Synthesis and Primary Activity Assay of Novel Benitrobenrazide and Benserazide Derivatives

Schiff bases attract research interest due to their applications in chemical synthesis and medicinal chemistry. In recent years, benitrobenrazide and benserazide containing imine moiety have been synthesized and characterized as promising inhibitors of hexokinase 2 (HK2), an enzyme overexpressed in most cancer cells. Benserazide and benitrobenrazide possess a common structural fragment, a 2,3,4-trihydroxybenzaldehyde moiety connected through a hydrazone or hydrazine linker acylated on an N′ nitrogen atom by serine or a 4-nitrobenzoic acid fragment. To avoid the presence of a toxicophoric nitro group in the benitrobenrazide molecule, we introduced common pharmacophores such as 4-fluorophenyl or 4-aminophenyl substituents. Modification of benserazide requires the introduction of other endogenous amino acids instead of serine. Herein, we report the synthesis of benitrobenrazide and benserazide analogues and preliminary results of inhibitory activity against HK2 evoked by these structural changes. The derivatives contain a fluorine atom or amino group instead of a nitro group in BNB and exhibit the most potent inhibitory effects against HK2 at a concentration of 1 µM, with HK2 inhibition rates of 60% and 54%, respectively.

Molecules 2024, 29, x FOR PEER REVIEW 2 of 18 Benserazide (BND) and benitrobenrazide (BNB) (Figure 2) are compounds belonging to the group of hydrazine derivatives containing common structural fragments, including a N-acyl rest of serine or 4-nitrobenzoic acid and a 2,3,4-trihydroxybenzaldehyde (pyrogallol-4-carboxaldehyde) fragment a ached to an N′ nitrogen atom by a methylene or methine carbon atom.Both compounds BNB and BND have promising inhibitory activity against hexokinase 2 (HK2) [13][14][15], an enzyme involved in the phosphorylation of glucose in glycolysis, a first step of glucose metabolism [16].An increased requirement for glucose is observed in cancer cells, especially in rapidly growing and drug-resistant tumors [ [17][18][19][20][21][22].Among all human hexokinase isoenzymes, HK2 shows overexpression in cancer cells, which makes it interesting in the context of molecularly targeted therapy [23][24][25][26][27][28].Currently, several HK2 potent inhibitors are recognized, like metformin, 2-deoxy-D-Glucose, or 3-bromopyruvate [24, [29][30][31][32].Benserazide is an FDA-approved drug for the treatment of Parkinson's disease, but it was recently also recognized as a strong HK2 inhibitor [14].Benitrobenrazide was identified as a potential HK2 inhibitor by structure-based virtual ligand screening [15].According to our previous study, the key structural feature is the presence of three hydroxy groups in a benzene ring, which occupies the same HK2-binding pocket as the natural substrate, glucose [29].The influence of other structural elements, namely -NH-NH-CH2and -NH=N-CH-, on HK2 enzymatic activity has not yet been determined and requires investigation to explain their impact on enzymatic activity.
Herein, we present our primary a empts to explore the influence of structural features observed in benserazide and benitrobenrazide in their biological activity against HK2.The serine originally present in benserazide has been exchanged with other amino acid fragments, e.g., glycine, tyrosine, and cysteine.Application of these amino acids that are different in structure, including glycine (achiral), threonine-containing benzene ring, and cysteine (containing a thiol group instead of a hydroxyl one), in the synthesis of benserazide analogues should deliver an answer regarding the importance of serine moiety on benserazide inhibitory activity.In the BNB molecule, the 4-nitrophenyl moiety has been substituted by an alkyl chain or aromatic rings of different molecular areas (benzene, naphthalene, and anthracene) to judge their downstream influence on HK2 activity and gauge HK2 active site volume.We decided to exchange the toxicophoric nitro group present in BNB moiety by two common substituents, namely the fluorine atom and the amino group [33,34].These substituents have the opposite effects on electron density in the benzene ring of BNB and were introduced to explain the eventual influence of electronic conditions on its activity.The fluorine atom, like the nitro group, decreases the electron density, whereas the amino group is typical of the electron-donating group.The novel analogues of benserazide and benitrobenrazide were examined for their inhibitory activity against HK2 to obtain information about the influence of structural variations on inhibition activity.Benserazide is an FDA-approved drug for the treatment of Parkinson's disease, but it was recently also recognized as a strong HK2 inhibitor [14].Benitrobenrazide was identified as a potential HK2 inhibitor by structure-based virtual ligand screening [15].According to our previous study, the key structural feature is the presence of three hydroxy groups in a benzene ring, which occupies the same HK2-binding pocket as the natural substrate, glucose [29].The influence of other structural elements, namely -NH-NH-CH 2and -NH=N-CH-, on HK2 enzymatic activity has not yet been determined and requires investigation to explain their impact on enzymatic activity.
Herein, we present our primary attempts to explore the influence of structural features observed in benserazide and benitrobenrazide in their biological activity against HK2.The serine originally present in benserazide has been exchanged with other amino acid fragments, e.g., glycine, tyrosine, and cysteine.Application of these amino acids that are different in structure, including glycine (achiral), threonine-containing benzene ring, and cysteine (containing a thiol group instead of a hydroxyl one), in the synthesis of benserazide analogues should deliver an answer regarding the importance of serine moiety on benserazide inhibitory activity.In the BNB molecule, the 4-nitrophenyl moiety has been substituted by an alkyl chain or aromatic rings of different molecular areas (benzene, naphthalene, and anthracene) to judge their downstream influence on HK2 activity and gauge HK2 active site volume.We decided to exchange the toxicophoric nitro group present in BNB moiety by two common substituents, namely the fluorine atom and the amino group [33,34].These substituents have the opposite effects on electron density in the benzene ring of BNB and were introduced to explain the eventual influence of electronic conditions on its activity.The fluorine atom, like the nitro group, decreases the electron density, whereas the amino group is typical of the electron-donating group.The novel analogues of benserazide and benitrobenrazide were examined for their inhibitory activity against HK2 to obtain information about the influence of structural variations on inhibition activity.

