Authors: Guang-Bin Shen, Shun-Hang Gao, Yan-Wei Jia, Xiao-Qing Zhu, Bao-Chen Qian
Categories: Article, dipine oxidative metabolism, elementary steps, thermodynamic cards
Source: Molecules
Dipines are a type of important antihypertensive drug as L-calcium channel blockers, whose core skeleton is the 1,4-dihydropyridine structure. Since the dihydropyridine ring is a key structural factor for biological activity, the thermodynamics of the aromatization dihydropyridine ring is a significant feature parameter for understanding the mechanism and pathways of dipine metabolism in vivo. Herein, 4-substituted-phenyl-2,6-dimethyl-3,5-diethyl-formate-1,4-dihydropyridines are refined as the structurally closest dipine models to investigate the thermodynamic potential of dipine oxidative metabolism. In this work, the thermodynamic cards of dipine models’ aromatization on 21 potential elementary steps in acetonitrile have been established. Based on the thermodynamic cards, the thermodynamic properties of dipine models and related intermediates acting as electrons, hydrides, hydrogen atoms, protons, and two hydrogen ions (atoms) donors are discussed. Moreover, the thermodynamic cards are applied to evaluate the redox properties, and judge or reveal the possible oxidative mechanism of dipine models.
Keywords: thermodynamic cards, dipine oxidative metabolism, elementary steps
1,4-dihydropyridines are a type of important antihypertensive drug known as L-calcium channel blockers [1,2,3,4,5,6]. Until now, over a dozen 1,4-dihydropyridine drugs have been extensively used in clinic, such as Nifedipine, Felodipine, Amlodipine, etc. (Figure 1) [7,8]. Because their drug names all contain the word “dipine”, they are also well-known as dipine drugs or dipines. The core skeleton of dipines is the 1,4-dihydropyridine structure, and the dihydropyridine ring is a key structural factor for their biological activity [7,8,9,10]. If the dihydropyridine ring is oxidized or aromatized into a pyridine structure in vivo, the resulting oxidative metabolites would lose the biological activity of blocking the L-calcium ion channel to lower blood pressure (Figure 2a) [1,2,3,4,5,6,7,8,9,10]. There exists various oxidoreductases in vivo (Figure 2b), such as CYP450 [11,12], flavin coenzyme [13,14,15], heme iron coenzyme [16,17,18,19], and coenzyme Q [20,21,22,23], as well as NADH coenzyme [24,25,26], and they are excellent electron, hydrogen atom, or hydride acceptors [27,28,29,30,31]. For example, it was reported that dipines were oxidated by cytochrome P-450 in human liver microsomes, and the metabolism process experienced a successive e + H^+^ + e + H^+^ mechanism [30]. Some highly active intermediates, including a radical cation, a radical, and a protonated pyridine, were involved in the oxidative process [30]. Therefore, the aromatized metabolism process of dipines may involve 21 possible thermodynamic elementary steps and six potential active intermediates (Figure 3), whose thermodynamic driving forces are the significant physical parameters for understanding and revealing the possible oxidative pathways of dipine metabolism in vivo.
Figure 1 The chemical structures of dipines and refined dipine models (DH
2).
Figure 2 (a) The oxidative metabolism process of Nifedipine in vivo. (b) Some common oxidoreductases in vivo.
Figure 3 Thermodynamic cards of dipine model (DH
2) aromatization on 21 potential elementary steps during oxidative metabolism.
From the view of structure, dipines are the analogues of the Hantzsch ester (YH2) [31] with the same core structure of 1,4-dihydropyridine, so dipines and the Hantzsch ester (YH2) all belong to NADH models. Further examining the structural characteristics of dipines in Figure 1, it can be discovered that they generally have the same parent structure of 4-aromatic-2,6-dimethyl-3,5-diformate-1,4-dihydropyridines. In this work, 4-substituted-phenyl-2,6-dimethyl-3,5-diethyl-formate-1,4-dihydropyridines (DH2) are refined as the structurally closest dipine models and represented all of the dipines to investigate the thermodynamic parameters of dipine oxidative metabolism (Figure 1).
Our group has long been committed to thermodynamic research on hydrogen transfer for NADH models, and determining the thermodynamic driving forces of over 200 organic hydrides releasing hydrides in non-aqueous media [27,31]. The previous works inspire us to further investigate and clarify the thermodynamic parameters of dipine aromatization in solution. Herein, the thermodynamic cards of dipine models’ aromatization on 21 potential elementary steps has been established (Figure 3). From the thermodynamic cards, the thermodynamic properties of dipine models and related intermediates acting as electrons, hydrides, hydrogen atoms, protons, and two hydrogen ions (atoms) donors are discussed. Furthermore, the thermodynamic cards are utilized to measure the redox properties, and diagnose the possible oxidative mechanism of dipine models.
The thermodynamics of five 4-substituted-phenyl-2,6-dimethyl-3,5-diethyl-formate-1,4-dihydropyridines (DH2), including 4-(4-methoxyphenyl)-2,6-dimethyl-3,5-di(ethylformate)-1,4-dihydropyridine (1H2), 4-(4-methylphenyl)-2,6-dimethyl-3,5-di(ethylformate)-1,4-dihydropyridine (2H2), 4-phenyl-2,6-dimethyl-3,5-di(ethylformate)-1,4-dihydropyridine (3H2), 4-(4-chlorophenyl)-2,6-dimethyl-3,5-di(ethylformate)-1,4-dihydropyridine (4H2), and 4-(4-nitrophenyl)-2,6-dimethyl-3,5-di(ethylformate)-1,4-dihydropyridine (5H2) in acetonitrile were investigated, along with the thermodynamics of the Hantzsch ester (YH2) for better comparison.
The thermodynamic potentials of 21 elementary steps are defined as the corresponding Gibbs free energies for Step 1, Steps 4–7, Steps 10–21 or potentials for Step 2, Step 3, Step 8, and Step 9, which are shown in Figure 3.
