JPS589116B2 - Method for isolating hydroxycholester-5,7↓-dienes - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for isolating hydroxycholester 5,7-dienes.

More specifically, hydroxycholester 5,7-diene is a useful intermediate that can lead to hydroxyvitamin D3 from a mixture containing hydroxycholester 5,7-dienes and hydroxycholester-4,6-dienes. Concerning a method for isolating species.

Conventionally, from a mixture containing hydroxycholester-5,7-dienes and hydroxycholester-4,6-dienes,
As a method for separating hydroxycholester-5,7-dienes, for example, the method described in JP-A-51-68559 is known.

In this method, a mixture containing hydroxycholester 5,7-dienes and hydroxycholester 4,6-dienes is subjected to chromatography using silica gel containing silver nitrate as an adsorbent. This is a method for separating and obtaining 5,7-dienes.

This method is different from other known methods, for example, by making use of the difference in chemical properties between hydroxycholester 5,7-dienes and hydroxycholester 4,6-dienes, and first preparing a mixture of the two with a dielectric constant of 6 or more. The hydroxycholester 4,6-dienes were converted only to the corresponding cholester 2,4,6-trienes, and then the hydroxycholester 5,7-dienes thus formed 2
・A method of separating hydroxycholester 5,7-dienes by recrystallization or chromatography using the difference in solubility or polarity from 4,6-triene (see JP-A-52-71457, etc.) ) compared to 5.7-
This method can be said to be superior in that it is a method in which a mixture containing dienes and 4,6-dienes is directly subjected to a separation operation.

However, the method described in JP-A No. 51-68559 uses a special adsorbent called silica gel containing silver nitrate, and it also uses a special adsorbent called silica gel containing silver nitrate. star 5
・In the method for producing 7-dienes, the protective groups for the hydroxyl groups at the 1α-position and the 3β-position that were intentionally added were removed again, and both the 1α-position and the 3β-position became free hydroxyl groups.・7-diene and hydroxycholester There are still some points that need to be improved, especially for industrial large-scale production, such as the need to convert to 4,6-diene and subject it to chromatography, but the method is still insufficient. It is hard to say that this is a satisfactory method.

On the other hand, 1α,3β-diacetoxycholest-5-ene was brominated using diglomodimethylhydantoin as a brominating agent to obtain 1α,3β-diacetoquine-7-bromocholesterol, and then 20 mg of it was added to trimethylphosphorol. By subjecting the resulting reaction mixture to column chromatography using Scherisolve-diethyl ether and neutral alumina as a carrier, 1α・3β-diacetoxycholester-5. A method for obtaining 600 μg of 7-diene is also known.

(Refer to Japanese Unexamined Patent Publication No. 48-62750) However,
This method has an extremely low yield, and there are points that need to be improved as an industrial production method.

From this point of view, the present inventors have discovered that hydroxycholest-
In a method for producing hydroxycholester 5,7-dienes from 5-enes, the inevitable hydroxycholester-4,6-dienes produced are separated and removed to produce hydroxycholester 5,7-dienes. As a result of intensive research to find an industrially advantageous method for isolating hydroxyl groups, we found that a bivaloyl group or an ethoxycarbonyl group was used as the protecting group for the hydroxyl group, and a mixture of a lower aliphatic alcohol and a lower aliphatic ether was used as the solvent. When using a solvent, hydroxycholester-5 can be obtained by a simple recrystallization operation.
・From a mixture containing 7-dienes and hydroxycholester-4,6-dienes, hydroxycholester 5,7-
The present invention was achieved by researching ways to isolate dienes in high yields.

That is, the present invention has 1. The following formula [I] [wherein R1 and R2 are each independently a pivaloyl group or an ethoxycarbonyl group, and R3 and R4 are each independently a hydrogen atom, a hydroxyl group, a pivaloyloxy group, or an ethoxycarbonyloxy group.

] Hydroxycholester 5,7-dienes represented by the following formula [■], [In the formula, the definitions of R1, R2, R3 and R4 are the same as above] By recrystallizing a mixture containing the above from a mixed solvent of a lower aliphatic alcohol and a lower ether, the above formula [■]
This is a method for isolating hydroxycholester 5,7-dienes from the above mixture, characterized by isolating hydroxycholester 5,7-dienes represented by:

The object to be separated used in the present invention has the above formula [I
] Hydroxycholester 5,7-dienes represented by the above formula [■] and hydroxycholester-4, represented by the above formula [■]
It is a mixture containing 6-dienes.

In the above formula [■], R1 and R2 are each independently a bivaloyl group or an etquin carbonyl group, and R3 and R4
are each independently a hydrogen atom, a hydroxyl group, a bivaloyloxy group or an ethoxycarbonyloxy group.

That is, specific examples of the hydroquincholester 5,7-dienes represented by the above formula [■] include the following.

