The replacement of hydrogen of hydroxyl group with fluorine is an important and effective strategy for designing analogues of biologically important molecules. The small size of fluorine and high electronegativty are factors that contribute to the value of many of these analogues as pharmacological tools and medicinal agents. Included in our research in this area has been the preparation and biological evaluation of ring fluorinated imidazoles, biogenic amines and amino acids and related compounds. We are continuing the work with ring-fluorinated analogues, and have extended this now to side chain fluorinated analogues of these important biological molecules. Side-chain fluorinated bioimidazoles: Ring-fluorinated imidazoles we previously prepared have been very valuable as biochemical and pharmacological agents. We now have developed syntheses of side-chain fluorinated biologically important imidazoles, including beta-fluoro- and beta,beta-difluorohistamine, beta-fluorourocanic acid, and alpha,beta-difluorourocanic acid. We are extending this to the corresponding histidine derivatives, and biosynthetic precursors of histidine. New approaches to side chain fluorinated histidines and histidinols involve addition of a Grignard reagent derived from a protected 4-iodoimidazole with optically active Garner aldehyde. This installs the enantiopure (S)-alpha-amino group and a beta-hydroxyl group. Deoxyfluorination and deprotection provides a route to optically active beta-fluorohistidinols, compounds previously prepared in racemic form. Oxidation of the ?benzylic? OH to a ketone and deoxyfluorination similarly provides a route to beta,beta-difluorohisitidinol. If oxidation of the histidinols to histidines is not successful, a carboxy-protected beta-oxo glycine derivative will be used in this sequence with the imidazole Grignard reagent. Similar chemistry is in progress in the indole series as a route to tryptophans. In another approach, diasteroselective alkylaion of protected imidazole and indole aldehydes produced chiral precursors having an OH group in the ?benzylic? position. These were subjected to deoxyfluoroination. To date, hydrolysis of the chiral auxiliary has not been achieved without loss of fluorine. A new synthesis of 4-fluoro-L-histidine: L-4-Fluorohistidine has been used as a substrate for incorporation into peptides. In order to take advantage of new molecular biological methods to do this incorporation, new supplies of this important amino acid analogue are needed. In the past we prepared this compound from ethyl 4-fluoroimidazole-5-carboxylate using a multi-step process that included side chain elaboration followed by enzymatic deacylation of the S-enantiomer of the N-acetyl precursor. In an alternative approach, we carried out in situ reduction of a protected 4-nitro-L-histidine, followed by diazotization, photolysis, and deprotection. The latter route was more direct, but proceeded in low yield. We have now carried out diastereoselective alkylation of an intermediate derived from ethyl 4-fluoroimidazole-5-carboxylate, followed by deprotection of the side chain. This route provides the S-isomer directly, obviating enzymatic resolution. Fluorinated cyclopropyl amines as inhibitors of amine oxidases: 1-Phenylcyclopropylamine is an irreversible inhibitor of MAO with selectivity for MAO B over MAO A. We have shown that E-2-fluoro-1-phenycyclopropylamine is a potent irreversible MAO A selective inhibitor. We now have shown that the Z-isomer has modestly greater activity. Additional examples of 1-aryl-2-fluorocyclopropylamnes have been prepared and evaluated (E- and Z-isomers, para-substituted with F, Cl, Me, MeO) and all show potent inhibition of MAO A. The most potent compound, Z-2-fluoro-1-(para-methylphenyl)cyclopropylamine, had an IC50 of 0.3 micromolar, or about 2400 times more potent than the parent 1-phenylcyclopropylamine. Cyclopropaaanne ring opening is an important part of the mechanism of inhibition of cyclopropylamines. Structural studies have shown tranylcypromine forms a ring-opened C4a adduct with the FAD coenzyme of MAO B as the mode of irreversible inhibition. Silverman has proposed a SET mechanism for oxidation that would lead to homolytic ring opening of the cyclopropy ring and subsequent covalent bonding and irreversible inhibition. Since fluorine is known to increase the strain energy of cyclopropane, this could facilitate ring opening, leading to the increased potency observed. Accordingly, we prepared 2,2-difluoro-1-phenylcyclopropyl amine, to see if the even greater ring strain would lead to a more potent inhibitor. This compound was four-fold less potent than Z-2-fluoro-2-phenylcyclopropylamine for MAO A, and showed no inhibition of MAO B. Since these inhibitors displayed only modest diastereoselectivity, we felt it would be important to examine the enantioselective of inhibition. Diastereomeric amides of (E)-2-fluoro-1-phenylcyclopropylamine were prepared and separated. A crystal structure of one diastereomer was determined, and shown to possess the (E)-1-R,2-S configuration. The amide groups of the diasteromeric amides have been removed, and the enantiopure amines are being examined as inhibitors of MAO A and B. Fluorophosphonate analogues of UDP-Glc-NAc as potential inhibitors of OGT transferase. The difluoromethylene group is an isosteric and isopolar replacement of oxygen in phosphate esters. Accordingly, difluorophosphonate analogues of biologically important phosphate esters have been prepared and studied extensively. We are using this strategy to prepare potential inhibitors of OGT transferase, the enzyme that catalyzes the transfer of GlcNAc to serine and threonine residues in proteins. We have prepared the phosphonate analogue of UDP GlcNAc from a key C-allyl glycoside of GlcNAc. Conversion to the phosphonic acid, coupling with a protected UMP-ribose, and deprotection produced the desired analogues. Inhibition studies are in progress, along with the preparation of the monofluoro- and difluorophosphonate analogue. The yields in certain steps of the above sequence unfortunately are quite low. In order to have more direct access to key intermediates we are exploring alternative routes. In one of these, the stereoselective syntheses of 3,4,6-tri-O-benzyl alpha- and beta-1-lithio-N-acetylglucosamines are being carried out. Direct alkylation with carbethoxy phosphonic acid diethyl ester will then be attempted in an effort to access directly the alpha-ketophosphonate intermediate needed for preparation of the difluorophosnonate anogue of UDP-Glc-NAc.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Intramural Research (Z01)
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U.S. National Inst Diabetes/Digst/Kidney
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Hajduch, Jan; Nam, Ghilsoo; Kim, Eun Ju et al. (2008) A convenient synthesis of the C-1-phosphonate analogue of UDP-GlcNAc and its evaluation as an inhibitor of O-linked GlcNAc transferase (OGT). Carbohydr Res 343:189-95
Hajduch, Jan; Cramer, John C; Kirk, Kenneth L (2008) An Enantioselective Synthesis of (S)-4-Fluorohistidine. J Fluor Chem 129:807-810
Hajduch, Jan; Dolensky, Bohumil; Yoshida, Shinichi et al. (2008) Synthesis of (E)- and (Z)-alpha,beta-Difluorourocanic Acid. J Fluor Chem 129:112-118
Fujiwara, Tomoya; Yin, Bin; Jin, Meixiang et al. (2008) Synthetic Studies of 3-(3-Fluorooxindol-3-yl)-l-alanine. J Fluor Chem 129:829-835
Heredia-Moya, Jorge; Kirk, Kenneth L (2007) Photochemical Schiemann Reaction in Ionic Liquids. J Fluor Chem 128:674-678
Wimalasena, D Shyamali; Cramer, John C; Janowiak, Blythe E et al. (2007) Effect of 2-fluorohistidine labeling of the anthrax protective antigen on stability, pore formation, and translocation. Biochemistry 46:14928-36
Nagasawa, Hideko; Uto, Yoshihiro; Kirk, Kenneth Lee et al. (2006) Design of hypoxia-targeting drugs as new cancer chemotherapeutics. Biol Pharm Bull 29:2335-42
Uto, Yoshihiro; Ae, Shutaro; Koyama, Daisuke et al. (2006) Artepillin C isoprenomics: design and synthesis of artepillin C isoprene analogues as lipid peroxidation inhibitor having low mitochondrial toxicity. Bioorg Med Chem 14:5721-8
Kirk, Kenneth L (2006) Selective fluorination in drug design and development: an overview of biochemical rationales. Curr Top Med Chem 6:1447-56
Eichler, Jack F; Cramer, John C; Kirk, Kenneth L et al. (2005) Biosynthetic incorporation of fluorohistidine into proteins in E. coli: a new probe of macromolecular structure. Chembiochem 6:2170-3

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