As can be seen from Figure 3, Step 1 and Step 7 are the hydride-releasing chemical processes, DH2 → DH^+^ + H^−^ and DH^−^ → D + H^−^, and their thermodynamic potentials are described by the related Gibbs free energies of DH2 and DH^−^ releasing hydrides, respectively, ΔGH^−^D(DH2) and ΔGH^−^D(DH^−^), which are also called hydricities [32,33,34]. Since the entropy change (TΔS) of organic hydrides releasing hydrides is estimated as 4.9 kcal/mol [35] in acetonitrile, so the ΔGH^−^D(DH2) and ΔGH^−^D(DH^−^) are derived from their corresponding enthalpy changes, ΔHH^−^D(DH2) and ΔHH^−^D(DH^−^), by Equations (1) and (5) in Table 1. The reliability was proved in previous literature [36,37]. ΔHH^−^D(DH2) and ΔHH^−^D(DH^−^) were available from our previous work [27,38].
Steps 2–3 and Steps 8–9 are the electron-releasing chemical processes, and their thermodynamic potentials are described by the related oxidation potentials, Eox(DH2) for Step 2, Eox(DH^•^) for Step 3, Eox(DH^−^) for Step 8, and Eox(D^•−^) for Step 9, respectively. The oxidation potentials were determined in our previous work [27,38].
Step 4–5, Steps 1–11, and Steps 17–18 are the hydrogen-atom-releasing chemical processes, and their thermodynamic potentials are described by the related Gibbs free energies, ΔGHD(DH2) for Step 4, ΔGHD(DH2^•+^) for Step 5, ΔGHD(DH^−^) for Step 10, ΔGHD(DH’^•^) for Step 11, ΔGHD(DH^•^) for Step 17, and ΔG’HD(DH2) for Step 18, respectively. Based on Hess’ law [27,37,38], ΔGHD(DH2), ΔGHD(DH2^•+^), ΔGHD(DH^−^), ΔGHD(DH’^•^), ΔGHD(DH^•^), and ΔG’HD(DH2) were calculated using Equations (2)–(3), (6)–(7), and (13)–(14), respectively, by constructing corresponding thermodynamic cycles, cycle Step 4–Step 3–Step 1 for ΔGHD(DH2), cycle Step 1–Step 2–Step 5 for ΔGHD(DH2^•+^), cycle Step 10–Step 9–Step 7 for ΔGHD(DH^−^), cycle Step 7–Step 8–Step 11 for ΔGHD(DH’^•^), cycle Step 4–Step 17–Step 16 for ΔGHD(DH^•^), and cycle Step 18–Step 11–Step 16 for ΔG’HD(DH2).
Step 6, Steps 12–13, Step 15, and Step 21 are the proton-releasing chemical processes, and their thermodynamic potentials are described by the related Gibbs free energies, ΔGPD(DH2^•+^) for Step 6, ΔGPD(DH’^•^) for Step 12, ΔGPD(DH^+^) for Step 13, ΔGPD(DH2) for Step 15, and ΔG’PD(DH2^•+^) for Step 21, respectively. ΔGPD(DH^+^) for Step 13 could be calculated by Equation (10) using the pKa(DH^+^) value, ΔGPD(DH^+^) = 1.37pKa(DH^+^) [39]. In this work, the pKa(DH^+^) values were predicted using the method developed by Luo and coworker at 2020 [40]. Based on Hess’ law [27,37,38], the ΔGPD(DH2^•+^), ΔGPD(DH’^•^), ΔGPD(DH2), and ΔG’PD(DH2^•+^) were calculated using Equations (4), (8), (11), and (17) in Table 1, respectively, by constructing corresponding thermodynamic cycles, cycle Step 4–Step 2–Step 6 for ΔGPD(DH2^•+^), cycle Step 8–Step 12–Step 10 for ΔGPD(DH’^•^), cycle Step 14–Step 15–Step 7 for ΔGPD(DH2), and cycle Step 6–Step 20–Step 21 for ΔG’PD(DH2^•+^).
Step 14 is the chemical process of DH2 releasing two hydrogen ions (H^−^ + H^+^), and the thermodynamic potential is described by the Gibbs free energy of DH2 releasing two hydrogen ions, ΔGH^−^P(DH2). ΔGH^−^P(DH2) could be obtained from ΔGH^−^D(DH2) and ΔGPD(DH^+^) via Equation (10) in Table 1, ΔGH^−^P(DH2) = ΔGH-D(DH2) + ΔGPD(DH^+^) (Equation (10)), by constructing the thermodynamic cycle [27] Step 1–Step 13–Step 14.
Step 16 is the chemical process of DH2 releasing two hydrogen atoms, and the thermodynamic potential is described by the Gibbs free energy of DH2 releasing two hydrogen atoms, ΔG2H(DH2). ΔG2H(DH2) could be obtained by Equation (12) in Table 1, ΔG2H(DH2) = ΔGH^−^P(DH2) + F[Ered(H^•^) − Eox(H^•^)] (Equation (12)). Among Equation (12), Ered(H^•^) and Eox(H^•^) were reported as −1.137 V and −2.307 V (vs. Fc) [27,38] in acetonitrile.
Step 19 is the chemical process of DH2 releasing hydrogen gas (H2), and the thermodynamic potential is described by the Gibbs free energy of DH2 releasing H2, ΔGH2(DH2). ΔGH2(DH2) could be obtained by Equation (15) in Table 1, ΔGH2(DH2) = ΔG2H(DH2) − ΔGH^−^D(H2) (Equation (15)). Among Equation (15), ΔGH^−^D(H2) refers to the Gibbs free energy of H2 releasing hydrides, which was reported as 76.0 kcal/mol [41,42] in acetonitrile.
Step 20 is the chemical process of a hydrogen atom transfer within a molecule from the N1-position to the C4-position, and the thermodynamic potential is described by the related Gibbs free energy, ΔGHT(DH^•^). ΔGHT(DH^•^) could be obtained by Equation (16) in Table 1, ΔGHT(DH^•^) = ΔGHD(DH^•^) − ΔGHD(DH’^•^) (Equation (16)) by constructing the thermodynamic cycle [27] Step 20–Step 11–Step 17.
Step 21 is the chemical process of DH2^•+^ releasing protons from the N1-position, and the thermodynamic potential is described by the related Gibbs free energy, ΔG’PD(DH2^•+^). ΔG’PD(DH2^•+^) could be obtained by Equation (17) in Table 1, ΔG’PD(DH2^•+^) = ΔGPD(DH2^•+^) + ΔGHT(DH^•^) (Equation (17)) by constructing the thermodynamic cycle [27] Step 6–Step 20–Step 21.
