SK278338B6 - Rebecamycine analogue, preparation method thereof and pharmaceutical agent containing its - Google Patents

Abstract

In the case, some special tryptophan analogues are supplied to the cultivation environment when the pehrmentation process is provided, new rebecamycine analogues, having favourable features enabling to apply them, when healing tumour diseases of mammals, are created as a result of that.

Description

Technical field

The invention relates to novel rebeccamycin analogs having antineoplastic properties and anti-tumor activity. The invention also relates to a process for their preparation and to pharmaceutical compositions containing them.

BACKGROUND OF THE INVENTION

U.S. Pat. Nos. 4,479,725 and 4,528,242 disclose an anticancer composition referred to as rebeccamycin, and its 5 ', 2', 3, 6-tetraacetate derivatives, and a method for producing the composition by culturing strains producing rebeccamycin Nocardia aerocolonigenes, preferably Nocardia aerocolonigenes ATCC 39 rebeccamycin producing mutant in an aqueous nutrient medium containing assimilable sources of carbon and nitrogen under flooding under aerobic conditions until a sufficient amount of rebeccamycin is produced. The latest Nocardia aerocolonigenes ATCC 39243 has been reclassified as Saccharothrix aerocolonigenes ATCC 39243 (Bush et al., J. Antibiotics, 40, 668-678, 1987).

SUMMARY OF THE INVENTION

SUMMARY OF THE INVENTION The present invention provides novel analogs of the anticancer agent referred to as rebeccamycin of formula (A),

H

(Λ2 where X 1 and X ₂ are independently of each other fluorine or hydrogen, provided that X 1 and X _{2 are} not both hydrogen and X 3 are hydroxy or methoxy and pharmaceutically acceptable acid addition salts thereof.

In particular, the present invention provides novel rebeccamycin analogs of formulas (Π) and (ΙΠ),

wherein X 1 and X ₂ are independently fluorine or hydrogen, provided that X 1 and X _{2 are} not both hydrogen.

Compounds of formula (H) and (ΙΠ) are produced by delivering a suitable tryptophan analogue to rebeccamycin producing Saccharothrix aerocolonigenes cultures.

The invention is illustrated by the following description and the accompanying drawings:

In FIG. 1 is an IR spectrum of a compound of formula IV. In FIG. 2 is 1 H-NMR for compounds of formula IV. In FIG. 3 is ¹³ C-NMR for the compound of formula IV. In FIG. 4 is an IR spectrum for the compound of formula V. FIG. 5 is a ^J H-NMR for the compound of formula V.

In FIG. 6 is ¹³ C-NMR for the compound of formula V. In FIG. 7 is an IR spectrum for a compound of formula VHI. In FIG. 8 is 1 H-NMR for the compound of formula VE. In FIG. 9 is ¹³ C-NMR for the compound of formula VIII. In FIG. 10 is a mass spectrum of a compound of formula IV.

In FIG. 11 is a UV spectrum of a compound of formula IV. In FIG. 12 is a mass spectrum of the compound of formula V. FIG. 13 is a UV spectrum of a compound of formula V. FIG. 14 is a mass spectrum of the compound of formula VIH. In FIG. 15 is a UV spectrum of a compound of formula VHI.

U.S. Pat

No. 5,528,442 discloses the production and isolation of an anti-tumor agent, referred to as rebeccamycin, of formula (1).

This rebeccamycin compound is a major component of the fermentation of the rebeccamycin producing Saccharothrix aerocolonigenes strain.

It has now surprisingly been found that the fermentation process described in U.S. Patent Nos. 4,489,725 and 4,558,242 can be carried out in the presence of certain tryptophan analogs, whereby new rebeccamycin analogs with valuable antitumor properties can be prepared. The Rebeccamycin analogs of the invention have the general formula 50 (H) and (E),

wherein X 1 and X _{2 are} fluorine or hydrogen, provided that X 1 and X _{2 are} not simultaneously hydrogen. The invention also includes their pharmaceutically acceptable acid addition salts.

⁶³ Preferred examples of the compounds of the invention are compounds of formula (IV).

SK 278338 Β6

H

H / IV /

OH

Other preferred exemplary compounds of the invention are compounds of formula (V), (VI), (VH), (VE), (IX), (X) and (XI).

at

he

H / VI /

OH / VII /

II

Λ1ΙΙ /

OCH

/ 11 / / X /

In the method of the invention, the tryptophan analog is added to the rebeccamycin fermentation medium and introduced into the rebeccamycin structure during fermentation to form the corresponding rebeccamycin analog.

