WO2006017761A2 - Analogues d'epothilone en tant qu'agents therapeutiques - Google Patents

Analogues d'epothilone en tant qu'agents therapeutiques Download PDF

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WO2006017761A2
WO2006017761A2 PCT/US2005/027942 US2005027942W WO2006017761A2 WO 2006017761 A2 WO2006017761 A2 WO 2006017761A2 US 2005027942 W US2005027942 W US 2005027942W WO 2006017761 A2 WO2006017761 A2 WO 2006017761A2
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compound
atom
alkyl
remark
heteroaryl
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WO2006017761A3 (fr
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James P. Snyder
James Nettles
Dennis C. Liotta
David George Ian Kingston
Ganesh Thota
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Emory University
Virginia Polytechnic Institute And State University
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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
    • C07D493/08—Bridged systems

Definitions

  • the present invention provides analogues of epothilone that are useful as microtubule stabilizing agents in the treatment of abnormal cell proliferation, methods of making the compounds, compositions containing the compounds, and their use as microtubule stabilizing agents, antineoplastic agents, and as therapeutic agents in treating abnormal cell proliferation. Also provided is a 3D binding model of epothilone A on ⁇ , ⁇ -tubulin and its use in predicting, designing, or selecting therapeutic epothilone analogs based on the model of interactions between epothilone analogs and ⁇ , ⁇ -tubulin.
  • Microtubules are polymeric structures that are an integral part of all eukaryotic cells. Drugs that target and disrupt microtubules are among the most prominently prescribed anticancer therapies in use today. There are two major classes of chemotherapeutic agents that induce mitotic arrest by disrupting tubulin dynamics—those that depolymerize tubulin and those that stabilize tubulin. This second class of chemotherapeutic agents operate by initiating tubulin polymerization as well as hyper-stabilizing existing microtubules (Schiff, P.B., et al., Proc. Natl. Acad. Sci. (USA), 77, pp. 1561-1565 (1980)).
  • Such drugs appear to arrest cells in mitosis by both increasing the microtubular polymer mass in cells and inducing microtubule "bundling" (Rowinsky, E.K., et al., Cancer Res., 49, pp. 4093-4100 (1988)).
  • tubulin-stabilizing agent is paclitaxel (Taxol®, Bristol- Myers Squibb; and Onxol®, IVAX) (1), which is currently a frontline anticancer agent approved for use by the FDA in 1992 for the treatment of advanced ovarian cancer, and now indicated for breast cancer chemotherapy.
  • Taxol® may bind to Bcl-2 in a second pathway which leads to programmed cell death (Chun, E., et ah, Biochem. Biophys. Res. Commun., 315, pp. 771-779 (2004)).
  • Taxol® and the related taxane docetaxel (Taxotere®; Aventis) (2) in combating a number of human carcinomas has suggested the potential efficacy of other, mechanistically similar but structurally distinct compounds.
  • microtubule-stabilizing agent (+)-discodermolide (6) isolated from the marine sponge Discodermia dissolute [Gunasekera, S.P., et ah, J. Org. Chem., 55, pp. 4912-4915 (1990)], has a higher affinity for the paclitaxel binding site on tubulin than does paclitaxel itself [Te Haar, E., et ah, Biochemistry, 35(1): pp. 243-250 (1996)], and is a promising candidate for clinical development either alone or in combination with paclitaxel.
  • epothilones Another recently identified family of natural products which demonstrate potent microtubule-stabilizing properties are the epothilones.
  • polyketides epothilones A and B (7 and 8, respectively) were isolated from the cell culture extract of the cellulose-degraded myxobacterium Sorangium cellulosum strain So ce90 found in soil collected from the banks of the Zambesi River in South Africa (Hofle, G, et ah, Chem. Abstracts, 120, 52841 (1993); H ⁇ fle, G, et ah, DE 3172925 CO; EP 064547B1).
  • Epothilone B is a three- to twenty-fold more potent inhibitor of human cancer cell growth than paclitaxel and is also effective against multidrug-resistant cell lines. Epothilone B has demonstrated potent in vivo antitumor activity [Altmann, K. -H., et al., Biochimica et Biophysica Acta, 1470, pp. M79-M91 (2000)] and is currently undergoing Phase I clinical trials by Novartis.
  • U.S. Patent No. 6,262,094 to Hoefle, et al. describes a series of C-21 modified epothilones, wherein the thiazole substitutent at the C-21 position of the macrocyclic ring have been modified but not replaced.
  • the epothilone analogs described are suggested to be useful as antifungal or therapeutic agents for the treatment of diseases such as cancer.
  • U.S. Patent No. 6,380,395 to Bristol-Myers Squibb describes a series of 12,13- position modified epothilone derivatives having a cyclopropane functionality in place of the epoxide found in the epothilone A and B ring systems. Also described are methods for their preparation and methods for their use as microtubule-stabilizing agents, especially in the treatment of a variety of cancers and as apoptosis inhibitors. Similarly, U.S. Patent No. 6,727,276, describes modifications at the C12-C13 positions as well as at the Cl 8 side chain position, suggests methods of treating tumors in a mammal, especially a mammal having demonstrated resistance to treatment with taxane oncology agents. Th epothilone derivatives are described as efficacious upon oral administration.
  • U.S. Patent No. 6,489,314 to Kosan Biosciences, Inc. offers a series of 16-membered macrocyclic compounds structurally related to the epothilones, with changes at the Cl -ether linkage, the C2-methyl, the C6-position, the introduction of a double bond at the ClO-CIl position, modifications to the C12 moiety, changes to the C 12-Cl 3 epoxide, and the introduction of a point of variability at the C14 position. These compounds are suggested as suitable for use in the treatment of cancer and non-cancerous disorders, such as psoriasis.
  • a series of 14-methyl epothilone derivatives is described in U.S. Patent No. 6,583,290.
  • the patent indicates that the preferred compounds of the invention are those that can be produced by altering the epothilone PKS genes as described. Also suggested are methods of altering the epothilone PKS genes by the action of epothilone modification enzymes and/or by chemically modifying the epothilones produced when those genes are expressed.
  • U.S. Patent No. 6,596,875 to White, et al. suggests a method for making the cis- and trans-9,10-dehydroepothilone D analogs having selective saturation at the 9,10 olefin position. Also described are methods of making such compounds using a convergent synthesis approach based upon a Wittig coupling of an ylide and an aldehyde.
  • the epothilones made according to the patent have a potency less than that of epothilone B or D alone in a panel of human cancer cell lines, but exhibited full anti-proliferative activity against a paclitaxel-resistant cell line overexpressing P-glycoprotein.
  • Non-hydroxy analogues of several epothilones having a cyano substitution have been reported in efforts to provide meaningful structure-activity relationship information, especially towards the dependence of the C1/C3 conformation on activity [Regueiro-Ren, A., et al., Organic Letters, 4: pp. 3815-3818 (2002)].
  • These C3-cyano epothilone derivatives were further described in U.S. Patent No. 6,719,540. It was found that only those analogs having the naturally-occurring (3S)-configuration displayed activity in vitro, and that analogues having a 12-cyano substitution had an improved pH stability over epothilone B.
  • the Nicolaou research group provided numerous evaluations of analogs of the epothilone ring system and its substituents. Examples of such probes include investigations of the ring size of the epothilones [Nicolaou, K.C., et al, Angew. Chem. Int. Ed., 37: pp. 81- 84 (1998)], synthesis and biological evaluation of a series of pyridine epothilone B analogues in order to explore the idea that the nitrogen atom of the C 18 -side chain needed to be strategically placed [Nicolaou, K.C., et al, Chemistry & Biology, 7: pp.
  • the Nicolaou group also developed a solid-phase synthesis of epothilone A and analogues thereof, and proceeded to apply Furka's "split-and-pool" concept of parallel synthesis to generate libraries of diverse structural congeners of epothilone A [Nicolaou, K.C., et al., Angew. Chem. Int. Ed. Engl, 36: pp. 2097-2100 (1997)].
  • New compounds, methods, compositions, and strategies for use in treating abnormal cell proliferation, including tumors, cancer and angiogenesis-related disorders are provided.
  • the compounds described herein, including in formulas (I) - (IX), and in particular including the eight principal embodiments set out below, are novel analogues of epothilones A and B which bear unique modifications via bridging regions of the molecule.
  • the new analogs have a bridging ring, e.g., at the C4 to C6 position or at the C6 to C8 position, and may exhibit greater long term stability than epothilone A or B itself.
  • analogues have a bridging ring at the C4 to ClO position, C2 to ClO position, C2 to C7 position, C4 to C26 position, C4 to C27 bridge position, or at the C19 to C22 position.
  • other heterocyclic and aromatic rings are substituted for the thiazole ring at the C17 position.
  • heteroatoms are inserted at the C9 and ClO positions of the macrocyclic ring.
  • Processes for in silico screening of compounds which bind to ⁇ , ⁇ -tubulin are also provided. More specifically, a process of identifying compounds which can bind to ⁇ , ⁇ - tubulin by comparing the 3-D structure of candidate compounds with the 3-D molecular model described herein is provided, wherein a) the coordinate data of the 3-D molecular model of ⁇ , ⁇ -tubulin described herein is input in a data structure such that the interatomic distances between the atoms of ⁇ , ⁇ -tubulin are easily retrieved, and b) the distances may be used to determine chemical complementarity between candidate compounds which would theoretically form the most stable complexes with the 3-D molecular model binding pocket of ⁇ , ⁇ -tubulm.
  • ⁇ , ⁇ -tubulin binding pockets and protein domains are also provided. These domains generally include an isolated and purified molecule including a binding pocket of ⁇ , ⁇ -tubulin defined by the structural coordinates of specific amino acid residues according to the Figures described herein. Similarly, crystalline forms of ⁇ , ⁇ -tubulin having unit cell dimensions as described herein are provided.
  • pharmacophores and pharmacophore-defined compounds are provided.
  • the pharmacophores have a spatial arrangement of atoms with a molecule defined by the formulas elaborated on herein, in which there are both electron donor atoms and at least one carbon atom that is part of a hydrophobic group, as well as defined distances between the centers of the respective atoms, hi a further embodiment of this aspect of the invention, isolated compounds or their salts defined by the pharmacophore are described.
  • the invention includes the following features:
  • Methods for the treatment of a host typically a mammal, and more typically a human suffering from a disorder of abnormal cellular proliferation that includes administering an effective amount of one or more of the epothilone A or B analogues described herein;
  • compositions comprising the epothilone A and B analogues and pharmaceutically acceptable salts, solvates, esters and prodrugs thereof with a pharmaceutically acceptable carrier or diluent, optionally for the treatment of a disorder of abnormal cellular proliferation in a host;
  • Figure 1 shows a general synthetic route to C9-functionalized epothilone analogues in accordance with the present invention.
  • Figure 2 shows a general synthetic route to ClO-functionalized epothilone analogues in accordance with the present invention.
  • Figure 3 shows a general synthetic route to C4-C6 bridged epothilone analogues.
  • Figure 4 illustrates the synthetic route to C6-C8 bridged epothilone analogues.
  • Figure 5 shows the synthesis of C4-C26 bridged epothilone analogues in accordance with the present disclosure.
  • Figure 6 illustrates an alternative synthesis of C4-C26 bridged epothilone analogues in accordance with the present invention.
  • Figure 7 shows the general synthetic route to C2-C10 bridged epothilone analogues in accordance with the present invention.
  • Figure 8 shows several of the heterocyclic stannanes envisioned for use in forming analogues of the present invention, and procedures for their synthesis.
  • Figure 9A illustrates the 2F 0bs -F calc density map and associated model derived from electron crystallographic analysis of epothilone A (7) bound to zinc-stabilized 2D crystals of tubulin.
  • Figure 9B illustrates the hydrophobic to hydrophilic properties mapped to the solvent accessible ⁇ -tubulin surface at the ligand binding site.
  • Epothilone A is shown with hydrogen bonds (dashes) to associated centers on the ⁇ -tubulin protein.
  • Figure 10 shows the superposition of epo A (blue) and T-Taxol (gold) in ⁇ -tubulin as determined by electron crystallography. Hydrogen atoms have been eliminated for clarity. Side chains terminating in aromatic rings occupy distinctly different regions of the binding site. The single common center ( ⁇ 1.1 A) between the molecules is C7-OH (blue arrows); right view corresponds to a 90° rotation of the left view about an axis approximately parallel to the blue side chain of epo-A. The alignment shown here without tubulin is identical to those structures illustrated in Figures 9A-B, 11 and SOM Figure 19.