Chemistry of Benitrobenrazide and Benserazide Derivatives
Benitrobenrazide and benserazide contain a N-acyl fragment, 4-nitrophenyl, or a serine moiety, respectively [13,15].We modified these regions of each molecule by incorporating aromatic rings of different molecular areas instead to probe the volume of the active site pocket of HK2.In further modifications, a nitro group suspected as a genotoxic substituent was replaced by substituents of different polarity, constituting either a fluorine atom or Molecules 2024, 29, 629 3 of 18 an amino group.The introduction of fluorine and amino substituent as pharmacophore groups into drugs molecules increases their therapeutic effectiveness [35,36].Additionally, the imine bond in hydrazone analogue was reduced to a single carbon-nitrogen bond to identify the effect of the imine double bond (C=N) on HK2 activity.
The final benitrobenrazide analogue synthesis is depicted below (Scheme 1).Commercially available methyl propionate and benzoate 2a and 2b were used.Methyl esters of other aromatic acids 1c, 1e, and 1f were obtained in a one-pot synthesis involving the primary transformation of each appropriate carboxylic acid into its respective acyl chloride by treatment with thionyl chloride in excess, followed by esterification in the presence of methanol (MeOH) [37] (Scheme 1; Pathway A).The ester of anthracene-9-carboxylic acid 1d was synthesized by a two-step synthesis.The first step was conversion of anthracene-9carboxylic acid into its acyl chloride in the presence of an excess of thionyl chloride and a small amount of dimethylformamide (DMF) as a catalyst.The separated acyl chloride was then subjected to esterification with ethanol (EtOH) in the presence of triethylamine, according to synthesis pathway B shown in Scheme 1.
Benitrobenrazide and benserazide contain a N-acyl fragment, 4-nitrophenyl, or a serine moiety, respectively [13,15].We modified these regions of each molecule by incorporating aromatic rings of different molecular areas instead to probe the volume of the active site pocket of HK2.In further modifications, a nitro group suspected as a genotoxic substituent was replaced by substituents of different polarity, constituting either a fluorine atom or an amino group.The introduction of fluorine and amino substituent as pharmacophore groups into drugs molecules increases their therapeutic effectiveness [35,36].Additionally, the imine bond in hydrazone analogue was reduced to a single carbon-nitrogen bond to identify the effect of the imine double bond (C=N) on HK2 activity.
The final benitrobenrazide analogue synthesis is depicted below (Scheme 1).Commercially available methyl propionate and benzoate 2a and 2b were used.Methyl esters of other aromatic acids 1c, 1e, and 1f were obtained in a one-pot synthesis involving the primary transformation of each appropriate carboxylic acid into its respective acyl chloride by treatment with thionyl chloride in excess, followed by esterification in the presence of methanol (MeOH) [37] (Scheme 1; Pathway A).The ester of anthracene-9-carboxylic acid 1d was synthesized by a two-step synthesis.The first step was conversion of anthracene-9-carboxylic acid into its acyl chloride in the presence of an excess of thionyl chloride and a small amount of dimethylformamide (DMF) as a catalyst.The separated acyl chloride was then subjected to esterification with ethanol (EtOH) in the presence of triethylamine, according to synthesis pathway B shown in Scheme 1.
Scheme 1. Preparation of benitrobenrazide derivatives.Reagent and conditions: (A) a: SOCl2 (1.2 equiv.),MeOH, 0 °C to RT, 24 h; b: N2H4•H2O (98%, 4 equiv.),MeOH, RT/reflux 24/72 h.; c: (HO)3C6H2CHO; (1.0 equiv.),MeOH, RT/reflux, 24 h.(B) d: SOCl2, (53.6 equiv.),DMF, RT, Ar, 4 h; e: EtOH, TEA (1.2 equiv.),RT, 24 h; g: N2H4•H2O (98%, 100 equiv.),reflux, 72 h; g: (HO)3C6H2CHO; (1.1 equiv.),MeOH, reflux; 24 h.Hydrazides 3a-3f were obtained with a good yield from appropriate esters in nucleophilic substitution on a carbonyl carbon atom in the presence of an excess of hydrazine monohydrate (four equivalents) in anhydrous boiling methanol [38,39].Conducting hydrazide synthesis 3b-3c and 3f at room temperature led to a purer product and a good yield (60-77%).The preparation of hydrazide 3d needs harsher conditions, so the reaction was conducted in an excess of boiling hydrazine hydrate, with a prolonged reaction time (Scheme 1; B pathway).Hydrazides 3b-3f were obtained in the form of white solids, which precipitated during the reaction process and crystallized from ethanol or aqueous Hydrazides 3a-3f were obtained with a good yield from appropriate esters in nucleophilic substitution on a carbonyl carbon atom in the presence of an excess of hydrazine monohydrate (four equivalents) in anhydrous boiling methanol [38,39].Conducting hydrazide synthesis 3b-3c and 3f at room temperature led to a purer product and a good yield (60-77%).The preparation of hydrazide 3d needs harsher conditions, so the reaction was conducted in an excess of boiling hydrazine hydrate, with a prolonged reaction time (Scheme 1; B pathway).Hydrazides 3b-3f were obtained in the form of white solids, which precipitated during the reaction process and crystallized from ethanol or aqueous ethanol.Compound 3a was purified via silica gel column chromatography using MeOH/CHCl 3 (1:1 v/v) as an eluent.The hydrazide structure was confirmed by 1 H NMR spectroscopy.The absence of a singlet assigned to the methyl group in the region of 3. .95ppm confirmed the conversion of the esters into hydrazides.
