Abstract:
Geldanamycin derivatives the following formula ##STR1## wherein R 1  represents a saturated alkylamino which should have at least two carbon atoms, an unsaturated alkylamino, a substituted lower alkylamino, cycloalkylamino, pyrrolidino or aralkylamino group or methoxy group; R 2  represents hydrogen atom, a halogen atom or a lower mono or dialkylamino; when R 1  is methoxy group, R 2  is a halogen atom or lower alkylamino group, or ##STR2## and Geldanamycin derivative is 8,9-epoxy-Geldanamycin. The compounds are effective as antitumor drugs.

Description:
BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention relates to novel Geldanamycin derivatives and antitumor drug containing the same as an active ingredient. 
     2. Description of the Prior Arts 
     Geldanamycin is the antiprotozoan antibiotic produced by streptomyces hygroscopicus var. geldanus var. nova strain (Journal of Antibiotics Vol. 23, Page 442 (1970)) and has the following formula ##STR3## (Journal of the American Chemical Society Vol. 92, Page 7591 (1970)). 
     SUMMARY OF THE INVENTION 
     It is an object of the present invention to provide novel antitumor drugs. 
     The novel Geldanamycin derivatives of the present invention have the following formula ##STR4## wherein R 1  represents a saturated alkylamino which should have at least two carbon atoms, an unsaturated alkylamino, a substituted lower alkylamino, cycloalkylamino, pyrrolidino or aralkylamino group or methoxy group; R 2  represents hydrogen atom, a halogen atom or a lower alkylamino group; when R 1  is methoxy group, R 2  is a halogen atom or a lower alkylamino group, or ##STR5## wherein R 3  represents ##STR6## R 4 , R 5  and R 6  respectively represent hydrogen atom or methyl group and at least one of R 4 , R 5  and R 6  is methyl group; or ##STR7## 
     In the formula (I), when R 1  is methoxy group, R 2  is a halogen atom or a mono- or di-lower alkylamino group. 
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     The Geldanamycin derivatives having the formula (I) can be produced by reacting Geldanamycin with an amine having the formula ##STR8## wherein R 7  and R 8  respectively represent hydrogen atom or a saturated alkyl group having at least two carbon atoms; an unsaturated alkyl group, a substituted lower alkyl group a cycloalkyl group or an aralkyl group and both R 7  and R 8  can not be hydrogen atom and R 7  and R 8  can bonded to form one alkylene group. 
     The typical groups as R 7  or R 8  include ethyl, propyl, butyl, pentyl, hexcyl, heptyl, octyl, decyl, dodecyl, allyl or substituted lower alkyl groups (substituted with hydroxy, amino, methylamino, pyrrolidino, pyridinyl, alkoxy, piperadino, morpholino, cycloalkyl or hydroxyalkoxy group or a halogen atom); or cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, benzyl, phenethyl group. The bonded R 7  and R 8  can be ethylene, tetramethylene, pentamethylene or hexamethylene group. 
     The typical amines include ethylamine, propylamine, butylamine, pentylamine, hexylamine, heptylamine, octylamine, decylamine, dodecylamine, allylamine, β-hydroxyethylamine, β-chloroethylamine, β-glycoxyethylamine, aminobutylamine, adamanthylmethylamine, cyclopropylamine, cyclopentylamine, cyclohexylamine, cycloheptylamine, benzylamine, phenethylamine, ethyleneimine, pyrrolidine, piperidine, dimethylamine, aminoethylamine, β-hydroxyethylamine, diglycolamine, β-morpholinoethylamine, β-piperadinoethylamine, 2&#39;-, or 3&#39;-, or 4&#39;-picolylamine, β-pyrrolidinoethylamine, 2&#39;-pyridinylethylamine, β-methoxyethylamine, β-N-methylaminoethylamine, etc. Methylamine can be used in the special condition. 
     The reaction of Geldanamycin with an amine can be carried out in the presence of an organic solvent. 
     Suitable organic solvents include alcohols, chlorinated hydrocarbons especially chloroform, dichloroethane, methanol etc. The solvent can be a mixture of two or more solvents. The components used in the reaction can be combined at a desired ratio such as an equimolar ratio or excess. It is preferable to use 1 to 50 mole equivalent of the amine per mole of Geldanamycin. The reaction is usually carried out at room temperature for 1 to 48 hours. 
     When excess of the amine is used in severe condition, both R 1  and R 2  are partially converted into the corresponding amino groups. 
     When excess of the di-lower alkylamine is used in the reaction, both R 1  and R 2  are partially converted into the corresponding dialkylamino groups, though R 1  is partially remained as methoxy group. 
     The Geldanamycin derivatives having the formula (I) wherein R 1  is methoxy group and R 2  is a halogen atom, such as Br, I, Cl or F, espcially Br, I or Cl can be obtained by a halogenation of Geldanamycin. The halogenation is carried out by using a halogenating agent such as pyridinum bromide, perbromide, iodine-pyridine, and lithium chloride etc. in a solvent such as ethanol-chloroform mixture, pyridine, ethyl acetate or methyl ethyl ketone, at 0° to 100° C. for 1 to 10 hours. 
     The Geldanamycin derivative having the formula (II) can be obtained by methylation of Geldanamycin. 
     Suitable methylating agents include methyl halides such as methyl chloride, methyl bromide and methyl iodide. 
