WO2010059858A1 - Composés de manassantin et leurs procédés de production et d'utilisation - Google Patents

Composés de manassantin et leurs procédés de production et d'utilisation Download PDF

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WO2010059858A1
WO2010059858A1 PCT/US2009/065194 US2009065194W WO2010059858A1 WO 2010059858 A1 WO2010059858 A1 WO 2010059858A1 US 2009065194 W US2009065194 W US 2009065194W WO 2010059858 A1 WO2010059858 A1 WO 2010059858A1
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independently selected
group
hydrogen
hydroxyl
alkyl
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PCT/US2009/065194
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Jiyong Hong
Mark Dewhirst
Hyoungsu Kim
Amanda C. Kasper
Eui Jung Moon
Yongho Park
Ceshea M. Wooten
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Duke University
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Priority to US13/130,237 priority Critical patent/US8946289B2/en
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    • 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
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A61K31/341—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide not condensed with another ring, e.g. ranitidine, furosemide, bufetolol, muscarine
    • 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
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
    • C07D307/04—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having no double bonds between ring members or between ring members and non-ring members
    • C07D307/10—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having no double bonds between ring members or between ring members and non-ring members with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D307/12—Radicals substituted by oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D407/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00
    • C07D407/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00 containing two hetero rings
    • C07D407/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00 containing two hetero rings linked by a chain containing hetero atoms as chain links

Definitions

  • the present invention is directed to novel manassantin compounds and analogues thereof and methods of making and using the same.
  • Hypoxia-inducible factors are transcription factors that respond to cellular changes in oxygen levels, and specifically, to a condition of low physiological oxygen levels known as hypoxia.
  • Most, if not all, oxygen-consuming species express HIF-1 , which is a heterodimeric transcriptional complex comprised of an alpha and a beta subunit.
  • the related protein HIF-2 ⁇ can also dimerize with HIF-1 ⁇ .
  • Heterodimers that contain HIF-1 ⁇ or HIF-2 ⁇ seem to have overlapping but distinct specificities, with regard to physiological inducers and target-gene activation.
  • HIF-3 ⁇ is a third related protein that may function primarily as an inhibitor of HIF-1 ⁇ .
  • HIF-1 upregulates several genes, including glycolysis enzymes and vascular endothelial growth factor (VEGF).
  • Hypoxia affects an organism in many ways, including inhibiting cellular differentiation, promoting formation of blood vessels, promoting the formation of the vascular system in embryos, and promoting the migration of karatinocytes and the restoration of the epithelium in wounds.
  • the present invention provides a method of treating a disease, the method comprising administering to a patient in need thereof an effective amount of a compound according to Formula I: with variables as detailed below.
  • the disease may be selected from the group consisting of leukemia, lung cancer, colon cancer, CNS cancer, melanoma, ovarian cancer, renal cancer, liver cancer, prostate cancer, breast cancer, stroke, heart disease, arthritis, ocular neovascular diseases, inflammation, kidney disease, and anemia.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound according to
  • the present invention provides methods of inhibiting HIF-1 comprising contacting a cell with a compound according to Formula I, in an amount effective to inhibit the HIF-1 pathway.
  • the invention provides a method of potentiating a cancer cell for treatment with ionizing radiation or chemotherapeutics, the method comprising contacting the cancer cell with an effective amount of a compound according to Formula I.
  • Figure 1 is a general scheme for the chemical synthesis of manassantin compounds according to the invention.
  • Figure 2 is a scheme for the retrosynthetic chemical synthesis of manaimpulsin compounds according to the invention.
  • Figure 3 is a scheme for the chemical synthesis of 2,3-c/s-3,4-frans-4,5-c/s- tetrahydrofuran and 2,3-c/s-3,4-frans-4,5-frans-tetrahydrofuran diastereomers in a ratio of 2:1 .
  • Figure 4 is a scheme for the chemical synthesis of tetrahydrofuran diastereomers using different nucleophiles.
  • Figure 5 is a scheme for the chemical synthesis of 2,3-c/s-3,4-frans-4,5-c/s- tetrahydrofuran diastereomer.
  • Figure 6 is a scheme for the chemical synthesis of manassantin compounds from the 2,3-c/ ' s-3,4-fra/7s-4,5-c/ ' s-tetrahydrofuran diastereomer.
  • Figure 7 is a graph of relative luciferase activity versus concentration of manaimpulsin compound concentration, demonstrating the HIF-1 inhibitory activity of three manaimpulsin compounds.
  • Figure 8 are Western blots of 4T1 cells or MDA-MB-231 cells under hypoxia and treated with and without different amounts of manassantin A.
  • Figure 9 is a graph of VEGF secretion in 4T1 cells after hypoxia treatment, with and without treatment of different amounts of manassantin A.
  • Figure 10 are graphs of manassantin A concentration versus cell survival rates using an MTS assay.
  • Figure 1 1 is a graph of the mean growth percent of various human cancer cell lines with one dose of manassantin A.
  • Figure 12 are graphs of the growth of various human cancer cell lines with various doses of manassantin A (trial 1 ).
  • Figure 13 shows the Gl 50 , TGI, and LC 50 determined for the effect of manassantin A on various human cancer cell lines (trial 1 ).
  • Figure 14 is a table of the Gl 50 , TGI, and LC 50 determined for the effect of manassantin A on various human cancer cell lines (trial 1 ).
  • Figure 15 are graphs of the growth of various human cancer cell lines with various doses of manassantin A (trial 2).
  • Figure 16 is shows the GI 50 , TGI, and LC 50 determined for the effect of manassantin A on various human cancer cell lines (trial 2).
  • Figure 17 is a table of the Gl 50 , TGI, and LC 50 determined for the effect of manassantin A on various human cancer cell lines (trial 2).
  • Figure 18 is a scheme of the chemical synthesis of manassantin A analogs with 2,3-cis-3,4-trans-4,5-trans- and 2,3-trans-3,4-trans-4,5-trans-tetrahydrofuran cores (31 and 33).
  • Figure 19 is a graph of luciferase activity as a function of the concentration of the manaimpulsin A analogs with 2,3-cis-3,4-trans-4,5-trans- and 2,3-trans-3,4-trans- 4,5-trans-tetrahydrofuran cores (31 and 33) to determine the HIF-1 inhibitory activity of 31 and 33.
  • Figure 20 shows the optimized conformations of truncated structures of manaimpulsin A, 31 , and 33.
  • Figure 21 is a graph of compound concentration versus accumulation of IL-2 for an inflammation inhibition assay.
  • Figure 22 is a scheme for the chemical synthesis of 2,3-cis-3,4-trans-4,5-trans- (52a) and 2,3-fr-ans-3,4-frar)s-4,5-f/-ans-tetrahydrofurans (53b).
  • Figure 23 is a scheme for the chemical synthesis of 2,3-cis-3,4-trans-4,5-trans- terahydrofuran analogue (60).
  • Figure 24 is a scheme for the chemical synthesis of 2,3-trans-3,4-trans-4,5- frans-terahydrofuran analogue (65).
  • Figure 25 is a scheme for the chemical synthesis of pyrrolidiine analogue (89).
  • Figure 26 is a scheme for the chemical synthesis of C9" mono-methyl analogue (93).
  • Figure 27 is a scheme for the chemical synthesis of C9' and C9" desmethyl analogue (102).
  • Figure 28 is a scheme for the chemical synthesis of extended chain analogue (110).
  • Figure 29 is a scheme for the chemical synthesis of keto analogue (111 ) and methoxy analogue (112).
  • Figure 30 is a scheme for the chemical synthesis of a C9 and C9"' desmethyl analogue (115).
  • Figure 31 is a scheme for the chemical synthesis of C7 and C7"' de-oxo analogues (117 and 119).
  • Figure 32 is a scheme for the chemical synthesis of a C4 and C4"' hydroxyl analogue (120).
  • the present invention is directed to novel manassantin compounds and stereospecific analogues thereof as well as methods of making and using the compounds of the present invention.
  • the inventors have discovered that manaimpulsins A, B, and analogues thereof may be chemically synthesized stereospecifically through nucleophilic addition of organozinc reagents to 2- benzenesulfonyl cyclic ethers to achieve the 2,3-c/s-3,4-frans-4,5-c/s-tetrahydrofuran moiety, to which side chains may be attached to form the manaimpulsin compounds.
  • the manassantin compounds are useful in methods of treating disorders or diseases such as cancer, stroke, heart disease, arthritis, ocular neovascular diseases, inflammation, kidney disease, tissue ischemia, and anemia.
  • Cancers include but are not limited to leukemia, lung cancer, colon cancer, CNS cancer, melanoma, ovarian cancer, renal cancer, liver cancer, prostate cancer, and breast cancer. Further, the methods of the present invention are useful for stereoselective synthesis of manassantin compounds and analogues.
