GB2225576A - Substituted azatricyclo derivatives and metabolites - Google Patents

Description

1 7 6 SANDOZ Ltd.

4000 Basle Case 900-9542 SUBSTITUTED AZATRICYCL0 DERIVATIVES AND METABOLITES, THEIR PREPARATION AND PECEUTICAL COMPOSITIONS CONTAINING THE14 The invention relates to substituted azatricyclo derivatives and metabolites, their preparation and pharmaceutical compositions containing them.

Compounds of formula II H CH 3 0 CH CH3 (CH 3 0 2)n 0 OH N Y 0 0 0 CH3 OH 0 OCH 3 OCH 3 II CH 3 CH wherein R, is methyl, ethyl or allyl and n is 1 or 2 900-9542 and their preparation and immunosuppressant and antimicrobial activity have been disclosed in the literature. They are isolated from culture media obtained in accordance with known methods by fermentation of Streptomyces strains. A preferred producing strain is ' Streptomyces tsukubaensis No. 9993 (FERM BP-927) as described in Fujisava EP 184162. This strain can be obtained from the Fermentation Research Institute, Tsukuba, Ibaraki 305, Japan in accordance with the provisions of the Budapest Treaty. This strain has been redeposited on April 27, 1989 with the Agricultural Research Culture Collection International Depository Authority, Peoria, Illinois 61604, USA under the conditions of the Budapest Treaty. The deposit number is NRRL 18488.

It has now been found that this fermentation broth contains a number of metabolites possessing valuable pharmacological activity. They may be obtained by a process comprisingp after separation of the compounds of formula II, isolation of the metabolites from the remaining solutions and washing liquors by methods such as chromatography.

This process may be effected in accordance with known methods, e.g. using chromatographic procedures. The working-up of the fermentation broth is e. g. effected as follows: 6200 1 of fermentation broth are stirred for 6 hours with 6000 1 of ethyl acetate at room temperature and then fractionated in a separator. The ethyl acetate fraction is then evaporated to dryness under reduced pressure. The resultant extract is then defatted by distribution between three 70 1 portions of methanol/water (9/1) and three 70 1 portions of hexane. The methanol/vater fraction gives after evaporation to dryness under reduced pressure further extract. This extract is chromatographed over a column containing 25 kg Sephadex LH20. The fractions which upon thin-layer and high pressure liquid chromatography contain the compounds of formula II are combined and chromatographed for further purification over a column 1 900-9542 (diameter 15 cm) of 20 kg Silicagel Merck (0.04 to 0.063 mm) using tertbutyl methyl ether as an eluant. After 50 1 have eluted, fractions of 6.2 1 are collected. Those fractions which upon chromatographic verification contain the compound of formula IIa HG...

,.. H CH 3 0 CH3 CH 3 0 O OH N 0 0 0 CH OH CH 3 H CH3 i OCH 3 OCH 3 IIa are combined and evaporated to dryness under reduced pressure. After crystallization from acetonitrile the compound of formula Ha is recovered as a crystalline product. The column is then washed out with 90 1 of methanol. 91 g of a mixture of metabolites is recovered, which is fractionated by further chromatography over 800 g Silicagel H using tertbutyl methyl ether/methanol/water (88/11/1, 80/17.5/2 and 70/25/4). Sephadex filtration followed by chromatography over Lichroprep RP18 leads to further fractionation of the metabolite mixture. The fractions which upon verification by thin-layer and high pressure liquid chromatography contain identical metabolites are combined. Three fractions with different retention times are obtained:

900-9542 Fraction Retention time (minutes) Gradient 1 Gradient 2 A B c 10.21 4.99 9.65 17.69 13.94 HPLC system: column: Lichrosorb RP18 Merck, 250x4 mm flow: 2 ml/min, detection UV 220 nm/0.1 Eluants: triethylamine-phosphate buffer pH 3.5 with 0.05 M 10 X acetonitrile; acetonitrile Gradient 1: in 20 min from 50:50 to 10:90 Gradient 2: in 20 min from 90:10 to 10:90.

Fraction A is further purified over Lichroprep RP18 using water/methanol (1/4) as an eluant and metabolite A is recovered as a colourless substance homogenous upon HPLC. Characterization data: IR spectrum: see diagramm 1 mass spectrum:see diagramm 5 M.P.: 70-75C 100D 20 = - 75.60 (c = 1.17 in methanol) UV spectrum:Xmax (methanol) 320 log s, - 1.3074.

