WO2003070719A1 - Analogues de bryostatine, procedes de synthese et utilisations de ces derniers - Google Patents

Analogues de bryostatine, procedes de synthese et utilisations de ces derniers Download PDF

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WO2003070719A1
WO2003070719A1 PCT/US2003/004599 US0304599W WO03070719A1 WO 2003070719 A1 WO2003070719 A1 WO 2003070719A1 US 0304599 W US0304599 W US 0304599W WO 03070719 A1 WO03070719 A1 WO 03070719A1
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Paul A. Wender
Blaise Lippa
Cheol-Min Park
Kevin W. Hinkle
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The Board Of Trustees Of The Leland Stanford Junior University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D407/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00
    • C07D407/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00 containing two hetero rings
    • C07D407/06—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D309/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings
    • C07D309/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having no double bonds between ring members or between ring members and non-ring members
    • C07D309/04—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having no double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
    • C07D309/06—Radicals substituted by oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D309/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings
    • C07D309/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having no double bonds between ring members or between ring members and non-ring members
    • C07D309/08—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D309/10—Oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D319/00—Heterocyclic compounds containing six-membered rings having two oxygen atoms as the only ring hetero atoms
    • C07D319/10—1,4-Dioxanes; Hydrogenated 1,4-dioxanes
    • C07D319/14—1,4-Dioxanes; Hydrogenated 1,4-dioxanes condensed with carbocyclic rings or ring systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D319/00—Heterocyclic compounds containing six-membered rings having two oxygen atoms as the only ring hetero atoms
    • C07D319/10—1,4-Dioxanes; Hydrogenated 1,4-dioxanes
    • C07D319/14—1,4-Dioxanes; Hydrogenated 1,4-dioxanes condensed with carbocyclic rings or ring systems
    • C07D319/16—1,4-Dioxanes; Hydrogenated 1,4-dioxanes condensed with carbocyclic rings or ring systems condensed with one six-membered ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
    • C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
    • C07D493/08—Bridged systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F7/00—Compounds containing elements of Groups 4 or 14 of the Periodic System
    • C07F7/02—Silicon compounds
    • C07F7/08—Compounds having one or more C—Si linkages
    • C07F7/18—Compounds having one or more C—Si linkages as well as one or more C—O—Si linkages
    • C07F7/1804—Compounds having Si-O-C linkages

Definitions

  • the present invention concerns biologically active compounds related to the bryostatin family of compounds, and to methods of preparing and utilizing the same.
  • Cancer is a major cause of death in the developed countries, with more than 500,000 human fatalities occurring annually in the United States. Cancers are generally the result of the transformation of normal cells into modified cells that proliferate excessively, leading to the formation of abnormal tissues or cell populations. In many cancers, cell proliferation is accompanied by dissemination
  • cancers can significantly impair normal physiological processes, ultimately leading to patient mortality. Cancers have been observed for many different tissue and cell types, with cancers of the lung, breast, and colorectal system accounting for about half of all cases. Currently, about one-third of cancer patients can be cured by surgical or radiation techniques. However, these approaches are most effective with cancerous lesions that have not yet metastasized to other regions of the body. Chemotherapeutic techniques currently cure another 17% of cancer patients. Combined chemotherapeutic and non-chemotherapeutic protocols can further enhance prospects for full recovery. Even for incurable cancer conditions, therapeutic treatments can be useful to achieve remission or at least extend patient longevity.
  • anthracyclines such as doxorubicin and daunorubicin
  • doxorubicin have been found to intercalate DNA, blocking DNA and RNA synthesis and causing strand scission by interacting with topoisomerase II.
  • the taxanes such as TaxolTM and TaxotereTM, disrupt mitosis by promoting tubulin polymerization in microtubule assembly.
  • Cis-platin forms interstrand crosslinks in DNA and is effective to kill cells in all stages of the cell cycle.
  • cyclophosphamide and related alkylating agents contain di-(2-chloroethyl)-amino groups that bind covalently to cellular components such as DNA.
  • the bryostatins are a family of naturally occurring macrocyclic compounds originally isolated from marine bryozoa. Currently, there are about 20 known natural bryostatins which share three six-membered rings designated A, B and C, and which differ mainly in the nature of their substituents at C7 (OR A ) and C20 (R B ) (Pettit, 1996).
  • the bryostatins exhibit potent activity against a broad range of human cancer cell lines and provide significant in vivo life extensions in murine xenograft tumor models (Pettit et al., 1982; Hornung et al., 1992; Schuchter et al., 1991; Mohammad et al., 1998). Doses that are effective in vivo are extremely low, with activities demonstrated for concentrations as low as 1 ⁇ g/kg (Schuchter et al., 1991).
  • the bryostatins have been found to promote the normal growth of bone marrow progenitor cells (Scheid, 1994; Kraft, 1996), provide cellular protection against normally lethal doses of ionizing radiation (Szallasi, 1996), and stimulate immune system responses that result in the production of T cells, tumor necrosis factors, interleukins and interferons (Kraft, 1996; Lind, 1993).
  • Bryostatins are also effective in inducing transformation of chronic lymphocytic leukemia cells to a hairy cell type (Alkatib, 1993), increasing the expression of p53 while decreasing the expression of bcl-2 in inducing apoptosis in cancer cells (Maki, 1995; Mohammad, 1995) or at least pre-disposing a cell towards apoptosis, and reversing multidrug resistance (MDR) (Spitaler, 1998).
  • MDR multidrug resistance
  • bryostatins have been shown to competitively inhibit the binding of plant-derived phorbol esters and endogenous diacyl glycerols to protein kinase C (PKC) at nanomolar to picomolar drug concentrations (DeVries, 1998), and to stimulate comparable kinase activity (Kraft, 1986; Berkow, 1985; Ramsdell, 1986). Unlike the phorbol esters, however, the bryostatins do not act as tumor promoters. Thus, the bryostatins appear to operate through a mode of action different from, and complementary to, the modes of action of established anticancer agents; human clinical trials are presently evaluating bryostatin combination therapy with cisplatin or taxol.
  • bryostatins have been known for some time, their low natural abundance, difficulties in isolation and severely limited availability through total synthesis have impeded efforts to elucidate their mode of action and to advance their clinical development.
  • synthetic analogues of bryostatin were reported wherein the C4-C14 spacer domain was replaced with simplified spacer segments using a highly efficient esterification-macrotransacetalization (Wender et al., 1998a, 1998b).
  • the reported analogues retained orientation of the C1-, C19-, C26-oxygen recognition domain as determined by NMR spectroscopic comparison with bryostatin and varying degrees of PKC-binding affinity.
  • the one analogue tested for in vitro inhibition in human tumor cell lines was reported to possess significant activity.
  • R zu is H, OH, or -T-U-V-R' where:
  • T is selected from -0-, -S-, -N(H)- or -N(Me ;
  • U is absent or is selected from -C(O)-, -C(S)-, -S(O)- or -S(0) 2 -;
  • V is absent or is selected from -0-, -S-, -N(H)- or -N(Me)-, provided that V is absent when U is absent;
  • R a and R b are independently H, C0 2 R', CONR c R d or R';
  • R c and R d are independently H, alkyl, alkenyl or alkynyl, or (CH 2 ) n C0 2 R' where n is 1,
  • R 26 is H, OH or R'; R' (each instance) being independently selected from the group: H, alkyl, alkenyl or alkynyl, or aryl, heteroaryl, aralkyl or heteroaralkyl; L is a straight or branched linear, cyclic or polycyclic moiety, containing a continuous chain of preferably from 6 to 14 chain atoms, which substantially maintains the relative distance between the Cl and C17 atoms and the directionality of the C1C2 and C16C17 bonds of naturally-occurring bryostatin; and Z is -O- or -N(H)-; and the pharmaceutically acceptable salt thereof.
  • R 26 is H.
  • a prefened upper limit on carbon atoms in any of R d , R e and R' is about 20, more preferably about 10 (except as otherwise specifically noted, for example, with reference to the embodiment of the invention where a preferred R 20 substituent has about 9 to 20 carbon atoms), h certain embodiments, R' is a straight-chain alkyl, alkenyl (having from 1 to 6, preferably 1 to 4 double bonds, preferably trans double bonds) or alkynyl group.
  • L contains a terminal carbon atom that, together with the carbon atom corresponding to C17 in the native bryostatin structure, forms a trans olef ⁇ n. It is further preferred that L contain a hydroxyl on the carbon atom corresponding to C3 in the native bryostatin structure.
  • Another aspect of the invention concerns the simplified bryostatin analogues represented by Formulae II - V:
  • R is H, OH or a protecting group
  • R 9 is H, OH or is absent
  • R 20 , R 21 , R 26 , R' and Z are as defined above with respect to Formula I; p is 1, 2 or 3; and
  • X is -CH 2 -, -0-, -S- or -N(R where R e is COH, C0 2 R' or S0 2 R', X preferably being -0-, and the pharmaceutically acceptable salts thereof.
  • Still another aspect of the invention concerns the simplified bryostatin analogues represented by Formulae II-A to V-A:
  • R 3 , R 6 , R 9 , R 20 , R 21 , R 26 , R', X, Z and p are as defined above with respect to Formulae ⁇ to V;
  • Still another aspect of the invention relates to the C26 des-methyl homologues of the native bryostatins, as illustrated in Formula VI:
  • compositions of matter and methods of treatment are the analogues of Formula 1998a where R 3 is H or OH and where R 20 is -0-C(0)-CH 3 or -0-C(0)-(CH 2 ) 6 -CH 3 , and those of Formula 1998b where R 8 is H or t-Bu:
  • the invention in another aspect, relates to a pharmaceutical composition containing a therapeutically effective amount of a compound of Formula I or a pharmaceutically acceptable salt thereof admixed with at least one pharmaceutically acceptable excipient.
  • the invention relates to a method of treating hyperproliferative cellular disorders, particularly cancer in a mammal by administering to a mammal in need of such treatment a therapeutically effective amount of a compound of Formula I or a pharmaceutically acceptable salt thereof, either alone or in combination with a second agent, preferably a second anti-cancer agent that acts by a distinct mechanism vis-a-vis the mechanism of the compound of Formula I.
  • a second agent preferably a second anti-cancer agent that acts by a distinct mechanism vis-a-vis the mechanism of the compound of Formula I.
  • the invention relates to methods of treatment for a mammal having an immune-related disease or receiving immunosuppressive therapy, by administering of a therapeutically effective amount of a compound of Formula I or a pharmaceutically acceptable salt thereof.
  • a method for the synthesis of bryostatin analogues including the steps of esterification and macrotrasacetylization of a protected recognition domain with a protected linker synthon, followed by deprotection. Particularly prefened is reduction of a C26 OBn protected precursor to give the corresponding C26 des-methyl bryostatin analogue.
