Novel chalcone/aryl carboximidamide hybrids as potent anti-inflammatory via inhibition of prostaglandin E2 and inducible NO synthase activities: design, synthesis, molecular docking studies and ADMET prediction

Abstract

Two series of chalcone/aryl carboximidamide hybrids 4a–f and 6a–f were synthesised and evaluated for their inhibitory activity against iNOS and PGE2. The most potent derivatives were further checked for their in vivo anti-inflammatory activity utilising carrageenan-induced rat paw oedema model. Compounds 4c, 4d, 6c and 6d were proved to be the most effective inhibitors of PGE2, LPS-induced NO production, iNOS activity. Moreover, 4c, 4d, 6c and 6d showed significant oedema inhibition ranging from 62.21% to 78.51%, compared to indomethacin (56.27 ± 2.14%) and celecoxib (12.32%). Additionally, 4c, 6a and 6e displayed good COX2 inhibitory activity while 4c, 6a and 6c exhibited the highest 5LOX inhibitory activity. Compounds 4c, 4d, 6c and 6d fit nicely into the pocket of iNOS protein (PDB ID: 1r35) via the important amino acid residues. Prediction of physicochemical parameters exhibited that 4c, 4d, 6c and 6d had acceptable physicochemical parameters and drug-likeness. The results indicated that chalcone/aryl carboximidamides 4c, 4d, 6c and 6d, in particular 4d and 6d, could be used as promising lead candidates as potent anti-inflammatory agents.

1. Introduction

Inflammation is a complex process in the host defence mechanism for the protection against injuries, microbial infections, and foreign substances^1,2. It includes various cellular and plasma regulators that limit its action at a critical time and place³. There are two types of inflammation, namely, acute and chronic inflammation. If the acute inflammation persists for a long time, it may lead to systemic or chronic serious inflammatory disorders that results in several damaging consequences on the host cells and tissues and consequently leading to the development of cardiovascular diseases and cancer³. Upon inflammatory stimulation, macrophages generate a diversity of inflammatory mediators such as prostaglandin E₂ (PGE₂) and nitric oxide (NO)⁴. PGE2 is an influential lipid inflammatory mediator produced via the COX pathway and distributed in human body^5,6. PGE2 is the most copious metabolic product which is responsible for the inflammatory-related disorders and shows a crucial role in vascular permeability, hyperalgesia and pyresis⁷. Moreover, nitric oxide (NO), an important free radical key inflammatory mediator in living organisms that has vital functions in the physiological and pathophysiological regulation mechanisms at cardiovascular, nervous and immunological systems⁸. The NO overproduction might responsible for the immunological pathology of macrophage-dependent inflammatory and degenerative diseases, together with cancer⁹. In mammals, NO is produced via l-arginine oxidation by NO synthase (NOS); additionally, there are three NOS enzyme isoforms have been detected: endothelial NOS, neuronal NOS and inducible NOS (iNOS)^10,11. Consequently, inhibition of iNOS-mediated NO and PGE₂ generation is a favourable therapeutic target in the discovery of effective anti-inflammatory agents for the therapy of inflammatory diseases¹⁰.

Notably, prolonged use of traditional non-steroidal anti-inflammatory drugs (NSAIDs), e.g. aspirin, indomethacin and ibuprofen promoted the appearance of several undesired side effects such as gastrointestinal irritation, ulceration and bleeding due to their inhibitory action on the gastroprotective prostanoids formed by COX-1 enzymes in the gastrointestinal tract^3,12 that highlighted the significance of the innovation of new potent and selective COX-2 inhibitors with less gastro-intestinal side effects and with more safety profile such as celecoxib and its analogues^13,14. Nevertheless, prolonged administration of selective COX-2 for inhibition of prostaglandins production stimulated the metabolism of arachidonic acid pathway to LOX enzyme leading to increased availability of substrate which consequently resulted in increased leukotriene production by lipoxygenase pathway that intensifies airway inflammation and exaggerates bronchoconstriction¹². Thus, efforts have been focussed on the design and innovation of novel anti-inflammatory drugs with minimum or no side effects as an alternative to non-selective NSAIDs that might be valuable for the managing of inflammatory diseases^3,12.

Chalcone is a type of open chain flavonoids with two aryl rings linked through a three-carbon propanone spacer. The α,β-unsaturated propanone fragment facilitates the conversion of chalcones to several classes of heterocyclic compounds¹⁵ such as flavonoids, iso flavonoids¹⁶, pyrazoles¹⁷, 2-pyrazolines^18,19, imidazoles and pyrimidines²⁰. Chalcones and their derivatives have attracted substantial research attention²¹ not only due to their ease of synthesis but also due to their varied and interesting biological activities such as HDAC inhibitory activity²², anticancer activities^23,24, antihypertensive²⁵, antimalarial^26,27, antioxidant^28,29, analgesic³⁰, anti-inflammatory³¹, antibacterial³², antifungal^33,34, antiulcer agents³⁵, parasitic protease inhibitors³⁶, antiviral³⁷, anti-tuberculosis³⁸ and as insulin mimetic in 3T3-L1 adipocyte³⁹.

Moreover, both natural and synthetic chalcones exert their the anti-inflammatory activities (Figure 1) against various therapeutic targets such as cyclooxygenase (COX-1 and COX-2), lipooxygenase (LOX), nitric oxide synthase (NOS), interleukins, expression of cell adhesion molecules (CAM) and prostaglandins (PGs)⁴⁰.

Figure 1.

Structures of anti-inflammatory chalcones (I–III), aryl carboximidamides (V–VII) and rational design of chalcone/aryl carboximidamides as novel iNOS/PGE2 inhibitors.

On the other hand, amidoxime derivatives exhibited notable biological activities as anti-inflammatory, antihyperglycemic, antimycobacterial, serotonergic inhibitory, muscarinic agonist and peptide inhibitory activities^12,41–45. It has been used as bioisostere for carboxylic and ester groups for the design of drugs having improved pharmacokinetic (PK) and pharmacodynamic (PD) properties⁴⁵. Recently, we have investigated the effects of a series of aryl carboximidamides appended Naproxen derivatives as dual acting COX-2/15-LOX inhibitors¹² (Figure 1). Among these estimated derivatives, IV and V were the targets having remarkable inhibitory potencies; furthermore, compound V is the most potent as COX-2 inhibitor with approximately 6.6-folds higher than the reference drug, celecoxib and compound V has bestowed with the strongest 15-LOX inhibitory activity. Moreover, and very recently we have studied the effects of aryl carboximidamides appended indomethacin as dual iNOS/PGE2 inhibitors¹⁰. Most of the target indomethacin/aryl carboximidamides displayed powerful inhibitory action against LPS-prompted NO production. Compounds VI and VII (Figure 1), showed significant in vivo anti-inflammatory activity. Notably, compound VI demonstrated inhibition to LPS-induced NO production, iNOS activity and PGE2 with IC₅₀ of 10.70 nM, 2.31 nM and 29 nM, respectively.

Encouraged by the previously mentioned information, herein, we report the synthesis and the in vivo anti-inflammatory screening of two series of chalcone/aryl carboximidamides 4a–f and 6a–f (Figure 1) as iNOS and PGE₂ inhibitors compared to indomethacin as standard well-known NSAID.

