LU86562A1 - NOVEL ANTIBIOTIC AND ANTITUMOR SUBSTANCES, METHOD FOR THEIR PREPARATION AND INSULATION; PHARMACEUTICAL COMPOSITIONS CONTAINING THEM - Google Patents

Description

v,

The present invention essentially relates to new antibiotic and anti-tumor substances, as well as a method for obtaining and isolating them.

The invention also relates to pharmaceutical 5-ceutical compositions containing these new substances which constitute an antimicrobial and antitumor agent.

The antitumor compounds according to the present invention have not yet been "identified in terms of structure. However, in view of their unique physiological, chemical and biological properties, the antibiotics BBM-1675C and BBM-1675D invention, constitute new substances.

British Patent Application No. 2,141,425, published December 19, 1984, discloses a new antibiotic and antitumor complex called BBM-1675, obtained by fermentation of a strain H964-92 (ATCC 39334) Actinomadura verrucosospora or a strain A1327Y (ATCC 39638)

Actinomadura verrucosospora. Two major bioactive components of the BBM-1675 complex described in this application have been designated BBM-1675A ^ and BBM-1675A2. The structures of the antibiotics BBM-1675A ^ and BBM-1675A2. also known as esperamicin A and esperamicin A2, respectively, have not been elucidated to date, but these two components exhibit excellent antimicrobial and antitumor activities.

US Patent No. 4,530,835, issued July 23, 1985 to Bunge et al, discloses a process 2 of fermentation of an isolated strain of unidentified Actinomycete WP-444 (ATCC 39363) to obtain anti-tumor antibiotics designated CL-1577A and CL-1577B. The structures of the CL-1577 antibiotics have so far not been elucidated, but their characteristic properties indicate that CL-1577A and CL-1577B are similar with respect to the structure of the BBM-1675 antibiotics, and in particular BBM- 1675A ^ and BBM-1675A2 mentioned in the aforementioned British patent application No. 2,141,425.

10 The publication J. Antibiotics, 37 (12), 1566-1571 (1984) (R. H. Bunge et al) reveals the fermentation of Actinomadura sp. (ATCC 39363) for obtaining a biologically active complex from which the two essential components PD 114 759 and PD 115 028 have been isolated. In pulbica-15 tion J_. Chem. Soc. Chem, Common. , 919-920 (1985), JH Wilton et al, the elucidated partial structure of the antibiotics PD 114 759 and PD 115 028 has been described. Obtaining, isolating and characterizing the antibiotics PD 114 759 and PD 115 028 appear be identical to that of the above-mentioned antibiotics CL-1577A and CL-1577B, respectively.

European patent application No. 95,154, published on November 30, 1983, reveals the fermentation of Actinomadura pulveraceus sp. Nov. No. 6049 (ATCC 39100) for obtaining anti-tumor antibiotics designated WS 6049-A and WS 6049-B. The structures of the antibiotics WS-6049 have not yet been elucidated, but the characteristic properties given for these antibiotics indicate that WS 6049-A and WS 6049-B are similar in structure to the antibiotics BBM-1675 revealed by British Patent Application No. 2,141,425 and antibiotics CL-1577 disclosed by US Patent No. 4,530,835. The spectral data show, however, that neither WS 6049-A nor WS 6049-B is the same as one of the components of BBM-1675. 35 In addition, the producing organism described in the 3 European patent application No. 95 154 can be clearly distinguished from Actinomadura verrucosospora used in the British patent application No. 2 141 425 by the color of the mycelium - aerial on ISP medium. N ° 2, 3 and 4, its positive peptonization in milk and its positive use of D-fructose, D-mannitol, · trehalose and cellulose.

The object of the present invention is to provide new antibiotic and anti-tumor substances designated BBM-1675C and BBM-1675D, also BMY-27305 and BMY-27307, respectively, said substances being obtained by selective chemical hydrolysis of the biologically active components BBM -1675A ^ (Esperamicin A ^) or BBM-1675A2 (Esperamicin Ag) which are themselves obtained by culture of a strain producing BBM-1675 Actino-15 madura verrucosospora. The biologically active substances BBM-1675C and BBM-1675D can be separated and purified by traditional chromatography processes and these two substances show excellent anti-tumor and antimicrobial activity.

The invention will be better understood, and other objects, characteristics, details and advantages thereof will appear more clearly during the explanatory description which follows, made with reference to the appended schematic drawings given solely by way of example. illustrating several embodiments of the invention and in which: - Figure 1 represents the ultraviolet absorption spectrum of BBM-1675C, - Figure 2 represents the ultraviolet absorption spectrum of BBM-1675D, - Figure 3 represents the infrared absorption spectrum of BBM-1675C (KBr, film), - Figure 4 represents the infrared absorption spectrum of BBM-1675D (KBr, film), 35 - Figure 5 represents the mass spectrum of 4 BBM-1675C, - Figure 6 shows the mass spectrum of BBM-1675D, “- Figure 7 shows the magnetic resonance spectrum of the proton of BBM-1675C in CDCl ^ (360 MHz), - Figure 8 shows the proton magnetic resonance spectrum of BBM-1675D in C DClg + 10% CD ^ OD (360 MHz), - Figure 9 represents the magnetic resonance spectrum 13 of BBM-1675C in CDClg (90.6 MHz), - Figure 10A represents the magnetic resonance spectrum 13 of BBM-1675C C (110-200 ppm) of BBM-1675D in CDC13 + 10% CDgOD (90.6 MHz), - Figure 10B represents the magnetic resonance spectrum 13 of C (0-110 ppm) of BBM-1675D in CDC13 + 10% CDgOD (90.6 MHz), - Figure 11A represents the magnetic resonance spectrum of the proton of compound 3A (-anomer) in CDClg (360 MHz), and - Figure 11B represents the magnetic resonance spectrum of proton of compound 3B (yG-anomer) in CDC13 (360 MHz).

