A method of preparing an abacavir metabolite

Abstract

The application discloses a preparation method of an abacavir metabolite, and belongs to the field of drug metabolite synthesis. The whole process is reasonable in design, strong in operability, and has high implementation value and social and economic benefits. The method uses compound I as a starting material, and synthesizes the abacavir metabolite through two-step reactions. The optimal synthesis step and reaction condition are screened through experiments. The abacavir metabolite synthesized by the method has a liquid-phase purity of 98.2% or above and a chiral purity of 99.9% or above, provides a test sample for abacavir research, and has important research reference value in clinical pharmacokinetic research and new drug research and development.

Description

Technical Field

[0001] This invention relates to a method for preparing abacavir metabolites, belonging to the field of drug metabolite synthesis. Technical Background

[0002] Abacavir is an antiviral drug with anti-HIV activity. Its English name is Abacavir, and its chemical name is ((1S,4R)-4-(2-amino-6-(cyclopropylamino)-9H-purine-9-yl)cyclopent-2-en-1-yl)methanol. Its CAS number is 136470-78-5, and its molecular formula is C2. 14 H 18 N6O has a molecular weight of 286.34 g / mol. Its structural formula is shown in Formula I.

[0003] Abacavir was first developed by Glaxo-Wellcome in the UK and approved for marketing by the US Food and Drug Administration (FDA) in 1998 under the brand name Ziagen. It is a nucleoside reverse transcriptase inhibitor (NRTI). Like other nucleoside reverse transcriptase inhibitors, it is an inactive prodrug that is converted into the active metabolite carbovir triphosphate by cellular kinases in the human body. Carbovir triphosphate is an analog of deoxyguanosine triphosphate (dGTP) and competes with or integrates into viral DNA, inhibiting the activity of HIV reverse transcriptase, causing defects in the integrated nucleoside analogue, and stopping viral DNA replication.

[0004] In February 2008, the U.S. Food and Drug Administration revised its guidelines for HIV antiretroviral therapy, making abacavir one of the first-line antiretroviral drugs for treatment-naïve patients. This is due not only to its similar antiretroviral efficacy to tenofovir and its less mitochondrial damage, resulting in fewer adverse reactions such as fat loss, pancreatitis, neuritis, and lactic acidosis, but also to the fact that the HLA-B5701 gene, used for pre-treatment screening for hypersensitivity reactions, is widely used clinically abroad. Individuals who test negative for HLA-B5701 generally do not experience hypersensitivity reactions after using the drug, significantly improving its safety.

[0005] Abacavir metabolites are metabolites produced during the metabolism of abacavir in the human body. Pharmacological and pharmacokinetic studies of abacavir metabolites can provide control samples for the quality control of the raw material abacavir, thereby advancing abacavir research. Currently, there are few reports on the synthesis of abacavir metabolites.

[0006]

[0007] The paper Chem. Res. Toxicol. 2011, 24, 2129–2141 explicitly states that during the oxidation of active pharmaceutical ingredients, double bonds are prone to rearrangement, and the carbonyl alpha position is also prone to racemization. Furthermore, the rearranged products contain isomers, making purification and identification very difficult.

[0008]

[0009] The literature also explicitly states that direct oxidation of the active pharmaceutical ingredient (API) fails to yield the target product, and conventional oxidation conditions are insufficient. The main reason is that double bonds are prone to rearrangement during oxidation, and the alpha position of the carbonyl group is susceptible to racemization, leading to product instability. After numerous trials, we finally found a solution, overcoming a major challenge in the industry. Currently, no methods for preparing bacavir metabolites have been reported.

[0010] In order to conduct a comprehensive analysis and study of the clinical, pharmacological, pharmacokinetic, and toxicological aspects of abacavir, it is necessary to design and develop a method for preparing abacavir metabolites based on existing technologies. This would provide a reference substance for the comprehensive analysis of the clinical, pharmacological, pharmacokinetic, and toxicological aspects of abacavir, as well as for new drug development. Summary of the Invention

[0011] Objective of the invention: To address the deficiencies of the prior art, the present invention provides a method for preparing abacavir metabolites.

