New Analogs of Phenacetin with Anti‐Inflammatory Activity

Abstract

Phenacetin is a compound with pharmacological activity used in the past with antipyretic and analgesic effects. Three new analogues were synthetized aiming improvements in efficacy and less adverse effects, named 4‐ethoxyaniline, R‐71 and R‐72. Nociception and inflammation have been assessed by the formalin‐induced licking response and carrageenan‐induced cell migration models, respectively. Male Swiss webster mice (22–30 g, n = 6–8) were used for both tests, following animal ethics guidelines. The protocol for animal use was approved by CEUA/UFRJ and received the number DFBCICB015‐04/16. Results are compared with vehicle‐treated group and expressed as mean ± SD and statistical analysis were performed by ANOVA followed by Bonferroni test (*p<0.05). Formalin‐induced licking response. The stimulus is given by the injection of 20 μL of formalin 2.5% in mice's left hind paw. The time the animal spends licking the injected paw is recorded. The animals received oral treatment of 4‐ethoxyaniline, R‐71 or R‐72 at 0.1, 1 or 10 mg/kg, acetylsalicylic acid (200 mg/kg) or morphine (2.5 mg/kg) 60 minutes before formalin injection. To access cell migration, the subcutaneous air pouch (SAP) was produced with a sterile air injection and 6 days later, animals received oral treatment with 4‐ethoxyaniline, R‐71 or R‐72 (0.1, 1 or 10 mg/kg) one hour before the injection of sterile carrageenan (1%) or saline into the SAP. After 24 h, all animals were euthanized, the SAP was washed with 1 mL of saline and the exudate was collected for mediators and protein measurements to investigate mechanism of action. Treatment with the three compounds significantly reduced licking time in neurogenic and inflammatory phases of the licking model. 1st phase: vehicle= 52.7 ± 11.3 seconds (sec); 4‐ethoxyaniline: 23.0 ± 5.5* sec; 24.8 ± 10.2* sec; 24.9 ± 3.5* sec; R‐71: 28.8 ± 6.6* sec; 24.2 ± 7.2* sec; 10.2 ± 2.9* sec; R‐72: 16.7 ± 5.6 * sec; 21.7 ± 7.0 * sec; 10.1 ± 3.8 * sec. 2nd phase: vehicle = 212.2 ± 56.3 sec; 4‐ethoxyaniline: 113.8 ± 11.0* sec; 118.2 ± 45.9* sec; 107.8 ± 32.2* sec; R‐71: 126.9 ± 33.3* sec; 137.8 ± 16.2* sec; 82.3 ± 15.9* sec; R72: 129.5 ± 19.6* sec; 109.7 ± 16.2* sec; 60.4 ± 16.6* sec, respectively at doses of 0.1, 1 and 10 mg/kg. Regarding cell migration, 4‐ethoxyaniline reduced 67%, 79% and 77%; R‐71: 66%, 70% and 71%; R‐72: 67%, 69% and 75% when compared to vehicle that also received carrageenan in the pouch. Protein levels were reduced in 4‐ethoxyaniline 31%, 48% and 64%; R‐71: 3%, 15% and 78%; R‐72: 9%, 28% and 70%. NO measurement of 4‐ethoxyaniline reduced 48%, 39% and 3%; R‐71: 56%, 23% and 18%; R‐72: 56%, 44% and 1%. Levels of IL‐1β were also reduced, 4‐ethoxyaniline: 49%, 41% and 55%; R‐71: 38%, 34% and 63%; R‐72: 59%, 16% and 71% respectively at doses of 0.1, 1 and 10 mg/kg. We can conclude that the analogues presented anti‐inflammatory effects since reduced cell migration, NO production and IL‐1β secretion and could be considered to further assays.Support or Funding InformationFinancial Support: This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil/Finance Code 001, CNPq and FAPERJ. Leukocyte migration, protein extravasation, Nitric oxide quantification and IL‐1β measurement of treatment with 4‐ethoxyaniline, R‐71 and R‐72. Leukocyte Migration Pre‐treatment 0.1 mg/kg 1 mg/kg 10 mg/kg Vehicle 93.4 ± 33.9 93.4 ± 33.9 93.4 ± 33.9 Dexamethasone 31.0* ± 5,5 31.0* ± 5,5 31.0* ± 5,5 4‐Ethoxyaniline 31.6* ± 5.0 20.4* ± 4.7 22.2* ± 6.9 R‐71 32.1* ±4.8 28.9* ± 16.3 27.6* ± 15.3 R‐72 31.7* ± 6.5 29.9* ± 16.6 24.8* ± 8.1 Protein Extravasation Pre‐treatment 0.1 mg/kg 1 mg/kg 10 mg/kg Vehicle 703.35 ± 163.9 703.35±163.9 703.35±163.9 Dexamethasone 453.59*±214.5 453.59* ± 214.5 453.59* ± 214.5 4‐Ethoxyaniline 460.8* ± 84.6 345.2*± 181.1 240.8* ± 45.9 R‐71 646.0 ± 147.9 564.7 ± 94.96 214.2* ± 24.6 R‐72 609.5 ± 158.1 483.4 ± 137.3 198.3* ± 28.9 NO Quantification Pre‐treatment 0.1 mg/kg 1 mg/kg 10 mg/kg Vehicle 248.63 ± 64.3 248.63 ± 64.3 248.63 ± 64.3 Dexamethasone 112.3* ± 34.9 112.3* ± 34.9 112.3* ± 34.9 4‐Ethoxyaniline 128.3* ± 59.5 149.7 ± 22.1 241.0 ± 86.0 R‐71 109.4* ± 33.48 190.3 ± 74.75 202.3 ± 101.3 R‐72 108.5* ± 36.4 138.2 ± 42.2 244.4 ± 34.9 Levels IL‐1β Pre‐treatment 0.1 mg/kg 1 mg/kg 10 mg/kg Vehicle 4016.2 ± 130.6 4016.2 ± 130.6 4016.2 ± 130.6 Dexamethasone 1109.8* ± 307.0 1109.8* ± 307.0 1109.8* ± 307.0 4‐Ethoxyaniline 2038.2 ± 1480.7 2354.9 ± 779.4 1786.7 ± 437.6 R‐71 2472.8 ± 1308.1 2622.4 ± 753.4 1470.5* ± 671.6 R‐72 1637.7 ± 805 3356.6 ± 246.9 1158.8* ± 419.4 This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.