SI9800141A - Now procedure for obtaining chromatographiccally pure antibiotics and their synthesis intermediates - Google Patents

Abstract

In the invention, a new procedure of purification of lincosanide antibiotics and their salts, addition salts or esters and synthesis intermediates by means of preparative HPLC is presented, by which a substance of purity higher or equal than 99% is obtained and the amount of each single known impurity does not excess 0,5% and of the unknown one 0,1%. The chromatography is carried out on a reverse stationery phase using various mobile phases. The end product is isolated according to the one of known procedures which may include evaporation and crystallization, as well as desalinization (either by electrodialysis or in a column). The so purified antibiotic is suitable to all kinds of application, especially to injections, because it contains essentially fewer impurities in comparison with commercially available products.

Description

A NEW PROCEDURE FOR THE OBTAINING OF CHROMATOGRAPHICALLY PURE ANTIBIOTICS AND THEIR SYNTHESIS INTERMEDIATES

The field of technology

MPK: C 07 H 15/16; B 01 D 15/08.

The present invention relates to the field of pharmaceutical organic chemistry and deals with antibiotics, namely lincomycin and its derivatives, synthesis intermediates to clindamycin and clindamycin itself, and their salts, addition salts or esters.

In a narrower sense, the present invention contemplates a novel process for the purification of linkosanide antibiotics, including lincomycin and clindamycin, and their salts, addition salts or esters and synthesis intermediates by preparative HPLC (High Performance Liquid Chromatography), to improve the chromatographic purity of the product finally, one of the known processes, including evaporation and crystallization, is isolated, and, if necessary, also desalination (either by electrodialysis or on a column).

View the problem

Linkomycin is a precursor in the synthesis of clindamycin, so the purity of clindamycin and its salts, addition salts or esters also depends on the purity of the incoming linkomycin.

Clindamycin is an antibiotic that can be synthesized, isolated and purified in different ways to produce a substance with the same or similar impurities, but in different amounts and proportions. Some of them can be removed by crystallization, while the proportion of some of them by this method cannot be noticeably reduced.

Recently, requirements for substances with a significantly lower proportion of individual and total impurities than those currently permitted by the pharmacopoeial standards are increasing in the field of drug production.

9.

This is especially the case for substances intended for formulation into injectable pharmaceutical forms, including e.g. clindamycin-2-phosphate.

For the effective functioning of the drug, stability is also very important: the basic properties of a substance should be as small as possible from the initial values during the shelf life, and in particular the quantity of related substances should not increase.

Our invention, therefore, arises from the task of finding the conditions for the purification of impurities from said antibiotics and their synthesis intermediates such that in the final product, no matter what the impurity profile of the feedstock, the total amount of impurities will not exceed 1% known impurities of 0.5% and unknown 0.1%. In this way, the prepared product will also be more stable than commercial products.

The state of the art

Linkomycin was isolated in 1962 from Streptomyces Lincolnensis and is a precursor to clindamycin synthesis.

Clindamycin is a semi-synthetic antibiotic produced by the 7 (S) -chloro-substitution of the 7 (R) -hydroxyl group of linkomycin. The synthesis has already been described in US Patents 3,475,407 and US 3,509,127 and in J. Med. Chem., 13 (1970) 616.

Clindamycin has Gram-positive bacteria, clostridia and some anaerobic germs. It is used to treat respiratory tract infections, skin and soft tissues.

Clindamycin is used as hydrochloride, palmitate hydrochloride and phosphate, and is marketed in the form of capsules, powder, vaginal cream, topical gel and injections.

Clindamycin hydrochloride is the hydrated addition salt of clindamycin. Clindamycin-2-phosphate is a water-soluble ester between clindamycin and phosphoric acid. The gross formula of clindamycin phosphate is C ₁₈ H3 ₄ CIN ₂ O ₂ PS, the molar mass is 504.96 g / mol, and the chemical name is (2S-trans) -methyl-7-chloro-6,7,8trideoxy-6 - [[( 1-methyl-4-propyl-2-pyrrolidinyl) carbonyl] amino] -1-thio-L-threo-a-Dgalacto-octopyranoside 2- (dihydrogen phosphate). Both appear to be white or almost white crystalline powder.

