AU774960B2 - Amino ceramide-like compounds and therapeutic methods of use - Google Patents

Amino ceramide-like compounds and therapeutic methods of use Download PDF

Info

Publication number
AU774960B2
AU774960B2 AU59296/00A AU5929600A AU774960B2 AU 774960 B2 AU774960 B2 AU 774960B2 AU 59296/00 A AU59296/00 A AU 59296/00A AU 5929600 A AU5929600 A AU 5929600A AU 774960 B2 AU774960 B2 AU 774960B2
Authority
AU
Australia
Prior art keywords
phenyl
pyrrolidino
propanol
palmitoylamino
ethylenedioxy
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
AU59296/00A
Other versions
AU5929600A (en
Inventor
Norman S. Radin
James A. Shayman
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Michigan
Original Assignee
University of Michigan
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US09/350,768 external-priority patent/US6255336B1/en
Application filed by University of Michigan filed Critical University of Michigan
Priority claimed from PCT/US2000/018935 external-priority patent/WO2001004108A1/en
Publication of AU5929600A publication Critical patent/AU5929600A/en
Application granted granted Critical
Publication of AU774960B2 publication Critical patent/AU774960B2/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07DHETEROCYCLIC COMPOUNDS
    • C07D295/00Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/04Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
    • C07D295/12Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms
    • C07D295/125Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms with the ring nitrogen atoms and the substituent nitrogen atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07DHETEROCYCLIC COMPOUNDS
    • C07D317/00Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms
    • C07D317/08Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3
    • C07D317/44Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D317/46Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 ortho- or peri-condensed with carbocyclic rings or ring systems condensed with one six-membered ring
    • C07D317/48Methylenedioxybenzenes or hydrogenated methylenedioxybenzenes, unsubstituted on the hetero ring
    • C07D317/50Methylenedioxybenzenes or hydrogenated methylenedioxybenzenes, unsubstituted on the hetero ring with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to atoms of the carbocyclic ring
    • C07D317/58Radicals substituted by nitrogen atoms
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07DHETEROCYCLIC COMPOUNDS
    • C07D319/00Heterocyclic compounds containing six-membered rings having two oxygen atoms as the only ring hetero atoms
    • C07D319/101,4-Dioxanes; Hydrogenated 1,4-dioxanes
    • C07D319/141,4-Dioxanes; Hydrogenated 1,4-dioxanes condensed with carbocyclic rings or ring systems
    • C07D319/161,4-Dioxanes; Hydrogenated 1,4-dioxanes condensed with carbocyclic rings or ring systems condensed with one six-membered ring
    • C07D319/18Ethylenedioxybenzenes, not substituted on the hetero ring
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07BGENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00Indexing scheme relating to specific properties of organic compounds
    • C07B2200/07Optical isomers

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
  • Heterocyclic Compounds That Contain Two Or More Ring Oxygen Atoms (AREA)
  • Hydrogenated Pyridines (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Description

