SE469632B - NEW ANTITUMOERANTIBIOTICS - Google Patents

Description

20 25 30 35 _2- 469 632 R.H. Bunge et al reveal in J. Antibiotics, 37 (12), 1566-1571 (1984) fermentation of Actinomadura sp. (ATCC 39363) for the preparation of a bioactive complex, from which two main components, PD 114 759 and PD 115 028, were isolated. I Q¿ Chem. Soc. Chem. Commun., 919-920 (1985) describes J.H.

Wilton et al. a partial structure of the antibiotic compounds PD 114 759 and PD 115 028. The preparation, isolation and characterization of the antibiotic compounds PD 114 759 and PD 115 028 appear to be identical to those of the above antibiotics CL-1577A respectively CL-1577B.

European Patent Application No. 95,154, published November 30, 1983, discloses fermentation of Actinomadura pulveraceus for the manufacture of antitumor antibiotics designated WS 6049-A and WS 6049-B. The structures of these WS 6049 antibiotics have not yet been determined, but the characteristics given for these antibiotics show that WS 6049-A and WS 6049-B are structurally related to the BBM-1675 antibiotics described in British Patent Application 2,141. 425 and the CL-1577 antibiotics described in U.S. Patent 4,530,835. However, spectral data show that neither WS 6049-A nor WS 6049-B is identical to any of the BBM-1675 components. Furthermore, the producing organism described in European Patent Application 95 154 can be clearly distinguished from the Actinomadura verrucosospora used in British Patent Application 2,141,425 as regards the fifigpn of these aerial myceliums on ISP media Nos. 2, 3 and 4, in terms of its positive milk peptonization and its positive utilization of D-fructose, D-mannitol, trehalose and cellulose.

Summary of the invention.

According to the present invention there are provided novel anti-tumor and antibiotic substances, which in the present context are designated BBM-1675C and BBM-1675D and are also "ß" 469 62.2 known as BMY-27305 and BMY-27307, respectively. wherein said substances are prepared by selective chemical hydrolysis of the bioactive components BBM-1675A1 (esperamicin A1) or BBM-1675A2 (esperamicin A2), which in turn are prepared by culturing a BBM-1675-producing strain of Actinomadura verrucosospora. The bioactive substances BBM-1675C and BBM-1675D can be separated and purified by conventional chromatographic methods and both substances show excellent antimicrobial and antitumor activity.

Description of the drawings.

FIG. 1 shows the ultraviolet absorption spectrum of BBM-175 ° C.

FIG. 2 shows the ultraviolet absorption spectrum of BBM-1675D.

FIG. 3 shows the infrared absorption spectrum of BBM-1675C (Kßr, film).

FIG. 4 shows the infrared absorption spectrum of BBM-1675D (KBr, film).

FIG. 5 shows the relative mass spectrum of BBM-16750.

FIG. 6 shows the relative mass spectrum of BBM-1675D.

FIG. 7 shows the proton magnetic resonance spectrum of BBM-1675C in CDCl 3 (360 MHz).

FIG. 8 shows the proton magnetic resonance spectrum of BBM-1675D in CDCl3 + 10% CD3OD (360 MHz).

FIG. 9 shows the 13C magnetic resonance spectrum of BBM-1675C 1 cDc13 (90.6 MHz). Fig. 10A shows the 13 C magnetic resonance spectrum (110-200 ppm) of BBM-1675D in CDCl 3 + 10% CD 3 OD (90.6 MHz).

FIG. 10B shows the 136 magnetic resonance spectrum (0-110 ppm) for BBM-1675D in CDCl 3 + 10% CD OD (90.6 MHz). FIG. 11A shows the proton magnetic resonance spectrum of compound 3A (K anomer) in CDCl 3 (360 MHz).

FIG. 11B shows the proton magnetic resonance spectrum of the compound 3B (Qß anomer) in CDCl3 (360 MHz).

Detailed description of the invention.

This invention relates to two novel antitumor and antibiotic substances, hereinafter referred to as BBM-16750 and BBM-1675D, which are also known as BMY-27305 and BMY-27307, respectively, said substances being prepared by selective chemical hydrolysis of the bioactive components BBM -1675A1 (esperamicin A1) or BBM-1675A2 (esperamicin A2), which in turn are prepared by culturing a BBM-1675-producing strain of Actinomadura verrucososopora, in particular Actinomadura verrucosospora strain H964-92 (ATCC 39 334 ) or Actinomadura verrucosospora strain Al327Y (ATCC 39638) or a mutant thereof. In another aspect, the present invention provides a process for the preparation of the substance BBM-1675C by selective hydrolysis of the bioactive components BBM-1675A1 or BBM-1675A2.

In a further aspect, the present invention provides a process for the preparation of BBM-1675D by selective hydrolysis of the substance BBM-1675C or, which is more preferred, starting from the bioactive components BBM-1675A1 or BBM-1675A2. The isolation and purification of BBM-175C and BBM-17575D from the reaction mixture can be accomplished by conventional chromatographic methods.

The bioactive substances BBM-1675C and BBM-1675D show "S" 469 632 antimicrobial activity against a wide range of microorganisms and have also been shown to show inhibitory activity with respect to various mouse tumor systems such as leukemia P -388 and melanoma B16. The novel substances of the present invention can therefore be used as antimicrobial agents or as antitumor agents for the inhibition of mammalian CLIIIIÖIEI.