(100) 1α, 3β-dipivaloyloxycholester 5,7-diene, (102) 1α, 3β, 24-tripivaloyloxycholester-5, 7-diene, (104) 1α, 3β, 25-tripivaloyloxycholester-5,7-diene, (106) 1α,3β,24,25-tetrabivaloyloxycholester 5,7-diene, (108)1α,3β-diethoxycarbonyloxy Cholesta-5,7-diene, (110)1α,3β,24-triethoxycarbonyloxycholester-5,7-diene, (112)1α,3β,25-triethoxycarbonyloxycholester-5,7- Diene, (114) 1α・3β・24・25-tetraethoxycarbonyloxycholester-5・7-diene, (116)
3β-ethoxycarbonyloxy-1α-pivaloyloxycholester-5,7-diene, (118) 1α-ethoxycarbonyloxy-3β-pivaloyloxycholester-5,7-diene, (120) 3β- Etquin carbonyloxy-1α, 24-dipivaloyloxycholester-5,7-diene, (122) 24-hydroxy-1α, 3β-dipivaloyloxycholester-5,7-diene, (124) 24-
Hydroxy-1α・3β-diethoxycarbonyloxycholest-5,7-diene, (126)24-Hydroquine-1α・3β・25-tripivaloyloxycholesta-5,7-diene, (128)24 -Hydroquine-1α,3β,25-triethoxycarbonyloxycholester-5,7-diene, Specific examples of hydroxycholester 4,6-dienes represented by the above formula [■] include the above formula [ (2) Compounds corresponding to the 5,7-dienes represented by:

The above hydroxycholester 5,7- used in the present invention
A mixture of dienes and hydroxycholester-4,6-dienes refers to a mixture of 5,7-dienes and 4,6-dienes that differ only in the position of the double bond in their chemical structures. It means.

Therefore, the mixture containing hydroquincholester 5,7-dienes and hydroxycholester 4,6-dienes used in the present invention has a higher concentration of hydroxycholecalciferol than hydroxycholester-5-enes. It is produced as an intermediate reaction product in a series of steps to produce products.

The reaction formula for this series of reactions is as follows.

In the above reaction formula, R31 and R41 are each independently a hydrogen atom or a hydroxyl group, and R1, R2, R3 and R4
The definition of is the same as above.

In the reaction of introducing a protecting group into hydroxycholest-5-enes having a free hydroxyl group to produce hydroxycholest-5-enes having a protected hydroxyl group, for example, hydroxycholest-5-enes having a free hydroxyl group are It can be easily produced by reacting -enes with pivaloyl chloride pivalic anhydride or ethyl chloroformate in the presence of a tertiary amine catalyst such as pyridine.

The reaction proceeds at room temperature to 80°C, but while the pivaloylation reaction completes in about 5 to 6 hours, the ethoxycarbonylation reaction requires caution due to the low boiling point of ethyl chloroformate. It is necessary to carry out a reaction operation, and it is usually desirable to carry out the reaction over a period of 20 to 30 hours.

Although this protecting group introduction reaction can proceed relatively quantitatively, it still usually contains a small amount of impurities such as amines used in the catalyst or Harz.
If used as is as a raw material for the next reaction, it will cause a decrease in yield in the subsequent reaction.

According to the research of the present inventor, especially when a pivaloyl group is introduced as a protecting group, the obtained pivaloyl-protected hydroxycholest-5-enes can be easily crystallized using, for example, methanol, ethanol, etc. as a recrystallization solvent. It has been found that this product can be purified by recrystallization as a crystal, which is extremely superior to the known acetyl-protected hydroxycholest-5-enes, which are difficult to purify as crystals.

Therefore, in this sense as well, the present invention provides a series of industrially advantageous reactions for producing hydroxycholecalciferols from hydroxycholest-5-enes.

Of course, if only one of pivaloyl chloride or ethyl chloroformate is used as a protecting group-introducing reagent, protection in which only either pivaloyl group or etquin carbonyl group is introduced as a protecting group will result. Hydroxycholest-5-enes having a hydroxyl group can be obtained, but by taking advantage of the slight difference in reactivity of each hydroxyl group of hydrochunkerest-5-ene, both protecting group-introducing reagents are used as a protecting group. Hydroxycholest-5-enes having a protected hydroxyl group containing both a pivaloyl group and an ethyloxinecarbonyl group can also be produced.

For example, 1α·3β-dihydroxycholest-5-ene is first introduced with a protecting group using ethyl chloroformate,
3β-ethoxycarbonyloxy- with two hydroxyl groups protected by different protecting groups by preparing 3β-ethoxycarbonyloquine-1α-hydroxycholest-5-ene and then acylating with pivaloyl chloride.
1α-pivaloyloxycholest-5-ene can be produced.

The thus obtained hydroxycholest-5-enes having a protected hydroxyl group are then brominated (Br).
and further dehydrobromated (deHBr) to form the object to be separated of the present invention.

Examples of the brominating agent include N-promosuccinimide, 1.3, which is well known as an allylic brominating agent.
-dipromo-5,5-dimethylhydantoin, N-promocaprolactam, etc. are preferably used, and as the dehydrogenating agent, for example, trimethyl phosphite, s-collidine, diethylaniline, etc. are preferably used.

As a reaction solvent for any reaction, for example, a hydrocarbon such as hexane, heptane, cyclohexane, ligroin, benzene, toluene, xylene, carbon tetrachloride, 1,2-dichloroethane, 1,2-dibromoethane, or a halogen. Hydrocarbons and the like are preferably used.

The bromination reaction proceeds at room temperature to 140°C, and the dehydrobromination reaction proceeds at 60° to 250°C, preferably 120 to 180°C.
Proceed with

These detailed reaction conditions are described in U.S. Patent No. 4,022,891.
It is stated in the specification of the No.

