Classifications

• • 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
  • C07D493/06—Peri-condensed systems
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D265/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom and one oxygen atom as the only ring hetero atoms
  • C07D265/04—1,3-Oxazines; Hydrogenated 1,3-oxazines
  • C07D265/12—1,3-Oxazines; Hydrogenated 1,3-oxazines condensed with carbocyclic rings or ring systems
• • 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
  • C07D309/12—Oxygen atoms only hydrogen atoms and one oxygen atom directly attached to ring carbon atoms, e.g. tetrahydropyranyl ethers
• • 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
  • C07D319/04—1,3-Dioxanes; Hydrogenated 1,3-dioxanes
  • C07D319/08—1,3-Dioxanes; Hydrogenated 1,3-dioxanes condensed with carbocyclic rings or ring systems
• • 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

Definitions

• the present invention concerns biologically active compounds related to the bryostatin family of compounds, and to methods of preparing and utilizing the same Introduction
• Cancer is a major cause of death in the developed count ⁇ es, with more than 500,000 human fatalities occurring annually in the United States Cancers are generally the result of the transfoiTtiation of normal cells into modified cells that proliferate excessively, leading to the formation of abnormal tissues or cell populations
• cell proliferation is accompanied by dissemination (metastasis) of malignant cells to other parts of the body which spawn new cancerous growths 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
• 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 m 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 d ⁇ -(2-chloroethyl)-am ⁇ no groups that bind covalently to
• 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 ,
• 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 (DeVnes, 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.
• R 20 is H, OH, or O 2 CR';
• R 21 CR a R b or R 2 ' represents independent moieties R c and R d where: 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 , 2 or 3; R 26 is H, OH or R'; each R' being independently selected from the group: alkyl, alkenyl or alkynyl, or aryl, heteroaryl, aralkyl or heteroaralkyl; and 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 the pharmaceutically acceptable salt thereof.
• a preferred 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).
• L contains a terminal carbon atom that, together with the carbon atom corresponding to C17 in the native bryostatin structure, forms a trans olefin.
• Another aspect of the invention concerns the simplified bryostatin analogues represented by Formulae II - V:
• R 3 is H, OH or a protecting group
• R 8 is selected from the group: H, OH, R', -(CH 2 ) n O(0)CR' or (CH 2 ) n C0 2 -haloalkyl where n is 0, 1, 2, 3, 4 or 5;
• R 9 is H or OH
• R 20 , R 2 ', R 26 and R' are as defined above with respect to Formula I; p is 1 , 2, 3 or 4; and
• X is C, O, S or N-R c where R e is COH, C0 2 R' or S0 2 R', and the pharmaceutically acceptable salts thereof.
• the invention relates to the C26 des-methyl analogue of Formula Ila
• Still another aspect of the invention relates to the C26 des-methyl homologues of the native bryostatins, as illustrated in Formula VI:
• 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
• 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 estenfication and macrotrasacetyhzation of a protected recognition domain with a protected linker synthon, followed by deprotection
• Particularly preferred is reduction of a C26 OBn protected predursor to give the corresponding C26 des-methyl bryostatin analogue
• the invention also includes pharmaceutical compositions containing one or more compounds m accordance with the invention
• the invention includes a method of inhibiting growth, or proliferation, of a cancer cell
• 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 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 ⁇ ng, 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-y
• “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 ⁇ ng structure of carbon atoms with no heteroatoms in the r ⁇ ng(s) Examples are phenyl, naphthyl, anthracyl, and phenanthryl Preferred examples are phenyl and napthyl
• heteroaryl is used herein to denote an aromatic ⁇ ng 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 nng structures
• non-carbon atoms such as oxygen, nitrogen, and sulfur
• Examples are furanyl, pyranyl, thienyl, lmidazyl, pyrrolyl, py ⁇ dyl, pyrazolyl, pyrazinyl, py ⁇ midinyl, indolyl, quinolyl, isoquinolyl, quinoxalyl, and quinazohnyl
• Preferred examples are furanyl, lmidazyl, pyranyl, pyrrolyl, and py ⁇ dyl
• 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 alkynyl 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 prefe ⁇ ed 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
• mice is intended to have its conventional meaning Examples include humans, mice, rats, guinea pigs, horses, dogs, cats, sheep, cows, etc.
