I. Continued studies on the reaction ofortho-diamines with perfluorobutane-2,3-dione and its application to the synthesis of riboflavin synthase inhibitors. II. Initial studies in the synthesis of protoberberine-antisense oligonucleotide conjugates

Donald Alan Patrick, Purdue University

Abstract

The inhibition of riboflavin synthase, an enzyme absent in mammals but present in microorganisms, is a rational basis for the design of antimicrobial agents, allowing, in principle, for toxicity to the parasite without harming the host. Previous studies in this laboratory have established the generality of the reaction of perfluorobutane-2,3-dione with ortho-diamines to produce fluorinated, fused pyrazine heterocycles or covalent hydrates thereof. In the present work, the reaction of this reagent with 5-amino-6-ribitylamino-pyrimidine-2,4(1H,3H)-dione hydrochloride gave two epimeric, covalently hydrated products, which are fluorinated analogs of a 7,8-dihydro derivative of the substrate which is believed to bind at the donor site of the enzyme. One epimer binds to the enzyme (K$\sb{\rm D}$ value of 13 $\mu$M) and is a competitive inhibitor (K$\sb{\rm i}$ value of 120 $\mu$M). This is the first example of inhibition of the enzyme by an analog of the substrate which is sp$\sp3$ hybridized at C-7. There is no evidence that the other epimer binds to the enzyme in the absence of substrate. Antisense oligonucleotides have shown activity against human immunodeficiency virus, the etiological agent of acquired immunodeficiency syndrome. Oligonucleotide-intercalator conjugates have shown, in some cases, greater biological activity than the corresponding unmodified oligonucleotides. Protoberberine alkaloids, some of which intercalate DNA and inhibit viral reverse transcriptase, are logical candidates for conjugation to oligonucleotides. In the present work, methodology has been worked out for the covalent attachment of terminally hydroxylated linker chains to three different positions on berberine. One pathway involves the introduction of a 13-amino group, which then undergoes acylation. A second pathway involves the introduction of side chain directly attached to C-8 via a Grignard reagent. A third pathway involves O-alkylation of a known demethylation product of berberine. Two unexpected rearrangements resulting in novel ring systems, as confirmed by X-ray crystallography, were encountered in this investigation. One rearrangement, a ring expansion, resulted in a 12a-homoazaprotoberberine; the other involved an intramolecular oxidative cycloaddition which resulted in a highly bridged product. The successful conjugation of the terminally hydroxylated protoberberine derivatives to oligonucleotides via phosphoramidite intermediates is expected in the near future.

Degree

Ph.D.

Advisors

Cushman, Purdue University.

Subject Area

Organic chemistry|Pharmacology

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