WO2015180549A1

WO2015180549A1 - I-type crystal of l-alanine-(14-oridonin) ester trifluoroacetate and preparation method therefor

Info

Publication number: WO2015180549A1
Application number: PCT/CN2015/077620
Authority: WO
Inventors: 孙飘扬; 武乖利; 邱振均; 陈永江; 沈灵佳
Original assignee: 江苏恒瑞医药股份有限公司
Priority date: 2014-05-30
Filing date: 2015-04-28
Publication date: 2015-12-03
Also published as: HK1223618A1; CN105636964A; CN105636964B; CN105131009A

Abstract

The present invention relates to an I-type crystal of L-alanine-(14-oridonin) ester trifluoroacetate and a preparation method therefor.The preparation method comprises crystallizing a L-alanine-(14-oridonin) ester trifluoroacetate solid in any crystal form or amorphous form in a single organic solvent or a mixed organic solvent thereof to obtain the I-type crystal of the L-alanine-(14-oridonin) ester trifluoroacetate.The I-type crystal of the L-alanine-(14-oridonin) ester trifluoroacetate obtained in the present invention has a good crystal form stability and chemical stability, and uses a crystallization solvent which has a low toxicity and low residue, and can be better used in clinical treatments.

Description

Form I crystal of L-alanine-(14-Rhoxedron A) ester trifluoroacetate and preparation method thereof

Technical field

The present invention relates to a type I crystal of L-alanine-(14-Rhoxantine) ester trifluoroacetate, a preparation method thereof and use thereof.

Background technique

The compound of formula (I) is L-alanine-(14-Rhoxedron A) ester trifluoroacetate, which is

The oridonin derivative obtained by the modification of the Rubescensine A as the mother nucleus; the oridonin is a kind of kauriene extracted from the rubescens Diterpenoid natural organic compounds, which account for more than 90% of the active ingredients of Rubescens, are the main active ingredients for anti-tumor.

However, the oral bioavailability of Rubescensine A is less than 5%, and the effective blood concentration cannot be achieved. Meanwhile, Rubescensine A is almost insoluble in water, and it is difficult to use intravenous administration. Therefore, the modification of its structure, improvement of its water solubility and activity, and its development into a new anti-tumor drug is an important direction of the research of Rubescensine A. After data investigation and experimental research, it was found that after the 14-position hydroxyl group of Rubescensine A was esterified with L-alanine, the product not only had good water solubility, but also the conversion rate in rats and dogs was 94.1% and 128%. The pharmacological and toxicological experiments confirmed that the compound not only has similar activity to Rubescensine A, but also can be rapidly transformed into Rubescensine A in plasma and in vivo in vitro, and exerts the pharmacodynamic activity of Rubescensine A; The toxicity study showed that the product is less toxic and has no fear of genotoxicity. In addition, this product overcomes the side effects caused by phlebitis and special preparations caused by the use of non-aqueous irritating solvents. Rabbit vascular irritation test shows that this product is correct. Vascular non-irritating, guinea pig There was no allergic phenomenon in the systemic allergy test, which effectively solved the above problems of the Rubescensine A drug.

Considering the high activity and instability of the Rubescensine A derivative, we further studied the crystal form and preparation of L-alanine-(14-Rhoxantine A) trifluoroacetate. method. The person skilled in the art knows that the crystal structure of the pharmaceutically active ingredient often affects the chemical stability of the drug, and the difference in crystallization conditions and storage conditions may lead to changes in the crystal structure of the compound, sometimes accompanied by other Form of crystal form. In general, amorphous drug products have no regular crystal structure and often have other defects, such as poor product stability, fine crystallization, difficult filtration, easy agglomeration, and poor fluidity. Therefore, it is necessary to improve the various aspects of the above products. We need to study in depth to find new crystal forms with high purity and good chemical stability.

Summary of the invention

It is an object of the present invention to provide a stable crystalline form of L-alanine-(14-Erymentum A) ester trifluoroacetate and a process for preparing the crystal form.

