Synthesis of New Series of 2-C-(β-D-glucopyranosyl)-Pyrimidines and Their Evaluation as Inhibitors of Some Glycoenzymes

Despite the substantial interest in C-glycosyl heterocycles as mimetics of biologically active native glycans, the appearance of C-glycopyranosyl derivatives of six-membered heterocycles, both in synthetic and biological contexts, is rather scarce. As part of our ongoing research program aimed at preparing hitherto barely known 2-C-glycopyranosyl pyrimidines, the goal of the present study was to synthesize new 5-mono- and multiply substituted derivatives of this compound class. Thus, 2-C-(β-D-glucopyranosyl)-5,6-disubstituted-pyrimidin-4(3H)-ones and 4-amino-2-C-(β-D-glucopyranosyl)-5,6-disubstituted-pyrimidines were prepared by base-mediated cyclocondensations of O-perbenzylated and O-unprotected C-(β-D-glucopyranosyl) formamidine hydrochlorides with methylenemalonic acid derivatives. The 2-C-(β-D-glucopyranosyl)-5-substituted-pyrimidines were obtained from the same amidine precursors upon treatment with vinamidinium salts. The deprotected derivatives of these pyrimidines were tested as inhibitors of some glycoenzymes. None of them showed inhibitory activity towards glycogen phosphorylase and α- and β-glucosidase enzymes, but some members of the sets exhibited moderate inhibition against bovine liver β-galactosidase.

Recently, as part of a systematic study on the syntheses of 2-C-glycopyranosyl pyrimidines (e.g., I and II in Figure 1), we published their first general synthesis from the corresponding O-perbenzylated (1) or O-unprotected C-glucopyranosyl formamidines (2) as well as in a one-pot three-step transformation of O-peracylated glycopyranosyl cyanides [17]. Some members of I and II exhibited moderate inhibition of some glycosidase enzymes [17], however, each proved inactive against glycogen phosphorylase [17]. Although these biological effects are not outstanding, these are the first investigations to reveal potential utilities of this novel compound class.
As a continuation of these studies, in this paper, we disclose the preparation of 4,5,6-tri-and 5-monosubstituted 2-C-glucopyranosyl pyrimidines (III, IV and V, respectively) by the reaction of amidines 1 and 2 with methylenemalonic acid derivatives and vinamidinium salts, respectively, and the evaluation of the resulting heterocycles as inhibitors of glycoenzymes.
Recently, as part of a systematic study on the syntheses of 2-C-glycopyranosyl pyrimidines (e.g., I and II in Figure 1), we published their first general synthesis from the corresponding Operbenzylated (1) or O-unprotected C-glucopyranosyl formamidines (2) as well as in a one-pot threestep transformation of O-peracylated glycopyranosyl cyanides [17]. Some members of I and II exhibited moderate inhibition of some glycosidase enzymes [17], however, each proved inactive against glycogen phosphorylase [17]. Although these biological effects are not outstanding, these are the first investigations to reveal potential utilities of this novel compound class.
As a continuation of these studies, in this paper, we disclose the preparation of 4,5,6-tri-and 5monosubstituted 2-C-glucopyranosyl pyrimidines (III, IV and V, respectively) by the reaction of amidines 1 and 2 with methylenemalonic acid derivatives and vinamidinium salts, respectively, and the evaluation of the resulting heterocycles as inhibitors of glycoenzymes.