  • Figure 11 illustrates the hydrogen bonding (violet) around epo A in ⁇ -tubulin. Oxygens from C1-C7 engage in network H-bonds with M-loop residues. The thiazole is anchored by His227. Disruption of primary or secondary hydrogen bonds would occur upon mutation of Ala231, Thr274, Arg282 or Gln292 to other residues as observed in epothilone resistant cells. ⁇
  • Figures 12A-12B depict an energy optimized composite model of a fictionalized epothilone compound illustrating diverse features of the structure activity relationships in the context of the EC derived model for tubulin (TB)/epothilone A.
  • FIGS 13A and 13B illustrate the Epothilone A stabilizing effect on Zn-sheets.
  • 13A is the control; no Epothilone A was added.
  • the crystalline sheets disassembled into aggregates after a 20 min incubation on ice.
  • 13B shows the Zn-sheets with Epothilone A; the crystalline sheets remain intact at control conditions when exposed to the microtubule stabilizer [scale bar, 1mm].
  • Figures 14A and 14B show the electron diffraction pattern of a Zn-sheet stabilized with epothilone A.
  • 14A illustrates the diffraction patter where reflections extend to the edge of the image at 2.5 A resolution.
  • 14B shows three lattice line curves.
  • the vertical axis represents intensity in an arbitrary unit, while the horizontal axis is z*, the height of the lattice rod in reciprocal space. Note the odd numbered curve (17,6). Although the intensities are weak, the peaks are well defined because of the large number of measurements.
  • Figure 15 shows the effect of diffraction resolution upon omit map projections.
  • Figures 15A- F illustrate the results obtained as less diffraction data is included in calculation of 2F Obs -F Ca ic omit maps from CNS.
  • Each map was phased from the same high temperature simulated annealing model of the protein structure, the only change being the number of reflections used.
  • the tubulin coordinates used for molecular replacement were obtained from the deposited structure, IJFF, with Taxol® ligand removed and B-factors uniformly set to 30 before annealing.
  • the ligand omit map using all data down to 2.89 A. Unoccupied volumes of density above His227 approximate the shape and total spatial volume of epothilone A.
  • Figure 16 shows the 2F O bs-F ca ic "omit" map used in the automated query.
  • the 2F O b s -F C aic omit map calculated in CNS with a) all reflections to 2.89 A, and b) phases from five "shaken" high temperature annealed protein models.
  • This omit map exhibits a similar spatial distribution and total volume by comparison with the map in Figure 15 A, but it possesses a single contiguous volume suitable for automated fitting of conformational databases as described in detail herein.
  • the maximum volume of density (highlighted in gold) was used to fit epothilone rotomers from the conformational database.
  • Figures 17A-D illustrate the effect of varied ligand orientations and conformational models upon 2F obs -Fcaic and F ob s-F ca i c projections of the 2.89 A diffraction data.
  • A) The same 2F Obs -F ca i c omit map shown in Figure 16 calculated in CNS with all reflections to 2.89 A and phases from "shaken" high temperature annealed protein models. This omit map exhibits a similar spatial distribution and total volume by comparison to the map in Figure 15 A, but possesses a single contiguous volume suitable for automated fitting of conformational data bases as described in text.
  • 17B-D are 2F 0bs -F cal c (sky-blue) and F 0bs -F C ai c (green +3 ⁇ , red -3 ⁇ ) maps calculated at 2.89 A in CCP4 using model derived phases from identical protein models, but different ligand models, hi all cases, green signifies diffraction data that is not being fit by the model and red represents atomic model positions that are not supported by the diffraction data.
  • the maximum negative difference (red) in this fit is associated with a poor positioning of Arg276 away from ligand and can also be seen in 17C and 17D.
  • a small negative difference peak is located under the ligand, but is >1.5 A from any model atoms.
  • the positive difference (green), diminished upon local refinement, can also be seen near Arg282 in all maps. Atoms of residues within an 8 A sphere of the ligand were refined by maximum likelihood in reciprocal space to the final solution pictured in Figure 9A.
  • Figure 19 shows site origins of differing resistance profiles between epothilones and Taxol® T-taxol (green) in its previously published binding site with (left) and without (right) superposition of epothilone A (orange carbons) from the current work.
  • the bound model of Taxol® interacts primarily through pairing of aromatic side chains at C2 and C3' with complementary residues along the bottom of the binding site.
  • Cells with a tubulin mutation of Phe270 to valine show 24-fold resistance to Taxol, but only 2.8-fold reduction in response to epothilone.
  • mutations at Arg282 and Thr274 that induce >30-fold resistance to epothilone A have a relatively small, ⁇ 10 fold, affect upon Taxol® activity.
  • the M-loop positions Arg282 away from direct interaction with bound Taxol® and suggests a reduced role for the hydroxyl of Thr274.
  • the change in position of His227 (left image) associated with epothilone A's binding (orange carbons) relative to Taxol' s (white carbons) is seen very clearly in the EC diffraction maps and suggests ligand specific interaction roles for this side chain.
  • the position of Ala231 on helix 7 near His227 may be associated with the similar loss of activity shared by the two ligands upon mutation to threonine.
  • Figure 20 illustrates specific structures discussed or referred to in the main text. Functional groups that differ from those found in naturally-occuring epothilone A or B are highlighted in red.
  • Compounds, pharmaceutical compositions, methods and uses are provided for the treatment of a disorder of abnormal cellular proliferation in a host is provided, comprising at least one compound of principal embodiments I-XI below, or a pharmaceutically acceptable salt, solvate, ester or prodrug thereof, optionally with a pharmaceutically acceptable carrier; and optionally with one or more therapeutic agents.
  • R 1 is hydrogen, halogen, C 1 -C 10 alkyl, C 1 -C 10 hydroxyalkyl, C 1 -C 10 haloalkyl, acyl, aryl, heteroaryl, trifluoromethyl, cyano, -C(O)R 7 , --C(O)OR 7 , or --NR 7 R 8 , wherein R 7 and R 8 are each independently hydrogen, C 1 -C 10 alkyl, C 2 -C 10 alkenyl, C 2 -C 10 alkynyl, halogen, aryl, or heteroaryl;
  • R 2 , R 3 , and R 4 are each independently hydrogen, C 1 -C 10 alkyl, C 2 -C 10 alkenyl, C 2 -C 10 alkynyl, acyl, aryl, or heteroaryl;
  • R 5 is selected from the group consisting of thiazolyl, oxazolyl, phenyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, furyl, thienyl, pyrrolyl, imidazolyl, pyrazolyl, quinolyl, isoquinolyl, quinoxalyl, indolyl, benzothiazolyl, benzoxazolyl, benzoimidazolyl, benzopyrazolyl, and substituted versions thereof;
  • R 6 is absent, O, CH 2 , NR 9 , CR 10 R 11 , or SH; each R 9 is H, C 1 -C 10 alkyl, C 2 -C 10 alkenyl, C 2 -C 10 alkynyl, acyl, aryl, or heteroaryl; each R 10 and R 11 are independently hydrogen, chlorine, bromine, or fluorine;
  • W is O, CH 2 , orNR 9 ;
  • X is CH 2 , O, NR 9 , or S
  • Y is CH 2 , O, NR 9 , or S; where, in the above structures, when X is O, NR 9 or S, Y is CH 2 ; when Y is O, NR 9 or S, X is CH 2 ; and "a" can be either a single or a double bond; “b” can be either absent or a single bond; “c” can be either absent or a single bond.
  • R 1 is C 1 -C 10 alkyl
  • R 6 is O
  • X is O
  • NR 9 or S
  • Y is CH 2 .
  • R 1 is C 1 -C 10 alkyl
  • R 6 is O
  • Y is O
  • NR 9 or S
  • X is CH 2 .
  • R 1 is C 1 -C 10 alkyl
  • R 6 is N
  • X is O, NR 9 , or S
  • Y is CH 2 .
  • R 1 is C 1 -C 10 alkyl
  • R 6 is N
  • Y is O
  • NR 9 or S
  • X is CH 2 .
  • R 1 is CF 3 , R 6 is O, X is O, NR 9 , or S and Y is CH 2 .
  • a compound of Formula I, or a pharmaceutically acceptable salt, solvate, ester, or prodrug thereof is provided, wherein:
  • R 1 is CF 3 , R 6 is O, Y is O, NR 9 , or S and X is CH 2 .
  • R 1 is CF 3 , R 6 is N, X is O, NR 9 , or S and Y is CH 2 .
  • R 1 is CF 3
  • R 6 is N 5
  • Y is O, NR 9
  • S is CH 2 .
  • a is a double bond of either (E)- or (Z)-orientation, "c” is absent, Ri is CF 3 , X is O, NR 9 , or S and Y is CH 2 .
  • R 6 is O, X is O, NR 9 , or S and Y is CH 2 .
  • R 6 is O, Y is O, NR 9 , or S and X is CH 2 .
  • R 6 is N, X is O, NR 9 , or S and Y is CH 2 .
  • R 6 is N, Y is O, NR 9 , or S and X is CH 2 .
  • a is a double bond of either (E)- or (Z)-orientation, "c” is absent, X is O, NR 9 , or S and Y is CH 2 .
  • n is 0 or 1 ;
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , W, "a”, "b”, and “c” are as defined previously, with the proviso that when n is 0, there is a bond between the C6 and the C8 substituent.
  • n is 0 or 1 ;
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , W, "a”, "b”, and “c” are as defined previously, with the proviso that when n is 0, there is a bond between the C4 and the C6 substituent.
  • W is O
  • R 1 is C 1 -C 10 alkyl
  • R 5 is thiazolyl or pyridyl
  • R 6 is O
  • n 0.
  • a compound of Formula III, or a pharmaceutically acceptable salt, solvate, ester, or prodrug thereof is provided, wherein:
  • W is O
  • R 1 is C 1 -C 10 alkylamino
  • R 5 is thiazolyl or pyridyl
  • W is O
  • R 1 is CF 3
  • R 5 is thiazolyl or pyridyl
  • W is O
  • R 1 is C 1 -C 10 alkyl
  • R 5 is thiazolyl or pyridyl
  • W is O
  • R 1 is C 1 -C 10 alkylamino
  • R 5 is thiazolyl or pyridyl
  • W is O
  • R 1 is CF 3
  • R 5 is thiazolyl or pyridyl
  • W is O
  • R 1 is C 1 -C 10 alkyl
  • R 5 is thiazolyl or pyridyl
  • W is O
  • R 1 is C 1 -C 10 alkylamino
  • R 5 is thiazolyl or pyridyl
  • W is O
  • R 1 is CF 3
  • R 5 is thiazolyl or pyridyl
  • W is O
  • R 1 is C 1 -C 10 alkyl
  • R 5 is thiazolyl or pyridyl
  • R 6 is O
  • n 1.
  • W is O
  • R 1 is C 1 -C 10 alkylamino
  • R 5 is thiazolyl or pyridyl
  • W is O
  • R 1 is CF 3
  • R 5 is thiazolyl or pyridyl
  • W is O
  • R 1 is C 1 -C 10 alkyl
  • R 5 is thiazolyl or pyridyl
  • W is O
  • R 1 is C 1 -C 10 alkylamino
  • R 5 is thiazolyl or pyridyl
  • W is O
  • R 1 is CF 3
  • R 5 is thiazolyl or pyridyl
  • W is O
  • R 1 is C 1 -C 10 alkyl
  • R 5 is thiazolyl or pyridyl
  • W is O
  • R 1 is C 1 -C 10 alkylamino
  • R 5 is thiazolyl or pyridyl
  • W is O
  • R 1 is CF 3
  • R 5 is thiazolyl or pyridyl
  • a compound of Formula III or a pharmaceutically acceptable salt, solvate, ester, or prodrug thereof is provided, wherein: a" is a double bond, "c" is absent, W is O, R 1 is C 1 -C 10 alkyl, R 5 is thiazolyl or pyridyl, and n — j
  • Ri, R 2 , R 3 , R 4 , R 5 , R 6 , W, "a", "b", and “c” are as defined previously;
  • V is CH 2 , OCH 2 , CH 2 O or CH 2 OCO;
  • Y is CH 2 , O, NR 9 , or S wherein V and Y are not simultaneously a heteroatom.
  • W is O
  • R 6 is O
  • R 1 is C 1 -C 10 alkyl
  • Y is CH 2
  • V is CH 2 , CH 2 O or CH 2 OCO.
  • W is O
  • R 6 is O
  • R 1 is C 1 -C 10 alkyl
  • Y is CH 2
  • V is OCH 2 .
  • W is O
  • R 6 is O
  • R 1 is CF 3
  • Y is CH 2
  • V is CH 2 .
  • W is O
  • R 6 is O
  • R 1 is CF 3
  • Y is CH 2
  • V is OCH 2, CH 2 O or CH 2 OCO.
  • W is O
  • R 6 is O
  • R 1 is C 1 -C 10 alkyl
  • Y is O
  • V is CH 2 , CH 2 O or CH 2 OCO.
  • W is O
  • R 6 is O
  • R 1 is CF 3
  • Y is O
  • V is CH 2 , CH 2 O or CH 2 OCO.
