CS242856B2 - Method of chlorambucile production - Google Patents

Abstract

NEW MATERIAL:An addition compound II composed of an esteradiol derivative and chlorambucil. USE:Anti-tumor agent, having selectivity to cancer cells having receptors, and low side effects. Especially effective to breast cancer, solenoma, prostatic cancer, nephritic cancer, thyroid cancer, etc. PROCESS:The objective compound X is prepared by reacting the 17-OH group of estradiol with a functional group-introducing agent (e.g. monobromoacetyl bromide) in a solvent such as THF, etc., and then reacting the reaction product with an anti- tumor agent (e.g. silver salt of chlorambucil) in a solvent such as DMSO at 0- 100 deg.C for 2-74 hours.

Description

This invention relates to novel anti-cancer derivatives of 4 ^ £ p / 2 ~ ^ bi.s- chh.oi: ethyl-amino / ^alkyl fenyimrnáselné petroleum jelly / called. c ^h lormbbuil. Ex ^and the STE is ^{characterized} in that n ^and falsehood ^ka th derivatives obtained from an ee chlorambuučl in přítomnosSi binding agent chemically bonds with estradiol or derivatives thereof and via the hydroxy at position 17th

It is well known that most finger and drug drugs act on cancer cells as well as on normban cells, and that these substances therefore have serious side effects. It is therefore difficult to administer such agents long enough to destroy all cancer cells.

The autooi have dreamed of overcoming these unrighteous drugs of known finger cancer drugs and developing a new drug that would have a high therapeutic effect. The result is:! new esteradiol derivatives which bind selectively to certain cancer cells and which have low side effects.

The present invention relates to a process for the preparation of chlorobenzil derivatives of the general formula I

(I) wherein R represents a hydrogen atom or an acyl group such as li

-С _СН-3, according to the invention consists in the fact that L and estradiol or acylated ^ e ^ 1 ^ rr ^ is; L is bound via the hydroxy at position 17 by treatment with a coupling agent bromaacey ^ Rohm then the product is optionally acylated. The above-mentioned derivatives of the general formula I are novel compounds.

The derivatives of the invention are conjugates and estradiol or derivatives thereof with a binding agent Lam. The derivatives of the invention have a specific affinity for cancer cells and therefore have a specific effect because they selectively attack cancer cells.

The particular cancer cells have a receptor for steroid hormones, especially estradiol derivatives, which are part of the derivatives of the invention.

These receptors can be used for the derivatives of the compounds of the invention. Accordingly, the chlorobenzyl derivatives of the invention write cancer cells having estradiol receptors. and derivatives thereof.

These cancers include breast, prostate, liver cancer, thyroid and uterine cancer. The products of the invention are particularly effective in treating breast, uterine and prostate cancer.

The chlorobenzyl scavengers of the present invention are selectively distributed to cancer cells in the body and thus attack cancer cells without side effects.

It is a particular feature of the invention that the binding of estradiol or derivatives thereof and chloro-Gibtle occurs without loss of the active centers of estradiol or derivatives thereof and without loss of the lipoprotective properties.

It is preferred to bring the hydroxy group of the estradio at the 3-position to an acloxy group such as it

0 0

II *

-OC-CH ₃ , -OC-C ₂ H 5 and -ocC ₃ 11 ₇ .

This acyloxy group in the body is readily converted to the hydroxyl group required by the body to bind to receptors in cells.

ChlorambucCl and estradiol or derivatives thereof may be bound by suitable methods. For example, in one of these methods, estradiol or a derivative thereof is first reacted with a binding agent and the thus obtained modified estradiol or its acylated derivative is exposed to chlorambutyl.

In another such procedure, the moodfluorinated chlorobutyl is reacted with estradic or acylated estradic.

In the first case, the coupling agent is reacted with an inactive position of estradiol or acylated estradiol to give an ester of formula X- / CH ₉ / _n -COOB wherein B is an estradiol or acylated estradiol radical resulting from the removal of the 17-hydroxyl group and x is halogen atom. Thereafter, the halogen of this ester is reacted with chloramucucir to produce the chlorambtiucl derivative of the invention.

These reactions are now described in more detail.

The binding agent (Uromaccryl bromide) reacts with the hydroxyl group at position 17 of estradiol or 3-acylated estradiol in solvents such as carbon tetrachloride, chloroform, tetrahydrofuran, dimethylsulfoxia (DMSO), dimethylformamide (DMF), pyridine and acetone.

The reaction product is then reacted with chlorine and chlorine in solvents such as dimethylformamide, dimethylformamide, pyridine, carbon tetrachloride, chloroform or tetrahydrofuran. CCloramruuCl can be used in the form of an acid or sodium salt / e.g. silver salts or aluminous metal salts.

^Yp ro JcaMou temperature reaction is chosen in the range of -30 °, ^and 100 ° C, preferably between -10 ° C and ⁸ 0 ° C. Reaction to ývvj ^b y ^{b j} 'in the ^0.5 and ^{4 h ≧} 7 o'clock. Reaction ^P ro ^d uct was purified defective purification procedures.

In the second process, the coupling reagent is reacted with a chloroambiud carboxyl group to give a compound of the formula АСОО / СНн / пСОХ in which A is a chlorambucil residue from which the COOH group at position 1 has been removed.