As we have seen, the present invention relates to two new anti-tumor antibiotic substances designated BBM-1675C and BBM-1675D, also designated BMY-27305 and BMY-27307, respectively, these substances being obtained by selective chemical hydrolysis of the biologically active components BBM-1675A1 (Esperamicin A) or BBM-1675A3 (Esperamicin Ag) which are themselves obtained by culture of a strain producing BBM-1675 of Actinomadura verrucosospora, and preferably of a strain

Actinomadura verrucosospora H964-92 (ATCC 39334) or an Actinomadura verrucosospora A1327Y (ATCC 39638) strain or a mutant of these strains. In another aspect, the present invention provides a process for obtaining the substance BBM-1675C by selective hydrolysis of the biologically active components BBM-1675A1 or BBM-1675A2. According to another characteristic, the present invention provides a method of separation of BBM-1675D by selective hydrolysis of the substance BBM-1675C or, more preferably, from the biologically active components BBM-1675A ^ or BBM-1675A2. Isolation and purification of BBM-1675C and BBM-1675D from the reaction mixture can be accomplished by conventional chromatography procedures.

10 Biologically active substances BBM-1675C

and BBM-1675D exhibit antimicrobial activity against a wide variety of microorganisms, and these substances have also been shown to exhibit inhibitory activity against various tumor systems of mice, such as P-388 leukemia and B16 melanoma. The newly described substances according to the present invention, therefore, can be used as antimicrobial agents or as anti-tumor agents to inhibit tumors in mammals.

20 In degradation studies performed to elucidate the structure of the anti-tumor antibiotics BBM-1675A ^ (esperamicin A ^) and BBM-1675A2 (esperamicin Ag), a mixture of components has led to the isolation and identification of two inactive fragments, composed of formulas 1 and 2, respectively. However, it has surprisingly been discovered that chemical degradation leads to the stepwise release of the two biologically active fragments BBM-1675C and BBM-1675D. Even more surprisingly, it was discovered that the two different antibiotics BBM-1675A ^ and BBM-1675A2 produced the same biologically active fragments as illustrated in Scheme 1. Also surprisingly, it was discovered that the fragments of molecular weight the lowest BBM-1675C and BBM-1675D (having approximately 70% and 55% 35 of the molecular weight of the parent antibiotics BBM-1675A ^ and 6 BBM-1675A2, respectively) were more effective as antimicrobial and anti-tumor agents than BBM-1675A2 and comparable to BBM-1675A ^.

Figure 1 5 BBM-1675A1 ^ (esperamicin A ^) 10

BBM-1675C-►BBM-1675D

BBM-1675A2 ^ (esperamicin A2

Substances BBM-1675C and BBM-1675D can be obtained by selective chemical hydrolysis of the antibiotic BBM-1675A ^ as outlined in Figure 2.

7

Diagram 2 och3 H0 q h Θ CH3 ° VOv ^ n BBM-1675A.; —_— * BBM-1675C + 10 1 CH3OH (weight

(molecular weight CHgO 'NH

molecular 855.) I rH

1248) 2 och3

Compound 1 (molecular weight 425) (mixture of the anomers o {and ß>

H © / CH30H

* ^ 3 V — o BBM-1675D + CH „S - (V''QChL · (molecular weight J \ / 3 \ - 'e95) at,

Compound 3 (molecular weight 192) (mixture of anomers o (and / 3) 8

The starting compound BBM-1675A ^ is prepared according to the process described in British patent application No. 2 141 425, published on December 19, 1984. The purified component BBM-1675A ^ is hydrolysed with an organic or inorganic acid such as hydrochloric acid, sulfuric acid, p-toluenesulfonic acid, benzenesulfonic acid

or the like, in an inert organic or mixed aqueous-organic solvent at a temperature of about 0 ° C at the reflux temperature of the solvent until a substantial amount of the desired compounds is obtained BBM-1675C

or BBM-1675D. Preferably, the hydrolysis is carried out in C ^-C d'alcool alcohol solvents, and more preferably, the alcoholysis is carried out in methanol. The reaction temperature is not critical, but it will be preferred to carry out the reaction at a temperature between about room temperature and 60 ° C, and preferably between 40 ° C and 60 ° C.

The selective hydrolysis of BBM-1675A ^ is carried out in stages, with the initial obtaining of the antibiotic BBM-1675C and the inactive fragment of formula 1. Subsequent or continuous treatment under hydrolysis conditions leads to the release of a mixture of the anomers o {and / 3 of thio sugar of formula 3 and the production of the antibiotic BBM-1675D. Those skilled in the art will understand that by varying the reaction conditions such as time, temperature and concentration of the acid, variable relative amounts of the antibiotics BBM-1675C and BBM-1675D are obtained. Thus, it is desirable to monitor the reaction by thin layer chromatography, as will be described in the examples given below.

When it is desired to prepare only the antibiotic BBM-1675D, the selective hydrolysis is preferably carried out with an organic acid such as p-toluenesulfonic acid, as described below, to obtain a large amount of BBM-1675D .

