Antitumor antibiotics (LL-E33288 complex)

Abstract

Antibacterial and antitumor agents designated LL-E33288 complex and their production by new strains of Micromonospora echinospora ssp. calichensis NNRL-15839, NRRL-15975 and NRRL-18149, are disclosed.

Description

Claims

1. A LL-E33288.alpha.-Br complex prepared by extracting LL-E33288.alpha..sub.1 -Br, LL-E33288.alpha..sub.2 -Br, LL-E33288.alpha..sub.3 -Br, LL-E33288.alpha..sub.4 -Br, LL-E33288.beta..sub.1 -Br, LL-E33288.beta..sub.2 -Br and LL-E33288.gamma..sub.1 -Br from the fermentation broth of Micromonospora echinospora NRRL-15839, NRRL-15975 or NRRL-18149 with an organic solvent and

purifying the extracted mixture by selective precipitation from lower hydrocarbons,

separating LL-E33288.alpha..sub.1 -Br, LL-E33288.alpha..sub.2 -Br, LL-E33288.alpha..sub.3 -Br and LL-E33288.alpha..sub.4 -Br from LL-E33288.beta..sub.1 -Br, LL-E33288.beta..sub.2 -Br and LL-E33288.gamma..sub.1 -Br by a series of column chromatography and

assaying and analyzing the LL-E33288.alpha.-Br complex by thin layer chromatography.

2. A compound LL-E33288a.sub.2 -Br, having the following R.sub.f values in the indicated solvent systems on TLC on silica gel sheets:

(a) ethyl acetate saturated with 0.1M aqueous potassium dihydrogen phosphate, R.sub.f =0.61;

(b) 3% isopropanol in ethyl acetate saturated with 0.1M aqueous potassium dihydrogen phosphate, R.sub.f =0.75; and

(c) ethyl acetate:methanol (95:5), R.sub.f =0.73;

and having the following structure: ##STR5##

3. A compound LL-E33288a.sub.3 -Br having the following R.sub.f values in the indicated solvent systems on TLC on silica gel sheets:

(a) ethyl acetate saturated with 0.1M aqueous potassium dihydrogen phosphate, R.sub.f =0.55;

(b) 3% isopropanol in ethyl acetate saturated with 0.1M aqueous potassium dihydrogen phosphate, R.sub.f =0.69; and

(c) ethyl acetate:methanol (95:5), R.sub.f =0.61;

and having the following structure: ##STR6##

4. A compound LL-E33288.beta..sub.1 -Br,

(a) having an approximate elemental analysis: C 48.6; H 5.6; N 2.9; S 9.1 and Br 5.5;

(b) having a melting point: 146.degree.-150.degree. C. (dec.);

(c) having a specific rotation: [.alpha.].sub.D.sup.26 =-49.degree..+-.10.degree. (0.1%, ethanol);

(d) having ultraviolet absorption spectra as shown in FIG. I of the drawings;

(e) having an infrared absorption spectrum as shown in FIG. II of the drawings;

(f) having a proton magnetic resonance spectrum as shown in FIG. III of the drawings;

(g) having a carbon-13 magnetic resonance spectrum as shown in FIG. IV of the drawings with significant peaks at:

______________________________________17.60(q) 17.64(q) 18.9(q) 19.7(q)22.4(q) 22.8(q) 23.5(q) 34.3(t)36.9(t) 39.2(t/d) 47.8(d) 51.7(q)52.7(q) 54.6(d) 56.3(q) 57.2(q)57.8(d) 61.0(q/d) 61.7(d) 62.4(t)66.9(d) 68.4(d) 69.1(d) 69.7(d)70.2(d) 71.1(d) 71.9(d) 72.1(s)76.1(d) 81.0(d) 83.3(s) 88.2(s)97.4(d) 99.7(d) 100.8(s) 102.5(d)115.1(s) 123.4(d) 124.4(d) 126.5(d)130.2(s) 130.8(s) 144.6(s) 149.3(s)149.5(s) 191.7(s) 192.4(s) and______________________________________

(h) having the following R.sub.f values in the indicated solvent systems on TLC on silica gel sheets:

(i) ethyl acetate saturated with 0.1M aqueous potassium dihydrogen phosphate, R.sub.f =0.24;

(ii) 3% isopropanol in ethyl acetate saturated with 0.1M aqueous potassium dihydrogen phosphate, R.sub.f =0.35;

(iii) ethyl acetate:methanol (95:5), R.sub.f =0.36.

