The pharmaceutical composition inhibiting bone resorption or promoting osteogenesis

 

(57) Abstract:

A pharmaceutical composition comprising a compound of formula (I): X - Y - Z, where Y is represented by the following formulas III, IV and V:

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X represents a monovalent group connection type of tetracycline, and Z represents a monovalent group of steroid compounds type, such as estrogen. The connection can concentrate on bone tissue and has features that any abscopal resorption and promoting bone formation. The invention expands the Arsenal of effective promoters of osteogenesis. 9 C.p. f-crystals, 7 ill., 10 table.

The invention relates to pharmaceutical compositions, which has new features, any abscopal bone resorption or enhance osteogenesis.

Normal preservation of the bones is due to the balance of bone resorption and osteogenesis, and if increases bone resorption, the components of the bones dissolve and decreases that leads to such a bone disease such as osteoporosis. It is known that sex hormones such as estrogen, in a manner that inhibit bone resorption, and therefore they are used in Europe and America as a preventive or therapeutic agent for the and we cannot exclude the possibility of carcinogenesis due to a single injection of these hormones.

On the other hand, antibiotics such as tetracycline have the ability to concentrate in the bones, but they are unable to inhibit bone resorption or to cause bone formation. Only in U.S. patent N 4925833 indicated that tetracycline stimulates the synthesis of bone proteins in experiments at the cellular level.

Although osteogenesis needed synthesis of bone proteins, only one synthesis of bone proteins cannot provide osteogenesis.

It is still not known materials having the ability to promote osteogenic that could be used for the prevention and treatment of bone diseases.

Accordingly, the present invention features a drug for bone diseases, which has the ability to inhibit bone resorption and promotes osteogenesis, preferably, synergistically, and can accumulate in the bones.

Conducted various studies to solve the previously described problems, and found that the compounds that are formed by covalent bonds between antibiotic type of tetracycline and that steroid type a hormone like estrogen, performed by the linker has the function osteogenesis object of the present invention.

Accordingly, the active component of the present invention can be represented by the formula (I):

X - Y - Z (I)

/where X represents a monovalent group represented by the formula (II):

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where R1represents hydrogen or a hydroxyl group, R2represents hydrogen or a hydroxyl group, R3represents hydrogen or methyl group, and R4represents hydrogen, halogen or dimethylaminopropyl/;

Y represents a divalent or trivalent group represented by the formulas /III/ IV/, or /V/:

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(2) -CH2- N - (CH2CH2O)n- CH2CH2- X - (3) (IV)

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where = O -4, and-X - represents a simple bond, -O - or-NH -/, and

represents a monovalent group formed by removing hydrogen or hydroxyl group of the compounds of formula /VI/:

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/where R1'is HO or O =; R2'represents a hydrogen atom or methyl group, R3'represents a hydrogen atom, phenyl group or substituted phenyl group; R4'represents methyl group or ethyl group; R5'represents a hydroxyl group, a ketone group or acetyl group; R6'represents hydrogen, hydroxyl group, ' represents hydrogen, a hydroxyl group or a =O, or R6'and R7'together connected with oxygens 2,2-dioxypurine group, and the symbol ... represents a simple bond or double bond/, which is connected group exists in the 2-position, 3-position, 4-position, 6-position, 7-position, or 17-position, or phenyl groups, linked in the 11-position; /1/ formula /II and /2/ formula /III/-/V/ is directly connected, and /3/ formula /III/-/V/ and any of the related group of the formula /VI/ are directly connected/.

In the formula (II) halide may be, for example, fluorine, chlorine, bromine or iodine, and preferably is chlorine.

Fig. 1 is a photograph showing the results of experiment No. 3.

Fig. 2 is a photograph showing the results of experiment No. 3.

Fig. 3 is a photograph showing the results of experiment No. 3.

Fig. 1-3 correspond to the results obtained from three replications of each experiment.

Fig. 4 is a graph showing the effect of compounds 1 - 3 on the body weight of castrated rats.

Fig. 5 is a graph showing the effect of compounds 1 - 3 density cost the I 1 - 3 bone density /BMD/ rat DVX.

Fig. 7 is a graph showing the effect of 0.5 mg/day of compound 1 - 3 on bone density /BMD/ rat DVX.

Examples of the monovalent group of formula (II), which is a fragment of the compounds of formula (I) is the active ingredient of the pharmaceutical compositions of the present invention is the following:

Formula /II - 1/:

Monovalent group tetracycline represented by formula (II-I/ /where in the formula (II), R1is hydrogen, R2represents a hydroxyl group, R3represents a methyl group, and R4represents a hydrogen/;

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Formula /II-2/

Monovalent group tetracycline represented by formula (II-2/ /where in formula II, R1represents a hydroxyl group, R2represents a hydroxyl group, R3represents a methyl group, and R4represents a methyl group/:

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Monovalent group chlortetracycline, represented by formula II-3/ /where in the formula (II), R1is hydrogen, R2represents a hydroxyl group, R3represents a methyl group, and R4represents chlorine/:

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Formula II-4/:

Monovalent group is the SCP, R2is hydrogen, R3represents a methyl group, and R4represents a hydrogen/:

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Formula II-5/:

Monovalent group aminotetraline represented by formula (II-5/ /where in formula II, R1is hydrogen, R2is hydrogen, R3represents hydrogen, and R4is dimethylaminopropyl/:

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Examples of the monovalent groups of the compounds of formula /VI/, which is a fragment of the compounds of formula (I) is the active ingredient of the pharmaceutical compositions of the present invention is the following:

Monovalent group of estrone represented by the formula /VI-1/ /where in the formula /VI/ R5'and R6'together form =O, and R7'represents a hydrogen/:

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Formula /VI 2/:

Monovalent group estradiol represented by the formula /VI 2/ /where in the formula /I/ R5'represents a hydroxyl group, R6'represents hydrogen, and R7'represents a hydrogen/:

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Formula /VI 3/:

Monovalent group extraordinal represented by the formula /VI 3/, where in the formula /VI/ R5'represents a hydroxyl group, R6'is etinilnoy group, and R7'represents a hydrogen/:

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Formula /VI-4/drakelow group, R6'represents hydrogen, and R7'represents a hydroxyl group/:

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A related group of compounds of formulas /VI/-/VI-4/ described earlier, there is a 3-position, 6-position or 17-position.

Connection /VI/ can be, in addition, monovalent groups that receive, removing the hydrogen or the hydroxyl group of the following compounds (see below).

Accordingly, the active ingredient of the pharmaceutical compositions of the present invention may be labeled, for example, by the following formula:

/II-1/-/III/-/3/ /VI-1/ /the number in parentheses/ /are the position of the group communication group of the formula /VI-1/: hereinafter the same/ (see the end of the description).

The above-described compounds per se can be obtained by known methods. For example, a binding fragment /linker/ represented by the formulas /III/-/V/ in the beginning, associated with steroid compound represented by the formula /VI/, and then the obtained product is associated with a material such as tetracycline.

The binding of the linker of the formula /III/-/V/ s position -3 steroid compounds of formula /VI/ is carried out, for example, in accordance with the following reaction scheme (see below).

Further, the connection in which to obtain for example, the following reaction (see below).

In order to link the linker formulas /III/-/V/ with position 6 of the steroid compound represented by the formula /VI/, group =O initially introduced in the 6-position of the steroid compounds, and then, carry out the following reaction (see below).

Then tetracycline compound can be associated with a reaction product of the linker and steroid compounds due to cross-linking the nitrogen atom of the linker and an atom of the amide nitrogen group of tetracycline compounds due to formaldehyde.

A pharmaceutical compound of the present invention can be administered orally or parenteral, for example, by intravenous, intramuscular, subcutaneous injection, intraperitoneal injection, etc., an Effective daily dose for humans is from 0.2 to 200 mg when administered orally, and from 0.1 to 100 mg by parenteral introduction. Compounds of the present invention have extreme low toxicity and LD50after oral administration of compound 1 obtained in example 1, for example, is for mice 143 mg/kg

The pharmaceutical compositions of the present invention may have a conventional form of preparations in accordance with the method wok, granules, powders, liquid preparations, etc. you can get Them in the usual ways. For example, liquid preparations can be obtained by dissolving or suspending the active ingredient of the present invention in a suitable medium such as an aqueous buffer or similar Preparations powders can be obtained by mixing the active ingredient of the present invention with a powdery filler, such as starch /for example, corn starch/ and/or saccharides such as lactose.

