JPH03128347A - 2-aminopentanoic acid compound and use thereof as immunosuppressor - Google Patents

Abstract

NEW MATERIAL:A compound shown by formula I {R is 1-30C alkyl, 1-6C hydroxyalkyl, group shown by formula II((n) and (m) are 1-10), group shown by formula III [C=X is group shown by formula IV (A is 1-4C alkylene), C=N-OB (B is H or 1-4C alkyl) or CHNH2; (p) and (q) are 1-10]}, a salt thereof, a gamma-lactone compound or a compound protected with a protecting group thereof. EXAMPLE:A compound shown by formula V. USE:A suppresser for rejection in organ or bone marrow transplantation, preventive and therapeutic agent for autoimmune diseases for mammals. PREPARATION:The title compound is obtained by using (2R, 3R, 4R)-(E)-2- amino-3,4-dihydroxy-2-hydroxymethyl-14-oxoeicose-6-enoic acid produced by fermentation method as a starting substance. The example compound is obtained by culturing a microorganism belonging to the genus Isaria or Myriococcum.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to 2-aminopencunic acid compounds useful as medicines, particularly immunosuppressants.

[Prior art]

In recent years, cyclosporine has been used to suppress rejection reactions that occur during organ transplants. So-called immunosuppressants, including compounds currently under development, are also expected to be used as therapeutic agents for rheumatoid arthritis and the like. However, cyclosporine has the problem of causing side effects such as renal damage.

On the other hand, JP-A-1-104.087 describes that an immunosuppressive substance is collected from a liquid culture of Cordyceps sinensis (Isaria 5inelairii), and this substance is disclosed in US Pat. No. 3,928,572. (2S, 3R, 41?) −(E)
-2-amino-3,4-dihydroxo-2-hydroxy/
It has been confirmed that it is methyl 14-oxoeicos-6-enoic acid.

[Problems to be Solved by the Invention] An object of the present invention is to provide novel 2-aminopencunic acid compounds that exhibit excellent immunosuppressive effects and have few side effects.

[Means to solve the problem]

The present invention is based on the general formula % [wherein R is alkyl, hydroxyalkyl, formula -CH]
=CH1(CH2), CH=CH-CI(OH)-(C
H2), Cfl3 (where n and m are each 1 to 10
indicates an integer.

-CH=CH(CHz)--C(・χ)-(CH2)
-CH3S (where the >C=X group represents >C<>A (A represents alkylene), >C=N-OB (B represents hydrogen or alkyl) or >CHN H□, p, q
each represents an integer from 1 to 10. )) or -CH=
This invention relates to a 2-aminopencunic acid compound represented by CH-Y (Y represents alkyl), a salt thereof, a T-lactone compound thereof, or a compound protected by a protecting group thereof.

Regarding R in this specification, alkyl includes methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, isopentyl, neopentyl,
Straight or branched alkyl having 1 to 30 carbon atoms such as hexyl, octyl, 2-ethylhexyl, decyl, dodecyl, tetradecyl, hexadecyl, octadecyl, eicosyl, and tocosyl are exemplified, and examples of hydroxoalkyl include hydroxymethyl, 2-hydroxoethyl, 3-hydroxypropyl, l-hydroquinemethylethyl, 4-hydroxybutyl, 6-hydroquinehexyl, etc. with 1 or more carbon atoms
Six hydroxyalkyl (the alkyl portion may be branched) is exemplified.

Regarding substituent = , methyl, ethyl, propyl, isopropyl, butyl,
Straight chain or branched alkyl having 1 to 4 carbon atoms such as tertiary butyl is exemplified.

Regarding Y in this specification, alkyl has the same meaning as the alkyl explained for R.

As a γ-lactone compound of the compound of general formula (1), the formula () (In the formula, R has the same meaning as above.) This is a compound represented by

In addition, in the compound of the general formula (H), the protecting group of the compound protected by a protecting group is one that is widely used in organic chemistry for a hydroxyl group or an amino group, and includes acyl such as acetyl, benzoyl, tertiary Examples include okinocarbonyl such as butoxycarbonyl, and tetrahydropyranyl.

Salts of the compound of general formula (1) include metal salts such as sodium salts, potassium salts, calcium salts, zinc salts, and aluminum salts, salts with amines such as triethylamine, and salts with amino acids such as lysine and ornithine. Examples include pharmacologically acceptable salts such as salt. It also includes hydrates and other solvates, as well as individual optical isomers, diastereoisomers, and racemates.

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

CO□■ H H CO□H 01 ( 5 U-) co', n H The compound of general formula (1) of the present invention can be produced by fermentation method etc. (25,3R,4R) -(E)-
2-amino-3,4-dihydroxy-2-hydroxymethyl-14-oxoeicosa-6-enoic acid (hereinafter referred to as l5
P-1) as a starting material. For example, compound (a) can be produced by fermentation, but microorganisms that can produce compound (a) include those belonging to Ascomycetes and Deuteromycetes, specifically, Isaria, which belongs to Deuteromycetes. The genus Myceria, and the genus Myriococcum (genus Cherabia), which belong to ascomycetes, are included in the American Type Culture Collection (American Type Culture Collection).
1ture Co11ection) and Isaria Sinclairii (Isaria 5inclairii) A
TCC No. 24400. Myriococcum albomyces
s) ATCC No. 16425, Mycelia 5terilia ATCCN
o, 20349. Also, Myriococcum albomyces ^TCC No. 1642
5 has been deposited with the Fermentation Research Institute (Osaka) as [FO32292].

Compound (a) can also be produced using a mutant strain obtained by modifying the above-mentioned bacterial strain by, for example, commonly used artificial mutagenesis means such as ultraviolet rays, high-frequency radiation, and chemicals.

Compound (a) producing bacteria can be cultured in a variety of culture media containing conventional mold nutrient sources. For example, glucose, starch, glycerin, sugar syrup, dextrin,
Molasses, maltose, xylose, etc., and cornstarch liquor peptone, yeast extract, as a nitrogen source,
Inorganic or organic nitrogen compounds such as potato decoction, meat juice, soybean flour, wheat germ, potassium nitrate, sodium nitrate, ammonium sulfate, casein, gluten meal, cottonseed meal, feather meal, etc., as well as other common inorganic salts and fungi. Additives commonly used in culturing, such as organic and inorganic substances and antifoaming agents, which aid growth and promote the production of compound (a), can be suitably added.

Although the culture method is not particularly limited, an aerobic deep culture method is suitable. The appropriate temperature for culturing is 20-35'C for bacteria belonging to the genus Isaria.

The temperature is preferably 25 to 30°C, and 30 to 50°C in the case of bacteria belonging to the genus Myrioconocum or Mycelia.
1 The temperature is preferably 35-45°C.

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 bacteria belonging to the genus Isaria such as Isaria cinclairi, insoluble substances such as bacterial bodies are separated from the culture solution by methods such as filtration or centrifugation, and the culture filtrate is passed through Amberly 1-XAD-2. The compound (a) is removed by adsorption of the compound (a). The thus obtained compound (a) is further eluted with, for example, methanol, and the eluted portion is further subjected to reverse phase chromatography and fractionated to obtain a highly purified compound (al). In the case of bacteria belonging to the genus Myrioconocum or Myceria, such as Albomyces and Mycelia stetulia, the bacterial cells are separated from the culture solution by a method such as a filtration or centrifugation, and the culture filtrate is treated in the same manner as for the genus Isaria.

On the other hand, from the isolated bacterial cells, compounds (
a), and the extract was mixed with Amberlite XA in the same manner as the filtrate.
D-2 and purification by chromatography or recrystallization to obtain compound (a).

Compound (b) is a γ-lactone derived from l5P-1 (protected by a protective group), for example, a compound represented by the formula (wherein, Ac represents an acetyl group) in the presence of 37 boron fluoride. The alkanedithiol is reacted to obtain a compound represented by the general formula (wherein Ac has the same meaning as above and A represents alkylene), and then Raneynifkel,
It is produced by subjecting it to a reduction reaction using palladium on carbon or the like to form a compound represented by the formula (wherein Ac has the same meaning as above), and then cleaving the lactone ring by alkaline hydrolysis.

