Insight on novel oxindole conjugates adopting different anti-inflammatory investigations and quantitative evaluation

Abstract

Background: A dual COX/5-LOX strategy was adopted to develop new oxindole derivatives with superior anti-inflammatory activity. Methods: Three series of oxindoles – esters 4a–p, 6a–l and imines 7a–o – were synthesized and evaluated for their anti-inflammatory and analgesic activities. Molecular docking and predicted pharmacokinetic parameters were done for the most active compounds. A new LC–MS/MS method was developed and validated for the quantification of 4h in rat plasma. Results: Compounds 4h, 6d, 6f, 6j and 7m revealed % edema inhibition up to 100.00%; also, 4l and 7j showed 100.00% writhing protection. Compound 4h showed dual inhibitory activity with IC₅₀ = 0.0533 and 0.4195 μM for COX-2 and 5-LOX, respectively. Molecular docking rationalized the obtained biological activity. The pharmacokinetic parameters of 4h from rat plasma were obtained.

Graphical abstract

Plain language summary

Summary points.

• Different oxindole-based derivatives were synthesized.

• The new compounds showed potent anti-inflammatory and analgesic activities.

• Compounds 4h, 6d, 6f, 6j and 7m were the most active as anti-inflammatory.

• Compounds 4l and 7g showed the best analgesic activity.

• Compound 4h showed high COX-2 inhibitory activity with selectivity index of 3.07.

• Compounds 6d and 6j revealed potent 5-LOX inhibitory activity.

• The molecular docking studies confirmed the structure–activity relationship study and the enzyme inhibition results.

• Promising pharmacokinetic parameters of compounds 4h, 6d and 6j were obtained using the free webserver Swiss Institute of Bioinformatics SwissADME^®.

• In vivo pharmacokinetic parameters of compound 4h were obtained from rat plasma using a newly developed MS/MS method.

The inflammatory process is controlled by different mechanisms that lead to the release of several mediators [1]. Metabolism of arachidonic acid through cyclooxygenase enzyme produces prostaglandin H₂, which is transformed into several products such as thromboxane (a potent vasoconstrictor), prostacyclin and prostaglandin. Both prostacyclin and prostaglandin act as vasodilators that increase blood flow at the area of inflammation, but on the other hand they have a gastroprotective role, as they increase mucus formation in the stomach [2,3]. There are two COX isoforms: COX-1, which is expressed in most tissues and is responsible for the physiological production of prostaglandin, and COX-2, which is induced through inflammatory stimuli. That is why it is believed that nonselective inhibition of COX by NSAIDs is the reason behind their side effects; therefore, COX-2 selective inhibitors were invented to avoid those side effects, but, in fact, many findings have pointed toward the overlapping actions of COX-1 and COX-2 [4,5]. COX-2 also was found to increase thrombosis risk by blocking prostacyclin biosynthesis, which is considered a potent antiaggregating agent, and leaving thromboxane A₂, which is mainly synthesized by the contribution of COX-1, which is a potent vasoconstrictor [6,7].

Leukotrienes that arise from the metabolism of arachidonic acid by the 5-LOX pathway also play an important role in the inflammatory process by stimulating the release of proinflammatory cytokines [8]. They have also been found to be involved in the pathogenesis of bronchial asthma, besides other inflammatory diseases. In addition, it is believed that leukotrienes may be related to cancer and cardiovascular diseases [9,10]. Zileuton (I), which is an N-hydroxyurea derivative, was approved by the US FDA in 1996 as the first 5-LOX inhibitor, which inhibits leukotriene biosynthesis and is used for the treatment of asthma [11]. However, the dosage regimen and the need for liver transaminase monitoring have limited the clinical use of zileuton (Figure 1) [12] .

Figure 1.

Reported compounds I–VI and the design strategy of the novel oxindole derivatives.

Studies also have shown that leukotrienes contribute to the damaging effects of NSAIDs on the gastrointestinal tract. COX pathway inhibition by NSAIDs results in switching the arachidonic acid metabolism to the 5-LOX pathway, leading to an increase in leukotriene production, which in turn increases the potential cardiovascular and gastrointestinal side effects [13,14].

Targeting only COX enzymes to control such a multifactorial process of inflammation seems to be insufficient and accompanied by many undesired side effects, which explains the need for multitarget anti-inflammatory agents that maintain the activity of NSAIDs and, at the same time, have a better safety profile by adapting the dual inhibition of the COX and 5-LOX strategy [15,16]. Several compounds have been developed and many indole and oxindole derivatives have been reported to have potent inhibitory activity of COX and 5-LOX enzymes [17,18]. Tenidap (II) is one of the representative oxindoles that was found to be more effective in the treatment of rheumatic arthritis in comparison with other traditional NSAIDs [19], although it was withdrawn due to the toxicity caused by the oxidative metabolites of its thiophene moiety. Many analogues have been developed to replace the thiophene moiety; those analogues have shown even higher anti-inflammatory activity (e.g., compound III, with IC₅₀ = 0.62, 0.18 and 9.87 μM, and compound IV, which exhibited excellent inhibitory activity, with IC₅₀ = 0.11, 0.10 and 0.56 μM for COX-1, COX-2 and 5-LOX, respectively [20]). On the other hand, it was reported that benzoic acid and benzoic acid esters have anti-inflammatory and analgesic effects [21]. Also, indolyl ester derivative V was reported as a selective COX-2 inhibitor, with IC₅₀ = 0.05 μM [22]; hydrazinyl indomethacin analogue VI was also reported as a selective COX-2 inhibitor, with IC₅₀ = 0.19 μM (Figure 1) [23].

On the basis of these findings and aiming to find new, potent anti-inflammatory agents with good tolerability, new oxindole candidates were designed, synthesized and biologically evaluated as anti-inflammatory agents with dual COX/5-LOX inhibitory activity. This includes N-unsubstituted oxindole esters 4a–d, which were synthesized from the reaction of hydroxymethylene oxindole with various derivatives of benzoyl chloride, in order to evaluate the effect of different substituents on the biological activity. In addition, N-benzyl oxindole esters 4e–h were synthesized to compare the biological activity between them and the N-unsubstituted esters 4a–d. Benzyl fluoro oxindole esters 4i–l and benzyl methoxy oxindole esters 4m–p were also synthesized to study the effect of 5-fluoro/methoxy-substitution on the biological activity. Moreover, ketoxime esters 6a–l were developed to compare the effect of a ketoxime ester side chain with the effect of an ester side chain in 4e–p. All the esters were intentionally designed and anticipated to exhibit activity both in their ester form and subsequent to hydrolysis, which results in the formation of benzoic acid along with hydroxymethylene/imino moieties. Schiff's bases 7a–o were also synthesized with different un-/substituted phenylhydrazines to investigate the electronic effect of different substituents on the biological activity. Additionally, the impact of their imine linkage on activity was contrasted with that of the ester linkage. All the derivatives were strategically engineered with lipophilic phenyl side chains to enhance their selectivity for COX-2 while promoting favorable binding to 5-LOX [24,25].

All synthesized compounds were screened for their in vivo anti-inflammatory activity, and selected compounds representative of each series were evaluated for their analgesic activity. The most active derivatives were further investigated for their toxicity, gastric safety and ulcerogenicity and gastric histopathological investigations were performed. In addition, the most active compounds in vivo as anti-inflammatories were tested for their in vitro COX and 5-LOX inhibitory activity.

Molecular docking simulations were carried out for 4h, 6d and 6j, in addition to the reference compounds (meloxicam and celecoxib), in order to study their binding pattern and binding affinity in the active sites of COX-1, COX-2 and 5-LOX and to rationalize their experimental biological activity. Moreover, their key physicochemical parameters were calculated to predict their pharmacokinetic properties and drug-likeness.

Furthermore, a new LC–MS/MS method was developed and validated for the quantification of compound 4h in rat plasma as one of the most active compounds with reasonable safety. Then the developed method was applied to obtain the in vivo pharmacokinetic parameters of compound 4h.

Experimental

Chemistry

The details regarding the experimental techniques and instruments employed have been incorporated into the supplementary data under Supplementary Section 14. Compounds 2a–d, 3a–d, 4a, 5a–c, 7a and 7e were prepared according to the reported methods [26–31].

General procedure for the synthesis of compounds 4a–l

To a stirred mixture of 3a–d (0.01 mol) and anhydrous triethylamine (TEA; 0.01 mol) in diethyl ether (30 ml), 0.02 mol of appropriate acid chloride was added dropwise at 0°C. The reaction mixture was stirred at 20–25°C for 24 h. The formed precipitate was filtered; washed with ether, water and saturated NaHCO₃ solution; left to dry; and recrystallized from dichloromethane.

(2-Oxoindolin-3-ylidene) methyl 4-chlorobenzoate (4b)

Light brown powder, yield (87%), melting point (m.p.) 214°–216°C; IR (KBr, ʋ cm^-1): 3163 (NH stretching), 3062 (CH aromatic), 1755, 1728 (2C=O), 1662 (NH bending), 1616–1585 (C=C); ¹H NMR (DMSO-d₆, 400 MHz): δ 6.90 (d, J = 7.72 Hz, 1H, aromatic H), 7.08 (t, J = 7.56 Hz, 1H, aromatic H), 7.29 (t, J = 8.00 Hz, 1H, aromatic H), 7.73–7.77 (m, 3H, aromatic H), 8.20 (d, J = 8.60 Hz, 2H, aromatic H), 8.31 (s, 1H, olefinic H), 10.63 (s, 1H, NH, D₂O exchangeable); ¹³C NMR (DMSO-d₆, 100 MHz): δ 110.35, 114.36, 122.01, 122.26, 129.20, 129.45, 131.60, 132.50, 138.24, 141.17, 141.32 (aromatic carbons), 152.78 (C-O), 166.92 (C=O), 169.16 (C=O); MS, m/z: 263.26 (M^-2); Anal. calcd. for C₁₆H₁₁NO₃ (265.27): C, 64.12; H, 3.36; N, 4.67. Found: C, 64.91; H, 3.57; N, 5.06.

(2-Oxoindolin-3-ylidene) methyl 4-fluorobenzoate (4c)

Brown powder, yield (91%), m.p. 190°–192°C; IR (KBr, ʋ cm^-1): 3425 (NH stretching), 3082 (CH aromatic), 1766, 1712 (2C=O), 1662 (NH bending), 1604 (C=C); ¹H NMR (DMSO-d₆, 400 MHz): δ 6.90 (d, J = 7.72 Hz, 1H, aromatic H), 7.08 (t, J = 7.56 Hz, 1H, aromatic H), 7.28 (t, J = 7.64 Hz, 1H, aromatic H), 7.52 (t, J = 8.80 Hz, 2H, aromatic H), 7.74 (d, J = 7.40 Hz, 1H, aromatic H), 8.27 (dd, J = 5.48, 8.76 Hz, 2H, aromatic H), 8.31 (s, 1H, olefinic H), 10.62 (s, 1H, NH, D₂O exchangeable); ¹³C NMR (DMSO-d₆, 100 MHz): δ 110.35, 114.25, 116.15, 117.19, 120.76, 122.22, 124.67, 127.87, 129.96, 132.50, 133.83, 161.87 (aromatic carbons), 164.12 (C=O), 166.84 (C-F), 169.19 (C=O); Anal. calcd. for C₁₆H₁₀FNO₃ (283.26): C, 67.84; H, 3.56; N, 4.94. Found: C, 67.97; H, 4.27; N, 5.42.

(2-Oxoindolin-3-ylidene) methyl 4-methoxybenzoate (4d)

Yellow powder, yield (93%), m.p. 205–207°C; IR (KBr, ʋ cm^-1): 3159 (NH stretching), 3028 (CH aromatic), 2970 (CH aliphatic), 1751, 1716 (2C=O), 1654 (NH bending), 1604 (C=C); ¹H NMR (DMSO-d₆, 400 MHz): δ 3.82, 3.90 (2s, 3H, OCH₃), 6. 90 (d, J = 7.68 Hz, 1H, aromatic H), 7.09 (t, J = 7.48 Hz, 1H, aromatic H), 7.21 (d, J = 8.64 Hz, 2H, aromatic H), 7.27 (t, J = 7.56 Hz, 1H, aromatic H), 7.74 (d, J = 7.36 Hz, 1H, aromatic H), 8.17 (d, J = 8.60 Hz, 2H, aromatic H), 8.34 (s, 1H, olefinic H), 10.59 (s, 1H, NH, D₂O exchangeable); ¹³C NMR (DMSO-d₆, 100 MHz): δ 55.85 (OCH₃), 114.25, 115.26, 119.42, 120.68, 121.27, 122.29, 123.37, 126.48, 129.82, 131.82, 133.07, 141.49 (aromatic carbons), 163.30 (C=O), 165.00 (C-O), 167.53 (C=O); Anal. Calcd. for C₁₇H₁₃NO₄ (295.29): C, 69.1 5; H, 4.44; N, 4.74. Found: C, 69.40; H, 4.63; N, 4.91.

