WO2018019188A1

WO2018019188A1 - Polymorph of nucleoside phosphoramidate prodrug and preparation method therefor

Info

Publication number: WO2018019188A1
Application number: PCT/CN2017/093925
Authority: WO
Inventors: 袁建栋; 顾家宁; 黄仰青; 缪琳峰; 梁朝华; 孙占莉
Original assignee: 博瑞生物医药（苏州）股份有限公司
Priority date: 2016-07-23
Filing date: 2017-07-21
Publication date: 2018-02-01
Also published as: CN107652342A

Abstract

Provided are a crystal form A of a nucleoside phosphoramidate prodrug as shown in formula I and a preparation method therefor. The crystal form A at least has an X-ray powder diffraction (XRPD) pattern with peaks at the 2θ angles of 6.9 ± 0.2°, 13.9 ± 0.2°, 20.8 ± 0.2° and 21.1 ± 0.2°. The crystal form A is stable in nature and suitable for being prepared into various dosage forms. The preparation method is simple in operation, and the prepared crystal form A is high in purity and low in industrialization costs.

Description

Polymorph of nucleoside phosphoramidate prodrug and preparation method thereof

Technical field

The invention relates to the field of medical technology. More specifically, the present invention relates to a polymorph of a nucleoside phosphoramidate prodrug and a process for the preparation thereof. The invention further relates to a pharmaceutical composition comprising a polymorph of a nucleoside phosphoramidate prodrug and at least one pharmaceutically acceptable carrier and the use of said pharmaceutical composition.

Background technique

NUC-1031 is a gemcitabine prodrug developed by NuCana BioMed and currently in clinical phase III for the treatment of advanced solid tumors, pancreatic cancer, breast cancer and other cancers. The CAS of NUC-1031 is 840506-29-8 and has the structure shown in the following formula:

Compound I is a NUC-1031 chiral P which is an S epimer, which has a CAS of 1562406-27-2 and has the following structure:

WO2014076490A1 discloses the following method for preparing NUC-1031,

3'-Boc protected gemcitabine (100 mg) was reacted with 2 mol equivalents of phenyl (benzyl-L alanine) chlorophosphate (150 mg) with 0.5 mol equivalent of tris(acetylacetonate) iron (III) (56 mg of) as a catalyst, 1.5 mol equivalents of DIPEA (55μL) as base, 10 ml of THF as a reaction solvent, under nitrogen, for 24 hours at room temperature, 45% yield, isomer wherein _{_{R p: S p = 3:}} 1.

The title of Application of ProTide Technology to Gemcitabine: A Successful Approach to Overcome the Key Cancer Resistance Mechanisms Leads to a New Agent (NUC-1031) in Clinical Development (Journal of Medicinal Chemistry, Volume 57, Issue 4, Pages 1531-1542) The literature reports that the compound of the formula 5f is deprotected with hydroxyl in the presence of TFA in methylene chloride as a solvent, and finally purified by silica gel column to prepare NUC-1031 in a yield of 70%, wherein the isomer content is 48%. , 52%.

At present, there are relatively few studies on the crystal form of Compound I, and only WO2016055769 discloses a separation method of two diastereomers of NUC1031 chiral P and a crystal form of S-epimer.

The present inventors have found through a large amount of research work that Compound I exhibits relatively strong cytotoxicity against BxPC-3, MIA Paca-2 and OVCAR-3 tumor cells compared with NUC-1031, and the IC ₅₀ value is about 0.01. Between nM and 0.05 nM; its inhibitory effect on tumor cell proliferation in vitro is about 10 times that of NUC-1031 chiral P in the R configuration; pharmacokinetic evaluation results show that compound I is active in tumor tissue. The product dFdCTP concentration was significantly higher than that of the NUC-1031 chiral P in the R configuration. Thus, there is a great difficulty in preparing a dosage form for an effective therapeutic dose, and the industrial expansion of production costs is very high. Moreover, the study found that NUC-1031 is very lipophilic, so it is difficult to dissolve in water (calculated solubility <0.1mg/mL), and the pH range of the pKas estimated by its ionic part is not suitable for parenteral administration. The S-epimer (Compound I) was found to have good solubility in a mixed solution of a plurality of polar organic solvents and water, and is suitable for preparation of a preparation. It is of great significance to develop a compound I with a stable crystalline form to meet the needs of the formulation and clinical needs.