Compound (C) can be obtained by subjecting a compound of the general formula ([[l) (where A represents ethylene) to an alkaline hydrolysis reaction, and compound (d) can be obtained by reacting l5P-1 with hydroxylamine. Manufactured.

Note that the alkoxime corresponding to compound (d) is l5
It can also be obtained by reacting P-1 with ○-alkylhydrokylamine, or reacting the hydroxyme compound with dialkyl sulfate, alkyl iodide, etc. Furthermore, the hydroxyme form can be converted into an azine compound by a conventional method. Compound (d) or the corresponding alkoxime has anti- and syn-geometric isomers, and the present invention also includes these individual isomers and mixtures thereof.

Compound (e) is obtained by reducing the 14th position of compound (II) to hydroxy with a metal hydrogen complex such as sodium borohydride or lithium aluminum hydride, and then reacting it to form an alcohol, which is then converted into 1 , 1-thiocarbonyldiimidazur to substitute 1-imidazolylthiocarbonyloxy at position 14, for example,
By radical reduction with tributyltin hydride,
It is produced by eliminating the substituent and then subjecting it to an alkaline hydrolysis reaction. Alternatively, a secondary hydroxyl group may be converted into trifluoromethanesulfonate, and a deoxygenation reaction using sodium borohydride or the like may be applied.

Compound (f) is obtained by subjecting the T-lactone form of 15P-1, that is, compound Obtained by reduction using sodium or the like.

In the compound of general formula (1), R is -CH=CH-Y
The compound is prepared by subjecting 15Pi to an ozonolysis reaction and then reacting with Witteich's reagent.

The compound thus obtained can be converted into the corresponding r-lactone form by treatment with an acid such as hydrochloric acid or acetic acid or a tertiary alcohol such as 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 general formula (1) of the present invention, that is, the compound of general formula (1), that is, the carboxylic acid compound, can be converted into the above-mentioned salt by a conventional method. Various isomers can also be produced by optically resolving racemic forms and diastereoisomers, or by using optically active raw materials.

[Action/Effect]

The 2-aminopencunic acid compounds of the present invention exhibit excellent immunosuppressive effects on humans, cattle, horses, and dogs.

For mammals such as mice and rats, it is used as an inhibitor of rejection during organ or bone marrow transplantation, and for rheumatoid arthritis, systemic lupus erythematosus, Schugren's disease, multiple sclerosis, myasthenia gravis, type I diabetes, etc. Endocrine dysgenesis, primary biliary cirrhosis, Crohn's disease, glomerulonephritis, sarcoid
It can be used as a prophylactic or therapeutic agent for autoimmune diseases such as cirrhosis, psoriasis, aplastic anemia, idiopathic thrombocytopenic purpura, and allergies, or as a reagent in medicine and pharmacy.

These compounds can be mixed with carriers, excipients, diluents, etc., formulated into powders, capsules, tablets, injections, etc., and administered to patients. Alternatively, it may be made into a lyophilized preparation by means known per se.

The dosage of these compounds can vary depending on the disease, symptoms, weight, sex, age, etc., but for example, for suppressing rejection in kidney transplantation, the dosage is usually 0.01-0.00% per day for adults.
1-1O titer) is administered in one to several divided doses per day.

[Experimental Examples/Example] The functions and effects of the present invention will be explained in further detail by giving experimental examples and the like below. The immunosuppressive activity was measured using the method described below.

Various immunoreactions using mouse, rat, or human lymphocytes can be used to measure the activity. For example, immunosuppressive activity can be measured using a mixed reaction (allogeneic MLR ), it is possible to measure with high sensitivity. Allogeneic MLR refers to immature lymphocytes induced by mixed culture of lymphocytes from two individuals of the same species but with different major histocompatibility antigens, such as tile cells, lymph node cells, peripheral blood lymphocytes, etc. It is a chemical reaction. This allogeneic MLR is a phenomenon that reflects and is induced by differences in major histocompatibility antigens between lymphocyte donors. It is not allowed. Allogeneic MLR is therefore a widely used method for donor-recipient selection, for example in organ transplants.

Normally, when performing allogeneic MLR, one lymphocyte is used as a stimulator cell after its division and proliferation is inhibited by X-ray irradiation or mitomycin C treatment, and the other lymphocyte (responsive cell) is immature. A method for measuring a rejuvenation reaction (one way MLR) can be used.

Furthermore, the immunosuppressive activity can also be measured as an activity that suppresses the induction of cytotoxic T cells with major histocompatibility antigen restriction induced during allogeneic MLR.

In addition, the immunosuppressive activity suppresses the lymphocyte blastogenesis induced by stimulation with various mitogenes (concanavalin A5 phytohemagglutinin, pork lyde mitogenes, etc.) in addition to allogeneic MLR. or induced by cytokines (such as interleukin 1.2.3.4.5.6) that have the activity of enhancing the division and proliferation or promoting the differentiation of lymphocytes such as T cells and B cells. It can also be evaluated as an activity to suppress lymphocyte division/proliferation response or functional expression. Furthermore, it is possible to evaluate the activity of suppressing the production of these cytotoxic cells from T$llI cells and macrophages.

Furthermore, by intraperitoneally, orally, intravenously, subcutaneously, or intramuscularly administering the compound to mice, etc., allogeneic cell-specific The activity of suppressing the induction of cytotoxic T cells, the activity of suppressing the production of allogeneic cell-specific antibodies produced in the serum of mice immunized with allogeneic cells, etc., or the rejection reaction during organ transplantation of allogeneic mice, or graft-versus-host reaction or delayed allergy;
It can also be evaluated as an adjuvant for its activity of suppressing arthritis and the like.

In addition, MRL/lpr, which is an autoimmune disease model animal,
Mouse, NZ B/WF + Mouse, BXS
By administering a compound to B mice, NOD mice, etc., it can be evaluated, for example, as an inhibitory activity on anti-DNA antibody production, rheumatoid factor production, nephritis, abnormal lymphocyte proliferation, proteinuria, etc., or as a survival effect. .

Experimental Example 1 (Immunosuppressive effect of immunosuppressive substances) The immunosuppressive activity of compounds was measured using mouse allogeneic lymphocyte mixed reaction (hereinafter sometimes referred to as MLR). Mouse allogeneic MLR has B A L B /
C7 mouse (H-2') (7) Lung cells were cultured in equal proportions using mitomycin C-treated C57BL/6 mouse (H2'') lung cells as stimulator cells.

The reaction cells were prepared by the following method. 5
Lungs were removed from ~6-week-old BAL B/c mice and cultured in RPM11640 medium (60 μg/g of kanamycin/g acid) supplemented with 5% heat-inactivated fetal bovine serum (hereinafter also referred to as Fe2). mL L-gluta n 2m
M5N-2-hydroxyethylpiperazine-N'-2-
Ethanesulfonate (HEPES) 10mM, 0.1%
(containing sodium bicarbonate) to obtain a single cell suspension of lung cells.

After hemolyzing treatment, the cells were adjusted to 107 cells/d using RPM11640 medium containing 10-'M 2-mercaptoethanol and 20% FC3, and used as a reaction cell suspension.

The stimulated cells were prepared as follows. Lungs were removed from 5-6 week old male C57BL/6 mice and RPM
A single cell suspension of lung cells was obtained using 11640 medium.

After cold blood treatment, the cells were treated with 40 μg/rn1 mitomycin C for 560 minutes at 37°C. After washing 3 times, t
o-'M 2-mercaptoethanol and 20% F
Using RPM11640 medium containing C3, 10' pieces/
mi and used as a stimulated cell suspension.

50 μl of the reaction cell suspension prepared by the method described above, 50 μE of the stimulated cell suspension, and 100 μl of the test substance,
The cells were added to a 96-well flat bottom microtest plate and cultured for 4 days at 37°C under conditions of 5% carbon dioxide gas and 95% air.

As a method for measuring the rejuvenation reaction of lymphocytes, a method was used in which the incorporation of 3H-thi and phthalate was used as an indicator.

