FI83422B - FOERFARANDE FOER FRAMSTAELLNING AV TUMOERBEKAEMPANDE ANTIBIOTIKA BBM-1675C OCH BBM-1675D. - Google Patents

Description

1 83422

This invention relates to novel anti-tumor antibiotics and to their preparation and isolation.

The antitumor compounds of the present invention have not yet been identified in structure. However, due to their unique physical, chemical and biological properties, the Applicant believes that BBM-1675 C and BBM-1675D antibiotics are new substances.

10 GB Patent Application No. 2,141,425, issued Dec. 19, 1984, discloses that fermentation of Actinomadura verrucosospora strain H964-92 (ATCC 39334) or Actinomadura verrucosospora strain A1327Y (ATCC 39638) yields a new antitumor complex, of which used ni-15 mimetic BBM-1675. The two major bioactive components of the BBM-1675 complex disclosed herein were designated BBM-1675A1 and BBM-1675A2 · BMM-1675A.J and BBM-1675A2, also known as esperamycin A1 and esperamycin A2, respectively. not yet elucidated, but both compo-. . show excellent, antimicrobial and antitumor activity.

U.S. Patent No. 4,530,835, issued July 23, 1985 to Bunge et al., Discloses that 25 unidentified Actinomycete isolates WP-444 (ATCC

39363) fermentation produces antibiotics designated CL-1577A and CL-1577B. The structures of the CL-1577 antibiotics have not yet been elucidated, but the characteristic properties obtained for the antibiotics show that CL-1577A 30 and CL-1577B are similar in structure to the BBM-1675 antibiotics and in particular BBM-1675A and A2, which are . . in GB Patent Application No. 2,141,425, cited above.

R. H. Bunge et al. have presented in the journal 35 j. Antibiotics, 37 (12), 1566-1571 (1984), that Actinomadura sp. Fermentation of (ATCC 39363) yields a bioactive complex 2 83422 from which two major components were isolated, PD 114 759 and PD 115 028. J.H. Wilton et al. presented in J. Chem. Soc. Chem. Commun., 919-920 (1985), a partial study of the structure of the antibiotics PD 114 759 and PD 115 028.

The preparation, isolation and characterization of PD 114 759 and PD 115 028 antibiotics appear to be identical to the aforementioned CL-1577A and CL-1577B antibiotics, respectively.

EP Patent Application No. 95,154, published November 30-10, 1983, discloses that Actinomadura pulveraceus sp. nov. No. 6049 (ATCC 39100) fermentation produces anti-tumor antibiotics designated WS 6049-A and WS 6049-B. The structures of the WS 6049 antibiotics have not yet been elucidated, but the characteristic properties of the antibiotics obtained for the antibiotics indicate that WS 6049-A and WS 6049-B are structurally related to the BBM-1675 antibiotics of GB Patent Application No. 2,141,425 and U.S. Pat. 4,530,835 for CL-1577 antibiotics. However, the spectral data show that neither WS 6049-A nor WS 6049-B is identical to any of the BBM-1675 components. Moreover, the producing organism described in EP patent application No. 95 154 can be clearly distinguished from Actinomadura verrucosospora1 used in GB patent application No. 2 141 425 in terms of its aerial mycelium color by ISP media Nos. 2, 3 and 25 4, its positive milk peptonization and its positive use of D-fructose, D-mannitol, trehalose and cellulose.

The present invention provides novel antitumor antibiotics, referred to herein as BBM-1675C and BBM-1675D, also known as BMY-27305 and BMY-27307, respectively, which are prepared from the bioactive components BBM-1675A. (esperamycin A2) or BBM-1675A2 (esperamycin A2) by selective chemical hydrolysis, which in turn are prepared by culturing a BBM-1675-producing strain of Actinomadura verrusosospora. The bioactive substances BBM-1675C and BBM-1675D can be isolated and purified by conventional chromatographic methods and both substances show excellent antimicrobial and antitumor activity.

Figure 1 shows the ultraviolet light absorption spectrum of BBM-1675C.

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

Figure 3 shows the infrared light absorption spectrum of BBM-1675C (KBr, membrane),

Figure 4 shows the infrared light absorption spectrum (KBr, membrane) of BBM-1675D.

Figure 5 shows the relative abundance mass spectrum of BBM-1675C.

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

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

Figure 8 shows the proton magnetic resonance spectrum of BBM-1675D in a mixture of CDCl 3 + 10% CD 2 OD. . (360 MHz).

13

Figure 9 shows the C-magnetic resonance spectrum of BBM-1675C in CDCl 3 (90.6 MHz).

· * 13

Figure 10A shows the C-magnetic re-: of BBM-1675D. 25 sonication spectra (110-200 ppm) in a mixture of CDCl 3 + 10% CD 2 OD (90.6 MHz).

^ 13

Figure 10B shows the C-magnetic resonance spectrum (0-110 ppm) of BBM-1675D in a mixture of CDCl 3 + 10% CD 3 OD (90.6 MHz).

