Organic amine salts, amino acids salts and combrestatin a-4 phosphate amino acid ester salts for modulating tumor or metastasis proliferation and benign vascular proliferative disorder growth

FIELD: organic chemistry, medicine, oncology.

SUBSTANCE: invention relates to organic amine salts, amino acid salts and combrestatin A-4 phosphate amino acid ester salts. Invention describes compound of the general formula (I):

wherein one of substitute -OR1 or -OR represents -O-QH+ and another one represents hydroxyl or -O-QH+; Q represents (A) optionally substituted aliphatic organic amine comprising at least one nitrogen atom that in common with proton forms quaternary ammonium cation QH+; (B) amino acid comprising at least two nitrogen atoms wherein one of nitrogen atoms in common with proton forms quaternary ammonium cation QH+; (C) amino acid comprising one or some nitrogen atoms wherein one of nitrogen atoms in common with proton forms quaternary ammonium cation QH+ and wherein, except for, all carboxyl groups of amino acids are in ester form. Also, invention describes pharmaceutical compositions used in modulation of tumor or metastasis proliferation and growth of benign vascular proliferative disorders, using compound of the formula (I) and a method for synthesis of compound of the formula (I). Invention provides preparing new combrestatin A-4 salts showing useful physicochemical properties that enhance solubility of combrestatin A-4.

EFFECT: valuable medicinal properties of compounds and pharmaceutical compositions.

27 cl, 13 dwg, 5 ex

 

This application claims the priority application U.S. registration No. 60/232568, filed September 14, 2000, and at the request of the USA, registration number №60/251921, filed December 7, 2000, both of these prior applications is incorporated herein by reference.

The technical field

The present invention relates to new and useful proletarienne mono - and di-salts of organic amines, mono - and di-salts of amino acids and mono - and di-salts of esters of amino acids phosphate combretastatin a-4 (CA4), and these salts have a higher solubility than natural combretastatin a-4 and easily regenerate combretastatin a-4 under physiological conditions.

The level of technology

Cancer is a serious and widespread disease. According to estimates of the National cancer Institute in the USA only 1 person out of 3 will be affected by cancer during his lifetime. Moreover, approximately 50 to 60% of people with cancer, will eventually die. As a consequence, since the Foundation of the National cancer Institute in the early 70's. the money spent on the study of cancer increased sharply.

Although cancer, as usually considered, is one disease, in fact it includes a family of diseases in which the normal differentiation of cells is modified in such a way that it becomes abnormal and uncontrolled. The result of such malignant cells rapidly proliferate. In the end, the cells spread and metastasize from their source and colonise other organs, eventually killing its host. Because of the wide diversity of types of cancer identified to date, developed a variety of strategies to eradicate cancer in the body. One of such ways is used cytotoxic chemotherapeutic tools. Such compounds are administered to cancer patients to destroy malignant cells without affecting live normal cells. Specific examples of such compounds are 5-fluorouracil, cisplatin and methotrexate.

Combretastatin a-4 was first isolated from the trunk of the African tree Combretum caffrum (Combretaceae) and it was found that it is an effective inhibitor microtraumatic structures. In addition, combretastatin a-4, as installed, has significant activity against the cell line of murine L1210 and R lymphocytic leukemia according to the classification of the National cancer Institute of the USA (NCI). In addition, combretastatin a-4, as it competes with combretastatin a-1, another compound isolated from Combretum caffrum as an inhibitor of the binding of colchicine to tubulin. It also significantly delays the development of VoLo, DLD-1 and HCT-15 colon cancer man (ED50<0,01 µg/ml) and is one of the stronger antimitoticescoy agents, agennix among components Combretum caffrum (U.S. patent 4996237).

Accordingly, studies have been conducted to determine the effectiveness of combretastatin a-4 as a chemotherapeutic agent in the treatment of various types of human cancer. Unfortunately, combretastatin a-4 is essentially not soluble in water. This property greatly hinders the development of pharmaceutical compositions containing combretastatin a-4. To improve the solubility and effectiveness, attempts have been made to create proletarienne derived combretastatin a-4, which will regenerate combretastatin a-4 under physiological conditions. For example, Koji Ohsumi with co-workers described the synthesis of the HCl-amino acid prodrugs of analogs combretastatin, in which the salt of the amino acids attached to the amino group derived combretastatin containing the basic amino group [such derivatives are described in the work Ohsumi et.al, Anti-Cancer Drug Design, 14, 539-548 (1999)]. Although such prodrugs may have an increased solubility compared to the solubility of natural combretastatin a-4, they are limiting their use of the property, namely, that the regeneration of combretastatin a-4 depends on endogenous aminopeptidase in the patient's blood, which introduced the prodrug.

The free acid phosphate combretastatin a-4 (free acid CAR", which has the following structures is:

exists only in the form of oil. The free acid CAR by nature very little soluble (as defined by the U.S. Pharmacopoeia) in water at 25°C, and the solubility increases with increasing pH. Acid has two acidic groups of values PKand1,2 and 6.2, which are capable of forming salts. Since in practice there are problems when working with the free acid CAR because of her physical condition, it is advisable to obtain crystalline, stable salt form of the compound.

Attempts to obtain the derivatives of combretastatin a-4 included obtaining salt derivatives of phosphate combretastatin a-4 (salt derivatives "SAR"). Specific examples of such salts are presented in U.S. patent 5561122. Although such proletarienne salts have a higher solubility than natural combretastatin a-4, they also suffer from inherent disadvantages, such as hygroscopicity.

Hygroscopicity is one of the very important selection criteria for salt. Cm. K. Morris et al, "An Integrated Approach to the Selection of Optimal Salt Form for a New Drug Candidate", Int. J. Pharm., 105, 209-217 (1994). The degree of hygroscopicity for many medicinal substances has a strong influence on them and their stability during the shelf-life of the drug product.

Thus, there is a need the ü in new and useful proletarienne salts of combretastatin a-4 with useful physical and chemical properties, which increase the solubility and preferably efficiency combretastatin a-4 in the treatment of a wide spectrum of tumors.

Also need new and useful proletarienne salt combretastatin a-4, which are easily regenerate natural combretastatin a-4 in vivo and does not produce undesirable or potentially harmful by-products during regeneration.

Citation of any reference herein should not be construed as an admission that such reference is suitable as prior art for this application.

The invention

In accordance with the present invention proposed a new and useful proletarienne mono - and di-salts of organic amines, mono - and di-salts of amino acids and mono - and di-salts of esters of amino acids phosphate combretastatin a-4, and these salts have a higher solubility than natural combretastatin a-4 and easily regenerate combretastatin a-4 in vivo. The present invention also relates to compounds, which are much less hygroscopic than known so far proletarienne salt combretastatin a-4 (for example, they do not change significantly in shape in conditions of normal temperature and humidity). These compounds are easier to work with, they are more stable and can form solutions at pH values that drive to mini the mind or eliminate pain at the injection site. Thus, the compounds of the present invention provide significant advantages for pharmaceutical use.

More specifically, the present invention relates to a compound having the General structure formula I:

where one of the substituents OR1or2represents-O-QH+and the other represents a hydroxyl or-O-QH+; and

Q represents

(A) an organic amine containing at least one nitrogen atom which, together with a proton, forms a Quaternary ammonium cation QH+;

(C) an amino acid containing at least two nitrogen atom, where one of the nitrogen atoms, together with a proton, forms a Quaternary ammonium cation QH+; or

(C) an amino acid containing one or more nitrogen atoms where one of the nitrogen atoms, together with a proton, forms a Quaternary ammonium cation QH+and where, in addition, all carboxyl groups of the amino acids are in the form of esters.

Throughout the description, when both Deputy OR1and-OR2represent-O-QH+, Q is preferably the same in both groups-OR1and-OR2.

When Q has a definition (A), the preferred organic amine, in this case represents tromethamine (i.e., Tris(hydroxymethyl)aminomethan, the which is hereinafter denoted by the reduction of TRIS (TRIS)).

Tromethamine salts of formula I are illustrated by the following formulas Ia and Ib, which are mono-tromethamine and di-tromethamine salt of formula I, respectively:

Mono-trometamina salt of the formula Ia are preferred.

When Q has a definition (In), any amino acid containing at least two nitrogen atom, can be used in this case. Any nitrogen atoms of the amino acids may form a Quaternary ammonium cation of the formula I, for example, any nitrogen atom of the side chain of the amino acid or nitrogen atom α-amino group. Amino acids that can be used in this case are, but certainly not limited to, ornithine, histidine, lysine, arginine, tryptophan and other

When Q has a definition (In), preferred amino acid is histidine. For example, or the nitrogen atoms of imidazole moiety of the side chain of histidine or, on the other hand, the nitrogen atom α-amino group of histidine may form a Quaternary ammonium cation of the formula I. As can be easily seen, due to the aromatic nature of the imidazole group any nitrogen atom of imidazole moiety of the side chain of histidine can form the structure of formula (I). Preferred mono-his-tag patterns form the s I are illustrated by the following formulae Ic or Id:

When Q has the definition (S), any amino acid can be used in this case, for example, such as glycine, but ETM is not limited. The preferred esters are alkalemia esters, such as methyl or ethyl esters.

In addition, the present invention relates to pharmaceutical compositions containing:

(a) compounds having the General structure formula I:

where:

one of the substituents OR1or2represents-O-QH+and the other represents a hydroxyl or-O-QH+; and

Q represents

(A) an organic amine containing at least one nitrogen atom which, together with a proton, forms a Quaternary ammonium cation QH+;

(C) an amino acid containing at least two nitrogen atom, where one of the nitrogen atoms, together with a proton, forms a Quaternary ammonium cation QH+; or

(C) an amino acid containing one or more nitrogen atoms where one of the nitrogen atoms, together with a proton, forms a Quaternary ammonium cation QH+and where, in addition, all carboxyl groups of the amino acids are in the form of esters; and

(b) a pharmaceutically acceptable carrier.

Specific examples of the compounds present, and the finding, used in such pharmaceutical compositions described above. In addition, any pharmaceutically acceptable carrier used in the pharmaceutical compositions of the present invention. Specific examples are described below. Particular pharmaceutical compositions of the present invention includes the compound of the present invention, in which tromethamine is an organic amine and which preferably is tromethamine salt, having the structure of formula Ia or Ib, most preferably Ia:

Another particular variant of the pharmaceutical compositions of the present invention includes the compound of the present invention, in which histidine is an amino acid and which preferably is a mono-his-tag salt, having the structure of formula Ic or Id:

Of course, such a pharmaceutical composition should also include pharmaceutically acceptable carrier.

In addition, the present invention also encompasses compositions containing the salt of the present invention. In particular, the composition according to the present invention can be obtained by mixing compounds, including:

(a) freedoms is th acid SAR, having the structure:

(b) compound Q, where Q is a

(A) an organic amine containing at least one nitrogen atom that is capable of together with a proton to form a Quaternary ammonium cation QH+;

(C) an amino acid containing at least two nitrogen atom, where one of the nitrogen atoms, together with a proton, forms a Quaternary ammonium cation QH+; or

(C) an amino acid containing one or more nitrogen atoms where one of the nitrogen atoms, together with a proton, forms a Quaternary ammonium cation QH+and where, in addition, all carboxyl groups of the amino acids are in the form of esters.

Optional composition of the present invention may also include pharmaceutically acceptable carrier.

In addition, when Q has a definition (A), any organic amine, which is defined herein, can be used in the compositions of the present invention. Specific examples include, but are not limited to, tromethamine, diethanolamine, glucamine, N-methylglucamine, Ethylenediamine and 2-(4-imidazolyl)ethylamine. When Q has a definition (In), any amino acid containing at least two nitrogen atom, can be used in the compositions of the present invention. Specific examples include ornithine, GIS is one, lysine, arginine, tryptophan, etc. When Q has the definition (S), any ether amino acids, as defined herein, can be used in the compositions of the present invention. A concrete example is glycine.

In another embodiment, the present invention relates to a method of modulating tumor growth or metastasis in an animal, which includes the introduction to be effective amount of compounds having the General structure:

where:

one of the substituents OR1or2represents a

-O-QH+and the other represents a hydroxyl or-O-QH+; and

Q represents

(A) an organic amine containing at least one nitrogen atom which, together with a proton, forms a Quaternary ammonium cation QH+;

(C) an amino acid containing at least two nitrogen atom, where one of the nitrogen atoms, together with a proton, forms a Quaternary ammonium cation QH+; or

(C) an amino acid containing one or more nitrogen atoms where one of the nitrogen atoms, together with a proton, forms a Quaternary ammonium cation QH+and where, in addition, all carboxyl groups of the amino acids are in the form of esters.

In a specific embodiment, the present invention relates to a method of modulating tumor growth or metasta is and the animal, which includes an introduction to be effective amount of compounds in which tromethamine is an organic amine which is preferably tromethamine salt, having the structure of formula Ia or Ib, most preferably, Ia:

In another specific embodiment, the present invention relates to a method of modulating tumor growth or metastasis in an animal, which includes the introduction to be effective amount of compounds in which histidine is an amino acid and which preferably is a mono-his-tag salt, having the structure of formula Ic or Id:

Thus, the present invention relates to new and useful proletarienne mono - and di-salts of organic amines, mono - and di-salts of amino acids and mono - and di-salts of esters of amino acids phosphate combretastatin A4, and these salts are more soluble in aqueous solutions than natural combretastatin a-4. Thus, the effectiveness of this medication may be increased.

The present invention also relates to proletarienne mono - and di-salts of organic amines, mono - and di-salts of amino acids and mono - and di-salts of esters of amino acids phosphate combretastatin A4, these salts Le is to regenerate combretastatin A4 in vivo, and these salts in the dissociation free organic amine, amino acid or ester of the amino acids in the form of a physiologically portable byproduct.

In the most preferred embodiment, the present invention relates to a new crystalline (1:1) proletarienne tromethamine (TRIS) salt antivascular antitumor agent phosphate combretastatin A4 having the structure of formula Ia. This connection is Palekastro phosphate salt of ester, in which the phosphate fragment undergoes dephosphorylation under physiological conditions, forming the active drug residue combretastatin a-4 (as mentioned below, olefinic group linking the phenyl bridge remains core combretastatin A4 is isconfigurable; olefinic group preferred mono-TRIS salt of phosphate combretastatin A4 also is isconfigurable). TRIS salt of 1:1 (mono) CAR has good properties in the solid state and unexpectedly almost nephroscopy. These and other preferred features make the TRIS-salt SAR the preferred connection for pharmaceutical dosage of the drug.

