CZ295121B6 - Chiral stationary phase for liquid chromatography - Google Patents

Abstract

The present invention relates to a chiral stationary phase for liquid chromatography wherein it is characterized in that it comprises as a chiral selector ergoline derivative of the general formula I, in which A represents a connecting chain through the mediation of which said chiral selector is bound to silica gel, Ri1 and Ri2 can represent methyl, ethyl, n-propyl, isopropyl, n-butyl or isobutyl and Ri3 can denote hydrogen, methyl, ethyl, n-propyl, isopropyl, benzyl or a cyano group. The connecting chain A, extending between the chiral selector and silica gel can have a structure given by the general formula II, in which n can represent 2 to 10, preferably 3, or a structure of the general formula III, in which m can denote 2 to 10, preferably 3.

Description

The invention relates to a novel chiral stationary phase for liquid chromatography. The chiral stationary phase allows separation of enantiomers by liquid chromatography and can therefore be used both analytically and preparatively.

BACKGROUND OF THE INVENTION

The classical liquid chromatography is not able to separate enantiomers, ie pairs of substances, one of which is a mirror image of the other. To separate the enantiomers, a chiral selector must be introduced into the liquid chromatography system. The chiral selector can be part of both the mobile phase and the stationary phase. Previously, chiral selectors that were part of the mobile phase were more widely used, since such a chiral selector simply dissolved in the mobile phase. In recent years, chiral stationary phases have been increasingly used, employing various relatively readily available chiral substances, both natural and synthetic, which are anchored to the stationary phase, usually by covalent bonding, as chiral selectors. Further information on this subject can be found in a number of monographs or review articles, for example: Beesley T. E. and Scott R. P. W .: Chiral Chromatography, John Wiley and Sons, Chichester, 1998; Maier N, M., Farnco, P. and Lindner, W., J. Chromatogr. A 906,3 (2001); Chankvetadze, C. E. and Blaschke, B .: J. Chromatogr. A 906,309 (2001); Zief M. aCraneL. J .: Chromatographic Chiral Separations, Marcel Dekker Inc, New York, 1988; Gillar M., Tesarova E., Patzelova V. and Deyl Z .: Chem Listy 88, 514 (1994).

Although a huge number of chiral stationary phases have been described, it is still desirable to seek new ones with targeted use in both analytical chemistry and preparative purposes. At the same time, universal stationary phases are sought for analytical use, which could be used for the widest possible spectrum of separated substances. Conversely, tailor-made stationary phases for a particular application are increasingly being used for preparative liquid chromatography, very often for industrial separation of racemic mixtures obtained by chemical synthesis and hence for the production of chiral products, usually pharmaceuticals.

Natural substances, as such or their analogues obtained by partial chemical modification of their structure, are relatively readily available chiral substances which are useful as chiral selectors and their practical use in liquid chromatography depends on whether the substance can be anchored to a stationary phase and retain its chiral selectivity. Examples of the use of macrolide antibiotics, cyclodextrins and polysaccharides as chiral selectors have been found in the literature: Ward T. J. and Farris A. B .: J. Chrom. A 906,73 (2001), Fadnavis N. W., Babu R. L., Sheelu G., and Deshpande A., J. Chrom. A 893, 189 (2000), Kartozia I., Kanyonyo M., Happaerts T., Lammbert D. M., Scriba G. K. E., and Chankvetadze B., J. Pharm. And Biomed. Analysis 27, 457 (2002), Gasparrini, F., Masiti, D., and Villani, C., J. Chrom. A 906,35 (2001).

Ergot alkaloids are natural chiral substances produced and used as pharmaceuticals. While most natural ergot alkaloids are unusable as a chiral selector because natural alkaloids readily isomerize at one chiral center, the partially synthetic derivatives are chiral stable and are therefore useful as a chiral selector. It has been found in the past how to anchor these derivatives on a stationary phase and maintain their chiral selectivity: Flieger M., Sinibaldi M., Cvak L. and Castelani L .: Chirality 6, 549 (1994). Of the many ergot alkaloid derivatives tested, the partially synthetic analogue terguride, anchored to the stationary phase via a linker bonded via the nitrogen atom at position 1 of the ergoline skeleton, showed the highest chiral selectivity - see for example: Padiglioni P., Polcaro CM, Marchese S., Sinibaldi M. Flieger M: J. Chrom. A, 756,119 (1996). However, it has now surprisingly been found that some analogs of terguride exhibit an even higher chiral selectivity and these findings are the essence of the present invention.

