JPS6233118A - Anti-inflammatory agent - Google Patents

Abstract

PURPOSE:An anti-inflammatory agent, containing dolichol and/or ester thereof as an active constituent, having mold anti-inflammatory action with slight side effect and useful particularly for treating inflammation of persons, e.g. aged people, or patients with serious diseases, etc. CONSTITUTION:An anti-inflammatory agent containing dolichol expressed by formula I (group expressed by formula II is trans-isoprene unit; group expressed by formula III is cis-isoprene unit; n is an integer 12-18) as an active constituent. The compound expressed by formula I is capable of inhibiting carrageenan induced edema in 300-600mg/kg dose depending on the dose. The agent has low toxicity without side effect of the conventional anti-inflammatory agents, etc. The above-mentioned agent is administered in the dosage form suitable for oral and injection administration, e.g. tablet, capsule, ampul, etc. The compound expressed by formula I can be synthesized from ginkgo and Himalayan ceder purely in large amount according to the method for extraction from internal organs of mammals or the method described in Patent Publication No.58-83643.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to anti-inflammatory agents. More specifically, regarding an anti-inflammatory agent containing dolichol and/or its ester as an active ingredient, the anti-inflammatory agent has few side effects and has a mild physiological effect, so it can be used to reduce physical strength, especially in the elderly and patients with severe diseases. It is useful in treating and treating inflammation in people who are suffering from cancer.

BACKGROUND OF THE INVENTION In recent years, with the increase in the number of people entering old age, various geriatric diseases or adult diseases have become apparent, and countermeasures against them have become important. especially,
Due to a decline in various biological control functions.

Unlike in healthy people, inflammation in the elderly does not heal easily, and there is a strong need for the development of effective anti-inflammatory agents for its treatment and treatment. Traditionally, steroid-based anti-inflammatory drugs are used to treat inflammation! A number of non-steroidal anti-inflammatory drugs have been developed and are in use, such as salicylic acid, pyrazolone, indomethacin, and aniline.

On the other hand, in 1960, J.F. Perznock et al. reported that Jottecht's kidney I11. Dolichol was isolated for the first time from pig liver (Nature (London))
, 186, 470 (1960)], and later the dolichol had the general formula % (wherein -CH2-C=C-CH2- is a trans-isobu aCH unit, and -CH2-C=C-CH2- is represents a cis-inprene unit (hereinafter the same shall apply hereinafter), wherein the number (n') of cis-inprene units in the above formula is generally from 12 to 18. It was revealed that three types of homologs, n=14, n=15, and n=16, were the main ones [for molecular structure, J, +3urgos
et al., Biochem, Journal.

88.470 (1963), Rl for homologue distribution
W. Keenan et al., Biochem, Journal
l, 165, 4Q5 (see 1977'l)
.

Dolichol is not limited to human kidneys, pig livers, etc.
It is known that they are widely distributed in the bodies of mammalian mammals and play extremely important functions in sustaining the life of living organisms. In particular, it plays an important role in the formation of sugar chains in glycoprotein synthesis.
z et al. recognized that when sea urchin cells differentiate and begin intestinal embryogenesis, the ability to synthesize dolichol within the cells increases and the intracellular dolichol content increases significantly. Also,
At this time, if compactin is added to the cell culture system to inhibit dolichol biosynthesis, the above differentiation is not achieved, and this suggests that the presence of dolichol is closely related to the intestinal embryogenic differentiation of sea urchin cells. suggested [P
roc, National Academy of
5science.

U.S.A., 76.5709 (1979) and J.
Biological Chemistry, 2
56, 4679 (1981)]. Also, S, S,
Mookerjea et al. injected turpentine subcutaneously into rats and found that when the site was inflamed, glycoprotein synthesis was activated in the liver, and at the same time, the liver's ability to synthesize dolichols was significantly enhanced. , suggested that dolichols have a close relationship with inflammation.

(J, Biological Chemtry,
256, 4529 (1981)].

However, until now, the only way to obtain Dolichol was through extraction from animal organs.
It is extremely difficult to secure a large amount of Dolichol, and therefore the effects of Dolichol, such as its pharmacological effects, when administered into living organisms have hardly been studied. There was no consideration at all as to what kind of phenomena would be observed.

Problems to be Solved by the Invention As mentioned above, drugs that have been developed and used to treat inflammation have excellent anti-inflammatory effects, but most of these drugs inhibit prostaglandin biosynthesis. Perhaps because its mechanism of action is inflammatory, it is well known that it has strong physiological effects in addition to its intended anti-inflammatory effect. For example, non-steroidal compounds such as aspirin and indomethacin are known to exert anti-inflammatory effects by inhibiting the synthesis of endoperoxide of arachidonic acid. By inhibiting it, it also inhibits the synthesis of prostaglandin 2, thromboxane A2, etc., which have important functions in the body, and reduces the regulation mechanism of blood vessels, so it should not be used for a long period of time. Requires sufficient consideration. Furthermore, steroidal compounds such as dexamethasone are disadvantageous because they reduce the immune function in the body.

