WO2007035734A2 - Composes de cytotoxines et procedes d'isolation - Google Patents

Composes de cytotoxines et procedes d'isolation Download PDF

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Publication number
WO2007035734A2
WO2007035734A2 PCT/US2006/036484 US2006036484W WO2007035734A2 WO 2007035734 A2 WO2007035734 A2 WO 2007035734A2 US 2006036484 W US2006036484 W US 2006036484W WO 2007035734 A2 WO2007035734 A2 WO 2007035734A2
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WIPO (PCT)
Prior art keywords
palmerolide
compound according
compound
alkyl
alkoxy
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PCT/US2006/036484
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English (en)
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WO2007035734A3 (fr
Inventor
Bill Baker
Thushara Diyabalanage
James B. Mcclintock
Charles D. Amsler
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University Of South Florida
Uab Research Foundation
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Application filed by University Of South Florida, Uab Research Foundation filed Critical University Of South Florida
Priority to AU2006292268A priority Critical patent/AU2006292268A1/en
Priority to CA002622912A priority patent/CA2622912A1/fr
Priority to EP06825017A priority patent/EP1934196A4/fr
Priority to US12/066,938 priority patent/US8669376B2/en
Priority to JP2008531434A priority patent/JP2009508878A/ja
Publication of WO2007035734A2 publication Critical patent/WO2007035734A2/fr
Publication of WO2007035734A3 publication Critical patent/WO2007035734A3/fr
Priority to US14/204,996 priority patent/US9394270B2/en
Priority to US15/190,679 priority patent/US10815212B2/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D313/00—Heterocyclic compounds containing rings of more than six members having one oxygen atom as the only ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • Tunicates have proven to be an important source of bioactive natural products.
  • the ecteinascidins and didemnins are derived from tunicates, and the eudistomins have potent antiviral activity.
  • S. adareanum is a circumpolar tunicate common in the shallow waters around Anvers island (64° 46'S, 64° 03 'W) on the Antarctic Peninsula from 15 to 796 meters depth.
  • S. adareanum colonies consist of large rounded or club-shaped heads with the bottom stalk being wrinkled and leathery and only slightly narrower than the head.
  • S. adareanum colonies can be up to eighteen centimeters high with a diameter of twelve centimeters.
  • S. adareanum colonies may comprise a single head or, up to six heads can arise from a single stalk.
  • the subject invention concerns extracts from S. adareanum comprising Palmerolide
  • Palmerolide G Palmerolide H, and/or Palmerolide K and uses thereof.
  • Compounds of the invention exhibit bioactivity in field-based feeding-deterrent assays.
  • novel, isolated polyketides Palmerolide A, Palmerolide B, Palmerolide C, Palmerolide D, Palmerolide E,
  • the present invention also concerns methods of treating a subject with cancer, comprising administering to the subject a therapeutically effective amount of at least one isolated compound obtained from extracts of a Synoicum species.
  • the Synoicum species is S. adareanum and the isolated compound is a Palmerolide.
  • the Palmerolide is chosen from Palmerolide Al , Palmerolide B, Palmerolide C, Palmerolide D, Palmerolide E, Palmerolide F, Palmerolide G, Palmerolide H, and Palmerolide K.
  • Figure 1 shows a perspective view of the chemical formula for Palmerolide A.
  • Figure 2 shows a chart showing the NMR Data for Palmerolide A.
  • Figure 3 shows selected ROE correlations relating the relative stereochemistry between C-I l and C-19.
  • Figure 4A shows a chart showing the National Cancer Institute (NCI) Developmental
  • Figure 4B shows a continued chart, showing the National Cancer Institute (NCI) Developmental Therapeutics Program In-Vitro Testing Results for Palmerolide A.
  • Figure 5 shows a graph showing National Cancer Institute (NCI) Developmental Therapeutics Program Dose Response Curves for all cell lines tested for Palmerolide A.
  • Figure 6 shows a graph showing National Cancer Institute (NCI) Developmental Therapeutics Program Dose Response Curves for Melanoma cell lines tested for Palmerolide A.
  • Figure 7 shows a graph showing National Cancer Institute (NCI) Developmental Therapeutics Program Dose Response Curves for Colon Cancer cell lines tested for Palmerolide A.
  • NCI National Cancer Institute
  • Figure 8 shows a graph showing National Cancer Institute (NCI) Developmental Therapeutics Program Dose Response Curves for Renal Cancer cell 'lines tested for Palmerolide A.
  • Figure 9 shows a perspective view of the chemical formula for Palmerolide C.
  • Figure 10 shows a chart showing the NMR Data for Palmerolide C.
  • Figure HA shows a chart showing the National Cancer Institute (NCI) Developmental Therapeutics Program In- Vitro Testing Results for Palmerolide C.
  • Figure HB shows a continued chart, showing the National Cancer Institute (NCI) Developmental Therapeutics Program In- Vitro Testing Results for Palmerolide C.
  • Figure 12 shows a graph showing National Cancer Institute (NCI) Developmental Therapeutics Program Dose Response Curves for all cell lines tested for Palmerolide C.
  • NCI National Cancer Institute
  • Figure 13 shows a perspective view of the chemical formula for Palmerolide D.
  • Figure 14 shows a chart showing the NMR Data for Palmerolide D.
  • Figure 15 shows a perspective view of the chemical formula for Palmerolide E.
  • Figure 16 shows a chart showing the NMR Data for Palmerolide E.
  • Figure 17A shows a chart showing the National Cancer Institute (NCI) Developmental Therapeutics Program In- Vitro Testing Results for Palmerolide E.
  • Figure 17B shows a continued chart, showing the National Cancer Institute (NCI) Developmental Therapeutics Program In- Vitro Testing Results for Palmerolide E.
  • Figure 18 shows a graph showing National Cancer Institute (NCI) Developmental Therapeutics Program Dose Response Curves for all cell lines tested for Palmerolide E.
  • NCI National Cancer Institute
  • FIG 19 shows the purification steps for isolating Palmerolides A-G.
  • Figure 20 shows a perspective view of the chemical formula for Palmerolide B.
  • Figure 21 shows a perspective view of the chemical formula for Palmerolide F.
  • Figure 22 shows a perspective view of the chemical formula for Palmerolide G.
  • Figure 23 shows a perspective view of the chemical formula for Palmerolide H.
  • Figure 24 shows a perspective view of the chemical formula for Palmerolide K.
  • the subject invention concerns extracts from S. adar ⁇ anum comprising compounds, referred to herein as Palmerolides, and uses thereof.
  • Palmerolides specifically exemplified herein include Palmerolide A, Palmerolide B, Palmerolide C, Palmerolide D, Palmerolide E,
  • R 1 is carboxaldehyde, -CHCHNHC(O)-Alkyl, -OC-Alkyl, -OC-aryl, -OC-amino, aryl, amino, -vinylamido, arylamido, alkyl, alkoxy, cycloalkyl, cycloalkoxy, aryloxy, alkylcarbonyl; alkoxycarbonyl, cycloalkylcarbonyl, cycloalkoxycarbonyl, heteroalkyl, heterocycloalkyl, heteroaryl, arylcarbonyl, heteroarycarbonyl, heterocycloalkylcarbonyl, aryloxycarbonyl, heteroaryloxycarbony, heterocycloalkoxycarbonyl,a halogen, or -CHO, any of which can be optionally substituted with H, alkyl, alkoxy, -OH, -NO 2 , -NH 2 , -COOH, a halogen, or
  • R 2 is, independently, OH, O-Acyl, carbamate, H, O-alkyl, amino, -OSO 3 H, alkyl, alkoxy, cycloalkyl, cycloalkoxy, aryloxy, alkylcarbonyl; alkoxycarbonyl, cycloalkylcarbonyl, cycloalkoxycarbonyl, heteroalkyl, heterocyclo alkyl, heteroaryl, arylcarbonyl, heteroarycarbonyl, heterocycloalkylcarbonyl, aryloxycarbonyl, heteroaryloxycarbony, heterocycloalkoxycarbonyl, a halogen, and/or oxo;
  • R 3 is H, alkyl, alkoxy, cycloalkyl, cycloalkoxy, aryloxy, alkylcarbonyl; alkoxycarbonyl, cycloalkylcarbonyl, cycloalkoxycarbonyl, heteroalkyl, heterocycloalkyl, heteroaryl, arylcarbonyl, heteroarycarbonyl, heterocycloalkylcarbonyl, aryloxycarbonyl, heteroaryloxycarbony, heterocycloalkoxycarbonyl, halogen, any of which can be optionally substituted with alkyl, alkoxy, -OH, -NO 2 , -NH 2 , -COOH, a halogen, and/or -CH 3 ;
