FR2639637A1 - CYCLIC DERIVATIVES AND THEIR METABOLITES, THEIR PREPARATION AND THEIR USE AS MEDICAMENTS - Google Patents

Abstract

The present invention relates to the compounds of formula I: (CF DRAWING IN BOPI) in which R1, R2 and X have various meanings, and four metabolites of unknown structure, designated metabolites A, B, C and D. These compounds can be obtained by synthesis and fermentation of strains of Streptomyces, for example the strain Spreptomyces tsukubaensis ndegre (s) 9993. The invention also relates to the use of these compounds as medicaments.

Description

The subject of the present invention is cyclic derivatives and their

metabolites, their preparation

  and their use as medicines.

The compounds of formula II Hem, CH

O; 0 AH

H3

<S) (0 OH C R III

O o

CH3 <H CH3

OCH 3 OC 3

3 3 CH

  wherein R 1 is methyl, ethyl or allyl and n is 1 or 2, and their preparation and activity as immunosuppressive and antimicrobial agents have been described in the literature. They are isolated from cultures obtained according to known methods by fermentation of strains of Streptomyces. A preferred strain is the strain Streptomyces tsukubaensis N 9993 (FERM BP-927) described by Fujisawa in European Patent 184162. This strain can be obtained by Fermentation Research Institute, Tsukuba, Ibaraki 305, Japan, in accordance with the provisions of the Budapest Treaty. This strain was resubmitted on April 27, 1989

  at the Agricultural Research Culture Collection Interna-

  Depository Authority, Peoria, Illinois 61604, USA, in accordance with the provisions of the Treaty of

  Budapest, under the deposit number NRRL 18488.

  The Applicant has now found that this fermentation broth contains metabolites

  possessing an interesting pharmacological activity.

  They can be obtained according to a process comprising

  after separation of the compounds of formula II, the

  metabolites from the remaining solutions and wash liquors by various methods.

like chromatography.

  This process can be carried out according to known methods, for example by chromatography. The treatment of the fermentation broth is carried out for example as follows: 6200 liters of the fermentation broth are stirred at room temperature for 6 hours with 6000 l of ethyl acetate and fractionated in a separator. The ethyl acetate fraction is then evaporated to dryness under reduced pressure. The resulting extract is defatted by partitioning between 3 portions of 70 l of a methanol / water mixture (9: 1) and 3 portions of 70 l of hexane. The methanol / water fraction gives another extract after evaporation to dryness under reduced pressure. This extract is chromatographed on a column containing 25 kg of Sephadex LH20. The fractions which after thin layer chromatography and liquid phase (HPLC) contain the compounds of formula II are combined and chromatographed for further purification on a column (15 cm in diameter) of 20 kg of silica gel Merck (0.04 to 0.063 mm) eluting with tert.butyl ether and methyl. After elution of 50 liters, fractions of 6.2 liters are collected. These fractions, which after

  verification by chromatography, contain the composition

  of formula IIa oH O IIa -CH3H CH OH3

OCH 3 OCH 3

  are combined and evaporated to dryness under reduced pressure. After crystallization from acetonitrile, the compound of formula IIa is recovered as a crystalline product. The column is then rinsed with 90 liters of methanol. 91 g of a mixture of metabolites are recovered which are fractionated by chromatography on 800 g of Silicagel H with a mixture of tert.-butyl and methyl / methanol / water as eluent (88/11/1, / 17 , 5/2 and 70/25/4). Filtration on Sephadex followed by chromatography on Lichroprep RP18 gives another fraction of the mixture of metabolites. Fractions which after verification by thin layer chromatography and liquid phase (HPLC) contain identical metabolites are combined. 3 fractions with different retention times are obtained: Fraction Retention time (minutes) Gradient 1 Gradient 2

  ______________________________________________________________

A 10.21 17.69

B 4.99 13.94

C, 9.65

  HPLC system: colornn -Lichroso.b RPI. fMerck, 250 x 4'mm-

  flow rate: 2 ml / minute, detection UV.220 nm / 0O1 Eluents: triethylamine-tamnon. phosphate-pH 3.5 with 0.05 mole; 10% acetonitrile; acetonitrile Gradient 1: in 20 minutes from 50:50 to 10:90 Gradient 2: in 20 minutes from 90:10 to 10:90 Fraction A is again purified on

  Lichroprep RP18 with a mixture of water and

  thanol (1/4) and metabolite A is recovered by liquid chromatography (HPLC) in the form of

  a colorless homogeneous substance. Characteristic data

  The following are the IR spectra: see Figure 1. Mass Spectrum: see Figure 5

F - 70-750C -

  [=] 20D - -75.6 (c - 1.17 in methanol)

  UV Spectrum: X max (methanol) 320 log ε -1.3074.