900-9542 Fraction B is further purified over a Sephadex LH20 column using methanol as an eluant and metabolite B is recovered as a colourless amorphous substance homogenous upon HPLC. Characterization data:

IR spectrum: see diagramm 2 mass spectrum: see diagramm 6 M.P.: 72-780C DMID 20 - - 71.10 (c - 1.22 in methanol) UV spectrum: Xmax (methanol) 225 log el - 1.1561; 323 log el - - 0.0907 IH-NMR (CDC13): about 2:1 mixture of conformers: characteristic signals (ppm): 6.70 (m,H-24); 6.11 (d,broad,J-15ft, H-23) 13C-NMR (CDC13): characteristic signals (ppm) of the conformer present in higher concentration: 199.9 (C-22); 148.8 (C-24); 137.6 (C-19); 131.8 (C- 23); 98.1 (C-10?); 128.1 (C-29); 134.0 (C-20) Metabolite B has a L23 double bond.

Fraction C is further purified over Lichoprep RP18 using methanol/water (4/1) as an eluant and metabolite C is recovered as an amorphous substance homogenous upon HPLC. Characterization data:

IR spectrum: see diagramm 3 mass spectrum: see diagramm. 7 M.P.: 55-60C IOCID 20 - + 7.5 (c - 1.0 in methanol) UV spectrum: Xmax (methanol) 321 log el - 1.2972 The residual fractions which upon chromatography over silicagel with tert- butyl methyl ether contain no compound of formula IIa are combined, further fractionated over silicagel H and Lichroprep RP18 and evaluated with HPLC analysis. The fractions with the retention time 8.01 in gradient 1 are combined. Metabolite D is recovered as a colourless amorphous substance. Characterization data:

900-9542 IR spectrum: see diagramm 4 mass spectrum: see diagramm 8 M.P.: 74-780C [CCID 20 - - 1310 (c - 1.02 in chloroform) UV spectrum: Max (methanol) 256 log el - 0.3716 'H-NMR (CDC13): characteristic signals (ppm) at 5.33 (d,J.4Hz,H-26); 5.22 (d,J-9Hz,H-29); 4.53 (d,broad,J.l5Hz,H-6e); 4.37 (d,broad,J.4Hz,H-2); 4.19 (ddd,Jl.2Hz, J2,5Hz, J3-7ft,H-24) 13C-NMR (CDC13): 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 thus also concerns a process for the preparation of metabolites A, B and C comprising, after separation from the fermentation broth of the compound of formula IIa, washing-out the column with methanol and fractionating the resultant mixture of metabolites in solution by repeated successive chromatographic steps.

It further concerns a process for the preparation of metabolite D comprising fractionating the minor fractions obtained during chromatography of the fermentation broth with tert-butyl methyl ether for separation of the compound of formula IIa.

During working-up of the fermentation runs side fractions are obtained which, based on HPLC analysis contain a further novel metabolite having the retention time 11.7 min (gradient 1). Upon chromatographic fractionation and purification over Silicagel H, Sephadex LH20 and Lichroprep RP18 the compound of formula Ia -7 HG...

.,,H CH 0 CH3 1 CH 3 2 4 0 OH 0 21 N 0 0 0 CH3 H H OCH3 OCH 3 900-9542 CH %% CH 3 Ia is obtained as an amorphous colorless substance homogenousupon HPLC. Characterization data:

M.P.: 103-1050C IOID 20 = - 41.8 (c - 0.95 in chloroform) H-NMR (CDC13 + CH30H 5:1): about 3:1 mixture of two conformers: characteristic signals at (ppm) 4.37 (d,broad,J-14Hz,H-6e); 3.91 (d,J=2Hz, H-26); 4.08 (H-261) 13C-NMR (CDC13): 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).

900-9542 The invention thus also concerns a process for the preparation of the compound of formula Ia comprising subjecting to repeated successive chromatographic steps the minor fractions which are produced during working-up of the fermentation broth to separate the compound of formula IIa [process variant a)].

As appears from formula Ia the lactone ring is not joined via the oxy group in position 26, but via the oxy group in position 24. This isomeric structure is hereinafter named Ilisoll.

The invention also comprises a process for the synthetic preparation of the compounds having this basic isomer structure, namely of formula 1 33 H PCH 3 CH 3 0 CH '3 6 X 22 OH 0 2 1 N 1 0 0 0 CH3 OH 22 0H 0 21 R CH H CH3 1 OCH 3 OCH 3 wherein X is oxo or optionally protected hydroxy, R,' is methyl, ethyl, n- propyl or allyl and R2 is optionally protected hydroxy, I comprising b) for the preparation of the compounds of formula Ib R 2 H CH3 CH 3 0 CH 3' X 0 H 0 21 N 0 0 0 CH3 0 H H i OCH 3 OCH 3 wherein X' is optionally protected hydroxy and R,' and R2 are as defined above, reacting a compound of formula IIal R 2 -.,.