9

Substances BBM-1675C and BBM-1675D can also be prepared by selective chemical hydrolysis of the antibiotic BBM-1675A2 as shown in Diagram 3. Diagram 3 CH3 ^^ 0 ^ 7 ~ OCH3 h © CHs ° '' ΤτλΎ '^ 0 BBM-1675A2— - ^ BBM- 1675C + fj (weight 3 (weight

molecular .molecular J I

1248) 855) 0CH3

Compound 2 (molecular weight 425) (mixture of anomers o <and / ¾)

H © / CH30H

: CH3

* j ^ Y

BBM-1675D + CH3S- \ Y * OCHg (weight \ _ / molecular /

695) OH

Compound 3 (molecular weight 192) (mixture of anomers c * and fi) 10

The starting compound BBM-1675A2 is prepared according to the process described in British patent application No. 2 141 425, published on December 19, 1984. The selective hydrolysis * of BBM-1675A2 is carried out in a similar manner in stages, with the initial obtaining of the antibiotic BBM-1675.C. and the inactive fragment of formula 2. The continuous treatment under hydrolysis conditions leads to the release of a mixture of the anomers and / 3, the thio sugar of formula 3 and the obtaining of the antibiotic BBM-1675D.

The reaction conditions used for the selective chemical hydrolysis of BBM-1675A2 are practically identical to those used for the hydrolysis of BBM-1675A ^ described above. In a similar way to obtaining BBM-1675D from BBM-1675A ^, when it is desired to obtain only the antibiotic BBM-1675D, the hydrolysis of BBM-1675A2 is carried out until substantially completely converted BBM-1675A2 and BBM-1675C in BBM-1675D. Preferably, the hydrolysis is carried out in an organic acid such as p-toluenesulfonic acid.

20 The discovery as described below, that the same

antibiotics BBM-1675C and BBM-1675D are produced from the two different antibiotics BBM-1675A ^ and BBM-1675A2 with the concomitant release of two inactive fragments of formulas 1 and 2, respectively and of the "thio-sugar" of formula 3 , provides an additional advantage to the present invention. Accordingly, according to another aspect of the present invention, there is provided a process for the selective hydrolysis of a mixture of BBM-1675A1 and BBM-1675A2 to obtain BBM-1675C and BBM-1675D

30 as illustrated by Diagram 4 »Diagram 4

BBM-1675A1 + BBM-1675A2-ψ BBM-1675C-► BBM-1675D

11

This advantage becomes apparent when one considers that the relative amounts of BBM-1675A ^ and BBM-1675A2 produced in the fermentation process are subject to variation. Obtaining BBM-1675C and BBM-1675D is therefore independent of the relative amounts of BBM-1675A ^ and BBM-1675A2 used as starting material in the present invention.

As described below, hydrolysis of the antibiotics BBM-1675A ^, BBM-1675A2 and BBM-1675C leads to the release of an inactive thiosugar fragment. This thio sugar has been isolated in order to obtain further information on the chemical structure of the antibiotic BBM-1675C and, therefore, of the antibiotics BBM-1675A ^ and BBM-1675A2 · The compound of formula 3 has been identified as being a mixture of the 15 anomers oc and fi of a thio sugar having the formula illustrated in Schemes 2 and 3. Other characterizations have been made possible when the products of alcoholysis, the anomers 0 (and fl, are separated. The proton magnetic resonance spectra (360 MHz) of compound 3A (anomer) and compound 3B (anomer ^ Q) are shown in Figures 11A and 11B, respectively. From an analysis of these spectral data, it is assumed that the methyl glycoside sugars of formula 3 have the relative stereochemistry the formula 25

OH

cv 30 Currently, absolute stereochemistry, i.e. D or L, has not yet been determined. Consequently, based on the current interpretation of the spectral data, it can be concluded that the thio sugar of formula 3 (minus the CH3 group of the methoxy anomeric compound which is incorporated during methanolysis) is a component of the structure of l BBM-1675C antibiotic and more, is a component of the structure of the starting antibiotics BBM-1675A ^ and BBM-1675A2.

5 Physico-chemical properties of BBM-1675C

Description ·: amorphous solid

Ultraviolet absorption spectrum: See figure 1

Spectrometer: Hewlett-Packard 8458

Solvent: methanol 10 Concentration: 0.0155 g / 1 X (nm) Absorbency

Hnax—- - 210 21,770 214 9,340 15,313 (shoulder) 4,190

No significant change is observed with an acid or a base. .

Infrared absorption spectrum: See figure 3

Instrument: Nicolet 5DX FT-IR

20 Main absorption bands (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 cm "1.

25 Mass spectrum: See Figure 5

Spectrometer: Finigan 4500 TSQ

Process: ionization by bombardment of accelerated atoms (FAB) 13 M. Ion Abundance - relative molecular ro / z glycerol 856 Cm + h] + 100% glycerol + NaCl 878 d ^ M + Na] | + 100% dithiothreitol: 5 dithioerythritol 856 £ m + HA + 100% '(3: 1) (weight / weight)

Instrument: Kratos MS-50 FAB High resolution (m / z): [M + h3 + = 856.3362 10 Molecular weight: apparent molecular weight = 855 (based on mass spectral data described above)

Elementary composition: OggHg ^ NgO ^ Sg (based on the aforementioned high resolution data)

Proton magnetic resonance spectrum: See figure 7 15 Instrument: WM 360 Bruker

Solvent: CDClg NMR. 1H 360 MHz S (ppm): 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, 20 J = 2.9); 4.94 (1H, dd, J = 10.3, 1.8); 4.61 (1H, d, 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 (2H, m); 1.37 (3H, d, J = 5.9); 1.32 (3H, d, J = 6.3); 1.08 (6H).

