JP3122161B2 - γ-lactone immunosuppressant - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an immunosuppressant containing a γ-lactone derivative. In particular, the present invention has an immunosuppressive action, angiogenesis inhibitory action, rejection at the time of organ transplantation,
The present invention relates to a medicine containing a γ-lactone derivative that can be used for treatment and prevention of various inflammatory diseases (rheumatism, psoriasis, etc.) and cancer.

[0002]

2. Description of the Related Art It has been disclosed that a .gamma.-lactone carboxylic acid derivative represented by the general formula (I) is useful as an antibacterial agent or a synthetic intermediate thereof (JP-A-1-3497).
6). However, the above patent document does not show that the compound represented by the general formula (I) is useful as an immunosuppressant.

[0003] Immunosuppressive drugs are indispensable drugs for suppressing rejection in organ transplantation such as kidney, heart and liver, and for suppressing graft-versus-host reaction in bone marrow transplantation. It is also used as a therapeutic drug in autoimmune diseases.
Immunosuppressants are divided into therapeutic and induction drugs and drugs for acute rejection in terms of treatment.

[0004] The transplantation immune response is said to consist of a primary immune response centered on T cells and a secondary immune response with humoral antibodies. In fact, the emergence of cyclosporin, which strongly suppresses the T cell-dependent immune response, has led to a remarkable improvement in the survival rate of primary transplant patients as compared to conventional treatment results with azathioprine and prednisolone. 7-
Eight years of long-term observations have revealed the efficacy and limitations of cyclosporin. The use of all immunosuppressive drugs, including cyclosporin, can lead to chronic rejection, resulting in a survival rate of about 65 years after transplantation.
%, And it cannot be said that stable engraftment over a long period has been sufficiently obtained. The reasons for this are: 1) differences in sensitivity of the patient's own drug (cyclosporin), 2) reduction in drug dosage due to side effects, and 3) transplantation immunoreactive systems that cannot be sufficiently suppressed by conventional immunosuppressants, for example, Presence of activated monocytes / macrophages. Steroids are effective in suppressing the production of activated monocyte / macrophage effectors, but long-term large-dose administration is not possible due to side effects, and cyclosporin also inhibits the production of activated monocyte / macrophage effectors. However, constant efficacy cannot be expected due to the difference in sensitivity of the drug. Therefore, suppression of rejection is insufficient, and chronic organ rejection causes transplant organ failure. In addition, side effects due to drugs cause serious complications due to side effects due to long-term administration, as is remarkably observed in steroids, and have a significant effect on long-term survival and survival rates.

[0005] That is, new problems of immunosuppressive drugs in the current organ transplantation are that the drug sensitivity and the limit of drug efficacy of cyclosporin and the side effects due to long-term use of steroids are stable and favorable. The grades have not been obtained. In particular, no immunosuppressant with few side effects has been found to replace steroids, which have an excellent effect on suppressing the production of activated monocyte / macrophage effectors which are thought to be involved in rejection.

[0006]

DISCLOSURE OF THE INVENTION The present invention provides an immunosuppressant as a transfection and maintenance agent which has a reduced side effect, replacing the immunosuppressive effect of steroids on activated monocyte / macrophage effectors. Things.

[0007]

Means for Solving the Problems In order to solve the above problems, the present inventors have conducted research on a novel immunosuppressant. As a result, surprisingly, the compound represented by the general formula (I) was found to be immunosuppressive. It has been found that it has an action and can be used as a medicine for preventing rejection at the time of organ transplantation. Moreover, they have found that the compound represented by the formula (I) has extremely low toxicity, and have completed the present invention.

[0008]

Embedded image

The optionally substituted phenyl group represented by R ¹ in the compound represented by formula (I) contained in the immunosuppressant provided by the present invention includes, for example, halogen (eg, fluorine, chlorine, Bromine, iodine), C
A phenyl group having 1 to 3 groups selected from _1-3 alkoxy is preferable, and a phenyl group optionally substituted with halogen is preferable, and 4-chlorophenyl, 4-fluorophenyl and 2,2 are particularly preferable. 4-Difluorophenyl is preferred.

