RUFOMYCINS AND DERIVATIVES THEREOF USEFUL AS INHIBITORS OF MULTI-DRUG RESISTANCE ASSOCIATED PROTEIN-1 (MRP-1)

Abstract: The present invention provides rufomycin factors and derivatives thereof useful as inhibitors of multi-drug resistant protein. As such these compounds are useful in combination therapy for the treatment of multi-drug resistant diseases. The present invention also provides novel rufomycin factors and derivatives thereof and their pharmaceutical compositions.

Description: RUFOMYCINS AND DERIVATIVES THEREOF USEFUL AS INHIBITORS OF MULTI-DRUG RESISTANCE ASSOCIATED PROTEIN-1 (MRP-1) The present invention relates to pharmaceutically active compounds, cyclic heptapeptides commonly known as rufomycins, and derivatives thereof, useful for inhibiting multi-drug resistance. These compounds enhance the efficacy of chemotherapeutic agents. Some of these compounds are new. The present invention also relates to these new compounds and to pharmaceutical compositions thereof. Along with surgery and radiation therapy, chemotherapy continues to be an effective therapy for many cancers. In fact, several types of cancer are now considered to be . curable by chemotherapy and include Hodgkin ' s disease, large cell lymphoma, acute lymphocytic leukemia, testicular cancer and early stage breast cancer. Other cancers such as ovarian cancer, small cell lung and advanced breast cancer, while not yet curable, are exhibiting positive response to chemotherapy. In addition, chemotherapy is used in the treatment of malaria. One of the most important unsolved problems in cancer and malaria treatment is drug resistance. Drug resistance may be intrinsic resistance at the time of treatment by chemotherapy or may develop, that is, be acquired during chemeotherapy . These conditions are referred to as multi- drug resistance (MDR) and are believed to arise by a number of different mechanisms. Medical Research Reviews, 11 (2), 185-217 (1991); Advances in Pharmacology, 21, 185-220 (1990); and Annu. Rev. Biochem. , 62:385-427 (1993). Multi-drug resistance has developed to a broad spectrum of oncolytic agents and is a major impediment to effective chemotherapy. For example, multi-drug resistance has developed to anthracyclines , vinca alkloids, epipodophyllotoxins, antibiotics, antimicrotubule drugs, protein synthesis inhibitors, DNA intercalators, and others. Anthracyclines represent an important class of oncolytic agents. Doxorubicin, an anthracycline, which is also known in the art as ADRIAMYCIN™, is a drug of choice in the clinical management of breast cancer. Therapy with anthracyclines such as doxorubicin is complicated by the appearance of the anthracycline resistant phenotype which limits or negates the oncolytic activity of doxorubicin. Taxol (PACLITAXE ™) is an oncolytic taxane derivative. This compound, and later derivatives thereof, are useful in the treatment of metastatic ovarian carcinoma which is refractory to first-line chemotherapy. Topoisomerase inhibitors represent a further class of oncolytic agents. Epipodophyllotoxins such as ETOPOSIDE® and TENIPOSIDE® are topoisomerase inhibitors which are useful in the therapy of neoplasms of the testis, small-cell lung and other lung, breast, Hodgkin ' s disease, non- Hodgkin ' s lymphomas, acute granulocytic leukemia and Karposi ' s sarcoma. The therapeutic utility of the epipodophylotoxins is limited by the appearance of the epipodophyllotoxin resistant phenotype. Multi-drug resistance is believed to arise by a number of different mechanisms. One form of multi-drug resistance is mediated by P- glycoprotein, an energy dependent, efflux membrane pump with broad substrate specificity. Ann. Rev. Biochem. , 62 , 385- 427, (1993) and Anticancer Res., 15, 331-336 (1995). P- glycoprotein is distributed in normal tissue, such as the gastrointestinal tract, the liver, the kidney, and the central nervous system. Apical expression of P-glycoprotein results in decreased drug absorption and enhanced elimination. Anticancer Res . , 15 , 331-336 (1995). Expression of P-glycoprotein at the level of the blood-brain barrier is a critical factor in preventing the some drugs from entering the central nervous system. J . Clin . Invest . , 97, 2517-2524 (1996). While P-glycoprotein associated MDR is a major determinant in tumor cell resistance to chemotherapeutic agents, it is clear that the phenomenon of MDR is multifactorial and involves a number of different mechanisms . One such alternative pathway for resistance to anthracyclines involves the emergence of a 190 kD protein (pl90) that is not P-gp, now commonly known as multi-drug resistance associated protein-1 (MRP-1) . See, T. McGrath, et al . , Biochemical Pharmacolology, 38:3611 (1989). The protein pl90 is not found exclusively on the plasma membrane but rather appears to be localized predominantly in the endoplasmic reticulu . See, e.g., D. Marquardt, and M.S. Center, Cancer Research, 52:3157 (1992). It possesses a nucleotide binding domain that is homologous with the ATP binding site of P-glycoprotein. See, D. Marquardt, et al . , Cancer Research, 50:1426 (1990). The mechanism(s) utilized by pl90 to confer resistance to ADRIAMYCIN™ is not well understood but may involve the intracellular redistribution of ADRIAMYCIN™ away from the nucleus. See, D. Marquardt and M.S. Center, supra . ADRIAMYCIN™ is an inhibitor of topoisomerase II. W.T. Beck, Bulletins in Cancer, 77:1131 (1990) . Redistribution of ADRIAMYCIN™ away from the nucleus would therefore be an important component in cellular resistance to this drug. The studies published to date on pl90 have utilized cell lines selected in vitro for resistance to ADRIAMYCIN™. T. McGrath, et al . , supra ; D. Marquardt and M.S. Center, supra ; and D. Marquardt, et al . , Cancer Research, supra . The association of pl90 with drug resistance was made by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of radioactive extracts prepared from Adriamycin- resistant HL60/Adr human leukemia cells labeled with 8- azido-alpha [32p]ATP. See, T. McGrath, et al . , supra . The drug-resistance phenotype conferred by pl90 is not limited to the anthracyclines. Epipodophyllotoxin resistance is linked to pl90 expression. H 60/S cells treated with ADRIAMYCIN™ and ETOPOSIDE™ had IC 50 of 0.011 μg/mL and 0.39 μg/mL, respectively. HL60/Adr cells (a HL60-derived cell line which is resistant to doxorubicin) treated with Adriamycin and Etoposide had IC 50 of 2.2 μg/mL and >10 μg/mL, respectively. HL60/S and HL60/Adr cell lines do not express P-glycoprotein. HL60/Adr expresses pl90. Thus, resistance to the anthracyclines and epipodophyllotoxins results from pl90 expression. Compounds capable of inhibiting multi-drug resistance enhance the efficacy of cancer chemotherapeutics, that is, oncolytic agents, and of malaria chemotherapeutics to which resistance has developed. Thus, there is a need for compounds which can treat, that is, decrease, reverse, inhibit and/or prevent multi-drug resistance. The rufomycins comprise a family of structurally unique cyclic peptides which are secondary metabolites of actinomycetes, particularly Strepto yces atratus and S. islandicus . The rufomycins are structurally characterized by a cyclic heptapeptide containing unusual amino acid residues such as nitrotyrosine and tryptophan in which the indole nitrogen is alkylated with an isoprene or a modified isoprene unit. The present invention provides a method of treating multi-drug resistant diseases comprising administering to a patient in need thereof an effective amount of a therapeutic agent and a multi-drug resistance inhibitory amount of a compound of the formula I wherein R is hydrogen, R' is a radical selected from the group consisting of wherein R 4 is selected from the group consisting of hydroxy, Cι-C 6 alkoxy, and -NRR 6 wherein R 5 is selected from the group consisting of hydrogen, Ci-Cε alkyl substituted Cι-C 6 alkyl, benzyl, and substituted benzyl, and R 6 is selected from the group consisting of hydrogen, Ci-Cε alkyl substituted Cι-C 6 alkyl, aralkyl, heteroaryl, heteroarylalkyl, and heterocyclic ; or R and R' taken together form a divalent radical selected from the group consisting of Ri and R 2 taken together with the atoms to which they are attached form a carbon-carbon double bond; or Ri and R 2 taken together with the atoms to which they are attached form an epoxide; or Ri is fluoro and R 2 is hydroxy; or Ri is hydroxy or ester and R 2 is hydroxy, fluoro, chloro, bromo, or a radical selected from the group consisting of -NR7R 8 and -X-R 9 wherein R 7 is selected from the group consisting of hydrogen, Ci-Cε alkyl, and benzyl; Rs is selected from the group consisting of hydrogen, Ci-Cg alkyl, substituted Ci-Cε alkyl, C 3 -C7 cycloalkyl, C 5 -C 7 cycloalkenyl , aryl, aralkyl, heteroaryl, heteroarylalkyl , and heterocyclic ; or R 7 and R 8 together with the nitrogen to which it is attached form a piperidine ring, pyrrolidine ring, morpholine ring, thiomorpholine ring, piperazine ring, or a 1,2,3,4- tetrahydroquinoline ring; X is O or S; Rg is selected from the group consisting of Ci- C s alkyl, substituted Cι-C 6 alkyl, C 3 -C 7 cycloalkyl, C5-C7 cycloalkenyl, aryl, aralkyl, heteroaryl, heteroarylalkyl; R 3 is selected from the group consisting of hydrogen, ester, Cι-C 6 alkyl, benzyl, substituted benzyl, and -CH 2 C(0)NH 2 ; and the stereoisomers and pharmaceutical salts thereof . More specifically, the present invention provides a method of treating cancer comprising administering to a patient in need thereof an effective amount of a oncolytic agent and a multi-drug resistance inhibitory amount of a compound of the formula I. That is, the present invention provides for the use of a compound of formula I for treating cancer by administering to a patient in need thereof an effective amount of a oncolytic agent and a multi-drug resistance inhibitory amount of a compound of the formula I. The present invention provides novel compounds of formula II: wherein R is hydrogen, R' is a radical selected from the group consisting of wherein R is selected from the group consisting of hydroxy, Cι-C 6 alkoxy, and -NR 5 R 6 wherein R 5 is selected from the group consisting of hydrogen, C I -C Θ alkyl substituted Cι-C 6 alkyl, benzyl, and substituted benzyl, and R 6 is selected from the group consisting of hydrogen, Ci-Cβ alkyl substituted Cι-C 6 alkyl, aralkyl, heteroaryl, heteroarylalkyl, and heterocyclic ; or R and R' taken together form a divalent radical selected from the group consisting of Ri and R taken together with the atoms to which they are attached form a carbon-carbon double bond; or Ri and R 2 taken together with the atoms to which they are attached form an epoxide; or Ri is fluoro and R 2 is hydroxy; or Ri is hydroxy or ester and R 2 is hydroxy, fluoro, chloro, bromo, or a radical selected from the group consisting of -NR 7 R 8 and -X-R 9 wherein R 7 is selected from the group consisting of hydrogen, Ci-Cε alkyl, and benzyl; Rs is selected from the group consisting of hydrogen, Cι-C 6 alkyl, substituted Ci-Cε alkyl, C 3 -C7 cycloalkyl, C 5 -C7 cycloalkenyl, aryl, aralkyl, heteroaryl, heteroarylalkyl, and heterocyclic; or R and R 8 together with the nitrogen to which it is attached form a piperidine ring, pyrrolidine ring, morpholine ring, thiomorpholine ring, piperazine ring, or a 1,2,3,4- tetrahydroquinoline ring; X is 0 or S; R 9 is selected from the group consisting of Ci- C 6 alkyl, substituted d-C 6 alkyl, C 3 -C 7 cycloalkyl, C 5 -C 7 cycloalkenyl, aryl, aralkyl, heteroaryl, heteroarylalkyl; R 3 is selected from the group consisting of hydrogen, ester, Cι-C 6 alkyl, benzyl, substituted benzyl, and -CH 2 C(0)NH 2 ; provided that when R 3 hydrogen, R is hydrogen, and R' is the radical then Ri and R 2 cannot be taken together with the atoms to which they are attached to form a carbon- carbon double bond or taken together with the atoms to which they are attached to form an epoxide; further provided that when R and R' are taken together to form the divalent radical then Ri and R 2 cannot be taken together with the atoms to which they are attached to form a carbon- carbon double bond or taken together with the atoms to which they are attached to form an epoxide; and the stereoisomers and pharmaceutical salts thereof. Also, the present invention provides pharmaceutical compositions comprising a compound of the present invention and a pharmaceutically acceptable dilutent. That is, the present invention provides for the use of a pharmaceutical compositions comprising a compound of formula I or II and a pharmaceutically acceptable dilutent for the treatment of multi-drug resistant diseases, in particular, cancer. In particular, the present invention provides pharmaceutical compositions comprising a compound of formula II and a pharmaceutically acceptable dilutent. As used herein the following terms have the meanings indicated: the term "Ci-Cβ alkyl" represents a straight or branched alkyl chain having from one to six carbon atoms, and includes methyl, ethyl, propyl , iso-propyl, butyl, iso- butyl, sec-butyl, t-butyl, pentyl , hexyl , and the like; the term "substituted Cι-C 6 alkyl" refers to a Cι-C 6 alkyl independently substituted with 1 to 2 substituents selected from the group consisting of hydrogen, hydroxy, amino, substituted amino, -NRioR wherein Rχ 0 and Rn are independently Cι-C 6 alkyl, and Cι-C 6 alkoxy; the term "C 3 -C7 cycloalkyl" refers to a saturated cyclic alkyl group having from three to seven carbon atoms and includes, cyclopropyl, cylcobutyl, cyclopentyl, cyclohexyl, and cycloheptyl; the term "C 5 -C7 cycloalkenyl" refers to an unsaturated cyclic alkyl group having from five to seven carbon atoms and includes, cyclopentenyl , cyclohexenyl , and cycloheptenyl ; the term "C 1 -C 6 alkoxy" represents a straight or branched alkyl chain having from one to six carbon atoms attached to an oxygen atom, and includes methoxy, ethoxy, propoxy, iso-propoxy, butoxy, iso-butoxy, sec-butoxy, t- butoxy, pentoxy, hexoxy, and the like; the term "halo" or "halogen" encompasses chloro, fluoro, bromo and iodo; the term "substituted benzyl" refers to a benzyl, having from 1 to 2 substituents independently selected from the group consisting of hydrogen, halogen, Ci-Cβ alkyl, and Cι-C 6 alkoxy; the term "aryl" refers aromatic hydrocarbon, specifically including phenyl and naphthyl having from 1 to 2 substituents independently selected from the group consisting of hydrogen, halogen, -OCF 3 , C 2 -C 5 -alkenyl , phenyl, substituted phenyl, -CF 3 , acetamido, Cι-C 6 alkyl, Ci- C δ alkoxy, and sulfona ide; the term "C -C 5 -alkenyl" refers to a unsaturated straight chain or branched alkenyl group having from two to five carbon atoms and includes, vinyl, allyl, prop-1-enyl, crotyl, and the like; the term "aralkyl" refers to aryl