Compounds detailed in 3, treated with 2,3,4-trihydroxybenzaldehyde in methanol, produced the products listed in 4 at a 50-100% yield.Hydrazones 4 can adopt an E or a Z configuration at the imine double bond (-N=CH-).In the case of the E conformation, the geometrical isomer can be stabilized by the formation of an intramolecular hydrogen bond between the 2-OH hydroxyl group and the nitrogen atom of the imine [40], as is depicted in Figure 3. Similar behavior is exhibited by compound 10.
For conformer E, the corresponding hydrogen bond length is 2.1 Å. Hydrogen bond formation confirms our DFT calculation of stretching vibrations for the O-H group at 3224.47 cm −1 .Additionally, our calculation data show higher stability of the E conformer over the Z conformer by 9 kcal/mol when hydrogen bond formation is included in simulations [41].We can assume that the E conformer is the predominant form of hydrazone 4a. 1 H NMR spectroscopy verified the results of our calculation based on DFT.The chemical shifts of the protons of the 3-OH and 4-OH groups create singlet peaks at δ H 8.4-9.6 ppm, while the peak for the proton of the 2-OH group is shifted to a lower field and was observed in the region of δ H 11. 3-12.4 ppm.We a ribute this down-field shift of the 2-OH group to the formation of an intramolecular hydrogen bond.Based on 1 H and 13 C NMR spectra of 4a in DMSO, we observed separate chemical signals for the imine proton (N=CH) at 8.09 and 8.17 ppm, respectively.In contrast, the protons of the ethyl group (CH2CH3) were present as two triplets in the region of 1.05-1.09ppm and two quartets in the region of 2.19-2.23 and 2.51-2.54.In the 13 C NMR spectrum, the carbon atom of the amide carbonyl groups (-C(O)NHN-) was detected from signals at 173.71 and 168.73 ppm.The signals for carbon from the ethyl groups (CH2CH3) were present at 8.47, 9.41 ppm, 25.24 and 26.97.These spectra suggest the existence of another structural feature for compounds 4, namely a keto-enol tautomerism (Scheme 2).
Quantum chemical calculations using DFT were used to quantify Gibbs free energy for the constitutional isomers of 4a, which confirms the possibility of the keto and enol forms' occurrence.According to the calculations performed on data collected without a solvent, the keto tautomer is preferred over the enol tautomer and is thermodynamically more stable than the enol form by about 11 kcal/mol.However, based on calculations performed in the presence of DMSO, the solvation process changed the Gibbs free energy of both tautomers.Our calculations clearly indicate that, in a polar aprotic solvent like DMSO, the enol form is more stable by 5 kcal/mol when compared with the keto form due to the interaction of the OH-enol group with the oxygen atom of dimethyl sulfoxide (DMSO).Using quantum chemical calculations based on Density Function Theory (DFT), we studied the possibility of the existence of equilibrium between conformers E and Z for 4a.For conformer E, the corresponding hydrogen bond length is 2.1 Å. Hydrogen bond formation confirms our DFT calculation of stretching vibrations for the O-H group at 3224.47 cm −1 .Additionally, our calculation data show higher stability of the E conformer over the Z conformer by 9 kcal/mol when hydrogen bond formation is included in simulations [41].We can assume that the E conformer is the predominant form of hydrazone 4a. 1 H NMR spectroscopy verified the results of our calculation based on DFT.The chemical shifts of the protons of the 3-OH and 4-OH groups create singlet peaks at δ H 8.4-9.6 ppm, while the peak for the proton of the 2-OH group is shifted to a lower field and was observed in the region of δ H 11. 3-12.4 ppm.We attribute this down-field shift of the 2-OH group to the formation of an intramolecular hydrogen bond.
Based on 1 H and 13 C NMR spectra of 4a in DMSO, we observed separate chemical signals for the imine proton (N=CH) at 8.09 and 8.17 ppm, respectively.In contrast, the protons of the ethyl group (CH 2 CH 3 ) were present as two triplets in the region of 1.05-1.09ppm and two quartets in the region of 2.19-2.23 and 2.51-2.54.In the 13  studied the possibility of the existence of equilibrium between conformers E and Z for 4a.
For conformer E, the corresponding hydrogen bond length is 2.1 Å. Hydrogen bond formation confirms our DFT calculation of stretching vibrations for the O-H group at 3224.47 cm −1 .Additionally, our calculation data show higher stability of the E conformer over the Z conformer by 9 kcal/mol when hydrogen bond formation is included in simulations [41].We can assume that the E conformer is the predominant form of hydrazone 4a. 1 H NMR spectroscopy verified the results of our calculation based on DFT.The chemical shifts of the protons of the 3-OH and 4-OH groups create singlet peaks at δ H 8.4-9.6 ppm, while the peak for the proton of the 2-OH group is shifted to a lower field and was observed in the region of δ H 11. 3-12.4 ppm.We a ribute this down-field shift of the 2-OH group to the formation of an intramolecular hydrogen bond.Based on 1 H and 13 C NMR spectra of 4a in DMSO, we observed separate chemical signals for the imine proton (N=CH) at 8.09 and 8.17 ppm, respectively.In contrast, the protons of the ethyl group (CH2CH3) were present as two triplets in the region of 1.05-1.09ppm and two quartets in the region of 2.19-2.23 and 2.51-2.54.In the 13 C NMR spectrum, the carbon atom of the amide carbonyl groups (-C(O)NHN-) was detected from signals at 173.71 and 168.73 ppm.The signals for carbon from the ethyl groups (CH2CH3) were present at 8.47, 9.41 ppm, 25.24 and 26.97.These spectra suggest the existence of another structural feature for compounds 4, namely a keto-enol tautomerism (Scheme 2).
