Improved Inhibitors of Glucosylceramide Synthase

Previous work has led to the identification of inhibitors of glucosylceramide synthase, the enzyme catalyzing the first glycosylation step in the synthesis of glucosylceramide-based glycosphingolipids. These inhibitors have two identified sites of action: the inhibition of glucosylceramide synthase, resulting in the depletion of cellular glycosphingolipids, and the inhibition of 1-O-acylceramide synthase, resulting in the elevation of cell ceramide levels. A new series of glucosylceramide synthase inhibitors based on substitutions in the phenyl ring of a parent compound, 1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (P4), was made. For substitutions of single functional groups, the potency of these inhibitors in blocking glucosylceramide synthase was primarily dependent upon the hydrophobic and electronic properties of the substituents. An exponential relationship was found between the IC50 of each inhibitor and the sum of derived hydrophobic (π) and electronic (ς) parameters. This relationship demonstrated that substitutions that increased the electron-donating characteristics and decreased the lipophilic characteristics of the homologues enhanced the potency of these compounds in blocking glucosylceramide formation. A novel compound was subsequently designed and observed to be even more active in blocking glucosylceramide formation. This compound,d-threo-4′-hydroxy-P4, inhibited glucosylceramide synthase at an IC50 of 90 nm. In addition, a series of dioxane substitutions was designed and tested. These included 3′,4′-methylenedioxyphenyl-, 3′,4′-ethylenedioxyphenyl-, and 3′4′-trimethylenedioxyphenyl-substituted homologues.d-threo-3′,4′-Ethylenedioxy-P4-inhibited glucosylceramide synthase was comparably active to thep-hydroxy homologue. 4′-Hydroxy-P4 and ethylenedioxy-P4 blocked glucosylceramide synthase activity at concentrations that had little effect on 1-O-acylceramide synthase activity. These novel inhibitors resulted in the inhibition of glycosphingolipid synthesis in cultured cells at concentrations that did not significantly raise intracellular ceramide levels or inhibit cell growth. Previous work has led to the identification of inhibitors of glucosylceramide synthase, the enzyme catalyzing the first glycosylation step in the synthesis of glucosylceramide-based glycosphingolipids. These inhibitors have two identified sites of action: the inhibition of glucosylceramide synthase, resulting in the depletion of cellular glycosphingolipids, and the inhibition of 1-O-acylceramide synthase, resulting in the elevation of cell ceramide levels. A new series of glucosylceramide synthase inhibitors based on substitutions in the phenyl ring of a parent compound, 1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (P4), was made. For substitutions of single functional groups, the potency of these inhibitors in blocking glucosylceramide synthase was primarily dependent upon the hydrophobic and electronic properties of the substituents. An exponential relationship was found between the IC50 of each inhibitor and the sum of derived hydrophobic (π) and electronic (ς) parameters. This relationship demonstrated that substitutions that increased the electron-donating characteristics and decreased the lipophilic characteristics of the homologues enhanced the potency of these compounds in blocking glucosylceramide formation. A novel compound was subsequently designed and observed to be even more active in blocking glucosylceramide formation. This compound,d-threo-4′-hydroxy-P4, inhibited glucosylceramide synthase at an IC50 of 90 nm. In addition, a series of dioxane substitutions was designed and tested. These included 3′,4′-methylenedioxyphenyl-, 3′,4′-ethylenedioxyphenyl-, and 3′4′-trimethylenedioxyphenyl-substituted homologues.d-threo-3′,4′-Ethylenedioxy-P4-inhibited glucosylceramide synthase was comparably active to thep-hydroxy homologue. 