JPS6340200B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to cyclosporine derivatives which are monocyclic undecapeptide derivatives, processes for their production and pharmaceutical compositions containing them.

The present invention is based on the formula [] [In the formula, A is or or It is. ] Cyclosporin G, dihydrocyclosporin G or isocyclosporin G shown in the following is provided.

The present invention provides the following steps: a. In the production of cyclosporin G, a cyclosporin G-producing strain belonging to the genus Tolypocladium is cultured in the presence of a nutrient medium, and then cyclosporin G is produced.
b. In the production of dihydrocyclosporin G, cyclosporin G is reduced; or c. In the production of iso-cyclosporin G,
Rearranges cyclosporin G.

Provided is a method for producing cyclosporin G, dihydrocyclosporin G or iso-cyclosporin G, comprising:

Cultivation according to method a may be carried out by known methods for culturing related bacterial strains, for example as described in Example 1.

A preferred cyclosporin G-producing strain is a strain of Tolypocladium inflatum Gams. A preferred strain is freely available and is described in British Patent Specification No. 1451509. This is the United States in Peoria, Illinois, United States.
States Department of Agriculture
(Northern Research and Development
Division) with the code NRRL8044, and the code FERM P No. 2796 with the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, Yatabe-cho, Tsukuba-gun, Ibaraki Prefecture, Japan. This strain was previously known as Trichoderma polysporium (Trichoderma polysporium).
polysporium (Link ex Pers) and has now been reclassified as the Tolypocladium inflatum gums strain.

Other cyclosporin G-producing strains of Tolypocladium inflatum gums can be obtained by, for example, screening, mutation of the above-mentioned preferred strains under the action of ultraviolet or X-ray radiation, or reaction of said strains with suitable chemicals. It may be manufactured by.

Cyclosporin G may be isolated by known methods for isolating fermentation products from culture broth, such as those described in Example 1.

Purification conditions may also include other natural products that may be present, such as the less polar cyclosporin D,
and the more polar cyclosporins A and B (S7481/F-1 and S7481/F-2, respectively)
The choice should be made to separate cyclosporin G from the more polar cyclosporin C), also known as cyclosporin C).

Method b may be carried out using conventional reducing methods, such as catalytic hydrogenation.

Suitable solvents include ethyl acetate or lower alcohols such as methanol, ethanol or isopropanol. It is advantageous to use neutral reaction conditions. A suitable reaction temperature is 20-30°C at atmospheric or slightly elevated pressure. Suitable catalysts include platinum or preferably palladium, such as palladium on activated carbon.

Method c may be carried out by conventional methods for the rearrangement of analogous cyclosporins, eg under acid conditions. Preference is given to using strong organic acids, such as trifluoroacetic acid, methanesulfonic acid or p-toluenesulfonic acid.

The amount of strong acid present is advantageously from 1 to 4 mol per mole of cyclosporin G.

As solvent, for example methanol, chloroform or dioxane may be used. Advantageously, the reaction temperature is between 20 and 65°C, preferably between 45 and 55°C.

In the examples below, all temperatures are in degrees Celsius and are uncorrected. All percentage ratios are in parts by volume unless otherwise noted. Silica gel is, for example, silica gel 60 (Merck, particle size 0.063-0.2
mm).

Example 1: Cyclosporin G Nutrient Solution 500 (Glucose 40g per 1
Sodium caesinate 2g,
Ammonium phosphate 2.5g, MgSO ₄ 7H ₂ O 5g,
KH ₂ PO ₄ 2g, NaNO ₃ 3g, KCl 0.5g,
Preculture of strain NRRL8044 in 0.01 g of FeSO ₄ and deionized water to 1
and then incubated for 13 days at 27° C. in a steel fermenter under agitation (170 rpm) and aeration (1 air/min/nutrient solution) (see also the UK patent specification mentioned above).

This culture broth is stirred and mixed with the same volume of n-butyl acetate. The organic layer is separated, concentrated in vacuo, then partitioned between methanol/water (9:1) and petroleum ether. In order to further remove fatty substances, the methanol phase is shaken two more times with petroleum ether. The methanol phase is concentrated in vacuo and water is added to precipitate the product, which is collected. The product is chromatographed on a 5-7 volume Sephadex LH20 with methanol as eluent. The main fraction was then chromatographed on silica gel using hexane/acetone (2:1) as eluent to obtain a first fraction containing mainly cyclosporins A and D (as well as some cyclosporin G). A fraction and a fraction containing cyclosporin C are obtained.