  • a compound of Formula IV or a pharmaceutically acceptable salt, solvate, ester, or prodrug thereof is provided, wherein: "a” is a double bond, "c” is absent, W is O, R 6 is O, R 1 is C 1 -C 10 alkyl, Y is CH 2 and V is OCH 2 , CH 2 O or CH 2 OCO.
  • Ri, R 2 , R 3 , R 4 , R 5 , R 6 , V, W, Y, "a", “b”, and “c” are as defined previously, and wherein V and Y are not simultaneously a heteroatom.
  • W is O
  • R 6 is O
  • R 1 is C 1 -Ci 0 alkyl
  • Y is CH 2
  • V is CH 2 .
  • W is O
  • R 6 is O 5
  • Ri is C 1 -C 10 alkyl
  • Y is CH 2
  • V is OCH 2 , CH 2 O or CH 2 OCO.
  • W is O
  • R 6 is O
  • R 1 is CF 3
  • Y is CH 2
  • V is CH 2 .
  • W is O
  • R 6 is O
  • R 1 is CF 3
  • Y is CH 2
  • V is OCH 2, CH 2 O or CH 2 OCO.
  • W is O
  • R 6 is O
  • R 1 is C 1 -C 10 alkyl
  • Y is O
  • V is CH 2 , CH 2 O or CH 2 OCO.
  • W is O
  • R 6 is O
  • R 1 is CF 3
  • Y is O
  • V is CH 2 , CH 2 O or CH 2 OCO.
  • R 2 , R 3 , R 4 , R 5 , W and Y are as defined previously; and X is O, CH 2 , NR 9 , or S, Z is O or H 2 , where R 9 is hydrogen, C 1 -C 1O alkyl, C 2 -C 10 alkenyl, C 2 -C 10 alkynyl, acyl, aryl, or heteroaryl.
  • X is O
  • W is O
  • Y is CH 2
  • Z is O or H 2 .
  • X is CH 2 , W is O, and Y is CH 2 and Z is O or H 2 .
  • X is S, W is O, and Y is CH 2 and Z is O or H 2 ,
  • X is NR 9 , W is O, and Y is CH 2 , wherein each R9 is independently hydrogen, C 1 -C 10 alkyl, C 2 -C 10 alkenyl, C 2 -C 10 alkynyl, acyl, aryl, or heteroaryl and Z is O or H 2 .
  • R9 is independently hydrogen, C 1 -C 10 alkyl, C 2 -C 10 alkenyl, C 2 -C 10 alkynyl, acyl, aryl, or heteroaryl and Z is O or H 2 .
  • X is O
  • W is O
  • Y is CH 2 and R 5 is thiazolyl and Z is O or H 2 .
  • X is CH 2 , W is O, Y is CH 2 and R 5 is thiazolyl, and Z is O or H 2 .
  • X is S, W is O, Y is CH 2 and R 5 is thiazolyl, and Z is O or H 2 .
  • X is NR 9 , W is O, Y is CH 2 and R 5 is thiazolyl, wherein each R9 is independently hydrogen, C 1 -C 10 alkyl, C 2 -Ci O alkenyl, C 2 -C 10 alkynyl, acyl, aryl, or heteroaryl, and Z is O or H 2 .
  • R 1 , R 2 , R 3 , R 4 , R 5 , V, W, and Y are as defined previously; and R 6 is N or CH.
  • R 1 is C 1 -C 10 alkyl
  • R 6 is N
  • V is OCH 2
  • Y is CH 2 .
  • R 1 is C 1 -C 10 alkyl
  • R 6 is N
  • V is CH 2
  • Y is CH 2 .
  • R 1 is C 1 -C 10 alkyl
  • R 6 is N
  • V is OCH 2
  • Y is O.
  • R 1 is C 1 -C 10 alkyl
  • R 6 is N
  • V is CH 2
  • Y is O.
  • R 1 is CF 3
  • R 6 is N
  • V is OCH 2
  • Y is CH 2 .
  • R 1 is CF 3
  • R 6 is N 5 V is CH 2 and Y is CH 2 .
  • R 1 is CF 3 , R 6 is N, V is OCH 2 and Y is O.
  • R 1 is CF 3 , R 6 is N, V is CH 2 and Y is O.
  • R 1 is C 1 -C 10 alkyl
  • R 6 is CH
  • V is OCH 2
  • Y is CH 2 .
  • R 1 is C 1 -C 10 alkyl
  • R 6 is CH
  • V is CH 2
  • Y is CH 2 .
  • R 1 is C 1 -C 10 alkyl
  • R 6 is CH
  • V is OCH 2
  • Y is O.
  • R 1 is C 1 -C 10 alkyl
  • R 6 is CH
  • V is CH 2
  • Y is O.
  • R 1 is CF 3
  • R 6 is CH
  • V is OCH 2
  • Y is CH 2 .
  • R 1 is CF 3
  • R 6 is CH
  • V is CH 2
  • Y is CH 2 .
  • R 1 is CF 3
  • R 6 is CH
  • V is OCH 2
  • Y is O.
  • Ri is CF 3 , R 6 is CH, V is CH 2 and Y is O.
  • R 1 is Ci-C 10 alkyl
  • R 5 is thiazolyl or pyridyl
  • R 5 is N
  • V is OCH 2
  • Y is CH 2 .
  • R 1 is Ci-Cio alkyl
  • R 5 is thiazolyl or pyridyl
  • R 6 is N
  • V is CH 2
  • Y is CH 2 .
  • n 1 or 2;
  • R 1 , R 3 , R 4 , R 5 , R 6 , W, Y, "a", “b”, and “c" are as defined previously;
  • Z is O, S, NR 9 or CH 2 , where R 9 is hydrogen, C 1 -C 10 alkyl, C 2 -C 10 alkenyl, C 2 -C 10 alkynyl, acyl, aryl, or heteroaryl.
  • W is O
  • Z is O
  • R 1 is C 1 -C 10 alkyl
  • R 6 is O
  • n is 1.
  • W is O
  • Z is CH 2
  • R 1 is C 1 -C 10 alkyl
  • R 6 is O
  • n is 1.
  • W is O
  • Z is S
  • R 1 is C 1 -C 10 alkyl
  • R 6 is O
  • n is 1.
  • W is O
  • Z is NR 9
  • R 1 is C 1 -C 10 alkyl
  • R 6 is O
  • n is 1, wherein each R9 is independently hydrogen, C 1 -C 10 alkyl, C 2 -C 10 alkenyl, C 2 -C 10 alkynyl, acyl, aryl, or heteroaryl.
  • a compound of Formula VIII, or a pharmaceutically acceptable salt, solvate, ester, or prodrug thereof is provided, wherein: W is O, Z is O, R 1 is CF 3 , R 6 is O, and n is 1.
  • W is O
  • Z is CH 2
  • R 1 is CF 3
  • R 6 is O
  • n is 1.
  • W is O
  • Z is S
  • R 1 is CF 3
  • R 6 is O
  • n is 1.
  • W is O
  • Z is NR 9
  • R 1 is CF 3
  • R 6 is O
  • n is 1, wherein each R9 is independently hydrogen, C 1 -C 1O alkyl, C 2 -C 10 alkenyl, C 2 -C 10 alkynyl, acyl, aryl, or heteroaryl.
  • W is O
  • Z is O
  • R 1 is C 1 -C 10 alkyl
  • R 5 is thiazolyl or pyridyl
  • R 6 is O
  • n is 1.
  • W is O
  • Z is CH 2
  • R 1 is C 1 -C 10 alkyl
  • R 5 is thiazolyl or pyridyl
  • R 6 is O
  • n is 1.
  • W is O
  • Z is S
  • R 1 is C 1 -C 10 alkyl
  • R 5 is thiazolyl or pyridyl
  • R 6 is O
  • n is 1.
  • W is O
  • Z is NR 9
  • R 1 is C 1 -C 10 alkyl
  • R 5 is thiazolyl or pyridyl
  • R 6 is O
  • n is 1, wherein each R9 is independently hydrogen, C 1 -C 10 alkyl, C 2 -C 10 alkenyl, C 2 -C 10 alkynyl, acyl, aryl, or heteroaryl.
  • W is O
  • Z is O
  • R 1 is CF 3
  • R 5 is thiazolyl or pyridyl
  • R 6 is O
  • n is 1.
  • W is O
  • Z is CH 2
  • R 1 is CF 3
  • R 5 is thiazolyl or pyridyl
  • R 6 is O
  • n is 1.
  • W is O
  • Z is S
  • R 1 is CF 3
  • R 5 is thiazolyl or pyridyl
  • R 6 is O
  • n is 1.
  • W is O
  • Z is NR 9
  • R 1 is CF 3
  • R 5 is thiazolyl or pyridyl
  • R 6 is O
  • n is 1, wherein each R9 is independently hydrogen, C 1 -C 10 alkyl, C 2 -C 10 alkenyl, C 2 -C 10 alkynyl, acyl, aryl, or heteroaryl.
  • a compound of Formula DC or a pharmaceutically acceptable salt, solvate, ester, or prodrug thereof, is provided,
  • W is O
  • R 6 is O
  • R 2 is H
  • R 3 is C 1 -C 10 alkyl
  • R 5 is thiazolyl or pyridyl
  • W is O
  • R 6 is O
  • R 2 is H
  • R 3 is C 1 -C 10 alkyl
  • R 5 is thiazolyl or pyridyl
  • W is O
  • R 6 is O
  • R 2 is H
  • R 3 is C 1 -C 10 alkyl
  • R 5 is thiazolyl or pyridyl
  • X is CH 2 O and Y is CO.
  • C 1 -C 10 alkyl independently includes C 1 , C 2 , C 3 , C 4 , C 5 , C 6 , C 7 , C 8 , C9 and C 10 alkyl.
  • alkyl alone or in combination, means an acyclic, saturated straight, branched, or cyclic, primary, secondary, or tertiary hydrocarbon, including those containing from 1 to 10 carbon atoms or from 1 to 6 carbon atoms.
  • Said alkyl radicals maybe optionally substituted with groups including but not limited to methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl, t-butyl, sec-butyl, pentyl, cyclopentyl, isopentyl, neopentyl, hexyl, isohexyl, cyclohexyl, cyclohexylmethyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3- dimethylbutyl, heptyl, octyl; nonyl, decyl, trifluoromethyl and difluoromethyl.
  • Moieties with which the alkyl group can be substituted include, for example, alkyl, hydroxyl, halo, nitro, cyano, alkenyl, alkynyl, heteroaryl, heterocyclic, carbocycle, alkoxy, oxo, aryloxy, arylalkoxy, cycloalkyl, tetrazolyl, heteroaryloxy; heteroarylalkoxy, carbohydrate, amino acid, amino acid esters, amino acid amides, alditol, haloalkylthi, haloalkoxy, haloalkyl, hydroxyl, carboxyl, acyl, acyloxy, amino, aminoalkyl, aminoacyl, amido, alkylamino, dialkylamino, arylamino, nitro, cyano, thiol, imide, sulfonic acid, sulfate, sulfonate, sulfonyl, alkylsulfon
  • alkenyl alone or in combination, means an acyclic, straight, branched, or cyclic, primary, secondary, or tertiary hydrocarbon, including those containing from 2 to 10 carbon atoms or from 2 to 6 carbon atoms, wherein the substituent contains at least one carbon-carbon double bond.
  • alkenyl radicals may be optionally substituted. Examples of such radicals include but are not limited to are ethylene, methylethylene, and isopropylidene.
  • alkynyl refers to an unsaturated, acyclic hydrocarbon radical, linear or branched, in so much as it contains one or more triple bonds, including such radicals containing about 2 to 10 carbon atoms or having from 2 to 6 carbon atoms.
  • the alkynyl radicals may be optionally substituted.
  • alkynyl radicals include but are not limited to ethynyl, propynyl, hydroxypropynyl, butyn-1-yl, butyn-2-yl, pentyn-1-yl, pentyn-2-yl, 4-methoxypentyn-2-yl, 3-methylbutyn-l-yl, hexyn-1-yl, hexyn-2-yl, hexyn-3-yl, 3,3-dimethylbutyn-l-yl radicals and the like.
  • acyl alone or in combination, means a carbonyl or thionocarbonyl group bonded to a radical selected from, for example, hydrido, alkyl, alkenyl, alkynyl, haloalkyl, alkoxy, alkoxyalkyl, haloalkoxy, aryl, heterocyclyl, heteroaryl, alkylsulfinylalkyl, alkylsulfonylalkyl, aralkyl, cycloalkyl, cycloalkylalkyl, cycloalkenyl, alkylthio, arylthio, amino, alkylamino, dialkylamino, aralkoxy, arylthio, and alkylthioalkyl.