  • Alkyl refers to a saturated or unsaturated hydrocarbon chain having 1 to 18 carbon atoms, suitably 1 to 12 carbon atoms, or 1 to 6 carbon atoms, or 1 to 4 carbon atoms.
  • “Lower alkyl” refers to a saturated or unsaturated hydrocarbon chain having 1 to 4 carbon atoms.
  • Alkyl groups may be straight or branched. In some embodiments, branched alkyl groups have one or two branches. Unsaturated alkyl groups have one or more double bonds and/or one or more triple bonds. Suitably, unsaturated alkyl groups have one or two double bonds or one triple bond.
  • Alkyl chains may be unsubstituted or substituted with from 1 to about 4 substituents unless otherwise specified.
  • alkyl groups are mono-, di-, or tri-substituted.
  • Suitable alkyl substituents include, but are not limited to, cyano, halo, hydroxy, aryl (e.g., phenyl, tolyl, alkyloxphenyl, alkyloxycarbonylphenyl, halophenyl), heterocyclyl, and heteroaryl.
  • Aromatic ring or "aryl” refers to an aromatic hydrocarbon ring system.
  • Aromatic rings are monocyclic or fused bicyclic ring systems. Monocyclic aromatic rings contain from about 5 to about 10 carbon atoms, suitably from 5 to 7 carbon atoms, or from 5 to 6 carbon atoms in the ring. Bicyclic aromatic rings contain from 8 to 12 carbon atoms, suitably 9 or 10 carbon atoms in the ring. Aromatic rings may be unsubstituted or substituted with from 1 to about 4 substituents on the ring. Suitable aromatic ring substituents include, but are not limited to, halo, cyano, lower alkyl, heteroalkyl, haloalkyl, phenyl, phenoxy or any combination thereof. Suitably, the aromatic ring substituents are lower alkyl, cyano, halo, or halo alkyl. "Carbocycle” refers to a saturated or unsaturated hydrocarbon ring.
  • Carbocycles are not aromatic. Carbocycles are monocyclic, or are fused, spiro, or bridged bicyclic ring systems. Monocyclic carbocycles contain from about 4 to about 10 carbon atoms, suitably from 4 to 7 carbon atoms, or from 5 to 6 carbon atoms in the ring. Bicyclic carbocycles contain from 8 to 12 carbon atoms, suitably from 9 to 10 carbon atoms in the ring. Carbocycles may be unsubstituted or substituted with from 1 to about 4 substituents on the ring.
  • Suitable carbocycle substituents include, but are not limited to, halo, cyano, lower alkyl, heteroalkyl, haloalkyl, phenyl, phenoxy or any combination thereof.
  • the carbocycle substituents are halo or haloalkyl.
  • Suitable carbocycles include, but are not limited to, cyclopentyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.
  • Halo or halogen refers to fluoro, chloro, bromo, or iodo.
  • Haloalkyl refers to a straight, branched, or cyclic hydrocarbon substituted with one or more halo substituents.
  • the haloalkyl is C 1 -C 12 , or Ci-C 6 , or C 1 -C 3 .
  • Suitable halo substituents include fluoro and chloro.
  • One suitable haloalkyl is trifluoromethyl.
  • ⁇ eteroalkyl refers to a saturated or unsaturated chain containing carbon and at least one heteroatom, wherein no two heteroatoms are adjacent.
  • Heteroalkyl groups contain from 1 to 18 member atoms (carbon and heteroatoms) in the chain, or 1 to 12 member atoms, or 1 to 6 member atoms, or 1 to 4 member atoms. Heteroalkyl groups may be straight or branched. Suitably, the branched heteroalkyl may have one or two branches. Unsaturated heteroalkyl have one or more double bonds and/or one or more triple bonds. Suitably, heteroalkyl groups have one or two double bonds or one triple bond. Heteroalkyl groups may be unsubstituted or substituted with from 1 to about 4 substituents unless otherwise specified.
  • Suitable heteroalkyl substituents include halo, aryl (e.g., phenyl, tolyl, alkyloxyphenyl, alkyloxycarbonylphenyl, halophenyl), heterocyclyl, heteroaryl.
  • alkyl chains substituted with the following substituents are heteroalkyl: alkoxy (e.g., methoxy, ethoxy, propoxy, butoxy, pentoxy), aryloxy (e.g., phenoxy, chlorophenoxy, tolyloxy, methoxyphenoxy, benzyloxy, alkyloxycarbonylphenoxy, acyloxyphenoxy), acyloxy (e.g., propionyloxy, benzoyloxy, acetoxy), carbamoyloxy, carboxy, mercapto, alkylthio, acylthio, arylthio (e.g., phenylthio, chlorophenylthio, alkylphenylthio, alkoxyphenylthio, benzylthio, alkyloxycarbonylphenylthio), amino (e.g., amino, mono- and di-C 1 -C 3 alkanylamino, methylphenylamino, methyl
  • Heteroatom refers to a nitrogen, sulfur, or oxygen atom. Groups containing more than one heteroatom may contain different heteroatoms. As used herein, halogens are not heteroatoms.
  • ⁇ eterocycle refers to a saturated or unsaturated ring containing carbon and from 1 to about 4 heteroatoms in the ring, wherein no two heteroatoms are adjacent in the ring and no carbon in the ring that has a heteroatom attached to it also has a hydroxyl, amino, or thiol group attached to it.
  • Heterocycles are not aromatic. Heterocycles are monocyclic, or are fused or bridged bicyclic ring systems. Monocyclic heterocycles contain from about 4 to about 10 member atoms (carbon and heteroatoms), suitably from 4 to 7 member atoms, or from 5 to 6 member atoms in the ring.
  • Bicyclic heterocycles contain from 8 to 12 member atoms, suitably 9 or 10 member atoms in the ring. Heterocycles may be unsubstituted or substituted with from 1 to about 4 substituents on the ring. Suitably, the substituents are halo or haloalkyl. Suitable heterocycle substituents include: halo, cyano, lower alkyl, heteroalkyl, haloalkyl, phenyl, phenoxy or any combination thereof. Suitable heterocycles include, but are not limited to, piperzyl, morpholinyl, tetrahydrofuranyl, tetrahydropyranyl, and piperdyl.
  • ⁇ eteroaryl refers to an aromatic ring system containing carbon and from 1 to about 4 heteroatoms in the ring.
  • Heteroaryls are monocyclic or fused bicyclic ring systems.
  • Monocyclic heteroaryls contain from about 5 to about 10 member atoms (carbon and heteroatoms), or from 5 to 7 member atoms, or from 5 to 6 member atoms in the ring.
  • Bicyclic heteroaryls contain from 8 to 12 member atoms, or 9 or 10 member atoms in the ring.
  • Heteroaryls may be unsubstituted or substituted with from 1 to about 4 substituents on the ring.
  • Suitable heteroaryl substituents include: halo, cyano, lower alkyl, heteroalkyl, haloalkyl, phenyl, phenoxy, or any combination thereof.
  • the substituents are halo, haloalkyl, or phenyl.
  • Suitable heteroaryls include, but are not limited to, benzothienyl, benzofuranyl, thienyl, thiazolo, purinyl, pyrimidyl, pyridyl, and furanyl.
  • “Lower alkyl” refers to an alkyl chain comprised of 1 to 4 carbon atoms, suitably
  • Lower alkyl groups may be saturated or unsaturated and substituted or unsubstituted.
  • Lower alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, and t-butyl.
  • Lower heteroalkyl refers to a heteroalkyl chain comprised of 1 to 4 member atoms. Lower heteroalkyl groups may be saturated or unsaturated and substituted or unsubstituted.
  • Manganese atom refers to a polyvalent atom (C, O, N, or S atom) in a chain or ring system that continues the chain or ring system. For example, in benzene the six carbon atoms are member atoms and the six hydrogen atoms are not member atoms.
  • Phenyl refers to a six-membered monocyclic aromatic ring which may or may not be substituted with from about 1 to about 4 substituents. The substituents may be substituted at the ortho, meta or para position on the phenyl ring, or any combination thereof.
  • Suitable phenyl substituents include: halo, cyano, lower alkyl, heteroalkyl, haloalkyl, phenyl, phenoxy or any combination thereof.
  • the above groups, whether alone or part of another substituent, may themselves optionally be substituted with one or more groups selected from themselves and the additional substituents listed below.
  • Ether -OR, wherein R is an ether substituent, for example, a Ci_ 7 alkyl group
  • R is an acyl substituent, for example, H, a C 1-7 alkyl group (also referred to as C 1-7 alkylacyl or Ci -7 alkanoyl), a C 3-2O heterocyclyl group (also referred to as C 3-2 O heterocyclylacyl), or a C 5-2 O aryl group (also referred to as C 5-2 O arylacyl), preferably a Cw alkyl group.
