JP2003034690A - Inositol derivative, method for producing the same, and cyclooxygenase-2 expression inhibitor - Google Patents

Abstract

(57) [Summary] [PROBLEMS] To synthesize a novel compound using inositol, which is a food ingredient and obtained industrially, as a main raw material, to produce COX-2
To provide a highly safe drug that is effective in suppressing the expression of cysteine. SOLUTION: An inositol derivative represented by the following formula (I). Embedded image (I) R1 in the formula (I) is H or the following formula (I)
A substituent selected from any of the substituents represented by I), (III) and (IV), wherein R2 and R3 in the formula (I) are H or the following formulas (III) and IV) represents a substituent selected from any of the substituents represented by the following formula (II), wherein Me in the following formula (II) is a methyl group, and the following formula (IV)
Ac in each represents an acetyl group. Embedded image (II) (III) (IV)

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel inositol derivative having a cyclooxygenase-2 expression inhibitory action, which is useful for preventing cancer and alleviating inflammatory symptoms, and more specifically, it relates to a myo-containing derivative of edible plants. The present invention relates to a highly safe inositol derivative containing inositol and ferulic acid as main raw materials, a method for producing the same, and a cyclooxygenase-2 expression inhibitor.

[0002]

2. Description of the Related Art Non-steroidal anti-inflammatory drug (hereinafter referred to as NSAID
Is a drug that is widely used as an anti-inflammatory agent or an analgesic agent. NSAIDs are said to exhibit anti-inflammatory effects by inhibiting the enzymatic activity of cyclosoxygenase (hereinafter referred to as COX), which is a prostaglandin synthase. Inflammatory chemical mediators such as prostaglandin E ₂ are synthesized by the action of COX using arachidonic acid as a substrate. COX includes cyclooxygenase-1 (hereinafter CO
X-1) and cyclooxygenase-2 (hereinafter referred to as C
(Described as OX-2) is known to exist. COX-1 is widely expressed in digestive organs, whereas COX-2 is locally expressed in inflammatory lesions. For the induction of COX-2, cytokines (J. Biol. Chem. 1990, 256: 10805-10
808), mitogen, or endotoxin (J. Biol. Chem.
1992, 264: 25934-25938), etc. are involved. Aspirin, which is an anti-inflammatory agent, is considered to cause side effects such as gastrointestinal disorders because it similarly inhibits enzyme activity against both COX-1 and COX-2 (Arch. Intern. Med. 1987,14
7: 85-88). Therefore, the recently developed activity inhibitor that selectively inhibits the activity of COX-2 (COX-2 selective activity inhibitor) reduces the side effects common to the conventional non-selective COX inhibitors. Is expected to be possible.

Furthermore, not only activity inhibitors but also agents capable of selectively inhibiting the expression itself of COX-2 are
It is considered to be useful as an anti-inflammatory agent with few side effects as well as a COX-2 selective activity inhibitor. However, COX-
There are few reports on compounds that suppress the expression of 2. For example, Fukuda et al. Established a method for screening COX-2 transcriptional repressor active substances using colon cancer cells transformed with a vector in which a reporter gene is located downstream of the COX-2 promoter (J. Ethnopharmacology, 1999. , 66: 227-23
3). However, the compounds for which COX-2 transcription inhibitory activity was confirmed by this method are limited to berberine, resacetophenone, etc. and flavonoids, and it is necessary to identify the basic structure required for COX-2 selective activity inhibitors. Is still lacking information. Identification of the basic structure and accumulation of information on the substituents involved in the suppressive action of expression are indispensable for enhancing the effectiveness as a drug.

For example, the derivative of ferulic acid recovered from the extraction residue of rice bran oil, Ethyl-3- (4'-geranylox).
y-3'-methoxyphenyl) -2-propenoate (EGMP) has been shown to be effective as a carcinogen in a screening system that uses the development of abnormal crypt foci, which are precancerous lesions in the rat large intestine, as an index. (Anticancer Res, 1999,19:
3779). This compound was tested for LPS and interferon-γ
It was found that the expression of COX-2 was suppressed in mouse macrophage cells RAW264.7 stimulated with C (C
ancer Lett., 2000, 157: 77.). Also, regarding ferulic acid derivatives other than EGMP, their COX-2 expression inhibitory action is of interest. So far, COX-
2 Northern blotting was used to confirm the expression-suppressing effect. This method is complicated in operation as compared with the reporter gene assay used in the present invention, and is not suitable for treating many compounds. In other words, it can be said that, with regard to the COX-2 expression inhibitor, it was difficult to accumulate information that enables the structure-correlated activity by a general method.

According to a recent epidemiological survey, NSAID
The long-term users of aspirin, a typical drug of the drug, have a 30-40% reduction in colorectal cancer mortality (Cancer Res.
1993, 53: 1322-1327). In addition, although COX-2 expression is not found in normal digestive tissue, it has been clarified that COX-2 expression is induced in colorectal cancer (Cance
r Res. 1995, 55: 2556-2559, Cancer Res. 1995, 55:37.
85-3789). Therefore, it is speculated that the reduction in colorectal cancer mortality due to NSAID is due to its COX-2 inhibitory action. Furthermore, it strongly suggests that inhibition of COX-2 expression will enable chemoprevention of colorectal cancer or treatment of polyps. Thus, there is a possibility that COX-2 expression inhibitors can be applied to the prevention and treatment of colorectal cancer, and it is desired to provide candidate compounds that can be used as medicines.

[0006]

A drug effective in suppressing the expression of COX-2 is, for example, epigallocatechin (Bioche) of green tea.
mPharmacol, 1997, 54, 1281-1286) and soybean genistein (Carcinogenesis, 1999, 20, 1945-1952) exist naturally, but it is difficult to industrially use it as a drug because it is a minor component. . By the way, inositol and ferulic acid are widely present in edible natural products, and are industrially manufactured and sold. However, a compound in which inositol and ferulic acid are bound is not known. In addition, the compound consisting of inositol and ferulic acid is COX-2.
It has not yet been reported that it has an expression suppressing effect.

