CA2011045A1 - Immunosuppressant - Google Patents

Abstract

ABSTRACT
The present invention relates to an immunosuppressive agent which comprises at least one compound selected from the compounds of formula wherein R represents a hydrogen atom or an acyl, Y represents carbonyl or hydroxymethylene and ----- represents a single bond or a double bond and their lactones, in an effective amount and a pharmaceutically acceptable carrier; a method of prophylaxis and therapy for suppressing rejection or autoimmune diseases which comprises administering the above-mentioned compound or its lactone in an effective amount; and a novel compound of formula

Description

202~o4~, SPECIFICATION
IMMUNOSUPPRESSANT
[Technical Field]
This invention relates to the new uses of the extracts from a certain kind of microorganisms including genus Isaria and their analogues, and their related novel compounds.
[Background Art and Disclosure of the Invention]
As an immunosuppressant known heretofore, ciclosporin can be mentioned. Ciclosporin has been used for suppression of rejection in transplantation of the kidney and possesses an excellent immunosuppressive effect.
Ciclosporin, however, has a drawback that it causes side effects (e.g. renal disturbances, hepatic disturbances).
Therefore, there has been demanded immunosuppressants which have potent immunosuppressive activities and the lowest possible side effects.
From such a viewpoint, the present inventors have con-ducted extensive studies to find that the compounds of the formula (I) mentioned below which include novel compound and their lactone possessed excellent immunosuppressive activi-ties while having lower side effects, and further studies have led to the completion of the present invention.
That is, this invention relates to immunosuppressive agents which comprise at least one compound selected from the compounds of formula 2~
HO \

RHN ~ ~ COOH
(I) HO ~ ~ (CH2)5-CH2-Y-(CH2)5CH3 OH

wherein R represents a hydrogen atom or an acyl, Y represents carbonyl or hydroxymethylene and _ - represents a single bond or a double bond [hereinafter referred to as compounds (I)3 and their lactones, in an effective amount and a pharma-ceutically acceptable carrier, to a use ~ for suppressing rejection or a use for prophylaxis and therapy for autoimmune diseases, of at least one compound selected from compounds (I) and their lactones in an effective amount, and to a compound of the formula (I-1) HG \
H2N ~ / COOH (I-1) OH
HO / ~ (CH2)5-CH2-CH(CH2)5CH3 OH

and its lactone.

Referring to R in the present specification, mention is made of, for example, alkanoyls having 2 to 5 carbon atoms such as acetyl, propionyl, butyryl, pivaloyl, etc. and aroma-tic acyls having 7 to 11 carbon atoms such as benzoyl, phenyl-2~ 4~

acetyl, etc as the acyl.
Preferred compounds (I) and their lactones are as shown below:
HO
H2N ~ COOH
O
HO ~ ~ (CH2)s-CH2-C-(CH2)5-CH3 OH
HO \ l H2N o \ ~0 0 HO / ~ (CH2)s-CH2-C-(CH2)5-CH3 HO \
H2N \ / COOH

HO'~ ~ (cH2)s-cH2-c-(cH2)5-cH3 OH

HO
H2N ~ COOH
OH
HO / ~ (CH2)5-CH2-CH-(CH2)5-CH3 OH

HO \
H2N o \ ~0 0 HO / ~ (C~2)5-CH2-C-(CH2)5-CH3 HO ~)11 04~, H 2N ~ , O OH

HO/ \ ~(CH2)5-CH2-CH-(CH2)5-CH3 O
CH3CONH ~0 Ho/~//~(cH2)5-cH2-c-(cH2)s-cH3 HO

H2N \ /COOH

OH

HO/ '1~-- (CH2~5-CH2-cH-(cH2)5 C 3 OH
HO~
H2N ~L a O OH
Ho~6\~ (CH2)5-CH2-CH-(CH2)5-CH3 Among the above-mentioned compounds, the last two com-pounds, namely, the compound of the formula (I--1) and its lactone are novel compounds.
The compound of the formula (I) wherein R is a hydrogen atom, ----- is a double bond and Y is carbonyl which is the compound obtained in accordance with Example 1 mentioned hereafter, namely, ISP-I is known as Myriocin or Thermozymo-cidin [See The Journal of Antibiotics, vol. XXV No. 2, 109 115 (1972), The Journal of Organic Chemistry, 38(7), 1253 -1260 (1973), J. Chem. Soc. Perkin Trans. I, 1613 - 1619 X01104~

(1983), J. Chem. Soc., Chem. Commun., 488 - 490 (1982), etc.], with its action being an antifungal action.
Compounds (I) and their lactones can be produced by fermentation method or synthesis method in accordance with, for example, the following Production Methods 1 - 5.
The production method for ISP-I has been disclosed in the above literatures as well.
Production Method 1 (Fermentation Method) ISP-I can be usually produced by fermenting an ISP-I-producing microorganism and collecting ISP-I from the culture.
As the microorganisms to be used, mention is made of, for example, those belonging to Ascomycotina and Deuteromycotina, more specifically, genus Isaria and genus Mycelia belonging to Deuteromycotina and genus Myriococcus (Thielavia) belonging to Ascomycotina, which are respectively deposited at American Type Culture Collection as Isaria sinclairii ATCC No. 24400, _yriococcum albomyces ATCC No. 16425 and Mycelia sterilia_ _ _ _ _ ATCC No. 20349.
ISP-I can also be produced by a mutant which can be obtained by modification of a strain mentioned above by way of a conventional artificial mutating means such as ultraviolet rays, microwave radioactive rays and chemicals.
ISP-I producing microorganisms can be cultivated in various culture-media comprising usual nutrient sources for fungi. For example, there can be suitably added glucose, starches, glycerine, sugar millet jelly, dextrin, molasses, ;~)11045 maltose, xylose, and the like as carbon sources; inorganic or organic nitrogen compounds such as corn steep liquor, peptone, yeast extract, potato extract, meat extract, soy bean meal, wheat germ, potassium nitrate, sodium nitrate, ammonium sulfate, casein, gluten meal, cotton seed meal, feather meal as nitrogen sources; other conventional inorganic salts; and conventional additives for cultivation, such as organic and inorganic sub-stances and antifoaming agents which help growth of micro-organisms and can promote the production of ISP-I.
Though there is no particular limitation to the cultivation method thereof, aero~ic submerged cultivation is more advan-tageous. The preferable culture temperature in the case of strains belonging to genus Isaria is in the range from 20C to 35C, more preferably 25C to 30C, and that in the case of strains of genus Myriococcum or Mycelia is in the range from 30C to 50C, preferably 35C to 45C.
The ISP-I produced in the culture can be harvested from the culture by conventional procedures such as extraction, adsorption or by combination of conventional procedures. For example, in the case of strains such as Isaria sinclairii belonging to genus Isaria, insoluble matters such as cells are removed by a separation method such as filtration or centri fugation, and the resulting culture filtrate is put in contact with Amberlite XAD-2 to adsorb the ISP-I for harvesting. The thus-obtained ISP-I is dissolved in methanol, and the dissolved objective ISP-I is subjected to reverse phase chromatography Trade-mark 6 ;~011~34~

for fractionation to obtain highly purified ISP-I. In the case of strains such as Myriococcu_ albomyces and Mycelia sterilia which belong to genus Myriococcum or genus Myc~lia, cells are removed from the culture by a separation method such as filtration and centrifugation, and the culture filtrate is subjected to the same procedure as that in the case of strains of genus Isaria. Meanwhile, ISP-I is extracted from the separated cells with methanol and the extract is subjected to Amberlite XAD-2 as for the filtrate, followed by further purification such as chromatography or recrystallization to give ISP-I.
The compounds (I) and their lactones other than ISP-I
can be produced, for example, by the following methods.
Production Method _ (Synthesis Method) The lactones of the compounds (I) can be produced by ~
treating the corresponding compounds ~I) including ISP-I with an inorganic acid such as hydrochloric acid or an organic acid such as acetic acid or ~ treating them with a tertiary alcohol such as tert-amyl alcohol.
The reaction ~ can be conducted usually in the presence of a solvent inert to the reaction (e.g. an alcohol such as methanol or ethanol). The reaction temperature is usually in the range from 0C to 50C, preferably about room temperature, and the reaction time is usually 10 to 30 hours, preferably about 20 hours.
The reaction temperature in the reaction ~ is usually ;~01104~

in the range from 80C to 150C, preferably 100C to 120C.
The reaction time is usually 10 to 30 hours, preferably about 20 hours.
Production Method 3 (Synthesis Method) Among the compounds (I) including ISP-I, the compounds (I) wherein ----- is a single bond and their lactones can be produced by hydrogenating the corresponding compounds (I) wherein ----- is a double bond or their lactones.
Such hydrogenation is conducted in the presence of a conventional catalyst such as a palladium compound, a nickel compound or a platinum compound. The reaction is usually conducted in the presence of a solvent (e.g. an alcohol such as methanol or ethanol). The reaction temperature usually ranges from 0C to 50C, preferably about room temperature, and the reaction time is usually 1 to 10 hours, preferably about several hours.
Production Method 4 (Synthesis Method) The compounds (I) wherein Y is hydroxymethylene and their lactones can be produced by reducing the corresponding compounds (I) wherein Y is carbonyl or their lactones.
The reduction reaction can be conducted by a metal hydride complex compound such as sodium borohydride or lithium aluminum hydride.
The reaction is conducted usually in the presence of a solvent (e.g. an alcohol such as methanol or ethanol). The reaction temperature is usually 0 - 50C, preferably about Xl~o~