  • R 4 is, independently, H, alkyl, alkoxy, cycloalkyl, cycloalkoxy, aryloxy, alkylcarbonyl; alkoxycarbonyl, cycloalkylcarbonyl, cycloalkoxycarbonyl, heteroalkyl, heterocycloalkyl, heteroaryl, arylcarbonyl, heteroarycarbonyl, heterocycloalkylcarbonyl, aryloxycarbonyl, heteroaryloxycarbony, heterocycloalkoxycarbonyl, halogen, any of which can be optionally substituted with alkyl, alkoxy, -OH, -NO 2 , -NH 2 , -COOH, a halogen, and/or -CH 3 ;
  • At least one R 2 is -OC(NH 2 )O.
  • R 3 is methyl.
  • at least one R 4 is methyl, hi a further embodiment, both R 4 are methyl.
  • R 1 is -CHCHNHC(O)CHC(CH 3 ) 2 . In one embodiment, R 1 is -CHCHNHC(O)CHC(CH 3 )CH 2 C(CH 3 )CH 2 .
  • At least one R 2 is -OSO 3 H.
  • At least two R 2 are -OH and at least one R 2 is -OC(NH 2 )O, and optionally R 3 and R 4 are -CH 3 .
  • R 1 is carboxaldehyde, -CHCHNHC(O)-Alkyl, -OC-Alkyl, -OC-aryl, -OC-amino, aryl, amino, -vinylamido, arylamido, alkyl, alkoxy, cycloalkyl, cycloalkoxy, aryloxy, alkylcarbonyl; alkoxycarbonyl, cycloalkylcarbonyl, cycloalkoxycarbonyl, heteroalkyl, heterocycloalkyl, heteroaryl, arylcarbonyl, heteroarycarbonyl, heterocycloalkylcarbonyl, aryloxycarbonyl, heteroaryloxycarbony, heterocycloalkoxycarbonyl,a halogen, or -CHO, any of which can be optionally substituted with H, alkyl, alkoxy, -OH, -NO 2 , -NH 2 , -COOH, a halogen, or
  • R is H, alkyl, alkoxy, cycloalkyl, cycloalkoxy, aryloxy, alkylcarbonyl; alkoxycarbonyl, cycloalkylcarbonyl, cycloalkoxycarbonyl, heteroalkyl, heterocycloalkyl, heteroaryl, arylcarbonyl, heteroarycarbonyl, heterocycloalkylcarbonyl, aryloxycarbonyl, heteroaryloxycarbony, heterocycloalkoxycarbonyl, halogen, any of which can be optionally substituted with alkyl, alkoxy, -OH, -NO 2 , -NH 2 , -COOH, a halogen, and/or -CH 3 ;
  • R 4 is, independently, H, alkyl, alkoxy, cycloalkyl, cycloalkoxy, aryloxy, alkylcarbonyl; alkoxycarbonyl, cycloalkylcarbonyl, cycloalkoxycarbonyl, heteroalkyl, heterocycloalkyl, heteroaryl, arylcarbonyl, heteroarycarbonyl, heterocycloalkylcarbonyl, aryloxycarbonyl, heteroaryloxycarbony, heterocycloalkoxycarbonyl, halogen, any of which can be optionally substituted with alkyl, alkoxy, -OH, -NO 2 , -NH 2 , -COOH, a halogen, and/or -CH 3 ;
  • At least one R 2 is -OC(NH 2 )O. In one embodiment, R 3 is methyl.
  • At least one R 4 is methyl. In a further embodiment, both R 4 are methyl. In one embodiment, R 1 is -CHCHNHC(O)CHC(CH 3 ) 2 .
  • At least two R 2 are -OH and at least one R 2 is -OC(NH 2 )O, and optionally R 3 and R 4 are -CH 3 .
  • a composition (or an isomer, racemate or racemic mixture thereof, or a pharmaceutically acceptable salt or crystalline form thereof) is provided comprising an isolated Palmerolide A compound of formula (III):
  • the present invention provides for a composition (or an isomer, racemate or racemic mixture thereof, or a pharmaceutically acceptable salt or crystalline form thereof) comprising an isolated Palmerolide C compound of formula (IV):
  • composition or an isomer, racemate or racemic mixture thereof, or a pharmaceutically acceptable salt or crystalline form thereof
  • a composition comprising an isolated Palmerolide D compound of formula (V):
  • the present invention also provides for a composition (or an isomer, racemate or racemic mixture thereof, or a pharmaceutically acceptable salt or crystalline form thereof) comprising an isolated Palmerolide E compound of formula VI:
  • the present invention also provides for a composition (or an isomer, racemate or racemic mixture thereof, or a pharmaceutically acceptable salt or crystalline form thereof) comprising an isolated Palmerolide B compound of formula VII:
  • the present invention also provides for a composition (or an isomer, racemate or racemic mixture thereof, or a pharmaceutically acceptable salt or crystalline form thereof) comprising an isolated Palmerolide F compound of formula (VIII):
  • the present invention also provides for a composition (or an isomer, racemate or racemic mixture thereof, or a pharmaceutically acceptable salt or crystalline form thereof) comprising an isolated Palmerolide G compound of formula (IX):
  • the present invention also provides for a composition (or an isomer, racemate or racemic mixture thereof, or a pharmaceutically acceptable salt or crystalline form thereof) comprising an isolated Palmerolide H compound of formula (X):
  • the present invention also provides for a composition (or an isomer, racemate or racemic mixture thereof, or a pharmaceutically acceptable salt or crystalline form thereof) comprising an isolated Palmerolide K compound of formula (XI):
  • the new compounds are substantially pure; ideally containing at least 95% of the compound as determined by established analytical methods, acceptably containing at least 90% of the desired compound, permissibly containing at least 75% of the compound.
  • Also provided by the discoveries of the invention are new pharmaceutical compositions between about 0.01% to 60% by weight, preferably 0.1% to 50% by weight, and more preferably 1% to 35% by weight based on the total weight of the composition, of one of the new compounds of the invention, or a mixture of two or more such compounds, and one or more pharmaceutically acceptable carriers or diluents.
  • alkyl means straight or branched chain, saturated or mono- or polyunsaturated hydrocarbon groups having from 1 to 20 carbon atoms and C 1-X alkyl means straight or branched chain alkyl groups containing from one up to X carbon atoms, and includes alkyls, alkenyl, and alkynyls.
  • Ci -6 alkyl means straight or branched chain alkyl groups containing from 1 up to 6 carbon atoms.
  • Alkoxy means an alkyl-O- group in which the alkyl group is as previously described.
  • Cycloalkyl includes a nonaromatic monocyclic or multicyclic ring system, including fused and spiro rings, of from about three to about 10 carbon atoms.
  • a cyclic alkyl may optionally be partially unsaturated.
  • Cycloalkoxy means a cycloalkyl-O- group in which cycloalkyl is as defined herein.
  • Aryl means an aromatic monocyclic or multicyclic carbocyclic ring system, including fused and spiro rings, containing from about six to about 14 carbon atoms.
  • Aryloxy means an aryl-O- group in which the aryl group is as described herein.
  • Alkylcarbonyl means a RC(O)- group where R is an alkyl group as previously described.
  • Alkoxycarbonyl means an ROC(O)- group where R is an alkyl group as previously described.
  • Cycloalkylcarbonyl means an RC(O)- group where R is a cycloalkyl group as previously described.
  • Cycloalkoxycarbonyl means an ROC(O)- group where R is a cycloalkyl group as previously described.
  • Heteroalkyl means a straight or branched-chain having from one to 20 carbon atoms and one or more heteroatoms selected from nitrogen, oxygen, or sulphur, wherein the nitrogen and sulphur atoms may optionally be oxidized, i.e., in the form of an N-oxide or an S-oxide.
  • Heterocycloalkyl means a monocyclic or multicyclic ring system (which may be saturated or partially unsaturated), including fused and spiro rings, of about five to about 10 elements wherein one or more of the elements in the ring system is an element other than carbon and is selected from nitrogen, oxygen, silicon, or sulphur atoms.
  • Heteroaryl means a five to about a 14-membered aromatic monocyclic or multicyclic hydrocarbon ring system, including fused and spiro rings, in which one or more of the elements in the ring system is an element other than carbon and is selected from nitrogen, oxygen, silicon, or sulphur and wherein an N atom may be in the form of an N-oxide.
  • Arylcarbonyl means an aryl-CO- group in which the aryl group is as described herein.
  • Heteroarylcarbonyl means a heteroaryl- CO- group in which the heteroaryl group is as described herein and heterocycloalkylcarbonyl means a heterocycloalkyl-CO- group in which the heterocycloalkyl group is as described herein.
  • Aryloxycarbonyl means an ROC(O)- group where R is an aryl group as previously described.
  • Heteroaryloxycarbonyl means an ROC(O)- group where R is a heteroaryl group as previously described.
