WO2019072005A1 - Azacitidine disaccharide impurity, preparation method therefor and use thereof - Google Patents

Abstract

The present invention relates to an azacitidine disaccharide impurity, a preparation method therefor and use thereof. A compound of formula III is obtained by reacting a compound represented by formula IV as a start raw material with a tetraacetyl ribose under the catalysis of a Lewis acid, and the compound of formula III is hydrolyzed to obtain a compound of formula II. The compound of formula II prepared can be used as a reference for the detection of related materials of azacitidine, and is used for quality control applications of azacitidine and related preparations thereof.

Description

Azacitidine disaccharide impurity and preparation method and application thereof Technical field

The invention relates to azacitidine disaccharide impurity and preparation method and application thereof.

Background technique

Azacitidine was first synthesized by Czechoslovakian scientists Piskala and Sorm, and later isolated from the fermentation broth of Streptoverticillium ladakanus.

Azacitidine is a 5-aza analog of cytidine and belongs to a class of epigenetic antineoplastic agents known as Hypomethylating agents. Abnormal DNA methylation inactivates key genes that regulate normal cell growth, differentiation, and apoptosis, and is associated with tumor development and progression. The efficacy of azacitidine in the treatment of MDS is mainly due to its DNA hypomethylation activity and direct cytotoxicity to abnormal hematopoietic cells in the bone marrow. Azacitidine is a DNA methyltransferase inhibitor that does not significantly inhibit DNA synthesis at the maximum inhibition of DNA methylation.

The structural formula of azacitidine is as shown in formula I:

A small amount of impurities such as the structure shown in Formula II may be produced during the production of azacitidine:

The preparation and separation method of this impurity has not been reported in the literature at home and abroad, and therefore the disaccharide impurity of azacitidine is an important compound for the quality study of the azacitidine bulk drug and related preparations.

Summary of the invention

In the study we found that azacitidine produced a small amount of compound impurities of formula II during the production process. Therefore, the synthesis of this compound has been studied in the present invention.

A first aspect of the invention provides a compound having the structure of formula II, III:

In a second aspect of the invention, there is provided a process for the preparation of a compound of formula II, the route of which is as follows:

The preparation method of the present invention comprises the following steps: Step 1: The compound of the formula IV is reacted with tetraacetyl ribose under the catalysis of a Lewis acid to prepare a compound of the formula III; Step 2: the compound of the formula III is subjected to a hydrolysis reaction. The compound of formula II is then prepared.

The Lewis acid in step 1 is selected from trimethylsilyl triflate or aluminum trichloride, preferably from aluminum trichloride.

The molar ratio of the Lewis acid to the tetraacetyl ribose is from 1 to 10:1, preferably from 2 to 5:1, preferably from 2.5 to 3:1.

The molar ratio of the compound of the formula IV to the tetraacetyl ribose is from 1.5 to 10:1, preferably from 2 to 6:1, further preferably from 3.5 to 5:1.

The reaction in the step 1 is carried out under an organic solvent, which may be an aprotic solvent, preferably one or more of dichloromethane, chloroform or 1,2-dichloroethane.

The reaction temperature in the step 1 is 20 to 60 ° C, preferably 20 to 50 ° C.

The hydrolysis reaction of the step 2 is carried out under a basic substance selected from the group consisting of sodium methoxide.

The hydrolysis reaction is carried out under a protic solvent selected from one or a mixture of two of methanol or ethanol.

The temperature of the hydrolysis reaction is 0 to 30 ° C, preferably 0 to 10 ° C, and more preferably 5 to 10 ° C.

In a third aspect of the invention, there is provided the use of a compound of formula II as a control for quality control of azacitidine and its related formulations. The compound of the formula II can be used as an impurity as a reference substance for the detection of azacitidine, and is used for quality control of azacitidine and its related preparations.

Detailed ways

The invention will be described in more detail below by way of examples, which are intended to be illustrative and not to be construed as limiting.

1H-NMR spectra were recorded at room temperature using a Bruker spectrometer. Deuterated dimethyl sulfoxide was used as a solvent, and the solvent included tetramethylsilane as an internal standard (if not otherwise mentioned). MS spectra were recorded using an Agilent 6100 LCTM. The relative signal strength is given (expressed as a percentage based on the main peak). Purity checks were performed using an Agilent 1200 HPLC. The conditions determined are given with the respective working examples.

