WO2018068757A1 - Agents cycloheptapeptidiques pour le traitement du cancer et des maladies de l'obésité - Google Patents

Agents cycloheptapeptidiques pour le traitement du cancer et des maladies de l'obésité Download PDF

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WO2018068757A1
WO2018068757A1 PCT/CN2017/106025 CN2017106025W WO2018068757A1 WO 2018068757 A1 WO2018068757 A1 WO 2018068757A1 CN 2017106025 W CN2017106025 W CN 2017106025W WO 2018068757 A1 WO2018068757 A1 WO 2018068757A1
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compound
independently selected
cancer
halogen
hydrocarbyl
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PCT/CN2017/106025
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Hongjie Zhang
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Hong Kong Baptist University
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Priority claimed from US15/293,516 external-priority patent/US9963485B2/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/64—Cyclic peptides containing only normal peptide links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D259/00—Heterocyclic compounds containing rings having more than four nitrogen atoms as the only ring hetero atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/50—Cyclic peptides containing at least one abnormal peptide link
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention is in the field of pharmaceuticals and chemical industries.
  • this invention relates to new anticancer and anti-obesity agents based on the cyclic peptide compounds.
  • the invention also includes its preparation and application method for treating cancer and obesity diseases.
  • the present invention relates to anticancer and anti-obesity agents based on cyclic peptide compounds. More particularly, the agents are derivatives of cycloheptapeptides. It is a goal of the present invention to provide cyclopeptide compounds having anticancer and anti-obesity activity.
  • cancer More than 10 million people are diagnosed with cancer every year in the world. Cancer has become a leading cause of death. According to the compiled statistics by WHO, cancer claimed the lives of more than 8.2 million people worldwide in 2012 (WHO: http: //www. who. int/mediacentre/factsheets/fs297/en/index. html; retrieved on 18 September, 2016) . Although numerous cancer chemotherapeutics are available today, they often have very narrow therapeutic indices and very severe side effects. In addition, cancers can and often do develop resistance to many of these drugs. The fact that there currently are no drugs available that are capable of curing cancer diseases, the discovery and development of new anticancer drugs are very much needed and the undertaking of such studies is imperative.
  • Obesity the metabolic disease, has become increasingly concerned in modern society. It affects nearly a third population of adults in the developed countries, and more than 1.9 billion adults were overweight in 2014 according to WHO report (http: //www. who. int/mediacentre/factsheets/fs311/en/, retrieved on 18 September, 2016) .
  • Many health problems such as cardiovascular diseases, type 2 diabetes, cancer and osteoarthritis are associated with obesity.
  • Obesity is largely preventable, and in fact, it is considered to be a leading preventable cause of death in the world. However, the number of people with obesity in the world is more than doubled since 1980. Obesity, the once considered a wealthy country problem is now on the rise in low-and middle-income countries.
  • anti-obesity drugs orlistat, lorcaserin hydrochloride and Qsymia TM ) approved by the FDA for long term use.
  • the drugs have side effects associated with high blood pressure, rapid heart eat, palpitations, drug addiction, hallucination, constipation and insomnia. To develop new anti-obesity drugs is thus needed.
  • Cyclopeptides are peptide compounds whose amino and carboxyl termini are linked together by a peptide bond to form a circular chain. Cyclodepsipeptides have at least one lactone linkage in place of one of the amides.
  • a cycloheptapeptide is the cyclopeptide compound containing seven amino acid residues.
  • the present invention relates to cycloheptapeptides.
  • the present invention is based, at least in part, on the discovery that cyclopeptide compounds such as compound MV-A is effective in the treatment of cancer such as colon cancer, breast cancer, prostate cancer, lung cancer, melanoma, leukemia, brain cancer, renal cancer, ovarian cancer, and oral epidermoid cancer as well as obesity diseases.
  • a first aspect of the invention is a cycloheptapeptide compound or a pharmaceutically acceptable salt or prodrug thereof, for use in the treatment, prevention or delay of progression of a cancer or an obesity in a patient.
  • the cycloheptapeptide may be a cyclohepta-depsipeptide compound.
  • a second aspect of the invention is a pharmaceutical formulation comprising a cycloheptapeptide compound, or a pharmaceutically acceptable salt or prodrug thereof, for use in the treatment, prevention or delay of progression of a cancer or an obesity in a patient.
  • the cycloheptapeptide may be a cyclohepta-depsipeptide compound.
  • a third aspect of the invention concerns the use of a combination of one or more cycloheptapeptide (s) based on the formula (I) or the formula (II) with one or more other clinically used anticancer or anti-obesity agent (s) , for use in the treatment, prevention or delay of progression of a cancer or an obesity in a patient.
  • a fourth aspect of the invention concerns the use of an extract or a fraction made from plant material or extraction material fermented from a microorganism containing one or more cycloheptapeptide (s) based on the formula (I) or the formula (II) for use in the treatment, prevention or delay of progression of a cancer or an obesity in a patient.
  • a fifth aspect of the present invention there is provided a method for use in the treatment, prevention or delay of progression of cancer or obesity in a subject in needs thereof by administering an effective dosage of a composition comprising a compound according to formula (II) :
  • composition comprising a compound having formula (I) :
  • a compound wherein said compound is an optically pure stereoisomer; an enantiomer; a racemate; a diastereomer; or a tautomer.
  • a compound is selected from compound MV-A, compound MV-B, compound MV-C or compound MV-D:
  • a method wherein said subject is a human.
  • a method wherein said cancer comprising colon cancer, breast cancer, prostate cancer, lung cancer, melanoma, leukemia, brain cancer, renal cancer, ovarian cancer, and oral epidermoid cancer.
  • the effective dosage is at least 0.0041 mg/kg per patient body weight, preferably about 0.0081 mg/kg per patient body weight, or preferably about 0.0162 mg/kg per patient body weight, or preferably about 0.0324 mg/kg per patient body weight, or preferably about 0.0649 mg/kg per patient body weight, or preferably about 0.0405 mg/kg per patient body weight, or preferably about 0.0811 mg/kg per patient body weight or preferably about 0.162mg/kg per patient body weight.
  • a sixth aspect of the present invention concerns an amide or amine that contains at least one substructure that is formed from the amino acid having the formula (III) .
  • Compounds of the invention may exist in different forms, such as free acids, free bases, enantiomers, racemates, diastereomers, esters and other prodrugs, salts and tautomers, and the disclosure includes all variant forms of these compounds.
  • the extent of protection includes counterfeit or fraudulent products which contain or purport to contain a compound of the invention irrespective of whether they do in fact contain such a compound and irrespective of whether any such compound is contained in a therapeutically effective amount.
  • packages which include a description or instructions which indicate that the package contains a species or pharmaceutical formulation of the invention and a product which is or comprises, or purports to be or comprise, such a formulation or species.
  • packages may be, but are not necessarily, counterfeit or fraudulent.
  • the invention includes all such variation and modifications.
  • the invention also includes all of the steps and features referred to or indicated in the specification, individually or collectively and any and all combinations or any two or more of the steps or features.
  • Patent law e.g., they can mean “includes” , “included” , “including” , and the like; and that terms such as “consisting essentially of” and “consists essentially of” have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention.
  • Figure1 (A) shows 1 H NMR data of 1 (CDCl 3 ) .
  • Figure 1 (B) shows 1 H NMR data of 1 (CDCl 3 ) .
  • Figure 1 (C) shows 1 H NMR data of 1 (CDCl 3 ) .
  • Figure 1 (D) shows 1 H NMR data of 1 (CDCl 3 ) .
  • Figure 1 (E) shows 1 H NMR data of 1 (CDCl 3 ) .