Tablets get by mixing the active ingredient with a filler, such as mentioned previously fillers and binder such as starch paste, and extruding the mixture in the car for tableting. Granules can be obtained by mixing the active ingredient with the filler, binder and so on, mixing the mixture with such a liquid, such as water and/or glycerin, passing the resulting product through a sieve to obtain granules, and drying the obtained granules. Capsules can be obtained by filling a standard size capsule, powder or granules obtained as described previously.

Doses for parenteral injection can be obtained by dissolving or suspending the active ingredient in physiological saline or this buffer, as the buffer phosphorus is th before use should be dissolved or suspended, and media for freeze-drying can serve sugars such as lactose, or common carriers for freeze-drying.

Examples

The following are specific examples of compounds of the present invention. However, the main scope of the invention, these examples are not limited.

An example of obtaining 1.

101. Obtaining 3-chloroethoxy-17-okestra-1,3,5(10)-triene /connection 1-1/

The NaOH solution is added to the toluene solution obtained by mixing at 27.1 g of estrone, 22,2 g sulfonate chloroethyl-n-toluene and a small amount of triethylenediamine. After pH was adjusted to 10, the reaction of lead within 4 hours and the solvent is evaporated. The solid product is recrystallized from alcohol, and get a connection /1-1, R7=H/. Yield 79%. So melting 86-88oC. Elemental analysis: C 72,40 H 43 C 10,71.

1-2. Obtaining N-/17-hydroxy-östra 1,3,5/10/-trien-3-oxyethyl/piperizine. /Connection 1-2/:

of 7.8 g of compound /1-1/ previously described and 46.6 g of anhydrous piperazine and 120 ml of dimethylformamide /DMF/ subject interaction at a temperature of 80-100oC for 5 hours. After evaporation of DMF obtained solid is again recrystallized from ethanol and acetone to obtain beh">

1-3. Obtaining N-4-/17-hydroxy-östra 1,3,5/10/-trien-3-oxyethyl/- piperazine-1-methylenedianiline /Connection 1-3/:

3.8 g of the compound /1-2/, previously described, 0.03 g of metatarsalgia and 15 ml of isopropanol is subjected to interaction with the 40oC for 2 hours. After adding 3.5 g of tetracycline, the resulting mixture is stirred, and the reaction of lead for 5 hours. After completion of the reaction, the reaction product is filtered off, washed with isopropanol and ethyl ether. Resulting in getting a solid yellow /Connection 1-3/ /R1= R4H; R2= OH, R3= CH3/

Yield 95%. So melting 160oC /decomposition/

Elemental analysis: C 67,21 H 7,12 N 6,67

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Example of receipt 2.

2-1. Obtaining N-/17 - hydroxy-östra-1,3,5(10)-trien-3-oxyethyl/piperazine. /Connection 2-1/:

3.8 g of the compound /1-2/ example 1 was dissolved in methyl alcohol. After 0.5 g of bergerat potassium is added to the alkaline solution, the reaction mixture was treated by heating and returning the stream for 3 hours. The reaction solution is neutralized with acid, and methyl alcohol is evaporated. The obtained solid component is recrystallized from alcohol. And finally, get white crystals /connection 2-1, R7of N-4-/17 - hydroxy-östra-1,3,5(10)-trien-3-oxyethyl/-piperazine-1-methylenedianiline /Connection 2-2/:

of 3.84 g of compound /2-1/, previously described, 0.03 g of metatarsalgia and 20 ml of isopropanol is subjected to interaction with the 40oC for 2 hours. Once you add 3.5 g of tetracycline, the reaction mixture is stirred, and the reaction of lead for 5 hours. After completion of the reaction, the reaction product is filtered and washed with isopropanol and ethyl ether. Then get a pale yellow solid /connection 2-2/ /R1=R4=H, R3= OH, R3=CH3/. Yield 95%.

So melting 165oC /decomposition/

Elemental analysis: C 67,30 H 7,34 N 6,54

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Example of getting 3

Obtaining N-4-/17 - hydroxy-östra-1,3,5-(10)-trien-3-oxyethyl/-1-piperazine-1 - methylene-oxytetracycline /connection 3-1/

3.8 g of the compound of example 2/2-1/, 0.03 g of metatarsalgia and 20 ml of isopropanol is subjected to interaction with the 40oC for 2 hours. Once you add 3.5 g of tetracycline, the reaction mixture is stirred and the reaction is carried out at a stirring for 5 hours. After completion of the reaction the mixture is treated according to the method of example 1-3, resulting in a gain solid pale yellow color of the decomposition/.

Elemental analysis: C 65,62 H 7,10 N 6,67

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Example 4

4-1. Obtaining bis-N,N-/17-hydroxy-östra-1,3,5-(10)-trien-3-oxyethyl/Amin /connection 4-1/

The NaOH solution is added to a mixture of 3.6 g of masterthread, 12 g of estrone, 4 g triethylamine, water and toluene with stirring. After the mixed solution is refluxed for 5 hours, the solvent is evaporated. The hard part is recrystallized from alcohol and get the right product. Yield 72%.

So melting = 256 - 259oC.

Elemental analysis: C 78,50 H at 8.60 N 2,31

4-2. Obtaining bis-N,N-/17-hydroxy-östra 1,3,5/10/-trien-3-oxyethyl/-aminomethylpyridine /connection 4-2/:

of 6.1 g of compound /4-1/ indicated previously, 0.03 g of metatarsalgia and 20 ml of isopropanol is subjected to interaction with the 40oC for 2 hours. After adding 3.5 g of tetracycline reaction mixture is stirred and the reaction is carried out at a stirring for 8 hours. After completion of the reaction receive the solid product is a pale yellow /connection 4-2/ /R1= R4= H, R2= OH, R3= CH3/. The output is 68%. So melting = 183oC /decomposition/.

Elemental analysis: C 71,10 H 7,21 N 3,89

Patterns obtained in example 4, the connection of bis-N,N-/17 - hydroxy-östra 1,3,5/10/-trien - 3-oxyethyl/Amin/ /connection 5-1/:

Methyl alcohol is added to 6,1 g previously specified connection /4 - 1/, and, after alkaline conditions add 0.5 g of bergerat potassium, the reaction is carried out at the boil under reflux for 5 hours. Then the reaction solution is neutralized with acid, and methyl alcohol is evaporated. The hard part is cleaned in acetone and ethanol solution. Estrone-17-ketone in the connection /4-1/ restore to the white product containing-17-hydroxyl group. The yield is 82%.

So melting = 193 - 197oC.

Elemental analysis: C 78,41 H 8,51 N 2,33

5-2. Obtaining bis-N,N-/17 - hydroxy-östra-1,3,5(10)-trien - 3-oxyethyl/aminomethylenemalonate /connection 5-2/:

to 5.4 g of compound /5-1/, previously described, 0.03 g of metatarsalgia and 20 ml of isopropanol is subjected to interaction with the 40oC for 2 hours. After adding 3.5 g of tetracycline reaction is carried out at a stirring for 8 hours. After completion of the reaction receive the solid product is a pale yellow /connection 5-2/ /R1= R4= H, R2= OH, R3= CH3/ by way of example 1-3. The yield is 94%. So melting = 171oC /s decomposition of the R receive 6

6-1. Obtaining bis-N,N - 17 - hydroxy and 17 - ethinyl-östra 1,3,5/10/-trien-3-oxyethyl/Amin/ connection 6-1/:

of 6.1 g of compound /4-1/ example 4 are dissolved in 100 ml of tetrahydrofuran and 1.0 g of powdered potassium hydroxide, and the resulting mixture is subjected to interaction until the end of the reaction at 0oC under vigorous stirring, introducing gaseous acetylene. The reaction mixture was neutralized to pH 4 acid, and the solvent is evaporated. Then the reaction product is recrystallized from alcohol and chloroform, resulting in a gain white solid product /6-1/. Yield 78%.

So melting 201-205oC.

