Inhibitors of phosphatidyl myo-inositol cycle Number:7,153,843 from the United States Patent and Trademark Office (PTO) owispatent The present invention relates to the preparation and biological activity of 3-deoxy-Dmyo-inositol ether lipid analogs as inhibitors of phosphatidylinositol-3-kinase signaling and cancer cell growth. The compounds of the present invention are useful as anti-tumor 5 agents which effectively inhibit the growth of mammalian cells.<H phosphatidyl, Inhibitors, Inhibitors, of, p, 7153843.html, Patent, pto, 7153843

Title: Inhibitors of phosphatidyl myo-inositol cycle

Abstract: The present invention relates to the preparation and biological activity of 3-deoxy-Dmyo-inositol ether lipid analogs as inhibitors of phosphatidylinositol-3-kinase signaling and cancer cell growth. The compounds of the present invention are useful as anti-tumor 5 agents which effectively inhibit the growth of mammalian cells.

Patent Number: 7,153,843 Issued on 12/26/2006 to Kozikowski, et al.

Inventors: Kozikowski; Alan P. (Princeton, NJ), Qiao; Lixin (Arlington, VA), Powis; Garth (Tuscon, AZ)
Assignee: Georgetown University (Washington, DC)
Arizona Board of Regents on behalf of the University of Arizona (Tuscon, AZ)

Appl. No.: 10/733,115

Filed: December 11, 2003

Related U.S. Patent Documents


Application Number	Filing Date	Patent Number	Issue Date
09879765	Jun., 2001	6667340
09339948	Jun., 1999	6245754
60090877	Jun., 1998
60235269	Sep., 2000
60235270	Sep., 2000
60223724	Aug., 2000
60223421	Aug., 2000

Current U.S. Class: 514/129 ; 558/276; 560/182

Current International Class: A61K 31/66 (20060101); C07C 69/66 (20060101); C07C 69/96 (20060101)

Other References

Hu et al, Molecular and Cellular Biology, Interaction of Phosphotidylinositol 3-Kinase-Associated p85 with Epidermal Growth Factor and Platelet-Derived Growth Factor Receptors, 1992, pp. 981-990. cited by examiner .
J Med Chem 43, pp. 3045-3051 by Hu et al Jul. 2000. cited by other .
Carpenter et al., J. Biol. Chem., "Phosphoinositide 3-Kinase is Activated by Phosphopeptides that Bind to the SH2 Domains of the 85kDa Subunit," 268: 9478-9483 (1993). cited by other .
Kozikowski et al., J. of Medical Chem., "Synthesis and Biology of 1D-3-Deoxyphosphatidylinositol: A Putative Antimetabolite of Phosphatidylinositol-3-Phosphate and an Inhibitor of Cancer Cell Colony Formation," 38: 1053-1056 (1995). cited by other .
Hu et al., Bioorganic & Medicinal Chemistry Letters 11(2001 173-176, "3-Deoxy-3substituted-d-myo-inositol Imidazolyl Ehter Lipid Phosphates and Carbonate as Inhibitors of the Phosphatidylinositol 3-Kinase Pathway and Cancer Celll Growth". cited by other .
Kozikowski, et al., Teirahedron, "Synthesis of 1D-3-Deoxy- and -2,3-Dideoxyphosphatidylinositol", vol. 53, No. 44, pp. 14903-14914, 1997. cited by other .
Schultz et al., Anticancer Res., "In Vitro and In Vivo Antitumor Activity of the Phosphatidylinositol-3-Kinase Inhibitor, Wortmannin," 15: 1135-1140 (1995). Abstract. cited by other .
Wymann et al., Mol. Cell Biol., "Wortmannin Inactivates Phosphoinositide 3-Kinase by Covalent Modification of Lys-802, a Residue Involved in the Phosphate Transfer Reaction" 16:1722-1733 (1996). cited by other .
Cross et al., J. Biol. Chem, "Wortmannin and its structural analogue demethoxyvirdin inhibit stimulated phospholipase A2 activity in swiss 3T3 cells Wortmannin is not a specific inhibitor orf phosphatidylinositol 3-kinase" 270: 25352-25355 (1995). cited by other .
Lemmon et al., Cell, "PH domains: diverse sequences with a common fold recruit signaling molecules to the cell surface" 85: 621-624 (1996). Abstract. cited by other .
Nakanishi et al., J. Biol Chem, "Activation of the zeta isozyme of protein kinase C by phosphatidylinositol 3,4,5-trisphospate" 268: 13-6 (1993). cited by other.

Primary Examiner: Zucker; Paul A.
Attorney, Agent or Firm: Pepper Hamilton LLP

Parent Case Text

RELATED APPLICATIONS

This application claims priority to, and is a continuation of, the U.S. patent application Ser. No. 09/879,765 filed Jun. 12, 2001, now U.S. Pat. No. 6,667,340, which is a continuation-in-part of application U.S. Ser. No. 09/339,948 filed Jun. 25, 1999, now U.S. Pat. No. 6,245,754, which claimed the benefit of priority from U.S. Provisional Patent Application Ser. No. 60/090,877 filed on Jun. 26, 1998; this application claims the benefit of U.S. Provisional Application Ser. No. 60/223,421 filed on Aug. 7, 2000, and U.S. Provisional Application No. 60/223,724 filed on Aug. 8, 2000, and U.S. Provisional Application No. 60/235,269 filed on Sep. 26, 2000, and U.S. Provisional Application No. 60/235,270 filed on Sep. 26, 2000 now abandoned.

Claims

What is claimed is:

1. A 3-deoxy-D-myo-inositol analog having the formula (I): ##STR00011## wherein X is O or CH.sub.2; R.sup.1 and R.sup.2 are individually, (C.sub.1 C.sub.25) alkyl, (C.sub.6 C.sub.10) aryl, (C.sub.3 C.sub.8) cycloalkyl, (C.sub.2 C.sub.22) alkenyl, (C.sub.5 C.sub.8) cycloalkenyl, (C.sub.7 C.sub.32) aralkyl, (C.sub.7 C.sub.32) alkylaryl, (C.sub.9 C.sub.32) aralkenyl, (C.sub.9 C.sub.32) alkenylaryl or C(O)R.sup.3; and R.sup.3 is (C.sub.1 C.sub.25) alkyl, (C.sub.6 C.sub.10) aryl, (C.sub.3 C.sub.8) cycloalkyl, (C.sub.2 C.sub.22) alkenyl, (C.sub.5 C.sub.8) cycloalkenyl, (C.sub.7 C.sub.32) aralkyl, (C.sub.7 C.sub.32) alkylaryl, (C.sub.9 C.sub.32) aralkenyl or (C.sub.9 C.sub.32) alkenylaryl, with the proviso that when X is O, R.sup.3 is not (C.sub.15) alkyl; R.sup.4 and R.sup.5 are individually hydrogen or a phosphate group; or when R.sup.4 or R.sup.5 is not hydrogen, a pharmaceutically acceptable salt thereof.

2. The 3-deoxy-D-myo-inositol analog of claim 1, wherein X is O.

3. The 3-deoxy-D-myo-inositol analog of claim 2, wherein R.sup.1 is methyl.

4. The 3-deoxy-D-myo-inositol analog of claim 2, wherein R.sup.2 is octadecyl.

5. The 3-deoxy-D-myo-inositol analog of claim 1, wherein X is Ch.sub.2.

6. The 3-deoxy-D-myo-inositol analog of claim 5, wherein O--R.sup.1 and/or O--R.sup.2 is palmitoyl.

7. A method of inhibiting cell growth in a subject in need of such inhibition comprising administering to a subject an effective amount of a 3-deoxy-D-myo-inositol analog according to claim 1.

8. The method of claim 7, wherein the compound is 1-O-octadecyl-2-O-methyl-sn-glycero-3-phospho-1D-3-deoxy-myo-inositol.

9. The method of claim 8, wherein 1-O-octadecyl-2-O-methyl-sn-glycero-3-phospho-1D-3-deoxy-myo-inositol is administered to a subject in a daily dose of between 0.1 and 500 mg for each kilogram of the subject's weight.