  • acyl are formyl, acetyl, benzoyl, trifluoroacetyl, phthaloyl, malonyl, nicotinyl
  • alkoxy and alkoxyalkyl embrace linear or branched oxy-containing radicals each having alkyl portions of, for example, from one to about ten carbon atoms, including the methoxy, ethoxy, propoxy, and butoxy radicals.
  • alkoxyalkyl also embraces alkyl radicals having one or more alkoxy radicals attached to the alkyl radical, that is, to form monoalkoxyalkyl and dialkoxyalkyl radicals.
  • Other alkoxy radicals are "lower alkoxy" radicals having one to six carbon atoms. Examples of such radicals include methoxy, ethoxy, propoxy, butoxy and tert-butoxy alkyls.
  • alkoxy radicals may be further substituted with one or more halo atoms, such as fluoro, chloro or bromo, to provide "haloalkoxy” radicals.
  • haloalkoxy radicals include fluoromethoxy, chloromethoxy, trifluoromethoxy, difluoromethoxy, trifluoroethoxy, fluoroethoxy, tetrafluoroethoxy, pentafluoroethoxy, and fluoropropoxy.
  • alkylamino includes "monoalkylamino" and "dialkylamino" radicals containing one or two alkyl radicals, respectively, attached to an amino radical.
  • arylamino denotes “monoarylamino” and “diarylamino” containing one or two aryl radicals, respectively, attached to an amino radical.
  • aralkylamino embraces aralkyl radicals attached to an amino radical, and denotes “monoaralkylamino” and “diaralkylamino” containing one or two aralkyl radicals, respectively, attached to an amino radical.
  • aralkylamino further includes "monoaralkyl monoalkylamino" containing one aralkyl radical and one alkyl radical attached to an amino radical.
  • alkoxyalkyl is defined as an alkyl group wherein a hydrogen has been replaced by an alkoxy group.
  • (alkylthio)alkyl is defined similarly as alkoxyalkyl, except a sulfur atom, rather than an oxygen atom, is present.
  • alkylthio and arylthio are defined as -SR, wherein R is alkyl or aryl, respectively.
  • alkylsulfmyl is defined as R-SO 2 , wherein R is alkyl.
  • alkylsulfonyl is defined as R-SO 3 , wherein R is alkyl.
  • aryl alone or in combination, includes a carbocyclic aromatic system containing one, two or three rings wherein such rings may be attached together in a pendent manner or may be fused.
  • aryl groups include phenyl, benzyl, naphthyl, and biphenyl.
  • the "aryl” group can be optionally substituted where possible with one or more of the moieties including but not limited to alkyl, hydroxyl, halo, nitro, cyano, alkenyl, alkynyl, heteroaryl, heterocyclic, carbocycle, alkoxy, oxo, aryloxy, arylalkoxy, cycloalkyl, tetrazolyl, heteroaryloxy; heteroarylalkoxy, carbohydrate, amino acid, amino acid esters, amino acid amides, alditol, haloalkylthi, haloalkoxy, haloalkyl, hydroxyl, carboxyl, acyl, acyloxy, amino, aminoalkyl, aminoacyl, amido, alkylamino, dialkylamino, arylamino, nitro, cyano, thiol, imide, sulfonic acid, sulfate, sulfonate, sulfony
  • halo includes fluoro, bromo, chloro, and iodo.
  • heterocyclic includes nonaromatic cyclic groups that may be partially (e.g., contains at least one double bond) or fully saturated and wherein there is at least one heteroatom, such as oxygen, sulfur, nitrogen, or phosphorus in the ring.
  • heteroaryl or heteroaromatic refers to an aromatic that includes at least one sulfur, oxygen, nitrogen or phosphorus in the aromatic ring.
  • heterocylics and heteroaromatics include pyrrolidinyl, tetrahydrofuryl, piperazinyl, piperidinyl, morpholino, thiomorpholino, tetrahydropyranyl, imidazolyl, pyrolinyl, pyrazolinyl, indolinyl, dioxolanyl, or 1,4-dioxanyl.
  • Suitable protecting groups can include but are not limited to trimethylsilyl (TMS), dimethylhexylsilyl (DMHS), t-butyldimethylsilyl (TBS or TBDMS), and t-butyldiphenylsilyl (TBDPS), trityl (Trt) or substituted trityl, alkyl groups, acyl (Ac) groups such as acetyl and propionyl, methanesulfonyl, and p-toluenelsulfonyl.
  • TMS trimethylsilyl
  • DMHS dimethylhexylsilyl
  • TBDMS t-butyldimethylsilyl
  • TBDMS t-butyldiphenylsilyl
  • Trt trityl
  • alkyl groups alkyl groups
  • acyl (Ac) groups such as acetyl and propionyl, methanesulfonyl, and
  • protecting group refers to a substituent that protects various sensitive or reactive groups present, so as to prevent said groups from interfering with a reaction. Such protection may be carried out in a well-known manner as taught by Greene, et al., Protective Groups in Organic Synthesis, John Wiley and Sons, Third Edition, 1999 or the like. The protecting group may be removed after the reaction in any manner known by those skilled in the art.
  • Non-limiting examples of protecting groups suitable for use within the present invention include but are not limited to allyl, benzyl (Bn), tertiary-butyl (t-Bu), methoxymethyl (MOM), ⁇ -methoxybenzyl (PMB), trimethylsilyl (TMS), dimethylhexylsily (TDS)I, t-butyldimethylsilyl (TBS or TBDMS), and t-butyldiphenylsilyl (TBDPS), tetrahydropyranyl (THP), trityl (Trt) or substituted trityl, alkyl groups, acyl groups such as acetyl (Ac) and propionyl, methanesulfonyl (Ms), and p-toluenesulfonyl (Ts).
  • allyl benzyl (Bn), tertiary-butyl (t-Bu), methoxymethyl (MOM),
  • Such protecting groups can form, for example in the instances of protecting hydroxyl groups on a molecule: ethers such as methyl ethers, substituted methyl ethers, substituted alkyl ethers, benzyl and substituted benzyl ethers, and silyl ethers; and esters such as formate esters, acetate esters, benzoate esters, silyl esters and carbonate esters, as well as sulfonates, and borates.
  • ethers such as methyl ethers, substituted methyl ethers, substituted alkyl ethers, benzyl and substituted benzyl ethers, and silyl ethers
  • esters such as formate esters, acetate esters, benzoate esters, silyl esters and carbonate esters, as well as sulfonates, and borates.
  • prodrug refers to compounds that are transformed in vivo to a compound of the present invention, for example, by hydrolysis. Prodrug design is discussed generally in Hardma et al. (Eds.), Goodman and Gilman's The Pharmacological Basis of Therapeutics, 9th ed., pp. 11-16 (1996). A thorough discussion is also provided by Higuchi, et al., in Prodrugs as Novel Delivery Systems, Vol. 14, ASCD Symposium Series, and in Roche (ed.), Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press (1987). Typically, administration of a drug is followed by elimination from the body or some biotransformation whereby the biological activity of the drug is reduced or eliminated.
  • a biotransformation process can lead to a metabolic by-product that is more or equally active compared to the drug initially administered.
  • Increased understanding of these biotransformation processes permits the design of so-called "prodrugs," which, following a biotransformation, become more physiologically active in their altered state.
  • Prodrugs therefore, as used within the scope of the present disclosure, encompass compounds that are converted by some means to pharmacologically active metabolites.
  • prodrugs can be converted into a pharmacologically active form through hydrolysis of, for example, an ester or amide linkages thereby introducing or exposing a functional group on the resultant product.
  • the prodrugs can be designed to react with an endogenous compound to form a water-soluble conjugate that further enhances the pharmacological properties of the compound, for example, increased circulatory half-life.
  • prodrugs can be designed to undergo covalent modification on a functional group with, for example, glucuronic acid, sulfate, glutathione, an amino acid, or acetate.
  • the resulting conjugate can be inactivated and excreted in the urine, or rendered more potent than the parent compound.
  • High molecular weight conjugates also can be excreted into the bile, subjected to enzymatic cleavage, and released back into the circulation, thereby effectively increasing the biological half-life of the originally administered compound.
  • a "therapeutically effective dose” refers to that amount of the compound that results in achieving the desired effect. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD 5O (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which is expressed as the ratio of LD 50 to ED 5O . Compounds that exhibit high therapeutic indices (i.e., a toxic dose that is substantially higher than the effective dose) are preferred. The data obtained can be used in formulating a dosage range for use in humans. The dosage of such compounds preferably lies within a range of circulating concentrations that include the ED 50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed, and the route of administration utilized.
  • the term "host”, as used herein, refers to a cell or organism that exhibits the properties associated with abnormal cell proliferation.
  • the hosts are typically vertebrates, including both birds and mammals.
  • the host is typically a mammal, and is typically from the family of Primates, Carnivora, Proboscidea, Perissodactyla, Artiodactyla, Rodentia, and Lagomorpha.
  • the mammal vertebrate of the present invention be Cards familiaris (dog), Felis catus (cat), Elephas maximus (elephant), Equus caballus (horse), Sus domesticus (pig), Camelus dromedarious (camel), Cervus axis (deer), Giraffa camelopardalis (giraffe), Bos taurus (cattle/cows), Copra hircus (goat), Ovis aries (sheep), Mus musculus (mouse), Lepus brachyurus (rabbit), Mesocricetus auratus (hamster), Cavia porcellus (guinea pig), Meriones unguiculatus (gerbil), and Homo sapiens (human). Birds can also be suitable as hosts within the confines of the present invention include Gallus domesticus (chicken) and Meleagris gallopavo (turkey). Most commonly, the host or patient is Homo sapiens (human).
  • stannane coupling partners which can be used in the Stille reaction are shown in Scheme 8 ( Figure 8).
  • Stannanes 14k, 141 and 14o can be obtained from commercial sources, whereas stannanes 14e-g and 14m,n can be prepared using established procedures [Dondoni, A., et al, Synthesis, pp. 757-759 (1986)].
  • the remaining coupling partners 14a-d, 14h-j, and 14p-r, as well as pyridine stannanes 14s-y were prepared from readily accessible 2,4- dibromothiazole (15) [Reynaud, P., et al., Bull. Soc. CHm. Fr., p.
  • Conversion of terminal olefin (24) to carboxylic acid (25) is accomplished through a two step process: a) ozonolysis in dichloromethane followed by exposure to triphenylphosphine to provide the aldehyde (not shown), and b) oxidation of the aldehyde with NaClO 2 (e.g, in the presence of NaH 2 PO 4 ) to give acid (25).
  • Aldehyde (29) is prepared as shown in Scheme 10 below, wherein TBS-protected alcohol (propane 1,2-diol), amine (2-amino-propan-l-ol), or thiol (2-mercapto-propan-l-ol) (27a-c) is reacted with allyl bromide (28) in the presence of base to generate the TBS-ether coupling product.
  • TBS-protected alcohol propane 1,2-diol
  • amine 2-amino-propan-l-ol
  • thiol 2-mercapto-propan-l-ol
  • Esterification of acid (25) with allylic alcohol (22) is effected using EDCI or DCC and 4-DMAP (although other known, suitable reagents are envisioned to work) to afford, after purification, pure iodo ester (26).
  • Merger of ester (26) with (29a-c) is accomplished by treating ester (26) with LDA in an ether solvent (such as tetrahydrofuran) for a period of time sufficient to effect deprotonation. This is followed by the addition of aldehyde 29a-c, leading to a facile aldol reaction and providing metathesis precursor 30 after selective protection of the primary alcohols.
  • olefins can be epoxidized into final analogues (33a) and (35a), respectfully, by the use of any suitable epoxidizing agent, such as in-situ generated methylperoxycarboximidic acid [Chaudhuri, N., et ah, J. Org. Chem., 47: pp. 5196-5198 (1982)], dimethyldioxirane, or methyl(trifluoromethyl)dioxirane [Nicolaou, K.C., et ah, J. Am. Chem. Soc, 119: pp. 7974-7991 (1997)], proceeds in high yield and good diastereoselectivity to yield the final stereocontrolled products.
  • any suitable epoxidizing agent such as in-situ generated methylperoxycarboximidic acid [Chaudhuri, N., et ah, J. Org. Chem., 47: pp. 5196-5198 (1982)], di
  • Olefinic macrolactones (32) and (34) are transformed into the respective aziridine (33b, 35b) and cyclopropane (33c, 35c) analogs by reacting the respective alkenes with an aziridination reagent such as those described by Evans, D. A., et al. [J. Am. Chem. Soc, 116: p. 2742 (1994)], or a known cyclopropanation method such as the Charette cyclopropanation [Charette, A.B., et ah, J. Am. Chem. Soc, 120: pp. 11943-11952 (1998)].
