CHIRAL SEPARATIONS OF PYRIMIDINES

Abstract: An enantiomeric mixture of a chiral compound containing a pyrimidine ring is separated into its respective enantiomers through chromatography on a chiral polysaccharide stationary phase eluting with a mobile phase comprising a polar solvent, preferably a lower alkanol or acetonitrile.

Description: Chiral Separations of Pyrimidines The present invention relates to the separation of chiral materials utilizing high performance liquid chromatography (HPLC) techniques. Detailed Description The present invention pertains to a method of separating an enantiomeric mixture of chiral pyrimidines, or a salt thereof, into their respective enantiomers. This method comprises subjecting the chiral mixture to chromatography on chiral polysaccharide stationary phase, eluting with a polar mobile phase, preferably a liquid lower alkanol. Pyrimidines constitute an important class of biologically active compounds, encompassing monocyclic nucleic acid bases such as uracil, cytosine, thymine, and 5- methylcytosine, bicyclopyrimidines such as the purine bases adenine, guanine, xanthine, and hypoxanthine, folates and folic acid antagonists, etc. Numerous compounds of this type have been synthesized as antiviral and anticancer drugs. Typical pyrimidine structures include for example: Al 0 so include ζ d arex the hy>droge Q nated fo O rms of th 0 e forego 0 ing struc 0 tures. Often the final compounds containing such ring systems will be substituted with a chiral group, thereby giving rise to the existence of enantiomers, and in such cases it generally is desirable to separate the enantiomers into chirally pure form. For example iodoxuridine, vidarabine, azidothymidine, sparsomycin, cytosine arabinoside, 5-fluorouridine, methotrexate, DDATHF, and aminopterin, to mention but a few, are biologically active pyrimidine compounds containing a center of chirality. The enantiomeric separations encompassed by the present invention utilize chiral polysaccharides as stationary phases. Typically these are aromatic carbamate or ester derivatives of cellulose or amylose which can be generically represented by the formula: in which the depicted glucosidic linkage is either (amylose) or β (cellulose). The depicted R groups can be for example a phenylcarbamate or α-phenethylcarba- mate. which itself is chiral, or a benzoate group. Typical R groups thus include 3,5- dimethylphenyl carbamate, α-phenethylcarbamate, and 4- methylbenzoate, e.g. : O H CH N - ^g) O -I -CH, Such chiral polysaccharide stationary supports are commercially available from Chiral Technologies, Inc., Exton, PA, under the trademarks CHIRALPAK ® amylosic stationary phase and CHIRALCEL cellulosic stationary phase. Suitable materials include CHIRALPAK AD , an amylose derivative in which each glucose monomer carries three 3,5-dimethylphenyl carbamate groups, CHIRALPAK" AS , an amylose derivative in which each glucose monomer carries three (S)- -phenethylcarbamate groups, CHIRALCEL OD , a cellulose derivative in which each glucose monomer carries three 3.5-dimethylphenyl carbamate groups, and CHIRALCEL OJ , a cellulose derivative in which each glucose monomer carries three 4-methylbenzoyl groups. Reference may be made to U.S. Patent Nos. 4,912,205 and 5,434,299 for further details, the disclosures of which are incorporated herein by reference. The amylose derivatives are preferred. The stationary phase conveniently can be packed in columns adapted for use with commercially available HPLC systems, as for example those available from 3 Shimadzu, Columbia, MD, and Jasco, Easton, MD. Generally the particle diameter will be from about 1 to about 100 μm, typically from about 5 to about 75 μm. Multiple or single columns can be employed. A simulated moving bed apparatus also can be employed, as described for example in U.S. Patent Nos. 5,434,298, 5,434,299, 5,456,825, and 5,498,752, the disclosures of which are incorporated herein by reference. The eluent or mobile phase comprises a liquid polar solvents such as methanol, ethanol, n-propanol, isopropanol, butanol, acetonitrile, supercritical carbon dioxide and the like, preferably acetonitrile or a liquid lower alkanol such as ethanol or methanol. Also of value is supercritical carbon dioxide, alone or in combination with at one or more of acetonitrile and a liquid lower alkanol. The separation will be conducted at ambient temperatures; e.g., 25-40°C. pH will vary depending upon the nature of the material being chromatographed but generally will be from about 2 to about 7. Typical flow rates are from about 0.2 mL/min. to about 25 mL/min., depending on the apparatus, column dimensions, and stationary phase. Separation can be monitored by measuring UV absorption, optical rotation, refractive index, evaporative light scattering, or a similar physical parameter. Detection can be conducted for example by measuring UN absorption at an appropriate wavelength of the eluted material, utilizing a UN spectrophotometer, or the optical activity of the eluted material, using for example a device such as the IBZ Chiralyser ® instrument (available from JM Science, Inc., Grand Island, ΝY) which monitors the rotation of plane polarized light. The parameter selected for detection will depend on the specific pyrimidine compound being eluted. The following examples will serve to further typify the nature of the invention but should not be construed as limitation on the scope thereof which is defined solely by the appended claims. Example 1 A chiral mixture of 2-amino-6-hydroxy-9-(2-hydroxymethylcyclopropylidene- methyl)purine: OH CH— CH 2 — OH CH =< CIH 2 was chromatographed on a 4.6 mm id x 250 mm column in which the stationary phase was CHIRALCEL AD , an amylose derivative in which each glucose monomer carries three 3,5-dimethylphenyl carbamate groups. The run was conducted at room temperature at a flow rate of 1.0 mL/min., utilizing 100% methanol as the mobile phase. Separation was measured by absorption at 254 n . Good separation into two distinct peaks was observed with the dextrorotatory enantiomer being eluted first. Example 2 A chiral mixture of 2-amino-6-chloro-9-(2-hydroxymethylcyclopropylidenemeth- yl)purine: Cl H 7 N- r N ' ^ Nx- * N> CH-CH,— OH CH =< CIH 2 was separated into its two enantiomers using the procedure of Example 1. Good separation into two distinct peaks was observed. The first enantiomer eluted was dextrorotatory. Example 3 A chiral mixture of 2,6-diamino-9-(2-hydroxymethylcyclopropylidenemeth- yl)purine: NHo CH 7 — OH was separated into its two enantiomers using the procedure of Example 1. Good separation into two distinct peaks was observed. The first enantiomer eluted was the dextrorotatory form. Example 4 A chiral mixture t 2-(2-methylpropionamido)-6-hydroxy-9-(2-hydroxymethyl- cyclopropylidenemet )p -rine: OH .N 3 (CH 3 ), C-NH X-^^NX^^N > CH-CH,— OH CH-C CH 2 was separated into _ two enantiomers using the procedure of Example 1. Good separation into tv,o istinct peaks was observed. Dextrorotatory 2-(2- methylpropionamic >)-6 droxy-9-(2-hydroxymethylcyclopropylidenemethyl)purine was eluted first. Example 5 A chiral mixture ι-( -methyl-5-benzoyloxymethylfur-2-yl))thymiine: OH L ^CH 3 N HO X^ ^ N ' * CH 3 was separated into its two enantiomers using the procedure of Example 1 but eluting with acetonitrile. Good separation into two distinct peaks was observed. Example 6 Other chiral pyrimidines which can be similarly separated include the following: Cl OH N N N HO HO' CH,OH CB,OR> X U° ^CH 2 OH R\ .C-C^<] N H. \ / R3 • HC1 HO O^^ ^NN' ^ R4 I O' H N. HOCHy Η 4 • in which R is for example t-butyldiphenylsilyloxy, and each of R , R . and R , is hydrogen, methyl, chloro, fluoro, trifluoromethyl, or trichloromethyl.

