EP1091978A2 - Synthetische spiroketalpyrane als wirkungsvolle antikrebsmittel - Google Patents

Synthetische spiroketalpyrane als wirkungsvolle antikrebsmittel

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Publication number
EP1091978A2
EP1091978A2 EP99932062A EP99932062A EP1091978A2 EP 1091978 A2 EP1091978 A2 EP 1091978A2 EP 99932062 A EP99932062 A EP 99932062A EP 99932062 A EP99932062 A EP 99932062A EP 1091978 A2 EP1091978 A2 EP 1091978A2
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Prior art keywords
compound
tubulin
independently
aryl
cycloalkyl
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English (en)
French (fr)
Inventor
Fatih M. Uckun
Chen Mao
Shyi-Tai M. Jan
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Parker Hughes Institute
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Parker Hughes Institute
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    • 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
    • C07D493/10—Spiro-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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

  • the invention relates to novel tubulin depolymerization agents, SPIKET, as potent anti-cancer agents.
  • Cancer is a major disease that continues as one ofthe leading causes of death at any age. In the United States alone, it is anticipated that more than a half a million
  • Cellular proliferation for example, in cancer and other cell proliferative disorders, occurs as a result of cell division, or mitosis.
  • Microtubules play a pivotal role in mitotic spindle assembly and cell division "5. These cytoskeletal elements are formed by the self-association ofthe ⁇ tubulin heterodimers 1 ⁇ 5.
  • Agents which induce depolymerization of tubulin and/or inhibit the polymerization of tubulin provide a therapeutic approach to the treatment of cell proliferation disorders such as cancer.
  • the structure of the ⁇ tubulin dimer was resolved by electron crystallography of zinc-induced tubulin sheets ⁇ .
  • each 46 x 40 x 65 A tubulin monomer is made up of a 205 amino acid N-terminal GTP/GDP binding domain with a Rossman fold topology typical for nucleotide-binding proteins, a 180 amino acid intermediate domain comprised of a mixed ⁇ sheet and five helices which contain the taxol binding site, and a predominantly helical C-terminal domain implicated in binding of microtubule-associated protein (MAP) and motor proteins 2> 5
  • MAP microtubule-associated protein
  • Novel tubulin-binding molecules which, upon binding to tubulin, interfere with tubulin polymerization, can provide novel agents for the inhibition of cellular proliferation and treatment of cancer.
  • Spongistatin (SP) ( Figure 1) is a potent tubulin depolymerizing natural product isolated from an Eastern Indian Ocean sponge in the genus Spongia?.
  • Spongistatins are 32-membered macrocyclic lactone compounds with a spongipyran ring system containing
  • SP spongistatin
  • Novel tubulin binding compounds which , upon binding to tubulin, interfere with tubulin assembly, for example by causing depolymerization of tubulin or by inhbiting tubulin polymerization, would provide novel agents for the prevention of cellular proliferation, for example in the inhibition of tumor cell growth and treatment of cancer.
  • a novel binding pocket has been identified in tubulin, which binding pocket accepts and binds novel, small molecule tubulin binding spiroketal pyrane compounds of the invention. Binding ofthe spiroketal pyranes (SPIKETs) to tubulin causes tubulin depolymerization, and/or inhibits tubulin polymerization.
  • the siroketal pyranes of the invention are therapeutically effective as cytotoxic agents, to inhibit cellular proliferation, and as effective anti-cancer agents.
  • the compounds of the invention have the general structure shown in formula I, and are designed to include moieties and/or substitutions capable of favorable interaction with amino acid residues in the SPIKET binding pocket of tubulin.
  • R 1 , R 2 , S 1 , and S 2 preferably contain functional groups and/or substitutions designed to favor hydrophobic interaction and/or Van der Waals interaction with hydrophobic residues of the SPIKET binding pocket, as described more fully below.
  • X 1 , X 2 , and X 3 are the same or different, and are each independently O, C, or S;
  • hydrocarbon moieties of R 1 , R 2 ,S', and S 2 may be substituted or unsubstituted.
  • Particular embodiments of the claimed compounds for example, those compounds depicted in formulae II-V and preferred compounds ofthe invention are described below in the Detailed Description, Examples, and Claims.