Syntheses
For the synthesis of the target trisubstituted 2-C-glucopyranosyl pyrimidines, the ring-closures of amidine hydrochloride 1 [18,19] with methylenemalonic acid derivatives 3-7 were investigated first ( Table 1). Treatment of 1 with compounds 3-7 in the presence of NaOMe in MeOH at 0 • C gave the desired pyrimidines 10a-f, respectively, in good yields. In the reaction of 1 with ethyl 2-cyano- 3-ethoxyacrylate 4, the nucleophilic amidine attacked both the cyano and the ester groups of the reagent. Thus, this cyclocondensation led to the formation of a mixture of ethyl 4-amino-pyrimidine- 5-carboxylate 10b and 6-oxo-1,6-dihydropyrimidine- 5-carbonitrile 10c. Surprisingly, the same reaction of 1 with ethyl 2-cyano-2-phenylacrylate 7 afforded only one product 11f, derived from a ring-closure involving the ester group of the reagent.         For the O-debenzylation of the new 2-glucosyl pyrimidines 10a-f, catalytic hydrogenolysis in an acidified EtOAc-EtOH solvent mixture at ambient temperature was attempted. Under the applied reductive conditions, the deprotection of compounds 10b and 10d was smoothly affected to get the test compounds 11b and 11d, respectively, in acceptable yields. Unfortunately, pyrimidines 10a,c,e,f with a 5-CN substituent remained intact under the same conditions. This might be due to a poisoning of the catalyst, caused by the coordination of the cyano group to the palladium.
In order to avoid the critical deprotection in the last step of the synthesis, the preparation of the unprotected pyrimidines 11 was also examined in a reversed sequence, wherein the formamidine salt 2, obtained from 1 by hydrogenolytic O-debenzylation [17], was cyclized with the corresponding methylenemalonic acid derivatives 3-7 ( Table 1). The ring-closure of 2 with compounds 3-7 proceeded similarly to that of amidine salt 1, providing each target test compound 11a-f in moderate to good yields.
The cyclocondensations of amidine salts 1 and 2 with dialkyl benzylidenemalonates 8 and 9, under the same ring-closing conditions used for compounds 3-7, did not directly provide the expected pyrimidinone derivatives 10g,h and 11g,h (Table 1). Similarly to a literature example [20], compounds 8 and 9, when cyclized with 2, furnished 6-oxo-1,4,5,6-tetrahydropyrimidines 12 (Scheme 1). Our attempts to achieve the spontaneous oxidation of compounds 12g,h to get 10g,h by using prolonged reaction times or higher temperatures, were unsuccessful. Finally, the transformation of 12g,h into 10g,h was carried out by applying DDQ as an oxidant in an additional step. The removal of the O-benzyl protecting groups of 12g,h was then performed by hydrogenolysis over Pd(OH) 2 to get the final products 11g,h in good yields.
the desired pyrimidines 10a-f, respectively, in good yields. In the reaction of 1 with ethyl 2-cyano-3ethoxyacrylate 4, the nucleophilic amidine attacked both the cyano and the ester groups of the reagent. Thus, this cyclocondensation led to the formation of a mixture of ethyl 4-amino-pyrimidine-5-carboxylate 10b and 6-oxo-1,6-dihydropyrimidine- 5-carbonitrile 10c. Surprisingly, the same reaction of 1 with ethyl 2-cyano-2-phenylacrylate 7 afforded only one product 11f, derived from a ring-closure involving the ester group of the reagent.
For the O-debenzylation of the new 2-glucosyl pyrimidines 10a-f, catalytic hydrogenolysis in an acidified EtOAc-EtOH solvent mixture at ambient temperature was attempted. Under the applied reductive conditions, the deprotection of compounds 10b and 10d was smoothly affected to get the test compounds 11b and 11d, respectively, in acceptable yields. Unfortunately, pyrimidines 10a,c,e,f with a 5-CN substituent remained intact under the same conditions. This might be due to a poisoning of the catalyst, caused by the coordination of the cyano group to the palladium.
