Crystalline etoposide 4`-phosphate diethanolamine and method thereof


(57) Abstract:

Usage: as protivoopujolevoe of the drug. The inventive product is crystalline etoposide 4-phosphate diethanolamine f-ly 1. So PL. 160 172°C. B. F. C29H33O16P2C2H6O. Exit 91% Reagent 1 4-phosphate etoposide. Reagent 2: absolute ethanol or methanol boiling under reflux. Reagent 3 (in the case of ethanol) water. Connection structure of f-crystals 1: (see below) 2 S. and 2 C.p. f-crystals, 3 ill. table 1.

The invention relates to crystalline etoposide 4'-phosphate, solvated two molecules of ethanol.

Etoposide is an anti-cancer agent, as currently approved by the United States for cancer treatment small cell lung and resistant tumors of the testicles. Since etoposide only moderately soluble in water to prepare the solution of etoposide requires an organic solvent or mixture of organic solvents. The drug etoposide for parenteral use, currently marketed system contains many solvents.

Etoposide 4'-phosphate (I) and its disodium salt (II) are described in U.S. patent N 4904768 as proletarienne

I: R=H

II: R=Na

While the solubility of etoposide in water is about 0.1 mg/ml, as etoposide 4'-phosphate, and disodium salt are soluble in water, 100 mg/ml, thereby allowing the preparation of pharmaceutical compositions with the use of a small amount or absence of organic solvents. The previously described forms of etoposide 4'-phosphate and disodium salts are loose amorphous substances that may be difficult to apply and which tend to decompose during storage. Thus, the aim of the invention is to provide a form of etoposide 4'-phosphate, which can more advantageously be used for pharmaceutical purposes than the preceding forms. The invention considers a new crystalline ethanol MES etoposide 4'-phosphate, which is easier to handle and which exhibits unexpected stability compared with known forms of etoposide 4'-phosphate.

The present invention is a stable crystalline etoposide 4'-phosphate, solvated two molecules of ethanol, subsequently called etoposide 4'-phosphate diethanolamine

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On the other hand present from adowanie saturated solution of etoposide 4'-phosphate in containing the ethanol solvent system.

In Fig.1 shows a picture of the x-ray diffraction on powder etoposide 4'-phosphate diethanolamine; Fig.2 is an NMR spectrum for the protons (D2Oh, 200 MHz) etoposide 4'-phosphate diethanolamine.

I. Receiving etoposide 4'-phosphate diethanolamine.

Crystalline etoposide 4'-phosphate diethanolamine can be obtained by forming a saturated solution of etoposide 4'-phosphate in a solvent system containing ethanol, allowing the crystals to be formed and collecting these crystals starting material etoposide 4'-phosphate can be obtained by the method described in U.S. patent N 4904768, this method involves interactions etoposide with diphenylchlorophosphine and exposure of the resulting product to catalytic hydrogenation. The product obtained by this method, is an amorphous substance, which can be converted into crystalline etoposide 4'-phosphate diethanolamine of the present invention.

Saturated ethanolic solution of etoposide 4'-phosphate can be obtained by mixing etoposide 4'-phosphate, ethanol and co-solvent; the specified co-solvent may constitute, for example, water or methanol. If necessary, the mixture can be heated to achieve full is mperature, suitable for crystal formation; temperature is not specifically limited and can vary from, for example, about 0aboutup to about 65aboutWith, but preferably the crystallization is carried out at ambient temperature, i.e. about 15aboutWith up to about 25aboutC. Crystallization of the desired product is usually quite slow, and the solution usually is stirred for about 18 to about 72 h before collecting the crystals. Optional solution can be hunted down by a small amount of pre-formed crystals of etoposide 4'-phosphate diethanolamine. The crystals are collected in a standard way, for example by filtration, and the crystalline substance was washed with absolute ethanol, and then dried, receiving etoposide 4'-phosphate diethanolamine in the form of a colorless crystalline solid. Drying is preferably carried out in vacuum at a temperature varying from 20 to 25aboutC.

In one typical method of etoposide 4'-foft mixed with absolute ethanol, preferably about 1 g of etoposide 4'-phosphate in about 15 ml of ethanol, and the mixture is heated to reverse the dripping liquid. To this mixture, water is added in sufficient quantity to complete the tee is filtered hot to remove undissolved impurities, and the filtrate is allowed to cool to ambient temperature, i.e. about 15 to 25aboutWith under stirring. The filtrate support at a temperature favorable for the formation of crystals, up until not formed enough crystals. Although crystallization may occur at about 55-65aboutWith, preferably, solution support at ambient temperature. Thus, the solution is stirred for about 18 to 72 h, the formed crystals are collected by filtration, washed with absolute ethanol and dried, obtaining the desired product as a colourless crystalline solid.