That is, after the completion of culture, 18.5KB of 3H-thymidine
q/well, and after culturing for 4 hours, use a cell harvester.
The cells were collected, and the radioactivity incorporated into the cells was measured using a liquid scintillation counter, which was used as an indicator of lymphocyte development in mouse allogeneic MLR. Mouse allogeneic ML
The suppression of R was evaluated by calculating the suppression rate using the following method. The results are shown in Table 1A.

Inhibition rate = (blank below) The test substance was dissolved in methanol and then diluted with RPMII640 medium for use. Note that methanol was used in an amount of 0.01% or less, and in this case no influence was observed on the homogeneous MLR.

For the compounds of Examples 1 to 6, 1tIg/! As shown in Table 1A, these compounds showed concentration-dependent effects on mouse allogeneic MLR at final concentrations ranging from 0.0001 μg/ml to 0.0001 μg/ml. However, it showed a suppressive effect.

The concentration (IC,.) of the compounds of Examples 1 to 6 that inhibits the mouse allograft MLR by 50% is 1.3×10−”t, respectively.
t g/d, 1.6 x 10-”tt g/r
a, 2.5X10-'μgod, 2-5XIQ-zμg
/lri, 2.5XlO-'μg/d and 4.5X1
Furthermore, as is clear from the results in Table 1B, the sodium salt of the compound of Example 5 also had a mouse conspecific MLR of approximately the same level as the compound of Example 5. It showed suppressive effect.

As a method for measuring the lymphocyte rejuvenation reaction, the following 3-(4,5-dimethylthiazuryl 2-(ru)-
25-diphenyltetrazoliu J, bromide (MTT
) can also be used.

After completing the culture, remove 1oOff of supernatant from each well,
Add 20 liters of 7 volumes of mg/m MTT to each well and incubate for 4 hours at 37°C. Then, 100 μl of a cold solution of 0.011A constant hydrochloric acid containing 10% sodium dodecyl sulfate was added, and the mixture was left at 37°C overnight to dissolve the formed purple formazan crystals. 57Q using
The intensity of light bite in nm is measured and used as an index of lymphocyte development in mouse allogeneic MLR. Suppression of mouse allogeneic MLR can be evaluated by calculating the suppression rate Cγ using the following formula.

(Suppression rate (%) - Experimental Example 2 (Suppression effect on human allo-MLR) The stamp effect on human allo-MLR was tested by the following method.

Human peripheral blood lymphocytes obtained by separating normal human peripheral blood by fluorocarbon density gradient centrifugation were suspended in RPM11640 medium supplemented with 10% FC3, plated on a plastic plate, and incubated with 5% carbon dioxide gas and 95% air. Under the conditions of 37°C
The cells were cultured for 2 hours. After the culture was completed, the supernatant was collected by gentle pipetting and centrifuged (1000 rpm, 5 minutes) to obtain plastic non-adherent cells.

The plastic non-adherent cells were further made into nylon non-adherent cells by passing through a nylon wool column, and then
4X106 pieces/in RPM11640 medium supplemented with %FC3
d and used as a reaction cell suspension.

Additionally, plastic adherent cells were treated with 5% FC3 and 0
．． 02% ethylenediaminetetraacetic acid disodium (EDT)
A) Obtained by adding phosphate buffered saline (PBS), vigorously pipetting, and peeling. In addition, 40 μg of mitomynon C was added to the plus-thick adherent cells.
After treatment at 37°C for 60 minutes, and washing three times, the cells were adjusted to 4 x 10 cells/ml using RPM11640 medium supplemented with 10% FC3 and used as a stimulated cell suspension. . Donor A prepared by the method described above
Alternatively, mix 50 μl of the reactive cell suspension derived from donor C and 50 μl of the stimulated cell suspension derived from donor B or D, add 100 μl of the test substance, and store at 37°C at 5% concentration.
Culture was carried out for 5 days under conditions of 95% carbon dioxide gas and air.

After the completion of the culture, 37 KBq/well of 3H-thiamine was added, and after culturing for 18 hours, the cells were collected using a cell bar Hester, and the radioactivity incorporated into the cells was measured using a liquid scintillation counter. This was used as an indicator of lymphocyte rejuvenation in MLR.

Suppression of human allo-MLR was evaluated by calculating the suppression rate using the following formula. The results are shown in Table 2A and Table 2B.

(Hereinafter in the margin) Inhibition rate (%) (Hereinafter in the margin) For the compounds of Examples 1.2.3 and 5, lμg
The imprint activity of lymph f, L blastogenesis in human allogeneic MLR was measured at final concentrations ranging from /ml to 0.0001 ug/rd. As a result, as shown in Tables 2A and 2B, these compounds showed a stamp effect on human homologs M1 and R in a concentration-dependent manner.

Experimental Example 3 (Mouse allogeneic lymphocyte mixed culture (M+-C: 1
B A l 13 prepared in the same manner as in Experimental Example 1)
/C7 mouse (H-2') spiny cell floating m? Night (2XlO'
cells/mi) 0.5 ml and mitomycin C-treated C57BL/6 mouse (H-21) lung cell suspension (2 x 10' cells/mN) 0.5- and test subject], O
mt! was added to a 2.1-well multi-dish and cultured for 6 days at 37°C under conditions of 5% carbon dioxide gas and 95% air.

After culturing, cells were collected by centrifugation and incubated at 5XlO' to 6.25X in RPM11640 medium containing 10% FC3.
The cells were prepared at 10' cells/d and used as effector cells.

Target cells include C5 syngeneic (H-2b) as the stimulator cell.
7BL/6 mouse-derived leukemia cells, El, 4 were used.

106 EL4 cells in 370MBq of N a Z”C
S I Cr was incorporated into the cytoplasm by incubation at 37°C for 1 hour using rOa, and the cells were washed and prepared at 10" cells/mQ, which were used as target cells.

For one-ill determination of cytotoxic activity, add 100 μl of effector cell suspension and 100 μl of target cell suspension to a 96-well flat-bottom microtest plate, incubate at 37°C for 4 hours, and then release into the supernatant. Cr1l was measured and cytotoxic activity was calculated using the following method.The results are shown in Table 3A.

Cytotoxic activity - (margin) The cytotoxic T cells induced by the above method are
Stimulator cells (H-2′) and syngeneic EL4 cells (H-2″″
) showed strong cytotoxic activity against allogeneic Me
th A cells (H-2'), P815 cells (H-2')
), showed no cytotoxic activity against BW5147 cells (H2''), suggesting that they were H-2'-restricted allogeneic cytotoxic T cells.Cytotoxic T cells The activity was expressed in l+7Lic unit.Lytic unit (LU) is the target cell 5X10
It is expressed as LU per 10' effector cells, where ILU is the number of effector cells required to destroy 3 cells by 25%.

As shown in Table 3A, it was revealed that the addition of the compounds of Examples 2 and 5 significantly suppressed the induction of allogeneic cytotoxic T cells.

In addition, C3H/He N mouse (H-2′) lung cell suspension was used as the reaction cell, and mitomycin C-treated C5
When 7BL/6 mouse (H-25) lung cell suspension was used as the stimulator cell, the induction of allogeneic cytotoxic T cells was also inhibited by the addition of the compound of Example 5 (Table 3B) .

(Margins below) Experimental Example 4 (Suppressive effect on interleukin-2 (IL-2) and IL-3 production in mitogen-stimulated mouse tile cells) Production of IL2 and IL-3 by mitogen-stimulated mouse tile cells The effect test was conducted in the following manner.

Lungs were removed from 5- to 8-week-old male C3H/He N mice, and a single cell suspension of tile cells was obtained using RPM11640 medium. RPM containing 20% FCS after hemolysis treatment
Prepared to 10″ cells/arl using I1640 medium,
Phytohemagglutinin (PHA) as mitogene
) 10tIg/d was added. This cell suspension 1d,
Add the subject 1 relative to the 24-hole multi-instrument, and
C, 5% carbon dioxide and 95% air for 24 hours. After the culture was completed, the supernatant was collected and used as a PHA-stimulated mouse tile cell culture supernatant.

