RU2017724C1 - Method of synthesis of taxol derivatives - Google Patents

Abstract

FIELD: organic chemistry. SUBSTANCE: product: derivatives of taxol of formula (I), where R₁ and R₂ - H or the group of formula (III)

, where n= 1-3; R₃ and R₄ is C₁-C₃-alkyl at condition that at least one of R₁ and R₂ is not hydrogen atom. Reagent 1: taxol. Reagent 2: compound of the general formula (II)

, where R₃ and R₄ as indicated above. Process conditions: in the presence of condensing agent and catalyst (the presence of latter does not obligatory). At the ratio of compound (II) and taxol 1:1 2-substituted compound of formula (I) is prepared, where R₁ is a group of formula (III) and R₂ - H. At the case of synthesis of 2,7-di(substituted) of compound of formula (I), where R₁ and R₂ - a group of formula (III), is reacted with compound of formula (II). If necessary, 2,7-di(substituted) compounds of formula (I) is split at pH 7-7.4, and 7-substituted compound of formula (I) is prepared, where R₁ - H, and R₂ - a group of formula (III). Compound of formula (I) shows high antitumor activity at the dose 1.88-7.50 mg/kg and water solubility. Synthesized compounds are used in medicine. EFFECT: enhanced antitumor activity. 6 cl, 2 tbl

Description

 The invention relates to derivatives of taxol, which have better water solubility compared to taxol and show good antitumor activity. These compounds are intended for use in the treatment of the same types of cancer in which taxol is used, namely lung tumors, melanoma, leukemia, breast cancer and colon cancer.

(I)
Таксол также обладает сильной ингибирующей активностью против широкого ряда опухолей. Однако это соединение плохо растворяется в воде, что создает ряд значительных трудностей при изготовлении фармацевтических композиций, используемых для лечения опухолей у человека. Некоторые композиции таксола для инъекций или внутривенных вливаний изготавливаются в основном с использованием кремофора EL^® в качестве наполнителя в целях устранения недостатка, связанного с низкой водорастворимостью таксола. Однако кремофор сам по себе является отчасти токсичным и вызывает идиосинкразическое высвобождение гистамина и анафиклатическиподобную реакцию, поэтому использование этого наполнителя, к сожалению, не является хорошим решением указанной выше проблемы разработки подходящих композиций таксола.Taxol is a known diterpenoid, sesquiterpene, which is obtained from the bark of the yew tree, Taxus brevi folia, and which has the following structure:

(I)
Taxol also has strong inhibitory activity against a wide range of tumors. However, this compound is poorly soluble in water, which creates a number of significant difficulties in the manufacture of pharmaceutical compositions used to treat tumors in humans. Some compositions of Taxol for injection or intravenous infusion are made mainly using Cremophor EL ^® as a filler in order to eliminate the disadvantage associated with the low water solubility of Taxol. However, cremophor itself is partly toxic and causes an idiosyncratic release of histamine and an anaphylactic-like reaction, so the use of this filler, unfortunately, is not a good solution to the above problem of developing suitable taxol compositions.

 In accordance with this, taxol derivatives were obtained as a result of studies, which, having better water solubility than taxol, at the same time show the same good antitumor and cytotoxic activity as taxol.

 The aim of the invention is to obtain derivatives of taxol with good antitumor activity and water solubility, the manufacture of compositions containing derivatives of taxol and non-toxic carriers, which will eliminate the use of toxic carriers such as cremophor, as well as the preparation of taxol derivatives that would have reasonably good stability at levels pH suitable for the manufacture of pharmaceutical compositions (pH 3-4), but would have the ability to quickly decompose in vivo with a physiologist other pH values (pH 7.4), i.e. which could act as a precursor of taxol.

 These objectives of the invention can be carried out by developing the preparation of taxol derivatives having good antitumor activity and water solubility.

(II)
где R и R' каждый является Н или остатком аминокислоты, выбранной из группы, состоящей из аланина, лейцина, изолейцина, валина, фенилаланина, пролина, лизина или аргинина, или группой формулы

(III)
где n является целым числом от 1 до 3;
R² и R³ - каждый является водородом или алкилом, имеющим от 1 до 3 атомов углерода, или R² и R³ вместе с атомом азота, с которым они связаны, образуют насыщенное гетероциклическое ядро, имеющее 4-5 атомов углерода, при условии, что, по крайней мере, один из R и R' не является водородом.New derivatives of taxol can be mainly represented as 2'- and / or 7-esters of taxol having the following formula:

(Ii)
where R and R 'each is H or an amino acid residue selected from the group consisting of alanine, leucine, isoleucine, valine, phenylalanine, proline, lysine or arginine, or a group of the formula

(III)
where n is an integer from 1 to 3;
R ² and R ³ - each is hydrogen or alkyl having from 1 to 3 carbon atoms, or R ² and R ³ together with the nitrogen atom to which they are attached form a saturated heterocyclic ring having 4-5 carbon atoms, provided that at least one of R and R 'is not hydrogen.

If in the indicated formula (III) R ² and / or R ³ are not hydrogen, then these compounds can be regarded as the so-called "alkylated amino acids". Thus, the compounds of formula (I), in a broader sense, can be considered as 2'- and / or 7-esters of taxol with amino acids or alkylated amino acids.

 Thus, the invention includes: derivatives esterified at the 2'-hydroxyl group of taxol, derivatives esterified at the 7-hydroxyl group of taxol, and derivatives esterified at the 2'- and 7-positions of hydroxyl groups.

Among the compounds described by the indicated general formula I, preferred compounds of the invention are the following compounds:
2 '(N, N-diethylaminopropionyl) taxol (compound 1)
2 '(N, N-dimethylglycyl) taxol (compound 2)
7 (N, N-dimethylglycyl) taxol (compound 3)
2 ', 7-di- (N, N-dimethylglycyl) taxol (compound 4)
7 (N, N-diethylaminopropionyl) taxol (compound 5)
2 ', 7-di (N, N-diethylaminopropionyl) taxol (compound 6)
2 '- (L-glycyl) taxol (compound 7)
7- (L-glycyl) taxol (compound 8)
2 ', 7-di (L-glycyl) taxol (compound 9)
2 '- (L-alanyl) taxol (compound 10)
7- (L-alanyl) taxol (compound 11)
2 ', 7-di (L-alanyl) taxol (compound 12)
2 '- (L-leucyl) taxol (compound 13)
7- (L-leucyl) taxol (compound 14)
2 ', 7-di (L-leucyl) taxol (compound 15)
2 '- (L-isoleucyl) taxol (compound 16)
7- (L-isoleucyl) taxol (compound 17)
2 ', 7-di (L-isoleucyl) taxol (compound 18)
2 '- (L-valyl) Taxol (Compound 19)
7- (L-valyl) Taxol (Compound 20)
2 ', 7-di (L-valyl) taxol (compound 21)
2 '- (L-phenylalanyl) taxol (compound 22)
7- (L-phenylalanyl) taxol (compound 23)
2 ', 7-di (L-phenylalanyl) taxol (compound 24)
2 '- (L-propyl) taxol (compound 25)
7- (L-propyl) taxol (compound 26)
2 ', 7-di (L-propyl) taxol (compound 27)
2 '- (L-lysyl) taxol (compound 28)
7- (L-lysyl) taxol (compound 29)
2 ', 7-di (L-lysyl) taxol (compound 30)
2 '- (L-glutamyl) taxol (compound 31)
7- (L-Glutamyl) Taxol (Compound 32)
2 ', 7-di (L-glutamyl) taxol (compound 33)
2 '- (L-arginyl) taxol (compound 34)
7- (L-arginyl) taxol (compound 35)
2 ', 7-di (L-arginyl) taxol (compound 36)
It has been found that the 2'- and 7-hydroxyl groups of taxol, especially the 2'-hydroxyl group, are more chemically active than the 7-hydroxyl group. And this fact formed the basis for obtaining the proposed derivatives.

