JIANG ZHI-DONG
GALLAGHER REX T
| 1. | A method of treating cancer, comprising administering to a patient an effective anticancer amount of a pharmaceutical composition including a compound of having the formula (I) : wherein A is C2_10 1methylalkyl, lfC^^ alkyloxy)ethyl, 1(C1_10 alkylthio)ethyl, C2_10 hydroxycarbonylalkyl, C2_10 alkenyl, C2 alkenylene, X1(CH)R1, or X^CJR^ wherein Xx is H, F, Cl, Br, I, C1_10 alkyl, C2_10 alkenyl, c2ιo alkenylene (where A is X^CJR.^, C^^ haloalkyl, 0Ri# SRi# NRjP^, or NRi(C=0)Rii, each of RL and Rii being independently selected from H, C^^ alkyl, C3_6 heteroaryl, and (C6_14 aryl) (C0_6 alkyl) ; and R^ is H or methyl, or =0 where A is X1(C)R1; each of Ra and Rb is selected independently from H, Cλ_6 alkyl, C^g haloalkyl, and Cλ_6 alkenyl, except that where A is C2_10 alkenylene, Rb is deleted; X2 is H, F, Cl, Br, I, N03, S04, C^Q alkyl, ciio haloalkyl, OR^, 0(C=0)Riii, SR^, NR^R^, or NRiii(C=0)Riv, wherein each of RLii and Riv is independently selected from H, CJ^Q alkyl, and (C6_14 aryl) (C0_6 alkyl) ; each of and Y2 is independently selected from H, Cλ_10 alkyl, OR,,, SR,., NRyR^, and NRv(C=0)Rvi, wherein each of Ry and R^ is independently selected from H, Cλ_10 alkyl, and (C6_14 aryl) (C0_6 alkyl); or Y and Y2, taken together, are =0, =S, =NRvii, ^NOR^i, or =CHRvii in the E or Z configuration, wherein R^ is H, ^^ alkyl, (C6_14 aryl) (C0_10 alkyl) , or (C^Q alkyl) (C6_14 aryl); and Z is H, C1_10 alkyl, C2_10 alkenyl, or a monovalent C1_30 organic moiety, said moiety being bonded to the carbon which is also bonded to Yχ and Y by a heteroatom selected from O, S, and N; or a pharmaceutically acceptable salt or ester thereof; and a pharmaceuticallyacceptable carrier, thereby treating said cancer. |
| 2. | A method of claim 1, wherein said organic moiety is a dehydro moiety of a C^Q natural product. |
| 3. | A method of claim 2, wherein said natural product is selected from the group consisting of monoterpenes, diterpenes, sesquiterpenes, steroids, and polyketides. |
| 4. | A method of claim 3, wherein said natural product is selected from sesquiterpenes. |
| 5. | A method of claim 3, wherein said natural product is selected from ere ophilanes. |
| 6. | A method of claim 3, wherein Z is selected from farnesol, geranylgeraniol, 3hydroxy9,11 eremophiladien8one (e.g., 3α,7αform, and 3α,70form) ; 3,12dihydroxy9,ll, (13)eremophiladien8one (3α,70H form) ; eremofortin B; 9,lleremophiladien8one; 6α,7α epoxy3α,13dihydroxy,9,12eremophiladien9one; 13 desoxyphomenone, phaseolinone, phomenone, 1,3dihydroxy 9,lleremophiladien8one (10,3α,7/3Hform) , petasin, S petasin, isopetasin, isoSpetasin, petasol, and isopetasol. |
| 7. | A method of claim 1, wherein Z is OR^^, SRviii' or NK iiΛx' wherein ^ϋ is H, C1_10 alkyl, c330 cycloalkyl, C3_10 heterocyclic radical, C3_10 heteroaromatic radical, a hydroxyl protecting group where Z is ORviii, a thiol protecting group where Z is SR^^, an amino protecting group where Z is NR^^R^, or a carboxyl protecting group where Yα and Y2 taken together are C=0 and Z is O ^^; and Riχ is H or C^Q alkyl. |
| 8. | A method of claim 7, wherein Z is 0Rviii or NRviiiRix, wherein ^ L is H, C _10 alkyl, C3_1S cycloalkyl, C3_10 heterocyclic radical, or C3_10 heteroaryl. |
| 9. | A method of claim 7, wherein Z is ORviii or NRviiiRiχf and Rviϋ is a hydroxyl protecting group where Z is O v , an amino protecting group where Z is NR^ϋ^^, or a carboxyl protecting group where Yλ and Y2 taken together are C=0 and Z is OR^^; and Rix is selected from H and Cie alkyl. |
| 10. | A method of claim 1, wherein j and Y2 taken together are =0. |
| 11. | A method of claim 10, wherein X2 is ORviii. |
| 12. | A method of claim l, wherein A is the side chain of threonine, C2_10 1methylalkyl, lCC^g alkyloxy) ethyl, lfc^g alkylthio)ethyl) , X1(CH)R1, or X1(C)R1, wherein χ is H, C _10 alkyl, C2_10 alkenyl, C2_10 alkenylene (where A is X1(C)R1) , C1_10 haloalkyl, ORif SRi, NRiRj^i, or NRi(C=0)Rii, each of R± and Rii being independently selected from H, C^g alkyl, and (C6_10 aryl) (C0_3 alkyl); and Rλ is H, C^g alkyl, or =0 (where A is X^CJRi); X2 is or Y1 and Y2, taken together, are =0, =S, =NRvii, or =NORvii. |
| 13. | A method of claim 1, wherein the compound has the formula (V) (V) . |