(1-Benzyl-2-oxoindolin-3-ylidene) methyl benzoate (4e)

White powder, yield (90%), m.p. 190°–192°C; IR (KBr, ʋ cm^-1): 3059 (CH aromatic), 2935 (CH aliphatic), 1759, 1701 (2C=O), 1608 (C=C); ¹H NMR (DMSO-d₆, 400 MHz): δ 4.98 (s, 1H, CH₂), 7.00 (d, J = 8.08 Hz, 1H, aromatic H), 7.16 (t, J = 7.50 Hz, 1H, aromatic H), 7.26–7.29 (m, 4H, aromatic H), 7.34 (d, J = 4.4 Hz, 4H, aromatic H), 7.50 (t, J = 7.57 Hz, 1H, aromatic H), 7.62 (t, J = 7.40 Hz, 1H, aromatic H), 7.81–7.86 (m, 1H, aromatic H), 7.95 (d, J = 7.04 Hz, 1H, aromatic H), 8.23 (d, J = 8.32 Hz, 1H, aromatic H), 8.46, 8.49 (2s, 1H, olefinic H); ¹³C NMR (DMSO-d₆, 100 MHz): δ 45.79 (CH₂), 105.05, 108.56, 109.84, 113.06, 119.88, 121.79, 121.98, 126.05, 127.62, 129.03, 129.71, 130.75, 131.22, 133.33, 135.53, 137.80, 139.36, 142.21, 156.62, 162.71 (aromatic carbons), 167.78, 169.15 (2C=O); Anal. calcd. for C₂₃H₁₇NO₃ (355.39): C, 77.73; H, 4.82; N, 3.94. Found: C, 77.61; H, 5.05; N, 4.15.

(1-Benzyl-2-oxoindolin-3-ylidene) methyl 4-chlorobenzoate (4f)

White powder, yield (89%), m.p. 198°–200°C; IR (KBr, ʋ cm^-1): 3035 (CH aromatic), 2974 (CH aliphatic), 1755, 1705 (2C=O), 1593 (C=C); ¹H NMR (DMSO-d₆, 400 MHz): δ 4.91–5.02 (m, 2H, CH₂), 7.20–7.33 (m, 4H, aromatic H), 7.54 (d, J = 7.84 Hz, 4H, aromatic H), 7.94 (d, J = 7.84 Hz, 4H, aromatic H), 8.22 (d, J = 7.88 Hz, 1H, aromatic H), 8.46, 9.11 (2s, 1H, olefinic H); ¹³C NMR (DMSO-d₆, 100 MHz): δ 65.38 (CH₂), 111.53, 117.60, 119.46, 120.55, 122.00, 124.95, 127.21, 127.62, 127.66, 127.79, 128.01, 128.73, 128.80, 129.11, 130.60, 131.59, 136.89, 138.04, 138.42, 138.90 (aromatic carbons), 167.07 (C=O); Anal. calcd. for C₂₃H₁₆ClNO₃ (389.08): C, 70.86; H, 4.14; N, 3.59; Found: C, 71.04; H, 4.29; N, 3.77.

(1-Benzyl-2-oxoindolin-3-ylidene) methyl 4-fluorobenzoate (4g)

Yellow powder, yield (84%), m.p. 202°–204°C; IR (KBr, ʋ cm^-1): 3062 (CH aromatic), 2927 (CH aliphatic), 1759, 1705 (2C=O), 1604 (C=C); ¹H NMR (DMSO-d₆, 400 MHz): δ 4.98 (s, 2H, CH₂), 7.00 (d, J = 7.84 Hz, 1H, aromatic H), 7.15 (q, J = 8.04 Hz, 1H, aromatic H), 7.24–7.32 (m, 7H, aromatic H), 7.53 (t, J = 8.84 Hz, 1H, aromatic H), 7.81 (d, J = 7.32 Hz, 1H, aromatic H), 8.00 (dd, J = 8.84, 5.64 Hz, 1H, aromatic H), 8.30 (dd, J = 8.84, 5.40 Hz, 1H, aromatic H), 8.46 (s, 1H, olefinic H); ¹³C NMR (DMSO-d₆, 100 MHz): δ 45.81 (CH₂), 104.96, 108.56, 115.97, 116.18, 121.81, 121.95, 122.60, 126.02, 127.61, 128.99, 132.52, 132.61, 137.78, 139.30, 156.77, 164.11 (aromatic carbons), 166.60 (C-F), 166.84 (C=O), 169.20 (C=O); Anal. calcd. for C₂₃H₁₆FNO₃ (373.38): C, 73.99; H, 4.32; N, 3.75. Found: C, 74.15; H, 4.25; N, 3.78.

(1-Benzyl-2-oxoindolin-3-ylidene) methyl 4-methoxybenzoate (4h)

Brown powder, yield (88%), m.p. 208°–210°C; IR (KBr, ʋ cm^-1): 3086 (CH aromatic), 2981 (CH aliphatic), 1743, 1712 (2C=O), 1604 (C=C); ¹H NMR (DMSO-d₆, 400 MHz): δ 3.91 (s, 3H, OCH₃), 4.98 (s, 1H, CH₂), 7.00 (t, J = 7.88 Hz, 2H, aromatic H), 7.14 (t, J = 7.00 Hz, 1H, aromatic H), 7.22 (d, J = 8.56 Hz, 1H, aromatic H), 7.27–7.34 (m, 6H, aromatic H), 7.81 (d, J = 7.24 Hz, 1H, aromatic H), 7.89 (d, J = 8.40 Hz, 1H, aromatic H), 8.19 (d, J = 8.44 Hz, 1H, aromatic H), 8.48 (s, 1H, olefinic H); ¹³C NMR (DMSO-d₆, 100 MHz): δ 43.03 (CH₂), 55.87 (OCH₃), 109.77, 112.63, 114.26, 115.30, 119.30, 123.00, 123.40, 124.45, 127.34, 128.85, 129.14, 129.73, 131.82, 133.15, 136.93, 141.80, 142.35, 162.17, 163.30 (aromatic carbons), 165.07 (C=O), 167.53 (C-O), 167.95 (C=O); MS, m/z: 386.32 (M⁺); Anal. calcd. for C₂₄H₁₉NO₄ (385.42): C, 74.79; H, 4.97; N, 3.63. Found: C, 75.01; H, 5.12; N, 3.89.

(1-Benzyl-5-fluoro-2-oxoindolin-3-ylidene) methyl benzoate (4i)

Yellow powder, yield (84%), m.p. 196°–198°C; IR (KBr, ʋ cm^-1): 3028 (CH aromatic), 2924 (CH aliphatic), 1759, 1712 (2C=O), 1600 (C=C); ¹H NMR (DMSO-d₆, 400 MHz): δ 4.93 (s, 2H, CH₂), 7.20–7.30 (m, 5H, aromatic H), 7.50 (t, J = 7.60 Hz, 3H, aromatic H), 7.63 (t, J = 7.40 Hz, 2H, aromatic H), 7.94–7.96 (m, 3H, aromatic H), 8.14 (s, 1H, olefinic H); ¹³C NMR (DMSO-d₆, 100 MHz): δ 42.71 (CH₂), 104.66, 108.87, 111.34, 127.61, 127.69, 129.02, 129.72, 131.20, 133.32, 135.25, 137.71, 139.05, 157.31, 159.53 (aromatic carbons), 163.51 (C=O), 167.82 (C-F), 169.06 (C=O); Anal. calcd. for C₂₃H₁₆FNO₃(373.38): C, 73.99; H, 4.32; N, 3.75. Found: C, 73.82; H, 4.53; N, 3.98.

(1-Benzyl-5-fluoro-2-oxoindolin-3-ylidene) methyl 4-chlorobenzoate (4j)

Yellow powder, yield (81%), m.p. 195°–197°C; IR (KBr, ʋ cm^-1): 3089, 3074 (CH aromatic), 2920 (CH aliphatic), 1759, 1716 (2C=O), 1593 (C=C); ¹H NMR (DMSO-d₆, 400 MHz): δ 4.93 (s, 2H, CH₂), 7.20–7.28 (m, 4H, aromatic H), 7.50 (t, J = 7.64 Hz, 3H, aromatic H), 7.63 (t, J = 7.28 Hz, 2H, aromatic H), 7.95 (d, J = 7.28 Hz, 3H, aromatic H), 8.14 (s, 1H, olefinic H); ¹³C NMR (DMSO-d₆, 100 MHz): δ 42.83 (CH₂), 109.22, 112.27, 127.27, 127.67, 128.78, 129.22, 130.09, 131.61, 135.68, 137.59, 138.27, 139.11, 157.31, 157.72, 159.25, 159.64, 163.53 (aromatic carbons), 165.65 (C=O), 166.94 (C-F), 168.88 (C=O); Anal. calcd. for C₂₃H₁₅ClFNO₃ (407.83): C, 67.74; H, 3.71; N, 3.43. Found: C, 67.98; H, 3.82; N, 3.67.

(1-Benzyl-5-fluoro-2-oxoindolin-3-ylidene) methyl 4-fluorobenzoate (4k)

Yellow powder, yield (87%), m.p. 180°–182°C; IR (KBr, ʋ cm^-1): 3082 (CH aromatic), 2978 (CH aliphatic), 1759, 1708 (2C=O), 1600 (C=C); ¹H NMR (DMSO-d₆, 400 MHz): δ 4.93 (s, 2H, CH₂), 6.37–6.43 (m, 1H, aromatic H), 6.64 (dd, J = 8.40, 4.64 Hz, 1H, aromatic H), 6.85 (dd, J = 10.12, 2.56 Hz, 1H, aromatic H), 7.20–7.35 (m, 7H, aromatic H), 7.98–8.03 (m, 2H, aromatic H), 8.14 (s, 1H, olefinic H); ¹³C NMR (DMSO-d₆, 100 MHz): δ 45.90 (CH₂), 107.02, 109.00, 115.97, 116.19, 127.26, 127.63, 127.67, 127.82, 127.8, 128.77, 129.02, 129.11, 132.52, 132.61, 137.67, 139.13, 156.96, 159.25 (aromatic carbons), 163.48 (C=O), 164.13 (C-F), 166.83 (C=O); Anal. calcd. for C₂₃H₁₅F₂NO₃ (391.37): C, 70.59; H, 3.86; N, 3.58. Found: C, 70.65; H, 4.03; N, 3.80.

(1-Benzyl-5-fluoro-2-oxoindolin-3-ylidene) methyl 4-methoxybenzoate (4l)

Yellow powder, yield (85%), m.p. 200°–202°C; IR (KBr, ʋ cm^-1): 3028 (CH aromatic), 2943 (CH aliphatic), 1747, 1712 (2C=O), 1600 (C=C); ¹H NMR (DMSO-d₆, 400 MHz): δ 3.82 (s, 3H, OCH₃), 4.98 (s, 2H, CH₂), 7.02 (d, J = 8.88 Hz, 1H, aromatic H), 7.14 (d, J = 8.96 Hz, 4H, aromatic H), 7.23–7.26 (dd, J = 8.92, 4.36 Hz, 1H, aromatic H), 7.33–7.34 (m, 1H, aromatic H), 7.88–7.90 (dd, J = 6.92, 1.96 Hz, 1H, aromatic H), 8.05–8.08 (m, 3H, aromatic H), 8.18 (d, J = 8.92 Hz, 1H, aromatic H), 8.51 (s, 1H, olefinic H); ¹³C NMR (DMSO-d₆, 100 MHz): δ 42.73 (CH₂), 55.87 (OCH₃), 109.04, 110.13, 112.15, 114.26, 114.98, 120.65, 123.40, 127.37, 127.62, 129.04, 131.82, 132.00, 135.73, 136.23, 137.55, 139.08, 139.90, 163.30 (aromatic carbons), 163.54 (C=O), 164.43 (C-F), 165.07 (C-O), 167.50 (C=O); Anal. calcd. for C₂₄H₁₈FNO₄ (403.41): C, 71.46; H, 4.50; N, 3.47. Found: C, 71.68; H, 4.61; N, 3.62.