[L-2 activity in PHA-stimulated mouse tile cell culture soil was measured as follows. IL-2 dependent mouse cell line C
TLL-2 was prepared at 2×10′ cells/Id using RPM11640 medium containing 30% FC3. 100 μl of the cell suspension and 100 μl of a 2-fold serial dilution of the PHA-stimulated mouse tile cell culture supernatant were added to a 96-well flat-bottomed microtest plate. 37°C 15% carbon dioxide,
After culturing under 95% air conditions for 44 hours, absorbance was measured by the dye quantitative method using MTT in the same manner as in Experimental Example 1, and was used as an index of IL-2-dependent cell proliferation.

IL-2 activity was measured at 50% of the maximum absorbance by the MTT method.
It was expressed in U/cell from the dilution concentration giving %.

! L-3 activity was measured as follows.

IL-3 dependent mouse cell line FDC-P2 was incubated with 10% F
2X 10' using RPM11640 medium containing C3
number/d. 100 μl of the cell suspension and the above P
Add 100μ2 of a 2-fold serial dilution of the HA-stimulated mouse tile cell culture supernatant to a 96-well flat-bottomed microtest plate and incubate for 44 hours at 37"C under conditions of 15% carbon dioxide and 95% air.
Cultured for hours. After the completion of the culture, perform MTT in the same manner as described above.
The absorbance was measured by the dye constant method using IL,
-3-dependent cell proliferation was used as an indicator. II,-3 activity was expressed in U/rd from the dilution concentration giving 50% of the maximum absorbance by the MTT method.

(Left space below) Table 4A Formula % Formula % () () (1 As shown in Tables 4A and B, the compounds of the present invention are
IL-2 and I in PHA-stimulated mouse tile cells
It was suggested that L-3 production was suppressed.

Experimental Example 5 (Suppressive effect on IL-2 and IL-3 production in mouse allogeneic lymphocyte confluence culture (MLC)) Mouse allogeneic MLC was carried out as follows.

That is, the reaction cell suspension tr prepared in the same manner as in Experimental Example 1
0.5 ml each of cell suspension and stimulated cell suspension,
The samples were added to a 24-well multi-dish containing the sample l-, and cultured for 2 days at 37°C and 55% carbon dioxide gas. After the culture is completed, the supernatant is collected and the mouse homologous M
This was used as the LC supernatant.

IL-2 and IL-3 in the supernatant of mouse allogeneic MLC
Activity was measured in the same manner as in Experimental Example 4.

(Leaving space below) Table 5A Sample IL-2 activity inhibition rate CU/m (χ) Culture supernatant of reactive cells only 1 LC supernatant 4.5 Table 5B Sample rL-3 activity inhibition rate (U # +4 (χ) Culture supernatant of reactive cells only 1000 LC supernatant 49.2 As shown in Tables 5A and 8, the compounds of the present invention
It was suggested that it suppresses the production of IL-2 and IL-3 in mouse allogeneic MLC.

Experimental Example 6 (Suppressive effect on proliferation of 1L-2 dependent mouse cell line CTLL-2 induced by interleukin 2 (rL-2)) 30% of IL-2 dependent mouse cell line CTLL-2
4XIO' cells/in RPM11640 medium containing FC5
Adjusted to mR. 50 μl of this cell suspension and 200 U/- of recombinant human IL-2 (rhlL2)
0 μl and 100 μl of analyte were added to a 96-well flat bottom microtest plate. 37°C 15% carbon dioxide 9
After culturing for 68 hours under 5% air conditions, absorbance was measured by the dye quantitative method using MTT in the same manner as in Experimental Example 1, and was used as an index of IL-2-dependent cell proliferation.

The inhibition rate (%) was calculated using the following formula.

(Margin below) Suppression rate (%) = The results are summarized in Tables 6A and 6B.

(Margin below) Table 6A IL-2 Subject dose absorbance Suppression rate (50Ll/mQ) (μg/ml) (0057°) (%) fo, ool in Example 1 Compound (0,01 0,1 From, 0 Example 2, o, oot Compound 0.01 0.1 (1,0 0,001 0,586 0,508 0,556 0,009 0,005 0,555 0,543 0,558 0,161 13,3 5,1 98,6 99,3 5,3 7,4 4,8 72,6 + + 10.I [1,.

0.471 ■9.7 0.152 74.2 Table 6B 0.026 + 0.961 As is clear from Tables 6A and B, the compound of the present invention has an effect on CTL induced by recombinant human IL-2.
It was suggested that it strongly suppresses the division and proliferation of L-2 cells.

Experimental Example 7 (Suppressive effect on IL-1, IL-2 and IL-4 responses of mouse thymocytes) Male 6-week-old C3H/
The thymus was removed from HeN mice and treated with serum-free RPM11.
A single cell suspension was obtained using 640 medium. After three washes, 10-'M 2-mercaptoethanol and 2
Using RPM11640 medium containing 0% FC3, 1.
Adjust the cell concentration to 5 x 10' cells/ml and suspend this cell! Add 100 μl of sample and 100 μl of test material to a 96-well flat bottom microtest plate, add phytohemagglutinin (P
I-IA) 1 μg/d and recombinant human IL4
-1α, in the presence of recombinant human IL-2 and Aluminum recombinant mouse IL-4 at 37°C 15%
The cells were cultured for 54 hours under conditions of 95% carbon dioxide and air. After the completion of the culture, 18.5 KBq/well of 3H-thymidine was added, and after culturing for 18 hours, the cells were collected using a cell bar Hester, and the radioactivity incorporated into the cells was measured using a liquid antitillation counter. This was used as an index of IL-1, IL-2 or IL-4 response. IL-1, IL
-2 or l L-4 response was evaluated by calculating the inhibition rate using the following formula.

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

As is clear from these tables, the compounds of the present invention
It was suggested that this product strongly suppresses the division and proliferation of mouse thymocytes induced by IL-1, IL-2 or IL-4.

Table 7A 172± 18 + 11617± 20 Table 7B IL-2 Subject dose 3■ Thymidine inhibition rate (07ml) (μg/ml) Uptake (cpm) (χ) 60 0 800 0 0183 Table 7C IL-4 Subject dose jI+ Thymidine inhibition rate (U/m1) (μg/m+) Uptake (cpm) (χ) 22 0 182 50 2761 Experimental example 8 (inhibitory effect on anti-sheep red blood cell antibody production in mice) 5-7 weeks old Male BALB/c mice were immunized with sheep red blood cells (5 x 10' cells/mouse, intravenously administered), and subjects were administered 3 to 10 mg/kg/day for 4 days from the day of immunization.
The drug was administered intraperitoneally every day. Four days after immunization, the lungs were removed.
The inhibitory effect on anti-sheep red blood cell antibody production was investigated by measuring the number of plaque-forming cells (PFC) by a direct plaque method using sheep red blood cells and Mormono l-j+i bodies. At this time, the body weight, wet weight of the thymus and lung, and number of tile cells were also measured. The results are shown in Table 8.

(Margin below) As shown in Table 8, the compound of Example 2 has an inhibitory effect on anti-sheep red blood cell antibody production, namely the number of PFC per unit number of tile cells and the number of PFC per total number of tile cells (I).
It is clear that it has the effect of reducing the number of XIO' pieces.

Experimental Example 9 (Suppressive effect on induction of allogeneic cytotoxic T cells by allogeneic cell immunization in mice) 5-7 week old male B
ALB/c mice (H-2') were transferred to C57BL
/6 mouse (H-2b)-derived leukemia cell EL4 (10
'pcs/mouse, intraperitoneal injection! -j), and from the day of immunization, the test subjects were administered intraperitoneally 5 times at a dose of 0.001 to 3 mg/kg/day or orally at a dose of 0.3 to 10 mg/kg/day. After 9 to 10 days of immunization, the lungs were removed from the mice and made into a single cell suspension, which was then used as effector cells. Similarly to Experimental Example 3, a 51Cr release test was conducted using EL4 cells as target cells to measure cytotoxic activity.

The activity of allogeneic cytotoxic T cells is 1yric unit
Displayed at. Lytic unit (LU) is the number of effector cells required to destroy 25% of 5 x 103 target cells in ILU, and is expressed in LU per lung. At this time, the body weight and number of tile cells of the mice were also measured.