33,,,. H CH 0 CH 3 CH 3 H: 3 j CH X, 1 i 22 0 OH 21 0 0 0 CH OH 3 3 CH 3 H CH 1 OCH 3 OCH 3 wherein X', R,' and R2 are as defined above, with a base such as imidazole or 900-9542 Ib IIal c) for the preparation of the compounds of formula Ic HCL., 33 ,,. H CH 30 CH3 CH 3- 1 1 22 X OH 0 21 N 0 0 0 CH3 H wherein CH3 H CH3 1 OCH 3 OCH 3 X" is oxo or hydroxy and R,' is as defined above, deprotecting a compound of formula Id R2 --..

33 CH, 3 CH 3 0 CH 1 C 13- i 1 22 X PG 0 21 R' N 0 0 0 CH3 OH 0 H CH 3 OCH 3 OCH 3 wherein PH3 1 J-1 C3 R3 is optionally protected hydroxy and R,', R2 and X are as defined above, with the proviso that at least one of X 2 and R2 is protected hydroxy.

f, J113 900-9542 Ic Id 900-9542 The compounds having this iso configuration are important intermediates for the preparation of novel, biologically active derivatives. When X is optionally protected hydroxy they can have In position 22 the S or the R configuration. The diastereoisomers named hereinafter lldiastereoisomers AN have the S configuration in position 22, those named hereinafter lldiastereoisomers Y' have the R configuration at that position. Protected hydroxy is e.g. hydroxy protected by a conventional protecting group such as tert-butyldimethylsilyl.

The above process variants are effected in accordance with conventional methods.

above.

Process variant a) preferably is effected as described Process variant b) is an isomerization reaction. It is e.g. effected by reacting a compound of formula Hal in an inert solvent, such as dimethy1formamide, with a base such as imidazole, carbony1diimidazole or 4-dimethylaminopyridine. Reaction is preferably effected at elevated temperature, e.g. at about 50 to about 60C. Separation of the desired end products from impurities, i.e. fractionation of mixtures which may have formed is effected in accordance with known methods, e.g. chromatographically.

Process variant c) is a deprotection reaction. It is also effected in conventional manner, e.g. using hydrofluoric acid/ acetonitrile, preferably at about room temperature. The reaction can be directed so that depending on the reaction conditions (duration, temperature) either all protecting groups or only a part therefrom are removed. Partial deprotection is especially preferred where in a subsequent step it is desired to react a specific hydroxy group.

The configuration remains unchanged when effecting the above process variants, particularly, when X is an optionally protected hydroxy group, at position 22. If diastereoisomeric mixtures are used as starting products, fractionation into the individual diastereoisomers can be effected in conventional manner at any stage of the reaction process, e.g. chromatographically.

900-9542 The starting materials can be obtained in accordance with known procedures. The compounds of formula Id' R2 --., 33 CH 3 0 CH,CH3 t 1 22 0 21 N 0 0 0 CH3 H H 1 OCH 3 OCH 3 nj j1 CH 3 %.. CH 3 wherein R,', R2 and R3 are as defined above, can be obtained by oxidation of the compounds of formula Idu R 2.-., 33 H jCH3 CH 3 0 CH OH r13 22 F S 0 21 N R 0 0 " 0 D:

CH 34h H H OCH 3 OCH 3 wherein R,', R2 and R3 are as defined above.

Id' Id' 900-9542 The fermentation broth used as a starting material can e.g. be obtained as follows: A) Starting culture on agar: an agar culture of strain Streptomyces tsukubaensis No. 9993 is grown for 14 days at 27C in the following medium: yeast extract malt extract dextrose agar 4 g1l 10 g/l 4 g1l 20 g1l pH 6.6 (NaOH/H2SO4)- B) Preculture: the spores and mycelium from 6 starting cultures are suspended in 90 ml of a 0.9 % solution of sodium chloride. 10 Erlenmeyer flasks containing each 1 liter of preculture medium are each inoculated with 7 ml of this suspension. The preculture medium has the following composition: glycerol starch glucose (Wollsamen?) extract yeast extract CaC03 pH 6.8.

g/1 10 g/1 5 g11 10 g/1 5 g11 2 g11 This preculture is incubated for 96 hours at 270C and 200 rpm rotation speed.

C) Intermediate culture: two 500 1 portions of preculture medium are each incubated in a 750 1 steel fermentor with 5 1 of preculture for 48 hours at 27C, 100 rpm rotation speed and a ventilation rate of 0.5 1/min per liter of medium.

D) Main culture: 6000 1 of main culture medium are inoculated in two 4500 1 steel fermentors with 600 1 of intermediate culture.

900-9542 The main culture medium has the following composition:

soluble starch 45 g/1 corn steep 10 g/1 yeast extract 10 g/l CaC03 1 g/1 pH 6.8 (NaOH).

Incubation is effected for 96 hours at 270C, 50 rpm rotation speed, 0.5 bar and a ventilation rate of 0.5 1/min per liter of medium.