2. Experimental

2.1. Chemistry

2.1.1. General details (see Supporting information)

2.1.1.1. General procedure for the synthesis of N’-{2-[1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl]acetoxy}arylcarboximidamide (4a–f)

To a suspension of chalcones 3 or 5 (1.5 mmole) in 30 ml acetonitrile, CDI (1.8 mmole) was added and the mixture was allowed to stir at room temperature for 30–60 min. Then, amidoximes 2a–f (1.5 mmole) was added and stirring continued for further 3 h. After completion of the reaction (as monitored by TLC), the formed precipitate was collected by filtration, washed several times with cold acetonitrile, dried and recrystallized from acetonitrile to afford 4a–f and 6a–f.

2.1.1.2. N’-(2-{4-[(2E)-3-(4-Methoxyphenyl)prop-2-enoyl]phenoxy}acetoxy) benzenecarboximidamide (4a)

Yield 86%; white solid; m.p.: 164–166 °C. IR (ATR) ν_max 3444, 3322 (NH₂), 3067 (C–H aromatic), 2996, 2967, 2834 (C–H aliphatic), 1755 (C=O), 1651 (C=O), 1627 (C=N) cm⁻¹. ¹H NMR (400 MHz, DMSO-d₆) δ 3.83 (s, 3H, OCH₃), 5.15 (s, 2H, CH₂), 6.96 (s, 2H, NH₂), 7.02 (d, J = 8.4 Hz, 2H, CH_arom.), 8.15 (d, J = 8.6 Hz, 2H, CH_arom.), 7.45–7.53 (m, 3H, CH_arom.), 7.68–7.85 (m, 6H, CH_arom.), 8.16 (d, J = 8.6 Hz, 2H, CH_arom.); ¹³C NMR (100 MHz, DMSO-d₆) δ 55.8, 64.8, 114.9, 115.0, 120.1, 127.3, 127.6, 127.9, 128.9, 129.8, 131.1, 131.7, 131.8, 143.7, 158.1, 161.7, 161.9, 167.2, 187.9; -ESI-MS (m/z): 429.4, [M-H]⁻; Anal. Calcd. for C₂₅H₂₂N₂O₅ (430.45): C, 69.76; H, 5.15; N, 6.51. Found: C, 69.58; H, 5.25; N, 6.38.

2.1.1.3. N’-(2-{4-[(2E)-3-(4-Methoxyphenyl)prop-2-enoyl]phenoxy}acetoxy)-4-chlorobenzenecarboximidamide (4b)

Yield 88%; white solid; m.p.: 180–182 °C. IR (ATR) ν_max 3508, 3379 (NH₂), 3073, 3052 (C–H aromatic), 2998, 2928, 2833 (C–H aliphatic), 1763 (C=O), 1654 (C=O), 1621 (C=N) cm⁻¹. ¹H NMR (400 MHz, DMSO-d₆) δ 3.83 (s, 3H, OCH₃), 5.14 (s, 2H, CH₂), 7.2 (d, J = 8.4 Hz, 4H, NH₂ + CH_arom.), 7.14 (d, J = 8.7 Hz, 2H, CH_arom.), 7.55 (d, J = 8.4 Hz, 2H, CH_arom.), 7.69 (d, J = 15.4 Hz, 1H, CH_arom.), 7.76–7.85 (m, 5H, CH_arom.), 8.16 (d, J = 8.7 Hz, 2H, CH_arom.); ¹³C NMR (100 MHz, DMSO-d₆) δ 55.8, 64.7, 114.9, 115.0, 120.0, 129.0, 129.1, 129.4, 130.0, 131.1, 131.3, 135.9, 137.1, 143.9, 157.2, 161.8, 161.9, 176.2, 187.9; -ESI-MS (m/z): 463.3, [M-H]⁻; Anal. Calcd. for C₂₅H₂₁ClN₂O₅ (464.89): C, 64.59; H, 4.55; N, 6.03. Found: C, 64.47; H, 4.39; N, 6.08.

2.1.1.4. N’-(2-{4-[(2E)-3-(4-Methoxyphenyl)prop-2-enoyl]phenoxy}acetoxy)-4-methoxybenzenecarboximidamide (4c)

Yield 89%; white solid; m.p.: 167–169 °C. IR (ATR) ν_max 3441, 3318 (NH₂), 3063, 3004 (C–H aromatic), 2968, 2935, 2840 (C–H aliphatic), 1754 (C=O), 1653 (C=O), 1629 (C=N) cm⁻¹. ¹H NMR (400 MHz, DMSO-d₆) δ 3.81 (s, 3H, OCH₃), 3.83 (s, 3H, OCH₃), 5.14 (s, 2H, CH₂), 6.85 (s, 2H, NH₂), 7.02 (d, J = 8.2 Hz, 4H, CH_arom.), 7.14 (d, J = 8.0 Hz, 2H, CH_arom.), 7.68–7.85 (m, 6H, CH_arom.), 8.16 (d, J = 8.0 Hz, 2H, CH_arom.); ¹³C NMR (100 MHz, DMSO-d₆) δ 55.7, 55.8, 64.8 (exchangeable with dept-135), 114.2, 114.9, 115.0, 120.1, 123.8, 127.9, 128.8, 131.1, 131.2, 131.8, 143.7, 157.7, 161.6, 161.7, 162.0, 167.2, 187.9; -ESI-MS (m/z): 459.2, [M-H]⁻; Anal. Calcd. for C₂₆H₂₄N₂O₆ (460.47): C, 67.82; H, 5.25; N, 6.08. Found: C, 67.70; H, 5.46; N, 6.14.

2.1.1.5. N’-(2-{4-[(2E)-3-(4-Methoxyphenyl)prop-2-enoyl]phenoxy}acetoxy)-3,4-dimethoxybenzenecarboximidamide (4d)

Yield 88%; white solid; m.p.: 150–152 °C. IR (ATR) ν_max 3451, 3329 (NH₂), 3072 (C–H aromatic), 2978, 2936, 2839 (C–H aliphatic), 1755 (C=O), 1654 (C=O), 1626 (C=N) cm⁻¹. ¹H NMR (400 MHz, DMSO-d₆) δ 3.78 (s, 9H, 3OCH₃), 5.08 (s, 2H, CH₂), 6.80 (s, 2H, NH₂), 6.98–7.01 (m, 3H, CH_arom.), 7.12 (d, J = 8.3 Hz, 2H, CH_arom.), 7.26 (s, 1H, CH_arom.), 7.31 (d, J = 8.4 Hz, 1H, CH_arom.), 7.67 (d, J = 6.0 Hz, 2H, CH_arom.), 7.76 (d, J = 8.2 Hz, 2H, CH_arom.), 8.10 (d, J = 8.3 Hz, 2H, CH_arom.); ¹³C NMR (100 MHz, DMSO-d₆) δ 55.8, 56.01, 56.03, 64.7, 110.5, 111.7, 114.9, 115.0, 119.9, 120.2, 123.8, 127.9, 131.18, 131.22, 131.7, 143.8, 148.8, 151.3, 157.9, 161.7, 162.0, 167.3, 187.8, 191.8; -ESI-MS (m/z): 489.5, [M-H]⁻; Anal. Calcd. for C₂₇H₂₆N₂O₇ (490.50): C, 66.11; H, 5.34; N, 5.71. Found: C, 66.01; H, 5.55; N, 5.62.

