IL39306A - 3-formylrifamycin sv hydrazone derivatives - Google Patents

Abstract

1336399 3-Formylrifamycin SV derivatives GRUPPO LEPETIT SpA 26 April 1972 [24 June 1971] 19458/72 Heading C2A The invention comprises 3-formylrifamycin SV compounds of general Formula I wherein R 1 and R 2 are independently selected from (a) hydrogen, (b) alkyl, (c) alkenyl, (d) alkynyl, (e) aryl, (f) aralkyl, (g) heterocyclic and (h) cycloalkyl, and are further characterized in that only one of R 1 and R 2 can be hydrogen or C 1-4 alkyl, and if hydrogen or lower alkyl is present, the other of R 1 and R 2 must be unsubstituted alkyl or hydroxyalkyl of at least 5 carbon atoms or a substituted alkyl other than dimethylaminoethyl or a member of groups (c), through (h), wherein aryl is other than phenyl and p-carboxyphenyl, and aralkyl is other than benzyl and where R 3 is -H or CH 3 CO- and the 16, 17, 18, 19, 28, 29 hexahydro and 27-demethoxy-27-hydroxy derivatives thereof. A process for the preparation of compounds of Formula I comprises reacting a 3-formylrifamycin of general Formula II or the corresponding hexahydro derivative with a hydrazine of the general formula H 2 N-NR 1 R 2 wherein R 1 and R 2 are as defined above. A therapeutic composition comprises as active ingredient a compound of Formula I in conjunction with a pharmaceutically acceptable carrier. [GB1336399A]