  • R is an acyl substituent, for example, H, a C 1-7 alkyl group (also referred to as C 1-7 alkylacyl or Ci -7 alkanoyl), a C 3-2O heterocyclyl group (also referred to as C 3-2 O heterocyclylacyl), or a C 5-2 O aryl group (also referred to as C 5-2 O arylacyl), preferably a Cw alkyl group.
  • R 1 and R 2 together with the nitrogen atom to which they are attached, form a heterocyclic structure as in, for example, piperidinocarbonyl, morpholinocarbonyl, thiomorpholinocarbonyl, and piperazinylcarbonyL
  • R 1 and R 2 are independently amino substituents, for example, hydrogen, a C- ⁇ _ 7 alkyl group (also referred to as Ci_ 7 alkylamino or di-Ci_ 7 alkylamino), a C 3 _ 20 heterocyclyl group, or a C 5 _ 20 aryl group, preferably H or a C 1-7 alkyl group, or, in the case of a "cyclic" amino group, R 1 and R 2 , taken together with the nitrogen atom to which they are attached, form a heterocyclic ring having from 4 to 8 ring atoms.
  • R 1 and R 2 are independently amino substituents, for example, hydrogen, a C- ⁇ _ 7 alkyl group (also referred to as Ci_ 7 alkylamino or di-Ci_ 7 alkylamino), a C 3 _ 20 heterocyclyl group, or a C 5 _ 20 aryl group, preferably H or a C 1-7 alkyl group, or, in the case of a
  • amino groups include, but are not limited to, -NH 2 , -NHCH 3 , - NHCH(CH 3 ) 2 , -N(CH 3 ) 2 , -N(CH 2 CH 3 ) 2 , and -NHPh.
  • cyclic amino groups include, but are not limited to, aziridinyl, azetidinyl, pyrrolidinyl, piperidino, piperazinyl, perhydrodiazepinyl, morpholino, and thiomorpholino.
  • the cyclic amino groups may be substituted on their ring by any of the substituents defined here, for example carboxy, carboxylate and amido.
  • Acylamido (acylamino): -NR 1 C( O)R 2 , wherein R 1 is an amide substituent, for example, hydrogen, a Ci -7 alkyl group, a C 3-20 heterocyclyl group, or a C 5 . 20 aryl group, preferably H or a C 1-7 alkyl group, most preferably H, and R 2 is an acyl substituent, for example, a C 1-7 alkyl group, a C 3-20 heterocyclyl group, or a C 5 . 20 aryl group, preferably a C 1-7 alkyl group.
  • R 1 and R 2 may together form a cyclic structure, as in, for example, succinimidyl, maleimidyl, and phthalimidyl.
  • R 2 and R 3 are independently amino substituents, as defined for amino groups, and R 1 is a ureido substituent, for example, hydrogen, a C 1-7 alkyl group, a C 3-2O heterocyclyl group, or a C 5 . 2 o aryl group, preferably hydrogen or a Cw alkyl group.
  • ureido groups include, but are not limited to, -NHCONH 2 , -NHCONHMe, -NHCONHEt, -NHCONMe 2 , -NHCONEt 2 , -NMeCONH 2 , -NMeCONHMe, -NMeCONHEt, NMeCONMe 2 , -NMeCONEt 2 , and -NHCONHPh.
  • acyloxy (reverse ester): -0C( 0)R, wherein R is an acyloxy substituent, for example, a C1.7 alkyl group, a C 3-2 O heterocyclyl group, or a C5. 2 o aryl group, preferably a C 1-7 alkyl group.
  • R is an acyloxy substituent, for example, a C1.7 alkyl group, a C 3-2 O heterocyclyl group, or a C5. 2 o aryl group, preferably a C 1-7 alkyl group.
  • acyloxy groups include, but are not limited to, -
  • Oxo: 0.
  • R is a thioether substituent, for example, a C 1 . 7 alkyl group (also referred to as a C 1-7 alkylthio group), a C 3-20 heterocyclyl group, or a C 5-20 aryl group, preferably a C 1 . ? alkyl group.
  • Examples of C 1 . ? alkylthio groups include, but are not limited to, -SCH 3 and -SCH 2 CH 3 .
  • R is a sulfoxide substituent, for example, a C 1-7 alkyl group, a C 3-20 heterocyclyl group, or a C 5-20 aryl group, preferably a Ci -7 alkyl group.
  • Sulfonyl (sulfone): -S( O) 2 R, wherein R is a sulfone substituent, for example, a C 1-7 alkyl group, a C 3-20 heterocyclyl group, or a C 5 . 20 aryl group, preferably a C- ⁇ _ 7 alkyl group.
  • R is a sulfone substituent, for example, a C 1-7 alkyl group, a C 3-20 heterocyclyl group, or a C 5 . 20 aryl group, preferably a C- ⁇ _ 7 alkyl group.
  • Thioamido (thiocarbamyl): -C( S)NR 1 R 2 , wherein R 1 and R 2 are independently amino substituents, as defined for amino groups.
  • R 1 is an amino substituent, as defined for amino groups
  • R is a sulfonamino substituent, for example, a C 1-7 alkyl group, a C 3 . 20 heterocyclyl group, or a C 5 . 20 aryl group, preferably a C 1-7 alkyl group.
  • Protected refers to a chemical structure wherein one or more of the chemically-sensitive groups in the molecule have been modified to reduce its activity and allow for better synthetic techniques to be used.
  • Protecting groups vary but are generally found in "Protecting Groups in Organic Synthesis” by Theadora Green.
  • Unprotected refers to a chemical structure that does not contain any groups that have been added to protect sensitive functional moieties such as hydroxy groups or carboxcylic acid groups.
  • “Pharmaceutically acceptable carrier” refers to a carrier that is useful for the preparation of a pharmaceutical composition that is generally compatible with the other ingredients of the composition, not deleterious to the recipient, and neither biologically nor otherwise undesirable.
  • “A pharmaceutically acceptable carrier” includes both one and more than one carrier. Embodiments include carriers for topical, ocular, parenteral, intravenous, intraperitoneal intramuscular, sublingual, nasal and oral administration.
  • “Pharmaceutically acceptable carrier” also includes agents for preparation of aqueous dispersions and sterile powders for injection or dispersions.
  • Effective amount refers to a dosage of the compounds or compositions effective for eliciting a desired effect. This term as used herein may also refer to an amount effective at bringing about a desired in vivo effect in an animal, preferably, a human, such as inhibition of HIF-1.
  • Excipient refers to a physiologically compatible additives useful in preparation of a pharmaceutical composition.
  • Examples of pharmaceutically acceptable carriers and excipients can for example be found in Remington Pharmaceutical Science, 16th Ed.
  • Administration refers to administration of the compounds as needed to achieve the desired effect. Administration may include, but is not limited to, oral, sublingual, intramuscular, subcutaneous, intravenous, transdermal, topical, parenteral, buccal, rectal, and via injection, inhalation, and implants.
  • contacting a cell is used to mean contacting a cell in vitro, ex vivo, or in vivo (i.e. within a subject, such as a mammal, including humans, mice, rats, rabbits, cats, and dogs). Contacting may occur as a result of administration to a subject.
  • Inhibiting HIF-1 refers to direct or indirect inhibition of HIF-1 , including but not limited to reducing the activity of HIF-1 , reducing the expression of HIF-1 , preventing activity of HIF-1.
  • Reducing proliferation of a cell refers to reducing, inhibiting, or preventing the growth or differentiation of a cell, including killing a cell.
  • Manassantins and “manassantin compounds” refer to manassantin compounds including manassantin A, manassantin B, manassantin B 1 , 4-0- demethylmanassantin B, and analogues thereof, whether synthetic or naturally occurring.
  • the present invention is directed to novel manassantin compounds.
  • the compounds according to the present invention are those according to Formula I below:
  • R4, R 6 , R7, and Rg are independently selected from alkyl, heteroalkyl, aryl, heteroaryl, ester, acyl, sulfonyl, heterocyclyl, amido, thioamido, acetyl, hydroxyl, amino, hydrogen, halogen, and cyano.
  • R4, Re, R 7 , and Rg are independently OH, hydrogen, oxo, methoxy, or lower alkyl.
  • R 4 , R 6 , R 7 , and Rg are independently lower alky.
  • R 4 , R 6 , R 7 , and R g are independently methyl.
  • R 4 , R 6 , R 7 , and R 9 are independently hydrogen.
  • X, Y, and Z are independently selected from the group consisting of CR 13 , O, S, and NR 14 , wherein R 13 and R 14 are independently selected from the group consisting of alkyl, heteroalkyl, aryl, heteroaryl, ester, acyl, sulfonyl, heterocyclyl, amido, thioamido, acetyl, hydroxyl, amino, hydrogen, halogen, and cyano.
  • R 13 and R 14 are independently selected from the group consisting of alkyl, heteroalkyl, aryl, heteroaryl, ester, acyl, sulfonyl, heterocyclyl, amido, thioamido, acetyl, hydroxyl, amino, hydrogen, halogen, and cyano.
  • X, Y, and Z are independently O.