The object of the present invention is a food ingredient, and
It is to provide a highly safe drug effective for suppressing the expression of COX-2 by synthesizing a novel compound using inositol obtained industrially as a main raw material.

[0008]

In view of the above situation, the present inventors have developed a novel COX-2 consisting mainly of inositol.
As a result of earnest research to find a substance having an expression-suppressing effect of COX-2, the inventors have found that an inositol derivative having a predetermined characteristic structure has COX-2 expression-suppressing activity, and completed the present invention.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The COX-2 expression inhibitor of the present invention is characterized by using myo-inositol obtained from rice bran as a main raw material, and these are derived from other plants such as corn and wheat. The one obtained is also available.
When ferulic acid is also used, a lignin decomposition product or a recovered product from pulp waste liquid can be used.

The compound as an active ingredient in the COX-2 expression inhibitor of the present invention is represented by the following formula (I): myo-
It is necessary to have an inositol-1,3,5-orthoformate backbone.

[Chemical 8] (I) R1 in the formula (I) is the following formula (II), (III),
(IV) represents a substituent selected from any of the substituents represented by (IV), R2 in the formula (I) is H, or a substituent represented by the following formulas (III) and (IV): R3 in formula (I) represents a substituent selected from any of the substituents represented by the following formulas (III) or (IV), and Me in formula (II)
Represents a methyl group, and Ac in the following formula (IV) represents an acetyl group.

[Chemical 9] (II)

[Chemical 10] (III)

[Chemical 11] (IV)

In order to maintain the COX-2 inhibitory activity, ferulic acid is added to the hydroxyl groups at the 4th and 6th positions of myo-inositol converted to myo-inositol-1,3,5-orthoformate. Bound compounds are preferred. Further, those in which the hydroxyl group of bound ferulic acid remains as it is are suitable because the effect of suppressing COX-2 expression is remarkable. Specifically, 2-O- (t-butyldimethylsilyl) -4,6-di-O- (feruloyl) -myo-
Inositol-1,3,5-orthoformate (shown in formula (V) below),

[Chemical 12] (V) 2,4,6-tri-O-feruloyl-myo-inositol 1,3,5-orthoformate (the following formula (VI)
Shown in)

[Chemical 13] The structure represented by (VI) is preferable for maintaining the COX-2 inhibitory activity. The expression of COX-2 in the present invention means COX-2
It means the action of inhibiting any stage of the process of gene transcription and translation.

The COX-2 expression inhibitor of the present invention can be produced, for example, by the following method. It can be manufactured by applying a synthesis method.

In the present invention, myo-inositol is my
It is important to use a compound converted to o-inositol-1,3,5-orthoformate. Such compounds are known and are shown in formula (VII) below.
Bull. Chem. Soc. Jpn., 67, 1058-1063 (1994).

[Chemical 14] (VII)

Further, a compound represented by the following formula (VIII) is also known, J. Org. Chem., 50, 4402-4404 (1985).
It can be manufactured according to.

[Chemical 15] (VIII) Further, O-acetylferulic acid chloride represented by the following formula (IX) is

[Chemical 16] (IX) It can be produced from ferulic acid represented by the following formula (X) by a known method represented by the following reaction formula 1.

[Chemical 17] (X)

(Reaction formula 1)

[Chemical 18]

Then, the compounds of the present invention can be obtained according to the following reaction formulas 2, 3, 4, and 5. (Reaction formula 2)

[Chemical 19] In the reaction formula 2, the reaction from the left side to the middle side is described in detail in Example 3 described later, and the reaction from the middle side to the right side is described in detail in Example 4 described later.

(Reaction formula 3)

[Chemical 20] In this reaction formula 3, the reaction from the left side to the right side through the middle side is described in detail in Example 5 below.

(Reaction formula 4)

[Chemical 21] In the reaction formula 4, the reaction from the left side to the middle side is described in detail in Example 6 described later, and the reaction from the middle side to the right side is described in detail in Example 7 described later.

(Reaction formula 5)

[Chemical formula 22] The reaction of Reaction Scheme 5 is described in detail in Example 8 below.

The COX-2 expression inhibitor according to the present invention is COX-2.
It is useful in the prevention and treatment of diseases associated with abnormal expression of a. Specifically, it can be used for the prevention of general tumors that overexpress COX-2 such as colon cancer, or for alleviation of inflammatory symptoms. It has already been mentioned that COX-2 expression is enhanced in colorectal cancer (Cancer Res., 1
995, 55: 2556-2559, Cancer Res., 1995, 55: 3789).
It has been pointed out that suppression of COX-2 expression may prevent canceration. Therefore, the COX-2 expression inhibitor is useful as a chemopreventive agent for colorectal cancer. In addition to colorectal cancer, gastric cancer (Cancer Res., 1997, 57: 127
6-1280), pancreatic cancer (Cancer Res., 1999, 59: 4356-436).
Since COX-2 overexpression is reported in 2) or in colorectal adenomatosis, etc., the cancer preventive agent according to the present invention is also effective against these cancers. CO according to the invention
Since the expression inhibitor of X-2 controls the expression of COX-2,
Higher selectivity can be achieved than with activity inhibitors.
Since COX-2 and COX-1 are products of different genes, it is unlikely that a compound that regulates the expression of one is involved in the expression of the other. In an activity inhibitor, it is difficult to selectively inhibit different enzymes having similar activities.