  • Figure 1 (F) shows 1 H NMR data of 1 (CDCl 3 ) .
  • Figure 1 (G) shows 1 H NMR data of 1 (CDCl 3 ) .
  • Figure 1 (H) shows 1 H NMR data of 1 (CDCl 3 ) .
  • Figure 2 (A) shows 13 C NMR data of 1 (CDCl 3 ) .
  • Figure 2 (B) shows 13 C NMR data of 1 (CDCl 3 ) .
  • Figure 2 (C) shows 13 C NMR data of 1 (CDCl 3 ) .
  • Figure 2 (D) shows 13 C NMR data of 1 (CDCl 3 ) .
  • Figure 3 (A) shows DEPT-135 NMR data of 1 (CDCl 3 ) .
  • Figure 3 (B) shows DEPT-135 NMR data of 1 (CDCl 3 ) .
  • Figure 4 (A) shows DEPT-90 NMR data of 1 (CDCl 3 ) .
  • Figure 4 (B) shows DEPT-90 NMR data of 1.
  • Figure 5 (A) shows 1 H NMR data of 2 (CDCl 3 ) .
  • Figure 5 (B) shows 1 H NMR data of 2 (CDCl 3 ) .
  • Figure 5 (C) shows 1 H NMR data of 2 (CDCl 3 ) .
  • Figure 5 (D) shows 1 H NMR data of 2 (CDCl 3 ) .
  • Figure 5 (E) shows 1 H NMR data of 2 (CDCl 3 ) .
  • Figure 5 (F) shows 1 H NMR data of 2 (CDCl 3 ) .
  • Figure 6 (A) shows 13 C NMR data of 2 (CDCl 3 ) .
  • Figure 6 (B) shows 13 C NMR data of 2 (CDCl 3 ) .
  • Figure 6 (C) shows 13 C NMR data of 2 (CDCl 3 ) .
  • Figure 6 (D) shows 13 C NMR data of 2 (CDCl 3 ) .
  • Figure 7 (A) shows DEPT-135 NMR data of 2 (CDCl 3 ) .
  • Figure 7 (B) shows DEPT-135 NMR data of 2 (CDCl 3 ) .
  • Figure 8 (A) shows DEPT-90 NMR data of 2 (CDCl 3 ) .
  • Figure 8 (B) shows DEPT-90 NMR data of 2.
  • Figure 9 (A) shows 1 H NMR data of 3 (CDCl 3 ) .
  • Figure 9 (B) shows 1 H NMR data of 3 (CDCl 3 ) .
  • Figure 9 (C) shows 1 H NMR data of 3 (CDCl 3 ) .
  • Figure 9 (D) shows 1 H NMR data of 3 (CDCl 3 ) .
  • Figure 9 (E) shows 1 H NMR data of 3 (CDCl 3 ) .
  • Figure 9 (F) shows 1 H NMR data of 3 (CDCl 3 ) .
  • Figure 9 (G) shows 1 H NMR data of 3 (CDCl 3 ) .
  • Figure 10 (A) shows 13 C NMR data of 3 (CDCl 3 ) .
  • Figure 10 (B) shows 13 C NMR data of 3 (CDCl 3 ) .
  • Figure 10 (C) shows 13 C NMR data of 3 (CDCl 3 ) .
  • Figure 10 (D) shows 13 C NMR data of 3 (CDCl 3 ) .
  • Figure 11 (A) shows DEPT-135 NMR data of 3 (CDCl 3 ) .
  • Figure 11 (B) shows DEPT-135 NMR data of 3 (CDCl 3 ) .
  • Figure 12 (A) shows DEPT-90 NMR data of 3 (CDCl 3 ) .
  • Figure 12 (B) shows DEPT-90 NMR data of 3 (CDCl 3 ) .
  • Figure 13 shows label of the atoms of the structure of compound 1 for X-ray crystallography.
  • Figure 14 shows the tereochemistry of the structure of compound 1 determined by X-ray crystallography.
  • Figure 15 shows the single-crystal X-ray structures of compound 1
  • Figure 16 shows the chemical structures of Boc 2 O (di-tert-butyl dicarbonate) and HSi-DES-PS (butyl diethylsilane polystyrene) .
  • Figure 17 shows the chemical structures of Boc-protected amino acids
  • Figure 18 shows label of the atoms of the structure of MV-A for X-ray crystallography.
  • Figure 19 shows stereochemistry of the structure of MV-A determined by X-ray crystallography.
  • Figure 20 shows the single-crystal X-ray structures of MV-A.
  • Figure 21 (A) shows inhibition of HCT116-tumor xenograft growth by MV-A. Tumor growth curve.
  • Figure 21 (B) shows inhibition of HCT116-tumor xenograft growth by MV-A.
  • Figure 22 (A) shows inhibition of MCF7-tumor xenograft growth by MV-A. Tumor growth curve.
  • Figure 22 (B) shows inhibition of MCF7-tumor xenograft growth by MV-A.
  • potent anticancer and anti-obesity compounds from a plant.
  • the promising compounds belong to cyclopeptides containing a novel amino acid residue, and they were isolated from the stem barks of Maytenus variabilis (Loes. ) C.Y. Cheng (Celastraceae) .
  • the novel cyclopeptide compounds (1, 2 and 3) demonstrated tumor cell killing activity against a panel of human cancer cell lines with IC 50 values in the range of 0.05-52 nM.
  • Compound 1 also demonstrated to be effective on reducing body weight of mice.
  • the inventor discovered several potent anticancer and anti-obesity compounds (1, 2 and 3) belonging to cycloheptapeptide molecules, which the inventor designated as “mavacyocines” .
  • the inventor has further synthesized several mavacyocine compounds (MV-A, MV-C and MV-D) , and one orientation of compound 2 is determined as MV-B.
  • MV-A mavacyocine compounds
  • MV-A mavacyocine compounds
  • MV-C mavacyocine compounds
  • MV-D mavacyocine compounds
  • MV-B mavacyocine compounds
  • These compounds demonstrated tumor cell killing activity against a panel of human cancer cell lines with IC 50 values in the range of 0.05-52 nM.
  • Compound MV-A also demonstrated to be effective on reducing body weight of mice.
  • the mavacyocines (MV-A, MV-B, MV-C and MV-D) have demonstrated potent biological activities, and warrant further study and development. Thus, mavacyocine compounds with an improved biological activities and low in toxicity are needed.
  • cycloheptapeptide as used herein includes reference to a cyclopeptide compound whose amino and carboxyl termini are linked together by a peptide bond to form a circular chain.
  • a cycloheptapeptide comprises the basic structure shown as below:
  • cyclohepta-depsipeptide as used herein includes reference to a cyclodepsipeptide compound, which has at least one lactone linkage in place of one of the amides.
  • a cyclohepta-depsipeptide comprises the basic structure shown as below:
  • Y 1 , Y 2 , Y 3 , Y 4 , Y 5 and Y 6 can be oxygen, sulfur or nitrogen substituted with an L group (L can be L 1 , L 2 , L 3 , L 4 , L 5 or L 6 ) .
  • T can be a hydrocarbyl or an alkoxy. At least one of the Y 1 , Y 2 , Y 3 , Y 4 , Y 5 and Y 6 is nitrogen.
  • mavacyocine as used herein includes reference to a compound comprising the basic structure shown as below:
  • the carbon and nitrogen numbering of a mavacyocine molecule as used herein includes reference to a compound comprising numbering system shown as below:
  • hydrocarbyl as used herein includes reference to a moiety consisting exclusively of hydrogen and carbon atoms; such a moiety may comprise an aliphatic and/or an aromatic moiety. The moiety may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms.
