Amino ceramide-like compounds and therapeutic methods of use

Novel amino ceramide-like compounds are provided which inhibit glucosyl ceramide (GlcCer) formation by inhibiting the enzyme GlcCer synthase, thereby lowering the level of glycosphingolipids. 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 glycosphingolipid levels.

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
 (GlcCer), which is enzymatically formed from ceramide and UDP-glucose. The
 enzyme involved in GlcCer formation is UDP-glucose:N-acylsphingosine
 glucosyltransferase (GlcCer synthase). The rate of GlcCer 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, I. Z. et al., "A Role for Glycosphingolipid Accumulation in the
 Renal Hypertrophy of Streptozotocin-Induced 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 cells. 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.sub.3, tumor necrosis
 factor-.alpha., 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 GlcCer 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
 (1R-phenyl-2R-decanoylamino-3-morpholino-1-propanol), previously
 identified 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 B., 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 as cites 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)).
 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 selecting, can, under certain conditions, bind to
 GSLs in the blood vessel walls and produce acute, life-threatening
 inflammation (Alon, R. et al., "Glycolipid Ligands for Selections 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 GlcCer 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 desirable 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.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
 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
 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:
 ##STR1##
 wherein
 R.sub.1 is a phenyl group, preferably a substituted phenyl group such as
 p-methoxy, hydroxy, dioxane substitutions such as methylenedioxy,
 ethylenedioxy, and trimethylenedioxy, cyclohexyl or other acyclic group,
 t-butyl or other branched aliphatic group, 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.sub.2 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 (e.g., a hydroxyl group).
 R.sub.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 (i.e., 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 (i.e.,
 DL-threo or DL-erythro).
 The preferred aliphatic compound of the present invention is
 D-threo-1-pyrrolidino-1-deoxyceramide, identified as IV-231B herein and
 also referred to as PD. The preferred aromatic compound of the present
 invention is 1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol,
 identified as BML-119 herein and also referred to as P4. The structures of
 the preferred compounds are as follows:
 ##STR2##
 An additional preferred compound of the present invention are
 D-t-3',4'-ethylenedioxy-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol
 , also referred to herein as D-t-3',4'-ethylenedioxy-P4, and
 D-t-4'-hydroxy-1-phenyl-2-palmitoylamino-3-pyrrolidino-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 GlcCer 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 chiral synthetic route, it was discovered that the
 isomers active against GlcCer synthase had the
 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 GlcCer
 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 GlcCer
 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 .mu.M), 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 GlcCer-derived GSLs due to the GSL hydrolases
 which normally destroy them. While the body will continue to make the more
 complex GSLs from available GlcCer, 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 GlcCer 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 (PD), does not
 produce ceramide accumulation at all, despite almost complete blockage of
 GlcCer 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 small 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 inborn genetic errors in the metabolism of GlcCer 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 GlcCer product (lactosylceramide). Oxidized
 serum lipoprotein, a material that normally circulates in the blood,
 stimulates the formation of plaques and 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 i.e., 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 GlcCer 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 (P4), 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 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 (e.g., 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 (e.g., 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 ("MDR") 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 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 clear 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. Lipid 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 e.g., 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:
 ##STR3##
 identified 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
 p-methoxyphenyl).
 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 (e.g., a hydroxyl group).
 R is morpholino, pyrrolidino, piperidino, azetidino (trimethyleneimino),
 N-methylethanolamino, diethylamino or N-phenylpiperazino. A small
 substituent, such as a hydroxyl group, is preferably included on the
 cyclic amine moiety.
 ##STR4##
 identified as (2R,3R)-2-palmitoyl-sphingosyl amine or 1-cyclic
 amino-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 (e.g., a hydroxyl group).
 R is morpholino, pyrrolidino, piperidino, azetidino (trimethyleneimino),
 N-methylethanolamino, 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 3).
 Hydroxyl groups can, with advantage, be substituted along the aliphatic
 chain, particularly on carbon 4 (as in the naturally occurring sphingol,
 phytosphingosine). The long chain can also be replaced by other aliphatic
 groups, such at t-butyl or cyclopentyl.
 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 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 (Gb.sub.3)," 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)]-sphingosylphosphorylc
 holine) was from Molecular Probes, Inc., Eugene, Oreg.
 Methods
 TLC of the amines was carried out with HPTLC plates (E. Merck silica gel
 60) and C-M-HOAc 90:10:10 (solvent A) or 85:15:10 (solvent B) or C-M-conc.
 ammonium hydroxide 30:10:1 (solvent C). 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 1S,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. Cancer Inst. 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.degree. C. in 5% CO.sub.2 for 24 h, then incubated another 24 h with
 medium containing the inhibitor-BSA complex. The control cells were also
 incubated in the presence of BSA. The cells were washed with
 phosphate/saline and trichloroacetic acid, then scraped off the dishes,
 dissolved in alkali, and analyzed for protein and DNA incorporated
 tritium. [Methyl-.sup.3 H]thymidine (10 .mu.Ci) was added 4 h prior to
 harvesting.