We investigated a series of crystalline products of L-alanine-(14-Rhoxantine) ester trifluoroacetate under different crystallization conditions. X-ray diffraction and DSC detection of the obtained crystalline product were found. L-Alanine-(14-Rhoxedron A) ester trifluoroacetate can obtain a crystal form with good stability under conventional crystallization conditions, which we call type I crystal. The DSC pattern of the type I crystal in the present application shows a melting endotherm peak near 234 ° C, and the X-ray powder diffraction pattern is shown in Fig. 1, using Cu-Ka radiation, at 2θ angle and interplanar spacing (d value) The X-ray powder diffraction pattern is represented by 6.85 (12.89), 8.09 (10.92), 11.30 (7.82), 13.71 (6.45), 14.08 (6.29), 14.35 (6.17), 16.40 (5.40), 17.41 (5.09). , 18.19 (4.87), 18.44 (4.81), 20.12 (4.41), 21.17 (4.19), 23.38 (3.80), 23.88 (3.72), 24.42 (3.64), 24.84 (3.58), 25.72 (3.46), 26.46 (3.37) , 27.22 (3.27), 28.40 (3.14), 29.38 (3.04), 31.26 (2.86) And 31.79 (2.81) have characteristic peaks.

In the method for producing Form I crystals of the present invention, the form of use which can be used as a raw material is not particularly limited, and any crystal form or amorphous solid can be used, and L-alanine-(14-Rhoxedron A) ester of the present invention can be used. The preparation method of the trifluoroacetate type I crystal is:

a certain polar organic solvent, preferably a polar organic solvent such as an alcohol, a ketone, an ester or a halogenated hydrocarbon having a small number of carbon atoms and capable of volatilization and being used as a crystallization solvent, or a mixed solution thereof; Preferred is acetone, ethyl acetate, methanol, ethanol, isopropanol, diisopropyl ether or a mixture thereof as a recrystallization solvent of L-alanine-(14-Rhoxantine) ester and trifluoroacetate . A single solvent may be used for crystallization, or a mixed solvent selected from the above organic solvents may be used.

Specifically, the method for preparing L-alanine-(14-Rhoxantine) ester trifluoroacetate Form I crystal provided by the present invention comprises the following steps:

(1) N-Boc-L-alanine-(14-Rhoxedron A) ester with trifluoroacetic acid, or L-alanine-(14-Rhoxedron A) ester trifluoroethyl The acid salt solid is dissolved in an appropriate amount of an organic solvent, and stirred and crystallized; or an anti-solvent is added, and the crystal is stirred and stirred;

(2) filtering, washing and drying;

Wherein the organic solvent is one or more selected from the group consisting of an alcohol having a carbon number of 3 or less, a halogenated hydrocarbon, a ketone, and an ester, or an alcohol selected from the group consisting of carbon atoms of 3 or less; A mixed solvent of any one or more of a halogenated hydrocarbon, a ketone, and an ester and an ether having a carbon number of 3 or less.

In a preferred embodiment of the present invention, the organic solvent is selected from one or more of ketones, alcohols, and esters having a carbon number of 3 or less, or is selected from the group consisting of ketones, alcohols, and esters. Any one or more mixed solvents with ethers.

Further, the most preferred single solvent is ethyl acetate.

In one embodiment of the present invention, a preferred mixed organic solvent is acetone/isopropyl ether, and the ratio of the two is not particularly limited. In a preferred embodiment of the present invention, the volume ratio of the two is 1:2.

The method of recrystallization is not particularly limited and can be carried out by a usual recrystallization operation method. For example, the raw material L-alanine-(14-Rhoxedron A) ester trifluoroacetate can be stirred and dissolved in an organic solvent, and then stirred and crystallized at room temperature. After the crystallization is completed, it can be obtained by filtration and drying. The crystals required. The crystals collected are usually vacuumed under reduced pressure at normal temperature. Drying can achieve the effect of removing the recrystallization solvent.

The crystal form of the obtained L-alanine-(14-Erymentum A) ester trifluoroacetate crystal was studied by differential scanning calorimetry (DSC) and X-ray diffraction spectrometry. The residual solvent of the crystal was examined.

The L-alanine-(14-Rhoxantine) ester trifluoroacetate salt prepared according to the method of the present invention contains no or only a low content of residual solvent, and is in compliance with the relevant Pharmacopoeia regulations. The solvent is required to be limited, and thus the crystal of the present invention can be preferably used as a pharmaceutically active ingredient.

Studies have shown that the preparation of L-alanine-(14-Rhoxedron A) ester trifluoroacetate type I crystal under the conditions of grinding, pressure and heat, the crystal form stability is good, can meet the production The medicinal requirements for transport and storage, the production process is stable and repeatable and controllable, and can be adapted to industrial production.

DRAWINGS

Figure 1 is an X-ray powder diffraction pattern of Form I crystal of L-alanine-(14-Rhoxedron A) trifluoroacetate.

Figure 2 is a DSC chart of Form I crystal of L-alanine-(14-Rhoxedron A) trifluoroacetate.