  • R 3 and R 10 are independently selected from the group consisting of hydroxyl, hydrogen, alkyl, heteroalkyl, and oxo. In some embodiments, R 3 and R 10 are independently OH, hydrogen, oxo, methoxy, or lower alkyl. In some embodiments, R 3 and R 10 are independently methyl or methoxy.
  • R 11 and R 12 are independently selected from the group consisting of alkyl, heteroalkyl, aryl, heteroaryl, ester, acyl, sulfonyl, heterocyclyl, amido, thioamido, acetyl, hydroxyl, amino, hydrogen, halogen, oxo, substituted alkylaryl, and cyano, or R 11 and R 12 combine to form a ring.
  • R 11 and R 12 combine to form a ring comprising -0-CH 2 -O-.
  • R 1 and R 2 are of Formula Il wherein R 11 and R 12 are methoxy.
  • R 1 and R 2 are of Formula Il with R 11 and R 12 methoxy groups; X, Y, and Z are suitably O; R 6 and R 7 are suitably methyl groups; R 5 and R 8 are suitably methoxy-substituted phenyl groups; and R 3 , R 4 , Rg, and R 10 are suitably independently hydroxyl, hydrogen, or methyl groups.
  • Suitable manassantin compounds according to the present invention include, but are not limited to, those shown below:
  • compounds of the invention do not include the following compound:
  • compounds of the invention do not include the compounds of manassantin A (1 ) and manassantin B (2) shown below:
  • the general process for stereoselective synthesis of manaimpulsin compounds according to the present invention is depicted in Figure 1 , and a retrosynthetic pathway is depicted in Figure 2.
  • the central five-membered heterocyclic ring may be synthesized first.
  • the 2,3-c/s-3,4-frans-4,5-frans-tetrahydrofuran or 2,3-trans-3,4- trans-4, 5-frans-tetrahydrofuran may be stereoselective ⁇ synthesized via BF 3 OEt 2 - promoted reductive deoxygenation of cyclic hemiketals (Kim, H.; Wooten, C. M.; Park, Y.; Hong, J. Org. Lett.
  • the resulting stereochemistry outcome may be rationalized based on Woerpel's "inside attack" model. Based on the same rationale, an organozinc reagent such as ArZnBr may be added to the sterically more favorable conformation of the 2-benzenesulfonyl cyclic ether from the inside face of the envelope conformer to stereoselective ⁇ provide the 2,3-cis-3,4-trans-4,5-cis-tetrahydrofuran.
  • organozinc reagent such as ArZnBr may be added to the sterically more favorable conformation of the 2-benzenesulfonyl cyclic ether from the inside face of the envelope conformer to stereoselective ⁇ provide the 2,3-cis-3,4-trans-4,5-cis-tetrahydrofuran.
  • model systems may be used where the postulated repulsive interaction (between the incoming nucleophile and the C-4 methyl group during addition of 4 to the oxocarbenium intermediate) is reduced by addition of a smaller nucleophile or removal of the C-4 methyl group ( Figure 4).
  • Addition of a sterically less demanding PhZnBr to 5 may yield a 3.5:1 diastereomeric mixture of 10a and 10b.
  • a sterically less demanding exo-methylene group as a precursor to the C-4 methyl group and stereoselective reduction of the double bond may be used.
  • alkylation of 8 with Eschenmoser's salt and m-CPBA oxidation may generate 12 (80% for 2 steps) (Schreiber, J.; Maag, H.; Hashimoto, N.; Eschenmoser, A. Angew. Chem., Int. Ed. Engl. 1971 , 10, 330-331 ; Mandal, M.; Yun, H.; Dudley, G. B.; Lin, S.; Tan, D. S.; Danishefsky, S. J. J.
  • This core tetrahydrofuran unit 2,3-c/s-3,4-fr"ans-4,5-c/s-tetrahydrofuran, may be coupled to the appropriate side arms via S N 2 reactions to complete the synthesis of manaimpulsin A and manaimpulsin B.
  • S N 2 reactions As shown in Figure 6 and detailed in Example 3, a BEMP-mediated S N 2 reaction of 16 and 17 (following the procedures reported in Lee, A.-L; Ley, S. V. Org. Biomol. Chem.
  • 17 and 20 were prepared from 1 ,2-dimethyl-4-(2-propen-1-yl)benzene and 5-(2-propen-1-yl)-1 ,3-benzodioxole, respectively) followed by stereocontrolled- reduction using polymer-supported BH 4 may be used to complete the synthesis of manaimpulsin A.
  • 16 may be subjected to the BEMP-mediated S N 2 reaction with 1 equivalent of 17 to form the mono-alkylation product 19 (58% BRSM) in addition to 18 (42% BRSM).
  • Compound 19 may be then subjected to a second BEMP-mediated S N 2 reaction with 20 (following the procedures reported in Lee, A.-L.; Ley, S. V. Org. Biomol. Chem. 2003, 7, 3957-3966, which is incorporated by reference, 17 and 20 were prepared from 1 ,2-dimethyl-4-(2-propen-1- yl)benzene and 5-(2-propen-1 -yl)-1 ,3-benzodioxole, respectively) to give 21 (77%). Reduction of 21 with polymer-supported BH 4 may be used to generate manassantin B.
  • the manassantin compounds can be converted into other analogs in various ways.
  • 4-f ⁇ rt-butyldimethylsilyloxy-3-methoxyphenyllithium may give a anomeric mixture of the cyclic hemiketal.
  • BF 3 « OEt 2 may preferentially provide the desired 2,3-c/s-3,4-frans-4,5-frans- tetrahydrofuran through the addition of hydride from the inside face of the envelope conformer. If the cyclic hemiketal is treated with BF 3 OEt 2 (-78 to -20 0 C) followed by reduction with NaBH 3 CN, it may provide the 2,3-frans-3,4-fr " ans-4,5-frans- tetrahydrofuran.
  • This core tetrahydrofuran units may be coupled to the appropriate side arms via S N 2 reactions to complete the synthesis of other manassantin analogues.
  • the invention provides methods of assessing the effectiveness of a compound for treatment of a disease or disorder.
  • the methods may comprise contacting a cell with a compound according to Formula I, determining the proliferation of the cell, and comparing the proliferation to a control, wherein the reduction in proliferation is indicative of the effectiveness of the compound to treat the disease or disorder.
  • Manassantin compounds may be useful in inhibiting the activity of HIF-1 or down regulating HIF-1 .
  • the manassantin compounds of the present invention may be used to treat various conditions, disorders or diseases such as cancer, stroke, heart disease, arthritis, ocular neovascular diseases, inflammation, kidney disease, tissue ischemia, and anemia.
  • Cancers include but are not limited to leukemia, lung cancer, colon cancer, CNS cancer, melanoma, ovarian cancer, renal cancer, liver cancer, prostate cancer, and breast cancer.
  • a cell is contacted with an amount of a manassantin compound effective to inhibit HIF-1 pathway.
  • an effective amount of a manassantin compound according to the present invention will vary with the particular condition being treated, the age and physical condition of the patient being treated, the severity of the condition, the duration of treatment, the nature of concurrent therapy, the route of administration, the particular pharmaceutically-acceptable carrier utilized, and like factors within the knowledge and expertise of the attending physician.
  • an effective amount of the manassantin compounds of the present invention for systemic administration may be from about 6 to about 100 mg/kg body weight.
  • Transdermal dosages may be designed to attain similar serum or plasma levels, based upon techniques known to those skilled in the art of pharmacokinetics and transdermal formulations.
  • the manassantin compounds of the present invention may also be administered at other intervals, such as twice per day, twice weekly, once weekly, or once a month.
  • One of ordinary skill in the art would be able to calculate suitable effective amounts for other intervals of administration.
  • compositions Comprising Manarialin Compounds
  • the manassantin compounds may be administered in a pharmaceutically acceptable composition, such as in or with a pharmaceutically acceptable carrier.
  • Compositions may include one or more of the isoforms of the manalichin compounds of the present invention.
  • each enantiomer or diastereomer may be separately used, or they may be combined in any proportion.
  • tautomers all possible tautomers are specifically contemplated.
  • compositions for use in accordance with the present invention may be formulated in a conventional manner using one or more physiologically acceptable carriers or excipients.
  • the manalichin compounds may be formulated for administration by, for example, solid dosing, in a topical oil-based formulation, injection, inhalation (either through the mouth or the nose), implants, or oral, buccal, parenteral, or rectal administration. Techniques and formulations may generally be found in "Remington's Pharmaceutical Sciences", (Meade Publishing Co., Easton, Pa.). The route by which the manassantin compounds of the present invention
  • component A will be administered and the form of the composition will dictate the type of carrier (component B) to be used.
  • the composition may be in a variety of forms, suitable, for example, for systemic administration (e.g., oral, rectal, nasal, sublingual, buccal, implants, or parenteral) or topical administration (e.g., local application on the skin, liposome delivery systems, or iontophoresis).