SUMMARY OF THE INVENTION

The invention is based on the discovery that ergoline derivatives of the formula I exhibit excellent chiral selectivity,

in which:

A represents a linker through which the chiral selector is bound to silica gel,

R 1 and R ₂ can be methyl, ethyl, n-propyl, isopropyl, n-butyl or isobutyl, and R ₃ can be hydrogen, methyl, ethyl, n-propyl, isopropyl, benzyl, or cyano

The ergoline derivatives of formula (I) may be attached to silica gel by means of linker A of formula (II) or (III),

II = - Si (OCH ₃ ) ₂ - (CH ₂ ) ₃ -S- (CH ₂ ) _n -

III = -Si (OCH ₃ ) ₂ -CH ₂ -CHOH-CH ₂ -NH- (CH ₂ ) _m - in which n and _m may be 2 to 10.

The ergoline derivatives of formula I wherein A is hydrogen are analogs of a partially synthetic ergot alkaloid terguride which is used as a medicament. These terguride analogs can be prepared by methods known per se. Thus, terguride analogues with altered R1 and R2 substituents can be prepared by converting dihydroisolysergic hydrazide to the corresponding azide by treatment with nitrous acid, the azide undergoing Curtius degradation, and the resulting isocyanate is converted to the terguride analogue by reaction with a corresponding secondary amine, for example dimethylamine. Another method of preparing said analogs of terguride with altered R1 and R2 substituents is by reacting 8α-aminoergoline with the corresponding dialkylcarbamoyl chloride, preferably diisopropylcarbamoyl chloride may be used.

Derivatives and analogs of terguride containing a substituent other than methyl on N6 can be prepared by von Braun demethylation. By reaction of terguride or its analog with bromocyanate, an N6-cyanoderivative is prepared which can be further converted to an N6-noderivative by hydrolysis with sodium in liquid ammonia and the obtained norderivative is alkylated with an appropriate alkyl halide or reductive alkylation

Alkylation of the 6-nor-derivative with propyl bromide. It has been found that intermediates of such synthesis, i.e. the corresponding 6-nor-6-cyanoergoline and 6-norergoline, can also be used as chiral selectors.

The coupling of the terguride derivative or analog to silica gel via linker A can be accomplished in two ways. The first consists in the preparation of the N1-co-alkenyl derivative of the ergot alkaloid, which is further coupled via this ω-alkenyl group to a silanized silica gel mercaptopropyltrimethoxysilane. The second process consists in the preparation of the-ω-aminoalkyl derivative of the corresponding terguride, which is then bound to glycidyltrimethoxysilane silanized silica gel. According to elemental analysis, silica gel coverage varies from 0.469 to 0.769 pmol / m ² depending on the type of coupling procedure of the derivative.

The prepared stationary phases were packed into metal and glass columns of various sizes (5-25 cm, 2-5 mm I.D.) and tested on many analytes. The use of short columns (especially 5 cm), which in many cases are sufficient (R > 1.05), reduces retention times and, at the same time, due to the low cost of the selector and the columns made therefrom, favor analysis. The columns showed many times higher stability and at the same time lower memory characteristics compared to other commercially available columns (Astec or ChiralBond). Individual selectors can be linked to different sizes of silica gel. For example, flobufen separation where baseline separation was not achieved using commercially available chiral columns, Tesarova E. Gilar M., Jegorov A., Uhrova M. Deyl Z .: Biomed. Chrome. 11 (1997) 321-324. Using the chiral stationary phases of the invention, complete separation is achieved in 5 min.

Examples

The preparation of the compounds according to the invention and examples of their use as chiral liquid chromatography selectors will be described in the examples, which are not intended to limit the scope of the invention in any way:

Example 1. 3- (1-Anyl-6-methyergolin-8a-yl) -1,1-dimethylurea and its use as a chiral selector

40.0 g of methyldihydroisolysergate was dissolved in 200 ml of ethanol, 200 ml of hydrazine monohydrate was added to the solution, and the mixture was heated on a water bath to boiling for 3 hours. The reaction mixture was then evaporated to a residue which was crystallized from methanol

31.8 g of dihydroisolysergic hydrazide.