WO nI/din R PCT/US00/18935 1 -1- AMINO CERAMIDE LIKE COMPOUNDS AND THERAPEUTIC METHODS OF USE RELATED APPLICATIONS The present application is a continuation-in-part of U.S. Serial No. 08/883,218.
filed June 26, 1997, which is a divisional of U.S. Serial No. 08/708,574, filed September 5, 1996, now U.S. Patent No. 5,916,911, which claims priority from U.S.
Serial No. 60/004,047, filed September 20, 1995, all of which are hereby expressly incorporated by reference.
SPONSORSHIP
The present invention was supported by grant nos. R01 DK41487, R01 DK69255 and R0139255 from the National Institutes of Health, contract R43 CA 58159 from the National Cancer Institute, grant GM 35712 from the National Institute of General Medical Sciences, and by the University of Michigan Comprehensive Cancer Center grant 2P30 CA 46592 from the National Cancer Institute, U.S. Public Health Service, DHHS. Grant number for Merit Award from Veteran's Administration? The government may have certain rights in this invention.
FIELD OF THE INVENTION The present invention relates generally to ceramide-like compounds and, more particularly, to ceramide-like compounds containing a tertiary amine group and their use in therapeutic methods.
BACKGROUND OF THE INVENTION Hundreds of glycosphingolipids (GSLs) are derived from glucosylceramide (GIcCer), which is enzymatically formed from ceramide and UDP-glucose. The enzyme involved in GIcCer formation is UDP-glucose:N-acylsphingosine glucosyltransferase (GIcCer synthase). The rate of GIcCer formation under physiological conditions may depend on the tissue level of UDP-glucose, which in turn depends on the level of glucose in a particular tissue (Zador, et al., "A Role for Glycosphingolipid Accumulation in the Renal Hypertrophy of Streptozotocin-lnduced Diabetes Mellitus," J. Clin. Invest 91:797-803 (1993)). In vitro assays based on endogenous ceramide yield lower synthetic rates than mixtures containing added ceramide, suggesting that tissue levels of ceramide are also normally rate-limiting (Brenkert, A. et al., "Synthesis of Galactosyl Ceramide and Glucosyl Ceramide by Rat Brain: Assay Procedures and Changes with Age," Brain Res. 36:183-193 (1972)).
It has been found that the level of GSLs controls a variety of cell functions, such as growth, differentiation, adhesion between cells or between cells and matrix proteins, binding of microorganisms and viruses to cells, and metastasis of tumor
II
WO 01/04108 PCT/US00/18935 -2cells. In addition, the GlcCer precursor, ceramide, may cause differentiation or inhibition of cell growth (Bielawska, A. et al., "Modulation of Cell Growth and Differentiation by Ceramide," FEBS Letters 307:211-214 (1992)) and be involved in the functioning of vitamin D, tumor necrosis factor-a, interleukins, and apoptosis (programmed cell death). The sphingols (sphingoid bases), precursors of ceramide, and products of ceramide catabolism, have also been shown to influence many cell systems, possibly by inhibiting protein kinase C (PKC).
It is likely that all the GSLs undergo catabolic hydrolysis, so any blockage in the GIcCer synthase should ultimately lead to depletion of the GSLs and profound changes in the functioning of a cell or organism. An inhibitor of GlcCer synthase, PDMP (1 R-phenyl-2R-decanoylamino-3-morpholino-1-propanol), previouslyidentified as the D-threo isomer (Inokuchi, J. et al., "Preparation of the Active Isomer of 1-Phenyl-2-Decanoylamino-3-Morpholino-1-Propanol. Inhibitor of Glucocerebroside Synthetase," J. Lipid Res. 28:565-571 (1987)), has been found to produce a variety of chemical and physiological changes in cells and animals (Radin, N.S. et al., "Use of 1-Phenyl-2-Decanoylamino-3-Morpholino-1-Propanol (PDMP), an Inhibitor of Glucosylceramide Synthesis," In NeuroProtocols, A Companion to Methods in Neurosciences, S. K. Fisher et al., Ed., (Academic Press, San Diego) 3:145-155 (1993) and Radin, N.S. et al., "Metabolic Effects of Inhibiting Glucosylceramide Synthesis with PDMP and Other Substances," In Advances in Lipid Research; Sphingolipids in Signaling, Part R.M. Bell et al., Ed. (Academic Press, San Diego) 28:183-213 (1993)). Particularly interesting is the compound's ability to cure mice of cancer induced by Ehrlich ascites carcinoma cells (Inokuchi, J. et al., "Antitumor Activity in Mice of an Inhibitor of Glycosphingolipid Biosynthesis," Cancer Lett.
38:23-30 (1987)), to produce accumulation of sphingosine and N,N-dimethylsphingosine (Felding-Habermann, B. et al., "A Ceramide Analog Inhibits T Cell Proliferative Response Through Inhibition of Glycosphingolipid Synthesis and Enhancement of N,N-Dimethylsphingosine Synthesis," Biochemistry 29:6314-6322 (1990)), and to slow cell growth (Shayman, J.A. et al., "Modulation of Renal Epithelial Cell Growth by Glucosylceramide: Association with Protein Kinase C, Sphingosine, and Diacylglyceride," J. Biol. Chem. 266:22968-22974 (1991)). Compounds with longer chain fatty acyl groups have been found to be substantially more effective (Abe, A. et al., "Improved Inhibitors of Glucosylceramide Synthesis," J. Biochem.
111:191-196 (1992)).
WO 01/04108 PCT/USOO00/18935 -3- The importance of GSL metabolism is underscored by the seriousness of disorders resulting from defects in GSL metabolizing enzymes. For example, Tay- Sachs, Gaucher's, and Fabry's diseases, resulting from enzymatic defects in the GSL degradative pathway and the accumulation of GSL in the patient, all have severe clinical manifestations. Another example of the importance of GSL function is seen in a mechanism by which blood cells, whose surfaces contain selectins, can, under certain conditions, bind to GSLs in the blood vessel walls and produce acute, lifethreatening inflammation (Alon, R. et al., "Glycolipid Ligands for Selectins Support Leukocyte Tethering Rolling Under Physiologic Flow Conditions." J. Immunol., 154:5356-5366 (1995)).
At present there is only one treatment available for patients with Gaucher disease, wherein the normal enzyme which has been isolated from normal human tissues or cultured cells is administered to the patient. As with any drug isolated from human material, great care is needed to prevent contamination with a virus or other dangerous substances. Treatment for an individual patient is extremely expensive, costing hundreds of thousands, or even millions of dollars, over a patient's lifetime.
It would thus be desirable to provide a treatment which includes administration of a compound that is readily available and/or producible from common materials by simple reactions.
Possibly of even greater clinical relevance is the role of glucolipids in cancer.
For example, it has been found that certain GSLs occur only in tumors; certain GSLs occur at abnormally high concentrations in tumors; certain GSLs, added to tumor cells in culture media, exert marked stimulatory or inhibitory actions on tumor growth; antibodies to certain GSLs inhibit the growth of tumors; the GSLs that are shed by tumors into the surrounding extracellular fluid inhibit the body's normal immunodefense system; the composition of a tumor's GSLs changes as the tumors become increasingly malignant; and, in certain kinds of cancer, the level of a GSL circulating in the blood gives useful information regarding the patient's response to treatment Because of the significant impact GSLs have on several biochemical processes, there remains a need for compounds having improved GlcCer synthase inhibition activity.
It would thus be desirable to provide compounds which inhibit GlcCer synthase activity. It would also be desirable to provide compounds which inhibit GIcCer synthase activity, thereby lowering the level of GSLs and increasing GSL precursor levels, e.g. increasing the levels of ceramide and sphingols. It would further be WO 01/04108 PCTUSO0/18935 -4desirable to provide compounds which inhibit GlcCer synthase activity and lower the level of GSLs without also increasing ceramide levels. It would also be desirable to provide compounds and therapeutic methods to treat conditions and diseases associated with altered GSL levels and/or GSL precursor levels.
SUMMARY OF THE INVENTION Novel compounds are provided which inhibit GlcCer formation by inhibiting the enzyme GlcCer synthase, thereby lowering the level of GSLs. The compounds of the present invention have improved GlcCer synthase inhibition activity and are therefore highly useful in therapeutic methods for treating various conditions and diseases associated with altered GSL levels, as well as GSL precursor levels. For example, the compounds of the present invention may be useful in methods involving cancer growth and metastasis, the growth of normal tissues, the ability of pathogenic microorganisms to bind to normal cells, the binding between similar cells, the binding of toxins to human cells, and the ability of cancer cells to block the normal process of immunological cytotoxic attack.
Additional objects, advantages, and features of the present invention will become apparent from the following description and appended claims, taken in conjunction with the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS The various advantages of the present invention will become apparent to one skilled in the art by reading the following specification and subjoined claims and by referencing the following drawings in which: Figure 1 is a graph showing the growth and survival of 9L gliosarcoma cells grown in medium containing different GIcCer synthase inhibitors; Figure 2 is a graph showing the protein content of MDCK cells cultured for 24 hr in medium containing different concentrations of the separated erythro- and threoisomers of a preferred compound of the present invention; Figure 3 is a graph showing 3 H]thymidine incorporation into the DNA of MDCK cells treated with a preferred compound of the present invention; Figures 4A and 4B are graphs showing the effects of P4 and p-methoxy-P4 on GIcCer synthase activity; Figure 5 is a graph showing the linear relationship between the inhibition of GlcCer synthase activity and electronic parameter and hydrophobic parameter Figure 6 is a graph showing the effects of dioxy P4 derivatives on GIcCer synthase activity; WO 01104108 PCT/US00/18935 Figure 7 is a bar graph showing the effects of D-t-3',4'-ethylenedioxy-P4 on GlcCer synthesis and cell growth; Figure 8 is a schematic of the synthetic pathway for 4'-hydroxy-1-phenyl-2palmitoylamino-3-pyrrolidino-1-propanol; Figure 9 is an illustration of the structures of P4 and of phenyl-substituted P4 homologues; Figure 10 is an HPLC chromatogram showing the separation of the enantiomers of P4 and p-methoxy-P4 by chiral chromatography; Figure 11 is a graph showing the effects of D-threo-4'-hydroxy-P4 as compared to D-threo-p-methoxy-P4 on GIcCer synthase activity; Figure 12 is a graph showing the effects of D-threo enantiomers of P4, 4'hydroxy-P4 and 3',4'-ethylenedioxy-P4 on 1-O-acyceramide synthase activity; Figure 13 is a graph showing the effect of D-threo-P4 on GIcCer synthesis and cell growth; Figure 14 is a graph showing the effect of D-threo-4'-hydroxy-P4 on GlcCer synthesis and cell growth; and Figure 15 is a graph showing the effect of D-threo-3',4'-ethylenedioxy-P4 on GlcCer synthesis and cell growth.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Novel compounds are provided which inhibit GIcCer formation by inhibiting the enzyme GIcCer synthase, thereby lowering the level of GSLs. The compounds of the present invention have improved GIcCer synthase inhibitory activity and are therefore highly useful in therapeutic methods for treating various conditions and diseases associated with altered GSL levels.
The compounds of the present invention generally have the following formula: R c-cH-R,
OHNH
I
R=
wherein R, is a phenyl group, preferably a substituted phenyl group such as p-methoxy, hydroxy, dioxane substitutions such as methylenedioxy, ethylenedioxy, and trimethylenedioxy, cydohexyl or other acydic group, t-butyl orother branched aliphatic WO 01/04108 PCT/US00/18935 -6group, or a long alkyl or alkenyl chain, preferably 7 to 15 carbons long with a double bond next to the kernel of the structure. The aliphatic chain can have a hydroxyl group near the two asymmetric centers, corresponding to phytosphingosine.
R, is an alkyl residue of a fatty acid, 10 to 18 carbons long. The fatty acid can be saturated or unsaturated, or possess a small substitution at the C-2 position a hydroxyl group).
R
3 is a tertiary amine, preferably a cyclic amine such as pyrrolidine, azetidine, morpholine or piperidine, in which the nitrogen atom is attached to the kernel a tertiary amine).
All four structural isomers of the compounds are contemplated within the present invention and may be used either singly or in combination DL-threo or DL-erythro).
The preferred aliphatic compound of the present invention is D-threo-1pyrrolidino-1-deoxyceramide, identified as IV-231B herein and also referred to as PD.
The preferred aromatic compound of the present invention is 1-phenyl-2palmitoylamino-3-pyrrolidino-l-propanol, identified as BML-119 herein and also referred to as P4. The structures of the preferred compounds are as follows: II CH,(CH)1 2 CH-CH-C-C-CHI-N \Ok tC I OHNH OHIN I I c=o yo
C
1 5
H
3 1
C
15
H
3 1 PD P4 An additional preferred compound of the present invention are ethylenedioxy-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol, also referred to hereinasD-t-3',4'-ethylenedioxy-P4, and D-t-4'-hydroxy-1-phenyl-2-palmitoylamino-3pyrrolidino-1-propanol, also referred to herein as D-t-4'-hydroxy-P4.
By increasing the acyl chain length of PDMP from 10 to 16 carbon atoms, the efficacy of the compounds of the present invention as GIcCer synthase inhibitors is greatly enhanced. The use of a less polar cyclic amine, especially a pyrrolidine instead of a morpholine ring, also increases the efficacy of the compounds. In addition, replacement of the phenyl ring by a chain corresponding to sphingosine yields a strongly inhibitory material. By using a chirai synthetic route, it was discovered that the isomers active against GlcCer synthase had the WO 01/04108 PCT/US00/18935 -7- R,R-(D-threo)-configuration. However, strong inhibition of the growth of human cancer cells in plastico was produced by both the threo and erythro racemic compounds, showing involvement of an additional factor beyond simple depletion of cell glycosphingolipids by blockage of GIcCer synthesis. The growth arresting effects could be correlated with increases in cellular ceramide and diglyceride levels.
Surprisingly, the aliphatic pyrrolidino compound of the present invention (identified as IV-231B), was strongly inhibitory toward the GIcCer synthase and produced almost complete depletion of glycolipids, but did not inhibit growth or cause an accumulation of ceramide. Attempts were made to determine if the differences in growth effects could be attributed to the influence of the inhibitors on related enzymes (ceramide and sphingomyelin synthase and ceramidase and sphingomyelinase).
While some stimulation or inhibition of enzyme activity was noted, particularly at high inhibitor concentrations (50 pM), these findings did not explain the differing effects of the different inhibitors.
By slowing the synthesis of GlcCer, the compounds of the present invention lower the levels of all the GIcCer-derived GSLs due to the GSL hydrolases which normally destroy them. While the body will continue to make the more complex GSLs from available GIcCer, the rate of synthesis will slow down as the level of GlcCer diminishes. The rate of lowering depends on the normal rate of destruction of each GSL. These rates however, are relatively rapid in animals and cultured cells.
At higher dosages, many of the compounds of the present invention produce an elevation in the level of ceramide. Presumably this occurs because cells continue to make ceramide despite their inability to utilize it for GlcCer synthesis. Ceramide is also normally converted to sphingomyelin, but this process does not seem to be able to handle the excess ceramide. It has been unexpectedly found however, that an additional process is also involved, since even those isomers that are inert against GIcCer synthase also produce an elevation in ceramide levels. Moreover, the blockage of GlcCer synthase can occur at low inhibitor dosages, yet ceramide accumulation is not produced. The preferred aliphatic compound of the present invention, D-threo-1-pyrrolidino-1-deoxyceramide does not produce ceramide accumulation at all, despite almost complete blockage of GIcCer synthesis.
This distinction between the aromatic and the aliphatic compounds of the present invention is important because ceramide has recently been proposed to cause cell death (apoptosis) by some still unknown mechanism. At lower dose levels, the aromatic compounds of the present invention cause GSL disappearance with only WO 01/04108 PCT/US00/18935 -8small accumulation of ceramide and inhibition of cell growth. Higher dosages cause much more ceramide deposition and very slow cell growth or cell death.
In one embodiment of the present invention, methods of treating patients suffering from inbor genetic errors in the metabolism of GIcCer and its normal anabolic products (lactosylceramide and the more complex GSLs) are provided. The presently known disorders in this category include Gaucher, Fabry, Tay-Sachs, Sandhoff, and GM1 gangliosidosis. The genetic errors lie in the patient's inability to synthesize a hydrolytic enzyme having normal efficiency. Their inefficient hydrolase allows the GSL to gradually accumulate to a toxic degree, debilitating or killing the victim. The compounds of the present invention slow the formation of GSLs, thus allowing the defective hydrolase to gradually "catch up" and restore the concentrations of GSLs to their normal levels and thus the compounds may be administered to treat such patients.
With respect to Gaucher disease, it has been calculated that much of the patient's accumulated GlcCer in liver and spleen arises from the blood cells, which are ultimately destroyed in these organs after they have reached the end of their life span. The actual fraction, lipid derived from blood cells versus lipid formed in the liver and spleen cells, is actually quite uncertain, but the external source must be important. Therefore it is necessary for the compounds of the present invention to deplete the blood cells as they are formed or (in the case of white blood cells) while they still circulate in the blood. Judging from toxicity tests, the white cells continue to function adequately despite their loss of GSLs. Although the toxicity studies were not of a long enough duration to produce many new red cells with low GSL content, it is possible that circulating red cells also undergo turnover (continual loss plus replacement) of GSLs.
In an alternative embodiment of the present invention, for the treatment of disorders involving cell growth and division, high dosages of the compounds of the present invention are administered but only for a relatively short time. These disorders include cancer, collagen vascular diseases, atherosclerosis, and the renal hypertrophy of diabetic patients. Accumulation or changes in the cellular levels of GSLs have been implicated in these disorders and blocking GSL biosynthesis would allow the normal restorative mechanisms of the body to resolve the imbalance.
With atherosclerosis, it has been shown that arterial epithelial cells grow faster in the presence of a GIcCer product (lactosylceramide). Oxidized serum lipoprotein, a material that normally circulates in the blood, stimulates the formation of plaques WO 01/04108 PCT/US00/18935 -9and lactosylceramide in the inner lining of blood vessels. Treatment with the compounds of the present invention would inhibit this mitogenic effect.
In an additional embodiment of the present invention, patients suffering from infections may be treated with the compounds of the present invention. Many types of pathogenic bacteria have to bind to specific GSLs before they can induce their toxic effects. As shown in Svensson, M. et al., "Epithelial Glucosphingolipid Expression as a Determinant of Bacterial Adherence and Cytokine Production," Infect and Immun.
