Design, Synthesis, In Vivo Anti-inflammatory, Analgesic Activities and Molecular Docking of Some Novel Pyrazolone Derivatives

1Medicinal Chemistry Department, National Research Center, Dokki, Cairo, Egypt 2Department of Chemistry of Natural and Microbial Products, National Research Center, Dokki, Cairo, Egypt 3Pharmaceutical Chemistry Department, College of Pharmacy, Taif University, Taif, KSA 4Pharmacology Department, National Research Center, Dokki, Cairo, Egypt 5Faculty of Medicine and Medical Sciences, Taif University, KSA


Introduction
The anti-inflammatory properties of Nonsteroidal antiinflammatory drugs (NSAIDs) have been attributed to their ability to inhibit the enzyme cyclooxygenase (COX) enzymes, which catalyzes the formation of arachidonic acid (AA) to prostaglandins H 2 (PGH 2 ) [1][2][3][4]. Many of (NSAIDs) have a wide clinical use in the treatment of acute or chronic inflammation [5,6]. There are two isoforms of cyclooxygenase (COX); COX-1 and COX-2 [7]. These isoforms are poorly distinguishable by most of the classical NSAIDs. They actually inhibit COX-1 extensively; COX-1 has housekeeping functions, including low-level production of gastro protective PGs, besides COX-2, leading to gastrointestinal injury, suppression of Thromboxane A2 (TXA2) formation and platelet aggregation. The combination of these interactions is probably the reason for gastrointestinal bleeding as the most serious complication of these drugs [8]. To prevent or decrease these side effects, a current strategy consists of designing selective COX-2 inhibitors with an improved gastric safety profile. The improved safety profile of COX-2 inhibitors may allow the use of these new agents for long-term prophylactic use in certain chronic diseases. This has led intense efforts in search for potent and selective COX-2 inhibitors, which could provide anti-inflammatory drugs with fewer risks. Several classes of compounds having selective COX-2 inhibitory activity have been reported in the literature such as SC-558 and celecoxib. Pyrazole, pyrazoline, and pyrazolone ring systems found in many non-steroidal anti-inflammatory drugs have been used for clinical application as NSAIDS like celecoxib [9] antipyrine, phenylbutazone, ramifenazone and famprofazone. Antipyrine is the first pyrazoline derivative used in the management of pain, inflammation and fever ( Figure 1) [10][11][12][13]. Pyrazoles are considered among the most important class of heterocyclic compounds having a broad spectrum of application in the field of medicinal chemistry [14]. Pyrazole derivatives were found to exhibit anti-inflammatory [15][16][17], analgesic [18], antitumor [19,20], antiviral [21,22], anticonvulsant [23] and antimicrobial activities [22,24]. The importance of pyrazole derivatives as antimicrobial agents attracted attention after the discovery of the natural pyrazole C-glycoside pyrazofurin which demonstrated a broad spectrum of antimicrobial activities [25]. Appreciation with the well-documented anti-inflammatory and analgesic properties associated with these heterocyclic cores and as part of our continuing work in the area of drug discovery including anti-inflammatory and analgesic compounds [26][27][28], herein we report the synthesis of new pyrazolone derivatives in combination with pyrazole and dihydropyrimidinone scaffold, in addition to heteroaryl and aryl pyrazole derivatives. The analgesic and anti-inflammatory activities of all novel compounds were investigated utilizing the acetic acid-induced writhing test and the carrageenaninduced hind paw edema test, respectively. Furthermore, a molecular docking study was carried out for the most potent anti-inflammatory new compounds against COX-1 and COX-2 crystal structures in an attempt to understand their binding mode to both enzymes in comparison to the reference drug indomethacin.

Chemistry
All chemicals were purchased from common commercial suppliers and used without further purification. All reactions were carried out under argon with dry solvents. Also all reactions were monitored by TLC carried out on Merck silica gel-coated plastic sheets (60 F254) by using UV light as visualizing agent. Thin layer chromatography (TLC) was performed on silica gel 60 F254 plastic plates (E.Merck, layer thickness 0.2 mm). Detection was achieved by treatment either with a solution of 20 g of ammonium molybdate and 0.4 g of cerium (IV) sulfate in 400 ml of 10% H 2 SO 4 or with 15% H 2 SO 4 , and heating at 150°C. Melting points were determined on a Gallenkamp melting point apparatus and were uncorrected. IR spectra (KBr) were recorded on a Perkin-Elmer 1650 spectrophotometer, NRC. 1 H and 13 C NMR were determined on a Varian Mercury (300 MHz) spectrometer (Varian, UK) and the chemical shifts were expressed in δ ppm relative to TMS as an internal reference, Faculty of science, Cairo University. Mass spectra were recorded on Thermo Finnigan LCQ Advantage spectrometer in ESI mode, I Spray Voltage 4.8 kV. Microanalyses were performed at the Micro analytical Center of Cairo University.

