An emtricitabine degradation impurities and preparation method thereof

Abstract

The present invention is relates to an identification and isolation of an emtricitabine degradation impurity compounds. The present invention also relates to process of preparing the emtricitabine impurity compounds by photocatalytic forced degradation study.

Description

[0001] AN EMTRICITABINE DEGRADATION IMPURITIES AND PREPARATION METHOD THEREOF

[0002] FIELD OF THE INVENTION

[0003] The present invention is relates to identification and synthesis of degraded impurity compounds of emtricitabine. The present invention particularly related to an emtricitabine degradation impurities and preparation of the same by photocatalytic forced degradation of emtricitabine drug.

[0004] BACKGROUND OF THE INVENTION

[0005] Emtricitabine is a nucleoside reverse-transcriptase inhibitor for the prevention and treatment of HIV infection in adults and children. Emtricitabine is a vital antiretroviral drug used in combination therapy to treat HIV / AIDS. Ensuring the purity and safety of emtricitabine is critical for effective HIV / AIDS treatment. The active pharmaceutical ingredient (API) in a drug product must be highly pure to ensure its safety and efficacy. Impurities, even in small amounts, can reduce the drug's effectiveness and causes harmful side effects.

[0006] An impurity is defined as any component that is not the chemical entity described in the formulation. Pharmaceutical impurities are tightly regulated. For every impurity, there exists chemistry and a safety aspect. The chemical aspect is purely technological as any impurity can cause secondary reactions, both directly and catalytically. However, assessment and control of the impurities in a drug substance and drug product are important aspects for a drug development team to obtain various marketing approvals. Indeed, it is extremely challenging for an organic chemist to identify the impurities, which are formed in very small quantities in a drug substance and wearisome if the product is non-pharmacopeial.

[0007] Emtricitabine is susceptible to degradation under various conditions, including heat, light and acidic or basic environments. Degradation products can form during various stages, including manufacturing, storage and shelf life of the drug product. These degradation products causes potential risks of reduced effectiveness of emtricitabine treatment and adverse side effects in patients.

[0008] Forced degradation studies are employed to stress the drug molecule and accelerate the formation of degradation products. While existing forced degradation studies on emtricitabine exist, they might not have identified the full spectrum of potential degradation products. This present invention aims to address the limitations of current knowledge by conducting a comprehensive forced degradation study on emtricitabine under various stress conditions. The present invention also identifying and isolating novel degradation products of emtricitabine which are not previously reported. The degradation products were characterized by employing analytical technique liquid chromatography-mass spectrometry (LC- MS).

[0009] The presence of impurities in pharmaceutical drug compounds, have raised toxicological concerns, including the potential for genotoxic, carcinogenic, and mutagenic characteristics. Impurities can arise at various stages of the synthetic process, including from reagents, by-products, starting materials, intermediates, or even during the stability testing of the drug substance, where they may manifest as degrades. As a result, the precise identification and quantification of these impurities, becomes imperative to ensure the safety and efficacy of the pharmaceutical product. Addressing this technical challenge is essential in advancing the development and production of Emtricitabine and similar compounds in the pharmaceutical industry.

[0010] However, isolating impurities from the final API is a very tedious and time consuming task. Hence, synthesis of impurity allows for the use of a pure form of impurities in the identification of their biological effects and can be used as a standard reference to detect the presence of similar impurities in the final product. There are no specific processes available which can produce such impurities that are used as a standard reference in the quality control of the drug emtricitabine. There is no any reported method of separation, identification and estimation of related substance or impurities of emtricitabine.

[0011] Accordingly, there exists a need to provide an identification, characterization and preparation of the impurity degradation compounds of emtricitabine. The present invention contributes significantly to the advancement of emtricitabine-based therapies by providing a deeper understanding of its degradation process and products and provide the way for safer and more effective HIV / AIDS treatment options.

[0012] The inventors of present invention have surprisingly found that ten degradation products were formed by forced degradation study of emtricitabine. All ten degradation impurity compounds of emtricitabine were characterized by LC-MS method.

[0013] OBJECT OF THE INVENTION

[0014] The main object of the present invention is to provide an emtricitabine degradation impurities.

[0015] Another object of the present invention is to provide an impurity compounds of emtricitabine by forced degradation study for use of quality control of emtricitabine.