(i) having a molecular weight: 1333/1335, respectively for .sup.79 Br/.sup.81 Br;

(j) having a molecular formula: C.sub.56 H.sub.76 N.sub.3 O.sub.21 S.sub.4 Br;

(k) and having the structure ##STR7##

5. A compound LL-E33288.beta..sub.2 -Br, having the following R.sub.f values in the indicated solvent systems on TLC on silica gel sheets:

(a) ethyl acetate saturated with 0.1M aqueous potassium dihydrogen phosphate, R.sub.f =0.32;

(b) 3% isopropanol in ethyl acetate saturated with 0.1M aqueous potassium dihydrogen phosphate, R.sub.f =0.41; and

(c) ethyl acetate:methanol (95:5), R.sub.f =0.45 prepared by extracting LL-E33288.alpha..sub.1 -Br, LL-E33288.alpha..sub.2 -Br, LL-E33288.alpha..sub.3 -Br, LL-E33288.alpha..sub.4 -Br, LL-E33288.beta..sub.1 -Br, LL-E33288.beta..sub.2 -Br and LL-E33288.gamma..sub.1 -Br from the fermentation broth of Micromonospora echinospora NRRL-15839, NRRL-15975 or NRRL-18149 with an organic solvent and

purifying the extracted mixture by selective precipitation from lower hydrocarbons,

separating LL-E33288.beta..sub.1 -Br, LL-E33288.beta..sub.2 -Br and LL-E33288.gamma..sub.1 -Br from LL-E33288.alpha..sub.1 -Br, LL-E33288.alpha..sub.2 -Br, LL-E33288.alpha..sub.3 -Br, and LL-E33288.alpha..sub.4 -Br by a series of column chromatography,

assaying, analyzing and working up the LL-E33288.beta.-Br complex containing LL-E33288.gamma..sub.1 -Br and

separating LL-E33288.beta..sub.2 -Br from the LL-E33288.beta.-Br complex containing LL-E33288.gamma..sub.1 -Br by column chromatography.

6. A compound LL-E33288.gamma..sub.1 -Br

(a) having ultraviolet absorption spectra as shown in FIG. V of the drawings;

(b) having an infrared absorption spectrum as shown in FIG. VI of the drawings;

(c) having a proton magnetic resonance spectrum as shown in FIG. VII of the drawings; and

(d) having the following R.sub.f values in the indicated solvent systems on TLC on silica gel sheets:

(i) ethyl acetate saturated with 0.1M aqueous potassium dihydrogen phosphate, R.sub.f =0.18;

(ii) 3% isopropanol in ethyl acetate saturated with 0.1M aqueous potassium dihydrogen phosphate, R.sub.f =0.28;

(iii) ethyl acetate:methanol (95:5), R.sub.f =0.27,

(e) having a carbon-13 magnetic resonance spectrum as shown in FIG. VIII of the drawings with significant peaks at:

______________________________________14.4 17.6 17.9 19.019.7 -- 22.8 ---- 34.0 37.6 39.542.1 -- 51.6 52.754.1 56.3 57.3 --59.3 61.1 61.8 61.967.2 68.18 68.23 69.770.1 70.8 71.1 71.771.8 76.1 -- 81.082.9 88.4 -- 97.8100.0 100.2 101.3 103.0115.3 123.0 124.9 126.9130.4 131.1 131.8 138.0144.7 -- 149.5 149.6155.6 192.5 192.9______________________________________

(f) having a molecular formula: C.sub.55 H.sub.74 N.sub.3 O.sub.21 S.sub.4 Br;

(g) having a molecular weight: 1319/1321, respectively for .sup.79 Br/.sup.81 Br;

(h) and having the structure ##STR8##

7. A compound LL-E33288.alpha..sub.1 -I

(a) having the following R.sub.f values in the indicated solvent systems on TLC on silica gel sheets:

(i) ethyl acetate saturated with 0.1 M aqueous potassium dihydrogen phosphate, R.sub.f =0.67;

(ii) 3% isopropanol in ethyl acetate saturated with 0.1M aqueous potassium dihydrogen phosphate, R.sub.f =0.80; and

(iii) ethyl acetate:methanol (95:5), R.sub.f =0.80;

