EP1192178A2 - Rufomycine und deren derivate als hemmer von multi-drug resistence assoziiert mit protein-1 (mrp-1) - Google Patents

Rufomycine und deren derivate als hemmer von multi-drug resistence assoziiert mit protein-1 (mrp-1)

Info

Publication number
EP1192178A2
EP1192178A2 EP00946770A EP00946770A EP1192178A2 EP 1192178 A2 EP1192178 A2 EP 1192178A2 EP 00946770 A EP00946770 A EP 00946770A EP 00946770 A EP00946770 A EP 00946770A EP 1192178 A2 EP1192178 A2 EP 1192178A2
Authority
EP
European Patent Office
Prior art keywords
group
alkyl
hydroxy
hydrogen
substituted
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00946770A
Other languages
English (en)
French (fr)
Inventor
Palaniappan Kulanthaivel
Venkatraghavan Vasudevan
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Eli Lilly and Co
Original Assignee
Eli Lilly and Co
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Eli Lilly and Co filed Critical Eli Lilly and Co
Publication of EP1192178A2 publication Critical patent/EP1192178A2/de
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/64—Cyclic peptides containing only normal peptide links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • 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.
  • 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.
  • 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).
  • MDR multi- drug resistance
  • Multi-drug resistance has developed to a broad spectrum of oncolytic agents and is a major impediment to effective chemotherapy.
  • 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 ADRIAMYCINTM, 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 TM) 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.
  • 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.
  • 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 .
  • MDR multi-drug resistance associated protein-1
  • ADRIAMYCINTM is an inhibitor of topoisomerase II. W.T.
  • HL60/Adr cells (a HL60-derived cell line which is resistant to doxorubicin) treated with Adriamycin and Etoposide had IC 50 of 2.2 ⁇ g/mL and >10 ⁇ g/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.
  • 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.
  • R is hydrogen
  • R' is a radical selected from the group consisting of
  • R 4 is selected from the group consisting of hydroxy, C ⁇ -C 6 alkoxy, and -NRR 6 wherein R 5 is selected from the group consisting of hydrogen, Ci-C ⁇ alkyl substituted C ⁇ -C 6 alkyl, benzyl, and substituted benzyl, and R 6 is selected from the group consisting of hydrogen, Ci-C ⁇ alkyl substituted C ⁇ -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 is fluoro and R 2 is hydroxy
  • 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-C ⁇ alkyl, and benzyl; Rs is selected from the group consisting of hydrogen, Ci-Cg alkyl, substituted Ci-C ⁇ 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 C ⁇ -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, C ⁇ -C 6 alkyl, benzyl, substituted benzyl, and -CH 2 C(0)NH 2 ;
  • 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:
  • R is hydrogen
  • R' is a radical selected from the group consisting of wherein
  • R is selected from the group consisting of hydroxy, C ⁇ -C 6 alkoxy, and -NR 5 R 6 wherein R 5 is selected from the group consisting of hydrogen, C I -C ⁇ alkyl substituted C ⁇ -C 6 alkyl, benzyl, and substituted benzyl, and R 6 is selected from the group consisting of hydrogen, Ci-C ⁇ alkyl substituted C ⁇ -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 is fluoro and R 2 is hydroxy
  • 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-C ⁇ alkyl, and benzyl;
  • Rs is selected from the group consisting of hydrogen, C ⁇ -C 6 alkyl, substituted Ci-C ⁇ 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-
  • R 3 is selected from the group consisting of hydrogen, ester, C ⁇ -C 6 alkyl, benzyl, substituted benzyl, and -CH 2 C(0)NH 2 ;
  • 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.
  • Ci-C ⁇ 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;
  • substituted C ⁇ -C 6 alkyl refers to a C ⁇ -C 6 alkyl independently substituted with 1 to 2 substituents selected from the group consisting of hydrogen, hydroxy, amino, substituted amino, -NRioR wherein R ⁇ 0 and Rn are independently C ⁇ -C 6 alkyl, and C ⁇ -C 6 alkoxy;
  • C 3 -C7 cycloalkyl refers to a saturated cyclic alkyl group having from three to seven carbon atoms and includes, cyclopropyl, c
  • 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, ⁇ -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,
  • 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.
  • the Cahn-Prelog-Ingold designations of (R) - and (S)- are used to refer to specific isomers.
  • 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.
  • 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-C ⁇ alkyl, substituted C ⁇ -C 6 alkyl, and aralkyl are preferred;