  • Heterocycloalkoxy means a heterocycloalkyl-O- group in which the heterocycloalkyl group is as previously described.
  • Heterocyclo alkoxycarbonyl means an ROC(O)- group where R is a heterocycloalkyl group as previously described.
  • saturated alkyl groups include, but are not limited to, methyl, ethyl, N- propyl, isopropyl, N-butyl, tert-butyl, isobutyl, sec-butyl, N-pentyl, N-hexyl, N-heptyl, and N-octyl.
  • An unsaturated alkyl group is one having one or more double or triple bonds.
  • Unsaturated alkyl groups include, for example, ethenyl, propenyl, butenyl, hexenyl, vinyl, 2- propynyl, 2-isopentenyl, 2-butadienyl, ethynyl, 1-propyny ⁇ , 3-propynyl, and 3-butynyl.
  • Cycloalkyl groups include, for example, cyclopentyl, " cyclohexyl, " 1-cyclohexenyl, 3- cyclohexenyl, and cycloheptyl.
  • Heterocycloalkyl groups include, for example, 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, • 3-morpholinyI, 4-morpholinyl, tetrahydrofuran-2-yl,
  • Aryl groups include, for example, phenyl, indenyl, biphenyl, 1- naphthyl, 2-naphthyl, anthracenyl, and phenanthracenyl.
  • Heteroaryl groups include, for example, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, furyl, thienyl, imidazolyl, oxazolyl, thiazolyl, pyrazolyl, pyridyl, indolyl, quinolinyl, isoquinolinyl, benzoquinolinyl, carbazolyl, and diazaphenanthrenyl.
  • halogen means the elements fluorine (F), chlorine (Cl), bromine (Br), and iodine (I).
  • the term embraces aldehydes, carboxylic acids, ketones, sulfonic acids, amides, and esters.
  • kits comprising in one or more containers a compound or composition of the invention.
  • a kit of the invention comprises one or more of a Palmerolide A, Palmerolide B, Palmerolide C, Palmerolide D, Palmerolide E, Palmerolide F, Palmerolide G, Palmerolide H, and Palmerolide K.
  • a kit further comprises a pharmaceutically acceptable carrier, such as a diluent, hi another embodiment, a kit further comprises an antitumor or anticancer compound.
  • the subject invention also concerns methods for inhibiting a vacuolar adenosine triphosphatase (V-ATPase) enzyme, comprising contacting or exposing a V-ATPase to an effective amount of a compound or composition of the present invention sufficient to inhibit activity or block function of the V-ATPase.
  • the compound or composition comprises one or more Palmerolides selected from Palmerolide A, Palmerolide B, Palmerolide C, Palmerolide D, Palmerolide E, Palmerolide F, Palmerolide G, Palmerolide H, and Palmerolide K.
  • the subject invention also concerns methods for inhibiting or killing a cancer cell, comprising contacting the cell with an effective amount of a compound or composition of the invention.
  • the compound or composition comprises one or more Palmerolides selected from Palmerolide A, Palmerolide B, Palmerolide C, Palmerolide D, Palmerolide E, Palmerolide F, Palmerolide G, Palmerolide H, and Palmerolide K.
  • Types of cancer cells that can be inhibited or killed according to the present invention include, but are not limited to cancer and/or tumor cells of bone, breast, kidney, mouth, larynx, esophagus, stomach, testis, cervix, head, neck, colon, ovary, lung, bladder, skin (e.g., melanoma), liver, muscle, pancreas, prostate, blood cells (including lymphocytes), and brain.
  • the subject invention also concerns methods for treating a condition associated with abnormal expression or overexpression of a V-ATPase enzyme comprising administering to a person or animal having the condition and in need of treatment of an effective amount of a compound or composition of the present invention.
  • Conditions that can be treated according to the present invention include, but are not limited to, cell proliferation disorders, such as cancer; diabetes; pancreatitis; and osteoporosis, hi one embodiment, the compound or composition comprises one or more Palmerolides selected from Palmerolide A, Palmerolide B, Palmerolide C, Palmerolide D, Palmerolide E, Palmerolide F, Palmerolide G, Palmerolide H, and Palmerolide K.
  • the present invention also provides methods of treating a person or animal with cancer or an oncological disorder.
  • a method comprises administering to the person or animal a therapeutically effective amount of at least one isolated compound obtained from extracts of a Synoicum species.
  • the animal can be, but is not limited to, a mammal, such as primate (monkey, chimpanzee, ape, etc.), dog, cat, cow, pig, or horse, or other animals having an oncological disorder.
  • Methods of the invention can optionally include identifying a person or animal who is or may be in need of treatment of cancer or an oncological disorder.
  • the Synoicum species is S. adareanum and the isolated compound is a Palmerolide.
  • the Palmerolide is chosen from Palmerolide A, Palmerolide B > Palmerolide C, Palmerolide D, Palmerolide E, Palmerolide F, Palmerolide G, Palmerolide H, and Palmerolide K.
  • Types of cancer that can be treated according to the present invention include, but are not limited to cancer and/or tumors of the bone, breast, kidney, mouth, larynx, esophagus, stomach, testis, cervix, head, neck, colon, ovary, lung, bladder, skin (e.g., melanoma), liver, muscle, pancreas, prostate, blood cells (including lymphocytes), and brain.
  • the compounds and compositions of this invention can be administered to a patient in need of treatment in combination with other antitumor or anticancer substances or with radiation and/or photodynamic therapy or with surgical treatment to remove a tumor.
  • these other substances or radiation treatments may be given at the same as or at different times from the compounds or compositions of this invention.
  • the compounds or compositions of the present invention can be used in combination with mitotic inhibitors such as taxol or vinblastine, alkylating agents such as cyclophosamide or ifosfamide, antimetabolites such as 5-fluorouracil or hydroxyurea, DNA intercalators such as adriamycin or bleomycin, topoisomerase inhibitors such as etoposide or camptothecin, antiangiogenic agents such as angiostatin, antiestrogens such as tamoxifen, and/or other anti-cancer drugs or antibodies, such as, for example, GLEEVEC (Novartis Pharmaceuticals Corporation) and HERCEPTIN (Genentech, Inc.), respectively.
  • mitotic inhibitors such as taxol or vinblastine
  • alkylating agents such as cyclophosamide or ifosfamide
  • antimetabolites such as 5-fluorouracil or hydroxyurea
  • DNA intercalators such as adriamycin or bleomycin
  • the subject invention also concerns methods for isolating and purifying a compound of the present invention.
  • the method comprises subjecting a Synoicum tunicate to solvent extraction; removing said solvent to provide an extract; and fractionating said extract to isolate said Palmerolide.
  • the subject invention also concerns methods for synthesizing a compound of the present invention.
  • “Pharmaceutically acceptable carrier” refers to any carrier, diluent, excipient, wetting agent, buffering agent, suspending agent, lubricating agent, adjuvant, vehicle, delivery system, emulsifier, disintegrant, absorbent, preservative, surfactant, colorant, flavorant, or sweetener, preferably non-toxic, that would be suitable for use in a pharmaceutical composition.
  • “Pharmaceutically acceptable equivalent” includes, without limitation, pharmaceutically acceptable salt or crystalline forms, hydrates, metabolites, prodrugs, and isosteres. Many pharmaceutically acceptable equivalents are expected to have the same or similar in vitro or in vivo activity as the compounds of the invention.
  • “Pharmaceutically acceptable salt or crystalline form” refers to a salt of the inventive compounds which possesses the desired pharmacological activity and which is neither biologically nor otherwise undesirable.
  • the salt can be formed with acids that include, without limitation, acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethane- sulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, ox
  • Examples of a base salt include ammonium salts, alkali metal salts such as sodium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases such as dicyclohexylamine salts, N- methyl-D-glucamine, and salts with amino acids such as arginine and lysine.