Insofar as their preparation is not described herein the starting materials are known or can be prepared in accordance with known procedures or analogously to procedures described in the Examples.

A subgroup of compounds of the invention is the compounds of formula Ie wherein X is as defined above.

HCl,' ,.,H CH 3 0 CH %' CH3 3 X 3 0 21 0 0 0 CH3 OH H OCH 3 CH 3 CH 3 Ie Explanation of the Figures:

Diagramms 1 to 4 are the IR spectra for, respectively, metabolites A to D.

Diagramms 5 to 8 are the mass spectra for, respectively, metabolites A to D.

900-9542 EXAMPLES:

900-9542 In the following Examples all temperatures are in degrees Centigrade. They illustrate the invention and are not meant to be limitative.

The compounds designated uFK 506u, "FR 5201, Oiso-FK 50P and lliso-FR 5201 have the following structural formula:

M 506:

FR 520:

HCL...

.,H CH3 CH 30 CH 3 0 OH 21.

N 0 0 0 H CH3 H OCH 3 OCH 3 HG...

H CH 0 1, CH3 3 CH 3 1.. CH CH 0 N 0 0 0 CH3 OH 0 H OCH 3 OCH 3 1 1 0 OH 21 CH 3 CH 3 iso-FK 506:

iso-FR 520:

HCL., 33 HH,PH3: c 3 CH 0 3 CH 0 G) n3- 22 00 H 0 21 N 0 0 CH34k OH H 1 OCH 3 OCH 3 HCL,, 33 H CH3 CH 0 3 CH 3 %.. CH, CH 3 0 22 6H 0 21 N 0 0 0 CH OH CH 3 H CH i OCH 3 OCH 900-9542 3 900-9542 Example 1: 33-0-tert-butyldimetMlsiAyl-22(S)-dihydro-iso-FK 506 [formula I: R11allyl; R2 - tert-butyldimethylsilyloxy; X - (S)-OH] [process variant b)] 92 mg 33-0-tert-butyldimethylsilyl-22(S)-dihydro-FK 506 (diastereoisomer A) and 12 mg imidazole are stirred for 120 hours in 1 ml of dimethylformamide at 60. The solution is evaporated to dryness and the residue purified by chromatography over silicagel using toluenelacetic acid ethyl ester (2/1) as an eluant.

In place of imidazole other bases, such as 4-dimethylaminopyridine or carbonyldlimidazole may also be used. With carbonyldiimidazole the reaction is e.g. effected as follows: to a solution of 459 mg of 33-0tert-butyldimethylsilyl22(S)-dihydro-FK 506 (diastereoisomer A) in 5 C of dry benzene is added 85 mg carbonyldiimidazole and the mixture is stirred for 12 hours at 501. The solution is evaporated to dryness under reduced pressure and the residue purified by chromatography over silicagel using toluene/acetic acid ethyl ester (3/1).

The title compound (together with the 22,24-cyclic carbonate of the starting material) is obtained as a colourless amorphous solid: 'H-NMR spectrum (CDC13, characteristic signals, ppm): 5.04 (m, H-24); 3.84 (d,J. 7Hz, H-26); 4.79 (d,broad,J-10Hz, H-20); 3.28, 3.38 and 3.41 (OCH3); 3. 655 (dd,J1.2Hz, J29HZ, H-14).

The following compounds of formula I are obtained in a manner analogous to Example 1:

900-9542 Example R, 1 X No.

R2 Characterization data 2 allyl (R)-OE tert-butyldimethylsilylcolourless amorphous oxy solid 3 ethyl (S)-OH tert-butyldimethylsilylcolourless amorphous oxy solid; NMR 4 ethyl (R)-OR tert-butyldimethylsilyl- colourless amorphous oxy solid 'H-NMR: (CDC13, characteristic signals, ppm): 3.29+3.39+3.42 (5+5+SP OCH3); 3.59 (dd,Jj-12Hz, J2.2.5Hz, H-15); 3.67 (d,J-10Hz, H-14); 3.85 (d, J.7Hz, H-26); 4.49 (d,broad, J.13Hz, H-6); 4.76 (d, Jm10Hz, H-20).

Example 5: 22(S)-dihydro-iso-FK 506 [formula I: R,'. allyl; R2 - OH; X (S)-OH] [process variant c)] 900-9542 To a solution of 1 ml acetonitrile and 20 P1 of 40 % hydrofluoric acid are added 106 mg 33-0-tert-butyldimethylsilyl22(S)-dihydro-iso-FK 506 (diastereoisomer A; compound of Example 1) in 1.4 ml of acetonitrile dropwise and the mixture is stirred for 1 hour at room temperature. The solution is then concentrated under reduced pressure and purified by chromatography over silicagel using acetic acid ethyl ester as an eluant. The title compound is obtained as an amorphous colourless solid: 'H-NMR spectrum (CDC13, characteristic signals, ppm): 3.895 (d, J=6Hz, H-26); 4. 80 (d,broad, J.10Hz, H-20); 3.67 (d,Jx9Hz, H-14); 3.295, 3.39 and 3.42 (OCH3).