  • an aziridination reagent such as those described by Evans, D. A., et al. [J. Am. Chem. Soc, 116: p. 2742 (1994)]
  • a known cyclopropanation method such as the Charette cyclopropanation [Charette, A.B., et ah, J
  • bromo-alcohol 36a is first protected as the tert-butyldimethylsilyl (TBS) ether using standard methodologies (e.g., TBS-Cl, imidazole). Coupling of protected bromide 36b with commercially available 37a-c using an appropriate base (e.g., K 2 CO 3 , N,N-diisopropylethylamine) is followed by the deprotection (HF-Pyridine complex) and oxidation of the alcohol to the appropriate aldehyde
  • TBS tert-butyldimethylsilyl
  • ring closing precursor (39) is prepared by the reaction of (38a-c) with ally iodide (26) using LDA in an ether solvent, followed by protection of the primary alcohol with TBSOTf.
  • Compound (39) is cyclized by ring-closing etal
  • olefins can be epoxidized into final analogues (42a) and (44a), respectfully, by the use of any suitable epoxidizing agent, such as in-situ generated methylperoxycarboximidic acid [Chaudhuri, N., et ah, J. Org. Chem., 47: pp. 5196-5198 (1982)], dimethyldioxirane, or methyl(trifluoromethyl)dioxirane [Nicolaou, K. C, et ah, J. Am. Chem. Soc, 119: pp. 7974- 7991 (1997)], to yield the final stereocontrolled products.
  • any suitable epoxidizing agent such as in-situ generated methylperoxycarboximidic acid [Chaudhuri, N., et ah, J. Org. Chem., 47: pp. 5196-5198 (1982)], dimethyldioxirane, or methyl(trifluor
  • Olefinic macrolactones (41) and (43) are transformed into the respective aziridine (42b, 44b) and cyclopropane (42c, 44c) analogs by reacting the respective alkenes with an aziridination reagent such as those described by Evans, D.A., [J. Am. Chem. Soc, 116: p. 2742 (1994)], or a known cyclopropanation method such as the Charette cyclopropanation [Charette, A.B., et ah, J. Am. Chem. Soc, 120: pp. 11943-11952 (1998)].
  • an aziridination reagent such as those described by Evans, D.A., [J. Am. Chem. Soc, 116: p. 2742 (1994)]
  • a known cyclopropanation method such as the Charette cyclopropanation [Charette, A.B., et ah, J. Am. Chem. Soc, 120
  • Epoxidation using mCPBA, dimethyldioxirane, or any other suitable epoxidizing agent will provide C6-C8 bridged analogues (68) and (70).
  • Olefins (67) and (69) can also be transformed to the aziridine or cyclopropane derivatives as described previously.
  • Known compound (77) is converted to allyl compound (78) by reacting 3- iodopropene with an appropriate base, followed by global deprotection using HF-pyridine complex and treatment with S ⁇ 3 -pyridine complex. Condensation of the dianion of (76) with (78) using LDA, followed by deprotection (HF-pyridine complex) and sequential oxidation to the alcohol using a Swern oxidation and NaClO 2 affords hydroxy acid cyclic precursor (79), which is converted to macrolactone (80) according to known esterification methods, such as the Yamaguchi method using 2,4,6-trichlorobenzoylchloride [Mulzer, J., et ah, Synthesis, pp.
  • intermediate (77) is alternatively converted to epoxide intermediate (83) using the Sharpless epoxidation.
  • Epoxide (83) is then converted to the target (82) in a manner similar to that described above for intermediate (77). That is, conversion of the alcohol to the terminal alkene is followed by condensation with intermediate (76), oxidation, maacrolactonization, and finally formation of the C4-C26 bridge using Grubbs catalyst.
  • C4-C26 Bridged, C17-pyridyl epothilone analogues such as compounds (90) and (95) can be prepared as shown in Scheme 6, similar to the methods described for the C4-C26 bridged thiazole analogues (81) and (82).
  • Readily accessible intermediate (84) is converted to the vinyl iodide (85) by reaction of 3-iodo-propene with alcohol (84) in the presence of a base.
  • Stille coupling of vinyl iodide (85) with an appropriate methyl pyridyl stannane (14t- w) in the presence of a palladium catalyst (Pd(AcCN) 2 Cl 2 ) furnishes pyridine intermediate (86).
  • intermediate (84) is first epoxidated using known procedures, followed by formation of the allyl vinyl iodide (91) using 3-iodopropene and an appropriate base. Stille coupling pyridyl stannane (14s-x) in the presence of a palladium catalyst (Pd(AcCN) 2 Cl 2 ) furnishes pyridine intermediate (92).
  • Scheme 7 illustrates the proposed synthesis of C2-C10 bridged epothilone analogs in accordance with the present invention.
  • 3-Hydroxymethyl-hex-5-en-2-one (96a) is protected as the TBS ether under standard conditions to afford (96b).
  • Coupling of (96b) with 57, followed by oxidation affords keto aldehyde (97).
  • Cyclization to the macrolactone alkene is accomplished using [RuCl 2 ((R)- BINAP) 2 ][Et 3 N], followed by reaction with Grubbs catalyst to afford cyclic compound (106).
  • Selective deprotection using HF-pyridine complex is followed by C2-C10 bridge formation using TsCl in the presence of a base such as triethylamine, producing bridged thiazolyl epothilone analogue (107).
  • the C17-pyridyl epothilone analogue (109) is prepared by reacting intermediate (103) with allylic alcohol pyridine derivative (108), followed by cyclization and bridge formation using synthetic sequences similar to those described above for the thiazole derivative (107).
  • compounds of the present invention have chiral centers and may exist in and be isolated in optically active and racemic forms. Some compounds may exhibit polymorphism. It is to be understood that the present invention encompasses any racemic, optically-active, diastereomeric, polymorphic, or stereoisomeric form, or mixtures thereof, of a compound of the invention, which possess the useful properties described herein. It is now well known in the art how to prepare optically active forms (for example, by resolution of the racemic form by recrystallization techniques, by synthesis from optically-active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase).
  • Examples of methods to obtain optically active materials include at least the following. i) physical separation of crystals - a technique whereby macroscopic crystals of the individual enantiomers are manually separated. This technique can be used if crystals of the separate enantiomers exist, i.e., the material is a conglomerate, and the crystals are visually distinct; ii) simultaneous crystallization - a technique whereby the individual enantiomers are separately crystallized from a solution of the racemate, possible only if the latter is a conglomerate in the solid state; iii) enzymatic resolutions - a technique whereby partial or complete separation of a racemate by virtue of differing rates of reaction for the enantiomers with an enzyme; iv) enzymatic asymmetric synthesis - a synthetic technique whereby at least one step of the synthesis uses an enzymatic reaction to obtain an enantiomerically pure or enriched synthetic precursor of the desired enantiomer; v)
  • the resulting diastereomers are then separated by chromatography or crystallization by virtue of their now more distinct structural differences and the chiral auxiliary later removed to obtain the desired enantiomer; vii) first- and second-order asymmetric transformations - a technique whereby diastereomers from the racemate equilibrate to yield a preponderance in solution of the diastereomer from the desired enantiomer or where preferential crystallization of the diastereomer from the desired enantiomer perturbs the equilibrium such that eventually in principle all the material is converted to the crystalline diastereomer from the desired enantiomer.
  • kinetic resolutions this technique refers to the achievement of partial or complete resolution of a racemate (or of a further resolution of a partially resolved compound) by virtue of unequal reaction rates of the enantiomers with a chiral, non-racemic reagent or catalyst under kinetic conditions; ix) enantiospecific synthesis from non-racemic precursors - a synthetic technique whereby the desired enantiomer is obtained from non-chiral starting materials and where the stereochemical integrity is not or is only minimally compromised over the course of the synthesis; x) chiral liquid chromatography - a technique whereby the enantiomers of a racemate are separated in a liquid mobile phase by virtue of their differing interactions with a stationary phase.
  • the stationary phase can be made of chiral material or the mobile phase can contain an additional chiral material to provoke the differing interactions; xi) chiral gas chromatography - a technique whereby the racemate is volatilized and enantiomers are separated by virtue of their differing interactions in the gaseous mobile phase with a column containing a fixed non-racemic chiral adsorbent phase; xii) extraction with chiral solvents - a technique whereby the enantiomers are separated by virtue of preferential dissolution of one enantiomer into a particular chiral solvent; xiii) transport across chiral membranes - a technique whereby a racemate is placed in contact with a thin membrane barrier.
  • the barrier typically separates two miscible fluids, one containing the racemate, and a driving force such as concentration or pressure differential causes preferential transport across the membrane barrier. Separation occurs as a result of the non-racemic chiral nature of the membrane which allows only one enantiomer of the racemate to pass through.
  • Hosts including mammals and particularly humans, suffering from any of the disorders described herein, including both diabetic vascular disorders and ocular inflammatory disorders, can be treated by administering to the host an effective amount of a compound described herein, or a pharmaceutically acceptable prodrug, ester, and/or salt thereof, optionally in combination with a pharmaceutically acceptable carrier or diluent.
  • the active materials can be administered by any appropriate route, for example, orally, parenterally, intravenously, intradermally, intramuscularly, subcutaneously, sublingually, transdermally, bronchially, pharyngolaryngeal, intranasally, topically such as by a cream or ointment, rectally, intraarticular, intracisternally, intrathecally, intravaginally, intraperitoneally, intraocularly, by inhalation, bucally or as an oral or nasal spray.
  • parenterally intravenously, intradermally, intramuscularly, subcutaneously, sublingually, transdermally, bronchially, pharyngolaryngeal, intranasally, topically such as by a cream or ointment, rectally, intraarticular, intracisternally, intrathecally, intravaginally, intraperitoneally, intraocularly, by inhalation, bucally or as an oral or nasal spray.
  • the compounds of the present invention can be used in the form of pharmaceutically acceptable salts derived from inorganic or organic acids.
  • pharmaceutically acceptable salt is meant those salts which are, within the scope of sound medical judgement, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like and are commensurate with a reasonable benefit/risk ratio.
  • Pharmaceutically acceptable salts are well-known in the art. For example, P. H. Stahl, et al. describe pharmaceutically acceptable salts in detail in "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" (Wiley VCH, Zurich, Switzerland: 2002).
  • the salts can be prepared in situ during the final isolation and purification of the compounds of the present invention or separately by reacting a free base function with a suitable organic acid.
  • Representative acid addition salts include, but are not limited to acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsufonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethansulfonate (isethionate), lactate, maleate, methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate
  • the basic nitrogen-containing groups can be quaternized with such agents as lower alkyl halides such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyl and diamyl sulfates; long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides; arylalkyl halides like benzyl and phenethyl bromides and others. Water or oil-soluble or dispersible products are thereby obtained.
  • lower alkyl halides such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides
  • dialkyl sulfates like dimethyl, diethyl, dibutyl and diamyl sulfates
  • long chain halides such as decyl
  • acids which can be employed to form pharmaceutically acceptable acid addition salts include such inorganic acids as hydrochloric acid, hydrobromic acid, sulphuric acid and phosphoric acid and such organic acids as oxalic acid, maleic acid, succinic acid and citric acid.
  • Base addition salts can be prepared in situ during the final isolation and purification of compounds of this invention by reacting a carboxylic acid-containing moiety with a suitable base such as the hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation or with ammonia or an organic primary, secondary or tertiary amine.
  • a suitable base such as the hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation or with ammonia or an organic primary, secondary or tertiary amine.
  • Pharmaceutically acceptable salts include, but are not limited to, cations based on alkali metals or alkaline earth metals such as lithium, sodium, potassium, calcium, magnesium and aluminum salts and the like and nontoxic quaternary ammonia and amine cations including ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, diethylamine, ethylamine and the like.
  • Other representative organic amines useful for the formation of base addition salts include ethylenediamine, ethanolarnine, diethanolamine, piperidine, piperazine and the like.
  • Typical salts of the compounds of the present invention include phosphate, tris and acetate.
  • compositions may be also obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion.
  • a sufficiently basic compound such as an amine
  • a suitable acid affording a physiologically acceptable anion.
  • Alkali metal for example, sodium, potassium or lithium
  • alkaline earth metal for example calcium or magnesium
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing into association a compound of the invention or a pharmaceutically acceptable salt or solvate thereof ("active ingredient") with the carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both and then, if necessary, shaping the product into the desired formulation. [0216]
  • the compound or a pharmaceutically acceptable ester, salt, solvate or prodrug can be mixed with other active materials that do not impair the desired action, or with materials that supplement the desired action, including other drugs against diabetic vascular disease or ocular inflammatory disease.
  • Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical application can include, for example, the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • the parental preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • carriers can be physiological saline or phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • compositions of this invention for parenteral injection comprise pharmaceutically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions and sterile powders for reconstitution into sterile injectable solutions or dispersions.
  • suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol, and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate.
  • Proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • compositions may also contain adjuvants including immunostimulating factors (including immunostimulatory nucleic acid sequences, including those with CpG sequences), preservative agents, wetting agents, emulsifying agents, and dispersing agents.
