Chemistry: molecular biology and microbiology – Process of utilizing an enzyme or micro-organism to destroy... – Resolution of optical isomers or purification of organic...
Reexamination Certificate
2001-04-17
2004-08-24
Marx, Irene (Department: 1651)
Chemistry: molecular biology and microbiology
Process of utilizing an enzyme or micro-organism to destroy...
Resolution of optical isomers or purification of organic...
C435S128000, C435S122000, C435S136000, C435S147000, C435S227000
Reexamination Certificate
active
06780634
ABSTRACT:
DESCRIPTION
The invention's method for preparing compounds of the general formulas
wherein R
1
is acyl , alkoxycarbonyl or aryloxycarbonyl and R
2
is a hydrogen atom or C
1-10
alkyl, comprises treating with a hydrolase and an effective amount of a nucleophile and a base in a constant pH range a racemic lactam of the formula
Formula I compounds, such as, for example, (1R,4S)-2-acetyl-2-azabicyclo[2.2.1]hept-5-ene-3-one are important intermediates for preparing (1R,4S)-1-amino4hydroxymethyl)-2-cyclopentene, which, in turn, is an important intermediate for preparing carbocyclic nucleosides, such as, for example, Carbovir (Campbell et al. J. Org. Chem. 1995, 60, 4602-4616). Formula II compounds, such as, for example, the propyl ester of (1S,4R)-acetylamino-2-cyclopentene-1-carboxylic acid, are an important intermediate for preparing (1S,4R)-1-amino-4-(hydroxymethyl)-2-cyclopentene, which similarly can be an important intermediate for preparing carbocyclic nucleosides.
Only the chemical preparation of (1R,4S)-2-acetyl-2-azabicyclo[2.2.1]hept-5-ene-3-one by acylating (1R,4S)-2-azabicyclo[2.2.1]hept-5-ene-3-one (Katagiri et al., Tetrahedron Letters, 1997, 38, 1961) is known. According to this method, (1R,4S)-2-acetyl-2-azabicyclo[2.2.1]hept-5-ene-3-one can be obtained only from the corresponding (1R,4S)-2-azabicyclo[2.2.1]hept-5-ene-3-one as an educt. This educt is too expensive.
The problem involved in this invention was to develop a method for preparing compounds of general formula I and II, which can be prepared from easily obtainable, inexpensive starting material with good enantiomer purity.
This problem is solved with the new biotechnological method according to claim
1
.
The invention's method for preparing compounds of the general formulas
wherein R
1
is acyl or acyloxy and R
2
is a hydrogen atom or C
1-10
alkyl, takes place by means of a hydrolase in the presence of a nucleophile and in the presence of a base in a constant pH range starting with a racemic lactam of the formula
The starting material, the lactam of the general formula III (substrate), can be prepared, for example, according to Taylor et al. (Tet. Asymmetry; 4, 1993,1117).
C
1-10
alkyl is linear or branched and substituted or unsubstituted. Examples of C
1-10
alkyl are methyl, ethyl, propyl, butyl, isobutyl, t-butyl, isopropyl, pentyl, hexyl, heptyl, octyl, nonyl or decyl and its isomers, as well as chloromethyl, bromomethyl, dichloromethyl, dibromomethyl, chloropropyl and bromobutyl.
Acyl means alkanoyl or arylcarbonyl. Alkanoyl is suitably C
1-4
alkanoyl, which can be substituted or unsubstituted. Substituted C
1-4
alkanoyl in the following is understood to be substituted with one or more halogen atoms. Examples of C
1-4
alkanoyl are acetyl, propionyl, butyryl, chloroacetyl, bromoacetyl and dichloroacetyl. Arylcarbonyl is suitably benzylcarbonyl or phenylcarbonyl, substituted or unsubstituted.
Acyloxy means alkoxycarbonyl or aryloxycarbonyl. Alkoxycarbonyl is suitably C
1-4
alkoxycarbonyl, such as methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl or t-butoxycarbonyl (BOC). Aryloxycarbonyl is suitably benzyloxycarbonyl or phenyloxycarbonyl.
R
1
is preferably C
1-4
alkanoyl or C
1-4
alkoxycarbonyl, in particular acetyl or ethoxycarbonyl.
