Synthesis of Ethyl Pyrimidine-Quinolincarboxylates Selected from Virtual Screening as Enhanced Lactate Dehydrogenase (LDH) Inhibitors

The inhibition of the hLDHA (human lactate dehydrogenase A) enzyme has been demonstrated to be of great importance in the treatment of cancer and other diseases, such as primary hyperoxalurias. In that regard, we have designed, using virtual docking screening, a novel family of ethyl pyrimidine-quinolinecarboxylate derivatives (13–18)(a–d) as enhanced hLDHA inhibitors. These inhibitors were synthesised through a convergent pathway by coupling the key ethyl 2-aminophenylquinoline-4-carboxylate scaffolds (7–12), which were prepared by Pfitzinger synthesis followed by a further esterification, to the different 4-aryl-2-chloropyrimidines (VIII(a–d)) under microwave irradiation at 150–170 °C in a green solvent. The values obtained from the hLDHA inhibition were in line with the preliminary of the preliminary docking results, the most potent ones being those with U-shaped disposition. Thirteen of them showed IC50 values lower than 5 μM, and for four of them (16a, 18b, 18c and 18d), IC50 ≈ 1 μM. Additionally, all compounds with IC50 < 10 μM were also tested against the hLDHB isoenzyme, resulting in three of them (15c, 15d and 16d) being selective to the A isoform, with their hLDHB IC50 > 100 μM, and the other thirteen behaving as double inhibitors.


Introduction
The lactate dehydrogenase enzyme (LDH, EC 1.1.1.27)is a tetramer ubiquitously present in the human body, constituted mainly by two different subunits, the M-type (muscle) and the H-type (heart), encoded by the genes Ldha and Ldhb, respectively.Amongst the five isoforms formed by the combination of those units, the most important ones are the homotetramers hLDHA (M 4 ), which are mostly found in the human liver and skeletal muscle, and hLDHB (H 4 ), which are mostly found in the human heart and brain [1,2].
One of the main reasons for cancer development is the alteration of cell metabolism.An example of such is the Warburg effect, which promotes cancer growth [3][4][5].This alteration leads to cells using glycolysis instead of oxidative phosphorylation, resulting in the production of lactate by the hLDHA enzyme.Targeting the hLDHA enzyme could be a promising approach for treating cancer, as it has been the most-researched isoenzyme in this field over the past few decades [6][7][8][9][10][11][12].In addition to this, the hLDHA enzyme has been associated with various diseases, such as epilepsy [13,14], osteoporosis [15], vascular diseases [16,17], primary hyperoxalurias (PHs) [18][19][20], and arthritis and other inflammatory disorders [21][22][23].
The hLDHB isoenzyme, while not as well documented as hLDHA, is still considered vital in the development of various cancer types [24,25].It has been associated with aggressive cancer phenotypes [26][27][28] and is known to be overexpressed in breast and lung cancers [27][28][29][30][31][32].Due to this, the hLDHB enzyme is seen as an ideal target for the prevention and treatment of several types of cancer [26].In addition to being present in cancer, the hLDHB isoform is also found in different inflammatory [33] and neurodegenerative [34] diseases, and it also acts as a biomarker in ischemic stroke [35].
The inhibition of both hLDHA and hLDHB isoenzymes has been shown to be safe with no severe side effects in humans [24,36].As a result, new therapeutic approaches are being developed to target these isoenzymes.While other biotechnological approaches, such as using small RNA (siRNA or miRNA), are under development [37][38][39], inhibiting LDH using small molecules has several advantages, including lower costs and an easier administration [40].However, despite the development of various hLDHA and hLDHB inhibitors (Figure 1) [8,24,26,[41][42][43][44], none have been approved for clinical use yet [45].
Int. J. Mol.Sci.2024, 25, x FOR PEER REVIEW 2 of 30 cancers [27][28][29][30][31][32].Due to this, the hLDHB enzyme is seen as an ideal target for the prevention and treatment of several types of cancer [26].In addition to being present in cancer, the hLDHB isoform is also found in different inflammatory [33] and neurodegenerative [34] diseases, and it also acts as a biomarker in ischemic stroke [35].
To carry out this objective, we made changes to the quinoline nucleus, including the replacement of the previous oxo group with a carboxylic group (Figure 2).The intention of this change was to enhance the hydrogen bonding ability of the quinolone moiety.The decision to use a carboxylic group was made following reports of hLDHA inhibitors having carboxylic moieties [75][76][77], which are essential for their enzymatic inhibitory activity [78][79][80][81][82].The new hybrids successfully passed the virtual screening using molecular docking.The Pfitzinger reaction was used to design the chemical synthesis as a strategic step to prepare the ethyl quinoline-4-carboxylate ester intermediates 7-12, which were coupled with 4-aryl-2-chloropyrimidines VIII(a-d).We are pleased to report the synthesis and biological evaluation of twenty novel ethyl pyrimidine-quinoline-4-carboxylate derivatives.Four of them are linked by 1,4-and 1,3-disubstituted aryl moieties in a non-U-shaped disposition (13,14)(a,b), and sixteen are linked by a 1,2-disubstituted aryl moiety in a Ushaped disposition (15-18)(a-d).Additionally, we have thoroughly evaluated the inhibitory activity of these compounds against both LDH isoforms (hLDHA and hLDHB).

Virtual Screening
The initial virtual screening was conducted through molecular docking using hLDHA-W31 complex (pdb code: 4R68), as previously documented [46].
A database containing a total of 672 structures of a new family of pyrimidine-quinoline hybrids was created for docking screening.This database was built using a strategic step that involved the Pfitzinger reaction to produce the quinoline-4-carboxylic intermediates.The structures were obtained by combining a variety of substituents to the quinoline, carboxylic derivatives, and aryl residues in the pyrimidine ring.These structures show a linkage between the pyrimidine and quinoline residues through an aminophenyl moiety (see Figure 3).
The analysis of the different substituents (R, X and Ar) was performed as follows: To streamline the process, and based on our previous experience in synthesizing pyrimidinequinolone hybrids as hLDHA inhibitors [46], all structures were docked in the W31 site of the enzyme.Once the docking was completed, the output database was analysed according to the raw affinity values and without any further minimization.
Based on the pre-screening results, we found that all structures produced promising results, with raw affinity values being quite good (S ≤ −7 kcal/mol).However, we prioritized the synthesis of structures containing the ester moiety, as they showed the highest number of structures with S ≤ −9 kcal/mol as well as the most similar affinity towards the active site compared to the reference compound W31 (S = −10.44kcal/mol).The new hybrids successfully passed the virtual screening using molecular docking.The Pfitzinger reaction was used to design the chemical synthesis as a strategic step to prepare the ethyl quinoline-4-carboxylate ester intermediates 7-12, which were coupled with 4-aryl-2-chloropyrimidines VIII(a-d).We are pleased to report the synthesis and biological evaluation of twenty novel ethyl pyrimidine-quinoline-4-carboxylate derivatives.Four of them are linked by 1,4-and 1,3-disubstituted aryl moieties in a non-U-shaped disposition (13,14)(a,b), and sixteen are linked by a 1,2-disubstituted aryl moiety in a Ushaped disposition (15-18)(a-d).Additionally, we have thoroughly evaluated the inhibitory activity of these compounds against both LDH isoforms (hLDHA and hLDHB).