Brief description of drawings

Figure 1 shows a plot of sorption/desorption of moisture mono-TRIS salt SAR (obtained in example 1) at 25°C. floor Data is obtained using the balance scale, graduated in percent humidity, VTI Model MB-300W, with limits of relative humidity from 10% to 90% range increase of 10%. Maximum time to establish equilibrium at each humidity value is set to 4 hours.

Figure 2 presents the spectra of powder x-ray diffraction samples of mono-TRIS salt SAR (obtained in example 1), which is in the form of a suspension in different solvents (water, isopropanol, ethanol, acetonitrile and acetone) initially at a temperature of from 70 to 75°C for 5-10 min and then at room temperature overnight. X-ray diffraction pattern recorded using x-ray diffractometer Rigaku Miniflex Model with a source of Cu-Kα if the scan speed of 1° per minute from 2° to 40° 2θ.

3 shows a thermogram of differential scanning calorimetry (DSC) (Model DSC 2910, TA) mono-TRIS salt SAR (obtained in example 1), resulting in a stream of nitrogen at a heating rate of 10 degrees per minute.

Figure 4 shows a graph of the pH-solubility of mono-TRIS salt SAR (obtained in example 1) at 25°C. the value of the pH is adjusted with sodium hydroxide.

Figure 5 presents thermogram of differential scanning calorimetry (DSC) mono-L-his-tag salt SAR (obtained in example 3) (example 2,0900 mg).

Figure 6 presents thermogram is differentsialnoi scanning calorimetry (DSC) mono-L-his-tag salt SAR (obtained in example 3).

Figure 7 presents thermogram of differential scanning calorimetry (DSC) mono-L-his-tag salt SAR (obtained in example 3).

On Fig presents powder x-ray diffraction spectra of mono-L-his-tag salt SAR (obtained in example 3).

Figure 9 presents powder x-ray diffraction spectra of mono-L-his-tag salt SAR (obtained in example 3).

Figure 10 presents thermogram of differential scanning calorimetry (DSC) of anhydrous mono-L-his-tag salt SAR (obtained in example 3).

Figure 11 presents the spectra of powder x-ray diffraction anhydrous mono-L-his-tag salt SAR (obtained in example 3).

On Fig presents a thermogram of differential scanning calorimetry (DSC) mono-salt of methyl ester of glycine and SAR (obtained in example 4).

On Fig presents powder x-ray diffraction spectra of the mono-salt of methyl ester of glycine and SAR (obtained in example 4).

Detailed description of the invention

The present invention is based on the surprising and unexpected discovery that can be obtained proletarienne mono - and di-salts of organic amines, mono - and di-salts of amino acids and mono - and di-salts of esters of amino acids phosphate combretastatin a-4, which have increased solubility in vivo compared to the solubility of natural combretastatin-4, easily regenerate combretastatin a-4 under physiological conditions, and regeneration secrete physiologically tolerated organic amines or physiologically tolerated acids or esters of amino acids that are easily metabolized in vivo.

Speaking more broadly, the present invention relates to a compound having the General structure:

where:

one of the substituents OR1or2represents-O-QH+and the other represents a hydroxyl or-O-QH+; and

Q represents

(A) an organic amine containing at least one nitrogen atom which, together with a proton, forms a Quaternary ammonium cation QH+;

(C) an amino acid containing at least two nitrogen atom, where one of the nitrogen atoms, together with a proton, forms a Quaternary ammonium cation QH+; or

(C) an amino acid containing one or more nitrogen atoms where one of the nitrogen atoms, together with a proton, forms a Quaternary ammonium cation QH+and where, in addition, all carboxyl groups of the amino acids are in the form of esters.

All isomers of the considered compounds (for example, the isomers that can exist due to the presence of asymmetric carbon atoms, for example, at different substituents), including anytime the forms and diastereomeric form, included in the scope of the present invention. Individual stereoisomers of compounds of the invention can, for example, essentially not contain other isomers (for example, pure or substantially pure optical isomers, having a specific activity) or can be mixed, for example, as racemates or with all other, or other selected stereoisomers. The chiral centers of the compounds of the present invention can have the S or R configuration in accordance with the recommendations of the IUPAC 1974. The racemic forms can be separated by physical means, such as, for example, fractional crystallization, separation or crystallization of diastereomeric derivatives or by separation using chiral column chromatography. The individual optical isomers can be obtained from the racemate by any suitable means. Olefinic group linking the phenyl bridge remains core combretastatin a-4, is isconfigurable, which is the preferred configuration of the compounds of the present invention. Using the definition of "combretastatin a-4" or "CA4" as the name or part of name of connection " means a connection in this isconfigurable. Also refers to a solvate of the compounds of formula I, such as hydrates.

Throughout the description of the group and the substituents may be ibrani so, to be obtained remains stable and connections.

Embodiments of the invention presented in the document as examples or as preferred options, are intended only to illustrate the invention and not limit it.

In another embodiment of the invention the present invention relates to pharmaceutical compositions containing the compound of the present invention and a pharmaceutically acceptable carrier. Of course, the compounds of the present invention can be used in any form such as a solid form or in solution (especially aqueous solution), which are also described below. For example, the compound of the present invention can be obtained and used in dried form, alone or with suitable auxiliary substances.

In yet another embodiment, the present invention relates to a method of modulating tumor growth or metastasis in an animal, which includes the introduction to be effective amount of compounds having the General structure formula I:

where:

one of the substituents OR1or2represents a

-O-QH+and the other represents a hydroxyl or-O-QH+; and

Q represents

(A) an organic amine containing at least Odie is a nitrogen atom, which, together with a proton, forms a Quaternary ammonium cation QH+;

(C) an amino acid containing at least two nitrogen atom, where one of the nitrogen atoms, together with a proton, forms a Quaternary ammonium cation QH+; or

(C) an amino acid containing one or more nitrogen atoms where one of the nitrogen atoms, together with a proton, forms a Quaternary ammonium cation QH+and where, in addition, all carboxyl groups of the amino acids are in the form of esters.

Of course, the connection of the present invention may be introduced separately or in pharmaceutical compositions.

The terms and expressions used in the document are defined below and have the specified values, unless otherwise noted.

In the description, the terms "modulate", "modulating" or "modulation" refers to the speed at which there is a specific process to inhibition of a specific process to address specific process in the other direction and/or the prevention of the beginning of a process. Thus, when a specific process includes, for example, tumor growth or metastasis, "modulation" process is to reduce the speed at which it develops a tumor and/or metastasis, inhibition of tumor growth and/or metastasis, in circulation in the opposite direction of tumor growth and/or metasta is a (including reduction and/or disappearance) and/or prevention of tumor growth and/or metastasis, especially for a subject that is predisposed to this process.

Used in the description, the expression "effective amount" or "effective amount" entered connection to an animal, means a quantity sufficient to modulate tumor growth or metastasis in an animal. The person skilled in the art can easily determine, for example, using conventional methods, an effective amount of the compounds of the present invention, which should be put animal. Examples of dosage amounts for an adult human of approximately from 0.05 to 1000 mg/kg of body weight of active compound per day, which can be entered as a single dose (for example, in the form of a striking single dose or in the form of infusion for some time to the maximum allowable dose or below) or in the form of individual divided doses (for example, by sequential injection of doses below the maximum permissible dose), for example from 1 to 4 times a day. It should be understood that the specific level of dose and frequency of dose any particular subject may vary and depends on several factors, including the activity of the specific compound, body weight, General health, sex and diet of the subject, method and time of administration, rate of excretion, the concentration of the drug and the severity of con the specific state.

As used herein, the term "animal" preferably includes such subjects as Pets and, most preferably, humans.

Used in the description, the term "prodrug" refers to a precursor compound that will undergo metabolic activation in vivo to form the active drug compounds. Thus, for example, the connection according to the invention, introduced the subject will undergo metabolic activation, and regenerate combretastatin a-4 due to the dissociation of the compounds of the present invention, for example, under the action of endogenous nonspecific phosphatase in plasma.

Used in the description of the concept of "organic amine" refers to organic (i.e. carbon-containing) compound containing at least one primary (i.e.- NH2), secondary (i.e.- NH-) or tertiary (that is,) amino group, can form a phosphate salt of the compounds of formula I of the present invention. When there is more than one primary, secondary and/or tertiary amino groups in the specified organic Amina, any such group that have the specified property may form a Quaternary ammonium group QH+formula I. the Present definition of "organic amine" not katyayni amino acid compounds (see previous application entitled "Combretastatin A-4 Phospate Mono - and Di-Amino Acid Salt Prodrugs", which was filed Venit in application U.S. registration No. 60/232568 September 14, 2000, included in the description by reference)or some compounds described in the publication WO 99/35150 (glucosamine, piperazine, piperidine, 6'-methoxyindole-9-ol, Cinchona-9-ol, pyrazole, pyridine, tetracycline, imidazole, adenosine, verapamil, morpholine), which entered into the description by reference. Used organic amine is preferably a physiologically tolerated a compound selected from the following groups:

(a) organic amines having the value of the PKandgreater than or equal to 7, more preferably the value of the PKandgreater than or equal to 8;

(b) organic amines where the nitrogen atom, forms a Quaternary ammonium cation QH+in formula I, is associated with optionally substituted aliphatic group, or optionally substituted nonaromatic heterocyclic group (or two or three such optional substituted aliphatic and/or heterocyclic non-aromatic groups in the case of secondary or tertiary amines, respectively). "Aliphatic group" is a linear or branched, saturated or unsaturated hydrocarbon (e.g., alkane, alkene or alkyne containing from 1 to 20,preferably from 1 to 12, more preferably from 1 to 6 carbon atoms in the chain. "Nonaromatic heterocyclic group" is a saturated or partially unsaturated ring containing the nitrogen atom, forms a Quaternary ammonium cation QH+in formula I, and optionally other heteroatoms in the ring, such as O, S or additional atoms N. "Optional substituents" is preferably represent one or more substituents present in organic Amina, which when used in the formula I of the present invention, lead to the phosphate salts of the formula I, which are crystalline and essentially non-hygroscopic or non-hygroscopic. Preferred optional substituents are a hydroxyl group, an amino group (NH2) or alkoxy group (O-alkyl), most preferred are one or more hydroxyl groups; and/or

(C) organic amines where the nitrogen atom, forms a Quaternary ammonium cation QH+in formula I, is a primary amine that is associated with the optionally substituted aliphatic group, or a secondary amine, associated with two optionally substituted aliphatic group, where preferred optional substituents are one or more hydroxyl or amino groups most preferably, hydroxyl group.

Of course, any given organic amine selected as the preferred amine for use in the present invention may have the characteristics of two or more groups (a)to(C)described above (for example, to have the value of the PKandgreater than or equal to 7, and can be optionally substituted aliphatic amine described in paragraph (C)).

Any organic amine, defined in this way are suitable for use in compounds of the formula I according to the present invention, as well as in the present pharmaceutical compositions and methods. The concept of "organic amine" includes compounds in salt form with other acidic and/or basic fragments (where, for example, one amino group forms a phosphate salt of the formula I, and the other amino group forms a salt with the acidic residue). Therefore, the remainder of the organic amine, a part of the compound of the present invention, may also contain salt residues.

Examples of organic amines include, but are not limited to, tromethamine, diethanolamine, glucamine, N-methylglucamine, Ethylenediamine and 2-(4-imidazolyl)ethylamine.

"Mono-salt of an organic amine" phosphate combretastatin a-4 of the formula I contains one organic amino group Q as part of the substituents R1or R2as defined above; di-salt bodies is practical Amin" phosphate combretastatin a-4 of the formula I contains two organic amino group Q, one as part of a substituent R1and as part of a substituent R2as set forth above. Preferred are mono-salts of organic amines of formula I. Appropriate definitions apply to mono-salts of amino acids", "di-salt aminosilane", "mono-salt of the ester of the amino acids and di-salt of the ester of the amino acids".

Any suitable amino acid finds application in this case, including many natural and unnatural amino acids which are used in the connection of the present invention. Specific examples include, but certainly are not limited to, ornithine, histidine, lysine, arginine and tryptophan.

As used herein, the term "amino acid" refers to a compound containing a primary amino group (NH2) and an acidic carboxyl group (COOH), including such compounds in the form of zwitter-ion battery (where the amino and carboxyl groups together form zwitter-ion or internal salt) or in salt form with other acidic and/or basic residues (where, for example, the amino acid contains a carboxyl group, in addition to α-COOH group, and the first is in salt form with an alkali metal). Thus, the remainder of the amino acids located in the compound of the present invention, may also contain salt fragments. The definition includes the em unnatural, as well as natural amino acids, such as α-amino acids (in particular L-amino acids), many of which are the building blocks of proteins. The definition of "natural amino acid" refers to the 20 amino acids that are common to all proteins, i.e., glycine, alanine, valine, leucine, isoleucine, Proline, serine, threonine, cysteine, methionine, asparagine, glutamine, phenylalanine, tyrosine, tryptophan, lysine, arginine, histidine, aspartate and glutamate. The definition of "unnatural amino acids" refers to amino acids that are not common in proteins, such as 4-hydroxyproline, 5-hydroxylysine, N-methyllysine, γ-carboxyglutamate, selenocysteine, ornithine and citrulline. Amino acids containing two or more nitrogen atoms, are suitable for use in compounds of the formula I according to the present invention, when Q has a definition (In), as well as in the present pharmaceutical compositions and methods.

Used in this description in connection with the amino acid expression "side chain" means a balance of amino acids, which is different for each amino acid, and especially to the group associated with the carbon atom connecting-NH2and-COOH group of the amino acids.

Used in the description of the definition of "essentially nephroscope", in relation to the connection means preferably less than 1 increase the mass of water to mass of the compounds (more preferably less than 0.5%increase in the mass of water per mass of compound), measured under the following conditions: a temperature of about 25°C, relative humidity 20 to 95% and the equilibrium state (for example, measured when the rate of sorption and desorption of moisture aligned) relative to measurements made at 25°and at a relative humidity of 0%. As used in this description, the definition of "nephroscope", in relation to the connection means preferably not measurable increase in the mass of water to mass of compounds in the above-described conditions.

Used in this description with reference to the amino acid definition of "ether" refers to a carboxylic acid group (i.e., to the group-COOH) and amino acids, which is in the form-soo(G), where G represents an organic residue, such as unsubstituted or substituted alkyl, Alchemilla, Alchemilla, cycloalkyl, cycloalkenyl, aryl or heterocyclic group. Preferred as the groups G are C1-6is an alkyl group such as methyl or ethyl. Most preferred as the esters of amino acids are1-6-alkalemia esters of glycine, such as methyl ester of glycine or ethyl ester of glycine.

As used in this description, the definition of "Sol" refers to the compound of the present invention, which is an ionic compounds is s, for example, having the ionic bond between the Quaternary nitrogen atom of the fragment QH+organic amine, amino acid or ester of the amino acids and residual phosphate phosphate combretastatin A4.