[0012] Technical solution: The present invention provides a method for preparing an abacavir metabolite, which is synthesized according to the following route:

[0013] .

[0014] The abacavir metabolite (Formula III) described in this invention is a metabolite produced during the metabolism of abacavir in the human body. It is also known as abacavir trifluoroacetic acid salt, with the free state CAS number 384380-52-3. Its chemical name is (1S,4R)-4-(2-amino-6-(cyclopropylamino)-9H-purin-9-yl)cyclopent-2-ene-1-carboxylic acid compound with 2,2,2-trifluoroacetic acid (1:1), a molecular weight of 300.32 (free product molecular weight) and 114.02 (trifluoroacetic acid molecular weight), and its molecular formula is C2. 14 H 16N6O2. (Free product molecular formula) C2HF3O2 (trifluoroacetic acid molecular formula), structure as shown in Formula III. The abacavir metabolite, compared to the active pharmaceutical ingredient, has its hydroxyl group oxidized to a carboxylic acid, simultaneously forming a salt with one molecule of trifluoroacetic acid. Currently, there are no reported methods for its synthesis that can be used chemically.

[0015] Furthermore, the specific steps include:

[0016] (1) Dissolve abacavir in acetone, add Jones reagent under ice bath to react, add isopropanol to quench the reaction, filter, wash, and dry to obtain intermediate product II;

[0017] (2) Dissolve intermediate product II in dichloromethane, add trifluoroacetic acid under ice bath to dissolve and clarify it, filter after restoring to room temperature, concentrate, purify, and crystallize to purify.

[0018] Furthermore, the structure of the abacavir metabolite is shown in Formula II:

[0019]

[0020] II.

[0021] Furthermore, the structure of the abacavir metabolite is shown in Formula III:

[0022]

[0023] III.

[0024] Further, in step (1), the volume ratio of abacavir to acetone is 1:45 to 1:90, preferably 1:45; the molar ratio of abacavir to Jones reagent is 1:2 to 1:4, preferably 1:2; and the concentration of Jones reagent is 2 mol / L. Maintaining the reaction in an ice bath for 30-60 minutes after 10-30 minutes can increase the reaction yield. The obtained intermediate product II is unstable; during processing, it should be protected from light and nitrogen, and heating should be avoided. Rapid processing and transfer are required.

[0025] Further, in step (2), the volume ratio of intermediate product II to dichloromethane is 1:10 to 1:20, preferably 1:20; the volume ratio of intermediate product II to trifluoroacetic acid is 1:1 to 1:3, preferably 1:3. After the reaction returns to room temperature, it is maintained at room temperature for 1-2 hours, preferably 1 hour. The room temperature is preferably 25°C. Purification is performed using C18 column chromatography, with the mobile phase being water and methanol containing 1% trifluoroacetic acid at a volume ratio of 10:1; the stationary phase of the C18 column chromatography is C18 powder, and the matrix is ​​200-300 mesh silica gel. The solvent for crystallization purification is acetonitrile and methanol at a volume ratio of 1:1.

[0026] Beneficial Effects: Compared with existing technologies, this invention has the following significant advantages: the synthesis process of this invention is rationally designed and highly operable; the reagents used in the preparation method are simple and readily available; the liquid phase purity of the abacavir metabolite obtained from the reaction can reach over 98%, and the chiral purity can reach over 99%. Through actual experimental operation, and characterization of the obtained product by MS, 1H NMR, HPLC, Chiral HPLC, OR, and IR, it is verified that the corresponding metabolite can be synthesized. The abacavir metabolite (compound III) synthesized by this invention provides a test sample for abacavir research and also provides a standard reference for quality monitoring of abacavir. Attached Figure Description

[0027] Figure 1 The mass spectrum of compound III;

[0028] Figure 2 The NMR spectrum of compound III;

[0029] Figure 3 The liquid phase spectrum of compound III;

[0030] Figure 4 The chiral liquid phase spectrum of compound III;

[0031] Figure 5 The optical rotation spectrum of compound III;

[0032] Figure 6 The image shows the infrared spectrum of compound III. Detailed Implementation

[0033] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0034] Compound I in the following experiment is the active pharmaceutical ingredient abacavir, which was purchased directly from the market.