Carbon 13 magnetic resonance spectrum: See Figure 9

Instrument: WM 360 Bruker 30 Solvent: CDClg NMR 13C 90.6 MHz Γ (ppm): 13.7, 17.5, 19.8, 22.3, 22.7, 23.5, 34.2, 35, 2, 39.5, 14 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.

Physico-chemical properties of BBM-1675D 5 Description: amorphous solid ~ Ultraviolet absorption spectrum: See Figure 2

Spectrometer: Hewlett-Packard 8458

Solvent: methanol

Concentration: 0.01 g / 1 10 (nm) absorption aptitudes ΓΠ α X 1 - -—— - - 214 27,000 274 12,800 325 5,400

No significant change is observed with an acid or a base.

Infrared absorption spectrum: See figure 4

Instrument: Nicolet 5DX FT-IR

Main absorption bands (KBr, film): 735, 755, 910, 960, 1000, 1020, 1085, 1150, 1195, 20 1250, 1310, 1335, 1365, 1385, 1445, 1510, 1685, 1720, 1735, 2880 , 2930, 2960, 3400 cm-1.

Mass spectrum: See Figure 6

Instrument: Finigan 4500 TSQ

Method: ionization by bombardment of accelerated atoms (FAB)

Matrix: thioglycerol • Molecular ion (m / z): [m + h] + = 696

Relative abundance: 100% 30 Instrument: Kratos MS-50 FAB high resolution (m / z): [m + h} + = 696.2794

Molecular weight: apparent molecular weight = 695 (based on the above-mentioned mass spectral data) 15

Basic composition: ^ 29H49 ^ 331232 (based on the aforementioned high resolution data)

The correlation of the relative abundances Cm + h] and £ (M + H) +2 3+ with respect to the calculated values 5 confirms the derived elementary composition - FAB-high resolution measurements.

Proton magnetic resonance spectrum: See figure 8

Instrument: WM 360 Bruker

Solvent: CDCl “+ 10% CD„ 0D

10 DD

H NMR 360 MHz S (ppm): 6.43 (1H, dd, J = 4.4, 10.3); 6.13 (1H, 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 (background of the solvent); 3.77 (1H, t, J = 9.0); 3.70-3.40 (11H, brm); 3.35 (1H, m); 3.28 (3H, s); 3.22 (3H, brs); 2.66-2.55 (2H, m); 2.38 (3H, s); 2.23-2.12 (2H, m); 1.42 (1H, brdt); 1.22 (3H, d, J = 5.9); 0.94 (3H, d, J = 6.6); 0.87 (3H, d, J = 5.9).

Magnetic resonance spectrum

20 of carbon 13: See Figures 10A and 10B

Instrument: WM 360 Bruker

Solvent: CDClg + 10% CDgOD

13C NMR 90.6 MHz $ (ppm): 17.5, 21.6, 22.2, 23.0, 33.4, 39.2, 46.4, 52.3, 55.8, 25 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 BBM-1675 Substances The antimicrobial activity of BBM-1675 substances has been determined for a variety of gram-positive and gram-negative microorganisms. Table 1 below provides the data as the results of a antimicrobial screening process comprising the parent compound BBM-1675A! and the substances BBM-1675C and BBM-1675D of 16. In the screening process, each compound tested, having a uniform concentration of 10 µg / ml of a solution impregnated on a paper strip, was placed in a culture medium, and the measurement of the antibiotic activity is given by the resulting zone of inhibition of the paper strip. As shown in Table 1, the substances BBM-1675C and BBM-1675D exhibit a wide range of antimicrobial activity and are at least as effective as the compound BBM-1675A ^; and in particular, substances BBM-1675C and BBM-1675D are more effective as inhibitors of 'o gram-negative organisms.

17 TABLE 1 ANTIMICRQBIENNE ACTIVITY OF BBM-1675 SUBSTANCES

Inhibition zone, mm

Microorganism tested BBM-1675A ^ BBM-1675C BBM-1675D

Escherichia coli AS 19 22 52 51

Escherichia coli K 12 13 36 35

Escherichia coli P 1373 12 34 33

Escherichia coli R Azasérine 14 35 34

Escherichia coli R Netopsin 11 32 32

Escherichia coli R Mitomycin 12 35 34

Escherichia coli R Bleomycin 16 38 36

Escherichia coli R Daunomycin 19 45 44

Escherichia coli R Neomycin 24 53 52

Escherichia coli R Sibiromycin 14 32 30

Escherichia coli R Hedamycin 14 30 25

Escherichia coli R Aclacinomycin 15 41 40

Bacillus subtilis ATCC 6633 34 43 41

Klebsiella pneumoniae 17 35 35

Staphylococcus 209 P 32 47 44

Staphylococcus R Actinoleucine 33 35 33

Staphylococcus R Streptonigrin 37 50 48

Staphylococcus faecalis P1377 30 39 38

Streptococcus aureus Smith P 36 47 45

Staphylococcus aureus 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

Saccharomyces cerevisiae petite 22 42 43 R = resistant to the antibiotic concerned, 18

Activity against P-388 leukemia Tables II and III contain the results of laboratory tests on CDF1 mice implanted intraperitoneally with a tumor inoculum of 10 cells of ascites cells of P-388 leukemia, and treated with various doses of BBM-1675A1, C or D. The substances were administered by intraperitoneal injection. Groups of six mice were used for each amount dosed, and were treated with a single dose of the substance on day 10 after inoculation. A group of ten standard mice treated with saline was included in each series of experiments. The group treated with BBM-1675A ^ in Table III was used for direct comparison.