During the degradation tests performed to determine the structure of the antitumor and antibiotic compounds BBM-1675A1 (esperamicin A1) and BBM-1675A2 (esperamicin A2), a mixture of components was obtained, which led to the isolation and identification of two inactive fragments, namely the compounds of formulas I and II, respectively. However, it was surprising to find that the chemical degradation led to the gradual release of two bioactive fragments BBM-1675C and BBM-1675D. What was more surprising was that the two different antibiotic substances BBM-1675A1 and A2 gave the same two fragments, as illustrated in Reaction Scheme 1 below. Even more surprising was the fact that the low molecular weight fragments BBM-1675C and D (with approximately 70 and 55% of the molecular weight, respectively, of the parent antibiotic compounds BBM-1675A1 and A2) were found to be more effective than BBM-1675A2 and comparable to BBM- l675A1 as antitumor and antimicrobial agents.

Reaction Scheme 1.

BBM-1675A1 (esperamicin A1) BBM-1675C '------- å BBM-1675D BBM-1675A2 (esperamicin A2) 469 652 "ö" The substances BBM-1675C and BBM-167SD can be prepared by selective chemical hydrolysis of the antibiotic BBM-1675A1 as outlined in Reaction Scheme 2.

Reaction Scheme 2. e ßBM-wvsil-H- »BBM-lsßc + CH3OH (molecular weight 1248) (molecular weight 855) _ oca3 Compound 1 (molecular weight 425) (A mixture of α and β-anomers) HQ / CH3OH / \ CH3 BBM-1675D t 0 (molecular weight 695) + C335 QCH3 OH Compound 3 (molecular weight 192) (A mixture of aë and lß anomers) The starting compound BBM-l675A1 is prepared by a process described in the British patent application 2,141,425, published December 19, 1984. The purified BBM-1675Al component is hydrolyzed with a mineral acid or organic acid, such as hydrochloric acid, sulfuric acid, p-toluenesulfonic acid, benzenesulfonic acid or the like, in an organic or mixed aqueous-organic inert solvent at a temperature of from about 0 ° C to the reflux temperature of the solvent. until a substantial amount of the desired compound BBM-1675C or BBM-1675D has been obtained.

Preferably the hydrolysis is carried out in C1-C6 alcohol solvents and in particular the lysis is carried out in methanol. The reaction temperature is not critical but it is preferable to carry out the reaction at a temperature from about ambient temperature to 60 ° C and in particular from about 40 ° to 11 ° C.

The selective hydrolysis of BBM-1675A1 takes place stepwise with the initial formation of the antibiotic BBM-1675C and the inactive fragment of formula 1. Subsequent or continued treatment under hydrolyzing conditions leads to the release of a mixture of AP and β anomers of ten the sugar of formula 3 and the formation of the antibiotic BBM-l675D. It will be appreciated by those skilled in the art that variation of the reaction conditions, such as time, temperature and acid concentration, gives varying relative amounts of the antibiotic substances BBM-175 ° C and D. Thus, it is desirable to monitor the course of the reaction by thin layer chromatography as described in the examples below.

In addition to preparing only the antibiotic BBM-17575D, the selective hydrolysis is preferably carried out with an organic acid, such as p-toluenesulfonic acid, as described below to obtain a quantitative amount of BBM-17575D.

The substances BBM-16750 and BBM-1675D can also be prepared by selective chemical hydrolysis of the antibiotic BBM-1675A2 - 469 632 as outlined in Reaction Scheme 3.

Reaction Scheme 3.

I OH V.

H69 C330 BBM-1675A2-Eš-öñ-9 BBM-16750 + 0. 3. (molecular weight 1248) (molecular weight 855) CHBO NH O caz f> ° fl = Compound 2 (molecular weight 425) (A mixture of Oα and IE anomers) HQ / CH 3 OH C33 \ / O BBM-1675D + CHBS 0CH3 (molecular weight 695 OH Compound 3 (molecular weight 192) (A mixture of OC and β anomers) The starting compound BBM-1675A2 is prepared by the method described in British Patent Application 2,141,425, which was published on December 19, 1984. The selective hydrolysis of purified BBM-1675A2 likewise takes place in a stepwise manner with the initial formation of the antibiotic BBM-16756 and the inactive fragment of formula II. Continued treatment under hydrolyzing conditions leads to the release of a mixture of M? and, the β-anomers of the thio sugar of formula 3 and formation of the antibiotic with BBM-1675D.

The reaction conditions used for the selective chemical hydrolysis of BBM-1675A2 are essentially the same as those used for hydrolysis of BBM-1675A1, as described above.

When it is preferred to prepare only the antibiotic BBM-167SD, in a manner similar to the preparation of BBM-1675D starting from BBM-1675A1, the hydrolysis of BBM-1675A2 is carried out until substantially all of the BBM-1675A2 and BBM-1675C have been converted to BBM-l675D. In particular, the hydrolysis is carried out with an organic acid such as p-toluenesulfonic acid.

The above-described discovery that the same antibiotics BBM-1675C and D are formed from two different antibiotics BBM-1675A1 and BBM-1675A2 with concomitant loss of two inactive fragments of formulas I and 2, respectively, and the thio sugar of formula III provides a further advantage of the present invention finding. Accordingly, in a further aspect of the present invention there is provided a process for the selective hydrolysis of a mixture of BBM-1675A1 and A2 to produce BBM-1675C and D as illustrated in Reaction Scheme 4.