group attached to a C2-C 8 straight chain alkylene; the term "C 2 -C 8 -alkenyl" refers to a straight chain alkylene group having from two to eight carbon atoms and includes, ethylene, propylene, butylene, pentylene, hexylene, heptylene, and octylene; the term "heteroaryl" refers to a 5 or 7 membered saturated heterocyclic group containing from one to four N, O or S atom(s) or a combination thereof, which is optionally substituted with 1 to 2 substituents independently selected from the group consisting of hydrogen, halogen, -OCF 3 , phenyl, -CF 3 , Cι-C 6 alkyl, and Cι-C 6 alkoxy, the term includes, but is not limited to thiophene, pyrrole, furan, oxazole, pyrazole, imidazole, thiazole, purine, triazole, pyridine, quinoline, isoquinoline, pyridazine; the term "heteroarylalkyl" refers to an heteroaryl group attached via a Cι-C 8 straight chain alkylene; the term "Cι-C 8 -alkenyl" refers to a straight chain alkylene group having from one to eight carbon atoms and includes, methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, and octylene; the term "heterocyclic" refers to a 5 or 6 membered saturated heterocyclic group containing from one to two N, 0 or S atom(s) or a combination thereof, which is optionally substituted with 1 to 2 substituents independently selected from the group consisting of hydrogen, benzyl, substituted benzyl, phenyl, substituted phenyl, carboxy, carbamoyl, Ci- Cβ alkyl, and Ci-Cβ alkoxy, the term heterocyclic specifically includes pyrrolidine, morpholine, thiomorpholine, piperidine, piperazine, dioxane, and the like; the term "ester" includes amino acid esters, such as the esters of glycine, alanine, phenylalanine, glutamine, aspartamine, lysine, arginine, glutamic acid, and aspartic acid, and the like; alkyl esters, such as acetyl, propionyl, butroyl, iso-butroyl, sec-butroyl, valeroyl, iso-valeroyl, pivavoyl, and the like; ether substituted alkyl esters, such as benzyloxy acetyl and methoxy acetyl, aryl esters, benzoyl and substituted benzoyl, having 1 to 2 substituents independently selected from the group consisting of hydrogen, benzyl, halogen, Ci-C δ alkyl, and Cι-C 6 alkoxy; esters of alkyl and aryl diacids, such as the esters of malonic acid, succinic acid, glutaric acid, adipic acid pimelic acid, maleic acid fumaric acid, phthalic acid, and the like; the term "pharmaceutically-acceptable addition salt" refers to an acid addition salt or a base addition salt. The present compounds and the intermediates thereof form pharmaceutically acceptable acid addition salts with a wide variety of organic and inorganic acids and pharmaceutically acceptable base addition salts with a wide variety of organic and inorganic bases. These salts include the physiologically acceptable salts which are often used in pharmaceutical chemistry. A pharmaceutically-acceptable salt is formed from a pharmaceutically-acceptable acid or a pharmaceutically-acceptable base as is well known in the art. Such salts are also part of this invention. Typical inorganic acids used to form acid addition salts include hydrochloric, hydrobromic, hydriodic, nitric, sulfuric, phosphoric, hypophosphoric , metaphosphoric, pyrophosphoric, and the like. Acid addition salts derived from organic acids, such as aliphatic mono and dicarboxylic acids, phenyl substituted alkanoic acids, hydroxyalkanoic and hydroxyalkandioic acids, aromatic acids, aliphatic and aromatic sulfonic acids, may also be used. Such pharmaceutically acceptable salts thus include acetate, phenylacetate, trifluoroacetate, acrylate, ascorbate, benzoate, chlorobenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, methylbenzoate, o-acetoxybenzoate, naphthalene-2-benzoate, bromide, isobutyrate, phenylbutyrate, β-hydroxybutyrate, butyne-1 , 4-dicarboxylate, hexyne-1, 4-dicarboxylate, caprate, caprylate, cinnamate, citrate, formate, fumarate, glycollate, heptanoate, hippurate, lactate, malate, maleate, hydroxymaleate, malonate, mandelate, mesylate, nicotinate, isonicotinate, nitrate, oxalate, phthalate, teraphthalate, propiolate, propionate, phenylpropionate, εalicylate, sebacate, succinate, suberate, benzene-sulfonate, p- bromobenzenesulfonate, chlorobenzenesulfonate, ethanesulfonate, 2-hydroxyethanesulfonate, methanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, p- toluenesulfonate, xylenesulfonate, tartarate, and the like. Typical bases commonly used for formation of salts include ammonium hydroxide and alkali and alkaline earth metal hydroxides and carbonates, as well as aliphatic and aromatic amines, aliphatic diamines and hydroxy alkylamines . Bases especially useful in the preparation of addition salts include ammonium hydroxide, sodium hydroxide, potassium hydroxide, sodium bicarbonate, sodium carbonate, potassium carbonate, potassium bicarbonate, calcium hydroxide, methylamine, diethylamine, ethylene diamine, cyclohexylamine and ethanolamine . The present invention relates to stereoisomers of the compounds of formula I and II. Where designated herein, the Cahn-Prelog-Ingold designations of (R) - and (S)- are used to refer to specific isomers. As used herein the designation " ' " indicates a bond that protrudes forward out of the plane of the paper and the designation " "' " indicates a bond that protrudes backwards out of the plane of the paper. The specific stereoisomers can be prepared by stereospecific synthesis or can be resolved and recovered by techniques known in the art, such as, chromatography on chiral stationary phases, and fractional recrystallization of addition salts formed by reagents used for that purpose. Useful methods of resolving and recovering specific stereoisomers are known in the art and described in Stereochemistry of Organic Compounds, E.L. Eliel and S.H. Wilen (Wiley-Interscience 1994) and Enantiomers, Racemates, and Resolutions, J. Jacques, A. Collet, and S.H. Wilen (Wiley-Interscience 1981) . As with any group of pharmaceutically active compounds, some groups are preferred in the end use application. Preferred embodiments of the present invention are given below: Compounds wherein R 3 is hydrogen are preferred; Compounds wherein R and R' taken together form the divalent radical are preferred; Compounds wherein R is hydrogen and R' is the radical are preferred; Compounds wherein Ri is fluoro and R 2 is hydroxy are preferred; Compounds wherein Ri is hydroxy and R 2 is fluoro, chloro, or bromo are preferred; Compounds wherein Ri is hydroxy and R 2 is -NR7R 8 wherein R 7 is hydrogen and R 8 is selected from the group consisting of Ci-Cε alkyl, substituted Cι-C 6 alkyl, and aralkyl are preferred; Compounds wherein Ri is hydroxy and R 2 is -X-R 9 wherein X is 0 and R 9 is selected from the group consisting of Ci-Cε alkyl, substituted Ci-Cε alkyl, aryl, and aralkyl are preferred; Compounds wherein Ri is hydroxy and R 2 is -X-R 9 wherein X is S and Rg is selected from the group consisting of Ci-Cβ alkyl, substituted Cι-C 6 alkyl, C 3 -C7 cycloalkyl, aryl, and aralkyl are preferred. For compounds wherein Ri is ester amino acid esters, such as the esters of glycine, alanine, phenylalanine, glutamine, aspartamine, lysine, arginine, glutamic acid, and aspartic acid, and the like; alkyl esters, such as acetyl, propionyl, butroyl , iso-butroyl, sec-butroyl, valeroyl, iso- valeroyl, pivavoyl, and the like; ether substituted alkyl esters, such as benzyloxy acetyl and methoxy acetyl, aryl esters, benzoyl and substituted benzoyl, having 1 to 2 substituents independently selected from the group consisting of hydrogen, benzyl, halogen, Cι-C 6 alkyl, and Ci- Ce alkoxy; esters of alkyl and aryl diacids, such as the esters of malonic acid, succinic acid, glutaric acid, adipic acid pimelic acid, maleic acid fumaric acid, phthalic acid, are preferred. For compounds wherein R 3 is ester alkyl esters, such as acetyl, propionyl, butroyl , iso-butroyl, sec-butroyl, valeroyl, iso-valeroyl , pivavoyl, and the like; ether substituted alkyl esters, such as benzyloxy acetyl and methoxy acetyl, aryl esters, benzoyl and substituted benzoyl, having 1 to 2 substituents independently selected from the group consisting of hydrogen, benzyl, halogen, Ci- C 6 alkyl, and Cι-C 6 alkoxy; are preferred. Several years ago the isolation, structure determination, and antibiotic properties, specifically inhibiting the growth of mycobacteria, of certain rufomycin factors were reported. See Kakazawa et al . , "Rufomycins", US Patent No. 3,655,879. The present invention encompasses rufomycin factors and derivatives thereof. While the rufomycins can be obtained by synthetic methods well known in the art, they are also conveniently obtained by fermentation. We have discovered and isolated a new strain of rufomycin producing organism from a soil sample. The rufomycin factors used in the present application are obtained by fermentation from this newly discovered, isolated, and biologically-purified Streptomyces macrosporeus , as described in detail below. As used herein the term "biologically-purified" means a bacterium that has been isolated or otherwise separated from its naturally occurring condition to give a purified composition which contains the bacterium. This new Streptomyces macrosporeus has been deposited in the Deutsche Sammlung von Mikroorgansimen und Zellkulturen GmgH (DSMZ) , Mascheroder Weg lb, D-38124 Braunschweig, Germany with the accession number DSM-12818. This particular strain is a robust producer of rufomycin factors, including newly discovered members of the class which were isolated and characterized as described below. The structures were determined largely by spectroscopic techniques and to a lesser extent by derivativization, that is, chemical transformations. The identification of such compounds using techniques such as optical rotation, UV, IR, NMR and mass spectral data and derivativization are well known in the art. See Takita et al . "New Antibiotics, Ila ycins", J. Antibiotics, Ser. A, 15: 46-48, 1962; Hara et al. "Further Studies on the Tryptophan-parts of Ilamycin" , J. Antibiotics, Ser. A 17: 264-266, 1964; Cary et al . "A Study of the Secondary Structure of Ilamycin Bi by 300 MHz Proton magnetic Resonance", FEBS Letters, 17: 145-148, 1971; Kakazawa et al . , "Rufomycins", US Patent No. 3,655,879. Fermentation gave compounds 1 and 2 while compound 3 was not found. 1 compound of formula I compound of formula I in which R is hydrogen and R' in which R is hydrogen and R' is the radical is the radical RI and R2 are taken together RI and R2 are taken together with the atoms to which they with the atoms to which they are attached to form an are attached to form an epoxide carbon-carbon double bond and R3 is hydrogen and R3 is hydrogen Compounds 1 and 2 were identified by UV, IR, NMR and mass spectral data (NMR data, below) . Compound 1: Yellow powder; [α] D -73.4 (c 0.095, MeOH); MS (ES) : m/z 1012.5 (M+H) + ; IR (CHC1 3 ): V max 3429 (br) , 3349 (br) , 3312 (br) , 1648, 1540 and 1505 cm "1 ; UV (MeOH): A^ 352 (ε 2837), 282 (ε 10875) and 221 (ε 50450) nm. Compound 2: Yellow powder; [α] D -95.1 (c 0.095, MeOH); MS (ES) : m/z 1028.4 (M+H) + ; IR (CHCI 3 ): V m a x 3345 (br) , 3312 (br) , 1649, 1540 and 1506 cm "1 ; UV (MeOH): ^ ax 357 (ε 2840), 278 (ε 11064) and 221 (ε 50865) nm. Compounds 4-7 were also isolated and characterized. Compounds 4 and 5 showed identical molecular composition C 54 H 75 N 9 Oi 2 as determined by high resolution FABMS (Calculated for C 54 H 76 N 9 Oi 2 1042.5613 (M+H) , observed 1042.5599 (Δ +1.4 mmu) and 1042.5629 (Δ -1.6 mmu) , respectively. compound of formula I in which R and R' are the divalent radical 4 has α-OH 5 has β-OH RI and R2 are taken together with the atoms to which they are attached to form an epoxide and R3 is hydrogen Detailed analysis of 1 H, 13 C and 2D NMR (COSY, TOCSY, HSQC, HMBC and ROESY) spectra and chemical transformations enabled elucidation of the structure for these factors. Compound 4: Yellow powder; [α] D -61.2 ( c 0.098, EtOH); MS (ES) : m/z 1064.4 (M+Na) + , m/z 1040.5 (M-H) " ; IR (CHC1 3 ): V max 3470 (br) , 3349 (br) , 1680 (sh), 1639 and 1540 cm "1 ; UV (EtOH): A™ ax 361 (ε 2674), 280 (ε 9996) and 222 (ε 46722) nm; 1 H and 13 NMR: Listed in Tables 1 and 2. Compound 5: Yellow powder; [α] D -86.6 (c 0.104, EtOH); MS (ES) : m/z 1042.5 (M+H) + , m/z 1040.5 (M-H) " ; IR (CHC1 3 ): V max 3419 (br) , 3304 (br) , 1689 (sh), 1661, 1638, 1540 and 1491 cm "1 ; UV (EtOH): λ max 361 (ε 2710), 276 (ε 10144) and 221 (ε 47808) nm. 1 H and 13 NMR for Compounds 4 and 5 are listed in Tables 1 and 2, below. The "" " H NMR spectra of 4 and 5 are very similar to each other and to the spectrum of 2. The results from the COSY and TOCSY spectra revealed the presence of spin systems for five amino acid residues: 2-amino hexenoic acid (AHA), prenylated tryptophan, N-methylleucine, nitrotyrosine and alanine that are found in the known rufomycins. The remaining two amino acids exhibited signals in the 1 H and 13 C NMR spectra reminiscent of modified leucine residues. The first of the two amino acids showed 1 H and 13 C NMR signals (4: δ αH 5.28 and δ αc 53.3, δ βH 2.02 , 1.74 and δ βc 35.7, δγ H 1.57 and δγ C 25.3, δ δH 0.98 and δδc 23.6 and δ εH 0.91 and δ εC 21.8 and 5: δαH 5.40 and δ αC 56.0, δ βH 2.05, 1.89 and δ βc 37.6, δγ H 1.83 and δγc 25.1, δ δH 0.95 and δ δc 23.8 and δ εH 0.93 and δ εc 21.9) characteristic of leucine with the exception of absence of the amide proton resonance. The second amino acid displayed H and 13 C NMR signals (4: δ αH 3.86 and δ αc 59.8, δ βH 2.52, 1.99 and δ βc 26.2, δγ H 2.24 and δγ C 34.6, δ δH 4.83 and δ^ 81.3 and δ εH 1.11 and δ εc 16.4 and 5: δ αH 3.81 and δ αC 59.8, δ βH 2.43, 1.74 and δ βc 31.9, δγ H 2.71 and δγ C 33.3, δ δH 4.75 and δβc 82.0 and δ εH 1.00 and δ εc 17.5) characteristic of another leucine moiety with the exceptions of absence of the amide and one of the methyl resonances . The 1 H and 13 C NMR spectra showed resonances (δ H 3.32 and 3.30, δ c 38.8 and 39.1 for 4 and 5, respectively) for one additional N-CH 3 group, unaccounted thus far, could be assigned to the leucine residue adjacent to the alanine by analogy with the known rufomycins. That this was so was evidenced by the HMBC cross peaks that were observed from the alanine amide carbonyl (δ c 172.9) to the leucine proton (δ H 3.86) and N-CH 3 (δ H 3.32) . The missing methyl group was replaced by a hydroxyl-bearing methine carbon (5 H 4.83 and 4.75, δ c 81.3 and 82.0 for 4 and 5, respectively) as revealed by the COSY and TOCSY sequence (Table 1) . The presence of a carbinol functionality was further confirmed by the acetylation of 4 and 5 with acetic anhydride, pyridine and catalytic amount of dimethylaminopyridine to diacetates 8 and 9, respectively. 