The optionally esterified carboxy group represented by R ² in the compound represented by formula (I) includes, for example, carboxy, methoxycarbonyl, ethoxycarbonyl, n-propoxycarbonyl, i-propoxycarbonyl Aralkyl having 8 to 13 carbon atoms, such as an alkoxycarbonyl group having 2 to 5 carbon atoms such as n-butoxycarbonyl, t-butoxycarbonyl, benzyloxycarbonyl, p-nitrobenzyloxycarbonyl, phenethyloxycarbonyl, and benzhydryloxycarbonyl; Oxycarbonyl and the like.

In the compound represented by formula (I), the optionally oxidized sulfur atom represented by X may be sulfoxide (-SO-) or sulfone (-SO _2- ) depending on the oxidation step. . Examples of the compound represented by the general formula (I) include the compounds shown in Table 1.

[0012]

[Table 1]

[Table 2] Ph: phenyl group PNB: p-nitrobenzyl group

The compounds ( 1 to 19 ) in Table 1 are described in
-34976 as an antibacterial agent or a synthetic intermediate thereof.

The compound represented by formula (I) contained in the immunosuppressant provided by the present invention may form a salt, and may be a pharmacologically acceptable salt such as an alkali metal (eg, sodium). , Potassium) and salts with alkaline earth metals (eg, magnesium and calcium).

Since the compound represented by the general formula (I) has an asymmetric carbon, at least two or more stereoisomers may exist. Therefore, the immunosuppressive agent of the present invention can contain either a single isomer thereof or a mixture thereof.

The compound of the general formula (I) contained in the immunosuppressant provided by the present invention is that [R ¹ is phenyl substituted with ¹ to 2 halogens, and R ² is ethoxycarbonyl or benzyloxy. X is oxygen or sulfur which may be oxidized, and particularly preferable compounds are (1) 2- (4-chlorophenyl) thio-5-oxo-
2-tetrahydrofurancarboxylic acid benzyl ester (compound 13 ) (2) 2- (4-fluorophenyl) oxy-5-oxo-2-tetrahydrofurancarboxylic acid ethyl ester (compound 39 ) (3) 2- (2,4-difluoro Phenyl) sulfonyl-5-oxo-2-tetrahydrofurancarboxylic acid benzyl ester (Compound 32 ).

The compound of the formula (I) can be produced, for example, by the method shown in the following reaction formula. In this reaction scheme, the compound (I) is represented by the general formulas (IV), (V), (VI) and (VII)
Is represented by

[0018]

Embedded image Wherein R ¹ and R ² are as defined above, and R ² ′
Represents an esterified carboxy group, X ′ represents an oxygen atom or a sulfur atom, and n represents an integer of 1-2. ]

[0019] Evaluation of the immunosuppressive effects of (utility) compound was carried out by the following experiment. 1. Immunosuppressive effect on the production of human activated monocytes / macrophages in vitro: The method of inducing the production of human activated monocytes in vitro is performed according to the method established by the present co-inventor, Ishibashi (References 1 and 2). Was. Spontaneou
The splaque-forming cell (SPFC) is a new activated monocyte that lyses allogeneic red blood cells without adding extraneous complement. This SP
FC can be induced to be produced in vitro under two model conditions. Human peripheral blood mononuclear cells including monocytes were cultured for 1-7 days in RPMI1640 supplemented with human AB serum without antigen stimulation (condition: innate immunity model). 2) Untreated human peripheral blood mononuclear cells were And the same number as the number of mitomycin C-treated stimulator cells, 6-7
By culturing in two experimental systems of allogeneic mixed culture (condition: acquired immune model) for one day, SPFC that forms hemolytic spots on monolayered human erythrocytes is induced.

The compound was added simultaneously with the start of culture in each of the conditions: innate immunity model and condition: acquired immunity model, and compared with the number of activated monocytes produced in the solvent used as a control to evaluate the immunosuppressive effect in vitro. investigated.

Compound (I) showed immunosuppressive activity in this test method. Table 2 shows the compounds ( 18 ), ( 13 ),
( 39 ), ( 36 ), ( 16 ), ( 32 ) and IC50s of cyclosporine, azathioprine, prednisolone, and mizoribine as controls are shown. These compounds
They were classified into two categories based on the mode of action of inhibition. That is, there were those showing suppression in both the natural and acquired immunity models, and those showing suppression in only the acquired immunity model.

Conditions that simultaneously suppress conditions:
Compound (18), (13), (39), (32). What suppresses only the condition: Compound (36), (1
6).

