DE1593569C - - Google Patents

Description

The monodesmethyl compound of ubiquinone (coenzyme Q) found in mitochondria and animal Tissues present has a relatively high biological activity similar to that of Ubiquinone of the formula

CH ₂ -CH = C-CH ₂ -CH ₃

In addition to the biological activity, the monodesmethyl compound can be based on a hydroxyl group, which reacts easily with other compounds into various therapeutically valuable compounds convert. *

The following two methods are available for converting an alkoxyl group into a hydroxyl group known:

1. heating the starting material with a mineral acid or an alkali hydroxide,

2. Irradiation of the starting material with artificial light or natural light.

The above process (1) is very unsatisfactory for carrying out on an industrial scale, taking into account that ubiquinone ^? especially to heat or acids or alkalis is very unstable. The process (2) has, inter alia, the disadvantage of a low yield of the desired compound.

The invention relates to a process for the preparation of monodesmethylubiquinone compounds the formula

40

45

or ubihydroquinone compounds of the general formula

OR

"5

where R ₁ , R ₂ and A have the same meaning as above, R _{3 is} an alkyl radical and R is a hydrogen atom or an acyl radical, is reacted with lithium aluminum hydride and / or sodium borohydride in an organic solvent, preferably an ether.

The reaction takes place at room temperature or at an elevated temperature. The response time depends on the starting materials and solvents used. The relationship between response time and product yield is exemplified in the following table.

Starting material:
O

1. CH ₃ O - / \ - CH ₃

CH ₃ OI / - / CH, -CH = C-CH ₂ ^ pH

I i i j

I i

2. Lithium aluminum hydride
Solvent: tetrahydrofuran

wherein R _{1 is} an alkyl radical, R _{2 is} hydrogen or an alkyl radical and A is the groupings

CH ₂ -CH = C-CH ₂
CH,

and or

-h CH ₂ - CH ₂ - CH - CH ₂

CH ₃

in which η is an integer from 1 to 10, which is characterized in that there are ubiquinone compounds of the general formula

Reaction period
Hours \ Yield of monodemethyl
ubiquinon
% 20th
25th
45 26th
32
33

The monodesmethylubiquinone compound was determined by column chromatography on silica. It has generally been found that the yield becomes almost constant as the reaction time increases than 25 hours. It should also be noted that the separation of the desired compound from The longer the reaction time, the more difficult the reaction mixture becomes, because of the formation of by-products increases.

The end point of the reaction can be determined by examining the reaction product, for example by Reverse phase paper chromatography or column chromatography can be determined.

The monodesmethylubiquinone compound thus produced is contaminated including the unconverted starting materials and by-products are separated. Preferably this is done for example by column chromatography. Suitable adsorbents for the column are, for example Aluminum oxide, silicon dioxide, silica salt or activated carbon. For example, when column chromatography is applied to silica for separation, the monodesmethylubiquinone compound in a certain fraction by increasing the concentration of the polar solvent in the non-polar Solvent to be eluted. For example, when a mixture of hexane and chloroform is used is, the Monodesmethylubiquinonver- ^ compound is eluted in a fraction in which the on hexane related chloroform content is 10 to 20 percent by volume. When using column chromatography on magnesium silicate, a polar solvent, e.g. B. methanol, used as the eluent. The so eluted monodesmethylubiquinone compound can optionally from a suitable organic Solvents such as methanol or ethanol are crystallized.

The product obtained can be determined by means of the infrared spectrum, the ultraviolet spectrum and the nuclear magnetic resonance spectrum compared to an authentic sample of monodesmethylubiquinone be identified. This is confirmed by methylation of the product with dimethyl sulfate to ubiquinone.

The monodesmethylubiquinone compound prepared according to the claimed process has a biological activity similar to that of ubiquinone, which has recently appeared as a new drug. The monodesmethylubiquinone compound can also be converted into various derivatives which are important for biochemical studies. For example, it can be converted into ubiquinone- ¹⁴ C, which is often used for studying ubiquinone metabolism.

In the following examples, the numbers (I) to PCI) correspond to the connections in the following Table are mentioned.