Elemental analysis: C 79,21 H 8,58 N 2,18

The compound obtained has the following structural formula:

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6-2. 6 g of compound /6-1/ obtained earlier, 0.03 g of metatarsalgia and 20 ml of isopropanol is subjected to interaction at 60oC for 2 hours and then added 3.4 g of tetracycline. The reaction mixture is stirred and left to react for 8 hours. After completion of the reaction receive solid pale yellow /connection 6-2/, i.e., bis-N,N-/17 - hydroxy-17 - ethinyl-östra-1,3,5(10)-trien-3-oxyethyl/aminomethylation /R1=R4=H, R2=OH, R3=CH3/ according to the method of example 1-the compound has the following structural formula:

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Example of getting 7

7-1. Obtaining N-/17-hydroxy-östra-1,3,5(10)-trien-3-oxyethyl/N-methyl-amine /connection 7-1/:

2.7 g of estrone, 1 g chloroethylamine and a small amount triethylamine mixed with a toluene solution, and add a solution of sodium hydroxide. After pH was adjusted to about 10, the reaction of lead within 4 hours. After that, the solvent is evaporated, the solid portion is recrystallized from ethanol to obtain compounds /7-1, R7=H/. Yield 71%/.

So melting = 262-266oC.

Elemental analysis: C 75,24 H 9,41 N 4,28

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7-2. 3,3 g of compound /7-1/ indicated previously, 0.03 g of metatarsalgia and 20 ml of isopropanol are interacting within 2 hours, and then add 3.5 g of tetracycline. The reaction mixture is stirred, and conduct a reaction for 8 hours under stirring/. After completion of the reaction, the solid pale yellow /connection 7-2/, i.e., N-/17-hydroxy-östra-1,3,5(10)-trien-3-oxyethyl/N-methylaminomethyl - tetracycline /R1=R4=H, R2=OH, R3=CH3/, receive by way of example 1-3. Output 90%.

So melting 190oC /decomposition/

Elemental analysis: C 68,8 H 7,22 N 3,62

The structure of the obtained compound represented by the following the Il/piperazine /Connection 8-1/:

to 5.1 g of compound /8-0/ dissolved in 120 ml of tetrahydrofuran, and added 3.2 g AMINOETHYLPIPERAZINE. The reaction mixture is refluxed for 2 hours. THF is evaporated and removed, and add 100 ml of methyl alcohol and 2.8 g of formic acid. Continue boiling under reflux, and the reaction of the lead within 3 hours. Methyl alcohol is evaporated and removed, and the residue is recrystallized from ethanol to obtain compound 8-1. So melting 172 - 177oC.

8-2. Obtaining N-4-/3,17 - dihydroxy-extra-1,3,5(10)- triene-6-amino-ethyl-piperazine-1-methylene-tetracycline /connection 8-2/:

4.1 g of compound /8-1/, 0.03 g of metatarsalgia and 20 ml of isopropanol are mixed and the reaction is conducted at 50oC for 2 hours. Then add 3.5 g of tetracycline and the reaction is carried out at a stirring for 5 hours. After completion of the reaction, the obtained product is filtered, washed with isopropanol and ethyl ether, then dried in vacuo to obtain a solid product, pale yellow /connection 8-2/. Melting point 167oC /decomposition/.

The output was 81,20.

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Connection /8-0/ obtained as follows.

1. Getting 17 - estradiol

4 g of estrone races the l of water at a temperature of about 30oC. After this reaction are within 2 hours, the reaction solution is neutralized with diluted acetic acid to neutral, and then diluted with water. After the formation of solids it is filtered off, washed with water and dried. The resulting product is recrystallized from ethanol containing water to obtain white crystals. So melting = 173-174oC.

Output 97,2%.

Getting 17 - estradiolvalerate

10 g of 17 - astroengine dissolved in pyridine, and to this add 35 ml of acetic acid. The reaction is carried out at boiling under reflux for one hour, then the reaction mixture was poured into ice-cold water to obtain a solid product, which is filtered off and dried, recrystallized from absolute ethanol to obtain white crystals. So melting point 126-128oC. the Yield is 97%.

3. Obtain 6-hydroxy-17 - estradiolvalerate /Conn. XII/.

5 g of 17 - estradiolvalerate dissolved in benzene and to this was added dropwise 0.45 g of chromium trioxide under cooling, then the mixture is dissolved in a mixed benzene solution of 30 ml of glacial acetic acid, 20 ml of acetic acid and 30 ml of benzene. After completion of the reaction, the reaction is washed with saturated sodium bicarbonate solution, water, then dried and concentrated, and then allocate on silica gel target product. So melting 173 - 175oC. the Yield is 40%.

Example of receipt 2.

Obtaining N-4-/17-hydroxy-extra-1,3,5(10)-trien-3-oxyethyl/- 1 - piperazine-1-methylene-doxycycline. HCl /Connection 9/:

2.2 g of the compound /1-2/, 0.20 g of polyformaldehyde and 100 ml of isopropanol is heated and stirred at 60oC for 1.5 hours, then add 3 g of doxycyclineonline. The reaction mixture was kept at 60oC under stirring for 2.5 hours. After completion of the reaction, the resulting product is filtered, washed with isopropanol and ethyl ether, then dried to obtain a solid product, pale yellow /connection 9/ with a melting point of 172oC /decomposition/. The yield is 87%.

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Example 10

Obtaining N-4-/17 hydroxy-extra-1,3,5(10)-trien-3-oxyethyl/-piperazine-1-methylene - oxytetracycline /connection 10/:

of 0.91 g of compound /1-3/, 80 mg polyformaldehyde and 50 ml of isopropanol is subjected to interaction with stirring at 60oC for 2 hours. Then add 1.0 g of terramicina, and the reaction mixture was kept under stirring at 60oC for 3 hours. Product is ledno yellow /connection 10/ with Tons of melting point 175oC /decomposition/. Yield 89%.

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Example of getting 11

Obtaining N-4/17 hydroxy-östra-1,3,5(10)-triene-3-ethoxyethyl/piperazine-1-methylenedianiline /Connection 11/:

4.1 g of N-/17-hydroxyestrone-1,3,5(10)-trien-3-oxyethyl/piperazine, 0.5 g of polyformaldehyde and 50 ml of isopropanol is subjected to interaction with stirring at 60oC for 2 hours. Then add 4 g of tetracycline, and the reaction is conducted at a temperature of 40 - 45oC for 3 hours. The resulting product is filtered, washed with isopropanol and ethyl ether to obtain a solid product, pale yellow /connection 11/ with a melting point 154oC /decomposition/. The output is 68,6%.

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Example 12

Getting 17-hydroxy-androst-4-EN-3-oxyethylenenitrilo

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3.3 grams of 3-aminoethoxy-17-hydroxyandrost-4-ene, 0.3 g metatarsalgia and 40 ml of isopropanol is subjected to interaction at 60oC for 4 hours. After adding 4.5 g of tetracycline reaction is carried out at a stirring for 5 hours. The reaction product is filtered off, washed with isopropanol and ethyl ether to obtain a solid product of pale yellow color. Yield 92%.

Elemental analysis is literacylink:

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3.6 g of 6-aminoethoxymethyl-17-hydroxy-androst-4-EN-3-one, 0.3 g metatarsalgia, 4.5 g of tetracycline and 30 ml of acetone was stirred at room temperature for 24 hours without light. After completion of the reaction, the obtained product is filtered, washed with acetone and ethyl ether to obtain a solid yellow color. Yield: 86%.

Elemental analysis: C 67,90 to 7.67 H N 5,18

Example of getting 14

Getting 17-hydroxy-androstane-3-oxyethylenenitrilo:

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at 3.35 g of 3-aminoethoxy-17-hydroxyandrost, 0.3 g metatarsalgia, 4.5 g of tetracycline and 30 ml of acetone is subjected to interaction with stirring at room temperature for 30 hours without light. After completion of the reaction, the obtained product is filtered, washed with acetone and ethyl ether to obtain a solid yellow color. Yield 85%.

Elemental analysis: C 66,61 H 7,66 N 5,40

Example get 15

Getting 17 - hydroxy-18-methyl-19-norandro-4-EN-3-one-17-butylaminoethyl:

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3.7 g of 17 - aminoethyl-17 - hydroxy-8-methyl-19-norandro-4-EN-3-one, 0.3 g metatarsalgia and 30 ml of isopropanol are interacting at 60oC for 4 hours. and the resulting product is filtered and washed with isopropanol and ethyl ether to obtain a solid yellow color.

The yield is 85%.