Quantum chemical calculations using DFT were used to quantify Gibbs free energy for the constitutional isomers of 4a, which confirms the possibility of the keto and enol forms' occurrence.According to the calculations performed on data collected without a solvent, the keto tautomer is preferred over the enol tautomer and is thermodynamically more stable than the enol form by about 11 kcal/mol.However, based on calculations performed in the presence of DMSO, the solvation process changed the Gibbs free energy of both tautomers.Our calculations clearly indicate that, in a polar aprotic solvent like DMSO, the enol form is more stable by 5 kcal/mol when compared with the keto form due to the interaction of the OH-enol group with the oxygen atom of dimethyl sulfoxide (DMSO).Quantum chemical calculations using DFT were used to quantify Gibbs free energy for the constitutional isomers of 4a, which confirms the possibility of the keto and enol forms' occurrence.According to the calculations performed on data collected without a solvent, the keto tautomer is preferred over the enol tautomer and is thermodynamically more stable than the enol form by about 11 kcal/mol.However, based on calculations performed in the presence of DMSO, the solvation process changed the Gibbs free energy of both tautomers.Our calculations clearly indicate that, in a polar aprotic solvent like DMSO, the enol form is more stable by 5 kcal/mol when compared with the keto form due to the interaction of the OH-enol group with the oxygen atom of dimethyl sulfoxide (DMSO).
The imine bond in derivative 4f was reduced using hydrogen in the presence of Pd(OH) 2 as a catalyst at elevated pressure (Scheme 3).The reaction progress was monitored by 1 HNMR spectrometry.The disappearance of the signal for the 8.39 ppm imine proton region (N=CH) indicated the consumption of substrate 4f.Scheme 2. Tautomeric equilibrium in 4a benitrobenrazide.
The imine bond in derivative 4f was reduced using hydrogen in the presence of Pd(OH)2 as a catalyst at elevated pressure (Scheme 3).The reaction progress was monitored by 1 HNMR spectrometry.The disappearance of the signal for the 8.39 ppm imine proton region (N=CH) indicated the consumption of substrate 4f.Scheme 3. Reduction of hydrazone 4f.
Continuing our study, we synthesized benserazide analogues, in which serine was replaced with a side chain moiety of another L-amino acid glycine, tyrosine, and cysteine (Scheme 4).Continuing our study, we synthesized benserazide analogues, in which serine was replaced with a side chain moiety of another L-amino acid glycine, tyrosine, and cysteine (Scheme 4).The imine bond in derivative 4f was reduced using hydrogen in the presence of Pd(OH)2 as a catalyst at elevated pressure (Scheme 3).The reaction progress was monitored by 1 HNMR spectrometry.The disappearance of the signal for the 8.39 ppm imine proton region (N=CH) indicated the consumption of substrate 4f.Continuing our study, we synthesized benserazide analogues, in which serine was replaced with a side chain moiety of another L-amino acid glycine, tyrosine, and cysteine (Scheme 4).Commercially available L-amino acids 6a-c were converted to methyl esters 7a-c, using thionyl chloride and methanol.A benzyloxycarbonyl group (Cbz) was used to protect the amino group of the amino acids by reacting esters 7 with benzyl chloroformate in the presence of triethylamine (TEA) in an anhydrous methylene chloride solution [42].This method of protection was used to facilitate the parallel deprotection of Cbz together with catalytic hydrogenation of the double bond.The reaction of N-Cbz-L-amino acid esters 8a-c with 98% hydrazine hydrate under the same conditions described in Scheme 4 provided products 9 as white crystals at a 36-97% yield.Hydrazones 10a and 10c were synthesized via the reaction of hydrazide 9a and 9c with 2,3,4-trihydroxybenzaldehyde in methanol at room temperature.In the case of 10b, as the above procedure failed, we repeated the condensation in tetrahydrofuran (THF) instead of methanol in an inert atmosphere of argon at reflux, which produced product 10b at a 55% yield.All hydrazones 10a-c were obtained with the E configuration, as confirmed by NMR spectroscopy.The best purification method for compounds 10 was crystallization with aqueous methanol (1:1 v/v).The last step in this synthesis was the elucidation of an efficient method for the reduction of the imine bond of hydrazone and deprotection of the Cbz group.For the corresponding hydrazone 10b, hydrogenation was performed in a Parr's autoclave with gaseous H 2 at 2.5 bar in the presence of palladium catalysts (mixture of 25% Pd/C and Pd(OH) 2 ) at room temperature in methanolic solution.We observed only cleavage of the Cbz group.
Compound 10a was reduced under modified conditions.Instead of gaseous hydrogen, ammonium formate was used as a hydrogen donor and the same palladium catalysts were used, according to the reported protocol [43].Product 11 was obtained as a hygroscopic powder, which rapidly liquefied after 5 min on air.For this reason, hydrazide 11 was lyophilized after the purification was complete.In the case of the cysteine analogue of benserazide 10c, hydrogenation did not occur under these conditions, or with the use of other catalysts such as Raney nickel and electrochemical reduction.The likely reason is that a compound containing sulfur in its structure causes catalyst poisoning, resulting in the loss of catalyst function [44].