(1-Benzyl-5-methoxy-2-oxoindolin-3-ylidene) methyl benzoate (4m)

White powder, yield (78%), m.p. 200°–202°C; IR (KBr, ʋ cm^-1): 3078 (CH aromatic), 2931 (CH aliphatic), 1759, 1701 (2C=O), 1597 (C=C); ¹H NMR (DMSO-d₆, 400 MHz): δ 3.78 (s, 3H, OCH₃), 4.95 (s, 2H, CH₂), 6.88–6.90 (m, 1H, aromatic H), 7.32–7.37 (m, 5H, aromatic H), 7.50 (t, J = 7.72 Hz, 2H, aromatic H), 7.71 (t, J = 7.72 Hz, 2H, aromatic H), 7.93–7.96 (d, J = 6.96 Hz, 1H, aromatic H), 8.21 (d, J = 7.16 Hz, 2H, aromatic H), 8.46 (s, 1H, olefinic H); ¹³C NMR (DMSO-d₆, 100 MHz): δ 45.89 (CH₂), 55.84 (OCH₃), 102.33, 103.83, 104.52, 107.85, 108.56, 110.27, 119.41, 127.60, 128.93, 129.12, 131.21, 133.21, 137.72, 138.31, 155.14 (aromatic carbons), 167.80 (C=O), 168.93 (C=O); Anal. calcd. for C₂₄H₁₉NO₄ (385.42): C, 74.79; H, 4.97; N, 3.63. Found: C, 75.03; H, 5.08; N, 3.84.

(1-Benzyl-5-methoxy-2-oxoindolin-3-ylidene) methyl 4-chlorobenzoate (4n)

White powder, yield (82%), m.p. 205°–207°C; IR (KBr, ʋ cm^-1): 3086 (CH aromatic), 2985 (CH aliphatic), 1755, 1712 (2C=O), 1593 (C=C); ¹H NMR (DMSO-d₆, 400 MHz): δ 3.67 (s, 3H, OCH₃), 4.88 (s, 2H, CH₂), 7.22–7.27 (m, 2H, aromatic H), 7.56 (d, J = 6.44 Hz, 5 H, aromatic H), 7.94 (d, J = 6.48 Hz, 5H, aromatic H), 8.35 (s, 1H, olefinic H); ¹³C NMR (DMSO-d₆, 100 MHz): δ 45.73 (CH₂), 55.80 (OCH₃), 102.18, 103.36, 107.12, 108.13, 109.38, 124.90, 125.03, 127.39, 128.86, 129.17, 130.34, 131.59, 132.28, 138.14, 138.42, 155.01 (aromatic carbons), 161.58 (C-O), 166.96 (C=O), 169.37 (C=O); Anal. calcd. for C₂₄H₁₈ClNO₄ (419.86): C, 68.66; H, 4.32; N, 3.34. Found: C, 68.95; H, 4.60; N, 3.45.

(1-Benzyl-5-methoxy-2-oxoindolin-3-ylidene) methyl 4-fluorobenzoate (4o)

Yellow powder, yield (80%), m.p. 190°–192°C; IR (KBr, ʋ cm^-1): 3078 (CH aromatic), 2958 (CH aliphatic), 1759, 1701 (2C=O), 1597 (C=C); ¹H NMR (DMSO-d₆, 400 MHz): δ 3.67 (s, 3H, CH₃), 4.98 (s, 2H, CH₂), 6.23 (d, J = 10.04 Hz, 1H, aromatic H), 7.22 (d, J = 7.08 Hz, 4H, aromatic H), 8.00 (dd, J = 8.16, 5.76 Hz, 7H, aromatic H), 9.22 (s, 1H, olefinic H); ¹³C NMR (DMSO-d₆, 100 MHz): δ 42.93 (CH₂), 55.49 (OCH₃), 105.52, 106.57, 115.65, 115.87, 127.19, 127.68, 127.75, 128.78, 129.04, 129.38, 132.49, 139.33, 153.98 (aromatic carbons), 163.80 (C-O), 166.28 (C=O), 167.40 (C=O); Anal. calcd. for C₂₄H₁₈FNO₄ (403.41): C, 71.46; H, 4.50; N, 3.47. Found: C, 71.70; H, 5.34; N, 3.63.

(1-Benzyl-5-methoxy-2-oxoindolin-3-ylidene) methyl 4-methoxybenzoate (4p)

White powder, yield (86%), m.p. 208°–210°C; IR (KBr, ʋ cm^-1): 3051 (CH aromatic), 2978 (CH aliphatic), 1759, 1720 (2C=O), 1604 (C=C); ¹H NMR (DMSO-d₆, 400 MHz): δ 3.82 (s, 6H, 2 OCH₃), 4.94 (s, 2H, CH₂), 7.02 (d, J = 8.80 Hz, 3H, aromatic H), 7.22–7.33 (m, 5H, aromatic H), 7.89 (d, J = 8.76 Hz, 3H, aromatic H), 8.16 (m, 1H, aromatic H), 8.45 (s, 1H, olefinic H); ¹³C NMR (DMSO-d₆, 100 MHz): δ 45.72 (CH₂), 55.91 (OCH₃), 110.40, 110.69, 110.86, 114.27, 115.37, 123.45, 127.68, 128.94, 129.12, 131.97, 132.97, 135.50, 135.70, 142.20, 155.64, 161.77 (C=O), 163.29 (C-O), 165.12 (C-O), 167.44 (C=O); Anal. calcd. for C₂₅H₂₁NO₅ (415.45): C, 72.28; H, 5.10; N, 3.37. Found: C, 72.09; H, 5.34; N, 3.63.

General procedure for the synthesis of compounds 6a–l

To a mixture of 5a–c (0.01 mol) and anhydrous TEA (0.01 mol) in diethyl ether (30 ml) at 0°C, 0.02 mol of appropriate acid chloride was added dropwise. The reaction mixture was stirred at room temperature for 24 h. The formed precipitate was filtered, washed with ether, water and saturated NaHCO₃ solution; left to dry; and recrystallized from dichloromethane.

3-([Benzoyloxy]imino)-1-benzylindolin-2-one (6a)

Yellow powder, yield (92%), m.p. 175°–177°C; IR (KBr, ʋ cm^-1): 3028 (CH aromatic), 2927 (CH aliphatic), 1759, 1732 (2C=O), 1606 (C=C); ¹H NMR (DMSO-d₆, 400 MHz): δ 4.99 (s, 2H, CH₂), 7.07 (d, J = 7.92 Hz, 1H, aromatic H), 7.22 (t, J = 7.56 Hz, 1H, aromatic H), 7.27–7.32 (m, 1H, aromatic H), 7.35 (t, J = 7.40 Hz, 2H, aromatic H), 7.40 (d, J = 7.12 Hz, 2H, aromatic H), 7.51–7.55 (m, 1H, aromatic H), 7.68 (t, J = 7.80 Hz, 2H, aromatic H), 7.81 (t, J = 7.40 Hz, 1H, aromatic H), 8.06 (d, J = 7.40 Hz, 1H, aromatic H), 8.15 (d, J = 7.24 Hz, 2H, aromatic H); ¹³C NMR (DMSO-d₆, 100 MHz): δ 43.43 (CH₂), 111.05, 114.81, 124.09, 127.76, 127.78, 128.09, 128.15, 129.21, 129.57, 129.89, 130.20, 130.20, 134.94, 135.47, 136.07, 145.55, 149.88 (aromatic carbons), 162.69 (C=N), 163.38 (C=O); Anal. calcd. for C₂₂H₁₆ N₂O₃ (356.38): C, 74.15; H, 4.53; N, 7.86. Found: C, 73.89; H, 4.67; N, 8.15.

1-Benzyl-3-([(4-chlorobenzoyl) oxy] imino) indolin-2-one (6b)

Yellow powder, yield (94%), m.p. 210°–212°C; IR (KBr, ʋ cm^-1): 3028 (CH aromatic), 2924 (CH aliphatic), 1766, 1728 (2C=O), 1593 (C=C); ¹H NMR (DMSO-d₆, 400 MHz): δ 4.98 (s, 2H, CH₂), 7.06 (d, J = 6.84 Hz, 1H, aromatic H), 7.20–7.39 (m, 5H, aromatic H), 7.50 (d, J = 6.52 Hz, 3H, aromatic H), 7.74 (d, 1H, J = 6.88 Hz, aromatic H), 7.91 (d, J = 6.72 Hz, 1H, aromatic H), 8.03 (d, J = 6.24 Hz, 1H, aromatic H), 8.14 (d, J = 6.92 Hz, 1H, aromatic H); ¹³C NMR (DMSO-d₆, 100 MHz): δ 43.44 (CH₂), 111.06, 114.76, 124.08, 126.68, 127.77, 128.08, 128.79, 129.20, 130.03, 131.51, 132.05, 132.44, 135.54, 136.06, 137.19, 139.91, 145.60, 150.01 (aromatic carbons), 162.64 (C=N), 167.50 (C=O); MS, m/z: 391.91 (M⁺); Anal. calcd. for C₂₂H₁₅ClN₂O₃ (390.82): C, 67.61; H, 3.87; N, 7.17; Found: C, 67.90; H, 4.01; N, 7.40.

1-Benzyl-3-([(4-fluorobenzoyl) oxy] imino) indolin-2-one (6c)

Yellow powder, yield (96%), m.p. 195°–197°C; IR (KBr, ʋ cm^-1): 3082 (CH aromatic), 2924 (CH aliphatic), 1766, 1732 (2C=O), 1604 (C=C); ¹H NMR (DMSO-d₆, 400 MHz): δ 4.99 (s, 2H, CH₂), 7.07 (d, J = 7.96 Hz, 1H, aromatic H), 7.21 (t, J = 7.60 Hz, 1H, aromatic H), 7.29–7.41 (m, 5H, aromatic H), 7.49–7.55 (m, 3H, aromatic H), 8.05 (d, J = 7.40 Hz, 1H, aromatic H), 8.23 (dd, J = 8.64, 5.48 Hz, 2H, aromatic H); ¹³C NMR (DMSO-d₆, 100 MHz): δ 43.43 (CH₂), 111.08, 114.79, 117.25, 124.43, 127.75, 128.11, 129.23, 129.39, 133.38, 135.54, 136.06, 136.73, 143.06, 145.59, 149.94 (aromatic carbons), 162.69 (C=N), 164.93 (C=O), 167.44 (C-F); Anal. calcd. for C₂₂H₁₅FN₂O₃ (374.37): C, 70.58; H, 4.04; N, 7.48. Found: C, 70.42; H, 4.24; N, 7.69.

1-Benzyl-3-([(4-methoxybenzoyl) oxy] imino) indolin-2-one (6d)

Yellow powder, yield (91%), m.p. 205°–207°C; IR (KBr, ʋ cm^-1): 3028 (CH aromatic), 2981 (CH aliphatic), 1759, 1728 (2C=O), 1600 (C=C); ¹H NMR (DMSO-d₆, 400 MHz): δ 3.90 (s, 3H, OCH₃), 4.99 (s, 2H, CH₂), 7.07 (d, J = 7.96 Hz, 1H, aromatic H), 7.20 (d, J = 8.56 Hz, 3H, aromatic H), 7.27–7.30 (m, 1H, aromatic H), 7.34–7.41 (m, 4H, aromatic H), 7.53 (t, J = 7.72 Hz, 1H, aromatic H), 8.06 (d, J = 7.52 Hz, 1H, aromatic H), 8.12 (d, J = 8.80 Hz, 2H, aromatic H); ¹³C NMR (DMSO-d₆, 100 MHz): δ 43.41 (CH₂), 56.19 (OCH₃), 111.04, 112.88, 115.26, 119.61, 122.96, 124.03, 127.76, 129.21, 131.13, 132.50, 135.33, 136.12, 145.49, 149.51 (aromatic carbons), 162.75 (C=N), 162.95 (C-O), 164.55 (C=O); Anal. calcd. for C₂₃H₁₈N₂O₄ (368.41): C, 71.49; H, 4.70; N, 7.25. Found: C, 71.26; H, 4.83; N, 7.44.

3-([Benzoyloxy]imino)-1-benzyl-5-fluoroindolin-2-one (6e)

Yellow powder, yield (89%), m.p. 190°–192°C; IR (KBr, ʋ cm^-1): 3062 (CH aromatic), 2912 (CH aliphatic), 1770, 1732 (2C=O), 1600 (C=C); ¹H NMR (DMSO-d₆, 400 MHz): δ 4.99 (s, 2H, CH₂), 7.08 (dd, J = 8.72, 4.08 Hz, 1H, aromatic H), 7.27–7.29 (m, 2H, aromatic H), 7.34–7.41 (m, 3H, aromatic H), 7.45 (dd, J = 9.12, 2.44 Hz, 1H, aromatic H), 7.70 (t, J = 7.84 Hz, 2H, aromatic H), 7.75 (dd, J = 7.92, 2.64 Hz, 1H, aromatic H), 7.82 (t, J = 7.44 Hz, 1H, aromatic H), 8.14 (d, J = 7.16 Hz, 2H, aromatic H); ¹³C NMR (DMSO-d₆, 100 MHz): δ 43.07 (CH₂), 110.79, 113.83, 116.54, 118.17, 121.98, 127.66, 128.30, 129.18, 129.59, 129.91, 131.23, 135.06, 135.85, 136.59, 139.07, 141.94, 149.59 (aromatic carbons), 162.57 (C-F), 163.27 (C=N), 163.93 (C=O), 169.29 (C=O); Anal. calcd. for C₂₂H₁₅FN₂O₃ (374.37): C, 70.58; H, 4.04; N, 7.48. Found: C, 70.74; H, 4.21; N, 7.70.