  • the basic nitrogen-containing groups can be quarternized with agents including lower alkyl halides such as methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dialkyl sulfates such as dimethyl, diethyl, dibutyl, and diamyl sulfates; long chain halides such as decyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides; and aralkyl halides such as benzyl and phenethyl bromides.
  • lower alkyl halides such as methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides
  • dialkyl sulfates such as dimethyl, diethyl, dibutyl, and diamyl sulfates
  • long chain halides such as decyl, lauryl, myristyl
  • Prodrug refers to a derivative of the inventive compounds that undergoes biotransformation, such as by metabolism, before exhibiting a pharmacological effect.
  • the prodrug is formulated with the objective of improved chemical stability, improved patient acceptance and compliance, improved bioavailability, prolonged duration of action, improved organ selectivity, improved formulation (for example, increased hydrosolubility), and/or decreased side effects (for example, toxicity).
  • the prodrug can be readily prepared from the inventive compounds using methods known in the art, such as those described by Burger's Medicinal Chemistry and Drug Chemistry, Fifth Ed., Vol. 1, pp. 172-178, 949-982 (1995).
  • “Palmerolide,” as used herein, refers to a multi-membered macrocyclic polyketide bearing carbonate and amide -functionality.
  • the Palmerolide is isolated from the tunicate Synoicwn adareanum; collected from the vicinity of Palmer Station on the Antarctic Peninsula.
  • Polyketides refers to any natural compound containing alternating carbonyl and methylene groups (' ⁇ -polyketones'), derived from repeated condensation of acetyl coenzyme A.
  • “”Macrocycle,” as use herein, refers to a large molecule arranged in a circle with various semi-compounds attached at various points. The point of attachment and the nature of the sub-molecule determine the nature and physiological effect of the compound which contains it.
  • Macrolide as used herein, refers to a class of antibiotics characterized by molecules made up of large-ring lactones.
  • Olefin as used herein, is synonymous with “alkene” and refers to an acyclic hydrocarbon containing one or more double bonds.
  • u a clinical response is the response of a cell proliferative disorder, such as melanoma, colon, and renal cancer, to treatment with novel compounds disclosed herein. Criteria for determining a response to therapy are widely accepted and enable comparisons of the efficacy alternative treatments (see Slapak and Kufe, Principles of Cancer Therapy, in Harrison's Principles of Internal Medicine, 13 th edition, eds. Isselbacher et al, McGraw-Hill, Inc. 1994).
  • a complete response (or complete remission) is the disappearance of all detectable malignant disease.
  • a partial response is an approximately 50 percent decrease in the product of the greatest perpendicular diameters of one or more lesions.
  • a "pharmaceutical composition" of the invention is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral or nasal (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, or phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion maximnf containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid
  • polyetheylene glycol and the like
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In 0 many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol, and/or sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier. They can be
  • the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules.
  • Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed.
  • Pharmaceutically compatible binding agents, and/or o adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be
  • ⁇ permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
  • the materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art.
  • Compounds and compositions of the invention can be delivered to a cell either through direct contact with the cell or via a carrier means.
  • Carrier means for delivering compositions to cells are known in the art and include, for example, encapsulating the compound or composition in a liposome moiety.
  • Another means for delivery of a compound of the invention to a cell comprises attaching the compound to a protein or nucleic acid that is targeted for delivery to the target cell.
  • U.S. Patent No. 6,960,648 and Published U.S. Patent Application Nos. 20030032594 disclose amino acid sequences that can be coupled to another compound and that allows the compound to be translocated across biological membranes. Published U.S. Patent Application No.
  • compositions for transporting biological moieties across cell membranes, for intracellular delivery can also be incorporated into polymers, examples of which include poly (D-L lactide-co-glycolide) polymer for intracranial tumors; poly[bis(p- carboxyphenoxy) propane:sebacic acid] in a 20:80 molar ratio (as used in GLIADEL); chondroitin; chitin; and chitosan.
  • compounds of the invention may contain one or more asymmetrically substituted carbon atoms which can give rise to stereoisomers.
  • AU such stereoisomers, including enantiomers, and diastereoisomers and mixtures, including racemic mixtures thereof, are contemplated within the scope of the present invention.
  • a “therapeutically effective amount” is the amount of a Palmerolide of the invention, including Palmerolides A, B, C, D, E, F, G, H, and K, or any combination thereof necessary to provide a therapeutically effective amount of the corresponding compound in vivo.
  • the amount of the compound must be effective to achieve a response, including but not limited to total prevention of (e.g., protection against and complete cure) and to improved survival rate or more rapid recovery, or improvement or elimination of symptoms associated with a cellular proliferative disease or other indicators as are selected as appropriate measures by those skilled in the art.
  • a suitable single dose size is a dose that is capable of preventing or alleviating (reducing or eliminating) a symptom in a patient when administered one or more times over a suitable time period.
  • One of skill in the art can readily determine appropriate single dose sizes for systemic administration based on the size of a mammal and the route of administration.
  • the Biological Testing Branch of the Developmental Therapeutics Program has adopted a preliminary in vivo screening tool for assessing the potential anticancer activity of compounds identified by the large scale in vitro cell screen.
  • This hollow fiber based assay has been in use since June, 1995 (http://dtp.nci.nih.gov/branches ⁇ b/hfa.html).
  • Each compound is tested against a standard panel of 12 human tumor cell lines including NCI-H23, NCI-H522, MDA-MB-231, MDA-MB-435, SW-620 COLO 205, LOX IMVI, UACC-62, OVCAR-3, OVCAR 5, U251 and SF-295.
  • the cell lines are cultivated in RPMI- 1640 containing 10% FBS and 2mM glutamine. On the day preceding hollow fiber preparation the cells are given a supplementation of fresh medium to maintain log phase growth.
  • the cells are harvested by standard trypsinization technique and resuspended at the desired cell density (varies by cell line between 2 - 10 X 10 6 cells/ml).
  • the cell suspension is flushed into 1 mm ID. polyvinylidene hollow fibers with a molecular weight exclusion of 500,000 Da.
  • the hollow fibers are heat-sealed at 2 cm intervals and the samples generated from these seals are placed into tissue culture medium and incubated at 37°C in 5% CO 2 for 24 - 48 hours prior to implantation.
  • a total of 3 different tumor lines are prepared for each experiment so that each mouse receives 3 intraperitoneal implants (1 of each tumor line) and 3 subcutaneous implants (1 of each tumor line).
  • mice On the day of implantation, samples of each tumor cell line are quantitated for viable cell mass by a stable endpoint MTT assay so that the time zero (0) cell mass is known. Thus, the cytostatic and cytocidal capacities of the test compound can be assessed.