• treatment means any treatment of a disease in a mammal, including • preventing the disease, that is, causing the clinical symptoms of the disease not to develop,
• an 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 pharmacophonc 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 arrows 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 refe ⁇ ed 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
• the linker region of of the bryostatin family can be varied significantly without eliminating activity
• the compounds of the present invention include a linker moiety L, which is a linear, cyclic, or polycychc 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
• L may consist solely of a linear chain of atoms that links C17 via Cl to C25, or alteratively, may contain one or more ⁇ ng structures which help link C17 via Cl to C25
• the compounds of the invention differ from known bryostatins and bryostatin analogues in that the present compounds contain a primary alcohol moiety at C26, l e , the present analogues lack a methyl group corresponding to the C27 methyl that is ordinarily present in naturally occurring bryostatins
• the C27 methyl moiety was previously believed to limit rotation of the C26 alcohol and contribute to PKC binding affinity, it has been found that this structural modification can significantly increase PKC binding and also increases efficacy against cancer cells
• Other modifications of R 26 are provided to further modulate these characte ⁇ stics, as are the C26 des-methyl homologues of the native 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 is H, OH or a protecting group
• R 8 is selected from the group H, OH, R', -(CH 2 ) n O(0)CR' or (CH 2 ) n C0 2 -haloalkyl, where n is 0, 1, 2, 3, 4 or 5,
• R 20 , R 21 , R 26 and R' are as defined above with respect to Formula I,
• X is C, O, S or N-R e where R e is a group that stabilizes the nitrogen's lone pair of electrons, such as COH, C0 2 R' or S0 2 R', and the pharmaceutically acceptable salts thereof Excluded from the present invention are the analogues of Formula 1998a (where R 3 is H or OH) and the analogue of Formula 1998b
• Formula Ila Formula IVa are also referred to as "C26 des-methyl", notwithstanding that the structures corresponding to L (in Formula I) or the corresponding 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 Synthetic Reaction Parameters
• 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 chlo ⁇ de (or dichloromethane), diethyl ether, methanol, py ⁇ dine and the like]
• solvents used in the reactions of the present invention are inert organic solvents
• protecting group refers to any group which when bound to a functional group such as one or more hydroxyl, thiol, amino or carboxyl groups of the compounds (including intermediates thereof) prevents reactions from occur ⁇ ng at these groups and which protecting group can be removed by conventional chemical or enzymatic steps to reestablish the hydroxyl, thiol, amino or carboxyl group
• the particular removable blocking group employed is not c ⁇ tical and preferred 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 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 descnbed herein take place at atmosphe ⁇ c 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)
• the 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 pu ⁇ fication procedure such as, for example, distillation, filtration, extraction, crystallization, column chromatography, thin-layer chromatography or thick-layer chromatography, or a combination of these procedures
• the compounds of the invention may be produced by any methods available in the art, inducing chemical and biological (e g , recombinant and in vitro enzyme-catalyzed) methods
• the present invention provides a convergent synthesis in which subunits primarily corresponding to the recognition and spacer domains are separately prepared and then joined by este ⁇ fication- 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.
• 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 5A illustrates the synthesis of linker synthons for preparing the compounds of
• Reaction Scheme 5B illustrates the synthesis of linker synthons for preparing the compounds of
• Reaction Scheme 6 illusttates the synthesis of linker synthons for preparing the compounds of Formula IV.
• 606 OTBS OSEM Reaction Scheme 7A illustrates the synthesis of the compounds of Formula II.
• Reaction Scheme 7B illustrates the synthesis of the compounds of Formula III.