Virtual Screening
The initial virtual screening was conducted through molecular docking using hLDHA-W31 complex (pdb code: 4R68), as previously documented [46].
A database containing a total of 672 structures of a new family of pyrimidine-quinoline hybrids was created for docking screening.This database was built using a strategic step that involved the Pfitzinger reaction to produce the quinoline-4-carboxylic intermediates.The structures were obtained by combining a variety of substituents to the quinoline, carboxylic derivatives, and aryl residues in the pyrimidine ring.These structures show a linkage between the pyrimidine and quinoline residues through an aminophenyl moiety (see Figure 3).
The analysis of the different substituents (R, X and Ar) was performed as follows: To streamline the process, and based on our previous experience in synthesizing pyrimidinequinolone hybrids as hLDHA inhibitors [46], all structures were docked in the W31 site of the enzyme.Once the docking was completed, the output database was analysed according to the raw affinity values and without any further minimization.
Based on the pre-screening results, we found that all structures produced promising results, with raw affinity values being quite good (S ≤ −7 kcal/mol).However, we prioritized the synthesis of structures containing the ester moiety, as they showed the highest number of structures with S ≤ −9 kcal/mol as well as the most similar affinity towards the active site compared to the reference compound W31 (S = −10.44kcal/mol).
Furthermore, nearly all of the highest-scoring structures were U-shaped hybrids with the naphth-2-yl, styryl, phenyl, and 4-chlorophenyl moieties in the lipophilic tails.These structures demonstrated strong interactions with Arg 168 and with the other main amino acid residues responsible for hLDHA activity: His 192 , Asn 137 , and Asp 194 .
Furthermore, nearly all of the highest-scoring structures were U-shaped hybrids with the naphth-2-yl, styryl, phenyl, and 4-chlorophenyl moieties in the lipophilic tails.These structures demonstrated strong interactions with Arg 168 and with the other main amino acid residues responsible for hLDHA activity: His 192 , Asn 137 , and Asp 194 .A thorough docking study was conducted using the selected structures containing ethyl carboxylate esters.This study involved an analysis of the aforementioned lipophilic tails and the definition of R as H, F, Cl, and Br for the substituents on the quinoline.The docking studies were performed at three sites: the active site (W31 location), the cofactor site (NADH), and the extended site (W31-NADH).
The results indicate that the only feasible docking position is the active site (W31 location) because the interactions in the NADH site are not strong enough to compete with it.When docked in the extended site, we observed interactions with the same residues as those described in the W31 site.For more detailed information, please refer to Tables S1-S3 in the Supplementary Materials.
Focusing on the docking in the W31 active site, we proceeded to conduct a detailed analysis of all the structures that were initially selected.The best pose for each hybrid was determined using our previously established filtering criteria [46].The results indicated that only the U-shaped esters (15-18)(a-d) advanced to the final stage.We then determined their affinity and energy values involved in the interactions with the main amino acid residues.For detailed information, please refer to Table S4 in the Supplementary Materials.
In light of the above, we decided to start synthesizing esters (13-15)(a,b) to test the accuracy of the in silico predictions regarding the inhibitory potency of the U-shaped vs. the non-U-shaped compounds.Afterwards, we would determine whether to synthesize the full series of U-shaped hybrids (15-18)(a-d), as shown in Figure 4.A thorough docking study was conducted using the selected structures containing ethyl carboxylate esters.This study involved an analysis of the aforementioned lipophilic tails and the definition of R as H, F, Cl, and Br for the substituents on the quinoline.The docking studies were performed at three sites: the active site (W31 location), the cofactor site (NADH), and the extended site (W31-NADH).
The results indicate that the only feasible docking position is the active site (W31 location) because the interactions in the NADH site are not strong enough to compete with it.When docked in the extended site, we observed interactions with the same residues as those described in the W31 site.For more detailed information, please refer to Tables S1-S3 in the Supplementary Materials.
Focusing on the docking in the W31 active site, we proceeded to conduct a detailed analysis of all the structures that were initially selected.The best pose for each hybrid was determined using our previously established filtering criteria [46].The results indicated that only the U-shaped esters (15-18)(a-d) advanced to the final stage.We then determined their affinity and energy values involved in the interactions with the main amino acid residues.For detailed information, please refer to Table S4 in the Supplementary Materials.
In light of the above, we decided to start synthesizing esters (13-15)(a,b) to test the accuracy of the in silico predictions regarding the inhibitory potency of the U-shaped vs. the non-U-shaped compounds.Afterwards, we would determine whether to synthesize the full series of U-shaped hybrids (15-18)(a-d), as shown in Figure 4.