Reaction scheme 4.

BBM-1675A + BBM-1675A2 --- åBBM-16750 '* --- 9BBM-1675D l This advantage becomes apparent when one considers that the relative amounts of BBM-1675A1 and A2 formed in the fermentation process are subject to variations. The production of BBM-1675C 469 632 "10" and D is therefore independent of the relative amounts of BBM-1675A1 related invention. and A2 used as starting material as described above, the hydrolysis of the antibiotic compounds BBM-1675A1, A2 and C results in the release of an inactive sugar sugar fragment. Said thio sugar has been isolated to provide further information on the chemical structure of the antibiotic BBM-1675C and consequently for the antibiotic compounds BBM-1675A1 and A2. The compound of formula 3 has been identified as a mixture of us and ß anomers of a thio sugar, which has the structure shown in Reaction Schemes 2 and 3. Further characterization has been made possible when the products of the alcoholysis, the ar and the f anomers, have been separated . The proton magnetic resonance spectrum (360 MHz) of compound 3A (Cranomer) and compound 3B (β-anomer) are shown in Figures 11A and 11B, respectively. Through an analysis of spectral data, the thio sugar methyl glycosides of formula 3 have been preliminarily attributed to the relative stereochemistry of the formula OH CH CH S At present, the absolute stereochemistry, ie. D or L, not yet determined. Consequently, on the basis of the present interpretation of spectral data, it has been concluded that the thio sugar of formula 3 (minus the CH3 group from the anomeric methoxy incorporated during the methanolysis) is a component in the structure of the antibiotic BBM-1667C and further is a component in the structure of the starting antibiotic compounds BBM-1675A1 and A2. 'u 10 15 20 25 30 35 V' .r »_-.....-...-_ n ^ _ ._- a¿ ....-» .. »-....-_ ' ..4w .. @ ...... / .. .fu-ß ._ _, .-., - 'W 469 632 Physico-chemical properties of BBM-l675C Description: amorphous solid Ultraviolet absorption spectrum: See FIG. 1 Instrument Hewlett-Packard 8458 Solvent: methanol Concentration: 0.0155 g / l iLmaX (nm) Absorbents 210 21.770 274 9.340 313 sk (shoulder) 4.190 No significant change is observed with acid or base.

Infrared Absorption Spectrum See FIG. 3 Instrument Nicolet 5DX FT-IR Main Absorption Band (KBr, film): 540, 740, 955, 990, 1017, 1065, 1080, 1118, 1150, 1250, 1305, 1325, 1340, 1370, 1385, 1440, 1690, 1705, 1735, 2900, 2920, 2930, 2970, 3450 @m "1.

Mass spectrum See FIG. 5 Instrument Finigan 4500 TSQ Method: ionization by rapid respiration (FAB) Matrix m / z Molecular ion Relative presence glycerol 856 [M + H] + 100% glycerol + NaCl 878 [M + Na7 + 100% dithiothreitol: dithioerythritol 856 [M + H¶ + 100% (weight ratio 3: 1) 10 15 20 25 30 35 469 632 "M" Instrument Kratos MS-50 High Resolution FAB (m / z) [M +: »; 7 * = sseßsez Molecular weight apparent MV = 855 (based on above described mass spectral data) Elemental composition: C36H61N301¿S3 (based on the high resolution data described above) Proton magnetic resonance spectrum: See FIG. 7 Instrument: WM 360 Bruker Solvent: CDCl 3 111 NMR 360 MHz δ (pmm 6.54 (1H, dd, J = 7.7, 7.0); 6.21 (1H, brs); 5.87 ( 1H, d, J = 9.6), 5.78 (1H, dd, J = 9.6, 1.5), 5.66 (1H, brd, J = 2.9), 4.94 (1H , da, J = 10, 3, 1.8), 4.61 (m, <1, J = 7.7), 4.25 (1H, s), 4.09 (1H, q, J = 2 , 6); 3.97 (1H, t, J = 9.6); 3.92-3.53 (10H), 3.45 (1H, dt, J = 10.3, 4.0); 3 .37 (3H, s); 2.77 (1H, m); 2.69 (1H, dt, J = 9.9, 5.2); 2.49 (1H, dd, J = 10.3, 2.6); 2.48 (3H, s); 2.30 (2H, m); 2.13 (1H, m); 2.09 (3H, s); 1.50 (ZH, m); 1.37 (3H, d, J = 5.9); 1.32 (3H, d, J = 6.3); 1.08 (6H) 13 C magnetic resonance spectrum: See FIG. 9 Instrument: WM 360 Uses Solvent: CDCl 3 mc NMR 90.6 MHz δ (ppmh 13.7, 17.5 19.8, 22.3, 22.7, 23.5, 34.2, 35.2, 39.5, 47.7, 52.7, 55.8, 56.1, 57.7, 62.4 64.7, 67.4, 69.3, 69.8, 71.9, 76.1, 77.1 , 77.7, 79.7, 83.2 88.4, 97.3, 99.7, 123.4, 124.6, 130.1, 193.1 10 15 20 25 30 35 _13- 469 632 Physicochemical properties of BBM-1675D Description: Amorphous solid Ultraviolet absorption spectrum: See FIG. ument Hewlett-Packard 8458 Solvent: methanol Concentration: 0.01 g / l ¿maX (nm) absorptivities 214 27,000 274 12,800 325 5,400 No significant change is observed with acid or base.