10. The method of claim 9, wherein 1-O-octadecyl-2-O-methyl-sn-glycero-3-phospho-1D-3-deoxy-myo-inositol is administered to a subject in a daily dose of about 50 100 mg for each kilogram of the subject's weight.

11. The method of claim 7, wherein said compound inhibits PtdIns-3-kinase signaling.

12. The method of claim 11, wherein inhibiting PtdIns-3-kinase signaling comprises inhibiting a src-homology 2 domain of a p85 regulatory subunit of PtdIns-3-kinase.

13. The method of claim 7, wherein inhibiting cell growth comprises inhibiting the activity of a PH domain in a PH domain containing enzyme.

14. The method of claim 13, wherein the PH domain activates the enzyme PKC-.zeta. and/or PKC-.gamma..

15. The method of claim 7, wherein inhibiting cell growth comprises promoting the activity of a PH domain in a PH domain containing enzyme.

16. The method of claim 15, wherein the PH domain activates the enzyme Akt.

17. A pharmaceutical composition which comprises a therapeutically effective amount of a compound according to claim 1, and a pharmaceutically acceptable carrier.

18. The composition of claim 17, which is suitable for administration via injection, orally, transdermally, intranasally, intraoculary, or rectally.

Description

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to specific compounds designed to inhibit cell growth signaling. In particular, PtdIns-3-kinase anti-metabolites are rationally designed to provide compounds that inhibit cell differentiation and/or cell proliferation, and/or which promote apoptosis by antagonizing myo-inositol cell growth signaling. The present invention also relates to therapeutic methods, e.g., treatment of cancer, including the administration of the compounds according to the invention.

2. Background of the Invention

For mammalian cells to survive, they must be able to respond rapidly to changes in their environment. Furthermore, for cells to reproduce and carry out other cooperative functions, they must be able to communicate efficiently with each other. Cells most frequently adapt to their environment and communicate with one another by means of chemical signals. An important feature of these signaling mechanisms is that in almost all cases a cell is able to detect a chemical signal without it being necessary for the chemical messenger itself to enter the cell. This permits the cell to maintain the homeostasis of its internal environment, thereby permitting the cell to respond to its external environment without being adversely affected by it.

These sensing functions are carried out by a variety of receptors, which are dispersed on the outer surface of the cell and function as "molecular antennae". These receptors detect an incoming messenger and activate a signal pathway that ultimately regulates a cellular process such as secretion, contraction, metabolism or growth. In the cell's cellular plasma membrane, transduction mechanisms translate external signals into internal signals, which are then carried throughout the interior of the cell by chemicals known as "second messengers."

In molecular terms, the process depends on a series of proteins within the cellular plasma membrane, each of which transmits information by inducing a conformational change in the protein next in line. At some point, the information is assigned to small molecules or even to ions within the cell's cytoplasm, which serve as the above-mentioned second messengers. The diffusion of the second messengers enables a signal to propagate rapidly throughout the cell.

Several major signal pathways are now known, but two seem to be of primary importance. One employs cyclic nucleotides as second messengers. These cyclic nucleotides activate a number of proteins inside the cell, which then cause a specific cellular response. The other major pathway employs a combination of second messengers that includes calcium ions as well as two substances whose origin is remarkable: myo-inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). These compounds are cannibalized from the plasma membrane itself, by enzymes which are activated by specific cellular membrane receptors. However, this pathway requires that myo-inositol, in its non-phosphorylated form, be initially synthesized by the cell from glucose or obtained from the extracellular environment. Recently, another phosphatidylinositol signaling pathway has been identified and linked to the action of some growth factors and oncogenes. Phosphatidylinositol-3'-kinase (also designated type 1 phosphatidylinositol kinase) is found associated with a number of protein tyrosine kinases including the ligand-activated receptors for insulin, platelet derived growth factor (PDGF), epidermal growth factor (EGF), and colony-stimulating factor-1 (CSF-1) as well as proto-oncogene and oncogene tyrosine kinases (Y. Fukui et al., Oncogene Res., 4, 283 (1989)). This enzyme phosphorylates the D-3 position of the myo-inositol ring of phosphatidylinositols to give a class of phosphafdylinositol-3'-phosphates that are not substrates for hydrolysis by phosphatidylinositol phospholipase C. Accordingly, these compounds apparently exert their signaling action independently of the inositol phosphate pathway.

Based on the potential effects thereof on cell proliferation, differentiation and apoptosis, it would be beneficial if compounds could be obtained which selectively block phosphatidylinositol signaling pathways. More specifically, it would be beneficial if compounds could be obtained which antagonize myo-inositol metabolites produced by PtdIns-3-Kinase. Such compounds have significant therapeutic potential, in particular for treatment of cancer and other conditions involving abnormal cell differentiation and proliferation. Compounds having improved selectivity, solubility and stability are particularly desirable.

SUMMARY AND OBJECTS OF THE INVENTION

It is an object of the invention to provide novel compounds which inhibit the phosphatidylinositol signaling pathway.

It is a more specific object of the invention to provide novel compounds which are antagonistic of myo-inositol metabolites provided by PtdIns-3-Kinase.

It is an even more specific object of the invention to provide novel analogs of 3-deoxy-D-myo-inositol which inhibit the phosphatidylinisitol signaling pathway.

It is still a more specific object of the invention to provide compounds having the formulae (I) and (II) set forth below:

##STR00001## wherein X is O or CH.sub.2; R.sup.1 and R.sup.2 are individually, (C.sub.1 C.sub.25) alkyl, (C.sub.6 C.sub.10) aryl, (C.sub.3 C.sub.8) cycloalkyl, (C.sub.2 C.sub.22) alkenyl, (C.sub.5 C.sub.8) cycloalkenyl, (C.sub.7 C.sub.32) aralkyl, (C.sub.7 C.sub.32) alkylaryl, (C.sub.9 C.sub.32) aralkenyl, (C.sub.9 C.sub.32) alkenylaryl or C(O)R.sup.3; and R.sup.3 is (C.sub.1 C.sub.25) alkyl, (C.sub.6 C.sub.10) aryl, (C.sub.3 C.sub.8) cycloalkyl, (C.sub.2 C.sub.22) alkenyl, (C.sub.5 C.sub.8) cycloalkenyl, (C.sub.7 C.sub.32) aralkyl, (C.sub.7 C.sub.32) alkylaryl, (C.sub.9 C.sub.32) aralkenyl or (C.sub.9 C.sub.32) alkenylaryl, with the proviso that when X is O, R.sup.3 is not (C.sub.16) alkyl; R.sup.4 and R.sup.5 are individually hydrogen or a phosphate group; or when R.sup.4 or R.sup.5 is not hydrogen, a pharmaceutically acceptable salt thereof; and

##STR00002## wherein X is O or CH.sub.2; R.sup.1 and R.sup.2 are individually, (C.sub.1 C.sub.25) alkyl, (C.sub.6 C.sub.10) aryl, (C.sub.3 C.sub.8) cycloalkyl, (C.sub.2 C.sub.22) alkenyl, (C.sub.5 C.sub.8) cycloalkenyl, (C.sub.7 C.sub.32) aralkyl, (C.sub.7 C.sub.32) alkylaryl, (C.sub.9 C.sub.32) aralkenyl, (C.sub.9 C.sub.32) alkenylaryl or C(O)R.sup.3; and R.sup.3 is (C.sub.1 C.sub.25) alkyl, (C.sub.6 C.sub.10) aryl, (C.sub.3 C.sub.8) cycloalkyl, (C.sub.2 C.sub.22) alkenyl, (C.sub.5 C.sub.8) cycloalkenyl, (C.sub.7 C.sub.32) aralkyl, (C.sub.7 C.sub.32) alkylaryl, (C.sub.9 C.sub.32) aralkenyl or (C.sub.9 C.sub.32) alkenylaryl; R.sup.4 and R.sup.5 are individually hydrogen or a phosphate group; or when R.sup.4 or R.sup.5 is not hydrogen, a pharmaceutically acceptable salt thereof.