Hydrolases that can be used are proteases or lipases, preferably proteases, such as serine proteases. Examples of serine proteases that can be used are chymotrypsin, trypsins and subtilisins (bacterial serine proteases). Subtilisins that can be used are commercial subtilisins, such as subtilisin A, subtilisin B, alcalases, ALK enzyme bacillopeptidase A, bacillopeptidase B, bioprases, colistinases, esperases, genenase I, kazusase, maxacal, maxatases, nagarses, peptidases, protease S, protease VIII, protease XXVII, proteinases, such as the alkaline proteinase of
Bacillus subtilis
or
Aspergillus oryzae
, proteinase K from Tritirachium album, savinases, subtilopeptidasen, superases, and thermoases. Conducting the biotransformation by means of savinases is preferred. Suitable savinases are savinase 12 Type W™, savinase 16.0 L Type EX™, savinase 32.0 L Type EX™, savinase 4.0 T Type W™, and savinase 8.0 L™. The lipase that can be used is, for example, lipase from Candida Antarctica.
If the hydrolases used are proteases, such as proteases from
Bacillus subtilis
, proteases from
Aspergillus oryzae
, proteinase K from Tritirachium album, the (1S, 4R) enantiomer in the racemic lactam of formula III is hydrolyzed suitably into the corresponding compound of general formula II, whereby the (1R, 4S) enantiomer of general formula I is obtained. If the hydrolases used are lipases, such as lipase from Candida Antarctica, the (1R, 4S) enantiomer in the racemic lactam of formula III is hydrolyzed suitably into the corresponding compound of general formula II, whereby the (1S, 4R) enantiomer of general formula I is obtained.
Water or C
1-10
alcohols can be used as the nucleophile. Suitable C
1-10
alcohols are methanol, ethanol, propanol, isopropanol, butanol, t-butanol, isobutanol, pentanol, hexanol, heptanol, octanol, nonanol or decanol. If the nucleophile used is a C
1-10
alcohol, the corresponding ester of general formula II (R
2
=C
1-10
alkyl) is formed, as the expert knows. If water is used as the nucleophile, obviously, the corresponding acid of general formula II (R
2
=H) is formed.
Depending on the hydrolase and the substrate (formula III lactam), the biotransformation is conducted suitably between pH 5 and 12, preferably between pH 6 and 8. In the invention, for a given hydrolase and a given substrate, the pH value is maintained constant in the presence of a base. The pH value is suitably maintained constant at +/−0.5 pH units by addition of a base. If, for example, the substrate is racemic 2-acetyl-2-azabicyclo[2.2.1]hept-5-ene-3-one (R
1
=acetyl) and savinase is used as the hydrolase, the pH value is held constant at preferably between pH 7.0 and pH 7.5.
An inorganic or organic base can be used as the base. For example, KOH and NaOH are suitable as inorganic bases. A suitable organic base can be, for example, triethanolamine dissolved in an organic solvent. If one of the aforesaid alcohols is used as the nucleophile, the corresponding alcoholate can serve as the base.
The biotransformation is suitably conducted in water, a buffer solution, a C
1-10
alcohol or in a mixture of these with an aprotic organic solvent. Suitable aprotic organic solvents are, for example, ether and aromatic hydrocarbons. Tetrahydrofuran, dioxane or t-butyl methyl ether can be used as the ether. Toluene and benzene are suitable aromatic hydrocarbons. The buffer solutions used can be, for example, low molarity, such as 10-100 mM sodium or potassium phosphate buffer, hepes buffer. The C
1-10
alcohols used can be those previously described.
The biotransformation can also be conducted so that the lactam of general formula III serves as solvent. Then the biotransformation is suitably conducted in the presence of half of the stoichiometric quantities of water or the corresponding alcohol.
Depending on the solvent, the biotransformation can be conducted in a two-phase or one-phase system. The biotransformation is advisedly conducted in a one-phase system.
After a usual conversion time of a few hours depending on the selected starting material, the desired optically active compounds of general formulas I and II are obtained in outstanding yields and enantiomer purity. The preferred starting materials are racemic 2-acetyl-2-azabicyclo-[2.2.1]hept-5-ene-3-one (R
1
=acetyl) and the racemic 2-ethoxycarbonyl-2-azabicyclo-[2.2.1]hept-5-ene-3-one (R
1
=ethoxycarbonyl). The preferred compounds of formula II are (1S, 4R)-4-acetylamino-2-cyclopentene-1-carboxylic acid (R
1
=acetyl, R
2
=H), (1S, 4R)-4-ethoxycarbonylamino-2-cyclopentene-1-carboxylic acid (R
1
=ethoxycarbonyl, R
2
=H), (1S, 4R)-4-acetylamino-2-cyclopentene-1-
Bernegger-Egli Christine
Brux Frank
Guggisberg Yves
Roduit Jean Paul
Werbitzky Oleg
Baker & Botts L.L.P.
Lonza AG
Marx Irene
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