RU2059631C1 - Derivatives of taxol and pharmaceutical composition showing antitumor activity - Google Patents

Abstract

FIELD: organic chemistry. SUBSTANCE: products: derivatives of taxol of the formula

where R and R′ - amino acid residue taken from the group consisting of alanine, leucine, isoleucine, valine, phenylalanine, proline, lysine or arginine, or the group of the formula

where n = 1-3; R² and R³ is every C₁-C₃-alkyl, or their acid additive salt. Condition: at least one of R² and R³ is not hydrogen atom and compound of the formula

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, leukemia, breast cancer and colon cancer.

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 Е1 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 анафиклактическиподобную реакцию, поэтому использование этого наполнителя, к сожалению, не является хорошим решением указанной выше проблемы разработки подходящих композиций таксола.Taxol is a well-known diterpenoid, sesquiterpene, which is obtained from the bark of the yew tree, Taxus brevifolia, and which has the following structure:

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 E1 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.

 Another objective of the invention is the manufacture of compositions containing derivatives of taxol and non-toxic carriers, which will eliminate the use of toxic carriers, such as cremophor.

 Another objective of the invention is to obtain derivatives of taxol, which would have sufficiently good stability at pH levels suitable for the manufacture of pharmaceutical compositions (pH 3-4), but would have the ability to quickly decompose in vivo at physiological pH values (7.4) , i.e. which could act as a precursor of taxol.

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

I
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 1 3 атомов углерода.The novel taxol derivatives of the present invention can generally be represented as 2'-and / or 7-taxol esters having the formula I:

I
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 are} each hydrogen or alkyl having 1 to 3 carbon atoms.

If in the indicated formula R ² and / or R ³ are not hydrogen, then these compounds can be considered 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: a) derivatives esterified at the 2'-hydroxyl group of taxol; b) derivatives esterified with the 7-hydroxyl group of taxol; and c) derivatives esterified at the 2'- and 7-positions of the hydroxyl groups.

Among the compounds described by the above general formula I, preferred compounds of the invention are the following compounds:
1.2 '- (N, N-diethylaminopropionyl) taxol
2.2 '- (N, N-dimethylglucyl) taxol
3. 7- (N, N-dimethylglycyl) taxol
4.2 ', 7-di- (N, N-dimethylglycyl) taxol
5.7- (N, N-diethylaminopropionyl) taxol
6.2 ', 7-di (N, N-diethylaminopropionyl) taxol
7.2 '- (L-glycyl) taxol
8.7- (L-glycyl) taxol
9.2 ', 7-di (L-glycyl) taxol
10.2 '- (L-Alanyl) Taxol
11.7- (L-Alanyl) Taxol
12.2'7-di (L-alanyl) taxol
13.2 '- (L-leucyl) taxol
14.7- (L-leucyl) taxol
15.2 ', 7-di (L-leucyl) taxol
16.2 '- (L-isoleucyl) taxol
17.7- (L-isoleucyl) taxol
18.2'-7-di (L-isoleucyl) taxol
19.2 '- (L-valyl) Taxol
20.7- (L-valyl) Taxol
21. 2 ', 7-di (L-valyl) taxol
22.2 '- (L-phenylalanil) taxol
23. 7- (L-phenylalanil) taxol
24. 2 ', 7-di (L-phenylalanyl) taxol
25.2 '- (L-propyl) taxol
26.7- (L-propyl) taxol
27.2 ', 7-di (L-propyl) taxol
28.2 '- (L-lysyl) taxol
29. 7- (L-lysyl) taxol
30.2 ', 7-di (L-lysyl) taxol
31.2 '- (L-Glutamyl) Taxol
32.7- (L-Glutamyl) Taxol
33.2'7-di (L-glutamyl) taxol
34.2 '- (L-arginyl) taxol
35.7- (L-Arginyl) Taxol
36. 2'-7-di (L-arginyl) taxol
In studies, it was 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 derivatives of the invention.