Summary of the invention

In order to solve the above problems, the present invention provides a crystal form A of a compound of the formula I.

A second aspect of the invention provides the use of Form A for the manufacture of a medicament for the treatment and/or prevention of cancer.

A third aspect of the invention provides a pharmaceutical composition comprising Form A of the invention and at least one pharmaceutically acceptable excipient.

A fourth aspect of the invention provides a process for the preparation of a crystalline form A of a compound of formula I.

First, the invention provides a crystalline form A of a compound of the formula I, characterized in that the crystal form A has an X-ray powder diffraction (XRPD) pattern having a peak at least at the following 2 theta angle: 6.9 ± 0.2 , 13.9.±0.2, 20.8±0.2, and 21.1±0.2,

Preferably, the Form A has an X-ray powder diffraction (XRPD) pattern having peaks at least at the following 2 theta angles: 6.9 ± 0.2, 13.9. ± 0.2, 18.4 ± 0.2, 18.8 ± 0.2, 20.8 ± 0.2, and 21.1 ± 0.2. .

Still more preferably, the crystal form A having an X-ray powder diffraction (XRPD) pattern is substantially as shown in FIG. 1, FIG. 3, FIG. 4, or FIG.

Another aspect of the invention also provides the use of Compound I having Form A for the manufacture of a medicament for the treatment and/or prevention of cancer. The cancer includes, but is not limited to, pancreatic cancer, advanced solid tumor, ovarian tumor, non-small cell lung cancer, breast cancer, bladder cancer, cervical cancer, mesothelioma, esophageal cancer, gastric cancer, colon cancer, liver cancer, cholangiocarcinoma, nasal Pharyngeal cancer, testicular cancer, lymphoma or head and neck cancer.

Another aspect of the present invention also provides a pharmaceutical composition comprising the crystalline form A of the present invention together with a pharmaceutically acceptable carrier. The pharmaceutical composition can be administered orally or parenterally, including intravenous, subcutaneous, intraperitoneal, intramuscular, inhalation, rectal, and topical (such as oral or sublingual). Administration).

Wherein, the pharmaceutical composition for oral administration includes a tablet, a capsule, a granule or a suspension; the tablet for oral administration comprises the composition provided by the present invention as an active ingredient, and may further comprise one or more Pharmaceutically acceptable excipients such as diluents, disintegrating agents, binders, lubricants, sweeteners, flavoring agents, coloring agents, and preservatives. When corn starch and alginic acid are used as disintegrants, suitable inert diluents include sodium carbonate, calcium carbonate, sodium phosphate, calcium phosphate and lactose. The binder comprises starch and gelatin. Optionally, the lubricant is magnesium stearate, stearic acid or talc. Optionally, the tablet may also be coated with glyceryl monostearate or glyceryl distearate to delay absorption in the stomach.

Capsules for oral administration include hard capsules and soft capsules, wherein the hard capsules comprise the composition provided by the present invention as an effective active ingredient and a solid diluent; the soft capsules comprise the pharmaceutical composition provided by the present invention as an effective active ingredient With water or oil (such as peanut oil, liquid paraffin or olive oil).

A dosage form of a suppository for rectal administration wherein the base of the suppository may be cocoa butter or a salicylate.

Formulations for vaginal administration are in the form of pessaries, cotton balls, creams, gels, pastes, foams, or sprays, which comprise compositions containing active ingredients and conventional carriers known in the art.

For use in dosage forms for intravenous, intraperitoneal, subcutaneous, and intramuscular administration, the compositions provided herein are typically sterile solutions or sterile suspensions, and have suitable pH and osmotic pressure. The preparation of such formulations can be prepared according to conventional methods well known in the art.

A fourth aspect of the invention provides a process for the preparation of a crystalline form A of a compound of formula I, the process comprising the steps of:

(1) adding a compound of formula I to a first organic solvent to obtain a mixture I;

(2) adding a second organic solvent to the mixture I, and filtering to obtain a filtrate II;

(3) The solution II was stirred and crystallized, and then filtered, and the obtained cake was dried to obtain a crystal form A.