Description

NEW SV HYDRAZONE DERIVATIVES invention is concerned with new derivatives of More particularly the invention relates to hydrazones of SV of general formula is CH3CO and of and is of 1 to 12 phenyl and and the other is alkyl of 5 to 12 carbons which may be optionally substituted with hydroxy alkenyl of 3 to 5 adament l phenyl substituted with one to three groups each selected from lower tr lower alkyl and alk alkyl whereih the alkyl is of 2 to 4 carbons and the phenoxy group may optionally have one to substituents each independently selected from halo and 5 to 15 an heterocyclic ring selected from pyridine substituted with groups each independently selected from lower alkyl and pyrimidine substituted with groups each selected from lower hydroxy and pyridazine substituted with and quinoline substituted with groups each independently selected lower lover and with the proviso that when one of and is or alkyl of 1 to 4 the other may not be and the and Certai hydrazones of am n are described in our patent These compounds although possessing a good antibacterial activity have practically no effect against bacteria which have become resistant to the other namely the most known and therapeutically useful SV It is well known by those which are expert in the antibiotic field that when a microorganism strain becomes resistant to a particular antibiotic it is rathe difficult to find another compound of the same antibiotic family which is capable to inhihit the growth of said resistant In some instances it is quite difficult to find compounds which are active against such a resistant strain even among the other different species of We have surprisingly found that representative compounds of this invention are able to inhibit at low the growth of strains resistant to the other In particular compounds and of example 3 at concentration of ml bout 10 or less inhibit the growth of a aureus Tour strain resistant to The invention compounds are generally very active also against the usual Gram positive and Gram negative In the new compounds shows a remarkable activity against Staphylococcus faecalis a aureus St eptococcus Streptococcus hemolyticus and pneumoniae In these cases the minimum inhibiting concentra tion ranges from about to about Another very important feature of the invention compounds is their inhibiting activity of which are charact erist of human leukemic blood lymphoblasts and against typical nucleotidyl transferases of virus not utilized by the normal story of breast cancer and from inbred et Priori et New 232 isolated a virus named containing reverse t anscriptase from cells from the pleural fluid of a child with lymphoma and have succesfully grown it in tissue The presence in human breas cancer of RNA homologous to mouse mammary tumor virus RNA has been Q demostrated through molecular experiments by Axel et At present there are no very effective drugs for treating viral diseases since viruses and cells have commo metabolic requirements and The most promising approach to v ral chemotherapy clearly is the design of suitable chemicals which c bine specifically with viral or virus transformed b not with host cell polymerases controlling the expression of genetic information of Specific inhibitors of the viral or virus tr sformed enzymes in inhibitors of polymerases o RNA tumor viruses may have an important role in proving drugs for le kemia and other cancer The inhibiting activity of the invention compounds has been tested o dependent DNA polymerase of murine sarcoma virus an DNA dependent DNA polymerase activity of purified The inhib tion was tested according to the methods described by Gurgo et al New The effect of different concentr bions of drugs on polymerase activity was determined by following thymine deoxyriboside incorporation int bhe insoluble A typical example of the experimental procedu Isolation of virus and purification of viral Virus was isolated and purified from murine sarcoma virus transformed rat cells and murine sarcoma rus transformed mouse cells as ously described et Rokutanda et The vi rion polymerase was purified fold by incubation of purified virus with in M Tris buffer M ΕΌΤΑ for 5 minutes at room temperature an zonal centrifugation in sucrose gradients in 10 mM sodium phosphate buffer 7 4 mM dithiothreitol and glycerol for 24 hours at 000 rpm in a Spinco SW41 Th peak fractions of enzyme activity of fractions were and stored at in DNA polymerase was Enzyme incubation performed for 1 hour at in 100 μΐ of reaction mixture containing mM Tris buffer 5 dithi mM mM M mM and 1 of as described by Green et in USA The reaction was minated by the addition of 150 μΐ of IN perchloric Calf was mus ΌΝΑ added as the radioactive DNA produc was processed as described in the two papers mentioned Endogenous activity was measured afte the addition of to purified virus at the time of The activity of purified viral polymerase was measu red with 2 μg of poly as template and no Test for inhibition by rifamycin Rifamycin derivatives were dissolved in a a concentration of 5 and stored at Inhibition of the e dogenous activity was tested by addin 2 μΐ of derivative appropriately diluted in DMSO or 2 μΐ of OMSO to the assay mixture prior to addition to disrupted virus wh contained 1 to g of viral Enzyme incubation was perf med for 60 minutes at Inhibition of purified enzyme was test by of 2 μΐ of derivative or with of enzy to 2 of for 10 minutes at then JO μΐ of subst te mixture were added and the mixture further incubated and proces as described In representative tests the invention compounds described in ples 16 at a concentration of or 3 less reduced the incorporation of to less than 10 per cent of that found in the control tests clearly demonstrating inhibition of mechanism of carcinogenesis by RNA tumor viruses according to the most recent biochemical points of The inhibiting effect of reverse transcriptases has been confirmed also by test of polymerase from murine leukemia Murine kemia virus was prepared from Triton X 100 disrupted virions as described by Gallo et in New Virus of both Rauscher and Moloney types were viously purified by banding in the region of a sucrose density gradient after initial low speed centrifugation to remove cellular debris and cushioning sucrose through 11 Final concentration of virus preparation was 10 As template endogenous was Concentrations of or less were found to be effective in inhibiting the For inhibitions of about per cent were obtained with concentrations of only about of representative Similar results were found by using tumor cell polymerases of human In this case the inhibiting activity was studied also on normal polymerases to characterize a selective Repre sentative rifamycin derivatives of formula I have been evaluated fo 1 their effects on two purified DNA polymerases isolated from man normal blood a lymphoblast cel line from a normal and human leukemic blood lym Synthetic native templates were A typical example of the experimental procedure is the Human Blood Lymphoblas s Leukemic lymphoblasts were isolated from the peripheral blood of patients with acute lymphocytic leukemia by leukophoresis The cells were washed and erythrocytes removed by hypotonic Normal lymphocytes were obtained from the peripheral blood from thy donors after removal of granulocytes by nylon column They were stimulated with phytohemagglutinin for hours as described before et Ga lo et 16 in order to maximize DNA polyme rase because of the logistic problems in obtaining sufficient mounts of these a human tissue culture cell line to supply less purified DNA polymerases for some of the initial survey Compounds of interest were then studie