Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Reexamination Certificate
1998-11-23
2001-07-17
Prats, Francisco (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
C435S136000, C435S117000, C435S123000, C435S072000
Reexamination Certificate
active
06261813
ABSTRACT:
BACKGROUND OF THE INVENTION
There are many pharmaceuticals which are not substantially soluble in water and this lace of solubility limits the usefulness of these pharmaceuticals. For example, in many cases a pharmaceutical which might otherwise be administered intramuscularly or intraperitoneally might be administered orally or intravenously if the pharmaceutical were water soluble.
SUMMARY
The present invention provides a method for modifying the solubility of pharmaceuticals through back to back acylation reactions using a bifunctional acylating agent. It has been found that compounds such as paclitaxel, bergenin, erythromycin and adenosine which contain a neucleophilic group in the molecule that is amenable to enzymatic acylation will react regiospecifically with a bifunctional acylating agent in the presence of a hydrolytic enzyme such as a lipase or protease enzyme. Preferably the bifunctional acylating agent is a di(vinyl) ester or carbonate. This yields a vinyl ester or vinyl carbonate derivative which can be subsequently hydrolyzed or reacted with nucleophiles such as alcohols, sugars and amines.
In accordance with a preferred embodiment of the invention, the method is used to produce derivatives of paclitaxel and, more particularly, water soluble derivatives of paclitaxel. The present invention also provides derivatives of paclitaxel, bergenin, erythromycin and adenosine.
DETAILED DESCRIPTION
Representative examples of bifunctional acylating agents include di(vinyl) or di(2,2,2-trifluoroethyl) esters of dicarboxylic acids or di(vinyl carbonates) of dialcohols.
The bifunctional acylating agents useful in the present invention include compounds of the formulas (I), (II) and (III) and their equivalents:
wherein L
1
and L
2
are linking groups, R is vinyl or trifluoroethyl. Those skilled in the art will recognize that a wide range of linking groups may be useful and that other R groups which activate the acyl group will also be useful. Typically, L
1
is a linking group such as a straight chain or a branched chain or a cyclic alkylene having 1 to 10 and preferably 1 to 8 carbon atones or a direct bond and L
2
is a linking group such as a straight chain, branched chain or cyclic alkylene having 2 to 10 and preferably 2 to 8 carbon atoms or a linkage including a saturated or unsaturated heterocyclic ring such as tetrahydrofuranyl or pyridinyl. Specific examples of L
1
include trimethylene, tetramethylene, hexamethylene, cyclohexylene, phenylene, and propylenylene. Specific examples of L
2
include trimethylene, tetramethylene hexamethylene, 2-butenylene, propylenylene, 1,3-dimethylene cyclohexylene, 1,4-dimethylene cyclohexylene, 2, 5-dimethylenefuranyl and 2,6-dimethylene pyridinyl.
Specific bifunctional acylating agents useful in the invention include: adipic acid divinyl ester; 1,3-propanediol di(vinyl carbonate); 1,4-butanediol di(vinyl carbonate); acetone oxime vinyl carbonate; 1,4-cyclohexane dimethanol di(vinyl carbonate); 1,6-hexanediol di(vinyl carbonate); 2,5-furandimethanol di(vinyl carbonate); 2,6-pyridine dimethanol di(vinyl carbonate); 1,4-but-2-enediol di(vinyl carbonate); oxalic acid di(trifluoroethyl) ester; 1,4-cyclohexane dicarboxylic acid di(trifluoroethyl) ester; terephthalic acid di(trifluoroethyl) ester and 1,3-propylenediol di(vinyl carbonate).
The back to back acylation reactions are conducted in the presence of a hydrolase, such as a lipase or a protease enzyme. Preferably the enzymes have been lyophilized in the presence of simple salts to enhance catalytic activity in organic solvents. U.S. Pat. No. 5,449,613 to Dordick et al. (incorporated herein by reference) discloses a method for reacting an enzyme in a non-aqueous media comprising the steps of first preparing a lyophilizate of an enzyme and a salt wherein the lyophilizate contains a salt in a weight ratio sufficient to activate the enzyme in an organic solvent and then dispersing the lyophilizate in a non-aqueous, organic solvent in the presence of a substrate for the enzyme. More preferably, the enzyme is activated by dissolving the enzyme and a salt capable of activating the enzyme in an aqueous solution and then lyophilizing for a period of time sufficient to maximize the activity of the enzyme.
