Glucopyranosylidene-spiro-imidazolinones, a New Ring System

Article Cite This: J. Med. Chem. 2019, 62, 6116−6136

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Glucopyranosylidene-spiro-imidazolinones, a New Ring System: Synthesis and Evaluation as Glycogen Phosphorylase Inhibitors by Enzyme Kinetics and X‑ray Crystallography

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Katalin E. Szabó,†,⊥ Efthimios Kyriakis,‡,⊥ Anna-Maria G. Psarra,‡ Aikaterini G. Karra,‡ Á dám Sipos,§ Tibor Docsa,§ George A. Stravodimos,‡ Elisabeth Katsidou,‡ Vassiliki T. Skamnaki,‡ Panagiota G. V. Liggri,‡,∥ Spyros E. Zographos,∥ Attila Mándi,† Sándor Balázs Király,† Tibor Kurtán,† Demetres D. Leonidas,*,‡ and László Somsák*,† †

Department of Organic Chemistry, University of Debrecen, P.O. Box 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: Epimeric series of aryl-substituted glucopyranosylidene-spiro-imidazolinones, an unprecedented new ring system, were synthesized from the corresponding Schiff bases of O-perbenzoylated (gluculopyranosylamine)onamides by intramolecular ring closure of the aldimine moieties with the carboxamide group elicited by N-bromosuccinimide in pyridine. Test compounds were obtained by Zemplén O-debenzoylation. Stereochemistry and ring tautomers of the new compounds were investigated by NMR, time-dependent density functional theory (TDDFT)-electronic circular dichroism, and DFT-NMR methods. Kinetic studies with rabbit muscle and human liver glycogen phosphorylases showed that the (R)-imidazolinones were 14−216 times more potent than the (S) epimers. The 2-naphthyl-substituted (R)-imidazolinone was the best inhibitor of the human enzyme (Ki 1.7 μM) and also acted on HepG2 cells (IC50 177 μM). X-ray crystallography revealed that only the (R) epimers bound in the crystal. Their inhibitory efficacy is based on the hydrogen-bonding interactions of the carbonyl oxygen and the NH of the imidazolinone ring.

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the easily available rabbit muscle GPb, rmGPb7 ): Cglucopyranosyl heterocycles, 8 N-acyl-N′-glucopyranosyl ureas,3−5 and glucopyranosylidene-spiro-heterocycles.5 Several studies conducted with glucose-derived GP inhibitors (GPIs) demonstrated the ex vivo and in vivo efficacy of these compounds. Thus, glucopyranosylidene-spiro-thiohydantoin (TH) diminished blood sugar levels in Zucker diabetic rats9 and also increased plasma insulin levels as well as restored whole body insulin sensitivity.10 A single dose administration of a spiro-isoxazoline to Zucker rats diminished the hepatic glucose output by ∼1/3, an effect that may be relevant for

INTRODUCTION

Hepatic glycogen phosphorylase (GP), the rate-determining enzyme of glycogen degradation, has been a validated target in the quest of new therapeutic possibilities for type 2 diabetes mellitus (T2DM). Since GP has a direct effect on blood sugar levels, its inhibition may diminish the production of glucose by the liver known to be elevated in T2DM patients.1,2 A large number of compounds were shown to inhibit GP,3 and among them, glucose derivatives, most often binding to the catalytic site of the enzyme as revealed by X-ray crystallographic studies, represent the most intensively investigated class of inhibitors.4−6 Three main groups of glucose analogues have been shown to exhibit submicromolar effects against glycogen phosphorylase (mainly studied with © 2019 American Chemical Society

Received: February 26, 2019 Published: June 18, 2019 6116

DOI: 10.1021/acs.jmedchem.9b00356 J. Med. Chem. 2019, 62, 6116−6136

Journal of Medicinal Chemistry

Article

therapeutic use.11 Treatment by TH and some N-acyl-N′glucopyranosyl ureas improved glucose tolerance in mice and resulted in unexpected metabolic effects in the liver such as enhanced oxygen consumption and elevation of mammalian target of rapamycin complex 2 levels.12 Furthermore, an Nacyl-N′-glucopyranosyl urea increased the size of pancreatic Langerhans islets and improved glucose-induced insulin secretion in mice, thereby raising the possibility of using GP inhibitors to preserve or even ameliorate the β-cell function.13 The glucopyranosylidene-spiro-(thio)hydantoins A (Chart 1) were the first glucose derivatives with low μM inhibition

After these experiences, we turned to imidazolinones C (X = NH), whose syntheses, kinetic tests with GP enzymes, ex vivo evaluation with hepatic cells, and X-ray crystallographic study of their enzyme−inhibitor complexes are described in this paper.

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RESULTS AND DISCUSSION Syntheses. A literature survey revealed several syntheses to get 2,5,5-trisubstituted imidazolinones inclusive spirocyclic structures (Scheme 1). The most often used procedure starts

Scheme 1. Literature Syntheses of 2,5,5-Trisubstituted Imidazolinones

Chart 1. Selected Glucopyranosylidene-spiro-heterocyclic Inhibitors of Glycogen Phosphorylase and the Target Compounds of This Work

with an α-amino-carboxamide I, which is N-acylated to II whose base induced ring closure results in the target compounds IV.21−24 Direct transformation of I with an orthoester21 and that of the analogous α-amino-ester V with an imidate25 to give IV have also been reported. Compounds I can also be reacted with aldehydes to yield imidazolidinones III, which upon oxidation lead to the target IV.26,27 Since the corresponding glucose-derived starting materials28 I, II, and V were available to us from earlier studies, several experiments were carried out on routes A, C, and D (Scheme 1) to ring-close these compounds under conditions suggested in the literature; however, these attempts resulted in either no reaction, decomposition, or the formation of products other than IV. Next, we turned to the transformations of the glucosederived α-amino-carboxamide 1 with aldehydes. The reaction of 128 with benzaldehyde in refluxing MeOH in the presence of catalytic AcOH as suggested in the literature26 resulted in a 50% conversion of the starting material and gave, instead of the expected imidazolidinone-type product, an inseparable mixture of the epimeric Schiff bases 2 and 20 (Scheme 2) (ratio 2:1) in 41% combined yield. Under base-catalyzed conditions, in the presence of 10 mol % pyrrolidine in CH2Cl2 at room temperature (rt),29 2 was formed as a single stereoisomer in 86% yield, and these conditions could be extended to other aldehydes to give condensation products 3−5 in good yields. Ring closure to get the target imidazolinones (e.g., 6) was attempted first by heating 2 in refluxing xylene for 3 days or in

constants.14,15 Based on X-ray crystallography investigations of both hydantoins’ complexes with rmGPb, the strong binding was ascribed to the rigid spirobicyclic structure, the hydrogen bond between the β-NH and His377 next to the binding site, and the participation of the hydantoin polar groups (e.g., the α-CO) in water-mediated H-bond networks to the protein.16,17 Later, other spirocyclic compounds such as the oxathiazoles18 (B, X = S) and isoxazolines19 (B, X = CH2) became available and showed nanomolar inhibition if the substituent was a 2-naphthyl group. In these compounds, the H-bridge toward His377 does not exist and also the α-CO group is absent; therefore, the strong binding must be due to the interactions of the aromatic moiety and the so-called βchannel of the enzyme. These, mainly van der Waals contacts, may even override the contributions of the above polar interactions. To unify the important features of these spirocycles, the syntheses of compounds C were envisaged. The preparation of aryl-thiazolinones (C, X = S) was achieved in O-peracetylated or O-perbenzoylated forms; however, on attempting deprotections, the addition of alcohols onto the CN double bond was observed. An analogous addition took place also with water under neutral conditions; therefore, the enzyme kinetic studies of these compounds, necessarily to be done in aqueous medium, failed.20 6117



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Scheme 2. Syntheses of Glucopyranosylidene-spiro-imidazolinones

To find a more straightforward route to imidazolinones 24− 27, the corresponding Schiff bases 20−23 were also prepared. This was envisaged in a Staudinger-type reaction of α-azidocarboxamide 14 28 based on earlier observations with glycopyranosyl azides to react with different phosphines to give phosphinimines whose reactions with aldehydes resulted in the corresponding imines.30 A D-galactose-derived analogue of 14 was also shown to react with PPh3 to produce the respective phosphinimines; however, no further reactions were performed.31 Reactions of compound 14 were carried out with different phosphines. Addition of the most often applied PPh3 to 14 followed by PhCHO did not result in the formation of the expected Schiff base at rt, while on heating, decomposition took place. More encouraging was the use of aliphatic phosphines since with PhCHO, the hoped-for imine 20 was formed at rt although in admixture with 2 (PMe3 24%; 20/2, 1:0.7; P(n-Bu)3 22%; 20/2, 1:0.2). No gas evolution could be observed in these reactions, and on addition of the aldehyde, a bright yellow compound appeared, which was transformed to the imines very slowly. Due to safety and also cost reasons, the further experiments were carried out with freshly opened P(nBu)3. Thus, 14 was reacted with P(n-Bu)3 for 10 min, then PhCHO was added, and the formed yellow compound was isolated after 2 h by column chromatography in 66% yield. The 1 H and 13C NMR spectra were consistent with an intact sugar moiety as well as the presence of the CONH2 substituent, while the mass spectrum exhibited a [M + Na]+ peak (m/z =

dimethylformamide (DMF) under microwave irradiation at 160 °C for 30 min; however, in these reactions, only a sugarannelated oxazoline derivative 32 could be isolated in 17 and 24% yields, respectively. In refluxing pyridine, only decomposition could be observed, while the treatment of 2 with BF3· OEt2 (20 mol %) in CH2Cl2 at rt gave again 32 (25%). The formation of 32 was investigated further, and those results including the syntheses of analogous derivatives will be published elsewhere. Treatment of 2 with N-bromosuccinimide (NBS) (1.1 equiv) in CH2Cl2 at rt for 2 days furnished the expected 6 in 41% yield. Addition of bases such as solid K2CO3 or pyridine (1.1 equiv) to the reaction mixtures to neutralize the HBr liberated raised the yields of 6 to 60 and 71%, respectively. Thus, the latter conditions were applied to the other Schiff bases 3−5 to obtain imidazolinones 7−9 in 60, 65, and 63% isolated yields, respectively (Scheme 2). In these reactions, the spiro-epimeric compounds 24−27 were also formed as minor byproducts to be isolated in 10, 9, 16, and 11% yields, respectively.

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Scheme 3. (A) Proposed Mechanism for the Ring Closures; (B) Possible Mechanism of Epimerizations

the tautomerization (vide infra a detailed spectroscopic and computational analysis). Thus, the tautomeric equilibria between e and f as well as g and h allow deprotonation by the bases present in the reaction mixtures (pyridine in the ring closures, MeO− in deprotections) to give intermediates i and k, respectively, which may equilibrate and thus epimerize via the ring-opened intermediate j. Structural Elucidation and Molecular Modeling. Structural elucidation of the new compounds was based on NMR measurements. Vicinal 1H−1H coupling constants indicated the 4C1 conformation of the sugar rings. In this conformation, the vicinal heteronuclear coupling constants (3JH,C, Figure 1) determined for the phenyl-substituted pairs of Schiff bases 2 and 20 as well as imidazolinones 6 and 24 proved the anomeric/spiro-epimeric configuration. This configurational assignment was corroborated by nuclear

777.215) corresponding to the structure of triazene 16. The decomposition of 16 to 20 was followed by 1H NMR at rt to show the appearance of 2 after 3 days and that of 1 after 5 days (the latter might be explained by traces of water in the solvent), while ∼60 and ∼20% of 16, respectively, was still present in the mixture (see Table S1 for a tabular presentation of these data). Formation of 2 could be suppressed at lower temperatures and, by keeping the reaction mixtures in a refrigerator at below 5 °C, 14 could be transformed into single epimers 20−23, though the column chromatographic isolation resulted in 31−60% yields only (Scheme 2). Oxidative ring closure of 20−23 by NBS in the presence of pyridine gave the expected imidazolinones 24−27, respectively, in good yields (Scheme 2) together with the spiro-epimers 6−9, which were isolated in 10, 13, 16, and 18% yields, respectively. In both series of imidazolinones 6−9 and 24−27, the protecting groups were removed under Zemplén conditions to give the corresponding 10−13 and 28−31, respectively. The formation of 28−31 was accompanied by the formation of 10−13 in 17, 11, 18, 20% yields, respectively. In the other series, thin-layer chromatography (TLC) indicated the formation of the spiro-epimeric products 28−31 in very low proportions; therefore, only the major products 10−13 were isolated in yields indicated in Scheme 2. The NBS-mediated ring closure of the Schiff bases to give the imidazolinones can be understood by the mechanistic proposal outlined in Scheme 3A depicting the formation of the (S) epimers (an analogous mechanism must work for the (R) isomers, too). A bromonium ion may attack the nitrogen atom of high electron density in Schiff base a to form the benzylic cation b. Ring formation may occur by an attack of the amide nitrogen on the positively charged carbon to furnish c, which, upon deprotonation, may result in d. Final loss of HBr may give the imidazolinone product e. The formation of both epimeric spirobicycles in each transformation including the removal of the O-benzoyl protecting groups can be understood by taking into account

Figure 1. Characteristic vicinal heteronuclear coupling constants (3JH,C) and nuclear Overhauser effects to establish the configuration of epimeric pairs 2, 20 and 6, 24. 6119



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B3LYP/TZVP, BH&HLYP/TZVP, CAM-B3LYP/TZVP, and PBE0/TZVP levels, while 13C NMR shifts were calculated at the mPW1PW91/6-311+G(2d,p) level of theory. Since three major transitions of the experimental ECD spectra of epimeric 10 and 28 (Figure 3A,B) had opposite Cotton effects (CEs) at ca. 300, 260, and 220 nm, it was plausible that the anomeric configuration can be safely elucidated by TDDFT-ECD calculations. As expected, computed ECD spectra for tautomers 10Ta and 10Tb were similar to the experimental ECD spectrum of 10, suggesting (1′R) absolute configuration in line with the NMR results. Similarly, the absolute configuration of the spiro center of 28 could be elucidated as (1′S). Tautomerism seemed to be manifested in less intense transitions, and the ECD computational results suggested excess of 10Ta in MeOH. The strongly coordinating nature of the DMSO solvent often causes difficulties for the proper computation of solution conformers and spectral data.43,44 Furthermore, quite small experimental differences were found between the 13C NMR chemical shift values of 10 and 28 and thus the computed NMR data cannot be utilized to verify the anomeric configuration. Large differences were found, however, among the 13C NMR chemical shifts for C-2, C-4, and C-1′ of the two tautomers of 10, suggesting an excess of 10Ta in DMSO (Table 1). As to the tautomers of 28, computed ECD spectra of 28Tb gave a better agreement with the experimental one than those of 28Ta, suggesting excess of tautomer 28Tb in MeOH (Figure 3B). On the other hand, NMR calculations showed preference for tautomer 28Ta in DMSO (Table 1). While for 10Ta and 10Tb the energy differences of the lowest-energy conformers were found to be 16.4 kJ/mol at B3LYP/6-31G(d) and 8.7 kJ/mol at ωB97XD/TZVP PCM/MeOH levels favoring 10Ta, these values for 28Ta and 28Tb were only 7.4 kJ/mol in the gas phase and 3.7 kJ/mol in MeOH, indicating that the tautomeric equilibrium of 28 can be more dependent on the solvent than that of 10. The ECD measurements and the above computational protocol were also performed for the tautomers of 12 and 30. To check the supposed solvent dependency, which can alter the tautomeric equilibrium, the ECD spectra of 12 and 30 were recorded in both MeOH and H2O (Figure 4). Although the experimental ECD spectra in the two solvents were quite similar for both molecules, there was a positive shoulder for 12 in H2O at about 275 nm, while this region had a negative CE in MeOH. Moreover, the region at around 230 nm had a positive CE in H2O and a negative one in MeOH. These ECD spectra indicated that the ratios of the tautomers and conformers of 12 can be different in MeOH and H2O, while only minor differences could be observed for 30. This also suggested that the tautomeric ratio found in MeOH for 30 can be safely transferred to H2O. ECD spectra computed at various levels for the DFT reoptimized geometries of the initial 35−105 OPLS-2005 conformers of 12Ta, 12Tb and 30Ta, 30Tb gave moderate to good agreement with the experimental ECD spectra (Figure 3C,D). This allowed elucidation of the absolute configuration of the spiro center as (1′R) for 12 and (1′S) for 30 in accordance with the conclusion from NMR and X-ray analyses. The computed ECD spectra of tautomer 12Ta reproduced the experimental ECD spectrum substantially better than those of 12Tb, suggesting the dominance of tautomers 12Ta in MeOH. The computed ECD spectra could also explain the experimental differences between the solvents MeOH and

Overhauser effect (NOE) measurements for 2 and 20 as illustrated in Figure 1. The configuration of the other members of the series was established by comparing the chemical shifts of the sugar ring protons to show characteristic downfield shifts32,33 of the H-3 (∼0.7 ppm) and H-5 (∼0.3 ppm) protons in 20 and H-3′ (∼0.1 ppm) and H-5′ (∼0.3 ppm) in 24, wherein the CO group is in an axial position as compared to those of 2 and 6, respectively, with an equatorial CO group [for a detailed presentation of these data, see Tables S2 and S3 in the Supporting Information (SI)]. This chemical shift difference was also observed in the 1H NMR spectra of the unprotected imidazolinones 10−13 and 28−31 (H-3′ ∼0.3 ppm, H-5′ ∼0.4 ppm, cf. Table S4 in the Supporting Information). It is worth noting that the 13C NMR spectra (in D2O, CD3OD, dimethyl sulfoxide (DMSO)-d6) of the unprotected imidazolinones 10−13 and 28−31 contained no characteristic signals for the heterocycle. This might be due to tautomerism, resulting in line broadening, a phenomenon observed in non-sugar-derived imidazolinones.34 Finally, recording these spectra in DMSO-d6 containing trifluoroacetic acid (TFA, 1 equiv) gave assignable signals for each carbon (see the Experimental Section). Since electronic circular dichroism (ECD) spectra of several types of protected and unprotected monosaccharides are greatly influenced by the aglycon part or the anomeric configuration,20,35,36 the solution time-dependent density functional theory (TDDFT)-ECD method was to be tested and the outcomes be compared with the NMR and X-ray results.37 Provided that different tautomeric forms may also have considerable impact on the ECD, the ECD computational studies, complemented by DFT-NMR calculations,38,39also aimed at identifying the major tautomers present in the solution. Application of ECD for the identification of ring tautomers is rather rare in the literature.40−42 Since the enzyme kinetic tests have detected significant inhibition by the phenyl and 2-naphthyl derivatives 28 and 30, respectively (vide infra), the ECD and computational results are shown here for the corresponding pairs of spiro-epimers (10, 28 and 12, 30, resp.), while the data for the 1-naphthyl compounds 11 and 29 can be found in the Supporting Information. Tautomers of the compounds are shown in Figure 2. The initial 18−52 OPLS-2005 (optimized potential for liquid simulations) conformers for tautomers 10Ta, 10Tb, 28Ta, and 28Tb were reoptimized at the B3LYP/6-31G(d), B3LYP/6-31+G(d,p), and ωB97XD/TZVP polarizable continuum model (PCM)/MeOH levels independently, and ECD spectra were computed for the low-energy conformers at the

Figure 2. Possible tautomers of 10−12 and 28−30. 6120



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Figure 3. Experimental ECD spectra of phenyl [(A) 10 and (B) 28]- and 2-naphthyl [(C) 12 and (D) 30]-substituted spiro-imidazolinones in MeOH compared with the Boltzmann-weighted B3LYP/TZVP PCM/MeOH or CAM-B3LYP/TZVP PCM/MeOH spectra of the respective tautomers Ta and Tb computed for the ωB97XD/TZVP PCM/MeOH conformers.

Table 1. Comparison of the Computed 13C NMR Data of Three Relevant Carbons of Tautomers Ta and Tb Corrected for DMSO with the Experimental 13C NMR Data of the Phenyl (10, 28)- and 2-Naphthyl (12, 30)-Substituted Spiro-imidazolinones in DMSO-d6 10

10Ta

10Tb

ΔδTa

ΔδTb

C-2 C-4 C-1′

167.3 179.5 93.4 28

163.8 178.0 94.5 28Ta

181.6 185.4 87.7 28Tb

3.5 1.5 1.1 ΔδTa

14.3 5.9 5.7 ΔδTb

C-2 C-4 C-1′

167.5 180.9 92.2 12

163.0 181.2 94.5 12Ta

180.6 187.3 87.7 12Tb

4.5 0.3 2.3 ΔδTa

13.1 6.4 4.5 ΔδTb

C-2 C-4 C-1′

165.4 181.6 94.0 30

164.1 178.0 94.6 30Ta

182.1 185.4 87.7 30Tb

1.3 3.6 0.6 ΔδTa

16.7 3.8 6.3 ΔδTb

C-2 C-4 C-1′

166.9 181.8 92.7

163.4 181.0 94.6

181.1 187.2 87.9

3.5 0.8 1.9

14.2 5.4 4.8

nm positive shoulder, however, could not be reproduced by any combinations of levels, and the 220 nm positive band could be reproduced only with some combinations of levels for 30Tb to suggest that besides tautomer 30Tb, 30Ta may also be present with a comparable population in MeOH. This is corroborated by the computed relative energies of the lowestenergy conformers of 12Ta, 12Tb, 30Ta, and 30Tb (Table 2) showing that tautomer 30Tb had 4.02 kJ/mol higher energy than 30Ta, while 12Tb had 9.22 kJ/mol higher energy than 12Ta. DFT 13C NMR calculations indicated the dominance of tautomer 30Ta in DMSO, which was a different situation from that in MeOH. DFT conformers of tautomers 30Ta and 30Tb could be divided into four groups on the basis of the orientation of the 2-naphthyl group. In conformer groups A and C of 30Ta (Figure 5), the 2-naphthyl group points downward with dihedral angles ωN3−C2−C2″−C1″ −22.3 and 4.6° for the lowestenergy conformers, respectively, and these make the majority (∼62%) of all conformers. In groups B and D, the 2-naphthyl moiety is directed upward with dihedral angles ωN3−C2−C2″−C1″ 159.4 and −160.6°, respectively. In groups A and C of 30Tb (Figure 6), the ωN3−C2−C2″−C1″ values were 14.5 and −20.5°, and in groups B and D, −159.3 and 160.2° angles were observed, respectively. None of the conformer groups of 30Tb dominated the population (A + C ∼ 38%; B + D ∼ 38%). The torsional angles are provided for the ωB97XD/TZVP PCM/ MeOH conformers. In solution also for tautomers 11Ta, 11Tb, 29Ta, and 29Tb, there are a large number of conformers with relatively small populations, which can be divided into four groups on the basis

H2O and indicated a higher ratio of tautomer 12Tb in H2O compared to that in MeOH. Similarly to 10, the 13C NMR DFT calculations also suggested the prevalence of tautomer 12Ta in DMSO (Table 1). Computed ECD spectra of 30Tb reproduced the experimental ECD better than those of 30Ta (Figure 3D). The 280 6121



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Figure 4. Comparison of the experimental ECD spectra of 12 (A) and 30 (B) measured in MeOH and H2O.

up to 625 μM, the highest concentration investigated in this study. The significantly different inhibition of the epimeric pairs is similar to that observed with epimeric spirohydantoins14,15,45 33 and 34 (Table 3). With respect to the aromatic substituents of the imidazolinone rings, the order of potency proved to be different in the two series: for the (S) epimers, the phenyl (10) > 2-naphthyl (12) > 1-naphthyl (11), while for the (R) epimers, the 2-naphthyl (30) > phenyl (28) > 1-naphthyl (29) sequence could be observed. The latter order is similar to those observed in other types of glucosederived GPIs (such as N-acyl-β-D-glucopyranosylamines, Nacyl-N′-β-D-glucopyranosyl ureas, and C-β-D-glucopyranosyl heterocycles).5,8 The most potent compound is 30 with Ki values of 2.1 and 1.7 μM for rmGPb and hlGPa, respectively, classifying it as one of the most potent GPIs and the third among the glucopyranosylidene-spiro-heterocyclic inhibitors5 discovered thus far. Furthermore, the difference between the Ki values of this inhibitor for rmGPb and hlGPa is the smallest among the differences between the Ki values of the other five inhibitors for rmGPb and hlGPa, indicating that 30 does not differentiate between the two enzymes. Further comparisons (Table 3) with known glucopyranosylidene-spiro-heterocyclic inhibitors revealed that in rmGPb tests the phenyl-substituted imidazolinone 28 (Ki = 9 μM) was more efficient than isoxazoline 35 (Ki = 19.6 μM) and oxathiazole 37 (Ki = 26 μM). Although the increase of binding strength by a factor of 2−3 is not as big as expected, this may

Table 2. Relative Energy Values of the Lowest-Energy Conformers of 12Ta, 12Tb, 30Ta, and 30Tb Computed at ωB97XD/TZVP PCM/MeOH compound

rel. E (kJ/mol)

12Ta 30Ta 12Tb 30Tb

0.00 7.55 9.22 11.57

of the orientation of the 1-naphthyl group. These conformer populations are shown in Figures S9−S12, respectively, in the SI. Enzyme Kinetics. The inhibitory potency of the new compounds was assessed by enzyme kinetics in the direction of glycogen synthesis using rabbit muscle GP enzymes b (rmGPb) and a (rmGPa) and human liver GP (hlGPa). The inhibition constants are shown in Table 3. All compounds displayed competitive inhibition with respect to the substrate glucose-1-phosphate (Glc-1-P) as revealed by the Lineweaver− Burk plots that intersect at the same point on the y axis (Figure S13). The potency pattern, as indicated by the Ki values, is similar for rmGPa and hlGPa, implying that the effect of each inhibitor is similar on both enzymes. A comparison within the compounds studied reveals that the (R) spiro-epimers 28−30 are more potent than their (S) counterparts 10−12, while this could not be established for the 4-CF3-phenyl derivatives 13 and 31 that showed no inhibition

Figure 5. Classification of the 19 low-energy (≥1%) ωB97XD/TZVP PCM/MeOH conformers of 30Ta. Group A (55.0%) contains conformers A, B, D, H, J, L, M, O, and S; group B (24.5%) contains conformers C, E, G, Q, and R; group C (7.4%) contains conformers F, K, and P; and group D (4.3%) contains conformers I and N. The individual conformers are presented in Figure S7 in the SI. 6122



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Figure 6. Classification of the 32 low-energy (≥1%) ωB97XD/TZVP PCM/MeOH conformers of 30Tb. Group A (12.4%) contains conformers B, H, O, X, and AC; group B (15.0%) contains conformers A, G, L, T, and Y; group C (25.6%) contains conformers D, F, J, K, N, P, S, U, V, Z, AB, and AF; and group D (22.7%) contains conformers C, E, I, M, Q, R, W, AA, AD, and AE. The individual conformers are presented in Figure S8 in the SI.

potency between the epimers and suggests that the protein, in the crystal, binds preferentially only one of the epimers. The slow appearance of both epimers in the soaking solutions is congruent with the possible spiro-epimerization of the imidazolinones also observed during the synthetic work. The binding of the compound of higher affinity depletes this epimer from the soaking solution, shifting thus the equilibrium toward this epimer and increasing its occupancy in the binding site in the crystal. Furthermore, 12 was not found bound at the active site, even after 96 hrs of soaking; instead, it was found bound at the new allosteric site, while the corresponding (R) epimer (30) was found bound at both sites. Consequently, we present the structural analysis of the binding of 28−30 at rmGPb. The electron density maps (Figure 8) clearly defined the position of each atom of inhibitors 28−30 bound at the catalytic site of rmGPb. At the rmGPb-30, there was additional density at the new allosteric site (Figure 8), indicating additional binding. The new allosteric site is located in the central cavity of the biologically active GP dimer, at the subunit interface.3 Occupancy refinement for 30 at the active and the new allosteric sites yielded values of 1.0 and 0.7, respectively, indicating that the active site is the primary binding site for this ligand. Secondary binding at the new allosteric site has been also observed for other glucose-derived inhibitors in the past.3 The root-mean-square deviation (r.m.s.d.) values from the superposition of the three inhibitor complexes onto the free rmGPb structure for well-defined residues (18−49, 262−312, and 326−829) were 0.75, 0.67, and 0.66 Å for the 28−30 complexes, respectively, indicating that the binding of the inhibitors did not cause any major conformational change to the overall structure. Inhibitor binding at the catalytic site occurs by anchoring the glucose moiety at the glucose-binding subsite (a location where α-D-glucose and all glucose-based inhibitors bind3,5,52). There, the glucose moiety of each of the three inhibitors engages in the typical hydrogen bond (Table 4) and van der Waals interactions network previously observed for glucose analogues. In addition, three conserved water molecules mediate hydrogen bond interactions between the glucopyranose moiety of each ligand and residues Asp283, Tyr573, Lys574, Thr671, Ala673, and Thr676 and the phosphate group of the cofactor pyridoxal 5′-phosphate (PLP) (Figure 9). A notable difference in the hydrogen-bonding network between the three inhibitor complexes is the hydrogen bond between O2′ of the glucose moiety and the side chain atoms of

prove that the assumption in the design of the imidazolinonetype compounds (cf. Introduction) has been correct. On the other hand, among the 2-naphthyl-substituted derivatives, imidazolinone 30 (Ki = 2.1 μM) showed the weakest inhibition (isoxazoline 36, Ki = 0.63 μM; oxathiazole 38, Ki = 0.16 μM). To understand this rather unexpected finding, we turned to Xray crystallography of the enzyme−inhibitor complexes. Ex Vivo Studies. The efficacy of the most potent inhibitor 30, to inhibit human GP in an ex vivo cellular system, was assessed in HepG2 hepatocarcinoma cells. HepG2 cells were precultured with 25 mmol/L glucose and 10 nM insulin for repletion of glycogen and subsequently with 5 mmol/L glucose for determination of glycogenolysis activation, in the presence of 30, at a concentration range of 80−516 μM for 2 h. The IC50 value determined spectrophotometrically in the whole homogenate was 176.8 ± 1.7 μM (Figure S14 in the Supporting Information), while no cytotoxicity of the inhibitor was detected. The potency of 30 to preserve its inhibitory effect on GP activity in a cell culture system is further indicative of its potential antihyperglycaemic effect. The observed inhibition is comparable to 40% inhibition of 20− 50 μM 4-dimethylaminoazobenzene as assessed in hepatocytes.46 Thus, it seems that 30 is a promising candidate for in vivo assessment of its therapeutic value. X-ray Crystallography Studies. To determine the structural basis of the inhibitory potency, we performed Xray crystallography studies of the rabbit muscle GPb (rmGPb) in complex with each one of the six new inhibitors. Although the relevant pharmaceutical target is hlGPa, structural studies with rmGPb are plausible to hlGPa as it has been shown in numerous previous studies46−50 since the catalytic site (targeted by these inhibitors) is totally conserved in all mammalian GPs.51 The most intriguing finding of the X-ray crystallography study was that when rmGPb crystals were soaked in a 10 mM solution of each of 10 and 11, we did not observe any binding in the crystal up to 10 h. However, in longer soaks (>14 h), binding of their epimeric compound (i.e., 28 and 29, respectively) was observed. In fact, gradually increasing the soaking time also increased the occupancy of the binding site for the latter compounds, as illustrated in Figure 7. Co-crystallization experiments with rmGPb and each of the six new inhibitors resulted in crystals with only the (R) epimer bound, as in the soaking experiments. This observation is consistent with the significant difference in the inhibitory 6123



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Table 3. Inhibition of Glycogen Phosphorylases and the Crystallographic Numbering of the New Compounds

No inhibition at 625 μM.

a

Figure 7. Electron density maps from rmGPb crystals soaked with a 10 mM solution of 11 for various times. The augmentation of the electron density map over soaking time is apparent. The electron density map of 29 is also shown for comparison.

Asn284. This hydrogen bond is not formed in the 28 complex due to a significant conformational change of the 280s loop (residues 282−289) at this complex. This loop adopts a different conformation from the one it adopts in the native and the other two inhibitor complex structures presented here. The

r.m.s. distances for all atoms of residues 282−289 between rmGPb-28−rmGPb-29 and rmGPb-28−rmGPb-30 complex structures are 2.0 and 2.1 Å, respectively, with Asn282, Asn284, Phe285, and Glu287 being the residues with the greatest difference. The new conformation of the 280s loop (Figure 10) 6124



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rmGPb-29 and rmGPb-30 complexes, respectively, of all residues constituting the 280s loop out of the active site with Asn284 being the residue with the most significant conformational change (Figure 11). This shift might be attributed to the presence of the hydrophobic naphthyl group of 29 and 30 that repulses polar residues out of the active site. Thus, Asn284 is no longer in hydrogen-bonding distance from the N2 of the imidazolinone ring in the 29 complex, while in the 30 complex (where this shift is smaller), this hydrogen bond is formed (Table 4). In all three complexes, the carbonyl oxygen O4 of the imidazolinone ring forms two hydrogen bonds with the main chain amides of Gly135 and Leu136 (Table 4) and participates in water-mediated hydrogen bond interactions with the main chain atoms of Arg569, the phosphate group of PLP, and the side chain atoms of Asp283. In addition, O4 in the 29 complex participates in water-mediated hydrogen bond interactions with side chain atoms of Glu88 and the main chain amide of Asn133. Upon binding at the catalytic site, 28−30 engage in a total of 49, 78, and 89 van der Waals interactions with protein residues, respectively (Table S5). Focusing on the aromatic substituent of the inhibitors, the phenyl ring of 28 is involved in five nonpolar/polar van der Waals interactions with residues Asp283, Asp339, His341, and His377. The naphthyl group of 29 engages in 25 van der Waals interactions; 15 nonpolar/ polar interactions with residues Glu88, Asn282, Asn284, His341, His377, Thr378, and Ala383; and 10 nonpolar/ nonpolar interactions with residues Asn284, His341, Thr378, and Ala383. The naphthyl moiety of 30 engages in 26 van der Waals interactions, 10 nonpolar/nonpolar interactions with residues Asn284 and His341, and 16 nonpolar/polar interactions with residues Asn284, Phe285, Arg292, His341, His377, Thr378, and Ala383. Superposition of the three complex structures may offer a structural explanation of their variance in inhibitory potency (Figure 11). The plane of the phenyl ring of 28 is inclined by 50° with respect to the planes of the naphthyl group of 29 and 30 whose planes have a difference of ∼17°. These conformations for the aromatic groups allow an edge-to-face (perpendicular) π-stacking interaction with the imidazole ring of His341 in the 30 complex, but not in the other two. This offers a possible structural explanation for the higher potency of 30 in comparison to the other two inhibitors 28 and 29. Upon binding of 30 at the new allosteric site (a solvent filled cavity formed at the interface of the two subunits, of the functionally active dimer,52 composed on each side by residues from the N-terminal domains of both subunits), participates in five hydrogen bond interactions with protein residues. O6′ forms a hydrogen bond with the side chain amide of Lys191 and O2′ with the side chain terminal amide of Arg60, while O3′ forms two hydrogen bonds with the side chain atoms of His57′ and Arg60′ from the symmetry subunit (Table S6). In addition, the imidazolinone ring (N1) forms a hydrogen bond with the side chain of Thr38′ of the symmetry subunit (Figure S15). The binding of 30 is further stabilized through 83 van der Waals interactions (64 with one subunit and 19 with its symmetrical subunit; Table S6). The majority of the van der Waals interactions are formed by the naphthyl group with Arg60, Val64, Trp67, Pro188, Trp189, Glu190, Lys191, and Pro229, and Phe37′, Thr38′, and Val40′ from the symmetry subunit (Figure S15). The binding of 30 at the new allosteric site triggered a shift by ∼3.0 Å of the side chain of Arg60 to make space for the 2-naphthyl group of 30. This shift causes a

Figure 8. REFMAC weighted 2Fo−Fc electron density maps of the bound ligands (28 and 29) at the catalytic site. The electron density map for 30 at the active site (left) and the new allosteric site (right) is also presented. Maps are contoured at 1.0σ before the incorporation of the ligand molecules in the refinement process, and the final models of the inhibitors are shown.

Table 4. Potential Hydrogen Bond Interactions of Inhibitors with rmGPb Residues at the Catalytic Site in the Crystala distances (Å) inhibitor atomsa

protein residues

O2′

Asn284 (ND2) Asn284 (OD1) Tyr573 (OH) Glu672 (OE1) Water321 (O) Glu672 (OE1) Ala673 (N) Ser674 (N) Gly675 (N) Gly675 (N) Water133 (O) Asn484 (ND2) His377 (ND1) His377 (O) Asn284 (ND2) Water311 (O) Gly135 (N) Leu136 (N) Water59 (O) Water311 (O)

O3′

O4′ O6′ N1 N2 O4

total

28

3.3 3.3 3.0 2.7 3.2 3.1 3.1 2.9 2.6 2.7 2.7 2.7 3.1 3.3 3.2 3.3 16

29

30

2.8

3.1 3.2 3.3

3.3 2.9 2.8 3.3 3.1 3.2 2.9 2.6 2.6 2.7 2.8 3.0 3.3 3.2 3.2 3.3 17

3.0 2.8 3.2 3.1 3.2 2.9 2.6 2.8 2.7 2.9 3.3 2.9 3.2 3.2 3.0 18

a

For the crystallographic numbering of atoms, see Table 3.

drives Asn284 out of the catalytic cavity, disrupting a hydrogen bond with O2′ of glucopyranose in the 28 complex (Table 4). This new conformation is stabilized by the formation of a hydrogen bond between His571 and Asp283. The loss of the hydrogen bond of O2′ with Asn284 in the rmGPb-28 complex is compensated by a hydrogen bond to the side chain of Glu672 (Table 4). The absence of the O2′-Asn284 H-bond present with strongly binding inhibitors may explain why this compound is only slightly but not much better than the corresponding isoxazoline 35 and oxathiazole 37. The conformation of the 280s loop in the rmGPb-29 and rmGPb-30 complex structures has also small but notable differences from that of the unliganded rmGPb structure. These differences occur as a shift of ∼0.4 and ∼0.3 Å in the 6125



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Figure 9. Binding of 28 (A), 29 (B), and 30 (C) at the active site of rmGPb. The inhibitor is shown as thick sticks, hydrogen bonds are shown as dashed lines, and water molecules are shown as blue spheres.

group in the two ligands in the enzyme active site differs by a 180° rotation along the C2−C5 bond (Table 3). There is no obvious reason for this change, but this leads to differences in the van der Waals interactions with protein residues in the two complexes. Thus, the 2-naphthyl group of inhibitor 30 forms 26 (10 nonpolar−nonpolar and 16 nonpolar−polar) van der Waals contacts (Table S5), while that of 36 forms 30 (9 nonpolar−nonpolar and 21 nonpolar−polar).19 Furthermore, the formation of a hydrogen bond between N1 of 30 and the carbonyl oxygen of His377 induces a small but significant conformational change of the 377−379 segment from the conformation it adopts in the 36 complex and which is more profound on Thr378 (Figure 12). The energy cost associated with this conformational change and smaller number of van der Waals contacts of the 2-naphthyl moiety of 30 with respect to those of 36 seem to be counterbalanced by the hydrogenbonding interactions of O4 and N1 and thus there is not a very big difference in compounds’ 30 and 36 efficiency. This observation further supports recent studies,48 where van der Waals interactions govern the inhibitory potency of glucosederived GPIs. The fact that 30 is not as potent as expected can also be attributed to the presence of tautomers 30Ta and 30Tb, of which only 30Tb of higher energy binds in the crystal, as well as the rather evenly distributed conformer population that may result in both enthalpic and entropic penalties during the binding event.

Figure 10. Superposition of the rmGPb-28 complex (brown) onto the free rmGPb structure (blue) highlighting the different conformations of the 280s loop.

small translocation of the helix (residues 60−64) with r.m.s.d. of 1.0 Å for all atoms, a change observed previously with other bound inhibitors at the new allosteric site.48,52−55 The imidazolinones 28 and 30 presented here were expected to be more potent than the corresponding spiro-isoxazolines 35 and 36 (Table 3) since they were anticipated to form additional hydrogen bonds through O4 and N1. Indeed, both 28 and 30 are able to form additional hydrogen bond interactions through O4 with main chain amides of Gly135 and Leu136 and through N1 with main chain carbonyl oxygen of His377. These hydrogen bond interactions could be the structural basis of the higher potency of 28 (Ki = 9.0 μM) with respect to that of 35 (Ki = 19.6 μM). This shows that although the binding of 28 triggered a significant conformational change of the 280s loop, which is not observed upon binding of 35 (Figure S16), not only the energy cost associated with this change is counterbalanced by these hydrogen bond interactions, but also they confer to 28 somewhat more than 2 times higher inhibitory potency than that of 35.19 However, the hydrogen bond interactions of O4 and N1 did not confer higher potency to 30 (Ki = 2.1 μM) with respect to that of the corresponding spiro-isoxazoline 36 (Ki = 0.6 μM).19 Structural comparison of the binding of 30 and 36 to rmGPb (Figure 12) reveals that the position of the 2-naphthyl

■

CONCLUSIONS

Synthetic methods were elaborated for both epimers of the new ring system glucopyranosylidene-spiro-imidazolinones via Schiff bases of (gluculopyranosylamine)onamides, which were cyclized by an unprecedented NBS-mediated oxidative ring closure. While the (S) spiro-epimers had rather weak or no inhibition, the (R) epimers of these imidazolinones with a phenyl or 2-naphthyl substituent were shown to have strong inhibitory potency against hlGPa and rmGPa with Ki values in the low μM range. The 2-naphthyl-substituted compound 30 belongs to the best 10 glucose-derived inhibitors of glycogen phosphorylases in vitro and ex vivo. X-ray crystallography revealed that only the (R) epimers bound at the crystal. All inhibitors participate in an extended network of interactions with residues of the 280s loop (albeit the binding of the phenyl inhibitor 28 triggers its significant conformational change) holding the enzyme’s closed T-state conformation, thus 6126



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Figure 11. Stereo diagram of the superposition of the rmGPb ligand complexes, 28 (coral), 29 (green), and 30 (brown).

Figure 12. Stereo diagram of the superposition of the rmGPb-30 complex onto the analogous spiro-isoxazoline 36.19

inhibiting access of the substrate to the catalytic site. Structural analysis revealed crucial interactions between the imidazolinone carbonyl oxygen and the main chain amides of Gly135 and Leu136, the NH of the imidazolinone ring to the main chain carbonyl oxygen of His377, and water-mediated Hbonds with Asp283, Arg569, and the phosphate group of PLP. These interactions seem to govern the inhibitory potency of the three inhibitors although the van der Waals interactions of the aryl moieties in the β-cavity also play an important role. While 28 induces a conformational change of the 280s loop, which is mainly counterbalanced by the hydrogen bond interactions of the carbonyl oxygen and the NH of the imidazolinone linker, 29 and 30 do not cause this change. The latter places its 2-naphthyl group in a more suitable place for π-

stacking interactions with His341 explaining the weaker than expected Ki value. Comparing the potency of the aryl imidazolinones to that of spirohydantoin 34 revealed that the addition of the 2-naphthyl group improved the potency of the inhibitor. Comparing the efficiency of imidazolinones 28 and 30 to that of the corresponding spiro-isoxazolines 35 and 36 and -oxathiazoles 37 and 38, respectively, showed that replacing the latter heterorings by an imidazolinone led to a better inhibitor with a phenyl substituent and a slightly weaker one with a 2-naphthyl group.

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EXPERIMENTAL SECTION

Syntheses. General Methods. Melting points were measured on a Kofler hot stage and are uncorrected. Optical rotations were

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determined with a PerkinElmer 241 polarimeter at rt. ECD spectra were recorded on a Jasco J-810 spectropolarimeter. Elemental analyses were performed on an Elementar Vario Micro Cube instrument to prove ≥95% purity of the test compounds 10−13 and 28−31. NMR spectra were recorded with Bruker 360 (360/90 MHz for 1H/13C), Bruker 400 (400/100 MHz for 1H/13C), or Avance DRX 500 (500/125 MHz for 1H/13C) spectrometers. Chemical shifts were referenced to internal tetramethylsilane (1H) or to the residual solvent signals (13C). In the NMR spectra, complete signal assignments were based on correlated spectroscopy, heteronuclear single quantum coherence, and heteronuclear single quantum multiple bond correlation (HSQMBC) correlations. Mass spectra were obtained by a Bruker micrOTOF-Q instrument. TLC was performed on DC-Alurolle Kieselgel 60 F254 (Merck), and the plates were visualized under UV light and by gentle heating. For visualization of bromine-containing sugars, the plate was sprayed with the following solutions: spray solution I (0.1% fluorescein solution in 50% ethanol) and spray solution II (mixture of equal parts of 30% hydrogen peroxide and glacial acetic acid) followed by gentle heating. For column chromatography, Kieselgel 60 (Merck, particle size 0.063−0.200 mm) was used. CH2Cl2 was distilled from P4O10 and stored over 4 Å molecular sieves. MeOH was purified by distillation after refluxing for a couple of hours with magnesium turnings and iodine. Organic solutions were concentrated under diminished pressure at 40−50 °C (water bath). Tri-n-butylphosphine, pyrrolidine, N-bromosuccinimide, and aldehydes were purchased from Sigma-Aldrich. C-(1-Amino-2,3,4,6-tetra-O-benzoyl-1-deoxy-βD-glucopyranosyl)formamide (1) and C-(1-azido-2,3,4,6-tetra-Obenzoyl-1-deoxy-α-D-glucopyranosyl)formamide (14) were synthesized according to published procedures.28 General Procedure I for the Synthesis of C-(1-Arylideneamino2,3,4,6-tetra-O-benzoyl-1-deoxy-β-D-glucopyranosyl)formamides (2−5). To a solution of amine 1 in anhydrous CH2Cl2 (15 mL/ mmol) 4 Å molecular sieves (1 g/mmol), the corresponding aldehyde (1.1 equiv) and 10 mol % pyrrolidine were added. The mixture was stirred at rt until disappearance of the starting material (TLC, 1:1 EtOAc/hexane). Then, the mixture was filtered on a Celite pad and the solvent was removed in vacuo. The residue was purified by column chromatography. General Procedure II for the Preparation of O-Perbenzoylated Glucopyranosylidene-spiro-imidazolinones (6−9, 24−27). An imine (2−5, 20−23) was dissolved in anhydrous CH2Cl2 (15 mL/ mmol), NBS (1.1 equiv) was added at 0 °C, and the reaction mixture was allowed to warm to rt. When TLC (2:3 EtOAc/hexane) showed complete transformation of the starting material into the brominated intermediate (∼2 h), pyridine (1.1 equiv) was added and stirring was continued for 2 days. The solvent was removed under diminished pressure, and the residue was purified by column chromatography. General Procedure III for the Removal of O-Benzoyl Protecting Groups (10−13, 28−31). An O-perbenzoylated compound (6−9, 24−27) was dissolved in dry MeOH (10 mL/mmol), and a few drops of a 1 M methanolic NaOMe solution were added. The mixtures were kept at rt (with compounds 6−9) or cooled to 0−5 °C (with compounds 24−27), and the progress of the reaction was monitored by TLC (3:7 MeOH/CHCl3). When the starting material and the partially debenzoylated products disappeared (1−3 days), the mixtures were neutralized with acetic acid and the solvent was removed. The residue was purified by column chromatography to give a syrup, which solidified to a crystalline product on addition of diethyl ether. General Procedure IV for the Synthesis of C-(1-Arylideneamino2,3,4,6-tetra-O-benzoyl-1-deoxy-α-D-glucopyranosyl)formamides (20−23). To a solution of the glycosyl azide 14 in anhydrous CH2Cl2 (15 mL/mmol), nBu3P (1.1 equiv) was added, and the mixture was stirred at rt for 10 min. Then, the reaction mixture was cooled in an ice bath and the corresponding aldehyde was added (1.1 equiv). The mixture was kept at low temperature for several days (practically stored in a fridge at 3 °C) until the intermediate triazene (16−19) had disappeared, as judged by TLC (1:1 EtOAc/hexane). Finally, the

solvent was evaporated and the obtained syrup was purified by column chromatography. Synthetic Details and Characterization of the Compounds. C(2,3,4,6-Tetra-O-benzoyl-1-benzylideneamino-1-deoxy-β- D glucopyranosyl)formamide (2). This was prepared from compound 1 (550 mg, 0.86 mmol) and benzaldehyde (96 μL, 0.95 mmol) according to general procedure I (reaction time: 2 h). Purified by column chromatography (1:2 EtOAc/hexane) to give 540 mg (86%) of a white amorphous solid. Rf = 0.40 (1:1 EtOAc/hexane); [α]D +162 (c 0.50, CHCl3); 1H NMR (500 MHz, CDCl3) δ (ppm): 8.91 (1H, s, CHN), 8.13 (2H, d, J = 7.1 Hz, aromatics), 7.98 (2H, d, J = 7.2 Hz, aromatics), 7.90−7.87 (4H, m, aromatics), 7.77 (2H, d, J = 7.2 Hz, aromatics), 7.61−7.20 (15H, m, aromatics), 6.84 (1H, d, J = 3.0 Hz, NH), 5.99 (1H, d, J = 9.8 Hz, H-2), 5.93 (1H, pseudo t, J = 9.4 Hz, H-3), 5.86 (1H, pseudo t, J = 9.6 Hz, H-4), 5.71 (1H, d, J = 3.0 Hz, NH), 5.25 (1H, ddd, J = 9.9, 4.1, 2.7 Hz, H-5), 4.93 (1H, dd, J = 12.3, 2.7 Hz, H-6a), 4.57 (1H, dd, J = 12.3, 4.1 Hz, H-6b); 13C NMR (90 MHz, CDCl3) δ (ppm): 169.3 (CONH2, 3J H‑2,CO = 2.3 Hz, from HSQMBC at 125 MHz), 166.8, 165.8, 165.4, 164.9 (CO), 164.3 (CHN), 135.7 (q, Ph), 133.5−128.3 (aromatics), 88.5 (C-1), 71.9 (C-3), 71.3 (C-2), 69.7, 69.6 (C-4, C-5), 62.7 (C-6). Electrospray ionization mass spectrometry (ESI-MS) positive mode (m/z): calcd for C42H34N2NaO10 ([M + Na]+): 749.211. Found: 749.209. C-[2,3,4,6-Tetra-O-benzoyl-1-deoxy-1-(naphth-1-ylmethylidene)amino-β-D-glucopyranosyl]formamide (3). This was prepared from compound 1 (350 mg, 0.55 mmol) and 1-naphthaldehyde (82 μL, 0.61 mmol) according to general procedure I (reaction time: 6 h). Purified by column chromatography (1:2 EtOAc/hexane) to give 322 mg (75%) of a white amorphous solid. Rf = 0.44 (1:1 EtOAc/ hexane); [α]D +114 (c 0.65, CHCl3); 1H NMR (400 MHz, CDCl3) δ (ppm): 9.67 (1H, s, CHN), 8.70 (1H, d, J = 8.4 Hz, aromatics), 8.19−8.13 (2H, m, aromatics), 8.04 (1H, d, J = 8.1 Hz, aromatics), 8.00−7.89 (5H, m, aromatics), 7.77 (2H, d, J = 7.2 Hz, aromatics), 7.65−7.19 (16H, m, aromatics), 6.91 (1H, d, J = 3.6 Hz, NH), 6.06 (1H, d, J = 9.8 Hz, H-2), 6.00 (1H, pseudo t, J = 9.4 Hz, H-3), 5.89 (1H, pseudo t, J = 9.4 Hz, H-4), 5.68 (1H, d, J = 3.6 Hz, NH), 5.28 (1H, ddd, J = 10.1, 4.4, 2.7 Hz, H-5), 4.95 (1H, dd, J = 12.3, 2.7 Hz, H-6a), 4.61 (1H, dd, J = 12.3, 4.4 Hz, H-6b); 13C NMR (90 MHz, CDCl3) δ (ppm): 169.5 (CONH2) 166.8, 165.8, 165.4, 165.0 (C O), 163.6 (CHN), 134.0−124.0 (aromatics), 88.8 (C-1), 71.8 (C3), 71.6 (C-2), 70.0, 69.7 (C-4, C-5), 62.9 (C-6). ESI-MS positive mode (m/z): calcd for C46H36N2NaO10 ([M + Na]+): 799.226. Found: 799.224. C-[2,3,4,6-Tetra-O-benzoyl-1-deoxy-1-(naphth-2-ylmethylidene)amino-β-D-glucopyranosyl]formamide (4). This was prepared from compound 1 (250 mg, 0.39 mmol) and 2-naphthaldehyde (67 mg, 0.43 mmol) according to general procedure I (reaction time: 6 h). Purified by column chromatography (1:2 EtOAc/hexane) to give 175 mg (58%) of a pale yellow amorphous solid. Rf = 0.42 (1:1 EtOAc/ hexane); [α]D +173 (c 0.65, CHCl3); 1H NMR (360 MHz, CDCl3) δ (ppm): 9.06 (1H, s, CHN), 8.20 (2H, d, J = 8.2 Hz, aromatics), 8.14 (2H, d, J = 7.3 Hz, aromatics), 8.09 (1H s, aromatics), 8.01−7.84 (6H, m, aromatics), 7.78 (2H, d, J = 7.2 Hz, aromatics), 7.59−7.18 (15H, m, aromatics), 6.86 (1H, d, J = 2.7 Hz, NH), 6.04 (1H, d, J = 9.6 Hz, H-2), 5.98 (1H, pseudo t, J = 9.3 Hz, H-3), 5.90 (1H, pseudo t, J = 9.4 Hz, H-4), 5.77 (1H, s, NH), 5.32 (1H, ddd, J = 10.0, 4.2, 2.7 Hz, H-5), 4.97 (1H, dd, J = 12.2, 2.7 Hz, H-6a), 4.60 (1H, dd, J = 12.3, 4.2 Hz, H-6b); 13C NMR (90 MHz, CDCl3) δ (ppm): 169.4 (CONH2) 166.7, 165.7, 165.4, 164.8 (CO), 164.2 (CHN), 135.4−123.5 (aromatics), 88.7 (C-1), 71.9 (C-3), 71.3 (C-2), 69.7, 69.7 (C-4, C-5), 62.8 (C-6). ESI-MS positive mode (m/z): calcd for C46H36N2NaO10 ([M + Na]+): 799.226. Found: 799.224. C-[2,3,4,6-Tetra-O-benzoyl-1-deoxy-1-(4trifluoromethylbenzylidene)amino-β-D-glucopyranosyl]formamide (5). This was prepared from compound 1 (300 mg, 0.47 mmol) and p(trifluoromethyl)benzaldehyde (71 μL, 0.52 mmol) according to general procedure I (reaction time: 2 h). Purified by column chromatography (1:2 EtOAc/hexane) to give 255 mg (68%) of a white amorphous solid. Rf = 0.45 (1:1 EtOAc/hexane); [α]D +127 (c 6128



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0.50, CHCl3); 1H NMR (360 MHz, CDCl3) δ (ppm): 9.01 (1H, s, CHN), 8.13 (2H, d, J = 7.4 Hz, aromatics), 8.00−7.96 (4H, m, aromatics), 7.87 (2H, d, J = 7.4 Hz, aromatics), 7.81−7.74 (4H, m, aromatics), 7.58 (2H, t, J = 7.4 Hz, aromatics), 7.52−7.43 (4H, m, aromatics), 7.38−7.29 (4H, m, aromatics), 7.22 (2H, t, J = 7.5 Hz, aromatics), 6.93 (1H, d, J = 2.6 Hz, NH), 6.03−5.98 (2H, m, H-2 and NH), 5.94−5.86 (2H, m, H-3 and H-4), 5.23 (1H, ddd, J = 9.8, 4.1, 2.4 Hz, H-5), 4.95 (1H, dd, J = 12.3, 2.4 Hz, H-6a), 4.60 (1H, dd, J = 12.3, 4.1 Hz, H-6b); 13C NMR (90 MHz, CDCl3) δ (ppm): 169.1 (CONH2) 166.8, 165.8, 165.4, 164.8 (CO), 163.0 (CHN), 138.6−122.4 (aromatics, CF3), 88.4 (C-1), 71.7 (C-3), 71.3 (C-2), 69.9, 69.5 (C-4, C-5), 62.7 (C-6). ESI-MS positive mode (m/z): calcd for C43H33F3N2NaO10 ([M + Na]+): 817.198. Found: 817.196. (1′R)-1′,5′-Anhydro-2′,3′,4′,6′-tetra-O-benzoyl-D-glucitol-spiro[1′,5]-2-phenyl-imidazolin-4-one (6). This was prepared from imine 2 (250 mg, 0.34 mmol) with NBS (67 mg, 0.37 mmol) and pyridine (30 μL, 0.37 mmol) according to general procedure II. Column chromatography (1:3 EtOAc/hexane) gave two fractions. Fraction I: 175 mg (70%) of 6 as a white amorphous solid. Rf = 0.32 (2:3 EtOAc/hexane); [α]D +87 (c 0.58, CHCl3); 1H NMR (500 MHz, CDCl3) δ (ppm): 10.16 (1H, s, NH), 8.09 (2H, d, J = 7.4 Hz, aromatics), 8.00 (2H, d, J = 7.3 Hz, aromatics), 7.95 (2H, d, J = 7.4 Hz, aromatics), 7.79 (2H, d, J = 7.4 Hz, aromatics), 7.67 (2H, d, J = 7.5 Hz, 2H, aromatics), 7.63 (1H, t, J = 7.4 Hz, aromatics), 7.56 (2H, t, J = 7.5 Hz, aromatics), 7.49 (2H, m, aromatics), 7.40−7.31 (6H, m, aromatics), 7.26−7.21 (2H, m, aromatics), 7.12 (2H, t, J = 7.8 Hz, aromatics), 6.40 (1H, pseudo t, J = 9.7 Hz, H-3′), 6.02 (1H, d, J = 9.9 Hz, H-2′), 5.95 (1H, pseudo t, J = 9.9 Hz, H-4′), 5.04 (1H, ddd, J = 9.9, 4.5, 2.8 Hz, H-5′), 4.61 (1H, dd, J = 12.4, 2.8 Hz, H-6′a), 4.52 (1H, dd, J = 12.4, 4.5 Hz, H-6′b); 13C NMR (100 MHz, CDCl3) δ (ppm): 179.3 (CONH, 3J H‑2′,CO = 2.8 Hz, from HSQMBC at 125 MHz), 166.4, 165.9, 165.3, 164.8 (CO), 163.3 (CN), 133.5− 127.4 (aromatics), 94.1 (C-1′), 72.7 (C-3′), 72.3 (C-5′), 71.4 (C-2′), 69.9 (C-4′), 63.3 (C-6′). ESI-MS positive mode (m/z): calcd for C42H32N2NaO10 ([M + Na]+): 747.195. Found: 747.191. Fraction II: 25 mg (10%) of 24 as a white amorphous solid. (1′R)-1′,5′-Anhydro-2′,3′,4′,6′-tetra-O-benzoyl-D-glucitol-spiro[1′,5]-2-(1-naphthyl)-imidazolin-4-one (7). This was prepared from imine 3 (320 mg, 0.41 mmol) with NBS (81 mg, 0.45 mmol) and pyridine (36 μL, 0.45 mmol) according to general procedure II. Column chromatography (1:3 EtOAc/hexane) gave two fractions. Fraction I: 190 mg (60%) of 7 as a pale yellow amorphous solid. Rf = 0.46 (1:1 EtOAc/hexane); [α]D +63 (c 0.40, CHCl3); 1H NMR (400 MHz, CDCl3) δ (ppm): 9.28 (1H, s, NH), 8.86 (1H, d, J = 8.2 Hz, aromatics), 8.05 (1H, d, J = 8.3 Hz, aromatics), 8.01 (2H, d, J = 7.5 Hz, aromatics), 7.94 (3H, t, J = 8.3 Hz, aromatics), 7.83−7.78 (5H, m, aromatics), 7.64−7.58 (2H, m, aromatics), 7.55 (1H, t, J = 7.7 Hz, aromatics), 7.51−7.44 (2H, m, aromatics), 7.41−7.30 (6H, m, aromatics), 7.24 (2H, t, J = 7.6 Hz, aromatics), 7.14 (2H, t, J = 7.7 Hz, aromatics), 6.45 (1H, pseudo t, J = 9.8 Hz, H-3′), 6.05 (1H, d, J = 9.9 Hz, H-2′), 5.95 (1H, pseudo t, J = 9.9 Hz, H-4′), 5.07 (1H, ddd, J = 10.0, 4.8, 3.0 Hz, H-5′), 4.63 (1H, dd, J = 12.4, 3.0 Hz, H-6′a), 4.52 (dd, J = 12.4, 4.8 Hz, H-6′b); 13C NMR (90 MHz, CDCl3) δ (ppm): 178.0 (CONH), 166.3, 165.8, 165.4, 164.8, 164.1 (CO, CN), 134.0−124.9 (aromatics), 94.2 (C-1′), 72.7, 72.7, 71.6, 69.8 (C-2′− C-5′), 63.3 (C-6′). ESI-MS positive mode (m/z): calcd for C46H34N2NaO10 ([M + Na]+): 797.211. Found: 797.212. Fraction II: 28 mg (9%) of 25 as a pale yellow amorphous solid. (1′R)-1′,5′-Anhydro-2′,3′,4′,6′-tetra-O-benzoyl-D-glucitol-spiro[1′,5]-2-(2-naphthyl)-imidazolin-4-one (8). This was prepared from imine 4 (155 mg, 0.20 mmol) with NBS (39 mg, 0.22 mmol) and pyridine (18 μL, 0.22 mmol) according to general procedure II. Column chromatography (1:3 EtOAc/hexane) gave two fractions. Fraction I: 100 mg (65%) of 8 as a pale yellow amorphous solid. Rf = 0.36 (2:3 EtOAc/hexane); [α]D +122 (c 0.63, CHCl3); 1H NMR (400 MHz, CDCl3) δ (ppm): 10.08 (1H, s, NH), 8.47 (1H, s, aromatics), 8.25 (1H, dd, J = 8.6, 1.2 Hz, aromatics), 8.07−7.89 (7H, m, aromatics), 7.81 (d, J = 7.3 Hz, 2H), 7.72−7.55 (4H, m, aromatics), 7.53−7.20 (10H, m, aromatics), 7.09 (2H, t, J = 7.8 Hz, aromatics), 6.46 (1H, pseudo t, J = 9.7 Hz, H-3′), 6.09 (1H, d, J = 9.9

Hz, H-2′), 5.99 (1H, pseudo t, J = 9.9 Hz, H-4′), 5.10 (1H, ddd, J = 9.9, 4.5, 2.9 Hz, H-5′), 4.62 (1H, dd, J = 12.4, 2.9 Hz, H-6′a), 4.54 (1H, dd, J = 12.4, 4.5 Hz, H-6′b); 13C NMR (90 MHz, CDCl3) δ (ppm): 179.3 (CONH), 166.4, 166.0, 165.4, 164.9 (CO), 163.4 (CN), 135.8−123.7 (aromatics), 94.2 (C-1′), 72.9 (C-3′), 72.4 (C5′), 71.5 (C-2′), 70.1 (C-4′), 63.4 (C-6′). ESI-MS positive mode (m/ z): calcd for C46H34N2NaO10 ([M + Na]+): 797.211. Found: 797.208. Fraction II: 25 mg (16%) of 26 as a pale yellow amorphous solid. (1′R)-1′,5′-Anhydro-2′,3′,4′,6′-tetra-O-benzoyl-D-glucitol-spiro[1′,5]-2-(4-trifluoromethylphenyl)-imidazolin-4-one (9). This was prepared from imine 5 (240 mg, 0.30 mmol) with NBS (59 mg, 0.33 mmol) and pyridine (27 μL, 0.33 mmol) according to general procedure II. Column chromatography (1:3 EtOAc/hexane) gave two fractions. Fraction I: 151 mg (63%) of 9 as a white amorphous solid. Rf = 0.42 (1:1 EtOAc/hexane); [α]D +91 (c 0.48, CHCl3); 1H NMR (360 MHz, CDCl3) δ (ppm): 10.58 (1H, s, NH), 8.23 (2H, d, J = 8.1 Hz, aromatics), 8.02−7.93 (4H, m, aromatics), 7.86−7.76 (4H, m, aromatics), 7.66 (1H, d, J = 7.9 Hz, aromatics), 7.55−7.46 (2H, m, aromatics), 7.41−7.30 (6H, m, aromatics), 7.23 (2H, t, J = 7.7 Hz, aromatics), 7.14 (2H, t, J = 7.7 Hz, aromatics), 6.40 (1H, pseudo t, J = 9.7 Hz, H-3′), 6.03 (1H, d, J = 9.9 Hz, H-2′), 5.98 (1H, pseudo t, J = 9.9 Hz, H-4′), 5.02 (1H, ddd, J = 9.9, 4.3, 2.6 Hz, H-5′), 4.65 (1H, dd, J = 12.4, 2.6 Hz, H-6′a), 4.52 (1H, dd, J = 12.5, 4.3 Hz, H-6′b); 13 C NMR (100 MHz, CDCl3) δ (ppm): 179.9 (CONH), 166.3, 165.9, 165.3, 164.9 (CO), 162.5 (CN), 135.1−126.3 (aromatics), 123.6 (q, CF3, 1JCF = 272.5 Hz), 94.3 (C-1′), 72.6 (C-3′), 72.6 (C-5′), 71.4 (C-2′), 69.7 (C-4′), 63.1 (C-6′). ESI-MS positive mode (m/z): calcd for C43H31F3N2NaO10 ([M + Na]+): 815.183. Found: 815.181. Fraction II: 26 mg (11%) of 27 as a white amorphous solid. (1′R)-1′,5′-Anhydro-D-glucitol-spiro-[1′,5]-2-phenyl-imidazolin4-one (10). This was prepared from compound 6 (130 mg, 0.18 mmol) according to general procedure III. Purified by column chromatography (15:1:0.01 EtOAc/MeOH/AcOH) to yield 50 mg (91%) of off-white crystals. mp: 146−148 °C; Rf = 0.39 (5:1:0.1 EtOAc/MeOH/AcOH); [α]D +80 (c 0.62, MeOH); 1H NMR (360 MHz, D2O) δ (ppm): 7.94 (2H, d, J = 7.6 Hz, Ph), 7.73 (1H, t, J = 7.4 Hz, Ph), 7.60 (2H, t, J = 7.7 Hz, Ph), 4.06−3.98 (1H, m, H-5′), 3.97−3.80 (4H, m, H-2′, H-3′, H-6′a, H-6′b), 3.68 (1H, pseudo t, J = 9.6 Hz, H-4′); 13C NMR (90 MHz, DMSO-d6 + TFA) δ (ppm): 179.5 (CON), 167.3 (CN), 134.5, 129.6, 129.3, 128.8 (Ph), 93.4 (C′-1), 78.2, 74.2, 73.0, 70.3 (C-2′−C-5′), 61.2 (C-6′). ECD [MeOH, λ (nm) (Δε), c 0.255 mM]: 290 (+2.55), 254 (−1.87), 219 (−0.58); ESI-MS positive mode (m/z): calcd for C14H16N2NaO6 ([M + Na]+): 331.090. Found: 331.089. Anal. Calcd for C14H16N2O6: C, 54.54; H, 5.23; N, 9.09. Found: 54.80; H, 5.02; N, 9.00. (1′R)-1′,5′-Anhydro-D-glucitol-spiro-[1′,5]-2-(1-naphthyl)-imidazolin-4-one (11). This was prepared from compound 7 (125 mg, 0.16 mmol) according to general procedure III. Purified by column chromatography (15:1:0.1 EtOAc/MeOH/AcOH) to yield 46 mg (81%) of yellowish crystals. mp: 152−154 °C; Rf = 0.36 (5:1:0.1 EtOAc/MeOH/AcOH); [α]D +49 (c 0.40, MeOH); 1H NMR (360 MHz, D2O) δ (ppm): 8.27 (1H, d, J = 7.6 Hz, aromatics), 8.04 (1H, d, J = 8.1 Hz, aromatics), 7.92 (1H, d, J = 7.2 Hz, aromatics), 7.75 (1H, d, J = 6.8 Hz, aromatics), 7.63−7.48 (3H, m, aromatics), 4.02 (1H, ddd, J = 10.0, 5.4, 1.7 Hz, H-5′), 3.95−3.79 (4H, m, H-2′, H-3′, H-6′a, H-6′b), 3.67 (1H, pseudo t, J = 8.7 Hz, H-4′); 13C NMR (90 MHz, DMSO-d6 + TFA) δ (ppm): 180.6 (CON), 164.8 (CN), 133.4, 132.4, 130.1, 128.5, 128.2, 127.5, 126.7, 126.0, 125.5, 124.9 (aromatics), 94.8 (C′-1), 77.8, 74.7, 73.1, 70.5 (C-2′−C-5′), 61.3 (C6′). ECD [MeOH, λ (nm) (Δε), c 0.166 mM]: 313sh (+3.00), 301 (+3.54), 274 (−2.90), 253 (+0.53), 225 (−3.14). ESI-MS positive mode (m/z): calcd for C18H18N2NaO6 ([M + Na]+): 381.106. Found: 381.107. Anal. Calcd for C18H18N2O6: C, 60.33; H, 5.06; N, 7.82. Found: C, 60.49; H, 4.93; N, 7.99. (1′R)-1′,5′-Anhydro-D-glucitol-spiro-[1′,5]-2-(2-naphthyl)-imidazolin-4-one (12). This was prepared from compound 8 (145 mg, 0.19 mmol) according to general procedure III. Purified by column chromatography (16:1:0.1 EtOAc/MeOH/AcOH) to yield 53 mg 6129



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(79%) of yellowish crystals. mp: 172−174 °C; Rf = 0.42 (4:1:0.1 EtOAc/MeOH/AcOH); [α]D +95 (c 0.44, MeOH); 1H NMR (360 MHz, D2O) δ (ppm): 7.60 (1H, s, aromatics), 7.44 (2H, t, J = 6.9 Hz, aromatics), 7.39−7.24 (4H, m, aromatics), 4.04−3.77 (4H, m, H-3′, H-5′, H-6′a, H-6′b), 3.80 (1H, d, J = 9.8 Hz, H-2′), 3.69 (1H, pseudo t, J = 9.3 Hz, H-4′); 13C NMR (90 MHz, DMSO-d6 + TFA) δ (ppm): 181.6 (CON), 165.4 (CN), 134.9, 132.1, 129.1, 128.6, 128.6, 127.9, 127.3, 125.2, 123.9 (aromatics), 94.0 (C′-1), 77.6, 74.4, 73.1, 70.5 (C-2′−C-5′), 61.3 (C-6′). ECD [MeOH, λ (nm) (Δε), c 0.186 mM]: 330sh (+0.58), 301 (+8.64), 290sh (+4.40), 279sh (−0.65), 255 (−8.63), 226sh (−1.30), 205 (+1.77); ECD [H2O, λ (nm) (Δε), c 0.118 mM]: 333sh (+0.31), 300 (+6.11), 290sh (+4.52), 274sh (+1.36), 255 (−10.12), 233 (+0.40), 225 (−0.70), 202 (+1.67). ESIMS positive mode (m/z): calcd for C18H18N2NaO6 ([M + Na]+): 381.106. Found: 381.106. Anal. Calcd for C18H18N2O6: C, 60.33; H, 5.06; N, 7.82. Found: C, 60.52; H, 5.10; N, 7.67. (1′R)-1′,5′-Anhydro-D-glucitol-spiro-[1′,5]-2-(4-trifluoromethylphenyl)-imidazolin-4-one (13). This was prepared from compound 9 (130 mg, 0.19 mmol) according to general procedure III. Purified by column chromatography (15:1:0.1 EtOAc/MeOH/AcOH) to yield 36 mg (51%) of yellowish crystals. mp: 159−161 °C; Rf = 0.40 (8:1:0.1 EtOAc/MeOH/AcOH); [α]D +41 (c 0.70, MeOH); 1H NMR (360 MHz, D2O) δ (ppm): 8.05 (2H, d, J = 7.0 Hz, aromatics), 7.87 (2H, d, J = 7.0 Hz, aromatics), 4.02 (1H, ddd, J = 9.9, 4.9, 2.0 Hz, H-5′), 3.95−3.77 (4H, m, H-2′, H-3′, H-6′a, H-6′b), 3.64 (1H, pseudo t, J = 9.5 Hz, H-4′); 13C NMR (90 MHz, DMSO-d6 + TFA) δ (ppm): 181.8 (CON), 165.8 (CN), 138.1, 132.2, 128.5, 125.9 (aromatics) 123.9 (q, CF3, 1JCF = 272.3 Hz), 94.7 (C′-1), 77.5, 74.5, 73.3, 70.6 (C-2′−C-5′), 61.3 (C-6′). ESI-MS positive mode (m/z): calcd for C15H15F3N2NaO6 ([M + Na]+): 399.077. Found: 399.075. Anal. Calcd for C15H15F3N2O6: C, 47.88; H, 4.02; N, 7.44. Found: C, 48.12; H, 4.16; N, 7.31. C-(2,3,4,6-Tetra-O-benzoyl-1-benzylidenetriazenyl-1-deoxy-α-Dglucopyranosyl)formamide (16). The azide 14 (260 mg, 0.39 mmol) was dissolved in anhydrous CH2Cl2 (5 mL) and cooled to 0 °C in an ice bath, and tri-n-butylphosphine (108 μL, 0.43 mmol) was added. After 10 min, benzaldehyde (41 μL, 0.43 mmol, 1.1 equiv) was added and the reaction mixture turned strong yellow within several minutes. The mixture was stirred for 2 h, and the solvent was then removed. The remaining syrup was purified by column chromatography (1:2 EtOAc/hexane) to give 194 mg (66%) of a yellow amorphous solid. Rf = 0.28 (1:1 EtOAc/hexane); [α]D −56 (c 0.58, CHCl3); 1H NMR (500 MHz, CDCl3) δ (ppm): 8.72 (1H, s, CHN), 8.06 (2H, d, J = 6.8 Hz, aromatics), 7.98 (2H, d, J = 6.9 Hz, aromatics), 7.93 (2H, d, J = 6.9 Hz, aromatics), 7.86 (2H, d, J = 6.9 Hz, aromatics), 7.81 (2H, d, J = 7.3 Hz, aromatics), 7.58−7.30 (13H, m, aromatics), 7.24 (2H, t, J = 7.6 Hz, aromatics), 6.96 (1H, s, NH), 6.48 (1H, pseudo t, J = 8.1 Hz, H-3), 6.20 (1H, s, NH), 6.10 (1H, d, J = 8.1 Hz, H-2), 5.83 (1H, dd, J = 9.7, 8.1 Hz, H-4), 5.18 (1H, ddd, J = 9.8, 4.6, 3.2 Hz, H-5), 4.81 (1H, dd, J = 12.3, 3.2 Hz, H-6a), 4.51 (dd, J = 12.3, 4.6 Hz, H6b); 13C NMR (100 MHz, CDCl3) δ (ppm): 172.2 (CHN), 168.2, 166.2, 165.3, 164.6 (CO), 133.8−128.4 (aromatics), 94.2 (C-1), 73.4, 71.9, 71.6, 69.1 (C-2−C-5), 63.1 (C-6). ESI-MS positive mode (m/z): calcd for C42H34N4NaO10 ([M + Na]+): 777.217. Found: 777.215. C-(2,3,4,6-Tetra-O-benzoyl-1-benzylideneamino-1-deoxy-α-Dglucopyranosyl)formamide (20). This was prepared from azide 14 (500 mg, 0.75 mmol), tri-n-butylphosphine (207 μL, 0.83 mmol), and benzaldehyde (84 μL, 0.83 mmol) according to general procedure IV (reaction time: 14 days) and purified by column chromatography (1:4 acetone/hexane) to give 172 mg (32%) of a white amorphous solid. Rf = 0.40 (1:1 EtOAc/hexane); [α]D −29 (c 0.53, CHCl3); 1H NMR (500 MHz, CDCl3) δ (ppm): 8.68 (1H, s, CHN), 8.14 (2H, d, J = 7.4 Hz, aromatics), 8.04 (2H, d, J = 7.5 Hz, aromatics), 7.97 (2H, d, J = 7.5 Hz, aromatics), 7.80 (2H, d, J = 7.5 Hz, aromatics), 7.62 (2H, d, J = 7.2 Hz, aromatics), 7.56 (1H, t, J = 7.5 Hz, aromatics), 7.51−7.32 (12H, m, aromatics), 7.22 (2H, t, J = 7.8 Hz, aromatics), 6.78 (1H, d, J = 4.0 Hz, NH), 6.64 (1H, pseudo t, J = 9.6 Hz, H-3), 6.40 (1H, d, J = 4.0 Hz, NH), 5.85 (1H, pseudo t, J = 9.8 Hz, H-4), 5.53 (1H, ddd, J = 9.8, 3.6, 2.7 Hz, H-5), 5.47 (1H, d, J = 9.8 Hz, H-2), 4.88 (1H, dd, J

= 12.3, 2.7 Hz, H-6a), 4.48 (dd, J = 12.3, 3.6 Hz, H-6b); 13C NMR (125 MHz, CDCl3) δ (ppm): 171.4 (CONH2, 3J H‑2,CO = 5.4 Hz, from HSQMBC at 125 MHz), 166.4, 165.6, 165.5, 164.9 (CO), 160.6 (CHN), 135.2−128.3 (aromatics), 89.6 (C-1), 75.1 (C-2), 73.5 (C-5), 71.7 (C-3), 69.3(C-4), 62.8 (C-6). ESI-MS positive mode (m/z): calcd for C42H34N2NaO10 ([M + Na]+): 749.211. Found: 749.208. C-[2,3,4,6-Tetra-O-benzoyl-1-deoxy-1-(naphth-1-ylmethylidene)amino-α-D-glucopyranosyl]formamide (21). This was prepared from azide 14 (500 mg, 0.75 mmol), tri-n-butylphosphine (207 μL, 0.83 mmol), and 1-naphthaldehyde (113 μL, 0.83 mmol) according to general procedure IV (reaction time: 28 days) and purified by column chromatography (1:4 acetone/hexane) to give 353 mg (60%) of a white amorphous solid. Rf = 0.44 (1:1 EtOAc/hexane); [α]D −48 (c 0.46, CHCl3); 1H NMR (360 MHz, CDCl3) δ (ppm): 9.36 (1H, s, CHN), 8.67 (1H, d, J = 8.6 Hz, aromatics), 8.18 (2H, d, J = 7.1 Hz, aromatics), 8.09 (2H, d, J = 7.2 Hz, aromatics), 8.00 (2H, d, J = 7.2 Hz, aromatics), 7.91 (1H, d, J = 8.1 Hz, aromatics), 7.84 (4H, m, aromatics), 7.56−7.26 (13H, m, aromatics), 7.21 (2H, t, J = 7.7 Hz, aromatics), 6.78 (1H, d, J = 3.1 Hz, NH), 6.71 (1H, pseudo t, J = 9.6 Hz, H-3), 6.34 (1H, d, J = 3.1 Hz, NH), 5.90 (1H, pseudo t, J = 9.8 Hz, H-4), 5.63−5.54 (2H, m, H-2 and H-5), 4.99 (1H, dd, J = 12.4, 2.5 Hz, H-6a), 4.52 (1H, dd, J = 12.4, 3.5 Hz, H-6b); 13C NMR (90 MHz, CDCl3) δ (ppm): 171.3 (CONH2), 166.5, 165.6, 165.5, 165.1 (CO), 160.2 (CHN), 133.8−123.9 (aromatics), 89.9 (C-1), 75.3 (C-2), 73.7 (C-5), 71.8 (C-3), 69.3 (C-4), 62.6 (C-6). ESI-MS positive mode (m/z): calcd for C46H36N2NaO10 ([M + Na]+): 799.226. Found: 799.225. C-[2,3,4,6-Tetra-O-benzoyl-1-deoxy-1-(naphth-2-ylmethylidene)amino-α-D-glucopyranosyl]formamide (22). This was prepared from azide 14 (500 mg, 0.75 mmol), tri-n-butylphosphine (207 μL, 0.83 mmol), and 2-naphthaldehyde (129 mg, 0.83 mmol) according to general procedure IV (reaction time: 5 days, room temperature) and purified by column chromatography (2:5 acetone/hexane) to give 182 mg (31%) of a white amorphous solid. Rf = 0.42 (1:1 EtOAc/ hexane); [α]D; −61 (c 0.48, CHCl3); 1H NMR (400 MHz, CDCl3) δ (ppm): 8.84 (1H, s, CHN), 8.19 (2H, d, J = 7.2 Hz, aromatics), 8.07 (2H, d, J = 7.3 Hz, aromatics), 7.98 (2H, d, J = 7.2 Hz, aromatics), 7.91 (1H, s, 1H, aromatics), 7.80 (5H, m, aromatics), 7.74 (1H, d, J = 8.6 Hz, aromatics), 7.57 (1H, t, J = 7.4 Hz, aromatics), 7.54−7.43 (6H, m, aromatics), 7.39−7.29 (5H, m, aromatics), 7.19 (2H, t, J = 7.8 Hz, aromatics), 6.91 (1H, d, J = 3.9 Hz, NH), 6.77− 6.65 (2H, m, H-3 and NH), 5.91 (1H, pseudo t, J = 9.7 Hz, H-4), 5.60−5.52 (2H, m, H-2 and H-5), 4.95 (1H, dd, J = 12.4, 2.8 Hz, H6a), 4.50 (1H, dd, J = 12.4, 3.5 Hz, H-6b); 13C NMR (90 MHz, CDCl3) δ (ppm): 171.4 (CONH2), 166.4, 165.6, 165.4, 164.9 (C O), 160.7 (CHN), 135.2−123.4 (aromatics), 89.7 (C-1), 75.2 (C2), 73.5 (C-5), 71.7 (C-3), 69.2 (C-4), 62.7 (C-6). ESI-MS positive mode (m/z): calcd for C46H36N2NaO10 ([M + Na]+): 799.226. Found: 799.223. C-[2,3,4,6-Tetra-O-benzoyl-1-deoxy-1-(4trifluoromethylbenzylidene)amino-α-D-glucopyranosyl]formamide (23). This was prepared from azide 14 (500 mg, 0.75 mmol), tri-nbutylphosphine (207 μL, 0.83 mmol), and 4-trifluoromethylbenzaldehyde (113 μL, 0.83 mmol) according to general procedure IV (reaction time: 21 days) and purified by column chromatography (1:3 acetone/hexane) to give 227 mg (38%) of a white amorphous solid. Rf = 0.45 (1:1 EtOAc/hexane); [α]D −11 (c 1.0, CHCl3); 1H NMR (360 MHz, CDCl3) δ (ppm): 8.72 (1H, s, CHN), 8.15 (2H, d, J = 7.3 Hz, aromatics), 8.05 (2H, d, J = 7.3 Hz, aromatics), 7.98 (2H, d, J = 7.2 Hz, aromatics), 7.80 (2H, d, J = 7.3 Hz, aromatics), 7.73 (2H, d, J = 8.1 Hz, aromatics), 7.64−7.54 (3H, m, aromatics), 7.52−7.42 (4H, m, aromatics), 7.41−7.30 (5H, m, aromatics), 7.22 (2H, t, J = 7.5 Hz, aromatics), 6.84−6.71 (2H, m, 2 × NH), 6.66 (1H, pseudo t, J = 9.6 Hz, H-3), 5.88 (1H, pseudo t, J = 9.7 Hz, H-4), 5.53 (1H, ddd, J = 9.8, 3.4, 2.4 Hz, H-5), 5.49 (1H, d, J = 9.7 Hz, H-2), 4.94 (1H, dd, J = 12.4, 2.4 Hz, H-6a), 4.48 (1H, dd, J = 12.4, 3.4 Hz, H-6b); 13C NMR (90 MHz, CDCl3) δ (ppm): 171.1 (CONH2), 166.4, 165.6, 165.4, 164.9 (CO), 159.4 (CHN), 138.1−125.8 (aromatics), 123.8 (q, CF3, 1JCF = 272.0 Hz), 89.9 (C-1), 74.9 (C-2), 73.7 (C-5), 6130



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Hz, aromatics), 7.74 (2H, d, J = 7.3 Hz, aromatics), 7.69 (1H, t, J = 7.4 Hz, aromatics), 7.62 (2H, d, J = 8.2 Hz, aromatics), 7.59−7.53 (3H, m, aromatics), 7.46−7.35 (3H, m, aromatics), 7.33−7.21 (5H, m, aromatics), 7.16 (1H, t, J = 7.8 Hz, aromatics), 6.66 (1H, pseudo t, J = 10.0 Hz, H-3′), 6.08 (1H, pseudo t, J = 10.0 Hz, H-4′), 6.00 (1H, d, J = 10.2 Hz, H-2′), 5.79 (1H, dd, J = 13.2, 2.7 Hz, H-6′a), 5.54 (1H, ddd, J = 10.0, 2.7, 1.8 Hz, H-5′), 4.36 (1H, dd, J = 13.1, 1.8 Hz, H-6′b); 13C NMR (90 MHz, CDCl3) δ (ppm): 180.1 (CONH), 168.1, 165.7, 165.5, 164.6, 163.5 (CO, CN), 134.1−125.2 (aromatics), 123.5 (q, CF3, 1JCF = 272.7 Hz), 93.4 (C-1′), 72.1, 71.3, 70.5, 68.6 (C-2′−C-5′), 61.6 (C-6′). ESI-MS positive mode (m/z): calcd for C43H31F3N2NaO10 ([M + Na]+): 815.183. Found: 815.179. (1′S)-1′,5′-Anhydro-D-glucitol-spiro-[1′,5]-2-phenyl-imidazolin4-one (28). This was prepared from compound 24 (180 mg, 0.25 mmol) according to general procedure III. Column chromatography (20:1:0.1 EtOAc/MeOH/AcOH) gave two fractions. Fraction I: 13 mg (17%) of 10 as a white amorphous solid. Fraction II: 48 mg (63%) of 28 as pale yellow crystals. mp: 165− 167 °C; Rf = 0.32 (5:1:0.1 EtOAc/MeOH/AcOH); [α]D +36 (c 0.56, MeOH); 1H NMR (360 MHz, D2O) δ (ppm): 7.87 (2H, d, J = 7.8 Hz, Ph), 7.70 (1H, t, J = 7.5 Hz, Ph), 7.56 (2H, t, J = 7.8 Hz, Ph), 4.43 (1H, ddd, J = 9.9, 5.0, 2.0 Hz, H-5′), 4.21 (1H, pseudo t, J = 9.5 Hz, H-3′), 3.87 (1H, dd, J = 12.6, 2.0 Hz, H-6′a), 3.81 (1H, d, J = 9.7 Hz, H-2′), 3.77 (1H, dd, J = 12.6, 5.0 Hz, H-6′b), 3.57 (1H, pseudo t, J = 9.7 Hz, H-4′); 13C NMR (90 MHz, DMSO-d6 + TFA) δ (ppm): 180.9 (CON), 167.5 (CN), 134.4, 129.2, 128.5, 125.6 (Ph), 92.2 (C′-1), 75.4, 73.0, 72.1, 69.5 (C-2′−C-5′), 61.1 (C-6′). ECD [MeOH, λ (nm) (Δε), c 0.215 mM]: 336 (+0.62), 301 (−2.44), 262 (+2.64), 237 (−1.12), 218 (+1.44), 202 (−0.90). ESI-MS positive mode (m/z): calcd for C14H16N2NaO6 ([M + Na]+): 331.090. Found: 331.090. Anal. Calcd for C14H16N2O6: C, 54.54; H, 5.23; N, 9.09. Found: C, 54.45; H, 5.31; N, 9.15. (1′S)-1′,5′-Anhydro-D-glucitol-spiro-[1′,5]-2-(1-naphthyl)-imidazolin-4-one (29). This was prepared from compound 25 (135 mg, 0.17 mmol) according to general procedure III. Column chromatography (20:1:0.1 EtOAc/MeOH/AcOH) gave two fractions. Fraction I: 7 mg (11%) of 11 as yellowish crystals. Fraction II: 52 mg (84%) of 29 as yellow crystals. mp: 167−169 °C; Rf = 0.30 (5:1:0.1 EtOAc/MeOH/AcOH); [α]D +39 (c 0.35, MeOH); 1H NMR (360 MHz, D2O) δ (ppm): 8.28 (1H, d, J = 6.4 Hz, aromatics), 7.97 (1H, d, J = 7.6 Hz, aromatics), 7.85 (1H, d, J = 6.0 Hz, aromatics), 7.70 (1H, d, J = 6.2 Hz, aromatics), 7.58−7.43 (3H, m, aromatics), 4.52−4.45 (1H, m, H-5′), 4.25 (1H, t, J = 9.5 Hz, H-3′), 3.94−3.76 (3H, m, H-2′, H-6′a, H-6′b), 3.60 (1H, t, J = 9.6 Hz, H-4′); 13C NMR (90 MHz, DMSO-d6 + TFA) δ (ppm): 181.3 (CON), 166.8 (CN), 133.3, 133.1, 129.9, 128.7, 128.6, 127.8, 126.9, 125.9, 125.0, 124.3 (aromatics), 93.4 (C′-1), 75.3, 73.2, 72.3, 69.7 (C-2′−C-5′), 61.2 (C-6′). ECD [MeOH, λ (nm) (Δε), c 0.199 mM]: 311 (−2.08), 284 (+4.66), 256 (−0.61), 227 (+1.15), 207 (−6.14). ESI-MS positive mode (m/z): calcd for C18H18N2NaO6 ([M + Na]+): 381.106. Found: 381.106. Anal. Calcd for C18H18N2O6: C, 60.33; H, 5.06; N, 7.82. Found: C, 60.49; H, 5.18; N, 7.60. (1′S)-1′,5′-Anhydro-D-glucitol-spiro-[1′,5]-2-(2-naphthyl)-imidazolin-4-one (30). This was prepared from compound 26 (170 mg, 0.22 mmol) according to general procedure III. Column chromatography (20:1:0.1 EtOAc/MeOH/AcOH) gave two fractions. Fraction I: 14 mg (18%) of 12 as yellowish crystals. Fraction II: 50 mg (64%) of 30 as yellow crystals. mp: 167−169 °C; Rf = 0.34 (4:1:0.1 EtOAc/MeOH/AcOH); [α]D +23 (c 0.32, MeOH); 1H NMR (360 MHz, D2O) δ (ppm): 7.76 (1H, s, aromatics), 7.70 (1H, d, J = 8.1 Hz, aromatics), 7.67−7.59 (2H, m, aromatics), 7.55−7.43 (3H, m, aromatics), 4.50 (1H, ddd, J = 9.7, 4.6, 1.8 Hz, H-5′), 4.27 (1H, pseudo t, J = 9.5 Hz, H-3′), 3.98 (1H, dd, J = 12.4, 1.8 Hz, H-6′a), 3.88 (1H, dd, J = 12.4, 4.6 Hz, H-6′b), 3.85 (1H, d, J = 9.9 Hz, H-2′), 3.64 (1H, pseudo t, J = 9.6 Hz, H-4′); 13C NMR (90 MHz, DMSO-d6 + TFA) δ (ppm): 181.8 (CON), 166.9 (CN), 135.2, 132.0, 130.0, 129.2, 129.1, 128.7, 127.9, 127.4, 123.8, 123.6 (aromatics), 92.7 (C′-1), 75.2, 73.1, 72.2, 69.7 (C-2′−C-5′), 61.2 (C6′). ECD [MeOH, λ (nm) (Δε), c 0.038 mM]: 339 (+0.44), 308 (−5.90), 283sh (+3.71), 258 (+5.51), 246sh (−2.11), 241 (−2.91),

71.5 (C-3), 69.1 (C-4), 62.6 (C-6). ESI-MS positive mode (m/z): calcd for C43H33F3N2NaO10 ([M + Na]+): 817.198. Found: 817.197. (1′S)-1′,5′-Anhydro-2′,3′,4′,6′-tetra-O-benzoyl-D-glucitol-spiro[1′,5]-2-phenyl-imidazolin-4-one (24). This was prepared from imine 20 (240 mg, 0.33 mmol) with NBS (65 mg, 0.36 mmol) and pyridine (30 μL, 0.36 mmol) according to general procedure II. Column chromatography (1:3 EtOAc/hexane) gave two fractions. Fraction I: 26 mg (10%) of 6 as a white amorphous solid. Fraction II: 163 mg (68%) of 24 as a white amorphous solid. Rf = 0.26 (2:3 EtOAc/hexane); [α]D +44 (c 0.65, CHCl3); 1H NMR (500 MHz, DMSO-d6) δ (ppm): 12.30 (1H, s, NH), 8.00 (2H, d, J = 7.3 Hz, aromatics), 7.92−7.86 (4H, m, aromatics), 7.74 (2H, d, J = 7.3 Hz, aromatics), 7.69−7.60 (4H, m, aromatics), 7.58−7.44 (9H, m, aromatics), 7.43−7.36 (4H, m, aromatics), 6.48 (1H, pseudo t, J = 9.9 Hz, H-3′), 5.96 (1H, d, J = 10.0 Hz, H-2′), 5.93 (1H, pseudo t, J = 9.9 Hz, H-4′), 5.34 (1H, dt, J = 10.0, 3.3 Hz, H-5′), 4.55 (2H, d, J = 3.3 Hz, H-6′a and H-6′b); 13C NMR (125 MHz, DMSO-d6) δ (ppm): 180.7 (CONH, 3J H‑2′,CO = 5.5 Hz, from HSQMBC at 125 MHz), 165.4, 165.3, 165.1, 164.7 (CO), 163.9 (CN), 134.0−126.9 (aromatics), 92.6 (C-1′), 71.7 (C-2′), 70.5 (C-3′), 69.6 (C-5′), 68.6 (C-4′), 62.6 (C-6′). ESI-MS positive mode (m/z): calcd for C42H32N2NaO10 ([M + Na]+): 747.195. Found: 747.191. (1′S)-1′,5′-Anhydro-2′,3′,4′,6′-tetra-O-benzoyl-D-glucitol-spiro[1′,5]-2-(1-naphthyl)-imidazolin-4-one (25). This was prepared from imine 21 (265 mg, 0.34 mmol) with NBS (68 mg, 0.38 mmol) and pyridine (31 μL, 0.38 mmol) according to general procedure II. Column chromatography (1:3 EtOAc/hexane) gave two fractions. Fraction I: 34 mg (13%) of 7 as a pale yellow amorphous solid. Fraction II: 177 mg (67%) of 25 as a pale yellow amorphous solid. Rf = 0.42 (1:1 EtOAc/hexane); [α]D +43 (c 0.61, CHCl3); 1H NMR (360 MHz, DMSO-d6) δ (ppm): 12.24 (1H, s, NH), 8.52 (1H, d, J = 8.5 Hz, aromatics), 8.09 (1H, d, J = 7.8 Hz, aromatics), 8.02 (2H, d, J = 8.0 Hz, aromatics), 7.98−7.88 (3H, m, aromatics), 7.83−7.72 (5H, m, aromatics), 7.65−7.33 (15H, m, aromatics), 6.60 (1H, pseudo t, J = 9.9 Hz, H-3′), 6.16 (1H, d, J = 9.9 Hz, H-2′), 6.03 (1H, pseudo t, J = 9.7 Hz, H-4′), 5.46 (1H, d, J = 9.8 Hz, H-5′), 4.64 (2H, s, H-6′a and H-6′b); 13C NMR (90 MHz, DMSO-d6) δ (ppm): 180.2 (CONH), 166.2, 165.5, 165.1, 164.7 164.2 (CO, CN), 134.1− 124.4 (aromatics), 92.8 (C-1′), 71.7 (C-2′), 70.5 (C-3′), 69.7 (C-5′), 68.7 (C-4′), 62.7 (C-6′). ESI-MS positive mode (m/z): calcd for C46H34N2NaO10 ([M + Na]+): 797.211. Found: 797.210. (1′S)-1′,5′-Anhydro-2′,3′,4′,6′-tetra-O-benzoyl-D-glucitol-spiro[1′,5]-2-(2-naphthyl)-imidazolin-4-one (26). This was prepared from imine 22 (170 mg, 0.22 mmol) with NBS (42 mg, 0.24 mmol) and pyridine (20 μL, 0.24 mmol) according to general procedure II. Column chromatography (1:3 EtOAc/hexane) gave two fractions. Fraction I: 28 mg (16%) of 8 as a pale yellow amorphous solid. Fraction II: 131 mg (77%) of 26 as a pale yellow amorphous solid. Rf = 0.30 (2:3 EtOAc/hexane); [α]D +16 (c 0.8, CHCl3); 1H NMR (400 MHz, DMSO-d6) δ (ppm): 12.45 (1H, s, NH), 8.53 (1H, s, aromatics), 8.04−7.88 (8H, m, aromatics), 7.75 (2H, d, J = 7.2 Hz, aromatics), 7.70−7.32 (16H, m, aromatics), 6.53 (1H, pseudo t, J = 9.9 Hz, H-3′), 6.01 (1H, d, J = 9.9 Hz, H-2′), 5.97 (1H, pseudo t, J = 9.8 Hz, H-4′), 5.39 (1H, dt, J = 9.9, 3.3 Hz, H-5′), 4.59 (2H, d, J = 3.3 Hz, H-6′a and H-6′b); 13C NMR (90 MHz, DMSO-d6) δ (ppm): 180.6 (CONH), 165.4, 165.3, 165.1, 164.7, 163.9 (CO, CN), 134.9−123.1 (aromatics), 92.6 (C-1′), 71.8 (C-2′), 70.5 (C-3′), 69.7 (C-5′), 68.6 (C-4′), 62.6 (C-6′). ESI-MS positive mode (m/z): calcd for C46H34N2NaO10 ([M + Na]+): 797.211. Found: 797.210. (1′S)-1′,5′-Anhydro-2′,3′,4′,6′-tetra-O-benzoyl-D-glucitol-spiro[1′,5]-2-(4-trifluoromethylphenyl)-imidazolin-4-one (27). This was prepared from imine 23 (200 mg, 0.25 mmol) with NBS (50 mg, 0.28 mmol) and pyridine (23 μL, 0.28 mmol) according to general procedure II. Column chromatography (1:3 EtOAc/hexane) gave two fractions. Fraction I: 36 mg (18%) of 9 as a white amorphous solid. Fraction II: 109 mg (55%) of 27 as a white amorphous solid. Rf = 0.36 (1:1 EtOAc/hexane); [α]D +40 (c 0.48, CHCl3); 1H NMR (400 MHz, CDCl3) δ (ppm): 10.58 (1H, s, NH), 8.24 (2H, d, J = 6.9 Hz, aromatics), 8.11 (2H, d, J = 7.0 Hz, aromatics), 7.82 (2H, d, J = 7.3 6131



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Table 5. Summary of the Diffraction Data Processing and Refinement Statistics for the rmGPb Complexesa rmGPb complex

10

data collection and processing statistics resolution (Å) 13.53−2.30 outermost shell (Å) 2.39−2.30 reflections measured 200 015 unique reflections 39 153 (3172) multiplicity 5.1 (3.5) Rsymm 0.082 (0.709) completeness (%) 90.4 (71.2) ⟨I/σI⟩ 11.4 (1.4) CC1/2 0.991 (0.784) refinement statistics Rcryst 0.150 (0.244) Rfree 0.202 (0.246) no of solvent molecules 261 r.m.s. deviation from ideality in bonds (Å) 0.008 angles (deg) 1.2 average B-factor (Å2) protein atoms 30.9 solvent molecules 28.9 inhibitor atoms 18.17 PDB entry

11

12

28

29

30

13.55−2.30 2.38−2.30 212 848 39 926 (3073) 5.3 (3.3) 0.086 (0.686) 91.7 (73.8) 11.9 (1.5) 0.993 (0.805)

13.74−2.28 2.36−2.28 202 859 41 141 (2962) 4.9 (2.9) 0.091 (0.769) 92.0 (702) 12.2 (1.4) 0.991 (0.727)

13.44−2.35 2.44−2.35 173 043 37 248 (3258) 4.6 (3.3) 0.073 (0.610) 91.6 (77.9) 12.4 (1.6) 0.993 (0.804)

13.70−2.36 2.45−2.36 204 077 35 956 (3168) 5.7 (4.1) 0.080 (0.583) 90.2 (76.8) 15.1 (2.3) 0.996 (0.859)

13.69−2.40 2.49−2.40 197 811 33 997 (2975) 5.8 (4.2) 0.074 (0.402) 90.3 (76.8) 16.5 (3.3) 0.997 (0.925)

0.157 (0.248) 0.213 (0.298) 227

0.158 (0.256) 0.211 (0.329) 251

0.157 (0.253) 0.211 (0.339) 221

0.141 (0.227) 0.191 (0.275) 229

0.141 (0.213) 0.200 (0.242) 228

0.009 1.3

0.008 1.5

0.007 1.1

0.008 1.2

0.012 1.5

30.5 29.0 30.3

21.4 30.7 55.3b

33.7 30.7 20.6 6QA8

31.2 28.2 20.1 6QA7

29.0 26.2 17.3/55.5b 6QA6

a

Values in parentheses are for the outermost shell. bValues for inhibitor molecules bound at the active and new allosteric site, respectively.

223 (+2.66), 205 (−3.37); ECD [H2O, λ (nm) (Δε), c 0.160 mM]: 362sh (+0.28), 334 (+1.37), 306 (−6.24), 284sh (+3.62), 259 (+6.90), 242 (−3.05), 236sh (−2.35), 220 (+3.36), 203sh (−2.81). ESI-MS positive mode (m/z): calcd for C18H18N2NaO6 ([M + Na]+): 381.106. Found: 381.106. Anal. Calcd for C18H18N2O6: C, 60.33; H, 5.06; N, 7.82. Found: C, 60.29; H, 5.21; N, 7.73. (1′S)-1′,5′-Anhydro-D-glucitol-spiro-[1′,5]-2-(4-trifluoromethylphenyl)-imidazolin-4-one (31). This was prepared from compound 27 (180 mg, 0.23 mmol) according to general procedure III. Column chromatography (20:1:0.1 EtOAc/MeOH/AcOH) gave two fractions. Fraction I: 17 mg (20%) of 13 as yellowish crystals. Fraction II: 48 mg (56%) of 31 as yellowish crystals. mp: 166−168 °C; Rf = 0.33 (8:1:0.1 EtOAc/MeOH/AcOH); [α]D +41 (c 0.70, MeOH); 1H NMR (360 MHz, D2O) δ (ppm): 7.93 (2H, d, J = 8.1 Hz, aromatics), 7.80 (2H, d, J = 8.4 Hz, aromatics), 4.41 (1H, ddd, J = 9.7, 5.1, 1.9 Hz, H-5′), 4.19 (1H, pseudo t, J = 9.5 Hz, H-3′), 3.87 (1H, dd, J = 12.6, 1.9 Hz, H-6′a), 3.80 (1H, d, J = 9.7 Hz, H-2′), 3.67 (1H, dd, J = 12.6, 5.1 Hz, H-6′b), 3.56 (1H, pseudo t, J = 9.7 Hz, H4′); 13C NMR (90 MHz, DMSO-d6 + TFA) δ (ppm): 183.0 (CON), 164.3 (CN), 132.5, 131.6, 128.5, 125.9 (aromatics) 123.7 (q, CF3, 1 JCF = 272.6 Hz), 93.8 (C′-1), 75.0, 73.3, 72.2, 69.7 (C-2′−C-5′), 61.2 (C-6′). ESI-MS positive mode (m/z): calcd for C15H15F3N2NaO6 ([M + Na]+): 399.077. Found: 399.078. Anal. Calcd for C15H15F3N2O6: C, 47.88; H, 4.02; N, 7.44. Found: C, 48.09; H, 4.10; N, 7.26. (3aR)-(1′,5′-Anhydro-3′,4′,6′-tri-O-benzoyl-2′-deoxy-D-arabinohexitol)-[1,2-d]-2-phenyl-oxazolin-3a-carboxamide (32). This was isolated as an unwanted product when compound 2 was heated in refluxing xylene (3 days) or in DMF at 160 °C under microwave irradiation (30 min). After solvent removal, the residual syrup was purified by column chromatography (1:3 EtOAc/hexane) to give 17 or 24%, resp., of 32 as a white foam. Rf = 0.34 (1:1 EtOAc/hexane); [α]D +37 (c 0.50, CHCl3); 1H NMR (360 MHz, CDCl3) δ (ppm): 8.13 (2H, dd, J = 8.1, 1.2 Hz, aromatics), 7.97−7.89 (4H, m, aromatics), 7.83 (2H, dd, J = 8.2, 1.2 Hz, aromatics), 7.59 (1H, t, J = 7.4 Hz, aromatics), 7.53−7.43 (4H, m, aromatics), 7.40 (1H, t, J = 7.5 Hz, aromatics), 7.33−7.22 (4H, m, aromatics), 7.15 (2H, t, J = 7.8 Hz, aromatics), 7.10 (1H, d, J = 3.7 Hz, NH), 6.62 (1H, d, J = 3.7 Hz, NH), 5.87 (1H, dd, J = 3.1, 2.1 Hz, H-3), 5.61 (1H, ddd, J = 6.7, 2.1,

1.5 Hz, H-4), 5.27 (1H, dd, J = 3.1, 1.5 Hz, H-2), 4.82 (1H, dd, J = 12.0, 3.0 Hz, H-6a), 4.59 (1H, dd, J = 12.0, 4.6 Hz, H-6b), 4.15 (1H, ddd, J = 6.7, 4.6, 3.0 Hz, H-5); 13C NMR (90 MHz, CDCl3) δ (ppm): 171.2 (CONH2), 168.5 (OCN), 166.3, 165.0, 164.5 (CO), 133.6−125.7 (aromatics), 99.5 (C-1), 75.4 (C-2), 70.9 (C-5), 68.0 (C-3), 66.7 (C-4), 64.4 (C-6). ESI-MS positive mode (m/z): calcd for C35H28N2NaO9 ([M + Na]+): 643.169. Found: 643.171. Kinetic Studies. All isoenzymes were produced as described earlier.50,56 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.57 rmGPb and rmGPa (3 μg/mL) or 1 μg/mL hlGPa were assayed in a 30 mM imidazole/ HCl buffer (pH 6.8) containing 60 mM KCl, 0.6 mM ethylenediaminetetraacetic acid (EDTA), and 0.6 mM dithiothreitol (DTT) using constant concentrations of glycogen (0.2% w/v) and adenosine 5′-monophosphate (AMP) (1 mM; only for the rmGPb experiments) and various concentrations of Glc-1-P (2, 3, 4, 6, and 10 mM for rmGP and 1, 2, 3, 4, and 6 mM for hlGPa) and inhibitors as described previously.47 Initial velocities were calculated from the pseudo-first-order rate constants using the first-order rate equation, and inhibition constant (Ki) values were calculated from the plot of Km(app.) versus [inhibitor] using nonlinear regression program GRAFIT58 and an explicit weighting. Ex Vivo Studies. Ex vivo studies with human HepG2 hepatocarcinoma cells were performed as described previously59 using a range of 30 of 80−516 μM. X-ray Crystallography. Tetragonal (space group P43212) T-state rmGPb crystals were grown by the batch method using AMP-free rmGPb (25−30 mg/mL) in the presence of 1 mM inosine 5′monophosphate, 1 mM spermine, and in a buffer of 10 mM N,Nbis(2-hydroxyethyl)-2-aminoethanesulfonic sodium salt titrated with 0.1 N HCl to pH 6.7, containing 3 mM DTT, 0.1 mM EDTA, and 0.02% (w/v) NaN3 and seeding from previous rmGPb crystals. X-ray crystallographic binding studies were performed by diffusion of either 10 (10 mM; 19 h), 11 (7.5 mM; 96 h), 28 (10 mM; 14 h), 12 (10 mM; 96 h), 29 (7.5 mM; 15 h), or 30 (1 mM; 29 h), solution in the crystallization media (see above) supplemented with 10% (v/v) DMSO in preformed rmGPb crystals at room temperature prior to data collection. X-ray diffraction data were collected using a Cu X-ray microfocus source (Oxford Diffraction SuperNova) equipped with a 4 6132



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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 were performed by the program CrysAlisPro.60 Scaling and merging of intensities were performed by Aimless,61 and the optimum resolution was selected based on the CC1/2 criterion.62 Crystallographic refinement of the complexes was performed by maximum-likelihood methods using REFMAC.61 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 AceDRG63 within Coot,64 and they were fitted to the electron density maps after adjustment of their torsion angles. A summary of the data processing and refinement statistics for the inhibitor complex structures is given in Table 5, and the validity of the refinement procedure was checked using the PDB_REDO server.65 As there were more than five reflections per atom available, both an isotropic and an anisotropic B-factor models were considered, and the isotropic B-factor model was selected based on the Hamilton R ratio test. A translation libration screw (TLS) model for grouped atom movement with one TLS group was used. The stereochemistry of the protein residues was validated by MolProbity.66 Hydrogen bonds and van der Waals interactions were calculated with the program CONTACT as implemented in CCP461 applying distance cutoff values of 3.3 and 4.0 Å, respectively. Figures were prepared with CCP4 Molecular Graphics.67 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 5.

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel: +30 2410 565278. Fax: +30 2410 565290 (D.D.L.). *E-mail: [email protected]. Tel: +3652512900 ext 22348. Fax: +3652512744 (L.S.). ORCID

Attila Mándi: 0000-0002-7867-7084 Demetres D. Leonidas: 0000-0002-3874-2523 László Somsák: 0000-0002-9103-9845 Author Contributions ⊥

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

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financed by the National Research, Development and Innovation Office of Hungary (OTKA 109450 and 120181) as well as the EU and co-financed by the European Regional Development Fund under the projects GINOP-2.3.215-2016-00008 and GINOP-2.3.3-15-2016-00004. The Hungarian Governmental Information-Technology Development Agency (KIFÜ ) is acknowledged for CPU time. This work was also supported in part by 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. E.K. would like to acknowledge support by Greece and the European Union (European Social Fund, ESF) through the Operational Programme “Human Resources Development, Education and Lifelong Learning” in the context of the project “Strengthening Human Resources Research Potential via Doctorate Research” (MIS-5000432), implemented by the State Scholarships Foundation (IKY).

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CONFORMATIONAL ANALYSIS AND TDDFT-ECD CALCULATIONS Mixed torsional/low-frequency mode conformational searches were carried out by means of Macromodel 10.8.011 software68 using the optimized potential for liquid simulations (OPLS)2005 force field69,70 with the implicit solvent model for CHCl3 applying a 21 kJ/mol energy window. Geometry optimizations [B3LYP/6-31G(d) and B3LYP/6-31+G(d,p) in vacuo and ωB97XD/TZVP71 with a PCM solvent model for MeOH] and TDDFT-ECD and DFT-NMR calculations were performed with Gaussian 09.72 ECD spectra were generated as the sum of Gaussians with 3000 and 3600 cm−1 half-height widths, using dipole-velocity computed rotational strengths.73 NMR calculations were performed at the mPW1PW91/6-311+G(2d,p)74 level for the B3LYP/6-31+G(d,p) conformers. Computed NMR data were corrected with I = 185.2853 and S = −1.0267.75,76 Boltzmann distributions were estimated from the B3LYP and ωB97XD energies. In the case of the B3LYP/631G(d)-level optimizations, the zero-point vibrational energycorrection was also applied.

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ABBREVIATIONS ECD, electronic circular dichroism; Glc-1-P, glucose-1phosphate; GP, glycogen phosphorylase; hlGPa, human liver glycogen phosphorylase; OPLS, optimized potentials for liquid simulations; PCM, polarizable continuum model; rmGP, rabbit muscle glycogen phosphorylase; r.m.s, root mean square; T2DM, type 2 diabetes mellitus; TH, glucopyranosylidenespiro-thiohydantoin; TZVP, triple ζ valence plus (a set of polarization function)

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ASSOCIATED CONTENT

S Supporting Information *

REFERENCES

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.9b00356. Tabular presentation of some NMR spectroscopic data; copies of NMR spectra; computed conformer distribution and classification of conformers; data of enzyme kinetics, ex vivo studies and X-ray crystallography (PDF) Molecular formula strings (CSV) Accession Codes

286QA8; 296QA7; 306QA6. Authors will release the atomic coordinates and experimental data upon article publication. 6133



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