The reaction mixture obtained by these bromination reactions and the subsequent dehydrobromination reaction is represented by the above formula [■]
Hydroxycholester 5,7-diene represented by the formula [■] and Hydroxycholester-4, represented by the above formula [■]
According to the research conducted by the present inventors, it is considered to be almost impossible to produce a reaction mixture containing 6-diene and not containing hydroxycholester-4,6-diene represented by the above formula [■]. It will be done.

Furthermore, according to the experiments of the present inventors, when attempting to carry out the bromination reaction thoroughly, not only the mono-Br compound at position 7 but also by-products such as dibromination products are produced.
It has also been confirmed that when carrying out a bromination reaction, it is best to complete the reaction when a small amount of hydroxycholest-5-enes having a protected hydroxyl group, which is the origin of this reaction, remains.

Therefore, the separation targets of the present invention obtained by bromination and dehydrobromination under desirable reaction conditions are 5,7-
In addition to dienes and 4,6-dienes, it contains hydroxycholest-5-enes having a protected hydroxyl group.

Of course, the present invention also covers such hydroxycholest-5
-It also includes substances to be separated that contain enes.

Now, the present invention provides a mixture containing a hydroxycholester 5,7-diene represented by the above formula [■] and a hydroxycholester 4,6-diene represented by the above formula [■]. This is carried out by recrystallization in a mixed solvent of alcohol and lower aliphatic ether.

Examples of lower aliphatic alcohols include methanol,
Preferred examples include ethanol, n-propanol, iso-propanol, n-butanol, methoxyethanol, and n-hexanol. Examples of lower aliphatic ethers include dimethyl ether, diethyl ether, diisopropyl ether, and tetrahydrofuran. , dioxane, 1,2-dimethoxyethane and the like are preferred.

According to the research of the present inventor, among these mixed solvents, aliphatic chain alcohols having 1 to 4 carbon atoms are preferable as lower aliphatic alcohols, and aliphatic chain alcohols having 2 to 6 carbon atoms are preferable as lower aliphatic ethers. It has been found that aliphatic linear ethers or 5- to 6-membered aliphatic cyclic ethers are preferred.

When these mixed solvents are used, the isolation yield and purity of the hydroxycholester 5,7-diene represented by the above formula [■] obtained by separation and isolation are particularly excellent.

In particular, the best results were obtained when a mixed solvent of methanol and ethyl ether was used.

In the present invention, the ratio of the lower aliphatic alcohol to the lower aliphatic ether may vary slightly depending on the skeletal structure of the object to be separated, the presence or absence of protective groups, the type, etc., but usually the lower aliphatic A mixed solvent containing a larger amount of lower aliphatic alcohol than a group ether is preferably used, and a mixed solvent containing about 0.2 to 0.05 parts by weight of lower aliphatic ether per 1 part by weight of lower aliphatic alcohol is particularly preferably used. It will be done.

The preferred quantitative range varies depending on the type of lower aliphatic alcohol and lower aliphatic ether used, but the combined use of lower aliphatic ethers improves the solubility of the separation target and slows down the precipitation of crystals during cooling. They are common in that they are thought to be well separated from the Harz component and 4,6-diene body contained as impurities.
It is not very difficult to determine the quantitative proportion in actual operation.

In the separation operation of the present invention, the substance to be separated is added to a predetermined amount of lower aliphatic ether to dissolve it, then a predetermined amount of lower aliphatic alcohol is added to create a slight turbidity, and then the temperature is raised again. Heat until completely transparent, then
It is preferable to use a cooling method.

Of course, a conventional method may be used in which a mixed solvent of a predetermined amount of a lower aliphatic alcohol and a predetermined amount of a lower aliphatic ether is prepared in advance, and then the substance to be separated is added thereto for recrystallization.

As described above, the object to be separated in the present invention has the above formula [■]
Hydroxycholester 5,7-dienes represented by and hydroxycholester 4,6 represented by the above formula [■]
- Dienes, but all of them have a hydroxyl group protected at the 1α-position and the 3β-position. It is different in that it includes things that do not.

When viewed as objects to be separated, among these common structures, those with a hydroxyl group or a protected hydroxyl group in the side chain and those without are still common, and can be separated by the method of the present invention. be able to.

The most preferred embodiment of the present invention is applied to the separation target of the present invention produced by the above-described reaction under the most favorable conditions than the corresponding hydroxycholest-5-enes.

In this sense, the most preferred embodiment of the present invention is to add hydroxycholester-4,6-represented by the above formula [■] to 1 part by weight of the hydroxycholester 5,7-diene represented by the above formula [I]. about 0.03 to 4 parts by weight of dienes,
Particularly preferably, by recrystallizing a mixture containing 0.1 to 1 part by weight from methanol or a mixed solvent of ethanol and diethyl ether once or multiple times as necessary, the hydroxycholester represented by the above formula [■] can be obtained. This is a method for isolating star 5,7-dienes.

All of the letahydroxycholesta-5,7-dienes represented by the above formula [■] whose hydroxyl groups at the 1α and 3β positions are protected by a pivaloyl group or an ethoxycarbonyl group obtained by isolation and purification in this way are new compounds. According to the reaction formula described above, useful nahydroxycholecalciferols can be obtained.

