阅读说明：本技术 一种骨化三醇的药物组合物 (Calcitriol pharmaceutical composition ) 是由 刘俊 吕飞龙 谭问非 张俊国 黄毅 于 2020-12-09 设计创作，主要内容包括：本发明提供了一种新化合物杂质I,还提供了包含杂质I的骨化三醇药物组合物。通过限定骨化三醇药物组合物中杂质I的含量,使骨化三醇药物组合物的安全性得到提高,产品质量得到进一步保障。(The invention provides a new compound impurity I and also provides a calcitriol pharmaceutical composition containing the impurity I. The content of the impurity I in the calcitriol pharmaceutical composition is limited, so that the safety of the calcitriol pharmaceutical composition is improved, and the product quality is further guaranteed.)

1. A compound impurity I characterized by the structure:

2. a calcitriol pharmaceutical composition is characterized in that the content of impurity I is not higher than 1.0%

3. The pharmaceutical composition of claim 2, comprising calcitriol and impurity I.

4. The pharmaceutical composition according to any one of claims 2 or 3, wherein the content of impurity I is not higher than 0.2%.

5. The pharmaceutical composition of claim 4, wherein the content of impurity I is not higher than 0.1%.

6. Use of impurity I according to claim 1, characterized by the use in quality control of calcitriol bulk drug substances or calcitriol preparations.

7. The process for the preparation of impurity I according to claim 1, characterized by comprising the steps of:

(1) dissolving calcitriol in a dichloromethane-chloroform mixed solution;

(2) and (2) cooling the solution obtained in the step (1) to about 0 ℃, adding a dichloromethane-chloroform mixed solution dissolved with a proper amount of m-chloroperoxybenzoic acid, and carrying out heat preservation reaction to generate an impurity I.

8. The high performance liquid chromatography detection method of impurity I according to claim 1, characterized by comprising the following parameters: a chromatographic column: phenylsilane bonded silica gel as filler (4.6 × 250mm,3 μm);

mobile phase: mobile phase A: acetonitrile-Tris buffer (40:60) (Tris buffer: 1.0g Tris (hydroxymethyl) aminomethane was weighed into 900mL water, pH was adjusted to 7.0-7.5 with phosphoric acid, diluted to 1000mL with water and mixed), mobile phase B: acetonitrile;

gradient program:

flow rate: 1.0 mL/min; detection wavelength: 230 nm; column temperature: 30 ℃; sample introduction amount: 100 μ L.

9. The detection method according to claim 8, wherein

The preparation method of the test solution comprises the following steps: taking a proper amount of sample, precisely weighing, placing in a glass test tube, adding a proper amount of diethyl ether, shaking up, adding a pre-activated solid phase extraction column, adjusting the pressure for elution until the eluent does not flow out, adding a proper amount of ethyl acetate-n-hexane eluent according to a certain proportion, eluting until the eluent does not flow out, naturally drying the column, adding a proper amount of ethanol, eluting until the eluent does not flow out, combining the eluates, placing in a measuring flask, and adding 20% ethanol solution for dilution to prepare a solution containing about 3.2 mu g of calcitriol per 1 mL;

the preparation method of the reference solution comprises the following steps: taking a proper amount of reference substance of the impurity I, precisely weighing, adding 60% ethanol for dissolving and diluting to prepare a reference substance solution of 32ng/mL, and shaking up;

the determination method comprises the following steps: precisely measuring reference solution and sample solution, injecting into high performance liquid chromatograph, recording chromatogram, and calculating according to external standard method.

Technical Field

The invention belongs to the field of medicinal chemistry, and particularly relates to a new impurity of calcitriol and a medicinal composition thereof.

