INTRODUCTION:

Inflammation is a vital protection system of the body against outside interferences such as tissue damage, microbial infections, and other dangerous conditions. Chronic or excessive inflammation reaction is the cause of several diseases, such as cancer, inflammatory bowel disease, Diabetes Mellitus (type1), arthritis, asthma, and coronary artery disease. The prevalence of illnesses caused by inflammation is predicted to increase over the next thirty years. Globally, three of five people died because of inflammation^1-5.

Non-steroidal anti-inflammatory drugs (NSAIDs) are ordinarily used to manage inflammation⁶. However, NSAIDs are associated with various interactions causing side effects such as gastrointestinal ulcers, cardiovascular disorders, hypertension, and acute kidney failure^2,7-8.

Curcumin has several activities: anti-inflammation, anticancer, antioxidant, and antidiabetic. Its anti-inflammatory potential has been studied for chronic disease therapy such as Alzheimer's, asthma, inflammatory bowel disease, and arthritis^9-12. However, its potential is confined because of its low bioavailability, rapid metabolism, poor absorption, poor solubility in gastrointestinal fluid, and physical instability¹³.

The alteration of curcumin structure through cyclization of its 1,3-diketones chain with hydrazine to be a pyrazole ring increases its stability^14,15. The curcumin pyrazole (CP) derivatives have anti-inflammatory (5-LOX and COX inhibitor), anti-angiogenic, and WtAS aggregation inhibitors^14-16. Substitution of curcumin pyrazole with bis-Mannich base increases its solubility¹⁷. To investigate their potential further, herein, we synthesized six novel bis-Mannich bases derivative of CP to assess their anti-inflammatory activity in-vitro. Besides, we performed an in-silico study via the molecular docking method to predict their binding interaction against COX-1 and COX-2 enzymes.

MATERIALS AND METHODS:

Materials:

Curcumin was isolated from curcuminoid (Java plant, Solo, Indonesia) using the reported method¹⁸. Bis-(2,6-dimethylmorpholine Mannich base) CP (2c) was obtained from our earlier research¹⁷. Reagents and solvents (Merck, Sigma-Aldrich, or Mallinckrodt) were purchased commercially. The TLC method was applied for products' purity and reaction rate monitoring. Stuart Scientific's melting point equipment was used to take melting points. FTIR Spectrophotometer (Shimadzu 8400S) and NMR Spectrometer (JEOL JNM 500) were used to take IR and NMR spectra. LC-MS/MS with the ESI+ method (UNIFI-Waters) was applied to determine mass spectra. A molecular docking experiment was performed using Auto Dock. The 2D and 3D structures were drawn and optimized using Marvin Sketch software, while ligand-macromolecular target interactions were visualized using LigPlot+ dan PyMOL software.

Synthesis of Curcumin Pyrazole (CP) (1):

CP (1) was prepared using a synthesis method described earlier with some modifications¹⁶. Curcumin in anhydrous acetic acid (13.6mmol/250mL) suspension was sonicated for 30 min, and hydrazine hydrate (6.94 mL, 122.4mmol) was added and stirred for 48 h at r.t. until completed. Then ice powder was added to the mixture while stirring and left overnight until the product was settled. The product was filtered off, washed with water, and dried in a vacuum oven to afford CP as a pale-yellow powder. Yield: 55%. M.P.: 206-208°C. FT-IR (KBr, ῡ/cm^-1): 3483, 3390, 3008, 1617, 1566, 1517, 1401 & 1354, 1235, 1033. ¹H-NMR (δ/ppm): 9.21 (s, 2H), 12.82 (s, 1H), 3.83 (s, 6H), 6.61 (s, 1H), 6.77 (d, 2H,J=8Hz), 6.92 (d, 2H,J=8Hz), 7.14 (s, 2H), 6.91 (d, 2H,J=15Hz), 7.03 (d, 2H,J=17Hz).

Synthesis of Bis-Mannich Base derivatives of CP (2a-b, 2d-f):

Bis-Mannich base derivatives of CP (2a-b, 2d-f) were synthesized according to the method for synthesis of bis-(2,6-dimethylmorpholine Mannich base) CP reported previously by replacing 2,6-dimethylmorpholine with corresponding sec-amines (morpholine, dibutyl amine, diethyl amine, pyrrolidine, and dimethylamine)¹⁷.

Bis-(Morpholine Mannich base) CP (2a). Yield: 47.1% as a pale-yellow powder, M.P.: 190–192^oC. FT-IR (ῡ/cm^-1): 2967 & 2848, 1535, 1458, 1518, 1155, 1267, 1400 & 1350, 1303.¹H-NMR (δ/ppm): 2.58 (s, 8H), 3.69 & 3.71 (s, 4H), 3.75 (s, 8H), 3.89 & 3.90 (s, 6H), 6.55 (s, 1H), 6.85 (d, 2H,J=16Hz), 6.98 (d, 2H,J=16Hz), 6.95 (d, 2H,J=1Hz), 6.72 (s, 2H). ¹³C-NMR (δ/ppm): 53.0 and 66.9 (8C), 56.2 (2C), 61,7 (2C), 99.7 and 128.1 (3C), 115.6 and 130.7 (4C), 108.9, 109.0, 120.0, 121.0, 147.5, and 148.3 (12C). Mass (m/z): found 563.2877 [M+H]⁺, calc mass for C₃₁H₃₉N₄O₆ = 563.2870 (error 1.2ppm).

Bis-(Dibutyl amine Mannich base) CP (2b). Yield: 66.5% as a brown powder, M.P.: 57,9^oC. FT-IR (ῡ/cm^-1): 2972, 2935, 2823, 1558, 1496, 1558, 1458, 1400, 1317, 1147, 1084, 1158, 1251. ¹H-NMR (δ/ppm): 0.89 (t, J=7, 12H), 1.30 (st; J=7, 8H), 1.54 (qt, J=7, 8H), 2.52 (t; J=7, 8H), 3.26 (s, 4H). 3.90 (s, 6H), 6.54 (s, 1H), 6.97 (d, 2H,J=16Hz), 6.86 (d, 2H, J=16Hz), 6.95 (s, 2H), 6.94 (s, 2H).¹³C-NMR (δ/ppm): 14.1, 20.7, 28.5, and 53.4 (16C), 56.1 (2C), 58.1 (2C), 99.5 and 127.6 (3C), 115.2 and 131.1 (4C), 106.4 (2C), 108.6, 119.7, 122.4, 148.3, 148.4 (12C). Mass (m/z): found 645.4382 [M-H]^-, calc mass for C₃₉H₅₇N₄O₄ = 6454380, Error 0.3 ppm.

Bis-(Diethyl amine Mannich base) CP (2d). Yield: 88.5% as a pale brown powder, M.P.: 150-151°C. FT-IR (ῡ/cm^-1): 3321, 2970, 2934, 2834, 1409, dan 1359, 1595, 1493, 1450, 1285, 1085, 1159. ¹H-NMR (δ/ppm): 1.09 (t, , 12H), 2.63 (q, 8H); 3.75 (s, 4H), 3.88 (s, 6H), 6.53 (s, 1H), 6.71 (s, 2H,J=3 Hz), 6.93 (s, 2H), 6.88 (s, 2H,J=15 Hz), 6.99 (s, 2H,J=15Hz, 2H).¹³C-NMR (δ/ppm): 11.31 (4C), 46.5 (4C), 56.0 (2C), 56.8 (2C), 99.4 and 127.5 (3C), 115.1 and 130.9 (4C), 108.5, 108.6, 119.7, 122.1, 148.3, 148.5 (12C). Mass (m/z): found 533.3147 [M+H]⁺, calc mass for C₃₁H₄₁N₄O₄ = 533.3128 (error 3.6 ppm).

Bis-(Pyrrolidine Mannich base) CP (2e). Yield: 15.7% as a brown powder, M.P.: 123-125°C. FT-IR (ῡ/cm^-1): 3200-2700, 2964, 2808, 1595, 1491, 1462, 1244, 1155, 1087.¹H-NMR (δ/ppm): 1.84 (m, 8H), 2.65 (m, 8H), 3.89 (s, 6H), 6.72 (s, 2H), 6.54 (s, 1H), 6.92 (d, 2H,J=1Hz), 6.89 (d; 2H,J=16Hz), 7.10 (d; 2H,J=16Hz, 2H). ¹³C-NMR (δ/ppm): 23.7 (4C), 53.8 (4C), 56.1 (2C), 58.7 (2C), 99,6 and 128.1 (2C), 115.0 and 130.6 (4C), 108.7, 117.1, 119.2, 122.6, 147.5, and 148,1 (12C). Mass (m/z): found 531.2695 [M+H]⁺, calc mass for C₃₁H₃₇N₄O₄ = 530.28931 (neutral mass) (error -0.2 ppm).

Bis-(Dimethylamine Mannich base) CP (2f). Yield: 25.6% as a pale-brown powder, M.P.: 161-163°C. FT-IR (ῡ/cm^-1): 3400-3000, 2949, 2833, 1361, 1595, 1496, 1458, 1153, 1085, 1026. ¹H-NMR (δ/ppm): 2.31 (s, 12H); 3.65 (s, 4H), 3.89 (s, 6H), 6.53 (s, 1H), 6.71 (s, 2H), 6.95 (s, 2H), 6.88 (s, 2H,J=15Hz), 7.12 (s, 2H,J=15Hz).¹³C-NMR (δ/ppm): 44.6 (4C), 56.0 (2C), 62.7 (2C), 99.8 and 127.60 (3C), 117.1 and 130.8 (4C), 108.7, 108.8, 119.7, 122.1, 147.5, and 148,1 (12C). Mass (m/z): found 479.2654 [M+H]⁺, calc mass for C₂₇H₃₄N₄O₄ = 478.2580 (neutral mass) (error 0.2 ppm).

Anti-inflammatory Activity Assay:

The compounds (2a-f) were assessed for their anti-inflammatory activity via the protein denaturation technique reported earlier using diclofenac sodium, curcumin, and CP as a reference and comparable compounds^19-21. The mixture of 0.5% BSA solution in PBS pH 6.4(4.5mL) and solution of the test/reference/ comparable compounds (various concentrations, μM) (0.5mL) was incubated at 37°C for 15 min, then heated at 70°C for 5min. The mixtures were cooled and measured its turbidity at 600nm against the blank. The formula: Inhibition (%) = 100 x (1 - A2/A1) was used to calculate the percent of inhibition, where A1 = absorption of the control and A2 = absorption of the sample.

Molecular Docking study:

The in-silico study was done according to the molecular docking protocol described by earlier researchers with some modifications²². The synthesized compounds, curcumin, and celecoxib were drawn using MarvinSketch (www.chemaxon.com) and converted into three-dimensional using the clean 3D mode to get the lowest energy conformation and saved in*. pdb format. Subsequently, water molecules were added to the polar part of the ligands, and the addition of Gasteiger charge was. Finally, the torque determination was done, and the structures obtained were saved in *.pdbqt format. The macromolecular targets: cyclooxygenase-1 (COX-1) (ID: 1EQG) and cyclooxygenase-2 (COX-2) (ID: 4PH9) structures co-crystallized both with ibuprofen (resolution 2.61Å and 1.81Å) were retrieved from the RCSB PDB website (http://www.rcsb.org/pdb)^23-24. The macromolecules were optimized by removing water molecules, separating co-crystal ligands and other non-standard residues, and adding polar hydrogen atoms and Gasteiger charges. The ligands were docked into the macromolecular targets using AutoDock (http://autodock.scripps.edu/). The docking protocol was validated by re-docking the native ligands and determined by the root mean square deviation (RMSD) values ≤ 2.0Å before being applied²⁵. Variations of a maximum number of short, medium, and long energy evaluations and the grid box sizes of 60 x 60 x 60 points, 70 x 70 x 70 points, and 80 x 80 x 80 points with 1 point equivalent to 0.375 Å were applied in the validation process. Ligand-macromolecule interactions in its binding pocket were visualized by LigPlot+ and PyMOL software.

RESULT:

Synthesis of Bis-Mannich Base derivatives of CP:

The compounds were prepared in two steps (Figure 1). The condensation of curcumin and hydrazine hydrate in anhydrous acetic acid afforded curcumin pyrazole (CP) (1). Aminomethylation of 1 by the addition of formaldehyde (HCHO) and corresponding 2^o amine (R₂NH) in ethanol afforded the title compounds (2a-f).

Figure 1: Synthesis route of bis-Mannich base derivatives of CP.

Anti-inflammatory Activity:

The result of the protein denaturation inhibition (anti-inflammatory) evaluation of the compounds is provided in Table 2.

Molecular Docking Study:

The study aimed to predict the interaction between the synthesized compounds and the macromolecule target. COX-1 and COX-2 were used as macromolecular targets. The optimum RMSD values obtained in the protocol’s validation are presented in Table 1. The superpositions of ibuprofen as a native ligand and native ligand re-docking at COX-1 and COX-2 are presented in Figure 2. The docking study found that all the synthesized compounds interacted with COX-1 and COX-2 well. The binding energy of all the synthesized compounds to COX-2 and COX-1 and the selectivity score are presented in Table 2. To evaluate the type of binding site interaction formed between the synthesized compounds and macromolecular, compound 2f to COX-1 and compound 2c to COX-2 were selected and compared to the binding site interaction of ibuprofen (native ligand/COX inhibitor nonselective) to COX-1 and celecoxib (COX-2 inhibitor selective) to COX-2. Visualization of compound 2f and ibuprofen interactions at binding pocket COX-1 and visualization of compound 2c and celecoxib interactions at binding pocket COX-2 are presented in Figures 3 and 4.

Table 1: The optimum RSMD of the docking protocol’s validation of COX-1 and COX-2 using AutoDock

Macromolecule target	Grid box size (points)	Maximum number of energy evaluations	Binding energy/ΔG (kcal/mol)	RMSD (Å)
1EQG	80x80x80	Short	-7.36	0.3699
4PH9	80x80x80	Medium	-8.15	0.3261

Table 2: Anti-inflammatory activity (IC₅₀) and binding energy interaction to COX-1 and COX-2 of the compounds.

Ligands (Compounds)	Anti-inflammatory	COX-1		COX-2		*S^)**
Ligands (Compounds)	IC₅₀(μM)	ΔG (kcal/mol)	Ki (µM)	ΔG (kcal/mol)	Ki x10^-3 (µM)	COX-2/ COX-1
2a	0.506 ± 0.005	-6.38	20.94	-10.14	36.94	1x10^-4
2b	0.593 ± 0.002	-3.57	2430.00	-8.90	298.90	1x10^-7
2c	0.545 ± 0.004	-6.19	29.25	-11.01	8.52	2x10^-4
2d	0.585 ± 0.004	-5.96	42.48	-9.15	197.56	5x10^-3
2e	0.666 ± 0.008	-6.70	12.33	-10.45	21.84	2x10^-3
2f	0.715 ± 0.006	-7.00	7.34	-9.09	12.67	2x10^-3
Curcumin	0.860 ± 0.005	-7.19	5.34	-8.92	289.65	5x10^-2
Ibuprofen	-	-7.36	4.00	-8.51	1060.00	3x10^-1
Celecoxib	-	-6.93	8.29	-10.79	12.25	1x10^-3
Diclofenac Sod	0.779 ± 0.006	-	-	-	-	-
Curc. Pyrazole	0.701 ± 0.008	-	-	-	-	-

^*) S = selectivity score

Figure 2: Superposition ibuprofen as a native ligand (blue) and native ligand re-docking (green) to COX-1 (a) and ibuprofen as a native ligand (yellow) and native ligand re-docking (pale red) to COX-2 (b) at the best parameter using AutoDock.

Figure 3. Visualization of compound 2f (a) and ibuprofen (b) interactions at COX-1 using LigPlot⁺.

Figure 4: Visualization of compound 2c (a) and celecoxib (b) interactions at COX-2 using LigPlot⁺.

DISCUSSION:

The preparation route of bis-Mannich Base derivatives of curcumin pyrazole (CP) is presented in Figure 1. The compounds’ structures were elucidated by FTIR, NMR (1H and ¹³C), and mass spectra. FTIR of 1 exhibited N–H and C=N peaks at 3390 and 1617 cm^-1, indicating the cyclization of 1,3-diketone occurred to afford the pyrazole ring. In the ¹H-NMR, the appearance of one proton of NH as a singlet at 12.82ppm, two protons of OH as a singlet peak at 9.21ppm, one proton as a singlet peak of CH pyrazole, four protons as doublet peak of the aromatic ring at 6.77 and 6.92ppm, two protons as a singlet peak of the aromatic ring, and four protons as a doublet of CH ethylenic confirmed the structure of curcumin pyrazole. The FTIR of bis-Mannich base derivatives of CP (2a-f) exhibited an N-H peak at 3321 cm^-1, C-H aliphatic peak at 2970, 2934, 2834, 1409, and 1359 cm^-1, C=C peak at 1493 cm^-1, C–O of phenol peak at 1285 cm^-1, and (C–O ether) peak at 1085 cm^-1. Peaks of C-H, C-N, and C-O of morpholine ring of 2a appear at 2967-2848, 1155, and 1267 cm^-1; peaks of alkylamine of 2b, 2d, and 2f appear at 2972-2830 and 1158-1159 cm^-1; a peak of C-N pyrrolidine ring of 2e appears at 1087 cm^-1. The ¹H-NMR of 2a showed 8 protons peaks of the N-CH₂ morpholine ring at δ 2.58 ppm and 8 protons of the OCH₂-morpholine ring at δ 3.75 ppm. The protons of dibutyl amine chains of 2b appear at δ: 0.89 ppm (12H, -CH₃), 1.3 ppm (8H, -CH₂-), 1.54 ppm (8H, -CH₂-), and 2.52 ppm (8H, N-CH₂-). The protons of diethyl amine chains of 2d appear at δ: 1.09 ppm (12H, -CH₃); 2.63 ppm (8H, N-CH2). The protons of the pyrrolidine ring of 2e appear as two peaks at δ: 1.77-1.85 ppm (8H, C-CH₂-CH₂-C) and 2.58-2.64 ppm (8H, C-CH₂-N-CH₂-C). The protons of dimethylamine chains of 2f appear at δ 2.32 ppm (12H, N-(CH₃)₂) The protons of CP appear at δ: 3.88 ppm (6H, Ar-OCH₃), 6.53 ppm (1H, CH pyrazole ring), 6.71 ppm (2H, phenyl ring), 6.93 ppm (2H, phenyl ring), 6.88 ppm (2H, -CH- trans ethylenic), 6.99 ppm (2H, CH- trans ethylenic), and 6.93 ppm (1H, NH). While protons of CH₂ connected N to the aromatic ring, appear at δ 3.26-3.65 ppm. Furthermore, the compounds' ¹³C-NMR and mass spectroscopic data justified the targeted structures entirely.

All the synthesized compounds showed higher inhibitory protein denaturation activity than diclofenac sodium, curcumin, and CP (Table 2). It indicated that the cyclization of the 1,3-diketone group to be a pyrazole ring in curcumin and the addition of the bis-Mannich base group to the CP through aminomethylation increased the anti-inflammatory activity¹⁴. Compound 2a having morpholine Mannich base substitution showed the best activity (IC₅₀ = 0.506 μM). It is in line with the activity of the Mannich base derivative of ibuprofen and the mono-carbonyl analogs of curcumin reported earlier^26-27. Denaturation of protein was the early step for the following protein transformations—for example, glycosylation. In chronic inflammation, specific abnormal glycosylation was observed²⁸. Some compounds that showed the ability to inhibit heat-induced protein denaturation have also shown anti-inflammatory activity^29-31. Therefore, compounds exhibiting to inhibit protein denaturation are the potential to be anti-inflammatory drugs. Indeed, the mechanism of action of NSAIDs through the inhibition of cyclooxygenase enzymes is more trustworthy. However, protein denaturation inhibition also has a significant role^32-34.

All the synthesized compounds have binding energy against COX-2 lower than COX-1. Their selectivity score (S) indicated that the compounds were very selective against COX-2 (Table 2)³⁵. The compounds, curcumin, and celecoxib to COX-1 showed binding energy greater than ibuprofen (native ligand). Compound 2f having dimethylamine Mannich base substitution showed the lowest binding energy. The compound was fixed at the COX-1's binding pocket through the formation of hydrogen bonds to Arg83 (3.1Å) and hydrophobic bonds with Leu115, Val116, Arg120, Trp387, Tyr385, Tyr348, Leu384, Leu359, Tyr355, Leu352, Val349, Ala527, Gly526, Glu524, Ile523, and Met522 (Figure 3a). Meanwhile, ibuprofen was fixed at the COX-1's binding pocket by forming two hydrogen bonds to the carboxyl group (3.05Å) and Arg120 (2.77Å). Ibuprofen also interacted via hydrophobic bonds with Leu531, Ser530, Ser353, Ala527, Gyl526, Ile523, Phe518, Trp387, Leu384, Tyr355, Val349, and Val116 (Figure 3b). These results align with previous studies^35-36. Leu352, Leu353, Tyr355, Phe518, and Ile523 are considered essential in COX-1 inhibition³⁷. Except for compound 2b, the synthesized compound to COX-2 exhibited binding energy lower than curcumin and ibuprofen (native ligand), but only compound 2c had binding energy lower than celecoxib (COX-2 inhibitor agent). Compound 2c having 2,6-dimetylmorpholine Mannich base substitution showed the lowest binding energy. The compound was fixed at the COX-2's binding pocket through hydrophobic interaction with His90, Thr94, Gln193, Val350, His352, Ser354, Leu353, Gly355, Tyr356, His357, Trp388, Tyr386, Phe382, Leu385, Pro515, Ala517, Ser531, Ala528, Gly527, Val524, Met523, and Phe519 (Figure 4a). Meanwhile, celecoxib was fixed at the COX-2's binding pocket with binding pocket through three hydrogen bonds to His90 (3.14Å), Gln193 (2.51Å), and Leu353 (2.96Å), as well as hydrophobic bonds with Val350, Ser354, Tyr356, Leu 385, Trp388, Phe519, Val523, Val524, Gly527, and Ala528 (Figure 4b). It follows an earlier study³⁸. His352, Leu353, Tyr356, Phe519, and Val524 are thought to have a role in COX-2 inhibition³⁹.

CONCLUSION:

A series of Mannich base derivatives of curcumin pyrazole (2a-f) was successfully prepared and investigated for the potential of its anti-inflammatory activity in-vitro and in-silico. All the compounds showed higher protein denaturation inhibitory activity than diclofenac sodium. The compounds were fixed at the binding pocket of COX-1 and COX-2 enzymes. The binding energy of all the compounds to COX-2 was lower than that of COX-1. Their selectivity score (S) indicated that the compounds were very selective against COX-2. So, all the compounds possess high potential as anti-inflammatory agents, and further study is necessary to identify these compounds' safety and activity in vivo.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

We thank the Research and Development Directorate Universitas Indonesia, which granted funding for the study (No.: NKB-2091/UN2.RST/HKP.05.00/2020) and the Molecular Structure Analysis Laboratory, Institute Technology Bandung (ITB), Indonesia, and the Chemical Research Center LIPI, Serpong, Indonesia, for recording NMR and mass spectra.

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Received on 19.01.2023 Modified on 08.08.2023

Research J. Pharm. and Tech 2024; 17(4):1537-1543.

DOI: 10.52711/0974-360X.2024.00243