Claims: What is claimed is: 1. The method of separating an enantiomeric mixture of a chiral pyrimidine compound, or a salt thereof, into its respective enantiomers which comprises subjecting said mixture to chromatography on a chiral polysaccharide stationary phase eluting with a mobile phase of at least one polar solvent. 2. The method of claim 1 wherein said polar solvent is a liquid lower alkanol.. 3. The method of claim 2 wherein said lower alkanol is methanol. 4. The method of claim 2 wherein said lower alkanol is ethanol. 5. The method of claim 1 wherein said polar solvent is acetonitrile. 6. The method of claim 1 wherein said polar solvent comprises supercritical carbon dioxide. 7. The method of claim 6 wherein said polar solvent comprises supercritical carbon dioxide in combination with at one of acetonitrile and a liquid lower alkanol. 8. The method of claim 1 wherein said chiral polysaccharide stationary phase is an amyl- ose derivative in which each glucose monomer carries three 3,5-dimethylphenyl carbamate groups or α-phenethylcarbamate groups. 9. The method of separating an enantiomeric mixture of a chiral nucleotide which com- prises subjecting said mixture to chromatography on chiral polyamylose stationary phase, in which each glucose monomer of the polyamylose carries three 3,5- dimethylphenyl carbamate groups or α-phenethylcarbamate groups, with a mobile phase comprising 100% methanol. -1 -

Inventors: GEISER FIONA O, GEISER, FIONA, O.
Applicants: CHIRAL TECHNOLOGIES INC, GEISER FIONA O, CHIRAL TECHNOLOGIES, INC., GEISER, FIONA, O.
Application year: 1998
Main classification: C07H1
IPC codes: C07D239, C07D405, C07D411, C07D413, C07D473, C07H19