Anticancer Miliusane Lactams

Abstract

The present invention is in the field of pharmaceuticals and chemical industries. In particular, the present invention relates to new anticancer miliusane lactam compounds. The present invention also includes its preparation and application method for treating cancer.

Claims

1. A compound represented by the formula (IX) or formula (X): ##STR00052## or a pharmaceutically acceptable salt, tautomer, and/or prodrug thereof, wherein R.sup.1 is alkyl; and R.sup.2 is aryl.

2. The compound of claim 1, wherein said compound is an optically pure stereoisomer.

3. The compound of claim 1, wherein said compound has formula (IX).

4. The compound of claim 1, wherein said compound has formula (X).

5. The compound of claim 3, wherein R.sup.1 is C.sub.1-6 alkyl.

6. The compound of claim 4, wherein R.sup.2 is phenyl.

7. The compound of claim 1, wherein the compounds of formula (IX) and (X) have the relative stereochemistry depicted below: ##STR00053##

8. The compound of claim 1, wherein the compound is selected from N-(n-butyl)-miliusol lactam or N-phenyl-miliusol lactam with the following formula: ##STR00054## or a pharmaceutically acceptable salt and/or prodrug thereof.

9. A pharmaceutical composition comprising a compound of claim 1 and at least one pharmaceutically acceptable carrier.

10. A method of treating cancer in a subject comprising administering a therapeutically effective amount of a compound of claim 1 to a subject in need thereof, wherein said cancer is colon cancer, breast cancer, prostate cancer, lung cancer, epidermoid cancer, or melanoma cancer.

11. (canceled)

12. The method of claim 10, wherein said subject is a human.

13. The method of claim 10, further comprising the step of administering a therapeutically effective amount of a second anti-cancer agent, wherein said compound of claim 1 and said second anti-cancer agent are administered sequentially or administered simultaneously.

Description

DETAILED DESCRIPTION OF INVENTION

[0042] The present invention is not to be limited in scope by any of the specific embodiments described herein. The following embodiments are presented for exemplification only.

Definitions

Miliusane and Core Structures

[0043] The term “miliusane” as used herein includes reference to a compound comprising the basic structure shown as below:

##STR00012##

[0044] The carbon numbering of miliusane molecule as used herein includes reference to a compound comprising the numbering system shown as below:

##STR00013##

[0045] The term “the core structure of miliusane” as used herein includes reference to a compound comprising the basic structure shown as below:

##STR00014##

[0046] In one class of the core structures of miliusane compounds, the methyl group and the ethyl group form a tetrahydrofuran ring (1-oxa-spiro [4.5] decane and 2-oxa-spiro [4.5] decane).

##STR00015##

[0047] In the second class of the core structures of miliusane compounds, the methyl group and the ethyl group form a tetrahydro-thiophene ring (1-thia-spiro [4.5] decane and 2-thia-spiro [4.5] decane).

##STR00016##

[0048] In the third class of the cores structure of miliusane compounds, the methyl group and the ethyl group form a pyrrolidine ring (1-aza-spiro [4.5] decane, 2-aza-spiro [4.5] decane, 1-aza-spiro [4.5] dec-1-ene, 2-aza-spiro [4.5] dec-1-ene and 2-aza-spiro [4.5] dec-2-ene).

##STR00017##

##STR00018##

Hydrocarbyl

[0049] The term “hydrocarbyl” as used herein includes reference to a moiety consisting 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. Examples of hydrocarbyl groups include C.sub.1-6 alkyl (e.g. C.sub.1, C.sub.2, C.sub.3 or C.sub.4 alkyl, for example methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl or tert-butyl); C.sub.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.

Alkyl

[0050] The terms “alkyl” and “C.sub.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. In particular, the alkyl moiety may have 1, 2, 3 or 4 carbon atoms.

Alkenyl

[0051] The terms “alkenyl” and “C.sub.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, 3-hexenyl, and the like.

Alkynyl

[0052] The terms “alkynyl” and “C.sub.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, 3-hexynyl, and the like.

Alkoxy

[0053] The terms “alkoxy” and “C.sub.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

[0054] The term “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

[0055] The term “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. 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

[0056] “Cyclic group” means a ring or ring system, which may be unsaturated or partially unsaturated but is usually saturated, typically containing 3 to 13 ring-forming atoms, for example a 3-, 4-, 5- or 6-membered ring. The ring system may be a bridged or polycyclic ring system. The ring or ring system may be substituted with one or more hydrocarbyl groups. Cyclic groups includes carbocyclyl and heterocyclyl moieties.

Carbocyclyl

[0057] The term “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. In particular, carbocyclyl includes a 3- to 10-membered ring or ring system and, in particular, 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

[0058] The term “heterocyclyl” as used herein includes reference to a non-aromatic (e.g. heterocycloalkyl) or an aromatic (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 sulfur. In particular, 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.

[0059] 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, morpholinyl, thiomorpholinyl, especially thiomorpholino, indolizinyl, isoindolyl, 3H-indolyl, indolyl, benzimidazolyl, cumaryl, indazolyl, triazolyl, tetrazolyl, purinyl, 4/V-quinolizinyl, isoquinolyl, quinolyl, tetrahydroquinolyl, tetrahydroisoquinolyl, decahydroquinolyl, octahydroisoquinolyl, benzofuranyl, dibenzofuranyl, benzothiophenyl, dibenzothiophenyl, phthalazinyl, naphthyridinyl, quinoxalyl, quinazolinyl, quinazolinyl, cinnolinyl, pteridinyl, carbazoiyl, β-carbolinyl, phenanthridinyl, acridinyl, perimidinyl, phenanthrolinyl, furazanyl, phenazinyl, phenothiazinyl, phenoxazinyl, chromenyl, isochromanyl, chromanyl, and the like.

Heterocycloalkyl

[0060] The term “heterocycloalkyl” as used herein includes reference to a 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 sulfur. Heterocycloalkyl can have one or more carbon-carbon double bonds or carbon-heteroatoms double bonds as long as the ring is not aromatic. 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

[0061] The term “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 sulfur. 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.

Halogen

[0062] The term “halogen” as used herein includes reference to F, Cl, Br, or I.

Halogen Containing Moiety

[0063] The expression “halogen containing moiety” as used herein includes reference to a moiety comprising 1 to 30 plural valence atoms selected from carbon, nitrogen, oxygen and sulfur which moiety includes at least one halogen. The moiety may be hydrocarbyl for example C.sub.1-6 alkyl or C.sub.1-6 alkoxy, or carbocyclyl for example aryl.

Substituted

[0064] The term “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. The term “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.

Enantiomer

[0065] The term “enantiomer” as used herein means one of two stereoisomers that have mirror images of one another.

Racemate

[0066] The term “racemate” as used herein means a mixture of equal amounts of enantiomers of a chiral molecule.

Diastereomer

[0067] The term “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

[0068] The term “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.

Tautomers

[0069] The term “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.

Prodrug

[0070] 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.

Independently

[0071] Where two or more moieties are described as being “each independently” selected from a list of atoms or groups, this means that the moieties may be the same or different. The identity of each moiety is therefore independent of the identities of the one or more other moieties.

[0072] Embodiments of the present invention are described below. Preferred features of each aspect of the present invention are as for each of the other aspects mutatis mutandis. Moreover, it will be appreciated that the features specified in each embodiment may be combined with other specified features, to provide further embodiments.