     The methylation is carried out by dissolving Geldanamycin into an organic solvent such as chloroform, methanol or a mixture thereof and adding excess especially 4 to 6 times of silver oxide into the solution in a form of suspension and then, adding 8 to 10 times of methyl halide and mixing them. In general, the reaction is accomplished at room temperature for 3 to 24 hours. 
     In the reaction, several compounds having the formula (I) can be simultaneously obtained. 
     The Geldanamycin derivative having the formula (III) can be obtained by an epoxidation. 
     The compound (II) can be obtained by an epoxidation with an oxidant in an organic solvent such as chloroform, benzene, or a mixture thereof. 
     Suitable oxidizing agent include organic or inorganic peroxides such as peracetic acid, perbenzoic acid, chloroperbenzoic acid, perphthalic acid etc.; or alkylhalide peroxides such as t-butylhydroperoxide or hydrogen peroxide. 
     Excess of the oxidizing agent preferably 1.1 to 1.5 mole-equivalent per Geldanamycin can be used. When hydroperoxide is used, it is preferable to incorporate a catalytic amount of vanadium (IV) oxyacetyl acetonate. When hydrogen peroxide is used, it is preferable to react 2 mole of hydrogen peroxide with 1 mole of Geldanamycin in the presence of 4 mole of para-chlorophenyl isocyanate. The reaction is carried out at from room temperature to the boiling point of the mixture for 2 to 80 hours. 
     In the epoxidation of Geldanamycin by the peroxide, only double bond at 8 and 9 positions is epoxidized. 
     The isolation and the purification of the object compound can be carried out by conventional methods. 
     When the amine (IV) is used, excess of the amine is removed by washing with a dilute mineral acid. The solution is concentrated and dried at 35° to 45° C. under a reduced pressure and the residue is purified by a chromatography or a recrystallization. 
     The geldanamycin derivatives having the formula (I) are crystalline compounds (red, reddish violet, brown, blue) which are not soluble in water but soluble in an organic solvent such as methanol, ethanol, acetone, ethyl acetate, halogenated hydrocarbon, tetrahydrofuran, dioxane, dimethylsulfoxide, dimethylformamide and pyridine. 
     The Geldanamycin derivatives (I) are reduced with a reducing agent such as hydrogensulfite, dithionite, zinc-acetic acid or ascorbic acid to be pale yellow or white, but the reduced products are easily oxidized by contacting with air or an oxidizing agent to be colored and to form the Geldanamycin derivatives (I). 
     When the methylation is carried out to obtain the Geldanamycin derivatives having the formula (II), the isolation and the purification from the reaction mixture can be carried out by separating insoluble silver iodide and excess of silver oxide and concentrating the filtrate to dryness under a reduced pressure and purifying the residue by a chromatography or a recrystallization. 
     The Geldanamycin derivatives (II) are crystalline compounds (yellow or yellowish brown) which are not soluble in water but soluble in said organic solvent. 
     When the oxidation is carried out to obtain the Geldanamycin derivatives having the formula (III), the isolation and the purification from the reaction mixture can be carried out by separating excess of the oxidizing agent by washing with 5% aqueous solution of sodium sulfite and dehydrating the solution over anhydrous sodium sulfate and concentrating the filtrate to dryness under a reduced pressure and purifying the residue by a chromatography or a recrystallization. 
     The Geldanamycin derivatives (II) are crystalline compounds (yellowish brown or yellow) which are not soluble in water but soluble in said organic solvent. 
     The novel Geldanamycin derivatives (I), (II) or (III) of the present invention have significant growth inhibition effect to cancer cell W-2K-11 which is well-known as the model of cancer cells, to be useful as antitumor drugs. 
     One or more kinds of the Geldanamycin derivatives (I), (II) or (III) of the present invention can be administered as an antitumor drug. 
     It is preferable to combine the active ingredient with a suitable adjuvant or an additive so as to form a pharmacological composition which is suitable for oral administration or non-oral administration. 
     The adjuvants and additives can be the following organic or inorganic solid or liquid. 
     Suitable adjuvants include water, gelatin, lactose, starch, calcium, carboxymethyl cellulose, microcrystalline cellulose, stearyl alcohol, magnesium stearate, talc, vegetable oil, benzyl alcohol, propyleneglycol, rubber, polyalkyleneglycol, kerosen, jelly and cholesterol. 
     Suitable additives includes preservatives, wetting agents, emulsifiers, dissolution accelerators, osmotic pressure adjusting salts, buffers, binders, suspending agents and dispersing agents. 
     The pharmacological compositions can be powder, granule, capsule, pellet, tablet, sugar coated tablet, injection, suppository and ointment. These compositions can be prepared by conventional methods. 
     The antitumor drug of the present invention can be used for human therapy but also used as an animal drug, in the same form. 
     In the therapy with the antitumor drug of the present invention, a dose of the antitumor drug is usually in a range of 0.5 to 80 mg./kg. preferably 1 to 40 mg./kg. in the non-oral administration (injection) and in a range of 1 to 100 mg./kg. preferably 2 to 50 mg./kg. in the oral administration. 
    