Used in the description the term "ionic bond" is a chemical bond formed by electrostatic attraction between positive and negative ions or chemical structures. This relationship can easily dissociates (or ionize) in water solution. The dissolution of the compounds of the present invention in a solvent, in particular in aqueous solvent and lyophilization of this solution, are variants of implementation covered by the present invention.

Used in the present invention, the expression "physiologically acceptable" when describing chemical sample that is present in vivo, such as an organic amine, amino acid or ester of the amino acids refers to the inability of the chemical sample to induce side effects, unacceptable in terms of animal treatment. Preferred physiologically acceptable chemical samples do not cause harmful side effects.

As explained above, the present invention relates to a method of modulating growth or metastasis of tumors, preferably solid tumors, using the soedineniya of the present invention. As used in this description, the term "tumor" or "tumor growth" can be used interchangeably, and it refers to an abnormal growth of tissue resulting from uncontrolled progressive increase in the number of cells and not performing any physiological function. Solid tumors can be malignant, for example, have a tendency to give metastases and be life-threatening or benign. Examples of solid tumors that can be treated in accordance with the method of the present invention include sarcomas and carcinomas such as, but not limited by them: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, lymphoma, such as non-Hodgkin's lymphoma, osteogenic sarcoma, chordoma, tumors of the esophagus, angiosarcoma, osteosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangiectasia sarcoma, sinovioma, synovial sarcoma, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, carcinoma of the colon, colorectal cancer, gastric cancer, pancreatic cancer, lung cancer, such as metastatic lung cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, adenocarcinoma of the colon and rectum, cancer of the sweat glands, cancer of the sebaceous glands, papilionaceae, cystadenocarcinoma, medullary carcinoma, bronchogenic cancer, renal cell the cancer, hepatoma, liver metastasis, cancer of the bile ducts, cardiocaryon, seminoma, embryonal cancer, thyroid cancer, such as nedifferencirovannaja small cell lung cancer, thyroid cancer, medullary thyroid cancer, Wilms ' tumor, cervical cancer, testicular tumor, lung tumors, such as non-small cell lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

Some tumors, including deproliferation changes (such as metaplasia and dysplasia), can be treated or prevented using the compounds or method according to the present invention in epithelial tissues such as the cervix, esophagus, and lungs. Thus, the present invention provides treatment of conditions for which it is known or assumed progressive development of tumors or cancer, in particular, when forming tumor cell growth, including hyperplasia, metaplasia, and particularly, dysplasia (for exploring conditions such abnormal growth, see Robbins, Angell, 1976, Basic Pathalogy, 2d Ed., W.B. Saunders Co., Philadelphia, pp.68-79). Hyperplasia is a form of uncontrolled about what operacii cells, which includes growth in the number of cells in a tissue or organ without substantially changing the structure or functions. One example is endometrial hyperplasia, often precedes endometrial cancer. Metaplasia is a form of controlled cell growth, in which one type of Mature or fully differentiated cells is replaced by another type of Mature cells. Metaplasia may occur in cells of epithelial or connective tissue. Atypical metaplasia creates a somewhat messy metaplastic epithelium. Dysplasia is often a precursor to cancer and identified mainly in the epithelium; it is the most chaotic form which does not form tumor cell growth, which causes the loss of the individual cell information and the architectural orientation of cells. Dysplastic cells often have abnormally large, highly colored kernels, and show pleomorphism. Usually dysplasia occurs when there is chronic irritation or inflammation, and is often identified on the neck, respiratory tract, oral cavity and gall bladder. A review of such disorders, see, for example, Fishman et al., 1985, Medicine, 2d Ed., J.B.Lippincott Co., Philadelphia.

Other types of tumors that are benign and can be treated with the compounds and method is about the present invention, are arteriovenous (AV) malformations, in particular in intracranial sites, and myoleomas.

Compounds of the present invention can also be useful for treatment of malignant vascular proliferative diseases such as macular degeneration, psoriasis and restenosis, and in General in the treatment of inflammatory diseases characterized by vascular proliferation. Such diseases and disorders described in the publication WO 00/48606, which is put into the description by reference.

The pharmaceutical composition

The present invention also relates to pharmaceutical compositions comprising a compound of the present invention, described above, and a pharmaceutically acceptable carrier. The expression "pharmaceutically acceptable" refers to molecular objects and compositions that are physiologically acceptable and preferably prevent an allergic or similar undesirable reactions, such as gastrointestinal disorders, dizziness and so on, with the introduction of man. Preferably, as used herein, "pharmaceutically acceptable" means approved by the regulatory Agency of the Federal or National government or a listing in the U.S. Pharmacopoeia or other generally known Pharmacopoeia for use in animals and particularly in chelovekoobrazniye "carrier" refers to a diluent, adjuvant, filler and solvent, which introduces a connection. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including oils, petroleum, animal, vegetable and synthetic origin, such as peanut oil, soybean oil, sesame oil, alcohols such as ethanol, propanol, ethylene glycol, propylene glycol, sorbitol, glycerin and other, Cremophor and other, including mixtures thereof. Water, aqueous salt solutions and aqueous dextrose and glycerol are also preferably used as carriers, particularly as solutions for injection. Suitable pharmaceutical carriers are described in the publication "Remington''s Pharmaceutical Sciences", E.W.Martin.

The pharmaceutical composition according to the present invention can be designed for administration by injection, or for oral, pulmonary, nasal, transdermal, intraocular injection or other forms of injection. Typically in the present invention refers to pharmaceutical compositions containing an effective amount of the compounds of the present invention, for example, together with pharmaceutically acceptable diluents, preservatives, solubilizers agents, emulsifying agents, adjuvants and/or other media. Such compositions include diluents with different content is the use of buffers (such as, for example, TRIS and other amines, carbonates, phosphates, amino acids, such as hydrochloride glycinamide (especially at pH values in the physiological range), N-glycylglycine, sodium phosphate or potassium (dibasic, rejonowy), etc. or TRIS-HCl or acetate), with different values of pH or ionic strength; additives such as detergents and solubilizing agents (e.g., surface-active substances, such as Pluronics, Tween 20, Tween 80 (Polysorbate 80), Cremophor, polyols such as polyethylene glycol, propylene glycol and others), antioxidants (e.g. ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimersol, benzyl alcohol, parabens and other) or creating volume substances (for example, sugars such as sucrose, lactose, mannitol, polymers, such as polyvinyl-pyrrolidone or dextran, and other); and/or require the introduction of material into the formulation in the form of particles of polymeric compounds such as polylactic acid, polyglycolic acid, etc. or into liposomes. Hyaluronic acid can also be used. Such compounds can be used to influence the physical state, stability, rate of release in vivo and the rate of cleavage in vivo to the compounds of the present invention. See, for example, the publication "Remington's Pharmaceutical Sciences, 18th Ed., (1990, Mack Publishing Co., Easton, PA 18042), pp.1435-1712, which is included in the description as SS is the CTL. Compositions, for example, can be obtained in liquid form or can be in the form of a dry powder, such as lyophilized form. Specific methods of administration of such compositions are described below.

If necessary, the pH value can be adjusted. For example, to increase the solubility of the compounds of the present invention, it is preferable to bring the pH value of the pharmaceutical compositions containing these compounds, to a pH greater than 7, more preferably to a pH greater than 8 (for example, to 8.5).

If proletarienne salts SAR formula I according to the present invention the formation of the active parent compound (CA4) occurs at low pH values. The applicants of the present invention have found that the addition of a buffer/(agent, regulating pH) prevents the fall in pH during freezing, resulting in a more stable lyophilic the drug. Also unexpectedly found that in the case of lyophilic drugs proletarienne salts SAR, such as salts of formula I of the present invention, used for regulating the pH of sodium hydroxide as a pH-regulating agent can lead to the formation of the active parent compound (CIS) CA4 (that is minimally soluble in water and can form undesirable precipitates in aqueous solution) and that the application of the pH-regulating agent, different from the sodium hydroxide may reduce the formation of CA4. The use of TRIS as a pH-regulating agent and buffer reduces, for example, the formation of the active parent compound (CIS) CA4 in comparison with the use of sodium hydroxide as a pH-regulating agent and, therefore, especially preferred for use in compositions of the present invention. For example, in the case of compounds of formula I where Q is a TRIS or histidine, noted that the lyophilic medicines, obtained with the use of NaOH as a pH-regulating agent, when storing hydrolyzed to the original CIS-CA4, while for regulating the pH of a suitable buffering agent, other than NaOH, for example TRIS, reduces the formation of insoluble educt. Thus, in accordance with another aspect of the present invention relates to pharmaceutical freeze compositions of the present invention and a pH-regulating agent other than sodium hydroxide, preferably containing organic base, such as an amino acid or organic amine, in particular TRIS, as pH-regulating agent. Thus, although the use of sodium hydroxide in lyophilic compositions of the present invention is included in the scope of the invention, such use of m which it is preferable, for example, in the case of pharmaceutical compositions, which must be administered intravenously, when the formation of solids is undesirable.

Can be obtained pharmaceutical compositions, for example, the solutions (in particular aqueous solutions, for example, at a concentration of 15 mg/ml, 30 mg/ml and 60 mg/ml)containing the compound of the present invention and a pH-regulating agent, including sodium hydroxide, preferably containing organic base, such as an amino acid such as arginine, glycine, or an organic amine, such as ethanolamine, in particular TRIS, as pH-regulating agent. The stability of aqueous solutions increases with the pH of the drug from 9.0 to 10.5. The stability of the solution becomes higher at higher values of ionic strength. For example, the drug in the form of a solution with NaOH as a pH-regulating agent at pH 10 can exhibit stability, comparable to the stability of lyophilized obtained with TRIS as a buffer agent at pH 8.5.

In the case of compositions according to the present invention preferably uses protect from light.

Methods of modulating tumor growth or metastasis

Combretastatin A-4 is a very effective antimitoticescoe agent, obtained from a tree trunk Combretum caffrum, and shows high t is cicnet against a wide spectrum lines cancerous human cells. Therefore, the introduction of the compounds or pharmaceutical compositions of the present invention to a subject may reduce tumor growth and/or metastasis in a patient and, on the other hand, if the patient has no detectable metastasis or tumor growth, will prevent metastasis and/or tumor growth. Of course, the connection of the present invention may be introduced separately or together with a pharmaceutically acceptable carrier.

Therefore, the present invention relates to a method of modulating tumor growth or metastasis, which comprises introducing an effective amount of proletarienne mono - or di-salt of the organic amine phosphate combretastatin a-4 of the present invention, which is easily regenerates combretastatin a-4 in vivo. As mentioned above, the expression "effective amount", as used in the description refers to the number of compounds of the present invention, which introduced the subject and which is sufficient for modulating tumor growth or metastasis to reduce tumor growth and metastasis in an animal or, on the other hand, to prevent the formation of cancerous tumors in an animal, which before the introduction of the compounds of education tumors were not observed. Qualified in the field technician can easily determine the effective amount of the compound really is obreteniyu for introduction, for example, using conventional techniques.

In addition, in the method according to the present invention find application in numerous means of introducing the compounds of the present invention. In particular, the compound or pharmaceutical composition of the present invention may be introduced parenterally, through mucous membranes, such as oral, nasal or rectal, or transdermal. Preferably the introduction is carried out parenterally, e.g. by intravenous injection, as well as, including, but without limitation, by vnutriarterialnah, intramuscular, intradermal, subcutaneous, intraperitoneal, intraventricular and intracranial injection. The compound or pharmaceutical composition can be, for example, introduced by injection into the tumor(s) (s) Podwale(Yu)treatment, or in the tissue surrounding the tumor(s). The term "agent that increases the permeability of mucosal"refers to a reagent that increases the speed or the ability to penetrate through the mucous membranes of the compounds of the present invention, such as, but without limitation, a salt of a bile acid, fatty acid, surfactant or alcohol. In a specific embodiment of the invention, the agent that increases the permeability can be Halat sodium, sodium dodecyl sulphate, deoxycholate intothree is, taurodeoxycholate, glycocholate sodium, dimethyl sulfoxide or ethanol. Suitable agents that increase the permeability also include glycyrrhetinic acid (U.S. patent No. 5112804, Kowarski) and Polysorbate-80, the latter in combination with the nonionic surface-active agent, such as nonoxynol-9, Laureth-9, poloxamer-124, octoxynol-9 or lauramid-DEA (European patent EP 0242643 B1, Stoltz).

In another embodiment, the method of the present invention, the compound or pharmaceutical composition of the present invention can be delivered in the vesicles, especially in the liposome (see Langer, Science 249:1527-1533 (1990); Treat et al., Liposomes in therapy of infectious disease and cancer, Lopez-Berestein and Fidler (eds.), Liss: New York, pp.353-365 (1989); Lopez-Berestein, ibid. pp. 317-327; see generally ibid.).

In yet another embodiment of the present invention such a connection and such pharmaceutical composition can be delivered through a system of controlled release, for example, using intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration. In a special embodiment of the invention can be used pump [see Larger, see above; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)]. In another embodiment, can be used polymeric materials [see Medical Applicatins of Controlled Release, Langer and Wise (eds.), CRC Press: Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley: New York (1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983; see also Levy et al., Science 228:190 (1985); During et al., Ann Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105 (1989)]. In yet another embodiment of the invention, the system controlled release can be placed in close proximity to the target tissue of the subject, consuming, thus, only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, Vol. 2, pp. 115-138 (1984)]. Preferably the device with controlled-release is administered to the animal in close proximity to the site with inappropriate immune activation or tumor. Other systems with controlled drug release are discussed in the review Langer [Science 249:1527-1533 (1990)].

Parenteral

As mentioned above, the compound or pharmaceutical composition of the present invention can be introduced to the patient parenterally and, therefore, eliminates the introduction through the gastrointestinal tract. Specific methods of parenteral administration, which are used include, but without limitation, intravenous injection (through the introduction of a single loading dose or infusion), intraperitoneal injection, subcutaneous injection, intramuscular injection or catheterization, and are called only some of the which of them. Examples of compositions for parenteral administration are injectable solutions or suspensions which can contain, for example, suitable non-toxic acceptable for parenteral diluents or solvents, such as mannitol, 1,3-butanediol, water, buffered water system, ringer's solution, isotonic sodium chloride or other suitable dispersing or wetting and suspendresume agents, including synthetic mono - or diglycerides, and fatty acids, including oleic acid, alcohols and/or Cremophor. If necessary, you can adjust the pH value.