The results are shown in Tables 9A and B.

(Here are blank spaces) As shown in Tables 9A and B, the compound of Example 2 inhibited lip reading of allogeneic cytotoxic T cells in the tile cells of allogeneic cell-immunized mice by intra- or oral injection five times. It is clear that it is strongly suppressed.

However, no suppression of body weight gain or significant decrease in the number of tile cells was observed.

Furthermore, as is clear from the results in Table 9C-F, the compound of Example 5 and its sodium salt also exhibited allocytotoxicity in the tile cells of allocell-immunized mice when administered 5 times intraperitoneally or transcutaneously. It strongly suppressed lip reading in Ta1l cells, and after 10 oral administrations, a significant suppressive effect was observed up to a dose of 0.03 m</kg.

As shown in Table 9F, the sodium salt of Example 5 showed allocytotoxicity in H11m cells of allogeneic cell-immunized mice.
When administered intraperitoneally, it has almost the same imprint effect as the compound other than the sodium salt of Example 5 on cell induction. Furthermore, a significant inhibitory effect was observed up to a dose of 0.03 mg/kg by intravenous administration.

Experimental Example 10 (Suppressive effect on allogeneic cytotoxic specific antibody production by allogeneic cell immunization in mice) Male BAI, B/c mice aged 5 to 7 weeks were treated with C57BL/6 mouse-derived leukemia cells EL4 in the same manner as in Experimental Example 7.
(107 mice/mouse, intraperitoneal injection F-j), and from the day of immunization, subjects were administered intraperitoneally or orally at a dose of 0.001 to 3 mg/kg/day.
Serum was collected from the mice one day later.

50 μR of a 2-fold serial dilution of the resulting serum, 50 μL of Mormont complement and target f'l! S as an I cell
EL4 cell suspension labeled with IC, 5i't (1 (5x 1
0' cells/mL 1OOttR) was added to a 96-well microtest plate, incubated at 37°C for 1 hour, and then the s+Cr9 released into the supernatant was measured.
Complement-dependent cytotoxic activity was calculated from the following.

Cytotoxic activity (%) - The allogeneic cytotoxic specific antibody titer in the serum measured by the above method is 5ICr labeled target cells at 0°I
81 when treated with NHC1 (:, 50% of the released amount
The titer was calculated and displayed from the diluted concentration giving .

(Left below) As shown in Table 1 OA-E, intraperitoneal or oral administration of the compound of the present invention strongly suppresses the production of allo-cytotoxic specific tA bodies in the serum of mice immunized with stroma cells. became clear.

As shown in Table 10E, the sodium salt of Example 5 has almost the same effect on allocytotoxic specific antibody production in the serum of allogeneic cell-immunized mice as the compounds other than the sodium salt of Example 5 when administered intraperitoneally. It has an inhibitory effect on In addition, a significant inhibitory effect was observed up to a dose of 0.01 mg/kg by intravenous administration.

Experimental Example 11 (Mouse acute graft-versus-host reaction (GVHR)
5-7 week old female BDF, C57B on the right foot of mice
L/6 mouse tile cell suspension'ii solution (2XIO' cells/mouse) was injected for induction, and 7 days after the injection, the right knee lymph node was removed, weighed, and compared with p, @ (saline injection). ) was used as an index of acute graft-versus-host reaction (GVHR) by comparing the weight of the left knee lymph node. From the day of challenge injection, the compound of Example 5 was administered intraperitoneally at 0.3 mg/kg, orally at 1 mg/kg, and 0.3.1 mg for 6 days.
/kg was administered intramuscularly, and the weight of the right knee lymph node was measured on the 7th day.

As a result, as shown in Table 1t, it was revealed that acute graft-versus-host reaction (GVHR) in mice was strongly suppressed by intraperitoneal, oral, or intramuscular administration of the compound of Example 5. .

(The following is a blank space) Experimental Example 12 (Examination of cytotoxicity to various cultured tumor cell lines) Cytotoxicity to various human-derived cultured tumor cell lines was examined as follows.

562, MOLT4, U9 in human-derived cultured tumor cell lines
37 and HL60 each containing 20% FC3
The number of cells/d was prepared in M11640 medium.

Add 50 μl of this cell suspension to 50 μl of test solution.
1 was added to each well of a 9G-well microtest plate containing 1. , 72 at 37°C and 15% carbon dioxide.
After culturing for an hour, the absorbance at 570 nm was measured at 7j using the same method as the dye quantitative method using MTT in Experimental Example 1.
It was fixed at 11.

The inhibition rate was calculated using the following formula and used as an index of cytotoxicity.

As is clear from Tables 12A-C showing the results,
The compounds of the present invention have weak cytotoxicity against various human-derived cultured am cell lines, with concentrations that give 50% inhibition (IC
so) was 10 μg/- or more.

Table 12A Compound of Example 2 0 1.086±0.045 4.4 0 1.045±0.041 9.4 0 0.673±0.111 1.3 Table 12B Compound of Example 4 Table 12C Compound of Example 5 0 1.083±0.033 4.7 0 0.684±0.038 Example 1 (i) Jar culture of Albomyces bacteria CCYi@if! ! (ip, per, glycose 20g
, cornstarch liquor 5g, yeast extract 3g, Mg
5Oa ・7HzOO-5g, pH 6) at 500 m
Dispense 100 ml into two R-neck shake flasks,
After sterilization in an autoclave at 121°C for 520 minutes, approximately 1 cJ of mycelium of Myrioconocum albomyces ATCC No. 16425 grown on potato dextrose agar medium was inoculated into each flask, and cultured with reciprocating shaking (135 rpm, Shakeback 1+3 cm
) was carried out. The above GC using the obtained culture solution as a seed mother
It was inoculated into a 1O1 jar fermentor containing Y medium and antifoam agent (F-18 manufactured by Dow Corning) Ig, and
'C 17 days, aeration agitation culture (0.5VVM, 300r
pm) was carried out.

(11) Compound (8) from culture solution of Albomyces bacteria
4.52 of the culture solution obtained in I-Kyo (i) was separated into bacterial cells and culture filtrate by filtration. Culture solution a (41) was passed through Amberlite XAD-2 (φ40 mm x h 750 mm) to adsorb compound (a), and then water 1e was passed through it for washing. Next, 30% 0% methanol Lumi IP, 50% methanol? Volume/night 12.80% methanol) Volume/fi, 3f were sequentially passed, and the 80% methanol elution fraction containing compound (a) was collected.

On the other hand, the bacterial cells were treated with methanol in an amount approximately 5 times their wet weight.
After extraction, water was added to the extract, and 30% methanol 7
Amber light XAD2 (φ40mm
h 750 mm) to adsorb compound (a),
30% methanol solution 71! l 12. 50% methanol solution IL 80% methanol 7 volume solution 3 was sequentially passed through the solution,
An 80% methanol elution fraction containing compound (a) was collected.

8 from the culture filtrate and bacterial cells obtained as above.
The 0% methanol elution fractions were combined, concentrated under reduced pressure, and then lyophilized to obtain 0.5 g of powder containing compound (a).

(iii) Dissolve 0.5 g of the powder obtained in purification (ii) of compound (al) in methanol, and add the same amount of water as methanol to make a 50% methanol solution. This solution is Amberly) XAD-2 (φ2
5mm + X h 300mm) for 7 nights, 30%, 5
0%, 60%, 70% and 75% methanol 500 each
d was sequentially passed through the plate, and a 70 to 75% methanol solution fraction containing compound (a) was collected. Add water to this elution fraction,
Make a 50% methanol solution and add Amberlite XAD again.
-2 was purified by column chromatography to obtain 28 mg of compound (il).

m, p, 145-147°C'H-NMRδ (ppm CD, On): 5.55
(m), 5.40 (m)3.87 (l11),
2.30 (t)IRp (KBr): 330
0 1625 970 cvn Example 2 1509.5 mg of compound (2-i) was poured into 12.5 d of dry chloromethane, 367 μl of 1,2-ethylenedithiol, and boron trifluoride (needle) 12.
5 μl was added and stirred in a chamber for 47 hours.