Representative examples of hydrolases useful in the present invention include proteases such as thermolysin from
Bacillus thermoproteolyticus rokko
and subtlisin Carlsberg from
Bacillus lichiniformis
. Additionally, pH adjusted proteases/esterases (Type XIII from
Aspergillus saitol
, Newlase, ficin, Solvay fungal protease 31000, alkaline protease, papain 16,000, Prozyme 6, Prozyme N, Prozyme S, Biozyme M2 and bromelain) may also be used.
Representative examples of lipases include those from
Candida antarctica, Pseudomonas cepacia, Rhizopus oryzae, Mucor meihei
, Pseudomonos sp.,
Humicola lanuginosa
(type CE), Alcaligences sp.,
Geotrichium candidum
(type GC4), Rhizopus sp.,
Rhizopus arrhizus, Rhizopus javanicus, Rhizopus delemar, Candida lipolytica
(type L-10),
Aspergillus niger
(type AP-12), porcine pancreas, and
Chromobacterium viscosum.
The reaction product of the bifunctional acylating agent can be hydrolyzed by reaction with water or it can be reacted with a nucleoplile. The activated ester or carbonate produced in the first acylation is reactive with a variety of nucleophiles. Representative examples of nucleophiles that can be acylated with the activated ester or carbonate of the present invention include water, sugars, alcohols, amines and aminoalcohols.
Representative examples of sugars include: glucose, galactose, mannose, fructose, 1,2:3,4-di-O-isopropylidene-D-galactopyranose, deoxynojirimycin, D-fucose, and N-acetyl-D-glucosamine.
Representative examples of alcohols include: (±)-1,3, butanediol, (±)-2-butanol, (±)-menthol, 1-(2-hydroxyethyl)-2-pyrrolidinone, 1,4-butanediol, 1-aziridineethanol, 1-butanol, 1-methyl-3-piperidinemethanol, 1-octanol, 2-(methylsulfonyl)ethanol, 2-naphthaleneethanol, 3,3-diethoxy-1-propanol, 3-thiophenemethanol, 4-chlorobenzyl alcohol, 4-hydroxybenzyl alcohol, 4-methoxybenzyl alcohol, 4-methyl-5-thiazoleethanol, 5-methyltetrahydrofuran-2-methanol, benzyl alcohol, furfuryl alcohol, glycidol, phenethyl alcohol and sec-phenethyl alcohol.
Representative examples of amines include: (aminomethyl)cyclopropane, 1-(2-aminoethyl)piperazine, 1-(2-aminoethyl)pyrrolidine, 1-(3-aminopropyl)imidazole, 1-butylamine, 1-(aminomethyl)naphthalene, 2-(2-aminoethyl)-1-methylpyrrolidine, 2-aminomethyl benzodioxane, 2-(aminomethyl)thiophene, 3-(aminomethyl)pyridine, 3,3-dimethylbutylamine, 3-dimethylamninopropylamine, 4-chlorobenzyl amine, 4-phenylbutylamine, benzylamine, cyclohexylamine, furfurylamine, hexylamine, and tetrahydrofurfurylamine.
Representative examples of aminoalcohols include: 2-amino-2-methyl-1-propanol, 3-amino-1-propanol, ethanolamine, 4-amino-2-butanol, and (±)-2-amino-1-butanol.
The reactions of the invention are typically carried out in a solvent. Representative examples of solvent useful in the present invention include tert-amyl alcohol, butyl acetate, acetonitrile, toluene, hexane, tert-butyl methyl ether, 2-pentanone, tetrahydrofuran, 1,4-dioxane, dimethylsulfoxide (DMSO) and mixtures thereof.
The method of the invention is typically conducted in accordance with the following procedure. The base compound and bifunctional acylating agent are admixed in solvent to form a solution. This solution is added to solid enzyme and reacted at 37° C. to 60° C. with 250 rpm shaking for 1 to 7 days to yield an activated acyl ester or carbonate. The activated acyl ester or carbonate is purified by preparative reversed phase HPLC and then dissolved in an appropriate solvent, typically acetonitrile or tert-butyl alcohol. The resulting solution is added to dry enzyme and nucleophile and allowed to react at 37° C. to 60° with 250 rpm shaking for 1 to 15 days to produce the derivatives of the present invention.
REFERENCES:
patent: WO9505475 (1995-02-01), None
patent: WO 97/00233 (1997-03-01), None
J. Am. Chem. Soc. 119(47):115
Arnold John M.
Budde Cheryl L.
Chen Sharon S.
Clark Douglas S.
Dordick Jonathan S.
Albany Molecular Research, Inc.
Nixon & Peabody LLP
Prats Francisco
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