[0016] Another object of the present invention is to provide a preparation method of emtricitabine forced degradation impurities.

[0017] Another object of the present invention is to provide an emtricitabine degradation impurities which can be used as a reference standard to detect the presence of these impurities during the stability studies and in vitro dissolution studies in the emtricitabine and its formulation products. SUMMARY OF THE INVENTION

[0018] The main aspect of the present invention is to provide an emtricitabine degradation impurities and preparation thereof.

[0019] Another aspect of the present invention is to provide an emtricitabine degradation impurities having the structure of formula (I) to formula (III).

[0020] Another aspect of the present invention is to provide a process of preparation of an emtricitabine degradation impurities having the structure of formula (I) to formula (III).

[0021] BRIEF DESCRIOPTION OF DRAWINGS

[0022] Figure 1: LC Chromatogram of developed method for determination of emtricitabine

[0023] Figure 2: LC Chromatogram of photocatalytic degradation of emtricitabine Figure 3: HPLC Chromatogram of degraded sample

[0024] Figure 4: LC-MS analysis of the degraded products of the emtricitabine

[0025] DETAILED DESCRIPTION OF INVENTION

[0026] The present invention overcomes the aforesaid drawbacks of the above, and other objects, features and advantages of the present invention will now be described in greater detail. Also, the following description includes various specific details and is to be regarded as merely exemplary. Accordingly, those of ordinary skill in the art will recognize that: without departing from the scope and spirit of the present disclosure and its various embodiments there may be any number of changes and modifications described herein.

[0027] As used herein, whether in a transitional phase or in the body of a claim, the terms “comprise(s)” and “comprising” are to be interpreted as having an open-ended meaning. That is, the terms are to be interpreted synonymously with the phrases “having at least” or “including at least”. When used in the context of a process, the term “comprising” means that the process includes at least the recited steps, but may include additional steps. When used in the context of a composition, the term “comprising” means that the process includes at least the recited features or components, but may also include additional features or components.

[0028] As used herein, the singular forms “a,” “an” and “the” specifically also encompass the plural forms of the terms to which they refer, unless the content clearly dictates otherwise.

[0029] As used herein the term “photocatalytic forced degradation” refers to an advanced oxidation process, which can be used to degrade compounds. Photocatalytic degradation uses light energy to drive compound degradation.

[0030] As used herein the term “degradation impurity compounds” are unwanted chemicals that can develop during the manufacturing, transportation, and storage of drug products and can affect the efficacy of pharmaceutical products.

[0031] As per main embodiment, the present invention is an emtricitabine impurity compounds and preparation method thereof.

[0032] As per one embodiment, the present invention is all about an emtricitabine impurity compounds identified during photocatalytic forced degradation of emtricitabine.

[0033] As per one embodiment of the present invention, an emtricitabine impurity compounds is having structural formula 2 to 11.

[0034] As per one embodiment, the process of preparation of emtricitabine degradation impurities is prepared by forced degradation study.

[0035] As per one embodiment, the process of preparation of emtricitabine degradation impurities is prepared photocatalytic forced degradation study. As per one embodiment of the present invention, the process for synthesizing and isolating the emtricitabine impurity compounds, comprising the step of, a) dissolving emtricitabine in 0.5 N NaOH solution under visible light (8 W) and stirring for 6 hours at room temperature. b) monitoring the step (a) solution on TLC using acetate: n-Hexane (1: 1, v / v) solvent system c) filtering the step (a) solution after completion of reaction and collecting solid mass as degradation product formula 11; d) extracting filtrate from step (c) solution with ethyl acetate (10 mL) and separating organic layer; e) drying organic layer from step (d) over anhydrous sodium sulphate and evaporating and collecting the solid product as formula 2.

[0036] The present invention is all about identification and quantification of emtricitabine and its Photolytic alkali degraded products which are identified as impurities of emtricitabine by LC-MS method. The said evaluated emtricitabine degradation impurities can be used as a standard reference to detect the presence of similar impurities in the formulation product of emtricitabine and for the quality control of the drug emtricitabine.

[0037] The invention is further illustrated by the following examples which are provided to be exemplary of the invention and do not limit the scope of the invention. While the present invention has been described in terms of its specific embodiments, certain modifications and equivalents will be apparent to those skilled in the art and are intended to be included within the scope of the present invention.