room temperature, and the reaction time is usually 0.5 - 4 hours, preferably about 1 hour.
Production Method 5 (Synthesis Method) -The compounds (I) wherein R is an acyl or their lactones can be produced by acylating the corresponding compounds (I) wherein R is a hydrogen atom or their lactones by a per se known means.
As the acylating agent to be used for the acylation, mention is made of, for example, acid anhydrides, acid halides, active esters and the like.
The acylation reaction can be carried out under per se known conditions.
The compounds (I) and their lactones possess excellent immunosuppressive actions, and therefore are usable as a suppressive agent for rejection in organ or marrow transplan-tation, as a prophylactic or therapeutic agent for autoimmune diseases such as rheumatoid arthritis, systemic lupus erythe-matosus, Sjogren's syndrome, multiple sclerosis, grave myas-thenia, I-type diabetes, endocrine ophthalmopathy, primary biliary cirrhosis, Crohn's diseases, glomerulonephritis, sarcoidosis, psoriasis, pemphigus, hypoplastic anemia, idio-pathic thrombocytopenic purpura and allergy, or as a medi-cal and pharmaceutical reagent to humans, cattles, horses, dogs, mice, rats and so on.
The compounds (I) or their lactones are admixed with carriers, excipients, diluents and the like to be formulated ~01~L04~

into dosage forms such as powders, capsules, tablets, injections for administration to patients. They may also be lyophilized into a pharmaceutical composition by a per se known means. The pharmaceutical composition in dosage unit form is usually packaged in a container for commercial purpose. The container may carry instructions that the composition be used as an immunosuppressive agent.
While the dosage of the compounds (I) or their lactones varies depending on diseases, symptoms, body weight, sex, age and so on, for the suppression of immune activities during or after an organ (such as kidney) transplantation, for example, they can be usually administered at the daily dosage per adult of 0.1 - 10 mg (potency), in one to several divided doses.
[Examples]
The following examples will illustrate this invention in more detail, though this invention should not be restricted to these examples as far as it comes within the scope of this invention. Immunosuppressive activities were assayed by the following methods.
Said activities are assayed based on various immune reactions using mouse, rat and human lymphocytes; for example, immunosuppressive activites are assayed with high sensitivity by using mouse, rat or human allogenic mixed lymphocyte reactions (allogenic MLR). Allogenic MLR is blastogenesis of lymphocytes induced by mixed culture of lymphocytes derived from two individuals that are allogenic but different in their major histo-compatibility antigens, such as spleen cells, lymphnode cells and peripheral blood lymphocytes. This allogenic MLR is a reaction representing a ;~o~o~

phenomenon which reflects the difference in the major histo-compatibility antigens among the donors; for example, blasto-genesis of lymphocytes cannot be observed by the mixed cul-ture of lymphocytes from monozygotic twins. Therefore, allo-genic MLR is widely used for selection of the donor and the recipient in organ transplantation.
It is usual for allogenic MLR that the lymphocytes from one of the two donors are used as stimulator cells after treatment with X-ray irradiation or with mitomycin C to inhibit their mitotic proliferation, while blastogenesis of the lymphocytes from the other donor (responder cells) is measured (one way-MLR).
Immunosuppressive activities can be determined also by measuring the activities to suppress the induction of major histocompatibility antigens-restricted cytotoxic T cells in allogenic MLR.
In addition, immunosuppressive activities can be eva-luated also as the activities to suppress blastogenesis of lymphocytes induced by stimulation with various mitogens (e.g. concanavalin A, phytohemagglutinin, pokeweed mitogen, etc.), or as the activities to suppress the mitotic prolife-ration or induction of functions of lymphocytes induced by cytokines (e.g. interleukin 1, 2, 3 ,4 ,5, 6, etc.) which enhance the mitotic proliferation or promote differentia-tion of lymphocytes such as T cells and B cells. Immunosup-pressive activities can also be evaluated as the activities to suppress the production of such cytokines from T cells, macrophages, etc.
Immunosuppressive activities can also be evaluated as the activities to suppress the induction of plasma cells producing the anti-xenogenic red blood cell antibodies in-duced within mouse spleen cells which have been immunized in advance with xenogenic red blood cells, etc. by intraperito-neal, oral, intravenous or intradermal injection to mice, or as the activities to suppress rejection in organ transplanta-tion from allogenic mice, graft-versus-host reaction, delayed allergy, adjuvant arthritis, etc.
Furthermore, immunosuppressive activities can be eva-luated as the suppression of production of anti-DNA antibody, production of rheumatoid factor, nephritis, abnormal prolife-ration of lymphocytes, or as the life-prolonging effect in MRL/Qpr mice, NZB/WF1 mice or BXSB mice, which are the model mice of autoimmune diseases, by the administration of the compounds (I) or their lactones.
Example 1 (i) Jar cultivation of Isaria sinclairii One hundred ml of the GPY medium (30 g of glucose, 5 g of peptone, 3 g of yeast extract, 0.3 g of KH2PO4, 0.3 g of K2HPO4 and 0.3 g of MgSO4 7H2O in one liter, pH 5.5) was placed into each of two 500 ml-long-neck shaking flasks, and autoclaved at 121C for 20 minutes, followed by inoculation of about 1 cm2 of mycelia of Isaria sinclairii ATCC No.24400 2 O ll O~ 5 27103-44 grown on the potato dextrose agar medium, which was then incubated at 25C for 6 days in a reciprocal shaker ~145 rpm, amplitude: 8 cm). The resultant culture was inoculated as the seed into the 10 l-fermentation jar in which 5 l of the GPY medium described above had been placed, which was then subjected to aerobic spinner culture (1 VVM, 300 rpm) at 25C
for 10 days.
(ii) Collection of ISP-I from the culture of Isaria sinclairii From 4.5 Q of the culture obtained in (i) cells and insoluble matters were removed by filtration to give 4.0 Q of culture filtrate. The obtained culture filtrate was allowed to pass through a column of Amberlite XAD-2 (~ mm x 750 mm) so that ISP-I could be adsorbed. The column was washed with 4.0 Q of water. Then 6 Q of methanol was allowed to pass to elute ISP-I. The eluate was concentrated under reduced pres-sure, dissolved in 200 ml of ethyl acetate, and extracted 3 times each with 200 ml of water in a separatory funnel.
The extract with water and that with ethyl acetate were separately concentrated under reduced pressure, and freeze-dried to give 2.23 g and 0.34 g, respectively, of ISP-I.
(iii) Purification of ISP-I
ISP-I (2.23 g) obtained in (ii~ by freeze-drying of the water extract was dissolved in 5 ml of water and fed tc the column for reverse phase chromatography (ODS DM-1020T manufa-ctured by Fuji-Devison Chemicals, Co.) (~ mm x h85 mm).
Elution was begun with water, and fractionation was carried Trade-mark 13 out by gradient elution with increasing methanol concentra-tion. The fractions eluted with 70% methanol were concen-trated to dryness under reduced pressure, dissolved in a small amount of hot methanol, and allowed to cool to give crystals of ISP-I. The crystals were dissolved again in hot methanol for recrystallization to give 40 mg of pure ISP-I.
Example 2 ~i) Jar cultivation of Myriococcum albomyces One hundred ml of the GCY medium (20 g of glucose, 5 g of corn steep liquor, 3 g of yeast extract and 0.5 g of MgSO4 7H2O in one liter, pH 6) was placed into each of two 500 ml-long-neck shaking flasks, and autoclaved at 121C for 20 minutes, followed by inoculation of about 1 cm2 of mycelia of Myriococcum albomyces ATCC No. 16425 grown on the potato dextrose agar medium, which was then incubated at 40C for 3 days in a reciprocal shaker (145 rpm, amplitude: 8 cm). The resultant culture was inoculated as the seed into the 10 l-fermentation jar in which the GCY medium described above and 1 g of an antifoaming agent (F-18 manufactured by Dow Corning Co.~ had been placed, which was then subjected to aerobic spinner culture (0.5 VVM, 300 rpm) at 40C for 7 days.
(ii) Collection of ISP-I from the culture of Myriococcum albomyces From 4.5 Q of the culture obtained in (i) cells were removed and the culture filtrate was obtained. The culture filtrate (4 Q) was allowed to pass through a column of Amber-0~

lite XAD-2 (~ 40mm x h750 mm) so that ISP-I could be adsorbed.
The column was washed with 1 Q of water. Then 1 Q of 30%
methanol, 1 Q of 50% methanol and 3 Q of 80% methanol were allowed to pass in this order, and the eluate with 80% methanol containing ISP-I was collected.
Separately, the cells were extracted 3 times with methanol of an amount about 5 times that of the wet weight of the cells, and water was added to the extract to give a 30%
methanol solution, which was then allowed to flow through a column of Amberlite XAD-2 ~40 mm x h750 mm) so that ISP-I
could be adsorbed. One liter of 30% methanol, 1 Q of 50%
methanol and 3 Q of 80% methanol were allowed to flow in this order, and the eluate with 80% methanol containing ISP-I was collected.
The fractions eluted with 80% methanol from the culture filtrate and from the cells obtained as described above were combined, concentrated under reduced pressure, and freeze-dried to give 0.5 g of powders containing ISP-I.
~iii3 Purification of ISP-I
The powders (0.5 g) containing ISP-I obtained in (ii) was washed with ethyl acetate and then with hot water (60C), dissolved in hot methanol, and allowed to cool to give crystals of ISP-I. Repeated recrystallization from methanol gave 250 mg of ISP~Ir Example 3 (i) Jar cultivation of Mycelia sterilia ~1104~

Mycelia sterilia ATCC No. 20349 was cultivated in the same manner as in Example 2 (i).
(ii) Collection of ISP-I from the culture of Mycelia sterilia In the same way as in Example 2 (ii), 1 g of powders containing ISP-I was obtained.
(iii) Purification of ISP-I
In the same way as in Example 2 (iii), 600 mg of ISP-I
was obtained.
The physical propertles of the ISP-I obtained in Exam-ples 1 to 3 are as follows.