Derivatives of 2'-ester-taxol
Derivatives of the 2'-ester can be obtained in one or two ways, depending on whether the derivative is a formed amino acid or an alkylated amino acid.

где n, R² и R³ определены выше.The following reaction scheme was used to obtain alkyl ester esters:
Scheme I
alkyl amino acid + taxol - >>2'-ester (taxol) -derivative

where n, R ² and R ^{3 are} defined above.

с

т

з

В обеих реакционных схемах (I и II) реакцию алкилированных или защитных аминокислот проводят в присутствии конденсирующего реагента и необязательно в присутствии катализатора, предпочтительно при комнатной температуре.The following reaction scheme was used to obtain amino acid esters:
Scheme II

with

t

s

In both reaction schemes (I and II), the reaction of alkylated or protective amino acids is carried out in the presence of a condensing reagent and optionally in the presence of a catalyst, preferably at room temperature.

 Suitable condensing agents are carbodiimides, such as dicyclohexylcarbodiimide (DCC).

 Suitable catalysts are 4-dimethylaminopyridine (DMAP) and pyridine.

 In the reaction scheme (II), various known amino protecting groups and commercially available protected amino acids can be used as starting materials. For this purpose, amino acids protected by t-BOC, FMOC or carbobenzyloxy group (CBZ) can be used. Amino acids protected by t-BOC or FMOC groups are preferred. Although the unlocking of the t-BOC group on 2'-esters using aqueous formic acid and other organic acids leads to degradation of the product, as well as stereochemical modification of this product, however, the use of 99% formic acid gives better results. In the case of esters of FMOC-protected amino acids, the recovery of the product depends on the reaction conditions. Thus, in the final unlocking step, the specific conditions used to remove the t-BOC-protecting group may cause undesirable modifications to the free hydroxyl group at the 7-position of the taxol molecules. These undesirable modifications are obviously the result of stereochemical modifications of the molecules.

NH

COOH t-ВОС-защищенные

FMOC-защищенные
Как уже указывалось, было обнаружено, что 2'-гидроксильная группа таксола является более химически активной, чем 7-гидроксильная группа. Таким образом, в обеих указанных схемах I и II замещение или этирификации направлены на 2'-положение и осуществляются путем взаимодействия алкилированной или N-защищенной аминокислоты с таксолом при молярном соотношении 1: 1 или слегка превышающем 1:1. Указанная реакция, проводимая с использованием равных молярных количеств, приводит к получению значительного избыточного количества производного 2'-сложный эфир-таксола, хотя при этом могут образовываться небольшое количество 7-сложный эфир-таксол-производного, как побочного продукта.Examples of N-protected alanine compounds are the following compounds:
CH

NH

COOH t-boc-protected

FMOC protected
As already indicated, it was found that the 2'-hydroxyl group of taxol is more chemically active than the 7-hydroxyl group. Thus, in both of these schemes I and II, substitution or etrification is directed to the 2'-position and is carried out by reacting an alkylated or N-protected amino acid with taxol at a molar ratio of 1: 1 or slightly greater than 1: 1. This reaction, carried out using equal molar amounts, results in a significant excess amount of the 2'-ester-taxol derivative, although a small amount of the 7-ester-taxol-derivative may be formed as a by-product.

 Obtaining 7-position esters.

Scheme III
Since the 2'-hydroxyl group of taxol is more active than the 7-hydroxyl group, this esterification requires the use of a procedure different from that used to obtain 2'-derivatives. Thus, to obtain the 7-esters, a procedure was used by which the 2-hydroxyl group was first blocked, then the 7-hydroxyl group was esterified, and finally the 2'-protective or blocking group was removed.

д

Для блокирования 2'-положения таксола могут быть использованы ряд известных защитных групп. В приведенной ниже схеме представлен пример использования защитной группы и алкилированной аминокислоты.

d

A number of known protecting groups can be used to block the 2'-position of taxol. The following diagram provides an example of the use of a protecting group and an alkylated amino acid.

В приведенной выше реакционной схеме, реакцию 2'-troc-таксола и алкилированной аминокислоты проводят в присутствии конденсирующего реагента и катализатора. Подходящими конденсирующими реагентами и катализаторами являются те же реагенты и катализаторы, которые были указаны для схем I и II.

In the above reaction scheme, the reaction of 2'-troc-taxol and an alkylated amino acid is carried out in the presence of a condensing reagent and a catalyst. Suitable condensing reagents and catalysts are the same reagents and catalysts that were indicated for schemes I and II.

 The unlocking of 2 '- (troc) -taxol can be carried out, for example, by using a mixture of zinc and acetic acid.

В этой процедуре, аналогичной процедуре схемы III, реакция таксола и защитной аминокислоты протекает в присутствии конденсирующего реагента и катализатора. Разблокирование аминокислот проводили методом, обычно используемoм для разблокирования аминокислот, например с помощью муравьиной кислоты.Scheme IV
Alternatively, derivatives of 7-substituted taxol can be obtained using the procedure, which is that first taxol is subjected to interaction with 2-3 equivalents of N-protected amino acids in order to obtain 2 ', 7-disubstituted taxol, then subjected to unlocking 2 'and 7-amino acids and the 2'-amino acid is cleaved. This procedure can be schematically represented as follows using t-BOC protected alanil:
Taxol + Nt

In this procedure, similar to that of Scheme III, the reaction of taxol and the protective amino acid proceeds in the presence of a condensing reagent and catalyst. The unlocking of amino acids was carried out by a method commonly used to unlock amino acids, for example with formic acid.

 The splitting of the 2'-amino acid was carried out by adjusting the pH of the solution of 2 ', 7- (amino acid) taxol to 7-7.4, for example, using a mixture of 2', 7-di (amino acid) taxol in phosphate buffer pH 7-7.4 . Adjusting the pH in this way leads to the cleavage of the 2'-amino acid with the formation of the target 7- (amino acid) taxol.

 The cleavage of the 2'-amino acid was carried out by adjusting the pH of the solution of 2 ', 7- (amino acid) taxol to 7-7.4, for example using a mixture of 2', 7-di (amino acid) taxol in phosphate buffer pH 7-7.4. Adjusting the pH in this way leads to the cleavage of the 2'-amino acid with the formation of the target 7- (amino acid) taxol.

 Thus, for example, taxol is reacted with 2-3 molar equivalents of an N-protected amino acid (t-BOC, CBZ or FMOC-protected) in methylene chloride in the presence of DCS and a catalytic amount of 4-dimethylaminopyridine. In this method, the protected amino acid is introduced at the 2'- and 7-position. Protecting groups are removed using an appropriate unlocking agent (e.g., acid, weak base, hydrogenolysis). The 2 ′, 7-bis-amino acid of taxol derivative is kept for 24-48 hours in phosphate buffer at neutral pH, resulting in selective unlocking at the 2′-position with the formation of a 7-substituted taxol derivative.

