WO2000015604A1

WO2000015604A1 - Malonic diester derivatives and process for producing the same

Info

Publication number: WO2000015604A1
Application number: PCT/JP1999/004914
Authority: WO
Inventors: Yasushi Kono; Masahiro Nomura; Takayuki Sawada; Naoki Ando; Yukie Takahashi; Kazuhiko Kuriyama
Original assignee: Kyorin Pharmaceutical Co., Ltd.
Priority date: 1998-09-11
Filing date: 1999-09-10
Publication date: 2000-03-23
Also published as: AU5648699A

Abstract

Malonic diesters derivatives represented by general formula (1) and pharmacologically acceptable salts thereof being capable of preventing ICAM-1 and VCAM-1, which play the major roles among cell adhesion molecules, from binding to leukocytes; and cell adhesion inhibitors containing as the active ingredient at least one of the above compounds and serving as excellent immunosuppressants, anti-inflammatory agents, antiallergic agents and tumor metastasis inhibitors.

Description

Satsuki Itoda β Muconic acid diester derivative and its production

TECHNICAL FIELD The present invention relates to a malonic acid diester derivative which has a binding inhibitory activity between cell adhesion molecules and is useful as an immunosuppressant, an anti-inflammatory agent, an anti-allergic agent, and a cancer metastasis inhibitor, and a method for producing the same. Background art

In immune and inflammatory responses, adhesion between vascular endothelial cells and leukocytes is an extremely important process (Mebio, Νο · 5, Vol. 10, (1993)). The main adhesion molecules on the vascular endothelial cell side involved in this adhesion are reported to be ICAM-1, VCAM-1, E-selectin, P-selectin, etc., and the expression of each adhesion molecule is inflammatory. (Diagnosis and treatment, Vol. 83, 1164, (1995)), Springer Semin Immunopathol, Vol. 11 163, (1989), Cell, Vol. 76, 301 (1994)). That is, P-selectin force s, and the expression peaks 2 to 6 hours (early), assuming that the expression peaks at 5 to 30 minutes (immediately) after the inflammation is induced and the expression decreases thereafter. E-selectin may show a decrease in expression after that, and ICAM-1 and VCAM-1 may show a beak of expression after an additional 12 to 48 hours (late). . In particular, adhesion to leukocytes via I CAM-1 and V CAM-1 which are expressed in large amounts in the late stage is the strongest, and these two adhesion molecules play an important role in various diseases. It is said that it is doing. Therefore, if it is possible to inhibit adhesion through adhesion molecules such as ICAM-1 and VCAM-1 which play a central role in inflammation, chronic It is considered to be effective as a therapeutic drug for autoimmune diseases such as arthritis, nephritis and knee osteoarthritis, allergic diseases such as chronic inflammatory diseases, asthma and dermatitis, and as a cancer metastasis inhibitor.

Cell adhesion inhibitors that have been reported to date include so-called expression inhibitors that inhibit adhesion by suppressing the expression of adhesion molecules, and so-called expression inhibitors that inhibit adhesion by inhibiting the bonding between adhesion molecules. Classified as a binding inhibitor. Most of the cell adhesion inhibitors for ICAM-1 and VCAM-1 are expression inhibitors (JP-A-9-110689, JP-A-8-28316, JP-A-8-156). 198,752, JP-A-Heisei 7-304,667, JP-A-Heisei 7-258,168), and for binding inhibitors, peptide properties such as antibody ligands of adhesion molecules Except for macromolecules, only non-peptide low molecular weight compounds are reported in J. Med. Chem., Vol. 38, 1057 (1995). Expression inhibitors often act on intracellular signal transduction systems, and are thought to suppress functions other than expression of adhesion molecules. Unlike such expression inhibitors, binding inhibitors only inhibit the binding between adhesion molecules, so they are considered to be excellent drugs in terms of safety, but they are still satisfactory. is not.

The present invention provides an excellent immunosuppressant, anti-inflammatory agent by providing a substance that inhibits the binding between ICAM-1, VCAM-1 and leukocytes, which plays a central role in cell adhesion molecules. An antiallergic agent and a cancer metastasis inhibitor. Disclosure of the invention

The present inventors have developed a compound that inhibits the binding between human monocyte-like cell line (U933) and human umbilical vein-derived vascular endothelial cells (HUVEC) 24 hours after IL-1 stimulation. As a result of intensive research on A novel malonate ester derivative with a different structure from the known cell adhesion inhibitors binds to cells via ICAM-I and VCAM-1 without showing the effect of suppressing the expression of adhesion molecules. Have been found, and the present invention has been completed.