It is a more specific object of the invention to treat cancer by the administration of at least one compound of the formulae (I) or (II):

##STR00003## wherein X is O or CH.sub.2; R.sup.1 and R.sup.2 are individually, (C.sub.1 C.sub.25) alkyl, (C.sub.6 C.sub.10) aryl, (C.sub.3 C.sub.8) cycloalkyl, (C.sub.2 C.sub.22) alkenyl, (C.sub.5 C.sub.8) cycloalkenyl, (C.sub.7 C.sub.32) aralkyl, (C.sub.7 C.sub.32) alkylaryl, (C.sub.9 C.sub.32) aralkenyl, (C.sub.9 C.sub.32) alkenylaryl or C(O)R.sup.3; and R.sup.3 is (C.sub.1 C.sub.25) alkyl, (C.sub.6 C.sub.10) aryl, (C.sub.3 C.sub.8) cycloalkyl, (C.sub.2 C.sub.22) alkenyl, (C.sub.5 C.sub.8) cycloalkenyl, (C.sub.7 C.sub.32) aralkyl, (C.sub.7 C.sub.32) alkylaryl, (C.sub.9 C.sub.32) aralkenyl or (C.sub.9 C.sub.32) alkenylaryl, with the proviso that when X is O, R.sup.3 is not (C.sub.16) alkyl; R.sup.4 and R.sup.5 are individually hydrogen or a phosphate group; or when R.sup.4 or R.sup.5 is not hydrogen, a pharmaceutically acceptable salt thereof; and

##STR00004## wherein X is O or CH.sub.2; R.sup.1 and R.sup.2 are individually, (C.sub.1 C.sub.25) alkyl, (C.sub.6 C.sub.10) aryl, (C.sub.3 C.sub.8) cycloalkyl, (C.sub.2 C.sub.22) alkenyl, (C.sub.5 C.sub.8) cycloalkenyl, (C.sub.7 C.sub.32) aralkyl, (C.sub.7 C.sub.32) alkylaryl, (C.sub.9 C.sub.32) aralkenyl, (C.sub.9 C.sub.32) alkenylaryl or C(O)R.sup.3; and R.sup.3 is (C.sub.1 C.sub.25 ) alkyl, (C.sub.6 C.sub.10) aryl, (C.sub.3 C.sub.8) cycloalkyl, (C.sub.2 C.sub.22) alkenyl, (C.sub.5 C.sub.8) cycloalkenyl, (C.sub.7 C.sub.32) aralkyl, (C.sub.7, C.sub.32) alkylaryl, (C.sub.9 C.sub.32) aralkenyl or (C.sub.9 C.sub.32) alkenylaryl; R.sup.4 and R.sup.5 are individually hydrogen or a phosphate group; or when R.sup.4 or R.sup.5is not hydrogen, a pharmaceutically acceptable salt thereof.

It is another object of the invention to provide pharmaceutical compositions comprising at least one novel compound that inhibits the phosphatidylinositol signaling pathway, and more preferably a compound that antagonizes myo-inositol metabolites produced by PtdIns-3-Kinase.

It is a more specific object of the invention to provide pharmaceutical compositions that comprise at least one compound having the formulae (I) or (II):

##STR00005## wherein X is O or CH.sub.2; R.sup.1 and R.sup.2 are individually, (C.sub.1 C.sub.25) alkyl, (C.sub.6 C.sub.10) aryl, (C.sub.3 C.sub.8) cycloalkyl, (C.sub.2 C.sub.22) alkenyl, (C.sub.5 C.sub.8) cycloalkenyl, (C.sub.7 C.sub.32) aralkyl, (C.sub.7 C.sub.32) alkylaryl, (C.sub.9 C.sub.32) aralkenyl, (C.sub.9 C.sub.32) alkenylaryl or C(O)R.sup.3; and R.sup.3 is (C.sub.1 C.sub.25) alkyl, (C.sub.6 C.sub.10) aryl, (C.sub.3 C.sub.8) cycloalkyl, (C.sub.2 C.sub.22) alkenyl, (C.sub.5 C.sub.8) cycloalkenyl, (C.sub.7 C.sub.32) aralkyl, (C.sub.7 C.sub.32) alkylaryl, (C.sub.9 C.sub.32) aralkenyl or (C.sub.9 C.sub.32) alkenylaryl, with the proviso that when X is O, R.sup.3 is not (C.sub.16) alkyl; R.sup.4 and R.sup.5 are individually hydrogen or a phosphate group; or when R.sup.4 or R.sup.5 is not hydrogen, a pharmaceutically acceptable salt thereof; or

##STR00006## wherein X is O or CH.sub.2; R.sup.1 and R.sup.2 are individually, (C19 C.sub.25) alkyl, (C.sub.6 C.sub.10) aryl, (C.sub.3 C.sub.8) cycloalkyl, (C.sub.2 C.sub.22) alkenyl, (C.sub.5 C.sub.8) cycloalkenyl, (C.sub.7 C.sub.32) aralkyl, (C.sub.7 C.sub.32) alkylaryl, (C.sub.9 C.sub.32) aralkenyl, (C.sub.9 C.sub.32) alkenylaryl or C(O)R.sup.3; and R.sup.3 is (C.sub.1 C.sub.25) alkyl, (C.sub.6 C.sub.10) aryl, (C.sub.3 C.sub.8) cycloalkyl, (C.sub.2 C.sub.22) alkenyl, (C.sub.5 C.sub.8) cycloalkenyl, (C.sub.7 C.sub.32) aralkyl, (C.sub.7 C.sub.32) alkylaryl, (C.sub.9 C.sub.32) aralkenyl or (C.sub.9 C.sub.32) alkenylaryl; R.sup.4 and R.sup.5 are individually hydrogen or a phosphate group; or when R.sup.4 or R.sup.5 is not hydrogen, a pharmaceutically acceptable salt thereof; which inhibit the phosphatidylinositol signaling pathway and thereby inhibit cell proliferation and/or differentiation and/or promote apoptosis.

It is another object of the invention to provide novel therapies based on inhibiting in vivo the phosphatidylinositol signaling pathway.

It is a more specific object of the invention to provide novel therapies that result in the inhibition of cell proliferation and/or differentiation and/or the promotion of cell apoptosis comprising the administration of a compound that antagonizes myo-inositol cell growth signaling.

It is an even more specific object of the invention to provide novel therapies that result in the inhibition of cell proliferation and/or differentiation and/or promotion of cell apoptosis by the administration of a compound having formulae (I) or (II):

##STR00007## wherein X is O or CH.sub.2; R.sup.1 and R.sup.2 are individually, (C.sub.1 C.sub.25) alkyl, (C.sub.6 C.sub.10) aryl, (C.sub.3 C.sub.8) cycloalkyl, (C.sub.2 C.sub.22) alkenyl, (C.sub.5 C.sub.8) cycloalkenyl, (C.sub.7 C.sub.32) aralkyl, (C.sub.7 C.sub.32) alkylaryl, (C.sub.9 C.sub.32) aralkenyl, (C.sub.9 C.sub.32) alkenylaryl or C(O)R.sup.3; and R.sup.3 is (C.sub.1 C.sub.25) alkyl, (C.sub.6 C.sub.10) aryl, (C.sub.3 C.sub.8) cycloalkyl, (C.sub.2 C.sub.22) alkenyl, (C.sub.5 C.sub.8) cycloalkenyl, (C.sub.7 C.sub.328) aralkyl, (C.sub.7 C.sub.32) alkylaryl, (C.sub.9 C.sub.32) aralkenyl or (C.sub.9 C.sub.32) alkenylaryl, with the proviso that when X is O, R.sup.3 is not (C.sub.16) alkyl; R.sup.4 and R.sup.5 are individually hydrogen or a phosphate group; or when R.sup.4 or R.sup.5 is not hydrogen, a pharmaceutically acceptable salt thereof; or

##STR00008## wherein X is O or CH.sub.2; R.sup.1 and R.sup.2 are individually, (C.sub.1 C.sub.25) alkyl, (C.sub.6 C.sub.10) aryl, (C.sub.3 C.sub.8) cycloalkyl, (C.sub.2 C.sub.22) alkenyl, (C.sub.5 C.sub.8) cycloalkenyl, (C.sub.7 C.sub.32) aralkyl, (C.sub.7 C.sub.32) alkylaryl, (C.sub.9 C.sub.32) aralkenyl, (C.sub.9 C.sub.32) alkenylaryl or C(O)R.sup.3; and R.sup.3 is (C.sub.1 C.sub.25) alkyl, (C.sub.6 C.sub.10) aryl, (C.sub.3 C.sub.8) cycloalkyl, (C.sub.2 C.sub.22) alkenyl, (C.sub.5 C.sub.8) cycloalkenyl, (C.sub.7 C.sub.32) aralkyl, (C.sub.7 C.sub.32) alkylaryl, (C.sub.9 C.sub.32) aralkenyl or (C.sub.9 C.sub.32) alkenylaryl; R.sup.4 and R.sup.5 are individually hydrogen or a phosphate group; or when R.sup.4 or R.sup.5 is not hydrogen, a pharmaceutically acceptable salt thereof.

