JPH0764797B2 - 2-Aminopentanoic acid compound and use thereof as immunosuppressant - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to 2-aminopentanoic acid compounds useful as a medicine, particularly as an immunosuppressant.

[Prior art]

In recent years, cyclosporine has been used to suppress the rejection reaction that occurs during organ transplantation. So-called immunosuppressants, including compounds under development, are expected to be further used as therapeutic agents for rheumatoid arthritis and the like. However, the cyclosporine has a problem that it causes side effects such as renal damage.

On the other hand, Japanese Patent Laid-Open No. 1-104087 discloses that Cordyceps sinensis (Isar)
ia sinclairii) is described in US Pat.
Formulas disclosed in the specification (2S, 3R, 4R)-(E) -amino-3,4 represented by
-Dihydroxy-2-hydroxymethyl-14-oxoeicosa-6-enoic acid has been confirmed.

[Problems to be Solved by the Invention]

An object of the present invention is to provide novel 2-aminopentanoic acid compounds which exhibit an excellent immunosuppressive action and have few side effects.

[Means for Solving the Problems]

The present invention has the general formula [Wherein, R is an alkyl, hydroxyalkyl, wherein _{-CH = CH (CH 2) n} CH = CH-CH (OH) - with (CH _{2) m} CH ₃ (wherein, n, m are each from 1 to 10 . represents an _{integer), -CH = CH (CH 2} ) p -C (= X) - (CH 2) q CH 3 ( wherein,> C = X group (A represents alkylene),> C = N-OB (B represents hydrogen or alkyl) or> CHNH ₂ .
p and q each represent an integer of 1 to 10. )) Or -CH
Represents a group represented by ═CH—Y (Y represents alkyl). ] The 2-aminopentanoic acid compound represented by these, its salt, or the compound protected by the protective group.

Or the present invention has the general formula (Wherein R has the same meaning as above).
It relates to a lactone compound or a compound protected by a protecting group thereof.

Regarding R in the present specification, as alkyl, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, isopentyl, neopentyl,
Hexyl, octyl, 2-ethylhexyl, decyl, dodecyl, tetradecyl, hexadecyl, octadecyl,
Examples of the linear or branched alkyl having 1 to 30 carbon atoms such as eicosyl and docosyl include hydroxymethyl, 2-hydroxyethyl, and 3 as hydroxyalkyl.
-Hydroxypropyl, 1-hydroxymethylethyl,
Examples thereof include hydroxyalkyl having 1 to 6 carbon atoms such as 4-hydroxybutyl and 6-hydroxyhexyl (the alkyl part may be branched).

With respect to the substituent = X in the present specification, examples of the alkylene represented by A include alkylene having 1 to 4 carbon atoms such as methylene, ethylene, trimethylene, propylidene and tetramethylene, and the alkyl represented by B is Examples thereof include linear or branched alkyl having 1 to 4 carbon atoms such as methyl, ethyl, propyl, isopropyl, butyl, and tertiary butyl.

Regarding Y in the present specification, alkyl has the same meaning as the alkyl described for R.

Further, in the compound of the general formula (I) and the γ-lactone compound of the general formula (Ia), the protecting group of the compound protected by the protecting group is widely used in organic chemistry with respect to a hydroxyl group or an amino group. Examples thereof include acyl such as acetyl and benzoyl, oxycarbonyl such as tertiary butoxycarbonyl, and tetrahydropyranyl.

Examples of the salt of the compound of the general formula (I) include metal salts such as sodium salt, potassium salt, calcium salt, zinc salt and aluminum salt, salts with amines such as triethylamine, salts with amino acids such as lysine and ornithine. Examples thereof include pharmacologically acceptable salts. It also includes hydrates and other solvates, and further includes individual optical isomers, diastereoisomers, and racemates.

Preferred compounds of the general formula (I) are as follows.

The compound of the general formula (I) of the present invention can be produced by a fermentation method or the like (2S, 3R, 4R)-(E) -2-amino-3,4-
It can be prepared by using dihydroxy-2-hydroxymethyl-14-oxoeicosa-6-enoic acid (hereinafter referred to as ISP-I) as a starting material. Further, for example, the compound (a) can be produced by a fermentation method, but the derivative capable of producing the compound (a) belongs to Ascomycetes and Imperfect fungi, specifically, to Imperfect bacteria. Examples include Isaria, Myceria, and Myriococcum (Chierabia), which belong to Ascomycetes, and are of the American Type Culture Collection (America).
an Type Culture Collection) Isaria sinclairii TACC No.24400, Myriococcum albomyces ATC
C No.16425, Mycelia ster
ilia) ATCC No.20349 has been deposited. Myriococcum arbomyces ATCC No. 16425 has been deposited as IFO32292 at the Fermentation Research Institute (Osaka).

The compound (a) can also be produced by a mutant strain obtained by modifying the above-mentioned strain by an artificial mutagenesis method using commonly used ultraviolet rays, high-frequency radiation, chemicals and the like.

The compound (a) -producing bacterium can be cultivated in various culture media containing a usual fungal nutrient source. For example, glucose, starch, glycerin, sugar syrup, dextrin as a carbon source,
Molasses, maltose, xylose, etc., and corn steep liquor, peptone, yeast extract, potato decoction, broth, soybean flour, wheat germ, potassium nitrate, sodium nitrate, ammonium sulfate, casein, gluten meal, cottonseed flour, feather dust as a nitrogen source. Inorganic or organic nitrogen compounds such as, and other usual inorganic salts and additives that are commonly used in culture such as organic and inorganic substances and defoamers that help the growth of fungi and promote the production of compound (a) It can be added appropriately.

The culture method is not particularly limited, but an aerobic deep culture method is suitable. The temperature suitable for culturing is 20 to 35 ° C., preferably 25 to 30 ° C. in the case of a bacterium belonging to the genus Isaria, and 30 to 50 in the case of a bacterium belonging to the genus Myriococcum or Myceria, preferably Is 35 to 45 ° C.

The compound (a) produced in the culture is extracted from the culture by appropriately combining commonly used operations such as extraction and adsorption as necessary. For example, in the case of a bacterium belonging to the genus Isaria such as Isaria cinclaili, the insoluble matter such as bacterial cells is filtered from the culture solution, separated by a method such as centrifugation, and the culture filtrate is passed through Amberlite XAD-2, It is taken out by adsorbing the compound (a). The compound (a) thus obtained is further eluted with, for example, methanol, and the eluate is further subjected to reverse phase chromatography to fractionate to obtain a highly purified product of the compound (a). Also, in the case of bacteria belonging to the genus Myriococcum or Mycelia such as Myriococcum arbomyces, Mycelia sterilia, the bacterial cells are separated from the culture solution by a method such as centrifugation and the culture filtrate is separated from the genus Isaria. Do the same. On the other hand, from the separated bacterial cells,
The compound (a) is extracted with methanol, the extract is treated with Amberlite XAD-2 in the same manner as the filtrate, and purified by chromatography and recrystallization to obtain the compound (a).

The compound (b) is a γ-lactone derivative derived from ISP-I (protected by a protecting group), for example, a compound represented by the formula: (In the formula, Ac represents an acetyl group.) The compound represented by the general formula is reacted with alkanedithiol in the presence of boron trifluoride. (Wherein Ac has the same meaning as above and A represents alkylene), and then Raney nickel,
By reacting with a reduction reaction using palladium carbon, the formula (Wherein Ac is as defined above), and then the lactone ring is cleaved by alkaline hydrolysis.

The compound (c) is produced by subjecting the compound of the general formula (III) (wherein A represents ethylene) to an alkaline hydrolysis reaction, and the compound (d) is produced by reacting ISP-I with hydroxylamine. It The alkoxime corresponding to the compound (d) can also be obtained by reacting ISP-I with O-alkylhydroxylamine or by reacting the oxime with dialkylsulfate, alkyl iodide or the like. Further, the oxime form can be converted into an amine compound by a conventional method. The compound (d) or the corresponding alkoxime has anti- and syn-geometric isomers, but the present invention also includes these individual isomers and a mixture thereof.