62:4404-4410 (1994), expressly incorporated by reference, PDMP treatment reduces the adherence of E. coli to mammalian cells. Several viruses, such as influenza type A, also must bind to a GSL. Several bacterial toxins, such as the verotoxins, cannot themselves act without first binding to a GSL. Thus, by lowering the level of GSLs, the degree of infection may be ameliorated. In addition, when a patient is already infected to a recognizable, diagnosable degree, the compounds of the present invention may slow the further development of the infection by eliminating the binding sites that remain free.
It has been shown that tumors produce substances, namely gangliosides, a family of GSLs, that prevent the host patient, from generating antibodies against the tumor. By blocking the tumor's ability to secrete these substances, antibodies against the tumor can be produced. Thus, by administering the GIcCer synthase inhibitors of the present invention to the patient, the tumors will become depleted of their GSLs and the body's normal immunological defenses will come into action and destroy the tumor. This technique was described in Inokuchi, J. et al., "Antitumor Activity in Mice of an Inhibitor of Glycosphingolipid Biosynthesis," Cancer Lett. 38:23- 30(1987), expressly incorporated by reference. The compounds of the present invention and in particular the aliphatic compounds require much lower doses than those previously described. This is particularly important because the lower dose may reduce certain side effects. Moreover, because the aliphatic compounds of the present invention do not produce ceramide accumulation, they are less toxic. In addition, 1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol may act via two pathways, GSL depletion and ceramide accumulation.
In an alternative embodiment, a vaccine-like preparation is provided. Here, cancer cells are removed from the patient (preferably as completely as possible), and the cells are grown in culture in order to obtain a large number of the cancer cells.
The cells are then exposed to the inhibitor for a time sufficient to deplete the cells of their GLSs (generally 1 to 5 days) and are reinjected into the patient These WO 01/04108 PCT/US00/18935 reinjected cells act like antigens and are destroyed by the patient's immunodefense system. The remaining cancer cells (which could not be physically removed) will also be attacked by the patient's antibodies. In a preferred embodiment, the patient's circulating gangliosides in the plasma are removed by plasmapheresis, since the circulating gangliosides would tend to block the immunodefense system.
It is believed that tumors are particularly dependent on GSL synthesis for maintenance of their growth (Hakomori, S. "New Directions in Cancer Therapy Based on Aberrant Expression of Glycosphingolipids: Anti-adhesion and Ortho-Signaling Therapy," Cancer Cells 3:461-470 (1991)). Accumulation of ceramide in treated tumors also slows their growth or kills them. Tumors also generate large amounts of GSLs and secrete them into the patient's body, thereby preventing the host's normal response by immunoprotective cells, which should generate antibodies against or otherwise destroy tumor cells tumors are weakly antigenic). It has also been shown that GSL depletion blocks the metastasis of tumor cells (Inokuchi, J. et al., "Inhibition of Experimental Metastasis of Murine Lewis Long Carcinoma by an Inhibitor of Glucosylceramide Synthase and its Possible Mechanism of Action," Cancer Res.
50:6731-6737 (1990). Tumor angiogenesis the production of blood capillaries) is strongly influenced by GSLs (Ziche, M. et al., "Angiogenesis Can Be Stimulated or Repressed in In Vivo by a Change in GM3:GD3 Ganglioside Ratio," Lab. Invest.
67:711-715 (1992)). Depleting the tumor of its GSLs should block the tumors from generating the new blood vessels they need for growth.
A further important characteristic of the compounds of the present invention is their unique ability to block the growth of multidrug resistant tumor cells even at much lower dosages. This was demonstrated with PDMP by Rosenwald, A.G. et al.,"Effects of the Glycosphingolipid Synthesis Inhibitor, PDMP, on Lysosomes in Cultured Cells," J. Lipid Res. 35:1232 (1994), expressly incorporated by reference.
Tumor cells that survive an initial series of therapeutic treatments often reappear some years later with new properties they are now resistant to a second treatment schedule, even with different drugs. This change has been attributed to the appearance in the tumor of large amounts of a specific MDR protein (P-glycoprotein).
It has been suggested that protein kinase C (PKC) may be involved in the action or formation of P-glycoprotein (Blobe, G.C. et al., "Regulation of PKC and Its Role in Cancer Biology," Cancer Metastasis Rev. 13:411-431 (1994)). However decreases in PKC have other important effects, particularly slowing of growth. it is known that PDMP does lower the cellular content of PKC (Shayman, J.A. et al., "Modulation of WO 01/04108 PCT/US00/18935 -11 Renal Epithelial Cell Growth by Glucosylceramide: Association with Protein Kinase C, Sphingosine, and Diacylglyceride," J. Biol. Chem. 266:22968-22974 (1991)) but it is not dear why it so effectively blocks growth of MDR cells (Rosenwald, A.G. et al., "Effects of the Glycosphingolipid Synthesis Inhibitor, PDMP, On Lysosomes in Cultured Cells," J. Lpid Res. 35:1232 (1994)). A recent report showed that several lipoidal amines that block MDR action also lower the level of the enzyme acid sphingomyelinase (Jaffrezou,J. et al., Inhibition of Lysosomal Acid Sphingomyelinase by Agents which Reverse Multidrug Resistance," Biochim. Biophys. Acta 1266:1-8 (1995)). One of these agents was also found to increase the cellular content of sphingosine 5-fold, an effect seen with PDMP as well. One agent, chlorpromazine, behaves like the compounds of the present invention, in its ability to lower tissue levels of GlcCer (Hospattankar, A.V. et al., "Changes in Liver Lipids After Administration of 2-Decanoylamino-3-Morpholinopropiophenone and Chlorpromazine," Lipids 17:538-543 (1982)).
It will be appreciated by those skilled in the art that the compounds of the present invention can be employed in a wide variety of pharmaceutical forms; the compound can be employed neat or admixed with a pharmaceutically acceptable carrier or other excipients or additives. Generally speaking, the compound will be administered orally or intravenously. It will be appreciated that therapeutically acceptable salts of the compounds of the present invention may also be employed.
The selection of dosage, rate/frequency and means of administration is well within the skill of the artisan and may be left to the judgment of the treating physician or attending veterinarian. The method of the present invention may be employed alone or in conjunction with other therapeutic regimens. It will also be appreciated that the compounds of the present invention are also useful as a research tool to further investigate GSL metabolism.
The following Specific Example further describes the compounds and methods of the present invention.
SPECIFIC EXAMPLE 1 The following formulas set forth preferred aromatic and aliphatic compounds: FORMULA I 1 Po R9 WO 01/04108 PCT/US00/18935 -12identified as (1R,2R)-1-phenyl-2-acylamino-3-cyclic amino-1-propanol, and referred to herein as the "aromatic inhibitors," wherein The phenyl group can be a substituted phenyl group (such as pmethoxyphenyl).
R' is an alkyl residue of a fatty acid, 10 to 18 carbons long. The fatty acid can be saturated or unsaturated, or possess a small substitution at the C-2 position a hydroxyl group).
R is morpholino, pyrrolidino, piperidino, azetidino (trimethyleneimino), Nmethylethanolamino, diethylamino or N-phenylpiperazino. A small substituent, such as a hydroxyl group, is preferably included on the cyclic amine moiety.
FORMULA II 3
NH
c=o
R'
identified as (2R,3R)-2-palmitoyl-sphingosyl amine or 1 -cyclicamino-1 -deoxyceramide or 1-cyclic amino-2-hexadecanoylamino-3-hydroxy-octadec-4,5-ene, and referred to herein as the "aliphatic inhibitors," wherein R' is an alkyl residue of a fatty acid, 10 to 18 carbons long. The fatty acid can be saturated or unsaturated, or possess a small substitution at the C-2 position a hydroxyl group).
R is morpholino, pyrrolidino, piperidino, azetidino (trimethyleneimino), Nmethylethanolamino, diethylamino or N-phenylpiperazino. A small substituent, such as a hydroxyl group, is preferably included on the cyclic amine moiety.
The long alkyl chain shown in Formula II can be 8 to 18 carbon atoms long, with or without a double bond near the asymmetric carbon atom (carbon Hydroxyl groups can, with advantage, be substituted along the aliphatic chain, particularly on carbon 4 (as in the naturally occurring sphlngol, phytosphingosine). The long chain can also be replaced by other aliphatic groups, such at t-butyl or cycopentyl.
The aromatic inhibitors (see Formula I and Table 1) were synthesized by the Mannich reaction from 2-N-acylaminoacetophenone, paraformaldehyde, and a secondary amine as previously described (Inokuchi, J. et al., "Preparation of the WO 01/04108 PCT/USO0/18935 -13- Active Isomer of 1-Phenyl-2-Decanoylamino-3-Morpholino-1-Propanol, Inhibitor of Glucocerebroside Synthetase," J. Lipid Res. 28:565-571 (1987) and Vunnam, R.R.
et al., "Analogs of Ceramide that Inhibit Glucocerebroside Synthetase in Mouse Brain," Chem. Phys. Lipids 26:265-278 (1980)). For those syntheses in which phenyl-substituted starting materials were used, the methyl group in the acetophenone structure was brominated and converted to the primary amine. Bromination of p-methoxyacetophenone was performed in methanol. The acetophenones and amines were from Aldrich Chemical Co., St. Louis, MO. Miscellaneous reagents were from Sigma Chemical Co. and the sphingolipids used as substrates or standards were prepared by methods known in the art. The reactions produce a mixture of four isomers, due to the presence of two asymmetric centers.
The aliphatic inhibitors (See Formula II and Table 2) were synthesized from the corresponding 3-t-butyldimethylsilyl-protected sphingols, prepared by enantioselective aldol condensation (Evans, D.A. et al., "Stereoselective Aldol Condensations Via Boron Enolates," J. Am. Chem. Soc. 103:3099-3111 (1981) and Abdel-Magid, A. et al., Metal-Assisted Aldol Condensation of Chiral A-Halogenated Imide Enolates: A Stereocontrolled Chiral Epoxide Synthesis," J. Am. Chem. Soc.
108:4595-4602 (1986)) using a modification of the procedure of Nicolaou et al.
(Nicolaou, K.C. et al., "A Practical and Enantioselective Synthesis of Glycosphingolipids and Related Compounds. Total Synthesis of Globotriaosylceramide J. Am. Chem. Soc. 110:7910-7912 (1988)). Each protected sphingol was first converted to the corresponding primary triflate ester, then reacted with a cyclic amine. Subsequent N-acylation and desilylation led to the final products in good overall yield (Carson. K.G. et al., "Studies on Morpholinosphingolipids: Potent Inhibitors of Glucosylceramide Synthase," Tetrahedron Lett. 35:2659-2662 (1994)). The compounds can be called 1-morpholino-(or pyrrolidino)-1-deoxyceramides.
Labeled ceramide, decanoyl sphingosine, was prepared by reaction of the acid chloride and sphingosine (Kopaczyk, K. C. et al., "In Vivo Conversions of Cerebroside and Ceramide in Rat Brain," J. Lipid Res. 6:140-145 (1965)) and NBD-SM (12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)]- sphingosylphosphorylcholine) was from Molecular Probes, Inc., Eugene, OR.
Methods TLC of the amines was carried out with HPTLC plates Merck silica gel and C-M-HOAc 90:10:10 (solvent A) or 85:15:10 (solvent B) or C-M-conc. ammonium WO 01/04108 PCT/US00/18935 -14hydroxide 30:10:1 (solvent The bands were stained with iodine or with Coomassie Brilliant Blue R-250 (Nakamura, K. et al., "Coomassie Brilliant Blue Staining of Lipids on Thin-Layer Plates," Anal. Biochem. 142:406-41 (1984)) and, in the latter case, quantified with a Bio-Rad Model 620 videodensitometer operated with reflected white light. The faster band of each PDMP analog, previously identified as the erythro form, corresponds to the 1 S,2R and 1R,2S stereoisomers, and the slower band, previously identified as the threo form, corresponds to the 1R,2R and 1S,2S stereoisomers.
TLC of the cell lipids was run with C-M-W 24:7:1 (solvent D) or 60:35:8 (solvent E).
Growth of cell lines. Comparisons of different inhibitors with regard to suppression of human cancer cell growth were made by the University of Michigan Cancer Center in vitro Drug Evaluation Core Laboratory. MCF-7 breast carcinoma cells, HT-29 colon adenocarcinoma cells, H-460 lung large cell carcinoma cells, and 9L brain gliosarcoma cells were grown in RPMI 1640 medium with 5% fetal bovine serum, 2 mM glutamine, 50 units/ml of penicillin, 50 mg/ml of streptomycin, and 0.1 mg/ml of neomycin. UMSCC-10A head and neck squamous carcinoma cells were grown in minimal essential medium with Earle salts and the same supplements.
Medium components were from Sigma Chemical Co. Cells were plated in 96-well microtiter plates (1000 cells/well for H-460 and 9L cells, and 2000 cells/well for the other lines), and the test compounds were added 1 day later. The stock inhibitor solutions, 2 mM in 2 mM BSA, were diluted with different amounts of additional 2 mM BSA, then each solution was diluted 500-fold with growth medium to obtain the final concentrations indicated in the Figures and Tables.
Five days after plating the H-460 and 9L cells, or 6 days for the other lines, cell growth was evaluated by staining the adhering cells with sulforhodamine B and measuring the absorbance at 520 nm (Skehan, P. et al., "New Colorimetric Cytotoxicity Assay for Anticancer-Drug Screening," J. Natl. Cancerlnst. 82:1107-1112 (1990)). The absorbance of the treated cultures is reported as percent of that of control cultures, to provide an estimate of the fraction of the cells that survived, or of inhibition of growth rate.
For the experiments with labeled thymidine, each 8.5 cm dish contained 500,000 Madin-Darby canine kidney (MDCK) cells in 8 ml of Dulbecco modified essential supplemented medium. The cells were incubated at 37'C in 5% CO 2 for 24 h, then incubated another 24 h with medium containing the inhibitor-BSA compiex.
The control cells were also incubated in the presence of BSA. The cells were washed WO 01/04108 PCT/US00/1 8935 with phosphate/saline and trichloroacetic acid, then scraped off the dishes, dissolved in alkali, and analyzed for protein and DNA incorporated tritium. [Methyl-'H]thymidine yCi) was added 4 h prior to harvesting.
Assay of sphingolipid enzymes. The inhibitors were evaluated for their effectiveness against the GicCer synthase of MDCK cell homogenates by incubation in a thermostatted ultrasonic bath (Radin N.S. et al., "Ultrasonic Baths as Substitutes for Shaking Incubator Baths," Enzyme 45:67-70 (1991)) with octanoyl sphingosine and uridinediphospho['H]glucose (Shukla, G.S. et al., "Glucosylceramide Synthase of Mouse Kidney: Further Characterization and Improved Assay Method," Arch.
Biochem. Biophys. 283:372-378 (1990)). The lipoidal substrate (85 pg) was added in liposomes made from 0.57 mg dioleoylphosphatidylcholine and 0.1 mg of Na sulfatide. Confluent cells were washed, then homogenized with a micro-tip sonicator at O'C for 3 x 30 sec; -0.2 mg of protein was used in each assay tube. In the case of the aromatic inhibitors, the test compound was simply evaporated to dryness from solution in the incubation tube. This method of adding the inhibitor was found to give the same results as addition as a part of the substrate liposomes. The aliphatic inhibitors, which appeared to be less soluble in water, were added as part of the substrate liposomes.
Acid and neutral ceramidases were assayed under conditions like those above, but the medium contained 110 pM [1-"C 1 4 decanoyl sphingosine (10 5 cpm) in 340 pM dioleoylphosphatidylcholine liposomes and 0.34 mg of MDCK cellular protein homogenate. The acid enzyme was incubated in 32.5 mM citrate-Na* (pH 4.5) and the neutral enzyme buffer was 40 mM Tris-Cl" (pH 7.1 at 37'C). After 60 min in the ultrasonic bath, 3 ml of C-M 2:1, carrier decanoic acid, and 0.6 ml of 0.9% saline were added and the lipids in the lower layer were separated by TLC with C-HOAc 9:1. The liberated decanoic acid was scraped off the glass plate and counted.
Ceramide synthase was assayed with 1 pM [3- 3 H]sphingosine (70,000 cpm, repurified by column chromatography), 0.2 mM stearoyl-CoA, 0.5 mM dithiothreitol, and -300 pg of MDCK homogenate protein in 25 mM phosphate-K* buffer, pH 7.4, in a total volume of 0.2 ml. The incubation (for 30 min) and TLC were carried out as above and the ceramide band was counted.
Sphingomyelin synthase was evaluated with 44 pM "C]Jdecanoyl sphingosine (105 cpm) dispersed with 136 pM dioleoyllecithin as in the ceramide synthase assay, and 5 mM EDTA and 50 mM Hepes-Na' pH 7.5, in a total volume of 0.5 ml. MDCK homogenate was centrifuged at 600 X g briefly, then at 100,000 X g for 1 h, and the WO 01/04108 PCI/US00/18935 -16pellet was suspended in water and sonicated with a dipping probe. A portion of this suspension containing 300 pg of protein was used. Incubation was at 37'C for min, after which the lipids were treated as above, using C-M-W 60:35:8 for the isolation of the labeled decanoyl SM.
Acid and neutral SMase assays were based on the procedures of Gatt et al.
(Gatt, S. et al., "Assay of Enzymes of Lpid Metabolism With Colored and Fluorescent Derivatives of Natural Lipids," Meth. Enzymol. 72:351-375 (1981)), using liposomes containing NBD-SM dispersed like the labeled ceramide (10 pM substrate and 30 pM lecithin). The assay medium for the neutral enzyme also contained 50 mM Tris-CI' (pH 25 mM KCI, 5 mM MgCI, and 0.29 mg of MDCK cell protein in a total volume of 0.25 ml. Incubation was at 37'C for 30 min in the ultrasonic bath, then the fluorescent product, NBD-ceramide, was isolated by partitioning the assay mixture with 0.45 ml 2-propanol, 1.5 ml heptane, and 0.2 ml water. After centrifugation, a trace of contaminating NBD-SM was removed from 0.9 ml of the upper layer by washing with 0.35 ml water. The upper layer was analyzed with a fluorometer (460 nm excitation, 515 nm emission).
Acid SMase was assayed with the same liposomes in 0.2 ml of assay mixture containing 125 mM NaOAc (pH 5.0) and 61 pg of cell protein, with 60 min of incubation at 37'C. The resultant ceramide was determined as above.
Results Table 1 lists the aromatic compounds (see Formula I) synthesized and their migration rates on silica gel TLC plates. Separation of the threo- and erythro-steroisomers by TLC was generally very good, except for BML-120, -121, and -122 in the acidic solvent. In the basic solvent BML-119 and BML-122 yielded poorly resolved double bands. BML-112 was unexpectedly fast-running, especially when compared with BML-120; both are presumably dihydrochlorides.
TABLE 1 Structures of the Aromatic Inhibitors BML Number R G Phenyl TLC hR, or Name Group Substituent Value" PDMP" morpholino 34(47) PPMP morpholino (53) 112 N-phenylpiperazino 56 113 morpholino p-fluoro WO 01/04108 PCT/US00/18935 -17- 114 diethylamino 115 piperidino (pentamethyleneimino) 29 116 hexamethyleneimino 34 117" morpholino p-fluoro 41 118 piperidino p-fluoro 26 119 pyrrolidino (tetramethyleneimino) 20-70(44) 120 1 -methylpiperazino 7-62 121 3-dimethylaminopiperidino 1-30 122 N-methylethanolamino 6-71 123 azetidino (trimethyleneimino) 12 124 amino 125 morpholino p-methoxy 37 126 pyrrolidino p-methoxy Only the relative R, value of the faster-moving band is shown. The first value was obtained with solvent A, the second with solvent C, and the numbers in parentheses, with solvent B. In the case of BML-117, -125, and -126, a 20-cm high TLC plate was used to improve the seperation.
b The fatty acid chain suggested by the R' group is decanoyl, not palmitoyl.
Table 2 describes four aliphatic inhibitors (see Formula II), which can be considered to be ceramide analogs in which the C-1 hydroxyl group is replaced by a cyclic amine. It should be noted that the carbon frameworks of compounds in Tables 1 and 2 are numbered differently (see Formulas I and II), thus affecting comparisons of stereochemical configurations. The threo- and erythro-isomers separated very poorly on TLC plates. Like the aromatic inhibitors, however, the morpholine compounds ran faster than the pyrrolidine compounds. The latter are presumably more strongly adsorbed by the silica gel because they are more basic.