In a preferred embodiment, such therapies will comprise treatment of cancer and other neoplastic conditions and/or will comprise treatment of arthritis, inflamation or modulation of platelet aggregation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of the signaling by PtdIns-3-kinase leading to cancer cell proliferation;

FIG. 2 is a schematic diagram of phosphatidylinositol (PtdIns);

FIG. 3 shows the structure of 3-deoxy-phosphatidyl-myo-inositol (30) (DPI);

FIG. 4 shows the generic structure of phosphonate analogs of DPI (30);

FIG. 5 is a schematic diagram of the synthesis of the 3-deoxy-phosphatidyl-myo-inositol phosphonate analog (50);

FIG. 6 shows a generic structure of D-3-deoxyPtdIns ether lipid analogs designed according to the invention;

FIG. 7 is a schematic diagram of the synthesis of OMDPI (60);

FIG. 8 illustrates the synthesis of 1-O-octadecyl-2-O-Me-sn-glycerol (84);

FIG. 9 is a schematic outline of the synthesis of the phosphonate analog (90) of OMDPI;

FIG. 10 shows the results for PH Domain inhibition; and

FIGS. 11 and 12 show the cell growth inhibiting ability of DPI (30) and OMDPI (60), respectively.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.

The present invention includes novel compounds which are rationally designed to inhibit cell growth. Rational design of the novel compounds of the present invention includes identifying a mechanism associated with cell growth. Information relating to the mechanism is then analyzed such that compound structures having possible activity in interfering with such mechanism are formulated. In particular, novel structures are synthesized based on "building blocks", wherein each building block has a feature potentially capable of interfering with a particular mechanism associated with cell growth. Compounds having different building block combinations are then synthesized and their activity in relation to the identified mechanism tested. Such tests are conducted in vitro and/or in vivo. The information obtained through such tests is then incorporated in a new cycle of rational drug design. The design-synthesis-testing cycle is repeated until a lead compound having the desired properties is identified. The lead compound is then clinically tested.

Identification of a Mechanism Associated with Cell Growth

Growth factors and certain oncogenes activate a range of phospholipid-mediated signal transduction pathways associated with cell proliferation. Phosphatidyl myoinositol (PI) occupies a unique position in that it can undergo reversible phosphorylation at multiple sites to generate five different phosphoinositides. PI metabolites regulate two pathways important for cell proliferation, the inositol phosphate/diacylglycerol signaling pathway and the phosphate/diacylglycerol 3-phosphate (PI-3-kinase) pathway.

In the first pathway, PI specific phospholipase C (PI-PLC) hydrolyses a minor membrane phospholipid, PI(4,5)P.sub.2 to give the water soluble Ins(1,4,5)P.sub.3, and a lipophilic diacylglycerol (DAG). Ins(1,4,5)P.sub.3 interacts specifically with membrane receptors to release Ca.sup.2+, a key event in cellular signal transduction. DAG is an endogenous activator of protein kinase C (PKC). lns(1,4,5)P.sub.3 is metabolized either by hydrolysis of the phosphate at position 5, giving Ins(1,4)P.sub.2 or phosphorylation at position 3 giving Ins(1,3,4,5)P.sub.4. Ins(1,4)P.sub.2 is not active as a Ca.sup.2+ mobilizing agent and is subsequently degraded by other phosphatases. However, it has been suggested that Ins(1,3,4,5)P.sub.4 may play a role in refilling the intracellular Ca.sup.2+ stores with extracellular Ca.sup.2+. Together, the increase in Ca.sup.2+ concentration and the increased activity of PKC lead to a sequence of events that culminate in DNA synthesis and cell proliferation.

In the second pathway, PI-3-kinase has been found to be associated with almost every growth factor receptor or oncogene transformation. PI-3-kinase phosphorylates PI at position 3 of the myo-inositol ring to give a class of PIs that are poor substrates for hydrolyses by PI-PLC, e.g., PI(3,4)P.sub.2 and PI (3,4,5)P.sub.3.

PtdIns-3-kinases are a family of enzymes that phosphorylate the D-3-OH position of the myo-inositol ring of the minor cell membrane phospholipid phosphatidylinositol (PtdIns). The most studied member of the PtdIns-3-kinase family is a heterodimer consisting of an 85 kDa regulatory subunit (p85) and a 110 kDa catalytic subunit (p110). All of the known isoforms of p110 are capable of phosphorylating both PtdIns and PtdIns(4)P in vitro, however, PtdIns (4,5)P.sub.2 is the preferred substrate in vivo. PtdIns-3-kinase is activated by a wide range of growth factor receptor and oncogene proteintyrosine kinases as well as by p21.sup.Ras.

The exact mechanism by which 3-phosphorylated PIs modulate cell growth is not known but they appear to be important modulators of protein interaction and enzyme activity through binding to specific sites on proteins. For example, binding of PI(3,4)P.sub.2 PI(4,5)P.sub.2 or PI(3,4,5)P.sub.3 to pleckstrin-homology (PH) domains on enzymes such as Akt (protein kinase B) leads to enzyme activation, whereas the Src-homology-2 (SH2) domain that mediates protein tyrosine phosphate binding binds specifically PI(3,4,5)P.sub.3. PtdIns-3-kinase is activated by binding of the src-homology 2 (SH2) domain of the p85 regulatory subunit of PtdIns-3-kinase to phosphorylated tyrosine residues on activated growth factor receptors and oncogenic protein tyrosine kinases which cause a conformational change at the active site of the p110 catalytic subunit and brings PtdIns-3-kinase from the cytoplasm to the inner surface of the plasma membrane where PtdIns substrates are located. Also, PtdIns-3-kinase itself becomes tyrosine phosphorylated, however, this phosphorylation apparently does not result in any increased activity of the enzyme.

FIG. 1 depicts schematically the means by which PtdIns-3-kinase signaling is theorized to enhance cancer cell proliferation. Essentially, activation of PtdIns-3-kinase (PI-3-K) leads to the formation of PtdIns-3-phosphates which bind to the PH domains of enzymes such as Akt, PtdIns PLC-.gamma. and activation of PKC-.zeta..

Several lines of evidence suggest an essential role for PtdIns-3-kinase in the modulation of cancer cell growth and the cancer phenotype. For example, cells transfected with a mutant PDGF receptor that retains protein tyrosine kinase activity, but which do not associate with or activate PtdIns-3-kinase, fail to show a mitogenic response to PDGF, unlike cells transfected with the wild-type PDGF receptor ("Role of phosphatidylinositol kinase in PDGF receptor signal transduction"; Coughlin et al.; Science, 243:1191 1194 (1989)). Also, it has been reported that a mutant CSF-1 receptor which contains a kinase-insert deletion results in significantly reduced association with PtdIns-3-kinase. Moreover, this mutant receptor is only capable of conferring CSF-1-dependent transformation to some cells and has lost the ability to transform other cells ("Phosphatidylinositol-3-kinase is necessary for 12-O-tetradecanoylphorbol-13-acetate-induced cell transformation and activated protein 1 activation"; Huang et al.; J. Biol. Chem., 272:4187 4194 (1997)).

Further, it has been reported that active PtdIns-3-kinase is necessary for phorbol ester mediated transformation of cells. In particular, it has been reported that polyoma middle T mutants which associate with and activate pp60.sup.c-src tyrosine kinase, but which fail to activate PtdIns-3-kinase are nontransforming ("Common elements in growth factor stimulation and oncogenic transformation: 85 kd phosphoprotein and phosphatidylinositol kinase activity"; Kaplan et al.; Cell, 50:1021 1029 (1987)). It is further known that the levels of cellular PtdIns-3-phosphates are elevated in transforming mutants of middle T but not by transformation of defective mutants, suggesting that these compounds play a significant role in transformation.