Chemistry
After obtaining promising results from virtual screening of quinoline-pyrimidine hybrids containing the ethyl ester group on the quinoline core, we proceeded with their synthesis.In order to accomplish this, we optimized the synthetic strategy to obtain the quin-

Chemistry
After obtaining promising results from virtual screening of quinoline-pyrimidine hybrids containing the ethyl ester group on the quinoline core, we proceeded with their synthesis.In order to accomplish this, we optimized the synthetic strategy to obtain the quinoline residue using the Pfitzinger reaction, which is based on the ring-opening/cyclocondensation sequence of the isatin derivative in its reaction with the acetophenones [83].Subsequently, the aminophenylquinoline residue would be coupled with the chloropyrimidine fragment through an aromatic nucleophilic substitution (aminolysis from here).
brids containing the ethyl ester group on the quinoline core, we proceeded with their synthesis.In order to accomplish this, we optimized the synthetic strategy to obtain the quinoline residue using the Pfitzinger reaction, which is based on the ring-opening/cyclocondensation sequence of the isatin derivative in its reaction with the acetophenones [83].Subsequently, the aminophenylquinoline residue would be coupled with the chloropyrimidine fragment through an aromatic nucleophilic substitution (aminolysis from here).
It is well known that maintaining a pH of around 6 is crucial in the process of isolating intermediates 1-3, which were obtained in moderate yields (59-80%).If the pH is higher than 6, the carboxylate anion is formed, and at pH lower than 6, the ammonium group is formed, making them soluble in water.This reaction was also extended to other haloisatins II-IV to produce novel intermediates 4 (F−), 5 (Cl−), and 6 (Br−) with yields of 31%, 50%, and 70%, respectively; in one attempt for 5, the yield reached 94%.The low yield in the synthesis of compound 4 is attributed to the electronegativity of the fluorine atom and the low reactivity of the amino group in this case, which is in agreement with related reactions between 5-haloisatin (I-IV) and 4′-aminoacetophenone V [85,86].Once the aminophenylquinolincarboxylic acid intermediates 1-6 were prepared, we tested the aminolysis reaction with the 2-(4-aminophenyl)quinoline 1 and 2-chloro-4-(4chloro)phenylpyrimidine VIIIa to adjust the reaction conditions.
We initially attempted to carry out the aminolysis reaction using the established conditions with ethanol as the solvent under microwave (MW) irradiation at 120 °C [46,49].However, the reaction did not proceed as expected to obtain the desired hybrid A (see Scheme 2).As a result, we investigated the use of different bases (K2CO3 and Et3N), acids Scheme 1. Synthesis of aminophenylquinolin-4-carboxylic acids 1-6 as intermediates to pyrimidinequinoline hybrids.
It is well known that maintaining a pH of around 6 is crucial in the process of isolating intermediates 1-3, which were obtained in moderate yields (59-80%).If the pH is higher than 6, the carboxylate anion is formed, and at pH lower than 6, the ammonium group is formed, making them soluble in water.This reaction was also extended to other haloisatins II-IV to produce novel intermediates 4 (F−), 5 (Cl−), and 6 (Br−) with yields of 31%, 50%, and 70%, respectively; in one attempt for 5, the yield reached 94%.The low yield in the synthesis of compound 4 is attributed to the electronegativity of the fluorine atom and the low reactivity of the amino group in this case, which is in agreement with related reactions between 5-haloisatin (I-IV) and 4 ′ -aminoacetophenone V [85,86].
We initially attempted to carry out the aminolysis reaction using the established conditions with ethanol as the solvent under microwave (MW) irradiation at 120 • C [46,49].However, the reaction did not proceed as expected to obtain the desired hybrid A (see Scheme 2).As a result, we investigated the use of different bases (K 2 CO 3 and Et 3 N), acids (AcOH and HCl), and solvents (H 2 O, EtOH, isopropanol, CH 3 CN, THF, and 1,4-dioxane) together with different heating conditions (reflux or MW) at different temperatures reaching to 160 • C. Despite our efforts, the reaction did not yield the desired product in most cases.In the few instances where it did proceed (EtOH, MW, 150 • C), a complex mixture was obtained, making purification of the desired product A very challenging.
After finding that the direct method to synthesize type-A carboxylic acid compounds from quinoline-4-carboxylic acid 1 was unsuccessful, we decided to convert the intermediate carboxylic acid 1 into the corresponding ester (7) to avoid by-products.This was also to determine if the purification of desired compounds could be easier.Initially, we attempted the classic Fischer's esterification conditions by carrying out the reaction in ethanol with sulfuric acid; however, this method did not yield good results in terms of purity and isolation simplicity.Furthermore, we also tested this reaction under microwave irradiation with intermediate 1, but a complex mixture along with the formation of its 4 ′ -N-ethyl derivative was observed.
to determine if the purification of desired compounds could be easier.Initially, we attempted the classic Fischer's esterification conditions by carrying out the reaction in ethanol with sulfuric acid; however, this method did not yield good results in terms of purity and isolation simplicity.Furthermore, we also tested this reaction under microwave irradiation with intermediate 1, but a complex mixture along with the formation of its 4′-Nethyl derivative was observed.We tested different reaction conditions and found that using thionyl chloride in ethanol, as reported in related studies [87], allowed us to produce compound 7 with a 50% yield.We then applied these conditions to other acids 2-6, resulting in the desired ester derivatives 8-12 (see Scheme 3).We isolated these compounds through a simple chromatographic separation, obtaining moderate yields of 51-62%, except for compound 12, which had a yield of 30% due to a more complex mixture.To set up the conditions for the aminolysis of aminoesters 7-12 to be coupled to 2chloropyrimidines VIII(a-d), we used the reaction between 2-chloro-4-(4-chlorophenyl)pyrimidine VIIIa and 7 as a model.The reaction was successfully performed under MW irradiation at 150 °C, yielding 13a at 85%.This method was then applied to 8-9, resulting in the ester hybrids 14a and 15a with good yields (78-81%) in less than 60 min (see Scheme 4).
Docking studies indicated that U-shaped esters such as 15a would have the highest affinities, which the bioassays later confirmed.Once we defined the synthetic pathway for producing the new ethyl pyrimidine-quinolincarboxylic esters, we proceeded with synthesizing the complete series of U-shaped esters.An overview of the defined synthetic pathway is presented in Scheme 5.The esterified intermediates 7-12 were combined with the corresponding 4-aryl-2-chloropyrimidines VII(a-d) to produce esterified hybrids 13, Scheme 2. Aminolysis reaction attempt to get pyrimidine-quinoline hybrid A by reaction of aminophenylquinolin-4-carboxylic acid 1 and VIIIa.
We tested different reaction conditions and found that using thionyl chloride in ethanol, as reported in related studies [87], allowed us to produce compound 7 with a 50% yield.We then applied these conditions to other acids 2-6, resulting in the desired ester derivatives 8-12 (see Scheme 3).We isolated these compounds through a simple chromatographic separation, obtaining moderate yields of 51-62%, except for compound 12, which had a yield of 30% due to a more complex mixture.
to determine if the purification of desired compounds could be easier.Initially, we attempted the classic Fischer's esterification conditions by carrying out the reaction in ethanol with sulfuric acid; however, this method did not yield good results in terms of purity and isolation simplicity.Furthermore, we also tested this reaction under microwave irradiation with intermediate 1, but a complex mixture along with the formation of its 4′-Nethyl derivative was observed.We tested different reaction conditions and found that using thionyl chloride in ethanol, as reported in related studies [87], allowed us to produce compound 7 with a 50% yield.We then applied these conditions to other acids 2-6, resulting in the desired ester derivatives 8-12 (see Scheme 3).We isolated these compounds through a simple chromatographic separation, obtaining moderate yields of 51-62%, except for compound 12, which had a yield of 30% due to a more complex mixture.To set up the conditions for the aminolysis of aminoesters 7-12 to be coupled to 2chloropyrimidines VIII(a-d), we used the reaction between 2-chloro-4-(4-chlorophenyl)pyrimidine VIIIa and 7 as a model.The reaction was successfully performed under MW irradiation at 150 °C, yielding 13a at 85%.This method was then applied to 8-9, resulting in the ester hybrids 14a and 15a with good yields (78-81%) in less than 60 min (see Scheme 4).
Docking studies indicated that U-shaped esters such as 15a would have the highest affinities, which the bioassays later confirmed.Once we defined the synthetic pathway for producing the new ethyl pyrimidine-quinolincarboxylic esters, we proceeded with synthesizing the complete series of U-shaped esters.An overview of the defined synthetic pathway is presented in Scheme 5.The esterified intermediates 7-12 were combined with the corresponding 4-aryl-2-chloropyrimidines VII(a-d) to produce esterified hybrids 13, Scheme 3. Esterification of aminophenylquinolin-4-carboxylic acids 1-6 to obtain the corresponding ethyl esters 7-12.
To set up the conditions for the aminolysis of aminoesters 7-12 to be coupled to 2chloropyrimidines VIII(a-d), we used the reaction between 2-chloro-4-(4-chlorophenyl)pyrimidine VIIIa and 7 as a model.The reaction was successfully performed under MW irradiation at 150 • C, yielding 13a at 85%.This method was then applied to 8-9, resulting in the ester hybrids 14a and 15a with good yields (78-81%) in less than 60 min (see Scheme 4).-d) with good yields ranging from 75% to 91%, except for 15d, which yielded 65%.All of the above structures and their reaction details are provided in Table 1.Docking studies indicated that U-shaped esters such as 15a would have the highest affinities, which the bioassays later confirmed.Once we defined the synthetic pathway for producing the new ethyl pyrimidine-quinolincarboxylic esters, we proceeded with synthesizing the complete series of U-shaped esters.An overview of the defined synthetic pathway is presented in Scheme 5.The esterified intermediates 7-12 were combined with the corresponding 4-aryl-2-chloropyrimidines VII(a-d) to produce esterified hybrids 13, 14(a,b), and (15-18)(a-d) with good yields ranging from 75% to 91%, except for 15d, which yielded 65%.All of the above structures and their reaction details are provided in Table 1.In the case of compound 16d, we obtained a crystalline solid from a DMF solution, enabling us to determine its structure unambiguously through single-crystal X-ray diffraction analysis (Figure 5), which is consistent with the spectroscopic characterization.In the case of compound 16d, we obtained a crystalline solid from a DMF solution, enabling us to determine its structure unambiguously through single-crystal X-ray diffraction analysis (Figure 5), which is consistent with the spectroscopic characterization.