Figure 11A shows the proton magnetic resonance spectrum of compound 3A (Qf anomer) in CDCl 3 (360 MHz).

. . Figure 11B shows the proto-nominal magnetic resonance spectrum of compound 3B (β-anomer) in CDCl 3 (360 MHz).

The present invention relates to two novel antitumor agents: 35 antibiotics, referred to herein as BBM-1675C and BBM-1675D, respectively, also known as «83422 as BMY-27305 and BMY-27307, wherein said agents are prepared from the bioactive components BBM-1675A1. (esperamycin) or by the selective chemical hydrolysis of BBM-1675A2 (esperamycin A2), which in turn is prepared by culturing the BBM-1675-producing strain of Actinomadura verrucosospora, most preferably Actinomadura verrucosospora (strain AT964) 39334) or Actinomadura verrucosospora strain A1327Y (ATCC 39638) or a mutant thereof. In another aspect, the present invention provides a process for preparing BBM-1675C by selective hydrolysis of bioactive components BBM-1675A.J or BBM-1675A2. In another aspect, the present invention provides a process for preparing BBM-1675D by selective hydrolysis of BBM-1675C-α-15 or, more preferably, from the bioactive components BBM-1675A1 or BBM-1675A2 * of BBM-1675C and BBM-1675D. isolation from the reaction mixture and purification can be performed by conventional chromatographic methods.

The bioactive agents BBM-1675C and BBM-1675d show antimicrobial activity against a broad sector of microorganisms and have also been shown to have inhibitory activity against various murine tumor systems, such as P-388 leukemia and B16. against melanoma.

The agents of the present invention just disclosed can then be used as antimicrobial agents or antitumor agents for controlling mammalian tumors.

During degradation studies to determine the structure of anti-tumor antibiotics, BBM-1675A1 (esperamycin A1) and BBM-1675A2 (esperamycin A2), a mixture of components formed which resulted in the isolation of two inactive fragments of formulas 1 and 2, respectively. and identification. Surprisingly, however, it was found that ... chemical degradation resulted in two bioactive fragments, BBM-1675C and BBM-1675D.

II

5 83422 for phased release. Even more surprising was the finding that two different antibiotics, BBM-1675A1 and -C2, produced the same bioactive fragments as described in Scheme 1. Even more surprising was that the lower molecular weight fragments BBM-1675C and -D (having molecular weights of about 70% and 55% of the molecular weights of the source antibiotics BBM-1675A1 and -A2, respectively) were found to be more potent than BBM-1675A2 and the same than BBM-1675A as antitumor and anti-10 antimicrobial agents.

Scheme 1 BBM-1675A1 (esperamycin

15 V

BBM-16 75C-BBM-1675D

20. . BBM-1675A2 ^ (esperaquizime a2) BBM-1675C and BBM-1675D can be prepared by selective chemical hydrolysis of antibiotic BBM-1675A ^ as shown in Scheme 2 6 83422

Chart 2 ch .__

HO O

5 β CH 2 Cl 2 ^ Ao

BBM-167SA1 —CH 011> BBM-1675C * IMI

(molecular weight J (molecular weight 855) CH 1248) 3 I

OCH3 1 0 1 Compound 1 (molecular weight 425) (mixture of β- and β-anomers)

© H / CH, OH

15 3 20

BBM-16 75D + CH 2 S / OCH.J

(molecular weight \ _ / no 695) /

0H

25 •. Compound 3 (molecular weight. · 192) (mixture of β and β anomers) The starting compound BBM-1675A 2 is prepared according to the method described in GB Patent Application No. 2,141,425, published December 19, 1984. The purified BBM-1675A 2 component is hydrolyzed with mineral acid or an organic acid such as hydrogen chloride, sulfuric acid, p-toluenesulfonic acid, benzenesulfonic acid. or the like in an organic solvent or aqueous organic inert solvent mixture at a temperature between about 0 ° C and the solvent reflux temperature until a substantial amount of the desired BBM-1675C or BBM-1675D is obtained. has formed. The hydrolysis is preferably performed in -C8 alcohol solvents, and most preferably the alcoholysis is performed in methanol. The reaction temperature is not critical, but it is preferred to carry out the reaction between about ambient temperature and 60 ° C, and most preferably between about 40-60 ° C.

The selective hydrolysis of BBM-1675A? Progresses stepwise to initially form the BBM-1675C antibiotic and an inactive fragment of formula 1. Subsequent or continued treatment under hydrolysing conditions results in the release of a mixture of thiosugar 06 * and / 3 anomers of formula 3 and the formation of the antibiotic BBM-1675D. It should be apparent to those skilled in the art that reaction conditions such as time, temperature and altering the acid concentration produces varying relative amounts of antibiotics BBM-1675C and -D. Therefore, it is desirable to monitor the progress of the reaction by thin layer chromatography, as exemplified herein.