It is also known that PtdIns-3-kinase prevents apoptosis and is necessary for the inhibition of apoptosis caused by nerve growth factor in PC 12 phemochromocytoma cells ("Requirement for phosphatidylinositol-3-kinase in the prevention of apoptosis by nerve growth factor"; Yao et al.; Science, 267:2003 2006 (1995)) and by IL-3 and IL-4 in 5 hematopoietic cells ("Signaling through the lipid products of phosphoinositide-3-OH kinase"; Toker et al.; Nature, 387:673 676 (1997)).

Based on the foregoing, PtdIns-3-kinase has generated considerable interest as a target for the development of anticancer drugs to block the activity of increased growth factor signaling or oncogene expression. More particularly, based on what has been reported about this enzyme, disease conditions that potentially would be susceptible to growth inhibition by PtdIns-3-kinase inhibitors include cancers that over-express PDGF receptors such as colon, pancreatic, prostate and head and neck tumors, and tumors overexpressing EGF receptor such as breast, gastric and prostate tumors. Also, tumors expressing mutant ras such as colon and pancreatic cancer and CML which is characterized by a Bcr/Abl (Philadelphia chromosome) translocation (where Bcr/Abl has been shown to require PtdIns-3-kinase for its effects) may also be amenable to treatment by compounds that affect PtdIns-3-kinase activity.

Essentially, because of the important role PtdIns-3-kinase apparently plays in effecting cell growth, it provides an exciting avenue for designing therapeutic protocols based on controlling PtdIns-3-kinase activity. More specifically, compounds which mediate PtdIns-3-kinase activity potentially may be used to control (inhibit) tumor cell growth.

A direct approach for modulating PtdIns-3-kinase and the biological pathways it affects is to design therapeutic protocols based on compounds having PtdIns-3-kinase inhibitory activity. Supplying such compounds to target cells potentially should reduce or block cell proliferation attributable to the inhibition of PtdIns-3-kinase.

An example thereof is the fungal metabolite wortmannin, which is an irreversible inhibitor of p110 PtdIns-3-kinase (having an IC.sub.50 of 4 nM) ("Wortmannin inactivates phosphoinositide 3-kinase by covalent modification of Lys-802, a residue involved in the phosphate transfer reaction"; Wymann et al.; Mol. Cell Biol, 16:1722 1733 (1996)). Because of this activity, wortmannin has been used extensively as a pharmacological probe of the functions of PtdIns-3-kinase. Based on such inhibitory activity, it was initially hoped that wortmannin might be a useful anticancer drug against tumors with activated PtdIns-3-kinase signaling ("In vitro and in vivo activity of the phosphatidylinositol-3-kinase inhibitor, wortmannin"; Schultz et al.; Anticancer Res., 15:1135 1140 (1995)).

Unfortunately, while although wortmannin has shown anti-tumor activity against a variety of tumors, it lacks target selectivity and is toxic to normal tissues, particularly the liver and hematopoietic system. This has precluded its further therapeutic development. Such lack of selectivity is apparently attributable to the fact that wortmannin inhibits other serine/threonine kinases of the PtdIns-3-kinase family, e.g., mTOR and DNA-dependent protein kinase, with IC.sub.50s of 2 to 4 nM. The unrelated enzyme phospholipase A2 is also inhibited by wortmannin, with an IC.sub.50 of 2 nM ("Wortmannin and its structural analogue demethoxyviridin inhibit stimulated phospholipase A.sub.2 activity in Swiss 3T3 cells"; Cross et al.; J. Biol. Chem., 270:25352 25355 (1995)).

The poor selectivity of PtdIns-3-kinase inhibition by wortmannin suggests that the binding site of PtdIns-3-kinase does not have unique structural features recognizable by this inhibitor. This, in turn suggests that designing PtdIns-3-kinase inhibitors having acceptable selectivity requires detailed structural analysis of the active sites of PtdIns-3kinase and related enzymes whose activity is indiscriminately inhibited by known inhibitors such as wortmannin.

Another potential approach for controlling PtdIns-3-kinase activity and thereby cell growth which is the focus of the present invention, is directed to PtdIns-3-kinase metabolites. More specifically, the subject approach is based on rationally designing compounds which are antagonists of myo-inositol second messengers produced by PtdIns-3-kinase which reduce or block cell growth by antagonizing myo-inositol cell growth signaling. Preferably, antagonists are designed which reduce or block cell proliferation while leaving other aspects of myo-inositol signaling unaffected. The designed antagonists should provide a novel basis for therapeutic protocols based on the selective control of cancer cell growth signaling which do not disrupt the function of normal cells.

Metabolic Products of, Phosphatidylinositol-3-kinase

The products of PtdIns-3-kinase, i.e., PtdIns-3-phosphates, are responsible for the effects of PtdIns-3-kinase on tumor growth and apoptosis. Only recently has their mechanism of action begun to be understood. PtdIns-3-phosphates are found in the cell as small amounts of PtdIns-3-phosphate and larger amounts of PtdIns(3,4)P.sub.2 and PtdIns(3,4,5)P.sub.3 ("Phosphoinositide 3-kinase is activated by phosphopeptides that bind to the SH2 domains of 84-kDa subunit"; Carpenter et al.; J. Biol. Chem., 268:9478 9483 (1993)). PtdIns-3-phosphates have the unique ability to bind to specific protein domains, a property not shared by non-3-phosphorylated PtdIns, resulting in the activation of key signaling proteins involved in cell growth and death. The pleckstrin homology (PH) domain is a protein module of approximately 120 amino acids found in a number of signaling proteins activated by PtdIns-3-phosphate binding. The PH domain of these proteins binds specifically to PtdIns-3-phosphates present in the inner plasma membrane resulting in the translocation of the signaling proteins from the cytosol to the plasma membrane where their substrates are located. Binding of PtdIns-3-phosphates to PH domains may also result in a direct increase in the catalytic activity of the enzyme ("PH domains: diverse sequences with a common fold recruit signaling molecules to the cell surfaces"; Lemmon et al.; Cell, 85:621 624 (1996)).

The most extensively studied examples of PH domain-regulated signaling are the PH domain dependent activation by PtdIns(3,4)P.sub.2 and PtdIns(3,4,5)P.sub.3 of the serine/threonine kinase Akt (PKB/Rac) and of PtdIns-PLC.gamma.. Binding of the PH domain to membrane PtdIns-3-phosphates causes the translocation of Akt to the plasma membrane bringing it into contact with membrane bound Akt kinase, which is itself activated by PtdIns(3,4,5)P.sub.3, which then phosphorylates and activates Akt ("Characterization of a 3-phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase Ba"; Alessi et al., Curr. BioL, 7:261 269 (1997)), ("Dual role of Phosphatidylinositol 3,4,5-triphosphate in the activation of protein kinase B"; Stokoe et al., Science, 277:567 570 (1997)). Akt is a proto-oncogene that inhibits apoptosis by phosphorylating Bad, thus, promoting its binding to, and blocking the activity of the cell survival factor Bcl-x ("A bad kinase makes good"; Franke et al.; Nature, 390:116 124 (1997)).

Accordingly, the inhibition of Akt activation potentiates cancer cell apoptosis. Translocation of PtdIns-PLC.gamma. to the plasma membrane brings it into contact with its substrate PtdIns(4,5)P.sub.2 resulting in more efficient hydrolysis to Ins(1,4,5)P.sub.3 and diacylglycerol. The binding of PtdIns(3,4,5)P.sub.3 to the SH2 domain of PtdIns-PLC.gamma., as well as the better recognized SH2 binding to tyrosine phosphate residues on autophosphorylated growth factor receptors, provides additional mechanisms for translocating PtdIns-PLC.gamma. to the plasma membrane (Id.). An increase in intracellular free Ca.sup.2+ caused by the release of intracellular stores of Ca.sup.2+ by Ins(1,4,5)P.sub.3 together with the activation of protein kinase C by diacylglycerol leads to a series of events that culminate in increased cell proliferation. PKC-.zeta. is also directly activated by PtdIns(3,4,5)P.sub.3 (Activation of zeta isozyme of protein kinase C by phosphatidylinositol 3,4,5-triphosphate; Nakanishi et al.; J. Biol. Chem., 268:13 16 (1993)). Thus, an increase in PtdIns-3-phosphates in the cell membrane results in the activation of two different pathways, one leading to increased cell proliferation, the other to inhibition of cell death. These separate pathways explain the growth stimulating and transformation related effects of PtdIns-3-kinase.