In order to avoid the critical deprotection in the last step of the synthesis, the preparation of the unprotected pyrimidines 11 was also examined in a reversed sequence, wherein the formamidine salt 2, obtained from 1 by hydrogenolytic O-debenzylation [17], was cyclized with the corresponding methylenemalonic acid derivatives 3-7 (Table 1). The ring-closure of 2 with compounds 3-7 proceeded similarly to that of amidine salt 1, providing each target test compound 11a-f in moderate to good yields.
The cyclocondensations of amidine salts 1 and 2 with dialkyl benzylidenemalonates 8 and 9, under the same ring-closing conditions used for compounds 3-7, did not directly provide the expected pyrimidinone derivatives 10g,h and 11g,h (Table 1). Similarly to a literature example [20], compounds 8 and 9, when cyclized with 2, furnished 6-oxo-1,4,5,6-tetrahydropyrimidines 12 (Scheme 1). Our attempts to achieve the spontaneous oxidation of compounds 12g,h to get 10g,h by using prolonged reaction times or higher temperatures, were unsuccessful. Finally, the transformation of 12g,h into 10g,h was carried out by applying DDQ as an oxidant in an additional step. The removal of the O-benzyl protecting groups of 12g,h was then performed by hydrogenolysis over Pd(OH)2 to get the final products 11g,h in good yields.  The formation of 2-C-glucopyranosyl-5-substituted-pyrimidines was also envisaged starting from the same carbohydrate precursors 1 and 2. To this end, NaOMe-mediated cyclisations of compounds 1 and 2 with vinamidinium salts 13-16 were accomplished to get the desired 2,5-disubstituted heterocycles 17 and 18, respectively, in good to high yields ( Table 2). Compound 18a was prepared both by the ring-closure of 1 with 13, followed by a BCl 3 -mediated O-debenzylation of the resulting pyrimidine 17a, and by a reversed debenzylation-cyclisation sequence 1→2→18a. In terms of the overall yields of 18a, the latter route proved to be more efficient (51% for 1→17a→18a vs. 80% for 1→2→18a). By applying this second synthetic pathway, high-yielding preparation of the test compounds 18b and 18c was also smoothly achieved (Table 2). Table 2. Ring-closure of C-(β-D-glucopyranosyl)formamidines with vinamidinium salts. from the same carbohydrate precursors 1 and 2. To this end, NaOMe-mediated cyclisations of compounds 1 and 2 with vinamidinium salts 13-16 were accomplished to get the desired 2,5disubstituted heterocycles 17 and 18, respectively, in good to high yields ( Table 2). Compound 18a was prepared both by the ring-closure of 1 with 13, followed by a BCl3-mediated O-debenzylation of the resulting pyrimidine 17a, and by a reversed debenzylation-cyclisation sequence 1→2→18a. In terms of the overall yields of 18a, the latter route proved to be more efficient (51% for 1→17a→18a vs. 80% for 1→2→18a). By applying this second synthetic pathway, high-yielding preparation of the test compounds 18b and 18c was also smoothly achieved (Table 2). In addition, further transformations of compounds 17c and 17d were carried out to get additional 2-C-glucopyranosyl-5-substituted-pyrimidines (Scheme 2). A Pd(PPh3)2Cl2-catalyzed cross-coupling of 5-bromopyrimidine 17c with phenylboronic acid furnished 5-phenylpyrimidine 17e in excellent yield, while the oxidation of 5-formylpyrimidine 17d with NIS in the presence of K2CO3 and MeOH resulted in methyl pyrimidine-5-carboxylate 17f in good yield. Finally, the cleavage of the O-benzyl protecting groups of 17e,f was performed with BCl3 to obtain the test compounds 18e,f in high yields. In addition, further transformations of compounds 17c and 17d were carried out to get additional 2-C-glucopyranosyl-5-substituted-pyrimidines (Scheme 2). A Pd(PPh 3 ) 2 Cl 2 -catalyzed cross-coupling of 5-bromopyrimidine 17c with phenylboronic acid furnished 5-phenylpyrimidine 17e in excellent yield, while the oxidation of 5-formylpyrimidine 17d with NIS in the presence of K 2 CO 3 and MeOH resulted in methyl pyrimidine-5-carboxylate 17f in good yield. Finally, the cleavage of the O-benzyl protecting groups of 17e,f was performed with BCl 3 to obtain the test compounds 18e,f in high yields.