HO\
H2N ~ COOH

HO ~ ~ ~ (CH2)s-CH2-c-tcH235 CH3 OH

melting point: 172 - 177C
H-NMR ~(ppm CD30D) : 5.52(m), 5.39(m), 3.99(d), 3.87(d), 3.83(m), 3.78(d) IR ~(KBr) : 3400, 1710, 1670, 1605, 970 cm~
Example 4 One hundred mg of ISP-I was dissolved in 20 ml of metha-nol, to which 0.4 ml of 44% methanolic hydrochloric acid was added, and kept standing overnight at room temperature, and thereafter the solvent was evaporated off under reduced pres-sure. The residue was purified by chromatography on silica 2~1104~3 gel (10 q) using a mixture of chroloform and methanol (9:1), followed by recrystallization from chloroform-petroleum ether to give 75 mg of the compound having the following structure formula.

~ O

HO ~ (CH2)5-CH2-C-(CH2)5-CH3 The physical properties of the compound thus obtained are as follows:
melting point: 75 - 76C
H-NMR ~(ppm CDCl3) : 5.62(m), 5.43(m), 4.48(m), 4.14(d), 3.75(d), 3.66(d) IR v(CHCl3) : 3400, 1770, 1705, 975 cm~
Example 5 Fifty mg of ISP-I was dissolved in 15 ml of methanol and the solution was subjected to hydrogenation using 5% palla-dium earbon (40 mg) as the catalyst. Palladium earbon was filtrated off, the solvent was evaporated under reduced pres-sure, and repeated reerystallization from methanol gave 38 mg of the eompound having the following struetural formula.

HO
H2N ~ COOH

HO ~ (CH2)s-CH2-C-(CH2)5-CH3 2~110~5 The physical properties of the compound are as follows:
melting point: 154 - 155.5C
1H-NMR ~(ppm CD30D) : 3.99(d), 3.87(d), 3.81(m), 3.87(d) IR v(KBr) : 3400, 1715, 1670 cm~
Example 6 Fifty mg of ISP-I was dissolved in 15 ml of methanol, to which sodium borohydride (20 mg) was added little by little.
Thirty minutes later, 1 ml of a saturated ammonium chloride solution was added to stop the reaction, followed by extrac-tion with chloroform and recrystallization from methanol to give 46 mg of the compound having the following structural formula.

HO
H2N \ ; COOH
OH
HO ~ ~ (CH2)5-CH2-CH-(CH2)5-CH3 OH

The physical properties of the compound thus obtained are as follows:
melting point: 162 - 165C
H-NMR ~(ppm CD30D) : 5.52(m), 5.38(m), 3.99(d) 3.87(d), 3.82(m), 3.78(s), 3.48(m) IR v(KBr) : 3300, 1665, 1635, 970 cm~
Example 7 2llo~,, The compound obtained in Example 5 was treated in the same way as in Example 4 and the compound having the follow-ing structural formula was obtained.

HO ~
H2N o \ ~0 0 HO ~ ~ (CH2)s-CH2-C-(CH2)5-CH3 The physical properties of the compound are as follows:
melting point: 96 - 98C
1H-NMR ~(ppm CDCl3) : 4.49(t), 4.13(s), 3.72(m) IR v(KBr) : 3400, 1770, 1720 cm 1 Example 8 The compound obtained in Example 6 was treated in the same way as in Example 4 and the compound having the follow-ing structural formula was obtained.

HO

H2N~
O OH

HO ~ ~ (cH2)5-cH2-cH-(cH2)s-cH3 The physical properties of the compound are as follows:

melting point: 55 - 56C

H-NMR ~(ppm CDCl3) : 5.61(m), 5.44(m), 4.48(m), 4.14(d), 3.72(d), 3.66(d), 3.57(m) IR v(KBr) : 3350, 1760~ 975 cm 1 20~1o4~.;, Example 9 The compound obtained in Example 5 was treated in the same way as in Example 6 and the compound having the follow-ing structural formula was obtained.

HO
H2N ~ COOH
¦ OH
HO ~(CH~)5-CH2-CH-(cH2)5-cH3 OH

The physical properties of the compound are as follows:
melting point: 161 - 162C
H-NMR ~(ppm CD30D~ : 4.00(d), 3.88~d), 3.81(m), 3.74(s), 3.49(m) IR vtKBr) : 3300, 1630 cm~
Example 10 The compound obtained in Example 9 was treated in the same way as in Example 4 and the compound having the follow-ing structural formula was obtained.

HO
H2N~ ~
O OH
HO ~ (CH2)5-CH2-lH-(CH2)5-CH3 The physical properties of the compound are as follows:

2~

melting point: 71.5 - 72.5C
H-NMR ~(ppm CDCl3) : 4.49(m), 4.11(d), 3.74(d), 3.69(d), 3.58(m) ~ v(KBr) : 3350, 1760 cm 1 Example 11 One hundred mg of ISP-I was dissolved in 30 ml of metha-nol, to which 10 ml of acetic anhydride was added, kept standing overnight at room temperature. Water was added to decompose acetlc anhydride, and the solvent was evaporated off under reduced pressure. The residue was purified by chromatography on silica gel (10 g) using chloroform-methanol (9:1) to give 60 mg of the compound having the following structural formula.

HO
CH CONH O
3 \ ~ O O
HO'' ~ (CH2)s-CH2-C-(CH2)5 3 The physical properties of the compound are as follows:
melting point: 105.5 - 107C
H-NMR ~(ppm CDCl3) : 6.59(s), 5.62(m), 5.42(m), 4066(m), 4.60(m) IR v(KBr) : 3300, 1760, 1710, 1650, 975 cm 1 [Experimental Example]
Experimental Example 1 (Immunosuppressive activity of immuno-suppressive compounds) 21104~, The immunosuppressive activity o~ the compounds (I) and their lactones was assayed by using mouse allogenic mixed lymphocyte reaction (hereinafter sometimes abbreviated as MLR). Mouse allogenic MLR was carried out by mixed culture of the BALB/c mouse (H-2d) spleen cells as the responder cells and the mitomycin C-treated C57BL/6 (H-2b) spleen cells as the stimulator cells in equal amounts.
The responder cells were prepared as follows: The spleen was resected from 5- to 6-week-old male BALB/c mice, and single cell suspension of spleen cells was prepared by using the RPMI1640 culture medium (containing 60 llg/ml of kanamycin sulfate, 2 mM of L-glutamine, 10 mM of N-2-hydroxyethylpipe-razine-N'-2-ethanesulfonate (HEPES) and 0.1% sodium hydrogen-carbonate) to which heat-inactivated fetal calf serum (here-inafter sometimes abbreviated as FCS) had been added to 5%.
After hemolytic treatment, the cell suspension was adjusted to a concentration of 107 cells/ml by using the RPMI1640 culture medium supplemented with 10-4 M 2-mercaptoethanol and 20% FCS, and was used as the responder cell suspension.
'rhe stimulator cells were prepared as follows:
The spleen was resected from 5- to 6-week-old male C57BL/6 mice, and single cell suspension of spleen cells was prepared by using the RPMI1640 culture medium. After hemolytic treatment, the cells were treated with 40 ~Ig/ml of mito-mycin C at 37C for 60 minutes. After washing three times, the concentration of the cell suspension was adjusted to 107 20110~, cells/ ml by using the RPMI1640 culture medium supplemented with 10-4 M 2-mercaptoethanol and 20~ FCS, and was used as the stimulator cell suspension.
Fifty ~l of the responder cell suspension, 50~1 of the stimulator cell suspension prepared by the above method, and 100~l of the test substance were placed in 96-well microcul-ture plates, and were cultured at 37C in an atmosphere of 5%
carbon dioxide for 4 days.
The blastogenesis of lymphocytes was assayed by a method of 3H-thymidine uptake as an index. That is, after termination of the culture, 0.5 ~Ci/well of 3H-thymidine was added and cultured for 4 hours. Cells were harvested by a cell-harvester, and the radioactivity incorporated into the cells was deter-mined by a liquid scintillation counter to obtain the index of blastogenesis of lymphocytes in the mouse allogenic MLR.
The suppression of the mouse allogenic MLR was evaluated by calculating the percent suppression by the following formula.
The results are summarized in Tables 1A to 1D.

cpm in MLRcpm with with test -responder percent substancecells alone suppression = 1 - x 100 (%) cpm in MLR cpm in without test -responder substancecells alone 2~

Table 1A

Responder Stimulator Test Dose 3H-thymidine Suppression cell cell substance (~gtml) uptake(cpm) (~) BALB/c - - - 963 BALB/cC57BL/6 - -14375 BALB/cC57BL/6compound of 0.001 14760 0 Example 1 compound of 0.016436 59.2 Example 1 compound of 0.1685 100.0 Example 1 compound of 1 698 100.0 Example 1 Table 1B

Responder Stimulator Test Dose 3H-thymidine Suppression cell cell substance (~g/ml) uptake(cpm) ~%) BALB/c - - - 2125 BALB/cC57BL/6 - -29716 BALB/cC57BL/6compound of 0.001 29598 0.4 Example 4 compound of 0.0110394 70.0 Example 4 compound of 0.11414 100.0 Example 4 Table 1C