Compounds of formula (IV) to (XI) are produced by culturing a rebeccamycin producing strain of Saccharothrix aerocolonigenes with DL-4-fluoro-tryptophan, DL-5-fluoro-tryptophan, DL-6-fluoro-tryptophan or DL-7-fluoro-tryptophan. A preferred production organism is a novel strain of Saccharothrix aerocolonigenes, originally referred to as Nocardia aerocolonigenes strain C38.383 RK2 (ATCC 39243) in U.S. Patent 4,489,225. More recently, this strain has been reclassified to Saccharothrix aerocolonigenes (Bush et al., J. Antibiotics, J. Antibiotics 40, J. Antibiotics. pp. 668-678 (1987) and is referred to herein as Saccharothrix aerocolonigenes strain C38,383-RK2 (ATCC 39243). This strain was isolated from a sample of soil taken in Panama. A biologically pure culture of strain C 38,383-RK2 is stored in the American Type Culture Collection, Rockville, Maryland and is maintained with a permanent collection of microorganisms as ATCC 39243. This culture, designated C38,383-RK2, is also maintained as a sleeping culture in lyophilized tubes. and in cryogenic vials at Bristol-Myers Squibb Co. Pharmaceutical Research and Development Division of Culture Collection, 5 Research Parkway, Wallingford, Connecticut 06492.

Taxonomic studies of strain C38,383-RK2 (ATCC 39243) are described in detail in U.S. Patent 4,489,225 and in J. Antibiotics 40, p. 668-678, 1987. The strain is designated as a new strain of Saccharothrix aerocolonigenes.

However, the invention is not limited to one particular preferred strain of ATCC 39243 or to the organisms fully described herein. In particular, it relates to other rebeccamycin-producing strains or mutants of the described organisms that can be produced by conventional methods such as X-ray irradiation, ultraviolet rays, chemical processing, and the like.

In the method of the invention, the rebeccamycin-producing Saccharothrix aerocolonigenes strain having the identifying characteristics of strain C38,383-RK2 (ATCC 39243) or a mutant or variant thereof is cultured in an aqueous conventional nutrient medium supplemented with a suitable tryptophan analogue. For optimal production of the compounds of formula (VI) and (X), the culture medium should be supplemented with DL-4-fluortryptophan. For optimal production of compounds of formulas (IV) and (IX), the culture medium should be supplemented with DL-5-fluortryptophan. For optimal production of the compounds of formulas (V) and (VE), the medium should be supplemented with DL-6-fluorothiyptophan. For optimal production of compounds of formulas (VII) and (X), the medium should be supplemented with DL-7-fluortryptophan. The organism grows in a nutrient medium containing known sources of nutrients for actinomycetes. The body grows in a culture medium containing an assimilable carbon source such as sucrose, lactose, glucose, rhamnose, fructose, glycerol or soluble starch. The culture medium should also contain an assimilable nitrogen source such as fish meal, peptone, peanut meal, cottonseed meal, corn soil, amino acids or ammonium salts. Nutrient inorganic salts may also be introduced into the culture medium to form sodium, potassium, ammonium, calcium, phosphate, sulfate, nitrate, carbonate and similar ions.

Optionally, trace elements such as copper, manganese, iron and zinc may be added to the culture medium or may be present as impurities of other media components. Preferably, submerged aerobic conditions are used to produce a large number of antibiotics, although surface cultures and cells may be used to produce limited amounts. For the method of the invention, general procedures customary for the cultivation of other actinomycetes can be used.

The method of producing the antibiotics of the invention can be carried out at any temperature resulting in sufficient growth of the production organism, for example, a temperature of 18 to 39 ° C is commonly used and the most suitable temperature is about 28 ° C. The fermentation can be carried out in banks or in laboratory or industrial fermenters of different capacity.

If tank fermentation is used, it is desirable to produce a vegetative inoculum in the culture medium by inoculating a small volume of culture medium with a cryopreservative or lyophilized culture of the production organism, slanted in a test tube. In order to obtain a viable and active inoculum in this manner, a septic inoculum is transferred to a fermentation vessel equipped with a culture medium for the production of the antibiotic according to the invention. The environment in which the vegetative inoculum grows may be the same or different from the environment in the container if good growth of the producing microorganism is achieved and is supplemented with a suitable fluortryptophan. Further mixing can be achieved with a mechanical stirrer. Optionally, anti-foaming agents such as pork fat or silicone oil may be added. Antibiotic production is monitored by high performance liquid chromatography or conventional bioassays. In general, optimal production of antibiotics by the method of the invention is achieved after incubation for 6 days. The isolation and purification of the derivatives thus obtained can be carried out by known conventional chromatographic methods.