• Reaction Scheme 8 illusttates synthesis of the Compounds of Formula IV, particularly where
• R is methyl, the C26 des-methyl analogues of Formula IV being obtained by like synthesis.
• 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 6-(Ter/-butyld ⁇ methyls ⁇ lylhydroxy)-5-d ⁇ methylhexane-2,4-d ⁇ one (101, Example IB) is stirred with 2 equivalents of LDA (lithium dnsopropylamine) in THF (tetrahydrofuran), followed by addition of 0 9 equivalents of 3R-p-methoxybenzyl-4R-benzylhydroxypentane-l-al (102, Example 1A) to afford diaste ⁇ omenc aldol mixture 103 after suitable purification To 103 is then added a catalytic amount of p-methylphenylsulfonic acid (p-TsOH) with stirring at room temperature followed by base quenching to produce pyranone condensation product 104 as a mixture of ⁇ and ⁇ -isomers at C23 (104a and 104b) The ⁇ -isomer (104a) is separated from the ⁇ -isomer and is reacted with NaBH 4 in the presence of CeC
• 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 carboxyhc acids
• the substituent e g , a desired C20 ester substituent
• the C20 octanoate substituent in synthon 111 can be replaced with an acetyl group by first protecting the base labile aldehyde group using t ⁇ methyl orthorformate to form the dimethyl acetal
• the C20 octanoate ester can then be cleaved using a basic solution, such as
• Addition of backbone atoms corresponding to C15 and C16 of the bryostatin backbone to 205 can be accomplished in four steps First, 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 t ⁇ ethylamine and 4- dimethylaminopy ⁇ dine (DMAP), to afford a diastereome ⁇ c mixture of homoallyhc C17 acetates This product mixture can be oxidized using N-methylmorphohne N-oxide and osmium tetraoxide, followed by neutralization with sodium bicarbonate After extraction, the residue is reacted with lead tettaacetate, 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
• 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 nngs that are analogous to the A and B ⁇ ngs of bryostatin, but lack the naturally occurring substituents at C7, C8, C9, and C13
• 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 transacerylization 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- ⁇ ng-like structure but lack an A- ⁇ ng, are prepared, for example, as illustrated with reference to Reaction Schemes 5A through 5C Examples 2C and 2D describe methods for prepa ⁇ ng synthons
• R 8 is a tert butyl group attached to C9
• the /-BuLi reactant can be replaced by R'Li to generate the corresponding 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 corresponding 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
• R 8 is
• the compounds of Formula IV can be made from pharmacophonc 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 dicarboxyhc acid (succinic anhyd ⁇ de) to give formula 903
• 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 descnbed in Example 1C
• Compounds of the invention having a naturally occurring bryostatin backbone e g , including a naturally occu ⁇ ng linker region
• 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 t ⁇ ethylamine, 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 transacetyhzation in step h, and saturation of the double bond upon removal of the benzyl group by hydrogenolysis in step i
• Synthesis of a C20 my ⁇ state ester analogue 48 (14 carbon atom chain) is lllustratred with reference to Reaction Scheme 11 and descnbed in Example 4C Reaction Scheme 11 and Example 4D descnbes synthesis of a bryostatin analogue containing an aryl ester group (benzoate) at
• a C19,C26 hydroxyl-protected, C26 des-methyl bryostatin recognition domaine precursor and an optionally protected linker synthon are este ⁇ fied, macrottansacetylated and de-protected to give the corresponding 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 corresponding compound of Formula I
• Serine is substituted for threomne m a Masamune's C17-C26 southern bryostatin synthesis to yield the corresponding C26 des-methyl sulfone, which in turn is employed in synthesis of a C26 desmethyl bryostatin homologue
• a compound of Formula I-VI is contacted with a pharmaceutically acceptable acid to form the corresponding acid addition salt
• a pharmaceutically acceptable acid addition salt of Formula I-VI is contacted with a base to form the corresponding compound of Formula I-VI
• R 26 is H
• R 26 is H
• X is oxygen
• R 20 is 0 2 CR'
• R 20 is 0 2 CR' and R' is alkyl (preferably about C 7 -C 20 alkyl), alkenyl (preferably about C 7 -C 20 alkenyl such as
• 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).