(I), (IV) to (IX)

CH,

CH ₇ -CH = C-CH,

55

60

(II), (III), (X), (XI)

CH,

CH ₂ -CH = C-CH ₂

links R. R ₂ R ₃ H (I) CH ₃ CH ₃ CH ₃ CO (Π) CH ₃ CH ₃ - (III) CH ₃ CH ₃ - (IV) CH ₃ H - (V) H CH ₃ - (VI) CH ₃ C ₂ H ₅ - (VII) C ₂ H ₅ CH ₃ - (VIII) CH ₃ CH ₃ CO CH ₃ (IX) CH ₃ CO CH ₃ CH ₃ (X) CH ₃ C ₂ H ₅ (XI) C ₂ H ₅ CH ₃

0-R ₃

example 1
Ubihydroquinone (II)

500 mg (I) in 20 ecm ethanol / ether (1: 1) were _{shaken vigorously with 10 ecm 10% aqueous Na 2} S ₂ O ₄ solution under N ₂ until the yellow color of the organic layer had disappeared. The organic phase was then separated off, washed with water, over Na ₂ SO ₄ . dried and evaporated in vacuo. 490 mg (98%) of light yellow oil were obtained - UV maximum: 290 πΐμ; IR absorption: 3500 cm " ¹ (OH).

Example 2
Diacetate (III)

500 mg (I) were refluxed for 30 minutes with 500 mg zinc dust, 10 ecm acetic anhydride and 2 ecm triethylamine (oil bath). Then ice / water (100 ecm) was added in order to decompose remaining acetic anhydride, and shaken out with 100 ecm of ether. The ether solution was washed with 5% NaHCO ₃ solution, then with water and, after drying (Na ₂ SO _4. ), Evaporated. The remaining oil was dissolved in ethanol and cooled. Yield 503 mg (89%) of colorless needles, mp. 24 to 26 ° C. - UV maximum: (EJ *); 268 πΐµ (9.08); IR absorption: 1775 cm " ¹ (CH ₃ CO).

C ₄₈ H ₇₂ O ₆ (745.0):

Calculated ... C 77.37, H 9.74;
found .... C 77.12, H 9.47.

B is ρ ie 1 3
Hydroxy homolog

250 mg (II) in 50 ecm purified tetrahydrofuran were mixed with 500 mg LiAlH ₄ . Boiled under reflux for 25 hours. After cooling, water was added in portions while cooling with ice, acidified with 1N HCl and exhaustively extracted with ether. The combined ether extracts were washed with water, _{dried over Na 2} SO ₄ and evaporated in vacuo. The orange-colored oil that remained was chromatographed on 10 g of magnesium silicate (74 to 149 μ, column 2 × 8 cm). The fraction eluted with 100 ecm of ether yielded 200 mg (I), the fraction eluted with 100 ecm of methanol afterwards gave 47 mg of violet oil. This was dissolved in 30 ecm of n-hexane / methanol (4: 1), the solution was _{shaken with 10 ml of 3N H 2} SO ₄ until its color had turned pure orange, then with

Washed 50 ecm of water over Na ₂ SO ₄ . dried and evaporated in vacuo. The orange-colored oil that remained was purified by chromatography using a column (1 × 4 cm) of 1.5 g of silica. The fractions eluted with 50 ml of n-hexane / chloroform (9: 1) and (8: 2) each gave 38 mg (15 , 6%) mixture of (IV) + (V) as an orange-red flakes of melting point 43 to 47 ° C (from methanol). the mixed melting point with a sample (mp. 35 to 40 ⁰ C) formed by photochemical reaction of (I) was obtained, was 38 to 41 ⁰ C. Leukomethylenblausowie FeCl ₃ / K ₃ Fe (CN) ₆ were positive reaction - UV maximum (e | £,): oxidized form 275 πΐμ (208); reduced. Form 350-> 295 πΐμ. IR absorptions: 1610, 1640, 1665 (quinone), 3350 cm " ¹ (OH).

C ₄₃ H ₆₄ O ₄ (644.9):

Calculated ... C 80.07, H 10.00, 1 CH ₃ O 4.81;
Found .... C 80.05, H 10.03, 1 CH ₃ O 4.22.

15th

Example 4

240 mg of (III) in 50 ml of purified tetrahydrofuran were _{reacted with 500 mg of LiAlH 4} as in Example 3. (IV) + (V) were obtained in a yield of 85 mg (41%).

Example 5

From (I) with LiAlH ₄ : 500 mg of (I) in 100 ecm purified tetrahydrofuran were refluxed with 1 g of LiAlH _{4 for} 25 hours and worked up as in Example 3. 268 mg of unchanged (I) and 130 mg (26.5%) of a mixture of (IV) · + (V) were obtained.