    
     The present invention will be further illustrated by certain examples of the preparations. 
     EXAMPLE 1 
     Into 50 ml. of chloroform, 280 mg. of Geldanamycin was dissolved and then, 1 ml. of n-propylamine was added. The mixture was stirred at room temperature for 1 hour and the mixture was charged into 50 ml. of cold water and then, pH was adjusted 3 with 6 N-HCl. The chloroform layer was separated and the water layer was extructed with 50 ml. of chloroform. Both chloroform layers were combined and dehydrated over anhydrous sodium sulfate for 2 hours. Chloroform was distilled off under a reduced pressure to remain a reddish violet solid product. The solid product was recrystallized from acetone-n-hexane to obtain 286 mg. (yield 98%) of 17-n-propylamino-Geldanamycin as reddish violet acicular crystal. 
     Melting point: 143°-145° C. 
     Elementary analysis: C 31  H 45  N 3  O 8 . 
     
         ______________________________________        C       H         N______________________________________Calculated (%) 63.35     7.72      7.15Found (%)      63.35     7.78      6.96______________________________________ 
    
     molecular weight (m/e): 587 (M + ) (mass spectrograph). 
     EXAMPLE 2 
     Into 100 ml. of chloroform, 560 mg. of Geldanamycin was dissolved and then, 2 ml. of ethyleneimine was added. The mixture was stirred at room temperature for 21 hours and was charged into 100 ml. of cold water and pH was adjusted to 3 with 6 N-HCl. The chloroform layer was separated. The water layer was extracted with 100 ml. of chloroform. Both chloroform layers were combined and dehydrated over anhydrous sodium sulfate and concentrated to dryness under a reduced pressure to obtain a red oily product. The oily product was purified by a silica gel column chromatography with 25% methanol-chloroform. The desired fraction was collected by a fraction collector and concentrated to dryness under a reduced pressure and the product was recrystallized from acetone-n-hexane to obtain 530 mg. (yield 93%) of 17-ethyleneimino-Geldenamycin as a reddish orange crystal. 
     Melting point: 261°-262° C. (decomposition). 
     Elementary analysis: C 30  H 41  N 3  O 8 . 
     
