Synthesis and pharmacological evaluation of aminoacetylenic isoindoline-1,3-dione derivatives as anti-inflammatory agents

2.1 Chemistry

Melting points were measured by Fischer–Johns melting Point Apparatus and DSC measured were carried out by using DSC-50 (Shimadzu, Japan). Infrared spectra (IR) were recorded, as potassium bromide (KBr) discs on a Nicolet Impact-400 FT-IR spectrophotometer. ¹H and ¹³C NMR were acquired with the aid of Bruker-DPX 300 MHz spectrometers with DMSO-d₆ as solvents and TMS as an internal standard. Elemental analysis was obtained using EU Elemental Analyzer.

2.2 Synthesis of 2-[prop-2-yn-1-yl]isoindoline-1,3-dione (ZM1)

A solution of potassium phthalimide (1.87 g, 0.01 mol) in 30 ml benzene was refluxed to 40 °C, propargyl bromide (1.4 g, 0.01 mol) was added drop wise to the solution during 30 min. The mixture was stirred for 2 h and then filtered. The solvent was removed under reduced pressure to afford the desired compound (1.2 g, 66.6%) as a white crystalline powder, m.p. (152–153) °C. IR (KBr, cm⁻¹): 3294 (≡CH, stretch), 3049 (ArH, stretch), 2245 (C≡C, stretch), 1766, 1720 (C⚌O, stretch), 1612, 1553, 1396 (Ar, C⚌C, stretch), 1000–900 (C⚌C, bending), 802, 694, 632, (ArH, bending). ¹H NMR (DMSO-d₆): δ, 3.24 (t, 1H, J = 2.01 Hz, C≡CH), 4.32 (d, 2H, J = 2.01 Hz, N–CH₂–C≡), 7.76–7.8 (m, 4H, ArH). ¹³C NMR (DMSO-d₆): δ, 27 (C⁵), 74 (C⁷), 78 (C⁶), 123 (C^1,1′), 131 (C^3,3′), 135 (C^2,2′), 167 (C^4,4′). Anal. Calcd. (C₁₁H₇NO₂): C, 73.50; H, 3.78; N, 7.56. Found: C, 73.4; H, 3.75; N, 7.52.

2.3 Synthesis of N-[4-(pyrrolidino-1-yl)-but-2-yn-1-yl]isoindoline-1,3-dione (ZM2)

A mixture of 2-[prop-2-yn-1-yl]isoindoline-1,3-dione (0.9 g, 0.1 mol), paraformaldehyde (0.15 g, 0.12 mol), pyrrolidine (0.78 g, 0.11 mol) and cuprous chloride catalytic amount (0.03 g) in peroxide-free dioxane 20 ml was refluxed for 1 h. After cooling, 100 ml of water was added and the crude product recrystallized from ethanol (5–10 ml) afforded the desired compound (1.6 g, yield 61.9%), m.p. (112–113) °C. IR (KBr, cm⁻¹): 2931, 2845 (ArH, stretch), 2260 (C≡C, stretch), 1720, 1755 (C⚌O, stretch), 1612.5, 1458 (Ar, C⚌C, stretch), 1080, 9412 (Ar, C⚌C, bending), 895, 725, 640 (ArH, bending). ¹H NMR (DMSO-d₆): δ, 1.9 (m, 4H, ¹⁰CH₂–^10′CH₂), 1.6 (m, 4H, ⁹CH₂–⁹CH₂), 3.05 (t, 2H, J = 2.4 Hz, ≡C–⁸CH₂–N), 3.66 (t, 2H, J = 2.4 Hz, ⁵CH₂), 7.7–8 (m, 4H, ArH). ¹³C NMR (DMSO-d₆): δ, 20 (C¹²), 24 (C¹¹), 26.2 (C^10′), 27.4 (C¹⁰), 54.4 (C^9′), 43.8 (C⁹), 52.9 (C⁸), 54.5 (C⁵), 78.2 (C⁶), 79.5 (C⁷), 123.8 (C^1,1′), 131.9 (C^3,3′), 135.2 (C^2,2′), 167.2 (C^4,4′). Anal. Calcd. (C₁₆H₁₆N₂O₂): C, 72.97; H, 6.75; N, 9.41. Found: C, 72.14; H, 6.72; N, 9.43.