  • hydrocarbyl groups include C 1-6 alkyl (e.g. C 1 , C 2 , C 3 or C 4 alkyl, for example methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl or tert-butyl) ; C 1-6 alkyl substituted by aryl (e.g.
  • benzyl or by cycloalkyl (e.g. cyclopropylmethyl) ; cycloalkyl (e.g. cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl) ; aryl (e.g. phenyl, naphthyl or fluorenyl) and the like.
  • cycloalkyl e.g. cyclopropylmethyl
  • cycloalkyl e.g. cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl
  • aryl e.g. phenyl, naphthyl or fluorenyl
  • alkyl and C 1-6 alkyl as used herein include reference to a straight or branched chain alkyl moiety having 1, 2, 3, 4, 5 or 6 carbon atoms. This term includes reference to groups such as methyl, ethyl, propyl (n-propyl or isopropyl) , butyl (n-butyl, sec-butyl or tert-butyl) , pentyl, hexyl and the like.
  • the alkyl moiety may have 1, 2, 3 or 4 carbon atoms.
  • alkenyl and C 2-6 alkenyl as used herein include reference to a straight or branched chain alkyl moiety having 2, 3, 4, 5 or 6 carbon atoms and having, in addition, at least one double bond, of either E or Z stereochemistry where applicable. This term includes reference to groups such as ethenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 1-hexenyl, 2-hexenyl and 3-hexenyl and the like.
  • alkynyl and C 2-6 alkynyl as used herein include reference to a straight or branched chain alkyl moiety having 2, 3, 4, 5 or 6 carbon atoms and having, in addition, at least one triple bond. This term includes reference to groups such as ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 1-hexynyl, 2-hexynyl and 3-hexynyl and the like.
  • alkoxy and C 1-6 alkoxy as used herein include reference to -O-alkyl, wherein alkyl is straight or branched chain and comprises 1, 2, 3, 4, 5 or 6 carbon atoms. In one class of embodiments, alkoxy has 1, 2, 3 or 4 carbon atoms. This term includes reference to groups such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, tert-butoxy, pentoxy, hexoxy and the like.
  • cycloalkyl as used herein includes reference to an alicyclic moiety having 3, 4, 5, 6, 7 or 8 carbon atoms.
  • the group may be a bridged or polycyclic ring system. More often cycloalkyl groups are monocyclic. This term includes reference to groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbomyl, bicyclo [2.2.2] octyl and the like.
  • aryl as used herein includes reference to an aromatic ring system comprising 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 ring carbon atoms.
  • the ring or ring system may be substituted with one or more hydrocarbyl groups.
  • Aryl is often phenyl but may be a polycyclic ring system, having two or more rings, at least one of which is aromatic. This term includes reference to groups such as phenyl, naphthyl, fluorenyl, azulenyl, indenyl, anthryl and the like.
  • Cyclic group means a ring or ring system, which may be unsaturated or partially unsaturated but is usually saturated, typically containing 5 to 13 ring-forming atoms, for example a 3, 4-, 5-or 6-membered ring.
  • the ring or ring system may be substituted with one or more hydrocarbyl groups. Cyclic group includes carbocyclyl and heterocyclyl moieties.
  • carbocyclyl as used herein includes reference to a saturated (e.g. cycloalkyl) or unsaturated (e.g. aryl) ring moiety having 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15 or 16 carbon ring atoms.
  • carbocyclyl includes a 3-to 10-membered ring or ring system and, in particular, 3-, 4-, 5-or 6-membered rings, which may be saturated or unsaturated.
  • the ring or ring system may be substituted with one or more hydrocarbyl groups.
  • a carbocyclic moiety is, for example, selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl, bicyclo [2.2.2] octyl, phenyl, naphthyl, fluorenyl, azulenyl, indenyl, anthryl and the like.
  • heterocyclyl as used herein includes reference to a saturated (e.g. heterocycloalkyl) or unsaturated (e.g. heteroaryl) heterocyclic ring moiety having from 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 ring atoms, at least one of which is selected from nitrogen, oxygen, phosphorus, silicon and sulphur.
  • heterocyclyl includes a 3-to 10-membered ring or ring system and more particularly a 5-or 6-membered ring, which may be saturated or unsaturated.
  • the ring or ring system may be substituted with one or more hydrocarbyl groups.
  • a heterocyclic moiety is, for example, selected from oxiranyl, azirinyl, 1, 2-oxathiolanyl, imidazolyl, thienyl, furyl, tetrahydrofuryl, pyranyl, thiopyranyl, thianthrenyl, isobenzofuranyl, benzofuranyl, chromenyl, 2H-pyrrolyl, pyrrolyl, pyrrolinyl, pyrrolidinyl, pyrrolizidinyl, imidazolyl, imidazolidinyl, benzimidazolyl, pyrazolyl, pyrazinyl, pyrazolidinyl, thiazolyl, isothiazolyl, dithiazolyl, oxazolyl, isoxazolyl, pyridyl, pyrazinyl, pyrimidinyl, piperidyl, piperazinyl, pyridazinyl, morph
  • heterocycloalkyl as used herein includes reference to a saturated heterocyclic moiety having 3, 4, 5, 6 or 7 ring carbon atoms and 1, 2, 3, 4 or 5 ring heteroatoms selected from nitrogen, oxygen, phosphorus and sulphur.
  • the group may be a polycyclic ring system but more often is monocyclic.
  • This term includes reference to groups such as azetidinyl, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, oxiranyl, pyrazolidinyl, imidazolyl, indolizidinyl, piperazinyl, thiazolidinyl, morpholinyl, thiomorpholinyl, quinolizidinyl and the like.
  • the ring or ring system may be substituted with one or more hydrocarbyl groups.
  • heteroaryl as used herein includes reference to an aromatic heterocyclic ring system having 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 ring atoms, at least one of which is selected from nitrogen, oxygen and sulphur.
  • the group may be a polycyclic ring system, having two or more rings, at least one of which is aromatic, but is more often monocyclic.
  • the ring or ring system may be substituted with one or more hydrocarbyl groups.
  • This term includes reference to groups such as pyrimidinyl, furanyl, benzo [b] thiophenyl, thiophenyl, pyrrolyl, imidazolyl, pyrrolidinyl, pyridinyl, benzo [b] furanyl, pyrazinyl, purinyl, indolyl, benzimidazolyl, quinolinyl, phenothiazinyl, triazinyl, phthalazinyl, 2H-chromenyl, oxazolyl, isoxazolyl, thiazolyl, isoindolyl, indazolyl, purinyl, isoquinolinyl, quinazolinyl, pteridinyl and the like.
  • amino acid as used herein includes reference to a compound comprising the basic structure shown as below:
  • R is selected from R 1 , -OR 1 , -C (O) R 1 and -C (O) OR 1 ;
  • R 1 is independently selected from hydrogen, halogen, trifluoromethyl, cyano, nitro, hydrocarbyl optionally substituted with 1, 2, 3, 4 or 5 R 2 , - (CH 2 ) k -heterocyclyl optionally substituted with 1, 2, 3, 4 or 5 R 2 , -OR 3 , -C (O) R 4 , -C (O) N (R 3 ) R 4 , -C (O) OR 3 , -OC (O) R 3 , -S (O) 2 R 3 , -S (O) 2 N (R 3 ) R 4 , -N (R 3 ) R 4 , -N (R 3 ) N (R 3 ) R 4 , -N (R 3 ) C (O) R 4 and -N (R 3 ) S (O) 2 R 4 ;
  • R 3 and R 4 are each independently hydrogen or selected from hydrocarbyl and - (CH 2 ) k -heterocyclyl, either of which is optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from halogen, cyano, amino, hydroxy, C 1-6 alkyl and C 1-6 alkoxy; wherein k is an integer between 1 and 6 (e.g. 1, 2 or 3) ;
  • the nitrogen atom of the amino acids may be alkylated to form N-alkylated amino acids.