1α-hydroxycholecalciferol, 1α24-dihydroxycholecalciferol, 1α25-dihydroxycholecalciferol, and further 1α・24・25-
Trihydroxycholecalciferol is a compound known as active vitamin D3, and is known to be useful for treating or preventing diseases such as rickets and osteomalacia.

The present invention provides the above-mentioned A, hydroxycholest-5
- In a series of steps for producing such useful active vitamin D3 from enes, hydroxycholester 4,6-dienes, which are generated in the middle of the series of steps and have an extremely detrimental effect on subsequent reactions, are separated and removed. However, it constitutes one of a series of industrially advantageous steps for producing active vitamin D3, and has great industrial significance.

Hereinafter, the present invention will be described in detail with reference to Examples.

Example 1 (1) 1α・3β-dipivaloyloxycholest-5-
Synthesis of ene; 1α・3β-dihydroxycholest-5-ene 2.01
g (5 mmol) in 10 m of pyridine (dried with NaOH)
1 and stirred under a nitrogen atmosphere to dissolve. Next, 3.02 g of pivaloyl chloride was added under ice cooling and stirred for 1 hour.

Furthermore, 3.01 g of pivaloyl chloride was added, and after addition 8
The mixture was heated to 0°C and stirred for 5 hours.

The resulting reaction mixture was poured into 30 ml of ice water, the pH was adjusted to 5 with acetic acid, and then extracted with 50 ml of ethyl acetate.

After separating the ethyl acetate layer, the aqueous phase was extracted again with 30 ml of fresh ethyl acetate, and the two ethyl acetate layers were combined.

Wash with a saturated aqueous sodium bicarbonate solution, then with water, dry over anhydrous sodium sulfate, filter, and concentrate under reduced pressure; 3.
9 g of residue (wet) was obtained.

This residue was subjected to column chromatography using 54 g of silica gel as a packing material (packing solvent: benzene) (developing solvent: benzene-ethyl acetate), and then reconstituted in a mixed solvent of 1 part by weight of ethyl acetate and 30 parts by weight of methanol. Crystallization was performed to obtain 2.41 g (yield: 85%) of 1α/3β-dipivaloyloxycholest-5-ene having the following properties.

Melting point: 132.5-133°C, IR (KBr, cm-1): 2960, 2870, 17
30, 1480, 1283, 1160, NMR (CDC
l3, δ (ppm): 0.66 (3H, s, C18CH3), 0.86 (6H, d, C-26·27-CH3), 1.
16 (9H,s, C-3 pivaloyl group CH3X3), 1.21 (9H,s, C-1 pivaloyl group CH3x3)
, 4.88, 5.00 (2H, b, C-1・3-H), 5
．． 52 (1H, b, C-6-H), (2) 1α・3β-dipivaloyloxycholester 5.
Synthesis of 7-diene; 1α・3β-dipivaloyloxycholest-5-ene 5
70ml (1mmol) of n-hexane (Na dry)7
．． 5 ml, heated in an aqueous solution at 95°C, and stirred for 1 hour.
・3-dibromo-5,5-dimethylhydantoin 171
．． Add 6 mg (0.6 mmol) and 15 minutes under infrared irradiation.
Stir for a minute.

After cooling to room temperature, the precipitate was filtered and washed with n-hexane.

The filtrate and washing liquid were concentrated under reduced pressure at 40°C to obtain a concentrated residue.

S-collidine 2 dissolved in 3 ml of xylene (Na dry)
．． 5 ml was heated at 170°C, and while stirring, a solution of the concentrated residue (crude bromine) dissolved in 3 ml of xylene was added dropwise.
．． (washed with 5 ml) was heated under reflux for 20 minutes.

After cooling to room temperature, the precipitate was filtered off and washed with xylene.

The filtrate and washing liquid were concentrated under reduced pressure at 40°C, and the concentrated residue (
The main component, diene body) was obtained.

This product was dissolved in 30 ml of ethyl acetate and washed twice with 10 ml of 1N hydrochloric acid.

The ethyl acetate layer was washed successively with a saturated aqueous sodium bicarbonate solution and water, dried over anhydrous sodium sulfate, filtered,
It was concentrated under reduced pressure to obtain 582 mg of a crude concentrated residue.

(3) 1α・3β-dipivaloyloxycholester 5.
Isolation of 7-diene: The crude concentrated residue obtained in (2) above (according to the UV spectrum, the ratio of 5,7-diene to 4,6-diene is approximately 2:1, and in addition, a small amount of 5 The entire amount of the solution (which was separately confirmed to contain the -ene isomer) was dissolved in 4 ml of ether, and after adding 20 ml of methanol, the mixture was heated under reflux at 80° C. for 30 minutes.

After cooling to room temperature for 30 minutes, it was left in a cool, dark place (5°C) for 1.5 hours.

The precipitated needle crystals were collected by filtration, washed with cold methanol at -20°C, and dried under reduced pressure in the dark to produce 1α/3β-dipivaloyloxycholester 5/7 as colorless needle crystals with the following properties. - 176 mg of diene was obtained.