  Fraction B is again purified on Sephadex LH20 with methanol as eluent and metabolite B is recovered by liquid chromatography (HPLC) as a colorless amorphous substance. The characteristic data are as follows: IR spectrum: see figure 2 Mass spectrum: see 1-a figure 6

Mp = 72-78 ° C .;

  [?] 2 OD - -71.1 (c - 1.22 in methanol) UV spectrum: Xmax (methanol 225 log -1 - 1,1561,323 log c '- - 0,0907 H-NMR (CDCl 3): a mixture of about 2: 1 conformers: characteristic signals (ppm): 6.70 (m, H-24), 6.11 (d, broad, J-15Hz, H-23) 3C-NMR (CDCl3): characteristic signals (ppm)

present in a concentration

higher rate: 199.9 (C-22);

148.8 (C-24); 137.6 (C19); 131.8

(C-23); 98.1 (C-10); 128.1 (C-29);

134.0 (C-20)

  Metabolite B has an A23 double bond.

  Fraction C is again purified on Lichoprep RP18 using as eluent a methanol / water mixture (4/1) and metabolite C is recovered by liquid chromatography (HPLC) in the form of

  of an amorphous homogeneous substance. Characteristic data

  The following are the IR spectra: see Figure 3. Mass Spectrum: see Figure 7

F - 55-60 C

  [] 2 D - + 7.50 (c - 1.0 in methanol) UV spectrum: X max (methanol 321 log e -1.972)

  The residual fractions which, after chroma-

  on silica gel with methyl tert-butyl ether, do not contain a compound of formula IIa, are combined, fractionated again on Silicagel H and Lichroprep RP18 and evaluated by liquid chromatography (HPLC) . Fractions with a retention time of 8.01 in gradient 1 are combined. Metabolite D is recovered as a colorless amorphous substance. The characteristic data are as follows: IR spectrum: see figure 4 Mass spectrum: see figure 8

F - 74-780C

  [] 20D - - 131 (c - 1.02 in chloroform) UV spectrum: Xmax (methanol) 256 log e '- 0.3716 H-NMR (CDCl3): characteristic signals (ppm) at 5.33 (d, J-4Hz, H-26); 5.22 (d, J-9 Hz, H-29); 4.53 (d, broad, J-15Hz, H-6e); 4.37 (d, broad, J-4Hz, H-2); 4.19 (ddd, J1-2Hz, J2-SHz, J3-7Hz, H-24) 13C-NMR (CDCl3): characteristic signals (ppm) at

208.4 (C-22); 202.6 (C-9); 169.6

(C-1); 167.5 (C-8); 131.6 (C-29);

124.2 (C-20); 135.1 (C-37); 116.6

(C-38); 81.4 (C-26); 68.3 (C-24).

  The invention therefore also relates to a process for the preparation of metabolites A, B and C, wherein, after separation of the compound of formula IIa from the fermentation broth, the column is rinsed with methanol and the resulting metabolite mixture is fractionated. solution by repeated chromatography. The invention also relates to a process for the preparation of metabolite D, in which the minor fractions obtained during the chromatography of the fermentation broth are fractionated with

  tert-butyl methyl ether for the separation of

of the compound of formula IIa.

  During the further processing of the fermentation products, secondary fractions are obtained which, according to liquid chromatography (HPLC), contain a new additional metabolite with a retention time of 11.7 min (gradient 1). By chromatographic fractionation and purification on Silicagel H, Sephadex LH20 and Lichroprep RP18, the compound of formula la is obtained.

CH3O H3

0s4> Ia

CH, H

0O CH3

O la

CH1 HC

CH3

H <; - 'CH3

OCH 3 OCH3

  in the form of a colorless and homogeneous amorphous substance

  born. The characteristic data are as follows:

Mp 103-105 ° C

  [=] 20D-41.8 (c-0.95 in chloroform) 1H-NMR (CDCl3 + CH3OH 5: 1): A mixture of about 3: 1 of

  two conformers: signals

  4.37 (ppm) (d, broad, J-14Hz, H-6e); 3.91 (d, J = 2 Hz,

H-26); 4.08 (H-26 ')

  13C-NMR (CDCl3): characteristic signals (ppm) at 168.8

  (C-1); 162.2 (C-8); 189.9 (C-9); 99.8

(C-10); 50.5 (C-18); 210.9 (C-22);

  41.2 (C-23); 71.5 (C-24); 37.7 (C-25);

136.3 (C-28); 127.8 (C-29).