Compounds of the Present Invention

[0073] In an exemplary embodiment, the present invention provides compounds of formula (I), or (II):

##STR00019##

[0074] wherein R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8, R.sup.9, R.sup.10, R.sup.11, R.sup.12, R.sup.13, R.sup.14, R.sup.15 and R.sup.16 are each independently selected from hydrogen, halogen or a moiety comprising 1 to 30 plural valence atoms selected from carbon, nitrogen, oxygen and sulfur; or these groups may be taken together with the carbon 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 sulfur; R.sup.1 and R.sup.2, R.sup.3 and R.sup.4, R.sup.5 and R.sup.6, R.sup.7 and R.sup.8, R.sup.9 and R.sup.10, R.sup.11 and R.sup.12, R.sup.13 and R.sup.14 or R.sup.15 and R.sup.16 may be taken together with the carbon atoms to which they are attached to form one or more carboxyl groups (G=0); or while one of R′ and R.sup.2, R.sup.3 and R.sup.4, R.sup.5 and R.sup.6, R.sup.7 and R.sup.8, R.sup.9 and R.sup.10, R.sup.11 and R.sup.12, R.sup.13 and R.sup.14 or R.sup.15 and R.sup.16 is hydrogen, halogen, hydrocarbyl or alkoxy, the other one of R.sup.1 and R.sup.2, R.sup.3 and R.sup.4, R.sup.5 and R.sup.6, R.sup.7 and R.sup.8, R.sup.9 and R.sup.10, R.sup.11 and R.sup.12, R.sup.13 and R.sup.14 or R.sup.15 and R.sup.16 is selected from R.sup.17, —OR.sup.17, —C(O)R.sup.17 and —C(O)OR.sup.17; R.sup.17 is independently selected from hydrogen, halogen, trifluoromethyl, cyano, nitro, hydrocarbyl optionally substituted with 1, 2, 3, 4 or 5 R.sup.18, heterocyclyl optionally substituted with 1, 2, 3, 4 or 5 R.sup.18, —(CH.sub.2).sub.k-heterocyclyl optionally substituted with 1, 2, 3, 4 or 5 R.sup.18, —OR.sup.19, —C(O)R.sup.20, —C(O)N(R.sup.19)R.sup.20, —C(O)OR.sup.19, —OC(O)R.sup.19, —S(O).sub.2R.sup.19, —S(O).sub.2N(R.sup.19)R.sup.20, —N(R.sup.19)R.sup.20, —N(R.sup.19)N(R.sup.19)R.sup.20, —N(R.sup.19)C(O)R.sup.20 and —N(R.sup.19)S(O).sub.2R.sup.20; wherein k is an integer between 1 and 6 (e.g. 1, 2 or 3); R.sup.18 is independently selected from halogen, trifluoromethyl, cyano, nitro, oxo, ═NR.sup.19, —OR.sup.19, —C(O)R.sup.20, —C(O)N(R.sup.19)R.sup.20, —C(O)OR.sup.19, —OC(O)R.sup.20, —S(O).sub.2R.sup.19, —S(O).sub.2N(R.sup.19)R.sup.20, —N(R.sup.19)R.sup.20, —N(R.sup.19)N(R.sup.19)R.sup.20, —N(R.sup.19)C(O)R.sup.20 and —N(R.sup.19)S(O).sub.2R.sup.20; R.sup.19 and R.sup.20 are each independently hydrogen or selected from hydrocarbyl, heterocyclyl or —(CH.sub.2).sub.k-heterocyclyl, which is optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from halogen, cyano, amino, hydroxy, C.sub.1-6 alkyl and C.sub.1-6 alkoxy; wherein k is an integer between 1 and 6 (e.g. 1, 2 or 3); X is oxygen or sulfur; R is hydrogen or selected from hydrocarbyl, heterocyclyl or —(CH.sub.2).sub.k-heterocyclyl, which is optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from halogen, cyano, amino, hydroxy, C.sub.1-6 alkyl and C.sub.1-6 alkoxy; wherein k is an integer between 1 and 6 (e.g. 1, 2 or 3); dashed line “----” denotes a single or double bond; or a pharmaceutically acceptable salt or prodrug thereof.

[0075] In certain embodiments, the compounds of the present invention are represented by formula (IX) or formula (X):

##STR00020##

or a pharmaceutically acceptable salt, tautomer, and/or prodrug thereof, wherein R.sup.1 is alkyl; and R.sup.2 is aryl.

[0076] In certain embodiments the compound is an optically pure stereoisomer.

[0077] In certain embodiments, the compound has formula (X).

[0078] In certain embodiments, the compound of formula (X) has the relative stereochemistry indicated below:

##STR00021##

[0079] In certain embodiments, the compound of formula (X) has the absolute stereochemistry indicated below:

##STR00022##

[0080] In certain embodiments, R.sup.2 is phenyl.

[0081] In certain embodiments, the compound has formula (IX).

[0082] In certain embodiments, the compound of formula (IX) has the relative stereochemistry indicated below:

##STR00023##

[0083] In certain embodiments, the compound of formula (IX) has the absolute stereochemistry indicated below:

##STR00024##

[0084] In certain embodiments, R.sup.1 is C.sub.1-6 alkyl, C.sub.2-6 alkyl, C.sub.3-6 alkyl, or C.sub.3-5 alkyl. In certain embodiments, R.sup.1 is C.sub.4 alkyl. In certain embodiments, R.sup.1 is n-butyl.

[0085] In certain embodiments, the compound is a tautomer of the compound of Formula (IX) or (X).

[0086] In certain embodiments, the compound has the same relative stereochemistry at the C-1, C-1′, and C-5 carbons as miliusane.

[0087] In certain embodiments, the compound has the same absolute stereochemistry at the C-1, C-1′, and C-5 carbons as miliusane.

[0088] In certain embodiments, the compound is an enantiomer.

[0089] In certain embodiments, the compound is a racemate.

[0090] In certain embodiments, the compound is a diastereomer.

[0091] In certain embodiments, the compound is a tautomer.

[0092] The present invention also provides a pharmaceutical composition comprising a compound of the present invention and at least one pharmaceutical acceptable carrier.

[0093] It should be noted that compounds having one of the following formulae should not be the compound of the present invention and should be removed from the compound of the present invention:

##STR00025## ##STR00026## ##STR00027## ##STR00028## ##STR00029##

wherein Ac is acetyl group (CH.sub.3C═O), and Me is methyl group (CH.sub.3);
Compounds having the following formula (V) are excluded in the present invention:

##STR00030##

[0094] wherein R.sup.1═R.sup.2═R.sup.3═R.sup.4═R.sup.5═H; or R.sup.1═R.sup.2═R.sup.3═R.sup.4═H, R.sup.5═OCH.sub.3; or R.sup.1═R.sup.3═R.sup.4═R.sup.5═H, R.sup.2═OCH.sub.3; or R.sup.1═R.sup.2═R.sup.4═R.sup.5═H, R.sup.3═OCH.sub.3; or R.sup.1═R.sup.2═R.sup.4═H, R.sup.3═R.sup.5═OCH.sub.3; or R.sup.1═R.sup.5═OCH.sub.3, R.sup.2═R.sup.3═R.sup.4═H; or R.sup.1═R.sup.4═R.sup.5═H, R.sup.2═R.sup.3═OCH.sub.3; or R.sup.1═R.sup.3═R.sup.5═H, R.sup.2═R.sup.4═OCH.sub.3; or R.sup.1═R.sup.5═H, R.sup.2═R.sup.3═R.sup.4═OCH.sub.3; or R.sup.1═R.sup.4═R.sup.5═H, R.sup.2 and R.sup.3 taken together with the carbon atoms to which they are attached to form a cyclic [1,3]dioxole group; or R.sup.1═R.sup.2═R.sup.3═R.sup.4═H, R.sup.5═CH.sub.3; or R.sup.1═R.sup.3═R.sup.4═R.sup.5═H, R.sup.2═CH.sub.3; or R.sup.1═R.sup.2═R.sup.4═R.sup.5═H, R.sup.3═CH.sub.3; or R.sup.1═R.sup.2═R.sup.3═R.sup.4═H, R.sup.5═F; or R.sup.1═R.sup.3═R.sup.4═R.sup.5═H, R.sup.2═F; or R.sup.1═R.sup.2═R.sup.4═R.sup.5═H, R.sup.3═F; or R.sup.1═R.sup.2═R.sup.3═H, R.sup.4═R.sup.5═F; or R.sup.1═R.sup.2═R.sup.4═H, R.sup.3═R.sup.5═F; or R.sup.1═R.sup.3═R.sup.4═H, R.sup.2═R.sup.5═F; or R.sup.1═R.sup.5═F, R.sup.2═R.sup.3═R.sup.4═H; or R.sup.1═R.sup.4═R.sup.5═H, R.sup.2═R.sup.3═F; or R.sup.1═R.sup.3═R.sup.5═H, R.sup.2═R.sup.4═F; or R.sup.1═R.sup.2═R.sup.3═R.sup.4═H, R.sup.5═Cl; or R.sup.1═R.sup.3═R.sup.4═R.sup.5═H, R.sup.2═Cl; or R.sup.1═R.sup.2═R.sup.4═R.sup.5═H, R.sup.3═Cl; or R.sup.1═R.sup.2═R.sup.4═H, R.sup.3═R.sup.5═Cl; or R.sup.1═R.sup.5═Cl, R.sup.2═R.sup.3═R.sup.4═H; or R.sup.1═R.sup.4═R.sup.5═H, R.sup.2═R.sup.3═Cl; or R.sup.1═R.sup.3═R.sup.5═H, R.sup.2═R.sup.4═Cl; or R.sup.1═R.sup.2═R.sup.3═R.sup.4═H, R.sup.5═Br; or R.sup.1═R.sup.3═R.sup.4═R.sup.5═H, R.sup.2═Br; or R.sup.1═R.sup.2═R.sup.4═R.sup.5═H, R.sup.3═Br; or R.sup.1═R.sup.2═R.sup.3═R.sup.4═H, R.sup.5═I; or R.sup.1═R.sup.2═R.sup.4═R.sup.5═H, R.sup.3═I; Compounds having the following formula (VI) are excluded in the present invention:

##STR00031##

wherein X.sup.1 and X.sup.2 are carbon atoms either joined by double bond or joined by a single bond constituents of an epoxide ring or a hydroxyethylene moiety; X.sup.3 and X.sup.4 are carbon atoms either joined by double bond or joined by a single bond comstituents of an epoxide ring or a hydroxyethylene moiety; R.sup.1 is selected from the group consisting of branched alkyl chains, unbranched alkyl chains, cycloalkyl groups, aromatic groups, alcohols, ethers, amines, and substituted or unsubstituted ureas, esters, aldehydes and carboxylic acids; R.sup.2 is selected from the group consisting of H, OH and NHR.sup.3, wherein R.sup.3 is a nitrogen protecting group; Compounds having the following formula (VII) are excluded in the present invention:

##STR00032##

wherein R.sup.3 and R.sup.4 are selected from the group consisting of branched alkyl chains, unbranched alkyl chains, cycloalkyl groups, aromatic groups, alcohols, ethers, amines, and substituted or unsubstituted ureas, esters, aldehydes and carboxylic acids; m is an integer between 0 and 5;

[0095] Compounds having the following formula (VIII) are excluded in the present invention:

##STR00033##

wherein Z is oxygen or sulfur or nitrogen substituted with D group; D represents hydrogen or represents a C.sub.1-C.sub.6-alkyl or C.sub.1-C.sub.6-alkoxy-C.sub.1-C.sub.6-alkyl radical or represents a C.sub.3-C.sub.7-cycloalkyl or 4- to 7-membered monocyclic heterocyclyl radical, where the radicals mentioned may optionally be mono- or polysubstituted by identical or different substituents selected from the group consisting of halogen and hydroxyl and C.sub.1-C.sub.3-alkyl, halo-C.sub.1-C.sub.3-alkyl, or C.sub.1-C.sub.3-alkoxy; X represents halogen, nitro or cyano or represents an optionally monohalogen- or polyhalogen-substituted C.sub.1-C.sub.6-alkyl, C.sub.1-C.sub.6-alkoxy, C.sub.1-C.sub.6-alkoxy-C.sub.1-C.sub.6-alkoxy, C.sub.3-C.sub.7-cycloalkyl or a C.sub.3-C.sub.7-cycloalkyl-C.sub.1-C.sub.6-alkoxy radical, and W and Y independently of one another represent hydrogen, nitro, cyano or halogen or represent an optionally monohalogen- or polyhalogen-substituted C.sub.1-C.sub.6-alkyl, C.sub.1-C.sub.6-alkoxy or C.sub.3-C.sub.7-cycloalkyl radical, and V.sup.1, V.sup.2 and V.sup.3 independently of one another represent hydrogen, halogen, nitro or cyano or represent a C.sub.1-C.sub.6-alkyl, halo-C.sub.1-C.sub.6-alkyl, C.sub.1-C.sub.6-alkoxy, halo-C.sub.1-C.sub.6-alkoxy, C.sub.1-C.sub.6-alkylthio, C.sub.1-C.sub.6-alkylsulphinyl, C.sub.1-C.sub.6-alkylsulphonyl, C.sub.1-C.sub.6-alkoxy-C.sub.1-C.sub.6-alkyl, C.sub.3-C.sub.10-cycloalkyl radical or represent a monocyclic heterocycloalkyl radical, and/or
V.sup.1 and V.sup.2 together with the carbon atoms to which they are attached form a saturated or unsaturated cycle T.sup.1 which optionally contains at least one further heteroatom and has 4 to 7-ring atoms and whose ring-forming atoms may be mono- or polysubstituted by identical or different substituents selected from the group consisting of halogen and a C.sub.1-C.sub.6-alkyl radical.

[0096] Examples of the compounds of the present invention include those shown below. It will of course be appreciated that, where appropriate, each compound may be in the form of the free compound, an enantiomer, an acid or base addition salt, or a prodrug.

[0097] The present invention provides 17 compounds with potent anti-cancer activity and synthesis thereof. The compounds of the present invention are synthesized and evaluated for their anticancer activity, namely 4β-(N-phenyl)miliusate, 4α-(N-phenyl)miliusate, 4β-(N-benzoyl-N-phenyl)miliusate, 4α-(N-benzoyl-N-phenyl)miliusate, hexahydro-miliusate, 3, 4-dihydro-miliusate, 2′, 3′, 6′, 7′-tetrahydro-miliusate, 2-hydroxy-3, 4-dihydro-miliusate, 2-acetoxy-3, 10′-hydroxy-8′-oxo-miliusate, 8′, 10′-dihydroxy-miliusate, 5β-(p-trimethylammonio-benzoyl)miliusol iodide, 5β-(p-dimethyl-allyl-ammonio-benzoyl)miliusol bromide, N-(n-butyl)-miliusol lactam and N-phenyl-miliusol lactam. The chemical formulae of the compounds of the present invention are as follows:

##STR00034## ##STR00035## ##STR00036## ##STR00037##

Examples

Synthesis of Novel Miliusane Derivatives

[0098] The two natural miliusane compounds (miliusol and miliusate), isolated from Miliusa balansae Finet & Gagnep. (Annonaceae), were used as the starting compounds to prepare new miliusane derivatives. By using different synthetic methods, 17 new miliusane analogs are synthesized.

##STR00038##

[0099] These new miliusane compounds have been evaluated for their anticancer activity against a panel of cancer cell lines comprising KB, HCT116, LNCaP, A549, MCF-7 and A375. 4β-(N-Phenyl)miliusate demonstrates cell killing activity with IC.sub.50 values ranging from 0.2-2.0 μM. 4α-(N-Phenyl)miliusate demonstrated cell killing activity with IC.sub.50 values ranging from 1.1-6.5 μM. 4β-(N-Benzoyl-N-phenyl)miliusate demonstrates cell killing activity with IC.sub.50 values ranging from 0.5-2.5 μM. 4α-(N-Benzoyl-N-phenyl)miliusate demonstrates cell killing activity with IC.sub.50 values ranging from 5.0-9.8 μM. 2′,3′,6′,7′-Tetradehydro-miliusate demonstrates cell killing activity with IC.sub.50 values ranging from 1.2-6.8 μM. 8′-Oxo-miliusate demonstrates cell killing activity with IC.sub.50 values ranging from 3.6-5.5 μM. 8′-Hydroxy-miliusate demonstrates cell killing activity with IC.sub.50 values ranging from 4.2-8.9 μM. 10′-Hydroxy-8′-oxo-miliusate demonstrates cell killing activity with IC.sub.50 values ranging from 2.5-4.9 μM. 5β-(p-Trimethylammonio-benzoyl)miliusol iodide demonstrates cell killing activity with IC.sub.50 values ranging from 0.05-1.2 μM. 5β-(p-Dimethyl-allyl-ammonio-benzoyl)miliusol bromide demonstrates cell killing activity with IC.sub.50 values ranging from 0.1-1.5 μM. N-(n-butyl)-miliusol lactam demonstrates cell killing activity with IC.sub.50 values ranging from 0.5-4.2 μM. 5β-(p-Trimethylammonio-benzoyl)miliusol iodide, 5β-(p-dimethyl-allyl-ammonio-benzoyl)miliusol bromide, and 4β-(N-phenyl)miliusate demonstrate more potent cell killing activity than that of miliusol. All other compounds show no cell killing activity at a concentration of 20 μM.