Melting point: 161.5-162°C (ether/methanol reconsolidation) IR (KBr, cm-1): 2960, 2870, 1730, 1480, 21285
, 1165 NMR (CDCl3, δ (ppm)): 0.61 (3H, s, C-18-CH3), 0.86 (
6H, d, C-26・27-CH3), 1.16 (9H
, s, C-3 pivaloyl group CH3×3), 1.22 (9H, s, C-1 pivaloyl group CH3×3)
, 4.92 (2H, b, C-1 3-H), 5.36 and 5.64 (2H, b, C-6-H and C-7-H
), UV (ether), λmax: 263 (7300), 2
72 (10500), 282 (11300), 294 (
(4) Synthesis of 1α-hydroxycholecalciferol; 142 mg (0.25 mmol) of 1α・3β-dipiparoyloxycholester 5,7-diene was added to benzene (N
a) Dissolved in 600 ml and degassed with argon gas for 30 minutes.

Bubble argon gas while cooling with ice water, 2
Light was irradiated with a 00W mercury lamp for 5 minutes (during this time, the internal temperature rose from 5°C to 15°C).

The obtained light irradiation liquid was concentrated under reduced pressure at 40°C to about 100ml.
It was concentrated to

After degassing again with argon gas for 30 minutes, the mixture was heated to 80° C. (bath temperature) for 2 and a half hours while stirring and degassing with argon gas.

The obtained thermal isomerization mixture was concentrated at 40°C, the obtained residue was dissolved in 5 ml of methanol, 1 ml of a 25% methanol solution of potassium hydroxide was added, and the mixture was heated for 60 minutes in the dark.
The pivaloyl group was removed by heating and stirring at ℃ (bath temperature) for 3 hours.

The obtained depivaloyl reaction solution was poured into 30 ml of cold water, extracted three times with 30 ml of ether, the ether layers were combined, washed twice with 10 ml of saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. A concentrated residue was obtained.

The obtained concentrated residue was dissolved in ethyl acetate, and 2% silver nitrate-
The crude 1α-hydroxy 18.5 mg of cholecalciferol was obtained.

This crude product was placed on a silica gel thin layer plate (20 cm x 20
cm x 0.5 mm) After separation and extraction on one plate, n-heptane 0
．． By recrystallizing with 3 ml, crystals of 1α-hydroxycholecalciferol were obtained.

The properties of this product were in good agreement with literature values.

Example 2 (1) Synthesis of 1α,3β-diethoxycarbonyloxycholest-5,7-diene; 1α,3β-diethoxycarbonyloxycholest-5
-ene 163.8 mg (0.3 mmol) was added to 3 ml of n-hexane (Na dry), heated to 95°C, and 1,3-dipromo-5,5-dimethylhydantoin 5
1.5 mg (0.18 mmol) was added and stirred for 15 minutes under infrared irradiation.

After cooling to room temperature, the precipitate was filtered off and washed with n-hexane.

The filtrate and washing liquid were combined and concentrated under reduced pressure at 40°C to obtain a concentrated residue (crude bromine compound).

A solution of 0.75 ml of S-collidine dissolved in 1 ml of xylene was heated to 170°C (bath temperature), and a solution of the above crude prome dissolved in 1 ml of xylene was added dropwise with stirring (
The dropping funnel was further washed with 0.25 ml of xylene)
, and heated under reflux for 20 minutes.

After cooling to room temperature, the precipitate was filtered off, washed with xylene, the filtrate and washing solution were combined, concentrated under reduced pressure at 40°C, and 1α.
3β-diethoxycarbonyloxycholester-5,7-
A residue containing diene was obtained.

Dissolve this residue in 10 ml of ethyl acetate and add 10 ml of 1N hydrochloric acid.
Extracted twice with ml.

The separated ethyl acetate layer was washed successively with saturated aqueous sodium hydrogen carbonate solution and water.

Dry over anhydrous sodium sulfate and concentrate under reduced pressure to obtain crude 1α and 3β
180 mg of a residue containing -diethoxycarbonyloxycholester-5,7-diene was obtained.

(2) Isolation of 1α,3β-diethoxycarbonyloxycholesta-5,7-diene: The above residue (by uV, 1α,3β-diethoxycarbonyloxycholesta-5,7-diene: 1α,3β- It was found that the ratio of diethoxycarbonyloxycholest-4,6-diene was 3:1, and it was separately confirmed that it contained a small amount of 1α,3β-diethoxycarbonyloxycholest-5-ene) 180mg, 1ml
Then, 10 ml of methanol was added, and the mixture was heated under reflux at 80° C. (bath temperature) for 30 minutes.

After cooling to room temperature for 30 minutes, it was left in a cool, dark place (5°C) for 4 hours.

The precipitated needle crystals were collected by filtration, washed with cold methanol at -20°C, and dried under reduced pressure in a dark place to obtain 1α/3β-diethoxycarbonyloxycholester-5 as colorless needle crystals with the following properties. - 66 mg of 7-diene was obtained.

Melting point (°C): 141-142.5 (ether/methanol reconsolidation), IR (KBr, cm-1): 2950, 2860, 17
40, 1465, 1370 NMR (CDCl3, δ (ppm)): 0.62 (3H
, s, C-18-CH3), 0.86 (6H, d, C-26·27-CH3), 1.
31 (6H, t, C-1, 3-ethoxycarbonyl group C
H3×2), 4.20 (4H, q, C-1・3-ethoxycarbonyl group CH2×2), 4.86 (2H, b, C-1・3-H), 5.37 and 5. 67 (2H, b, C-6-H and C-7-H)
, UV (ethanol, λmax): 263 (6500),
272 (9300), 282 (10000), 294 (
6000) nm, Comparative Example 1 (1) Synthesis of 1α/3β-diacetoxycholest-5/7-diene: 1α/3β-diacetoxycholest-5-ene 972m
g (2 mmol) was dissolved in 15 ml of n-hexane (Na dry), and 343 mg (1
．． 2 mmol) was added thereto, and the mixture was stirred for 15 minutes under infrared irradiation.

