currently enormous costs are associated with enzyme therapy and the efficacy of this approach proves to be intra - individually highly variable . the present alternatives for therapeutic intervention either can be applied only for a limited number of cases ( bone marrow transplantation ), or have in fact not yet been shown to be effective and safe ( gene therapy and substrate deprevation ). this has prompted us to search for a novel option for therapeutic intervention that may be used in addition to enzyme therapy . according to our view on the pathogenesis of gaucher disease ( see fig3 ), the activation of macrophages that leads to the release of hydrolases and cytokines forms an ideal target for intervention . an agent that is capable of preventing the activation of gaucher cells should have a therapeutic value , and moreover should be able to augment the efficacy of enzyme therapy . in order to be able to develop the desired therapeutic agent , first the mechanism by which gaucher cells are driven in their characteristic activated state had to be elucidated . two crucial findings were made by us in the course of our investigations that allowed us to develop the envisioned agent . in the first place , we discovered a sensitive marker for the characteristic activated state of gaucher cells , i . e . the massive synthesis and secretion of chitotriosidase by these cells ( see above ). importantly , the potential of agents to prevent the relevant activation of macrophages can be sensitively tested by the analysis of their capacity to inhibit the production and secretion of chitotriosidase by macrophages in cell culture . in the second place , we discovered that human cells contain , besides the lysosomal glucocerebrosidase , a distinct enzyme that is capable of hydrolyzing glucosylceramide into glucose and ceramide [ 17 ]. the glucosylceramidase differs in many respects from the lysosomal glucocerebrosidase . the enzyme is not located in lysosomes in contrast to glucocerebrosidase ; it is not deficient in gaucher disease in contrast to glucocerebrosidase ; it behaves as an integral membrane protein whilst glucocerebrosidase shows the features of a membrane - associated protein ; and finally , it differs from glucocerebrosidase in specificity towards artificial substrates , inhibitors and activators . for example , the glucosylceramidase is not able to hydrolyze artificial b - xylosidic substrates contrary to glucocerebrosidase . glucocerebrosidase is irreversibly inhibitable by conduritol b - epoxide in contrast to the glucosylceramidase that is insensitive for this compound . the lysosomal activator protein saposin c potently stimulates glucocerebrosidase in its enzymatic activity but is without effect on the glucosylceramidase . concerning the function of glucosylceramidase , an important observation was made . using a relatively novel technique for subcellular fractionation it was found that the glucosylceramidase is present at the plasma membrane or in early endosomal structures . in other words , the enzymatic activity of the glucosylceramidase results in the generation of ceramide in the plasma membrane or specific invaginations of this membrane . it is known that significant amounts of glucosylceramide are indeed present in the plasma membrane . consequently the activity of glucosylceramidase might result in relevant changes in ceramide concentration in those cellular membranes that are involved in ceramide - mediated signalling . it was furthermore observed using membrane suspensions prepared from cells and tissues that the ceramide formed from glucosylceramide by the activity of the lysosomal glucocerebrosidase is hardly converted into sphingomyelin ; in sharp contrast to this is the efficient conversion of the ceramide formed by the action of glucosylceramidase into sphingomyelin . apparently , the ceramide generated by the glucosylceramidase activity is rapidly further metabolized within the same membranes , as can be expected for a lipid that acts as transient second messenger . on the basis of these findings we postulate a new mechanism for the pathological activation of macrophages in gaucher disease . in this model , as depictured in fig4 it is proposed that the lysosomal impairment to degrade glucosylceramide in glucocerebrosidase - deficient individuals also leads to an increased concentration of this glycolipid