Infrared Absorption Spectrum Mass Spectrum Instruments See FIG. Nicolet 5DX FT-IR Main Absorption Band (KBr, film): 735, 755, 910, 960, 1000, 1020, 1085, 1150, 1195, 1250, 1310, 1335, 1365, 1385, 1445, 1510, 1685, 1720, 1735 , 2880, 2930, 2960, 3400 6m'1.

Instrument Method Matrix.

Molecular ion (m / z): Relative occurrence: Instrument High resolution FAB (m / z) Molecular weight: apparent MW = 695 (based on mass spectral data described above) See FIG. 6 Finigan 4500 TSQ ionization by fast atom bombardment (FAB) thioglycerol [M + H] + = 696 100% Kratos MS-50 ¿M + ny * = 696,2794 10 15 20 25 30 35 469 632 "u" Elemental composition: C29H49N3012S2 ( based on the high-resolution data described above) Correlation of the relative occurrences of [M + H] + and [(M + H) +2] + to their calculated values confirms the elemental composition derived from the high-resolution FAB measurements.

See FIG. 8 Instrument: WM 360 Bruker Solvent: CDCl 3 + 10% CD 3 OD 1 H NMR 360 MHz δ (ppm): 6.43 (1H, ad, J = 4.4, 10, 3); 6.13 (1H, s); 5.81 (in, d, J = 8.8); 5.70 (1H, d, J = 8.8); 5.48 (1H, 6 brs); 4.48 (1H, d, J = 8.1); 4.02 (1H, d, J = 2.0); 3.95-3.80 (solvent background); 3.77 (1H, t, J = 9.0); 3.70 - 3.40 (11H, brm); 3.35 (1H, m); 3.28 (3 H, s); 3.22 (3 H, brs); 2.66-2.55 (2H, m); 2.38 (3 H, S); 2.23-2.12 (2H, m); 1.42 (1H, brdw; 1.22 (3H, d, J = 5.9); 0.94 (SH, d, J = 6.6); 0.87 (3H, d, J = 5, 9).

PIOtOI1 fl18 gI1etIêSOIlaIlSSpêktICHID.I 1 13 se FIG. 10A and 1013 Instruments WM 360 Uses Solvent: CDCl3 + 10% CD3OD C Magnetic Resonance Spectrum BC NMR 90.6 MHz δ (ppm) = 17.5 21.6, 22.2, 23.0, 33.4, 39 , 2, 46.4, 52.3, 55.8, 62.1, 67.8, 69.8, 70.1, 71.3, 75.8, 77.1, 78.1, 82.4 , 83.3, 88.2, 97.4, 99.6, 122.6, 124.8, 130.1, 130.8, 134.3, 148.7, 192.8.

Biological properties of the BBM-1675 substances.

The antimicrobial activity of the BBM-1675 substances was determined for a variety of gram-positive and gram-negative microorganisms. Table 1 below provides data in the form of the results of an antimicrobial screening procedure comprising "IS" 469 652 comprising the parent component BBM-1675A1 and the substances BBM-1675C and BBM-1675D of the present invention. In the screening method, each test compound was applied in a uniform concentration of 10 pg / ml solution and impregnated on a paper strip on the growth culture and the measure of the antibiotic activity is the obtained inhibition zone on the paper strip. As shown in Table 1, the substances BBM-16750 and D showed a broad antimicrobial spectrum of activity, which was at least as effective as for the BBM-1675A1 component; in particular, the substances BBM-175C and D were more effective as inhibitors of gram-negative organisms. _16- TABLE I.

ANTIMICROBIAL ACTIVITY OF BBM-1675 SUBSTANCES Inhibition zone, mm Test microorganism BBM-1675A1 BBM-16750 BBM-1675Q Escherichia 5211 AS 19 22 52 51 Escherichia coli K 12 13 36 35 Escherichia coli P 1373 12 34 33 Escherichia coli 34 Azaser coli R Netropsin 11 32 32 Escherichia coli R Mitomycin C 12 35 34 Escherichia 231; R Bleomycin 16 38 36 Escherichia 321; R Daunomycin 19 45 44 Escherichia coli R Neomycin 24 53 52 Escherichia E91; R Sibir0mYCi fl 14 32 30 Escherichia ggli R HedamYCi fl 14 30 25 Escherichia E21; R AClaCi fl0 mYCi fl 15 41 40 Bacillus subtilis ATCC 6633 34 43 41 Klebsiella pneumoniae 17 35 35 Staphzlococcus 209 P 32 47 44 Staphylococcus R Actinoleukin 33 35 33 Staphílococcus R Streptonigrin 37 50 48 Staphylococcus 30 Pccalisus 38 Smith R 40 55 53 Actinomycin D Staphylococcus aureus Smith R 17 32 31 Aureolic acid Acinetobacter 16 33 32 Micrococcus luteus 35 57 55 Saccaromyces cerevisiae petite 22 42 43 R = Resistant to the said antibiotic. 10 15 20 25 30 35 '”' 469 652 Activity against leukemia P-388.