2.4 Synthesis of N-[4-piperidino-1-yl]-but-2-yn-1-yl]isoindoline-1,3-dione (ZM3)

ZM3 was prepared following the same procedure for the synthesis of ZM2 afforded (1.7 g, 63%) as a white crystalline compound, m.p. (83–84) °C. IR (KBr, cm⁻¹): 2931 (ArH, stretch), 2252 (C≡C, stretch), 1720, 1755 (C⚌O, stretch), 1612, 1458 (Ar, C⚌C, stretch), 995, 941.2 (C⚌C, bending), 864, 786, 717, (Ar, H, bending). ¹H NMR (DMSO-d₆): δ, 1.53 (m, 2H, ¹¹CH₂–C), 1.59 (m, 4H, C–¹⁰CH₂–^10′CH₂), 1.64 (m, 4H, ⁹CH₂–^9′CH₂), 3.39 (t, 2H, J = 2.4 Hz, ⁸CH₂–N), 3.66 (t, 2H, J = 2.4 Hz, ⁵CH₂), 7.7–8 (m, 4H, ArH). ¹³C NMR (DMSO-d₆): δ, 24.03 (C¹¹), 25.8 (C^10,10′), 27.1 (C^9,9′), 52.9 (C⁸), 66.6 (C⁵), 74.2 (C⁶), 79.3 (C⁷), 123.8 (C^1,1′), 131.9 (C^3,3′), 135.2 (C^2,2′), 167 (C^4,4′). Anal. Calcd. (C₁₇H₁₈N₂O₂): C, 72.34; H, 6.30; N, 9.90. Found: C, 72.31; H, 6.27; N, 9.92.

2.5 Synthesis of N-[4-(2-methyl piperidino-1-yl)-but-2-yn-1-yl]isoindoline-1,3-dione (ZM4)

ZM4 was prepared following the same procedure described for the synthesis of ZM2 afforded (1.8 g, 63.1%) as a white crystalline compound, m.p. (105–107) °C. IR (KBr, cm⁻¹): 2931, 2845 (ArH, stretch), 2260 (C≡C, stretch), 1720, 1755 (C⚌O, stretch), 1612.5, 1458 (Ar, C⚌C, stretch), 1080, 9412 (Ar, C⚌C, bending) 895, 725, 640 (ArH, bending). ¹H NMR (DMSO-d₆): δ, 1.12 (d, 3H, J = 4.2 Hz, ¹²CH₃), 1.5 (m, 6H, ¹⁰CH₂–^10′CH₂–¹¹CH₂), 1.5 (m, 2H, ⁹CH₂), 1.64 (m, 1H, ^9′CH), 3.05 (t, ⁵CH₂, J = 2.4 Hz), 3.66 (t, 2H, ⁵CH₂, J = 2.4 Hz), 7.7–8.0 (m, 4H, ArH). ¹³C NMR (DMSO-d₆): δ, 20.0 (C¹²), 24 (C¹¹), 26.2 (C^10′), 27.4 (C¹⁰), 43.8 (C^9′), 52.9 (C⁸), 54.5 (C⁵), 78.2 (C⁶), 79.5 (C⁷), 123.8 (C^1,1′), 131.9 (C^3,3′), 135.2 (C^2,2′), 167.2 (C^4,4′). Anal. Calcd. (C₁₈H₂₀N₂O₂): C, 72.97; H, 6.75; N, 9.41. Found: C, 72.92; H, 6.73; N, 9.42.

2.6 Synthesis of N-[4-(2-azepan-1-yl)-but-2-yn-1-yl]isoindoline-1,3-dione (ZM5)

The title compound was prepared to according to synthetic procedure described for ZM2, affording (1.8 g, 62.5%) as a white crystalline product, m.p. (72–75) °C. IR (KBr, cm⁻¹): 2924, 2831 (ArH, stretch), 2225 (C≡C, stretch), 1712, 1755 (C⚌O, stretch), 1612, 1465, 1319 (Ar, C⚌C, stretch), 1111, 1087, 941 (Ar, C⚌C, bending), 840, 794, 717 (ArH, bending). ¹H NMR (DMSO-d₆): δ, 1.2–1.4 (m, 8H, ¹⁰CH₂–^10′CH₂–¹¹CH₂–^11′CH₂), 1.6 (m, 4H, ⁹CH₂–^9′CH₂), 3.03 (t, 2H, J = 2.4 Hz, –⁸CH₂), 3.46 (t, 2H, J = 2.4 Hz, –⁶CH₂–), 3.66 (t, 2H, J = 4.04 Hz, N–CH₂–C≡), 7.7–8 (m, 4H, ArH). ¹³C NMR (DMSO-d₆): δ, 26.8 (C^11,11′), 27.4 (C^10,10′), 28.15 (C^9,9′), 47.7 (C⁸), 54.7 (C⁵), 78.5 (C⁷), 80.8 (C⁶), 123.8 (C^1,1′), 131.9 (C^3,3′), 135.1 (C^2,2′), 167.2 (C^4,4′). Anal. Calcd. (C₁₈H₂₀N₂O₂): C, 72.97; H, 6.75; N, 9.45; O, 10.81. Found: C, 72.94; H, 6.79; N, 9.4.