[0038] EXAMPLES:

[0039] EXAMPLE 1: Method Development for the estimation of emtricitabine

[0040] For develop the ideal conditions of the method, primarily the numbers of mobile phase variations, pH of the mobile phase, polarities of the organic solvents and the pressure of the instrument were applied and analysed for the sharp, single and clear chromatogram. The detailed developmental trials and changes has been occurring for the development of the unique mobile phase composition was mentioned below.

[0041] Example 2: Force degradation studies

[0042] The stability of the drug was evaluated by applying different stress conditions of the emtricitabine. Acidic was evaluated by applying 0.5N HC1 solution and it was further mixed with emtricitabine, with the interval of time; the sample were applied on the LC system and chromatogram was analysed. For the basic degradation 0.5N NaOH solution was prepared and mixed with the powdered emtricitabine, with the interval of time; the sample was carried out the analysed. Thermal degradation was performed at three different temperatures in oven. The pure form of API was weighed and transferred into glassware, further the glassware was put in to oven at 50°C, 75°C, 100°C after that the sample was analysed on the LC instrument. Moreover, the oxidative stress studies were carried out by applying the 10% H2O2 solution on the sample and it was further evaluated. Photolytic conditions were analysed by applying short UV, long UV and Visible light on the API and the samples were evaluated. The photo catalytic (photolytic + catalyst) degradation was performed by applying the 0.5N NaOH as a catalyst under the photolytic condition. The mixture was continuously stirred and samples were analysed at the intervals of time. The detailed results of the stress degradation studies were mentioned in table 2 and figure 2:

[0043] Example 3: LC-MS analysis of the degraded products of the emtricitabine

[0044] For detailed force degradation studies of emtricitabine, 7 mg of powdered drug was employed to 100 mL 0.1N NaOH solution (0.4 gm sodium hydroxide in to 100 mL distilled water) under visible white light (8 W). The samples were collected after each an hour and it was further analyzed on HPLC. Chromatogram showed three different peaks as shown in figure 3. Further the aliquot of the same sample was analyzed on LC-MS.

[0045] Example 4: Synthesis of emtricitabine impurities by photocatalytic degradation 1 gm of the drug was employed to 10 mL 0.5N NaOH solution (2 gm sodium hydroxide in to 100 mL distilled water) under visible white light (8 W). After the completion of reaction, (monitored on TLC using ethyl acetate: n-Hexane (1: 1, v / v) solvent system) the solution was filtered and solid (10 mg) was collected as product formula 11. The filtrate was extracted with ethyl acetate (10 mL) and organic layer was separated dried over anhydrous sodium sulphate and evaporated to dryness. The solid product (15 mg) collected as formula 2. LC-MS chromatogram is shown in figure 4.

[0046] From the mass obtained in LC-MS chromatograph, below are the possible structures of the degraded products based on molecular weight, which are identified as degradation impurities of emtricitabine.

[0047]

[0048] Example 5: Mechanism of formation of degraded products (Emtricitabine)

[0049] Example 5.1: Formation of dimer (Molecular Weight: 494.49 g / mol)

[0050] g . 4-amino-5-fluoro-1-((2R,5S)-2-(hydroxymethyl)-1,3- oxathiolan-5-yl)pyrimidin-2(1H)-one

[0051] NaOH hv

[0052] 1 ,1'-(2,7-bis(hydroxymethyl)-1,6,3,8-dioxadithiecane-5,10- diyl)bis(4-amino-5-fluoropyrimidin-2(1H)-one) Molecular Weight: 494.49

[0053] The solution of formula 1 (Emtricitabine) in the presence of NaOH and light usually promote free radical mediated degradation pathways. Thus, the oxathiolane ring opening lead to formation of oxygen (O) and carbon (C) free radical due to their stability. This free radical further form dimerization of formula 1 via radical recombination.

[0054] Example 5.2: Formation of formula 3 (Molecular Weight: 159.20 g / mol)

[0055]

[0056] The possible mechanism for the formation of Formula 3 is explained above.

[0057] Oxathiolane ring opening of formula 1 followed by radical recombination of isocyanate intermediate 3 may have produced acrylamide derivatives of formula 4. Further degradation of acrylamide side chain in the presence of hv (Light) and NaOH generated 3-allyl-4-hydroxythiazolidin-2-one (Molecular Weight: 159.20, Formula 3) through elimination of ammonia and water.