If it is desired to prepare only the BBM-1675D antibiotic, it is preferable to perform selective hydrolysis with an organic acid such as p-toluenesulfonic acid as described herein to obtain a quantitative amount of BBM-1675D.

BBM-1675C and BBM-1675D can also be prepared by selective chemical hydrolysis of antibiotic BBM-1675A2, as shown in Scheme 3.

8 83422

Figure 3

o 0H

5 J © CH3 ° VÄ> Ao BBM-16 75A2—> BBM-16 7SC + JC 7 (molecular weight 3 (molecular weight 855)

1248) CH 3 NH

oA ^ ch2 och3 10

Compound 2 (molecular weight 425) (mixture of c <and / J anomers)

ΐβ / CH-OH

15 3 20 CH3 l. \ 1 Jr \ BBM-16 75D + CH3S N. VoCH3 · * · (molecular weight! - \ _ /

J no 695) OH

Compound 3 (molecular weight - 192) (mixture of of and / or J anomers) The starting compound BBM-1675A2 is prepared according to the method disclosed in GB Patent Application No. 2,141,425, published December 19, 1984. Selective hydrolysis of purified BBM-1675Δ2 also proceeds in a gradual manner, initially forming the antibiotic BBM-1675C and an inactive fragment of formula 2. Further treatment under hydrolysing conditions results in the release of a mixture of thio sugar O (r and Of anomers 9 83422 of formula 3 and the formation of the antibiotic BBM-1675D.

The reaction conditions used for the selective chemical hydrolysis of BBM-1675A2 are substantially the same as those used for the hydrolysis of BBM-1675A2 described above. If it is preferred to prepare only the antibiotic BBM-1675D, the hydrolysis of BBM-1675A2 is performed in the same manner as for the preparation of BBM-1675D from BBM-1675A2 until BBM-1675A2 and BBM-1675C10 are substantially complete. changed to BBM-1675D. The hydrolysis is most preferably performed using an organic acid such as p-toluenesulfonic acid.

The finding described herein that the aforementioned BBM-1675C and D antibiotics are composed of two different antibiotics BBM-1675A1 and BBM-1675A2, with the simultaneous loss of two inactive fragments of formulas 1 and 2 and thiocytes of formula 3, respectively. provides an additional advantage of the present invention. Thus, according to a further aspect of the present invention, there is provided a method of mixing a mixture of BBM-1675A1 and -A2. . for selective hydrolysis of BBM-1675C and -D; as shown in Figure 4.

Figure 4

. 25 BBM-1675A1 + BBM-1675A2 —-> BBM-1675C -> BBM-1675D

This advantage is evident from the fact that the relative amounts of BBM-1675A- and -A2 formed during the fermentation process are subject to variability.

The production of BBM-1675C and -D is therefore independent of the relative amounts of BBM-1675A1 and -A2 used as starting materials in the present invention.

As described herein, hydrolysis of the antibiotics BBM-1675A1-, -A2- and '/ · -C results in the release of an inactive thio-: 35 sugar fragment. This thiosugar was isolated for further information on the chemical structure of the antibiotic BBM-1675C and thus 10 83422 BBM-1675A and -A2 antibiotics. The compound of formula III was identified as a mixture of the Cr and β anomers of thiosugar having the structure described in Schemes 2 and 3. Further characterization became possible when the products of alcoholysis, ofr and β-anomers were separated. The proton magnetic resonance spectra (360 MHz) of compound 3A (ε-anomer) and compound 3B (β-anomer) are shown in Figures 11A and 11B, respectively. Based on the analysis of the spectral data, the relative stereochemistry of the 10 thiosugar methyl glycosides of formula 3 was reported with the following formula

OH

CH3 ^ - \ 0 ^ __ ^ - OCH ,.

There is currently no absolute stereochemistry, i.e.

The D or L form has not yet been determined. Thus, based on the current interpretation of the spectral data, it is concluded that the thiosugar of formula 3 (minus the CH 2 group from the anomeric moieties attached during methanolysis) is a component in the structure of the antibiotic BBM-1675C and further is a component in the construction of the BBM-1675A1 and A2 antibiotics used as starting materials.

• Physicochemical properties of BBM-1675C. · Characteristic: amorphous solid

Ultraviolet light absorption spectrum: see Figure 1 Assay device: Hewlett-Packard 8458 30 Solvent: methanol

Concentration: 0,0155 g / l Λ-max absorbances 210 21 770 274 9 340 35 313 sh (shoulder) 4 190 11 83422

No significant change was observed with the use of acid or base.

Infrared light absorption spectrum: see Figure 3 Assay device: Nicolet 5DX FT-IR 5 Main absorption bands (KBr, membrane): 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.