4′-Hydroxy-P4 and ethylenedioxy-P4 blocked glucosylceramide synthase activity at concentrations that had little effect on 1-O-acylceramide synthase activity. These novel inhibitors resulted in the inhibition of glycosphingolipid synthesis in cultured cells at concentrations that did not significantly raise intracellular ceramide levels or inhibit cell growth. GlcCer 1The abbreviations used are: GlcCer, glucosylceramide; PDMP, 1-phenyl-2-decanoylamino-3-morpholino-1-propanol; P4, 1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol; HPLC, high performance liquid chromatography; MDCK, Madin-Darby canine kidney. is the precursor of hundreds of different glycosphingolipids. This cerebroside is synthesized from uridine diphosphate-glucose and ceramide by a glucosyltransferase, GlcCer synthase. GlcCer-based sphingolipids have been identified as important mediators of a variety of cellular functions, including proliferation, differentiation, development, and cell-cell recognition (1Hakomori S. Igarashi Y. Adv. Lipid Res. 1993; 23: 147-162Google Scholar). The (R,R)-(d-threo)-isomer of 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) and its homologues are potent inhibitors of GlcCer synthase. These compounds have been used extensively to study the metabolism and function of glycosphingolipids in living cells (2Inokuchi J. Radin N.S. J. Lipid Res. 1987; 28: 565-571Abstract Full Text PDF PubMed Google Scholar, 3Abe A. Inokuchi J. Jimbo M. Shimeno H. Nagamatsu A. Shayman J.A. Shukla G.S. Radin N.S. J. Biochem. (Tokyo). 1992; 111: 191-196Crossref PubMed Scopus (142) Google Scholar, 4Abe A. Radin N.S. Shayman J.A. Wotring L.L. Zipkin R.E. Sivakumar R. Ruggieri J.M. Carson K.G. Ganem B. J. Lipid Res. 1995; 36: 611-621Abstract Full Text PDF PubMed Google Scholar, 5Rani C.S.S. Abe A. Chang Y. Rosenwzeig N. Saltiel A.R. Radin S.A. Shayman J.A. J. Biol. Chem. 1995; 270: 2859-2867Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 6Abe A. Radin N.S. Shayman J.A. Biochim. Biophys. Acta. 1996; 1299: 331-341Google Scholar). In previously reported work, a series of PDMP homologues and analogues was synthesized (4Abe A. Radin N.S. Shayman J.A. Wotring L.L. Zipkin R.E. Sivakumar R. Ruggieri J.M. Carson K.G. Ganem B. J. Lipid Res. 1995; 36: 611-621Abstract Full Text PDF PubMed Google Scholar). Replacing the decanoyl moiety with a palmitoyl moiety enhanced the effectiveness of PDMP. In addition, replacing the morpholino ring with a pyrrolidino ring, formingdl-threo-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (dl-threo-P4), also enhanced the inhibitory activity. It was also noted that the dl-threo-P4 derivative possessing a p-methoxy substituent on the phenyl group increased the inhibitory activity further. This latter observation led to the present study, an evaluation of other phenyl group substitutions in which the phenyl group of the P4 compound was modified by various electron-donating or -withdrawing groups. As expected, only the d-threo-enantiomers among P4 or P4 derivatives specifically inhibited the enzyme activity. The potency of these compounds in inhibiting GlcCer synthase was quantitatively related to the hydrophobic and electronic properties of the phenyl group substitutions of single substituents. This association resulted in the design of a new PDMP homologue (4′-hydoxy-P4) that was significantly more potent than those studied to date. The acetophenones and amines were from Aldrich, Lancaster Synthesis Inc., and Maybridge Chemical Co. Silica gel for column chromatography (70–230 mesh ASTM) and silica gel thin-layer chromatography plates were purchased from Merck. The reagents and their sources included non-hydroxy fatty acid ceramide from bovine brain and delipidated bovine serum albumin from Sigma, dioleoylphosphatidylcholine from Avanti, dl-dithiothreitol from Calbiochem, and uridine diphosphate-[1-3H]glucose from NEN Life Science Products. Octanoylsphingosine was prepared as described previously (7Abe A. Wu D. Shayman J.A. Radin N.S. Eur. J. Biochem. 