Thus, anti-inflammatory agents for the treatment of inflammation are not in high demand from a clinical standpoint, effective compounds are few, and
The current situation is that even if they are effective, they cannot be put into practical use due to strong side effects, or only those that must be used carefully with due consideration for side effects.

Therefore, an object of the present invention is to provide a medicament that cures inflammation slowly and effectively without such major side effects.

Means for Solving the Problems According to the present invention, the above object is achieved by preparing a compound represented by the general formula % (wherein n represents an integer from 12 to 18) and/or its pharmacologically acceptable This is achieved by providing an anti-inflammatory agent containing an ester as an active ingredient.

Compounds represented by the general formula <11 (i.e. dolichol)
can be obtained by extraction from the mammalian organs mentioned above (e.g., J. Burgos et al., Bi.
ochem, Journal, 88, 470 (1
963), 'R, W. Keenan et al., Bioche
m, Journal, 165.

405 (1977), etc.], and US Signa
It is commercially available from the company, but preferably according to the method previously discovered by one of the present inventors and his co-researchers, for example, as described in Japanese Patent Application Laid-Open No. 58-83643. Ginkg.

biloba), Himalayan cedar (Cedrus da
It can also be synthesized in a large amount and in a pure form by C5 elongation of the polyprenyl fraction extracted from the leaves of plants such as P. odara ). Dolichol exists in the mammalian body in a distribution with respect to the value of n from 12 to 18, but when the compound represented by the general formula (1) is used as an active ingredient in the present invention, the compound has almost the same value as in the living body. It can be used as a mixture with a distribution or as a mixture in any proportion of 2 m or more,
Alternatively, if necessary, it is also possible to isolate and use them by molecular weight. Separation into individual products according to molecular weight can be carried out, for example, by silica gel column chromatography as described in JP-A-58-83643.

Examples of pharmacologically acceptable esters of the compound represented by general formula (1) include esters of lower fatty acids such as acetic acid and propionic acid; esters of higher fatty acids such as palmitic acid and oleic acid; phosphoric acid, Examples include esters such as monomannosyl phosphate. The synthesis of these esters can be carried out according to conventionally known methods of esterifying higher alcohols (for example, L. Danilova et al.
ndT.

Chojnacki, Febs Letters,
131, 310 (1981); Japanese Patent Publication No. 58-83
No. 643; see Japanese Unexamined Patent Publication No. 59-62599, etc.). For example, the acetate ester of dolichol can be easily obtained by reacting dolichol with acetic anhydride in the presence of pyridine in a hexane solvent.

The compound represented by the general formula (1) and its pharmacologically acceptable esters (hereinafter collectively referred to as "dolichols" unless otherwise specified) have the effect of curing inflammation that occurs in the human body. It is useful as an anti-inflammatory agent.

In general, inflammation is caused by some kind of organic change (P
It is a defense reaction of the living body that tries to arbitrarily repair and regenerate the damaged area due to the force of mutual injury, and this defense reaction includes local body fluids and vasculature.

Various types of tissue cells, phagocytes, immune cells, etc. are thought to be involved. The body uses this defense response to suppress the spread of damage to tissues and cells, eliminate the cause of the damage, and repair the tissue, so it is not desirable to suppress inflammation needlessly. Inflammation itself contains factors that are unfavorable to the living body, such as excessive pain (1) swelling and fever (91) and tissue damage (91) which may even be amplified.Therefore, it is important to suppress excessive inflammation. is necessary to alleviate the patient's pain, and administration of anti-inflammatory drugs can be cited as a means of suppressing inflammation.

A common method for evaluating the efficacy of anti-inflammatory drugs is to examine their healing effects on various inflammation models. Models that reproduce the pathology of inflammation include pain models that consider the behavior exhibited by animals when physical or chemical stimulation is applied locally as a pain response, and examine the effects on this response;
An edema model that reproduces the edema associated with the leakage of plasma components due to the normalization of vascular permeability at the site of inflammation, and an ultraviolet irradiation erythema model that reproduces the inflammatory redness associated with increased blood flow due to vasodilation at the site of inflammation. and so on. Among these, the edema model, which is created by inoculating carrageenan, a polysaccharide, into the paws of rats, is an inflammation caused by non-specific stimulation that various chemical mediators are involved in over time, and various anti-inflammatory drugs Known to be well inhibited [eg, M, Dirosa.