The following compounds of formula I are obtained in a manner analogous to Example 5:

Example No. R,' X R2 Characterization data 6 allyl (R)-OH OH colourless amorphous solid 7 ethyl (S)-OH OH colourless amorphous solid 8 ethyl (R)-OH OH colourless amorphous solid 9 ethyl OX0 OH colourless amorphous solid allyl OX0 OH colourless amorphous solid The starting materials are obtained as follows:

A) 33-0-tert-Butyldizethylsilyl-FR 520 900-9542 To a solution of 80 mg FR 520 in 3 ml of dichloromethane are added 15 mg imidazole and 17 mg tert-butyldimethylsilyl chloride under stirring. The reaction mixture ist stirred for 2 hours at room temperature, diluted with saturated aqueous ammonium chloride solution and extracted thrice with diethyl ether. The extract is washed with water and saturated sodium chloride solutionp dried over sodium sulfate, concentrated under reduced pressure and chromatographically purified.

B) 33-0-tert-Butyldimethylsilyl-22(S)-dihydro-FK 506 and 33-0-tertbutyldimethylsilyl-22(R)-dihydro-FK 506 3.9 g tetramethylammonium-triacetoxyborohydride and 2.75 g 33-0-tertButyldimethylsilyl-FK 506 are dissolved at -20 in 24 C of acetonitrile/glacial acetic acid (l/1) and the mixture is stirred for 2 hours at 00 to 50. Then water is added, the solution is stirred for 15 minutes at 100 to 150 and extracted with methylene chloride. The organic phase is washed with water and saturated aqueous sodium chloride solution, dried over magnesium sulfate and evaporated to dryness. The two diastereoisomeric title compounds are isolated from the crude product in the proportion of about 10:1 (S/R) as amorphous colourless solids by chromatography over silicagel using toluene/acetic acid ethyl ester (5/1) as an eluant: Diastereoisomer A (main product):

H-NMR (CDC13)' two conformers in the proportion of about 2:1: characteristic signals (ppm) of the major conformer at 5.30 (H-26); 4.66 (m,H-2); 4.44 (d,broad,J.13Hz, B-6e). Signals are found for the OCH3groups of both conformers at 3.31, 3.32, 3.335, 3.38, 3.39 and 3.42 ppm; 900-9542 13C-MR (CDC13): characteristic signals (ppm) of the major 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):

13C-MR (CDC13): characteristic signals (ppm) of the major conformer at 169.1 (C-1); 165.8 (C-8); 136.2 (C-19); 125.5 (C-20); 98.7 (C-10); 43.3 (C-21) and 36.8 (C-23).

C) 33-0-tert-Butyldimethylsilyl-22(S)-dihydro-FR 520 and 33-0-tertbutyldimethylsilyl-22(R)-dihydro-FR 520 The title compounds are obtained in a manner analogous to that described under B) above, starting from PR 520.

D) 33-0-tert-Butyldimethylsilyl-26-0-trimethylsilyl-iso-FK 506 a) 33-0-tert-Butyldimethylsilyl-26-0-trimethylsilyl22(S)-dihydro-iso-FK 506 460 mg 33-0-tert-butyldimethylsilyl-22(S)-dihydro-iso-FK 506 and 120 mg bis-trimethylsilylacetamide are stirred for 24 hours at room temperature in 5 ml of dry toluene. The solution is evaporated to dryness and the title compound, together with 33-0-tert-butyldimethylsiiyl22-0trimethylsilyl-22(S)-dihydro-iso-FK 506, is obtained upon chromatographic fractionation of the residue over silicagel using toluene/acetic acid ethyl ester (4/1) as an eluant. The title compound is a colourless, amorphous substance: IH-NMR (CDC13): 3.30 + 3.40 + 3.44 (s+s+s, OCHO; 3. 67 (d,J=9Hz, H-26); 4.52 (d,broad,J=13ft, H-6e); 4.81 (d,J=lOHz, H-20); 900-9542 b) 33-0-tert-Butyldimethylsilyl-26-0-trimethylsilyl-iso-FK 506 To a solution of 60 mg 33-0-tert-butyldimethylsilyl26-0-trimethylsilyl22(S)-dihydro-iso-FK 506 in 2 ml of dry methylene chloride are added 100 mg powdered molecular sieve 4A and 12 mg N-methylmorpholin-N-oxide. After stirring for 30 minutes at room temperature the solution is reacted with about 10 mg tetrapropylammonium-perruthenate and stirred further for 3 hours. The reaction mixture is evaporated to dryness, dissolved in acetic acid ethyl ester, washed with aqueous NaHC03 solution and water, dried and evaporated to dryness. The crude product can be purified by chromatography over silicagel using toluene/acetic acid ethyl ester (611) as an eluant. The title compound is obtained as a colourless amorphous substance.