The first fractions are each treated with 2-2.5 volumes of acetone to form crystals at -15°C. The crystals are chromatographed twice on silica gel using hexane/acetone (2:1) as eluent, resulting in a first fraction containing cyclosporin D (as well as some cyclosporin G).
Dissolve these fractions in twice the volume of acetone and -
Crystallize at 15℃. The crystals are crude cyclosporin D. The mother liquor contains cyclosporin G in addition to cyclosporin D, and is concentrated and dried. The residue is chromatographed on silica gel using water-saturated ethyl acetate as eluent. After eluting cyclosporin D, the fraction further contains cyclosporin G, which was first eluted on silica gel with chloroform/methanol (98:2).
and then chromatographed using hexane/acetone (2:1) for separation. The latter fraction contains cyclosporin G in purified form, which is crystallized twice from ether/petroleum ether (1:1) at room temperature. The final product is
Uniform and 95% pure by thin layer chromatography
% (melting point (after drying the crystals at 80° C. for 2 hours in high vacuum)) of 193-194° C., colorless polyhedrons.

[α] ²⁰ _D = -245° (C = 1, in CHCl ₃ ) [α] ²⁰ _D = -191° (C = 1.04, in CH ₃ OH) UV spectrum, in CH ₃ OH: edge absorption IR spectrum, In CH ₂ Cl ₂ : see Figure 1 ¹ H-NMR spectrum, in CDCl ₃ , 90 MHz (tetramethylsilane as internal standard): see Figure 2 Chemical formula: C ₆₃ H ₁₁₃ N ₁₁ O ₁₂ Example 2: Hydrocyclosporin G Palladium/Activated Carbon (Palladium 10W/W%)
700 mg are hydrogenated in 20 ml of ethanol for 30 minutes. Cyclosporine G 4.55g and ethanol 60g
Add ml of solution. The mixture was heated to 21℃ and 742mmH.
Hydrogenation is carried out at a pressure of g until hydrogen absorption is complete. The catalyst is removed and the liquid is evaporated to dryness in vacuo at 20-40°C. Residual dihydrocyclosporin G
is obtained in the form of a white amorphous powder, pure by TLC and having a melting point of 150-153°C (after drying at 80°C in high vacuum for 4 hours).

[α] ²⁰ _D = -232° (C = 0.64, in CHCl ₃ ) UV spectrum, in CH ₃ OH: edge absorption IR spectrum, in CH ₂ Cl ₂ : see Figure 3 ¹ H-NMR spectrum, in CDCl ₃ , 90MHz (Tetramethylsilane as internal standard solution): See Figure 4 Chemical formula: C ₆₃ H ₁₁₅ N ₁₁ O ₁₂ Example 3: Iso-Cyclosporin G 6.08 g of Cyclosporin G and 40 ml of anhydrous dioxane
A solution of 1.2 g of methanesulfonic acid and 20 ml of anhydrous dioxane was added to the solution, and then heated at 50°C under anhydrous conditions.
to hold. Thin layer chromatography on silica gel [Polygram SILG foil,
CHCl ₃ /CH ₃ OH/acetic acid (90:6:4), detected with iodine]. After 16 hours, the mixture is cooled to room temperature. Add 1.13 g of anhydrous sodium acetate to neutralize the above acid. After 45 minutes, the mixture is filtered and the liquid is evaporated in vacuo at 45°C. 8.1 g of the residue was added to 500 g of silica gel as an eluent.
Chromatograph using CHCl ₃ /CH ₃ OH (98:2). The fractions containing substantially pure isocyclosporin G are collected and evaporated to dryness in vacuo at 50°C, and the residue is then crystallized from ether at 7°C to give the title compound, mp 143-146°C.

[α] ²⁰ _D = -196° (C = 0.72, in CHCl ₃ ) [α] ²⁰ _D = -128° (C = 0.73, in CH ₃ OH) UV spectrum, in CH ₃ OH: edge absorption IR spectrum, In CH ₂ Cl ₂ : see Figure 5 ¹ H-NMR spectrum, in CDCl ₃ , 90 MHz (tetramethylsilane as internal standard solution) : see Figure 6 The compound of formula [ ] exhibits pharmacological action. especially,
The compound exhibits anti-inflammatory and anti-arthritic effects as shown by suppression of swelling in the Freund-Adjuband supplemental arthritis test in rats at oral doses of 30-100 mg/Kg.

