Stereoselective synthesis of nucleoside analogues

Details Stereoselective synthesis of nucleoside analogues Stereoselective synthesis of nucleoside analogues

2001-02-09

2003-04-01

Barts, Samuel (Department: 1623)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C435S041000, C435S087000, C435S088000, C435S280000, C536S022100

Reexamination Certificate

active

06541625

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates generally to a novel method for the preparation of nucleoside analogues and their precursors and more particularly to a method of preparing a nucleoside analogue by the use of specific enzymes to stereoselectively produce dioxolane nucleoside analogues or their precursors.
BACKGROUND OF THE INVENTION
An important class of pharmacological agents relate to 3′-oxa-substituted 2′,3′-dideoxynucleoside analogues (“dioxolane nucleoside analogues”). These compounds include 9-(&bgr;-D-2-hydroxymethyl-1,3-dioxolan-4-yl)-2-aminopurine (&bgr;-D-DAPD); 9-(&bgr;-D-2-hydroxymethyl-1,3-dioxolan-4-yl)-guanine (&bgr;-D-DXG); 1-(&bgr;-L-2-hydroxymethyl-1,3-dioxolan-4-yl)-thymine (Dioxolane-T); and 1-(&bgr;-L-2-hydroxymethyl-1,3-dioxolan-4-yl)-cytidine (&bgr;-L-OddC) which have known antiviral and antitumor activity.
As shown in the following dioxolane structure, dioxolanes have two chiral centers corresponding to the substituted carbons 2 and 4 of the dioxolane ring (C2 and C4 respectively). Thus each compound can exist as four different stereoisomers depending on the position of both substituents with respect to the dioxolane ring.
The stereoisomers of a dioxolane nucleoside analogue are represented by the following diagrams where the letter B represents a purine or pyrimidine base or an analogue or derivative of a purine or pyrimidine base as defined herewith.
For the purpose of consistency, the same stereochemical designation will be used even when the hydroxymethyl moiety or the base moiety (B) is replaced with another substituent group.
Chiral synthetic methods have improved over the past several years with respect to synthetic techniques that result in single stereoisomer compounds. However, there is a present need to find novel synthetic methods which can be widely used to form a particular stereoisomer with greater efficiency and purity.
For example, for many years a person of ordinary skill in the art could use enzymes to separate enantiomers of dioxolane compounds. However, there is still a need in the art to produce a dioxolane nucleoside analogue using a step of separating an anomeric mixture of certain dioxolane precursors to produce an end product with greater efficiency and purity.
Because stereochemically pure dioxolane nucleosides are an important class of compounds due to their known antiviral activity and anticancer activity, there is a need for other inexpensive and efficient stereoselective methods for their preparation. The present invention satisfies this and other needs.
SUMMARY OF THE INVENTION
The present invention provides a novel process for making dioxolane nucleoside analogues with a high degree of steric purity, greater efficiency and higher yields.
Specifically, the present invention provides a process for making dioxolane nucleoside analogues with a high degree of steric purity which includes the use of certain hydrolytic enzymes for separating &bgr; and &agr; anomers from an anomeric mixture represented by the following formula A or formula B:
wherein R
1
is selected from the group consisting of C
1-6
alkyl and C
6-15
aryl; Bz is Benzoyl.
The process involves the step of hydrolyzing the mixture of compounds represented by formula A and/or formula B with an enzyme selected from the group consisting of Protease N (Bacillus subtilus protease), Alcalase® (Subtilisin Carlsberg protease), Savinase® (Bacillus lentus subtilisin protease), ChiroCLEC™-BL (Bacillus licheniformis Subtilisin protease), PS-30 (Pseudomonas cepacia lipase), and ChiroCLEC™-PC (Pseudomonas cepacia lipase). The process stereoselectively hydrolyses predominantly one anomer to form a product where R
1
of formula A and formula B is replaced with H. The other anomer remains substantially unhydrolysed. The process also comprises separating the hydrolyzed product from unhydrolysed starting material.