 A similar reaction scheme can also be used to prepare 7-substituted taxol derivatives using alkylated amino acids. This reaction is similar to that described above, except that the N-protected amino acid was replaced with the desired alkylated amino acid, and the unlocking step was not performed. This scheme can be represented as follows.

>>2

Получение производных 2',7-двузамещенного таксола.Scheme V
taxol + alkyl

>> 2

Obtaining derivatives of 2 ', 7-disubstituted taxol.

 Disubstituted derivatives can be obtained using the following procedures or part of these procedures.

о

кисло

з

Схема VII: Замещение аминокислотами
таксол + N-

щ

и

Схема VI, в основном, аналогична схеме I, за исключением того, что реакция протекает с 2 эквивалентами алкилированной аминокислоты. Кроме того реакция схемы I не требует обязательного присутствия катализатора, а реакция схемы VI должна протекать в присутствии катализатора вследствие меньшей химической активности 7-гидроксильной группы таксола.Scheme VI: Substitution with Alkyl Amino Acids
Taxol + Alc

about

sour

s

Scheme VII: Substitution with Amino Acids
Taxol + N-

u

and

Scheme VI is basically the same as Scheme I, except that the reaction proceeds with 2 equivalents of an alkylated amino acid. In addition, the reaction of Scheme I does not require the presence of a catalyst, and the reaction of Scheme VI must proceed in the presence of a catalyst due to the lower reactivity of the 7-hydroxyl group of taxol.

 Scheme VII is basically the same as Scheme II. However, in this regard, it should be noted that in Scheme VI, the preferred protective group is the FMOC group, which avoids stearin modifications in the 7-position during the unlocking step. Such a problem, however, does not exist in the reaction of Scheme VII, since the 7-hydroxyl group is free. Thus, various known protecting groups can be used in this scheme, including both t-BOC and FMOC groups.

 PRI me R 1. 2 '- (N, N-Dimethylglycyl) taxol or its salt.

.

=H
Соединение 1-2'-(N,N-диметилглицил) таксол.

.

= H
Compound 1-2 '- (N, N-dimethylglycyl) taxol.

To a solution of taxol (0.21 g, 0.246 mmol) and N, N-dimethylglycine (0.0264 g, 0.246 mmol) in anhydrous methylene chloride (12 ml) was added 1,3-dicyclohexylcarbodiimide (0.15 g, 0.72 mmol) ) and 4-dimethylaminopyridine (0.025 g, 0.2 mmol). The reaction mixture was stirred for 1 day in an anhydrous medium. Then an additional 50 g of DCC was added and stirring continued for another 6 hours. The reaction mixture was filtered and the filtrate was evaporated in the presence of nitrogen. The residue was chromatographed on silanized silica gel (35 g, 26 cm) and subsequently eluted with a mixture of ethyl acetate and petroleum ether (1: 1) and ethyl acetate. After which the ethyl acetate: petroleum ether fractions were slowly evaporated and the resulting solid was filtered. The mother liquor was concentrated, and petroleum ether was added until turbidity appeared, after which it was discarded. There was obtained 0.14 g (61%) of compound (m.p. 168-171 ^{° C,} dec.). In the NMR spectrum of the compound (300 MHz, CDCl ₃ ), the resonances of the 2′-proton gave a shift from 4.71 ppm in taxol to 5.59 ppm, which corresponds to esterification at the 2-position. All other resonances of the spectrum are in accordance with the proposed structure. HPLC purity was 98-99.5%. Mass spectroscopy: (FAB) m / e 939 (M + H) ⁺ .

Elemental analysis: C ₅₁ H ₅₈ N ₂ O ₁₅
Calculated,%: C 65.26; H, 6.22; N, 2.98.

 Found,%: C 65.16; H 6.28; N, 3.13.

 Compound 2-methanesulfonic acid salt of 2 '- (N, N-dimethylglycyl) taxol.

2 '- (N, N-Dimethylglycyl) taxol (0.06 g, 0.064 mmol) was dissolved in t-butanol (2 ml) and water (1.5 ml). The mixture was cooled to ^-5 ° C and added dropwise methanesulfonic acid (3.1 ml, 2 mg / ml, 0.0645 mmol), and the mixture was stirred at 0-5 ^{° C} for one minute and filtered through a 20 micron filter (milliporous) and placed in a flask cooled in a mixture of ice and isopropanol. The solution was freeze-dried and 0.058 g of product (88%) was obtained, mp. 170-173 ^about S.

Elemental analysis: C ₅₂ H ₆₂ N ₂ SO ₁₈ x x2H ₂ O
Calculated,%: C 57.83; H, 6.27; N, 2.6.

 Found,%: C 57.49; H, 6.06; N, 2.73.

Physical properties Mol.m. 1035 mp 170-173 ^C. (dec.) Solubility 15 mg / ml (slightly hazy)
2 mg / ml (light)
Chemical Stability Studies
The resulting compound was subjected to a stability test in accordance with the following procedures.

Derivatives were studied at various pH ^at 25 ° C and 37 ° ^C. The plasma studies were conducted at ³⁷ ° C using human and rat plasma. Human plasma was obtained from Watkins Hospital, and rat plasma was obtained from the University of Kansas (Animal Care Unit). The concentration of the derivative was about 20-25 μg / ml. A mother solution of the compound was obtained in an amount of 0.8-1.0 mg / ml and added to the plasma to the desired concentration (20-25 μg / ml), 100 μl of the samples were removed and sharply cooled with 250 μl of acetonitrile and centrifuged to precipitate plasma proteins. The kinetics of degradation was investigated using HPLC with plotting the peak area versus time. T ₉₀ and t ₅₀ were calculated _. Plasma and chemical studies were performed using high-performance liquid chromatography using a P-8 column (15 cm) and a forcolumn (5 cm). The detector was installed at 227 nm. The mobile phase consisted of 0.02 M acetate (pH 5) and acetonitrile at a ratio of 50:50 or 35:65 and the flow rate varied between 1-1.5 ml / min or this phase consisted of the same solvents containing 0.001 M tetrabutylammonium bisulfate, and the flow rate was 1 ml / min.

When studying stability, the disappearance of the peak of the compound leads to the formation of a peak having a retention time corresponding to taxol. The identification of this peak was also confirmed by studies of the degradation of new derivatives. Thus, 2 '(DMS) taxol was incubated and purified using preparative TLC. After purification, the product was analyzed by HPLC and spectroscopic methods. The product showed a peak of the molecular ion at m / e 860 (M + Li) ⁺ , which confirms the fact that the resulting product is taxol.

Соединение 3.HPLC operating conditions. Column R-8, 150 mm long,
4.6 mm inside Mobile phase 0.02 M acetate (pH 5)
and acetonitrile 50:50. Kratos Spectroflow 757 detector Flow rate 1 ml / min Retention time 1 1.2 ml (Compound 2) 5.5 ml (Taxol)
The results of the study of chemical stability Conditions t 1/2 h 0.02 M acetate 0.1 mg / ml (pH 3.5, 25 ^° C) 96.2 (pH 4.5, 25 ^° C) 55.4 Water 2 mg / ml (pH 3.8, 37 ^° C) 89.8
Plasma Stability, 37 ^{° C} t 1/2 min Condition Rat Plasma 20 ug / ml 3.05 Dog Plasma 20 / ml human plasma was 121.6 20 ug / ml 198.6
PRI me R 2. Salts 2 '(N, N-diethylaminopropionyl) taxol

Compound 3.