Enzyme Inhibition Studies
The new unprotected compounds 11 and 18 were tested as inhibitors of some glycoenzymes. Similarly to the previously tested 2-C-glucopyranosyl pyrimidines (I and II in Figure 1) [17], none of them exhibited inhibition against rabbit muscle glycogen phosphorylase b (rmGPb) and almond βglucosidase.

Enzyme Inhibition Studies
The new unprotected compounds 11 and 18 were tested as inhibitors of some glycoenzymes. Similarly to the previously tested 2-C-glucopyranosyl pyrimidines (I and II in Figure 1) [17], none of them exhibited inhibition against rabbit muscle glycogen phosphorylase b (rmGPb) and almond β-glucosidase.

11a
Depending on the substitution pattern of the pyrimidine ring, varied inhibitory potencies of compounds 11 and 19 were observed towards bovine liver β-galactosidase (Table 3). The enzyme kinetic data of the comparable pairs 11a and 11e, 11c and 11f, and 11d and 11h clearly indicated the beneficial effect of the presence of a phenyl substituent at the C-6 position of the pyrimidine ring: while compounds 11a, 11c, and 11d did not inhibit the β-galactosidase, their phenyl-substituted counterparts 11e, 11f, and 11h, respectively, displayed a weak but noticeable inhibition in similar mM concentration ranges. The inhibitory activity of 11f-h in comparison to that of 19 showed that the introduction of a cyano group into the C-5 position of the heterocycle did not cause any significant effect on the potency (19 vs. 11f), but switching to the ester groups resulted in some strengthening of the inhibition (19 vs . 11g and 11h). A similar slight improvement was also observed in the pair 11a and 11b. Compound 11h, bearing both the phenyl and the ester substituent, proved to be the best inhibitor of the series, displaying submillimolar inhibitory effect against this β-galactosidase enzyme.
Among the 2-(β-D-glucopyranosyl)-5-substituted-pyrimidines, the unsubstituted 18a and the 5halogen-substituted heterocycles 18b,c proved to be inactive, while pyrimidines, having the phenyl (18e) and the methyl ester (18f) group, showed weak inhibition against the β-galactosidase enzyme ( Table 3). Although compounds 18e and 18f had no significant effects against this enzyme, their moderate activities indicated that the introduction of these substituents, not only at position 6 but also at 5 of the pyrimidine ring could also be advantageous. Depending on the substitution pattern of the pyrimidine ring, varied inhibitory potencies of compounds 11 and 19 were observed towards bovine liver β-galactosidase (Table 3). The enzyme kinetic data of the comparable pairs 11a and 11e, 11c and 11f, and 11d and 11h clearly indicated the beneficial effect of the presence of a phenyl substituent at the C-6 position of the pyrimidine ring: while compounds 11a, 11c, and 11d did not inhibit the β-galactosidase, their phenyl-substituted counterparts 11e, 11f, and 11h, respectively, displayed a weak but noticeable inhibition in similar mM concentration ranges. The inhibitory activity of 11f-h in comparison to that of 19 showed that the introduction of a cyano group into the C-5 position of the heterocycle did not cause any significant effect on the potency (19 vs. 11f), but switching to the ester groups resulted in some strengthening of the inhibition (19 vs . 11g and 11h). A similar slight improvement was also observed in the pair 11a and 11b. Compound 11h, bearing both the phenyl and the ester substituent, proved to be the best inhibitor of the series, displaying submillimolar inhibitory effect against this β-galactosidase enzyme.