| 14. | A method of treating cancer, comprising administering to a patient an effective amount of a pharmaceutical composition including a compound of the formula (VI) : wherein each of R1 and R2 is independently selected from H, C^^ alkyl, Cχ_10 alkoxy, C2_10 alkenyl, c2ιo alkenoxy, C6_10 aryl, substituted or unsubstituted c6ιo arylcarbonyloxy, and C2_10 acyloxy; or R1 and R2 taken together are =0 or substituted or unsubstituted C _ 10 alkylenedioxy, carbonyldioxy, and other carbonyl protecting groups; when there is no double bond between C7 and Cll, each of R3, R4, and R5 is independently selected from C _ 10 alkyl, Cj.^ alkoxy, and C^Q acyloxy; or R4 and R5 taken together are 0, C^^ alkylidene, or CJ^Q alkenylidene; and when there is a double bond between C7 and Cll, R5 is absent; R6 is λ^10 alkylamino or C1 >10 alkylthio when there is no double bond between C9 and C10; R6 is absent when there is a double bond between C9 and C10; X is OH, ORp where Rp is CJ^Q alkyl, C6_10 aryl, c2ιo ac 1» C610 arylcarbonyl, tri(C1_6 alkyl)silyl, and other hydroxyprotecting groups, or Y having the formula: Z is (C=0)NH, (C=0)0, or 0; and each of ra rh is independently selected from H, methyl, halomethyl, hydroxy, and ORp, where Rp is C1_10 alkyl, C6_ 10 aryl, C2_10 acyl, C6_10 arylcarbonyl, and other hydroxy protecting groups, provided that at least two of ra rh are hydroxyl groups which do not form a gre_ndiol;or a pharmaceutically acceptable salt thereof; and a pharmaceuticallyacceptable carrier, thereby treating said cancer. |
| 15. | The method of claim 14, provided that where X is OH, C(R3) (R4) (R5) is neither isopropenyl nor isopropylidene. |
| 16. | The method of claim 14, provided that where X is O(trialkylsilyl) , C(R3) (R4) (R5) is not 2hydroxyl isopropyl. |
| 17. | The method of claim 14, provided that when R1, R2, and X are H, C(R3) (R4) (R5) is not isopropenyl. |
| 18. | The method of claim 14, where one of ra and rb is methyl, one of rc and rd is hydroxyl, one of re and rf is H, and one of r„ and rh is hydroxyl and the other is methyl. |
| 19. | The method of claim 14, where at least three of ra rh are hydroxyl and at least two of the remaining of ra rh are methyl. |
Summary of the Invention The invention features anticancer agents and methods of treating cancer with these agents. The methods include administering to a patient an effective anticancer amount of a pharmaceutical composition containing one or more purified or synthetic compounds having the formula (I) :
In formula (I), A is C 2 _ 10 1-methylalkyl, l- (C λ _ 10 alkyloxy)ethyl, l-fC j .^ alkylthio)ethyl, C 2 _ 10 hydroxycarbonylalkyl, C 2 _ 10 alkenyl, C 2 alkenylene, XifCH-JRi, or X^C-JR^ wherein X j^ is H, F, Cl, Br, I, C λ _ 10 alkyl, C 2 _ 10 alkenyl, C 2 _ 10 alkenylene (where A is X } (C- )R l ), C-,..,^ haloalkyl, ORi, SR if KR R LL , or NR i (C=0)R ii , each of R i and R L being independently selected from H, Ci-io alkyl, C 3 . 6 heteroaryl, and (C 6 _ 14 aryl) (C 0 _ 6 alkyl); and R α is H or methyl, or =0 where A is X 1 (C-)R 1 . Each of R a and R b is selected independently from H, C^g alkyl, C 1-6 haloalkyl, and C^ 6 alkenyl, except that where A is 2 - 10 alkenylene, R b is deleted. X 2 is H, F, Cl, Br, I, N0 3 , S0 4 , C 1 _ 10 alkyl, C-^ Q haloalkyl, 0R i , 0(C=0)R iii7
SR j ^, NR i;Li R iv , or NR iii (C=0)R iv , wherein each of R iii and R iv is independently selected from H, C^.^ alkyl, and (C 6 _ 14 aryl) (C 0 _ 6 alkyl) .
Each of Y 2 and Y 2 is independently selected from H, C^ Q alkyl, OR.,, SR_., NR y R vL , and NR v (C=0)R vi , wherein each of R.. and R vi is independently selected from H, C l . 10 alkyl, and (C 6 _ 14 aryl) (C 0 _ 6 alkyl); or Y x and Y 2 , taken together, are =0, =S, =NR vii , =NOR vii , or =CHR vii in the E or Z configuration, wherein R vii is H, CJ^ Q alkyl, (C 6 _ 14 aryl)-(C 0 _ 10 alkyl), or (C^ Q alkyl) (C 6 _ 14 aryl). z is H, ι-ιo alkyl, C 2 _ 10 alkenyl, or a monovalent 0 2 . 30 organic moiety, said moiety being bonded to the carbon which is also bonded to Y and Y 2 by a heteroatom selected from O, S, and N. The invention also features pharmaceutically acceptable salts or esters of formula (I) . Compounds for use in this method include, for example, the compound 3390 (formula V) , and synthetic intermediates of the disclosed compounds, such as formulae (II) and (III) .
The invention also features anticancer agents and methods of treating cancer with the agent, comprising administering to a patient an effective amount of a pharmaceutical composition including a compound of the formula (VI) and a pharmaceutically acceptable carrier.
(VI)
In formula VI, each of R 1 and R 2 is independently selected from H, C 1 _ 10 alkyl, C-^ Q alkoxy, C 2 _ 10 alkenyl, c 2 -ιo alkenoxy, C 6 _ 10 aryl, substituted or unsubstituted c 6 - i o arylcarbonyloxy, and C 2 _ 10 acyloxy; or R 1 and R 2 taken together are =0 or substituted or unsubstituted c 2 _ 10 alkylenedioxy, carbonyldioxy, and other carbonyl- protecting groups. When there is no double bond between C-7 and C-ll, each of R 3 , R 4 and R 5 is independently selected from CJ^ Q alkyl, 0 2 . 0 alkoxy, and C^^ acyloxy; or R 4 and R 5 taken together are =0, C^^ alkylidene, or i - i o alkenylidene. When there is a double bond between C-7 and C-ll, R 5 is absent.