1-Benzyl-3-([(4-chlorobenzoyl) oxy] imino)-5-fluoroindolin-2-one (6f)

Yellow powder, yield (93%), m.p. 205°–206°C; IR (KBr, ʋ cm^-1): 3032 (CH aromatic), 2974 (CH aliphatic), 1770, 1732 (2C=O), 1593 (C=C); ¹H NMR (DMSO-d₆, 400 MHz): δ 4.48 (s, 2H, CH₂), 7.08 (dd, J = 8.72, 4.00 Hz, 1H, aromatic H), 7.27–7.31 (m, 1H, aromatic H), 7.34–7.47 (m, 7H, aromatic H), 7.73–7.78 (m, 1H, aromatic H), 7.90 (d, J = 8.32 Hz, 2H, aromatic H), 8.14 (d, J = 8.40 Hz, 2H, aromatic H); ¹³C NMR (DMSO-d₆, 100 MHz): δ 43.10 (CH₂), 126.63, 127.69, 127.97, 128.81, 129.22, 130.08, 131.52, 132.07, 132.40, 135.87, 136.55, 137.21, 139.36, 140.03 (aromatic carbons), 162.52 (C-F), 163.68 (C=N), 167.44 (2C=O); Anal. calcd. for C₂₂H₁₄ClFN₂O₃ (408.81): C, 64.64; H, 3.45; N, 6.85. Found: C, 64.39; H, 3.62; N, 6.93.

1-Benzyl-5-fluoro-3-([(4-fluorobenzoyl) oxy] imino) indolin-2-one (6g)

Yellow powder, yield (92%), m.p. 196°–198°C; IR (KBr, ʋ cm^-1): 3082 (CH aromatic), 2974 (CH aliphatic), 1770, 1732 (2C=O), 1600 (C=C); ¹H NMR (DMSO-d₆, 400 MHz): δ 4.99 (s, 2H, CH₂), 7.08 (dd, J = 8.64, 4.12 Hz, 1H, aromatic H), 7.29–7.39 (m, 4H, aromatic H), 7.46 (dd, J = 8.96, 2.44 Hz, 2H, aromatic H), 7.53 (t, J = 8.84 Hz, 2H, aromatic H), 7.74 (dd, J = 7.84 Hz, J = 2.60 Hz, 1H, aromatic H), 8.21 (dd, J = 8.88, 5.40 Hz, 2H, aromatic H); ¹³C NMR (DMSO-d₆, 100 MHz): δ 43.54 (CH₂), 110.89, 112.26, 115.15, 116.14, 117.25, 118.45, 122.02, 127.67, 128.15, 129.23, 132.14, 133.34, 135.84, 136.53, 141.96, 149.58, 157.28 (aromatic H), 159.66 (C-F), 162.54 (C=N), 163.74 (C=O), 167.50 (C=O); Anal. calcd. for C₂₂H₁₄F₂N₂O₃ (392.36): C, 67.35; H, 3.60; N, 7.14. Found: C, 67.18; H, 3.47; N, 7.42.

1-Benzyl-5-fluoro-3-([(4-methoxybenzoyl) oxy] imino) indolin-2-one (6h)

Yellow powder, yield (92%), m.p. 212°–214°C; IR (KBr, ʋ cm^-1): 3039 (CH aromatic), 2943 (CH aliphatic), 1759, 1732 (2C=O), 1600 (C=C); ¹H NMR (DMSO-d₆, 400 MHz): δ 3.90 (s, 3H, OCH₃), 4.99 (s, 2H, CH₂), 7.07 (dd, J = 8.72, 4.12 Hz, 1H, aromatic H), 7.21 (d, J = 8.88 Hz, 2H, aromatic H), 7.29–7.31 (m, 1H, aromatic H), 7.34–7.41 (m, 4H, aromatic H), 7.43 (d, J = 8.96, 2.48 Hz, 1H, aromatic H), 7.74 (dd, J = 7.88, 2.64 Hz, 1H, aromatic H) 8.10 (d, J = 8.88 Hz, 2H, aromatic H); ¹³C NMR (DMSO-d₆, 100 MHz): δ 42.96 (CH₂), 55.60 (OCH₃), 110.33, 113.28, 115.32, 119.49, 121.60, 127.66, 127.76, 129.14, 131.33, 132.50, 135.91, 136.92, 138.23, 141.87, 143.34, 149.23 (aromatic carbons), 161.43 (C=N), 162.62 (C=O), 164.67 (C=O), 169.48 (C-O); Anal. calcd. for C₂₃H₁₇FN₂O₄ (404.40): C, 68.31; H, 4.24; N, 6.93. Found: C, 68.45; H, 4.37; N, 6.79.

3-([Benzoyloxy]imino)-1-benzyl-5-methoxyindolin-2-one (6i)

Orange powder, yield (93%), m.p. 172°–174°C; IR (KBr, ʋ cm^-1): 3078 (CH aromatic), 2931 (CH aliphatic), 1759, 1701 (2C=O), 1554 (C=C); ¹H NMR (DMSO-d₆, 400 MHz): δ 3.75 (s, 3H, OCH₃), 4.95 (s, 2H, CH₂), 6.97 (d, J = 8.48 Hz, 1H, aromatic H), 7.12 (d, J = 7.48 Hz, 1H, aromatic H), 7.28–7.37 (m, 6H, aromatic H), 7.60 (s, 1H, aromatic H), 7.67 (t, J = 7.12 Hz, 2H, aromatic H), 7.80 (t, J = 4.44 Hz, 1H, aromatic H), 8.14 (d, J = 7.48 Hz, 1H, aromatic H); ¹³C NMR (DMSO-d₆, 100 MHz): δ 43.44 (CH₂), 55.04 (OCH₃), 110.38, 111.86, 113.07, 114.79, 115.22, 116.65, 120.77, 127.66, 129.20, 130.05, 134.98, 136.14, 136.94, 139.10, 143.67, 150.34, 155.68 (aromatic carbons), 162.48 (C-O), 163.22 (C=N), 164.06 (C=O), 170.19 (C=O); Anal. calcd. for C₂₃H₁₈N₂O₄ (386.41): C, 71.49; H, 4.70; N, 7.25. Found: C, 71.76; H, 4.82; N, 7.43.

1-Benzyl-3-([(4-chlorobenzoyl) oxy] imino)-5-methoxyindolin-2-one (6j)

Orange powder, yield (96%), m.p. 179°–181°C; IR (KBr, ʋ cm^-1): 3086 (CH aromatic), 2943 (CH aliphatic), 1755, 1712 (2C=O), 1593 (C=C); ¹H NMR (DMSO-d₆, 400 MHz): δ 3.76 (s, 3H, OCH₃), 4.95 (s, 2H, CH₂), 6.98 (d, J = 8.65 Hz, 1H, aromatic H), 7.12 (dd, J = 8.68, 2.64 Hz, 1H, aromatic H), 7.29 (d, J = 6.76 Hz, 1H, aromatic H) 7.33–7.39 (m, 2H, aromatic H), 7.54 (d, J = 8.52 Hz, 2H, aromatic H), 7.76 (d, J = 8.56 Hz, 1H, aromatic H), 7.93 (d, J = 8.56 Hz, 3H, aromatic H), 8.14 (d, J = 8.60 Hz 1H, aromatic H); ¹³C NMR (DMSO-d₆, 100 MHz): δ 43.44 (CH₂), 56.03 (OCH₃), 111.90, 114.74, 115.15, 120.99, 126.80, 127.74, 128.07, 128.95, 129.20, 130.03, 131.54, 131.89, 136.12, 137.63, 139.16, 139.98, 150.47 (aromatic carbons), 155.84 (C=N), 162.44 (C=O), 167.25 (C=O); Anal. calcd. for C₂₃H₁₇ClN₂O₄ (420.85): C, 65.64; H, 4.07; N, 6.66. Found: C, 65.85; H, 4.26; N, 6.92; HRMS (ESI/Q-TOF) m/z: [M + Na]⁺ Calcd. for C₂₃H₁₇ClN₂O₄: 443.0773; Found: 443.0771.

1-Benzyl-3-([(4-fluorobenzoyl) oxy] imino)-5-methoxyindolin-2-one (6k)

Orange powder, yield (97%), m.p. 190°–192°C; IR (KBr, ʋ cm^-1): 3032 (CH aromatic), 2954 (CH aliphatic), 1770, 1728 (2C=O), 1597 (C=C); ¹H NMR (DMSO-d₆, 400 MHz): δ 3.76 (s, 3H, OCH₃), 4.96 (s, 2H, CH₂), 6.98–7.06 (m, 2H, aromatic H), 7.13 (dd, J = 8.68 Hz, J = 2.32 Hz, 1H, aromatic H), 7.31–7.37 (m, 5H, aromatic H), 7.51–7.58 (m, 2H, aromatic H), 7.89 (dd, J = 8.60, 6.20 Hz, 1H, aromatic H), 8.20–8.24 (dd, J = 8.80, 5.40 Hz, 1H, aromatic H); ¹³C NMR (DMSO-d₆, 100 MHz): δ 42.95 (CH₂), 56.05 (OCH₃), 110.31, 111.91, 112.82, 114.35, 116.45, 117.25, 120.88, 127.66, 129.12, 131.68, 133.09, 136.15, 136.60, 137.02, 139.15, 143.71, 155.67 (aromatic carbons), 162.21 (C=N), 164.17 (C=O), 164.64 (C=O), 169.27 (C-F); Anal. calcd. for C₂₃H₁₇FN₂O₄ (404.40): C, 68.31; H, 4.24; N, 6.93. Found: C, 68.40; H, 4.39; N, 7.18.

1-Benzyl-5-methoxy-3-(([4-methoxybenzoyl] oxy) imino ) indolin-2-one (6l)

Orange powder, yield (91%), m.p. 180°–182°C; IR (KBr, ʋ cm^-1): 3028 (CH aromatic), 2939 (CH aliphatic), 1759, 1720 (2C=O), 1581 (C=C); ¹H NMR (DMSO-d₆, 400 MHz): δ 3.76 (s, 3H, OCH₃), 3.88 (s, 3H, OCH₃), 4.94 (s, 2H, CH₂), 6.97 (d, J = 8.64 Hz, 1H, aromatic H), 7.10 (dd, J = 8.64 Hz, 1H, aromatic H), 7.19 (d, J = 8.76 Hz, 2H, aromatic H), 7.28–7.39 (m, 5H, aromatic H), 7.57 (s, 1H, aromatic H), 8.08 (d, J = 8.76 Hz, 2H, aromatic H); ¹³C NMR (DMSO-d₆, 100 MHz): δ 43.41 (CH₂), 56.03 (OCH₃), 56.22 (OCH₃), 111.80, 112.88, 114.69, 115.22, 115.27, 119.66, 120.55, 127.64, 127.74, 128.05, 129.08, 129.19, 131.14, 132.31, 136.17, 138.99, 149.91, 155.82 (aromatic carbons), 162.52 (C=N), 162.78 (C=O), 164.58 (C=O); Anal. calcd. for C₂₄H₂₀N₂O₅ (416.43): C, 69.22; H, 4.84; N, 6.73. Found: C, 69.47; H, 4.91; N, 6.86.

General procedure for the synthesis of compounds 7a–o

To a solution of 1b–d (0.01 mol) in methanol in the presence of a few drops of glacial acetic acid, appropriate phenyl hydrazine derivative (0.01 mol) was added and the mixture was left to stir at room temperature for 2 h. The formed precipitate was filtered, washed with methanol and recrystallized from ethanol.