The compounds therefore find use in the treatment and prevention of chronic inflammations such as arthritis and rheumatic diseases.

Furthermore, the compounds exhibit an immuno-suppressive effect, for example due to the effects of humoral and cellular immunity, which is demonstrated, for example, in the following standard tests.

a Proliferation rate, blast formation and H ³ − of mouse splenic lymphocytes stimulated with concanavalin A in a lymphocyte stimulation test using the Janossy method at concentrations of 0.01 to 10.0 μg/ml in vitro.
Strong inhibition of thymidine uptake was confirmed.

b Oxazolone test in mice: A reduction in ear swelling is seen upon oral administration of 5 x 70 mg/Kg of the compound.

c Local hemolysis of gels: Jerne test [JFBorel, Agents and Actions (1974),
Volume 4, page 277]. Suppression of hemolytic plug-forming cells, immunoglobulin antibodies and immunoglobulin G ₂ antibodies is seen at a dose of 3 x 50 mg/Kg.

The compounds therefore find use in the treatment of autoimmune diseases.

For these uses, the indicated daily dosage ranges from about 50 to about 900 mg, in unit dosage forms containing from about 12 to about 450 mg in divided doses two to four times per day, and in sustained release form. , it is advantageous to administer.

The present invention also provides a pharmaceutical composition comprising a compound of formula [ ] in combination with a pharmaceutical carrier or diluent.

Such compositions may be in the form of solutions or tablets, for example.

Its preferred effect is immunosuppressive effect.

The compound of Example 2 exhibits a particularly interesting effect.

[Brief explanation of the drawing]

FIG. 1, FIG. 3 and FIG. 5 show the IR spectra of the compounds of the present invention obtained in Examples 1, 2 and 3, respectively, and FIGS. 1 shows the ¹ H-NMR spectra of the compounds of the present invention obtained in steps 2 and 3.

Claims (1)