[0035] Example 1

[0036] (1) Synthesis of Compound II: 9.50 g of Compound I (abacavir, CAS: 136470-78-5) was suspended in 427.5 mL of acetone. 33.2 mL of 2 mol / L Jones reagent was slowly added dropwise under ice bath until the solution dissolved and became clear. A pale green solid gradually precipitated from the reaction solution until stirring was stopped after 10 min. The reaction was continued under ice bath for 30 min, and then 40 mL of isopropanol was added to quench the reaction. The precipitated solid was filtered and washed with a small amount of isopropanol, then dried to obtain 6.50 g of off-white solid, Compound II, with a yield of 65.24%. During the process, care was taken to avoid light and nitrogen protection, avoid heating, and ensure rapid handling and transfer. The structure of Compound II is as follows:

[0037]

[0038] II

[0039] (2) Synthesis of Compound III: 6.50 g of Compound II was suspended in 97.5 mL of dichloromethane. 6.5 mL of trifluoroacetic acid was added under ice bath conditions to dissolve and clarify the compound. The mixture was then slowly brought to room temperature and maintained at room temperature for one hour. The reaction solution was filtered, and the filtrate was concentrated to obtain the crude product. The crude product was further purified by C18 column purification. The stationary phase of the C18 column was commercially available C18 silica gel, and the mobile phase was water and methanol (containing 0.1% trifluoroacetic acid) in a volume ratio of 10:1. The resulting off-white solid was further purified by crystallization with acetonitrile and methanol in a volume ratio of 1:1, finally yielding 3.80 g of off-white solid III, with a yield of 42.37%.

[0040] like Figure 1 The mass spectrum of compound III is [M+H]. + : 657.0.

[0041] like Figure 2 The 1H NMR spectrum data of compound III are as follows: 1 H NMR (400 MHz, DMSO-d6) δ 12.63+11.72(br+br, 1H), 9.61 (br, 1H), 7.81 (brs, 1H), 7.32 (br, 2H), 6.23 (m, 1H), 6.03(m, 1H), 5.44 (m, 1H), 3.69 (m, 1H), 2.88 (br, 1H), 2.77 (m, 1H), 2.07 (m,1H), 0.88+0.76 (m+m, 4H).

[0042] like Figure 3 The liquid phase purity of compound III is 98.21%.

[0043] like Figure 4 The chiral liquid phase purity of compound III is 99.98%.

[0044] like Figure 5 The optical rotation of compound III is -43.57°.

[0045] like Figure 6 The IR spectrum of compound III is as follows: 3389.61-3120.15 cm-1 (-COOH, -NH); 1685.58-1628.75 cm-1 (C=O); 1582.36 cm-1 (C=C).

[0046] Based on the above analysis, the structure of compound III is shown in formula III below:

[0047]

[0048] III

[0049] Example 2

[0050] (1) Synthesis of compound II: 8.20 g of compound I (CAS: 136470-78-5) was suspended in 492.0 mL of acetone. 43.0 mL of 2 mol / L Jones reagent was slowly added dropwise under ice bath until the solution dissolved and became clear. A pale green solid gradually precipitated from the reaction solution until stirring was stopped. After maintaining the ice bath for another 60 minutes, 50 mL of isopropanol was added to quench the reaction. The precipitated solid was filtered and washed with a small amount of isopropanol, and dried to obtain 5.20 g of off-white solid compound II, with a yield of 60.46%.