[0100] Since 4β-(N-phenyl)miliusate shows better cell killing activity than that of miliusol in the in vitro evaluation system, the compound is further evaluated for its in vivo antitumor activity in HCT116 xenograft mouse model study. In the experiment, HCT116 tumors in mice treated with 4β-(N-phenyl)miliusate grow much slower than those mice treated with vehicle. After 21 days administration, the measured average tumor size (L×W×W) is suppressed by 30.5% with treatment of 4β-(N-phenyl)miliusate at the dose of 20 mg/kg (p<0.01) in comparison with the vehicle control group (paclitaxel, the clinically used anticancer drug, shows 23.3% inhibition of tumor growth at the dose of 10 mg/kg). In addition, no weight loss is observed for the mice in the 4β-(N-phenyl)miliusate treatment group. On the contrary, 3 out of 10 mice died for the paclitaxel group. These results clearly indicate the superior antitumor efficacy of 4β-(N-phenyl)miliusate with low toxicity in comparison with the clinically used drug paclitaxel.

General Method for Preparation of 4-Amino Substituted Miliusane Derivatives

[0101] Bismuth nitrate [Bi(NO.sub.3).sub.3] (70 mg, 0.5 eq) is added to a mixture of an amine such as aniline (1.35 g, 50 eq) and a miliusane such as miliusate (100 mg, 0.29 mmol) in dichloromethane (CH.sub.2Cl.sub.2, 2 mL). The reaction mixture is stirred at room temperature for 48 hours. CH.sub.2Cl.sub.2 (30 mL) is added to the reaction mixture, and the CH.sub.2Cl.sub.2 solution is filtered through filter paper to remove Bi(NO.sub.3).sub.3. The filtrate is washed with saturated NaHCO.sub.3 (2×10 mL) and brine (1×10 mL), and dried over Na.sub.2SO.sub.4. The CH.sub.2Cl.sub.2 solution is concentrated, and is further purified using chromatography on a silica gel column eluting with a mixture of solvent such as petroleum ether:EtOAc (ethyl acetate) 4:1 to afford 4-amino substituted miliusane derivative(s). Unreacted, partially reacted or unwanted compounds as disclosed herein can be removed from the reaction product.

##STR00039##

[0102] The new molecules 4β-(N-phenyl)miliusate and 4α-(N-phenyl)miliusate are thus synthesized (see Scheme 1).

General Method for Preparation of Amino Substituted Miliusane Amide Derivatives

[0103] To a solution of an amino substituted miliusane derivative such as 4α-(N-benzyl)miliusate and 4β-(N-benzyl)miliusate (30 mg, 0.068 mmol) in anhydrous pyridine (5 mL) is added benzoyl chloride (BzCl) (19 mg, 2 eq) and a catalytic amount of 4-dimethylaminopyridine (DMAP). The reaction is stirred overnight at room temperature, and quenched with H.sub.2O (30 mL). The reaction mixture is then extracted with ethyl acetate (3×20 mL). The combined ethyl acetate solution is washed with H.sub.2O (20 mL) and brine (20 mL), and dried over Na.sub.2SO.sub.4. The solution is then concentrated in vacuo, and chromatographed on a silica gel column, eluted with a solvent system such as petroleum ether:EtOAc 2:1 to afford amino substituted miliusane amide derivative. Unreacted, partially reacted or unwanted compounds as disclosed herein can be removed from the reaction product.

##STR00040##

[0104] The new molecules 4β-(N-benzoyl-N-phenyl)miliusate, and 4α-(N-benzoyl-N-phenyl)miliusate are thus synthesized (see Schemes 2 and 3).

General Method for Preparation of Hexahydro-Miliusane Derivatives

[0105] A solution of a miliusane such as miliusate (20 mg, 0.058 mmol) in EtOAc (5 mL) is treated with 10% of the catalyst palladium on activate charcoal (Pd/C) (5 mg). The resulting black suspension is stirred under hydrogen (H.sub.2) (1 atmosphere) at room temperature for 3 hours. The catalyst is removed by filtration through Celite and washed with EtOAc (20 mL). The filtrate is evaporated in vacuo and the residue is subjected to chromatography separation on silica gel, eluted with a solvent system such as petroleum ether:EtOAc 4:1 to afford hexahydro-miliusane derivative. Unreacted, partially reacted or unwanted compounds as disclosed herein can be removed from the reaction product.

##STR00041##

[0106] The new molecule hexahydro-miliusate is thus synthesized (see Scheme 4).

General Method for Preparation of 3, 4-Dihydro-Miliusane Derivatives

[0107] A solution of a miliusane such as miliusate (10 mg, 0.029 mmol) in EtOAc (3 mL) is treated with 10% Pd/C (5 mg). The resulting black suspension is stirred under H.sub.2 (1 atmosphere) at −78° C. for 48 hours. The catalyst is removed by filtration through Celite and washed with EtOAc (20 mL). The filtrate is evaporated in vacuo and the residue is subjected to chromatography separation on silica gel, eluted with a solvent system such as petroleum ether:EtOAc 4:1 to afford the 3,4-dihydro-miliusane derivative. Unreacted, partially reacted or unwanted compounds as disclosed herein can be removed from the reaction product.

##STR00042##

[0108] The new molecule 3, 4-dihydro-miliusate is thus synthesized (see Scheme 5).

General Method for Preparation of 2′,3, 6′,7′-Tetrahydro-Miliusane Derivatives

[0109] To a solution of a miliusane such as hexahydro-miliusate (20 mg, 0.056 mmol) in toluene: DMSO (2:1, 0.1 M) is added 2-iodoxybenzoic acid (IBX) (20.7 mg, 1.3 eq). The resulting solution is heated to 100° C. for stirring 3 hours. The reaction mixture is then diluted with CH.sub.2Cl.sub.2 (30 mL), and washed sequentially with 5% NaHCO.sub.3 (1×10 mL), H.sub.2O (1×10 mL), and brine (1×10 mL), followed by removal of solvent in vacuo, leading to crude products, which can be purified using silica gel column chromatography to afford the 2′,3′,6′,7′-tetrahydro-miliusane derivative. Unreacted, partially reacted or unwanted compounds as disclosed herein can be removed from the reaction product.

##STR00043##

[0110] The new molecules 2′,3′,6′,7′-tetrahydro-miliusate is thus synthesized (see Scheme 6).

General Method for Preparation of 2-Hydroxy-Miliusane Derivatives

[0111] To a stirred solution of a miliusane such as miliusate (20 mg, 0.058 mmol) in MeOH (5 mL) is added sodium borohydride (NaBH.sub.4) (2.2 mg, 1 eq) at 0° C. The reaction mixture is stirred at 0° C. for 1 hour, quenched with saturated ammonium chloride (NH.sub.4Cl) (10 mL), and then extracted with EtOAc (3×20 mL). The combined EtOAc solution is washed sequentially with H.sub.2O (10 mL) and brine (10 mL), dried over Na.sub.2SO.sub.4, and concentrated in vacuo. The reaction mixture is subjected to chromatography separation on a silica gel column, eluted with a solvent system such as petroleum ether:EtOAc 2:1 to afford the 2-hydroxy-miliusane derivative. Unreacted, partially reacted or unwanted compounds as disclosed herein can be removed from the reaction product.