Design of PtdIns-3-Kinase Anti-Metabolites

As disclosed supra, the focus of the subject invention is to produce by rational methods antagonists of PtdIns-3-kinase metabolites. In order do so, the present inventors have elected to rationally designs antagonists of PtdIns-3-kinase metabolites such as PtdIns-3-phosphates, by utilizing the structure of the PtdIns-3-kinase substrate as a starting structure for modification. In particular, different modifications to this starting structure are judiciously selected and the effects thereof on activity evaluated, so that ideally an effective antagonist is produced. One method for obtaining effective antagonists is to maintain a high structural similarity between the antagonist and the substrate. That is, the modification is based on a balance between the new features providing the desired antagonistic effect and maintaining sufficient structural similarity such that metabolites are not produced. These antagonists will advantageously be sufficiently similar in structure to the metabolites such that they effectively interfere with the processing of the metabolites in the signaling cycle, down stream of the PtdIns-3-kinase step. Effective antagonists should have sufficient structural similarity to these metabolites so that they effectively compete with the metabolites for interaction with sites available for the step following phosphorylation by PtdIns-3-kinase, while at the same time being unaffected by this interaction. This should block the signaling cycle both upstream and downstream of the PtdIns-3-kinase mediated phosphorylation.

The starting structure of the modified metabolite phosphatidylinositol (PtdIns) is contained in FIG. 2. As explained, PtdIns 20 is utilized as the starting structure for designing PtdIns-3-kinase cell growth signaling antagonists. More specifically, the present inventors elected to focus on three specific sites in the PtdIns structure as candidates to be modified in order to obtain analogs of PtdIns-3-kinase metabolites which function as effective antagonists. Ideally, such antagonists will exhibit the desired pharmaceutical properties in vivo and will selectively antagonize myo-inositol metabolites produced by PtdIns-3-kinase while not disrupting other cell signaling pathways, in particular of normal cells. The rationale for selecting these specific modifications is based on the present inventors' analysis and understanding of the chemistry associated with cell growth signaling.

A) The 3 Position of the mvo Inositol Ring

The first position selected for modification was the 3 position of the myo-inositol ring. This was selected because analogs of myo-inositol in which the 3-hydroxyl group is removed or replaced can not be phosphorylated by PtdIns-3-kinase and appear to act as inhibitors of PtdIns-3-kinase signaling. D-3-deoxy-substituted myo-inositols are taken up by the myo-inositol transporter of cells and incorporated into cellular PtdIns by PtdIns synthetase leading to the selective growth inhibition of (some) transformed relative to normal cells. However, the affinity of the D-3-deoxy-substituted myo-inositols for uptake and PtdIns synthesis is less than that of myo-inositol itself and at physiological concentrations myo-inositol inhibits their growth inhibitory activity. D-3-deoxysubstituted PtdIns inhibits the growth of cancer cells in the presence of myo-inositol. In fact, D-3-deoxy-PtdIns and a more active analog have been reported to exhibit anti-tumor activity against human tumor xenografts in SCID mice ("Synthesis and Biology of 1D-3-Deoxyphosphatidylinisitol: A Putative Anti-metabolite of phosphatidylinositol-3-phosphate and an Inhibitor of Cancer Cell Colony Formation", Kozikowski, A. P. et al. J. of Medicinal Chem., Vol. 38, 7:1053 1056 (1995)), the contents of which are hereby incorporated by reference. Also, treatment of NIH 3T3 cells with D-3-deoxy-PtdIns blocks the activation of Akt due to inhibition of PH domain binding. Moreover, D-3deoxy-myo-inositols kill cells by inducing apoptosis which is consistent with the role of PtdIns-3-kinase and Akt in preventing apoptosis (Id.).

The position for a first modification, site 21, corresponds to the 3 position on the inositol ring. As discussed above, PtdIns-3-kinase phosphorylates the D-3-OH position of the myo inositol ring. Modifying the 3 position of the inositol ring to preclude phosphorylation should interrupt the PtdIns-3-kinase signaling cycle. Precluding phosphorylation by PtdIns-3-kinase is achieved by removing the oxy group at the 3 position of the inositol ring.

The resultant modified structure, 3-deoxy-phosphatidyl-myo-inositol 30 (DPI) is contained in FIG. 3. In fact, DPI is recalcitrant to phosphorylation by PtdIns-3-kinase and therefore possesses cell growth inhibiting activity. Assays of the biological activity of DPI show that the compound inhibits colony formation by HT-29 human colon carcinoma cells. DPI exhibits an IC.sub.50 of 35 .mu.M (Id.).

Also, it has been demonstrated by the inventors that the 3 position of the myo-inositol ring can be modified to include non-phosphorylable substituents. In particular, the 3 position hydrogen atom in DPI can be substituted by a halogen, such as fluorine or chlorine. The synthesis and biological activity of such substituted DPI analogs is the subject of U.S. Pat. No. 5,227,508, the contents of which are hereby incorporated in their entirety. For example, it has been shown that the PtdIns analog bearing a fluorine atom in place of the 3-hydroxy group inhibits colony formation by HT-29 human colon carcinoma cells with an IC.sub.50 of 37 .mu.M.

B) The DAG sn-3 Oxygen Position

A second site selected by the inventors for the rational drug design of PtdIns antagonists is the sn-3 oxygen of the DAG. This was chosen because in the PtdIns signaling cycle, PtdIns-3-kinase metabolites are hydrolyzed by PI-PLC at the sn-3 oxo position. Therefore, precluding hydrolysis by substituting the sn-3 oxygen by a non hydrolyzable group should allow the concentration of PtdIns-3-kinase anti-metabolites to remain at a high level, thereby inhibiting PtdIns-3-kinase activity.

More specifically, the present inventors elected to modify the 3-sn oxygen position of the PtdIns analog, preferably to preclude hydrolysis by PI-PLC, by replacing the oxygen with a methylene group (CH.sub.2). This modification was made because it is hypothesized that maintaining a high concentration of PtdIns-3-kinase anti-metabolites requires that such metabolites be present in the environment of PtdIns-3-kinase. Moreover, it is believed that the low potency of these compounds may be due to their hydrolysis by phospholipases including PI-PLC. Also, DAG produced by hydrolysis can activate PKC, which may lead to tumor cell proliferation. By contrast, the present inventors seek to obtain novel antagonists which act as PtdIns-3-kinase anti-metabolites which are not hydrolyzable at the 3-sn oxygen by PI-PLC. These antagonists are designed based on a double modification of the starting structure. In particular, both the 3 position 21 of the myo-inositol ring and the sn-3 oxygen position 23 of the DAG were modified.

FIG. 4 shows the generic structure of phosphonate analogs of DPI. Also, the synthesis of the 3-deoxy-phosphatidyl-myo-inositol phosphonate analog 1-O-[(3S)-3,4-bis(palmitoyloxy)butylphosphonyl]-1D-3-deoxy-myo-inositol (50) is schematically outlined in FIG. 5. For the synthesis of the phosphonate analog (50), the dichloride (48) is prepared from (S)-3,4-bis (palmitolyloxy)butyl-hosphonic acid (47) with oxalyl chloride in the presence of a catalytic amount of DMF at room temperature. The inositol component, ID-2,4,5,6-tetra-O-benzyl-3-deoxy-myo-inositol (49) is obtained as reported before. Phosphorylation of (49) with (48) in the presence of a base provides monoesterchloride intermediate which is transformed into (40) by hydrolysis, a reaction which proceeds in a surprisingly sluggish manner. After purification by preparative TLC, catalytic hydrogenation of (40) using Pd(OH).sub.2/C in tert-butanol provides the target phosphonatel-O-[(3S)-3,4-bis(palmitoyloxy)butyl-phosphonyl]-1D-3-deoxy-my- o-inositol (50) in good yield.

