JPS5842880B2 - Novel estradiol derivatives and their antitumor agents - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to novel antitumor steroid hormone conjugates.

Specifically, the present invention relates to a 17β-estradiol-chlorambutyl conjugate, which is a chemical combination of 17β-estradiol and the antitumor agent chlorambutyl, and an antitumor agent containing this conjugate as a main component.

As is well known, many of the known antitumor drugs destroy cancer cells and at the same time have a significant effect on some normal cells, have strong side effects, and are difficult to administer over a long period of time, making it difficult to eradicate cancer cells. It is considered difficult to do so.

As a result of our research to resolve the shortcomings of conventional anti-tumor agents and develop anti-tumor agents with high therapeutic efficacy, we discovered that when a certain tissue or cell becomes cancerous, The present invention was achieved by discovering a novel compound that can be intensively attacked and eliminated.

The structure of the conjugate of this new compound is represented by the following general formula (I).

General Formula This conjugate is characterized by selectively acting on tissues or cells that have estradiol receptors, and is made by using estradiol as a carrier and chemically bonding a known anticancer drug with strong cell-killing power to the carrier.

Therefore, if tissue cells become cancerous, this conjugate can be selectively distributed to that site and destroyed without causing any other side effects.

Furthermore, the conjugate of the present invention can also convert the OH group at the 3-position of estradiol to acyloxy, such as acyl t-0-C-C4H9, so that estradiol can fully serve its purpose as a carrier for anticancer agents.

These acyloxy groups naturally decompose in the body and return to OH groups, making it possible to bind to receptors.

When binding estradiol and an antitumor agent, it is important to do so so that the active site of estradiol is not inhibited.On the other hand, the site of the antitumor agent that binds to estradiol does not inhibit the antitumor activity due to the binding. Must be a body part.

Next, reaction conditions will be specifically explained.

When R is an acyl group in formula (2), the OH group at the 3-position of estradiol is reacted with an alkali in a solvent such as tetrahydrofuran (THF) to form -0Na or -OK, and further, the An acid chloride or an acid anhydride such as benzoyl chloride, acetyl chloride, or glopionyl chloride is reacted with the ester in a solvent to form an ester of the acid.

Another method is to dissolve a specified alkali (for example, NaOH1KOH) in a mixture of acetone and water, add estradiol, and then react with an acid chloride or an acid anhydride such as benzoyl chloride, acetyl chloride, or glopionyl chloride. It can also be obtained by doing

This product was then dissolved in dimethyl sulfoxide (DMSO)
, in a solvent such as dimethylformamide (DMF), pyridine, acetone, THF, benzene, toluene, carbon tetrachloride, chloroform, tetrahydrofuran (THF), etc.
React with chlorambutyl.

In this case, chlorambutyl can be used as an acid chloride or its hydrochloride.

The reaction temperature is usually 3°C to 150°C, preferably 0 to 100°C, and the reaction time is 0.1 to 90 hours.

The estradiol derivative conjugate of the present invention can be obtained by purifying the obtained reaction product by a conventional method.

Purification is performed through column chromatography (silica gel) using a mixed solvent of cyclohexane and ethyl acetate.

Alternatively, recrystallization may be used.

Next, when R in formula (1) is H, it can be easily obtained by hydrolyzing the present conjugate in which R is an acyl group in formula I) using an alkali at a low temperature of -30 to 50'C. .

It can also be easily obtained by reacting estradiol with excess chlorambutylic acid chloride or its hydrochloride to form a diester, and then hydrolyzing the diester with an alkali at a low temperature of -30 to 50°C.

Details of this type of manufacturing method will be easily understood from the examples below.

Of course, the examples are merely illustrative of specific embodiments, and various reaction conditions may be considered in the above-mentioned reactions.

The structure of the thus obtained conjugate of the present invention was confirmed by means such as infrared absorption spectrum, ultraviolet absorption spectrum, magnetic resonance, elemental analysis, melting point, etc. As a result, it was found that the conjugate of the present invention is suitable for general use.
It was confirmed that it was a conjugate of the estradiol derivative shown in

Further, as a result of acute toxicity and anticancer tests on the conjugate of the estradiol derivative of the present invention, it was revealed that the toxicity was extremely low and the anticancer effect was remarkable.

Since the conjugate of the present invention acts particularly effectively when tissues or cells sensitive to estradiol become cancerous, it is applicable to uterine cancer, breast cancer, prostate cancer, testicular cancer, thyroid cancer, etc.

Therefore, it is effective against other cancers such as gastric cancer, rectal cancer, oral cancer, esophageal cancer, lung cancer, skin cancer, leukemia cancer, etc., and is far less toxic than known anticancer drugs.

The reason for this will have to wait for future research, but it is thought to be due to a mechanism other than the expression of drug efficacy based on the receptor concept.

When the conjugate is used as a therapeutic agent, it can be prepared for administration in any conventional manner similar to known anticancer agents.

For example, tablets, granules, powders, capsules, etc. for oral administration may contain such things as binders, excipients, encapsulating agents, lubricants, surfactants, and disintegrants in the composition.

Oral liquid preparations may also be aqueous or oily suspensions, solutions, syrups, and shaken mixtures.

Suppositories may contain a lipophilic or hydrophilic base, stabilizers, decomposing agents, coloring agents, and the like.

The injection solution may be aqueous or may contain solubilizers, nutrients, stabilizers, surfactants, and the like.

In some cases, alkalis, acids, salts, etc. may be added within permissible limits in order to maintain or enhance drug activity.

Examples of these additives (carriers) include the following.

Lactose, sucrose, sorbitol, mannitol, potato starch, corn starch, amylopectin, other various starches, crystalline cellulose, cellulose derivatives (e.g. carboxymethyl cellulose, methyl cellulose), gelatin, magnesium stearate, polyvinyl alcohol, sodium alginate, Calcium stearate, polyethylene glycol, propylene glycol, wax, gum arabic, Merck, titanium dioxide, silicic acid, vegetable oils such as olive oil, peanut oil, and sesame oil, paraffin oil, cacao butter, alcohols (e.g. evaporated ethanol, benzyl alcohol), physiological These include saline, sterile water, glycerol, petrolatum, polysorbate, sodium chloride, potassium chloride, etc.

The conjugate can be contained in unit dosage form from 0.01 to 90%, preferably from 0.0-5 to 60%.

The conjugate thus formulated according to the purpose is administered by various routes such as oral, transdermal, intramuscular, intraperitoneal, intravenous, intrarectal, and topical.

The dosage varies depending on the administration method and the degree of treatment, but is roughly as follows.

For adults, oral administration per day approximately o, 1 mg/- ~ 50
mg/kg.

For adults, subcutaneous injection per day is approximately 0. O1■/kg~2
0■/kg.

The present invention will be explained below with reference to Examples, but the present invention is not limited thereto.

Example 1 Synthesis of 3-benzoyloxy-1,3,5(10)estratriene-17β-C(4-(P-(bis(2-chloroethyl)aminocophenyl)phthylate)) 1.0?(3 , 29mM) of 4-(P-(bis(2
-Chloroethyl)aminocophenyl)butyric acid (chlorambutyl) 2. Of (16,8mM) of 5
It was dissolved in OC12, left at room temperature for 5 minutes, concentrated under reduced pressure on a 40°C water bath, and after scattering as much of 5OC12 as possible, 20-dehydrated benzene was added and stirred until a homogeneous solution was obtained. .

The solution was again concentrated under reduced pressure to precipitate white-yellow crystals.

After drying under reduced pressure, 20 rul of dehydrated benzene was added to the crystals again to dissolve the crystals, and then dried under reduced pressure again to obtain the 4-
P-(bis(2-chloroethyl)aminocophenyl)butyryl chloride hydrochloride was obtained.