HCl-salt 2 '- (3-N, N-diethylaminopropionyl) taxol
To a solution of taxol (0.12 g, 0.14 mmol) in CH ₂ Cl ₂ (12 ml) containing N, N-diethylaminopropionic acid hydrochloride (0.025 g, 0.145 mmol) was added 1,3-dicyclohexylcarbodiimide (0.08 g, 0.38 mmol) and 4-dimethylaminopyridine (0.01 g, 0.081 mmol). The resulting mixture was stirred for 24 hours at room temperature. Then the reaction mixture was filtered and the filtrate was evaporated in the presence of nitrogen. The crude material was chromatographed on silanized silica gel columns (18 g, 22 cm) and subsequently eluted with a mixture of ethyl acetate and petroleum ether (1: 2), a mixture of ethyl acetate and petroleum ether (1: 1) and ethyl acetate. Fractions of a mixture of ethyl acetate and petroleum ether on evaporation formed a solid precipitate, which was then filtered. Stock solutions containing the product were combined, concentrated, and added until the solution became cloudy, after which it was discarded. After filtration, 0.068 g of product (48%) was obtained. Mp 188-191 ^C. MS (FAB) m / e 981 (M ⁺ H) +.

The NMR spectrum (300 Hz, CDCl ₃ ) showed that 2′-proton resonances from 4.71 ppm in taxol to 5.53 ppm ethyl groups showed methyl resonance Ar 1.0 ppm and CH ₂ resonance Ar 2.52 ppm All other resonance characteristics corresponded to the expected compound.

Elemental analysis: C ₅₄ H ₆₅ ClN ₂ O ₁₅
Calculated,%: C 63.73; H, 6.43; N, 2.75.

 Found,%: C 64.84; H 6.84; N, 2.89.

Physical properties of compound 3 1017.56 mp 186-189 ^о С (decomp.) Solubility ≈0.8 mg / ml HPLC conditions R-8 column, 150 mm (length),
4.6 mm (inner diameter). Mobile phase 0.02 M acetate (pH 5)
and acetonitrile 35: 65 Kratos Spectroflow 757 detector Flow rate 1.5 ml / min Retention time 16.71 ml (compound 3) Chemical stability Conditions t 1/2 h 0.02 M acetate (0.01 mg / ml pH 3.5, 25 ^C) 438.6 0.02 M phosphate (0.02 mg / ml pH 7.4, 25 ° ^C) 0.25 The stability of the plasma, 37 ^{° C} t 1/2 min Condition human plasma 4 , 2
Compound 4-methanesulfonic acid salt of 2 '(N, N-diethylaminopropionyl) taxol
To obtain the methanesulfonic acid salt of N, N-dimethylaminopropionic acid, 10 g of QAE-Sephadex (Pharmacia) was wetted with 0.1 M NaCl, 75% of the obtained material was poured into a column. The column was washed with distilled water (700 ml). Then the column was balanced with 500 ml of CH ₃ SO ₃ Na (obtained from 0.5 M methanesulfonic acid and 0.5 M sodium hydroxide and titrated for pH 6). The complete disappearance of Cl ^- was evaluated by collecting the eluate and testing for Cl ^- by adding a few drops of 1% silver nitrate in a few drops of nitric acid. Then the column was washed with distilled water to a neutral pH.

 2.5 g of the HCl salt of N, N-diethylaminopropionic acid in 15 ml of water was poured into a column and eluted with water. 4 fractions of 50 ml were collected. The first few fractions contained the product. These fractions were combined and the solvent was removed. The residue was dissolved in a mixture of methylene chloride and ethanol and dried in the presence of magnesium sulfate, after which the solvent was removed and 3.1 g of product was obtained. This product was precipitated from ethanol and ether, whereby 2.6 g of the methanesulfonic acid salt of N, N-diethylaminopropionic acid was obtained.

To a solution of taxol (0.05 g, 0.058 mmol) and methanesulfonic acid salt of N, N-diethylaminopropionic acid (0.014 g, 0.058 mmol) in methylene chloride (10 ml) was added 1,3-dicyclohexylcarbodiimide (0.061 g, 0.3 mmol) . The mixture was stirred for 24 hours at room temperature, then the reaction mixture was filtered, and the filtrate was evaporated in a nitrogen atmosphere. The residue was chromatographed on a silanized silica gel column and eluted with a mixture of ethyl acetate and petroleum ether (1: 1) and ethyl acetate. After slowly evaporating the ethyl acetate: petroleum ether fractions, 0.048 g of product was obtained (74%). Mp 170-174 ^about S.

2'-(troc) таксол (соединение 5).Physical properties Mol.m. 1077.12 mp 170-174 ^C. The solubility> 10 mg / ml of HPLC-purity:> 99% Chemical stability. Conditions t 1/2 (hour) 0.02 M acetate (pH 4.5, 25 ^° C) 305 0.02 M acetate (pH 5.5, 25 ^° C) 20.7
PRI me R 3. Obtaining 7- (N, N-dimethylglycyl) taxol or its salt

2 '- (troc) taxol (compound 5).

Taxol (0.27 g, 0.316 mmol) was dissolved in CH ₂ Cl ₂ (10 ml) and pyridine (1.5 ml). The reaction mixture was cooled to -20 - (- 25 ^° ) C, and 2,2,2-trichloroethyl chloroformate (80 μl) was added. Then the reaction mixture was stirred at this temperature for 3 hours. After that, another 25 μl of chloroformate was added and the resulting mixture was stirred overnight. Then the reaction mixture was diluted with CH ₂ Cl ₂ (50 ml) and washed successively with 0.1 N. HCl (25 ml x 2), 0.05 M cold NaHCO ₃ (25 ml x 1) and water. The organic extract was dried over anhydrous MgSO ₄ , and the solvent was removed. The resulting material was dried using preparative TLC on plates with silanized silica gel, developed in a mixture of ethyl acetate and petroleum ether (1: 3), the fraction above taxol was separated, eluted with ethyl acetate, and the solvent was removed, resulting in 0.32 g (97%) product, so pl. 221-226 (decomp., Soft. 160 ^about C).

2 '- (troc) -7-N, N-dimethylglycyl) taxol (compound 6)
A mixture of 2 '- (troc) taxol (0.27 g, 0.262 mmol) and N, N-dimethylglycine (0.054 g, 0.524 mmol) was dissolved in CH ₂ Cl ₂ (15 ml). To this solution were added 1,3-dicyclohexylcarbodiimide (0.215 g, 1.04 mmol) and 4-dimethylaminopyridine (0.025 g, 0.2 mmol), and the resulting mixture was stirred for two days at room temperature. Then the mixture was filtered and the solvent was removed. The resulting product was purified using preparative TLC on plates with silanized silica gel and developed in a mixture of ethyl acetate and petroleum ether (1: 1). The fraction below taxol (Rf 0.47, ethyl acetate and petroleum ether (1: 1) was separated and eluted with ethyl acetate, after which the solvent was removed and 0.26 g of product was obtained (89%).

Mp 176-180 ^{° C} (dec.).

Elemental analysis: C ₅₄ H ₆₀ Cl ₃ N ₂ O ₁₇
Calculated,%: C 58.16; H 5.42; N, 2.51.

 Found,%: C 58.68; H, 6.00; N, 3.18.

 7- (N, N-Dimethylglycyl) taxol (compound 7).