  • Mice are treated with experimental agents starting on day 3 or 4 following fiber implantation and continuing once daily for a total of 4 doses. Each agent is assessed by intraperitoneal injection at 2 dose levels with 3 mice/dose/experiment. Vehicle controls consist of 6 mice receiving the compound diluent only. The fibers are collected from the mice on the day following the fourth compound treatment and subjected to the stable endpoint MTT assay.
  • the optical density of each sample is determined spectrophotometrically at 540 nm and the mean of each treatment group is calculated.
  • the percent net cell growth in each treatment group is calculated and compared to the percent net cell growth in the vehicle treated controls.
  • Compounds are selected for further testing (for example, time/dose exposure studies preliminary pharmacology studies, subcutaneous xenograft efficacy studies) on the basis of several hollow fiber assay criteria. These include: (1) a reduction in net cell growth of 50% or greater in 10 of the 48 possible test combinations (12 cell lines X 2 sites X 2 compound doses); (2) a reduction in net cell growth of 50% or greater in a minimum of 4 of the 24 distant site combinations (intraperitoneal drug/subcutaneous culture); and/or (3) cell kill of 1 or more cell lines in either implant site (reduction in the viable cell mass below the level present at the start of the experiment).
  • the results of individual cell lines are not reported since the statistical power of the assay is based 5 on the impact of a compound against the entire panel of cells.
  • other factors for example, unique structure, mechanism of action, etc. may result in referral of a compound for further studies without the compound meeting these hollow fiber assay criteria.
  • Extracts from S. adareanum, Palmerolide A, Palmerolide C, Palmerolide D, and Palmerolide E displayed bioactivity in field-based feeding-deterrent assays, leading the inventors to investigate the chemical nature of the activity.
  • NCI National Cancer Institute
  • More polar palmerolides or other melanoma-bioactive compounds present in the hydrophilic extracts can be fractionated by reversed phase vacuum chromatography.
  • C-18 modified silica gel is packed, in water, in a vacuum funnel, then the extract applied and two bed volumes of water, 1 :9, 2:8, 4:6, 8:2 methanol/water, then 100% methanol pulled through under vacuum, collecting one fraction per bed volume.
  • polar constituents can be adsorbed onto polystyrene resins (HP-20 or XAD) and eluted with a similar gradient profile of methanol or acetone. Chromatographic fractions thus obtained are then concentrated and bioassayed.
  • This HPLC instrumentation includes analytical, semi-preparative and preparative capabilities (four instruments, 0.1 mL/min to 300 mL/min) and our LC/MS can be integrated with any of the instruments to add MS (total ion chromatogram, TIC) detection when necessary, with the added advantage of securing mass spectral profiles of the isolates.
  • MS total ion chromatogram
  • CCC and CPC have the advantage of being based on the partition of the analyte between two immiscible phases, a physical property that lends itself to higher recovery than adsorption chromatography.
  • Instrumentation available today includes multiple volume rotors (both techniques utilize a rotary-induced gravity gradient) for developmental through preparative scale separations. Foucault et al.
  • Palmerolide A was isolated as a white solid from the 1:1 methanol/ethyl acetate fraction eluting from silica gel chromatography of the crude lipophilic (1 :1 methanol/dichloromethane) extract. Mass spectroscopic analysis provided a molecular formula Of C 33 H 48 N 2 O 7 (HRFABMS mlz 585.3539, ⁇ 0.1 mmu for [M + + I]). The C-I to C- 24 carbon backbone of palmerolide A could be unambiguously assigned based on 1 H- 13 C connectivity assignments from gHMBC spectra. The macrocycle was completed by observation of a correlation between the C-19 ( ⁇ 73.69) methine to the C-I ester carbonyl.
  • Hydroxy methines at C-7 and C-IO were conclusively assigned based on observation of coupling of the hydroxyl protons in both the gHMBC and COSY spectra: in the gHMBC spectrum, the hydroxy protons correlated to the respective ⁇ - and ⁇ - carbons, while in the COSY spectrum correlations were observed between the hydroxyl protons and the hydroxy methine protons.
  • An isopentenoyl amide established by 2D NMR analysis, completed the gross structure with the exception of CO 2 NH 2 remaining unassigned from the molecular formula. This remaining carbon was correlated with the proton on the oxygen-bearing C-Il ( ⁇ 75.25), but no further connectivity was evident. The last remaining valence must be occupied by the -NH 2 , resulting in a carbamoyl group at C-Il and completing the planar structure of palmerolide A.
  • the four olefins in the macrocycle constrain the flexibility often found in macrolides, facilitating stereochemical analysis by NOE studies.
  • Further analysis of the ROESY spectrum revealed the macrolide to adopt two largely planar sides of a tear-drop shaped cycle, one side consisting of C-I through C-6, the other C-Il through C-19, with C-7 through C-IO providing a curvilinear connection.
  • H-19, H 3 -27, H-U and H 2 - 13 are sequentially correlated in the ROESY spectrum, as are H 3 -26, H 2 -18, H-16, H- 14 and H-12, defining the periphery of the top and bottom face of the western hemisphere.
  • H-I l correlates only to the top series of protons, a result consistent only with C- 19 and C-I l both adopting the R configuration.
  • Tunicates are not well known as producers of type I polyketides, though the patellazoles and iejimalides are significant, bioactive, representatives.
  • Palmerolide A is unusual in bearing a small macrocycle, with 20 members, compared to 24 in the patellazoles and iejimalides, and a vinyl amide, a feature more commonly associated with cyanophyte- derived macrolides such as tolytoxin.
  • Palmerolide A displays cytotoxicity toward several other melanoma cell lines, Figure 2, [M14(LC 50 0.076 ⁇ M), SK-MEL-5 (6.8 ⁇ M) and LOX IMVI (9.8 ⁇ M)] as well as the previously mentioned UACC-62.
  • Figure 3 one colon cancer cell line (HCC-2998, 6.5 ⁇ M), FIG. 4A, and one renal cancer cell line (PvXF 393, 6.5 ⁇ M)
  • Palmerolide A was largely devoid of cytotoxicity (LC 50 >10 ⁇ M), representing a selectivity index among tested cell lines of 10 3 for the most sensitive cells.
  • Palmerolide A is COMPARE.-negative against the NCI database, suggestive of a previously un-described mechanism of action.
  • Figures 4A and 4B indicate the National Cancer Institutes Developmental Therapeutics Program In- Vitro Testing Results for Palmerolide A.
  • Figure 5 shows the National Cancer Institute (NCI) Developmental Therapeutics Program Dose Response Curves for all cell lines tested for Palmerolide A. hi comparison, individual results are shown for Melanoma ( Figure 6), Colon Cancer ( Figure 7) and Renal Cancer ( Figure 8).
  • EXAMPLE IV - CYTOTOXICITY OF PALMEROLIDE C Palmerolide C shown below and in FIG. 9, has the chemical formula C 33 EUgN 2 O 7 (for
  • NMR data see Figure 10). NCI cytotoxicity is shown in Figure HA and Figure HB. NCI Dose Response Curves for all cell lines are presented in Figure 12.
  • EXAMPLE V - CYTOTOXICITY OF PALMEROLIDE D Palmerolide D shown below and in Figure 13, has the chemical formula C 36 H 53 N 2 O 7 .
  • Palmerolide E shown below and in Figure 15, has the chemical formula C 27 H 39 NO 7 (for NMR data see Figure 16).
  • NCI cytotoxicity is shown in Figure 17A and Figure 17B.
  • NCI Dose Response Curves for all cell lines are presented in Figure 18.
  • EXAMPLE VII BIOASSAY Established cell lines (UACC-62, SK-MEL-5 and MKl 4, which are all sensitive to palmerolide A) have been obtained from the NCI Standard protocols for cell culture are used.
  • UACC-62 Amundson et al. 2000
  • MELl 4 (Lin et al, 2003) Cells, will be grown in RPMI 1640 medium supplemented with 10 % fetal bovine serum and glutamine and treated with antibiotics (100 units/mL penicillin, 100 nig/mL streptomycin) in a humidified atmosphere of 95 % air with 5 % CO 2 at 37 °C.