hLDH Inhibitory Assays and Preliminary Structure-Activity Relationship
In our study, we initially chose to synthesize and evaluate different ethyl pyrimidinequinolincarboxylate esters using a spectrofluorometric assay [46] to confirm the reliability of our docking simulations.The results confirmed our predictions, showing that the Ushaped esters 15 had lower IC50 values compared to the non-U-shaped esters (13-14) (see Table 2).Furthermore, among the U-shaped esters, those with the naphth-2-yl group exhibited the best IC50 values, with 15b showing a value of less than 5 µM.In the case of compound 16d, we obtained a crystalline solid from a DMF solution, enabling us to determine its structure unambiguously through single-crystal X-ray diffraction analysis (Figure 5), which is consistent with the spectroscopic characterization.

hLDH Inhibitory Assays and Preliminary Structure-Activity Relationship
In our study, we initially chose to synthesize and evaluate different ethyl pyrimidinequinolincarboxylate esters using a spectrofluorometric assay [46] to confirm the reliability of our docking simulations.The results confirmed our predictions, showing that the Ushaped esters 15 had lower IC50 values compared to the non-U-shaped esters (13-14) (see Table 2).Furthermore, among the U-shaped esters, those with the naphth-2-yl group exhibited the best IC50 values, with 15b showing a value of less than 5 µM.