Example 6

From (I) with NaBH ₄ : 250 mg of (I) in 50 ml of purified tetrahydrofuran were refluxed with 500 mg of NaBH _{4 for} 25 hours. After cooling, it was worked up as in Example 3. The yellow oily reaction product (250 mg) was chromatographed on magnesium silicate. The fractions eluted with ether contained (II), as was shown by oxidation with 1% ethanolic FeCl ₃ solution to (I). The fractions then eluted with methanol gave 48 mg of crude (IV) -t (V), which was purified on silica; Yield 10 mg (4%).

50

Example 7

Methyl ether: 28 mg mixture of (IV) + (V) in 5 ecm acetone were kept on the boiling water bath for 30 minutes _{with 0.5 g K 2} CO _{3 and 0.5 ecm dimethyl sulfate.} After the acetone had been distilled off, the mixture was diluted with 10 ecm of water and extracted with 30 ecm of η-hexane. The hexane solution was washed with water and evaporated in vacuo. The remaining oil (28 mg) was purified by chromatography using a column (1 × 4 cm) of 1.5 g of silica. The fraction eluted with 100ccm n-hexane / chloroform (4: 1) yielded 22 mg (77%) (I) as yellow leaflets, mp 27 to 28 ° C.

C ₄₄ H ₆₆ O ₄ (658.9):

Calculated ... C 80.19, H 10.10, 2CH ₃ O 9.42;
Found .... C 80.18, H 9.98, 2 CH ₃ O 8.99.

Example8

Ethyl ether (VI) + (VII): 55 mg mixture of (IV) 4- (V) in 5 ecm acetone were kept on the boiling water bath for 1.5 hours _{with 0.5 g K 2} CO _{3 and 0.5 ecm diethyl sulfate} . After the acetone had been distilled off, it was dissolved in 30 ecm η-hexane, washed 3 times with 30 ecm methanol / water (1: 1), _{dried over Na 2} SO ₄ and freed from the solvent. The oil obtained crystallized from a little methanol on cooling. Yield 50 mg (87%). - UV maximum (E | ^,): oxidized form 275 ΐημ (222), reduced form 290ηΐμ. IR absorptions: 1620, 1650, 1660 cm " ¹ (quinone).

C ₄₅ H ₆₈ O ₄ (672.9):

Calculated ... C 80.31, H 10.18;
found .... C 80.43. H 10.19.

Example 9

Acetate (VIII) + (IX): 129 mg mixture of (IV) + (V) was left to stand for 6 hours at room temperature with 0.8 ecm acetic anhydride and 0.8 ecm pyridine; then ice / water was added and extracted with 50 ecm η-hexane. The Hcxanlösung was washed with 30 cm 3 η-H ₂ SO ₄ , 30 cm 5% NaHCO ₃ solution and 30 cm water and evaporated after drying. The orange-colored oil that remained was purified by chromatography using a column (2 × 5 cm) of 5 g of silica. The oil eluted with η-hexane chloroform (4: 1) crystallized from a little methanol. Yield 78 mg (57%) of yellow needles. F. 18 to 21 ° C. - UV maximum (EJ ^ J: oxidized form 273 ηΐμ (178), reduced form 354-> 295 ΐτίμ. IR absorption: 1180, 1780 cm- ¹ (CH ₃ CO).

C ₄₅ H ₆₆ O ₅ (686.9):

Calculated ... C 78.67, H 9.68;
found .... C 79.00, H 9.57.

Example 10

Dimethyl ether (X) + (XI): 500 mg mixture of (VI) + (VII) was converted into its hydroquinone compound _{with Na 2} S ₂ O _4. Then it is shaken in an N ₂ atmosphere with 2 ecm dimethyl sulfate and 2 g K ₂ CO ₃ in 30 ecm acetone for 65 hours at room temperature, and the acetone is distilled off. The residue was treated with 40 ecm of water and extracted twice with 50 ecm of η-hexane each time. The hexane solution was washed with water, _{dried over Na 2} SO ₄ and evaporated. The pale yellow residue was chromatographed on a column (2 × 7 cm) of 7 g of silica. The fraction eluted with η-hexane yielded 440 mg (84%) (X) + (XI) as a colorless oil (. '• max - 275 ma) and fraction 40 eluted with n-hexane / chloroform (4: 1) mg unchanged starting material.