The compound (e) is obtained by reducing the 14-position of the compound (II) to hydroxy with a metal hydride complex compound such as sodium borohydride and lithium aluminum hydride, and then subjecting it to an alcohol to give 1 It is reacted with 1,1′-thiocarbonyldiimidazole to form a 1-imidazolylthiocarbonyloxy substituent at the 14-position, and the substituent is eliminated by radical reduction with, for example, tributyltin hydride, followed by alkaline hydrolysis. It is produced by subjecting it to a decomposition reaction. Alternatively, a secondary hydroxyl group can be converted into trifluoromethanesulfonate and a deoxygenation reaction with sodium borohydride or the like can be applied.

Compound (f) is a γ-lactone form of ISP-I, that is, compound I
It can be obtained by subjecting I to an ozonolysis reaction or a 1,2-diol formation reaction with osmium oxide, followed by reaction with sodium periodate, and then reduction with sodium borohydride or the like.

The compound of the general formula (I) in which R is —CH═CH—Y is produced by subjecting ISP-I to an ozonolysis reaction and then reacting it with a Wittig reagent.

The thus obtained compound can be converted into the corresponding γ-lactone form by treating with an acid such as hydrochloric acid or acetic acid or a tertiary alcohol such as a tertiary pentyl alcohol.

The above reaction proceeds in the absence of a solvent or in the presence of a solvent inert to the reaction, under cooling, at room temperature or under heating for about 5 minutes to 50 hours.

The compound of the general formula (I) of the present invention, that is, the compound of the general formula (I), that is, the carboxylic acid compound can be converted into the above-mentioned salt by a conventional method. Further, various isomers can also be produced by optically resolving a cerami body or a diastereomer, or by using an optically active raw material.

[Action / effect]

The 2-aminopentanoic acid compounds of the present invention exhibit an excellent immunosuppressive action, and suppress rejection of mammals such as humans, cows, horses, dogs, mice and rats, for example, in organs or bone marrow transplantation. Agents and rheumatoid arthritis, systemic lupus erythematosus, Sjogren's disease, multiple sclerosis, myasthenia gravis, type I diabetes, endocrine eye disorders, primary biliary cirrhosis, Crohn's disease, glomerulonephritis, sarcoidosis, psoriasis, pemphigus It can be used as a preventive or therapeutic agent for autoimmune diseases such as aplastic anemia, idiopathic thrombocytopenic purpura, and allergies, or as a reagent in medicine and pharmacy.

These compounds can be mixed with a carrier, an excipient, a diluent and the like to be formulated into a powder, a capsule, a tablet, an injection, etc. and administered to a patient. Alternatively, a freeze-dried preparation may be prepared by means known per se.

The dose of these compounds may vary depending on the disease, symptoms, body weight, sex, age, etc. For example, in order to suppress the rejection reaction in renal transplantation, it is usually 0.01 to 10 mg per day for an adult.
The (titer) is administered once or several times a day.

[Experimental Example / Example]

Hereinafter, the operation and effects of the present invention will be described in more detail with reference to experimental examples and the like. The immunosuppression activity was measured by the following method.

As the activity measuring method, various immune reactions using mouse, rat or human lymphocytes can be used. For example, the immunosuppressive activity is the mixed allogeneic lymphocyte reaction of mouse, rat and human (allogeneic MLR ), It is possible to measure with high sensitivity. Allogeneic MLR is a lymphocyte derived from two individuals of the same species but different in major histocompatibility antigen,
For example, it is a blast transformation reaction induced by mixed culture of splenocytes, lymph node cells, peripheral blood lymphocytes and the like. This type of MLR is a phenomenon that is induced by reflecting differences in major histocompatibility antigens among donors of lymphocytes. For example, lymphocyte blastogenesis by monoculture of monozygotic twin lymphocytes. It is not allowed. So homogeneous ML
R is a widely used method for donor-recipient selection in organ transplants, for example.

Normally, when performing allogeneic MLR, one lymphocyte is
Use as a stimulating cell in a state where mitotic proliferation is blocked by performing radiation or mitomycin C treatment, and using a method (one way-MLR) for measuring the blastogenic response of the other lymphocyte (reactive cell) You can

Furthermore, the immunosuppressive activity can also be measured as the activity of suppressing the induction of cytotoxic T cells having major histocompatibility antigen-restricted properties induced by allogeneic MLR.

In addition, immunosuppressive activity, in addition to allogeneic MLR, activity to suppress the blastogenic reaction of lymphocytes induced by stimulation of various mitogens (concanavalin A, phytohemagglutinin, porkweed mitogen, etc.), Alternatively, lymphocytes induced by cytokines (interleukin 1, 2, 3, 4, 5, 6 etc.) having an activity of enhancing mitotic proliferation or promoting differentiation of lymphocytes such as T cells and B cells. It can also be evaluated as the activity of suppressing the division-proliferation reaction or expression of the function. Further, it can be evaluated as the activity of suppressing the production of these cytokines from T cells, macrophages and the like.

Furthermore, by intraperitoneally, orally, intravenously, intradermally, subcutaneously or intramuscularly administering a compound to a mouse or the like, for example, allogeneic cell-specific cells induced in splenocytes of a mouse previously immunized with allogeneic cells or the like. Activity of suppressing the induction of impaired T cells, and activity of suppressing the production of allogeneic cell-specific antibodies produced in the serum of mice immunized with allogeneic cells, or rejection upon organ transplantation of allogeneic mice,
Alternatively, it can also be evaluated as the activity of suppressing graft-versus-host reaction, delayed allergy, adjuvant arthritis, or the like.

In addition, by administering the compound to MRL / lpr mouse, NZB / WF ₁ mouse, BXSB mouse, NOD mouse, etc., which are model animals of autoimmune disease, for example, production of anti-DNA antibody, production of rheumatoid factor, nephritis, lymph It can also be evaluated as abnormal proliferation of spheres, inhibitory activity against urinary protein, etc. or life-prolonging effect.

Experimental Example 1 (Immunosuppressive action of immunosuppressive substance) The immunosuppressive activity of a compound was measured using a mouse allogeneic lymphocyte mixed reaction (hereinafter, also referred to as MLR). The mouse allogeneic MLR used BALB / c mouse (H-2 ^d ) splenocytes as responsive cells and C57BL / 6 mouse (H-2 ^b ) splenocytes as stimulator cells treated with mitomycin C. It was carried out by mixed culture with.

The reaction cells were prepared by the following method. 5
Spleens were excised from BALB / c mice aged 6 weeks and heat-inactivated fetal calf serum (hereinafter also referred to as FCS) was 5%.
Added RPMI1640 medium (kanamycin sulfate 60 μg / ml, L
-Glutamine 2 mM, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonate (HEPES) 10 mM, 0.1
% Sodium bicarbonate) was used to obtain a single cell suspension of splenocytes. After hemolysis treatment, using the RPMI1640 medium containing 10 ^-4 M 2-mercaptoethanol and 20% FCS, 10 ⁷
The cells / ml were prepared and used as a reaction cell suspension.

The stimulated cells were prepared by the following method. The spleen was extracted from 5 to 6-week-old male C57BL / 6 mice, and a single cell suspension of splenocytes was obtained using RPMI1640 medium. 40 after hemolysis
It was treated with μg / ml mitomycin C at 37 ° C. for 60 minutes. After washing 3 times, the cells were prepared at 10 ⁷ cells / ml using RPMI1640 medium containing 10 ⁻⁴ M 2-mercaptoethanol and 20% FCS, and used as a stimulator cell suspension.

50 μl of reaction cell suspension, 50 μm of stimulatory cell suspension, and 100 μm of test substance prepared by the above method were added to a 96-well flat-bottom microtest plate, and 5% carbon dioxide gas was added at 37 ° C.
Culture was performed for 4 days under the condition of% air.

As a method for measuring the blast transformation reaction of lymphocytes, a method using ³ H-thymidine incorporation as an index was used. That is, after the completion of the culture, ³ H-thymidine 18.5 KBq / well was added, and after 4 hours of culture, cells were collected by a cell harvester, and radioactivity incorporated into the cells was measured by a liquid scintillation counter. It was used as an index of lymphocyte blastogenesis in allogeneic MLR. The suppression of mouse allogeneic MLR was evaluated by calculating the suppression rate by the following formula. The results are shown in Table 1A.

