CA1247028A - Microbiological process for the preparation of anthracycline derivatives - Google Patents

Abstract

Abstract of the disclosure:

The present application relates to a microbio-logical process for the preparation of compounds of the formula I

I

in which R denotes hydrogen or hydroxyl and R1 and R2 are identical or different and represent sugar combina-tions of the following composition:
- Roa-dF-CinA
- Roa-dF-Acu - Roa-dF=CinB
- Roa-Rod-CinA or - Roa-Rod-Acu or R2 has the abovementioned meanings and R1 represents sugar combinations of the following composition:
- Roa-dF-Rod - Roa-dF-CinB
- Roa-dF-Acu - Roa-Rod-Acu - Roa-dF-CinA
- Roa-Rod-Rod or - Roa-Rod-CinA
in which Roa denotes rhodosamine; dF denotes deoxyfucose;
Rod denotes rhodinose; Acu denotes aculose; CinA denotes cinerulose A and CinB denotes cinerulose B, and dF = CinB
means that the two sugar units deoxyfucose and cinerulose B are linked by an extra ether bridge, in addition to the usual glycosidic bond, which comprises reacting a compound of the formula II

II

in which R denotes hydrogen or hydroxyl and R3 and R4 are identical or different and represent sugar combinations of the following composition:
-Roa-dF-Rod -Roa-Rod-Rod -Roa-dF-CinA or -Roa-Rod-CinA
or only R3 or only R4 has the meanings given and the other substituent in each case denotes -Roa-dF-Rod -Roa-dF=CinB
-Roa-dF-Acu -Roa-Rod-Acu -Roa-dF-CinA
-Roa-Rod-Rod or -Roa-Rod-CinA
with a specific oxidoreductase, isolating the resulting compound from the reaction medium by extraction in a manner which is known per se and purifying the product.

Description

- 2 - ~OE 84~F D46 The present Application relates to a microbio-logical process for the preparation of compounds of the f D r~ula I
~1 R o Hp ~OH

OH O HO O~

in ~hich R denotes hydrogen or hydroxyl and R1 and R2 are identical or different and represent sugar combina-tions of the following composition:
-- Roa-dF-CinA
- Roa dF-Acu - Roa-dF=CinB
- Roa-Rod-CinA or - Roa-Rod-Acu or R2 has the abovement;oned meanings and R1 represents su~ar combinations of the follo~ing composition:
- Roa-dF-Rod - Roa-dF-Cin~
- Roa-dF-Acu - Roa-Rod-Acu - Roa-dF-CinA
- Roa-Rod-Rod or ~ Roa~Rod-CinA
in ~hich Roa denotes rhodosamine; dF denotes deoxyfucose;
Rod denotes rhodinose; Acu denotes aculose; CinA denotes cinerulose A and CinB denotes cinerulose ~, and dF=CinB
neans that the t~o sugar units deoxyfucose and cinerulose B are l;nked by an extra ether bridge, in addition to the usual 0lycosidic bond, ~hich comprises reacting a com-pound of the formula II