in more detail with the more purified enzymes from the normal and leukemic blood These tissue culture cells were ned from Associated Biomedic DNA Polymerase I I Cellular DNA polymerase were extracted and purified from normal blo and leukemic blood lymphocytes and 17 lymphoid cells by homogenization in hypotonic buffer followed by Tr ton X 100 high salt extraction of the ext ralysosomal After differential cent ifugation cellular extracts were further pu Iified by DEAE and Sephadex G 200 colu chromatography ΏΝΑ Polymerase DNA polymerase assays were carried out in a final volume of 100 μΐ The assay mixture contained pH 60 Adjustment of pH was carried out after addition of inhibitors which were previously dis solved in dimethyl sulfoxide The final concentration of DMSO was and all control samples included of An enzyme concen ration that catalyzes an incorporation of Mmately was used in the The enzyme was in most cases preincuba ed for minutes with the The reaction was then initiated by the addition of template either synthetic DNA Miles and RNA hybrid at 5 or native activated salmon sperm DNA at μ and endogenous 70S viral 10 μθχ of Englan lyophilized and redissolved in prior to and dATP x 10 with synthetic or all three deoxynucleoside triphosphates x 10 with RNA or DNA templated In some there was no preincu bation of enzyme with In these cases reactions were initiated by adding enzyme to the com plete reaction mixture which included the Samples were withdrawn at the start of incubation and after minutes and termi nated by the addition of 2 of M sodium and precipitated in cold trichloroacetic acid with yeast RNA as The products were collected on Millipore washed extensively with TCA and 1 of M NaCl mixture dried and counted in 2 of and 10 of England in a Packard liquid scintillation In representative concentrations varying from 5 to 10 of compounds 11 and 12 were found to provoke a inhibition S of leukemic polymerase with a synthetic DNA Reaction tern plated by a synthetic RNA template were even more I Representative experiments carried out with native template on g mal and tumor cells polymerase showed a higher susceptibility of th tumor enzymes to the tested For a concentration of about of compound 12 gives a per cent inhibition of tu polymerase while it is practically inactive on normal polymerase Other biological characteristics displayed by the new rifamycin deri vatives include inhibition of focus formation on rat and man cells by the Moloney Kirsten strain of murine sarcoma selective inhibition of virus production by already transformed mous I and human detection of revertant cells using the murine sarco ma virus transformed mouse and rat cell systems The hydrazone compounds of the present invention have moreover confirmed their selective toxicity for virus transformed cells of rat and human origin when tested for colony forming In studies to determine the effect of the compounds in inhibiting fo cus formation by Moloney sarcoma virus on tissue cultures the following procedure is cell cultures are grown in 250 plastic flasks in growth medium consisting of minimal essential medium with fetal bovine Cell counts are made with a Coulter counter after pending the cells with and diluting in growth Moloney murine sarcoma as a tumor is It is passaged four times in a high passage mouse embryo cell line and assayed for units in In I I conducting the a modification of the method described by from 10 minutes to some the crude compound is recovered by concentrating or evaporating the The purification of these derivatives does not represent a particular problem for those skilled in the organic field and is generally effected by lizing from a suitable solvent which for instance may be selected from lower lower acyl esters of lower akanols or As it appears from the structure formula of the invention compounds a quite large number of derivatives falling within the scope of the invention can be synthetized by selecting appropriate hydrazine In some instances when the hydrazine contains a strongly acidic group such as the final rifamycin derivative is hydro lized in the position 27 to the corresponding droxy compound during the hydrazone The preparation of the following compounds is given as example of a convenient performing the invention and is not to be intended as limitative of the scope of the General method of preparation of the To a tetrahydrofuranK solution of moleiC of SV or its derivative or the co pound mole of a hydrazone is added at room temperature under After agitation for a period of time varying from 10 minu tes to 3 hours a drop of the solution is tested by thin layer chroma tography on control the disappearing of the starting compound and the formation of the end ance After complete of the carbonyl compound the solution is concentrated to dryness and the crude compound is then recovered an purified by crystallizing from a solvent or by column In table I the data of some representative compoun are In the table only compounds in which is acetyl are and no hydrogenated when not otherwise The starting compound for preparing hexahydro derivatives of the rifamycins of formula 29 hexahydro formylrifamycin S is obtained in the following Twenty grams of rifamycin S suspended in 600 of dry ethanol are hydrogenated in a Parr bomb with 2 of as the for 3 hours at room temperature under a hydrogen pressure of about 5 at After filtering off the catalyst the solution is evaporate to dryness and the crude product dissolved in t is maintained under stirring with 18 of MnO at room The inorganic precipitate is filtered off and after concentration of the filtrate to a small volume the mixture is taken up with ethy acetate and washed with The organic layer is drie over Na SO and after evaporation gives 8 of hexahydrorifamycins 2 4 The product is then converted into the corresponding by following essentially the same method described in example 5 of British Patent The crude product may be purified by column chromatography of its chloroform solution through and by eluting with chloroform containing of The compound recovered by evaporation of the chromatographed solution is SV melting at O o H t Crystallization or chromatographic solvent H Ethyl acetate Carbon etrachloride Methanol Carbon tetrachlo CI Methanol Ligroin Methanol 37 3 H Methanol Crystallization Yield chromatographic solvent methanol 90 methanol 63 methanol 80 methanol 73 Crystallization or chromatographic solvent Methanol compounds listed in the foregoing table are given by way of illustration it being intended that other hydrazones are comprised vithin the meaning of the generic formula and are also useful for the purposes which have been For hydrazones are advantageously prepared starting from SV or its derivative or the corresponding hexahydro compounds and hydrazines of the formula Example 1 2 H 3 C cyclohexyl 4 H quinolyl 5 6 H 7 H 8 H 9 12 13 14 15 H 16 H Example H H phenyl H 2 H n l CH3 H H H H H H cy H H H insufficientOCRQuality

Claims (1)

1. 3 A t 1 A cin SV of the formula of and is of 1 to selected from pyridine substituted with 1 groups each independently selected lower alkyl and alk pyrimidine tuted with groups each independently selected from lower hydroxy and pyridazine substituted with groups each independently selected from and substituted with groups one of and is hydrogen or of 1 to 4 the other not be and the hexahydro and A process for preparing compounds of claim 1 vhich consists in contacting a ormylrifamycin of the formula where has the same above or the corresponding hexahydro derivative with a hydrazine of the therein and have the same significance as above April 1972 26 insufficientOCRQuality