10

Mass spectrum: see Figure 5 Assay device: Finigan 4500 TSQ

Method: ionization by fast atom bombardment (FAB) 15 Molecules- Relative

Matrix m / z rion ion abundance glycerol 856 Jm + HJ + 100% glycerol + NaCl 878 / M + Na7 + 100% dithiothreitol: dithioerythritol (3: 1) (w / w) 856 fH + Uj + 100% Assay device; Kratos MS-50

High power resolution FAB (m / z): / M + H} +

Molecular weight; apparent molecular weight = 856

Based on the mass spectral data presented above); - 25 Elemental composition:, Ν-, Ο.,. S0. . 36 61 3 14 3 (based on the high power separation data above)

Proton magnetic resonance spectrum: see Figure 7 Assay device: WM 360 Bruker 30 Solvent: CDCl 3 12 83422 ΧΗ NMR 360 MHz δ (ppm): δ τ 54 (1H, dd, J = 7f7, 7.0); 6.21 (1 H, 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 J = 7.7); 4.25 (1 H, 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 (3 H, s); 2.77 (1 H, m); 2 ^ 69 (1H, dt, J = 9.9, 5.2); 2.49 (1H, dd, J = 10.3, 2.6); 2.48 (3 H, s); 2.30 (2 H, m); 2.13 (1 H, m); 2.09 (3 H, s); 1.50 (2 H, m); 1.37 (3H, d, 10 J = 5.9); 1.32 (3H, d, J = 6.3); 1.08 (6 H).

1 3 C-magnetic resonance spectrum: see Figure 9 Assay device; WM 360 Bruker Solvent: CDCl 2 15 1 H NMR 90.6 MHz δ (ppm): 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.

Physico - chemical properties of BBM-1675D, - ·· Characteristic: amorphous solid

Ultraviolet light absorption spectrum: see Figure 2 Assay device: Hewlett-Packard 8458 • · 25 Solvent: methanol

Concentration: 0,01 g / l iaks absorbances 214 27 000 274 12 800 30 325 5 400 ·. ·· No significant change was observed with the use of acid or base.

• · Infrared light absorption spectrum: see Figure 4

'·' Assay device: Nicolet 5DX FT-IR

35 Main absorption tapes (KBr), film):

II

13 83422 735, 755, 910, 960, 1000, 1020, 1085, 1150, 1195, 1250, 1310, 1335, 1365, 1385, 1445, 1510, 1685, 1720, 1735, 2880, 2930, 2960, 3400 cm-1 .

5 Mass spectrum: see Figure 6

Assay device: Finigan 4500 TSQ

Method: ionization using fast atom bombardment (FAB)

Matrix: thioglycerol 10 Molecular ion (m / z): / M + H7 + = 696

Relative abundance: 100% Assay device: Kratos MS-50

High power separation FAB (m / z): / M + HJ + = 696.2794 Molecular weight: apparent molecular weight = 695 15 (based on mass spectral data above)

Elemental composition: C29H49N3 ° 12S2 (based on the high power separation data presented above) The correlation of the relative abundances of / M + H7 + and / ^ (M + H) + 2_7 + with their calculated values confirms this al-. . substance composition derived from high power separation FAB measurements.

Proton magnetic resonance spectrum: see Figure 8 Assay device: WM 360 Bruker: 25 Solvent: CDC13 + 10% CD3OD

1 H NMR 360 MHz δ (ppm): 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 solution ~ 30 inks); 3.77 (1H, t, J = 9.0); 3.70 - 3.40 (11H, brm); 3.35 (1 H, m); 3.28 (3 H, s); 3.22 (3 H, br s); 2.66-2. 55 (2H, m); 2.38 (3 H, s); 2.23-2.12 (2 H, m); 1.42 (1H, brdt); 1.22 (3H, d, J = 5.9); 0.94 (3H, d, J = 6.6); 0.87 (3H, d, J = 5r 9).

i4 83422 1 3 C-magnetic resonance spectrum: see figures

10A and 10B

Configuration device: WM 360 Bruker

Solvent: CDCl 3 + 10% CD-iOD

Δ 13 C 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.

10 Biological Properties of BBM-1675 The antimicrobial potency of BBM-1675 was determined for several gram-positive and gram-negative microorganisms. Table 1 below shows data in the form of results obtained by an antimicrobial separation method using the starting BBM-1675A1 component and the BBM-1675C and BBM-1675D agents of the present invention. In the separation method, a paper strip saturated with a solution containing a uniform solution was placed in the growth culture. . seven concentrations of 10 μg / ml of each test compound, and the * · prevention zone obtained from the paper strip is a measure of antibiotic efficacy. As shown in Table 1, BBM-1675C and D showed broad antimicrobial activity:. 25 regions and agents were at least as effective as the BBM-1675A.J component; and in particular BBM-1675C and D were more effective as inhibitors of gram-negative organisms.

II

*

Table I

Antimicrobial activity of BBM-1675 agents 15 83422

Perhaps the zone, e.g.