The halogen atom (X) of this compound is then reacted with a hydroxyl group in the 17-position of estradiol or 3-acylated estradiol to form the chloramblucine derivatives of the invention.

The solvents used for these reactions are the same as those of chlorambucel or estradiol or acylated estradiol.

The reaction temperatures and reaction times are selected within the same range.

The 3-hydroxy group of estradiol bound in the described conjugate of the invention may be acylated before or after binding to chlooamruud in the presence of a binder. These reactions are described in more detail below.

The hydroxyl group at position 3 of the lstoadiol reacts with an alkali metal hydroxide in a solvent such as TH to convert it to an ONa or OK group, and then this product is subjected to further reaction with acylchloride, e.g. ) rdeem.

The product is 3-acylated estradM1. Then, a coupling agent such as bromoacetyl boomide is treated with the d-hydroxy group of this acylated estradiol in a suitable solvent (DMSO, DMF, pyridine, toluene, carbon tetrachloride, chloroform or THF).

The reaction temperature of each step is selected from temperatures between -30 ° C and 100 ° C, preferably between 10 ° C and 100 ° C

-10 ° C and 80 ° C. The reaction times range from 0.5 to 74 hours. The resulting product is purified by appropriate methods to provide the chlorambucil derivative of the invention.

Certain processes for the production of chlorambucil derivatives will be described by way of illustration, and the reaction conditions may be selected at will.

The chlorambucil derivatives of the invention have the general formula I, i.e. they are the chlorambucil and estradiol or acylated estradiol conjugates. This is confirmed by IR spectrum, UF spectrum, NMR spectrum, thin layer chromatography, mass spectrum, elemental analysis and melting points of the products.

According to acute toxicity tests, by binding of substances to estrogen-sensitive cells, by anticancer effect, it is evident that the chlorambucil derivatives of the invention exhibit remarkably low toxicity, very high affinity for estrogen-sensitive cells and high anti-cancer activity.

The chlorambucil derivatives of the invention are particularly effective in treating cancer tissues and cells that are sensitive to estradiol and thus can be used to treat cancer of the breast, prostate, liver, thyroid and uterus.

They are also effective in the treatment of gastrointestinal cancer, rectal cancer, larynx, esophagus, lung, skin and leukosarcoma and have remarkably low toxicity compared to known anticancer drugs including chlorambucil alone.

The chlorambucil derivatives of the invention do not have specific sexual functions of estradiol, although they are estradiol derivatives. The reason for this is unknown at this stage. It is contemplated that these effects are supported by certain unknown mechanisms in addition to the therapeutic effect based on conventional receptor design.

If the chlorambucil derivative according to the invention is to be used in medicine, the drug delivery formulations can be manufactured by conventional procedures common to anticancer drugs.

The chlorambucil derivatives of the invention may be formulated in the desired form as an injection, for oral administration such as a suppository or an ointment. When formulated in solid form (oral preparations: tablets, pills, granules, powders, capsules), a binder, diluent, filler, lubricant, oil, surfactant or disintegrant may be added to the drug.

When formulated in liquid form for oral administration, these may be aqueous suspensions, oily suspensions, solutions, syrups, and biphasic mixtures that need to be shaken before use. When formulated as a suppository, a hydrophobic or hydrophilic support may be used together with a stabilizer, a disintegrant and a colorant.

When formulated as an injection, an aqueous solution, solvent, nutrient, stabilizer and surfactant may be added. If desired, a base, acid or salt may be added to maintain or increase the therapeutic effect.

The amount of active ingredient contained in the formulation is generally from 0.001% to 90% by weight, preferably from 0.01% to 60% by weight of the formulation.

The chlorambucil derivatives of the invention may be administered orally, by percutaneous absorption, muscle, intraperitoneal, subcutaneous, intravenous or intrarectal injection, or topically.

The dose of the chlorambucil derivative according to the invention is selected in the range from 0.01 to 50 mg / kg / day for an adult for oral administration and from 0.001 to 20 mg / kg / day for an adult for intravenous injection.

The chlorambucil derivatives of the invention have the following characteristics:

1. To the extent that the cancer produced in the tissue contains receptors, the product selectively attacks the cancer cells of that tissue and destroys them. It is therefore effective in small doses.

2. has lower side effects compared to effects after administration of chlorambucclc alone. It can therefore be administered for a long UoUc and thus cancer cells can be completely destroyed.

3. Eslradiol or acylated estradiol, which is to be used as a carrier in a chlorambucio conjugate, has a simple structure and its physiological activity is well known. Thus, the conjugate can be used without fear.

4. The structure and efficacy of the anti-cancer component of the conjugate are also known. The product can therefore be administered without fear.

5. It is possible to study the cancer cell recipe and select the corresponding steroid hormone or its roar accordingly, and then to use the carrier. Thus, the medicament for different types of cancers can be selected according to a suitable carrier component. ·

6. The chloaambuuil derivative can be administered by conventional means: oral, injectable and suppository.

The products according to the invention have unique properties which contribute excellently. developing medicine and helping people. These chlorocarbonyl derivatives can also be used as stabilizers for high molecular weight polymers, especially polyolefins.

The invention will be elucidated in some cases, which are intended only for the purpose of the invention and do not imply a limitation of the scope of the invention.

Example 1

Transport of 3-nitrobenzaldehyde) oy ^l1beUre-R 4 - pPb ^{u S} '2 ^ ^j chlorrthy aIninoO rnyl ^f / ^u utya ^y lox ^y] acetoxy-1,3, ^5/10 / -estratreenu.

1. Preparation of 3-hydroxy-17-bura-Uromaacrooyyl, 3,5 (10) -stirerrence:

1,3,5 / 10/51 51 ^ ^ ^ 1 ^ 0, 3, β-diol; (1) (g) is dissolved in anhydrous tetrahydrofuran (THF, 400 ml) and 8.8 g of pyridine are added to the solution. To this solution was slowly added dropwise at tejiotě -5 ° C to -7 ° C iozeo ^kb aomar! C ^r yl romi ^{u u} u / ^22, 5 ^{g /} in uhiič chloride-EM / 74 ^{g /.}

The mixture is allowed to stand overnight, then the precipitate formed and the filtrate is concentrated to dryness. The residue is taken up in ether and crystallized from ether to give 3.17-butanil (boomacrtoxy) = 3.5 (WO). ^ ta ^ im.

Two grams TAHO ^ ^p ro ^d uktu dissolved in mmthanolu / ^9/0 ml / and expand the ^ ^ ^C ^ l ^l ^ J and ^{I at -5} ° C. To this solution was added a solution of potassium carbonate (0.24 g) in water (20 mL). After 30 minutes, the mixture is diluted with 1 ml of water and the precipitate formed is drained and dried.

Elemetary analysis and IR spectrum confirm that the product is 3-hydroxy-17beta-bromoacetoxy-1,3,5 (10H) -ra ariene.

2. Preparation of hydroxy- ^4- ( ^4- ( ⁴ -phenylethyl) methyl) acetoxo-1,3,5 (10) -triethylsulfamate conjugate and estradiol.

A silver salt of 4- Uil ^ p / 2-ih oireh ^{i /} i / well jm ^m / ^f rn ^t / ^m lm SELT sowed ⁱⁿ ^ ^ / ² 00 mg / cc the ratio ^ ^í mL of DMSO was converted into lap-white solution of to which is added 190.8 mg of 3-hydroxy-17Uetk-Uromarcroxy-1,3 1 / ΙΟ / θ ^^ γ ^^.

The mixture was allowed to stir for 64 hours in the dark, whereupon the precipitate turned yellow-green. Small amounts of acetone were added and the precipitate was removed by filtration through a G4 filter. The precipitate darkens to a black-green color due to daylight, but the filtrate remains colorless and clear.

For sn ^h o ^iz ene tkku to ⁸ vocfoí bath at 0 ° C, the steamed DMSO ^{d p} of 100 ml in a strictly ^dy product precipitates in the form of white crystals. After an hour (DMSO removal), the crystals were aspirated on a filter and washed thoroughly with distilled water. The product was dried under vacuum in a desiccator, yield 330.5 g.

Product cleaning:

330.5 mg of crude crystals were dissolved in a mixture of 50 parts by volume of cyclohexane and 10 parts by volume of ethyl acetate.

The solution was slowly filtered through a column of silica gel and successive separation gave 188.2 mg (62.86%) of pure product.

Elemeenární analýza:

found : 66 ₍ , 0 % C, 7.0 % H, 2.3 % N, 11.0 % Cl; calculated: 66, , 22 % C, 6.98 % H, 2.27 % N, 11.52 % Cl Temperature thaw: softens at 25 Deň: 32 ° C. IR spectrum ^ µm / cm ^- 1 /: 3,420, 2 920, 2 840, 1,750, . 74 (0, 1 612, 1 582, 1 516, 1 450, 1 380, 1 350, 1,280, 1 250, 1 210, 1 175, 1 142, 1,070, 1 000, 960 917 867, 810, 800, 740, 655 klad 2

3-ben2oyloxy l7beta-1,3,5 / 10 / -estratrěenu ^{EXAMPLE beta.} rava

4- £ ^p- bis / ^2- c ^UL lrtt ^Uy l / amio ⁰ 7. tnyl -but ^{i y} y ^y lox ^y acetoxy1,3,5 / 10 / -Esttаarieo-3,17bbetadiol / 10g / rozpuusí with 100 ml THF and 10 ml of a solution containing 1.47 g of sodium hydroxide in water are added. The mixture was stirred at room temperature for 30 minutes. pa ti and evaporated to ^the sum of ^the va uu / te ^p lota used ^^ l, ^and waste water is ⁸⁰ ° C /.

The residue was dissolved in anhydrous THF, and a solution of 5.5 g of benzyl chloride in 50 ml of ether was slowly added dropwise. After 16 hours at room temperature, the sodium chloride was separated by conventional means, the filtrate was evaporated to dryness in vacuo, and the unreacted benzyl chloride was removed by adding 200 ml of aqueous sodium hydroxide solution (0.1 N).

After stirring at room temperature for 15 minutes, the white crystals were collected using G3, washed with water and dried in a desiccator under vacuum.

The product is analyzed by silica gel thin film / developing system; 50 parts of ethyl acetate and 30 parts of cyclohexane, the major spot being at Rp = 0.34.

The crude crystalline product was recrystallized from ethyl acetate to give 8.6 g of white crystals.

Elemeeneal analysis and IR spectrum confirm that the product is 1-benzooloxy-.beta.-hydroxy-1,3,5 (10) estratriene.

All product was dissolved in THF (7 g) and pyridine (2 g) was added and the solution was cooled to -5 ° C. To this solution was slowly added a 30% solution of bromoactyl bromide in carbon tetrachloride (15.5 ^g ) ^ Z ^ ga tttruhydli / uαotem / 50 ^m j /. ^p o p ^ ailments clavan mixture MICs are ≤ ^P s ^- 5 ° C for 2 hours and then stirred for another 4 hours in an ice bath.

After 16 hours of standing in lednňci was filtered on G4 ^ u fil white precipitate formed which when dried ^{in uu p} s t ^^ I la ^Zn of 30 ° C. Furthermore smenina MIC ^{hook 200} ml Etter and ^Z ^^ ^5.3 g of white crystals.

Elementary analysis:

Found: C 64.3%, H 5.8%, Br 15.7%; calculated: 64.23% C, 5.78% H, 15.8% Br. Melting point: ¹⁴⁵ to ¹⁴⁶ ° C.

Analysis of the product by thin layer chromatography with silica gel and using a mixed developing system (50 parts by volume of ethyl acetate and 30 parts by volume of cyclohexane) shows the product as a single spot at Rf, 0.77.

IR spectrum shows absence! hydroxy and thus it is shown that the product is 3-benzoxy-17beta-bromoacnoxy-1,3,3,10,10-brethrenes.

^IR spket.rum / cm ¹ /:

2 920, 1 735, 1 728, 1 595, 1 579, L 490 1 448, 1 412, 1 382, 1 286, 1 280, 1 260 1 210, 1 200, 1 170, 1 145, 1 09 5, 1 075 1 019, 1 004, 897, 7 80, 700, 680

3-Bentouloxy-17beta-brumafououl 1,3,5 (100-62%) 6% / 182.2 mg / mmA The reaction was silver and to 5 ml.

DMSO was mixed. . . . .

and a silver salt θΙ Κ ^ ^ ^- 4- £ р - [^ a / 2-ch ontt ^{u - f} y / ^t uoJft- am ^l l ^m m ^A snlté / М ^ ⁵ interferes with místnooti temperature in the dark for 3 days , then bromide bromide is filtered off, 400 ml of water are added.

The resulting white precipitate was collected by filtration, the precipitate was dissolved in 50 ml of acetone, and the insoluble material was separated by filtration through a G4 filter. The filtrate was concentrated to dryness in vacuo to give 165 mg of an oily product.

TLC analysis of the tert-fafrafrafram product using ethyl acetate / cyclohexane (10:50 parts by volume) showed a major component at Rf = 0.44.

r

Since the starting solvent remained in the mixture, the product was purified by chromatography to give a silica gel in a mixture of solvents (ethyl acetate and cyclohexene in a ratio of 10:50 parts by volume).

^p RECIST ^{currency P} ro ^d uct ⁱ s e ^{p 20} C ^b i ^la ^ Cr ^ taHctá weave. It ^ hardened, ^made for e ^ ^b ktem 3- mntou ^- -OX ^--17 beta- -4- | -p- ^p bib / 2 c ^{2 d} Lour ^nt y ^l / -amino 13.5 / ¹ ^ ° ^/ -estratrien.

ELe ^^ ent ^ i ^ r ^^ í analysis:

found: 68.5% c, 6.60% H, 1.99% N, 9.79% Cl; H, 6.53; N, 1.94; Cl, 9.86. Melting point: 110-111 ° C.

IR spectrum (cm ^-1 ):

2 920, 2 860, 1 755, 1 735, 1 612, 1 582 1 516, 1 491, 1 450, 1 420, 1 380, 1 355 1 260, 1 224, 1 210, 1 174, 1 145, 1 079 1 022, 1 005, 960, 915, 890, 800

740, 705. ·

Example 3

Preparation

-1,3,5 / 10 / -estratreens

3- ^P r (u i ^p (Una ^{l (l-7} UXY BE TA / ^l - ^p ^ ^£ - ^£ ^ ^B _/ ^{/ 2} -lH urпЪЬ ^{L -} 1 / г ^- ^ ^- acetoxymethyl ^y 1.3, 5 (10) -EEtrafrtnt-3,1 ⁷ bbnt-cfol (10 g) was suspended in 100 ml of uranium and 10 ml of an aqueous solution containing 1.47 g of sodium hydroxide was added and the mixture was stirred at room temperature for 30 minutes. the reaction mixture was zahuutí to dryness under vacuum, to remove water water oužije ^{p la} Zen ⁸ 0 ° C te ^pL.

The residue was dissolved in anhydrous tetrahydrofuran and a solution containing 3.4 g of propylstylsulfide in 50 ml of anhydrous tetrahydrofuran was slowly added to the solution. After 16 hours at room temperature the precipitate of sodium chloride is separated off and the filtrate is dried to dryness in vacuo. Crystallization of the residue from ethanol gave 9 g of white crystals.

exacerbates

It has been found, ie the product is 3- ^p ro ^p PostУosχyl, 3.5 / 10 / -estthtrěen 17beth-sl ~ h analysis by IR /.

Elemental g of this product was dissolved in 70 ml of anhydrous tetrahydrofuran. The mixture was cooled to ^-5 ° C and ^to this extended ^t ^ o ^to u ^p omal bromide chloride dropwise u ^ where ^ Citta / -7.3 g / diluted with 50 ml THF.

and add

^The organic solution 30% cheese sauce ^ ^ YSE 3 g ^{of p} axes ^to ončeném Plus a wealth mixture ааШ ^ 2 ^h Odin ^p s ^- 5 ° C and ^P and ^K

Upon completion of reaction as to the ^d 30 s ^p láíinw

1 ^{6 hrs} . vznj-monasteries sSífltrsjě precipitate, the filtrate was concentrated in ° C ^{and P} waste RID ^and Etter / ² ° 0 MLA with ^the esters of the vacuum to dryness ^P ro ^d ^ uL IT MIC ^Hooks

6 g of white crystals are obtained. Concentration of the fittalate yielded additional tall (3.5 g). The product was recrystallized from a mixture of ether and ethanol.

white crystalsElemenium analysis: found: 61.5% C, 6.5% calculated: 61.43% C, 6.45%

H, 17.9

H, 17.78% Br;

% Br.

The structure of 3-prspistyl-sxy-7beta-b-methoxys-1,3,5 / 10 / -021-triene was not found in the IR spectrum.

thus, the absorption of the hydroxyl group and the product is per gram of this product, and 0.91 g of buttery is dispersed or dissolved for 3 days in the dark.

silver SOJ-i ^ Sehne ⁴ - ^- £ P ^P-2b / 2-c ^hl srět ^Uy lhamino ^3f et ^yl in 50 ml of DMSO and the reaction was allowed to proceed at místPo completion of the reaction, the silver bromide oddfltruje and fur-carboxylate was diluted water (4 liters). The precipitate was separated by centrifugation and dissolved in 50 ml of acetone and the insoluble mea-gel. is separated from this solution by fixing through a G4 filter. The filtrate was dried to dryness under vacuum to give 1.3 g of an oily product.

The product was cUromahosgafuje to tilihagěls Smee in ethyl acetate / cyclohexane 10: ⁵ 0 o ^ ^ ii emove ^1U /. Unrefined ^P ro ^d uct s ^{2 p} 0 vs ^ Ozni oil.

Elemeenární analýza;

found: C 67.1%, H 7.0%, N 2.1%, 11.0% Cl;

calculated: 66.0% C, 6.99% H, 2.08% N, 10.56% cl.

IR spectrum (cm ^-1 ):

2 916, 2 840, 1 750, 1 740, 1 6 10, 1 512, 1 488, 1 · 141 1 415, 1 379, 1 361, 1 270, 1 210, 1 200, 1 170, 1 140, 1 06 8, 1 00 4, 956, 931, 885, 817, 793, 735 with ^г-1

has a ^t rs ^kt SRS-propwi- .c ^ ^y ^ y ^y 17beěth ^{/ 4} £ ppjbist2 c ^cu l> stitches ^at У / is thus confirmed, ie product and Inoj ^f ^ en ^the l st ^{b y} t ^y. oxy ^^ ation that ^x - 1, ^3, 5/1 O / -estratriene.

Example 4 Treatment of ^3-yl acětsx - beta ^ ^4/10/16 ^ ^^^^.

^ - £ Bis / by Wani ^ ^^ l ^ -Butyl-oxy-acetoxy ^{1, 3>} 59

^3-yl Aceeox -l7betř-bromařeeoxχy1,3,5 / 10 / -estrřtrSenu / 1 g /, at ^{p p} Raven Ostu ^are converted by at ≤ 2 ACLU and STI-silver salt ^to yselin ^{y 4} - ^<p - [bit / ^{2-chloroethyl} H ^{r JL} / rιmin <^ ^j ^ phenylthio butyric ^t / 0 ^{9 g} / are mixed in 50 ml of DMSO.

The reaction for NY ^Biham 3 ^{d p} s at ²⁵ ° C ^{of T} ^ m. ^P o skonaní reaction was filtered OCl ^ ^BR precipitated silver bromide and fitráátu with 4 1 of water. The resulting precipitate was isolated by centrifugation, dissolved in 50 ml of acetone and filtered with G4 to remove the product from insoluble impurities.

Evaporation of the solvent in vacuo gave 1.2 g of an oily product which was chromatographed on silica in a mixture of ethyl acetate and cyclohexane (10:50 by volume). The purified product is a viscous oil at 20 ° C.

»

Elemmenární analýza:

found: 66.0% C, 6.9% calculated: 65.64% C, 6.84%

Cl;

Cl.

No 3-acetoxy-17butane was found in the IR spectrum

IR spectrum / cm H:

absorption of hydroxyl

- (4- £ ρ - [bis- / 2-chlorethyJ / ⁰ amino fag out -phenyl ^{b y} lox ^y ^ group, the product thus has the structure f acetoxy-1,3,5 / 10 / -estrařrie-

2 915, 2 840, 1 750, 1 740, 1 610, 1 512 1 488, 1 442, 1 415, 1 378, 1 360, 1 2 70 1 210, 1 200, 1 170, 1 140, 1 06 8, 1 005 956, 931 i 885, 817, 793, 735 Example 1 a d 5

^3-acetoxy -Асе WBE ta ^ Romm aeeoxy-3 ^{3.5 5 1} 00e e ^ ^e ATR ene ^/ 1 ^{g / f,} and with a salt ^to yseiin ^{y 4/2-chloroeth} lřím ^{y /} o ^[f nnyl máteln ^{m s} / ^0, 8 ^{g /} are mixed with ⁵⁰ ml of ^ ^ ro ^ trahi wound. The reaction renders the ^{hook p p} s ⁶ 0 DEG ^C to about 24 hours at ^b.

After completion of the reaction, the precipitate formed is filtered and the filtrate is concentrated to dryness in vacuo. Separate from ^the plates and purify by silica gel column chromatography using a solvent mixture (ethyl acetate and cyclohexaa). The purified product / 0.09 gl has the structure of ^3-acetoxy-yl -17.beta. - ρ- £ ^{£ i} b s / ² · -chlot lrami eth ^y / ^{0] phenoxy-yl-butyl} ^^ LOX-acetoxy-1 3,5 / 110-eetratriene.

EXAMPLE 6 ^EXAMPLE 3 rava ^{beta.-acetoxy-17} ss- (4 - ^ p- ^ -chlorethy bis ^- phenyl Aamin ^of j ^ -but-yloxy acetoxy ^y 1,3,5 / 10 / -estrrtrSenu

STRI ^b rn ^and salt Lora c ^{h b c} lu IIu that is 4- {p- | bi s / ² ~ c ^hl oreth ^{ylamino] f} enyljm numerical ^{á é} k ^y n ^{y i} sei / 200 mg / are added to 10 ml DMSO to give a white colloidal solution. To this solution was added 190.8 mg of 3-Hydroxy h ^{y y b} -17 ^b bea- romaaceoo ^y j- 3.5 / 1O / -estratreenu and the mixture was stirred at místnoosi in the dark for 64 hours.

After this time, the color of the precipitate becomes yellow-green. A small mnoossví acetone to precipitate odffltruje fitrru G4, film formers, which is colorless and clear, freed DMSO DEET demethylating ^under vacuum ^at pH oo ^{at 80} ° C APPLICATION Voch Ш / х.

After adding 100 ml of water to the residue, white crystals precipitated. After one hour, DMSO was distilled off. The white crystals were collected by filtration on G4 and then washed with distilled water. Drying is carried out under vacuum in a desiccator. The yield of the crude material was 330.5 mg.

330.5 mg of this product was dissolved in a mixture of ethyl acetate and cyclohexane (10:50 by volume), and the solution was then passed through a column of silica gel (40 g). Gradually the product is collected: 188.2 mg / 62.86% pure product.

Elementary analysis:

Found: C 66.0%, H 7.0%, N 2.3%, Cl 11.0;

C, 66.22; H, 6.98; N, 2.27; Cl, 11.52.

Melting point: softens at ²⁵ ° C.

It was found that ^p roducts is 3- ^{hydroxy-17bett- (4} - _L p- [bis / 2-chlonehhyl / ^t am no] -phenyl) butyryloxyy tcetoxy-1,3 ^ / loz-estratrienes.

mg of this product was dissolved in 1 ml of anhydrous pyridine and 1 ml of acetic anhydride, allowed to stand in the fridge for 16 hours. After completion of the reaction, and guess at tmět concentrated in vacuo at rt ^{t h} e ^pl ^ ^tl ^ ^and Zn ^{of 30} ° C.

K by ^P Arc distilled water is added and tmět allowed to stand for 1 hour to te vylouščl oily product as a white foam colloid. The residue of pyridine and acetic acid is removed with de-distilled water and the product is washed out with water until neeurrait. The oily product was isolated and dried in vacuum, yield: 45 mg.

The product was analyzed chromatoorafií thin layer silifarelu mixture of ethyl acetate and cyclohexane in a ratio of 30:50 by volume: TE shows that the product shows a single spot at _Rf 0.78.

To purify the product, the chroaatooratin on the silifarel was carried out at a ratio of 10:50 (v / v) to ethyl acetate / cyclohexane. The purified product consisting ^{of p} s ² 0 C ^ Lskozní oil.

Elemmenámí analýza:

found: C 66.0, H 6.5, N 2.0, Cl 10.9;

calculated: 65.64% C, 6.84% H, 2.13% N, 10.79% Cl.

In the IR spectrum absorption was found and the hydroxyl groups of the product thus corresponds to the structure of 3-N-ťetoxy 17bett- (4- ^ p- ^and bis / 2-chlornthy ^{/ f i} aain0 nnyУ ^lj bst ^y r ^y l), x ^{1 yj} acetoxy- ^3, 5: LO -nstrft.rnens.

IR spectrum (cm ^-1 ):

915, 2 840, 1 750, 1 740, 1 610, 1 512 488, 1 4 42, 1 415, 1 378, 1 360, 1 2 70 210, 1 200, 1 170, 1 140, 1 068, 1 005

Example 7

Preparation of 3-propilnyloxy-17beta-1,3,5 (10) -tratene.

«- {p- ^{[bis /} 2 ™ -chořethyiam !! п ^ ^ ^- ^ ^ -but ^-:! ^ ^-), and ^ (^ ^xy too Э-ох Ну ^ ^ ^- 17 ~ (4 -. ^ P- Qis / 2 c lornth ^{t - i} lfaainO phenyl] -but-r lDx- 3.5 / 0 ^ // 50mg / rlZFUStí was in 1 mL of pyridine and 1.5 ml tnhydridu KYSE ^ ny propane. Roz flow leave in the fridge for one day, then 30 ^o c.

To the residue was added distilled water and a flashed product. The β-idine and acids, the product is washed with water until iR? Aqueous in the desiccator: 40 mg of oil.

The temperature of the bath is allowed to stand for 2 hours in order to eliminate the oleoacetic solution with distilled water, crushed and the oily phase dried under vacuum.

The product te ctroaafoorafsjn on Si Utapeli: in a mixture. nt-lntetyl acetate and cyclotextin in a ratio of 10:50 (parts by volume). The purified product is an oily substance. The IR spectrum is not tlatorpce at 3600 t 3200 cm ^{f 1st}

This confirms that the product is 3-propionyloxy-17beta-4- [beta] -bis (2-chloroethyl) aminobiphenyl} butyryloxy] acetoxy-1,3,5 (10) -estratriene.

Example 8

Preparation of 3-benzolpxy-17beta- (4- {p-Qbis (2-chloroethyl) amino] phenyl} butyryloxy) acetoxy-1,3,5 (10) -estratriene.

3-Hydroxy-17-beta- (4- [beta] - [bis (2-chloroethyl) (amino) phenylbutyryloxy) acetoxy-1,3,5 (10) -estratriene (50 mg) was dissolved in 1 ml of anhydrous pyridine and benzoové. The mixture is left to stand in the refrigerator for one day, then the bath is 30 ° C.

Mix with 2 g of the acid anhydride and concentrate to dryness / stand temperature for minutes to precipitate an oily colloidal product. Pyridine and benzoic acid are removed with distilled water and

The residue is mixed with distilled water and the product is left with the desiccator washed with water to neutrality. The aqueous phase is separated and the oily phase is dried under vacuum. Yield:

mg of oil.

is chromatographed

Product ratio 10:50 (parts by volume).

on silica gel in a mixture of ethyl acetate and cyclohexane The purified product is a viscous oily substance.

No absorption between 3600 and 3200 cm ^-1 was found in the IR spectrum. Thus, it is confirmed that the product belongs to the structure of 3-benzoyloxy-17beta- (4- [beta] -bis (2-chloroethyl) amino) phenylbutyryloxy-acetoxy-1,3,5 (10) -estratriene.

Test 1

Acute toxicity and anti-cancer activity (in vivo) of chlorambucil derivatives according to the invention.

1. Acute toxicity (LD _5q )

Groups of 8 ICF-JC1 female mice at 5 weeks of age were placed in a transparent cage to measure LD40. Each drug was dissolved in olive oil and administered in a single dose by intraperitoneal injection, subcutaneous injection and orally.

Values were subtracted using Litchfield-Wilcoxon plotting method after 7 days. It was found that Chlorambucil has LD 20 ^ ₀ mg / kg for intraperitoneal administration of 80 mg / kg by oral administration and 26 mg / kg by subcutaneous administration.

^{The LD} 50 ^of Product # 2 (see Table 1) is greater than 3000 mg / kg for intraperitoneal, greater than 6000 mg / kg for oral and greater than 3000 mg / kg for subcutaneous administration.

2. In vivo anti-cancer test.

Pieces of human breast cancer cells having steroid hormone receptors were implanted subcutaneously in mouse armpits, (BALB / C-nu / nu, 5 weeks old) to induce tumor formation. 24 hours after implantation, the active substances were started in the oil dispersion of the solution: the application was performed subcutaneously or orally and every other day for a total of 10x.

On day 25 after implantation, tumors were resected. Tumor growth inhibition efficacy was always calculated by comparing the total tumor weight of the 10 mice treated with the active substance (/) with the total tumor weight of the 10 control mice (/).

Inhibition efficiency /% / = / 1 - ^ / x 100

В

In both the subcutaneous and oral chlorambucil (15 mg / kg) treated mice, the activity was about 70%, while the efficacy of greater than 90% was found when the chlorambutyl derivatives of the invention were administered. All mice remained alive when the chlorambilcil derivatives of the invention were administered.

Serious spleen, uterine, and thyroid disorders were found in PPi autopsy in mice treated with β-β-β, but in those treated with the chloramycin derivatives of the invention, no changes were found on these organs.