  • Particularly preferred compounds ofthe invention are:
  • the SPIKET binding pocket on tubulin is useful for designing and screening tubulin binding molecules, anti-cell proliferation agents, and cancer therapeutic agents.
  • Useful agents are designed to fit the pocket and to favorably interact with the pocket for enhanced binding and anti-tubulin activity.
  • Figure 1 is a diagram showing the structure of spongistatin (SP) and of SPIKET-P1.
  • Figure 2A is a photograph of a space filling model of beta-tubulin, shown with ball and stick models of GDP and Taxoter molecules binding to their respective binding sites as labeled.
  • the spongistatin binding site (SBP) is marked and shown in orange in the color photograph for all aromatic residues in close range.
  • Tubulin residue N101, shown in red, is loctaed near the GDP binding site.
  • Figure 2B is a ribbon representation of bea-tubulin, shown with an aromatic residue cluster as a remarkable characteristic of the spongistatin binding site.
  • Figure 3 is a schematic representation of SPIKET-P1 docked in its binding site in tubulin. Shown are the tubulin residues available for interaction with SPIKET-P1.
  • Figure 4 is a schematic representation ofthe X-ray structure of SPIKET-P1.
  • Figure 5 is a graph showing the anti-tubulin activity of the compounds of the invention as analyzed in turbidity assays.
  • Figures 6 A-6E are electron micrographs showing induced apoptosis in control ( Figures 6A and 6B), SPIKET-Pl ( Figure 6C), SPIKET-P1P ( Figure 6D), and NP-25 ( Figure 6E)treated human breast cancer cells.
  • Figures 7A and 7B are confocal microscopic images showing the effects of SPIKET-Pl on microtubules in human breast cancer cells.
  • Control Figure 7A
  • treated cells Figure 77B
  • microtubules as green fluoresence
  • DNA as blue.
  • the Bar indicates a size of 20 microns.
  • Figures 8A-8C are graphs showing the anti-tubulin effects o the library HHL2C ( Figure 8A), HHL2f (Figure 8B), and HHL4c ( Figure 8C).
  • alkyl includes both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms. As a preferred embodiment, chains of 1 to 22 carbon atoms are included.
  • alkene includes both branched and straight chain aliphatic hydrocarbon groups that have at least one double bond.
  • alkoxy includes, saturated and unsaturated, branched and straight chain aliphatic hydrocarbon groups having a specified number of carbon atoms where at least one carbon atom forms a single-bond to an oxygen atom.
  • amine includes primary, secondary, and tertiary amines.
  • halogen or “halo” substituent includes fluoro, chloro, bromo, and iodo.
  • pharmaceutically acceptable salt thereof includes an acid addition salt or a base salt.
  • pharmaceutically acceptable carrier includes any material which, when combined with a compound of the invention, allows the compound to retain biological activity, such as the ability to induce apoptosis of leukemia or breast tumor cells, and is non-reactive with the subject's immune system.
  • examples include, but are not limited to, any ofthe standard pharmaceutical carriers such as a phosphate buffered saline solution, water, emulsions such as oil/water emulsions, and various types of wetting agents.
  • Compositions comprising such carriers are formulated by well known conventional methods (see, for example, Remington's Pharmaceutical Sciences, Chapter 43, 14th Ed., Mack Publishing Co., Easton, PA).
  • Substituted cycloalkyl includes cyclic hydrocarbons having substituents including halo, alkyl, alkenyl, oxyalkyl, oxyalkenyl, haloalkyl, haloalkenyl, and aryl.
  • “Substituted cycloalkenyl” includes cyclic hydrocarbons having at least one double bond where substituents include halo, alkyl, alkenyl, oxyalkyl, oxyalkenyl, haloalkyl, haloalkenyl, and aryl.
  • Substituted aryl includes aromatic hydrocarbons having substituents including hydroxyl, amino, aminomethyl, halo, alkyl, alkenyl, oxyalkyl, oxyalkenyl, haloalkyl, haloalkenyl, and aryl.
  • Treating in the context of this invention means the prevention or reduction in severity of symptoms or effects of a pathological condition, including prolonging life expectancy.
  • treatment includes prevention of tumor growth, reduction of tumor size, enhanced tumor cell death, and increased apoptosis.
  • the binding pocket determined for spongistatin located near the GDP binding site on the surface of tubulin has dimensions of approximately ⁇ A wide x 18A long x 1 lA deep.