15.0 g of dihydroisolysergic hydrazide were dissolved in 500 ml of 0.2 M hydrochloric acid and the solution was cooled to 0 ° C. To the cooled solution, 50 ml of 1M sodium nitrite solution was added over 15 minutes with stirring, and then, within 10 minutes, 250 ml of cooled saturated sodium bicarbonate solution. The resulting mixture was then extracted with 500 mL of a 4: 1 mixture of ethyl acetate and 1,2-dichloroethane. The organic phase was separated, dried over magnesium sulfate and slowly added dropwise to 200 ml of boiling toluene. After 15 minutes at reflux, the solution was cooled to 40 ° C and 52 mL of a 2M solution of dimethylamine in tetrahydrofuran was added with stirring. After concentrating the solution, 10.1 g of 3- (6-methylergolin-8a-yl) -1,1-dimethylurea was obtained.

10.0 g of 3- (6-methylergolin-8oc-yl) -1,1-dimethylurea was dissolved in 400 ml of dichloromethane. 20 ml of a 20% aqueous solution of tetraethylammonium hydroxide, 50 ml of 50% aqueous sodium hydroxide and 12.5 ml of allyl bromide were added with vigorous stirring. After stirring for 10 minutes, the organic phase was separated, washed three times with 30 ml of water and evaporated to a residue. Chromatography on 50 g of silica gel using methylene chloride sequentially treated with up to 5% methanol and crystallization from diethyl ether gave 8.8 g of 3- (1-allyl-6-methylergolin-8a-yl) -1,1-dimethylurea.

To 10.0 g of silica gel for HPLC (Biospher ® PSI 200, 5 µm) suspended in 200 ml of toluene was added 10 ml of trimethoxy (3-sulfanylpropyl) silane and the mixture was heated to quark for 24 hours. Then the silica gel was filtered off and filtered and washed with 300 ml of toluene and dried under vacuum (1 mbar) at 40 ° C. The dry product was suspended in 100 ml of chloroform, 1.77 g of 3- (1-allyl-6-methylergolin-8a-yl) -1,1-dimethylurea and 0.07 g of 2,2'-azobisisobutyronitrile were added to the suspension, and the mixture was heated to boiling for 24 hours. Then, the resulting chiral stationary phase was washed with 300 mL of chloroform and 300 mL of methanol and dried under vacuum (1 mbar) at 40 ° C. Elemental analysis determined the chiral selector concentration of 0.39 mmol / g.

The chiral stationary phase was suspended in methanol and packed into a metal chromatography column (50 x 4.6 mm) under constant pressure (60 MPa). The enantioseparation properties of the column were tested on the racemate of flobufen, dichloroprop and dansylthreonine. The record of separations is shown in FIG. 1.

Example 2. 3- (1-Allyl-6-methylergolin-8a-yl) -1,1-diisopropyl urea and its use as a chiral selector

10.0 g of 8α-amino-6-methylergoline was suspended in 1000 ml of dichloromethane, 10.0 g of diisopropylcarbamoyl chloride and 40 ml of triethylamine were added thereto, and the mixture was stirred for 24 hours. 250 ml of water were then added and after stirring for 30 minutes the phases were separated and the dichloromethane phase was extracted twice more with 100 ml of water. The refined dichloromethane phase was evaporated to a residue which was crystallized from acetone. 13.8 g of 3- (6-methylergolin-8a-yl) -1,1-diisopropylurea were obtained. This intermediate was further converted to the 1-allyl derivative and bound to silica gel in a manner analogous to that described in Example 1. The chiral stationary phase containing 3- (1-allyl-6-methylergolin-8a-yl) -1,1-diisopropylurea was loaded onto a metal chromatography column (50 x 4.6 mm) and its enantioseparation properties were tested on the haloxyfop, fluazifop and dansylserine racemate. The separation record is shown in FIG. 2.

Example 3. 3- (1-Allyl-6-nor-6-propylergolin-8a-yl) -1,1-diethylurea and its use as a chiral selector

88.0 g of cyanogen bromide was dissolved in 4000 ml of dichloromethane and 50.0 g of calcined potassium carbonate and 50.0 g of terguride were added to the solution, and the suspension was stirred at room temperature for 3 hours. Then, 1000 ml of concentrated aqueous ammonia was added to the suspension over 1 hour, and the reaction mixture was stirred at room temperature for 16 hours, the phases were separated and the aqueous phase was extracted with a further 300 ml of dichloromethane. The combined organic phases were concentrated to a residue which was crystallized from ethyl acetate. 30.0 g of 3- (6-nor-6-cyanoergolin-8a-yl) -1,1-diethylurea were obtained.