The molecular weight of each compound during the experiment is as follows:

Example 1: Preparation of a compound of formula III

20 g (62.9 mmol) of tetraacetyl ribose was mixed with 57 g (222.7 mmol) of the compound of the formula IV, dichloromethane was added thereto, the temperature was raised to 30 ° C, and 21 g (157.9 mmol) of aluminum trichloride was added thereto, and the reaction was carried out for 23 hours. The temperature was lowered to a normal temperature of 25 ° C, and the reaction liquid was poured into ice water of sodium hydrogencarbonate, and dichloromethane was added thereto, and the insoluble matter was removed by filtration, and the organic layer was concentrated to dryness. Purification by column chromatography on silica gel eluting elut elut elut elut elut

Example 2: Preparation of a compound of formula III

17 g (53.4 mmol) of tetraacetyl ribose was mixed with 55 g (214.5 mmol) of the compound of the formula IV, 1,2-dichloromethane was added, the temperature was raised to 50 ° C, and 18 g (133.5 mmol) of aluminum trichloride was added thereto, and the reaction was carried out for 19 hours. . The temperature was lowered to a normal temperature of 25 ° C, and the reaction liquid was poured into ice water of sodium hydrogencarbonate, and dichloromethane was added thereto, and the insoluble matter was removed by filtration, and the organic layer was concentrated to dryness. Purification by column chromatography on silica gel eluting elut elut elut elut elut

Example 3: Preparation of a compound of formula III

15 g (47.1 mmol) of tetraacetyl ribose was mixed with 60 g (235.6 mmol) of the compound of the formula IV, 1,2-dichloromethane was added, the temperature was raised to 30 ° C, and trimethylsilyl trifluoromethanesulfonate 42 g (188.4) was added. Methanol.), reaction for 19 hours. The temperature was lowered to a normal temperature of 25 ° C, and the reaction liquid was poured into ice water of sodium hydrogencarbonate, and dichloromethane was added thereto, and the insoluble matter was removed by filtration, and the organic layer was concentrated to dryness. Purification by column chromatography on silica gel eluting elut elut elut

The structural confirmation data of the compound of Formula III are shown in Tables 1 and 2.

Table 1 Hydrogen spectrum measurement results

chemical shift	Number of protons	Peak shape	Related proton chemical shift	Attribution	Remarks
9.18	1	d	5.70	H 15	J=9.2Hz
8.50	1	s	/	H 17	/
5.76	1	d	5.59	H 8	J=3.6Hz
5.70	1	Dd	5.21	H 25	J=9.2, 5.7 Hz
5.59	1	Dd	5.44, 5.76	H 6	J=6.2, 3.6Hz
5.44	1	t	4.26, 5.59	H 4	J=6.5Hz
5.29	1	m	4.16, 5.21	H 21	/
5.21	1	t	5.29	H 23	J=5.6Hz
4.36	1	Dd	4.26	H 2	J=11.7, 3.1Hz
4.26	1	m	4.10, 4.36, 5.44	H 3	/
4.24	1	m	4.16	H 19	/
4.16	1	m	5.29	H 20	/
4.11	1	m	4.16	H 19	/
4.10	1	m	4.26	H 2	/
2.06	18	s	/	H 28 , H 31 , H 34 , H 38 , H 40 , H 43	/

Table 2 Carbon spectrum measurement results

Example 4: Preparation of a compound of formula II

31 g (49.3 mmol) of the compound of the formula III was dissolved in 50 mL of methanol and 50 mL of ethanol, and the mixture was cooled to 5 ° C, and 1 mL of a sodium methoxide methanol solution (30 wt%) was added thereto, and the mixture was stirred for 30 minutes, and a large amount of pale yellow solid was precipitated, filtered, and dried white. 16.4 g of solid. Preparative HPLC separation and purification of white solid 10.2 g (27.1 mmol, yield 55%).

Example 5: Preparation of a compound of formula II

29 g (46.1 mmol) of the compound of the formula III and 500 mL of ethanol were mixed and dissolved, and the mixture was cooled to 5 ° C, and 2 mL of a sodium methoxide methanol solution (30 wt%) was added thereto, and the mixture was stirred for 30 minutes, and a large amount of pale yellow solid was precipitated, filtered, and dried to give a white solid. . The white solid 11.1 g (29.5 mmol, yield 64%) was purified by preparative HPLC.