  • the amino acids can be L-amino acids, or D-enantiomers of all of the above.
  • An amino acid is, for example, selected from twenty genetically encoded L-amino acids (Table 1) , common non-encoded amino acids (Table 1) , and the like. It also includes ⁇ -amino-cyclic-acetic acids (see below the term “ ⁇ -Amino-cyclic-acetic acid” .
  • ⁇ -amino-cyclic-acetic acid as used herein includes reference to a compound comprising the basic structure shown as below:
  • R is selected from cyclic group and - (CH 2 ) k -cyclic group, either of which is optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from R 1 , -OR 2 , -C (O) R 3 , -C (O) N (R 2 ) R 3 , -C (O) OR 2 , -OC (O) R 2 , -S (O) 2 R 2 , -S (O) 2 N (R 2 ) R 3 , -N (R 2 ) R 3 , -N (R 2 ) N (R 2 ) R 3 , -N (R 2 ) C (O) R 3 and -N (R 2 ) S (O) 2 R 3 ;
  • R 2 and R 3 are each independently hydrogen or selected from hydrocarbyl and - (CH 2 ) k -heterocyclyl, either of which is optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from halogen, cyano, amino, hydroxy, C 1-6 alkyl and C 1-6 alkoxy; wherein k is an integer between 1 and 6 (e.g. 1, 2 or 3) .
  • amide as used herein includes reference to a compound comprising the basic structure shown as below:
  • X 1 is selected from R 1 , -OR 1 , -C (O) R 1 and -C (O) OR 1 ;
  • X 2 and X 3 are each independently selected from R 1 , -C (O) R 1 and -C (O) OR 1 ;
  • R 1 is independently selected from hydrogen, halogen, trifluoromethyl, cyano, nitro, hydrocarbyl optionally substituted with 1, 2, 3, 4 or 5 R 2 , - (CH 2 ) k -heterocyclyl optionally substituted with 1, 2, 3, 4 or 5 R 2 , -OR 3 , -C (O) R 4 , -C (O) N (R 3 ) R 4 , -C (O) OR 3 , -OC (O) R 3 , -S (O) 2 R 3 , -S (O) 2 N (R 3 ) R 4 , -N (R 3 ) R 4 , -N (R 3 ) N (R 3 ) R 4 , -N (R 3 ) C (O) R 4 and -N (R 3 ) S (O) 2 R 4 ;
  • R 3 and R 4 are each independently hydrogen or selected from hydrocarbyl and - (CH 2 ) k -heterocyclyl, either of which is optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from halogen, cyano, amino, hydroxy, C 1-6 alkyl and C 1-6 alkoxy; wherein k is an integer between 1 and 6 (e.g. 1, 2 or 3) .
  • amine as used herein includes reference to a compound comprising the basic structure shown as below:
  • X 1 and X 2 are each independently selected from R 1 , -C (O) R 1 and -C (O) OR 1 ;
  • R is independently selected from hydrocarbyl optionally substituted with 1, 2, 3, 4 or 5 R 2 , - (CH 2 ) k -heterocyclyl optionally substituted with 1, 2, 3, 4 or 5 R 2 , -OR 3 , -C (O) R 4 , -C (O) N (R 3 ) R 4 , -C (O) OR 3 , -OC (O) R 3 , -S (O) 2 R 3 , -S (O) 2 N (R 3 ) R 4 , -N (R 3 ) R 4 , -N (R 3 ) N (R 3 ) R 4 , -N (R 3 ) C (O) R 4 and -N (R 3 ) S (O) 2 R 4 ;
  • R 1 is independently selected from hydrogen, halogen, trifluoromethyl, cyano, nitro, hydrocarbyl optionally substituted with 1, 2, 3, 4 or 5 R 2 , - (CH 2 ) k -heterocyclyl optionally substituted with 1, 2, 3, 4 or 5 R 2 , -OR 3 , -C (O) R 4 , -C (O) N (R 3 ) R 4 , -C (O) OR 3 , -OC (O) R 3 , -S (O) 2 R 3 , -S (O) 2 N (R 3 ) R 4 , -N (R 3 ) R 4 , -N (R 3 ) N (R 3 ) R 4 , -N (R 3 ) C (O) R 4 and -N (R 3 ) S (O) 2 R 4 ;
  • R 3 and R 4 are each independently hydrogen or selected from hydrocarbyl and - (CH 2 ) k -heterocyclyl, either of which is optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from halogen, cyano, amino, hydroxy, C 1-6 alkyl and C 1-6 alkoxy; wherein k is an integer between 1 and 6 (e.g. 1, 2 or 3) .
  • peptide bond as used herein includes reference to a covalent chemical bond formed between two amino acids when the carboxylic acid group of one molecule reacts with the amino group of the other molecule.
  • halogen as used herein includes reference to F, Cl, Br or I.
  • halogen containing moiety includes reference to a moiety comprising 1 to 30 plural valence atoms selected from carbon, nitrogen, oxygen and sulphur which moiety includes at least one halogen.
  • the moiety may be hydrocarbyl for example C 1-6 alkyl or C 1-6 alkoxy, or carbocyclyl for example aryl.
  • enantiomer as used herein means one of two stereoisomers that have mirror images of one another.
  • racemate as used herein means a mixture of equal amounts of enantiomers of a chiral molecule.
  • diastereomer as used herein means one of a class of stereoisomers that are not enantiomers, but that have different configurations at one or more of the equivalent chiral centers.
  • Example of diasteromers are epimers that differ in configuration of only one chiral center.
  • stereoisomer as used herein means one of a class of isomeric molecules that have the same molecular formula and sequence of bonded atoms, but different three-dimensional orientations of their atoms in space.
  • tautomer means isomeric molecules that readily interconvert by a chemical reaction. The reaction commonly results in the migration of a hydrogen atom, which results in a switch of a single bond and adjacent double bond.
  • a prodrug is a medication that is administered as an inactive (or less than fully active) chemical derivative that is subsequently converted to an active pharmacological agent in the body, often through normal metabolic processes.
  • substituted as used herein in reference to a moiety means that one or more, especially up to 5, more especially 1, 2 or 3, of the hydrogen atoms in said moiety are replaced independently of each other by the corresponding number of the described substituents.
  • optionally substituted as used herein means substituted or un-substituted. It will, of course, be understood that substituents are only at positions where they are chemically possible, the person skilled in the art being able to decide (either experimentally or theoretically) without inappropriate effort whether a particular substitution is possible.
  • the invention involves the use of cycloheptapeptide and cyclohepta-depsipeptide compounds including derivatives of 1, 2 or 3.
  • the compounds are cycloheptapeptide and cyclohepta-depsipeptide compounds of which at least one of the peptide bonds is resulted from the coupling of the carbolic acid group of an amino acid and the amino group of an ⁇ -amino-cyclic-acetic acid.
  • cycloheptapeptide and cyclohepta-depsipeptide compounds of which the nitrogen atom of at least one of the peptide bonds may be alkylated.