Co-microorganism BBM- ^ SAj ^ BBM-1675C BBM-16 75D

5

Escherichia coli AS 19 22 52 51

Escherichia coli K 12 13. 36 35

Escherichia coli P 1373 12 34 33

Escherichia coli R Azaserine 14 35 34

Escherichia coli R Netropsin 11 32 32

Escherichia coli R Mitomycin C 12 35 34 io-

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 Actinoleukin 33 35 33

Staphylococcus R Streptonigrin 37 50 48

Staphylococcus faecali s P1377 30 39 3¾ 20 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 '25 Micrococcus luteus 35 57 55. Saccharomyces cerevisiae Petite 22 42 43 R = resistant to the designated antibiotic

Efficacy against P-388 leukemia 30 Tables II and III contain the results of laboratory experiments using CDF1 mice with. . was implanted intraperitoneally with a tumor inoculum comprising 10 P-388 leukemia ascites cells: - which mice were treated with different doses of BBM-35 1675A.J, -C or -D. The agents were administered by intraperitoneal injection. For each dose, groups of six 83,8422 mice were used and the mice were treated with a single dose of the substance on the day after inoculation. Ten saline-treated control mice were included in each set of experiments. The 5 groups treated with BBM-1675A® shown in Table III were included in the experiment as a direct control. The test protocol was maintained for 30 days and the mean survival time in days was determined for each group of mice and the number of survivors was recorded at the end of the five-day period. Mice were weighed-10 before treatment and again on the fourth day. Weight gain was considered a measure of drug toxicity. Mice weighing 20 g each were used and weight loss of up to about 2 g was not considered excessive. Solvent-treated control animals generally died within nine days. The results were determined as% T / C, which is the ratio of the mean survival time of the treated group to the mean survival time of the solvent-treated control group multiplied by 100. A power expressed as% T / C equal to or greater than 125 indicates that the achieved-: significant antitumor activity. The separation results in Table II show a level of antitumor efficacy of BBM-1675C that was not initially expected. Table III shows the results obtained by direct comparison of BBM-1675A ^ (esperamycin A ^) and BBM-1675C and BBM-1675D. The data suggest that the BBM-1675C is approximately equivalent to the BBM-1675A in potency and antitumor potency and is not time dependent, while the BBM-30 1675D is only slightly less potent.

. .. The present invention further shows that the same substances BBM-1675C and BBM-1675D can also be obtained from the BBM-1675A2 (esperamycin A2) component. Comparing the data reported in this specification for BBM-1675C 35 and BBM-1675D with the data presented in published GB patent application il 17 83422 No. 2 141 425 for component BBM-1675A2, it is surprisingly found that the substances BBM-1675A -1675C and -D are more potent antitumor agents than the source BBM-1675A2 ~ component from which they are derived.

5 Table II

Efficacy of BBM-1675C in P-388 Leukemia (Day 1 Treatment)

Power AWC Alive

Dose, IP MST MST g in ^ q Compound mq / kq / inj. pv% T / C pv 4 pv 5_ 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 15 0.003125 13.5 150 -2.5 6/6

Solvent - 9.0 100 10/10 g

Tumor inoculum: 10 gastric cells implanted intraperitoneally-20 tonally

Oriental animal: male CDF1 mice

Judging Criterion: Median Survival Time (MST)

Efficacy:% T / C = (MST treated / MST ratio) x 100 25 Criteria:% T / C £ 125 was considered significant, anti-tumor efficacy AWC: average weight change (treated ratio) in grams (day 4)

Table III

Efficacy of BBM-1675 agents in P-388 leukemia 18 83422

Power AWC Alive Treatment Dose, IP MST MST g available g Compound timing mq / kq / inj. pv. % T / C day 4 day 5_ BBM-16 75 day 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 day 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 0.3 676 20 0, 0004 11.5 128 0.8 6/6 0.0002 11.0 122 1.4 6/6 f · 0.0001 11.0 122 0.8 6/6 0.00005 10.5 117 1.3 6 / 6 19 83422

Table III (continued) AWS Alive Treatment Dose, IP MST m ST g

Compound timing mq / kq / inj. pv% T / C pv 4 days 5_ 5 BBM-1675D day 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 1Q 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 Day 1-5 0.0032 16.0 178 -1, 3 6/6 15 il * 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 20 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 ·· Solvent - 9.0 100 2.4 10/10. .. ^

Tumor inoculum: 10 ul of peritoneal cells implanted intraperitoneally

Host animal: female CDF ^ mice Judging Criterion: Mean Survival Time MST 30

Efficacy:% T / C = (MST treated / MST ratio) x 100 Criteria:% T / C * 125 was considered a significant anti-tumor efficacy AWC: average weight change 35 grams treated (day 4) 20 8 3 4 2 2

Efficacy against B16 melanoma

Table IV contains the results of tumor control experiments using mouse-grown B16 melanoma. BDF 2 mice were used and inoculated subcutaneously with a tumor implant. The minutes were kept for 60 days. For each dose tested, groups of 10 mice were used and the mean survival time of each group was determined. Control animals were inoculated in the same manner as the experimental animals and treated with an injection of solvent and no drug at all, and these animals had an average survival time of 22.5 days. For each dosing amount, experimental animals were treated with the test compound by intraperitoneal injection on days 1, 5, and 9. A potency expressed as% T / C of equal to or greater than 125 indicates that a significant antitumor effect was achieved. The results shown in Table IV show that, in a direct comparison, BBM-1675C was also effective in treating mice with B16 melanoma and was approximately equivalent in intensity to BBM-1675Aβ.

