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1.
Mol Biosyst ; 9(11): 2816-22, 2013 Nov.
Article in English | MEDLINE | ID: mdl-24056940

ABSTRACT

The main objectives of this work were to characterise a range of purified recombinant sterol 3ß-glucosyltransferases and show that rational sampling of the diversity that exists within sterol 3ß-glucosyltransferase sequence space can result in a range of enzyme selectivities. In our study the catalytically active domain of the Saccharomyces cerevisiae 3ß-glucosyltransferase was used to mine putative sterol 3ß-glucosyltransferases from the databases. Selected diverse sequences were expressed in and purified from Escherichia coli and shown to have different selectivities for the 3ß-hydroxysteroids ergosterol and cholesterol. Surprisingly, three enzymes were also selective for testosterone, a 17ß-hydroxysteroid. This study therefore reports for the first time sterol 3ß-glucosyltransferases with selectivity for both 3ß- and 17ß-hydroxysteroids and is also the first report of recombinant 3ß-glucosyltransferases with selectivity for steroids with a hydroxyl group at positions other than C-3. These enzymes could therefore find utility in the pharmaceutical industry for the green synthesis of a range of glycosylated compounds of medicinal interest.


Subject(s)
Glucosyltransferases/metabolism , Sterols/metabolism , Testosterone/metabolism , Amino Acid Sequence , Catalysis , Catalytic Domain , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Glucosyltransferases/chemistry , Glucosyltransferases/genetics , Kinetics , Molecular Sequence Data , Protein Interaction Domains and Motifs , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Sequence Alignment , Substrate Specificity
2.
J Med Chem ; 54(6): 1847-59, 2011 Mar 24.
Article in English | MEDLINE | ID: mdl-21366300

ABSTRACT

Membrane transport of nucleosides or nucleobases is mediated by transporters including the equilibrative nucleoside transporters (ENTs), and resistance to antitumor antimetabolite drugs may arise via salvage of exogenous purine or pyrimidine nucleosides or nucleobases by ENT transporters. The therapeutic utility of dipyridamole (3), a potent ENT inhibitor, is compromised by binding to the serum protein α(1)-acid glycoprotein (AGP). Derivatives and prodrugs of the ENT inhibitor 4,8-bis[(3,4-dimethoxybenzyl)amino]-2,6-bis[(2-hydroxypropyl)amino]pyrimido[5,4-d]pyrimidine (6, NU3108) are described, with improved in vivo pharmacokinetic properties and reduced AGP binding relative to dipyridamole. The mono- and diglycine carbamate derivatives were at least as potent as 6 and showed no reduction in potency by AGP. In a [(3)H]thymidine incorporation assay, employing COR-L23 cells, the diastereoisomers of 6 (IC(50) = 26 nM) exhibited activity comparable with 3 (IC(50) = 15 nM). The monophenyl carbamate and mono-4-methoxyphenyl carbamate exhibited the best ENT-inhibitory activity in the COR-L23 assay (IC(50) = 8 and 4 nM, respectively). All of the new prodrugs were also highly effective at reversing thymidine/hypoxanthine rescue from pemetrexed cytotoxicity in the COR-L23 cell line.


Subject(s)
Antineoplastic Agents/chemical synthesis , Glutamates/pharmacology , Guanine/analogs & derivatives , Nucleosides/metabolism , Orosomucoid/pharmacology , Prodrugs/chemical synthesis , Pyrimidines/chemical synthesis , Antimetabolites, Antineoplastic/pharmacology , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/pharmacology , Biological Transport/drug effects , Cell Line, Tumor , Drug Synergism , Guanine/pharmacology , Humans , Orosomucoid/metabolism , Pemetrexed , Prodrugs/pharmacokinetics , Prodrugs/pharmacology , Protein Binding , Pyrimidines/pharmacokinetics , Pyrimidines/pharmacology , Stereoisomerism , Structure-Activity Relationship
3.
Org Biomol Chem ; 8(10): 2457-64, 2010 May 21.
Article in English | MEDLINE | ID: mdl-20448906