##STR00044##

[0112] The new molecule 2-hydroxy-3, 4-dihydro-miliusate is thus synthesized (see Scheme 7).

General Method for Preparation of Ester Derivatives of 2-Hydroxy-Miliusanes

[0113] A solution of a 2-hydroxy-miliusane such as 2-hydroxy-3, 4-dihydro-miliusate (8 mg, 0.023 mmol) in CH.sub.2Cl.sub.2 (5 mL) is treated with triethylamine (TEA) (16.0 μL, 5 eq), anhydrous acetic acid (Ac.sub.2O) (6.5 μL, 3 eq) and a catalytic amount of DMAP. The reaction mixture is stirred at room temperature for 3 hours, quenched with saturated NaHCO.sub.3 (20 mL), and then extracted with EtOAc (3×10 mL). The combined EtOAc solution is washed with H.sub.2O (10 mL) and brine (10 mL), dried over Na.sub.2SO.sub.4, and concentrated in vacuo. The mixture is chromatographed on a silica gel column, eluting with petroleum ether:EtOAc 4:1 to afford the ester derivative of 2-hydroxy-miliusane. Unreacted, partially reacted or unwanted compounds as disclosed herein can be removed from the reaction product.

##STR00045##

[0114] The new molecule 2-acetoxy-3, 4-dihydro-miliusate is thus synthesized (see Scheme 8).

General Method for Preparation of Aldehyde and Hydroxy Derivatives on The Side Chain of Miliusanes

[0115] To a 25-mL flask is introduced 16 mg (0.5 eq) of selenium dioxide (SeO.sub.2), 10 mL of CH.sub.2Cl.sub.2 and 80 μL (2 eq) of 70% tert-butyl hydroperoxide (TBHP). After the mixture is stirred for 0.5 hours at 25° C. (water bath), a miliusane such as miliusate (100 mg, 0.29 mmol) is added in several minutes. The mixture is stirred at 25° C. for 48 hours. The solvent is then removed in vacuo, and the residue is subjected to silica gel column chromatography separation to give side chain aldehyde and hydroxy derivatives of miliusanes. Unreacted, partially reacted or unwanted compounds as disclosed herein can be removed from the reaction product.

##STR00046##

[0116] The new molecules 8′-oxo-miliusate, 8′-hydroxy-miliusate, 10′-hydroxy-8′-oxo-miliusate, and 8′, 10′-dihydroxy-miliusate are thus synthesized (see Scheme 9).

General Method for Preparation of Miliusol Ester Derivatives

[0117] The solution of 5.0 mg of miliusane such as miliusol (0.016 mmol) in 1.0 mL of dry pyridine is pipetted into a solution of selected acyl chloride reagent such as p-dimethyl-benzyol chloride (0.2 mmol) in 1.0 mL of dry pyridine at 0° C. The reaction is allowed to proceed at 0° C. for 2 hours, and additional 20 hours at room temperature. The reaction product is evaporated in vacuo to dryness to afford a mixture, which is subjected to a silica gel column chromatography to afford the acyl-miliusane ester. Unreacted, partially reacted or unwanted compounds as disclosed herein can be removed from the reaction product. The molecule 5β-(p-dimethylamino-benzoyl)miliusol is thus synthesized (see Scheme 10).

##STR00047##

General Method for Preparation of Miliusane Ammonium Salts

[0118] A chloroform (CHCl.sub.3) solution of an amino group containing miliusane derivative such as 5β-(p-dimethylamino-benzoyl)miliusol (10 mg, 0.022 mmol) and an alkyl halide such as methyl iodide or allyl bromide (10 mL) is refluxed under N.sub.2 atmosphere for 72 hours. Products can be detected by TLC, HPLC and LC-MS. The reaction product is evaporated in vacuo to dryness to afford a mixture, which is subjected to a Si gel column separation to afford miliusane ammonium salt product. Unreacted, partially reacted or unwanted compounds as disclosed herein can be removed from the reaction product.

##STR00048##

##STR00049##

The new molecules 5β-(p-trimethylammonio-benzoyl)miliusol iodide and 5β-(p-dimethyl-allyl-ammonio-benzoyl)miliusol bromide are thus synthesized (see Schemes 11 and 12).

General Method for Preparation of Miliusane Lactam

[0119] A mixture of a miliusane such as miliusol (6.1 mg, 0.02 mmol), a primary amine such as n-butylamine (5.9 μL, 0.06 mmol) or aniline (5.5 μL, 0.06 mmol) and 1-butyl-3-methylimidazolium tetrafluoroborate ([bmim]BF.sub.4, 3.7 μL, 0.02 mmol) is vortexed in a sealed vessel for 30 seconds prior to microwave irradiation at 220° C. for 35 minutes. The cooled reaction mixture is then diluted with 5 mL of EtOAc and washed with saturated aqueous NH.sub.4Cl. The collected EtOAc solution is concentrated in vacuo, and purified over a silica gel column to afford miliusane lactam product. Unreacted, partially reacted or unwanted compounds as disclosed herein can be removed from the reaction product.

##STR00050##

##STR00051##

The new molecules N-(n-butyl)-miliusol lactam and N-phenyl-miliusol lactam are thus synthesized (see Schemes 13 and 14).

[0120] 4β-(N-Phenyl)miliusate is obtained as a white powder with a molecular formula of C.sub.26H.sub.33NO.sub.5 determined by positive HRESIMS and NMR studies. .sup.1H NMR (400 MHz, CDCl.sub.3, J in Hz) 6 (in ppm with reference to the signal of CDCl.sub.3 at δ 7.24 ppm) phenyl protons [7.18 (2H, brt, J=7.5), 6.79 (1H, brt, J=6.7), 6.60 (2H, brs)], 5.58 (1H, d, J=10.6, H-1′), 5.48 (1H, brs, H-5), 4.96 (1H, brd, J=10.8, H-2′), 4.94 (1H, m, H-6′), 3.81 (1H, brdt, J=13.1, 3.5, H-4), 3.57 (1H, d, J=18.1, H-7β), 2.76 (1H, brdd, J=13.1, 3.1, H-3α), 2.66 (1H, dd, J=15.7, 2.7, H-6β), 2.48 (1H, brt, J 13.1, H-3β), 2.24 [3H, s, OC(═O)CH.sub.3], 2.00 (1H, d, J=18.0, H-7α), 2.02 (4H, m, H.sub.2-4′ and H.sub.2-5′), 1.84 (3H, d, J=1.3 Hz, CH.sub.3-9′), 1.75 (1H, dd, J=15.6, 3.3, H-6α), 1.63 (3H, s, CH.sub.3-8′), 1.55 (3H, s, CH.sub.3-10′). HRTOF positive ESIMS m/z calcd for C.sub.26H.sub.33NO.sub.5: 440.2432 [M+1].sup.+, found: 440.2425 [M+1].sup.+.

[0121] 4α-(N-Phenyl)miliusate is obtained as a white powder with a molecular formula of C.sub.26H.sub.33NO.sub.5 determined by positive HRESIMS and NMR studies. .sup.1H NMR (400 MHz, CDCl.sub.3, J in Hz) 8 (in ppm with reference to the signal of CDCl.sub.3 at δ 7.24 ppm) phenyl protons [7.21 (2H, brt, J=7.8), 6.79 (3H, m)], 5.81 (1H, d, J=10.6, H-1′), 5.38 (1H, brq, J=2.9, H-5), 5.01 (1H, brd, J=10.7, H-2′), 4.95 (1H, m, H-6′), 4.04 (1H, m, H-4), 3.57 (1H, d, J=18.1, H-7β), 2.81 (1H, dd, J=14.0, 4.6, H-3β), 2.40 (1H, brd, J=15.3, H-6β), 2.39 (1H, brd, J=13.2, H-3α), 2.23 [3H, s, OC(═O)CH.sub.3], 2.16 (1H, dd, J=15.4, 3.8, H-6α), 2.08 (1H, d, J=18.3, H-7α), 2.04 (4H, m, H.sub.2-4′ and H.sub.2-5′), 1.86 (3H, d, J=1.3 Hz, CH.sub.3-9′), 1.66 (3H, s, CH.sub.3-8′), 1.57 (3H, s, CH.sub.3-10′). HRTOF positive ESIMS m/z calcd for C.sub.26H.sub.33NO.sub.5: 440.2432 [M+1].sup.+, found: 440.2436 [M+1].sup.+.