C) The Diacylglycerol Position

A third site of interest for rational drug design of PtdIns antagonists selected by the inventors was the diacylglycerol at position 25 (the lipid ester moiety in the DAG). The diacylglycerol position was selected for modification in order to potentially enhance the PtdIns-3-kinase anti-metabolite properties of the compounds designed according to the invention.

Specifically, rational modification of the diacylglycerol ester lipid at position 25 was effected by substituting the diacylglycerol group with a lipid moiety of a compound having known PtdIns-3-kinase inhibition properties and/or anti-tumor properties. It is noted that diacylglycerol, which is an endogenous activator of PKC and tumor cell growth, and which is liberated upon PtdIns analog hydrolysis, has antagonizing effects against the inhibition of PtdIns-3-kinase signaling. By contrast, the lipid moiety which was incorporated in the designed compounds lacks the antagonist effects of diacylglycerol against PtdIns-3-kinase signaling inhibition.

Thus, novel compounds were designed potentially to increase the potency of D-3 deoxyPtdIns and to reduce the possibility of unwanted side effects stemming from the metabolic production of diacylglycerol.

FIG. 6 shows a generic structure of D-3-deoxyPtdIns ether lipid analogs designed according to the invention. Design of 3-deoxy-PI ether lipid analogs is of especial interest because of the potential enhanced stability of these compounds to phospholipases. This will potentially occur because the 3-deoxy-PI ether lipid should not function as a substrate for PI-PLC. A further advantage of the ether lipids is that they have previously been shown to possess intrinsic anti-tumor activity against a variety of tumor types. In fact, some ether lipid analogs which have undergone clinical testing as anti-tumor agents are inhibitors of PI-3-kinase. They affect several aspects of lipid intracellular signaling, and their anti-tumor activity may arise, from a combination of effects on the signaling pathway. In this regard, 1-O-octadecyl-2-O-methylglycero-phosphocholine (edelfosfine) and a number of related compounds are known inhibitors of PI-PLC with IC.sub.50s in the low .mu.M range.

In particular, the compound 1-O-(2-O-methyl-1-O-octadecyl-sn-glycero-3-phospho)1D-3-deoxy-myo-inosito- l (OMDPI) was synthesized by modifying DPI to replace the dipalmitoylglycerol group with 1-O-octadecyl-2-O-methyl-sn-glycerol. The synthesis of OMDPI (60) is schematically outlined in FIG. 7.

The starting material for the D-3-deoxy-PtdIns ether lipid analog is the regioisomeric mixture of viburnitol (i.e., 3-deoxy-myo-inositol) 1,2:4,5- and 1,2:5,6-diacetonides (62), (63), obtained from L-quebrachitol. Controlled acidic hydrolysis of the more labile trans acetonide moieties in this mixture provides monoacetonide (64) in 79% yield. All of the three required O-benzyl groups are then introduced simultaneously with benzyl bromide and NaH in DMF (74% yield), and the remaining cis-acetonide gas removed by acidic hydrolysis (96% yield). The resulting diol (66) is protected selectively at the equatorial 1-hydroxyl by reacting its cyclic dibutylstannylene derivative with chloromethyl methyl ether. Following benzylation of the 2-hydroxyl (73% yield) and acidic hydrolysis of the MOM ether (77% yield) resulted in the formation of the key intermediate, 2, 4, S, 6-tetra-O-benzylburnitol (69), in crystalline form.

Another component for the synthesis of 3-deoxy-phosphatidylinositol ether lipid analog is 1,2 disubstituted-sn-glycerol, which is obtained in high yield and in high enantiomeric excess by carrying out the asymmetric dihydroxylation of allyl 4-methoxyphenyl ether (80). 1-O-octadecyl-2-O-Me-sn-glycerol (84) is illustrated in FIG. 8 as an example. Selective monoalkylation with 1bromooctadecane is achieved via the 1-2-O-stannylene intermediate, and the resulting secondary alcohol (82) is then methylated to provide (83). This particular strategy allows facile manipulation of the size of the alkyl side chains, a feature which was found to significantly affect the solubility of the resultant PtdIns analogs under the assay conditions. Final removal of the 3-O-(4-methoxyphenyl) group with ceric ammonium nitrate (CAN) provides the desired glycerol (84).

Subsequently phosphitylation of intermediates (69) with O-benzyl N,N,N'N'-tetraisopropyl-phosphorodiamidite catalyzed by diisopropylammonium tetrazolide provided the phosphoramidite (75) in quantitative yield which was then coupled with ether lipid (84) in the presence of tetrazole. The resulting phosphates were oxidized to the phosphates (76) with tert-butyl hydroperoxide (74% yield for 3 steps). Final hydrogenolysis then provides the desired ether lipid analog 1-O-(2-O-methyl-1-O-octadecyl-sn-glycero-3-phospho)-1D-3-deoxy-myo-inosit- ol (60) in 96% yield.

The synthesis of the phosphonate analog 1-O-[(3S)-methoxy-4-(octadecyloxy)butylphosphonyl]-1D-3-deoxy-myo-inosito- l (90) of OMDPI is schematically outlined in FIG. 9. Methyl phosphonate (94) underwent an S.sub.N2 reaction with glyceryl triflate to yield the phosphonate (95). Lastly, hydrogenation of (95) delivered (90).

Biological Activity

To confirm the efficacy of the subject analogs in inhibiting PtdIns-3-Kinase, and in particular the inhibition of cell proliferation and/or differentiation and/or induction of apoptosis of cancer cells, the following experiments were controlled. These experiments were effected in particular to assess the anti-tumor activity and the binding properties of compounds according to the invention.

A) Anti-Tumor Activity

Specifically, anti-tumor activity was assessed by growing HT-29 colon cancer cells colonies in soft agarose which were then exposed to compounds according to the invention for 7 days and the colonies then counted. Values were expressed as the IC.sub.50 for inhibition of colony formation and are the mean of 3 determinations.+-.S.E. The results of these experiments are contained in Table 1 below.

TABLE-US-00001 TABLE 1 In Vitro Anti-Tumor Activity Compound Soft Agarose IC.sub.50 (.mu.M) 3-deoxy-PtdIns (30) 35 .+-. 9 3-flouro-PtdIns (35) 37 .+-. 3 2,3-dideoxy-PtdIns 50 .+-. 7 3-deoxy-PtdIns phosphonate (50) 10 .+-. 2 OMDPI (60) 2.1 .+-. 0.1 OMDPI phosphonate (90) 45 .+-. 7

As shown in Table 1, 3-deoxy-PtdIns (30) and 3-Fluoro-3-deoxy-PtdIns (35) inhibited colony formation of HT-29 human colon carcinoma cells with IC.sub.50 values of 35 and 37 .mu.M, respectively, while 3-chloro-3-deoxy-PtdIns (not shown) was virtually inactive (<20% growth inhibition at maximum tested concentration). The ether lipid analog OMDPI (60) was found to be 15-fold more active in its growth inhibitory activity (2 .mu.M) compared to DPI. Replacement of the phosphate group of DPI by a phosphonate was found to increase the growth inhibition by over a 3-fold (1C.sub.50 for (50) is 10 .mu.M). However, the same modification decreased the activity of OMDPI (IC.sub.50 for (90) is 45 .mu.M).

These results indicate that replacement of the diacylglycerol moiety with an ether 20 lipid group resulted in an over 15-fold increase in growth inhibition activity (compare compounds (30) and (60)). Replacement of phosphate by phosphonate increased the growth inhibiting activity of 3-deoxy-PI by almost 3-fold (compare compounds (30) and (50)). However, it decreased the growth inhibiting activity of the 3-deoxy ether lipid analog (compare (60) and (90)).

