlated with variations in cytotoxicity. Furthermore, depletion of this enzyme in a resistant, O6-AGAT proficient cell line (Raji), by pre-treatment with the free base O6-methylguanine resulted in 54% sensitisation to the effects of temozolomide. These observations have been extended to 3 glioma cell lines; results that support the view that the cytotoxicity of temozolomide is related to alkylation at the O6-position of guanine and that resistance to this drug is determined by efficient repair of this lesion. It is clear, however, the other factors may influence tumour response since temozolomide showed little differential activity towards 3 established solid murine tumours in vivo, despite different tumour O6-AGAT levels. Unlike mitozolomide, temozolomide is incapable of cross-linking DNA and a mechanism by which O6-methylguanine may exert lethality is unclear. The cytotoxicity of the methyl group may be due to its disruption of DNA-protein interactions, or alternatively cell death may not be a direct result of the alkyl group itself, but manifested by DNA single-strand breaks. Enhanced alkaline elution rates were found for the DNA of Raji cells treated with temozolomide following alkyltransferase depletion, suggesting a relationship between O6-methylguanine and the induction single-strand breaks. Such breaks can activate poly(ADP-ribose) synthetase (ADPRT) an enzyme capable of rapid and lethal depletion of cellular NAD levels. However, at concentrations of temozolomlde relevant in vivo little change in adenine nucleotides was detected in cell lines, although this enzyme would appear important in modulating DNA repair since inhibition of ADPRT potentiated temozolomide cytotoxicity in Raji cells but not O6-AGAT deficient GM892A cells. Cell lines have been reported that are O6-AGAT deficient yet resistant to methylating agents. Thus, resistance to temozolomide may arise not only by removal of the methyl group from the O6-position of guanine, but also from another mechanism involving caffeine-sensitive post-replication repair or mismatch repair activity. A modification of the standard Maxam Gilbert sequencing technique was used to determine the sequence specificity of guanine-N7 alkylation. Temozolomide preferentially alkylated runs of guanines with the intensity of reaction increasing with the number of adjacent guanines in the DNA sequence. Comparable results were obtained with a polymerase-stop assay, although neither technique elucidates the sequence specificity of O6-guanine alkylation. The importance of such specificity to cytotoxicity is uncertain, although guanine-rich sequences are common to the promoter regions of oncogenes. Expression of a plasmid reporter gene under the control of the Ha-ras proto~oncogene promoter was inhibited by alkylation with temozolomide when transfected into cancer cell lines, However, this inhibition did not appear to be related to O6~guanine alkylation and therefore would seem unimportant to the chemotherapeutic activity of temozolomide.

Studies on the mode of cytotoxicity of imidazotetraziones

The irnidazotetrazinones are a novel group of anti tumour agents which have demonstrated good activity against a range of murine tumours and human xenografts. They possess a structure activity relationship similar to the anti tumour triazenes, with the chloroethyl (mitozolomide) and methyl (temozolomide) analogues being active antitumour agents, whilst the ethyl (CCRG 82019) and higher homologues are inactive. This thesiS attempts to elucidate the biological mechanisms responsible for the strict structure-activity relationship observed amongst the imidazotetrazinones. Mitozolomide is the only agent chemically capable of cross-linking DNA , which has been suggested to be responsible fo r the cytotoxicity of this group of agents. Only mitozolomide and ternozolornide Exhibit a marked ditferential toxicity towards the 0 -alkylguanine-DNA alkyltransferase deficient GM892A (Mer-) cell line rather than the proficient Raji cell line (Mer+). The rate of uptake of imidazotetrazinones into cells is similar for all three agents in both cell lines, and does not explain the differing sensitivities to these agents. The effect of drug treatment on the incorporation of precursors into macromolecules, and their pool sizes, was examined. Temozolomide administration was found to alter de novo protein synthesis in both GM892A and Raji cells. Flow cytometric analysis revealed that temozolomide and CCRG 82019 block cells in late S/G2/M phase of the cell cycle , similar to that observed with mitozolomide. The extent of reaction of all three drugs with isolated macromolecules and cellular macromolecules was determined, and differences found, with cellular repair processes influencing the number of alkyl lesions remaining bound to macromolecules. The specific bases formed in calf thymus DNA after treatment with either temozolornide and CCRG 82019 was measured, and it was found that the types and relative amounts of lesions formed, differed, as well as the total level of alkylation. Whereas DNA extracted from imidazotetrazinone treated cells is not affected in its ability to support RNA polymerase activity, an effect is observed on the ability to extract DNA polymerase from drug treated cells. This may suggest that the alkylated DNA must be in intact chromatin for the lesion to manifest its effects. Temozolomide and methyl methanesulphonate do got appear to act with a synergistic mode of action. The 0 -position of guanine is suspected to be a critical site for the action of these types of drugs.