P R I m e R 1. Receiving etoposide 4'-phosphate diethanolamine of etoposide 4'-phosphate.

To amorphous etoposide phosphate (18.0 g) add absolute ethanol (270 ml). The mixture is heated to reverse draining the fluid and add deionized water until until complete dissolution occurs (requires 5 to 10 ml). The solution is filtered hot, then he is allowed to cool to approximately 20aboutC and stirred for 18 hours, the Product is collected by filtration, washed with absolute ethanol (2x20 ml) and dried in vacuum at 20aboutWith over 18 hours of Etoposide phosphate diethanolamine the methodology crystalline etoposide 4'-phosphate diethanolamine can be obtained, adding the final stage of crystallization to a method for etoposide 4'-phosphate, is described in U.S. patent N 4904768. The usual method of obtaining crystalline etoposide 4'-phosphate diethanolamine on the basis of etoposide, described below. Etoposide 4'-phosphate get through a modified version of the method described in U.S. patent N 4904768.

P R I m m e R 2. Receiving etoposide 4'-phosphate diethanolamine based on it - posed.

A. Obtain 4'-dimensionful etoposide.

To a stirred solution of dibenzylamine (446,5 g, 1.70 mol, 2 EQ.) in methylene chloride (3.5 l) at 20aboutTo add one portion N-chlorosuccinimide (227,5 g, 1.70 mol, 2 EQ.). The reaction mixture was stirred for 3 h at 30aboutC, cooled to 0aboutS, and incubated at 0aboutC for 12-72 h, and then filtered. Part of the thus obtained solution dibenzoylmorphine (62,5% to 1.25 EQ.) during the time interval 1-1,5 h are added to a solution of etoposide (500 g, 0.85 mol), 4-dimethylaminopyridine (10.5 g, 0,086 mol, 0.1 EQ.) and N,N-diisopropylethylamine (311,5 ml, 1,79 mol, 2.1 EQ.) in dry acetonitrile (5 l), stir at (-15)-(-20)aboutC in an atmosphere of inert gas. The reaction mixture is stirred for 20 the percentage chromatography high resolution will not show that spent at least 98% of etoposide. To this reaction mixture was then added 0.5 mol/l KN2PO4(5 l) and the mixture is allowed to warm to room temperature. The organic phase is separated and washed twice with 10% such as NaCl (2 l), dried over sodium sulfate and then the solvent is evaporated in vacuum. The crude solids chromatographic on silica gel using 20% EtOAc/CH2Cl2, 40%EtOAc/CH2Cl2then EtOAc as solvent for elution; the fractions containing the desired product are mixed and evaporated in vacuo to 2 liters of Solution slowly with good stirring to heptane (5 l), the reaction mixture is stirred at 20aboutC for 1-3 h, filtered and dried in vacuum at 40aboutC for 18 h, receiving indicated in the title compound (505 g, 70% yield).

C. Obtaining crystalline etoposide 4'-phosphate diethanolamine.

A solution of 4'-dimensionful etoposide (500 g, 0.59 mol) in methanol (2 l) is added to a suspension of 10% Pd/C (50 g) in methanol (1 l), and the suspension is heated to about 37aboutC. To this suspension is added slowly a solution of 1-methyl-1,4-cyclohexadiene (555 g, 660 ml of 5.89 mol, 10 EQ.) in methanol (1 l), and the suspension is stirred at 40-45aboutWith as long of ptrace adjusted to about 1 l by concentration or by adding additional amounts of methanol, the resulting solution is added then in absolute ethanol (4 l). The solution tatrallyay reference etoposide 4'-phosphate and concentrate to about 2.5 liters of suspension add absolute ethanol (3.5 l) and stirred at about 20aboutC for 18-72 hours, the Solids are collected by filtration, washed with absolute ethanol (2x250 ml) and dried under vacuum for 18 h at about 20aboutWith getting 300-350 g of product (yield 80-90% ).

II. Physico-chemical properties of crystalline etoposide 4'-phosphate diethanolamine

Melting point: 141-150aboutWith its



Ethanol residue: 11,8% (by

NMR); 13,2% (pore-

by the rotary thermogravi-

metric analysis-

for). Designed for


12.1% of

Moisture content: 0,22% through

method of Charles the fish


NMR: corresponds to the one presented in Fig.2.

The diffraction pattern of x-ray radiation on the powder in accordance with the shown in Fig.1 were obtained on the system of diffraction on the powder Philips model DGS 3720, equipped with a vertical goniometer in geometry /2. With the x-ray generator izlucheniya. Each sample was scanned between 2 and 32 degrees 2, using a step size of 0.04 degrees 2 and the scanning speed of 0.04 C 2/c. To gather and analyze all the data used execution Philips DGS software 4,00.