J, Pharm, Pharmacol, 24.
89 (1979), C.A.

Winter, lNon-5teroidal An
ti-InflammatoryDrugs”ed
, by S., Garaffini et al., E.
xcetptaMedica Foundation,
Amsterdam, 1965+ p. 190]. Below, a test on the anti-inflammatory effect of dolichols using this model and the results thereof will be shown.

(1) Carrageenin-induced edema suppression test test method Holfzman male rats (body weight 125-1659
) were used in the test with 4 animals per group. Dolichol synthesized according to Reference Example 1 described below was added to corn oil at a rate of 100 Q/
It was dissolved in bath to a concentration of rnl and intraperitoneally administered to rats at a rate of 100 Vζ body weight. One hour after administration, carrageenin (Moxibustion, A1gin, USA) suspended in physiological saline to a concentration of 1% was added. OsJ was subcutaneously administered to the right hind leg of the rat, and the constant volume was immediately measured. Three hours after the administration of carrageenan, the constant volume was measured again, and the edema rate was calculated according to the following formula.

In the control group, the same procedure as above was carried out except that (c) 1 corn oil not containing dolichol was administered intraperitoneally.

Test results The edema rate of each group is shown in Table 1 as the average Hanato standard error,
In addition, the suppression rate calculated based on the edema rate of the control group is also listed in the same table.

Table 1 Note) *Significant difference compared to control group (P<0.05 tested) As is clear from Table 1, dolichol significantly suppresses the formation of edema induced by carrageenil.

(2) Carrageenin-induced edema suppression test Test method Wistar male rats (body weight 120-14of) were used in the test, with 6 rats per group. Dolichol, which was synthesized according to Reference Example 1 described below, was 6
0- (manufactured by Nikko Chemical Co., Ltd.) was suspended in distilled water, and a predetermined amount of the suspension was intraperitoneally administered to rats. One hour after administration, 1 ml of carrageenan O suspended in physiological saline at a concentration of 0.1% was subcutaneously administered to the right hind paw of the rat. After administration, constant volume was measured over time, and the edema rate was calculated according to the following formula.

In the control group, the same procedures as above were performed except that a suspension of neo-6o, a nonionic surfactant containing no dolichol, was intracavitally administered to the rats.

Test results Table 2 shows the edema rates at 1 hour, 4 hours, and 6 hours after administration of carrageenan for each group as the standard error above the mean value, and calculated based on the edema rate of the control group after the next administration of carrageenan. The inhibition rate at 6 hours is also shown in the same table.

Table 2 As is clear from Table 2, Dolichol inhibits carrageenan-induced edema formation by 300-600 M9/ky.
suppresses in a dose-dependent manner over a range of doses.

-1. Dolichols do not have the side effects seen with the conventional anti-inflammatory agents mentioned above, and have low toxicity.

The absence of side effects of dolichols can be confirmed by the following experiment using dolichol synthesized in Reference Example 1, which will be described later.

In addition, since the drug used is an oily liquid that is insoluble in water, in the experiment, it was dissolved in sesame oil for oral administration, and a nonionic surfactant 1 (for intraperitoneal administration and in vitro). The suspension was made using Co-60 (trade name, Nikko Chemical Co., Ltd.) and used.

In addition, in this experiment, ddY male mice, Wist
Arbor male rats (Shizuoka Experimental Animals), Japanese white male rabbits (Cary), and Hartley male guinea pigs (Nippon Talea) were used. Animals at a temperature of 23±1°C1
Humidity 55±10%, lighting time (7:00AM-9:QQ
PM), the ventilation rate is 15 times/hour, and the food is solid feed MF (Oriental Yeast) for mice and rats, and solid feed GM for guinea pigs and rabbits.
-3 and RM-1 (Funabashi Farm), and tap water was provided ad libitum. The animals were preliminarily bred for at least one week and were in a healthy state.

(1) Neuropharmacological test using mice ddY male Maunus were used with 6 mice per group.

Mouse behav according to Irwtn's multidimensional cherry blossom viewing method
Changes in tor, neurological symptoms, autonomic nervous symptoms, and toxic symptoms were observed and analyzed from multiple angles and recorded. 15 dorikor
0, 30 o, 600 mg/9 times were administered intraperitoneally.

Dolichol administration at 150.300.600Q/kg resulted in behavioral and neurological symptoms of 1 mouse, autonomic nervous symptoms and 4
No model number was observed in symptoms etc. 0 (2) Peripheral effect test using removed organs 1 Guinea pig removed ileum specimen Hartley male guinea pig (350-40C1)
were used in groups of 3 animals. The extracted ileum was suspended in Tyrode's solution (at a moderate temperature of 32°C) through air.

