JP4206070B2 - Cell adhesion inhibitor - Google Patents

Description

  The present invention relates to a cell adhesion inhibitor useful as an anti-inflammatory agent or the like.

  As anti-inflammatory agents, steroid agents, chemical transmitter production / release inhibitors typified by arachidonic acid metabolites and histamine, receptor antagonists and the like have been widely used. As immunosuppressants, antimetabolites such as azathioprine and mizoribine, steroids such as prednisolone, various antibodies, cyclosporine, FK506 and the like are used. There is no substance effective as a cancer metastasis inhibitor. Thus, until now, no clear relevance has been recognized for these anti-inflammatory agents, immunosuppressive agents, and cancer metastasis inhibitor.

  However, recent studies on various inflammations, immune responses, and cancer metastasis have progressed, and it has been reported that cell-cell adhesion is greatly involved in these diseases (Non-patent Documents 1 to 8). It has also been found that cell surface adhesion molecules such as ICAM-1, ELAM-1, and VCAM-1 are involved in adhesion between cells (Non-Patent Documents 1 to 10).

Known substances that suppress cell adhesion include antibodies and ligands to cell surface adhesion molecules, N- (fluorenyl-9-methoxycarbonyl) amino acids, 3-deazaadenosine, and the like (Non-patent Document 11). -14), its activity is not yet satisfactory, and there are drawbacks such as safety problems.
"Adhesion molecule expression regulation and clinical application" (1991, Medical View) Nature, Vol. 364, 149-151 (1993) Science, Vol. 247, 456-459 (1990) Annual Review Immunity 1989, 175-185 Trends in Glycoscience and Glycotechnology, Vol. 4, no. 19,405-414 (1992) Experimental Medicine Vol. 10, no. 11, 1402-1413 (1992) Experimental Medicine Vol. 11, no. 16, 2168-2175 (1993) Science, Vol. 255, 1125 to 1127 (1992) Annual Review Immunity 1989, 175-185, Infection, inflammation, immunity Vol. 19 (2), 129-153 (1989) Proc. Natl. Acad. Sci. USA, Vol. 88, 355-359 (1991) Immunopharmacology, 23 (1992) 139-149. Journal of Biological Chemistry, Vol. 267, no. 13, 9376-9382 (1992) Journal of Immunology, Vol. 144, no. 2,653-661 (1990)

  Accordingly, an object of the present invention is to provide an excellent anti-inflammatory agent that is highly safe and based on inhibition of cell adhesion.

  Therefore, the present inventor conducted not only the cell adhesion inhibitory action but also various inflammatory models, immune reaction models, cancer cell metastasis models and safety tests on many compounds. As a result, the following general formula (1) It was found that the benzoyl anilides represented have excellent cell adhesion inhibitory action, anti-inflammatory action, immunosuppressive action, and cancer metastasis inhibitory action, and are also highly safe.

  Of these, the present invention is represented by the general formula (1):

[Wherein, R ¹ represents a hydrogen atom or a t-butyl group, and R ² represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms]
The cell adhesion inhibitor and the anti-inflammatory agent which use the benzoic acid anilide represented by these, or its salt as an active ingredient are provided.

  Benzoic acid anilides (1) or salts thereof have excellent cell adhesion inhibitory action, anti-inflammatory action, and high safety. Therefore, various inflammatory diseases, bronchial asthma, rheumatism, atopic skin It is useful for the treatment and prevention of inflammation, hay fever, psoriasis, ischemia-reperfusion injury suppression, nephritis, encephalomyelitis, acute respiratory distress syndrome and the like.

In the general formula (1), R ² is a hydrogen atom, methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, t-butyl group, n-pentyl. Group, isopentyl group and the like, among which hydrogen atom is particularly preferable.

  The benzoic acid anilides (1) may be stabilized in the form of a hydrate, a solvate, or a salt depending on the production means or the like, and any of these can be used in the present invention. Examples of the salt include alkali metal salts such as sodium and potassium, alkaline earth metal salts, and amine salts.

  Benzoic acid anilides (1) can be produced, for example, according to the following reaction formula.

[Wherein R ³ represents an acyl group or a hydrogen atom, and R ¹ and R ² are the same as described above]

  That is, the benzoic acid anilide (1) is produced by reacting the benzoic acid (2) or a reactive derivative thereof with the aniline (3) and removing the hydroxy protecting group as necessary.

Examples of the reactive derivatives of benzoic acids include acid halides and acid anhydrides. These reactive derivatives can be subjected to a condensation reaction without isolation. The protecting group represented by R ³ is preferably an acetyl group.