[0023]

[Table 3]

Reference 1. M.Ishibashi, Y.Kokado, S.Takahara, Y.Ichikawa,
and T. Sonoda, Cellular immune response against hum
an red blood cell antigens andrenal allograft reje
ction, Transplant Proc, 19: 4511-4515, 1987. M.Ishibashi, S.Jiang, Y.Kokado, S.Takahara, an
d T.Sonoda, Immunopharmacologic effects of immunosu
ppressive agents exploredby a new effector generat
ion assay.Transplant Proc, 21: 1854-1858,1989.

2. Activated monocytes and macrophages that form hemolytic spots on allogeneic erythrocytes are similarly found in allograft skin infiltrated cells during acute rejection in humans as well as in rats. It is. Examination of an immunosuppressant using a rat allogeneic skin transplantation model is effective in knowing the rejection suppression effect in humans.

The immunosuppressive effect of the compound was examined using the combination of the strongest tissue incompatibility among the inbred rat conspecifics. The donor is ACI, the recipient is Lewis,
Allogeneic skin transplants were performed at 9 weeks for males. The skin graft was placed on the recipient's anterior thorax with the donor's skin 3
X3 cm size was transplanted and observed every day from the 5th postoperative day, and the day when the skin piece became 50% or more necrotic was regarded as the rejection day. The compound was suspended in a 5% gum arabic solution, and 1 day from the day of transplantation.
Oral administration for 4 consecutive days. Results: As shown in Table 3, the compound ( 13 ) and the compound ( 39 ) exhibited an effect of prolonging the survival of the allogeneic skin graft.

[0027]

[Table 4]

[Table 5]

3. Acute toxicity The acute toxicity of compound ( 13 ) was examined using Jcl: ICR mice and Jcl: Wist-ar rats. Compound ( 13 ) 1500 mg / kg and 3000 mg / kg
When kg is orally administered to the aforementioned mice and rats,
Since there was no death in any case, it is clear that the compound ( 13 ) has low toxicity and can be safely administered.

[0029]

Industrial Applicability The compounds according to the present invention strongly suppress rejection by activated monocytes / macrophages, and are therefore expected to suppress chronic rejection as well as acute rejection. It is useful as a drug with less side effects.

The daily dose of the immunosuppressant containing the compound represented by the general formula (I) or a salt thereof is the same as that of the compound (I)
Is about 0.1 to 100 mg / kg, more preferably about 0.2 to 40 mg / kg.

To administer compound (I), compound (I) or a pharmaceutically acceptable salt thereof is mixed with an appropriate pharmaceutically acceptable carrier, excipient or diluent by conventional means. For example, tablets, granules, capsules, drops and the like, and can be administered orally, or molded into injections by conventional means, for example,
It can be formulated in a sterile carrier prepared by conventional means and administered parenterally.

When producing the above oral preparations, for example, tablets, a binder (eg, hydroxypropylcellulose, hydroxypropylmethylcellulose, macrogol, etc.), a disintegrant (eg, starch, carboxymethylcellulose calcium, etc.), excipient (E.g., lactose, starch, etc.), lubricants (e.g., magnesium stearate, talc, etc.) and the like can be appropriately compounded.

When a parenteral preparation such as an injection is produced, an isotonic agent (eg, glucose, D-sorbitol,
D-mannitol, sodium chloride, etc.), preservatives (eg, benzyl alcohol, chlorobutanol, methyl paraoxybenzoate, propyl paraoxybenzoate, etc.), buffers (eg, phosphate buffer, sodium acetate buffer, etc.) Can be appropriately blended.

Next, the contents of the present invention will be described in more detail by reference examples and examples, but the present invention is not limited thereto.

Reference Example 1 2-phenoxy-5-oxo-2-tetrahydrofuran
Carboxylic acid benzhydryl ester [compound (2
0)]]: Phenol (2.1 g), 2-oxog
With rutalic acid 1-benzhydryl ester (6.2 g)
DCC (4.6 g) in dichloromethane (100 ml)
In addition, the resulting mixture was stirred at room temperature for 12 hours. Precipitation
The crystals that formed were filtered off. The filtrate was concentrated under reduced pressure, and the residue was silica.
Gel column chromatography, dichlorometa
: Hexane (3: 2). Concentrate the target fraction under reduced pressure
The resulting oil was crystallized from isopropyl ether.
To give the title compound (20) As a colorless prism
Obtained. Yield 2.0 g (26%) Melting point 115-117 ° C¹ H-NMR (CDCl_Three) Δ: 2.43-2.87
(4H, m), 6.84 (1H, s), 6.94-7.
37 (15H, m) Elemental analysis: C_{twenty four}H₂₀O_FiveCalculated: C, 74.21; H, 5.19 Found: C, 74.04; H, 5.18

[0036] The In the same manner as in Reference Example 2-8 Reference Example 1 compound shown in Table 4, when reacted under the conditions shown in Table, Compound 21 - 27 were obtained.