The reaction solution was washed with 5% sodium bicarbonate and saturated sodium chloride, dried over magnesium sulfate, and concentrated under reduced pressure to obtain an oily residue. This was separated using a silica gel (50 g) column (developing solvent: ethyl acetate: n-hexane-6:4), and then subjected to thin layer chromatography (developing solvent: ethyl acetate: n-hexane-7:
The fraction showing an Rf value of 0.4 in step 3) was concentrated under reduced pressure to obtain 1538.9 mm of compound (2-ii) as a colorless and transparent oil.

IR Shinji': Kure''(cm-'): 3400.2950
．． 2850.27001780, 1750.1680.
1500.1370.1230.1050'HNMR(
200MHz, in CDC1z, Ref, TM
S) δ:IH,6,1(br,s,, -N)IA
c), IH, 5, 8 (d.

J=4.64 3-1t), IH,5,6(dL-
t, 1ike.

J=15.38 6.47 7-11), III,
5.4 (dt-t, 1ikeJ=15.38
6.60. 6-11) III 4.7 (
dddJ=8.18,5.37 4.64.4-11)
, 2tl, 4.5 (s.

21) 1z), 4H,3,3(s,, 1',2
'-11□), ii, 2.4(ddd,
J=14.89, 8.18, 6.60.5-11),
IH.

2.3 (ddd, J= 14.89.6.6
0,5,37.5-11)311,2.1 (s,
, Ac), 311. 2.05 (s,
8c), 311(s, 2.03) 211. 2
．． 0 (m,, 811z), 411. 1.
9(m,,13.15-H=), 16H,1,55
(m), 1.30 (br, s,), 1.29 (
br, s, ) (9-12,1619-tlz)3H
, 0,9 (t,, J Eng G, 47. 20-fiz)
El-MS mHz: 585,500.440
398. 189. 160.43(2) Compound (2-ii) ↓ Compound (2-ii) 1422.0■ was dissolved in 7 volumes of 40 ml of dry ethanol, and after adding 22 g of Ufuichi Nisokel, the mixture was heated under reflux for 90 minutes. . After the reaction solution was cooled to room temperature, Raney-Ninkel was filtered off. This Lane nickel was washed with 200 ml of ethanol, and the 7 days of washing was combined with the filtrate.
It was concentrated under reduced pressure to obtain 1062.7ff1g of solid residue.

To this were added 5.0 ml of acetic anhydride and 5.0 ml of pyridine, 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 chloride, and dried over magnesium sulfate. 946.1 mg of a white foamy residue obtained by applying reduced pressure to 4'4v1 was separated using a silica gel (33 g) column. Developing solvent: ethyl acetate: n-hexane-7
= 3? Ejection, thin layer chromatography (developing solvent:
Fractions exhibiting an Rf value of 0.4 were collected using ethyl acetate:n-hexane (7:3) and concentrated under reduced pressure to obtain 862.2 mg of the target compound (2-iIi) as a white foamy substance.

[Rν::dan” (cn+-'): 3400.3010
．． 2910.28501775, 1750.1680.
1500.1460.1370 12701250, 1
190.1030'HNMR (200MHz, in CDCl3.R
ef, TMS) δ:1H,6,0(br,s,,
-NHAc), IH,5,8(d,.

J・4.39. 3-H), III, 4.7
(dt,, J・9.03. 4.40°4-H)
, 2)1. 4.53 (s, 2l-Hz
), 3L 2.10 (s,.

Ac), 3tl, 2.05 (s, Ac
)+ 3H12,03Cs-+ Ac)4H,1,
65(m,, 5.6-Hri, 26H,1,26(b
r, s,.

7-19-H), 311. 0.88 (L,,
J・6.47. 20-)1) MS (El-MS)
, mHz: 497. 454. 365.
322. 129. 43(3) Compound (2-iii
)→Compound (b) step Compound (2-iii) 8
Dissolve 1.9■ in 4.0 ml of methanol, add 0.99-lN aqueous solution of sodium hydroxide, and refrigerate overnight under nitrogen temperature.The 6 reaction mixture was neutralized with 1N hydrochloric acid water and precipitated. The white insoluble material is filtered off with suction and washed with methanol and water.

After drying, the residue was recrystallized from methanol to obtain Compound (b) 16.1■ as white crystals.

m, p, 159.0-161.0°C IR 2',
(c ``'): 3380.3200.2910.1
620°1460, 1100.1060.960) IN
MR (200MHz, in CDzOD) δ:
1) 1.4.0 (d.

J=11.12.2l-)1), IH,3,9(d,
, J・11.12°2l-H), 1)1. 3.
8 (br, t,, J=7.08.4-H),
18°3.7 (d,, J=1.22.3
-H), 4)1. 1.6 (m), 26H
.

1.3 (br, s,, 5-19-H), 3
H, 0,9 (t,, J=6.47°20-H) MS (FAB), mHz: 390. 372.
291. 277, 165. 104 compounds (2-
i) is produced as follows.

65 ml of pyridine was added to 10 g of 15P-18 at room temperature, and 10 ml of acetic anhydride was added with vigorous stirring. Approximately 1
Stirring was continued for an hour, and after 15P-1 was completely dissolved, stirring was stopped and the apparatus was heated overnight. Ice water was added to the reaction mixture, and the mixture was extracted with ethyl acetate, washed successively with IN hydrochloric acid, 5% sodium bicarbonate, and saturated aqueous sodium chloride, and the organic layer was dried. 10.08 g of the oily residue obtained by distilling off the solvent was mixed with ethyl acetate:
Silica gel column (with hexane = 7:3 as developing solvent)
300g). The fractions eluted from 900 to 1700 m were combined and concentrated under reduced pressure to obtain 9.29 g of compound (2-i) as a colorless transparent oil (yield 91.4%).

IRy□, (CHCh) (cm-'): 2930
．． 1760.1690°1380, 1220 11NMR (200MIIz, in CDC1z, R
ef: TMS), δ: 6.05 (18br
,s,,-Nil8c, 5.79 (18,d
, J, 4.411z 3-11) 5.57
(IH, dt, like, J=15.4.
6.3Hz, 7-tl) 5.38 (Ill,
dL, t-1ike, J-15,4,6,4
Hz, 6-H) 4.72 (III, ddd
, J=7.6. 5.1. 4.611z 4-
H) 4.51 (2L s, 2l-H2),
2.5-2.2 (2)1. m, 5-L)
2.38 (4H,t+ -1'7.511z+
13-. 15-H2), 2.10311, s
, -Ac), 2.05 (3H,s, -
Ac), 2.03 (311°s, -Ac,),
2.1-1.9 (2L m, 8-112),
1.55 (411, qui, J=6.811
z, 12.16-11z), 1.27 (1
211br, s, 9 ~11.17 ~19-11
z), 0.88 (3tl, t.

J=6.511z, 2O-111) 'CNMR (100M) Iz, in CDC1z,
Ref: TMS), δ: 211.37 (s, l4-
C), 172.32 (s > C=O), 170.
08 (s>C=O), 169.27 (s,>C
=O), 168.75 (s >C=O) 135.
04 (d, 6-C), 123.16 (d,
7, C) 81.62 (a, 4-C), 72
．． 01 (d, 3-C), 62.75 (
t, 2l-C)62.73 (s, 2-C)
, 42.85. 42.77 (each t
, 13゜l5-C), 32.46, 32.20, 3
1.63 (each t, s-g-18-C),
29.1+, 29.00 28.97 28.
9123.9L 23.85 (each t+
9-12. 16. 17-C) 22.76 (
q, -COMe), 22.50 <t,
l9-C), 20.5.5(q, -COMe)
, 20.31 (q, -GO?Ie),
14.00 (q20-C) El-MS mHz: 509.49L 382
, 348, 279. 129.43HREI-MS:
Calculated value as CzyllaJOn: 509.2990
, Actual value = 509°2998 Example 3 Synthesis of compound (C) 72.6 mg of compound (2-ii) was dissolved in methanol 2
．． (to 8 ml), IN sodium hydroxide 0.75
．． After the sword 2 was washed once, it was refluxed under a nitrogen stream at night. reaction,
The mixture was neutralized with IN hydrochloric acid, and the solvent was distilled off under reduced pressure. Water was added to the mixture, and insoluble matter was filtered off with suction. This was washed with water, air-dried, and then dried under reduced pressure in a desiccator. The white substance (23.3cm) thus obtained was dissolved in methanol while heating, and after filtering off insoluble matter, the solvent of the filtrate was distilled off. Ethyl acetate was added to the resulting white residue, and the insoluble matter was filtered off with suction.
After drying, the compound (C112,8) was obtained as a white substance.

m, p, 167.0~170.0°C'IINMR
(200MHz, in CD30D) δ: IH
,5,5(dt-L.