L7~Z~3 ~< 2 CH3 C~3 0 Off OR4 in ~hich R has the meanin~ given ;n the case of formula I and R3 and R4 are identical or different and represent sugar combinations of the follouing composition:
-Roa-dF-Rod -Roa-Rod-Rod -Uoa-dF-CinA or -Roa-Rod-CinA
or only R3 or only R4 has the ~ean;ngs given and the other substituent in each case denotes -Roa-dF-Rod -Roa-dF=CinB
-Roa-dF-Acu -Roa-Rod~Acu -Roa-dF-rinA
-Roa-Rod-Rod or -Roa-Rod-CinA
~ith a spec;fic ox;doreductase, isolating the resulting compound from the reaction medium by ex~raction in a manner which ;s kno~n per se and pur;fy;ng the product.
The oxidat;on of term;nal sugar molecules ;n anthracycline tr;~lycos;des by react;on ~ith an oxido-reductase is already kno~n (A. Yoshimoto et al.~ The Journal of Ant;b;otics Vol n X X X II D 472 (1979))~ However, the anthracycl;ne derivat;ve here is subst;tuted w;th a tri~lycoside on only one side.
Surpris;n~ly, however~ if this process is appl;ed to anthracycline derivatives uith tuo tr;0lycoside chains on the ~olecule, selective ox;dation of terminal su~ar molecules is poss;ble. Thus, for example, uhen a cyto-rhod;n of the formula II ;s reacted w;th un oxidoreduc-tase product which has been obtained from the culture filtr~te of Streptomyces galilaeus ~ATCC 31133), only the lower triglycoside side chain (= R3) ;s oxidized.
Suitable oxidoreductases are ox;d;z;ng enzymes and en7yme products" such as, for example, cytoplasma membrane suspens;ons which are isolated in the customary manner from the culture filtrate of microorganisms, preferably of Streptomyces purpurascens (DSM 2658) or of Streptomyces galilaeus ~ATCC 31133)~
The enzymatic process according to the invention ;s carried out at temperatures of 20 - 45C, preferably at 37C, and at a pH of 4-7, preferably 5-6. The reaction time can be from a few hours up to three days. Further enzyme can be subsequently metered in to increase the yields. When the reaction has ended, the desired com-pounds are isolated from the reaction medium by extrac-tion and, ;f appropriate, separated by column chromato-graphy, preferably on silica gel or on chemically bonded reversed phase adsorbents, or by droplet countercurrent chromatography, and are purified.
The following compounds, for example, are obtained by the process according to the invention:
cytorhodin U (compound of the formula I in which R1 is -Roa-Rod-Rod and R2 is -Roa-dF=CinB3 cytorhodin Y (compound of the formula 1 in which R1 iS
-Roa-Rod-Rod and R2 is -Roa-Rod-Acu) cytorhodin P (compound of the formula I in which R1 is -Roa-Rod-Acu and R2 is -Roa-dF-CinA) cytorhodin D (compound of the formula I in which R1 iS
-Roa-Rod-Acu and R2 is -Roa-dF=CinB) 1-hydroxy-cytorhodin V
1-hydroxy-cytorhodin P
1-hydroxy-cytorhodin D and 1~hydroxy-cytorhodin B (compound of the formula I in ~hich R1 is -Roa-Rod-Rod and R2 is -Roa-dF-CinA) The cytorhodins obtained according to the inven-tion are distinguished by a high cytostatic activity.
The starting compounds of the formula II ~here R=H can be obtained in accordance with ~ C~c~
, ~.
, 76~

Pa-tent Application 457,301 br ~er~entDt1~n of the ~icroor0an~s~ Strep~o~yces purpur~scens (DS~ 2658) ~nd ~ubsequent ~ol~tlon.
Accord~n~ to th~s application the stro1n S Strepto~yc~s purpur~cens t3S~ 2658) ~s fer~entod 1n ~
nutr1ent ~ed1um ~n the cust3~ary ~nnerD teQper~t~res of ~bout 24 - ~0C~ ~ pH of ~.5 - 8.5 ~nd erob1c condi-tions be1ng ~a1nt~ned. Fron the ~ycel1u~ ~ep~r~ted off, ~he onthr~cycl1ne co~pounds tcytorhod~ns) ~re extracted, ~D for exDnple, ~1th aqueous ncetone at a pH of 3,5, the ~rctone 18 re~oved ~nd the aqueous ph~se ls extr~cted ~lth othyl ocet~te at a pH of 7.5. Thc culture tlltrate ~s ~xtracted t pH of 7.5 ~th, preter~bly, ethyl eetote. 7he Qthyl ~c2t-te extrAct~ fro~ the ~ycel~un~
~5 ond the eulture f~ltr-te ~re co~lned and, ~hen evsporo-ted to dryn~sg, ~ve n crude res1duo. The rcs1due 1s pref2r~bly further worked up a~ 1~ llkæ~ e ~escr1bed ln th~ 6er~n pat~nt ~ppl~c-t10n ~ent~oned.
The erude re$~due 18 ~ecord~n0ly d1ssolved 1n ZO toluene ~nd extrac~d ~th an ~eet3~e bufter ~pH 3.5)~
toluene ~ha~e ~nd 4n ~queous phase bo~ng o~ta1ne~ The ~queous phose 18 fur~her e2orked up ln the follo~ng - ~nner- ~tt~r brlnoln~ the pH to 7.5, ît 1s a~n extr~eted s~leh ethyl acetate and the ~thyi 2cel:~2 ph~se 25 il~ eancentrst~d" ~ soocall~d ertorhe~d~n erud~ ~xtlJre be1ng obta1ne~ fro0 ~hleh ~nthr~cycllne der1vat~ves (cytorhodln~) of the fornula II ean b~ obtalned by chro~to~r~phy.
The ~t~rtlng coapounds o~ the torcula II Mherc Q ~ OH sre prep~r~ ~1n accoPd~nc~ ~ith Canadian Patent Application 485,958 ) by ~ proeess ~nalogous to th~t ~e~erlbe~ ~bove, ~1th the diff~rence th~t the t~r~ent~-tlon 1~ e3rrl~d out at ~n lncr~ d ~er~tion r~te of 0.8 to 1.2 vv~ ond ov~r a perlod of 70 to 130, pr~fer~bly 35 90~110, houris.
The pr~ent 1nvent10n 1~ 1llu~trstod ln ~ore det~11 by th~ tollo~1ng ~x~ples.