[0037]

[Table 6]

[Table 7]

[Table 8]

[Table 9]

[Table 10]

[Table 11]

Reference Example 9 Compound21(4.2 g) in dichloromethane (80 ml)
And anisole (4 ml) and trifluoro
Loacetic acid (4.5 ml) was added. The reaction mixture was cooled for 2 hours under ice cooling.
After stirring, the mixture was distilled off under reduced pressure.
00 ml) and ethyl acetate (50 ml), and the aqueous layer was separated.
I took it. The pH of the aqueous layer was adjusted to 3.0 with 2N hydrochloric acid, and
Extract twice with ethyl (60 ml) and combine the ethyl acetate layers.
And wash with water (40 ml) and dry (MgSO_Four) After decompression
After distillation, 2- (4-fluorophenyl) thio-5-o
Oxo-2-tetrahydrofurancarboxylic acid (compound2
8) Was obtained as a colorless oil. Yield 2.3 g (86%)¹ H-NMR (CDCl_Three) Δ: 2.41-2.90
(4H, m), 7.02-7.10 (2H, m), 7.
54-7.61 (2H, m), 8.48 (1H, bs) SIMS (m / z): 257 (M + H)⁺

Reference Example 10 Compound 22 (1.9 g) was treated with trifluoroacetic acid in the same manner as in Reference Example 9 to give 2- (4-methoxyphenyl) thio-5-oxo-2-tetrahydrofurancarboxylic acid (Compound 29) ) Was obtained as a colorless oil. Yield 1.1 g (95%) ¹ H-NMR (CDCl ₃ ) δ: 2.41-2.90
(4H, m), 3.81 (3H, s), 6.86-6.
90 (2H, m), 7.47-7.52 (2H, m),
8.47 (1H, bs) SIMS (m / z): 269 (M + H) ⁺

Reference Example 11 Compound 23 (7.9 g) was treated with trifluoroacetic acid in the same manner as in Reference Example 9 to give 2- (2,4-difluorophenyl) thio-5-oxo-2-tetrahydrofurancarboxylic acid ( Compound 30 ) was obtained. Crystallization from isopropyl ether gave colorless prism crystals. Yield 4.23 g (86%) Melting point 88-89 ° C NMR (CDCl ₃ ) δ: 2.40-3.00 (4H,
m), 6.79-7.02 (2H, m), 7.50-
7.70 (1H, m). Elemental analysis: C ₁₁ H ₈ F ₂ O ₄ S Calculated: C, 48.18; H, 2.94 Found: C, 48.46; H, 3.22

Reference Example 12 Compound 26 (2.4 g) was added to dichloromethane (60 ml).
And m-chloroperbenzoic acid (4.4 g) was added thereto, followed by stirring at room temperature for 3 hours. The reaction solution was filtered to remove insolubles, and 5% sodium bicarbonate water (100 ml) and dichloromethane (60 ml) were added to the filtrate. Separate the dichloromethane layer,
After washing with water (30 ml) and drying (MgSO ₄ ), the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (2.9 × 50 cm, eluent, dichloromethane). The desired fraction was concentrated and isopropyl ether was added to the residue to give 2- (4-fluorophenyl) sulfonyl-5-oxo-2-tetrahydrofurancarboxylic acid
The benzyl ester (compound 31 ) was obtained as colorless needles. Yield 0.82 g (32%) Melting point 129-131 ° C. ¹ H-NMR (CDCl ₃ ) δ: 2.61-3.38
(4H, m), 5.10 (1H, d, J = 12 Hz),
5.21 (1H, d, J = 12 Hz), 6.99-7.
07 (2H, m), 7.23-7.42 (5H, m),
7.66-7.72 (2H, m) Elemental analysis: C ₁₈ H ₁₅ FO ₆ S · １ ／ H ₂ O Calculated: C, 56.46; H, 4.04 Actual: C, 56 .45; H, 3.94

REFERENCE EXAMPLES 13-16 In the same manner as in Reference Example 12, the compounds shown in Table 5 were oxidized with m-chloroperbenzoic acid to give the corresponding sulfones (compounds 32 , 33 , 42 , 45). )was gotten.