  • the invention provides compounds of the formulae (I) and (II) :
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 and R 14 are each independently hydrogen, halogen or a moiety comprising 1 to 30 plural valence atoms selected from carbon, nitrogen, oxygen and sulphur;
  • At least one of R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 and R 14 is a cyclic group;
  • R 15 is independently selected from hydrogen, halogen, trifluoromethyl, cyano, nitro, hydrocarbyl optionally substituted with 1, 2, 3, 4 or 5 R 16 , - (CH 2 ) k -heterocyclyl optionally substituted with 1, 2, 3, 4 or 5 R 16 , -OR 17 , -C (O) R 18 , -C (O) N (R 17 ) R 18 , -C (O) OR 17 , -OC (O) R 17 , -S (O) 2 R 17 , -S (O) 2 N (R 17 ) R 18 , -N (R 17 ) R 18 , -N (R 17 ) N (R 17 ) R 18 , -N (R 17 ) C (O) R 18 and -N (R 17 ) S (O) 2 R 18 ;
  • R 17 and R 18 are each independently hydrogen or selected from hydrocarbyl and - (CH 2 ) k -heterocyclyl, either of which is optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from halogen, cyano, amino, hydroxy, C 1-6 alkyl and C 1-6 alkoxy; wherein k is an integer between 1 and 6 (e.g. 1, 2 or 3) ;
  • L 1 , L 2 , L 3 , L 4 , L 5 , L 6 and L 7 are each independently selected from R 15 , -C (O) R 15 and -C (O) OR 15 ;
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 R 14 , L 1 , L 2 , L 3 , L 4 , L 5 , L 6 and L 7 may be taken together with the carbon atoms and the nitrogen atoms to which they are attached to form one or more cyclic groups which is optionally substituted with halogen or a moiety comprising 1 to 30 plural valence atoms selected from carbon, nitrogen, oxygen and sulphur;
  • Z is selected from oxygen, nitrogen, hydrocarbyl, or alkoxy
  • Y 1 , Y 2 , Y 3 , Y 4 , Y 5 and Y 6 are each independently selected from oxygen, sulfur, nitrogen with substitution of an L 1 , L 2 , L 3 , L 4 , L 5 or L 6 group, C 1-6 alkyl or C 1-6 alkoxy. At least one of Y 1 , Y 2 , Y 3 , Y 4 , Y 5 and Y 6 is nitrogen with substitution of an L 1 , L 2 , L 3 , L 4 , L 5 or L 6 group when Z is oxygen, hydrocarbyl or alkoxy.
  • each compound may be in the form of the free compound, an enantiomer, an acid or base addition salt, or a prodrug.
  • the dry stem barks (3.0 kg) from the decaying wood of Maytenus variabilis (Loes. ) C.Y. Cheng (Celastraceae) were collected through purchase from Guizhou, China.
  • a primary screening showed that the methanol extract made from a small of amount of the plant materials (5 g) exhibited complete inhibition activity against HCT116 cancer cells at a concentration of 0.625 ⁇ g/mL. All the collected barks are thus submitted to phytochemical study in order to identify anticancer compounds.
  • three novel cyclic peptides (1, 2 and 3) are isolated from the methanol extract of this plant.
  • the cyclopeptides 1 and 2 demonstrate potent activity against a panel of cancer cell lines (Tables 2 and 3) .
  • Compound 1 displays more potent cell killing activity than those of paclitaxel and vinblastine. It also shows comparative activity to the anticancer compound maytansine. Furthermore, compound 1 is able to inhibit cancer cells by more than 90 %at much lower concentration than those of maytansine, paclitaxel and vinblastine (Table 3) .
  • the structures of compounds 1, 2 and 3 are containing peptide bond (s) made from a novel amino acid.
  • the novel amino acid is identified as ⁇ -amino-2, 3-dimethyl-cyclopropaneacetic acid (DMCPA) , which has not been reported in the literature.
  • DMCPA 3-dimethyl-cyclopropaneacetic acid
  • the inventor believes that the novel amino acid forms key substructural component (s) responsible for the cancer cell killing activity of the isolated cyclopeptides.
  • the inventor has evaluated the effects of compounds 1 and 2 on murine P388 leukemia cells, which show that 1 and 2 are able to inhibit cell growth of murine P388 leukemia cells by 100 %at a concentration of 10 ng/mL.
  • the result demonstrates that the cyclopeptides having the DMCPA residue (i.e. 1 and 2) possess much more potent biologically activity than ternatin.
  • the activity is affected by the presence of an adjacent functional group that may have interference with the DMCPA residue.
  • compound 2 has comparative activity to paclitaxel, it shows much lower activity than 1.
  • the only structural difference between the two compounds is that the leucine residue near the DMCPA residue in 1 is substituted by a hydroxy group in 2. This hydroxy substitute might have greatly disrupted the binding interaction between the DMCPA residue and the DMCPA targeted protein.
  • mice In an animal study, the inventor demonstrate that the body weights of mice are significantly decreased after drug treatment of 1.
  • the average weight of mice (10 mice) drops 1.5 g and 2.2 g after the first treatment (i.p. injection) with 1 at a dose of 1 mg/kg and 2 mg/kg, respectively, but the weight of the mice is kept relatively stable after the first treatment.
  • the average weight of mice drops 1.7 g and 4.7 g at 1 mg/kg and 2 mg/kg, respectively. No mice die during the six treatments, and the average weight of mice gains back after the treatment stopped, which showed that the effects of compound 1 on the mice are reversible. No sign of toxicity is observed from dissection of the mice.
  • the inventor has discovered potent anticancer compounds from a plant, and further showed that one of the compounds is able to inhibit fat-accumulation in mice.
  • the present invention provides a synthesis compound with potent anticancer and anti-obesity activity having a formula (IV) :
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 and R 18 are each independently selected from hydrogen, halogen and a moiety comprising 1 to 30 plural valence atoms selected from carbon, nitrogen, oxygen and sulphur;
  • R 1 and R 2 , R 3 and R 4 , R 5 and R 6 , R 7 and R 8 , R 9 and R 10 , R 11 and R 12 , R 13 and R 14 or R 15 and R 16 is hydrogen, halogen, hydrocarbyl or alkoxy
  • R 19 is independently selected from hydrogen, halogen, trifluoromethyl, cyano, nitro, hydrocarbyl optionally substituted with 1, 2, 3, 4 or 5 R 20 , heterocyclyl optionally substituted with 1, 2, 3, 4 or 5 R 20 , - (CH 2 ) k -heterocyclyl optionally substituted with 1, 2, 3, 4 or 5 R 20 , -OR 21 , -C (O) R 22 , -C (O) N (R 21 ) R 22 , -C (O) OR 21 , -OC (O) R 21 , -S (O) 2 R 21 , -S (O) 2 N (R 21 ) R 22 , -N (R 21 ) R 22 , -N (R 21 ) N (R 21 ) R 22 , -N (R 21 ) C (O) R 22 and -N (R 21 ) S (O) 2 R 22 ;
  • R 21 and R 22 are each independently hydrogen or selected from hydrocarbyl, heterocyclyl and - (CH 2 ) k -heterocyclyl; each of the hydrocarbyl, heterocyclyl and - (CH 2 ) k -heterocyclyl is optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from halogen, cyano, amino, hydroxy, C 1-6 alkyl and C 1-6 alkoxy; wherein k is an integer between 1 and 6;
  • L 1 , L 2 , L 3 , L 4 , L 5 , L 6 and L 7 are each independently selected from R 19 , -C (O) R 19 and -C (O) OR 19 ;
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , L 1 , L 2 , L 3 , L 4 , L 5 , L 6 and L 7 forms one or more cyclic groups with a carbon atom and a nitrogen atom to which they are attached to, the one or more cyclic groups is optionally substituted with halogen or a moiety comprising 1 to 30 plural valence atoms selected from carbon, nitrogen, oxygen and sulphur;
  • Y 1 , Y 2 , Y 3 , Y 4 , Y 5 , Y 6 and Y 7 are each independently selected from oxygen, sulfur, nitrogen with substitution of an L 1 , L 2 , L 3 , L 4 , L 5 , L 6 or L 7 group, C 1-6 alkoxy, or C 1-6 alkyl optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from halogen, cyano, amino, hydroxy, C 1-6 alkyl and C 1-6 alkoxy; at least one of Y 1 , Y 2 , Y 3 , Y 4 , Y 5 , Y 6 and Y 7 is nitrogen with substitution of an L 1 , L 2 , L 3 , L 4 , L 5 , L 6 or L 7 group;
  • the present invention further provides four embodiments of compounds MV-A, MV-B, MV-C and MV-D with potent anticancer and anti-obesity activity and synthesis further to the compound of formula (V) .