  In the condensation reaction, when the benzoic acid (2) is reacted as it is, a condensing agent such as N, N'-dicyclohexylcarbodiimide (DCC) is used, and in the presence of a base such as pyridine or dimethylaniline in an inert organic solvent. It can be performed at −30 ° C. to room temperature. Moreover, when using reactive derivatives, such as an acid halide, it can carry out at -30 degreeC-room temperature in presence of bases, such as a pyridine and a dimethylaniline, in an inert organic solvent.

The benzoic acid anilides (1) or salts thereof thus obtained suppress the expression of cell surface molecules such as ICAM-1 and ELAM-1 involved in cell adhesion, and are represented by excellent leukocyte-vascular endothelial cells. Has the effect of suppressing cell adhesion. In addition, it has an excellent anti-inflammatory action against allergic inflammation, inflammation caused by ultraviolet irradiation, and the like. Furthermore, it has an excellent immunosuppressive action and cancer metastasis inhibitory action. Furthermore, cytotoxicity, skin irritation, etc. are weak and safety is high.
Therefore, a medicine containing benzoic acid anilides (1) or a salt thereof as an active ingredient is based on suppression of cell adhesion, and various inflammations, rheumatism, bronchial asthma, atopic dermatitis, hay fever, psoriasis, ischemia reperfusion injury It is useful for treatment and prevention of suppression, nephritis, encephalomyelitis, acute respiratory distress syndrome, transplant organ rejection suppression, autoimmune disease and the like.

  The medicament of the present invention can be administered to humans by any route such as oral, enteral, parenteral and topical administration for the treatment or prevention of the above-mentioned diseases. The dose is appropriately determined according to the age, disease state, body weight, etc. of the patient, but is usually selected within the range of 0.1 to 1000 mg / kg body weight, preferably 1 to 100 mg / kg body weight per day. It is administered once or divided into several times.

  The medicament of the present invention is usually used as a composition containing excipients and other additives usually used in medicine. Examples of these include solids such as lactose, kaolin, sucrose, crystalline cellulose, corn starch, talc, agar, pectin, stearic acid, magnesium stearate, lecithin, sodium chloride and the like; Examples include glycerin, peanut oil, polyvinyl pyrrolidone, olive oil, ethanol, benzyl alcohol, propylene glycol, and water.

  The dosage form can take any form, for example, solid preparations such as tablets, powders, granules, capsules, suppositories, lozenges; syrups, emulsions, soft gelatin capsules, creams, gels, pastes, sprays, Examples include liquid preparations such as injection.

  EXAMPLES Next, although an Example is given and this invention is demonstrated in detail, this invention is not limited to this at all.

Synthesis example 1
o-Aminophenol (10.99 g, 100.0 mmol) was dissolved in a mixed solvent (180 ml) of pyridine-chloroform (5: 1), stirred at −20 ° C. and stirred with p-acetoxybenzoic acid chloride (10.0 g, 50 0.0 mmol) dissolved in chloroform (200 ml) was slowly added dropwise over 3.5 hours. Stirring was further continued for 1 hour from the end of dropping to complete the reaction. The reaction solution was concentrated at room temperature, ethanol (100 ml) was added to the residue and dissolved, methylamine 40% methanol solution (10.0 g, 140.0 mmol) was added dropwise, and the mixture was stirred at 50 ° C. for 1 hr.
The reaction solution was concentrated again under reduced pressure, and the residue was extracted with ethyl acetate. The organic layer was washed successively with 12% hydrochloric acid, distilled water, aqueous sodium hydrogen carbonate solution and saturated brine, and dried over anhydrous sodium sulfate. After filtration and concentration under reduced pressure, a brown oily substance was obtained.
This was subjected to silica gel column chromatography, eluted with a chloroform-methanol mixed solvent, concentrated under reduced pressure, and the residue was recrystallized with ethyl acetate-n-hexane to give 4-hydroxy-n as white powder crystals. -(2-Hydroxyphenyl) benzoic acid amide (5.02 g, 45%) was obtained.
mp.159.1-159.7 ℃
NMR: (DMSO-d ₆ ): δ; ppm
6.78-7.06 (m, 2H), 6.88 (d, 2H, J = 10Hz), 7.04 (d, 1H, J = 7Hz), 7.71 (d, 1H, J = 7Hz), 7.87 (d, 2H, J = 10Hz), 9.37 (s, 1H), 9.76 (s, 1H), 10.13 (s, 1H)
IR: (KBr): cm ^-1
3616,3340,2956,1641,1515,1434,828

Synthesis example 2
In the same manner as in Synthesis Example 1, 4-hydroxy-N- (3-hydroxyphenyl) benzamide was obtained (35%).
mp.230-231 ℃
NMR: (DMSO-d ₆ ): δ; ppm
6.87 (d, 1H, J = 8Hz), 6.93 (d, 2H, J = 8Hz), 7.38 (t, 1H, J = 8Hz), 7.64 (d, 1H, J = 8Hz), 7.76 (s, 1H) , 7.86 (d, 1H, J = 8Hz), 8.00 (d, 1H, J = 8Hz), 10.00 (br.s, 2H), 10.13 (s, 1H)