1-Benzyl-3-(2-[4-chlorophenyl] hydrazineylidene) indolin-2-one (7b)

Yellow powder, yield (96%), m.p. 202°–204°C; IR (KBr, ʋ cm^-1): 3240 (NH stretching), 3028 (CH aromatic), 2939 (CH aliphatic), 1689 (C=O), 1666 (C=O), 1589 (C=C); ¹H NMR (DMSO-d₆, 400 MHz): δ 5.03 (s, 2H, CH₂), 7.06 (d, J = 7.88 Hz, 1H, aromatic H), 7.11 (d, J = 7.48 Hz, 1H, aromatic H), 7.25–7.29 (m, 2H, aromatic H), 7.32–7.38 (m, 4H, aromatic H), 7.43 (d, J = 8.92 Hz, 2H, aromatic H), 7.53 (d, J = 8.92 Hz, 2H, aromatic H), 7.62 (d, J = 7.28 Hz, 1H, aromatic H), 12.68 (s, 1H, NH, D₂O exchangeable); ¹³C NMR (DMSO-d₆, 100 MHz): δ 42.81 (CH₂), 110.25, 116.38, 117.11, 119.14, 120.89, 123.03, 127.10, 127.64, 127.82, 128.00, 129.08, 129.17, 129.73, 136.74, 140.72, 141.98 (aromatic carbons), 161.50 (C=O); Anal. calcd. for C₂₁H₁₆N₃O (361.83): C, 69.71; H, 4.46; N, 11.61. Found: C, 69.88; H, 4.70; N, 11.89.

1-Benzyl-3-(2-[4-fluorophenyl] hydrazineylidene) indolin-2-one (7c)

Yellow powder, yield (98%), m.p. 155°–157°C; IR (KBr, ʋ cm^-1): 3232 (NH stretching), 3032 (CH aromatic), 2974 (CH aliphatic), 1666 (C=O), 1562 (C=C); ¹H NMR (DMSO-d₆, 400 MHz): δ 5.03 (s, 2H, CH₂), 7.06 (d, J = 7.88 Hz, 1H, aromatic H), 7.11 (d, J = 7.44 Hz, 1H, aromatic H), 7.22–7.29 (m, 4H, aromatic H), 7.32–7.38 (m, 4H, aromatic H), 7.54 (dd, J = 9.04, 4.76 Hz, 2H, aromatic H), 7.61 (d, J = 7.24 Hz, 1H, aromatic H), 12.70 (s, 1H, NH, D₂O exchangeable); ¹³C NMR (DMSO-d₆, 100 MHz): δ 42.79 (CH₂), 110.22, 116.33, 116.41, 116.47 116.70, 118.95, 121.04, 122.97, 126.92, 127.82, 127.99, 128.81, 129.17, 136.81, 139.59, 139.61, 140.51, 157.66 (aromatic carbons), 160.04 (C-F), 161.50 (C=O); Anal. calcd. for C₂₁H₁₆FN₃O (345.38): C, 73.03; H, 4.67; N, 12.17. Found: C, 72.89; H, 4.62; N, 12.35.

1-Benzyl-3-(2-[p-tolyl] hydrazineylidene) indolin-2-one (7d)

Yellow powder, yield (93%), m.p. 177°–179°C; IR (KBr, ʋ cm^-1): 3221 (NH stretching), 3024 (CH aromatic), 2916 (CH aliphatic), 1689 (C=O), 1666 (C=O), 1554 (C=C); ¹H NMR (DMSO-d₆, 400 MHz): δ 2.29 (s, 3H, CH₃), 5.03 (s, 2H, CH₂), 7.05 (d, J = 7.84 Hz, 1H, aromatic H), 7.10 (d, J = 7.48 Hz, 1H, aromatic H), 7.20–7.22 (m, 2H, aromatic H), 7.24–7.29 (m, 2H, aromatic H), 7.32–7.39 (m, 6H, aromatic H), 7.61 (d, J = 7.32 Hz, 1H, aromatic H), 12.72 (s, 1H, NH, D₂O exchangeable); ¹³C NMR (DMSO-d₆, 100 MHz): δ 20.88 (CH₃), 42.76 (CH₂), 110.19, 114.79, 118.79, 121.11, 122.92, 126.31, 127.82, 127.97, 128.54, 129.16, 130.40, 132.83, 136.86, 140.29, 140.59 (aromatic carbons), 161.66 (C=O); Anal. calcd. for C₂₂H₁₉N₃O (341.41): C, 77.40; H, 5.61; N, 12.31. Found: C, 77.23; H, 5.80; N, 12.49.

1-Benzyl-5-fluoro-3-(2-phenylhydrazineylidene) indolin-2-one (7f)

Yellow powder, yield (96%), m.p. 135°–137°C; IR (KBr, ʋ cm^-1): 3452 (NH stretching), 3032 (CH aromatic), 2927 (CH aliphatic), 1674 (C=O), 1562 (C=C); ¹H NMR (DMSO-d₆, 400 MHz): δ 5.03 (s, 2H, CH₂), 7.03–7.12 (m, 3H, aromatic H), 7.26–7.30 (m, 1H, aromatic H), 7.32–7.36 (m, 4H, aromatic H), 7.37–7.42 (m, 2H, aromatic H), 7.45 (dd, J = 8.36, 2.48 Hz, 1H, aromatic H), 7.52 (d, J = 7.76 Hz, 2H, aromatic H), 12.76 (s, 1H, NH, D₂O exchangeable); ¹³C NMR (DMSO-d₆, 100 MHz): δ 42.86 (CH₂), 106.01, 106.27, 111.17, 111.25, 114.72, 114.96, 115.08, 122.43, 124.05, 126.40, 126.43, 127.82, 128.03, 129.13, 129.19, 129.95, 136.64, 142.66 (aromatic carbons), 160.31 (C-F), 161.69 (C=O); Anal. calcd. for C₂₁H₁₆FN₃O (345.38): C, 73.03; H, 4.67; N, 12.17. Found: C, 72.91; H, 4.85; N, 12.41.

1-Benzyl-3-(2-[4-chlorophenyl] hydrazineylidene)-5-fluoroindolin-2-one (7g)

Yellow powder, yield (92%), m.p. 205°–207°C; IR (KBr, ʋ cm^-1): 3232 (NH stretching), 3028 (CH aromatic), 2947 (CH aliphatic), 1662 (C=O), 1599 (C=C); ¹H NMR (DMSO-d₆, 400 MHz): δ 5.02 (s, 2H, CH₂), 7.03–7.13 (m, 2H, aromatic H), 7.27–7.29 (m, 1H, aromatic H), 7.32–7.37 (m, 4H, aromatic H), 7.42–7.47 (m, 3H, aromatic H), 7.57 (d, J = 8.92 Hz, 2H, aromatic H), 12.71 (s, 1H, NH, D₂O exchangeable); ¹³C NMR (DMSO-d₆, 100 MHz): δ 42.89 (CH₂), 106.23, 106.49, 111.22, 111.32, 115.01, 115.25, 116.72, 122.32, 127.03, 127.54, 127.84, 128.03, 129.18, 129.72, 136.62, 136.88, 141.82, 157.95 (aromatic carbons), 160.31 (C-F), 161.53 (C=O); MS, m/z: 380.88 (M⁺); Anal. calcd. for C₂₁H₁₅ClFN₃O (379.82): C, 66.41; H, 3.98; N, 11.06. Found: C, 66.57; H, 4.17; N, 11.28.

1-Benzyl-5-fluoro-3-(2-[4-fluorophenyl] hydrazineylidene) indolin-2-one (7h)

Yellow powder, yield (95%), m.p. 189°–190°C; IR (KBr, ʋ cm^-1): 3244 (NH stretching), 3035 (CH aromatic), 2974 (CH aliphatic), 1674 (C=O), 1566 (C=C); ¹H NMR (DMSO-d₆, 400 MHz): δ 5.02 (s, 2H, CH₂), 7.02–7.11 (m, 2H, aromatic H), 7.22–7.29 (m, 3H, aromatic H), 7.32–7.35 (m, 4H, aromatic H), 7.44 (dd, J = 8.36, 2.52 Hz, 1H, aromatic H), 7.57 (dd, J = 9.08, 4.76 Hz, 2H, aromatic H), 12.73 (s, 1H, NH, D₂O exchangeable); ¹³C NMR (DMSO-d₆, 100 MHz): δ 42.86 (CH₂), 106.00, 106.25, 111.13, 114.69, 114.93, 116.45, 116.68, 122.46, 122.56, 126.33, 127.68, 127.83, 128.01, 129.17, 136.63, 136.67, 139.41, 157.93 (aromatic carbons), 160.29 (C-F), 161.55 (C=O); Anal. calcd. for C₂₁H₁₅F₂N₃O (363.37): C, 69.41; H, 4.16; N, 11.56. Found: C, 69.63; H, 4.25; N, 11.74.

1-Benzyl-5-fluoro-3-(2-[p-tolyl] hydrazineylidene) indolin-2-one (7i)

Yellow powder, yield (93%), m.p. 178°–180°C; IR (KBr, ʋ cm^-1): 3224 (NH stretching), 3028 (CH aromatic), 2916 (CH aliphatic), 1658 (C=O), 1593 (C=C); ¹H NMR (DMSO-d₆, 400 MHz): δ 2.30 (s, 3H, CH₃), 5.03 (s, 2H, CH₂), 7.02–7.10 (m, 2H, aromatic H), 7.21 (d, J = 8.28 Hz, 2H, aromatic H), 7.27–7.29 (m, 1H, aromatic H), 7.32–7.35 (m, 4H, aromatic H), 7.41–7.44 (m, 3H, aromatic H), 12.76 (s, 1H, NH, D₂O exchangeable); ¹³C NMR (DMSO-d₆, 100 MHz): δ 20.89 (CH₃), 42.83 (CH₂), 105.78, 106.04, 111.13, 114.39, 114.63, 115.05, 122.52, 125.71, 127.68, 127.83, 127.99, 129.16, 130.35, 133.26, 136.38, 136.72, 140.36, 157.93 (aromatic carbons), 160.29 (C-F), 161.73 (C=O); Anal. calcd. for C₂₂H₁₈FN₃O (359.40): C, 73.52; H, 5.05; N, 11.69; Found: C, 73.69; H, 5.17; N, 11.47; HRMS (ESI/Q-TOF) m/z: [M + Na]⁺ Calcd. for C₂₃H₁₇ClN₂O₄: 382.1330. Found: 382.1322.

4- (2-[1-Benzyl-5-fluoro-2-oxoindolin-3- ylidene] hydrazineyl) benzenesulfonamide (7j)

Yellow powder, yield (97%), m.p. 270°–272°C; IR (KBr, ʋ cm^-1): 3379, 3317, 3259 (NH, NH₂ stretching), 3070 (CH aromatic), 2924 (CH aliphatic), 1681 (C=O), 1573 (C=C), 1350, 1157 (SO₂); ¹H NMR (DMSO-d₆, 400 MHz): δ 5.03 (s, 2H, CH₂), 6.99, 7.06 (2dd, J = 8.64, 4.44 Hz, 1H, aromatic H), 7.11–7.23 (m, 1H, aromatic H), 7.28 (d, J = 7.28 Hz, 3H, aromatic H), 7.33 (d, J = 4.68 Hz, 2H, aromatic H), 7.36, 7.38 (2s, 2H, NH₂, D₂O exchangeable), 7.50 (dd, J = 8.96, 2.64 Hz, 1H, aromatic H), 7.68 (dd, J = 8.96, 2.64 Hz, 2H, aromatic H), 7.81–7.86 (m, 2H, aromatic H), 10.91, 12.78 (2s, 1H, NH, D₂O exchangeable); ¹³C NMR (DMSO-d₆, 100 MHz): δ 42.94 (CH₂), 106.65, 106.91, 111.46, 114.82, 115.57, 115.80, 122.11, 122.20, 127.68, 127.83, 128.07, 128.46, 129.21, 136.47, 137.29, 138.65, 145.41, 157.98 (aromatic carbons), 160.34 (C-F), 161.51 (C=O); Anal. calcd. for C₂₁H₁₇FN₄O (424.45): C, 59.43; H, 4.04; N, 13.20. Found: C, 59.69; H, 4.12; N, 13.49.

1-Benzyl-5-methoxy-3-(2-phenylhydrazineylidene) indolin-2-one (7k)

Yellow powder, yield (97%), m.p. 155°–157°C; IR (KBr, ʋ cm^-1): 3232 (NH stretching), 3024 (CH aromatic), 2939 (CH aliphatic), 1662 (C=O), 1593 (NH bending), 1558 (C=C); ¹H NMR (DMSO-d₆, 400 MHz): δ 3.77 (s, 3H, OCH₃), 4.99 (s, 2H, CH₂), 6.83 (dd, J = 8.60, 2.56 Hz, 1H, aromatic H), 6.95 (d, J = 8.60 Hz, 1H, aromatic H), 7.07 (t, J = 7.28 Hz, 1H, aromatic H), 7.20 (d, J = 2.52 Hz, 1H, aromatic H), 7.27–7.29 (m, 1H, aromatic H), 7.32–7.41 (m, 6H, aromatic H), 7.50 (d, J = 7.68 Hz, 2H, aromatic H), 12.75 (s, 1H, NH, D₂O exchangeable); ¹³C NMR (DMSO-d₆, 100 MHz): δ 42.79 (CH₂), 56.04 (OCH₃), 104.54, 110.97, 114.68, 114.79, 121.91, 123.66, 127.27, 127.79, 127.96, 129.15, 129.94, 134.32, 136.84, 142.82 (aromatic carbons), 156.07 (C-O), 161.72 (C=O); Anal. calcd. for C₂₂H₁₉N₃O₂ (357.15): C, 73.93; H, 5.36; N, 11.76. Found: C, 73.93; H, 5.36; N, 11.76.

