Nanomolar Inhibitors of Glycogen Phosphorylase Based on Î²-d

Article Cite This: J. Med. Chem. 2017, 60, 9251-9262

pubs.acs.org/jmc

Nanomolar Inhibitors of Glycogen Phosphorylase Based on β‑D‑Glucosaminyl Heterocycles: A Combined Synthetic, Enzyme Kinetic, and Protein Crystallography Study Éva Bokor,†,# Efthimios Kyriakis,‡,# Theodora G.A. Solovou,‡ Csenge Koppány,† Anastassia L. Kantsadi,‡,⊥ Katalin E. Szabó,† Andrea Szakács,† George A. Stravodimos,‡ Tibor Docsa,§ Vassiliki T. Skamnaki,‡ Spyros E. Zographos,∥ Pál Gergely,§ Demetres D. Leonidas,*,‡ and László Somsák*,† †

Department of Organic Chemistry, University of Debrecen, POB 400, H-4002 Debrecen, Hungary Department of Biochemistry and Biotechnology, University of Thessaly, Biopolis, 41500 Larissa, Greece § Department of Medical Chemistry, Faculty of Medicine, University of Debrecen, Egyetem tér 1, H-4032 Debrecen, Hungary ∥ Institute of Biology, Pharmaceutical Chemistry and Biotechnology, National Hellenic Research Foundation, 48 Vassileos Constantinou Avenue, 11635 Athens, Greece ‡

S Supporting Information *

ABSTRACT: Aryl substituted 1-(β-D-glucosaminyl)-1,2,3-triazoles as well as C-β-D-glucosaminyl 1,2,4-triazoles and imidazoles were synthesized and tested as inhibitors against muscle and liver isoforms of glycogen phosphorylase (GP). While the N-β-Dglucosaminyl 1,2,3-triazoles showed weak or no inhibition, the C-β-D-glucosaminyl derivatives had potent activity, and the best inhibitor was the 2-(β-D-glucosaminyl)-4(5)-(2-naphthyl)-imidazole with a Ki value of 143 nM against human liver GPa. An Xray crystallography study of the rabbit muscle GPb inhibitor complexes revealed structural features of the strong binding and offered an explanation for the differences in inhibitory potency between glucosyl and glucosaminyl derivatives and also for the differences between imidazole and 1,2,4-triazole analogues.

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low micromolar values similarly to that of benzimidazole33−35 7. The most efficient submicromolar inhibitors are the Cglucosyl 1,2,4-triazoles36,37 3 and 4 as well as imidazoles38,39 5 and 6 (compound 6 represents the most efficient glucose analogue inhibitor of muscle and liver GPs known to date). In many of these glucosyl heterocyclic inhibitors, the substituents of the heterocycle had a strong bearing on the efficiency: the larger aromatic substituents lent higher efficacy to the compounds (compare the pairs 1/2, 3/4, and 5/6). The inhibitors were also modified beyond the heterocyclic parts and their substituents to get information on the variability of the sugar moiety. A formal removal of the CH2OH group from compounds 1−7 to give 12−18,40−42 respectively (actually a change from glucose to xylose), resulted in a significant weakening or even a complete loss of the inhibition. Pyrimidine derivative 8 was modified by introducing either an equatorial fluorine43 (19) or an axial hydroxymethyl44 (20) substituent in place of the 3′-OH of the glucose unit, again

INTRODUCTION Glycogen phosphorylase (GP), the main regulatory enzyme of hepatic glucose output, is a validated target in the search for new therapeutic possibilities toward type 2 diabetes mellitus (T2DM).1−3 Inhibitors of GP (GPIs) have been shown to exert in vivo blood sugar diminishing activity,4−8 to have potential in other diseased states like myocardial9,10 and cerebral11,12 ischemias and tumors,13−18 and to exhibit further unexpected physiological effects such as modifying hepatic metabolism19 and improving pancreatic β-cell function.20 Among the large variety of GPIs21,22 the most populated class consists of glucose derivatives23−25 which primarily bind to the catalytic site of the enzyme. Extensive studies into the structure−activity relationships of glucose-derived compounds revealed among others β-D-glucopyranosyl heterocycles as very efficient GPIs. Thus, N-glucopyranosyl heterocycles like 1,2,3triazoles26,27 1 and 2 (Table 1) and pyrimidine28,29 8 are low micromolar inhibitors. A large series of C-glucopyranosyl heterocycles30 has shown a wide range of inhibition. The activity of some of the isomeric oxadiazoles,31,32 9−11, strongly depending on the constitution of the heterocycle, reaches the © 2017 American Chemical Society

Received: August 8, 2017 Published: September 19, 2017 9251

DOI: 10.1021/acs.jmedchem.7b01056 J. Med. Chem. 2017, 60, 9251−9262

Journal of Medicinal Chemistry

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Table 1. Selected Inhibitors of Rabbit Muscle Glycogen Phosphorylase b (rmGPb) with β-D-Glucopyranosyl Heterocyclic Motifs and Their Counterparts with Modified Sugar Units (Ki [μM])

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ending up with a weakening of the inhibitory activity. Modification of the oxadiazoles 9−11 by the introduction of a double bond in the 1′,2′-positions to furnish 21−23 (formally replacing the glucopyranosyl unit by a glucal moiety to model the probably glycosylium ion-like transition state45 of the reaction) also gave inactive compounds.46 In this paper, a new direction of structure−activity relationship studies of glucose derived GPIs is disclosed wherein the replacement of the 2′-OH of the glucose unit by an isosteric NH2 group has been investigated. The target compounds of the study (Table 1, bottom line) comprise 2amino-2-deoxy-β-D-glucopyranosyl heterocycles with phenyl and 2-naphthyl appendages, whose inhibitory effect against rabbit muscle and recombinant human liver GPs as well as peculiarities of their binding to the protein have been determined to discover the first nanomolar active site inhibitors of GP with a modified glucose moiety.

RESULTS AND DISCUSSION

Syntheses. 1-(β-D-Glucosaminyl)-1,2,3-triazoles 25 were prepared first, and to this end, we turned back to an earlier work describing the synthesis of the fully protected derivatives 24.47 The O-acetyl and N-phthaloyl protecting groups were removed simultaneously (Scheme 1) by using 20 equiv of hydrazine hydrate in MeOH to give the required test compounds 25 in very good yields. For the preparation of 3-(β-D-glucopyranosyl)-5-substituted 1,2,4-triazoles (e.g., 3, 4), several methods were elaborated in our laboratory;36,37,48,49 however, all of these failed to give the protected intermediates toward the target compounds 29 or the removal of the protecting groups was unsatisfactory. Therefore, a most recent method50 was applied in which the O,Nprotected β-D-glucosaminylformyl chloride51 27 (Scheme 2), obtained from the corresponding acid51 26 with a slight modification48 of the literature procedure by using SOCl2 9252



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Scheme 3a

Scheme 1

Scheme 2a a

Reagents and conditions: (a) 3 equiv of Et3OBF4, dry CH2Cl2, Ar, rt; (b) 2 equiv of ArCOCH2NH2·HCl, dry pyridine, rt; (c) 2 equiv of 1,2diaminobenzene, dry CH2Cl2, reflux; (d) 20 equiv of NH2NH2·H2O, MeOH, reflux.

compound is 33b whose Ki value classifies it to be among the top five most potent active site GPIs,30 and 33b is the best inhibitor with a modified glucopyranose unit reported thus far. A comparison of the analogous compounds (Table 2) derived from glucose (R = OH) and glucosamine (R = NH2) reveals that the substitution of the 2′-OH by an NH2 group leads to a decrease in the inhibitory potency by a factor of ∼5−40 in favor of the glucose derivatives. Furthermore, compounds with an imidazole linker (5, 6, 33) are more potent than the ones with a 1,2,4-triazole (3, 4, 29) in both series. Structural Studies. X-ray crystallography studies have revealed the architecture of the catalytic site of GP and the existence of six regulatory peripheral sites: the allosteric, the inhibitor, the new allosteric, the quercetin, the benzimidazole, and the glycogen storage site.25 Toward elucidating the structural basis of inhibition and most importantly the differences in the inhibition constants, we have determined the crystal structures of 29a, 29b, 33a, and 33b in complex with rmGPb. Although the pharmacologically relevant target is hlGPa, our studies were performed with the easily available rabbit muscle isoform since the catalytic site is identically conserved in all mammalian GPs;54 furthermore, it has been discovered that compounds inhibiting rmGP by binding at this site inhibit hlGPa similarly.39 The 2Fo − Fc and Fo − Fc electron density maps (Figure 1) clearly defined the position of each atom of the inhibitors and specifically showed that all the studied compounds were bound at the catalytic site of rmGPb. For compound 33b, some additional electron density (Figure 1) was located at the new or indole-binding allosteric site located in the region of the central cavity of the biologically active GP dimer at the subunit interface21 indicating that this inhibitor was also partially bound at this site (occupancy refined to 0.70). The lower occupancy value for the bound ligand at this site indicates that the new allosteric site is not the primary binding site for this ligand. Secondary binding at the new allosteric site has been also observed for the glucopyranosyl compounds 4 and 6.52 This binding might be attributed to the excess of the inhibitor (10 mM) used in forming the rmGPb−inhibitor complex in the crystal. Each of the inhibitors binds at the catalytic site with practically no disturbance of the native structure; rmsd between all main chain atoms of the native

a

Reagents and conditions: (a) SOCl2, reflux; (b) 1.5 equiv of ArCSNH2, 1.5 equiv of dry pyridine, dry CH3CN, rt; (c) 1.2 equiv of NH2NH2·H2O, MeOH, rt, then 20 equiv of NH2NH2·H2O, reflux.

instead of PCl5, was used to acylate thiobenzamide and 2thionaphthamide to give N-acyl-thioamides 28 in good yields. Subsequent reaction of 28 with somewhat more than one equivalent of hydrazine hydrate in MeOH at rt to form the 1,2,4-triazole ring was followed by total deprotection in the same pot by an excess of the same reagent at reflux temperature to give acceptable yields for the desired compounds 29. Toward imidazole type C-glucosyl heterocycles, amide51 30 was transformed to imidate 31 by Meerwein’s salt (Et3OBF4), which was then reacted with 2-amino-ethanones to give the fully protected 4(5)-aryl-2-(β-D-glucosaminyl)-imidazoles 32 (Scheme 3). Reaction of 31 with 1,2-diaminobenzene furnished 2-(β-D-glucosaminyl)-benzimidazole 34. Removal of the O- and N-protecting groups from 32 and 34 was effected in one operation by an excess of hydrazine hydrate in MeOH, and the target compounds 33 and 35, respectively, were obtained in good yields. Kinetic Studies. The inhibitory potency of the deprotected β-D-glucosaminyl heterocycles (25, 29, 33, 35) was determined first against rmGPb (Table 2) to show no or very weak inhibition for the N-glycosyl 1,2,3-triazoles 25 and benzimidazole 35. 1,2,4-Triazoles 29 as well as imidazoles 33 exhibiting low micromolar or even stronger inhibition were evaluated further through kinetic experiments in the direction of glycogen synthesis using rmGPa and hlGPa. All compounds proved to be competitive inhibitors with respect to the substrate Glc-1-P as revealed by the Lineweaver−Burk plots that intersected at the same point on the y-axis. Each of the inhibitors displayed similar Ki values for the three enzymes. The most potent 9253



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Table 2. Inhibition of Glycogen Phosphorylases (Ki [μM])a

a

Asterisk indicates Ki calculated from the IC50 value (650 μM) by a web-based tool.53

total of 502 Å2 (29a), 302 Å2 (29b), 283 Å2 (33a), and 306 Å2 (33b) solvent accessible surface area becomes inaccessible on binding of the ligand. The total buried surface area (protein plus ligand) for the 29a, 29b, 33a, and 33b complexes is 981, 850, 754, and 850 Å2, respectively. Although in the 29a, 29b, 33a, and 33b rmGPb complexes the 2′-NH2 group of the glucosamine engages in the same interactions as the 2′-OH of the analogous compounds 3, 4, 5, and 6,39,52 the glucosamine derived GPIs are less potent. Based on the absence of any other significant structural difference between the 29a, 29b, 33a, and 33b rmGPb complexes and the 3, 4, 5, and 639,52 rmGPb complexes, this can be attributed to the strength of the hydrogen bond interactions of the group in the 2′ position of the glucopyranose moiety. Thus, O−H···X hydrogen bonds are known to be stronger than N−H···X ones with an energy difference of ∼2 kcal/mol on average depending on the nature of X.62 Thus, the potency of 29a, 29b, 33a, and 33b follows the inhibitory potency pattern of their 2′-OH counterpart compounds 3−6, respectively, but with an order of magnitude difference. Overall, compounds 29a, 29b, 33a, and 33b form 76, 87, 71, and 85 van der Waals interactions with active site protein residues, respectively. The structural basis of the significant inhibitory potency difference between 29a and 29b and between 33a and 33b might be also attributed to the larger number of the van der Waals interactions that are formed by 29b and 33b in comparison those of 29a and 29b. Structural comparison of the 29a and 29b rmGPb complexes with a 1,2,4triazole linker to the 33a and 33b complexes that have an imidazole linker reveals that, although the additional nitrogen atom (N3) of the triazole group does not engage in any hydrogen bond interaction with protein residues, it triggers a rotation by ∼50° of the Asn284 side chain. This rotation results in small shifts of Asn284 from its respective position in the 29a and 29b complexes (Figure 3) disrupting a hydrogen bond

structure and the 29a, 29b, 33a, and 33b rmGPb complexes are 0.16, 0.17, 0.15, and 0.15 Å, respectively. The glucose moiety participates in hydrogen bonds (Table 3) and van der Waals interactions almost identical not only among the four inhibitors but also to those that have been observed for α-D-glucose and other glucose derived inhibitors.24,39,52 The 2′-NH2 group forms three hydrogen bonds with the side chains of Asn284, Tyr573, and Glu672, while it participates in water mediated hydrogen bond interactions with side chain atoms of Asp283, Thr378, and Lys574 and the main chain carbonyl of Thr671 (Figure 2). The imidazole or the triazole linker participates in a hydrogen bond interaction with the main chain oxygen of His377 (Table 3). Similar interactions have been previously observed with other C-glucosyl derivatives bearing 1,2,4-triazole or (benz)imidazole linkers34,39,52 and N-acyl β-D-glucopyranosylamine,55−57 as well as glucopyranosylidene-spiro-(thio)hydantoin type inhibitors.58−60 The strong inhibitory potency of these compounds was ascribed to the hydrogen-bond forming capacity of the NH moieties with the main chain oxygen of His377. The aromatic substituents of 29a, 29b, 33a, and 33b engage in van der Waals interactions within the βpocket (a cleft adjacent to the enzyme’s active site formed by nonpolar residues61). The structural basis of the inhibitory potency of the four compounds seems to lie in their interactions with the side chain atoms of Asn282, Asp283, and Asp284, which hold the 280s loop in its closed T-state conformation, therefore inhibiting access of the substrate to the catalytic site. The solvent accessibilities of the free and bound ligand molecules are 494 Å2 and 15 Å2 (29a), 562 Å2 and 15 Å2 (29b), 486 Å2 and 16 Å2 (33a), 558 Å2 and 14 Å2 (33b), respectively, indicating that 97% (29a, 29b, 33a, 33b) of the ligand surface becomes buried. The greatest contribution comes from the nonpolar residues that contribute 56% (29a) or the polar residues that contribute 54% (29b, 33a, 33b) of the surface, which becomes inaccessible. On the protein surface, a 9254



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interaction of Asn284 to the side chain hydroxyl group of Tyr573. This small but significant perturbation in the 280s loop may offer an explanation for the differences in the Ki values between compounds with an imidazole (33a and 33b) and those with a 1,2,4-triazole (29a and 29b). This perturbation of Asn284 has been observed before in the 3 and 4 rmGPb complexes.52 Compound 33b is the most potent compound with a modified glucopyranose ring, and it seems that the van der Waals interactions of its naphthyl moiety are able to counter significantly the negative impact of the 2′-NH2 group in the glucopyranose moiety. This might be attributed to the 11 van der Waals interactions (Asn282 (1), Phe285 (3), Phe286 (1), Arg292 (2), His341 (3), and Ala383 (1)) of the naphthyl moiety, which were also responsible for the strong inhibitory potency of 6,39 the 2′-OH counterpart of 33b. Based on the strong potency displayed by 29a, 29b, 33a, and 33b, it seems that the negative impact of substitution of the 2′ position of glucopyranose is less profound and relatively easier to counter since in previous studies derivatization of the 3′ position led to significant increment of the Ki value.43,44

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CONCLUSION Syntheses of a series of β-D-glucosaminyl heterocycles allowed study and comparison of their inhibitory efficiency against isoforms of GP. N-β-D-Glucosaminyl 1,2,3-triazoles 25 proved practically inefficient; however, C-β-D-glucosaminyl 1,2,4triazoles 29 and especially imidazoles 33 showed significant inhibition. The 2-(β-D-glucosaminyl)-4(5)-(2-naphthyl)-imidazole 33b with its 191, 125, and 143 nM inhibition constants belongs to the top five best known glucose analogue inhibitors of rmGPb, rmGPa, and also of hlGPa, the physiologically relevant enzyme. This compound has thus far proven to be the most potent glucose based inhibitor with a modified glucopyranose unit. However, glucosamine derived inhibitors are less potent than their glucopyranosyl counterparts. X-ray crystallography revealed that the structural basis of this potency difference lies with the strength of the hydrogen bond interactions formed by the group at the 2′-position of the glucopyranose moiety with active site protein residues. At the same time, the structural studies highlighted the perturbation of Asn284 caused by compounds with a 1,2,4-triazole linker in comparison to the ones with an imidazole linker providing a structural explanation for the stronger potency of the latter.

Figure 1. Crystallographic numbering of the studied inhibitors and the REFMAC weighted 2Fo − Fc electron density maps of bound ligands at the catalytic site contoured at 1.0σ before the refinement process. The final refinement models of bound inhibitor molecules are shown as ball-and-stick models.

Table 3. Potential Hydrogen Bond Interactions of Inhibitors with rmGPb Residues at the Catalytic Site in the Crystala inhibitor atoms

protein structure atoms

29a

29b

33a

33b

N2′

Asn284(OD1) Tyr573(OH) Glu672(OE2) Water261(O) Water280(O) Glu672(OE2) Ala673(N) Ser674(N) Gly675(N) Gly675(N) Water121(O) His377(ND1) Asn484(ND2) His377(O) Asn284(OD1) Water258(O)

2.7 3.1 3.2 3.4 2.8 2.7 3.3 3.1 3.1 2.9 2.6 2.7 2.8 2.9

3.0 3.2 3.0 3.4

3.2 3.2 3.2 3.3 2.9 2.7 3.3 3.1 3.2 2.9 2.6 2.7 2.8 2.8

3.1 3.1 3.2 3.2 2.9 2.7 3.4 3.1 3.2 2.9 2.7 2.7 2.8 2.8

2.8 15

2.8 15

O3′

O4′ O6′ N2 N5 total a

2.8 15

2.8 3.2 2.9 2.8 2.8 2.7 2.7 2.8 2.9 3.2 2.9 15

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EXPERIMENTAL SECTION

Synthesis. General Methods. Melting points were measured on a Kofler hot-stage and are uncorrected. Optical rotations were determined with a PerkinElmer 241 polarimeter at rt. NMR spectra were recorded with Bruker 360 (360/90 MHz for 1H/13C) or Bruker 400 (400/100 MHz for 1H/13C) spectrometers. Chemical shifts are referenced to the internal Me4Si (1H), or to the residual solvent signals (13C). Mass spectra were obtained by Thermo Scientific LTQ XL or MicroTOF-Q type Qq-TOF MS (Bruker Daltonik, Bremen, Germany) instruments. Microanalyses were performed on an Elementar Vario Micro cube instrument. TLC was performed on DC-Alurolle Kieselgel 60 F254 (Merck) plates, visualized under UV light and by gentle heating. For column chromatography Kieselgel 60 (Merck, particle size 0.063−0.200 mm) was used. Toluene, CH2Cl2, and CH3CN were distilled from P4O10 and stored over pressed sodium plates or 4 Å molecular sieves. Pyridine (VWR) and Et3OBF4 (SigmaAldrich) were purchased from the indicated suppliers. The 4-aryl-1(2′-deoxy-2′-phthalimido-3′,4′,6′-tri-O-acetyl-β-D-glucopyranosyl)1,2,3-triazoles47 (24a,b), C-(2-deoxy-2-phthalimido-3,4,6-tri-O-acetyl-

Numbers shown are distances in Å.

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Figure 2. Stereodiagrams of the binding of 29a (a), 29b (b), 33a (c), and 33b (d) at the catalytic site of rmGPb. Water molecules are drawn as cyan spheres, and hydrogen bonds are indicated as dashed lines. β-D-glucopyranosyl)formic acid51 (26), C-(2-deoxy-2-phthalimido3,4,6-tri-O-acetyl-β-D-glucopyranosyl)formamide51 (30), and the 2amino-1-arylethanone hydrochlorides39 were synthesized according to published procedures. Purity of the test compounds 25, 29, 33, and 35 (≥95%) was determined by elemental analysis. General Procedure I for Removal of the O- and N-Acyl Protecting Groups to Get Compounds 25, 29, 33, and 35. To a solution of the corresponding peracylated sugar derivative in MeOH (3 mL/100 mg), hydrazine monohydrate (20 equiv) was added, and the reaction mixture was heated at reflux temperature. When the TLC showed total consumption of the starting material, the solvent was removed under

reduced pressure and the residue was purified by column chromatography. General Procedure II for the Preparation of N-(2-Deoxy-2phthalimido-3,4,6-tri-O-acetyl-β-D-glucopyranosylcarbonyl) Arenethiocarboxamides (28). C-(2-Deoxy-2-phthalimido-3,4,6-tri-O-acetylβ-D-glucopyranosyl)formic acid51 (26, 0.20 g, 0.43 mmol) was boiled in thionyl chloride (4 mL) for 2 h. The excess SOCl2 was evaporated under reduced pressure, and then the traces were removed by repeated coevaporations with anhydrous toluene. The residual crude C-(3,4,6tri-O-acetyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl)formyl chloride51 (27) was dissolved in anhydrous CH3CN (5 mL), and a solution of the corresponding aromatic thioamide (0.65 mmol, 1.5 9256



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1-(2′-Amino-2′-deoxy-β-D-glucopyranosyl)-4-phenyl-1,2,3-triazole (25a). Prepared from 1-(2′-deoxy-2′-phthalimido-3′,4′,6′-tri-Oacetyl-β-D-glucopyranosyl)-4-phenyl-1,2,3-triazole47 (24a, 0.20 g, 0.36 mmol) according to general procedure I. Reaction time: 6 h. Purified by column chromatography (8:1 CHCl3−MeOH) to give 0.10 mg (95%) of white solid. Mp: 220−222 °C. [α]D = −13 (c 0.51, DMSO). 1 H NMR (CD3OD): δ (ppm) 8.57 (1H, s, triazole CH), 7.85 (2H, d, J = 8 Hz, Ar), 7.47−7.34 (3H, m, Ar), 5.62 (1H, d, J = 9.2 Hz, H-1′), 3.91 (1H, dd, J = 12.3, < 1 Hz, H-6′a), 3.75 (1H, dd, J = 12.3, 4.9 Hz, H-6′b), 3.62 (1H, ddd, J = 9.2, 4.9, < 1 Hz, H-5′), 3.56−3.46 (2H, 2 pseudo t, J = 8.6, 8.6 Hz in each, H-2′ and/or H-3′ and/or H-4′), 3.38 (1H, pseudo t, J = 9.2, 8.6 Hz, H-2′ or H-3′ or H-4′). 13C NMR (CD3OD): δ (ppm) 149.1 (triazole C-4), 131.6, 130.0 (2), 129.5, 126.8 (2) (Ar), 121.7 (triazole C-5), 90.4 (C-1′), 81.3, 78.1, 71.2 (C-3′ − C-5′), 62.5 (C-6′), 57.6 (C-2′). ESI-MS (positive mode) (m/z) calcd for C14H19N4O4+ [M + H]+: 307.140 and C14H18N4NaO4+ [M + Na]+: 329.122. Found: 307.140 [M + H]+, 329.123 [M + Na]+. Anal. Calcd for C14H18N4O4: C, 54.89; H, 5.92; N, 18.29. Found: C, 55.06; H, 5.96; N, 18.22. 1-(2′-Amino-2′-deoxy-β-D-glucopyranosyl)-4-(2-naphthyl)-1,2,3triazole (25b). Prepared from 1-(2′-deoxy-2′-phthalimido-3,4,6-tri-Oacetyl-β-D-glucopyranosyl)-4-(2-naphthyl)-1,2,3-triazole47 (24b, 0.20 g, 0.33 mmol) according to general procedure I. Reaction time: 6 h. Purified by column chromatography (8:1 CHCl3−MeOH) to give 88 mg (76%) of white solid. Mp: 232−234 °C. [α]D = −18 (c 0.51, DMSO). 1H NMR (DMSO-d6): δ (ppm) 8.97, 8.48 (2 × 1H, 2 s, Ar, triazole CH), 8.09−7.54 (6H, m, Ar), 5.58 (1H, d, J = 9.2 Hz, H-1′), 5.50−4.40 (br signals, OH, NH2) 3.77−3.74, 3.52−3.50, 3.39−3.28 (5H, m, H-2′ and/or H-3′ and/or H-4′, H-5′, H-6′a, H-6′b), 3.21 (1H, pseudo t, J = 9.2, 9.2 Hz, H-2′ or H-3′ or H-4′). 13C NMR (DMSO-d6): δ (ppm) 146.4 (triazole C-4), 133.1−123.5 (Ar), 120.8 (triazole C-5), 88.7 (C-1′), 80.0, 76.9, 69.6 (C-3′ − C-5′), 60.7 (C-6′), 56.4 (C-2′). ESI-MS (positive mode) (m/z) calcd for C18H21N4O4+ [M + H]+: 357.16. Found: 357.17. Anal. Calcd for C18H20N4O4: C, 60.66; H, 5.66; N, 15.72. Found: C, 60.79; H, 5.76; N, 15.67. N-(2-Deoxy-2-phthalimido-3,4,6-tri-O-acetyl-β-D-glucopyranosylcarbonyl) Benzenethiocarboxamide (28a). Prepared from C-(2deoxy-2-phthalimido-3,4,6-tri-O-acetyl-β-D -glucopyranosyl)formic acid51 (26, 0.2 g, 0.43 mmol) and thiobenzamide (0.09 g, 0.65 mmol) according to general procedure II. Purified by column chromatography (1:2 EtOAc−hexane) to yield 0.21 g (82%) red amorphous solid. Rf: 0.33 (1:1 EtOAc/hexane). [α]D = +2 (c 0.54, CHCl3). 1H NMR (CDCl3) δ (ppm): 10.41 (1H, s, NH), 7.82−7.80 (2H, m, Ar), 7.70− 7.65 (4H, m, Ar), 7.50−7.45 (1H, m, Ar), 7.36−7.31 (2H, m, Ar), 5.95 (1H, pseudo t, J = 10.3, 9.3 Hz, H-3 or 4), 5.25 (1H, pseudo t, J = 10.3, 9.2 Hz, H-3 or H-4), 5.14 (1H, d, J = 10.8 Hz, H-1), 4.62 (1H, pseudo t, J = 10.5, 10.5 Hz, H-2), 4.38−4.35 (2H, m, H-6a, H-6b), 4.09−4.02 (1H, m, H-5), 2.11, 2.06, 1.89 (3 × 3H, 3 s, CH3). 13C NMR (CDCl3) δ (ppm): 201.7 (CS), 170.5, 169.9, 169.4 (COCH3), 167.5 (NPhtCO), 163.9 (NHCO), 141.7−123.7 (Ar), 76.1, 72.7, 70.9, 68.4 (C-1, C-3 − C-5), 61.6 (C-6), 51.5 (C-2), 20.7, 20.5, 20.4 (CH3). ESI-MS (positive mode) (m/z) calcd for C28H26N2NaO10S+ [M + Na]+: 605.12. Found: 605.11. N-(2-Deoxy-2-phthalimido-3,4,6-tri-O-acetyl-β-D-glucopyranosylcarbonyl) naphthalene-2-thiocarboxamide (28b). Prepared from C(2-deoxy-2-phthalimido-3,4,6-tri-O-acetyl-β-D-glucopyranosyl)formic acid51 (26, 0.2 g, 0.43 mmol) and naphthalene-2-thiocarboxamide (0.12 g, 0.65 mmol) according to general procedure II. Purified by column chromatography (1:2 EtOAc−hexane) to yield 0.20 g (74%) of red amorphous solid. Rf: 0.33 (1:1 EtOAc−hexane). [α]D = +4 (c 0.52, CHCl3). 1H NMR (CDCl3) δ (ppm): 10.46 (1H, s, NH), 8.14 (1H, s, Ar), 7.97−7.49 (10H, m, Ar), 5.98 (1H, pseudo t, J = 9.8, 9.8 Hz, H-3 or H-4), 5.27 (1H, pseudo t, J = 9.7, 9.7 Hz, H-3 or H-4), 5.13 (1H, d, J = 10.8 Hz, H-1), 4.68 (1H, pseudo t, J = 10.6, 9.8 Hz, H2), 4.41 (1H, dd, J = 12.6, 4.7 Hz, H-6a), 4.34 (1H, dd, J = 12.4, 1.7 Hz, H-6b), 4.09−4.02 (1H, m, H-5), 2.09, 2.06, 1.90 (3 × 3H, 3 s, CH3). 13C NMR (CDCl3) δ (ppm): 202.2 (CS), 170.5, 169.9, 169.4 (COCH3), 167.6 (NPhtCO), 163.8 (NHCO), 138.7−123.7 (Ar), 76.2, 72.8, 71.0, 68.5 (C-1, C-3 − C-5), 61.7 (C-6), 51.6 (C-2), 20.7, 20.5,

Figure 3. Stereodiagrams of superposition of the 29a onto 33a (a) and 29b onto 33b (b) complexes at the catalytic site of rmGPb. The inhibitors are drawn as ball-and-stick models and the protein residues as thin cylinders. equiv) in anhydrous CH3CN (5 mL), in the presence of anhydrous pyridine (52 μL, 0.65 mmol, 1.5 equiv), was added dropwise over 10 min. The reaction mixture was then stirred at rt and monitored by TLC (1:1 EtOAc−hexane). After completion of the reaction, the mixture was concentrated under diminished pressure, and the residue was purified by column chromatography. General Procedure III for the Preparation of 5-Aryl-3-(2′-amino2′-deoxy-β-D-glucopyranosyl)-1,2,4-triazoles (29). To a solution of the corresponding N-(2-deoxy-2-phthalimido-3,4,6-tri-O-acetyl-β-Dglucopyranosylcarbonyl) arenethiocarboxamide (28) in MeOH (3 mL/100 mg), hydrazine hydrate (1.2 equiv) was added, and the reaction mixture was stirred at rt. When the TLC (2:1 EtOAc− hexane) showed complete conversion of the starting material into a ring-closed product (an intermediary fully protected glucosyl 1,2,4triazole), an excess of hydrazine hydrate (20 equiv) was added to the reaction mixture. After the reaction mixture was heated at reflux temperature for 3 h, the solvent was removed in vacuo, and the residue was purified by column chromatography. General Procedure IV for the Preparation of 4(5)-Aryl-2-(2′deoxy-2′-phthalimido-3′,4′,6′-tri-O-acetyl-β-D-glucopyranosyl)-imidazoles (32). Ethyl C-(2-deoxy-2-phthalimido-3,4,6-tri-O-acetyl-β-Dglucopyranosyl)formimidate (31) and the corresponding 2-amino-1arylethanone hydrochloride (2 equiv) were dissolved in anhydrous pyridine (4 mL/100 mg of 31), activated molecular sieves were added, and the reaction mixture was stirred at rt under Ar. After completion of the reaction monitored by TLC (3:2 EtOAc−hexane), molecular sieves were removed by filtration, and the filtrate was evaporated. The residue was dissolved in CH2Cl2 and extracted with water (2×). The organic phase was dried over MgSO4, filtered, and evaporated. The remaining crude product was purified by column chromatography. 9257



Article

123.7 (Ar), 111.3 (imidazole CH), 76.4, 71.0, 70.8, 69.1, (C-1′, C-3′ − C-5′), 62.1 (C-6′), 53.9 (C-2′), 20.8, 20.6, 20.5 (CH3). ESI-MS (positive mode) (m/z) calcd for C29H28N3O9+ [M + H]+: 562.2. Found: 562.4. 2-(2′-Deoxy-2′-phthalimido-3′,4′,6′-tri-O-acetyl-β-D-glucopyranosyl)-4(5)-(2-naphthyl)-imidazole (32b). Prepared from imidate 31 (0.29 g, 0.60 mmol) and 2-amino-1-(naphthalen-2-yl)ethanone hydrochloride (0.27 g, 1.22 mmol) according to general procedure IV. Purified by column chromatography (1:1 EtOAc−hexane) to obtain 0.15 g (41%) of yellow syrup. Rf: 0.22 (1:1 EtOAc−hexane). [α]D = +8 (c 1.10, CHCl3). 1H NMR (CDCl3): δ (ppm) 9.93 (1H, s, NH), 7.95−7.36 (12H, m, Ar, imidazole CH), 6.14 (1H, pseudo t, J = 10.2, 9.4 Hz, H-3′ or H-4′), 5.64 (1H, d, J = 10.2 Hz, H-1′), 5.28 (1H, pseudo t, J = 10.2, 9.4 Hz, H-3′ or H-4′), 4.37 (1H, dd, J = 12.5, 4.7 Hz, H-6′a), 4.26 (1H, dd, J = 12.5, 1.6 Hz, H-6′b), 4.14−4.07 (2H, m, H-2′, H-5′), 2.09, 2.07, 1.92 (3 × 3H, 3 s, CH3). 13C NMR (CDCl3): δ (ppm) 170.9, 170.0, 169.6 (COCH3), 168.4, 167.5 (NPhtCO), 143.3, (imidazole C-2), 134.1−122.3 (Ar, imidazole CH), 76.3, 71.0, 70.9, 69.0 (C-1′, C-3′ − C-5′), 62.2 (C-6′), 53.9 (C-2′), 20.7, 20.6, 20.5 (CH3). ESI-MS (positive mode) (m/z) calcd for C25H25N2O7+ [M + H]+: 612.2. Found: 612.8. 2-(2′-Amino-2′-deoxy-β-D-glucopyranosyl)-4(5)-phenyl-imidazole (33a). Prepared from 32a (0.30 g, 0.53 mmol) according to general procedure I. Reaction time: 3 h. Purified by column chromatography (7:2 CHCl3−MeOH) to give 0.12 g (74%) of white amorphous solid. Rf: 0.27 (7:2 CHCl3−MeOH). [α]D = +4 (c 0.49, MeOH). 1H NMR (CD3OD): δ (ppm) 7.71−7.21 (6H, m, Ar, imidazole CH), 4.43 (1H, d, J = 10.0 Hz, H-1′), 3.92 (1H, dd, J = 11.8, 1.8 Hz, H-6′a), 3.73 (1H, dd, J = 11.8, 5.4 Hz, H-6′b), 3.51−3.40 (3H, m, H-2′ and/or H-3′ and/or H-4′, H-5′), 3.12 (1H, pseudo t, J = 9.5, 9.5 Hz, H-2′ or H-3′ or H-4′). 13C NMR (CD3OD): δ (ppm) 147.6 (imidazole C-2), 139.3 (imidazole C-4), 134.1, 129.8, 128.0, 125.9 (Ar), 117.9 (imidazole CH), 82.4, 78.2, 76.3, 71.7 (C-1′, C-3′ − C-5′), 62.9 (C-6′), 57.5 (C-2′). ESI-MS (positive mode) (m/z) calcd for C15H20N3O4+ [M + H]+: 306.14. Found: 306.17. Anal. Calcd for C15H19N3O4: C, 59.01; H, 6.27; N, 13.76. Found: C, 59.20; H, 6.39; N, 13.79. 2-(2′-Amino-2′-deoxy-β-D-glucopyranosyl)-4(5)-(2-naphthyl)-imidazole (33b). Prepared from 32b (0.10 g, 0.16 mmol) according to general procedure I. Reaction time: 5 h. Purified by column chromatography (7:2 CHCl3−MeOH) to give 45 mg (78%) of white amorphous solid. Rf: 0.19 (7:2 CHCl3−MeOH). [α]D = +3 (c 1.25, MeOH). 1H NMR (CD3OD) δ (ppm): 8.11 (1H, s, Ar), 7.79− 7.72 (4H, m, Ar) 7.43 (1H, s, imidazole CH) 7.40−7.32 (2H, m, Ar), 4.39 (1H, d, J = 10.2 Hz, H-1′), 3.89 (1H, dd, J = 11.7, 1.6 Hz, H-6′a), 3.73 (1H, dd, J = 11.7, 5.5 Hz, H-6′b), 3.50−3.43 (3H, m, H-2′ and/or H-3′ and/or H-4′, H-5′), 3.07 (1H, pseudo t, J = 9.4, 9.4 Hz, H-2′ or H-3′ or H-4′). 13C NMR (CD3OD): δ (ppm) 148.0 (imidazole C-2), 139.2 (imidazole C-4), 135.2, 134.0, 131.4, 129.3, 128.9, 128.7, 127.4, 126.6, 124.6, 123.7 (Ar), 118.5 (imidazole CH), 82.3, 78.7, 76.8, 71.6, (C-1′, C-3′− C-5′), 62.8 (C-6′) 57.6 (C-2′). ESI-MS (positive mode) (m/z) calcd for C19H22N3O4+ [M + H]+: 356.2. Found: 356.3. Anal. Calcd for C19H21N3O4: C, 64.21; H, 5.96; N, 11.82. Found: C, 64.29; H, 5.87; N, 11.84. 2-(2′-Deoxy-2′-phthalimido-3′,4′,6′-tri-O-acetyl-β-D-glucopyranosyl)-benzimidazole (34). A solution of imidate 31 (0.60 g, 1.2 mmol) and 1,2-diaminobenzene (0.26 g, 2.4 mmol, 2 equiv) in anhydrous CH2Cl2 (12 mL) was stirred at reflux temperature, and the reaction was monitored by TLC (2:1 EtOAc−hexane). After 1 day, the solvent was removed under diminished pressure, and the title compound was obtained from the residue by crystallization from Et2O to yield 0.49 g (75%) of white solid. Mp: 135−138 °C. [α]D = −53 (c 0.50, CHCl3). 1H NMR (CDCl3): δ (ppm) 9.87 (1H, s, NH), 7.80−7.05 (8H, m, Ar), 6.10 (1H, pseudo t, J = 9.9, 9.2 Hz, H-3′ or H4′), 5.79 (1H, d, J = 9.9 Hz, H-1′), 5.30 (1H, pseudo t, J = 9.9, 9.2 Hz, H-3′ or H-4′), 4.61 (1H, pseudo t, J = 9.9, 9.9 Hz, H-2′), 4.42 (1H, dd, J = 11.9, 5.3 Hz, H-6′a), 4.30 (1H, dd, J = 11.9, 2.6 Hz, H-6′b), 4.17 (1H, ddd, J = 9.9, 5.3, 2.6 Hz, H-5′), 2.12, 2.09, 1.88 (3 × 3H, 3 s, CH3). 13C NMR (CDCl3): δ (ppm) 170.9, 170.0, 169.6 (COCH3), 168.2, 167.0 (NPhtCO), 148.9 (benzimidazole C-2), 142.8 (benzimi-

20.4 (CH3). ESI-MS (positive mode) (m/z) calcd for C32H29N2O10S+ [M + H]+: 633.2. Found: 633.3. 3-(2′-Amino-2′-deoxy-β-D-glucopyranosyl)-5-phenyl-1,2,4-triazole (29a). Prepared from 28a (0.2 g, 0.35 mmol) according to general procedure III. Purified by column chromatography (1:3 MeOH− CHCl3) to yield 0.04 g (40%) of pale yellow amorphous solid. Rf: 0.21 (1:3 MeOH−CHCl3). [α]D = +11 (c 0.48, MeOH). 1H NMR (CD3OD) δ (ppm): 8.01−7.98 (2H, m, Ar), 7.48−7.46 (3H, m, Ar), 4.47 (1H, d, J = 9.9 Hz, H-1′), 3.93 (1H, dd, J = 12.0, 1.9 Hz, H-6′a), 3.73 (1H, dd, J = 12.0, 5.4 Hz, H-6′b), 3.51−3.43 (3H, m, H-2′ and/or H-3′ and/or H-4′, H-5′), 3.09 (1H, m, H-2′ or H-3′ or H-4′). 13C NMR (CD3OD) δ (ppm): 159.2, 158.7 (triazole C-3, C-5), 129.9, 128.8, 126.3 (Ar), 81.2, 77.6, 75.3, 70.4 (C-1′, C-3′ − C-5′), 61.7 (C6′), 56.3 (C-2′). ESI-MS (positive mode) (m/z) calcd for C14H19N4O4+ [M + H]+: 307.14. Found: 307.17. Anal. Calcd for C14H18N4O4: C, 54.89; H, 5.92; N, 18.29. Found: C, 55.12; H, 6.09; N, 18.37. 3-(2′-Amino-2′-deoxy-β-D-glucopyranosyl)-5-(2-naphthyl)-1,2,4triazole (29b). Prepared from 28b (0.2 g, 0.31 mmol) according to general procedure III. Purified by column chromatography (1:3 MeOH−CHCl3) to yield 0.075 g (68%) of pale yellow amorphous solid. Rf: 0.20 (1:3 MeOH−CHCl3). [α]D = +10 (c 0.48, MeOH). 1H NMR (CD3OD) δ (ppm): 8.47 (1H, s, Ar) 8.05−7.49 (6H, m, Ar), 4.53 (1H, d, J = 9.9 Hz, H-1′), 3.95 (1H, dd, J = 12.0, 1.9 Hz, H-6′a), 3.76 (1H, dd, J = 12.0, 5.3 Hz, H-6′b), 3.56−3.48 (3H, m, H-2′ and/or H-3′ and/or H-4′, H-5′), 3.14 (1H, m, H-2′ or H-3′ or H-4′). 13C NMR (CD3OD) δ (ppm): 160.3, 160.0 (triazole C-3, C-5), 135.4, 134.6, 129.7, 129.5, 128.8, 128.2, 127.8, 127.2, 127.1, 124.6 (Ar), 82.4, 78.6, 76.4, 71.6 (C-1′, C-3′ − C-5′), 62.9 (C-6′), 57.5 (C-2′). ESI-MS (positive mode) (m/z) calcd for C18H21N4O4+ [M + H]+: 357.2. Found: 357.3. Anal. Calcd for C18H20N4O4: C, 60.66; H, 5.66; N, 15.72. Found: C, 60.91; H, 5.70; N, 15.83. Ethyl C-(2-Deoxy-2-phthalimido-3,4,6-tri-O-acetyl-β- D glucopyranosyl)formimidate (31). To a solution of C-(2-deoxy-2phthalimido-3,4,6-tri-O-acetyl-β-D-glucopyranosyl)form-amide51 (30, 1.0 g, 2.16 mmol) in anhydrous CH2Cl2 (15 mL), Et3OBF4 (1.23 g, 6.49 mmol, 3 equiv) and activated 4 Å molecular sieves (powder, 500 mg) were added, and the reaction mixture was stirred at rt under Ar. When the TLC (3:2 EtOAc−hexane) showed complete conversion of the starting material (3 h), molecular sieves were filtered off. The filtrate was diluted with CH2Cl2 (40 mL) and extracted with satd NaHCO3 solution (2 × 30 mL), then with water (30 mL). The organic phase was dried over MgSO4, filtered, and evaporated. The resulting amorphous solid (1.03 g, 97%) was used without further purification. Rf: 0.36 (1:1 EtOAc−hexane). [α]D = +34 (c 0.50, CHCl3). 1H NMR (CDCl3) δ (ppm): 7.88−7.77 (5H, m, Ar, NH), 5.96, 5.17 (2 × 1H, 2 pseudo t, J = 9.9, 9.2 Hz in each, H-3, H-4), 4.78 (1H, d, J = 9.9 Hz, H-1), 4.38−4.33 (2H, m, H-2, H-6a), 4.23 (1H, dd, J = 12.6, < 1 Hz, H-6b), 3.98 (1H, ddd, J = 9.2, 5.3, < 1 Hz, H-5), 3.91−3.88 (2H, m, CH2), 2.13, 2.05, 1.89 (3 × 3H, 3 s, CH3), 0.60 (3H, t, 7.9 Hz, CH3). 13C NMR (CDCl3) δ (ppm): 170.6, 170.0, 169.4 (COCH3), 167.9 (NPhtCO), 167.6 (CN), 167.0 (NPhtCO), 134.3, 131.7, 131.2, 123.5 (Ar), 75.4, 71.2, 71.1, 68.6 (C-1, C-3 − C-5), 61.9, 61.7 (C-6, CH2), 52.6 (C-2), 20.7, 20.5, 20.4 (3 × CH3), 13.1 (CH3). ESI-MS (positive mode) (m/z) calcd for C23H26N2NaO10+ [M + Na]+: 513.149. Found: 513.146. 2-(2′-Deoxy-2′-phthalimido-3′,4′,6′-tri-O-acetyl-β-D-glucopyranosyl)-4(5)-phenyl-imidazole (32a). Prepared from imidate 31 (0.85 g, 1.73 mmol) and phenacylamine hydrochloride (0.59 g, 3.47 mmol) according to general procedure IV. Reaction time: 3 d. Purified by column chromatography (1:1 EtOAc−hexane) to obtain 0.60 g (62%) of yellow syrup. Rf: 0.47 (3:2 EtOAc−hexane). [α]D = +20 (c 0.50, CHCl3). 1H NMR (CDCl3): δ (ppm) 9.62 (1H, s, NH), 7.93−7.07 (10H, m, Ar, imidazole CH), 6.11 (1H, pseudo t, J = 9.9, 9.2 Hz, H-3′ or H-4′), 5.59 (1H, d, J = 9.9 Hz, H-1′), 5.26 (1H, pseudo t, J = 9.9, 9.2 Hz, H-3′ or H-4′), 4.53 (1H, pseudo t, J = 9.9, 9.9 Hz, H-2′), 4.36 (1H, dd, J = 11.9, 2.6 Hz, H-6′a), 4.26 (1H, dd, J = 11.9, < 1 Hz, H6′b), 4.08 (1H, J = 9.9, 2.6, < 1 Hz, H-5′), 2.11, 2.07, 1.92 (3 × 3H, 3 s, CH3). 13C NMR (CDCl3): δ (ppm) 170.9, 170.0, 169.6 (COCH3), 168.5, 167.6 (NPhtCO), 142.9, 141.6 (imidazole C-2, C-4), 134.1− 9258



Article

Table 4. Summary of the Diffraction Data Processing and Refinement Statistics for the rmGPb Complexesa

a

rmGPb complex

29a

resolution (Å) outermost shell (Å) reflections measured unique reflections multiplicity Rsymm completeness (%) ⟨I/σI⟩

13.91−2.45 2.55−2.45 338576 33526 (3345) 10.1 (7.3) 0.130 (0.775) 94.2 (84.9) 17.5 (2.9)

Rcryst Rfree no. solvent molecules

0.144 (0.205) 0.187 (0.221) 238

bond lengths (Å) angles (deg)

0.009 1.3

protein atoms solvent molecules inhibitor atoms PDB entry

18.3 25.0 17.0 5O54

29b Data Collection and Processing Statistics 13.56−2.45 2.56−2.45 175278 35487 (4119) 4.9 (3.4) 0.122 (0.793) 98.1 (94.8) 11.1 (1.3) Refinement Statistics 0.164 (0.269) 0.229 (0.285) 199 rms Deviation from Ideality 0.022 2.2 Average B Factor (Å2) 19.2 25.6 17.8 5O56

33a

33b

40.86−1.90 2.00−1.90 352273 76499 (10969) 4.6 (4.6) 0.073 (0.468) 99.6 (99.2) 12.0 (3.2)

38.42−1.90 2.00−1.90 303742 75575 (10839) 4.0 (4.0) 0.073 (0.450) 98.4 (97.7) 11.8 (3.5)

0.130 (0.219) 0.161 (0.244) 321

0.130 (0.212) 0.158 (0.231) 304

0.009 1.3

0.010 1.3

22.0 37.0 18.4 5O50

20.6 35.3 17.6/48.9b 5O52

Values in parentheses are for the outermost shell. bValues for inhibitor molecules bound at the catalytic and the new allosteric site, respectively. NaOH in a ratio 4:1; 700 μL of this solution was then added to reaction samples and samples were left for 15 min at 30 °C. The resultant chromophore produced was measured at 850 nm.65 The optical density measured was corrected by subtracting the optical density of samples that contain (i) the enzyme with glycogen and (ii) Glc-1-P with the inhibitor, at the concentrations used for the assay. The amount of orthophosphate ions produced was calculated using a plot of optical density measurements vs solutions of standard orthophosphate concentrations. Initial velocities were calculated from the pseudo-first order rate constants using five time intervals. Initial velocities were then plotted using the Michaelis−Menten equation.66 This procedure was repeated for each inhibitor using concentrations of 10, 20, 30, and 40 μM for 29a; 2, 3, 5, and 7 μM for 29b; 1, 2, 3, and 4 μM for 33a; 0.05, 0.10, 0.15, and 0.20 μM for 33b; and 312.5, 400, 475, 550, and 625 μM for 35. Inhibition constant (Ki) values were then calculated from the plot of 1/v vs [inhibitor]. For the calculation and statistical evaluation of the kinetic parameters, the nonlinear regression program GRAFIT67 was used by employing explicit weighting at each stage (i.e., a propagation of errors for each value was used to derive the next). The results of kinetic experiments have been calculated form three independent experiments. The inhibitory constants (Ki) are expressed as a mean ± SD of the independent experiments. To calculate IC50 for 25a and 25b, this procedure (at constant, 4 mM concentration of Glc-1-P) was repeated by using the inhibitor concentrations of 6.25, 12.5, 31.25, 62.5, 125, 312.5, and 625 μM. X-ray Crystallography. X-ray crystallographic binding studies were performed by diffusion of a 1 (29a, 29b) or 10 mM (33a, 33b) solution of the inhibitors in the crystallization media supplemented with DMSO (10−15%, v/v) in preformed rmGPb crystals at room temperature for 3 h prior to data collection. Tetragonal T state rmGPb crystals were grown as described previously.64 These crystals belong to space group P43212 with unit cell dimensions a = b = 128.6 Å and c = 116.3 Å. X-ray diffraction data were collected using synchrotron radiation (on station ID911−2 at MAX-Lab Synchrotron Radiation Source in Lund, Sweden) or using a Cu X-ray microfocus source (Oxford Diffraction SuperNova) equipped with a 4 kappa goniometer and the ATLAS CCD (135 mm) detector at room temperature. Crystal orientation, integration of reflections, interframe scaling, partial reflection summation, and data reduction was performed by the program Mosflm68 or CrysalisPro.69 Scaling and merging of intensities

dazole C-3a, C-7a), 134.0, 133.5, 131.3, 130.6, 123.2, 121.9, 119.3, 111.1 (NPht, benzimidazole C-4 − C-7), 76.3, 71.4, 71.1, 69.0 (C-1′, C-3′ − C-5′), 62.4 (C-6′), 53.9 (C-2′), 20.6 (2), 20.3 (CH3). ESI-MS (positive mode) (m/z) calcd for C27H26N3O9+ [M + H]+: 536.2. Found: 536.4. 2-(2′-Amino-2′-deoxy-β-D-glucopyranosyl)-benzimidazole (35). Prepared from 34 (0.15 g, 0.28 mmol) according to general procedure I. Reaction time: 3 h. Purified by column chromatography (7:2 CHCl3−MeOH) to yield 70 mg (90%) of white solid. Mp: 151−153 °C. [α]D = −2 (c 0.50, DMSO). 1H NMR (CD3OD): δ (ppm) 7.56, 7.23 (2 × 2H, 2 br s, Ar), 4.50 (1H, d, J = 9.2 Hz, H-1′), 3.94 (1H, dd, J = 11.9, < 1 Hz, H-6′a), 3.77 (1H, dd, J = 11.9, 5.3 Hz, H-6′b), 3.47 (3H, m, H-2′ and/or H-3′ and/or H-4′, H-5′), 3.03 (1H, pseudo t, J = 9.2, 9.2 Hz, H-2′ or H-3′ or H-4′). 13C NMR (CD3OD): δ (ppm) 153.7 (benzimidazole C-2), 139.4 (benzimidazole C-3a, C-7a), 123.8, 116.1 (benzimidazole C-4 − C-7), 82.4, 79.0, 77.6, 71.5 (C-1′, C-3′ − C-5′), 62.8 (C-6′), 57.9 (C-2′). ESI-MS (positive mode) (m/z) calcd for C13H18N3O4+ [M + H]+: 280.13. Found: 280.17. Anal. Calcd for C13H17N3O4: C, 55.91; H, 6.14; N, 15.05. Found: C, 56.16; H, 6.09; N, 15.08. Protein Production and Purification. rmGPb was purified from rabbit skeletal muscle following the protocol developed by Fischer and Krebs63 with a slight modification (L-cysteine was replaced with 2mercaptoethanol).64 Human liver GPa was purified as described earlier.39 Kinetic Studies. Kinetic studies were performed at 30 °C in the direction of glycogen synthesis by measuring the inorganic phosphate released in the reaction using the method by Saheki et al.65 rmGPb or rmGPa (3 μg/mL) or hlGPa (1 μg/mL) was assayed in a 30 mM imidazole/HCl buffer (pH 6.8) containing 60 mM KCl, 0.6 mM EDTA, and 0.6 mM dithiothreitol using constant concentrations of glycogen (0.2% w/v) and AMP (1 mM; only for the rmGPb experiments), and various concentrations of Glc-1-P (2, 3, 4, 6, and 10 mM for rmGPb and 1, 2, 3, 4, and 6 mM for hlGPa) and inhibitors. Enzyme and glycogen were preincubated for 15 min at 30 °C before initiating the reaction with Glc-l-P. As the reaction took place, at time intervals of 1, 2, 3, and 4 min from the start 40 μL was removed from the reaction mix and were added to 10 μL of an SDS solution (1% w/ v) to stop the reaction. A solution was then prepared by mixing 15 mM ammonium molybdate, 100 mM zinc acetate, pH 5.0, solution with ascorbic acid (10% w/v) solution adjusted to pH 5 with 10 N 9259