The test sample was dissolved in methanol and then diluted with RPMI1640 medium before use. Methanol was used at 0.01% or less, and in this case, no influence was observed on the same-type MLR.

For the compound of Example 1, the compound (b) of Example 2, the compounds of Examples 3 and 4, the compound (e) of Example 5 and the compound (f) of Example 6 from 1 μg / ml to 0.0001 μg / ml
As a result of measuring lymphocyte blastogenic activity in mouse allogeneic MLR at a final concentration in the range of, as shown in Table 1A,
These compounds were concentration-dependent on mouse allogeneic MLR,
The inhibitory effect was shown.

The concentration of the compound of Example 1, the compound of Example 2 (b), the compounds of Examples 3 and 4, the compound of Example 5 (e), and the compound of Example 6 (f) that suppresses mouse allogeneic MLR by 50%. (IC ₅₀ ) is 1.3 × 10 ^-2 μg / ml and 1.6 × 10 ^-2 μg / ml, respectively.
ml, 2.5 × 10 ^-1 μg / ml, 2.5 × 10 ^-2 μg / ml, 2.5 × 10 ^-4 μg
/ ml and 4.5 × 10 ^-2 μg / ml. In addition, as is clear from the results in Table 1B, Example 5
The sodium salt of the compound (e) in Example 5 also showed a mouse allogeneic MLR inhibitory effect to the same extent as the compound (e) in Example 5.

As a method for measuring the lymphocyte blast transformation reaction, the following 3- (4,5-dimethylthiazol-2-yl) -2,5 was further added.
A dye quantification method using diphenyltetrazolium bromide (MTT) can also be used.

After culturing, remove 100μ of supernatant from each well, and add 5mg / mlM
Add 20 μ each of TT solution to each well and incubate at 37 ° C. for 4 hours. Then containing 10% sodium dodecyl sulfate
Add 100μ of 0.01N hydrochloric acid solution and let stand at 37 ℃ overnight.
Dissolve the purple formazan crystals that formed and use a microplate absorptiometer (immunoreader) to measure 570n
The absorbance at m is measured and used as an index of lymphocyte blastogenesis in mouse allogeneic MLR. The suppression of allogeneic MLR in the mouse can be evaluated by calculating the suppression rate by the following formula.

Experimental Example 2 (inhibitory effect on human allogeneic MLR) The test for inhibitory effect on human allogeneic MLR was carried out by the following method.

RPMI1 supplemented with 10% FCS from human peripheral blood lymphocytes obtained by separating normal human peripheral blood by Ficoll-pack density gradient centrifugation
Suspend in 640 medium, spread on a plastic dish, 5
The cells were cultured at 37 ° C. for 2 hours under the condition of 95% carbon dioxide gas.
After completion of the culture, light pipetting was performed, and the supernatant was collected and centrifuged (1000 rpm, 5 minutes) to obtain plastic non-adherent cells. Plastic non-adherent cells
After making the cells non-adherent to nylon by the nylon wool column passage method, 4x1 in RPMI1640 medium containing 10% FCS.
0 was prepared in ⁶ / ml, was used as a reaction cell suspension.

Plastic-adherent cells also contained 5% FCS and 0.02
It was obtained by adding phosphate-buffered saline (PBS) containing 1% disodium ethylenediamine tetraacetate (EDTA), vigorously pipetting, and peeling. In addition, plastic-adherent cells were mitomycin C 40 μg / ml at 6 ° C at 6 ° C.
After treatment for 0 minutes, washed 3 times, then RPMI with 10% FCS
It was adjusted to 4 × 10 ⁶ cells / ml using 1640 medium and used as a stimulator cell suspension. Donor A prepared by the method described above
Alternatively, 50 μl of the reaction cell suspension from C and 50 μl of the stimulation cell suspension from donor B or D were mixed respectively, and 100 μl of the test substance was added to the mixture, and 5% carbon dioxide gas was added at 37 ° C.
The culture was carried out for 5 days under the condition of 5% air.

After completion of the culture, add 37 KBq / well of ³ H-thymidine, and add 18
After culturing for a period of time, cells were collected by a cell harvester and the radioactivity incorporated into the cells was measured by a liquid scintillation counter, which was used as an index of human allogeneic MLR lymphocyte blastogenesis. The suppression of human allogeneic MLR was evaluated by calculating the suppression rate using the following formula. The results are shown in Tables 2A and 2B.

Compound of Example 1, Compound (b) of Example 2, Example 3
1 μm for the compound of Example 1 and the compound (e) of Example 5
Human allogeneic MLR at final concentrations ranging from g / ml to 0.0001 μg / ml
The inhibitory activity of the lymphocyte blastogenic reaction in the was measured. As a result, as shown in Tables 2A and 2B, these compounds showed an inhibitory effect on human allogeneic MLR in a concentration-dependent manner.

Experimental Example 3 (inhibitory effect on allogeneic cytotoxic T cells induced by mixed culture of mouse allogeneic lymphocytes (MLC)) BALB / c mouse (H- prepared by the same method as in Experimental Example 1
2 ^d ) 0.5 ml of splenocyte suspension (2 × 10 ⁷ cells / ml) and 0.5 ml of mitomycin C-treated C57BL / 6 mouse (H-2 ^b ) splenocyte suspension (2 × 10 ⁷ cells / ml) and 1.0 ml of the sample was added to a 24-well multi-dish and cultured at 37 ° C. for 6 days under the conditions of 5% carbon dioxide and 95% air.

After culturing, collect cells by centrifugation and add R containing 10% FCS.
It was adjusted to 5 × 10 ^{6 to} 6.25 × 10 ⁵ cells / ml in PMI1640 medium and used as effector cells.

As target cells, C57BL / that is syngeneic (H-2 ^b ) with the stimulator cells
6 mouse-derived leukemia cells, EL4 were used. 10 ⁶ EL4 cells
After incorporating ⁵¹ Cr into the cytoplasm by incubating at 37 ° C. for 1 hour with 370 MBq of Na ₂ ⁵¹ CrO ₄ ,
The cells were washed, adjusted to 10 ⁴ cells / ml and used as target cells.

Effector cell suspension 100μ is used to measure the cytotoxic activity.
Then, 100 μl of the target cell suspension was added to a 96-well flat-bottomed microtest plate and incubated at 37 ° C. for 4 hours, and the amount of ⁵¹ Cr released in the supernatant was measured. It was calculated. The results are shown in Table 3A.

The cytotoxic T cells induced by the above method are
Stimulated cells (H-2 ^b) and syngeneic LE4 cells (H-2 ^b) with respect showed strong cytotoxic activity, allogeneic Meth A cells (H-2 ^d), P815 cells (H- 2 ^d), BW5147 cells (H-
Since it did not show any cytotoxic activity against 2 ^k ), it was suggested to be H-2 ^b- restricted allogeneic cytotoxic T cells. The activity of cytotoxic T cells was indicated by lytic unit. Lytic unit (LU) contains 5 × 10 ³ target cells 25
% LU is shown per 10 ⁶ effector cells when the number of effector cells required for destruction is 1 LU.

As shown in Table 3A, the addition of the compound (b) of Example 2 and the compound (e) of Example 5 resulted in homologous cytotoxic T
It was revealed that the induction of cells was remarkably suppressed.

Also, the C3H / HeN mice (H-2 ^k) reaction cells Spleen cell suspensions, mitomycin C-treated C57BL / 6 mice (H
-2 ^b ) The induction of allogeneic cytotoxic T cells induced when the splenocyte suspension was used as stimulator cells was also suppressed by the addition of the compound (e) of Example 5 (Table 3B).

Experimental Example 4 (inhibitory effect on interleukin 2 (IL-2) and IL-3 production in mitogen-stimulated mouse splenocytes) IL-2 and IL by mitogen-stimulated mouse splenocytes
The test of the effect on the production of -3 was performed by the following method.