To a solution of 2 '- (troc) -7- (N, N-dimethylglycyl) taxol (0.335 g, 0.3 mmol) in a mixture of methanol and acetic acid (9: 1) (12 ml) was added zinc dust (0.275 g) and the resulting mixture was stirred for 25 minutes at room temperature. Then the mixture was filtered, the filtrate was concentrated to 1 ml and diluted with CH ₂ Cl ₂ (35 ml), and then washed sequentially with 0.01 N. HCl (20 ml x 2), 0.01 M cold NaHCO ₃ and water. The organic extract was dried with anhydrous NaSO ₄ , the solvent was removed and 0.24 g of product was obtained. This compound was purified using preparative TLC on plates with silanized silica gel (20 x 20, 3 Nos) and developed in a mixture of CH ₂ Cl ₂ and ethyl acetate (7: 1). The fraction related to 7- (DMS) taxol (Rf 0.35) was separated and eluted with ethyl acetate and ethanol, then the solvent was removed and 0.19 g of product (68%) was obtained, MS (FAB) m / e 939 (M + H ) ⁺ .

In the NMR spectrum (300 MHz, CDCl ₃ ), 7-H resonances at 4.33 ppm in taxol appeared as a double doublet at 5.65 ppm N- (CH ₃ ) ₂ resonance appeared as a singlet at 2.35 ppm . The glycinate methyl group was detected at 3.16 ppm.

7- (Dimethylglycyl) Taxol methanesulfonic acid salt (compound 8)
7- (Dimethylglycyl) taxol (0.065 g, 0.069 mmol) was dissolved in t-butanol (2.5 ml) and water (1 ml). The solution was cooled to 5-10 ^{° C} and methanesulfonic acid (3.36 ml, 2 mg / ml, 0.0697 mmol). The mixture was stirred for 2 minutes, filtered through a millipore filter into an ice-cooled tube. The filtrate was dried by freezing and received 0,066 g of the product (94%). Mp 164-168 (decomp.)
Elemental analysis: C ₅₂ H ₆₂ N ₂ O ₁₈ ˙ 2H ₂ O
Calculated,%: C 58.29; H, 6.19; N, 2.6.

 Found,%: C 58.05; H 6.00; N 1.72.

Physical properties Like m. 1035 mp 164-168 ^C. Solubility> 2 mg / ml of HPLC conditions. Column R-8, 150 mm (long),
4.6 mm (internal diameter) Mobile phase 0.02 M acetate (pH 5) and acetonitrile 35:65 Kratos Spectroflow 757 detector Flow rate 1.5 ml / min Retention time 15.07 ml (compound
8)
Chemical stability t 1/2 h Condition 0.02 M acetate (pH 3.5, 25 ° ^C) 3397 0.02 M acetate (pH 4.5, 25 ° ^C) 1719
0.02 M phosphate (pH 7.4, 25 ^° C) 33.8
Plasma Stability, 37 ^{° C} t 1/2 h Condition rat plasma 20 ug / ml human Plasma 17.3 20 ug / ml human Plasma 27.7 10 ug / ml 24.4
PRI me R 4. Obtaining 2 ', 7- (dimethylglycyl) taxol.

Таксол (0,06 г, 0,0702 ммоль) растворяли в безводном метиленхлориде (5 мл) и добавляли N, N-диметилглицин (0,015 г, 0,145 ммоль). К полученной смеси добавляли 1,3-дициклогексилкарбодиимид (0,08 г, 0,388 ммоль) и 4-диметиламинопиридин (0,008 г, 0,065 ммоль). Реакционную смесь перемешивали в течение 24 ч при комнатной температуре и фильтровали. Затем растворитель удаляли из фильтрата. Остаток очищали с помощью препаративной ТСХ на тарелках с силанизированным силикагелем и проявляли в смеси этилацетата и петролейного эфира (1:1). Фракцию (Rf 0,17) выше диметиламинопиридина отделяли и элюировали смесью этилацетата и этанола, а растворитель удаляли. Остаток перекристаллизовывали из смеси этилацетата и петролейного эфира, в результате чего получали 0,046 г продукта (64%). Т.пл. 194-198^оС. МС: m/e 1024 (М⁺).

Taxol (0.06 g, 0.0702 mmol) was dissolved in anhydrous methylene chloride (5 ml) and N, N-dimethylglycine (0.015 g, 0.145 mmol) was added. To the resulting mixture were added 1,3-dicyclohexylcarbodiimide (0.08 g, 0.388 mmol) and 4-dimethylaminopyridine (0.008 g, 0.065 mmol). The reaction mixture was stirred for 24 hours at room temperature and filtered. Then, the solvent was removed from the filtrate. The residue was purified using preparative TLC on plates with silanized silica gel and developed in a mixture of ethyl acetate and petroleum ether (1: 1). A fraction (Rf 0.17) above dimethylaminopyridine was separated and eluted with a mixture of ethyl acetate and ethanol, and the solvent was removed. The residue was recrystallized from a mixture of ethyl acetate and petroleum ether, whereby 0.046 g of product was obtained (64%). Mp 194-198 ^about C. MS: m / e 1024 (M ⁺ ).

In the NMR spectrum (300 MHz, C DCl ₃ ), the C ₂ proton of 4.71 ppm and the C ₇ proton of 4.33 ppm are shifted to 5.5 ppm and 5.6 ppm, respectively, which indicates an esterification at 2 '- and 7-position. A -CH ₃ -proton was also recorded as a singlet at 2.3 ppm.

Elemental analysis: C ₅₅ H ₆₆ ON ₃ O ₁₆
Calculated,%: C 63.39; H 6.48; N, 4.03.

 Found,%: C 63.00; H 6.98; N, 3.98.

Synthesis of dimethanesulfonic acid salt of 2 ', 7- (N, N-dimethylglycyl) taxol
To a solution of 2,7-dimethylglycyl taxol (60 mg, 0.0585 mmol) in t-butanol (2 mL) and water (1 ml) cooled to 0-5 ^{° C,} was added methanesulfonic acid (11.37 mg, 3 79 ml, 0.117 mmol). The mixture was stirred at ^0-5 ° C for 2 min, and filtered through a millipore filter (0.2 micron) and the filtrate freeze dried to give 62 g of product. Mp 160-163 ^about S.

Physical properties Mol.m. 1217 Mp 160-163 ^о С (decomp.) Solubility> 10 mg / ml HPLC purity ≈96% HPLC operating mode RP8 column, 150 mm, 4.6 mm
(internal dia.) Mobile phase 0.02 M acetate mixture
pH 5 and acetonitrile
(50:50) containing
0.005 M TBA Kratos Spectroflow 757 Detector Flow rate 1 ml / min Retention time 6.64 ml
5.8 ml Taxol
PRI me R 5. Obtaining 7- (L-alanyl) taxol or its salt.

2 ', 7-di (t-BOC-L-alanyl) taxol
To a solution of taxol (0.021 g, 0.246 mmol) and t-BOC-L-alanine (0.14 g, 0.739 mmol) in methylene chloride (15 ml) was added 1,3-dicyclohexylcarbodiimide (0.25 g, 1.21 mmol) and dimethylaminopyridine (0.025, 0.20 mmol). The mixture was stirred for 24 hours at room temperature and filtered. The residue was chromatographed on silanized silica gel columns (20 g, 14 cm) and eluted with ethyl acetate and petroleum ether (1: 1) and ethyl acetate. The ethyl acetate-petroleum ether fractions containing the distributed derivative were poured and the solvent was removed, whereby 0.27 g of compound (92%) was obtained. Mp 158-161 ^{° C} (dec.)
7-L-Alanyl Taxol
2 ', 7-Di- (t-BOC-L-alanyl) taxol (0.29 g, 0.242 mmol) and formic acid (2 ml) were mixed at room temperature for 40 minutes and excess formic acid was removed in the presence of nitrogen. The residue was dissolved in ethanol and petroleum ether was added. The solid was filtered off to give 0.27 g, 2 ', 7-di (alanyl) taxol.