2.1.1.6. N’-(2-{4-[(2E)-3-(4-Methoxyphenyl)prop-2-enoyl]phenoxy}acetoxy)-4-nitrobenzenecarboximidamide (4e)

Yield 84%; pale yellow solid; m.p.: 184–186 °C. IR (ATR) ν_max 3508, 3374 (NH₂), 3074, 3019 (C–H aromatic), 2979, 2935, 2840 (C–H aliphatic), 1774 (C=O), 1655 (C=O), 1627 (C=N) cm⁻¹. ¹H NMR (400 MHz, DMSO-d₆) δ 3.83 (s, 3H, OCH₃), 5.18 (s, 2H, CH₂), 7.02 (d, J = 8.6 Hz, 2H, CH_arom.), 7.15 (d, J = 8.7 Hz, 2H, CH_arom.), 7.25 (s, 2H, NH₂), 7.67–7.84 (m, 4H, CH_arom.), 8.03 (d, J = 8.7 Hz, 2H, CH_arom.), 8.16 (d, J = 8.8 Hz, 2H, CH_arom.), 8.32 (d, J = 8.8 Hz, 2H, CH_arom.); ¹³C NMR (100 MHz, DMSO-d₆) δ 55.8, 64.7, 114.9, 115.0, 120.1, 124.0, 127.9, 128.8, 131.1, 131.2, 131.9, 137.8, 143.7, 149.3, 156.6, 161.7, 161.9, 167.1, 187.9; -ESI-MS (m/z): 474.2, [M-H]⁻; Anal. Calcd. for C₂₅H₂₁N₃O₇ (475.45): C, 63.15; H, 4.45; N, 8.84. Found: C, 63.42; H, 4.23; N, 8.78.

2.1.1.7. N’-(2-{4-[(2E)-3-(4-Methoxyphenyl)prop-2-enoyl]phenoxy}acetoxy) naphthalene-2-carboximidamide (4f)

Yield 85%; beige solid; m.p.: 176–178 °C. IR (ATR) ν_max 3437, 3319 (NH₂), 3056 (C–H aromatic), 2995, 2966, 2933, 2834 (C–H aliphatic), 1754 (C=O), 1652 (C=O), 1636 (C=N) cm⁻¹. ¹H NMR (400 MHz, DMSO-d₆) δ 3.83 (s, 3H, OCH₃), 5.20 (s, 2H, CH₂), 7.02 (d, J = 8.1 Hz, 2H, CH_arom.), 7.11 (s, 2H, NH₂), 7.17 (d, J = 8.3 Hz, 2H, CH_arom.), 7.59–7.61 (m, 2H, CH_arom.), 7.69–7.88 (m, 5H, CH_arom.), 7.97–8.03 (m, 3H, CH_arom.), 8.18 (d, J = 8.3 Hz, 2H, CH_arom.), 8.37 (s, 1H, CH_arom.); ¹³C NMR (100 MHz, DMSO-d₆) δ 55.8, 64.8, 114.9, 115.1, 120.1, 124.4, 127.1, 127.2, 127.8, 127.9, 128.1, 128.4, 129.0, 129.1, 131.1, 131.2, 131.9, 132.8, 134.4, 143.7, 158.0, 161.7, 162.0, 167.2, 187.9; -ESI-MS (m/z): 479.3, [M-H]⁻; Anal. Calcd. for C₂₉H₂₄N₂O₅ (480.51): C, 72.49; H, 5.03; N, 5.83. Found: C, 72.58; H, 4.89; N, 5.78.

2.1.1.8. N’-(2-{4-[(1E)-3-(4-Methoxyphenyl)-3-oxoprop-1-en-1-yl] phenoxy}acetoxy) benzenecarboximidamide (6a)

Yield 82%; white solid; m.p.: 172–173 °C. IR (ATR) ν_max 3399, 3302 (NH₂), 3045 (C–H aromatic), 2984, 2896 (C–H aliphatic), 1745 (C=O), 1666 (C=O), 1626 (C=N) cm⁻¹. ¹H NMR (400 MHz, DMSO-d₆) δ 3.81 (s, 3H, OCH₃), 5.04 (s, 2H, CH₂), 7.07 (s, 2H, NH₂), 7.20–7.22 (d, J = 7.9 Hz, 2H, CH_arom.), 7.29–7.72 (m, 6H, CH_arom.), 7.81–7.87 (m, 5H, CH_arom.), 8.01–8.02 (d, J = 7.8 Hz, 2H, CH_arom.); ¹³C NMR (100 MHz, DMSO-d₆) δ 55.2, 63.8, 113.9, 115.2, 121.2, 125.2, 125.9, 127.9, 128.8, 130.2, 131.1, 131.6, 131.9, 144.6, 158.5, 161.3, 163.0, 167.1, 179.8; -ESI-MS (m/z): 429.3, [M-H]⁻; Anal. Calcd. for C₂₅H₂₂N₂O₅ (430.45): C, 69.76; H, 5.15; N, 6.51. Found: C, 69.65; H, 5.00; N, 6.41.

2.1.1.9. N’-(2-{4-[(1E)-3-(4-Methoxyphenyl)-3-oxoprop-1-en-1-yl] phenoxy}acetoxy) -4-chlorobenzenecarboximidamide (6b)

Yield 85%; white solid; m.p.: 192–193 °C. IR (ATR) ν_max 3406, 3343 (NH₂), 3062 (C–H aromatic), 2983, 2875 (C–H aliphatic), 1758 (C=O), 1663 (C=O), 1629 (C=N) cm⁻¹. ¹H NMR (400 MHz, DMSO-d₆) δ 3.82 (s, 3H, OCH₃), 4.98 (s, 2H, CH₂), 7.00–7.29 (m, 6H, NH₂ + CH_arom.), 7.49–7.51 (d, J = 8.1 Hz, 2H, CH_arom.), 7.70–7.91 (m, 6H, CH_arom.), 8.21–8.23 (d, J = 8.4 Hz, 2H, CH_arom.); ¹³C NMR (100 MHz, DMSO-d₆) δ 55.5, 64.7, 113.5, 114.8, 115.7, 118.6, 122.3, 127.4, 130.4, 131.5, 132.7, 133.4, 139.8, 147.2, 147.8, 161.3, 162.9, 169.3, 179.9; -ESI-MS (m/z): 463.4, [M-H]⁻; Anal. Calcd. for C₂₅H₂₁ClN₂O₅ (464.89): C, 64.59; H, 4.55; N, 6.03. Found: C, 64.69; H, 4.27; N, 6.11.

2.1.1.10. N’-(2-{4-[(1E)-3-(4-Methoxyphenyl)-3-oxoprop-1-en-1-yl] phenoxy} acetoxy)-4-methoxybenzenecarboximidamide (6c)

Yield 83%; white solid; m.p.: 186–187 °C. IR (ATR) ν_max 3422, 3365 (NH₂), 3055, 3010 (C–H aromatic), 2989, 2873 (C–H aliphatic), 1751 (C=O), 1659 (C=O), 1631 (C=N) cm⁻¹. ¹H NMR (400 MHz, DMSO-d₆) δ 3.83 (s, 3H, OCH₃), 3.85 (s, 3H, OCH₃), 5.06 (s, 2H, CH₂), 6.97–7.07 (m, 6H, NH₂ + CH_arom.), 7.12–7.14 (d, J = 8.3 Hz, 2H, CH_arom.), 7.27–7.29 (d, J = 8.0 Hz, 2H, CH_arom.), 7.92–8.05 (m, 6H, CH_arom.); ¹³C NMR (100 MHz, DMSO-d₆) δ 55.8, 55.9, 64.5, 113.5, 114.8, 115.2, 117.1, 124.2, 127.2, 128.8, 131.0, 131.4, 131.9, 153.1, 158.4, 159.4, 161.4, 162.7, 169.9, 180.1; -ESI-MS (m/z): 459.4 [M-H]⁻; Anal. Calcd. for C₂₆H₂₄N₂O₆ (460.47): C, 67.82; H, 5.25; N, 6.08. Found: C, 67.62; H, 5.08; N, 6.02.