         ______________________________________        C       H         N______________________________________Calculated (%) 63.03     7.23      7.35Found (%)      62.98     7.34      7.11______________________________________ 
    
     Molecular weight (m/e): 571 (M + ) (mass spectrograph). 
     In accordance with the process of Example 1 or 2, the compounds shown in Table 1 were produced. 
     
                       TABLE 1______________________________________Ex-am-  Compound having formula(I)                   Melting   MW. by massple  R.sup.1            point(°C.)                             spect. (m/e)______________________________________3    ethylamino          226-227  5734    β-aminoethylamino                   225-227   5885    n-butylamino       153-155   6016    n-pentylamino      162-164   6157    n-hexylamino       201-203   6298    n-heptylamino      208-210   6439    n-octylamino       215-217   65710   n-decylamino       198-200   68511   n-dodecylamino     207-208   71312   isobutylamino      147-149   60113   cyclopropylamino   210-212   58514   cyclopentylamino   160-163   61315   cyclohexylamino    187-189   62716   cycloheptylamino   200-202   64117   adamanthylmethylamino                   153-155   69318   β-hydroxyethylamino                   150-152   58919   diglycolamino      128-130   63320   β-chloroethylamino                   150-152   609 60721   δ-amino-n-butylamino                   152-155   61622   allylamino         212-214   58523   benzylamino        187-188   63524   phenethylamino     185-187   64925   pyrrolidino        150-153   59926   β-morphorimoethylamino                   167-169   65827   β-piperadinoethylamino                   188-190   65728   2&#39;-picolylamino    201-203   63629   3&#39;-picolylamino    235-236   63630   4&#39;-picolylamino    230-231   636                   (decomp.)31   2&#39; -pyridinylethylamino                   210-211   650                   (decomp.)32   β-pyrrolidinoethylamino                   144-146   64233   β-methoxyethylamino                   128-130   60334   β-N-methylethylamino                   152-155   602                   (decomp.)______________________________________ 
    
     EXAMPLE 35 
     Into 100 ml. of chloroform-methanol (3:2), 560 mg. of Geldanamycin was dissolved and then, 30 ml. of 50% aqueous solution of dimethylamine was added. The mixture was stirred at room temperature for 2 hours and was charged into 100 ml. of cold water and pH was adjusted to 3 to 4 with 6N-HCl. The product was extracted twice with chloroform and the extracted chloroform layers were washed with water and dehydrated over anhydrous sodium sulfate and concentrated to dryness under a reduced pressure to obtain a blue oily product. 
     The oily product was purified by a silica gel column chromatography with 2% methanol-chloroform. The fractions (14th to 32th) were collected and the solvent was distilled off and the product was recrystallized from ether to obtain 52 mg. of 17-demethoxy-17,19-bisdimethylamino-Geldenamycin as brown crystal. 
     Melting point: 135°-137° C. 
     Elementary analysis: C 32  H 50  N 4  O 8 . 
     
         ______________________________________        C       H         N______________________________________Calculated (%) 62.11     8.15      9.05Found (%)      62.01     8.00      8.87______________________________________ 
    
     NMR spectrum (100 MHz: (DCl 3 ). 
     δ(ppm): 2.92 (dimethylamino); 2.93 (dimethylamino); 3.31 (methoxy); 3.34 (methoxy). 
     UV spectrum: λ max   CH .sbsp.3 OH  (nm): 246, 380, 540. 
     Molecular weight (m/e): 618 (M + ) (mass spectrograph). 
     EXAMPLE 36 
     The blue oily product obtained by the process of Example 35 was purified by a silica gel column chromatography with 2% methanol-chloroform. The fractions (34th to 70th) were collected and the solvent was distilled off and the product was recrystallized from ether-n-hexane to obtain 317 mg. of 19-dimethylamino-Geldanamycin as blue crystal. 
     Melting point: 153°-156° C. 
     Elementary analysis: C 31  H 45  N 3  O 9 . 
     