-

Согласно схеме II реакцию 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
N protected amino acid + taxol __ → 2

-

According to Scheme II, the protective amino acid reaction 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-dimethylamino-pyridine (DMAP) and pyridine.

 In 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. Preferred are amino acids protected by t-BOC or FMOC groups. 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.

Как указывалось выше, было обнаружено, что 2'-гидроксильная группа таксола является более химически активной, чем 7-гидроксильная группа. Таким образом, в обеих схемах, указанных выше (схема I и cхема II), замещение или этерификация направлены на 2'-положение и осуществляются путем взаимодействия алкилированной или N-защищенной аминокислоты с таксолом при молярном соотношении 1:1 или слегка превышающим 1:1. Указанная реакция, проводимая с использованием разных 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:
C

As indicated above, it was found that the 2'-hydroxyl group of taxol is more chemically active than the 7-hydroxyl group. Thus, in both schemes indicated above (Scheme I and Scheme II), substitution or esterification is directed to the 2'-position and is carried out by reacting an alkylated or N-protected amino acid with taxol in a molar ratio of 1: 1 or slightly greater than 1: 1 . This reaction, carried out using different 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 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.

о

и

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

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 выше для схем I и II.

about

and

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

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 mentioned above 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 используемым 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 finally, the 2'-amino acid is cleaved. This procedure can be schematically represented as follows using t-BOC protected alanyl:

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 the method commonly used to unlock amino acids, for example, using formic acid.

 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 subjected to a reaction of interaction with 2-3 mol. equivalent of an N-substituted amino acid (t-BOC, CBZ or FMOC-protected) in methylene chloride in the presence of DCC 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 may 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.

2,7-(двузаме

2′
С. Получение производных 2',7-двузамещенного таксола
Двузамещенные производные могут быть получены с помощью процедур, указанных ниже, или части этих процедур.Scheme V
Taxol +

2.7- (dvuzame

2 ′
C. Preparation of 2 ', 7-disubstituted Taxol Derivatives
Disubstituted derivatives can be obtained using the procedures indicated below, or part of these procedures.

е

алкилированная)
Схема VII: Замещение аминокислотами
Таксол+

__→

а

Схема VI, в основном, аналогична схеме I (см.выше), 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 +

e

alkylated)
Scheme VII: Substitution with Amino Acids
Taxol +

__ →

a

Scheme VI is basically the same as Scheme I (see above), 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 chemical activity 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 protecting 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.

 It is obvious that any specialist, using the above descriptions, can obtain the compounds of this invention in full.

 Examples of a preferred embodiment of the invention.

а) Соединение 1: 2'-(N,N-Диметиглицил)таксол
К раствору таксола (0,21 г, 0,246 ммоль) и N,N-диметиглицина (0,0264 г, 0,246 ммоль) в безводном метиленхлориде (12 мл) добавляли 1,3-дициклогексилкарбодиимид (0,15 г, 0,72 ммоль) и 4-диметиламинопиридин (0,025 г, 0,2 ммоль). Реакционную смесь перемешивали в течение 1 дня в безводной среде, после чего добавляли еще 50 г DCC и продолжали перемешивать еще 6 ч. Реакционную смесь отфильтровывали и фильтрат выпаривали в присутствии азота. Остаток хроматографировали на силанизированном силикагеле (35 г, 26 см) и последовательно элюировали смесью этилацетата и петролейного эфира (1:1) и этилацетатом, после чего фракции этилацетата петролейного эфира медленно выпаривали и полученное твердое вещество фильтровали. Маточный раствор концентрировали и добавляли петролейный эфир до тех пор, пока не появлялось помутнение, после чего его отбрасывали. В результате получали 0,14 г (61%) соединения (т. пл. 168-171^оС, разл. ). В ЯМР-спектре соединения (300 МГц, CDCl₃) резонансы 2'-протона давали сдвиг от 4,71 ppm в таксоле до 5,59 ppm, что соответствует этерификации в 2-положении. Все остальные резонансы спектра находятся в соответствии с предполагаемой структурой. БЭЖХ-чистота составляла 98-99,5% Масс-спектроскопия: (FAB) m/e 939 (M + H)⁺.PRI me R 1. 2 '- (N, N-dimethiglycyl) taxol or its salt