2.1.1.11. N’-(2-{4-[(1E)-3-(4-Methoxyphenyl)-3-oxoprop-2-en-1-yl] phenoxy} acetoxy) -3,4-dimethoxybenzenecarboximidamide (6d)

Yield 82%; white solid; m.p.: 172–174 °C. IR (ATR) ν_max 3462, 3378 (NH₂), 3058 (C–H aromatic), 2987, 2947, 2845 (C–H aliphatic), 1744 (C=O), 1652 (C=O), 1627 (C=N) cm⁻¹. ¹H NMR (400 MHz, DMSO-d₆) δ 3.85 (s, 3H, OCH₃), 3.89 (s, 3H, OCH₃), 3.92 (s, 3H, OCH₃), 5.15 (s, 2H, CH₂), 6.96 (s, 2H, NH₂), 7.08 (t, J = 8 Hz, 3H, CH_arom), 7.13 (t, J = 8 Hz, 3H, CH_arom.), 7.34 (s, 1H, CH_arom.), 7.39 (d, J = 8 Hz, 1H, CH_arom), 7.69–7.79 (m, 1H, CH_arom.), 7.93 (d, J = 8 Hz, 2H, CH_arom.), 8.22 (d, J = 8 Hz, 2H, CH = CH); ¹³C NMR (100 MHz, DMSO-d₆) δ 56.0, 56.1, 64.6, 110.5, 111.7, 114.5, 115.4, 116.7, 120.1, 120.3, 123.8, 128.6, 131.1, 131.3, 132.2, 135.6, 137.3, 143.4, 148.8, 151.2, 157.8, 160.1, 163.6, 167.4, 187.7; -ESI-MS (m/z): 489.5, [M-H]⁻; Anal. Calcd. for C₂₇H₂₆N₂O₇ (490.50): C, 66.11; H, 5.34; N, 5.71. Found: C, 66.23; H, 5.27; N, 5.64.

2.1.1.12. N’-(2-{4-[(1E)-3-(4-Methoxyphenyl)-3-oxoprop-2-en-1-yl] phenoxy} acetoxy)-4-nitrobenzenecarboximidamide (6e)

Yield 89%; pale yellow solid; m.p.: 193–194 °C. IR (ATR) ν_max 3452, 3364 (NH₂), 3075, 3022 (C–H aromatic), 2989, 2932, 2844 (C–H aliphatic), 1769 (C=O), 1660 (C=O), 1628 (C=N) cm⁻¹. ¹H NMR (400 MHz, DMSO-d₆) δ 3.84 (s, 3H, OCH₃), 5.12 (s, 2H, CH₂), 6.99–7.01 (d, J = 8.3 Hz, 2H, CH_arom.), 7.12–7.20 (m, 4H, NH₂ + CH_arom.), 7.71–7.80 (m, 4H, CH_arom.), 8.07–8.09 (d, J = 8.4 Hz, 2H, CH_arom.), 8.13–8.15 (d, J = 8.7 Hz, 2H, CH_arom.), 8.29–8.31 (d, J = 8.7 Hz, 2H, CH_arom.); ¹³C NMR (100 MHz, DMSO-d₆) δ 55.6, 64.8, 114.9, 115.4, 123.1, 125.5, 128.3, 128.9, 131.1, 131.6, 132.4, 135.3, 143.7, 148.6, 156.6, 161.8, 163.2, 167.4, 178.7; -ESI-MS (m/z): 474.4, [M-H]⁻; Anal. Calcd. for C₂₅H₂₁N₃O₇ (475.45): C, 63.15; H, 4.45; N, 8.84. Found: C, 63.36; H, 4.30; N, 8.90.

2.1.1.13. N’-(2-{4-[(1E)-3-(4-Methoxyphenyl)-3-oxoprop-2-en-1-yl] phenoxyacetoxy)naphthalene-2-carboximidamide (6f)

Yield 86%; beige solid; m.p.: 178–179 °C. IR (ATR) ν_max 3427, 3353 (NH₂), 3057 (C–H aromatic), 2992, 2952, 2854 (C–H aliphatic), 1752 (C=O), 1653 (C=O), 1633 (C=N) cm⁻¹. ¹H NMR (400 MHz, DMSO-d₆) δ 3.80 (s, 3H, OCH₃), 5.15 (s, 2H, CH₂), 7.05–7.12 (m, 4H, NH₂ + CH_arom.), 7.18–7.20 (d, J = 8.1 Hz, 2H, CH_arom.), 7.60–7.76 (m, 7H, CH_arom.), 7.94–8.00 (m, 3H, CH_arom.), 8.16–8.18 (d, J = 8.5 Hz, 2H, CH_arom.), 8.39 (s, 1H, CH_arom.); ¹³C NMR (100 MHz, DMSO-d₆) δ 55.8, 64.9, 114.2, 114.9, 118.3, 125.4, 126.7, 126.9,127.5, 127.6, 128.3, 128.5, 129.0, 129.6, 131.1, 131.3, 131.9, 132.8, 134.6, 143.3, 149.6, 160.0, 162.0, 167.6, 180.1; -ESI-MS (m/z): 479.2, [M-H]⁻; Anal. Calcd. for C₂₉H₂₄N₂O₅ (480.51): C, 72.49; H, 5.03; N, 5.83. Found: C, 72.64; H, 4.82; N, 6.01.

2.2. Biology

2.2.1. Cytotoxicity assay

MTT assay was used to assess the cell viability of RAW 264.7 cells cultured in RPMI-1640 complete media. The cells were treated with the investigated compounds two hours prior to 1 µg/ml of lipopolysaccharide (LPS) stimulation for 18 h. Afterwards, 5 µl of MTT solution were added and incubated for further 4 h. Finally, 150 ul of dimethyl sulfoxide (DMSO) were added and the optical density was assessed at 570 nm using an ELISA plate reader⁴⁶.

2.2.2. Griess assay for NO release determination

Equal volumes of Griess reagent and the supernatant, obtained from treated RAW264.7 cells with the compounds 2 h before LPS induction, were mixed for 10 min at room temperature in the dark. The absorbance was measured at 540 nm using ELISA plate reader and the nitrite concentration was calculated from sodium nitrite standard curve. The percentage inhibition (%) is calculated as absorbance at 540 nm of (LPS-compounds)/absorbance at 540 nm of (LPS-control) ×100^47,48.

2.2.3. Determination of iNOS enzymatic activity

Two hours after treatment with 2–50 µM of 4c, 4d, 6a, 6c, 6d, 6e, indomethacin, and 1 µg/ml of LPS at 37 °C, the culture media was replaced by 100 µl of NOS assay buffer (1×). Then, the NOS assay reaction solution (100 µl/well) was added and incubated for an extra 2 h at 37 °C. The fluorescence with excitation wavelength at 485 nm and emission wavelength at 528 nm was measured using a fluorescent microplate reader⁴⁹.

2.2.4. Assessment of prostaglandin E2 concentration

After seeding and incubation of RAW 264.7 cells for 24 h, then the cells were treated with the compounds (4c, 4d, 6a, 6c, 6d and 6e) at different concentrations and with LPS (1 µg/ml) for another 24 h. The concentration of prostaglandin E2 (PGE2) was measured in the culture media using ELISA kit (R&D Systems, Minneapolis, MN)⁵⁰.

2.2.5. In vitro cyclooxygenase (COX) inhibition assay

The colorimetric COX-1/COX-2 inhibition assay kit (kit catalogue number 560101, Cayman Chemical, Ann Arbour, MI) was used following the manufacturer’s instructions to test the ability of the test compounds and celecoxib to inhibit COX-1/COX-2 isozymes^3,12.