  The invention therefore also relates to a process for the preparation of the compound of formula Ia, the process according to which the minor fractions obtained are subjected to repeated chromatographies.

  during the subsequent treatment of the fermentation broth

  to separate the compound of formula IIa [variant

a) of the process].

  As can be seen from formula Ia, the lactone ring is not fixed via the oxy group at position 26, but via the oxy group at position 24. This

  isomeric structure is hereinafter referred to as "iso".

  The invention also relates to a process for the synthesis preparation of compounds having this basic isomer structure, namely the compounds of formula I

R2 '' 1

CHCH3 CH3

  wherein X is oxo or optionally protected hydroxy, R1 'is methyl, ethyl, n-propyl or allyl and

  R2 means a hydroxy group which may be

  embedded image, method according to which b) for the preparation of the compounds of formula Ib R2. o H o I

CH3 CH3

  In which 3 represents an optionally protected hydroxy group and R 1 'and R 2 are as defined above, a compound of formula IIIa is reacted.

OH O 2

P; Ib O C CH3 y "CH3 H

OCH3 0CH3

  wherein X 'signifies an optionally protected hydroxy group and R1', and R2 are as defined above, a compound of formula IIa, H3 is reacted;

CH XI

  In which X ', R 1' and R 2 are as defined above, with a base such as imidazole or c) for the preparation of the compounds of formula Ic .HO., H CH - jCH C 33r 8 H 3, X'I '

OCH 3 OC 3

  wherein X "means an oxo or hydroxy group and R1 'is as defined above, deprotecting a compound of the formula Id R2' 3,, R * O R

I I

OCH30CH3

CH3

OCH 3 OCH 3

  in which R3 is an optionally protected hydroxy group and R1 ', R2 and X are as defined above, at least one of X, R3 and R2 being meant to mean

a protected hydroxy group.

  Compounds having this iso configuration are important intermediates for the

  preparation of new biologically active derivatives.

  When X signifies an optionally protected hydroxy group, they may have the S or R configuration at position 22. The diastereoisomers designated hereinafter "diastereoisomers A" have the S configuration in

  22 and those referred to below as "diastereo-

  B-isomers have the R configuration at this position.The protected hydroxy group is for example a hydroxy group protected by a usual protecting group such as

  than the tert.-butyldimethylsilyl group.

  The above variants of the process are

  performed according to the usual methods.

  Variant a) is preferably performed

as described above.

  Variant (b) is an isomerization reaction

  tion. It may for example be carried out by reacting a compound of formula IIa 'in an inert solvent such as dimethylformamide, with a base such that

  imidazole, carbonyldiimidazole or 4-dimethyl-

  aminopyridine. The reaction is preferably carried out at a high temperature, for example between about 50 and about 60 C. The separation of products

  desired end-products of impurities, that is, the fraction

  mixtures which may have formed are carried out according to known methods, for example by chromatography.

  Variant c) is a reaction of

  protection. It is also carried out according to the usual methods, for example using

  hydrofluoric acid and acetonitrile, preferably

  at a temperature close to room temperature. The reaction can be carried out so that depending on the reaction conditions (time, temperature) all protecting groups or only a part of them are removed. Partial deprotection is especially preferred when in a subsequent step it is desired to react a group

specific hydroxy.

  The configuration remains unchanged when the above variants of the process are carried out, especially when X is an optionally protected hydroxy group, at position 22. When

  mixtures of diastereoisomers are used as

  starting point, the fractionation into diastereo-

  individual i-someros can be carried out so

  usual at any stage of the process of

  action, for example by chromatography.

  The starting products can be obtained according to the known methods. The compounds of the formula ## STR2 ## úÉ Ra: I 'a, Tu aI! anbei suep

HOH HO0

0, ú1

Zú96ú93

  above. The fermentation broth used as starting material can for example be obtained as follows: A) Initial culture on agar medium: Strain Streptomyces tsukubaensis NO 9993 is cultured for 14 days at 27 ° C. in the following medium: Yeast extract 4 g Malt extract 10 g / l Dextrose 4 g / l Agar 20 g / l

pH 6.6 (NaOH / H2SO4).