1ike, L=15°14. JL=6.27.
7-H), 11(, 5, 4(dt-t, 1ik
e,, L=14.89. JtJ, 60.6-14) Il
l 4.0 (d, J=10.99.2l-
II), IL 3.9 (d.

J=IO,99,2l-H), 18. 3.84
(br, t,, J=7.004-H), II
I, 3.79 (d,, J・0.97.3
-H), 41(, 3, 3(s, 1', 2'-1
0,2H,2,3(br,t,, J=6.47.5-
H) 211 2.0 (dt,, Ja・
6.35. Jt・6.35. 8-) 1n 4N
1.9 (m,, 13. 15-H),
4H,1,5(+a,, 1216-It), 1
2tl, 1.3 (m,, 9-11. 17
-19-H), 3H0,9(t-+ J, 6.6
0. 2O-H) Example 4 Synthesis of Compound (d) 26.9 ff1 g of sodium carbonate was added to 1.5 ml of water, and 35.0 ml of hydroxylamine hydrochloride and then 3.5 trtl of ethanol were added. 70% of this mixture
Warm in a water bath at °C, l5P-1200, 9tl1g
was added and shaken until dissolved. After completely dissolving 15I-1, this was further heated at the same temperature for 6 hours. After confirming that the pH of the 0 reaction solution was 7, it was directly concentrated to dryness under reduced pressure. Water is added to this residue, and the residual material is filtered off with suction. 183.6 mg of the obtained white substance was added to 9 ml of methanol, then the mixture was stirred to room temperature and allowed to stand overnight. Precipitated white insoluble matter (unreacted l5P-1
) is filtered off, if! The residue obtained from the liquid was recrystallized to obtain white substance Compound (d) (87.8 mg).

Quiet, p, 179.5~l 80.5°CC(r)
"E = -5,78° (cm0.509.) Tanol) IRvA2', (cm-'): 3200
2910.1620.14601320, 1100.1
050.965MS (El-MS) mHz: 39
B, 328.256.128.104.18 Example 5 11 589.4 ml of compound (2-i) was dissolved in 726-dioxa-, and 10 ml of water was added. Several small pieces of dry ice were added while stirring at room temperature, and after the solids had disappeared, 116 mg of sodium borohydride was added.

The same operation was repeated once more, and after 20 minutes, IN hydrochloric acid was added to make it acidic (pH 2 or less). Stir for 10 minutes! After washing, the weak acid was made neutral with IN sodium hydroxide, and then condensed under reduced pressure.
Dioxane was removed. Water was added to the residue, extracted with ethyl acetate, and the ethyl acetate layer was washed successively with 5% sodium chloride and saturated sodium chloride, and then dried over magnesium sulfate. The residue obtained by condensing the mixture under reduced pressure (1,589.6 cm) was separated using a silica gel (30 g) column. Chloroform = Mechnol = 98:2? The fractions showing RrO,4 were collected by thin layer chromatography (developing solvent: chloroform dimethanol = 9:l) and evaporated under reduced pressure to obtain the target compound (5i) 386.3■ as a colorless transparent oily substance. On the other hand, for the before and after fractions containing the target substance, chloroform:methanol = 95:5, ethyl acetate: n
- Subjected to preparative thin layer chromatography using hexane = 8 = 2 as a developing solvent, and from the band of Rf O, 4, the target product? ? 30.2■ was obtained as a colorless transparent oil.

IRvg:g"(cm-'): 2910.
2850. 1775. 17501685.150
0,1370. 1220. 1030 745HNM
R(200MIIz, in CDCl3.Ref
TMS) δun.

6.1 (br, s,, -NIIAc), I
H,5,8(d,, J=4.64°3-H),
ltl, 5.6 (dt-t, 1ike,
J=15.38. 6.47'.

7-H), ill, 5.4 (dt-t,
1ike J=15.38 6.596-H),
III, 4.7 (ddd,, J.7.5
6. 5.37. 4.64. 411) 21+,
4.5 (S,, 2l-112), Ill,
3.6 (m, 14-It)Ill, 2
．． 5 (ddd,, J-14,77,7,56,
6,59,5-11) Ill, 2.4 (ddd
,, J・+4.77.6.59,5.37.5-1
+)3ft, 2.10 (s,, Ac),
3tl, 2.05 (s,, Ac)3H,
2,03(s,, Ac), 2H,1,99Cm
, 8-H) 20L 1.3 (m, 9-13.
15-19-H), 3HO,9(t.

J・6.42.2O-1 (*) EtoMS m/z: 511.493.426.36
6, 332.263°129.43 (blank below) (2) Compound (5-i) Dissolve 386.6■ of compound (5-i) in 2.1 ml of dry 1.2-dichloroethane, and dissolve 1.1'- The weight of thiocarponyldiimidaso-/done was 273.7 cm. This solution was salted in an oil bath at 70°C for 2 hours, then in an ice water bath for 1 hour.
Cooled for 0 minutes. Add 1. to the yellow transparent reaction solution. 2 Add 20W1 of dichloroethane, 10mff1 of water. IN, salt #1
0d (twice), 10ae of 5% sodium bicarbonate, and LOml of water, and the 1,2-dichloroethane layer was dried over magnesium sulfate. The residue obtained by concentration under reduced pressure (451.9 cm) was separated using a silica gel (18 g) column. Elute with chloroform:methanol=98:2 and perform thin layer chromatography (
Rf with developing solvent chloroform:methanol-9:1)
The fractions showing 0.6 were collected and condensed under reduced pressure to obtain 265.7 mg of the target compound (5-ii) as a colorless and transparent oily substance.On the other hand, for the front and rear fractions containing the target substance, chloroform:methanol= 95=
The target substance (5-
ii) 121.0 mm was obtained as a colorless transparent oil.

UVλL:'A'': 274nm (ε=12800
)TR+z::tan"(cm-'): 2910.17
80.1?60.1680°1460, 1380.12
90.1230.1100.1040.970'HNM
R (200MHz, in CDCl3. Ref,
TMS) δ: lH8,4Cbr-s, + imidazole ring), 1) 1.7.6 (br, s, + imidazole ring), IH,7,0 (d,.

J=0.74. imidazole ring) + LH,6,3(b
r, s.

NHAc), 18.5.8 (d,, J=4.64
．． 3-H), l115.62 (qui,, J-5
,94,14-tl), IH,5,56(br,dt
,, J-15,38,6,35,7-H), IH,
5,4(dt, 1ike, J=15.38.6.6
6, 6-H), Ill, 4.7(dt,, J=8
．． 06.4.95.4-H), 2B, 4.5 (s
,.

2l-11z), 2H,2,4(m,, 511
2), 3+1. 2.10 (5,.

Ac), 311 2.05 (s,, Ac)
, 31L 2.03 (sAc), 28.
2.00 (m, 8-Hz), 4)1.
1.55 (m,.

13.15-Hz), 16H,1,31(br,s,,
9-12. ! 619-tlz), 3H,0,88(
L,, J・6.48.2O-1h) El-phylum S
m/z: 4931,133,374,332.
263. 141129.43 (3) Compound (5-ii) 60.0 mg of compound (5-ii) was dissolved in dry light Hensen 45d.
41 μ2 of tributyltin hydride and 5.0 + ng of 2.2′-azo(isobutyronitrile) were added, and the mixture was refluxed. The yellow oily residue obtained by condensation under reduced pressure was partitioned between n-hexane and acetonitrile, and the acetonitrile layer was condensed under reduced pressure. This residue a58.6cm was separated by pre-varatip thin layer chromatography (developing solvent: diethyl ether 20-hexyl J+7=9:1, double development), and the target compound (5-
iii) 21.2n+g was obtained as a colorless transparent oil.