All of the chemical processes, thermodynamic potentials, computed equations, or data sources of the 21 elementary steps for DH2 aromatization are listed in Table 1. Moreover, the thermodynamic results of 1H2–5H2 and YH2 aromatization are shown in Table 2. Accordingly, the thermodynamic cards [27,31,38] of 1H2–5H2 (Figures S1–S5) and YH2 (Figure S6) on 21 potential elementary steps are established and presented in the Supporting Information to make them more convenient to query and use. Obviously, the thermodynamic cards (Figure 3 and Figures S1–S5) can visually exhibit the mutual transformations among dipine models DH2, aromatic products D, and the related six active intermediates, as well as the thermodynamic driving forces of the related 21 potential elementary steps. Naturally, the thermodynamic cards could be employed to quantitatively measure and predict the characteristic chemical or thermodynamic properties of the dipine models and involved intermediates.
In our previous work [39], the pKa of 78 protonated pyridines in acetonitrile were predicted using the method developed by Luo and coworker at 2020 [40], and the predicted accuracy was estimated as ±1 pKa. Herein, the pKa of DH^+^ and YH^+^ with typical structures of protonated pyridine in acetonitrile are also predicted using the same method. To better understand the acidities of DH^+^ and YH^+^ in acetonitrile, the pKa values of DH^+^ and YH^+^, along with the pKa of common organic acids in acetonitrile, are displayed in Figure 4. From Figure 4, it is found that the pKa(DH^+^) scale ranges from 12.83 for 1H^+^ to 14.54 for 5H^+^, and the acidities of DH^+^ (12.83–14.54) are between PyH^+^ (12.3) [43,44,45] and protonated 2,4,6-trimethylpyridine (15.0) [43,44,45], which further verify the accuracy of the predicted pKa(DH^+^) values.
Figure 4 The pK
aof DH^+^ and YH^+^, along with the pKaof common organic acids in acetonitrile.
What is more, several interesting conclusions could be drawn from Figure 4. (1) Since the pKa of BINOL-derived phosphoric acids (BPA) are reported as 12–14 in MeCN [46,47,48,49], DH^+^ (12.83–14.54) have comparable acidities to BPA. (2) DH^+^ (12.83–14.54) generally belongs to medium–strong organic acids, which could be applied in acid-catalyzed chemical reactions. (3) The acidity of 3H^+^ (13.66) is almost equal to that of YH^+^ (13.65), and when the 4-phenyl group of DH^+^ is substituted by an electron-withdrawing group, the acidity of new DH^+^ is stronger than 3H^+^ (13.66) or YH^+^ (13.65). In contrast, if the 4-phenyl group of DH^+^ is substituted by an electron-donating group, the acidity of new DH^+^ is weaker than 3H^+^ (13.66) or YH^+^ (13.65). (4) Sometimes, after DH2 are oxidized by hydride acceptors, the acidities of resulting DH^+^ should be considered when the organic bases are involved in the reaction system. (5) D (pKa(DH^+^) = 12.83 − 14.54) could be used as alternative organic bases of pyridine (pKa of conjugate acid is 12.3) [43,44,45], 2,6-trimethylpyridine (pKa of conjugate acid is 14.1) [43,44,45], and 2,4,6-trimethylpyridine (pKa of its conjugate acid is 15.0) [43,44,45] in acetonitrile.
To clearly compare the electron-donating properties of DH2 and related intermediates (DH^−^, DH^•^, and D^•−^), the oxidation potentials (Eox vs. Fc) of DH2, DH^−^, DH^•^, and D^•−^, as well as the reduction potentials (Ered vs. Fc) of common coenzyme models (BNA^+^ for NADH coenzyme, PQ for coenzyme Q, Asc for oxidated ascorbate, Fl^+^ for flavin coenzyme, and Ru^IV^O^2+^ for heme enzyme) [40,42] and electron acceptors (H^+^, H^•^, O2, and PTZ^•+^) [29,50] in acetonitrile are exhibited in Figure 5. From Figure 5, it is clear that the oxidation potential scale is from 0.712 V to 0.781 V for DH2, from −0.382 V to −0.497 V for DH^−^, from −0.980 V to −0.655 V for DH^•^, and from −2.611 V to −2.289 V for D^•−^, indicating that DH^•−^ are the thermodynamically best electron donors, even better than H^•^ (Eox = −2.307 V) [38], and DH2 are the thermodynamically worse electron donors than BNAH (0.219 V) [27]. The electron-donating abilities increase in the following order of DH2 < DH^−^ < DH^•^ < D^•−^. According to their thermodynamic range, DH2 (0.712–0.781 V), DH^−^ (−0.382–−0.497 V), DH^•^ (−0.980–−0.655 V), and D^•−^ (−2.611–−2.289 V) are recognized as the weak electron donors, medium–strong electron donors, strong electron donors, and very strong electron donors, respectively.
Figure 5 Oxidation potentials (E
ox) of DH2, DH^−^, DH^•^, and D^•−^, as well as the reduction potentials (Ered) of common coenzyme models and electron acceptors in acetonitrile (V vs. Fc).
Since ascorbate is a good single-electron donor in vivo, the Eox(DH2) (0.712–0.781 V) is greater than 5,6-isopropylidene ascorbate (iAscH^−^, −0.425 V) [27], and the Eox(DH^•^) (−0.980–−0.655 V), Eox(DH^−^) (−0.382–−0.497 V), and Eox(D^•−^) (−2.611–−2.289 V) are more negative than iAscH^−^ (−0.425 V) [27], which means that DH2 are thermodynamically worse electron donors than iAscH^−^, and DH^•^, DH^−^, and D^•−^ are thermodynamically better electron donors than iAscH^−^. In addition, Ru^IV^O^2+^ is known as the model of high-valence metal oxides, such as heme or non-heme iron coenzyme [16,17,18,19], and cytochrome P450 [11,12,13,14,15]. The reduction potential of Ru^IV^O^2+^ is determined as −0.250 V (vs. Fc) [51] in acetonitrile; therefore, the electron transfers from DH^−^ (−0.382–−0.497 V), DH^•^ (−0.980–−0.655 V), and D^•−^ (−2.611–−2.289 V) to Ru^IV^O^2+^ are thermodynamically favorable with large thermodynamic driving forces, while the electron transfers from DH2 (0.71–0.781 V) to Ru^IV^O^2+^ are thermodynamically unfavorable with ΔGET(DH2/Ru^IV^O^2+^) ≥ 22.2 kcal/mol.