  • SK-MEL-5 (Miracco et al, 2003). Cells are grown in Eagle's minimal essential medium with Earle's BSS, adjusted to contain 1.5 g/L sodium bicarbonate, 0.1 mM non-essential amino acids and 1.0 mM sodium pyruvate in a humidified atmosphere of 95 % air with 5 % CO 2 at 37 °C.
  • Our bioassays use a 96-well format MTT-based (Vogt et al, 2004) method for the quantification of cell growth inhibition or cell lethality caused by palmerolides and their derivatives.
  • metabolically active cells cleave methylthiazol tetrazolium (MTT), which is yellow, to form a purple formazan pigment with a concomitant ultraviolet shift which can be quantified using a plate reader monitoring absorbance at 540 nm.
  • MTT methylthiazol tetrazolium
  • One-dimensional proton and carbon NMR spectroscopy in conjunction with mass spectral analysis (from LC/MS or electrospray ionization (ESI) or matrix assisted laser desorption ionization (MALDI) mass spectrometers) can secure assignment of the molecular formula of a new compound.
  • Two- and three-bond proton-proton connectivity can be established using a combination of two- dimensional NMR techniques such as COSY (Aue et al, 1976), or extended spin systems with TOCSY (Braunschweiler et al, 1983).
  • Proton-carbon connectivity can be established using HMQC or HSQC (Bax et al, 1986a; Bodenhausen et al, 1980) (one bond) and HMBC (Bax et al, 1986b) (two or three bond). These NMR experiments are most often acquired in gradient (Maudsley et al, 1978; Ruiz-Cabello et al, 1992) mode for maximal sensitivity, using solvent suppression where necessary. If sufficient material is available, carbon-carbon connectivity can be established by obtaining the 2D-INADEQUATE spectrum (Bax et al, 1980); the direct measure of 13 C- 13 C couplings enable significant portions of the structure to be readily assessed.
  • Stereochemical assignments in conformationally constrained systems are based on coupling constant data and the results of nuclear Overhauser enhancement or NOESY (Crews et al, 1998; Silverstein et al, 1998) techniques where possible, or by derealization or degradation.
  • Stereochemical analysis of flexible systems can be achieved using Murata's method (Murata et al, 1999) of analyzing 3 J HH and
  • the palmerolides provide ample functionalization for degradative studies. Reductive ozonolysis of palmerolides A, D, and/or E will lead to three hexane polyols (11-13, Scheme 1). Stereochemical conformation of C-7 in these palmerolides is achieved by comparison of the optical rotation of 11 to that of authentic (i?,+)-l,2,6-trihydroxyhexane (Wu et al, 2000). Positions C-10 and C-11 can be verified by comparison to tetra-ol 12, a compound not reported in the chemical literature. Tetra-ol 12 can be readily prepared from D-galactose (Scheme 2) based on a scheme modeled after the preparation of a similar compound (Zhu et al, 2001).
  • V-ATPases vacuolar adenosine triphosphatases
  • These are multi -protein trans-membrane enzymes responsible for translocation of protons out of the cytoplasm, resulting in pH regulation of intracellular compartments, a critical function for homeostasis (Sun-Wadaa et al, 2004). They are ubiquitous in eukaryotic cells and found occasionally in prokaryotes. The two domains of the enzyme are each composed of roughly half a dozen subunits and ultimately comprise a 20- protein assemblage.
  • V 0 domain which is imbedded in the membrane, is the locus of proton transfer while the sub-membrane Vi domain bears catalytic (ATP to ADP) activity (Nishi et al, 2002).
  • V-ATPases play a role in endocytosis, membrane fusion (Morel et al, 2003), bone resorption (Nomiyama et al., 2005) and other functions which are not simply functions of acidity (Nishi et al., 2002).
  • V-ATPases have been implicated in a number of disease states, including type 1 diabetes (Myers et al, 2003a; Myers et al, 2003b), osteoporosis (Nomiyama et al, 2005; Sundquist et al, 1990) and several cancers (Sennoune et al, 2004) such as cervical (Ellegaard et al, 1975), breast (Martinez-Zaguilan et al, 1999b) and melanoma (Martinez- Zaguilan et al, 1998).
  • Baf ⁇ lomycin A 1 (4, Figure 3) is the prototypical V-ATPase inhibitor (Zhang et al., 1994) although its binding and specificity are not nearly as great as more recently discovered inhibitors such as salicylihalamides (vis 5) (Erickson et al, 1997; Boyd et al, 2001) oxirhidines (Kim et al, 1999), lobatamides (McKee et al, 1998; Shen et al.,2002) and palmerolides.
  • bafilomycins the concanamycins (Huss et al, 2002), are similarly configured macrocycles while the poecillastrins and chondropsins (Xie et al, 2004) are considerably larger macrolides comprised of, in addition to a lactone linkage, a lactam linkage.
  • V-ATPase inhibitors are poised to become a new target of cancer intervention (Chene et al., 2003; Beutler et al., 2003). Key discoveries leading to first-in-class drugs, such as taxol was for inhibition of tubulin depolymerization (Horwitz et al., 2004), ultimately lead to a proliferation of drugs for the indication and greatly enhance treatment options.
  • Histone deacetylase (HDAC) inhibitors (Arts et al., 2003) are similarly undergoing a serge in interest as the Food and Drug Administration (FDA) has recently approved several Investigational New Drug (IND) applications.
  • IND Investigational New Drug
  • the subject functional group manipulation approach begins with the 'Rule of 5' principles elaborated by Lipinski et al., 2001 to address Absorption, Distribution, Metabolism, Excretion and Toxicity (ADMET) issues.
  • ADMET Absorption, Distribution, Metabolism, Excretion and Toxicity
  • MLogP is approximately 1.8 to 2.76 (calculated using SimulationsPlus, Inc. (Lancaster, CA) ADMET Predictor software) and there are less than 10 H-bond acceptors.
  • the molecular weights are a bit high, spanning the mid-500's to 610; palmerolide E is the only member less than 500, but it lacks the vinyl amide required for V-ATPase activity.
  • solubility and permeability are areas to focus derivatization studies and the two appear to track in opposing trends.
  • solubility improves with the addition of polar functional groups while permeability improves with removal of polar functional groups.
  • Reconciliation of this dichotomy will take place in the biological evaluation: compounds prepared according to teachings herein can be evaluated for melanoma and/or V-ATPase activity, as described above, and those retaining sufficient bioactivity (sub-millimolar) and displaying promising ADMET properties (based on evaluation by ADMET Predictor) can be subjected to the hollow fiber and/or xeno graph assays.
  • Derivatization studies can be conducted to increase polar groups in each region, to decrease polar groups in each region, and to make modifications without changing the number of polar groups. Permutations of polar group manipulations on different parts of the core structure lead to other possible modifications. As biological data accumulates, one selects the desired bioactivity profiles (potency and ADMET Predictor properties), rather than merely generating large numbers of derivatives. Described below are manipulations focused by area as well as manipulations to add, remove, or leave unchanged, the number of polar groups.
  • the SARl Region The region designated as SARl can be evaluated by at least four natural products.
  • Palmerolides A (1), D (8), E (9) and F (10) differ only in the nature of the terminus of the palmerolide parent chain.
  • Synthetic modifications to be introduced to further probe the C-24 terminus can include the following chain terminating groups:
  • Preparation of derivatives 30 to 45 can be accomplished by the copper-catalyzed vinyl amidation reaction (Scheme 8) (Shen et al, 2000).
  • High yields of stereocontrolled E vinyl amide analogues of palmerolides can be prepared by coupling of the desired amide with a vinyl iodide 46 using copper (I) thiophenecarboxylate (CuTC) as catalyst.
  • the necessary E- vinyl iodide 46 can be prepared in one of two ways.
  • the most direct route involves chromium-catalyzed (Takai olefmation, Takai et ah, 1986) homologation of tri-t-butylsilyl-protected palmerolide E ⁇ vis 47, Scheme 9).