In the present invention, the compounds found to have COX-2 expression-suppressing activity can be used alone or in combination of a plurality of compounds to prepare a pharmaceutical preparation by using a known formulation technique. That is, a pharmaceutical preparation can be obtained by mixing these compounds with a pharmaceutically acceptable excipient. The compound of the present invention can be administered to animals and humans as it is or together with a conventional pharmaceutical preparation alone. The dosage form is not particularly limited and may be appropriately selected and used as necessary. Specific examples thereof include oral agents such as tablets, capsules, granules, fine granules and powders, and parenteral agents such as injections and suppositories.

The oral preparation is produced by a conventional method using an excipient such as starch, lactose, sucrose, mannitol, carboxymethyl cellulose, corn starch, inorganic salts and the like. In addition to the above-mentioned excipients, binders, disintegrants, surfactants, lubricants, fluidity promoters, flavoring agents, coloring agents, fragrances, etc. can be appropriately used in this type of formulation. . Also,
The compound of the present invention can also be administered as a suspension, emulsion, syrup, or elixir, and these various dosage forms may contain a flavoring agent and a coloring agent.

Parenteral agents are manufactured according to a conventional method, and are generally used as diluents such as distilled water for injection, physiological saline, glucose solution, vegetable oil for injection, sesame oil, peanut oil, soybean oil,
Corn oil, propylene glycol, polyethylene glycol and the like can be used. Furthermore, if necessary, a bactericide, a preservative, and a stabilizer may be added. Other parenteral agents include external preparations, coating agents such as ointments, suppositories for rectal administration, and the like, which are also manufactured according to a conventional method.

The content of the active ingredient in the COX-2 expression inhibitor according to the present invention can be appropriately adjusted so that the required dose can be given according to the selected administration route. Specifically, the usual dose can be 0.01 μg to 1 mg, preferably 0.1 μg to 0.1 mg, and more preferably 0.1 μg to 0.01 mg per kg body weight. The final dose depends on the patient's weight, age, sex,
Then, it is appropriately adjusted in consideration of the symptoms and the like.

[0025]

The present invention will be described in more detail with reference to the following examples, but the technical scope of the present invention is not limited to these examples.

[Example 1] "Production of myo-inositol-1,3,5-orthoformate (shown by the above-mentioned formula (VII)) myo-inositol (22 g, 0.12 mol) was added to DMF (200 mL). ), 36 mL (0.22 mol) of triethyl orthoformate and 6 g of p-toluenesulfonic acid monohydrate were added, and the mixture was added under a nitrogen atmosphere to 12
The mixture was stirred at 0 to 130 ° C for 6 hours. During this time, the produced ethanol was removed from the system by a Dean Stark trap. After allowing to cool to room temperature, 40 mL of 10% NaHCO ₃ aqueous solution was added and stirred for 30 minutes. The formed precipitate was filtered off, and the filtrate was concentrated under reduced pressure and dried. The dried solid was recrystallized from methanol-chloroform to obtain known myo-inositol-1,3,5-orthoformate (15.2 g, 69%) as colorless needle crystals. Melting point 282-285 ° C. ¹ H-NMR (400MHz, DMSO- d ₆ ) δ 3.
94 (m, 2H, H1 & H3), 3.99 (d, 1H, J = 6.4 Hz, H-2), 4.
05 (m, 1H, H-5), 4.26 (m, 2H, H4 & H6), 5.30 (d, 1H,
J = 6.4Hz, OH), 5.40-5.50 (m, 3H, CH & OH).

Example 2 "2-Ot-butyldimethylsilyl-myo-inositol-1,3,5-orthoformate (the above-mentioned formula (VI
II)) ”, myo-inositol-1,3,5-orthoformate obtained in Example 1 (6.39 g, 33.6 mmo)
l) was dissolved in 60 mL DMF and the solution was kept at 0-5 ° C.
To this solution was added imidazole (2.97g, 43.7mmol) and t-
Butyldimethylsilyl chloride (6.08 g, 40.3 mmol) was added, and the mixture was stirred for 4 hours. After that, the temperature was slowly returned to room temperature, and the mixture was stirred at room temperature for 20 hours. After stopping the reaction by adding methanol, add 150 mL of ethyl acetate and transfer to a separating funnel.
It was washed with saturated KHSO ₄ , water, saturated NaHCO ₃ , and saturated saline in this order. After drying over MgSO ₄ , the solvent was distilled off and the residue was purified by column chromatography. The purified product was recrystallized from methanol to obtain the known 2-O-t-butyldimethylsilyl-myo-inositol-1,3,5-orthoformate (7.08 g, 69%) as colorless needle crystals. Melting point 179-181 ° C; ¹ H-NMR (400MHz, CDCl ₃ ) δ 0.14 (s, 6H)
, 0.93 (s, 9H), 4.13 (m, 2H), 4.24 (m, 2H), 4.
56 (m, 2H), 5.48 (d, 1H, J = 1.6 Hz).