Inhibitory Effect of Benserazide and Benitrobenrazide Derivatives on HK2 Enzymatic Activity
We conducted in vitro studies of representative derivatives, namely N-acylhydrazones 4a-4f as benitrobenrazide derivatives and a benitrobenrazide derivative with a single carbon-nitrogen bond 5.The benserazide derivatives, hydrazones 10, 12 with imine bonds, and hydrazide 11 with a single carbon-nitrogen bond, which, like benserazide, have an L-amino acid fragment (Figure 4), were selected for in vitro experiments.The selected intermediates of hydrazide 9a-c, the potential peptidomimetics, were also used to evaluate any inhibition of enzyme activity (Figure 4).The chosen compounds subjected to this enzyme activity assay were purified by preparative HPLC.
For the inhibitory activity estimation, we used a colorimetric method.The most popular HK2 enzymatic activity assays reported in the literature are tests based on spectroscopy, measuring/detecting the changes in NADPH absorbance at 320-490 nm [ [13][14][15]31].An alternative assay to determine the HK2 activity by reverse-phase high-performance liquid chromatography (RP-HPLC) was reported by Guan et al. [45].According to this method, the concentration of released ADP during the conversion of glucose into 6-glucose phosphate is measured at 254 nm.We excluded RP-HPLC as an alternative assay because tested compounds contain chromophores exhibiting absorption in UV-Vis in a region of 200-250 nm, which renders the assay results unreliable.To evaluate the potential of hexokinase 2 inhibitors, we used a commercially available Hexokinase II Inhibitor Screening Kit, which uses a spectrophotometric method by measurement of absorbance at 450 nm, where tested compounds exhibit no interference with the assay.This HK2 activity assay is based on HK2's ability to convert glucose into glucose-6-phosphate.Glucose-6-phosphate is oxidized by glucose-6-phosphate dehydrogenase to produce NADPH, which reduces the probe, showing strong absorbance at 450 nm.
Figures 5 and 6 show the results of the HK2 activity inhibition assay of synthesized compounds.Primary measurements were performed at 50 and 5 µM concentrations.We have decided that the concentration of 50 µM is the highest concentration accepted for the investigated compounds as inhibitors.Compounds 4a, 4b, 4c, 4e, and 4f, which showed the best inhibition of HK2 activity, were selected for the next assessment at a lower concentration of 1 µM.The data in Figure 5 illustrate the inhibition of HK2-mediated phosphorylation of glucose by the test compounds through changes in absorption detection at λ max 450 nm observed in kinetic mode after 5-45 min.
As shown in Figure 6, at a concentration of 50 µM, most of the compounds show significant HK2 inhibition activity, apart from hydrazides 9, which show moderate HK2 inhibition.According to the in vitro study, which considered the effect of HK2 inhibition by the tested compounds at a concentration of 5 µM, derivatives 4a-4f exhibited stronger HK2 inhibition activity than the benserazide derivatives 10c, 11, and 12, which have a modified L-amino acid fragment in their structure.The maximum inhibition by benserazide derivatives 10c, 11, and 12 was approximately 50%.Hydrazides 9 have no significant effect on HK2 activity at a concentration of 5 µM, which confirms that the presence of three hydroxyl groups is required for the inhibition of HK2 activity.
A comparison of the inhibition results for derivatives 4f and 5, for which HK2 inhibition was 92% and 15%, respectively, clearly indicates that the essential feature causing the HK2 inhibitory effect of benitrobenrazide derivatives is the presence of the imine bond (- CH=N-).Referring to studies on the pocket size of the HK2 binding site, it can be assumed that a bulky group, like the anthracenyl group in the structure of the Schiff base 4d, does not fit efficiently into the active site of HK2.Compared to other Schiff bases with smaller volume substituents 4a-c, compound 4d did not exhibit inhibitory activity against HK2 at a 5 µM concentration.
The most promising HK2 inhibitors assayed, 4a, 4b, 4c, 4e, and 4f, were evaluated against human HK2 at a concentration of 1 µM.The derivatives 4e and 4f, which contain a fluorine atom or amino group instead of a nitro group, exhibited the most potent inhibitory effects against HK2 at a concentration of 1 µM, with HK2 inhibition rates of 60% and 54%, respectively.The highest efficacy among the derivatives assessed against HK2 was recorded for Schiff base 4e, in which the fluorine atom acts as an electron-withdrawing substituent in the para position of the benzene ring.Figures 5 and 6 show the results of the HK2 activity inhibition assay of synthesized compounds.Primary measurements were performed at 50 and 5 µM concentrations.We have decided that the concentration of 50 µM is the highest concentration accepted for the investigated compounds as inhibitors.Compounds 4a, 4b, 4c, 4e, and 4f, which showed the best inhibition of HK2 activity, were selected for the next assessment at a lower concentration of 1 µM.The data in Figure 5 illustrate the inhibition of HK2-mediated phosphorylation of glucose by the test compounds through changes in absorption detection at λmax 450 nm observed in kinetic mode after 5-45 min.
As shown in Figure 6, at a concentration of 50 µM, most of the compounds show a fluorine atom or amino group instead of a nitro group, exhibited the most potent inhibitory effects against HK2 at a concentration of 1 µM, with HK2 inhibition rates of 60% and 54%, respectively.The highest efficacy among the derivatives assessed against HK2 was recorded for Schiff base 4e, in which the fluorine atom acts as an electron-withdrawing substituent in the para position of the benzene ring.

Chemistry
The 1 H NMR and 13 C NMR spectra were recorded using a Varian NMR system 600 spectrometer at 600 MHz in , with tetramethylsilane (TMS) as the reference standard.NMR chemical shifts are reported in ppm (δ) and coupling constants (J) in Hz.Melting points were measured on a Boethius PHMK apparatus (VEB Analytik Dresden, Dresden, Germany).The progress of the reaction was monitored by thin-layer chromatography (TLC) using Merck TLC silica gel 60 F254 plates and the following developing systems: A: 5% MeOH/CHCl 3 , B: 10% MeOH/CHCl 3 , and C: 20% MeOH/CHCl 3 .Column chromatography was conducted using silica gel 40-60 µm 60A with methanol-chloroform mixtures as eluents.Preparative high-performance liquid chromatography was performed with the LaboACE LC-5060 system (Japan Analytical Industry Co., Ltd., Tokyo, Japan), with an ODS column (JAIGEL-ODS-AP, model SP-12-10, Japan Analytical Industry, Co., Ltd.Tokyo, Japan).The compounds were eluted with a mobile phase of MeOH at a flow rate of 9 mL/min.High-resolution mass spectroscopy (HRMS) was measured on a Waters Corporation Xevo G2 QTOF apparatus (Waters Corporation, Milford, MA, USA) using electrospray ionization (ESI).