Preferably, the first organic solvent is selected from the group consisting of ethyl acetate, isopropyl acetate, propyl acetate, isobutyl acetate, and a mixed solution thereof; more preferably, the first organic solvent is selected from ethyl acetate.

Preferably, the second organic solvent is selected from the group consisting of methanol, ethanol, isopropanol, and a mixed solution thereof; more preferably, the second organic solvent is selected from the group consisting of methanol.

Preferably, the ratio of the first solvent to the second solvent is from 6:1 to 20:1 (v/v), more preferably from 6:1 to 15:1 (v/v), still more preferably 8:1. ~12:1.

In another preferred embodiment of the present invention, the total solvent amount ratio of the compound of the formula I to the first solvent and the second solvent is from 1:5 to 1:40 (w/v); preferably, the formula I The total solvent ratio of the compound to the first solvent and the second solvent is 1:10-1:30 (w/v); more preferably, the compound of the formula I is combined with the first solvent and the second solvent. The total solvent usage ratio is 1:15-1:25 (w/v).

Further preferably, in the above method, the mixture I is stirred and heated to 20 ° C to 80 ° C, preferably to 60 ° C ± 10 ° C, and then a second organic solvent is added, and the solid is dissolved and then filtered hot to obtain a filtrate. II. The filtrate II was cooled to room temperature, stirred and crystallized, and then filtered, and the filter cake was washed with a first organic solvent and dried to obtain a crystal form A.

Further, the method for preparing the crystal A of the compound of the formula I according to the present invention is as follows: ethyl acetate, a compound of the formula I is added to the reaction flask, the temperature is raised to 60 ± 2 ° C, and methanol is added thereto, and the solid is dissolved. Hot filtration. The filtrate is slowly cooled to 25 ± 2 ° C, stirred for 2 to 24 hours, preferably 4-12 hours, more preferably 6-8 hours, then filtered, the filter cake is rinsed with ethyl acetate, and the filter cake is at 25 ± 5 ° C Drying in vacuo to obtain Form A, wherein the ratio of the weight of the compound of Formula I to the total amount of ethyl acetate and methanol is 1:15 to 25 (w/v), and the ratio of ethyl acetate to methanol is 8-12. :1(v/v).

The compound I of the crystalline form A provided by the present invention is in a substantially pure crystalline form. Preferably, the crystalline form is characterized by being free or substantially free of water.

The invention provides a novel crystal form of the compound I, that is, the crystal form A, and the crystal form A provided by the invention has good stability and high purity, is suitable for preparing various preparations, and provides the crystal form A. The preparation method is simple, the solvent used is a conventional solvent, no special equipment is required, the industrial production cost is small, and the purity of the crystal form A prepared by the method provided by the invention is remarkably improved.

DRAWINGS

Figure 1 shows an XRPD (X-ray powder diffraction) pattern of Form A of the compound of Formula I prepared in Example 1; wherein the diffraction peak data is as follows:

Fig. 2 is a view showing three samples which were respectively determined by an X-ray powder diffractometer after being taken out after 3 months, 6 months, and 12 months in the stability study experiment of the crystal form A in Example 27. The XRPD comparison chart of the measurement time, from bottom to top, was taken out for 3 months, 6 months, and 12 months, respectively.

Figure 3 is an XRPD pattern of Form A taken in Example 27 for 3 months.

Figure 4 is an XRPD pattern of Form A taken in Example 27 for 6 months.

Figure 5 is an XRPD pattern of Form A taken in Example 27 for 12 months.

Specific embodiment

The content of the present invention is further illustrated and described in conjunction with the embodiments, which are intended to illustrate the invention and not to limit the scope of the invention. The experimental methods in the following examples which do not specify the specific conditions are usually carried out according to conventional conditions or according to the conditions recommended by the manufacturer. All percentages, ratios, ratios, unless otherwise stated Or the number of parts by weight. The compound of the formula I (abbreviated as the compound I) used in the present invention can be obtained by the method disclosed in WO2016055769, or can be obtained by the method disclosed in the patent application No. 201510586447.6, and the HPLC purity of the compound I used is above 95%. .

The XRPD test in the present invention uses a PAERT-type XPERT-3 X-ray diffractometer from PANalytical. Approximately 10 mg of the sample was evenly spread on a single crystal silicon sample pan and subjected to XRPD testing using the parameters described below.