  • the binding pocket is lined with an unusual cluster of aromatic residues situated in close proximity to each other, including Y108, W103, Y185, W407, Y408, F399, F404, F395, F418, and H406 (See Figure 2B).
  • the synthetic spiroketal pyrans (SPIKET-P) compounds of the invention when bound to the tubulin pocket, have the spiroketal ring sandwiched between aromatic residues F404 and W407, providing favorable hydrophobic interactions and van der Waals contacts with these residues (See Figure 3).
  • Unoccupied volume surrounding the SPIKET-Pl molecule in the binding pocket in the vicinity of the aromatic residues with some hydrophillic residues also nearby provide a basis for modification ofthe SPIKET-P molecules to design more potent tubulin binding compounds, and more potent inhibitors of tubulin assembly.
  • Compounds ofthe invention are designed to interact favorably with the binding pocket.
  • the aromatic ring substituents on the SPIKET-P compounds provide favorable contacts with aromatic residues such as F399, Y408, Y185, and H406.
  • Hydrophilic substituents added to the ring offer hydrogen binding capability with hydrophilic residues such as H406, K105, E411, and N101. (see, Figure 3).
  • the invention thus includes compounds designed to interact favorably with the binding pocket, methods for designing and screening such compounds, and methods for screening useful tubulin inhibitors and anti-cancer therapeutic agents.
  • the compounds of the invention include those having spiroketal subunits suitable for binding to the spongistatin binding pocket of tubulin.
  • Compounds ofthe invention include those having the following structural formula:
  • X 1 , X 2 , and X 3 are the same or different, and are each independently O, C, or S;
  • spiroketal ring substituents and substitutions are of a size and chemical functionality so as to permit favorable interaction with the SP binding pocket of tubulin.
  • Preferred compounds ofthe invention are shown in the data tables provided in the Examples below, and include the following compounds:
  • Base salts are formed with metals or amines, such as alkali and alkaline earth metals or organic amines.
  • metals used as cations are sodium, potassium, magnesium, calcium, and the like.
  • heavy metal salts such as for example silver, zinc, cobalt, and cerium.
  • suitable amines are N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamene, N-methylglucamine, and procaine.
  • Pharmaceutically acceptable acid addition salts are formed with organic and inorganic acids.
  • suitable acids for salt formation are hydrochloric, sulfuric, phosphoric, acetic, citric, oxalic, malonic, salicylic, malic, gluconic, fumaric, succinic, ascorbic, maleic, methanesulfonic, and the like.
  • the salts are prepared by contacting the free base form with a sufficient amount ofthe desired acid to produce either a mono or di, etc. salt in the conventional manner.
  • the free base forms may be regenerated by treating the salt form with a base. For example, dilute solutions of aqueous base may be utilized.
  • Dilute aqueous sodium hydroxide, potassium carbonate, ammonia, and sodium bicarbonate solutions are suitable for this purpose.
  • the free base forms differ from their respective salt forms somewhat in certain physical properties such as solubility in polar solvents, but the salts are otherwise equivalent to their respective free base forms for the purposes of the invention.
  • the compounds of the invention inlcude their salts.
  • the compounds of the invention bind to tubulin at a unique, novel binding pocket of tubulin. Upon binding the tubulin binding compounds, tubulin is caused to depolymerize and/or inhibitition of tubulin assembly results. Suitable assays for the anti- tubulin acitivity of the inventive compounds are disclosed in the Examples below.
  • the compounds of the invention can be used in methods of tumor treatment, for example, by administering to a subject a compound of the invention in order to achieve an inhibition of tumor cell tubulin assembly and/or depolymerization of tumor cell tubulin, inhibition of tumor cell growth, a killing of tumor cells, induced apoptosis, and/or increased patient survival time.
  • the anti-cancer tubulin binding compounds ofthe invention are suitable for use in mammals.
  • mammals means any class of higher vertebrates that nourish their young with milk secreted by mammary glands, including, for example, humans, rabbits, and monkeys.
  • the compounds ofthe present invention can be formulated as pharmaceutical compositions and administered to a mammalian host, including a human patient, in a variety of forms adapted to the chosen route of administration.
  • the compounds are preferably administered in combination with a pharmaceutically acceptable carrier, and may be combined with or conjugated to specific delivery agents, including targeting antibodies and/or cytokines.