2.7 Synthesis of N-[4-(2,6-dimethylpiperidino-1-yl)-but-2-yn-1-yl]isoindoline-1,3-dione (ZM6)

ZM6 was prepared following the same procedure described for the synthesis of ZM2 afforded (1.6 g, 52%) as a white crystalline compound, m.p. (132–134) °C. IR (KBr, cm⁻¹): 3030, 2931 (ArH, stretch), 2250 (C≡C, stretch), 1750, 1720 (C⚌O, stretch), 1612, 1465, 1390 (Ar, C⚌C, stretch), 1100, 1085, 942 (Ar, C⚌C, bending), 840, 794, 717 (ArH, bending). ¹H NMR (DMSO-d₆): δ, 1.14 (d, 3H, J = 4.4 Hz, ¹²CH₃), 1.14 (d, 3H, J = 4.2 Hz, ^12′CH₃), 1.5 (m, 6H, ¹⁰CH₂–¹¹CH₂–^10′CH₂), 1.80 (m, 2H, ⁹CH–^9′CH), 3.4 (t, 2H, J = 2.4 Hz, ⁸CH₂), 3.68 (t, 2H, J = 2.4 Hz, ⁵CH₂), 7.6–7.8 (m, 4H, ArH). ¹³C NMR (DMSO-d₆): δ, 22.02 (C¹²), 24.03 (C¹¹), 25.8 (C^10,10′), 27.1 (C^9,9′), 52.9 (C⁸), 66.6 (C⁵), 74.2 (C⁶), 79.3 (C⁷), 128.8 (C^2,2′), 131.9 (C^3,3′), 135.2 (C^1,1′), 167.0 (C^4,4′). Anal. Calcd. (C₁₉H₂₂N₂O₂): C, 73.54; H, 7.09; N, 9.03. Found: C, 73.52; H, 7.07; N, 9.06.

3.1 Animals

Male Sprague–Dawley rats (7–9 weeks old) were obtained from Yarmouk University animal house unit (Irbid, Jordan). Animals were housed at the Petra University animal facility in a 12 h light/dark cycle and a constant temperature of 22 °C. All animals were acclimatized for 10 days prior to experiments with free access to standard diet and drinking water. All animal experiments were performed in compliance with relevant laws and institution guidelines.

3.2 Anti-inflammatory activity

The paw edema was induced by subcutaneous injection of 0.1 ml of 1% carrageenan solution into the plantar region of the left hind paw of rats. The thickness of edema was measured and recorded for each rat at 1, 3 and 5 h intervals, using an electronic caliper (Mitutouo Corp., Japan). Different doses of the tested compounds (0, 5, 10 and 20 mg/kg) in comparison with Ibuprofen (5 and 10 mg/kg), Diclofenac (5 and 10 mg/kg), and Celecoxib (3, 6 and 10 mg/kg) were given to the rats by oral gavages 1 h prior to the administration of carrageenan. The percent inhibition of paw edema thickness was calculated using the following formula: $$\mathrm{Percent}\mathrm{inhibition}=100\times [1-(x_2-x_1)/(y_2-y_1)]$$ where x₁ is the thickness of paw of rats before administration of carrageenan and test or reference compounds, x₂ is the thickness of paw of rats after administration of carrageenan in the test group, y₁ is the thickness of paw of rats before the administration of carrageenan in the control group and y₂ is the thickness of paw of rats after administration of carrageenan in the control group.