1992; 210: 765-773Crossref PubMed Scopus (48) Google Scholar). The aromatic inhibitors were synthesized by the Mannich reaction from 2-N-acylaminoacetophenone, paraformaldehyde, and pyrrolidine, followed by reduction with sodium borohydride as described previously (2Inokuchi J. Radin N.S. J. Lipid Res. 1987; 28: 565-571Abstract Full Text PDF PubMed Google Scholar, 4Abe A. Radin N.S. Shayman J.A. Wotring L.L. Zipkin R.E. Sivakumar R. Ruggieri J.M. Carson K.G. Ganem B. J. Lipid Res. 1995; 36: 611-621Abstract Full Text PDF PubMed Google Scholar). The reaction produced a mixture of four isomers, due to the presence of two asymmetric carbons. For syntheses in which phenyl-substituted starting materials were used, the chloroacetophenone, methoxyacetophenone, methylenedioxyacetophenone, and methylacetophenone were brominated and converted to the primary amine. Brominations of the methoxyacetophenone, dimethoxyacetophenone, and 3′,4′-(methylenedioxy)acetophenone were performed in chloroform at room temperature, and the products were recrystallized from ethyl acetate and hexane. The synthesis of 1-(4′-hydroxy)phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol is described in detail in Fig. 1. This synthesis differed from those of the other compounds because of the need for the placement of a protecting group on the free hydroxyl (product 2) and its subsequent removal (product 8). All other syntheses employed a similar synthetic scheme. 4′-Hydroxyacetophenone (compound 1; 13.62 g, 100 mmol), benzyl bromide (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 F of 0.42 was observed when resolved by thin-layer chromatography using methylene chloride. 1H NMR (ppm, CDCl3) δ 7.94 (2H, d, 8.8 Hz, O-Ar-C(O)), 7.42 (5H, m,Ar′CH2O-), 7.01 (2H, d, 8.8 Hz, O-Ar-C(O)), 5.14 (2H, s, Ar′CH2O-), and 2.56 (3H, s, CH3). Bromine (80 mmol) was added dropwise over 5 min to a stirred solution of 4′-benzyloxyacetophenone (70 mmol) in 40 ml of 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 MgSO4, 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. AnR F of 0.62 was observed when resolved by thin-layer chromatography using methylene chloride. 1H NMR (ppm, CDCl3) δ 7.97 (2H, d, 9.2 Hz, O-Ar-C(O)), 7.43 (5H, m, Ar′CH2O-), 7.04 (2H, d, 9.0 Hz, O-Ar-C(O)), 5.15 (2H, s, Ar′CH2O-), and 4.40 (2H, s, CH2Br). Hexamethylenetetramine (methenamine; 3.8 g, 23 mmol) was added to a stirred solution of 2-bromo-4′-benyloxyacetophenone (6.8 g, 23 mmol) in 100 ml of chloroform. After 4 h, the crystalline adduct was filtered and washed with chloroform. The product was dried and heated with 150 ml of methanol and 8 ml of concentrated HCl in an oil bath at 85 °C for 3 h. Upon cooling, the precipitated hydrochloride salt (2.5 g) was removed by filtration. The filtrate was left at −20 °C overnight, and additional product (2.1 g) was isolated. The yield was 4.6 g (82.6%). [M + H]+: 242 for C15H16NO2. 1H NMR (ppm, CDCl3) δ 8.38 (2H, bs, NH2), 7.97 (2H, d, 8.8 Hz, O-Ar-C(O)), 7.41 (5H, m,Ar′CH2O-), 7.15 (2H, d, 8.6 Hz, O-Ar-C(O)), 5.23 (2H, s, Ar′CH2O-), and 4.49 (2H, s, CH2NH2). 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 of a and 150 ml) was added to the organic which was washed with water ml). The aqueous was with chloroform ml). The organic were and until dryness. The was in chloroform ml) and by the of hexane ml). The was to 4 °C for h. The were washed with and dried in a overnight. The product yield was g AnR F of was observed when resolved by thin-layer chromatography using methylene chloride. [M + H]+: for 1H NMR (ppm, CDCl3) δ (2H, d, 8.8 Hz, O-Ar-C(O)), (5H, m, Ar′CH2O-), (2H, d, 8.8 Hz, O-Ar-C(O)), bs, NH2), 5.14 (2H, s, Ar′CH2O-), (2H, s, (2H, Hz, (2H, m, m, and (3H, Hz, g, mmol), g, to of mmol), and (70 ml) were stirred HCl ml) was added the and the mixture was heated to for h. The brown solution was on and sodium borohydride g, mmol) was added in three The mixture was stirred at room temperature overnight, and the product was dried in a The was in ml) and with 3 HCl The aqueous was with ml). The organic layers were washed with water ml) and with saturated sodium chloride and dried over The solution was to a and purified by of a silica using a of methanol in This a mixture g, F of for and for were observed when resolved by thin-layer chromatography using chloride [M + H]+: for A of in acid ml) was stirred at room temperature a for 15 mmol) was and the solution was stirred overnight. The