a) Compound 1: 2 '- (N, N-Dimethiglycyl) Taxol
To a solution of taxol (0.21 g, 0.246 mmol) and N, N-dimethiglycine (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 anhydrous medium, after which another 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 fractions of petroleum ether 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. Mp. 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
b) 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 mol), 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 to give 0.058 g of product (88%), 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
turbid)
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 lare Unit). The concentration of the derivative was about 20-25 μg / ml. A mother liquor 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 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 the construction of a graph of the dependence of the area of cic on time. T ₉₀ and t ₅₀ were calculated. Plasma and chemical studies were performed using high-performance liquid chromatography using a PP-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 (pH5): acetonitrile 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 flow rate was 1 ml / min.

In the study of 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 has also been confirmed by studies of the degradation of new derivatives. Thus, 2 '(DMC) taxol incubated with water at ³⁷ ° C and the resulting product was concentrated and purified by 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.

а) Соединение 3: HCl-соль 2'-(3-N,N-диэтиламинопропионил)таксола
К раствору таксола (0,12 г, 0,14 ммоль) в CH₂Cl₂ (12 мл) содержащий гидрохлоид N, N-диэтиламинопропионовой кислоты (0,025 г; 0,145 ммоль) добавляли 1,3-дициклогексилкарбодиимид (0,08 г, 0,38 мМ) и 4-диметиламинопиридин (0,01 г, 0,081 ммоль). Полученную смесь перемешивали в течение 24 часов при комнатной температуре. Затем реакционную смесь фильтровали и фильтрат выпаривали в присутствии азота. Неочищенный материал хроматографировали на колонках с силанизированным силикагалем (18 г, 22 см) и последовательно элюировали смесью этилацетата и петролейного эфира (1:2), смесью этилацетата и петролейного эфира (1: 1) и этилацетатом. Фракции смеси этилацетата и петролейного эфира при медленном выпаривании образовывали твердый преципитат, который затем отфильтровывали. Маточные растворы, содержащие продукт, объединяли, концентрировали и добавляли до тех пор, пока раствор не становился мутным, после чего его отбрасывали. После фильтрования получали 0,068 г продукта (48% ), Т. пл. 188-191^оС, MC (FAB) m/e 981 (M+H)⁺. ЯМР (330 Гц, CDCl₃) показал, что 2'-протонные резонансы от 4,71 ppm в таксоле до 5,53 ppm N-этильные группы показывали метиловый резонанс А 1,0 ppm и СН₂-резонанс An 2,52 ppm. Все остальные характеристики резонансов соответствовали ожидаемому соединению.HPLC operating conditions. Column RP-8, 150 mm (length),
4.6 mm inner diameter) Mobile phase 0.02 M acetate (pH 5);
acetonitrile 50:50. Kratos spectroflow detector Flow rate 1 ml / min Retention time 11.2 ml (Compound 2)
5.5 ml (taxol)
The results of a 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

a) Compound 3: HCl salt of 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 with slow 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,} MC (FAB) m / e 981 (M ⁺ H) +. NMR (330 Hz, CDCl ₃ ) showed that 2'-proton resonances from 4.71 ppm in taxol to 5.53 ppm N-ethyl groups showed methyl resonance A 1.0 ppm and CH ₂ resonance An 2.52 ppm . All other resonance characteristics corresponded to the expected compound.