  • the compounds of the present invention are evaluated for their anticancer and anti-obesity activity, namely compounds MV-A, MV-B, MV-C and MV-D, wherein MV-A is one orientation of compound 1; MV-B is one orientation of compound 2; and MV-C is one orientation of compound 3.
  • the chemical formulae of the compound of the present invention are as follows:
  • the amino acid resin is prepared based on the methods depicted in SCHEME 1, and the general experimental procedures are described as following.
  • NMP N-methylpyrrolidinone
  • HSi-DES-PS 1 g, 1.45 mmol/g
  • KOAc 300 mg, 0.3 mmol
  • the reaction mixture is deaerated by bubbling by passing a slow stream of argon through it for 15 min.
  • Pd 2 (dba) 3 ⁇ CHCl 3 M.W.
  • the resin After being washed with DMF, 0.1 N HCl/THF, MeOH, and DMF, the resin is cycled through the same set of conditions for deprotection, washing, coupling, and washing as above using Boc-D-Leu-OH (5 eq) , Boc-D-MeAla-OH (5 eq) , Boc-L-MeAla-OH (5 eq) , Boc-L-AA (5 eq) , and Boc-L-MeLeu-OH (5 eq) successively in the peptide elongation.
  • the diprotected linear peptide bound to the resin (8) is shaken with LiOH (5 eq) in THF/H 2 O (7: 1, 20 mL) at room temperature for 12 h.
  • the resin is treated with 50%TFA in CH 2 Cl 2 (20 mL) for 15 min, then washed with CH 2 Cl 2 , 0.1 N HCl/THF, MeOH, and DMF.
  • Cyclization to prepare 9 is carried out by treatment of the resin in NMP (20 mL) with PyBOP (5 eq) , and DIPEA (15 eq) for 24 hours followed by washing with DMF, 0.1 N HCl/THF, MeOH, and CH 2 Cl 2 .
  • the resin is then treated with neat TFA for 24 hours at room temperature to release the cyclic peptide.
  • the cleavage solution is filtered, and the resin is rinsed with CH 2 Cl 2 (20 mL) . Concentration of the combined filtrates gives the crude product, which is filtered through a short silica gel plug with ethyl acetate to afford the desired compound 10.
  • MV-A is obtained as a white powder with a molecular formula of C 39 H 67 N 7 O 7 determined by positive HRESIMS and NMR studies .
  • Crystal data for MV-A (from acetone/n-hexane 3: 1) belong to the monoclinic spacnoclinic space group P2 1 2 1 2.
  • MV-B was obtained from the decaying wood of Maytenus variabilis.
  • the inventor first collected the stem barks of the decayed woods of Maytenus variabilis. Then a chloroform extract and a methanol extract were prepared from the collected stem barks immediately after the collection. These extracts are herein identified as the chloroform extract (MVSB-CLE) and the methanol extract (MVSBMTE) , respectively. These extracts were stored in a -20°C freezer for later analysis and study against the chloroform extract and the methanol extract taken from the stem barks after further treatment. The inventor then processed the remaining part of the collected stem barks was soaked into water for 24 hours at room temperature.
  • the soaked stem barks were then taken out of the water and placed on the floor.
  • the stem barks were kept moistened by spraying with water from time to time.
  • the stems were kept in the wet status at room temperature for 60 days, after which time all the stem barks were rotten.
  • the stem barks were then put in an oven at 40 °C for 3 days.
  • the stem barks were then milled and extracted with methanol to afford an extract, which was defatted with n-hexane and then partitioned with chloroform to afford a chloroform extract. Subsequent separation of the chloroform extract led to the isolation of the inventor’s target compounds (e.g. MV-B) .
  • target compounds e.g. MV-B
  • MV-B is not present in MVSB-CLE and MVSB-MTE, the extractions taken after collection of the stem barks and before further laboratory treatment.
  • MV-B is obtained as a white powder with a molecular formula of C 39 H 67 N 7 O 8 determined by positive HRESIMS and NMR studies .
  • MV-C is obtained as a white powder with a molecular formula of C 38 H 67 N 7 O 7 determined by positive HRESIMS and NMR studies .
  • MV-D is obtained as a white powder with a molecular formula of C 35 H 59 N 7 O 7 determined by positive HRESIMS and NMR studies .
  • MV-A cycloheptapeptide compounds
  • MV-A demonstrates cell killing activity with IC 50 values ranging from 0.05-1.5 nM.
  • MV-B demonstrates cell killing activity with IC 50 values ranging from 0.7-9.1 nM.
  • MV-C demonstrates cell killing activity with IC 50 values ranging from 12-60 nM.
  • MV-D demonstrates no inhibition activity against the cancer cells at 2 ⁇ M.
  • MV-A is evaluated in the murine hollow fiber tumor animal model, which demonstrates MV-A has potent anticancer activity against lung, colon and breast cancers (Table 6) .
  • the experiments reveal that i.p. (intraperitoneal) , i.v. (intravenous) , or oral administration of MV-A at the doses of 0.05 and 0.1 mg/kg of MV-A result in 18.2%and 56.6%inhibition of cell growth of a lung cancer cell line (A549) implanted at the i.p. compartments relative to the control, respectively.
  • MV-A inhibits the growth of a colon cancer cell line (HCT116) by 33.1%, 53.3%and 65.0%at the i.p. sites, respectively.
  • HCT116 colon cancer cell line
  • MV-A inhibits the growth of a breast cancer cell line (MCF7) by 76.8%, 68.3%and 66.7%at the i.p. sites, respectively.
  • MCF7 breast cancer cell line
  • MV-A also inhibits the growth of the breast cancer cell line by 56.0%at the s.c. (subcutaneous) sites.
  • MV-A inhibits the growth of a melanoma cancer cell line (A375) by 35.3%and 67.4%at the i.p. sites, respectively.
  • MV-A inhibits the growth of a cervical cancer cell line (Hela) by 45.8%and 82.7%at the i.p. sites, respectively.
  • MV-A also inhibits the growth of the cervical cancer cell line by 52.8%at the s.c. sites.
  • 0.1 mg/kg per mouse body weight corresponds to 0.0081 mg/kg per patient weight (weight in humans) ; 0.2 mg/kg per mouse body weight corresponds to 0.0162 mg/kg per patient weight (weight in humans) ; 0.4 mg/kg per mouse body weight corresponds to 0.0324 mg/kg per patient weight (weight in humans) ; and 0.8 mg/kg per mouse body weight corresponds to 0.0649 mg/kg per patient weight (weight in humans) .
  • MV-A is further evaluated for its anticancer activity in HCT116 and MCF7 xenograft mouse models.
  • the treatment started on the 6 th day when the HCT116-tumor reached approximately 100 mm 3 (L ⁇ W ⁇ W) .