  • a related aspect of the invention entails the novel products made by the foregoing process. Intermediates and steps for preparing them or converting them to bryostatin analogues are also included in the invention.
  • the invention also includes pharmaceutical compositions containing one or more compounds in accordance with the invention.
  • the invention includes a method of inhibiting growth, or proliferation, of a cancer cell, hi the method, a cancer cell is contacted with a bryostatin analogue compound in accordance with the invention in an amount effective to inhibit growth or proliferation of the cell.
  • the invention includes a method of treating cancer in a mammalian subject, especially humans. In the method, a bryostatin analogue compound in accordance with the present invention is administered to the subject in an amount effective to inhibit growth of the cancer in the patient.
  • alkyl As used herein, the terms “alkyl”, “alkenyl” and “alkynyl,” refer to saturated and unsaturated monovalent moieties in accordance with their standard meanings, including straight-chain, branched- chain and cyclic moieties, optionally containing one or more intervening heteroatoms, such as oxygen, sulfur, and nitrogen in the chain or ring, respectively.
  • exemplary alkyl groups include methyl, ethyl, isopropyl, cyclopropyl, 2-butyl, cyclopentyl, and the like.
  • Exemplary alkynyl groups include CH 3 C ⁇ CCH 2 -, 4-pentyn-l-yl, and the like.
  • cyclic moieties include cyclopentyl, cyclohexyl, furanyl, pyranyl, tetrahydrofuranyl, 1,3-dioxanyl, 1,4-dioxanyl, pyrrolidyl, piperidyl, morpholino, and reduced forms of furanyl, imidazyl, pyranyl, pyridyl, and the like.
  • “Lower alkyl”, “lower alkenyl”, and “lower alkynyl” refer to alkyl, alkenyl, and alkynyl groups containing 1 to 4 carbon atoms.
  • aryl denotes an aromatic ring or fused ring structure of carbon atoms with no heteroatoms in the ring(s). Examples are phenyl, naphthyl, anthracyl, and phenanthryl. Preferred examples are phenyl and naphthyl.
  • heteroaryl is used herein to denote an aromatic ring or fused ring structure of carbon atoms with one or more non-carbon atoms, such as oxygen, nitrogen, and sulfur, in the ring or in one or more of the rings in fused ring structures.
  • non-carbon atoms such as oxygen, nitrogen, and sulfur
  • examples are furanyl, pyranyl, thienyl, imidazyl, pynolyl, pyridyl, pyrazolyl, pyrazinyl, pyrimidinyl, indolyl, quinolyl, isoquinolyl, quinoxalyl, and quinazolinyl.
  • Preferred examples are furanyl, imidazyl, pyranyl, pyrrolyl, and pyridyl.
  • Alkyl and heteroarylkyl refer to aryl and heteroaryl moieties, respectively, that are linked to a main structure by an intervening alkyl group, e.g., containing one or more methylene groups.
  • Alkoxy”, “alkenoxy”, and “alkynoxy” refer to an alkyl, alkenyl, or alkynyl moiety, respectively, that is linked to a main structure by an intervening oxygen atom.
  • each X is independently a halogen (F, Cl, Br, or I, preferably F or Cl) and each
  • R' is independently hydrogen, alkyl, alkenyl, or allcynyl. In one embodiment, R' is lower alkyl, lower alkenyl, or lower alkynyl.
  • NR'R' also includes moieties wherein the two R' groups form a ring with the nitrogen atom.
  • alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, and heteroaralkyl moieties containing up to about 40 carbon atoms, more preferably up to about 20 carbon atoms and most preferably up to about 10 carbon atoms (except as otherwise specifically noted, for example, with reference to the embodiment of the invention where a preferred R 20 substituent has about 7 to 20 carbon atoms).
  • pharmaceutically acceptable salt refers to salts which retain the biological effectiveness and properties of the compounds of this invention and which are not biologically or otherwise undesirable.
  • the compounds of this invention are capable of forming acid and/or base salts, derived from a variety of organic and inorganic counter ions well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate and the like.
  • Relatively lipophilic describes a molecule, moiety, or region which is uncharged at neutral pH, and, taken alone, is only partially soluble in water. Relatively lipophilic moieties preferably have no more than one OH or NH bond for every five carbon atoms, even more preferably for every eight carbon atoms. Relatively lipophilic means that the molecule, moiety or region facilitates therapeutic use, helping to maintain a balance between lipophilicity (e.g., to permit cellular uptake) and hydrophilicity (e.g., to permit aqueous formulation).
  • mice is intended to have its conventional meaning. Examples include .humans, mice, rats, guinea pigs, horses, dogs, cats, sheep, cows, etc.
  • treatment or “treating” means any treatment of a disease in a mammal, including:
  • the term "effective amount” means a dosage sufficient to provide treatment for the disease state being treated. This will vary depending on the patient, the disease and the treatment being effected.
  • the present invention provides new analogues of bryostatin that can be synthesized conveniently in high yields and which have useful biological activities.
  • the compounds of the invention can be broadly described as having two main regions that are referred to herein as a "recognition domain” (or pharmacophoric region) and a relatively lipophilic "spacer domain” (or linker region).
  • the recognition domain contains structural features that are analogous to those spanning C17 through C26 to Cl, including the C ring formed in part by atoms C19 through C23, and the lactone linkage between Cl and C25 of the native bryostatin macrocycle.
  • the spacer domain joins the atoms corresponding to Cl through C17 of the native bryostatin macrocycle to substantially maintain the relative distance between the Cl and C17 atoms and the directionality of the C1C2 and C16C17 bonds, as illustrated by the areows and distance "d" in Formula la (in which the substituent groups are as defined with reference to Formula I).
  • the spacer domain (shown as "L" in Formula la and sometimes also refened to as a linker region) provides a moiety that can be readily derivatized according to known synthetic techniques to generate analogues having improved in vivo stability and pharmacological properties (e.g., by modulating side effect profiles) while retaining biological activity. It has been found in the present invention that the linker region of the bryostatin family can be varied significantly without eliminating activity. Thus, a wide variety of linkers can be used while retaining significant anticancer and PKC-binding activities.
  • the compounds of the present invention include a linker moiety L, which is a linear, cyclic, or polycyclic linker moiety containing a continuous chain of from 6 to 14 chain atoms, one embodiment of which defines the shortest path from C25 via Cl to C17.
  • Distance "d" should be about 2.5 to 5.0 angstroms, preferably about 3.5 to 4.5 angstroms and most preferably about 4.0 angstroms, such as about 3.92 angstroms (as experimentally determined, for example, by NMR spectroscopy).
  • L may consist solely of a linear chain of atoms that links C17 via Cl to C25, or alternatively, may contain one or more ring structures which help link C17 via Cl to C25.
  • the linker include a hydroxyl group analogous to the C3 hydroxyl found in naturally occurring bryostatins, to permit formation of an intramolecular hydrogen bond between the C3 hydroxyl of the linker and the C19 hydroxyl group of the recognition region (and optionally with the oxygen of the native B ring).
  • R 26 is H
  • the compounds of the invention differ from known bryostatins and bryostatin analogues in that the present compounds contain a primary alcohol moiety at C26, i.e., the present analogues lack a methyl group conesponding to the C27 methyl that is ordinarily present in naturally occurring bryostatins.
  • the present invention provides bryostatins and bryostatin analogues in which R 20 is longer (e.g., having 9 to 20 or more carbon atoms) than the corresponding substituents at C20 in the native bryostatins (e.g., Bryostatin 3 having an 8-carbon atom moiety).
  • R 3 is H, OH or a protecting group
  • R 9 is H, OH or is absent
  • R 20 , R 21 , R 26 , R' and Z are as defined above with respect to Formula I; p is 1, 2 or 3; and
  • X is -CH 2 -, -O-, -S- or -N(R e )- where R e is COH, C0 2 R' or S0 2 R', X preferably being -O-, and the pharmaceutically acceptable salts thereof.
  • R 3 , R 6 , R y , R , R , R , R', X, Z and p are as defined above with respect to Formulae H to V;
  • R 12 and R 12a are independently for each occurrence, H, OH, lower alkyl, lower alk
  • the analogues illustrated in Formulae II-A to V-A have one or more, preferably all of the following stereochemical dispositions:
  • Formula IV-B Formula V-B
  • R 26 is H or lower alkyl, preferably H or Me, and most preferably H.
  • R 21 CHC0 2 R', where R' is preferably lower alkyl, especially Me or Et.
  • R 20 represents a carbonate, urea, thiourea, thiocarbamate or carbamate substituent.
  • R 3 is OH.
  • Particularly prefened are those compounds, respectively having one or more of the stereochemical configurations illustrated in Formulae I throught V.
  • compositions of matter and methods of treatment are the analogues of Formula 1998a where R 3 is H or OH and where R 20 is -0-C(0)-CH 3 or -0-C(0)-(CH 2 ) 6 -CH 3 , and those of Formula 1998b where R 8 is H or t-Bu:
  • the compounds of Formulae I-V are named and numbered herein as conesponding to the naturally occuning bryostatin macrocycle, described above with reference to Formula A.
  • the C26 des-methyl homologue of native bryostatin 1 (a compound of the present invention) has the structure illustrated in Formula Via:
  • Formula Ila Formula IVa are also refened to as "C26 des-methyl", notwithstanding that the structures conesponding to L (in Formula I) or the conesponding spacer domain (in Formulae II-V), or even the recognition domain, contain fewer carbon atoms than native bryostatin such that the "C26" position would be assigned a lower number were these analogues to be named without reference to the native structure.
  • solvent inert under the conditions of the reaction being described in conjunction therewith [including, for example, benzene, toluene, acetonitrile, tetrahydrofuran (“THF”), dimethylformamide (“DMF”), chloroform, methylene chloride (or dichloromethane), diethyl ether, methanol, pyridine and the like].
  • solvents used in the reactions of the present invention are inert organic solvents.
  • protecting group or “blocking group” refer to any group which when bound to a functional group such as one or more hydroxyl, thiol, amino or carboxyl groups of the compounds
  • removable blocking group is not critical and prefened removable hydroxyl blocking groups include conventional substituents such as allyl, benzyl, acetyl, chloroacetyl, thiobenzyl, benzylidine, phenacyl, t-butyl-diphenylsilyl and any other group that can be introduced chemically onto a hydroxyl or like functionality and later selectively removed either by chemical or enzymatic methods in mild conditions compatible with the nature of the product.
  • q.s means adding a quantity sufficient to achieve a stated function, e.g., to bring a solution to the desired volume (i.e., 100%).