  • the compounds can be administered by known techniques, such as orally, parentally (including subcutaneous injection, intravenous, intramuscular, intrasternal or infusion techniques), by inhalation spray, topically, by absorption through a mucous membrane, or rectally, in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants or vehicles.
  • Pharmaceutical compositions ofthe invention can be in the form of suspensions or tablets suitable for oral administration, nasal sprays, creams, sterile injectable preparations, such as sterile injectable aqueous or oleagenous suspensions or suppositories.
  • the compositions can be prepared according to techniques well-known in the art of pharmaceutical formulation.
  • the ⁇ compositions can contain microcrystalline cellulose for imparting bulk, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity enhancer, and sweeteners or flavoring agents.
  • the compositions can contain microcrystalline cellulose, starch, magnesium stearate and lactose or other excipients, binders, extenders, disintegrants, diluents and lubricants known in the art.
  • the compositions can be prepared according to techniques well-known in the art of pharmaceutical formulation.
  • compositions can be prepared as solutions in saline, using benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons or other solubilizing or dispersing agents known in the art.
  • the compositions can be formulated according to techniques well-known in the art, using suitable dispersing or wetting and suspending agents, such as sterile oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid.
  • compositions can be prepared by mixing with a suitable non-irritating excipient, such as cocoa butter, synthetic glyceride esters or polyethylene glycols, which are solid at ambient temperatures, but liquefy or dissolve in the rectal cavity to release the drug.
  • a suitable non-irritating excipient such as cocoa butter, synthetic glyceride esters or polyethylene glycols, which are solid at ambient temperatures, but liquefy or dissolve in the rectal cavity to release the drug.
  • Preferred administration routes include orally, parenterally, as well as intravenous, intramuscular or subcutaneous routes.
  • the compounds of the present invention are administered parenterally, i.e., intravenously or intraperitoneally, by infusion or injection.
  • the compounds may be administered directly to a tumor by tumor injection; or by systemic delivery by intravenous injection.
  • Solutions or suspensions of the compounds can be prepared in water, isotonic saline (PBS) and optionally mixed with a nontoxic surfactant.
  • Dispersions may also be prepared in glycerol, liquid polyethylene, glycols, DNA, vegetable oils, triacetin and mixtures thereof. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical dosage form suitable for injection or infusion use can include sterile, aqueous solutions or dispersions or sterile powders comprising an active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions.
  • the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage.
  • the liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol such as glycerol, propylene glycol, or liquid polyethylene glycols and the like, vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size, in the case of dispersion, or by the use of nontoxic surfactants.
  • the prevention ofthe action of microorganisms can be accomplished by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, buffers, or sodium chloride.
  • Prolonged absorption ofthe injectable compositions can be brought about by the inclusion in the composition of agents delaying absorption—for example, aluminum monosterate hydrogels and gelatin.
  • Sterile injectable solutions are prepared by incorporating the conjugates in the required amount in the appropriate solvent with various other ingredients as enumerated above and, as required, followed by filter sterilization.
  • the preferred methods of preparation are vacuum drying and freeze-drying techniques, which yield a powder ofthe active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.
  • the compound ofthe invention can be targeted for specific delivery to the cells to be treated by conjugation of the compounds to a targeting moiety.
  • Targeting moiety useful for conjugation to the compounds ofthe invention include antibodies, cytokines, and receptor ligands expressed on the cells to be treated.
  • conjugation means a complex formed with two or more compounds.
  • targeting moiety means a compound which serves to deliver the compound ofthe invention to a specific site for the desired activity.
  • Targeting moieties include, for example, molecules which specifically bind molecules present on a cell surface.
  • Such targeting moieties useful in the invention include anti-cell surface antigen antibodies.
  • Cytokines, including interleukins, factors such as epidermal growth factor (EGF), and the like, are also specific targeting moieties known to bind cells expressing high levels of their receptors.
  • Particularly useful targeting moieties for targeting the compounds of the invention to cells for therapeutic activity include those ligands that bind antigens or receptors present on the tumor cells to be treated.
  • antigens present on B-lineage cancer cells such as CD 19, can be targeted with anti-CD 19 antibodies such as B43.
  • Antibody fragments, including single chain fragments, can also be used.
  • IL4 can also be used to target B-cells.