Elemental analysis C ₅₄ H ₆₅ CIN 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 the compound 3. Mol.m. 1017.56 mp 186-189 ^о С (decomp.) Solubility ≈ 0.8 mg / ml
HPLC conditions RP-8 column, 150 mm (length),
4.6 mm (internal diameter) Movable phase 0.02 M acetate (pH5): 35:65 acetonitrile Kratos Spectroflow detector Flow rate 1.5 ml / min
Retention time 16.71 ml (compound 3)
Chemical Stability Condition t _1/22, 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
Plasma stability, 37 ^о С Conditions t _1/2 , min Human plasma 4.2
b) 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 OAB-Sephadex (Pharmacia) was wetted with 0.1 M NaCl for 75 hours and 75% of the resulting 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, and 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.

a) 2'-(troc) таксол (соединение 5)
Таксол (0,27 г, 0,316 ммоль) растворяли в CH₂Cl₂ (10 мл) и пиридине (1,5 мл). Реакционную смесь охлаждали до-20-(-25^о)С и добавляли 2,2,2-трихлороэтилхлороформат (80 мкл). Затем реакционную смесь перемешивали при этой температуре в течение 3 ч. После добавляли еще 25 мкл хлороформата и полученную смесь перемешивали в течение ночи. Затем реакционную смесь разбавляли СН₂Cl₂ (50 мл) и последовательно промывали 0,1 н. HCl (25 мл х 2), 0,05 М холодного NaHCO₃ (25 мл х 1) и водой. Органический экстракт осушали безводным MgSO₄, а растворитель удаляли. По- лученный материал осушали с помощью препаративной ТСХ на тарелках с силанизированным силикагелем, проявляли в смеси этилацетата и петролейного эфира (1:3), фракцию выше таксола отделяли, элюировали этилацетатом, а растворитель удаляли, в результате чего получали 0,32 г (97%) продукта, т.пл. 221-226 (разл. размяг. 160^оС).Physical properties Mol.m. 1077.12 mp 170-174 ^C. The solubility> 10 mg / ml of HPLC-purity> 99%
Chemical stability. Terms t _1/2, h 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 salts

a) 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 with 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, mp 221-226 (decomp. Soft. 160 ^about C).

b) 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: 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} (again.).

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.

c) 7- (N, N-dimethylglycyl) taxol (compound 7)
To a solution of 2 '(troc) -7- (N, N-dimethylglycide) 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. The mixture was then filtered, the filtrate was concentrated to 1 ml and diluted with CH ₂ Cl ₂ (35 ml), and then washed sequentially with 0.01 M HCl (20 ml X 2), 0.01 M cold NaHCO ₃ and water. The organic extract was dried with anhydrous Na ₂ SO 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 : ethyl acetate (7: 1). The fraction related to 7- (DMG) taxol (R _f 0.35) was separated and eluted with ethyl acetate and ethanol, then the solvent was removed and 0.19 g of product was obtained (68%). 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 doublet at 5.65 ppm - (CH ₃ ) ₂ resonance appeared as a singlet at 2.35 ppm. The glycinate methyl group was detected at 3.16 ppm.

d) Methanesulfonic acid salt of 7- (dimethiglycyl) taxol (compound 8)
7- (Dimethylglycyl) taxol (0.065 g, 0.069 mmol) was dissolved in I-butanol (2.5 ml) in 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%). T. pl. 164-168 (decomp.).