Method for preparing Form A of Compound I of Example 1

1 L of ethyl acetate and 50.50 g of Compound I (HPLC: 96.8%) were added to a 2 L reaction flask, and the mixture was heated to 60 ± 2 ° C with stirring, and 115 mL of methanol was added thereto, and the solid was dissolved and filtered. The filtrate was slowly cooled to 25 ± 2 ° C, stirred for 20 hours, filtered, and the filter cake was rinsed with 100 ml of ethyl acetate. The cake was dried under vacuum at 25 ± 5 ° C to give 41.41 g, yield: 82.0%, XRPD spectrum Figure 1 shows the HPLC; 99.8%.

Preparation method of Form A of Compound I of Example 2

1 L of ethyl acetate and 55.00 g of Compound I were added to a 2 L reaction flask, and the mixture was stirred and heated to 60 ± 5 ° C, and 100 mL of methanol was added thereto, and the solid was dissolved and filtered. The filtrate was slowly cooled to 25 ± 2 ° C, stirred and decanted for 12 hours, filtered, and the filter cake was rinsed with 100 ml of ethyl acetate. The filter cake was vacuum dried at 25 ± 5 ° C to obtain crystal form A, and its XRPD spectrum is shown in Fig. 1. Yield: 82.3%, HPLC: 99.8%.

Method for preparing crystal form A of compound I of Example 3

1 L of ethyl acetate and 73.50 g of Compound I (HPLC: 96.8%) were added to a 2 L reaction flask, and the mixture was heated to 70 ± 2 ° C with stirring, and 110 mL of methanol was added thereto, and the solid was dissolved and filtered. The filtrate was slowly cooled to 25 ± 2 ° C, stirred and decanted for 18 hours, filtered, and the filter cake was rinsed with 100 ml of ethyl acetate. The filter cake was vacuum dried at 25 ± 5 ° C to obtain crystal form A, and its XRPD spectrum is shown in Fig. 1. Yield: 83.4%, HPLC: 99.5%.

Examples 4-9:

Referring to the preparation method of Example 1, using ethyl acetate as the first solvent and methanol as the second organic solvent, the temperature of the mixture of ethyl acetate and compound I in Example 1 was replaced with different temperatures, and the experimental results were obtained. The effect is as follows:

Table 1:

Examples 10-15: Study the effect of different solvent dosages on experimental results

Referring to the preparation method of Example 1, using ethyl acetate as the first solvent and methanol as the second organic solvent, the following results were obtained:

Table 2:

Examples 16-21: Study the effect of different crystallization time on experimental results

table 3:

Examples 22-24: Study the effect of crystallization of different solvents on crystal form and yield:

The first organic solvent and the compound I were added to a 2 L reaction flask, and the mixture was heated to 60 ± 10 ° C with stirring, and a second organic solvent was added thereto. The solid was dissolved and then filtered. The filtrate was slowly cooled to 25 ± 5 ° C, stirred for 4 to 12 hours, filtered, using the first The filter cake is rinsed with an organic solvent, and the filter cake is dried under vacuum at 25±5° C., wherein the ratio of the first solvent to the second solvent is 8:1 to 12:1 (v/v), and the compound I and the two solvents are used. The total dosage ratio is 1:15 to 25 (w/v). The experimental results are as follows:

Table 4:

Example 25: Solubility determination:

HPLC content and related substance analysis selected methanol-water as solvent, GC residual solvent selected N, N-dimethylformamide as solvent, and the compound I crystal form A of the present invention was investigated in the solvent methanol, ethanol, N, N-di Solubility in methylformamide, ethyl acetate, water, 0.1 mol/L hydrochloric acid, and 0.1 mol/L sodium hydroxide. According to the Chinese Pharmacopoeia 2015 edition of a routine project and requirements inspection, in view of the degradation of this product in acid and alkali, so the solubility in acid and alkali is not determined into the standard, the solubility of this product is set to: soluble in methanol, in N It is easily soluble in N-dimethylformamide, slightly soluble in ethanol, very slightly soluble in ethyl acetate, and almost insoluble in water.

Compound I crystal form A three batches of laboratory batch (batch 150801, 150901, S151001) and three batches of amplified batch (batch: S151201, S160101, S160102) in methanol, N, N-dimethylformamide, ethanol, acetic acid The solubility results for ethyl ester and water are shown in the table below.