E) 33-0-tert-Butyldimethylsilyl-26-0-trimethylsilyl-iso-FR 520 The title compound is obtained as a colourless amorphous solid in a manner analogous to that described under D) above.

The compounds and metabolites according to the invention possess pharmacological activity. They are indicated for use as pharmaceuticals.

In particular they possess antiinflammatory and immunosuppressant activity.

Antiinflammatory activity may e.g. be determined in the following test methods:

900-9542 1. Oxazolone allergic contact dermatitis in the mouse in vivo upon topical application: F.M. Dietrich and R. Hess, Int.Arch.Allergy 38 (1970) 246-259.

The following results are obtained upon topical application of a 0.01 % solution of test substance in ethanol:

Compound % inhibition Metabolite A Metabolite B Metabolite C Metabolite D 44 47 48 44 The compounds and metabolites elicit in this test an activity between about 20 % and about 70 % upon topical administration at a concentration of 0.01 X.

2. Inhibition of the oxidative burst in human neutrophil polymorphonuclear leukocytes (inhibition of the FMLP- or A23187- stimulated chemiluminescence) in vitro: the preparation of polymorphonuclear leucocytes (PMNL) and the measurement of chemiluminescence are effected as described in the literature (M. Schaude et al., Mycoses 31 [1988] 259-267), whereby the chemiluminescence reaction is started by addition of the peptide 900-9542 N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLPy 4x10-6M) or of the calcium ionophore A23187 (4x10-6M). The minimum inhibitory concentration is defined as the lowest concentration of active substance which causes a clear inhibition of all three parameters; The following results are obtained in the above test (MIC, minimum inhibitory concentration, uM):

Compound Stimulant FMLP A23187 Metabolite A 0.001 Metabolite B 0.5 Metabolite D 0.5 0.5 3. Inhibition of the TPA (12-0-tetradecanoylphorbol-13-acetate) stimulated release f PGE2 (prostaglandin E2) from mouse macrophages in vitro (inhibition of macrophage activation): the inhibition of PGE2 synthesis by peritoneal mouse macrophages is effected using TPA as stimulant in analogy to A. Haselberger et al., Circ.Shock Res. 26 (1988) 185-192.

The following results are obtained in the above test:

Compound Concentration (pM) % inhibition Metabolite A 0.5 40 Metabolite B 1 30 iso-FK 506 1 35 900-9542 Immunosuppressant activity may e.g. be determined in the following test methods:

4 4. froliferative response of lymphocytes to allogen stimulation in the mixed lymphocyte reaction (MLR) in vitro: T. Meo, "The MLR in the Mouse", Immunological Methods, L. Lefkovits and B. Pernis, Eds., Academic Press, N.Y. (1979), 227-239; The following results are obtained in the above test:

Compound ICS0 (ligIM1) Metabolite A Metabolite D 0.1 <O. 008 The compounds -and metabolites elicit in this test suppression of mixed lymphocytes at a dosage of from about 0.15 nM to about 10 nM.

Inhibition of the primary humoral immune response to sheep erythrocytes in vitro: 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.

The following results are obtained in the above test:

Compound ICS0 (Pglml) Metabolite A Metabolite D 0.1 0.036 The compounds and metabolites are active in this test at a concentration of from about 0.5 nM to about 10 nM.

900-9542 The compounds and metabolites of the invention are therefore indicated as immunosuppressant and antiinflammatory agents for use in the prevention and treatment of conditions requiring immunosuppression and of inflammatory conditions, such as a) the prevention and treatment of resistance in situations of organ or tissue transplantation, e.g. of heart, kidney, liver, bone marrow and skin, - graft-versus-host disease, such as following bone marrow grafts, autoimmune diseases such as rheumatoid arthritis, systemic Lupus erythematosus, Hashimotols thyroidis, multiple sclerosis, Myasthenia gravis, diabetes type I and uveitis, cutaneous manifestations of immunologically-mediated illnesses, such as Alopecia areata, and b) the treatment of inflammatory and hyperproliferative skin diseases, such as psoriasis, atopical dermatitis, contact dermatitis and further eczematous dermatitises, seborrhoeic dermatitis, Lichen planus, Pemphigus, bullous Pemphigoid, Epidermolysis bullosa, urticaria, angioedemas, vasculitides, erythemas, cutaneous eosinophilias, Lupus erythematosus and acne.

The compounds may be administered systemically or topically.

