Organic compounds -- part of the class 532-570 series – Organic compounds – Heterocyclic carbon compounds containing a hetero ring...
Reexamination Certificate
2000-09-05
2002-11-05
Solola, T. A. (Department: 1626)
Organic compounds -- part of the class 532-570 series
Organic compounds
Heterocyclic carbon compounds containing a hetero ring...
C549S406000
Reexamination Certificate
active
06476241
ABSTRACT:
FIELD OF THE INVENTION
This invention, in general, relates to polyphenolic products, particularly proanthocyanidins. The invention further relates to synthetic processes for preparing polyphenolic natural products and other related compounds.
BACKGROUND OF THE INVENTION
Proanthocyanidins (non-hydrolyzable tannins) are a group of polyphenolic natural products, of current interest because of their numerous biological activities, their widespread occurrence in foodstuffs, and their resulting relevance for human health.
Proanthocyanidins are dimeric or oligomeric flavanoids ch have one or several hydroxyl groups on their aromatic rings and often an additional hydroxyl group in the 3 position. Eleven different hydroxylation patterns of the A and B rings have been found in nature. Representative proanthocyanidins include:
Substitution Pattern
Class
Monomer
3
5
7
8
3′
4′
5′
Proapigeninidin
Apigeniflavan
H
OH
OH
H
H
OH
H
Proluteolinidin
Luteoliflavan
H
OH
OH
H
OH
OH
H
Protricetinidin
Tricetiflavan
H
OH
OH
H
OH
OH
OH
Propelargonidin
Afzelechin
OH
OH
OH
H
H
OH
H
Procyanidin
Catechin
OH
OH
OH
H
OH
OH
H
Prodelphinidin
Gallocatechin
OH
OH
OH
H
OH
OH
OH
Proguibourtinidin
Guibourtinidol
OH
H
OH
H
H
OH
H
Profisetinidin
Fisetinidol
OH
H
OH
H
OH
OH
H
Prorobinetinidin
Robinetinidol
OH
H
OH
H
OH
OH
OH
Proteracacinidin
Oritin
OH
H
OH
OH
H
OH
H
Promelacacinidin
Prosopin
OH
H
OH
OH
OH
OH
H
The stereochemistry of the substituents on a polyphenol monomeric unit of a proanthocyanidin may be described in terms of their relative stereochemistry, “alpha/beta” or “cis/trans”. The term “alpha” (&agr;) indicates that the substituent is oriented below the plane of the flavan ring, whereas, “beta” (&bgr;) indicates that the substituent is oriented above the plane of the ring. The term “cis” indicates that two substituents are oriented on the same face of the ring, whereas “trans” indicates that two substituents are oriented on opposite faces of the ring.
The isolation of pure proanthocyanidins from natural sources becomes increasingly difficult with increasing degree of oligomerization. Degradation by thiolysis permits identification of the underlying monomeric units but the tasks of elucidating the position and stereochemistry of the interflavan linkages is nontrivial. Both of these factors have resulted in few defined oligomers above the tetramer being described in the prior art. Proanthocyanidins and their parent monomers also occur naturally in the form of a variety of derivatives, for example, glycosides or esters with hydroxylated aromatic carboxylic acids, such as gallic or hexahydroxydiphenic acid.
Among the proanthocyanidins, two subtypes, the procyanidins (5,7,3′,4′-hydroxylation) and prodelphinidins (5,7,3′,4′,5′-hydroxylation), are widespread in human foodstuffs, e.g., cocoa. Cocoa procyanidins consist predominantly of epicatechin (the C-3 epimer of catechin) building blocks. Oligomers up to the size of the decamer have been identified. From the pentamer on, these oligomers exhibit growth inhibitory activity against various cancer cell lines. (Romanczk, L. J. Jr.; Hammerstone, J. F., Jr.; Buck, M. M. U.S. Pat. No. 5,554,645, Sep. 10, 1996.) Flavan-3-ols are biosynthetically derived from (2S)-phenylalanine via flavan-3,4-diols. These latter intermediates readily form a highly stabilized carbenium ion (or qu iinone methide) in position C-4 which attacks the A ring of a flavan-3-ol in what is essentially a Friedel-Crafts alkylation process, forming an interflavan bond. This process can be repeat once or several times, resulting in chain-type oligomers which together with the dimers are known as non-hydrolyzable tannins, condensed tannins, or proanthocyanidins. As one skilled in the art will realize, the structural complexity of these compounds rapidly increases with their chain length as a consequence of different hydroxylation patterns and C-3 stereochemistry in the monomer unit and different regio- and stereochemistries of the interflavan linkages, as well as additional structural modifications. In addition, chain branching may occur by alkylation of a monomer unit in both its 6- and 8-positions.