Figure 3 is an X-ray powder diffraction pattern of an amorphous solid of L-alanine-(14-Rhoxedron A) trifluoroacetate.

Figure 4 is a DSC chart of an amorphous solid of L-alanine-(14-Rhoxedron A) trifluoroacetate.

detailed description

The invention is explained in more detail below with reference to the embodiments, which are intended to illustrate the technical scope of the invention and not to limit the scope and scope of the invention.

Test instrument used in the experiment

1, DSC spectrum

Instrument model: Mettler Toledo DSC 1 Stare System

Purge gas: nitrogen

Heating rate: 10.0 ° C / min

Temperature range: 40-250 ° C

2. X-ray diffraction spectrum

Instrument model: Bruker D8Focus X-ray powder diffractometer

Ray: Monochrome Cu-Kα ray

Scanning mode: θ/2θ, scanning range: 2-40°

Voltage: 40KV, current: 40mA.

Example 1: Preparation of L-alanine-(14-Rhoxedron A) Trifluoroacetate

Step 1: Preparation of N-Boc-L-alanine-(14-Rhoxedron A) Ester

150 g (0.41 mol) of Rubescensine A (II), 195 g (1.04 mol) of Boc-L-Ala was suspended in 1.35 kg of dichloromethane, cooled to 0-5 ° C in an ice bath, and 213 g (1.04 mol) of DCC was added. After stirring for 0.5 h, the ice bath was removed and the mixture was stirred at room temperature for 5 h, and the reaction was confirmed by TLC (dichloromethane:methanol = 10:1, starting material R _f = 0.4, product R _f = 0.6). The reaction mixture was cooled to 0 ° C. EtOAc (EtOAc)EtOAc. The silica gel column chromatography (dichloromethane:methanol = 100:1 to 60:1; v/v) was collected. The product fractions were collected and concentrated under reduced pressure to dryness to give a white solid powder of about 140 to 165 g. The above white powder was added with 600 g of isopropyl ether, and the mixture was stirred and beaten for 2 hours, filtered, and the filter cake was washed with isopropyl ether (60 g), and blast dried at 25 ° C for 4 h to obtain a white powder of 110-137 g, yield 50-62%. . (HPLC>98%) ¹ H-NMR (400MHz, CDCl ₃ ): 6.12 (m, 2H), 5.90 (s, 1H), 5.49 (s, 1H), 5.10-5.08 (d, 1H), 4.30-4.19 (m, 3H), 4.06-4.04 (q, 1H), 3.74-3.70 (m, 1H), 3.49-3.44 (m, 1H), 3.12-3.10 (d, 1H), 2.60-2.57 (m, 1H) , 2.25-2.20 (m, 1H), 1.95-1.91 (dd, 1H), 1.78-1.73 (m, 1H), 1.69-1.53 (m, 4H), 1.47-1.38 (m, 10H), 1.31-1.29 ( d,3H), 1.25-1.23 (m, 2H), 1.09-1.08 (m, 6H). ¹³ C-NMR (100 MHz, CDCl ₃ ): 206.2, 171.7, 155.0, 149.9, 120.1, 96.1, 79.9, 76.2, 74.5, 73.4, 68.3, 63.4, 62.0, 59.5, 54.7, 49.6, 41.3, 41.3, 38.7, 33.7, 32.6, 30.5, 29.9, 28.2, 22.8, 21.7, 19.9, 18.1.