In the patent and other literature in the field, only individual articles are described describing the determination or identification of individual impurities by HPLC; however, we did not find any literature sources describing the purification of clindamycin or lincomycin and their salts, addition salts or esters using preparative HPLC.

Description of the new solution with implementation examples

The present invention is a novel process for the purification of clindamycin or lincomycin and their salts, addition salts or esters and their synthesis intermediates by preparative HPLC.

Following this process, the final antibiotic or its synthesis intermediates can be purified at any stage of the synthesis, which can be continued after completion of preparative HPLC, and crystallization is only required to obtain the final product with adequate purity.

Compared to the prior art, the main advantage of the process of the present invention is the improved chromatographic purity of the final product, which is also more stable and also has a good yield.

The invented process is particularly effective for removing three types of impurities that could not be separated from the above antibiotics by known methods, in particular crystallization, and have the following characteristics:

• Ethyl group analogue instead of propyl at site 4 (code: B), • Different epimers (for clindamycin: 7-epi) (code: epi), • Until now unknown double-chain analogues in the propyl chain (for clindamycin only exocyclic -> propylidene, with linkomycin may also be at the site of 1-> 1-propenyl), the structure of which was determined by NMR spectroscopy and mass spectrometry (MS) (code: en).

The invented process can be performed using the following stationary or mobile phases:

The stationary phase is a reverse phase, which may be natural (eg silica gel with different alkyl chains) or it may be a synthetic crosslinked polymer (eg styrene and divinylbenzene).

The stationary phase particle size is from a few to 20 pm.

Prior to application to the chromatographic column, the pH of the sample should be around neutral and therefore adjusted if necessary, e.g. with dilute aqueous ammonia solution.

The pH of the mobile phase, which must be in the narrow range from about 5.5 to about 8, preferably from about 6.5 to about 7.5, is very important for the successful removal of certain types of impurities. In this context, the mobile phase may be a dilute organic, halogenated organic or inorganic acid, e.g. formic, acetic, propionic, hydrochloric, boron, phosphorus, sulfur and other or their buffers with alkali metal cations, with ammonia or amines or as an aqueous solution of an organic solvent.

Separation on a HPLC column gives a pure product solution, which is then isolated by evaporation and subsequent crystallization, and, if necessary, desalination (either by electrodialysis or column).

The impurities in the individual samples and the purity were determined by HPLC, for clindamycin hydrochloride and phosphate according to the procedures of the European Pharmacopoeia, and for other substances by analogous methods.

Antibiotic substances from various sources, in our case clindamycin phosphate, either crude (directly from synthesis) or commercially available, may have very different amounts of each of the aforementioned and other impurities (in%) and their ratios, as indicated in Table 1 :

Table 1

substance CMF-B CMF-7epi CMF-3 CMF-en CM LY-F max. Σ crude 1,28 1.09 1.13 0.08 0.04 0.07 0.22 4.36 komerc. 1 1.35 0.10 0.04 0.10 0.04 0.27 0.28 2.89 komerc. 2 1.44 0.61 0.10 0.16 0.03 0.22 0.27 2.58

CMF-3 is clindamycin-3-phosphate; CM is clindamycin;

LY-F is linkomycin phosphate.

From the crude substance of Table 1, purification according to the invented process (using three combinations of parameters Samples 1, 2 and 3) yields a purified substance which, in comparison with the crude substance, as well as purified commercial substances, is the most reduced of those particular types of impurities which they cannot be essentially removed by other known processes. The profile is listed in Table 2:

Table 2

vz. CMF-B CMF-7-epi CMF-3 CMF-en CM LY-F max. Σ 1 0.03 0.11 0.06 0.02 0.04 0.02 0.06 0.37 2 0.02 0.18 0.06 0.02 0.03 0.02 0.06 0.44 3 0.01 0.11 0.05 0.01 0.03 0.02 0.05 0.35

The accompanying figure shows a chromatogram of a sample of clindamycin-2-phosphate before use of the invented process (A) and subsequent crystallization (B).