was filtered a and the was with chloride 5 ml). The filtrate was concentrated in and to yield a An R F of was observed when resolved by thin-layer chromatography using chloride [M + H]+: for NMR (ppm, CDCl3) δ d, Hz, d, m, (2H, m, m, m, (3H, m, and m, (2H, m, m, and (3H, Hz, Hexamethylenetetramine g, mmol) was added to a stirred solution of bromide g, mmol) in ml of chloroform. After h, the crystalline adduct was filtered and washed with chloroform. The product was dried and heated with methanol (200 ml) and concentrated HCl ml) in an oil bath at 85 °C for h. cooling, the precipitated chloride was removed by and the filtrate was left in a overnight. After the HCl was washed with and with The yield of product was g HCl g, mmol) and tetrahydrofuran ml) were in a with a Sodium acetate (50% in water, ml) was added in three portions to Palmitoyl chloride mmol) in tetrahydrofuran (25 ml) was added dropwise over 20 min to yield a dark brown solution. This mixture was stirred for an additional at room temperature. The mixture was a to the aqueous solution. 150 ml) was added to the organic and washed with water ml). The aqueous was with chloroform ml). The organic were and until The was in chloroform ml) and by the of hexane ml). The was to 4 °C for h. The were filtered and washed with hexane until were white and dried in a overnight. The yield of the product was g, mmol), g, mmol), mmol), and ml) were added to a HCl ml) was added to mixture the and the mixture was for h. The brown solution was in an Sodium borohydride g, mmol) was added in three This mixture was stirred at room temperature for 3 and The was in ml of and the was with HCl until the pH was The aqueous was with ml of The organic layers were washed with ml) and saturated and dried over The solution was to a which was purified by of a with a of methanol in to a mixture of and g, 4 performance liquid chromatography of was performed using a column 20 eluted with acid at a of 8 The column was at in the and The products were until by using an column (4.6 and the mixture at a of The enzyme activity was as described previously Shukla A. Inokuchi J. Radin N.S. Biochim. Biophys. Acta. PubMed Scopus Google Scholar). Madin-Darby canine cell of were with uridine and of 85 of of and 100 of sodium in a reaction mixture and for at P4 and P4 derivatives in was the reaction mixture the The of was was inhibition of the enzyme activity. cells were 8 ml of modified J.A. S. Inokuchi J. Radin N.S. J. Biol. Chem. Full Text PDF PubMed Google Scholar). After h, the was with 8 ml of the or The GlcCer synthase inhibitors were added to the as a with delipidated bovine serum albumin (4Abe A. Radin N.S. Shayman J.A. Wotring L.L. Zipkin R.E. Sivakumar R. Ruggieri J.M. Carson K.G. Ganem B. J. Lipid Res. 1995; 36: 611-621Abstract Full Text PDF PubMed Google Scholar, 6Abe A. Radin N.S. Shayman J.A. Biochim. Biophys. Acta. 1996; 1299: 331-341Google Scholar). The cells were for or with the After the the cells were washed with 8 ml of and with ml of The cells were and to a An additional ml of methanol was used to the cells in the ml of chloroform were added to the and using a water bath After at for 5 min, the was The were with After the the was with the first The were and for by the acid was added to the and the of was to After at for 5 min, the was The was washed with in an to that of the The was a and dried a of A of the was used for Scopus Google Scholar). The was using high performance thin-layer The of the parent compound, and the phenyl-substituted homologues including the new and homologues are in Fig. the effect of each P4 by chromatography on GlcCer synthase activity was were observed for the of and the and 3 and 4 the as by a of the P4 mixture by chromatography followed by the The enzyme activity was specifically inhibited by the the This for was with the observed in PDMP and PDMP homologues (2Inokuchi J. Radin N.S. J. Lipid Res. 1987; 28: 565-571Abstract Full Text PDF PubMed Google Scholar, 3Abe A. Inokuchi J. Jimbo M. Shimeno H. Nagamatsu A. Shayman J.A. Shukla G.S. Radin N.S. J. Biochem. (Tokyo). 