  • Carriers for systemic administration typically comprise at least one of a) diluents, b) lubricants, c) binders, d) disintegrants, e) colorants, f) flavors, g) sweeteners, h) antioxidants, j) preservatives, k) glidants, m) solvents, n) suspending agents, o) wetting agents, p) surfactants, combinations thereof, and others. All carriers are optional in the systemic compositions.
  • Ingredient a) is a diluent.
  • Suitable diluents for solid dosage forms include sugars such as glucose, lactose, dextrose, and sucrose; diols such as propylene glycol; calcium carbonate; sodium carbonate; sugar alcohols, such as glycerin; mannitol; and sorbitol.
  • the amount of ingredient a) in the systemic or topical composition is typically about 50 to about 90%.
  • Ingredient b) is a lubricant.
  • Suitable lubricants for solid dosage forms are exemplified by solid lubricants including silica, talc, stearic acid and its magnesium salts and calcium salts, calcium sulfate; and liquid lubricants such as polyethylene glycol and vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, com oil and oil of theobroma.
  • the amount of ingredient b) in the systemic or topical composition is typically about 5 to about 10%.
  • Ingredient c) is a binder.
  • Suitable binders for solid dosage forms include polyvinyl pyrrolidone; magnesium aluminum silicate; starches such as corn starch and potato starch; gelatin; tragacanth; and cellulose and its derivatives, such as sodium carboxymethylcellulose, ethyl cellulose, methylcellulose, microcrystalline cellulose, and sodium carboxymethylcellulose.
  • the amount of ingredient c) in the systemic composition is typically about 5 to about 50%, and in ocular solid dosing forms up to 99%.
  • Ingredient d) is a disintegrant.
  • Suitable disintegrants for solid dosage forms include agar, alginic acid and the sodium salt thereof, effervescent mixtures, croscarmelose, crospovidone, sodium carboxymethyl starch, sodium starch glycolate, clays, and ion exchange resins.
  • the amount of ingredient d) in the systemic or topical composition is typically about 0.1 to about 10%.
  • Ingredient e) for solid dosage forms is a colorant such as an FD&C dye.
  • the amount of ingredient e) in the systemic or topical composition is typically about 0.005 to about 0.1 %.
  • Ingredient f) for solid dosage forms is a flavor such as menthol, peppermint, and fruit flavors.
  • the amount of ingredient f), when used, in the systemic or topical composition is typically about 0.1 to about 1.0%.
  • Ingredient g) for solid dosage forms is a sweetener such as aspartame and saccharin.
  • the amount of ingredient g) in the systemic or topical composition is typically about 0.001 to about 1 %.
  • Ingredient h) is an antioxidant such as butylated hydroxyanisole (“BHA”), butylated hydroxytoluene (“BHT”), and vitamin E.
  • BHA butylated hydroxyanisole
  • BHT butylated hydroxytoluene
  • the amount of ingredient h) in the systemic or topical composition is typically about 0.1 to about 5%.
  • Ingredient j) is a preservative such as benzalkonium chloride, methyl paraben and sodium benzoate.
  • the amount of ingredient j) in the systemic or topical composition is typically about 0.01 to about 5%.
  • Ingredient k) for solid dosage forms is a glidant such as silicon dioxide.
  • the amount of ingredient k) in the systemic or topical composition is typically about 1 to about 5%.
  • Ingredient m) is a solvent, such as water, isotonic saline, ethyl oleate, glycerine, hydroxylated castor oils, alcohols such as ethanol, and phosphate buffer solutions.
  • the amount of ingredient m) in the systemic or topical composition is typically from about 0 to about 100%.
  • Ingredient n) is a suspending agent. Suitable suspending agents include
  • Avicel® RC-591 from FMC Corporation of Philadelphia, PA
  • sodium alginate The amount of ingredient n) in the systemic or topical composition is typically about 1 to about 8%.
  • Ingredient o) is a surfactant such as lecithin, Polysorbate 80, and sodium lauryl sulfate, and the TWEENS® from Atlas Powder Company of Wilmington, Delaware.
  • Suitable surfactants include those disclosed in the C.T.F.A. Cosmetic Ingredient
  • the amount of ingredient o) in the systemic or topical composition is typically about 0.1 % to about 5%.
  • system compositions comprise 0.01 % to 50% of component A and 50 to 99.99% of component B.
  • compositions for parenteral administration typically comprise A) 0.1 to 10% of the compounds of the present invention and B) 90 to 99.9% of a carrier comprising a) a diluent and m) a solvent.
  • component a) comprises propylene glycol and m) comprises ethanol or ethyl oleate.
  • compositions for oral administration can have various dosage forms.
  • solid forms include tablets, capsules, granules, and bulk powders.
  • These oral dosage forms comprise a safe and effective amount, usually at least about 5%, and more particularly from about 25% to about 50% of component A).
  • the oral dosage compositions further comprise about 50 to about 95% of component B), and more particularly, from about 50 to about 75%.
  • Tablets can be compressed, tablet triturates, enteric-coated, sugar-coated, film-coated, or multiple-compressed. Tablets typically comprise component A, and component B a carrier comprising ingredients selected from the group consisting of a) diluents, b) lubricants, c) binders, d) disintegrants, e) colorants, f) flavors, g) sweeteners, k) glidants, and combinations thereof.
  • Specific diluents include calcium carbonate, sodium carbonate, mannitol, lactose and cellulose.
  • Specific binders include starch, gelatin, and sucrose.
  • Specific disintegrants include alginic acid and croscarmelose.
  • Specific lubricants include magnesium stearate, stearic acid, and talc.
  • Specific colorants are the FD&C dyes, which can be added for appearance.
  • Chewable tablets preferably contain g) sweeteners such as aspartame and saccharin, or f) flavors such as menthol, peppermint, fruit flavors, or a combination thereof.
  • Capsules typically comprise component A, and a carrier comprising one or more a) diluents disclosed above in a capsule comprising gelatin.
  • Granules typically comprise component A, and preferably further comprise k) glidants such as silicon dioxide to improve flow characteristics.
  • Implants can be of the biodegradable or the non- biodegradable type. Implants may be prepared using any known biocompatible formulation.
  • ingredients in the carrier for oral compositions depends on secondary considerations like taste, cost, and shelf stability, which are not critical for the purposes of this invention. One skilled in the art would know how to select appropriate ingredients without undue experimentation.
  • the solid compositions may also be coated by conventional methods, typically with pH or time-dependent coatings, such that component A is released in the gastrointestinal tract in the vicinity of the desired application, or at various points and times to extend the desired action.
  • the coatings typically comprise one or more components selected from the group consisting of cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropyl methyl cellulose phthalate, ethyl cellulose, EUDRAGIT® coatings (available from Rohm & Haas G. M. B. H. of Darmstadt, Germany), waxes, and shellac.
  • compositions for oral administration can also have liquid forms.
  • suitable liquid forms include aqueous solutions, emulsions, suspensions, solutions reconstituted from non-effervescent granules, suspensions reconstituted from non- effervescent granules, effervescent preparations reconstituted from effervescent granules, elixirs, tinctures, syrups, and the like.
  • Liquid orally administered compositions typically comprise component A and component B, namely, a carrier comprising ingredients selected from the group consisting of a) diluents, e) colorants, f) flavors, g) sweeteners, j) preservatives, m) solvents, n) suspending agents, and o) surfactants.
  • Peroral liquid compositions preferably comprise one or more ingredients selected from the group consisting of e) colorants, f) flavors, and g) sweeteners.
  • compositions useful for attaining systemic delivery of the manaimpulsin compounds include sublingual, buccal, and nasal dosage forms.
  • Such compositions typically comprise one or more of soluble filler substances such as a) diluents including sucrose, sorbitol, and mannitol; and c) binders such as acacia, microcrystalline cellulose, carboxymethyl cellulose, and hydroxypropyl methylcellulose.
  • Such compositions may further comprise b) lubricants, e) colorants, f) flavors, g) sweeteners, h) antioxidants, and k) glidants.
  • the manassantin compounds of the present invention are topically administered.
  • Topical compositions that can be applied locally may be in any form known in the art, non-limiting examples of which include solids, gelable drops, sprays, or ointments.
  • Topical compositions that can be applied locally to the skin may be in any form including solids, solutions, oils, creams, ointments, gels, lotions, shampoos, leave-on and rinse-out hair conditioners, milks, cleansers, moisturizers, sprays, skin patches, and the like.
  • Topical compositions comprise: component A, the manassantin compounds described above, and component B, a carrier.
  • Component B may further comprise one or more optional components.
  • each component in the topical composition depends on various factors.
  • the amount of component A added to the topical composition is dependent on the IC 50 of component A : typically expressed in nanomolar (nM) units.
