LASSBio-1524 was designed as inhibitor of the IKK-β (kappa β kinase inhibitor) enzyme, which participates in the activation of the nuclear factor κB (NF-κB) canonical pathway, and its three N-acylhydrazone new analogues, LASSBio-1760, LASSBio-1763 and LASSBio-1764 are now being tested on their anti-inflammatory potential. The activity of these compounds was evaluated with the subcutaneous air pouch induced by carrageenan and by subsequent measurement of tumor necrosis factor-α (TNF-α), nitric oxide (NO) and reactive oxygen species (ROS). In the acute inflammation model, the oral pretreatment with doses from 0.3 to 30 mg/kg of N-acylhydrazone derivatives was able to significantly reduce leukocyte migration to the cavity. Pretreatment with LASSBio-1524 and its analogues also decreased NO, TNF-α and ROS biosynthesis an events closely involved with NF-kB pathway. The tetrahydronaphthyl-N-acylhydrazone derivative LASSBio-1764 was the most promising compound from this series, surpassing even LASSBio-1524. Additionally, none of the compounds demonstrated myelotoxicity or cytotoxicity. Cell viability was assayed and these compounds demonstrated to be safe at different concentrations. Western blot analysis demonstrated that LASSBio-1524 and LASSBio-1760 inhibited NF-κB expression in RAW 264.7 cell lineage. Our data indicate that the tested compounds have anti-inflammatory activity, which may be related to inhibition of leukocyte migration, reducing the production of NO, TNF-α and ROS. LASSBio-1524 and LASSBio-1760, in addition to these features, also reduced p65 nuclear expression assessed by western blot in RAW 264.7 murine cells.

Protein kinases constitute attractive therapeutic targets for development of new prototypes to treat different chronic diseases. Several available drugs, like tinibs, are tyrosine kinase inhibitors; meanwhile, inhibitors of serine/threonine kinases, such as mitogen-activated protein kinase (MAPK), are still trying to overcome some problems in one of the steps of clinical development to become drugs. So, here we reported the synthesis, the in vitro kinase inhibitory profile, docking studies, and the evaluation of anti-inflammatory profile of new naphthyl-N-acylhydrazone derivatives using animal models. Although all tested compounds (3a-d) have been characterized as p38α MAPK inhibitors and have showed in vivo anti-inflammatory action, LASSBio-1824 (3b) presented the best performance as p38α MAPK inhibitor, with IC = 4.45 μm, and also demonstrated to be the most promising anti-inflammatory prototype, with good in vivo anti-TNF-α profile after oral administration.

Inhibitor of nuclear factor κB kinase 2 (IKK2) is suggested to be a potential target for the development of novel anti-inflammatory and anticancer drugs. In this work, we applied structure-based drug design to improve the potency of the inhibitor (E)-N'-(4-nitrobenzylidene)-2-naphthohydrazide (LASSBio-1524, 1 a: IC50 =20 μm). The molecular model built for IKK2 together with the docking methodology employed were able to provide important and consistent information with respect to the structural and chemical inhibitor characteristics that may confer potency to IKK2 inhibitors, providing important guidelines for the development of a new N-acylhydrazone (NAH) derivative. (E)-N'-(4-(1H-pyrrolo[2,3-b]pyridin-4-yl)benzylidene)-2-naphthohydrazide hydrochloride (LASSBio-1829 hydrochloride, 10) is a 7-azaindole NAH able to inhibit IKK2 with an IC50 value of 3.8 μm. LASSBio-1829 hydrochloride was found to be active in several pharmacological inflammation tests in vivo, showing its potential as an anti-inflammatory prototype.

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.