ABSTRACT

The attenuated S(N)2 reactivity of the 2,2,2-trifluoroethyl group has been exploited for the synthesis of a series of 6-cyclohexylmethoxy-2-arylaminopurines in which a sulfonamide moiety was attached to the aryl ring via a methylene group. These were required as potential inhibitors of serine-threonine kinases of interest for the treatment of cancer. 3-Nitrophenylmethanesulfonyl chloride was converted into the corresponding 2,2,2-trifluoroethoxysulfonyl ester by reaction with 2,2,2-trifluoroethanol in the presence of triethylamine/4-dimethylaminopyridine. Catalytic hydrogenation of the nitro group employing 2,2,2-trifluoroethanol as solvent gave 2,2,2-trifluoroethyl 3-aminophenylmethanesulfonate, which was reacted with 6-cyclohexylmethoxy-2-fluoropurine in 2,2,2-trifluoroethanol/trifluoroacetic acid to afford 2,2,2-trifluoroethyl 3-(6-cyclohexylmethoxy-9H-purin-2-ylamino)phenylmethanesulfonate. 3-(6-Cyclohexylmethoxy-9H-purin-2-ylamino)phenylmethanesulfonamides were synthesised by microwave heating of the trifluoroethoxysulfonate with an amine and 1,8-diazabicycloundec-7-ene in tetrahydrofuran. The mechanism of this process was shown to involve an intermediate sulfene by a deuterium-labelling experiment. 3-(6-Cyclohexylmethoxy-9H-purin-2-ylamino)phenylmethanesulfonamide derivatives were assayed as inhibitors of human cyclin-dependent kinase 2. Previous structure-activity studies demonstrated that relocating the sulfonamide group of O(6)-cyclohexylmethoxy-2-(4'-sulfamoylanilino)purine from the 4- to the 3-position on the 2-arylamino ring resulted in a 40-fold reduction in potency against CDK2. In the present study, no further loss of activity was observed on introducing a methylene group between the sulfonamide and the aryl ring, 3-(6-cyclohexylmethoxy-9H-purin-2-ylamino)phenylmethanesulfonamide proving equipotent with O(6)-cyclohexylmethoxy-2-(3'-sulfamoylanilino)purine (IC(50) = 0.21 microM). N-Alkylation of the sulfonamide reduced CDK-2 inhibitory activity, while a substituted benzyl or 3-phenylpropyl group on the sulfonamide resulted in a loss of potency compared with 3-(6-cyclohexylmethoxy-9H-purin-2-ylamino)phenylmethanesulfonamide. The dimethylaminopropyl derivative, 1-[3-(6-cyclohexylmethoxy-9H-purin-2-ylamino)phenyl]-N-(3-dimethylaminopropyl)methanesulfonamide was only 2-fold less potent than 3-(6-cyclohexylmethoxy-9H-purin-2-ylamino)phenylmethanesulfonamide, suggesting an interaction between the basic dimethylamino group and the kinase. The presence of alicyclic groups on the pendant sulfonamide showed IC(50) values in the 0.5-1.5 microM range. N-(4-tert-Butylphenyl)-1-[3-(6-cyclohexylmethoxy-9H-purin-2-ylamino)phenyl]methanesulfonamide was markedly less active (IC(50) = 34 microM), suggesting a steric effect within the ATP-binding domain.


Subject(s)
Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacology , Sulfonamides/chemical synthesis , Sulfonamides/pharmacology , Sulfonic Acids/chemistry , Cyclin-Dependent Kinase 2/antagonists & inhibitors , Humans , Protein Kinase Inhibitors/chemistry , Purines/chemistry , Sulfonamides/chemistry
4.
Mol Cancer Ther ; 8(7): 1828-37, 2009 Jul.
Article in English | MEDLINE | ID: mdl-19509274