  • the reactions described herein take place at atmospheric pressure within a temperature range from about 5°C to 100°C (preferably from 10°C to 50°C; most preferably at "room” or “ambient” temperature, e.g., 25°C).
  • reaction times and conditions are intended to be approximate, e.g., taking place at about atmospheric pressure within a temperature range of about 5°C to about 100°C (preferably from about 10°C to about 50°C; most preferably about 25°C) over a period of about 0.5 to about 10 hours (preferably about 1 hour). Parameters given in the Examples are intended to be specific, not approximate.
  • Isolation and purification of the compounds and intermediates described herein can be effected, if desired, by any suitable separation or purification procedure such as, for example, distillation, filtration, extraction, crystallization, column chromatography, thin-layer chromatography or thick-layer chromatography, or a combination of these procedures.
  • suitable separation and isolation procedures can be had by reference to the general description and examples. However, other equivalent separation or isolation procedures can, of course, also be used.
  • the compounds of the invention may be produced by any methods available in the art, including chemical and biological (e.g., recombinant and in vitro enzyme-catalyzed) methods.
  • the present invention provides a convergent synthesis in which subunits primarily conesponding to the recognition and spacer domains are separately prepared and then joined by esterification-macrotransacetalization (Wender et al., 1998a, 1998b, 1998c). Additional syntheses of the compounds of Formulae I-VI are described below with reference to the Reaction Schemes.
  • the stereochemical relationships illustrated for the various substituents should be taken to be optional, but individually and collectively prefened; the stereochemical relationships conesponding to the native bryostatins are particularly prefened.
  • Reaction Scheme 1 illustrates synthesis of precursors for the recognition domain in compounds of the invention.
  • Reaction Scheme 2 illustrates the further synthesis of recognition domains for C26 des-methyl compounds of the invention.
  • Reaction Scheme 3 illustrates synthesis of the protected alcohol precursor to many of the hyl analogues of the invention.
  • Reaction Scheme 4 illustrates the synthesis of linker synthons for preparing the compounds of Formula II.
  • Reaction Scheme 5B illustrates the synthesis of linker synthons for preparing the compounds of Formula ⁇ i where R 8 and/or R 9 are H.
  • Reaction Scheme 6 illustrates the synthesis of linker synthons for preparing the compounds of
  • Reaction Scheme 7A illustrates the synthesis of the compounds of Formula II and II-A.
  • R' OTBS or OBn
  • Reaction Scheme 7B illustrates the synthesis of the compounds of Formula m.
  • Reaction Scheme 8 illustrates synthesis of the Compounds of Formula IV, particularly where R 2 ⁇ is methyl, the C26 des-methyl analogues and compounds of Formula IV where R 12 and R 12a are other than H being obtained by like synthesis.
  • Reaction Schemes 10 and 11 illustrate the synthesis of compounds of the invention that are further derivatized at the C20 position, as discussed in Examples 4B, 4C and 4D.
  • Reaction Scheme 1 illustrates a method for forming a synthon designated herein as 111 which is useful for providing the recognition domain in compounds of the invention, for example as detailed in Example 1.
  • p-TsOH p-methylphenylsulfonic acid
  • the ⁇ -isomer (104a) is separated from the ⁇ -isomer and is reacted with NaBH 4 in the presence of CeCl 3 -7H 2 0, followed by quenching with aqueous brine to form an allylic alcohol (not shown)that can then be epoxidized with r ⁇ -chloroperbenzoic acid (CPBA) in 2:1 CH 2 Cl 2 :Me0H containing sodium bicarbonate as a buffer to yield a C19-methoxylated C20,C21 syn-diol 105.
  • CPBA r ⁇ -chloroperbenzoic acid
  • substituents can be introduced in synthon 111 to generate substituent R 20 at C20 by substituting any of a variety of carboxylic acids for the octanoic acid reacted with axial alcohol 110 (as in the last step of Example 1C), including other saturated, unsaturated, aryl, and carboxylic acids.
  • the substituent e.g., a desired C20 ester substituent, carbonate, urea, thiourea, thiocarbamate or carbamate
  • the C20 octanoate substituent in synthon 111 can be replaced with an acetyl group by first protecting the base labile aldehyde group using trimethyl orthoformate to form the dimethyl acetal.
  • the C20 octanoate ester can then be cleaved using a basic solution, such as K 2 C0 3 in methanol, to afford the free C20 alcohol, followed by reaction with an activated form of acetic acid, such as acetic anhydride or acetyl chloride, to obtain the C20 acetate product.
  • the product can then be deprotected at the C15 aldehyde, C19 oxygen, and C25 oxygen using benzoquinone compound DDQ (to remove the p-methoxybenzyl group and cleave the dimethyl acetal) followed by aqueous HF (demethylation at C19) to afford the conesponding C19 alcohol.
  • This product can then be condensed with an appropriately substituted linker synthon to produce a desired bryostatin analogue, such as analogue 702.1, as detailed in Example 4A.
  • the protected alcohol precursor to many of the C26-desmethyl bryostatin analogues of the invention can be made as illustrated in Reaction Scheme 2.
  • Difbenzyl ether) 111 can be hydrogenated over Pearlman's catalyst to produce the conesponding C25,C26 diol 201.
  • Treatment of the diol with lead tetraacetate yields the conesponding C25 aldehyde (not shown), with the release of C26 and C27.
  • Reaction of the aldehyde with Cp 2 Ti(Cl)CH 2 Al(CH 3 ) 2 (Tebbe's reagent) yields C25,C26 olefin 202.
  • sodium periodate can be used in place of lead tetraacetate.
  • Treatment of olefin 202 with HF/pyridine is effective to remove the silyl protecting group, followed by treatment with Dess-Martin periodinane (supra) to oxidize the C17 alcohol to an aldehyde group, affording aldehyde 203.
  • the C25,C26 olefin of 203 can be converted to C25,C26 diol 204 by reaction with chiral dihydroxylating reagent (DHQD) 2 AQN in the presence of K 3 Fe(CN) 6 , K 2 C0 3 and K 2 Os0 2 (OH) 4 in t-butanol.
  • DHQD chiral dihydroxylating reagent
  • Product 204 is obtained as a 2:1 ( ⁇ : ) mixture of 25-hydroxy diastereomers.
  • the ⁇ -diastereomer can be removed later in the synthesis.
  • Treatment of 204 with triethylsilyl chloride yields protected diol 205, which can be employed in the synthesis of the compounds of Formula V.
  • Addition of backbone atoms conesponding to C15 and C16 of the bryostatin backbone to 205 can be accomplished in four steps.
  • the C17 aldehyde is allylated with allyl diethylborane.
  • the reaction is quenched with saturated sodium bicarbonate to yield the desired C17 allyl adduct.
  • the C17 hydroxyl group can then be acylated with acetic anhydride in the presence of triethylamine and 4- dimethylaminopyridine (DMAP), to afford a diastereomeric mixture of homoallylic C17 acetates.
  • DMAP 4- dimethylaminopyridine
  • This product mixture can be oxidized using N-methylmorpholine N-oxide and osmium tetraoxide, followed by neutralization with sodium bicarbonate. After extraction, the residue is reacted with lead tetraacetate, followed by addition of DBU to cause elimination of the acetate group, yielding enal 206.
  • the C25 hydroxyl group of 206 can be unmasked in preparation for closure of the macrocycle as follows. First, enal 206 is treated with aqueous hydrofluoric acid to provide a crude diol intermediate in which the C19 methoxy group is converted to a free hydroxyl.
  • the diol product is reacted with tert-butyldimethylsilyl chloride (TBSCl) in the presence of imidazole to produce alcohol 207 containing a C25 hydroxyl group and C26 OTBS group as a 2:1 ( ⁇ :cc) mixture of C25 diastereomers.
  • Silica gel chromatography can be used to resolve the diastereomers, affording the ⁇ diastereomer in 50-60% yield.
  • the protected alcohol precursor to many of the C26-methyl bryostatin analogues of the invention (Formula I where R 2 ⁇ is methyl) can be made as illustrated in Reaction Scheme 3, via methods analogous to the preparations for 205 and 206. Deprotection and acylation of formula 111 may be accomplished, for example, by following the procedures described in Wender et al., (1998a).
  • Linker synthons for the compounds of Formula II can be prepared, for example, as illustrated with reference to Reaction Scheme 4, and later described in Examples 2A and 2B. These compounds contain two rings that are analogous to the A and B rings of bryostatin, but lack the naturally occuning substituents at C7, C8, C9, and C13. The presence of a heteroatom, such as an oxygen, sulfur or nitrogen atom (the lone electron pair of which is stabilized) in place of C14 does not adversely affect activity of the end product, but is required for transacetylization in the later synthetic steps.
  • the compounds of formulae 406 and 408 differ in that 402 provides a protecting group precursor for a hydroxyl group attached to C3, whereas 406 does not provide for a hydroxyl at C3.
  • linker synthons for the compounds of Formula III (in which X is a heteroatom), which contain a B-ring-like structure but lack an A-ring, are prepared, for example, as illustrated with reference to Reaction Schemes 5A through 5C.
  • Examples 2C and 2D describe methods for preparing synthons 504 and 508.
  • R 8 is a tert butyl group attached to C9.
  • the t-BuLi reactant can be replaced by R'Li to generate the conesponding linker synthons of 508 where R 8 is R'.
  • 504 additionally contains a TMS protecting group for synthesis of the compounds where R 9 is a hydroxyl attached to C9, rather than hydrogen.
  • both synthons contain a
  • Example 2E describes the conesponding method for making synthon 507, which is unsubstituted at C9.
  • Example 2G describes a method for preparing linker synthons in which C5 is provided as an ester carbonyl.
  • the synthons in this Example contain an R 6 substituent that is preferably a saturated or unsaturated substituent containing 1 to 20 carbon atoms and optionally (1) one or more oxygen atoms and (2) optionally one or more nitrogen atoms.
  • other R 6 substituents can be introduced by suitable modification of the procedure as will be evident to one of ordinary skill in the chemical arts.
  • the ketal portion of 408 is then joined (in a process refened to as macrotranacetylization) to C15 of 701 by adding 70% HF/pyridine hydrofluoric acid to catalyze cleavage of the menthone ketal, cleavage of the TBS ethers at C3 and C26, and formation of a new ketal between the C15 aldehyde group and the linker diol moiety generated by release of the menthone (where X is oxygen), to afford desired analogue of Formula II where X is a heteroatom and R 26 is H (starting with alcohol 207) or methyl (starting with 303 or 304), i.e., compound of formula 702.
  • the compounds of Formula IV can be made from pharmacophoric synthon 801 and linker synthon 606 from Example.2F.
  • Reaction Scheme 9 illustrates synthesis of the compounds of Formula V, e.g., as further described in Example 3D, from synthon 111 and an activated dicarboxylic acid (succinic anhydride) to give formula 903.