That is, the present invention relates to the general formula (1)

〇

I "-IR ² CO ₂ R ¹

ヽ

, People

H —0 ₂ R

[Wherein, W is an optionally substituted benzene ring, an optionally substituted pyridine ring, an optionally substituted quinoline ring, an optionally substituted benzothiazole ring, or an optionally substituted benzothiazole ring; A pyrimidine ring, a quinazoline ring which may be substituted, a chenopyrimidine ring which may be substituted, a benzimidazole ring which may be substituted, X represents an amino group, One CONH—, Y is an optionally substituted benzene ring, naphthalene ring, pyridine ring, chroman ring, 1,3-thiazolyl ring, Z is one CH2 CH—, — OCH ₂ —, -0 C (CH 3) _2- , one NHC 0 (CH ₂ ) _2- , one (CH 2) n-(n is an integer of 0 to 3) R ¹ is a lower alkyl group having 1 to 4 carbon atoms And R ² represents a hydrogen atom, a lower alkyl group having 1 to 4 carbon atoms, or a lower alkoxycarbonyl group having 1 to 4 carbon atoms. A malonic acid diester derivative and a pharmacologically acceptable salt, and a cell adhesion inhibitor containing at least one or more thereof as an active ingredient.

Pharmaceutically acceptable salts of the compound represented by the general formula (1) ′ in the present invention include, for example, hydrochloride, hydrobromide, methanesulfonate, citrate, and tartrate. Acid addition salts. In the general formula (1) of the present invention, the “lower alkyl group” such as a lower alkyl group and a lower alkoxycarbonyl group refers to, for example, a linear or branched chain such as methyl, ethyl, propyl, and isoprovir. Represents a hydrocarbon having 1 to 4 carbon atoms, and may be a `` optionally substituted benzene ring, an optionally substituted pyridine ring, an optionally substituted quinoline ring, or an optionally substituted Benzothiazole ring, optionally substituted pyrimidine ring, optionally substituted quinazoline ring, optionally substituted chenobilimidine ring, optionally substituted benzoimidazole The term `` ring '' means a halogen atom such as a lower alkyl group, a lower alkoxy group, a trifluoromethyl group, a methoxymethyl group, a Cl atom, a Br atom, an I atom, a F atom, a nitro group, an a C Group, a lower Ashiruami amino group such as Asechiruami amino group, piperidine group, Jimechiruami amino group, can be mentioned, et al are those having a lower Jiarukiruami amino group such as di Echiruami cyano group.

According to the present invention, the compound represented by the general formula (1) is a compound represented by the general formula (5)

, _Z / ^C0R3 (5)

[Wherein, W is an optionally substituted benzene ring, an optionally substituted pyridine ring, an optionally substituted quinoline ring, an optionally substituted benzothiazole ring, or an optionally substituted benzothiazole ring. An optionally substituted pyrimidine ring, an optionally substituted quinazoline ring, an optionally substituted chenopyrimidine ring, an optionally substituted benzimidazole ring, X represents an amino group, one CONH —, And Y is an optionally substituted benzene ring, naphthylene ring, pyridine ring, chroman ring, 1,3 —thiazole ring , Z is one CH = CH—, _ 0 CH ₂ —, 0 C (CH ₃ ) ₂ —, NHCO (CH ₂ ) ₂ —, one (CH 2) n-(n is an integer of 0 to 3) R ³ represents a hydroxy group or a halogen atom], and a compound represented by the general formula (6) is condensed. '

[In the formula, R ¹ represents a lower alkyl group having 1 to 4 carbon atoms, R ² represents a hydrogen atom, a lower alkyl group having 1 to 4 carbon atoms, or a lower alkoxy group having 1 to 4 carbon atoms.]

In the case of an acid halide in which R ³ in the formula (5) is a halogen atom, the reaction is carried out by using methylene chloride, 1,4-dioxane or the like as a solvent in the presence of an organic base such as triethylamine or pyridine. C to room temperature. When R ³ in the formula (5) is a hydroxy group, the compound can be produced by a mixed acid anhydride method or an active ester method used in a general peptide bond formation reaction, and preferably a condensing agent is used. The method used is appropriate. Reactions include dihexyl carbyl imide (DCC), diisopropyl carbodiimide (DIPC), diphenylphosphonyl azide (DPPA), getyl phosphonyl cyanide (DEPC), and 1-ethyl. 1-3- (3-Dimethylaminopropyl) In the presence of a condensing agent such as 1-carposimid (WSC), 4-dimethylaminoviridine (DMAP) may be added as a catalyst in some cases, and the reaction solvent is added. Examples include tetrahydrofuran (THF), methylene chloride, dimethylsulfoxide (DMSO), and preferably dimethylformamide (DMF). The reaction can be carried out at a reaction temperature of 0 ° C. to room temperature.