After cooling the obtained reaction mixture to room temperature, the precipitate was filtered,
Washed with n-hexane.

The filtrate and washing liquid were combined and concentrated under reduced pressure at 40°C to obtain a residue (crude bromine compound).

A solution of 5 ml of S-collidine dissolved in 6 ml of xylene was heated to 170°C (bath temperature), and a solution of the above crude bromine dissolved in 6 ml of xylene was added dropwise to this while stirring (the dropping funnel was further heated with 3 ml of xylene). (washed) and heated under reflux for 20 minutes.

After cooling to room temperature, the precipitate was separated and washed with xylene, and the filtrate and washing liquid were combined and concentrated under reduced pressure at 40°C to obtain the residue.

This residue was dissolved in 30 ml of ethyl acetate, and 1N-hydrochloric acid 1
Washed twice with 0ml.

The separated ethyl acetate layer was washed successively with a saturated aqueous sodium bicarbonate solution and water, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 908 mg of a concentrated residue.

(2) Isolation of 1α,3β-diacetoxycholester-5,7-diene; The above concentrated residue (1α,3β-diacetoxycholester-5,
・7-Diene: 1α・3β-diacetoxycholester-4
- 908 mg of 6-diene (2:1) was dissolved by heating in ether-methanol (1:10), allowed to cool to room temperature for 30 minutes, and then left in a cool, dark place (5°C) for 14 hours.

The precipitated candy-like substance (amorphous substance) was decanted and washed with cold methanol.

It could not be obtained as a crystal.

Also, in this candy-like substance, 5,7-diene vs. 4,6-
The diene composition ratio was approximately 2:1, which was almost the same as the recrystallized composition.

Reference Example 1 (1) Synthesis of 1α,3β-diacetoxycholest-5,7-diene; 1α,3β-diacetoxycholest-5-ene 22.1
g (45.5 mmol) was dissolved in 550 ml of n-hexane, and while stirring at 95°C (bath temperature), 7.1 g (24.8
mmol) and stirred for 20 minutes under infrared irradiation.

After cooling the resulting reaction mixture to room temperature, the precipitate was filtered and filtered.
- Washed with hexane.

The filtrate and washing liquid were combined and concentrated under reduced pressure at 40°C to obtain a residue (crude bromine compound).

s-collidine (114.4 ml) in 300 ml of xylene
A solution containing the above crude bromine dissolved in 200 ml of xylene was added dropwise to the solution under stirring at 170°C (bath temperature) (the dropping funnel was further washed with 50 ml of xylene), and the mixture was heated under reflux for 25 minutes. .

After cooling to room temperature, the precipitate was concentrated under reduced pressure at 60° C. without being filtered.

150 ml of dimethylformamide was added to this residue, heated to 180°C (bath temperature), and stirred for 15 minutes.

After cooling to room temperature, it was concentrated under reduced pressure at 60°C.

Add 500 ml of benzene to the residue and precipitate (main component, S-
Collidine/HBr salt) was filtered out, and the filtrate was concentrated under reduced pressure.
1α・3β-diacetoxycholester-5・7-diene,
1α・3β-diacetoxycholester-5・7-gene,
1α・3β-diacetoxycholester-4・6-diene 1
α・3β-diacetoxycholest-5-ene and 1α
- Residue containing acetoxycholester-2,4,6-triene (5,7-diene form: 4,6-diene form ≒ 20:1)
I got it.

(2) 1α・3β-diacetoxycholester-5・7,-
Isolation of diene: Prepare a column packed with 200 g of silica gel (
The above residue was chromatographed using benzene-ethyl acetate as the filling solvent and benzene-ethyl acetate as the developing solvent.

As the main component, 1α・3β-diacetoxycholester-5
・7-diene, 1α・3β-diacetoxycholester-4
- 15.5 g of material containing 6-diene and 1α,3β-diacetoxycholest-5-ene was obtained.

This product was further heated and dissolved in 100 ml of ethanol at 50°C (bath temperature) and left in a cool, dark place for 24 hours.

The precipitate was filtered off and washed with cold ethanol.

1α and 3β with a melting point of 113-115℃ (ethanol)
7.6 g of -diacetoxycholester-5,7-diene was obtained.

Reference example 2 1α・3β-dipivaloyloxycholest-5-ene 7
0 mg (1 mmol), 1,3-dipromo-5,5-dimethylhydantoin 157 mg (0.55 mmol),
n-hexane 12.1ml, s-collidine 2.52ml
Bromination and dehydrobromination reactions were carried out in the same manner as in Example 1 using 13.9 ml of xylene and 13.9 ml of xylene.

The obtained dehydrobromination reaction mixture was allowed to cool to room temperature, and then heated to 60°C.
It was concentrated under reduced pressure.

4.86 ml of dimethylformamide was added to the resulting residue to dissolve it, and then stirred at 180°C (bath temperature) for 15 minutes.

The reaction solution was concentrated under reduced pressure at 60° C., dissolved in benzene, and insoluble materials were filtered off, followed by column chromatography purification using 10 g of silica gel.