  B) Preculture: in 90 ml of a 0.9% solution of sodium chloride, the spores and mycelium of 6 initial cultures are suspended. 10 Erlenmeyer flasks each containing 1 liter of preculture medium are each inoculated with 7 ml of this

  suspension. The preculture milieu has the composi-

  following: Glycerol 10 g / l starch 10 g / l Glucose 5 g / l Cottonseed extract 10 g / l Yeast extract 5 g / 1 CaCO3 2 g / 1 pH 6.8 This preculture is incubated for 96 hours at 27 C

and 200 laps per minute.

  C) Intermediate culture: 2 500-liter portions of the preculture medium are each incubated in a 750-liter steel fermenter with 5 liters of pre-culture for 48 hours at 27 ° C. at 100 rpm and at a ventilation rate of

0.5 l / min. per liter of medium.

  D) Main culture: 6000 liters of the main culture medium are inoculated in 2 steel fermenters of 4500 liters, with 600 liters of the intermediate culture. The main culture medium has the following composition: Soluble starch 45 g / 1 Corn soaking liquor 10 g / l Yeast extract 10 g / 1 CaCO3 1 g / 1 pH 6.8 (NaOH) The incubation is carried out for 96 hours at 27 C, at 50 rpm, at 0.5 bar and at one speed

  ventilation rate of 0.5 l / min. per liter of medium.

  When the preparation of the starting compounds is not described, these are known or can be prepared according to known methods or in a similar manner to the processes described in

examples.

  A subgroup of compounds of the invention consists of the compounds of formula Ie HCLe

- "CH3CH <C

± ^ "C

OCH3 CH 3

  wherein X is as defined above.

EXAMPLES:

  The following examples illustrate the invention without limiting its scope. In these examples, the

  temperatures are in degrees Celsius.

  The compounds designated "FK 506", "FR 520", "iso-FK 506" and "iso-FR 520" have the following structures:

FK 506:

HCHC3

CHH "

0 OH

cHO3h

H / "" CH3

OCH 3 OCH 3

FR 520:

| H oSg

C0 <CH 3

CH "CH iso-FK 506: mH ...., H

CH XOH

CH 3CH CH

- "0CH

OCH 3CCH 3

iso-FR 520: o3 ost

CH 3 CH 3

OH CH3

  Example 1: 33-O-tert-butyldimethylsilyl-22 (S) -dihydro-

  iso-FK 506 [Formula I: R1 '- an allyl group;

  R2 - a tert.-butyldimethyl group

silyloxy;

X - (S) -OH]

  [variant b) of the process] In 1 ml of dimethylformamide, the mixture is stirred

  for 120 hours at 92 mg of 33-O-tert.-butyl-

  dimethylsilyl-22 (S) -dihydro-FK 506 (diastereoisomer A) and 12 mg of imidazole. The solution is evaporated to dryness and the residue is purified by chromatography on silica gel using a mixture of

toluene / ethyl acetate (2/1).

  Instead of imidazole, you can also

  use other bases such as 4-dimethylamino-

  pyridine or carbonyldiimidazole. With carbonyl-

  diimidazole, the reaction is for example carried out

  as follows: To a solution of 459 mg of 33-O-tert.-bu-

  tyldimethylsilyl-22 (S) -dihydro-FK 506 (diastereo-

  isomer A) in 5 ml of anhydrous benzene, mg carbonyldiimidazole is added and the mixture is stirred for 12 hours at 50. The solution is evaporated to dryness under reduced pressure and the residue is purified by chromatography on silica gel using

  as eluent a toluene / ethyl acetate mixture (3/1).

  The title compound (together with the cyclic carbonate at the 22,24 position of the starting product) is obtained in the form of a colorless amorphous solid: 1 H-NMR spectrum (CDCl3, characteristic signals, ppm): 044 (m, H24 ); 3.84 (d, J-7 Hz, H-26); 4.79 (d, broad, J-10 Hz, H-20); 3.28, 3.38 and 3.41 (OCH3); 3,655

(dd, JI-2Hz, J2-9Hz, H-14).

  The following compounds of formula I are obtained in a manner analogous to that described in

example 1:

Example R1 'X R2 Characteristic Data

  2 allyl (R) -OH tert.-butyldisolid amorphous methylsilyloxy colorless 3 ethyl (S) -OH tert.-butyldisolid amorphous methylsilyloxy colorless; 1H NMR (ethyl) (1H-NMR) tert-butyldisolid amorphous methylsilyloxy colorless * 1H-NMR: (CDCl3, characteristic signals, ppm): 3.29 + 3.39 + 3.42 (s + s + s, OCH3); 3.59 (dd, J1-12Hz, J2-2.5Hz, H-15); 3.67 (d, J-10 Hz, H-14); 3.85 (d, J7Hz, H-26); 4.49 (d, broad, J-13Hz, H-6); 4.76 (d, J-10Hz,

H-20).

