Regioselective synthesis and anti-inflammatory activity of novel dispiro[pyrazolidine-4,3'-pyrrolidine-2',3"-indoline]-2",3,5-triones

Novel dispiro[pyrazolidine-4,3'-pyrrolidine-2',3"-indoline]-2",3,5-triones 5a-j were obtained regioselectively by 1,3-dipolar cycloaddition reaction of 4-arylidene-1-phenylpyrazolidine-3,5-diones 2a-e as dipolarophiles with non-stabilized azomethine ylides, generated in situ via decarboxylative condensation of isatins 3a,b and sarcosine 4 in dry ethanol. The prepared compounds were screened for their anti-inflammatory activity "at a dose of 10 mg/kg body weight", especially 5d , 5f , 5h , and 5j which reveal remarkable activities relative to indomethacin which was used as a reference standard in this study.


Introduction
Cycloadditions are an important group of reactions in organic synthesis. 1The azomethine ylide represents one of the most reactive and versatile classes of 1,3-dipoles and is trapped readily by a range of dipolarophiles, either inter or intramolecularly, forming substituted pyrrolidines. 24][5] N-Arylpyrazoles are a very interesting class of heterocyclic compounds that have remarkable pharmacological activities in areas such as antibacterialantifungal, 6 hypoglycemic, 7 tumor necrosis inhibition, 8 anti-thrombo-embolic disorders, as antiangiogenic-, 9 and anti-inflammatory effects. 10,11  the present work, it is intended to utilize a natural product isatin scaffold for formation of non-stabilized azomethine ylides following the previously described and successful methods, [12][13][14][15] through decarboxylative condensation with α-amino acids and trapping the generated reactive intermediate via 1,3-dipolar cycloaddition reaction with the exocyclic olefinic linkage derived from 1-phenylpyrazolidine-3,5-dione.The anti-inflammatory properties of the prepared compounds will be screened.[18][19] Figure 1.Representative naturally occurring spiropyrrolidinyl-oxoindole alkaloids.
Along with the experimental investigation, and for further confirmation of the structures of the synthesized compounds 5a-j, we also carried out some theoretical studies on selected prepared compounds, with the help of molecular modeling software.For example, the calculated heat of formation (∆H) of 5b (Figure 3) showed that the most stable form (with the lowest heat of formation) is 2'S, 3'R, 4'R (∆H= 68.622 kcal/mol).

Anti-inflammatory activity
The anti-inflammatory activity of the target compounds 5a-j (at a dose of 10 mg/kg body weight) was determined in vivo by the acute carrageenan-induced paw oedema standard. 24The antiinflammatory properties were recorded at successive time intervals 0.5, 1, 2, 3, and 4h and compared with that of indomethacin (at a dose of 10 mg/kg body weight) which was used as a reference standard.From the obtained results (Table 1) it was noticed after 1 h that many of the tested compounds exhibit considerable anti-inflammatory properties (especially 5d, 5f, 5h, and 5j) which reveal remarkable activities, with potency (percentage oedema inhibition of the tested compounds relative to percentage oedema inhibition of indomethacin) of 0.72, 0.64, 0.56, and 0.72 respectively.The structure-activity relationships based on the obtained results indicated that the type of substituents attached to N-1" and C-4' are controlling factors in developing the total pharmacological properties.The best observed anti-inflammatory property is that in which N-1'' bears a methyl group and C-4' is attached to a phenyl group substituted with an electron-donating group (methoxy) in 5j (potency 0.72).However, with an unsubstituted phenyl group, or substitution with electron-withdrawing groups (chlorine and bromine) the function was relatively decreased.It was also noticed that when N-1" is attached to a hydrogen atom, the antiinflammatory activity is increased only when C-4' is attached to a phenyl group substituted with an electron-donating hydroxyl group in 5d (potency 0.72).

Acute toxicity (LD50)
The median lethal dose (LD50) of the most active compounds 5d, 5f, 5h, and 5j was determined in mice, according to reported procedures. 25The results showed that the tested compounds 5d, 5f, 5h, and 5j were non-toxic at doses up to 200 mg / kg.

Experimental Section
General.The time required for completion of each reaction was monitored by TLC.All melting points are uncorrected, and were measured on a Gallenkamp apparatus.The IR spectra were recorded on a Shimadzu 470 IR spectrometer (KBr) νmax /cm -1 .The 1 H-, and 13

Compound
Mean swelling volume ± S.E.M a (% inhibition of oedema)