5.3 g of sodium was dissolved in 200 ml of liquid ammonia and a solution of 15.0 g of 3- (6-nor-6-cyanoergolin-8a-yl) -1,1-diethylurea in 200 ml of tetrahydrofuran and the resulting blue solution were added dropwise. was bleached with iron nitrate. The reaction mixture was then stirred in a fume hood for 16 hours to evaporate the ammonia. 300 ml of water and 300 ml of dichloromethane were added to the residue and after stirring for 10 minutes the phases were separated and the aqueous phase was extracted three more times with 100 ml of dichloromethane. The combined dichloromethane fractions were evaporated to a residue which, after chromatography (150 g silica gel, eluting with dichloromethane successively polarized to 10% methanol) gave crystallization from acetone 11.4 g of 3- (6-nor-6-cyanoergolin-8a-yl) -1. , 1-diethylurea.

-4GB 295121 B6

6.3 g of 3- (6-nor-6-ergolin-8a-yl) -1,1-diethylurea were dissolved in 38 ml of dimethylformamide and 4.5 g of potassium carbonate, 0.2 g of potassium iodide and 1.0 ml of propyl bromide. The mixture was stirred at 100 ° C for 1 hour, cooled, filtered and the filtrate evaporated to a residue. The residue was purified by chromatography (50 g silica gel, eluting with dichloromethane successively polarized to 1.5% methanol) and crystallized from acetone to give 5.6 g of 3- (6-nor-6-propylergolin-8a-yl) -1,1- diethylureas.

5.0 g of 3- (6-nor-6-propylergolin-8a-yl) -1,1-diethylurea was dissolved in 200 ml of dichloromethane. To the solution was added 10 mL of a 20% aqueous solution of tetraethylammonium hydroxide, 25 mL of 50% aqueous sodium hydroxide, and with vigorous stirring, 6.3 mL of allyl bromide. After stirring for 10 minutes, the organic phase was separated, washed three times with 20 ml of water and evaporated to a residue. After chromatography on 30 g of silica gel using methylene chloride sequentially treated with up to 5% methanol and crystallization from diethyl ether, 3.8 g of 3- (1-allyl-6-nor-6-propylergolin-8a-yl) -1,1- diethylureas.

3- (1-allyl-6-nor-6-ergolin-8cc-yl) -1,1-diethylurea was loaded onto silica gel as described in Example 1 and the chiral phase was packed into a column.

Example 4. 3- [1- (6-Aminohexyl) -6-nor-6-cyanoergolin-8a-yl] -1,1-diethylurea and its use as a chiral selector

15.0 g of 3- (6-nor-6-cyanoergolin-8a-yl) -1,1-diethylurea (prepared in Example 3) was dissolved in 300 ml of methylene chloride. To the solution was added 30 mL of a 20% aqueous solution of tetraethylammonium hydroxide, 75 mL of 50% aqueous sodium hydroxide, and with vigorous stirring 30 g of 1,6-dibromohexane. After stirring for 10 minutes, the organic phase was separated, washed three times with 100 ml of water and evaporated to a residue. Chromatography on 150 g of silica gel using methylene chloride sequentially treated with up to 5% methanol gave 13.0 g of a residue which contained predominantly 3- [1- (6-bromohexyl) -6-nor-6-cyanoergolin-8a-yl) - 1,1-diethylurea. The obtained residue was dissolved in 130 ml of dioxane, 5.0 g of potassium phthalimide was added to the solution, and the solution was heated to boiling for 5 hours and then evaporated to a residue (10.4 g). The residue was dissolved in 100 ml of ethanol, 5 ml of hydrazine monohydrate was added to the solution, and the solution was heated to boiling for 2 hours and then evaporated to a residue. The residue was purified by chromatography (130 g silica gel, eluting with dichloromethane successively polarized to 6% methanol) and fractions containing pure 3- [1- (6-aminohexyl) -6-nor-6-cyanoergolin-8a-yl] -1,1- the diethyl urea was evaporated to a residue (6.9 g).