  Example 5: 22 (S) -dihydroiso-FK 506 [Formula I: R1 '- an allyl group;

R2 - OH;

X - (S) -OH]

  [variant c) of the process] To a solution of 1 ml of acetonitrile and 40% of hydrofluoric acid is added dropwise to

  drop 106 mg of 33-O-tert-butyldimethylsilyl-22 (S) -

  dihydroiso-FK 506 (diastereoisomer A, compound of Example 1) in 1.4 ml of acetonitrile and the mixture is stirred for 1 hour at room temperature. The solution is then concentrated under reduced pressure and purified by chromatography on silica gel using ethyl acetate as eluent. The title compound is obtained in the form of a colorless amorphous solid: 1 H-NMR spectrum (CDCl 3, characteristic signals, ppm): 3.895 (d, J-6 Hz, H-26); 4.80 (d, broad, J-10 Hz, H-20);

  3.67 (d, J-9Hz, H-14); 3,295, 3.39 and 3.42 (OCH3).

  The following compounds of formula I are obtained in a manner analogous to that described in the example: EXAMPLE R1 X R2 Characteristic data no. 6 allyl (R) -OH OH colorless amorphous solid 7 ethyl (S) -OH OH colorless amorphous solid 8 ethyl (R) -OH OH amorphous solid colorless 9 ethyl oxo OH colorless amorphous solid allyl oxo OH colorless amorphous solid The starting materials can be obtained as follows: A) 33-O-tert.butyldimethylsilyl-FR 520 A solution 80 mg of FR 520 in 3 ml of dichloromethane is added with stirring 15 mg

  of imidazole and 17 mg of tert.-butyldi

  methylsilyl. The reaction mixture is stirred for 2 hours at room temperature, diluted with saturated aqueous ammonium chloride solution and extracted 3 times with diethyl ether. The extract is washed with water and saturated sodium chloride solution, dried over sodium sulfate, concentrated under reduced pressure and purified.

by chromatography.

  B) 33-O-tert.-butyldimethylsilyl-22 (S) -dihydro-FK 506 and 33-O-tert.butyldimethylsilyl-22 (R) -dihydro-FK 506

  3.9 g of triacetoxyboron are dissolved in

  tetramethylammonium hydride and 2.75 g of 33-O-

  tert.-butyldimethylsilyl-FK 506 in 24 ml of acetonitrile / glacial acetic acid (1/1) and the mixture is stirred for 2 hours at 0-5. Water is then added, and the solution is stirred for 15 minutes at

  -15 and extracted with methylene chloride.

  The organic phase is washed with water and a solution of

  Saturated aqueous solution of sodium chloride is dried over magnesium sulfate and evaporated to dryness. The two title diastereoisomers are isolated from the crude product in a ratio of about 10: 1 (S / R).

  in the form of colorless amorphous solids, by chromatographic

  Silica gel graphite using toluene / ethyl acetate (5/1) as eluent: Diastereoisomer A (main product): 1H-NMR (CDCl3): two conformers in a ratio of about 2: 1 : characteristic signals (ppm) of the main conformer at 5.30 (H-26); 4.66 (m, H-2); 4.44 (d, broad, J-13Hz, H-6e). Signals for OCH 3 groups of both conformers were found at 3.31, 3.32, 3.355, 3.38, 3.39 and 3.42 ppm; 13C-NMR (CDCl3): characteristic signals (ppm) of the main conformer at 195.9 (C-9);

169.1 (C-1); 165.0 (C-8); 137.4

(C-37); 136.4 (C-19); 132.2 (C-28);

128.9 (C-29); 126.1 (C-20); 115.7

  (C-38); 98.4 or 97.0 (C-10); Diastereoisomer B (minor product): 13CRMN (CDCl3): characteristic signals (ppm) of the main conformer at 169.1 (C-1);

, 8 (C-8); 136.2 (C-19); 125.5

(C-20); 98.7 (C-10); 43.3 (C-21) and

36.8 (C-23).

  C) 33-O-tert-butyldimethylsilyl-22 (S) -dihydro-

FR 520 and

  33-O-tert-butyldimethylsilyl-22 (R) dihydro

FR 520

  The title compounds are obtained analogously to that described under B) above, starting from

FR 520.