Table 5: Solubility results for Compound A Form A of the present invention

Two batches of the crystal form of Compound 3 (i.e., Compound I of the present invention) in WO2016055769A1 obtained by the method disclosed in Patent Document 1 (WO2016055769A1) were measured under the same conditions, and the solubility results obtained are shown in the following table.

Table 6: Crystal Form Solubility Data in WO2016055769A1

When the compound I of the present invention is used in clinical practice, it is usually required to be an injection or a lyophilized powder. The solubility of the compound I is very significant for the preparation of such a preparation, and it is apparent from the comparison of Table 5 and Table 6, The solubility of Form A provided by the present invention is significantly better than that of WO2016055769A1. Therefore, the Form A of Compound I provided by the present invention will be more advantageous for the research and development of subsequent preparations.

Example 26: Studying the long-term stability of Form A provided by the present invention

The test was carried out using Form A prepared according to the method of Example 1:

According to the "Chinese Pharmacopoeia" 2015 edition of the four general principles of 9001 "Guidelines for the stability of APIs and pharmaceutical preparations" and "Guidelines for the Study of Stability of Chemical Drugs", the stability investigation was carried out, and the A crystal form of the compound I of the present invention was placed in a closed state. In the container, placed at 5 °C ± 3 °C, respectively, after 0 days, 3 months, 6 months, 9 months, take out the specific rotation, traits, related substances, water and bacterial endotoxin, microbial limits, and content The results of the long-term placement stability obtained by the measurement method are shown in the following table. As can be seen from the results in the table, the long-term placement of the A crystal form of the compound I provided by the present invention has good stability.

Table 7: Stability test results

Example 27: Using XRPD to study whether crystal form A provided by the present invention undergoes crystal transformation:

The crystal form A prepared according to the method of Example 1 was separately placed in a closed container and placed at 25 ° C ± 2 ° C, 60% ± 10% RH, and taken out after 3 months, 6 months, and 12 months, respectively. The measurement is carried out by using an X-ray powder diffractometer, and the measurement results are as shown in FIGS. 2 to 5, wherein FIG. 2 is a comparison chart of three measurement times, and FIG. 3 is an XRPD chart measured for 3 months. 4 is the XRPD pattern measured for 6 months, and FIG. 5 is the XRPD pattern measured for 12 months.

Using the same method, it is investigated whether the crystal form disclosed in the patent document (WO2016055769A1) is crystallized under the same conditions, and the test comparison results are shown in Table 8 below.

The XRPD test in this example used a PANalytical XPERT-3 type X-ray diffractometer. Approximately 10 mg of the sample was evenly spread on a single crystal silicon sample pan and subjected to XRPD testing using the parameters described below.

XRPD scan parameters

Table 8: Comparison results of crystal form stability studies

As can be seen from the above Table 8, the crystal form of the present invention is a more stable crystal form, and the XRPD test is carried out at 25 ° C for 12 months without crystal transformation, and the crystal form disclosed in the patent document (WO2016055769A1) is placed for a long time. Partial crystal transformation occurred.

Example 28: Comparison of the stability of different crystal forms by HPLC:

The crystal forms disclosed in the crystal form A and the patent document (WO2016055769A1) prepared according to the method of the first embodiment of the present invention are respectively placed in a closed container and placed at 5 ° C ± 3 ° C, respectively, at 0 days, 3 months, 6 After 9 months, take out the purity measurement after 9 months. The HPLC test conditions were as follows: high performance liquid chromatography (Chinese Pharmacopoeia 2015 edition four general rules 0512), silica gel column (YMC-hydrosphere C18, 150mm × 4.6mm, 3μm or equivalent performance column); 0.1% phosphoric acid solution Mobile phase A with methanol-acetonitrile (30:70) as mobile phase B; flow rate of 1.0 ml per minute; column temperature of 25 ° C; detection wavelength of 220 nm. The results of the measurements are shown in the stability test comparison results in the table below.

Table 9: Stability test comparison results

As can be seen from the above Table 9, the crystal form A long-term stability ratio of the present invention has a significant advantage over the crystal form disclosed in WO2016055769A1.