Among the 2-(β-D-glucopyranosyl)-5-substituted-pyrimidines, the unsubstituted 18a and the 5halogen-substituted heterocycles 18b,c proved to be inactive, while pyrimidines, having the phenyl (18e) and the methyl ester (18f) group, showed weak inhibition against the β-galactosidase enzyme ( Table 3). Although compounds 18e and 18f had no significant effects against this enzyme, their moderate activities indicated that the introduction of these substituents, not only at position 6 but also at 5 of the pyrimidine ring could also be advantageous. Depending on the substitution pattern of the pyrimidine ring, varied inhibitory potencies of compounds 11 and 19 were observed towards bovine liver β-galactosidase (Table 3). The enzyme kinetic data of the comparable pairs 11a and 11e, 11c and 11f, and 11d and 11h clearly indicated the beneficial effect of the presence of a phenyl substituent at the C-6 position of the pyrimidine ring: while compounds 11a, 11c, and 11d did not inhibit the β-galactosidase, their phenyl-substituted counterparts 11e, 11f, and 11h, respectively, displayed a weak but noticeable inhibition in similar mM concentration ranges. The inhibitory activity of 11f-h in comparison to that of 19 showed that the introduction of a cyano group into the C-5 position of the heterocycle did not cause any significant effect on the potency (19 vs. 11f), but switching to the ester groups resulted in some strengthening of the inhibition (19 vs . 11g and 11h). A similar slight improvement was also observed in the pair 11a and 11b. Compound 11h, bearing both the phenyl and the ester substituent, proved to be the best inhibitor of the series, displaying submillimolar inhibitory effect against this β-galactosidase enzyme.
Among the 2-(β-D-glucopyranosyl)-5-substituted-pyrimidines, the unsubstituted 18a and the 5halogen-substituted heterocycles 18b,c proved to be inactive, while pyrimidines, having the phenyl (18e) and the methyl ester (18f) group, showed weak inhibition against the β-galactosidase enzyme ( Table 3). Although compounds 18e and 18f had no significant effects against this enzyme, their moderate activities indicated that the introduction of these substituents, not only at position 6 but also at 5 of the pyrimidine ring could also be advantageous. effects against this enzyme. Depending on the substitution pattern of the pyrimidine ring, varied inhibitory potencies of compounds 11 and 19 were observed towards bovine liver β-galactosidase (Table 3). The enzyme kinetic data of the comparable pairs 11a and 11e, 11c and 11f, and 11d and 11h clearly indicated the beneficial effect of the presence of a phenyl substituent at the C-6 position of the pyrimidine ring: while compounds 11a, 11c, and 11d did not inhibit the β-galactosidase, their phenyl-substituted counterparts 11e, 11f, and 11h, respectively, displayed a weak but noticeable inhibition in similar mM concentration ranges. The inhibitory activity of 11f-h in comparison to that of 19 showed that the introduction of a cyano group into the C-5 position of the heterocycle did not cause any significant effect on the potency (19 vs. 11f), but switching to the ester groups resulted in some strengthening of the inhibition (19 vs . 11g and 11h). A similar slight improvement was also observed in the pair 11a and 11b. Compound 11h, bearing both the phenyl and the ester substituent, proved to be the best inhibitor of the series, displaying submillimolar inhibitory effect against this β-galactosidase enzyme.
Among the 2-(β-D-glucopyranosyl)-5-substituted-pyrimidines, the unsubstituted 18a and the 5halogen-substituted heterocycles 18b,c proved to be inactive, while pyrimidines, having the phenyl (18e) and the methyl ester (18f) group, showed weak inhibition against the β-galactosidase enzyme ( Table 3). Although compounds 18e and 18f had no significant effects against this enzyme, their moderate activities indicated that the introduction of these substituents, not only at position 6 but also at 5 of the pyrimidine ring could also be advantageous.