  D) 33-O-tert.-butyldimethylsilyl-26-O-trimethyl-

silyl-iso-FK 506

  a) 33-O-tert.-butyldimethylsilyl-26-O-trimer

Thylsilyl-22 (S) -dihydroiso-FK 506 In 5 ml of anhydrous toluene, the mixture is stirred for 24 hours at room temperature.

  460 mg of 33-O-tert.-butyldi

  methylsilyl-22 (S) -dihydroiso-FK 506 and mg of bis-trimethylsilylacetamide. The solution is evaporated to dryness; we get the title compound together with the

  33-O-tert-butyldimethylsilyl-22-O-tri-

  methylsilyl-22 (S) -dihydroiso-FK 506, by chromatographic fractionation of the residue on silica gel using a toluene / ethyl acetate mixture (4/1) as eluent. The title compound is presented

  in the form of an amorphous substance

  color: 1H-NMR (CDCl3): 3.30 + 3.40 + 3.44 (s + s + s, OCH3); 3.67 (d, J-9Hz, H-26); 4.52 (d, broad, J-13Hz, H-6e); 4.81 (d, J-10 Hz,

H-20);

  b) 33-O-tert.-butyldimethylsilyl-26-O-tri-

methylsilyl-iso-FK 506

  To a solution of 60 mg of 33-O-tert.-bu-

  tyldiméthylsilyl-26-O-trimethylsilyl

  22 (S) -dihydroiso-FK 506 in 2 ml of anhydrous methylene chloride is added 100 mg of a 4A molecular sieve

  powdered and 12 mg of N-methyl-N-oxide

  morpholine. After stirring for 30 minutes at room temperature, the solution is reacted with about 10 mg of tetrapropylammonium perruthenate and stirred for a further 3 hours. The reaction mixture was evaporated to dryness, dissolved in ethyl acetate, washed with aqueous NaHCO 3 solution and water, dried and evaporated to dryness. The crude product can be purified by chromatography on silica gel using a toluene / ethyl acetate mixture (6/1) as eluent. The title compound is obtained in the form of a

colorless amorphous substance.

  E) 33-O-tert-butyldimethylsilyl-26-O-tri

  methylsilyl-iso-FR 520 The title compound is obtained in the form of a colorless amorphous solid by proceeding in a

  similar to that described under D) above.

  The compounds and metabolites of the invention

  have interesting pharmacological properties.

  and therefore can be used therapeutically as medicines. In particular, they exercise

  anti-inflammatory and immunosuppressive activity.

  The anti-inflammatory activity of these compounds

  The following assays can be demonstrated: 1. Allergic contact dermatitis with oxazolone in mice in vivo after topical application, according to the method described by F.M. Dietrich and R. Hess, in Int. Arch. Allergy 38 (1970)

246-259.

  The following results are obtained after topical application of a 0.01% solution of the test substance in ethanol: Compound% inhibition Metabolite A 44 Metabolite B 47 Metabolite C 48 Metabolite D 44 - It is apparent of this test that the compounds and

  their metabolites exert anti-

  between about 20% and about% after topical administration at a

concentration of 0.01%.

  2. Inhibition of oxidative respiratory explosion

  in neutrophil polymorphonuclear leukocytes

  human philes (inhibition of chemiluminescence stimulated by FMLP or A23187) in vitro:

  Preparation of polymorphous leucocytes

  (PMNL) and the measurement of chemilumi-

  nescence are carried out as described in the literature (see M. Schaude et al., Mycoses 31 [1978] 259-267), the chemiluminescence reaction.

  being initiated by addition of the N-formyl peptide

  L-methionyl-L-leucyl-L-phenyl-alanine (FMLP, 4x10 -6M) or calcium ionophore A23187 (4x10-6M). The minimum inhibitory concentration is defined as the lowest concentration of active substance which causes a clear inhibition of the three parameters; The following results were obtained in the above test (MIC - Minimum Inhibitory Concentration, #M): Stimulant Compound

