Acridine ester and acridinium ester synthesis

Claim 2

A process for the preparation of an acridine ester of general formula III wherein R 2 , R 3 , n and p are as defined below for general formula I; R S and q are as defined below for general formula II; and Z represents a group able to react with an amino group of a derivatised nucleic acid or protein; the process being characterised by reacting a compound of general formula I wherein each of R 2 and R 3 independently represent one or more substituents which may be the same as or different from each other and which will not interfere with the desired specific coupling chemistry of the compound of general formula I; each of n and p is independently an integer of from 1 to 4; and R 4 represents a leaving group; with a compound of general formula II wherein R 5 represents one or more substituents which may be the same as or different from each other and which are unreactive with compounds of general formula I and unreactive with compounds of general formula II; q is an integer of from 1 to 4; and Z is as defined for general formula III.

Claim 3

A process for the preparation of an acridinium ester of general formula IV wherein R 2 , R 3 , n and p are as defined below for general formula I; R 5 and q are as defined below for general formula II; Z represents a group able to react with an amino group of a derivatized nucleic acid or protein; R A represents a hydrogen atom or a C 1 -C 6 alkyl radical; and X- represents an anion or a monovalent portion of an anion; the process being characterised by reacting a compound of general formula I wherein each of R 2 and R 3 independently represent one or more substituents which may be the same as or different from each other and which will not interfere with the desired specific coupling chemistry of the compound of general formula I; each of n and p is independently an integer of from 1 to 4; and R 4 represents a leaving group; with a compound of general formula II wherein R 5 represents one or more substituents which may be the same as or different from each other and which are unreactive with compounds of general formula I and unreactive with compounds of general formula II; q is an integer of from 1 to 4; and Z is as defined for general formula IV, to form an acridine ester of general formula III wherein R2 , R3 , n and p are as defined for general formula I R 5 and q are as defined for general formula II; and Z is as defined for general formula I and subsequently either forming an acid addition salt of the compound of general formula III by reaction with an appropriate acid to form a compound of general formula IV wherein R A represents a hydrogen atom or N-alkylating the compound of general formula III with an N-alkylating agent containing the radical R AI wherein R A represents a C 1 -C 6 alkyl radical to form a compound of general formula IV wherein R A represents a C 1 -C 6 alkyl radical.

Claim 4

A process for the preparation of an acridinium ester of general formula IV wherein R 2 , R 3 , n and p are as defined below for general formula I; R 5 and q are as defined below for general formula II; Z represents a group able to react with an amino group of a derivatized nucleic acid or protein; R A represents a hydrogen atom or a C 1 -C 6 alkyl radical; and X- represents an anion or a monovalent portion of an anion; the process being characterised by reacting a compound of general formula I wherein each of R 2 and R 3 independently represent one or more substituents which may be the same as or different from each other and which will not interfere with the desired specific coupling chemistry of the compound of general formula I; each of n and p is independently an integer of from 1 to 4; and R 4 represents a leaving group; with a compound of general formula II wherein R 5 represents one or more substituents which may be the same as or different from each other and which are unreactive with compounds of general formula I and unreactive with compounds of general formula II; q is an integer of from 1 to 4; and Z is as defined for general formula IV, to form an acridine ester of general formula III wherein R2 , R3 , n and p are as defined for general formula I R 5 and q are as defined for general formula II; and Z is as defined for general formula I and subsequently either forming an acid addition salt of the compound of general formula III by reaction with an appropriate acid to form a compound of general formula IV wherein R A represents a hydrogen atom or N-alkylating the compound of general formula III with an N-alkylating agent containing the radical R AI wherein R A represents a C 1 -C 6 alkyl radical to form a compound of general formula IV wherein R A represents a C 1 -C 6 alkyl radical.

Claim 5

A process as claimed in Claim 1 or 2, wherein the acridine ester is purified.

Claim 6

A process as claimed in Claim 3 wherein, the purifying comprises extraction with ethyl acetate.

Claim 7

A process as claimed in Claim 3 or 4 wherein the purifying further comprises flash chromatography or reverse phase chromatography.

Claim 8

A process as claimed in any one of Claims 1 to 5 wherein the compound of general formula I and the compound of general formula II are mixed for a period of 10 to 60 minutes at 15 to 30°C.

Claim 9

A process as claimed in Claim 2, wherein the compound of general formula III and the alkylating reagent are mixed for a period of 15 to 25 hours at 15 to 30°C.

Claim 10

A process as claimed in Claim 2 or 7, wherein the alkylating reagent is methyl fluorosulphonate, methyliodide, or dimethyl sulphate.

Claim 11

A process as claimed in any one of Claims 1 to 8, wherein R 5 represents: ethyl, 0-methyl, S -methyl, carboxymethyl, N,N-dimethylamide, halide, azide, NCS, or where R 1 represents a hydrogen atom or an aliphatic chain containing from 1 to 30 carbon atoms, inclusive, which may be substituted with one or more alkyl, alkenyl, alkynyl or aryl groups; X represents nothing, an anion such as a halide, or one of the leaving groups: perchlorate, fluorosulphonate, iodide and sulphate and each of R 6 and R 7 independently represent a hydrogen atom or an alkyl group of from 1 to 30 carbon atoms, where R 6 and R 7 may be the same or different.

Claim 12

A process as claimed in any one of Claims 1 to 9, wherein the substituent represented by Z includes one of the following amino-reactive functionalities: N 3 ; where V represents an alkyl or phenyl group, 1 ≤ n ≤ 30, each of R 6 and R 7 independently represent a hydrogen atom or an alkyl group of from 1 to 30 carbon atoms, wherein R 6 and R 7 may be the same or different, and X is nothing, an anion such as halide or a leaving group. where 1 ≤ n ≤ 30; where 1 < n < 30; or where W represents a halogen.

Claim 13

A process as claimed in any one of Claims 1 to 10 wherein the ester formation reaction is carried out in anhydrous pyridine.

Claim 14

A process as claimed in any one of Claims 1 to 11, wherein R 4 represents N-hydroxysuccinimide, imidazole or analogue thereof (e.g. tetrazole), or a halide.