~ , :3 Z4~
6 ~
Example 1:
Isolation of the ox;doreductase from Streptomyces galilaeus - ATCC 31133.
Streptomyces galilaeus - ATCC 31133 ~as sub-cultured as a spore suspension in a 2~000 ml conicalflask w;th 500 ml of the follo~;ng nutrient solut;on~
soluble starch 15 g glucose 10 9 soybean flour 10 g yeast extract 1 g NaCl 3 9 distilled water 1 l pH 7.4 15 and incubated on a rotary shaker at 28C and 200 rpm for 48 hours.
900 ml of this preculture ~as used as the ;nocu-lum for a 12 l fermenter filled with 9 l of culture solution:
soluble starch 15 g glucose 10 9 soybean flour 30 g yeast extract 1 g MgS04 x 7 H20 1 9 NaCl 1 9 trace elements 1 ml d;st;lled water 1 l pH 7.4 Trace element solut;on CoCl2 x 6 ~2 0.25 9 NiCl2 x 6 H2~ 0.01 9 CuClz x 2 ~2 0.01 9 ZnCl2 0.1 9 H3B03 (bor;c ac;d) 0.5 9 Na2MoO4 x 2 H20 9 NaSeO3 x 3 ~l2 0.1 9 FeS04 x 7 HzO 0.2 g bring to pH 2 - 3 with HCl EDTA (Titr;plex III) 0.05 g distilled ~ater 1 l After a fermentat;on period of 3 - 4 days at 28C, 400 rpm and an aeration rate of 0.5 vvm, the cul-ture solution ~as harvested and centrifu~ed and the cul-ture f;ltrate was concentrated 10-fold~ This concentrate was brought to 50~ saturation with ammonium sulfate, the supernatant was discarded and the precipitate was taken up in tr;s-HCl buffer of pH 7.4 (10 mM) and d;alyzed aga;nst 100 times the volume of the same buffer. The enzyme solut;on was then st;rred batchw;se w;th the an;on . exchanger DE 52 (Whatman)~kunbound protein was removed '~' w;th the above buffer by wash;ng, w;th addit;on of NaCl ~20 mM3~ and the specific oxidoreductase ~as then eluted w;th the same buffer, w;th addit;on of NaCl (250 mM) and concentrated by prec;pitation w;th ammon;um sulfate (80%).
The result;ng enzyme solut;on conta;n;ng 130 mU/mg of prote;n ~as lyoph;l;zed and stored at -18C. The oxido-reductase act;vity was determined by measuring the H202which ;s formed ~;th the substrate cytorhod;n B and ;s transferred to o-phenylened;am;ne by means of perox;dase in a l;nked r~act;on. The concentrat;on of the yellow dyestuff formed is d;rectly proportional to the amount of H22 formed by the enzyme. 1 International Un;t of enzyme act;vity corresponds to the convers;on of 1 ~mol of substrate/m;nute.
Example 2:
Preparat;on of an ox;doreductase product from Strepto-myces purpurascens - DSM 2658.
Streptomyces purpurascens - DSM 2658 ~as intro-duced onto yeast-malt agar of the following compos;t;on:
malt extract 10 9 yeast extract 4 glucose 4 9 agar 15 9 d;stilled water 1 l pH 7Ø
~eno~e5 tr~ ~c~rk ~ ~7~