A 30 day protocol was used and the average survival time in days was determined for each group of mice as well as the number of survivors at the end of 5 day periods. The mice were weighed before treatment and - also every other day. Weight change values were used as a measure of drug toxicity. Mice each weighing 20 grams were used, and weight loss greater than about 2 grams was not considered excessive. The treated standard animals died during the first nine days.

The results were determined relative to the percentage T / C which is the ratio of the mean survival time of the treated group to the mean survival time of the standard group treated by the vehicle compared to 100. A T / C percentage equal to or greater than 125 indicates that a significant anti-tumor effect has been obtained. The screening results in Table II show the unexpected initial level of antitumor activity of the substance BBM-1675C. In Table III, the results of a direct comparison of BBM-1675A1 (esperamicin A ^) and the substances BBM-1675C and BBM-1675D are reported. The data suggest that BBM-1675C is roughly comparable to BBM-1G75A ^ in terms of its potential and anti-tumor efficacy, while BBM-1675D is only slightly less effective.

In addition, in accordance with the present invention, the same substances BBM-1675C and BBM-1675D can also be obtained from the component BBM-1675A2 (esperami-cine A2). By comparing the data reported here for BBM-1675C and BBM-1675D and the data reported in published British Patent Application No. 2,141,425 for the component BBM-1675A2, it was surprisingly found that the substances BBM-1675C and BBM-1675D are more effective as anti-tumor agents than the parent component BBM-1675A2 from which it is derived.

TABLE II

EFFECT OF BBM-1675C ON LEUKEMIA P-388 15 (Daily treatment)

AWC Survivors effect

Dose, IP MST MST 9m

Compound mg / kg / inj, days% T / C day 4 day 5 20 BBM-1675C 3.2 TOX TOX - 0/6 0.8 TOX TOX - 0/6 0.2 TOX TOX - 0/6 0.05 TOX TOX -1.8 1/6 0.0125 11.0 122 -2.5 5/6 25 0.003125 13.5 150 -2.5 6/6 Vehicle - 9.0 100 0.4 10/10 6

Tumor inoculum: 10 ascites cells implanted intraperitoneally (i.p.) Host: male miceCDF1

Assessment: STD = mean survival time

Effect: T / C (%) = (STD treated / STD sample) x 100

Criterion: T / C% ^ 125 is considered to be a significant anti-tumor activity AWC: average change in weight (sample-treated) in grams (on day 4)

TABLE III

20 EFFECT OF SUBSTANCES BBM-1675 ON LEUKEMIA P-388

Tnwonfaî Effect AWC SUT- Dose inventory, IP MST MST gro alive

Compound treatment mg / kg / inj. days% T / C Day 4 Day 5 BBM-1675A1 d. 1 0.0512 TOX TOX - 0/6 0.0256 TOX TOX - 0/6 0.0128 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 11.5 128 1.4 6/6 BBM-1675C d. 1 0.0256 TOX TOX - 0/6 0.0128 TOX TOX -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 TABLE III (continued) 21

AWC Overdose Effect Inventory, IP MST MST gm live

Compound treatment mg / kg / inj. days% T / C Day 4 Day 5 BBM-1675D d. 1 0.0256 9.0 100 0.1 6/6 v 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 0.00005 9.0 100 2.0 6/6 BBM-1675C d. 1 5 0.0032 16.0 178 -1.3 6/6 0.0016 13.5 150 -1.0 6/6 0.0008 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 0.00005 11.0 122 0.9 6/6 0 , 000025 8.5 94 2.2 6/6 0.0000125 8.0 89 2.4 6/6 0.00000625 8.0 89 2.4 6/6 Vehicle - 9.0 100 2.4 10/10 g

Tumor inoculum: 10 ascites cells implanted peritoneally (i.p.) Host: female CDF ^ mice

Assessment: STD = mean survival time

Effect: T / C (%) = (STD treated / STD sample) x 100

Criterion: T / C% 125 is considered to be a significant anti-tumor activity AWC: average variation in weight (treated sample) in grams (on day 4) "s 22 *

Activity against melanoma B16 Table IV contains the results of anti-tumor tests using the growth of melanoma B16 in mice. BDF ^ mice were used and inoculated subcutaneously with a tumor implant. A 60-day protocol was used. Groups of ten mice were used for each assay amount tested, and the mean survival time for each group was determined. Control animals inoculated in the same manner as the test animals were treated with a drug-free injection vehicle, and had an average survival rate of 22.5 days. For each dosage level, the animals tested were treated with the test compound on days 1, 5 and 9 by intraperitoneal injection. A percent T / C equal to or greater than 125 indicates that a significant anti-tumor effect is obtained. The results of Table IV show by direct comparison that BBM-1675C was also effective in the treatment of mice carrying a B16 melanoma and is practically comparable to BBM-1675Ar 23

TABLE IV

EFFECT OF SUBSTANCES BBM-1675 ON MELANOME B16 (Treatments performed on days 1, 5 and 9)

AWC effect

Dose, IP MST MST gm Survivors

Compound mg / kg / inj. days% T / C Day 12 Day 10 BBM-1675A1 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

Tumor inoculum: 0.5 ml of 10% brei, PI

Host: female BDF mice ^

Assessment: STD = mean survival time T Effect: T / C (%) = (treated STD / sample STD) x 100

Criterion: T / C%? / - 125 is considered to be a significant anti-tumor activity AWC: average change in weight (treated sample) in grams (on day 12).