hLDH Inhibitory Assays and Preliminary Structure-Activity Relationship
In our study, we initially chose to synthesize and evaluate different ethyl pyrimidinequinolincarboxylate esters using a spectrofluorometric assay [46] to confirm the reliability of our docking simulations.The results confirmed our predictions, showing that the U-shaped esters 15 had lower IC 50 values compared to the non-U-shaped esters (13-14) (see Table 2).Furthermore, among the U-shaped esters, those with the naphth-2-yl group exhibited the best IC 50 values, with 15b showing a value of less than 5 µM.The superior inhibitory effects of U-shaped 15 compared to 13 and 14 are due to the better positioning of 15a and 15b in the active site, similar to the reference molecule W31.In contrast, the non-U-shaped (13,14)(a,b) are located outside of the active site pocket.This information is illustrated in Figure 6.
Data are presented as the mean ± SD of n = 3 replicates for IC50 values.R values are given from the single dose-response curve of the average percentage of activity for each concentration.
The superior inhibitory effects of U-shaped 15 compared to 13 and 14 are due to the better positioning of 15a and 15b in the active site, similar to the reference molecule W31.In contrast, the non-U-shaped (13,14)(a,b) are located outside of the active site pocket.This information is illustrated in Figure 6.Due to this finding, we continued to focus only on the evaluation of the U-shaped hybrids 15c, 15d, and (16-18)(a-d).The results are shown in Table 3, which also includes the data for hLDHB to help with their comparison.
All the hybrid compounds exhibited interesting inhibition data.Thirteen out of sixteen hybrids had IC50 values of less than 5 µM.Overall, the hybrids showed good affinity values (S < −9 kcal/mol) and moderate interactions with Arg 168 (<−5 kcal/mol).Some hybrids also displayed interactions with other key amino acid residues, such as His 192 and Asp 194 .For specific docking parameters for each U-shaped hybrid, please refer to Table S4 in the Supplementary Materials.
Compound 15a is the only one with an IC50 value greater than 10 µM.This can be attributed to its weak interaction with the crucial Arg 168 amino acid residue.Unlike compound 18a, which interacts with additional amino acid residues such as His 192 , 15a does not demonstrate any other interactions beyond Arg 168 .Due to this finding, we continued to focus only on the evaluation of the U-shaped hybrids 15c, 15d, and (16-18)(a-d).The results are shown in Table 3, which also includes the data for hLDHB to help with their comparison.All the hybrid compounds exhibited interesting inhibition data.Thirteen out of sixteen hybrids had IC 50 values of less than 5 µM.Overall, the hybrids showed good affinity values (S < −9 kcal/mol) and moderate interactions with Arg 168 (<−5 kcal/mol).Some hybrids also displayed interactions with other key amino acid residues, such as His 192 and Asp 194 .
For specific docking parameters for each U-shaped hybrid, please refer to Table S4 in the Supplementary Materials.
Compound 15a is the only one with an IC 50 value greater than 10 µM.This can be attributed to its weak interaction with the crucial Arg 168 amino acid residue.Unlike compound 18a, which interacts with additional amino acid residues such as His 192 , 15a does not demonstrate any other interactions beyond Arg 168 .
The majority of hybrids (15-18)(a-d) are positioned in the active site of the hLDHA enzyme similar to the reference compound W31.This allows them to interact with the different amino acid residues located in the hLDHA active site.This can be seen with 18c in Figure 7a.Additionally, Figure 7b includes its 2D interaction diagram.For a detailed comparison of the placement of each compound in the hLDHA active site, please refer to Figures S1-S16    However, there are some hybrids (15b, 16c, 17c, and 18a) which are located differently in the enzyme compared to W31 (Figure 8).This difference makes them to have stronger interactions with Arg 168 (15b) or to establish another type of interaction with other amino acid residues, as in case of 16c, 17c, and 18a, which are positioned similarly to each other.These differences may be the reason for the good activities displayed by these structures.
After analysing the bioactivity of hybrids (15-18)(a-d) against the hLDHA enzyme, we proceeded to evaluate their inhibitory activity towards the other isoform (hLDHB).Consequently, those hybrids having an IC 50 < 10 µM for the hLDHA enzyme (all hybrids but 15a) were tested against the B isoform.See Table 3 for the inhibition results (IC 50 ).
The results from Table 3 show that three of the hybrids (15c, 15d, and 16d) selectively inhibit the A isoform, as their IC 50 values for hLDHB inhibition are greater than 100 µM.On the other hand, the remaining twelve hybrids showed low IC 50 values for the inhibition of both isoenzymes.This double inhibition is considered an interesting strategy for treating cancer and other proliferative diseases, as it enhances the cytotoxicity of conventional chemotherapeutic drugs.This contributes to demonstrating that the Warburg effect is not solely based on high LDHA expression, as both isozymes need to be targeted to avoid fermentative glycolysis [25,88,89].
Int. J. Mol.Sci.2024, 25, x FOR PEER REVIEW 11 of 30 However, there are some hybrids (15b, 16c, 17c, and 18a) which are located differently in the enzyme compared to W31 (Figure 8).This difference makes them to have stronger interactions with Arg 168 (15b) or to establish another type of interaction with other amino acid residues, as in case of 16c, 17c, and 18a, which are positioned similarly to each other.These differences may be the reason for the good activities displayed by these structures.In this context, compounds 17a, 17c, 18b, and 18d exhibited very similar IC 50 values in both isoforms.The other hybrids (15b, 16(a-c), 17b, 17d, 18a, and 18c) showed a slight preference for the A isoform, with an IC 50(B) /IC 50(A) ratio ranging between 1.5 and 4.0.
To further understand the observed bioactivity against the hLDHB isoenzyme, we conducted a detailed docking analysis of the evaluated hybrids 15(b-d) and (16-18)(a-d).
The reference complex hLDHB-OXM (PDB code: 1I0Z) was chosen, and hybrids 15(b-d) and (16-18)(a-d) were docked because the active site of hLDHB where oxamate is located is similar to hLDHA.Both complexes were aligned, sharing 75% of residues, with an RMSD of 1.405 Å.They have the central 1,3-cyclohexandione residue in W31 located in the same place as oxamate and have common interactions with main amino acid residues Arg 168 (9) , His 192 (3) , and Asn 137 (8) .
However, the pocket of the active site in hLDHA is bigger than in hLDHB, with noninteracting Arg 105 being present outside the pocket (Figure 9) in the latter, affecting the docking analysis and maybe the inhibitory results.
The reference complex hLDHB-OXM (PDB code: 1I0Z) was chosen, and hybrids 15(b-d) and (16-18)(a-d) were docked because the active site of hLDHB where oxamate is located is similar to hLDHA.Both complexes were aligned, sharing 75% of residues, with an RMSD of 1.405 Å.They have the central 1,3-cyclohexandione residue in W31 located in the same place as oxamate and have common interactions with main amino acid residues Arg 168 (9) , His 192 (3) , and Asn 137 (8) .
However, the pocket of the active site in hLDHA is bigger than in hLDHB, with noninteracting Arg 105 being present outside the pocket (Figure 9) in the latter, affecting the docking analysis and maybe the inhibitory results.
The docking studies in the OXM site did not explain the inhibitory results because the size of the hybrids analysed did not fit into the pocket, resulting in positive affinity values, which indicates their repulsion in the active site.Consequently, all hybrids were evaluated in the extended site to see if they could inhibit the enzyme by displacing the NADH.The docking studies in the OXM site did not explain the inhibitory results because the size of the hybrids analysed did not fit into the pocket, resulting in positive affinity values, which indicates their repulsion in the active site.Consequently, all hybrids were evaluated in the extended site to see if they could inhibit the enzyme by displacing the NADH.
We selected poses with an RMSD of less than 1.8 Å. Next, we minimized all the selected poses and chose the one with the best or highest number of interactions with the amino acid residues that interact with NADH.However, the results did not explain the inhibitory activity of hybrids (15-18)(a-d) because the affinity values were not strong enough to displace the NADH, and no strong interactions were observed.(Please refer to Table S5 in the Supplementary Materials for more details).
We proceeded to explore other potential allosteric pockets where hybrids (15-18)(a-d) could be positioned, as has been reported previously [24].We found that there is a selective inhibitor of the hLDHB enzyme with an IC 50 of 42 nM [26] located in an allosteric site of the enzyme (PDB code: 7DBJ) in the complex hLDHB-H1U.
The selected pose was determined based on the best affinity and energy values in interaction with Glu 214 , which is the main interaction in the reference H1U (−40 kcal/mol).
Once the best pose was determined for each structure, we observed that all compounds were positioned in the allosteric site close to the reference H1U, with compounds 16a, 17a, and 18a having a very similar pose to H1U.Furthermore, compounds 15d, 16b, 16d, and 17d exhibited very similar positioning among themselves.Detailed comparisons of their positioning in the hLDHB allosteric site can be found in Figures S17-S32 in the Supplementary Materials.
Additionally, we observed that the affinity values were quite similar (−8.1 ≤ S ≤ −6.7 kcal/mol) to that of the reported structure (S H1U = −8.667kcal/mol).These compounds also formed moderate interactions with Glu 214 and Lys 310 along with some weaker interactions with Ser 211 .For more detailed information, please refer to Table S6 in the Supplementary Materials.
Although the interactions of hybrids (15-18)(a-d) with Glu 214 were not as strong, as in the case of reference H1U, they were of a similar magnitude to those demonstrated with different amino acid residues in the case of hLDHA.Considering the IC 50 values obtained for both isoforms, these findings provide a reasonable explanation for the experimental results.
Additionally, compounds 15c, 15d, and 16d did not interact with either of the two possible Lys 310 residues (Figure 10a), which could explain their selectivity towards the A isoform.Therefore, this interaction seems crucial in these hybrid systems for inhibiting the hLDHB enzyme, either with the Lys 310 residue of one chain or another.