Table IV

Efficacy of BBM-1675 agents in B16 melanoma 21 83422

Power ÄWC Alive

Dose, xp MST MST 9 in 5 Compound mq / kq / inj. pv% T / C pv_12 Day 10_ ΒΒΜ-1675Αχ 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 1 0 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 15 0, 00005 27.5 122 0.8 10/10 0.000025 25.0 111 0.5 10/10

Solvent - 22.5 100 0.3 10710 20 Tumor inoculum: 0.5 ml of 10% "porridge", intraperitoneally

Host animal: female BDF ^ mice

Judging by: MST (= media Survival time) average survival time 25 Power:% T / C = (MST processed / MST compared to you) x 100

Criteria:% T / C £ 125 was considered significant antitumor activity AWC: (average weight change) average weight change (treated ratio) in grams (day 12) 30 As shown by the antimicrobial and mouse tumor data presented above, BBM-1675C and BBM-1675C 1675D are then useful as antibiotics for the therapeutic treatment of mammals and other animals in infectious diseases, as well as as antitumor agents for the therapeutic prevention of the growth of tumors in mammals.

22 8 3 4 2 2

The present invention thus provides a method of therapeutically treating a living organism suffering from a microbial infection or malignancy, comprising administering to said subject an effective antimicrobial or antitumor dose of BBM-1675C or BBM-1675D or a pharmaceutical composition thereof. .

The invention encompasses within its scope pharmaceutical compositions comprising an effective antimicrobial or antitumor amount of BBM-1675C or BBM-1675D in association with an inert, pharmaceutically acceptable carrier! with a diluent. Such compositions may also contain other active antimicrobial or antitumor agents and may be formulated into any pharmaceutical form suitable for the desired route of administration. Examples of such compositions include solid compositions for oral administration such as tablets, capsules, pills, powders and granules, liquid compositions for oral administration such as solutions, suspensions, syrups or elixirs, and preparations for parenteral administration such as sterile non-aqueous solutions, suspensions or emulsions. They may also be prepared in the form of sterile, solid compositions which can be dissolved in sterile water, physiological saline, or other sterile injectable medium immediately before use.

For use as an antimicrobial agent, BBM-30 1675C or BBM-1675D or a pharmaceutical composition thereof is administered in such a way that the concentration of the active ingredient is higher than the minimum contraceptive concentration for the particular organism to be treated. For use as an antitumor agent, one skilled in the art can readily determine the optimal dosages and treatment guidelines for BBM-1675C or BBM-1675D for the 23 83422 mammal in question. Of course, it should be noted that the particular dose of BBM-1675C or BBM-1675D used will vary depending on the particular composition prepared, the mode and site of administration, the subject and the disease being treated. Many factors that affect the effect of a drug must be considered, such as the patient's age, weight, sex, diet, time of administration, route of administration, rate of excretion, patient's condition, drug combinations, reaction sensitivities, and severity of the disease. The drug can be administered continuously or intermittently within the maximum tolerated dose. Optimal application rates for a particular combination of conditions can be determined by those skilled in the art using standard dosing assays, taking into account the instructions above.

The following examples are intended only to illustrate the invention without limiting its scope in any way.

Chemical Preparation and Isolation of BBM-1675C and BBM-1675D Example 1 A sample of BBM-1675A.J (50 mg) was dissolved in 2.5 ml of methanol and treated with 2.5 ml of hydrogen chloride. 1 molar methanol solution. The reaction was allowed to proceed at about 50 ° C and the disappearance of starting material (about 25 over 30 minutes) was monitored every 5-10 minutes by thin layer chromatography (TLC) using silica gel plates (Analtech, 250 microns, GF) and toluene as eluent. : acetone (3: 2, v / v). After the starting material was consumed, the reaction mixture was neutralized with saturated methanolic NaHCO 3 solution, then evaporated under reduced pressure to give a dry residue containing bioactive fragments. BBM-1675C was isolated from the residue by flash column chromatography using a column-35 with an inner diameter of 2 cm and a length of 10 cm packed on Woelm silica gel (particle size 24 83422 32-63 microns). The column was eluted with toluene: acetone (3: 2, v / v) and 3 ml fractions were collected. Each fraction was analyzed by TLC on silica gel eluting with toluene: acetone (3: 2, v / 5) and the TLC spots were visualized with a UV 254 nm light source and cerium sulfate spray (1% cerium sulfate and 2.5 % molybolic acid in 10% sulfuric acid). Fractions 6-12 (R f value for BBM-1675C is 0.28) were combined and evaporated to dryness to give 12 mg (35%) of substantially pure BBM-1675C.