ABSTRACT

Antifolates have been used to treat cancer for the last 50 years and remain the mainstay of many therapeutic regimes. Nucleoside salvage, which depends on plasma membrane transport, can compromise the activity of antifolates. The cardiovascular drug dipyridamole inhibits nucleoside transport and enhances antifolate cytotoxicity in vitro, but its clinical activity is compromised by binding to the plasma protein alpha(1)-acid glycoprotein (AGP). We report the development of a novel pyrimidopyrimidine analogue of dipyridamole, NU3153, which has equivalent potency to dipyridamole, remains active in the presence of physiologic levels of AGP, inhibits thymidine incorporation into DNA, and prevents thymidine and hypoxanthine rescue from the multitargeted antifolate, pemetrexed. Pharmacokinetic evaluation of NU3153 suggested that a soluble prodrug would improve the in vivo activity. The valine prodrug of NU3153, NU3166, rapidly broke down to NU3153 in vitro and in vivo. Plasma NU3153 concentrations commensurate with rescue inhibition in vitro were maintained for at least 16 hours following administration of NU3166 to mice at 120 mg/kg. However, maximum inhibition of thymidine incorporation into tumors was only 50%, which was insufficient to enhance pemetrexed antitumor activity in vivo. Comparison with the cell-based studies revealed that pemetrexed enhancement requires substantial (> or =90%) and durable inhibition of nucleoside transport. In conclusion, we have developed non-AGP binding nucleoside transport inhibitors. Pharmacologically active concentrations of the inhibitors can be achieved in vivo using prodrug approaches, but greater potency is required to evaluate inhibition of nucleoside rescue as a therapeutic maneuver.


Subject(s)
Dipyridamole/analogs & derivatives , Folic Acid Antagonists/pharmacology , Lung Neoplasms/drug therapy , Nucleosides/metabolism , Xenograft Model Antitumor Assays , Animals , Antimetabolites, Antineoplastic/pharmacology , Cell Proliferation/drug effects , Dipyridamole/pharmacokinetics , Dipyridamole/pharmacology , Drug Synergism , Female , Folic Acid Antagonists/pharmacokinetics , Glutamates/pharmacology , Guanine/analogs & derivatives , Guanine/pharmacology , Humans , Leukemia L1210/drug therapy , Leukemia L1210/metabolism , Leukemia L1210/pathology , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Mice , Mice, Inbred BALB C , Mice, Nude , Orosomucoid/metabolism , Pemetrexed , Phosphorylation/drug effects , Prodrugs/pharmacokinetics , Prodrugs/pharmacology , Thymidine/metabolism , Thymidylate Synthase/antagonists & inhibitors , Tissue Distribution , Tumor Cells, Cultured
5.
J Med Chem ; 49(21): 6209-21, 2006 Oct 19.
Article in English | MEDLINE | ID: mdl-17034127

ABSTRACT

From a set of weakly potent lead compounds, using in silico screening and small library synthesis, a series of 2-alkyl-3-aryl-3-alkoxyisoindolinones has been identified as inhibitors of the MDM2-p53 interaction. Two of the most potent compounds, 2-benzyl-3-(4-chlorophenyl)-3-(3-hydroxypropoxy)-2,3-dihydroisoindol-1-one (76; IC(50) = 15.9 +/- 0.8 microM) and 3-(4-chlorophenyl)-3-(4-hydroxy-3,5-dimethoxybenzyloxy)-2-propyl-2,3-dihydroisoindol-1-one (79; IC(50) = 5.3 +/- 0.9 microM), induced p53-dependent gene transcription, in a dose-dependent manner, in the MDM2 amplified, SJSA human sarcoma cell line.


Subject(s)
Antineoplastic Agents/chemical synthesis , Indoles/chemical synthesis , Proto-Oncogene Proteins c-mdm2/metabolism , Tumor Suppressor Protein p53/metabolism , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Combinatorial Chemistry Techniques , Drug Screening Assays, Antitumor , Humans , Indoles/chemistry , Indoles/pharmacology , Models, Molecular , Protein Binding , Stereoisomerism , Structure-Activity Relationship , Transcription, Genetic , Tumor Suppressor Protein p53/genetics
6.
Bioorg Med Chem Lett ; 15(5): 1515-20, 2005 Mar 01.
Article in English | MEDLINE | ID: mdl-15713419

ABSTRACT

A series of 2-N-alkyl-3-aryl-3-alkoxyisoindolinones has been synthesised and evaluated as inhibitors of the MDM2-p53 interaction. The most potent compound, 3-(4-chlorophenyl)-3-(4-hydroxy-3,5-dimethoxybenzyloxy)-2-propyl-2,3-dihydroisoindol-1-one (NU8231), exhibited an IC50 of 5.3 +/- 0.9 microM in an ELISA assay, and induced p53-dependent gene transcription in a dose-dependent manner, in the SJSA human sarcoma cell line.