Tables II and III contain the results of laboratory experiments with CDF mice with intraperitoneally implanted tumor inoculum of 10% ascites cells of leukemia P-388 and treated with different doses of BBM-1675A1, C or D. The substances were administered by intraperitoneal injection. Groups of six mice were used at each dose level and were treated with a single dose of the substance the day after tumor inoculation. A group of ten saline-treated control mice was incorporated into each experimental series. The BBM-1675A1-treated group according to Table III has been incorporated for direct comparison. A 30-day protocol was used, with the mean survival time in days being determined for each group of mice and the number of survivors at the end of the 5-day period being noted. The mice were weighed before treatment and again on day 4. The weight change was taken as a measure of the drug's toxicity. Mice weighing 20 g each were used and a weight loss of up to about 2 g was not considered. The vehicle-treated control animals usually died within 9 days.

The results were determined expressed in% B / K, which is the ratio between the survival time of the treated group and the mean survival time of the vehicle-treated control group, multiplied by 100. A value of% B / K equal to or greater than 125 indicates that a significant antitumor effect has been achieved. The screening results in Table II show the initially unexpected level of antitumor activity of the BBM-1675C substance. Table III gives the results of a direct comparison between the substances BBM-1675A1 (esperamicin A1) and BBM-1675C and BBM-1675D. The data suggest that BBM-1675C is approximately comparable to BBM-1675A1 in potency and antitumor efficacy and that it is not dependent on the dosing schedule, while the substance BBM-1675D is only slightly less effective.

Furthermore, it has been stated above that the same substances BBM-1675C and BBM-1675D can also be obtained starting from the BBM-1675A2 (esperamicin A2) component. In comparison with the data reported in this application for BBM-1675C and BBM-1675D and the data given in published British patent application 2,141,425 for the component BBM-1675A2, it has surprisingly shown the substances BBM-1675C and D are more effective as antitumor agents than the parent component BBM-1675A2 from which they are derived.

TABLE 11 EFFECT OF BBM-16750 ON P-388 LEUKEM1 (Treatment on day 1) Effect MVÄ Dose, IP MEET MEET g Survivor Compound mg / kg / inj. days% B / K day 4 day 5 BBM-167sc 3.2 Tax mox - o / 6 0.8 Iox Iox - 0/6 0.2 Iox Iox - o / 6 0.05 TOX TOX -1.8 1 / 6 0.0125 11.0 122 -2.5 5/6 0.003125 13.5 150 -2.5 6/6 Vehicle - 9.0 100 0.4 10/10 Tumor inoculum: 106 ascites cells implanted ip

Host animal: CDF1 male mice Evaluation: MÖI = mean survival time Effect:% B / K = (MÉ5_T '_' for treated group / bíÖT for control group) x 100 Criteria:% B / K 2 125 is considered to mean significant antitumor activity MVÄ: mean weight change ( treated group / control group) ig (on day 4). "lg" 469 632 TABLE III EFFECT OF BBM-1675 SUBSTANCES ON P-388 LEUKEMIA Effect MvÄ Överle Treatment- Dose, IP MEET g g vande Compound schedule mg / kg / inj. days% B / K Day 4 Day 5 BBM-1675A1 d. 1 _ 0.0512 TOX TOX - 0/6 0.0256 Iox Tox - 0/6 0, o128 TOX TOX -1.8 3/6 0.0064 15 .5 172 -0.3 6/6 0.0032 15.0 167 -0.6 6/6 0.0016 15.5 172 0.6 6/6 0.0008 12.5 139 0.3 6/6 0.0004 12.0 133 1.4 6/6 0.0002 11.0 122 0.8 6/6 0.0001 11.5 120 1.4 6/6 BBM-16750 d. 1 0, o256 TOX IOX - 0/6 0,012s Iox rox -0,8 3/6 0,0064 11,5 128 -0,3 6/6 0,0032 14,5 161 -0,1 6/6 0,0016 10,5 117 0.0 6/6 0.0008 12.0 133 0.3 6/6 0.0004 11.5 128 0.8 6/6 0.0002 11.0 122 1.4 6/6 0.0001 11, 0 122 0.8 6/6 0.00005 10.5 117 1.3 6/6 BBM-1675D d. 1 0.0256 9.0 100 0.1 6/6 0.0128 11.5 128 0.3 6/6 0.0064 12.5 139 0.3 6/6 0.0032 12.0 133 0.1 6/6 0.0016 11.5 128 0.8 6/6 0.0008 10.0 111 0 , 2 6/6 0.0004 10.0 111 0.5 6/6 0.0002 9.5 106 1.7 6/6 0.0001 9.5 106 1.7 6/6 0.0000s 9.0 100 2.0 6/6 10 15 20 25 30 35 469 632 "zo" TABLE III (continued) Effect MVÄ Survival- Treatment- Dose, IP MEET MEET g vande Compound schedule mg / kg / inj. days ZB / K Day 4 Day 5 BBM-16750 d. 1-es 0.0032 16.0 178 -1.3 6/6 0.0016 13.5 150 -1.0 6/6 0.000s 13.5 150 -0.3 6/6 0.0004 12.0 133 -0.4 6/6 0.0002 12.0 133 -0.4 6/6 0.0001 11.0 122 -0.4 5/6 0 , 0000s 11.0 122 0.9 6/6 0.0000z5 8.5 94 2.2 6/6 0.000012s 8.0 89 2.4 6/6 0.0000062s 8.0 89 2.4 6 / 6 vehicle - 9.0 100 2.4 10/10 Tumor inoculum: 106 ascites cells implanted ip

Host animal: CDF1 female mice Evaluation: MEET = mean survival time Effect:% B / K = (MEET for treated group / MEET for control group) x 100 Criteria:% B / K 2 125 is considered to mean significant antitumor activity MVÄ: mean weight change (treated group / control group) ig (on day 4).