[α)o=+63.8° (C=0.994.CH(1
3) IRv:;j,"(cm-'): 2910.
2840.1780.17551685, 1500.1
370.1220.1036'IINMR(200MI
IZ, in CDCl□ Ref, MS)
δ: 1116.0 (br, s, + -NHAc),
ill, 5.8 (d,, J=4.393-H)
, IH, 5, 6 (dt-t, 1ike, J=15
．． 26 6.597-1t), IH,5,4(dt-
t, 1ike, J=15.26 7.086-H)
, IIL 4.7 (ddd,, J.7.08.5
．． 62.4.4104-H), 2N, 4.5 (b
r, s,, 2LL), 211.2.4 (m,, 5
-11z), 3H,2,10(s,, Ac), 3
1L 2.05 (s,, Ac), 38.2.03
(s,, Ac), 2tl, 2.01(m, 8-
Hri, 228. 1.3 (br, s,, 9-19
-tlz), 3HO,9(t,, J・6.35.2
O-)1. ) El-MS m/z: 495.436
376 334 265 151129, 43 (4) Compound (5-iii) → Compound (e) Compound 'j
Dissolve 40.4 mg of jh (5-iii) in 2 ml of methanol and add 0.50 d of IN sodium hydroxide.
was added, and the mixture was refluxed under a nitrogen stream. After neutralization with IN hydrochloric acid, the precipitated white substance was collected by suction filtration and washed with water. On the other hand, the organic matter was condensed under reduced pressure to dryness, water was added, and insoluble materials were collected by suction filtration, washed with water, and recrystallized from methanol.

Both insoluble materials were combined to form compound (e) 1 as a white powder substance.
9.4■ was obtained.

m, p, 186°C [α]o+3.2° [C=lOO, MeOII-0,
2N:1aOH(1:1))IRv command S (cm-'
): 3400.3200.3100.290016
60, 1605. +565.1525.1,165.
1405.1360°1260 1200 1100
1070 1030 960 720'IINMR(2
00MHz in CIl, 00. Ref, TM
S) δ: 1115.5 (dt-t, 1ike, J
=15.38.5.86.7-11), IH.

5.4 (dt-t, 1ike, J=15.38.
6.10.6-11), IH4,0(d,, J・1
0.98.2l-H), IH,3,9(d.

J=10.99.21)1), LH,3,83(br
,t,, J=7.334-H), 18.3.78
(br,s,, 3-11), 2H,2,3(br
,t,, J=6.84.5-Hz), 2H,2,0
(m, 8-)1z).

22H1,3 (br, s, 9-19-H□), 3
1L O,9 (t.

J・6.47.20-)1) FAB-MS m/z: 388,104 57
, 45.29 The physical properties of the sodium salt of compound (e) are as follows.

m, p, 182-185°C IRv2:S (cm-'): 3400 290
0.2850.15801470, 1405.1040
．． 970.720HNMR (400MHz, in
CDxOD, Ref, TMS) δ; 2115.5
(m, 6-H, 7-H), IH, 3,8 (br,
t, J=6.44-H), IH,3,75(br
, s, 3-H), IH, 3,7 (d.

J=10.6.2l-H), IH,3,6(d, J
=10.6.2l-H)28,2.3 (br,, 5
-Hz), 2H,2,0(m, 8-tlz).

228, 1.3 (br, s,, 9~19-fiz
), 3tl, 0.9 (tJ=6.47.2O-L
) HPLC retention time: 19.5 minutes Measurement conditions; Column: Y? IC pack AQ-312 (ODS) 6
ff+ffi diameter Xl 50mm eluent Niacetonitrile-
0.05 perchlorate buffer (pH 2, 5) (6:4) Flow rate: 1 ml/min; Column temperature = 40°C Example 6
Compound (fl's precept (1) Compound (2 i) Compound (2-i) 351.3 ■ dichloromethane 25 m
2 and passed through ozone at -60°C for 6 minutes. Excess ozone was removed by blowing oxygen, and the reaction solution was concentrated under reduced pressure in a water bath at room temperature. Add 21.2 ml of dioxane to the obtained colorless transparent oily residue to dissolve it, and then add 8 ml of water.
added. Several small pieces of dry ice were added while stirring at room temperature, and when the solids disappeared, 80.0 μ of sodium borohydride was added. After 30 minutes, the reaction solution was made acidic (pH 2 or less) with IN hydrochloric acid, and after another 10 minutes, it was made from weakly acidic to neutral with IN sodium hydroxide and concentrated to dryness under reduced pressure in a 50'C water bath. After completely removing moisture by suctioning with an aspirator, about 100 ml of chloroform was added to the partially white solid residue, salt was added, and insoluble matter was filtered off under suction. Approximately 350 mg of the residue obtained by concentrating the filtrate under reduced pressure was subjected to preparative thin layer chromatography, and the target compound (6) was determined from the Rf 0.3 band using a developing solvent of chloroform:methanol = 9:1. was obtained as a white foam.

IRy"jdan"(Cm-'): 3400.300
0.2900.17801755, 1680.1500
．． 1370.1225.1050'HNMR (200M
Hz, in CDCl3. Ref, TMS) δ:
1■6.3 (br, s, -NHAc), IL
5.8 (d,, J=4.64°3-11), 1B
, 5.1 (ddd,, J.9.03.4.64.
4.394-8) 211.4.5 (br, s,,
7-Hz), 311.2.11 (s.

Ac), 3H,2,07(s,, Ac), 311
．． 2.0 (s,, Ac)4H,2,1-1,8(m
, 5.6-H2) El-MS mHz: 317.
274.185.167, 43(2) Compound (6) → Compound ('') Dissolve 70.0 mg of compound (6) in 1.33 d of methanol, add 3.99 d of water, and further add 1.33 d of IN sodium hydroxide. was added, and refluxed overnight in an oil bath at 105°C under a nitrogen stream.After cooling, IRC-50 (H' type) 6
#+1! The sample was charged onto a column packed with the following, and eluted with water-methanol (8:2). Eluate (30m0 at 50°C
It was concentrated under reduced pressure in a water bath and the resulting tan residue was suctioned into a desiccant over sodium hydroxide to remove acetic acid. A little less than 2 ml of water was added, a slight amount of insoluble matter was filtered off, and the mother liquor was condensed under pressure and water was added to bring the total volume to a little less than 2 ml. Add methanol at room temperature until the liquid turns slightly white, then leave it for 3 hours to form a colorless transparent scale-like compound (f) l 4.0
I got ■.

Claims (3)

[Claims] (1) General formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ [In the formula, R is alkyl having 1 to 30 carbon atoms, 1 to 30 carbon atoms
6 hydroxyalkyl, formula -CH=CH(CH_2)_nCH=CH-CH(O
H)-(CH_2)_mCH_3 (where n and m each represent an integer of 1 to 10), -CH=CH(CH
_2)_p-C(=X)-(CH_2)_qCH_3(
Here, >C=X group is >C<>A (A has 1 to 4 carbon atoms
represents alkylene. ), >C=N-OB (B represents hydrogen or alkyl having 1 to 4 carbon atoms), or >
CHNH_2 is shown. (p and q each represent an integer of 1 to 10)) or -CH=CH-Y (Y has 1 carbon number)
Indicates ~30 alkyls. ) represents a group represented by ] A 2-aminopentanoic acid compound represented by
A salt thereof, a γ-lactone compound thereof or a compound protected by a protecting group thereof. (2) A compound according to claim (1) selected from the following formulas (a) to (f), an optical isomer thereof, or a pharmacologically acceptable salt thereof. (a)▲There are mathematical formulas, chemical formulas, tables, etc.▼ (b)▲▲There are mathematical formulas, chemical formulas, tables, etc.▼ (c)▲There are mathematical formulas, chemical formulas, tables, etc.▼ (d)▲▲There are mathematical formulas, chemical formulas, tables, etc. Yes ▼ (e) ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (f) ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (3) An immunosuppressant comprising the compound according to claim (1) or (2).