1-Benzyl-3-(2-[4-chlorophenyl] hydrazineylidene)-5-methoxyindolin-2-one (7l)

Yellow powder, yield (92%), m.p. 175°–177°C; IR (KBr, ʋ cm^-1): 3294 (NH stretching), 3032 (CH aromatic), 2943 (CH aliphatic), 1689 (C=O), 1631 (NH bending), 1562 (C=C); ¹H NMR (DMSO-d₆, 400 MHz): δ 3.77 (s, 3H, OCH₃), 4.98 (s, 2H, CH₂), 6.82–6.95 (m, 2H, aromatic H), 7.20 (d, J = 2.44, 1H, aromatic H), 7.25–7.29 (m, 1H, aromatic H), 7.32–7.35 (m, 4H, aromatic H), 7.41–7.44 (m, 2H, aromatic H), 7.52–7.57 (m, 2H, aromatic H), 12.70 (s, 1H, NH, D₂O exchangeable); ¹³C NMR (DMSO-d₆, 100 MHz): δ 42.80 (CH₂), 56.02 (OCH₃), 104.68, 110.98, 114.93, 116.36, 121.75, 127.10, 127.79, 127.90, 129.15, 129.69, 134.51, 136.77, 141.89 (aromatic carbons), 156.07 (C-O), 161.50 (C=O); Anal. calcd. for C₂₂H₁₈ClN₃O₂ (391.86): C, 67.43; H, 4.63; N, 10.72. Found: C, 67.28; H, 4.80; N, 10.91.

1-Benzyl-3-(2-[4-fluorophenyl] hydrazineylidene)-5-methoxyindolin-2-one (7m)

Yellow powder, yield (93%), m.p. 185°C; IR (KBr, ʋ cm^-1): 3244 (NH stretching), 3032 (CH aromatic), 2954 (CH aliphatic), 1662 (C=O), 1597 (NH bending), 1566 (C=C); ¹H NMR (DMSO-d₆, 400 MHz): δ 3.77 (s, 3H, OCH₃), 4.99 (s, 2H, CH₂), 6.81 (dd, J = 8.60, 2.56 Hz, 1H, aromatic H), 6.95 (d, J = 8.56 Hz, 1H, aromatic H), 7.19–7.29 (m, 4H, aromatic H), 7.32–7.35 (m, 4H, aromatic H), 7.52–7.56 (dd, J = 9.04, 4.76 Hz, 2H, aromatic H), 12.72 (s, 1H, NH, D₂O exchangeable); ¹³C NMR (DMSO-d₆, 100 MHz): δ 42.78 (CH₂), 56.05 (OCH₃), 104.53, 110.97, 114.68, 116.45, 116.68, 121.91, 127.18, 127.78, 127.97, 129.16, 134.30, 136.83, 139.52 (aromatic carbons), 156.06 (C-O), 161.60 (C=O); Anal. calcd. for C₂₂H₁₈FN₃O₂ (375.40): C, 70.39; H, 4.83; N, 11.19. Found: C, 70.15; H, 5.01; N, 11.28.

1-Benzyl-5-methoxy-3-(2-[p-tolyl] hydrazineylidene) indolin-2-one (7n)

Yellow powder, yield (90%), m.p. 172°–174°C; IR (KBr, ʋ cm^-1): 3226 (NH stretching), 3028 (CH aromatic), 2943 (CH aliphatic), 1658 (C=O), 1589 (NH bending), 1558 (C=C); ¹H NMR (DMSO-d₆, 400 MHz): δ 2.29 (s, 3H, CH₃), 3.77 (s, 3H, OCH₃), 4.99 (s, 2H, CH₂), 6.81 (dd, J = 8.56, 2.48 Hz, 1H, aromatic H), 6.94 (d, J = 8.56 Hz, 1H, aromatic H), 7.18–7.21 (m, 3H, aromatic H), 7.25–7.29 (m, 1H, aromatic H), 7.33–7.34 (m, 4H, aromatic H), 7.39 (d, J = 8.32 Hz, 2H, aromatic H), 12.75 (s, 1H, NH, D₂O exchangeable); ¹³C NMR (DMSO-d₆, 100 MHz): δ 20.89 (CH₃), 42.78 (CH₂), 56.05 (OCH₃), 104.34, 110.96, 114.52, 114.82, 122.00, 126.61, 127.81, 127.93, 129.15, 130.38, 132.83, 134.15, 136.94, 140.56 (aromatic carbons), 156.06 (C-O), 161.76 (C=O); Anal. calcd. for C₂₃H₂₁N₃O₂ (371.44): C, 74.37; H, 5.70; N, 11.31. Found: C, 74.24; H, 5.62; N, 11.47.

4-(2-[1-Benzyl-5-methoxy-2-oxoindolin-3-ylidene] hydrazineyl) benzenesulfonamide (7o)

Yellow powder, yield (95%), m.p. 263°–265°C; IR (KBr, ʋ cm^-1): 3360, 3182 (NH, NH₂ stretching), 3078 (CH aromatic), 2935 (CH aliphatic), 1701 (C=O), 1627 (NH bending), 1581 (C=C), 1319, 1153 (SO₂); ¹H NMR (DMSO-d₆, 400 MHz): δ 3.78, 3.80 (2s, 3H, OCH₃), 4.96 (s, 2H, CH₂), 6.85–6.98 (m, 2H, aromatic H), 7.25 (s, 2H, NH₂, D₂O exchangeable), 7.27 (d, J = 2.04 Hz, 1H, aromatic H), 7.33–7.36 (m, 4H, aromatic H), 7.65 (t, J = 8.72 Hz, 2H, aromatic H), 7.83 (t, J = 8.84 Hz, 2H, aromatic H), 7.95 (d, J = 2.04 Hz, 1H, aromatic H), 10.87 (s, 1H, NH, D₂O exchangeable); ¹³C NMR (DMSO-d₆, 100 MHz): δ 42.85 (CH₂), 56.10 (OCH₃), 105.11, 111.17, 114.50, 115.20, 115.49, 121.55, 127.66, 127.81, 127.85, 128.00, 129.17, 129.33, 134.93, 136.68, 138.28, 145.55 (aromatic carbons), 156.16 (C-O), 161.55 (C=O); Anal. calcd. for C₂₂H₂₀N₄O₄S (436.49): C, 60.54; H, 4.62; N, 12.84. Found: C, 60.72; H, 4.83; N, 13.06.

Biological activity

In vivo screening

The procedures followed for the in vivo screening were done according to the standard guidelines. The treatment and handling protocol with the animals that was used was approved by the Animal Rights Committee of Faculty of Pharmacy, Cairo University (PC 3161).

The procedures implemented for in vivo studies are outlined in the supplementary data under Supplementary Section 16.

In vitro screening

The procedures followed for in vitro screening are outlined in the supplementary data under Supplementary Sections 17 & 18.

Molecular docking study

The detailed methods are provided in the supplementary data under Supplementary Section 20.

In vivo pharmacokinetic study

The details regarding the instrumentation used for chromatographic analysis and the experimental procedure are incorporated into the supplementary data under Supplementary Section 21.

Results & discussion

Chemistry

Synthesis of the target compounds proceeded as illustrated in Figure 2. The key intermediates 3a–d were obtained by refluxing commercially available isatin derivatives 1a–d with hydrazine hydrate for 4 h [26]; then the resultant oxindoles, 2a–d, were reacted with ethyl formate and in the presence of sodium ethanolate to obtain known 3a–d [27]. Targeted ester derivatives 4a–p were synthesized from the reaction of the obtained intermediates, 3a–d, with appropriate benzoyl chloride derivatives in the presence of TEA using anhydrous diethyl ether as solvent (Figure 2A).

Figure 2.

Synthetic pathways for the target compounds.

(A) Synthesis of target compounds 4a–p, reagents and reaction condition. (i) Hydrazine hydrate, reflux. (ii) Ethyl formate, sodium ethanolate, reflux, 30 min. (iii) Diethyl ether, triethylamine, stir, room temperature (r.t.), 24 h. (B) Synthesis of target compounds 6a–l and 7a–o, reagents and reaction condition. (i) Hydroxyl amine, water and ethanol mix, reflux. (ii) Diethyl ether, triethylamine, stir, r.t., 24 h. (iii) Methanol, stir, r.t., 2 h.

Figure 2B represents two synthetic pathways, one of them starting from the reaction of benzyl isatin derivatives 1b–d with hydroxyl amine in water/ethanol mixture to get ketoximes 5a–c [29], followed by the reaction with appropriate benzoyl chloride derivatives in diethyl ether with TEA to finally afford the target compounds 6a–l, while the other pathway shows direct reaction of 1b–d with appropriate phenyl hydrazine derivatives in methanol with a few drops of acetic acid to get 7a–o derivatives. Structures of all newly synthesized compounds were confirmed by spectral and elemental analyses, as mentioned in detail in the experimental part.

Biological activity

In vivo screening

Anti-inflammatory activity

The anti-inflammatory activity of all synthesized compounds was tested using the carrageenan-induced rat paw edema method [32]. The extent to which the tested compounds could reduce edema induced by injection of rat paw with carrageenan was evaluated using indomethacin as the reference standard. Almost all the tested compounds showed more potent anti-inflammatory effect than indomethacin except for isatin esters bearing 5-methoxy group 4m–o, which had low activity, while 4p revealed moderate activity compared with the used reference standard, indomethacin, at 2 h. On the other hand, ketoxime esters 6a–l exhibited high and prolonged protection from inflammation that reached to 100% inhibition, such as 6f and 6l after 1 h and compound 6j after 2 h (Supplementary Table 1).

Structure–activity relationship

Based on the in vivo anti-inflammatory activity results of the tested compounds and indomethacin, the activity was studied at 2 h (the time at which the reference showed its maximum anti-inflammatory activity), and the following structure–activity relationship was revealed: N-unsubstituted esters 4a–d showed anti-inflammatory activity higher than that of indomethacin with % inhibition ranging from 57.00 to 68.60%; also, it was noticed that p-fluoro and methoxy benzoyl derivatives 4c and 4d revealed higher inhibition than other congeners. The presence of the hydrophobic benzyl moiety was found to be preferable for the activity, as shown in N-benzyl oxindole esters 4e–h, which revealed higher activity than N-unsubstituted esters 4a–d; their % inhibition ranged between 64.90 and 73.84%, and compound 4h with p-methoxy substituent revealed the best activity (73.84%) compared with the other synthesized esters. Nonetheless, benzyl oxindole esters bearing 5-fluoro substituent 4i–l revealed lower activity than esters 4a–h, while 4m–p esters with 5-methoxy substituent almost lost activity compared with the reference standard, indomethacin and the other synthesized esters.

Replacing the benzyl oxindole ester side chain with ketoxime ester in 6a–l was found to be most preferable for activity compared with the others; 6a–l exhibited very potent anti-inflammatory activity and prolonged action, especially 5-fluoro and 5-methoxy oxindoles 6e–l; their inhibition ranged from 75.15 to 100.00%. Interestingly, compound 6a bearing an unsubstituted benzoyl moiety showed better activity than 6b and c with chloro or fluoro benzoyl moiety, but its activity was equipotent to that of 6d with a methoxy benzoyl moiety. Moreover, compound 6f with a chloro benzoyl moiety revealed 97.38% inhibition which surpassed that of other 5-fluoro oxindole ketoxime esters, while its 5-methoxy oxindole analogue, 6j, showed 100% inhibition, so the presence of p-chloro substituent at the benzoyl moiety enhanced the activity. In addition, hydrophobic substitution of oxindole in 5-position was found to increase the activity.