For these indications the appropriate dosage will, of course, vary depending upon, for example, the host, the mode of administration and the nature and severity of the condition being treated. However, in general, satisfactory results are indicated to be obtained systemically at daily dosages of from about 0.15 mg/kg to about 1.5 mg/kg animal body weight. An indicated daily dosage is in the range from about 0.01 mg to about 100 mg, conveniently administered, for example, in divided doses up to four times a day.

For topical use satisfactory results are obtained with local administration of a 1-3 % concentration of active substance several times daily, e.g. 2 to 5 times daily. Examples of indicated galenical forms are lotions, gels and creams.

900-9542 The compounds and metabolites of the invention may be administered by any conventional route, in particular enterally, e.g. orally, e.g. in the form of tablets or capsules, or topically, e.g. in the form of lotions, gels or creams.

Pharmaceutical compositions comprising a compound or metabolite as defined above in association with at least one pharmaceutical acceptable carrier or diluent may be manufactured in conventional manner by mixing with a pharmaceutically acceptable carrier or diluent. Unit dosage forms contain, for example, from about 0.0025 mg to about 50 mg of active substance.

Topical administration is e.g. to the skin. A further form of topical administration is to the eye, for the treatment of immune-mediated conditions of the eye, such as: auto-immune diseases, e.g. uveitis, keratoplasty and chronic keratitis; allergic conditions, e.g. vernal conjunctivitis; inflammatory conditions and corneal transplants, by the topical administration to the eye surface of a compound or metabolite of the invention in a pharmaceutically acceptable ophthalmic vehicle. The ophthalmic vehicle is such that the compound or metabolite is

maintained in contact with the ocular surface for a sufficient time period to allow the compound to penetrate the corneal and internal regions of the eye, e.g. the anterior chamber, posterior chamber, vitreous body, aqueous humor, vitreous humor, cornea, iris/ciliary, lens, choroid/retina and sclera.

The pharmaceutically acceptable ophthalmic vehicle may be e.g. an ointment, vegetable oil, or an encapsulating material.

c L A I M S:

1. A compound of formula I 33 H CH 3 CH 0 3 CH 13- X 22 OH,, 0 21 N R 0 0 0 CH3 OH 1 0 CH 3 H CH 3 OCH 3 CH 3 wherein R,' is methyl, ethyl, n-propyl or allyl, R2 is optionally protected hydroxy and X is oxo or optionally protected hydroxy.

900-9542 I 2. The compound according to claim 1 of formula Ia HG,..

,.. H CH,0 I CH3 IH 13- 26 24 0:1 0 1 1 UN 0 21 0 0 0 CH OH CH 3 H "'CH 3 i OCH 3 OCH 3 3. Metabolite A having a melting point of 70-75Cp [a]D20. -75.611 (c = 1. 17 in methanol) and the IR and mass spectra indicated in diagramms 1 and 5.

4. Metabolite B having a melting point of 72-780C, 1441D 20. -71.10 (c 1.22 in methanol), the IR and mass spectra indicated in diagramms 2 and 6 and the following characteristic signals (ppm) in the 'H-NMR spectrum (CDC13): 6.70 (m,H-24) and 6.11 (d,broad,J=15Hz, H-23).

900-9542 Ia 900-9542 5. Metabolite C having a melting point of 55-60C, ICCID 20= +7.5 (c = 1.0 in methanol) and the IR and mass spectra indicated in diagramms 3 and 7.

6. Metabolite D having a melting point of 74-780C, [OOD 20. -13111 (c - 1. 02 in chloroform), the IR and mass spectra indicated in diagramms 4 and 8 and the following characteristic signals (ppm) in the IH-NMR spectrum (CDC13)3 5.33 (d,Jm4Hzt H-26); 5.22 (d,J=9Hz, H-29); 4.53 (d,broad,J-15ft, H-6e); 4.37 (d,broad, J-4Hz, H-2); 4.19 (ddd,J1-2HZ,J2'5HZPJ3=7HZ, H-24).

7. A process for the preparation of a compound of formula I as defined in claim 1 comprising a) for the preparation of the compound of formula Ia 0 CH3 CH 3 0 -CH 3 1 24 0 OH 0 21 N 0 0 0 CH3 OH CH H CH 3 OCH 3 OCH 3 Ia 3 subjecting to repeated successive chromatographic steps the minor fractions which are produced during working up of the fermentation broth to separate the compound of formula IIa H CH 0 CH3 3 CH 3 0 0 OH21 N if' 0 0 1: 0 CH34k H H)CH 3 i OCH3 OCH 3 CH 3 b) for the preparation of a compound of formula Ib R 2-,. H "..I CH 0 P H3 1 3 CH3,,- X N 1 0 0 0 CH3 OH 1 21, R1 CH 3 H CH 3 OCH 3 OCH 3 wherein X' is optionally protected hydroxy and R,' and R2 are as defined in claim 1, 900-9542 IIa Ib reacting a compound of formula IIal R 2.... 33 ,,. H CH 3 0 CH3 CH 3 X 22 0 OH 21 CH3 0 0 0 CH3 H H.... CH3 wherein OCH 3 OCH 3

