Fluorinated Analogues: Biochemistry/Pharmacology

用氟取代羟基的氢是设计生物学重要分子类似物的重要且有效的策略。氟的小尺寸和高电负性是许多这些类似物作为药理学工具和药剂的价值的因素。 我们在这一领域的研究包括环氟化咪唑、生物胺和氨基酸及相关化合物的制备和生物学评价。我们正在继续研究环氟化类似物，现在已将其扩展到这些重要生物分子的侧链氟化类似物。 侧链氟化生物咪唑： 我们之前制备的环氟化咪唑作为生化和药理学试剂非常有价值。我们现在已经开发出具有重要生物学意义的侧链氟化咪唑的合成方法，包括β-氟组胺和β,β-二氟组胺、β-氟尿刊酸和α,β-二氟尿刊酸。我们将其扩展到相应的组氨酸衍生物和组氨酸的生物合成前体。 侧链氟化组氨酸和组氨醇的新方法涉及添加源自受保护的 4-碘咪唑和光学活性加纳醛的格氏试剂。这会安装对映体纯 (S)-α-氨基和 β-羟基。脱氧氟化和脱保护提供了获得光学活性β-氟组氨醇（之前以外消旋形式制备的化合物）的途径。 ？苯甲基？的氧化？ OH 生成酮和脱氧氟化类似地提供了生成 β,β-二氟组氨酸酚的途径。如果组氨醇氧化成组氨酸不成功，则在此序列中将使用羧基保护的β-氧代甘氨酸衍生物与咪唑格氏试剂。作为生产色氨酸的途径，类似的化学反应正在吲哚系列中进行。在另一种方法中，受保护的咪唑和吲哚醛的非对映选择性烷基化产生在“苄基”中具有OH基团的手性前体。位置。将它们进行脱氧氟化。迄今为止，尚未实现手性助剂的水解而不损失氟。 4-氟-L-组氨酸的新合成： L-4-氟组氨酸已被用作掺入肽的底物。为了利用新的分子生物学方法进行这种掺入，需要这种重要氨基酸类似物的新供应。过去，我们采用多步工艺从 4-氟咪唑-5-甲酸乙酯制备该化合物，其中包括侧链精制，然后对 N-乙酰基前体的 S-对映体进行酶促脱酰化。在另一种方法中，我们对受保护的 4-硝基-L-组氨酸进行了原位还原，然后进行重氮化、光解和脱保护。后一条路线更为直接，但产量较低。我们现在对衍生自 4-氟咪唑-5-甲酸乙酯的中间体进行了非对映选择性烷基化，然后对侧链进行脱保护。该路线直接提供S-异构体，避免了酶促拆分。 作为胺氧化酶抑制剂的氟化环丙胺： 1-苯基环丙胺是一种不可逆的 MAO 抑制剂，对 MAO B 的选择性高于 MAO A。我们已经证明，E-2-氟-1-苯基环丙胺是一种有效的不可逆 MAO A 选择性抑制剂。我们现在已经证明，Z 异构体具有稍强的活性。已制备并评估了 1-芳基-2-氟环丙胺的其他实例（E-和 Z-异构体，对位被 F、Cl、Me、MeO 取代），并且均显示出对 MAAO A 的有效抑制作用。最有效的化合物 Z-2-氟-1-（对甲基苯基）环丙胺的 IC50 为 0.3 微摩尔，或比母体 1-苯基环丙胺强约 2400 倍。 环丙胺开环是环丙胺抑制机制的重要组成部分。结构研究表明反苯环丙明与 MAO B 的 FAD 辅酶形成开环 C4a 加合物，作为不可逆抑制的模式。 Silverman 提出了一种 SET 氧化机制，该机制将导致环丙基环均裂开环以及随后的共价键合和不可逆抑制。由于已知氟会增加环丙烷的应变能，因此这可能会促进开环，从而导致观察到的效力增加。因此，我们制备了 2,2-二氟-1-苯基环丙胺，以观察更大的环张力是否会产生更有效的抑制剂。该化合物对 MAO A 的作用比 Z-2-氟-2-苯基环丙胺低四倍，并且对 MAO B 没有抑制作用。 由于这些抑制剂仅表现出适度的非对映选择性，因此我们认为检查抑制的对映选择性非常重要。制备并分离(E)-2-氟-1-苯基环丙胺的非对映酰胺。确定了一种非对映异构体的晶体结构，并显示其具有(E)-1-R,2-S构型。非对映体酰胺的酰胺基团已被除去，对映体纯胺正在作为 MAO A 和 B 的抑制剂进行研究。 UDP-Glc-NAc 的氟磷酸盐类似物作为 OGT 转移酶的潜在抑制剂。 二氟亚甲基是磷酸酯中氧的等排和等极取代。因此，已制备并广泛研究了生物学上重要的磷酸酯的二氟膦酸酯类似物。我们正在使用这种策略来制备 OGT 转移酶的潜在抑制剂，该酶催化 GlcNAc 转移到蛋白质中的丝氨酸和苏氨酸残基。我们从 GlcNAc 的关键 C-烯丙基糖苷制备了 UDP GlcNAc 的膦酸酯类似物。转化为膦酸，与受保护的UMP-核糖偶联，然后脱保护产生所需的类似物。抑制研究以及单氟膦酸盐和二氟膦酸盐类似物的制备正在进行中。 不幸的是，上述序列的某些步骤的产率相当低。为了更直接地获得关键中间体，我们正在探索替代路线。其中一项研究正在进行3,4,6-三-O-苄基α-和β-1-锂-N-乙酰葡糖胺的立体选择性合成。然后将尝试用乙酯氧基膦酸二乙酯进行直接烷基化，以直接获得制备UDP-Glc-NAc的二氟膦酸酯类似物所需的α-酮膦酸酯中间体。

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.