1992; 111: 191-196Crossref PubMed Scopus (142) Google Scholar, 4Abe A. Radin N.S. Shayman J.A. Wotring L.L. Zipkin R.E. Sivakumar R. Ruggieri J.M. Carson K.G. Ganem B. J. Lipid Res. 1995; 36: 611-621Abstract Full Text PDF PubMed Google Scholar). The IC50 of was for GlcCer synthase activity in the cell of P4 on GlcCer synthase of P4 or were by chromatography as described The were in the or presence of concentrations of P4 In work, the of a p-methoxy group was found to the effect of the inhibitor on the enzyme activity (4Abe A. Radin N.S. Shayman J.A. Wotring L.L. Zipkin R.E. Sivakumar R. Ruggieri J.M. Carson K.G. Ganem B. J. Lipid Res. 1995; 36: 611-621Abstract Full Text PDF PubMed Google Scholar). This was for the The enzyme activity was inhibited chromatography of the four to to each A inhibition of the enzyme activity in a mixture and was have been due to of these is not on the a series of derivatives other single on the phenyl group was The potency of these phenyl-substituted compounds as GlcCer synthase inhibitors was to that For the effect of aromatic substitutions on the activity has been and is the be described by the H. A of and a + + + are the between the electronic and properties of phenyl-substituted compounds and the inhibition of GlcCer + were from the in for and for and for and for and were derived from Fig. For other the as in Fig. 3 was to the IC50 not were derived from Fig. For other the as in Fig. 3 was to the IC50 not These were from the in H. A of and for and for and for and for and These were derived from Fig. For other the as in Fig. 3 was to the IC50 not in a new The hydrophobic effect (π) is described by the is the of the derivative and is that of the parent compound, as the between and The electronic substituent (ς) was by and is as are the for or derivative and and electron-donating The potency of and P4 derivatives as inhibitors is dependent upon two and electronic of a substituent of the phenyl group a relationship was observed between and + These that the more the of + the more potent derivatives as GlcCer synthase The association between + and GlcCer synthase inhibition that a more potent inhibitor be produced by the electron-donating properties and the lipophilic properties of the phenyl group A + be observed for the homologue. This compound was synthesized and was by An IC50 of 90 for GlcCer synthase inhibition was observed that thep-hydroxy homologue was as active as the relationship between and + was the of similar more derivatives with and substitutions on the phenyl group were designed The enzyme activity was inhibited by with an IC50 of 100 the other the IC50 were and was to these even or as an inhibitor As the and for the and and for a single substituent are and H. A of and the of + is to be based only on the electronic substituent from the observed in Fig. This be due to a between two in the that a effect that was in derivatives studied in Fig. GlcCer synthase is to a that and PDMP homologues and that the enzyme activity (2Inokuchi J. Radin N.S. J. Lipid Res. 1987; 28: 565-571Abstract Full Text PDF PubMed Google Scholar, 6Abe A. Radin N.S. Shayman J.A. Biochim. Biophys. Acta. 1996; 1299: 331-341Google Scholar). The effect by an additional group or more of these As a the potency of the homologue as a GlcCer synthase inhibitor was on PDMP and related homologues that the were of cell ceramide and inhibiting cell the observation that only the d-threo-enantiomers blocked GlcCer synthase (4Abe A. Radin N.S. Shayman J.A. Wotring L.L. Zipkin R.E. Sivakumar R. Ruggieri J.M. Carson K.G. Ganem B. J. Lipid Res. 1995; 36: 611-621Abstract Full Text PDF PubMed Google Scholar). An for ceramide metabolism was subsequently identified of ceramide at the that was blocked by of PDMP. The for GlcCer synthase were studied by the was a in activity in inhibiting GlcCer synthase, of three compounds demonstrated activity in blocking 1-O-acylceramide synthase inhibition of 1-O-acylceramide synthase was the for ceramide the ceramide and levels of cells were in ceramide and only in cells with inhibitor concentrations in of This was than the for inhibition of the 1-O-acylceramide synthase in the cellular This in the of the more potent homologues to cells A. Radin N.S. Shayman J.A. Biochim. Biophys. Acta. 1996; 1299: 331-341Google and of cells with by by by by by by by by by by by by cells were a and for h. the the cells were for or with or P4 or the phenyl-substituted ceramide and were by the of J. R. J. Biol. Chem. Full Text PDF PubMed Google Scholar). GlcCer was from glycosphingolipids by a and of as previously described J.A. S. Inokuchi J. Radin N.S. J. Biol. Chem. Full Text PDF PubMed Google Scholar). only two were by in a new cells were a and for h. the the cells were for or with or P4 or the phenyl-substituted ceramide and were by the of J. R. J. Biol. Chem. Full Text PDF PubMed Google Scholar). GlcCer was from glycosphingolipids by a and of as previously described J.A. S. Inokuchi J. Radin N.S. J. Biol. Chem. Full Text PDF PubMed Google Scholar). only two were made. the cellular as with cells were with different concentrations of the and of the GlcCer was from the cells with and only depletion of GlcCer in cells A of GlcCer in the cells with a or of This be due to the by of more sphingolipids or the of GlcCer synthase that is to the the other was little in the between cells and those with or A in was observed in the cells with a of P4 homologue. In addition, the of ceramide in the cells with was two than that in the cells was in ceramide or levels in cells with and concentrations of for GlcCer levels and were observed not The was at the concentrations of The of cell to cellular of of were and for and and and for and that the in GlcCer were related to inhibition of GlcCer synthase activity. These that two new are to and specifically inhibit GlcCer synthesis in cells at concentrations inhibition of cell growth. the of an inhibitor of GlcCer synthesis by and Radin Radin N.S. Chem. PubMed Scopus Google the of glycosphingolipid synthesis has to be a to the metabolism and function of glycosphingolipids. Previous work with the parent GlcCer synthase inhibitor (PDMP) identified two in These included the depletion of GlcCer-based glycosphingolipids and the of the ceramide was to be the of was that of compounds cell ceramide of GlcCer depletion (4Abe A. Radin N.S. Shayman J.A. Wotring L.L. Zipkin R.E. Sivakumar R. Ruggieri J.M. Carson K.G. Ganem B. J. Lipid Res. 1995; 36: 611-621Abstract Full Text PDF PubMed Google Scholar). In addition, homologues with substitutions GlcCer with on ceramide levels. The with the new phenyl-substituted inhibitors are with inhibition was observed at concentrations of was observed at concentrations at which ceramide levels These a of for these inhibitors that is of the inhibition of GlcCer synthase. The for of inhibition of ceramide metabolism in the presence of of P4 led to the identification of a novel for ceramide the of ceramide at the The of is by a novel In the presence of ceramide as an 1-O-acylceramide synthase ceramide the acid of or A. Shayman J.A. Radin N.S. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). This has been purified A. Shayman J.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). using of inhibitors that ceramide to the of blocking GlcCer has been that the inhibitory of these homologues are by ceramide and not GlcCer The of GlcCer depletion from ceramide is an important and is to the of GlcCer synthase inhibitors as for glycosphingolipid a little or cellular The inhibitory and of ceramide be for the of by inhibition of GlcCer synthase was reported by the of the in with a inhibitor of GlcCer B. PubMed Scopus Google Scholar). This inhibitor is significantly potent and than the compounds in the present Previous of the parent of PDMP have resulted in compounds with activity GlcCer synthase. these substitutions of the fatty and were designed In the present study, single phenyl substitutions the of inhibitor activity based on derived by and Adv. Res. Scholar). of and were observed to be of inhibitory activity. the design and synthesis the potent GlcCer synthase inhibitor to date. These be only to substitutions and were to the activity of more ring the of these in synthase inhibitors with even activity and the of Radin in