Physical and chemical properties:

The compounds of formula (IV), (V) and (VIH) have the following physical and chemical properties:

Compound of formula (IV)

Description: Bright yellow amorphous solid Molecular Formula: C20HWF2N3O7 Molecular Weight: 523454

Mass Spectrum: Kratos MS 25 FABMS 524 (M + H) ⁺ , 361 (M-162, glucose loss) mass spectrometer

Ultraviolet spectrum: Diode Array Hewlett Packard 845 A spectrophotometer: 1.0 mg / 100 mL MeOH, neutral max. nm (E 1% / 1 cm): 405, 322 (457), 288,277,259,230 (348)

Infrared spectrum: spectrometer Perkin-Elmer 1800

FTIR; KBr pellet (cm ^-1 ): 3340, 2915, 1752, 1708, 1628, 1591, 1484, 1462, 1392, 1331, 1292, 1247, 1191, 1112, 1081, 1052, 947, 904, 795, 762, 752, 752 , 511

360 MHz * H-NMR spectrometer Bruker Model AN-

-3000: dual probe for IH and 13C measurement, 5 mm, dil-DMSO solvent: observed chemical shifts (ppm) 11.76 (s, IH), 11.23 (s, IH), 8.85 (dd, IH) ), 8.77 (dd, 1H), 8.01 (dd, IH), 7.68 (dd, 1H), 7.46 (m, 10 2H), 6.29 (d, 1H), 6, 12 (t, 1H); 5.45 (d, 1H); 5.17 (d, 1H);

1H), 4.95 (d, 1H), 4.07 (d, 1H), 3.95 (m, 2H), 3.81 (d, 1H), 3.59 (m, 2H)

MHz ¹³ C-NMR: Bruker Model AM-3000: proton decoupled spectrum; dual probe 15 for IH and 13C measurement, 5 mm, cU-DMSO solvent;

observed chemical shifts (ppm): 170.9, 170.8, 157.0 (d), 157.0 (d), 138.6, 137.2, 130.7, 129.2, 121.7 (d), 121.4 (d), 121.3, 119.6, 116.6, 115.1, 115.0 (d), 114.6 (d), 113.3 (d), 113.2 (d) , 109.1 (d), 109.1 (d), 20 84.8, 78.7, 76.5, 73.2, 67.6, 58.3.

Solubility: soluble in DMSO, DMF, THF, acetone, EtOAC, MeOH

Thin layer chromatography (Rf values): normal phase (silica gel 60): EtOAC: 0.10. EtoAc-MeOH (9: 1 25 v / v): 0.41

Reverse phase (C _R ), 0.1 M NFLiOAc-MeOH-CH 3 CN (2: 4: 4 v / v): 0.67

Compound of formula (V)

Description: Bright yellow amorphous solid Molecular Formula: C26H19F2N3O7 Molecular Weight: 523454

Mass spectrum: Kratos MS 35 25 FABMS 524 (M + H) ⁺ , 361 (M-162, glucose loss) mass spectrometer

Ultraviolet spectrum: Diode Array Hewlett Packard 8452A Spectrophotometer: 1.0 mg / 100 mL MeOH, neutral max. nm (E 1% / 1 cm): 398, (60), 40,316 (640), 280 (259), 256 (313), 226 (526), 204 (497)

Infrared spectrum: Perkin-Elmer 1800 FTIR spectrometer; KBr pellet (cm ^-1 ): 3324, 2927, 1745, 1701, 1623, 1580, 1491, 1471, 1452, 1412, 1384, 1330, 1233, 1172, 1116, 1075, 1048, 1016, 963, 916, 45 829 , 764, 745, 646, 635, 617,498,490

360 MHz 1 H-NMR spectrometer Bruker Model AN-3000: dual probe for IH and 13C measurement, 5 mm, solvent <L-DMSO: observed chemical shifts (ppm) 11.77 (s, IH), 11.18 (s 1H, 9.12 (dd, IH), 50 9.05 (dd, IH), 7.85 (dd, IH), 7.43 (dd, IH), 7.23 (t,

2H), 6.26 (d, 1H), 6.24 (s, 1H), 5.31 (d, 1H), 5.04 (d, 1H), 3.98 (m, 2H), 3, 87 (m, 1H); 3.66 (s, 2H)