General
All chemicals and solvents were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany) unless stated otherwise.
Melting points were collected using a Barnstead Electrothermal 9100 melting point apparatus (Dubuque, IA, USA), and the acquired data are uncorrected.IR spectra were recorded on a Fourier Bruker Tensor 27 spectrophotometer (Leipzig, Germany) using the ATR dura Sample IR accessory.
NMR spectra were recorded in Bruker Avance NEO 400 spectrometer (Karlsruhe, Germany) at 400 MHz ( 1 H) and 100 MHz ( 13 C) at 298 K (and 393 K, if specified) using as solvent CDCl3 and DMSO-d6 and, as the internal reference, tetramethylsilane (0 ppm), or the residual 1 H/ 13  It is worth noting that all of the evaluated hybrids interacted with the Glu 214 amino acid residue of chain C through the amino group directly linked to the pyrimidine core.In contrast, the interaction with Lys 310 occurred with either of the residues from chain A or C, typically through the ester group or the nitrogen heteroatom belonging to the pyrimidine or quinoline ring, as illustrated with compounds 15a and 16a in Figure 10b.

General
All chemicals and solvents were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany) unless stated otherwise.
Melting points were collected using a Barnstead Electrothermal 9100 melting point apparatus (Dubuque, IA, USA), and the acquired data are uncorrected.IR spectra were recorded on a Fourier Bruker Tensor 27 spectrophotometer (Leipzig, Germany) using the ATR dura Sample IR accessory.
NMR spectra were recorded in Bruker Avance NEO 400 spectrometer (Karlsruhe, Germany) at 400 MHz ( 1 H) and 100 MHz ( 13 C) at 298 K (and 393 K, if specified) using as solvent CDCl 3 and DMSO-d 6 and, as the internal reference, tetramethylsilane (0 ppm), or the residual 1 H/ 13 C solvent signals, that is, 7.26/77.16for CDCl 3 and 2.50/39.52 for DMSO-d 6 .DEPT-135 and 2D-NMR (HSQC, HMBC, and COSY) experiments were used for the assignment of carbon and hydrogen signals.Chemical shifts (δ) are given in ppm, and coupling constants (J) are given in Hz.The following abbreviations are used for multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, ps = pseudosinglet, pd = pseudodoublet, and pt = pseudotriplet.Full assignation of all hydrogen and carbon atoms in the structures is shown in SI.
All the equipment used in the spectroscopic and spectrometric analysis belong to "Centro de Instrumentación Científico y Técnico", (CICT) in "Universidad de Jaén" (UJA).
All reactions were TLC monitored on a 0.2 mm pre-coated aluminium plate of silica gel (Merck 60 F 254 ), and spots were visualized by UV irradiation (254 nm).
All reagents were purchased from commercial sources and used without further purification.All starting materials were weighed and handled in air at room temperature.Precursor 4-aryl-2-chloropyrimidines VIII(a-d) were prepared according to the reported procedure [48].
Ethyl 2-(4-aminophenyl)quinoline-4-carboxylate (7).a-d) (1 mmol) were dissolved in ethanol (1.5 mL), and then the mixture was subjected to microwave irradiation at 150 • C with a setting of 250 psi and 250 W. Once the reaction was completed (TLC monitored using Hex:AcOEt, 8:2), the precipitated solid was filtered off and washed with ethanol; if no solid was formed, then the solvent was removed under reduced pressure, and the residue was dissolved with ethyl acetate (10 mL) and water (10 mL) was added.The aqueous phase was washed several times with ethyl acetate (3 × 10 mL).The organic phases were joined, dried over anhydrous sodium sulphate, and the solvent was removed under vacuum.If needed, compounds (13-18)(a-d) were further purified by FCC.   13 C NMR (100 MHz,