8 having α OAc 9 having β OAc Comparison of the 1 H NMR data of 8 and 9 with those of 4 and 5 revealed significant down field shift (-1 ppm) for the hydroxy-bearing methine (δ-proton) due to acylation. Complete 1 H and 13 C NMR resonance assignments of 8 and 9 are summarized in Tables 3 and 4, below. Treatment of 4 and 5 with 4 M hydrochloric acid at room temperature afforded a single dehydro product, 10 in which the epoxide was also converted to a chlorohydrin (FABMS: calculated for C 54 H 75 N 9 Oι 2 Cl m/z 1060.527468 (M+H) , observed m/z 1060.526600 (Δ +0.9 mmu). 10 the comound of formula I and II in which R and R' are taken together to form the divalent radical RI is hydroxy, R2 is chloro, and R3 is hydrogen The signals in the 1 H and 13 C NMR spectra (Tables 5 and 6, below) remained the same for all the amino acid residues, except for the modified leucine unit located next to the alanine residue. The most obvious difference in 10 when compared to 4 and 5 was the replacement of the carbinol functionality (δ H 4.83 and δ c 81.3 in 4 and δ H 4.75 and δ c 82.0 in 5) by a trisubstituted double bond (δ H 5.81 and δ c 118.4). Accordingly, the resonance due to the methyl doublet (δ H 1.11 and 1.00 in 4 and 5, respectively) adjacent to the carbinol carbon was replaced by an vinylic methyl resonance (δ H 1.73). Reduction of 4 and 5 with sodium cyanoborohydride in the presence of acetic acid produced a single deoxy product 11. 11 the comound of formula I and II in which R and R' are taken together to form the divalent radical RI and R2 are taken togehter to form an epoxide and R3 is hydrogen This product was fully characterized by mass (M+H m/z 1026.8) and 1-and 2-DNMR data (Tables 3 and 4, below). When compared to 4 and 5 the NMR spectra of 11 showed a set of diastereotopic methylene protons at δ H 3.37, 3.01 48.6) in the place of the carbinol proton. The HMQC spectrum correlated these signals to a carbon resonance at 48.6 ppm. The above 1 H and 13 C shifts suggest that the methylene is attached to a nitrogen. On the basis of these observations, it was concluded that 4 and 5 have an amino acetal moiety, presumably formed between the amino group of the leucine adjacent to AHA and the aldehyde group derived by the oxidation of one of the methyl groups of N-methylleucine adjacent to alanine (occurrence of such aldehyde in this class of compounds has been documented, e.g. 3) resulting in the construction of a six-membered ring in addition to the macrocyclic ring. The HMBC correlations (Fig 1) involving these two amino acid units augmented the above observation; a three bond correlation observed between the amino acetal carbon (δ 81.3) and the α-proton (δ 5.28) of leucine adjacent to AHA unit is of significance. With amino acid AHA as a starting point, the long range 13 C to 1 H correlations observed in the HMBC spectrum between the carbonyl and the adjacent amino acid amide proton and or proton attached to the α carbon (Figure 1) unambiguously established the amino acid sequence in 4 and 5 as found in known rufomycins . Figure 1 Selected HMBC Correlations (C to H) for compound 5 The additional six-membered ring present in 4 and 5 induced significant conformational changes to the macrocycle as indicated by the dramatic proton and carbon chemical shift differences observed for the N-CH 3 group and α-proton of the N-methylleucine residue next to alanine. The magnitude of shielding experienced by one of the methylene protons of the other N-methylleucine residue adjacent to tryptophan also showed significant changes between the mono- and the bi-cyclic series. (Tables 1 and 2, below) . The origin of 4 and 5 is most probably rufomycin A (3), which was not isolated in the present work. Considering that 4 and 5 are derived from the intermediate aldehyde 3, it is logical to interpret that both the compounds differ only in the stereochemistry at the amino acetal carbon. This was evidenced by the conversion of 4 and 5 to 11 as described above. The stereochemical distinction at the amino acetal carbon of 4 and 5 will be addressed after discussing the structure determination of the new peptide 6. compound of formula I in which R and R' are the divalent radical RI and R2 are taken together with the atoms to which they are attached to form an epoxide and R3 is hydrogen Compound 6 has the identical molecular formula C 5 H7 5 N 9 Oi 2 as that of 4 and 5 as determined by high resolution FABMS (Calculated for C 54 H7 6 N 9 Oi 2 1042.5613 (M+H), observed 1042.5589 (Δ +2.4 mmu) . Compound 6: Yellow powder ; [α] D -64.1 (c 0.097, EtOH); MS (ES): m/z 1064.4 (M+Na) + , m/z 1040.5 (M-H) " ; IR (CHCl 3 : 3471 (br) , 3355 (br. 3286 (br) , 1685 (sh) , 1638 and 1540, 1490 cm "1 ; UV (EtOH): λ ma χ 362 (ε 2758), 280 (ε 10121) and 222 (ε 47011) nm. X H and 13 NMR of Compound 6 is listed in Tables 1 and 2, below. Detailed analysis of 1-and 2-D NMR data revealed identical amino acid composition for 6 as in 4 and 5. Compound 6 also contained an amino acetal functionality as demonstrated by the 1 H and 13 C NMR data and conversion to the diacetate 12. \ 12 Treatment of 6 with hydrochloric acid produced a dehydro derivative which is identical in all respects (MS, X H NMR) with 10, a product obtained from 4 and 5 under identical conditions. Treatment of 6 with sodium cyanoborohydride and acetic acid, however, produced 13 which is chromatographically and spectroscopically different from 11 obtained from 4 and 5 under identical conditions. 13 the comound of formula I and II in which R and R' are taken together to form the divalent radical RI and R2 are taken togehter to form an epoxide and R3 is hydrogen The difference between 11 and 13 were mostly associated with the X H and 13 C shifts (Tables 5 and 6, below) of N- methylleucine next to alanine. The combined results of dehydration and reduction established that the N- methylleucine of 6 possesses opposite stereochemistry at the γ-carbon when compared to 4 and 5. The relative stereochemistry at the γ-and δ-carbons of the amino acetal containing N-methylleucine unit were assigned by proton coupling constant analysis and interpretation of differential NOE data. All three isomeric peptides showed a very small coupling constant between the γ-and δ-protons, suggesting axial-equatorial, equatorial- equatorial and equatorial-axial relationships between these protons. Further stereochemical distinction between 4 and 5 and 6 at the γ-carbon became evident by the coupling constant analysis of the reduction products 11 and 13. In the case of 11, the γ-proton showed two small couplings (4 and 4.5 Hz) to the δ-protons indicating that this proton is oriented equatorial or α and the methyl group is oriented axial or β . In the case of 13, the γ-proton showed one large (11.5 Hz) and one small (3.5 Hz) coupling to the δ-protons indicating that this proton is oriented axial, or β, and the methyl group is oriented equatorial or α. Accordingly, in the differential NOE spectrum of 11, irradiation of the α-proton of the amino acid produced a significant enhancement of the γ-methyl protons. Conversely, in the differential NOE spectrum of 13, irradiation of the α-proton of the amino acid produced a significant enhancement in the γ-proton (Table 10) . These results established that in 11 the α-proton and the γ-methyl are in cis-relationship and in 13 the α-proton and the γ- hydrogen are in cis-relationship. Previously, the absolute stereochemistry of rufomycin Bi as depicted in 1 was established by chemical methods and X-ray crystallography (see Iitaka et al . , "An X-ray Study of Ilamycin BI, a Cyclic Heptapeptide Antibiotic", Acta Cryst . , B30: 2817-2825, 1974) . In these studies, all amino acids were shown to have stereochemistry as derived from L-amino acids. Assuming that the new peptides, obtained by fermentation, also are derived from L-amino acids (the sign of the optical rotation of the new peptides are same as that reported for 1-3), the absolute stereochemistry at the γ- carbon could be assigned the (S) -configuration for 4 and 5 and the (R) -configuration for 6. With regard to stereochemistry at the amino acetal carbon of 6, the small coupling constant observed between the γ-and δ-protons warranted axial orientation, or α, of the hydroxyl group and hence the absolute configuration at the amino acetal carbon could be assigned the (R) -configuration. The information derived from the coupling constants has little or no value in assigning the relative stereochemistry at the δ-carbon of 4 and 5 as the γ- and δ-protons are disposed either equatorial-equatorial or equatorial-axial. However, inspection of the Dreiding model indicated that in the diastereomer in which the δ-H is oriented axial it should show a NOE interaction with the axial β-H, provided the six membered ring adopts a perfect chair conformation. Neither 4 nor 5 showed significant NOE interaction between these protons. However, upon close examination of the chemical shifts of β-carbon and the coupling constants displayed by the α-proton of 4, 5 and 6 and their acetates 8, 9 and 12 revealed similarities between 4 and 6 (Tables 7 and 8, below). On this basis the absolute stereochemistry at the amino acetal carbon could be assigned the (R) - configuration for 4 as in 6 and the (S) -configuration for 5 Another rufomycin factor of the present invention is compound 7. 7 the comound of formula I and II in which R is hydrogen and R' is the radical ?OR 4 RI and R2 are taken togehter to form an epoxide, R3 is hydrogen, and R4 is hydroxy Compound 7: Yellow powder; [α] D -68.0 (c 0.097, EtOH); MS (ES) : m/z 1058.5 (M+H) + , m/z 1056.4 (M-H) " ; IR (CHC1 3 ): v max 3432 (br) , 3344 (br) , 3298 (br) , 1683 (sh), 1646 and 1540, 1506 cm "1 ; UV (EtOH): ^ x 354 (ε 3646), 281 (ε 18383) nm. 1 H and 13 NMR of compound 7 are listed in Tables 1 and 2 The molecular formula C 5 H 75 N 9 Oi 3 of new compound 7 was deduced by the high resolution FABMS (Calculated for C 5 H 7 6N 9 Oi 3 m/z 1058.5563 (M+H), observed 1058.5573 (Δ -1.0 mmu) . Analysis of X H, TOCSY and HMQC data (Tables 1 and 2, below) provided evidence for the presence of six of the seven amino acids: 2-aminohexenoic acid, prenylated tryptophan, N-methylleucine, nitrotyrosine, alanine and leucine that are commonly found in known rufomycins. The seventh amino acid showed signals at δ αH 4.93 (δ αc 59.9), δ βH 2.21, 1.87 (δ βc 34.0), δγ H 2.58 (δγ C 36.7), δ εH 1.25 (δ εc 16.7), in addition to signals { 2.70 and δ c 28.8) due to an N-CH 3 group. The mass difference of 16 when compared 3 in conjunction with the chemical shifts observed for the γ- carbon and δ-methyl protons suggested that one of the geminal dimethyl groups of leucine is replaced by a carboxylic acid group. The presence of a carboxylic acid group in 7 was further corroborated by the formation of amides (compounds 14-24) with various amines. Table 2. C NMR data of 4-7 in acetone-d e *Shifts were obtained from a HMQC experiment and reported only for carbons bearing hydrogens . Table 3. 1 H NMR data of 8, 9 and 12 in Acetone-dg Table 4. 13 CNMR Data of 8, 9 and 12 in Acetone-d 6 Table 5 1 H NMR Data of 10, 11 and 13 in Acetone-d 6 Table 6 13 C NMR data of 10 , 11 and 13 in Acetone-d 6 Table 7 Chemical Shift and Coupling Constant Comparison of 4, 5 and 6 Table 8 Chemical Shift and Coupling Constant Comparison of acetates of 8, 9 and 12 Table 9 Chemical shift and Coupling Constant Comparison of 11 and 13 Table 10. Differential NOE data of NMeleu residue of 11 and 13 As is appreciated by those skilled in the art the compounds of formula I encompass the compounds of formula II. Thus, the teaching below relating to the compounds of formula I also provides the compounds of formula II. The rufomycin derivatives of the present invention are prepared according to Reaction Scheme A below. In Reaction Scheme A, all substituents, unless otherwise indicated, are as previously defined. In Reaction Scheme A all reagents are well known and appreciated in the art. Reaction Scheme A The reaction in Reaction Scheme A encompasses the reduction of an epoxide of formula (a) to an olefin and the reaction of an epoxide of formula (a) with nucleophiles . In a reaction to form an olefin, for example, an epoxide of formula (a) is contacted with a suitable epoxide deoxygenating reagent to give a compound of formula I in which Ri and R 2 together with the atoms to which they are attached form a carbon-carbon double bond. Such suitable epoxide deoxygenating reagents are known in the art and include triphenylphosphine, triethylphosphite, and others, see pages 140-142 Larock, Comprehensive Organic Transformations, (VCH New York 1989). The reaction is carried out in a suitable solvent, such as diethyl ether, tetrahydrofuran, and the like. Typically the reaction is carried out at temperatures of from about 0°C to the refluxing temperature of the solvent and require about 1 hour to 48 hours. The product can be isolated and purified by techniques well known in the art, such as filtration, evaporation, extraction, trituration, chromatography, and crystallization. A reaction with a suitable nucleophile, to open an epoxide, are carried out under acidic, neutral, and basic conditions and are well known in the art. For example, under acidic conditions, an epoxide of formula (a) is contacted with a suitable nucleophile to give a compound of formula I. Suitable nucleophiles for epoxide openings under acidic conditions include, hydrochloric acid, hydrobromic acid, hydrogen fluoride-pyridine, and triethylamine trihydrofluoride, and the like. The reaction is carried out in a suitable solvent, such as water, dimethylformamide, dimethylacetamide, acetic acid, dimethylformamide/acetic acid mixtures, water/ dimethylformamide mixtures, and the like. Typically the reaction is carried out at temperatures of from about 0°C to the refluxing temperature of the solvent, with temperatures of about 45°C to 75°C being preferred. Generally, the reaction requires about 1 hour to 4 days. The product can be isolated and purified by techniques well known in the art, such as filtration, evaporation, extraction, trituration, chromatography, and crystallization. For example, under neutral conditions, an epoxide of formula (a) is contacted with a suitable nucleophile to give a compound of formula I. Such suitable nucleophiles for epoxide openings under neutral conditions include, amines of the formula HNR 7 R 8 , which give rise to compounds in which Ri is hydroxy and R 2 is -NR 7 RsR 2 as desired in the end product of formula I. Typically, a five to ten fold molar excess of the amine is used. The reaction is generally carried out in a suitable solvent, such as tetrahydrofuran, methanol, ethanol, isopropanol, butanol, tetrahydrofuran/water mixtures, methanol /water mixtures, ethanol /water mixtures, and the like. Typically the reaction is carried out at temperatures of from about 0°C to the refluxing temperature of the solvent with temperatures of about 40°C to 80°C being