  • immunostimulating factors including immunostimulatory nucleic acid sequences, including those with CpG sequences
  • preservative agents wetting agents, emulsifying agents, and dispersing agents.
  • wetting agents including those with CpG sequences
  • emulsifying agents include dispersing agents.
  • dispersing agents include various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like.
  • isotonic agents for example, sugars, sodium chloride and the like.
  • Prolonged absorption of the injectable pharmaceutical form may be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Suspensions in addition to the active compounds, may contain suspending agents, as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, macrocrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, tragacanth, and mixtures thereof.
  • suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, macrocrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, tragacanth, and mixtures thereof.
  • the formulation compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
  • the active compounds can also be in micro-or nano-encapsulated form, if appropriate, with one or more excipients.
  • Injectable depot forms are made by forming microencapsulated matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues.
  • biodegradable polymers such as polylactide-polyglycolide.
  • Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues.
  • the injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium just prior to use.
  • Injectable preparations for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic, parenterally acceptable diluent or solvent such as a solution in 1,3-butanediol.
  • Suitable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil can be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid are used in the preparation of injectables.
  • Formulations for parenteral (including subcutaneous, intradermal, intramuscular, intravenous and intraarticular) administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • the formulations may be presented in unit-dose or multi-dose containers, for example sealed ampules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline, water-for-injection, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
  • Yet another aspect of the present invention involves formulating the inventive compounds of Formulas I-DC using polymers such as biopolymers or biocompatible (synthetic or naturally occurring) polymers.
  • Biocompatible polymers can be categorized as biodegradable and non-biodegradable. Biodegradable polymers degrade in vivo as a function of chemical composition, method of manufacture, and implant structure.
  • Illustrative examples of synthetic polymers include polyanhydrides, polyhydroxyacids such as polylactic acid, polyglycolic acids and copolymers thereof, polyesters polyamides polyorthoesters and some polyphosphazenes.
  • Illustrative examples of naturally occurring polymers suitable for use with the present invention include proteins and polysaccharides such as collagen, hyaluronic acid, albumin, and gelatin.
  • Another method of formulation of the present invention involves conjugating the compounds of Formulas I-IX to a polymer that enhances aqueous solubility.
  • suitable polymers include but are not limited to polyethylene glycol, poly-(d-glutamic acid), poly-(l -glutamic acid), poly-(l -glutamic acid), poly-(d-aspartic acid), poly-(l-aspartic acid), poly-(l-aspartic acid) and copolymers thereof.
  • Polyglutamic acids having molecular weights between about 5,000 to about 100,000 are typical, with molecular weights between about 20,000 and 80,000 being more typical and with molecular weights between about 30,000 and 60,000 being most common.
  • the polymer is conjugated via an ester linkage to one or more hydroxyls of an inventive epothilone using a protocol as essentially described by U.S. Pat. No. 5,977,163 which is incorporated herein by reference.
  • Typical conjugation sites include the hydroxyl off carbon-21 in the case of 21-hydroxy-derivatives of the present invention.
  • Other conjugation sites include but are not limited to the hydroxyl off carbon 3 and/or the hydroxyl off carbon 7.
  • inventive compounds of general Formulas I-DC are conjugated to a monoclonal antibody. This strategy allows the targeting of the inventive compounds to specific targets.
  • General protocols for the design and use of conjugated antibodies are described in "Monoclonal Antibody-Based Therapy of Cancer” [by Michael L. Grossbard, ed. (1998)], which is incorporated herein by reference.
  • the methods of the invention can be practiced using pharmaceutical formulations containing compounds of Formula I, Formula II, Formula III, Formula IV, Formula V, Formula VI, Formula VII, Formula VIII, Formule IX or combinations thereof.
  • Treatment of abnormal cellular prolilferation disorders in a host, such as a human, may be therapeutic by administering a compound of Formula I-DC to treat an existing condition so as to mitigate the effects of that event.
  • treatment of abnormal cellular proliferation diseases or disorders in a human may be prophylactic by administering a compound of Formula I-Formula IX in anticipation of a worsening condition of a specific abnormal cellular proliferation disease, for example, in a patient whose occupation, lifestyle, or exposure to irritants will expectedly worsen an existing condition of the abnormal cellular proliferation disease or disorder.
  • the underlying cause of the disease state will not be prevented or cured, but may be reduced in severity or extent and its symptoms ameliorated by administration of compounds of Formula I, Formula II, Formula ITI, Formula IV, Formula V, Formula VI, Formula VII, or Formula VIII, or Formula IX (and their formulations) using the method of the present invention.
  • the compounds of the invention are typically administered by any appropriate administration route, for example, orally, parenterally, intravenously, intradermally, intramuscularly, subcutaneously, sublingually, transdermally, bronchially, pharyngolaryngeal, intranasally, topically such as by a cream or ointment, rectally, intraarticular, intracisternally, intrathecally, intravaginally, intraperitoneally, intraocularly, by inhalation, bucally or as an oral or nasal spray.
  • the route of administration may vary, however, depending upon the condition and the severity of the diabetic vascular disease or ocular inflammation.
  • the precise amount of compound administered to a host or patient will be the responsibility of the attendant physician. However, the dose employed will depend on a number of factors, including the age and sex of the patient, the precise disorder being treated, and its severity.
  • a formulation for intravenous use can comprises an amount of an inventive compound of Formula I-IX ranging from about 1 mg/mL to about 25 mg/mL, or from about 5 mg/mL to 15 mg/mL, and more typically about 10 mg/mL.
  • a dose range of from about 0.001 mg/kg per day to about 2500 mg/kg per day is typical.
  • the dose range is from about 0.1 mg/kg per day to about 1000 mg/kg per day.
  • the dose range is from about 0.1 mg/kg per day to about 500 mg/kg per day, including 1 mg/kg, 2 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg, kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 100 mg/kg, 200 mg/kg, 300 mg/kg, 400 mg/kg, 500 mg/kg per day, and values between any two of the values given in this range.
  • the dose range for humans is generally from about 0.005 mg to 100 g/day.
  • the dose range in accordance with the present invention is such that the blood serum level of compounds of the present invention is from about 0.01 ⁇ M to about 100 ⁇ M, and typically from about 0.1 ⁇ M to about 100 ⁇ M.
  • Suitable values of blood serum levels in accordance with the present invention include but are not limited to about 0.01 ⁇ M, about 0.1 ⁇ M, about 0.5 ⁇ M, about 1 ⁇ M, about 5 ⁇ M, about 10 ⁇ M, about 15 ⁇ M, about 20 ⁇ M, about 25 ⁇ M, about 30 ⁇ M, about 35 ⁇ M, about 40 ⁇ M, about 45 ⁇ M, about 50 ⁇ M, about 55 ⁇ M, about 60 ⁇ M, about 65 ⁇ M, about 70 ⁇ M, about 75 ⁇ M, about 80 ⁇ M, about 85 ⁇ M, about 90 ⁇ M, about 95 ⁇ M and about 100 ⁇ M, as well as any blood serum level that falls within any two of these values (e.g, between about 10 ⁇ M and about 60 ⁇
  • the compounds and formulations of the present invention can be administered in any of the known dosage forms standard in the art; in solid dosage form, semi-solid dosage form, or liquid dosage form, as well as subcategories of each of these forms.
  • Solid dosage forms for oral administration include capsules, caplets, tablets, pills, powders, lozenges, and granules.
  • the active compound is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and salicylic acid; b) binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia; c) humectants such as glycerol; d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; e) solution retarding agents such as paraffin; f) absorption accelerators such as quaternary ammonium compounds; g) wetting agents such as cetyl alcohol and
  • compositions of a similar type may also be employed as fillers in soft and hard- filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
  • the solid dosage forms of tablets, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes.
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, lubricating, surface active or dispersing agent.
  • Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein.
  • compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the compounds of this invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.
  • suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.
  • Semi-liquid dosage forms include those dosage forms that are too soft in structure to qualify for solids, but to thick to be counted as liquids. These include creams, pastes, ointments, gels, lotions, and other semisolid emulsions containing the active compound of the present invention.
  • the ointments, pastes, creams and gels may contain, in addition to an active compound of this invention, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and
  • Dosage forms for topical or transdermal administration of a compound of this invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches, optionally mixed with degradable or nondegradable polymers.
  • the active component is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required.
  • Ophthalmic formulation, ear drops, eye ointments, powders and solutions are also contemplated as being within the scope of this invention.
  • Formulations containing compounds of the invention may be administered through the skin by an appliance such as a transdermal patch.
  • Patches can be made of a matrix such as polyacrylamide, polysiloxanes, or both and a semi-permeable membrane made from a suitable polymer to control the rate at which the material is delivered to the skin.
  • Other suitable transdermal patch formulations and configurations are described in U.S. Pat. Nos. 5,296,222 and 5,271,940, as well as in Satas, D., et al, "Handbook of Pressure Sensitive Adhesive Technology, 2 nd Ed.”, Van Nostrand Reinhold, 1989: Chapter 25, pp. 627-642.
  • Powders and sprays can contain, in addition to the compounds of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances.
  • Sprays can additionally contain customary propellants such as chlorofluorohydrocarbons.
  • excipients are described, for example, in "Handbook of Pharmaceutical Excipients, 3 rd Ed.”, A.H. Kibbe, Ed. (American Pharmaceutical Association and Pharmaceutical Press, Washington, DC, 2000), the entire contents of which are included herein by reference.
  • the active compounds of the present invention are prepared with carriers that will protect the compound against rapid elimination from the body or rapid release, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylacetic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
  • Compounds of the present invention can be used in combination with radiation and chemotherapy treatment, including induction chemotherapy, primary (neoadjuvant) chemotherapy, and both adjuvant radiation therapy and adjuvant chemotherapy, hi addition, radiation and chemotherapy are frequently indicated as adjuvants to surgery in the treatment of cancer.
  • radiation and chemotherapy in the adjuvant setting is to reduce the risk of recurrence and enhance disease-free survival when the primary tumor has been controlled.
  • Chemotherapy is utilized as a treatment adjuvant for lung and breast cancer, frequently when the disease is metastatic.
  • Adjuvant radiation therapy is indicated in several diseases including lung and breast cancers.
  • Compounds of the present invention also are useful following surgery in the treatment of cancer in combination with radio- and/or chemotherapy.
  • Chemotherapeutic agents that can be used in combination with a microtubule stabilizer of the present invention include, but are not limited to, alkylating agents, antimetabolites, hormones and antagonists, microtubule stabilizers, radioisotopes, antibodies, as well as natural products, and combinations thereof.
  • a compound of the present invention can be administered with antibiotics, such as doxorubicin and other anthracycline analogs, nitrogen mustards, such as cyclophosphamide, pyrimidine analogs such as 5-fluorouracil, cisplatin, hydroxyurea, and the like.
  • the compound in the case of mixed tumors, such as adenocarcinoma of the breast, where the tumors include gonadotropin-dependent and gonadotropin-independent cells, the compound can be administered in conjunction with leuprolide or goserelin (synthetic peptide analogs of LH- RH)
  • Other antineoplastic protocols include the use of an inhibitor compound with another treatment modality, e.g., surgery or radiation, also referred to herein as "adjunct anti ⁇ neoplastic modalities.”
  • chemotherapeutic agents useful for combination with compounds of the present invention include but are not limited to alkylating agents, such as nitrogen mustards (e.g., mechlorethanmine, cyclophosphamide, ifosfamide, melphalan, and chlorambucil); nitrosureas, alkyl sulfonates, such as busulfan; triazines, such as dacarbazine (DTIC); antimetabolites; folic acid analogs, such as methotrexate and trimetrexate; pyrimidine analogs, such as 5-fluorouracil, fluorodeoxyuridine, gemcitabin, cytosine arabinoside (AraC, cytarabine), 5-azacytidine, and 2,2 l -difluorodeoxycytidine; purine analogs, such as 6- mercaptopurine, 6-thioguanine ,
  • alkylating agents such as nitrogen mustards (e.g., mech
  • the complex accommodates the extensive epothilone SAR data developed for microtubules composed of wild type tubulin (TB) and the more limited resistance data from mutant tubulins.
  • TB wild type tubulin
  • the structure demonstrates that, while epo-A and Taxol overlap in their occupation of a rather expansive common binding site on tubulin, the expectation of a common pharmacophore is unfulfilled as each ligand exploits the binding pocket in a unique and qualitatively independent manner (cf. Figure 10).
  • Complementing the epoxide fold is a near parallel orientation of the C-O bonds at C3, C5 and C7, an alignment ideal for the ligand-tubulin interaction (see below).