To prove definitively the structures assigned to the compounds purified from cocoa, comparisons must be made to epicatechin dimers and oligomers of defined structure prepared synthetically. Synthetic monomers, dimers and oligomers are useful to develop structure-activity relationships in various in vitro and ultimately in vivo models of anticancer activity.
The synthetic challenge posed by procyanidins is related to the difficulty in controlling the interflavan regio- and stereochemistry, as well as the sensitivity of the nonprotected compounds to acids, bases, and oxidizing agents. The condensation between flavan-3-ols and 4-substituted, electrophilic flavans has traditionally been performed without the use of phenol protecting groups in a mildly acidic medium or recently, with AgBF
4
for benzylthio as the 4-substituent. The products are mixtures of regio- and sometimes stereoisomers, as well as higher oligomers despite the application of an excess of the nucleophilic building block. They have usually been separated by gel chromatography on Sephadex LH-20, a process that requires a considerable investment of time to develop for each particular se paration task because of the unavailability of fast analytical tools such as HPLC columns or thin layer plates for this adsorbent. In addition, optically pure, nonprotected 4-substituted catechins and epicatechins are not readily available, being prepared by reduction of the expensive natural product, (+)-taxifolin (the 4-ketone) or by in situ degradation or thiolytic degradation of natural proanthocyanidin oligomic fractions for which commercial sources are difficult to identify or nonexistant.
It is therefore not surprising that prior art syntheses have used protected oligomeric procyanidins as building blocks. As an additional incentive, prote ction of the phenolic but not of the alcoholic hydroxyls would permit the regioselective elaboration of derivatives such as 3-esters and -glycosides, as has been done in the case of catechin using acetyl protecting groups. An interesting approach has been reported in which the 8-bromo derivative of 3-O-benzyl-5,7,3′,4′-tetra-O-methylcatechin was subjected to halogen-lithium exchange and reacted with an O-methylated 4-ketone, thus ensuring complete regio control. However, the methyl O-blocking groups cannot be removed to obtain the free dimer. The remaining published work has made use of the above-described electrophilic substitution process with inclusion of phenol protecting groups on one or both of the reaction partners.
Our own previous work directed toward the synthesis of defined epicatechin oligomers used the TiCl
4
-mediated alkylation of 5,7,3′,4′-tetra-O-benzyl-(−)-epicatechin with 5,7,3′,4′-tetra-O-benzyl-4-(2-hydroxyethoxy)epicatechin. Besides higher oligomers, whose yields rapidly decrease with increased molecular mass, a single dimeric product having beta stereochemistry of the interflavan bond (a procyanidin B
2
derivative) was obtained.
Until quite recently, the analytical methods employed for the assignment of interflavan bond regio- and stereochemistry in these compounds were not validated by an independent confirmation of the structure of any dimeric proanthocyanidin. The application of X-ray crystallography has been prevented by the poor crystallizability of proanthocyanidins and their derivatives. Assignments of stereochemistry on the basis of
1
H NMR coupling constants and circular dichroism disregard the basic fact that the C rings are conformationally flexible. From a conservative point of view, postulates of specific conformations of flexible molecules, regardless of their source (intuitive or computational), cannot be considered a prudent approach to structure elucidation.
Advances in synthetic methodology, which have taken place after the isolation of numerous proanthocyanidins from natural source
Kozikowski Alan P.
Romanczyk Jr. Leo J.
Tückmantel Werner
Clifford Chance (US) LLP
Kelley Margaret B.
Mars Incorporated
Solola T. A.
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