Method for purifying rifampicin from nitrosamine

Abstract

The present invention relates to the control of the amount of genotoxic impurities in rifampicin, in particular the control of the amount of nitrosamines. In particular, the present invention relates to a process for the preparation of rifampicin that is substantially free of 1-methyl-4-nitrosopiperazine (MeNP) impurities. The present invention also relates to rifampicin that is substantially free of 1-methyl-4-nitrosopiperazine (MeNP) impurities.

Description

Technical Field

[0001] The present invention relates to the control of the amount of genotoxic impurities in rifampicin, in particular the control of the amount of nitrosamines.

[0002] In particular, the present invention relates to a process for preparing rifampicin substantially free of 1-methyl-4-nitrosopiperazine (MeNP) impurity. The present invention also relates to rifampicin substantially free of 1-methyl-4-nitrosopiperazine (MeNP) impurity. Prior art

[0003] Nitrosamines are compounds with the general formula R 1 R 2 Organic compounds with NN=O are classified as Category 1 impurities, “known mutagenic carcinogens” by EMA (European Medicines Agency) ICH M7 (R1) (Evaluation and control of DNA-reactive (mutagenic) impurities in pharmaceuticals to limit potential carcinogenic risk), and as 2A- “possible carcinogens” by the International Agency for Research on Cancer (IARC).

[0004] Such compounds are known by-products of various synthetic processes, for example in the presence of nitrosating agents (such as nitrites under acidic conditions) and amines (secondary, tertiary or quaternary), and can be difficult to isolate: thus, even after purification, these trace impurities may be present in the finished product.

[0005] Trace amounts of nitrosamines have been found in many active pharmaceutical ingredients for humans. In recent years, this has led global regulatory agencies (such as the US Food and Drug Administration (FDA) and EMA) to impose strict limits on the tolerance thresholds of these impurities. Therefore, companies holding marketing authorizations for drugs containing active pharmaceutical ingredients (APIs) of chemical or biological origin need to conduct increasingly accurate and rigorous reviews of their drug products and their production methods to prevent or limit nitrosamine content. The attention of regulatory agencies to the problem of genotoxic impurities (especially for nitrosamines) represents a preventive approach aimed at ensuring patient safety and health (in line with the measures introduced by the amendment of Article 5, paragraph 3 of EMA Regulation (EC) No 726 / 2004 to limit the presence of nitrosamines in medicinal products for human use).

[0006] Rifampicin (pictured below) belongs to the rifamycin class and is an antibiotic used primarily to treat tuberculosis, as well as to treat serious infections such as bloodstream infections and leprosy.

[0007] For example, rifampicin can be obtained starting from rifamycin-S by the usually unisolated Oxazino intermediates are prepared and subsequently reacted with 1-amino-4-methylpiperazine (1-AMP) to obtain rifampicin (see the synthesis scheme below). This type of synthesis is known in the literature, see for example US4174320 and CN101486716.

[0008]

[0009] Traces of nitrosamines, specifically 1-methyl-4-nitrosopiperazine (MeNP), have recently been detected in commercially available batches of rifampicin. The presence of this genotoxic impurity is known to originate from the use of 1-AMP in the rifampicin production process, as well as from the oxidation of degradation products of rifampicin itself.

[0010] The FDA recommends a maximum daily intake of MeNP of 96 ng / day. Specifically for rifampicin, based on this data and considering the maximum daily dose corresponding to 600 mg / day, the maximum MeNP threshold considered acceptable in the active ingredient corresponds to 0.16 parts per million (ppm; Sandrine and Mike Frick, N-nitrosamines and TuberculosisMedicines Rifampicin and Rifapentine, February 2021).

[0011] In fact, in August 2020, the FDA issued such a clarification, which set the acceptable limit for MeNPs in rifampin at 0.16 ppm, yet stipulated acceptance of interim distributions of rifampin containing less than 5 ppm MeNPs to maintain patient access to these life-saving medicines.

[0012] To the best of the applicant's knowledge, methods for obtaining rifampicin comprising MeNPs in an amount equal to or lower than 0.16 ppm are not known in the art.

[0013] Patent document CN111018887 describes a rifampicin purification method that allows obtaining rifampicin with a purity of about 99.5% and a single impurity content of less than 0.1%. However, this document does not mention at all the presence of genotoxic impurities (especially MeNP) and therefore does not mention the control of their amount. Summary of the invention

[0014] The Applicant has therefore solved the problem of obtaining a rifampicin substantially free of 1-methyl-4-nitrosopiperazine (MeNP), and in particular a rifampicin having a MeNP content equal to or lower than 0.16 ppm.