Test report

The following tests show that the monodesmethylubiquinone obtained by the claimed process ubiquinone derivatives known to reactivate the succinate oxidase fermentation system is superior.

The experiments were carried out on mitochondria which contain the fermentation system succinate oxidase. The mitochondria were determined by the method of C r a η e et al (F. L. C r a n'e et al (»Biochimica et Biophysica Acta ", 22, 475 [1956]) obtained from bovine heart muscle and were in 0.25 molar Solution of sucrose to a concentration of 20 mg enzyme protein per millimeter and suspended stored at - 15 ° C. The suspension was used for 10 days after its preparation.

The ferment system present in the mitochondria was then made according to the method of Lester et al ("Biochimica et Biophysica Acta", 47,358 [1961]) by extraction with acetone in the following manner inactivated: 2 ml of the mitochondrial suspension were added to 50 ml of acetone each during a period of Added dropwise for 5 minutes with stirring. Stirring was continued for an additional 5 minutes and centrifuging the suspension at 20,000 g for 10 minutes. The sedimented precipitate was * resuspended in 50 ml of acetone, the suspension was treated as described above and the obtained Precipitate resuspended in 0.25 molar sucrose (50 ml). The suspension obtained last was again stirred for 5 minutes and centrifuged at 20,000 g for 10 minutes; the obtained precipitate became stored under ice-cooling as a suspension in 0.25 mol of sucrose (1 ml) and within 6 hours continued to be used after manufacture. The entire process was carried out at 3 ° C and the acetone previously cooled to -15 ° C.

The measurement of the activity of the succinate oxidase system was carried out in a standard Warburg apparatus. The specific activity was calculated from the oxygen consumption of the gas phase (air) at 37.5 ⁰ C for 30 minutes and than / <- Mol succinate, consumed in 1 minute per milligram enzyme protein expressed. The reaction mixture in the apparatus has the following composition:

connection Addition (μ§) Relative activity,
related to
Ubiquinone (250 μg) Ubiquinone Q ₉
Ubiquinone Q ₁₀
Perhydroubiquinone Q ₇
Monodesmethyl
ubiquinon Q ₇ 250
125
250
125
250
125
250
125 95
72
96
70
46
18th
100
75

connection

Ubiquinone Q ₇

Encore (jzg)

250
125

Relative activity,

related to

Ubiquinone (250 μg)

100
71 ± 15 It follows from the table that monodesmethylubiquinone has a greater activity than other ubiquinone derivatives, especially in the diluted state.

Table 2

connection Addition (ag) Spcz. activity
(μΜοΙ)
Succinate / min / mg
protein Ubiquinone Q ₇
Monodesmethyl
ubiquinon Q ₇ 200
100
200
100 0.184
0.114
0.205
0.185

In the main compartment: phosphate buffer (pH 7.5)
(100 μ MoI), cytochrome C (2 mg),
Enzyme protein (2 mg),
Side arm: sodium succinate (150 μ mol),
Central space: 6 normal KOH (0.2 ml),
Total liquid volume: 3.2 ml.

The measurements of the reactivation of the enzyme system were carried out after im Main compartment of the apparatus has added 0.03 ml of an ethanolic solution of the ubiquinone derivative. The results are shown in the following tables:

Table 1

Reactivation of succinate oxidase from extracted mitochondria with ubiquinone and related links

40

The higher activity of the monodesmethylubiquinone Q ₇ according to the invention from Table 2 _{compared with ubiquinone Q 7} becomes particularly clear when the added amounts are lower.

Claims (1)

Claim: Process for the preparation of monodesmethylubiquinone compounds of the formula 55 6o where R _{1 is} an alkyl radical, R _{2 is} hydrogen or an alkyl radical and A is the groupings CH ₇ -CH = C-CH, CH, and or - / - CH ₂ -CH ₂ -CH-CH ₂ ^ -H
I CH ₃ Y _n 109524/378 in which η is an integer from 1 to 10, characterized in that ubiquinone compounds of the general formula IO or ubihydroquinone compounds of the general formula where Ri, R ₂ and A have the same meaning as above, R _{3 is} an alkyl radical and R is a hydrogen atom or an acyl radical, is reacted with lithium aluminum hydride and / or sodium borohydride in an organic solvent, preferably an ether.