• L be a group having from about 6 to about 14 carbon atoms.
• X is oxygen.
• R 3 is OH, and especially preferred that X is oxygen in the case of Formulae II and III.
• R 20 is 0 2 CR' where R' is alkyl (preferably about C 7 -C 20 alkyl), alkenyl (preferably about C 7 -C 20 alkenyl such as
• R 2 ' 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 ⁇ o alkyl, most preferably lower alkyl such as methyl).
• 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 lnherrent 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), 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
• CLL chronic lymphocytic leukemia
• 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
• non-small cell lung cancer The compounds appear to operate by a mechanism distinct from the mechanisms of other
• 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, rumor necrosis factors, mterleukins, mterferons, and the like
• Effective dosages may be comparable to those for anticancer uses, and can be optimized with the aid of va ⁇ ous 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 prophyllactically, e g , for subjects who are about to undergo anticancer therapies, as well as therapeutically, e g , for subjects suffering from microbial infection, bum victims, subjects with diabetes, anemia, radiation treatment, or anticancer chemotherapy
• 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
• any assay protocol that is predictive of activity in vivo
• a variety of convenient assay protocols are available that are generally predictive of anticancer activity in vivo
• anticancer activity of compounds of the invention can be assessed using the protein kinase C assay detailed in Example 5
• K values are determined for analogues based on competition with radiolabeled phorbol 12,13-d ⁇ butyrate for binding to a mixture of PKC lsoenzymes
• PKC enzymes are implicated in a variety of cellular responses which may be involved in the activity of the bryostatins
• Example 6 descnbes 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 vanety of tumor types (Grever et al , 1992, Monks et al , 1991), such as leukemia, non-small cell lung, colon, central nervous system (CNS), melanoma, ovanan, renal, prostate, and breast cancers
• Antitumor activites can be expressed in terms of ED 50 (or GI 50 ), where 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 desc ⁇ bes a P388 munne 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 aqdministered 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 concenttations of the therapeutic composition to the target site
• nanomolar will be effective to deliver picomolar to micromolar concenttations of the therapeutic composition
• Administration of compounds of the invention in an appropnate pharmaceutical form can be carried out by any approp ⁇ ate mode of administration
• administration can be, for example, intravenous, topical, subcutaneous, ttansocular ttanscutaneous, intramuscular, oral, intta-joint, parenteral, pe ⁇ toneal, lnttanasal, or by inhalation
• the formulations may take the form of solid, semi -solid, lyophihzed powder, or liquid dosage forms, such as, for example, tablets, pills, capsules, powders, sustained-release formulations, solutions, suspensions, emulsions, suppositones, retention enemas, creams, ointments, lotions, aerosols, and the like
• the formulation has a unit dosage form suitable for administration of a precise dose
• Pharmaceutical compositions of the invention typically include a conventional pharmaceutical earner or excipient and may additionally include other medicinal agents, earners, adjuvants, antioxidants, and 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, polyvinylpy ⁇ o done, gelatin, cellulose and/or derivatives thereof
• 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
• binder such as a starch, gum acacia, polyvinylpy ⁇ o done, gelatin, cellulose and/or derivatives thereof
• the compounds of the invention may also be formulated into a suppository compnsing, 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 0 5% to about 20% of final volume), and optional pharmaceutical adjuvants in a carrier, such as, for example, aqueous saline, aqueous dextrose, glycerol, ethanol and the like, to form a solution or suspension
• 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 administration