MHz ¹³ C-NMR: Bruker Model AM-3000: proton decoupled spectrum; dual probe 55 for IH and 13C measurement, 5 mm, ds-DMSO solvent;

observed chemical shifts (ppm): 170.8, 170.7,

161.8 (d), 161.8 (d), 143.2 (d), 141.5 (d), 130.1, 128.7, 126.0 (d), 125.9 (d), 120.9, 119.3, 118.2, 118.1, 117.7, 116.5, 108.8 (d), 108.6 (d), 98.9 (d), 60 98.3 ( d), 84.7, 78.6, 76.5, 73.1, 67.5, 58.3

Solubility: soluble in DMSO, DMF, THF, acetone, EtOAC, MeOH

Thin layer chromatography (Rf values): normal phase (silica gel 60), EtOAC: 0.40. EtoAc-MeOH (9: 1 65 v / v): 0.63

Reverse phase (C 18), 0.1 M NFL / EtOAc-MeOH-CH 3 CN (2: 4: 4 v / v): 0.60

SK 278338 Β6

Compound of formula (VHI)

Description: Bright yellow amorphous solid Molecular formula C27H21F2N3O7 Molecular weight: 537481 5

Mass spectrum: Kratos MS mass spectrometer

FABMS 538 (M + H) &lt; ^{+ &gt};, 361 (M -176, 4-0-methylglucose loss)

Ultraviolet spectrum: Hewlett Packard 8452A Diode Array Spectrophotometer: 1.2 mg / 100 n concentration 10 MeOH, neutral max. nm (E 1% / 1 cm): 398, (103), 316 (1050), 280 (387), 256 (454), 228 (780).

Infrared Spectrometer: Perkin-Elmer 1800 FTIR KBr Pellet (cm ⁴ ): 3324.2938, 1747, 1703.1623, 1580, 1491, 1471, 1452, 1412, 1384, 1330, 1233, 1172, 15

1140, 1116, 1087, 1054, 963, 918, 828, 764, 649, 635

618.498

360 MHz 1 H-NMR spectrometer Bruker Model AN-3000: dual probe for 1 H and 13 C, 5 mm, solvent D-DMSO: observed chemical shifts (ppm) 20 11.77 (s, 1 H), 11.18 ( s, 1H), 9.12 (dd, 1H), 9.05 (dd, 1H), 7.88 (dd, 1H), 7.41 (dd, 1H), 7.23 (m, 2H), 6.25 (d, 1 H), 6.24 (s, 1 H), 5.36 (dd, 1 H), 5.31 (d, 1 H), 5.04 (d, 1 H), 3.97 (m) (2H), 3.60-3.95 (m, 4H)

MHz ¹³ C-NMR: Bruker Model AM-25 -3000: proton decoupled spectrum; dual probe for 1H and 13C measurements, 5 mm, d-DMSO solvent; observed chemical shifts (ppm): 170.8, 170.7, 161.8 (d), 161.7 (d), 143.1 (d), 141.5 (d), 130.1, 128.7 , 126.0 (d), 125.9 (d), 120.9, 119.4, 118.2, 118.1, 117.7, 30

116.5, 108.8 (d), 108.6 (d), 98.7 (d), 98.3 (d), 84.8, 77.2, 77.2, 76.2, 73, 2, 59.9, 58.5 ppm

Solubility: soluble in DMSO, DMF, THF, acetone, EtOAc, MeOH

Thin layer chromatography (Rf values): normal 35 phase (silica gel 60); EtOAc: 0.72. EtOAc-MeOH (9: 1 v / v): 0.89. Reverse phase (C 18), 0.1 M N-EtOAc-MeOH-CH 2 CN (2: 4: 4 v / v): 0.59.

Biological properties

Representative compounds of the invention are tested for transplanted leukemia of P388 mice to determine in vivo anti-tumor efficacy (Tables I to ΠΊ). CDF mice were implanted intraperitoneally 45 (ip ") ^with 1 ° P388 leukemia cells obtained from DBA / 2 donor mice having transplantable murine leukemia. CDFi leukemia mice are treated ip either with saline (control mouse) or with a dose of the compound of formula (IV), (V) and (VIH) once, one day after tumor inoculation. These mice are then observed daily and their death is recorded. Changes in mean body weight (from day of leukemia implantation to day of last treatment) were determined as a drug reflecting drug toxicity in all mice. Live mice in each group are recorded 5 days after tumor implementation as an additional means of assessing drug toxicity. No therapeutic result is considered significant if more than 1 mouse in the treatment group dies on day 5. Each treatment group consists of 4 to 6 mice; the control group comprises 10 mice. Any number of mice surviving until day 30 (from day 1 of the test) is also recorded.