Structural confirmation:

The structure confirmation data of the compound represented by Formula II are shown in Tables 3 and 4.

Table 3 Hydrogen spectrum measurement results

chemical shift	Number of protons	Peak shape	Related proton chemical shift	Attribution	Remarks
8.67	1	s	/	H 2	/
5.99	1	Br s	/	H 6	Heavy water exchange disappears
5.68	1	d	4.08	H 11	J=3.5Hz
5.40	1	d	3.89	H 19	J=4.0Hz
4.08	1	t	3.87, 4.02, 5.68	H 10	J=4.0Hz
4.02	1	t	3.89	H 17	J=4.0Hz
3.91	1	d	3.74, 4.02, 5.40	H 18	J=4.0Hz
3.89	1	d	3.91, 4.08	H 9	J=4.0Hz
3.87	1	t	3.58, 3.71, 3.87	H 13	J=4.0Hz
3.74	1	t	3.39, 3.47	H 21	J=4.0Hz
3.71	1	Dd	3.58, 3.91	H 15-1	J=12.2, 2.8 Hz
3.58	1	Dd	3.71, 3.91	H 15-2	J=12.2, 2.8 Hz
3.47	1	Dd	3.39, 3.74	H 24-1	J=11.7, 4.6Hz
3.39	1	Dd	3.47, 3.74	H 24-2	J=11.7, 4.6Hz

Table 4 Carbon spectrum measurement results

chemical shift	Carbon type	Attribution	Related proton chemical shift	Remotely related proton chemical shift
164.44	C	C 5	/	5.40, 8.67
156.22	CH	C 2	8.67	5.68
152.90	C	C 3	/	5.69, 8.67
89.42	CH	C 11	5.68	4.02, 8.67
85.01	CH	C 19	5.40	3.74, 3.91, 4.01
84.23	CH	C 21	3.74	3.39, 3.47, 3.91
83.65	CH	C 13	3.87	3.89
73.98	CH	C 10	4.08	3.87, 5.67
73.38	CH	C 18	3.91	3.74, 5.40
70.22	CH	C 13	3.87	3.58, 3.71, 5.40

68.69	CH	C 17	4.02	3.39, 3.47, 5.68
61.97	CH 2	C 24	3.39, 3.47	3.91
59.68	CH 2	C 15	3.58, 3.71	3.87, 4.02

Claims (12)

1. Compounds of formula II and formula III:

   
2. A method for preparing a compound of the formula II, which comprises the steps of:

   

   Step 1: a compound of the formula IV is reacted with a tetraacetyl ribose under the catalysis of a Lewis acid to prepare a compound of the formula III;

   Step 2: The compound of the formula III is subjected to a hydrolysis reaction to prepare a compound of the formula II.
3. The process according to claim 2, wherein the Lewis acid in the step 1 is selected from trimethylsilyl trifluoromethanesulfonate or aluminum trichloride, preferably from aluminum trichloride.
4. The process according to claim 3, wherein the molar ratio of the Lewis acid to the tetraacetyl ribose is from 1 to 10:1, preferably from 2 to 5:1, further preferably from 2.5 to 3:1.
5. The process according to claim 2, wherein the molar ratio of the compound of the formula IV to the tetraacetyl ribose is from 1.5 to 10:1, preferably from 2 to 6:1, further preferably from 3.5 to 5:1.
6. The preparation method according to claim 2, wherein the reaction in the step 1 is carried out in an organic solvent, and the organic solvent may be an aprotic solvent, preferably dichloromethane, chloroform or 1,2- One or more of dichloroethane.
7. The process according to claim 2, wherein the reaction temperature in the step 1 is from 20 to 60 ° C, preferably from 20 to 50 ° C.
8. The process according to claim 2, wherein the hydrolysis reaction of the step 2 is carried out under a basic substance.
9. The process according to claim 8, wherein the basic substance is selected from the group consisting of sodium methoxide.
10. The process according to claim 8, wherein the reaction is carried out in a protic solvent selected from one or a mixture of two of methanol or ethanol.
11. The process according to claim 8, wherein the temperature of the reaction is from 0 to 30 ° C, preferably from 0 to 10 ° C, more preferably from 5 to 10 ° C.
12. Use of a compound of formula II according to claim 1 as a control for quality control of azacitidine and its related formulations.