Anti‐inflammatory drugs are among the most prescribed in the world. Ibuprofen is well known as a non‐steroidal anti‐inflammatory drug (NSAID) that has proven anti‐inflammatory effects, but like all NSAID, has adverse effects. Our aim is to evaluate effects of new Ibuprofen analogs (R‐60 and R‐65) on acute inflammation models, which are formalin‐induced paw licking and cell migration to the subcutaneous air pouch with exudate evaluation for mediator's production. Experimental groups (n=6–8, 25–35g) male Swiss webster mice were treated 1 h prior inductions at doses of 0.01, 0.1, 1 or 10 mg/kg. Standard drugs used were Ibuprofen (10 mg/kg, p.o.), acetylsalicylic acid (ASA,200 mg/kg, p.o.) or morphine (2.5 mg/kg, i.p.). The analogues reduced paw licking time in the 1st phase: groups treated with R‐60 0.01 mg/kg licked 41.5 ± 21.6 seconds (sec), 0.1 mg/kg: 33 ± 6.4 sec, 1 mg/kg: 32.1 ± 6.9* sec, 10 mg/kg: 29.7 ± 8.4* sec. In 2nd phase reduced licking: 0.01 mg/kg: 184,4 ± 80,1 sec, 0.1 mg/kg: 198,1 ± 54,2 sec, 1 mg/kg: 207,2 ± 23 sec and 89.4 ± 16.4 *sec when treated with 10 mg/kg. R‐65 reduced licking: 34.9 ± 15* sec at the dose of 0.01 mg/kg in the 1st phase, 0.1 mg/kg: 28 ± 5.7* sec, 1 mg/kg: 25.4 ± 7.5* sec and 10 mg/kg: 17 ± 5.8 * sec. In 2nd phase, 10 mg/kg reduced: 56.1 ± 25.1* sec. Ibuprofen reduced licking time in the 1st phase: 29.3 ± 11.6* sec and second: 97.8 ± 48.4* sec. when compared to vehicle‐treated group: 1st phase 53.8 ± 11.9 sec and 2nd phase 202.9 ± 56.6 sec. Regarding cell migration, treatment reduced leukocyte migration at all doses comparing to the vehicle‐treated group: 73.1±11 × 103 cell/mL. The R‐60 0.01 mg/kg treatment led to a migration of 45.7±13 × 103 cell/mL*; 0.1 mg/kg: 46.8 ± 14.7 × 103 cell/mL*, 1 mg/kg: 42.4 ± 12.3 × 103 cell/mL*, 10 mg/kg: 46.6 ± 13.9 × 103 cell/mL*. When treated with R‐65 0.01 mg/kg 47 ± 15.1 ×103 cell/mL* migrated, 0.01 mg/kg: 42 ± 13.1 × 103 cells/mL*, 1 mg/kg: 45.2 ± 14.8 × 103 cell/mL* and 10 mg/kg: 40.9 ± 6.5 × 103 cell/mL*. Ibuprofen: 40.3 ± 9 ×103 cell/mL*. Nitric oxide production was reduced with 0.01 mg/kg treatment of R‐60: 125.5 ± 90.5 μM, 0.1 mg/kg: 103.6 ± 97.9 μM, 1 mg/kg: 91.8 ± 51.6 μM* and 10 mg/kg: 108.9 ± 34.1 μM. R‐65: 0.01 mg/kg: 163.6 ± 84 μM, 0.1 mg/kg: 129.2 ± 85.4 μM, 1 mg/kg: 70.1 ± 52.5 μM* and 10 mg/kg: 89.2 ± 29.7 μM*. Ibuprofen did not reduce nitrite levels: 141.7 ± 14.6 μM when compared to vehicle group: 193.8 ± 110.4 μM. The analogs reduced IL‐1β production at the highest tested dose (10 mg/kg) when comparing to the vehicle‐treated group (605.4 ± 323.1 ng/mL). R‐60: 255.9 ± 103.1 ng/mL* and R‐65: 237 ± 142.7 ng/mL*. Ibuprofen did not reduce IL‐1β levels at any tested dose. R‐60 and R‐65 analogues demonstrated activity in the peripheral nociception in the formalin‐induced paw licking model, as well as in the reduction of cell migration, nitric oxide and IL‐1β production. This work points its anti‐inflammatory effects, which supports the continuity of the tests and that the compounds may become potential candidates to treat inflammation and pain.Support or Funding InformationThis study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES)/Finance Code 001, CNPq and FAPERJ.Alan Minho (technical support)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.