Spleen was removed from 5-8 week old male C3H / HeN mouse and RPM was used.
A single cell suspension of splenocytes was obtained using I1640 medium. After hemolytic treatment, 10 ⁷ cells / ml using RPMI1640 medium containing 20% FCS
And phytohemagglutinin (PHA) 10 μg / ml was added as a mitogen. 1 ml of this cell suspension,
Add 1 ml of the sample to a 24-well multi-dish, 37 ℃, 5
The cells were cultured for 24 hours under the conditions of 95% carbon dioxide and 95% air. After the culture was completed, the supernatant was collected and used as a PHA-stimulated mouse splenocyte culture supernatant.

IL-2 activity in the culture supernatant of PHA-stimulated mouse splenocytes was measured as follows. IL-2-dependent mouse cell line CTLL-2
Was prepared at 2 × 10 ⁵ cells / ml using RPMI1640 medium containing 30% FCS. 96 of the cell suspension (100 μ) and 100 μ of the above-mentioned PHA-stimulated mouse splenocyte culture supernatant diluted 2-fold serially
Wells were added to flat bottom microtest plates. After culturing for 44 hours under the conditions of 37 ° C., 5% carbon dioxide gas and 95% air, the absorbance was measured by the dye quantification method using MTT in the same manner as in Experimental Example 1, and the index of IL-2 dependent cell proliferation was obtained. And IL
-2 activity was expressed in U / ml from the dilution concentration giving 50% of the maximum value of absorbance by the MTT method.

IL-3 activity was measured as follows.

RPMI containing 10% FCS in the IL-3-dependent mouse cell line FDC-P2
It was adjusted to 2 × 10 ⁵ cells / ml using 1640 medium. 100 µl of the cell suspension and 100 µl obtained by serially diluting the culture supernatant of the PHA-stimulated mouse spleen cells in two cultures were added to a 96-well flat-bottomed micro test plate, and the conditions were 37 ° C, 5% carbon dioxide gas 95% air for 44 hours. Cultured. After culturing, MT as in the above method.
Absorbance was measured by a dye quantification method using T, and IL-3
It was used as an index of dependent cell proliferation. IL-3 activity was expressed in U / ml from the dilution concentration giving 50% of the maximum value of the absorbance by the MTT method.

As shown in Tables 4A and 4B, the compounds of the present invention have PH
It was suggested to suppress the production of IL-2 and IL-3 in A-stimulated mouse splenocytes.

Experimental Example 5 (Inhibitory effect on IL-2 and IL-3 production in mouse allogeneic lymphocyte mixed culture (MLC)) Mouse allogeneic MLC was performed as follows.

That is, 0.5 ml each of the reaction cell suspension and the stimulating cell suspension prepared in the same manner as in Experimental Example 1 was added to a 24-well multi-dish in which 1 ml of the test sample had been placed in advance, and 37 ° C, 5% carbon dioxide gas was added. The cells were cultured under the conditions for 2 days. After completion of the culture, the supernatant was collected and used as the supernatant of mouse allogeneic MLC.

IL-2 and IL-3 activity in the supernatant of mouse allogeneic MLC was
It measured by the method similar to Experimental example 4.

As shown in Tables 5A and B, the compounds of the present invention were suggested to suppress the production of IL-2 and IL-3 in mouse allogeneic MLC.

Experimental Example 6 (Inhibitory effect on proliferation of IL-2 dependent mouse cell line CTLL-2 induced by interleukin 2 (IL-2)) IL-2 dependent mouse cell line, CTLL-2 at 30% FCS Including R
It was adjusted to 4 × 10 ⁵ cells / ml in PMI1640 medium. 50 μl of this cell suspension and recombinant human IL-2 (rhIL-2)
50 μ of 200 U / ml and 100 μ of a liquid sample were added to a 96-well flat bottom micro test plate. 37 ° C, 5% carbon dioxide 95
After culturing for 68 hours under the condition of% air, the absorbance was measured by the dye quantification method using MTT by the same method as in Experimental Example 1, and used as an index of IL-2 dependent cell proliferation. The inhibition rate (%) was calculated by the following formula.

The results are summarized in Tables 6A and 6B.

As is clear from Tables 6A and B, the compounds of the present invention are
It was suggested to strongly suppress the mitotic proliferation of CTLL-2 cells induced by recombinant human IL-2.

Experimental Example 7 (IL-1, IL-2 and IL- of mouse thymocytes
Suppressive effect on 4 response) Serum-free RPM by removing thymus from male 6-week-old C3H / HeN mice
A single cell suspension was obtained using I1640 medium. After washing 3 times, 1
RPM containing 0 ^-4 M 2-mercaptoethanol and 20% FCS
I1640 medium was used to prepare a cell concentration of 1.5 × 10 ⁷ cells / ml, 100 μl of this cell suspension and 100 μl of a test substance were added to a 96-well flat-bottom microtest plate, and phytohemagglutinin (PHA) 1 μg / ml And recombinant human IL-1
In the presence of α, recombinant human IL-2, or recombinant mouse IL-4, the cells were cultured for 54 hours under the conditions of 37 ° C., 5% carbon dioxide gas 95% air. After completion of the culture, ³ H-thymidine 18.5 KBq / well was added, and after 18 hours of culture, the cells were collected by a cell harvester, and the radioactivity incorporated into the cells was measured by a liquid scintillation counter, and IL
-1, IL-2 or IL-4 was used as an index of response. IL-
1, suppression of IL-2 or IL-4 response was evaluated by calculating the suppression rate by the following formula.

The results are shown in Tables 7A, B and C.

As is clear from these tables, the compounds of the present invention have IL-
It was suggested to strongly suppress the mitotic proliferation of mouse thymocytes induced by 1, IL-2 or IL-4.

Experimental Example 8 (inhibitory effect on mouse anti-sheep erythrocyte antibody production) Male BALB / c mice aged 5 to 7 weeks were treated with sheep erythrocytes (5 x 10
⁷ mice / mouse, intravenous administration)
G / kg / day was intraperitoneally administered for 4 consecutive days from the day of immunization. Four days after immunization, the spleen was extracted, and the inhibitory effect on anti-sheep erythrocyte antibody production was examined by measuring the number of plaque forming cells (PFC) by the direct plaque method using sheep red blood cells and guinea pig complement. At this time, the weight of mice, the wet weight of thymus and spleen, and the number of splenocytes were also measured. The results are shown in Table 8.

As shown in Table 8, the compound (b) of Example 2 had an inhibitory effect on anti-sheep erythrocyte antibody production, that is, PFC number per unit splenocyte number and PFC number per total splenocyte number (1
It is clear that it has the effect of reducing (× 10 ⁷ pieces).

Experimental Example 9 (inhibitory effect on induction of allogeneic cytotoxic T cells by allogeneic cell immunization of mice) Male BALB / c mice (H-2 ^d ) aged 5 to 7 weeks were treated with C57BL / 6.
Mice (H-2 ^b) origin of leukemia cells EL4 (10 ⁷ cells / mouse, i.p.) were immunized with the subject from the immune day 0.00
Intraperitoneally 5 times at a dose of 1 to 3 mg / kg / day or 0.3 to 10 mg / kg
Orally administered at a dose of / day. The spleen was excised from the mouse 9 to 10 days after immunization to give a single cell suspension, which was then used as an effector cell. In the same manner as in Experimental Example 3, a ⁵¹ Cr release test using EL4 cells as target cells was carried out to measure the cytotoxic activity.

The activity of allogeneic cytotoxic T cells was represented by lytic unit. For the Lytic unit (LU), the number of effector cells required to destroy 5 × 10 ³ target cells by 25% was set to 1 LU, and LU per spleen was shown. At this time, the weight of the mouse and the number of splenocytes were also measured.

The results are shown in Tables 9A and B.

As shown in Tables 9A and B, the compound (b) of Example 2 strongly induces allogeneic cytotoxic T cells in splenocytes of allogeneic immunized mice by intraperitoneal or oral administration 5 times. Obviously it suppresses. On the other hand, at this time, neither suppression of body weight increase nor significant decrease in splenocyte count was observed.

Also, as is clear from the results of Tables 9C to F, the compound (e) of Example 5 and its sodium salt were allergic cell damage in splenocytes of allogeneic immunized mice by intraperitoneal or oral administration 5 times. Orally suppresses the induction of sex T cells 10
A single dose showed a significant inhibitory effect up to a dose of 0.03 mg / kg.