         ______________________________________        C       H         N______________________________________Calculated (%) 61.67     7.51      6.96Found (%)      61.81     7.98      6.88______________________________________ 
    
     NMR spectrum (100 MHz-CDCl 3 ). 
     δ(ppm): 3.00 (dimethylamino), 3.26 (methoxy), 3.33 (methoxy), 3.88 (methoxy). 
     UV spectrum: λ max   CH .sbsp.3 OH  (nm): 244, 295, 535. 
     Molecular weight (m/e): 603 (M + ) (mass spectrograph). 
     EXAMPLE 37 
     Into 60 ml. of ethanol and 60 ml. of chloroform, 1.12 g. of Geldanamycin was dissolved. The mixture was stirred while cooling with ice water and 960 mg. of pyridinium bromide perbromide was added. The mixture was stirred further for 1 hour. After the reaction, it was diluted with large amount of chloroform and repeatedly washed with chloroform and with saturated aqueous solution of sodium chloride and dehydrated over anhydrous sodium sulfate. The chloroform layer was concentrated to dryness under a reduced pressure to obtain yellowish orange oily product. The oily product was purified by a silica gel column chromatography with 3% methanolchloroform and then, recrystallized from ethyl ether to obtain 630 mg. of 19-bromo-Geldanamycin as orange crystal. 
     Melting point: 224°-226° C. (decomposed). 
     Elementary analysis: C 29  H 39  N 2  O 9  Br. 1/2(C 2  H 5 ) 2  O. 
     
         ______________________________________       C      H        N        Br______________________________________Calculated (%)         55.03    6.55     4.14   11.81Found (%)     55.24    6.85     3.92   11.62______________________________________ 
    
     IR spectrum (KBr): 1747, 1684, 1590 cm-1 . 
     UV spectrum: λ max   CH .sbsp.3 OH  (nm): 257, 312, 390 (sh). 
     Molecular weight (m/e): 640 and 638 (M + ) (mass spectrograph). 
     EXAMPLE 38 
     Into 20 ml. of pyridine, 762 mg. of iodine was dissolved and 1.12 g. of Geldanamycin was dissolved. The mixture was stirred at room temperature for 1 hour. The reaction mixture was diluted with ethyl acetate. The diluted solution was washed with 5% aqueous solution of sodium thiosulfate, with 10% aqueous solution of acetic acid and with saturated aqueous solution of sodium chloride and then dehydrated over anhydrous sodium sulfate. The ethyl acetate solution was concentrated to dryness under a reduced pressure to obtain an orange red oily product. The oily product was purified by a silica gel column chromatography with 3% methanol-chloroform and recrystallized from chloroform-n-hexane to obtain 988 mg. of 19-iodo-Geldanamycin as orange crystal. 
     Melting point: 152°-154° C. 
     Elementary analysis: C 29  H 39  N 2  O 9  I.CHCl 3 . 
     
         ______________________________________       C     H       N       halogen______________________________________Calculated (%)         44.71   5.00    3.48  27.94Found (%)     45.16   5.03    3.47  27.68______________________________________ 
    
     IR spectrum (KBr); 1740, 1673, 1580 cm-1 . 
     UV spectrum: λ max   CH .sbsp.3 OH  (nm): 255, 312, 405 (sh). 
     Molecular weight (m/e): 686 (M + ) (mass spectrograph). 
     EXAMPLE 39 
     Into 60 ml. of methyl ethyl ketone, 726 mg. of 19-bromo-Geldanamycin was dissolved and 3.5 g. of lithium chloride was added. The mixture was refluxed for 5 hours with stirring. After the reaction, the reaction mixture was cooled to room temperature and diluted with a large amount of chloroform and washed with 5% aqueous solution of sodium thiosulfate, with water, with saturated aqueous solution of sodium chloride and dehydrated over anhydrous sodium sulfate. The chloroform solution was concentrated to dryness to obtain orange oily product. The oily product was purified by a silica gel column chromatography with 3% methanol-chloroform and recrystallized from chloroform-n-hexane to obtain 580 mg. of 19-chloro-Geldanamycin as reddish orange crystal. 
     Melting point: 157°-159° C. 
     Elementary analysis: C 29  H 39  N 2  O 9  Cl.CHCl 3 .1/2H 2  O. 
     