Nasal delivery

For compounds or compositions of the present invention also provides for nasal delivery or delivery via the mucous membranes. Nasal delivery allows such a connection to enter the blood stream directly after the introduction of the effective number of connections in the nose without the need for precipitation of the product in the lung. Medications for nasal delivery include drugs with dextran or a cyclodextrin, as well as with other polymers, such as polyvinylpyrrolidone, methyl cellulose or other cellulose.

In the case of nasal introduction of an acceptable device is a small, solid bottle, attached to the dosing dispenser. In one embodiment, measured to the and released by promoting compounds or pharmaceutical compositions of the present invention in a camera of a certain volume, which has a hole having dimensions such to release the aerosol formulation by forming a spray when the liquid in the chamber is compressed. The camera compresses to enter the compound or pharmaceutical composition. In a specific embodiment of the invention the chamber with a piston device. Such devices are commercially available.

On the other hand, can be used plastic crushed relentlessly vial having a hole or slit with dimensions to secure the release of the aerosol product. The formation of aerosol occurs when the squeeze bottle. The hole is usually located in the upper part of the bottle, and the top part is usually narrowed to enter the nasal passages to ensure the effective introduction of the aerosol product. Preferably, nasal aspirator will give a measured amount of the aerosol drug for introducing a metered dose of the compounds or pharmaceutical compositions of the present invention.

For release through the mucous also refers to the use of agents that increase the permeability.

Pulmonary delivery

Also refers to pulmonary delivery of a compound or pharmaceutical composition of the present invention. The compound or pharmaceutical composition for the present and the gain can be delivered to the lungs of a mammal while inhaling and pass through the inner epithelial layer of the lung into the bloodstream. Other messages on this topic include the publication Adjei et al., [Pharmaceutical Research, 7:565-569 (1990); Adjei et al., International Journal of Pharmaceutics, 63:135-144 (1990)(acetate leuprolide), Braquet et al., Journal of Cardiovascular Pharmacology, 13 (suppl. 5):143-146 (1989) (endothelin-1-); Hubbard et al., Annals of Internal Medicine, Vol.III, pp.206-212 (1989) (α1-antitripsin); Smith et al., J. Clin. Invest., 84:1145-1146 (1989) (α-1-proteinase); Oswein et al., "Aerosolization of Proteins", Proceedings of Symposium on Respiratory Drug Delivery II, Keystone, Colorado, March (1990) (recombinant growth factor, human); Debs et al., J. Immunol. 140:3482-3488 (1988) (γ-interferon and tumor necrosis factor), Platz et al., U.S. patent No. 5284656 (factor stimulating the growth of colonies of granulocytes)]. Method and composition for pulmonary delivery of drugs for systemic effects is described in U.S. patent No. 5451569, issued September 19, 1995 (Wong et al.).

In the practice of the present invention assumes the use of a wide range of mechanical devices, for pulmonary delivery of therapeutic products, including, but without limitation, dispensers, dosing inhalers, and powder inhalers, all of which are well-known qualified in this area specialists. With regard to the design of the device for delivery, in the practice of the present invention can be used in any form of aerosols, known in this field, including, but without limitation, spray bottles, spraying, dispersing, or the creation of the Aer is Zola with a pump of liquid compositions as well as the dispersion of the dry powder drug.

Some specific examples of commercially available devices that are acceptable in the practice of the present invention, include the Ultravent nebulizer, manufactured Mallinckrodt, Inc., St. Louis, Missouri; spray Acorn II, manufactured by Marquest Medical Products, Englewood, Colorado; dosing Ventolin inhaler, manufactured by Glaxo Inc., Research Triangle Park, North Carolina; and powder inhaler Spinhaler manufactured by Fisons Corp., Bedford, Massachusetts.

Such devices require the use of drugs suitable for dispersion of the pharmaceutical composition of the present invention. Usually this drug is specific for the type of device and may include the use of a suitable propellant, in addition to the usual diluents, adjuvants and/or other media which are used in therapy. Also refers to the use of liposomes, microcapsules or microspheres, inclusion complexes, or other types of media. Can also be obtained by chemically modified pharmaceutical composition of the present invention in various preparations depending on the type of chemical modification or the type of device.

Drugs suitable for use in the spray or jet or ultrasonic, will typically include connection pharmaceutical compositions of this is the invention, dissolved in water at a concentration of from about 0.1 to 25 mg of biologically active ingredients per ml of solution. The drug may include a buffer or a simple sugar (for example, to stabilize or regulate the osmotic pressure of the pharmaceutical compositions of the present invention). Preparation for spraying may also contain a surfactant, to reduce or eliminate the surface, inducing the formation of aggregates of the compounds or pharmaceutical compositions of the present invention, caused by atomization of the solution in forming the aerosol.

Preparations for use in the dosing inhaler will usually contain, for example, finely ground powder containing the compound or pharmaceutical composition of the present invention, suspended in propellant using surfactants. Propellant may be any suitable material used for these purposes, such as chlorofluorocarbons, HCFC, ftoruglevodorodnye or a hydrocarbon, including Trichlorofluoromethane, DICHLORODIFLUOROMETHANE, dichlorotetrafluoroethane and 1,1,1,2-Tetrafluoroethane, or combinations thereof. Suitable surfactants are sarbatorile and soy lecithin. Oleic acid can also be used as a stand is Resto-active substances.

Liquid aerosol preparations containing the compound or pharmaceutical composition of the present invention and a dispersing agent in a physiologically acceptable diluent. Dry powder aerosol preparations may contain finely dispersed solid form of the compound or pharmaceutical composition of the present invention and a dispersing agent. Regardless of whether the drug is a liquid or dry powder aerosol drug, it should be sprayed. That is, it must be partitioned to a liquid or solid particles, to ensure that sprayed dose actually reaches the mucous membranes of the nasal passages or lungs. The definition of "aerosol particle" is used here to describe the liquid and solid particles suitable for nasal or pulmonary administration, that is, who will reach the mucous membranes. Other noteworthy issues are the design of the device for delivery of additional components in the formulation and characteristics of the particles. These aspects of the nasal or pulmonary administration of medication is also well known in the field and handling of medicines, retrieve aerosol and designs of the delivery devices can be easily implemented qualified in this area specialists.

In concr the coherent version of the invention, the bulk dynamic diameter is 5 μm or less, to ensure that the particles of the drugs reach the pulmonary alveoli (Wearley L.L., 1991, 1991, Crit. Rev. in Ther. Drug Carrier Systems, 8:333).

As mentioned above, in a particular embodiment of the invention the device for spraying is a dosing inhaler. Dosing inhaler delivers a specific dose in the application, and not different doses depending on the application. Such dosing inhaler can be used either with a liquid or dry powder aerosol drug. Dosing inhalers are also well known in this field.

Often the spraying of liquid or dry powder preparation for inhalation into the lungs requires the use of propellant. Propellant may be any suitable material commonly used in this field. Specific non-limiting examples of such useful propellants are chlorofluorocarbons, HCFC, ftoruglevodorodnye or a hydrocarbon, including Trichlorofluoromethane, DICHLORODIFLUOROMETHANE, dichlorotetrafluoroethane and 1,1,1,2-Tetrafluoroethane, or combinations thereof. The system of delivery of aerosols, such as working under pressure dosing inhalers and inhalers dry powder described in the publication S.P. Newman, Aerosols and the Lung, Clarke S.W., Davia D., editors, pp. 197-22, and they can be used in accordance with the present invention.

Liquid aeroso the performance communications drugs

The present invention provides a liquid aerosol medications and dosage forms of the compounds or pharmaceutical compositions of the present invention. In General such dosage forms contain the compound or pharmaceutical composition of the present invention in a pharmaceutically acceptable diluent. Pharmaceutically acceptable diluents include, but are not limited to, sterile water, saline, buffered saline, dextrose, etc. In a specific embodiment of the invention the diluent, which can be used in the present invention or the pharmaceutical preparation of the present invention, is a saline solution with a phosphate buffer or a buffered saline solution usually with a pH of 7.0 to 8.0 or water.

Liquid aerosol preparation of the present invention may include, as optional ingredients, pharmaceutically acceptable carriers, diluents, solubilizing or emulsifying agents, surfactants and fillers.

Drugs of this version of the invention can also include other agents that are useful for maintaining the pH of the stabilizing solution or regulation of osmotic pressure. Examples of such agents include, but are not ogranichivayutsya only them, salts such as sodium chloride or potassium chloride, and carbohydrates such as glucose, galactose or mannose, and other

Aerosol dry powder drugs

It also assumes that the aerosol product can be obtained in the form of a dry powder preparation containing finely powdered form compounds or pharmaceutical compositions of the present invention and dispersant.

Preparations for the dispersion of the device for inhalation of powder may contain finely ground dry powder containing the compound or pharmaceutical composition of the present invention, and may also include creating volume agent, such as lactose, sorbitol, sucrose, or mannitol in amounts which facilitate dispersal of the powder from the device, for example, from 50 to 90% of the mass. based on the drug. The compound or pharmaceutical composition of the present invention should preferably be obtained in the form of particles with an average particle size less than 10 microns, most preferably from 0.5 to 5 μm, for most effective delivery to remote areas of the lung.

In another embodiment, the invention is a dry powder preparation may contain finely ground dry powder containing the compound or pharmaceutical composition according to the present izobreteny is, dispersing agent and creates volume of the agent. Creating the amount of agents that can be used in combination with this composition include such agents as lactose, sorbitol, sucrose, or mannitol in amounts which facilitate dispersal of the powder from the device.

Transdermal introduction

In this area there are various and diverse ways transdermal injection of a medicinal product, for example, using transdermal patches. Transdermal patches are described, for example, in U.S. patent No. 5407713, issued April 18, 1995 Rolando et al.; U.S. patent No. 5352456, issued October 4, 1994 Fallon et al.; U.S. patent No. 5332213, issued August 9, 1994 D'angelo et al.; U.S. patent No. 5336168, issued August 9, 1994 Sibalis; U.S. patent No. 5290561, issued March 1, 1994 Farhadieh et al.; U.S. patent No. 5254346, issued October 19, 1993 Tucker et al.; U.S. patent No. 5164189, issued November 17, 1992 Berger et al.; U.S. patent No. 5163899, issued November 17, 1992 Sibalis; U.S. patent No. 5088977 and 5087240, issued February 18, 1992 Sibalis; U.S. patent No. 5008110, issued April 16, 1991 Benecke et al.; and U.S. patent No. 4921475, issued may 1, 1990 Sibalis; a description of each of the patents introduced entirely by reference.

You can easily notice that the transdermal route of administration may be improved by agent, reinforcing the dermal permeability, such reinforcing agents, which are described in the patent is U.S. No. 5164189 (see above), U.S. patent No. 5008110 (see above) and U.S. patent No. 4879119 (see above), issued November 7, 1989 Aruga et al., moreover, the description of each of the patents is incorporated as references.

Moreover, compound and pharmaceutical composition of the present invention can be introduced locally. For example, the connection may be mixed with the ointment vehicle with obtaining compositions, which can be rubbed into the skin. On the other hand, the compound of the present invention may be dissolved in the solvent, which is known as a well penetrating through the skin. A specific example of such a solvent is dimethyl sulfoxide (DMSO). It also assumes a gel preparations.

Oral delivery

In this case, means the use of oral solid dosage forms, which are described in the publication Remington''s Pharmaceutical Sciences, 18-th Ed., 1990 (Mack Publishing Co., Easton PA 18042), Chapter 89, which is incorporated in this description by reference. Solid dosage forms include, for example, tablets, capsules, pills, lozenges, or cakes, wafers or pellets. In addition, for preparation of the compounds or pharmaceutical compositions according to the present invention can be used liposomal or proteinoid encapsulation (as, for example, proteinoid microspheres, presented the in U.S. patent No. 4925673). Can be used liposomal encapsulation, and liposomes can be derivationally different polymers (see, for example, U.S. patent No. 5013556). Description possible dosage forms for therapeutic applications described in the publication: Marshall K. "Modern Pharmaceutical", Ed. G.S. Banker, C.T. Rhodes Chapter 10, 1979, which entered into the description by reference. The preparation may contain a compound or pharmaceutical composition of the present invention and inert ingredients that provide protection against the stomach environment, and release of the biologically active material in the intestine.

Also refers to oral dosage forms of the compounds of the present invention, in which the connection can be chemically modified so that oral delivery of the derivative was effective. Usually considered chemical modification is a connection, at least one fragment of the molecule, where the fragment provides (a) inhibition of proteolysis; and (b) uptake into the blood stream from the stomach or intestines. Also preferably increase the overall stability of the compounds of the present invention and an increase in circulation time in the body. Examples of such fragments include: polyethylene glycol, copolymers of ethylene glycol and propylene glycol, the carb is climatically, dextran, polyvinyl alcohol, polyvinylpyrrolidone and polyproline (Abuchowski and Davis, 1981, "Soluble Polymer-Enzyme Adducts", Enzymes as Drugs, Hocenberg and Roberts, eds., Wiley-Interscience, New York, NY, pp.367-383; Newmark, et al., 1982, J. Appl. Biochem., 4:185-189. Other polymers that may be used are poly-1,3-dioxolane and poly-1,3,6-dioxolan. Preferred for the aforementioned pharmaceutical applications are polietilenglikolya fragments.

For compounds of the present invention the area of the release may be the stomach, small intestine (duodenum, small intestine, or the ileum) or the large intestine. Qualified in this field specialist can easily make drugs that will not dissolve in the stomach, but, nevertheless, will release the connection of the present invention in the duodenum or in any place of the intestine. Preferably this release will eliminate the negative effects stomach environment, either by protection of the connection or result in the release of biologically active material with lower gastrointestinal environment, for example, in the gut.

To ensure full stomach sustainability is important coverage, impervious, at least at pH 5.0. Examples of the more common inert ingredients that can be used as enteric coatings are what I trimellitate cellulose acetate (TAC), phthalate of hydroxypropylmethylcellulose (GPMC), GPMC 50, GPMC 55, polyvinyl acetate phthalate (FCA), Eudragit L30D, Aquateric, phthalate cellulose acetate (FAC), Eudragit L, Eudragit S, and Shellac. Such coatings can also be used in the form of mixed films.

The tablets can also be used for coating or mixture of coatings, which are not intended for protection against gastric environment. They may include a coating of sugar or coatings that make tablets more legkoperevarivaemye. Capsules may consist of a hard shell (such as gelatin shell) for the dry release a therapeutic agent, such as a powder; in the case of liquid forms can be used in soft gelatin shell. The sheath material of the wafers can be a thick starch or other food paper. In the case of pills, cakes, molded tablets or powdered tablets can be used in methods of humidification of the.