Responder Stimulator Test Dose 3H-thymidine Suppression cell cell substance (~glml) uptake(cpm) (%) BALB/c - - -6195 BALB/cC57BL/6 - -27267 BALB/cC57BL/6compound of0.001 22321 23.5 Example 5 compound of 0.018182 90.6 Example 5 compound of 0.11952 100.0 Example 5 compound of 0.001 16237 52.3 Example 6 compound of 0.019276 85.4 Example 6 compound of 0.13017 100.0 Example 6 compound of 0.001 24446 13.4 Example 7 compound of 0.0134378 0 Example 7 compound of 0.13546 100.0 Example 7 compound of 0.001 22815 21.1 Example 8 compound of 0.013081 100.0 Example 8 compound of 0.11586 100.0 Example 8 compound of 0.001 25682 7.5 Example 9 ZO~10~5 compound of 0.0123668 17.1 Example 9 compound of 0.1 3167 100.0 Example 9 compound of 0.00119595 36.4 Example 10 compound of 0.0119019 39.1 Example 10 compound of 0.1 2405 100.0 Example 10 compound of 0.00132597 0 Example 11 compound of 0.0130777 0 Example 11 compound of 0.1 23430 18.2 Example 11 The test substance was dissolved in methanol or suspended in a mixture of methanol and acetic acid, and then diluted with the RPMI1640 culture medium. Methanol and acetic acid were used at a concentration of less than 0.01%, and they did not affect the allogenic MLR at all.
The compounds t1) and their lactones in the final con-centration range of 1 ~g/ml to 0.001 ~g/ml were examined for their suppressive activity for blastogenic response of lym-phocytes in mouse allogenic MLR. As shown in Tables 1A to 1D, ISP-I was suppressive in mouse allogenic MLR, with the 50%-inhibition concentration (IC50) being 7.1 x 10~3 ~g/ml.
The activities of other compounds (I) and their lactones were comparable to that of ISP-I, with the IC50 values being about 1/10 that of ciclosporin A or less. Mowever, these compounds 201104~

even at the concentration of 10 ~g/ml were not cytotoxic to mouse L929 cells (IC50 was 10 ~g/ml or more).
Blastogenesis of lymphocytes can be evaluated also by the following colorimetry using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT).
Colorimetry using MTT
The supernatant (100 ~l) is removed from each well after termination of the culture, and 20 ~l of the 5 mg/ml MTT
solution is added to each well, which is cultured at 37C for 4 hours. Thereafter, 100 ~l of a 0.01 N hydrochloric acid solution containing 10% sodium dodecylsulfate is added thereto and cultured at 37C overnight to dissolve the resultant purple crystals of formazan. The absorbancy at 550 nm is measured using a microplate absorption spectrophotometer as an index of blastogenic response of lymphocytes in mouse allogenic MLR. Suppression of mouse allogenic MLR is evaluated by caleulating the percent suppression by the following formula:

absorbancy absorbancy in MLR with - in responder pereenttest substance cells alone suppression = 1 - x 100 (%) absorbancy absorbancy in MLR without - in responder test substance cells alone Experimental Example 2 (Human allogenic MLR-suppressive activity) The human allogenic MLR-suppressive activity was assayed as follows:
Human peripheral blood lymphocytes obtained by Ficoll-~~.104~J

Paque density gradient centrifugation of normal human perip-heral blood were suspended in the RPMI1640 medium supplemented with 1o% FCS, placed in plastic dishes, and incubated at 37C
in an atmosphere of 5% carbon dioxide for 2 hours. After termination of incubation, the supernatant after gentle pipe-tting was harvested and centrifuged (1000 rpm, for 5 minutes) to obtain plastic-nonadherent cells. The plastic-nonadherent cells were allowed to pass through a nylon-wool column to give nylon-nonadherent cells, and the concentration of the cell suspension was adjusted to 4 x 106 cells/ml by using the RPMI1640 culture medium supplemented with 10% FCS, and used as the responder cell suspension.
The plastic-adherent cells were removed from the plastic dish by vigorous pipetting after addition of phosphate buf-fered saline supplemented with 5g FCS and 0.02% disodium ethylenediamine-tetraacetic acid (EDTA). The plastic-adhe-rent cells were treated with 40 ~g/ml of mitomycin C at 37C
for 60 minutes, washed three times, and suspended to the concentration of 4 x 106 cells/ml in the RPMI1640 culture medium supplemented with 10% FCS. The resultant suspension was used as the stimulator cell suspension. Fifty ~l of the responder cell suspension from the donor A or C was mixed with 50 ~l of the stimulator cell suspension from the donor B
or D, to which 100 ~l of the test substance was added, and cultured at 37C in the an atmosphere 5% carbon dioxide for 5 days.

20110~

After termination of the culture, 1.0 ~Ci/well of 3H-thymidine was added, and after 18 hours of culture, the cells were harvested by a cell-harvester. The radioactivity incor-porated into the cells was measured by a liquid scintillation counter as the index of blastogenic response of lymphocytes in human allogenic MLR. The suppression of human allogenic MLR was evaluated by calculating the percent suppression by the following formula.

cpm in MLR cpm in with test - responder percent substance cells alone suppression = 1 - x 100 (%) cpm in MLR cpm in without test - responder substance cells alone The compounds (1) and their lactones in the final con-centration range of 10 ~g/ml to 10 5 ~g/ml were examined for their suppressive activity for blastogenic response of lym-phocytes in human allogenic MLR. As shown in Tables 2A and 2B, the 50%-inhibition concentration (IC50) of ISP-I in Example 1 of this invention in the human allogenic MLR was 1.0 x 10 4 ~g/ml, IC50 of the compound in Example 4 was 7.9 x 10-4 ~g/ml, IC50 of the compound in Example 6 was 2.2 x 10-4 ~g/ml, and IC 50 of the compound in Example 5 was 3.5 x 10-4 ~g/ml.
Based on the results shown in Tables 2A and 2B, the IC50 values of the compounds (I) and their lactones in human allo-genic MLR were found to be lower than that of ciclosporin A.

20~104L5 Table 2A

Respon- Stimula- Test Dose 3H-thymidine Suppres-der cell tor cel substance (~g/ml~ uptake (cpm) sion (%) DonorA - - -2408 - DonorB - - 118 DonorA DonorB - - 23891 DonorA DonorB compound of0.0001 12769 51.8 Example 1 compound of 0.001719077.7 Example 1 compound of 0.018138 73.3 Example 1 compound of 0.16922 79.0 Example 1 compound of 1 6690 80.1 Example 1 compound of 10 1082 100.0 Example 1 compound of 0.0001 16963 32.3 Example 4 compound of 0.0011371547.4 Example 4 compound of 0.019754 65.8 Example 4 compound of 0.14734 89.2 Example 4 compound of 1 5954 83.5 Example 4 compound of 10 5332 86.4 Example 4 compound of 0.0001 20789 14.4 Example 5 ~10~

compound of 0.00114690 42.8 Example 5 compound of 0.0111130 59.4 Example 5 compound of 0.15181 87.1 Example 5 compound of 1 5548 85.4 Example 5 compound of 10 2439 99.8 Example 5 compound of 0.000114191 45.2 Example 6 eompound of 0.00111753 56.2 Example 6 compound of 0.016335 81.7 Example 6 eompound of 0.16418 81.3 Example 6 compound of 1 5877 83.9 Example 6 eompound of 10 4564 90.0 Example 6 Table 2B

Respon- Stimula- Test Dose 3H-thymidine Suppres-der eell tor cel substanee ~g/ml) uptake (epm) sion (%) DonorC - - - 89 - DonorD - - 56 DonorC DonorD - - 17427 DonorC DonorD eompound of 0.00001 10896 37.7 Example 1 eompound of 0.00019806 44.0 Example 1 2!1)11045 compound of 0.001 5646 67.9 Example 1 compound of 0.01 4460 74.8 Example 1 compound of 0.1 3613 79.2 Example 1 compound of 1 4167 76.5 Example 1 compound of 10 2018 88.8 Example 1 compound of 0.00001 7746 55.8 Example 4 compound of 0.0001 6700 61.9 Example 4 compound of 0.001 7278 58.5 Example 4 compound of 0.01 3417 80.8 Example 4 compound of 0.1 2708 84.9 Example 4 compound of 1 3703 79.2 Example 4 compound of 10 Example 4 compound of 0.00001 11827 32.3 Example 5 compound of 0.0001 7941 54.7 Example 5 compound of 0 n 001 9057 48.3 Example 5 compound of 0.01 7346 58.1 Example 5 compound of 0.1 2746 84.7 Example 5 ;~Q1104S

compound of 1 3466 80.5 Example 5 compound of 10 Example 5 compound of 0.0000116241 6.8 Example 6 compound of 0.0001 14996 14.0 Example 6 compound of 0.001 8468 51.7 Example 6 compound of 0.01 4082 77.0 Example 6 compound of 0.1 3501 80.3 Example 6 compound of 1 2448 86.4 Example 6 Experimental Example 3 [Suppression of induction of allo-reactive cytotoxic T cells in mouse allogenic mixed lymphocyte culture (MLC)]
The spleen cells suspension, 0.5 ml, (2 x 107 cells/ml) of BALB/c mouse (H-2d) prepared in the same way as in Experimen-tal Example 1, 0.5 ml of a suspension of mitomycin C-treated C57BL~6 mouse (H-2b) spleen cells (2 x 107 cell/ml) and 1.0 ml of the test substance were added to 24-well multidishes, and cultured at 37C in an atmosphere of 5% carbon dioxide for 6 days.