R 6 is C j .i Q alkyla ino or C _ 10 alkylthio when there is no double bond between C-9 and C-10; R 6 is absent when there is a double bond between C-9 and C-10. X is OH, OR p where p is C^^ alkyl, C 6 _ 10 aryl, C 2 _ 10 acyl, C 6 _ 10 arylcarbonyl, tri(C 2 _ 6 alkyl)silyl, and other hydroxy-protecting groups, or Y having the formula:
Z is -(C=0)-NH-, -(0=0)-O-, or -O-. Each of r a - r h is independently selected from H, methyl, halomethyl, hydroxy, and OR_, where R^ is 0 2 . 20 alkyl, C 6 _ 2 o aryl, C 2 _ 10 acyl, C 6 _ 2 o arylcarbonyl, and other hydroxy-protecting groups, provided that at least two of r a - r h are hydroxyl groups which do not form a grem-diol. The invention includes a pharmaceutically acceptable salt or ester of a disclosed compound.
Treating cancer in a patient includes achieving, partially or substantially, one or more of the following:
arresting the growth or spread of a cancer, reducing the extent of a cancer (e.g., reducing size of a tumor or reducing the number of affected sites) , inhibiting the growth rate of a cancer, and ameliorating or improving a clinical symptom or indicator associated with a cancer (such as tissue or serum components) .
Other features and advantages of the present invention will be apparent from the following detailed description, and also from the appended claims. Detailed Description of the Invention
A. Abbreviations
AIBN, 2,2'-azobis(isobutyronitrile) ; DCC, dicyclohexylcarbo-diimide; DMAP, dimethylaminopyridine; HOBT, 1-hydroxy-benzotriazole hydrate; PMB, p- methoxybenzyl; DMF, dimethyl-formamide; MS, ethanesulfonyl; Tf, trifluoromethane-sulfonyl; and Perkin (AIBN, RSH) .
B. Terms
Acetogenins are naturally derived from polyketones having a 0-dicarbonyl structure. Examples of acetogenins which contain at least one hydroxyl group include chrysin, protocoin, emodin, atrovenetin, visamminol, alternariol, and α-sorigenin.
An alkyl group is a branched or unbranched hydrocarbon that may be substituted or unsubstituted.
Examples of branched alkyl groups include isopropyl, sec- butyl, isobutyl, tert-butyl, sec-pentyl, isopentyl, tert- pentyl, isohexyl. Substituted alkyl groups may have one, two, three or more substituents, which may be the same or different, each replacing a hydrogen atom. Substituents are halogen (e.g., F, Cl, Br, and I), hydroxyl, protected hydroxyl, amino, protected amino, carboxy, protected carboxy, cyano, methylsulfonylamino, alkoxy, acyloxy, nitro, and lower haloalkyl. Similarly, cycloalkyl, aryl
(e.g., phenyl, naphthyl, biphenylyl, 2,4-dihalophenyl, 2,6-dihalophenyl) , arylalkyl (e.g., benzyl), alkylaryl (e.g., 2,4-dimethylphenyl) , heteroaryl, and heterocyclic radical groups may be subsituted with one or more of the above substituting groups. Examples of cycloalkyl groups are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.
An alkylene is a bivalent radical derived from alkanes by removing two hydrogens from two different carbon atoms. Examples of alkylenes include -CH 2 -CH(R)- CH 2 and 1,4-cyclohexylene. An alkylidene is a bivalent radical derived from alkenes by removing two hydrogens from the same carbon atom, such as l-propanyl-3-ylidene (=CH-CH 2 -CH 2 -) . Unless otherwise indicated, complex moieties such as arylalkyl or alkylcarbonyloxy groups are bonded to the rest of the molecule via the last-named radical moiety, in these examples, alkyl and oxy, respectively. Exceptions include moieties such as X 2 (CH-)R 2 and X (C- )R χ , which are bonded to the rest of the molecule via the central carbon atom, as indicated by the radical- indicating convention "-". Thus, in the former case, the central carbon atom C is bonded to H, X l f R l r and finally the carbon atom which is also bonded to R a and R b as shown in formula (I) .
An aryl group is a C 6 _ 2 o aromatic ring, wherein the ring is made of carbon atoms (e.g., C 6 _ 14 , C 6 _ 10 aryl groups) . Examples of haloalkyl include fluoromethyl, dichloromethyl, trifluoromethyl, 1,1-difluoroethyl, and 2,2-dibromoethy1.
A dehydro moiety of a C λ _ 30 organic compound (e.g., a natural product) is the organic compound with a hydrogen atom removed. The removed hydrogen leaves a single valence on a carbon, nitrogen, oxygen, sulfur, or silicon atom of the organic compound. The dehydro moiety
is bonded to a compound disclosed herein via the single valence. Examples include (i) natural products selected from the group consisting of monoterpenes, diterpenes, sesguiterpenes, steroids, and polyketides; and (ii) naturally occurring amino acids.
A heterocyclic radical contains at least one ring structure which contains carbon atoms and at least one heteroatom (e.g., N, O, S, or P) . A heteroaryl is an aromatic heterocyclic radical. Examples of heterocyclic radicals and heteroaryl groups include: thiazolyl, thienyl, thiophenyl, furyl, 1-isobenzofuranyl, 2H- chromen-3-yl, 2H-pyrrolyl, N-pyrrolyl, imidazolyl, pyrazolyl, isothiazolyl, isooxazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyradazinyl, indolizinyl, isoindolyl, indolyl, indazolyl, purinyl, phthalazinyl, cinnolinyl, and pteridinyl. A heterocylic radical or heteroaryl may be attached to another moiety via a carbon atom or a heteroatom of the radical.
A (C n alkyl)oxycarbonyl group has the formula R-O- (C=0)-. For example, (C 1-6 alkyl)oxycarbonyl, therefore, includes methoxycarbonyl and hexyloxycarbony1. A C 2 _ 10 acyl group as used herein is of the formula -(C=0)-L 3 and contains 1 to 10 carbon atoms and 1-5 heteratoms. Examples of such acyl groups include for yl, acetyl, benzyloxycarbonyl, tert-butoxycarbonyl, trifluoroethyloxy-carbonyl, thiobenzoyl, phenylamidocarbonyl, and 4-nitrophenoxycarbo yl.