  • an activated dicarboxylic acid succinic anhydride
  • the bryostatin analogues produced in Examples 3B, 3C and 3D all contain a C27 methyl group
  • analogous C26 desmethyl analogues can be readily synthesized using an appropriate C26 desmethyl synthon, such as C26 desmethyl synthon 207 described in Example lC.
  • Example 4B synthesis of a C20 heptanoate ester 43 is described in Example 4B, using a similar reaction scheme to that employed in Example 4A, except that heptenoic acid in the presence of triethylamine, DMAP, and Yamaguchi's agent is used in place of acetic anhydride.
  • Yamaguchi's reagent is again employed in step f to activate the COOH group of formula 6, followed by removal of the TBS group in step g, hydrolysis of the menthone and transacetylization in step h, and saturation of the double bond upon removal of the benzyl group by hydrogenolysis in step i.
  • the lactam analogues of the invention are obtained by converting the C25 hydroxyl group (e.g., of formula 207) to an amine under Mitsonobu conditions, after first protecting the aldehyde (and the C19 hydroxyl group in the conesponding compounds in Reaction Schemes 10 and 11) followed by formation of the macrocycle and de-protection under conditions analogous to those employed for the lactone analogues, as will be apparent to one skilled in the art.
  • Lactam embodiments can also be prepared by performing an aminohydroxylation reation, such as has been disclosed by Sharpless et al., instead of dihydroxylation with Os0 .(employing protection/deprotection as described above).
  • Chiral ligands for this reaction are known, and can be used to influence the stereochemical and/or regiochemical outcomes of the aminohydroxylation. This strategy can be employed on substrates in which the terminal alkene of the above scheme is further substituted, thereby providing access to compounds wherein R 26 is other than hydrogen.
  • Such starting materials can be prepared by cleaving the olefin to the aldehyde (e.g., by Os0 /periodate or ozonolysis) and performing a Wittig or other olefination reaction to obtain a desired secondary alkene.
  • the C26 des-methyl bryostatin homologues of the invention can be obtained by substituting homologous des-methyl starting materials for the starting materials employed in published bryostatin syntheses (e.g., Masamune 1988a, 1988b, Evans et al. 1998, Kageyama et al. 1990).
  • homologous des-methyl starting materials e.g., Masamune 1988a, 1988b, Evans et al. 1998, Kageyama et al. 1990.
  • Serine is substituted for threonine in a Masamune's C17-C26 southern bryostatin synthesis to yield the conesponding C26 des-methyl sulfone.
  • Other synthetic methodology will be apparent to those skilled in the art given the objective of providing such C26 des-methyl bryostatin homologues.
  • the fragment conesponding to the recognition domain (or C-ring portion) of bryostatin is prepared by a method that includes one or more of the steps illustrated with regard to Reaction Schemes 12 to 15, wherein:
  • R', R 20 and R 26 are as defined above (for example, R 20 being -0 2 CR' or -0 2 CNHR'); R* represents, independently for each occurrence, H or a lower alkyl group such as methyl or ethyl; q represents 0 or 1;
  • E represents an aldehyde, hydroxymethyl, carboxyl group, or a protected form thereof, such as CHO, CH 2 OP, CH(OP) 2 , or C0 2 P;
  • P represents H or a protecting group, including protecting groups wherein two occunences of P, taken together, form a ring having 5-7 members including the atoms through which they are connected (e.g., CH(OP) 2 may represent a cyclic acetal, e.g., with ethylene glycol or pinacol); and
  • G is absent or represents P.
  • R' is preferably hydrogen or lower alkyl (such as methyl or ethyl).
  • P represents a triallcylsilyl, dialkylarylsilyl, benzyl, substituted benzyl, benzhydryl, substituted benzhydryl,
  • 5-dibenzosuberyl triphenylmethyl, substituted triarylmethyl, naphthyldiphenylmethyl, 2- or 4-picolyl, 3 -methyl-2-picolyl-N-oxide, 4-(4 ' -bromophenacyloxyphenyl)diphenylmethyl, 4,4 ' ,4 " -tris(4,5 -dichloro- phthalamidophenyl)methyl, 4,4 ' ,4 " -tris(levulinoyloxyphenyl)methyl, 4,4 ' ,4 " -tris(benzoyloxy- phenyl)methyl, 9-(9-phenyl)xanthenyl, pivaloyl, adamantoyl, and 2,2,2-trichloroethylcarbonyl.
  • P is a protecting group for an acetal
  • P represents lower alkyl
  • P is a protecting group for a carboxylic acid
  • P represents dialkylarylsilyl, benzyl, substituted benzyl, trichloroethyl, t-butyl, trimethylsilylethyl, lower alkyl, lower alkenyl (such as allyl), or other suitable protecting group.
  • E represents a carboxylic acid derivative that can be readily converted to an aldehyde, such as a thioester (reduction by triethylsilane in the presence of a transition-metal catalyst), CONMe(OMe) (reduction by a hydride reagent), an ester (reduction by diisobutylaluminum hydride (DIBAL-H)), etc.
  • aldehyde such as a thioester (reduction by triethylsilane in the presence of a transition-metal catalyst), CONMe(OMe) (reduction by a hydride reagent), an ester (reduction by diisobutylaluminum hydride (DIBAL-H)), etc.
  • aldehyde such as a thioester (reduction by triethylsilane in the presence of a transition-metal catalyst), CONMe(OMe) (reduction by a hydride reagent
  • Reaction Scheme 12 The starting materials for Reaction Schemes 13 and 14 can be prepared, for example, as illustrated in Reaction Scheme 12. Reaction Scheme 12
  • compound 12A can be converted to compound 12B by reaction with an acetone enolate or equivalent thereof, such as a lithium or magnesium anion of the N,N- dimethylhydrazone of acetone.
  • an acetone enolate or equivalent thereof such as a lithium or magnesium anion of the N,N- dimethylhydrazone of acetone.
  • the methyl ester of 12A can be reduced to an aldehyde
  • aldol addition with an acetone enolate or equivalent and oxidation of the aldol product to the diketone.
  • the dienolate of diketone 12B can then be reacted with aldehyde 12C to give aldol product 12F, which can be subsequently protected and/or reduced at the C 21 ketone selectively (e.g., with tetramethylammonium triacetoxyborohydride, with an aldehyde in the presence of a Lewis acid via an intramolecular hydride transfer, etc.).
  • ester 12A can be converted to ketone 12E, e.g., by reduction to the aldehyde, reaction with methyllithium or methyl magnesium halide, and oxidation to the ketone.
  • Aldehyde 12C can be extended to aldehyde 12D by aldol addition of an acetaldehyde equivalent (e.g., reaction with an allylsilane, allylborane, or allyltin reagent followed by ozonolysis or dihydroxylation/periodate cleavage, reaction with a silyl ketene acetal of an acetate ester in the presence of a Lewis acid catalyst such as TiCl 3 (OiPr), TiCl 2 (OiPr) 2 , SnCl 4 , or Sn(OTf) 2 followed by reduction to the aldehyde, reaction of an enolate of an acetate ester followed by reduction to the aldehyde, etc.
  • chiral reagents or ligands are available that may be used to favor a desired diastereomer of the aldol product, while others may be sufficiently stereoselective without use of a chiral reagent.
  • An enolate of ketone 12E can then be added to aldehyde 12D to anive at aldol 12F by a different route.
  • Step a can be performed by treating the diketone compound 12F (produced as described in Reaction Scheme 12, where G at C 2 ⁇ is absent) with acid, preferably under conditions that favor the removal of the water generated, such as distillation of the generated water (optionally as an azeotrope), addition of a dehydrating agent (such as molecular sieves, a carboxylic acid anhydride, or sodium or calcium sulfate), or other suitable means.
  • a dehydrating agent such as molecular sieves, a carboxylic acid anhydride, or sodium or calcium sulfate
  • Step b can be performed by a series of reactions.
  • step b may begin with reduction of the ketone 13 A with a hydride source, such as lithium or sodium borohydride, lithium aluminum hydride, etc.
  • a reduction selective for the ketone over the unsaturation is performed, such as a Luche reduction (sodium borohydride in the presence of cerium (III) chloride heptahydrate).
  • Oxidation of the alkene can then be performed with monoperoxyphthalate hexahydrate (MMPP), by epoxidation (e.g., with mCPBA, or dimethyldioxirane), or by dihydroxylation (e.g., with osmium tetroxide, optionally with an asymmetric ligand as is well known in the art).
  • MMPP monoperoxyphthalate hexahydrate
  • epoxidation e.g., with mCPBA, or dimethyldioxirane
  • dihydroxylation e.g., with osmium tetroxide, optionally with an asymmetric ligand as is well known in the art.
  • the product of the oxidation if performed in the presence of water, will be the hemiketal, and if performed in the presence of an alcohol, will be the conesponding mixed ketal.
  • the present invention also encompasses the intermediate compounds useful in executing this strategy, including thecompounds of Formulae 13 A and 13B (with and without regard to the illustrated stereochemical relationships).
  • Compounds where R 20 is H can be prepared by a method including one or more steps of the following sequence:
  • Step a can be performed starting with a compound of Formula 12F (produced as described in
  • Reaction Scheme 12 where G at C 2 ⁇ is hydrogen) by an acid-catalyzed cyclization in a reaction mixture including an alcohol HOR*, preferably as a solvent or cosolvent, in order to form the mixed ketal.
  • HOR* preferably as a solvent or cosolvent
  • the cyclization can be performed under conditions that selectively remove this protecting group (and possibly also any protecting group at C21), or one or both of these protecting groups can be removed prior to the cyclization.
  • Step b can be performed by oxidation of the C21 alcohol (e.g., by Swern oxidation, Dess-Martin periodinane, etc.), after removing any protecting group at this position, followed by reaction with a reagent such as R'0 2 CCH 2 SiMe 3 or R'0 2 CCH 2 P0 3 Me 2 .
  • a reagent such as R'0 2 CCH 2 SiMe 3 or R'0 2 CCH 2 P0 3 Me 2 .
  • a chiral phosophonoacetate e.g., a phosphonate ester of BINOL, or other chiral diols or alcohols
  • phosphonamidoacetate e.g., a phosphonate derivative of ephedrine or another chiral aminoalcohol
  • Subsequent steps may be performed in analogy to well known procedures as discussed above.
  • the present invention also encompasses the intermediate compounds useful in executing this strategy, including the compounds of Formulae 14A and 14B (with and without regard to the illustrated stereochemical relationships).
  • alcohol 15A can be oxidized to an aldehyde (e.g., by Swern or TPAP/NMO oxidation), followed by addition of a Grignard or lithium reagent derived from a 4-halo-l-butanol, such as 4-chloro- or 4-bromo-l-butanol.
  • an aldehyde e.g., by Swern or TPAP/NMO oxidation
  • a Grignard or lithium reagent derived from a 4-halo-l-butanol, such as 4-chloro- or 4-bromo-l-butanol.
  • Both alcohols of 15B can then be oxidized (e.g., by Swern or TPAP/NMO oxidation), followed by selective addition of an allyl group to the aldehyde (e.g., by treatment with an allylstannane or allylsilane in the presence of a Lewis acid catalyst, optionally in the presence of a chiral ligand for the Lewis acid, such as BINOL, Ti(OPr) 4 , and B(OMe) 3 together) to give ketoalcohol 15C.
  • a Lewis acid catalyst optionally in the presence of a chiral ligand for the Lewis acid, such as BINOL, Ti(OPr) 4 , and B(OMe) 3 together
  • Cyclization and elaboration of this piece can be performed in analogy with the scheme presented above, for example, by cyclizing in the presence of an acid under dehydrating conditions, followed by oxidation of the enol ether with magnesium monoperoxyphthalate hexahydrate (MMPP), and oxidation of the resulting alcohol (e.g., by Swern or TPAP/NMO oxidation) to give ketone 15D.
  • MMPP magnesium monoperoxyphthalate hexahydrate
  • oxidation of the resulting alcohol e.g., by Swern or TPAP/NMO oxidation
  • the exocyclic enoate can then be installed by an aldol condensation with methyl glyoxylate, e.g., by forming the lithium anion of ketone 15D with lithium diisopropylamide (LDA) under anhydrous conditions, or in an alcoholic solvent (such as methanol) in the presence of a base (such as sodium, potassium, or cesium carbonate).
  • LDA lithium diisopropylamide
  • a base such as sodium, potassium, or cesium carbonate
  • the terminal alkene can then be oxidized to the diol (e.g., by Os0 or certain hypervalent iodine reagents), optionally in the presence of a chiral ligand, as is well known in the art.
  • Embodiments wherein q is 0 can be converted to embodiments wherein q is 1 as described in detail above.
  • the present invention also encompasses the intermediate compounds useful in executing this strategy, including the compounds of Formulae 15C, 15D, 15E and 15F (with and without regard to the illustrated stereochemical relationships).
  • a fragment analogous to the A and B rings of bryostatin can be prepared for inco ⁇ oration into an analog of the present invention by a method including the steps illustrated in Reaction Scheme 16, where the substituents R*, R 7 and P are as defined above:
  • a glutaric diester 16A can be condensed with a dienolate of an acetoacetate ester to provide diketoester 16B, followed by reduction of the two ketones to give diol 16C.
  • One ketone can be reduced by a Noyori hydrogenation in the presence of a chiral catalyst to impart a first asymmetric center, and the second can be reduced stereoselectively using the newly formed stereocenter (tetramethylammoniumtriacetoxyborohydride for anti, Et 2 BOMe andNaBH for syn, for example).
  • Acid-catalyzed lactonization followed by protection of the remaining alcohol to give lactone 16D provides a substrate for a second reaction with a dienolate of an acetoacetate ester to give ketoester 16E.
  • Acid-catalyzed reduction of the hemiketal with triethylsilane, or dehydration of the hemiketal followed by hydrogenation generates the tetrahydropyran of the A-ring analog.
  • the remaining ketone can be reduced using a Noyori hydrogenation for a second time, or by hydride reduction in the presence of a chelating Lewis acid to take advantage of the tetrahydropyran stereochemistry, thereby producing alcohol 16F.
  • the terminal 1,3-diol can be converted to a ketal 16G in the presence of acid and a ketone, ketal, or enol ether.
  • the present invention also encompasses the intermediate compounds useful in executing this strategy, including thecompounds of Formulae 16E, 16F and 16G (with and without regard to the illustrated stereochemical relationships).
  • Compounds useful in executing this strategy also include compounds represented by Formulae 16H, 161 and 16J:
  • a C19,C26 hydroxyl-protected, C26 des-methyl bryostatin recognition domaine precursor and an optionally protected linker synthon are esterified, macrotransacetylated and de-protected to give the conesponding C26 des-methyl bryostatin analogue.
  • a bryostatin analogue precursor having the C26 hydroxyl substituted by a protecting group (particularly OBn) is reduced to give the conesponding compound of Formula I.
  • Serine is substituted for threonine in a Masamune's C17-C26 southern bryostatin synthesis to yield the conesponding C26 des-methyl sulfone, which in turn is employed in synthesis of a C26 des- methyl bryostatin homologue.
  • a pyran-4-ol of Formula 404 is converted to the conesponding pyran-2-yl-acetaldehyde of Formula 405 under reaction conditions including the presence of isobutylvinyl ether and Hg(IT) diacetate.
  • the reaction conditions further include carrying the crude vinylated pyran forward without delay, contacting it with anhydrous decane.
  • a diketone of Formula 12F is converted to the conesponding dihydropyranone of Formula 13 A under reaction conditions including the presence of an acid, particularly where R 26 represents H or to C 6 alkyl.
  • the reaction conditions can further include means for removing water.
  • a ketone of Formula 12F is converted to a tetrahydropyran of Formula 14A under reaction conditions including the presence of an acid and an alcohol (R*OH), particularly where R 26 represents H or Ci to C ⁇ alkyl.
  • the alcohol can be present as a solvent or cosolvent making up at least 20% of total solvent.
  • a ketone of Formula 15C is converted to the conesponding dihydropyranone of Formula 15D under reaction conditions including the presence of an acid.
  • the reaction conditions can further include means for removing water.
  • a ketone of Formula 15D is converted to the conesponding ketoenoate of Formula 15E under reaction conditions including the presence of an alkyl glutarate ester.
  • An optionally protected lactone of Formula 16E is contacted with a dienolate of an ester of acetoacetate under conditions that provide the conesponding tetrahydropyran of Formula 16F.
  • a compound of Formula I- VI is contacted with a pharmaceutically acceptable acid to form the conesponding acid addition salt.
  • a pharmaceutically acceptable acid addition salt of Formula I-VI is contacted with a base to form the conesponding compound of Formula I-VI. Also prefened is a stereospecific synthesis for preparing a bryostatin analog, having a step selected from the group:
  • the starting material for Step 13a, 14a, 15c, 15d or 16d has one or more of the stereochemical configurations represented by formulae 12F, 12F, 15C, 15D or 16D, respectively, and especially where:
  • R 7 is absent;
  • R 20 is R 20 is H, OH, -0 2 C-lower alkyl or -0 2 C-alkenyl;
  • R 26 is H or OH;
  • R' is independently selected from: H and methyl
  • R* is independently selected from: H and methyl
  • E is OPMB, TBSO-CH 2 - or -C(0)H; and/or P is H, benzyl, OPMB or TBSO. Further prefened are those processes where:
  • Step 13a takes place under reaction conditions including the presence of an acid
  • Step 14a takes place under reaction conditions including the presence of an acid and an alcohol of the formula R*-OH, where R* is lower alkyl
  • Step 15c takes place under reaction conditions including the presence of an acid
  • Step 15d takes place under reaction conditions including the presence of an alkyl glutarate ester; and/or • Step 16d takes place under reaction conditions including the presence of a dienolate of an ester of acetoacetate.
  • R 26 is H
  • R 20 is 0 2 CR'
  • R 21 CR a R b (especially where one of R a or R b is
  • R 3 is OH
  • R 3 is OH, R 2 ⁇ is H and/or Z is -O-.
  • R 21 CR a R b (especially where one of R a or R b is H and the other is C0 2 R', and preferably where R' is C ⁇ -C ⁇ 0 alkyl, most preferably lower alkyl such as methyl). Further prefened are the compounds where R 3 is OH, R 26 is H and/or Z is -0-.
  • L be a group having from about 6 to about 14 carbon atoms. Also prefened are those compounds where distance "d" (in Formula la) is about 2.5 to 5.0 angstroms, preferably about 3.5 to 4.5 angstroms and most preferably about 4.0 angstroms, such as about 3.92 angstroms. Further prefened are those compounds where L contains a hydroxyl on the carbon atom conesponding to C3 in the native bryostatin structure.
  • R 21 CR a R b (especially where one of R a or R b is H and the other is C0 2 R', and preferably where R' is C ⁇ -C ⁇ 0 alkyl, most preferably lower alkyl such as methyl).
  • R 3 is OH
  • R 26 is H and or Z is -0-.
  • R 9 is H. It is further prefened that R 8 is H, alkyl (especially t-butyl), aralkyl, -CH 2 (CH 3 ) 2 -CH 2 -0-R [particularly where R is COCH 2 Cl,
  • the compounds of the present invention are useful as bryostatin-like therapeutic agents, and in pharmaceutical formulations and methods of treatment employing the same.
  • Other compounds of the invention are useful a precursors in the synthesis of such agents.
  • the compounds of the present invention can be readily synthesized on a large scale, and thus can be made readily available for commercial purposes as compared to the low yields and environmental problems inherent in the isolation of bryostatins from natural sources.
  • the compounds of the invention find use as anticancer agents in mammalian subjects.
  • representative cancer conditions and cell types against which the compounds of the invention may be useful include melanoma, myeloma, chronic lymphocytic leukemia (CLL), AJDS- 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.
  • the compounds appear to operate by a mechanism distinct from the mechanisms of other anticancer compounds, and thus can be used synergistically in combination with other anticancer drugs and therapies to treat cancers via a multimechanistic approach.
  • the compounds of the invention exhibit potencies comparable to or better than previous bryostatins against many human cancer types.
  • the compounds of the invention can be used to strengthen the immune system of a mammalian subject, wherein a compound of the invention is administered to the subject in an amount effective to increase one or more components of the immune system.
  • strengthening of the immune system can be evidenced by increased levels of T cells, antibody-producing cells, tumor necrosis factors, interleukins, interferons, and the like.
  • Effective dosages may be comparable to those for anticancer uses, and can be optimized with the aid of various immune response assay protocols such as are known in the art (e.g., see Kraft, 1996; Lind, 1993; US Patent 5,358,711, all incorporated herein by reference).
  • the compound can be administered prophylactically, e.g., for subjects who are about to undergo anticancer therapies, as well as therapeutically, e.g., for subjects suffering from microbial infection, burn victims, subjects with diabetes, anemia, radiation treatment, or anticancer chemotherapy.
  • the immunostimulatory activity of the compounds of the present invention is unusual among anticancer compounds and provides a dual benefit for anticancer applications. First, the immunostimulatory activity allows the compounds of the invention to be used in greater doses and for longer periods of time than would be possible for compounds of similar anticancer activity but lacking immunostimulatory activity. Second, the compounds of the present invention can offset the immunosuppressive effects of other drugs or treatment regimens when used in combination therapies.
  • PKC enzymes are implicated in a variety of cellular responses which may be involved in the activity of the bryostatins.
  • Example 6 describes another protein kinase C assay which can be used to assess the binding affinities of compounds of the invention for binding to the C1B domain 'of PKC ⁇ .
  • PKC ⁇ appears to be protected against down regulation by bryostatin 1.
  • Overexpression of PKC ⁇ inhibits tumor cell growth and induces cellular apoptosis, whereas depleting cells of PKC ⁇ can cause tumor promotion. Accordingly, this assay provides useful binding data for assessing potential anticancer activity.
  • Another 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, 1989).
  • This 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, Monks et al.,
  • ED 50 or GI 50
  • ED 50 is the molar concentration of compound effective to reduce cell growth by 50%.
  • Compounds with lower ED 50 values tend to have greater anticancer activities than compounds with higher ED 50 values.
  • Example 7 describes a P388 murine lymphocytic leukemia cell assay which measures the ability of compounds of the invention to inhibit cellular growth.
  • the invention includes a method of inhibiting growth, or proliferation, of a cancer cell, or enhancing the effectiveness of other drugs.
  • a cancer cell is contacted with a bryostatin analogue compound in accordance with the invention in an amount effective to inhibit growth or proliferation of the cell
  • the invention includes a method of treating cancer in a mammalian subject, especially humans.
  • a bryostatin analogue compound in accordance with the invention is administered to the subject in an amount effective to inhibit growth of the cancer in the patient.
  • a compound of the invention is administered to a subject in need thereof, in an amount herapeutically effective for bolstering of the immune system predisposed toward apoptosis
  • compositions and methods of the present invention have particular utility in the area of human and veterinary therapeutics.
  • administered dosages will be effective to deliver picomolar to micromolar concentrations of the therapeutic composition to the target site.
  • nanomolar to micromolar concentration at the target site should be adequate for many applications.
  • Appropriate dosages and concentrations will depend on factors such as the particular compound or compounds being administered, the site of intended delivery, and the route of administration, all of which can be derived empirically according to methods well known in the art.
  • Administration of compounds of the invention in an appropriate pharmaceutical form can be carried out by any appropriate mode of administration.
  • administration can be, for example, intravenous, topical, subcutaneous, transocular transcutaneous, intramuscular, oral, intra-joint, parenteral, peritoneal, intranasal, or by inhalation.
  • the formulations may take the fonn of solid, semi-solid, lyophilized powder, or liquid dosage forms, such as, for example, tablets, pills, capsules, powders, sustained-release formulations, solutions, suspensions, emulsions, suppositories, retention enemas, creams, ointments, lotions, aerosols, and the like.
  • the formulation has a unit dosage form suitable for administration of a precise dose.
  • compositions of the invention typically include a conventional pharmaceutical carrier or excipient and may additionally include other medicinal agents, carriers, adjuvants, antioxidants, and the like.
  • the composition may comprise from about 1% to about 75% by weight of one or more compounds of the invention, with the remainder consisting of suitable pharmaceutical excipients.
  • excipients include pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, gelatin, sucrose, magnesium carbonate, for example.
  • Appropriate excipients can be tailored to the particular composition and route of administration by methods well known in the art, e.g., (Gennaro, 1990).
  • the compositions will take the form of a pill, tablet or capsule.
  • the composition will contain, along with active drug, a diluent such as lactose, sucrose, dicalcium phosphate, and/or other material, a disintegrant such as starch or derivatives thereof; a lubricant such as magnesium stearate and the like; and a binder such a starch, gum acacia, polyvinylpynolidone, gelatin, cellulose and/or derivatives thereof.
  • the compounds of the mvention may also be formulated into a suppository comprising, for example, about 0.5% to about 50% of a compound of the invention, disposed in a polyethylene glycol (PEG) carrier (e.g., PEG 1000 [96%] and PEG 4000 [4%]).
  • PEG polyethylene glycol
  • Liquid compositions can be prepared by dissolving or dispersing compound (e.g., from about
  • aqueous saline aqueous dextrose, glycerol, ethanol and the like
  • a carrier such as, for example, aqueous saline, aqueous dextrose, glycerol, ethanol and the like
  • useful vehicles also include polyoxyethylene sorbitan fatty acid monoesters, such as TWEENTM 80, and polyethoxylated castor oils, such as Cremophor ELTM available from BASF (Wyandotte, MD), as discussed in PCT Publ. No. WO 97/23208 (which is incorporated herein by reference), which can be diluted into conventional saline solutions for intravenous administration.
  • Such liquid compositions are useful for intravenous adniinistration.
  • PET diluent which is a 60/30/10 v/v/v mixture of PEG 400, dehydrated ethanol, and TWEENTM-80.
  • Liquid compositions may also be formulated as retention enemas.
  • the compounds of the invention may also be formulated as liposomes using liposome preparation methods known in the art. Preferably, the liposomes are formulated either as small unilamellar vesicles or as larger vesicles.
  • composition to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, such as, for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate, and antioxidants.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering agents, such as, for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate, and antioxidants.
  • the composition is administered in any suitable format, such as a lotion or a transdermal patch.
  • the composition can be delivered as a dry powder (e.g., Inhale Therapeutics) or in liquid form via a nebulizer.
  • compositions to be administered will, in any event, contain a quantity of one or more compounds of the invention in a phannaceutically effective amount for relief of the condition being treated.
  • the compounds of the invention may also be introduced in a controlled-release form, for long- term delivery of drug to a selected site over a period of several days or weeks.
  • the compound of the invention is incorporated into an implantation device or matrix for delayed or controlled release from the device.
  • the compounds can be incorporated in a biodegradable material, such as a biodegradable molded article or sponge.
  • exemplary biodegradable materials include matrices of collagen, polylactic acid- polyglycolic acid, and the like, hi preparing bryostatin compounds in matrix form, the compounds may be mixed with matrix precursor, which is then crosslinked by covalent or non-covalent means to form the desired matrix. Alternatively, the compound can be diffused into a preformed matrix. Examples of suitable materials for use as polymeric delivery systems have been described e.g., Aprahamian, 1986; Emmanuel, 1987; Friendenstein, 1982; andUchida, 1987.
  • compounds of the invention are administered in a therapeutically effective amount, i.e., a dosage sufficient to effect treatment, which may vary depending on the individual and condition being treated.
  • a therapeutically effective daily dose is from 0.1 ⁇ g kg to 100 mg/kg of body weight per day of drug.
  • daily dosages of from about 1 ⁇ g/kg and about 1 mg/kg of body weight may be adequate, although dosages greater than or less than this range can also be used. It will be appreciated that the compounds of the invention may be administered in combination
  • the compounds of the invention may be used in combination with other anticancer drugs such as vincristine, cisplatin, ara-C, taxanes, edatrexate, L-buthionine sulfoxide, tiazofurin, gallium nitrate, doxorubicin, etoposide, podophyllotoxins, cyclophosphamide, camptothecins, dolastatin, and auristatin-PE, for example, and may also be used in combination with radiation therapy.
  • the combination therapy entails co-administration of an agent selected from: ara-C, taxol, cisplatin and vincristine.
  • Tetrahydrofuran (THF) and diethyl ether (Et 2 0) were distilled from sodium benzophenone ketyl under a nitrogen atmosphere.
  • Benzene, dichloromethane (CH 2 C1 2 ), acetonitrile (CH 3 CN), triethylamine (Et 3 N) and pyridine were distilled from calcium hydride under a nitrogen atmosphere.
  • Chloroform (CHC1 3 ), carbon tetrachloride (CC1 4 ) and deuterated NMR solvents were dried over 1/16" bead 4A molecular sieves.
  • 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.
  • the prefix app is occasionally applied in cases where the true signal multiplicity was unresolved and br indicates the signal in question was broadened.
  • Proton decoupled 13 C NMR spectra are reported in ppm ( ⁇ ) relative to residual CHC1 ( ⁇ 77.25) unless noted otherwise.
  • Infrared spectra were recorded on a Perkin-Elmer 1600 series FTIR using samples prepared as thin films between salt plates.
  • High-resolution mass spectra HRMS
  • FAB Fast Atom Bombardment
  • Combustion analyses were performed by Desert Analytics, Arlington, AZ, 85719 and optical rotations were measured on a Jasco DIP-1000 digital polarimeter.
  • Flash chromatography was performed using E. Merck silica gel 60 (240-400 mesh) according to the protocol of Still et al. (1978). 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.
  • Example 1 Exemplary Precursors 1A.
  • the crude C21 monobenzoate was taken up in 45 mL CH 2 C1 2 and treated with solid 1,1,1- triacetoxy-l,l-dihydro-l,2-benziodoxol-3(lH)-one (Dess-Martin periodinane or DMP, 1.78 g, 4.20 mmol) at rt.
  • the solution was stined for 10 h at rt after which a second portion (0.50 g, 1.18 mmol) of DMP was added.
  • the opaque white mixture was stined for another 1.5 h and quenched with 30 mL sat. NaHC0 3 /Na 2 S 2 0 3 .
  • the two phase system was vigorously stined until the organic layer cleared ( ⁇ 25 min).
  • the suspension was stined for 1 h, then the temperature was slowly raised to rt and 3N NaOH (240 ⁇ L), 30% hydrogen peroxide (100 ⁇ L) and diethyl ether (400 ⁇ L) was added.
  • the biphasic mixture was refluxed for 1 h and then quenched with water (9 mL) and extracted with ethyl acetate (4 x 10 mL).
  • the combined organics were washed with brine (10 mL), dried over sodium sulfate and the solvent was removed in vacuo. Chromatography on silica gel (12.5% EtOAc/ hexane) afforded the crude homoallylic alcohol as a yellow oil.
  • Undesired isomer 509a can be recycled by the following procedure: To 509a (320.8mg, 0.98 mmol) in methylene chloride (3 mL) was added Dess-Martin Periodinane (707 mg, 1.67 mmol) and stined for 1 h at room temperature. The reaction was diluted with methylene chloride (3 mL), saturated sodium bicarbonate (3 mL) and sodium thiosulfate (3 mL) and stined for 1 h. The layers were separated and the aqueous layer was re-extracted with EtOAc (4 x 5 mL). The combined organics were dried over sodium sulfate and the solvent was removed in vacuo.
  • Example 3 Exemplary Bryostatin Analogues 3A.
  • Formula II C26 Des-Methyl Bryostatin Analogue (702)
  • the crude alcohol was dissolved in CH 2 C1 2 (2 mL) and treated with a single portion of 1,1,1 - triacetoxy-l,l-dihydro-l,2-benziodoxol-3(lH)-one (Dess-Martin periodinane, 117 mg, 0.275 mmol) at room temperature.
  • the mixture was stined for 45 min and quenched with saturated aqueous NaHC0 3 /Na 2 S 2 0 3 (2 mL).
  • the two phase system was vigorously stined until the organic layer has cleared (90 min).
  • the layers were then separated and the aqueous phase was extracted with CH 2 C1 2 (4 x 5 mL).
  • a dihydroxylating stock solution was generated by dissolving (DHQD) 2 AQN (3.6 mg, 0.00425 mmol), K 3 Fe(CN) 6 (425 mg, 1.275 mmol), K 2 C0 3 (175 mg, 1.275 mmol) and K 2 0s0 2 (OH) 4 (0.65 mg,
  • Enal 206.1 (22 mg, 0.03 mmol) was dissolved m acetonitrile (2 mL) and water (205 ⁇ L) at room temperature. 48% aqueous HF (388 ⁇ L, 12.1 mmol) was added dropwise and the resulting clear solution was stined at room temperature for 75 mm. and was then quenched with a saturated aqueous solution of sodium bicarbonate (15 mL) and water (3 mL). The mixture was extracted with ethyl acetate (5 x 10 mL). The combined organic layers were dried over sodium sulfate, the solution was decanted and then the solvent was removed m vacuo to provide a crude diol which was taken immediately to the next step.
  • Ester 803.1 (27 mg, 0.0233 mmol) in wet methylene chloride (2 mL) was treated with DDQ (11 mg, 0.0466 mmol) and stined for 1 h.
  • the mixture was directly purified by silica gel to give the expected alcohol silyl ether product of Formula 804.
  • This silyl ether in acetonitrile-water (10:1, 1.5 mL) was treated with aqueous HF (100 :L, 48 %). After 4 h, the mixture was neutralized with aqueous sodium bicarbonate. The aqueous layer was extracted with ethyl acetate. The combined organic layer was dried over sodium sulfate.
  • the crude hydroxy acid product 805.1 was used for the next step.
  • R 8 is H, R 20 is -O-CO-C Hi j .
  • R 21 CH-CO,Me and R 26 is Me)
  • IR (film) 3256.8, 2915.8, 2845.2, 1746.4, 1722.9, 1158.4, 1029.0, 864.4, 793.8 cm “1 .
  • the crude macrocycle from the preceding step was dissolved in ethyl acetate (2.6 mL) and Pd(OH)2/C (2.4 mg, 20% wt. on carbon) was added.
  • the resulting suspension was evacuated and refilled with 1 Atm. hydrogen gas (x5) and was vigorously stined under a hydrogen atmosphere for 3 hours.
  • Alcohol 303.1 (10 mg, 0.02 mmol) was dissolved in 1.1 mL CH 3 CN / H 2 0 (9:1) and treated with 48% aqueous HF (200 ⁇ L, 300 mol % excess) at rt. The resulting mixture was stined for 1 h, quenched with sat. NaHC0 3 and diluted with 10 mL EtOAc. The aqueous layer was separated and extracted with EtOAc (2 x). The combined organics were dried over Na 2 S0 and concentrated in vacuo to afford crude hemiketal enal 304.1 as a colorless oil.
  • Carboxylic acid 407 (Example 2B, 15 mg, 0.03 mmol) and Et 3 N (16.5 ⁇ L , 0.12 mmol) were dissolved in 300 ⁇ L toluene and treated with 2,4,6-trichlorobenzoylchloride (4.8 ⁇ L, 0.03 mmol) dropwise at rt. After 1 h at rt, a toluene solution of freshly prepared 304.1 and 4-dimethylammopyridine
  • Heptenoic acid (6 mg, 0.05 mmol) and Et 3 N (21 ⁇ L, 0.16 mmol) were dissolved in 600 ⁇ L toluene and treated with 2,4,6-trichlorobenzoylchloride (7.0 ⁇ L, 0.05 mmol) dropwise at rt.
  • 2,4,6-trichlorobenzoylchloride 7.0 ⁇ L, 0.05 mmol
  • the intermediate alcohol (11 mg, 0.02 mmol) was dissolved in 1.0 mL CH 3 CN / H 2 0 (9:1) and treated with 48% aqueous HF (200 ⁇ L, 300 mol% excess) at rt. The resulting mixture was stined for 1 h, quenched with sat. NaHC0 3 and diluted with 10 mL EtOAc. The aqueous layer was separated and extracted with EtOAc (2x). The combined organics were dried over Na 2 S0 4 and concentrated in vacuo to afford the conesponding crude hemiketal enal of Formula 303a as a colorless oil.
  • Carboxylic acid 407 (21 mg, 0.04 mmol) and Et 3 N (19 ⁇ L , 0.12 mmol) were dissolved in 400 ⁇ L toluene and treated with 2,4,6-trichlorobenzoylchloride (6.0 ⁇ L, 0.04 mmol) dropwise at rt. After 1 h at rt, a toluene solution of freshly prepared C20 heptenoate hemiketal enal (16 mg, 0.03 mmol) and 4- dimethylaminopyridine (17 mg, 0.13 mmol) was added gradually and stining was continued for 40 min.
  • Carboxylic acid 6 (6 mg, 0.01 mmol) and Et 3 N (6 ⁇ L , 0.04 mmol) were dissolved in 300 ⁇ L toluene and treated with 2,4,6-frichlorobenzoylchloride (2.0 ⁇ L, 0.01 mmol) dropwise at rt. After 1 h at rt, a toluene solution of freshly prepared enal 44 and 4-dimethylaminopyridine (5 mg, 0.04 mmol) was added gradually and stirring was continued for 40 min. The crude mixture was pipetted directly onto a column of silica gel and the product eluted with 20% EtOAc / hexanes to provide ester-enal 46 as a colorless oil (9 mg, 90%).
  • ester-enal 46 (8.0 mg, 0.001 mmol) in THF (0.5 mL) was added 70% HF/ pyridine (0.3 mL, 0.3 mmol) and stined for 2 hours. The reaction was then quenched with a saturated solution of sodium bicarbonate. The biphasic mixture was extracted with ethyl acetate (x4) and the combined organics were dried over sodium sulfate. The solvent was removed in vacuo to provide crude macrocycle.
  • Enal 45 was prepared following the procedure for compound 111 in Example 1C except that benzoic acid was substituted for octanoic acid, to form the conesponding protected benzoate product.
  • Carboxylic acid 6 (6 mg, 0.01 mmol) and Et 3 N (6 ⁇ L , 0.04 mmol) were dissolved in 300 ⁇ L toluene and treated with 2,4,6-frichlorobenzoylchloride (2 ⁇ L, 0.01 mmol) dropwise at rt. After 1 h at rt, a toluene solution of freshly prepared 45 and 4-dimethylaminopyridine (5 mg, 0.01 mmol) was added gradually and stirring was continued for 40 min. The crude mixture was pipetted directly onto a column of silica gel and the product eluted with 20% EtOAc / hexanes to provide the expected ester product as a colorless oil (8 mg, 89%).
  • An assay buffer solution is prepared by the addition of TRIS (IM, pH 7.4, 1 mL), KC1 (IM, 2 mL), CaCl 2 (0.1M, 30 ⁇ L), bovine serum albumin (40 mg), diluted to 20 mL with deionized water and stored on ice.
  • Phosphatidyl serine vesicles are prepared by the addition of phosphatidyl serine (10 mg/mL in chloroform, 0.4 mL) to a glass test tube followed by removal of the chloroform under a stream of nitrogen (5 min). To this viscous liquid is added a portion of the prepared assay buffer (4 mL) and the resulting mixture is then transfened to a plastic tube with washing.
  • the resulting solution is stored over ice.
  • PKC is prepared by addition of cooled assay buffer (10 mL) to PKC (25 ⁇ L) purified from rat brain by the method of Mochly-Rosen and Koshland (1986) and then stored on ice. Stock solutions of compounds are diluted with absolute ethanol in glass in serial fashion.
  • Each plastic assay incubation tube is made to contain prepared phosphatidyl serine vesicles (60 ⁇ L), prepared PKC solution (200 ⁇ L) and analogue (0-20 ⁇ L) plus EtOH (20-0 ⁇ L) for a total volume of 20 ⁇ L).
  • phosphatidyl serine vesicles 60 ⁇ L
  • prepared PKC solution 200 ⁇ L
  • analogue 0.-20 ⁇ L
  • EtOH 20-0 ⁇ L
  • EtOH 20-0 ⁇ L
  • tritiated phorbol 12,13-dibutyrate (PDBU) (30 nM, 20 ⁇ L) is added to each tube.
  • the assay is carried out using 7-10 analogue concentrations, each in triplicate.
  • Non-specific binding is measured in 1-3 tubes by the substitution of phorbol myristate acetate (PMA) (1 mM, 5 ⁇ L) and EtOH (15 ⁇ L) for the analogue / EtOH combination.
  • PMA phorbol
  • the tubes are incubated at 37°C for 90 min. and then put on ice for 5 min. Each tube is then filtered separately through a pre-soaked filter disc. Each tube is rinsed with cold 20 mM TRIS buffer (500 ⁇ L) and the rinseate is added to the filter. The filter is subsequently rinsed with cold 20 mM TRIS buffer (5 mL) dropwise. The filters are then put in separate scintillation vials and Universol ® scintillation fluid is added (3 mL). The filters are immediately counted in a scintillation counter (Beckman LS 6000SC). Counts per minute are averaged among three trials at each concentration.
  • a scintillation counter Beckman LS 6000SC
  • the data is then plotted using a least squares fit algorithm with the Macintosh version of Kaleidagraph ® (Abelbeck Software) and an IC 50 (defined as the concenfration of analogue required to displace half of the specific PDBU binding to PKC) is calculated.
  • the K d of [H 3 ]-PDBu was determined under identical conditions to be 1.55 nM.
  • PKC ⁇ -ClB assay All aspects of the PKC ⁇ -ClB assay are identical to the PKC isozyme mix assay from Example 5 except the following features:
  • assay buffer is made without CaCl 2 .
  • PKC ⁇ -ClB 200 ⁇ g, 34.14 nmol
  • ZnCl 2 5 mM, 40 ⁇ L
  • the resulting solution is allowed to stand at 4°C for 10 min.
  • An aliquot (10 ⁇ L) of this solution is diluted to 2 mL with deionized water.
  • Example 7 P388 Murine Lymphocytic Leukemia Cell Assay
  • Cells from a P388 cell line (CellGate, Inc., Sunnyvale, CA) are grown in RPMI 1640 cell medium containing fetal calf serum (10%), L-glutamine, penicillin, streptomycin and are split twice weekly. All compounds are first diluted with DMSO. Later serial dilutions are done with a phosphate buffer solution (HYQ DPBS modified phosphate buffered saline). All dilutions are done in glass vials and the final DMSO concentration is always below 0.5% by volume. Final two-fold dilutions are done in a 96 well plate using cell media so that each well contains 50 ⁇ L.
  • a phosphate buffer solution HYQ DPBS modified phosphate buffered saline
  • All compounds are assayed in quadruplicate over 12 concentrations.
  • Cell concentration is measured using a hemacytometer and the final cell concenfration is adjusted to 1 x 10 4 cells/mL with cell medium.
  • the resulting solution of cells (50 ⁇ L) is then added to each well and the plates are incubated for 5 days in a 37°C, 5% C0 2 , humidified incubator (Sanyo C0 2 incubator).
  • MTT solution (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, 10 ⁇ L) is then added to each well and the plates are re-incubated under identical conditions for 2 h.
  • Anticancer data were obtained in vitro for C26 desmethyl bryostatin analogue 702.1 (Example 3A) tested against a spectrum of different NCI human cancer cell-lines associated with various cancer conditions. The results are shown in Table 3. Data obtained with Bryostatin-1 are included for comparison. Growth inhibition (GI50) values are expressed as the log of molar concenfration at half- maximum inhibition. As can be seen, the C26 desmethyl compound was at least as potent, on average, as bryostatin-1 for all cell groups tested.
  • the C26 desmethyl compound was more active than bryostatin-1 by more than 2 orders of magnitude for several cell lines: K-562 and MOLT-4 (leukemia), NCI-H460 (NSC lung), HCC-2998 (colon), TK-10 (renal), and MDA-MB-435 (breast). These results are significant and surprising since the C27 methyl group attached to C26 was previously believed to be necessary for activity. Table 3
  • reaction can be quenched with saturated NH 4 C1 solution.
  • aqueous layer was then extracted with Et 2 0 (3x) and the combined layers were dried over MgS0 4 and concentrated in vacuo.
  • the resulting oil was purified by flash chromatography (EtOAc 20%) to provide a mixture of 6a & 6b, the methyl and ⁇ -methoxybenzyl esters.
  • aldehyde 10 (164 mg, 0.5 mmol) was added dropwise and stining was continued for 15 minutes.
  • the reaction was quenched by addition of saturated NH 4 C1 and the mixture was warmed to rt and diluted with Et 2 0. The mixture was diluted with Et 2 0 and the layers separated. The aqueous layer was then extracted with EtOAc (3x). The combined organics were washed with brine, dried over MgS0 4 , and concentrated in vacuo. Flash chromatography on silica gel (EtOAc/hexane 1/2) provided 12 (169 mg, 87 %). The diastereoselectivity was determined to be 81%, favoring the desired isomer, after coupling with acid chloride 7 and cyclization to pyranone 14.
  • the layers were separated, and the aqueous phase extracted three times with ethyl acetate.
  • the combined organic phases were concentrated under reduced pressure.
  • the resultant oil was redissolved in 3 ml 2:1 tetrahydrofuran/water, to which was added sodium periodate (0.052 g, 0.243 mmol).
  • the reaction mixture was stined 6 hours at rt before being diluted with water and ethyl acetate.
  • the layers were separated, and the aqueous phase extracted three times with ethyl acetate, and the combined organic phases concentrated under reduced pressure.
  • the separated aqueous layer was extracted four times with ethyl acetate, and the combined organics were dried over Na 2 S0 4 and concentrated under reduced pressure to afford the crude allylic alcohol.
  • This moderately stable oil was reacted further without purification.
  • the crude allylic alcohol was dissolved in 6 ml CH 2 Cl 2 /MeOH (2:1) and cooled to 0 °C before being treated with solid NaHC0 3 (0.042 g, 0.5 mmol). Purified ⁇ r ⁇ -chloroperoxybenzoic acid (0.046 g, 0.370 mmol) was added in a single portion and the reaction mixture was stined for 30 minutes, then warmed to rt over a period of 15 minutes.
  • the filtrate was evaporated and taken up in 8 ml CH 2 C1 2 and treated with solid 1,1,1-friacetoxy- l,l-dihydro-l,2-benziodoxol-3(lH)-one (Dess-Martin periodinane, 113 mg, 0.267 mmol) at rt.
  • the solution was stined for 4 hours at rt after which a second portion (113 mg, 0.267 mmol) of DMP was added.
  • the opaque white mixture was stined for another 1.5 hours and quenched with 4 ml saturated NaHC0 3 / Na 2 S 2 0 3 .
  • the layers were separated and the aquous phase was extracted with CH 2 C1 2 (2 x).
  • the solution was stined for 30 minutes and treated with a solution of freshly distilled OHCC0 2 Me (88 mg, 0.74 mmol) in 5 ml THF, kept at -78 °C for 30 minutes and quenched with 3 ml saturated NH 4 C1.
  • the reaction mixture was brought to rt and diluted with 200 ml EtOAc.
  • the organic layer was washed with H 2 0 (2x) and brine, dried over Na 2 S0 and concentrated in vacuo.
  • the crude residue was chromatographed on silica gel (EtOAc/hexanes 35/65) to afford the aldol product (319 mg, 88%) as an inconsequential mixture of diastereomers.
  • Octanoic acid (93 mg, 0.64 mmol) and Et 3 N (117 ⁇ l, 0.88 mmol) were dissolved in 8 ml toluene and treated with 2,4,6-frichlorobenzoylchloride (92 ⁇ l, 0.59 mmol) dropwise at rt.
  • a toluene solution (5 ml) of freshly prepared alcohol was added gradually via syringe and stirring was continued for 1 h.
  • the reaction mixture was quenched with 10 ml saturated NaHC0 3 , diluted with EtOAc and washed successively with saturated NH 4 C1 and brine.
  • the reaction was stined 30 min at -78 °C and the cold bath removed.
  • MTBE 500 mL was added when the reaction reached -30 °C followed by aq. citric acid (87 g, 0.41 mol in 500 mL H 2 0).
  • the organic layer was collected and the aqueous layer was extracted with MTBE (200 mL).
  • the combined organic layers were washed with brine (2 x 400 mL), dried over Na 2 S0 4 , filtered, and concentrated under reduced pressure to a light orange oil.
  • the oil was dissolved in EtOAc and filtered through silica.
  • the filtrate was concenfrated under reduced pressure to yield 49.7 g of a light yellow oil which was used without further purification.
  • the resulting mixture was stined 1 h at -78 °C and NEt 3 (250 mL, 1.79 mol) was added dropwise via addition funnel over 15 min to keep T ⁇ -60 °C.
  • the cold bath was removed and the reaction was allowed to warm to -30 °C and then poured into a mixture of H 2 0 (500 mL) and CH 2 C1 2 (800 mL).
  • the resulting organic layer was collected and washed with H 2 0 (500 mL), satd. aq. NH 4 C1 (2 x 400 mL), satd. aq. NaHC0 3 (500 mL), and brine (500 mL).
  • aldehyde 14 (0.948 g, 2.2 mmol dissolved in Et 2 0 (22 mL, 0.1 mmol) was added dropwise, via syringe, and the reaction was stined for 2 h at -78°C. The reaction was quenched with a 1.0 M solution of HCl (40 mL) and allowed to warm to rt. The mixture stined vigorously for 19 h and was quenched with sat'd aq. NaHC0 3 (60 mL), diluted with EtOAc (35 mL) and H 2 0 (35 mL).
  • K 2 Os0 2 (OH) 4 (2.0 mg, 0.0054 mmol), (DHQD) 2 PYR (12.0 mg, 0.0136 mmol), K 3 Fe(CN) 6 (1.34 g, 4.07 mmol), and K 2 C0 3 (563 mg, 4.07 mmol) were combined in a round-bottom flask, to which was added 6.75 mL of H 2 0 and 6.75 mL of tBuOH. The two-phase system was vigorously stined at rt for 2h.
  • Enal 16 (300 mg, .646 mmol) was cooled to 0 °C, and a 6.4 mL aliquot of the stock solution was added.
  • the yellow-orange colored reaction mixture was stined at 0-4 °C for 60h (in cold room). After diluting with water (50 mL) the aqueous phase was extracted with ethyl acetate (3 x 250 mL). The combined organic phases were dried over MgS0 4 , and then filtered. The solvents were removed in vacuo. Purification via column chromatography (silica gel, hexane/ethyl acetate 1:9) yielded diol 17 (233 mg, 72%) as a 2.5:1 inseparable mixture of diastereomers.
  • a 0.5M stock solution of 1:3 TBSCkimidazole was prepared by dissolving TBSCl (754 mg, 5.0 mmol) and imidazole (1.02 g, 15.0 mmol) in CH 2 C1 2 (10.0 mL). A total of 9 eq. of this stock solution were added to the reaction over 6 hrs. (added in 3 eq. aliquots, 2 hrs. apart). Reaction was quenched with sat. aq. NH 4 C1 and the aqueous phase was exfracted with EtOAc (x3mL). The combined organic phases were dried over Na 2 S0 , filtered, and concentrated in vacuo. Purification via column chromatography (silica gel, Hexanes:EtOAc, 75:25) revealed pure product (5.6 mg, 47% over 2 steps).
  • reaction mixture was stined for lh and then directly poured onto a column (silica gel, hexane/ethyl acetate 85:15) and diluted with this solvent mixture to afford 24 mg (87%) of ester 32 as a colorless oil.

Abstract

La présente invention concerne des composés biologiquement actifs apparentés à la famille de composés de bryostatine, qui comportent des domaines espaceurs simplifiés et/ou des domaines de reconnaissance améliorés, ainsi que des procédés de préparation et d'utilisation de ces derniers.
PCT/US2003/004599 2002-02-15 2003-02-14 Analogues de bryostatine, procedes de synthese et utilisations de ces derniers WO2003070719A1 (fr)

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Cited By (2)

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Publication number Priority date Publication date Assignee Title
US8735609B2 (en) 2007-10-19 2014-05-27 The Board Of Trustees Of The Leland Stanford Junior University Bryostatin analogues, synthetic methods and uses
US9816066B2 (en) 2012-04-24 2017-11-14 The Regents Of The University Of California Method for delivery of small molecules and proteins across the cell wall of algae using molecular transporters

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8735609B2 (en) 2007-10-19 2014-05-27 The Board Of Trustees Of The Leland Stanford Junior University Bryostatin analogues, synthetic methods and uses
US9816066B2 (en) 2012-04-24 2017-11-14 The Regents Of The University Of California Method for delivery of small molecules and proteins across the cell wall of algae using molecular transporters

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