Article

were performed by SCALA and Aimless,70 and the optimum resolution was selected based on the CC1/2 criterion.71 Crystallographic refinement of the complexes was performed by maximumlikelihood methods using REFMAC.70 The starting model employed for the refinement of the complexes was the structure of the native T state rmGPb complex determined at 1.9 Å resolution (Leonidas et al., unpublished results). Ligand molecule coordinates and topologies were constructed using JLigand,72 and they were fitted to the electron density maps after adjustment of their torsion angles. Alternate cycles of manual rebuilding with the molecular graphic program COOT70 and refinement with REFMAC73 improved the quality of the models. A summary of the data processing and refinement statistics for the inhibitor complex structures is given in Table 4, and the validity of the refinement procedure was checked using the PDB_REDO server.74 As there were more than 5 reflections per atom available, both an isotropic and an anisotropic B-factor model were considered, and the isotropic B-factor model was selected based on the Hamilton R ratio test. A TLS model for grouped atom movement with one TLS group was used. The stereochemistry of the protein residues was validated by MolProbity.75 Hydrogen bonds and van der Waals interactions were calculated with the program CONTACT as implemented in CCP470 applying a distance cut off 3.3 and 4.0 Å, respectively. Figures were prepared with CCP4 Molecular Graphics.76 The coordinates of the new structures have been deposited with the RCSB Protein Data Bank (http://www.rcsb.org/pdb) with codes presented in Table 4.

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(OTKA PD105808); the project GINOP-2.3.2-15-2016-00008 funded by the EU and cofinanced by the European Regional Development Fund; the Postgraduate Programmes ‘‘Biotechnology-Quality assessment in Nutrition and the Environment”, ‘‘Application of Molecular Biology-Molecular Genetics-Molecular Markers”, Department of Biochemistry and Biotechnology, University of Thessaly. Work at the Synchrotron Radiation Sources, MAX-lab, Lund, Sweden was supported from the EU FP7 Programme (FP7/2007-2013) under BioStruct-X (grant agreement N°283570). E.K. acknowledges financial support from the Hellenic State Scholarships Foundation funded by the “Operational Programme Education and Lifelong Learning” cofunded by the European Social Fund (ESF) and National Resources.

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ABBREVIATIONS USED GP, glycogen phosphorylase; rmGP, rabbit muscle glycogen phosphorylase; hlGP, human liver glycogen phosphorylase; GPIs, glycogen phosphorylase inhibitors; T2DM, type 2 diabetes mellitus; Glc-1-P, glucose-1-phosphate; rmsd, rootmean-square deviation; SDS, sodium dodecyl sulfate

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ASSOCIATED CONTENT

S Supporting Information *

(1) Henke, B. R. Inhibition of glycogen phosphorylase as a strategy for the treatment of type 2 diabetes. RSC Drug Discovery Ser. 2012, 27, 324−365. (2) Gaboriaud-Kolar, N.; Skaltsounis, A.-L. Glycogen phosphorylase inhibitors: a patent review (2008−2012). Expert Opin. Ther. Pat. 2013, 23, 1017−1032. (3) Donnier-Maréchal, M.; Vidal, S. Glycogen phosphorylase inhibitors: a patent review (2013−2015). Expert Opin. Ther. Pat. 2016, 26, 199−212. (4) Martin, W. H.; Hoover, D. J.; Armento, S. J.; Stock, I. A.; McPherson, R. K.; Danley, D. E.; Stevenson, R. W.; Barrett, E. J.; Treadway, J. L. Discovery of a human liver glycogen phosphorylase inhibitor that lowers blood glucose in vivo. Proc. Natl. Acad. Sci. U. S. A. 1998, 95, 1776−1781. (5) Furukawa, S.; Murakami, K.; Nishikawa, M.; Nakayama, O.; Hino, M. FR258900, a novel glycogen phosphorylase inhibitor isolated from Fungus No. 138354 - II. Anti-hyperglycemic effects in diabetic animal models. J. Antibiot. 2005, 58, 503−506. (6) Docsa, T.; Czifrák, K.; Hüse, C.; Somsák, L.; Gergely, P. The effect of glucopyranosylidene-spiro-thiohydantoin on the glycogen metabolism in liver tissues of streptozotocin-induced and obese diabetic rats. Mol. Med. Rep. 2011, 4, 477−481. (7) Docsa, T.; Marics, B.; Németh, J.; Hüse, C.; Somsák, L.; Gergely, P.; Peitl, B. Insulin sensitivity is modified by a glycogen phosphorylase inhibitor: glucopyranosylidene-spiro-thiohydantoin in streptozotocininduced diabetic rats. Curr. Top. Med. Chem. 2015, 15, 2390−2394. (8) Goyard, D.; Kónya, B.; Chajistamatiou, A. S.; Chrysina, E. D.; Leroy, J.; Balzarin, S.; Tournier, M.; Tousch, D.; Petit, P.; Duret, C.; Maurel, P.; Somsák, L.; Docsa, T.; Gergely, P.; Praly, J.-P.; AzayMilhau, J.; Vidal, S. Glucose-derived spiro-isoxazolines are antihyperglycemic agents against type 2 diabetes through glycogen phosphorylase inhibition. Eur. J. Med. Chem. 2016, 108, 444−454. (9) Tracey, W.; Treadway, J.; Magee, W.; McPherson, R.; Levy, C.; Wilder, D.; Li, Y.; Yue, C.; Zavadoski, W.; Gibbs, E.; Smith, A.; Flynn, D.; Knight, D. A novel glycogen phosphorylase inhibitor, CP-368296, reduces myocardial ischemic injury. Diabetes 2003, 52, A135. (10) Tracey, W. R.; Treadway, J. L.; Magee, W. P.; Sutt, J. C.; McPherson, R. K.; Levy, C. B.; Wilder, D. E.; Yu, L. J.; Chen, Y.; Shanker, R. M.; Mutchler, A. K.; Smith, A. H.; Flynn, D. M.; Knight, D. R. Cardioprotective effects of ingliforib, a novel glycogen phosphorylase inhibitor. Am. J. Physiol.-Heart Circul. Physiol. 2004, 286, H1177−H1184.

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b01056. Copies of NMR spectra of the synthesized compounds (PDF) Molecular formula strings (CSV) Structure of 29a (PDB) Structure of 29b (PDB) Structure of 33a (PDB) Structure of 33b (PDB) Accession Codes

PDB codes: 29a 5O54, 29b 5O56, 33a 5O50, 33b 5O52. Authors will release the atomic coordinates and experimental data upon article publication.

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*Prof. László Somsák. Phone: +36 52512900, ext 22348. Fax: +36 52512744. E-mail: [email protected]. *Prof. Demetres D. Leonidas. Phone: +30 2410 565278. Fax: +30 2410 565290. E-mail: [email protected]. ORCID

Demetres D. Leonidas: 0000-0002-3874-2523 László Somsák: 0000-0002-9103-9845 Present Address ⊥

A.L.K.: Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, U.K. Author Contributions #

E.B. and E.K. have equally contributed to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support of the studies is gratefully acknowledged from the following sources: Hungarian Scientific Research Fund 9260



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