The therapeutic efficacy is evaluated by determining the mean survival time (MST) of the mice treated with the compound of formula (IV), (V) and (VRI) and compared to the MST of the parallel control group of mice. This comparison is performed by dividing the MST of the treated group by the MST of the untreated group and multiplying by one hundred to obtain a parameter called the percentage T / C value. A percent T / C value of greater than or equal to 125% is considered to be a significant increase in lifetime and thus an active result.

As shown in Table L, the compound of formula (IV) is active with respect to P388 leukemia at a dose of 10 to 90 mg / kg. The best results are obtained at a dose of 30 mg / kg, resulting in a T / C percentage of 175%. Toxicity was not observed even at the highest dose tested (90 mg / kg).

As shown in Table R, the compound of formula (V) is effective with respect to P388 leukemia at a dose of 0.8 to 102.4 mg / kg.

The best results are obtained at a dose of 102.4 mg / kg as expressed as a T / C percentage of 178%. Toxicity is not observed even at the highest dose (102.4 mg / kg).

As shown in Table ΠΙ, the compound of formula (VRI) is active against P388 leukemia at doses of 0.8 to 102.4 mg / kg. The best results are obtained at a dose of 51.2 mg / kg, expressed as a T / C percentage of 206%. Toxicity was not observed even at the highest dose tested (102.4 mg / kg).

Table I Effect of Compound of Formula (IV) on P388 Leukemia ³ (Day 1 Treatment)

Dose ip mg / kg / inj. Mean survival of the day T / C % Changing mean g Number of mice survived Day 5 Day 30 90 16.5 165 -0.9 4/4 0/4 30 17.5 175 0.6 4/4 0/4 10 14.5 145 0.7 4/4 0/4 inspection 10.0 100 10/10 010

³ implemented mice 10 ⁶ cells of P388 leukemia and treated with compound of formula (IV) started the next day. Control mice were injected with saline.

In table H below, note (a) has the following meaning:

a) Mice implemented 10 ^with P388 leukemia cells and treatment with the compound of formula (V) started the day after treatment. Control mice were injected with saline.

SK 278338 Β6

Table Π Effect of Compound of Formula (V) on P388 Leukemia ”(Day 1 of Treatment)

Dose, ip mg / kg / inj. Medium time; survival days T / C % Changing mean g Number of mice survived Day 5 Day 30 102.4 16.0 178 -0.1 6/6 0/6 51.2 15.0 167 -0.2 6/6 0/6 25.6 14.5 161 -0.2 6/6 0/6 12.8 15.0 167 0.8 6/6 0/6 6.4 15.0 167 - 1,1 6/6 0/6 3.2 14.0 156 0.0 6/6 0/6 1.6 14.0 156 -0.7 6/6 0/6 0.8 13.0 144 0.1 6/6 0/6 inspection 9.0 100 1.3 10/10 0/10

Table IH Effect of Compound of Formula (VIH) on P388 Leukemia ^and (Day 1 Treatment)

Dose, ip ing / kg / inj. Mean survival of the day T / C % Changing mean g Number of mice survived Day 5 Day 30 102.4 14.5 161 0.2 6/6 0/6 51.2 18.5 206 0.3 6/6 0/6 25.6 14.0 156 0.1 6/6 0/6 12.8 14.5 161 -o l 6/6 0/6 6.4 16.0 178 0.1 6/6 0/6 3.2 16.5 183 -0.3 6/6 0/6 1.6 14.0 156 -0.1 6/6 0/6 0.8 14.0 156 -0.4 6/6 0/6 inspection 9.0 100 1.3 10/10 0/10

^and Mice implanted with 10 ⁶ P388 leukemia cells and treatment with the compound of formula (VM) started the day after treatment. Control mice were injected with saline.

The invention also relates to pharmaceutical compositions comprising an effective tumor growth inhibiting amount of the rebeccamycin analog of the invention, or a pharmaceutically acceptable acid addition salt thereof, together with an inert, pharmaceutically acceptable carrier or diluent.

The invention also relates to a method of pharmaceutical treatment of animals (preferably mammals) with a malignant tumor, comprising administering to the treated animal an effective, tumor-inhibiting dose of the antibiotic of the invention, or a pharmaceutically acceptable acid addition salt thereof.