A compound in which R ² is a lower alkoxycarbonyl group having 1 to 4 carbon atoms in the general formula (6), that is, a compound represented by the general formula (4)

H ₂ NC (C0 ₂ R ¹ ) ₃ (4)

[Wherein R ′ is as described above] The compound represented by the general formula (2) and the compound represented by the formula (3) are produced by reacting the compound represented by the general formula (2) with the compound represented by the formula (3). Can be.

HC (C0 ₂ R ¹ ) ₃ (2)

[Where R ¹ is as described above]

The reaction is carried out using DMF, 1,4-dioxane, THF or the like as a solvent, in the presence of a base such as potassium butoxide, sodium alkoxide or sodium hydride. It can be carried out at a temperature between ° C and room temperature. A compound in which X is —C 0N H— in the above general formula (1), that is, the general formula (9) (9)

[Wherein W, Y, Z, R ′ and R ² are as described above], the compound represented by the general formula (7) is condensed with the compound represented by the general formula (8) This can also be manufactured.

[Wherein, R ⁴ represents a hydroxy group or a halogen atom, and W represents the above-mentioned ^]

( ⁸⁾

[Y, Ζ, R ', R 2 are as described above]

In the case of the acid halide in which in the formula (7) is a halogen atom, the reaction is carried out by using methylene chloride, 1,4-dioxane or the like as a solvent in the presence of an organic base such as triethylamine or pyridine. It can be carried out at a temperature between ° C and room temperature. Further, when R ⁴ in the formula (7) is a hydroxy group, it can be produced by a mixed acid anhydride method or an active ester method used in a usual peptide bond formation reaction, preferably using a condensing agent. The method is suitable. The reaction is DCC, DIPC ;, DPPA, DEPC, WS In some cases, DMAP is added as a catalyst in the presence of a condensing agent such as C, and THF, methylene chloride, DMSO, and DMF are used as reaction solvents, and the reaction temperature is 0. C to room temperature. BEST MODE FOR CARRYING OUT THE INVENTION

Next, the present invention will be described with reference to specific examples, but the present invention is not limited to these examples. Reference example 1

4-(Benzothiazol-2-yl) amino 2

A mixture of 2-chloromouth benzothiazol (2.12 g) and 4-amino 2-chloro-2-ethyl benzoate (2.50 g) was heated and stirred at 140 C for 30 minutes. After cooling, the reaction product was dissolved in ethanol, water was added, and the precipitated crystals were collected by filtration. 4- (Benzothiazol-1-yl) amino-2-chloroethyl benzoate (3.78 g) was obtained as a pale yellow powder. The obtained ester (1.28 g) was dissolved in ethanol (2 () ml), a 1% aqueous sodium hydroxide solution (5 ml) was added, and the mixture was heated under reflux for 1 hour. After cooling, ethanol was distilled off under reduced pressure, water was added to the residue, the reaction solution was adjusted to pH 3 with dilute hydrochloric acid, and the precipitated crystals were collected by filtration. After washing with water and drying, the desired product (1.05 g) was obtained as a colorless powder. Reference example 2

4 [(Benzothiazole-2-yl) amino] phenyloxyacetic acid

2-1,3-Dimethyl-1-2-imidazolidinone (300 ml) solution of benzothiazole (19.2 g) and ethyl phenylaminophenyl acetate (22.Og) in a 300 ml solution of pyridinium paratoluenesulfonic acid (2.83 g) g) was added and the mixture was stirred at 140 ° C for 2 hours. Water and a saturated aqueous solution of sodium hydrogen carbonate were added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic layer was washed with water and saturated saline, and then dried over anhydrous sodium sulfate. After evaporating the solvent under reduced pressure, the crystals obtained by purification by silica gel column chromatography (developing solvent n-hexane: ethyl acetate = 2: 1) are washed with methanol, and purified with methanol. 2- (amino) phenylethyl acetate (21.5 g) was obtained as a colorless powder. The obtained ester was subjected to alkaline hydrolysis in the same manner as in Reference Example 1 to obtain the desired product as a colorless powder. Reference Examples 3 to 6 1