Compounds of the present invention, for example, can be included in the preparation is in the form of finely ground multicystic in the form of granules or pellets with a particle size of approximately 1 mm, the Preparation of material for introduction into the capsules can also be in the form of a powder, slightly compressed mass or even in pill form. In addition, the compound or pharmaceutical composition for the present and the gain can be obtained by pressing.

Can also be used dyes and substances corrective taste and smell. For example, the connection may be prepared in the form of the drug (for example, by encapsulation in liposomes or microspheres)and then further introduced into the food product, such as a refrigerated beverage containing colorants and flavoring.

You can dilute or increase the volume of the compounds of the present invention or a pharmaceutical composition with an inert material. Such diluents may include carbohydrates, especially mannitol, α-lactose, anhydrous lactose, cellulose, sucrose, modified dextrans and starch. Some inorganic salts can also be used as fillers, including triphosphate calcium, magnesium carbonate and sodium chloride. Some commercially available diluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress and Avicell.

In the preparation of pharmaceutical compositions of the present invention in solid dosage form can be included leavening agents. The materials used as leavening agents, represent, but are not limited to, starch, including commercial baking powder, based on starch, Explotab. Can be used the sodium salt of glycolate starch, Amberlite, sodium carboxymethyl cellulose, ultraprobe the tin, sodium alginate, gelatin, orange peel, acid carboxymethyl cellulose, natural sponge and bentonite. Another form of leavening agents are insoluble cation exchange resin. As leavening agents and binders can be used powdered gum, and they include powdered gums such as agar, Karaya or tragakant. Also, as baking powder can be used alginic acid and its sodium salt.

Binders can be used to hold the compounds or pharmaceutical compositions of the present invention together in the form of a hard tablet and include materials from natural products such as Arabian gum, tragakant, starch and gelatin. Other examples are methyl cellulose (MC), ethylcellulose (EC) and carboxymethyl-cellulose (CMC). Polyvinylpyrrolidone (PVP) and hydroxypropyl-methylcellulose (HPMC) can be used in alcoholic solutions for granulating the drug.

To prevent sticking in the process of obtaining the drug in the preparation of pharmaceutical compositions of the present invention may be added to the agent that reduces friction. Lubricants can be used as a layer between the medicinal product and the walls of the die, and they may include, but without limitation, stearine the second acid, including its magnesium and potassium salts, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and waxes. Can also be used soluble lubricants such as sodium lauryl sulfate, lauryl sulfate, magnesium, polyethylene glycols with different molecular weights, Carbowax 4000 and 6000.

Can be added to the agents contributing to the slide, which can improve the fluid characteristics of the compounds of the present invention upon receipt of the product and contribute to the redistribution in the process of pressing. Contributing slip agents may include starch, talc, pyrogenic silica and hydrated aluminosilicate.

Supplements that effectively increase the absorption of the compounds of the present invention, administered orally, are, for example, fatty acids, oleic acid, linoleic acid and linolenic acid.

May be desirable oral drugs controlled release. Medication may be introduced into an inert matrix which provides the release or due to the diffusion or through the mechanism of leaching, for example, into the gums. The drug may also be introduced slowly disintegrating matrix. Some intersolubility coatings also have a slow release effect.

Other forms of con the controlled release of the compounds of the present invention lie in the way, based on the Oros therapeutic system (Alza Corp.), that is, the drug is enclosed in a semipermeable membrane which allows water to enter and push the medication through a single small opening due to osmotic effects.

The drug can be used with other coatings. These include a number of sugars that can be put in the pan to coat. The compound of the present invention can also be obtained in the form of film-coated tablets, and used in this case, the materials can be separated, for example, into two groups. The first group consists of not intersolubility materials, and it includes methylcellulose, ethylcellulose, hydroxyethyl cellulose, methylhydroxyethylcellulose, hydroxypropylcellulose, hypromellose, sodium carboxymethyl cellulose, provide and glycols. The second group includes intersolubility materials, which are typically esters of phthalic acid.

For optimum coverage, you must use a mixture of materials. Applying a film coating may be carried out in a machine for coating pan or fluidized bed or by coating under pressure.

Ways to get

The compounds of formula I, described above, can be obtained Luba the appropriate way for example, when contacting the desired compound Q (especially organic amine, amino acid or ester of the amino acids) with a free acid phosphate combretastatin a-4 (free acid SAR"), which has the structure:

in respective amounts sufficient to obtain a mono - or di-salt of an organic amine, mono - or di-salt of the amino or mono - or di-salt of the ester of the amino acids of the formula I according to the present invention (for example, when the molar ratio of 1:1 to obtain a mono-salt of an organic amine or mono-salt of an amino acid, or when a molar excess of an organic amine in a suitable solvent (for example, in a solvent selected according to the desired pKa) to obtain the di-salt of an organic amine). The free acid SAR can be obtained from the disodium salt SAR, for example, as described below in the examples. Connection Q, such as an organic amine or amino acid and free acid CAR may be, for example, was put into contact in a suitable solvent (preferably in C1-C6-alcohol, such as isopropanol, or their aqueous mixtures), then preferably should collect the compounds of formula I in the form of crystalline compounds, for example by filtration. The definition of "solvent" includes a single solvent, a mixture of two or more solution of the residents with the formation of mixed or biphasic mixtures of solvents. If desirable, the connection Q can be added in the form of salts, preferably pharmaceutically acceptable salts, as described below in the examples.

Thus, the present invention includes a method of obtaining compounds having the General structure formula I:

where one of the substituents OR1or2represents-O-QH+and the other represents a hydroxyl or-O-QH+; and

Q represents

(A) an organic amine containing at least one nitrogen atom which, together with a proton, forms a Quaternary ammonium cation QH+;

(C) an amino acid containing at least two nitrogen atom, where one of the nitrogen atoms, together with a proton, forms a Quaternary ammonium cation QH+; or

(C) an amino acid containing one or more nitrogen atoms where one of the nitrogen atoms, together with a proton, forms a Quaternary ammonium cation QH+and where, in addition, all carboxyl groups of the amino acids are in the form of esters.

This method according to the present invention involves the step of contacting in a solvent free acid CAR having the structure:

connection Q, where Q is a

(A) an organic amine containing at least one nitrogen atom, which is together with a proton, forms a Quaternary ammonium cation QH +;

(C) an amino acid containing at least two nitrogen atom, where one of the nitrogen atoms, together with a proton, forms a Quaternary ammonium cation QH+; or

(C) an amino acid containing one or more nitrogen atoms where one of the nitrogen atoms, together with a proton, forms a Quaternary ammonium cation QH+and where, in addition, all carboxyl groups of the amino acids are in the form of esters.

Optional connection of the present invention obtained by the method described in the document, you may fall out of the solvent to precipitate in crystalline form.

In addition, the present invention encompasses a method of obtaining compounds having the General structure formula I:

as described above, and this method comprises a stage of contacting the free acid CAR preferred connectivity Q (such as histidine,1-6-alkilany ester of glycine, or most preferably tromethamine) in the solvent and the subsequent gathering of the solvent obtained in crystalline salt form CAR with histidine,1-6-alkylbis ester of glycine or most preferably with tromethamine. Of course, as explained above, in the method according to the present invention is used a number of solvents. Specific examples include, but are not limited to, t is like them With1-6-alcohols, such as isopropanol, or their aqueous mixtures. In a preferred embodiment of the present invention described method gives mono-tromethamine salt SAR (mono-salt of TRIS CAR or mono-TRIS salt SAR). In another preferred embodiment of the present invention described method provides mono-L-his-tag salt CAR.

Mixture of compound Q (organic amine, amino acid or ester of the amino acid and free acid SAR, preferably in solution, for example in aqueous solution, are also considered as embodiments of the invention. Therefore, the present invention additionally provides a composition obtained by mixing compounds, including:

(a) free acid CAR having the structure:

(b) compound Q, where Q is a

(A) an organic amine containing at least one nitrogen atom which, together with a proton, forms a Quaternary ammonium cation QH+;

(C) an amino acid containing at least two nitrogen atom, where one of the nitrogen atoms, together with a proton, forms a Quaternary ammonium cation QH+; or

(C) an amino acid containing one or more nitrogen atoms where one of the nitrogen atoms, together with a proton, forms a Quaternary ammonium cation QH+and where, in addition, the CE carboxyl group of the amino acids are in the form of esters.

Optional composition of the present invention also includes pharmaceutically acceptable carrier.

The present invention will become more clear upon reference to the following not limiting examples, which are the illustration of the invention. The following examples are presented for a more complete explanation of the preferred embodiments of the invention. They should not in any way be considered as limiting the invention.

Example 1

Proletarienne mono-trometamina salt SAR

In this embodiment, the present invention is described proletarienne mono-trometamina salt SAR as a non-limiting example, the compounds of the present invention. Using the document information, qualified in this field specialist can easily get various proletarienne mono - or di-salts of organic amines CAR, for example, by adding the desired organic amine to the free acid CAR according to the method similar to that described below, and all that is required in this invention and the attached claims.

Reagents and methods

All reagents and chemicals obtained from commercial sources and used without additional purification: Tris(hydroxymethyl)aminomethan (TRIS, TRIS) (ldrich Chemical Co. category 99.9 +%, batch # 01404PU), isopropyl alcohol (grade B&J, a solvent of high purity). Multi-NMR spectra are recorded on a spectrometer Bruker DRX 400. Chemical shifts1H and13Given in ppm relative to tetramethylsilane. (Chemical shifts NMR13To determine, using methanol (Meon) as an external standard). The NMR spectra of13Recorded with isolation of protons {1N}. Experiments on two-dimensional NMR HMQC [spectroscopy heteronuclear multiplet quantum correlation, inverse correlation between chemical shift to determine which of the1N molecules associated with any kernel13(Or other kernel X)]; and HMBC [spectroscopy heteronuclear multiplet correlation relationships, modification HMQC used to detect distant interactions1H13C, and to determine the structure and classification1H and13With in the molecule] spend for the assignment of NMR signals1H and13With the structure. "The disodium salt of CAR" is a compound with the following structure:

(see above-mentioned U.S. patent No. 5561122).

Mono-trometamina salt SAR

An aqueous solution of isopropyl alcohol-TRIS (0,19 M). 0,19 M TRIS solution prepared by dissolving of 1.61 g of TRIS (13.3 mmol) in 7 ml of deionized water and the resulting solution on billaut 63 ml of isopropyl alcohol (IPA) (10% water in IPA).

The original solution of the free acid CAR in isopropyl alcohol (0,19 M). The disodium salt of SAR (12,15 g, 27.6 mmol) was dissolved in 70 ml of deionized water. To the resulting solution with rapid stirring in ethyl acetate (250 ml) and saturated aqueous sodium chloride solution (150 ml). A white precipitate is formed. Portions add 0,5 N. hydrochloric acid (325 ml) to dissolve the precipitate (final pH value of the aqueous phase is approximately 1 on the indicator paper). The organic phase is separated. The aqueous phase is extracted with ethyl acetate (CH ml). The organic phases are combined and dried with anhydrous Na2SO4. After filtration and evaporation of the solvent (rotary evaporator, the temperature of the water bath is 40° (C) receive a thick film free acid SAR, which is dissolved in 100 ml of isopropyl alcohol. The concentration of the resulting solution, which should be 0,19 M, determined by NMR1N. Use the following methodology titration: using a pipette into a vessel for HPLC with a volume of 4 ml contribute 45 μl of a solution of L-histidine (0.17 M) and 30 μl of a solution of the free acid CAR. The solvents are evaporated to dryness on a rotary evaporator. The solid is dissolved in 0.7 ml of D2O and analyzed by NMR1N, get the ratio of histidine to the free acid SAR 1:0.75 in. Just get a 19 mmol of the free acid SER (yield 69%).

Mo is about-TRIS salt CAR. In a round bottom flask with a volume of 200 ml load 70 ml of a solution of the free acid SAR obtained above (0,19 M, 13.3 mmol). With vigorous stirring to a solution of the free acid CAR portions add an aqueous solution of isopropyl alcohol-TRIS prepared above (0,19 M, 13.3 mmol). The resulting suspension is stirred at room temperature for 18 h (overnight), then cooled to 0°C (ice bath) for 30 minutes Crystalline solid is filtered off with suction through filter paper Whatman #54, washed with cold isopropyl alcohol and dried in a stream of air for 5 hours, then in a vacuum (vacuum desiccator) for 113 hours, get 7,01 g mono-TRIS salt SAR in the form of a white solid. The results of the analysis by NMR1N. show that the final product contains isopropyl alcohol (approximately 0.9% wt.) as residual solvent (a 13.4 mmol, quantitative yield). Mono-TRIS salt CAR has the structure of formula Ia, in which the olefin group linking the phenyl fragments, is isconfigurable, as in the original connection - phosphate combretastatin a-4.

Characterization of mono-TRIS salt SAR

Analysis of NMR and elemental analysis

1H NMR (400 MHz, D2O) δ to 3.52 (s, 6H, C(15)H3and C(17)H3), of 3.56 (s, 6H, C(20)H2C(21)H2C(22)H2)and 3.59 (s, 3H, C(16)H3), to 3.67 (s, 3H, C(18)H3), 6,38 (d, J=11.7 Hz, H, C(8)H), 6,46 (d, J=11.7 Hz, 1H, C(7)H), 6.48 in (s, 2H, C(10)H and C(14)H), 6,79 (d, J=8,8 Hz, 1H, C(3)H), 6,85 (userd, J=8,8 Hz, 1H, C(4)H), 7,06 (users, 1H, C(6)H);13C NMR (100 MHz, {1N}, D2O) δ 56,9 (2C, C15, C17), 57,0 (CIS)60,3 (3, C20, C21, C22), 62,0 (C16), 62,4 (C19), 107,5 (2C, C10, C14), 113,9 (C3), 122,6 (l, JPC=3.1 Hz, C6), 125,9 (C4), 130,0 (C8), 130, 8mm (C7), 131,2 (C5), 134,7 (C9), 136,8 (C12), 142,2 (l, JPC=7,7 Hz, C1), to 150.6 (d, Jpc=6,1 Hz, C2), 153,2 (2C, Cl1 and C13), Calculated for C22H32NO11P: S, 51,06; H, 6,23; N,2,70; P, 5,98, Found: C, 51,07; H, To 6.39; N, 2,58; P, 5,93.

Hygroscopicity

Mono-TRIS salt SAR this example, as it turns out, is almost non-hygroscopic at 25°at room conditions and in conditions of high humidity. This result is unexpected, since other salt forms, free acid CAR hygroscopic under the same conditions. Schedule sorption moisture mono-TRIS salt SAR is shown in figure 1. The x-ray diffraction at different relative humidity ("RH-XRD) indicates that the powder diffraction pattern remains unchanged during curing at different relative humidities at 25°C.