It is well-known that O2 often functions as the electron acceptor in vivo, and the reduction potential of O2 was determined as −1.050 V (vs. Fc) [29] in acetonitrile. The thermodynamic analyses indicate that the electron transfers from DH^−^ (−0.382–−0.497 V), DH^•^ (−0.980–−0.655 V) and DH2 (0.712–0.781 V) to O2 are thermodynamically unfavorable, while electron transfers from D^•−^ (−2.611–−2.289 V) to O2 are thermodynamically feasible with ΔGET(D^•−^/O2) ≤ −28.6 kcal/mol. Further considering the thermodynamic data, the Gibbs free energies of electron transfers from DH2 (0.712–0.781 V) to O2, DH2 + O2 → DH2^•+^ + O2^•−^, are estimated to be as high as 40.6–42.2 kcal/mol [ΔGET(DH2/O2)], meaning that DH2 is stable in the air, and the direct electron transfer from DH2 to O2 is thermodynamically unfeasible. In contrast, the Gibbs free energies of electron transfers from DH^−^ (−0.832–−0.479 V) and DH^•^ (−0.980–−0.655 V) to O2, are calculated as 5.0–13.2 kcal/mol and 1.6–9.1 kcal/mol separately, which are slightly large energy barriers to surpass.
For a better comparison, the hydride-donating properties of DH2 and DH^−^ and the hydricities of DH2, DH^−^, and common hydride donors (iAscH^−^, BNAH, and AcrH), as well as the hydride-affinities of common coenzyme models (BNA^+^ for NADH coenzyme, PQ for coenzyme Q, iAsc for oxidated ascorbic acid, Fl^+^ for flavin coenzyme, and Ru^IV^O^2+^ for heme enzyme) and hydride acceptors (H^+^ and PTZ^•+^) in acetonitrile are displayed in Figure 6. As can be seen from Figure 6, it is found that the hydricity ranges from 63.9 to 69.0 kcal/mol for DH2, and from 31.8 to 37.9 kcal/mol for DH^−^. If the hydricities between DH2 and DH^−^ are compared, it is easy to discover that the hydricities of DH^−^ are greatly improved by the negative charge at the N1-atom compared with their parents DH2, and the hydride-donating abilities of DH^−^ (31.8–37.9 kcal/mol) are greater than DH2 (63.9–69.0 kcal/mol) by more than 30 kcal/mol. Based on their thermodynamic range, DH2 (63.9–69.0 kcal/mol) belong to thermodynamically medium–strong hydride donors, while DH^−^ (31.8–37.9 kcal/mol) belong to thermodynamically strong hydride donors, respectively.
Figure 6 Hydricities of DH
2, and DH^−^, as well as H^−^-affinities of common coenzyme models and hydride acceptors in acetonitrile (kcal/mol).
DH2 are thermodynamically worse hydride donors than BNAH and thermodynamically better hydride donors than iAscH^−^, due to the hydricities of DH2 (63.9–69.0 kcal/mol) being more negative than BNAH (59.3 kcal/mol) [27] by 4.6–9.7 kcal/mol, and greater than iAscH^−^ (75.7 kcal/mol) [27] by 6.7–11.8 kcal/mol. DH^−^ are much better hydride donors than BNAH and iAscH^−^, and the hydricities of DH^−^ (31.8–37.9 kcal/mol) are much greater than BNAH (59.3 kcal/mol) and iAscH^−^ (75.7 kcal/mol) by more than 20 kcal/mol. The above thermodynamic data indicate that DH2 could not be oxidated by BNA^+^ through hydride transfer with related Gibbs free energies greater than 0, 4.6 kcal/mol ≤ ΔGH-T(DH2/BNA^+^) ≤ 9.7 kcal/mol, but the anion intermediates of DH2 (DH^−^) could be oxidated by BNA^+^ through hydride transfer with Gibbs free energies less than 0, −27.5 kcal/mol ≤ ΔGH-T(DH^−^/BNA^+^) ≤ −21.4 kcal/mol. In addition, DH2 and DH^−^ could be oxidated by iAsc (−75.7 kcal/mol) by hydride transfer with Gibbs free energies less than 0, −9.7 kcal/mol ≤ ΔGH-T(DH2/iAsc) ≤ −4.6 kcal/mol and −43.9 kcal/mol ≤ ΔGH-T(DH^−^/iAsc) ≤ −37.8 kcal/mol. What is more, since all of the Gibbs free energies of hydride transfer processes from DH2 to Ru^IV^O^2+^ (−114.1 kcal/mol), from DH2 to Fl^+^ (−78.5 kcal/mol), from DH2 to PQ (−70.0 kcal/mol), and from DH2 to H^+^ (−76.0 kcal/mol) are less than 0, it can be inferred that dipines may be oxidated by heme enzyme, cytochrome P450, flavin coenzyme, coenzyme Q, and H^+^ under suitable oxidoreductase by hydride oxidation in vivo.
Due to the N-H bond and C-H bond at the 1-position and 4-position of DH2, there are six possible elementary steps to release hydrogen atoms during the aromatization. Herein, the thermodynamic hydrogen-atom-donating abilities of DH2, DH^−^, DH2^•+^, DH^•^, and DH’^•^, as well as the hydrogen-atom affinities of 12 common radicals (involving H^•^, ^t^BuO^•^, CumO^•^, PhCH2^•^, PINO^•^, ^t^BuO2^•^, CumO2^•^, PhO^•^, DPPH, PhS^•^, 2,4,6-^t^Bu3PhO^•^, and TEMPO) [29] and coenzyme models (BNA^+^ for NADH coenzyme, PQ for coenzyme Q, iAsc for oxidated ascorbic acid, Fl^+^ for flavin coenzyme, and Ru^IV^O^2+^ for heme enzyme) in acetonitrile are shown in Figure 7.
Figure 7 Thermodynamic hydrogen-atom-donating abilities of DH
2, DH^−^, DH2^•+^, DH^•^, and DH’^•^, as well as hydrogen atom affinities of common radicals and coenzyme models in acetonitrile (kcal/mol).