[Example 3] "2-Ot-butyldimethylsilyl-4,6-di-O"
-[3- (4'-acetyloxy-3'-methoxyphenyl) -2-propenoyl] -myo-inositol-
1,3,5-orthoformate "2-O-obtained in Example 2
t-butyldimethylsilyl-myo-inositol-1,
3,5-Orthoformate (1.20 g, 3.94 mmol) was dissolved in 50 mL of anhydrous pyridine, and 4-dimethylaminopyridine (catalytic amount) and O-acetylferulic acid chloride (3.01 g,
11.83 mmol) was added and the mixture was stirred at room temperature overnight. After water was added to stop the reaction, 80 mL of ethyl acetate was added to the reaction solution, the mixture was transferred to a separating funnel, and washed with saturated KHSO ₄ , saturated NaHCO ₃ , and saturated saline in this order. After drying over anhydrous Na ₂ SO ₄ , the solvent was distilled off, and the residue was fractionated by column chromatography to obtain a reaction product. The reaction product was ethyl acetate / n-
The crystals were recrystallized from hexane and purified to give colorless needle crystals. The reaction product after recrystallization had a melting point of 148-150 ° C and a yield of 88% (yield 2.
It was 58 g). Then, when the reaction product after recrystallization was subjected to instrumental analysis, the following results were obtained. IR (KBr) ν 3119, 3065, 2958, 2858, 1763, 1716, 16
35, 1601, 1508, 1419, 1371, 1163, 1032, 1006, 987,
895 cm ^-1 ; ¹ H NMR (400MHz, CDCl ₃ ) δ 7.60 (d, 1H, J = 16.0 Hz,
CH =), 6.78-6.93 (m, 6H, aromatic), 6.22 (d, 1H, J = 1
6.0 Hz, CH =), 5.62 (d, 1H, J = 1.2 Hz, CH), 5.59 (t,
2H, J = 4.0 Hz, H-4 & 6), 4.76 (m, 1H, H-2), 4.29 (m,
3H, H-1, 3 & 5), 3.69 (s, 6H, OCH ₃ ), 2.30 (s, 6H, ac
etyl), 0.95 (s, 9H, CH ₃ ), 0.16 (s, 6H, SiCH ₃ ) ppm; ¹³ C NMR (CDCl ₃ ) δ 168.5, 164.8, 151.5, 145.7, 14
2.0, 132.3, 123.6, 120.7, 116.6, 111.5, 103.1, 71.
9, 68.6, 66.3, 61.7, 55.8, 26.0, 20.6, 18.6, -4.6
ppm; Anal. Calcd for C ₃₇ H ₄₄ O ₁₄ Si: C, 59.99; H, 5.9
9; Found: C, 59.96; H, 5.98.

From the above measurement results, the reaction product obtained in Example 3 was a novel inositol derivative (2-Ot-butyldimethylsilyl-4,6-di-O- [3- (4'-acetyl). Oxy-3′-methoxyphenyl) -2-propenoyl] -myo-inositol-1,3,5-orthoformate).

[Embodiment 4] "2-Ot-butyldimethylsilyl-4,6-di-O
-[3- (4'-hydroxy-3'-methoxypheny
Le) -2-propenoyl] -myo-inositol-1,
3,5-Orthoformate "2-Ot-butyl dimethyl
Rusilyl-4,6-di-O- [3- (4'-acetylo
Xy-3'-methoxyphenyl) -2-propenoyl]
-Myo-inositol-1,3,5-orthoformate
Chloroform / methanol mixture of (0.30g, 0.41 mmol)
Dissolve in solvent (7mL / 3mL), NH₂NH₂・ H₂O (44.6mg, 0.891m
mol) was added and stirred at room temperature for 3 hours for reaction. Reaction liquid
Was transferred to a separatory funnel and washed with saturated brine, and MgSO_FourTo dry
After drying, the solvent was distilled off. Recrystallize the residue with ethanol
Purification gave a white solid. The reaction product after recrystallization has a melting point of 21.
The yield was 92% (0.253 g) at 0-212 ° C. And
By subjecting the reaction product after recrystallization to instrumental analysis, the following results were obtained.
The fruit was obtained. IR (KBr) ν 3450, 3072, 2949, 2856, 1715, 1630, 15
16, 1466, 1431, 1250, 1165, 1000, 955, 881, 818 cm
^-1;¹ H NMR (400MHz, CDCl₃) δ 7.54 (d, 1H, J = 16.0 Hz,
CH =), 6.69-6.79 (m, 6H, aromatic), 6.09 (d, 1H, J = 1
6.0 Hz, CH =), 5.87 (s, 2H, OH), 5.62 (d, 1H, J = 1.2
Hz, CH), 5.57 (t, 2H, J = 4.0 Hz, H-4 & 6), 4.76 (m, 1
H, H-2), 4.35 (m, 1H, H-5), 4.29 (m, 2H, H-1 & 3),
3.67 (s, 6H, OCH₃), 0.96 (s, 9H, CH ₃), 0.18 (s, 6
H, SiCH₃) ppm;¹³ C NMR (CDCl₃) δ 165.3, 148.4, 146.7, 146.3, 12
6.2, 122.8, 114.7, 113.9, 109.5, 103.1, 72.0, 68.
4, 66.3, 61.8, 55.6, 26.0, 18.6, -4.6 ppm. Anal. C
alcd for C₃₃H₄₀O₁₂Si: C, 60.35; H, 6.14; Found: C,
 60.28; H, 6.10.

From the above measurement results, the reaction product obtained in Example 4 was a novel inositol derivative (2-Ot-butyldimethylsilyl-4,6-di-O- [3- (4'-hydroxy). -3'-Methoxyphenyl) -2-propenoyl] -myo-inositol-1,3,5-orthoformate)).