Ileal contractions were recorded using an ink oscilloscope (Nihon Kohden EMP-3004) via an isotonic transducer (Nihon Kohden zTp-1x28). Dorikor's 10. 10. The presence or absence of direct action and antagonistic action against acetylcholine, histamine, and nicotine at io-'t/ml was investigated.

Dolichol had no direct effect on isolated guinea pig ileum specimens at 10r/In1i and no antagonistic effect on each agonist.

ij Rat excised and exported f# tube specimen Wistar male rats (180-200 r) were used in groups of three. The extracted vas deferens was passed through the air and injected with Locke-Ringer solution (W temperature: 32°C).
I hung up inside.

Constriction of the vas deferens was recorded in the same manner as in the previous section. Dorikor's 10, 10, 10? The presence or absence of a direct effect on /rnl and an antagonistic effect on noradrenaline was investigated.

Dolichol had no direct effect on isolated rat vas deferens specimens and no antagonistic effect on noradrenaline up to 10 v/rnl.

111 Guinea pig isolated atrial specimen) Iartley male guinea pig (350-400?
) were used in groups of 3 animals. An atrial specimen consisting of both the left and right atria was prepared, and a mixture of 95% 02
0°C). The contraction of the atrium is detected by FD pickup (
The data were recorded using an ink oscillograph (Nihon Kohden EMP-3004) via a Nihon Kou zTB-sxxT). Dorikor's 10. 10. The inotropic and chronotropic effects of 10′f/rnl administration were investigated.

Dolichol had no inotropic or chronotropic effects on isolated guinea pig atrial specimens up to 1Qf/ml.

1 ■ Guinea pig isolated trachea specimen HartlBy male guinea pig (350-4oor)
were used in groups of 3 animals. The excised trachea was suspended in Loclce-Ringer solution (liquid temperature 37°C) through which air was passed. Tracheal constriction was recorded in the same manner as in the section of the isolated ileum. Dorikor's to, 10, 10
The presence or absence of an antagonistic effect on isoproterenol, which is a direct effect of administration of f/rnl, was investigated.

Drecall is to? /rrLl''t had no direct effect on isolated guinea pig trachea specimens or an antagonistic effect on isoproterenol.

(3) Effects on the blood g coagulation system in rats
Istar male rats (180-21C1') were used in groups of 6 rats. Two hours after dolichol administration, blood was collected from the abdominal vena cava, and prothrombin time was measured using the Quick one-step method.

Dorikor's 150.300. No significant difference was observed in the 600Q/ky administration group compared to the control group.

(4) Wistar glycolysis level lowering effect in rats
Male rats (180-210r) were used in groups of 6 rats. Two hours after administration of Dolichol, the sample was taken from the abdominal vena cava, and the sucrose level was measured using the glucose oxidase method. 300. No significant difference was observed in the 600 mg/kq administration group compared to the control group.

(6) Diuretic effect in rat VC WiStar male rats (80-90y) were used in a group of 6 rats. Immediately after dolichol administration, take 2 saline saline
．． 5rnl/100? The animals were administered orally, and each animal was placed in a substitute cage, and urine was collected 6 hours after administration of the sample.

The urine volume was measured.

No significant difference was observed in the 150.300.60071 g/7 administration group of Dolichol compared to the control group.

(6) Analgesic effect in mice (acetic acid writhi)
ng method) ddY male mice (27-30?) per group 6
It was used as a fish. One hour after dolichol administration, 0.6% acetic acid was administered intraperitoneally to the mice at o, 1i/xor.

After 10 minutes, observe for 10 minutes, add acetic acid
The inhibitory effect on g was investigated.

No significant difference was observed in the Dolichol administration group at 150.300.600 JIg/day compared to the control group.

(7) Reserpine [1, action ddY in mice
Lineage male-? Two C (27-30f) were used in groups of 6. Reserpine 4 mg/kq was administered intraperitoneally, 3 hours later Tru7-L was administered, and 1 hour later, the degree of ptosis of the mouse was evaluated according to Rubin's criteria (0: normal eye opening, 1 :1/4 blepharoptosis, 2:
Observations were recorded according to the following: 1/2 ptosis, 3: 3/4 ptosis, 4: complete ptosis).

In the 150.300.600 g/auxiliary administration group of Dolichol, no antagonistic effect on blepharoptosis caused by reserpine was observed compared with the control group, and fC.

(8) Anticonvulsive effect in mice (maximum electroconvulsive effect) ddY male mice (27-307) were set at 6 mice per group.
used. 4 on the cornea of mice 1 hour after dolichol administration.
QrnA, 0.4m5ec, 5Q) Iz, Q,
Electricity was applied for 5 seconds to induce tonic extensor spasm (T.

