# Patent

## Title
Apoptolidin analogs and derivatives for inducing apoptosis in transformed cells

## Abstract

The invention provides novel apoptosis-inducing compounds that include isolated, purified isoapoptolidin, and selectively functionalized isoapoptolidin derivatives and stereoisomers thereof; selectively functionalized apoptolidin derivatives and stereoisomers thereof; and deglycosylated isoapoptolidin and selectively functionalized derivatives and stereoisomers thereof. The isoapoptolidin, apoptolidin, and deglycosylated isoapoptolidin derivatives may be functionalized by substituting any or all of the methoxyl or hydroxyl groups of the parent molecule. Pharmaceutical compositions and methods for using the compounds are also provided.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority under 35 U.S.C. § 119(e)(1) to provisional U.S. patent applications Ser. No. 60/439,954, filed Jan. 13, 2003, and 60/472,657, filed May 21, 2003.


GOVERNMENT RIGHTS
This invention was made with United States Government support under contract CA31845 awarded by the National Cancer Institute. The United States Government has certain rights in this invention.


TECHNICAL FIELD
This invention relates generally to apoptolidin, and more particularly relates to novel apoptolidin analogs and derivatives that are effective in inducing apoptosis in transformed cells. The invention also pertains to pharmaceutical compositions and methods for treating a patient in need of anticancer therapy or therapy for the treatment of other disorders that are responsive to selective apoptosis, also known as programmed cell death.
BACKGROUND
Apoptolidin is a 20-membered macrolide that selectively induces apoptosis in certain cancer cell lines. Apoptosis is an important mechanism in the treatment of cancer and consequently, agents that can selectively induce apoptosis in cancer cells have great therapeutic use. The success of apoptolidin as a potentially powerful anti-cancer agent is demonstrated by the molecule's status as among the top 0.1% most selective cytotoxic agents of 37,000 compounds tested by the National Cancer Institute on 60 human cancer cell lines (NCI 60).




Apoptolidin
Apoptolidin was first isolated by Seto et al., who, in 1997, found that apoptolidin, isolated from Nocardiopsis sp. was capable of inducing apoptosis in rat glial cells transformed with the adenovirus E1A or E1A/E1B19K/E1B54K oncogenes but that the macrolide had no cytotoxic effect on untransformed rat glial cells or on normal fibroblasts. Kim et al. (1997) J. Antibiot., 50:628–630; Kim et al. (1998) J. Am. Chem. Soc., 120:3524–3525. It has been hypothesized that the cell death induced by apoptolidin results from the macrolide's ability to inhibit oxidative phosphorylation by targeting mitochondrial F0F1-ATP synthetase. Salomon et al. (2000) Proc. Natl. Acad. Sci., 97(26):14766–14771; Salomon et al. (2000) Chem. & Biol., 54:1–10. The ability of apoptolidin to inhibit eukaryotic mitochondrial ATP synthetase places this molecule in a growing family of structurally related macrolide antibiotics including the anti-fungal agents oligomycin and ossamycin.
While apoptolidin has been found to be an effective apoptosis-inducing agent in some cancer cell lines, its effectiveness is not universal. Salomon et al. have presented experiments that suggest that macrolide inhibitors of mitochondrial F0F1-ATP synthetase (F0F1-ATPase) selectively kill metabolically active tumor cells that do not exhibit a high level of anaerobic carbon metabolism in the presence of oxygen (a phenomenon known as the Warburg effect). For those cell lines that exhibit the Warburg effect, it has been demonstrated that treatment of the tumor cell lines with the lactase dehydrogenase inhibitor oxamate and the Embden-Meyerhof pathway inhibitor 2-deoxyglucose sensitizes the tumor cells to apoptolidin treatment. Salomon et al., PNAS, supra. Salomon et al. have hypothesized that these two inhibitors modulate apoptolidin activity by channeling carbon flux into the mitochondria and consequently, initiate mitochondrially derived aerobic energy. The foregoing finding, however, does not explain apoptolidin's selectivity against different cell lines.
Accordingly, there is a need in the art to determine the structure-activity relationship of apoptolidin in order to obtain information on the reasons for the cell-line specificity of this important molecule in its natural state and, consequently, be able to prepare analogs and derivatives of apoptolidin, including compounds that are broadly active as well as those that are cell-line specific (and thereby target specific types of abnormal or diseased cells). Through the preparation of such analogs and derivatives, therapeutic anti-cancer agents may be prepared using apoptolidin analogs and derivatives.
The present invention is the result of extensive, systematic research into apoptolidin analogs, including isolated and purified isomers of apoptolidin, and apoptolidin derivatives, prepared through modification of the functional groups of the apoptolidin molecule. To the best of the inventors' knowledge, the compounds, compositions, and methods of the invention are completely unknown and completely unsuggested by the art.
SUMMARY OF THE INVENTION
The present invention is directed to the aforementioned need in the art, and provides novel compounds that induce apoptosis in cancerous cells but not in normal cells. The compounds are apoptolidin analogs and derivatives thereof, including an apoptolidin isomer, “isoapoptolidin,” as well as functionalized isoapoptolidin, functionalized apoptolidin, “truncated” (deglycosylated) isoapoptolidin, and functionalized “truncated” (deglycosylated) isoapoptolidin.
In a first embodiment, the invention provides isolated, purified isoapoptolidin as a new composition of matter. Isoapoptolidin is a ring-expanded macrolide isomer of apoptolidin, and has the structure of formula (I)




Isoapoptolidin
The compounds of the invention also include functionalized isoapoptolidin, functionalized apoptolidin, functionalized deglycosylated isoapoptolidin, deglycosylated isoapoptolidin, and stereoisomers thereof. The functionalized compounds have the structure of isoapoptolidin, apoptolidin, and deglycosylated isoapoptolidin, respectively, wherein at least one hydroxyl group within the molecule is replaced with a substituent selected from:

    
    
        (a) C1–C24 hydrocarbyloxy, optionally substituted with one or more halogen atoms, e.g., C1–C24 alkoxy (—O—(C1–C24 alkyl)), C6–C24 aralkoxy (—O—(C6–C24 aralkyl)), C 6–C24 alkaryloxy (—O—(C6–C24 alkaryl)), C1–C24 haloalkoxy (—O—(C1–C24 haloalkyl)), and C6–C24 haloaralkoxy (—O—(C6–C24 haloaralkyl);
    
    


(b) C2–C25 acyloxy (—O—(CO)—(C1–C24 hydrocarbyl)), e.g., —O—(CO)—(C1–C24 alkyl), —O—(CO)—(C5–C24 aryl), —O—(CO)—(C6–C24 aralkyl), and —O—(CO)—(C6–C24 alkaryl));
(c) C2–C25 haloacyloxy (—O—(CO)—(C2–C24 halohydrocarbyl)), e.g., —O—(CO)—(C1–C24 haloalkyl), —O—(CO)—(C5–C24 haloaryl), —O—(CO)—(C5–C24 haloaralkyl), and —O—(CO)—(C5–C24 haloalkaryl);
(d) C2–C25 thioacyloxy (—O—(CS)—(C2–C24 hydrocarbyl)), e.g., —O—(CS)—(C1–C24 alkyl), —O—(CS)—(C5–C24 aryl), —O—(CS)—(C5–C24 aralkyl), and —O—(CS)—(C5–C24 alkaryl);
(e) C2–C25 thiohaloacyloxy (—O—(CS)—(C2–C24 hydrocarbyl)), e.g., —O—(CS)—(C1–C24 haloalkyl), —O—(CS)—(C5–C24 haloaryl), —O—(CS)—(C5–C24 haloaralkyl), and —O—(CS)—(C5–C24 haloalkaryl),
(f) C2–C25 carbonato (—O—(CO)—O—(C1–C24 hydrocarbyl)), e.g., C2–C25 alkylcarbonato (—O—(CO)—O—(C1–C24 alkyl)), C6–C25 arylcarbonato (—O—(CO)—O—(C5–C24 aryl)), C6–C25 aralkylcarbonato (—O—(CO)—O—(C5–C24 aralkyl)), and C6–C25 alkarylcarbonato (—O—(CO)—O—(C5–C24 alkaryl));
(g) halogenated C2–C25 carbonato (—O—(CO)—O—(C1–C24 halohydrocarbyl)), e.g., C2–C25 haloalkylcarbonato (—O—(CO)—O—(C1–C24 haloalkyl)), C6–C25 haloarylcarbonato (—O—(CO)—O—(C5–C24 haloaryl)), C6–C25 haloaralkylcarbonato (—O—(CO)—O—(C5–C24 haloaralkyl)), and C6–C25 haloalkarylcarbonato (—O—(CO)—O—(C5–C24 haloalkaryl));
(h) C2–C25 thiocarbonato (—O—(CS)—O—(C1–C24 hydrocarbyl)), e.g., C2–C25 alkylcarbonato (—O—(CS)—O—(C1–C24 alkyl)), C6–C25 arylcarbonato (—O—(CS)—O—(C5–C24 aryl)), C6–C25 aralkylcarbonato (—O—(CS)—O—(C5–C24 aralkyl)), and C6–C25 alkarylcarbonato (—O—(CS)—O—(C5–C24 alkaryl));
(i) halogenated C2–C25 thiocarbonato (—O—(CS)—O—(C1–C24 halohydrocarbyl)), e.g., C2–C25 haloalkylthiocarbonato (—O—(CS)—O—(C1–C24 haloalkyl)), C6–C25 haloarylthiocarbonato (—O—(CS)—O—(C5–C24 haloaryl)), C6–C25 haloaralkylthiocarbonato (—O—(CS)—O—(C5–C24 haloaralkyl)), and C6–C25 haloalkarylthiocarbonato (—O—(CS)—O—(C5–C24 haloalkaryl));
(j) carbamoyloxy (—O—(CO)—NH2), mono-(C1–C24 alkyl)-substituted carbamoyloxy (—O—(CO)—NH(C1–C24 alkyl)), di-(C1–C24 alkyl)-substituted carbamoyloxy (—O—(CO)—N(C1–C24 alkyl)2), mono-(C6–C24 aryl)-substituted carbamoyloxy (—O—(CO)—NH-aryl), di-(C6–C24 aryl)-substituted carbamoyloxy (—O—(CO)—N(aryl)2), and di-N—(C1–C24 alkyl), N—(C6–C24 aryl)-substituted carbamoyloxy;
(k) thiocarbamoyloxy (—O—(CS)—NH2), mono-(C1–C24 alkyl)-substituted thiocarbamoyloxy (—O—(CS)—NH(C1–C24 alkyl)), di-(C1–C24 alkyl)-substituted thiocarbamoyloxy (—O—(CS)—N(C1–C24 alkyl)2), mono-(C6–C24 aryl)-substituted thiocarbamoyloxy (—O—(CS)—NH-aryl), di-(C6–C24 aryl)-substituted carbamoyloxy (—O—(CS)—N(aryl)2), and di-N—(C1–C24 alkyl), N—(C6–C24 aryl)-substituted thiocarbamoyloxy;
(l) sulfamoyloxy (—O—SO2—NH2), mono-(C1–C24 alkyl)-substituted sulfamoyloxy (—O—SO2—NH(C1–C24 alkyl)), di-(C1–C24 alkyl)-substituted sulfamoyloxy (—O—SO2—N(C1–C24 alkyl)2), mono-(C6–C24 aryl)-substituted sulfamoyloxy (—O—SO2—NH-aryl), di-(C6–C24 aryl)-substituted sulfamoyloxy (—O—SO2—N(aryl)2), and di-N—(C1–C24 alkyl), N—(C6–C24 aryl)-substituted sulfamoyloxy; and
(m) protected hydroxyl groups such as tri-(C1–C12 hydrocarbyl)-substituted silyloxy and substituted methyl ethers.
The functionalized compounds of the invention also encompass other isoapoptolidin, apoptolidin, and deglycosylated isoapoptolidin analogs in which the core compounds have been modified so that: (a) a double bond is converted to a single bond via catalytic hydrogenation or nucleophilic addition; (b) a 1,3-diene functionality is converted to a cyclic group by a Diels-Alder reaction with a dienophile; and/or (c) a 1,2-diol functionality has been converted to a cyclic ether by reaction with a suitable reagent. The compounds of the invention may be functionalized to contain any or all of the modifications (a), (b), or (c), in addition to or in lieu of the hydroxyl group substitutions enumerated above.
In one preferred embodiment, a functionalized isoapoptolidin compound is provided having the structure of formula (II)




or is a stereoisomer thereof, wherein Q1, Q2, Q3, Q4, Q5, Q6, Q7, and Q8 are selected from H, C1–C12 hydrocarbyl, acyl of the formula —(CO)—R5 wherein R5 is C1–C12 hydrocarbyl, and hydroxyl-protecting groups, and R1, R2, R3, and R4 are independently selected from C1–C12 alkyl and H.

In another preferred embodiment of the invention, a functionalized apoptolidin compound is provided having the structure of formula (III)




or is a stereoisomer thereof, wherein Q1, Q2, Q3, Q4, Q5, Q6, Q7, Q8, R1, R2, R3,and R4 are defined as for the functionalized isoapoptolidin compounds of formula (II), with the proviso that at least one of Q1, Q2, Q3, Q4, Q5, Q6, Q7, and Q8 is other than H when R1, R2, R3, and R4 are methyl and the compound has the stereoisomeric configuration of formula (III).

In a further preferred embodiment, a functionalized deglycosylated isoapoptolidin compound is provided having the structure of formula (IV)




or is a stereoisomer thereof, wherein Q1, Q2, Q4, Q5, Q6, Q8, and Q9 are independently selected from H, C1–C12 hydrocarbyl, acyl of the formula —(CO)—R5, and hydroxyl-protecting groups, and R1, R2, and R3 are independently selected from C1–C12 alkyl and H.

In yet another embodiment, the invention encompasses pharmaceutical compositions containing a therapeutically effective amount of an apoptolidin analog or derivative as provided herein in combination with a pharmaceutically acceptable carrier. The compositions are generally “unit dosage” forms in which the therapeutically effective amount is suitable for a single dosage. The compositions may be immediate release or controlled release, and, if controlled release, are preferably sustained release. For those compounds that are orally active, oral dosage forms are preferred, in which case the carrier is one that is suitable for oral ingestion.
The invention also provides a method for treating a mammalian patient in need of anti-cancer treatment by administering a therapeutically effective amount of an apoptolidin analog or derivative as provided herein. Generally, the analog or derivative is administered in a pharmaceutical composition as described above.



BRIEF DESCRIPTION OF THE DRAWING
FIG. 1 shows a representative solution conformation of deglycosylated isoapoptolidin in the region from C-1 to C-26.
FIG. 2 shows a graph of kinetics toward equilibrium for the conversion of isoapoptolidin to apoptolidin and for the conversion of apoptolidin to isoapoptolidin in aqueous solution where Q=[2]/[1].
FIG. 3 shows some common (+)ESIMS fragmentation sites for derivatives of apoptolidin.



DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
I. Definitions and Nomenclature
Unless otherwise indicated, the invention is not limited to specific synthetic methods, analogs, substituents, pharmaceutical formulations, formulation components, modes of administration, or the like, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.
As used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a substituent” includes a single substituent as well as two or more substituents that may be the same or different, reference to “a compound” encompasses a combination or mixture of different compounds as well as a single compound, reference to “a pharmaceutically acceptable carrier” includes two or more such carriers as well as a single carrier, and the like.
In this specification and in the claims that follow, reference will be made to a number of terms, which shall be defined to have the following meanings:
The term “isomer” is used to describer a chemical compounds having the same number and kind of atoms (and hence the same molecular weight) as a parent compound but differing in the arrangement or configuration of the atoms. The term “stereoisomer” refers to chemical compounds that have identical atomic constituents but differ in the spatial arrangement of the atoms.
The term “analog” is used to describe a structural derivative of a parent compound that often differs from it by a single element. As used herein, the term analog includes isomers of a parent compound, e.g., isoapoptolidin is an analog as well as an isomer of apoptolidin.
As used herein, the term “derivative” is used to mean a compound derived or obtained from another and containing essential elements of the parent substance, e.g., the functionalized apoptolidin compounds of the invention are derivatives of apoptolidin, and the functionalized isoapoptolidin compounds of the invention are derivatives of isoapoptolidin.
As used herein, the phrase “having the formula” or “having the structure” is not intended to be limiting and is used in the same way that the term “comprising” is commonly used.
The term “alkyl” as used herein refers to a branched or unbranched saturated hydrocarbon group typically although not necessarily containing 1 to about 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, octyl, decyl, and the like, as well as cycloalkyl groups such as cyclopentyl, cyclohexyl, and the like. Generally, although again not necessarily, alkyl groups herein contain 1 to about 12 carbon atoms. The term “lower alkyl” intends an alkyl group of 1 to 6 carbon atoms. Preferred lower alkyl substituents contain 1 to 4 carbon atoms, and particularly preferred such substituents contain 1 or 2 carbon atoms (i.e., methyl and ethyl). “Substituted alkyl” refers to alkyl substituted with one or more substituent groups, and the terms “heteroatom-containing alkyl” and “heteroalkyl” refer to alkyl in which at least one carbon atom is replaced with a heteroatom, as described in further detail infra. If not otherwise indicated, the term “alkyl” includes linear, branched, cyclic, unsubstituted, substituted, and/or heteroatom-containing alkyl.
The term “aryl” as used herein, and unless otherwise specified, refers to an aromatic substituent containing a single aromatic ring or multiple aromatic rings that are fused together, directly linked, or indirectly linked (such that the different aromatic rings are bound to a common group such as a methylene or ethylene moiety). Preferred aryl groups contain 5 to 24 carbon atoms, and particularly preferred aryl groups contain 5 to 12 carbon atoms. Exemplary aryl groups contain one aromatic ring or two fused or linked aromatic rings, e.g., phenyl, naphthyl, biphenyl, diphenylether, diphenylamine, benzophenone, and the like. “Substituted aryl” refers to an aryl moiety substituted with one or more substituent groups, and the terms “heteroatom-containing aryl” and “heteroaryl” refer to an aryl substituent in which at least one carbon atom is replaced with a heteroatom, as will be described in further detail infra. If not otherwise indicated, the term “aryl” includes unsubstituted, substituted, and/or heteroatom-containing aromatic substituents.
The term “aralkyl” refers to an alkyl group with an aryl substituent, and the term “alkaryl” refers to an aryl group with an alkyl substituent, wherein “alkyl” and “aryl” are as defined above. In general, aralkyl and alkaryl groups herein contain 6 to 25 carbon atoms, while preferred aralkyl and alkaryl groups contain 6 to 13 carbon atoms. Examples of aralkyl groups include, without limitation, benzyl, 2-phenyl-ethyl, 3-phenyl-propyl, 4-phenyl-butyl, 5-phenyl-pentyl, 4-phenylcyclohexyl, 4-benzylcyclohexyl, 4-phenylcyclohexylmethyl, 4-benzylcyclohexylmethyl, and the like. Alkaryl groups include, for example, p-methylphenyl, 2,4-dimethylphenyl, p-cyclohexylphenyl, 2,7-dimethylnaphthyl, 7-cyclooctylnaphthyl, 3-ethyl-cyclopenta-1,4-dienyl, and the like.
When a functional group is termed “protected,” this means that the group is in modified form to preclude undesired side reactions at the protected site. Suitable protecting groups for the compounds of the present invention will be recognized from the present application taking into account the level of skill in the art, and with reference to standard textbooks, such as Greene et al., Protective Groups in Organic Synthesis (New York: Wiley, 1991).
The term “hydrocarbyl” refers to univalent moieties composed of hydrogen and carbon and containing 1 to about 24 carbon atoms, preferably about 1 to 12 carbon atoms, including linear, branched, cyclic, saturated, and unsaturated species, such as alkyl groups, alkenyl groups, aryl groups, aralkyl groups, alkaryl groups, and the like. Unless otherwise indicated, the term “hydrocarbyl” is to be interpreted as including substituted and/or heteroatom-containing hydrocarbyl moieties as well.
By “substituted” as in “substituted alkyl,” “substituted aryl,” and the like, as alluded to in some of the aforementioned definitions, is typically meant that in the alkyl, aryl, or other moiety, at least one hydrogen atom bound to a carbon (or other) atom is replaced with one or more non-hydrogen substituents. Examples of such substituents include, without limitation: functional groups such as halo, hydroxyl, sulfhydryl, C1–C24 alkoxy, C5–C24 aryloxy, acyl (including C2–C24 alkylcarbonyl (—CO-alkyl) and C6–C24 arylcarbonyl (—CO—C5–C24 aryl)), haloacyl (including C2–C24 haloalkylcarbonyl (—C)—haloalkyl) and C6–C24 haloarylcarbonyl (—CO-aryl)), C2–C24 thioacyloxy (including —O—(CS)-alkyl and —O—(CS)-aryl), C2–C24 thiohaloacyloxy (including —O—(CS)-haloalkyl and —O—(CS)-haloaryl)), acyloxy (—O-acyl), C2–C24 alkoxycarbonyl (—(CO)—O-alkyl), C6–C24 aryloxycarbonyl (—(CO)—O-aryl), C7–C24 alkaryloxycarbonyl (—(CO)—O-alkaryl), C7–C24 aralkyloxycarbonyl (—(CO)—O-aralkyl), C2–C24 haloalkylcarbonato (—O—(CO)—O-haloalkyl)), C6–C24 haloarylcarbonato (—O—(CO)—O-haloaryl), C2–C24 alkylthiocarbonato (—O—(CS)—O-alkyl)), C6–C24 arylthiocarbonato (—O—(CS)—O-aryl), C2–C24 haloalkylthiocarbonato (—O—(CS)—O-haloalkyl), and C6–C24 haloarylthiocarbonato (—O—(CS)—O-haloaryl), C6–C24 aryloxycarbonyl (—(CO)—O-aryl), halocarbonyl (—CO)—X where X is halo), C2–C24 alkylcarbonato (—O—(CO)—O-alkyl), C6–C24 arylcarbonato (—O—(CO)—O-aryl), carboxy (—COOH), carboxylato (—COO−), carbamoyl (—(CO)—NH2), mono-(C1–C24 alkyl)-substituted carbamoyl (—(CO)—NH(C1–C24 alkyl)), di-(C1–C24 alkyl)-substituted carbamoyl (—(CO)—N(C1–C24 alkyl)2), mono-(C6–C24 aryl)-substituted carbamoyl (—(CO)—NH-aryl), di-(C6–C24 aryl)-substituted carbamoyl (—(CO)—N(aryl)2), di-N—(C1–C24 alkyl), N—(C6–C24 aryl)-substituted carbamoyl, thiocarbamoyl (—(CS)—NH2), carbamido (—NH—(CO)—NH2), formyl (—(CO)—H), thioformyl (—(CS)—H), amino (—NH2), mono-(C1–C24 alkyl)-substituted amino, di-(C1–C24 alkyl)-substituted amino, mono-(C5–C24 aryl)-substituted amino, di-(C5–C24 aryl)-substituted amino, C2–C24 alkylamido (—NH—(CO)-alkyl), C6–C24 arylamido (—NH—(CO)-aryl), imino (—CR═NH where R=hydrogen, C1–C24 alkyl, C5–C24 aryl, C6–C24 alkaryl, C6–C24 aralkyl, etc.), alkylimino (—CR=N(alkyl), where R=hydrogen, C1–C24 alkyl, C5–C24 aryl, C6–C24 alkaryl, C6–C24 aralkyl, etc.), arylimino (—CR═N(aryl), where R=hydrogen, C1–C24 alkyl, C5–C24 aryl, C6–C24 alkaryl, C6–C24 aralkyl, etc.), nitro (—NO2), nitroso (—NO), sulfo (—SO2—OH), sulfonamido (—SO2—NH2), sulfonato (—SO2—O−), C1–C24 alkylsulfanyl (—S-alkyl; also termed “alkylthio”), arylsulfanyl (—S-aryl; also termed “arylthio”), C1–C24 alkylsulfinyl (—(SO)-alkyl), C5–C24 arylsulfinyl (—(SO)-aryl), C1–C24 alkylsulfonyl (—SO2-alkyl), C5–C24 arylsulfonyl (—SO2-aryl), phosphono (—P(O)(OH)2), phosphonato (—P(O)(O−)2), phosphinato (—P(O)(O−)), phospho (—PO2), and phosphino (—PH2); and the hydrocarbyl moieties C1–C24 alkyl (preferably C1–C18 alkyl, more preferably C1–C12 alkyl, most preferably C1–C6 alkyl), C2–C24 alkenyl (preferably C2–C18 alkenyl, more preferably C2–C12 alkenyl, most preferably C2–C6 alkenyl), C2–C24 alkynyl (preferably C2–C18 alkynyl, more preferably C2–C12 alkynyl, most preferably C2–C6 alkynyl), C5–C24 aryl (preferably C5–C14 aryl), C6–C24 alkaryl (preferably C6–C18 alkaryl), and C6–C24 aralkyl (preferably C6–C18 aralkyl).
The term “heteroatom-containing” as in a “heteroatom-containing alkyl group” (also termed a “heteroalkyl” group) or a “heteroatom-containing aryl group” (also termed a “heteroaryl” group) refers to a molecule, linkage or substituent in which one or more carbon atoms are replaced with an atom other than carbon, e.g., nitrogen, oxygen, sulfur, phosphorus or silicon, typically nitrogen, oxygen or sulfur, preferably nitrogen or oxygen. Similarly, the term “heteroalkyl” refers to an alkyl substituent that is heteroatom-containing, the term “heterocyclic” refers to a cyclic substituent that is heteroatom-containing, the terms “heteroaryl” and “heteroaromatic” respectively refer to “aryl” and “aromatic” substituents that are heteroatom-containing, and the like. Examples of heteroalkyl groups include alkoxyaryl, alkylsulfanyl-substituted alkyl, N-alkylated amino alkyl, and the like. Examples of heteroaryl substituents include pyrrolyl, pyrrolidinyl, pyridinyl, quinolinyl, indolyl, pyrimidinyl, imidazolyl, 1,2,4-triazolyl, tetrazolyl, etc., and examples of heteroatom-containing alicyclic groups are pyrrolidino, morpholino, piperazino, piperidino, etc.
The terms “halo” and “halogen” are used in the conventional sense to refer to a chloro, bromo, and fluoro or iodo substituent.
In addition, the aforementioned functional groups may, if a particular group permits, be further substituted with one or more additional functional groups or with one or more hydrocarbyl moieties such as those specifically enumerated above. Analogously, the above-mentioned hydrocarbyl moieties may be further substituted with one or more functional groups or additional hydrocarbyl moieties such as those specifically enumerated.
When referring to a compound of the invention as a therapeutic or otherwise biologically active agent, applicants intend the term “compound” or “active agent” to encompass not only the specified molecular entity but also its pharmaceutically acceptable, pharmacologically active analogs, including, but not limited to, salts, esters, amides, prodrugs, conjugates, and active metabolites.
The terms “treating” and “treatment” as used herein refer to reduction in severity and/or frequency of symptoms, elimination of symptoms and/or underlying cause, prevention of the occurrence of symptoms and/or their underlying cause, and improvement or remediation of damage. Thus, “treating” a patient with a compound of the invention includes prevention of a particular disorder or adverse physiological event in a susceptible individual as well as treatment of a clinically symptomatic individual by inhibiting or causing regression of a disorder or disease. For example, treatment of cancer encompasses chemoprevention in a patient susceptible to developing cancer (e.g., at a higher risk, as a result of genetic predisposition, environmental factors, or the like) and/or in cancer survivors at risk of cancer recurrence, as well as treatment of a cancer patient dual by inhibiting or causing regression of a disorder or disease.
By the terms “effective amount” and “therapeutically effective amount” of a compound of the invention is meant a nontoxic but sufficient amount of the drug or agent to provide the desired effect.
The term “dosage form” denotes any form of a pharmaceutical composition that contains an amount of active agent sufficient to achieve a therapeutic effect with a single administration. When the formulation is a tablet or capsule, the dosage form is usually one such tablet or capsule. The frequency of administration that will provide the most effective results in an efficient manner without overdosing will vary with the characteristics of the particular active agent, including both its pharmacological characteristics and its physical characteristics, such as hydrophilicity.
The term “controlled release” refers to a drug-containing formulation or fraction thereof in which release of the drug is not immediate, i.e., with a “controlled release” formulation, administration does not result in immediate release of the drug into an absorption pool. The term is used interchangeably with “nonimmediate release” as defined in Remington: The Science and Practice of Pharmacy, Nineteenth Ed. (Easton, Pa.: Mack Publishing Company, 1995). In general, the term “controlled release” as used herein includes sustained release and delayed release formulations.
The term “sustained release” (synonymous with “extended release”) is used in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that preferably, although not necessarily, results in substantially constant blood levels of a drug over an extended time period.
The term “pharmaceutically acceptable” means that a material that is not biologically or otherwise undesirable, i.e., the material may be incorporated into a pharmaceutical composition administered to a patient without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained. When the term “pharmaceutically acceptable” is used to refer to a pharmaceutical carrier or excipient, it is implied that the carrier or excipient has met the required standards of toxicological and manufacturing testing or that it is included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug administration. “Pharmacologically active” (or simply “active”) as in a “pharmacologically active” derivative or analog, refers to a derivative or analog having the same type of pharmacological activity as the parent compound and approximately equivalent in degree.
II. Compounds of the Invention
The present invention provides new compounds useful as therapeutic agents for the treatment of cancer or other disorders responsive to selective apoptosis. One of the novel compounds provided by the present invention is a purified, isolated form of isoapoptolidin, which is a ring-expanded macrolide isomer of apoptolidin having the structure of formula (I)




Although apoptolidin can be purified chromatographically, isoapoptolidin does not elute without some apoptolidin contamination. Example 1 describes an enrichment procedure required to purify isolated isoapoptolidin and the techniques that permit analysis of the resulting purified crystals.
Other compounds of the present invention include functionalized derivatives of isoapoptolidin, apoptolidin, and deglycosylated isoapoptolidin, as well as stereoisomers thereof and deglycosylated isoapoptolidin per se.
Functionalized isoapoptolidin compounds of the invention are compounds that correspond to isoapoptolidin or a stereoisomer thereof in which at least one hydroxyl group within the molecule is replaced with a substituent selected from:
(a) C1–C24 hydrocarbyloxy, optionally substituted with one or more halogen atoms, e.g., C1–C24 alkoxy (—O—(C1–C24 alkyl)), C6–C24 aralkoxy (—O—(C6–C24 aralkyl)), C6–C24 alkaryloxy (—O—(C6–C24 alkaryl)), C1–C24 haloalkoxy (—O—(C1–C24 haloalkyl)), and C6–C24 haloaralkoxy (—O—(C6–C24 haloalkoxy),
preferably C1–C12 hydrocarbyloxy, optionally substituted with one or more halogen atoms, e.g., C1–C12 alkoxy (—O—(C1–C12 alkyl)), C6–C12 aralkoxy (—O—(C6–C12 aralkyl)), C6–C12 alkaryloxy (—O—(C6–C12 alkaryl)), C1–C12 haloalkoxy (—O—(C1–C12 haloalkyl)), and C6–C12 haloaralkoxy (—O—(C6–C12 haloaralkyl);
(b) C2–C25 acyloxy (—O—(CO)—(C1–C24 hydrocarbyl)), e.g., —O—(CO)—(C1–C24 alkyl), —O—(CO)—(C5–C24 aryl), —O—(CO)—(C6–C24 aralkyl), and —O—(CO)—(C6–C24 alkaryl)),
preferably C2–C13 acyloxy (—O—(CO)—(C1–C12 hydrocarbyl)), e.g., —O—(CO)—(C1C12 alkyl), —O—(CO)—(C5–C12 aryl), —O—(CO)—(C6–C12 aralkyl), and —O—(CO)—(C6–C12 alkaryl));
(c) C2–C25 haloacyloxy (—O—(CO)—(C2–C24 halohydrocarbyl)), e.g., —O—(CO)—(C1–C24 haloalkyl), —O—(CO)—(C5–C24 haloaryl), —O—(CO)—(C5–C24 haloaralkyl), and —O—(CO)—(C5–C24 haloalkaryl),
preferably C2–C13 haloacyloxy (—O—(CO)—(C2–C12 halohydrocarbyl)), e.g., —O—(CO)—(C1–C12 haloalkyl), —O—(CO)—(C5–C12 haloaryl), —O—(CO)—(C5–C12 haloaralkyl), and —O—(CO)—(C5–C12 haloalkaryl);
(d) C2–C25 thioacyloxy (—O—(CS)—(C2–C24 hydrocarbyl)), e.g., —O—(CS)—(C1–C24 alkyl), —O—(CS)—(C5–C24 aryl), —O—(CS)—(C5–C24 aralkyl), and —O—(CS)—(C5–C24 alkaryl),
preferably C2–C13 thioacyloxy (—O—(CS)—(C2–C12 hydrocarbyl)), e.g., —O—(CS)—(C1–C12 alkyl), —O—(CS)—(C5–C12 aryl), —O—(CS)—(C5–C12 aralkyl), and —O—(CS)—(C5–C12 alkaryl);
(e) C2–C25 thiohaloacyloxy (—O—(CS)—(C2–C24 hydrocarbyl)), e.g., —O—(CS)—(C1–C24 haloalkyl), —O—(CS)—(C5–C24 haloaryl), —O—(CS)—(C5–C24 haloaralkyl), and —O—(CS)—(C5–C24 haloalkaryl),
preferably C2–C13 thiohaloacyloxy (—O—(CS)—(C2–C12 hydrocarbyl)), e.g., —O—(CS)—(C1–C12 haloalkyl), —O—(CS)—(C5–C12 haloaryl), —O—(CS)—(C5–C12 haloaralkyl), and —O—(CS)—(C5–C12 haloalkaryl);
(f) C2–C25 carbonato (—O—(CO)—O—(C1–C24 hydrocarbyl)), e.g., C2–C25 alkylcarbonato (—O—(CO)—O—(C1–C24 alkyl)), C6–C25 arylcarbonato (—O—(CO)—O—(C5–C24 aryl)), C6–C25 aralkylcarbonato (—O—(CO)—O—(C5–C24 aralkyl)), and C6–C25 alkarylcarbonato (—O—(CO)—O—(C5–C24 alkaryl)),
preferably C2–C13 carbonato (—O—(CO)—O—(C1–C12 hydrocarbyl)), e.g., C2–C13 alkylcarbonato (—O—(CO)—O—(C1–C12 alkyl)), C6–C13 arylcarbonato (—O—(CO)—O—(C5–C12 aryl)), C6–C13 aralkylcarbonato (—O—(CO)—O—(C5–C12 aralkyl)), and C6–C13 alkarylcarbonato (—O—(CO)—O—(C5–C12 alkaryl));
(g) halogenated C2–C25 carbonato (—O—(CO)—O—(C1–C24 halohydrocarbyl)), e.g., C2–C25 haloalkylcarbonato (—O—(CO)—O—(C1–C24 haloalkyl)), C6–C25 haloarylcarbonato (—O—(CO)—O—(C5–C24 haloaryl)), C6–C25 haloaralkylcarbonato (—O—(CO)—O—(C5–C24 haloaralkyl)), and C6–C25 haloalkarylcarbonato (—O—(CO)—O—(C5–C24 haloalkaryl)),
preferably halogenated C2–C13 carbonato (—O—(CO)—O—(C1–C12 halohydrocarbyl)), e.g., C2–C13 haloalkylcarbonato (—O—(CO)—O—(C1–C12 haloalkyl)), C6–C13 haloarylcarbonato (—O—(CO)—O—(C5–C12 haloaryl)), C6–C13 haloaralkylcarbonato (—O—(CO)—O—(C5–C12 haloaralkyl)), and C6–C13 haloalkarylcarbonato (—O— (CO)—O—(C5–C12 haloalkaryl));
(h) C2–C25 thiocarbonato (—O—(CS)—O—(C1–C24 hydrocarbyl)), e.g., C2–C25 alkylcarbonato (—O—(CS)—O—(C1–C24 alkyl)), C6–C25 arylcarbonato (—O—(CS)—O—(C5–C24 aryl)), C6–C25 aralkylcarbonato (—O—(CS)—O—(C5–C24 aralkyl)), and C6–C25 alkarylcarbonato (—O—(CS)—O—(C5C24 alkaryl)),
preferably C2–C13 thiocarbonato (—O—(CS)—O—(C1–C12 hydrocarbyl)), e.g., C2–C13 alkylcarbonato (—O—(CS)—O—(C1–C12 alkyl)), C6–C13 arylcarbonato (—O—(CS)—O—(C5–C12 aryl)), C6–C13 aralkylcarbonato (—O—(CS)—O—(C5–C12 aralkyl)), and C6–C13 alkarylcarbonato (—O—(CS)—O—(C5–C12 alkaryl));
(i) halogenated C2–C25 thiocarbonato (—O—(CS)—O—(C1–C24 halohydrocarbyl)), e.g., C2C25 haloalkylthiocarbonato (—O—(CS)—O—(C1–C24 haloalkyl)), C6–C25 haloarylthiocarbonato (—O—(CS)—O—(C5–C24 haloaryl)), C6–C25 haloaralkylthiocarbonato (—O—(CS)—O—(C5–C24 haloaralkyl)), and C6–C25 haloalkarylthiocarbonato (—O—(CS)—O—(C5–C24 haloalkaryl)),
preferably halogenated C2–C13 thiocarbonato (—O—(CS)—O—(C1–C12 halohydrocarbyl)), e.g., C2–C13 haloalkylthiocarbonato (—O—(CS)—O—(C1–C12 haloalkyl)), C6–C13 haloarylthiocarbonato (—O—(CS)—O—(C5–C12 haloaryl)), C6–C13 haloaralkylthiocarbonato (—O—(CS)—O—(C5–C12 haloaralkyl)), and C6–C13 haloalkarylthiocarbonato (—O—(CS)—O—(C5–C12 haloalkaryl));
(j) carbamoyloxy (—O—(CO)—NH2), mono-(C1–C24 alkyl)-substituted carbamoyloxy (—O—(CO)—NH(C1–C24 alkyl)), di-(C1–C24 alkyl)-substituted carbamoyloxy (—O—(CO)—N(C1–C24 alkyl)2), mono-(C6–C24 aryl)-substituted carbamoyloxy (—O—(CO)—NH-aryl), di-(C6–C24 aryl)-substituted carbamoyloxy (—O—(CO)—N(aryl)2), and di-N—(C1–C24 alkyl), N—(C6–C24 aryl)-substituted carbamoyloxy,
preferably carbamoyloxy (—O—(CO)—NH2), mono-(C1–C12 alkyl)-substituted carbamoyloxy (—O—(CO)—NH(C1–C12 alkyl)), di-(C1–C12 alkyl)-substituted carbamoyloxy (—O—(CO)—N(C1–C12 alkyl)2), mono-(C6–C12 aryl)-substituted carbamoyloxy (—O—(CO)—NH-aryl), di-(C6–C12 aryl)-substituted carbamoyloxy (—O—(CO)—N(aryl)2), and di-N—(C1–C12 alkyl), N—(C6–C12 aryl)-substituted carbamoyloxy
(k) thiocarbamoyloxy (—O—(CS)—NH2), mono-(C1–C24 alkyl)-substituted thiocarbamoyloxy (—O—(CS)—NH(C1–C24 alkyl)), di-(C1–C24 alkyl)-substituted thiocarbamoyloxy (—O—(CS)—N(C1–C24 alkyl)2), mono-(C6–C24 aryl)-substituted thiocarbamoyloxy (—O—(CS)—NH-aryl), di-(C6–C24 aryl)-substituted carbamoyloxy (—O—(CS)—N(aryl)2), and di-N—(C1–C24 alkyl), N—(C6–C24 aryl)-substituted thiocarbamoyloxy,
preferably thiocarbamoyloxy (—O—(CS)—NH2), mono-(C1–C12 alkyl)-substituted thiocarbamoyloxy (—O—(CS)—NH(C1–C12 alkyl)), di-(C1–C12 alkyl)-substituted thiocarbamoyloxy (—O—(CS)—N(C1–C12 alkyl)2), mono-(C6–C12 aryl)-substituted thiocarbamoyloxy (—O—(CS)—NH-aryl), di-(C6–C12 aryl)-substituted thiocarbamoyloxy (—O—(CS)—N(aryl)2), and di-N—(C1–C12 alkyl), N—(C6–C12 aryl)-substituted thiocarbamoyloxy;
(l) sulfamoyloxy (—O—SO2—NH2), mono-(C1–C24 alkyl)-substituted sulfamoyloxy (—O—SO2—NH(C1–C24 alkyl)), di-(C1–C24 alkyl)-substituted sulfamoyloxy (—O—SO2—N(C1–C24 alkyl)2), mono-(C6–C24 aryl)-substituted sulfamoyloxy (—O—SO2—NH-aryl), di-(C6–C24 aryl)-substituted sulfamoyloxy (—O—SO2—N(aryl)2), and di-N—(C1–C24 alkyl), N—(C6–C24 aryl)-substituted sulfamoyloxy,
preferably sulfamoyloxy (—O—SO2—NH2), mono-(C1–C12 alkyl)-substituted sulfamoyloxy (—O—SO2—NH(C1–C12 alkyl)), di-(C1–C12 alkyl)-substituted sulfamoyloxy (—O—SO2—N(C1–C12 alkyl)2), mono-(C6–C12 aryl)-substituted sulfamoyloxy (—O—SO2—NH-aryl), di-(C6–C12 aryl)-substituted sulfamoyloxy (—O—SO2—N(aryl)2), and di-N—(C1–C12 alkyl), N—(C6–C12 aryl)-substituted sulfamoyloxy and
(m) protected hydroxyl groups such as tri-(C1–C12 hydrocarbyl)-substituted silyloxy, preferably tri-(C1–C4 alkyl)silyloxy, and substituted methyl ethers such as (C1–C6 alkoxy)methyl ethers (e.g., methoxymethyl ether (—O—CH2—O—CH3) and t-butoxymethyl ether (—O—CH2—O—C(CH3)3)) and (C1–C6 alkylthio)methyl ethers such as methylthiomethyl ether (—O—CH2—S—CH3),
wherein any of (a) through (m) may be substituted and/or heteroatom-containing where the functional group permits.
Preferred functionalized isoapoptolidin compounds of the invention are those wherein at least one hydroxyl group of isoapoptolidin per se is replaced with C1–C12 hydrocarbyl, C1–C13 acyl, or a protected hydroxyl group. Of these, the more preferred functionalized isoapoptolidin compounds are exemplified by the structure of formula (II)




or are stereoisomers thereof, wherein Q1, Q2, Q3, Q4, Q5, Q6, Q7, and Q8 are selected from H, C1–C12 hydrocarbyl (including substituted and/or heteroatom-containing C1–C12 hydrocarbyl), acyl of the formula —(CO)—R5 wherein R5 is C1–C12 hydrocarbyl (including substituted and/or heteroatom-containing C1–C12 hydrocarbyl), and hydroxyl-protecting groups. In formula (II), R1, R2, R3, and R4, which are methyl groups in isoapoptolidin, are independently selected from C1–C12 alkyl (including substituted and/or heteroatom-containing C1–C12 alkyl) and H.

More preferred functionalized isoapoptolidin derivatives of the invention are those of formula (II) wherein Q1, Q2, Q3, Q4, Q5, Q6, Q7, and Q8 are selected from H, C1–C6 hydrocarbyl (including substituted and/or heteroatom-containing C1–C6 hydrocarbyl), —(CO)—R5 wherein R5 is C1–C6 hydrocarbyl (including substituted and/or heteroatom-containing C1–C6 hydrocarbyl), and —Si(R6R7R8) wherein R6, R7, and R8 are C1–C6 hydrocarbyl, and R1, R2, R3, and R4 are C1–C4 alkyl (including substituted and/or heteroatom-containing C1–C4 alkyl).
Still more preferred functionalized isoapoptolidin derivatives of the invention are those of the formula (II) wherein Q1, Q2, Q3, Q4, Q5, Q6, Q7, and Q8 are independently selected from H, C1–C6 alkyl (including substituted and/or heteroatom-containing alkyl), —(CO)—R5 wherein R5 is C1–C4 or benzyl, and —Si(R6R7R8) wherein R6, R7, and R8 are all methyl or are all ethyl, and R1, R2, R3, and R4 are methyl.
Representative functionalized isoapoptolidin derivatives of the invention which are particularly preferred herein are compounds of formula (II) wherein:
Q1 is —(CO)—R5 and Q2, Q3, Q4, Q5, Q6, Q7, and Q8 are H;
Q2 and Q3 are —(CO)—R5 and Q1, Q4, Q5, Q6, Q7, and Q8 are H;
Q3 is —(CO)—R5 and Q1, Q2, Q4, Q5, Q6, Q7, and Q8 are H;
Q4 is —(CO)—R5 and Q1, Q2, Q3, Q5, Q6, Q7, and Q8 are H;
Q5 is —(CO)—R5 and Q1, Q2, Q3, Q4, Q6, Q7 and Q8 are H;
Q6 is —(CO)—R5 and Q1, Q2, Q3, Q4, Q5, Q7 and Q8 are H;
Q6 is C1–C6 alkyl and Q1, Q2, Q3, Q4, Q5, Q7 and Q8 are H; or
Q8 is C1–C6 alkyl and Q1, Q2, Q3, Q4, Q5, Q6 and Q7 are H, and the most preferred functionalized isoapoptolidin derivatives of the invention are compounds of formula (II) wherein:
Q1 is benzoyl and Q2, Q3, Q4, Q5, Q6, Q7, and Q8 are H;
Q2 and Q3 are acetyl and Q1, Q4, Q5, Q6, Q7, and Q8 are H;
Q3 is acetyl and Q1, Q2, Q4, Q5, Q6, Q7 and Q8 are H;
Q4 is acetyl and Q1, Q2, Q3, Q5, Q6, Q7 and Q8 are H;
Q5 is acetyl and Q1, Q2, Q3, Q4, Q6, Q7, and Q8 are H;
Q6 is acetyl and Q1, Q2, Q3, Q4, Q5, Q7, and Q8 are H;
Q6 is methyl and Q1, Q2, Q3, Q4, Q5, Q7, and Q8 are H; or
Q8 is methyl and Q1, Q2, Q3, Q4, Q5, Q6, and Q7 are H.
The functionalized apoptolidin compounds of the invention correspond to apoptolidin per se or a stereoisomer thereof, in which at least one hydroxyl group within the molecule is replaced with a substituent selected from (a) through (j) above. Preferred functionalized apoptolidin compounds of the invention have the structure of formula (III)




wherein Q1, Q2, Q3, Q4, Q5, Q6, Q7, Q8, R1, R2, R3, and R4 are defined as for the functionalized isoapoptolidin compounds of formula (II), with the proviso that at least one of Q1, Q2, Q3, Q4, Q5, Q6, Q7, and Q8 is other than H when R1, R2, R3, and R4 are methyl and the functionalized apoptolidin compound has the stereoisomeric configuration shown in formula (III).

More preferred functionalized apoptolidin derivatives of the invention, as with the functionalized isoapoptolidin compound of formula (II), are, accordingly, those of formula (III) wherein Q1, Q2, Q3, Q4, Q5, Q6, Q7, and Q8 are selected from H, C1–C6 hydrocarbyl, —(CO)—R5 wherein R5 is C1–C6 hydrocarbyl, and —Si(R6R7R8) wherein R6, R7, and R8 are C1–C6 hydrocarbyl, with the proviso that at least one of Q1, Q2, Q3, Q4, Q5, Q6, Q7, and Q8 is other than H, and R1, R2, R3, and R4 are C1–C4 alkyl.
Still more preferred functionalized apoptolidin derivatives of the invention are those of the formula (III) wherein Q1, Q2, Q3, Q4, Q5, Q6, Q7, and Q8 are selected from H, C1–C6 alkyl, —(CO)—R5 wherein R5 is lower alkyl or benzyl, and —Si(R6R7R8) wherein R6, R7, and R8 are all methyl or are all ethyl, with the proviso that at least one of Q1, Q2, Q3, Q4, Q5, Q6, Q7, and Q8 is other than H, and R1, R2, R3, and R4 are methyl.
Representative functionalized apoptolidin derivatives of the invention which are particularly preferred herein are compounds of formula (III) wherein:
Q1 is —(CO)—R5 and Q2, Q3, Q4, Q5, Q6, Q7, and Q8 are H;
Q2 and Q3 are —(CO)—R5 and Q1, Q4, Q5, Q6, Q7, and Q8 are H;
Q3 is —(CO)—R5 and Q1, Q2, Q4, Q5, Q6, Q7, and Q8 are H;
Q4 is —(CO)—R5 and Q1, Q2, Q3, Q5, Q6, Q7, and Q8 are H;
Q5 is —(CO)—R5 and Q1, Q2, Q3, Q4, Q6, Q7, and Q8 are H;
Q6 is —(CO)—R5 and Q1, Q2, Q3, Q4, Q5, Q7, and Q8 are H;
Q6 is C1–C6 alkyl and Q1, Q2, Q3, Q4, Q5, Q7, and Q8 are H; or
Q8 is C1–C6 alkyl and Q1, Q2, Q3, Q4, Q5, Q6, and Q7 are H, and the most preferred functionalized apoptolidin derivatives of the invention are compounds of formula (III) wherein:
Q1 is benzoyl and Q2, Q3, Q4, Q5, Q6, Q7, and Q8 are H;
Q2 and Q3 are acetyl and Q1, Q4, Q5, Q6, Q7, and Q8 are H;
Q3 is acetyl and Q1, Q2, Q4, Q5, Q6, Q7, and Q8 are H;
Q4 is acetyl and Q1, Q2, Q3, Q5, Q6, Q7, and Q8 are H;
Q5 is acetyl and Q1, Q2, Q3, Q4, Q6, Q7, and Q8 are H;
Q6 is acetyl and Q1, Q2, Q3, Q4, Q5, Q7, and Q8 are H;
Q6 is methyl and Q1, Q2, Q3, Q4, Q5, Q7 and Q8 are H; or
Q8 is methyl and Q1, Q2, Q3, Q4, Q5, Q6 and Q7 are H.
The deglycosylated isoapoptolidin compounds of the invention include deglycosylated isoapoptolidin per se as well as functionalized analogs thereof, wherein “deglycosylated” refers to a truncated structure in which the oleandrose and olivomycose sugars are absent. Deglycosylated apoptolidin is described in U.S. Patent Application Publication No. 2002/0049168 to Khosla et al. and U.S. Patent Application Publication No. 2002/0077300 to Khosla et al., now U.S. Pat. No. 6,548,485. The deglycosylated isoapoptolidin compounds of the invention have the structure of formula (IV)




wherein Q1, Q2, Q4, Q5, Q6, Q8, and Q9 are independently selected from H, C1–C12 hydrocarbyl, —(CO)—R5 wherein R5 is C1–C12 hydrocarbyl, and hydroxyl-protecting groups, and R1, R2, and R3 are independently selected from C1–C12 alkyl and H.

More preferred deglycosylated isoapoptolidin compounds of the invention are those of formula (IV) wherein Q1, Q2, Q4, Q5, Q6, Q8, and Q9 are selected from H, C1–C6 hydrocarbyl, —(CO)—R5 wherein R5 is C1–C6 hydrocarbyl, and —Si(R6R7R8) wherein R6, R7, and R8 are C1–C6 hydrocarbyl, and R1, R2, R3, and R4 are C1–C4 alkyl.
Still more preferred deglycosylated isoapoptolidin compounds of the invention are those of formula (IV) wherein Q1, Q2, Q4, Q5, Q6, Q8, and Q9 are selected from H, C1–C6 alkyl, —(CO)—R5 wherein R5 is lower alkyl or benzyl, and —Si(R6R7R8) wherein R6, R7, and R8 are all methyl or are all ethyl.
Representative deglycosylated isoapoptolidin compounds of the invention which are particularly preferred herein are compounds of formula (IV) wherein:
Q1 is —(CO)—R5 and Q2, Q4, Q5, Q6, Q8, and Q9 are H;
Q2 is —(CO)—R5 and Q1, Q4, Q5, Q6, Q8, and Q9 are H;
Q4 is —(CO)—R5 and Q1, Q2, Q5, Q6, Q8 and Q9 are H;
Q5 is —(CO)—R5 and Q1, Q2, Q4, Q6, Q8 and Q9 are H;
Q6 is —(CO)—R5 and Q1, Q2, Q4, Q5, Q8, and Q9 are H;
Q6 is C1–C6 alkyl and Q1, Q2, Q4, Q5, Q8, and Q9 are H; or
Q8 is C1–C6 alkyl and Q1, Q2, Q4, Q5, Q6, and Q9 are H, and the most preferred deglycosylated isoapoptolidin compounds of the invention are compounds of formula (IV) wherein:
Q1 is benzoyl and Q2, Q4, Q5, Q6, Q8, and Q9 are H;
Q2 and are acetyl and Q1, Q4, Q5, Q6, Q8, and Q9 are H;
Q4 is acetyl and Q1, Q2, Q5, Q6, Q8 and Q9 are H;
Q5 is acetyl and Q1, Q2, Q4, Q6, Q8 and Q9 are H;
Q6 is acetyl and Q1, Q2, Q3, Q4, and Q6 are H;
Q6 is methyl and Q1, Q2, Q3, Q4, and Q5 are H;
Q8 is methyl and Q1, Q2, Q4, Q5, Q6, and Q9 are H.
In addition to the foregoing, it is also to be understood that within the context of the present invention, adjacent (1,2-)diols in any one of the compounds of formulae (II), (III), and (IV) may be linked to form cyclic ethers. For example, cyclic ethers may be prepared by linking together Q1 and Q5. As will be appreciated by those of ordinary skill in the art, the reaction is readily carried out by treating the diol form of the compound with triphosgene or acetone to yield a cyclic carbonate or acetonide group, respectively.
The compounds of the invention also include derivatives in which at least one of the carbon-carbon double bonds of the compounds of formulae (II), (III), and (IV) is be rendered saturated through, for example, catalytic hydrogenation or nucleophilic addition. In another embodiment, the C-10/C-13 diene of the compounds of formulae (II), (III), and (IV) is reacted with a dienophile to yield Diels-Alder adducts. In one example, the reaction of the C-10/C-13 diene of apoptolidin with an N-halosuccinimide results in a compound having the structure of formula (V)




wherein Rx is a halogen atom. The dienophile may be, for example, N-bromosuccinimide (NBS), N-iodosuccinimide (NIS), or N-chlorosuccinimide (NCS).

Additional compounds within the scope of the invention result from the cleavage or decomposition of apoptolidin. Such structures serve to facilitate a divide-and-diversify strategy for determining the structural basis of apoptolidin's biological activity. For example, when the C-20/C-21 bond of the apoptolidin derivative of formula (III) is oxidatively cleaved, and the macrocyclic aldehyde is reduced, the cyclic ether of formula (VI) and the δ-lactone of formula (VII) result.




The procedure for separating apoptolidin into the structures of formulas (VI) and (VII) is described in Example 6. When the C-19/C-20 diol is activated by formation of the stanylidine acetal, it undergoes a retro-aldol reaction to cleave the C-22/C-23 carbon-carbon bond as shown in formulae (VIII) and (IX)




wherein R9 is selected from H, C1–C12 hydrocarbyl, —(CO)—R5 wherein R5 is C1–C12 hydrocarbyl, and hydroxyl-protecting groups.

III. Utility, Testing, and Administration:

As previously mentioned, the compounds of the present invention are useful as therapeutic agents for the treatment of cancer or other disorders responsive to selective apoptosis. The compounds can be administered to a human patient by themselves or in pharmaceutical compositions in which they are mixed with suitable carriers or excipients. Compounds of the invention may also be administered in combination, in which case they may be administered separately, in different dosage forms, or simultaneously, either in one dosage form or in two different dosage forms. Pharmaceutically acceptable dosage forms are described infra.
Representative cancer conditions and cell types against which the compounds of the invention may be useful include melanoma, myeloma, chronic lymphocytic leukemia (CLL), AIDS-related lymphoma, non-Hodgkin's lymphoma, colorectal cancer, renal cancer, prostate cancer, cancers of the head, neck, stomach, esophagus, anus, or cervix, ovarian cancer, breast cancer, peritoneal cancer, and non-small cell lung cancer. A compound of the invention can be administered alone, i.e., in monotherapy, or in combination with one or more other active agents, e.g., other anticancer agents.
In practicing various aspects of the present invention, compounds in accordance with the invention can be tested for a biological activity of interest using any assay protocol that is predictive of activity in vivo.
A useful method for assessing anticancer activities of compounds of the invention involves the multiple-human cancer cell line screening assays run by the National Cancer Institute (e.g., Boyd, “Status of the NCI Preclinical Antitumor Drug Discovery Screen” in Cancer: Principles and Practice of Oncology Updates, DeVita et al., eds, pp. 1–12 (1989)). The screening panel, which involves approximately 60 different human cancer cell lines, is a useful indicator of in vivo antitumor activity for a broad variety of tumor types (Grever et al. (1992) Seminars Oncol. 19:622; Monks et al. (1991) J. Natl. Cancer Inst. 83:757–766), such as leukemia, non-small cell lung, colon, central nervous system (CNS), melanoma, ovarian, renal, prostate, and breast cancers. Antitumor activities can be expressed in terms of ED50 (or GI50), where ED50 is the molar concentration of compound effective to reduce cell growth by 50%. Compounds with lower ED50 values tend to have greater anticancer activities than compounds with higher ED50 values. Example 7 of U.S. Patent Application Publication No. 2002/0137789, noted above, describes a P388 murine lymphocytic leukemia cell assay that measures the ability of compounds of the invention to inhibit cellular growth.
To study the biological activities of the claimed apoptolidin analogs and derivatives, mitochondrial F0F1-ATPase inhibition assays were performed; the results of these experiments are set forth in Example 5. Upon the confirmation of a compound's potential activity in vitro, further evaluation is typically conducted in vivo in laboratory animals, for example, by measuring reduction of lung nodule metastases in mice with B16 melanoma (e.g., Schuchter et al, (1991) Cancer Res. 51(2):682–687). The efficacy of drug combination chemotherapy can be evaluated, for example, using the human B-CLL xenograft model in mice (e.g., Mohammad et al., (1996) Leukemia 10(1): 130–137). Ultimately, the safety and efficacy of compounds of the invention are evaluated in human clinical trials.
Administration of a compound of the invention may be carried out using any appropriate mode of administration. Thus, administration can be, for example, oral, parenteral, transdermal, transmucosal (including rectal and vaginal), sublingual, by inhalation, or via an implanted reservoir in a dosage form. The term “parenteral” as used herein is intended to include subcutaneous, intravenous, and intramuscular injection.
Depending on the intended mode of administration, the pharmaceutical formulation may be a solid, semi-solid or liquid, such as, for example, a tablet, a capsule, a caplet, a liquid, a suspension, an emulsion, a suppository, granules, pellets, beads, a powder, or the like, preferably in unit dosage form suitable for single administration of a precise dosage. Suitable pharmaceutical compositions and dosage forms may be prepared using conventional methods known to those in the field of pharmaceutical formulation and described in the pertinent texts and literature, e.g., in Remington: The Science and Practice of Pharmacy (Easton, Pa.: Mack Publishing Co., 1995). For those compounds that are orally active, oral dosage forms are generally preferred, and include tablets, capsules, caplets, solutions, suspensions and syrups, and may also comprise a plurality of granules, beads, powders, or pellets that may or may not be encapsulated. Preferred oral dosage forms are tablets and capsules.
Tablets may be manufactured using standard tablet processing procedures and equipment. Direct compression and granulation techniques are preferred. In addition to the active agent, tablets will generally contain inactive, pharmaceutically acceptable carrier materials such as binders, lubricants, disintegrants, fillers, stabilizers, surfactants, coloring agents, and the like. Binders are used to impart cohesive qualities to a tablet, and thus ensure that the tablet remains intact. Suitable binder materials include, but are not limited to, starch (including corn starch and pregelatinized starch), gelatin, sugars (including sucrose, glucose, dextrose, and lactose), polyethylene glycol, waxes, and natural and synthetic gums, e.g., acacia sodium alginate, polyvinylpyrrolidone, cellulosic polymers (including hydroxypropyl cellulose, hydroxypropyl methylcellulose, methyl cellulose, microcrystalline cellulose, ethyl cellulose, hydroxyethyl cellulose, and the like), and Veegum. Lubricants are used to facilitate tablet manufacture, promoting powder flow and preventing particle capping (i.e., particle breakage) when pressure is relieved. Useful lubricants are magnesium stearate, calcium stearate, and stearic acid. Disintegrants are used to facilitate disintegration of the tablet, and are generally starches, clays, celluloses, algins, gums, or crosslinked polymers. Fillers include, for example, materials such as silicon dioxide, titanium dioxide, alumina, talc, kaolin, powdered cellulose, and microcrystalline cellulose, as well as soluble materials such as mannitol, urea, sucrose, lactose, dextrose, sodium chloride, and sorbitol. Stabilizers, as well known in the art, are used to inhibit or retard drug decomposition reactions that include, by way of example, oxidative reactions.
Oral dosage forms, whether tablets, capsules, caplets, or particulates, may, if desired, be formulated so as to provide for gradual, sustained release of the active agent over an extended time period. Generally, as will be appreciated by those of ordinary skill in the art, sustained release dosage forms are formulated by dispersing the active agent within a matrix of a gradually hydrolyzable material such as a hydrophilic polymer, or by coating a solid, drug-containing dosage form with such a material. Hydrophilic polymers useful for providing a sustained release coating or matrix include, by way of example: cellulosic polymers such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, methyl cellulose, ethyl cellulose, cellulose acetate, and carboxymethylcellulose sodium; acrylic acid polymers and copolymers, preferably formed from acrylic acid, methacrylic acid, acrylic acid alkyl esters, methacrylic acid alkyl esters, and the like, e.g. copolymers of acrylic acid, methacrylic acid, methyl acrylate, ethyl acrylate, methyl methacrylate and/or ethyl methacrylate; and vinyl polymers and copolymers such as polyvinyl pyrrolidone, polyvinyl acetate, and ethylene-vinyl acetate copolymer.
Preparations according to this invention for parenteral administration include sterile aqueous and nonaqueous solutions, suspensions, and emulsions. Injectable aqueous solutions contain the active agent in water-soluble form. Examples of nonaqueous solvents or vehicles include fatty oils, such as olive oil and corn oil, synthetic fatty acid esters, such as ethyl oleate or triglycerides, low molecular weight alcohols such as propylene glycol, synthetic hydrophilic polymers such as polyethylene glycol, liposomes, and the like. Parenteral formulations may also contain adjuvants such as solubilizers, preservatives, wetting agents, emulsifiers, dispersants, and stabilizers, and aqueous suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, and dextran. Injectable formulations are rendered sterile by incorporation of a sterilizing agent, filtration through a bacteria-retaining filter, irradiation, or heat. They can also be manufactured using a sterile injectable medium. The active agent may also be in dried, e.g., lyophilized, form that may be rehydrated with a suitable vehicle immediately prior to administration via injection.
The compounds of the invention may also be administered through the skin using conventional transdermal drug delivery systems, wherein the active agent is contained within a laminated structure that serves as a drug delivery device to be affixed to the skin. In such a structure, the drug composition is contained in a layer, or “reservoir,” underlying an upper backing layer. The laminated structure may contain a single reservoir, or it may contain multiple reservoirs. In one embodiment, the reservoir comprises a polymeric matrix of a pharmaceutically acceptable contact adhesive material that serves to affix the system to the skin during drug delivery. Alternatively, the drug-containing reservoir and skin contact adhesive are present as separate and distinct layers, with the adhesive underlying the reservoir which, in this case, may be either a polymeric matrix as described above, or it may be a liquid or hydrogel reservoir, or may take some other form. Transdermal drug delivery systems may in addition contain a skin permeation enhancer.
In addition to the formulations described previously, the compounds may also be formulated as a depot preparation for controlled release of the active agent, preferably sustained release over an extended time period. These sustained release dosage forms are generally administered by implantation (e.g., subcutaneously or intramuscularly or by intramuscular injection).
Although the present compositions will generally be administered orally, parenterally, transdermally, or via an implanted depot, other modes of administration are suitable as well. For example, administration may be rectal or vaginal, preferably using a suppository that contains, in addition to the active agent, excipients such as a suppository wax. Formulations for nasal or sublingual administration are also prepared with standard excipients well known in the art. The pharmaceutical compositions of the invention may also be formulated for inhalation, e.g., as a solution in saline, as a dry powder, or as an aerosol.
It is to be understood that while the invention has been described in conjunction with the preferred specific embodiments thereof, the description above as well as the examples that follow are intended to illustrate and not limit the scope of the invention. Other aspects, advantages, and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.
All patents, patent applications, journal articles, and other reference cited herein are incorporated by reference in their entireties.
Experimental
Unless noted otherwise, materials were obtained from commercially available sources and used without further purification.
All operations involving moisture-sensitive materials were conducted in oven- and/or flame-dried glassware under an atmosphere of anhydrous nitrogen. Hygroscopic solvents and liquid reagents were transferred using dry GastighTM syringes or cannulating needles. In cases where rigorous exclusion of dissolved oxygen was required, solvents were degassed via consecutive freeze, pump, thaw cycles, or inert gas purge.
Nuclear magnetic resonance (NMR) spectra were recorded on either a Varian UNITY INOVA-500, XL400 or a Gemini-300 magnetic resonance spectrometer. 1H chemical shifts are given in parts per million (δ) downfield from tetramethylsilane (TMS) using the residual solvent signal (CHC3=δ 7.27, benzene=δ 7.15, methanol=δ 3.30) as internal standard. Proton (1H) NMR information is tabulated in the following format: number of protons, multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; sept, septet, m, multiplet), coupling constant(s) (J) in hertz and, in cases where mixtures are present, assignment as the major or minor isomer, if possible. Proton decoupled 13C NMR spectra are reported in ppm (δ) relative to residual CHCl3 (δ 77.25) unless noted otherwise. Deuterated NMR solvents were dried over 1/16′′ bead 4 Å molecular sieves.
Flash chromatography was performed using E. Merck silica gel 60 (240–400 mesh) according to the protocol of Still et al. (1978) J. Org. Chem. 43:2923. Thin layer chromatography was performed using precoated plates purchased from E. Merck (silica gel 60 PF254, 0.25 mm) that were visualized using either a p-anisaldehyde or Ce(IV) stain. In a typical procedure, 1.0 g cell extract was purified using a 40 cm×2.5 cm flash column of 230–400 mesh normal phase silica eluting with 7% methanol in chloroform where applicable.
Rotating-frame Overhauser Effect Spectroscopy (ROESY) correlations were used in conjunction with an MC conformational search to determine the solution structure of apoptolidin, isoapoptolidin, and derivatives and analogs of same. Calculations performed using the GB/SA water solvation model were performed according to the protocol in Still et al., (1990) J. Am. Chem. Soc. 112:6127.
EXAMPLE 1
Structural Characteristics of Isoapoptolidin
Isoapoptolidin, which has a slightly lower Rƒ on normal phase silica than apoptolidin, was isolated from apoptolidin in crude cell extracts by flash column chromatography. An enriched solution of isoapoptolidin in either chloroform or acetonitrile over several hours at room temperature produced pure isoapoptolidin as white filamentous crystals (mp 134–146° C.). While even very dilute solutions of isoapoptolidin results in crystallization, the presence of apoptolidin in the solution will hinder the crystallization of isoapoptolidin. Mass recovery of apoptolidin and isoapoptolidin from crude cell extracts indicates that the two isomers are present in roughly equal amounts.
The crystals of isoapoptolidin were found unsuitable for X-ray analysis due to a poor aspect ratio and consequently, the structure of isoapoptolidin was determined by analysis of one- and two-dimensional NMR spectra (500 MHz). In deuterated methanol (CD3OD), the chemical shifts of most proton signals in isoapoptolidin remain very similar to those reported for apoptolidin (see, Kim et al., supra). In double quantum filtered correlated spectroscopy (DQF-COSY) experiments, isoapoptolidin and apoptolidin showed the same spin systems; however, the Heteronuclear Multi-Bond Connectivity (HMBC) spectrum of isoapoptolidin, showed a strong correlation between the carbonyl carbon (C-1) and 20-H. Further, the chemical shift of 20-H shifted downfield, from 3.57 ppm in apoptolidin to 4.98 in apoptolidin, suggesting that the 20-H is geminal to the lactone functionality.
The proton (1H (500 MHz)) and carbon (13C (100 MHz)) NMR data for isoapoptolidin in CD3OD are summarized in Table 1.







TABLE 1







NMR DATA FOR ISOAPOPTOLIDIN IN CD3OD










ISOAPOPTOLIDIN
APOPTOLIDIN
ISOAPOPTOLIDIN
APOPTOLIDIN














NO.
δC
δH (J = Hz)
δH
NO.
δC
δH (J = Hz)
δH

















 1
169.92


2-Me
13.92
2.04
2.14


 2
124.86


4-Me
17.82
2.07
2.21


 3
147.08
7.31
7.41
6-Me
17.63
1.72
1.97








(1.0)


 4
132.76


8-Me
18.26
1.17
1.17








(6.4)


 5
143.65
5.93
6.23
12-Me
12.00
1.70
1.71


 6
132.90


17-OMe
59.87
3.47
3.40


 7
136.75
5.06
5.27
22-Me
12.13
1.03
1.06




(9.1)



(6.7)


 8
39.41
2.74
2.79
24-Me
5.38
0.86
0.92








(6.9)


 9
83.98
3.77
3.87
28-OMe
59.48
3.35
3.30




(9.0, 9.0)


10
125.77
5.26
5.26
1′
96.23
4.78
4.85




(9.6, 4.8)



(3.8)


11
141.01
6.04
6.21
2′
73.32
3.38
3.44




(15.8)



(9.8, 3.8)


12
134.05


3′
74.92
3.71
3.76


13
133.80
5.48
5.71
4′
87.53
2.70
2.76




(9.6, 4.8)



(9.0)


14α
25.49
1.93
2.09
5′
68.05
3.73
3.78


14β

2.48
2.50
6′
18.38
1.22
1.29








(6.2)


15α
33.37
1.24
1.44
4′-OMe
61.01
3.56
3.61


15β

1.39
1.52
1′′
99.88
5.05
4.97








(4.1)


16
73.10
3.51
3.47
2′′α
45.29
2.01
1.96




(10.3, 5.0, 2.3)


17
81.89
3.36
2.75
2′′β

1.77
1.84


18α
35.05
1.36
1.78
3′′
73.67


18β

1.84
2.20
4′′
85.91
3.32
3.37


19
67.44
4.41
5.32
5′′
68.62
3.73
3.70




(11.3, 3.7, 2.1)


20
74.13
4.98
3.57
6′′
18.35
1.22
1.25




(3.7)



(6.2)


21
103.27


3′′-Me
22.76
1.34
1.36


22
36.79
1.77
2.08
1′′′
101.91
4.82
4.86








(9.7, 1.8)


23
73.20
3.72
3.76
2′′′α
37.51
2.43
2.47








(12.3, 5.0, 1.8)


24
40.52
1.74
1.76
2′′′β

1.28
1.32


25
68.17
4.21
3.99
3′′′
81.98
3.16
3.21




(10.3, 2.5, 2.0)



(11.6, 9.0, 5.0)


26α
36.82
1.76
1.62
4′′′
77.12
2.95
3.01








(9.0, 9.0)


26β

1.53
1.49
5′′′
73.45
3.19
3.24




(14.7, 10.7, 3.0)


27
76.24
3.82
3.48
6′′′
18.87
1.27
1.31








(6.2)


28
77.07
3.44
3.36
3′′′-OMe
57.32
3.42
3.46









The conformation of the isoapoptolidin ring was determined by way of ROESY correlations. Specifically, strong ROESY correlations were observed between 1,3-diaxial substituents on the C-21 to C-25 cyclic hemiketal in isoapoptolidin, indicating that the ring in the C-21 to C-25 puranyl moiety is in a chairlike conformation, and ROESY correlations were observed from 20-H to 22-H and 22-Me-H3, indicating that C-20 is equatorial on the ring. Using the coupling constant data for the anomeric proton signals (Table 1), it was determined that the stereochemistry of the glycosidic bonds in isoapoptolidin is the same as in apoptolidin. The geometry of the olefins in isoapoptolidin were all determined to be in the (E) geometry through high field carbon shift for the allylic methyl signals and the large vicinal coupling constant across the C-10/C11 double bond; these findings were confirmed via ROESY correlations.
ROESY correlations were also used to determine the solution conformation of isoapoptolidin. Of 51 ROESY correlations for the C-1 to C-26 portion of isoapoptolidin, 23 were found nontrivial and applied in subsequent analyses. Strong ROESY correlations were observed between 3-H and 5-H and between 5-H and 7-H, indicating that the three methyl groups on the triene line of isoapoptolidin lie on the same face of the macroline as they do in apoptolidin. It was also noted that isoapoptolidin had transannular correlations between 3-H and both 18-Hβ and 15-Hβ. The hypochromic shift in the enoate chromophore from a λmax of 319 nm in apoptolidin to 304 nm in isoapoptolidin, along with a decrease in the extinction coefficient for this absorption, from 22 800 in apoptolidin to 19 400 in isoapoptolidin, suggests a decrease in the degree of conjugation of the triene system in isoapoptolidin relative to apoptolidin.
Also observed was a high chemical shift of 19-H in isoapoptolidin, which is probably attributable to a new hydrogen bond between the C-19 alcohol and the C-25 oxygen. Significant ROESY correlations between the 20-H and the 22-H proton suggest an arrangement of the C-20/C-21 torsion that would favor this hydrogen bond. A representative structure from the conformational search is depicted in FIG. 1 and is fully consistent with date from the ROESY experiment.
EXAMPLE 2
Kinetic Parameters of Isoapoptolidin
To study the kinetic parameters of isoapoptolidin, the conversion between apoptolidin and isoapoptolidin under biological assay and chemical isolation conditions was observed. Apoptolidin has been found to be stable for up to three months at −20° C. when dissolved in chloroform or methylene chloride. During this time, no conversion to isoapoptolidin is observed, either by NMR or HPLC. Similarly, isoapoptolidin is stable as a solution in methanol at −20° C. for up to six months. In contrast, a dilute aqueous solution of apoptolidin at ambient temperature can be observed to convert to isoapoptolidin when monitored by HPLC or UV-vis as will a dilute aqueous solution of isoapoptolidin at ambient temperature will convert to apoptolidin when monitored by HPLC and UV-vis. Given that the conversion between apoptolidin and isoapoptolidin goes in both directions, it is likely that these two compounds are in equilibrium.
To measure the kinetic parameters of apoptolidin and isoapoptolidin, solutions of both compounds in PBS (Dulbecco's phosphate buffered saline) were prepared and incubated at 37° C. in sealed reaction vessels. Periodically, the components present in the solutions were quantified using reverse-phase HPLC. Baseline separation was achieved using a 150×4.6 mm C18/5μ column eluting with a water/acetonitrile gradient from 40% to 90% over 30 min. After normalization for the difference in extinction coefficients (λobs=254 nm), these values were plotted and the rate constants k1 and k−1 were extracted by fitting the data to the integrated rate expression for a simple equilibrium. The kinetic parameters for apoptolidin and isoapoptolidin equilibrium over time in PBS at 37° C. (where Q(t)=[2]/[1]) are set forth in Table 2. The relative amounts of apoptolidin and isoapoptolidin at various time points are plotted in FIG. 2.







TABLE 2







KINETIC PARAMETERS FOR


APOPTOLIDIN-ISOAPOPTOLIDIN EQUILIBRIUM








 










Q(t) = 0 at t = 0
Q(t) = ∞ at t = 0













k1
0.0656 h−1
0.0626 h−1


k−1
0.106 h−1
0.0982 h−1


Keq
0.0616
0.638





Q(t) = [2]/[1] at time t.






The similar rate parameters derived from solutions starting with either isoapoptolidin or apoptolidin suggests the absence of any long-lived intermediate structure in the interconversion between the two molecules. Further, the value of k1 suggests that under conditions of most cell-based assays, a significant conversion of apoptolidin to isoapoptolidin will occur within several hours. These findings indicate that the presence of isoapoptolidin must be taken into account when interpreting the results of cell-based assays.
EXAMPLE 3
Selective Functionalization of the Eight Hydroxyl Groups of Apoptolidin
To study the structure-activity relationship of apoptolidin and its analogs, the eight hydroxyl functionalities of apoptolidin were substituted to produce apoptolidin derivatives. The hydroxyl groups were selected for this study because of their potential role as pharmacophoric hydrogen bond donors and acceptors.
The functional selection of the eight hydroxyl groups of apoptolidin is shown schematically in Table 3. Of the eight hydroxyl groups of apoptolidin, the sole cyclic hemi-ketal C-21 was selectively derivatized by treatment with AmberlystTM-15 sulfonic acid resin in methanol to result in a ketal in 80% yield (compound 1). The hydroxyl at C-2′ was found to be the most reactive toward acylation and thus was selectively esterified by using pivaloyl anhydride (“Piv2O”; compound 2). The C-2′/C-3′ diol was activated through formation of a dibutylstannylidene acetal followed by treatment with benzoyl chloride to produce a C-2′ benzoate (compound 3). The remaining hydroxyl groups were selectively functionalized by using a strategy of progressive selective protection with triethylsilyl ether, followed by acetylation and deprotection. For protection, triethylsilyl ether was found to be most effective as it was easily installed with good chemoselectivity and removed under mild conditions. By contrast, tert-butyldimethylsilyl ether was found to both install and remove with difficulty. By altering the number of equivalents of triethylsilyl triflate used in the protection step, each of the corresponding silyl ethers of apoptolidin identified as compounds 4–8 in Table 3 were selectively formed. Careful acetylation of the silyl ethers of compounds 4–8 produced compounds 9–13 of Table 3, respectively. Finally, silyl deprotection of each derivative with buffered hydrogen fluoride (HF) produced compounds 14–18 of Table 3. The functional groups referenced in Table 3 correspond to the groups shown in the compound of formula (II).







TABLE 3







FUNCTIONAL SELECTION OF HYDROXYL GROUPS OF APOPTOLIDIN











POSITION OF

CMPD

FINAL


OH GROUP
REAGENT
NO.
FUNCTIONAL GROUPS
PRODUCT














C-21
MeOH
1
Q1 = Q2 = Q3 = Q4 = Q5 = Q6 = Q7 = H, Q8 = CH3
KETAL



Amberlyst-15


C-2′
Piv2O pyr,
2
Q1 = Piv, Q2 = Q3 = Q4 = Q5 = Q6 = Q7 = Q8 = H
ESTER



DMAP


C-2′/C-3′
Bu2SnO, 4Å
3
Q1 = Bz, Q2 = Q3 = Q4 = Q5 = Q6 = Q7 = Q8 = H
BENZOATE



M.S.



then BzCl


REMAINING
TESOTf,
4
Q1 = TES, Q2 = Q3 = Q4 = Q5 = Q6 = Q7 = Q8 = H
SILYL ETHERS


HYDROXYL
DCM, THF,
5
Q1 = Q2 = TES, Q3 = Q4 = Q5 = Q6 = Q7 = Q8 = H


GROUPS
pyr or 2,6-lut
6
Q1 = Q2 = Q3 = TES, Q4 = Q5 = Q6 = Q7 = Q8 = H


C-16

7
Q1 = Q2 = Q3 = Q4 = TES, Q5 = Q6 = Q7 = Q8 = H


C-20

8
Q1 = Q2 = Q3 = Q4 = Q5 = TES, Q6 = Q7 = Q8 = H


C-23
Ac2O, DCM,
9
Q1 = TES, Q2 = Q3 = Ac, Q4 = Q5 = Q6 = Q7 = Q8 = H
ACETYLATED


C-3′′
pyr, DMAP
10
Q1 = Q2 = TES, Q3 = Ac, Q4 = Q5 = Q6 = Q7 = Q8 = H
SILYL ETHERS


C-4′′

11
Q1 = Q2 = Q3 = TES, Q4 = Ac, Q5 = Q6 = Q7 = Q8 = H




12
Q1 = Q2 = Q3 = Q4 = TES, Q5 = Ac, Q6 = Q7 = Q8 = H




13
Q1 = Q2 = Q3 = Q4 = Q5 = TES, Q6 = Ac, Q7 = Q8 = H



HF-pyr, pyr,
14
Q1 = H, Q2 = Q3 = Ac, Q4 = Q5 = Q6 = Q7 = Q8 = H
ACETATES



THF
15
Q1 = Q2 = H, Q3 = Ac, R4 = R5 = Q6 = Q7 = Q8 = H




16
Q1 = Q2 = Q3 = H, Q4 = Ac, Q5 = Q6 = Q7 = Q8 = H




17
Q1 = Q2 = Q3 = Q4 = H, Q5 = Ac, Q6 = Q7 = Q8 = H




18
Q1 = Q2 = Q3 = Q4 = Q5 = H, Q6 = Ac, Q7 = Q8 = H





In Table 3, the abbreviations used are as follows:


Ac = acetyl;


Ac2O = acetyl acetone;


DCM = dichloromethane;


DMAP = dimethylaminopyridine;


THF = tetrahydrofuran;


TES = triethylsilyl;


Bz = benzoyl; and


Piv = pivaloyl.






The location of the acetyl group in the acetates at compounds 14–18 was determined by analysis of their proton chemical shift assignments based on 2D-NMR experiments. The downfield chemical shift of the proton geminal to the newly formed ester was used to identify the site of derivatization. Proton chemical shifts also served to confirm that macrolide ring expansion did not occur. The location of the silyl ethers in compounds 4–13 was inferred by the identities of compounds 14–18. When analyzed by tandem mass spectrometry, apoptolidin and its derivatives fragmented in a consistent way that allowed for a cross-check of the NMR assignments, and thus served as an independent confirmation of structure. FIG. 3 shows the fragmentation sites for the apoptolidin derivatives produced herein. On the basis of the fragmentation patterns of compounds 9–13, it was concluded that silyl transfer did not occur during the acetylation of compounds 4–8.
EXAMPLE 4
Functionalization of the C-20 Hydroxyl Group of Apoptolidin
To determine whether apoptolidin or isoapoptolidin is primarily responsible for apoptosis, the C-20 alcohol of apoptolidin was functionalized in order to prepare an apoptolidin derivative that does not isomerize to isoapoptolidin.
The functional selection of the C-20 alcohol of apoptolidin is shown schematically in Table 4. For this experiment, the penta-(triethylsilyl)-protected apoptolidin of Table 3 (compound 8) was reacted with Meerwein's salt in the presence of 2,6-di-tert-butyl-4-methylpyridine to produce the C-20 methyl-ether identified as compound 19 in Table 4. Compound 19 was subsequently deprotected to yield the apoptolidin derivative identified as compound 20 in Table 4. The location of the methyl ether was confirmed by observation of the strong HMBC correlations between C-20 and the C-20 methoxy protons.







TABLE 4







FUNCTIONAL SELECTION OF THE


C-20 ALCOHOL OF APOPTOLIDIN










CMPD
FUNCTIONAL




NO.
GROUPS
FINAL PRODUCT
REAGENT













8
Q1 = Q2 = Q3 =
SILYL ETHER




Q4 = Q5 = TES,



Q6 = Q7 = Q8 = H


19
Q1 = Q2 = Q3 =
METHYL ETHER
Me3OBF4, DCM,



Q4 = Q5 = TES 

2.6-di-tert-butyl-



Q6 = Me, Q7 = Q8 = H

4-methylpyridine


20
Q1 = Q2 = Q3 =
METHYL ETHER
HF-pyr, pyr, THF



Q4 = Q5 = Q7 =



Q8 = H, Q6 = Me









The stability of compound 20 was studied in PBS, under the same conditions described in Example 1 that were found to promote the isomerization of isoapoptolidin to apoptolidin. For compound 20, the macrolactone could not undergo ring expansion; however, this compound was found to partially convert to a second isomeric compound over a period of several hours, as observed by liquid chromatography and mass spectrometry (LC-MS). Similarly, the C-20 acetate derivative (compound 18) was also found to be unstable under these conditions, producing several new compounds over a five hour period. This experiment leads to the conclusion that while derivatization of apoptolidin at C-20 blocks isomerization to isoapoptolidin, the resulting derivatives (compounds 18 and 20) undergo other transformations.
EXAMPLE 5
F0F1-ATPase Studies
The ability of isoapoptolidin and the apoptolidin derivatives described in Examples 3 and 4 to inhibit F0F1-ATPase in isolated yeast mitochondria was studied. The F0F1-ATPase assay is a rapid 20 minute cell-free assay. The short duration of the assay ensures that isomerization of isoapoptolidin to apoptolidin is minimal and the cell-free nature of the assay ensures that the acetate derivatives of apoptolidin will be stable and not subject to hydrolysis.
To test the ability of isoapoptolidin to inhibit F0F1-ATPase activity, solutions of isoapoptolidin and apoptolidin including all assay components were analyzed for isomerization. After 20 minutes at 37° C., less than 12% conversion was detected.
To test the ability of apoptolidin derivatives to inhibit F0F1-ATPase activity, compound 16 from Table 3 was exposed to all of the assay components for up to 24 hours. The assay solution was then analyzed for hydrolysis products using Electrospray Ionization Mass Spectrometry (ESIMS), and no hydrolysis was detected.
The data for the F0F1-ATPase inhibition assay for apoptolidin, isoapoptolidin, and the apoptolidin derivatives identified as compounds 1, 3, 14, 15, 16, 17, 18, and 20 above is provided in Table 5. The results of the assay show that while isoapoptolidin has reduced F0F1-ATPase inhibitory activity over apoptolidin, many of the apoptolidin derivatives show F0F1-ATPase inhibitory activity similar to that of apoptolidin.







TABLE 5







F0F1-ASSAY FOR APOPTOLIDIN, ISOAPOPTOLIDIN,


AND APOPTOLIDIN DERIVATIVES











IC50 (μM)







APOPTOLIDIN
0.7 ± 0.5



ISOAPOPTOLIDIN
 17 ± 5.0



COMPOUND 1
2.3 ± 0.5



COMPOUND 3
0.3 ± 0.5



COMPOUND 14
0.4 ± 0.5



COMPOUND 15
0.8 ± 0.5



COMPOUND 16
0.8 ± 0.5



COMPOUND 17
0.4 ± 0.5



COMPOUND 18
1.1 ± 0.5



COMPOUND 20
2.8 ± 0.5










EXAMPLE 6
Oxidative Cleavage of Apoptolidin
In a preliminary experiment to cleave apoptolidin, the molecule was treated with sodium periodate in a 1:1 mixture of methanol and water; this reaction yielded a considerable amount of lactone and trace amounts of the macrolide fragment. After investigating various protection strategies and oxidant screenings, it was found that reaction of pentatriethylsilyl protected apoptolidin (Table 4, compound 8) with lead tetraacetate under mild anhydrous conditions results in the rapid cleavage of apoptolidin at the C-20/C-21 bond and yields excellent quantities of lactone (87%) and aldehyde (89%). Referring to the formulas (V) and (VI), the resulting compounds had the following scheme:

Q1=Q2=Q3=Q4=Q5=TES; Q7=H; R1=R2=R3=R4=CH3 

While the C-19/C-20 bond of formula (V) shows an alcohol, the moiety at C-20 of the resulting aldehyde was CHO. To evaluate the stabilities and activities of the derivatives, the fully protected macrocyclic aldehyde was smoothly reduced using sodium borohydride in THF to produce a primary alcohol having the following scheme:

Compound 21: Q1=Q2=Q3=Q4=Q5=TES; Q6=Q7=H; R1═R2=R3=R4=CH3 

The macrocylic alcohol was fully desilyated using buffered HF/pyridine to produce a compound with the following scheme:

Compound 22: Q1=Q2=Q3=Q4=Q5=Q6=Q7=H; R1=R2=R3=R4=CH3 

The desilylated macrocyclic alcohol did not isomerize during the desilylation process but slowly isomerized to its ring expanded isomer in basic CD3OD as determined by LCMS and NMR. To prevent ring expansion, the primary alcohol may be efficiently converted to a methyl ether or benzoate ester by way of treatment with Meerwein's salt or benzoyl anhydride followed by deprotection, under this procedure, the following macrocycles resulted, respectively:

Compound 23: Q1=Q2=Q3=Q4=Q5=Q7=H; Q6=R1=R2=R3=R4=CH3 

Compound 24: Q1=Q2=Q3=Q4=Q5=Q7=H; Q6=COC6H6; R1=R2=R3=R4=CH3 

Under the conditions of these transformation, acyl migration was not observed.
The functionalized macrolide and δ-lactone fragments were assayed for inhibitory activity against mitochondrial F0F1-ATPase. While the δ-lactone fragment had greatly reduced potency as compared with apoptolidin, the macrolide fragments (compounds 21–24) retained significant activity in the assay. The results of the F0F1-ATPase assay for the apoptolidin oxidative cleavage products are set forth in Table 6.







TABLE 6







F0F1-ASSAY FOR APOPTOLIDIN


OXIDATIVE CLEAVAGE PRODUCTS











IC50 (μM)







APOPTOLIDIN
0.7 ± 0.5



ISOAPOPTOLIDIN
 17 ± 5.0



COMPOUND 8
 190 ± 50.0



COMPOUNDS 21
13.0 ± 5.0 



COMPOUND 22
 16 ± 5.0



COMPOUND 23
 32 ± 5.0



COMPOUND 24
 34 ± 5.0










Compound 21 and compound 22 (the methyl ester) were both found to have a potency comparable to isoapoptolidin whereas compound 23 (the benzoate derivative) and compound 24 were found to be less active than compounds 21 and 22 as well as apoptolidin and isoapoptolidin. These results are consistent with the observation of decreased potency upon functionalization of the C-20 bond of apoptolidin, either as an acetate or a methyl ester.

## Claims

1. A functionalized isoapoptolidin compound consisting essentially of an isoapoptolidin core or a stereoisomer thereof, in which:s
(a) at least one hydroxyl group in the isoapoptolidin core is replaced with a substituent selected from C1–C24 hydrocarbyloxy, C2–C25 acyloxy, C2–C25 haloacyloxy C2–C25 thioacyloxy, C2–C25 thiohaloacyloxy, C2–C25 carbonato, halogenated C2–C25 carbonato, C2–C25 thiocarbonato, halogenated C2–C25 thiocarbonato, carbamoyloxy, N—(C1–C24 hydrocarbyl)-substituted carbamoyloxy, N,N-di(C1–C24 hydrocarbyl)-substituted carbamoyloxy, thiocarbamoyloxy, N—(C1–C24 hydrocarbyl)-substituted thiocarbamoyloxy, N,N-di(C1–C24 hydrocarbyl)-substituted thiocarbamoyloxy, sulfamoyloxy, N—(C1–C24 hydrocarbyl)-substituted sulfamoyloxy, N,N-di(C1–C24 hydrocarbyl)-substituted sulfamoyloxy, and protected hydroxyl groups;
(b) at least one 1,3-diene functionality in the isoapoptolidin core is replaced by the product of a Diels-Alder reaction with a dienophile;
(c) at least one carbon-carbon double bond in the isoapoptolidin core is replaced with a carbon-carbon single bond; and/or
(d) at least one 1,2-diol functionality in the isoapoptolidin core is replaced with a cyclic ether.

2. The functionalized isoapoptolidin compound of claim 1, in which at least one hydroxyl group in the isoapoptolidin core is replaced with a substituent selected from C1–C24 hydrocarbyloxy, C2–C25 acyloxy, C2–C25 haloacyloxy C2–C25 thioacyloxy, C2–C25 thiohaloacyloxy, C2–C25 carbonato, halogenated C2–C25 carbonato, C2–C25 thiocarbonato, halogenated C2–C25 thiocarbonato, carbamoyloxy, N—(C1–C24 hydrocarbyl)-substituted carbamoyloxy, N,N-di(C1–C24 hydrocarbyl)-substituted carbamoyloxy, thiocarbamoyloxy, N—(C1–C24 hydrocarbyl)-substituted thiocarbamoyloxy, N,N-di(C1–C24 hydrocarbyl)-substituted thiocarbamoyloxy, sulfamoyloxy, N—(C1–C24 hydrocarbyl)-substituted sulfamoyloxy, N,N-di(C1–C24 hydrocarbyl)-substituted sulfamoyloxy, and protected hydroxyl groups.

3. The functionalized isoapoptolidin compound of claim 2, wherein the substituent is selected from C1–C12 hydrocarbyloxy, C2–C13 acyloxy, C2–C13 haloacyloxy C2C13 thioacyloxy, C2–C25 thiohaloacyloxy, C2–C13 carbonato, halogenated C2–C13 carbonato, C2–C13 thiocarbonato, halogenated C2–C13 thiocarbonato, carbamoyloxy, N—(C1–C12 hydrocarbyl)-substituted carbamoyloxy, N,N-di(C1–C12 hydrocarbyl)-substituted carbamoyloxy, thiocarbamoyloxy, N—(C1–C12 hydrocarbyl)-substituted thiocarbamoyloxy, N,N-di(C1–C12 hydrocarbyl)-substituted thiocarbamoyloxy, sulfamoyloxy, N—(C1–C12 hydrocarbyl)-substituted sulfamoyloxy, N,N-di(C1–C12 hydrocarbyl)-substituted sulfamoyloxy, (C1–C6 alkoxy)methyl ether, (C1–C6 alkylthio)methyl ether, and tri(C1–C12 hydrocarbyl)-substituted silyloxy.

4. The functionalized isoapoptolidin compound of claim 3, wherein the substituent is selected from C1–C12 hydrocarbyloxy, C2–C13 acyloxy, and tri(C1–C12 hydrocarbyl)-substituted silyloxy.

5. A compound having the structure of formula (II)




wherein:
Q1, Q2, Q3, Q4, Q5, Q6, Q7, and Q8 are independently selected from H, C1–C12 hydrocarbyl, acyl of the formula —(CO)—R5 in which R5 is C1–C12 hydrocarbyl, and hydroxyl-protecting groups; and
R1, R2, R3, and R4 are C1–C12 alkyl or H, or a stereoisomer thereof.

6. The compound of claim 5, having the stereoisomeric configuration of formula (I).

7. The compound of claim 5, wherein:
Q1, Q2, Q3, Q4, Q5, Q6, Q7, and Q8 are independently selected from H, C1–C6 hydrocarbyl, —(CO)—R5 wherein R5 is C1–C6 hydrocarbyl, and —Si(R6R7R8) wherein R6, R7, and R8 are C1–C6 hydrocarbyl; and
R1, R2, R3, and R4 are C1–C4 alkyl.

8. The compound of claim 7, wherein:
Q1, Q2, Q3, Q4, Q5, Q6, Q7, and Q8 are independently selected from H, C1–C6 alkyl, —(CO)—R5 wherein R5 is C1–C6 alkyl, and —Si(R6R7R8) wherein R6, R7, and R8 are all methyl or all ethyl; and
R1, R2, R3, and R4 are methyl.

9. The compound of claim 6, wherein:
Q1, Q2, Q3, Q4, Q5, Q6, Q7, and Q8 are independently selected from H, C1–C6 hydrocarbyl, —(CO)—R5 wherein R5 is C1–C6 hydrocarbyl, and —Si(R6R7R8) wherein R6, R7, and R8 are C1–C6 hydrocarbyl; and
R1, R2, R3, and R4 are C1–C4 alkyl.

10. The compound of claim 9, wherein:
Q1, Q2, Q3, Q4, Q5, Q6, Q7, and Q8 are independently selected from H, C1–C6 alkyl, —(CO)—R5 wherein R5 is C1–C6 alkyl, and —Si(R6R7R8) wherein R6, R7, and R8 are all methyl or all ethyl; and
R1, R2, R3, and R4 are methyl.

11. The compound of claim 5, wherein:
Q1is —(CO)—R5 and Q2, Q3, Q4, Q5, Q6, Q7, and Q8 are H;
Q2 and Q3 are —(CO)—R5 and Q1, Q4, Q5, Q6, Q7, and Q8 are H;
Q3 is —(CO)—R5 and Q1, Q2, Q4, Q5, Q6, Q7, and Q8 are H;
Q4 is —(CO)—R5 and Q1, Q2, Q3, Q5, Q6, Q7, and Q8 are H;
Q5 is —(CO)—R5 and Q1, Q2, Q3, Q4, Q6, Q7, and Q8 are H;
Q6 is —(CO)—R5 and Q1, Q2, Q3, Q4, Q5, Q7, and Q8 are H;
Q6 is C1–C6 alkyl and Q1, Q2, Q3, Q4, Q5, Q7, and Q8 are H; or
Q8 is C1–C6 alkyl and Q1, Q2, Q3, Q4, Q5, Q6, and Q7 are H.

12. The compound of claim 11, wherein:
Q1is benzoyl and Q2, Q3, Q4, Q5, Q6, Q7, and Q8 are H;
Q2 and Q3 are acetyl and Q1, Q4, Q5, Q6, Q7, and Q8 are H;
Q3 is acetyl and Q1, Q2, Q4, Q5, Q6, Q7, and Q8 are H;
Q4 is acetyl and Q1, Q2, Q3, Q5, Q6, Q7, and Q8 are H;
Q5 is acetyl and Q1, Q2, Q3, Q4, Q6, Q7, and Q8 are H;
Q6 is acetyl and Q1, Q2, Q3, Q4, Q5, Q7, and Q8 are H;
Q6 is methyl and Q1, Q2, Q3, Q4, Q5, Q7, and Q8 are H; or
Q8 is methyl and Q1, Q2, Q3, Q4, Q5, Q6, and Q7 are H.

13. The compound of claim 6, wherein:
Q1is —(CO)—R5 and Q2, Q3, Q4, Q5, Q6, Q7, and Q8 are H;
Q2 and Q3 are —(CO)—R5 and Q1, Q4, Q5, Q6, Q7, and Q8 are H;
Q3 is —(CO)—R5 and Q1, Q2, Q4, Q5, Q6, Q7, and Q8 are H;
Q4 is —(CO)—R5 and Q1, Q2, Q3, Q5, Q6, Q7, and Q8 are H;
Q5 is —(CO)—R5 and Q1, Q2, Q3, Q4, Q6, Q7, and Q8 are H;
Q6 is —(CO)—R5 and Q1, Q2, Q3, Q4, Q5, Q7, and Q8 are H;
Q6 is C1–C6 alkyl and Q1, Q2, Q3, Q4, Q5, Q7, and Q8 are H; or
Q8 is C1–C6 alkyl and Q1, Q2, Q3, Q4, Q5, Q6, and Q7 are H.

14. The compound of claim 13, wherein:
Q1 is benzoyl and Q2, Q3, Q4, Q5, Q6, Q7, and Q8 are H;
Q2 and Q3 are acetyl and Q1, Q4, Q5, Q6, Q7, and Q8 are H;
Q3 is acetyl and Q1, Q2, Q4, Q5, Q6, Q7, and Q8 are H;
Q4 is acetyl and Q1, Q2, Q3, Q5, Q6, Q7, and Q8 are H;
Q5 is acetyl and Q1, Q2, Q3, Q4, Q6, Q7, and Q8 are H;
Q6 is acetyl and Q1, Q2, Q3, Q4, Q5, Q7, and Q8 are H;
Q6 is methyl and Q1, Q2, Q3, Q4, Q5, Q7, and Q8 are H; or
Q8 is methyl and Q1, Q2, Q3, Q4, Q5, Q6, and Q7 are H.

15. A compound having the structure of formula (III)




wherein:
Q1, Q2, Q3, Q4, Q5, Q6, Q7, and Q8 are selected from H, C1–C12 hydrocarbyl, acyl of the formula —(CO)—R5 in which R5 is C1–C12 hydrocarbyl, and hydroxyl-protecting groups; and
R1, R2, R3, and R4 are C1–C12 alkyl or H, with the proviso that at least one of Q1, Q2, Q3, Q4, Q5, Q6, Q7, and Q8 is other than H when R1, R2, R3, and R4 are methyl and the compound has the stereoisomeric configuration of formula (III),
or a stereoisomer thereof.

16. The compound of claim 15, having the stereoisomeric configuration of formula (III).

17. The compound of claim 15, wherein:
Q1, Q2, Q3, Q4, Q5, Q6, Q7, and Q8 are selected from H, C1–C6 hydrocarbyl, —(CO)—R5 wherein R5 is C1–C6 hydrocarbyl, and —Si(R6R7R8) wherein R6, R7, and R8 are C1–C6 hydrocarbyl; and
R1, R2, R3, and R4 are C1–C4 alkyl.

18. The compound of claim 17, wherein:
Q1, Q2, Q3, Q4, Q5, Q6, Q7, and Q8 are selected from H, C1–C6 alkyl, —(CO)—R5 wherein R5 is C1–C6 alkyl, and —Si(R6R7R8) wherein R6, R7, and R8 are all methyl or all ethyl; and
R1, R2, R3, and R4 are methyl.

19. The compound of claim 16, wherein:
Q1, Q2, Q3, Q4, Q5, Q6, Q7, and Q8 are selected from H, C1–C6 hydrocarbyl, —(CO)—R5 wherein R5 is C1–C6 hydrocarbyl, and —Si(R6R7R8) wherein R6, R7, and R8 are C1–C6 hydrocarbyl; and
R1, R2, R3, and R4 are C1–C4 alkyl.

20. The compound of claim 19, wherein:
Q1, Q2, Q3, Q4, Q5, Q6, Q7, and Q8 are selected from H, C1–C6 alkyl, —(CO)—R5 wherein R5 is C1–C6 alkyl, and —Si(R6R7R8) wherein R6, R7, and R8 are all methyl or all ethyl; and
R1, R2, R3, and R4 are methyl.

21. The compound of claim 15, wherein:
Q1 is —(CO)—R5 and Q2, Q3, Q4, Q5, Q6, Q7, and Q8 are H;
Q2 and Q3 are —(CO)—R5 and Q1, Q4, Q5, Q6, Q7, and Q8 are H;
Q3 is —(CO)—R5 and Q1, Q2, Q4, Q5, Q6, Q7, and Q8 are H;
Q4 is —(CO)—R5 and Q1, Q2, Q3, Q5, Q6, Q7, and Q8 are H;
Q5 is —(CO)—R5 and Q1, Q2 Q3, Q4, Q6, Q7, and Q8 are H;
Q6 is —(CO)—R5 and Q1, Q2, Q3, Q4, Q5, Q7, and Q8 are H;
Q6 is C1–C6 alkyl and Q1, Q2, Q3, Q4, Q5, Q7, and Q8 are H; or
Q8 is C1–C6 alkyl and Q1, Q2, Q3, Q4, Q5, Q6, and Q7 are H.

22. The compound of claim 21, wherein:
Q1 is benzoyl and Q2, Q3, Q4, Q5, Q6, Q7, and Q8 are H;
Q2 and Q3 are acetyl and Q1, Q4, Q5, Q6, Q7, and Q8 are H;
Q3 is acetyl and Q1, Q2, Q4, Q5, Q6, Q7, and Q8 are H;
Q4 is acetyl and Q1, Q2, Q3, Q5, Q6, Q7, and Q8 are H;
Q5 is acetyl and Q1, Q2, Q3, Q4, Q6, Q7, and Q8 are H;
Q6 is acetyl and Q1, Q2, Q3, Q4, Q5, Q7, and Q8 are H;
Q6 is methyl and Q1, Q2, Q3, Q4, Q5, Q7, and Q8 are H; or
Q8 is methyl and Q1, Q2, Q3, Q4, Q5, Q6, and Q7 are H.

23. The compound of claim 16, wherein:
Q1 is —(CO)—R5 and Q2, Q3, Q4, Q5, Q6, Q7, and Q8 are H;
Q2 and Q3 are —(CO)—R5 and Q1, Q4, Q5, Q6, Q7, and Q8 are H;
Q3 is —(CO)—R5 and Q1, Q2, Q4, Q5, Q6, Q7, and Q8 are H;
Q4 is —(CO)—R5 and Q1, Q2, Q3, Q5, Q6, Q7, and Q8 are H;
Q5 is —(CO)—R5 and Q1, Q2, Q3, Q4, Q6, Q7, and Q8 are H;
Q6 is —(CO)—R5 and Q1, Q2, Q3, Q4, Q5, Q7, and Q8 are H;
Q6 is C1–C6 alkyl and Q1, Q2, Q3, Q4, Q5, Q7, and Q8 are H; or
Q8 is C1–C6 alkyl and Q1, Q2, Q3, Q4, Q5, Q6, and Q7 are H.

24. The compound of claim 23, wherein:
Q1 is benzoyl and Q2, Q3, Q4, Q5, Q6, Q7, and Q8 are H;
Q2 and Q3 are acetyl and Q1, Q4, Q5, Q6, Q7, and Q8 are H;
Q3 is acetyl and Q1, Q2, Q4, Q5, Q6, Q7, and Q8 are H;
Q4 is acetyl and Q1, Q2, Q3, Q5, Q6, Q7, and Q8 are H;
Q5 is acetyl and Q1, Q2, Q3, Q4, Q6, Q7, and Q8 are H;
Q6 is acetyl and Q1, Q2, Q3, Q4, Q5, Q7, and Q8 are H;
Q6 is methyl and Q1, Q2, Q3, Q4, Q5, Q7, and Q8 are H; or
Q8 is methyl and Q1, Q2, Q3, Q4, Q5, Q6, and Q7 are H.

25. A compound having the structure of formula (IV)




wherein:
Q1, Q2, Q4, Q5, Q6, Q8, and Q9 are independently selected from H, C1–C12 hydrocarbyl, acyl of the formula —(CO)—R5 in which R5 is C1–C12 hydrocarbyl, and hydroxyl-protecting groups; and
R1, R2, and R3 are independently selected from C1–C12 alkyl and H,
or a stereoisomer thereof.

26. The compound of claim 25, having the stereoisomeric configuration of formula (IV).

27. The compound of claim 25, wherein:
Q1, Q2, Q4, Q5, Q6, Q8, and Q9 are independently selected from H, C1–C6 hydrocarbyl, —(CO)—R5 wherein R5 is C1–C6 hydrocarbyl, and —Si(R6R7R8) wherein R6, R7, and R8 are C1–C6 hydrocarbyl; and
R1, R2, and R3 are C1–C4 alkyl.

28. The compound of claim 27, wherein:
Q1, Q2, Q4, Q5, Q6, Q8, and Q9 are independently selected from H, C1–C6 alkyl, —(CO)—R5 wherein R5 is C1–C6 alkyl, and —Si(R6R7R8) wherein R6, R7, and R8 are all methyl or all ethyl; and
R1, R2, and R3 are methyl.

29. The compound of claim 26, wherein:
Q1, Q2, Q4, Q5, Q6, Q8, and Q9 are independently selected from H, C1–C6 hydrocarbyl, —(CO)—R5 wherein R5 is C1–C6 hydrocarbyl, and —Si(R6R7R8) wherein R6, R7, and R8 are C1–C6 hydrocarbyl; and
R1, R2, and R3 are C1–C4 alkyl.

30. The compound of claim 29, wherein:
Q1, Q2, Q4, Q5, Q6, Q8, and Q9 are independently selected from H, C1–C6 alkyl, —(CO)—R5 wherein R5 is C1–C6 alkyl, and —Si(R6R7R8) wherein R6, R7, and R8 are all methyl or all ethyl; and
R1, R2, and R3 are methyl.

31. A functionalized apoptolidin compound consisting essentially of anapoptolidin core in which:
(a) at least one hydroxyl group in the apoptolidin core is replaced with a substituent selected from C1–C24 hydrocarbyloxy, C2–C25 acyloxy, C2–C25 haloacyloxy C2–C25 thioacyloxy, C2–C25 thiohaloacyloxy, C2–C25 carbonato, halogenated C2–C25 carbonato, C2–C25 thiocarbonato, halogenated C2–C25 thiocarbonato, carbamoyloxy, N—(C1–C24 hydrocarbyl)-substituted carbamoyloxy, N,N-di(C1–C24 hydrocarbyl)-substituted carbamoyloxy, thiocarbamoyloxy, N—(C1–C24 hydrocarbyl)-substituted thiocarbamoyloxy, N,N-di(C1–C24 hydrocarbyl)-substituted thiocarbamoyloxy, sulfamoyloxy, N—(C1–C24 hydrocarbyl)-substituted sulfamoyloxy, N,N-di(C1–C24 hydrocarbyl)-substituted sulfamoyloxy, and protected hydroxyl groups;
(b) at least one 1,3-diene functionality in the apoptolidin core is replaced by the product of a Diels-Alder reaction with a dienophile;
(c) at least one carbon-carbon double bond in the apoptolidin core is replaced with a carbon-carbon single bond; and/or
(d) at least one 1,2-diol functionality in the apoptolidin core is replaced with a cyclic ether.

32. The functionalized apoptolidin compound of claim 31, in which at least one hydroxyl group in the isoapoptolidin core is replaced with a substituent selected from C1–C24 hydrocarbyloxy, C2–C25 acyloxy, C2–C25 haloacyloxy C2–C25 thioacyloxy, C2–C25 thiohaloacyloxy, C2–C25 carbonato, halogenated C2–C25 carbonato, C2–C25 thiocarbonato, halogenated C2–C25 thiocarbonato, carbamoyloxy, N—(C1–C24 hydrocarbyl)-substituted carbamoyloxy, N,N-di(C1–C24 hydrocarbyl)-substituted carbamoyloxy, thiocarbamoyloxy, N—(C1–C24 hydrocarbyl)-substituted thiocarbamoyloxy, N,N-di(C1–C24 hydrocarbyl)-substituted thiocarbamoyloxy, sulfamoyloxy, N—(C1–C24 hydrocarbyl)-substituted sulfamoyloxy, N,N-di(C1–C24 hydrocarbyl)-substituted sulfamoyloxy, and protected hydroxyl groups.

33. The functionalized apoptolidin compound of claim 32, wherein the substituent is selected from C1–C12 hydrocarbyloxy, C2–C13 acyloxy, C2–C13 haloacyloxy C2–C13 thioacyloxy, C2–C25 thiohaloacyloxy, C2–C13 carbonato, halogenated C2–C13 carbonato, C2–C13 thiocarbonato, halogenated C2–C13 thiocarbonato, carbamoyloxy, N—(C1–C12 hydrocarbyl)-substituted carbamoyloxy, N,N-di(C1–C12 hydrocarbyl)-substituted carbamoyloxy, thiocarbamoyloxy, N—(C1–C12 hydrocarbyl)-substituted thiocarbamoyloxy, N,N-di(C1–C12 hydrocarbyl)-substituted thiocarbamoyloxy, sulfamoyloxy, N—(C1–C12 hydrocarbyl)-substituted sulfamoyloxy, N,N-di(C1–C12 hydrocarbyl)-substituted sulfamoyloxy, (C1–C6 alkoxy)methyl ether, (C1–C6 alkylthio)methyl ether, and tri(C1–C12 hydrocarbyl)-substituted silyloxy.

34. The functionalized apoptolidin compound of claim 33, wherein the substituent is selected from C1–C12 hydrocarbyloxy, C2–C13 acyloxy, and tri(C2–C12 hydrocarbyl)-substituted silyloxy.

35. A composition of matter consisting essentially of isoapoptolidin in isolated, purified form.

36. A compound prepared by reaction of the compound of claim 5 with a dienophile, wherein the C-10/C-13 diene functionality is converted to a cyclic group.

37. A compound prepared by reaction of the compound of claim 15 with a dienophile, wherein the C-10/C-13 diene functionality is converted to a cyclic group.

38. A compound prepared by reaction of the compound of claim 25 with a dienophile, wherein the C-10/C-13 diene functionality is converted to a cyclic group.

39. The compound of any one of claims 36, 37, and 38, wherein the dienophile is an N-halosuccinimide.

40. A compound prepared by catalytic hydrogenation of the compound of claim 5, wherein at least one carbon-carbon double bond of the compound of claim 5 is converted to a single bond.

41. A compound prepared by catalytic hydrogenation of the compound of claim 15, wherein at least one carbon-carbon double bond of the compound of claim 15 is converted to a single bond.

42. A compound prepared by catalytic hydrogenation of the compound of claim 25, wherein at least one carbon-carbon double bond of the compound of claim 15 is converted to a single bond.

43. A compound prepared by nucleophilic addition of the compound of claim 5, wherein at least one carbon-carbon double bond of the compound of claim 5 is converted to a single bond.

44. A compound prepared by nucleophilic addition of the compound of claim 15, wherein at least one carbon-carbon double bond of the compound of claim 15 is converted to single bond.

45. A compound prepared by nucleophilic addition of the compound of claim 25, wherein at least one carbon-carbon double bond of the compound of claim 25 is converted to single bond.

46. The compound of claim 5, wherein at least one 1,2-diol functionality of the compound of claim 5 is converted to a cyclic ether.

47. The compound of claim 15, wherein at least one 1,2-diol functionality of the compound of claim 15 is converted to a cyclic ether.

48. The compound of claim 25, wherein at least one 1,2-diol functionality of the compound of claim 26 is converted to a cyclic ether.

49. A pharmaceutical composition comprising a therapeutically effective amount of the compound of claims 1, 5, 15, 25, or 35 and a pharmaceutically acceptable carrier.

50. The composition of claim 49, wherein the therapeutically effective amount is a unit dosage and the composition is composed of a unit dosage form.

51. The composition of claim 49, comprising a sustained release formulation.

52. A method for inducing apoptosis in cancer cells, wherein the compound of anyone of claims 1, 5, 15, 25, or 35 is administered to the cancer cells.

53. A composition of matter consisting essentially of deglycosylated isoapoptolidin in isolated, purified form.

54. A compound having the structure of formula (V)




wherein:
Q1, Q2, Q3, Q4, Q5, Q6, Q7, and Q8 are independently selected from H, C1–C12 hydrocarbyl, acyl of the formula —(CO)—R5 in which R5 is C1–C12 hydrocarbyl, and hydroxyl-protecting groups; and
R1, R2, R3, and R4 are C1–C12 alkyl or H,
or a stereoisomer thereof.

55. The compound of claim 54 oxidatively cleaved at the C-20/C-21 bond.

56. The compound of claim 54 oxidatively cleaved at the C-22/C-23 bond.