Molecular Modelling
The molecular modelling and docking analysis were performed using the MOE 2022 suit from Chemical Computing Group's Molecular Operating Environment, and the minimization of the energy of molecules and complexes was performed under molecular mechanics using the Amber14:EHT force field.
The complexes of the hLDHA protein with the inhibitor W31 (PDB code 4R68) and hLDHB protein with the inhibitor Oxamate (PDB code 1I0Z) and Oxamate-H1U (PDB code 7DBJ) were downloaded from the Protein Data Bank (PDB) and prepared as follows: For 4R68, 1I0Z, and 7DBJ at the OXM site, all the chains along with their respective ligands and solvents, save for chain A, were deleted using the sequence editor (SEQ); for 7DBJ at the HIU site, which is located between two chains (A-C and B-D), chains B-D along with their respective ligands and solvents were deleted using the sequence editor (SEQ).Then, quick preparation was completed, performing the structure preparation for docking, including the following steps: (i) fixing the issues in the protein structure.There were structural problems that needed to be addressed before proceeding with a simulation, and most items were identified and corrected automatically by the application.In all of these instances, no additional actions needed to be taken.(ii) Protonation, which also analysed residues in which it was possible to have multiple protomers and/or a tautomer and checked for the right charge in any heteroatom, ultimately minimizing the complex system using the force field Amber14.(iii) It was confirmed that no warnings that were not automatically corrected and the user should be aware of were displayed.Then, the complexes were submitted to the general energy minimization mode, in which force field minimization was performed.No restraints were applied, and tethers of different strengths were set on the receptor, ligand, and solvent atoms.Constraints were selected in order to maintain rigid water molecules.The gradient was of 0.1 RMS, meaning that the energy minimization was finished when the root mean square gradient fell below the specified value (0.1).
The input database of screened molecules was prepared from builder editor and imported in the corresponding database file (*.mdb), which was used as the input file in the docking process.To prepare the database input file, we followed a similar preparation process that included a first wash (a set of cleaning rules to ensure that each structure is in a suitable form for subsequent modelling steps, such as conformational enumeration and protein-ligand docking), checking for the right partial charges, and finally, minimizing the energy of the molecules using the force field Amber14.
For hLDHA docking, three pharmacophoric models were created from the Pharmacophore Query Editor tool: (i) W31 site, (ii) NADH site, and (iii) extended site W31-NADH site.Three features were defined so as to interact with the main amino acid residues: Arg 168 , His 192 , Asn 137 , and Asp 194 .All three features were defined with a radius of 1.2 Å, and none of them were classified as essential nor ignored.When establishing the search criteria, the partial match was clicked on and defined as at least one interaction with one of those features.The docking screening was carried out with the following settings: (i) receptor, MOE (the previously prepared complex) receptor atoms; (ii) site, ligand atoms, (iii) pharmacophore, on; (iv) conformation import for analysis of ligand conformation; (v) placement, pharmacophore; (vi) number of returned poses (poses returned by each ligand's placement), 3000; (vii) placement score, London dG; (viii) placement poses, 100; (ix) refinement method, rigid receptor; (x) refinement score, GBI/WSA dG; refinement poses (number of poses retained to be written in the output file), 10.
Once the docking was complete, the best pose score for each ligand determined by a further minimization process (in the output file) was required using molecular mechanics and the specified forcefield.The best pose was determined by the following criteria: (i) RMSD < 1.8 Å [99], (ii) affinity (S) [100] values < −9 kcal/mol, and (iii) energy values involved in the interactions with the main amino acid residues [100], selecting those interacting with Arg168 first, followed by those with a higher number of interactions.In the case that they all interacted with the same amino acids, the ones with the highest energy values involved in the interactions with those amino acid residues were chosen.
For hLDHB docking, no pharmacophoric models were created neither for the OXM nor the extended (OXM-NADH) or the H1U sites.The docking screening was carried out with the following settings: (i) receptor, MOE (the previously prepared complex), receptor atoms; (ii) site, ligand atoms; (iii) conformation import for analysis of ligand conformations; (iv) placement: triangle matcher; (v) number of returned poses (poses returned by each ligand's placement), 3000; (vi) placement score, London dG; placement poses, 100; (vii) refinement method, rigid receptor; (viii) refinement score, GBI/WSA dG; (ix) refinement poses (number of poses retained to be written in the output file), 10.
For the initial screening, the best pose was determined, in both complexes, according to the best raw affinity value.For the in-depth analysis, the best pose was determined as follows: Those poses with an RMSD < 1.8 Å were selected [99] for further minimization using molecular mechanics and the specified forcefield.Then, the best pose was defined according to the best (or higher number of) interactions with the amino acid residues [98] that interacted with reference NADH (in case of complex 1I0Z) or with Glu 214 (in complex 7DBJ).

Human Lactate Dehydrogenase a Enzymatic Activity Assay
The ability of the synthesized compounds to inhibit hLDHA enzyme was measured using recombinant human LDHA (95%, specific activity > 300 units/mg, and concentration 0.5 mg/mL, Abcam, Cambridge, UK) with sodium pyruvate (96%, Merck) as substrate and β-NADH (≥97%, Merck) as cofactor in potassium phosphate buffer (100 mM, pH 7.3).The enzymatic assay was conducted on 96-well microplates, and the decrease in the β-NADH fluorescence (λ excitation = 340 nm; λ emission = 460 nm) was detected in a TECAN Infinite 200 Pro M Plex fluorescent plate reader (Tecan Instrument, Inc., Männedorf, Switzerland) at 28 • C. The activity was determined according to our previously described protocol [44,46].The final volume in each well was set to 200 µL using 100 mM potassium phosphate buffer, 0.041 units/mL LDHA, 155 µM β-NADH, 1 mM pyruvate (saturated conditions), and DMSO solutions (5%, v/v) of pure compounds at concentrations in the range of 0.048 to100 µM.The addition of pyruvate enabled the beginning of the reaction, and fluorescence was registered every 60 s over the course of 10 min.A lineal time interval was selected to calculate the slope at every single concentration.The establishment of the 0% and 100% enzymatic activity was performed by controls and by the use of the inhibitor 3-[ [3[(cyclopropylamino)sulfonyl]-7-(2,4-dimethoxy-5-pyrimidinyl)-4quinolinyl]amino]-5-(3,5-difluorophenoxy)benzoic acid (GSK 2837808 A, Tocris, Minneapolis, MN, USA) at 1 µM [41].The slope obtained at each concentration was compared to the one obtained for the 100% enzymatic activity control to determine the corresponding enzymatic activity.A nonlinear regression analysis in GraphPad Prism version 9.00 for Windows (GraphPad Software, La Jolla, CA, USA) was used for the dose-response curve, fitting of the logarithm of inhibitor concentration vs. normalized enzymatic activity, and to calculate IC 50 values (see Figures S33-S64 in Supplementary Materials).All measurements were taken in triplicate, and data were expressed as the mean ± SD.

Human Lactate Dehydrogenase B Enzymatic Activity Assay
The ability of the synthesized compounds to inhibit hLDHB enzyme was determined using recombinant human LDHB (95%, specific activity > 300 units/mg, and concentration 1.0 mg/mL, Abcam, Cambridge, UK) following the same fluorimetric protocol described in Section 3.4.

Conclusions
We have designed and synthesised a new family of ethyl pyrimidine-quinolinecarboxylate derivatives based on our previous pyrimidine-quinolone hybrids, which showed moderate inhibition of hLDHA.Our primary modification to the quinoline moiety consisted of adding a carboxylic group, which acted as a good hydrogen bonding acceptor/donor fragment.This new family successfully passed virtual docking screening and was synthesized using the Pfitzinger reaction to produce key ethyl 2-aminophenylquinoline-4carboxylate intermediates (7)(8)(9)(10)(11)(12).These intermediates were then coupled with a set of 4aryl-2-chloropyrimidines VIII(a-d) using microwave irradiation at 150-170 • C and ethanol as a green solvent, affording the ethyl pyrimidine-quinolinecarboxylate derivatives in moderate to good yields.
The hLDHA inhibition assay results agreed with the docking, showing that the most potent compounds were those with a 2-aminophenyl group linking both quinoline and pyrimidine scaffolds in a U-shaped arrangement that fit well in the enzyme's active site.
As a result, we identified thirteen compounds with IC 50 values of less than 5 µM, and four of them (16a, 18b, 18c, and 18d) with IC 50 values of approximately 1 µM.This represents an average of a 20-fold improvement in their inhibitory activity compared to the result obtained with the previous family of compounds.
In addition, this study evaluated all hybrids with IC 50 < 10 µM against the hLDHB isoenzyme.Three of the hybrids (15c, 15d, and 16d) showed preference towards the A isoform, with an hLDHB IC 50 > 100 µM.The other thirteen hybrids acted as double inhibitors.

Figure 1 .
Figure 1.Examples of some reported double hLDHA and hLDHB, hLDHA selective, and hLDHB selective inhibitors with their corresponding IC50 values.

Figure 1 .
Figure 1.Examples of some reported double hLDHA and hLDHB, hLDHA selective, and hLDHB selective inhibitors with their corresponding IC 50 values.

Figure 2 .
Figure 2. Overview of the previous pyrimidine-quinolones and current optimized ethyl pyrimidinequinoline carboxylates.

Figure 2 .
Figure 2. Overview of the previous pyrimidine-quinolones and current optimized ethyl pyrimidinequinoline carboxylates.

Figure 3 .
Figure 3. Structural combination of the novel pyrimidine-quinoline hybrids for virtual screening selection.

Figure 3 .
Figure 3. Structural combination of the novel pyrimidine-quinoline hybrids for virtual screening selection.

73 0. 9477 a
Data are presented as the mean ± SD of n = 3 replicates for IC 50 values.b R 2 values are given from the single dose-response curve of the average percentage of activity for each concentration.

Figure 6 .
Figure 6.Comparison of the poses of esters 13b (yellow), 14b (pink), 15b (blue), and reference W31 (black) in the hLDHA active site.The molecular surface around receptor atoms in the enzyme pocket has been represented in grey with green and red colors showing lipophilic and hydrophilic sites, respectively.Main amino acid residues have been labelled in yellow.

Figure 6 .
Figure 6.Comparison of the poses of esters 13b (yellow), 14b (pink), 15b (blue), and reference W31 (black) in the hLDHA active site.The molecular surface around receptor atoms in the enzyme pocket has been represented in grey with green and red colors showing lipophilic and hydrophilic sites, respectively.Main amino acid residues have been labelled in yellow.

a
Data are presented as the mean ± SD of n = 3 replicates for IC50 values.b R 2 values are given from the single dose-response curve of the average percentage of activity for each concentration.The majority of hybrids (15-18)(a-d) are positioned in the active site of the hLDHA enzyme similar to the reference compound W31.This allows them to interact with the different amino acid residues located in the hLDHA active site.This can be seen with 18c in Figure 7a.Additionally, Figure 7b includes its 2D interaction diagram.For a detailed comparison of the placement of each compound in the hLDHA active site, please refer to Figures S1-S16 in the Supplementary Materials.

Figure 7 .Figure 7 .
Figure 7. (a) Placement of compound 18c (yellow) and reference W31 (black) in the hLDHA active site.The molecular surface around receptor atoms in the enzyme pocket is represented in grey with green and red colors indicating lipophilic and hydrophilic sites, respectively.Main amino acid residues have been labelled in yellow.(b) 2D diagram of the ligand-protein interaction of compound 18c in the hLDHA active site.

Figure 8 .
Figure 8. 2D diagram of the ligand-protein interaction of compounds 15b (a), 16c (b), 17c (c), and 18a (d) in the hLDHA active site.The hLDHA pocket has been represented with a grey dashed line.

Figure 8 .
Figure 8. 2D diagram of the ligand-protein interaction of compounds 15b (a), 16c (b), 17c (c), and 18a (d) in the hLDHA active site.The hLDHA pocket has been represented with a grey dashed line.

Figure 9 .
Figure 9. Superposition of hLDHA 4R68 complex (ligand: W31, light red) and hLDHB 1I0Z complex (ligand: OXM, cyan).Arg 106 residue of 1I0Z is labelled in purple.Arg 105 residue of 4R68 is labelled blue.The other 4R68 and 1I0Z amino acid residues have been labelled in red and cyan, respectively.

30 Figure 10 .
Figure 10.(a) Placement of hybrids 15c (red), 15d (blue), and 16d (green) in the allosteric H1U site between chains A-C in 1I0Z.Amino acid chains have been represented with white sticks save for Glu 214 and Lys 310 , which are represented in orange and have been labelled.(b) Placement of hybrids 15a (black) and 16a (yellow).Amino acid chains have been represented in orange (chain A) and blue (chain C).Amino acid residues of Glu 214 and Lys 310 have been labelled.

Figure 10 .
Figure 10.(a) Placement of hybrids 15c (red), 15d (blue), and 16d (green) in the allosteric H1U site between chains A-C in 1I0Z.Amino acid chains have been represented with white sticks save for Glu 214 and Lys 310 , which are represented in orange and have been labelled.(b) Placement of hybrids 15a (black) and 16a (yellow).Amino acid chains have been represented in orange (chain A) and blue (chain C).Amino acid residues of Glu 214 and Lys 310 have been labelled.

Table 3 .
IC 50 (µM) values against hLDHA and hLDHB of the selected pyrimidine-quinoline esters 15(b-d) and (16-18)(a-d).Data are presented as the mean ± SD of n = 3 replicates for IC 50 values.b R 2 values are given from the single dose-response curve of the average percentage of activity for each concentration. a in the Supplementary Materials.