Examples of suitable pharmaceutical compositions are solid oral compositions such as tablets, capsules, pills, powders or granules, liquid oral compositions such as solutions, suspensions, syrups and elixirs, and parenteral compositions such as sterile solutions, suspensions or emulsions. The pharmaceutical compositions may also be prepared in the form of sterile solids, which may be dissolved in sterile water, saline or other sterile injectable medium immediately prior to use.

A possible preferred dosage form of the rebeccamycin analogs of the invention is governed by the particular compound employed, the dosage form thereof, the mode of administration and the condition of the subject being treated. Many factors may modify the action of the drug, so that the skilled artisan must consider, for example, age, body weight, sex, diet, time of administration, rate of excretion, condition of the subject being treated, drug combination, reaction sensitivity and disease severity. Administration can be continuous or periodic at the maximum tolerated dose. The optimum rate of administration for a variety of conditions is readily determined by those of ordinary skill in the art using conventional dosing dose tests.

The following practical examples illustrate the invention but do not limit it in any way.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

Preparation of the Saccharothrix 15 aerocolonigenes cryopreservative culture strain C38,383-RK2 (ATCC 39243)

Saccharothrix aerocolonigenes strain C38,383-RK2 is maintained as a cryopreservative culture stored at -80 ° C in an ultra low temperature 20 Revco freezer. To prepare this cryopreservative culture, strain C38,383-RK2 is transferred to a test tube on sloping yeast-malt agar supplemented with calcium carbonate and having the following composition:

25 dextrose 4.0 g yeast extract 4.0 g malt extract 10,0 g calcium carbonate 1.5 g agar 15,0 g 30 demineralized water q. with up to 1 liter

The agar slopes were incubated at 28 ° C for 7-10 days. Prepare a vegetative culture by transferring the surface growth from the slanted culture to a 500 ml Erlenmayer flask containing 100 ml of sterile vegetative medium of the following composition:

Cerelosa (corn product) 30g

Pharmamedia (Trades Oil Milli Co.) 10 g Nutrisoy (Trades Oil Milli Co.) 10g calcium carbonate3 g demineralized water q. s up to 1 liter

This vegetative culture is incubated at 28 [deg.] C. for 48 hours on a rotary shaker at 250 rpm. The vegetative curl is mixed with an equal volume of cryoprotecting solution with the following composition:

sucrose 100 g glycerol 200 g demineralized water q. s up to 1 liter

Aliquots of 4 ml of this mixture are transferred to a sterile cryogenic tube (5 ml capacity, Coming) and frozen in a dry acetone / ice bath. Frozen vegetative cultures are then stored at -80 ° C in a Revco ultra low temperature freezer.

Example 2

Preparation of Saccharothrix aerocolonigenes vegetative culture - strain C38,383-RK2 (ATCC 39243)

A vegetative culture is prepared by transferring 4 ml of a cryopreservative culture to a 500 ml Erlenmeyer flask containing 100 ml of a sterile vegetative medium of the same composition as described for the vegetative environment of Example 1. The vein culture is incubated at 28 ° C for 48 hours in a rotary shaker at 250 rpm.

Example 3

Preparation of the compound of formula (IV) ml of the vegelative culture of Example 2 is inoculated into a 500 ml Erlenmeyer flask containing 100 ml of production medium each having the following composition:

starch Staclipsc J-UB (A. E. Staley) 10 g potassium dihydrogen phosphate2 g magnesium sulphate1 g

L-threonine 2.5 g calcium carbonate2 g demineralized water q. s up to 1 liter

The production culture is incubated at 28 ° C on a rotary shaker at 250 rpm. After 48 hours of fermentation, DL-5-fluortryptophan is added to the culture to a final concentration of 1 mg / ml. The culture is incubated at 28 ° C and shaken at 250 rpm. for another 4 days. Production of the compound of formula (IV) is monitored by HPLC chromatography. Optimum production of compound of formula (IV) is generally achieved at a concentration of 23 to 32 mg / ml on day 6 of fermentation (i.e. 4 days after addition of DL-5-fluortryptophan).

Example 4

Preparation of the compound of formula (V) and formula (VHI) ml of the vegetative culture of Example 2 is inoculated into a 500 ml Erlenmeyer containing 100 ml of production medium of the same composition as in Example 3.

The production culture is incubated at 28 ° C on a rotary shaker at 250 rpm. After 48 hours of fermentation, DL-6-fluorotryptophan is added to the culture to a final concentration of 1 mg / ml. The culture is incubated at 28 ° C and shaking at 250 rpm. for another 4 days. Production of the compound of formula (V) and formula (VIH) is monitored by HPLC chromatography. Optimal production of the compound of formula (V) and formula (VIH) is generally obtained on day 6 of fermentation (i.e. 4 days after addition of DL-6-fluortryptophan) at concentrations of 36 to 58 pg / ml and 31 to 42 pg / ml.