FMLP A23187

  Metabolite A 0.001 Metabolite B 0.5 Metabolite D 0.5 0.5 3. In vitro inhibition of the release of PGE2 (prostaglandin E2), stimulated by TPA (12-O-tetradecanoylphorbol-13-acetate), by macrophages mouse (inhibition of macrophage activation): Inhibition of PGE2 synthesis by mouse peritoneal macrophages is performed according to a method analogous to that described by A. Haselberger et al., in Circ. Schock Res. 26 (1988) 185-192, using TPA as a stimulant. In this test, the following results are obtained: Compound Concentration (#M) Inhibition% Metabolite A 0.5 40 Metabolite B 1 Iso-FK 506 1 The immunosuppressive activity can be demonstrated in the following tests: 4. In vitro proliferative response of lymphocytes to allogeneic stimulation in the mixed lymphocyte reaction (MLR) according to the method described by T. Meo, "The MLR in the Mouse", Immunological Methods, L. Lefkovits and B. Permis, Eds, Academic Press, NY (1979), 227239: In this test, the following results are obtained: Compound C15 (g / ml) Metabolite A 0.1 Metabolite D <0.008 This test shows that the compounds and the metabolites cause the suppression of the proliferative response at a dose

about 0.15 nM and about 10 nM.

  5. In vitro inhibition of the primary humoral immune response to sheep erythrocytes: a method described by R.I. Mishell and R.W. Dutton,

  Science 153 (1966) 1004-1006; R.I. Mishell and R.W.

  Dutton, J. Exp. Med. 126 (1967) 423-442.

  In this test, the following results are obtained: Compound IC50 (μg / ml) Metabolite A 0.1 Metabolite D 0.036 This test shows that compounds and metabolites are active at a concentration of

  from about 0.5 nM to about 10 nM.

  The compounds and metabolites of the invention can therefore be used therapeutically as

  immunosuppressive and anti-inflammatory agents of

  for the prevention or treatment of conditions requiring immunosuppression or inflammatory conditions such as a) prevention and treatment - resistance to organ or tissue transplants, for example for heart, kidney, liver, marrow and skin, - graft-versus-host disease, such as following marrow transplants, - autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, Hashimoto thyroiditis , multiple sclerosis, myasthenia gravis, type I diabetes and uveitis,

  - cutaneous manifestations of immunological diseases

such as alopecia areata, and

  (b) the treatment of inflammatory and hyper-

  proliferating skin conditions, such as psoriasis, atopic dermatitis, contact dermatitis and other eczematous dermatitis, seborrheic dermatitis, lichen planus, pemphigus, bullous pemphigus, epidermolysis bullosa, urticaria, Quincke's edema, vasculitis, erythema, cutaneous eosinophilia, lupus

erythematosus and acne.

  The compounds can be administered by

  systemic or topical route.

  For the above indications, the appropriate dose of course depends for example on the patient, the mode of administration and the nature and severity of the condition to be treated. However, satisfactory results are obtained systemically with daily doses ranging from about 0.15 mg / kg to about 1.5 mg / kg. A suitable daily dose is between about 0.01 mg and about 100 mg, advantageously administered for example in doses

  Fractional up to 4 times a day.

  For topical application, satisfactory results are obtained after local administration of the active substance at a concentration of 1-3% several times daily, for

  example of 2 to 5 times. As an example of galenic forms

  suitable substances include lotions, gels

and creams.

  The compounds and metabolites of the invention

  may be administered in any of the usual ways, in particular enterally, for example orally, for example in the form of tablets or capsules, or topically, by way of

  example in the form of lotions, gels or creams.

  The pharmaceutical compositions containing a compound or a metabolite as defined above, in combination with at least one pharmaceutically acceptable carrier or diluent, may be prepared according to the usual methods, by mixing with a pharmaceutically acceptable diluent or carrier. Unit doses contain, for example, about

  0.0025 mg to about 50 mg of active substance.

  The topical administration is for example intended for the skin. It can also be intended for the eye, for example for the treatment of

  conditions of the eye involving an immune response

  such as autoimmune diseases of the eye eg uveitis, keratoplasty and chronic keratitis, allergic conditions, for example

  spring conjunctivitis, inflam-

  and corneal transplants, by topical application to the surface of the eye of a compound or metabolite of the invention in an ophthalmic vehicle

pharmaceutically acceptable.

  The carrier is such that the compound or metabolite is maintained in contact with the ocular surface for a period of time sufficient to allow the compound to enter the cornea or the internal regions of the eye, for example the anterior chamber, the posterior chamber, vitreous body, aqueous humor, vitreous humor, cornea, iris / ciliary body, lens,

  the choroid / retina complex and the sclera.

  The pharmaceutically acceptable ophthalmic vehicle may be, for example, an ointment, an oil or

  plant or material intended for encapsulation.