Background

Calcitriol (Calcitriol), also known as 1, 25-dihydroxycholecalciferol or 1, 25-dihydroxyvitamin D₃Is vitamin D in the human body₃The most important metabolismOne of the active products has the functions of promoting the absorption of calcium by intestinal tracts, regulating the transport of inorganic salt in bone, and the like. It is mainly used for treating rickets, postmenopausal osteoporosis, senile osteoporosis, renal osteodystrophy, idiopathic hypoparathyroidism, pseudopathy, postoperative parathyroid gland function disorder, osteomalacia, etc. of chronic renal failure patients. Calcitriol preparation developed by Roche and approved by FDA to be marketed in 1978 and 8 months and approved to be imported in China in 1999, and the trade name of calcitriol preparation is

Calcitriol is important as a highly active drug that needs to be taken for a long time to conduct related safety studies such as impurity studies. At present, the calcitriol variety is not collected in the Chinese pharmacopoeia (2020 edition), and the variety is collected in the European pharmacopoeia (EP9.0) and the United states pharmacopoeia (USP40), and impurities including: calcitriol precursors, trans-calcitriol, 1 β -calcitriol, methylene calcitriol, and calcitriol precursor triazoline adducts.

The inventor takes a compound S as an initial raw material and prepares calcitriol through a Grignard reaction, a hydroxyl deprotection reaction and a photoreaction. During the preparation process, the inventor unexpectedly found that although an oxidizing agent enough to oxidize the double bond is not used and argon gas is adopted for protection in many places during the preparation process, the prepared calcitriol is inevitably subjected to oxidative degradation to generate an impurity I. This indicates that the production of this impurity is of low relevance to the synthetic route itself and is difficult to avoid by route optimization. However, the impurities are not included in the pharmacopoeia or standards, and are not reported or disclosed. Therefore, the determination of the chemical structure and preparation method of the impurity I, the establishment of a detection method, the analysis of the impurity content and the determination of a reasonable impurity limit are necessary to ensure the product quality and the medication safety of calcitriol.

Disclosure of Invention

The invention provides a compound impurity I, which has the following structure:

the impurities I have an epoxidation structure, and the epoxidation structure belongs to an alarm structure of genotoxic impurities (see the literature: Mali Lei, Malayu, Chenzhao, Jianyu. the alarm structure of genotoxic impurities [ J ]. China New medicine journal, 2014,23(18):2106-2111), and belongs to class 3 genotoxic impurities, has potential genotoxicity risks and is necessary to be analyzed, detected and controlled according to ICH M7 [ mutagenicity impurity assessment and control ] issued by the International harmonization society (ICH) for registration of human drugs.

The present invention also provides a pharmaceutical composition of calcitriol, wherein the content of impurity I is not higher than 1.0%, in some embodiments not higher than 0.2%; in some embodiments, not higher than 0.1%.

The invention also provides a pharmaceutical composition of calcitriol, which comprises calcitriol and an impurity I, wherein the content of the impurity I is not higher than 1.0%; in some embodiments, no higher than 0.2%; in some embodiments, not higher than 0.1%.

The invention also provides the application of the impurity I in the quality control of calcitriol bulk drugs or calcitriol preparations.

The invention also provides a preparation method of the impurity I, which comprises the following steps:

(1) dissolving calcitriol in a dichloromethane-chloroform mixed solution;

(2) cooling the solution obtained in the step (1) to about 0 ℃, adding a dichloromethane-chloroform mixed solution dissolved with a proper amount of m-chloroperoxybenzoic acid, and carrying out heat preservation reaction to generate an impurity I; wherein, in some embodiments, the method further comprises a washing and purifying step of the product impurity I; in some embodiments, a washing purification step of product impurity I is further included, and the eluent used for purification is dichloromethane methanol (30: 1).

The invention also provides a high performance liquid chromatography detection method of the impurity I, which comprises the following parameters:

a chromatographic column: phenylsilane bonded silica gel as filler (4.6 × 250mm,3 μm);

mobile phase: mobile phase A: acetonitrile-Tris buffer (40:60) (Tris buffer: 1.0g Tris (hydroxymethyl) aminomethane was weighed into 900mL water, pH was adjusted to 7.0-7.5 with phosphoric acid, diluted to 1000mL with water and mixed), mobile phase B: acetonitrile;

gradient program:

flow rate: 1.0 mL/min; detection wavelength: 230 nm; column temperature: 30 ℃; sample introduction amount: 100 mu L of the solution; in some embodiments, the method of calculating the impurity I content is external standard.