. \ 24

As indicated by the antimicrobial and tumor data in the aforementioned mice, BBM-1675C and BBM-1675D are thus useful as antibiotics in the therapeutic treatment of mammals and other animals suffering from infections and also as anti-tumor agents for inhibiting therapeutically the growth of tumors in mammals.

Also, the present invention also relates to pharmaceutical compositions containing an effective antimicrobial or antitumour amount of BBM-1675C or BBM-1675D and an inert pharmaceutically acceptable diluent or carrier. Such compositions may also contain other antimicrobial or antitumor agents and may be made in any suitable pharmaceutical form depending on the chosen route of administration. Examples of such compositions include solid compositions for oral administration such as cachets, capsules, pills, powders and granules, liquid compositions for oral administration such as solutions or suspensions, syrups or elixirs and preparations for parenteral administration such as solutions sterile aqueous or non-aqueous, suspensions or emulsions. They can also be produced in the form of sterile solid compositions which can be dissolved in sterile water, physiological saline solution or other sterile injectable media immediately before use.

For use as an antimicrobial agent, BBM-1675C or BBM-1675D, or a pharmaceutical composition containing them is administered so that the concentration of the active substance is greater than the minimum inhibitory concentration for the particular organism to be treated. For use as an anti-tumor agent, the optimum doses and regimes of BBM-1675C or BBM-1675D for a given host can be readily established for those skilled in the art. It will be understood, of course, that the actual dose of BBM-16755C or BBM-1675D to be used will vary depending on the particular composition formulated, the mode of application and the particular sites, as well as the host and the disease to be treated. Many factors that modify the action of the drug will need to be considered, including age, weight, gender, diet, time of administration, mode of administration, excretion rate, condition of the patient, combination of different drugs, sensitivity to reactions and severity of the disease. Administration can be carried out continuously or periodically within the maximum tolerated range. Optimal application rates for a given set of conditions can be determined by those skilled in the art, using traditional dose determination tests in light of the guidelines given herein.

The following examples are given to illustrate the present invention, without limiting its scope.

20 Chemical preparation and isolation of BBM-1675C

and BBM-1675D EXAMPLE 1

A sample of BBM-1675A ^ (50 mg) 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 carried out at a temperature of about 50 ° C, and the consumption of the starting material (approximately 30 minutes) was monitored every 5 to 10 minutes by thin layer chromatography (TLC) on

30 ml silica gel (Analtech, 250 microns, GF) with a mixture of toluene: acetone (3: 2, volume / volume) as elution solvent. When the starting material was consumed, the reaction mixture was neutralized with a saturated solution of NaHCOg in methanol, then evaporated under reduced pressure to yield a dry residue containing the 26 t fragments

. H

biologically active. The substance BBM-1675C was isolated from the residue by flash column chromatography, on a column of 2 cm (internal diameter) x 10 cm filled with gel, · of Woelm silica (particle size 32-63 microns). The column was eluted with a toluene: acetone mixture (3: 2, volume / volume), recovering 3 ml fractions.

Each fraction was analyzed by thin layer chromatography [silica gel with toluene: acetone (3: 2, volume / volume) as eluent ^, and the spots were visualized with a UV light source of 154 nm and vaporization ceric sulfate (1% ceric sulfate and 2.5% molybolic acid in 10% sulfuric acid).

Fractions 6-12 (Rf value for BBM-1675C = 0.28) were pooled and evaporated to dryness to give 12 mg (35%) of substantially pure BBM-1675C.

The physico-chemical properties of BBM-1675C have been given previously and the UV, infrared, mass, proton NMR and carbon 13 NMR spectra of the compound appear in Figures 1, 3, 5, 7 and 9, respectively.

20 EXAMPLE 2

When the reaction time of the process of Example 1 is extended, the quantity of BBM-1675C decreases, and two new products called compound 3 (R ^ = 0.65) and BBM-1675D (R ^ identical to the baseline) £ thin layer chromatography: silica, toluene-acetone (3: 2, volume / volume) appear and become all the more important as time passes.

The BBM-1675D compound which accompanies the production of *. BBM-1675C was isolated by the chromatography column described in Example 1 by elution with a chloroform: methanol mixture (5: 1, volume / volume). The appropriate fractions pooled and evaporated to dryness to yield 18 mg of substantially pure BBM-1675D from the reaction described in Example 1.

27

The substance BBM-1675D has a major spot at = 0.37 in reverse phase thin layer chromatography (Whatman MKC ^ gF, 200 microns) using 30% water in methanol as eluent and R ^ = 0, 22 by 5 thin layer chromatography on silica gel in normal phase using a chloroform: methanol mixture (5: 0, volume / volume) as eluent.

EXAMPLE 3

A substantial improvement in the yield of BBM-1675D can be obtained by using p-toluenesulfonic acid instead of hydrogen chloride in the chemical hydrolysis of BBM-1675A2 or BBM-1675A ^, as the processes show. Examples 3 and 5, respectively.