Example 29: Humidity study

According to the "Guidelines for Drug Hygroscopicity" (Chinese Pharmacopoeia 2015 Edition, Part 4, 9103), the crystal form A of the compound I provided by the present invention and the crystal form disclosed in the patent document (WO2016055769A1) were tested. The hygroscopicity results are shown in Table 10. The wet weight gain is less than 0.2%, that is, the Form A material of Compound I has no or almost no hygroscopicity.

Table 10: Humidity and melting point results

As can be seen from the above Table 10, the crystal form A of the present invention and the patent document (WO2016055769A1) provide a small difference in the wettability of the crystal form, and the raw materials of the two crystal forms have no or almost no hygroscopicity.

Claims

A crystal form A of a compound of the formula I, characterized in that the crystal form A has an X-ray powder diffraction (XRPD) pattern having peaks at least at the following 2 theta angles: 6.9 ± 0.2, 13.9. ± 0.2 , 20.8 ± 0.2 and 21.1 ± 0.2,
Crystalline Form A of the compound of Formula I according to Claim 1, wherein the Form A has an X-ray powder diffraction (XRPD) pattern having peaks at least at the following 2 theta angles: 6.9 ± 0.2, 13.9 .±0.2, 18.4±0.2, 18.8±0.2, 20.8±0.2, and 21.1±0.2.
The crystalline form A of the compound of formula I according to claim 1, wherein the crystalline form A has an X-ray powder diffraction (XRPD) pattern substantially as shown in Fig. 1, Fig. 3, Fig. 4 or Fig. 5. Shown.
Use of Form A according to any one of claims 1 to 3 for the preparation of a medicament for the treatment and/or prevention of cancer.
A pharmaceutical composition comprising Form A according to any one of claims 1 to 3 and at least one pharmaceutically acceptable excipient.
A method of preparing a crystalline form A of a compound of formula I according to any one of claims 1 to 3, characterized in that the method comprises the steps of:

(1) adding a compound of formula I to a first organic solvent to obtain a mixture I;

(2) adding a second organic solvent to the mixture I, and filtering to obtain a filtrate II;

(3) The solution II was stirred and crystallized, and then filtered, and the obtained cake was dried to obtain a crystal form A.
The method according to claim 6, wherein said first organic solvent is selected from the group consisting of ethyl acetate, isopropyl acetate, propyl acetate, isobutyl acetate, and a mixed solution thereof.
The method of claim 7 wherein said second organic solvent is selected from the group consisting of methanol, ethanol, isopropanol, and mixtures thereof.
The method of claim 6 wherein said first organic solvent is selected from the group consisting of ethyl acetate and the second organic solvent is selected from the group consisting of methanol.
The method of claim 6 wherein the ratio of the first solvent to the second solvent is from 6:1 to 15:1 (v/v).
The method according to any one of claims 7 to 10, wherein the ratio of the first solvent to the second solvent is from 8:1 to 12:1. (v/v).
The method according to claim 11, wherein the total solvent ratio of the compound of the formula I to the first solvent and the second solvent is from 1:5 to 1:40 (w/v), preferably in the formula I. The total solvent ratio of the compound to the first solvent and the second solvent is 1:10-1:30 (w/v), more preferably the total of the compound of formula I and the first solvent and the second solvent. The solvent usage ratio is 1:15‐1:25 (w/v).
The method according to claim 6, wherein the mixture I is first stirred and heated to 20 ° C to 80 ° C, and then a second organic solvent is added, and the solid is dissolved and then hot filtered to obtain a filtrate II, and the filtrate II is cooled to The mixture was stirred at room temperature, then filtered, and the filter cake was washed with a first organic solvent and dried to give crystal form A.
The method according to claim 6, characterized in that ethyl acetate, a compound of the formula I is added to the reaction flask, the temperature is raised to 60 ± 2 ° C, the methanol is added, the solid is dissolved and then hot filtered; the filtrate is slowly cooled to 25 ± After 2 ° C, stirring and crystallization for 2 to 24 hours, filtration, washing the filter cake with ethyl acetate, and drying the filter cake under vacuum at 25 ± 5 ° C to obtain crystal form A, wherein the weight of the compound represented by formula I and ethyl acetate The ratio of total amount to methanol is 1:15 to 25 (w/v), and the ratio of ethyl acetate to methanol is 8 to 12:1 (v/v).