in membranes containing the glucosylceramidase . consequently , the glycolipid is there at an abnormal high rate hydrolysed to ceramide . this ceramide activates protein kinases and phosphatases , resulting in the characteristic activation of the macrophage and the corresponding production and release of pathogenetic factors . experimental proof for this model in which the constitutively stimulated glucosylceramidase activity promotes macrophage activation is described below . the glucosylceramidase is an ideal and novel target for prevention of the activation of macrophages in gaucher disease . specific inhibition of the enzyme activity would prevent further release of pathogenetic factors and disrupt the pathological cascade , resulting in a therapeutic effect . it may be envisioned that the combination of this approach with that of enzyme supplementation can improve markedly the efficacy of therapeutic intervention and meanwhile will result in a significant reduction of associated costs . the properties of the glucosylceramidase present in membrane suspensions and intact cells were carefully analysed in order to identify a suitable inhibitor for the enzyme . a number of important findings were made in this connection . it was observed that the enzyme is tightly integrated in the membrane and most likely hydrolyzes its substrate glucosylceramide while this is also inserted in the membrane . the identification of the location of the glucosylceramidase in ( invaginations of ) the plasma membrane is also of importance . furthermore , a number of known glucosidase inhibitors ( d - gluconolacton , castanospermine , deoxynojirimycin and butyl - deoxynojirimycin ) were tested on their capacity to inhibit the glucosylceramidase activity . the most promising inhibitors were deoxynojirimycin and in particular butyl - deoxynojirimycin . however their specificity as well as that of the other inhibitors tested was poor . for example , the lysosomal glucocerebrosidase is also quite sensitive to the inhibitors , rendering them unattractive for administration to the already glucocerebrosidase - deficient gaucher patients . the inhibitors would moreover seriously interfere with enzyme therapy of patients due to their inhibitory effect on the administered alglucerase or imiglucerase . it was noted that incubation of intact cells with deoxynojirimycin or butyl - deoxynojirimycin at their ic50 concentration for glucosylceramidase ( 20 and 0 . 3 um , respectively ), resulted also in a significant inhibition of glucocerebrosidase activity ( about 20 and 10 %, resp .) and in an inhibition of glucosylceramide synthase activity ( about 30 and 20 %, respectively ). in the same concentration range a marked inhibition of er a - glucosidase i activity ( n - linked glycan trimming ) has also been reported for several cell types [ 15 ]. the negative results with known glucosidase inhibitors prompted us to design a novel , more specific inhibitor for glucosylceramidase , exploiting the generated information on the features of the enzyme . it was conceived that the desired potent and specific inhibitor for the glucosylceramidase should have the following features : n - alkylation of deoxynojirimycin was found to increase its capacity to inhibit glucosylceramidase . 3 — coupled to the spacer a large hydrophobic group that tends to insert in a lipid bilayer , preferably ( invaginations of ) the plasma membrane ; preferential insertion of the inhibitor in glucosylceramidase - containing membranes should increase the in vivo capacity and specificity of the inhibitor . a series of deoxynojirimycin - derivatives was made by chemical synthesis in order to test the concept and develop the ideal inhibitor for the glucosylceramidase . based on the abovementioned features , a series of deoxynojirimycin ( dnm ) derivatives of the following type were synthesized ( fig1 ): in this structure , x is a saturated alkane chain and r is a large apolar group . compounds of this type can be synthesized by reacting dnm . hcl , which is readily available in seven steps from the commercially available tertabenzyl - glucopyranose [ 18 ], with the appropriate aldehyde , in a reductive amination [ 19 ] reaction ( scheme 1 ). the strategy is exemplified by the synthesis of the following two compounds : n -( 5 - cholesteroloxy - pentyl )- deoxynojirimycin 9 and n -( 5 - adamantane - 1 - yl - methoxy - pentyl )- deoxynojirimycin 10 ( scheme 2 ). thus , glutaric aldehyde 1 was first converted into the monoacetal 2 [ 20 ] using an ion exchange catalyst . after reduction of the monoacetal to the corresponding alcohol 3 and transformation to the mesviate 4 , reaction with the appropriate alcohol , in which roh is cholesterol and adamantanemethanol respectively , under basic conditions , afforded the acetals 5 and 6 . after liberation of the second aldehyde function , that is formation of compounds 7 and 8 , reductive amination with dnm . hcl afforded the target compounds 9 and 10 . the inhibitory effects of the various deoxynojirimycin - analogues on relevant enzyme activities were analysed in vitro and in intact cells . firstly , in vitro experiments were performed with purified human enzymes and membrane suspensions of human tissue . attention was focussed to the inhibition of lysosomal glucocerebrosidase and glucosylceramidase activities and that of the lysosomal a - glucosidase . as source for glucosylceramidase were used a membrane fraction from human spleen . the glucosylceramidase activity was measured as the hydrolytic activity towards 4mu - b - glucoside in the membrane suspension that was pre - treated with conduritol b - epoxide to abolish the activity of glucocerebrosidase . as source of glucocerebrosidase served human placental enzyme ( ceredase , genzyme corp . boston , usa ) that is used in enzyme therapy . alternatively , glucocerebrosidase activity was determined in a membrane fraction from human spleen . glucocerebrosidase activity was measured as the hydrolysis of 4mu - b - glucoside that is inhibitable by conduritol b - epoxide . lysosomal a - glucosidase activity was measured as the hydrolytic activity towards 4mu - a - glucosidase shown by a purified a - glucosidase preparation . tables 6 and 7 show the structures of the tested compounds . table 3 gives an overview of the apparent ki values of the inhibitors . it can be seen in table 3 that glucosylceramidase is potently inhibited by n - alkyl derivatives of deoxynojirimycin . optimal inhibition was noted for the n - pentyl derivative . n - hexyl - deoxynojirimycin was a less potent inhibitor ( not shown in table 3 ). the presence of a carbonyl moiety in the spacer negatively influences the inhibitory capacity . coupling of a large hydrophobic group such as adamantane p21 ) or cholesterol ( p24 ) to a n - pentyl spacer dramatically increases the capacity of the compound to inhibit the glucosylceramidase activity . ic50 values were also determined in the case of some inhibitors . the apparent ic50 values for p21 and p24 are extremely low , 1 nm and 0 . 1 um , respectively . for a comparison , the ic50 values for deoxynojirimycin and butyl - deoxynojirimycin are 20 um and 0 . 3 um , respectively . table 3 shows that glucocerebrosidase is in general less sensitive to deoxynojirimycin derivatives than glucosylceramidase . pure glucocerebrosidase in solution ( ceredase ) and enzyme associated to membranes show a different sensitivity for the inhibitors . apparently , the kinetic properties of the enzyme in these two different states differ , as is also suggested by the difference in apparent km for 4mu - b - glucoside . both the soluble and membrane - associated glucocerebrosidase are most potently inhibited by deoxynojirimycin - analogues with a n - pentyl spacer and coupled to it a large hydrophobic group . for soluble glucocerebrosidase ( ceredase ) the apparent ics50 value of p21 was 0 . 2 um , and that of p24 was 0 . 8 um ; for the membrane - associated glucocerebrosidase the apparent ic50 values of p21 and p24 were 0 . 06 and 0 . 7 um , resp . with respect to the lysosomal a - glucosidase it was found that substitutions in deoxynojirimycin generally exerted relatively little effect . however , the compounds p4 , p11 , p16 , p9 and p13 were very poor inhibitors . next , the capacity of the deoxynojirimycin - analogues to inhibit the glucosylceramidase and the glucocerebrosidase activities in intact cells was investigated . enzyme activities were measured as described in ref . 