Activity against melanoma B16 Table IV contains the results of antitumor tests using melanone B16 which has been grown in mice. BDF1 mice were used and inoculation of the tumor implant was performed subcutaneously. A 60-day protocol was used. Groups of ten mice were used at each dose level and the mean survival time of each group was determined. Control animals, which had been inoculated with "Zl" 469 632 in the same manner as the experimental animals and treated with the injection vehicle and no drug, had a mean survival time of 22.5 days. At each dose level, the test animals were treated with the test compound on days 1, 5 and 9 by intraperitoneal injection. An effect expressed as% B / K equal to or greater than 125 indicates that a significant antitumor effect has been achieved. The results in Table IV show that in a direct comparison, BBM-1675C was also effective in treating mice with melanoma B16 and was approximately comparable to BBM-1675A1 in potency. 10 15 20 25 30 469 632 _22- TABLE IV EFFECT OF BBM-1675 SUBSTANCES ON B16 MELANOMOM (Treatment days 1, 5 and 9) Effect MVÄ Dose, IP MEET MEET g surviving Compound mg / kg / inj. days% B / K Day 12 Day 10 BBM-1675Al 0.0032 37.5 167 0.3 10/10 0.0016 37.5 167 0.3 10/10 0.0008 38.5 171 1.4 10 / 10 0.0004 37.0 164 1.8 10/10 0.0002 34.5 153 2.0 10/10 0.0001 32.0 142 1.9 10/10 BBM-1675C 0.0008 31.5 140 0.6 10/10 0.0004 37.0 164 1.2 10/10 0.0002 31.0 138 0.6 10/10 0.0001 31.5 140 1.0 10/10 0.00005 27, 5 122 0.8 10/10 0.000025 25.0 111 0.5 10/10 Vehicle - 22.5 100 0.3 10/10 Tumörinokulatz 0.5 ml of a 10% porridge, IP Host animal: BDF1 female mice Evaluation: MEET = mean survival time Effect:% B / K = (MEAT in treated group / MEET in control MVÄ: mean weight change (treated group / control group) in group) x 100 Criteria:% B / K 2 125 is considered to mean significant anti-tumor activity (on day 12). As shown by the above antimicrobial data and mouse tumor data, BBM-1675C and BBM-1675D are useful as antibiotics in the therapeutic treatment of mammals and other animals for infectious diseases and also as antitumor agents for to therapeutically inhibit the growth of mammalian tumors.

The present invention therefore also provides a method of therapeutically treating a host animal which has been infected with a microbial infection or of a malignant tumor, which method comprises administering to the host animal an effective antimicrobial or tumor inhibiting dose of BBM-167SC or BBM-1675D. or a pharmaceutical preparation thereof.

The present invention also relates to a pharmaceutical preparation which contains an effective antimicrobial or tumor inhibitory amount of BBM-1675C or BBM-1675D in combination with an inert pharmaceutically acceptable carrier or diluent. Such preparations may also contain other active antimicrobial or antitumor agents and may be prepared in any pharmaceutical form appropriate to the intended route of administration. Examples of such formulations include solid formulations for oral administration such as tablets, capsules, pills, powders and granules, liquid formulations for oral administration such as solutions, suspensions, syrups or tinctures and mixtures and preparations for parenteral administration such as sterile aqueous or anhydrous solutions, suspensions or emulsions. They can also be prepared in the form of sterile solid preparations which can be dissolved in sterile water, physiological saline or any other sterile injectable medium immediately before use.

For use as an antimicrobial agent, BBM-1675C or BBM-1675D or a pharmaceutical preparation thereof is administered so that the concentration of the active ingredient is greater than the minimum inhibitory concentration of the particular organism in question being treated. For use as an antitumor agent, optimal dosages and dosing schedules for BBM-1675C or BBM-1675D for a given host mammal can be readily determined by those skilled in the art. It will of course be appreciated that the actual dose of BBM-1675C or BBM-1675D will vary depending upon the particular formulation, method of administration and the host and disease being treated and the particular site of disease.

Many factors that modify the effects of the drug may be considered, including age, weight, sex, diet, time of administration, mode of administration, rate of excretion, patient's condition, drug combinations, reaction sensitivities and severity of the disease. The administration can be performed continuously or periodically within the maximum tolerated dose. Optimal administration for a given set of conditions can be determined by those skilled in the art using conventional dosage determination tests in light of the above guidelines.

The invention is further illustrated by the following working examples.

Chemical preparation and isolation of BBM-1675C and BBM-1675D.

Example 1

A 50 mg sample of BBM-1675A1 was dissolved in 2.5 ml of methanol and treated with 2.5 ml of a 0.1 molar solution of hydrogen chloride in methanol. The reaction was allowed to proceed at a temperature of about 50 ° C and the disappearance of the starting material (took about 30 minutes) was monitored every 5 to 10 minutes by thin layer chromatography (TLC) on silica gel plates (Analtech, 250 microns, GF) with toluene acetone in a volume ratio of 3: 2 as elution solvent. After the starting material was consumed, the reaction mixture was neutralized with a saturated solution of sodium hydrogencarbonate in methanol and evaporated under reduced pressure to give a dry residue containing the bioactive fragments.