[0058] Example 5.3: Formation of formula 4 (Molecular Weight: 136.17 g / mol)

[0059] 2-(hydroxymethyl)-1 ,3-

[0060] 4-amino-5-fluoro-1 -((2R,5S)-2- 4-amino-5-fluoropyrimidin-2(1H)-one oxathiolan-5-ol (hydroxymethyl)-1 ,3-oxathiolan-5- Molecular Weight: 129.09 Molecular Weight: yljpyrimid in-2( 1 H)-one 136.17 Molecular Weight: 247.24 hv NaOH, H2O

[0061] NH3+ CO2+ H2O

[0062] 4-amino-5-hydroxypyrimidin-2(1H)-one Molecular Weight: 127.10

[0063] The possible mechanism for the formation of formula 4 is explained above. The NaOH mediated hydrolysis of formula 1 generate the two-building block (A) and (B, Formula 4). Further building block A decomposed in the presence of hv (Light) and generate ammonia, carbon dioxide and water.

[0064] Example 5.4: Formation of formula 5 to 11

[0065] 7

[0066] 3-allyl-4-hydroxythiazolidin-2-one

[0067] Molecular Weight: 160.2 Molecular Weight: 159.20 1 thiazolidin-2-one Molecular Weight: 295.37 hv )- ne

[0068] 4-hydroxy-3-(2-((10,15,20-trihydroxy-2,7,12,17- 3-(2-((10,15-dihydroxy-2,7,12-tris(hydroxymethyl)- tetrakis(hydroxymethyl)-1 ,6,11,16-tetraoxa-3,8, 13,18- 1 ,6,11-trioxa-3,8,13-trithiacyclopentadecan-5-yl)oxy)propyl) tetrathiacycloicosan-5-yl)oxy)propyl)thiazolidin-2-one -4-hydroxythiazolidin-2-one Molecular Weight: 703.86 Molecular Weight: 567.70

[0069] The possible mechanism for the formation of formula 5 to 11 is represented above. Photochemical activation of alkene present in the formula 3 generated alkyl radical which started chain polymerization reaction with formula 4 and formed addition product formula 5. Subsequently, the monomer addition of formula 4 might have produced formula 6 to 11. The mass spectrum showed incremental addition of mass (136 g / mol) which corroborate the hypothesis of chain polymerization of formula 3 with 4.

[0070] Example 6: Toxicity study

[0071] 5 In order to estimate the toxicity of generated products (formula 2, 3, 4 & 11), a in silico studies were carried out using online web server pKCSM [2] and data are tabulated as follows. As evident this degraded product formula 2 might have hepatotoxicity, while formula 3 may have skin sensitization and formula 11 could be hERG II inhibitor. Further all the degraded products possessed possible oral rate 10 acute toxicity due to lower LD50. In summary presence of these degraded products along with API formula 1 might be suggested as toxic impurities and their monitoring would be necessary. The results are shown below:

[0072] Table 4: Predicted toxicity properties of degraded products

[0073] Conclusion:

[0074] The process of the present invention provides degraded impurity compounds of emtricitabine which can be used as reference standard to detect the presence of these impurities during the stability studies and in vitro dissolution studies in the 20 emtricitabine and its formulation products.

Claims

CLAIMSWE CLAIM;1. An emtricitabine degradation impurities, wherein the impurity compounds having the following formula (2) to (11);2. The emtricitabine degradation impurities as claimed in claim 1, wherein the impurity compounds are detected by LC-MS with3. The process for preparing an emtricitabine degradation impurities comprising the steps of, a) dissolving emtricitabine in 0.5 N NaOH solution under visible light (8 W) and stirring for 6 hours at room temperature. b) monitoring the step (a) solution on TLC using acetate: n-Hexane (1: 1, v / v) solvent system c) filtering the step (a) solution after completion of reaction and collecting solid mass as degradation product formula 11; d) extracting filtrate from step (c) solution with ethyl acetate (10 mL) and separating organic layer; e) drying organic layer from step (d) over anhydrous sodium sulphate and evaporating and collecting the solid product as formula 2.

4. The process of preparation of emtricitabine degradation impurities as claimed in claim 3, wherein the process is a forced degradation study.The process of preparation of emtricitabine degradation impurities as claimed in claim 3, wherein the process is a photocatalytic forced degradation study.

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