WO 01/04108 PCT/US00/18935 18- TABLE 2 Characterization of the Sphingosyl Inhibitors Number R Group Sphingol TLC hR, GroupStructure Value' IV-181A morpholino 2R,3S 43 IV-206A morpholino 2R,3R IV-230A pyrrolidino 2R,3S 31 IV-231B pyrrolidino 2R,3R 31 TLC solvent: C-M-HOAc 90:5:10. Similar but faster migrations were obtained with solvent A.
Structure-activity correlations. The results of testing the compounds in an assay system for GIcCer synthase are listed in Table 3. Each inhibition determination SD) shown in Table 3 was carried out in triplicate. Some of the inhibitors were tested as mixtures of DL-erythro- and DL-threo-isomers (see column Only the D-threo enantiomer in each mixture was predicted to be the actual enzyme inhibitor (Inokuchi, J. et al., "Preparation of the Active Isomer of 1-Phenyl-2-Decanoylamino-3-Morpholino-1-Propanol, Inhibitor of Glucocerebroside Synthetase," J. Lipid Res. 28:565-571 (1987)); the content of this Isomer was calculated by measuring the proportions of the threo- and erythro- racemic mixtures by quantitative TLC. The DL-threo contents were found to be in the range of 40 to 72%. The comparisons, in the case of the mixtures, are therefore approximate (most of the samples were not purified to remove the three less-active isomers and the observed data were not corrected for the level of the primary enantiomers). The separation of the threo- and erythro- forms is most conveniently accomplished by crystallization, but the specific conditions vary for each substance; thus only BML-119, a strong inhibitor, was separated into its threo- and erythro- forms. BML-112 is not included in Table 3 because it had no inhibitory activity against GlcCer synthase of rabbit liver microsomes.
WO 01/04108 PCF[USOO/18935 19 TABLE 3 Inhibition of Ceramide Glucosyltransferase of MDCK cell Homogenates by Different Compounds Inhibitor Inhibitio at 80P inhibition at I Active Number 5 fl'Ol 8 a pM isomer'~ BML-1 13 60+.-478 29 BML-114 31 2.98 BML-1 15 84 +0.8a 12.4 27 820. b BML-1 16 28 +3.28 27 13ML-1 17 350. b 36 BML-1 18 62 0.4b 8.3 32 B3ML-1 19 94 1.4" 51 +2.30 29 97 .1c 49±+0.8' 96 01 BML-120 11 +4 3.Oc 26 BML-121 11 0.4c 28 BML-122 581. d 26 BML-123 86 0.d15 0.8' 33 BMVL-124 BML-1 25 9+3.00 26 BML-126 60 1.80 34 54 0.3' POMP 90 +0.88 16 1.8' 100 PPMP 32+ 1.80 100 32 0.7' IV-1 81A ___12+0.29 100 IV-206A 73+ 1.59 100 IV-230A 100 LIV-231 B 87+0.49 100 'Different samples were assayed as parts of different experiments.
"Percent of the active D-stereolsomer in the synthesized sample, estimated by scanning the two stained bands, assuming the slower one was the (raoemic) active form.
Comparison of POMP (1 R,2R-decanoate) and PPMP (1 R,21R-palmitate), when evaluated at the same time in Expt. f, shows that an Increase In the chain length of WO 01/04108 PCT/US00/18935 the N-acyl group from 10 to 16 carbon atoms distinctly improved the inhibitory activity against GlcCer synthase, as noted before (Abe, A. et al., "Improved Inhibitors of Glucosylceramide Synthesis," J. Biochem. 111:191-196 (1992)). Accordingly, most of the other compounds were synthesized with the palmitoyl group for comparison with PPMP. The comparisons between the best inhibitors are clearer at the 5 pM level.
Replacing the oxygen in the morpholine ring of PPMP with a methylene group (BML-115) improved activity -1.4-fold (calculated from the inhibitions at 5pM in Expt.
f and relative purities, and assuming that the percent inhibition is proportional to concentration in this region: 12.4/27 x 100/32 Previous comparison with mouse brain, human placenta, and human Gaucher spleen glucosyltransferase also showed that replacing the morpholino ring with the piperidino ring in a ketone analog of PDMP (1-phenyl-2-decanoylamino-3-piperidino-1-propanone) produced a much more active inhibitor (Vunnam, R.R. et al., "Analogs of Ceramide that Inhibit Glucocerebroside Synthetase in Mouse Brain," Chem. Phys. Lipids 26:265-278 (1980)).
Replacing the piperidine group with a 7-membered ring (BML-116) greatly decreased the activity, while use of a 5-membered ring (BML-119) quadrupled the effectiveness (50 vs 12.4% inhibition). A 4-membered ring (BML-123) yielded a compound about as effective as the piperidino compound. The parent amine (BML-124), its N,N-diethyl analog (BML-114), and the sterically bulky N-phenylpiperazine analog (BML-112) displayed little or no activity.
Replacing a hydrogen atom with a fluorine atom in the p-position of the phenyl ring decreased the inhibitory power (BML-117 vs PDMP and BML-118 vs BML-115).
Substitution of the p-position with an electron-donating moiety, the methoxy group, had a similar weakening effect in the case of the morpholino compound (BML-125 vs PPMP). Comparison of the pyrrolidino compounds, which are more basic than the morpholino compounds, showed that the methoxy group enhanced the inhibitory power (BML-126 vs BML-119).
Preparations of BML-119 were separated into threo and erythro racemic mixtures by HPLC on a Waters Microbondapak column, using M-W-conc. NH 4
OH
90:10:0.2 as the elution solvent. The material eluting earlier (but migrating more slowly on a TLC plate) was called BML-130; the later eluting material (faster by TLC) was called BML-129. Assay of GicCer synthase with each preparation at 5 pM showed 15% inhibition by BML-129 and 79% inhibition by BML-130. TLC analysis of WO 01/04108 PCT/US00/18935 -21 the two preparations revealed incomplete separation, which could explain the minor inhibition by BML-129. When the two stereoisomers were separated by preparative TLC, the difference in effectiveness was found to be somewhat higher, evidently due to the better separation by this method. Thus the slower-migrating stereoisomer accounted for all or nearly all of the inhibitory activity, as noted with PDMP (Inokuchi, J. et al., "Preparation of the Active Isomer of 1-Phenyl-2-Decanoylamino-3-Morpholino-1-Propanol, Inhibitor of Glucocerebroside Synthetase," J. Upid Res. 28:565-571 (1987)).
Comparison of the two pairs of aliphatic inhibitors (bottom of Table 3) showed that the 2R,3R (D-threo) form is the primary inhibitor of glucosyltransferase. This finding is in agreement with previous identification of the active PDMP isomer as being the D-threo enantiomer. However, unlike the aromatic analog, BML-129 (2R,3S/2S,3R), there was a relatively small but significant activity in the case of the (erythro) 2R,3S stereoisomer. The erythro form of PDMP was found to inhibit cell proliferation of rabbit skin fibroblasts almost as well as R,R/S,S-PDMP but it did not act on the GSLs (Uemura, K. et al., "Effect of an Inhibitor of Glucosylceramide Synthesis on Cultured Rabbit Skin Fibroblasts," J. Biochem. (Tokyo) 108:525-530 (1990)). As noted with the aromatic analogs, the pyrrolidine ring was more effective than the morpholine ring (Table 3).
Comparison of the aliphatic and corresponding aromatic inhibitors can be made in the case of the optically active morpholine compounds PPMP and IV-206A, both of which have the R,R structure and the same fatty acid. Here it appears that the aliphatic compound is more effective (Table However in a second comparison, at lower concentrations with the inhibitors incorporated into the substrate liposomes, the degree of inhibition was 77 0.9% with 3 pM IV-231B and 89 0.6% with 6 pM DL-threo BML-119.
Evaluations of cultured cell growth. Exposure of five different cancer cell lines to inhibitors at different concentrations for 4 or 5 days showed that the six BML compounds most active against GlcCer synthase were very effective growth inhibitors (Table The IC5 values (rounded off to one digit in the table) ranged from 0.7 to 2.6 pM.
WO 01/04108 PCT/USOO/18935 -22- TABLE 4 Inhibition of Tumor Cell Growth In Vitro by Various Inhibitors Cell BML- BML- BML- BML- BML- BML- BML- Type 115 118 119 123 126 129 130 MCF-7 2 2 2 2 1 3 2 H-460 2 2 1 1 1 2 3 HT-29 2 1 2 1 2 2 9L 2 2 1 2 2 2 2
UMSCC
UMSCC 1 1 1 2 2 -10A Figure 1 shows growth and survival of 9L gliosarcoma cells grown in medium containing different GlcCer synthase inhibitors, as described above. The BML compounds were used as synthesized (mixtures of DL-threo and -erythro stereoisomers) while the PDMP and PPMP were optically resolved R,R isomers. The concentrations shown are for the mixed racemic stereoisomers, since later work (Table 4) showed that both forms were very similar in effectiveness. Figure 1 illustrates the relatively weak effectiveness of R,R-PPMP and even weaker effectiveness of R,R-PDMP. The three new compounds, however, are much better inhibitors of GIcCer synthase and growth. These differences in growth inhibitory power correlate with their effectiveness in MDCK cell homogenates as GIcCer synthase inhibitors. Some differences can be expected due to differences in sensitivity of the synthase occurring in each cell type (the synthases were assayed only in MDCK cells).
Growth inhibition by each of the most active BML compounds occurred in an unusually small range of concentrations the slopes of the cytotoxic regions are unusually steep). Similar rapid drop-offs were seen in another series of tests with 9L cells, in which BML-119 yielded 71% of the control growth with 1 pM inhibitor, but only 3% of control growth with 3 pM. Growth was 93% of control growth with 2 pM BML-130 but only 5% of controls with 3 pM inhibitor. While some clinically useful drugs also show a narrow range of effective concentrations, this is a relatively uncommon relationship.
When the erythro- and threo-stereoisomeric forms of BML-119 (-129 and -130) were compared, they were found to have similar effects on tumor cell growth (Table This observation is similar to the results with PDMP isomers in fibroblasts cited WO 01/04108 PCT/US00/18935 -23above (Uemura, K. et al., "Effect of an Inhibitor of Glucosylceramide Synthesis on Cultured Rabbit Skin Fibroblasts," J. Biochem. (Tokyo) 108:525-530 (1990)). Since enzymes are optically active and since stereoisomers and enantiomers of drugs can differ greatly in their effect on enzymes, it is likely that BML-129 and BML-130 work on different sites of closely related metabolic steps.
Figure 2 shows the amount of cellular protein per dish for MDCK cells cultured for 24 h in medium containing different concentrations of the separated erythro- and threo- isomers of BML-119, as percent of the incorporation by cells in standard medium. Each point shown in Figure 2 is the average of values from three plates, with error bars corresponding to one standard deviation.
Figure 3 shows 3 H]thymidine incorporation into DNA of MDCK cells incubated as in Figure 2. The values in Figure 3 are normalized on the basis of the protein content of the incubation dishes and compared to the incorporation by cells in standard medium.
Figures 2 and 3 thus provide comparison of the two stereoisomers with MDCK cells. The isomers were found to inhibit growth and DNA synthesis with similar effectiveness. Thus the MDCK cells behaved like the human tumor cells with regard to ICo and the narrow range of concentrations resulting in inhibition of protein and DNA synthesis.
Surprisingly, the aliphatic inhibitor IV-231B exerted no inhibitory effect on MDCK cell growth when incubated at 20 pM for 1 day or 1 pM for 3 days. Tests with a longer growth period, 5 days, in 5 pM inhibitor also showed no slowing of growth.
The dishes of control cells, which contained BSA as the only additive to the medium, contained 3.31 0.19 mg of protein, while the IV-231B/BSA treated cells contained 3.30 0.04 mg.
Lipid changes induced in the cells. Examination by TLC of the alkali-stable MDCK lipids after a 24 h incubation disclosed that BML-130 was more effective than BML-129 in lowering GIcCer levels, as expected from its greater effectiveness in vitro as a glucosyltransferase inhibitor. The level of GIcCer, estimated visually, was greatly lowered by 0.3pM BML-130 or 0.5 pM BML-129. The levels of the other lipids visible on the plate (mainly sphingomyelin cholesterol, and fatty acids) were changed little or not at all. BML-129 and the GIcCer synthase inhibitor, BML-130, were readily detected by TLC at the various levels used, showing that they were taken up by the cells during the incubation period at dose-dependent rates. Lactosylceramide WO 01/04108 PCT/US00/18935 -24overlapped the inhibitor bands with solvent D but was well separated with solvent E, which brought the inhibitors well above lactosylceramide.
Ceramide accumulation was similar for both stereoisomers (data not shown).
An unexpected finding is that noticeable ceramide accumulation appeared only at inhibitor concentrations that were more than enough to bring GIcCer levels to a very low point at 2 or 4 pM). The changes in ceramide concentration were quantitated in a separate experiment by the diglyceride kinase method, which allows one to also determine diacylglycerol (DAG) concentration (Preiss, J.E. et al., "Quantitative Measurement of SN-1,2-Diacylglycerols Present in Platelets, Hepatocytes, and Ras- and Sis-Transformed Normal Rat Kidney Cells," J. Biol. Chem.
261:8597-8600 (1986)). The results (Table 5) are similar to the visually estimated ones: at 0.4 pM BML-129 or -130 there was little effect on ceramide content but at 4 pM inhibitor, a substantial increase was observed. (While the duplicate protein contents per incubation dish were somewhat erratic in the high-dose dishes, in which growth was slow, the changes were nevertheless large and clear.) Accumulation of ceramide had previously been observed with PDMP, at a somewhat higher level of inhibitor in the medium (Shayman, J.A. et al., "Modulation of Renal Epithelial Cell Growth by Glucosylceramide: Association with Protein Kinase C, Sphingosine, and Diacylglyceride," J. Biol. Chem. 266:22968-22974 (1991)). From the data for cellular protein per incubation dish, it can be seen that there was no growth inhibition at the 0.4 pM level with either compound but substantial inhibition at the 4 pM level, especially with the glucosyltransferase inhibitor, BML-130. This finding is similar to the ones made in longer incubations with human cancer cells.
TABLE Effects of BML-129 and -130 on MDCK Cell Growth and the Content of Ceramide and Diacylglycerol Growth Medium Protein Ceramide Diglyceride _g/dish nmol/mg protein Controls 490 1.04 4.52 560 0.96 5.61 0.4 pm BML-129 500 1.29 5.51 538 0.99 5.13 0.4 pm BML-130 544 0.94 4.73 __538 0.87 5.65 4 pm BML-129 396 3.57 9.30 _311 3.78 9.68 ii WO 01/04108 PCT/US00/18935 4 pm BML-130 160 5.41 11.9 268 3.34 8.71 In a separate study of ceramide levels in MDCK cells, BML-130 at various concentrations was incubated with the cells for 24 h. The ceramide concentration, measured byTLC densitometry, was 1.0 nmol/mg protein at 0.5 pM, 1.1 at 1 pM, at 2 pM, and 3.3 at 4 pM. The results with BML-129 were virtually identical.
It is interesting that the accumulation of ceramide paralleled an accumulation of diacylglycerol (DAG), as observed before with PDMP (Shayman, J.A. et al., "Modulation of Renal Epithelial Cell Growth by Glucosylceramide: Association with Protein Kinase C, Sphingosine, and Diacylglyceride," J. Biol. Chem. 266:22968-22974 (1991)). DAG is ordinarily considered to be an activator of protein kinase C and thus a growth stimulator, but the low level of GlcCer in the inhibited cells may counteract the stimulatory effect Ceramide reacts with lecithin to form SM and DAG, so it is possible that the increased level of the latter reflects enhanced synthesis of the phosphosphingolipid rather than an elevated attack on lecithin by phospholipase D.
Arabinofuranosylcytosine (ara-C), an antitumor agent, also produces an elevation in the DAG and ceramide of HL-60 cells (Strum, J.C. et al., "1-P-D-Arabinofuranosylcytosine Stimulates Ceramide and Diglyceride Formation in Cells," J. Biol. Chem. 269:15493-15497 (1994)).
TLC of MDCK cells grown in the presence of 0.02 to 1 pM IV-231B for 3 days showed that the inhibitor indeed penetrated the cells and that there was a great depletion of GlcCer, but no ceramide accumulation. The depletion of GlcCer was evident even at the 0.1 pM level and virtually no GIcCer was visible at the 1 JpM level; however the more polar GSLs were not affected as strongly. After incubation for days in 5 pM inhibitor, all the GSLs were virtually undetectable. The ceramide concentrations in the control and depleted cells were very similar 13.5 1.4 vs 13.9 0.2 pg/mg protein.
The lack of ceramide accumulation in cells exposed to the aliphatic inhibitors was examined further to see if it might be due to differential actions of the different inhibitors on additional enzymes involving ceramide metabolism. For example, IV-231B might block ceramide synthase and thus prevent accumulation despite the inability of the cells to utilize ceramide for GlcCer synthesis. However, assay of ceramide synthase in homogenized cells showed it was not significantly affected by WO 01/04108 PCT/US0/1 8935 -26pM inhibitors (Table There did appear to be moderate inhibition at the 50 pM level with PDMP and the aliphatic inhibitor.
TABLE 6 Effect of Inhibitors on Acid and Neutral Ceramldases and Ceramide Synthase of MDCK Cells Enzyme Activity of control) Inhibitor Tested Ceramidase Ceramidase Ceramide pH 4.5 pH 7.4 Synthase D-threo-PDMP, 5 pM 97 4 116+ 19 99 D-threo-PDMP, 50 /M 133 13' 105 11 66 9" BML-129, 5 M 108 8 100 0 97 0 BML-129, 50 pM 171 26' 99 2 102 1 BML-130, 5 pM 107 11 100 15 108+ BML-130, 50 pM 160 21' 100+ 15 106+ 29 IV-231B, 5 pM 106 3 116 20 90 8 IV-231B, 50 pM 113 8 112 3 71 188 "Notable differences.
Assay of the two kinds of ceramidase (Table 6) showed that there was no effect of either the aliphatic oraromatic inhibitors at the 5 pM level, at which point cell growth is completely stopped in the case of the pyrrolidino compounds. At the 50 pM level, however, the acid enzyme was stimulated markedly by the aromatic inhibitors, particularly the two stereoisomeric forms of the pyrrolidino compound.
Sphingomyelin synthase was unaffected by PDMP or the aliphatic inhibitor but BML-129 and -130 produced appreciable inhibition at 50 pM (54% and 61%, respectively) (Table 7).
WO 01/04108 PCT/US00/18935 -27- TABLE 7 Effect of Inhibitors on Acid and Neutral Sphingomyelinases and Sphingomyelin Synthase Enzyme Activity of control) Inhibitor Tested Sphingomyelinase Sphlngomyelinase Sphlngomyelinase Inhibir T d pH 4.5 pH 7.1 Synthasea D-threo-PDMP, 1023 121 5102 3 121 13 D-threo-PDMP, 1003 108 pM BML-129, 5 M 108 +4 105+11 84 +27 BML-129, 50pM 97 +3 142 11b 4 6 11" BML-130, 5 pM 109 +1 110 +7 87 +14 BML-130, 50 pM 114 +2 152 14 39+ 18b IV-231B, 5 pM 101 7 131 3_ IV-231B, 50 pM 112+ 11 120 3" Data for PDMP and IV-231B are not shown here as they were tested in other experiments; no effect was seen.
Notable differences.
Neutral sphingomyelinase (SMase) was distinctly stimulated by the aliphatic inhibitor, IV-231B, even at 5 pM (Table From this one would expect that the inhibitor would produce accumulation of ceramide, yet it did not. The two pyrrolidino compounds produced appreciable stimulation at the 50 pM level. No significant effects were obtained with acid SMase.
Discussion The present invention shows that the nature and size of the tertiary amine on ceramide-like compounds exerts a strong influence on GIcCer synthase inhibition, a ring being most active. It also shows that the phenyl ring used previously to simulate the trans-alkenyl chain corresponding to that of sphingosine could, with benefit, be replaced with the natural alkenyl chain.
Findings with the most active GIcCer synthase inhibitors in growth tests compare favorably with evaluations of some clinically useful chemotherapeutic agents on three of the tumor cell lines in the same Drug Evaluation Core Laboratory. The ICs values were 0.2 to 6 pM for cisplatin, 0.02 to 44 pM for carboplatin, 0.03 to 0.2 pM for methotrexate, 0.07 to 0.2 pM for fluorouracil, and 0.1 to 1 pM for etoposide. Unlike WO 01/04108 PCT/US00/18935 -28these agents, the compounds of the present invention yielded rather similar effects with all the cell types, including MDCK cells, and thus have wider potential chemotherapeutic utility. This uniformity of action is consistent with the idea that GSLs play a wide and consistent role in cell growth and differentiation.
An important observation from the MDCK cell study is that strong inhibition of cell growth and DNA synthesis occurred only at the same concentrations of aromatic inhibitor that produced marked ceramide accumulation. This observation supports the assertion that ceramide inhibits growth and enhances differentiation or cell death (Bielawska, A. et al., "Modulation of Cell Growth and Differentiation by Ceramide," FEBS Letters 307:211-214 (1992)). It also agrees with previous work with octanoyl sphingosine, a short chain ceramide that produced greatly elevated levels of natural ceramide and slowed growth (Abe, A. et al., "Metabolic Effects of Short-Chain Ceramide and Glucosylceramide on Sphingolipids and Protein Kinase Eur. J.
Biochem. 210:765-773 (1992)). It is also in agreement with a finding that some synthetic, nonionic ceramide-like compounds did not inhibit GIcCer synthase even though they behave like ceramide in blocking growth (Bielawska, A. et al., "Ceramide-Mediated Biology. Determination of Structural and Stereospecific Requirements Through the Use of N-Acyl-Phenylaminoalcohol Analogs," J. Biol.
Chem. 267:18493-18497 (1992)). Compounds tested included 20 pM D-erythro-N-myristoyl-2-amino-1-phenyl-1-propanol, its L-enantiomer, the four stereoisomers of N-acetylsphinganine, and N-acetylsphingosine. Furthermore, the lack of growth inhibition and ceramide accumulation in cells treated with the aliphatic inhibitor IV-231B is also consistent with the correlation between ceramide level and growth rate.
The accumulation of ceramide that occurred at higher levels of GIcCer synthase inhibitors could be attributed not only to blockage of ceramide utilization, but also to blockage of SM synthesis or ceramide hydrolase. This possibility is especially relevant to the and S,S-isomers, which seem to exert effects on sphingolipids without strongly inhibiting GIcCer synthesis. The tests with both the DL-etythro-pyrrolidino inhibitor (BML-129) and the DL-threo-pyrrolidino inhibitor (BML-130), at a level producing strong growth inhibition, showed that neither material at a low concentration inhibited the enzymes tested in vitro (Tables 6 and 7) but they did cause growth inhibition as well as accumulation of ceramide. PDMP, at relatively high concentrations (50 pM), was found to inhibit SM synthase in growing CHO cells (Rosenwald, A.G. et al., "Effects of a Sphingolipid Synthesis Inhibitor on Membrane WO 01/04108 PCT/US00/18935 -29- Transport Through the Secretory Pathway," Biochemistry 31:3581-3590 (1992)). In the test with MDCK homogenates, it did not inhibit this synthase, in agreement with the finding that labeled palmitate incorporation into SM was stimulated by POMP (Shayman, J.A. et al., "Modulation of Renal Epithelial Cell Growth by Glucosylceramide: Association with Protein Kinase C, Sphingosine, and Diacylglyceride," J. Biol. Chem. 266:22968-22974 (1991)).
Retinoic acid is a growth inhibitor of interest in cancer chemotherapy and a possible adjunct in the use of the inhibitors of the present invention. It has been found to elevate ceramide and DAG levels (Kalen, A. et al., "Elevated Ceramide Levels in GH4C1 Cells Treated with Retinoic Acid," Biochim. Biophys. Acta 1125:90-96 (1992)) and possibly lower lecithin content (Tang, W. et al., "Phorbol Ester Inhibits 13-Cis-Retinoic Acid-Induced Hydrolysis of Phosphatidylinositol 4,5-Bisphosphate in Cultured Murine Keratinocytes: a Possible Negative Feedback Via Protein Kinase C-Activation," Cell Bioch. Funct. 9:183-191 (1991)).
D-threo-PDMP was found to be rather active in delaying tumor cell growth or in producing complete cures in mice (Inokuchi, J. et al., "Antitumor Activity in Mice of an Inhibitor of Glycosphingolipid Biosynthesis," CancerLett. 38:23-30 (1987)) but high doses were needed. From the data in Figure 1, the inhibitors of the present invention are approximately 30 times as active, so the dosage levels are typical of clinically useful drugs. The need to use high doses with PDMP was attributed to rapid inactivation by cytochrome P450 (Shukla, A. et al., "Metabolism of D-[3H]PDMP, an Inhibitor of Glucosylceramide Synthesis, and the Synergistic Action of an Inhibitor of Microsomal Monooxygenase," J. Lipid Res. 32:713-722 (1991)). Cytochrome P450 can be readily blocked by various nontoxic drugs such as cimetidine, therefore high levels of the compounds of the present invention can be maintained.
SPECIFIC EXAMPLE 2 A series of inhibitors based on substitutions in the phenyl ring of P4 were synthesized and studied. It was found that the potency of the inhibitors in blocking GIcCer synthase was mainly dependent upon hydrophobic and electronic properties of the substituent. Surprisingly, a linear relationship was found between log [ICJ and hydrophobic parameter electronic parameter This correlation suggested that electron donating and hydrophilic characters of the substituent enhance the potency as an inhibitor. This observation resulted in the synthesis of novel compounds that are more active in blocking glucosylceramide formation. Two compounds, dioxy D-t- P4 compounds, D-t-3',4'-ethylenedioxy-P4 and D-t-4'-hydroxy-P4, were observed to WO 01/04108 PCT/US00/18935 be significantly more potent than other tested inhibitors. In particular, at 11.3 nM D-t- 3',4'-ethylenedioxy-P4, 80% of glucosylceramide in MDCK cell was depleted without any ceramide accumulation and cell growth inhibition. The potency of ethylenedioxy-P4 appears to be not only regulated by hydrophobic and electronic properties but also by stearic properties of the substituents on the phenyl group.
Materials and Methods Materials. The acetophenones and amines were from Aldrich Chemical Co., SL Louis, MO., Lancaster Synthesis Inc., Windham, NH. and Maybridge Chemical Co., Corwall, UK. Silica gel for column chromatography (70-230 mesh ASTM) and Silica gel thin layer chromatography plates were purchased from Merck Co. The reagents and their sources were: non-hydroxy fatty acid ceramide from bovine brain and delipidated bovine serum albumin (BSA) from Sigma; dioleoyphosphatidylcholine from Avanti; DL-dithiothreitol from Calbiochem; 1-['H]-glucose uridine diphosphate from NEN. Octanoylsphingosine, glucosylceramide and sodium sulfatide were prepared as previously described. Abe, A. et al., Eur. J. Biochemistry 210:765-773 (1992).
General synthesis of inhibitors. The aromatic inhibitors were synthesized by the Mannich reaction from 2-N-acylaminoacetophenone, paraformaldehyde, and pyrrolidine, and then the reduction from sodium borohydride as described before.
Inokuchi, J. et al., J. Lipid. Res. 28:565-571 (1987); Abe, A. et al., J. Lipid. Res.
36:611-621 (1995). The reaction produces a mixture of four isomers, due to the presence of two asymmetric centers. For these syntheses in which phenyl-substituted starting materials were used, the chloro, methoxy, methylenedioxy, methyl groups in the acetophenone structure were brominated and converted to the primary amine.
Bromation of the methoxyacetophenone, dimethyoxyacetophenone, (methylenedioxy)acetophenone were performed in chloroform at room temperature and recrystallized from ethyl acetate and hexane.
Synthesis of 1-(4'-hydroxy)phenyl-2-palmitoylamino-3-pyrrolidino-1propanol. The synthesis of 1-(4'-hydroxy)phenyl-2-palmitoylamino-3-pyrrolidino-1propanol is described in detail in Figure 8. This synthesis differs from those of the other compounds because of the need for the placement of a protecting group on the free hydroxyl (step 1) and its subsequent removal (step All other syntheses employ a similar synthetic scheme (steps 2 to 6).
4'-Benzyloxyacetophenone formation (step 4'-Hydroxyacetophenone (13.62 g, 100 mmol), benzyibromide (17.1g, 100 mmol), and cesium carbonate (35.83 g, 100 mmol) were added to tetrahydrofuran at room temperature and stirred WO 01/04108 PCT/US00/18935 -31 overnight. The product was concentrated to dryness and recrystallized from ether and hexane to yield 15 g of4'-benzyloxyacetophenone which appeared as a white powder.
An R, of 0.42 was observed when resolved by thin layer chromatography using methylene chloride. 'H nmr ppm, CDCI,), 7.94 (2H, 6, 8.8 Hz, 7.42 (5H, m, Ar'CHO-), 7.01 (2H, 6, 8.8 Hz, 5.14 (2H, s, Ar'CHO-), 2.56 (3H, S, CH,).
Bromination of 4'-benzyloxyacetophenone (step Bromine (80 mmol) was added dropwise over 5 min to a stirred solution of 4'-benzyloxyacetophenone mmol) in 40 ml chloroform. This mixture was stirred for an additional 5 min and quenched with saturated sodium bicarbonate in water until the pH reached 7. The organic layers were combined, dried over MgSO 4 and concentrated to dryness. The crude mixture was purified over a silica gel column and eluted with methylene chloride to yield 2-bromo-4'-benyloxyacetophenone. An R, of 0.62 was observed when resolved by thin layer chromatography using methylene chloride. 1H nmr ppm, CDCI 7.97 (2H, 6, 9.2 Hz, 7.43 (5H, m, Ar'CH,0-), 7.04 (2H, 6, 9.0 Hz, 5.15 (2H, s, Ar'CH 2 4.40 (2H, s, CH 2 Br).
2-Amino-4'-benzyloxyacetophenone HCI formation (step 3): Hexamethylenetetramine (methenamine, 3.8 g, 23 mmol) was added to a stirred solution of 2-bromine-4'-benyloxyacetophenone (6.8 g, 23 mmol) in 100 ml chloroform. After 4 h the crystalline adduct was filtered and washed with chloroform.
The product was dried and heated with 150 ml methanol and 8 ml of concentrated HCI in an oil bath at 85 0 C for 3 h. Upon cooling the precipitated hydrochloride salt g) was removed by filtration. The filtrate was left at -20 0 C overight and additional product (2.1 g) was isolated. The yield was 4.6 g 242 for
C,,H,,NO
2 'H nmr ppm, CDCI,), 8.38 (2H, bs, NH 2 7.97 (2H, 6, 8.8 Hz, O-Ar- 7.41 (5H, m, Ar'CH20-), 7.15 (2H, 6, 8.6 Hz, 5.23 (2H, s, Ar'CH 2
O-
4.49 (2H, s, CHNH,).
2-Palmitoylamino-4'-benyloxyacetophenone formation (step Sodium acetate (50% in water, 29 ml) was added in three portions to a stirred solution of 2amino-4'-benzyloxyacetophenone HCI (4.6 g,17 mmol) and tetrahydrofuran (200 ml).
Palmitoyl chloride (19 mmol) in tetrahydrofuran (25 ml) was added dropwise over min yielding a dark brown solution. The mixture was stirred ovemight at room temperature. The aqueous fraction was removed by use of a separatory funnel and chloroform/methanol 150 mi) was added to the organic layer which was then washed with water (50 ml). The yellow aqueous layer was extracted once with WO 01/04108 PCT/US00/18935 -32chloroform (50 ml). The organic solutions were then pooled and rotoevaporated until near dryness. The residue was redissolved in chloroform (100 ml) and crystallized by the addition of hexane (400 ml). The flask was cooled to 4°C for 2 h. The crystals were filtered and washed with cold hexane and dried in a fume hood overnight. The product yield was 3.79 g (8 mmol). An R, of 0.21 was observed when resolved by thin layer chromatography using methylene chloride. 479 for C,,H 4 NO,. 'H nmr ppm, CDCI 3 7.96 (2H, 6, 8.8 Hz, 7.40 (5H, m, Ar'CHO-), 7.03 (2H, 6, 8.8 Hz, 6.57 (1H, bs, NH 5.14 (2H, s, Ar'CH20-), 4.71 (2H, s,
C(O)CH
2 NHC(O)), 2.29 (2H, t, 7.4 Hz, C(O)CH,(CH 2 3
CH
3 1.67 (2H, m,
C(O)CH
2
(CH
2 13
CH
3 0.87 (3H, t. 6.7 Hz, C(O)CH 2 1-(4'-Benzyloxy)phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol formation (steps 5 and 2-Palmitoylamino-4'-benyloxyacetophenone (3.79 g, mmol), paraformaldehyde (0.25 g, 2.7 mmol), pyrrolidine (0.96 ml, 11.4 mmol) and ethanol (70 ml) were stirred under nitrogen. Concentrated HCI (0.26 ml) was added through the condensor and the mixture was heated to reflux for 16 h. The resultant brown solution was cooled on ice and then sodium borohydride (1.3 g, 34 mmol) was added in three portions. The mixture was stirred at room temperature ovemight, and the product was dried in a solvent evaporator. The residue was redissolved in dichloromethane (130 ml) and hydrolyzed with 3N HCI The aqueous layer was extracted twice with dichloromethane (50 ml). The organic layers were pooled and washed twice with water (30 ml), twice with saturated sodium chloride (30 ml), and dried over anhydrous magnesium sulfate. The dichloromethane solution was rotoevaporated to a semisolid and purified by use of a silica rotor using a solvent consisting of 10% methanol in dichloromethane. This yielded a mixture of DL-threoand DL-erythro enantiomers (2.53 g, 4.2 mmol). An R, of 0.43 for the erythro diastereomers and 0.36 for the threo diastereomers was observed when resolved by thin layer chromatography using methanol:methylene chloride 565 for C3eHN03,.
1-(4'-Hydrxy)phenyl-2-palmitoylamino-3-pyrolidino-1-propanol formation (step A suspension of 20% Pd/C (40 mg) in acetic add (15 ml) was stirred at room temperature under a hydrogen balloon for 15 min. 1-(4'-Benzyloxy)phenyl-2hexadecanoylamino-3-pyrrolidino-l -propanol (420 mg, 0.74 mmol) was added and the solution was stirred ovemight The suspension was filtered through a glass frit, and the filter was rinsed with acetic acid:methyiene chloride 5 mi). The filtrate was concentrated in vacuo and crystallized to yield a pale yellow semisolid (190 mg, 0.4 WO 01/04108 PCT/US00/18935 -33mmol). An R of 0.21 was observed when resolved by thin layer chromatography using methanol:methylene chloride 475 for C,,H.oNO,. 'H nmr ppm, CDCI 7.13 (4H, m, ArCHOH-), 7.14 (1H, 6, 6.9 Hz, 5.03 (1H, 6, 3.3 Hz, CHOH-), 4.43 (1H, m, c-(CH 2
CH
2 2
NCH
2 CH), 3.76 (2H, m, c-(CHCH 2 2 3.51 (1H, m, c-(CH 2
CH)
2 NCH-), 3.29 (1H, m, c-(CH 2 2.97 (3H, m, c-(CH 2
CH
2 2
N-
and ArC(OH)H-), 2.08 (6H, m, -C(O)CH,(CH 2 3
CH
3 and c-(CH 2
CH
2 2 1.40 (2H, m,
C(O)CHCH(CH
2 2
CH
3 1.25 (2H, m, -C(O)CHCH 2
(CH,),,CH
3 0.87 (3H, t, 6.7 Hz,
C(O)CH
2
(CH),
3
CH
3 Synthesis of D-threo-1-(3',4'-ethylenedioxy)phenyl-2-palmitoylamino- 3 pyrrolidino-1-propanol.
2-Amino-3',4'-(ethylenedioxy)acetophenone HCI: Hexamethylenetetramine (methenamine, 5.4 g, 0.039 mol) was added to a stirred solution of phenacylbromide (10.0 g, 0.039 mol) in 200 ml chloroform. After 2 h, the crystalline adduct was filtered and washed with chloroform. The product was then dried and heated with methanol (200 ml) and concentrated HCI (14 ml) in an oil bath at 85 0 C for 2 h. On cooling, the precipitated ammonium chloride was removed by filtration and the filtrate was left in a freezer overnight. After filtration the crystallized phenacylamine HCI was washed with cold isopropanol and then with ether. The yield of this product was -7.1 g 2-Palmitoylamino-3',4'-(ethylenedioxy)acetophenone: Aminoacetophenone HCI (7.1 g, 31 mmol) and tetrahydrofuran (300 ml) were placed in a 1 liter three-neck round bottom flask with a large stir bar. Sodium acetate (50% in water, 31 ml) was added in three portions to this suspension. Palmitoyl chloride (31 ml, 10 excess, 0.036 mol) in tetrahydrofuran (25 ml) was then added dropwise over 20 min to yield a dark brown solution. This mixture was then stirred for an additional 2 h at room temperature. The resultant mixture was poured into a separatory funnel to remove the aqueous solution. Chloroform/methanol 150 ml) was then added to the organic layer and washed with water (50 ml). The yellow aqueous layer was extracted once with chloroform (50 ml). The organic solutions were pooled and rotoevaportated until almost dry. The residue was redissolved in chloroform (100 ml) and crystallized by the addition of hexane (400 ml). The flask was then cooled to 4°C for 2 h. The crystals were filtered and washed with cold hexane until they were almost white and then dried in a fume hood ovemight. The yield of the product was 27 mmol (11.6 g).
D-threo-1-(3',4'-ethylenedioxy)phenyl-2-palmitoylamino-3-pyrrolidino-lpropano almitoylaminoacetophenone (11.6 g, 0.027 mol), paraformaldehyde (0.81 g, 0.009 mol), pyrrolidine (3.6 ml, 0.042 mol) and ethanol (250 ml) were added to a WO 01/04108 PCT/US00/18935 -34- 500 ml round flask under nitrogen flow. Concentrated HCI (0.8 ml) was added to this mixture through the reflux condenser and the mixture was refluxed for 16 h. The brown solution was cooled in an ice-bath. Sodium borohydride (2.28 g, 0.06 mol) was added in three portions. This mixture was stirred at room temperature for 3 h and then rotoevaporated. The residue was dissolved in 130 ml of dichloromethane and the borate complex hydrolyzed with HCI (3N) until the pH was approximately 4. The aqueous layer was extracted twice with 50 ml dichloromethane. The organic layers were pooled and washed twice with H 2 0 (30 ml), saturated NaCI (30 ml) and dried over anhydrous MgSO 4 The dichloromethane solution was rotoevaporated to a viscous oil which was purified by use of a Chromatotron with a solvent consisting of methanol in dichloromethane to obtain a mixture of DL-threo and erythro enantiomers (2.24 g, 0.004 mol).
Resolution of inhibitor enantiomers. High performance liquid chromatography (HPLC) resolution of the enantiomers of DL-threo and DL-erythro are performed using a preparative HPLC column (Chirex 3014: naphtyl)ethylamine, 20 x 250 mm: Phenomenex], eluted with hexane-1,2dichloroethane-ethanol-trifluroacetic acid 64:30:5.74:0.26, at a flow rate of 8 ml/min.
The column eluent was monitored at 254 nm in both the preparative and analytical modes. Isolated products were reinjected until pure by analytical HPLC analysis, determined using an analytical Chirex 3014 column (4.6 x 250 mm) and the above solvent mixture at flow rate of 1 ml/min.
Glycosylceramidesynthase activity. The enzyme activity was measured by the method previously described in Skukla, G. et al., Biochim. Biophys. Acta 1083:101-108 (1991). MDCK cell homogenate (120jg of protein) was incubated with uridinediphosphate [H]glucose (100,000 cpm) and liposomes consisting of 85 pg octanoylsphingosine, 570pg dioleoyphosphatidylcholine and 100 pg sodium sulfatide in 200 p1 of reaction mixture and kept for 1 h at 37 P4 and P4 derivatives dissolved in dimethyl sulfoxide were dispersed into the reaction mixture after adding liposomes. The final concentration of dimethyl sulfoxide was kept 1% under which the enzyme activity was not at all inhibited.
Cell culture and lipid extraction. One half million of MDCK cells were seeded into 10 cm style dish containing 8 ml serum free DMEM supplemented medium. Shayman, J.A. et al., J. Biol. Chem. 265:12135-12138 (1990). After 24 h the medium was replaced with 8 ml of the medium containing 0, 11.8, 118 or 1180 nM D-t-P4, D-t-3',4'-ethylenedioxy-P4 or D-4'-hydroxy)-P4. The GIcCer synthase WO 01/04108 PCT/USOO/18935 inhibitors were added into the medium as a one to one complex with delipidated BSA.
Abe, A. et al., J. Lipid. Res. 36:611-621 (1995); Abe, A. et al., Biochim. Biophys. Acta 1299:331-341 (1996). The cells were incubated for 24 h or 48 h with the inhibitors.
After the incubation, the cells were washed twice with 8 ml of cold PBS and fixed with 2 ml of cold methanol. The fixed cells were scraped and transferred to a glass tube.
Another one ml of methanol was used to recover the remaining cells in the dish.
Three ml of chloroform was added to the tube and briefly sonicated using a water bath type sonicator. After centrifugation at 800g for 5 min, the supematant was transferred into another glass tube. The residues were reextracted with chloroform/methanol After the centrifugation, the resultant supematant was combined with the first one. The residues were air-dried and kept for protein analysis.
Adding 0.9% NaCI to the supernatant combined, the ratio of chloroform/methanolaqueous was adjusted to 1/1/1. After centrifugation 800g for min, the upper layer was discarded. Methanol/water with the same amount of volume of the lower layer was used to wash. The resultant lower layer was transferred into a small glass tube and dried down under a stream of nitrogen gas.
A part of the lipid was used for lipid phosphate determination. Ames, Methods Enzymol. 8:115-118 (1966). The remainder was analyzed using HPTLC (Merck).
Results Synthesis of P4 and P4 derivatives. The preparation of P4 derivatives utilized the Mannich reaction from 2-N-acylaminoacetophenone, paraformaldehyde, and pyrrolidine, and then the reduction of DL-pyrrodino ketone from sodium borohydride. In most cases, no isolation of DL-pyrrodino ketones were performed to maintain solubility. The overall yields of the DL-threo and DL-erythro syntheses were 10-30%. These derivatives were purified by the either silica gel column or rotors with solvent 5-12% methanol in dichloromethane to optimize the separation from the chiral column. To obtain the best separation, each injection contains no more than 150 mg, and fractions were pooled to obtain sufficient quantity of isomer of D-threo for further biological characterization.
Resolution of PDMP homologues by chiral chromatography. The structures of the parent compound, D-threo-P4 and the phenyl-substituted homologues including the new dioxy-substituted and 4'-hydroxy-P4 homologues are shown in Figure 9. Initially the effect of each P4 isomer separated by chiral chromatography on GlcCer synthase activity was determined (Figure 10). Four peaks were observed for the chiral separation of P4. Peaks 1 and 2 represented the erythro WO 01/04108 PCT/US00/18935 -36diastereomers and 3 and 4 represented the threo diastereomers as determined by a sequential separation of the P4 mixture by reverse phase chromatography followed by the chiral separation. The enzyme activity was specifically inhibited by the fourth peak, the D-threo isomer (Figure 4A). This specificity for the D-threo enantiomer was consistent with the previous results observed in PDMP and PDMP homologues The IC, of D-threo-P4 was 0.5 mM for GlcCer synthase activity measured in the MDCK cell homogenates.
Effects of P4 andP4 derivatives with a single substituent of phenyl group on GlcCer synthase activity. The effect of each P4 isomer on GIcCer synthase activity was analyzed. The reaction was carried out in the absence or presence of 0.1, 1.0 or 10 pM P4 (Figure 4A) or p-methoxy-P4 (Figure 4B). As shown in Figure 4A, the enzyme activity was specifically inhibited by D-threo isomer. In Figure 4A, the symbols are denoted as follows: D-threo D-erythro L-threo and L-erythro This specificity is consistent with previous results observed in PDMP and PDMP homologs. Inokuchi, J. et al., J. Lipid. Res. 28:565-571 (1987); Abe, A. et al., J. Lipid.
Res. 36:611-621 (1995). The IC5 of D-t-P4 was 500 nM.
As set forth herein, the addition of a p-methoxy group to DL-t-P4 was found to enhance the effect of the inhibitor on the enzyme activity. Abe, A. et al., J. Lipid.
Res. 36:611-621 (1995). As shown in Figure 4B, it was confirmed that the enzyme activity was potently inhibited by D-threo-p-methoxy-P4 whose IC5 was 200 nM. In Figure 4B, o denotes a mixture of D-erythro and L-threo isomers contaminated with a small amount of the D-threo isomer. Chiral chromatography of the four p-methoxy- P4 enantiomers failed to completely resolve to baseline each enantiomer (Figure A slight inhibition of the enzyme activity by p-methyoxy-P4 in a combined D-erythro and L-threo mixture (peaks 2 and 3, Figure 10) was observed; this was due to contamination of the D-threo isomer (peak 4, Figure 10) into these fractions.
A series of D-t-P4 derivatives containing a single substituent on the phenyl group were investigated. As shown in Table 8, the potency of the derivatives as inhibitors were inferior to that of D-t-P4 or p-methoxy-D-t-P4. In many drugs, the influence of an aromatic substituent on the biological activity has been known and predicted. Hgberg, T. et al., Theoretical and experimental methods in drug design applied on antipsychotic dopamine antagonists. Larsen, and Bundgaard, H., "Textbook of Drug Design and Development," pp. 55-91 (1991). Generally IC, is described as the following equation: WO 01/04108 PCT/US00/1893S -37log a (hydrophobic parameter b (electronic parameter c (stearic parameter) d (other descriptor) e where a, b, c, d and e are the regression coefficients. Hogberg. T. et al., Theoretical and experimental methods in drug design applied on antipsychotic dopamine antagonists. Larsen, and Bundgaard, "Textbook of Drug Design and Development," pp. 55-91 (1991).
The hydrophobic effect, n, is described by the equation n IogPx log P, where Px is the partition coefficient of the substituted derivative and P. is that of the parent compound, measured as the distribution between octanol and water.
The electronic substituent parameter, o, was originally developed by Hammett (Hammett, In Physical Organic Chemistry, McGraw-Hill, New York (1940)) and is expressed as a logK x logK, where Kx and KH are the ionization constants for a para or meta substituted derivative and benzoic acid respectively. Positive uvalues represent electron withdrawing properties and negative a values represent electron donating properties.
The potency of D-threo-P4 and P4 derivatives as an inhibitor is mainly dependent upon two factors, hydrophobic and electronic properties, of a substituent of phenyl group (Table Surprisingly, a linear relationship was observed between log (IC 5 and a a (Figure These findings suggest that the more negative the value of n a, the more potent is D-threo-P4 derivatives made as GIcCer synthase inhibitor.
The data in Table 8 indicate that the potency of D-t-P4 and P4 derivatives as an inhibitor is mainly dependent upon two properties, hydrophobic and electronic properties, of a substituent of the phenyl group. Surprisingly, a linear relationship was observed between log(IC,) and nr a (Figure These findings suggest that the more negative the value of ar a, the more potent the D-t-P4 derivative as a GIcCer synthase inhibitor.
WO 01/04108 PCT/US00/18935 -38- Table 8 D-threo-P4 derivative a W ICo (pM)" p-methoxy -0.29 0.2 P-4 0.00 m-methoxy-P4 0.10 0.6 p-methyl-P4 0.39 2.3 p-chloro-P4 0.94 7.2 These values were estimated from the Table in H6gberg, T. et al., Theoretical and experimental methods in drug design applied on antipsychotic dopamine antagonists. Larsen, and Bundgaard, "Textbook of Drug Design and Development," pp. 55-91 (1991), for methoxy, 0.12, o, -0.27, a= -0.02; hydro, a= 0, n= 0; methyl, a, -0.17, n 0.56; chloro, op 0 2 3 n= 0 7 1 These values were derived from Figures 4A and 4B. For other compounds the same analytical approach as shown in Figures 4A and 4B was carried out to obtain the IC,.
The p-hydroxy-substituted homologue was a significantly better GlcCer synthase inhibitor. The strong association between n a and GlcCer synthase inhibition suggested that a still more potent inhibitor could be produced by increasing the electron donating and decreasing the lipophilic properties of the phenyl group substituent A predictably negative n o value would be observed for the p-hydroxy homologue. This compound was synthesized and the D-threo enantiomer isolated by chiral chromatography. An IC., of 90 nM for GlcCer synthase inhibition was observed (Figure 11), suggesting that the p-hydroxy homologue was twice as active as the pmethoxy compound. Moreover, the linear relationship between the log (IC50) and n a was preserved (open circle, Figure 4).
Effects of 3,4'-dioxy-D-threo-P4 derivatives on GicCersynthase activity.
The result in Figure 5 suggested that an electron donating and hydrophilic substituent of phenyl group makes the GIcCer synthase inhibitor potent. To attain further improvement of the inhibitor, another series of P4 derivatives with methylenedioxy, ethylenedioxy and trimethyldioxy substitutions on the phenyl group were designed (Figure 9).
As shown in Figure 6, the enzyme activity was markedly inhibited by ethylenedioxy-P4 whose IC 5 was 100 nM. In Figure 6, o denotes methylenedioxy-P4, o denotes D-t-3',4'-ethylenedioxy-P4, a denotes WO 01/04108 PCT/USOO/18935 -39trimethylenedioxy-P4 and denotes D-t-3',4'-dimethyoxy-P4. One the other hand, the ICas for D-t-3',4'-methylenedioxy-P4 and D-t-3',4'-trimethylenedioxy-P4 were about 500 and 600 nM, respectively. These results suggest that the potency of ethylenedioxy-P4 is not only regulated by hydrophobic and electronic properties but also by other factors, most likely stearic properties, induced from the dioxy ring on the phenyl group.
Interestingly, D-t-3',4'-dimethoxy-P4 was inferior to these dioxy derivatives, even to D-t-P4 or m- or D-t-p-methoxy-P4, as an inhibitor (Figure As the parameters, op and n, for methoxy substituent are 0.12, -0.27 and -0.02, respectively (Hbgberg, T. et al., Theoretical and experimental methods in drug design applied on antipsychotic dopamine antagonists. Larsen, and Bundgaard, H., "Textbook of Drug Design and Development," pp. 55-91 (1991)), the value of o of D-t-dimethoxy P4 is presumed to be negative. Therefore the dimethoxy-P4 is thought to deviate quite far from the correlation as observed in Figure 5. There may be a repulsion between two methoxy groups in the dimethoxy-P4 molecule that induces a stearic effect that was negligible in mono substituent D-t-P4 derivatives studied in Figure 5. GIcCer synthase is thought to possess a domain that interacts with D-t-PDMP and PDMP homologs and that modulates the enzyme activity.
Inokuchi, J. et al., J. Lipid. Res. 28:565-571 (1987); Abe, A. et al., Biochim. Biophys.
Acta 1299:331-341 (1996). The stearic effect generated by an additional methoxy group may affect the interaction between the enzyme and the inhibitor. As a result, the potency as an inhibitor is markedly changed.
Distinguishing between inhibition of GlcCer synthase and acylceramide synthase inhibition. Prior studies on PDMP and related homologues revealed that both the threo and erythro diastereomers were capable of increasing cell ceramide and inhibiting cell growth in spite of the observation that only the Dthreo enantiomers blocked GlcCer synthase. An alternative pathway for ceramide metabolism was subsequently identified, the acylation of ceramide at the 1-hydroxyl position, which was blocked by both threo and erythro diastereomers of PDMP. The specificities of D-threo-P4, D-threo-3',4'-ethylenedioxy-P4, and D-threo-(4'-hydroxy)- P4 for GlcCer synthase were studied by assaying the transacylase. Although there was an ca. 100 fold difference in activity between D-threo-3',4'-ethylenedioxy-P4, Dthreo-(4'-hydroxy)-P4, and D-threo-P4 (IC, 0.1 mM versus 10 mM) in inhibiting GicCer synthase, the D-threo enantiomers of ail three compounds demonstrated comparable activity in blocking 1-O-acylceramide synthase (Figure 12).
WO 01/04108 PCT/US00/18935 In order to determine whether inhibition of 1-O-acylceramide synthase was the basis for inhibitor mediated ceramide accumulation, the ceramide and diradylglycerol levels of MDCK cells treated D-threo-P4, D-threo-3',4'-ethylenedioxy-P4, and D-threo- (4'-hydroxy)-P4 were measured (Table MDCK cells (5 x 10 5 were seeded into a 10 cm dish and incubated for 24 h. Following the incubation, the cells were treated for 24 or 48 h with or without P4 or the phenyl substitute homologues. Both ceramide and diradylglycerol contents were determined by the method of Preis, J. et al., J. Biol.
Chem. 261:8597-8600 (1986). GIcCer content was measured densitometrically by a video camera and use of NIH image 1.49. Significant increases in both ceramide and diradylglycerol occurred only in cells treated with inhibitor concentrations in excess of 1 mM. This was approximately 30-fold lower than the concentration required for inhibition of the 1-O-acylceramide synthase assayed in the cellular homogenates. This disparity in concentration effects most likely reflects the ability of the more potent homologues to accumulate within intact cells. Abe, A. et al., Biochim. Biophys. Acta 1299:331-341 (1996).
Table 9 GlcCer, ceramide and diradylglycerol content of MDCK cells treated with D-threo-P4, D-threo-3,4'-ethylenedioxy-P4, and D-threo-(4'-hydroxy)-P4 Condition Ceramide Diradylglycerol (pmol/nmol (pmol/nmol phospholipid) phospholipid) Control 24 h 4.53 0.12 24.2 2.36 48 h 6.68 0.49 32.3 3.11 D-threo-P4 11.3 nM 24 h 5.33 0.41' 24.1 1.66 48 h 5.68 0.27* 29.6 0.73 113 nM 24 h 4.64 0.38 26.6 1.56 48 h 7.08 0.29 33.0 2.63 1130 nM 24 h 5.10 0.35 27.1 0.67 48 h 9.74 0.53* 38.8 1.11 WO 01/04108 PCT/US00/18935 -41 D-threo-4'-hydroxy-P4 11.3 nM 24 h 48 h 113 nM 24 h 48 h 1130 nM 24 h 48 h D-threo-3',4'-ethylenedioxy-P4 11.3 nM 24 h 113 nM 24 h 1130 nM 24 h 4.29 0.71 6.70 0.29 5.09 0.95 7.47 0.29 7.38 0.13 13.4 1.03* 5.24 5.04 5.21 5.21 9.64 13.0 30.9 2.01* 38.4 1.44* 31.5 3.84* 41.5 0.66* 38.5 3.84* 47.2 2.51* 22.0 24.7 32.5 41.6 32.5 41.6 *Denotes p 0.05 by the Student t test. For the D-threo- (ethylenedioxy)-P4 only two determinations were made.
Effects of D-threo-P4, D-threo-4'-hydroxy-P4 and D-threo-3,4'ethylenedioxy-P4 on GlcCer synthesis and cell growth. To confirm the cellular specificity of D-thro-3',4'-ethylenedioxy-P4 and D-threo-(4'-hydroxy)-P4 as compared to D-threo-P4, MDCK cells were treated with different concentrations of the inhibitors.
The total protein amount in each sample was determined by the BCA method. In GlcCer analysis, lipid samples and standard lipids were applied to the same HPTLC plate pre-treated with borate and developed in a solvent consisting of C/M/W (63/24/4). The level of GlcCer was estimated from a standard curve obtained using a computerized image scanner. The values were normalized on the basis of the phospholipid content The results are shown in Figure 7, wherein each bar is the average values from three dishes, with error bars corresponding to one standard WO 01/04108 PCT/US00/18935 -42deviation. In the control, the total protein and GIcCer were 414 47.4 pg/dish and 24.3 1.97 ng/nmol phosphate, respectively.
Approximately 66 and 78% of the GlcCer was lost from the cells treated by 11.3 nM D-threo-4'-hydroxy-P4 and D-threo-3',4'-ethylenedioxy-P4 respectively (Figures 7, 14 and 15). By contrast, only 27 percent depletion of GlcCer occurred in cells exposed to D-threo-P4 (Figure 13). A low level of GlcCer persisted in the cells treated with 113 or 1130 nM of either compound. This may be due to the contribution, by degradation, of more highly glycosylated sphingolipids or the existence of another GlcCer synthase that is insensitive to the inhibitor.
On the other hand, there was little difference in the total protein content between untreated and treated cells with 11.3 or 113 nM nM D-threo-4'-hydroxy-P4 and D-threo-3',4'-ethylenedioxy-P4 (Figures 14 and 15). A significant decrease in total protein was observed in the cells treated with 1130 nM of either P4 homologue.
In addition, the level of ceramide in the cells treated with 1130 nM D-threo-3',4'ethylenedioxy-P4 and D-threo-(4'-hydroxy)-P4 was two times higher than that measured in the untreated cells (Table There was no change in ceramide or diradylglycerol levels in cells treated with 11.3 nM or 113 nM concentrations of either compound. Similar pattems for GlcCer levels and protein content were observed at 48 h incubations.
The phospholipid content was unaffected at the lower concentrations of either D-threo-3',4'-ethylenedioxy-P4 or D-threo-(4'-hydroxy)-P4. The ratios of cell protein to cellular phospholipid phosphate (mg protein/nmol phosphate) were 4.94 0.30, 5.05 0.21, 4.84 0.90, and 3.97 0.29 for 0, 11.3, 113, and 1130 nM D-threo- 3'.4'-ethylenedioxy-P4 respectively, and 4.52 0.39, 4.35 0.10, and 3.68 0.99 for 11.3, 113, and 1130 nM D-threo-4'-hydroxy-P4 suggesting that the changes in GIcCer content were truly related to inhibition of GIcCer synthase activity. These results strongly indicate that the inhibitors D-threo-4'-hydroxy-P4 and D-threo-3',4'ethylenedioxy-P4, are able to potently and specifically inhibit GIcCer synthesis in intact cells at low nanomolar concentrations without any inhibition of cell growth.
SPECIFIC EXAMPLE 3 Compositions within the scope of invention include those comprising a compound of the present invention in an effective amount to achieve an intended purpose. Determination of an effective amount and intended purpose is within the skill of the art. Preferred dosages are dependent for example, on the severity of the disease and the individual patient's response to the treatment.
WO 01/04108 PCT/US00/18935 -43- As used herein, the term "pharmaceutically acceptable salts" is intended to mean salts of the compounds of the present invention with pharmaceutically acceptable adds, inorganic acids such as sulfuric, hydrochloric, phosphoric, etc.
or organic acids such as acetic.
Pharmaceutically acceptable compositions of the present invention may also include suitable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which may be used pharmaceutically. Such preparations can be administered orally tablets, dragees and capsules), rectally suppositories), as well as administration by injection.
The pharmaceutical preparations of the present invention are manufactured in a manner which is itself known, using the conventional mixing, granulating, dragee-making, dissolving or lyophilizing processes. Thus, pharmaceutical preparations for oral use can be obtained by combining the active compounds with solid excipients, optionally grinding a resulting mixture and processing the mixture of granules, after adding suitable auxiliaries, if desired or necessary, to obtain tablets or dragee cores.
Suitable excipients are, in particular, fillers such as sugars, lactose or sucrose, mannitol or sorbitol, cellulose preparations andlor calcium phosphates, e.g., tricalcium diphosphate or calcium hydrogen phosphate, as well as binders such as starch paste, using, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose and/or polyvinylpyrrolidone. If desired, disintegrating agents may be added such as the above-mentioned starches and also carboxymethyl starch, cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate. Auxiliaries are, above all, flow-regulating agents and lubricants, silica, talc, stearic acid or salts thereof, such as magnesium stearate or calcium stearate, and/or polyethylene glycol. Dragee cores are provided with suitable coatings which, if desired, are resistant to gastric juices. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and/ortitanium dioxide, lacquersolutions and suitable organic solvent or solvent mixtures. In order to produce coatings resistant to gastric juices, solutions of suitable cellulose preparations, such as acetylcellulose phthalate or hydroxypropylmethylcellulose phthalate, are used. Dyestuffs or pigments may be added to the tablets or dragee coatings, for identification or In order to characterize different combinations of active compound doses.
44/1 Other pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol. The push-fit capsules may contain the active compounds in the form of granules which may be mixed with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds are preferably dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be used.
Possible pharmaceutical preparations which can be used rectally include, e.g., suppositories, which consist of a combination of the active compounds with a suppository base. Suitable suppository bases are, natural or synthetic triglycerides, paraffin hydrocarbons, polyethylene glycols or higher alkanots. It is also possible to use gelatin rectal capsules which consist of a combination of the active compounds with a base. Possible base materials include, liquid triglycerides, polyethylene glycols or paraffin hydrocarbons.
Suitable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form, water-soluble salts. In addition, suspension of the active compounds as appropriate oily injection suspensions may be administered. Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, ethyl oleate or triglycerides. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension such as sodium carboxymethylcellulose, sorbitol and/or dextran.
Optionally, the suspension may also contain stabilizers.
Attematively, the active compounds of the present invention may be 25 administered in the form of liposomes, pharmaceutical compositions wherein the active compound is contained either dispersed or variously present in corpuscles consisting of aqueous concentrate layers adherent to hydrophobic lipidic layer. The active compound may be present both in the aqueous layer and in the lipidic layer or in the non-homogeneous system generally known as a lipophilic suspension, 30 The foregoing discussion discloses and describes merely exemplary embodiments of the present invention. One skilled in the art will readily recognize from such discussion, and from the accompanying drawings, that various changes, modifications and variations can be made therein without departing from the spirit and scope of the invention.
35 All publications cited herein are expressly incorporated by reference.
44/2 Throughout the specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.
*000 0 *0 *0 0 0 o go

Claims (24)

1. A compound comprising D-t-3',4'-ethylenedioxy-1-phenyl-2-palmitoylamino-3- pyrrolidino-l-propanol and stereoisomers and pharmaceutically acceptable salts thereof.
2. A compound comprising D-t-4'-hydroxy-l-phenyl-2-palmitoylamino-3-pyrrolidino- 1 -propanol and stereoisomers and pharmaceutically acceptable salts thereof.
3. A composition comprising a compound selected from the group consisting of D-t- 3',4'-ethylenedioxy- I -phenyl-2-palmitoylamino-3-pyrrolidino- I -propanol, D-t-4'- hydroxy- 1 -phenyl-2-palmitoylamino-3-pyrrolidino- I -propanol, and stereoisomers and pharmaceutically acceptable salts thereof.
4. The composition of claim 3, wherein the compound is D-t-3',4'-ethylenedioxy-1- phenyl-2-palmitoylamino-3-pyrrolidino- 1 -propanol.
The composition of claim 3, wherein the compound is a pharmaceutically acceptable salt of D-t-3',4'-ethylenedioxy- 1 -phenyl-2-palmitoylamino-3-pyrrolidino- 1 -propanol.
6. The composition of claim 3, wherein the compound is D-t-4'-hydroxy-l-phenyl-2- palmitoylamino-3-pyrrolidino- I1-propanol.
The composition of claim 3, wherein the compound is a pharmaceutically acceptable S salt of D-t-4'-hydroxy- 1 -phenyl-2-palmitoylamino-3 -pyrrolidino- I -propanol.
8. A method for inhibiting the growth of cancer cells in a mammal, wherein said cancer 20 cells are sensitive to the compounds below, comprising the step of administering to ~the mammal a therapeutically effective amount of a composition comprising a compound selected from the group consisting of D-t-3',4'-ethylenedioxy- I-phenyl-2- palmitoylamino-3-pyrrolidino- 1 -propanol, D-t-4'-hydroxy- 1 -phenyl-2- palmitoylamino-3-pyrrolidino- I1-propanol and stereoisomers and pharmaceutically 25 acceptable salts thereof. -46-
9. The method of claim 8, where the growth of cancer cells is inhibited by increasing ceramide levels in the cancer cells to a toxic level.
A method for treating a patient having sphingolipidosis by reducing glycosphingolipid synthesis comprising the step of administering to the patient a therapeutically effective amount of a composition comprising a compound selected from the group consisting of D-t-3',4'-ethylenedioxy-l-phenyl-2-palmitoylamino-3- pyrrolidino-1 -propanol, D-t-4'-hydroxy-1 -phenyl-2-palmitoylamino-3-pyrrolidino-1- propanol and stereoisomers and pharmaceutically acceptable salts thereof.
11. The method of claim 10, wherein the patient is diagnosed as having Gaucher disease.
12. The method of claim 10, wherein the patient is diagnosed as having Tay-Sachs disease.
13. The method of claim 10, wherein the patient is diagnosed as having Fabry disease.
14. A method for treating a patient having a microbial or viral infection comprising the step of administering to the patient a therapeutically effective amount of a composition comprising a compound selected from the group consisting of ethylenedioxy- 1 -phenyl-2-palmitoylamino-3-pyrrolidino- 1 -propanol, D-t-4'-hydroxy- l-phenyl-2-palmitoylamino-3-pyrrolidino-l-propanol and stereoisomers and pharmaceutically acceptable salts thereof.
15. A method for treating a patient having a drug resistant tumour sensitive to the 20 compounds below, comprising the step of administering to the patient a therapeutically effective amount of a composition comprising a compound selected from the group consisting of D-t-3',4'-ethylenedioxy-1-phenyl-2-palmitoylamino-3- S. pyrrolidino-1 -propanol, D-t-4'-hydroxy- I -phenyl-2-palmitoylamino-3-pyrrolidino- 1- propanol and stereoisomers and pharmaceutically acceptable salts thereof. 25
16. A method for reducing tumor angiogenesis in a patient, wherein said angiogenesis is sensitive to the compounds below, comprising the step of administering to the patient a therapeutically effective amount of a composition comprising a compound selected -47- from the group consisting of D-t-3',4'-ethylenedioxy-l-phenyl-2-palmitoylamino-3- pyrrolidino-1 -propanol, D-t-4'-hydroxy-1 -phenyl-2-palmitoylamino-3-pyrrolidino-1- propanol and stereoisomers and pharmaceutically acceptable salts thereof.
17. A vaccination method comprising the steps of: removing cancer cells sensitive to the compounds below, from a patient; treating the cancer cells in vitro with an effective amount of a composition comprising a compound selected from the group consisting of ethylenedioxy- -phenyl-2-palmitoylamino-3-pyrrolidino- 1-propanol, D-t-4'- hydroxy-1 -phenyl-2-palmitoylamino-3-pyrrolidino-1 -propanol and stereoisomers and pharmaceutically acceptable salts thereof; and administering treated cells to the patient.
18. A compound according to claim 1 or 2 substantially as herein before described with reference to any one of the examples.
19. A composition according to claim 3 substantially as herein before described with reference to any one of the examples. S:
20. Use of a compound selected from the group consisting of D-t-3',4'-ethylenedioxy-1- phenyl-2-palmitoylamino-3-pyrrolidino- -propanol, D-t-4'-hydroxy- -phenyl-2- palmitoylamino-3-pyrrolidino- -propanol and stereoisomers and pharmaceutically acceptable salts thereof to prepare a medicament to inhibit the growth of cancer cells in a mammal.
21. Use of a compound selected from the group consisting of D-t-3',4'-ethylenedioxy-1- *O. S* phenyl-2-palmitoylamino-3-pyrrolidino- -propanol, D-t-4'-hydroxy- -phenyl-2- palmitoylamino-3-pyrrolidino- -propanol and stereoisomers and pharmaceutically acceptable salts thereof to prepare a medicament to treat sphinolipidosis. o• -48-
22. Use of a compound selected from the group consisting of D-t-3',4'-ethylenedioxy- phenyl-2-palmitoylamino-3-pyrrolidino- 1 -propanol, D-t-4'-hydroxy- 1 -phenyl-2- palmitoylamino-3-pyrrolidino- I-propanol and stereoisomers and pharmaceutically acceptable salts thereof to prepare a medicament to treat a microbial or viral infection.
23. Use of a compound selected from the group consisting of D-t-3',4'-ethylenedioxy-1- phenyl-2-palmitoylamino-3-pyrrolidino- 1 -propanol, D-t-4'-hydroxy- 1 -phenyl-2- palmitoylamino-3-pyrrolidino- I1-propanol and stereoisomers and pharmaceutically acceptable salts thereof to prepare a medicament to treat a drug resistant tumour sensitive to a compound selected from the group consisting of ethylenedioxy- 1 -phenyl-2-palmitoylamino-3-pyrrolidino- I -propanol, D-t-4'-hydroxy- 1 -phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol and stereoisomers and pharmaceutically acceptable salts thereof.
24. Use of a compound selected from the group consisting of D-t-3',4'-ethylenedioxy-l- phenyl-2-palmitoylamino-3-pyrrolidino- I -propanol, D-t-4'-hydroxy- 1 -phenyl-2- palmitoylamino-3-pyrrolidino-1-propanol and stereoisomers and pharmaceutically acceptable salts thereof to prepare a medicament to treat a patient by reducing tumour angiogenesis in a patient, wherein said angiogenesis is sensitive to a compound selected from the group consisting of D-t-3',4'-ethylenedioxy- I-phenyl-2- palmitoylamino-3-pyrrolidino- I -propanol, D-t-4'-hydroxy- I -phenyl-2- 20 palmitoylamino-3-pyrrolidino- I-propanol and stereoisomers and pharmaceutically acceptable salts thereof. Dated this TWENTY-FIFTH day of MAY 2004. Resents of the University of Michigan Applicant Wray Associates Perth, Western Australia Patent Attorneys for the Applicant
AU59296/00A 1999-07-09 2000-07-07 Amino ceramide-like compounds and therapeutic methods of use Ceased AU774960B2 (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US09/350,768 US6255336B1 (en) 1995-09-20 1999-07-09 Amino ceramide-like compounds and therapeutic methods of use
US09/350678 1999-07-09
PCT/US2000/018935 WO2001004108A1 (en) 1999-07-09 2000-07-07 Amino ceramide-like compounds and therapeutic methods of use
CA002454453A CA2454453A1 (en) 2000-07-07 2003-12-24 Amino ceramide-like compounds and therapeutic methods of use