[0015] The Applicant has also tackled the object of developing a process for purifying rifampicin from genotoxic substances, in particular from 1-methyl-4-nitrosopiperazine (MeNP).

[0016] Thus, in a first aspect, the present invention relates to a process for the preparation of rifampicin substantially free of 1-methyl-4-nitrosopiperazine (MeNP), and preferably having a MeNP content equal to or lower than 0.16 ppm, said process comprising the steps of:

[0017] a) providing a mixture of crude rifampicin in an organic solvent, wherein the mixture is obtained by dissolving solid crude rifampicin in an organic solvent;

[0018] b) washing the mixture with an aqueous solution having a pH of 2 to 7 to obtain an organic layer and an aqueous layer, which are then separated;

[0019] c) adding at least one antioxidant to the separated organic layer obtained from step b);

[0020] d) distilling the organic solvent under an inert atmosphere;

[0021] e) crystallization under an inert atmosphere; and

[0022] f) isolating the rifampicin thus obtained under an inert atmosphere.

[0023] The present invention also relates to rifampicin having a 1-methyl-4-nitrosopiperazine (MeNP) content equal to or lower than 0.16 ppm. DETAILED DESCRIPTION

[0024] definition

[0025] In this document, the terms "crude active ingredient" and "crude rifampicin" are intended to mean an active ingredient obtained from a preparation process prior to the process according to the invention, in particular rifampicin. In particular, it is intended to mean rifampicin comprising one or more impurities, in particular rifampicin comprising 1-methyl-4-nitrosopiperazine (MeNP).

[0026] For the purposes of the present invention, the term "substantially free of 1-methyl-4-nitrosopiperazine (MeNP)" is intended to mean an active ingredient, in particular rifampicin, comprising 1-methyl-4-nitrosopiperazine (MeNP) in an amount equal to or lower than 0.16 ppm.

[0027] For the purposes of the present invention, the term "process for the preparation of rifampicin substantially free of 1-methyl-4-nitrosopiperazine (MeNP)" is intended to refer to a purification process, the purpose of which is to remove or reduce the amount of impurities, in particular the amount of MeNP, from the crude active ingredient. Such a process is applied downstream of the process for the preparation of the crude active ingredient.

[0028] Furthermore, such methods may also be used to control the levels of impurities formed by degradation of the crude active ingredient.As used herein, the term "control" and variations thereof are also intended to include the terms "prevent" and "inhibit".

[0029] Unless otherwise stated, percentage values ​​(%) indicated herein are intended to be percentages by weight.

[0030] For the purpose of the present invention, the term "aqueous solution having a pH of 2 to 7" is intended to mean an aqueous solution at a controlled pH, which may contain a pH adjusting agent, or may be pure water when the pH is equal to 7.

[0031] Detailed Description

[0032] Therefore, in a first aspect, the present invention relates to a process for preparing rifampicin substantially free of 1-methyl-4-nitrosopiperazine (MeNP), the process comprising the following steps:

[0033] a) providing a mixture of crude rifampicin in an organic solvent, wherein the mixture is obtained by dissolving solid crude rifampicin in an organic solvent;

[0034] b) washing the mixture with an aqueous solution having a pH of 2 to 7 to obtain an organic layer and an aqueous layer, which are then separated;

[0035] c) adding at least one antioxidant to the separated organic layer obtained from step b);

[0036] d) distilling the organic solvent under an inert atmosphere;

[0037] e) crystallization under an inert atmosphere; and

[0038] f) isolating the rifampicin thus obtained under an inert atmosphere.

[0039] Preferably, said method allows obtaining a MeNP content of rifampicin equal to or lower than 0.16 ppm.

[0040] According to a preferred aspect, the method allows obtaining a rifampicin having a MeNP content ranging from 0.01 to 0.16 ppm, preferably from 0.01 to 0.10 ppm, even more preferably from 0.01 to 0.05 ppm.

[0041] According to another preferred aspect, the method allows obtaining a rifampicin having a MeNP content ranging from 0.02 to 0.16 ppm, preferably from 0.02 to 0.10 ppm, even more preferably from 0.02 to 0.05 ppm.

[0042] According to a preferred aspect of the present invention, the mixture provided in step a) is a crude mixture obtained at the end of the process for the preparation of rifampicin.

[0043] According to an alternative aspect of the present invention, the mixture provided in step a) is a mixture obtained by dissolving solid crude rifampicin in an organic solvent.