E,) duration was measured.

No significant difference was observed in the Dolichol 150.300.600'*9/moan administration group compared to the control group.

(9) Local anesthetic effect Hartl in guinea pigs
Male guinea pigs (370-440f) of the Ey strain were used in groups of 3 animals. Each concentration of dolichol (0,001%,
0.01%, 0.1%) was instilled into the eyes, and 1.
At 2.3.4.5.10.15.20 minutes, the presence or absence of corneal reflexes due to stimulation was observed.

Dolichol did not cause any loss of corneal reflex in guinea pigs up to 0.1%, and no local anesthetic effect was observed.

[Acute toxicity]

(1) For oral administration of Dolichol synthesized in Diagnosis Example 1 below, use a solution dissolved in sesame oil, and for intraperitoneal administration, use ICO-60 (manufactured by Nikko Chemical Co., Ltd.), a nonionic active agent. It was used as a suspension.

ddY mice (21-271, male) were used in groups of six. Vi5,000II9/ for oral administration
In the case of intraperitoneal administration, each drug was administered to 1,000 door q/moaning mice, and antenatal symptoms and whether or not they were alive or dead were observed on day 1, 7 days after administration. Body weight was measured before administration, on days 1 and 7 of administration. Ushita. Sesame oil and l-1cO-5Q, which are the respective solvents, were administered to the control group, and the same observations and measurements as for the sample administration group were performed. On the 7th day after administration, all animals were dissected and macroscopically observed.

There were no deaths in all cases, and no significant difference in body weight change was observed compared to the control group.

No abnormalities were observed in the autopsy findings on the 7th day of administration.

(2) After dissolving the dolichol phosphoric acid synthesized in Reference Example 3 described later in the same amount of sesame oil, 5% dolichol phosphoric acid was dissolved in a 11% disodium carboxyl cellulose water bath solution.
It was added and dispersed to form an emulsion and used so that the ratio of %W/V was 9 degrees.

BDFz mice (20-232, female) were used in a group of 6 mice.
The animals were administered intraperitoneally at a dose of 2,000 Q/ky and 2000 Q/ky, and the presence or absence of survival was observed on the second day after administration. There were no deaths in all cases.

As mentioned above, dolichols not only have a mild anti-inflammatory effect, but also have few side effects and low toxicity, and are useful for the treatment of inflammation caused by various yuzu factors, especially in patients with impaired function, such as the elderly and patients with kaede disease. It is useful as a drug for the treatment of inflammation.

When using dolichols for the above diseases, the dosage can vary widely depending on the method of administration, symptoms, weight, age, gender, and judgment of the treating physician of the patient to whom it is administered. Yes, but generally 0.05
11 g/kf/day ~ 1,000 mg/k/day, preferably o, iη/kf/day ~ 100 m9/kg/
day, more preferably 0.21Ag/m2/day to 5ony
/kr/day, this dosage once a day'! Or it can be administered in several doses.

The method of administration may be either oral or parenteral; examples of parenteral administration include intravenous, intravascular, intraarterial, intramuscular, intraperitoneal, intramedullary, rectal administration, etc. The following method can be used.

Dolichols are available in various dosage forms suitable for oral administration, such as tablets, granules, powders, coatings, hard capsules, soft capsules, and oral liquid preparations. can be. Furthermore, it may be in a form suitable for injection administration, such as a suspension, solution, oily or aqueous emulsion.

Dolichols can contain various pharmaceutically acceptable liquid or solid diluents or carriers.

Examples of such diluents or carriers are, for example, syrup, acacia, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone, magnesium stearate, talc.

Examples include polyethylene glycol, silica, lactose, sugar, corn starch, calcium phosphate, glycine, potato starch, carboxymethyl cellulone calcium, sodium lauryl sulfate, water, ethanol 1 glycerin, mannitol, phosphate buffer, etc. .

In addition to the pharmaceutically acceptable diluent or carrier as exemplified above, the anti-inflammatory agent of the present invention contains other adjuvants commonly used in the pharmaceutical field, such as coloring agents, flavoring agents, flavoring agents, preservatives, and solubilizing agents. Other adjuvants such as suspending agents, dispersing agents, etc. may further be included.

The anti-inflammatory agent of the present invention includes tablets, capsules, etc. as exemplified above.
In addition to being in the form of fixed-dose dosage forms such as coatings, ampoules, etc., it can also be in the form of multiple-dose, 4-dose containers.

The present invention will be further explained below using reference examples and examples.