2.2.6. In vitro 5-lipoxygenase (LOX) inhibition assay

The 5-LOX inhibition assay kit (kit catalogue number 760700, Cayman Chemical, Ann Arbour, MI) was used following the manufacturer’s instructions to test the ability of the test compounds and NDGA to inhibit 5-LOX enzyme^3,12.

2.2.7. Carrageenan-induced paw edoema

The anti-inflammatory activity of the inspiring active compounds in vitro (4c, 4d, 6a, 6c, 6d and 6e) were further estimated in vivo using the carrageenan-induced paw edoema test. The paw edoema was induced by a single injection of 1% w/v carrageenan (1 g dissolved in 100 ml saline) into the left hind paw. The paw thickness was assessed using Vernier calliper after carrageenan injection for 1,2, 3 and 4 h. Wistar albino rats (males weighing 120–140 g, six rats per group) were grouped as follows: Group 1 (Control) rats were received the vehicle. Groups 2 and 3 (standard-treated) rats were given 50 mg/kg oral dose of celecoxib or indomethacin as standard anti-inflammatory drugs. Groups 4–9 (compound-treated) each group of rats has orally administered one compound (4c, 4d, 6a, 6c, 6d or 6e) at a dose of 50 mg/kg 1 h before paw edoema induction. The percentage inhibition of edoema thickness was calculated at each time interval in comparison to the control group⁵¹.

2.2.8. Acute oral toxicity experiment

The compounds (4c, 4d, 6a, 6c, 6d and 6e) were examined for the possible acute oral toxicity after their oral administration to male mice at doses of 100, 200, 300, 400 and 500 mg/kg, respectively. Twenty-four hours later, mice were observed for any signs of toxicity, and dead mice were recorded. The median lethal dose (LD₅₀) was calculated according to Litchfield and Wilcoxon method⁵².

2.3. Docking methodology

For molecular docking analysis, Discovery Studio 2.5 software (Accelrys Inc., San Diego, CA) was used. The crystal structures of iNOS protein (PDB code: 1r35) was retrieved from protein data bank^1,10,53–56. See Supplementary file.

2.4. In silico prediction of physicochemical properties and pharmacokinetic profile

For Lipinski’s rule (rule of five) and molecular property prediction, the free accesses to website (https://www.molsoft.com/servers.html) was used. Also, for Pre-ADMET estimation, the free access of website (https://preadmet.bmdrc.kr/) was utilised for estimation.

Approval and the corresponding ethical approval code.

3. Results and discussion

3.1. Chemistry

The chemical synthetic approach of the target chalcone/aryl carboximidamides 4a–f and 6a–f is outlined in Scheme 1. The intermediate aryl cyanides 1a–f, amidoximes 2a–f and the key chalcones acid 3 and 5 were prepared according to the previously reported procedure^10,12,22,57. Reacting the synthesised amidoximes 2a–f with the carboxylic acid group of chalcone 3 and 5 using carbonyldiimidazole (CDI) in acetonitrile gave the chalcone/aryl carboximidamides 4a–f and 6a–f, respectively, in good yield. The structure of the newly synthesised chalcone/aryl carboximidamides 4a–f and 6a–f was characterised by IR, ¹H NMR,¹³CNMR spectra and elemental analyses. The IR spectrum of 4b, as an example, displayed characteristic absorption bands at 3379, 3508 (NH₂); 1763 (C=O), 1654 (C=O_enone) and 1621(C=N) cm⁻¹. While its ¹H NMR spectrum indicates two singlet signals at δ 3.83 and 5.14 ppm assigned to methoxy and methylene protons, respectively. The amidoxime NH₂ appeared at 7.01–7.03 ppm and the aromatic protons appeared in their expected chemical shifts. In addition, a characteristic doublet signal at δ 7.67–7.71 ppm assigned to be one of olefin hydrogen with coupling constant 15.4 Hz, which confirm the E configuration. The ¹³C NMR spectra as well as elemental analyses results are consistent with the suggested structures (see Supporting Information).

Scheme 1.

Synthesis of the target compounds 4a–f and 6a–f. Reagents and conditions: (i) I₂, NH₃, THF, rt, 1–2 h; (ii) NH₂OH.HCl, K₂CO₃, MeOH, reflux; 5–8 h; (iii) CDI, CH₃CN, rt, 3 h.

The IR spectrum of 6d as a typical example of this sets revealed characteristic bands at 3462, 3378 (NH₂); 1744 (C=O), 1652 (C=O_enone) and 1627 (C=N) cm⁻¹. ¹H NMR spectrum revealed two singlet signals at δ 3.81 and 3.87 three methoxy and methylene protons, and 5.14 ppm assigned to methylene protons. The aromatic protons appeared in their expected chemical shifts while the amidoxime NH₂ appeared at 6.84–6.86 ppm. In addition, a characteristic doublet signal at δ 7.67–7.71 ppm assigned to be one of olefin hydrogen with coupling constant 15.4 Hz, which confirm E configuration. The ¹³C NMR data and elemental microanalyses data are consistent with the expected structures.

3.2. Biology

3.2.1. Determination of the cytotoxicity

First, the cytotoxic effect of the synthesised target compounds 4a–f and 6a–f on cell growth and proliferation were evaluated. As illustrated in Figure 2, treatment of RAW 264.7 cells with 5 µM of compounds 2 h before induction with LPS did not show any noticeable cytotoxicity in comparison with untreated control cells as well as LPS-treated cells. Accordingly, other bioactivities were further assessed.

Figure 2.

Cytotoxicity assessment of treatment with 4a–f and 6a–f compounds on RAW 264.7 cells, two hours before the induction with LPS. Cell viability was performed using MTT assay. Data are shown as mean ± SD (n = 3). *^,** (p < 0.05) compared to untreated control cells and LPS-induced cells, respectively.

3.2.2. Inhibitory activity of NO production

The important pro-inflammatory mediator, nitric oxide (NO), plays a vital role in the pathogenesis of several inflammatory diseases. Moreover, studies have demonstrated a positive link between the exaggerated concentration of NO and the severity of the disease serving NO as a potential biomarker in the evaluation of the inflammatory process. Therefore, NO inhibitors are essential therapies in the management of inflammatory disorders^58–60.

Hence, we investigated the potential inhibitory effect of the two series on the over-production of NO from LPS-stimulated macrophages. As demonstrated in Table 1, compounds 4a–f and 6a–f at 5 µM showed more potent inhibition of NO production over the used standard drug, indomethacin (inhibitory rate= 25.5 ± 1.6%). Among the two series, compounds 4d and 6d were the most effective NO inhibitors with inhibition rates of 73.5 ± 2.1 and 78.2 ± 3.4%, respectively. On the contrary, compounds 4f and 6f were the least effective NO inhibitors displaying inhibitory rates of 31.2 ± 2.2 and 29.4 ± 2.5%, respectively. Compounds 4a, 4b, 4c, 4e, 6a, 6b, 6c and 6e displayed promising NO inhibition activity with inhibitory rates varying from 41.9% to 67.1%.

Table 1.

The inhibition rates of NO release from RAW 264.7 cells after treatment with compounds 4a–f and 6a–f.