Synthesis example 3
In the same manner as in Synthesis Example 1, 4-bidoxy-N- (4-hydroxyphenyl) benzamide was obtained (61%).
mp.276.1 ℃ (Automatic melting point measurement)
NMR: (DMSO-d ₆ ): δ; ppm
6.71 (d, 2H, J = 9Hz), 6.82 (d, 2H, J = 8Hz), 7.43 (d, 2H, J = 9Hz), 7.81 (d, 2H, J = 8Hz), 9.17 (s, 1H) , 9.74 (s, 1H), 10.00 (s, 1H)

Synthesis example 4
3,5-di-tert-butyl-4-hydroxybenzoic acid (6.26 g, 25.0 mmol) and p-aminophenol (2.73 g, 25.0 mmol) mixed with ethyl acetate-dimethylformamide (4: 1) Dissolved in a solvent (60 ml), N, N ¹ -dicyclohexylcarbodiimide (5.16 g, 25.0 mmol) dissolved in ethyl acetate (20 ml) was added with stirring at 0 ° C.
After 1 hour, the temperature was returned to room temperature, and the mixture was further stirred for 2 hours to complete the reaction.
The reaction solution was filtered to remove urea and then extracted with chloroform. The organic layer was washed successively with 3% hydrochloric acid, aqueous sodium hydrogen carbonate solution and saturated brine, and concentrated under reduced pressure.
The residue was dissolved in methanol-ether, the free urea was filtered off, the solution was concentrated again, and recrystallized with a mixed solvent of ethyl acetate-ether-n-hexane to give 3,5-diethyl as white crystals. -T-Butyl-4-hydroxy-N- (4-hydroxyphenyl) benzamide (5.18 g, 61%) was obtained.
mp.237-241 ℃
NMR: (DMSO-d ₆ ): δ; ppm
1.43 (s, 18H), 6.73 (d, 2H, J = 9Hz), 7.45 (d, 2H, J = 9Hz), 7.50 (br.s, 1H), 7.65 (s, 2H), 9.18 (br.s) , 1H), 9.80 (s, 1H)
IR: (KBr): cm ^-1
3616,3340,2956,1641,1515,1430,828

Synthesis example 5
In the same manner as in Synthesis Example 4, 3,5-di-t-butyl-4-hydroxy-N- (3-hydroxyphenyl) benzamide was obtained (20%).
mp.237-241 ℃
NMR: (DMSO-d ₆ ): δ; ppm
1.44 (s, 18H), 6.42-6.51 (m, 1H), 7.13-7.04 (m, 2H), 7.29 (s, 1H), 7.47 (br.s, 1H), 7.64 (s, 2H), 9.33 ( br.s, 1H), 9.90 (s, 1H)

Synthesis Example 6
In the same manner as in Synthesis Example 4, 3,5-di-t-butyl-4-hydroxy-N- (2-hydroxyphenyl) benzamide was obtained (50%).
mp.237-241 ℃
NMR: (DMSO-d ₆ ): δ; ppm
1.44 (s, 18H), 6.79-7.06 (m, 2H), 7.00 (d, 1H, J = 8Hz), 7.53 (br.s, 1H), 7.64 (d, 1H, J = 8Hz), 7.72 (s , 2H), 9.47 (s, 1H), 9.67 (br.s, 1H)
IR: (KBr): cm ^-1
3620,3334,2962,1638,1605,1533,1455,1368,1239,750

Example 1
Leukocyte-vascular endothelial cell adhesion inhibition test:
A test compound is added to a human vascular endothelial cell confluent on a 96-well flatbottom plate to a final concentration of 10 ⁻⁵ M. After 18 hours, human IL-1α is added to a final concentration of 5 units / ml and cultured for 6 hours. After removing the culture solution, the plate is washed twice with a new culture solution, and 200 μl of human peripheral leukocytes (10 ⁶ cells / ml) previously labeled with ⁵¹ Cr is added according to a conventional method and cultured. After 30 minutes, non-adherent cells are removed, and the radioactivity is measured after lysing the adherent cells.
As a result, as shown in Table 1, benzoic acid anilides (1) were found to have an excellent cell adhesion inhibitory effect.