preferred. Generally, the reaction requires about 1 hour to 4 days. The product can be isolated and purified by techniques well known in the art, such as filtration, evaporation, extraction, trituration, chromatography, and crystallization. For example, under basic conditions, an epoxide of formula (a) is contacted with a suitable nucleophile to give a compound of formula I. in which Ri is hydroxy and R 2 is -XR 9 . Such suitable nucleophiles for epoxide openings under basic conditions include, alcohols and thiols of the formula HXR 9 , which give compounds in which Ri is hydroxy and R 2 as desired in the end product of formula I. Typically, a two to five fold molar excess of the alcohol or thiol is used. The reaction is generally carried out using a suitable base and in a suitable solvent, such as tetrahydrofuran, methanol, ethanol, isopropanol, butanol, tetrahydrofuran/water mixtures, methanol /water mixtures, ethanol/water mixtures, dimethylformamide, dimethylformamide/water mixtures, dimethylsulfoxide, acetonitrile, and the like. Suitable bases, include sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, potassium t-butoxide, sodium hydroxide, potassium hydroxide, lithium hydroxide, sodium hydride, potassium hydride, and the like. Typically, a slight molar excess to about to five fold molar excess of base over alcohol or thiol is used. The reaction may advantageously be carried out in the presence of a catalyst, such as benzyltriethylammonium chloride, tetrabutylammonium chloride, benzyltriethylammonium hydrosulfate, and the like. Typically the reaction is carried out at temperatures of from about 0°C to about 90°C. Generally, the reaction requires about 8 hour to 120 hours. The product can be isolated and purified by techniques well known in the art, such as filtration, evaporation, extraction, trituration, chromatography, and crystallization. As will be appreciated by those skilled in the art, in an additional step, compounds in which R 3 is other than hydrogen can be prepared by alkylation of the nitrophenol moiety with a molar excess of an alkylating agent. The reaction is generally carried out using a suitable base and in a suitable solvent, such as tetrahydrofuran, methanol, ethanol, isopropanol, butanol, acetone, acetone/water mixtures, dimethylformamide, dimethylformamide/water mixtures, dimethylsulfoxide, acetonitrile, and the like. Suitable bases, include sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, potassium t-butoxide, sodium hydride, sodium hydroxide, potassium hydroxide, lithium hydroxide, and the like. Typically, a two to ten fold molar excess of the base is used. In the case that a strong base, such as potassium t- butoxide or sodium hydride, is used then a slight molar excess of base is preferred. Typically the reaction is carried out at temperatures of from about 20°C to about 100°C. Generally, the reaction requires about 1 hour to 48 hours. The product can be isolated and purified by techniques well known in the art, such as filtration, evaporation, extraction, trituration, chromatography, and crystallization. Also, as will be appreciated by those skilled in the art, in an additional step, compounds in which R 2 is an ester can be prepared by acylation of a compound in which R 2 is hydroxy. The reaction can be carried out using an activated acid or and acid and a coupling reagent. This reaction uses, where necessary, a suitable protected acid, such as a protected amino acid or the half ester of a diacid. Activated acids include acid halides, anhydrides, and activated esters and amides, such as N-hydroxysuccinate esters and imidazole amides . Such acylation reactions using activated acids or coupling reagents are well known and appreciated in the art. The reaction is generally carried out in a suitable solvent, such as tetrahydrofuran, dimethylformamide, dichloromethane, and the like. Typically the reaction is carried out at temperatures of from about 0°C to about 100°C. Generally, the reaction requires about 1 hour to 48 hours. The product can be isolated and purified by techniques well known in the art, such as filtration, evaporation, extraction, trituration, chromatography, and crystallization. In Reaction Scheme A, in an optional step, also not shown, an pharmaceutically-acceptable salt is formed using a pharmaceutically-acceptable acid or base. The formation of pharmaceutically acceptable salts is well known and appreciated in the art. Starting materials for Reaction Scheme A, compounds of formula (a) , are obtained by synthesis of the required heptapeptide or by fermentation or by fermentation and subsequent manipulation as described in Reaction Scheme B, below. In Reaction Scheme B, all substituents, unless otherwise indicated, are as previously defined. In Reaction Scheme B all reagents are well known and appreciated in the art. Reaction Scheme B In Reaction Scheme B, step a, a rufomycin factor in which R is hydrogen and R' is the radical of the formula COR. wherein R 4 is hydroxy undergo an ester formation or amide formation to give a compound of formula (ai) in which R 4 is Ci-Cε alkoxy or -NR 5 R 6 . Such ester and amide formations are well known in the art. For example, the compound of formula (b) in which R 4 is hydroxy is contacted with a suitable alcohol or amine. Suitable alcohols are one which give rise to Ci-Cβ alkoxy as desired in the final product. Suitable amines are ones which give rise to -NR 5 R 6 as desired in the final product. The reaction can be carried out using a coupling reagent, such as dicyclohexylcarbodiimide, 1- (3- (dimethylaminopropyl) -3- ethylcarbocdiimide hydrochloride, ethyl ethoxydihydroquinoline, and other coupling reagents which are well known and appreciated in the art. The reaction is generally carried out in a suitable solvent, such as tetrahydrofuran, dimethylformamide, dichloromethane, and the like. Typically the reaction is carried out at temperatures of from about 0°C to about 100°C. Generally, the reaction requires about 1 hour to 48 hours. The product can be isolated and purified by techniques well known in the art, such as filtration, evaporation, extraction, trituration, chromatography, and crystallization. In Reaction Scheme B, step b, a rufomycin factor in which R and R' are the divalent radical of the formula undergoes dehydration to give a compound of formula (a 2 ) in which R and R' are the divalent radical of the formula For example, the compound of formula (b) as described above is contacted with a suitable acid under dehydrating conditions to give a compound of formula (a 2 ) - Suitable acids include, hydrochloric acid, hydrobromic acid, p- toluenesulfonic acid, acetic acid, and the like. The reaction is generally carried out in a suitable solvent, such as tetrahydrofuran, toluene, dimethylformamide, water, acetic acid, dimethylformamide/ acetic acid mixtures, dimethylformamide/acetic acid/water mixtures, and the like. The reaction may be advantageously carried out under conditions that remove water from the reaction mixture, such as the action of a Dean-stark trap, molecular sieves, sodium sulfate, etc. Typically the reaction is carried out at temperatures of from about 0°C to the refluxing temperature of the solvent. Generally, the reaction requires about 1 hour to 48 hours. The product can be isolated and purified by techniques well known in the art, such as filtration, evaporation, extraction, trituration, chromatography, and crystallization. As will be appreciated by those skilled in the art, such a dehydration reaction under acidic conditions may also open the epoxide. For instance, in the case where hydrochloric acid or hydrobromic acid are used the chlorohydrin or bromohydrin may be obtained. In such a case the epoxide can be reformed by treating the chlorohydrin or bromohydrin with base, such as sodium carbonate or potassium carbonate. The epoxide is generally reformed in a solvent, such as acetonitrile at temperatures of from about 0°C to the refluxing temperature of the solvent. Generally, the reaction requires about 1 hour to 48 hours. The product can be isolated and purified by techniques well known in the art, such as filtration, evaporation, extraction, trituration, chromatography, and crystallization. In Reaction Scheme B, step c, a rufomycin factor in which R and R' are the divalent radical of the formula undergoes reduction to give a compound of formula (a 3 ) in which R and R' are the divalent radical of the formula For example, the compound of formula (b) as described above is contacted with a suitable reducing reagent to give a compound of formula (a 3 ) . Suitable reducing agents are well known in the art and include, sodium borohydride, lithium borohydride, sodium cyanoborohydride, and the like. The reaction is generally carried out in a suitable solvent, such as tetrahydrofuran, methanol, ethanol, isopropanol, methanol/water mixtures, ethanol/water mixtures, and the like. The reaction may be advantageously be buffered or carried out under slightly acidic conditions. Typically the reaction is carried out at temperatures of from about 0°C to about 80°C. Generally, the reaction requires about 1 hour to 24 hours. The product can be isolated and purified by techniques well known in the art, such as filtration, evaporation, extraction, trituration, chromatography, and crystallization. In Reaction Scheme B, step d, a rufomycin factor in which R and R' are the divalent radical of the formula undergoes oxidation to give a compound of formula (a 4 ) in which R and R' are the divalent radical of the formula For example, the compound of formula (b) as described above is contacted with a suitable oxidizing reagents to give a compound of formula (a ) . Suitable oxidizing reagents are well known in the art and include, pyridine- dichromate, and dimethylsulfoxide under Swern conditions, and the like. The reaction is generally carried out in a suitable solvent, such as tetrahydrofuran, dichloromethane, chloroform, and the like. Typically the reaction is carried out at temperatures of from about -60°C to about 50°C. Generally, the reaction requires about 1 hour to 8 hours. The product can be isolated and purified by techniques well known in the art, such as filtration, evaporation, extraction, trituration, chromatography, and crystallization . The present invention is further illustrated by the following examples and preparations. These examples and preparations are illustrative only and are not intended to limit the invention in any way. The terms used in the examples and preparations have their normal meanings unless otherwise designated. For example "°C" refers to degrees Celsius; "N" refers to normal or normality; "M" refers to molar or molarity; "mM" refers to millimolar; "nM" refers to nanomolar; "mol" refers to mole or moles; "mmol" refers to millimole or millimoles; "kg" refers to kilogram or kilograms; "g" refers to gram or grams; "mg" refers to milligram or milligrams; "μg" refers to microgram or micrograms; "mL" refers milliliter or milliliters; "L" refers to liter or liters; "bp" refers to boiling point; "mp" refers to melting point; "brine" refers to a saturated aqueous sodium chloride solution; "HPLC" refers to high performance liquid chromatography; "MS" refers to mass spectrometry; "IR" refers to infrared spectroscopy; "UV" refers to ultraviolet spectroscopy; "NMR" refers to nuclear magnetic resonance spectroscopy; " [CC]D" refers to specific rotation of the sodium D line in a 1 decimeter cell; " [α] 365 " refers to specific rotation at 365 nanometers in a 1 decimeter cell, "c" refers to concentration in g/mL; "TLC refers to thin layer chromatography on silica gel; "R f " refers to retention factor; "R t " refers to retention time, "min" refers to minute; "TFA" refers to trifluoroacetic acid; "DMF" refers to dimethylformamide; "HBr" refers to hydrobromic acid; "EtOH" refers to ethanol; "CHCI 3 " refers to chloroform; "MeOH" refers to methanol; "MgS0 " refers to magnesium sulfate; "rpm" refers to revolutions per minute; "psi" refers to pounds per square inch; etc. In the examples and preparations the progress of the reactions were monitored by analytical HPLC using a Waters Symmetry (Cis, 5 μ , 3.9 X 150 mm) column. Two HPLC methods were employed to study the entire polarity range; method 1 employed a mobile phase of 60-90% (linear gradient) of acetonitrile containing 0.1% trifluoroacetic acid and method 2 employed 10-60% (linear gradient) of acetonitrile containing 0.1% trifluoroacetic acid. Purification of reaction mixtures at the product level of 0.025 mmol were carried out using a Waters SymmetryPrep column (Cis, 7 μm, 19 X 300 mm) and reaction mixtures at the product level of 0.2 mmol and higher were carried out using a Rainin column (Cis, 8 μm, 41.4 X 250 mm) eluted at about 40 mL/minute with a linear gradient similar to those described above using 0.1% TFA in water to 0.1%TFA in acetonitrile. The products were identified by observing M+H or other adducts by electro-spray mass spectrometry . EXAMPLE 1.1 Fermentation: 150 liter Fermentation Stock cultures of present Streptomyces macrosporeus (DSM-12818) were maintained in a suspension of 10% glycerol - 5% lactose in the vapor phase of liquid nitrogen. Thawed aliquots (0.5 mL) were used to inoculate 50 mL portions of CS medium containing 3% Trypticase Soy Broth (BBL) , 0.5% glucose, 0.4% maltose, 0.3% yeast extract, and 0.2% MgS0 4 • 7H 2 0 in deionized water (no pH adjustment) . Each portion of inoculated medium was contained in a 250 mL wide-mouth Erlenmeyer flask and was incubated at 250 rpm for 72 hours at 30°C in a rotary shaker with a 5.1 cm throw. The resulting culture was homogenized for 10 sec. in a Waring blender (two 5 sec pulses) . The homogenized culture (13.0 mL aliquots) was transferred to 900 mL of the same medium contained in 2.5 liter non-baffled Tunair™ flasks. After an incubation period of 72 hours under the same conditions described above, the contents of two Tunair™ flasks were combined to inoculate a 150 liter fermentor containing 115 liters of production medium. The production medium consisted of G-2 medium (Table A) and 1 mL/liter of M&M salts solution (Table B) . The pH of the reactor contents was 6.6 after steam sterilization and was adjusted to 6.9 before inoculation. During fermentation, temperature was controlled at 30°C and dissolved oxygen was maintained at 30% by agitation and air flow. The pH of the culture dropped to 6.2 after 24 hours of fermentation and remained in the range of 5.9 to 6.2 and until the fermentation was terminated at 90 to 120 hours. TABLE A. Components of G-2 Medium pH adjust to 7.0 TABLE B. M&M Salts Stock Solution Fermentation: Shake-Flask Studies Initial flask studies showed that the present Streptomyces macrosporeus (DSM-12818) was a better producer of rufomycins than the