As shown in Table 9F, the sodium salt of the compound (e) of Example 5 induced intraperitoneal administration of the compound of Example 5 (e) against the induction of allogeneic cytotoxic T cells in spleen cells of allogeneic cell-immunized mice. ) Has almost the same inhibitory action as compounds other than the sodium salt. In addition, a significant inhibitory effect was observed up to a dose of 0.03 mg / kg by intravenous administration.

Experimental Example 10 (inhibitory effect on allocytotoxic specific antibody production by allogeneic cell immunization of mice) Male BALB / c mice aged 5 to 7 weeks were treated with C5 in the same manner as in Experimental Example 7.
Immunize with 7BL / 6 mouse-derived leukemia cells EL4 (10 ⁷ cells / mouse, intraperitoneal injection), and subject 0.001 to 3 mg / k from the day of immunization
Intraperitoneal or oral administration was performed at a dose of g / day, and serum was collected from the mouse 9 to 10 days after immunization. 50 μ of the obtained serum diluted 2-fold and guinea pig complement of 50 μ
And ⁵¹ Cr-labeled EL4 cell suspension (5 × 10 ⁴ cells / ml, 100μ) as target cells were added to a 96-well microtest plate, incubated at 37 ° C for 1 hour, and then released into the supernatant. The amount of ⁵¹ Cr produced was measured, and the complement-dependent cytotoxic activity was calculated from the following formula.

The homologous cytotoxic specific antibody titer in serum, which was measured by the above method, was calculated from the dilution concentration that gave 50% of ⁵¹ Cr release when ⁵¹ Cr-labeled cells were treated with 0.1N HCl.
It was displayed by calculating r).

As shown in Tables 10A to 10E, it was revealed that intraperitoneal or oral administration of the compound of the present invention strongly suppressed the production of allogeneic cytotoxic specific antibody in serum of allogeneic cell-immunized mice. .

As shown in Table 10E, the sodium salt of the compound (e) of Example 5 against the production of the homologous cytotoxic specific antibody in the serum of allogeneic cell-immunized mice was administered intraperitoneally to the compound (e) of Example 5 It has almost the same inhibitory action as compounds other than the sodium salt of. In addition, 0.01 mg by intravenous administration
A significant inhibitory effect was observed up to a dose of / kg.

Experimental Example 11 (Suppressive effect on mouse acute graft-versus-host reaction (GVHR)) C57BL / 6 mouse splenocyte suspension (2 × 10 ⁷ cells / mouse) was applied to the right footpad of 5 to 7-week-old female BDF ₁ mice. 7 days after the injection, the right knee extremity lymph node was removed and weighed.
It was used as an index of acute graft-versus-host reaction (GVHR) by comparing with the weight of the left knee popliteal lymph node of the control (saline injection). From the day of the induction injection, the compound (e) of Example 5 was added to 6
Intraperitoneal administration of 0.3 mg / kg daily, oral administration of 1 mg / kg, and
Intramuscular administration of 0.3 and 1 mg / kg was performed, and on the 7th day, the right knee extremity lymph node weight was measured.

As a result, as shown in Table 11, it was revealed that intraperitoneal, oral or intramuscular administration of the compound (e) of Example 5 strongly suppressed the mouse acute graft-versus-host reaction (GVHR). Became.

Experimental Example 12 (Study of Cytotoxicity to Various Cultured Tumor Cell Lines) The study of cytotoxicity to various human-derived cultured tumor cell lines was carried out as follows.

Human-derived cultured tumor cell lines K562, MOLT4, U937 and HL6
0 × 4 × 10 ⁵ / RPMI1640 medium containing 20% FCS
Prepared to ml. 50 μL of this cell suspension was added to each well of a 96-well microtest plate in which 50 μL of the test liquid had been put in advance. After culturing at 37 ° C. under 5% carbon dioxide gas for 72 hours, the absorbance at 570 nm was measured by the same method as the dye quantification method using MTT in Experimental Example 1. The inhibition rate was calculated by the following formula and used as an index of cytotoxicity.

As is clear from the results shown in Tables 12A to 12C, the compounds of the present invention have weak cytotoxicity to various human-derived cultured tumor cell lines, and the concentration (IC ₅₀ ) that gives 50% inhibition is
It was 10 μg / ml or more.

Example 1 (i) Jar culture of arbomyces bacterium GCY medium (per one, 20 g of glucose, 5 g of corn steep liquor, 3 g of yeast extract, 0.5 g of MgSO ₄ .7H ₂ O, pH 6)
Dispense 100 ml into two 500 ml cervix long shake flasks, and
After sterilizing in an autoclave for 20 minutes at 1 ° C, about 1 cm ² of mycelium of Myriococcum arbomyces ATCC No.16425 grown on potato dextrose agar was inoculated into each flask, and 40 ° C for 3 days reciprocal shaking culture ( 135 rpm, swing width
8 cm). Using the obtained culture solution as a seed mother, the above G
A CY medium and a defoaming agent (F-18 manufactured by Dow Corning Co., Ltd.) (1 g) were inoculated into a 10-volume jar fermenter, and aerated agitated culture (0.5 VVM, 300 rpm) was performed at 40 ° C. for 7 days.

(Ii) Collection of Compound (a) from Culture Solution of Arbomyces bacterium The culture solution 4.5 obtained in (i) was separated into bacterial cells and culture filtrate by filtration. Culture filtrate (4) is Amberlite XA
After passing through D-2 (φ40 mm × h750 mm) to adsorb the compound (a), water 1 was passed through for washing. Next, 30% methanol solution 1, 50% methanol solution 1 and 80% methanol solution 3 were sequentially passed through to collect an 80% methanol elution fraction containing compound (a).

On the other hand, the bacterial cells should be mixed with about 5 times the wet weight of methanol.
After extraction twice, water is added to the extract to make a 30% methanol solution, and the solution is passed through Amberlite XAD-2 (φ40 mm x h750 mm) to adsorb the compound (a) and 30% methanol solution 1
, 50% methanol solution 1, 80% methanol solution 3
Was sequentially passed through and 80% methanol elution fraction containing compound (a) was collected.

80 from the culture filtrate and cells obtained as described above
The% methanol elution fractions were combined, concentrated under reduced pressure and freeze-dried to obtain 0.5 g of a powder containing the compound (a).

(Iii) Purification of compound (a) 0.5 g of the powder obtained in (ii) is dissolved in methanol, and the same amount of water as methanol is added to make a 50% methanol solution.
This solution is Amberlite XAD-2 (φ25mm × h300mm)
To 50%, 60%, 70%, and 75% methanol (500 ml each) in sequence, and 70 to 75% containing compound (a).
The methanol solution fraction was collected. Water was added to this eluted fraction to make a 50% methanol solution, and Amberlite XAD was added again.
-2 column chromatography for purification to obtain 28 mg of compound (a).

mp 145 to 147 ° C. ¹ H-NMR δ (ppm CD ₃ OD): 5.55 (m), 5.40 (m), 3.87
(M), 2.30 (t) IR ν (KBr): 3300, 1625, 970 cm ^-1 Example 2 Compound (2-i) (1509.5 mg) in dry dichloromethane (12.5 ml)
1,2-ethylenedithiol (367 μm), boron trifluoride etherate (12.5 μm) were added, and the mixture was stirred at room temperature for 47 hours. The reaction mixture was washed with 5% sodium bicarbonate and saturated sodium chloride, dried over magnesium sulfate, and concentrated under reduced pressure to give an oily residue. This is a silica gel (50g) column (developing solvent:
Separation with ethyl acetate: n-hexane = 6: 4, thin layer chromatography (developing solvent: ethyl acetate: n-hexane = 7:
The fraction showing Rf value 0.4 in 3) was concentrated under reduced pressure to obtain 1538.9 mg of a colorless transparent oily compound (2-ii).