Reference Example 1 Synthesized according to the method described in JP-A No. 58-83643. Yellowed ginkgo leaves (100K, undried weight) collected in Sanshiki City in November were mixed at about 40° 10 in C
After drying with hot air for an hour, it was immersed in chloroform 800j at room temperature (approximately 15° C.) and extracted for one week. In the concentrate obtained by distilling off chloroform from this extract, 50% of bexane was added.
After adding 1 and separating the insoluble components, the solution was concentrated, and then subjected to silica gel column chromatography using a hexane/ethyl acetate mixture as a developing solvent. Hexane/ethyl acetate was mixed at a ratio of 9/1 (volume ratio). Silica gel thin layer chromatography using liquid (TLC plate manufactured by Merck & Co.)
5ilica 5 Q F254, precoate
d (10z expansion using 1 layer thickness of 0.25 m) and separate the fraction with Rf = 0.52 to approximately 275
A liquid product of No. 2 was obtained. Mix this with 21° methanol and 20° water.
0 punishment and potassium hydroxide 15 (2 hours 65 with 1'
After heating to ℃, hexane 21 was added to extract the organic layer, washed 5 times with water, dried over anhydrous magnesium sulfate, and the solvent was distilled off. The product was purified by silica gel column chromatography to obtain about 227f polyprenol. Next, this material was dissolved in 51 hexane with 252 f of pyridine and 50 f of acetic anhydride, and 1
Stirred for 2 hours. The resulting reaction mixture was washed with saturated brine, dried over anhydrous magnesium sulfate, and concentrated to give 2
282 polyprenylacetate was obtained.

In a three-bottle flask purged with argon, magnesila 4 pieces (3,16 ml, 130 mmol), anhydrous tetrahydrofuran (5x/) and 1,2-dibromoethane (0,8
tl) and heat it with a hair dryer until it bubbles violently. Then (R) -2-(4-bromo-3-methylbutoxy-2H-pyry(25,1
? , 100 mmol s Cα)p−3,61°
, C=4.0, CHCla) in anhydrous tetrahydrofuran (3 omg) was added dropwise to the activated magnesium at such a rate that the solvent just boiled. After the addition was completed, the mixture was stirred at 70°C for 15 minutes. Anhydrous tetrahydrofin (60011IA') was added to this to make a Grignard solution. The polyprenyl acetate (64,2F, 50 mmol) prepared earlier was placed in a three-bottle flask that had been replaced with argon.
Anhydrous tetrahydrofura/(150+x/) solution and Ll
A solution of zCuC14 in anhydrous tetrahydrofuran (0.1 molar solution, 200 fi/) was charged. The previously prepared Guineal solution was added dropwise to this at 0°C over 4 hours, and the reaction was continued at 0°C for another 4 hours. Thereafter, saturated ammonium chloride water was added to the reaction mixture for hydrolysis, followed by extraction with ether. The ether layer was washed with saturated brine, dried over anhydrous magnesium sulfate, and the solvent was distilled off using a rotary evaporator to obtain a pale yellow liquid. Next, this product was dissolved in hexane (400d), and p-toluenesulfonic acid pyridine (1,3F, 5 mmol) and ethanol (
200 yd') was added. This solution was heated and stirred at 55° C. for 3 hours. After cooling to room temperature, add sodium carbonate (2.5F
) was added for neutralization, washed with saturated brine, dried over anhydrous magnesium sulfate, and the solvent was distilled off. Q the remaining liquid
, 5 Torr, heated at 150°C for 30 minutes to remove low-boiling components, and the residue was granulated by silica gel column chromatography using a hexane/ethyl acetate mixture as a developing solution to obtain a colorless and transparent liquid of 56.8F. I got it. This product was confirmed by IR and NMR analysis to be dolichol represented by the above-mentioned general formula (1). this)"
IJ near-k rise μm Bondapak
-C1g (silica gel surface treated with C18O hydrocarbon compound) is used as filler, acetone/methanol =
The area ratio of each peak in a chromatogram obtained by high performance liquid chromatography using 90710 (volume ratio) as a developing solution and a differential refractometer as a detector was determined, and the content ratio regarding the value of n in general formula (I) was determined. and is described below.

n=12 1.2% 13 6.7 14 24.6 1.5 40.4 16 20.0 17 5.9 18 1.2 Reference Example 2 General formula (1 ) in n
Dolichol 10f distributed from = 12 to 18 was treated with acetone/methanol = 9 using a Merck semi-preparative high performance liquid chromatography column (C1s type) RP18-10.
Using a mixed solvent of 0/10 (volume ratio) as a developing solution, the components were separated for each value of n to obtain the following.

n=12 0.1? 13
0.65 14 2.6 16 1.9 17 0.6 18 0.1 All of these separated substances have the value of n in general formula (1) according to the results of LH-NMR, 13C-NMR and FD-MASS analysis. It was confirmed that these have chemical structures corresponding to 12 to 18, respectively.