From Figure 7, the thermodynamic hydrogen-atom-donating ability scale ranges from 92.9 to 93.9 kcal/mol for DH2 releasing hydrogen atoms from N1-H, from 58.6 to 60.3 kcal/mol for DH2 releasing hydrogen atoms from C4-H, from 64.4 to 65.7 kcal/mol for DH^−^, from 21.3 to 34.9 kcal/mol for DH2^•+^, from 50.5 to 55.7 kcal/mol for DH^•^, and from 17.1 to 20.5 kcal/mol for DH’^•^, and the thermodynamic hydrogen-atom-donating abilities increase in the order of DH2 (N1-H, 92.9–93.9 kcal/mol) < DH^−^ (64.4–65.7 kcal/mol) < DH2 (C4-H) (58.6–60.3 kcal/mol) < DH^•^ (50.5–55.7 kcal/mol) < DH2^•+^ (21.3–34.9 kcal/mol) < DH’^•^ (17.1–20.5 kcal/mol). According to their thermodynamic ranges, DH^•^, DH2^•+^, and DH’^•^ belong to thermodynamically strong hydrogen atom donors, DH^−^ and DH2 generally belong to thermodynamically medium–strong hydrogen atom donors to break C4-H bonds, while DH2 belong to thermodynamically weak hydrogen atom-donors to break N1-H bonds. Based on the above analysis, whether DH2 are medium–strong hydrogen atom donors or weak hydrogen atom donors depends on which hydrogen atoms DH2 releases, N1-H bonds or C4-H bonds. Generally, DH2 prefer to release hydrogen atoms from C4-H bonds (58.6–60.3 kcal/mol) instead of N1-H bonds (92.9–93.9 kcal/mol) from thermodynamics.
Since ascorbate is an excellent antioxidant to quench radicals in vivo [52], it is necessary to compare the thermodynamic properties of iAscH^−^, DH2, and related intermediates releasing hydrogen atoms. Several interesting conclusions could be made as follows. It is revealed that (1) the thermodynamic antioxidant potentials of DH^−^ (64.4–65.7 kcal/mol) are comparable to iAscH^−^ (64.1 kcal/mol) [27,52]. (2) DH2 (C4-H homolysis, 58.6–60.3 kcal/mol), DH^•^ (50.5–55.7 kcal/mol), DH2^•+^ (21.3–34.9 kcal/mol), and DH’^•^ (17.1–20.5 kcal/mol) are thermodynamically better antioxidants than iAscH^−^ (64.1 kcal/mol). (3) Unlike hydricities, the thermodynamic hydrogen-atom-donating abilities of DH^−^ decrease as a result of the negative charge at the N1-atom compared with their parents DH2, and the thermodynamic hydrogen-atom-donating abilities of DH^−^ (64.4–65.7 kcal/mol) are more negative than DH2 (58.6–60.3 kcal/mol) by 4.1–7.1 kcal/mol. (4) Because the final products of DH^•^ and DH’^•^ releasing hydrogen atoms are the same (D and H^•^), DH^•^ → D + H^•^ and DH’^•^ → D + H^•^. When the hydrogen-atom-donating Gibbs free energies of DH^•^ (50.5–55.7 kcal/mol) and DH’^•^ (17.1–20.5 kcal/mol) are compared, it can also be deduced that DH^•^ are more thermodynamically stable radicals than DH’^•^, and the Gibbs free energies of hydrogen atom transfer within DH^•^ from the N1-position to the C4-position [ΔGHT(DH^•^) for Step 20 in Figure 3] are computed as 33.4–35.2 kcal/mol, which further verify the relative stability of DH^•^ and DH’^•^ in solution.
BNAH is the structurally closest model of the NADH coenzyme, and the hydrogen-atom-donating Gibbs free energy of BNAH is 66.0 kcal/mol [27]. It is clear that DH2 (C4-H homolysis, 58.6–60.3 kcal/mol), DH^•^ (50.5–55.7 kcal/mol), DH2^•+^ (21.3–34.9 kcal/mol), and DH’^•^ (17.1–20.5 kcal/mol) are thermodynamically better hydrogen atom donors than BNAH (66.0 kcal/mol), while DH2 are thermodynamically much weaker hydrogen atom donors than BNAH (66.0 kcal/mol) if the DH2 break N1-H bonds (92.9–93.9 kcal/mol).
As for the common 11 organic radicals collected in Figure 7, involving ^t^BuO^•^, CumO^•^, PhCH2^•^, PINO^•^, ^t^BuO2^•^, CumO2^•^, PhO^•^, DPPH, PhS^•^, 2,4,6-^t^Bu3PhO^•^, and TEMPO [29,53,54], the hydrogen atom affinity scale ranges from −66.5 for TEMPO to −104.40 kcal/mol for ^t^BuO^•^. All of the 11 radicals (−66.5–−104.40 kcal/mol) could thermodynamically oxidize DH^−^ (64.4–65.7 kcal/mol), DH2 (breaking C4-H) (58.6–60.3 kcal/mol), DH^•^ (50.5–55.7 kcal/mol), DH2^•+^ (21.3–34.9 kcal/mol), and DH’^•^ (17.1–20.5 kcal/mol) via hydrogen atoms transfer. Only ^t^BuO^•^ (−104.40 kcal/mol) and CumO^•^ (−110.73 kcal/mol) could oxidize DH2 by N1-H bonds homolysis (92.9–93.9 kcal/mol) from the point of thermodynamics. In addition, H^•^ (−102.3 kcal/mol) [28,29], Ru^IV^O^2+^ (−80.3 kcal/mol) [51], and PQ (−69.4 kcal/mol) [29] have the thermodynamic abilities to oxidize DH^−^ (64.4–65.7 kcal/mol), DH2 (breaking C4-H) (58.6–60.3 kcal/mol), DH^•^ (50.5–55.7 kcal/mol), DH2^•+^ (21.3–34.9 kcal/mol), and DH’^•^ (17.1–20.5 kcal/mol) via hydrogen atoms transfer from thermodynamics, meaning that dipines may be oxidated by heme enzyme, cytochrome P450, coenzyme Q (Co Q), and H^•^ under suitable oxidoreductase, by hydrogen atoms oxidation in vivo.