[0044] According to a preferred aspect of the present invention, the organic solvent used in step a) is selected from polar and non-polar aprotic solvents, such as dichloromethane, ethyl acetate, 2-methyltetrahydrofuran, methyl isobutyl ketone, toluene and mixtures thereof. According to a preferred aspect, the organic solvent is a non-polar aprotic solvent. Preferably, the non-polar aprotic solvent is dichloromethane.

[0045] Step b) of the process according to the invention is a liquid-liquid extraction stage, in which the aqueous solution is added to the mixture of step a), obtaining a two-phase mixture; the two organic layers and the aqueous layer are mixed (for example by stirring) and subsequently separated. The aqueous layer is then discarded and the organic layer is used in step c) of the above-described process. Preferably, step b) can be repeated several times (for example 2 to 5 times): in this case, at the end of each step b), the organic layer obtained after the first washing is recovered and washed again with the aqueous solution.

[0046] Step b) is carried out to remove any excess unreacted 1-AMP and MeNP, both water-soluble compounds, so that at the end of this step a mixture comprising the organic solvent and rifampicin is obtained, wherein the impurity content is reduced, but not null.

[0047] According to another preferred aspect of the present invention, the method further comprises one or more additional washing stages subsequent to step b). Preferably, the one or more additional washing steps are carried out with an aqueous solution, more preferably with water.

[0048] According to a preferred aspect of the present invention, the aqueous solution used has a neutral pH, preferably it is pure water at pH 7.

[0049] According to another preferred aspect of the present invention, the aqueous solution used in step b) is pH controlled water, also defined as acidic water, with a pH of 2 to 6, preferably 3 to 6, even more preferably 3 to 5.

[0050] Preferably, the aqueous solution comprises a pH adjusting agent selected from organic acids and inorganic acids. Preferably, the pH adjusting agent is an organic acid or a mixture of organic acids, more preferably selected from acetic acid, formic acid, oxalic acid, and mixtures thereof. Preferably, the pH adjusting agent is acetic acid.

[0051] Advantageously, without being bound by any theory, the addition of at least one antioxidant in step c) before the distillation (step d) allows the formation of oxygen radicals to be suppressed, thus avoiding new formation of MeNPs.

[0052] Preferably, the at least one antioxidant is selected from ascorbic acid, ascorbic acid-2-glucoside, ascorbic acid-6-octanoate, ascorbic acid-6-palmitate, cysteine, sodium pyrosulfite, propyl gallate, butylhydroxyanisole, butylhydroxytoluene (butylhydroxytoluene, BHT) and combinations thereof. In a particularly preferred aspect of the present invention, the at least one antioxidant is ascorbic acid. According to another particularly preferred aspect of the present invention, the at least one antioxidant is a mixture of ascorbic acid and butylhydroxytoluene (BHT).

[0053] Preferably, the amount of the antioxidant added is 0.3% to 2% by weight, preferably 0.5% to 1% by weight. The amount of the antioxidant is calculated as the weight percentage relative to the weight of rifamycin S (the starting material for synthesizing rifampicin). Alternatively, in some embodiments, wherein the mixture a) is provided from solid crude rifampicin, the percentage is calculated by weight relative to the weight of the crude rifampicin used.

[0054] Steps d) to f) and preferably also other optional steps before or after any one of steps d) to f) are carried out in an inert atmosphere to prevent or limit the new formation of MeNPs. Preferably, the inert atmosphere is an atmosphere substantially free of oxygen, for example substantially saturated with an inert gas (e.g. nitrogen or argon), or under vacuum.

[0055] According to a preferred aspect, the inert atmosphere is an O 2 The amount is lower than 3% by volume, preferably lower than 1% by volume, even more preferably equal to or lower than 0.5% by volume of the atmosphere.

[0056] According to another preferred aspect, the relative oxygen density of the inert atmosphere is <0.1.

[0057] Steps d) to f) are performed according to methods known in the art.

[0058] In particular, the distillation step d) comprises using a boiling temperature that depends on the organic solvent chosen for step a).Generally speaking, at the end of step d), a concentrated residue is obtained from which substantially all the organic solvent has been eliminated.

[0059] According to a preferred aspect of the invention, after step d), the process comprises a further step d'), preferably also carried out in an inert atmosphere, in which an aprotic polar solvent, preferably acetone, is added to the residue obtained from the distillation step d) and the resulting solution is distilled again. This step can be used, for example, to eliminate possible traces of organic solvent used in step a) before carrying out the crystallization step e).