After termina-tion of the culture, the cells were harvested by centrifugation, and the concentration of the cell suspension was adjusted to 5 x 1o6 _ 6.25 x 105 cells/ml by using the RPMI1640 culture medium supplemented with 10~ FCS, and used as the effector cell suspension.
The target cells used were leukemia cells EL4 from the syngenic (H-2b) C57BL/b mouse as used for preparation of the stimulator cells. By incubating 106 EL4 cells in the presen-ce of 100 ~Ci of Na251CrO4 (1 mCi/ml) at 37C for 1 hour to incorporate 51Cr into the cytoplasm. The cell were washed, adjusted to the concentration of 104 cells/ml and used as the target cell suspension.
For the assay of the cytotoxic activity, 0.1 ml of the effector cell suspension and 0.1 ml of the target cell suspension were added to 96-well flat-bottomed plates, and cultured at 37C for 4 hours. The amount of 51Cr released into the supernatant was determined and the cytotoxic activity was calculated by the following formula.

cpm, effec-tor cells +- cpm, target cytotoxic target cells cells alone activity = 1 - _ x 100 (%) cpm, totalcpm, target radio-- cells alone activity The cytotoxic T cells induced by the method described above exhibited strong cytotoxic activity to the EL4 cells (H-2b) which are syngenic with the stimulator cells (H-2b) whereas they were not cytotoxic to the allogenic Meth A cells (H-2d~, and thus it was suggested that they were H-2b-restri-cted allo-reactive cytotoxic T cells.
As shown in Table 3, addition of the compound (I) or a 2()11~4a5 lactone thereof markedly inhibited the induction of the allo-reactive cytotoxic T cells and at the same time cytotoxic activity was hardly observed.

Table 3 Test Dose Amo5u~t of Cytotoxic substance (~g/ml)Cr activity release (%) (cplr.) -Total radioactivity - - 2179 of target cells Target cells alone - - 788 0 Effector cells + - - 1392 43.4 target cells (100:1) (50:1) - - 983 14.0 (25:1) - - 996 15.0 Effector cells + compound of 0.01 808 1.4 target cells Example 1 (100:1) (50:1) 0.01 783 0 (25:1) 0.01 816 2.0 Effector cells + compound of 0.1 771 0 target cells Example 1 ( 1 0 0 : 1 ) (50:1) 0.1 773 0 (25:1) 0.1 764 0 Effector cells + compound of 0.1 761 0 target cells Example 4 (100:1) (50:1) 0.1 780 0 (25:1) 0.1 767 0 2~1~045 Experimental Example 4 (Suppression of blastogenic response of mouse spleen cells by mitogen stimulation) The effect on the mouse blastogenic response of mouse spleen cells stimulated with phytohemagglutinin (PHA) or with pokeweed mitogen (PWM) was examined as follows:
The spleen was resected from 5 to 8-week-old male spleen cells BALB/c mice, and single cell suspension of spleen cells was prepared by using the RPMI1640 culture medium supplemented with 5% FCS. After hemolytic treatment, the concentration of the suspension was adjusted to 5 x 106 cells/ml by using the RPMI1640 culture medium supplemented with 10~4 M 2-mercaptoethanol and 20% FCS, to which PHA or PWM was added. One hundred ~l of the cell suspension was added to each well of 36-well microculture plates to which 100 ~l per well of the test solution had been added (5 x 105 mouse spleen cells per well). After culturing at 37C in an atmosphere of 5% carbon dioxide for 72 hours, 0.5 ~Ci/well of 3H-thymidine was added and cultured under the same conditions for further 4 hours. After termination of the culture, the cells were harvested by a cell harvester, and the radioactivity incorporated into the cells was measured by a liquid scinti-llation counter, which was used as the index of blastogenic response of mouse spleen cells. The results are summarized in Table 4.

20~04~

Table 4 Mitogen Test Dose 3H-thymidine Suppres-substance (~g/ml) uptake (cpm) sion (%) PHA(1/100) - - 8693 PHA(1/100) + eompound of0.01 3238 71.9 Example 1 0.13624 66.8 1 1686 92.4 PHA~1f100) + compound of0.01 6074 34.5 Example 4 0.14312 57.8 1 3308 71.0 _ _ - 1916 PWM(1/100) - - 31646 PWM(1/100) + eompound of0.01 13499 61.0 Example 1 0~19895 73.2 1 6529 84.5 PWM(1/100) + eompound of0.01 19335 41.4 Example 4 0.19325 75.1 1 8712 77.1 As shown in Table 4, the compounds (I) and their lae-tones strongly inhibited the ineorporation of 3H-thymidine indueed by PHA or PWM as compared with the eontrol without the eompounds.
Experimental Example 5 (Suppression of the interleukin 1 (IL1) response of mouse thymocytes) The thymus was resected from 7-week-old male C3H/HeN
mice, and single cell suspension was prepared using the serum-free RPMI1640 culture medium. After three times washing with the medium, the cells were suspended to a concentration of 1.5 x 107 cells/ml in the RPMI1640 culture medium supple-mented with 20% fetal calf serum, 5 x 10-5 M 2-mercaptoethanol, 2 x 10~3 M L-glutamine,1 x 10~3 M sodium pyruvate, 1 ~g/ml of phytohemagglutinin (PHA, Wellcome Co., HA16t17) and 2 units/ml of human ultrapure interleukin 1 (Genzyme Co., GUPi-1). One hundred ~l of this cell suspension and 100 ~l of the solution containing ISP-I were mixed in each well of the 96-well flat-bottomed microculture plate, cultured at 37~C in an atmosphere of 5% carbon dioxide for 66 hours, and cultured for further 6 hours after addition of 0.5 ~Ci/well of 3H-thymidine. After termination of the culture, the cells in each well were harvested onto a filter by a multiple cell harvester, and the radioactivity incorporated into the cells was measured by liquid scintillation method using a toluene-base scintillator.
The results obtained are summarized in Table 5. In the table, SD means standard deviation. The percent suppression (%) was calculated by the following formulation.

201~04~

3H-thymidine 3H--thymidine incorporated - incorporated with PHA + IL1 with PHA alone + test sub-percent stance suppression = 1 - x 100 (%) 3H-thymidlne3H-thymldine incorporated - lncorporated wlth PHA + IL1 wlth PHA alone Table 5 Test Dose3H-thymi.dlne Suppres-substance (~g/ml)uptake sion (%) (cpm + SD) - - 1389 + 42 PHA - 3270 + 316 PHA + IL1 + -11803 + 1740 0 compound of Example 1 2 3973 + 39 91.8 0.24646 + 826 83.9 As shown ln Table 5, lt is evident that the compound (I) and the lactones thereof show suppression of the IL1 response in a dose-dependent manner.
Experlmental Example 6 (Suppression of IL2 production in mouse allogenic mixed lymphocyte culture (MLC) and ln PHA-stimulated mouse spleen cells) Mouse allogenic MLC was carried out as follows:
The respondor cell suspension and the stimulator cell suspension (0.5 ml each) prepared in the same way as in Experimental Example 1 together with 1 ml of test substance were added to 24-well multidishes, and cultured at 37C in an atmosphere of 5% carbon dioxide for 2 days. After termi-nation of the culture, the supernatant was collected and used as the supernatant of mouse allogenic MLC.
Culture of PHA-stimulated mouse spleen cells was carried out as follows: PHA (1/100 dilution) was added to the BALB/c mouse spleen cells suspension prepared in the same way as in Experimental Example 4. The cell suspension (1 ml) and 1 ml of the test substance were added to 24-well multidishes, and cultured at 37C in an atmosphere of 5% carbon dioxide for 24 hours. After termination of the culture, the supernatant was collected and used as the supernatant of PHA-stimulated cultures.
The IL2 activity in the supernatants of mouse allogenic MLC and the supernatants of PHA-stimulated mouse spleen cells culture was assayed as follows: IL2-dependent mouse cell line CTLL-2 cells were suspended in the RPMI1640 culture medium supplemented with 30% FCS to the concentration of 105 cells/ml, and 100 ~l of the suspension was added to each well of 96-well microculture plates in which 100 ~l of the super-natant of the MLC described above had been placed. After culturing at 37C in an atmosphere of 5~ carbon dioxide for 20 hours, 0.5 ~Ci/well of 3H-thymidine was added, and incu-bated for 4 hours under the same conditions. After termination of the culture, the cells were harvested by a cell harvester, and the radioactivity incorporated into the cells was measured by a liquid scintillation counter. IL2 actlvity was expressed in U/ml as the titer at various concentrations at which the ~01~0~5 3H-thymidine incorporation was 50% of the maximum. The results are summarized in Table 6.

Table 6 Sample IL2 activity Suppres-(U/ml) sion (%) . _ _ _ . _ _ culture supernatant of responder <1 cells alone Culture supernatant of Untreated 9.8 MLC
Supernatant of MLC treated with compound of Example 1 (0.01 ~g/ml) 4.0 59.2 (0.1 ~g/ml) 2.0 79.6 (1 ~g/ml) 1.1 38.8 (10 ~g/ml) <1 >90 Supernatant of MLC treated with compound of Example 4 (0.01 ~g/ml) 4.0 59.2 (0.1 ~g/ml) 1.7 82.7 (1 ~g/ml) <1 >90 (10 ~g/ml) <1 >90 Unstimulated culture supernatant <1 Culture supernatant stimulated 12.1 with PHA
PHA-stimulated culture supernatant treated with compound of Example 1 (0.01 ~g/ml)6.5 46.3 (0.1 ~g/ml)2.5 79.3 (1 ~g/ml) 1.1 90.9 ~10 ~g/ml) <1 >92 2~)11045 PHA-stimulated culture supernatant treated with compound of Example 4 (0.01 ~g/ml) 4.9 59.5 (0.1 ~g/ml) 2.6 78.5 (1 ~g/ml) <1 >92 (10 ~g/ml) <1 >92 As shown in Table 6, it was suggested that the compounds (I) and their lactones suppress the production of IL2 in mouse allogenic MLC and in the PHA-stimulated mouse spleen cells.
Experimental Example 7 [Suppression of IL-2 induced 3H-thymi-dine incorporation of IL2-dependent mouse cell line CTLL-2]
IL2-dependent mouse cell line CTLL-2 cells were suspended in the ~PMI1640 culture medium supplemented with 30% FCS to a concentration of 2 x 105 cells/ml. The suspension (50 ~l) and 50 ~l of concanavalin A-stimulated rat spleen cell culture supernatants containing IL2 were added to each well of 96-well microculture plates in which 100 ~l of the test substance had been placed. After culturing at 37C in an atmosphere of 5% carbon dioxide for 20, 44, or 68 hours, 0.5 ~Ci/well of 3H-thymidine was added, and cultured for 4 hours under the same conditions. After termination of the culture, the cells were harvested by a cell harvester, and the radioactivity incorporated into the cells was measuered by a liquid scinti-llation counter. The results are summerized in Table 7.