Таксол (0,06 г, 0,00702 ммоль) растворяли в безводном метиленхлориде (5 мл) и добавляли N,N-диметиглицин (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, счищали с помощью препаративной ТСХ на тарелках с силанизированным силикагелем и проявляли в смеси этилацетата и петролейного эфира (1: 1). Фракцию (R_f0,17) выше диметиламинопиридина отделяли и элюировали смесью этилацетата и этанола, а растворитель удаляли. Остаток перекристаллизовывали смеси этилацетата и петролейного эфира, в результате чего получали 0,046 г продукта (64%). Т.пл. 194-198^оС. MC: m/e 1024 (М⁺). В ЯМР-спектре (300 МГц, CDCl₃) С₂-протон 4,71 ppm и С₇-протон 4,33 ppm сдвинуты к 5,5 ppm и 5,6 ppm соответственно, что свидетельствует о этерификации в 2'- и 7-положении. Зафиксирован также -СН₃-протон в виде синглета при 2,3 ppm.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 164-168 ^{° C} Solubility:> 2 mg / ml
HPLC conditions RP-8 column, 150 mm (length),
4.6 mm (internal diameter) Mobile phase 0.02 M acetate (pH 5):
acetonitrile 35: 65 Kratos Spectroflow detector Flow rate 1.5 ml / min Retention time 15.07 ml (compound 8)
Chemical stability. Terms t _1/2, h 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 h
Plasma Stability, 37 ^{° C} Condition t _1/2, h Rat Plasma 20 / ml human plasma 20 17.3 ug / ml 27.7 Human Plasma 10 / ml 24.4
PRI me R 4. Obtaining 2 ', 7- (dimethylglycyl) taxol

Taxol (0.06 g, 0.00702 mmol) was dissolved in anhydrous methylene chloride (5 ml) and N, N-dimethiglycine (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 cleared using preparative TLC on plates with silanized silica gel and developed in a mixture of ethyl acetate and petroleum ether (1: 1). A fraction (R _f 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. MC: m / e 1024 (M ⁺ ). In the NMR spectrum (300 MHz, CDCl 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 the esterification in 2'- and 7-position. A -CH ₃ -proton was also recorded as a singlet at 2.3 ppm.

Elemental analysis: C ₅₅ H ₆₆ N ₃ O ₁₆
Calculated, C 63.39; H 6.48; N 4.03
Found, C 63.00; H 6.98; N 3.98%
HPLC conditions RP-8 column, 150 mm (length),
4.6 mm (internal diameter) Mobile phase 0.02 M acetate (pH 5):
acetonitrile 35: 65 Kratos Spectroflow detector Flow rate 1.5 ml / min Retention time 15.07 ml (compound 8)
Chemical stability. Terms t _1/2, h 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 h
Plasma Stability, 37 ^{° C} Condition t _1/2, h Rat Plasma 20 / ml human plasma 20 17.3 ug / ml 27.7 Human Plasma 10 / ml 24.4
PRI me R 5. Obtaining 7- (L-alanyl) taxol or its salt
a) 2'-7-di (t.BOC-L-alanyl) taxol
To a solution of taxol (0.021 g, 0.246 mmol) and Nt, 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: 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.).

b) 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 of 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) 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-spectrometer: (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 HC ₅₀ H ₅₈ N ₂ O ₁₆ xx 2.5 H ₂ O
Calculated, C 61.91; H 6.54; N, 2.89.

Found, C, 61.41; H 6.59; N 22.78%
c) Methanesulfonic acid salt of 7- (alanine) taxol
To a solution of 7- (Anan) 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 min, after which the mixture was filtered through a millipore filter (0.2 μmol). 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 (inner diameter) Mobile phase 0.02 M acetate mixture
(pH 5) and acetonitrile
(50:50) containing
0.001 M bisulfate
tetrabutylammonium Kratos Spectroflow detector Flow rate 1 ml / min Retention time 8.7 ml 7.3 Taxol Plasma stability 37 ^° C Conditions t _1/2 , h Human plasma 20 μg / ml 11.9
PRI me R 6. Obtaining 2- (alanine) taxol
a) Synthesis of 2 '(CBZ-L-alaninyl) 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) was added DCC (45 mg) and 4-dimethylaminopyridine (4 mg), and the resulting mixture was stirred for 3 days at room temperature. The reaction mixture was filtered and the solvent was removed from the filtrate. The residue was purified by preparative TLC on silanized silica gel plates 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 and 28 g of 2 '- (CBZ alanyl) taxol was obtained.

 b) Synthesis of 2 '(alanyl) taxol by unlocking 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 as a low or moderate yield of format or acetate.