The compounds shown in Table 1 were synthesized in the same manner as in Reference Example 1 or 2. table 1

Υ C0 ₂ H

\ 、 Ζ

1

Reference example, 6 2

[2- (Benzothiazolu-2-ol) amino 1,3-thiazo-ro 41 acetic acid

Chioshian acid Anmoniumu (9. () 9 g) in THF (200 ml) Atsushi Muro under stirring to a solution, adding Benzoiruku port Lai de (14.5 _g), was heated under reflux for between 1 0 minutes (Preparation of Benzoirui Sochioshianeto). 2-Aminobenzothiazole (13.6 g) was added to the reaction solution, and the mixture was further heated under reflux for 3 hours. The solvent was distilled off under reduced pressure, water was added to the residue, and the precipitated crystals were collected by filtration. The crystals were washed with hot ethanol to obtain N-benzoyl-N, 1- (benzothiazole-2-yl) thiourea (17.5 g) as a pale yellow powder. The obtained thiourea (17.5g), lithium hydroxide monohydrate

(6.03 g) was dissolved in water (200 ml) and heated under reflux for 20 minutes. After cooling, dilute hydrochloric acid was added to the reaction solution to adjust the pH to pH 10, then the pH was adjusted to pH 10 with aqueous ammonia, and the mixture was allowed to cool on a water bath and then allowed to cool. The precipitated crystals were collected by filtration and recrystallized from THF-isopropyl ether. N- (benzothiazoyl-2-yl) thiourea (3.62 _g ) was obtained as colorless needles. The obtained crystals (l.72 g) were dissolved in THF (50 ml), 4-ethyl chloroacetate acetate (6.30 g) and DMAP (0.12 g) were added, and the mixture was heated under reflux for 20 hours. The solvent was distilled off under reduced pressure, isopropyl ether was added to the residue, and the precipitated crystals were collected by filtration. [2- (Benzothiazol-1-yl) amino-1,3-thiazol-1-ethyl 41-ethyl acetate Hydrochloride (2.69g) as light brown powder Obtained. The obtained ester was subjected to alkaline hydrolysis in the same manner as in Reference Examples 1 and 2, and the target product was obtained as a colorless powder. Reference example 6 3

2-ano-1-ethyl methyl malonate

2- (t-butoxycarbonylamino) getyl malonate

To a solution of (5.1 ml) in THF (80 ml) was added 60% sodium hydride oily (() .96 g) at room temperature, and the mixture was stirred for 30 minutes. Chloromethane (1.5 ml) was added, and the mixture was further stirred for 8 hours. The reaction solution was poured into an ice-cooled 5% aqueous solution of citrate, and extracted with ethyl acetate. The organic layer was washed with water and saturated saline in that order, and then dried over anhydrous sodium sulfate. After evaporating the solvent under reduced pressure, the residue was purified by silica gel column chromatography (developing solvent: ethyl acetate: hexane = 1: 1: 10). Getyl 2-(-t-butoxycarbonylamino) -2-methylmalonate (5.16 g) was obtained as a colorless oil. The obtained oil (5.16 g) was dissolved in 1 M ethyl acetate monohydrochloride (8 () ml) and stirred at room temperature for 10 minutes. A saturated aqueous sodium hydrogen carbonate solution was added, the organic layer was separated, and dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure to obtain the desired product (2.70 g) as a colorless oil. Reference example 6 4

(4-aminophenyl) oxyacetylamimalonate getyl

(4-Nitrophenyl) oxyacetic acid (() .92g) and dimethyl aminomalonate hydrochloride (l.llg) were dissolved in DMF (20ml), and triethylamine (0.72ml), WSC ( 0.98 g) and DMAP (0.06 g) were added, and the mixture was stirred at room temperature for 9 hours. The reaction solution was poured into water and extracted with ethyl acetate. The organic layer was washed with water, a saturated aqueous solution of sodium hydrogen carbonate, water, dilute hydrochloric acid, water, and saturated brine in that order, and dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and the obtained residue was suspended in isopropyl ether, filtered and dried. 2 _ [(412-Trophenyl) -aged xyacetylamino] Jethyl malonate (l.OOg) was obtained as milky white crystals. Mp l () r) -l () 8 ° C Elemental analysis (%): as a C 〖5 H, _₈ N ₂ 0 ₈