         ______________________________________       C      H        N        Cl______________________________________Calculated (%)         49.80    5.71     3.87   19.60Found (%)     49.74    5.60     3.77   20.67______________________________________ 
    
     IR spectrum (KBr): 1730, 1680, 1588 cm-1 . 
     UV spectrum: λ max   CH .sbsp.3 OH  (nm); 257, 310, 395 (sh). 
     Molecular weight (m/e): 596 and 594 (M + ) (mass spectrograph). 
     EXAMPLE 40 
     Into 200 ml. of chloroform and 200 ml. of methanol, 5 g. of Geldanamycin was dissolved and then, 20 g. of silver oxide was added and then 40 g. of methyl iodide was added with stirring and the mixture was stirred for 5 hours. The insoluble materials were separated by a filtration and the filtrate was concentrated to dryness under a reduced pressure at 40° C. The resulting reddish orange oily product was purified by a silica gel column chromatography with 2.5% methanol-chloroform. The fractions (42th to 60th) were collected and concentrated to dryness and recrystallized from ether-n-hexane to obtain 600 mg. of 1-O-methyl-Geldanamycin as yellowish orange crystal. 
     Melting point: 213°-215° C. 
     Elementary analysis: C 30  H 42  N 2  O 9 .1/2H 2  O. 
     
         ______________________________________        C       H         N______________________________________Calculated (%) 61.73     7.42      4.80Found (%)      62.09     7.37      4.73______________________________________ 
    
     NMR spectrum: (100 MHz: CDCl 3 ). 
     δ(ppm): 3.85 (iminomethyl ether). 
     UV spectrum: λ max   CH .sbsp.3 OH  (nm): 262, 305, 390 (sh). 
     Molecular weight (m/e): 574 (M + ). 
     EXAMPLE 41 
     The fractions (77th to 128th) obtained by the silica gel column chromatography in the process of Example 40 were collected and concentrated to dryness and the product was recrystallized from chloroform-n-hexane to obtain 2.42 g. of 21-N-methyl-Geldanamycin as yellow crystal. 
     Melting point: 141°-143° C. 
     Elementary analysis: C 30  H 42  N 2  O 9 .1/2H 2  O. 
     
         ______________________________________        C       H         N______________________________________Calculated (%) 61.73     7.42      4.80Found (%)      61.39     7.28      4.80______________________________________ 
    
     NMR spectrum: (100 MHz: CDCl 3 ). 
     δ(ppm): 3.12 (N-methyl). 
     UV spectrum: λ max   CH .sbsp.3 OH  (nm): 254, 316, 390 (sh). 
     Molecular weight (m/e): 574 (M + ) (mass spectrograph). 
     EXAMPLE 42 
     The fractions (13th to 34th) obtained by the silica gel column chromatography in the process of Example 40 were collected and concentrated to dryness and the product was purified by a chromatography with 2% methanol-chloroform and recrystallized from chloroform-n-hexane to obtain 452 mg. of 21-N-methyl-7-OCONH-methyl-Geldanamycin as yellow crystal. 
     Melting point: 130°-132° C. 
     Elementary analysis: C 31  H 44  N 2  O 9 . 
     