Schiff's bases 7a–o also showed good anti-inflammatory activity. As can be noticed, derivatives 7b and 7c with chloro or fluoro substituent (electron-withdrawing group at para position) showed very high potency with 85.00 and 81.60% inhibition, respectively; which surpassed that of other derivatives and the reference, indomethacin. In addition, benzenesulfonamide derivative 7e revealed 76.40% inhibition with prolonged action. Nevertheless, 5-fluoro isatin phenylhydrazineyl derivatives 7f–h showed lower activity than their unsubstituted analogues, 7a–c, except that 7i with p-tolyl substituent revealed activity higher than its analogue, 7d. Also, benzenesulfonamide derivative 7j with 76.47% inhibition was found to be equipotent to its analogue, 7e. 5-Methoxy isatin Schiff's derivatives 7k–m were nearly equipotent with their unsubstituted analogues, 7a–c, while 5-methoxy oxindole 7n bearing a p-tolyl benzoyl moiety showed 82.35% inhibition, which exceeded that of the other p-tolyl derivatives, 7d and 7i, with 42.43 and 69.90% inhibition, respectively. This series with imine linkage showed higher activity than ester series 4a–p but not higher than ketoxime esters 6a–l.

Analgesic activity

Selected compounds from each series (4d, 4h, 4l, 4n, 6a, 6d, 6f, 7b, 7e, 7j and 7m) were screened for their analgesic activity using the reported method of acetic acid-induced abdominal writhing in mice [33], and indomethacin was used as the reference drug. All compounds revealed promising analgesic potentiality which surpassed that of indomethacin (77.14%), as their protection percentages ranged from 78.20 to 100%. All tested esters revealed very potent analgesic activity, especially 5-fluoro oxindole ester 4l, which showed 100% protection. Moreover, both ketoxime esters 6a and 6f showed interesting analgesic activity with 98.28% protection. Benzenesulfonamide derivative 7j with imine linkage also revealed 100% protection, as presented in Supplementary Table 2.

Toxicological studies

The most active anti-inflammatory agents, 4h, 6d, 6f, 6j and 7m, were investigated for their toxicological effect using the standard method [34]. All tested compounds showed a high safety margin with no mortality after a 24 h observation period following intraperitoneal injection of the animals with doses up to tenfold of the used anti-inflammatory dose (0.28 mmol/kg).

Ulcerogenic effect

The most active compounds, 4h, 6d, 6f, 6j and 7m, were also investigated for their ulcerogenicity and compared with the reference, indomethacin, using the standard method [35]. Their ulcer index was calculated according to the reported method [36] using indomethacin as reference. All tested compounds showed lower ulcer indices than indomethacin, with decreased ulcer incidence and severity, except for compound 7m, which showed an ulcer index very close to that of indomethacin. Methoxy ester 4h and ketoxime esters 6d and 6j exhibited much better gastrointestinal tract tolerance with ulcer indexes of 4.20, 4.36 and 6, respectively (Supplementary Table 3).

Histopathological examination

For further investigation of ulcer severity of the most active compounds, 4h, 6d, 6f and 6j, microscopic examination of the fundic region of the rats' gastric mucosa was carried out and compared with the ulcer severity of the reference, indomethacin [37]. In addition, mucosal and submucosal inflammation was evaluated, and the intensity of the inflammation was scored from 0 to 3 [38].

Representative tissue sections of fundic region from several treatment groups were prepared and examined, as shown in Figure 3. The control group was found with the normal architecture of the fundic region lined by pale with basal oval nuclei mucosal cells. The fundic glands were lined with mucous neck cells, oxyntic cells and peptic cells and they opened to the surface in gastric pits. The mucous neck cells were simple columnar epithelium with pale staining. The oxyntic cells were large and rounded with extensive acidophilic cytoplasm and central rounded nuclei. The peptic cells had basophilic cytoplasm; their nuclei were basal and rounded, score 0 (Figure 3A).

Figure 3.

Effect of the treatment with different compounds on histopathological alterations of the gastric mucosal tissue.

Photomicrograph of hematoxylin and eosin-stained sections of stomach from the control group (A), indomethacin-treated group (B), 4h-treated group (4h), 6d-treated group (6d), 6f-treated group (6f) and 6j-treated group (6j) with 200× magnification power (inside box 400×). (A) Intact gastric mucosal epithelial lining (black arrow – 200×) and normal histological profile of gastric glands (black arrow – 400×). (B) Exfoliation of epithelium lining and leukocytic infiltration (blue arrow – 200×) and deep ulceration reach to lamina muscularis mucosa (blue arrow – 400×). (4h) Intact surface mucosal epithelium with low numbers of leukocytic infiltration (red arrow – 200×) and gastric glands with mild swelling of mucous cells (red arrow – 400×). (6d) Desquamation of surface epithelial lining with intact subepithelial glands (green arrow – 200×) and submucosal and muscular edema and congestion with low numbers of leukocytic infiltration (green arrow – 400×). (6f) Desquamation of surface epithelial lining with intact subepithelial glands (yellow arrow – 200×) and submucosal edema and severe congestion with leukocytic infiltration (yellow arrow – 400×). (6j) Intact surface mucosal epithelial lining (orange arrow – 200×) and edema with low numbers of leukocytic infiltration (orange arrow – 400×).

The group that received indomethacin revealed marked damage and exfoliation of the epithelial surface. Erosions and deep ulcerations reach lamina muscularis mucosa were noticed, score 3. The gastric glands showed disturbance in architecture with necrosis. Infiltration of leukocytes, mainly lymphocytes and macrophages in the mucosal and submucosal layers, was seen, score 3, with necrosis of the gastric glands, atrophy of the gastric folds, submucosal edema and congestion (Figure 3B).

The methoxy ester 4h-treated group showed intact surface mucosal epithelium in comparison with the reference group, score 0. Submucosal edema and congestion with low numbers of leukocytic infiltration, mainly lymphocytes and macrophages, score 1. On the other hand, the gastric glands appeared intact with mild swelling of mucous cells, which appeared as pale with central round nuclei (Figure 3, 4h).

Examination of tissues from the group treated with ketoxime ester 6d showed desquamation of the surface epithelial lining with intact subepithelial glands, score 2. Submucosal edema and low numbers of leukocytic infiltration, mainly lymphocytes and macrophages, was noticed, score 1 (Figure 3, 6d).

Treatment with the other ketoxime derivative, 6f, with a chloro benzoyl moiety showed desquamation of the surface mucosal epithelial lining with intact subepithelial glands, score 2. Submucosal edema and leukocytic infiltration, score 1, and severe congestion of blood capillaries with leukocytic infiltration, mainly in lymphocytes and macrophages, was noticed, (Figure 3, 6f). Moreover, the gastric mucosa of rats treated with 6j showed more intact surface mucosal epithelium in comparison with previous groups, score 0. Submucosal and muscular edema and congestion with low numbers of leukocytic infiltration, mainly lymphocytes and macrophages, also were noticed, score 1. The gastric glands appeared to be intact, which were pale with central round nuclei (Figure 3, 6j). The histopathological features score is recorded in Supplementary Table 4.

From the aforementioned results, it is obvious that all examined groups of the newly synthesized compounds showed a lower score than the indomethacin group, which supported the calculated ulcer index values of the compounds and confirmed their superior safety and tolerability over the reference drug. Also, ester 4h and ketoxime ester 6j were found to have the most tolerable effect on gastric mucosa.

In vitro COX enzyme inhibition assay

The most active compounds 4h, 6d and 6j were examined for their ability to inhibit COX-1 and COX-2 in vitro. As presented in Table 1, the methoxy ester 4h was found to have potent inhibitory activity for COX-2 with IC₅₀ = 0.0533, and it showed better selectivity index (3.07) than that of indomethacin (0.53). On the other hand, ketoxime esters 6d and 6j exhibited inhibitory activity toward COX-1 and COX-2 but with lower potency than that of reference standards indomethacin and celecoxib. Also, 6d was preferentially selective toward COX-2 with selectivity index = 1.38, which was also higher than that of indomethacin.

Table 1.

IC₅₀ and selectivity index values of the tested compounds, celecoxib and indomethacin as COX-1, COX-2 and 5-LOX inhibitors.

Compound number	IC50 (μM)† ± SD		Selectivity index‡	IC50 (μM) ± SD
	COX-1	COX-2		5-LOX
Celecoxib	0.4016 ± 0.0050	0.0445 ± 0.0040	9.02	0.8373 ± 0.0080
Indomethacin	0.0386 ± 0.0050	0.0726 ± 0.0060	0.53	2.6080 ± 0.0080
4h	0.1641 ± 0.0040	0.0533 ± 0.0040	3.07	0.4195 ± 0.0050
6d	0.1213 ± 0.0050	0.0875 ± 0.0060	1.38	0.2925 ± 0.0050
6j	0.07408 ± 0.0060	0.1750 ± 0.0050	0.42	0.2577 ± 0.0040

^†IC₅₀ (presented as mean ± SD) is the needed concentration of a compound to inhibit 50% of COX-1, COX-2 and 5-LOX enzymatic activity.

^‡Selectivity index = COX-1 IC₅₀ divided by COX-2 IC₅₀.

In vitro 5-LOX enzyme inhibition assay

Compounds 4h, 6d and 6j were also examined in vitro for their ability to inhibit 5-LOX. Both 6d and 6j showed potent 5-LOX inhibitory activity with IC₅₀ of 0.2925 and 0.2577 μM, which surpassed that of indomethacin and celecoxib. In addition, compound 4h with IC₅₀ = 0.4195 μM showed higher activity than that of the reference standards indomethacin and celecoxib (IC₅₀ = 2.6080 and 0.8373, respectively; Table 1), so their promising in vivo anti-inflammatory activity may be due to their potent inhibitory activity of 5-LOX.

 Molecular docking study

To justify the obtained COXs/5-LOX inhibition results, molecular docking simulations for 4h, 6d and 6j was performed using Protein Data Bank 4O1Z [39], 3LN1 [40] and 3O8Y [41] for COX-1, COX-2 and 5-LOX, respectively. The molecular docking protocols were validated before proceeding to the actual simulation process by redocking the corresponding cocrystallized ligand to get the lowest possible root-mean-square deviation (Supplementary Figures 1 & 2). The webserver DockRMSD [42] was used to calculate the corresponding validation root-mean-square deviation values, which were 0.75 and 0.73 Å for COX-1 and COX-2, respectively. Both the protein crystals and the tested derivatives were prepared and protonated as detailed in the experimental section, giving the results illustrated in Supplementary Tables 5–7 & Supplementary Figures 3–7 & Figures 4 and 5.

Figure 4.

Binding interactions of 4h.

2D interactions of 4h (A). The 3D conformation of 4h as a green stick model relative to the red-colored celecoxib with the hydrophobic surface appearing as solid brown-colored surrounding the compounds at (B).

Figure 5.

2D interaction of 6d (A) and 6j (B) with 5-LOX (Protein Data Bank: 3O8Y).

As revealed by the COX-1 docking results, the best binding score was achieved by ketoxime ester 6j followed by 6d, which complied with their IC₅₀ values of 0.074 and 0.121 μM, respectively. The ketoxime ester 6j showed -7.20 kcal/mol, while the other derivative, 6d, demonstrated -6.80 kcal/mol binding energy with COX-1 (Supplementary Table 5). Moreover, the three tested derivatives, 4h, 6d and 6j, exhibited a hydrophobic binding pattern similar to the cocrystallized meloxicam (Supplementary Figures 3 & 4). Both 6d and 6j managed to form hydrogen bonds with Arg120, which is located at the entrance of the main hydrophobic channel of COX-1 with average bond distance of 2.50 Å (Supplementary Figure 4A & B) [43]. This binding would block the way against the natural substrate arachidonic acid, justifying their inhibition potentials. Moreover, the benzyl moiety of 6j managed to bind with the crucial Tyr385 in a pi–pi interaction that hindered its electron transferee role in enzyme activity. It is well established that upon arachidonic acid binding to COXs, ionic bonds are formed with Tyr355 and Arg120 by its carboxylate group where the electron-transfer process is maintained by Tyr385 [44]. Also, this benzyl moiety of 6j formed additional hydrophobic interactions with the main channel Leu352 and Trp387, which justified its astonishing inhibition (Supplementary Figure 4B) [45]. Furthermore, the para-chlorophenyl substitution of 6j formed three hydrophobic interactions with Val116, Leu 357 and Leu359. Also, the 6j binding conformation was maintained by the two H-bonds formed between the oxindole carbonyl group, imine nitrogen and Ser530 in addition to the hydrophobic interaction with Val349 (Supplementary Figure 4C). In a similar fashion, the carbonyl oxygen of the oxindole scaffold of 6d formed H-bonds with Ser530 and Leu531 beside the Van der Waals interactions with the hydrophobic residues, maintaining its conformation inside the COX-1 active site (Supplementary Figure 4A). On the other hand, 4h showed the crucial hydrophobic interaction with Leu523 and interactions with the main hydrophobic channel residues but at less favorable binding energy (-6.40 kcal/mol) than 6d and 6j, which accounted for its higher IC₅₀ value of 0.164 μM (Supplementary Figure 3B). Several pi–alkyl and alkyl interactions were observed with Leu535, Leu117, Leu531 and Ile345, among other residues. An additional pi–sigma bond was formed between the benzyl moiety of 4h and Val116 (Supplementary Figure 3B).