This was almost a theoretical amount. 1.1f (3.1mM)
4-(P-(bis(2chloroethyl)amine)phenyl)butyryl chloride hydrochloride was dissolved in 201 rLl of dehydrated benzene, and 0.82 f?
Pentyloxyto β-ol was added, and the mixture was refluxed at the boiling point for 1 hour. After the reaction, benzene was removed under reduced pressure to obtain 1.4f of crude crystals.

The crude crystals were dissolved in 70 ml of benzene and filtered using 20 f Florisil (60-100 mesh of magnesium silicate).
Lysyl was washed with 140 TLl of benzene and concentrated to dryness under reduced pressure to obtain white crystals.

The white crystals were mixed with cyclohexane/ethyl acetate in a solution of 50:1.
Column chromatography using silica gel (developing solvent dichlorohexane/
It was purified with a mixed solution of ethyl acetate in a 50:10 volume ratio.

A fraction showing Rf = 0.46 (TLC silica gel: 50:20 volume ratio mixed solvent of cyclohexane/ethyl acetate) was collected, dried under reduced pressure, and recrystallized with ethyl ether to give 1.25S' white crystals. I got it.

Yield: 86.8% based on estradiol benzoate
Met.

The properties of the obtained product were as follows.

Melting point = 69-70℃ (Yazawa micromelting point B
Y-2 (manufactured by Yazawa Seisakusho)) IR spectrum: 2960, 2930, 2860, 1745 (173 to shoulder
0, 1715, 1612, 1517, 1491, 145
0,1365,1310,1262,1244,122
2, 1 2 1 011175, 1150, 1084,
1063, 1040, 1024, 1005, 9 1 5
, 878, 799, 740, 705cIrL' (KB
r method) Elemental analysis value: C H N Cl Actual value (to) 70.60 6.90 2.20
10.50 Theoretical value (3) 70.62 6.79
2.11 10.71 Example 2 3-acetoxylate 3.5(10) estratriene-
Synthesis of 17β-(4-(P-(bis(2-chloroethyl)amino)phenyl)butyrate] Chloride hydrochloride was dissolved in 30 TrLl of dehydrated benzene by heating, and 0
．． 925f (2,95mM) 3-7) key 71.3
- 5(10)-Estratrien-1-fluor β-ol was added, and the mixture was refluxed at the boiling point for 1 hour. After the reaction was completed, benzene was removed under reduced pressure to obtain crude crystals of 1.7i.

The crude crystals were dissolved in 50 ml of benzene and filtered using 20 t of Florisil (60-100 mesh).
Further washing with 150 ml of benzene gave a clear oily substance.

This oil was dissolved in 50% of cyclohexane/ethyl acetate:
It was dissolved in a mixed solvent at a volume ratio of 1:0 and purified by column chromatography using silica gel.

When a single spot fraction was collected and dried under reduced pressure, no crystals were precipitated, and a transparent semi-molten material at room temperature of 1.51 was obtained.

The properties of the obtained product were as follows.

IR spectrum (cIrL-1): 2960.2920, 2860, 1730°1725.
1700, 1611.1515.1445.1380.
1351, 128011245.1216.1179.
1149.1037.1005.960,918.86
8800.783 (NaC1 plate) Elemental analysis value CHN C1 Actual measurement f 67.8 7.2 10.9 11
．． 1 theory f Harrei 68.00 7.17 10.67
11.83 Therefore, it was confirmed that this was the desired compound.

Example 3 Base of 3-propionyloxy-1,3,5(10)-estratriene-17β-(4-(P-C bis(2-J'chloroethylaminocophenyl)phthylate) 1.1? (3.1mM) of 4-(P-Cbis(2chloroethyl)aminocophenyl)butyryl chloride hydrochloride was dissolved in 30TLl of dehydrated benzene under heating.
68y (2,95mM) of 3-propionyloxy-
1,3,5(10)-estratrien-17β-ol was added, and the mixture was refluxed at the boiling point for 1 hour to obtain crude crystals of 1.61.

The crude crystals were purified by column chromatography using silica gel to obtain a colorless and transparent semi-molten material at room temperature of 1.5'f.

The properties of the obtained product were as follows.

IR spectrum (CrrL-1): 2960.2920, 2860, 173011725.
1700, 1611.1515.1444.1376.
1350, 1282.1245.1216.1179.
1150°1039.1005.960,918.86
8.799.784 (NaC1 plate) Elemental analysis value CHN C1 Real side @', 4 68.00 7.50 10.10 1
1.90 theory (i% 68.40 7.33 10.
42 1156 Therefore, it was confirmed that this was the desired compound.

Example 4 3-Hydroxy-1,3,5(10)-estratriene-17β-[(4-(P-[bis(2chloroethyl)
8rnf? (1,49x 10'mol) of 3
-benzoyloxy-1,3,5(10)-estratriene-17β-(4-(P-(bis(2-chloroethyl)aminocophenyl)butyrate) in acetone 10r
It was dissolved in a mixed solvent of rtl and methanol (10 ml).

The solution was cooled with ice and, without stirring, was dissolved in an aqueous solution of 2Co3 (8,27%'/77'') (0,6X 10'
mol) was added dropwise over about 30 minutes.

The reaction is a homogeneous hydrolysis.

After standing for approximately 16 hours, the solvent was removed at room temperature.

Separation and purification was carried out using silica gel to obtain 63.7 cm of the target substance.

The yield was 76.65%.

The IR band of this material was as follows.

3370 (crfL' included 2920.2860.17
25.1700.1611.1515.1442.13
80.135011280゜1245.1210.11
77.1148.1037.1005.960,918
．． 868.800.783 The elemental analysis values were as follows.

CHN C1 Actual value 1'J 67.70 7.70 2.20 1
2.50 theoretical value 68.83 7.35 2.51
12.71 Example 5 Synthesis of 3-Hydroxy-1,3,5(10)-estratriene-17β-C4-(1-(bis(2chloroethyl)aminocophenyl)phthylate) 47ft (13,1mM) 4-(P-(bis(2-chloroethyl)aminocophenyl)butyryl chloride hydrochloride was dissolved under heating in 301 rLl of dehydrated benzene, and 5
．． 51 mM estradiol-17β was added and refluxed at boiling point for 1 hour.

After the reaction was completed, benzene was removed under reduced pressure to obtain a slurry-like residue of 5.51%.

This residue was heated and dissolved in 701 rLl of benzene, and
Filtered through 0r Florisil (60-100 mesh of magnesium silicate), washed the Florisil with 700ml of benzene, and concentrated the benzene solution under reduced pressure to give a pale yellow oil.

This was mixed with 50ml of cyclohexane/ethyl acetate of 50:
It was dissolved in a mixed solvent of 10 volume ratio and purified by column chromatography using silica gel (developing solvent dichlorohexane/ethyl acetate mixed solvent of 50:10 volume ratio).

Rf=0.28 (TLC silica gel: cyclohexane/
The fraction containing ethyl acetate (50:10 volume ratio mixed solvent) was collected and dried under reduced pressure in a desiccator to obtain a semi-solid material at room temperature of 2.22.

2. This material was recrystallized from an ether-petroleum ether mixed solvent. A colorless semi-solid material was obtained at room temperature.

The yield was 43% based on estradiol.

The properties of the obtained product were as follows.

IR spectrum (crrL-1); 2920.2860.1750.1725.1613.
1515.1490, 1445.1350.1245.
1205.1175.1143.1133.1o35.
1005.800, 746 (KBr method) Elemental analysis value; CHN C1 Actual value 65.50 7.10 3.40 16.4
0 theoretical value 65.34 6.87 3.31 16.
81125.7■ (1,49X 10'mol
) was dissolved in a mixed solvent of 1011 liters of acetone and 10 liters of methanol.

This solution was cooled with ice and stirred, and a 2CO3 aqueous solution (s, 2r
v/1 m0 (0.6×10'mol) was added dropwise over about 30 minutes.

The reaction is a homogeneous hydrolysis.

After standing for approximately 16 hours, the solvent was removed at room temperature.

64.5TnII was separated and purified using silica gel.
The target substance was obtained.

The properties of the product obtained were the same as those of the product obtained in Example 4.

Example 6 Acute toxicity and anticancer activity test (in vivo) of this conjugate
I will describe the results of this.

(1) Acute toxicity Acute toxicity was measured using ICR-JCL mice (4 weeks old).
Eight animals per group were placed in a transparent polycage, and a sample dissolved in olive oil was administered via a syringe in an amount of 3 t1 kg via the intraperitoneal route.

After administration, poisoning symptoms and death were observed for one week, but there was no difference from the olive oil administration group.

No cases of death were observed.

On the other hand, the group receiving the same amount of chlorambutyl alone died immediately after total loss.

Considering that the LD5 value of chlorambutyl is 201n9/kg, it is understood how low the toxicity of this conjugate is.

(2) Cancer control test Human breast cancer cells were transferred to nude mice (BALB/C1-nu/
nu) (5 weeks old) The tumor was subcutaneously transplanted into the lower armpit to form a solid tumor.

Twenty-four hours after transplantation, a predetermined amount of the conjugate of the present invention and olive oil, well dispersed or dissolved, was administered subcutaneously or orally.

Administration was carried out at a predetermined dose 10 times every other day, and the tumors were excised on the 25th day after transplantation. ,
The tumor growth inhibition rate was calculated from the following formula.

When chlorambutyl was administered orally 10 to 15 days per nine days, the growth inhibition rate was 50 to 70%.

On the other hand, all of the conjugates of the present invention were 70% or more.

Furthermore, the survival rate of the conjugate of the present invention was 100%.

Example 7 Formulation example 1 3-benzoyloxy-■・3・5(10)-estratriene-17β -C4-(P-(bis(2-chloroethyl)aminocophenyl)buty2-19 40 parts mannitol 35 parts sorbitol Carboxymethylcellulose stearate Magnesium Merck 25 parts 5 parts 5 parts 40 parts The above composition was thoroughly mixed, powdered, and compressed into tablets with a diameter of 10 mm.

Formulation Example 2 3-Glopionyl-1,3,5 (10) Estratriene-17β-[4 (P-[bis(2-chloroethyl)aminocophenyl)butyrate] ■ 0 parts lactose 50 parts sucrose fatty acid ester 10 parts starch
20 parts water (methyl cellulose 0.
10 parts (containing 4%) A mixture having the above composition was prepared, kneaded, and then extruded using an extrusion pelleter to form granules.

This was dried and sorted between 10 meshes and 24 meshes to prepare granules for oral administration.

Formulation Example 3 The granules obtained in Formulation Example 2 were filled into commercially available capsule containers to form 0.5CC capsules.

Prescription example 3-benzoyloxy-1, 3, 5 (10)-Estratriene-17β -(4-(p-C bis(2-chloroethane) thylaminocophenyl)buti2 19 Part 3 benzyl alcohol 5th part macro goal 300+ 92 copies is heated and sterilized to make an injection.

Prescription example 5 3-hydroxy-1, 3, 5 (10) -Estratriene-17β-[4 (P-[bis(2-chloroethyl)a minocophenyl)butyrate] Part 1 olive oil 99 copies was added, heated, and sterilized to make an injection.

Claims (1)

[Scope of Claims] 1. A 17-β-estradiol-chlorambutyl conjugate represented by the general formula [where R represents a hydrogen atom or an acyl group]. 2 acyl group hapenzoyl, phenylacetyl, acetyl, propionyl, butyryl, valeryl group) *17β- according to claim 1
Estradiol-chlorambutyl conjugate 3 An antitumor agent characterized by containing a 17-β-estradiol-chlorambutyl conjugate represented by the general formula [where R represents a hydrogen atom or an acyl group]. 4. The antitumor agent according to claim 3, wherein the acyl group is selected from benzoyl, phenylacetyl, acetyl, propionyl, butyryl, and valeryl groups.