Table 1

Sample no. 1 LD50 / mg / kg Peeoral application 80 LD5 ₀ / mg / k // Subtaneous application 26 Table 2

LD50 2 3 4 6 000 3 000 3 000 2 000 2 000 2 000

Anticancer efficacy

Sample No. 1 2345

Protiratovinný test

Ppeorální aplikace

Dose / mg / k // 10 15 Dec 10 15 Dec 10 15 Dec 10 15 Dec 15 Dec Korefcient is readable 8/10 7/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 Inhibition efficiency (%) 55 70 92 98 9 1 93 93 97 0 Subbutaneous application Dose / mg / k // 5 10 5 10 5 10 5 10 10 Korefcient Survival 9/10 7/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10 Inhibition efficiency (%) 53 71 90 98 93 95 92 94 0

Comment:

chlorine

Sample C. 1 Sample C. 2 Sample C. 3 Sample C. 4 Sample C. 5

3-benzorlrxy-17ss- (4- ^ S ^"£ bis / 2-ihlorlthy ^/ Rmin] -phenyl ^ ^{ь и гу1ох} у) -1,3,5 acetoxymethyl ^y / LO / stratreen ^{3-acetoxy Y-} 17ss- (4 - ^[beta] - [ ^b ii] 2-chloro-thiophenyl ^] -methoxy-1,3,5 (H) -ef-s-jratrkn

- ⁽⁴ - (p- [ ^b is / 2-i: hloleth ^{y /} aamino] ^^ 1 ^ bctyloxy) s ^ t ^ y-1,3,5 / 10 / -estratriene olive oil (control).

Test 2:

Tests of austerity were carried out as in Test 1, except that each active substance was applied in the form of a dispersion in Polysolvate 80 (emulsion / emulsifying agent) and injected three times with 100 µl injection. , the results are summarized in the table below:

Chlorambucil

Dose / mg / kg /

Inhibition efficiency (%)

Acute toxicity test (LD _5Q )

0.5

000

Test 3

In this assay affinity zjisřuje 3- Hydroxy-17 ~ ^H-4- {p- [b IS / 2 c ^h Loret yiaam ^{h i} n (yloxy) acetoxy-1,3,5 / 10 / -estratrien ^e oxalate ^To estrogen-sens tive ^to the cells for ^the Am.

^EC Radiol labeled tnUem / ³ И / B / L in ^the case of ^B alkylated ^d of ^d elo ^{hy k} r ^AL I ^to U, and ^when from ^{D Oslo} binding of labeled estradiol, b ^y L to the system introduced sample and was measured nmnosSví free ³ He ^e Sradiolu, ^kt era was ^{characterized} seal řidaným esturadl ^p ^ o ^ h ^ m.

The results are summarized in the graph of FIGⁱⁿ nM and in axis^y mine tie^3H- ^ e^withi: and i^lu in%. Gra^F at^toazuje^{, p}e mn ^ UI³H-estra the diol released by estradioe (curve 1) and the chlorambuccinate-estradiol conjugate (curve 2) are comparable and the dependence on the amount of added drug is practically the same in both cases. This shows that the chloramethyl ester conjugate exhibits high estrogen receptor activity. Curve 3 shows the ratios for chlorambulation alone.

Preparations: parts by weight

Formulation 1: active ingredient obtained according to example 350 maaot35 soobit25 carboxymmethyacelulose5 magnesium stearate5 talc 40

The monolithic constituents are mixed, sprayed and mixed to a boil of 10 mm diameter tablets.

parts hmc) t.

Formulation 2: active ingredient prepared from Example 2 lactose sugar and fatty acid ester starch water / 1% sodium carbohydrate cellulose

100 ALIGN!

500

100 ALIGN!

100 ALIGN!

The mono-constituents are kneaded and squeezed, and the resulting granules are then dried and sieved to obtain a size of 10 and 24 mesh for the preparation of grants for oral administration.

Formulation 3: The granules of Formulation 2 are filled into commercially available capsules containing

0. ⁵ cm ³ .

Formulation 4: active ingredient prepared according to Case 1 olive oil * wt. parts by weight parts

The components are concentrated, mixed and injectable.

Claims (5)

OBJECT OF THE INVENTION 1. Method of production of chlorambucil derivatives -ec ₃ H ₇ vyznaaučíií said chlorambbcíl and estradiol or acylated estradiol bind via hydroxyskcpinc in position 17 by treatment bromaaceylbromidc whereupon the optionally acyl CoA product. 2. The process of claim 1, wherein the bromaaceyl bromide is first reacted with 17-hydroxyscpinoc estradiol or acylated estradiol in a solvent, and then the thus modified estradiol or acylated ethyl ester is reacted with a carboxyl group. ^ incO ^ οι ^^ Ιη in a solvent. 3. The process of claim 1, wherein the bromoacetyl bromide is first reacted with a carboxylic acid group in a solvent and then reacted with the 17-hydroxy group of estradiol or acylated estradiol in the solvent. 4. A process according to claim 1, wherein the compound of formula I wherein R is hydroxyalkyl is present with an anhydrite or an aliphatic acid halide containing from 1 to 4 carbon atoms or benzoic acid in a solvent. 5. A process according to claim 1, wherein the chloramethylsulfamate is chloroform. converts the salt or the salt of the di-metal to the striatum and then reacts with or modified estradiol or acylated estradiol in a solvent.