Towards safer anti-inflammatory therapy: synthesis of new thymol–pyrazole hybrids as dual COX-2/5-LOX inhibitors

Abstract New thymol − 1,5-disubstitutedpyrazole hybrids were synthesised as dual COX-2/5-LOX inhibitors. Compounds 8b, 8g, 8c, and 4a displayed in vitro inhibitory activity against COX-2 (IC50 = 0.043, 0.045, 0.063, and 0.068 µM) nearly equal to celecoxib (IC50 = 0.045 µM) with high SI (316, 268, 204, and 151, respectively) comparable to celecoxib (327). All target compounds, 4a–c and 8a–i, showed in vitro 5-LOX inhibitory activity higher than reference quercetin. Besides, they possessed in vivo inhibition of formalin-induced paw oedema higher than celecoxib. In addition, compounds 4a, 4b, 8b, and 8g showed superior gastrointestinal safety profile (no ulceration) as celecoxib and diclofenac sodium in the population of fasted rats. In conclusion, compounds 4a, 8b, and 8g achieved the target goal. They elicited in vitro dual inhibition of COX-2/5-LOX higher than celecoxib and quercetin, in vivo potent anti-inflammatory activity higher than celecoxib and in vivo superior gastrointestinal safety profile (no ulceration) as celecoxib.


13C-NMR of Compound 8i
Biological Screening

Anti-inflammatory activity
In vitro COX-1 and COX-2 inhibitory assay Compounds 4a-c and 8a-i were screened for their ability to inhibit COX-1 and COX-2 enzymes in vitro. This was carried out using Cayman colorimetric COX (ovine) inhibitor screening assay kit (Catalog No. 560131) supplied by Cayman chemicals, Ann Arbor, MI, USA according to reported method 1 . The colorimetric COX inhibitor screening assay utilizes the peroxidase component of cyclooxygenase. The peroxidase activity was assayed calorimetrically by monitoring the appearance of oxidized N,N,N',4phenylenediamine (TMPD) at 590nm. The appearance of reagent was carried out following the instructions given with the assay kit (Catalog No. 560131). The half-maximal inhibitor concentrations (IC50µM) were determined and the selectivity index (SI) values were calculated as IC50 (COX-1)/IC50 (COX-2).

Procedure:
1) Background wells-160 µL of assay buffer and 10 µL of heme were added to background wells.
3) Inhibitor well-150 µL of assay buffer, 10 µL of heme and 10 µL of enzyme were added. 4) 10 µL of inhibitor (3 concentrations) in DMSO was added to the inhibitor wells to yield a final concentration of 25, 50 and 100 µM. 5) 10 µL of solvent (DMSO) was added to the 100% Initial activity wells and background wells.
6) The plate was shaken for a few seconds and incubated for 5 min at 25°C. 7) 20 µL of colorimetric substrate solution (TMPD) was added to all wells.
8) 20 µL of arachidonic acid was added to all wells to yield a final concentration of 100 µM.
9) The plate was shaken for a few seconds and incubated for 5 min at 25°C.
10) The absorbance was measured at 590 nm using a plate reader.
The calculations were carried out in the following manner: 1) The average absorbance of all samples was determined.
2) The absorbance of background wells was subtracted from absorbance of the 100% Initial activity and the inhibitor wells.

In vitro LOX inhibitory assay
Compounds 4a-c and 8a-i were screened for their ability to inhibit lipoxygenase enzymes. This was carried out using Abnova lipoxygenase inhibitor screening assay kit (Catalog No. 760700). 2 The Lipoxygenase Inhibitor Screening Assay Kit detects and measures the hydroperoxides produced in the lipoxygenation reaction using a purified LOX. The detection reaction is equally sensitive to hydroperoxides at various positions within the fatty acid and will work with fatty acids of any carbon length. It is thus a general detection method for 5-LOX and can be used to screen libraries of compounds for those which inhibit 5-LOX enzymes.

Procedure:
1) Blank Wells-add 100 µL of assay buffer to at least two wells.

3)
100% Initial Activity Wells-add 90 µL 15-LO and 10 µL of solvent (DMSO) (the same solvent to dissolve the inhibitor) to two wells. The 100% initial activity wells should result in approximately 10 nmol/min/mL of activity.

4)
Inhibitor Wells-add 90 µL of sample and I10 µL of inhibitor to two wells.

5)
Initiate the reaction by adding 10 µL of substrate (either arachidonic acid or linoleic acid) to all the wells. Place the 96-well plate on a shaker for at least five minutes.

6)
Add 100 µL of chromogen to each well to stop enzyme catalysis and develop the reaction. Cover with a plate cover and place the 96-well plate on a shaker for at least five minutes.

7)
Remove the cover and read the absorbance at 490-500 nm using a plate reader. Inhibitors were dissolved in DMSO. The inhibitors were added to the assay in a final volume of 10 µL before initiating with substrate. Three concentrations were prepared (25, 50 and 100 µM) to determine the concentration produced 50% enzyme inhibition.
The calculations were carried out in the following manner: 1) The average absorbance of all samples was determined.

2)
The absorbance of blank wells was subtracted from absorbance of the 100% Initial activity and the inhibitor wells.