[0051] (3) Synthesis of Compound III: 5.20 g of Compound II was suspended in 52.0 mL of dichloromethane. 10.4 mL of trifluoroacetic acid was added under ice bath conditions to dissolve and clarify the solution. The solution was then slowly brought to room temperature and maintained at room temperature for two hours. The reaction solution was filtered, and the filtrate was concentrated to obtain the crude product. The crude product was further purified by C18 column purification. The stationary phase of the C18 column was commercially available C18 silica gel, and the mobile phase was water and methanol (containing 0.1% trifluoroacetic acid) in a volume ratio of 10:1. The resulting off-white solid was further purified by crystallization with acetonitrile and methanol, finally yielding 3.20 g of off-white solid Compound III, with a yield of 44.60%. The structure of Compound III was confirmed by characterization as shown in Formula III.

[0052] Example 3

[0053] (1) Synthesis of Compound II: 9.00 g of Compound I (CAS: 136470-78-5) was suspended in 810.0 mL of acetone. Under ice bath conditions, 62.9 mL of 2 mol / L Jones reagent was slowly added dropwise until the solution dissolved and became clear. A pale green solid gradually precipitated from the reaction solution until stirring was stopped. After maintaining the ice bath for another 45 minutes, 80 mL of isopropanol was added to quench the reaction. The precipitated solid was filtered and washed with a small amount of isopropanol. After drying, 5.50 g of off-white solid Compound II was obtained, with a yield of 58.27%.

[0054] (2) Synthesis of Compound III: 5.50 g of Compound II was suspended in 110.0 mL of dichloromethane. 16.5 mL of trifluoroacetic acid was added under ice bath conditions to dissolve and clarify the solution. The solution was then slowly brought to room temperature and maintained at room temperature for one hour. The reaction solution was filtered, and the filtrate was concentrated to obtain the crude product. The crude product was further purified by C18 column purification. The stationary phase of the C18 column was commercially available C18 silica gel, and the mobile phase was water and methanol (containing 0.1% trifluoroacetic acid) in a volume ratio of 10:1. The resulting off-white solid was further purified by crystallization with acetonitrile and methanol, finally yielding 3.60 g of off-white solid III, with a yield of 47.44%. The structure of Compound III was confirmed by characterization as shown in Formula III.

[0055] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method of preparing a metabolite of abacavir, characterized in that, Synthesis according to the following route: 。 2. The production method according to claim 1, characterized by, Comprising the following steps: (1) Abacavir is dissolved in acetone, and Jones reagent is added under ice bath to react, and isopropanol is added to quench the reaction, and then the mixture is filtered, washed, and dried to obtain intermediate product II; (2) Intermediate product II is dissolved in dichloromethane, and trifluoroacetic acid is added under ice bath to dissolve and clarify, and then the mixture is filtered, concentrated, purified, and crystallized.

3. The production method according to claim 2, characterized by, The structure of the abacavir metabolite is shown in formula II: II.

4. The preparation method according to claim 2, characterized in that, The structure of the abacavir metabolite is shown in formula III: Ⅲ。 5. The preparation method according to claim 2, characterized in that, In step (1), the volume ratio of abacavir to acetone is 1:45-1:90, and the molar mass ratio of abacavir to Jones reagent is 1:2-1:4, and the concentration of Jones reagent is 2 mol / L.

6. The method of claim 2, wherein, In step (1), after the reaction is continued for 10-30 min, the ice bath is maintained for 30-60 min.

7. The preparation method according to claim 2, characterized in that, In step (2), the volume ratio of intermediate product II to dichloromethane is 1:10-1:20, and the volume ratio of intermediate product II to trifluoroacetic acid is 1:1-1:

3.

8. The preparation method according to claim 2, characterized in that, In step (2), after the reaction is continued at room temperature for 1-2 h, the mixture is maintained at room temperature.

9. The preparation method according to claim 2, characterized in that, In step (2), C18 column chromatography is used for purification, and the mobile phase is water and methanol containing 1% trifluoroacetic acid at a volume ratio of 10:

1.

10. The method of claim 1, wherein, In step (2), the solvent for crystallization purification is acetonitrile and methanol at a volume ratio of 1:1.

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