  • the amount of component A will be from about 0.001 to about 0.3%. If the IC 50 of the medicament is 10 nM, the amount of component A) will be from about 0.01 to about 1 %. If the IC 50 of the medicament is 100 nM, the amount of component A will be from about 0.1 to about 10%. If the IC 50 of the medicament is 1000 nM, the amount of component A will be 1 to 100%, preferably 5% to 50%. If the amount of component A is outside the ranges specified above (i.e., lower), efficacy of the treatment may be reduced. One skilled in the art understands how to calculate and understand an IC50. The remainder of the composition, up to 100%, is component B.
  • the amount of the carrier employed in conjunction with component A is sufficient to provide a practical quantity of composition for administration per unit dose of the medicament.
  • Techniques and compositions for making dosage forms useful in the methods of this invention are described in the following references: Modern Pharmaceutics, Chapters 9 and 10, Banker & Rhodes, eds. (1979); Lieberman et al., Pharmaceutical Dosage Forms: Tablets (1981 ); and Ansel, Introduction to Pharmaceutical Dosage Forms, 2nd Ed., (1976).
  • Component B may comprise a single ingredient or a combination of two or more ingredients.
  • component B comprises a topical carrier.
  • Suitable topical carriers comprise one or more ingredients selected from the group consisting of phosphate buffered saline, isotonic water, deionized water, monofunctional alcohols, symmetrical alcohols, aloe vera gel, allantoin, glycerin, vitamin A and E oils, mineral oil, propylene glycol, PPG-2 myristyl propionate, dimethyl isosorbide, castor oil, combinations thereof, and the like.
  • carriers for skin applications include propylene glycol, dimethyl isosorbide, and water, and even more particularly, phosphate buffered saline, isotonic water, deionized water, monofunctional alcohols, and symmetrical alcohols.
  • the carrier of the topical composition may further comprise one or more ingredients selected from the group consisting of q) emollients, r) propellants, s) solvents, t) humectants, u) thickeners, v) powders, w) fragrances, x) pigments, and y) preservatives.
  • Ingredient q) is an emollient.
  • the amount of ingredient q) in a skin-based topical composition is typically about 5 to about 95%.
  • Suitable emollients include stearyl alcohol, glyceryl monoricinoleate, glyceryl monostearate, propane-1 ,2-diol, butane-1 ,3-diol, mink oil, cetyl alcohol, isopropyl isostearate, stearic acid, isobutyl palmitate, isocetyl stearate, oleyl alcohol, isopropyl laurate, hexyl laurate, decyl oleate, octadecan-2-ol, isocetyl alcohol, cetyl palmitate, di-n-butyl sebacate, isopropyl myristate, isopropyl palmitate, isopropyl stearate, butyl stearate, polyethylene glycol, Methylene glycol, lanolin, sesame oil, coconut oil, arachis oil, castor oil, acetylated lanolin alcohols, petroleum, mineral oil
  • Specific emollients for skin include stearyl alcohol and polydimethylsiloxane.
  • Ingredient r) is a propellant.
  • the amount of ingredient r) in the topical composition is typically about 0 to about 95%.
  • Suitable propellants include propane, butane, isobutane, dimethyl ether, carbon dioxide, nitrous oxide, and combinations thereof.
  • Ingredient s) is a solvent.
  • the amount of ingredient s) in the topical composition is typically about 0 to about 95%.
  • suitable solvents include water, ethyl alcohol, methylene chloride, isopropanol, castor oil, ethylene glycol monoethyl ether, diethylene glycol monobutyl ether, diethylene glycol monoethyl ether, dimethylsulfoxide, dimethyl formamide, tetrahydrofuran, and combinations thereof.
  • Specific solvents include water, ethyl alcohol and propylene glycol.
  • Ingredient t) is a humectant. The amount of ingredient t) in the topical composition is typically 0 to 95%.
  • Suitable humectants include glycerin, sorbitol, sodium 2-pyrrolidone-5-carboxylate, soluble collagen, dibutyl phthalate, gelatin, and combinations thereof.
  • Specific humectants include glycerin.
  • Ingredient u) is a thickener.
  • the amount of ingredient u) in the topical composition is typically about 0 to about 95%.
  • Ingredient v) is a powder.
  • the amount of ingredient v) in the topical composition is typically 0 to 95%.
  • Suitable powders include beta-cyclodextrins, hydroxypropyl cyclodextrins, chalk, talc, fullers earth, kaolin, starch, gums, colloidal silicon dioxide, sodium polyacrylate, tetra alkyl ammonium smectites, trialkyl aryl ammonium smectites, chemically-modified magnesium aluminum silicate, organically- modified Montmorillonite clay, hydrated aluminum silicate, fumed silica, carboxyvinyl polymer, sodium carboxymethyl cellulose, ethylene glycol monostearate, and combinations thereof.
  • Ingredient w) is a fragrance.
  • the amount of ingredient w) in the topical composition is typically about 0 to about 0.5%, particularly, about 0.001 to about 0.1%.
  • Ingredient x) is a pigment.
  • Suitable pigments for skin applications include inorganic pigments, organic lake pigments, pearlescent pigments, and mixtures thereof.
  • Inorganic pigments useful in this invention include those selected from the group consisting of rutile or anatase titanium dioxide, coded in the Color Index under the reference Cl 77,891 ; black, yellow, red and brown iron oxides, coded under references Cl 77,499, 77,492 and, 77,491 ; manganese violet (Cl 77,742); ultramarine blue (Cl 77,007); chromium oxide (Cl 77,288); chromium hydrate (Cl 77,289); and ferric blue (Cl 77,510) and mixtures thereof.
  • the organic pigments and lakes useful in this invention include those selected from the group consisting of D&C Red No. 19 (Cl 45, 170), D&C Red No. 9 (Cl 15,585), D&C Red No. 21 (Cl 45,380), D&C Orange No. 4 (Cl 15,510), D&C Orange No. 5 (Cl
  • Red No. 30 (Cl 73,360), D&C Red No. 3 (Cl 45,430), the dye or lakes based on Cochineal Carmine (Cl 75,570) and mixtures thereof.
  • the pearlescent pigments useful in this invention include those selected from the group consisting of the white pearlescent pigments such as mica coated with titanium oxide, bismuth oxychloride, colored pearlescent pigments such as titanium mica with iron oxides, titanium mica with ferric blue, chromium oxide and the like, titanium mica with an organic pigment of the above-mentioned type as well as those based on bismuth oxychloride and mixtures thereof.
  • the amount of pigment in the topical composition is typically about 0 to about 10%.
  • Examples of z) cellulose derivatives suitable for use in the topical pharmaceutical composition for ocular administration include sodium carboxymethylcellulose, ethylcellulose, methylcellulose, and hydroxypropyl- methylcellulose, particularly, hydroxypropyl-methylcellulose.
  • Examples of aa) salts suitable for use in the topical pharmaceutical composition for ocular administration include mono-, di- and trisodium phosphate, sodium chloride, potassium chloride, and combinations thereof.
  • Examples of cc) pH adjusting additives include HCI or NaOH in amounts sufficient to adjust the pH of the topical pharmaceutical composition for ocular administration to 5.0-7.5.
  • Component A may be included in kits comprising component A, a systemic or topical composition described above, or both; and information, instructions, or both that use of the kit will provide treatment for cosmetic and medical conditions in mammals (particularly humans).
  • the information and instructions may be in the form of words, pictures, or both, and the like.
  • the kit may comprise the medicament, a composition, or both; and information, instructions, or both, regarding methods of application of medicament, or of composition, preferably with the benefit of treating or preventing cosmetic and medical conditions in mammals
  • the 2:1 mixture of 2,3-cis-3,4-trans-4,5-cis- tetrahydrofuran 3a and 2,3-cis-3,4-trans-4,5-transtetrahydrofuran 3b was a colorless oil (102.1 mg, 88%), and was purified again by column chromatography (silica gel, hexanes/EtOAc, 7/1 ) to afford 2,3-cis-3,4-trans-4,5-cis-tetrahydrofuran 3a (59.2 mg, 52%) and 2,3-cis-3,4-trans-4,5-trans-tetrahydrofuran 3b (28.3 mg, 25%) and a mixture of 3a and 3b (12.0 mg, 1 1 %).
  • Manassantin A was generated by adding side chains to the desired tetrahydrofuran 3a described in Example 1.
  • a BEMP-mediated S N 2 reaction of 16 and 17 (following the procedures reported in Lee, A.-L; Ley, S. V. Org. Biomol. Chem. 2003, 1, 3957-3966, which is incorporated by reference), 17 and 20 were prepared from 1 ,2-dimethyl-4-(2-propen-1-yl)benzene and 5-(2-propen-1-yl)- 1 ,3-benzodioxole, respectively) followed by stereocontrolled-reduction using polymer- supported BH 4 completed the synthesis of manassantin A (1 ).