The content in the invention refers to the weight percentage content.

The content of the impurities in the invention refers to the weight percentage of the impurities in the calcitriol.

The temperature of the invention is about 0 ℃, which means 0 +/-4 ℃.

The invention has the beneficial effects that:

(1) provides a novel compound impurity I;

(2) the pharmaceutical composition of calcitriol is provided, the content of the impurity I in the pharmaceutical composition is limited, so that the content of the impurity I is simultaneously lower than the acceptable intake (calculated according to the TTC method) of the 3 rd genotoxic impurity and the control limit of related substances, the safety of the calcitriol pharmaceutical composition is improved, and the product quality is further ensured;

(3) provides the application of the impurity I in the quality control of calcitriol bulk drugs or calcitriol preparations;

(4) provides a preparation method of the impurity I;

(5) a method for detecting an impurity I is provided, which provides assurance for the detection and control of the impurity.

Detailed Description

In order to further illustrate the present invention and to facilitate an understanding thereof, only some examples are provided and will be described in detail. It will be understood by those skilled in the art that the following examples are not intended to limit the scope of the present invention.

EXAMPLE 1 Process for the preparation of impurity I

0.28g of calcitriol and 200mL of a mixed solution (dichloromethane: chloroform: 1) were added to a single-neck flask, the temperature was reduced to about 0 ℃ with stirring, 20mL of a mixed solution (dichloromethane: chloroform: 1) in which 0.16g of m-chloroperoxybenzoic acid was dissolved was added dropwise to the flask, and the mixture was reacted for 2.5 hours with heat preservation after completion of the dropwise addition. TLC (dichloromethane: methanol ═ 20:1) monitored completion of the reaction, and then 100mL of 1N aqueous sodium bicarbonate solution was added to the flask and washed 1 time. Drying with anhydrous sodium sulfate, filtering, and spin-drying the filtrate under reduced pressure. Purifying by column chromatography (dichloromethane: methanol: 30:1), eluting, and spin-drying the eluent under reduced pressure to obtain 80.0mg of impurity I. MS M/z 455.31[ M + Na ]]⁺。

¹H-NMR(400MHz,CDCl₃):

Example 2 method for detecting impurity I

The content of the impurity I was measured according to high performance liquid chromatography (chinese pharmacopoeia 2015 edition four parts general rules 0512).

Tris buffer: weighing 1.0g of tris (hydroxymethyl) aminomethane in 900mL of water, adjusting the pH value to 7.0-7.5 by using phosphoric acid, diluting to 1000mL of water, and uniformly mixing;

test solution: taking a proper amount of sample, precisely weighing, placing in a glass test tube, adding a proper amount of diethyl ether, shaking up, adding a pre-activated solid phase extraction column, adjusting the pressure for elution until the eluent does not flow out, adding a proper amount of ethyl acetate-n-hexane eluent according to a certain proportion, eluting until the eluent does not flow out, naturally drying the column, adding a proper amount of ethanol, eluting until the eluent does not flow out, combining the eluates, placing in a measuring flask, and adding 20% ethanol solution for dilution to prepare a solution containing about 3.2 mu g of calcitriol per 1 mL.

Control solution: taking a proper amount of the impurity I reference substance, precisely weighing, adding 60% ethanol for dissolving and diluting to prepare a 32ng/mL reference substance solution, and shaking up.

Chromatographic conditions are as follows: a chromatographic column: phenyl silane bonded silica gel as filler (4.6 x 250mm,3 μm);

mobile phase: mobile phase A: acetonitrile-Tris buffer (40:60), mobile phase B: acetonitrile;

gradient program:

time (min)	Mobile phase A (%)	Mobile phase B (%)
			0	100	0
20	100	0
			20.1	20	80
35	20	80
			35.1	100	0
50	100	0

Flow rate: 1.0 mL/min; detection wavelength: 230 nm; column temperature: 30 ℃; sample introduction amount: 100 μ L.

The determination method comprises the following steps: precisely measuring reference solution and sample solution, injecting into high performance liquid chromatograph, recording chromatogram, and calculating according to external standard method. The content of impurity I was found to be 0.08%.