Interestingly, 4h showed the best COX-2 inhibition (IC₅₀ = 0.053 μM) among the three derivatives, which showed a binding pattern similar to its congener 6d (IC₅₀ = 0.087 μM) but with two additional hydrophobic interactions with Met99 and Tyr371 resembling the cocrystallized celecoxib (Figure 4A, B and Supplementary Figure 4A). Moreover, the binding energy of 4h was comparable to 6d, demonstrating -9.60 and -9.50 kcal/mol, respectively (Supplementary Table 6). On top of that, the phenyl group of both 4h and 6d oriented inside the side hydrophobic pocket similar to the benzenesulfonamide moiety of celecoxib exhibiting multiple interactions with Ser339, Leu338 and Val509 (Supplementary Figure 6A & B) [45]. In contrast, the other phenyl moieties of 4h showed hydrophobic interactions with the main hydrophobic cavity residues of COX-2, Tyr371, Trp373, Leu345 and Leu517 (Figure 4A). Furthermore, the carbonyl oxygen of both 4h and 6d linker formed an H-bond with Arg106 of average distance 1.85 Å to support their orientation inside the active site. A decrease in COX-2 inhibition was observed by adding a methoxy substitution to the phenyl group of the oxindole scaffold in 6j (IC₅₀ = 0.175 μM). As revealed from its predicted binding conformation, the methoxy group of 6j hindered the hydrophobic interactions with the side pocket residues Ser339, Leu338 and Val509 (Supplementary Figure 5B), which justified the decline in its COX-2 inhibition. Apparently, the achieved COX-2 inhibition of 6j resulted from the H-bond formation with Arg106 and the hydrophobic interaction with the main channel Tyr371 and Trp373.

In the same context, the 5-LOX inhibition of the promising derivatives was justified by their predicted binding conformation inside the 5-LOX active site (Figure 5, Supplementary Table 7 & Supplementary Figure 7). Certain inhibition of 5-LOX was observed with both 6d and 6j with IC₅₀ 0.293 and 0.257 μM. A similar binding pattern was revealed for both derivatives with close binding energy of -9.29 and -9.41 kcal/mol for 6d and 6j, respectively, explaining their comparable IC₅₀ values (Figure 5A and B). The linker moiety of both 6d and 6j formed an H-bond with the crucial Arg138 of the allosteric site of 5-LOX, hindering its activity (Figure 5A and B). In addition to the hydrophobic interactions with Ala388 and Leu111, both derivatives exhibited pi–cation interaction with Arg101 by their oxindole and benzyl moieties. Furthermore, the para-chloro substitution of 6j was involved in halogen interaction with Arg112, while the para-methoxy substitution of 6d bound to it through H-bonds. Unfavorable binding energy was obtained with 4h, demonstrating -8.29 kcal/mol, maintaining the H-bond with the crucial Arg138 and pi–cation bond with Arg101 that increased its IC₅₀ by twofold into 0.419 μM (Supplementary Figure 7).

In silico physicochemical, pharmacokinetic & drug-likeness evaluation

An in silico assessment of the physicochemical and pharmacokinetic properties of the promising derivatives 4h, 6d and 6j was implemented using the Swiss Institute of Bioinformatics SwissADME^® interface (Supplementary Table 8) [46]. All derivatives obeyed Lipinski's rule of five in terms of molecular weight and number of H-bond donor and acceptor groups. Lipinski's rule states that acceptable criteria are achieved by molecular weight less than 500 g/mol; H-bond donor and acceptor groups should not exceed 5 and 10, respectively [47,48]. Moreover, the partition coefficient values for all derivatives did not exceed 5, despite the use of several methods of calculation complying with Lipinski's rule (Supplementary Table 8). Furthermore, the number of rotatable bonds of the three derivatives was six, which obeyed Veber's rule for oral bioavailability and should not surpass 10 [49]. Another compliance with Veber's rule was observed in terms of topological polar surface area being less than 140 Å² for optimum oral absorption [50]. As predicted from their logP and topological polar surface area values, the three derivatives were expected to exhibit high gastric absorption for preferable oral administration. Nonetheless, they might show inhibitory effect on some of the hepatic cytochrome P-450 isoenzymes such as 1A2, 2C19, 2C9 and 3A4 but not CYP2D6. Additionally, their physicochemical properties could assist their blood–brain barrier penetration, which could guide their usage for CNS inflammation treatment. Fortunately, all derivatives were expected to be a poor substrate of the efflux P-glycoprotein pump that ultimately would favor their intracellular drug accumulation [51]. Despite that, there was no violation observed for the drug-likeness rules such as Lipinski's, Veber's and Egan's [47,49,52]. Egan's rule states that a drug-like property is achieved by a chemical derivative when possessing wLogP <5.88 and topological polar surface area <131.6 Å2 [52]. In addition, the high bioavailability scores of 0.85, 0.55 and 0.55 for 4h, 6d and 6j, respectively, supported the assumption of having good absorption [53] together with other rules.

In vivo pharmacokinetic study

Development of LC–MS/MS method

A new method was developed and validated using MS/MS for the quantification of compound 4h in rat plasma as being one of the most active compounds that showed anti-inflammatory activity with a high safety margin and its application to a pharmacokinetic study in rat plasma.

Method development

Choice of internal standard

Due to the loss that may happen during sample transfer, extraction and ionization effects, using an internal standard (IS) is required to compensate for any variation. In the present work, indomethacin was used as the IS due to structure similarity with the analyzed compound, 4h.

Mass spectrometric & chromatographic conditions

Spectrometric parameter optimization is the first step in the development of any analytical method to achieve the best shape and intensity of the signals of the analyzed compound and IS. ESI conditions were optimized for compound 4h and the IS by carrying out quadrupole full scans in positive ion determination mode. Collision energies of 15 and 20 eV for compound 4h and IS, respectively, were used for fragmentation. The most stable product ions for compound 4h and IS were at m/z 386.32→135.13 and 358.23→139.14, respectively (Figure 6).

Figure 6.

Mass spectrum for compound 4h.

Parent ion scan of compound 4h at m/z 386.32 and its product ion scan at m/z 135.13 (A). Parent ion scan of indomethacin (internal standard) at m/z 358.23 and its product ion scan at m/z139.14 (B).

In order to obtain the best peak shape and intensity, chromatographic conditions were optimized. As several mixtures of solvents of different percentages as methanol or acetonitrile and different buffers of different percentages as ammonium formate or 0.1% formic acid were tried to select the most appropriate mobile phase. It was found that a mixture of methanol:0.1% formic acid in water (95:5, v/v) gave the most sensitive response, best analyte elution and sharp peaks. Isocratic elution at a flow rate of 0.3 ml/min was applied. The optimized mass spectrometric conditions for the determination of compound 4h and the IS in rat plasma are summarized in Supplementary Table 9. The retention times of compound 4h and the IS were 2.85 and 2.18, respectively (Figure 7).

Figure 7.

Representative multiple reaction monitoring chromatograms of compound 4h and indomethacin (internal standard) in rat plasma.

A blank plasma sample (A); a plasma sample spiked with compound 4h at lower limit of quantification (10 ng/ml) (B); and the internal standard (zero sample) (C).

Extraction of plasma samples

Several sample preparation techniques were tried such as protein precipitation with acetonitrile or methanol; liquid–liquid extraction using different solvents as diethyl ether, ethyl acetate and dichloromethane; and mixtures of solvents with different volumes. Finally, the most appropriate and efficient technique was liquid–liquid extraction using a mixture of ethyl acetate:dichloromethane (55:45, v/v), as it was very efficient for the extraction of compound 4h and the IS from rat plasma with the highest response.

Method validation results

According to European Medicines Agency guidelines [54], full validation for the quantification of compound 4h in rat plasma was carried out.

Selectivity

The mass chromatograms of blank samples revealed no significant interference from matrix endogenous substances at the retention time of compound 4h and the IS (Figure 7A), which confirmed the selectivity of the method.

Linearity & lower limit of quantification

Evaluation of linearity for compound 4h was carried out over the concentration range of 10–1500 ng/ml. Six calibration standards were used to construct a calibration curve between peak area ratios (compound 4h/IS) and the corresponding concentrations. The regression coefficient was found to be 0.9991, which indicates good linear response. The intercept and slope were found to be 0.0106 and 0.0008, respectively.

Precision & accuracy

Intraday and interday precision and accuracy results for compound 4h are presented in Supplementary Table 10. The obtained values were found to be within the accepted range, displaying good accuracy and precision.

Carryover

No significant carryover was observed after the injection of high-concentration samples at the retention times of compound 4h and the IS.

Recovery (extraction efficiency)

The recovery values obtained by the developed method for compound 4h were 99.31, 91.03 and 96.91% at lower quality control, middle quality control and higher quality control concentrations, respectively. The results indicate that the developed extraction method was rationally efficient.

Matrix effect

The plasma components can influence the ionization of the analyte or the IS, which is why the matrix effect must be evaluated. The results of matrix effects, shown in Supplementary Table 11, indicate that the matrix components did not interfere with the analytes. It was found to be 1 and 1.12 for compound 4h at lower quality control and higher quality control, respectively, while the IS matrix factor was 1.01 at a concentration of 500 ng/ml. In addition, the IS normalized matrix factor was calculated for compound 4h.

Dilution effect

To check the dilution integrity of the method, samples with concentration above upper limit of quantification were diluted by two- and fourfold with blank plasma. The final concentrations obtained were found to be within the accepted range of accuracy and precision (107.71, 5.47%) after twofold dilution and (96.63, 15%) after fourfold dilution.

Stability studies

As can be concluded from the stability results of compound 4h presented in Supplementary Table 12, the analyte was found to be stable in rat plasma after being left for 6 h on benchtop (short-term stability), stored for 21 days at -80°C (long-term satbility), subjected to three freeze–thaw cycles and stored at autosampler for 24 h before being analyzed.

In vivo pharmacokinetic study in rats

The developed method was found specific and sensitive for the quantification of compound 4h in rat plasma, so it was used for pharmacokinetic study of compound 4h as one of the most active synthesized compounds with reasonable safety. The mean plasma concentration versus time profile of compound 4h after a single intraperitoneal injection of a 1 mg/kg dose is shown in Supplementary Figure 8.

Good pharmacokinetic parameters were obtained, as the maximum concentration (C_max) in rat plasma was achieved after 6 h (T_max) and found to be 171.37 ng/ml, while half-life was at 2.46 h and Area under the curve (AUC_0-t ) and (AUC_0-o)were 2069.33 and 2073.59 ng.h/ml, respectively. Also, the volume of distribution was 1.71 ml/kg. In addition, the clearance value was found to be 0.48 ml/h.kg, as it is represented by the sum of the individual organs' clearance value.

Conclusion

Novel oxindole derivatives were designed, synthesized and evaluated for their in vivo anti-inflammatory activity and analgesic potentiality. All the new target compounds, except for 5-methoxy oxindole esters 4m–p, showed very potent anti-inflammatory activity that ranged from 35 to 100% inhibition. The ketoxime ester 6a–l series was the most potent one as anti-inflammatory, especially 6j, which showed 100% inhibition. Also, promising analgesic activity was revealed, especially for 4l and 7j, which showed 100% protection with no writhing recorded in their mouse groups. Moreover, ulcer index calculation revealed superior safety of the newly invented compounds over the reference, as methoxy ester 4h and ketoxime ester 6h revealed a higher safety margin than that of indomethacin. Additionally, methoxy ester 4h exhibited dual inhibitory activity against COX-2/5-LOX, with IC₅₀ values of 0.0533 and 0.4195 μg for COX-2 and 5-LOX, respectively. It demonstrated a superior selectivity index (3.07) compared with indomethacin (0.53). Ketoxime esters 14d and 14j showed potent 5-LOX inhibitory activity with IC₅₀ of 0.2925 and 0.2577 μg, respectively.

Molecular docking simulations that were carried out for the most active compounds 4h, 6d and 6j in addition to the reference compounds (meloxicam and celecoxib), predicted the binding pattern and the binding affinity, also it rationalized the obtained experimental biological activity of the most active compounds in the active sites of COX-1, COX-2 and 5-LOX. Furthermore, their key physicochemical parameters were calculated, and the three compounds possessed promising predicted pharmacokinetic properties and drug-likeness.

Further, a new method was developed and validated for the quantification of compound 4h in rat plasma using MS/MS, that was found specific and sensitive to be applied to a pharmacokinetic study in rats.

Financial disclosure

The authors have no financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending or royalties.

Competing interests disclosure

The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending or royalties.

Writing disclosure

No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research

The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations.