2. Results and Discussion
The synthesis of coumarin derivatives was initially optimized using a range of different conditions, as indicated in
Table 1. Following the successful optimization of the initial reaction, we proceeded to synthesize various coumarin derivatives. Additionally, we carried out further optimization for the second step of this process, as outlined in
Table 2.
Light yellowish powder; % age yield: 59%; melting point range = 226–227 °C; Rf = 0.51 (CH3COOC2H5:CH3OH:: 1.5:1); UV-vis (THF) λmax (nm) = 305; FT-IR: ν (cm−1) = 3370 (N-H stretching), 1596 (C=O vibration), 3196 (=C-H stretching vibration), 1455 (C=C vibration), 1321 (C-N stretching vibration); 1HNMR: (400 MHz, DMSO-d6); δ: 2.51 (s, 2H, H-11), 5.22 (s, 1H, H-9), 7.98 (d, J = 8 Hz, 1H, H-6), 7.59 (m, 1H, H-1), 7.31 (t, J = 8 Hz, 2H, H-2, 3). 13C NMR δ:159.58, 153.47, 152.63, 132.53, 125.74, 122.64, 114.66, 114.45, 86.62.
The structures (
Figure 1) and spectral data of synthesized derivatives of 4-aminocoumarin are given below.
White solid; yield = 69%; Rf = 0.5 (CH3COOC2H5:C6H14:: 1.5:1); melting point range = 295–298 °C; UV-vis (THF) λmax (nm) = 307; FT-IR: ν (cm−1) = 1568 (C=N vibrations), 1701 (C=O stretch), 3048 (=C-H stretching), 1383 (C-F vibration), 3248 (OH vibration); 1H-NMR: (400 MHz, CDCl3); δ: 10.43 (s, 1H, H-13), 5.37 (s, 1H, H-9), 8.16 (d, J = 8 Hz, 1H, H-6), 7.53 (m, 1H, H-1), 7.67 (m, 1H, H-2), 8.06 (d, J = 8 Hz, 1H, H-3), 7.49 (s, 1H, H-15), 7.03 (m, 1H, H-17), 6.87 (dd, J = 8 Hz and 4 Hz, 1H, H-18), 3.51 (s, 1H, H-20). 13C NMR δ: 164.44, 164.41, 163.28, 156.23, 156.16, 156.14, 154.45, 154.22, 151.59, 133.22, 128.76, 123.92, 121.28, 121.21, 119.59, 119.43, 118.50, 118.25, 118.18, 117.69, 116.66, 116.50, 102.80
White powder; yield = 64%; Rf = 0.6 (CH3COOC2H5:C6H14:: 1.5:1); melting point range = 200–202 °C; UV-vis (THF) λmax (nm) = 304; FT-IR: ν (cm−1) = 1560 (C=N stretching), 1650 (C=O stretching vibration), 3047 (=C-H vibrations), 1524 and 1341 (NO2 stretching), 1443 (C=C, Ar stretching vibrations); 1H-NMR: (400 MHz, CDCl3); δ: 8.10 (s, 1H, H-13), 6.15 (s, 1H, H-9), 7.58 (d, J = 8 Hz, 1H, H-6), 7.47 (m, 2H, H-1, 3), 7.54 (t, J = 8 Hz, 1H, H-2), 8.02 (d, J = 8 Hz, 1H, H-15), 8.12 (d, J = 8 Hz, 1H, H-16), 8.17 (d, J = 8 Hz, 1H, H-17), 7.69 (s, 1H, H-19). 13C NMR δ: 163.28, 163.22, 154.56, 151.39, 146.84, 136.79, 133.55, 130.37, 129.03, 128.60, 123.69, 122.65, 122.13, 118.52, 117.61, 103.03.
Pale-yellow solid; yield = 57%; Rf = 0.6 (CH3COOC2H5:C6H14:: 1.5:1); melting point range = 185–188 °C; UV-vis (THF) λmax (nm) = 368; FT-IR: ν (cm−1) = 1543 (C=N stretching), 1596 (C=O vibration), 3061 (=C-H stretching vibration), 3218 (OH stretch), 1453 (C=C vibrations); 1H-NMR: (400 MHz, DMSO-d6); δ: 7.33 (s, 1H, H-13), 5.22 (s, 1H, H-9), 7.99 (d, J = 8 Hz, 2H, H-6, 15), 7.59 (m, 2H, H-1, 2), 7.30 (d, J = 8 Hz, 2H, H-2, 16), 7.38 (s, 1H, H-18), 5.76 (s, 1H, H-20), 3.34 (s, 3H, H-21).
Off-white solid; yield = 63%; Rf = 0.6 (CH3COOC2H5:C6H14:: 1.5:1); melting point range = 255–257 °C; UV-vis (THF) λmax (nm) = 374; FT-IR: ν (cm−1) = 1577 (C=N stretching vibration), 1670 (C=O stretch), 3050 (=C-H vibrations), 1509 and 1340 (NO2 stretching vibrations), 1436 (C=C vibrations); 1H-NMR: (400 MHz, CDCl3); δ: 12.54 (s, 1H, H-13), 6.12 (s, 1H, H-9), 8.03 (d, J = 8 Hz, 1H, H-6), 7.64 (m, 1H, H-1), 7.69 (t, J = 8 Hz, 1H, H-2), 7.73 (d, J = 8 Hz, 1H, H-3), 8.17 (d, J = 8 Hz, 2H, H-15, 19), 8.19 (d, J = 8 Hz, 2H, H-16, 18). 13C NMR δ: 164.73, 163.76, 163.28, 158.34, 154.56, 151.48, 133.55, 130.31, 128.60, 123.67, 118.50, 117.55, 114.58, 107.24, 102.80, 101.85, 55.57.
White solid; yield = 61%; Rf = 0.7 (CH3COOC2H5:C6H14:: 1.5:1); melting point range = 280–282 °C; UV-vis (THF) λmax (nm) = 305; FT-IR: ν (cm−1)= 1582 (C=N stretching vibrations), 1517 (C=O stretching), 3191 (=C-H vibration), 3337 (OH stretching), 1436 (C=C, Ar stretch); 1H-NMR: (400 MHz, DMSO-d6); δ: 9.28 (s, 1H, H-13), 4.98 (s, 1H, H-9), 8.51 (d, J = 8 Hz, 1H, H-6), 7.46 (t, J = 8 Hz, 1H, H-1), 7.68 (t, J = 8 Hz, 1H, H-2), 7.41 (d, J = 8 Hz, 1H, H-3), 7.11 (d, J = 0 Hz, 2H, H-15, 19), 6.64 (d, J = 8 Hz, 2H, H-16, 18), 9.71 (br.s, 1H, H-20, -OH). 13C NMR δ: 164.05, 163.28, 154.56, 151.58, 148.24, 139.10, 133.55, 128.60, 128.41, 124.30, 123.69, 118.52, 117.61, 103.03.
Yellow solid; yield = 79%; Rf = 0.6 (CH3COOC2H5:C6H14:: 1.5:1); melting point range = 253–255 °C; UV-vis (THF) λmax (nm) = 368; FT-IR: ν (cm−1) = 1545 (C=N stretch), 1608 (C=O stretching vibration), 3150 (=C-H stretching), 3374 (OH stretching vibrations), 2959 (C-H vibration), 1454 (C=C, Ar stretching vibration); 1H-NMR: (400 MHz, DMSO-d6); δ: 10.22 (s, 1H, H-13), 5.22 (s, 1H, H-9), 7.99 (d, J = 8 Hz, 1H, H-6), 7.33 (m, 1H, H-1), 7.59 (t, J = 8 Hz, 1H, H-2), 6.90 (d, J = 8 Hz, 1H, H-3), 7.31 (s, 1H, H-15), 7.45 (d, J = 8 Hz, 1H, H-17), 7.35 (d, J = 8 Hz, 1H, H-18), 7.38 (br.s, 1H, H-20, -OH), 2.24 (s, 3H, H-21, -CH3). 13C NMR δ: 164.31, 163.28, 157.14, 154.45, 151.59, 133.27, 132.49, 131.98, 129.44, 128.76, 123.92, 120.58, 118.50, 117.69, 116.51, 102.80, 20.61.
White solid; yield = 60%; Rf = 0.6 (CH3COOC2H5:C6H14:: 1.5:1); melting point range = 265–268 °C; UV-vis (THF) λmax (nm) = 304; FT-IR: ν (cm−1) = 1571 (C=N stretching vibrations), 1667 (C=O vibration), 3249 (=C-H stretching), 1517 and 1348 (NO2 stretching vibrations), 1439 (C=C, Ar stretching), 1H-NMR: (400 MHz, DMSO-d6); δ: 8.88 (s, 1H, H-13), 6.49 (s, 1H, H-9), 7.75 (d, J = 8 Hz, 1H, H-6), 7.40–7.46 (m, 2H, H-1,3), 7.50 (t, J = 8 Hz, 1H, H-2), 7.92 (d, J = 8 Hz, 1H, H-15), 7.66 (t, J = 8 Hz, 1H, H-16), 7.62 (m, 1H, H-17), 8.12 (d, J = 8 Hz, 1H, H-18). 13C NMR δ: 163.28, 159.16, 154.56, 151.06, 147.42, 133.55, 131.35, 130.56, 130.00, 128.60, 128.03, 125.27, 123.69, 118.53, 117.61, 102.80.
Yellowish powder; yield = 57%; Rf = 0.6 (CH3COOC2H5:C6H14:: 1.5:1); melting point range = 255–257 °C; UV-vis (THF) λmax (nm) = 301; FT-IR: ν (cm−1) = 1524 (C=N stretch), 1669 (C=O vibrations), 3047 (=C-H stretching vibration), 1437 (C=C vibrations); 1H-NMR: (400 MHz, CDCl3); δ: 11.34 (s, 1H, H-13), 6.07 (s, 1H, H-9), 8.08 (m, 1H, H-6), 7.44 (d, J = 8 Hz, 2H, H-1, 3), 7.66 (t, J = 8 Hz, 1H, H-2), 7.32 (d, J = 8 Hz, 2H, H-15, 19), 7.18 (d, J = 8 Hz, 2H, H-16, 18). 13C NMR δ: 164.10, 163.28, 154.30, 151.72, 135.21, 133.39, 133.26, 129.32, 128.92, 128.76, 123.92, 118.56, 117.69, 103.16.
White solid; yield = 75%; Rf = 0.6 (CH3COOC2H5:C6H14:: 1.5:1); melting point range = 276–278 °C; UV-vis (THF) λmax (nm) = 321; FT-IR: ν (cm−1) = 1524 (C=N stretching vibration), 3342 (O-H stretching vibration), 1670 (C=O stretching vibration), 3208 (=C-H stretching vibration), 1509 (C=C, Ar stretching vibration); 1H-NMR: (400 MHz, DMSO-d6); δ: 8.18 (s, 1H, H-13), 5.54 (s, 1H, H-9), 8.08 (d, J = 8 Hz, 1H, H-6), 7.56 (t, J = 8 Hz, 1H, H-1), 7.69 (t, J = 8 Hz, 1H, H-2), 7.36 (t, J = 8 Hz, 1H, H-3), 7.43 (s, 1H, H-15), 7.31 (d, J = 8 Hz, 1H, H-17), 7.22 (d, J = 8 Hz, 1H, H-18), 7.85 (br.s, 1H, H-20, -OH). 13C NMR δ: 164.29, 163.28, 157.95, 154.56, 151.59, 136.03, 133.25, 133.20, 128.61, 123.83, 120.65, 118.50, 117.93, 117.80, 112.47, 102.94.
White powder; yield = 69%; Rf = 0.6 (ethyl acetate:n-hexane:: 1.5:1); m.p. = 264–266 °C; UV-vis (DCM) λmax (nm) = 305 nm; FT-IR:ν = 1509 cm−1 (C=N stretching vibration), 1650 cm−1 (C=O stretching vibration), 3125 cm−1 (=C-H stretching vibration), 1443 cm−1 (C=C, Ar stretching vibration), 1099 cm−1 (C-O stretching vibration); 1H-NMR: (400 MHz, DMSO-d6); δ: 9.7 (s, 1H, H-13), 5.04 (s, 1H, H-9), 8.54 (d, J = 8 Hz, 1H, H-6), 7.50 (t, J = 8 Hz, 1H, H-1), 7.67 (t, J = 8 Hz, 1H, H-2), 7.45 (d, J = 8 Hz, 1H, H-3), 7.21 (d, J = 8 Hz, 2H, H-15, 19), 6.79 (d, J = 8 Hz, 2H, H-16, 18), 3.92 (q, J = 8 Hz, 2H, H-20), 1.26 (t, J = 8 Hz, 3H, H-21). 13C NMR δ: 164.14, 163.28, 160.88, 154.56, 151.58, 133.55, 130.24, 129.16, 128.60, 123.69, 118.52, 117.61, 114.73, 103.03, 63.56, 14.67.
2.1. UV-Vis Analysis
Absorbance spectra of all the newly synthesized Schiff bases under investigation were recorded from 200 nm to 800 nm using a quartz cuvette (10 mm), and THF was used as solvent. All the newly synthesized compounds possess a coumarin ring in their chemical structure. The maximum absorbance of compound 2a was recorded at 305 nm, whereas spectra for compounds 3a–11a showed maximum absorbance at 307, 304, 368, 305, 374, 368, 304, 310, 301, 321, and 305 nm, respectively. The compounds 4a, 7a, and 8a showed a blue shift (hypsochromic), whereas the compounds 3a, 5a, 6a, 9a, 10a, and 11a showed a red shift (bathochromic) compared to the starting material (compound 2). The shift in maximum absorbance can be attributed to extended conjugation and provides evidence in favor of the successful synthesis of the anticipated products.
2.2. FT-IR Analysis
The FT-IR spectrum of newly synthesized compound
2a shows a characteristic absorption peak at 3370 cm
−1 with a shoulder band at 3484 cm
−1, which corresponds to the primary amine group (N-H). These characteristic bands disappear in the FT-IR spectra of synthesized Schiff bases. Also, a characteristic absorption peak at around 1517–1575 cm
−1 appears in each FT-IR spectrum of Schiff bases, which corresponds to strong C=N vibrations [
16]. This analysis further confirms that the amino group of compound
2a has successfully been converted to an imine group in each succeeding Schiff base compound. Furthermore, the FT-IR spectra of newly synthesized compounds exhibit a strong absorption peak at 3047–3214 cm
−1, which can be attributed to the presence of a methylene group (=C-H) [
17]. The appearance of this particular signal indicates that this moiety did not get involved in the reaction with substituted aromatic aldehydes (
Figure S1).
2.3. NMR Spectral Analysis
The NMR spectra of newly synthesized compounds were obtained using a Bruker AM-400 MHz NMR spectrometer. All of the compounds were either soluble in DMSO-d6 or CDCl3. The 1H-NMR data of all the newly synthesized compounds confirmed their proposed structures.
In the case of compound 2a, a singlet appearing at δ 2.51 ppm accounted for the two protons of the amino group (NH2). The active methylene proton in compound 2a at position 9 has no neighboring proton; therefore, it showed a singlet at δ 5.22 ppm. The proton at position 6 shows strong coupling with its ortho proton, does not show long-range coupling, and gives a doublet signal at δ 7.98 ppm. The proton at position 1 shows a multiplet signal at δ 7.59 ppm, which is due to a long-range coupling. Protons at positions 2 and 3 are neighboring protons and show a triplet signal at δ 7.31 ppm. All of these signals confirm the successful synthesis of compound 2a (4-aminocoumarin).
The singlet peak appearing at
δ 2.51 ppm due to two protons of the amino group (NH
2) in 4-aminocoumarin disappears in the
1H-NMR spectrum of Schiff bases (
2a–
11a); instead, a singlet due to azomethine proton (N=CH) appears at 7.43–12.5 ppm. The shielding and deshielding of the imine proton are due to the mesomeric effect of pi-electrons of aromatic and aldehydic moiety. The appearance of a characteristic singlet peak due to methylene proton at
δ 4.98–6.49 ppm in the
1H-NMR spectra is strong evidence for the formation of Schiff bases (
Figure S1).
2.4. Antibacterial Activity
All the newly synthesized Schiff bases of 4-aminocoumarin (
2a–
11a) were tested against two bacterial strains:
Salmonella typhimurium (Gram-negative) and
Staphylococcus aureus (Gram-positive). Zones of inhibition (mm) of all the synthesized compounds against these two strains are shown in
Figure 2.
The zones of inhibition were measured in millimeters. The 50 μg/mL concentration of ciprofloxacin (reference drug) showed 62.9 ± 0.94 and 54.4 ± 0.50 mm zones of inhibition against Staphylococcus aureus and Salmonella typhimurium, respectively. The negative control (DMSO) did not show any activity against these two strains. The synthesized compounds were also tested against the two bacterial strains (concentration: 200 μg/mL). The compounds 3a and 7a showed higher zones of inhibition against these two bacterial strains compared to other synthesized derivatives. Compound 3a showed antimicrobial activity against Staphylococcus aureus with a zone of inhibition of 41.5 ± 0.96 mm and against Salmonella typhimurium with a zone of inhibition 35 ± 0.90. Also, compound 7a showed antibacterial activity against Staphylococcus aureus with a zone of inhibition of 40.6 ± 0.76 mm and against Salmonella typhimurium with a zone of inhibition of 42.6 ± 0.76 mm. Hence, it can be deduced that compound 3a is highly active against Staphylococcus aureus compared to compound 7a, whereas compound 7a is comparatively more effective against Salmonella typhimurium compared to compound 3a. The antibacterial bioassay proves that compound 3a can be used as a lead for the development of antibacterial drugs against Staphylococcus aureus and compound 7a can be taken as an antibiotic against the Gram-negative bacterial strain Salmonella typhimurium.
2.5. In Vitro Urease Inhibition
In continuation of the in silico studies, compounds
3a and
5a were subjected to in vitro urease inhibition assay. The results of this assay further confirm the findings obtained as a result of in silico studies. The inhibition potential of compounds
3a and
5a is comparable to that of the standard used (thiourea). Further optimization of
3a and
5a may lead to the development of new standards for future use in urease inhibition assays (
Table 3).
2.6. In Silico Studies
Urease has the tendency to catalyze the hydrolysis of urea into ammonia and carbon dioxide. It is a key virulence factor for a wide range of human infections: 10 of the 12 antibiotic-resistant priority pathogens designated by the World Health Organization (WHO) in 2017 are ureolytic bacteria that colonize and thrive in the host organism using urease activity [
1,
18].
All of the synthesized compounds were screened against a minimized crystal structure and the active site was identified by using the site finder tool embedded in MOE; the rest of the parameters were kept as default, with each molecule sampled in 10 conformations. All of the compounds docked into the urease’s active pocket showed a strong binding affinity with the active site residues which is evident from ∆G
bind score (
Table 4). All of the compounds have molecular weights within 300–350 and calculated logP values of 0.3–3.2, which places them within the lead-like range. Although all reported compounds are topologically similar to one another, binding affinity and docked poses suggest that compounds
3a (−8.67 Kcal/mol),
5a (−7.44 Kcal/mol) and
9a (−6.96 Kcal/mol) fall into the best candidates for activity against the urease enzyme. In the docked poses, all the compounds showed van der Waal interaction as well as H–arene interaction through an unprecedented coumarin functional group.
The lead compounds identified as a result of the molecular docking study were subjected to MD simulations in order to understand the structural dynamics, which is essential for identifying potential inhibitors associated with protein inhibition mechanisms. For all trajectories, RMSD calculations were performed to investigate the time-evolved behavior of the protein. Amplitude perturbations of about >2Å were detected in free protein, thereby illustrating the complicated structural changes involved with protein expansion over time. The bound protein has reduced dynamics relative to the free protein, as evidenced by the RMSD result. The average RMSD for all
3a and
5a complexed proteins revealed dynamics ranging from 1.2 to 1.4 Å. The attachment of
3a and
5a in the active site shifts the protein to a more rigid texture, whereas the free protein is more flexible in its motions. The RMSF is used to analyze the flexibility of individual residues in bound and free proteins. The orientation of the helix-turn-helix motif has a significant impact on urease enzyme binding to compounds (
Figure 3A).
The RMSFs of ligand-bound urease are lower than those of free protein, and they are more prominent in the active site. An MMPBSA study was carried out to determine the thermodynamic parameters of the protein–ligand complexes, such as binding free energy and van der Waals, electrostatic, and polar solvation energies for the last 50 ns of the MD simulation trajectories. The binding energy of 3a and 5a bound urease complexes remained stable during the explored time scale, as evidenced by ∆Gbind.
The MMPBSA data analysis aids in calculating the contribution of individual amino acid residues to the total binding energy. The binding of both compounds is adjacent to the nickel ion, in a similar fashion to that of either urea or thiourea fragment which were being complexed by nickel (II) ion. The determination of trajectories that were gained also exposes that binding of 3a and 5a to the target protein occurs. It also shows effectively that the compounds under consideration efficiently take up the active sites of the target protein. It has also been observed that tight attachment of the helix-turn-helix motif occurs as a cover on the active site space. This results in obstruction of the flap closure of the urease active site and consequently leads towards inhibition of the urease enzyme. The studies of interaction showed that binding of the sample compounds within the active pocket leads towards the generation of Ni-O electrostatic bonds, whereby nickel-bound oxygen of the compounds 3a and 5a forms H-bonds with His492. Apart from this—the key residues (Arg609, Asp494, Arg439, Met697) facilitating the binding of the compounds 3a and 5a to the active site residues—a group of histidines (His593, His594, His492) encapsulated the active site, forming a highly charged atmosphere. Ni atom facilitates the binding of the substrate (urea) to the active site; however, the compounds 3a and 5a block the entry of the substrate via strongly coordinating with metal at one end and histidine at the other end. The formation of H-bonds between compounds 3a and 5a to that of active-site charged residues act as an additional force confirming the strong binding of compounds inside the active pocket.
2.7. In Vivo Pharmacology
2.7.1. Pylorus Ligation Activity
The pyloric ligation ulcer was induced in the rat’s stomach, and the stomach was ligated for approximately 6 h. The insertion of compound
5a resulted in a reduction in the ulcer index (14.30 ± 0.34 vs. 6.80 ± 0.24, Group I vs. Group V; see
Figure 4). In terms of pH, fewer changes were observed (
Figure 5), while the gastric content volume after the administration of
5a was 2.75 ± 0.10 vs. 1.40 ± 0.14 (Group I vs. Group V;
Figure 4). The total acidity was 43.10 ± 0.52 meq/L/100 g vs. 25.10 ± 0.16 meq per liter per 100 g (Group I vs. Group V;
Figure 6), while the free acidity was 23.34 ± 0.42 meq per liter per 100 g vs. 9.50 ± 0.46 meq per liter per 100 g (Group I vs. Group V;
Figure 6), and both were reduced upon the insertion of compound
5a. Similarly, lipid peroxidation (0.70 ± 0.02; Group I) was reduced (0.42 ± 0.046; Group V, ***
p < 0.001;
Figure 7) upon the administration of compound
5a.
2.7.2. Ethanol-Induced Ulcer
To investigate ethanol-induced ulcers in the rat model, we administered compound
5a, which exhibited promising results by significantly decreasing the ulcer index (***
p < 0.001; 14.50 ± 0.32 vs. 7.20 ± 0.36, Group I vs. Group V; see
Figure 5). Gastric content pH is shown in
Figure 4. Similarly, the gastric content volume (**
p < 0.01) decreased after the insertion of the potent compound
5a (2.70 ± 0.10 vs. 1.32 ± 0.14, Group I vs. Group V;
Figure 6). The pH of the gastric contents (**
p < 0.01, Group V) also increased (1.94 ± 0.26 in Group I, 3.60 ± 0.52 in Group V). The total acidity, initially at 45.10 ± 0.52 meq per liter per 100 g (Group I), was reduced to 27.10 ± 0.28 meq per liter per 100 g (Group V, ***
p < 0.001;
Figure 7), while the free acidity levels (19.45 ± 0.43 meq per liter per 100 g; Group I;
Figure 8) decreased to 10.01 ± 0.16 meq per liter per 100 g (Group V, **
p < 0.01). Similarly, the rate of malondialdehyde formation (0.65 ± 0.040; Group I) was reduced to 0.38 ± 0.048 (Group V, ***
p < 0.001;
Figure 7).
Conclusively, the administration of compound 5a demonstrated promising results in reducing ethanol-induced ulcers in a rat model, as indicated by a significant decrease in the ulcer index and alterations in gastric pH, content volume, total acidity, free acidity, and malondialdehyde formation. These findings suggest the potential therapeutic value of compound 5a for ulcer treatment.
2.7.3. Aspirin-Induced Ulcer
In the dose range of 20 and 40 mg/kg, compound
5a demonstrated a significant reduction in the ulcer index (***
p < 0.001; 9.60 ± 0.26 vs. 14.40 ± 0.28, Group IV vs. Group I; 6.60 ± 0.34 vs. 14.40 ± 0.28, Group V vs. Group I; see
Figure 5) in rats treated with aspirin, along with pH alterations (
Figure 4). Similarly, compound
5a elevated gastric pH from 2.22 ± 0.34 (Group I) to 2.94 ± 0.42 (Group IV, **
p < 0.01) and 3.58 ± 0.22 (Group V, ***
p < 0.001). A significant decrease in gastric content volume was observed with the treatment of compound
5a (**
p < 0.01; Group IV and Group V;
Figure 6). Total acidity, initially at 44.23 ± 0.17 meq per liter per 100 g (Group I), decreased to 26.34 ± 0.34 meq per liter per 100 g (Group V, ***
p < 0.001;
Figure 7), while free acidity values decreased from 24.23 ± 0.53 meq per liter per 100 g (Group I) to 8.10 ± 1.16 meq per liter per 100 g (Group V, **
p < 0.01;
Figure 6). Correspondingly, the rate of malondialdehyde formation decreased from 0.63 ± 0.022 (Group I) to 0.44 ± 0.042 (Group V, ***
p < 0.001;
Figure 8).
2.7.4. Histamine-Induced Ulcer
Administration of histamine was shown to be ulcerogenic in the rats. The ulcer index in the group I animals was (14.78 ± 0.44;
Figure 5). Compound
5a was administered, and displayed a decline in ulcer severity and ulcer index (9.44 ± 0.42 in Group IV, **
p < 0.01; 6.52 ± 0.42 in Group V, ***
p < 0.001;
Figure 3). The pH of the gastric content (2.24 ± 0.42 in Group I;
Figure 4) was elevated (2.62 ± 0.42 in Group III, **
p < 0.01; 2.98 ± 0.22 in Group IV, **
p < 0.01; 3.42 ± 0.12 in Group V, ***
p < 0.001;
Figure 4). A noteworthy decline in the gastric contents volume was observed (2.73 ± 0.11 versus 1.41 ± 0.14., Group I versus Group V; ***
p < 0.001 in Group V;
Figure 6). The total acidity was 41.24 ± 0.66 meq per liter per 100 g (Group I) and reduced to 22.46 ± 0.42 meq per liter per 100 g (Group V, ***
p < 0.001;
Figure 5), while the free acidity was 22.78 ± 0.78 meq per liter per 100 g (Group I;
Figure 7) decreased to 7.60 ± 0.44 meq per liter per 100 g (Group V, **
p < 0.01). Likewise, the rate of the formation of the malondialdehyde (0.67 ± 0.010; Group I) was reduced (0.46 ± 0.022; Group V, ***
p < 0.001;
Figure 7).
2.7.5. H+–K+ ATPase Assay
Compound
5a was also demonstrated to significantly (*
p < 0.05) affect gastric mucosal homogenate in rats. Its inhibitory potential was concentration-dependent, and the tested drug showed a comparable effect to omeprazole. Specifically, compound
5a significantly reduced the hydrolysis of ATP (see
Figure 9) through gastric ATPase, with an IC
50 of 30 μg/mL, which was very similar to the IC
50 of omeprazole, used as a positive control (IC
50 of 22 μg/mL).
In the rat model with pyloric ligation-induced ulcers, the animal stomachs were ligated for approximately 6 h. This resulted in a notable increase in total acidity (43.10 ± 0.52 meq/L/100 g vs. 25.10 ± 0.16 meq per liter per 100 g) and free acidity (23.34 ± 0.42 meq per liter per 100 g vs. 9.50 ± 0.46 meq per liter per 100 g) in Group I compared to Group V (as shown in
Figure 7b). However, upon the introduction of compound
5a, these acidity levels were significantly reduced.
Likewise, lipid peroxidation, which was at 0.70 ± 0.02 in Group I, showed a significant reduction (0.42 ± 0.046) in Group V upon the administration of compound
5a. This suggests the potential effectiveness of compound
5a in reducing ulceration induced by ethanol in the rat model. Furthermore, the gastric content pH, as depicted in
Figure 4, was notably affected. The insertion of compound
5a resulted in a significant increase in gastric pH from 2.22 ± 0.34 (Group I) to 2.94 ± 0.42 (Group IV, **
p < 0.01) and 3.58 ± 0.22 (Group V, ***
p < 0.001). Additionally, the volume of gastric content decreased significantly with the treatment of compound
5a. Moreover, compound
5a demonstrated a significant reduction in the hydrolysis of ATP, as shown in
Figure 9, with an IC
50 of 30 μg/mL, which is comparable to the positive control omeprazole (IC
50 of 22 μg/mL). This suggests the potential of compound
5a as a gastric ATPase inhibitor.
The purity of two best-acting derivatives was confirmed using HPLC (
Figure S1, Compound
3a and
5a).