  • functionalization of the palmerolides is not unlike that of discodermolide (Gunasekera et ah, 1990), their stability to conditions described herein can be verified by synthetic procedures applied to discodermolide as are well known in the art (Paterson et ah, 2001; Smith et ah, 2000).
  • vinyl iodide 46 can be prepared from Palmerolide A; while more steps are involved, this may be desired since Palmerolide A is more predominant in the tunicate.
  • palmerolide A can be suitably protected (Scheme 10) to differentiate the C-7 and C-IO oxygen functions from the C-I l oxygen function, then the amide selectively hydrolyzed (Eaton et ah, 1988) and the resultant phthalimide hydrolyzed to produce aldehyde 48.
  • Aldehyde 48 is amenable to direct conversion to the vinyl iodide using Danishefsky's method (Di Grandi et ah, 1993), the latter of which produces a vinyl iodide directly from a ketone.
  • thermodynamically E vinyl iodide results.
  • Palmerolide analogues 39 - 41 can be prepared from the Wittig reaction of protected palmerolide E (47) with methyl triphenyl-phosphonium bromide/BuLi (yielding 39), NaBH 4 ZCoCl 2 reduction of hydroxymethyl derivative 48 (yielding 40) or acetylation of 40 (yielding 41).
  • the C-7 to C- 12 SAR2 region can be probed by natural products including palmerolides A (1), B (6) and C (7), which differ only in the SAR2 region.
  • Palmerolide C is less potent than palmerolide A.
  • Further synthetic derivatizations can assess the role of the alcohol groups based on modification of template 51 by retention, reduction and addition of polar functional groups. Alternate modifications are readily obtained.
  • C-7 mono-derivatized compounds
  • C-IO mono-derivatives will be prepared by a protection/deprotection of C-7 (tri-t-butylsilyl ether) sequence.
  • the des- carbamato (55) reaction is described in Scheme 10; suitably protected C-7 and C-IO alcohols will provide access to modification of C-11.
  • Carbamates can be interchanged among C-7, C- 10 and C-Il (Cl 3 CC(O)NCHO, then K 2 CO 3 ) (Kocovsky 1986) if warranted by bioactivity profiles of the acetates 63 - 65).
  • Valine esters 62 and 63 are modeled after the similar valaciclovir prodrug, which demonstrated significantly improved pharmacodynamic properties (Guglielmo et al, 2004).
  • Compounds 67 to 69 can be prepared from osmimum tetroxide dihydroxylation of 63 — 65 (forming diastereomeric products such that two products from each of 63 to 65 will result, after separation).
  • the total synthesis of natural products is a major tool in the determination of the unambiguous structure of a compound.
  • Total synthesis also serves as a successful strategy for the formation of analogues of the natural product by simple variation of starting materials or reactions in the synthetic scheme.
  • Our proposed synthetic studies toward the palmerolides detail a convergent approach whereby each component can be varied to provide analogues inspired by X-ray co-crystallization studies with V-ATPase and by computational predictions using ADMET.
  • the synthesis will also be used to confirm the structure and absolute stereochemistry of the palmerolides. This is especially important for less abundant palmerolides such as palmerolide D, E, and F due to the trace quantities that can be isolated.
  • Amide 80 could be inserted into the C-21 side chain by means of a copper-catalyzed amidation reaction directly related to work the Co-PI helped develop (Shen et al., 2000; Klapars et al., 2001; Jiang et al., 2003).
  • the vinyl iodide 82 required for the amidation reaction could be synthesized by employing the Takai olefmation of the corresponding aldehyde precursor. (Takai et al., 1986)
  • the C-22 double bond could, in turn, be formed by using the Schlosser modification of the Wittig reaction. (Schlosser et al., 1966) This should lead to selective formation of the ( ⁇ )-alkene.
  • the stereochemistry of C-20 and C-21 could be established by means of the highly predictive diastereoselective aldol reaction that was developed by Evans. (Evans et al., 1981)
  • the chiral diol 79 could, in turn, be prepared enantioselectively using the Sharpless asymmetric dihydroxylation (Jacobsen et al., 1988).
  • the asymmetric dihydroxylation could also be employed to form the chiral alcohol fragment 78. This leads to the enantioselective formation of the chiral secondary alcohol on C-8.
  • the vinylogous ester in fragment 78 may be prepared by a Horner-Wadsworth-Emmons olefmation reaction (Stocksdale et al., 1998).
  • Asymmetric dihydroxylation of 88 with Sharpless conditions should lead to the formation of desired diol 89 with high enantioselectivity (Jacobsen et al., 1988). It should be noted at this point that the Sharpless asymmetric aminohydroxylation could be employed at this stage of the synthesis (Li et al., 1996). This would lead to the formation of an enantiopure 1,2-amino alcohol.
  • the amide nucleophile generally prefers the less hindered carbon of the alkene. Based upon that observation, the protected amine should be installed on C-Il of palmerolide A. This aminohydroxylation route could provide an aza variant of palmerolides.
  • the two secondary alcohol groups of compound 89 can be selectively protected as an acetonide upon treatment with acetone and TsOH (Coe et al., 1989). This would protect the two secondary alcohols in favor of the primary alcohol due to the thermodynamic stability of the product.
  • the primary alcohol in 90 can be protected with a TIPS group (Cunico et al., 1980).
  • the acetonide could then be opened with FeCl 3 and SiO 2 in chloroform (Kim et al., 1986). This should open the acetonide and not affect the silyl protected alcohols.
  • the secondary alcohol on C-12 can now be protected with TBS-Cl and imidazole.
  • the C-12 alcohol protection should be the major product due to the bulk of the TIPS protecting that is present on the C-IO hydroxyl group.
  • the C-I l alcohol can now be protected with MOM-Cl, NaH in THF (Kluge et al, 1972).
  • the ability to protect all of the hydroxyl groups with different protecting groups is vital to this study because it allows for our main goal, the derivatization of palmerolide A.
  • the aldehyde 92 can be prepared by selectively deprotecting the TBDPS group followed by oxidation with Dess-Martin periodinane (Dess et al, 1983).
  • the aldehyde can then be converted to vinyl iodide 93 via Takai olefmation (Jiang et al, 2003). This olefmation procedure forms the (E)-vinyl iodide selectively. This vinyl iodide would have the correct stereochemistry required for the Heck reaction later in the synthetic route
  • the next major step in the synthesis of palmerolide A is the preparation of fragment 78 from the retrosynthesis.
  • the synthesis of 78 begins with the oxidation of 94 (Scheme 14) with Dess-Martin periodinane (Dess et al, 1983). Sharpless asymmetric dihydroxylation of the alkene would lead to the enantioselective formation of diol 95 (Jacobsen et al, 1988).
  • the primary alcohol would be selectively protected with TBDPS-Cl and imidazole (Hanessian et al, 1975).
  • the secondary hydroxyl group can be converted to SEM ether 96 with SEM-Cl and DIPEA in dichloromethane (Lipshutz et al, 1980).
  • the last fragment of the molecule that needs to be prepared is the C-17 through C-25 amide side chain.
  • the first step (Scheme 15) in the synthesis of this fragment is the PDC oxidation of the commercially available geraneol, to yield 103. (Reiter et al, 2003).
  • the use of Evans diastereoselective Aldol condensation at this point would allow for the formation of the 104 (Evans et al, 1981).
  • This Evans methodology has proven to be one of the most dependable tools available to a synthetic chemist for the formation of syn-selective Aldol products.
  • the R group of the oxazolidinone can be varied in order to obtain the product with the highest diastereomeric excess.
  • the final step in the synthesis of palmerolide A is the global deprotection of the remaining protecting groups with MgBr 2 and BuSH in Et 2 O (Kim et al., 1991). This is a mild deprotection procedure that should not open the lactone or cleave the carbamate functional groups present.

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  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Epidemiology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Pyrane Compounds (AREA)

Abstract

La présente invention concerne des groupes de composés tirés de tuniqués de l'espèce Synoicum, ainsi que des compositions pharmaceutiques comprenant ces composés, et leurs utilisations. Des extraits de tuniqués font preuve d'une toxicité sélective contre différentes lignées de cellules cancéreuses du panneau des lignées cellulaires NCI 60. Ces composés conviennent au traitement de cancers, et notamment de mélanomes malins, du cancer du colon, et des lignées cellulaires cancéreuses du rein.
PCT/US2006/036484 2004-02-17 2006-09-18 Composes de cytotoxines et procedes d'isolation WO2007035734A2 (fr)

Priority Applications (7)

Application Number Priority Date Filing Date Title
AU2006292268A AU2006292268A1 (en) 2004-02-17 2006-09-18 Cytotoxin compounds and methods of isolation
CA002622912A CA2622912A1 (fr) 2005-09-16 2006-09-18 Composes de cytotoxines et procedes d'isolation
EP06825017A EP1934196A4 (fr) 2005-09-16 2006-09-18 Composes de cytotoxines et procedes d'isolation
US12/066,938 US8669376B2 (en) 2004-02-17 2006-09-18 Cytotoxin compounds and methods of isolation
JP2008531434A JP2009508878A (ja) 2005-09-16 2006-09-18 細胞毒化合物および単離法
US14/204,996 US9394270B2 (en) 2004-02-17 2014-03-11 Cytotoxin compounds and methods of isolation
US15/190,679 US10815212B2 (en) 2004-02-17 2016-06-23 Cytotoxin compounds and methods of isolation

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US71759805P 2005-09-16 2005-09-16
US60/717,598 2005-09-16

Related Parent Applications (1)

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US10/906,386 Continuation-In-Part US7625885B2 (en) 2004-02-17 2005-02-17 Cytotoxin compound and method of isolation

Related Child Applications (2)

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US12/066,938 A-371-Of-International US8669376B2 (en) 2004-02-17 2006-09-18 Cytotoxin compounds and methods of isolation
US14/204,996 Continuation US9394270B2 (en) 2004-02-17 2014-03-11 Cytotoxin compounds and methods of isolation

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WO2007035734A3 WO2007035734A3 (fr) 2007-05-03

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JP (1) JP2009508878A (fr)
KR (1) KR20080059213A (fr)
CN (1) CN101277942A (fr)
AU (1) AU2006292268A1 (fr)
CA (1) CA2622912A1 (fr)
WO (1) WO2007035734A2 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008124072A1 (fr) * 2007-04-05 2008-10-16 The Board Of Regents Of The University Of Texas System Procédés de préparation de palmerolides et dérivés de ceux-ci
US20110213182A1 (en) * 2008-07-08 2011-09-01 Solvay Solexis S.P.A. Process for the manufacture of fluorosurfactants
WO2012045451A1 (fr) 2010-10-05 2012-04-12 Ludwig-Maximilians-Universitaet Muenchen Nouveau traitement thérapeutique de maladies dépendantes de la progranuline

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2556550A1 (fr) * 2004-02-17 2005-09-01 University Of South Florida Compose de cytotoxine et procede d'isolement

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of EP1934196A4 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008124072A1 (fr) * 2007-04-05 2008-10-16 The Board Of Regents Of The University Of Texas System Procédés de préparation de palmerolides et dérivés de ceux-ci
US7838691B2 (en) 2007-04-05 2010-11-23 Board Of Regents, Of The University Of Texas System Palmerolides: methods of preparation and derivatives thereof
US20110213182A1 (en) * 2008-07-08 2011-09-01 Solvay Solexis S.P.A. Process for the manufacture of fluorosurfactants
US9416085B2 (en) * 2008-07-08 2016-08-16 Solvay Specialty Polymers Italy S.P.A. Process for the manufacture of fluorosurfactants
WO2012045451A1 (fr) 2010-10-05 2012-04-12 Ludwig-Maximilians-Universitaet Muenchen Nouveau traitement thérapeutique de maladies dépendantes de la progranuline

Also Published As

Publication number Publication date
JP2009508878A (ja) 2009-03-05
EP1934196A2 (fr) 2008-06-25
KR20080059213A (ko) 2008-06-26
WO2007035734A3 (fr) 2007-05-03
EP1934196A4 (fr) 2011-05-18
AU2006292268A1 (en) 2007-03-29
CN101277942A (zh) 2008-10-01
CA2622912A1 (fr) 2007-03-29

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