For th;s, the medium was distributed among Roux bottles, sterilized at 121C for 30 minutes, cooled~
inoculated with a spore suspension and ;ncubated at 25C
for 7 - 14 days. Thereafter, it was possible to detect good gro~th and good spore format;on. A spore suspens;on with 107 spores/ml in sterile NaCl (0.8X strength) was prepared from this stock culture. 2 ml ~as used as the inoculum of 50 ml of the following preculture medium:
glucose 15 9 10 soybean flour 15 9 cornsteep tsolid) 5 9 CaC03 2 9 NaCl 5 9 distilled ~ater 1 l 15 pH 7Ø
50 ml portions of the above medium were distr;bu-ted amongst 300 ml conical flasks and sterilized at 121C
for 30 minutes. The inoculated flasks were incubated on a rotary shaker at 25C and 200 rpm for 2 - 3 days.
10 ml of the preculture yrown were used as the ;noculum for the follow;ng main culture:
glucose 20 9 malt extract 10 9 yeast extract 4 9 25 d;stilled water 1 l pH 6.8.
5D0 ml portions of the above medium were distri-buted amongst 2~000 ml conical flasks and steril;zed at 121C for 30 minutes. The inoculated flasks were incubated on a rotary shaker at 25C and 200 rpm for 4 - 5 days.
The cultures grown ~ere harvested and centrifuged and the cells were washed w;th NaCl.
Either the whole cells or membrane fractions of these cells were used for the enzymatic reactions.
Example 3:
~reparation of cytorhodin U.
35 mg of cytorhodin a (compound of the formula _ 9 _ II in uhich R3 denotes -Roa-Rod-Rod and R4 denotes -Roa-dF-CinA) were dissolved in 3 ml of 96% strength e~hanol and filled up with 30 ml of citrate~NaOH buffer, pH 5.5~ and, after adding 1.4 ml of ox;doreductase from Str. galilaeus (1.6 U/ml), the mixture was stirred at 150 rpm at 37C. The reaction was monitored by HPLC
(LiChrosorb(R) Si 60, 7~ ~Merck), chloroform : methanol :
acetic acid (96X) : water : triethylamine ;n a ratio of 80 : 10 : ln: 2 : 0.01, flow of 0.5-1.5 ml, detection by means of a flo~ photometer at 495 nm). After 48 hours, the starting compound uas no longer to be detected.
200 Jul of 0.1 M EDTA solution were added to the reaction solution, the pH was brought to 7.5 with 1 N
NaOH and the m;xture was extracted 3 times with 10 ml of ethyl acetate each time. The ethyl acetate phase was dried with Na2S04, freed from the drying agent and gently evaporated in vacuo.
The residue ~25 mg) was dissolved in the above-mentioned mobile phase and chromatographed on 35 9 of ZO sil;ca gel L;ChrosorbtR) Si 60, 7 ~ ~Merck) in a 16 x 250 mm steel column at a flo~ of 10 ml/minute. Fractions 60 - 70 ~in each case 2 ml), whichy according to analyti-cal HPLC, conta;ned the compound cytorhodin U~ were combined and concentrated in vacuo and the residue was taken up in 4 ml of Na acetate solution, pH 3~5. 0.1 ml of 1 mM EDTA solution was added to the solution, the mix-ture ~as washPd free of fats and plasticizers w;th toluene and brought to pH 7.5 with 1 N NaOH and the crude pro-duct was extracted with methylene chloride. After cus-tomary drying over Na2so4 and evaporation ;n vacuo,pure cytorhodin U (1 mg) was obta;ned.
Cytorhodin U: red amorphous substance, read;ly soluble ;n methanol, ethyl acetate~ chloroform and toluene, ;n-soluble in water and hexane~ soluble ;n 1X strength aqueous D-yluconic ac;d or D-lactobionic acid.
Th;n layer chromato~raphy: silica gel 60 F2s4 ~ pre-coated plates "Merck"; system chloroform : methanol :
acet;c ac;d (96X) : water 80 : 10 : 10 : 2, RF = 0.25.

~2~7~

1H - NMR: figure 1 Absorption spectrum:
a) 234 (4.b7) 253 tSH) 295 t3.86) 495 t4.10) b) 235 t4.68) 253 tSH) 295 t3.92) 495 (4.18) 5 c) ~44 t4~70) - 290 tSH 3.84) 580, 610 (4.18) tconfirmed by FAB-MS) C60H84N221~ calculated molecular we;ght: 1~168 Cytorhodin U has the formula I in which R1 denotes -Roa-Rod-Rod and R2 denotes -Roa-dF=Cin~.
Example 4:
Preparation of cytorhodin V.
69 mg of cytorhodin A tcompound of the formula II in which R3 and R4 each denote -Roa-Rod-Rod) as the D-gluconate tcorresponds to 54 mg of cytorhodin A
free base) were dissolved in 4 ml of c;trate-NaOH buffer, pH 5.5, and~ after addit;on of 2 ml of a solut;on of the oxidoreductase from Streptomyces gal;laeus, the mix-ture ~as stirred at 150 rpm at 37C~ The react;on ~as monitored by HPLC. After 48 hours, 35X of the starting ZO compound had reacted. The react;on solution was stored in the frozen state. After thaw;ng, 0.2 ml of a 0.1 mM
EDTA solut;on ~as added, the pH was brought to 7.5 with 1 N NaOH and the mixture was extracted with ethyl acetate ~3 portions of 30 ml each~ The organic phase separated off ~as dried with Na2S04 and gently evaporated under reduced pressure.
The res;due t33 mg) was dissolved ;n the mobile phase, as in Example 3~ and separated by preparative HPLC.
Fractions 30 - 45 contained the reaction product and fract;ons 90 - 1ZO conta;ned unreacted starting com-pound.
The react;on product ~as ~ashed free of fats and softeners as ;n Example 3 and was isolated as an amor-phous red powder t1.0 mg).
Cytorhod;n V_ red amorphous substance, readily soluble ;n methanol, ethyl acetate, chloroform and toluene, ;n-soluble ;n water and hexane; soluble ;n 1X strength aqueous D-gluconic acid or D-lactob;onic acid.