Takeda patent strain, Streptomyces atra tus ATCC-14046. This was true in a variety of media, including G-2 and media described in the Takeda patent (US 3,655,879). Shake-flask studies demonstrated improved growth and rufomycin production by adding cottonseed flour at 0.5% w/v to G-2 medium. Increasing the fermentation time from 4 to 7 days also improved rufomycin titer. Cottonseed flour results were scaled to 150 liter fermentors and, after a single trial, factors 4 and 5 reached a total, combined titer of 212 mg/1 in 5 days. Addition of L-leucine (putative precursor) did not enhance factor production in four different shake-flask media. MOPS buffer (pH 7.0) was routinely used in shake-flask studies to control culture pH . Key nutrients in the modified G-2 medium (G-2 plus cottonseed flour) were tested for main effects using a Plackett-Burman screening design. Cane molasses, glycerol, phytic acid, potato dextrin, soluble starch, and soybean flour were tested at zero and control levels in shake-flask trials. Results showed that potato dextrin had the strongest, positive main effect on rufomycin production (Table C) . Corn steep liquor, soluble starch, and soybean flour could be eliminated from the medium without significantly affecting rufomycin titers. TABLE C. Plackett-Burman Screen Parameter Estimates for Major Nutrients λ r 2 = 0.981 'Parameter estimate = one-half the main effect Flask Optimization Study A Box-Wilson central composite design was used to optimize the following factors (each at five levels) : glucose, potato dextrin, glycerol, cottonseed flour, and phytic acid. Factors held constant included cane molasses, CaC0 3 , MgS0 4 • 7H 2 0, M&M salts, and MOPS buffer (control levels) . Analysis of the data showed that optimal 4 and 5 production occurred with the highest levels of potato dextrin used (5.0% w/v) and glucose, glycerol, cottonseed flour, and phytic acid at intermediate levels, 3, 1, 3, and 0.1% w/v, respectively (Table D) . TABLE D. Box-Wilson Optimization 1 Parameter Estimates for Key Nutrients x r = 0.883 2 Only the five main effects and significant interactions and quadratic effects are shown. Prediction profiles for Box-Wilson central composite design indicating the organism response (factor 4 + 5 titer) to various concentrations of glucose, potato dextrin, glycerol, cottonseed flour, and phytic acid; nutrient concentrations are in grams/liter. EXAMPLE 1.2 Fermentation: 150 liter Fermentors The optimized medium contained glucose at 3% w/v, potato dextrin at 5% w/v, glycerol at 1% w/v, cottonseed flour at 3% w/v, and phytic acid at 0.1% w/v, with the remaining ingredients held at control levels. The results showed a combined factor potency of over 600 mg/liter, about 6-fold higher than the best G-2 control. No problems with broth viscosity were encountered although spin solids nearly doubled in the optimized medium (56% v/v vs. 30% v/v) . No feeds were used during these fermentations and the seed trains were free of any animal-derived nutrients. pH control was not used for the new process and pH ranged from 5.9 to 7.6 during the course of the optimized fermentations. EXAMPLE 1.3 Fermentation: Fermentation at 5000 liters The present culture was inoculated into 2 X 2 liter flasks each containing 400 mL of CS medium consisting of 0.3% yeast extract, 3% Trypticase Soy Broth, 0.5% dextrose, 0.2% magnesium sulfate and 0.4% maltose and the flasks were incubated at 30°C for 72 hours. The contents of the two flasks were combined ( 800 mL) to inoculate a 100 liter seed tank containing the same CS medium. The fermentation was performed at 30°C with air flow at 0.5 scfm with agitation at 150 rpm. No additional controls (e.g. pH) were used. The fermentation was continued until the spin solids reached 10-15% (average time 72 hours) . The total volume of the seed tank was transferred into a fermentor containing 5000 liters of production medium. The production medium consisted of 5% potato dextrin, 3% dextrose, 1% cane molasses, 1% glycerol, 0.01% phytic acid, 0.05% magnesium sulfate, 1 mL of M&M salts, 0.3% calcium carbonate and 3% cottonseed flour. During fermentation, temperature was controlled at 30°C , pH was maintained at 7.0 ± 1.0 using 30% sulfuric acid and 28% sodium hydroxide solution and the dissolved oxygen content was controlled at 30% using air flow and agitation. The fermentation was harvested at 168 hours . ISOLATION OF FACTORS Isolation of rufomycin factors Fermentations were carried out at different scales (1, 150 and 5000 liters) in order to isolate and characterize all the rufomycin factors described in this invention. Isolation from 1 liter fermentation The whole broth (5 X 800 mL) was centrifuged using a Beckman J6B at 2100 rpm for 15 min. The cell mass was separated from the supernatant, extracted with 2.5 liters of methanol and the extract thus obtained was combined with the supernatant (total volume 4.9 liters). The combined solution was diluted with equal volume of water and chromatographed over a TosoHaas CG300sd column (50 X 250 mm, flow rate 190 mL/min) equilibrated with 76:24 water- methanol . After charging the extract, the column was eluted with 2-90% of acetonitrile over a period of 20 min followed by final wash with methanol for 10 min. Fractions containing rufomycins were combined (534 mg) and rechromatographed over a Kromasil column (Cg, 25 X 250 mm, flow rate 40 mL/min) using a 65% acetonitrile-0.05% ammonium acetate to yield 228 mg of a mixture containing 4 and 5, 32.9 mg of rufomycin Bi (1) and 31.1 mg of rufomycin B 2 (2) . The mixture containing 4 and 5 was repurified over a Rainin column (Cι 8 , 21.4 X 250 mm, mobile phase: a linear gradient of 50-70% triethylamine phosphate (pH 3.0 ) -acetonitrile for 45 min with a flow rate of 15 mL/min) . Fractions containing individual compounds were desalted on the same column to yield 37 mg of 4 and 23 mg of 5. Alternate Isolation from fermentation The rufomycin factors 1, 2, and 4-7 described herein are also analyzed by HPLC using a Vydac Cis column (catalog number, 218TP54) 0.46 cm X 25 cm, 5 micron; eluted with solvent A (degassed 0.1% TFA in water) and solvent B (0.1% TFA in acetonitrile) in the composition and gradient described by Table 1, below Table 1 eluted at 1 mL/minute; at 40°C; and with detection at 220 nm. Rufomycin whole broth (15 mL) was mixed with water (30 mL) adjusted to pH 11.4 using solid sodium hydroxide. The sample was kept chilled and rotated for 50 minutes. At the end of this time the pH was 11.0. The solids were subsequently removed using centrifugation and the supernatant were analyzed by reversed phase chromatography for the presence of rufomycin factors 1, 2, and 4-7. The sample were rotated for a further 7 hours and assayed again as mentioned above. After 50 minutes the yield of rufomycin factors 5, 4 and 6 was about 85% of that achieved by a 67% acetone extract. After this the rufomycin product, isolated from the broth solids in the supernatant, can either be supplemented with organic solvent to conduct preparative reversed phase chromatography, or alternatively, the pH of the aqueous solution can be adjusted to pH 4 upon which the rufomycin factors become insoluble and can be collected as a solid. The solid can be further purified by using reversed phase chromatography on a chromatographic support such as TosoHaas CG161 reversed phase resin as described above. Isolation of rufomycin factors 1, 2 , 4 and 5 Rufomycin whole broth (15 mL) was mixed with acetone (30 mL, 67% final concentration) or water (30 mL) or water (30 mL) and the final pH adjusted to 11.4 using an aqueous 10% (w/v) sodium hydroxide solution. The samples were kept chilled and rotated for 50 minutes. At the end of this time the pH of the third sample was 11.0 and the pH of the second sample was 7.7. The solids were subsequently removed using centrifugation and the supernatants were analyzed by reversed phase chromatography for the presence of rufomycin factors 1, 2, and 4-7. The three samples were rotated for a further 7 hours and assayed again as mentioned above. The following chromatogram illustrates that the high pH sample released all the rufomycin factors into the aqueous phase while the neutral pH sample did not. Only the polar factor 7 was released at pH 7.7. After 50 minutes the yield of rufomycin factors 5, 4 and 6 was about 85% of that achieved by the 67% acetone. After this the rufomycin product, isolated from the broth solids in the supernatant, can either be supplemented with organic solvent to conduct preparative reversed phase chromatography, or alternatively, the pH of the clarified aqueous solution can be adjusted to pH 4 upon which the rufomycin factors become insoluble and can be collected as a solid. The solid can be further purified by using reversed phase chromatography on a chromatographic support such as TosoHaas CG161 reversed phase resin; eluting with an increasing solvent gradient. Selective Release of Rufomycin Factors as a Function of pH Fermentation broth (700 mL) was added incremental amounts of sodium hydroxide. After each addition the pH of the solution was read and an aliquot was removed. Removed samples were placed in the chillroom and rotated for about 3 hours and 15 minutes. The supernatants were obtained by centrifugation and analyzed by the reversed phase chromatographic assay. The data indicates that at intermediate levels of pH between 7 and 11 that selective removal of factors from the solid phase is seen as a function of pH. Isolation from 150 liter fermentation The whole broth (2 X 115 liter) was filtered through a pad of Hi-Flo to obtain 38 liter of a semi-solid cell mass. To this was added 100 liters of acetone and the mixture was agitated for 1 hr and filtered through Hi-Flo to yield 72 liters of acetone extract. The remaining cell mass was extracted as above to yield additional 82 liter of acetone extract. The two acetone extracts were processed separately as detailed below. The extract was diluted with an equal volume of water and loaded on a 10 liter HP20ss column. The column was eluted at a flow rate of 1 liter/min starting from triethylamine phosphate buffer (pH 3) followed by increasing concentration of acetonitrile. Fractions from each chromatography were combined based on their HPLC profile, concentrated near aqueous and extracted with ethyl acetate to afford fractions A-F. Fraction A was loaded on a 4 liter Kromasil (Cι 8 , 4 mm) column. The column was equilibrated with 50% triethylamine phosphate buffer (pH 3.0) -acetonitrile, and after loading the sample the column was eluted with a linear gradient of 50-70% triethylamine phosphate (pH 3.0 ) -acetonitrile for 67 min with a flow rate of 1 liter/min. Each 1 liter fraction was extracted with 500 mL of ethyl acetate, and the ethyl acetate soluble material was dissolved in t-butanol and lyophilized to yield 27 fractions. Fraction 6 (849 mg) was repurified on a Waters SymmetryPrep column (Ciβ. 19 X 300 mm, 7 micron, mobile phase: linear gradient 50-70% triethylamine phosphate- acetonitrile, flow rate: 15 mL/min, fractions collected at an interval of 1 min) . Fractions 26-28 containing 7 were combined and desalted using the above column to yield 44.8 mg of a light yellow powder. Fraction 9 of the Kromasil column (2.01 g) was chromatographically pure 5. Fraction 11 from the Kromasil column (5.37 g) was partitioned between equal volumes of ethyl acetate and water (total volume 350 mL) . The ethyl acetate layer was separated and the aqueous layer was additionally extracted with 175 mL of ethyl acetate. The combined ethyl acetate layer was dried, evaporated, dissolved in t-butanol and freeze-dried to yield 2.9 g of 4 as a light yellow powder. Isolation from 5000 liter fermentation The whole 5000 liter fermentation broth was mixed with 5000 liter of acetone. The mixture was centrifuged to remove substantial amount of solids and the supernatant was further filtered over Cuno 30S filters to yield ~ 8000 liter of a clear acetone solution. To this solution water was added to adjust the final concentration of acetone to 33%. This solution was filtered over a 1 micron filter, and then loaded on a TosoHaas CG161c resin column (30 X 70 cm ) which was previously equilibrated with 35% acetone. After loading the entire solution, the column was washed with 100 liter of 50% acetone followed by elution with 500 liters of 50-60% acetone using a linear gradient. Fractions were assayed by analytical HPLC, and fractions containing compounds 4-6 were combined. The pH of the solution was adjusted to 4 and diluted with 3 volumes of water. The precipitated material was collected by filtration over a 1 micron polypropylene filter and dried in a vacuum oven to yield 500 g of a solid. Ten grams of this solid was dissolved in 40 mL of acetonitrile-water (9:1) mixture and approximately 4 mL of this solution was injected on to a Dynamax 60A Cis column (41.4 X 250 mm, flow rate 40 mL/min) . The column was eluted with 0.1% trifluoroacetic acid-acetonitrile (42:58). This procedure was repeated until all the material was purified (a total of 10 injections) . The column fractions were then analyzed by analytical HPLC and combined to yield 4.09 g of a mixture of 4 and 5 (94% purity) and 1.43 g of 6 (93% purity) . The presence of compound 6 was observed in significant quantities only in the 5000 liter fermentation. EXAMPLE 2 General procedure for acetylation of 4, 5 and 6 A solution of alcohol (0.1 mmol, 104 mg) in 1 mL of pyridine was stirred with dimethylaminopyridine (0.01 mmol, 1.2 mg) and acetic anhydride (1 mmol, 102 mg) at room temperature overnight. The reaction mixture was purified by HPLC to afford the diacetate. EXAMPLE 3 General procedure for the preparation compounds 14-24 To a solution of 7 (26.5 mg, 0.025 mmol) in 0.6 mL of 1:2 dichloromethane-dimethylformamide were added diisopropylethylamine (0.025 mL) , hydroxyazatriazole (0.018 mmol in 0.1 mL of dimethylformamide), 2-chloro-l , 3- dimethhylimidazolidium hexafluorophosphate (0.0625 mmol in 0.1 mL of dimethylformamide) and an amine (0.05 mmol) in 0.1 mL of dichloromethane and the mixture was kept at room temperature for 24 h. Purification by HPLC afforded the amide derivatives (14-24). EXAMPLE 3 Preparation of 25 A mixture of 2 (0.025 mmol, 25 mg) and triethylamine trihydrofluoride (1 mL) was heated at 75°C. After 60 hours, 0.5 mL of triethylamine trihydrofluoride was added and heating was continued for 60 h more. Purification by HPLC afforded 10 mg of 25. EXAMPLE 4 Preparation of 26 To a solution of 2 (0.025 mmol, 25 mg) in 0.5 mL of DMF was added 0.15 mL of 1 M HCI in acetic acid. The reaction mixture was left at room temperature for 24 h by which time HPLC indicated consumption of >90% starting material. The mixture was purified by HPLC to yield 13 mg of 26. EXAMPLE 5 Preparation of 27 