[0043]

[Table 12]

[Table 13]

[Table 14]

Reference Example 17 Compound 24 (1.2 g) was dissolved in methanol (30 ml), and 5% palladium-carbon (0.8 g) was added. The catalytic reduction was carried out at room temperature under normal pressure for 45 minutes. The catalyst was removed by filtration through Celite, and the catalyst was washed with a small amount of methanol. The methanol portion was combined and evaporated under reduced pressure. Dichloromethane (20 ml) was added to the residue, followed by drying (MgSO ₄ ). After distillation under reduced pressure, hexane was added to the residue to give 2- (4-fluorophenoxy) -5-oxo-2-tetrahydrofurancarboxylic acid (compound 34 ) as colorless needles. Yield 0.64 g (74%) Melting point 122-124 ° C ¹ H-NMR (CDCl ₃ ) δ: 2.54-2.83
(4H, m), 6.15 (1H, bs), 6.93-
7.12 (4H, m) Elemental analysis: C ₁₁ H ₉ FO ₅ .1 / 4H ₂ O Calculated: C, 54.00; H, 3.88 Found: C, 54.22; H, 3.88

[0045] Reference Example 18-20 in the same manner as in Reference Example 17, and catalytic reduction of compounds shown in Table 6 by deprotection reaction when the carboxylic acid derivative (Compound 35 -
37 ) was obtained.

[0046]

[Table 15]

[Table 16]

Reference Example 21 Compound3(0.55 g) in dimethylformamide (15
ml), ethyl iodide (0.6 ml) and potassium carbonate.
Add lithium (3.42 g) and stir at room temperature for 3 hours
Was. The reaction solution was added to water (100 ml), and ethyl acetate was added.
(100 ml) twice. Combine the ethyl acetate layers
And washed with water (100 ml) and dried (MgSO _Four) After decompression
Distilled off. Silica gel column chromatography of the residue
-(2.9 × 30 cm, eluate, ethyl acetate: hexane)
= 1: 1). Concentrate the target fraction and concentrate on the residue.
Addition of xane gives 2-phenoxy-5-oxo-2-te
Trahydrofurancarboxylic acid ethyl ester (compound3
8) Was obtained as colorless needle crystals. Yield 0.48 g (79%) Melting point 82-83 ° C¹ H-NMR (CDCl_Three) Δ: 1.12 (3H, t,
J = 7 Hz), 2.49-2.91 (4H, m), 4.
09-4.31 (2H, m), 7.07-7.12 (3
H, m), 7.24-7.32 (2H, m) Elemental analysis: C₁₃H₁₄O_FiveCalculated: C, 62.39; H, 5.64 Found: C, 62.37; H, 5.61

[0048] is reacted with ethyl iodide compound shown in the same manner as in Reference Example 22-26 Example 21 in Table 7, the corresponding ethyl ester (compound <br/> compound 39 - 41, 43 - 44) was gotten.

[0049]

[Table 17]

[Table 18]

[Table 19]

[Table 20]

Embodiment 1 Ingredients (1), (2) and 17 g of ingredient (3) are mixed and granulated with a paste made from 7 g of ingredient (3), and 5 g of ingredient (3) and ingredient (4) are added to the granules. After mixing, the mixture was compressed with a compression tablet machine, and 100 mg of a tablet having a diameter of 7 mm containing 10 mg of the component (1) per tablet.
0 pieces are manufactured.

Embodiment 2 After mixing the above components (1), (2), (3), and (4), the mixture is granulated according to a conventional method. The component (5) is added thereto, and the mixture is encapsulated in a gelatin capsule according to a conventional method to prepare a capsule.

──────────────────────────────────────────────────続 き Continuation of front page (56) References JP-A-64-34976 (JP, A) JP-A-60-58973 (JP, A) JP-A-2-193987 (JP, A) JP-A-2- 40323 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) A61K 31/365 A61P 37/06 C07D 307/33 CA (STN) REGISTRY (STN)

Claims (1)

(57) [Claims] 1. A compound of the general formula (I): (Wherein R ¹ represents a phenyl group which may be substituted,
² represents a carboxy group which may be esterified, and X represents an oxygen atom or a sulfur atom which may be oxidized)
An immunosuppressant comprising a compound represented by the formula: