In vitro inhibition of glycogen-degrading enzymes and glycosidases by six-membered sugar mimics and their evaluation in cell cultures

We investigated in vitro inhibition of mammalian carbohydrate-degrading enzymes by six-membered sugar mimics and their evaluation in cell cultures. 1-Deoxynojirimycin (DNJ) showed no significant inhibition toward glycogen phosphorylase (GP) but was a potent inhibitor of another glycogen-degrading enzyme, amylo-1,6-glucosidase (1,6-GL), with an IC(50) value of 0.16 microM. In primary rat hepatocytes, the inhibition of glycogen breakdown by DNJ reached plateau at 100 microM with 25% inhibition and then remained unchanged. The potent GP inhibitor 1,4-dideoxy-1,4-imino-D-arabinitol (D-AB1) inhibited hepatic glucose production with an IC(50) value of about 9 microM and the inhibition by D-AB1 was further enhanced in the presence of DNJ. DNJ and alpha-homonojirimycin (HNJ) are very potent inhibitors of rat intestinal maltase, with IC(50) values of 0.13 and 0.08 microM, respectively, and also showed a similar strong inhibition toward maltase in Caco-2 cell model system, with IC(50) value of 0.05 and 0.10 microM, respectively. D-Isofagomine (D-IFG) and L-IFG are competitive and noncompetitive inhibitors of human lysosomal beta-glucosidase (beta-GL), respectively, with K(i) values of 8.4 nM and 6.9 microM. D-IFG increased intracellular beta-GL activity by twofold at 10 microM in Gaucher N370S cell line as an 'active-site-specific' chaperone, and surprisingly a noncompetitive inhibitor L-IFG also increased intracellular beta-GL activity by 1.6-fold at 500 microM.     

Synthesis of 2-deoxy-2-fluoro and 1,2-ene derivatives of the naturally occurring glycosidase inhibitor, salacinol, and their inhibitory activities against recombinant human maltase glucoamylase

2-Deoxy-2-fluorosalacinol and a 1,2-ene derivative of the naturally occurring glycosidase inhibitor salacinol were synthesized for structure activity studies with human maltase glucoamylase (MGA). 2-Deoxy-2-fluorosalacinol was synthesized through the coupling reaction of 2-deoxy-2-fluoro-3,5-di-O-p-methoxybenzyl-1,4-anhydro-4-thio-D-arabinitol with 2,4-O-benzylidene-l-erythritol-1,3-cyclic sulfate in hexafluoroisopropanol (HFIP) containing 0.3 equiv of K(2)CO(3). Excess of K(2)CO(3) resulted in the elimination of HF from the coupled product, and the formation of an alkene derivative of salacinol. Nucleophilic attack of the 1,4-anhydro-4-thio-D-arabinitol moiety on the cyclic sulfate did not proceed in the absence of K(2)CO(3). No reaction was observed in acetonitrile containing K(2)CO(3). The target compounds were obtained by deprotection with TFA. The 2-deoxy-1-ene derivative of salacinol and 2-deoxy-2-fluorosalacinol inhibited recombinant human maltase glucoamylase, one of the key intestinal enzymes involved in the breakdown of glucose, with an IC(50) value of 150 microM and a K(i) value of 6+/-1 microM, respectively.     

Purification and properties of inositol-1,4-bisphosphate 4-phosphohydrolase from rat brain

Inositol-1,4-bisphosphate 4-phosphohydrolase (inositol-1,4-bisphosphatase) was highly purified from a soluble fraction of rat brain. On SDS-polyacrylamide gel electrophoresis, the purified enzyme gave a single protein band and its molecular weight was estimated to be 42000. The isoelectric point of the enzyme was 4.3. The enzyme specifically hydrolyzed the 4-phosphomonoester linkage of inositol 1,4-bisphosphate. The Km value for inositol 1,4-bisphosphate was 30 microM, and it required Mg2+ for activity. Ca2+ was a competitive inhibitor with a Ki value of 60 microM as regards the Mg2+ binding. Li+, which is known to be a strong inhibitor of inositol 1-phosphatase (EC 3.1.3.25), inhibited the enzyme activity and caused 50% inhibition at a concentration of 1 mM (IC50 = 1 mM). Li+ was an uncompetitive inhibitor of substrate binding with a Ki value of 0.6 mM. These inhibitory parameters of Li+ were quite similar to those for inositol 1-phosphatase (IC50 = 1 mM, Ki = 0.3 mM). Thus, the effect of Li+ on decreasing the free inositol level with a subsequent decrease in agonist-sensitive phosphoinositides, is caused by its inhibition of multiple enzymes involved in conversion of inositol 1,4-bisphosphate to inositol.     

In vitro inhibition and intracellular enhancement of lysosomal alpha-galactosidase A activity in Fabry lymphoblasts by 1-deoxygalactonojirimycin and its derivatives.

Fabry disease is a lysosomal storage disorder caused by deficient lysosomal alpha-galactosidase A (alpha-Gal A) activity. Deficiency of the enzyme activity results in progressive deposition of neutral glycosphingolipids with terminal alpha-galactosyl residue in vascular endothelial cells. We recently proposed a chemical chaperone therapy for this disease by administration of 1-deoxygalactonojirimycin, a potent inhibitor of the enzyme, at subinhibitory intracellular concentrations [Fan, J.-Q., Ishii, S., Asano, N. and Suzuki, Y. (1999) Nat. Med. 5, 112-115]. 1-Deoxygalactonojirimycin served as a specific chaperone for those mutant enzymes that failed to maintain their proper conformation to avoid excessive degradation. In order to establish a correlation between in vitro inhibitory activity and intracellular enhancement activity of the specific chemical chaperone, a series of 1-deoxygalactonojirimycin derivatives were tested for activity with both alpha-Gal A and Fabry lymphoblasts. 1-Deoxygalactonojirimycin was the most potent inhibitor of alpha-Gal A with an IC50 value of 0.04 microM. alpha-Galacto-homonojirimycin, alpha-allo-homonojirimycin and beta-1-C-butyl-deoxygalactonojirimycin were effective inhibitors with IC50 values of 0.21, 4.3 and 16 microM, respectively. N-Alkylation, deoxygenation at C-2 and epimerization at C-3 of 1-deoxygalactonojirimycin markedly lowered or abolished its inhibition toward alpha-Gal A. Inclusion of 1-deoxygalactonojirimycin, alpha-galacto-homonojirimycin, alpha-allo-homonojirimycin and beta-1-C-butyl-deoxygalactonojirimycin at 100 microM in culture medium of Fabry lymphoblasts increased the intracellular alpha-Gal A activity by 14-fold, 5.2-fold, 2.4-fold and 2.3-fold, respectively. Weaker inhibitors showed only a minimum enhancement effect. These results suggest that more potent inhibitors act as more effective specific chemical chaperones for the mutant enzyme, and the potent competitive inhibitors of alpha-Gal A are effective specific chemical chaperones for Fabry disease.     

Design, synthesis, and biological evaluation of N-acetyl-2-carboxybenzenesulfonamides: a novel class of cyclooxygenase-2 (COX-2) inhibitors

N-Acetyl-2-carboxybenzenesulfonamide (11), and a group of analogues possessing an appropriately substituted-phenyl substituent (4-F, 2,4-F(2), 4-SO(2)Me, 4-OCHMe(2)) attached to its C-4, or C-5 position, were synthesized for evaluation as selective cyclooxygenase-2 (COX-2) inhibitors. In vitro COX-1/COX-2 inhibition studies showed that 11 is a more potent inhibitor (COX-1 IC(50)=0.06microM; COX-2 IC(50)=0.25microM) than aspirin (COX-1 IC(50)=0.35microM; COX-2 IC(50)=2.4microM), and like aspirin [COX-2 selectivity index (S.I.)=0.14], 11 is a nonselective COX-2 inhibitor (COX-2 S.I.=0.23). Regioisomers having a 2,4-difluorophenyl substituent attached to the C-4 (COX-2 IC(50)=0.087microM; COX-2 S.I. >1149), or C-5 (COX-2 IC(50)=0.77microM, SI>130), position of 11 exhibited the most potent and selective COX-2 inhibitory activity relative to the reference drug celecoxib (COX-1 IC(50)=33.1microM; COX-2 IC(50)=0.07microM; COX-2 S.I.=472). N-Acetyl-2-carboxybenzenesulfonamide (11, ED(50)=49 mg/kg), and its C-4 2,4-difluorophenyl derivative (ED(50)=91 mg/kg), exhibited superior antiinflammatory activity (oral dosing) in a carrageenan-induced rat paw edema assay compared to aspirin (ED(50)=129 mg/kg). These latter compounds exhibited comparable analgesic activity to the reference drug diflunisal, and superior analgesic activity compared to aspirin, in a 4% NaCl-induced abdominal constriction assay. A molecular modeling (docking) study indicated that the SO(2)NHCOCH(3) substituent present in N-acetyl-2-carboxy-4-(2,4-fluorophenyl)benzenesulfonamide, like the acetoxy substituent in aspirin, is suitably positioned to acetylate the Ser(530) hydroxyl group in the COX-2 primary binding site. The results of this study indicate that the SO(2)NHCOCH(3) pharmacophore present in N-acetyl-2-carboxybenzenesulfonamides is a suitable bioisostere for the acetoxy (OCOMe) group in aspirin.     

Synthesis, cytotoxicity, and anti-inflammatory evaluation of 2-(furan-2-yl)-4-(phenoxy)quinoline derivatives. Part 4

A number of 2-(furan-2-yl)-4-phenoxyquinoline derivatives have been synthesized and evaluated for anti-inflammatory evaluation. 4-[(2-Furan-2-yl)quinolin-4-yloxy]benzaldehyde (8), with an IC(50) value of 5.0 microM against beta-glucuronidase release, was more potent than its tricyclic furo[2,3-b]quinoline isomer 3a (>30 microM), its 4'-COMe counterpart 7 (7.5 microM), and its oxime derivative 13a (11.4 microM) and methyloxime derivative 13b (>30 microM). For the inhibition of lysozyme release, however, oxime derivative 12a (8.9 microM) and methyloxime derivative 12b (10.4 microM) are more potent than their ketone precursor 7 and their respective tricyclic furo[2,3-b]quinoline counterparts 4a and 4b. Among them, 4-[4-[(2-furan-2-yl)-quinolin-4-yloxy]phenyl]but-3-en-2-one (10) is the most active against lysozyme release with an IC(50) value of 4.6 microM, while 8 is the most active against beta-glucuronidase release with an IC(50) value of 5.0 microM. (E)-1-[3-[(2-Furan-2-yl)quinolin-4-yloxy]phenyl] ethanone oxime (11a) is capable of inhibiting both lysozyme and beta-glucuronidase release with IC(50) values of 7.1 and 9.5 microM, respectively. For the inhibition of TNF-alpha formation, 1-[3-[(2-furan-2-yl)quinolin-4-yloxy]phenyl]ethanone (6) is the most potent with an IC(50) value of 2.3 microM which is more potent than genistein (9.1 microM). For the inhibitory activity of fMLP-induced superoxide anion generation, 11a (2.7 microM), 11b (2.8 microM), and 13b (2.2 microM) are three of the most active. None of above compounds exhibited significant cytotoxicity.     

Synthesis of N-substituted piperidine-4-(benzylidene-4-carboxylic acids) and evaluation as inhibitors of steroid-5alpha-reductase type 1 and 2

The synthesis of N-substituted piperidine-4-(benzylidene-4-carboxylic acids) is described [benzoyl (1), benzyl (2), adamantanoyl (3), cyclohexanoyl (4), cyclohexylacetyl (5), diphenylacetyl (6), dicyclohexylacetyl (7), 2-propylpentanoyl (8), diphenylcarbamoyl (9), trimethylacetyl (10), 3,3-dimethylacryloyl (11), dicyclohexylacetyl derivative of the benzyl compound (12)]. Compounds were tested for inhibitory activity toward 5alpha-reductase isozymes 1 and 2 in human and rat. The test compounds inhibited 5alpha-reductase, showing a broad range of inhibitory potencies. In rat, compounds 6 (IC50 = 3.44 and 0.37 microM for type 1 and 2, respectively) and 9 (IC50=0.54 and 0.69 microM for type 1 and 2, respectively) displayed the best inhibition toward both isozymes. Compound 7 showed a strong inhibition toward type 2 human and rat enzyme (IC50 = 60 and 80 nM) but only a moderate activity versus type 1 enzyme (IC50 approximately 10 microM for rat and human enzyme). In vivo, selected compounds reduced prostate weights in castrated testosterone treated rats.     

Identifying 6,7,4'-trihydroxyisoflavone as a potent tyrosinase inhibitor

A known biotransformed compound, 6,7,4'-trihydroxyisoflavone, was identified as a potent tyrosinase inhibitor. It inhibited mushroom tyrosinase with an IC50 value of 9.2 microM, which is six times the anti-tyrosinase activity of kojic acid (IC50 = 54.4 microM). The inhibition kinetics, analyzed by Lineweaver-Burk plots, indicated 6,7,4'-trihydroxyisoflavone to be a competitive inhibitor of tyrosinase when L-tyrosine was used as a substrate. Its biosynthesis precursors and analogs, including glycitein, daidzein, and genistein, showed little anti-tyrosinase activity. The results suggest that hydroxyl groups at the C-6 and C-7 positions of the isoflavone skeleton might play an important role in the expression of tyrosinase inhibitory activity.     

Polyhydroxylated pyrrolidine and piperidine alkaloids from Adenophora triphylla var. japonica (Campanulaceae)

Adenophora triphylla var. japonica (Campanulaceae) yielded two new alkaloids, the 6-C-butyl derivative of 2R,5R-bis(hydroxymethyl)-3R,4R-dihydroxypyrrolidine (DMDP) and alpha-1-C-ethyl-fagomine, together with the known alkaloids 1,4-dideoxy-1,4-imino-D-arabinitol, 1-deoxynojirimycin, and 1-deoxymannojirimycin. 6-C-Butyl-DMDP showed inhibitory activity toward almond beta-glucosidase (IC50 = 68 microM), whereas alpha-1-C-ethyl-fagomine inhibited bovine liver beta-galactosidase (IC50 = 29 microM).     

Selective inhibition of beta-1,4- and alpha-1,3-galactosyltransferases: donor sugar-nucleotide based approach

A combined rational and library approach was used to identify bisphosphonates (IC50 = 20 microM) and galactose type 1-N-iminosugar (IC50=45 microM) as novel motifs for selective inhibition of beta-1,4-galactosyltransferase (beta-1,4-GalT) and alpha-1,3-galactosyltransferase (alpha-1,3-GalT), respectively. Our results demonstrate that, though these two galactosyltransferases both utilize the same donor sugar-nucleotide (UDP-Gal), the difference in their mechanisms can be utilized to design donor sugar or nucleotide analogues with inhibitory activities selective for only one of the galactosyltransferases. Investigation of beta-1,4-GalT inhibition using UDP-2-deoxy-2-fluorogalactose (UDP-2-F-Gal), UDP, and bisphosphonates, also led to the observation of metal dependent inhibition of beta-1,4-GalT. These observations and the novel inhibitor motifs identified in this study pave the way for the design and identification of even more potent and selective galactosyltransferase inhibitors.     

Non-peptidic anti-AIDS agents: inhibition of HIV-1 proteinase by disulfonates.

Based upon an earlier observation that sodium docosanedioate (NaO2C-(CH2)20-CO2NA) weakly inhibits HIV-1 proteinase (IC50 12 microM), we have identified a class of more potent inhibitors (sulfonic acids) of this enzyme which are likewise dianionic at pH 5-6.5. Many of the compounds were moderately strong inhibitors of the enzyme (IC50 40nM-10 microM) and some have previously been shown to have anti-HIV activity in lymphocytes. Proteinase inhibition was dependent on the separation between sulfonate/carboxylate substituents, consistent with the hypothesis that negative charged ends of an inhibitor might form ionic bonds with Arg 8 and Arg 108 located at either end of the substrate-binding groove of the enzyme. The binding mode remains to be established by structure elucidation. Results for enzyme inhibition are presented along with structure-activity relationships and evidence for pH dependent inhibition. The general observations reported here may be useful for developing more potent and selective non-peptidic proteinase inhibitors.     

Biological evaluation of de-O-sulfonated analogs of salacinol, the role of sulfate anion in the side chain on the alpha-glucosidase inhibitory activity

De-O-sulfonated analogs (10a, Y(-)=CH(3)OSO(3) and 10b, Y(-)=Cl) of salacinol, a naturally occurring glycosidase inhibitor, and its diastereomer (12a, Y(-)=CH(3)OSO(3)) with L-thiosugar moiety (1,4-dideoxy-1,4-epithio-L-arabinitol) were prepared. Their inhibitory activities against intestinal maltase and sucrase were examined and compared with those of the parent alpha-glycosidase inhibitor, salacinol (1a). Compounds 10a and 10b showed a potent inhibitory activity equal to that of 1a against both enzymes, although 12a was a weak inhibitor against sucrase and maltase. These results indicated that the O-sulfonate anion moiety of 1a is not essential for the inhibitory activity.     

Structure-activity relationship of aza-steroids as PI-PLC inhibitors

A number of aza-steroids were synthesized as potent phosphatidylinositol phospholipase C (PI-PLC) inhibitors. The epimeric mixtures 22,25-diazacholesterol (8a) and 3beta-hydroxy-22,25-diazacholestane (8b) were among the most active of these inhibitors, with IC(50) values of 7.4 and 7.5 microM, respectively. The 20alpha epimer, 8a2 (IC(50)=0.64 microM), whose stereochemistry at C-20 coincides with that of cholesterol, was found 50 times more potent than the 20beta epimer, 8a1 (IC(50)=32.2 microM). In diaza-estrone derivatives, the 3-methoxy group on the aromatic A-ring of 23 exhibited moderate PI-PLC inhibitory activity (IC(50)=19.7 microM), while compound with a free hydroxyl group (21) was inactive. However, in diaza-pregnane derivatives, epimers with a 3-hydroxyl group (8a, IC(50)=7.4 microM) exhibited more potent PI-PLC inhibitory activity than their counterparts with 3-methoxyl group on the non-aromatic A-ring (26, IC(50)=17.4 microM). We have illustrated in our previous publication that 3-hydroxyl-6-aza steroids are potent PI-PLC inhibitors.(3) However, simultaneous presence of the 6-aza and 22,25-diaza moieties in one molecule as in 13, led to loss of activity. Epimeric mixture 8a showed selective growth inhibition effects in the NCI in vitro tumor cell screen with a mean GI(50) value (MG-MID) of 5.75 microM for 54 tumors.     

Novel 3-phenylpropane-1,2-diamine derivates as inhibitors of aminopeptidase N (APN)

Aminopeptidase N (APN) is an essential peptidase involved in the process of tumor invasion and metastasis. Here we describe a novel class of inhibitor with 3-phenylpropane-1,2-diamine as scaffold to APN. Preliminary activity evaluation with enzyme inhibition studies showed that compound 12i exhibited potent and selective inhibitory activity towards APN with the IC(50) value 15.5+/-1.2microM.     

Development of a new inhibitor of glucosylceramide synthase

Analogs of the potent inhibitor of glucosylceramide (GlcCer) synthase, D-threo-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (P4), based on substitutions in the palmitoyl group were made by means of a stereo-selective synthetic method in order to elucidate the role of the hydrophobic portion in both the inhibitory action toward the enzyme and the biological effects. While P4 strongly inhibited GlcCer synthase with an IC(50) of 0.5 microM in vitro, it also inhibited cell growth by 50% at the concentration of 7 microM. The shorter N-acyl chain analogs including decanoyl, octanoyl, and hexanoyl groups showed similar IC(50) values for GlcCer synthase (around 2 microM) but the hexanoyl analog exhibited only a slight inhibitory effect on cell growth, showing the dissociation between GlcCer depletion and cell growth. Several compounds which exhibit similar hydrophobicity to the hexanoyl analog of P4 were subsequently designed. We found that D-threo-1-phenyl-2-benzyloxycarbonylamino-3-pyrrolidino-1-pr opanol (PBPP) was a most potent inhibitor, showing an IC50 of 0.3 microM. In cultured cells, PBPP was able to deplete glycosphingolipids without affecting cell growth or the ceramide level.     

Structure-activity study of L-amino acid-based N-type calcium channel blockers

Synthesis and structure-activity relationship (SAR) study of L-amino acid-based N-type calcium channel blockers are described. The compounds synthesized were evaluated for inhibitory activity against both N-type and L-type calcium channels focusing on selectivity to reduce cardiovascular side effects due to blocking of L-type calcium channels. In the course of screening of our compound library, N-(t-butoxycarbonyl)-L-aspartic acid derivative 1a was identified as an initial lead compound for a new series of N-type calcium channel blockers, which inhibited calcium influx into IMR-32 human neuroblastoma cells with an IC(50) of 3.4 microM. Compound 1a also exhibited blockade of N-type calcium channel current in electrophysiological experiment using IMR-32 cells (34% inhibition at 10 microM, n=3). As a consequence of conversion of amino acid residue of 1a, compound 12a, that include N-(t-butoxycarbonyl)-L-cysteine, was found to be a potent N-type calcium channel blocker with an IC(50) of 0.61 microM. Thus, L-cysteine was selected as a potential structural motif for further modification. Optimization of C- and N-terminals of L-cysteine using S-cyclohexylmethyl-L-cysteine as a central scaffold led to potent and selective N-type calcium channel blocker 21f, which showed improved inhibitory potency (IC(50) 0.12 microM) and 12-fold selectivity for N-type calcium channels over L-type channels.     

Chemical modification of the beta-glucocerebrosidase inhibitor N-octyl-beta-valienamine: synthesis and biological evaluation of 4-epimeric and 4-O-(beta-D-galactopyranosyl) derivatives

N-Octyl-beta-valienemine (1), a potent beta-glucocerebrosidase inhibitor, was chemically transformed into two biologically interesting compounds: the 4-epimer, beta-galacto-type N-octyl-valienamine, and the 4-O-(beta-D-galactopyranosyl) derivative, a carba-lactosylceramide analogue. The former, interestingly, could be demonstrated to act as a very effective inhibitor (IC(50)=0.3 microM) of human beta-galactosidase. The latter exhibited moderate inhibitory activity (IC(50)=20 microM) against beta-glucocerebrosidase (mouse liver).     

Synthesis and structure-activity relationship studies on tryprostatin A, an inhibitor of breast cancer resistance protein

Tryprostatin A is an inhibitor of breast cancer resistance protein, consequently a series of structure-activity studies on the cell cycle inhibitory effects of tryprostatin A analogues as potential antitumor antimitotic agents have been carried out. These analogues were assayed for their growth inhibition properties and their ability to perturb the cell cycle in tsFT210 cells. SAR studies resulted in the identification of the essential structural features required for cytotoxic activity. The absolute configuration L-Tyr-L-pro in the diketopiperazine ring along with the presence of the 6-methoxy substituent on the indole moiety of 1 was shown to be essential for dual inhibition of topoisomerase II and tubulin polymerization. Biological evaluation also indicated the presence of the 2-isoprenyl moiety on the indole scaffold of 1 was essential for potent inhibition of cell proliferation. Substitution of the indole N(a)-H in 1 with various alkyl or aryl groups, incorporation of various L-amino acids into the diketopiperazine ring in place of L-proline, and substitution of the 6-methoxy group in 1 with other functionality provided active analogues. The nature of the substituents present on the indole N(a)-H or the indole C-2 position influenced the mechanism of action of these analogues. Analogues 68 (IC(50)=10 microM) and 67 (IC(50)=19 microM) were 7-fold and 3.5-fold more potent, respectively, than 1 (IC(50)=68 microM) in the inhibition of the growth of tsFT210 cells. Diastereomer-2 of tryprostatin B 8 was a potent inhibitor of the growth of three human carcinoma cell lines: H520 (IC(50)=11.9 microM), MCF-7 (IC(50)=17.0 microM) and PC-3 (IC(50)=11.1 microM) and was equipotent with etoposide, a clinically used anticancer agent. Isothiocyanate analogue 71 and 6-azido analogue 72 were as potent as 1 in the tsFT210 cell proliferation and may be useful tools in labeling BCRP.     

Inhibitory activity of linoleic acid isolated from proso and Japanese millet toward histone deacetylase

Linoleic acid was isolated from both the methanol extracts of proso and Japanese millet as a histone deacetylase inhibitor. It showed uncompetitive inhibitory activity toward histone deacetylase (IC(50)=0.51 mM) and potent cytotoxicity toward human leukemia K562 (IC(50)=68 microM) and prostate cancer LNCaP cells (IC(50)=193 microM). Millet containing linoleic acid might have anti-tumor activity.     

Discovering benzamide derivatives as glycogen phosphorylase inhibitors and their binding site at the enzyme

A series of novel benzamide derivatives was designed, synthesized, and their inhibitory activities against glycogen phosphorylase (GP) in the direction of glycogen synthesis by the release of phosphate from glucose-1-phosphate were evaluated. The structure-activity relationships (SAR) of these compounds are also presented. Within this series of compounds, 4m is the most potent GPa inhibitor (IC(50)=2.68 microM), which is nearly 100 times more potent than the initial compound 1. Analysis of mapping between pharmacophores of different binding sites and each compound demonstrated that these benzamide derivatives bind at the dimer interface of the rabbit muscle enzyme, and possible docking modes of compound 4m were explored by molecular docking simulation.