III. The stability studies.

A. the substance Used.

A. Crystalline etoposide 4'-phosphate diethanolamine received in accordance with the above.

b. Crystalline etoposide 4'-phosphate (dissolved) was obtained by the following method. Etoposide 4'-phosphate (300 mg) in ethanol (175 ml) was heated under stirring at 70aboutWith getting a clear solution. The solution was filtered, and the filtrate was heated at 70aboutWith up until the volume was reduced to approximately 34 ml. portion of the solution (approximately 17 ml) was placed in a vial and was made fun of a small amount of pre-obtained crystals etoposide 4'-phosphate having a needle shape. The solution was placed in a freezer at -12aboutWith 4 days, and the resulting needle-like crystals form was collected in the form of a white powder substance. NMR analysis of the substance indicated on the ethanol content of approximately 1.6 wt.

C. Amorphous disodium salt etoposide 4'-Locale, dissolving crystalline etoposide 4'-phosphate diethanolamine (125 mg) in methanol (50 ml), viparita the solution to dryness on a rotary evaporator and drying the residue at 25aboutC in vacuum for 1 h

Century Technique.

Accurately weighed samples of the tested substances were placed in ampoules of glass flint type 1 and corked tubes with Teflon and aluminum caps. The ampoule was kept at 50aboutC. the Effectiveness of these samples was determined by liquid chromatography high resolution at 2, 4 and 8 weeks. The samples were stored dried at 4aboutWith, served as the control (100% of expected performance).

C. Conditions of analysis by liquid chromatography high pressure (HPLC) (ghvd)

Column: Jones Apex Octadecyl,

5 μm, h,6 mm

Flow rate: 1 ml/min

The mobile phase A: 0.02 mol/l ammonium phosphate (monobasic)

in water MiII-Q (pH 4.5):

Acetonitrile (85:15)

Mobile phase b: Acetonitrile

Gradient: 0-3 min isocratic

at 0% B;

3-18 min linear

gradient 0 to 30% B;

18-23 min isocratic

at 30%;

23-25 min linear

gradient 30 to 0% B;

25-30 min again urav-

namasivaya when

100% Adigna phase AND

Volume of sample: 20 ál

Retention time: Etoposide 4'-phosphate of 5.4 min; a lignin P

11.5 min; etoposide

17,5 min

The peak area for etoposide 4'-phosphate is linearly correlated with the concentration in the region of 0.05-0.20 mg/ml (correlation coefficient 0,9999).

D. The Results.

The results of the above stability studies are presented in the table in which the name of "ER" represents etoposide 4'-phosphate.

The results of stability studies at the 50aboutTo show that crystalline etoposide 4'-phosphate diethanolamine undergoes very little loss of efficiency for a period of time equal to 8 weeks, and clearly show the stability of the crystalline etoposide 4'-phosphate diethanolamine exceeding the stability of other forms of etoposide 4'-phosphate.

Crystalline etoposide 4'-phosphate diethanolamine of the present invention can be used directly in pharmaceutical preparations. For example, it can be prepared by mixing with inert fillers, such as lactose, mannitol, dextran, or any other suitable filler; sodium citrate for pH adjustment. The solid mixture can then be used to populate ampelman diluent, for example, a glucose solution or normal saline. The solid mixture can also be used to fill gelatin capsules suitable for oral administration of etoposide 4'-phosphate.

Or crystalline etoposide 4'-phosphate diethanolamine can be used for the preparation of lyophilised drugs etoposide 4'-phosphate. So, crystalline etoposide 4'-phosphate diethanolamine dissolved in water for injection, and the solution brought to a pH in the range from about 4 to about 5, adding a pharmaceutically acceptable base, such as sodium hydroxide or sodium citrate; optionally, the solution may also contain other pharmaceutical components such as a filler, for example, lactose or dextran. One milliliter of the solution was placed in a glass ampoule and lyophilizers. The lyophilisate can be mixed with physiologically acceptable diluent before application to the patient.

It should be noted that crystalline etoposide in the form of a pharmaceutical preparation, and the bulk of the dosage form should be stored in conditions with low relative humidity, preferably at a relative humidity of less than about 33% and Aibolit unwanted exposure to moisture.

1. Crystalline etoposide 4-phosphate diethanolamine formula I

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2. The method of obtaining crystalline etoposide 4-phosphate diethanolamine, characterized in that it comprises the formation of a saturated solution of etoposide 4-phosphate in the system of solvents containing ethanol.

3. The method according to p. 2, characterized in that the solvent system comprises water and ethanol.

4. The method according to p. 2, characterized in that the solvent system comprises methanol and ethanol.


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