General Procedure for the Synthesis of Esters 2e-f
Methyl esters 2a and 2b were purchased from Merck.Methyl esters 2c and 2e-2f were obtained by the well-known method for esterification of acids using thionyl chloride in methanol [37].To a stirred and cooled (0 • C) solution of the required acid 1c, 1e-1f (20 mmol) in anhydrous methanol (30 mL), thionyl chloride (1.10 equiv.) was added dropwise while stirring.The mixture was warmed up to room temperature and stirred for 24 h.After that, the excess of methanol was removed under diminished pressure and then dried under reduced pressure.In the case of 2f synthesis, after completion of the esterification reaction, the solution was neutralized by adding saturated aqueous NaHCO 3 solution until no further gas evolution was observed.Solid 2f was filtered under reduced pressure.
Synthesis of ethyl anthracene-9-carboxylate 2d: to SOCl 2 (7 mL), we added anthracene-9-carboxylic acid (1.80 mmol) and DMF (0.4 mL) as a catalyst.The reaction mixture was stirred at room temperature under argon atmosphere for 4h.The excess of SOCl 2 was removed under reduced pressure.The residue was washed with toluene.To obtain orange solid acid chloride, EtOH (10 mL) and TEA (1.3 equiv.)were added sequentially.The reaction mixture was stirred at room temperature for 24 h.EtOH was removed under reduced pressure; then, chloroform (10 mL) was added to the residue and the organic layer was washed with water (2 × 8 mL) and dried over anhydrous Na 2 SO 4 .After filtration and evaporation under reduced pressure, the residue was purified on a silica gel packed column using (AcOEt:n-hexane 1:1, v/v) as an eluent, obtaining solid 2d at a yield of 90%.