In vivo anti-inflammatory activity
Approved by AlexU-IACUC. 3

Animals:
Animals Adult female albino rats weighing 150-250 g were used (Experimental Animal Centre in Alexandria University). All animals accessed to food and water ad libitum and were housed in 12 h dark/light cycle in a controlled condition at 23-25 0 C. They were allowed to acclimatize for 1 week prior to experimentation. Procedures involving animals and their care were conducted in conformity with the Guide for the Care and Use of Laboratory Animals published by US National Institute of Health 4 (NIH publication No. 83-23, revised) and following the ethical guidelines of Alexandria University on laboratory animals. In all tests, adequate considerations were adopted to reduce pain or discomfort of animals (Approved by AlexU-IACUC) 3 . .

Compounds:
Celecoxib and Diclofenac sodium (European Egyptian Pharmaceutical industries, Alexandria, Egypt), formalin 5% made from formaldehyde 37% and saline (Merck, Germany) were used. The novel compounds were synthesized based on the previously described methods. Compounds that showed in vitro selectivity indices higher or nearly equivalent to reference drugs towards COX 2 enzyme, were further evaluated for their in vivo antiinflammatory activity applying the formalin-induced paw edema screening protocol as an acute inflammation model. 5, 6 Celecoxib (5 mg/kg) and Diclofenac sodium (5 mg/kg) were used as reference drugs. Animals were divided into groups of six rats each treated with test compounds. Groups treated with Celecoxib and Diclofenac sodium served as references and rats which were given the vehicle (DMSO) served as control.

Formalin-induced paw edema test (acute inflammation model):
Procedure: A solution of freshly prepared formalin 5% was used as a phlogistic agent. A mark was made on the lateral malleolus of the rats' paws delineating the injection sites. The initial volume of paw was measured by means of digital calibrated Vernier caliper. Then, the novel test compounds (5 mg/kg body weight), Celecoxib (5 mg/kg body weight), Diclofenac sodium (5 mg/kg body weight), or DMSO, as the control solution, were administered orally. After 45 minutes, 40 μl formalin were injected subcutaneously into the sub plantar tissue of the right hind paw of all groups under light ether anesthesia. An equal volume of saline was injected into the left hind paw and served as internal control for the degree of inflammation in the right hind paw. 5, 6 The volume of paw was measured in different treatment groups, 4 h following the formalin injection and the amount of increase in paw volume (edema volume) was calculated by subtracting the volumes before and 4 h after the injection of formalin. Edema was expressed as an increase in the volume of paw, and the percentage of edema inhibition (or percent protection against inflammation) for each rat and each group was calculated according to the following equation: Where Vt is the mean volume of edema at specific time interval (4 h) and Vo is the mean volume of edema at zero-time interval.
Relative potency of the tested compounds was expressed as % inhibition of edema for the tested compounds relative to % inhibition of edema for the reference drugs at 4 h from the induction of inflammation.
% Relative potency = % inhibition of edema for the test compound after 4h x 100 % inhibition of edema for the reference after 4h (Approved by AlexU-IACUC) 3 .

Gastric ulcerogenic activity:
Procedure: Compounds were evaluated for acute gastric ulcerogenic effect in adult female Wistar rats. Rats (150-250 g) were divided into groups of six rats each and test compounds, references or DMSO as control were administered orally at a dose of 60 mg/kg body weight (three times the previously used dose). Six hours after the treatment they were sacrificed under deep ether anesthesia and their stomachs were removed and opened through greater curvature, washed under running water and fixed in saline solution. Gross examination was performed for any evidence of hyperemia, hemorrhage, definite hemorrhagic erosion or ulcer 6, 7 (Approved by AlexU-IACUC) 3 .

Ulcerogenic activity:
The degree of ulcerogenicity was determined by viewing the gastric epithelial ulceration using a 5x magnifying lens and rated by ulcer score.
Ulcer score was used to grade the incidence and severity of the lesions such as: 1) Shedding of epithelium-10 2) Petechial and frank hemorrhages-20 3) One or more ulcers-30 4) More than two ulcers-40

Molecular Modeling
The molecular modeling studies were performed using the Molecular Operating Environment (MOE 2016.08) software (Chemical Computing Group, Montreal, Canada). 8 and the crystal structures of the proteins were downloaded from the Protein Data Bank (PDB) website.

Steps for preparation of the compounds and enzymes for docking:
The ligand molecules were constructed using the builder module in MOE and collected in a database. The database was adapted by using the option "Protonate 3D" to add hydrogens, calculate partial charges and minimize energy (using Force Field MMFF94x). In addition, the downloaded proteins were prepared by deleting the repeated chains, water molecules and any surfactants, then hydrogens were added to the atoms of the receptor and the partial charges were calculated. The protocol in the MOE application was used to calculate the best score between the ligands and the enzymes' binding sites using triangle matcher as placement method and London dG as the scoring function. The output database contained the energy scores between the ligands' conformers and the enzyme binding sites in kcal/mol. The docking poses for each ligand were visually examined and the interactions with active site residues were analyzed. The highest scoring pose was selected to compute the ligand interactions using the Ligand Interactions module in MOE.
To confirm the validity of our docking results, the pose selection method was adopted to validate our docking protocol 9 . For all the used proteins, their co-crystallized ligand were drawn in MOE, prepared as the targeted compounds (hydrogens, partial charges and energy minimization), and then docked into the active site of the protein using our protocol. The Root Mean Square Deviation (RMSD) between the original and docked conformers was less than 2 Å for all the ligands. It was reported that values less than 2 Å were a sign of a successful and reliable docking protocol 9 . S Crystal structures of Cyclooxygenase-2 enzyme co-crystallized with Celecoxib (PDB: 3LN1) and 5-LOX in complex with Arachidonic acid (PDB: 3V99) were used for prediction of mode of binding of the active antiinflammatory compounds.