Compounds (5 µM)	NO Inhibition rates (%) ± SD	Compounds (5 µM)	NO Inhibition rates (%) ± SD
Indomethacin 25.5 ± 1.6
4a	41.9 ± 1.4	6a	55.5 ± 2.4
4b	47.6 ± 3.6	6b	49.6 ± 2.3
4c	62.3 ± 1.9	6c	67.1 ± 2.4
4d	73.5 ± 2.1	6d	78.2 ± 3.4
4e	44.1 ± 2.8	6e	57.9 ± 1.3
4f	31.2 ± 2.2	6f	29.4 ± 2.5

3.2.3. Inhibitory activity of iNOS and PGE2 production

NO is a core signalling mediator involved in the inflammation through iNOS up-regulation with the subsequent triggering of PGE2 induction and overstated inflammation^10,61. Therefore, compounds (4c, 4d, 6a, 6c, 6d and 6e) demonstrated an excellent inhibition rate of NO release from RAW 264.7 cells, were evaluated for potential inhibition on iNOS activity, NO, and PGE2 production.

As shown in Table 2, the treatment of LPS-induced RAW 264.7 cells with the selected six compounds resulted in a remarkable inhibition of iNOS activity (IC₅₀ range from 1.91 to 7.15 µM) and NO (IC₅₀ range = 4.36–15.80 µM) compared to indomethacin (IC₅₀ 24.57 µM and 45.69 µM, respectively. Interestingly, the six compounds presented more inhibitory activity on the PGE2 production (IC₅₀ range = 21.24–48.11 nM) in comparison with indomethacin (IC₅₀ = 76.58 nM).

Table 2.

NO, iNOS, PGE2 inhibitory activity (IC₅₀) of compounds 4c, 4d, 6a, 6c, 6d and 6e and their cytotoxicity on RAW 264.7 cells.

Compound	NO IC50 (µM)	iNOS IC50 (µM)	PGE2 IC50 (nM)	Cytotoxicity IC50 (µM)
				without LPS	with LPS
Indomethacin	45.69	24.57	76.58	>80	>80
4c	8.61	3.62	32.30	>80	>80
4d	5.73	2.22	24.72	>80	>80
6a	13.55	6.32	48.11	>80	>80
6c	5.92	2.45	27.51	>80	>80
6d	4.36	1.91	21.24	>80	>80
6e	15.80	7.15	45.14	>80	>80

Finally, the six active compounds were further evaluated for potential cytotoxicity using their inhibitory concentrations on NO, iNOS and PGE2 and the cell viability was assessed by MTT assay. Fortunately, the six compounds at the same inhibitory concentrations did not show notable cytotoxicity against RAW 264.7 cells with or without LPS presenting IC₅₀ values >80 µM. This study proved that these compounds had promising inhibitory effects on LPS-stimulated inflammatory response without exerting cytotoxicity.

3.2.4. In vitro COX-1/COX-2 inhibition assay

The six active compounds 4c, 4d, 6a, 6c, 6d and 6e were evaluated against both bovine COX-1 and COX-2 subtypes using enzyme immunoassay (EIA) kit using celecoxib as a reference drug. The IC₅₀ values of the tested compounds along with their selectivity index (SI)^3,12 are listed in Table 3. The obtained results revealed that compounds 4c, 6a, 6c and 6e are the most active COX-2 inhibitors with IC₅₀ values of 3.279, 1.103, 8.263, 1.714 µM, respectively, compared to their COX-1 inhibitory activity (IC₅₀ values of 19.88, 20.91, 64.41, 12.73 µM, respectively). Additionally, 6a displayed the highest SI value of 19 compared to that of celecoxib (SI = 175.49). Unexpectedly, compounds 4d and 6d were proved to be more selective COX-1 inhibitor (IC₅₀ = 19.88 and 17.82 µM, respectively) compared to their COX-2 inhibitory activity (IC₅₀ = 23.4 and 102.7 µM, respectively).

Table 3.

In vitro COX1, COX2 and 5LOX inhibitory activity (IC₅₀) of compounds 4c, 4d, 6a, 6c, 6d and 6e.

Compound	COX 1 IC50 (µM)	COX 2 IC50 (µM)	SI	5LOX IC50
				(µM)
4c	19.88 ± 1.03	3.279 ± 0.16	6.06	8.136 ± 0.5
4d	5.824 ± 0.3	23.4 ± 1.13	0.25	16.47 ± 1
6a	20.91 ± 1.09	1.103 ± 0.05	19	6.126 ± 0.4
6c	64.41 ± 3.35	8.263 ± 0.4	7.79	3.186 ± 0.2
6d	17.82 ± 0.93	102.7 ± 4.97	0.17	9.877 ± 0.6
6e	12.73 ± 0.66	1.714 ± 0.08	7.43	54.15 ± 3.2
Celecoxib	35.8 ± 1.25	0.204 ± 0.06	175.49	89.4 ± 1.16
NDGA	na	na	na	2.96 ± 0.2

3.2.5. In vitro 5-LOX inhibition assay

The six active compounds 4c, 4d, 6a, 6c, 6d and 6e were evaluated for their 5-LOX inhibitory activity. The IC₅₀ values of the six compounds were determined and illustrated in Table 3. The results disclosed that compound 6c exhibited good inhibitory activity towards 5-LOX enzyme (IC₅₀ = 3.186 µM) compared to nordihydroguaiaretic acid (NDGA) (IC₅₀ = 2.96 µM). Additionally, compounds 4c, 4d, 6a and 6d showed moderate ability to inhibit 5-LOX enzyme with IC₅₀ values of 8.136, 16.4, 6.126 and 9.877 µM, respectively. Moreover, compound 6e showed the weakest 5-LOX inhibitory activity (IC₅₀ =54.15 µM).

3.2.6. In vivo anti-inflammatory activity

Compounds that displayed promising in vitro bioactivities (4c, 4d, 6a, 6c, 6d and 6e) were further assessed for in vivo anti-inflammatory activity using the carrageenan-induced rat paw edoema model. Celecoxib and indomethacin were used as standard anti-inflammatory drugs. As shown in Table 4, the paw thickness was measured at 1, 2, 3 and 4 h following oedema induction, and the results were demonstrated as percentage oedema inhibition.

Table 4.

The anti-inflammatory activities of compounds 4a, 4d, 6a, 6c, 6d, 6e, indomethacin, and celecoxib against in vivo model of carrageenan-induced paw edoema.

% of edoema inhibition (% mean ± SEM)a
Compound	1 h	2 h	3 h	4 h
Control	0.00	0.00	0.00	0.00
4c	11.21 ± 2.41	33.62 ± 2.69	57.71 ± 2.21	69.86 ± 3.51
4d	11.65 ± 2.26	34.78 ± 3.34	69.50 ± 1.49	54.21 ± 3.47
6a	14.36 ± 3.11	24.33 ± 2.26	45.83 ± 2.74	55.74 ± 2.59
6c	11.42 ± 1.75	28.44 ± 2.75	54.36 ± 2.34	62.21 ± 3.46
6d	13.25 ± 2.04	38.43 ± 3.62	65.31 ± 2.64	78.53 ± 3.28
6e	10.23 ± 3.17	19.34 ± 2.45	39.26 ± 18	33.21 ± 3.15
Indomethacin	28.03 ± 1.35	45.27 ± 2.48	63.71 ± 3.48	56.27 ± 2.14
Celecoxib	10.61 ± 2.59	25.43 ± 3.29	46.42 ± 2.87	12.32 ± 2.42

^aData are presented as means ± SEM (n = 6). The anti-inflammatory activity represented as percentage edoema inhibition was calculated as follows: the increase of paw thickness in the control group – (the increase of paw thickness in the treated group/the increase of paw thickness in the control group)×100.