[0060] Preferably, the crystallization step e) is carried out using an aprotic polar solvent, preferably acetone or an acetone / ethyl acetate mixture. Preferably, in step e), the residue obtained from step d) or d') is dissolved in the selected aprotic polar solvent, the aprotic polar solvent is preferably preheated, usually heating the obtained mixture at the reflux temperature of the selected solvent, and then slowly cooling.

[0061] Advantageously, separation step f) is carried out by filtration, for example optionally under vacuum. At the end of this step, solid rifampicin is obtained.

[0062] According to a preferred aspect of the present invention, the method comprises another step f') after step f), which comprises washing the solid rifampicin obtained after crystallization with an aprotic polar solvent (preferably acetone) and isolating the rifampicin thus obtained again. Preferably, step f') is carried out in an inert atmosphere.

[0063] According to another preferred aspect of the present invention, the method comprises a further step g) after the separation step f) or f'), wherein the obtained rifampicin is preferably dried in an inert atmosphere.

[0064] Thus, in a second aspect, the present invention relates to rifampicin characterized in that it is substantially free of 1-methyl-4-nitrosopiperazine (MeNP) and preferably comprises MeNP in an amount lower than or equal to 0.16 ppm.

[0065] Another aspect of the invention relates to Rifampicin obtainable by the above process, characterized by the fact that it is substantially free of 1-methyl-4-nitrosopiperazine (MeNP) and preferably comprises MeNP in an amount lower than or equal to 0.16 ppm.

[0066] Preferably, the rifampicin comprises 1-methyl-4-nitrosopiperazine (MeNP) in an amount of 0.01 to 0.16 ppm, preferably 0.01 to 0.10 ppm, even more preferably 0.01 to 0.05 ppm.

[0067] According to another preferred aspect, the amount of MeNP contained in the rifampicin is 0.02 to 0.16 ppm, preferably 0.02 to 0.10 ppm, even more preferably 0.02 to 0.05 ppm.

[0068] The above is intended to be exemplary rather than limiting. In addition, those skilled in the art will appreciate that modifications may be made without departing from the scope of the present invention.

[0069] Experimental Section

[0070] The present description will be better illustrated in the following examples, which are intended to be illustrative only and not limiting.

[0071] Example 1 - Preparation of crude rifampicin starting from rifamycin-S

[0072] Rifamycin S (105 g, 0.1509 mol) was dissolved in N, N-dimethylformamide (53.0 g) and dichloromethane (40 ml), heated to a temperature of about 35 to 40° C. and stirred until completely dissolved. Acetic acid (25 g, 0.4163 mol) and N-methyl-tert-butylamine (32 g, 0.3671 mol) were then added, and the mixture was maintained at a temperature of about 32 to 36° C. for 6 hours.

[0073] The mixture was then cooled to room temperature, dichloromethane (108 mL), piperazine (20 g, 0.2322 mol) and 1-amino-4-methylpiperazine (32 g, 0.2778 mol) were added, and the mixture was maintained at a temperature of about 32 to 36 °C for about 3 hours until the reaction was complete (as evidenced by TLC).

[0074] The mixture was then washed with an aqueous solution at pH 5 to 6. 1-Amino-4-methylpiperazine (0.32 g) was added to the organic layer, and the mixture was brought to reflux temperature and stirred for 2 hours.

[0075] This gave a mixture of crude rifampicin in dichloromethane.

[0076] Example 2 - Purification of crude rifampicin (according to the invention)

[0077] The mixture of crude rifampicin obtained according to Example 1 in dichloromethane was washed with an aqueous acetic acid solution (540 ml) at pH 3.9 to 4.2 and then stirred for 30 minutes. The mixture was then kept still for 60 minutes and then the organic phase was separated from the aqueous phase.

[0078] The mixture thus obtained was heated to a temperature of about 45° C., and butylated hydroxytoluene (0.1 g, 4.5*10^ -4 mol) and ascorbic acid (0.52 g, 0.003 mol, 0.5 wt % relative to the weight of rifamycin S used in Example 1). Dichloromethane was then distilled at about 53° C. in an inert atmosphere (under nitrogen saturation).

[0079] The residue thus obtained was then dissolved again in preheated acetone (200 ml) in an inert atmosphere, and the mixture was distilled a second time in an inert atmosphere at a temperature of about 50 to 58° C.

[0080] The residue thus obtained is then crystallized from acetone in an inert atmosphere (under nitrogen saturation conditions). Specifically, under an inert atmosphere, the residue is dissolved in acetone (520 ml) preheated to 53° C., the mixture is heated to reflux (about 55 to 59° C.) and maintained at this temperature for 40 to 45 minutes, then slowly cooled to a temperature between -5 and -20° C.

[0081] Pure rifampicin was then isolated by filtration in an inert atmosphere (under nitrogen saturation conditions) and washed with 80 ml of cold acetone. The solid rifampicin obtained was then dried in vacuo at 55°C.

[0082] Example 3 - Purification of crude rifampicin (according to the invention)

[0083] The procedure of Example 2 was repeated, but using 1% ascorbic acid (1.04 g, 0.006 mole relative to the weight of rifamycin S used in Example 1) instead of 0.5%.

[0084] Example 4 - Purification of crude rifampicin (comparative)

[0085] The procedure of Example 2 was repeated without using ascorbic acid or an inert atmosphere in either of the steps.

[0086] MeNP content in purified rifampicin

[0087] Table 1 below shows the MeNP contents detected in the products obtained from Examples 2 to 4 as determined by liquid chromatography-mass spectrometry (LC-MS).

[0088] Table 1

[0089] sample MeNP content determined by LC-MS 2 0.033ppm 3 0.028ppm 4 0.250ppm

[0090] It is evident from the data summarized in Table 1 that the use of ascorbic acid and an inert atmosphere in the purification of rifampicin is necessary to obtain rifampicin containing very low amounts of MeNPs (Samples 2 to 3), in particular amounts well below the limit of 0.16 ppm set by regulatory agencies.

[0091] In fact, by carrying out the same purification method without the use of ascorbic acid or inert atmosphere (Comparative Sample 4), a rifampicin was obtained containing a MeNP content that was 1.5 times higher than the limit established by the FDA and almost 10 times higher than the MeNP content of the rifampicin obtained by the method according to the present invention.

Claims

1. A method for preparing rifampicin substantially free of 1-methyl-4-nitrosopiperazine (MeNP), the method comprising the following steps: a) providing a mixture of crude rifampicin in an organic solvent, wherein the mixture is obtained by dissolving solid crude rifampicin in an organic solvent; b) washing the mixture with an aqueous solution having a pH of 2 to 7 to obtain an organic layer and an aqueous layer, which are then separated; c) adding at least one antioxidant to the separated organic layer obtained from step b); d) distilling the organic solvent under an inert atmosphere; e) crystallization under an inert atmosphere; and f) isolating the rifampicin thus obtained under an inert atmosphere.

2. The method according to claim 1, wherein the rifampicin comprises 1-methyl-4-nitrosopiperazine in an amount less than or equal to 0.16 ppm.

3. The method according to claim 2, wherein the amount of 1-methyl-4-nitrosopiperazine contained in the rifampicin is 0.01 ppm to 0.16 ppm, preferably 0.01 ppm to 0.10 ppm.

4. The process according to any one of claims 1 to 3, wherein the organic solvent used in step a) is selected from polar and non-polar aprotic solvents, such as dichloromethane, ethyl acetate, 2-methyltetrahydrofuran, methyl isobutyl ketone, toluene and mixtures thereof.

5. The method according to claim 4, wherein the organic solvent is a non-polar aprotic solvent, preferably dichloromethane.

6. The method according to any one of claims 1 to 5, wherein the at least one antioxidant is selected from ascorbic acid, ascorbic acid-2-glucoside, ascorbic acid-6-octanoate, ascorbic acid-6-palmitate, cysteine, sodium metabisulfite, propyl gallate, butylated hydroxyanisole, butylated hydroxytoluene (BHT), and combinations thereof.

7. The method of claim 6, wherein the antioxidant is ascorbic acid.

8. The method according to any one of claims 1 to 7, wherein the inert atmosphere is a substantially nitrogen-saturated or argon-saturated atmosphere, or under vacuum, preferably with an amount of O2 lower than 3% by volume.

9. The process according to any one of claims 1 to 8, wherein step f) of separating is performed by filtration.

10. The process according to any one of claims 1 to 9, further comprising a step f'), wherein the rifampicin obtained in step f) is washed with an aprotic polar solvent, preferably acetone, and separated.

11. The method according to any one of claims 1 to 10, further comprising a step g) in which the rifampicin obtained from step f) or from step f') is dried.

12. Rifampicin obtainable by the process according to claims 1 to 11, characterized in that 1-Methyl-4-nitrosopiperazine was contained in an amount lower than or equal to 0.16 ppm.

13. Rifampicin, characterized in that 1-Methyl-4-nitrosopiperazine was contained in an amount lower than or equal to 0.16 ppm.

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