  • Two doses of MV-A (0.5 and 1.0 mg/kg) , paclitaxel (5 mg/kg) , maytansine (0.1 mg/kg) and vehicle were administered via i.p. (intraperitoneal) , i.v. (intravenous) , or oral, respectively, to the tumor-bearing mice every other day for 21 days (10 mice/group) . All mice died for the maytansine group after the first two treatments.
  • the HCT 116 xenograft experiment showed that MV-A inhibits tumor growth by 31.1 %and 52.8 %at the doses of 0.5 and 1.0 mg/kg, respectively ( Figures 21 (A) –21 (B) ) .
  • a weight loss of 1-2 g is observed for the MV-A groups after the first two treatments, but the weights of the mice are kept relatively stable after the first two treatments. All mice died for the maytansine group after the initial two treatments at the dose of 0.1 mg/kg, indicating the highly toxicity of maytansine.
  • the antitumor response by MV-A at the dose of 1.0 mg/kg is apparently superior to that of paclitaxel at the dose of 5.0 mg/kg (37.6%inhibition) .
  • Human equivalent dosage (mg/kg) animal dosage (mg/kg) ⁇ (animal Km/human Km) , wherein mouse Km is 3 and human Km is 37.
  • 0.5 mg/kg per mouse body weight corresponds to 0.0405 mg/kg per patient weight (weight in humans)
  • 1.0 mg/kg per mouse body weight corresponds to 0.0811 mg/kg per patient weight (weight in humans) .
  • the treatment started on the 12 th day when the MCF7-tumor size reached approximately 100 mm 3 (L ⁇ W ⁇ H) .
  • Two doses of MV-A (1.0 and 2.0 mg/kg) , paclitaxel (5 mg/kg) , and vehicle were administered via i.p. (intraperitoneal) , i.v. (intravenous) , or oral respectively, to the tumor-bearing mice twice a week for 3 weeks (8 mice/group) .
  • the MCF7 xenograft experiment showed that MV-A inhibited tumor growth by 48.1 %and 72.6 %at the doses of 1.0 and 2.0 mg/kg, respectively ( Figures 22 (A) -22 (B) ) .
  • a weight loss of 1-2 g is observed for the MV-A groups after the first two treatments, but the weights of the mice are kept relatively stable after the first two treatments.
  • the antitumor response by MV-A at the two doses is apparently superior to that of paclitaxel at the dose of 5.0 mg/kg (28.2%inhibition) .
  • MV-A, MV-B, MV-C and MV-D are evaluated for their anti-obesity effects in 3T3-L1 murine adipocytes.
  • 3T3-L1 cells are cultured in the presence of insulin to induce adipocyte differentiation.
  • triglyceride levels as indication of fat accumulation in the differentiation cells are measured to determine the anti-obesity effects of MV-A, MV-B, MV-C and MV-D.
  • MV-A demonstrates the anti-obesity activity with an EC 50 value of 0.3 nM (the cell viability with an IC 50 value of 12.9 nM.
  • MV-B demonstrates the anti-obesity activity with an EC 50 value of 2.6 nM (the cell viability with an IC 50 value of 83.7 nM.
  • MV-C demonstrates the anti-obesity activity with an EC 50 value of 38.9 nM (the cell viability with an IC 50 value of 689.4 nM) .
  • MV-D demonstrates the anti-obesity activity with an EC 50 value of 13.3 nM (the cell viability with an IC 50 value of more than 5.0 ⁇ M) .
  • MV-D shows potent anti-obesity activity with low toxicity. In the animal studies, the inventor demonstrates that the body weights of mice are significantly decreased after drug treatment of MV-A.
  • mice The average weight of mice (10 mice) drops 1-2 g after the first two treatments (intravenous, intraperitoneal or oral administration) with MV-A at the doses of 1 mg/kg and 2 mg/kg, respectively, but the weight of the mice is kept relatively stable after the first two treatments. No mice die during the treatments, and the average weight of mice gains back after the treatment stopped, which showed that the effects of compound MV-A on the mice are reversible. No sign of toxicity is observed from dissection of the mice.
  • the compound Since the low toxicity of MV-D, the compound is evaluated for its antiviral activity against HIV (human immunodeficiency virus) . It displays anti-HIV potential with an IC 50 value of 1.24 ⁇ M.
  • the inventor has discovered the anticancer, anti-obesity and anti-HIV compounds in in vitro and in vivo studies.
  • Cytotoxicity assays involving oral epidermoid (KB) , colon (HCT116) , prostate (LNCaP) , breast (MCF7) , Hela (cervical) , leukemia (HL-60) melanoma (A375) and lung (A549) carcinoma cell lines, are performed using sulforhodamine B according to established protocols (Zhang et al., Journal of Medicinal Chemistry 2006; 49: 693-708; and Jutiviboonsuk A et al., Phytochemistry 2005; 66: 2745-2751. ) .
  • KB and A375 cells are maintained in DMEM (Dulbecco’s modified Eagle medium) medium.
  • LNCaP and HL60 cells are maintained in RPMI1640 medium with hormone-free 10%heat-activated FBS (fetal bovine serum) supplemented with 0.1 nM testosterone.
  • MCF7 and Hela cells are maintained and assayed in MEME (Minimum Essential Medium Eagle) medium containing 10 mg/L of insulin.
  • HCT116 cells are maintained in McCoy’s 5A medium supplemented with 10%fetal bovine serum.
  • A549 cells are maintained in RPMI-1640 medium supplemented with 10%FCS. Serial dilutions of the compounds are prepared using 10%aqueous DMSO as solvent.
  • the 190 ⁇ L cell suspension (3 ⁇ 10 4 cells in 1 ml media) is incubated with10 ⁇ L sample solutions, in triplicate, in 96-well tissue culture plate at 37°C in a humidified atmosphere of 5%CO 2 in air for 72 hours. 10 ⁇ L 10%aqueous DMSO is used as control group. Then the cells are fixed to plastic substratum by the addition of 100 ⁇ L cold 20%aqueous trichloroacetic acid and washing with water after incubation at 4°C for 30 min. After staining cells with 100 ⁇ L of 0.4%sulforhodamine B in 1%aqueous AcOH for 30 min, unbound dye is removed by rinsing with 1%aqueous AcOH.
  • the bound dye is solubilized with 200 ⁇ L 10 mM unbuffered Tris base, pH 10, and the optical density is measured at 515 nm using an ELISA plate reader. The average data are expressed as a percentage, relative to the control.
  • the IC 50 values, the dose that inhibit cell growth by 50%, are calculated using nonlinear regression analysis (percent survival versus concentration) .
  • 3T3-L1 Cell Culture Bioassay Pure compounds are evaluated against 3T3-L1 cell line for their potential anti-obesity activity.
  • the preadipocyte murine cell line 3T3-L1 is cultured in normal DMEM medium containg with 10%FCS and 90 U/mL penicillin-streptomycin at 37°C in a humidified atmosphere of 5%CO 2 in air for 72 hours.
  • the culture medium is changed to a differentiation DMEM medium containing 10%FCS, 1 mM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX) , 90 U/mL penicillin, 90 mg/mL streptomycin and 10 ⁇ g/mL insulin for induction of the adipocyte form of the 3T3-L1 cells.
  • the differentiation medium is changed back to the normal DMEM medium and refreshed with the normal medium every 2 days. After incubation of additional 7 days, the cells are seeded in two sets of 96-well plates (about 10,000 cells per well) .
  • test compounds in different concentrations are then added to the wells with 0.5 %aqueous DMSO as control group.
  • the differentiated 3T3-L1 adipocytes are treated with 2%Triton-X 100 (10 mL/well) for 30 min at room temperature followed by sonication for 1 min.
  • Fat accumulation is determined by measuring the liberated triglyceride using a Wato Triglyceride E-test Kit with the absorbance at 630/690 nm. The fat-accumulation rate is calculated as a percentage of the DMSO control.