Polymorphism

The study of single crystal mono-TRIS salt SAR the sample using x-ray diffraction analysis shows that the salt is an achiral compact form (N-1), which does not contain any areas with solvent, and Thu is she has a centrally symmetric monoclinic crystal structure. The simulated diffractogram of the powder, which is obtained from the refined atomic parameters of monoclinic single crystal at room temperature, consistent with the observed diffraction pattern. Several portions of mono-TRIS salt, obtained from a mixture of isopropyl alcohol/water, as it turned out, according to NMR1N., differential scanning calorimetry (DSC), thermogravimetry (TG) and x-ray diffraction the powder x-ray phase analysis XRD) was reproducible. Mono-TRIS salt SAR suspended in various solvents such as ethanol, isopropanol, acetone, acetonitrile and water, at 70-75°C for 5-10 min and then at room temperature overnight. The obtained solid phase analyzed by means of DSC, TG and XRD. None of the suspended samples not detected the presence of a solvent or in suspension or in dry form. In thermograms of DSC and waxs diffractograms (see Figure 2) is not detected difference of the samples compared to the substance as such. This shows that N-1 form is relatively stable, the only polymorphic form.

Other physico-chemical properties

The DSC thermogram of mono-TRIS salt CAR there is a single endotherm melting at 196°With (Figure 3). Thermogravimetric analysis shows no weight loss due to evaporation at a temperature below 150 C. the Equilibrium solubility of mono-TRIS salt SAR in water is 3.37 mg/ml (pH of 4.8) at 25°C. the solubility in water increases with increasing pH and reaches a value of 191 mg/ml at pH of 8.2. This property is particularly suitable for the preparation of dosage forms of the compounds of the present example in the range of pH 8-9 for intravenous injection (including but not limited to, solutions, directly enter the patient ("Ready-to-use solutions"), or boot solution for lyophilization). A graph of the solubility of the mono-TRIS salt SAR shown in Figure 4. Mono-TRIS salt also exhibits good chemical stability in the solid state during curing at ambient conditions and under more severe conditions of temperature and humidity.

Mono-TRIS salt SAR of this example has excellent physical and chemical properties for use in pharmaceutical formulations, for example, intended or for oral or parenteral administration. Although other salt forms SAR not considered here, mono-TRIS salt has unexpectedly beautiful properties in the solid state, in particular nephroscopes. This is particularly unexpected from the point of view of the degree of water solubility of the TRIS.

Mono-trometamina salt SAR (large-scale transition)

The solution of the free acid CAR the IPS. Three-neck round bottom flask with a volume of 12 l provide a mechanical stirrer and addition funnel. Into the flask was placed a solution disodium salt SAR (99,92 g, 0,227 mol) in 1.5 l of deionized water, and the flask was added ethyl acetate (2.0 l). With rapid stirring from a dropping funnel add a solution of hydrochloric acid (0.5 n, 950 ml, 0.475 mol, the final pH value of the aqueous phase is approximately 1 on the indicator paper). The organic phase is separated. The aqueous phase is extracted with ethyl acetate (5×1.6 l). The combined organic phases are dried with Na2SO4. The ethyl acetate evaporated on a rotary evaporator to yield a thick oil which was dissolved in isopropyl alcohol (800 ml).

Mono-TRIS salt CAR. In a three-neck round bottom flask with a volume of 12 liters was placed a solution of TRIS (25,0 g, 0,206 mol) in 800 ml of deionized water. With vigorous stirring via an addition funnel is added slowly cooked above solution of the free acid CAR in the IPS. After adding the resulting solution is brought priming mono-TRIS salt SAR and stirred with a mechanical stirrer at room temperature for 1 hour. Then to the suspension slowly add a further quantity of isopropyl alcohol (2.0 l) and stirring is continued for another hour. The crystalline solid is filtered off with suction, washed with isopropyl alcohol (800 ml) and dried in a vacuum oven at about 40°t is the significance of 4 days get 101,55 g mono-TRIS salt CAR. The results of the analysis by NMR1N. show that the final product contains isopropyl alcohol (about 0.4% wt.) as residual solvent (0,196 mol, the total yield of 86%). Elemental analysis for C22H32NO11P. Calculated, %: 51,06, N 6,23, 2,70 N, P 5,98. Found: 50,95, N 6,14, N 2,69, R Of 5.82.

Example 2

TRIS-Drug proletarienne mono-tromethamine salt SAR

Pharmaceutical compositions in the form of an aqueous solution and freeze-dried (i.e. dried by freezing) of the compound of example 1 (mono-TRIS salt SAR) was obtained as follows.

An aqueous solution of the compound of example 1 is obtained by dissolving the compounds in water for injection (USP) to a concentration of 60 mg/ml with the addition of TRIS (tromethamine in the form of a base in a quantity sufficient to obtain a pH of 8.5. The dissolution is carried out under protection from light.

Then get the lyophilisate according to the following procedure. The above solution is filtered through a suitable sterilizing filter 0.2 micron, and aliquots placed in sterile glass vials. Solutions lyophilizer in liofilizadora Virtis at -35°C in high vacuum for 24 to 72 hours and then further dried at 5°C in high vacuum for 24 to 48 hours, get the lyophilisate.

In accordance with these methods can be obtained from other FA the pharmaceutical composition, containing the compound of example 1. For example, as shown previously, the above-described solution can be added to create the volume of agents (for example, amino acids such as arginine or lysine, sugars such as sucrose, lactose, mannitol, polymers, such as polyvinylpyrrolidone or dextran, and the like) or other excipients.

For example, get water solution of the compound of example 1 by dissolving the compounds in water for injection (USP) to a concentration of 30 mg/ml with the addition of creating a suitable amount of an agent, such as mannitol, dextran, or combinations thereof, to a concentration of 100 mg/ml) and TRIS (tromethamine in the form of a base in a quantity sufficient to obtain a pH of 8.6. The dissolution is carried out under protection from light.

Then get the lyophilisate according to the following procedure. The above solution is filtered through a suitable sterilizing filter 0.2 micron, and aliquots placed in sterile glass vials. Solutions lyophilizer in liofilizadora Virtis at -10°in moderate vacuum for 24 to 72 hours and then further dried at 5°C in high vacuum for 24 to 48 hours, get the lyophilisate.

As described above, any suitable organic amines, including, but not limited to, diethanolamine, glucamine, N-methylglucamine, Ethylenediamine and 2-(4-imidazolyl)ethylamine can easily replace trometamin in the examples described above to obtain compounds or compositions of the present invention. Can be similarly obtained composition, in which Q is other definitions within formula I.

Example 3

Proletarienne mono-L-his-tag salt SAR

In this embodiment, the present invention is described proletarienne mono-L-his-tag salt SAR as a non-limiting example, the compounds of the present invention. Using the description information, qualified in this field specialist can easily get various proletarienne mono - or di-salts of amino acids SER, for example, by adding the required amino acid to the free acid CAR according to the method similar to that described below, and all that is required in the present invention and the attached claims.

Reagents and methods

All reagents and chemicals obtained from commercial sources and used without additional purification: L-histidine (Aldrich Chemical Co. category 98%, batch # 04821JR), methanol and isopropyl alcohol (grade B&J, a solvent of high purity). "The disodium salt of CAR" is a compound having the formula:

(see above-mentioned U.S. patent No. 5561122).

Multi-NMR spectra are recorded on a spectrometer Bruker DPX 300 and DRX 400. Chemical shifts in NMR spectra1H and13Given in ppm from siteline tetramethylsilane (chemical shifts NMR 13With determined with the use of methanol as an external standard). Chemical shifts NMR31R are given in ppm relative to 85% H3PO4(external standard). The NMR spectra of13C and31R recorded with isolation of protons {1N}. Experiments on two-dimensional NMR (HMQC and HMBC) were conducted for the assignment of NMR signals1H and13With the structure. DSC is performed on a differential scanning calorimeter DSC/2920, TA Instruments.

Hydrated mono-L-his-tag salt SAR

The original aqueous solution of L-histidine (0.2 M). In 10 ml of deionized water dissolve L-histidine (0,3167 g, 2.0 mmol), get a 0.2 M solution.

The original solution SAR in the form of the free acid in methanol (0.6 M). The disodium salt of SAR (1,9194 g) dissolved in 5 ml of deionized water. To the resulting solution was added a saturated aqueous solution of sodium chloride (40 ml). A white precipitate is formed. Add ethyl acetate (50 ml)and the resulting suspension is stirred with a magnetic stirrer, acidified with 0.5 G. hydrochloric acid up until a two-phase mixture becomes transparent (aqueous phase is acidic reaction of the indicator paper). The organic phase is separated. The aqueous phase is extracted with ethyl acetate (2×50 ml). The combined organic phases are dried Na2SO4. After filtration and evaporation of the solvent (rotary evaporator, the the temperature of the water bath 37° C) get a thick film free acid SAR, which is dissolved in 10 ml of methanol. The methanol is evaporated on a rotary evaporator (37° (C)receive almost white foam (1.52 g), which re-dissolve in the Meon (4,79 ml), get a solution with an expected concentration of 0.8 M.

Certain higher concentration further acknowledge by mixing 100 μl of 0.2 M solution of L-histidine (0.20 mmol) with 25 μl of a solution of the free acid CAR. The solvent from the resulting solution evaporated to dryness. The solid is analyzed by NMR1N receive the molar ratio of histidine to the free acid SAR 1:0.75 in. Therefore, the concentration of free acid CAR is 0.6 M (this suggests that the foam of the free acid SAR contains solvent).

Hydrated mono-L-his-tag salt CAR. In the vessel for HPLC with a volume of 4 ml add L-histidine (900 μl, 0.2 M, 180 mmol), a solution of the free acid SER (300 μl, 0.6 M, 180 mmol) and isopropyl alcohol (1.0 ml). The resulting solution was evaporated on a rotary evaporator (the temperature of the water bath 39-40° (C) to reduce the volume of about 1.5 ml Add 1.0 ml of isopropyl alcohol, and the volume is again reduced to approximately a 1.5 ml solution observe the formation of small crystal. The process of evaporation ceased, the vessel closed and leave when ControlTemplate for 5 hours. The crystalline solid is filtered off with suction through filter paper Whatman #54, washed with isopropyl alcohol (approximately 2 ml) and dried in a stream of nitrogen overnight, get 82,7 mg of mono-L-his-tag salt SAR in the form of a white solid. Received proletarienne mono-L-his-tag salt SAR is a crystalline solid; analysis by Karl-Fischer solids shows that the water content is 4.66%, that is, according to the calculations, crystalline solid gidratirovana 1.5 molecules of water per molecule of salt (143 mmol, yield 79%).1H NMR (300 MHz, D2O) δ to 3.33 (d, J=6,59 Hz, 2H, C(21)H2), to 3.67 (s, 6H, C(15)H3and C(17)H3), 3,74 (s, 3H, C(16)H3), 3,82 (s, 3H, C(18)H3), to 4.01 (t, J=6,50 Hz, 1H, C(20)H), 6,53 (d, J=12,25 Hz, 1H, C(8)H), 6,62 (d, J=12,25 Hz, 1H, C(7)H), 6,62 (s, 2H, C(10)H and C(14)H)6,94 (d, J=8,00 Hz, 1H, C(3)H), 7,01 (d, J=8,00 Hz, 1H, C(4)H), 7,21 (s, 1H, C(6)H), 7,37 (s, 1H, C(23)H, 8,63 (s, 1H, C(24)H);13C NMR (100 MHz, {1N}, D2O) δ 26,12 (C21), 53,93 (C20), 56.26 vertical (2C, C15, C17), 56,35 (C18), 61,32 (C16), 106,86 (2C, CIO, C14), 113,26 (C3), 118,03 (C23), 122,04 (l, Jpc=2.3 Hz, C6), 125,52 (C4), 127,80 (C22), 129,40 (C8), 130,05 (C7), 130,57 (C5), 133,99 (C9), 134,34 (C24), 136,18 (C12), 141,37 (l, Jpc=6,90 Hz, Cl), 150,01 (l, Jpc=4,60 Hz, C2), 152,52 (2C, C11 and C13), 172,87 (C19);31P NMR (121 MHz, {1N}, D2O) δ - 2,61 (C)Calculated for C24H30N3O10P.1,5H2O: C, 49,83; H, Of 5.75; N, 7,26, Found: C, 50,13; H, 5,78; N, 7,26.

Analysis of the Karl-Fischer and elemental analysis of the product of this method show that the crystalline salt is a single hydrate. The DSC analysis shows the presence of one major crystalline forms endothermal melting 158,6°With (see Figure 5). Data powder diffraction pattern obtained for this product are listed on Fig, upper graph.

Differences in the polymorphism observed at room temperature as a function of the number of mmol of the free acid CAR in relation to the total volume of the crystallization mixture. In the above method using 0.2 mmole free acid SAR 1 ml total volume of the crystallization mixture. Modification of the above method using 0.03 mmole free acid SAR 1 ml total volume crystallization of the mixture gives the mono-L-his-tag salt CAR containing 1.8 molecules of water per molecule of salt (see Fig.6, the sample size 3,8500 mg); the analysis of the DSC indicates the presence of one crystalline form with endothermal melting at 184,9°C. Analysis by NMR1N gives the ratio SAR: histidine = 1:1. Data of powder x-ray diffraction analysis, were obtained for this substance, found on Fig, lower graph. In another modification of the above-described techniques are used 0.07 mmole free acid SAR 1 ml total volume of crystallization the th mixture, get the mixture forms a mono-L-his-tag salt SAR, one of which contains 1.5 molecules of water per molecule of salt, other 1.8 molecules of water per molecule of salt (see Fig.7, the sample size 4,4500 mg); analysis by the method of Karl Fischer and elemental analysis of the mixture shows that the crystalline salt is a single hydrate. The analysis of the DSC shows the presence of two crystalline forms. Endothermy like endothermal shown in Figure 5 and 6, respectively. Data of powder x-ray diffraction analysis is shown in Fig.9. The data of DSC and powder x-ray diffraction analysis show that the shape with the ratio of water: salt of 1.5:1 and 1.8:1 are different crystalline forms. As also indicated above, the mixture of these two forms is formed easily. When making seed each form can be obtained in a pure state.

Polpithigama form of mono-L-his-tag salt SAR can also be obtained in the presence of water. However, this form turns into a Queen-hydrate, mono-L-his-tag salt CAR.

As mentioned above, various natural or unnatural amino acids, including, but not limited to, ornithine, lysine, arginine and tryptophan, can be used in place of histidine in the above description, with the formation of compounds of the present invention.

Hydrated mono-L-g is steinova salt SAR (large-scale transition)

The original aqueous solution of L-histidine (0.2 M). In 60 ml of deionized water dissolve L-histidine (1.90 g, 12,0 mmol), get a 0.2 M solution. (The solution can also be prepared in situ).