Example 2
Inhibition test of cell surface molecules involved in cell adhesion (FACScan):
In a 25 cm ² culture flask, a test compound is added to a confluent human vascular endothelial cell to a final concentration of 10 ⁻⁵ M. After 18 hours, human IL-1α or TNFα is added to a final concentration of 2.5 ng / ml and cultured for 6 hours. After removing the culture solution, the cells are washed with PBS (−), and the cells are detached and collected with trypsin-EDTA. Anti-ICAM-1 and anti-ELAM-1 antibodies (mouse IgG) were used as primary antibodies, and anti-mouse IgG-FITC was used as a secondary antibody, followed by staining with FACScan after staining the cells according to a conventional method.
As a result, as shown in Table 2, benzoic acid anilides (1) exhibited the expression of ICAM-1 and ELAM-1, which are known as cell surface adhesion molecules and cell surface factors involved in immune reactions. It turned out to be strongly suppressed.

Example 3
Action on allergic inflammation:
The back of 7-week-old Balb / c male mice is shaved and sensitized by applying 100 μl of a 7% picryl chloride / acetone olive oil (4: 1) solution. 6 days after sensitization, 20 μl of 1% solution was applied to both sides of the right auricle and sacrificed 24 hours after inception, punched with a punch (7 mm) after cutting both auricles, the right and left auricle weights were measured, and the difference was edema The amount. The test compound was applied to the right auricle by 20 μl of a 100 mM solution on the day before the induction, at the time of induction, and 6 hours after the induction, and in the control group, only the solvent was applied instead of the test substance.
As a result, as shown in Table 3, it was found that the benzoic acid anilides (1) have an excellent allergic inflammation inhibitory action.

Example 4
Action on UV inflammation:
The back of a Hartley white guinea pig was shaved and a sample (100 mM, 25 μl) was applied. Two hours later, the sample was wiped with 70% ethanol, irradiated with UVB (1.7 mW / cm ² × 9 min), and immediately after that, the test compound (100 mM, 25 μl) was applied. Reapplied 6 hours after UVB irradiation, and at 6 and 24 hours after irradiation, the degree of erythema was determined according to the criteria of the Japanese Dermatological Association.
As a result, as shown in Table 4, benzoic acid anilides (1) were found to have a strong inhibitory action against inflammation caused by ultraviolet irradiation.

Example 5
Cancer cell-vascular endothelial cell adhesion inhibition test:
A test compound is added to a human vascular endothelial cell confluent on a 96-well flatbottom plate to a final concentration of 10 ⁻⁵ M. After 18 hours, human IL-1α is added to a final concentration of 5 units / ml and cultured for 6 hours. After removing the culture solution, the plate is washed twice with a new culture solution, and then 200 μl of 10 ⁶ cells / ml of ⁵¹ Cr-labeled human bone marrow tumor cells (HL-60) is added and cultured according to a conventional method. After 30 minutes, non-adherent cells are removed, and the radioactivity is measured after lysing the adherent cells.
As a result, as shown in Table 5, it was found that the benzoic acid anilides (1) strongly suppress the adhesion between cancer cells and vascular endothelial cells, which is important for cancer cell metastasis.

Example 6
Toxicity to vascular endothelial cells (cell morphology, DNA synthesis):
Morphological changes were visually evaluated by an inverted microscope, and DNA synthesis was performed according to a conventional method, and the amount of uptake in the last 8 hours of culture for 24 hours after addition of the sample was quantitatively evaluated using a liquid scintillation counter in accordance with ³ H-TdR uptake. . The test compound concentration was 10 ⁻⁵ M.
As a result, as shown in Table 6, the benzoic acid anilides (1) had almost no toxicity to vascular endothelial cells.

Example 7
Toxicity to immunocompetent cells (lymph node cells) (DNA synthesis):
Mouse lymph node cells were used. For DNA synthesis, the amount of uptake in the last 24 hours after 72 hours of culture after addition of the sample and lymphocyte stimulating substance (IL-2: 5 U / ml) was quantified using a liquid scintillation counter, using ³ H-TdR uptake as an indicator. evaluated. The test compound concentration was 10 ⁻⁵ M.
As a result, as shown in Table 7, benzoic acid anilides (1) had almost no toxicity to immunocompetent cells.

Example 8
Skin irritation:
The test compound (10 ⁻³ M / ethanol: glycerin = 9: 1) or solvent (ethanol: glycerin = 9: 1) was applied to Balb / c mouse right auricle for 14 days at a time for 14 days. The difference in thickness was taken as the amount of inflammation.
As a result, the benzoic acid anilides (1) did not cause any inflammation even when applied to the skin continuously for 2 weeks, and were highly safe.

Claims (2)

General formula (1):
[Wherein, R ¹ represents a t-butyl group, and R ² represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms]
The cell adhesion inhibitor which uses the benzoic acid anilide represented by these, or its salt as an active ingredient.   The anti-inflammatory agent which uses the benzoic acid anilides or its salt of Claim 1 as an active ingredient.