The dialanyl derivative thus obtained was diluted in acetonitrile (4 ml) and phosphate buffer (0.02 M, pH 7.4, 50 ml) and the mixture was stirred at room temperature for 12 hours. The pH of the solution was raised to 6.8 using a small amount (ml) of 5% Na ₂ HPO ₄ . The turbid solution was stirred at room temperature for another 8 hours. Then the reaction mixture was diluted with methylene chloride (50 ml) and cold NaHCO ₃ (0.05 M, 50 ml) was added. Then the reaction mixture was extracted with methylene chloride (50 ml x 3), and the resulting organic extract was washed once with water and dried with anhydrous sodium sulfate. After which the solvent was removed and 0.24 g of product was obtained. This compound was purified by silica gel column chromatography to obtain 0.135 g of the product (63%). Purity 95%. Mp 159-163 ^about S.

Mass spectrum: (FAB) m / e 925 (M + H). In the NMR spectrum (300 MHz, CDCl ₃ ) 7-H at 4.33 ppm in taxol was recorded as a doublet of doublets at 5.65 ppm. The CH ₃ group on the alanine molecule is fixed as a doublet at 1.27 ppm.

Elemental analysis: C ₅₀ H ₅₈ N ₂ O ₁₆ x x 2.5H ₂ O
Calculated,%: C 61.91; H 6.54; N, 2.89.

 Found,%: C 61.41; H 6.59; N, 2.78.

7- (Alanine) Taxol Methanesulfonic Acid Salt
To a solution of 7- (alanyl) taxol (62 mg, 0.0658 mmol) in t-butanol (2 mL) and water (1 ml) cooled to 0-5 ^{° C,} was added methanesulfonic acid (6.39 mg, 2 , 13 ml, 3 mg / ml) and the resulting mixture was stirred at this temperature for 2 minutes, after which the mixture was filtered through a millipore filter (0.2 μM). The filtrate was freeze dried and 66 mg of product was obtained. Mp 180-184 ^about S.

Physical properties Mol.m. 1021 mp 180-184 ^C. (dec.) Solubility> 2 mg / ml
Operating conditions HPLC Column RP-8, 150 mm (length),
4.6 mm (internal dia.) Mobile phase 0.02 M acetone mixture
ta (pH 5)
and acetonitrile (50:50),
containing 0.001 M
tetrabutyl bisulfate
ammonium detector Kratos Spectroflow 757 Flow rate 1 ml / min Retention time 8.7 ml 7.3 ml Taxol
Plasma stability, 37 ^° C Conditions: t 1/2 h Human plasma 20 mcg / ml 11.9
PRI me R 6. Obtaining 2 '- (alanine) taxol
Synthesis of 2 '(CBZ-L-Alanyl) Taxol
To a solution of taxol (30 mg, 0.0036 mmol) and CBZ-L-alanine (8.5 mg, 0.036 mmol) in methylene chloride (5 ml) were added DSS (45 mg) and 4-dimethylaminopyridine (4 mg) and the resulting mixture stirred for 3 days at room temperature. The reaction mixture was filtered and the solvent was removed from the filtrate. The residue was purified using preparative TLC on plates with silanized silica gel and developed in a mixture of ethyl acetate and petroleum ether (1: 1), the layer above taxol was separated and eluted with ethyl acetate, after which the solvent was removed to obtain 28 g of 2 '- (CBZ-L-alanyl ) taxol.

Synthesis of 2 '(Alanyl) Taxol by Unblocking 2' (CBZ-L-Alanyl) Taxol
2 '(CBZ-L-alanyl) taxol was dissolved in ethanol in the presence of an organic acid, such as formic acid, and stirred for 2 hours at room temperature in the presence of 5% palladium carbon. The reaction mixture was filtered to remove the catalyst, and then the solvent was removed. The crude product was dissolved in ethanol and petroleum ether was added, whereby 2 '(alanyl) taxol was obtained in the form of a format or acetate with a low to moderate yield.

PRI me R 7. Obtaining 2 '- (lysyl) taxol
Synthesis of 2 '(N-di-t-BOC-lysyl) Taxol
To a mixture of taxol (30 mg, 0.035 mmol) and N-di-t-BOC-L-lysine (19 mg, 0.0368 mmol) in methylene chloride (10 ml) were added DSS (100 mg) and 4-dimethylaminopyridine (10 mg ) and stirred for 2 days at room temperature. After which the mixture was filtered and the solvent was removed. The residue was purified using preparative TLC on silanized silica gel plates and developed in a mixture of acetate and petroleum ether (1: 1), the layer above taxol was separated and eluted with ethyl acetate, then the solvent was removed and 20 mg of product was obtained.

Synthesis of 2 '- (Lysyl) Taxol by Unblocking the t-BOC Group
The taxol derivative of the Nt-BOC-protected amino acid was reacted with formic acid (99%, Sigma) for 30-40 minutes at room temperature, the excess formic acid was removed by evaporation in the presence of nitrogen. The resulting product was purified by crystallization or chromatography, whereby a taxol derivative of an N-unblocked amino acid was obtained as a format (salt).

PRI me R 8. Obtaining 2 '- (L-alanyl) taxol
Synthesis of 2 '- (FMOC-L-Alanyl) Taxol
To a solution of taxol (60 mg, 0.072 mmol) and N-FMOC-L-alanine (22.4 mg) in methylene chloride (6 ml) was added DCC (60 mg) and 4-dimethylaminopyridine (2 mg), after which the mixture was stirred in for two days at room temperature and filtered. The solvent was removed from the filtrate. The resulting product was purified using preparative TLC on plates with silanized silica gel and developed in a mixture of ethyl acetate and petroleum ether (1: 2). The layer above taxol was separated, the solvent was removed, and 48 mg of a product containing 2 '(FMOC-L-alanyl) taxol was obtained. Mp 162-164 ^{° C} (dec.).

Unlocking the FMOC group of the protected amino acid derivative of taxol
The N-FMOM protected amino acid derivative of taxol was reacted with piperidine in methylene chloride for 2 hours, after which the solvent was removed. The residue was purified by chromatography, whereby an unlocked amino acid derivative of taxol was obtained.

 Pharmaceutical Compositions

 The compounds of the proposed method can be included in the pharmaceutical compositions in pure form or in the form of their pharmaceutically acceptable salts, in particular in the form of non-toxic pharmaceutically acceptable acid addition salts or acceptable basic salts. These salts can be obtained from the compounds of this invention in accordance with standard chemical technology.

 Typically, salts are prepared by reacting the free base or acid with stoichiometric amounts or with excess amounts of the desired salts to form an organic or inorganic acid in an appropriate solvent or in a combination of solvents. For example, the free base can be dissolved in an aqueous solution of the corresponding acid and the salt isolated using standard techniques such as evaporation of the solution. Alternatively, the free base can be dissolved in an organic solvent, such as lower alkanoyl, ether, alkyl ester, or mixtures thereof, for example, methanol, ethanol, ether, ethyl acetate, a solution of ethyl acetate and ether, and the like, after which the solution treated with the appropriate acid to form the corresponding salt. The resulting salt is isolated using standard techniques, for example, by filtering the target salt after self-isolation from the solution, or by precipitation by adding a solvent in which this salt does not dissolve, and then isolating it.