¹ HNMR (200MHz, in CDCl ₃ , Ref.TMS) δ: 1H, 6.1 (br.s.,
-NHAc), 1H, 5.8 (d., J = 4.64,3-H), 1H, 5.6 (dt-t.
like, J = 15.38,6.47,7-H), 1H, 5.4 (dt-t.like, J = 1
5.38,6.60,6-H), 1H, 4.7 (ddd., J = 8.18,5.37,4.64,4
-H), 2H, 4,5 (s ., 21-H 2), 4H, 3.3 (s., 1 ', 2'-
H ₂ ), 1H, 2.4 (ddd., J = 14.89,8.18,6.60,5-H), 1H, 2.
3 (ddd., J = 14.89,6.60,5,37,5-H), 3H, 2.1 (s., A
c), 3H, 2.05 (s, Ac), 3H (s., 2.03), 2H, 2.0 (m., 8−
H ₂ ), 4H, 1.9 (m., 13,15−H ₂ ), 16H, 1.55 (m), 1.30 (b
rs), 1.29 (br.s.) (9-12,16-19-H 2), 3H, 0.9
EI-MS m / z (t , J = 6.47,20-H 3.): 585,500,440,398,198,160,43 1422.0 mg of compound (2-ii) was dissolved in 40 ml of dry ethanol, 22 g of Raney nickel was added, and the mixture was heated under reflux for 90 minutes. After cooling the reaction solution to room temperature, Raney nickel was filtered off. This Raney nickel was washed with 200 ml of ethanol, and the washings were combined with the filtrate and concentrated under reduced pressure to give a solid residue 1062.7.
to obtain mg. To this, 5.0 ml of acetic anhydride and 5.0 ml of pyridine were added, and the mixture was stirred overnight at room temperature. The reaction solution was diluted with water and extracted with ethyl acetate. The ethyl acetate layer was washed successively with 1N hydrochloric acid, 5% sodium bicarbonate and saturated sodium chloride, and dried over magnesium sulfate. White foamy residue obtained by concentration under reduced pressure 946.
1 mg was separated on a silica gel (33 g) column. Developing solvent:
Elution with ethyl acetate: n-hexane = 7: 3, thin layer chromatography (developing solvent: ethyl acetate: n-hexane = 7:
Fractions showing an Rf value of 0.4 in 3) were collected and concentrated under reduced pressure to obtain 862.2 mg of the target compound (2-iii) as a white foamy substance.

¹ HNMR (200MHz, in CDCl ₃ , Ref.TMS) δ: 1H, 6.0 (br.s.,
-NHAc), 1H, 5.8 (d., J = 4.39,3-H), 1H, 4.7 (dt., J
= 9.03,4.40,4-H), 2H, 4.53 (s., 21-H 2), 3H, 2.10
(S., Ac), 3H, 2.05 (s, Ac), 3H, 2.03 (s., Ac), 4H, 1.65
_{(M., 5,6-H 2} ), 26H, 1.26 (br.s., 7-19-H), 3H, 0.88
(T., J = 6.47,20-H) MS (EI-MS), m / z: 497,454,365,322,129,43 (3) Compound (2-iii) → step of compound (b) Compound (2-iii) 81.9 mg Dissolved in 4.0 ml of methanol,
After adding 0.99 ml of 1N aqueous sodium hydroxide solution, the mixture was refluxed overnight under a nitrogen stream. The reaction solution is neutralized with 1N aqueous hydrochloric acid, the white insoluble matter that has precipitated is suction filtered, and washed with methanol and water. After drying, recrystallization from methanol gave 16.1 mg of compound (b) as white crystals.

mp 159.0-161.0 ℃ ¹ HNMR (200MHz, in CD ₃ OD) δ: 1H, 4.0 (d., J = 11.12,21
-H), 1H, 3.9 (d., J = 11.12,21-H), 1H, 3.8 (br.t.,
J = 7.08,4-H), 1H, 3.7 (d., J = 1.22,3-H), 4H, 1.6
(M), 26H, 1.3 (br.s., 5-19-H), 3H, 0.9 (t., J = 6.
47,20-H) MS (FAB), m / z: 390,372,291,277,165,104 Compound (2-i) is produced as follows.

To 8.10 g of ISP-I, 65 ml of pyridine was added at room temperature, and 70 ml of acetic anhydride was added with vigorous stirring. Continue stirring for about 1 hour,
After ISP-I was completely dissolved, stirring was stopped and the mixture was left overnight. Ice water was added to the reaction solution, and the mixture was extracted with ethyl acetate, washed successively with 1N hydrochloric acid, 5% sodium hydrogen carbonate and saturated aqueous sodium chloride solution, and the organic layer was dried. The oily residue (10.08 g) obtained by distilling off the solvent was separated by a silica gel column (300 g) using ethyl acetate: hexane = 7: 3 as a developing solvent. 900-170
When the 0 ml eluate was combined and concentrated under reduced pressure, compound (2-i) 9.2
9 g was obtained as a colorless transparent oily substance. (Yield 91.4%).

IR ν _max (CHCl ₃ ) (cm ⁻¹ ): 2930,1760,1690,1380,1220 ¹ HNMR (200MHz, in CDCl ₃ , Ref: TMS), δ: 6.05 (1H, br.
s., -NHAc), 5.79 (1H, d, J = 4.4Hz, 3-H), 5.57 (1H, d
t, t−like, J = 15.4,6.3Hz, 7−H), 5.38 (1H, dt, t−lik
e, J = 15.4,6.4Hz, 6-H), 4.72 (1H, ddd, J = 7.6,5.1,4.
6Hz, 4-H), 4.51 (2H, s, 21-H 2), 2.5~2.2 (2H, m, 5-
H ₂ ), 2.38 (4H, t, J = 7.5Hz, 13−, 15−H ₂ ), 2.10 3H, s,
-Ac), 2.05 (3H, s, -Ac), 2.03 (3H, s, -Ac), 2.1 to 1.
9 (2H, m, 8−H ₂ ), 1.55 (4H, qui., J = 6.8Hz, 12,16−
H ₂ ), 1.27 (12H, br.s, 9〜11,17〜19−H ₂ ), 0.88 (3H,
t., J = 6.5 Hz, 20−H ₃ ) ¹³ C-NMR (100 MHz, in CDCl ₃ , Ref: TMS), δ: 211.37 (s,
14-C), 172.32 (s,> C = 0), 170.08 (s,> C =
0), 169.27 (s,> C = 0), 168.75 (s,> C = 0), 13
5.04 (d, 6-C), 123.16 (d, 7-C), 81.62 (d, 4-
C), 72.01 (d, 3-C), 62.75 (t, 21-C), 62.73 (s, 2
-C), 42.85,42.77 (each t, 13,15-C), 32.46,32.2
0,31.63 (each t, 5-, 8-18-C), 29.11,29.00,28.97,
28.91,23.91,23.85 (each t, 9-12,16,17-C), 22.76
(Q, -COMe), 22.50 (t, 19-C), 20.55 (q, -COMe), 2
0.31 (q, -COMe), 14.00 (q, 20-C) EI-MS m / z: 509,491,282,348,279,129,43 HREI-MS: C ₂₇ H ₄₃ NO ₈ , calculated value: 509.2990, measured value:
509.2998 Example 3 Synthesis of compound (c) 72.6 mg of compound (2-ii) was dissolved in 2.8 ml of methanol, and 1
After adding 0.75 ml of N sodium hydroxide, the mixture was refluxed overnight under a nitrogen stream. The reaction solution was neutralized with 1N hydrochloric acid, and the solvent was evaporated under reduced pressure. Water was added to this, and the insoluble matter was collected by suction filtration. This was washed with water, air-dried, and then dried in a desiccator under reduced pressure. The white substance (23.3 mg) thus obtained was dissolved in methanol while heating, the insoluble matter was filtered off, and the solvent of the filtrate was distilled off. Ethyl acetate was added to the obtained white residue, the insoluble matter was filtered off with suction, and dried to obtain 12.8 mg of compound (c) as a white substance.