To a solution of 2 (0.025 mmol, 25 mg) in 0.5 mL of DMF was added 0.04 mL of 10% HBr in 2 : 1 DMF-acetic acid at room temperature. After 3 h, the reaction mixture was purified by HPLC to give 12 mg of 27. EXAMPLE 6 Preparation of 28 To a solution of 2 (0.025 mmol, 25 mg) in 0.5 mL of dioxane was added 0.05 mL of 70% perchloric acid in 0.5 mL of 3:2 dioxane-water mixture at room temperature. After 24 h additional 0.02 mL of 70% perchloric acid was added and the reaction was left for 36 h. Purification by HPLC gave 4 mg of 28. EXAMPLE 7 General procedure for the preparation of compounds 29-54 To a solution of 2 (0.025 mmol, 25 mg) in 0.5 mL of methanol in a 4 mL glass vial was added an amine (0.15 mmol) . The mixture was heated at about 65°C for 2-4 days and purified by HPLC to yield β-hydroxy ethanolamine derivatives 29-54. EXAMPLE 8 Preparation of 10 A mixture of 4, 5 and 6 (1 mmol, 1.042 g) was dissolved in 6 mL of dimethylformamide. To this solution 3 mL of 1 M hydrogen chloride in acetic acid was added slowly at room temperature. After 3 h an additional 1 mL of 1 M hydrogen chloride was added and the reaction mixture was stirred over night. Purification by HPLC gave 0.782 g of 10 (74% yield). EXAMPLE 9 Preparation of 55 A mixture of 4 and 5 (4.09 mmol, 4.26 g) was dissolved in 25 mL of dimethylformamide. To this solution 9 mL of 10% hydrogen bromide (3 mL of commercially available 30% hydrogen bromide in acetic acid was diluted with 6 mL of dimethylformamide) was added slowly at room temperature. After 16 h HPLC analysis indicated >95% disappearance of starting material. The reaction mixture was purified by reversed-phase chromatography to yield the bromohydrin, 56. The bromohydrin was dissolved in 40 mL of acetonitrile and to this solution 2 g of potassium carbonate in 6 mL of water was added. The mixture was stirred vigorously at room temperature for 16 h by which time no starting material was detected by HPLC. The reaction mixture was purified by HPLC to afford 3.3 g of 55 (overall yield 79%) . EXAMPLE 10 Preparation of 57 and 59 A mixture of 55 (0.791 mmol, 0.81 g) and 12 mL of triethylamine trihydrofluoride was heated at about 75°C for 7 days. The mixture was cooled and purified on a reversed- phase column to yield 0.431 g of 59 (51% yield), 0.148 g 57 (18 % yield) and 0.084 g of starting material (10%). EXAMPLE 11 Preparation of 58 A mixture of 55 (0.02 mmol, 20 mg) and 0.20 mL of hydrogen fluoride-pyridine was stirred at room temperature for 30 min. Saturated sodium bicarbonate solution was added, the mixture was extracted with 20 mL of ethyl acetate, evaporated and purified by HPLC to afford 58. EXAMPLE 12 General procedure for the preparation of compounds 61-79 To a 4 mL glass vial were added 55 (0.025 mmol, 25 mg) 0.5 mL of methanol and amine (0.15 mmol) . The mixture was heated at about 65°C for 2-4 days and purified by HPLC to yield β-hydroxy ethanolamine derivatives 61-79 in 50-80% yield. EXAMPLE 14 General procedure for the preparation of compounds 80-101 A mixture of phenol (0.175 mmol) in 0.25 mL of ethanol, potassium carbonate (0.175 mmol) in 0.1 mL of water and benzyltriethylammonium chloride (0.018 mmol) in 0.1 mL of water was added to 55 (0.025 mmol, 25 mg) in 0.25 mL of ethanol in a 4 mL glass vial . The vial was heated at about 75°C for 3 days and purified by HPLC to afford β-hydroxy arylether derivatives 80-101. EXAMPLE 15 General procedure for the preparation of compounds 102-113 To a solution of thiophenol (0.13 mmol) in 0.25 mL of acetonitrile were added tetrabutylammonium fluoride hydrate (0.01 mmol) in 0.1 mL of acetonitrile and 55 (0.026 mmol, 26 mg) in 0.25 mL of acetonitrile. The mixture was heated at about 75°C for 3 days and purified by HPLC to give β-hydroxy thioarylether derivatives 102-113. EXAMPLE 16 General procedure for the preparation of compounds 114-116 To a solution of thiol (0.0625 mmol) in 0.2 mL of methanol were added sodium carbonate (0.0325 mmol) in 0.1 mL of water and 55 (0.025 mmol, 25 mg) in 0.5 mL of methanol. The mixture was heated at about 75°C for 6 h and purified by HPLC to furnish β-hydroxy thioether derivatives 114-116. EXAMPLE 17 Preparation of 117 To a solution of 55 (0.025 mmol, 25 mg) in 1 mL of dimethylformamide were added potassium t-butoxide (0.0375 mmol, 4.2 mg) and 2-bromoacetamide (0.05 mmol, 6.9 mg) . The mixture was heated at about 70°C for 18 h. Additional potassium t-butoxide (0.0125 mmol, 1.4 mg) and 2- bromoacetamide (0.025 mmol, 3.5 mg) were added, the mixture was heated for 3 h and purified by HPLC to yield 117. EXAMPLE 18 Preparation of 118 To a 4 mL glass vial containing 55 (0.025 mmol, 25 mg) were added 1 mL of dimethylformamide and potassium t- butoxide (0.05 mmol, 5.6 mg) and 4-fluorobenzyl bromide (0.075 mmol, 14.2 mg) . The reaction mixture was heated at about 70°C for 24 h and purified by HPLC to yield 118. EXAMPLE 19 General procedure for the preparation of compounds 119, 121- 123 To a solution of a corresponding phenol (0.025 mmol) in 1.5 mL of acetone were added potassium carbonate (0.1 mmol, 13.8 mg) and dimethyl sulfate (0.25 mmol, 0.025 mL) . The reaction mixture was stirred for 16 h at room temperature and purified by HPLC to afford 119,121-123. EXAMPLE 20 Preparation of 120 To a solution 10 (0.025 mmol, 27 mg) in 1 mL of ethanol was added SnCl 2 -dihydrate (0.125 mmol, 28 mg) and the reaction mixture was heated at about 70°C for 24 h. Purification by HPLC gave 22 mg of 120. EXAMPLE 21 Preparation of 127 To a solution of 5 (1.03 g, 0.99 mmol) in 4 mL of dimethylformamide was added pyridiniumdichromate (0.76g, 5 mmol) and the reaction mixture was stirred at 0°C . After 5 h the mixture was diluted with 70 mL of water, extracted with 2 X 70 mL of ethyl acetate. The ethyl acetate solution was washed with 50 mL of water, dried over magnesium sulfate and evaporated to yield a brown residue. Purification of the residue by HPLC afforded 395 mg of 127 and 238 mg of starting material . EXAMPLE 22 General procedure for the preparation of 128-140 Compounds 128-140 were prepared by the procedures described above using compound 127. EXAMPLE 23 Preparation of 11 A mixture of 4 and 5 (1 mmol, 1.04 g) , sodium cyanoborohydride (6 mmol, 0.377 g) and 3 mL of acetic acid was stirred overnight at room temperature. HPLC indicated -50% of unreacted starting material and several products. An additional 2 mmol of the reducing agent was added and the reaction continued for another 24 h. After this the mixture was diluted with 20 mL of water and extracted with 50 mL of ethyl acetate. The ethyl acetate solution was back washed with water, dried over magnesium sulfate and purified by HPLC to afford 204 mg of 11 (yield: 20%) . EXAMPLE 24 General procedure for the preparation of compounds 141-149 Compounds 141-149 were prepared according to the procedures above using compound 11. EXAMPLE 25 General procedures for the preparation of compounds 150-162 i) A mixture of 106 (0.3 mmol, 353.5 mg) , t- butoxycarbonylglycine (0.9 mmol, 157.7 mg) , dimethylaminopyridine (0.03 mmol, 3.7 mg) was dissolved in 4 mL of tetrahydrofuran. To this solution 1,3- dicyclohexylcarbodiimde (0.9 mmol, 185.7 mg) was added and the mixture was stirred at room temperature for 16 h. HPLC indicated consumption of all starting material, but showed mono- and di-acylated product. The mixture was purified by HPLC, the mono- and di-acylated products were combined and dissolved in 4 mL of dichloromethane. To this solution at about 0°C was added 1 mL of 4 M HCI in dioxane and the mixture was stirred for two h at 0°C . Solvents were removed in vacuo and HPLC analysis of the resulting residue showed -20% of starting material. The sample was dissolved in 3 mL of dichloromethane and stirred with 0.5 mL of 4 M HCI in dioxane at room temperature for 2 h. After usual work-up the product showed a mixture of mono- and di-glycinates. The glycinates were dissolved in 2 mL of methanol and stirred with 1.5 mL of 0.1 M sodium bicarbonate at room temperature for 16 h. Purification by HPLC afforded 152 (243 mg) . ii) To a mixture of 10 (796 mg, 0.75 mmol), 9- fluorenylmethoxycarbonylglycine (2.25 mmol, 669 mg) , dimethylaminopyridine (0.075 mmol, 9.2 mg) in 5 mL of tetrahydrofuran 1 , 3-dicyclohexylcarbodiimde (2.25 mmol, 464 mg) was added. The mixture was stirred at room temperature for 16 h and purified by HPLC. The product thus obtained was stirred with 30% piperidine in dimethylformamide for 30 min at room temperature and purified by HPLC to afford 739 mg of 151. iii)To a solution of 97 (0.25 mmol, 292 mg) in 2 mL of pyridine were added dimethylaminopyridine (0.025 mmol, 3.1 mg) and succinic anhydride (12.5 mmol, 1.25 g) . The mixture was heated at about 50°C for 60 h by which time HPLC indicated disappearance of starting material. After removing most of the pyridine under a stream of nitrogen, 50 mL of water was added and the mixture was extracted with 2 X 50 mL of ethyl acetate. The ethyl acetate solution was back washed with 25 mL of water, dried over magnesium sulfate, evaporated and purified by HPLC to afford 260 mg of 161. To an ice-cold solution of 161 (240 mg) in 1 mL of ethanol, 0.1 N sodium hydroxide (0.3 mL) was added and the mixture was diluted with large excess of ethyl acetate. The ethyl acetate solution was evaporated to near dryness and again diluted with excess ethyl acetate. The precipitated disodium salt was collected, re-dissolved in water and lyophilized to yield 238 mg of 162. EXAMPLE 26 Procedures for the preparation of compounds 163-173 Preparation of 163 A solution of 10 (0.05 mmol, 53.5 mg) in 1 ml of dichloromethane was stirred with dimethylaminopyridine (0.005 mmol, 0.6 mg) and benzoyl chloride (0.5 mmol, 70.4 mg) at room temperature for 36 h. HPLC of the reaction mixture indicated -60% of unreacted starting material. To the reaction mixture triethylamine (100 ml) was added and the mixture was stirred for 2 h by which time HPLC indicated disappearance of starting material. The reaction mixture was purified by HPLC to afford 163 (45.8 mg) . Preparation of 164 To a solution of 10 (0.025 mmol, 26.5 mg) in 800 μl of dichloromethane were added dimethylaminopyridine (0.0025 mmol, 0.3 mg) in 100 μl of dichloromethane, isobutyryl chloride (0.5 mmol, 53.3 mg) and triethylamine (100 μl) . The mixture was stirred for 16 h at room temperature and purified by HPLC to yield 164 (18.2 mg) . Preparation of 165 and 166 To a solution of 10 (0.025 mmol, 26.5 mg) in 800 μl of dichloromethane were added dimethylaminopyridine (0.0025 mmol, 0.3 mg) in 100 μl of dichloromethane, isovaleryl chloride (0.5 mmol, 60.3 mg) and triethylamine (100 μl). The mixture was stirred for 16 h at room temperature and purified by HPLC to yield 165 (5.6 mg) and 166 (16 mg) . Preparation of 167 To a solution of 166 (0.025 mmol, 31 mg) in 1 ml of methanol was added saturated bicarbonate solution (250 μl) at room temperature and stirred for 16 h. Purification by HPLC gave 167 (23.2 mg) Preparation of 168 and 169 To a solution of 10 (0.025 mmol, 26.5 mg) in 800 μl of dichloromethane were added dimethylaminopyridine (0.0025 mmol, 0.3 mg) in 100 μl of dichloromethane, benzyloxyacetyl chloride (0.5 mmol, 92.3 mg) and triethylamine (100 μl ) . The mixture was stirred for 16 h at room temperature and purified by HPLC to yield 168 (11.4 mg) and 169 (10.8 mg) . Preparation of 170 and 171 To a solution of 10 (0.025 mmol, 26.5 mg) in 800 μl of dichloromethane were added dimethylaminopyridine (0.0025 mmol, 0.3 mg) in 100 μl of dichloromethane, methoxyacetyl chloride (0.5 mmol, 54.3 mg) and triethylamine (100 μl). The mixture was stirred for 16 h at room temperature and purified by HPLC to yield 170 (19.4 mg) and 171 (5 mg) . Preparation of 172 Compound 172 was prepared from 171 as described in preparation of 167. Preparation of 173 To a solution of 10 (0.025 mmol, 26.5 mg) in 500 μl of pyridine were added dimethylaminopyridine (0.005 mmol, 0.6 mg) and butyric anhydride (0.5 mmol, 79.1 mg) at room temperature. After 16 h the mixture was purified by HPLC to yield 173 (25.2 mg) . 54- By substantially following the procedures described above one skilled in the art can prepare the other compounds of formula I . The present invention provides a method of treating multi-drug resistance diseases by administering to a patient in need thereof an effective amount of a therapeutic agent and a multi-drug resistance inhibitory amount of a compound of the present invention. In particular, the present invention provides a method of treating cancer by administering to a patient in need thereof an effective amount of a oncolytic agent and a multi-drug resistance inhibitory amount of a compound of the formula I. In making the neoplasm less resistant, the compounds of the invention may be used on neoplasms having intrinsic and/or acquired resistance. Such neoplasms include those which have a pathway for resistance which includes MRP-1. The treatment of the resistant and susceptible neoplasm will result in a reversal or inhibition of resistance, or in other words, will cause the neoplasm to be more sensitive to the appropriate therapeutic agent. Cancer chemotherapy with such oncolytic agents includes treatment with vinblastine, vincristine, vindesine, navelbine, daunorubicin, doxorubicin, mitroxantrone, etoposide, teniposide, mitomycin C, actinomycin D, taxol, topotecan, mithramycin, colchicine, puromycin, podophyllotoxin, ethidium bromide, emetine, gramicidin D, and valinomycin. The compounds of the invention may be used for many resistant neoplasms, including colon cancer, mesothelioma, melanoma, prostate cancer, ovarian cancer, non-small cell lung cancer, small-cell lung cancer, bladder cancer, endometrial cancer, leukemia, renal cancer, liver cancer, neurological tumors, testicular cancer, breast cancer, and large cell lymphoma. More particular types of cancer are Hodgkin ' s disease, Karposi ' s sarcoma, and acute granulocytic leukemia . As used herein the following terms have the meanings indicated: the terms "multi-drug resistance" or "MDR" include both intrinsic resistance and acquired drug resistance; the terms "treatment" or "treating" refer to preventing a disease, that is causing the clinical manifestations of the disease not to develop and/or inhibiting a disease, that is, arresting or decreasing the clinical manifestations of the disease, and/or curing a disease; the term "patient" refers to a mammal and specifically includes, humans, horses, cattle, sheep, dogs, cats, guinea pigs, mice, rats, monkeys, chimpanzees, apes; the term "patient in need thereof" refers to a patient which has a disease or condition which manifests multi-drug resistance; the term, "therapeutic agent" refers an chemotherapeutic agent which has decreased efficacy due to multi-drug resistance, and specifically includes oncolytic agents or anti-malarial