Three hours after oedema induction, compounds 4c, 4d, 6a, 6c and 6d displayed outstanding anti-inflammatory activity with oedema inhibition of 57.71%, 69.50%, 54.36% and 65.31%, respectively, compared to indomethacin (63.71%) and celecoxib (46.42%), while compound 6a showed moderate anti-inflammatory activity with 45.83% decrease in oedema thickness. Consistent with the in vitro studies, compound 4e demonstrated the least activity of oedema inhibition with 39.26%.

Strikingly, the anti-inflammatory activity of 4c, 6c and 6d was remarkably increased four hours after carrageenan injection indicating a long-lasting inhibition activity of these compounds with 69.86%, 62.21% and 78.53% inhibition, respectively, compared to celecoxib (12.32%) and indomethacin (56.27%). Compound 6d was the most potent one in this research showing nearly 7-fold and 1.5-fold more activity than the two used standard drugs celecoxib and indomethacin, respectively.

3.2.7. Acute toxicity study

The most active compounds 4c, 4d, 6c and 6d, representing the maximum anti-inflammatory activity were administered p.o. to mice at doses of 100, 200, 300, 400 and 500 mg/kg. No treatment-associated toxic signs or deaths were detected or recorded at the tested concentrations suggesting the safety as well as the well-tolerability of these compounds.

4. Molecular docking study of iNOS (PDB ID: 1r35)

Discovery Studio 2.5 software was used to explore and better understand the potency and the ability of the evaluated compounds to fit nicely into the active site of iNOS protein, the most active compounds 4c, 4d, 6c and 6d were selected to inspect their binding with iNOS protein whose 3D crystal structure (PDB ID: 1r35) was downloaded from the Protein Data Bank¹. The virtually docked compounds were built using ChemBioDraw Ultra 12.0 and finally to get the minimum lowest energy structure, the force fields were applied on compounds 4c, 4d, 6c and 6d. Then, the binding site sphere has been defined automatically by the software. The best obtained studied poses were chosen for docking using CDOCKER energy and were inspected in 3D and 2D styles^54–56.

From the inspection of docking results, it is obvious that compounds 4a, 4d, 6c and 6d have the ability to nicely fit into iNOS (PDB ID: 1r35) catalytic binding pocket, demonstrating good uniformity between the in vitro iNOS screening and the in silico prediction.

The docking results of compound 4c (CDOCKER energy = −37.0199 and CDOCKER interaction energy = −56.5283) (Figure 3(A,B)), revealed that it formed 5-H bonds; the oxygen of 4-methoxy formed one hydrogen bond with Gly196, the oxygen of the 1,3-propenone moiety engaged in one hydrogen bond with Arg375, the oxygen adjacent to methylene group incorporated in one hydrogen bond with Arg382 and finally the carbonyl oxygen of the carboximidamide group formed 2-H bond with Tyr367 and Asp376 amino acid residues. Additionally, 4c showed many hydrophobic interactions such as Pi–Sigma interaction with Val346, Pi–sulphur interaction with Cys194, Salt Bridge and Pi–anion interactions with Glu371 and two Pi–alkyl interactions with Ile195 and Arg375 amino acid residues.

Figure 3.

Binding mode of compounds 4c and 4d into iNOS pocket (PDB code: 1r35). (A) 3D structure of 4c (yellow), (B) 2D structure of 4c (yellow), (C) 3D structure of 4d (blue) and (D) 2D structure of 4d (blue).

The found result of compound 4d (Figure 3(C,D)) exhibited that 4d has better binding scores than 4c (CDOCKER energy = −40.445 and CDOCKER interaction energy = −66.5266). Moreover, 4d formed 7-H bonds; the oxygen atom of one of the dimethoxy groups incorporated in one hydrogen bond with Arg260, the proton of the amino group of the carboximidamide formed one hydrogen bond with Gln275, while the carbonyl oxygen of the carboximidamide group engaged in 4-H bonds with Gln275, Tyr341, Tyr367 and Asp376 amino acid residues. Further, 4d showed many hydrophobic interactions such as van der Waals, Carbon Hydrogen Bond, Pi–cation, Pi–anion, Pi–Pi T-shaped and Pi–alkyl interactions with Gly196, Glu371, Asp379, Arg382, Trp366, Cys194 and Val346 amino acid residues, respectively. These finding in agreement with the iNOS inhibition assay and explain the higher activity of compound 4d more than 4c.

Concerning compound 6c (Figure 4(A,B)), (CDOCKER energy = −43.0261 and CDOCKER interaction energy = −64.8146), involved into 6-H bonds; the carbonyl oxygen of the 1,3-propenone moiety formed 4-H bonds with Gln275, Tyr341, Tyr367 and Asp376 amino acid residues, the proton of the amino group of the carboximidamide engaged in 2-H bonds with Cys194 and Ile195 amino acid residues. Also, 6d showed many hydrophobic interactions as van der Waals, Pi-Cation, Pi-Anion and Pi-Alkyl interactions with Glu371, Arg382, Val346 amino acid residues.

Figure 4.

Binding mode of compounds 6c and 6d iNOS pocket (PDB code: 1r35). (A) 3D structure of 6c (pink), (B) 2D structure of 6c (pink), (C) 3D structure of 6d (cyan) and (D) 2D structure of 6d (cyan).

Finally, compound 6d (Figure 4(C,D)), with −35.5756 CDOCKER energy and −67.008 CDOCKER interaction energy, incorporated in 8-H bonds; Arg260 formed one hydrogen bond with the nitrogen atom of the of the carboximidamide group, Glu257 engaged in 2-H bond with the proton of the amino group of the carboximidamide group and, the oxygen adjacent to methylene group, Arg382 incorporated in 2-H bond with the oxygen atom of the carboximidamide group, wherein Tyr341, Tyr367 and Asp376 each engaged in one hydrogen bond with carbonyl oxygen of the carboximidamide group.

From the inspection of the docking results, it could be concluded that, compounds 4c, 4d, 6d and 6d, particularly, 4d and 6d fit nicely into the pocket of iNOS protein and they are entitled to be used as future lead template for identifying more potent anti-inflammatory candidates.

5. In silico prediction of physicochemical properties and pharmacokinetic profile

5.1. Lipinski rule calculations and ADMET analysis

Prediction of the physicochemical characters, pharmacokinetics and toxicity is an important tool in drug discovery of biologically active agents^10,62. Thus, the most active derivatives 4c, 4d, 6c and 6d were analysed for prediction of Lipinski’s Rule of Five⁶³ and Veber’s standard⁶⁴. Therefore, utilising the online application Pre-ADMET, theoretical calculations of the pharmacokinetic parameters as well as the theoretical agreement of the most potent compounds 4c, 4d, 6c and 6d to both Veber’s criteria and Lipinski’s rule of five were carried out⁶⁵.

The obtained results as illustrated in Table 5 showed that compounds 4c, 4d, 6c and 6d are in full accordance to Lipinski’s rule without any violation. Moreover, all the tested compounds had TPSA values < 140 Ų which used to calculate the percentage of oral absorption (%ABS) using the following equation: (%ABS = 109–(0.345 TPSA)⁶⁶. The tested compounds 4c, 4d, 6c and 6d exhibited %ABS of 78.96, 76.30, 78.96 and 76.30, respectively.

Table 5.

Calculated parameters of Veber's and Lipinski's rule of five for compounds 4c, 4d, 6c and 6d and indomethacin.