465 mg of the obtained crude product was 4:1 of 1α,3β-dipivaloyloxycholester-5,7-diene:1α,3β-dipivaloyloxycholester-4,6-diene. The same ratio of the above residue before dimethylformamide treatment was about 2:1, so when the protecting group is a pivaloyl group, the dimethyl It was found that the conversion efficiency of 4,6-diene to 2,4,6-triene by formamide is low.

Example 3 (1) 1α・3β. Synthesis of 24(R)-triethoxycarbonyloxycholest-5-ene; 1α・3β・24(R)-trihydroxycholest-5
-ene 3.63 gr and 4-dimethylaminopyridine 6.
25 gr was added to 40 ml of methylene chloride, dissolved by stirring under a nitrogen atmosphere, and then added dropwise to 24.75 ml of ethyl chlorocarbonate under ice cooling.

After dropping, the mixture was heated at a bath temperature of 60°C and stirred for 27 hours.

The resulting reaction mixture was poured into ice water and extracted with methylene chloride.

The methylene chloride extract was washed with 2N hydrochloric acid, then with a saturated aqueous sodium bicarbonate solution, and with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to yield 4.98 gr.
A residue was obtained.

This residue was recrystallized in 10 ml of methanol to obtain 1α
・3.64 gr of crystals of 3β・24(R)-triethoxycarbonyloxycholest-5-ene were obtained.

(2) Synthesis of 1α・3β・24(R)-triethoxycarbonyloxycholest-5,7-diene; 1α・3β
・Add 3.49 gr (5.5 mmol) of 24(R)-triethoxycarbonyloxycholest-5-ene to 58 ml of n-hexane (Na dry), heat in a 95°C oil bath, and add 1. - 998 mg (3.3 mmol) of 3-dibromo-5,5-dimethylhydantoin was added and stirred for 15 minutes under infrared irradiation.

After cooling to room temperature, the precipitate was filtered and washed with n-hexane.

The filtrate and washing liquid were concentrated under reduced pressure at 40°C to obtain a concentrated residue.

14.5 ml of S-collidine dissolved in 19 ml of xylene (Na dry) was heated in an oil bath at 170°C, and a solution of the above concentrated residue (crude bromine) dissolved in xylene was added dropwise while stirring. The dropping funnel was further washed with xylene) and heated under reflux for 20 minutes.

After cooling to room temperature, the precipitate was filtered off and washed with xylene.

The filtrate and washing liquid were concentrated under reduced pressure at 40°C, and the concentrated residue (
The main component, diene body) was obtained.

This product was dissolved in 200 ml of ethyl acetate and washed three times with 100 ml of 1N hydrochloric acid.

The ethyl acetate layer was washed successively with a saturated aqueous sodium bicarbonate solution and water, dried over anhydrous sodium sulfate, filtered,
It was concentrated under reduced pressure to obtain 3.68 gr of crude concentrated residue.

(3) Isolation of 1α・3β・24(R)-triethoxycarbonyloxycholesta-5・7-diene; (2) above
The entire amount of the crude concentrated residue obtained in step 1 was dissolved in a mixed solution of 7 ml of methanol and 1 ml of ether and left in a cool, dark place (5° C.).

The precipitated crystals (1.95 gr) were collected by filtration and further dissolved in 13 ml of methanol by heating under reflux at 70° C. for 30 minutes.

After cooling to room temperature for 30 minutes, it was left in a cool, dark place (5°C) for 1.5 hours.

The precipitated needle crystals were collected by filtration, washed with cold methanol at -20°C, and dried under reduced pressure in the dark to produce 1α・3β・24(R)-triethoxycarbonyloxy, which showed the following properties as colorless needle crystals. 1.69 gr of cholesta-5,7-diene was obtained.

Melting point: 116-7°C (methanol) U. V. (ethanol, λmaX); 262 (8110), 271 (11410), 281 (
12130), 293(7200)N. M. R(CDC
l3, δ (ppm)): 0.62 (3H, S.C-18
-CH3) 1.31 (9H, t, C-1, 3, 24-ethoxycarbonyl group CH3 x 3) 4.18 (6H, q, C-1, 3, 24-ethoxycarbonyl group CH3 x 3) 4 .50 (1H, bm, C-24-H) 4.80 (2H, bm, C-1・3-H×2) 5.35
and 5.65 (2H, bm, C-6-H and C-7
-H) Reference example 3 (1) 1α・3β・24(R)-to! J Synthesis of acetoxycholest-5,7-diene 1α,3β,24(R)-triacetoxycholest-5
- 4.7 gr (8.63 mmol) of ene in 69 ml of n
-Dissolved in hexane and stirred at 95°C (bath temperature) for 1.
3-dipromo-5,5-dimethylhydantoin 1.36
gr (4.74 mmol) was added, and 15
Stir for a minute.

The resulting reaction mixture was left to cool to room temperature, and the precipitate was filtered off.
- Washed with hexane.

The filtrate and solution were combined and concentrated under reduced pressure at 40°C to obtain a residue (crude bromine compound).

A solution of 13 ml of S-collidine dissolved in 43 ml of xylene was heated to 170°C (bath temperature), and a solution of the above crude bromine dissolved in 44 ml of xylene was added dropwise to this while stirring (the dropping funnel was further washed with 5 ml of xylene). ) The mixture was heated under reflux for 25 minutes.