The invention is explained, but not limited in any way, by the following examples:

Example 1

Parameters for HPLC:

Stationary phase: octadecylsilicagel.

Column: Amicon, dimensions 150x10 mm, particle size: 15 pm.

Mobile phase: 47% methanol solution in water.

Application to the column: 3 ml.

Mobile phase flow: 7.5 ml / min.

The combined major clindamycin-2-phosphate fractions obtained by HPLC column separation were evaporated to a concentration of 120-170 g / l.

The resulting concentrate was evaporated after evaporation. With concentrated H ₃ PO _{4 we} adjust the pH to 3.5-4.5. Let's cool down. Add as much methanol as the volume of the concentrate. Crystallize for 5 hours at 5 ° C. The product is filtered off, washed with methanol and dried in a vacuum oven.

Evaporate the liquors, and salt the concentrated concentrate if necessary. We also evaporate the lateral fractions and return them to the column with the alkalis.

Electrodialysis desalines alkaline, lateral or major fractions when the concentration of ammonium acetate is too high after evaporation.

The major fractions were desalinated on a column of industrial HPLC with water, and clindamycin-2-phosphate was then eluted with methanol.

Example 2

Mobile phase: phosphate buffer (c = 20 mmol / l, pH = 7.5), equal volume of methanol.

The procedure is the same as in Example 1 except that the purified antibiotic is linkomycin hydrochloride.

Example 3

Mobile phase: ammonium acetate (c = 50 mmol / l, pH = 7.5), equal volume of methanol.

The procedure is the same as in Example 1 except that the purified antibiotic is clindamycin hydrochloride.

Example 4

Stability of clindamycin-2-phosphate

Comparatively, we tested the three-month accelerated stability of clindamycin-2phosphate purified by an ion exchanger and the subsequent crystallization in methanol and the invented process. The following parameters were monitored: appearance, pH, and content of water and related substances (individually and collectively) by pharmacopoeia. The essential differences listed in Table 3 are, in particular, in the content of related substances:

Table 3

cleaning process - » ion-izm.kr. crystal. invented process Φ parameter / time -> initial 3 мес. initial 3 мес. related substances - total 0.75% 0.92% 0.81% 0.81% CMF-B 0.65% 0.57% p.m.d. p.m.d. CMF-3 <0.05% 0.10% p.m.d. p.m.d. clindamycin. HCI p.m.d. 0.15% 0.14% 0.13% linkomycin. HCI <0.05% <0.10% p.m.d. p.m.d.

p.m.d. = below detection limit (<0.01%)

Lek, a pharmaceutical and chemical factory d.d.

Claims (20)