In order to better understand the acidities of DH2 and related active intermediates, the thermodynamic proton-donating abilities of DH2, DH^+^, DH2^•+^, and DH’^•^, as well as the proton-donating abilities of common acids (PhSO3H, iAscH2, Et3NH^+^, PhCO2H, AcOH, AcrH, and H2) [43,44,45] in acetonitrile are presented in Figure 8.
Figure 8 Thermodynamic proton-donating abilities of DH
2, DH^+^, DH2^•+^, and DH’^•^, as well as proton-donating abilities of common acids in acetonitrile (kcal/mol).
Obviously, the proton-donating Gibbs free energy scale ranges from −13.2 to −9.3 kcal/mol for DH2^•+^ releasing protons from C4-H bonds, from 22.0 to 24.1 kcal/mol for DH2^•+^ releasing protons from N1-H bonds, from 17.6 to 19.9 kcal/mol for DH^+^, from 20.1 to 24.1 kcal/mol for DH’^•^, and from 48.7 to 52.0 kcal/mol for DH2, which discloses that the thermodynamic proton-donating abilities increase according to the order of DH2 (N1-H, 48.7–52.0 kcal/mol) < DH2^•+^ (N1-H, 22.0–24.1 kcal/mol) ≈ DH’^•^ (20.1–24.1 kcal/mol) < DH^+^ (17.6–19.9 kcal/mol) < DH2^•+^ (C4-H, −13.2–−9.3 kcal/mol). Based on their thermodynamic ranges, it could be suggested that DH2 (N1-H, 48.7–52.0 kcal/mol) belong to weak proton donors, DH2^•+^ (N1-H, 22.0–24.1 kcal/mol), DH’^•^ (20.1–24.1 kcal/mol), and DH^+^ (17.6–19.9 kcal/mol) belong to medium–strong proton donors, and DH2^•+^ (C4-H, −13.2–−9.3 kcal/mol) belong to strong proton donors.
The above thermodynamic analyses result in the following conclusions.
Examining the structural characteristics of NADH models, they are generally N-alkyl-1,4-dihydropyridines and act as hydride carriers [27,55]. Unlike common NADH models, the biggest difference between common NADH models (such as BNAH and AcrH) and DH2 or the Hantzsch ester (YH2) is that DH2 and YH2 have two hydrogen atoms at the N1-position and C4-position. During the aromatization process, DH2 and YH2 could release two hydrogen ions (atoms) or hydrogen gas from both N1-H and C4-H bonds. Therefore, the Hantzsch ester (YH2) has been widely used as an excellent hydrogenation reagent to reduce unsaturated compounds by offering two hydrogen ions (atoms) [56,57,58]. Furthermore, many studies also focused on the hydrogen storage properties of YH2 and related N-heterocycles [42]. In addition, some oxidoreductases, for example flavin coenzyme, coenzyme Q (Co Q), heme or nonheme iron coenzyme, and pyrroloquinoline quinone (PQQ) are also the two hydrogen ions (atoms) carriers in vivo. As a result, the thermodynamic abilities of DH2 releasing two hydrogen ions (atoms) or H2, ΔGH^−^P(DH2), ΔG2H(DH2), and ΔGH2(DH2), are vital thermodynamic parameters to evaluate the comprehensive reduction properties, and judge the oxidation feasibility of DH2 by two hydrogen ions (atoms) carrier enzymes in vivo.
The thermodynamic abilities of DH2 and common hydrogen carriers (HCO2H, H2, YH2, F420H2, PQH2, and iAscH2) releasing two hydrogen ions (H^−^ + H^+^), two hydrogen atoms (2H^•^), or H2 in acetonitrile are shown in Figure 9 [27,43]. It is found from Figure 9 that the Gibbs free energies of DH2 releasing two hydrogen ions range from 83.8 to 86.6 kcal/mol, which belong to medium–strong two hydrogen ion donors.
Figure 9 Thermodynamic abilities of DH
2and common hydrogen carriers releasing two hydrogen ions (H^−^ + H^+^) in the red brackets, releasing two hydrogen atoms (2H^•^) in the purple brackets, and releasing H2in the blue brackets in acetonitrile (kcal/mol).
According to the thermodynamic data, several valuable conclusions could be drawn. (1) The ΔGH^−^P(DH2) values (83.8–86.6 kcal/mol) are greater than those of H2 (76.0 kcal/mol) [41,42], meaning that DH2 are thermodynamically worse two hydrogen ions (atoms) reductants than H2, and H2 could hydrogenate D to regenerate DH2 in solution under suitable catalysts. (2) The ΔGH^−^P(DH2) values (83.8–86.6 kcal/mol) are slightly greater than ΔGH^−^P(YH2) (83.1 kcal/mol), illustrating that DH2 are thermodynamic alternatives of YH2 as two hydrogen ions (atoms) reductants in chemical reactions. (3) Since p-hydroquinone (PQH2) is a close model of Co Q, if the ΔGH^−^P(DH2) values (83.8–86.6 kcal/mol) are compared with the ΔGH^−^P(PQH2) value (96.2 kcal/mol) [29], it is discovered that DH2 are thermodynamically better two hydrogen ion donors than PQH2, and DH2 may be oxidated by Co Q via two hydrogen ions (atoms) transfer in vivo. (4) Due to the fact that ΔGH^−^P(DH2) values (83.8–86.6 kcal/mol) are much more negative than ΔGH^−^P(iAscH2) (100.8 kcal/mol) [27], DH2 are thermodynamically worse two hydrogen ions (atoms) donors, and DH2 may be oxidated by the oxidation state of ascorbic acid (Asc) in vivo. (5) Because the ΔGH2(DH2) values (7.8–10.6 kcal/mol) are greater than ΔGH2(H2) (0.0 kcal/mol), H2 release from DH2 is thermodynamically unfavorable, and DH2 are not hydrogen storage chemicals unless extra energy is being provided, such as light, electron, heat, or gas pressure.