General Procedure for the Synthesis of Hydrazides 3a-3f
Hydrazides 3a-3f were synthesized from their corresponding esters 2a-2f, followed by reaction with hydrazine according to the method reported by Khan et al. [10].

General Procedure for the Synthesis of N-Benzyloxycarbonyl-L-amino Acid Hydrazides 9a-9c
To a solution of N-Cbz-amino acid methyl esters 8a-8c (16.5 mmol) in anhydrous methanol (30 mL), hydrazine hydrate (98% 4.0 equiv.) was added.The reaction mixture was stirred for 24 h at room temperature.The insoluble product formed was filtered off and was recrystallized from a solution of ethanol: H 2 O, 1:1 v/v [25].

Computational Methods
Calculations were performed by the Orca 4.2.1 package on the DFT level (B3LYP/def2-SVP) [53].The accuracy of the optimization process was determined using the Hessian eigenvalue analysis.All the calculated Hessian eigenvalues were positive for the compounds evaluated.In the case of calculations in a solvent environment, the CPCM continuous solvation model for DMSO was used.

Hexokinase Activity Assay
For studying the potential of HK2 inhibitors, a commercially available assay test (ab211114) Hexokinase II Inhibitor Screening (colorimetric) was used.The in vitro hexokinase activity assay was conducted according to the manufacturer's instructions.Briefly, the enzyme and substrate solution were prepared.Next, the enzyme solution was added to the wells containing sample compounds and incubated for 5 min at 25 • C.Then, the substrate solution was added to the wells and the absorbance was measured at 450 nm every 5 min for 45 min using the Thermo Scientific™ Varioskan™ LUX multimode microplate reader (ThermoFisher Inc.Waltham, MA, USA).The test compounds were dissolved in DMSO at 50 µM, 5 µM, and 1 µM concentrations, with the final concentration of solvent not exceeding 1% by volume.

Conclusions
In our research, we synthesized benitrobenrazide and benserazide analogues.We identified that some of these compounds, namely compounds 4e and 4f, represent a promising class of HK2 inhibitors, inhibiting HK2 at a concentration of 5 µM by 98% and 82%, respectively.At the lower concentration of 1 µM, 4e and 4f inhibited HK2 by 60% and 54%, respectively.We have confirmed that the presence of a bulky anthracenyl group in 4d has no significant effect on HK2 enzymatic activity.The exchanging of serine by glycine or threonine in benserazide analogues has a minor effect on their inhibition activity against HK2.Compounds 11 and 12 reduce the enzymatic activity of HK2 by approx.40% in comparison with the negative control.The presented findings suggest that the imine scaffold -CH=N-, in the structure of the potent HK2 inhibitors, helps to enhance and regulate their biological activities.The -CH=N-core is responsible for the possible binding of various groups with nucleophilic and electrophilic properties and, thus, can interact with targeted enzymes and inhibit their enzymatic activity.

Figure 2 .
Figure 2. The chemical structures of reported HK2 inhibitors.

Figure 2 .
Figure 2. The chemical structures of reported HK2 inhibitors.
C NMR spectrum, the carbon atom of the amide carbonyl groups (-C(O)NHN-) was detected from signals at 173.71 and 168.73 ppm.The signals for carbon from the ethyl groups (CH 2 CH 3 ) were present at 8.47, 9.41 ppm, 25.24 and 26.97.These spectra suggest the existence of another structural feature for compounds 4, namely a keto-enol tautomerism (Scheme 2).

Figure 4 .
Figure 4. Structure of the final derivatives evaluated for inhibition of HK2.

Figure 4 .
Figure 4. Structure of the final derivatives evaluated for inhibition of HK2.

Figure 5 .
Figure 5. Inhibition of HK2 activity, demonstrated as a reduction in rate or extent of generation of HK2-dependent absorbance (OD) at 450 nm by a synthesized compound.Ctrl-is an enzyme control without inhibitor, which shows the normal rate of phosphorylation by HK2.All experiments were performed independently in triplicate.

Figure 5 .
Figure 5. Inhibition of HK2 activity, demonstrated as a reduction in rate or extent of generation of HK2-dependent absorbance (OD) at 450 nm by a synthesized compound.Ctrl-is an enzyme control without inhibitor, which shows the normal rate of phosphorylation by HK2.All experiments were performed independently in triplicate.

Figure 6 .
Figure 6.Inhibitory effect of synthesized compounds on HK2 enzyme activity.Bromopyruvic acid was used as a positive HK2 inhibitor control.The relative activity of the negative enzyme control (without inhibitor) was set at 100%.Results are displayed as the mean ± SD from three independent experiments.

Figure 6 .
Figure 6.Inhibitory effect of synthesized compounds on HK2 enzyme activity.Bromopyruvic acid was used as a positive HK2 inhibitor control.The relative activity of the negative enzyme control (without inhibitor) was set at 100%.Results are displayed as the mean ± SD from three independent experiments.