  • 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-C ⁇ alkyl, substituted Ci-C ⁇ alkyl, aryl, and aralkyl are preferred;
  • Ri is hydroxy and R 2 is -X-R 9 wherein X is S and Rg is selected from the group consisting of Ci-C ⁇ alkyl, substituted C ⁇ -C 6 alkyl, C 3 -C7 cycloalkyl, aryl, and aralkyl are preferred.
  • 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, C ⁇ -C 6 alkyl, and Ci- Ce alkoxy; esters of alkyl and aryl diacids, such as the esters of malonic acid, succinic acid, glutaric acid,
  • 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 C ⁇ -C 6 alkoxy; are preferred.
  • 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 acet
  • 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.
  • 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 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 ( ⁇ +1.4 mmu) and 1042.5629 ( ⁇ -1.6 mmu) , respectively.
  • RI and R2 are taken together with the atoms to which they are attached to form an epoxide and R3 is hydrogen
  • H NMR spectra of 4 and 5 are very similar to each other and to the spectrum of 2.
  • AHA 2-amino hexenoic acid
  • prenylated tryptophan N-methylleucine
  • nitrotyrosine and alanine that are found in the known rufomycins.
  • the first of the two amino acids showed 1 H and 13 C NMR signals (4: ⁇ ⁇ H 5.28 and ⁇ ⁇ c 53.3, ⁇ ⁇ H 2.02 , 1.74 and ⁇ ⁇ c 35.7, ⁇ H 1.57 and ⁇ C 25.3, ⁇ ⁇ H 0.98 and ⁇ c 23.6 and ⁇ ⁇ H 0.91 and ⁇ ⁇ C 21.8 and 5: ⁇ H 5.40 and ⁇ ⁇ C 56.0, ⁇ ⁇ H 2.05, 1.89 and ⁇ ⁇ c 37.6, ⁇ H 1.83 and ⁇ c 25.1, ⁇ ⁇ H 0.95 and ⁇ ⁇ c 23.8 and ⁇ ⁇ H 0.93 and ⁇ ⁇ c 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: ⁇ ⁇ H 3.86 and ⁇ ⁇ c 59.8, ⁇ ⁇ H 2.52, 1.99 and ⁇ ⁇ c 26.2, ⁇ H 2.24 and ⁇ C 34.6, ⁇ ⁇ H 4.83 and ⁇ 81.3 and ⁇ ⁇ H 1.11 and ⁇ ⁇ c 16.4 and 5: ⁇ ⁇ H 3.81 and ⁇ ⁇ C 59.8, ⁇ ⁇ H 2.43, 1.74 and ⁇ ⁇ c 31.9, ⁇ H 2.71 and ⁇ C 33.3, ⁇ ⁇ H 4.75 and ⁇ c 82.0 and ⁇ ⁇ H 1.00 and ⁇ ⁇ c 17.5) characteristic of another leucine moiety with the exceptions of absence of the amide and one of the methyl resonances .
  • the missing methyl group was replaced by a hydroxyl-bearing methine carbon (5 H 4.83 and 4.75, ⁇ 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.
  • RI is hydroxy
  • R2 is chloro
  • R3 is hydrogen
  • 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 ( ⁇ +2.4 mmu) .
  • Compound 6 Yellow powder ; [ ⁇ ] D -64.1 (c 0.097, EtOH); MS (ES): m/z 1064.4 (M+Na) + , m/z
  • the ⁇ -proton showed two small couplings (4 and 4.5 Hz) to the ⁇ -protons indicating that this proton is oriented equatorial or ⁇ and the methyl group is oriented axial or ⁇ .
  • the ⁇ -proton showed one large (11.5 Hz) and one small (3.5 Hz) coupling to the ⁇ -protons indicating that this proton is oriented axial, or ⁇ , and the methyl group is oriented equatorial or ⁇ . Accordingly, in the differential NOE spectrum of 11, irradiation of the ⁇ -proton of the amino acid produced a significant enhancement of the ⁇ -methyl protons.
  • the absolute stereochemistry at the ⁇ - carbon could be assigned the (S) -configuration for 4 and 5 and the (R) -configuration for 6.
  • stereochemistry at the amino acetal carbon of 6 the small coupling constant observed between the ⁇ -and ⁇ -protons warranted axial orientation, or ⁇ , 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 ⁇ -carbon of 4 and 5 as the ⁇ - and ⁇ -protons are disposed either equatorial-equatorial or equatorial-axial.
  • inspection of the Dreiding model indicated that in the diastereomer in which the ⁇ -H is oriented axial it should show a NOE interaction with the axial ⁇ -H, provided the six membered ring adopts a perfect chair conformation. Neither 4 nor 5 showed significant NOE interaction between these protons.
  • RI and R2 are taken to togetherer to form an epoxide, R3 is hydrogen, and R4 is hydroxy
  • the compounds of formula I encompass the compounds of formula II.
  • 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.
  • 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 .
  • 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.
  • epoxide deoxygenating reagents 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.
  • 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.
  • 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.
  • 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.
  • an epoxide of formula (a) is contacted with a suitable nucleophile to give a compound of formula I.
  • 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.
  • 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.
  • 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.
  • a suitable solvent such as tetrahydrofuran, methanol, ethanol, isopropanol, butanol, tetrahydrofuran/water mixtures, methanol /water mixtures, ethanol /water mixtures, and the like.