17 . briefly , the hydrolysis of 4mu - b - glucoside by cultured melanoma cells that were pre - incubated with and without conduritol b - epoxide was determined . the conduritol b - epoxide sensitive activity can be ascribed to glucocerebrosidase and the insensitive activity to glucosylceramidase . the results of this study are shown in table 4 . a comparison of table 3 and table 4 reveals that the inhibition by deoxynojirimycin analogues of the glucosylceramidase activity in intact melanoma cells is similar to that observed in in vitro experiments using splenic membrane preparations . the most potent inhibitors are p21 and p24 with ic50 values of about 0 . 3 nm and 50 nm . at these or ten - fold higher concentrations no significant inhibition of the glucocerebrosidase activity is detectable , see table 4 . the inhibitory constants of deoxynojirimycin analogues were also determined by analysis of the metabolism of c6 - nbd glucosylceramide in melanoma cells , employing again conduritol b - epoxide to discriminate between the activities of the insensitive glucosylceramidase and the sensitive glucocerebrosidase . the results obtained with c6 - nbd glucosylceramide as substrate were almost identical to those obtained with the fluorogenic 4mu - b - glucoside substrate ( not shown ). it was studied to which extent other reactions were inhibited by incubating cells with p21 or p24 at their ic50 concentration for the glucosylceramidase activity . under these conditions no inhibition of glycogen synthase was noted in rat hepatocytes ; no inhibition of glucosylceramide synthase activity or lysosomal a - glucosidase was noted in cultured melanoma cells . because of the extreme sensitivity of the glucosylceramidase for p21 , it was examined whether the inhibition by this compound might be not reversible . to test this , melanoma cells were preincubated with or without p21 , and subsequently washed extensively . next , the glucocerebrosidase and glucosylceramidase activities were determined with 4mu - b - glucoside as substrate . it was found that the pretreatment with inhibitor was without significant effect on the glucocerebrosidase activity , but led to an irreversible loss of the glucosylceramidase activity . proof of concept : value of deoxynojirimycin analogues for intervention in macrophage activation the effect of p21 ( n -( 5 - adamantane - 1 - yl - methoxy - pentyl ) deoxynojirimycin ) and butyldeoxynojirimycin on macrophages in culture was examined . the iminosugars , dissolved in dmso at a concentration of 10 mm , were added to cultured macrophages at various concentrations by dilution in culture medium . it was checked that the minor amounts of dmso introduced in this manner were without effect . table 5 shows the inhibition by the deoxynojirimycin analogues of the glucocerebrosidase and glucosylceramidase activities in macrophages , as measured with c6 - nbd glucosylceramide as substrate ; the effects are quite comparable to those noted for the enzymes in melanoma cells . table 5 shows furthermore the effect of the deoxynojirimycin analogues on the secretion of chitotriosidase by the cells . it can be seen that chitotriosidase secretion is reduced concomitantly with inhibition of the activity of glucosylceramidase , but not of that of the lysosomal glucocerebrosidase . using c6 - nbd ceramide as substrate , glucosylceramide synthase activity in cultured macrophages was also determined . it was noted that this enzyme activity is not significantly inhibited by the presence of 5 um butyldeoxynojirimycin in the culture medium , a condition causing a reduced chitotriosidase secretion . moreover it was found that inhibition of glucosylceramide synthase by the presence of pdmp or ppmp was without effect on chitotriosidase secretion . in conclusion , the experiments show that low concentrations of butyldeoxynojirmycin and particularly of p21 are able , by virtue of their specific inhibition of glucosylceramidase activity , to de - activate macrophages that massively secrete chitotriosidase ( and concomitantly other hydrolases and cytokines ). thus , experimental proof of concept has been obtained . one application for the newly developed , highly specific inhibitors is to be found in therapeutic intervention of gaucher disease . as discussed above , the effects of the inhibitors on macrophage activation may be expected to favourably interfere with the pathogenesis of gaucher disease . the administration of inhibitors may improve the efficacy of enzyme therapy , and consequently result in an improved clinical response and a marked reduction of associated costs . a beneficial effect might also be exerted