 Derivatives of taxol of the present invention can be used in the treatment of cancer due to their cytotoxicity and antitumor activity. New compounds can be introduced in the form of tablets, pills, powder mixtures, capsules, injectable solutions, suppositories, emulsions, dispersions, food additives, and other suitable forms. A pharmaceutical preparation containing the inventive compound is usually mixed with a non-toxic pharmaceutical organic excipient or non-toxic pharmaceutical inorganic excipient in an amount of about 0.01 mg to 2500 mg or higher for a single dosage form, and preferably about 50-500 mg. Examples of pharmaceutically acceptable carriers include mannitol, urea, dextran, lactose, potato or corn starches, magnesium stearate, talc, vegetable oils, polyalkylene glycols, ethyl cellulose, poly (vinylpyrrolidone), calcium carbonate, ethyl oleate, isopropyl myristate, benzene bilobenzo potassium carbonate, silicic acid, and other commonly used acceptable carriers. Pharmaceutical preparations may also contain non-toxic additives, such as emulsifying, preservative, wetting agents and others, for example, sorbitan monolaurate, triethanolaminoleate, polyoxyethylene monostearate, glycerol trinmitate, diocyl sodium sulfoccinate and the like.

 A standard method for preparing a tablet preparation containing the active ingredient is that the active agents are first mixed with a non-toxic binder such as gelatin, acacia resin, ethyl cellulose, etc. Mixing is usually carried out in a standard V-mixer and subject to an anhydrous environment. Then, the freshly obtained mixture is compressed using a tabletting machine. Freshly prepared tablets may be coated or left uncoated. Typical suitable coatings are non-toxic coatings containing shellac, carnauba wax, copolymers of styrene and maleic acid, and the like. For oral administration, compressed tablets containing 0.01 mg, 5 mg, 25 mg, 50 mg, 500 mg, etc. up to 2500 mg, are made in accordance with the description of the invention and using standard techniques.

 For the manufacture of tablets, the active compound, corn starch, lactose, dicalcium phosphate, calcium carbonate are usually mixed under anhydrous conditions and using a V-mixer until a uniform mixture is obtained. Then a corn starch paste is prepared in the form of a 10% paste, and the mixture just obtained is mixed with this paste until a uniform mixture is obtained. This mixture is then passed through a standard fine sieve, dried in an anhydrous atmosphere and then mixed with calcium stearate, after which the mixture is compressed into tablets and coated if desired. In a similar manner, tablets are prepared containing 10, 50, 100 and 150 mg of the active substance.

 The following is an example of the manufacture of preparations containing the compound of the present invention in the form of tablets.

 The preparation I contains the following ingredients, mg per tablet: active compound 50.0; corn starch 15.0; corn starch paste 4.0; calcium carbonate 15.0; lactose 67.0; calcium stearate 2.0; dicalcium phosphate 50.0.

 The manufacture of capsules containing 10 mg to 2500 mg of a compound of the present invention for oral administration consists in mixing said active compound with a non-toxic carrier and introducing the resulting mixture into a polymer shell, usually gelatin or the like. It is known that capsules can be of soft form, which are made by incorporating the compound in carefully mixed dispersion mixtures together with food compatible carriers, or these capsules can be of hard form, which mainly contain the compound of the present invention mixed with non-toxic solid carriers such as talc calcium stearate, calcium carbonate and the like. An example of capsules containing 25 mg, 75 mg, 125 mg, etc. of a new compound in its pure form or in the form of mixtures of one or more new compounds, is preparation II.

 The preparation II contains the following ingredients, mg per capsule: active ingredient 50.0; calcium carbonate 100.0; lactose, U.S.P. 200.0; starch 130.0; magnesium stearate 4.5.

 These ingredients are mixed in a standard mixer and commercially available capsules are filled into the resulting mixture. The higher the concentration of the active component, the less lactose should be used in this composition.

 The compounds of this invention can be freeze-dried, and if necessary, can be combined with other pharmaceutically acceptable excipients, commonly used in the preparation of compositions intended for parenteral administration by injection. For such an introduction, the compositions may be diluted in water (normal or saline) or in a mixture of water and an organic solvent such as propylene glycol, ethanol, etc.

The administered dose may be in the form of a single or divided dose and vary depending on the particular compound used in each case, the method of administration, the weight of the patient and his physical condition. The administered dose is not a strictly defined parameter, however, as a rule, the indicated dose is an effective amount or an equivalent (on a molar basis) amount of the pharmacologically active free form produced from the drug as a result of the metabolic release of the active drug in order to achieve the desired pharmacological or physiological effect. Typically, the administered dose is about 0.8-8 mg / kg body weight or about 50-275 mg / m ² of the patient’s body surface, and preferably about 230-275 mg / m ² .

 Biological activity.

 The proposed derivatives of taxol are valuable drugs due to their antitumor activity, in particular, these compounds can be used in the treatment of certain types of cancer, for example lung tumors, melanoma, leukemia, breast tumors and colon cancer.

 The biological activity of these derivatives of taxol was tested through in vitro studies and measurement of the kinetics of accumulation of microtubes; in vitro kinetics studies on B16 melanoma cell culture; in vivo studies of an MX-1 graft grafted onto an SRC renal capsule.

In vitro microtubule accumulation kinetics study
Microtubules are an integral part of eukaryotic cells, and microtubule complexes play an important role in cell division and proliferation. It is known that the polymerization of microtubules is very susceptible to calcium, which has the ability to inhibit the accumulation of tubulin and depolymerization of already accumulated microtubules. Known antitumor compounds have been investigated for their effect on microtubule accumulations.

 Vinca alkaloids, such as vinblastine and vincristine, contributed to the destruction of cell microtubules, i.e. in vitro they showed the ability to inhibit the accumulation of microtubules and to depolymerize microtubules in a stable state.

 Similarly, colchicine also showed the ability to depolymerize microtubules in cells.

On the other hand, it was found that taxol has a completely unique mechanism of action, which consists in the fact that it stimulates the accumulation of microtubules, but inhibits their separation, thereby interfering with the G ₂ and M phases of cell cycles and cell division. In vitro studies have shown that microtubules already polymerized in the presence of taxol become resistant to depolymerization caused by other agents, such as CaCl ₂ or low temperatures, which usually promote microtubule depolymerization.

 A study was conducted of the effect of taxol derivatives on microtubule accumulation. This study was carried out using 2'- and 7-derivatives in accordance with known in vitro procedures. The ability of these compounds to stimulate the accumulation of microtubules can be traced as follows: taxol> 7- (N, N-dimethylglycyl) taxol (11)> 2 '- (N, N-diethylaminopropionyl) taxol (8)> 2' (N, N- dimethylglycyl) taxol (2). This study showed that the free 2'-hydroxyl group plays a major role in the accumulation of microtubules, 2'-derivatives were active only if the 2'-hydroxyl group remained free throughout the entire experimental procedure. 7-derivatives have a free 2'-hydroxyl group and, therefore, are active. The result obtained is in accordance with the results of the activities of 2'- and 7-acetyl taxol.

 In vitro study of B16 cell culture.

 In vitro melanoma B-16 cell culture studies were performed to confirm the activity of the taxol derivatives of the present invention. These studies were carried out in accordance with standard procedures.