According to one embodiment of the invention, the aforementioned steps of hydrolyzing and separating results in an isolated starting material having an anomeric purity of at least 97% &bgr;-anomer. In an additional embodiment, the aforementioned steps of hydrolyzing and separating results in an isolated starting material having an anomeric purity of at least 98% &bgr;-anomer. In an additional embodiment, the aforementioned steps of hydrolyzing and separating results in an isolated starting material having an anomeric purity of at least 98.5% &bgr;-anomer. In an additional embodiment, the aforementioned steps of hydrolyzing and separating results in an isolated starting material having an anomeric purity of at least 98.8% &bgr;-anomer.
According to one embodiment of the invention, the aforementioned steps of hydrolyzing and separating results in an isolated product having an anomeric purity of at least 97% &agr;-anomer. In an additional embodiment, the aforementioned steps of hydrolyzing and separating results in an isolated product having an anomeric purity of at least 98% &agr;-anomer. In an additional embodiment, the aforementioned steps of hydrolyzing and separating results in an isolated product having an anomeric purity of at least 98.5% &agr;-anomer. In an additional embodiment, the aforementioned steps of hydrolyzing and separating results in an isolated product having an anomeric purity of at least 98.8% &agr;-anomer.
In one embodiment, the &bgr;-anomer is the predominant product. In another embodiment, the &agr;-anomer is the predominant product. In yet another embodiment, the &bgr;-L-enantiomer is the predominant product. In an additional embodiment, the &bgr;-D-enantiomer is the predominant product. In yet another embodiment, the &agr;-L-enantiomer is the predominant product. In an additional embodiment, the &agr;-D-enantiomer is the predominant product.
In one embodiment, the invention is a process for stereoselectively preparing a dioxolane nucleoside analogue by separating &bgr; and &agr;-nomers from an anomeric mixture represented by formula A or formula B according to one of the above embodiments. The process further includes the step of stereoselectively replacing the functional group at the C4 position (COOR
1
) with a purinyl or pyrimidinyl or analogue or derivative selected from the group consisting of:
In this embodiment, R
2
, R
9
and R
11
are independently selected from the group consisting of hydrogen, C
1-6
alkyl, C
1-6
acyl and R
8
C(O) wherein R
8
is hydrogen or C
1-6
alkyl. Additionally, R
3
, R
4
and R
10
are each independently selected from the group consisting of hydrogen, C
1-6
alkyl, bromine, chlorine, fluorine, iodine and CF
3
; and R
5
, R
6
and R
7
are each independently selected from the group consisting of hydrogen, bromine, chlorine, fluorine, iodine, amino, hydroxyl and C
3-6
cycloalkylamino. The process results in the production of a stereochemical isomer of the dioxolane nucleoside analogue.
According to one embodiment, the process further includes the step of stereoselectively replacing the functional group at the C4 position (COOR
1
) with a purinyl or pyrimidinyl or derivative selected from the group consisting of:
In this embodiment, R
2
is selected from the group consisting of hydrogen, C
1-6
alkyl, C
1-6
acyl and R
8
C(O) wherein R
8
is hydrogen or C
1-6
alkyl. Additionally, R
3
and R
4
are each independently selected from the group consisting of hydrogen, C
1-6
alkyl, bromine, chlorine, fluorine, iodine and CF
3
; and R
5
, R
6
and R
7
are each independently selected from the group consisting of hydrogen, bromine, chlorine, fluorine, iodine, amino, hydroxyl and C
3-6
cycloalkylamino. The process results in the production of a stereochemical isomer of a dioxolane nucleoside analogue.
In another embodiment, the process further includes the step of stereoselectively replacing the functional group at the C4 position (COOR
1
) with a pyrimidinyl or analogue or derivative selected from the group consisting of:
In this embodiment, R
9
and R
11
are independently selected from the group consisting of hydrogen, C
1-6
alkyl, C
1-6
acyl and R
8
C(O). Additionally,

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