Step 2: Preparation of L-alanine-(14-Rhoxedron A) ester trifluoroacetate

120 g (0.224 mol) of HAO472-01 was dissolved in 600 g of dichloromethane and cooled to 0 °C. A mixed solution of 2.5 kg of trifluoroacetic acid/dichloromethane (1:1; w/w) was slowly added dropwise at 0 to 5 ° C, and the addition was completed within 20 minutes. After stirring at 0 to 5 ° C for 30 min, the reaction was confirmed by TLC (dichloromethane:methanol = 10:1, material: R _f = 0.6, product R _f = 0.3), and the reaction mixture was concentrated to give a pale yellow to pale red oil. . After stirring with 7.2 kg of isopropyl ether, a large amount of pale yellow solid was precipitated, stirred for 1 hour, filtered, and the filter cake was washed with isopropyl ether (145 g × 3). The mixture was dried in a vacuum oven to obtain 105-116 g of a white or light yellow solid, and the yield was 85 to 94%. (HPLC>98%) ¹ H-NMR (400MHz, MeOH): 6.16 (s, 1H), 6.10 (s, 1H), 5.63 (s, 1H), 4.28-4.31 (d, 1H), 4.05-4.07 ( d, 1H), 3.92-3.97 (q, 1H), 3.67-3.69 (d, 1H), 3.45-3.49 (dd, 1H), 3.13 - 3.15 (d, 1H), 2.58-2.66 (m, 1H), 2.21-2.26(m,1H), 1.97-2.02(dd,1H),1.81-1.88(m,1H),1.57-1.69(m,H),1.42-1.47(m,4H),1.31-1.36(dd , 1H), 1.28-1.29 (d, 1H), 1.12 (s, 3H), 1.08 (s, 3H). ¹³ C-NMR (100MHz, MeOD): 208.08, 169.75, 163.51, 163.17, 168.22, 162.49, 151.87 , 120.75, 122.60, 119.69, 116.77, 113.86, 97.29, 77.35, 75.98, 73.75, 64.63, 63.71, 60.76, 56.24, 50.21, 43.06, 42.46, 39.77, 34.59, 33.23, 31.74, 30.46, 22.15, 21.13, 15.68. /MS:[M+H]=436.2328.

Example 2: Determination of the crystal form of the sample of Example 1.

The X-ray powder diffraction pattern of the solid sample prepared in Example 1 is shown in Fig. 3, showing the characteristic peak of the amorphous form, the DSC spectrum is 4, and the melting characteristic absorption peak is not observed below 300 ° C, and the product is determined to be amorphous. solid.

Example 3

Weigh 500 mg of L-alanine-(14-Rhoxedron A) ester trifluoroacetate amorphous solid in a 25 ml round bottom flask, add 5 ml of acetone, stir to dissolve, and slowly add 10 ml of isopropyl ether at room temperature. The crystals were stirred for 2.5 hours, suction filtered, and the filter cake was washed with a small solvent mixture of acetone/isopropyl ether (V/V = 1/2) and dried to give 370 mg of white solid. The X-ray powder diffraction pattern of the crystalline sample is shown in Figure 1. The crystals are at about 6.85 (12.89), 8.09 (10.92), 11.30 (7.82), 13.71 (6.45), 14.08 (6.29), 14.35 (6.17), 16.40 (5.40), 17.41 (5.09), 18.19 (4.87), 18.44. (4.81), 20.12 (4.41), 21.17 (4.19), 23.38 (3.80), 23.88 (3.72), 24.42 (3.64), 24.84 (3.58), 25.72 (3.46), 26.46 (3.37), 27.22 (3.27), 28.40 (3.14), characteristic peaks at 29.38 (3.04), 31.26 (2.86), and 31.79 (2.81). The DSC spectrum is shown in Figure 2. There is a sharp melting endotherm peak of 232.59 ° C. This crystal form is defined as the I crystal form.

Example 4

500 mg of N-Boc-L-alanine-(14-anthracycline) ester was dissolved in 1.5 ml of dichloromethane and cooled to 0 ° C in an ice water bath. 8 ml of a trifluoroacetic acid/dichloromethane (1:1) mixed solution was slowly added dropwise at less than 5 ° C, and the addition was completed within 15 min. Stir under ice water, and the reaction was complete by TLC. 20 ml of isopropyl ether was slowly added to the reaction solution. After stirring at room temperature for 24 hours, it was filtered, and the filter cake was washed with isopropyl ether (8 ml × 3) and dried in vacuo to give 372 mg of white solid. The X-ray powder diffraction and DSC patterns of the crystalline sample were compared by study to confirm that the product was in Form I.

Example 5

Weigh 500 mg of L-alanine-(14-Rhoxedron A) ester trifluoroacetate amorphous solid in a 25 ml round bottom flask, add acetone 5 ml, stir to dissolve, stir and crystallize at room temperature for 18 hours, pump After filtration, the filter cake was washed with a small amount of acetone and dried to give a white solid, 300 mg, yield 60.0%. The X-ray powder diffraction and DSC patterns of the crystalline sample were compared by study to confirm that the product was in Form I.

Example 6

Weigh 500mg L-alanine-(14-Rhoxantine A) ester trifluoroacetate amorphous solid in a 25ml round bottom flask, add 4.5ml of ethyl acetate, stir to dissolve, stir and crystallize at room temperature 4 After a while, suction filtration, the cake was washed with a small portion of ethyl acetate and dried to yield white crystals (yield: The X-ray diffraction and DSC spectra of the crystalline sample were compared by study to confirm that the product was in Form I.