Reference Example 3 (Phosphoric acid esterification) Method of L, L, Danilov et al.
tters, vol. 131, p. 310, 198.1).

Phosphorus oxytrichloride (1,92ml) in hexane (75+
+! /) Triethylamine (2,8711!/) in solution
After stirring, a hexane (75+t/') solution of dolichol (5) synthesized in Reference Example 1 was added dropwise at room temperature, and the mixture was stirred for 30 minutes. Pour the reaction solution into a mixture of acetone/methanol/water = 88/10/2 (volume ratio),
After stirring overnight at room temperature, place in a separatory funnel and separate the upper layer.
Washed with saturated saline, dried over anhydrous magnesium sulfate,
The solvent was distilled off under reduced pressure, and the resulting yellow liquid was evaporated with DEAE.
-Using cellulose (acetate ester type cellulose ion exchanger s 3.5 crn id x 12α), a mixture of chloroform/methanol = 2/1 (volume ratio) with a small amount of ammonium acetate was used as a developing solution. A fraction containing dolichol phosphate was obtained by column chromatography. Without it, this thing is 5 ephadex
Ammonium acetate was removed by gel filtration using LH-20 (dextran gel 140?) with chloroform/methanol = 2/1 (volume ratio) as the developing solution.
The resulting solution was concentrated and treated with dolichol phosphoric acid (3, On
I got it. NMR analysis of this product revealed a signal (δ, = 3.66) due to -CH20H of the raw material dolichol.
) disappears, -Cdan20P: The signal caused by (
Almost the same signal as the raw material was observed, except that δ=3.90) was observed. From this, it was confirmed that this compound was dolichol phosphoric acid.

Reference example 4 (acetic acid esterification) Dolichol obtained in reference example 1 (13,1F, 10 m
mol) in anhydrous methylene chloride (100 shoulder g), added pyridine (3,2F, 4 Q mmol) and 4-dimethylaminopyridine (5 (1m9), and dissolved acetic anhydride (2,04f) with stirring under water cooling. , 20 mmol
) was added dropwise. After stirring at room temperature for 30 minutes, the mixture was poured into ice water and extracted with methylene chloride. The organic layer was washed with diluted hydrochloric acid and water, dried over anhydrous magnesium sulfate, and the solvent was distilled off under reduced pressure to obtain a yellow liquid. This product was purified by silica gel column chromatography using hexane/ethyl acetate = 99/1 (volume ratio) as a developing solution to obtain a colorless and transparent liquid (12,21).This product was analyzed by NMR. , -C) of raw material dolichol!
The signal caused by 20H (δ=3.66) disappeared;
-CH2(X!:CHs oyo(j -CHzOCCH
s K due to f signal (δ = 4.04 and 1
．． 97) was observed, and other than this, almost the same signal as the raw material was observed.

The following results were obtained by IR analysis.

3030.2950, 2910.2845.1740°1660.1440, 1370, 1230.1020.
830c! ! From the fact that it was greater than L-1°, it was confirmed that this product was dolichol acetic acid.

Reference example 5 (palmitic acid esterification) Dolichol (1,31F, 1 mmo
1) was dissolved in anhydrous diethyl ether (2 WL/), pyridine (80 µ, 1 mmol) was added, and palmitic acid chloride (275 µ, 1 mmol) was added with stirring at room temperature.
added. After stirring for 3 hours, the mixture was poured into water, extracted with diethyl ether, washed with diluted hydrochloric acid and water, dried over anhydrous magnesium sulfate, and the solvent was distilled off under reduced pressure to obtain a 1.4f yellow liquid. This product was subjected to silica gel column chromatography using hexane as a developing solution to obtain a colorless and transparent liquid 1.32. NMR analysis of this material revealed that the raw material was Dolichol 10! (The signal (δ = 3.66) caused by 20H disappeared, and -CH2-O-C-CH
Signals due to 2(CH2)13 (δ=4.04 and δ=1.28) were observed. The following results were obtained by IR analysis.

IR analysis: 3040.2970, 2935.2860.
1740.1665.1450.1380,1170.
835C1l 0 or more, it was confirmed that this product was dolichol palmitic acid.