Claims (12)

1. The compounds of formula I OCH30 O CH3 OO "" CH CHC H 3   wherein R1 'is methyl, ethyl, n-propyl or allyl, R2 is optionally protected hydroxy and X is oxo or hydroxy possibly protected. 2. The compound of formula Ia H 3 C OHHA, N I </ Ia 0 & CO CH 30CH O CH3 H C H OCH 3 OCH 3   3. The metabolite A, characterized in that it has a melting point of 7075 C, a ["] 2 OD - -75.6 (c - 1.17 in methanol) and the IR spectra and   mass as indicated in Figures 1 and 5.   4. The metabolite B, characterized in that it has a melting point of 7278 [deg.] C, a [=] 20D-71.10 (c - 1.22 in methanol), IR and mass spectra such as indicated in FIGS. 2 and -6 and the following characteristic signals (ppm) in the 1H-NMR spectrum (CDCl3): 6.70 (m, H24) and 6.11 (d, broad, J-15 Hz, H-23).   5. Metabolite C, characterized in that it has a melting point of 5560 C, a [α] 20: = +7.50 (c-1.0 in methanol) and the IR spectra and   mass as indicated in FIGS. 3 and 7.   6. The metabolite D, characterized in that it has a melting point of 7478 C, a [a] 20D - -131 (c = 1.02 in chloroform), the IR and mass spectra as indicated in FIGS. 4 and 8 and the following characteristic signals (ppm) in the 1 H-NMR spectrum (CDCl 3): 5.33 (d, J 4 Hz, H-26); 5.22 (d, J-9 Hz, H-29); 4.53 (d, broad, J-15Hz, H-6e); 4.37 (d, broad, J-4Hz, H-2); 4.19 (ddd, J1-2Hz, J2-5Hz, J3-7Hz, H-24).   7. A process for the preparation of the compounds of formula I as defined in claim 1, characterized in that a) for the preparation of the compound of formula Ia HCHH OH jH 04 4 OCH 3CH 3 CCH3 CHH   the minor fractions which are obtained during the subsequent treatment of the fermentation broth are subjected to repeated chromatography in order to separate the compound of formula IIa aeTTenbeT suep HOO ú HOO úHOO H qI A ? - 'HO   qI elnumio; ep ssod = oD sep uoTqevedead and inod (q úHOO I HOO úHH t'II6: -Irv HOO Hú Zú96ú9Z   X 'signifies an optionally protected hydroxy group and R 1' and R 2 are as defined in claim 1, a compound of formula IIa 'R C <O OH <R lia' o o CH <H'oCH is reacted; OCH3 OCH3   in which X 'is as defined above and R 1' and R 2 are as defined in claim 1, with a base such as imidazole or c) for the preparation of compounds of formula Ic) CH3H h3 X in which X ' "means an oxo or hydroxy group and R 1 'is as defined in claim 1, deprotecting a compound of formula Id R2 .... é 0 <'OHCH 2 N Pl o iOH3 CH OH 30OCH-3   wherein R 1 ', R 2 and X are as defined in claim 1 and R 3 is an optionally protected hydroxy group, at least one of X, R 3 and R 2 in front of the formula mean a protected hydroxy group.   8. A method for recovering new metabolites from the fermentation broth used for the preparation of compounds of formula II 0 4 I I CH3 OH 3 CH3 OCH 3 OCH 3   wherein R 1 is methyl, ethyl or allyl and n is 1 or 2, by usual fermentation of strains of Streptomyces, characterized in that, after separation of the compounds of formula II, the metabolites are isolated from the solutions and remaining washing baths according to methods such as chromatography.   9. A process according to claim 8 for the preparation of metabolites A, B and C as   specified in claims 3, 4 and 5, characterized in   after separation of the compound of formula IIa HO ... C $ H 3 H 210I H 3 A s_0 oO H the CH3 H 2 1 C ô O H 3 AT CH 3P00H CH3 OCH3 OCH 3   fermentation broth, the column is rinsed with methanol and the mixture of metabolites   resulting in solution by repeated chromatography.   10. A process according to claim 8 for the preparation of the metabolite D as specified in claim 6, characterized in that the minor fractions obtained during the chromatography of the fermentation broth are fractionated with sodium ether.   tert.-butyl and methyl for the separation of   compound of formula IIa specified in claim 9.   11. A compound according to any of   1 to 6, for use as a medi-   CAMENT. 12. A pharmaceutical composition containing   a compound according to any one of claims 1   to 6, in combination with a pharmaceutical vehicle or diluent ceutically acceptable.