C H N

Calculated value: 50.85 5.12 7.91

Measured value: 50.57 5.02 8.05

The above nitro form (0.67 g) was dissolved in ethanol (3 () ml), 10% Pd / C (0.07 g) was added, and the mixture was stirred at room temperature under a hydrogen atmosphere for 2.5 hours. After removing the catalyst by filtration, the solvent was distilled off under reduced pressure to obtain the desired product (() .61 g) as a purple-brown powder. Example 1

Triethoxycarbonylmethylamine

H ₂ NC (C 0 ₂ E t) 3 J. Org. Chem., 44, 4836 (1979), a 60% sodium hydride oil (0.22 _g ) suspension in THF (7.5 ml) was synthesized as follows. A solution of ethoxycarbonylmethane (1.16 g) in THF (7.5 ml) was added dropwise at room temperature over 10 minutes. After stirring for further 10 minutes, a solution of 0- (2,4-dinitrophenyl) hydroxyamine (1 (0) g) in THF (10 ml) was added dropwise. After stirring at room temperature overnight, the solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (developing solvent: hexane: ethyl acetate = 2: 1) to obtain the desired product (() .80 g). Obtained as a yellow oil.

_{H- NM R (CDC1 3):} . 1.33 (9H, t, j = 7.3Hz) 4.34 (6H, q, j = 7.3Hz) 5.05 (2H, br) Example 2

[4- (Benzothiazole-2-ylano) benzoylamino] getyl malonate

In a DMF (10 ml) solution of the compound of Reference Example 3 (0.27 g) and getyl aminomalonate hydrochloride (0.23 g), triethylamine (0.15 ml), WSC (0.29 g)-DMAP (0.05 g) ) And stirred at room temperature for 18 hours. After adding water to the reaction mixture, the mixture was extracted with ethyl acetate, and the organic layer was washed with water, a saturated aqueous solution of sodium hydrogen carbonate, water, dilute hydrochloric acid, water, and saturated saline in this order, and then sodium sulfate anhydride. And dried. The solvent is distilled off under reduced pressure. Purification by column chromatography (developing solvent: ethyl acetate: n-hexane 2: 1: 1) gave the desired product (0.38 g) as a colorless powder. Melting point 196-197 ° C

Elemental analysis (%): Calculated as 2 1 h ₂ , N 305 S, 1/5 H ₂₀

C H N

: 58.51 5.00 9.75

Measured value: 58.52 4.87 9.92 Example 3 — 6 7

The compounds of Reference Example and the compound of Example 1 were reacted in the same manner as in Example 2 to synthesize the compounds shown in Table 2 (Examples 3 to 63). In Examples 64 to 67, after reacting in the same manner as in Example 2, crystals precipitated when the organic layer extracted in the post-treatment stage was washed with diluted hydrochloric acid were collected by filtration.

Table 2

Elemental analysis Example W X Y Melting point (.C)

Calculated value Z Actual value (recrystallization solvent) C, H N

169-170.5 C22H23 3O5S

NH -CH ₂ -H (AcOEt) 59.85, 5.25, 9.52

59.72 5.16, 9.42

C23H23 3O5S

205-206.5

NH-^-H 60.91, 5.11, 9.27

(AcOEt) 60.88, 5.02, 9.22

C ₂₄ H _{26 4} 0 ₆ S

NH — -NHCO (CH ₂ ) ₂ -H 179-181 57.82, 5.26, 11.24

C23H25 3O5S r ₅ H NH — ^ — CH ₂ CH ₂ H 138.5-140.5 60.64, 5.53, 9.22

C23H25 3O6S

N

NH 173-176 58.59, 5.34, 8.91 eO ^^ S.-Q- CH ₂ H

58.39, 5.33, 8.88

N C23H25N3O5S

10 NH 182.5-183.5 60.64, 5.53, 9.22

60.62, 5.48, 9.26

C23H25 3O5S

11 众. 181-183 60.64, 5.53 9.22

60.53, 5.49, 9.28

Me C ₂₃ H ₂₅ I ₃ 0 ₅ S

12 N NH-CH ₂ H 142.5-144.0 60.64, 5.53, 9.22

60.41, 5.48, 9.23 eOH ₂ C C24H27N3O6S

13 N NH ~ ~ ^CH 2 H 158.0-159.5 HRMS 485.1621

485.1621

N. 23 22 3 30 ₅

CH H 164-166 54.22, 4.35, 8.25

¹⁴ F ₃ C '' NH

Fifteen

16

17

18

CI Η22 ΙΝ3〇6θ

19 NH "OCH ₂ -H 179.0-180.5 53.71, 4.51, 8.54

53.79, 4.65, 8.27

O

} 30

S9 SIAIHH ειη-οιη H ² HO ^s HO HN S9

Next, the results supporting the usefulness of the compound of the present invention are shown by experimental examples.