• the compounds of the invention may also be formulated as liposomes using liposome preparation methods known in the art
• the liposomes are formulated either as small unilamellar vesicles or as larger vesicles
• the 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, ttiethanolamine oleate, and antioxidants
• auxiliary substances such as wetting or emulsifying agents, pH buffering agents, such as, for example, sodium acetate, sorbitan monolaurate, ttiethanolamine oleate, and antioxidants
• the composition is administered in any suitable format, such as a lotion or a ttansdermal patch
• the composition can be delivered as a dry powder (e g , Inhale Therapeutics) or in liquid form via a nebulizer
• composition to be administered will, in any event, contain a quantity of one or more compounds of the invention in a pharmaceutically effective amount for relief of the condition being treated
• the compounds of the invention may also be introduced in a conttolled-re lease form, for long-term delivery of drug to a selected site over a penod 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 matenal, such as a biodegradable molded article or sponge
• Exemplary biodegradable materials include matrices of collagen, polylactic acid- polyglycohc acid, and the like
• the compounds may be mixed with matnx precursor, which is then crosslinked by covalent or non-covalent means to form the desired matnx
• the compound can be diffused into a preformed matnx Examples of suitable matenals for use as polymenc delivery systems have been descnbed e g , Aprahamian, 1986, Emmanuel, 1987, Fnendenstein, 1982, and Uchida, 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
• the compounds of the invention may be administered in combination (I e , together in the same formulation or in separate formulations administered by the same or different routes) with any other anti-cancer regimen deemed appropriate for the patient
• the compounds of the invention may be used in combination with other anticancer drugs such as vincnstine, cisplatin, ara-C, taxanes, edafrexate, L-buthionine sulfoxide, tiazofu ⁇ n, gallium nitrate, doxorubicin, etoposide, podophyllotoxins, cyclophosphamide, camptothecins, dolastatin, and au ⁇ statin-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 vincnstine
• Proton ( ⁇ ) 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 C NMR spectra are reported in ppm ( ⁇ ) relative to residual CHC1 3 ( ⁇ 77.25) unless noted otherwise.
• Infrared spectra were recorded on a Perkin-Elmer 1600 senes FTIR using samples prepared as thin films between salt plates
• HRMS High-resolution mass spectra
• FAB Fast Atom Bombardment
• high-resolution mass spectra were recorded at the University of California, Riverside Combustion analyses were performed by Desert Analytics, Arlington, AZ, 85719 and optical rotations were measured on a Jasco DIP-1000 digital polanmeter.
• Example 1 Exemplary Precursors 1A Protected Diol Aldehyde 102
• the mixture was diluted with 50 mL EtOAc and acidified with 10 mL of a 0.1 N aqueous NaHS0 4 solution.
• the phases were separated and the aqueous phase was extracted with EtOAc (3x).
• the combined organic extracts were washed with brine, dried over MgS04, and concenttated to give a colorless oil.
• Rapid filtration through a short pad of silica gel (20% acetone/benzene) gave a crude product which was dissolved in 2 mL methanol.
• Palladium on activated charcoal (10%, ⁇ 5 mg) was added and the mixture was sti ⁇ ed at room temperature for 18 h under balloon pressure of hydrogen gas.
• a stock solution of Ipc2B(allyl) was prepared by first dissolving (-)-Ipc2BOMe (700 mg, 2.22 mmol) in ether (4.15 mL) at 0 °C and adding 1M allyl magnesium bromide (1.78 mL, 1.78 mmol). The mixture was warmed to rt and sti ⁇ ed for 30 min. In a separate flask, the aldehyde was dissolved in ether (4 mL) and tteated with the stock solution of Ipc2B(allyl) (0.3 M, 3.9 mL, 1.17 mmol) at -78 °C.
• the mixture was treated with hydrogen peroxide (30 %, 2 mL) and sodium hydroxide (15 %, 2 mL) and warmed to rt. After another 2 h, the mixture was partitioned between ethyl acetate and brine. The aqueous layer was extracted with ethyl acetate. The combined organic layers were dried over sodium sulfate, and concentrated to give the co ⁇ esponding crude homoallylic alcohol, which was directly used in the next step.