Example 7

The sample of the Form I obtained in Example 3 and the sample of the amorphous L-alanine-(14-Rhoxedron A) trifluoroacetate obtained in Example 1 were separately placed in an open position and examined. The stability of the sample under illumination (4500 Lux), heating (60 ° C), and high humidity (RH 90%) conditions, the sampling time was 5 days and 10 days, and the purity of HPLC detection is shown in Table 1.

Table 1. Comparison of the stability of L-alanine-(14-Rhoxedron A) ester trifluoroacetate type I crystal and amorphous sample

The results of stability investigation showed that L-alanine-(14-Rhodoxin A) trifluoroacetate type I crystal and amorphous sample were placed under open conditions, under illumination, high temperature and high humidity conditions. The comparison of stability shows that the effect of illumination on the two is not significant, but under high temperature and high humidity conditions, the stability of type I crystal is significantly better than that of amorphous sample.

Example 8

The L-alanine-(14-Rhoxedron A) trifluoroacetate type I crystal obtained by the method of Example 1 was subjected to special stability studies such as grinding, pressure and heat, and the results showed that the crystal form was stable. For detailed experimental data, see Table 2 below:

Table 2. Study on the special stability of HAO472I crystal form

Claims

Form I crystal of L-alanine-(14-Rhoxantine) ester trifluoroacetate characterized by using Cu-Ka radiation to obtain X-rays expressed in 2θ angle and interplanar spacing a powder diffraction pattern having an X-ray powder diffraction pattern as shown in Figure 1, wherein at about 6.85 (12.89), 8.09 (10.92), 11.30 (7.82), 13.71 (6.45), 14.08 (6.29), 14.35 (6.17), 16.40 (5.40), 17.41 (5.09), 18.19 (4.87), 18.44 (4.81), 20.12 (4.41), 21.17 (4.19), 23.38 (3.80), 23.88 (3.72), 24.42 (3.64), 24.84 (3.58), 25.72 (3.46), 26.46 (3.37), 27.22 (3.27), 28.40 (3.14), 29.38 (3.04), 31.26 (2.86) and 31.79 (2.81) have characteristic peaks.
A method of preparing Form I crystal of L-alanine-(14-Rhoxedron A) trifluoroacetate according to claim 1, the method comprising the steps of:

1) Any crystalline or amorphous N-Boc-L-alanine-(14-Rhoxedron A) ester with trifluoroacetic acid, or any crystalline or amorphous L-alanine-( 14-Erythromycin A ester trifluoroacetate solid is dissolved in an organic solvent at room temperature, stirred and crystallized, or added to an anti-solvent, and the organic solvent is selected from the group consisting of alcohol having a carbon number of 3 or less One or more of a class, a halogenated hydrocarbon, a ketone, an ester, or any one or more selected from the group consisting of an alcohol having a carbon number of 3 or less, a halogenated hydrocarbon, a ketone, and an ester. a mixed solvent of an ether having a carbon number of 3 or less; preferably, the organic solvent is one or more selected from the group consisting of a ketone, an alcohol, and an ester, or is selected from the group consisting of a ketone, an alcohol, and an ester. Any one or more mixed solvents with ethers;

2) Filter the crystals, wash and dry.
The method according to claim 2, wherein the organic solvent in the step 1) is acetone, ethyl acetate, dichloromethane/diethyl ether, acetone/isopropyl ether, dichloromethane/isopropyl ether, ethanol. /isopropyl ether, isopropanol / diisopropyl ether, methanol / isopropyl ether.
The method according to claim 3, wherein the organic solvent in the step 1) is acetone/isopropyl ether.
A pharmaceutical composition comprising the Form I crystal of L-alanine-(14-oxalyl) ester trifluoroacetate according to claim 1 and a pharmaceutically acceptable carrier.
The use of Form I crystal of L-alanine-(14-oxalyl) ester trifluoroacetate according to claim 1 or the pharmaceutical composition according to claim 5 for the preparation of a medicament for treating leukemia; The leukemia gene target is an acute myeloid AML1-ETO fusion gene.
The use of the type I crystal of L-alanine-(14-oxalyl) ester trifluoroacetate according to claim 1 or the pharmaceutical composition according to claim 5 for the preparation of a medicament for treating cancer, The cancer is esophageal cancer, gastric cancer, primary liver cancer, pancreatic cancer, cardiac cancer, colorectal cancer, bladder cancer, breast cancer, and acute myeloid leukemia.