A sample of BBM-1675A2 (15.2 mg) was hydrolyzed with a 0.03 molar solution of p-toluene sulfonic acid in methanol (1 ml) at a temperature of about 63 ° C for about one hour. The reaction mixture was then evaporated to dryness under reduced pressure at 30 ° C. BBM-1675D was isolated from the dry residue by flash column chromatography on a column filled with Woelm silica gel (particle size 32-63 microns). The column was eluted with a chloroform: methanol mixture (5: 0.5, volume / volume), and the recovered fractions were analyzed by thin layer chromatography \ silica gel with chloroform: methanol (5: 0.5 , volume / volume) as eluent ^. The chromatography conditions used allowed the separation of the mixture of the two inactive compounds 2 and 3 (7 mg) from the biologically active substance BBM-1675D having an R ^ value of 0.22. The appropriate fractions were pooled and evaporated to dryness to obtain 8 mg of substantially pure BBM-1675D in practically quantitative yield.

The physico-chemical properties of BBM-1675D have been given and the ultraviolet, infrared, mass spectra, * h 28 proton NMR and carbon 13 NMR of this compound appear in FIGS. 2, 4, 6, 8 and 10A and 10B, respectively.

EXAMPLE 4 A sample of BBM-1675A2 (40 mg) was treated with 5 ml of a 0.5 molar solution of hydrogen chloride in methanol at about 50 ° C for about 2 hours according to the general process and the isolation process described in Example 1. After neutralization with NaHCOg and evaporation to dryness, the substance BBM-1675C was isolated from the residue by column chromatography, on a column filled with Woelm silica gel (size 32-63 micron) using a toluene: acetone mixture (3: 2, volume / volume) as the eluent. The appropriate fractions combined and evaporated to dryness to give 8.4 mg of substantially pure BBM-1675C which is a product identical to that isolated in Example 1.

The above chromatographic column was then eluted with a chloroform: methanol mixture (5: 0.25, volume / volume) and the collected fractions were pooled and evaporated to dryness to yield BBM-1675D.

Substance BBM-1675D was then purified using a flash chromatography column with silica gel using a chloroform: methanol mixture (5: 0.5, volume / volume) as the eluent. The required fractions were combined and evaporated to dryness to give 6.3 mg of substantially pure BBM-1675D which is a product identical to that isolated in Example 3.

EXAMPLE 5

A sample of BBM-1675A ^ (49.3 mg) was hydrolyzed with a 0.037 molar solution of p-toluenesulfonic acid in methanol (1.5 ml) at a temperature of about 60 ° C for about 1 hour. 5 o'clock. 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 substance

"I

V \ 29 biologically active BBM-1675D was isolated from the residue by flash column chromatography, on a column filled with Woelm silica gel (particle size 32-63 microns) using a chloroform: methanol mixture (5: 0.25, 5 volume / volume) as eluent. The appropriate fractions were combined and evaporated to dryness to give 27 mg of practically pure BBM-1675D which is a product identical to that isolated in Example 3.

EXAMPLE 6 A sample of BBM-1675C (5.1 mg) was hydrolyzed with a 0.5 molar solution of hydrogen chloride in methanol (1 ml) at about 40-50 ° C overnight.

After neutralization with NaHCOg and evaporation to dryness, the biologically active substance BBM-1675D was isolated from the residue by flash column chromatography on a column filled with Woelm silica gel (particle size 32-63 microns) using a chloroform: methanol mixture (5: 0.25, volume / volume) as eluent.

The appropriate fractions led to practically pure BBM-1675D which is a product identical to that isolated in Example 3.

EXAMPLE 7

When the general process of Examples 1 and 2 is repeated, except that the starting material BBM-1675A ^ is replaced by an equimolar amount of a mixture containing BBM-1675A1 and BBM-1675A2, the substances BBM-1675C and BBM-1675D.

EXAMPLE 8 When the general process of Example 5 is repeated using an equimolar amount of a mixture containing BBM-1675A ^ and BBM-1675A2 as the starting material in place of BBM-1675A ^, substance BBM-1675D.

Note that BBM-1675 C is soluble in methanol, ethanol, ethyl acetate, acetone, tetrahydofuran and chloroform.

BBM 1675 0 is soluble in methanol, ethanol, acetone and tetrahydrofuran and sparingly soluble in chloroform.

Claims (9)