Example 5 “2-Ot-butyldimethylsilyl-4-O- [3-
(4'-Hydroxy-3'-methoxyphenyl) -2-
Propenoyl] -myo-inositol-1,3,5-orthoformate ”2-Ot-butyldimethylsilyl-m
yo-inositol-1,3,5-orthoformate (0.5
g, 1.6 mmol) was dissolved in 50 mL of anhydrous pyridine, 4-dimethylaminopyridine (catalytic amount) and O-acetylferulic acid chloride (0.63 g, 2.46 mmol) were added, and the mixture was stirred at room temperature overnight. After adding water to stop the reaction, add 80 mL of ethyl acetate to the reaction mixture, transfer to a separating funnel, and saturate KHSO ₄ ,
It was washed with saturated NaHCO ₃ and saturated saline in this order. Anhydrous Na
After drying over ₂ SO ₄ , the solvent was distilled off and the residue was purified by column chromatography. The obtained 2-Ot-butyldimethylsilyl-4-O- [3- (4'-acetyloxy-3'-methoxyphenyl) -2-propenoyl] -myo-inositol (81.7 mg, 0.156 mmol) was added. It was dissolved in a mixed solvent of chloroform / methanol (3 mL / 2 mL), NH ₂ NH ₂ · H ₂ O (10 μL, 0.2 mmol) was added, and the mixture was stirred at room temperature overnight. Chloroform (15 mL) was added to the reaction solution, and the mixture was transferred to a separating funnel, washed with saturated brine and dried over MgSO ₄ , and the solvent was evaporated. The residue was recrystallized from ethyl acetate / n-hexane for purification. The purified reaction product had a melting point of 154-156 ° C and a yield of 88% (amount 66 mg). When this reaction product was subjected to instrumental analysis, the following results were obtained. IR (KBr) ν 3450, 3300, 2968, 2932, 2858, 1668, 16
08, 1514, 1435, 1267, 1161, 1062, 1010, 976, 947,
851 cm ^-1 ; ¹ H NMR (400MHz, CDCl ₃ ) δ 7.62 (d, 1H, J = 16.0 Hz,
CH =), 6.89-7.07 (m, 3H, aromatic), 6.18 (d, 1H, J = 1
6.0 Hz, CH =), 5.98 (s, 1H, OH), 5.62 (m, 1H, H-4),
5.55 (d, 1H, J = 1.2 Hz, CH), 4.57 (m, 1H), 4.41 (m,
1H), 4.24 (m, 2H), 4.16 (m, 1H), 3.92 (s, 3H), 2.
54 (d, 1H, J = 7.2 Hz, OH), 0.92 (s, 9H, CH ₃ ), 0.13
(s, 6H, SiCH ₃ ) ppm; ¹³ C NMR (CDCl ₃ ) δ 165.2, 148.6, 147.2, 146.8, 12
6.3, 123.6, 114.8, 113.1, 109.5, 102.8, 74.5, 72.
1, 69.1, 68.3, 68.0 61.0, 55.9 25.9, 18.4, -4.70 p
pm; Anal. Calcd for: C ₂₃ H ₃₂ O ₉ Si: C, 57.48; H, 6.7
1; Found: C, 57.55; H, 6.77.

From the above measurement results, the reaction product obtained in Example 5 was a novel inositol derivative (2-Ot-butyldimethylsilyl-4-O- [3- (4'-hydroxy-3'- Methoxyphenyl) -2-propenoyl] -m
yo-inositol-1,3,5-orthoformate)).

[Example 6] "2,4,6-tri-O- [3- (4'-acetyloxy-3'-methoxyphenyl) -2-propenoyl]-"
myo-inositol-1,3,5-orthoformate "
From the myo-inositol-1,3,5-orthoformate obtained in Example 1, a reaction product having a pale yellow solid and a melting point of 175 to 177 ° C. was obtained in the same manner as in Example 3 (yield 67%, yield 2.
25 g) was obtained. When this reaction product is subjected to instrumental analysis,
The following results were obtained. IR (KBr) ν 3070, 3010, 2939, 2841, 1766, 1720, 16
36, 1601, 1508, 1419, 1369, 1259, 1155, 1003, 949,
903, 833 cm ^-1 ; ¹ H NMR (400MHz, CDCl ₃ ) δ 7.77 (d, 1H, J = 16.0 Hz,
CH =), 7.67 (d, 2H, J = 16.0 Hz, CH =), 6.87-7.27 (m,
9H, aromatic), 6.58 (d, 1H, J = 16.0 Hz, CH =), 6.33
(d, 2H, J = 16.0 Hz, CH =), 5.71 (d, 1H, J = 1.2 Hz, C
H), 5.68 (t, 2H, J = 4.0 Hz, H-4 & 6), 5.54 (m, 1H, H-
2), 4.89 (m, 1H, H-5), 4.56 (m, 2H, H-1 & 3), 3.88
(s, 3H, OCH ₃ ), 3.71 (s, 6H, OCH ₃ ), 2.33 (s, 3H, ac
etyl), 2.32 (s, 6H, acetyl) ppm; ¹³ C NMR (CDCl ₃ ) δ 168.7, 168.5, 166.2, 165.0, 15
1.5, 151.4, 146.1, 145.8, 142.0, 141.8, 132.9, 13
2.3, 123.5, 123.3, 121.7, 121.2, 117.2, 116.4, 11
1.3, 111.1, 103.2, 69.4, 67.9, 66.4, 63.4, 55.9, 5
5.8, 20.6 ppm; Anal.Calcd for C ₄₃ H ₄₀ O ₁₈ : C, 61.14;
H, 4.77; Found: C, 61.03; H, 4.73.

From the above measurement results, the reaction product obtained in Example 6 was a novel inositol derivative (2,4,6-tri-O- [3- (4'-acetyloxy-3'-methoxyphenyl)]. 2-propenoyl] -myo-inositol-1,3,5-orthoformate)).