 When studying the proliferation of B-16 melanoma cells, efficiencies were established in the following increasing order: taxol> 2 '- (N, N-dimethylglycyl) taxol> 2- (N, N-diethylaminopropionyl) taxol> 7- (N, N-dimethylglycyl) taxol. These studies and studies of other kinetics have shown that the activity of 2'-derivatives is due to taxol. In all likelihood, the 2'-derivatives act as a prodrug. However, 7-derivatives have their own activity and, obviously, are not a prodrug.

 In vivo studies.

 The third type of experiment, conducted to confirm the biological activity of taxol derivatives, was an in vivo study, which was carried out by introducing an MX-1 human breast carcinoma transplant into the mouse capsule. The tests were carried out using a known technique.

 Procedure.

 In the described procedure used test groups of animals (6 mice in each group) and control groups (12 mice in each group). A tumor fragment (transplant of human breast carcinoma MX-1) was implanted into the membrane membrane of the kidney of each mouse.

 The study was carried out as follows.

 Day 0. Animals were anesthetized. Fixed body weight (weight of day 1). The implantable tumor was measured and the results were recorded. After recovery from anesthesia, the animals were randomized. A series of bacterial cultures was prepared. The solubility of the test agents was determined. Mortality was recorded daily.

 Day 1. Controlled culture. In case of contamination, cultures were discarded. Prepared materials for the test. Initial injections of the test agent (in the scruff of the neck) were determined based on body weight. Treatment Q, 4D on day 1, 5 and 9. For each day, a fresh injection of the test agent was prepared and administered according to the body weight of the mouse on that day.

 Day 2. Again checked the culture. In case of contamination, the test was terminated and a recording was made accordingly.

 Days 5 and 9. For each day, a fresh test agent for injection was prepared, which was administered according to body weight on that day.

 Day 11. The experiment was completed and the results were evaluated. Body weight was recorded (mass of day 2). The tumor was measured in OM. (Eyepiece-micrometer-unit - 10 OMe = 1 mm) and measurements were recorded.

, где L представляет более 2-х измерений;
3) При расчете изменения (дельта) средней массы опухоли для каждой группы мышей. Изменение среднего веса опухоли = средняя масса опухоли конечная - средняя масса опухоли;
4) При расчете изменения начальной (дельта) средней массы опухоли для тестируемых (Т) и контрольных (С) групп;
5) При расчете Т/C (%) для всех тестируемых групп с процентом выживания более 65% на последний день эксперимента:
Т/С(% ) = T/C% =

× 100 - если Δ массы Т положительно x 100 - если Δ массы Т положительно.The change in tumor mass (delta) was recorded based on measurements of length and width (mm). The average weight of animals was calculated for day 1 and day 11, and T / C was calculated for all test groups with more than 65% survival on day 11. Dynamics of excess body weight (test minus control) can also be used to assess toxicity. The sizes were measured in OMed. The computer was used in the following calculations:
1) When converted to OMed in millimeters (mm);
2) When calculating the mass of the tumor (mg) based on the size of the tumor mm x mm according to the formula for the volume of the elongated ellipsoid

where L represents more than 2 dimensions;
3) When calculating the change (delta) of the average tumor mass for each group of mice. Change in the average tumor weight = average tumor mass; final - average tumor mass;
4) When calculating the change in the initial (delta) average tumor mass for the test (T) and control (C) groups;
5) When calculating T / C (%) for all tested groups with a survival rate of more than 65% on the last day of the experiment:
T / C (%) = T / C% =

× 100 - if Δ of mass T is positive x 100 - if Δ of mass T is positive.

× 100 - если Δ массы Т отрицательно 100 - если Δ массы Т отрицательно.T / C (%) = T / C% =

× 100 - if Δ of mass T is negative 100 - if Δ of mass T is negative.

 Activity Criteria.

 The initial T / C ≅ 20% value indicates moderate activity. The reproducible T / C ≅ 10% indicates significant activity.

 The test results for several derivatives of taxol are presented in tables 1 and 2.

 From these results it is seen that the proposed taxol derivatives show excellent antitumor activity. Therefore, these compounds can be used as antitumor agents, due to their good biological activity and better water solubility compared to taxol.

где R₁ и R₂ - каждый водород или группа общей формулы II

где n = 1 - 3 - целое число,
R₃ и R₄-C₁-C₃-алкил,
при условии, что по крайней мере один из R₁ и R₂ не является водородом,
отличающийся тем, что таксол подвергают реакции взаимодействия с соединением общей формулы III

где n, R₃ и R₄ имеют указанные значения.1. METHOD FOR PRODUCING TAXOL DERIVATIVES OF GENERAL FORMULA I

where R ₁ and R ₂ are each hydrogen or a group of the general formula II

where n = 1 - 3 is an integer,
R ₃ and R ₄ -C ₁ -C ₃ -alkyl,
provided that at least one of R ₁ and R _{2 is} not hydrogen,
characterized in that taxol is reacted with a compound of general formula III

where n, R ₃ and R ₄ have the indicated meanings.

2. The method according to claim 1, characterized in that taxol and a compound of formula III are reacted in an equivalent ratio of 1: 1 to obtain a 2-substituted compound of general formula 1, where R ₁ is a group of formula II and R ₂ is hydrogen.

3. The method according to p. 1, characterized in that taxol is reacted with a compound of the general formula III to obtain a 2,7-di (substituted) compound of the formula 1, wherein R ₁ and R ₂ are a group of the formula II.

4. The method according to claim 3, characterized in that the 2,7-di (substituted) compound of general formula 1 is subjected to a cleavage reaction to form a 7-substituted compound of general formula 1, where R ₁ is hydrogen, R ₂ is a group of formula II.

 5. The method according to claim 1, characterized in that the process is carried out in the presence of a condensing reagent and not necessarily in the presence of a catalyst.

 6. The method according to claim 4, characterized in that the 2,7-di (substituted) compound of the general formula 1 is subjected to a cleavage reaction at pH 7-7.4.

Claims (6)

1. METHOD FOR PRODUCING TAXOL DERIVATIVES OF GENERAL FORMULA I

where R ₁ and R ₂ are each hydrogen or a group of the general formula II

where n = 1 - 3 is an integer,
R ₃ and R ₄ -C ₁ -C ₃ -alkyl,
provided that at least one of R ₁ and R _{2 is} not hydrogen,
characterized in that taxol is reacted with a compound of general formula III

where n, R ₃ and R ₄ have the indicated meanings. 2. The method according to claim 1, characterized in that taxol and a compound of formula III are reacted in an equivalent ratio of 1: 1 to obtain a 2-substituted compound of general formula 1, where R ₁ is a group of formula II and R ₂ is hydrogen. 3. The method according to p. 1, characterized in that taxol is reacted with a compound of the general formula III to obtain a 2,7-di (substituted) compound of the formula 1, wherein R ₁ and R ₂ are a group of the formula II. 4. The method according to claim 3, characterized in that the 2,7-di (substituted) compound of general formula 1 is subjected to a cleavage reaction to form a 7-substituted compound of general formula 1, where R ₁ is hydrogen, R ₂ is a group of formula II.  5. The method according to claim 1, characterized in that the process is carried out in the presence of a condensing reagent and not necessarily in the presence of a catalyst.  6. The method according to claim 4, characterized in that the 2,7-di (substituted) compound of the general formula 1 is subjected to a cleavage reaction at pH 7-7.4.

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