  • the EC 50 values, the dose that inhibit fat accumulation by 50% are calculated using nonlinear regression analysis (percent survival versus concentration) .
  • the cell viability of the differentiated 3T3-L1 adipocytes in another 96-well plate is treated with a Wako Cell Counting Kit-8 Test and measured with the absorbance at 450 nm. The cell viability is calculated as a percentage of the control.
  • the IC 50 values, the dose that inhibit cell growth by 50%, are calculated using nonlinear regression analysis (percent survival versus concentration) .
  • Hollow fiber animal study All animal studies are approved and performed according to Animal Care and Use Guidelines of the Animal Ethics Committee at Hong Kong Institution University and performed following Animal Care and Use guidelines set by NIH (National Institute of Health, USA) . Hollow fiber tests are well known in the art for providing preliminary indications of therapeutic efficacy (Mi et al. J. Nat. Prod. 2002, 65: 842-850) .
  • human tumor cell lines currently employed in cell cultures are grown inside semipermeable hollow fibers to form heterogeneous solid tumor models.
  • the hollow fibers containing the human tumor cells are implanted in the intraperitoneal or subcutaneous compartments of host mice, and the mice treated with the test compound of interest administered via i.p. (intraperitoneal) , i.v.
  • mice SPF class, male or female, 5-6 weeks old, were purchased from Charles River Laboratories. Before the experiment, it is one week of acclimatization to SPF class laboratory conditions.
  • MV-A is tested for its anticancer activity against HCT116 and MCF7 cancer cells using a number of nude mice (Balc/nu/nu, female) in comparison of paclitaxel and maytansine.
  • HCT116 or MCF7 cancer cells are subcutaneously implanted with 5 ⁇ 10 6 cells in the rear flank of each mouse.
  • Female mice receiving MCF-7 cells are implanted with an s.c. pellet of 17 ⁇ -estradiol (0.72 mg/pellet) a few days prior to injection to induce tumors. After 10 days, solid tumors with average size of about 80-100 mm 3 appear at the implanted sites.
  • mice are divided into 5 groups for HCT116 cancer cells: one high dose (1.0 mg/kg: 10 mice) group of MV-A, one low dose (0.5 mg/kg: 10 mice) group of MV-A, one dose (5.0 mg/kg: 10 mice) of paclitaxel, one dose of maytansine (0.1 mg/kg) and one dose of vehicles (negative control: 10 mice) .
  • the mice are then divided into 4 groups for MCF7 cancer cells: one high dose (2.0 mg/kg: 10 mice) group of MV-A, one low dose (1.0 mg/kg: 10 mice) group of MV-A, one dose (5.0 mg/kg: 10 mice) of paclitaxel, and one dose of vehicles (negative control: 10 mice) .
  • tumor size length ⁇ width ⁇ height (L ⁇ W ⁇ H) .
  • HIV/VSV-G are produced by co-transfecting 3 g of VSV-G envelope expression plasmid with 21 g of a replication-defective HIV vector (pNL4-3-Luc-RE) [34, 35] into Human embryonic kidney 293T cells (90%confluent) in 10-cm plates with PEI (Invitrogen) . Eight hours post-transfection, all media is replaced with fresh, complete DMEM.
  • Target A549 cells are seeded at 104 cells per well (96-well plate) in complete DMEM.
  • Ten microliter compound for serial concentrations (20, 10, 5, 2.5, 1.25, 0.625 and 0.3125 ⁇ g/mL) and 190 ⁇ L of the pseudovirus are incubated with target cells.
  • Forty-eight hours post-infection cells are lysed and prepared for luciferase assay (Promega) .
  • references cited herein are incorporated by reference herein in their entirety to indicate the state of the art as of their publication or filing date and it is intended that this information can be employed herein, if needed, to exclude specific embodiments that are in the prior art.
  • composition of matter are claimed, it should be understood that compounds known and available in the art prior to Applicant's invention, including compounds for which an enabling disclosure is provided in the references cited herein, are not intended to be included in the composition of matter claims herein.
  • This invention is in the field of pharmaceuticals and chemical industries.
  • this invention relates to new anticancer and anti-obesity agents based on the cyclic peptide natural products.
  • the invention also includes its preparation and application method for treating cancer and obesity diseases.
  • the different functions discussed herein may be performed in a different order and/or concurrently with each other. Furthermore, if desired, one or more of the above-described functions may be optional or may be combined.
  • the embodiments disclosed herein may be implemented using general-purpose or specialized computing platforms, computing devices, computer processors, or electronic circuitries including but not limited to digital signal processors (DSP) , application specific integrated circuits (ASIC) , field programmable gate arrays (FPGA) , and other programmable logic devices configured or programmed according to the teachings of the present disclosure.
  • DSP digital signal processors
  • ASIC application specific integrated circuits
  • FPGA field programmable gate arrays
  • Computer instructions or software codes running in the general-purpose or specialized computing platforms, computing devices, computer processors, or programmable logic devices can readily be prepared by practitioners skilled in the software or electronic art based on the teachings of the present disclosure.
  • the present invention includes computer storage media having computer instructions or software codes stored therein which can be used to program computers or microprocessors to perform any of the processes of the present invention.
  • the storage media can include, but are not limited to, floppy disks, optical discs, Blu-ray Disc, DVD, CD-ROMs, and magneto-optical disks, ROMs, RAMs, flash memory devices, or any type of media or devices suitable for storing instructions, codes, and/or data.

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  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
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  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne de nouveaux agents anticancéreux et anti-obésité basés sur les composés peptidiques cycliques, leur préparation et leur procédé d'application pour le traitement du cancer et des maladies de l'obésité.
PCT/CN2017/106025 2016-10-14 2017-10-13 Agents cycloheptapeptidiques pour le traitement du cancer et des maladies de l'obésité WO2018068757A1 (fr)

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US15/293,516 US9963485B2 (en) 2012-10-16 2016-10-14 Cycloheptapeptide agents for treatment of cancer and obesity diseases

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999047489A1 (fr) * 1998-03-19 1999-09-23 F. Hoffmann-La Roche Ag Elaboration de la (2s,2'r,3'r)-2-(2,3-dicarboxylcyclopropyl)-glycine
WO2000075102A1 (fr) * 1999-06-03 2000-12-14 Lilly, S.A. Modulateurs du recepteur d'acide amine excitateur
CN104640873A (zh) * 2012-10-16 2015-05-20 香港浸会大学 抗癌和抗肥胖的环状肽剂
US20170096454A1 (en) * 2012-10-16 2017-04-06 Hong Kong Baptist University Cycloheptapeptide agents for treatment of cancer and obesity diseases

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999047489A1 (fr) * 1998-03-19 1999-09-23 F. Hoffmann-La Roche Ag Elaboration de la (2s,2'r,3'r)-2-(2,3-dicarboxylcyclopropyl)-glycine
WO2000075102A1 (fr) * 1999-06-03 2000-12-14 Lilly, S.A. Modulateurs du recepteur d'acide amine excitateur
CN104640873A (zh) * 2012-10-16 2015-05-20 香港浸会大学 抗癌和抗肥胖的环状肽剂
US20170096454A1 (en) * 2012-10-16 2017-04-06 Hong Kong Baptist University Cycloheptapeptide agents for treatment of cancer and obesity diseases

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LACKNER G. ET AL.: "Endofungal bacteria as producers of mycotoxins", TRENDS IN MICROBIOLOGY, vol. 17, no. 12, 1 October 2009 (2009-10-01), pages 570 - 576, XP026778834 *

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