  • a suitable solvent such as tetrahydrofuran, methanol, ethanol, isopropanol, butanol, tetrahydrofuran/water mixtures, methanol /water mixtures, ethanol /water mixtures, and the like.
  • 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.
  • 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
  • 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.
  • reaction may advantageously be carried out in the presence of a catalyst, such as benzyltriethylammonium chloride, tetrabutylammonium chloride, benzyltriethylammonium hydrosulfate, and the like.
  • a catalyst such as benzyltriethylammonium chloride, tetrabutylammonium chloride, benzyltriethylammonium hydrosulfate, and the like.
  • 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.
  • 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.
  • a two to ten fold molar excess of the base is used.
  • a strong base such as potassium t- butoxide or sodium hydride
  • a slight molar excess of base is preferred.
  • 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.
  • 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.
  • a suitable solvent such as tetrahydrofuran, dimethylformamide, dichloromethane, and the like.
  • 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.
  • 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.
  • step a a luciomycin factor in which R is hydrogen and R' is the radical of the formula
  • R 4 is hydroxy undergo an ester formation or amide formation to give a compound of formula (ai) in which R 4 is Ci-C ⁇ alkoxy or -NR 5 R 6 .
  • ester and amide formations are well known in the art.
  • 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-C ⁇ 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.
  • a suitable solvent such as tetrahydrofuran, dimethylformamide, dichloromethane, and the like.
  • 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.
  • step b a lucidation factor in which R and R' are the divalent radical of the formula
  • 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.
  • the reaction is carried out at temperatures of from about 0°C to the refluxing temperature of the solvent.
  • 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.
  • such a dehydration reaction under acidic conditions may also open the epoxide.
  • the chlorohydrin or bromohydrin may be obtained.
  • the epoxide can be reformed by treating the chlorohydrin or bromohydrin with base, such as sodium carbonate or potassium carbonate.
  • 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.
  • 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.
  • step c a lucidation factor in which R and R' are the divalent radical of the formula
  • 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.
  • step d a lucidogen factor in which R and R' are the divalent radical of the formula
  • 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 .
  • reaction mixtures at the product level of 0.025 mmol were carried out using a Waters SymmetryPrep column (Cis, 7 ⁇ m, 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 ⁇ m, 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 .
  • 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 TunairTM flasks. After an incubation period of 72 hours under the same conditions described above, the contents of two TunairTM 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.
  • 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.
  • 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
  • MOPS buffer pH 7.0 was routinely used in shake-flask studies to control culture pH .
  • Key nutrients in the modified G-2 medium were tested for main effects using a
  • 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) .
  • 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.
  • 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.
  • 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.
  • 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 .
  • 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.
  • the lucimycin 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
  • 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.
  • the yield of rufomycin factors 5, 4 and 6 was about 85% of that achieved by a 67% acetone extract.
  • 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.
  • 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.
  • 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.
  • pH 3 triethylamine phosphate buffer
  • 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 (C ⁇ 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.
  • 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.
  • 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.
  • 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 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.
  • 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 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.