Polyketides are a class of chemicals including linear polyketides, marine halogenated acetogenins, annonaceae acetogenins, macrolide polyketides, lactone polyketides, ansamycins, polyenes, linear tetracyclines, angucyclines, polyether antibiotics, and aflatoxins.
A salt is an ionic derivative of a compound of the invention wherein a hydrogen atom is removed (e.g., deprotonation of a carboxylic acid to form a carboxylate
anion) or a hydrogen atom is added (e.g., protonation of an amino group to form an ammonium cation) , and one or more suitable counterions are added. Counterions may be organic or inorganic, and are, in general, biologically acceptable (e.g., pharmaceutically or agriculturally acceptable) . Examples of counter-cations include primary, secondary, or tertiary ammoniums or organic amines, group IA alkaline metal ions, and group IIA alkaline earth metal ions. Examples of counter-anions include halogen, nitrate, sulfate, citrate, salicylate, acetate, succinate, and acetylsalicylate. In certain embodiments, salts are formed by reacting a carboxylic acid- or amine-containing compound of the invention with hydrogen bromide, hydrogen chloride, or trifluoroacetic acid.
Unless otherwise indicated, in the structures provided herein, double bonds between r and r 2 , and r 3 and r , may be present (cis or trans, preferably trans) or absent (e.g., hydrogenated, alkylated, or halogenated) .
According to convention, a structure drawn with single, uniform lines includes both the (R) and (S) stereoisomers of any chiral carbon atom. Unless otherwise indicated, the compounds of the invention include both the (R) and (S) forms, although in certain embodiments it may be desirable to separate the various stereoisomers due to differences in activity. Such resolution is accomplished by means known to those skilled in asymmetric synthesis and chiral separation (e.g., chromatography) . Steroids include aromatic ring A C 18 estrane, C 19 androstanes, C20 19-norpreganes, C 21 pregnanes, C 23 24- norcholan-23-oic-acid steroids, cardanolide steroids (C 23 ) , cholan-24-oic acid steroids (C 24 ) , other cholane steroids (C 2 ) , bufanolide steroids (C 24 ) , homocholane steroids (26,27-dinorcholestanes) (C 25 ) , 27-norcholestane
steroids (C 26 ) > neutral cholestane steroids (C 27 ) , cholestanoic acid steroids (C 27 ) , ecdysteroids (C 27 ) , spirostan steroids (C 27 ) , furostan steroids (C 27 ) , ergostane steroids (excluding withanolides and brassinolides) (C 28 ) , withanolide and brassinolide steroids (C 28 ) , stigmastane steroids (C 29 ) , gorgostane and other cyclopropacholestane steroids (C 30 ) , vitamin D 3 (cholecalciferol) metabolites and analogues, and vitamin D 2 (ergocalcifero) metabolites and analogues. Terpenes are natural plant products having a multiple of five carbon atoms. Skeletal types of monoterpenes contain 10 carbon atoms and are acyclic (e.g., myrcane, artemisane, santolinane, lavandulane, secoiridane, senecic acid, and incanic acid) , monocyclic (e.g., eucarbone, α-thujaplicin, p-menthane, o-menthane, m-menthane, cyclogeraniolane, 3-cyclolavandulal, ferulol, chondrocole, cantharidin, α-campholene aldehyde, polyzonimine, fragranol, junioine, and chrysanthemane) , bicyclic or tricyclic (e.g., thujane, carane, pinane, bornane, isocamphane, fenchene, photocitral, filifolone, or tricyclene) . Specific examples include geraniol, myrcene, menthol, α-pinene, and camphene. See, also, CRC Handbook of Terpenoids , ed. Sukh Dev (1982) vols. I and II. Sesquiterpenes are C 15 compounds. Skeletal types include mono- and bicyclofarnesane, trans, trans- farnesyl diphosphate, nerolidyl diphosphate, cis, trans- farnesyl diphosphate, farnesane, bicycloger acrane, maaliane, aristolane, aromadendrane, germacrane, eudesmane, guaiane, elemane, elemophilane, valencane, valerane, hu ulane, caryophyllane, proto-illudane, illudane, hirsutane, marasmane, bisabolane, santalene, bergamotane, acorane, cedrane, chamigrane, thujopsane, widdrane, cuparane, laurane, trichothecane, cadinane, amorphane, muurolane, bulgarane, sativane, copaane, cubebane,
ylangane, himachalane, longifolane, longibornane, longipinane, furamoid farnesane sesquiterpenoids, irregular acyclic sesquiterpenoids, miscellaneous cyclobutane sesquiterpenoids, cyclopentane sesquiterpenoids, cyclofarnesane sesquiterpenoids, rearranged cyclofarnesane sesquiterpenoids, bisabolane sesquiterpenoids, miscellaneous cyclohexane sesquiterpenoids, cycloheptane sesquiterpenoids, cyclobisabolane sesquiterpenoids, elemane sesquiterpenoids, simple germacrane sesquiterpenoids, 12,6-germacranolide sesquiterpenoids, 12,8- germacranolides and furanogermacrane sesquiterpenoids, nor- and horoogerroacrane sesquiterpenoids, secogermacrane sesguiterpenoids, cyclogermacrane sesquiterpenoids, lepidozanes and bicyclogermacrane sesquiterpenoids, humulane sesquiterpenoids, caryophyllane sesquiterpenoids, bicyclohumulane sesquiterpenoids, cuparane sesquiterpenoids, syslolaurane sesquiterpenoids, herbertane sesquiterpenoids, laurane sesquiterpenoids, trichothecane sesquiterpenoids, simple eudesmane sesquiterpenoids , 12,6-eudesmanolide sesquiterpenoids, 12,8-eudesmanolides and furanoeudesmane sesquiterpenoids, agarofuran eudesmane sesquiterpenoids, secoeudesmane sesquiterpenoids, noreudesmane sesquiterpenoids, emmotin sesquiterpenoids, oppositane sesquiterpenoids, farfugin sesquiterpenoids, cycloeudesmane sesquiterpenoids, gorgonane sesquiterpenoids, simple eremophilane sesquiterpenoids, eremophilanolide and furanoeremophilane sesquiterpenoids, seco- and abeoeremophilane sesquiterpenoids, noreremophilane sesquiterpenoids, chiloscyphane sesquiterpenoids, aristolane sesquiterpenoids, nardosinane sesquiterpenoids, brasilane sesquiterpenoids, cacalol sesquiterpenoids, valerane sesquiterpenoids, miscellaneous rearranged eudesmane sesquiterpenoids, cadinane sesquiterpenoids, nor- and