  • a direct nucleophilic addition of the organozinc reagent 4 was applied to the 2-benzenesulfonyl cyclic ether 5 followed by an asymmetric hydrogenation to synthesize the 2,3-c/s-3,4-frans-4,5-c/s-tetrahydrofuran moiety manaimpulsin A and B.
  • the stereoselectivity of the nucleophilic addition reaction was improved by introduction of the sterically less demanding exo-methylene group as a surrogate for the C-9' methyl group in manassantin A and B.
  • Manassantin B (2) was generated as in Example 2 and Figure 6 to first generate compound 18 and compound 19.
  • BEMP 0.027 ml_, 0.0
  • reaction mixture was allowed to warm to 25 0 C for 1 h with stirring, quenched with saturated aqueous NH 4 CI, and diluted with CH 2 CI 2 .
  • the layers were separated and the aqueous layer was extracted with CH 2 CI 2 (2 x 5 ml_).
  • the combined organic layers were washed with brine, dried over anhydrous Na 2 SO 4 , and concentrated in vacuo.
  • the residue was purified by column chromatography, (silica gel, hexanes/EtOAc, 2/1 ) to afford 21 as a colorless oil (24.8 g, 77%).
  • HIF-1 inhibitory of compound 1 , 18, and the anf/-diol diastereomer 22 were assessed with the ODD-Luc assay (Li, F.; Sonveaux, P.; Rabbani, Z. N.; Liu, S.; Yan,
  • 4T1-ODD-Luc cells stably transfected with the oxygen-dependent-degradation (ODD) domain of HIF-1 ⁇ and a firefly luciferase reporter, were seeded in the 24-well plate at a density of 10 5 cells/well. After 16-hour incubation, cells were treated with 240 ⁇ M of Cobalt (II) Chloride (Sigma-Aldrich, St. Louis, MO, USA) and serially diluted compounds for 24 h.
  • ODD oxygen-dependent-degradation
  • HIF-1 ⁇ expression induced by CoCI 2 was inhibited by manaimpulsin A (100 nM) ( Figure 8b) indicating that manassantin A inhibits chemically induced HIF-1 ⁇ expression as well as hypoxia-induced HIF-1 ⁇ expression.
  • VEGF Vascular endothelial growth factor
  • HIF-1 regulated VEGF secretion were examined in 4T1 cells using ELISA.
  • Cells were incubated under hypoxia (0.5% O 2 for 24 h) with various concentrations (0, 1 , 10, 100 nM, and 1 ⁇ M) of manassantin A.
  • Cell culture supernates were collected, and VEGF levels in supernates were measured by a commercially available ELISA kit (R&D systems, Minneapolis, MN).
  • VEGF induced by hypoxia was significantly inhibited by manassantin A at concentrations higher than 10 nM ( Figure 9).
  • MTS MTT
  • PMS phenazine methosulfate
  • NCI-60 DTP human tumor cell line screen One Dose Mean Graph, trial 1 : The effects of manassantin A (1) on cancer cell lines were evaluated using the NCI-60 DTP Human Tumor Cell Line Screen.
  • the NCI-60 DTP Human Tumor Cell Line Screen utilized 60 different human tumor cell lines, representing leukemia, melanoma, and cancers of the lung, colon, brain, ovary, breast, prostate, and kidney. The screening was a two-stage process. First, manassantin A was evaluated against all 60 cell lines at a single dose of 10 ⁇ M. Second, manassantin A was evaluated against the 60 cell panel at five concentration levels.
  • the human tumor cell lines of the cancer screening panel were grown in RPMI 1640 medium containing 5% fetal bovine serum and 2 mM L-glutamine.
  • cells were inoculated into 96 well microtiter plates in 100 ⁇ L at plating densities ranging from 5,000 to 40,000 cells/well depending on the doubling time of individual cell lines.
  • the microtiter plates were incubated at 37 0 C, 5% CO 2 , 95% air and 100% relative humidity for 24 h prior to addition of manassantin A.
  • two plates of each cell line were fixed in situ with TCA, to represent a measurement of the cell population for each cell line at the time of drug addition (Tz).
  • Manassantin A was solubilized in dimethyl sulfoxide at 400-fold the desired final maximum test concentration and stored frozen prior to use. At the time of drug addition, an aliquot of frozen concentrate was thawed and diluted to twice the desired final maximum test concentration with complete medium containing 50 ⁇ g/mL gentamicin. Additional four, 10-fold, or V_ log serial dilutions were made to provide a total of five drug concentrations plus control. Aliquots of 100 ⁇ L of these different drug dilutions were added to the appropriate microtiter wells already containing 100 ⁇ L of medium, resulting in the required final drug concentrations.
  • the plates were incubated for an additional 48 h at 37°C, 5% CO 2 , 95% air, and 100% relative humidity.
  • the assay was terminated by the addition of cold TCA.
  • Cells were fixed in situ by the gentle addition of 50 ⁇ L of cold 50% (w/v) TCA (final concentration, 10% TCA) and incubated for 60 minutes at 4°C. The supernatant was discarded, and the plates were washed five times with tap water and air dried.
  • Sulforhodamine B (SRB) solution 100 ⁇ L) at 0.4% (w/v) in 1 % acetic acid was added to each well, and plates were incubated for 10 minutes at room temperature.
  • GI 5 O Three dose response parameters were calculated for each experimental agent: GI 5 O, TGI, and LC 50 .
  • SRB Sulphorhodamine-B
  • TGI total growth inhibition
  • the LC 50 concentration of drug resulting in a 50% reduction in the measured protein at the end of the drug treatment as compared to that at the beginning
  • a single dose (10 ⁇ M) of manalichin A reduced the growth of all tumors tested, including leukemia, non-small cell lung cancer, colon cancer, CNS cancer, melanoma, ovarian cancer, renal cancer, prostate cancer, and breast cancer. Further, a single dose (10 ⁇ M) of manalichin A was more than 50% lethal to leukemia, melanoma, and ovarian cancer. Results shown in Figures 12-14 further support this finding.
  • Example 10 NCI-60 DTP human tumor cell line screen One Dose Mean Graph, trial 2
  • the effects of manassantin A on cancer cell lines were evaluated as described in Example 9 for a second trial.
  • manassantin A reduced the growth of all tumors tested, including leukemia, non-small cell lung cancer, colon cancer, CNS cancer, melanoma, ovarian cancer, renal cancer, prostate cancer, and breast cancer.
  • a single dose (10 ⁇ M) of manassantin A was more than 50% lethal to leukemia, melanoma, and ovarian cancer.
  • TMD maximum tolerated dose
  • mice were allowed ad libitum feed and water. Injections were administered IP. Dose volumes were generally 0.1 mL/10 grams body weight but may be up to 0.2 mL/10 grams of body weight. As shown in Table 1 , manassantin A was non-toxic at levels of 6.25, 3.13, and 1.56 mg/kg/dose but resulted in death at levels of 12.50 and 100.00 mg/kg/dose.
  • manassantin A is largely determined by the 2,3-cis-3,4- trans-4,5-cis-configuration of the tetrahydrofuran core, and to test whether the overall conformation is an important determinant for the binding mode and affinity toward molecular target(s), potency, and HIF-1 signaling specificity of manassantin A, manassantin A analogues with modifications in tetrahydrofuran configuration were prepared (Kim, H.; Wooten, C. M.; Park, Y.; Hong, J. Org. Lett. 2007, 9, 3965; Kim, H.;
  • 27a was reacted (d) with H2, Pd/C, EtOAc/EtOH (3:1 ) at 25°C for 2h and then (e) with TBAF and THF at 25°C for 1 h to make 29 with 88% yield.
  • 29 was reacted (f) with BEMP and CHCI 2 at 25°C for 18 h with 66% yield and then reacted (g) with (polystyrylmethyl)trimethylammonium borohydride and MeOH at 25°C for 48 h to yield 31 with 75% yield.
  • 28b was reacted (d) with H 2 , Pd/C, EtOAc/EtOH (3:1 ) at 25°C for 2h and then (e) with TBAF and THF at 25°C for 1 h to make 32 with 88% yield.
  • 32 was reacted (h) with BEMP and CHCI 2 at 25 0 C for 20 h with 92% yield and then (i) with (polystyrylmethyl)trimethylammonium borohydride and MeOH at 25°C for 30 h to make 33 with 86% yield.
  • HIF-1 inhibition To determine HIF-1 inhibitory activity of 31 and 33, a luciferase-reporter based assay was used as a primary screen.
  • 4T1-ODD-Luc cells Li, F.; Sonveaux, P.; Rabbani, Z. N.; Liu, S.; Yan, B.; Huang, Q.; Vujaskovic, Z.; Dewhirst, M. W.; Li, C. Y. MoI. Cell 2007, 26, 63, incorporated by reference
  • ODD oxygen-dependent-degradation
  • This ODD-Luc reporter contained a CMV promoter, which is constitutively active. Since its ODD domain is identical to that of HIF-1 , it enabled the direct detection of the stability of HIF-1.