  • 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.
  • 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 ⁇ l 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) .
  • 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.
  • 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.
  • the compounds of the invention may be used on neoplasms having intrinsic and/or acquired resistance.
  • 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 .
  • multi-drug resistance include both intrinsic resistance and acquired drug resistance
  • 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
  • patient refers to a mammal and specifically includes, humans, horses, cattle, sheep, dogs, cats, guinea pigs, mice, rats, monkeys, chimpanzees, apes
  • patient in need thereof refers to a patient which has a disease or condition which manifests multi-drug resistance
  • therapeutic agent refers an chemotherapeutic agent which has decreased efficacy due to multi-drug resistance, and specifically includes oncolytic agents or anti-malarial agents
  • effective amount refers to that amount of a therapeutic agent that is effective in treating
  • 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.
  • 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 .
  • 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.
  • 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.
  • a carrier which can be in the form of a capsule, sachet, paper or other container.
  • 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.
  • 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.
  • 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.
  • the active compound 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.
  • 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.
  • a solid preformulation composition containing a homogeneous mixture of a compound of the present invention.
  • 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.
  • 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.
  • 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.
  • 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.
  • the compound of the present invention may be incorporated into a solution, suspension, emulsion, or microemulsion.
  • 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
  • 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.
  • 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.
  • 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.
  • active ingredient means a compound according to the present invention or a pharmaceutically acceptable salt thereof.
  • Capsules each containing 40 mg of medicament are made as follows:
  • Suspensions each containing 50 mg of medicament per 5.0 mL dose are made as follows:
  • Quantity Ingredient (mg/capsule)
  • 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 .
  • composition Example 4 An intravenous formulation may be prepared as follows:
  • Phosphatidylcholine (Phospholipon ® 90G) 1.4 g Dextrose 1.75 g
  • 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.
  • phosphate buffer 0.05 M sodium dihydrogen phosphate hydrate adjusted to pH 5.5 using 0.05 M disodium hydrogen phosphate, about 80 mL
  • a topical formulation may be prepared as follows:
  • 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.
  • An intravenous formulation may be prepared as follows :
  • An intravenous formulation may be prepared as follows:
  • 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.
  • test compounds were 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
  • test compound 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) •
  • 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, 0.2m
  • 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 ⁇ g of membrane preparation with 4 mM ATP (Sigma), 10 mM MgCl 2 , 1 mM glutathione (Sigma) an ATP regenerating system consisting of 100 ⁇ g/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 .
  • test compounds were added to give a final highest concentration of 50 ⁇ M 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 ⁇ M and showed no effect in HL60S and HL60VCR cells at and up to 2.5 ⁇ M demonstrating that the compounds are selective for MRP-1-mediated drug resistance.
  • 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.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
EP00946770A 1999-06-21 2000-06-08 Rufomycine und deren derivate als hemmer von multi-drug resistence assoziiert mit protein-1 (mrp-1) Withdrawn EP1192178A2 (de)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US14036299P 1999-06-21 1999-06-21
US140362P 1999-06-21
PCT/US2000/015020 WO2000078795A2 (en) 1999-06-21 2000-06-08 Rufomycins and derivatives thereof useful as inhibitors of multi-drug resistance associated protein-1 (mrp-1)