Example 1 (Exfoliation: Injection) Dolichol obtained in Reference Example 1 10 m 9 Polyoxyethylene hydrogenated castor oil 70 Tnf Sorbitan trioleate 5.0 ■ Propylene glycol 20 ■ Distilled water for injection Approximately 1 l Polyoxyethylene hydrogenated castor oil and sorbitan trioleate were respectively weighed, heated and dissolved, and the separately weighed dolichol obtained in Reference Example 1 was added, followed by rapid stirring for a while while warm. from now on,
Add propylene glycol and distilled water for injection and bring the total volume to l.
ml, then filtered according to the usual method for manufacturing injections, and l#! !
The solution was filled into a brown ampoule with nitrogen gas. Sterilization was carried out at 100° C. for 40 minutes by the flow steam method.

Example 2 (splitting and peeling: bowl agent) Dolichol 102 beeswax obtained in Reference Example 1
1 Tahydroxybrobylcellulose 3 Risui
30a/crystalline cellulose
302 lactose
302 Corn starch 20
y Carboxymethyl cellulose calcium 5y Weighed each of Tetokudolichol, beeswax and hydroxyglobil cellulose in Reference Example 1, and added water to give approximately 70%
The mixture was heated to ℃ to form an emulsion. Crystalline cellulose, lactose, and corn starch were mixed, and the emulsion was added to the mixture for adsorption. After drying, the particles are sized and mixed with carboxymethyl cellulose calcium to form 6mm in diameter, 3.3mm in thickness, 1
Lock 100TWt! Compression molded into tablets.

Example 3 (m-ablation: injection) Dolichol with n=15 obtained in Reference Example 2 1f Polyoxyethylene hydrogenated castor oil 77 Propylene glycol 101 Glucose
2.5f distilled water for injection
Approximately 100 d of dolichol, polyoxyethylene hydrogenated castor oil, and propylene glycol with n=15 obtained in Reference Example 2 were each weighed out, heated and dissolved, and stirred rapidly for a while. Thereafter, distilled water for injection was added to bring the total volume to 100 at, followed by filtration in the usual manner for manufacturing injections, and filled into brown ampoules each having a volume of 1 d and sealed with nitrogen. Sterilization was carried out at 100° C. for 40 minutes by the flow steam method.

Example 4 (Formulation Example: Injection) Dolichol phosphoric acid obtained in Reference Example 3 12 Dolichol phosphoric acid obtained in Reference Example 3 and polyoxyethylene sorbitan monooleate were mixed, and further diluted with triglyline. The volume was made 100d. Next, the mixture was filtered according to a conventional method for manufacturing injections, filled into brown ampoules each having a capacity of 1, and sealed with nitrogen.

Sterilization was carried out at 100° C. for 40 minutes by the flow steam method.

Example 5 (Formulation Example: Powder) Dolichol obtained in Reference Example 1 51 Microcrystalline cellulose 40F corn starch 552 Dolichol obtained in Reference Example 1 was dissolved in acetone, then adsorbed on microcrystalline cellulose, and then dried. did. This was mixed with corn starch and a 20-fold powder of the main drug was prepared as a powder by a conventional method.

Example 6 (Formulation Example: Capsule) Dolichol obtained in Reference Example 1 52 Microcrystalline cellulose 80 Corn starch 209 Milk shield
222 Polyvinylpyrrolidone
3? After the above ingredients are granulated by a conventional method f), hard gelatin capsules are prepared. Filled into ooo capsules. One capsule contains Dolichol 5M.

Example 7 (Liposomal formulation) Dolichol obtained from reference ψ111 45+1? Egg yolk lecithin 135 ■ 1 Gt of Dolichol 45 ~ obtained in Reference Example 1 and egg yolk lecithin 135 ■
A total of 30 ml of chloroform was added to a t500d eggplant-shaped flask, and the mixture was sufficiently dissolved and mixed. This eggplant-shaped flask was attached to a rotary evaporator whose atmosphere had been previously replaced with nitrogen gas, and chloroform was distilled off at about 40° C. under reduced pressure with a water aspirator, thereby forming a thin film on the inner surface of the flask. The inside of the flask system was returned to normal pressure under a nitrogen gas atmosphere, the flask was removed from the rotary evaporator, and then the remaining chloroform was completely distilled off under reduced pressure of about 0.1 UHf using a vacuum pump. Distilled water 2Q m! in a flask with a U membrane on the inside! was added and vigorously stirred for 60 minutes using an ultrasonic crusher while cooling with water to obtain a water emulsion. After removing dissolved oxygen (by bubbling gas into the water emulsion), it was left to stand at about 4°C for 12 hours. This water emulsion was again stirred for 5 minutes using an ultrasonic crusher under water cooling, and then freeze-dried to obtain a white spongy substance 180''17.

Effect of the invention

Claims (1)

[Claims] General formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (In the formula, ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ is the trans-isoprene unit, ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ is a cis-isoprene unit, and n represents an integer of 12 to 18.