Inhibitory effect of test compound on adhesion between human vascular endothelial cells and U933 cells (human monocyte cell line)

By stimulating human umbilical vein-derived vascular endothelial cells (HUVEC) with human IL-1 (IL-1), ICAM-1, VCAM-1, ELAM-1 And the like are induced. At 24 hours after stimulation, expression of ICAM-1 and VCAM-1 is mainly observed (J. Immunol. 144, 2558 (1990), ibid, 149, 698 (1992)) ₀ IL-1 By performing a cell adhesion test using HUV EC stimulated for 24 hours, the adhesion reaction via ICAM-1VCAM-1 can be tested. Furthermore, by dividing the time of addition of the test compound into the time of stimulating HUVEC with IL-11 and the time of adhesion between HUVEC and U937, the adhesion inhibitory effect of the test compound is mainly It can be evaluated whether it is due to inhibition of the binding of the adhesion molecule or suppression of the expression of the adhesion molecule. Experimental example 1 Binding inhibition test of adhesion molecule

HUVEC suspended in M199 medium (culture) containing 20% fetal serum and 1 ng / ml vascular endothelial cell growth factor (ECGF) was transferred to a 96-well collagen plate (flat bottom). Seed 2 x 10 ⁴ / ゥ wells and 37. C, and cultured under 5% C ₂ . After culturing for about 24 hours, HUV ECs were washed twice with M199 medium (for washing) containing no fetal serum and ECGF. Next, Hytointer and Leukin

(L-1?) Was cultured for 24 hours in a culture medium Ml99 containing 10 U / ml. On the other hand, U 9 3 7 cell suspension ^{(lxl 0 7 / ml) 1} mM BCECF _ AM solution Ri per 1 ml of (Wako Pure Chemical Industries, Ltd.) 1 0 // 1 not a One added, under ice-cooling for 1 hour Incubated and fluorescently labeled. Fluorescent marker After washing twice with phosphate buffered saline (PBS (-)), the cells were washed twice with RPMI-164 medium containing 10% fetal calf serum. ^{1 X 1 0 7 / m 1} ). HUVEC were washed three times with M199 medium for washing. The test compound was dissolved in dimethyl sulfoxide, and diluted 100-fold with M199 medium for culture, and added in 80/1 / well. Subsequently, the fluorescence-labeled U933 cell suspension was added at a concentration of 201 / well (final concentration of the test compound was 10〃M). Min 1 0 0 0 rotation, at room temperature, was centrifuged for 1 min, and incubated 3 7 ° C, 5% C 0 2 3 0 minutes under. Each well was washed twice with PBS (-) 1001 to remove non-adherent cells. The cells were solubilized by adding 0.1% aqueous sodium dodecyl sulfate at 100 / l / well. The fluorescence intensity of each well was measured (Excitation 490 nm, Emission 530 nm), and the number of adhered U933 cells was determined from the calibration curve force. The adhesion inhibition rate was calculated according to the following equation.

Number of adherent cells in one test compound-Number of adherent cells in control without iL-Ιβ Negative leakage rate (¾) X100

Number of adherent cells in control with IL-1P addition |

Table 3 shows the results. In addition, the suppression rate exceeding 100% was shown as 100%. Experimental Example 2 Test for suppressing the expression of adhesion molecules