The BBM-1675C substance was isolated from the residue by flash column chromatography on a column having an inner diameter of 2 cm and a length of 10 cm and packed with Woelm silica gel (particle size 32-63 microns). The column was eluted with toluene acetone in a volume ratio of 3: 2 and 3 ml fractions were collected. Each fraction was analyzed by TLC (silica gel with toluene acetone in a volume ratio of 3: 2 as eluent) and the TLC spots were visualized with an ultraviolet light source with a wavelength of 254 nm and a cerium sulphate spray (1% cerium sulphate and 2.5% molybdenic acid in 10 % sulfuric acid). Fractions 6-12 (Rf value for BBM-16750 is 0.28) were pooled and evaporated to dryness to give 12 mg (35%) of substantially pure BBM-175 ° C.

The physicochemical properties of BBM-175 ° C are shown in the description and the ultraviolet spectrum, infrared spectrum, mass spectrum, 1 H-NMR spectrum and 13C-NMR spectrum of the compound are shown in Figures 1, 3, 5, 7 and 9, respectively.

Example 2.

When the reaction time of the procedure of Example 1 is extended, the amount of BBM-175 ° C and two new products designated Compound 3 (Rf = 0.65) and BBM-1675D (Rf remains at baseline) decrease (TLC: silica, toluene acetone in volume). the attitude 3: 2) appears and becomes more dominant with time.

The compound BBM-1675D, which usually accompanies the formation of BBM-16750, was isolated from the chromatographic column described in Example 1 by eluting the column with chloroform-methanol in a volume ratio of 5: 1. The appropriate fractions were pooled and evaporated to dryness to give 18 mg of the substantially pure BBM-1675D from the reaction described in Example 1.

The BBM-1675D substance shows a major spot at Rf = 0.37 on reverse phase TLC (Whatman MKC18F, 200 microns) using 30% water in methanol as eluent and Rf chloroform-methanol in the volume ratio 5: 0.5 as elution- = 0.22 in normal phase silica gel TLC using agent.

Example 3.

A significant improvement in the yield of BBM-1675D can be achieved by using p-toluenesulfonic acid instead of hydrogen chloride in the chemical hydrolysis of EBM-1675A2 or BBM-1667SA1, as illustrated by the procedures of Examples 3 and 5 below, respectively.

A sample of 15.2 mg of BBM-1675A2 was hydrolyzed with a 0.03 molar solution of p-toluenesulfonic acid in methanol (1 ml) at a temperature of about 6300 for about 1 hour. The reaction mixture was then evaporated to dryness under reduced pressure at about 30 ° C. The BBM-1675D substance was isolated from the dry residue by flash column chromatography on a column packed with Woelm silica gel (particle size 32-63 microns). The column was eluted with chloroform-methanol in a volume ratio of 5: 0.5 and the recovered fractions were analyzed by TLC (silica gel with chloroform-methanol in a volume ratio of 510.5 as eluent). The chromatography conditions used allowed separation of the mixture of inactive compounds 2 and 3 (7 mg) from the bioactive substance BBM-1675D, which has an Rf value of 0.22.

The appropriate fractions were pooled and evaporated to dryness to give 8 mg of substantially pure BBM-1675D in near quantitative yield.

The physicochemical properties of BBM-1675D are apparent from the specification and ultraviolet, infrared, mass, 1 H-NMR-13 and C-NMR spectra of the compound are shown in Figures 2, 4, 6, 8 and 10A and 10B, respectively. combination. 10 15 20 25 30 35 _27- 469 652 Example 4.

A 40 mg sample of BBM-1675A2 was treated with 5 ml of a 0.5 molar solution of hydrogen chloride in methanol at about 50 ° C for about 2 hours by the general procedure and isolation method of Example 1. After neutralization with sodium bicarbonate and evaporation to dryness, the BBM-16750 substance was isolated from the residue by flash column chromatography on a column packed with Woelm silica gel (particle size 32-63 microns) using toluene acetone in a volume ratio of 3: 2 as eluent. The appropriate fractions were combined and evaporated to dryness to give 8.4 mg of substantially pure BBM-175 DEG C., which is identical to the product isolated in Example 1.

The chromatography column as above was then eluted with chloroform-methanol 5: 0.25 by volume and the recovered fractions were pooled and evaporated to dryness to give BBM-1675D. The BBM-1675D substance was further purified by a further flash chromatography column on silica gel using chloroform: methanol in a volume ratio of 5: 0.5 as eluent. The appropriate fractions were combined and evaporated to dryness to give 6.3 mg of substantially pure BBM-1675D, which is identical to the product isolated in Example 3.

Example 5.

A sample of 49.3 mg of BBM-1675A1 was hydrolyzed with a 0.037 M solution of p-toluenesulfonic acid in 1.5 ml of methanol at a temperature of about 60 ° C for about 1.5 hours. The reaction mixture was evaporated to dryness under reduced pressure at about 30 ° C to give a residue containing BBM-1675D and the inactive compounds 1 and 3. The bioactive BBM-1675D substance was isolated from the residue by flash column chromatography on a column. packed with Woelm silica gel (particle size 32-63 microns) using chloroform-methanol in the volume ratio 5: 0.25 as eluent. The appropriate fractions were combined and evaporated to dryness to give 27 mg of substantially pure BBM-1675D, which is identical to the product isolated in Example 3.