After cooling to room temperature, the precipitate was concentrated under reduced pressure at 60° C. without being filtered.

Dimethylformamide was added to this residue, heated to 175°C (bath temperature), and stirred for 25 minutes.

After cooling to room temperature, concentrate under reduced pressure at 60°C to obtain 1α, 3β, 24
A mixture containing (R)-triacetoxycholester-5,7-diene was obtained.

(2) Synthesis of 1α.3β.24(R)-trihydroxycholester 5.7-diene The mixture obtained in (1) above was dissolved in a mixed solution of 43 ml of methanol and 43 ml of benzene.

129.5 ml of 2N potassium hydroxide-methanol solution was added to the obtained mixed solution and heated to 60°C (bath temperature).
The mixture was stirred for 1.5 hours.

After the reaction was completed, an appropriate amount of water was added to the reaction solution, and the mixture was extracted with ethyl acetate.

The ethyl acetate extract was washed with 1N hydrochloric acid and then with water, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 1α, 3β, 24(R)-trihydroxycholester 5,7-diene and 1α.・Contains 3β・24(R)-trihydroquincholester 4,6-diene (5,7-
Diene body: 4.6-diene body≒3.3=1) mixture Og
I got r.

(3) Isolation of 1α, 3β, 24(R) monotrihydroxycholester 5,7-diene Silica gel thin layer plate (20cm x 20cm x 2mm) 20
The mixture obtained in (2) above was subjected to chromatography using benzene-acetone as a developing solvent.

Main components: 1α, 3β, 24(R)-trihydroxycholester 5,7-diene, 1α, 3β, 24(R)
monotrihydroxycholester 4,6-diene and 1α
- 1.83 gr of material containing 3β.24(R)-trihydroxycholest-5-ene was obtained.

This was further treated with a 2% silver nitrate-acetonitrile solution to form a thin silica gel plate (20 cm x 20 cm x 0.5 cm).
By purifying by chromatography on a 76 mm) plate using chloroform-methanol as a developing solvent,
1α・3β・24(R)-trihydroxycholester 5
- 1.1 gr (30.6%) of 7-diene was obtained.

Claims (1)

[Scope of Claims] 1 The following formula [■] [In the formula, R1 and R2 are each independently a pivaloyl group or an ethoxycarbonyl group, and R3 and R4 are each independently a hydrogen atom, a hydroxyl group, a pivaloyl group, a quinone group, or an ethoxy It is a carbonyloxy group. ] Hydroxycholester-5,7-dienes represented by the following formula [■] [In the formula, the definitions of R1, R2, R3 and R4 are the same as above] The hydroxycholester 5,7-dienes represented by the above formula [■] are isolated by recrystallizing a mixture containing the above from a mixed solvent of a lower aliphatic alcohol and a lower aliphatic ether. A method for isolating hydroxycholester 5,7-dienes from the above mixture. 2 Hydroquincholester 5,7-dienes in which either R3 or R4 is a hydrogen atom in the above formula [I] and hydroquincholester 4.7-dienes in which either R3 or R4 is a hydrogen atom in the above formula [■] A method for isolating hydroxycholester-5,7-dienes according to claim 1, which comprises recrystallizing a mixture containing hydroxycholester-5,7-dienes. 3 Hydroxycholester-5,7-dienes in which R3 and R4 are both hydrogen atoms in the above formula [I] and hydroxycholester-4,6-dienes in which R3 and R4 are both hydrogen atoms in the above formula [■] A method for isolating hydroxycholester-5,7-dienes according to claim 1, which comprises recrystallizing a mixture containing hydroxycholester-5,7-dienes. 4 The lower aliphatic alcohol is an aliphatic chain alcohol having 1 to 4 carbon atoms, and the lower aliphatic ether is an aliphatic chain alcohol having 2 to 6 carbon atoms.
A method for isolating hydroxycholester 5,7-dienes according to any one of claims 1 to 3, which is an aliphatic chain ether or a 5- to 6-membered aliphatic cyclic ether. 5. A method for isolating hydroxycholester-5,7-dienes according to any one of claims 1 to 4, wherein the lower aliphatic alcohol is methanol and the lower aliphatic ether is diethyl ether. . 6. The statement according to any one of claims 1 to 5, which uses a mixed solvent in which the lower aliphatic cyclic ether is about 0.2 to 0.05 parts by weight per 1 part by weight of the lower aliphatic alcohol. A method for isolating hydroquincholester 5,7-dienes by. 7 Hydroxycholester 5 represented by the above formula [■]
・Hydroxycholester 4,6-diene represented by the above formula [■] is about 0.03 to 1 part by weight of 7-diene
By recrystallizing the mixture containing 4 parts by weight from methanol or a mixed solvent of ethanol and diethyl ether several times as necessary, the hydroxycholester 5,7-diene represented by the above formula [■] can be isolated. Hydroxycholester 5 and 7- according to any one of claims 1 to 6, characterized in that they are separated.
Method for isolating dienes. 8 The following formula [■], [wherein R1 and R2 are each independently a pivaloyl group or an ethoxycarbonyl group, and R3 and R4 are each independently a hydrogen atom, a hydroxyl group, a pivaloyloxy group, or an ethoxycarbonyloxy group. ]Hydroxycholester-5,7-dienes.