Elemental analysis: C 68,51 H 7,11 N 5,17

Example of 16

Obtaining 16, 17 - dihydroxy-östra-1,3,5(10)-triene-3 - oxyethylenenitrilo:

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3.3 grams of 3-aminoethoxy-16 , 17 - östra-1,3,5(10)-triens, 0.3 g metatarsalgia and 50 ml of isopropanol is subjected to interaction with 80oC for 2 hours and then cooled to 40oC. After adding 4.5 g of tetracycline reaction are within 6 hours. After completion of the reaction, the resulting product is filtered, washed with isopropanol and ethyl ether to obtain a solid yellow color. Yield 85%.

Elemental analysis: C 65,48, H 6,82 N 5,13

Example of getting 17

Getting 18-methyl-17-hydroxy-östra-1,3,5(10)-triene-3 - oxyethylenenitrilo

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3.3 grams of 3-aminoethoxy-18-methyl-östra-1,3,5(10)-triene-17-she 0.3 g metatarsalgia and 50 ml of acetone is subjected to interaction with the 30oC for 48 hours without light. After completion of the reaction the resulting product is filtered and washed with isopropanol and ethyl ether to obtain a solid yellow color. Yield 82%. Elemental analysis: C 67,45 H 6,86 N 5,27

Example of getting 18

Getting 17 - hydroxy-pregna-4-EN-20-one-3 - acetylaminophenol heated and at 60oC reaction are within 2 hours. The reaction mixture is cooled to 40oC and add 4.5 g of tetracycline. The reaction is carried out at 60oC for 5 hours. After completion of the reaction the resulting product is filtered and washed with acetone and ethyl ether to obtain a solid yellow color.

The yield is 87%.

Elemental analysis: C 66,52 H 7,37 N 5,01

Example of getting 19

Getting pregna-5-ene-20-one-3-oxyethylenenitrilo:

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3.6 g of 3-aminoethoxy-pregna-5-ene-20-she, 0.6 g of metatarsalgia and 40 ml of isopropanol is heated to 80oC and reaction are within 2 hours. The reaction mixture is cooled to 40oC and add 4.5 g of tetracycline. The resulting mixture was left to react at 40oC for 6 hours. After completion of the reaction the resulting product is filtered and washed with isopropanol and ethyl ether to obtain a solid product. The output is 93%.

Elemental analysis: C 67,70 H of 7.36 N OF 5.05

Example of getting 20

17-hydroxy-androst-1,4-Dien-3-acetylenedicarboxylic:

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of 3.3 g of 3-amino-ethoxy-17 - hydroxyandrost-1,4-diene, 0.3 g metatarsalgia and 50 ml of isopropanol is subjected to interaction with 80oC in the FLS for 4 hours. After completion of the reaction, the obtained product is washed with isopropanol and ethyl ether to obtain a solid yellow color. The output is 89%.

Elemental analysis: C 67,23 H 7,25 N 5,28

Example of getting a 21

Getting 17 - methyl-17 - hydroxy-androst-4-EN-3-one-6 - methylenedioxymethylamphetamine:

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3.8 g of 6-amino-ethoxymethylene-17 - methyl-17 - hydroxyandrost-4-EN-3-one, 0.3 g metatarsalgia and 25 ml of isopropanol is subjected to interaction at 60oC for 4 hours. After completion of the reaction, the reaction mixture is cooled to 40oC and add 4.5 g of doxycyclineonline, and reaction are within 8 hours. After completion of the reaction, the obtained product is washed with isopropanol and ethyl ether to obtain a solid yellow color. Yield: 86%.

Elemental analysis: C 63,62 H 7,02 N 5,13

Example of getting 22

Getting 17 - methyl-17 - hydroxyandrost-3-one-2 - acetylenedicarboxylic:

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3.6 g of 2-aminoethoxy-17 - methyl-17 - hydroxyandrost-3-one, 0.3 g metatarsalgia and 30 ml of isopropanol are interacting at 60oC for 2 hours, and then add 4.5 g of doxycyclineonline. The reaction of the lead further. After the plant's color.

The output is 91%.

Elemental analysis: C 65,91 H 7,51 N 5,07

An example of retrieving 23

Getting 17 - methyl-17 - hydroxy-19-norandro-4-EN-3-one-methylenedioxyethylamphetamine:

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3.7 g of 6-aminoethoxymethyl-17 - methyl-17 - hydroxy-19-nor - androst-4-EN-3-one, 0.3 g metatarsalgia and 50 ml of acetone is subjected to interaction with the 30oC for 2 hours and then add 4.5 g of doxycyclineonline. Reactions are then back in for 30 hours. After completion of the reaction the resulting product is filtered and washed with isopropanol and ethyl ether to obtain a solid yellow color.

The output is 87%

Elemental analysis: C 63,64 H 7,03 N 5,18

Example of getting 24

Getting 17 - ethinyl-17 - hydroxyandrost-5-(10)-EN-3-one-6 - methylenedioxymethylamphetamine:

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3.7 g of 6-amino-ethoxymethylene-17 - ethinyl-17 - hydroxy - androst-5-/10/-EN-3-one, 0.3 g metatarsalgia and 50 ml of isopropanol is subjected to interaction at 60oC for 2 hours, and then add 4.5 g of doxycyclineonline. The reaction is carried out at 40oC for 8 hours. After completion of the reaction the resulting product is filtered and washed with isopropanol and ethyl ether to obtain a solid which treatment 25

17 - propylene-17 - hydroxy-11-dimethylaminopropanol-4,9-Dien-3-one-6 - methylenedioxymethylamphetamine:

< / BR>
5 g of 6-amino-ethoxymethylene-11-/4'-dimethylaminophenyl/-17 propylene-17 - hydroxyandrost-4,9-Dien-3-one, 0.3 g metatarsalgia and 40 ml of isopropanol is subjected to interaction with 80oC for 2 hours. Then the reaction mixture is cooled to 40oC and add 4.5 g of doxycyclineonline. The reaction of conduct within 6 hours. After completion of the reaction the resulting product is filtered and washed with isopropanol and ethyl ether to obtain a solid yellow color. Output 90%.

Elemental analysis: C 68,76 H 6,88 N 5,72

Example of getting 26

Getting 16,17-isopropylidene-16,17-dixisti-1,3,5(10)-triene-3-acetylenedicarboxylic:

< / BR>
3.7 g 16,17-isopropylidene-16,17-dixisti-1,3,5(10)-triene-3-aminoethylamide ether, 0.3 g of metatarsalgia and 50 ml of isopropanol is subjected to interaction at 60oC for 2 hours, and then add 4.5 g of doxycyclineonline. The reaction of lead within 8 hours. After completion of the reaction the resulting product is filtered and washed with isopropanol and ethyl ether to obtain a solid yellow color. Output sostavljae,3,5(10)-triene-3-17-acetate-7 - methylenedioxymethylamphetamine:

< / BR>
3.8 g aminoethoxyethanol-3,17-Dien-17-acetate, 0.3 g of metatarsalgia and 50 ml of isopropanol is subjected to interaction at 60oC for 4 hours, and then add 4.5 g of doxycyclineonline. The reaction of lead within 4 hours. After completion of the reaction the resulting product is filtered and washed with isopropanol and ethyl ether to obtain a solid yellow color. The output is 88%.

Elemental analysis: C 65,34 H 6,68 N 4,95

Example of getting a 28

Getting 17-hydroxy-pregna-4-EN-20-one-3-acetylenedicarboxylic:

< / BR>
3.7 g of 3-aminoethoxy-17 - hydroxypregn-4-EN-20-she 0.3 g metatarsalgia and 40 ml of isopropanol is subjected to interaction at 60oC for 2 hours, and then add 4.5 g of doxycyclineonline. Reactions are further for 4 hours. After completion of the reaction, the obtained product is filtered, washed with isopropanol and ethyl ether to obtain a solid yellow color.

The yield is 92%.

Elemental analysis: C 66,41 H 7,40 N 5,14

An example of obtaining 29

Getting pregna-4-ene-3,20-dione-6-methylene-acetylenedicarboxylic:

< / BR>
of 3.9 g of 6-aminoethoxymethyl-pregna-4-ene-3,20-diem add 4.5 g of doxycyclineonline. The reaction of lead within 4 hours. After completion of the reaction, the reaction mixture is allowed to react for another 4 hours. After completion of the reaction the resulting product is filtered and washed with isopropanol and ethyl ether to obtain a solid yellow color. The output is 93%.