Based on the observation that replacement of the diacylglycerol moiety of D-3-deoxy-PtdIns with ether lipid provided over 15-fold increase in in vitro growth inhibitory potency against HT-29 tumor cells, the activity of 1-O-octadecyl-2-O-methyl-sn-glycero3-phospho-myo-inositol (that is the ether lipid analog of PtdIns with a myo-inositol group instead of a D-3-deoxymyo-inositol group) was then tested. 1-O-octadecyl-2-O-methyl-sn-glycero-3-phospho-myo-inositol was found to be a poor inhibitor of PtdIns-PLC and only a weak inhibitor of cancer cell growth. Based on these results, it appears that the increase in anti-tumor activity of OMDPI is not solely attributable to the incorporation of the ether lipid moiety. Rather, the enhanced anti-tumor activity of OMDPI apparently is the synergistic result of both the modification of the 3 position of the myo-inositol ring 21 and the diacylglycero position 25.

B) PH Domain Inhibition

PtdIns-3-phosphates bind to and activate PH domain containing enzymes. Accordingly, the ability of the D-3-deoxy-PtdIns analogs to inhibit the activation of the PH domain dependent enzyme Akt was investigated. NIH 3T3 cells were transiently transfected with human Akt with a hemagglutinin (HA) epitope tag. The cells were exposed to the D-3-deoxy-PtdIns analogs for 6 hours and stimulated with PDGF to activate Akt. The Akt was immunoprecipitated with anti-HA antibody and its ability to phosphorylate histone-H2B measured using [.gamma..sup.32P]-ATP. Histone H2B was separated by SDS PAGE and bands on the gel quantitated using a phosphorimager. The results are shown in FIG. 10. PDGF resulted in a marked increase in Akt activity in the cells and both D-3-deoxy-PtdIns and D-3-deoxy-PtdIns ether lipid blocked Akt activation. Wortmannin, an inhibitor of PtdIns-3-kinase, blocked Akt activation as expected.

Also, the in vitro inhibition of bovine PI-PLC and of bovine brain p110/p85 PI-3-kinase were measured as previously described ("In vitro and in vivo activity of the phosphatidylinositol-3-kinase inhibitor, wortrnannin"; Schultz et al.; Anticancer Res., 15:1135 1140 (1995)). The results of this binding experiment are summarized in Table 2:

TABLE-US-00002 TABLE 2 -PH Domain Inhibition PtdIns-PLC PI-3-K IC50 W) IC50 (gM) (30) N/A >250 (35) 8 30 (50) N/A N/A (60) 19.9 2.5 (90) 10 5.3 N/A = not active, with <20% inhibition at 100 gM.

The results show that all the compounds are only weak inhibitors of PI-PLC compared to 1-O-octadecyl-2-O-methyl glycerophosphocholine which has an IC.sub.50 under the same assay conditions of around 1 .mu.M. However, the 3-deoxy ether lipid PIs are relatively potent inhibitors of PI-3-kinase with IC.sub.50 values of 2 to 5 .mu.M. 1-O-octadecyl-2-O-methylglycerophosphocholine has previously been found to be an inhibitor of PI-3kinase with an IC.sub.50 of 35 .mu.M, while the myo-inositol containing analog is a much weaker inhibitor with an IC.sub.50 of 90 .mu.M. Thus, the presence of a 3-deoxy-myo-inositol moiety appears to impart PI-3-kinase inhibiting activity to the compounds.

Based on the unexpectedly low anti-tumor and PI-3-kinase inhibition IC.sub.50 values obtained with the lead compound OMDPI, in vivo and human clinical tests are being designed to establish anticancer protocols based on OMDPI.

Example 1 illustrates the synthesis of compounds (50), (60) and (90). Example 2 shows in vivo activity and toxicity studies comparing OMDPI (60) and DPI (30) as anti-tumor agents.

EXAMPLE 1

Preparation of Compound 50:

To a suspension of 110 mg (170 gmol) of (47) in 2 mL of CH.sub.2Cl.sub.2, and 5 .mu.L of DMF under N.sub.2, was added 119 .mu.L (1.36 mmol) of oxalyl chloride. The white solid disappeared in five minutes, the resulting solution is then stirred at room temperature for another 4 hours. After removal of the solvent in vacuum, the residue (compound (48)) is dried and used directly in the next step without further purification.

To a solution of (48) and 89 mg (170 .mu.mol) of (49) in 2 mL of CH.sub.2Cl.sub.2, was added 89 .mu.L (0.51 mmol) of i-pr.sub.2NEt and 5 mg of DMAP. The resulting mixture was allowed to stir at room temperature overnight followed by hydrolysis with water. The product was then extracted with CDCl.sub.3 and dried over MgS0.sub.4. After concentration, the residue was purified by preparative TLC developed by CH.sub.2Cl.sub.2/MeOH (v/v 9/1), giving 105 mg (54%) of (40) as a yellow syrup.

99.1 mg of (40) in 11 mL of tert-butanol is hydrogenated under 5 bar H.sub.2 over 56 mg of 20% Pd(OH).sub.2/C for 12 hours. After filtration, the filtrate is concentrated and dried in vacuum, leaving 64.9 mg (95%) of 1-O-[(3S)-3,4-bis(palmitoyloxy)butylphosphonyl]-1D-3-deoxy-myo-inosito- l (50) as a white solid.

Preparation of Compound 60:

A solution containing 1.80 g (1.74 mmol) of (69) in 50 ML of tert-butanol was hydrogenated in a Parr shaker under 70 psi of H.sub.2 for 36 h, using 1.0 g of 20% Pd(OH).sub.2/C (Aldrich, .ltoreq.50% H.sub.2O) as catalyst. The catalyst was filtered out and the filter cake was washed with 100 nl of MeOH/CHCl.sub.3 (v/v=1/1). The filtrate was concentrated and dried in vacuo leaving 0.98 g (96%) of (60) as a white powder.

Preparation of Compound 90:

To a solution of 262 mg (0.5 mmol) of (99), 113 mg (0.6 mmol) of ammonium O-benzyl-H-phosphonate, 0.2 mL of pyridine in 2 mL of CH.sub.2Cl.sub.2, was added 74 .mu.L (0.6 mmol) of pivaloyl chloride. The mixture was stirred at room temperature for 10 minutes, then diluted with 50 mL of EtOAc. The organic layer was washed with 10 mL.times.2 saturated aqueous CUS04, dried over MgS0.sub.4. After evaporation, the residue was purified by column chromatography on silica gel with EtOAc/hexane 1:1, affording 335 mg (94%) of (93) as a colorless oil.

Under N.sub.2, to a solution of 170 mg (0.25 mmol) of (93) and 20 mg (60%, 0.5 mmol) of NaH in 2 mL anhydrous THF, is added 31 .mu.L (0.5 mmol) of MeI. The resulting mixture was allowed to stir at room temperature overnight and then partitioned in 50 mL of EtOAc and 5 mL of H.sub.2O. The organic layer was washed with brine and dried over MgSO.sub.4. After evaporation, the residue was purified by column chromatography on silica gel with EtOAc/hexane 2:1, giving 112 mg (65%) of (94) as a colorless oil.

Under N.sub.2, to a solution of 77 mg (0.11 mmol) of (94) in 2 mL of anhydrous THF at -78.degree. C., is added 56 .mu.L (0.11 mmol, 2.0 N in Hexane) of n-BuLi. After stirring at -78.degree. C. for 30 min, a solution of triflate in 1 mL of THF was dropped in. The resulting reaction mixture was warmed to room temperature slowly and stirred overnight. 1 mL of MeOH was added, and the reaction mixture was then concentrated. After column chromatography on silica gel with EtOAc/hexane 1/1, 57.9 mg (51%) of (95) is obtained as a colorless oil.

A solution of 49 mg (47 .mu.mol) of (95) in 3 mL of EtOH was hydrogenated over 25 mg of 20% Pd(OH).sub.2/C under atm. of H.sub.2 at room temperature for 20 hours. After filtration, the filter cake was washed with 20 mL of CHCl.sub.3/MeOH (v/v=1/1). Evaporation and drying in vacuo left 25.7 mg (93%) of 1-O-[(3S)-methoxy-4-(octadecyloxy)butylphosphonyl]-1D-3-deoxy-myo-inosito- l (90) as a white powder.

In Vivo Activity of DPI and OMDPI

Preliminary studies of in vivo anti-tumor activity were conducted in SCID (severe combined immunodeficient) mice implanted subcutaneously with 10 HT-29 human colon adenocarcinoma cells. Injection of compoun