  • Cancer cells expressing EGF or IGF receptors can be targeted with the binding ligand.
  • Other such ligand-receptor binding pairs are known in the scientific literature for specific cancers. Methods for producing conjugates ofthe compounds of the invention and the targeting moieties are known.
  • the administered dose is that effective to have the desired effect, such as is sufficient to reduce or eliminate tumors.
  • Appropriate amounts can be determined by those skilled in the art, extrapolating using known methods and relationships, from the in vitro data provided in the Examples.
  • the dose ofthe novel tubulin binding compounds effective to achieve tumor cell apoptosis, reduction in tumors, and increased survival time is 1-100 mg/kg body weight/dose for a direct targeted administration.
  • the effective dose to be administered will vary with conditions specific to each patient. In general, factors such as the disease burden, tumor location (exposed or remote), host age, metabolism, sickness, prior exposure to drugs, and the like contribute to the expected effectiveness of a drug.
  • One skilled in the art will use standard procedures and patient analysis to calculate the appropriate dose, extrapolating from the data provided in the Examples.
  • a dose which delivers about 1-100 mg/kg body weight is expected to be effective, although more or less may be useful.
  • compositions of the invention may be administered in combination with other anti-tumor therapies.
  • the administered dose ofthe tubulin binding compounds may be less than for single drug therapy.
  • Example 1 Spongistatin Binding Pocket on Tubulin In a rational drug design effort intended to determine the minimal molecular architecture ofthe SP structure necessary for biologic activity, we used the 3-D atomic model of the ⁇ ⁇ tubulin dimer for the identification of the potential tubulin binding sites for SP. Our integrated effort to identify the SP binding site involved "cavity searching", analysis ofthe binding environment, docking procedures based on the electron crvstallographic coordinates of tubulin, and the calculation of the binding constant by a modified score function (LUDI score function).
  • LODI score function modified score function
  • SP is a macrocyclic molecule with dimensions larger than lOA wide and lOA long.
  • the large molecular volume of SP may contribute to its remarkable potency as a tubulin depolymerizing agent and suggests that it may bind to a deep pocket on or near the surface of tubulin, providing a highly hydrophobic environment for molecular interactions.
  • GRASP A. Nicholls, GRASP, Graphical Respresentation and Analysis of Surface Properties, 1992, New York
  • INSIGHTII Molecular Simulations Inc., 1996, San Diego, California
  • the SP molecule was then docked into this candidate binding site using the Affinity module within the Insight II program (Molecular Simulations Inc., 1996, San Diego, California).
  • the docking results which utilized different conformations of SP were analyzed and ranked based on their interaction scores, as shown in the examples below.
  • the SP model which was assigned the highest interaction score and lowest potential energy was chosen for further calculations.
  • the pocket consists of an unusual cluster of aromatic residues situated in close proximity including Y108, W103, Y185, W407, Y408, F399, F404, F395, F418, and H406 ( Figure 2B).
  • the proposed tubulin binding site for SP is in close proximity to the GDP exchange site on the ⁇ subunit of the tubulin heterodimer ( Figure 2).
  • This location for the SP binding site would provide a cogent explanation for the ability of SP to inhibit the displacement ofthe bound GDP molecules from tubulin° > 9
  • this binding pocket contacts the longitudinal interdimer interface of the microtubule; the existence of bound SP in this binding pocket may therefore hinder interdimer interactions of tubulin and contribute to the tubulin depolymerizing activity of SP.
  • Advanced modeling studies of the interactions of SP with this putative SP binding pocket indicated that the two spiroketal groups of SP are in close contact with protein residues lining the binding pocket and may therefore serve as the critical binding components of SP.
  • SPIKET-P compounds simple synthetic spiroketal pyrans representing these subunits, such as compound SPIKET-Pl ( Figure 3), could serve as SP pharmacophores.
  • Docking studies indicated that, when bound to tubulin, the spiroketal ring of SPIKET-Pl would be sandwiched between aromatic residues F404 and W407 in the binding pocket and would provide favorable hydrophobic interactions and van der Waals contacts with these residues ( Figure 3).
  • an analysis of the SPIKET-Pl binding environment indicated some unoccupied volume surrounding the docked SPIKET-Pl molecule (see Figure 3) in the vicinity of mostly hydrophobic aromatic residues mixed with a few hydrophilic residues.