The physicochemical properties of BBM-1675C are apparent from the description of the invention, and the ultraviolet, infrared, mass, 1 H-NMR, and NMR spectra of the compound are shown in Figures 1, 3, 5, 7, and 9, respectively.

15 Example 2

As the reaction time of the process in Example 1 is extended, the amount of BBM-1675C decreases and two new products, termed Compound 3 (R 2 = 0.65) and BBM-1675D (R 2 remains baseline) / TLC: silica-20, are detected. po, toluene-acetone (3: 2, v / v) mixture and they become more striking over time.

BBM-1675D, which is usually formed during the preparation of BBM-1675C, was isolated from the chromatographic column described in Example 1 by eluting with p-chloroform: methanol (5: 1, v / v). The appropriate fractions were combined and evaporated to dryness to give 18 mg of substantially pure BBM-1675D from the reaction described in Example 1.

BBM-1675D gives one major spot with an Rf value = 0.37 by reverse phase TLC (Whatman MKCl 2 gF, 200 microns) using 30% aqueous methanol in eluent to give Rf value = 0.22 in normal-phase silica gel TLC using chloroform-ethanol (5: 0.5, v / v) as eluent.

Il 25 83422

Example 3

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 BBM-1675A2 or BBM-1675A2, as described in Examples 3 and 5, respectively.

A sample of BBM-1675A2tn (15.2 mg) was hydrolyzed with a 0.03 molar methanolic solution of p-toluenesulfonic acid (1 mL) at about 63 ° C for about one hour. The reaction mixture was then evaporated to dryness under reduced pressure at about 30 ° C. BBM-1675D was isolated from the dry residue by flash column chromatography using a column packed with Woelm silica gel (particle size 32-63 microns). The column was eluted with chloroform: ethanol (5: 0.5, v / v / v) and the combined fractions were analyzed by TLC / silica gel using chloroform: methanol (5: 0.5 v / v) as eluent. the mixture was ^. The chromatographic conditions used allowed the separation of a mixture of inactive compounds 2 and 3 (7 mg) from bioactive BBM-1675D-α-20 with an Rf value of 0.22. The appropriate fractions were combined and evaporated to dryness to give 8 mg of substantially pure BBM-1675D, which corresponded to an almost quantitative yield.

The physicochemical properties of BBM-1675D are shown in the description of the invention and the ultraviolet, 113 infrared, mass, 1 H-NMR and C-NMR spectra of the compound are shown in Figures 2, 4, 6, 8 and combined Figures 10A, respectively. and 10B.

Example 4 A sample of BBM-1675A2 (40 mg) was treated with 5 ml of a 0.5 molar methanolic solution of hydrogen chloride at about 50 ° C for about two hours according to the general preparation method and isolation procedure set forth in Example 1. After neutralization with NaHCO 3 and evaporation to dryness, BBM-1675C was isolated from the residue by flash column chromatography using a column packed with Woelm silica gel (particle size 32-63 microns) and toluene: acetone (3: 2) as eluent (3: 2). vol./vol.) - mixture. The appropriate fractions were combined and evaporated to dryness to give 8.4 mg of substantially pure BBM-1675C, which is identical to the product isolated in Example 1.

The above chromatographic column was then eluted with chloroform-methanol (5: 0.25 v / v) and the combined fractions were combined and evaporated to dryness to give BBM-1675D. BBM-1675D was then further purified using another flash column packed with silica gel eluting with chloroform: methanol (5: 0.5, v / v). 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 BBM-1675A.J (49.3 mg) was hydrolyzed with a 0.035 molar methanolic solution of p-toluene-20-sulfonic acid (1.5 ml) at 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 inactive compounds 1 and 3. Bioactive BBM-1675D was isolated from the residue by flash column chromatography using a packed column. Woelm's silica gel (particle size 32-63 microns) and chloroform: methanol (5: 0.2, v / v) as eluent. The appropriate fractions were combined and evaporated to dryness to give 27 mg of substantially pure BBM-1675D, which was identical to the product isolated in Example 3.

Example 6 A sample of BBM-1675C (5.1 mg) was hydrolyzed with a 0.5 M methanolic solution of hydrogen chloride (1 mL) at about 35-40-50 ° C overnight. After neutralization with NaHCO 3 and evaporation to dryness, the bioactive BBM-1675D-27 83422 was isolated from the residue by flash column chromatography using a column packed with Woelm silica gel (particle size 32-63 microns) and chloroform: methanol (5: 25, methanol) as eluent. /til.)-seosta. The appropriate 5 fractions gave essentially pure BBM-1675D, which was identical to the product isolated in Example 3.

Example 7

When the general procedure of Examples 1 and 2 is repeated, except that the starting material 10 BBM-1675A.J is replaced by an equimolar amount of a mixture containing BBM-1675A1 and BBM-1675A2, BBM-1675C and BBM are obtained. -1675D materials.