 Assay of sphingolipid enzymes.
 The inhibitors were evaluated for their effectiveness against the GlcCer
 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[.sup.3 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 .mu.g) 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 0.degree. C.
 for 3.times.30 sec; .about.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 .mu.M [1-.sup.14 C]decanoyl
 sphingosine (10.sup.5 cpm) in 340 .mu.M dioleoylphosphatidylcholine
 liposomes and 0.34 mg of MDCK cellular protein homogenate. The acid enzyme
 was incubated in 32.5 mM citrate-Na.sup.+ (pH 4.5) and the neutral enzyme
 buffer was 40 mM Tris-Cl.sup.- (pH 7.1 at 37.degree. 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 .mu.M [3-.sup.3 H]sphingosine (70,000
 cpm, repurified by column chromatography), 0.2 mM stearoyl-CoA, 0.5 mM
 dithiothreitol, and .about.300 .mu.g of MDCK homogenate protein in 25 mM
 phosphate-K.sup.+ 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 .mu.M [.sup.14 C]decanoyl
 sphingosine (10.sup.5 cpm) dispersed with 136 .mu.M dioleoyllecithin as in
 the ceramide synthase assay, and 5 mM EDTA and 50 mM Hepes-Na.sup.+ pH
 7.5, in a total volume of 0.5 ml. MDCK homogenate was centrifuged at
 600.times.g briefly, then at 100,000.times.g for 1 h, and the pellet was
 suspended in water and sonicated with a dipping probe. A portion of this
 suspension containing 300 .mu.g of protein was used. Incubation was at
 37.degree. C. for 30 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 Lipid 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 .mu.M substrate and 30 .mu.M lecithin). The assay medium for
 the neutral enzyme also contained 50 mM Tris-Cl.sup.- (pH 7.4), 25 mM KCl,
 5 mM MgCl.sub.2 and 0.29 mg of MDCK cell protein in a total volume of 0.25
 ml. Incubation was at 37.degree. 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 .mu.g of cell protein, with 60 min
 of incubation at 37.degree. 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 Phenyl TLC hR.sub.f
 or Name R Group Substituent Value.sup.a
 PDMP.sup.b morpholino 34(47)
 PPMP morpholino (53)
 112 N-phenylpiperazino 56
 113 morpholino .rho.-fluoro 25
 114 diethylamino 25
 115 piperidino 29
 (pentamethyleneimino)
 116 hexamethyleneimino 34
 117.sup.b morpholino .rho.-fluoro 41
 118 piperidino .rho.-fluoro 26
 119 pyrrolidino 20-70(44)
 (tetramethyleneimino)
 120 1-methylpiperazino 7-62
 121 3-dimethylaminopiperidino 1-30
 122 N-methylethanolamino 6-71
 123 azetidino (trimethyleneimino) 12
 124 amino 15
 125 morpholino .rho.-methoxy 37
 126 pyrrolidino .rho.-methoxy (50)
 .sup.a Only the relative R.sub.f 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.
 .sup.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.
 TABLE 2
 Characterization of the Sphingosyl Inhibitors
 Sphingol TLC hR.sub.f
 Number R Group Structure Value.sup.a
 IV-181A morpholino 2R,3S 43
 IV-206A morpholino 2R,3R 40
 IV-230A pyrrolidino 2R,3S 31
 IV-231B pyrrolidino 2R,3R 31
 .sup.a 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 GlcCer 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 4). 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.
 TABLE 3
 Inhibition of Ceramide Glucosyltransferase of
 MDCK cell Homogenates by Different Compounds
 Inhibitor Inhibition at Active
 Number % Inhibition at 80 .mu.M 5 .mu.M Isomer.sup.h
 BML-113 60 .+-. 4.7.sup.a 29
 BML-114 31 .+-. 2.9.sup.a 20
 BML-115 84 .+-. 0.8.sup.a 12.4 .+-. 0.7.sup.f 27
 82 .+-. 0.3.sup.b
 BML-116 28 .+-. 3.2.sup.a 27
 BML-117 35 .+-. 0.6.sup.b 36
 BML-118 62 .+-. 0.4.sup.b 8.3 .+-. 1.4.sup.f 32
 BML-119 94 .+-. 1.4.sup.b 51 .+-. 2.3.sup.e 29
 97 .+-. 0.1.sup.c 49 .+-. 0.8.sup.f
 96 .+-. 0.1.sup.d
 BML-120 11 .+-. 3.0.sup.c 26
 BML-121 11 .+-. 0.4.sup.c 28
 BML-122 58 .+-. 1.6.sup.d 26
 BML-123 86 .+-. 0.1.sup.d 15 .+-. 0.8.sup.f 33
 BML-124 -2 .+-. 1.6.sup.d 15
 BML-125 9 .+-. 3.0.sup.e 26
 BML-126 60 .+-. 1.8.sup.e 54 .+-. 0.3.sup.f 34
 PDMP 90 .+-. 0.8.sup.a 16 .+-. 1.8.sup.e 100
 PPMP 32 .+-. 1.8.sup.e 100
 32 .+-. 0.7.sup.f
 IV-181A 12 .+-. 0.2.sup.g 100
 IV-206A 73 .+-. 1.5.sup.g 100
 IV-230A 19 .+-. 2.1.sup.g 100
 IV-231B 87 .+-. 0.4.sup.g 100
 .sup.a-g Different samples were assayed as parts of different experiments.