Example 7 "2,4,6-tri-O- [3- (4'-hydroxy-
3'-methoxyphenyl) -2-propenoyl] -my
o-inositol-1,3,5-orthoformate "2,4,6-tri-O- [3- (4'-acetyloxy-3'-methoxyphenyl) -2-propenoyl obtained in Example 6 ] -Myo-inositol-1,3,5-orthoformate was used to obtain a reaction product (yield 50%, yield 0.36 g) having a melting point of 225-227 ° C as a pale yellow solid in the same manner as in Example 4. Obtained. When this reaction product was subjected to instrumental analysis, the following results were obtained. IR (KBr) ν 3402, 3067, 3010, 2938, 2842, 1715, 16
32, 1593, 1516, 1431, 1375, 1269, 1161, 1030, 893,
847, 820 cm ^-1 ; ¹ H NMR (400MHz, CDCl ₃ ) δ 7.72 (d, 1H, J = 16.0 Hz,
CH =), 7.58 (d, 2H, J = 16.0 Hz, CH =), 6.69-7.10 (m,
9H, aromatic), 6.46 (d, 1H, J = 16.0 Hz, CH =), 6.15
(d, 2H, J = 16.0 Hz, CH =), 5.90-5.95 (m, 3H, OH), 5.
68 (d, 1H, J = 1.2 Hz, CH), 5.63 (t, 2H, J = 4.0 Hz, H
-4 & 6), 5.54 (m, 1H, H-2), 4.88 (m, 1H, H-5), 5.53
(m, 2H, H-1 & 3), 3.91 (s, 3H, OCH ₃ ), 3.65 (s, 6H, OC
H ₃ ) ppm; ¹³ C NMR (CDCl ₃ ) δ 166.7, 165.5, 148.4, 146.8, 14
6.7, 146.6, 146.5, 126.6, 126.2, 123.8, 123.2, 11
4.7, 114.5, 114.3, 113.8, 109.1, 103.2, 69.5, 67.
8, 66.3, 63.3, 55.9, 55.6 ppm; Anal. Calcd for C ₃₇
H ₃₄ O ₁₅ : C, 61.84; H, 4.77; Found: C, 61.40; H, 4.71.

From the above measurement results, the reaction product obtained in Example 7 was a novel inositol derivative (2,4,6-tri-O- [3- (4'-hydroxy-3'-methoxyphenyl)- 2-propenoyl] -myo-inositol-
1,3,5-orthoformate).

Example 8 [4,6-di-O- [3- (4'-hydroxy-3'-
Methoxyphenyl) -2-propenoyl] -myo-inositol-1,3,5-orthoformate ester "2-Ot
-Butyldimethylsilyl-4,6-di-O- [3- (4
'- hydroxy-3'-methoxyphenyl) -2-propenoyl]-myo-inositol-1,3,5-orthoformate (70 mg, 0.1 mmol) was dissolved in tetrahydrofuran _{10mL, n-Bu 4 NF ·} 3H ₂ O (0.11g, 0.35 mmol) was added to 0
Stir at ~ 5 ° C for 5 hours and room temperature overnight. After adding 0.1 mL of acetic acid and 30 mL of ethyl acetate, transfer to a separating funnel and saturate.
It was washed with KHSO ₄ and saturated saline in this order. Anhydrous Na ₂ SO ₄
After drying with, the solvent was distilled off, and the residue was recrystallized from tetrahydrofuran / methanol to obtain a white solid. The reaction product after recrystallization had a melting point of 248-250 ° C and a yield of 98% (yield 56 mg).
Met. When the reaction product after recrystallization was subjected to instrumental analysis, the following results were obtained. IR (KBr) ν 3481, 3340, 3069, 2958, 2843, 1728, 17
09, 1634, 1601, 1514, 1433, 1281, 1192, 991, 953, 8
91 cm ^-1 ; ¹ H NMR (400MHz, THF-d ₈ ) δ 8.39 (s, 2H, OH), 7.62
(d, 2H, J = 16.0 Hz, CH =), 6.60-7.05 (m, 6H, aromati
c), 6.25 (d, 2H, J = 16.0 Hz, CH =), 5.53 (d, 1H, J = 1.
2 Hz, CH), 5.50 (t, 2H, J = 4.0 Hz, H-4 & 6), 4.97 (d,
1H, J = 10.4 Hz, OH), 4.65 (m, 1H, H-5), 4.21 (m, 2
H, H-1 & 3), 4.07 (m, 1H, H-2), 3.68 (s, 6H, OCH ₃ ) pp
m; ¹³ C NMR (THF-d ₈ ) δ 163.4, 148.2, 146.1, 144.6, 12
4.1, 121.1, 113.6, 112.1, 111.6, 108.8, 101.7, 70.
6, 66.5, 58.9, 53.3, ppm; Anal. Calcd for: C ₂₇ H ₂₆ O
₁₂ C, 59.78; H, 4.83; Found: C, 59.66; H, 4.85.

From the above measurement results, the reaction product obtained in Example 8 is a novel inositol derivative (4,6-di-O-
[3- (4'-hydroxy-3'-methoxyphenyl)
-2-Propenoyl] -myo-inositol-1,3,5-
Orthoformate ester).

CO of inositol derivative obtained according to the present invention
The X-2 expression inhibitory activity was confirmed by a screening system using the COX-2 promoter transcription inhibitory activity in human colon cancer cells as an index. For example, it was confirmed by the screening system described below.

[Measurement of COX-2 Promoter Transcriptional Activity] A reporter gene assay system developed by Fukuda et al. Was used for 7 kinds of myo-inositol derivatives (Examples 2 to 8) obtained in the above examples. (J. Ethnopharm
acol. 1999 66 (2): 227-33) was used to examine the inhibitory effect on the COX-2 promoter transcription activity in human colon cancer cells.

As a comparative example, ferulic acid, the following formula (X
With respect to the compound represented by I) and the compound represented by the following formula (XII), the inhibitory effect on the above COX-2 promoter transcription activity was also examined. The formulas (XI), (XII)
The compound represented by is used as it is without converting myo-inositol to myo-inositol-1,3,5-orthoformate and adding ferulic acid thereto.

[Chemical formula 23] (XI)

[Chemical formula 24] (XII)

In the cell culture method, human colon adenocarcinoma cells
(human colon adenocarcinoma cell line) of DLD-1
Transformed cells with the LacZ gene derived from Escherichia coli introduced downstream of the COX-2 promoter region DLD-1 / COX-2 / B-2-β-Gal-BSD
Was created by Fukuda et al. (J. Ethnopharmacol. 199
9 66 (2): 227-33). Cells were maintained in RPMI1640 medium supplemented with 5% heat-inactivated fetal bovine serum (GIBCO BRL, Grand Island, NY) at 37 ° C. in an atmosphere of 5% CO ₂ . 20000 cells per well were seeded on a 96-well tissue culture plate and precultured for 24 hours. Then, the cells were treated with the test compounds of Examples 2 to 8 and Comparative Example 3, respectively.