7~

Th;n layer chromatography as ;n Example 4: RF = 0.21.
1H-NMR: Figure 2 Absorption spectrum:
a~ 235 (4.72) 254 ~SH 4.48) 290 (4~04) 495 ~4.20) b) 235 ~4.73) 254 tSH 4.48) 290 t4.0~) 494 ~4.24 c) 244 (4.6~) 268 (4.~3) - 570 (4.22 601 (4.19 C60H84N2020~ calculated molecular ~eight: 1,152 (conf;rmed by FAa-MS).
Cytorhodin V has the formula I in wh;ch R1 denotes -Roa-Rod-Rod and R2 denotes Roa-Rod-Acu.

Preparation of cytorhodin P and D.
25 mg of cytorhodin ~ tcompound of the formula 15 II in which R3 denotes -Roa-Rod-Rod and R4 denotes -Roa-dF-CinA) were d;ssolved ;n 3 ml of 90X strength ethanol, 20 ml of c;trate-NaOH bufferO pH 5.5, were added and the compound was ox;d;zed at 37C with 2.5 ml of a suspension of cell membranes ~0.5 g/ml) from Streptomyces 2n purpurascens - DSM 2658 ~Example 2)~ w;th stirring ~150 rpm). According to HPLC, 21X of the start;ng com-pound was still present after 24 hours, and the compounds cytorhod;n P and cytorhod;n D had been newly formed ;n a ratio of about 5 : 1~
As described in Examples 3 and 4, the mixture of the products was ;solated from the react;on solut;on and separated by preparat;ve HPLC and the products were ;so-lated ;n pure form.
The pure compounds obta;ned were cytorhodin D
(0.7 mg), cytorhod;n P (1.9 mg) and unreacted cyto-rhodin ~ (1.Z mg).
Cytorhod;ne P: red amorphous substance, read;ly soluble ;n methanol, ethyl acetate, chloroform and toluene, in-soluble ;n ~ater and hexane; soluble ;n 1% strength aqueous D-gluconic ac;d or D-lactob;on;c ac;d.
Th;n layer chromatography as in Example 4: RF = 0.27.
1~ - NMR Figure 3 ~ ~7~

Absorpt;on spectrum-a) 235 54.~3) 254 tSH 4~333 293 (3r9U) 494 (4~13) b) 235 (4~60) 254 (SH 4.31) 293 (3.~5) 494 (4.12) c) 244 t4.33) _ 570 t3 5 620 (3.71) C60H82N20z1, calculated molecuLar weight: 1,164 (confirmed by FAB-MS).
Cytorhodin P has ~he formula I in which denotes Roa-Rod-Acu and R2 denotes -Roa-dF-CinA.
10 Cytorhodin C has the formula I in which R1 denotes -Roa-Rod-Acu and R2 denotes -Roa-dF=Cin8.
Example 6 Preparation of 1-hydroxy-cytorhodin U and 1-hydroxy~
cytorhodin B
15 50 mg of a 1.1 mixture of 1-hydroxy-cytorhodin N tcompound of the formula II in uhich R3 is Roa-dF-Rod and R4 is Roa-Rod-Rod3 and 1-hydroxy-cytorhodin 0 (compound of the formula II in ~hich R3 ;s Roa-Rod-Rod and R4 is Roa dF-Rod) were dissolved with 3 ml of 96%
strength ethanol and f;lled up w;th 30 ml of citrate-NaOH
buffer~ pH 5~5, and, after addit;on of 2 ml of oxido-reductase from Str. galilaeus (1.6 U/ml), the mixture was stirred at 150 rpm at 37C. The react;on ~as monitored by HPLC (LiChrosorb(R) Si 60, 71u (Merck), chloroform :
methanol : acetic acid (96X) : ~ater : triethylamine in a ratio of 80 : 10 : 10 : 2 : 0~01, flo~ of 0.5 - 1.5 ml, detection by means of a flow photometer at 495 nm). After 17 hours, the starting compounds were no longer to be de-tected and the compounds 1-hydroxy-cytorhodin ~ and 1-hyd-roxy-cytorhodin U had been newly formed in a ratio of 3:6.
200 ~ul of 0.1 M EDTA solut;on ~ere added to the reaction solution, the pH was brought to 7.5 ~ith 1 N
NaOH and the mixture was extracted 3 times with 10 ml of ethyl acetate each time. The ethyl acetate phase ~as dried ~ith Na2so4, freed fror the drying agent and gently evaporated in vacuo.
The residue (45 mg) was dissolved in the above-mentioned phase and chromatographed on 35 9 of s;lica gel ~2~2~