201~)4~

Table 7 Incubation IE2TestDose 3H-thymidine Suppres-time substance(~g/ml) uptake (cpm) sion (~) .. . ..
24 hrs. - - - 185 + - - 6671 +compound of 0.016003 10.3 Example 1 + 0.13722 45.5 + 1 3977 41~5 + 10 1968 72.5 +compound of 0.015907 11.8 Example 4 + 0.14240 37.5 + 1 5400 19.6 + 10 2602 62.7 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ . _ _ _ _ _ _ _ _ _ _ _ _ _ _ 48 hrs. - - - 436 + - -25216 +compound of 0.0119592 22.7 Example 1 + 0.17324 72.2 + 1 4794 82.4 + 10 1649 95.1 +compound of 0.0121930 13.3 Example 4 + 0.19202 64.6 + 1 7202 72.7 + 10 7465 71.6 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 2~o~3 72 hrs. - - - 683 ~ - - 78515 + compound of 0.0156688 28.0 Example 1 + 0.1 2436 97.7 + 1 846 99.8 + 10 478100.0 + compound of 0.0169355 11.8 Example 4 + 0.1 16705 79.4 + 1 4088 95.6 + 10 11806 85.7 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 72 hrs. - - - 56 + - - 30168 + compound of 0.126108 13.5 Example 5 + 1 1532 95.1 + compound of 0.1 7237 76.2 Example 6 + 1 1346 95.7 compound of 0.137960 0 Example 7 1 2088 93.3 + compound of 0.1 7262 76.1 Example 8 + 1 1591 94 9 + compound of 0.133835 0 Example 9 2~)1104., + 1 2247 92.7 + compound of 0.1 28083 6.9 Example 10 + 1 1844 94.1 + compound of 0.1 33948 0 Example 11 + 1 4876 84.0 As shown in Table 7, the compounds (I) and their lactones strongly suppressed IL2-induced 3H-thymidine incorporation of CTLL-2 cells.
Experimental Example 8 (Suppression of induction of interleukin 2 receptor (IL-2R, Tac) expression in mouse allogenic MLC and PHA-stimulated mouse spleen cells) Mouse allogenic MLC and PHA-stimulated mouse spleen cells were prepared in the same as in Experimental Example 3.
The IL-2R (Tac) induced in mouse allogenic MLC and PHA-stimulated mouse spleen cells was assayed as follows:
After culturing the mouse allogenic MLC and the PHA-stimulated mouse spleen cells at 37C in an atmosphere of 5%
carbon dioxide for 24 hours, the cells were collected by centrifugation (1000 rpm, 5 minutes, 4C), and about 106 cells were cultured in 10 ~l of phosphate buffered saline (PBS), to which 0.02% sodium azide-containing rat anti-mouse IL-2R monoclonal antibody (manufactured by Boehringer Co., 40 ~g/ml) had been added, for 30 minutes with ice-cooling.
After washing three times with ice-cooled PBS containing 0.02% sodium azide, 50 ~l of fluorescein isothiocyanate 2(:~10~

(FITC)-labeled goat anti-rat immunoglobulin G antibody was added and cultured for 30 minutes with ice-cooling. To the cell pellet after washing three times with ice-cooled 0.02%
sodium azide-added PBS was added, a few drops of PBS containing 0.2% sodium azide and 50% glycerin. Prepared specimens on non~fluorescent slide glasses were made and the IL-2R positive cells were counted under a fluorescent microscope to determine the ratio of numbers of IL-2R positive cells in the total numbers of the cells.

Table 8 IL-2R positive Suppres-cells ~%) sion (%) . _ . .
Respondor cells 0 Untreated MLC cells 24.4 MLC cells treated with compound 22.4 8.2 of Example 1 (0.001 ~g/ml) (0.01 ~g/ml~17.6 27.9 (0.1 ~g/ml)13.1 44,3 (1 ~g/ml) 11.4 53.3 Unstimulated spleen cells 0 Untreated PHA-stimulated 46.2 spleen cells PHA-stimulated spleen cells 36.6 20.8 treated with compound of Example 1 (0.1 ~g/ml) (1 ~g/ml) 26.6 42~4 (lQ ~g~ml) 13.4 71.0 ~01104~

As shown in Table 3, it was suggested that the compounds (I) and their lactones suppress the expression of IL-2R
induced by stimulation with the alloantigen and with PHA in a concentration-dependent manner .
Experimental Example 9 (Suppression of IL3 production in mouse allogenic MLC and PHA-stimulated mouse spleen cells) The supernatant of mouse allogenic MLC and the supernatant of PHA-stimulated mouse spleen cells culture were prepared in the same manner as in Experimental Example 6.
The IL3 activity in the supernatant described above was assayed as follows: IL3-dependent mouse cell line FDC-P2 cells were suspended in the RPMI1640 culture medium supplemented with 10% FCS to the concentration of 105 cells/ml, and 100 ~l of the suspension was added to each well of 96-well microculture plates in which 100 ~l per well of the two-fold serial dilutions of the supernatant described above had been placed. After cul-turing at 37C in an atmosphere of 5% carbon dioxide for 20 hours, 0.5 ~Ci/well of 3H-thymidine was added, and cultured for further 4 hours under the same conditions. After termi-nation of the culture, the cells were harvested by a cell harvester, and the radioactivity incorporated into the cells was measured by a liquid scintillation counter. The IL3 activity was expressed in U/ml as the titer at the concentra-tions at which the amount of 3H-thymidine incorporated was 50% of the maximum. The results are summarized in Table 9.

2~1104~

Table 9 Sample IL3 activity Suppres-(U/ml) sion (%) ... _ . . .. _ culture supernatant of responder <2 cells alone Culture supernatant of Untreated MLC 36.8 Supernatant of MLC treated with compound of Example 1 (0.01 ~g/ml) 13.9 62.2 (0.1 ~g/ml) 12.1 67.1 (1 ~g/ml) 9.8 73.4 (10 ~g/ml) <2 ~95 Supernatant of MLC treated with compound of Example 4 (0.01 ~g/ml) 26.0 29.3 (0.1 ~g/ml) 16.0 56.5 (1 ~g/ml) 8.6 76.6 (10 ~g/ml) 6.1 83.4 Unstimulated culture supernatant <2 Culture supernatant stimulated 17.1 with PHA
PHA-stimulated culture supernatant treated with compound of Example 1 (0.01 ~g/ml) 4.0 76.6 (0.1 ~g/ml) <2 >90 (1 ~g/ml) <2 >90 (10 ~g/ml) <2 >90 PHA-stimulated culture supernatant treated with compound of Example 4 (0.01 ~g/ml)9.1 46.8 (0.1 ~g/ml)2.8 83.6 (1 ~g/ml) 2.3 86.5 (10 ~g/ml) <2 >90 2~1104~

As shown in Table 9, it was suggested that the compounds (I) and their lactones suppress the IL3 production in mouse allogenic MLC and PHA-stimulated mouse spleen cells.
Experimental Example 10 [Suppression of proliferation of IL3-dependent mouse cell line FDC-P2 induced by interluekin 3 (IL3)]
IL3-dependent mouse cell line FDC-P2 cells were suspended in the RPMI1640 culture medium supplemented wi~h 10% FCS to the concentration of 2 x 105 cells/ml. One hundred ~l of the suspen-sion and 50 ~l of the supernatant of the culture of mouse lueke-mia cells WEHI3 containing IL3 were added to each well of 96-well microculture plates in which 100 ~l of the test substance had been placed. After culturing at 37C in an atmosphere of 5%
carbon dioxide for 20 hours, 0.5 ~Ci/well of 3H-thymidine was added, and cultured for 4 hours under the same conditions.
After termination of the culture, the cells were harvested by a cell harvester, and the radioactivity incorporated into the cells was determined by a liquid scintillation counter, and was used as an index of the IL3-dependent proliferation.
As shown in Table 10, the compounds (I) and their lactones suppressed the increase of IL3-induced 3H-thymidine incorpo-ration into FDC-P2 cells. It was thus suggested that the immunosuppressive compounds of this invention have the activity to suppress the IL3-dependent proliferation.