Elemental analysis: C 67,77 of 7.36 H N 4,59

Example 30

Getting 3,17-dihydroxy-östra 1,3,5/10/-trien-11-/4-phenoxyethylamine/-methylene-doxycycline:

< / BR>
4 g 11-/4'-aminoethoxyethanol/-3,17-dihydroxyethane-1,3,5(10)- triens, 0.3 g metatarsalgia and 50 ml of isopropanol is subjected to interaction at 60oC for 2 hours, and then add 4.5 g of doxycyclineonline. Reactions are another 8 hours. After completion of the reaction the resulting product is filtered and washed with isopropanol and ethyl ether to obtain a solid yellow color.

The output is 89%.

Elemental analysis: C 68,13 H 6,69 N 4,73

An example of retrieving 31

Getting 17 - hydroxy-17 - methylandrostan-/3,2-C/pyrazole-N-methylenedianiline:

< / BR>
3,29 g of 17 - hydroxy-17 - methylandrostan-/3,2-c/-pyrazole, 0.3 g metatarsalgia and 30 ml of isopropanol is subjected to interaction with the 40oC for 2 hours, and then dobavlyayut filtered off, washed with isopropanol and ethyl ether to obtain a solid yellow color. The output is 89%.

Elemental analysis: C 67,48 H 7,07 N 7,30

Experiment 1: Intracorporeal distribution connection

Connection 1 - 3 of example obtaining 1 - 3 have been labelled radioactively3H /0,34 of mccoury /mg/ and injected into the tail vein of mice at a dose of 20 mccoury/x /20, Each group consisting of 5 mice, which destroy after 1 minute, 5 minutes, 15 minutes, 30 minutes, 1 hour, 4 hours, 6 hours, 24 hours, 48 hours and 72 hours after injection and take 50 ál of blood from the eye of each mouse. Then take heart, ovaries, uterus, small intestine, bone /hip/ and so on, from each mouse and 50 mg of each tissue /blood 50 ál/, placed in a plastic test tube. Then in each test vial is placed 0.2 ml of perchlorate, 0.4 ml of hydrogen peroxide and a drop of n-oktilovom alcohol, and the test ampoule is placed in a water bath at 75oC for 45 minutes. Then 0.1 ml of this pass is selected and placed in a vial with scintillation solution, and even mixed with 5 ml of 0.5% of the scintillation solution. After the solution becomes transparent, it is placed in a liquid scintillation counter Fy 2105 for determination of radioactivity and determine CPM clicks the S="ptx2">

The amount of drug in the tissue is determined as follows. The number of drugs in fabric /CPM/mg/ = /CPM sample/ - / number-digested tissue /mg//

The amount of drug in the tissue /mg/mg/ = /CPM/mg of sample x 6 /-/ effectiveness account /E/ x 2,22 x 107x 0,34 of mcurie/mg/specific radioactivity//

The following results are obtained (see tab. 1-10 at the end of the description).

Changes in the concentration of drugs in blood /each time the average value for 5 animals/

Changes in the concentration of drugs in blood /CPM/50 μl/ after intravenous injection3H-link 1 - 3.

Experiment 2: Test for acute toxicity

/1/ Sample

Connection 1 - 3 is a crystalline powder pale yellow color, and the number of his lot 930113. His solution is a clear liquid pale yellow color, at a concentration of 50 mg/ml at a pH of about 5. He presents Osteoporosis Research Laboratory, School of Pharmacy, West China University of Medical Sciences (WCUMC).

/2/ Animals:

Mouse type Kunming health: first class, weighing from 18 to 21 kg, half males, half females. Mice were obtained from the Experimental animal center WCUMS.

/3/ the definition of the half lethal dose /LD50/:

Prepared 4 - 5 dose gstach. Medicine for oral administration get the suspending solid connection 1 - 3 1% CMCNa2(Na CMC) before formation of the suspension. For injection prepare solutions of drug with different concentrations, which get by dissolving compound 1 - 3 in physiological saline solution by the method of cultivation to low specific weight. Animals do not give food to /but are not limited to water and after 20 hours in each group select the 10 mice regardless of gender and weight. The drug is administered once daily at a dose of 0.2 mg/10 g, and animal watching. Dead animals anatomist and determine any changes that led to the death.

/4/ the results of the test:

/a/ to determine the maximum tolerated dose of the compounds 1 - 3 for mice.

Determine the maximum tolerant dose, when death does not occur by the time of the preliminary tests. Once a test compound is administered directly into the maximum concentration and the maximum amount for oral administration of 20 mice /10 males and 10 females/ for animals observed within 7 days. As a result, the mice show no abnormalities and mortality also occurs. The maximum tolerant dose /Me mice, the following (see table. 4).

Calculation method: the method of bliss

LD50= 143,11 mg/kg

LD50: inside the interval 95% of the limit of validity:

132,95 to 154,0 mg/kg

/5/ Conclusions:

The maximum tolerant dose /MTD/ connection 1 - 3 for a single injection to mice is at least 6 g/kg, and its toxicity is very low. LD50for intravenous injection into the tail vein of mice is 143,11 mg/kg After intravenous activity of mice is reduced, and then the mouse starts jumping up and are in spasm. The eyeball becomes convex and white; there is a continuous selection of urine and faeces. Although much of poisoned animals are killed instantly, a few survivors also die within 24 hours. Those that survived after 24 hours, was killed after 7 days. Not detected differences in dead animals depending on gender, and the dead animals failed to detect with the naked eye any changes in the anatomy. The temperature in the room where tested, was the 17oC.

Experiment 3: Test osteogenesis /1/

As the test cells using the first generation inkubiruemykh system osteoblasts obtained from krislet on Wednesday once daily at a dose of 10-5M, or 10-8M, or 10-9M on the second and third days /breeding/. In another embodiment, the connection 1 - 3 in these quantities added to the medium for 4 days /calcification/ period, starting from the seventh day from the beginning of the incubation. On the fourteenth day after the start of incubation of the cells stained by the method of Von Kossa and spend the determination of phosphates. The area of bone sections, which are painted in brown color, evaluate the naked eye and is used as an indicator of osteogenesis. The following results are obtained (see tab. 5).

In Fig. 1 - 3 presents the results of the same procedure, with three replications, the symbol A represents A variant without the addition of drugs, B represents the addition of 10-9M, C represents the addition of 10-8M, and D represents the addition of 10-6M,

As can be seen from the presented results, the compounds of the present invention demonstrate the function of promotion of osteogenesis.

Experiment 4: Experiment on osteogenesis 2

The first generation of the incubation system bone marrow cells obtained from the femur of rats Whister /female, age 6 months/, used as cells for experiments, and connect edcity days from the beginning of the incubation period calcification/. The evaluation is made according to the method of example 3. The following results are obtained (see tab. 6).

As can be seen from the table, the compounds of the present invention demonstrate the function of promotion of osteogenesis

Experiment 5: Pharmacological study antiosteoporotic action of compounds 1 - 3

1. Scheme of the experiment:

Clinical observations in the experiment on the study of pharmacological effects of compounds 1 - 3 against osteoporosis in castrated rats often show that bone loss due to deficiency of estrogen in women after menopause can cause osteoporosis. A satisfactory therapeutic effect is achieved by treatment with estrogen, but its prolonged use involves the risk of provoking endometrial cancer, breast cancer, and so on, and accordingly, this method has limited clinical application. To overcome these shortcomings and improve therapeutic effect at the School of Pharmacy, West China University of Medical Sciences improved the structure of estrogen and synthesized antiosteoporotic connection 1 - 3. After completion of the toxicity tests used model in castrated rats with osteoporosis and counterpunching effect and efficiency of the connection 1 - 3 against osteoporosis and to determine the basis for the clinical use of drugs.

11. Materials and methods

/i/ Drugs:

Connection 1 - 3 /received at the School of Pharmacy WCUMS/. of 0.1 n HCl is used for obtaining an initial solution concentration of 10 mg/ml, and the concentration in the experiments get diluting the original solution with distilled water.