[0122] 4β-(N-Benzoyl-N-phenyl)miliusate is obtained as a white powder with a molecular formula of C.sub.33H.sub.37NO.sub.6 determined by positive HRESIMS and NMR studies .sup.1H NMR (400 MHz, CDCl.sub.3, J in Hz) 6 (in ppm with reference to the signal of CDCl.sub.3 at δ 7.24 ppm) aromatic protons [7.06-7.25 (8H, m), 6.93-7.00 (2H, m)], 5.66 (1H, brtd, J=8.3, 4.9, H-5), 5.22 (1H, brd, J=10.0, H-2′), 5.10 (1H, d, J=10.0, H-1′), 4.99 (1H, m, H-6′), 4.60 (1H, brtd, J=8.0, 5.9, H-4), 3.33 (1H, d, J=17.4, H-7β), 2.87 (1H, dd, J=16.6, 5.8, H-3α), 2.74 (1H, dd, J=16.6, 8.4, H-313), 2.42 (1H, d, J=17.5, H-7α), 2.19 (1H, dd, J=13.9, 4.9, H-6β), 2.11 (1H, dd, J=14.0, 8.9, H-6α), 2.09 [3H, s, OC(═O)CH.sub.3], 2.05 (4H, m, H.sub.2-4′ and H.sub.2-5′), 1.76 (3H, d, J=1.3 Hz, CH.sub.3-9′), 1.64 (3H, s, CH.sub.3-8′), 1.57 (3H, s, CH.sub.3-10′). HRTOF positive ESIMS m/z calcd for C.sub.33H.sub.37NO.sub.6: 544.2694 [M+1].sup.+, found: 544.2690 [M+1].sup.+.

[0123] 4α-(N-Benzoyl-N-phenyl)miliusate is obtained as a white powder with a molecular formula of C.sub.33H.sub.37NO.sub.6 determined by positive HRESIMS and NMR studies. .sup.1H NMR (400 MHz, CDCl.sub.3, J in Hz) S (in ppm with reference to the signal of CDCl.sub.3 at δ 7.24 ppm) aromatic protons [7.06-7.22 (8H, m), 6.97-7.01 (2H, m)], 5.94 (1H, brq, J=2.2, H-5), 5.58 (1H, d, J=10.6, H-1′), 5.00 (1H, brddd, J=14.8, 3.9, 2.7, H-4), 4.92 (1H, brdd, J=10.6, 1.0, H-2′), 4.88 (1H, m, H-6′), 3.57 (1H, d, J=18.0, H-7β), 2.79 (1H, dd, J=14.9, 13.1, H-3β), 2.55 (1H, brdd, J=15.6, 2.9, H-6μ), 2.48 (1H, brdd, J=13.0, 3.9, 1.2, H-3α), 2.05 [3H, s, OC(═O)CH.sub.3], 1.96 (4H, m, H.sub.2-4′ and H.sub.2-5′), 1.88 (1H, dd, J=15.6, 3.4, H-6α), 1.98 (1H, d, J=18.2, H-7α), 1.67 (3H, d, J=1.3 Hz, CH.sub.3-9′), 1.62 (3H, s, CH.sub.3-8′), 1.52 (3H, s, CH.sub.3-10′). HRTOF positive ESIMS m/z calcd for C.sub.33H.sub.37NO.sub.6: 544.2694 [M+1].sup.+, found: 544.2699 [M+1].sup.+.

[0124] Hexadehydro-miliusate is obtained as a white powder with a molecular formula of C.sub.20H.sub.32O.sub.5 determined by positive HRESIMS and NMR studies .sup.1H NMR (400 MHz, CDCl.sub.3, J in Hz) δ (in ppm with reference to the signal of CDCl.sub.3 at δ 7.24 ppm) 5.29 (1H, brquintet, J=3.2, H-5), 5.01 (1H, brdd, J=11.6, 2.1, H-1′), 3.43 (1H, d, J=17.9, H-7β), 2.41-2.64 (3H, m), 2.28 (1H, m), 2.13 [3H, s, OC(═O)CH.sub.3], 2.04 (1H, m), 2.01 (1H, d, J=17.9, H-7α), 1.83 (1H, dd, J=15.2, 3.7, H-6α), 1.68 (1H, m), 1.27-1.56 (4H, m), 0.94-1.28 (5H, m), 0.88 (3H, d, J=6.7 Hz), 0.84 (3H, d, J=6.6 Hz), 0.83 (3H, d, J=6.6 Hz). HRTOF positive ESIMS m/z calcd for C.sub.20H.sub.32O.sub.5: 353.2323 [M+1].sup.+, found: 353.2318 [M+1].sup.+.

[0125] 3,4-Didehydro-miliusate is obtained as a white powder with a molecular formula of C.sub.20H.sub.28O.sub.5 determined by positive HRESIMS and NMR studies. .sup.1H NMR (400 MHz, CDCl.sub.3, J in Hz) 8 (in ppm with reference to the signal of CDCl.sub.3 at δ 7.24 ppm) 5.74 (1H, d, J=10.6, H-1′), 5.27 (1H, brquintet, J=3.0, H-5), 4.98 (1H, brdd, J=10.6, 1.0, H-2′), 4.94 (1H, m, H-6′), 3.54 (1H, d, J=18.0, H-7β), 2.55 (1H, brdt, J=15.3, 3.0), 2.42 (1H, brtd, J=14.6, 6.1), 2.30 (1H, brdd, J=13.4, 5.8, 2.9), 2.25 (1H, m), 2.15 [3H, s, OC(═O)CH.sub.3], 2.01 (4H, m, H.sub.2-4′ and H.sub.2-5′), 2.00 (1H, d, J=18.0, H-7α), 1.96 (2H, m), 1.82 (3H, d, J=1.3 Hz, CH.sub.3-9′), 1.64 (3H, s, CH.sub.3-8′), 1.55 (3H, s, CH.sub.3-10′). HRTOF positive ESIMS m/z calcd for C.sub.20H.sub.28O.sub.5: 349.2010 [M+1].sup.+, found: 349.2005 [M+1].sup.+.

[0126] 2′,3′,6′,7′-Tetradehydro-miliusate is obtained as a white powder with a molecular formula of C.sub.26H.sub.33NO.sub.5 determined by positive HRESIMS and NMR studies. .sup.1H NMR (400 MHz, CDCl.sub.3, J in Hz) 6 (in ppm with reference to the signal of CDCl.sub.3 at δ 7.24 ppm) 6.87 (1H, brddd, J=10.2, 4.2, 1.8, H-4), 6.16 (1H, brdd, J=10.2, 1.1, H-3), 5.52 (1H, brqd, J=4.0, 1.1, H-5), 4.72 (1H, brdd, J=10.8, 2.6, H-1′), 3.20 (1H, d, J=17.5, H-7β), 2.37 (1H, brdtd, J=14.9, 3.9, 0.9), 2.26 (1H, brdt, J=14.7, 4.2), 2.25 (1H, d, J=17.6, H-7α), 2.11 [3H, s, OC(═O)CH.sub.3], 1.83 (1H, dd, J=15.2, 3.7, H-6α), 1.40-1.60 (2H, m), 0.93-1.40 (7H, m), 0.88 (3H, d, J=6.7 Hz), 0.83 (6H, d, J=6.5 Hz). HRTOF positive ESIMS m/z calcd for C.sub.20H.sub.30O.sub.5: 351.2166 [M+1].sup.+, found: 351.2156 [M+1].sup.+.