The HUV EC were suspended in M 1 9 9 culturing medium, 9 6 seeded well collagen Coat plate (flat bottom) one by 2 x 1 0 ⁴ / Ueru were cultured at 3 7 ° C, 5% C 0 2 below . After culturing for about 24 hours, wash M 199 medium The HUVEC was washed twice. The test compound was dissolved in dimethylsulfoxide, and a 100-fold dilution in M199 medium for culture was added at 80 81 / ゥ, and the mixture was cultured for 1 hour. Next, a culture medium containing IL-11 was added at a rate of 20 1 / well, and cultured for 24 hours (final concentration of IL-11 was 10 U / m 1, Final concentration 10〃M). On the other hand, add 10 mM of ImM BCECF-AM solution (Wako Pure Chemical Industries) per 1 ml of U937 cell suspension (lxl O ⁷ / ml), and incubate for 1 hour under ice-cooling. The cells were incubated and fluorescently labeled. Fluorescently labeled U 9 3 7 cells, PBS (-) After twice washing, RPMI containing 1 0% © sheet calf serum - were suspended in 1 6 4 0 medium ^{(1 xl 0 7 / ml)} . After washing the HUVEC three times with the washing M199 medium, the culture M199 medium was added at 80〃1 / ゥ and the fluorescently labeled U937 cell suspension was added at 201 1 / ゥ. After centrifugation at 100 rpm, room temperature and 1 minute per minute, 37. C, and cultured 5% C_〇 3 0 minutes at ₂ under. Each well was washed twice with PBS (—) 100〃1 to remove non-adherent cells. The cells were solubilized by the addition of 0.1% sodium dodecyl sulfate aqueous solution at 100 / l / well. The fluorescence intensity of each well was measured (Excitation 490 nm, Emission 530 nm), and the number of adhered U933 cells was determined from the calibration curve. The adhesion inhibition rate was calculated according to the following equation.

,. C. Number of adherent cells of test compound-Number of adherent cells of control without IL-1p, Inhibition rate of Xiao Xiao (%) I X100

Number of adherent cells in IL-1p-added control-Adhesion in IL-Ιβ-free control

Table 4 shows the results. Table 3.Adhesion molecule binding inhibition test

Inhibition rate (%) ΊΗ 'υ ⁶ ^-, / 0 No.Example number 10μΜ Example number 10μΜ

Q-γ

Ό Of 4U 78

7 88 88

8 75 45 95

1 Ί 69 46 87

13 65 48 51

22 75 58 60

23 100 60 Ο 1

24 79 63 75

31 57 66 65

34 74

Table 4 Expression suppression test for adhesion molecules

Inhibition rate (%) 细 (κ) 巾 Width 睦, Example number 10μΜ Example number 10μ

6 7 40 Q C7

7 -9 44 I I

8 -5 45-9

11 -20 46

13 -4 48 0

22 7 58 13

23 -3 60 -14

24 5 63 12

31 7 66 6

34 9 Experimental example 3 Rat delayed type hypersensitivity test

Eight or nine-week-old Louis female rats (Japanese charles 'river') were divided into 5 animals per group. .05 ml of Mycobacterium 'butyricum-killed bacteria (Difco) () .6 _mg bag) suspended in liquid paraffin was injected into the skin of the right hind foot of the rat. Seven days later, the back was shaved with an electric hair clipper, and the skin thickness of the back (two places on the left and right) was measured using a dial thickness gauge (Ozaki Seisakusho). Next, the antigen solution 501 was injected intradermally into the skin thickness measurement part. As the antigen solution, use the supernatant obtained by suspending Mycobacterium butyricum at a concentration of 200 μg / ml in physiological saline and centrifuging at 3,000 rpm for 10 minutes at 4 ° C. did. At 24 hours after injection of the antigen solution, the skin thickness at the injection site was measured, and the amount of increase in skin thickness was calculated. The test compound was suspended in a 3% aqueous solution of gum arabic and orally administered once daily, 0.5 ml per 100 g of rat weight once a day from the day of injection of the killed Mycobacterium petilicum. . The control group received only a 3% gum arabic aqueous solution. The results were expressed as a percentage of the increase in skin thickness of the test compound relative to the increase in skin thickness of the control group. Table 5 shows the results.

Table 5 Effect on delayed type hypersensitivity reaction

Example No.

(mg / kg / day. p.o.) Percentage (%)

• P <0.05 "There is a significant difference at p <0.01 Industrial applicability

As described above, the compound of the present invention represented by the general formula (1) does not exhibit the inhibitory effect on the expression of cell adhesion molecules such as ICAM-1 and VCAM-1 and inhibits the cell-mediated binding therebetween. However, its efficacy was also confirmed in a delayed type hypersensitivity reaction test.

Claims

General expression (1)

[Wherein, W is an optionally substituted benzene ring, an optionally substituted pyridine ring, an optionally substituted quinoline ring, an optionally substituted benzothiazole ring, An optionally substituted pyrimidine ring, an optionally substituted quinazoline ring, an optionally substituted chenopyrimidine ring, an optionally substituted benzimidazole ring, and X represents an amino group , One CONH—, Y is an optionally substituted benzene ring, naphthalene ring, pyridine ring, chroman ring, 1,3-thiazole ring, and Z is one CH2CH—, -0CH2-,-. 0 C (CH) 2 —, one NHC 0 (CH ₂ ) ₂ —, — (CH ₂ ) n-(n is an integer of 0 to 3) R ¹ is a lower alkyl group having 1 to 4 carbon atoms, R ² is a hydrogen atom, this represented a lower alkyl group having 1 to 4 carbon atoms, a lower alkoxycarbonyl group having 1 to 4 carbon atoms] Malonic acid diester derivatives and pharmacologically acceptable cormorants Ru salt characterized.