Without doubt, the thermodynamic cards of DH2 display the redox properties of DH2 to reveal the possible oxidative process of DH2 (Figure 3). For DH2, there are five possible initial oxidation pathways (Figure 10), including hydride oxidation (Step 1), single-electron oxidation (Step 2), hydrogen atom oxidation from the C4-H bond (Step 4), hydrogen atom oxidation from the N1-H bond (Step 18), and proton release from the N1-H bond (Step 15). As hydrogen atom donors or antioxidants, DH2 could release hydrogen atoms from the C4-H (Step 4) or N1-H (Step 18) bond. The ΔG’HD(DH2) values of Step 18 (N1-H, 92.9–93.9 kcal/mol) are 30.0 kcal/mol larger than the ΔG’2~) values of Step 4 (CHD(DH4-H, 58.6–60.3 kcal/mol). Thereby, it is reasonable to deduce that DH2 generally release hydrogen atoms from C4-H instead of N1-H during the antioxidant processes from thermodynamics.
Figure 10 Thermodynamic abilities of five possible elementary steps for DH
2oxidation.
Based on their thermodynamic results (Steps 1–2, Step 4, Step 18, and Step 15), DH2 are medium–strong hydride donors (*Step * 63.9–69.0 kcal/mol), weak single electron donors (*Step * 0.712–0.781 V vs. Fc), strong hydrogen atom donors for C4-H bond breaking (*Step * 58.6–60.3 kcal/mol), weak hydrogen atom donors for N1-H bond breaking (*Step * 92.9–93.9 kcal/mol), and weak proton donors (*Step * 48.752.0 kcal/mol). It is supposed that (a) DH2 generally release hydrogen atoms from the C4-H bond instead of the N1-H bond in the radical oxidation process. (b) Generally, DH2 undergoing hydride or radical oxidations is thermodynamically favorable. (c) Since DH2 are thermodynamically very weak proton donors, the concerted or successive proton and hydride (H^+^ + H^−^) transfer mechanism is thermodynamically practicable. (d) The single-electron oxidation of DH2 is thermodynamically disadvantageous, and a high single-electron oxidant is needed.
The thermodynamic cards of DH2 also could reveal the redox properties of key intermediates (Figure 3). For example, if the oxidation of DH2 is initiated by single oxidation, the thermodynamic data of resulting DH2^•+^ are noteworthy physical parameters to diagnose possible oxidation pathways. As organic acids, DH2^•+^ have two different pathways to donate protons (Figure 11), consisting of releasing protons from C4-H bonds (Step 6) and N1-H bonds (Step 21). When the thermodynamic potentials of Step 6 and Step 21 are compared, it is easy to find that the ΔGPD(DH2^•+^) values (−9.3–−13.2 kcal/mol) are 3and −35 kcal/mol more negative than ΔG’PD(DH2^•+^) (22.0–24.1 kcal/mol), indicating that, as proton donors, DH2^•+^ thermodynamically prefer to release protons from C4-H bonds instead of N1-H bonds, and the proton release from C4-H bonds in DH2^•+^ occurs spontaneously.
Figure 11 Thermodynamic analysis of possible oxidative process for intermediate DH
2^•+^.
For DH2^•+^, there are three possible pathways to release hydrogen ions or atoms in all (Figure 11), including proton release from C4-H bonds (Step 6), proton release from N1-H bonds (Step 21), and hydrogen atom release from C4-H bonds (Step 5). According to their thermodynamic results (Step 6, Step 21, and Step 5), DH2^•+^ belong to strong proton donors for C4-H bond breaking (*Step * −9.3–−13.2 kcal/mol), medium–strong proton donors for N1-H bond breaking (*Step * 22.0–24.1 kcal/mol), and strong hydrogen atom donors for C4-H bond breaking (*Step * 21.3–24.9 kcal/mol).
DH2^•+^ may act as proton donors or hydrogen atom donors, which depends on the properties of substrates. If the substrates are radicals, DH2^•+^ act as hydrogen atom donors. If the substrates are bases, DH2^•+^ act as proton donors. Most importantly, with a combination of the oxidation potentials of DH2 and the properties of resulting DH2^•+^ releasing hydrogen ions (atoms), the initial slight energy barrier (10–20 kcal/mol) of electron oxidation could be overcome by the subsequent protons transfer or hydrogen atoms transfer owning extreme thermodynamic driving forces (ΔG << 0).
Since the thermodynamic cards of dipine models’ (DH2) aromatization on 21 potential elementary steps have been established, they provide a precious opportunity to investigate the possible oxidative mechanism of DH2 in vivo. Ascorbic acid (AscH2) is known as an excellent electron and hydrogen atom donor [52]; however, the oxidated ascorbic acid (Asc) is actually a potential two hydrogen ions (atoms) oxidant. Because of the ortho dicarbonyl feature, Asc has the property of o-benzoquinone [59], which is confirmed by the fact that ΔGH^−^P(iAscH2) (100.8 kcal/mol) [27] is slightly greater than ΔGH^−^P(PQH2) (96.2 kcal/mol) [29].
Herein, the oxidative mechanism between 3H2 and iAsc is taken as an example to represent the application of the thermodynamic cards of DH2 to oxidative mechanism diagnosis. First of all, the thermodynamic card of iAsc accepting two hydrogen ions (atoms) on nine potential elementary steps is constructed based on our previous work (Figure 12). Subsequently, according to the thermodynamic cards of 3H2 and iAsc, a thermodynamic analysis platform of elementary steps for the redox process between 3H2 and iAsc without any catalyst in acetonitrile is established and shown in Figure 13.
Figure 12 Thermodynamic card of iAsc accepting two hydrogen ions (atoms) on nine potential elementary steps.
Figure 13 Thermodynamic analysis platform of elementary steps for the redox process between 3H
2and iAsc without any catalyst in acetonitrile.
From Figure 13, Step 13 is the concerted two hydrogen ions (atoms) transfer step from 3H2 to iAsc, 3H2 + iAsc → 3 + iAscH2, and the overall Gibbs free energy of two hydride ions (atoms) transfer from 3H2 to iAsc for Step 13 is −16.1 kcal/mol, which means the oxidation of 3H2 by iAsc is thermodynamically feasible. Moreover, as can be seen from Figure 13, six possible elementary steps are involved in the initial oxidation of 3H2 by iAsc. Step a is the hydride transfer step from 3H2 to iAsc, 3H2 + iAsc → 3H^+^ + iAscH^−^, and the Gibbs free energy of Step a is −9.7 kcal/mol. Step b is the electron transfer step from 3H2 to iAsc, 3H2 + iAsc → 3H^•+^ + iAsc^•−^, and the Gibbs free energy of Step b is 31.4 kcal/mol. Steps c and d are the hydrogen atom transfer step from C4-H and N1-H bonds in 3H2 to iAsc, respectively, 3H2 + iAsc → 3H^•^ + iAscH^•^ and 3H2 + iAsc → 3H’^•^ + iAscH^•^, and the Gibbs free energy of Steps c and d are 0.0 and 34.2 kcal/mol. Step e is the proton transfer step from 3H2 to iAsc, 3H2 + iAsc → 3H^−^ + iAscH^+^, and the Gibbs free energy of Step e is 46.5 kcal/mol. Finally, Step f is the concerted two hydrogen ions (atoms) transfer step from 3H2 to iAsc, and the Gibbs free energy of Step f is −16.1 kcal/mol.