 .sup.h Percent of the active D-stereoisomer in the synthesized sample,
 estimated by scanning the two stained bands, assuming the slower one was
 the (racemic) active form.
 Comparison of PDMP (1R,2R-decanoate) and PPMP (1R,2R-palmitate), when
 evaluated at the same time in Expt. f, shows that an increase in the chain
 length of 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 .mu.M level.
 Replacing the oxygen in the morpholine ring of PPMP with a methylene group
 (BML-115) improved activity .about.1.4-fold (calculated from the
 inhibitions at 5 .mu.M in Expt. f and relative purities, and assuming that
 the percent inhibition is proportional to concentration in this region:
 12.4/27.times.100/32=1.4). 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 C.sub.18 column, using
 M-W-conc. NH.sub.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 GlcCer synthase with each preparation at 5 .mu.M showed 15%
 inhibition by BML-129 and 79% inhibition by BML-130. TLC analysis of 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. Lipid 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 3).
 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 .mu.M IV-231B and 89.+-.0.6% with 6 .mu.M
 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 4). The IC.sub.50 values (rounded off
 to one digit in the table) ranged from 0.7 to 2.6 .mu.M.
 TABLE 4
 Inhibition of Tumor Cell Growth In Vitro by Various Inhibitors
 Cell
 Type BML-115 BML-118 BML-119 BML-123 BML-126 BML-129
 BML-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 1 1 1 1 2 2
 -10A
 FIG. 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. FIG. 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 GlcCer synthase and
 growth. These differences in growth inhibitory power correlate with their
 effectiveness in MDCK cell homogenates as GlcCer 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 (e.g., 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 .mu.M inhibitor, but only 3% of control growth with 3 .mu.M.
 Growth was 93% of control growth with 2 .mu.M BML-130 but only 5% of
 controls with 3 .mu.M 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 4). This observation is similar to the results with PDMP
 isomers in fibroblasts cited above (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.
 FIG. 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 FIG. 2 is
 the average of values from three plates, with error bars corresponding to
 one standard deviation.
 FIG. 3 shows [.sup.3 H]thymidine incorporation into DNA of MDCK cells
 incubated as in FIG. 2. The values in FIG. 3 are normalized on the basis
 of the protein content of the incubation dishes and compared to the
 incorporation by cells in standard medium.
 FIGS. 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 IC.sub.50 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 .mu.M for 1 day or 1 .mu.M for 3
 days. Tests with a longer growth period, 5 days, in 5 .mu.M 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 GlcCer levels, as expected from its
 greater effectiveness in vitro as a glucosyltransferase inhibitor. The
 level of GlcCer, estimated visually, was greatly lowered by 0.3 .mu.M
 BML-130 or 0.5 .mu.M BML-129. The levels of the other lipids visible on
 the plate (mainly sphingomyelin (SM), cholesterol, and fatty acids) were
 changed little or not at all. BML-129 and the GlcCer 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 overlapped 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
 GlcCer levels to a very low point (e.g., at 2 or 4 .mu.M). 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 .mu.M BML-129 or -130 there was little effect on
 ceramide content but at 4 .mu.M 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 .mu.M
 level with either compound but substantial inhibition at the 4 .mu.M
 level, especially with the glucosyltransferase inhibitor, BML-130. This
 finding is similar to the ones made in longer incubations with human
 cancer cells.
 TABLE 5
 Effects of BML-129 and -130 on MDCK Cell Growth
 and the Content of Ceramide and Diacylglycerol
 Protein Ceramide Diglyceride
 Growth Medium .mu.g/dish nmol/mg protein
 Controls 490 1.04 4.52
 560 0.96 5.61
 0.4 .mu.m BML-129 500 1.29 5.51
 538 0.99 5.13
 0.4 .mu.m BML-130 544 0.94 4.73
 538 0.87 5.65
 4 .mu.m BML-129 396 3.57 9.30
 311 3.78 9.68
 4 .mu.m 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 by TLC densitometry, was 1.0 nmol/mg protein at
 0.5 .mu.M, 1.1 at 1 .mu.M, 1.5 at 2 .mu.M, and 3.3 at 4 .mu.M. 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-.beta.-D-Arabinofuranosylcytosine Stimulates Ceramide and Diglyceride
 Formation in HL-60 Cells," J. Biol. Chem. 269:15493-15497 (1994)).
 TLC of MDCK cells grown in the presence of 0.02 to 1 .mu.M 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 .mu.M level and virtually
 no GlcCer was visible at the 1 .mu.M level; however the more polar GSLs
 were not affected as strongly. After incubation for 5 days in 5 .mu.M
 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 .mu.g/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 5 .mu.M
 inhibitors (Table 6). There did appear to be moderate inhibition at the 50
 .mu.M level with PDMP and the aliphatic inhibitor.