3.3 COX-1 and COX-2 inhibition assay

This assay measures directly prostaglandin (PGF_2α) produced by Sncl₂ reduction of Cox-derived prostaglandin H synthase (Caynman, Chemical Co., MI, USA). Briefly, the PGF_2α are produced using ovine Cox 1 and human recombinant Cox 2, arachidonic acid and heme in a reaction buffer (0.1 M Tris–HCl, pH 8.0 containing 5 mM EDTA and 2 mM phenol). The reaction is stopped by adding 1 M HCl followed by adding saturated stannous chloride solution which is used to reduce PGH₂ produced in the COX reaction to more stable PGF₂. The produced PGF_2α was then assayed by an enzyme immunoassay using a capture assay mouse anti-rabbit IgG (captured antibody) for rabbit anti-prostaglandin antiserum. Prostaglandin standards (15.6–2000 pg/ml) were used to construct a standard curve, and prostaglandin tracer was used to establish a competitive type of assay. After 18 h of incubation, plates were washed and Ellman’s Reagent was added. The intensity of color was measured at a 405 nm using SCO GmbH (Dingelstadt, Germany) ELISA Plate Reader. The absorbance was transformed to pg/ml of PGF_2α using standard curve computed on excel after transforming values to % binding or (B/Bo) and present them on log–log graph paper.

3.4 Data analysis

The overall differences between the treated groups were analyzed using one way ANOVA with Dunnett’s post hoc test. The level of significant difference was defined as p < 0.05.

4.1 Chemistry

The desired compounds listed in Table 2 were synthesized through the following steps: potassium phthalimide was treated with propargyl bromide in benzene under reflux yielded the appropriate 2-(prop-2-yn-1-yl)isoindoline-1,3-dione. The Mannich reaction of 2-(prop-2-yn-1-yl)isoindoline-1,3-dione (ZM1) with paraformaldehyde and appropriate amines in peroxide-free dioxane with catalytic amount of cuprous chloride generated the designed aminoacetylenic isoindoline derivatives: ZM2, ZM3, ZM4, ZM5 and ZM6 (Table 2). The IR, ¹H NMR, ¹³C NMR spectra, DEPT 135, DEPT 90, and elemental analyses were consistent with the all assigned structures as shown in the experimental part (see Scheme 1).

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Scheme 1

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4.2 Pharmacology

The acute anti-inflammatory activity of ZM2, ZM3, ZM4 and ZM5 showed to be effective, with varying potencies. ZM6 was inactive in reducing any inflammatory caused by carrageenan-induced edema, for this reason it was excluded from testing its inhibitory activity against COX-1 and COX-2. ZM2 at doses of 10 and 20 mg/kg produced significant dose-dependent inhibition of inflammation (in the range from 22% to 57%) after 3 and 5 h post-carrageenan administration (p < 0.05). This compound at 20 mg/kg, showed equipotent activity (p > 0.05) to that of Ibuprofen (10 mg/kg), Diclofenac (10 mg/kg) and Celecoxib (12 mg/kg) at 3 and 5 h post-administration (Table 3). On the other hand, ZM3 exhibited more inhibitory activity than ZM2 since the inhibition of inflammation induced by carrageenan is more pronounced and independent to dose. Its activity was comparable to Ibuprofen and Diclofenac (5 and 10 mg/kg), and Celecoxib (12 mg/kg) (p < 0.05).

The anti-inflammatory activity of ZM4 was dose- and time-dependent. A significant reduction of carrageenan-induced inflammation was detected only at 20 mg/kg dose of ZM4 at 1, 3 and 5 h post-administration (p < 0.01). On the other hand, ZM5 produced a varying degree of inhibition of inflammation at 1, 3 and 5 h intervals post-carrageenan-induced inflammation. The maximal inhibition of inflammation was observed with 20 mg/kg after 3 and 5 h intervals, which was equipotent to Diclofenac and Celecoxib (Table 3).

The COX-1 and COX-2 inhibition assay (IC₅₀ values) for the tested compounds are presented in Table 4. The results showed that all the compounds ZM2, ZM3, ZM4, and ZM5 showed inhibition of COX-1 and COX-2 with a maximum inhibition at 5 μM (see Table 5).

However, the inhibition values were less than Diclofenac and Celecoxib, as COX-1 and COX-2 inhibitors. All of the ZM compounds exhibited a bell-shaped inhibition curve being the maximum at 5 μM and to a lesser extent at 2 and 10 μM. The best response was by ZM4 for COX-2 inhibition ranging from 27.5%, 90.5%, and 44.0%, for 2, 5, and 10 μM, respectively. Other ZM compounds such as ZM2, ZM3 and ZM5 exhibited inhibitory responses for COX-2 more than COX-1 at 10 μM.