[Measurement method of live cells (MTT assay)] When administration of a test compound impairs cell growth, whether expression of only target COX-2 is suppressed, or Careful consideration must be given to whether the overall function of protein synthesis in the cell is reduced.
Therefore, the cell viability of each culture medium was measured using 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) assay (Mosmann, J. Immunol. Methods.,
65, 1983: 55-63). After treatment with the test compounds, the cells were further incubated for 1 hour in medium containing 0.5 m / mL MTT. MTT formazan, which is produced by the action of the respiratory enzyme system of living cells, is added to dimethyl sulfoxide.
Dissolve with (DMSO), Microplate reader (TECAN)
The absorbance at 570 nm was measured at 630 nm as a control wavelength. For each cell, the cell viability of the cells not treated with the test compound was defined as 100%.

[COX-2 by the reporter gene assay system]
[Measurement of Promoter Transcriptional Activity] The COX-2 promoter transcriptional activity was measured by the method described below using the β-galactosidase gene as a reporter gene, not the COX-2 activity or the COX-2 protein itself. 96 cells
20000 cells per well in well tissue culture plates
The seeds were sown in such a manner as to become individual, and precultured for 24 hours. Next, the cells were treated with a test compound, and the total β-galactosidase activity of DLD-1 cells in each well was measured using o-nitrophenyl-β-D.
-Measured by a colorimetric method using galactopyranoside (ONPG). The standard β-galactosidase activity of DLD-1 cells is shown by DLD-1 / B2 untreated with a test compound as a control.
It was measured with -β-Gal-BSD (cells transformed with a plasmid containing no COX-2 promoter region), and the value was set to 0. In addition, DLD-1 / COX-2 / B2-β-Ga without the test compound
The β-galactosidase activity of l-BSD was set as 100%. The percentage of β-galactosidase activity when treated with each test compound was calculated from three wells. The β-galactosidase activity value was corrected by the cell viability evaluated by the MTT assay. The final maximum dose was 100 μM, all experiments were performed in triplicate, and the cell proliferation rate (cytotoxicity) and β-galactosidase activity were determined as mean ± SD.

“By a novel myo-inositol derivative
Inhibition of COX-2 Promoter Transcriptional Activity "The results of examining the inhibitory effect on COX-2 promoter transcriptional activity of the novel myo-inositol derivative synthesized in the above Example at a dose concentration of 100 μM are shown in FIGS. 1 to 8. Show. 2-O-t
-Butyldimethylsilyl-4,6-di-O- [3- (4
'-Acetyloxy-3'-methoxyphenyl) -2-
Propenoyl] -myo-inositol-1,3,5-orthoformate (FIG. 2, Formula (V) described above, Reaction Formula 2, Example 3), 2-Ot-butyldimethylsilyl-4,6. −
Di-O- [3- (4'-hydroxy-3'-methoxyphenyl) -2-propenoyl] -myo-inositol-1,3,5-orthoformate (Fig. 3, reaction formula 2 already described, implementation. Example 4), 2-Ot-butyldimethylsilyl-
4-O- [3- (4'-Hydroxy-3'-methoxyphenyl) -2-propenoyl] -myo-inositol-1,3,5-orthoformate (Fig. 4, reaction formula 3 described above, implementation. Example 5), 2,4,6-tri-O- [3- (4
'-Acetyloxy-3'-methoxyphenyl) -2-
Propenoyl] -myo-inositol-1,3,5-orthoformate (FIG. 5, reaction formula 4 described above, Example 6),
2,4,6-tri-O- [3- (4'-hydroxy-
3'-methoxyphenyl) -2-propenoyl] -my
o-inositol-1,3,5-orthoformate (FIG. 6, formula (VI) described above, reaction formula 4, Example 7), 4, 6
-Di-O- [3- (4'-hydroxy-3'-methoxyphenyl) -2-propenoyl] -myo-inositol-1,3,5-orthoformate (Fig. 7, reaction formula 5 described above, Regarding Example 8), the effect of suppressing the COX-2 promoter transcription activity was newly found.

As is apparent from FIGS. 2 to 7, 2-O-t
-Butyldimethylsilyl-4,6-di-O- [3- (4
'-Hydroxy-3'-methoxyphenyl) -2-propenoyl] -myo-inositol-1,3,5-orthoformate (Fig. 3, previously described formula (V)) shows the strongest inhibitory effect, Concentration-dependent in the concentration range of 5 μM to 100 μM
It inhibited the transcriptional activity of COX-2 promoter, and the inhibition rate at 100 μM was about 60%. On the other hand, the inhibition rate of cell proliferation at this time was as small as about 30%, which was preferable. Also, 2,
4,6-Tri-O- [3- (4'-hydroxy-3'-
Methoxyphenyl) -2-propenoyl] -myo-inositol-1,3,5-orthoformate (FIG. 6, previously described formula (VI)) has the next strongest inhibitory effect after the inositol derivative of formula (V). The inhibition rate at 100 μM was about 70%. The inhibition rate of cell proliferation at this time was also small, about 30%. The inositol derivatives obtained in Examples 3, 5, 6, and 8 are also less inferior to the inositol derivatives of the formulas (V) and (VI), but have a corresponding inhibitory effect as shown in FIGS. The cell growth inhibition rate was low (not shown).

On the other hand, 2-O-t-butyldimethylsilyl-myo-inositol-1,3,5-orthoformate of Example 2 (FIG. 1) or ferulic acid itself as a comparative example (FIG. 8), myo-inositol-1,
The above-mentioned formula (X having no 3,5-orthoformate structure
Regarding the compounds of formula I) and formula (XII), the COX-2 promoter transcription activity was hardly decreased and the suppressive effect was not observed even when the dose concentration was increased.