mp 167.0-170.0 ° C ¹ HNMR (200MHz, in CD ₃ OD) δ: 1H, 5.5 (dt−t.like, J _d ＝
15.14, J _t = 6.27,7-H), 1H, 5.4 (dt-t.like, J _d = 14.8
9, J _t = 6.60,6-H), 1H, 4.0 (d., J = 10.99,21-H), 1
H, 3.9 (dJ = 10.99,21-H), 1H, 3.84 (br.t., J = 7.00,
4-H), 1H, 3.79 (d., J = 0.97, 3-H), 4H, 3.3 (s.,
1 ', 2'-H), 2H, 2.3 (br.t., J = 6.47,5-H), 2H, 2.0
(Dt., J _d = 6.35, J _t = 6.35,8−H), 4H, 1.9 (m., 13,15−
H), 4H, 1.5 (m., 12,16-H), 12,1.3 (m., 9-11,17-1)
9-H), 3H, 0.9 (t., J = 6.60,20-H) Example 4 Synthesis of compound (d) Sodium carbonate 26.9 mg was dissolved in water 1.5 ml, hydroxylamine hydrochloride 35.0 mg, and ethanol 3.5 ml. Was added. This mixture is heated in a water bath at 70 ° C and ISP-1200.9m
g was added and shaken until dissolved. After ISP-I was completely dissolved, it was further heated at the same temperature for 6 hours. After confirming that the pH of the reaction solution was 7, it was concentrated under reduced pressure to dryness. Water is added to this residue and the insoluble matter is filtered off with suction. 183.6 mg of the obtained white substance is dissolved in 9 ml of methanol while heating, and the mixture is returned to room temperature and left standing overnight. The precipitated white-white insoluble matter (unreacted ISP-I) was filtered off, and the residue obtained from the filtrate was recrystallized to give a white substance compound (d) (8
7.8 mg) was obtained.

mp 179.5 to 180.5 ° C [α ▲] ²⁸ _D ▼ = −5.78 ° (c = 0.509, methanol), MS (EI-MS) m / z: 398,328,256,128,104,18 Example 5 589.4 mg of compound (2-i) was dissolved in 26 ml of dioxane,
10 ml of water was added. While stirring at room temperature, add a few small pieces of dry ice, and after the solid disappeared, sodium borohydride
116 mg was added. Do the same operation again, 20 minutes later, 1N
Hydrochloric acid was added to make it acidic (pH 2 or less). After stirring for 10 minutes, 1N
After neutralizing the weak acid with sodium hydroxide, the mixture was concentrated under reduced pressure to remove dioxane. Water was added to the residue and the mixture was extracted with ethyl acetate. The ethyl acetate layer was washed successively with 5% sodium bicarbonate and saturated sodium chloride, and dried over magnesium sulfate. 589.6 mg of the residue obtained by concentration under reduced pressure was separated by a silica gel (30 g) column. Elute with chloroform: methanol = 98.2,
Fractions showing Rf0.4 were collected by thin layer chromatography (developing solvent chloroform: methanol = 9: 1) and concentrated under reduced pressure to obtain 386.3 mg of the target compound (5-i) as a colorless transparent oily substance. For the fractions before and after containing the target substance, chloroform: methanol =
Rf0.4 was applied to preparative thin layer chromatography using 95: 5, ethyl acetate: n-hexane = 8: 2 as a developing solvent.
The target substance (30.2 mg) was obtained as a colorless transparent oily substance from the band.

¹ HNMR (200MHz, in CDCl ₃ , Ref TMS) δ: 1H, 6.1 (br.s.,
-NHAc), 1H, 5.8 (d., J = 4.64,3-H), 1H, 5.6 (dt-t,
like, J = 15.38,6.47,7-H), 1H, 5.4 (dt-t.like, J = 1
5.38,6.59,6-H), 1H, 4.7 (ddd., J = 7.56,5.37,4.64,4
H), 2H, 4.5 (s ., 21-H 2), 1H, 3.6 (m, 14-H), 1H, 2.5
(Ddd., J = 14.77,7.56,6.59,5-H), 1H, 2.4 (ddd., J =
14.77,6.59,5.37,5-H), 3H, 2.10 (s., Ac), 3H, 2.05
(S., Ac), 3H, 2.03 (s., Ac), 2H, 1.99 (m, 8-H), 20H,
1.3 (m, 9-13, 15-19-H), 3H, 0.9 (t., J = 6.42, 20-H
₃ ) EI-MS m / z: 511,493,426,366,332,263,129,43 386.6 mg of compound (5-i) was dried with 1,2-dichloroethane 2.
Dissolve in 1 ml, 1,1'-thiocarbonyldiimidazole 27
3.7 mg was added. The solution was warmed in a 70 ° C. oil bath for 2 hours and then cooled in an ice water bath for 10 minutes. 20 ml of 1,2-dichloroethane was added to the yellow transparent reaction solution, 10 ml of water, 10 m of 1N hydrochloric acid.
l (twice), washed successively with 5% baking soda 10ml, water 10ml, 1,2-
The dichloroethane layer was dried over magnesium sulfate. The residue (451.9 mg) obtained by concentration under reduced pressure was separated by a silica gel (18 g) column. Elute with chloroform: methanol = 98: 2,
Fractions showing Rf0.6 were collected by thin layer chromatography (developing solvent chloroform: methanol = 9: 1) and concentrated under reduced pressure to obtain 265.7 mg of the target compound (5-ii) as a colorless transparent oily substance. For the fractions before and after containing the target substance, chloroform: methanol =
Preparative thin-layer chromatography was performed using 95: 5 as a developing solvent, and the target substance (5-ii)
21.0 mg was obtained as a colorless transparent oily substance.

¹ HNMR (200MHz, in CDCl ₃ , Ref.TMS) δ: 1H, 8.4 (br.s.,
Imidazole ring), 1H, 7.6 (br.s., imidazole ring), 1
H, 7.0 (d., J = 0.74, imidazole ring), 1H, 6.3 (br.s.,
-NHAc), 1H, 5.8 (d., J = 4.64,3-H), 1H, 5.62 (qui.,
J = 5.94, 14-H), 1H, 5.56 (br, dt., J = 15.38, 6.35, 7-
H), 1H, 5.4 (dt.like, J = 15.38,6.66,6-H), 1H, 4.7
(Dt., J = 8.06,4.95,4- H), 2H, 4.5 (s., 21-H 2), 2H,
2.4 (m., 5-H 2), 3H, 2.10 (s., Ac), 3H, 2.05 (s., Ac),
3H, 2.03 (s., Ac ), 2H, 2.00 (m, 8-H 2), 4H, 1.55 (m., 1
3,15−H ₂ ), 16H, 1.31 (br.s., 9-12,16-19−H ₂ ), 3H,
0.88 EI-MS m / z ( t, J = 6.48,20-H 3.): 493,433,374,332,263,141,129,43 Compound (5-ii) (60.0 mg) was dissolved in dry benzene (4.5 ml), tributyltin hydride (41 µ) and 2,2'-azo (isobutyronitrile) (5.0 mg) were added, and the mixture was refluxed overnight. The yellow oily residue obtained by concentration under reduced pressure was partitioned between n-hexane and acetonitrile, and the acetonitrile layer was concentrated under reduced pressure. 58.6 mg of this residue was subjected to preparative thin layer chromatography (developing solvent diethyl ether: n-hexane = 9:
(1 double development), and 21.2 mg of the desired compound (5-iii) was obtained as a colorless transparent oily substance from the band of Rf0.4.

[Α] _D = + 63.8 ° (C = 0.994, CHCl ₃ ) ¹ HNMR (200MHz, in CDCl ₃ , Ref TMS) δ: 1H, 6.0 (br.s.,
-NHAc), 1H, 5.8 (d., J = 4.39,3-H), 1H, 5.6 (dt-t.
like, J = 15.26,6.59,7-H), 1H, 5.4 (dt-t.like, J = 1
5.26,7.08,6-H), 1H, 4.7 (ddd., J = 7.08,5.62,4.40,4
-H), 2H, 4.5 (br.s. , 21-H 2), 2H, 2.4 (m., 5-H 2), 3
H, 2.10 (s., Ac), 3H, 2.05 (s., Ac), 3H, 2.03 (s., Ac),
2H, 2.01 (m, 8- H 2), 22H, 1.3 (br.s., 9-19-H 2), 3H,
0.9 EI-MS m / z ( t, J = 6.35,20-H 3.): 495,436,376,334,265,151,129,43 (4) Compound (5-iii) → Compound (e) the compound (5-iii) 40.4mg methanol 2ml Melted, 1N
0.50 ml of sodium hydroxide was added, and the mixture was refluxed overnight under a nitrogen stream. The mixture was neutralized with 1N hydrochloric acid, and the precipitated white substance was collected by suction filtration and washed with water. On the other hand, the mother liquor was concentrated under reduced pressure and dried to dryness, water was added thereto, insoluble matter was collected by suction filtration, washed with water and recrystallized from methanol. Both insoluble materials were combined to obtain 19.4 mg of compound (e) as a white powder substance.