Example 6.

A sample of 5.1 mg BBM-1675C was hydrolyzed with a 0.5 molar solution of hydrogen chloride in 1 ml of methanol at about 40-SOOC overnight. After neutralization with sodium bicarbonate and evaporation to dryness, the bioactive BBM-1675D substance was isolated from the residue by flash column chromatography on a column packed with Woelm silica gel (particle size 32-63 microns) using chloroform: methanol in the volume ratio S: O. 2S as eluent. The appropriate fractions in question gave essentially pure BBM-1675D, which is identical to the product isolated in Example 3.

Example 7.

When the general procedure of Examples 1 and 2 is repeated but with the difference that the starting material BBM-1675A1 is replaced by an equimolar amount of a mixture containing BBM-1675A1 and BBM-1675A2, the substances BBM-1675C and BBM-1675D are obtained.

Example 8.

When the general procedure of Example 5 was repeated but with the difference that the starting material BBM-1675A1 is replaced by an equimolar amount of a mixture containing BBM-1675A1 and BBM-1675A2, the substance BBM-1675D is obtained.

Claims (6)

10 15 203 25 30 35 29 469 632 PATENT CLAIMS The antitumor and antibiotic compound BBM-1675D as in substantially pure form; (a) acts as an amorphous solid; (b) (c) is soluble in methanol, ethanol, acetone and tetrahydrofuran and sparingly soluble in chloroform; in silica gel thin layer chromatography exhibits an Rf value of 0.22 with the solvent system chloroform: methanol in the volume ratio 5105 and in reverse phase silica gel thin layer chromatography exhibits an Rf-. value of 0.37 with the solvent system methanol water (d) (e) (f) (s) (h) in the volume ratio 70:30; has an apparent molecular weight of 695 when determined by high resolution FAB mass spectroscopy; has an ultraviolet absorption spectrum in methanol solution essentially as shown in Fig. 2 with ultraviolet absorption maxima and absorptivities at 214 nm (a = 27,000), 274 nm (a = 12,800) and 325 nm (a = 5,400) without any significant change when adding acid or base; has an infrared absorption spectrum (KBr, film) essentially as shown in FIG. 4 with main absorption peaks at 735, 755, 910, 960, 1000, 1020, 1035, 1150, 1195, 1250, 1310, 1335, 1365, 1335, 1445, 1510, 1685, 1720, 1735, zaao, 2930, 2960 and 3400 6m '1; has a low resolution mass spectrum_ substantially as shown in Fig. 6 with a molecular ion_ [M + H] + of 696; has a 360 MHz proton magnetic resonance spectrum in CDCl 3 + 10% CD 3 OD essentially as shown in Fig. 8 with signals at 469 652 6.43 (1H, dd, J = 4.4, 10.3), 6.13 (1 H, s); 5.81 (1H, d, J = 8.8); 5.70 (1H, d, J = 8.8); 5.48 (1H, 6 brs); 4.48 (1H, d, J = 8.1); 4.02 (1H, d, J = 2.0); 3.95-3.80 (solvent background); 3.77 (1H, t, J = 9.0); 3.70 - 3.40 (11H, brm); 3.35 (1H, m); 3.28 (3 H, s); 3.22 (3 H, brs); 2.66 - 2.55 - (2H, m), 2.38 (3H, s); 2.23-2.12 (211, m), 1.42 (m, bmw; 1.22 (3H, d, J = 5.9); 0.94 (3H, d, J = 6.6) and 0.87 (3H, d, J = 5.9) ppm precipitated as tetramethylsilane; (i) has a 90.6 MHz carbon-13 magnetic resonance spectrum in CDCl 3 + 10% CD 3 OD substantially as shown in Fig. 10 ( 10A + 10B) with signals at, 17.5, 21.6, 22.2, 23.0, 33.4, 39.2, 46.4, 52.3, 55.8, 62.1, 67, 8, 69.8, 70.1, 71.3, 75.8, 77.1, 78.1, 82.4, 83.3, 88.2, 97.4, 99.6, 122.6, 124.8, 130.1, 130.8, 134.3, 148.7 and 192.8 ppm precipitated as tetramethylsilane. Process for the preparation of the antitumor and antibiotic compound BBM-1675D according to claim 1, characterized in that BBM-1675A1 or BBM-1675A2 is hydrolyzed with a mineral acid or organic acid until a substantial amount of BBM-1675D has formed and then BBM-1675D extracts from the reaction medium. 3. A process for the preparation of the antitumor and antibiotic compound BBM-1675D according to claim 1, characterized in that EBM-1675C is hydrolyzed with a mineral acid or organic acid until a substantial amount of BBM-1675D has been formed and then BBM-1675D recovers from the reaction medium. 4. A process for the preparation of the antitumor and antibiotic compound BBM-1675D according to claim 1, characterized in that a mixture of BBM-1675A1 and BBM-1675A2 is hydrolyzed with a mineral acid or organic acid until a substantial amount of BBM-1675D has been formed and then EBM-1675D recovers from the reaction medium. 10 31 469 652 A pharmaceutical preparation comprising an effective antimicrobial amount of BBM-1675D according to claim 1 in combination with a pharmaceutical carrier or diluent. A pharmaceutical formulation comprising an effective tumor inhibiting amount of BBM-1675D according to claim 1 in combination with a pharmaceutical carrier or diluent.