General method for preparation of 10 and 12
An equimolar amounts of 2-(bis(methylthio)methylene) propanedinitrile 1 (10 mmol) and pyridine-4-carbohydrazide or 4-methylbenzenesulfonohydrazide (10 mmol) and 2-4 drops of triethylamine in (25 ml) methanol was heated for 8 h. The reaction mixture was cooled, poured onto ice-water and the solid formed was collected by filtration, dried under suction and crystallized from ethanol absolute to give (10 or 12).

General method for preparation of 11 and 13
An equimolecular amounts of (ethoxy-methylidene) propanedinitrile 9 (10 mmol) and pyridine-4-carbohydrazide or 4-methylbenzenesulfonohydrazide (10 mmol) and 4-6 drops of triethylamine in (25 ml) methanol was heated for 8hrs. The reaction mixture was cooled, poured onto ice-water and the solid formed was collected by filtration, dried under suction and crystallized from ethanol absolute to give (11 or 13).

Materials and methods
Animals: Albino mice and rats used in this experiment were obtained from the Animal House Colony at the National Research Centre (NRC), Egypt. Albino mice of both sexes (25-30 g b. wt) and Wistar rats of both sexes (150-200 g b. wt) were utilized. All animals were housed under standard conditions and were reserved in polyethylene cages under standard conditions (temperature 25± 3, and relative humidity 60 ± 10%) of natural 12 h light and dark cycle with free access to food and water. Animals were allowed to adapt to the laboratory environment for one week before experimentation. Mice and rats will be used only once in this study. All animal procedures were performed after approval from the Ethics Committee of The National Research Centre-Egypt and in accordance with the recommendations of the proper care and use of laboratory animals.
Analgesic activity: Analgesic activity of the selected Compounds was carried out in mature Albino mice (25-30 g body weight) by using two different models.

a. Central analgesic activity (Hot plate test):
The central analgesic activity of the selected Compounds was tested in mice as described by Turner [29] using hot-plate apparatus. Seventy two mice were divided into 12 groups of 6 animals each. Mice of the 1 st (normal control) and 2 nd (reference one) groups were treated orally with the vehicle (5 ml/kg) and tramadol (40 mg/kg), respectively. Animals of the 3 rd till the 12 th groups were orally given the selected Compounds at doses of (20 mg/kg, p.o.). One h post-medication, mice were placed individually on a hot plate maintained at 53 ± 0.5°C. The time taken by the animals to lick the fore or hind paw or jump out of the place was taken as the reaction time for the thermal stimulus. The reaction time was measured at 0, 30, 60 and 90 min after treatment. The cutoff time for the response to the thermal stimulus was set at 60 Sec. to avoid tissue damage to the mouse paws. All drugs were dissolved in DMSO (20 mg/kg, orally), except tramadol (was dissolved in DMSO, 40 mg/kg, orally).

b. Peripheral analgesic activity (Writhing test):
The peripheral analgesic activity of the selected Compounds was determined in mice as described by Collier [30]. Seventy two mice were divided into 12 groups of 6 animals each. Mice of the 1 st (normal control) and 2 nd (reference one) groups were treated orally with the vehicle (5 ml/kg)

Anti-inflammatory activity: Carrageenan-induced mouse paw edema model:
The anti-inflammatory testing was performed according to the method of Winter [31] in Wistar rats. Paw edema was induced in rats by subcutaneous (s.c.) injection of 0.1 ml of 1% (w/v) carrageenan in distilled water in the sub-plantar region of their left hind paws. A group of six rats was left without any treatment, but orally given a respective volume of the solvent (DMSO), and was kept as control. The selected Compounds were administered at doses of (20 mg/kg, p.o.). Indomethacin (20 mg/kg, p.o.) was used as a reference drug. The paw volumes of the rats were measured using plethysmometer, before and after injection of 1% carragennan at different time intervals (1, 2, 3 and 4 h). Edema and inhibition rates of each group were calculated at the above-mentioned time intervals as follows:

Edema (%)=[Vt-Vo/Vo] × 100 Inhibition (%)=[Ec-Et/Ec] × 100
Where, Vo is the volume before carrageenin injection [8], Vt is the volume at t hour after carrageenin injection [8], Ec is the edema rate of the control group, and Et is the edema rate of the treated group. All drugs were dissolved in DMSO (20 mg/kg, orally), except indomethacin (was dissolved in DW).