by inhibitors of glucosylceramidase activity in the case of other disease states that are characterized by elevated plasma chitotriosidase , such as the niemann - pick disease and sarcoidosis [ 4 , 5 ]. furthermore it is known to us that foam cells in atherosclerosis are over - producing chitotriosidase . it is likely that ceramide - mediated signalling processes are directly or indirectly effected by inhibition of glucosylceramidase activity . the potential applications for the developed inhibitors can therefore be extremely diverse . of particular interest are inflammatory states that are provoked by tnf - α or other proinflammatory cytokines that signal through ceramide . examples in this connection are septic shock , rheumatoid arthritis and crohn &# 39 ; s disease . the selection of a suitable route of administration and suitable formulations of pharmaceutical compositions is within the normal skills of the persons skilled in the art . examples of suitable administration routes are parenteral ( intravenous , subcutaneous , intramuscular ) injections or infusions , oral ingestion , and topical application . in particular attractive is the use of oily vehicles allowing slow and sustained release from the repository preparation . the use of an oily vehicle will not be feasible in the case of oral ingestion or intravenous administration . in the case of oral ingestion , absorption of the lipophilic compound will occur spontaneously in the gastro - intestinal tract upon the solubilization of the compound in mixed micelles followed by passive diffusion across the enterocyte membrane . in the case of intravenous administration use can be made of liposomes in which the lipophilic compound has prior been incorporated . a mixture of 5 , 5 - diethoxy - pentanal 1 ( 3 g , 17 mmol ) and nabh 4 ( 0 . 65 g , 17 mmol ) in 30 ml etoh was stirred at room temperature for 3 h . the solvent was evaporated and the residue was triturated with 10 % naoh and extracted with ch 2 cl 2 . the organic layers were collected , dried ( na 2 so 4 ) and the solvent evaporated to give 2 purified by silica gel flash chromatography eluting with petroleum ether 60 - 80 / ethyl acetate 1 : 1 ( yield 59 %). 1h nmr ( cdcl 3 ): d 4 . 47 ( t , 1h , j = 5 . 7 hz , c - 1 ), 3 . 70 - 3 . 58 ( m , 4h , c - 5 , ch 2 acetal ), 3 . 46 ( dq , 2h , j = 7 . 1 hz , 2 . 3 hz , ch 2 acetal ), 1 . 65 - 1 . 50 ( m , 4h , c - 2 , c - 4 ), 1 . 41 ( m , 2h , c - 3 ), 1 . 18 ( t , 6h , j = 7 . 1 hz , ch 3 acetal ). to an ice cooled solution of 2 ( 0 . 3 g , 1 . 7 mmol ) and triethylamine ( 0 . 21 g , 2 . 0 mmol ) in 3 ml ch 2 cl 2 , methanesulfonyl chloride ( 0 . 21 g , 1 . 9 mmol ) was added . after stirring for 1 h at rt the mixture was washed with water and the solvent , dried on na 2 so 4 , evaporated in vacuo to give 4 ( 0 . 43 g , 1 . 7 mmol , 100 %), which was used for the subsequent reaction without further purification . 1h nmr ( cdcl 3 ): d 4 . 47 ( dt , 1h , j = 5 . 5 hz , 2 . 5 hz , c - 1 ), 4 . 21 ( dt , 2h , j = 6 . 5 hz , 2 . 7 hz , c - 5 ), 3 . 63 ( m , 2h , ch 2 acetal ), 3 . 48 ( m , 2h , ch 2 acetal ), 3 . 00 ( s , 3h , oso 2 ch 3 ), 1 . 77 ( m , 2h , c - 2 ), 1 . 63 ( m , 2h , c - 4 ), 1 . 47 ( m , 2h , c - 3 ), 1 . 20 ( t , 6h , j = 7 . 0 hz , ch 3 acetal ). nah ( 60 % disp ., 0 . 108 g , 2 . 7 mmol ) was washed with pentane , and stirred with adamantanemethanol ( 0 . 3 g , 1 . 8 mmol ) in 5 ml dmf , for 1 h at rt yielding a suspension of the sodium salt . compound 4 ( 0 . 4 g , 1 . 6 mmol ) was added and the mixture was heated at 70 ° c . for 4 h and stirred at rt overnight . the mixture was treated with few drops of meoh , poured into ice and extracted with diethyl ether ( 3 × 15 ml ). the organic solvent , dried on na 2 so 4 , was evaporated and the residue purified by flash chromatography with petroleum ether 60 - 80 / ethyl acetate 7 : 3 giving the desired compound 5 as a viscous syrup . 5 : yield 34 %. 1h nmr ( cdcl 3 ): d 4 . 48 ( t , 1h , j = 5 . 7 hz , c - 1 chain ), 3 . 61 ( dq , 2h , j = 7 . 1 hz , 2 . 3 hz , ch 2 acetal ), 3 . 49 ( dq , 2h , j = 7 . 1 hz , 2 . 2 hz , ch 2 acetal ), 3 . 37 ( t , 2h , j = 6 . 5 hz , c - 5 chain ), 2 . 94 ( s , 2h , ch 2 adamant . ), 1 . 94 ( m , 3h , adamant . ), 1 . 73 - 1 . 51 ( m , 16h , c - 2 , c - 4 chain , adamant . ), 1 . 45 - 1 . 35 ( m , 2h , c - 3 chain ), 1 . 19 ( t , 6h , j = 7 . 1 hz , ch 3 acetal ). nah ( 60 % disp ., 0 . 