LiChrosorbR Si 60, 71U (Merck) in a 16 x 250 mm steel column at a flo~ of 10 mllminute. The fractions tin each case 2 ml) ~hich, according to analytical HPLC, contained the new compounds were combined and concentrated in vacuo and the residue ~as taken up in 4 ml of Na acetate solu-tion, pH 3~5. 0.1 ml of 1 mM EDTA solution was added to the solution, the mixture was ~ashed free from fats and softeners ~ith toluene and brought to pH 7.5 ~ith 1 N NaOH and the crude product ~as extracted with methyl-ene chloride. After customary dryin~ over Na2504 andevaporation in vacuo, pure 1-hydroxy-cytorhodin U S3.5 mg) and 1-hydroxy-cytorhodin B ~2 mg) were obtained.
1-Hydroxy-cytorhodin U and 1-hydroxy-cytorhodin B:
red-violet, amorphous substances, readily soluble in methanol~ ethyl acetate, chloroform and toluene, in-soluble in water and hexane; soluble in 1X strength aqueous D-gluconic ac;d or 9-lactob;on;c ac;d.
Thin layer chromatography: silica gel 60 F2s4 pre~coated plates "Merck"; system chloroform : mPthanol : acet;c 20 acid t9bX) ;n water 80 : 10 : 10 : 2~ RF = 0~25 (1-hydroxy-cytorhodin U) and RF = 0.18 t1-hydroxy-cyto-rhodin B) 1-Hydroxy-cytorhodin U:
Absorption spectrum:
25 a) 240 ~4.63) 296 ~3.82) 523 (4.1Z) 550 (4.10) 562 (4.11) b) 240 (4.64) 296 (3.80) 522 (4u20) 550 (4.15) 562 (4.16) c) 243 (4.70) 270 tSH) 590 (4.23) 635 (4.30) C60H84N2oz2~ calculated molecular ~eight: 1,184 (confirmed by FAB-MS) 1-Hydroxy-cytorhodine U has the formula I in ~hich R1 denotes -Roa-Rod-Rod and R2 denotes -Roa-dF=Cin~.
oxy-cytorhodin Absorption spectrum:
35 a) 259 (4.42) Z90 (3.66) 5ZZ ~3.95) 549 (3.82) 562 (3.85) b) 238 ~4.45~ 298 (3.57) 522 (4.03) 549 (3.91) 562 (3.93) c) 244 (4.41) 280 (3.62) 590 (3.93) 63Z ~4.01) C60H86N2o22~ calculated molecular weight: 1,186 ~7~28 ~ 14 -(confirmed by FA8-MS~.
1-Hydroxy-cytorhodin ~ has the formula I ;n ~hich R~ deno~es -Roa-Rod-Rod and R2 denotes -Roa-dF-CinA.
Example 7 Preparation of 1-hydroxy-cytorhodin P and 1-hydroxy-cytorhodin D
25 mg of 1-hydroxy-cytorhodin B tcompound of the formula II in ~hich R3 denotes -Roa-Rod-Rod and R4 denotes -Roa-dF-CinA) were dissolved in 3 ml of 90%
strength ethanol, 20 ml of citrate-NaOH buffer, pH 5.5, ~ere added and the compound was oxid;zed at 37C ~;th 2.5 ml of a suspens;on o~ cell membranes tO.5 g/ml) from Streptomyces purpurascens - DSM 2658 tExample 2), hith st;rring t150 rpm). Accord;ng to HPLC, about 22~ of the start;ng compound was st;ll present after 24 hours, and the compounds cytorhod;n P and cytorhod;n D had been newly formed ;n a ratio of about 4.5 ~ 1.
As described in Examples 3 - 6, the mixture of the products ~as isolated from the reaction solution and separated by preparat;ve UPLC and the products were iso~
lated ;n pure form.
The pure compounds obtained were 1-hydroxy-cyto-rhod;n D (009 mg), 1-hydroxy-cytorhod;n P t3.2 mg) and unreactsd cytorhod;n B (1.5 mg).
1-Hydroxy-cytorhodin P and D: vioLet~ amorphous sub-stances, readily soluble in methanol, ethyl acetate, chloroform and toluene, insoluble ;n ~ater and hexane;
soluble in 1% strength aqueous D-glucon;c ac;d or D-lactobionic ac;d.Thin layer chromatography as ;n Example 4: RF = O.Z7 tl hydroxy-cytorhodin P) and 0.35 t1-hydroxy-cytorhodin D) 1-OH-P:
Absor~ion spectrum:
a) 238 t4.58~ 292 t3.77~ 522 t4.03) 549 t3.92) b) Z37 S~.63) 296 t3.77) 522 t4.17) 549 t4.03) 561 t4.03) c) 243 (4.65) 275 t3.85) 590 t4.11~ 632 (4.17~