2. General formula (1)

? R ² , C0 ₂ R ¹

(ΞΜ Λ C0 ₂ R '("

Hapi [Wherein, W is an optionally substituted benzene ring, an optionally substituted pyridine ring, an optionally substituted quinoline ring, an optionally substituted benzothiazole ring, or an optionally substituted benzothiazole ring. An optionally substituted pyrimidine ring, an optionally substituted quinazoline ring, an optionally substituted chenopyrimidine ring, an optionally substituted benzimidazole ring, X represents an amino group, one CONH - a, Y is a benzene ring which may be substituted, naphtha Ren ring, pyridinium Jin ring, chroman ring, 1, 3-thiazole Ichiru ring, Z is one CH two CH-, one OCH ₂ _, - 0 C (CH 3) ₂ —, one NHCO (CH 2) ₂ —,-(CH 2) n — (n is an integer of 0 to 3) R j is a lower alkyl group having 1 to 4 carbon atoms, R ² Represents a hydrogen atom, a lower alkyl group having 1 to 4 carbon atoms, or a lower alkoxycarbonyl group having 1 to 4 carbon atoms.] Cell adhesion inhibitors as an active ingredient one or more at least malonic acid diester derivatives and pharmacologically acceptable salts, characterized in that.

3. General formula (2)

HC (C0 ₂ R ¹ ) ₃ (2)

[Wherein, R ¹ represents a lower alkyl group having 1 to 4 carbon atoms].

A compound represented by the general formula (4):

H ₂ NC (C0 ₂ R ¹ ) ₃ (4)

[Wherein R ¹ is as described above]. 4. General formula (5)

[Wherein, W is an optionally substituted benzene ring, an optionally substituted pyridine ring, an optionally substituted quinoline ring, an optionally substituted benzothiazole ring, or an optionally substituted benzothiazole ring; A pyrimidine ring, a quinazoline ring which may be substituted, a chenopyrimidine ring which may be substituted, a benzimidazole ring which may be substituted, X represents an amino group, one CONH —, Y is an optionally substituted benzene ring, naphthylene ring, pyridine ring, chroman ring, 1,3-thiazolyl ring, and Z is one CH2 CH—,-0 CH 2-,- 0 C (CH 3) ₂ _, one NHC 0 (CH 2) ₂ —, one (CH 2) n — (n is an integer of 0 to 3), and R ³ represents a hydroxy group or a halogen atom. And the compound represented by the general formula (6)

( ⁶⁾

[Wherein, R ′ is a lower alkyl group having 1 to 4 carbon atoms, R ² is a hydrogen atom, a lower alkyl group having 1 to 4 carbon atoms, and a lower alkoxy force having 1 to 4 carbon atoms. A compound represented by the general formula (1):

Says: (1)

Wherein, W, X, Y, Ζ , RR 2 are as described above] The method for producing a compound represented by.

5 General formula (7)

[Wherein, W is an optionally substituted benzene ring, an optionally substituted pyridine ring, an optionally substituted quinoline ring, an optionally substituted benzothiazole ring, or an optionally substituted benzothiazole ring; A pyrimidine ring, a quinazoline ring which may be substituted, a chenopyrimidine ring which may be substituted, a benzimidazole ring which may be substituted, R ⁴ is a hydroxy group, a halogen And a compound represented by the general formula (8):

Y,-

H ₂ N ⁽⁸⁾

[In the formula, Y is a benzene ring which may be substituted, naphtha Ren ring, pin lysine ring, chroman ring, a 1,3-thiazole ring, Zeta one CH two CH foremost, one OCH ₂ -., - 0 C (CH a) ₂ —, -NHC 0 (CH ₂ ) ₂ one, one (CH 2) n — (n is an integer of 0 to 3), and R ¹ is a lower alkyl group having 1 to 4 carbon atoms. And R ² represents a hydrogen atom, a lower alkyl group having 1 to 4 carbon atoms, or a lower alkoxycarbonyl group having 1 to 4 carbon atoms]. 9)

[Wherein W, Y, Z and R ¹ R ² are as described above].