agents; the term "effective amount" refers to that amount of a therapeutic agent that is effective in treating a patient in combination with a multi-drug resistance inhibitory amount of a compound of the present invention ; the term "multi-drug resistance inhibitory amount" refers to an amount effective to make the neoplasms less resistant to chemotherapy by the chosen therapeutic agent. An effective amount of a therapeutic agent and a multi- drug resistance inhibitory amount can be readily determined by the attending diagnostician, as one skilled in the art, by the use of conventional techniques and observing results obtained under analogous circumstances. In determining the effective amount of a therapeutic agent, the dose of the therapeutic agent, and a multi-drug resistance inhibitory amount, the dose of the compound of the present invention, a number of factors are considered by the attending diagnostician, including, but not limited to: the species of mammal; its size, age, and general health; the specific disease involved, including the extent to which that disease has become resistant; the degree of involvement or the severity of the disease; the response of the individual patient; the particular therapeutic agent administered, the particular compound of this invention which is administered; the mode of administration of the drugs; the bioavailability of the preparations used; the dose regimens selected; the use of concomitant medications; and other relevant circumstances . An effective amount of a therapeutic agent is expected to vary from about 0.1 milligram per kilogram per day (mg/kg/day) to about 200 milligram per kilogram per day (mg/kg/day) . Preferred amounts are to be determined by one skilled in the art. A multi-drug resistance inhibitory amount is expected to vary from about 0.1 milligram per kilogram per day (mg/kg/day) to about 100 milligram per kilogram per day (mg/kg/day) . Preferred amounts are to be determined by one skilled in the art. The compounds of the present invention are usually administered in the form of pharmaceutical compositions, that is, in admixture with pharmaceutically acceptable carriers or diluents, the proportion and nature of which are determined by the solubility, chemical properties, the chosen route of administration, and standard pharmaceutical practice . These compounds can be administered by a variety of routes including oral and parenteral, including intravenous, rectal, transdermal, subcutaneous, intramuscular, and intranasal . The preferred route of administration is parenteral with intravenous being more preferred. Such compositions are prepared in a manner well known in the pharmaceutical art and comprise at least one active compound. The present invention also includes methods employing pharmaceutical compositions which contain, as the active ingredient the compounds of the present invention, associated with pharmaceutically acceptable carriers for the treatment of neoplasms, comprising the use of such pharmaceutical compositions for treating multi-drug resistant cancer by treating a patient in need thereof with an effective amount of a oncolytic agent and a multi-drug resistance inhibitory amount of a compound of the present invention. In making the compositions of the present invention the active ingredient is usually mixed with an excipient, diluted by an excipient, or enclosed within such a carrier which can be in the form of a capsule, sachet, paper or other container. When the excipient serves as a diluent, it can be a solid, semi-solid, or liquid material, which acts as a vehicle, carrier or medium for the active ingredient. Thus, the compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium) , ointments containing for example up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders. Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. The formulations can additionally include: lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl- and propylhydroxybenzoates ; sweetening agents; flavoring agents, surfactants, buffers, and solubilizers . The compositions of the invention can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures known in the art. In preparing a formulation, it may be necessary to mill the active compound to provide the appropriate particle size prior to combining with the other ingredients. If the active compound is substantially insoluble, it ordinarily is milled to a particle size of less than 200 mesh. If the active compound is substantially water soluble, the particle size is normally adjusted by milling to provide a substantially uniform distribution in the formulation, e.g. about 40 mesh. For preparing solid compositions such as tablets the principal active ingredient is mixed with a pharmaceutical excipient to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention. When referring to these preformulation compositions as homogeneous, it is meant that the active ingredient is dipsersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules. This solid preformulation is then subdivided into unit dosage forms of the type described above containing from 0.1 to about 500 mg of the active ingredient of the present invention. The tablets or pills of the present invention may be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by enteric layer which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate. The liquid forms in which the novel compositions of the present invention may be incorporated for administration orally, by injection, or intravenously include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles. More specifically, for the purpose of parenteral administration, the compound of the present invention may be incorporated into a solution, suspension, emulsion, or microemulsion. Such, compositions are well known in the art . Pharmaceutical Dosage Forms, Parenteral Medications (Eds. Kenneth E. Avis, Leon Lachman, and Herbert A. Lieberman, Marcel Dekker, Inc. New York and Basel) ; Journal of Parenteral Schience & Technology, 40, 34-41 (1986); and Pharmaceutical Technology, 46-54 (March 1987). The preparations should containing at least 0.1% of a compound of the invention, but may vary between about 0.1% and about 50% of the weight thereof. The amount of the compound of the present invention present in such compositions is such that a suitable dosage will be obtained. Preferred compositions and preparations are able to be determined by those skilled in the art. The compositions are preferably formulated in a unit dosage form, each dosage containing from about 5 to about 3000 mg, more usually about 10 to about 300 mg, of the active ingredient. It is understood that the amount of the compound actually administered will be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered, the age, weight, and response of the individual patient, and the severity of the patient's symptoms, and therefore the above dosage ranges are not intended to limit the scope of the invention in any way. In some instances dosage levels below the lower limit of the aforesaid range may be more than adequate, while in other cases still larger doses may be employed without causing any harmful side effect, provided that such larger doses are first divided into several smaller doses for administration throughout the day. As used herein, the term "unit dosage form" refers to physically discrete units suitable as unitary dosages dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient. The following formulation examples are illustrative only and are not intended to limit the scope of the invention in any way. "Active Ingredient" means a compound according to the present invention or a pharmaceutically acceptable salt thereof. Formulation Example 1 Capsules, each containing 40 mg of medicament are made as follows: Quantity Ingredient (mg/capsule) Active Ingredient (s) 40.0 mg Starch 109.0 mg Magnesium stearate 1.0 mg Total 150.0 mg Formulation Example 2 Suspensions, each containing 50 mg of medicament per 5.0 mL dose are made as follows: Ingredient Amount Active Ingredient (s) 50.0 mg Xanthan gum 4.0 mg Sodium carboxymethyl cellulose (11%) Microcrystalline cellulose (89%) 50.0 mg Sucrose 1.75 g Sodium benzoate 10.0 mg Flavor and Color q.v. Purified water to 5.0 mL The medicament, sucrose and xanthan gum are blended, passed through a No . 10 mesh US sieve, and then mixed with a previously made solution of the microcrystalline cellulose and sodium carboxymethyl cellulose in water. The sodium benzoate, flavor, and color are diluted with some of the water and added with stirring. Sufficient water is then added to produce the required volume . Formulation Example 3 Capsules, each containing 15 mg of medicament, are made as follows: Quantity Ingredient (mg/capsule) Active Ingredient (s) 15.0 mg Starch 407.0 mg Magnesium stearate 3.0 mg Total 425.0 mg The active ingredient, cellulose, starch, and magnesium stearate are blended, passed through a No. 20 mesh US sieve, and filled into hard gelatin capsules in 425 mg quantities . Formulation Example 4 An intravenous formulation may be prepared as follows: Ingredient Quantity Active Ingredient ( s) 100 mg Polyethylene glycol hydroxysterate (Solutol HS1 155))33..44 gg Propylene glycol 1.7 g Fractionated coconut oil 3.5 g Phosphatidylcholine (Phospholipon ® 90G) 1.4 g Dextrose 1.75 g Phosphate buffer (pH 5.5) Q.S. The polyethylene glycol hydroxysterate (Solutol HS15), and propylene glycol, and fractionated coconut oil are mixed. While mixing the phosphatidylcholine (Phospholipon 90G) is added. Heat to about 40°C and continue mixing until clear. Add the active ingredient and mix until clear. Combine with a solution of dextrose in phosphate buffer (0.05 M sodium dihydrogen phosphate hydrate adjusted to pH 5.5 using 0.05 M disodium hydrogen phosphate, about 80 mL) and mix well. Q.S. to 100 mL with the phosphate buffer to give an almost transparent microemulsion . Formulation Example 5 A topical formulation may be prepared as follows: Ingredient Quantity Active Ingredient ( s) 1-10 g Emulsifying Wax 30 g Liquid Paraffin 20 g White Soft Paraffin to 100 g The white soft paraffin is heated until molten. The liquid paraffin and emulsifying wax are incorporated and stirred until dissolved. The active ingredient is added and stirring is continued until dispersed. The mixture is then cooled until solid. Formulation Example 6 An intravenous formulation may be prepared as follows : Ingredient Quantity Active Ingredient (s) 100 mg PEG-35 Castor Oil (Cremophor ® EL) 3.4 g Propylene glycol 1.7 g Fractionated coconut oil 3.5 g Phosphatidylcholine (Phospholipon ® 90G) 1.4 g Dextrose 1.75 g Phosphate buffer (pH 5.5) Q.S. Prepare by the procedure of Formulation Example 5 to give a microemulsion . Formulation Example 7 An intravenous formulation may be prepared as follows: Ingredient Quantity Active Ingredient (s) 100 mg Polyethylene glycol hydroxysterate (Solutol™ HS15)5.0 g Ethanol 5.0 g 0.9% Sodium Chloride Injection, USP Q.S. Mix polyethylene glycol hydroxysterate (Solutol™ HS15), ethanol, and the active ingredient. Q.S. to 100 mL with 0.9% sodium chloride injection, USP to give a solution. The compounds of the present invention can be evaluated employing the assays below. Other assays useful for evaluating this reversing capability are well known in the art. See, e.g. , T. McGrath, et al. , Biochemical Pharmacology, 38:3611, (1989); D. Marquardt and M.S. Center, Cancer Research, 52:3157, (1992); and D. Marquardt, et al . , Cancer Research, 50:1426, (1990). EXAMPLE A Assay for reversal of MRP-1-mediated doxorubicin resistance and MRP-1 mediated vincristine resistance HL60/ADR and HL60/VCR are continuous cell lines which were selected for doxorubicin and vincristine resistance, respectively, by culturing HL60, a human acute myeloblastic leukemia cell line, in increasing concentrations of doxorubicin or vincristine until a highly resistant variant was obtained. These cell lines were obtained from the laboratory of Dr. Melvin center, Kansas State University. The cells were grown in RPMI 1640 (Gibco) containing 10% fetal bovine serum and 250 mg/mL gentamycin (Sigma) . Cells were harvested , washed twice with assay medium (same as culture medium) , counted and diluted to 2 X 10 5 cells/mL in assay medium. The stock solution of test compounds was made in dimethyl sulfoxide (DMSO) . This solution was further diluted with assay medium to represent 1 nM to 10 mM final concentrations with one-half log dose intervals. The assay was performed in a 96 well plate containing 10,000 cells/well, test compounds and either doxorubicin or vincristine or etoposide. The concentrations of the oncolytic were selected such that they alone inhibit 10-20% of the growth of cells. Appropriate controls were incorporated in the study. Following 72 h incubation at 37 °C, Ala arBlue (15 mL/well) was added and additionally incubated for 4 h. The fluorescence (550 nm exitation and 590 nm emission) was measured using a Tecan SpectaFluor Plus fluorescent plate reader. The ability of a test compound to reverse the resistance of HL60/ADR and HL60/VCR cells to either doxorubicin or vincristine or etoposide was determined by comparison of the absorbance of the wells containing a test compound plus to the oncolytic (doxorubicin or vincristine or etoposide) with the absorbance of wells containing the oncolytic alone. The effectiveness of test compounds to modulate the cytotoxicity of doxorubicin, vincristine and etoposide to is expressed as EC 50 (values expressed as % control were plotted against concentration of test compounds. Curve fitting of the points is accomplished using the Bravo/SAS software package. The midpoint between the upper and lower limits of the curve is the EC 5 0) • EXAMPLE B Assay for reversal of MRP-1-mediated doxorubicin resistance in HeLa T5 cells The HeLa transfectants, Cl (vector control) and T5 (MRP transfectant ) , were obtained from the laboratory of Drs . Susan Cole and Roger Deeley, Queens University, Kingston, Ontario. Both cell lines were cultured with and assays performed in RPMI 1640 containing 25 mM HEPES supplemented with 5% iron supplemented calf serum and 400 mg/mL geneticin (Gibco-BRL) Cells were harvested using trypsin-EDTA, washed once with culture medium and centrifuged at 1000 rpm for 5 min. Supernatant was removed and the cells were resuspened in assay medium. The cells were counted and adjusted with assay medium to give a concentration of 2 X 10 5 cells/mL. Test compounds were serially diluted to represent final concentrations of 10-0.039 μM. Doxorubicin was added at the concentration of 0.5 μg/mL to the wells containing different concentrations of the test compound. Appropriate controls were used. The cells were incubated at 37 °C for 72 h. Then 20 mL of MTS reagent was added to all wells and additionally