2~1104~

Table 10 IL3 Test Dose 3H-thymldineSuppres-substance (llg/ml)uptake (cpm)sion (%) + - - 8315 + compound of 0.001 6858 21.5 Example 1 + 0.01 6572 25.7 + 0.1 5203 45.9 + 1 5029 48.5 + 10 2750 82.1 + compound of 0.001 7754 8.3 Example 4 0.01 6557 25.9 + 0.1 5861 36.2 + 1 4922 50.1 + 10 2902 79.9 (In the table, the marks '~' and '-' mean the presence of IL3 and the absence of IL3, respectively.) Experimental Example 11 (Suppression of interleukin 6 (IL6) res-ponse of mouse spleen cells) The spleen was resected from 8-week-old male BALB/c mice, and single cell suspension in the serum-free RPMI1640 culture medium was prepared, and thereafter cell pellets were obtained by centrifugation (1000 rpm, 5 minutes) of the suspension.
Then a mixture of 9 parts of a 0.16 M ammonium chloride solution and 1 part of a 0.17 M Tris solution (pH 7.65) was added to lyse red blood cells, and the suspension was washed three times with the serum-free RPMI1640 culture medium. The obtained cells were suspended to the concentration of 5 x 1 o6 cells/ml in the RPMI1640 culture medium supplemented with 20% FCS, 5 x 10 5 M 2-mercaptoethanol, 2 x 10~3 M L-gluta-mine, 1 x 10-3 sodium pyruvate and 25% of the supernatant of the culture of T24 human bladder carcinoma cell line as a source of interluekin 6. One hundred ~l of this cell suspen-sion and 100 ~l of the test substance were mixed in each well of 96-well microculture plates, and cultured at 37C in an atmosphere of 5% carbon dioxide for 72 hours. After termina-tion of the culture, 50 ~l of the cell suspension was collected from each well. Three hundred ~l of the RPMI1640 culture medium supplemented with 0.7% agarose gel, 20 ~l of physiolo-gical saline containing 40% protein A-bound sheep red blood cells and 20 ~l of 300-fold dilution of anti-mouse IgG anti-serum diluted in physiological saline were mixed in a test tube, and extended uniformly on Rohduck plates (Falcon Co.
1034). After gel formation by keeping still at room tempera-ture for a few mïnutes, the plates were cultured at 37C in an atmosphere of 5% carbon dioxide for about 4 hours.
Three hundred ~l of a 40-fold dilution of guinea pig comple-ment in the serum-free RPMI1640 culture medium was added to each plate, which was then incubated for further 2 hours. The number of plaques formed was counted under stereoscopic microscope.
The results of this procedure are summarized in Table 11.

XOl~O~

SD in the Table means standard deviation. The percent suppres-sion (%) was calculated by the following formula.

No. of plaque with IL6 + test - No. of plaque percent substance without IL6 suppression = 1 ~ x 100 (%) No. of plaque No. of plaque with IL6 - without IL6 alone Table 11 Test substance Dose Number of Suppression (~g/ml) plaque (~) (plaque/1x106 cells+SD) - - 12 + 2 IL6 - 206 + 12 0 IL6 + compound of Example 1 2 60 _ 8 75.3 0.2 78 + 6 66.0 0.02 108 + 10 50.5 0.002 130 + 4 39.2 As shown in Table 11, it is evident that the compounds (I) and their lactones have the suppressive effect of the IL6 response dose-dependent.
Experimental Example 12 (Suppression ~or mouse anti-sheep red blood cell antibody production) Male 5- to 7-w~ek-old BALB/c mice were immunized with sheep red blood cells (SRBC), and the spleen was resected three or four days later. By counting the plaque forming cells (PFC), suppressive effect on the pxoduction of the anti-sheep red blood cell antibody was examined as follows.
Experiment 1:
BALB/c mice were immunized with SRBC (1 x 107 cells/mouse, intravenous administration), and ISP-I in the Example 1 was administered intraperitoneally for 4 consecutive days from the day of immunization. Four days after immunization (the next day of the final administration), the spleen was resected and the anti-sheep red blood cell antibody-producing cells were determined by the direct plaque method. At the same time the body weight of the mouse, wet weight of the thymus and the spleen, and numbers of spleen cells were also determined.
Experiment 2:
BALB/c mice were immunized with SRBC (5 x 107 cells/mouse, intravenous administration), and the compound in the Example 5 was administered intraperitoneally for 4 consecutive days from the day of immunization. Four days after immunization (the next day of the final administration), the spleen was resected, and the anti-sheep red blood cell antibody-produ-cing cells were determined by the direct plaque method. At the same time, the body weight of the mouse, wet weight of the thymus and the spleen, and numbers of spleen cells were also determined.

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As shown in Table 12A and B, it is evident that the com-pound (I) and the lactones thereof have a suppressive effect on anti-sheep red blood cell antibody production, that is, they decrease PFC numbers per unit spleen cells numbers (1 x 107 cells) and PFC number per total spleen cells numbers.
Experimental Example 13 (Suppression of allo-reactive cytotoxic T cells induced by immunization of mouse with allogenic cells) Male BALB/c mice were immunized with spleen cells of C57BL/6 mice (5 x 107 cells/mouse, intraperitoneal admini-stration), and ISP-I in Example 1 was administered intraperi-toneally 6 times at the doses of 0.1 mg/kg/day from the next day of immunization. Eight days after immunization, the spleen was resected to prepare effector cells, with which the 51Cr release test was carried out as in Experimental Example 3, using EL4 cells as the target cells to measure the cytotoxic activity.
As shown in Table 13, it is evident that administration of the compound (I) or a lactone thereof suppresses the induction of allo-reactive cytotoxic T cells.

Table 13 Test substance Amg~nt of Cytotoxic Cr activity release (cpm~ (%) _ .
Total radioactivity - 7235 of target cells Target cells alone - 720 ~l~o~

Effector cells + - 2618 29 target cells (100:1) (50:1) - 2340 25 (25:1) - 1436 11 Effector cells +
target cells (100:1) compound of 1543 13 Example 1 (50:1) 1258 8 (25:1) 1046 5 Experimental Example 14 (Cytotoxicity to mouse L929 cells) Cytotoxicity to mouse L929 cells was assayed as follows:
L929 cells were suspended to the concentration of 1.5 x 105 cells/ml in the F12 culture medium supplemented with 10%
FCS. One hundred ~l of the suspension was added to each well of 96-well microculture plates, and cultured at 37C in an atmosphere of 5% carbon dioxide for 24 hours, to which 100 ~l of the test solution was added and cultured for further 48 hours. After the culture, 100 ~l of the supernatant was removed and the absorbancy at 550 nm was measured in the same way as the colormetry using MTT in Experimental Example 1.
The percent suppression was calculated by the following formula used as the index of cytotoxicity.

percent absorbancy with test substance suppression = 1 - x 100 (%) absorbancy without test substance The results are summarized in Table 14A and B.

20110~.

Table 14A

Test substance Dose Absorbancy Suppression (,ug/ml) ~%) - - 0.669 Compound of Example 1 1 0.699 0 0.693 0 100 0.321 52.0 Table 14B

Test substanceDose Absorbancy Suppression (~g/ml) (%) . _ _ _ _ 0.573 Compound of Example 5 0.1 0.775 0 1 0.805 0 0.749 0 Compound of Example 6 0.1 0.732 0 1 0.714 0 0.733 0 Compound of Example 7 0.1 0.799 0 1 0.811 0 0.696 0 Compound of Example 8 1 0.817 0 - 10 0.738 0 100 0.023 96.0 Compound of Example 9 1 0.749 0 0.747 0 100 0.567 l.0 Compound of Example 10 1 0.722 0 0.661 0 100 0.023 96.0 Compound of Example 11 1 0.710 0 0.669 0 100 0.047 91.8 As shown in Tables 14A and B, it is evident that the compounds (I) and their lactones show very low cytotoxicity to mouse L929 cells.
Experimental Example 15 (Cytotoxicity to various tumor cell lines) Cytotoxicity to human tumor cell lines was examined as follows:
Cells of human cell lines, K5~2, MOLT4, U937, HL60, KATOIII, KB, PC-6, PC-14 and CCRF-CEM were separately suspended to the concentration of 2 x 105 cells/ml in the RPMI1640 cul-ture medium supplemented with 20% FCS. Fifty lll of this sus-pension was added to each well of 96-well microculture plates to which 50 ~l of the test solution had been added. After culturing at 37C in an atmosphere of 5% carbon dioxide for 72 hours, the absorbancy at 550 nm was measured in the same way as the colorimetry using MTT in Experimental Example 1, and the percent suppression was calculated in the same manner as in Experimental Example 14, which was used as the index of cytoto-xicity. As shown in Tables 15A to 15F showing the results of XOlio~5 the calculation, the cytotoxicity of the compounds (I) and their lactones to the various cultured human tumor cell lines is weak, with the concentration of 50% inhibition (IC50) being 10 ug/ml or more.

Table 15A

Compound of Example 1 Cell lines Dose AbsorbancySuppression (~g/ml) (%) 0.1 0.886 10.6 1.0 0.905 8.6 10.0 0.929 6.2 100.0 0.583 41.1 MOLT4 0 0.618 0.1 0.560 9.4 1.0 0.559 9,5 10.0 0.499 19.3 100.0 0.074 88.0 U937 0 0.642 0.1 0.619 3.6 1.0 0.567 11.7 10.0 0.~97 22.6 100.0 0.156 75.7 HL60 0 0.631 0.1 0.402 36.3 1.0 0.390 38.2 ~

10.0 0.377 40-3 100.Q 0.101 84.0 KATOIII 0 0.961 0.01 0.990 0 0.1 0.972 0 1.0 0.961 0 10.0 0.869 9.6 100.0 0.041 95~7 Table 15B
Compound of Example 1 Cell linesDoseAbsorbancySuppression ~g/ml) (%) KB 0 0.888 0.01 0.898 0 0.1 0.865 2.6 1.0 0.879 1.0 10.0 0.886 0.2 100.0 0.025 97.2 PC-6 0 0.272 0.01 0.244 10.3 0.1 0.245 9.9 1.0 0.209 23.2 10.0 0.202 25.7 100.0 0.000 100.0 PC-14 0 0.787 0.01 0.801 0 2~04~;, 0.1 0.770 2.2 1.0 0.732 7.0 10.0 0.751 4.6 100.0 0.074 90.6 CCRF-CEM 0 0.708 0.01 0.678 4.2 0.1 0.709 0 1.0 0.660 6.8 10.0 0.637 10.0 100.0 0.013 98.2 Table 15C
Compound of Example 4 Cell linesDoseAbsorbancySuppression (~g/ml~ (%) K562 0 0.614 0.1 0.524 14~7 1.0 0.563 8.3 10.0 0.525 14.5 100.0 0.007 98.9 MOLT4 0 0.408 0.1 0.351 14.0 1.0 0.350 14.2 10.0 0.315 22.8 100.0 0.012 97.1 U937 0 0.373 201104~,, 0.1 0.313 16.1 1.0 0.384 0 10.0 0.362 2.9 100.0 0.004 99 0 HL60 0 0.346 0.1 0.265 23.4 1.0 0.300 13.3 10.0 0.273 21.1 100.0 0.008 g7.7 KATOIII 0 0.430 0.1 0.407 5,3 1.0 0.401 6.7 10.0 0.319 25.8 100.0 0.009 97.9 Table 15D
Compound of Example 4 Cell linesDoseAbsorbancySuppression (~g/ml) (%) KB 0 1.092 0.1 0.884 19.0 1.0 1.065 2.5 10.0 0.726 33.5 100.0 0.012 99.0 PC-6 0 0.291 0.1 0.247 15.1 1.0 0.273 6.2 2~iO4~