According to the thermodynamic potentials of Steps a–f, the Gibbs free energies of Step b (31.4 kcal/mol), Step d (34.2 kcal/mol), and Step e (46.8 kcal/mol) are greater than 30 kcal/mol, which implies that the oxidation of 3H2 could not occur through electron transfer (Step b), hydrogen transfer from the N1-H bond (Step d), and proton transfer (Step e). Since the Gibbs free energies of Step a (−9.7 kcal/mol), Step c (0.0 kcal/mol), and Step f (−16.1 kcal/mol) are less than or equal to zero, the oxidation of 3H2 may experience an initial hydride (Step a), hydrogen atom from C4-H (Step c), and concerted two hydrogen ions (atoms) transfer step (Step f).
Further investigating the thermodynamics of possible hydrogen atom oxidation processes, there are two different pathways for the oxidation of 3H2 by iAsc overall. The first is the H^•^ + e + H^+^ pathway (Step c–Step j–Step g), and the second is the H^•^ + H^•^ pathway (Step c–Step k). Due to the fact that all of the Gibbs free energies of the above five elementary steps are less than or equal to zero (−16.1–0.0 kcal/mol), it can be assumed that these two hydrogen-atom-initiated pathways, Step c–Step j–Step g and Step c–Step k, are thermodynamically feasible.
Additionally, the Gibbs free energies of direct hydride transfer from 3H2 to iAsc and proton transfer from 3H^+^ to iAscH^−^, Step a (hydride transfer, −9.7 kcal/mol) and Step g (proton transfer, −6.4 kcal/mol), are much less than zero, and it can be inferred that the oxidation process of 3H2 and iAsc undergoing successive hydride (Step a, −9.7 kcal/mol) and proton transfer (Step g, −6.4 kcal/mol) is thermodynamically feasible.
Similarly, the oxidation of 3H2 by iAsc processing concerted two hydrogen ions (atoms) transfer (Step f, −16.1 kcal/mol) is also thermodynamically feasible. Although the concerted two hydrogen ions (atoms) transfer (Step f) is thermodynamically feasible, 3H2 and iAsc molecules do not completely match each other in space structure and binding site during transition state. Therefore, the concerted two hydrogen ions (atoms) transfer (Step f) process is reasonably ruled out. It may be that in complex environments within the body, the hydride-acceptor/amino-acid residue pairs, two radicals, or benzoquinones could oxidize dipines through a concerted two hydrogen ions (atoms) transfer step.
In summary, the oxidation of 3H2 by iAsc may experience three possible pathways, that is, H^−^ + H^+^ (Step a–step g), H^•^ + e + H^+^ (Step c–Step j–Step g), and H^•^ + H^•^ (Step c–Step k). But which one pathway or pathways are the real oxidation mechanism needs further experimental verification in the lab. Beyond a doubt, the thermodynamic analyses of the redox process between 3H2 and iAsc provide us with a unique perspective into the dipines’ aromatization by quinones in vivo.
Prediction Methods. The pKa values of DH^+^ and YH^+^ in acetonitrile were predicted by the method developed by Luo and coworkers in 2020 at http://pka.luoszgroup.com/prediction (accessed on 9 January 2024). Prediction XGBoost with RMSE = 1.79 and r^2^ = 0.918 (80:20 train test split).
For dipines, their core skeleton is the 1,4-dihydropyridine structure, which is a key structural factor for biological activity. During the metabolism of dipines in vivo, 1,4-dihydropyridine rings are oxidized to release formal two hydrogen ions (atoms) and generate the aromatic pyridines, and six possible intermediates and 21 potential elementary steps are involved in the process. In this work, 4-substituted-phenyl-2,6-dimethyl-3,5-diethyl-formate-1,4-dihydropyridines (DH2) are refined as the structurally closest dipine models, and the thermodynamic cards of these dipine models’ aromatization on 21 potential elementary steps in acetonitrile have been established.
Based on the thermodynamic cards, the thermodynamic properties of dipine models and related intermediates acting as electrons, hydrides, hydrogen atoms, protons, and two hydrogen ions (atoms) donors are discussed. Several valuable conclusions are made as follows.
Moreover, the thermodynamic cards were applied to evaluate the redox properties and judge the possible oxidative mechanism of dipine models. The important conclusions are drawn as follows.
The thermodynamic data of dipine models’ aromatization suggests that the oxidative metabolism of dipine drugs is unexpectedly complex, and may involve many active intermediates and potential elementary steps under the conditions of various oxidoreductases in vivo. Therefore, the effects and levels of electrons and hydrogen atoms, as well as hydride oxidoreductases may need sustained attention or detection during the treatment period. Without a doubt, the thermodynamic cards of dipine models could help us to understand the redox properties of DH2 and related intermediates, and diagnose the possible mechanism and pathways of dipine metabolism in vivo.
The following supporting information can be downloaded https://www.mdpi.com/article/10.3390/molecules29153706/s1. The prediction method of pKa, original data of pKa prediction, and thermodynamic cards of DH2 and YH2 are available in Supporting Information. Reference [40] are cited in the supplementary materials.
Formal analysis, Y.-W.J.; Investigation, S.-H.G.; Writing—original draft, B.-C.Q.; Supervision, X.-Q.Z.; Project administration, G.-B.S. All authors have read and agreed to the published version of the manuscript.
The data underlying this study are available in the published article.
The authors declare no conflict of interest.
This study was supported by Health Commission of Shandong Province (202313051336) and the doctoral scientific research foundation of Jining Medical University.
The data underlying this study are available in the published article.