  • Cells were seeded in the 24-well plate at a density of 10 5 cells/well. After 16-h incubation, cells were treated with 240 ⁇ M of C0CI2 and serially diluted compounds for 24 h. Since luciferase requires O2 for its activity but the ODD-Luc is highly sensitive to reoxygenation, HIF-1 expression was induced by CoCI 2 , not by hypoxia, to accurately determine the effect of the compounds on HIF-1 stability. Luciferase signals were detected and quantified as relative light units (RLUs).
  • RLUs relative light units
  • Example 13 Optimized conformations: To further characterize the effect of tetrahydrofuran conformation of manaimpulsin A, 31 , and 33 on the HIF-1 inhibition, conformations of truncated structures (to avoid any unnecessary complications and/or exaggeration by the flexible side chains, truncated structures of 1 , 31, and 33 were used instead of the full structures.
  • Initial geometries were determined by conformational search based on molecular mechanics (MMFF)) were optimized using density functional theory (B3LYP)(Lee, C; Yang, W.; Parr, R. G. Phys. Rev. B 1988, 37, 785; Vosko, S.; WiIk, L.; Nusair, M. Can. J. Phys.
  • Figure 20 shows the optimized conformations of truncated structures of manaimpulsin A, 31 , and 33, wherein Figure 20a are the structures, Figure 20b is Overlay I, Figure 20c is Overlay Il front view, and Figure 2Od is Overlay Il side view.
  • compound 34 adopted a nearly linear conformation, but compound 35 adopted a bent-shaped conformation remarkably different from that of 34.
  • the conformation was relatively close to that of 34 ( Figure 20c and 2Od), indicating that the linear-shaped conformation resulting from the 2,5- transconfiguration may be important to the HIF- 1 inhibition.
  • designing a ligand that mimics the overall conformation of mana repetin A may improve the potency and selectivity toward the hypoxia signaling pathway.
  • Jurkat T cells were cultured in RPMI-1640 with 10% fetal bovine serum (FBS), 2 mM l-glutamine, and 1 mM Na-pyruvate. After serum starvation for 7 h, Jurkat cells were plated in 96 well culture plates at 1.25 * 105 cells/mL in RPMI- 1640 (without phenol red) with 10% FBS. Cells were treated with phorbol 12-myristate 13-acetate (PMA, 1.25 ng/mL) and phytohemaglutinin (PHA, 0.25 ⁇ g/mL) to stimulate secretion of IL-2. Testing compound or DMSO vehicle was added to appropriate wells.
  • FBS fetal bovine serum
  • PMA phorbol 12-myristate 13-acetate
  • PHA phytohemaglutinin
  • the testing compound was either manaimpulsin A (1 ), compound 22, or Psora4.
  • Psora4 was used as a positive control, i.e., it inhibited release of IL-2. Plates were then incubated for 18 h. Cell supernatants were collected (100 ⁇ l_) and assayed for interleukin-2 (IL-2) by ELISA (Human IL-2 ELISA Kit, available from BD Biosciences, San Jose, CA). All test conditions were assayed in triplicate and verified with repeated experiments three times. As shown in Figure 21 , manassantin A (1 ) and 22 both inhibited the release of interleukin-2. In Figure 21 , SU2-SU6 are test compounds not relevant to the invention.
  • the oxygen atom in the tetrahydrofuran core may play an important role not only as a determinant for the conformation of the THF core, but also a hydrogen bond acceptor.
  • the synthesis of pyrrolidine analogue 86 of manassantin A is achieved as with the synthesis of 1.
  • lactam 86 is synthesized from the known anf/-adol adduct 78 (Evans, D. A.; Downey, C. W.; Shaw, J. T.; Tedrow, J. T. Org. Lett. 2002, 4, 1 127- 1130, incorporated by reference).
  • the repulsive 1 ,2-interactions of C3 and C4 methyl groups with the C2 and C5 substituents may affect the conformation of the tetrahydrofuran core.
  • the mono- methyl analogue 93 and the desmethyl analogue 102 are prepared as described in Figures 26 and 27.
  • the mono-methyl tetrahydrofuran 90 is coupled to 63 followed by polymer-supported BH 4 reduction to complete the synthesis of 93 ( Figure 26).
  • the 2-benzenesulfonyl ether 98 is prepared from the acetate-aldol adduct 95
  • Shorter side chains may decrease or abolish the HIF-1 inhibitory activity of manaimpulsins, and an extended side chain analogue 110 is prepared as described in
  • keto analogue 111 is a synthetic intermediate to manassantin A (1 ). Treatment of 1 with NaH and MeI provides 112, as shown in Figure 29.
  • De-oxo analogues 117 and 119 are prepared by coupling of 116 and 118, respectively ( Figure 31 ). Since the tosylates 116 and 118 are less reactive than 63, the coupling reaction may require harsher conditions (e.g. higher reaction temperature).
  • mice Female Fisher-344 rats, C57/BI6 mice, and Balb/C mice are housed and treated in accordance with approved guidelines from the Duke University Institutional
  • Skinfold window chambers Mice are anesthetized with sodium pentobarbitol (80 mg/kg, Lp.), and a 1-cm diameter circular incision is made in the dorsal skin flap, over which a titanium chamber is surgically implanted. A 10- ⁇ L suspension of tumor cells (5 * 10 3 cells) is then injected into the opposing flap of skin. A circular cover slip is placed over the incision, through which the vasculature, tissue, and tumor cells are visualized. Observations of window chamber tumors are performed daily on restrained, unanaesthetized mice with an inverted Zeiss fluorescence microscope (Carl Zeiss, Jena, Germany).
  • Tumor volume and vascular length densities are calculated as follows. Briefly, tumor volumes are calculated with the formula: d 2 (3.14/2), where the diameter is determined from low- power (2.5*) microscopy images by comparing pixel dimensions with micrometer values. Vascular length densities are measured from medium-power (5*) fields by using image analysis software (Scion Image) to trace the vascular network. Measurement of the sum length of all vessels within each tumor is then determined (in pixels) and converted to metric length by comparing pixel dimensions with micrometer values.
  • PBS sterile phosphate buffered saline
  • the Dose Enhancing Factor is a ratio of the enhancement of cell growth inhibition elicited by the test compound in the presence of bleomycin compared to bleomycin alone.
  • the test compounds (manaimpulsin compounds and analogues) are used at a fixed concentration of 25 ⁇ M.
  • Bleomycin is used at a concentration of 0.5 ⁇ g/mL.
  • the DEF is calculated from the formula:
  • Growthcontroi is cell growth of control cells; Growth b i eo is cell growth in presence of bleomycin; and
  • Growth(bieo+ ⁇ c) is cell growth in presence of bleomycin and the test compound.
  • SRB sulforhodamine B
  • 2,000 HeLa cells are seeded into each well of a flat-bottomed 96-well microtiter plate in a volume of 100 ⁇ l_ and incubated for 6 hours at 37 0 C. Cells are either replaced with media alone or with media containing the test compound at a final concentration of 25 ⁇ M. Cells are allowed to grow for a further 1 hour before the addition of bleomycin to either untreated cells or test compound treated cells. Cells untreated with either bleomycin or test compound are used as a control. Cells treated with test compound alone are used to assess the growth inhibition by the test compound.
  • SRB sulforhodamine B
  • Cells are left for a further 16 hours before replacing the media and allowing the cells to grow for a further 72 hours at 37°C.
  • the media is then removed and the cells fixed with 100 ⁇ l_ of ice cold 10% (w/v) trichloracetic acid.
  • the plates are incubated at 4°C for 20 minutes and then are washed four times with water.
  • Each well of cells is then stained with 100 ⁇ l_ of 0.4% (w/v) SRB in 1 % acetic acid for 20 minutes before washing four times with 1 % acetic acid. Plates are then dried for 2 hours at room temperature.
  • the dye from the stained cells is solubilized by the addition of 100 ⁇ l_ of 10 mM Tris Base into each well. Plates are gently shaken and left at room temperature for 30 minutes before measuring the optical density at 564 nM on a Microquant microtiter plate reader.

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Abstract

L'invention porte sur des composés de manassantin et sur des procédés d'utilisation des composés. L'invention porte sur des procédés de traitement d'une maladie, le procédé comprenant l'administration d'un composé selon la Formule I. L'invention porte en outre sur des compositions pharmaceutiques comprenant des composés selon la Formule I. L'invention porte également sur des procédés consistant à inhiber HIF-1 dans une cellule, les procédés comprenant l'administration à la cellule d'un composé selon la Formule I.
PCT/US2009/065194 2008-11-19 2009-11-19 Composés de manassantin et leurs procédés de production et d'utilisation WO2010059858A1 (fr)

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WO2012163264A1 (fr) * 2011-05-27 2012-12-06 中国医学科学院药物研究所 Substance de saucernétine de structure simplifiée et son procédé de préparation et sa composition pharmaceutique et son utilisation
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