General Methods
Optical rotations were measured on a Jasco P-2000 polarimeter (Jasco, Easton, MD, USA) at rt, and the data were calculated as an average of three parallel measurements. NMR spectra were recorded with Bruker DRX360 (360/90 MHz for 1 H/ 13 C) and Bruker DRX400 (400/100 MHz for 1

General Methods
Optical rotations were measured on a Jasco P-2000 polarimeter (Jasco, Easton, MD, USA) at rt, and the data were calculated as an average of three parallel measurements. NMR spectra were recorded with Bruker DRX360 (360/90 MHz for 1 H/ 13 C) and Bruker DRX400 (400/100 MHz for 1

General Methods
Optical rotations were measured on a Jasco P-2000 polarimeter (Jasco, Easton, MD, USA) at rt, and the data were calculated as an average of three parallel measurements. NMR spectra were recorded with Bruker DRX360 (360/90 MHz for 1 H/ 13 C) and Bruker DRX400 (400/100 MHz for 1

General Methods
Optical rotations were measured on a Jasco P-2000 polarimeter (Jasco, Easton, MD, USA) at rt, and the data were calculated as an average of three parallel measurements. NMR spectra were recorded with Bruker DRX360 (360/90 MHz for 1 H/ 13 C) and Bruker DRX400 (400/100 MHz for 1  Depending on the substitution pattern of the pyrimidine ring, varied inhibitory potencies of compounds 11 and 19 were observed towards bovine liver β-galactosidase (Table 3). The enzyme kinetic data of the comparable pairs 11a and 11e, 11c and 11f, and 11d and 11h clearly indicated the beneficial effect of the presence of a phenyl substituent at the C-6 position of the pyrimidine ring: while compounds 11a, 11c, and 11d did not inhibit the β-galactosidase, their phenyl-substituted counterparts 11e, 11f, and 11h, respectively, displayed a weak but noticeable inhibition in similar mM concentration ranges. The inhibitory activity of 11f-h in comparison to that of 19 showed that the introduction of a cyano group into the C-5 position of the heterocycle did not cause any significant effect on the potency (19 vs . 11f), but switching to the ester groups resulted in some strengthening of the inhibition (19 vs .  11g and 11h). A similar slight improvement was also observed in the pair 11a and 11b. Compound 11h, bearing both the phenyl and the ester substituent, proved to be the best inhibitor of the series, displaying submillimolar inhibitory effect against this β-galactosidase enzyme.
Among the 2-(β-D-glucopyranosyl)-5-substituted-pyrimidines, the unsubstituted 18a and the 5-halogen-substituted heterocycles 18b,c proved to be inactive, while pyrimidines, having the phenyl (18e) and the methyl ester (18f) group, showed weak inhibition against the β-galactosidase enzyme ( Table 3). Although compounds 18e and 18f had no significant effects against this enzyme, their moderate activities indicated that the introduction of these substituents, not only at position 6 but also at 5 of the pyrimidine ring could also be advantageous.

General Procedure 1 for the Synthesis of 2-(β-D-Glucopyranosyl)-pyrimidines (10 or 11) by Cyclisation of C-β-D-Glucopyranosyl Formamidines (1 or 2) with Substituted Methylenemalonic Acid Derivatives
To a solution of the corresponding C-(β-D-glucopyranosyl)formamidine hydrochloride (1 or 2) in dry MeOH (2 mL/100 mg amidine),~1M solution of NaOMe in dry MeOH (3 equiv.) was added at 0 • C. After stirring the reaction mixture at this temperature for 10 min, the appropriate methylenemalonic acid derivative (2 equiv.) was added. The completion of the reaction was monitored by TLC (CHCl 3 -MeOH = 9:1 and EtOAc-hexane = 1:1 in the case of O-perbenzylated derivatives and CHCl 3 -MeOH = 7:3 in the case of unprotected derivatives). After the disappearance of the starting amidine (1 or 2), the reaction mixture was neutralized with glacial acid, the solvent was evaporated under reduced pressure, and the residue was purified by column chromatography. To a solution of amidine hydrochloride 1 (400 mg, 0.66 mmol) in dry MeOH or EtOH (2.5 mL/100 mg substrate),~1M solution of sodium alkoxide in MeOH or EtOH (2 equiv.) was added and the mixture was stirred at rt for 10 min. To this mixture, the corresponding 2-benzylidenemalonate derivative (2 equiv.) was added and stirred at rt until the TLC (EtOAc-hexane = 1:2 and CHCl 3 -MeOH = 9:1) showed the complete conversion of 1 (~6 h). The reaction mixture was then neutralized with glacial acid, concentrated under diminished pressure, and the residue was purified by column chromatography.

Enzyme Assays
The inhibition of rmGPb by the test compounds was investigated with a maximal inhibitory concentration of 625 µM by applying a general protocol described earlier [17,27].
A 10 µL aliquot for each of the different inhibitor stock solutions was mixed with 370 µL of the buffer and 20 µL of the enzyme stock solution in a plastic UV cuvette. After equilibration at 37 • C for 5 min, a 100 µL aliquot of the substrate stock solution was added. The resulting solutions were thoroughly mixed, and the change in absorbance was followed at 400 nm over 240 s in 2 s intervals using the Parallel Kinetics Analysis program of a JASCO V550 (JASCO Tokyo, Japan) spectrophotometer. Progress curves were plotted and fitted to a straight line. ∆A/min values, proportional to initial rate, were considered to be enzyme activities. In a control experiment, the aliquot of the inhibitor solution was replaced by the same amount of buffer. The initial rate data for the enzymatic substrate hydrolysis in the presence and absence of inhibitor were transferred into percentages of overall inhibition and plotted against the inhibitor concentration in logarithmic scale for IC 50 determination.

Conflicts of Interest:
The authors declare no conflict of interest.