Claims (1)

1. X, is as defined in this claim and

   R,, and R2 are as defined in claim 1, with a base such as imidazole or, 900-9542 IIal c) for the preparation of a compound of formula Ic HCL,, 33 H jCH3 CH 3 0 CH3 X_ 22 0 0 21 N Ir,-- R; 0 0 CH3 OH CH3 H CH 3 i wherein CH 3 OCH 3 T' is oxo or hydroxy and R,, is as defined in claim 1, deprotecting a compound of formula Id R2.., 33..,H jCH1 CH 30 1 wherein ,,CH X r13-- 722 21 Fb 0 21 N 0 0 CH3 H 0 3 H CH 3 0 H CH 3 OCH 3 OCH 3 R,', R2 and X are as defined in claim 1 and R3 is optionally protected hydroxy, with the proviso that at least one of Xand R2 is protected hydroxy.

   900-9542 Ic Id 1C3 1 Process for the recovery of novel metabolites from the fermentation broth used in the production of the compounds of formula II H - CH3 CH 3 0 CH 0 (CH 2) n 3 0 OH 0 0 0 CH3 OH OCH 3 OCH 3 A-- CH 3 wherein R, is methyl, ethyl or allyland n is 1 or 2 by fermentation in conventional manner of Streptomyces strains, comprising, after separation of the compounds of formula II, isolation of the metabolites from the remaining solutions and washing liquors by methods such as chromatography.

   900-9542 II 900-9542 9. A process according to claim 8 for the preparation of metabolites A, B and C as defined in claims 3, 4, and 5 comprising, after separation from the fermentation broth of the compound of formula IIa HCL., H CH 3 0 CH 3 0 0 OH 21, N 0 0 0 D CH3 OH H OCH 3 CH 3 jCH3 CH ". CH., washing-out the column with methanol and fractionating the resultant mixture of metabolites in solution by repeated successive chromatographic steps.

   IIa 10. A process according to claim 8 for the preparation of metabolite D as defined in claim 6 comprising fractionating the minor fractions obtained during chromatography of the fermentation broth with tert-butyl methyl ether for separation of the compound of formula IIa defined in claim 9.

   11. A pharmaceutical composition containing a compound according to any one of claims 1 to 6 together with a pharmaceutically acceptable carrier or diluent.

   12. A compound according to any one of claims 1 to 6 for use as a pharmaceutical.

   1 900-9542 13. Use of a compound according to any one of claims I to 6 in the preparation of a pharmaceutical composition comprising mixing a compound according to any one of claims 1 to 6 with a pharmaceutically acceptable carrier or diluent.

   14. A method for the prevention or treatment of conditions requiring immunosuppression or of inflammatory conditions, such as a) the prevention and treatment of resistance in situations of organ or tissue transplantation, e.g. of heart, kidney, liver, bone marrow and skin, graft-versus-host disease, such as following bone marrow grafts, autoimmune diseases such as rheumatoid arthritis, systemic Lupus erythematosus, Hashimoto's thyroidis, multiple sclerosis, Myasthenia gravis, diabetes type I and uveitis, cutaneous manifestations of immunologically-mediated illnesses, such as Alopecia areata, and b) the treatment of inflammatory and hyperproliferative skin diseases, such as psoriasis, atopical dermatitis, contact dermatitis and further eczematous dermatitises, seborrhoeic dermatitis, Lichen planus, Pemphigus, bullous Pemphigoid, Epidermolysis bullosa, urticaria, angioedemas, vasculitides, erythemas, cutaneous eosinophilias, Lupus erythematosus and acne, comprising administering a therapeutically effective amount of a compound according to - - - - - - clairn 1 - -- together with a pharmaceutically acceptable carrier or diluent to a subject in need of such treatment.

   9009542 15. A method of treatment of immune-mediated conditions of the eye, such as: auto-immune diseases, e.g. uveitis, keratoplasty or chronic keratitis; allergic conditions, e.g. vernal conjunctivitis; inflammatory conditions or corneal transplants, which comprises topically administering to the eye surface a therapeutically effective amount of a compound according to claim 1 in a pharmaceutically acceptable ophthalmic vehicle.

   z 3700/VA 1091 VASP131VASP23 Published 1990 at The Pa:er,Oljice,SatcHouse.66 71 High Holbcrr Lznd--,- WC1R4TP-Purther copies maybe obtained Offcl Sa-eE Branch. 51 Ma:; Cra_y. Orping-.on Ken BR5 3RD. Printed by Multplex techniques ltd- St M=Y CraY. Ke---t- CO: 1 8'