12 g , 3 mmol ) was washed with pentane and heated with cholesterol ( 1 . 16 g , 3 mmol ) in dmf ( 6 ml ) at 65 - 70 ° c . during 45 min , yielding a suspension of the sodium salt . 5 , 5 - diethoxy - o - methanesulfonylpentanol 4 ( 0 . 508 g , 2 mmol ) was added and the mixture was heated at 70 - 75 ° c . during 20 h . the dmf was evaporated and the residue was extracted with ether and water . the ether extracts were dried ( na 2 so 4 ) and the residue after evaporation was purified by flash chromatography ( pe 60 / 80 — ethyl acetate 5 / 1 ) giving the product as a viscous syrup ( 0 . 63 g , 58 %). 1h nmr ( cdcl 3 ): d 5 . 31 ( m , 1h , c - 6 chol . ), 4 . 45 ( t , j = 5 . 5 hz , 1h , c - 1 chain ), 3 . 61 ( dq , 2h , j = 7 . 1 hz , 2 . 4 hz , ch 2 acetal ), 3 . 45 ( m , 4h , ch 2 acetal , c - 5 chain ), 3 . 10 ( m , 1h , c - 3 chol . ), 2 . 34 ( m , 1h , chol . ), 2 . 16 ( m , 1h , chol . ), 2 . 07 - 1 . 70 ( bm , 4h ), 1 . 70 - 0 . 75 ( bm , 46h , chol ., ch 3 acetal ), 0 . 67 ( s , 3h , ch 3 chol .). a mixture of the appropriate acetal 5 , 6 , ( 0 . 2 mmol ) in 3 ml acetone and 1 ml 5 % hcl was stirred at rt for 1h . evaporation of the acetone , extraction of the residue with ether ( 3 × 7ml ), drying on na 2 so 4 and evaporation yielded the aldehyde ( quant .) used for the next step without further purification . 7 : yield 100 %. 1h nmr ( cdcl 3 ): d 9 . 77 ( s , 1h , cho ), 5 . 33 ( m , 1h , c - 6 chol . ), 3 . 46 ( t , 2h , j = 6 . 1 hz , c - 5 chain ), 3 . 11 ( m , 1h , c - 3 chol . ), 2 . 46 ( dt , 2h , j = 7 . 2 hz , 1 . 4 hz , c - 2 chain ) 2 . 34 ( m , 1h , chol . ), 2 . 16 ( m , 1h , chol . ), 2 . 05 - 1 . 75 ( bm , 42h , c - 3 c - 4 chain , chol ), 0 . 67 ( s , 3h , ch 3 chol .). 8 : yield 100 %. 1h nmr ( cdcl 13 ): d 9 . 76 ( t , 1h , j = 1 . 7 hz , cho ), 3 . 38 ( t , 2h , j = 6 . 2 hz , c - 5 chain ), 2 . 94 ( s , 2h , ch 2 adamant . ), 2 . 46 ( dt , 2h , j = 7 . 2 hz , 1 . 7 hz , c - 2 chain ), 1 . 95 ( m , 3h , adamant . ), 1 . 80 - 1 . 45 ( m , 16h , c - 3 , c - 4 chain , adamant .). the aldehyde 7 ( 0 . 118 g , 0 . 25 mmol ) was dissolved in a small amount of hot ethyl acetate , diluted with ethanol ( about 4 ml ) and added to a solution of dnm which was prepared by stirring dnj . hcl ( 0 . 050 g , 0 . 2 mmol ) and sodium acetate ( 0 . 020 g , 0 . 25 mmol ) in methanol ( 0 . 4 ml ) at rt for 1 h . nacnbh 3 ( 0 . 016 g , 0 . 25 mmol ) was added at 0 ° c . and the suspension was stirred vigorously at rt during 18 h , and finally at 60 ° c . for 1 h . after cooling to rt the mixture was acidified ( ph & lt ; 1 ) with hcl ( 5 %), stirred during 1 h and evaporated to dryness in vacuo . the remaining solids were suspended in a mixture of ch 2 cl 2 and methanol ( 1 / 1 , 20 ml ) and methanolic ammonia ( 20 %, 2 ml ) was added followed by silica ( about 5 g ). the mixture was ( carefully ) evaporated in vacuo to a free flowing solid , which was applied to a column of silica , pretreated with the eluent : ch 2 cl 2 / meoh / nh 3 in meoh ( 20 %)= 80 / 15 / 5 . the column was eluted with 80 / 15 / 5 , 75 / 20 / 5 and 70 / 25 / 5 mixtures of this eluent , resp ., yielding pure 9 ( solid , 0 . 081 g , 0 . 13 mmol , 65 %) after evaporation . hydrochloride : compound 9 ( 0 . 050 g ) was dissolved in hot ethanol ( 20 - 30 ml ) and treated with 3 drops of conc . hcl . evaporation of the solvents yielded the hydrochloride as a crystalline solid ( quant . ), mp 235 - 238 ° c . ( sublimates from ca 190 ° c .). 1h nmr ( d 2 o ): d 5 . 36 ( m , 1h , c - 6 chol . ), 4 . 10 ( d , 1h , j = 12 . 6 hz , c - 6 dnm ), 3 . 92 ( d , 1h , j = 12 . 6 hz , c - 6 dnm ), 3 . 73 ( m , 1h , c - 2 dnj ), 3 . 61 ( m , 1h , c - 4 dnj ), 3 . 52 ( t , 2h , j = 6 . 2 hz , c - 5 chain ), 3 . 47 ( dd , jc - 1 / c - 1 = 12 . 1 , jc - 1 / c - 2 = 4 . 8 , c - 1 eq . dnm ), 3 . 39 ( m , 2h , c - 3 dnm , c - 1 chain ), 3 . 29 - 3 . 05 ( m , 3h , c - 1 chain , c - 3 chol ., c - 5 dnm ), 3 . 00 ( app . t , 1h , j = 11 . 6 hz , c - 1 ax . dnm ), 2 . 34 ( m , 1h , c - 4 eq chol . ), 2 . 15 ( m , 1h , c - 4 eq chol . ), 2 . 03 ( m , 1h , c - 2 chol . ), 1 . 97 - 0 . 80 ( bm , 43h , c - 2 c - 4 c - 3 chain , chol ), 0 . 72 ( s , 3h , ch 3 chol .). hrms ( fab ) obs mass 640 . 4979 ( mna + ), calcd for c 38 h 67 no 5 na 640 . 4917 ; obs mass 618 . 5086 ( mh + ), calcd for c 38 h 68 no 5 618 . 5097 . a solution of deoxynojirimycin hydrochloride ( 0 . 030 g , 0 . 15 mmol ) and few μl of ch 3 cooh in meoh ( 2 ml ) was added to 8 ( 0 . 056 g , 0 . 22 mmol ) in meoh ( 1 ml ) at 0 ° c ., followed by addition of nacnbh 3 ( 0 . 014 g , 0 . 22 mmol ). after stirring overnight at room temperature the reaction was concentrated , treated with 5 % hcl ( 2 ml ), stirred for 1 h at rt and solid na 2 co 3 was added . the aqueous suspension was extracted with ch 2 cl 2 ( 3 × 7ml ), the extracts combined , dried ( na 2 so 4 ) and evaporated in vacuo . the product was purified by silica gel flash chromatography ( ch 2 cl 2 / meoh / 8n nh 3 in meoh 70 : 30 : 4 ), yielding pure 10 ( 0 . 030 g , 0 . 08 mmol , 50 %). the resulting oil was dissolved in 5 ml meoh and 1 ml 30 % hydrochloric acid was added dropwise . the solvents and the excess of hcl were removed by coevaporation with methanol . 1h nmr ( d 2 o ): d 4 . 11 ( d , 1h , j = 12 . 5 hz , c - 6 dnm ), 3 . 98 ( d , 1h , j = 11 . 6 hz , c - 6 dnm ), 3 . 82 ( m , 1h , c - 2 dnm ), 3 . 69 ( t , 1h , j = 9 . 6 hz , c - 4 dnm ), 3 . 62 - 3 . 45 ( m , 4h , c - 1 eq ., c - 3 dnm , c - 5 chain ), 3 . 36 ( m , 1h , c - 1 chain ), 3 . 21 ( m , 2h , c - 5 dnm , c - 1 chain ), 3 . 08 ( m , 3h , c - 1 ax . dnm , ch 2 adamant . ), 1 . 95 ( m , 3h , adamant . ), 1 . 90 - 1 . 56 ( m , 10h , c - 2 , c - 4 chain , adamant . ), 1 . 52 - 1 . 30 ( m , 8h , c - 3 chain , adamant .). hrms ( fab ) obs mass 420 . 2745 ( mna + ), calcd for c 22 h 39 no 5 na 420 . 2726 ; obs mass 398 . 2905 ( mh + ), calcd for c 22 h 40 no 5 398 . 2906 . ki values were determined by variation of substrate concentration at fixed inhibitor concentration and assuming competitive inhibition and michaelis - menten kinetics . all constants are expressed in um . (−) implies that no inhibition was noted at an inhibitor concentration of 100 um . the structures of the tested inhibitors are depictured in tables 6 and 7 . the activity of ceredase towards 4mu - b - glucoside was determined in the presence of 0 . 25 % ( w / v ) sodium taurocholate and 0 . 1 % ( v / v ) triton x - 100 in citrate / phosphate buffer ( ph 5 . 2 ). the activities of glucocerebrosidase and glucosylceramidase in membrane suspensions towards 4mu - b - glucoside were determined in citrate / phosphate buffer ( ph 5 . 2 ). conduritol b - epoxide was employed to discriminate between the two enzymes . the activity of lysosomal a - glucosidase towards 4mu - a - glucoside was determined in citrate / phosphate buffer at ph 4 . 0 . apparent ic50 values of p21 and p24 for soluble glucocerebrosidase ( ceredase ) were 0 . 2 and 0 . 8 um . apparent ic50 values of p21 and p24 for glucocerebrosidase in membrane suspension were 0 . 06 and 0 . 7 um . apparent ic50 values of p21 and p24 for glucosylceramidase were 1 nm and 0 . 1 um . melanoma cells were incubated with various concentrations of inhibitors to determine their ic50 value ( i . e . inhibitor concentration resulting in 50 % inhibition ). activities of glucosylceramidase and glucocerebrosidase were determined as described in ref . 17 . ni = no significant inhibition detectable at 1 um inhibitor . human macrophages , obtained and cultured as described in ref . 4 , were incubated with different concentrations butyldeoxynojirimycin ( bdnj ) or n - 5 - adamantane - 1 - yl - methoxy - pentyl ) deoxynojirimycin ( p21 ). after 4 days preincubation with inhibitor , glucosylceramidase and glucocerebrosidase activities were determined with c6nbd - glucosylceramide as substrate [ 17 ] and at the same time the released chitotriosidase in the medium was determined [ 4 ]. enzyme activities and chitotriosidase secretion in the presence of dnjs are related to those in the absence of inhibitor ( 100 %). note : glucosylceramide synthase activity is not significantly inhibited at 5 um b - dnj or 1 nm p21 . the presence of pdmp or ppmp , while potently inhibiting glucosylceramide synthase activity , does not result in reduced chitotriosidase secretion . in structure 1 - 5 the large apolar groups are linked to dnj by a chain bearing two carbonyl groups . these two groups are replaced by methylene groups in structure 6 and 7 . 1 . heller , r . a ., kronke , m . ( 1994 ) j . cell biol . 126 , 5 - 9 . tumor necrosis factor receptor - mediated signalling pathways . 2 . hannun , y . a . ( 1994 ) j . biol . chem . 269 , 3125 - 3128 . the sphingomyelin cyclin and the second messenger function of ceramide . 3 . barranger , j . a ., ginns , e . i . ( 1989 ). glucosylceramide lipidoses : gaucher &# 39 ; s disease . in : the metabolic basis of inherited diseases . c . r . scriver , a . l . beaudet , w . s . sly & amp ; d . valle , editors ; mcgraw - hill inc . new york , 1677 - 1698 . 4 . hollak , c . e . m ., van weely , s ., van oers , m . h . j ., aerts , j . m . f . g . ( 1994 ) j . clin . invest . 93 , 1288 - 1292 . marked elevation of plasma chitotriosidase activity . a novel hallmark of gaucher disease . 5 . guo , y ., he , w ., boer , a . m ., wevers , r . a ., de bruyn , a . m ., groener , j . e . m ., hollak , c . e . m ., aerts , j . m . f . g ., galjaard , h ., van diggelen , o . p . ( 1995 ) j . inher . metab . dis . 18 , 717 - 722 . elevated plasma chitotriosidase activity in various lysosomal storage disorders . 6 . renkema , g . h ., boot , r . g ., muysers , a . o ., donker - koopman , w . e ., aerts , j . m . f . g . ( 1995 ) j . biol . chem . 270 , 2198 - 2202 . purification and characterization of human chitotriosidase , a novel member of the chitinase family of proteins . 7 . boot , r . g ., renkema , g . h ., strijland , a . h ., van zonneveld , a . j ., aerts , j . m . f . g . ( 1995 ) j . biol . chem . 270 , 26252 - 26256 . cloning of a cdna encoding chitotriosidase , a human chitinase produced by macrophages . 8 . barton , n . w ., furbish , f . s ., murray , g . j ., garfield , m ., brady , r . o . ( 1990 ) proc . natl . acad . sci . usa 87 , 1913 - 1916 . therapeutic response to intravenous infusions of glucocerebrosidase in a patient with gaucher disease . 9 . hollak , c . e . m ., aerts , j . m . f . g ., goudsmit , r ., phoa , s . s . k . s ., ek , m ., van weely , s ., von dem borne , a . e . g . kr ., van oers , m . h . j . ( 1995 ) lancet 345 , 1474 - 1478 . individualised low - dose alglucerase therapy for type 1 gaucher &# 39 ; s disease . 10 . grabowski , g . a ., barton , n . w ., pastores , g ., dambrosia , j . m ., banerjee , t . k ., mckee , m . a ., parker , c ., schiffmann , r ., hill , s . c ., brady , r . o . ( 1995 ) ann . int . medicine 122 , 33 - 39 . enzyme therapy in type 1 gaucher disease : comparative efficacy of mannose - terminated glucocerebrosidase from natural and recombinant sources . 11 . aerts , j . m . f . g ., boot , r . g ., renkema , g . h ., van weely , s ., jones , s ., hollak , c . e . m ., van oers , m . h . j . ( 1995 ) sem . hematol . 32 , suppl . 1 , 10 - 13 . molecular and biochemical abnormalities of gaucher disease : chitotriosidase , a newly identified biochemical marker . 12 . nih technology assessment panel on gaucher disease ( 1996 ) jama 275 , 548 - 553 . gaucher disease . current issues in diagnosis and treatment . 13 . marshall , e . ( 1995 ) science 269 , 1050 - 1055 . gene therapy &# 39 ; s growing pains . 14 . abe , a ., radin , n . s ., shayman , j . a . ( 1996 ) biochim . biophys . acta 1299 , 333 - 341 . induction of glucosylceramide synthase by synthase inhibitors and ceramide . 15 . platt , f . m ., neises , g . r ., dwek , r . a ., butters , t . d . ( 1994 ) j . biol . chem . 269 , 8362 - 8365 . n - butyldeoxynojirimycin is a novel inhibitor of glycolipid biosynthesis . 16 . platt , f . m ., neises , g . r ., karlsson , g . b ., dwek , r . a ., butters , t . d . ( 1994 ) j . biol . chem . 269 , 27108 - 27114 . n - butyldeoxygalactonojirimycin inhibits glycolipid bio - synthesis but does not affect n - linked oligosaccharide processing . 17 . van weely , s ., brandsma , m ., strijland , a ., tager , j . m ., aerts , j . m . f . g . ( 1993 ) biochim . biophys . acta 1181 , 53 - 62 . demonstration of the existence of a second , non - lysosomal glucocerebrosidase that is not deficient in gaucher disease . 18 . overkleeft , h . s ., van wiltenburg , j ., pandit , u . k . ( 1994 ) tetrahedron 34 , 4215 - 4224 . 19 . baxter , e . w ., reitz , a . b . ( 1994 ) j . org . chem . 59 , 3175 - 3185 . 20 . wanner , m . j ., koomen , g . j . ( 1995 ) j . org . chem . 60 , 5634 - 5637 .
deoxynojirimycin derivatives containing a large hydrophobic moiety , such as cholesterol or adamantame - methanol , linked through a spacer , such as pentamethylene , to the nitrogen atom of deoxynojirimycin , and salts thereof , inhibit glucosylceramidase and may be useful in the treatment of diseases involving a ceramide - mediated signaling process , such as gaucher disease .