incubated at 37°C for 90 min. Absorbance was determined at 490 nm. The above experiment was repeated with two other oncolytics, vincristine (final concentration 0.002 μg/mL) and etoposide (final concentration 10 μg/mL) . The effectiveness of test compounds to modulate the cytotoxicity of doxorubicin, vincristine and etoposide in HeLaT5 cells are expressed as EC 50 (values expressed as % control were plotted against concentration of test compounds . Curve fitting of the points is accomplished using the Bravo/SAS software package. The midpoint between the upper and lower limits of the curve is the EC 50 ) . Plasma membrane binding assay: HeLaT5 cells were grown for 18-21 days in RPMI with 5% Fe supplemented bovine calf serum and 400mg/mL geneticin. The medium was replaced every two to three days . Membrane vesicles were prepared using a modified procedure of Doige, C.A., and Sharom, F . J . (Biochem. Biophys. Acta, 1109: 161- 171, 1992) and Cornwell, M.M. , Gottesman, M.M. , and Pastan, I.H. (J. Biol. Chem., 261: 7921-7928, 1986). Cells grown in monolayers were washed once with of ice cold PBS (pH 7.4) and scraped with Corning cell scraper. For frozen cells, cells were quickly thawed and slowly added with RPMI 1640 supplemented with 10%FBS at 37°C. The cells were washed once in cold buffer (250mM sucrose, 50mM Tris-HCl, 0.2mM CaC12, pH 7.5) with protease inhibitors ( 0.1 mM PMSF, 1.0 mM leupeptin, 0.3 mM aprotinin) . The cells were disrupted by nitrogen cavitation with a 1850 mL bomb (Parr Instrument Company, Moline, IL) at 350 psi for 1 hour. The homogenate was centrifuged in a Sorvall centrifuge at 2000 rpm for 5 min to remove unbroken cells. To the supernatant 0. ImM EDTA was added and layered over a 38% sucrose gradient. The interface was collected and centrifuged at 25,000 rpm in a Beckman Ultra centrifuge with an SW28 rotor for 1 hour. The resulting pellet was resuspended in 50mM Tris-HCl buffer and passed through a 27-gauge needle. The membranes were stored under argon in liquid nitrogen until use. The transport assay was performed using a modified procedure described by Leier, Jedlitschky, Buchholz and Keppler (European J. of Biochem. , 220, 599-606,1994). The assay was initiated by mixing 3-5 μg of membrane preparation with 4 mM ATP (Sigma), 10 mM MgCl 2 , 1 mM glutathione (Sigma) an ATP regenerating system consisting of 100 μg/mL creatine kinase (Sigma) and lO M creatine phosphate (Sigma) , 50 nM ( 3 H)LTC 4 (110 Ci/mmol purchased from DuPont NEN) and test compounds . The test compounds were added to give a final highest concentration of 50 μM and logarithmically diluted to give a lowest concentration of 1 nM. After incubation at 37°C for 60 seconds the uptake was terminated by 3 X 200 mL washes followed by 3 X 1 mL washes with a 1 mL 96well plate using the Packard Filtermate 196 attached to a vacuum pump. The membrane vesicles are collected onto a Packard unifilter-96 GF/B plate and 40 mL of Packard Microscint 20 were added to each well. The radioactivity associated with the filter was then counted on a Packard Top Count. The ability of test compounds to inhibit the uptake of radio labeled LTC 4 is expressed as EC 50 values. The compounds 1, 2 and 4-7 were tested in combination with a clinically used oncolytic, doxorubicin, in a panel of three cell lines: HL60S (drug-sensitive parental cell line), HL60VCR (cell line expressing P-glycoprotein-mediated multidrug resistance) and HL60ADR (cell line expressing MRP- 1-mediated multidrug resistance) . Compounds 1, 2 and 4-6 showed reversal of resistance to doxorubicin in HL60ADR cells at 1 μM and showed no effect in HL60S and HL60VCR cells at and up to 2.5 μM demonstrating that the compounds are selective for MRP-1-mediated drug resistance. In order to determine the affinity of MRP-1 for these compounds, the compounds were tested in a transporter assay with plasma membranes prepared from MRP-1-transfected HeLaT5 cells. Compounds 4 and 5 inhibited the transport of radiolabeled LTC 4 , a substrate of MRP-1, with an IC 50 value of -5 mM. Compounds 1 and 2 showed much weaker activity at this concentration . The compounds of the present invention demonstrate a significant effect in reversing the multi-drug resistance. Many of the compounds showed very significant enhancement of activity in combination with the oncolytic agent as opposed to the oncolytic agent alone. Results for representative compounds of formula I are given in the tables below. Modulation of Vincristine, Doxorubicin and Etoposide in HL60ADR Cells Modulation of Vincristine, Doxorubicin and Etoposide in HL60VCR Cells Table A.3 Modulation of Vincristine, Doxorubicin and Etoposide in HeLaT5 Cells

Claims: WE CLAIM: 1. A method of treating multi-drug resistant diseases comprising administering to a patient in need thereof an effective amount of a therapeutic agent and a multi-drug resistance inhibitory amount of a compound of the formula wherein R is hydrogen, R' is a radical selected from the group consisting of wherein R 4 is selected from the group consisting of hydroxy, Cι-C 6 alkoxy, and -NR 5 R 6 wherein R 5 is selected from the group consisting of hydrogen, Cι-C 6 alkyl substituted Cι-C 6 alkyl, benzyl, and substituted benzyl, and R s is selected from the group consisting of hydrogen, Ci-Cε alkyl substituted Cι-C 6 alkyl, aralkyl, heteroaryl, heteroarylalkyl, and heterocyclic ; or R and R' taken together form a divalent radical selected from the group consisting of Ri and R 2 taken together with the atoms to which they are attached form a carbon-carbon double bond; or Ri and R 2 taken together with the atoms to which they are attached form an epoxide; or Ri is fluoro and R 2 is hydroxy; or Ri is hydroxy or ester and R 2 is hydroxy, fluoro, chloro, bromo, or a radical selected from the group consisting of -NR 7 Rs and -X-R 9 wherein R 7 is selected from the group consisting of hydrogen, Ci-Cβ alkyl, and benzyl; Rs is selected from the group consisting of hydrogen, Ci-Cε alkyl, substituted Cι-C 6 alkyl, C 3 -C7 cycloalkyl, C 5 -C7 cycloalkenyl, aryl, aralkyl, heteroaryl, heteroarylalkyl, and heterocyclic ; or R 7 and R 8 together with the nitrogen to which it is attached form a piperidine ring, pyrrolidine ring, morpholine ring, thiomorpholine ring, piperazine ring, or a 1,2,3,4- tetrahydroquinoline ring; X is 0 or S; R 9 is selected from the group consisting of Ci- C 6 alkyl, substituted Cι-C 6 alkyl, C 3 -C 7 cycloalkyl, C 5 -C 7 cycloalkenyl, aryl, aralkyl, heteroaryl, heteroarylalkyl; R 3 is selected from the group consisting of hydrogen, ester, Cι-C 6 alkyl, benzyl, substituted benzyl, and -CH 2 C(0)NH 2 ; and the stereoisomers and pharmaceutical salts thereof. 2. A method according to Claim 1 wherein R 3 is hydrogen. 3. A method according to Claim 2 wherein R and R' taken together form the divalent radical 4. A method according to Claim 3 wherein Ri is fluoro and R2 is hydroxy . 5. A method according to Claim 3 wherein Ri is hydroxy and R 2 is fluoro, chloro, or bromo. 6. A method according to Claim 3 wherein Ri is hydroxy and R 2 is a radical selected from the group consisting of -NR 7 Rs and -X-Rg. 7. A method according to Claim 6 wherein R is -NR 7 Rs wherein R 7 is hydrogen and R 8 is selected from the group consisting of Cι-C 6 alkyl, substituted Ci-C δ alkyl, and aralkyl. 8. A method according to Claim 6 wherein R is -X-R 9 wherein X is 0. 9. A method according to Claim 8 wherein Rg is selected from the group consisting of Ci-Cε alkyl, substituted Ci-Cδ alkyl, aryl, and aralkyl. 10. A method according to Claim 6 wherein R 2 is -X-R 9 wherein X is S. 11. A method according to Claim 10 wherein R 9 is selected from the group consisting of Ci-Cε alkyl, substituted Ci-Cε alkyl, aryl, and aralkyl. 12. A method according to Claim 1 wherein the therapeutic agent is an oncolytic agent. 13. A method according to Claim 12 wherein the oncolytic agent is selected from the group consisting of vinblastine, vincristine, vindesine, navelbine, daunorubicin, doxorubicin, mitroxantrone, etoposide, teniposide, mitomycin C, actinomycin D, taxol, topotecan, mithramycin, colchicine, puromycin, podophyllotoxin, ethidium bromide, emetine, gramicidin D, and valinomycin. 4. A compound of the formula wherein R is hydrogen, R' is a radical selected from the group consisting of wherein R is selected from the group consisting of hydroxy, Ci-Cβ alkoxy, and -NR 5 R 6 wherein R 5 is selected from the group consisting of hydrogen, Cι-C 6 alkyl substituted Cι-C 6 alkyl, benzyl, and substituted benzyl, and Re is selected from the group consisting of hydrogen, Cι-C 6 alkyl substituted Ci-Cβ alkyl, aralkyl, heteroaryl, heteroarylalkyl, and heterocyclic; or R and R' taken together form a divalent radical selected from the group consisting of Ri and R 2 taken together with the atoms to which they are attached form a carbon-carbon double bond; or Ri and R taken together with the atoms to which they are attached form an epoxide; or Ri is fluoro and R 2 is hydroxy; or Ri is hydroxy or ester and R 2 is hydroxy, fluoro, chloro, bromo, or a radical selected from the group consisting of -NR 7 R 8 and -X-R 9 wherein R 7 is selected from the group consisting of hydrogen, Ci-Cε alkyl, and benzyl; R 8 is selected from the group consisting of hydrogen, Ci-Cβ alkyl, substituted Ci-Cε alkyl, C 3 -C7 cycloalkyl, C 5 -C 7 cycloalkenyl, aryl, aralkyl, heteroaryl, heteroarylalkyl, and heterocyclic ; or R 7 and R 8 together with the nitrogen to which it is attached form a piperidine ring, pyrrolidine ring, morpholine ring, thio orpholine ring, piperazine ring, or a 1,2,3,4- tetrahydroquinoline ring; X is 0 or S; Rg is selected from the group consisting of Ci- Cβ alkyl, substituted Cι-C 6 alkyl, C 3 -C7 cycloalkyl, C 5 -C 7 cycloalkenyl, aryl, aralkyl, heteroaryl, heteroarylalkyl; R 3 is selected from the group consisting of hydrogen, ester, Cι-C 6 alkyl, benzyl, substituted benzyl, and -CH 2 C(0)NH 2 ; provided that when R 3 hydrogen, R is hydrogen, and R' is the radical then Rx and R 2 cannot be taken together with the atoms to which they are attached to form a carbon- carbon double bond or taken together with the atoms to which they are attached to form an epoxide; further provided that when R and R' are taken together to form the divalent radical then Ri and R 2 cannot be taken together with the atoms to which they are attached to form a carbon- carbon double bond or taken together with the atoms to which they are attached to form an epoxide; and the stereoisomers and pharmaceutical salts thereof 15. A compound according to Claim 14 wherein R 3 is hydrogen. 16. A compound according to Claim 15 wherein R and R' taken together form the divalent radical 17. A compound according to Claim 16 wherein Ri is fluoro and R 2 is hydroxy . 18. A compound according to Claim 16 wherein Ri is hydroxy and R 2 is fluoro, chloro, or bromo. 19. A compound according to Claim 16 wherein Ri is hydroxy and R 2 is -NR 7 R 8 . 20. A compound according to Claim 16 wherein Ri is hydroxy and R 2 is -X-R 9 wherein X is 0. 21. A compound according to Claim 16 wherein Ri is hydroxy and R 2 is -X-R 9 wherein X is S. 22. A compound according to Claim 15 wherein R is hydrogen and R' is the radical 23. A compound according to Claim 24 wherein Ri is fluoro and R 2 is hydroxy. 24. A compound according to Claim 24 wherein Ri is hydroxy and R 2 is fluoro, chloro, or bromo. 25. A compound according to Claim 24 wherein Ri is hydroxy and R 2 is -NR 7 R 8 - 26. A compound according to Claim 24 wherein Ri is hydroxy and R 2 is -X-Rg wherein X is 0. 27. A compound according to Claim 24 wherein Ri is hydroxy and R 2 is -X-Rg wherein X is S. 28. A compound according to Claim 15 wherein R is hydrogen and R' is the radical Ri is hydroxy and R 2 is selected from the group consisting of fluoro, chloro, bromo, hydroxy, benzylamino, phenethy1amino, 3-phenylpropylamino, 4-phenylbutylamino, 2- methylbenzylamino, 2-methoxybenzylamino, N- methylbenzylamino, N-ethylbenzylamino, N- isopropylbenzylamino, N-methylphenethylamino, cc- methylbenzylamino, 3 , 4-methylenedioxybenzylamino, 4- sulfonylaminophenethyamino, α-phenylbenzylamino, 3- (hydroxymethyl) anilino, cyclopentylamino, 1-azacyclohept-l- ylamino, 3 , 5-dimethoxyanilino, 6-hydroxyhexylamino, 3- (butoxy) propylamino, decylamino, 2- (cyclohex-l-en-1- yl) ethylamino, 3- (N-morpholino) propylamino, dibenzylamino, napth-1-ylmethylamino, and 2-carbamoyl-N-pyrrolidino . 29. A compound according to Claim 15 wherein Ri is hydroxy and R 2 is selected from the group consisting of fluoro, chloro, bromo, hydoxy, p-toluenesulfonate, benzylamino, N- methylbenzylamino, napth-1-ylmethylamino, diethyamino, 2- hydroxyethyamino, 3- (N,N-dimethylamino) propylamino, 3- (butoxy) propylamino, dibenzylamino, N-isopropylbenzylamino, N-methylphenethylamino, 2-carbamoyl-N-pyrrolidino, 2-(lH- imidizol-5-yl) ethylamino, 4- (4H-1, 2 , 4-triazol-4-yl) amino, 2- chlorobenzylamino, 3 , 4-dimethoxyphenethylamino, 2- fluorophenoxy, 4-trifluoromethoxyphenoxy, 3-fluorophenoxy, 3-chlorophenoxy, 4-methylphenoxy, 4-iodophenoxy, 2- ethylphenoxy, 3-methoxphenoxy, phenoxy, 4-methoxyphenoxy, 4- ethyoxyphenoxy, 3 , 4 , 5-trimethoxyphenoxy, 3,4- dimethoxyphenoxy, 2 , 3-dimethoxyphenoxy, 3,5- dimethoxyphenoxy, 2 , 6-dimethoxyphenoxy, naphty-2-yloxy, 2- (prop-1-en-1-yl) phenoxy, 4-phenylphenoxy, 4- (4- chlorophenyl) phenoxy, 2-fluorothiophenoxy, 3- fluorothiophenoxy, 2-methylthiophenoxy, 4- trifluoromethoxythiophenoxy, 2, 5-dimethoxythiophenoxy, thiophenoxy, 4-acetamidothiophenoxy, 4-methoxythiophenoxy, 4- (t-butyl) thiophenoxy, 3-methoxythiophenoxy, 2- methoxythiophenoxy, 3 , 4-dimethoxythiophenoxy, propythio, cyclopentylthio, and cyclohexylthio . 30. A compound according to Claim 15 wherein R and R' taken together form the divalent radical Ri is hydroxy, and R 2 is selected from the group consisting of fluoro, chloro, bromo, hydoxy, benzylamino, N- methylbenzylamino, 2-chloeobenzylamino, 2- methoxybenzylamino, 2-fluorothiophenoxy, 4- fluorothiophenoxy, 2-methylthiophenoxy, 3- trifluoromethoxythiophenoxy, 2, 5-dimethoxythiophenoxy. 31. A compound according to Claim 15 wherein R and R' taken together form the divalent radical Ri is hydroxy, and R 2 is selected from the group consisting of fluoro, chloro, hydroxy, 2-fluorothiophenoxy, 2,5- dimethoxythiophenoxy, 2-methylthiophenoxy, 3 -fluorophenoxy, 3-chlorophenoxy, and 3-methoxyphenoxy . 32. A pharmaceutical composition comprising a compound of Claim 13 and a pharmaceutically acceptable dilutent.

Inventors: KULANTHAIVEL PALANIAPPAN, VASUDEVAN VENKATRAGHAVAN, KULANTHAIVEL, PALANIAPPAN, VASUDEVAN, VENKATRAGHAVAN
Applicants: KULANTHAIVEL PALANIAPPAN, LILLY CO ELI, VASUDEVAN VENKATRAGHAVAN, ELI LILLY AND COMPANY, KULANTHAIVEL, PALANIAPPAN, VASUDEVAN, VENKATRAGHAVAN
Application year: 2000
Main classification: C07K7
IPC codes: A61K38, C07K7