 TABLE 6
 Effect of Inhibitors on Acid and Neutral
 Ceramidases and Ceramide Synthase of MDCK Cells
 Enzyme Activity (% of control)
 Ceramidase Ceramidase Ceramide
 Inhibitor Tested pH 4.5 pH 7.4 Synthase
 D-threo-PDMP, 5 .mu.M 97 .+-. 4 116 .+-. 19 99 .+-. 5
 D-threo-PDMP, 50 .mu.M 133 .+-. 13.sup.a 105 .+-. 11 66 .+-.
 9.sup.a
 BML-129, 5 .mu.M 108 .+-. 8 100 .+-. 0 97 .+-. 0
 BML-129, 50 .mu.M 171 .+-. 26.sup.a 99 .+-. 2 102 .+-. 1
 BML-130, 5 .mu.M 107 .+-. 11 100 .+-. 15 108 .+-. 10
 BML-130, 50 .mu.M 160 .+-. 21.sup.a 100 .+-. 15 106 .+-. 29
 IV-231B, 5 .mu.M 106 .+-. 3 116 .+-. 20 90 .+-. 8
 IV-231B, 50 .mu.M 113 .+-. 8 112 .+-. 3 71 .+-. 18.sup.a
 .sup.a Notable differences.
 Assay of the two kinds of ceramidase (Table 6) showed that there was no
 effect of either the aliphatic or aromatic inhibitors at the 5 .mu.M
 level, at which point cell growth is completely stopped in the case of the
 pyrrolidino compounds. At the 50 .mu.M 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 .mu.M (54% and
 61%, respectively) (Table 7).
 TABLE 7
 Effect of Inhibitors on Acid and Neutral
 Sphingomyelinases and Sphingomyelin Synthase
 Enzyme Activity (% of control)
 Inhibitor Sphingomyelinase Sphingomyelinase Sphingomyelinase
 Tested pH 4.5 pH 7.1 Synthase.sup.a
 D-threo- 102 .+-. 3 121 .+-. 13
 PDMP, 5 .mu.M
 D-threo- 100 .+-. 3 108 .+-. 8
 PDMP, 50 .mu.M
 BML-129, 108 .+-. 4 105 .+-. 11 84 .+-. 27
 5 .mu.M
 BML-129, 97 .+-. 3 142 .+-. 11.sup.b 46 .+-. 11.sup.b
 50 .mu.M
 BML-130, 109 .+-. 1 110 .+-. 7 87 .+-. 14
 5 .mu.M
 BML-130, 114 .+-. 2 152 .+-. 14.sup.b 39 .+-. 18.sup.b
 50 .mu.M
 IV-231B, 101 .+-. 7 131 .+-. 3.sup.b
 5 .mu.M
 IV-231B, 112 .+-. 11 120 .+-. 3.sup.b
 50 .mu.M
 .sup.a Data for PDMP and IV-231B are not shown here as they were tested in
 other experiments; no effect was seen.
 .sup.b Notable differences.
 Neutral sphingomyelinase (SMase) was distinctly stimulated by the aliphatic
 inhibitor, IV-231B, even at 5 .mu.M (Table 7). 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
 .mu.M 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 GlcCer synthase
 inhibition, a 5-membered 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 GlcCer 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 IC.sub.50 values were 0.2 to 6 .mu.M for
 cisplatin, 0.02 to 44 .mu.M for carboplatin, 0.03 to 0.2 .mu.M for
 methotrexate, 0.07 to 0.2 .mu.M for fluorouracil, and 0.1 to 1 .mu.M for
 etoposide. Unlike these 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 C," 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 GlcCer 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 .mu.M
 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 GlcCer
 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 R,S-, S,R-, and
 S,S-isomers, which seem to exert effects on sphingolipids without strongly
 inhibiting GlcCer synthesis. The tests with both the
 DL-erythro-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
 .mu.M), was found to inhibit SM synthase in growing CHO cells (Rosenwald,
 A. G. et al., "Effects of a Sphingolipid Synthesis Inhibitor on Membrane
 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 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)).
 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,"
 Cancer Lett. 38:23-30 (1987)) but high doses were needed. From the data in
 FIG. 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-[.sup.3 H]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 GlcCer synthase was mainly dependent upon hydrophobic and
 electronic properties of the substituent. Surprisingly, a linear
 relationship was found between log [IC.sub.50 ] and hydrophobic parameter
 (.pi.)+electronic parameter (.delta.). 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 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 D-t-3',4'-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., St.
 Louis, Mo., Lancaster Synthesis Inc., Windham, N.H. and Maybridge Chemical
 Co., Cornwall, 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-[.sup.3 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,
 3',4'-(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-1-propanol.
 The synthesis of
 1-(4'-hydroxy)phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol is
 described in detail in FIG. 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 7).
 All other syntheses employ a similar synthetic scheme (steps 2 to 6).