The original solution of the free acid CAR in isopropyl alcohol (IPA) (0.17 M). The free acid SAR can be prepared by the following method. Equivalent of the acid can be reduced to 2.1; adding sodium chloride is not necessary. In an example, the following technique: the disodium salt of SAR (8,94 g, 20.3 mmol) is dissolved in 50 ml of deionized water. To the resulting solution with rapid stirring in ethyl acetate (200 ml) and saturated aqueous solution of sodium chloride (100 ml). A white precipitate is formed. Portions are added to 0.5 N. hydrochloric acid (220 ml) to dissolve the precipitate (final pH value of the aqueous phase is approximately 1 on the indicator paper). The organic phase is separated. The aqueous phase is extracted with ethyl acetate (1×200 ml) and then 2×150 ml). The combined organic phases dried over Na2SO4. After filtration and evaporation of the solvent (rotary evaporator, the temperature of the water bath is 40° (C) receive a thick film free acid SAR, which is dissolved in 100 ml of isopropyl alcohol. The concentration of the solution determined by NMR1N, and it is 0.17 M

To confirm is definitely the higher concentration with the help of a pipette into a vessel for HPLC with a volume of 4 ml contribute 60 μl of a solution of histidine (0.2 M) and 70 μl of a solution of the free acid CAR. The solvents are evaporated to dryness on a rotary evaporator. The solid is dissolved in 0.7 ml of D2O and analyzed by NMR1N, get the ratio of histidine to the free acid SAR 1:1. Just allocate 17 mmol of the free acid SER (yield 84%).

Hydrated mono-L-his-tag salt SAR (large-scale transition). In a round bottom flask of 250 ml is added a solution of the free acid SER (0.17 M, 12,0 mmol) and 50 ml of isopropyl alcohol. To a solution of the free acid CAR with rapid stirring portions add a solution of L-histidine (60 ml, 0.2 M) of 12.0 mmol). The resulting suspension is stirred at 40°C for 30 min, at room temperature for 3 h, then cooled at 0°C (ice bath) for 1 hour. The crystalline solid is filtered off with suction through filter paper Whatman #54, washed with cold isopropyl alcohol and dried in vacuum (vacuum desiccator) for 88 hours, get 6,07 g of mono-L-his-tag salt SAR in the form of a white solid. Analysis by the method of Karl Fischer solids shows that the water content is 4,48%, which corresponds to a crystalline solid substance, gidratirovannom 1.5 molecules of water per molecule of salt (10.5 mmol, yield 87%).1H NMR (300 MHz, D2O) δ of 3.32 (d, J=6.6 Hz, 2H, C(21)H2), 3,68 (s, 6H, C(15)H3and C(17)H3), 3,74 (s, 3H, C(16)H3), 3,82 (s, 3H, C(8)H 3), of 4.00 (t, J=6,6 Hz, 1H, C(20)H), 6,53 (d, J=12.1 Hz, 1H, C(8)H), 6,62 (d, J=12.1 Hz, 1H, C(7)H), only 6.64 (s, 2H, C(10)H and C(14)H), to 6.95 (d, J=8,3 Hz, 1H, C(3)H), 7,02 (d, J=8,3 Hz, 1H, C(4)H), 7,20 (users, 1H, C(6)H), of 7.36 (users, 1H, C(23)H, to 8.62 (d, J=1.3 Hz, 1H, C(24)H);13C NMR (100 MHz, {1N}, D2O) δ 26,11 (C21), 53,92 (C20), 56,22 (2C, C15, C17), 56,32 (C18), 61,28 (C16), 106,82 (2C, C10, C14), 113,20 (C3), 118,03 (C23), 122,01 (l, Jpc=2.3 Hz, C6), 125,48 (C4), 127,78 (C22), 129,38 (C8), 129,97 (C7), 130,54 (C5), 133,92 (C9), 134,31 (C24), 136,16 (C12), 141,38 (l, JPC=6,1 Hz, C1), 149,98 (l, Jpc=5.4 Hz, C2), 152,48 (2C, C11 and C13), 172,86 (C19)Calculated for C24H30N3O10P.1,5H2O: C, 49,82; H, Of 5.75; N, 7,26; P, 5,35, Found: C, 49,92; H, Of 5.84; N, 7,26; P, 5,44. In addition, using differential scanning calorimetry determined that the connection has the main endotherm at 158°and minor endotherm at 174°C.

This methodology can be easily used with any suitable natural or unnatural amino acid with other compounds of the present invention.

During crystallization of mono-L-his-tag salt SAR at room temperature usually obrazu(Yu)Garat(s). Carrying out the crystallization at temperatures above room temperature, especially at temperatures above 70°allows to obtain the anhydrous salt. The his-tag hydrate salt may be converted to the anhydrous crystalline form (especially with a melting point 210° (C)by, for example, when spendelove hydrated form in such a solvent, as ethanol, methanol, isopropanol or acetone, at these temperatures, 40°With (for example, within 2 days), followed by filtration, washing and drying in vacuum at a temperature of 45°With (during the night). Practically non-hygroscopic anhydrous form is preferred.

Anhydrous mono-L-his-tag salt SAR

In a round bottom flask with a volume of 200 ml download L-histidine (0,2620 g of 1.65 mmol) and 16.5 ml of deionized water. The resulting solution was heated at 74-76°C (oil bath temperature) with stirring with a magnetic stirrer. Add a solution of the free acid SER (8,7 ml to 0.19 M solution in isopropyl alcohol, of 1.65 mmol), then add isopropyl alcohol (90 ml). Obtained after approximately 1 min, the solution becomes milky. Stirring is continued at 75-76°C for 2 h, then at room temperature for 1 hour. By filtration through Whatman paper #4 with separate suction needle crystalline substance and dried in a stream of air (suction) during the night (19,5 hours, then in a vacuum desiccator for 24 hours, get 0,7788 g of mono-L-his-tag salt SAR in the form of a white solid (1,41 mmol, yield 86%), TPL 211,49° (DSC).1H NMR (400 MHz, D2O) δ 3,30 (d, J=6.5 Hz, 2H, H-21), the 3.65 (s, 6H, H-15 and H-17), and 3.72 (s, 3H, H-16), of 3.80 (s, 3H, H-18), to 3.99 (t, J=6.5 Hz, 1H, H-20), of 6.50 (d, J=12.3 Hz, 1H, H-8), 6,59 (d, J=12.3 Hz, 1H, H-7), 6,60 (s, 2H, H-10 and H-14), 6,92 (d, J=8.5 Hz, 1H, H3), 6,97 (userd, J=8.5 Hz, 1H, H-4), 7,19 (users, 1H, H-6), 7,33 (users, 1H, H-23), 8,58 (users, 1H, H-24);13C NMR (100 MHz, {1N}, D2O) δ 27,11 (C-21), 54,88 (C-20), 57,17 (2C, C-15 and C-17), 57,24 (C-18), 62,24 (C-16), 107,77 (2C, C-10 and C-14), 114,17 (C-3), 118,90 (C-23), 122,93 (l, Jpc=2.3 Hz, C-6), 126,40 (C-4), 128,88 (C-22), 130,29 (C-8), 131,00 (C-7), 131,47 (C-5), 134,93 (C-9), 135,32 (C-24), 137,08 (C-12), 142,31 (l, Jpc=6,1 Hz, C-l), 150,91 (l, Jpc=4,6 Hz, C-2), 153,45 (2C, C-11 and C-13), 173,84 (C-19), Calculated for C24H30N3O10P: S, 52,27; H, Of 5.48; N, A 7.62; P,5,61, Found: C, 52,03; H, 5,43; N, EUR 7.57; P, 5,57.

Anhydrous mono-L-his-tag salt SAR (large increase)

Round-bottom three-neck flask with a volume of 2000 ml supplied with a mechanical stirrer, addition funnel 500 ml and a thermocouple, connected to the device Therm-O-Watch L7-1100SA/28T, which regulates the heater casing. In the flask is charged with L-histidine (3.42 g, 21.6 mmol), then 216 ml of deionized water. The resulting solution was heated to 74-80°under stirring. Add a solution of the free acid SER (120 ml, 0.18 M in IPS, 21.6 mmol), then from the dropping funnel add isopropyl alcohol (1176 ml), maintaining the temperature of the solution 73-74° (requires 14 min). After adding IPS in a clear solution make a seed crystal of anhydrous L-his-tag salt SAR (trace amounts). The temperature of the solution was raised to 80°and crystallization starts after approximately 3 minutes after introduction of the seed. Temperature the Roux slowly reduced to 74° C for 30 min and maintain at 73-74°C for another 1.5 hour. The reaction mixture is left to cool slowly until 31°C for 3.5 hours. By filtration through Whatman paper #4 with separate suction needle crystalline substance, washed with isopropyl alcohol (100 ml) and dried in a stream of air (suction) overnight (16 h), then in a vacuum desiccator for 21.5 hours. Get 10,11 g anhydrous mono-L-his-tag salt SAR in the form of a white solid substance (to 18.3 mmol, yield 85%), TPL 213,65° (DSC).1H NMR (400 MHz, D2O) δ 3,30 (d, J= 6.5 Hz, 2H, H-21), the 3.65 (s, 6H, H-15 and H-17), and 3.72 (s, 3H, H-16), of 3.80 (s, 3H, H-18), to 3.99 (t, J=6.5 Hz, 1H, H-20), of 6.49 (d, J=12.0 Hz, 1H, H-8), to 6.58 (d, J=12.0 Hz, 1H, H-7), 6,59 (s, 2H, H-10 and H-14), 6,91 (d, J=8.5 Hz, 1H, H-3), 6,97 (DD, J=8,3, 1.7 Hz, 1H, H-4), 7,19 (ushort, J=1.7 Hz, 1H, H-6), 7,34 (users, 1H, H-23), at 8.60 (d, J=1.4 Hz, 1H,H-24);13C NMR (100 MHz, {1N}, D2O) δ 27,07 (C-21), 54,86 (C-20), 57,18 (2C, C-15 and C-17), 57,26 (C-18), 62,24 (C-16), 107,78 (2C, C-10 and C-14), 114,18 (C-3), 118,94 (C-23), 122,95 (l, Jpc=2.3 Hz, C-6), 126,43 (C-4), 128,79(C-22), 130,31 (C-8), 130,98 (C-7), 131,49 (C-5), 134,91 (C-9), 135,29 (C-24), 137,10 (C-12), 142,31 (l, JPC=6,9 Hz, C-l), 150,92 (l, JPC=4,6 Hz, C-2), 153,45 (2C, C-l1 C-13), 173,82 (C-19), Calculated for C24H30N3O10P: S, 52,27; H, Of 5.48; N, A 7.62; P,5,61, Found: C, 52,23; H, To 5.35; N, 7,60; P, 5,55.

Anhydrous L-his-tag salt SAR can be obtained reproducibly in a single crystalline form. Figure 10 shows a graph of the DSC (sample size 2,3600 mg); figure 11 shows Dan is haunted powder x-ray diffraction for this substance.

Example 4

Getting a mono-salt of methyl ester of glycine CIS-SAR

To obtain the mono-salt of methyl ester of glycine CAR the following preferred method of receipt of the disodium salt CAR. The methodology used hydrochloride methyl ester of glycine directly in the presence of N,N-diisopropylethylamine that provides improved stability compared to the use of methyl ester of glycine in the form of free base. In addition, significantly improves the free acid SAR, as by neutralization using concentrated sulfuric acid (as, for example, diluted hydrochloric acid, the result can be used, for example, ethyl acetate for extraction and evaporation). According to an improved method eliminates the formation of free acid TRANS-SAR.

Reagents and methods

The following reagents and chemicals obtained from commercial sources and used without additional purification: isopropyl alcohol (IPA) (grade B&J, a solvent of high purity), sulfuric acid (EM Science, 95-98%, batch #35310), the hydrochloride of the methyl ester of glycine (Aldrich Chemical Co. category 99%, batch # 03214MU), N,N-diisopropylethylamine (Aldrich Chemical Co. category 99.5%pure, batch #02819ER). Multi-NMR spectra are recorded on a spectrometer Bruker DRX 400. Chemical add the guy in NMR spectra 1H and13Given in ppm relative to tetramethylsilane. (Chemical shifts NMR13To determine using the Meon as an external standard). Experiments on two-dimensional NMR HMQC [spectroscopy heteronuclear multiplet quantum correlation, inverse correlation between chemical shift to determine which of the1N molecules associated with any kernel13(Or other kernel X)]; and HMBC [spectroscopy heteronuclear multiplet correlation relationships, modification HMQC used to detect distant interactions1H13C, and to determine the structure and classification1H and13With in the molecule] spend for the assignment of NMR signals1H and13With the structure. DSC is performed on a differential scanning calorimeter DSC/2920, TA Instruments.

Mono-salt of methyl ester of glycine CIS-SAR

In a round bottom flask of 100 ml download the sodium salt of CIS-SAR (2,866 g, 6,51 mmol) and IPA (30 ml). The resulting suspension is stirred with a magnetic stirrer at room temperature. To the suspension are added in several portions a solution of sulfuric acid (0,365 ml, 6,51 mmol) in isopropyl alcohol (60 ml). The mixture is continuously stirred for 10 min and filtered with suction using filter paper Whatman #1. Solid (Na2SO4insoluble in isopropyl alcohol) was washed with IPA (10 ml). The filtrate is leaching portion, containing free acid SAR, merge into another round bottom flask with a volume of 100 ml For the combined solution was added the hydrochloride of the methyl ester of glycine (0,826 g, 6,51 mmol) and N,N-diisopropylethylamine (1,254 ml, 7,16 mmol). The resulting mixture is heated on a water bath with stirring with a magnetic stirrer. At 60°With the mixture becomes a clear solution. At 65°the solution turns into suspension. When 78°the suspension is dissolved, forming a clear solution. The heating is stopped, and the solution is left to cool slowly in an oil bath. At 60°to the solution add priming mono-salt of methyl ester of glycine CIS-SAR, formed suspension. Stirring is continued at a temperature of from 60°C to room temperature for about 1 hour and then at room temperature overnight. The white crystalline substance is filtered off with suction using filter paper Whatman #1, washed with isopropyl alcohol (3×10 ml) and dried in a stream of air for 6 hours, get to 2.609 g mono-salt of methyl ester of glycine CIS-SAR (5,38 mmol, yield of 82.6%). Analysis by HPLC: 100% CIS-SAR, TPL 136,40° (DSC).1H NMR (400 MHz, D2O) δ to 3.52 (s, 6H, H-15 and H-17), 3,61 (s, 3H, H-16), 3,71 (s, 3H, H-18), of 3.77 (s, 3H, H-21), a 3.87 (s, 2H, H-20), of 6.26 (d, J=12.1 Hz, 1H, H-8), 6.42 per (s, 2H, H-10 and H-14), to 6.43 (d, J= 12.1 Hz, 1H, H-7), 6,70 (d, J=8,8 Hz, 1H, H-3), 6,79 (userd, J=8,8 Hz, 1H, H-4), 7,16 (users, 1H, -6); 13C NMR (100 MHz, {1N}, D2O) δ 41,27 (C-20), 54,58 (C-21), 56,99 (2C, C-15 and C-17), 57,21 (C-18), 62,09 (C-16), 107,60 (2C, C-10 and C-14), 113,89 (C-3), 122,98 (C-6), 126,22 (C-4), 130,25 (C-8), 130,69 (C-7), 131,37 (C-5), 134,61 (C-9), 137,09 (C-12), 142,42 (d, 2 jpc=6,9 Hz, C-l), 150,89 (d3jpc=4,6 Hz, C-2), 153,33 (2C, C-l1 C-13), 169,95 (C-19), Calculated for C21H28NO10P: C, 51,96; H, Of 5.81; N, Is 2.88; P, 6,38, Found: C, 51,74; H, 5,79; N, 2,87; P, 6,30.