Patent claims A process for the removal of impurities from clindamycin or lincomycin and their salts, addition salts or esters and their synthesis intermediates by preparative HPLC, characterized in that, by subsequent evaporation and crystallization, a purity of ^ 99% is obtained, containing less than 0.5% of epimers and 4-propylidenals of 4- (1-propenyl) -alogues and less than 0.2% of 4-ethyl analogue each with less than 0.1% of unknown impurities. Process according to claim 1, characterized in that preparative HPLC, evaporation and crystallization can be followed by desalination. Method according to claim 2, characterized in that the desalination can be carried out by electrodialysis or on a column. The method of claim 1, characterized by the purification of clindamycin hydrochloride. by so, that it applies to 5. A process according to claim 1, characterized by so, Yes se used for purification of clindamycin-2-phosphate. The process of claim 1, characterized Po so, Yes se used for purification of lincomycin hydrochloride. The process of claim 1, characterized PO so, Yes se used for purification of intermediates in the synthesis of clindamycin-2-phosphate. Method according to claim 1, characterized in that a reverse phase is selected for the stationary phase. A method according to claim 8, characterized in that the average particle diameter of which the stationary phase consists is between about 2 and about 20 μπη. A method according to claim 9, characterized in that the average particle diameter of which the stationary phase consists is between about 10 and about 15 μηη. 11. The method of claim 1, wherein the pH of the antibiotic sample is between about 6.5 and about 7.5 before being applied to the chromatographic column. A process according to claim 11, characterized in that the mobile phase is a solution of methanol in water. Process according to claim 1, characterized in that the pH of the organic or inorganic acid buffer in the mobile phase is between about 5.5 and about 8. Process according to claim 13, characterized in that the pH of the organic or inorganic acid buffer in the mobile phase is between about 6.5 and about 7.5. 15. Linkosanide antibiotic prepared according to any one of the methods of claims 1-14, characterized in that it contains a total of less than 1% impurities, less than 0.5% of epimers and 4-propylidene- or 4- (1- propenyl) analogs and less than 0.2% of 4-ethyl analogue. Clindamycin hydrochloride according to claim 15, prepared according to any of the methods of claims 1-14, characterized in that it contains less than 1% impurities in total, less than 0.5% of 7-epianalog and 4-propylidene analog and at the same time less than 0.2% of 4-ethylanalog. 17. Clindamycin-2-phosphate according to claim 15, prepared according to any of the methods of claims 1-14, characterized in that it contains a total of less than 1% of impurities, less than 0.5% of the 7-epi-analogue, and Of 4-propylidene-analog and less than 0.2% of 4-ethyl-analog at the same time. 18. Linkomycin hydrochloride according to claim 15, prepared according to any of the methods of claims 1-14, characterized in that it contains in total less than 1% of impurities, less than 0.5% of epimers and 4propylidene or 4- (1 -propenyl-analogs, and at the same time less than 0.2% of 4-ethyl analogue. 19. Compound (2S-trans) -methyl-7-chloro-6,7,8-trideoxy-6 - [[(1-methyl-4-propyl-2-pyrrolidinyl) carbonyl] amino] -1-thio-L-threo -aD-galacto-octopyranoside 2 (dihydrogen phosphate) according to claim 15, prepared according to any of the processes of claims 1-14, characterized in that it contains a total of less than 1% impurities, less than 0.5% 7- epi-analogue and 4-propylidene-analogue, and at the same time less than 0.2% of 4-ethyl-analogue. 20. Linkosanide antibiotic, characterized in that it contains a total of less than 1% of impurities, less than 0.5% of epimers and 4propylidene or 4- (1-propenyl) analogues and less than 0.2% of 4- etilanaloga. 21. Clindamycin hydrochloride according to claim 20, characterized in that it contains a total of less than 1% of impurities, less than 0.5% of 7-epi-analogue and 4-propylidene-analogue at the same time and less than 0.2% 4 -ethyl-analogue. 22. Clindamycin-2-phosphate according to claim 20, characterized in that it contains a total of less than 1% of impurities, less than 0.5% of 7-epi40 each. analogue and 4-propylidene analogue, and at the same time less than 0.2% of 4-ethylanalog. 23. Linkomycin hydrochloride according to claim 20, characterized in that it contains a total of less than 1% of impurities, less than 0.5% of epimers and 4-propyl-den- or 4- (1-propenyl) analogues and at the same time less than 0.2% of 4-ethyl-analogue. 24. (2S-trans) -methyl-7-chloro-6,7,8-trideoxy-6 - [[(1-methyl-4-propyl-2-pyrrolidinyl) carbonyl] amino] -1-thio-L-threo compound - <xD-galacto-octopyranoside 2- (dihydrogen phosphate) according to claim 20, characterized in that it contains less than 1% impurities in total, less than 0.5% of 7-epi-analogue and 4-propylidene analog and at the same time less than 0.2% of 4-ethyl analogue.