1.- Antibiotic and antitumor consisting of a substantially pure substance, characterized in that it is a substance called BBM-1675C which: 5 (a) is an amorphous solid; (b) is soluble in methanol, ethanol, ethyl acetate, acetone, tetrahydrofuran and chloroform; (c) presents, by thin layer chromatography on silica gel, a value of 0.28 with the toluene: acetone mixture (3: 2, volume / volume) as solvent; (d) has an apparent molecular weight of 855 determined by high resolution accelerated atom bombardment mass spectroscopy; (e) has an ultraviolet absorption spectrum in a methanol solution as shown in FIG. 1, and having the absorption maxima and the absorption capacities at 210 nm (a = 21,770); 274 nm (a = 9.340) and 313 nm (shoulder) (a = 4.190) without significant change by addition of acid or base); 20 (f) has an infrared absorption spectrum (KBr, film) as shown in FIG. 3 and showing the main absorption peaks at 540, 740, 955, 990, 1017, 1065, 1080, 1118,. 1150, 1250, 1305, 1325, 1340, 1370, 1385, 25 1440, 1690, 1705, 1735, 2900, 2920, 2030, 2970, and 3450 cm; (g) has a low resolution mass spectrum as shown in FIG. 5, having a CM + HÜ + of 856; 30 (h) presents a proton magnetic resonance spectrum at 360 MHz in CDCl ^ as represented in FIG. 7 * 31 and presenting signals at 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, dd, J = 10.3, 1.8); 4.61 (1H, d, J = 7.7); 4.25 (1H, s); 4.09 (1H, q, J = 2.6); 5 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, 9.2, 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 (2H, m); 1.37 (3H, d, J = 5.9); 1.32 (3H, d, J = 6.3) ; and 1.08 (6H) parts per 10 million from tetramethylsilane; (i) has a magnetic resonance spectrum of carbon 13 at 90.6 MHz in CDCl ^ as shown in Figure 9 and presenting signals at 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 , 15 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 and 193.1 parts per million from tetramethylsilane. 2. Antibiotic and antitumor in practically pure form, characterized in that it is the substance BBM-1675D which: (a) is an amorphous solid; (b) is soluble in methanol, ethanol, acetone and tetrahydrofuran, and sparingly soluble in chloroform; (C) has an R ^ value of 0.22 by thin layer chromatography on silica gel using a chloroform: methanol mixture (5: 0.5, volume / volume) as solvent and present in thin layer chromatography on reverse phase silica gel an Rf value of 0.37 with a methanol water mixture as solvent (70:30, volume / volume); (d) has an apparent molecular weight of 695 determined by high resolution accelerated atom bombardment mass spectroscopy; (e) has an ultraviolet absorption spectrum in a methanol solution as shown in FIG. 2,, s 32 N and having the following ultraviolet absorption maxima and absorbent powers (a) at 214 nm (a = 27,000), at 274 nm (a = 12,800), and at 325 nm (â = 5,400) without significant change by addition of acid or base; 5 (f) has an infrared absorption spectrum (KBr, film) as shown in FIG. 4 and showing main absorption peaks at 735, 755, 910, 960, 1000, 1020, 1085, 1150, 1195 1250 , 1310, 1335, 1365, 1385, 1445, 1510, 1685, 10 1720, 1735, 2880, 2930, 2960, and 3400 cm "1; (g) presents a mass spectrum in low resolution as shown in figure 6 and having an M + H + of 696; (h) presents a proton 15 magnetic resonance spectrum at 360 MHz in CDClg + 10% CD ^ OD as shown in FIG. 8 and presenting the signals at 6.43 ( 1H, dd, J = 4.4, 10.3); 6.13 (1H, s); 5.81 (1H, d, J = 8.8); 5.70 (1H, d, J = 8 , 8); 5.48 (1H, 6brs); 4.48 (1H, d,, \ J = 8.1); 4.02 (1H, d, J = 2.0); 3.95-3 , 80 (solvent JES); 20 3.77 (1H, t, J = 9.0); 3.70-3.40 (11H, brm); 3.35 (1H, m); 3.28 ( 3H, s); 3.22 (3H, brs); 2.66-2.55 (2H, m); 2.38 (3H, s); 2.23-2.12 (2H, m); 1 , 42, brdt); 1.22 (3H, d, J = 5.9); 0.94 (3H, d, J = 6.6) and 0.87 (3H, d, J = 5.9) parts per million apart ir tetramethylsilane; (I) has a magnetic resonance spectrum of carbon 13 at 90.6 MHz in CDCl ^ + 10% CD ^ OD as shown in FIGS. 10A and 10B and presenting signals at 17.5, 21.6, 22, 2, 23.0, 33.4, 39.2, 46.4, 52.3, 55.8, 62.1, 30 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 parts per million from tetramethylsilane. 3.- Method for obtaining the anti-tumor antibiotic BBM-1675C, defined in claim 1, characterized in that it comprises the hydrolysis of BBM-1675A ^ or BBM-1675A2 by c 33 a mineral or organic acid until an amount of BBM-1675C is formed, then the recovery of BBM-1675C in the reaction medium. 4. Method for obtaining the anti-tumor antibiotic BBM-1675D according to claim 2, characterized in that it comprises the hydrolysis of the substance BBM-1675A ^ or BBM-1675A2 with a mineral or organic acid up to obtaining a substantial amount of BBM-1675D and then recovering BBM-1675D from the reaction medium. 10 5, - Method for obtaining the anti-tumor antibiotic BBM-1675D according to claim 2, characterized in that it comprises the hydrolysis of the substance BBM-1675C according to claim 1 with a mineral or organic acid up to 'obtaining a substantial amount of BBM-1675D and recovering BBM-1675D from the reaction medium. 6. Method for obtaining the anti-tumor antibiotic BBM-1675C, as defined in claim 1, characterized in that it comprises the hydrolysis of a mixture of BBM-1675A ^ and BBM-1675A2 with an acid mineral or organic until a substantial amount of BBM-1675C is obtained and BBM-1675C is recovered from the reaction medium. 7. Method for obtaining the anti-tumor antibiotic BBM-1675D as defined in claim 2, characterized in that it comprises the hydrolysis of a mixture "of BBM-1675A ^ and BBM-1675A2 with an organic or mineral acid until a substantial amount of BBM-1675D is obtained, then recovery of BBM-1675D from the reaction medium. 8. Antimicrobial pharmaceutical composition, characterized in that it comprises an effective amount of BBM-1675C as defined in claim 1 with a pharmaceutically acceptable diluent or vehicle. * “34 9. Entimicrobial pharmaceutical composition, characterized in that it comprises an effective amount of BBM-1675D as defined in claim 2 with a pharmaceutically acceptable diluent or vehicle. 10. Anti-tumor pharmaceutical composition, "characterized in that it comprises an effective amount of BBM-1675C or BBM-1675D with a pharmaceutically acceptable diluent or vehicle.