~4~

C60H82N2022~ calculated molecular ~eight: 1,182 tconfirmed by FAB-MS).
Example 8:
Preparation of 1-hydroxy-cytorhodin V:
50 mg of 1-hydroxy-cytorhod;n A (compound of the formula II in uhich R3 and R4 each denote -Roa-Rod~Rod), as the D-gluconate (corresponds to 37 mg of 1-OH-cyto-rhod;n A free base), were dissolved in 40 ml of citrate-NaOH buffer, pH 5.5, and, after addition of 2 ml of a solution of the oxidoreductase from Streptomyces galilaeus, the mixture ~as stirred at 150 rpm at 37C. The reaction was mon;tored by HPLC. After 48 hours, about 35X of the starting compound had reacted~ 0.2 ml of a 0.1 mM EDTA
solution ~as added to the reaction solution, the pH was brought to pH 7.5 with 1 N NaOH and the mixture ~as extrac-ted with ethyl acetate (3 portions of 30 ml each). The organic phase separated off ~as dried with Na2S04 and gently evaporated under reduced pressure.
The residue (35 mg) ~as dissolved in the msbile phase, as in Example 3~ and separated by preparative HPLC.
Fractions 32-47 contained the reaction product and fractions 88-110 contained unreacted starting com-pound.
The reaction product was washed free from fats and softeners as in Example 3, and isolated as an amor-phous red powder (2~0 mg)O
l-Hydroxy-c~torhodin V: red-violet, amorphous substance, readily soluble in methanol, ethyl acetate, chloroform and toluene, insoluble ;n water and hexane; soluble in 1X
strength aqueous D-gluconic acid or D-lactobionic acid.
Thin layer chromatography as in Example 4: RF = 0.21.
Absorption spectrum:
a) 237 (4.54~ 295 t3.72) 522 (4.03) 548 t4.86) 561 (3.68) b) 237 t4.57) 295 (3.79) 522 (4.10) 548 (3.93) 561 (3.93) c) 244 (4.46) 28D (3.76) 590 t3.99) 630 t4.04) C60H88N2o21~ calculated molecular ~eight: 1,168 (confirmed by FAB-MS).
The components in the above Examples were ~917~

;dentified under the measurement condit;ons described belo~:
The proton resonance spectra ~1H--NMR spectra) were measured in an HX-270 ~RUKER Fourier-Transform nuclear magnetic resonance spectrometer at 270 MH2. The concentrations ~ere 2 - 4 mg/0.5 ml of 99.~X pure CDCl3;
immediately after preparation, the solutions were shaken with 0.1 ml of SX stren~th Na2C03 in 99~5X pure D2~.
The signals marked ;n the figures w;th an aster;sk orig;nate from low molecular we;ght ;mpurities in the 10-3 range and from solvent residues.
The mass spectra were measured on an MS-902 S, AEI mass spectrometer using a FAP (fast atom bombardment) ion source. The substances were introduced into the ion source in a matrix of thioglycerol, in some cases with the addit;on of ammonium chloride.
The absorption spectra were measured in the 200 -700 nm range in:
a) water/methanol 1 : 9 b) 10X strength 1 N HCl in methanol c) 10X strength 1 N NaOH in methanol The substance concentration was 10 - 30 mg/l; the absorption rnax;ma in nm and the molar extinction co-efficients (log ~ 3 are given~
Determination of the cytotox;c act;vity The cytostat;c act;v;ty of the compounds de-scribed here ~as determined on L1210 leukemia cells from mice. Specifically, the follow;ng test systems were used:
a) Prol;ferat;on assay In this method, the extent to wh;ch the cells can ;ncorporate radioactive DNA precursors ~for example 14C-labeled thym;dine) is determ;ned in v;tro after incuba-t;on of the cells ~;th different concentrations of the test substance. Untreated L1210 cells are subjected to the same test conditions and serve as a control. The method is briefly described belo~:
L121û cells in the exponential gro~th phase (5 x 103/ml in RPMI 1640) are incubated in a microtiter plate with different concentrations of the test substance Z~3Z~