Comp.	MW	Log P	HBD	HBA	nVs	TPSA	%ABS
Lipinskia	≤500	≤5	≤5	≤10	≤1	–	–
Veberb	–	–	–	–	–	≤140	–
4c	460.16	3.57	2	7	0	87.08 A2	78.96
4d	490.17	3.17	2	8	0	94.79 A2	76.30
6c	460.16	3.57	2	7	0	87.08 A2	78.96
6d	490.17	3.17	2	8	0	94.79 A2	76.30
Indomethacin	357.08	4.00	1	4	0	51.31 A2	91.30

^aReference values of Lipinski.

^bReference values of Veber; MW: molecular weight; LogP: lipophilicity (O/W); HBD: number of hydrogen bond donors; HBA: number of hydrogen bond acceptors; nVs: number of Lipinski rule violations; TPSA: topological polar surface area (TPSA) (Å2); %ABS: percentage of oral absorption.

Furthermore, the results as shown in Table 6 revealed that compounds 4c, 4d, 6c and 6d had intermediate cell permeability in the CaCO₂ cell model they are expected to be excellently absorbed through the intestine with HIA values close to 1. Notably, all the tested compounds 4c, 4d, 6c and 6d were predicted to be non-toxic in Ames test and to be non-carcinogenic as shown in Table 6. From these results, it could be concluded that compound 4c, 4d, 6c and 6d had acceptable physicochemical properties and reasonable drug-likeness, hence, can be used as a promising drug candidate for development of new anti-inflammatory agents that act as dual iNOS/PGE2 inhibitors.

Table 6.

Predicted ADMET data of compounds 4c, 4d, 6c, 6d and indomethacin.

Compound	HIA	CaCO2 value	Rule of five	MDDR-like rule	PPB	BBB	AMES toxicity	Carcinogenicity
4c	98.46	20.69	Suitable	Drug-like	90.97	0.212	Non-mutagen	Negative
4d	98.64	25.07	Suitable	Drug-like	86.73	0.202	Non-mutagen	Negative
6c	98.46	21.67	Suitable	Drug-like	90.97	0.161	Non-mutagen	Negative
6d	98.64	25.80	Suitable	Drug-like	86.73	0.164	Non-mutagen	Negative
Indomethacin	97.90	20.03	Suitable	Drug-like	89.55	0.027	Non-mutagen	Negative

HIA: human intestinal absorption (%); CaCO₂: permeability through CaCO₂ (human colorectal carcinoma) cells in vitro; PPB: plasma protein binding; BBB: blood brain barrier penetration.

6. Structure–activity relationship

Study of the SAR showed that, in general, the chalcone/aryl carboximidamides 6a–f proved to be more potent inhibitor of LPS induced NO production than chalcone/aryl carboximidamides 4a–f. Ongoing throughout the results, it is obvious that the presence of donating groups leads to increase the suppression of LPS induced NO production. For instant, compounds 4d and 6d, with two methoxy group, exhibited the highest activity (73.5%, 78.2% inhibition, respectively). Removal of the 3-methoxy group of 4d and 6d (Figure 5) gave 4c and 6c with slight decrease in activity (62.3%, 67.1.8% inhibition, respectively). Removing the electron-donating two methoxy groups (as in 4a and 6a) or introducing electron-withdrawing such as Cl (4b and 6b) or NO₂ (4e and 6e) resulted in decreased the NO release inhibition potency (41.9%, 55.5%, 47.6%, 49.6%, 44.1% and 57.9% inhibition, respectively). Replacing the phenyl group with the bulky naphthyl one yielded compounds 4f and 6f with dramatic decrease in activity (31.2%, 29.4% inhibition, respectively). The same SAR correlation could be applied for the obtained results of the in vitro iNOS, PGE2 inhibitory activity and the in vivo anti-inflammatory activity.

Figure 5.

Chemical structures of the most active compounds 4c, 4d, 6c and 6d.

Regarding the COX inhibitory activity, it is obvious that the unsubstituted phenyl group is the optimal for COX2 inhibitory activity as in compound 6a (1.103 ± 0.05 µM). introducing the strong electron withdrawing group NO2 retain the activity as in compound 6e (1.714 ± 0.08 µM) while introducing one donating group as methoxy results in a moderate decrease in the COX2 inhibitory activity as in compounds 4c (3.279 ± 0.16 µM) and 6c (8.263 ± 0.4 µM). On the other hand, introducing two methoxy groups leads to a dramatic decrease in the COX2 inhibitory activity as in compounds 4d (23.4 ± 1.13 µM) and 6d (102.7 ± 4.97 µM). Concerning the 5LOX inhibitory activity, compound 6c with one methoxy group displayed the best inhibitory activity towards 5-LOX enzyme (IC₅₀ = 3.186 µM). Converting the propanone moiety as in compound 4c (8.136 ± 0.5 µM), removing the methoxy group as in compound 6a (6.126 ± 0.4 µM), or adding another methoxy group as in compounds 4d (16.47 ± 1 µM) and 6d (9.877 ± 0.6 µM) result in a decrease in the 5LOX inhibitory activity. Finally, replacing the methoxy group with the strongly deactivating NO₂ group as in compound 6e (54.15 ± 3.2 µM) leads to a dramatic decrease in the 5LOX inhibitory activity. from all these results it is obvious that both the substituent and the propanone moiety affect the bioactivity of these compounds. Moreover, it is clear that propanone moiety in chalcones 6a–f exhibited better bioactivity compared to its corresponding chalcones 4a–f.

7. Conclusions

In summary, two series of chalcone linked to aryl carboximidamides 4a–f and 6a–f were designed, synthesised and evaluated for iNOS. PGE2 inhibitory activity as well as for their in vivo anti-inflammatory activity using carrageenan-induced paw oedema method. All the synthesised compounds (4a–f and 6a–f) displayed significant iNOS inhibitory activity with IC₅₀ values ranging from 2.31 to 9.48 µM) and NO (IC₅₀ range of 6.5–13.01 µM) in comparison to indomethacin (IC₅₀ 24.57 µM and 45.69 µ M, respectively), against LPS-induced RAW 264.7 cells. Compounds 4c, 4d, 6c and 6d, with one or two methoxy groups, proved to be the most potent LPS induced NO over-production inhibitors with 62.3%, 73.5%, 67.1% and 78.2%, respectively. Additionally, 4c, 6a and 6e exhibited good COX2 inhibitory activity while 4c, 6a and 6c showed the highest 5LOX inhibitory activity. Moreover, 4c, 4d, 6c and 6d exhibited significant in vivo anti-inflammatory activity with oedema inhibition of 69.86% (after 4 h), 69.5% (after 3 h), 62.21% (after 4 h) and 78.51% (after 4 h), respectively, compared to indomethacin (56.27%) (after 4 h) and celecoxib (12.32%) (after 4 h). Notably, compounds 4d and 6d were the most LPS-induced NO production, iNOS activity and PGE2 inhibitors. The docking study revealed that compounds 4c, 4d, 6c and 6d fit nicely into the iNOS protein pocket (PDB ID: 1r35) through the important amino acid residues and these results were in agreement with the obtained biological results of iNOS inhibition assay. Furthermore, the predicted parameters of Lipinski’s rule of five and ADMET analysis showed that 4c, 4d, 6c and 6d had acceptable physicochemical properties and good drug-likeness scores. Therefore, compound 4c, 4d, 6c and 6d, in particular 4d and 6d, could serve as promising lead as anti-inflammatory candidate which merit further structural optimisation for more precise SAR and more potent derivatives.

Funding Statement

This project was funded by the Deanship of Scientific Research (DSR), King Abdulaziz University, Jeddah, under Grant no. RG-8-166-41. The authors, therefore, gratefully acknowledge DSR technical and financial support.

Disclosure statement

The authors declare no conflict of interest.