[0127] 2-Hydroxy-3,4-didehydro-miliusate is obtained as a white powder with a molecular formula of C.sub.20H.sub.30O.sub.5 determined by positive HRESIMS and NMR studies. .sup.1H NMR (400 MHz, CDCl.sub.3, J in Hz) 8 (in ppm with reference to the signal of CDCl.sub.3 at δ 7.24 ppm) 5.24 (1H, brd, J=10.4, H-2′), 4.99 (1H, m, H-6′), 4.82 (1H, d, J=10.5, H-1′), 4.76 (1H, brseptet, J=4.7, H-5), 3.70 (1H, brs, H-2), 2.47 (1H, d, J=17.3, H-7β) 2.33 (1H, brdd, J=17.3, 1.4, H-7α), 2.07-2.24 (5H, m), 2.02 [3H, s, OC(═O)CH.sub.3], 1.98 (1H, brt, J=11.4), 1.77-1.92 (4H, m), 1.74 (3H, d, J=1.3 Hz, CH.sub.3-9′), 1.67 (3H, s, CH.sub.3-8′), 1.60 (3H, s, CH.sub.3-10′). HRTOF positive ESIMS m/z calcd for C.sub.20H.sub.30O.sub.5: 351.2166 [M+1].sup.+, found: 351.2160 [M+1].sup.+.

[0128] 2-Acetoxy-3,4-didehydro-miliusate is obtained as a white powder with a molecular formula of C.sub.22H.sub.32O.sub.6 determined by positive HRESIMS. HRTOF positive ESIMS m/z calcd for C.sub.22H.sub.32O.sub.6: 393.2272 [M+1].sup.+, found: 393.2266 [M+1].sup.+.

[0129] 8′-Oxo-miliusate is obtained as a white powder with a molecular formula of C.sub.20H.sub.24O.sub.6 determined by positive HRESIMS and NMR studies. .sup.1H NMR (400 MHz, CDCl.sub.3, J in Hz) 8 (in ppm with reference to the signal of CDCl.sub.3 at δ 7.24 ppm) 9.37 (1H, s, H-8′), 6.81 (1H, brddd, J=10.2, 4.0, 0.8, H-4), 6.33 (1H, brtq, J=7.1, 1.3, H-6′), 6.00 (1H, brdd, J=10.2, 1.2, H-3), 5.55 (1H, brq, J=4.2, H-5), 5.45 (1H, d, J=9.9, H-1′), 5.19 (1H, brdq, J=9.9, 1.3, H-2′), 3.31 (1H, d, J=17.6, H-7β), 2.43 (1H, brt, J 7.5, H-5′a), 2.41 (1H, brt, J=7.3, H-5′b), 2.36 (1H, brddd, J=14.6, 4.3, 0.9, H-6β), 2.27 (1H, d, J=17.6, H-7α), 2.24 (1H, dd, J=14.7, 5.5, H-6α), 2.19 (2H, brt, J=7.3, H.sub.2-4′), 2.13 [3H, s, OC(═O)CH.sub.3], 1.70 (3H, brd, J=1.1 Hz, CH.sub.3-10′), 1.69 (3H, d, J=1.3 Hz, CH.sub.3-9′). HRTOF positive ESIMS m/z calcd for C.sub.20H.sub.24O.sub.6: 361.1646 [M+1].sup.+, found: 361.1640 [M+1].sup.+.

[0130] 8′-Hydroxy-miliusate is obtained as a white powder with a molecular formula of C.sub.20H.sub.2606 determined by positive HRESIMS and NMR studies. .sup.1H NMR (400 MHz, CDCl.sub.3, J in Hz) δ (in ppm with reference to the signal of CDCl.sub.3 at δ 7.24 ppm) 6.83 (1H, brddd, J=10.3, 4.3, 1.3, H-4), 6.08 (1H, brdd, J=10.2, 1.2, H-3), 5.57 (1H, d, J=10.1, H-1′), 5.56 (1H, m, H-5), 5.17 (1H, m, H-6′), 5.05 (1H, brdq, J=10.1, 1.0, H-2′), 4.01 (1H, ABbrd, J=13.5, H-8′a), 3.95 (1H, ABbrd, J=13.4, H-8′b), 3.43 (1H, d, J=17.7, H-7β), 2.44 (1H, brddd, J=15.0, 3.2, 1.4, H-6β), 2.23 (1H, dd, J=15.1, 5.5, H-6α), 2.18 (1H, d, J=17.7, H-7α), 2.14 [3H, s, OC(═O)CH.sub.3], 2.12 (2H, m, H.sub.2-5′), 2.07 (2H, m, H.sub.2-4′), 1.65 (3H, d, J=1.3 Hz, CH.sub.3-9′), 1.61 (3H, s, CH.sub.3-10′). HRTOF positive ESIMS 172/Z calcd for C.sub.20H.sub.26O.sub.6: 363.1802 [M+1].sup.+, found: 363.1808 [M+1].sup.+.

[0131] 10′-Hydroxy-8′-oxo-miliusate is obtained as a white powder with a molecular formula of C.sub.20H.sub.24O.sub.7 determined by positive HRESIMS and NMR studies.

[0132] .sup.1H NMR (400 MHz, CDCl.sub.3, J in Hz) δ (in ppm with reference to the signal of CDCl.sub.3 at δ 7.24 ppm) 9.42 (1H, s, H-8′), 6.82 (1H, brddd, J=10.2, 3.9, 0.8, H-4), 6.45 (1H, brt, J=7.4, H-6′), 6.02 (1H, brdd, J=10.2, 1.2, H-3), 5.56 (1H, brq, J=4.2, H-5), 5.45 (1H, d, J=9.9, H-1′), 5.21 (1H, brdq, J=9.9, 1.2, H-2′), 4.35 (1H, ABd, J=12.8, H-10′a), 4.30 (1H, ABd, J=12.7, H-10′b), 3.32 (1H, d, J=17.6, H-713), 2.53 (1H, brtd, J=7.6, 2.3, H-5′a), 2.51 (1H, brt, J=7.4, H-5′b), 2.39 (1H, brddd, J=14.6, 4.3, 0.7, H-6β), 2.26 (1H, d, J=17.7, H-7α), 2.23 (2H, brt J=7.1, H.sub.2-4′), 2.23 (1H, overlap, H-6α), 2.14 [3H, s, OC(═O)CH.sub.3], 1.70 (3H, d, J=1.3 Hz, CH.sub.3-9′). HRTOF positive ESIMS m/z calcd for C.sub.20H.sub.24O.sub.7: 377.1595 [M+1].sup.+, found: 377.1600 [M+1].sup.+.

[0133] 8′, 10′-Dihydroxy-miliusate is obtained as a white powder with a molecular formula of C.sub.20H.sub.26O.sub.7 determined by positive HRESIMS and NMR studies .sup.1H NMR (400 MHz, CDCl.sub.3, J in Hz) δ (in ppm with reference to the signal of CDCl.sub.3 at δ 7.24 ppm) 9.42 (1H, s, H-8′), 6.83 (1H, brddd, J=10.2, 4.1, 1.1, H-4), 6.10 (1H, brdd, J=10.2, 1.1, H-3), 5.56 (1H, brq, J=4.1, H-5), 5.50 (1H, d, J=10.0, H-1′), 5.31 (1H, brt, J=7.1, H-6′), 5.08 (1H, brdq, J=10.0, 1.2, H-2′), 4.25 (1H, ABd, J=12.8, H-10′a), 4.21 (1H, ABd, J=12.8, H-10′b), 4.19 (1H, ABd, J=13.0, H-8′a), 4.15 (1H, ABd, J=13.0, H-8′b), 3.36 (1H, d, J=17.7, H-7β), 1.63 (2H, brs, 2×OH), 2.40 (1H, brddd, J=14.9, 3.7, 1.1, H-6β), 2.23 (1H, dd, J=14.9, 5.5, H-6α), 2.22 (1H, d, J=17.9, H-7α), 2.17 (1H, brt, J=6.7, H-5′a), 2.16 (1H, brt, J=7.2, H-5′b), 2.13 [3H, s, OC(═O)CH.sub.3], 2.07 (2H, brt J=6.7, H.sub.2-4′), 1.65 (3H, d, J=1.3 Hz, CH.sub.3-9′). HRTOF positive ESIMS m/z calcd for C.sub.20H.sub.26O.sub.7: 379.1751 [M+1]+, found: 379.1750 [M+1].sup.+.