Publications (1)

Publication Number Publication Date
EP1192178A2 true EP1192178A2 (de) 2002-04-03

Family

ID=22490896

Family Applications (1)

Application Number Title Priority Date Filing Date
EP00946770A Withdrawn EP1192178A2 (de) 1999-06-21 2000-06-08 Rufomycine und deren derivate als hemmer von multi-drug resistence assoziiert mit protein-1 (mrp-1)

Country Status (4)

Country Link
EP (1) EP1192178A2 (de)
AU (1) AU6047300A (de)
PE (1) PE20010305A1 (de)
WO (1) WO2000078795A2 (de)

Families Citing this family (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2385829B1 (de) 2009-01-09 2018-08-01 Board of Regents of the University of Texas System Proneurogene verbindungen
US8362277B2 (en) 2009-01-09 2013-01-29 Board Of Regents Of The University Of Texas System Pro-neurogenic compounds
US9162980B2 (en) 2009-01-09 2015-10-20 Board Of Regents Of The University Of Texas System Anti-depression compounds
US9962368B2 (en) 2009-01-09 2018-05-08 Board Of Regents Of The University Of Texas System Pro-neurogenic compounds
FR2949063B1 (fr) * 2009-08-11 2011-09-30 Pf Medicament Composition pharmaceutique comprenant un ester de dha destinee a etre administree par voie parenterale
CN103415289B (zh) 2010-07-07 2017-04-12 得克萨斯州大学系统董事会 前神经原性化合物
WO2014031986A1 (en) 2012-08-24 2014-02-27 Board Of Regents Of The University Of Texas System Pro-neurogenic compounds
EP3068388A4 (de) 2013-11-11 2017-04-12 Board of Regents of the University of Texas System Nervenschützende verbindungen und verwendung davon
WO2015070237A1 (en) 2013-11-11 2015-05-14 Board Of Regents Of The University Of Texas System Neuroprotective chemicals and methods for identifying and using same
CN106397544B (zh) * 2016-09-06 2019-09-27 中国科学院南海海洋研究所 环肽类化合物在制备抗肿瘤药物中的应用
CN106279370B (zh) * 2016-09-06 2019-09-27 中国科学院南海海洋研究所 一种海洋链霉菌、及其环肽化合物在制备抗结核分枝杆菌药物中的应用
CN106497827B (zh) * 2016-10-09 2019-08-02 中国科学院南海海洋研究所 一种定向生产抗结核活性和抗肿瘤活性化合物的基因工程菌株及其应用
CN111303247B (zh) * 2019-11-21 2022-03-22 中国科学院南海海洋研究所 海洋环肽化合物及其在制备抗结核分枝杆菌药物中的应用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CH401357A (de) * 1960-02-08 1965-10-31 Takeda Chemical Industries Ltd Verfahren zur Herstellung von Rufomycin

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0078795A2 *

Also Published As

Publication number Publication date
WO2000078795A2 (en) 2000-12-28
PE20010305A1 (es) 2001-03-12
WO2000078795A3 (en) 2001-07-05
AU6047300A (en) 2001-01-09

Similar Documents

Publication Publication Date Title
US6358957B1 (en) Phenylahistin and the phenylahistin analogs, a new class of anti-tumor compounds
EP0735819B1 (de) cryptophycine
US4879278A (en) Isolation and structural elucidation of the cytostatic linear depsipeptide dolastatin 15
EP0462531A2 (de) Zyklisches Polypeptid mit antibiotischer Aktivität, dessen Herstellung und Reinkultur eines Coelomycetes Stammes
EP1192178A2 (de) Rufomycine und deren derivate als hemmer von multi-drug resistence assoziiert mit protein-1 (mrp-1)
AU723652B2 (en) New cryptophycins from synthesis
US7098186B2 (en) Depsipeptide compound
WO1996039829A1 (en) New cryptophycins
KR100349485B1 (ko) 시클로헵타펩티드항염증제
AU628855B2 (en) Pipecolic acid-containing peptolides, their preparation and pharmaceutical compositions containing them
KR100482402B1 (ko) 액타가르딘과관련된신규랜티바이오틱,이의제조방법및이를포함하는약제학적조성물
WO2000078797A1 (en) Rufomycin derivatives useful as antibiotics
EP1142905B1 (de) Neue depsipeptid-verbindung
JP4554606B2 (ja) 新規な細胞障害性デプシペプチド
US5885959A (en) Cyclic peptide compounds and their production process
EP0894092B1 (de) Zyklisches heptapeptidderivat der kolonialen ascidians, lissoclinum sp
EP0535968B1 (de) Hydroxypropionitril-Cyclohexapeptide
EP1718631A2 (de) Cytotoxinverbindung und isolationsverfahren
US5494893A (en) Stylostatin 2
US6277860B1 (en) Furopyridine antibacterials
EP1896018A2 (de) Medizinische verwendungen von 39-desmethoxyrapamycin und seinen analogen
US20030158117A1 (en) Coniosulfides and their derivatives, processes for preparing them, and their use as pharmaceuticals
USH1638H (en) Use of the polypeptide compound
US20040033940A1 (en) Cyclic hepta-peptide derivative from colonial ascidians, lissoclinum SP
AU716025B2 (en) New cryptophycins

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20020121

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT

AX Request for extension of the european patent

Free format text: AL;LT;LV;MK;RO;SI

GRAH Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOS IGRA

GRAH Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOS IGRA

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20030416