10.0 0.271 6.9 100.0 0.014 95.2 PC-14 0 0.633 0.1 0.656 0 1.0 0.679 0 10.0 0.657 100.0 0.030 95.3 CCRF-CEM 0 0.636 0.1 0.564 11.3 1.0 0.556 12.6 10.0 0.389 37.4 100.0 0.0~4 99.4 Table 15E

CellTest substance Dose Absorbancy Suppression lines (~g/ml) (~) K562 - - 0.836 Compound of Example 5 0.1 0.823 1.6 1.0 0.8103.1 10.0 0.7806.7 Compound of Example 6 0.1 0.823 1.6 1.0 0.7984.5 10.0 0.8113.0 MOLT4 - 0.620 Compound of Example 5 0.1 0.591 4.7 1.0 0.5885.2 10.0 0.50818.1 ~03LlC~

Compound of Example 6 0.1 0.615 0.8 1.0 0.5993.4 10.0 0.5678.5 U937 - 0.484 Compound of Example 5 0.1 0.462 4.5 1.0 0.485 0 10.0 0.416 0 Compound of Example 6 0.1 0.439 14.0 1.0 0.4369.9 10.0 0.43510.1 HL60 - 0.746 Compound of Example 5 0.1 0.593 20.5 1.0 0.65711.9 10.0 0.59020.9 Compound of Example 6 0.1 0.619 16.0 1.0 0.58921.0 10.0 0.62717.0 CCRF-CEM - 0.503 Compound of Example 5 0.1 0.485 3.6 1.0 0.4667.4 10.0 0.39421.7 Compound of Example 6 0.1 0.463 8.0 1.0 0.4579.1 10.0 0.4755.6 KB _ 0,730 Compound of Example 5 0.1 0.761 0 1.0 0.734 0 10.0 0.711 2.6 Compound of Example 6 0.1 0.771 0 1.0 0.728 0.3 10.0 0.707 3.2 Compound of Example 5 0.1 0.420 11.4 1O0 0.396 16.5 10.0 0.352 25.7 Compound of Example 6 0.1 0.372 21.5 1.0 0.405 14.6 10.0 0.351 25.9 Table 15F

Cell ~est substance Dose Absorbancy Suppression lines (~g/ml) (%) .
K562 - 0.385 Compound of Example 7 0.1 0.387 0 1.0 0.332 13.8 10.0 0.302 21.6 Compound of Example 8 0.1 0.327 15.1 1.0 0.321 16.6 10.0 0.298 22.6 Compound of Example 9 0.1 0.378 1.8 1.0 0.364 5.5 10.0 0.317 17.7 ~:Oli(~4~

Compound of Example 10 0.1 0.390 0 1.0 0.34310.9 10.0 0.31119.2 Compound of Example 11 0.1 0.317 17.7 1.0 0.33712.5 10.0 0.25734.5 MOLT4 - 0.145 Compound of Example 7 0.1 0.107 26.2 1.0 0.12315.2 10.0 0.10031.0 Compound of Example 8 0.1 0.142 2.1 1.0 0.137 5.5 10.0 0.133 8.3 Compound of Example 9 0.1 0.111 23.4 1.0 0.132 9.0 10.0 0.142 2.1 Compound of Example 10 0.1 0.148 0 1.0 0.159 0 10.0 0.170 0 Compound of Example 11 0.1 0.182 0 1.0 0.150 0 10.0 0.12811.7 U937 - 0.583 Compound of Example 7 0.1 0.588 0 1.0 0.593 0 10.0 0.551 5.5 Compound of Example 8 0.10.590 0 1.0 0.5800.5 10.0 0.51312.0 Compound of Example 9 0.10.640 0 1.0 0.592 0 10.0 0.584 0 Compound of Example 10 0.10.545 6.5 1.0 0.5417.2 10.0 0.51711.3 Compound of Example 11 0.10.598 0 1.0 0.583 0 10.0 0.44923.0 HL60 - 0.522 Compound of Example 7 0.10.432 17.2 1.0 0.42718.2 10.0 0.42818.0 Compound of Example 8 0.10.434 16.9 1.0 0.44514.8 10.0 0.41819.9 Compound of Example 9 0.10.450 13.8 1.0 0.43516.7 I0.0 0.41320.9 Compound of Example 10 0.10.447 14.4 1.0 0.41520.5 10.0 0.40622.3 Compound of Example 11 0.10.475 9.0 2~

1.0 0.45612.6 10.0 0.38127.0 KB
Compound of Example 7 0.10.726 0.5 1.0 0.6954.8 10.0 0.49332.5 Compound of Example 8 0.10.741 0 1.0 0.749 0 10.0 0.7063.3 Compound of Example 9 0.10.778 0 1.0 0.762 0 10.0 0.6964.7 Compound of Example 10 0.10.743 0 1.0 0.735 0 10.0 0.61016.4 Compound of Example 11 0.10.679 7.0 1.0 0.6885.8 10.0 0.51030.1 PC-14 _ 0,474 Compound of Example 7 0.10.446 5.9 1.0 0.42410.5 10.0 0.31533.5 Compound of Example 8 0.10.452 4.6 1.0 0.42510~3 10.0 0.32631.2 Compound of Example 9 0.10.404 14.8 04~

1.0 0.441 7.0 10.0 0.37421.1 Compound of Example 10 0.1 0.433 8.6 1.0 0.41113.3 10.0 0.31733.1 Compound of Example 11 0.1 0.443 6.5 1.0 0.41312.7 10.0 0.28440.0 CCRF-CEM
Compound of Example 7 0.1 0.681 1.6 1.0 0.653 5.6 10.0 0.58715.2 Compound of Example 8 0.1 0.720 0 1.0 0.694 0 10.0 0.665 3.9 Compound of Example 9 0.1 0.702 0 1.0 0.696 0 10.0 0.680 1.7 Compound of Example 10 0.1 0.672 2.9 1.0 0.647 6.5 10.0 0.645 6.8 Compound of Example 11 0.1 0.694 0 1.0 0.663 4.2 10.0 0.60312.9 Formulation Example (1) Soft capsules (in one capsule) compound of Example 1 30mg polyethyleneglycol-300 300mg polysorbate 80 20mg total 35Omg Procedure of preparation Polyethyleneglycol-300 and polysorbate 30 are added to the compound of Example 1, and the mixture is filled in soft capsules.
(2) Injections (in one ampoule, 10 ml) compound of Example 1 0.3%
polyethyleneglycol-300 20%
ethanol 60%
A sufficient quantity of distilled water is added to make the total amount 10 ml.
Procedure of preparation Ethanol and polyethyleneglycol-300 are added to the com-pound of Example 1 for dissolution and a sufficient quantity of distilled water is added thereto to make the whole volume.
Thus, an injection containing 30 ml of the compound of Example 1 in 10 ml in an ampoule is obtained.

Claims (14)

1. An immunosuppressive agent which comprises at least one compound selected from the compounds of formula (I) (wherein R represents a hydrogen atom or an acyl, Y represents carbonyl or hydroxymethylene and ----- represents a single bond or a double bond)and their lactones, in an effective amount and a pharmaceutically acceptable carrier.

2. An immunosuppressive agent as claimed in claim 1 which comprises at least one compound selected from the compounds of formula , and in an effective amount.

3. A use for suppressing rejection or a use for prophy-laxis and therapy for autoimmune diseases of at least one compound selected from compounds of formula (I) (wherein R represents a hydrogen atom or an acyl, Y represents carbonyl or hydroxymethylene and ----- represents a single bond or a double bond)and their lactones.

4. A use as claimed in claim 3 wherein the compound is at least one compound selected from the compounds of formula , and .

5. A compound of formula or its lactone.

6. A compound as claimed in claim 5, having the formula appearing in claim 5.

7. A compound as claimed in claim 5, which is a lactone having the formula:

.

8. A process for producing a compound defined in claim 5, which comprises:
reducing the carbonyl group in a compound corresponding to the compound defined in claim 5 but having a carbonyl group in place of the hydroxymethylene group, into a hydroxymethylene group by using a metal hydride complex reducing agent.

9. An immunosuppressive agent which comprises the compound defined in claim 5 in an immunosuppressive effective amount and a pharmaceutically acceptable carrier.

10. An immunosuppressive agent as claimed in claim 1, wherein R represents a hydrogen atom or an acyl group selected from the class consisting of C2-5alkanoyl and C7-11aromatic acyl.

11. An immunosuppressive agent as claimed in claim 1, wherein R represents hydrogen, acetyl, propionyl, butyryl, pivaloyl, benzoyl or phenylacetyl.

12. A commercial package containing an immunosuppressive agent defined in any one of claims 1, 2, 9, 10 and 11 in dosage unit form and bearing instructions that the agent be used for suppressing immune activity of human.

13. A commercial package as claimed in claim 12, which bears instructions that the agent be used for the suppression of immune activity during or after an organ transplantation.

14. A commercial package as claimed in claim 13, wherein the dosage unit contains from 0.1 to 10 mg of the active compound/per day.