[Claims] 1 Formula [], [In the formula, A is or or It is. ] Cyclosporin G, dihydrocyclosporin G or isocyclosporin G represented by these. 2 The following summary properties: [α] ²⁰ _D = -245° (C = 1, in CHCl ₃ ) [α] ²⁰ _D = -191° (C = 1.04, in CH ₃ OH) UV spectrum, in CH ₃ OH : Edge absorption IR spectrum, in CH ₂ Cl ₂ : See Figure 1 ¹ H-NMR spectrum, in CDCl ₃ , 90MHz (tetramethylsilane as internal standard solution): See Figure 2 Chemical formula: C ₆₃ H ₁₁₃ N ₁₁ O ₁₂ , with a melting point of approximately 193-194℃ in crystalline form
The compound according to claim 1, which is cyclosporin G, which is more polar than cyclosporin D and less polar than cyclosporin A. 3. The compound according to claim 1, which is cyclosporin G substantially free of cyclosporin D. 4. The compound according to claim 1, which is cyclosporin G with a purity of over 95%. 5 The following general properties: [α] ²⁰ _D = -232° (C = 0.64, in CHCl ₃ ) UV spectrum, in CH ₃ OH: edge absorption IR spectrum, in CH ₂ Cl ₂ : see Figure 3 ¹ H- NMR spectrum, 90 MHz in CDCl ₃ (tetramethylsilane as internal standard): see Figure 4 Chemical formula: C ₆₃ H ₁₁₅ N ₁₁ O ₁₂ , melting point 150-153 in amorphous powder form
The compound according to claim 1, which is dihydrocyclosporin G obtained by hydrogenating cyclosporin G as defined in claim 2 at °C. 6. The compound according to claim 1, which is isocyclosporin G. 7a In the production of cyclosporin G,
A cyclosporin G-producing strain belonging to the genus Tolypocladium was cultured in the presence of a nutrient medium, and then cyclosporin G
b. In the production of dihydrocyclosporin G, cyclosporin G is reduced; or c. In the production of iso-cyclosporin G,
By rearranging cyclosporin G, the formula [], [In the formula, A is or or It is. ] A method for producing a cyclosporin derivative, which comprises obtaining cyclosporin G, dihydrocyclosporin G or isocyclosporin G shown in the following. 8 formula [], [In the formula, A is or or It is. ] A pharmaceutical composition having an immunosuppressive effect comprising a combination of a cyclosporin derivative of cyclosporin G, dihydrocyclosporin G or isocyclosporin G represented by the following formula and a pharmaceutical carrier or diluent. 9 Cyclosporin derivatives have the following general properties: [α] ²⁰ _D = −245° (C = 1, in CHCl ₃ ) [α] ²⁰ _D = −191° (C = 1.04, in CH ₃ OH) UV spectrum; In CH ₃ OH: edge absorption IR spectrum, in CH ₂ Cl ₂ : see Figure 1 ¹ H-NMR spectrum, in CDCl ₃ , 90MHz (tetramethylsilane as internal standard solution): see Figure 2 Chemical formula: C ₆₃ H ₁₁₃ N ₁₁ O ₁₂ with a melting point of approximately 193-194°C in crystalline form
The composition according to claim 8, wherein the composition is cyclosporin G, which is more polar than cyclosporin D and less polar than cyclosporin A. 10. The composition according to claim 8, wherein the cyclosporin derivative is cyclosporin G substantially free of cyclosporin D. 11. The composition according to claim 8, wherein the cyclosporin derivative is cyclosporin G with a purity of over 95%. 12 The cyclosporin derivative has the following general characteristics: [α] ²⁰ _D = -232° (C = 0.64, in CHCl ₃ ) UV spectrum, in CH ₃ OH: edge absorption IR spectrum, in CH ₂ Cl ₂ : Fig. 3 Reference ¹ H-NMR spectrum, 90 MHz in CDCl ₃ (tetramethylsilane as internal standard): see Figure 4 Chemical formula: C ₆₃ H ₁₁₅ N ₁₁ O ₁₂ , melting point 150 ~ in amorphous powder form 153
9. The composition according to claim 8, which is dihydrocyclosporin G obtained by hydrogenating cyclosporin G as defined in claim 2 at .degree. 13. The composition according to claim 8, wherein the cyclosporin derivative is isocyclosporin G. 14 formula [], [In the formula, A is or or It is. ] A pharmaceutical composition having an anti-inflammatory effect, comprising a combination of a cyclosporin derivative of cyclosporin G, dihydrocyclosporin G, or isocyclosporin G represented by the following formula and a pharmaceutical carrier or diluent. 15 The cyclosporin derivative has the following general properties: [α] ²⁰ _D = −245° (C = 1, in CHCl ₃ ) [α] ²⁰ _D = −191° (C = 1.04, in CH ₃ OH) UV spectrum; In CH ₃ OH: edge absorption IR spectrum, in CH ₂ Cl ₂ : see Figure 1 ¹ H-NMR spectrum, in CDCl ₃ , 90 MHz (tetramethylsilane as internal standard solution): see Figure 2 Chemical formula: C ₆₃ H ₁₁₃ N ₁₁ O ₁₂ with a melting point of approximately 193-194°C in crystalline form
The composition according to claim 14, which is cyclosporin G, which is more polar than cyclosporin D and less polar than cyclosporin A. 16. The composition according to claim 14, wherein the cyclosporin derivative is cyclosporin G substantially free of cyclosporin D. 17 Claim 14, wherein the cyclosporin derivative is cyclosporin G with a purity of over 95%
Compositions as described in Section. 18 The cyclosporin derivative has the following general properties: [α] ²⁰ _D = -232° (C = 0.64, in CHCl ₃ ) UV spectrum, in CH ₃ OH: edge absorption IR spectrum, in CH ₂ Cl ₂ : Figure 8 Reference ¹ H-NMR spectrum, 90 MHz in CDCl ₃ (tetramethylsilane as internal standard): see Figure 4 Chemical formula: C ₆₃ H ₁₁₅ N ₁₁ O ₁₂ , melting point 150 ~ in amorphous powder form 153
15. The composition according to claim 14, which is dihydrocyclosporin G obtained by hydrogenating cyclosporin G as defined in claim 2 at °C. 19. The composition according to claim 14, wherein the cyclosporin derivative is isocyclosporin G.