• 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 Penodmane (707 mg, 1 67 mmol) and sti ⁇ ed for 1 h at room temperature The reaction was diluted with methylene chlo ⁇ de (3 mL), saturated sodium bicarbonate (3 mL) and sodium thiosulfate (3 mL) and stirred for 1 h The layers were separated and the aqueous layer was re -exttacted with EtOAc (4 x 5 mL) The combined organics were dried over sodium sulfate and the solvent was removed in vacuo The crude ketone was dissolved in MeOH (17 3 mL) and CeCl 3 «7H 2 0 (1 83 g, 4 9 mmol) was added and sti ⁇ ed for 5 min The solution was cooled to - 50°C and NaBH 4 (74
• 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, 0.00175 mmol) in /-BuOH (2.1 mL) and water (2.1 mL). The resulting solution was sti ⁇ ed at room temperature for 3 h.
• Enal 206.1 (22 mg, 0 03 mmol) was dissolved in 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 sti ⁇ ed at room temperature for 75 min 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 in vacuo to provide a crude diol which was taken immediately to the next step.
• a 0 75 mM silylating solution was generated by the addition of lmidazole (62 mg, 0 91 mmol) and TBSC1 (45.6 mg, 0.3 mmol) to methylene chlonde (3 9 mL) at room temperature under nitrogen.
• lmidazole 62 mg, 0 91 mmol
• TBSC1 45.6 mg, 0.3 mmol
• 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 preceeding 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 stirred under a hydrogen atmosphere for 3 hours.
• This example illustrates methods for preparing bryostatin compounds and analogues that contain selected ester substituents at C20.
• Alcohol 303.1 (10 mg, 0.02 mmol) was dissolved in 1.1 mL CH 3 CN / H 2 0 (9: 1) and tteated with 48% ⁇ aqueous HF (200 ⁇ L, 300 mol % excess) at rt. The resulting mixture was stirred 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 4 and concentrated in vacuo to afford crude hemiketal enal 304.1 as a colorless oil.
• R 2 ' CH-CO,Me.
• R 26 is Me and X is oxygen
• 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 tteated 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-dimethylaminopyridine (14 mg, 0.12 mmol) was added gradually and stirring was continued for 40 min.
• 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 sti ⁇ ed 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 co ⁇ esponding 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 tteated 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 stirring 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-ttichlorobenzoylchloride (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 sti ⁇ ing was continued for 40 min.
• 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 sti ⁇ ed for 2 hours. The reaction was then quenched with a saturated solution of sodium bicarbonate. The biphasic mixture was exttacted with ethyl acetate (x4) and the combined organics were dried over sodium sulfate. The solvent was removed in vacuo to provide crude macrocycle.
• Benzoate C20 Ester (702.5) 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 co ⁇ esponding 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-trichlorobenzoylchloride (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%).
• Example 5 Protein Kinase C (Isozyme Mix) Assay Protocol The following procedure was used, based on a modification of a previous procedure described by Tanaka et al. (1986). Filters (Whatman GF-B, 21 mm diam.) are soaked for 1 h in a solution containing deionized water (97 mL), and 10% polyethyleneamine (3 mL).
• a filtering buffer solution containing TRIS (1M, pH 7 4, 10 mL) and water (490 mL) is prepared and cooled on ice
• An assay buffer solution is prepared by the addition of TRIS (1M, pH 7 4, 1 mL), KC1 (1M, 2 mL), CaCl 2 (0 1M, 30 ⁇ L), bovine serum albumin (40 mg), diluted to 20 mL with deionized water and stored on ice
• 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 concenttation 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.
• HYQ DPBS modified phosphate buffered saline phosphate buffer solution
• All compounds are assayed in quadruplicate over 12 concentrations.
• Cell concenttation is measured using a hemacytometer and the final cell concentration is adjusted to 1 x 10 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-diphenyltettazolium 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 concentration 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

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