secocadinane sesquiterpenoids, alliacane sesquiterpenoids, oplopane sesquiterpenoids, mutisianthol sesquiterpenoids, drimane sesquiterpenoids, coloratane sesquiterpenoids, nor- and secodrimane sesquiterpenoids, xanthane sesquiterpenoids, carabrane sesquiterpenoids, simple guaiane sesquiterpenoids, 12,6-guaianolide sesquiterpenoids, 12,8-guaianolide, dimeric guaiane sesquiterpenoids, seco-, cyclo-, abeo-, and norguaiane sesquiterpenoids, pseudoguaiane sesquiterpenoids, seco-, cyclo-, abeo and norpseudoguaiane sesquiterpenoids, aro adendrane sesquiterpenoids, cycloaromadendrane sesquiterpenoids, secoaro adendrane sesquiterpenoids, cubebane sesquiterpenoids, ivaxillarane sesquiterpenoids, patchoulane sesquiterpenoids, rearranged patchoulane sesquiterpenoids, valerenane sesquiterpenoids, africanane sesquiterpenoids, hi achalane sesquiterpenoids, longipinane sesquiterpenoids, longifolane sesquiterpenoids, pinguisane sesquiterpenoids, thapsane sesquiterpenoids, fukinane sesquiterpenoids, picrotoxane sesquiterpenoids, daucane sesquiterpenoids, isodaucane sesquiterpenoids, perforane sesquiterpenoids, pacifigorgiane sesquiterpenoids, asteriscane sesquiterpenoids, illudane sesquiterpenoids, protoilludane sesquiterpenoids, sterpurane sesquiterpenoids, illudalane sesquiterpenoids, isolactarane sesquiterpenoids, lactaran sesquiterpenoids, marasmane sesquiterpenoids, furodysin sesquiterpenoids, furodysinin sesquiterpenoids, botrydial sesquiterpenoids, spriovetivane sesquiterpenoids, acorane sesquiterpenoids, cha igrane sesquiterpenoids, secochamigrane sesquiterpenoids, miscellaneous spirosesquiterpenoids, cedrane sesquiterpenoids, isocedrane sesquiterpenoids, zizaane sesquiterpenoids, prezizaane sesquiterpenoids, clovane sesquiterpenoids, precapnellane sesquiterpenoids, capnellane sesquiterpenoids, hirsutane sesquiterpenoids.
rearranged hirsutane sesquiterpenoids, pentalenane sesquiterpenoids, silphinane sesquiterpenoids, silphiperfoliane sesquiterpenoids, presilphiperfoliane sesquiterpenoids, isocomane sesquiterpenoids, panasinsane sesquiterpenoids, modhephane sesquiterpenoids, quadrane sesquiterpenoids, campherenane sesquiterpenoids, α- santalane sesquiterpenoids, 0-santalane sesquiterpenoids, sativane sesquiterpenoids, copacamphane sesquiterpenoids, sinularane sesquiterpenoids, capaane sesquiterpenoids, ishwarane sesquiterpenoids, rotundane sesquiterpenoids, thujopsane sesquiterpenoids, bourbonane sesquiterpenoids, gymnomitrane sesquiterpenoids. Specific examples include farnesol, bisabolene, α-cadinol, and hinesol. Eremophilanes include 3-hydroxy-9,ll-eremophiladien-8-one (e.g., 3α,7α-form, and 3α,7,9-foπn) ; 3,12-dihydroxy-
9,11, (13)-eremophiladien-8-one (3α,7,9H-form) ; eremofortin B; 9,ll-eremophiladien-8-one; 6α,7α-epoxy-3α,13- dihydroxy,-9,12-eremophiladien-9-one; 13-desoxyphomenone, phaseolinone, phomenone, 1,3-dihydroxy-9,11- eremophiladien-8-one (13,3α,7ϊH-form) , petasin, S- petasin, isopetasin, iso-S-petasin, petasol, and isopetasol.
Diterpenes are C 20 compounds biogenetically derived from geranylgeranyl pyrophosphate, and include resin acids and gibberellins. Examples of diterpenoid skeletons include irregular linear diterpenoids, linear homo- and nor- diterpenoids, prenylbisabolane, 10,15- cyclophytane, labdane, secolabdane, norlabdane, halimane, rearranged labdane, colesane, clerodane, nor-, seco- and abeoclerodane, abietane, furanoabietane, furanoabietane, secoabietanes and secofriedoabietane, nor- and homoabietane, abeoabietane, dimeric abietane, 13,16- cycloabietane, 17(15-»16) abeoabietane, totarane, nagilactone, pimarane, rosane, erythroxylane, paraguarane, devadarane, isopimarane, rearranged pimarane
and isopimarane, norpimarane and morisopimarane, cassane, vouacapane, cleistanthane and isocleistanthane, isolane and spongiane, seco-, nor- and abeospongiane, podocarpane, kaurane, phyllocladane, norkaurane, secokaurane, rearranged kaurane, beyerane, nor- and secobeyerane, villanovane, atisane, trachylobane, helifulvane, aphidicolane, gibberellins, rearranged gibberellins, leucothol, grayanotoxane, cembrane, norcembrane, rearranged cembrane, eunicellane, asbestinane, sphaerane, briarane, dolabellane, modified dolabellane, dolastane, modified dolastane, cyathane, sphaeroane, verrucosane, modified verrucosane, casbane, jatrophane, 9,13-cyclojatrophane, lathyrane, rhamnofolane, daphnane, tigliane, ingenane, jatropholane and secojatropholane, fusicoccane, valparane, spatane, seco- and abeospatane, verticillane, taxane, trinervitane, kempane, amphilectane, cycloamphilectane, adociane, xenicane, nor-, seco- and cycloxenicane, phyllane, viscidane, eremane, prenyleudesmane, prenylgermacrane, prenylbicyclogermacrane, lobane, prenylguaiane, cneorubine, serrulatane and biflorane, decipiane, sacculatane, obtusane, irieol, sphenolobane, geranylgeraniol, beyerane, and gibber1lane. Specific diterpenes include geranylgeraniol and dextropimaric acid.