mp 186 ° C [α] _D + 3.2 ° [C = 100, MeOH-0.2N NaOH (1: 1)] ¹ HNMR (200MHz, in CD ₃ OD, Ref.TMS) δ: 1H, 5.5 (dt-tl
ike, J = 15.38,5.86,7−H), 1H, 5.4 (dt−t.like, J = 1
5.38,6.10,6-H), 1H, 4.0 (d., J = 10.98,21-H), 1H,
3.9 (d., J = 10.99,21-H), 1H, 3.83 (br.t., J = 7.33,4
-H), 1H, 3.78 (br.s., 3-H), 2H, 2.3 (br.t., J = 6.8
4,5-H ₂ ), 2H, 2.0 (m, 8-H ₂ ), 22H, 1.3 (br.s., 9-19-
H ₂ ), 3H, 0.9 (t., J = 6.47,20-H) FAB-MS m / z: 388,104,57,45,29 The physical properties of the sodium salt of compound (e) are as follows.

mp 182-185 ° C ¹ HNMR (400MHz, in CD ₃ OD, Ref.TMS) δ: 2H, 5.5 (m, 6-H,
7-H), 1H, 3.8 (br, t, J = 6.4,4-H), 1H, 3.75 (br.s,
3-H), 1H, 3.7 (d., J = 10.6, 21-H), 1H, 3.6 (d, J = 1
0.6,21-H), 2H, 2.3 (br., 5-H 2), 2H, 2.0 (m, 8-
H ₂ ), 22H, 1.3 (br.s., 9 to 19−H ₂ ), 3H, 0.9 (t., J = 6.4
7,20-H ₃ ) HPLC retention time: 19.5 minutes Measurement conditions; Column: YMC pack AQ-312 (ODS) 6 mm diameter 150 mm Eluent: acetonitrile-0.05 M perchlorate buffer (pH 2.
5) (6: 4) Flow rate: 1 ml / min; Column temperature: 40 ° C. Example 6 Synthesis of compound (f) 351.3 mg of compound (2-i) was dissolved in 25 ml of dichloromethane, and ozone was passed through at -60 ° C for 6 minutes. Excess ozone was blown out by blowing oxygen and the reaction solution was concentrated under reduced pressure in a room temperature water bath. 21.2 ml of dioxane was added to the resulting colorless transparent oily residue.
Was added to dissolve, and 8 ml of water was further added. While stirring at room temperature, several pieces of dry ice were added, and when the solid disappeared, 80.0 mg of sodium borohydride was added. After 30 minutes, the reaction solution was made acidic (pH 2 or less) with 1N hydrochloric acid, and after 10 minutes, made weakly acidic to neutral with 1N sodium hydroxide and concentrated to dryness under reduced pressure in a 50 ° C water bath. After suctioning with an aspirator to completely remove water, about 10 ml of chloroform was added to a part of the white solid residue and the mixture was heated, and then insoluble matter was filtered off with suction. About 350 mg of the residue obtained by concentrating the filtrate under reduced pressure was subjected to preparative thin layer chromatography, and 106.0 mg of the target compound (6) was white from the band of Rf0.3 with the developing solvent chloroform: methanol = 9: 1. Obtained as a foam.

¹ HNMR (200MHz, in CDCl ₃ , Ref.TMS) δ: 1H, 6.3 (br.s.,
-NHAc), 1H, 5.8 (d., J = 4.64,3-H), 1H, 5.1 (ddd., J
= 9.03,4.64,4.39,4-H), 2H, 4.5br.s., 7-H 2), 3H, 2.
11 (s., Ac), 3H, 2.07 (s., Ac), 3H, 2.0 (s., Ac), 4H, 2.
1-1.8 (m, 5,6-H ₂ ) EI-MS m / z: 317,274,185,167,43 (2) Compound (6) → Compound (f) Compound (6) 70.0 mg was dissolved in methanol 1.33 ml, and After adding 3.99 ml of water and 1.33 ml of 1N sodium hydroxide, the mixture was refluxed overnight in an oil bath at 105 ° C under a nitrogen stream. After cooling, the column was charged with 6 ml of IRC-50 (H ⁺ type) and eluted with water-methanol (8: 2). Eluate (30m
l) was concentrated under reduced pressure in a 50 ° C. water bath, and the obtained air brown residue was suctioned on a sodium hydroxide in a desiccator to remove acetic acid. A little less than 2 ml of water was added, the slightly formed insoluble matter was filtered off,
The mother liquor was concentrated under reduced pressure and water was added to reduce the total volume to 2 ml.
Methanol was added at room temperature until the liquid became slightly cloudy, and the mixture was allowed to stand for 3 hours to obtain 14.0 mg of a colorless transparent scaly compound (f).

mp 209.5-219.0 ° C (decomposition) ¹ HNMR (200MHz, in D ₂ O, Ref.DSS) δ: 1H, 4.03 (d., J = 1
1.72,7H), 1H, 3.95 (ddd., J = 8.30,5.12,1.22,4-H),
1H, 3.9 (d., J = 11.72,7-H), 1H, 3.8 (d., J = 1.23,3-
H), 2H, 3.7 (br.dd. , J = 7.08,6.35,6-H 2), 1H, 1.87
(Ddt., J = 14.16,8.30,6.10,5-H), 1H, 1.74 (dtd., J
= 14.16,7.08,5.12,5-H)

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location C07C 229/26 251/40 C07D 307/33 339/06 405/12 233 409/06 307 // C12P 13/04 2121-4B 17/00 7432-4B 17/04 7432-4B 17/16 7432-4B (C12P 13/04 C12R 1: 645) (C12P 17/00 C12R 1: 645) (C12P 17/04 C12R (1: 645) (C12P 17/16 C12R 1: 645) (72) Inventor Kenji Chiba 3-6-14 Oyama, Higashi-Kurume City, Tokyo Koyamadai Elegant House 105 (72) Inventor Yukio Hoshino Kiyose, Tokyo 2-12-19 City Suido

Claims (4)

[Claims] 1. A general formula [In the formula, R is alkyl having 1 to 30 carbons and 1 to 6 carbons.
Number of hydroxyalkyl the formula _{-CH = CH (CH 2) n} CH = CH-
_{CH (OH) - (CH 2} ) m CH 3 ( wherein, n, m are each an integer of 1~10.), -CH = CH ( CH 2) p -C (= X) - (CH 2 ) _Q CH ₃ (where> C = X is (A represents an alkylene having 1 to 4 carbon atoms),> C =
N-OB (B represents hydrogen or alkyl having 1 to 4 carbon atoms) or> CHNH ₂ . (P and q are 1
Indicates an integer from -10. )) Or -CH = CH-Y (Y represents alkyl having 1 to 30 carbon atoms). ] The 2-aminopentanoic acid compound represented by these, its salt, or the compound protected by the protecting group. 2. A compound according to claim 1 selected from the following formulas (a) to (f), an optical isomer thereof, or a pharmaceutically acceptable salt thereof. 3. General formula [In the formula, R is alkyl having 1 to 30 carbons and 1 to 6 carbons.
Number of hydroxyalkyl the formula _{-CH = CH (CH 2) n} CH = CH-
_{CH (OH) - (CH 2} ) m CH 3 ( wherein, n, m are each an integer of 1~10.), -CH = CH ( CH 2) p -C (= X) - (CH 2 ) _Q CH ₃ (where> C = X is (A represents an alkylene having 1 to 4 carbon atoms),> C =
N-OB (B represents hydrogen or alkyl having 1 to 4 carbon atoms) or> CHNH ₂ . (P and q are 1
Indicates an integer from -10. )) Or -CH = CH-Y (Y represents alkyl having 1 to 30 carbon atoms). ] The γ-lactone compound represented by or a compound protected by a protecting group thereof. (4) An immunosuppressive agent comprising the compound according to (1), (2) or (3).