The replacement of hydrogen of hydroxyl group with fluorine is an important and effective strategy In the design of 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. Fluorinated cis-urocanic acids are being used to study the mechanism of cis-urocanic acid-mediated photo-immunosuprression. 4-Alkynylimidazoles have been prepared and their behavior as substrates for ?FBr? addition (NBS + Et3N?3HF) have been studied. Facile Markownikov addition to 1-trityl-4-ethylnylimidazole gave the 1-bromo-2-fluoro-olefin, a potential synthon for the 2-imidazolyl-2-fluoro-1-ethenyl moiety. Addition of ?FBr? to 3-(1-trityl-4-imidazoyl)propargyl alcohol and reductive removal of bromine from the product produced E- and Z- 2-bromo-3-fluoro-3-(1-trityl-4-imidazoyl)-propene-1-ol, precursors to E- and Z-beta-fluorourocanic acids. The same fluoroolefins can be used as intermediates in the synthesis of beta-fluorohistidinols. Side-chain fluorinated biooindoles: Tryptamines disubstituted at the beta-position with fluorine have been synthesized as part of our research program to study the effects of fluorine substitution on the biological activities of neuroactive amines. Treatment of N-Boc-3-azidoacetyl indoles, prepared from readily available 2-chloracetylindoles, with dimethoxyethylamino sulfurtrifluoride produced the corresponding 3-(2-azido-1,1-difluoroethyl)indoles. Reduction of the azide to amine with hydrogen over Pd-C and careful removal of the N-Boc protecting group produced beta,beta-difluorotryptamines. Studies include the use of these analogues as mechanistic probes of tryptamine N-acetyltransferase. Approaches to the corresponding tryptophans are being developed. 2-Fluoroadenosine derivatives as P-site inhibitors of adenylyl cyclase: Glycosylation of 2-fluoroadenine with the appropriate protected thioglycoside derivatives, followed by deprotection and anomer separation, produced the alpha- and beta-anomers of 2?,5?-dideoxy-2-fluoro-adenosine, 2?,5?-dideoxy-2,5?-difluoro-adenosine and 2?-deoxy-2-fluoro-adenosine. These were examined as P-site inhibitors of adenylyl cyclase. The presence of fluorine on the purine ring increased potency of inhibition, and the most potent compound, beta-2?,5?-dideoxy-2-fluoro-adenosine was three times more potent than beta-2?,5?-dideoxyadenosine. Unexpectedly, there was weak, but significant, inhibition shown by the alpha-anomer of 2?,5?-dideoxy-2,5?-difluoro-adenosine. Preparation of the 3?-phosphates and 3?-polyphosphates of these analogues is in progress. Fluorinated cyclopropyl amines as inhibitors of amine oxidases: Two series of diastereopure phenylcyclopropylamine analogues, 2-fluoro-2-phenylcyclopropylamines and alkylamines, and 2-fluoro-1-phenylcyclopropylamines and methylamines were synthesized in order to study the effects of fluorine substitution on monoamine oxidase inhibition. Inhibitory activity was assayed using commercially available microbial tyramine oxidase. Characterization of tyramine oxidase, carried out prior to the inhibition experiments, confirmed earlier suggestions that this enzyme is a semicarbazide-sensitive copper-containing monoamine oxidase (CAO). The most potent competitive inhibitor was trans-2-fluoro-2-phenylcyclopropylamine, which had an IC50 value 10 times lower than that of the non-fluorinated compound, tranylcypromine. 2-Fluoro-1-phenylcyclopyl methylamine was found to be a weak non-competitive inhibitor of tyramine oxidase. The presence of a free amino group, directly bonded to the cyclopropane ring, and a fluorine atom in a cis-relationship to the amino group were structural features that increased tyramine oxidase inhibition. A series of p-substituted diastereopure cis- and trans-2-fluoro-2-arylcyclopropylamines also were synthesized and these were investigated as inhibitors of microbial tyramine oxidase. All compounds were shown to be competitive inhibitors of this enzyme. The nature of the p-substituents in the more potent trans-isomer (cis-relationship between fluorine and the amino group) of 2-fluoro-2-arylcyclopropylamine influenced the inhibitory potency in a consistent fashion. Thus, electron-withdrawing groups (F, Cl) slightly decreased the activity, while the methyl group (+ I substituent) increased the activity by factor of ca. 7 compared to trans-2-fluoro-2-phenylcyclopropylamine and by factor 90 to tranylcypromine. Activity also was strongly dependent on the absolute configuration. The (1S,2S)-enantiomer of 2-fluoro-2-phenylcyclopropylamine was an excellent inhibitor of tyramine oxidase whereas the (1R,2R)-enantiomer was essentially devoid of activity. The fluorinated phenylcyclopropylamines and alkyllamines also were examined as inhibitors of recombinant human liver monoamine oxidase A (MAO A) and B (MAO B). In the trans- and cis-2-fluoro-2-phenylcyclopropylamine series, the presence of F in the cyclopropane ring was found to result in an increase of the inhibitory toward MAO A and B. In addition, p-aromatic substitution of the aromatic ring of the trans-isomer by electron-withdrawing groups, such as Cl and F, increased the inhibition of both enzymes. (1S,2S)-2-Fluoro-2-phenylcyclopropylamine had stronger inhibitory for both MAO A and B than did the (1R,1R)-enantiomer. Interestingly, the presence of fluorine at the 2-position of 1-phenycyclopropylamine, which is known as selective inhibitor of MAO B relative to MAO A, reversed the selectivity to produce a potent MAO A selective inhibitor. These inhibitors showed time- and concentrate-dependent inhibition for both enzymes, with the exception of trans-2-fluoro-2-phenylcyclopropyl ethylamine, which acts as a competitive and reversible inhibitor. Fluorophosphonate analogues of UDP-Glc-NAc as potential inhibitors of OTG transferase. The difluoromethylene group is an isosteric and isopolar replacement of oxygen in phosphate esters. Accordingly, difluorophoshonate 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 and monfluorophoshonate analogues of UDP GlcNAc from a key C-allyl glycoside of GlcNAc. Conversion to the phosphonic acid, and alpha-fluorophosphonic acid, coupling with a protected UMP-ribose, and deprotection produced the desired analogues. Inhibition studies are in progress, along with the preparation of the difluorophosphonate analogue.
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