Triterpenes are C 30 compounds. Examples of triterpene skeletal structures include linear triterpenoids, botryococcenes, protostanes, fusidanes, lanostanes, cycloartane, cucurbitane, dammarane, triucallane/euphane, apotirucallane, intact tetranortriterpenoids, ring cleaved tetranortriterpenoids, rearranged tetranortriterpenoids, quassinoid nortriterpenoids, baccharane, lupane, 3(2→1) abeolupane, nor- friedo- and secolupane, oleanane, nor-, seco- and abeooleanane, taraxerane, nor-, seco- and
cyclotaraxerane, multiflorane, glutinane, friedelane, nor- and secofriedelane, pachysanane, taraxastane, ursane, nor-, seco- and abeoursane, bauerane, hopane, nor- seco, and abeohopane, neohopane, fernane, squalene, onocerane, serratane, ambrane, alabaricane, gammacerane, protostane, lanostane, cycloartane, arborane, hopane, fernane, filicane, dammarane, apo-euphane, euphane, shionane, germanicane, taraxsterane, and phyllanthane. C. Natural product isolation and purification 1. Extraction
A supernatant (7 L) was extracted from a pine root (Deutero Mycetes) fungal culture with about an equal volume of ethyl acetate (6-10 L) . The ethyl acetate extract was concentrated by rotary evaporation under vacuum at 32°C to yield between 1.0 and 2.5 g green syrup from the supernatant. The mycelium was extracted with 3 L methanol; methanol filtrate was concentrated by rotary evaporation under vacuum at 32°C to yield about 1 aqueous solution. Extraction with an equal volume of ethyl acetate and concentration as above yielded material which was combined with the supernatant extract.
The combined extracts were separated on a C-18 (EM Science, Gibbstown, NJ) 40-63 /xm column 4.5 cm h x 3.5 cm diameter. After eluting with a step gradient of methanol/water (50%, 75%, 85%, and 100%, 250 ml each step) , each fraction was analzyed on silica gel TLC precoated with Kieselgel 60 F254 on aluminium sheet (EM Separations) . The plates were developed with 60% ethyl acetate/hexanes and visualized with p-anisaldehyde. After concentrating the fraction containing compound 3390 in 75% methanol/water at 32°C under vacuum, the resulting residue was separated by a silica gel 60 (EM Science, < 0.063 mm) column 5.0 cm high x 1.5 cm diameter, eluting with a step gradient of ethyl acetate/hexanes (25%, 50%, 75%, and 100%, 20 ml for each step). Both the 75% ethyl
acetate/hexanes fraction and the 100% ethyl acetate fraction contained compound 3390. After combining these fractions, and purification by eluting with a step gradient of methanol/water through a C-18 (EM Science 40- 63 μm) column 5.0 cm high x 1.5 cm diameter yielded 7 mg of pure compound in the 80% methanol/water fraction. 2. Structural characterization
Compound 3390 (formula V) showed a UV maχ at 231 nm (e = 16,340 in methanol), indicative of a conjugated diene. Electrospray mass spectroscopy gave a [M + Na] + ion at m/z 453 in positive mode. Lithium chloride exchange gave a [M+Li] + ion at m/z 437, suggesting a molecular weight of 430. Proton and 13 C NMR signals were assigned (Table 1) .
Chemical Structure of A A03390
Table 1. NMR Signal Assignment for 3390
Carbon # λ i(δ) .multiplicity. J in Hz
1J cm
2.40 ddd, 15.0, 3.5, 3.0) 31.7 2.60 ddd, 15.0, 5.0, 1.0) 2.17 m) 32.8 1.49 m)
3 4.93 m) 74.9 4 1.64 dq, 7.5, 7.0) 48.6 5 41.5 6 1.94 dd, 15.0, 14.5) 43.1 2.05 dd, 15.0, 5.0)
7 3.22 dd, 10.0, 4.5) 51.6
8 201.3
9 5.76 br. s) 125.2
10 170.3
11 144.9
12 115.0
13 20.4 14 10.9 15 17.5 1' 176.3 2' 48.0 3' 75.7 4' 132.4 5' 134.0 6' 133.1 7' 132.7 8' 43.5
9' 68.5
10' 23.2 11' 14.0
D. Synthesis
1. Semisvnthesis
Representative derivatizations of the isolated natural product (compound 3390) are presented below, as well as semisynthetic methods concerning the generally linear portion of compound 3390 containing a conjugated diene, and the ring-containing or sesquiterpene portion of compound 3390. It will be easily apparent to one skilled in organic synthesis that the following transformations may be operated, in general, upon either the ester compound 3390 itself or upon the individually resolved alcohol and carboxylic acid hydrolysis products of the ester parent compound.
Derivatization of the hydroxyl group includes alkylation, reduction, oxidation, esterification, and silylation. Derivatization of the carboxylic acid group includes forming both esters and amides. A naturally- occurring amino acid includes the 20 common α-amino acids (Gly, Ala, Val, Leu, lie, Ser, Thr, Asp, Asn, Lys, Glu, Gin, Arg, His, Phe, Cys, Trp, Tyr, Met and Pro) , and other amino acids that are natural products, such as norleucine, ethylglycine, ornithine, methylbutenylmethylthreonine, and phenylglycine. Examples of amino acid side chains include H (glycine) , methyl (alanine) , -(CH 2 -(C=0)-NH 2 (asparagine) , -CH 2 -SH (cysteine) , and -CH(OH)CH 3 (threonine) .
Regarding the sesquiterpenoid portion, a wide variety of carboxylic acids or activated acyl groups can be reacted to form esters, RC0 2 H, wherein R is alkyl, cycloalkyl, arylalkyl, branched arylalkyl, alkyl, amino acid esters with N-protection such as R0(C=0)NH- and R- (C=0)-NH-. The extracyclic vinyl and enone groups can also be derivatized. These and other transformations are provided in the following schemes.
SCHEME 1
111 Vteit-HuSiCl
Imidazolc P B esicr
(or R 3 SiX for other si lanes)
DCC / DMAP t ROM
2a estcs
R' ■ various alcohols
(alkyl. cvloalkvl. aialkyl. etc)
2. R'R"NH
R" « H. alkvl. etc F-
2b. amides
SCHEME
2b. Amides w/arnino acids
Selecttve oxidation I.R3SiCl/imidazole
SCHEME 3
2. F-
H 1" anisole
,H
NaOH
R
SCHEME 4
lb. reduction
lb. reduction
SCHEME 5
le. oxidation
Cl 2
OH-
H
SCHEME 6
alkyl, aralkvl etc.
etc.
- 24 -
SCHEME 7
I.LDA 2. RX
SCHEME 8
SCHEME 9
Der i va t ization ot ' cmpd 3390(11)
I.R3S.CI/ Imidazole
SCHEME 10
SCHEME 1 1
(fast rxn, control condition to prevent double oxidation )
Scheme 12
I.NH1NHCSNH2
2.F-
2. Protecting groups
Numerous thiol-, amino-, hydroxy- and carboxy- protecting groups are well-known to those in the art. In general, the species of protecting group is not critical, provided that it is stable in the conditions of any subsequent reaction(s) on other positions of the compound and can be removed at the appropriate point without adversely affecting the remainder of the molecule. In addition, one protecting group may be substituted for another after substantive synthetic transformations are complete. Clearly, where a compound differs from a compound disclosed herein only in that one or more protecting groups of the disclosed compound has been substituted with a different protecting group, that compound is within the invention. Further examples and conditions are found in T.W. Greene and P.G.M. uts, Protective Groups in Organic Chemistry, (2nd ed. , 1991) .
The invention also encompasses isotopically- labelled counterparts of compounds disclosed herein. An isotopically-labelled compound of the invention has one or more atoms replaced with an isotope having a detectable particle- or x-ray-emitting (radioactive) nucleus or a magnetic nucleus. Examples of such nuclei include 2 H, 3 H, 13 C, 15 N, 19 F, 29 Si, 31 P, 32 P and 125 I. Isotopically-labelled compounds of the invention are particularly useful as probes or research tools for spectrometeric analyses, radioimmunoassays, binding assays based on γ- or / -"-scintillation, fluorography, autoradiography, and kinetic studies such as inhibition studies or determination of primary and secondary isotope effects.
3. Total Synthesis
In another aspect, the invention includes additional functional and structural analogs of the parent compound 3390. In this aspect, the conjugated
diene skeleton can be replaced by isosteric monocyclic or bicyclic structures, or other sp 2 -hybridized moieties such as carbonyls, amides, tertiary or secondary amines.
In formula (II) above, for example, each of r 1# r 2 , r 3 and r 4 is selected independently from H, methyl, and halomethyl; or r 1 and r 3 , or r 2 and r 4 , taken together are a bivalent moiety which forms a C 3 _ 20 substituted or unsubstituted five- or six-membered cycloalkyl, cycloalkenyl, aromatic, heterocyclic or heteroaromatic ring.
In this aspect, A includes any side chain of a naturally-occurring amino acid, C 2 _ 10 hydroxy-alkyl, c ι-ιo mercaptoalkyl, C 2 _ 10 alkenylene, X 1 (CH-)R 1 , or X α (C- )R lf wherein X χ is H, F, Cl, Br, I, C 1-10 alkyl, C 2 _ 10 alkenyl, C 2 _ 10 alkenylene (where A is X^C-)!^), C 1 _ 10 haloalkyl, 0R ir SR^ NR L R LL , or NR i (C=0)R ii , each of R L and R i± being independently selected from H, CJ^ Q alkyl, and ( c 6 -i 4 aryl) (C 0 _ 6 alkyl); and ΕL λ is H, C^g alkyl, c λ _ 10 alkenyl, or =0 (where A is X 1 (C-)R 1 ). It will be easily apparent to those in the art that some categories of A overlap. Thus, to avoid redundancy within a single claim, it will be understood that where A is X 1 (C-)R 1 , either R A is =0 or X 1 is C 2 _ 10 alkenylene; and that where A is X 1 (CH-)R 1 , Xi and R 2 are not such that A is a side chain of a naturally-occurring amino acid. Other variables are as described above in formula (I) .
The invention also features a compound of the formula (VI) . Based on the synthetic pathways provided in the disclosed schemes, the compounds of formula VI can be easily prepared from isolated natural products by one of ordinary skill in synthetic organic chemistry. In addition, the C-ll methylidene group can be oxidized to form a methyl ketone, a highly versatile moiety which allows chain extension by a variety of reactions.
In formula (VI), Z is -(C=0)-NH-, -(C=0)-0-, -0-, -NH-(C=0)-, or -0-(C=0)-. As shown, the Z moieties have a specific orientation, where left-most valence, e.g. , the carbonyl carbon of the first amide bond (or the amino nitrogen of the second amide bond) , is bonded to the carbon atom attached to r a and r b . In some embodiments, where X is OH, -C(R 3 ) (R 4 ) (R 5 ) is neither isopropenyl nor isopropylidene; when X is O-(trialkylsilyl) , -C(R 3 ) (R 4 ) (R 5 ) is not 2-hydroxylisopropyl; or when R 1 , R 2 , and X are H, -C(R 3 ) (R 4 ) (R 5 ) is not isopropenyl. The invention includes a pharmaceutically acceptable salt or ester of the disclosed compounds. In one embodiment of formula (VI), one of r a and r b is methyl, one of r c and r d is hydroxyl, one of r e and r f is H, and one of r g and r h is hydroxyl and the other is methyl. In another embodiment, at least three of r a - r h are hydroxyl and at least two of the remaining of r a - r h are methyl. In other embodiments, X is H; X is not H; or Z is -(C=0)-NH-, -(C=0)-0-, or -0-.
E. use
The disclosed compounds are useful in the treatment of cancer, and for making medicaments for treating cancer. The methods of the invention contemplate treatment of animal subjects, such as mammals (e.g., higher primates, and especially humans), animal cell and tissue cultures, plants (e.g., food crops), and
plant cell and tissue cultures. The invention encompasses pharmaceutical (or veterinary or agricultural) compositions which include novel compounds described herein. Where the compound contains a basic group, pharmaceutically acceptable salts may be formed, for example, with 1, 2, 3, or more equivalents of hydrogen chloride, hydrogen bromide, trifluoroacetic acid, and others known to those in the art of drug formulation. Sodium, potassium, ammonium, calcium, aluminium, or magnesium salts can be prepared where the compound contains an acid group, using a hydroxide compound or other base. Compounds of the invention can be formulated into pharmaceutical compositions by admixture with pharmaceutically acceptable non-toxic excipients and carriers. A pharmaceutical composition of the invention may contain more than one compound of the invention, and/or may also contain other therapeutic compounds not encompassed by the invention, such as anti-inflammatory, analgesic, or other agents. A compound of the invention may be administered in unit dosage form, and may be prepared by any of the methods well known in the pharmaceutical art, for example, as described in -Remington's Pharmaceutical Sciences (Mack Pub. Co., Easton, PA, 1980) . The invention also encompasses a packaged drug, containing a pharmaceutical composition formulated into individual dosages and printed instructions for self-administration.
Compounds disclosed herein may also be formulated for topical or parenteral administration, particularly in the form of solutions or liquid suspensions; for oral administration, particularly in the form of tablets or capsules; or intranasally, particularly in the form of powders, gels, oily solutions, nasal drops, aerosols, or mists. Formulations for parenteral administration may
contain as common excipients sterile water or sterile saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes, and the like. Controlled release of a compound of the invention may be obtained, in part, by use of bioco patible, biodegradable polymers of lactide, and copolymers of lactide/glycolide or polyoxyethylene/polyoxypropylene. Additional parental delivery systems include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation administration contain lactose, polyoxyethylene-9-lauryl ether, glycocholate, or deoxycholate. Formulations for buccal administration may include glycocholate; formulations for vaginal administration may include citric acid.
The concentration of a disclosed compound in a pharmaceutically acceptable mixture will vary depending on several factors, including the dosage of the compound to be administered, the phar acokinetic characteristics of the compound(s) employed, and the route of administration. In general, the compounds of this invention may be provided in an aqueous physiological buffer solution containing about 1-10% w/v of compound for parenteral administration. Typical dose ranges are from about 1-100 mg/kg of body weight per day, and preferably 2-10 mg/kg, given in 1-4 divided doses. Each divided dose may contain the same or different compounds of the invention. The dosage will be an effective amount depending on several factors including the overall health of a patient, and the formulation and route of administration of the selected compound(s) .
The effective amount of the active compound used to practice the present invention for treatment of conditions directly or indirectly mediated by cancer
varies depending upon the manner of administration, the age and the body weight of the subject and the condition of the subject to be treated, and ultimately will be decided by the attending physician or veterinarian. Such an amount of the active compound as determined by the attending physician or veterinarian is referred to herein as an "effective amount."
One aspect of the invention is a method of treating cancer in a patient (e.g., a mammal, e.g., a human, a mouse, a rat, or a cow) . This method includes administering to the patient an effective anti-cancer amount of a pharmaceutical composition containing one or more compounds having a formula (I) or (VI) ; and a pharmaceutically acceptable carrier, thereby treating the cancer in the patient. A pharmaceutical composition can include a plurality of anticancer compounds, whether disclosed herein or otherwise known, or a combination of a compound disclosed herein and another therapeutic, in any beneficial proportion. Without further elaboration, it is believed that one skilled in the art can, based on this disclosure, realize the present invention to its fullest extent. The following example is, therefore, merely illustrative. All publications cited herein are hereby incorporated by reference in their entirety.
Example l Cγtotoxicitv
Discard the transport media from two flasks 150 cm 3 of MRC-5 human fibroblast cells (Bio Whittaker, Walkersville, MD) . The following wash protocol was repeated once. Cells were washed twice with 40 ml PBS pH 7.2 (GIBCO, Gaithersburg, MD) and treated at 37°C for 3-4 minutes with 4 ml/flask of 1 x trypsin (JRH, Lenexa, KS) diluted with PBS. After centrifugation in a ClonicaL Centrifuge IEM at 5 min x 2000 rpm at room temperature.
the collected cells were resuspended in 120 ml fresh Dulbecco's modified Eagle's medium (DME/H) (JRH, supplemented with 4500 mg/L glucose and 4.0 mM L- glutamate without sodium pyruvate) , warmed to 37°C, and supplemented with 10% fetal bovine serum (GIBCO) . After repetition, the cells were resuspended in 20 ml fresh DME/H medium without FBS.
Cells stained with neutral red (1:1000 v/v) for 1 minute were counted in a hemocytometer. A cell suspension containing 5 x 10 4 cells/ml in DME/H medium supplemented with penicillin/streptomycin solution (Sigma Chemical Co., St. Louis, MO, 10,000 units, 10 mg/ l diluted 1:100). Microtiter plates were prepared with 100 μl suspension per well, and incubated at 37°C, 5% C0 2 for 43-48 hours. After incubation, old medium was shaken out and replaced with 2100 ml DME/H containing 10% Alamar Blue solution (BioSource, Camarillo, CA) before incubating for 4 hours. Alamar Blue contains a redox indicator which is reduced from the nonfluorescent form to the fluorescent form. Fluorescence intensity was read on a Perkin Elmer LS 50B reader with excitation at 535 nm and emission at 584 nm.
Row Hl-12 of each plate served as an untreated control. The highest and lowest values of the control row were dropped; the average of the remaining rows was used to calculate cell death in the test wells as a percentage of the control well. A sufficiently cytotoxic substance kills between 20% and 100% of the cells in the test well, and preferably kills between 50% and 100% of the cells. In view of the above, anti-cancer activity was shown.
Other Embodiments From the above description, the essential characteristics of the present invention can be easily ascertained. Without departing from the spirit and scope
thereof, various changes and modifications of the invention can be made to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.