(Note: the numbering system above, and wherever in the description of this numbering system is not operated, introduced only for convenience, and may not correspond to the IUPAC nomenclature).

On Fig is a graph of DSC mono-salt of methyl ester of glycine CIS-SAR (sample size 3,7400 mg); Fig shows powder x-ray diffraction data for this substance (2 lots).

Example 5

Getting salt of ethyl glycine ester and free acid CAR

In the vessel for HPLC placed in ethyl acetate (2 ml), the solution SAR in isopropanol (150 μl of 0.42 M solution, 63 μmol) and a solution of ethyl ester of glycine tert-butyl ether (800 ál 0.08 M solution, 64 mmol) and intensively stirred for approximately 3 minutes In the obtained transparent solution make a seed with a drop of suspension from another experience, and the mixture is left at room temperature overnight. The resulting white solid, as it turns out, is not crystal according to mi is roscopically research, and so the mixture is left at room temperature for another three days. Add methyl tert-butyl ether (1 ml), and the mixture is stirred for 10 minutes Study under a microscope shows that the mixture has turned into a needle crystals. Needles is filtered under vacuum and dried, are salt of the ethyl ester of glycine CAR (of 22.4 mg, yield 66%). Analysis of the proton NMR spectrum shows that the ratio of the ethyl ester of glycine to CAR is 1.7:1. These NMR1N. salt of the ethyl ester of glycine and SAR:1H NMR (300 MHz, D2O) δ of 1.20 (t, J - 7.2 Hz, 3H, CH3), of 3.60 (s, 6H, H-15 and H-17), 3,66 (s, 3H, H-16), 3,74 (s, 3H, H-18), 3,79 (s, 2H, CH2N), is 4.21 (q, J=7.2 Hz, 2H, CH2CH3), 6,44 (d, J=and 12.2 Hz, 1H, H-8), 6,55 (d, J=and 12.2 Hz, 1H, H-7), to 6.57 (s, 2H, H-10 and H-14), to 6.80 (d, J=8.7 Hz, 1H, H-3), 6,85 (userd, J=8.7 Hz, 1H, H-4), 7.23 percent (users, 1H, H-6).

The present invention is not limited to specific cases described here. Indeed, various modifications of the invention, in addition to those described here, will be apparent to the person skilled in the art from the preceding description. It is assumed that such modifications fall within the scope of the attached claims.

In the document cited various publications and patent documents described entirely as references.

1. The compound having the General structure of formula I

where one of the substituents OR1or2represents-O-QH+and the other represents a hydroxyl or-O-QH+; and Q represents a

(A) optionally substituted aliphatic organic amine containing at least one nitrogen atom which, together with a proton, forms a Quaternary ammonium cation HE+;

(B) an amino acid containing at least two nitrogen atom, where one of the nitrogen atoms, together with a proton, forms a Quaternary ammonium cation QH+;

(C) an amino acid containing one or more nitrogen atoms where one of the nitrogen atoms, together with a proton, forms a Quaternary ammonium cation QH+and where, in addition, all carboxyl groups of the amino acids are in the form of esters.

2. The compound according to claim 1, where Q is an optionally substituted aliphatic organic amine containing at least one nitrogen atom which, together with a proton, forms a Quaternary ammonium cation QH+.

3. The compound according to claim 2, where the nitrogen Q, forms a Quaternary ammonium cation QH+in formula I, is a primary amine that is associated with the optionally substituted aliphatic group, or a secondary amine, associated with two optionally substituted aliphatic group, where the optional what's the substituents are one or more hydroxyl groups or amino groups.

4. The compound according to claim 2, where the specified optionally substituted aliphatic organic amine is a tromethamine and its stereometria form.

5. The compound according to claim 2, where the specified connection has the structure of formula Ia

6. The compound according to claim 1, where Q is an amino acid containing at least two nitrogen atom, where one of the nitrogen atoms, together with a proton, forms a Quaternary ammonium cation QH+.

7. The connection according to claim 6, where the specified amino acid is a histidine and its stereometria form.

8. The connection according to claim 6, where the specified connection has one of the following structures:

9. The compound according to claim 1, where Q is an amino acid containing one or more nitrogen atoms where one of the nitrogen atoms, together with a proton, forms a Quaternary ammonium cation QH+and where, in addition, all carboxyl groups of the amino acids are in the form of esters.

10. The connection according to claim 9, where Q represents C1-6-alkilany ester of glycine.

11. Pharmaceutical composition for modulating tumor growth or metastasis and growth of benign vascular proliferative disorders, comprising

(a) a compound according to any one of PR the previous paragraphs, and

(b) a pharmaceutically acceptable carrier.

12. The use of the compounds of formula (I) according to any one of claims 1 to 10 in the manufacture of drugs for modulating tumor growth and metastasis in an animal.

13. Pharmaceutical composition for modulating tumor growth or metastasis and growth of benign vascular proliferative disorders, formed by mixing compounds, including

(a) free acid CAR having the structure

(b) compound Q, where Q is a

(A) optionally substituted aliphatic organic amine containing at least one nitrogen atom that is capable of together with a proton to form a Quaternary ammonium cation QH+;

(B) an amino acid containing at least two nitrogen atom, where one of the nitrogen atoms, together with a proton, forms a Quaternary ammonium cation QH+; or

(C) an amino acid containing one or more nitrogen atoms where one of the nitrogen atoms, together with a proton, forms a Quaternary ammonium cation QH; and where, in addition, all carboxyl groups of the amino acids are in the form of esters.

14. The composition according to item 13, where Q is an optionally substituted aliphatic organic amine containing at least, one of the n atom of nitrogen, which is able, together with a proton to form a Quaternary ammonium cation QH+.

15. The composition according to 14, where specified optionally substituted aliphatic organic amine is a tromethamine and its stereometria form.

16. The composition according to item 13, where Q is an amino acid containing at least two nitrogen atom, where one of the nitrogen atoms, together with a proton capable of forming a Quaternary ammonium cation QH+.

17. The composition according to item 16, where this amino acid is a histidine and its stereometria form.

18. The composition according to item 13, where Q is an amino acid containing one or more nitrogen atoms where one of the nitrogen atoms, together with a proton, forms a Quaternary ammonium cation QH+and where, in addition, all carboxyl groups of the amino acids are in the form of esters.

19. The composition according to p, where Q represents C1-6-alkilany ester of glycine.

20. Pharmaceutical composition for modulating tumor growth or metastasis and growth of benign vascular proliferative disorders, formed by mixing compounds, including

(a) free acid CAR having the structure

(b) compound Q, where Q is a

(A) is not necessarily alseny aliphatic organic amine, containing at least one nitrogen atom that is capable of together with a proton to form a Quaternary ammonium cation QH+;

(B) an amino acid containing at least two nitrogen atom, where one of the nitrogen atoms, together with a proton, forms a Quaternary ammonium cation QH+; or

(C) an amino acid containing one or more nitrogen atoms where one of the nitrogen atoms, together with a proton, forms a Quaternary ammonium cation QH+and where, in addition, all carboxyl groups of the amino acids are in the form of esters;

and (C) a pharmaceutically acceptable carrier.

21. A method of obtaining a compound according to claim 1, comprising a stage of contacting in a solvent free acid CAR having the structure

connection Q, where Q is a

(A) optionally substituted aliphatic organic amine containing at least one nitrogen atom that is capable of together with a proton to form a Quaternary ammonium cation QH+;

(B) an amino acid containing at least two nitrogen atom, where one of the nitrogen atoms, together with a proton, forms a Quaternary ammonium cation QH+; or

(C) an amino acid containing one or more nitrogen atoms where one of the nitrogen atoms, together with p is the Otho forms a Quaternary ammonium cation QH +and where, in addition, all carboxyl groups of the amino acids are in the form of esters.

22. The method according to item 21, where the specified connection according to claim 1 forms in the specified solvent residue in the form of crystals.

23. The method according to item 21, where the specified optionally substituted aliphatic organic amine is a tromethamine and its stereometria form.

24. The method according to item 21, where this free acid SAR contact with tromethamine in aqueous isopropanol as specified solvent, and subsequent collection of mono-tromethamine salt CAR in crystalline form from the specified solvent.

25. The method according to item 21, where said amino acid is a histidine or stereometria form,

26. The method according to item 22, where this free acid SAR contact with histidine in isopropanol as solvent, with subsequent collection of mono-L-his-tag salt CAR in crystalline form from the specified solvent.

27. The method according to item 21, where this free acid SAR contact with C1-6-alkylbis ester of glycine.



 

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The invention relates to medicine and relates to means for inhibiting endothelialised enzymes, which is a product of the formula (I), method of its production and pharmaceutical compositions containing the product of formula (I) as active principle

The invention relates to new derivatives thioamides formula (I), where X denotes the group-CO2H or -- CONHOH, Y and Z denotes sulfur or oxygen, R1denotes hydrogen, hydroxy, alkyl, alkenyl, R2denotes alkyl, phenylalkyl, phenylalkyl-alkyl, R3means defining a side chain of natural-amino acid in which any functional group may be protected, alkyl, cycloalkyl, R4denotes alkyl, phenylalkyl, optionally substituted phenyl, or a group of the formula -(Q-O - Q, where Q denotes alkyl

The invention relates to acylaminocinnamic derivative of the formula (I), where R denotes phenyl which is not substituted or may be substituted with halogen, alkyl, trifluoromethyl, hydroxy and alkoxygroup, R1is hydrogen, alkyl, R2is hydrogen, alkyl or phenyl which is not substituted or may be substituted with halogen, alkyl, trifluoromethyl, hydroxy and alkoxygroup, R3is phenyl which is not substituted or may be substituted with halogen, alkyl, trifluoromethyl, hydroxy and alkoxygroup, or represents naphthyl, lH-indol-3-yl or 1-alcheringa-3-yl, R4' and R4"is hydrogen, alkyl, and one of the radicals R4' and R4"is hydrogen, and R5- cycloalkyl, D-azacycloheptan-2-he-3-yl or L-azacycloheptan-2-he-3-yl, or its salt

The invention relates to derivatives of heterocyclic compounds, as well as agricultural and horticultural fungicides containing these compounds as active ingredients

FIELD: pharmaceutical industry, medicine.

SUBSTANCE: invention relates to compounds of formula I , wherein A, L, Y, and k are as defined in specification. Compounds of formula I have value pharmacological activity, in particular potent antithrombosis action and are useful in treatment and prophylaxis of cardiovascular diseases such as thromboembolia. They represent also reversible inhibitors of factor X and factor VIIa (blood coagulation enzymes). Also disclosed are methods for production of compounds I, uses thereof, in particular as active ingredients in pharmaceutical compositions, as well as medicines containing the same.

EFFECT: new pharmaceutical compositions for treatment and prophylaxis of cardiovascular diseases.

10 cl, 50 ex

The invention relates to pharmaceutical compositions containing as active ingredient a compound of General formula (I)

in which: R1choose from one of the following groups: - H; - (C1-C20)alkyl, unsubstituted or substituted (C1-C5)alkyl; R2choose from: - (C3-C8)cycloalkyl - (C6-C14)aryl, unsubstituted or substituted with halogen, (C1-C5)alkyl, (C1-C5)alkylthiol, (C5-C6)heteroaryl containing a nitrogen atom which may be substituted by one or more (C1-C5) and alkyl (C1-C5)alkylthio groups, R3denotes hydrogen, and a is chosen from the groups: - CH2-, - CH2-CH2-, - CHR4-, R4choose from: - H; - (C1-C20)alkyl, unsubstituted or substituted (C1-C5)alkyl, and their solvate and a pharmaceutically acceptable salt, which can be used to treat pathologies associated with syndrome of insulin resistance, because it has anti-diabetic and hypoglycemic effect; the compounds of General formula I, 2-m methods for their preparation

FIELD: organic chemistry, chemical technology.

SUBSTANCE: invention relates to novel 1,2,3-tris-{[aminopoly(ethyleneamino)ethylammonio]-methylcarbonyloxypoly(alkyleneoxy)]}propane trichlorides of the formula:

wherein at a + c + e (the general degree of oxypropylation) = 49; b + d + f (the general degree of ethylation) = 0, n = 1-6; at a + c + e = 55, b + d + f = 0, n = 1-6; at a + c + e = 49, b + d + f = 9, n = 1-6; at a + c + e = 10, b + d + f = 10, n = 1-6; at a + c + e = 66, b + d + f = 15, n = 1-6; at a + c + e = 76, b + d + f = 18, n = 1-6, and to a method for their synthesis. Method involves interaction of 1,2,3-tris-[hydroxypoly(alkyleneoxy)]propanes of the formula:

wherein a + c + e = 49-76, b + d + f = 0-18 with monochloroacetic acid in the presence of acidic catalysts, in boiling organic solvent medium, azeotropic removing water formed and the following treatment at heating the synthesized reaction product with polyethylenepolyamines of the formula: H2N(CH2CH2NH)nCH2CH2NH2 wherein n = 1-6, and in the following mole ratios of reagents - hydroxyl derivatives of propane : monochloroacetic acid : polyethylenepolyamines = 1:(3.0-3.2):(3.0-3.2), respectively. New compounds possess the fungicide activity, properties of emulsifiers of cationic bitumen emulsions, capacity to enhance adhesion of bitumen to mineral materials.

EFFECT: improved preparing method, valuable properties of compounds.

6 cl, 3 tbl, 6 ex

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