To 100 mg of estradiol /West China University of Medical Sciences (WCUMS) add 5 ml of 90% alcohol and 20 ml of polyethylene glycol 400, the resulting mixture is placed in water and heated to 90oC to dissolve. Then it is diluted to 100 ml with distilled water to obtain an initial solution 1 mg/ml

The calcium /as calcium carbonate and citric acid obtained from Beijing Chemical Factory/. To 100 mm of calcium carbonate was added 100 mm citric acid /2.1 g of citric acid are added to 1 g of calcium carbonate and placed in 100 ml of distilled water. Prepare before use/. Mark3H-TdR /Chimese Academy of Sciences, Radiation Laboratory/.

All other reagents and drugs were AR purity /Beijing Chemical Reagents Co./

/ii/ Animals:

Rat strain Whistar 80 females, 20 males, age 5 - 6 months, weighing 260 10 g /China University of Medical Sciences, Animal Laboratory/, being the experiment

1. Healthy female rats of Wistar strain divided into 10 groups of 6 animals in the group, which assign the following numbers. These 10 groups:

/1/ 7-stage surgical group

/2/ reference surgical group

/3/ the group with low doses /connection 1 - 3, 50 μg/rat/day;

/4/ the group with average doses /connection 1 - 3, 500 μg/rat/day/

/5/ the group with high doses /connection 1 - 3, 5 mg/rat/day/

/6/ the connection 1 - 3, 500 μg/rat + 0.5 ml Ca

/7/ Estradiol, 0.5 mg/rat/day/

/8/ Estradiol, 0.5 mg/rat/day/ + 0.5 ml Ca

/9/ the Connection 1 - 3, 500 μg/rat/day + 0.5 ml Ca

/introduction spend 5 weeks after surgery/

/10/ the Connection 1 - 3, 500 µg /5th week after surgery/, intraperitoneal administration.

Groups 1 - 8 the drug is administered orally for 1 week after surgery. Group 9 administered orally for 5 weeks after surgery. 10 the drug is administered intraperitoneally, beginning 5 weeks after the operation.

2. Healthy male rats of Wistar strain divided into 3 groups, which are designated as group 11 /simulating operation/ group 12 as a control after blindness, and group 13, which were injected with 500 μg/rat/day of compound 1 - 3 within 1 week after Orgy distilled water, and 13 week all animals killed, by cutting off the head and simultaneously taken samples of blood and tissue.

/iv/ Preparation of animal models

Experimental female rats of Wistar strain at the age of 5 - 6 weeks with body weight 260 10 g get from China University of Medical Sciences, Animal Lab. Before the operation they are not given food for 12 hours. Each of the rats anaesthetize intraperitoneal injection of sodium phenobarbital /35 mg/kg and fixed in a supine on the surgical Board; in the centre of Podkrepa open hole approximately 33 mm in order to open the pink Y-shaped uterus. Dark red cilicie globules in the upper part of the uterus are the ovaries. The lower part of the tie silk thread, and both ovaries cut off with surgical scissors. After confirming the absence of bleeding muscles and skin dried up, the uterus return to its original position. In the group with simulating operation the uterus just naked, but do not produce excision. After surgery in the abdominal cavity is injected penicillin /80000 IU/rat/ to prevent infection. After several hours, the rats are returned to normal.

In male rats of Wistar strain ovaries removed with the same anesthesia, and the males simulating group is/ Indicators to monitor:

1. The weight of the body. Measure once a week.

2. Vaginal smears females observed continuously for 6 days for 10 weeks /proestrus, ASTRUM, interval/.

3. Urine is collected for 24 hours /12 weeks/ and define the content of Ca, P and creatine.

4. 13 week determine the density of the bone tissue from each rat, just before the beheading.

5. 13 week animals kill and define the following parameters.

/1/ Selected serum and define the content of Ca, P and alkaline phosphatase.

/2/ Determine the weight esecanna females.

/3/ the Excised liver and kidneys and determine the toxicity and uptake of medicines for each of them.

/4/ Determine the strength of the right femur.

/5/ To the left femur add 20% decalcification nitrate, and observe morphological changes.

/6/ Bone right lower leg burn, and ash is weighed to determine the concentration of Ca and p

/7/ Receptor test is performed on the bones of the left tibia.

/vi/ cell Cultivation

UMR106 cells were cultured in DMEM/Ham F-12 medium containing 10% FCS, at 37oC and 5% CO2. Digestion is carried out once every three days parivara centrifuged at a speed of 2000 rpm for 10 minutes, and add DMEM/HamF12 (without phenol red) containing medium with 7.5% CS - FCS /4 g Norita charcoal + 100 ml FCS/, then 3 of 104/ml of cells was determined by the culture plate with 24 holes /1 ml/hole/. After 24 hours, the medium replaced DMEM/HamF-12 (without phenol red) containing 0.2% FSA. Test connection /estradiol connection 1-3/ enter after 48 hours of cultivation. 32 hours enter 0.5 mccoury /hole3H-TdR. At 48 hours after injection washed 5 to 6 times with PBS solution, add 0.2 n NaOH (1 ml of cell and after 24 hours, the toluene-tritanopia scintillation solution is used to determine the magnitude by radiation.

2. Determining the number of cells

48 hours after injection of the cells into the culture plate with cells, the cells are digested by trypsin-EDTA and count.

3. The activity of the ATP secreted from the cells, determine.

48 hours after introduction into the cells on the plate with 24 cells Wednesday sucked off by pipette, and the content of ATP was determined by biochemical colorimetry.

III. Results

/i/ is There a change cells in vaginal smears 1. Standards for evaluation.

10 weeks after surgery vaginal mA is but a large number of major definitely keratinized epithelial cells, and a small number of epithelial cells.

The indicator of the duration of oestrus:

There is an unusual amount of multicore white cells and a small number of epithelial cells.

2. The results are shown in table. 7.

Conclusions: the Change of oestrus is observed three times per week in a group with the simulated operation. Change in estrus occurs 1-2 times per 1 week after surgery, but all groups, which were injected medication after surgery, showed a change of oestrus 4 to 6 times in one week. This confirms the fact that the connection and E2affect the change of oestrus in rats.

/ii/ Change weight

Between the body weight of the operated groups of rats and groups with simulated operation no significant differences, but in the group treated after surgery E2, there is a greater weight gain than for the operated group. The weight of the bodies in the group, which was administered 0.05 mg/day of compound 1-3, and in the group which was administered 0.5 mg/day of compound 1-3 5 weeks after surgery, and in the group which was administered intraperitoneally 5 weeks after the operation, clearly increased more than in the operated group. The increase in the weight of bodies in groups, which iteratio.

The effect of compounds 1-3 on the body weight of castrated male rats are shown in table. 8.

Note to table 8: comparison with the operated group,

xP less than 0.05xxP less than 0.01

The weight of the bodies of male rats in the group which was administered 0.5 mg of compound 1-3 oral and operated the control group, clearly shows more gain /P is less than 0.01/ than for stimulirovano-operated group, but for the control group and groups treated with compound 1-3, there is not much difference in weight gain.

The effect of compounds 1-3 on the weight of the bodies castrated male rats (see Fig. 4/.

/iii/ Change the weight of the uterus:

1. Compared with a group with the simulated operation.

After the operation on the operated rats groups, compound 1-3 orally administered for 12 weeks at doses of 5 mg/day, 0.5 mg/day and 0.05 mg/day intraperitoneally for 6 weeks at a dose of 0.5 mg/day, and this is complemented by E2. Weight of all females show a clear decline.

2. Compared with the operated group.

After the operation there has been some increase in weights in the groups that were administered 0.05 mg/day of compound 1 - 3 orally for 12 weeks. This indicates that t is a group of bone density: see table 9.

1. Bone density in the operated group is significantly lower than the group with simulating operation.

2. 0.5 mg E2introduced through the stomach, and when compared with the operated group after 12 weeks, it was found that bone density is clearly higher, whereas it is not observed large differences when compared with the group with the simulated operation.

3. Connection 1 - 3 has a distinct effect on changes in bone density after surgery. There is a dose-dependent relationship (see Fig. 5/.

4. 0.5 mg of the compound 1 - 3 leads to clearly visible greater bone density than 0.5 mg E2/see Fig. 5/.

5. If the connection 1 - 3 administered orally and intraperitoneally, both methods lead to a significant increase in bone density, which is similar for both methods (see Fig. 6/.

6. With the introduction in 1 week and 5 weeks after surgery, both methods give a clear increase in the density of bone tissue. /see Fig. 7/.