         ______________________________________        C       H         N______________________________________Calculated (%) 63.25     7.53      4.77Found (%)      63.01     7.48      4.83______________________________________ 
    
     NMR spectrum (100 MHz: CDCl 3 ). 
     δ(ppm): 3.12 (N-methyl), 2.77 (N-methyl-carbamate). 
     UV spectrum: λ max   CH .sbsp.3 OH  (nm): 252, 310, 410 (sh). 
     Molecular weight (m/e): 588 (M + ) (mass spectrograph). 
     EXAMPLE 43 
     The fractions (3rd to 12th) obtained by the silica gel column chromatography in the process of Example 40 were collected and concentrated to dryness and the product was purified by a chromatography with % methanol-chloroform to collect the fractions (1st to 40th) and the fraction was crystallized from acetone-n-hexane to obtain 192 mg. of 7-OCONH-methyl Geldanamycin as yellow acicular crystal. 
     Melting point: 230°-232° C. 
     Elementary analysis: C 30  H 42  N 2  O 9 .1/2H 2  O. 
     
         ______________________________________        C       H         N______________________________________Calculated (%) 61.73     7.42      4.80Found (%)      62.01     7.35      4.73______________________________________ 
    
     NMR spectrum: (100 MHz: CDCl 3 ). 
     δ(ppm): 2.80 (N-methyl carbamate), 8.75 (N-H). 
     UV spectrum: λ max   CH .sbsp.3 OH  (nm): 258, 303, 404. 
     Molecular weight (m/e): 574 (M + ) (mass spectrograph). 
     EXAMPLE 44 
     The fractions (61st to 82nd) obtained by the second gel column chromatography in the process of Example 43 were collected and recrystallized from chloroform-n-hexane to obtain 343 mg. of 1-O-methyl-7-OCONH-methyl-Geldanamycin as yellow crystal. 
     Melting point: 119°-121° C. 
     Elementary analysis: C 31  H 44  N 2  O 9 .H 2  O. 
     
         ______________________________________        C       H         N______________________________________Calculated (%) 61.36     7.64      4.62Found (%)      61.10     7.58      4.50______________________________________ 
    
     NMR spectrum: (100 MHz; CDCl 3 ). 
     δ(ppm): 2.75 (N-methyl-carbamate), 3.85 (iminomethyl ether). 
     UV spectrum: λ max   CH .sbsp.3 OH  (nm): 263, 295 (sh), 410 (sh). 
     Molecular weight (m/e): 588 (M + ) (mass spectrograph). 
     EXAMPLE 45 
     Into 800 ml. of 20 % benzene-chloroform, 5.6 g. of Geldanamycin was dissolved and then, 1.9 g. of methachlorobenzoic acid was added. The mixture was stirred at room temperature for 5 hours. The reaction mixture was washed with 5% aqueous solution of sodium sulfite and with water and with saturated aqueous solution of sodium chloride and dehydrated over anhydrous sodium sulfate and concentrated to dryness under a reduced pressure. The resulting orange red oily reside was purified by a silica gel column chromatography with 3%-methanol-chloroform. The desired fractions were collected and concentrated to dryness under a reduced pressure and recrystallized from ether-n-hexane to obtain 4.84 g. of 8,9-epoxy-Geldanamycin as yellowish orange crystal. 
     Melting point: 148°-149° C. 
     Elementary analysis: C 29  H 40  N 2  O 10 .1/2H 2  O. 
     
         ______________________________________        C       H         N______________________________________Calculated (%) 59.47     7.06      4.78Found (%)      59.54     6.96      4.71______________________________________ 
    