Example 5

Isolation and purification of compounds of formula (IV), (V) and (VIH)

(a) General method

Solvents are not distilled prior to use. Methanol, acetone, ethyl acetate, isopropyl ether, chloroform, tetrahydrofuran, ethyl ether and hexanes are reagents of ACS purity. Water for HPLC chromatography is tap water demineralized from Bemstead Nonopure Π. Tetrahydrofuran, methanol and acetonitrile for HPLC chromatography are solvents B and J Brand HPLC grade. Ammonium acetate is a product of Fisher HPLC purity.

Normal thin-layer phase chromatography is used and chromatography is performed on silica gel 60, on F-254 plates (EM reagent, catalog number 5765, 5 x 10 cm at 0.25 mm thickness). Thin layer reverse phase chromatography was performed on Whatman MKCis plates (catalog number 4803-110, 0.2 mm thick). The plates are developed in Whatman cylindrical flasks with a 10 ml eluent, Rcbeccamycin analogs are detectable as yellow zones under normal light or as yellow fluorescent zones in ultraviolet light 254 nm or 366 nm.

Dicalite (speed plus) filter aid is added to all broth. After briefly mixing the broth, filtration is performed on a large Buchner funnel or on a Tolhurst Centerslung centrifugal filter unit (Model 1B15, Ametex, Inc.). The filtrates are discarded. The mycelial doormat is slurried in tetrahydrofuran or tetrahydrofuran / acetone mixture for one hour, filtered and Dicalite is further rinsed with acetone until yellow fluorescence in ultraviolet light is no longer observed. The combined filtrates were concentrated under reduced pressure to give yellow extracts.

The liquid chromatography vacuum unit (VBC) consists of a Buchner funnel (Kontes, type number K-954100) comprising a mounted sintered glass disc (porosity M) with a side hose connection to the vacuum and a lower 24/40 connection for receiving the receiving flasks. Initially, polar eluting solvents are poured over it under vacuum to form

2786 sealed absorbent layer 5 cm high. The samples are pre-adsorbed on the adsorbent and placed on the funnel as a suspension or used in a solution of at least a polar eluting solvent. Gradients are set, whereby predetermined volumes of eluant with increasing polarity form fractions. The funnel is aspirated to dryness after each volume of eluant. The fractions were concentrated and pooled by thin layer chromatography analysis.

The chromatographic apparatus is as follows: Column 10 Glenco (2.5 ID x 100 cm), equipped with Teflon and solvent resistant plates; Flowmeter Inc. FM1 laboratory pump (Model RP-G150; Glenco glass reservoir (500 ml), Iaco Model 328 fraction collector. Columns are filled with Sephadex LH-20 15 (Pharmacia) pre-swelled in elution solvent. laboratory pump.

The following components are used to construct the semi-preparative HPLC system: 20 Waters Associates Model 590 solvent pump; wavelength detector Knauer Model 87; Water Associates Model SR-204 Recorder; Whatman Partisil 10 ODS-3 column (10mm x 50cm); Stainless steel 316 pipe (0.2 3 mm internal diameter). 25

b) Isolation and purification of compounds of formula (TV)

The entire culture medium (5 liters) is filtered with Dicalit and the mycelial mat is extracted by stirring in 30 tetrahydrofuran (2 liters) for one hour. After re-filtering and rinsing again with tetrahydrofuran (1 liter), the combined filtrates were concentrated under reduced pressure to give 4.5 g of crude extract. The extract is adsorbed onto 6.5 g of Lichroprep 35 Si 60 silica gel (EM Science, type 9336, 15 to 25 microns) and placed on a 60 ml VLC funnel containing additional

24.5 g of silica gel. Rinse with hexane-ethyl acetate (200 mL volume) and then rinse with 200 mL tetrahydrofuran. The tetrahydrofuran extract (156 mg) was dissolved in 4 ml of tetrahydrofuran and placed on a column containing 160 g of Sepluidex LH-20 balanced with tetrahydrofuran (layer height 90 cm, layer volume 430 ml). The flow rate is 3.75 ml / min. As can be seen visually, the main yellow band (62 mg) elutes in the 45 first quarter of the second volume of the layer. Final purification was performed by reverse lazy (Cu) HPLC chromatography at a flow rate of 4 mL / min (0.1 M NH 4 CCa-THF (60-40)). Detected at 320 nm. The eluent at 39 minutes is the major analog (23 mg), designated 50 compound of formula (IV).

c) Isolation and purification of the compound of formula (V) and formula (VHI)

All nutrient broth (2 liters) is filtered using Dicalit filter aid. The mycelial mat, after stirring in tetrahydrofuran (2 liters) for 45 minutes, is filtered and rinsed with an additional amount (1.5 liters) of tetrahydrofuran. The filtrate was concentrated under reduced pressure to give 3.39 g of crude product. The crude extract was triturated five times with 50 ml of tetrahydrofuran each, the volumes obtained were combined and concentrated to give 0.74 g of an insoluble residue in tetrahydrofuran. This mass contains a yellow fluorescent material. The 65 tetrahydrofuran-soluble material is adsorbed onto 2 g of silica gel H (Merck, 10 to 40 microns) and placed in a 30 ml VLC funnel containing an additional 11 g of silica gel H. A hexane-ethyl acetate gradient is used with 100 ml of eluent volume. The two major rebeccamycin analogs are separated in this pure manner. The less polar yellow zone (141 mg) was eluted with hexane-ethyl acetate (1: 1) and the more polar analog (105 mg) with hexane-ethyl acetate (: 3). The more polar analog (105 mg) was dissolved in 2 ml of tetrahydrofuran and applied to a column containing 160 g of Sephadex LH-20 (layer height 90 cm, layer volume 430 ml) swollen in tetrahydrofuran. The flow rate is 4 ml / min. The main yellow band, eluting in the 1.25 volume layer, is visually determined as the desired compound of formula (V) (77 mg). The less polar liquid-chromatographic (VLC) analogue (141 mg) was dissolved in 2 ml of methanol and applied to a column containing 110 g of Sephadex LH-20 swollen in methanol (layer height 80 cm, layer volume 400 ml). The flow rate is 3.5 ml / min. The main yellow zone, eluting at the end, yields the compound of formula (VIH) (70 mg).

Claims (17)

PATENT CLAIMS 1. Rebeccamycin analog of general formula (A) In which X 1 and X ₂ are independently fluorine or hydrogen, provided that X 1 and X _{2 are} not both hydrogen and X 3 are hydroxy or methoxy and pharmaceutically acceptable acid addition salts thereof. 2. rebeccamycin analog of claim 1 of formula (H) wherein X, and X _2, independently of one another F or H, provided that X and X ₂ are not simultaneously each hydrogen, and the pharmaceutically acceptable acid addition salt thereof. Rebeccamycin analogue according to claim 2 of the general formula (E) H H OCH Wherein X 1 and X ₂ are independently fluorine or hydrogen, provided that X 1 and X _{2 are} not both hydrogen and pharmaceutically acceptable acid addition salts thereof. The Rebeccamycin analog according to claim 2 of formula (IV) clay I OH The Rebeccamycin analog according to claim 2 of formula (V) au 6. The Rebeccamycin analog of claim 2 of formula (VI): OH Rebeccamycin analogue according to claim 2 of the general formula (VH) H OH Rebeccamycin analogue according to claim 3 0CH ₃ Rebeccamycin analogue according to claim 3 of the general formula (IX) Rebeccamycin analogue according to claim 3 of the general formula (X) Rebeccamycin analog according to claim 3 of the general formula (XI) A process for preparing a rebeccamycin analog according to claims 1 to 11 and a pharmaceutically acceptable acid addition salt thereof, comprising culturing a rebeccamycin producing Saccharothrix aerocolonigenes ATCC 39243 strain in an aqueous nutrient medium in the presence of a tryptophan analogue until a substantial amount of the desired rebeccam is produced. the organism used in the culture medium and the desired rebeccamycin derivative are obtained from the culture medium in pure form. The method of claim 12, wherein the tryptophan analog is DL-4-fluortryptophan. 14. The method of claim 12 wherein the tryptophan analog is DL-5-fluortryptophan. The method of claim 12, wherein the tryptophan analog is DL-6-fluortryptophan. The method of claim 12, wherein the tryptophan analog is DL-7-fluorothiyptophan, A pharmaceutical composition for the therapeutic treatment of a mammal having a cancer sensitive to rebeccamycin analogue according to claims 1 to 11, comprising an effective tumor inhibiting amount of at least one compound according to claims 1 to 11, together with a pharmaceutically acceptable, non-toxic carrier or excipient.