Example 8

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

Claims (2)

28 83 422 A process for the preparation of the antitumor antibiotic BBM-1675C, characterized in that hydrolysis of BBM-1675A !, BBM-1675A2 or a mixture thereof with a mineral acid or an organic acid is carried out until a substantial amount of BBM-1675C is formed and BBM-1675C is then recovered from the reaction medium, which in its substantially pure form has the following characteristics: (a) it appears as an amorphous solid; (b) it is soluble in methanol, ethanol, ethyl acetate, acetone, tetrahydrofuran and chloroform; (C) it gives an Rf value of 0.28 in silica gel thin layer chromatography using toluene: acetone (3: 2, v / v) as the solvent system; (d) its apparent molecular weight is 855 as determined by a high-resolution FAB mass spectroscopy: (e) its ultraviolet light absorption spectrum in methanol solution is substantially as shown in Figure 1 and shows the ultraviolet light absorption maxima and absorbances at 210 nm ), 274 nm 25 (a * 9 340) and 313 nm (shoulder) (a = 4 190) without any significant change on addition of acid or base; (f) its infrared light absorption spectrum (KBr, film) is substantially as shown in Figure 3 and shows the main absorption peaks at 540, 740, 955, 990, 1017, 1065, 1080, 1118, 1150, 1250, 1305, 1325, 1340 , 1370, 1385, 1440, 1690, 1705, 1735, 2900, 2920, 2930, 2970 and 3450 cm -1; (g) its mass spectrum obtained by a low power separator is substantially as shown in Fig. 5 29 8 3 4 2 2 and shows a molecular ion [M + H] + of 856; (h) its proton magnetic resonance spectrum (360 MHz) in CDCl 3 is substantially as shown in Figure 7 and shows signals at δ 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), 3.97 (1H, t, J-9.6), 3.92-3.53 (10H), 3.45 (1H, dt, 10 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 (3 H, s); 2.30 (2 H, m); 2.13 (1 H, m); 2.09 (3 H, s); 1.50 (2 H, m); 1.37 (3H, d, J = 5.9); 1.32 (3H, d, J = 6.3); and 1.08 (6H) ppm; tetramethylsilane; (I) the magnetic resonance spectrum (90.6 MHz) of its carbon-13 in CDCl 3 is substantially as shown in Figure 9 and shows the 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, 57.7, 62.4, 64.7, 20 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 ppm; tetramethylsilane. 2. A process for the preparation of the antitumor antibiotic BBM-1675D, characterized in that hydrolysis of BBM-1675AJ, BBM-1675A2 or a mixture thereof or BBM-1675C with a mineral acid or an organic acid until a substantial amount of BBM-1675D has formed and BBM-1675D is then recovered from the reaction medium, which in its substantially pure form has the following characteristics: (a) it appears as an amorphous solid; (b) it is soluble in methanol, ethanol, acetone and tetrahydrofuran and sparingly soluble in chloroform; 35 (c) gives an Rf value of 0.22 in silica gel thin layer chromatography using a chloroform: methanol (5: 0.5, v / v) solvent system and gives an Rf value of 0 in reverse phase silica gel thin layer chromatography, 37 using methanol: water (70:30 5 v / v) as the solvent system; (d) its apparent molecular weight is 695 as determined by high resolution FAB mass spectroscopy; (e) its ultraviolet light absorption spectrum in the matano-10 solution is substantially as shown in Figure 2 and shows ultraviolet light absorption maxima and absorbances at 214 nm (a = 27,000), 274 nm (a = 12,800) and 325 nm (a = 5,400 ), without any significant change in the addition of acid or base; (F) its infrared light absorption spectrum (KBr, membrane) is substantially as shown in Figure 4 and shows major absorption peaks at 735, 755, 910, 960, 1000, 1020, 1085, 1150, 1195, 1250, 1310, 1335, 1365, 1385, 1445, 1510, 1685, 20, 1720, 1735, 2880, 2930, 2960 and 3400 cm-1; (g) its mass spectrum obtained by a low power separator is substantially as shown in Figure 6 and shows a molecular ion [M + H] + of 696; (h) its proton-magnetic resonance spectrum (360 25 MHz) in the (CDCl 3 + 10% CD 3 OD) mixture is substantially as shown in Figure 8 and shows signals at 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 (1 H, m); 3.28, (3 H, s,); 3.22 (3 H, br s); 2.66-2. 55 (2H, m); 2.38 (3 H, s); 2.23-2.12 (2 H, m); 1.42 (1H, brdt); 1.22 (3H, d, J-5.9); 0.94 35 (3H, d, J = 6.6); δ 0.87 (3H, d, J = 5.9) ppm; tetrail 3i 83422 from methylsilane; (i) its carbon-13 magnetic resonance spectrum (90.6 MHz) in the (CDCl 3 + 10% CD 3 OD) mixture is substantially as shown in Figure 10 (Figures 10A + 10B) and shows signals at position 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; tetramethylsilane. 32 8 3 4 2 2