(b) having a molecular weight: 1145 prepared by extracting LL-E33288.alpha..sub.1 -I, LL-E33288.alpha..sub.2 -1, LL-E33288.alpha..sub.3 -I, LL-E33288.beta..sub.1 -I, LL-E33288.beta..sub.2 -I, LL-E33288.gamma..sub.1 -I and LL-E33288.delta..sub.1 -I from the fermentation broth of Micromonospora echinospora NRRL-15839, NRRL-15975 and NRRL-18149 with an organic solvent,

purifying the extracted mixture by selective precipitation from lower hydrocarbons,

separating LL-E33288.alpha..sub.1 -I from a complex including LL-E33288.alpha..sub.2 -I, LL-E33288.alpha..sub.3 -I, LL-E33288.beta..sub.1 -I, LL-E33288.beta..sub.2 -I, LL-E33288.gamma..sub.1 -I and LL-E33288.delta..sub.1 -I by a series of column chromatography.

8. A compound LL-E33288.alpha..sub.2 -I

(a) having the following R.sub.f values in the indicated solvent systems on TLC on silica gel sheets:

(i) ethyl acetate saturated with 0.1M aqueous potassium dihydrogen phosphate, R.sub.f =0.61;

(ii) 3% isopropanol in ethyl acetate saturated with 0.1M aqueous potassium dihydrogen phosphate, R.sub.f =0.75; and

(iii) ethyl acetate:methanol (95:5), R.sub.f =0.73;

(b) containing only the following elements: C, H, N, O, S and I;

(c) having a molecular weight: 1207;

(d) having ultraviolet absorption spectra as shown in FIG. IX of the drawings;

(e) having an infrared absorption spectrum as shown in FIG. X of the drawings;

(f) having a proton magnetic resonance spectrum as shown in FIG. XI of the drawings;

(g) having a carbon-13 magnetic resonance spectrum as shown in FIG. XII of the drawings with significant peaks at:

______________________________________17.7 56.3 71.8 122.718.8 60.7 73.7 125.122.7 61.4 79.0 126.324.7 60.7 82.6 127.133.1 67.7 87.4 133.137.4 67.9 85.4 137.039.6 69.6 98.0 137.141.4 69.7 100.4 149.251.3 70.3 100.1 151.553.3 71.0 100.8 192.853.8 73.4 98.2 193.2______________________________________

(h) having a molecular formula: C.sub.48 H.sub.62 N.sub.3 O.sub.17 S.sub.4 I.sub.9 ;

(i) and having the structure ##STR9##

9. A compound LL-E33288.alpha..sub.3 3-I

(a) having the following R.sub.f values in the indicated solvent systems on TLC on silica gel sheets:

(i) ethyl acetate saturated with 0.1M aqueous potassium dihydrogen phosphate, R.sub.f =0.55;

(ii) 3% isopropanol in ethyl acetate saturated with 0.1M aqueous potassium dihydrogen phosphate, R.sub.f =0.69; and

(iii) ethyl acetate:methanol (95:5), R.sub.f =0.61;

(b) having a molecular weight: 1210;

(c) having ultraviolet absorption spectra as shown in FIG. XIII of the drawings;

(d) having an infrared absorption spectrum as shown in FIG. XIV of the drawings;

(e) having a proton magnetic resonance spectrum as shown in FIG. XV of the drawings;

(f) having a carbon-13 magnetic resonance spectrum as shown in FIG. XVI of the drawings with significant peaks at:

______________________________________17.5(q) 69.5(d) 103.1(d)18.0(q) 70.0(d) 123.4(d)19.0(q) 70.1(d) 124.7(d)22.7(q) 70.8(d) 127.3(d)25.3(q) 70.9(d) 131.137.4(t) 72.1(s) 130.4(s)39.2(t) 71.3(d) 133.5(s)51.5(d) 74.5(d) 136.8(s)53.5(q) 80.8(d) 143.0(s)53.7(t) 83.1(s) 145.857.2(q) 87.6(s) 150.5(s)61.0(q) 93.7(s) 151.6(s)61.8(q) 98.6(s) 154.7(s)67.1(d) 100.1(d) 192.4(s)67.3(d) 101.0(s) 192.6(s)68.0(d) 103.4(d)______________________________________

(g) having a molecular formula: C.sub.47 H.sub.59 N.sub.2 O.sub.19 S.sub.4 I;