  • the aromatic ring substituents on the inhibitor would provide favorable contacts with aromatic residues lining the tubulin binding site (residues F399, Y408, Y185 and H406) whereas hydrophilic substituents added to the ring would offer opportunities for hydrogen bonding with hydrophilic residues that are slightly more distant (residues H406, K105, E41 1 and N101 ( Figure 3)).
  • SPIKET-Pl interaction with the tubulin SP binding site is illustrated in a surface model ofthe SP binding site and stick-and-ball model for SPIKET-Pl molecule.
  • the spiroketal ring is sandwiched between phenylalanine 404 and tryptophan 407 (shown in the color photograph of Figure 3 in light blue and magenta) and close to a histidine (colored in green).
  • the Spiroketal ring ofthe SPIKET-Pl is in close contact with the surrounding hydrophobic residues with 75% of 218 A 2 total molecular surface buried.
  • the calculation and figure was prepared using GRASP 1 .
  • Two hydroxyl groups on SPIKET-Pl point toward unoccupied pockets which have room for larger substituents on SPIKET-Pl.
  • the inspection of SPIKET-P 1 aromatic-residue-rich environment indicates that substitution of the hydroxyl groups by hydrophobic rings would provide more favorable interactions with surrounding aromatic residues.
  • SPIKET-Pl has a molecular surface of 218 A 2 , 75% of which would be covered by the aforementioned two aromatic rings. Therefore, SPIKET- PI ( Figure 3) was selected as our first synthetic target. Retro-synthetic analysis of
  • SPIKET-P l (Scheme 1) shown in Example 2 below, indicated that SPIKET-Pl could be prepared using a versatile multi-step synthetic scheme in a stereocontrolled fashion.
  • the binding position was determined based on the position ofthe A-B spiroketal group of spongistatin in SP binding site.
  • the coordinates of SPIKET-Pl used in the following docking procedure were taken from the X-ray crystal structure (Example 3, Figure 4).
  • SPIKET-P 1-P and NP-25 were built on the SPIKET-Pl coordinates using the Sketcher module in the Insightll program (Molecular Simulation Inc., 1996, San Diego. California). Fixed docking in the Affinity module within Insightll was used for docking small molecules to the binding site of tubulin which was determined from the electron crystal structure and further defined from visual inspection.
  • the docking program has the ability to define a radius of residues within a 7 A distance from the ligand molecule. As the modeling calculations progressed, the residues within the defined radius were allowed to move in accordance with energy minimization. Ten final target positions were defined for each molecule which had starting positions randomly assigned. The final docked position ofthe molecule was chosen based on both the lowest energy estimation and the highest score rank which was defined by a modified LUDI function I ->> 16 (described below) for the search target. Calculations were carried out on a SGI INIDIGO2 using the CVFF force field in the Discover program and a Monte Carlo search strategy in Affinityl . No solvation procedures were used. Since the total number of movable atoms exceeded 200, conjugated gradient minimization was used instead of the Newton minimization method to conserve CPU time.
  • SPIKET-P1 (a) vinylMgBr, CuBr, 2h, 0°C. (b). TBDMSC1, imidazole, DMAP, lh, 0°C, 3h, rt.
  • the synthesis was initiated by opening the commercially available epoxide 5 using vinylmagnesium bromide to obtain the alcohol 6 which was protected as tert- butyldimethylsilyl ether to form 7. Hydroboration of the terminal olefin in 7 yielded the primary alcohol as in 8, which was than converted to the mesylate 4.
  • the mesylate group in 4 was substituted by bromide to form 3.
  • the mesylate in 4 was substituted by cyanide to form 9 which was further converted to aldehyde 2 by DIBAL reduction followed by acid catalyzed hydrolysis in one pot reaction.
  • Compound 3 was coupled with 2 by first converting 5 to a Grignard reagent and then reacting it with aldehyde 2 to form 10.
  • Swern oxidation converted 10 to 1.
  • the reaction was carried out in a one pot reaction by treating 1 with 5% HF in acetonitrile at room temperature for 30 minutes.
  • SPIKET-Pl was obtained by treating SPIKET-P 1-P with lithium 4,4'-di-tert-butylbiphenylide (LDBB).
  • LDBB 4,4'-di-tert-butylbiphenylide
  • the solution was stirred at 0 °C for lh and then at room temperature for 2 h.