[0049]

INDUSTRIAL APPLICABILITY The present invention provides a novel inositol derivative. All of the inositol derivatives provided by the present invention have low toxicity and are useful for future drug discovery, and can be particularly suitably used as a cyclooxygenase-2 expression inhibitor. The cyclooxygenase-2 expression inhibitor of the present invention can be expected as a drug with excellent selectivity, and can be applied to a preventive agent for tumors that overexpress cyclooxygenase-2. That is, since it acts on the expression of cyclooxygenase-2 encoded by a gene different from cyclooxygenase-1, it theoretically has no effect on the activity of cyclooxygenase-1 and is accompanied by side effects such as gastrointestinal disorders. Can be no drug.

[Brief description of drawings]

FIG. 1 is a graph showing the COX-2 promoter transcription activity with respect to the administration concentration of 2-Ot-butyldimethylsilyl-myo-inositol-1,3,5-orthoformate obtained according to Example 2 of the present invention. FIG.

FIG. 2 shows 2-Ot-butyldimethylsilyl-4,6-di-O- [3- (4′-acetyloxy-3′-methoxyphenyl) -2-obtained according to Example 3 of the present invention. FIG. 3 is a graph showing the COX-2 promoter transcription activity with respect to the administration concentration of propenoyl] -myo-inositol-1,3,5-orthoformate.

FIG. 3 shows 2-Ot-butyldimethylsilyl-4,6-di-O- [3- (4′-hydroxy-3′-methoxyphenyl) -2-propenoyl obtained according to Example 4 of the present invention. ] -Myo-inositol-1,3,5-orthoformate ester administration concentration versus COX-2 promoter transcriptional graph.

FIG. 4 shows 2-Ot-butyldimethylsilyl-4-O- [3- (4′-hydroxy-3′-methoxyphenyl) -2-propenoyl] -m obtained according to Example 5 of the present invention.
FIG. 3 is a graph showing the COX-2 promoter transcription activity with respect to the administration concentration of yo-inositol-1,3,5-orthoformate.

FIG. 5 2,4,6-Tri-O- [3- (4′-acetyloxy-3′-methoxyphenyl) -2-propenoyl] -myo-inositol-1 obtained according to Example 6 of the present invention. COX vs. Dose Concentration of 3,3,5-Orthoformate
FIG. 2 is a graph showing the transcription activity of the -2 promoter.

FIG. 6: 2,4,6-tri-O- [3- (4′-hydroxy-3′-methoxyphenyl) -2-propenoyl] -myo-inositol-obtained according to Example 7 of the present invention.
It is a figure of a graph showing COX-2 promoter transcription activity with respect to the administration concentration of 1,3,5-orthoformate.

FIG. 7 4,6-di-O-obtained according to Example 8 of the present invention
[3- (4'-hydroxy-3'-methoxyphenyl)
-2-Propenoyl] -myo-inositol-1,3,5-
FIG. 3 is a graph showing the COX-2 promoter transcription activity with respect to the administration concentration of orthoformate.

FIG. 8 is a graph showing the COX-2 promoter transcription activity against the administration concentration of ferulic acid used as a raw material in the present invention.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Asao Hosoda             92-3 Gobu City, Wakayama Prefecture (72) Inventor Yoshihiko Ozaki             1501-2 Tsubo, Shimotsu-cho, Kaiku-gun, Wakayama Prefecture (72) Inventor Kuji Taniguchi             170 Nakoso, Koyaguchi-cho, Ito-gun, Wakayama Prefecture (72) Inventor Nakayo Michiyo             242-7 Kano, Wakayama City, Wakayama Prefecture (72) Inventor Shigeaki Ikemoto             158 Nogamichu, Kainan City, Wakayama Prefecture (72) Inventor Himeko Yamanishi             17 Fujishiro, Hainan City, Wakayama Prefecture (72) Inventor Keiji Wakabayashi             2-5-28 Higashigaoka, Meguro-ku, Tokyo National cancer             Center Dormitory RG-401 (72) Inventor Norihiro Muto             2-34-8 Umezono, Tsukuba City, Ibaraki Prefecture (72) Inventor Ayumu Kashida             74-5 Nakajima, Wakayama Leopalace Alice             No. 202 F term (reference) 4C071 AA03 AA07 BB02 BB05 CC13                       EE06 FF16 HH05 LL01                 4C086 AA01 AA02 AA03 CA01 MA01                       MA04 ZB11 ZB26 ZC20

Claims (5)

[Claims] 1. An inositol derivative represented by the following formula (I): [Chemical 1] (I) R1 in the formula (I) is represented by the following formulas (II) and (II
I) and (IV) each represent a substituent selected from the substituents, and R2 in the formula (I) is represented by H or the following formulas (III) and (IV). Represents a substituent selected from any of the substituents, and R3 in the formula (I) represents a substituent selected from any of the substituents represented by the following formulas (III) or (IV). Me in the following formula (II) represents a methyl group, and Ac in the following formula (IV) represents an acetyl group. [Chemical 2] (II) (III) embedded image (IV) 2. An inositol derivative represented by the following formula (V): [Chemical 5] (V) 3. An inositol derivative represented by the following formula (VI): [Chemical 6] (VI) 4. A myo-represented by the following formula (VII):
Reacting an inositol derivative with an orthoformate ester to obtain myo-inositol orthoformate ester;
A method for producing an inositol derivative, which comprises reacting o-inositol orthoformate with ferulic acid to obtain the inositol derivative according to any one of claims 1 to 3. [Chemical 7] (VII) 5. A cyclooxygenase-2 expression inhibitor containing the inositol derivative according to any one of claims 1 to 3 as an active ingredient.