To 10.0 g of silica gel for HPLC (Biospher® PSI 200, 5 μη) suspended in 200 ml of toluene was added 5 ml of glycidoxypropyltrimethoxysilane and the suspension was stirred at 100 ° C for 12 hours. After cooling, the silanized silica gel was filtered off, washed successively with 500 ml of toluene, 200 ml of isopropyl alcohol and 200 ml of diethyl ether and dried under vacuum (1mbar, 40 ° C). The dried silanized silica gel was suspended in 60 ml of methanol and 0.7 g of 3- [1- (6-aminohexyl) -6-nor-6-cyanoergolin-8a-yl] -1,1-diethylurea was added to the suspension. The suspension was stirred at room temperature for 48 hours, then the silica gel was filtered off, washed with 400 ml of methanol and 100 ml of diethyl ether and dried under vacuum (1mbar, 40 ° C). Elemental analysis determined the concentration of the chiral selector bound to silica gel 0.19 mmol / g.

The chiral stationary phase was then packed into a column, the efficacy of which was tested for separation of racemic dansylated aspartic acid and dansyltryptophan. The separation record is shown in FIG. 3.

Example 5. 3- [1- (10-Aminodecyl) -6-nor-6-ergolin-8a-yl] -1,1-diethylurea are used as chiral selector

5.0 g of 3- (6-nor-6-ergolin-8oc-yl) -1,1-diethylurea (prepared in Example 3) was dissolved in 100 ml of methylene chloride. To the solution was added 10 mL of a 20% aqueous solution of tetraethylammonium hydroxide, 25 mL of 50% aqueous sodium hydroxide, and with vigorous stirring 10 g of 1,10-dibromodecane. After stirring for 10 minutes, the organic phase was separated, washed three times with 20 ml of water and evaporated to a residue. Chromatography on 50 g of silica gel using methylene chloride sequentially treated with up to 5% methanol gave 3.7 g of a residue which contained predominantly 3- [1- (10-bromo-decyl) -6-nor-6-ergolin-8a-yl] - 1,1-diethylurea. The obtained residue was dissolved in 40 ml of dioxane, 2.0 g of potassium phthalimide was added to the solution, and the solution was heated to boiling for 5 hours and then evaporated to a residue. The residue was dissolved in 30 ml of ethanol, 2 ml of hydrazine monohydrate was added to the solution, and the solution was heated to boiling for 2 hours and then evaporated to a residue. The residue was purified by chromatography (40 g silica gel, eluting with dichloromethane sequentially polarized to 10% methanol) and the fractions containing pure 3- (1- (10-aminodecyl) -6-nor-ergolin-8a-yl] -1,1-diethylurea were Evaporate to a residue (2.1 g).

The chiral selector was then coupled to silica gel as described in Example 4. The chiral stationary phase contained 0.12 mmol / g chiral selector and was tested after loading into a racemic dansylated aspartic acid / dansyltryptophan separation column. The separation record is shown in FIG. 4.

Industrial applicability

The chiral stationary phase according to the invention can be used to control the optical purity of substances, especially drugs. Preparative applications can be employed in the preparation and manufacture of optically active compounds from their racemic mixtures prepared by synthetic procedures.

Claims (8)

PATENT CLAIMS Chiral stationary phase for liquid chromatography, characterized in that it contains as an chiral selector an ergoline derivative of the general formula I, -6GB 295121 B6 in which: A represents a linker through which the chiral selector is bound to silica gel, R 1 and R ₂ are methyl, ethyl, n-propyl, isopropyl, n-butyl or isobutyl, and R ₃ is hydrogen, methyl, ethyl, n-propyl, isopropyl, benzyl, or cyano. Chiral stationary phase for liquid chromatography according to claim 1, characterized in that the linker A between the chiral selector and silica gel has the structure given by the general formula II, - Si (OCH ₃ ) ₂ - (CH ₂ ) ₃ -S- (CH ₂ ) _n - (II) is 2 to 10. The chiral stationary phase for liquid chromatography according to claim 2, characterized in that n is 3. Chiral stationary phase for liquid chromatography according to claim 1, characterized in that the linker A between the chiral selector and silica gel has the structure given by the general formula III, -Si (OCH ₃ ) ₂ -CH ₂ -CHOH-CH ₂ -NH- (CH ₂ ) _m - (III) wherein m is 2 to 10. 5. The chiral stationary phase for liquid chromatography according to claim 4, wherein m is 3. 6. A chiral stationary phase for liquid chromatography according to claims 1 to 5, characterized in that R, and R ₂ is isopropyl and R ₃ is methyl. 7th chiral stationary phase for liquid chromatography according to claims 1 to 5, characterized in that R], R ₂ and R ₃ represent ethyl and R ₃ is n-propyl. 8. A chiral stationary phase for liquid chromatography according to claims 1-5, c h en p i m, R], R ₂ and R ₃ are methyl.