(h) and having the structure ##STR10##

10. A compound LL-E33288.beta..sub.1 -I

(a) having the following R.sub.f values in the indicated solvent systems on TLC on silica gel sheets:

(i) ethyl acetate saturated with 0.1M aqueous potassium dihydrogen phosphate, R.sub.f =0.24;

(ii) 3% isopropanol in ethyl acetate saturated with 0.1M aqueous potassium dihydrogen phosphate, R.sub.f =0.35; and

(iii) ethyl acetate:methanol (95:5), R.sub.f =0.36;

(b) having ultraviolet absorption spectra as shown in FIG. XVII of the drawings;

(c) having an infrared absorption spectrum as shown in FIG. XVIII of the drawings;

(d) having a proton magnetic resonance spectrum as shown in FIG. XIX of the drawings;

(e) having a carbon-13 magnetic resonance spectrum as shown in FIG. XX of the drawings with significant peaks at:

______________________________________-- 17.5 17.6 18.9-- 22.4 22.8 23.425.4 34.3 36.9 39.2-- 47.9 51.6 52.854.8 56.3 57.2 57.960.9 -- 61.6 62.267.0 68.4 68.4 69.169.6 70.4 71.1 71.872.2 76.2 -- 80.883.3 88.1 93.6 97.499.6 99.6 -- 102.6112.4 123.4 124.4 126.4-- -- 133.4 ---- -- -- ---- 192.2 192.6 --______________________________________

(f) having a molecular formula: C.sub.56 H.sub.76 N.sub.3 O.sub.21 S.sub.4 I;

(g) having a molecular weight: 1381;

(h) and having the structure ##STR11##

11. A compound LL-E33288.beta..sub.2 -I having the following R.sub.f values in the indicated solvent systems on TLC on silica gel sheets:

(a) ethyl acetate saturated with 0.1M aqueous potassium dihydrogen phosphate, R.sub.f =0.32;

(b) 3% isopropanol in ethyl acetate saturated with 0.1M aqueous potassium dihydrogen phosphate, R.sub.f =0.41; and

(c) ethyl acetate:methanol (95:5), R.sub.f =0.45 prepared by

extracting LL-E33288.alpha..sub.1 -I, LL-E33288.alpha..sub.2 -I, LL-E33288.alpha..sub.3 -I, LL-E33288.beta..sub.1 -I, LL-E33288.beta..sub.2 -I, LL-E33288.gamma..sub.1 -I and LL-E33288.delta..sub.1 -I from the fermentation broth of Micromonospora echinospora NRRL-15839, NRRL-15975 and NRRL-18149 with an organic solvent,

purifying the extracted mixture by selective precipitation from lower hydrocarbons; and

separating LL-E33288.beta..sub.2 -I from LL-E33288.alpha..sub.1 -I, LL-E33288.alpha..sub.2 -I, LL-E33288.alpha..sub.3 -I, LL-E33288.beta..sub.1 -I, LL-E33288.gamma..sub.1 -I and LL-E33288.delta..sub.1 -I by a series of column chromatography.

12. A compound LL-E33288.gamma..sub.1 -I

(a) having the following R.sub.f values in the indicated solvent system on TLC on silica gel sheets:

(i) ethyl acetate saturated with 0.1M aqueous potassium dihydrogen phosphate, R.sub.f =0.18;

(ii) 3% isopropanol in ethyl acetate saturated with 0.1M aqueous potassium dihydrogen phosphate, R.sub.f =0.28; and

(iii) ethyl acetate: methanol (95:5), R.sub.f =0.27;

(b) containing only the following elements: C, H, N, O, S and I;

(c) having an approximate elemental analysis: C 48.8; H 5.4; N 2.8; S 9.0; and I 9.2;

(d) having a molecular weight: 1367;

(e) having a molecular formula: C.sub.55 H.sub.74 N.sub.3 O.sub.21 S.sub.4 I;

(f) having an ultraviolet absorption spectra as shown in FIG. XXI of the drawings;

(g) having an infrared absorption spectrum as shown in FIG. XXII of the drawings;

(h) having a proton magnetic resonance spectrum as shown in FIG. XXIII of the drawings; and

(i) having a carbon-13 magnetic resonance spectrum as shown in FIG. XXIV of the drawings, significant peaks as listed below:

______________________________________ 14.5(q) 17.6(q) 17.6(q) 18.9(q) -- -- 22.8(q) -- 25.4(q) 34.1(t) 37.0(t) 39.1(t) 42.3(t/s) -- 51.5(d) 52.8(q) 54.8(t) 56.3(q) 57.2(q) -- 60.4(d) 60.9(q) 61.3(t) 61.7(q) 67.0(d) 68.4(d) 68.5(d) 69.2(d) 69.7(d) 70.5 71.1(d) 71.8(d) 72.1(s) 75.7(d) 75.8(d) 80.9(d) 82.8(s) 88.1(s) 93.5(s) 97.3(s) 99.6(d) 99.7(d) 100.8(s) 102.6(d) -- 123.4(d) 124.4(d) 126.2(d)103.2(s) 131.0(s) 133.4(s) 139.1(s)143.0(s) 145.1 150.6(s) 151.5(s)154.5 192.0(s) 192.5(s)______________________________________

(j) and having the structure ##STR12##

13. A compound LL-E33288.delta..sub.1 -I

(a) having the following R.sub.f values in the indicated solvent system on TLC on silica gel sheets:

(i) ethyl acetate saturated with 0.1M aqueous potassium dihydrogen phosphate, R.sub.f =0.11; and

(ii) 3% isopropanol in ethyl acetate saturated with 0.1M aqueous potassium dihydrogen phosphate, R.sub.f =0.19;

(b) having ultraviolet absorption spectra as shown in FIG. XXV of the drawings;

(c) having an infrared absorption spectrum as shown in FIG. XXVI of the drawings;

(d) having a proton magnetic resonance spectrum as shown in FIG. XXVII of the drawings; and

(e) having a carbon-13 magnetic resonance spectrum as shown in FIG. XXVIII of the drawings, significant peaks as listed below:

______________________________________17.2 60.4 99.617.6 66.8 103.018.5 67.7 122.722.4 69.5 124.725.0 69.8 126.633.0 70.6 130.737.3 70.7 130.139.0 71.1 133.233.5 71.3 137.751.0 76.0 142.852.4 80.4 150.353.6 82.5 151.556.1 88.1 155.557.0 93.4 192.761.0 97.5 192.860.6 100.961.4 99.8______________________________________

(f) and having the structure ##STR13##

14. A method of treating bacterial infections in warm-blooded animals which comprises administering to said animals an antibacterially effective amount of a compound selected from the group consisting of LL-E33288.alpha..sub.[1] -Br complex; LL-E33288.alpha..sub.2 -Br; LL-E33288.alpha..sub.2 -I; LL-E33288.alpha..sub.3 -Br; LL-E33288.alpha..sub.3 -I; LL-E33288.beta..sub.1 -Br; LL-E33288.beta..sub.1 -I; LL-E33288.beta..sub.2 -Br; LL-E33288.beta..sub.2 -I; LL-E33288.gamma..sub.1 -Br; LL-E33288.gamma..sub.1 -I; and LL-E33288.delta..sub.1 -I.

15. A method of treating the growth of tumors in a mammal comprising administering to said mammal an effective amount of a compound selected from the group consisting of LL-E33288.alpha..sub.[1] -Br complex LL-E33288.alpha..sub.2 -Br; LL-E33288.alpha..sub.2 -I; LL-E33288.alpha..sub.3 -Br; LL-E33288.alpha..sub.3 -I; LL-E33288.beta..sub.1 -Br; LL-E33288.beta..sub.1 -I; LL-E32288.beta..sub.2 -Br; LL-E33288.beta..sub.2 -I; LL-E33288.gamma..sub.1 -Br; LL-E33288.gamma..sub.1 -I; and LL-E33288.delta..sub.1 -I.

16. A method of regressing leukemia in a mammal comprising administering to said mammal an effective amount of a compound selected from the group consisting of LL-E33288.alpha.-Br complex; LL-E33288.alpha..sub.2 -Br; LL-E33288.alpha..sub.2 -I; LL-E33288.alpha..sub.3 -Br; LL-E33288.alpha..sub.3 -I; LL-E33288.beta..sub.1 -Br; LL-E33288.beta..sub.1 -I; LL-E33288.beta..sub.2 -Br; LL-E33288.beta..sub.2 -I; LL-E33288.gamma..sub.1 -Br; LL-E33288.gamma..sub.1 -I; and LL-E33288.delta..sub.1 -I.