Subject(s)
Indoles/chemistry , Indoles/pharmacology , Nuclear Proteins/drug effects , Proto-Oncogene Proteins/drug effects , Tumor Suppressor Protein p53/drug effects , Cell Line, Tumor , Dose-Response Relationship, Drug , Humans , Indoles/chemical synthesis , Models, Molecular , Molecular Structure , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Protein Binding/drug effects , Proto-Oncogene Proteins/chemistry , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-mdm2 , Structure-Activity Relationship , Transcription, Genetic/drug effects , Tumor Suppressor Protein p53/chemistry , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism
7.
Beilstein J Org Chem ; 1(1): 13, 2005 Oct 17.
Article in English | MEDLINE | ID: mdl-16542024

ABSTRACT

BACKGROUND: Asymmetric introduction of fluorine alpha-to a carbonyl has become popular recently, largely because the direct fluorination of enolates by asymmetric electrophilic fluorinating reagents has improved, and as a result such compounds are becoming attractive synthons. We have sought an alternative but straightforward asymmetric method to this class of compounds, utilising the zwitterionic aza-Claisen rearrangement by reacting alpha-fluoroacid chlorides and homochiral N-allylpyrrolidines as starting materials. RESULTS: Treatment of N-allylmorpholine with 2-fluoropropionyl chloride under Yb(OTf)3 catalysis generated the zwitterionic aza-Claisen rearrangement product in good yield and demonstrated the chemical feasibility of the approach. For the asymmetric reaction, N-allyl-(S)-2-(methoxymethyl)pyrrolidine was treated with either 2-fluoropropionyl chloride or 2-fluorophenylacetic acid chloride under similar conditions and resulted in N-(alpha-fluoro-gamma-vinylamide)pyrrolidine products as homochiral materials in 99% de. These products were readily converted to their corresponding alpha-fluoro-gamma-lactones by iodolactonisation and in good diastereoselectivity. CONCLUSION: Molecules which have fluorine at a stereogeneic centre are finding increasing utility in pharmaceutical, fine chemicals and materials research. The zwitterionic aza-Claisen rearrangement proved to be an effective and competitive complement to asymmetric electrophilic fluorination strategies and provides access to versatile synthetic intermediates with fluorine at the stereogenic centre.

8.
J Nat Prod ; 67(9): 1476-82, 2004 Sep.
Article in English | MEDLINE | ID: mdl-15387645

ABSTRACT

The yellow cedar tree, Chamaecyparis nootkatensis, collected in southeast Alaska was evaluated as a potential source of new anticancer agents. Two new diterpene anticancer constituents termed nootkastatins 1 (4) and 2 (5) were isolated along with three previously known diterpene cancer cell growth inhibitors where two were reported as synthetic modifications of totarol and not previously found in nature. All five diterpene structures were established by HRMS and 1D and 2D NMR spectroscopic analyses combined with three X-ray crystal structure determinations (2, 3, and 5). Against a panel of six human cancer cell lines, this series of diterpenes exhibited inhibition over the range GI(50) 0.75-2.0 microg/mL, and all inhibited the growth of Gram-positive bacteria and fungi.


Subject(s)
Antineoplastic Agents, Phytogenic/isolation & purification , Chamaecyparis/chemistry , Diterpenes/isolation & purification , Plants, Medicinal/chemistry , Alaska , Animals , Antineoplastic Agents, Phytogenic/chemistry , Antineoplastic Agents, Phytogenic/pharmacology , Diterpenes/chemistry , Diterpenes/pharmacology , Drug Screening Assays, Antitumor , Fungi/drug effects , Gram-Positive Bacteria/drug effects , Humans , Leukemia P388 , Mice , Molecular Structure , Nuclear Magnetic Resonance, Biomolecular , Trees/chemistry , Tumor Cells, Cultured
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