  • the reaction was quenched with a small amount of water, the two layer separated and the aqueous layer was extracted with ethyl ether (2 x 100 mL).
  • the combined organic layers were washed with brine, dried over anhydrous MgSO 4 , and the solvent was removed under reduced pressure.
  • the compound, SPIKET-Pl, made via the above synthetic pathway was then analyzed with x-ray crystallography.
  • the crystal structure was utilized along with the above characterization data to confirm the structure of SPIKET-Pl .
  • SPIKET-Pl The structure of SPIKET-Pl was confirmed by small molecule X-ray crystallography, as shown in Figure 4.
  • NP-25 Another spiroketal was synthesized, NP-25, according to Scheme 3, shown below.
  • the known compound 20, a synthetic intermediate for the synthesis of A&B spiroketal unit of SP,* 2 was treated with hydrofluoric acid to afford NP-25.
  • Scheme 3 following monoalkylation of 1,3-propandiol 11 by benzyl chloride, Swern oxidation of the alcohol 12 gave the aldehyde 13.
  • Addition ofthe allylborane to 13 gave, after oxidative workup, the homoallylic alcohol 14.
  • Silyl protection of 14 as the TES ether gave 15. Reaction of 15 with osmium tetroxide and sodium periodate, gave the (S)-aldehyde 16.
  • (+)-Ipc 2 BCH 2 CH CH 2 2,6-lutidine OTES
  • NP-25 The detailed method for the synthesis of NP-25 is given below. The step is referred to by the intermediate product that it produces. Characterization data for each intermediate product is also given. The structures can be seen in Scheme 3 above.
  • the mixture was partitioned between diethyl ether (430 mL) and pH 7 buffer (310 mL). The organic layer was concentrated and the residue was dissolved in 310 mL of methanol. To the solution at 0 °C were added pH 7 buffer (62 mL) and 30% hydrogen peroxide (94 mL) and stirred at room temperature for 1-2 h. The mixture was diluted with 500 mL of water and extracted with dichloromethane (3 x 500 mL). The organic layer was washed with sodium bicarbonate solution and brine, dried over anhydrous sodium sulfate and concentrated.
  • NP25 l,13-dibenzyloxy-5,9-dihydroxy-spiroketal
  • Bovine brain tubulin (Sigma, St. Louis, MO) was used in standard turbidity assays to test the effects of SPIKET-Pl, SPIKET-P 1-P and NP- 25 on GTP-induced tubulin polymerization. Compounds (in 1% DMSO) were added to tubulin (lmg/ml, 0.1M MES, ImM
  • EGTA 0.5mM MgCI2, O.lmM EDTA, 2.5M glycerol, lmg/ml leupeptin, lmg/ml aprotinin, pH 6.5) followed by stimulation of polymerization with ImM GTP at 2 minutes.
  • Optical density was measured using a Becton Dickinson UV spectrophotometer at 350nm using a thermostated cuvette holder to keep the reaction at 37° C. Readings obtained from the spectrophotometer were standardized by subtracting the background absorbance of the compound in water from the sample reading following drug addition.
  • Figure 6 shows human BT-20 breast cancer cells that were treated with SPIKET- Pl, SPIKET-P 1-P, or NP-25 by transmission electron microscopy ⁇ . The data provide direct evidence that these compounds induce apoptosis in human breast cancer cells.
  • HHL2a fixed one ester group to Rl, vary the other ester group from Rl to R10
  • HHL2b fixed one ester group to R2
  • HHL2j fixed one ester group to R10, vary the other ester group from Rl to R10
  • HHL4a fixed one ester group to Rl, vary the other ester group from Rl to R4, HHL4b: fixed one ester group to R2, vary the other ester group from Rl to R4, HHL4c: fixed one ester group to R3, vary the other ester group from Rl to R4, HHL4d: fixed one ester group to R4, vary the other ester group from Rl to R4,
  • the molecular weight in Table 5 was symmetrical along the diagonal axis.
  • SPIKET-P compounds generated in the combinatorial libraries above include those listed below in order ofthe combinatorial table.
  • Turbidity assays were carried out on some ofthe libraries that were created in Example 6. The tubidity assays were completed using the materials and methods described in the Examples above. The results ofthe assays with the three libraries tested are shown in Figure 8.

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