 4'-Benzyloxyacetophenone formation (step 1)
 4'-Hydroxyacetophenone (13.62 g, 100 mmol), benzylbromide (17.1 g, 100
 mmol), and cesium carbonate (35.83 g, 100 mmol) were added to
 tetrahydrofuran at room temperature and stirred overnight. The product was
 concentrated to dryness and recrystallized from ether and hexane to yield
 15 g of 4'-benzyloxyacetophenone which appeared as a white powder. An
 R.sub.f of 0.42 was observed when resolved by thin layer chromatography
 using methylene chloride. .sup.1 H nmr (.delta., ppm, CDCl.sub.3), 7.94
 (2H, .delta., 8.8 Hz, O--Ar--C(O)), 7.42 (5H, m, Ar'CH.sub.2 O--), 7.01
 (2H, .delta., 8.8 Hz, O--Ar--C(O)), 5.14 (2H, s, Ar'CH.sub.2 O--), 2.56
 (3H, S, CH.sub.3).
 Bromination of 4'-benzyloxyacetophenone (step 2)
 Bromine (80 mmol) was added dropwise over 5 min to a stirred solution of
 4'-benzyloxyacetophenone (70 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.sub.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.sub.f of 0.62 was
 observed when resolved by thin layer chromatography using methylene
 chloride. 1H nmr (.delta., ppm, CDCl.sub.3), 7.97 (2H, .delta., 9.2 Hz,
 O--Ar--C(O)), 7.43 (5H, m, Ar'CH.sub.2 O--), 7.04 (2H, .delta., 9.0 Hz,
 O--Ar--C(O)), 5.15 (2H, s, Ar'CH.sub.2 O--), 4.40 (2H, s, CH.sub.2 Br).
 2-Amino-4'-benzyloxyacetophenone HCl 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 HCl in an oil bath at 85.degree. C. for 3 h. Upon cooling
 the precipitated hydrochloride salt (2.5 g) was removed by filtration. The
 filtrate was left at -20.degree. C. overnight and additional product (2.1
 g) was isolated. The yield was 4.6 g (82.6%). [M.sup.+ H].sup.+ : 242 for
 C.sub.15 H.sub.16 NO.sub.2. .sup.1 H nmr (.delta., ppm, CDCl.sub.3), 8.38
 (2H, bs, NH.sub.2), 7.97 (2H, .delta., 8.8 Hz, O--Ar--C(O)), 7.41 (5H, m,
 Ar'CH2O--), 7.15 (2H, .delta., 8.6 Hz, O--Ar--C(O)), 5.23 (2H, s,
 Ar'CH.sub.2 O--), 4.49 (2H, s, CH.sub.2 NH.sub.2).
 2-Palmitoylamino-4'-benyloxyacetophenone formation (step 4)
 Sodium acetate (50% in water, 29 ml) was added in three portions to a
 stirred solution of 2-amino-4'-benzyloxyacetophenone HCl (4.6 g, 17 mmol)
 and tetrahydrofuran (200 ml). Palmitoyl chloride (19 mmol) in
 tetrahydrofuran (25 ml) was added dropwise over 20 min yielding a dark
 brown solution. The mixture was stirred overnight at room temperature. The
 aqueous fraction was removed by use of a separatory funnel and
 chloroform/methanol (2/1, 150 ml) was added to the organic layer which was
 then washed with water (50 ml). The yellow aqueous layer was extracted
 once with chloroform (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.degree. 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.sub.f of 0.21 was observed when
 resolved by thin layer chromatography using methylene chloride. [M.sup.+
 H].sup.+ : 479 for C.sub.31 H.sub.45 NO.sub.3. .sup.1 H nmr (.delta., ppm,
 CDCl.sub.3), 7.96 (2H, .delta., 8.8 Hz, O--Ar--C(O)), 7.40 (5H, m,
 Ar'CH.sub.2 O--), 7.03 (2H, .delta., 8.8 Hz, O--Ar--C(O)), 6.57 (1H, bs,
 NH.sub.2), 5.14 (2H, s, Ar'CH.sub.2 O--), 4.71 (2H, s, C(O)CH.sub.2
 NHC(O)), 2.29 (2H, t, 7.4 Hz, C(O)CH.sub.2 (CH.sub.2).sub.13 CH.sub.3),
 1.67 (2H, m, C(O)CH.sub.2 (CH.sub.2).sub.13 CH.sub.3), 0.87 (3H, t, 6.7
 Hz, C(O)CH.sub.2 (CH.sub.2).sub.13 CH.sub.3).
 1-(4'-Benzyloxy)phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol formation
 (steps 5 and 6)
 2-Palmitoylamino-4'-benyloxyacetophenone (3.79 g, 8.0 mmol),
 paraformaldehyde (0.25 g, 2.7 mmol), pyrrolidine (0.96 ml, 11.4 mmol) and
 ethanol (70 ml) were stirred under nitrogen. Concentrated HCl (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 overnight, and the product was dried in a
 solvent evaporator. The residue was redissolved in dichloromethane (130
 ml) and hydrolyzed with 3N HCl (pH.about.4). 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-threo- and DL-etythro
 enantiomers (2.53 g, 4.2 mmol). An R.sub.f 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
 (1:9). [M.sup.+ H].sup.+ : 565 for C.sub.36 H.sub.56 N.sub.2 O.sub.3.