     UV spectrum: λ max   CH .sbsp.3 OH  (nm): 254, 303, 407. 
     Molecular weight (m/e): 576 (M + ) (mass spectrograph). 
     EXAMPLE 46 
     Into 1000 ml. of benzene, 1.4 g. of Geldanamycin was dissolved and a catalytic amount of vanadium (IV) oxyacetyl acetonate was added and the mixture was refluxed on an oil bath and 360 mg. of 70% t-butylhydroperoxide was added during the reflux. After refluxing for 6 hours, the reaction mixture was cooled to room temperature and was washed with 0.1N-HCl, with 5% aqueous solution of sodium sulfite, with water and with saturated aqueous solution of sodium chloride and dehydrated over anhydrous sodium sulfate and concentrated to dryness under reduced pressure. The resulting residue was purified by a silica gel column chromatography with 2.5% methanol-chloroform. The desired fractions were collected and concentrated to dryness and recrystallized from ether to obtain 280 mg. of 8,9-epoxy-Geldanamycin as yellowish orange crystal. 
     According to the silica gel thin layer chromatography, the melting point and the IR spectrum, it was confirmed that the product was the same as the product of Example 45. 
     Composition 
     2500 G. of each product, 1375 g. of lactose, 775 g. of microcrystalline cellulose and 375 g. of calcium carboxymethyl-cellulose were sieved by a 16 mesh sieve to mix uniformly them. The mixture was charged into a kneader and 3 liter of 3% solution of hydroxypropyl-cellulose (isopropyl alcohol:water=3:7), was added and the mixture was kneaded. The mixture was granulated by an extrusion-granulating machine and air-dired at 50° C. for 6 hours. The granule was dressed in a range of 16 to 60 mesh and magnesium stearate was admixed with the granule at a ratio of 0.3 wt. % and the mixture was tabletted to obtain tablet. 
     Test 
     Cancer cell W-2K-11 was obtained by transforming fibroblast C3H-2K clone derived from kidney of mouse by cancerogenic virus. The cancer cell W-2K-11 was cultured by the following method. 
     (1) Preparation of culture medium 
     9.4 Gram of Eagle&#39;s MEM culture media (manufactured by Nissui Seiyaku K. K.) was dissolved in 900 ml. of distilled water and the solution was sterilized under high pressure at 120° C. for 15 minutes and cooled. 100 Ml. of calf serum and 3-5 ml. of 10% aqueous solution of sodium bicarbonate were added to the solution to adjust pH to 7.1-7.2. 10 Ml. of an aqueous solution of L-glutamine (2.92 g./100 ml.) filtered by a millipore filter was added just before the use of the solution as culture medium. 
     (2) Preparation of transplanted cell solution 
     The cancer cell W-2K-11 stored in a freezer at -80° C. was melted at room temperature and treated by a centrifugal separator at 670 g. for 5 minutes. The precipitated cell was dispersed in 50 ml. of the culture medium (1). The suspension was charged into Roux flask and cultured at 37° C. The multiplication of the cells was initiated and accomplished after 3 to 4 days. The cultured mixture was decanted and 10 ml. of 0.2% trypsin solution was added and the mixture was kept at room temperature for 2 to 3 minutes and the trypsin mixture was decanted and then, 50 ml. of the culture medium (1) was added to prepare a suspension of cells. 
     (3) Incubation of cells and addition of sample 
     1.8 Ml. of the suspension of cells (2) was charged into each Petri dish and incubated in a carbon dioxide gas incubator (5% of CO 2  ; 95% of air) at 37° C. Twenty four hours after the initiation of the incubation, 0.2 ml. of each ethanol solution of each sample was added, and the incubation was continued. Forty eight hours after the addition of the sample, numbers of survival cells were counted under a microscopic observation and each percent inhibition of cell multiplication was calculated. The results are shown in Table 2. ##EQU1## 
     
                       TABLE 2______________________________________Compound No.  Concentration Percent(Example No.) (μg/ml.)   inhibition (%)______________________________________1             1             922             1             973             1             904             5             575             1             856             1             907             1             908             1             969             10            6710            10            4811            10            7912            1             9013            1             9414            1             9814            10            9715            1             9816            10            9817            10            9818            1             9819            10            8820            1             9821            10            5322            1             9323            1             8824            1             8225            10            9726            5             9327            5             5728            5             9029            5             9230            5             9531            5             9332            5             8433            0.5           9534            5             6735            10            9735            1             9236            10            8436            1             7037            5             8438            5             9539            5             9140            10            9540            1             8041            10            8242            10            9442            1             8243            10            9643            1             6044            10            8044            1             9445            10            10045            1             95Geldanamycin  1.2           61______________________________________ 
    
     Acute toxicity 
     LD 50  of each sample to mouse was measured by intraperitoneal injection of each sample. The results are shown in Table 3. 
     
                       TABLE 3______________________________________Compound No.(Example No.)       LD.sub.50______________________________________ 3                  120 mg./kg.36                  &gt;240 mg./kg.41                  120 mg./kg.45                  240 mg./kg.Geldanamycin         15 mg./kg.______________________________________