  • This arrangement leads to a very different set of backbone torsion angles from Cl to C9 for epothilone A in comparison with either its single crystal X-ray structure or the transfer NOE NMR structure of tubulin complex of epo-A [Carlomagno, T., et al, Angew. Chem. Int. Ed. Engl, 42: pp. 2511 (2003)] , the latter being locked in an unpolymerized soluble form of the protein.
  • Taxol® experiences significantly less cross-resistance to Thr274 (10-fold) and Arg282 (7-fold) mutations in strong contrast to cellular response to epo-A (40 and 57-fold, respectively) [He, L., et ah, Molecular Cancer Therapeutics, 1: pp. 3-10 (2001)]).
  • Ala231 is within hydrogen bonding contact of His227 which anchors epothilone in the binding pocket.
  • introduction of the polar threonine is predicted to perturb the His-anchor and to compromise ligand binding.
  • the binding model for epo-A accommodates a range of epothilone structure-activity data [Wartman, M., et ah, Current Medicinal Chemistry: Anti-Cancer Agents, 2: pp. 123-148 (2002)]. This is illustrated using six diverse bioactive modifications, as shown in Figure 20. [0254] First, it should be noted that alkyl chain extension at C 12 from methyl (epo-B) to hexyl does not eliminate in vitro activity, though in general methyl appears optimal for biological activity [Taylor, R.E., et al, J. Am. Chem.
  • the two phenyl Cl 3 side chain termini and the C2' OH group associate with TB centers distant from epothilone (i.e. Ser234/Pro358,Val21 and Gly368, respectively), while the C2 and C3' phenyl groups sandwich His227 in 3-ring stack, neither of which are observed for epothilone.
  • the ether oxygen of Taxol's oxetane ring interacts weakly with Thr274 at one end of the M-loop, while an indirect hydrophobic chain to the other end operates via the Cl 8 methyl ( Figure 16).
  • the only significant common non-bonded contact for the two ligands appears to occur through C7-OH in each molecule.
  • the double arginine relocation reflects a subtle reorganization of M-loop residues not previously seen with taxanes, but both epothilone and Taxol bridge the M-loop and helix H7 adjacent to the nucleotide binding site and thereby promote tubulin polymerization and microtubule stability.
  • tubulin displays a promiscuous binding pocket with the bound molecules exploiting contacts with an optimal subset of binding pocket residues.
  • the methods for using the associated three-dimensional coordinates to predict, design, or select therapeutic analogs results from the chemical information inherently encoded in them.
  • the unique orientation of epothilone within this coordinate system provides a template for further development.
  • Those skilled in the art of computational modeling and/or drug design will be able to read these coordinates into most modern computational modeling/graphics software to visualize the special relationships of the chemical feature as a catalyst for development of new ideas.
  • the coordinated may be transformed mathematically through, dynamics, manual modeling and rebuilding, in silico mutation, and used for docking and or pharmacophore design as needed for high throughput screening.
  • the model may be used to predict, and or select specific drugs most appropriate for treatment of the mutated cancers. Examples of its use are provided below.
  • a method for identifying compounds which can mimic epothilone binding to tubulin by comparing the 3-D structure of candidate compounds with the 3-D molecular model shown in Figs 9, 11 and 12.
  • the method comprises inputting the coordinate data of the molecular model in a data structure such that the interatomic distances between the atoms of tubulin and epothilone A are easily retrieved, and comparing these distances with those of the 3D structure of the candidate compounds.
  • the coordinate data is, or is derived firom, data described in Appendix 1.
  • distances between hydrogen-bonding heteroatoms of candidate compounds and the heteroatoms that form the binding pocket in the 3D molecular model of epothilone and tubulin are compared, allowing the identification of those candidate compounds which form the most stable complexes with the 3-D molecular model binding pocket.
  • the coordinates of the model are adjusted to increase efficiency of identification.
  • a method of identifying compounds that can mimic epothilone binding to tubulin comprising the steps of: applying a 3-dimensional molecular modeling algorithm to the atomic coordinates shown in Figure 9, 11 or 12 to determine the spatial coordinates of the binding pocket of tubulin for epothilone; and electronically screening the stored spatial coordinates of a set of candidate compounds against these spatial coordinates.
  • these of the protein P binding pocket to identify compounds that can bind to protein P.
  • a method of identifying compounds that can mimic binding of epothilone A to tubulin comprising the steps of: applying a 3-dimensional molecular modeling algorithm to the atomic coordinates of shown in Figures 9, 11 and 12 and in Table 1 to determine the spatial coordinates of the binding pocket; and electronically screening the stored spatial coordinates of a set of candidate compounds against the spatial coordinates of the epothilone/tubulin binding pocket to identify compounds.
  • R5 H, Methyl, Ethyl, Propyl, Butyl, isobutyl, N O S
  • EpothiloneA bound to tubulin is used as the structural template for positioning of these modifications.
  • the mutation specific modifications would also apply to analogs EpoB, C, D, and can be used in combination with modifications at other positions.
  • the following examples include compounds with modification of epothilone template at C7 position for Arg282 mutations. Cancer cells with a single mutation Arg282Gln have been grown which exibit 57-fold resistance to treatment with EpoA. Our model suggest that this is due to loss of hydrogen bonding with the C7-OH. Extension of the C7 substitution and maintaining correct stereo chemistry should restore hydrogen bonding and activity in drug resistant tumors.
  • R (n)CH 2 -OH
  • Gln282 n can be 2.
  • the compounds described herein are useful to treat abnormal cellular proliferation.
  • Cellular differentiation, growth, function and death are regulated by a complex network of mechanisms at the molecular level in a multicellular organism. In the healthy animal or human, these mechanisms allow the cell to carry out its designed function and then die at a programmed rate.
  • Abnormal cellular proliferation notably hyperproliferation, can occur as a result of a wide variety of factors, including genetic mutation, infection, exposure to toxins, autoimmune disorders, and benign or malignant tumor induction.
  • Illustrative disorders of abnormal cell proliferation include but are not limited to tumors and cancers; unwanted angiogenesis, psoriasis, chronic eczema, atopic dermatitis, lichen planus, warts, pemphigus vulgaris, actinic keratosis, basal cell carcinoma and squamous cell carcinoma, blood vessel proliferation disorders, fibrotic disorders, autoimmune disorders, graft-versus-host rejection, disorders brought about by abnormal proliferation of mesangial cells (including human renal diseases, such as glomerulonephritis, diabetic nephropathy, malignant nephrosclerosis, thrombotic micro-angiopathy syndromes, transplant rejection, and glomerulopathies), rheumatoid arthritis, Behcet's syndrome, acute respiratory distress syndrome (ARDS), ischemic heart disease, post-dialysis syndrome, leukemia, acquired immune deficiency syndrome, vasculitis, lipid hist
  • a tumor also called a neoplasm, is a new growth of tissue in which the multiplication of cells is uncontrolled and progressive.
  • a benign tumor is one that lacks the properties of invasion and metastasis and is usually surrounded by a fibrous capsule.
  • a malignant tumor i.e., cancer
  • Malignant tumors also show a greater degree of anaplasia (i.e., loss of differentiation of cells and of their orientation to one another and to their axial framework) than benign tumors.
  • Nonlimiting examples of neoplastic diseases or malignancies include the abnormal cellular proliferation of malignant or non-malignant cells in various tissues and/or organs, including, non-limitatively, muscle, bone and/or conjunctive tissues; the skin, brain, lungs and sexual organs; the lymphatic and/or renal system; mammary cells and/or blood cells; the liver, digestive system, and pancreas; and the thyroid and/or adrenal glands.
  • pathological conditions can also include psoriasis; solid tumors; ovarian, breast, brain, prostate, colon, stomach, kidney, urothelial, oesophageal, lung, and/or testicular cancer; Karposi's sarcoma; cholangiocarcinoma; choriocarcinoma; neuroblastoma; Wilm's tumor; hematopoietic tumors of lymphoid lineage; hematopoietic tumors of myeloid lineage; tumors of mesenchymal origin; tumors of the central and peripheral nervous system Hodgkin's disease; melanomas; multiple myelomas; seminomas; chronic lymphocytic leukemias; and acute or chronic granulocytic lymphomas.
  • novel compounds in accordance with the invention are particularly useful in the treatment of non-Hodgkin's lymphoma, multiple myeloma, melanoma, and ovarian, urothelial, oesophageal, lung, and breast cancers.
  • the compounds can be utilized to prevent or delay the appearance or reappearance, or to treat these pathological conditions.
  • the compounds may be used as antiangiogenesis inhibitors for both anticancer activities or for abnormal wound healing or other hyperproliferative diseases dependent on blood vessel formation.
  • the inventive compounds are used to treat non-cancer disorders that are characterized by cellular hyperproliferation.
  • the compounds of the present invention are used to treat psoriasis, a condition characterized by the cellular hyperproliferation of keratinocytes which builds up on the skin to form elevated, scaly lesions.
  • the method comprises administering a therapeutically effective amount of an inventive compound to a subject suffering from psoriasis. The method may be repeated as necessary either to decrease the number or severity of lesions or to eliminate the lesions.
  • practice of the method will result in a reduction in the size or number of skin lesions, diminution of cutaneous symptoms (pain, burning and bleeding of the affected skin) and/or a reduction in associated symptoms (e.g., joint redness, heat, swelling, diarrhea, abdominal pain, Pathologically, practice of the method will result in at least one of the following: inhibition of keratinocyte proliferation, reduction of skin inflammation (for example, by impacting on: attraction and growth factors, antigen presentation, production of reactive oxygen species and matrix metalloproteinases), and inhibition of dermal angiogenesis.
  • Psoriasis is a benign disease of human skin generally characterized by plaques covered by thickened scales. The disease is caused by increased proliferation of epidermal cells of unknown cause. Chronic eczema is also associated with significant hyperproliferation of the epidermis. Other diseases caused by hyperproliferation of skin cells include atopic dermatitis, lichen planus, warts, pemphigus vulgaris, actinic keratosis, basal cell carcinoma and squamous cell carcinoma. Li a further embodiment of the present invention, methods of treating skin disorders associated with cellular hyperproliferation are provided.
  • hyperproliferative cell disorders include blood vessel proliferation disorders, fibrotic disorders, autoimmune disorders, graft- versus-host rejection, tumors and cancers.
  • Blood vessel proliferative disorders include angiogenic and vasculogenic disorders. Proliferation of smooth muscle cells in the course of development of plaques in vascular tissue cause, for example, restenosis, retinopathies and atherosclerosis. The advanced lesions of atherosclerosis result from an excessive inflammatory-proliferative response to an insult to the endothelium and smooth muscle of the artery wall (Ross, R. Nature, 362:801-809 (1993)). Both cell migration and cell proliferation play a role in the formation of atherosclerotic lesions.
  • the compounds of the present invention are used to treat multiple sclerosis, a condition characterized by progressive demyelination in the brain.
  • multiple sclerosis a condition characterized by progressive demyelination in the brain.
  • the method comprises administering a therapeutically effective amount of an inventive compound to a subject suffering from multiple sclerosis.
  • the method may be repeated as necessary to inhibit astrocyte proliferation and/or lessen the severity of the loss of motor function and/or prevent or attenuate chronic progression of the disease.
  • practice of the method will result in in improvement in visual symptoms (visual loss, diplopia), gait disorders (weakness, axial instability, sensory loss, spasticity, hyperreflexia, loss of dexterity), upper extremity dysfunction (weakness, spasticity, sensory loss), bladder dysfunction (urgency, incontinence, hesitancy, incomplete emptying), depression, emotional lability, and cognitive impairment.
  • practice of the method will result in the reduction of one or more of the following, such as myelin loss, breakdown of the blood-brain barrier, perivascular infiltration of mononuclear cells, immunologic abnormalities, gliotic scar formation and astrocyte proliferation, metalloproteinase production, and impaired conduction velocity.
  • the compounds of the present invention are used to treat rheumatoid arthritis, a multisystem chronic, relapsing, inflammatory disease that sometimes leads to destruction and ankyiosis of affected joints.
  • Rheumatoid arthritis is characterized by a marked thickening of the synovial membrane which forms villous projections that extend into the joint space, multilayering of the synoviocyte lining (synoviocyte proliferation), infiltration of the synovial membrane with white blood cells (macrophages, lymphocytes, plasma cells, and lymphoid follicles; called an "inflammatory synovitis"), and deposition of fibrin with cellular necrosis within the synovium.
  • pannus The tissue formed as a result of this process is called pannus and, eventually the pannus grows to fill the joint space.
  • the pannus develops an extensive network of new blood vessels through the process of angiogenesis that is essential to the evolution of the synovitis.
  • digestive enzymes matrix metalloproteinases (e.g., collagenase, stromelysin)
  • other mediators of the inflammatory process e.g., hydrogen peroxide, superoxides, lysosomal enzymes, and products of arachadonic acid metabolism
  • the pannus invades the articular cartilage leading to erosions and fragmentation of the cartilage tissue.