/V/ is the result of the determination of calcium and phosphorus

After removal of the ovaries in rats the level of calcium in the blood increases, and the rate of content of phosphorus is reduced, but the difference Nevel 'the ptx2">

1. Ca2+in blood Levels of calcium in the blood is stored in the norm with the introduction of 5 mg/day of compound 1 - 3. There is a clear difference between the treated group and the operated control group when P is less than 0.01. No action is not observed for the group, which is administered 0.05 mg/day of compound 1 - 3.

Processing E2: As with the introduction of only one E2and in combination with calcium, the calcium level in the blood OVX rats maintained. There is a clear difference between the groups E2+ Ca and the operated control group with P < 0.01.

2. The phosphorus level in blood Levels of phosphorus in the blood is stored for a dose of 0.5 mg/day and 5 mg/day of compound 1 - 3, but this saving even more clearly to 0.05 mg/day. For the treated group there is a distinct difference with the operated control group with P < 0.01.

/vi/ Results determine osteocalcin /BGP/ serum:

After oophorectomy serum BGP slightly pinkish, and after treatment with compound 1, 3 - levels BGP reduced dependent on the dose way in all rats, which were injected with 5 mg/day of compound 1 to 3, there is a more noticeable decrease compared with the group with simulating the operation and OVX group, with P the reduction, than the OVX control group, with P less than 0.05.

/Vii/ Changes ALP in serum:

The content of ALP in serum OVX rats decreased slightly greater than in rats from stimulirovano operated group, while the group that was treated with 0.5 mg/day E2, there is a slightly greater reduction than in the operated group, the difference is insignificant. Processing connection increases the levels of ALP in serum OVX rats dependent on dose. The group treated with 0.5 mg/day and 5 mg/day of the compounds 1 to 3 are respectively differences P less than 0.05 and P less than 0,001 compared with stimulirovano operated by the group.

/Viii/ Changes in bone strength:

After the animals cut off the head of the femur sorted and placed in small vials with saline solution, which are marked with corresponding numbers. Conduct testing using electronic universal tester.

Determine the conditions under which dynamic index thighs.

1. Method: 3xspot test on a bend with a simple beam.

2. Direction: front to back.

3. Test length: 25 mm

4. Tensile strength is defined as the development of the working point, expressed in kg/mm

Each group studied in the t test and analysis of variance to determine the effect of compounds 1 - 3 dynamic indicators of femurs of male and female rats; not found any difference.

Connection 1 - 3 administered orally to groups 1 - V, and determine the dynamic performance of the thighs, but it was necessary to repeat the test, as in the selection of material thighs errors occurred. The other groups were satisfactorily analyzed, and found no differences. This was due to the small number of animals. This test will be repeated.

Note: bending Strength is defined as the maximum bending matrix at break and the amount is calculated using the following formula:

< / BR>
/i x/ Action connection on the cells of the cell line UMR106 osteosarcoma

1. Add3H-TdR: 10-10m E2no effect on adding3H-TdR in UMR106 cells. 10-9up to 10-7M E2clearly lead to a peak value after adding3H-TdR to UMR106 cells, and this peak value is changed to 10-7M, giving a 63% increase compared with the control group. When compared with group E-9M and 10-7M give values of 13% and 9% higher than the E2respectively.

2. Determining the number of cells: E2does not affect the number of cells in 10-10M, but at the 10-9M - 10-7M the number of cells increases, and the peak in the 10-7M Connection 1 - 3 clearly leads to an increase in the number of cells in 10-10M - 10-7M, with peak concentrations accounted for 10-7M. the Effect of compounds 1 - 3 is stronger than the effect of E2but the difference between them is small.

3. Changes in ALP activity:

/1/ Changes in ALP activity in amniotic fluid:

E2no effect on amniotic ALP activity at a concentration of 10-10M. At a concentration of 10-9M - 10-7M E2clearly increases ALP activity independent of dose. Connection 1 - 3 also increases ALP activity in amniotic fluid at concentrations of 10-10M - 10-7M dependent on dose. These effects appear to be stronger than for E2in all concentrations, with marked differences /P less than 0,001/.

/2/ Changes in ALP activity secreted from the cult is all about concentrations of 10-9M to 10-7M, and, although E2does not affect the enzyme at a concentration of 10-10M, the connection 1-3 at a concentration of 10-10M and 10-7M produces the enzyme activity, which is 29% and 23% higher, respectively, than the activity of E2group. Connection 1-3 at a concentration of 10-9and 10-8M also has a much stronger effect than E2.

Experiment 6: the Message about the experiment on the effects of drug compounds 1-3 on osteoporosis in castrated rats.

1. Materials 1-1 Drugs. All drugs used in this experiment were obtained from the group of Professor Zheng Hu and have been prepared in accordance with the requirements.

1-2. Animals. Female SD rats, aged 3 months, weight 180-220 g, provided by West China University of Medical Sciences, Laboratory Animal Center

2. Experimental method

2-1. The selection of groups of animals and the introduction of drugs.

Animals observed within 1 week prior to the experiment, for which selected 150 rats and break them down into 10 groups of 15 rats each.

Group 1 /stimulated/: Temporarily operated group.

Spend laparotomy, and in the stomach of each animal pour in 1 ml of physiological solution 3 times the provide 1 ml of saline 3 times a week.

Group 3 /oral E2/: Oral administration of E2. For each animal to spay and pour in the stomach 2 times a week, and only 0.8 mg/ml of 17 beta-estradiol injected each time.

Group 4: /Oral, low dose/: Oral administration of compounds 1-3 in low doses. For each animal to spay and pour in the stomach 3 times a week, and only 5 mg/ml each time.

Group 5 /average oral dose/: Orally administered compound 1-3 in moderate doses. For each animal to spay and pour in the stomach 2 times a week, each time enter is only 20 mg/ml

Group 6 /oral, high-dose/: Orally administered compound 1-3 in high doses. For each animal to spay and pour in the stomach 3 times a week for 80 mg/ml each time.

Group 7 /injection E2/: Enter the injection of E2. For each animal to spay and 200 μg /0.2 ml 17-estradiol is administered twice a week, each time during the first 4 weeks and then to 7.5 μg /0.2 ml 17 - estradiol each rat over the next 6 weeks.

Group 8 /injection of low doses/: Animals injected injection of low doses of each animal to remove the ovaries, and 1 mg/0.2 ml of compounds 1-3 enter twice and medium doses/: Animals injected injection medium doses. For each animal to spay and 3.75 mg/0.2 ml of compounds 1-3 injection twice a week during the first 4 weeks and then 0.375 mg/0.2 ml over the next 6 weeks.

Group 10 /injection high doses. Animals injected injection of high doses of the drug. For each animal to remove the ovaries, and 15 mg/0.2 ml of compounds 1-3 injection twice a week during the first 4 weeks, then 1.5 mg/0.2 ml over the next 6 weeks.

2-2. Observation of the animals.

On the 11th week of the animals killed by dissection of the femoral artery and remove the uterus. Then spend the definition of AMD right Shin bone, patrologicheskoe study the bones of the left tibia, a biomechanical study of bone of left femur, the determination of the ash content of the bones of the right thigh and biochemical examination and weighing of the uterus.

2-3. The statistical analysis.

All parameters are expressed as X SD. The parameters of each group are compared with the parameters of the castrated group, and the statistical level of 0.05.

3. Results.

3-1. The effect of compounds 1-3 on the body weight and overall health castrated rats.

4 weeks after the start of the experiment in rats dose by injection and in the group, which has introduced E2who are experiencing the loss of hair, inactive movement of hair removal and so on, and so the introduction is prescribed for 1 week all test animals. As a result of introducing a test compound with a low dose over the next 6 weeks, the above effects are reduced or disappear. After completion of the experiment, all animals show a weight gain of about 30 - 40%, with the exception of animal groups, which were administered high doses of compounds 1-3 oral. In this group the increase in weight is suppressed /1,4%/.

3-2. Biodynamic effect of compounds 1-3 on the bone castrated rats.