Molecular docking
All docking studies were performed using "Internal Coordinate Mechanics (Molsoft ICM 3.8)". A set of three compounds 10, 12 and 16 designed to inhibit cyclooxygenases was compiled and 3D structures were constructed using Chembio3D ultra 13

Pharmacology
Analgesic activity: a. Central analgesic activity (Hot plate test): All tested compounds as well as the reference drug tramadol significantly prolonged the reaction time against the thermal stimulus as compared to the control one after 30, 60 and 90 min of administration (Table 1). Maximum protection against the thermal stimulus was observed at 90 min following the administration of the compound (5) (105.8%), which was statistically significant comparable to the reference drug tramadol (148.7%). As shown in Table 1, compounds (5, 6, 11 and 13) revealed The analgesic activity of the tested compounds after 90 min, as compared to the reference drug tramadol, arranged in descending order, were 105. 8
Anti-Inflammatory Activity: The selected compounds were evaluated for their possible anti-inflammatory activities in a rat model of carrageenan-induced paw edema. Table 3 shows the effect of selected compounds (4,5,6,7,8,10,11,12,13 and 16) on carrageenan-induced paw edema in rats in comparison to indomethacin, as a reference drug. Intra-plantar injection of carrageenan in rats led to increase in paw volume denoting edema in the control non-treated group as shown in Table 3. It was noticed that compounds (6, 8, 10, 11, 12 and16) in oral doses of 20 mg/kg significantly decreased the paw edema rate all over the four hours in comparison to the control non-treated group. The anti-inflammatory potencies of selected compounds were calculated by comparing their inhibition rate at different time intervals; with those obtained from animals receiving indomethacin, as standard anti-inflammatory drug. Administration of indomethacin significantly decreased the carrageenin-induced edema starting from the first hour and was persistent till the end of the experiment. The inhibitory effect of indomethacin on paw edema was 32.78, 26.35, 29.02, and 27.45% at the 1 st , 2 nd , 3 rd and 4 th hour, respectively. It was noticeable that compound 13 failed to decrease inflammation all over the experimental period. Moreover, the compounds (7 and 5) failed to decrease inflammation at the 1 st hour, while compound 4 failed to decrease inflammation at the 1 st and 2 nd hours. It is noteworthy to mention that the derivatives 10, 12, and 16showed anti-inflammatory potency after 4 hours greater than that of indomethacin and reached the maximum effect at the 2 nd h.

Molecular docking:
In an attempt to understand both the antiinflammatory and analgesic data on a structural basis, molecular docking studies were carried out using Mol soft ICM 3.8 software. The aim of the flexible docking calculations is prediction of correct binding geometry for each binder. The scoring functions and hydrogen bonds formed with the surrounding amino acids of the receptor. The new compounds which scores the highest anti-inflammatory activates 10, 12 and 16 were docked against the active site of COX-1 and COX-2 enzymes. Indomethacin was also docked against both COX-1 and COX-2 and used as reference drug. The scoring functions of the compounds were calculated from minimized ligand protein complexes. The docking results revealed that all the tested compounds showed   5, 6, 7, 8, 10, 11, 12, 13 and 16) in mice. Central pain was induced in Albino mice by thermal stimulation as detailed in the Materials and Methods section. Animals were treated with the test and control compounds and the analgesic activity was determined after 30, 60 and 90 min and compared to the controls. Data are shown as mean ± SEM.    -2d and 3a-3d). The ICM score values show good agreement with predicted binding affinities obtained by molecular docking studies as verified by pharmacological screening.

Conclusion
In this work, we have prepared a series of new pyrazolone derivatives in combination with pyrazole and dihydropyrimidinone scaffold, as well as heteroaryl and aryl pyrazole derivatives. The analgesic and anti-inflammatory activities were investigated for the title compounds utilizing the acetic acid-induced writhing test and the carrageenaninduced hind paw edema test, respectively.
The newly synthesized pyrazolone derivatives were found to possess potent analgesic and anti-inflammatory activities. The results showed that the central analgesic potencies of the tested compounds  8) which was greater than that of acetyl salicylic acid (71.5%). The selected Compounds were evaluated for their possible anti-inflammatory effects in a rat model of carrageenan-induced paw edema. It was noticed that compounds, (6, 8, 10, 11, 12, and16) in oral doses of 20 mg/kg significantly decreased the paw edema rate all over       the four hours in comparison to the control non-treated group. It is noteworthy to mention that the derivatives 10, 12 and 16 showed antiinflammatory potency after 4 hours greater than that of indomethacin and reached the maximum effect at the 2 nd h.