 1-(4'-Hydroxy)phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol formation
 (step 7)
 A suspension of 20% Pd/C (40 mg) in acetic acid (15 ml) was stirred at room
 temperature under a hydrogen balloon for 15 min.
 1-(4'-Benzyloxy)phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol (420
 mg, 0.74 mmol) was added and the solution was stirred overnight. The
 suspension was filtered through a glass frit, and the filter was rinsed
 with acetic acid:methylene chloride (1:1, 5 ml). The filtrate was
 concentrated in vacuo and crystallized to yield a pale yellow semisolid
 (190 mg, 0.4 mmol). An R.sub.f of 0.21 was observed when resolved by thin
 layer chromatography using methanol:methylene chloride (1:9). [M.sup.+
 H].sup.+ : 475 for C.sub.29 H.sub.50 N.sub.2 O.sub.3. .sup.1 H nmr
 (.delta., ppm, CDCl.sub.3), 7.13 (4H, m, ArCHOH--), 7.14 (1H, .delta., 6.9
 Hz, --NH--), 5.03 (1H, .delta., 3.3 Hz, CHOH--), 4.43 (1 H, m, c-(CH.sub.2
 CH.sub.2).sub.2 NCH.sub.2 CH), 3.76 (2H, m, c-(CH.sub.2 CH.sub.2).sub.2
 N--), 3.51 (1H, m, c-(CH.sub.2 CH.sub.2).sub.2 NCH.sub.2 --), 3.29 (1 H,
 m, c-(CH.sub.2 CH.sub.2).sub.2 NCH.sub.2 --), 2.97 (3H, m, c-(CH.sub.2
 CH.sub.2).sub.2 N-- and ArC(OH)H--), 2.08 (6H, m, --C(O)CH.sub.2
 (CH.sub.2).sub.13 CH.sub.3 and c-(CH.sub.2 CH.sub.2).sub.2 N--, 1.40 (2H,
 m, C(O)CH.sub.2 CH.sub.2 (CH.sub.2).sub.12 CH.sub.3), 1.25 (2H, m,
 --C(O)CH.sub.2 CH.sub.2 (CH.sub.2).sub.12 CH.sub.3), 0.87 (3H, t, 6.7 Hz,
 C(O)CH.sub.2 (CH.sub.2).sub.13 CH.sub.3).
 Synthesis of
 D-threo-1-(3',4'-ethylenedioxy)phenyl-2-palmitoylamino-3-pyrrolidino-1-pro
 panol
 2-Amino-3',4'-(ethylenedioxy)acetophenone HCl
 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 HCl (14 ml) in an oil bath at 85.degree. 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 HCl was washed with cold isopropanol and then
 with ether. The yield of this product was .about.7.1 g (81%).
 2-Palmitoylamino-3',4'-(ethylenedioxy)acetophenone
 Aminoacetophenone HCl (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 (2/1, 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.degree. 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 overnight. The yield of
 the product was 27 mmol (11.6 g).
 D-threo-1-(3',4'-ethylenedioxy)phenyl-2-palmitoylamino-3-pyrrolidino-1-prop
 anol
 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 500 ml round flask under nitrogen flow. Concentrated HCl (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 HCl (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.sub.2 O (30 ml), saturated NaCl
 (30 ml) and dried over anhydrous MgSO.sub.4. The dichloromethane solution
 was rotoevaporated to a viscous oil which was purified by use of a
 Chromatotron with a solvent consisting of 10% 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:
 [(S)-val-(R)-1-(a-naphtyl)ethylamine, 20.times.250 mm: Phenomenex], eluted
 with hexane-1,2-dichloroethane-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.times.250 mm) and
 the above solvent mixture at flow rate of 1 ml/min.
 Glycosylceramide synthase 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 (120 .mu.g of protein) was
 incubated with uridinediphosphate [.sup.3 H]glucose (100,000 cpm) and
 liposomes consisting of 85 .mu.g octanoylsphingosine, 570 .mu.g
 dioleoyphosphatidylcholine and 100 .mu.g sodium sulfatide in 200 .mu.l of
 reaction mixture and kept for 1 h at 37.degree. C. 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 GlcCer synthase 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 800 g for 5 min, the
 supernatant was transferred into another glass tube. The residues were
 reextracted with chloroform/methanol (1/1). After the centrifugation, the
 resultant supernatant was combined with the first one. The residues were
 air-dried and kept for protein analysis. Adding 0.9% NaCl to the
 supernatant combined, the ratio of chloroform/methanol/aqueous was
 adjusted to 1/1/1. After centrifugation 800 g for 5 min, the upper layer
 was discarded. Methanol/water (1/1) 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, B. N.,
 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 .about.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 FIG. 9. Initially the effect of each P4 isomer separated by chiral
 chromatography on GlcCer synthase activity was determined (FIG. 10). Four
 peaks were observed for the chiral separation of P4. Peaks 1 and 2
 represented the erythro diastereomers 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 (FIG. 4A). This specificity for the D-threo enantiomer was
 consistent with the previous results observed in PDMP and PDMP homologues
 (2-4). The IC.sub.50 of D-threo-P4 was 0.5 mM for GlcCer synthase activity
 measured in the MDCK cell homogenates.
 Effects of P4 and P4 Derivatives with a Single Substituent of Phenyl Group
 on GlcCer Synthase Activity
 The effect of each P4 isomer on GlcCer synthase activity was analyzed. The
 reaction was carried out in the absence or presence of 0.1, 1.0 or 10
 .mu.M P4 (FIG. 4A) or p-methoxy-P4 (FIG. 4B). As shown in FIG. 4A, the
 enzyme activity was specifically inhibited by D-threo isomer. In FIG. 4A,
 the symbols are denoted as follows: D-threo (.cndot.), D-erythro
 (.quadrature.), L-threo and (.multidot.), L-erythro (.DELTA.). 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 IC.sub.50 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 FIG. 4B, it was
 confirmed that the enzyme activity was potently inhibited by
 D-threo-p-methoxy-P4 whose IC.sub.50 was 200 nM. In FIG. 4B, .quadrature.
 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 (FIG. 10). A slight inhibition of the enzyme activity by
 p-methyoxy-P4 in a combined D-erythro and L-threo mixture (peaks 2 and 3,
 FIG. 10) was observed; this was due to contamination of the D-threo isomer
 (peak 4, FIG. 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. Hogberg, T. et
 al., Theoretical and experimental methods in drug design applied on
 antipsychotic dopamine antagonists. Larsen, P. K., and Bundgaard, H.,
 "Textbook of Drug Design and Development," pp. 55-91 (1991). Generally
 IC.sub.50 is described as the following equation:
 log(1/IC.sub.50)=a(hydrophobic parameter (.pi.)+b(electronic parameter
 (.sigma.))+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, P. K., and Bundgaard, H.,
 "Textbook of Drug Design and Development," pp. 55-91 (1991).
 The hydrophobic effect, .pi., is described by the equation .pi.=log P.sub.X
 -log P.sub.H where P.sub.X is the partition coefficient of the substituted
 derivative and P.sub.H is that of the parent compound, measured as the
 distribution between octanol and water.
 The electronic substituent parameter, .sigma., was originally developed by
 Hammett (Hammett, L. P., In Physical Organic Chemistry, McGraw-Hill, New
 York (1940)) and is expressed as .sigma.=log K.sub.X -log K.sub.H, where
 K.sub.X and K.sub.H are the ionization constants for a para or meta
 substituted derivative and benzoic acid respectively. Positive .sigma.
 values represent electron withdrawing properties and negative .sigma.
 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 8). Surprisingly, a linear relationship
 was observed between log (IC.sub.50) and .pi.+.sigma. (FIG. 5). These
 findings suggest that the more negative the value of .pi.+.sigma., the
 more potent is D-threo-P4 derivatives made as GlcCer 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.sub.50) and .pi.+.sigma.
 (FIG. 5). These findings suggest that the more negative the value of
 .pi.+.sigma., the more potent the D-t-P4 derivative as a GlcCer synthase
 inhibitor.
 TABLE 8
 D-threo-P4 derivative .sigma. + .pi.* IC.sub.50 (.mu.M)**
 p-methoxy -0.29 0.2
 P-4 0.00 0.5
 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 Hogberg, T. et al.,
 Theoretical and experimental methods in drug design applied on
 antipsychotic dopamine antagonists. Larsen, P. K., and Bundgaard, H.,
 "Textbook of Drug Design and Development," pp. 55-91 (1991), for methoxy,
 .sigma..sub.m = 0.12, .sigma..sub.p = -0.27, .pi. = -0.02; hydro, .sigma.
 = 0, .pi. = 0; methyl, .sigma..sub.p = -0.17, .pi. = 0.56; chloro,
 # .sigma..sub.p = 0.23, .pi. = 0.71.
 **These values were derived from FIGS. 4A and 4B. For other compounds the
 same analytical approach as shown in FIGS. 4A and 4B was carried out to
 obtain the IC.sub.50.
 The p-hydroxy-substituted homologue was a significantly better GlcCer
 synthase inhibitor. The strong association between .pi.+.sigma. 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
 .pi.+.sigma. value would be observed for the p-hydroxy homologue. This
 compound was synthesized and the D-threo enantiomer isolated by chiral
 chromatography. An IC.sub.50 of 90 nM for GlcCer synthase inhibition was
 observed (FIG. 11), suggesting that the p-hydroxy homologue was twice as
 active as the p-methoxy compound. Moreover, the linear relationship
 between the log (IC50) and .pi.+.sigma. was preserved (open circle, FIG.