Bending strength thighs stimulirovano operated rats is 97 Newton, and this value is reduced to 80 Newton castrated rats. There is a clear difference between the two groups /P less than 0.05/. Bending strength of femurs of animals in those groups, which is administered orally E2and due to injection is increased to 101 and 90 Newton, respectively, and there is a clear difference with animals castrated group. Bending strength of femurs of rats in groups, which is administered orally, low, medium, and high doses soedinenii doses compared to the castrated group, with a distinct difference. Bending strength of femurs of rats that orally introduced low, medium and high doses by injection of compounds 1-3, comprise 83, 103 and 97 Newton, respectively, and groups with medium and high doses, demonstrate a distinct difference compared to the castrated group. This experiment was performed in collaboration with Biohimicheskoe laboratory South West Jiao Tong University.

3-3. The effect of compounds 1-3 on the bone density of the tibia in castrated rats.

Bone density in the group of castrated rats is 0,037 g/cm and it is significantly lower than the density of temporarily operated group /0,072 g/cm/, and bone density groups of rats, which were injected E2oral and injections, increases to 0,057 and 0,065 g/cm, respectively. Bone density in rats in groups, which were injected compound 1-3 orally in low medium and high doses, is 0,056, 0,062 and 0,064 g/cm, respectively.

Bone density in rats in groups, which were injected connection through injections of low, medium and high doses, is 0,054, of 0.066 and of 0.085 g/cm, respectively, and for all of them is higher than for the castrated group, and the ratio of strength hung the Average weight of the ash of the femur in rats with simulated operation is 0,246 g/rat, and lower in castrated group, where this value is 0,227 g/rat, which represents significant difference /P less than 0.05/. The weight of ash femur in groups, which were injected compound 1-3 orally and by injection, clearly higher than in rats castrated group, and either the same or higher levels in rats in stimulirovano operated group.

3-5. The effect of compounds 1-3 on the femur castrated rats.

The calcium content in the femoral bones castrated rats clearly lower than the content of stimulirovano operated rats, or in any of the treated group, P less than 0.05/. The calcium content in the femoral bones from stimulirovano operated rats is 0,308 mg/g, compared to 209 mg/g castrated group, 319 mg/g and 330 mg/g, respectively, for groups that have introduced E2orally or by injection, 315, 321 and 322 mg/g, respectively, for groups, which were injected compounds 1-3 orally in low, medium and high doses, and 312, 315 and 322 mg/g, respectively, for groups, which were administered injections of compounds 1-3 in low, medium and high doses. If the connection 1-3 administered orally or by injection, there is a constant dependent on the dose ratio ical changes of the Shin bone castrated rats.

Bone trabecula is normal in the group with pseudoeurycea. Bone trabecula in castrated rats and somewhat less already, and bone trabeculotomy bone increased. Extensive depression found in some parts of the trabecular epiphyseal plate, and the cavity of the bone marrow explicitly expanded. Absorption lacunae on the surface of the trabeculae and the number of osteoclasts are markedly increased and become active. Increase the osteoblasts, but the action of osteoclasts much more. The density and width of trabeculae in the group treated with E2and XW 630, increases slightly, the trabeculae type buttons obviously reduced, and this reduces the number of active osteoclasts and osteoblasts.

3-7. The effect of compounds 1-3 on the weight of the uterus in castrated rats.

Uterine castrated rats, obviously, atrophic and their weight lowered /0.17 g/, which is much lower than the weight of stimulirovano operated group /0,37 g/. In the group orally and by injection was administered E2weight females significantly increased /to 0.40 and 0.21/. In the group that was administered the compound 1-3 orally in low, medium and high doses, the weight of females is 0.33, 0.45 and of 0.48 g, respectively. In groups, which was introduced soedinenii in groups, which was introduced medium and high doses of compounds 1-3, significantly higher than the weight of the uterus in the castrated group, unlike groups, which were administered low-dose orally and by injection.

3-8. See table 10 on changes of indicators for compounds 1-3 when comparing all test groups with the castrated group.

4. Preliminary conclusions.

4-1. If the connection 1-3 administered orally or by injection, there is a noticeable effect on the contents of the bone trabeculae, bone density, biomechanism bones and the calcium content in the bones castrated rats, and is able to effectively maintain the stability of the bone tissue after castration.

There is a notable difference when compared to the castrated group. Some of the numerical values reach or exceed the corresponding values for stimulirovano operated group. This experiment shows that the group, which is injected doses of compounds 1-3 orally, usually shows an increase in bone mass, although if you experience symptoms of toxicity, but the transparency of the bending of the bones is very low.

4-2. If the connection 1-3 be administered orally, or by Sceta and the content of calcium in the bones castrated rats there is a constant, a dose-dependent relationship.

4-3. The effect of compounds 1-3 on the bone tissue of castrated rats in these experimental conditions the same, or superior therapeutic effects of E2.

4-4. If the compound is administered orally or by injection in low doses, there is practically no noticeable effects or improvements of the entire set of parameters of bone in castrated rats. If the compound is administered orally in low doses, slightly increases the uterus, but it is not much smaller than the characteristic stimulirovano operated group.

If the connection 1-3 injected by the injection of low doses, the effect on the weight of the uterus in castrated rats was not observed. This shows that the effective dose of compounds 1-3 with a preventive effect on bone loss in castrated rats or have a weak stimulating effect on the uterus, or does not have any steps.

1. The pharmaceutical composition inhibiting bone resorption and enhances osteogenesis, comprising an active agent and a pharmaceutically acceptable excipient or solvent, wherein the active agent it contains the
where R1represents a hydrogen or hydroxyl group;

R2represents a hydrogen or hydroxyl group;

R3represents hydrogen or methyl group;

R4represents hydrogen, halogen or dimethylaminopropyl;

Y represents a divalent or trivalent group represented by formula III, IV or V:

< / BR>
< / BR>
< / BR>
where n = from 0 to 4;

-X - represents a simple bond, -O - or-NH-;

Z represents a monovalent group formed by removing a hydrogen atom or hydroxyl group of the compounds represented by formula VI

< / BR>
where R'1is HO or O =;

R'2represents a hydrogen atom or methyl group;

R'3represents a hydrogen atom, phenyl group or substituted phenyl group;

R'4represents methyl group or ethyl group;

R'5represents a hydroxyl group, ketone group or acetyl group;

R'6represents hydrogen, hydroxyl group, methyl group, etinilnoy group or propenyloxy group;

or R'5and R'6together form = O;

R'7represents hydrogen, a hydroxyl group or a = O,

or R'

2. The pharmaceutical composition under item 1, characterized in that the monovalent group represented by the formula II, is a monovalent group tetracycline compounds, where R1is hydrogen, R2represents a hydroxyl group, R3represents a methyl group, and R4represents hydrogen.

3. The pharmaceutical composition under item 1, characterized in that the monovalent group represented by the formula II, is a monovalent group terramicina, where R1represents a hydroxyl group, R2represents a hydroxyl group, R3represents a methyl group, and R4represents methyl.

4. The pharmaceutical composition under item 1, characterized in that the monovalent group represented by the formula II, is a monovalent group chlortetracycline, where R1is hydrogen, R2the submitted rmaceuticals composition on p. 1, characterized in that the monovalent group represented by the formula II, is a monovalent group doxytetracycline, where R1represents a hydroxyl group, R2is hydrogen, R3represents a methyl group, and R4represents hydrogen.

6. The pharmaceutical composition under item 1, characterized in that the monovalent group represented by the formula II, is a monovalent group aminotetraline, where R1is hydrogen, R2is hydrogen, R3represents hydrogen, and R4is dimethylaminopropyl.

7. The pharmaceutical composition according to any one of paragraphs.1 - 6, characterized in that the monovalent group represented by the formula VI, a is a monovalent group of estrone, where R'5and R'6together form = O, and R'7represents hydrogen.

8. The pharmaceutical composition according to any one of paragraphs.1 - 6, characterized in that the monovalent group represented by the formula VI, a is a monovalent group of estradiol, where R'5represents a hydroxyl group, R'6represents hydrogen, and R'7represents hydrogen.

9. Pharmaceutically VI, is a monovalent group astrolgical, where R'5represents a hydroxyl group, R'6is etinilnoy group, and R'7represents hydrogen.

10. The pharmaceutical composition according to any one of paragraphs.1 - 6, characterized in that the monovalent group represented by the formula VI, a is a monovalent group Astola, where R'5represents a hydroxyl group, R'6represents hydrogen, and R'7represents a hydroxyl group.

 

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< / BR>
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< / BR>
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< / BR>
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< / BR>
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< / BR>
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< / BR>
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< / BR>
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< / BR>
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