for 7Z hours (37C, 5X C02, 95X relative atmospheric humidity). Controls consist of cells which are incubated only with fresh medium. All the determinations were carried out as ~-fold determinations~ After 65 hours, 50~ul of 14C-1-thymidine ~1.5 uc/ml) are added in order to radioactively label the DN~ of the cells. After incubation ~or 7 hours, the cells are filtered off ~ith suction and the DNA is precipitated with 5X strength tri-chloroacetic ac;d and ~ashed successively w;th ~ater and methanol.
After drying at 50C, the radioactivity incor-porated into the DNA is determined, after addition of 5 ml of scintillation liquid.
The results are g;ven as the scintillation ;nde~
after incubation ~ith the test substance in percent of that of the untreated control. The dose/effect curve is determined from the measurement values thus obtained, and the IC50, i.e. the concentration uh;ch reduces the incorporat;on of radioactive thymidine under test condi-tions by 50X in comparison ~ith the control, is deter-mined graphically~ The ICso values of the compounds described here in comparison with those of adriamycin (A~M~ are summar;zed ;n Table 1.
b) Colony formation of L1210 leukemia cells ;n soft agar This method serves to detect an influence of the test subs~ance on the growth properties of the cells over several generat;ons (u;th a cell cycle t;me of 10 - 12 hours, about 14 success;ve generat;ons are observed in the test period of 7 days)~
In this test, cytostatically act;ve substances cause a reduct;on in the colony count to be observed in comparison with an untreated control~ Specifically, the test is carried out as follo~s:
500 leukemia cells per plate are incubated with different concentrations of test substance at 37C for 1 hour. The cells are then uashed t~ice with McCoy 5A
medium and finally poured into Petri dishes, after addi-tion of 0.3% of agar. Controls are incubated only ~;th ~247U ~B

fresh med;um. Instead of incubation for 1 hour~ in some cases different concentrations and test substance are mixed to the upper layer of agar, in order thus to achieve continuous exposure of the cells throughout the entire in~ubation period. After solidification of the agar, the plates are incubated in an incubating cabinet at 37C for 7 days (5X C02, 95% relative atmospheric hum;d;ty). The number of colon;es formed ~;th a d;ameter of 60~u is then counted. The results are g;ven as the colony count ;n treated agar plates in percent to that in the untreated control. From the dose/effect curve thus obta;ned, the ICso ;s determined as a measure of the act;v;ty of the substance. The results for the com-pounds descr;bed here are summarized in comparison with those for adr;amyc;n in Table 1.
Table 1 Proliferation test Colony format;on test IC50 (~g/ml) IC50 (~g/ml) 7 days 1 hour Incubat;on Incubat;on ADM 6.0 x 10-3 2.2 x 10-2 4.4 x 10-2 Cytorhodin U 2.5 x 10-3 2.7 x 10-3 2.8 x 10-~
2~8 x 10-4 3 x 10-3 1.1 x 10-2 " P 2.6 x 10-3 3.Z x 10-4 2.8 x 10-3 25 1-OH " U 2~8 x 10-3 4.5 x 1D-4 2.4 x ~0~3 1-9H " V 2.8 x 10-3 4.5 x 10-3 2.4 x 10-3 1-OH " 8 5 x 10-3 3 x 10 3 2~5 x 10-3

Claims (3)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A process for the preparation of a compond of the formu-la I

wherein R denotes hydrogen or hydroxyl, R1 is Roa-Rod-Rod and R2 is Roa-dF-CinB, Roa-Rod-Acu or Roa-dF=CinB, where Roa denotes rhodosamine, Rod denotes rhodinose, Acu denotes acylose, dF denotes desoxyfucose, CinA denotes cinerulose A and CinB denotes cinerulose B, and df=CinB
means that the two sugar units deoxyfucose and cinerulo-se B are linked by an extra ether bridge, in addition to the usual glycosidic bond, which comprises reacting a compound of the formula II

II

wherein R is ad defined above, R3 is Roa-Rod-Rod, and R4 is Roa-dF-CinA, Roa-Rod-Rod or Roa-dF-Rod, where Roa, Rod, df and CinA are as defined above, with the oxidore-ductase obtained from the culture filtrate of Streptomy-ces galileus (ATCC 31133), isolating the resulting com-pound or compounds from the reaction medium by extrac-tion in a manner which is known per se, and purifying the product.

2. A process for the preparation of a compound of the for-mula I
wherein R denotes hydrogen or hydroxyl, R1 is Roa-Rod-Acu, R2 is Roa-dF-CinA, or Roa-dF=CinB, in which Roa denotes rhodosamine;dF denotes deoxyfucose; Rod denotes rhodinose; Acu denotes aculose; CinA denotes ci-nerulose A and CinB denotes cinerulose B, and dF=CinB means that the two sugar units deoxyfucose and cinerulose B are linked by an extra ether bridge, in addition to the usual glycosidic bond, which comprises reacting a compound of the formula II

II

wherein R is as defined above, R3 is Roa-Rod-Rod and R4 is Roa-dF-CinA where Roa, Rod, dF and CinA are as defi-ned above, with the oxidoreductase obtained from the culture filtrate of Streptomyces purpurascens (DSM
2658), isolating the resulting compund or compounds from the reaction medium by extraction in a manner which is known per se, and purifying the product.

3. The process as claimed in claim 1 or 2, wherein the en-zymatic reaction is carried out at a temperature of 20°
to 45°C and a pH of 4-7 over a reaction period of a few hours up to three days.