Publications (2)

Publication Number Publication Date
AU5929600A AU5929600A (en) 2001-01-30
AU774960B2 true AU774960B2 (en) 2004-07-15

Family

ID=56290039

Family Applications (1)

Application Number Title Priority Date Filing Date
AU59296/00A Ceased AU774960B2 (en) 1999-07-09 2000-07-07 Amino ceramide-like compounds and therapeutic methods of use

Country Status (6)

Country Link
EP (1) EP1196406A1 (en)
JP (1) JP2003521479A (en)
AU (1) AU774960B2 (en)
BR (1) BR0012318A (en)
CA (1) CA2378600A1 (en)
MX (1) MXPA02000296A (en)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8003617B2 (en) * 2004-11-10 2011-08-23 Genzyme Corporation Methods of treating diabetes mellitus
BRPI0823522A2 (en) * 2007-10-05 2014-01-07 Genzyme Corp USE OF CERAMIDE DERIVATIVE COMPOUND
WO2010039256A1 (en) * 2008-10-03 2010-04-08 Genzyme Corporation 2-acylaminopropoanol-type glucosylceramide synthase inhibitors
SI3133070T1 (en) * 2009-11-27 2019-11-29 Genzyme Corp Eliglustat (genz 112638) as inhibitor of glucosylceramide synthase for use in a method of treating fabry's or gaucher's disease, the method comprising adjusting the individual therapeutical dose to the p-450 metabolism of the patient
CN116239513B (en) * 2023-05-05 2023-08-18 天津凯莱英制药有限公司 Preparation method of MMAE key intermediate, preparation method of MMAE and antibody coupling drug

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997010817A1 (en) * 1995-09-20 1997-03-27 The Regents Of The University Of Michigan Amino ceramide-like compounds and therapeutic methods of use

Also Published As

Publication number Publication date
CA2378600A1 (en) 2001-01-18
EP1196406A1 (en) 2002-04-17
BR0012318A (en) 2002-05-28
AU5929600A (en) 2001-01-30
MXPA02000296A (en) 2004-05-21
JP2003521479A (en) 2003-07-15

Similar Documents

Publication Publication Date Title
US7253185B2 (en) Amino ceramide-like compounds and therapeutic methods of use
US6255336B1 (en) Amino ceramide-like compounds and therapeutic methods of use
US5945442A (en) Amino ceramide-like compounds and therapeutic methods of use
US20050239862A1 (en) Amino ceramide-like compounds and therapeutic methods of use
US7335681B2 (en) Amino ceramide-like compounds and therapeutic methods of use
Abe et al. Structural and stereochemical studies of potent inhibitors of glucosylceramide synthase and tumor cell growth.
WO2001004108A1 (en) Amino ceramide-like compounds and therapeutic methods of use
US20030073680A1 (en) Amino ceramide-like compounds and therapeutic methods of use
US20060217560A1 (en) Amino ceramide-like compounds and therapeutic methods of use
Lee et al. Improved inhibitors of glucosylceramide synthase
US7148251B2 (en) Amino ceramide-like compounds and therapeutic methods of use
Inokuchi et al. Preparation of the active isomer of 1-phenyl-2-decanoylamino-3-morpholino-1-propanol, inhibitor of murine glucocerebroside synthetase
JP2009520031A (en) Use of benzo-fused heterocyclic sulfamide derivatives as neuroprotective agents
Shayman et al. [38] Inhibitors of glucosylceramide synthase
US20040260099A1 (en) Amino ceramide-like compounds and therapeutic methods of use
AU774960B2 (en) Amino ceramide-like compounds and therapeutic methods of use
CN1805933A (en) 2-hydroxymethyl-3,4,5-trihydroxy-1benzylpiperidine derivatives as inhibitors of glucosylceramide synthase (gcs)
AU2002249942B2 (en) Amino ceramide-like compounds and therapeutic methods of use
AU2002249942A1 (en) Amino ceramide-like compounds and therapeutic methods of use
JP6422018B2 (en) Glycolipid metabolic disorder treatment