PRI me R 7. Obtaining 2 '- (lysyl) taxol
a) Synthesis of 2 '(N-di-t-BOC-lysyl) taxol
To a mixture of taxol (30 mg, 0.035 mmol) and N-di-t-BOC-1-lysine (19 mg, 0.0368 mmol) in methylene chloride (10 ml) were added DCC (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.

c) Synthesis of 2 '- (lysyl) taxol by unlocking 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. 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-unlocked amino acid was obtained in the form of a salt.

PRI me R 8. Obtaining 2 '- (L-alanyl) taxol
a) Synthesis of 2 '- (FMOC-L-alanyl) Taxol
To a solution of taxol (60 mg, 0.072 mol) and N-FMOV-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 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.).

c) Unlocking the FMOC group of the protected amino acid derivative of taxol
The N-FMOC-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 invention may be formulated 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 the present invention in accordance with standard chemical technology.

 Typically, salts are prepared by reacting a 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 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 technology, such as evaporation of the solution. Alternatively, the free base can be dissolved in an organic solvent such as lower alkanol, 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 is 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 the salt does not dissolve, and then isolating it.

 The taxol derivatives 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 / 2500 mg or higher for a single dosage form, and preferably about 50-500 mg. Examples of pharmaceutically acceptable carriers include lure, urea, dextran, lactose, potato or corn starches, magnesium stearate, talc, vegetable oils, polyalkylene glycols, ethyl cellulose, poly (vinylpyrrolidone), calcium carbonate, ethyl oleate, isopropyl myristate, benzylbenzene benzylbenzene , 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 tripalmitate, 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 above description using standard techniques known to those skilled in the art (see Remington Pharmaceutical Science, Chapter 39, Mack Publishing Co. 1965).

 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. A corn starch paste is then 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 pressed 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 proposed compound in the form of tablets.

Drug I
Ingredients, mg / tab. 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 of 2500 mg of the proposed compound for oral administration, is that the specified active compound is mixed with a non-toxic carrier and the resulting mixture is introduced into a polymer shell, usually gelatin or the like. As is known, capsules can be soft in shape and are made by incorporating the compound in carefully mixed dispersion mixtures together with food compatible carriers, or these capsules may be in a rigid form, which mainly contain a compound of the 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. new compounds in pure form or in the form of mixtures of one or more new compounds, the following preparation II can serve.

Drug II
Ingredients, mg / capsule: Active ingredient 50.0 Calcium carbonate 100.0 Lactose, USR 200.0 Starch 130.0 Magnesium stearate 4.5
The above ingredients are mixed in a standard mixer and commercially available capsules are filled with the resulting mixture. The higher the concentration of the active component, the less lactose should be used in this composition.

 The compounds of the invention can be freeze-dried, and if necessary, can be combined with other pharmaceutically acceptable excipients commonly used in formulating compositions for parenteral administration by injection. For such administration, 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, and the like.

The administered dose can 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 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.

 As indicated above, 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, melanoma, leukemia, breast and rectal cancers.

 The biological activity of these taxol derivatives was tested by (A) in vitro studies and measurement of the kinetics of microtubule accumulation; (B) in vitro studies of an MX-1 graft grafted onto an SRC renal capsule.

A. In vitro study of the kinetics of cluster accumulation
Microtubules are an inseparable part of po-kriotic 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 turbulin 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. Studies in vitro have shown that microtubules are already polymerized, in the presence of Taxol are stable to depolymerization caused by other agents, such as CaC _l2 or low temperatures, which typically contribute to depolymerization of microtubules.