 4).
 Effects of 3',4'-dioxy-D-threo-P4 Derivatives on GlcCer Synthase Activity
 The result in FIG. 5 suggested that an electron donating and hydrophilic
 substituent of phenyl group makes the GlcCer synthase inhibitor potent. To
 attain further improvement of the inhibitor, another series of P4
 derivatives with methylenedioxy, ethylenedioxy and trimethyidioxy
 substitutions on the phenyl group were designed (FIG. 9).
 As shown in FIG. 6, the enzyme activity was markedly inhibited by
 D-t-3',4'-ethylenedioxy-P4 whose IC.sub.50 was 100 nM. In FIG. 6,
 .quadrature. denotes D-t-3',4'-methylenedioxy-P4, .omicron. denotes
 D-t-3',4'-ethylenedioxy-P4, .DELTA. denotes D-t-3',4'-trimethylenedioxy-P4
 and.cndot.denotes D-t-3',4'-dimethyoxy-P4. One the other hand, the
 IC.sub.50 s 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 D-t-3',4'-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
 (FIG. 6). As the parameters, .sigma..sub.m, .sigma..sub.p and .pi., for
 methoxy substituent are 0.12, -0.27 and -0.02, respectively (Hogberg, T.
 et al., Theoretical and experimental methods in drug design applied on
 antipsychotic dopamine antagonists. Larsen, P. K., and Bundgaard, H.,
 "Textbook of Drug Design and Development," pp. 55-91 (1991)), the value of
 .pi.+.sigma. 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 FIG. 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 FIG. 5.
 GlcCer 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 1-O-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 D-threo
 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, D-threo-(4'-hydroxy)-P4, and D-threo-P4
 (IC.sub.50 0.1 mM versus 10 mM) in inhibiting GlcCer synthase, the D-threo
 enantiomers of all three compounds demonstrated comparable activity in
 blocking 1-O-acylceramide synthase (FIG. 12).
 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 9). MDCK cells (5.times.10.sup.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). GlcCer 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
 Ceramide Diradylglycerol
 (pmol/nmol (pmol/nmol
 Condition 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
 D-threo-4'-hydroxy-P4
 11.3 nM
 24 h 4.29 .+-. 0.71 30.9 .+-. 2.01*
 48 h 6.70 .+-. 0.29 38.4 .+-. 1.44*
 113 nM
 24 h 5.09 .+-. 0.95 31.5 .+-. 3.84*
 48 h 7.47 .+-. 0.29 41.5 .+-. 0.66*
 1130 nM
 24 h 7.38 .+-. 0.13 38.5 .+-. 3.84*
 48 h 13.4 .+-. 1.03* 47.2 .+-. 2.51*
 D-threo-3',4'-ethylenedioxy-P4
 11.3 nM
 24 h 5.24 22.0
 5.04 24.7
 113 nM
 24 h 5.21 32.5
 5.21 41.6
 1130 nM
 24 h 9.64 32.5
 13.0 41.6
 *Denotes p &lt; 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-threo-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 FIG. 7,
 wherein each bar is the average values from three dishes, with error bars
 corresponding to one standard deviation. In the control, the total protein
 and GlcCer were 414.+-.47.4 .mu.g/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 (FIGS. 7, 14 and 15). By contrast, only 27 percent depletion
 of GlcCer occurred in cells exposed to D-threo-P4 (FIG. 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 (FIGS. 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 9). There was no change in ceramide or
 diradylglycerol levels in cells treated with 11.3 nM or 113 nM
 concentrations of either compound. Similar patterns 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 GlcCer content
 were truly related to inhibition of GlcCer 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 GlcCer 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.
 As used herein, the term "pharmaceutically acceptable salts" is intended to
 mean salts of the compounds of the present invention with pharmaceutically
 acceptable acids, e.g., 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
 (e.g., tablets, dragees and capsules), rectally (e.g., suppositories), as
 well as administration by injection.
 The pharmaceutical preparations of the present invention are manufactured
 in a manner which is itself known, e.g., 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, e.g.,
 lactose or sucrose, mannitol or sorbitol, cellulose preparations and/or
 calcium phosphates, e.g., tricalcium diphosphate or calcium hydrogen
 phosphate, as well as binders such as starch paste, using, e.g., 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, e.g., 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/or
 titanium dioxide, lacquer solutions 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 hydroxypropylmethyl cellulose phthalate, are
 used. Dyestuffs or pigments may be added to the tablets or dragee
 coatings, e.g., for identification or in order to characterize different
 combinations of active compound doses.
 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, e.g.,
 natural or synthetic triglycerides, paraffin hydrocarbons, polyethylene
 glycols or higher alkanols. 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, e.g., liquid triglycerides,
 polyethylene glycols or paraffin hydrocarbons.
 Suitable formulations for parenteral administration include aqueous
 solutions of the active compounds in water-soluble form, e.g.,
 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, e.g., 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.
 Alternatively, the active compounds of the present invention may be
 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.
 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.
 All publications cited herein are expressly incorporated by reference.