  • the method comprises administering a therapeutically effective amount of an inventive compound to a subject suffering from rheumatoid arthritis.
  • the method may be repeated as necessary to accomplish to inhibit synoviocyte proliferation and/or lessen the severity of the loss of movement of the affected joints and/or prevent or attenuate chronic progression of the disease.
  • practice of the present invention will result in one or more of the following: (i) decrease in the severity of symptoms (pain, swelling and tenderness of affected joints; morning stiffness, weakness, fatigue, anorexia, weight loss); (ii) decrease in the severity of clinical signs of the disease (thickening of the joint capsule, synovial hypertrophy, joint effusion, soft tissue contractures, decreased range of motion, ankylosis and fixed joint deformity); (iii) decrease in the extra-articular manifestations of the disease (rheumatic nodules, vasculitis, pulmonary nodules, interstitial fibrosis, pericarditis, episcleritis, ulceris, Felty's syndrome, osteoporosis); (iv) increase in the frequency and duration of disease remission/symptom-free periods; (v) prevention of fixed impairment and disability; and/or (vi) prevention/attenuation of chronic progression of the disease.
  • practice of the present invention will produce at least one of the following: (i) decrease in the inflammatory response; (ii) disruption of the activity of inflammatory cytokines (such as IL-I, TNOFa, FGF, VEGF); (iii) inhibition of synoviocyte proliferation; (iv) inhibition of matrix metalloproteinase activity, and/or (v) inhibition of angiogenesis.
  • inflammatory cytokines such as IL-I, TNOFa, FGF, VEGF
  • the compounds of the present invention are used to threat atherosclerosis and/or restenosis, particularly in patients whose blockages may be treated with an endovascular stent.
  • Atherosclerosis is a chronic vascular injury in which some of the normal vascular smooth muscle cells ("VSMC") in the artery wall, which ordinarily control vascular tone regulating blood flow, change their nature and develop "cancer-like” behavior.
  • VSMC normal vascular smooth muscle cells
  • These VSMC become abnormally proliferative, secreting substances (growth factors, tissue- degradation enzymes and other proteins) which enable them to invade and spread into the inner vessel lining, blocking blood flow and making that vessel abnormally susceptible to being completely blocked by local blood clotting.
  • VSMC vascular smooth muscle cells
  • the compounds of the invention can be useful for the treatment or prevention of restenosis. Restenosis is the overgrowth of cells, particularly smooth muscle cells after intervention in, for example, coronary angioplasty.
  • the compounds of the invention can be administered systemically, or can be administered locally. In one embodiment of local administration, the compounds are administered via a stent.
  • the stent can be coated or can be impregnated with the compounds. In one embodiment, the stent is coated or impregnated with a polymeric carrier that contains the compound of the invention. The concentration of compounds can be adjusted to deliver a gradient of antiproliferative compound over time.
  • the compounds can be delivered in a vehicle that is biodegradable and is degraded over a time course of from about one week to about three months, or from about one week to about two or one month.
  • the drug can be delivered over this time at steady concentrations or the concentration can vary over time.
  • a method of treating atherosclerosis and/or restenosis comprises coating a therapeutically effective amount of an inventive compound on a stent and delivering the stent to the diseased artery in a subject suffering from atherosclerosis.
  • Methods for coating a stent with a compound are described for example by U.S. Pat. Nos. 6,156,373 and 6,120, 847.
  • practice of the present invention will result in one or more of the following: (i) increased arterial blood flow; (ii) decrease in the severity of clinical signs of the disease; (iii) decrease in the rate of restenosis; or (iv) prevention/attenuation of the chronic progression of atherosclerosis.
  • practice of the present invention will produce at least one of the following at the site of stent implanataion: (i) decrease in the inflammatory response, (ii) inhibition of VSMC secretion of matrix metalloproteinases; (iii) inhibition of smooth muscle cell accumulation; and (iv) inhibition of VSMC phenotypic dedifferentiation.
  • diseases or disorders associated with uncontrolled or abnormal cellular proliferation including, but not limited to, a disease process which features abnormal cellular proliferation, e.g., benign prostatic hyperplasia, familial adenomatosis polyposis, neuro-fibromatosis, atherosclerosis, pulmonary fibrosis, arthritis, psoriasis, glomerulonephritis, restenosis following angioplasty or vascular surgery, hypertrophic scar formation, inflammatory bowel disease, transplantation rejection, endotoxic shock, and fungal infections.
  • a disease process which features abnormal cellular proliferation e.g., benign prostatic hyperplasia, familial adenomatosis polyposis, neuro-fibromatosis, atherosclerosis, pulmonary fibrosis, arthritis, psoriasis, glomerulonephritis, restenosis following angioplasty or vascular surgery, hypertrophic scar formation, inflammatory bowel disease, transplantation rejection, endotoxic shock, and fungal infections.
  • diseases or disorder associated with abnormal cellular proliferation which may be treated with compounds of the present invention include defective apoptosis-associated conditions, such as cancers (including but not limited to those types mentioned hereinabove), viral infections (including but not limited to herpesvirus, poxvirus, Epstein-Barr virus, Sindbis virus and adenovirus), prevention of AIDS development in HIV-infected individuals, autoimmune diseases (including but not limited to systemic lupus erythematosus, rheumatoid arthritis, psoriasis, autoimmune mediated glomerulonephritis, inflammatory bowel disease and autoimmune diabetes mellitus), neurodegenerative disorders (including but not limited to Alzheimer's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, Parkinson's disease, AJDS-related dementia, spinal muscular atrophy and cerebellar degeneration); myelodysplastic syndromes; aplastic anemia; ischemic injury associated with myocardial infarctions; stroke
  • Epothilone A has been incorporated in zinc sulfate stabilized tubulin sheets ( Figure 13) that diffract electrons at a resolution below 3 A.
  • Figure 13 the use of real-space molecular modeling, electron crystallographic (EC) difference mapping, and reciprocal space annealing is described to elucidate the atomic interactions of the drug with its tubulin receptor. Mapping reveals a model with an unexpected mode of binding that is consistent with the SAR of a wide range of epothilone analogs, but one that differs qualitatively from current pharmacophores based upon taxanes.
  • Three dimensional coordinates and associated structure factors for the EC solution have been placed on deposit at the Protein Data Bank (PDB; http://www.rcsb.org/pdb/ )- (PDB E) - ITVK). Electron crystallography.
  • Tubulin crystals were formed as described previously [Nogales, E., et ah, J. Struct. Biol, 115: pp. 199 (1995)]. Briefly, 25 ⁇ l of tubulin at a concentration of 10 mg/ml in 80 mM PIPES, 1 mM EGTA, 1 mM GTP, and 10 % glycerol, pH 6.8, was mixed with 50 ⁇ l 80 mM MES, 200 mM NaCl, 3 mM GTP 3 1.25 mM MgSO 4 , 1.25 mM ZnSO 4 , 0.025 mg/mL pepstatin, pH 5.3. The samples were incubated at 32 °C for 20 hr.
  • Epothilone A was added at a 2:1 molar ratio of drug:tubulin-dimer and incubated at 32 °C for 15 min.
  • samples were prepared on conventional carbon-coated grids and stained with 2% uranyl acetate aqueous solution.
  • samples were embedded in a tannin-glucose mixture. Grids were held at -170 0 C and examined in a JEOL-4000 electron microscope operating at 400 kV. Electron diffraction data were recorded using a Gatan 794 2k CCD camera with typical exposures of 10 electrons per square Angstrom, low enough to avoid serious effects of radiation damage.
  • a weak electron beam and long exposure time (40-6Os) were used to minimize the vertical blooming streak in the diffraction pattern recorded with the CCD camera. Processing of diffraction data followed procedures outlined previously [Downing, K.H., et ah, Microsc. MicroanaL, 7: p. 407 (2001)]. Following subtraction of the radially symmetric diffuse and inelastic scattering component of the diffraction pattern and correction for geometric distortions that arise from the positioning of the CCD detector, intensities were summed in foreground and background boxes around each diffraction spot for calculation of the background-corrected spot intensity. Special care was taken to remove x-ray events from the patterns, which can produce large errors in the data.
  • Example 1 Effect of resolution upon ligand omit mapping.
  • the modified protein system was subjected to torsion angle annealing starting at 5000 K using the epo-A reflection data ranging from 2.89 to 50 A resolution. This procedure was repeated five times using different random number seeds.
  • the resultant tubulin models differed very little from the Taxol derived predecessor except in the position of M-loop residues and orientation of His227 within the binding site.
  • the five models were used to calculate phases and to produce 2F O bs-F C aic "omit" maps for ligand fitting and for further protein refinement.
  • F Obs -F ca i c difference maps were also used to evaluate and correct model bias during structure determination.
  • the new omit map from the shaken/annealed model shown in Figures 16 and 17A displayed a more contiguous volume needed for automated ligand fitting with the same general shape and position as the map displayed in Figure 15 A.
  • Example 4 Comparative evaluation ofligand binding mode and conformation.
  • epo-A conformers were obtained from structures of epo-B obtained by X-ray crystallography (2 structures) [H ⁇ fle, G., et ah, Angew. Chem. Int. Ed. Engh, 35: p. 1567 (1996)] by replacing the methyl substituent at C 12 with a hydrogen and minimizing geometry with the MMFF force field in Sybyl [Tripos Inc., (www.tripos.com), St. Louis, MO, 2002].
  • An additional 17 conformers came from the NAMFIS deconvolution analysis described above.
  • the gem-dimethyl center (C4) required readjustment.
  • the resulting three conformers were combined with the original 20 conformations and subjected to one further round of ligand analysis which provided a final optimal alignment of each within the omit density and the static tubulin model. From the thousands of automated fits, only two binding modes of epo-A were found to best match the experimental omit maps. These correspond to orientations with the side chain directed toward either the M-loop or His227 ( Figure 16). Within the 23 starting ring conformations, only those allowing near planar alignment of the side chain with the average plane of the macrolide ring could achieve such orientations.
  • Each ligand/tubulin model was refined as a rigid body in Refmac5 [Collaborative Computational Project Number 4, Acta Cryst., D50: p. 760 (1994)] and analyzed by difference mapping as illustrated in Figure 16.
  • the corresponding CCP4 maps favored models with side chains oriented toward His227 and main ring groups in contact with M-loop side chains ( Figure 17C compared to Figure 17D).
  • the modified NAMFIS conformation ( Figure 17B) provided the superior match of all combinations tested.
  • Figure 19 illustrates molecular features associated with cell lines expressing resistance bearing mutations in response to Taxol® or epothilone A exposure.
  • REMARK 3 COMPLETENESS FOR RANGE (%) : 67.03 REMARK 3 NUMBER OF REFLECTIONS 18321 REMARK 3 REMARK 3 FIT TO DATA USED IN REFINEMENT.
  • REMARK 3 TOTAL NUMBER OF BINS USED 10 REMARK 3 BIN RESOLUTION RANGE HIGH 2.890 REMARK 3 BIN RESOLUTION RANGE LOW 3.046 REMARK 3 REFLECTION IN BIN (WORKING SET) 1467 REMARK 3 BIN R VALUE (WORKING SET) 0.519 REMARK 3 BIN FREE R VALUE SET COUNT 66 REMARK 3 BIN FREE R VALUE 0.469 REMARK 3 REMARK 3 NUMBER OF NON-HYDROGEN ATOMS USED IN REFINEMENT.
  • REMARK 3 ALL ATOMS : 6672 REMARK 3 REMARK 3 B VALUES.
  • R REEMMAARRKK 229900 2555 -X,l/2+Y,-Z
  • HELIX 5 5 GLY A 111 GLN A 128 1 18
  • HELIX 21 21 ASN B 100 HIS B 105 1 6
  • HELIX 32 ASN B 337 PHE B 341 5 5

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Abstract

La présente invention a trait à des analogues d'épothilone de formules I-IX utiles comme agents stabilisateurs de microtubules et dans le traitement de maladies et de troubles à prolifération cellulaire anormale. L'invention a également trait à des procédés de fabrication des composés, à des compositions contenant les composés, à des modèles de liaison tridimensionnels d'analogues d'épothilone sur la tubuline α,β, et à des procédés pour son utilisation dans la prédiction, la conception, ou la sélection d'analogues d'épothilone thérapeutiquement utiles.
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7893268B2 (en) 2005-07-27 2011-02-22 University Of Toledo Epithiolone analogues
US8435983B2 (en) 2007-03-23 2013-05-07 The University Of Toledo Conformationally restrained epothilone analogues as anti-leukemic agents

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7893268B2 (en) 2005-07-27 2011-02-22 University Of Toledo Epithiolone analogues
US8435983B2 (en) 2007-03-23 2013-05-07 The University Of Toledo Conformationally restrained epothilone analogues as anti-leukemic agents

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