 The authors of this application conducted a study of the effect of taxol derivatives on the accumulation of microtubules. This study was performed using 2'- and 7-derivatives in accordance with in vitro procedures (see Mellado et al. Biochemical and Biophysical Research Communication, vol. 124, No. 2, 1984, pp. 329-336; Magri et al. J. Org. Chem. 51, 797-802, 1986; and Parness et al. Biochemical and Biophysical Research Communication, vol. 105, No. 3, pp. 1082-1091, 1982). The ability of these compounds to stimulate the accumulation of microtubules can be traced as follows: taxol> 7- (N, N-dimethiglycyl) taxol (II)> 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 good agreement with the results of the activities of 2'- and 7-acetyl taxol (see et al. Biochemical and Biophysical Research Communication, vol. 124, 1984, pp. 324-336).

 B. In vitro study of cell culture B16.

 To confirm the activity of the proposed derivatives of taxol, in vitro studies of the culture of melanoma B-16 cells were conducted. These studies were carried out in accordance with standard procedures (see Himes, Lancer Biochem. Biophys. Vol. 7. 1984, p.133).

 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, 2'-derivatives act as prodrugs. However, 7-derivatives have their own activity and, obviously, are not a prodrug.

 C. 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 known methods (see NLH Publication N 84-2635, In vivo lancer Model. 1984, S. 23-24).

 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 according to the following scheme:
Day 0. Animals were anesthetized. The body weight was fixed (weight of day 1). The implantable tumor was measured and the results were recorded. After recovery from anesthesia, 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 the case of contamination, the 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. Q4D treatment on days 1,5 and 9. For each day, a fresh injection of the test agent was prepared and administered according to the mouse body weight on that day.

 Day 2. Again checked the culture. In case of contamination, the test was stopped and a recording was made in accordance with this.

 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 (day 2 weights). The tumor was measured in OM. (Eyepiece-micrometer-unit 10 Omed 1 mm) and measurements were recorded.

 Evaluation of the results.

The change in tumor mass (Δ) was recorded based on measurements of length and width (mm). The average animal weight was calculated for day 1 and day 11, and T / C was calculated for all test groups with> 65% survival on day 11. The dynamics of changes in 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 translating OMed. in millimeters (mm).

где L представляет более 2-х измерений.2. When calculating the weight 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 (Δ) in the average tumor weight for each group of mice:
Change in average tumor weight. Average tumor weight. Final. Average tumor weight. Initial.

 4. When calculating the change (Δ) in the average tumor weight for the test (T) and control (C) groups.

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л

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Критерии активности.5. When calculating T / C (%) for all tested groups with a survival rate of more than 65% on the last day of the experiment:

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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.

Claims (7)

1. Derivatives of Taxol of General Formula I

where R and R ₁ are each an amino acid residue selected from the group consisting of alanine, leucine, isoleucine, saline, phenylalanine, proline, lysine or arginine, or a group of general formula II

where n 1, 2 or 3
R ₂ and R _{3 are} each a C ₁ -C ₃ alkyl or acid addition salt, provided that at least one of R and R _{1 is} not hydrogen, and also the compound of general formula I is not 2 ′ - (β - Alanyl) Taxol.  2. The compound according to claim 1, which is 2 ′ - (N, N-dimethylglycyl) taxol or its acid addition salt.  3. The compound of claim 1, which is 2 ′ - (N, N-dimethylaminopropionyl) taxol or an acid addition salt thereof.  4. The compound according to claim 1, which is 7- (N, N-dimethylglycyl) taxol or its acid addition salt.  5. The compound according to claim 1, which is 2 ′, 7-di (N, N-dimethylglycyl) taxol or its acid addition salt.  6. The compound according to claim 1, which is 7- (alanyl) taxol or its acid addition salt.  7. A pharmaceutical composition having antitumor activity, comprising an active substance and a pharmaceutically acceptable carrier, characterized in that the active substance contains a taxol derivative of the general formula I in an amount of 0.01 to 2500.0 mg.

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