[0134] 5β-(p-Trimethylammonio-benzoyl)miliusol iodide is obtained as a white powder with a molecular formula of C.sub.28H.sub.36NO.sub.5I determined by positive HRESIMS. HRTOF positive ESIMS m/z calcd for C.sub.28H.sub.36NO.sub.5: 466.2588 [M].sup.+, found: 466.2579 [M].sup.+.

[0135] 5β-(p-Dimethyl-allyl-ammonio-benzoyl)miliusol bromide is obtained as a white powder with a molecular formula of C.sub.30H.sub.38NO.sub.5Br determined by positive HRESIMS. HRTOF positive ESIMS m/z calcd for C.sub.30H.sub.38NO.sub.5: 492.2745 [M].sup.+, found: 492.2735 [M].sup.+.

[0136] N-(n-Butyl)-miliusol lactam is obtained as a white powder with a molecular formula of C.sub.22H.sub.33NO.sub.3 determined by positive HRESIMS. HRTOF positive ESIMS m/z calcd for C.sub.22H.sub.34NO.sub.3: 360.2539 [M+1].sup.+, found: 360.2530 [M+1].sup.+.

[0137] N-Phenyl-miliusol lactam is obtained as a white powder with a molecular formula of C.sub.24H.sub.29NO.sub.3 determined by positive HRESIMS. HRTOF positive ESIMS m/z calcd for C.sub.24H.sub.34NO.sub.3: 380.2226 [M+1].sup.+, found: 380.2235 [M+1].sup.+.

[0138] Cell Culture Panel Bioassays.

[0139] All pure compounds of the present invention are evaluated against human cancer cell lines using a cytotoxicity screening panel. Cytotoxicity assays involving oral epidetmoid (KB), colon (HCT116), prostate (LNCaP), breast (MCF-7), lung (A549) and melanoma (A375) carcinoma cell lines, are performed using sulforhodamine B according to established protocols (Zhang H J, Ma C Y, Hung N V, Cuong N M, Tan G T, Santarsiero B D, Mesecar A D, Soejarto D D, Pezzuto J M, Fong H H S. Miliusanes, a class of cytotoxic agents from Miliusa sinensis. Journal of Medicinal Chemistry 2006; 49: 693-708; and Jutiviboonsuk A, Zhang H J, Tan G T, Ma C M, Hung N V, Cuong N M, Bunyapraphatsara N, Soejarto D D, Fong H H S. Bioactive constituents from the roots of Bursera tonkinensis. Phytochemistry 2005; 66: 2745-2751.). KB cells are maintained in DMEM medium. LNCaP cells are maintained in RPMI1640 medium with hormone-free 10% heat-activated FBS (fetal bovine serum) supplemented with 0.1 nM testosterone. MCF-7 cells are maintained and assayed in MEME medium containing 10 mg/L of insulin. HCT116 cells are maintained in McCoy's 5A medium supplemented with 10% fetal bovine serum. A549 and A375 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 μL cell suspension (3×10.sup.4 cells in 1 ml media) is incubated with 10 μL sample solutions, in triplicate, in 96-well tissue culture plate at 37° C. in a humidified atmosphere of 5% CO.sub.2 in air for 72 hours. 10 μL 10% aqueous DMSO is used as control group. Then the cells are fixed to plastic substratum by the addition of 100 μL cold 20% aqueous trichloroacetic acid and washing with water after incubation at 4° C. for 30 min. After staining cells with 100 μL 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 μL 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.sub.50 values, the dose that inhibit cell growth by 50%, are calculated using nonlinear regression analysis (percent survival versus concentration).

[0140] Antitumor Animal Study.

[0141] 4β-(N-Phenyl)miliusate shows potent cell killing activity in our in vitro evaluation system. In order to determine its antitumor activity, HCT116 xenograft animal study is used to evaluate the anticancer activity of 4β-(N-phenyl)miliusate in comparison with the clinically used anticancer drug paclitaxel. All animal studies are approved and performed according to Animal Care and Use Guidelines of the Animal Ethics Committee at Hong Kong Baptist University and performed following Animal Care and Use guidelines set by NIH (National Institute of Health, USA). BALB/c nude mice, SPF class, male or female, 7-8 weeks old, are purchased from Charles River Laboratories. Before the experiment, the mice are kept for one week of acclimatization to SPF class laboratory conditions.

[0142] 4β-(N-Phenyl)miliusate was tested for its antitumor activity against HCT116 cancer cells using a number of nude mice (Balc/nu/nu, female) in comparison of paclitaxel. HCT116 cancer cells are subcutaneously implanted with 5×10.sup.6 cells in the rear flank of each mouse. After 10 days, solid tumors with average size of about 80 mm.sup.3 appeared at the implanted sites. The mice are then divided into three groups: one dose (20 mg/kg: 10 mice) group of 4β-(N-phenyl)miliusate, one dose (10 mg/kg: 10 mice) of paclitaxel and one dose of vehicle (negative control: 10 mice). Every other day injections at i.p. sites are scheduled for 21 days. Weights of mice and tumor diameters are measured twice a week until the end of the experiment. The tumor size in mm.sup.3 is calculated by the formula: tumor size=length×width×height (L×W×H).

[0143] Having now fully described the present invention in some detail by way of illustration and examples for purposes of clarity of understanding, it will be obvious to one of ordinary skill in the art that the same can be performed by modifying or changing the present invention within a wide and equivalent range of conditions, formulations and other parameters without affecting the scope of the present invention or any specific embodiment thereof, and that such modifications or changes are intended to be encompassed within the scope of the appended claims. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the present invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of the present invention.

[0144] As used herein, “comprising” is synonymous with “including,” “containing,” or “characterized by,” and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, “consisting of” excludes any element, step, or ingredient not specified in the claim element. As used herein, “consisting essentially of” does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. In each instance herein any of the terms “comprising”, “consisting essentially of” and “consisting of” may be replaced with either of the other two terms.

[0145] When a group of materials, compositions, components or compounds is disclosed herein, it is understood that all individual members of those groups and all subgroups thereof are disclosed separately. When a Markush group or other grouping is used herein, all individual members of the group and all combinations and subcombinations possible of the group are intended to be individually included in the disclosure. Every formulation or combination of components described or exemplified herein can be used to practice the present invention, unless otherwise stated. Whenever a range is given in the specification, for example, a temperature range, a time range, or a composition range, all intermediate ranges and subranges, as well as all individual values included in the ranges given are intended to be included in the disclosure. In the disclosure and the claims, “and/or” means additionally or alternatively. Moreover, any use of a term in the singular also encompasses plural forms.

[0146] All references cited herein are hereby incorporated by reference in their entirety to the extent that there is no inconsistency with the disclosure of this specification. Some references provided herein are incorporated by reference to provide details concerning sources of starting materials, additional starting materials, additional reagents, additional methods of synthesis, additional methods of analysis, additional biological materials, additional cells, and additional uses of the present invention. All headings used herein are for convenience only. All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the present invention pertains, and are herein incorporated by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference. 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. For example, when 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.

[0147] If desired, 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.

[0148] While the foregoing invention has been described with respect to various embodiments and examples, it is understood that other embodiments are within the scope of the present invention as expressed in the following claims and their equivalents. Moreover, the above specific examples are to be construed as merely illustrative, and not limitative of the reminder of the disclosure in any way whatsoever. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest extent. All publications recited herein are hereby incorporated by reference in their entirety.

INDUSTRIAL APPLICABILITY

[0149] The present invention discloses new anticancer agents based on the miliusane natural products. The present invention also includes its preparation and application method for treating cancer.