Utility of novel 2-furanones in synthesis of other heterocyclic compounds having anti-inflammatory activity with dual COX2/LOX inhibition

Abstract Inflammation is associated with the development of several diseases comprising cancer and cardiovascular disease. Agents that suppress cyclooxygenase (COX) and lipoxygenase (LOX) enzymes, besides chemokines have been suggested to minimise inflammation. Here, a variety of novel heterocyclic and non-heterocyclic compounds were prepared from novel three furanone derivatives. The structures of all synthesised compounds were confirmed by elemental and spectral analysis including mass, IR, and 1H-NMR spectroscopy. Anti-inflammatory activities of these synthesised compounds were examined in vitro against COX enzymes, 15-LOX, and tumour necrosis factor-α (TNF-α), using inhibition screening assays. The majority of these derivatives showed significant to high activities, with three pyridazinone derivatives (5b, 8b, and 8c) being the most promising anti-inflammatory agents with dual COX-2/15-LOX inhibition activities along with high TNF-α inhibition activity.


Introduction
Inflammation is a protective physiological defence mechanism provided by the body immune system in response to toxins, infectious pathogens, and local injury 1 . It occurs as a result of biosynthesis of pro-inflammatory mediators (leukotrienes [LTs] and prostaglandins [PGs]) from arachidonic acid (AA) by the action of the enzymes lipoxygenase (LOX) and cyclooxygenase (COX), respectively. Although inflammation is a normal defence mechanism, persistent/untreated inflammation leads to complicated events with release of many mediators that can turn the condition to be harmful and may lead to the development of certain diseases, as asthma, rheumatoid arthritis, atherosclerosis, diabetes, and cancer 2 . Therefore, anti-inflammatory agents may be helpful in the management of inflammatory disorders 3 . Anti-inflammatory effects of various compounds may result mainly from their ability to inhibit some of the key enzymes involved in inflammation and/ or cell signalling pathways such as COX and LOX 4 .
Thus, inhibition of these enzymes may be valuable treatment for inflammatory conditions. In the human system, COX occurs in two isoforms: COX-1 and COX-2 5 . Both isoforms catalyse a COX reaction in which they act on AA as substrate. COX isoforms are haem containing enzymes that demonstrate distinctive expression profiles and roles in numerous physiological processes. 6 COX-1 is constitutive isoform and is found in the gastrointestinal (GI)-tract, renal collecting tubules, and platelets and is believed to be responsible for the maintenance of physiological homeostasis such as renal function and GI integrity through production of gastroprotective PGs. On the other hand, the inducible isoform; COX-2 is released during tissue injury and induced by many kinds of inflammatory mediators; playing an important role in the proinflammatory PGs biosynthesis 7 .
Inhibition of both isoforms of COX by classical nonsteroidal anti-inflammatory drugs (NSAIDs) leads to inhibition of gastroprotective PGs produced via the COX-1 pathway; which occurs along with suppression of the pathological COX effects; resulting in the GI toxicities accompanying the use of numerous NSAIDs as GI irritation, bleeding, and ulceration 8 . Several studies revealed that COX-2 is highly expressed in a wide range of cancer tissues, such as colon, breast, and prostate, suggesting that it may control several cellular processes. Thus, selective COX-2 inhibitors have been extensively investigated for the treatment and prevention of a variety of cancers 9 .
Also, COX-2 is found in wide range of tissues e.g. brain, spinal cord, and kidneys, as well as many cells like vascular endothelium, suggesting that this isoenzyme may play a more complex physiological role than was expected 10,11 .
However, potent selective COX-2 inhibitors, which were used instead of NSAIDs; also showed disadvantages as incidence of vascular-diseases 12 . AA; which is the substrate of COX enzymes, is also converted by LOX enzyme to several lipid mediators recognised as eicosanoids 13 . LOXs are an exceptional group of nonhaem iron-containing enzymes that catalyse the peroxidation of polyunsaturated fatty acids viz. AA and linoleic acid to their hydroperoxides 14 . 15-LOXs are implicated in a variety of human diseases, like the oxidative alteration of low-density lipoproteins and thus, the progression of atherosclerosis 15 . In addition to many neurodegenerative diseases as Alzheimer's disease 16 , 15-LOX-1 inhibition has been reported to be a focal point to decrease the biosynthesis of eoxines, which are known to be pro-inflammatory mediators 17 and promotors for cancer disease 18 . Thus, some literature work has been targeting 15-LOX-1.
Yet, the use of LOX-inhibitors might represent an insufficient single therapeutic model in inflammatory diseases other than asthma 19,20 . It was discovered that dual inhibition of the COX and LOX pathways could produce a wider spectrum of anti-inflammatory effects and can limit the vascular-changes seen during inflammation and leukocyte-induced GI damage 21 .
Many pyridazinone derivatives were also reported as Va,b; which have superior anti-inflammatory activities over celecoxib and indomethacin, while derivatives VIa-c were more potent than aminopyrine, mebirizole, phenylbutazone, and mefenamic acid 37,38 . Some of oxadiazole derivatives VIIa,b showed superior activity over celecoxib besides compounds VIIIa,b; which were reported to be comparable with ibuprofen as dual COX/LOX inhibition activity 42,43 .
The anti-inflammatory activity shown by the above derivatives has drawn our interest to continue our research [44][45][46] for development of new anti-inflammatory agents. The synthesis and biological evaluation of new 2-furanone derivatives are reported herein. Also 2-furanone derivatives were used for synthesis of other heterocyclic and non-heterocyclic derivatives, which were tested as for their potential as anti-inflammatory agents against COX enzymes, LOX, and tumour necrosis factor-a (TNF-a).

Synthesis of lead compounds
All commercial chemicals used as starting materials and reagents in this study were purchased from Merck (Darmstadt, Germany) and were of reagent grade. All melting points were uncorrected and measured using Electro-thermal IA 9100 apparatus (Shimadzu, Japan); IR spectra were recorded as potassium bromide pellets on a Perkin-Elmer 1650 spectrophotometer (  2.1.2. General procedure for the synthesis of compounds 3a-c A solution of the furanone derivatives 2a-c (0.01 mol) and ammonium acetate (7.7 g, 0.1 mol) in acetic acid (10 ml) was refluxed for 3 h. The reaction mixture was left to cool at room temperature and the product obtained was filtered off, recrystallised from ethanol to give compounds 3a-c.  2.1.4. General procedure for the synthesis of compounds 5a-c A solution of the furanone 2a-c (0.01 mol) and phenyl hydrazine 3 ml in Na ethoxide (10 ml) was refluxed for 3 h. The product obtained was filtered, washed with water, and recrystallised from ethanol to give compounds 5a-c.    2.1.8. General procedure for the synthesis of compounds 9a-c A solution of hydrazides 6a-c (0.01 mol) and carbon disulphide (3 ml) in pyridine (10 ml) was refluxed for 3 h. The reaction mixture was left to cool at room temperature and poured onto ice water; the product obtained was filtered, washed with water and recrystallised from ethanol to give compounds 9a-c.

Cyclooxygenase (COX-1 and COX-2) inhibition assay
Tested compounds were dissolved in DMSO. Each compound was tested in triplicates using a COX inhibitory screening assay kit according to the manufacturer (Cayman test kit-560131, Cayman Chemical, Ann Arbor, MI). The COX inhibitor screening assay depends on direct measurement of the amount of PG2a produced in the COX reaction. Celecoxib, rofecoxib, and indomethacin were used as the positive control for inhibition of COX-1 and COX-2. An aliquot of 20 ml of each test compound or solvent (100% initial activity) was added to 950 ml of Reaction Buffer (0.1 M Tris-HCl, pH 8.0, containing 5 mM EDTA and 2 mM phenol), 10 ml of haem, and 10 ml of COX-1 or COX-2, then incubated with the enzymes at 37 C for 10 min. The reaction was initiated by addition of 10 ml AA to all the test tubes and incubation at 37 C for an additional 2 min. Enzyme catalysis was terminated by addition of 50 ml 1 M HCl and 100 ml of the saturated stannous chloride solution. The PGs are quantified by enzyme immunoassay (EIA) at 410 nm.

Lipoxygenase (15-LOX) inhibition assay
The experiment was performed in triplicates using Cayman's LOX inhibitor screening assay kit (Cayman test kit-760700, Cayman Chemical, Ann Arbor, MI) according to the manufacturer's protocol. DMSO was used as 100% initial activity and Quercetin was used as the positive control. Briefly, in a 96-well plate, 10 ml of each test compound (dissolved in DMSO) or vehicle were pre-incubated with 90 ml of 15-LOX enzyme. The reaction was started by addition of 10 ml of substrate (AA) and the plate was shaken for at least 5 min. Then, 100 ml of chromogen supplied with the kit was added to each well to stop the enzymatic reaction and develop the colour. The absorbance was measured at 490 nm using microplate reader.

TNF-a [biotinylated] inhibition assay
The assay was performed in triplicates using TNFR2: TNF-a [biotinylated] Inhibitor Screening Assay Kit (BPS bioscience, San Diego, CA, Catalog #79756), following the manufacturer's protocol. First, TNFR2 is coated on a 96-well plate. Next, biotinylated TNF-a is incubated with TNFR2 on the plate. Finally, the plate was treated with streptavidin-HRP followed by addition of an HRP substrate to produce chemiluminescence, which can be measured using a chemiluminescence reader.
The required 4-oxobutanoic derivative 1 was prepared by condensing dry dichlorobenzene with succinic anhydride in presence of anhydrous aluminium chloride, following Friedel-Crafts acylation reaction conditions.
As revealed in Scheme A, 2-furanone derivatives 2a-c proved to be useful precursors in the synthesis of several heterocyclic and non-heterocyclic derivatives. When they were allowed to react, separately, with ammonium acetate; pyrrol-2-one derivatives 3a-c were produced. 4-oxobutanamides 4a-c were prepared by refluxing 2-furanones 2a-c with benzylamine and 1-phenylpyridazinone 5a-c were also prepared from 2-furanones by refluxing with phenyl hydrazine according to the reported procedure 29,31 .
Pyridazinone derivatives 7a-c can be prepared by first stirring of 2-furanone derivatives 2a-c with hydrazine hydrate to obtain hydrazide derivatives 6a-c which can be cyclised by refluxing in HCl to obtain the desired pyridazinone derivatives 7a-c. They can be prepared directly by refluxing 2-furanone derivatives 2a-c with hydrazine hydrate.

Anti-inflammatory activity results
Inflammation is a protective defence response of the body. However, during inflammation, several pathological changes occur, which involve the release of common mediators of inflammation like PGs, histamine, nitric oxide, leukotrienes (LTB4), platelet-activation factor, lipoxins, and cytokines 50 . The inflammatory response must be terminated; using anti-inflammatory compounds, when no more needed to prevent avoidable harmful biological processes 51 . Inhibition of eicosanoids generation, in addition to the release of the pro-inflammatory cytokine; TNF-a from macrophages are used for in vitro inflammation tests 52 .
Thus, the newly synthesised compounds were tested for their anti-inflammatory activity against isozymes COX-1 and COX-2, which were determined by the COX-catalysed transformation of AA into PGH2 that was reduced to PGF2a and detected by the EIA 53 , as shown in Table 1.
Compound 2c was the most potent COX-1 inhibitor (IC 50 3.93 ± 0.06 mM) among test compounds, being 39 times less active than the reference drug indomethacin (IC 50 ¼0.10 mM) indicating the selective COX-2 inhibition activity of all test compounds. Pyridazinone derivatives 5b, 8b, and 8c were the most potent selective COX-2 inhibitors (IC 50 ¼0.04 mM for the three compounds), being superior to the reference drug (celecoxib IC 50 ¼0.05 mM). While amide derivatives; 4a-c, hydrazides; 6a, 6b, N-phenyl and N-benzoylpyridazinones; 5a, 5c, 8a and oxadiazole; 9b (IC 50 ¼0.05-0.09 mM range), showed comparable activity to celecoxib as selective COX-2 inhibitors. These results indicate the high activity of hydrazides, pyridazinones, and oxadiazole; which is in consistent with previously reported activities of similar scaffolds as selective COX-2 inhibitors 37,42,[54][55][56][57] . Studies showed that soybean LOX enzyme has a wide substrate specificity, and that the oxygenation sites for the soybean LOX has been demonstrated to be at C-13 of a-linolenic acid and C-15 of AA 58 . Therefore, the soybean LOX is most like the mammalian 15-LOX 59 .
Consequently, the in vitro inhibitory effect of all synthesised compounds against 15-LOX was determined using a lipoxygenation reaction to transform AA into hydroperoxides, which were detected by the addition of a chromogen, whose results are displayed in Table 1 60 .
Moreover, the ability to inhibit TNF-a, which is a potent proinflammatory chemokine was tested; results are shown in Table 2. The 15-LOX pathway was reported to induce inflammation through increased expression of IL-6, IFN-c, IL-12, and TNF-a 62 .
To analyse the structure-activity relationship (SAR) of the tested compounds from the previous results, it is obvious that our three newly synthesised furanones are totally inactive towards all the tested enzymes. But upon their conversion to other heterocyclic and non-heterocyclic compounds the biological activities appear as revealed in Figure 2 and explained as follows: 3 Pyrrolone derivatives 3a-c acquired high activity as LOXinhibitors, only one of them having 3-nitrobenzylidene substituent (3b) showed high activity as TNF-a inhibitor. They still have no significant activities as COX-2 inhibitors.
Upon ring opening and formation of amide derivatives 4a-c, a high TNF-a inhibition appears with significant selective COX-2 inhibition, but no significant LOX-inhibition observed.
Conversion of furanones to pyridazinines 7a-c did not affect their biological activities as they were still inactive. But formation of N-phenylpyridazinones 5a-c and N-benzoylpyridazinones 8a-c was a perfect pathway for highly potent derivatives against all the tested enzymes with desirable dual COX-2/15-LOX inhibition activities for compounds 5b, 8b, and 8c indicating the important influence of presence of 3-nitrobenzylidene substituent in both pyridazinones (5b, 8b) and 4-dimethylaminobenzylidene in N-benzoylpyridazinone (8c).

Conclusion
In conclusion, as presented in this study, novel 2-furanone derivatives were synthesised and used to prepare novel hydrazides, 2-pyrrolone, 2-pyridazinone, and oxadiazole derivatives. All the synthesised compounds were investigated for their anti-inflammatory activity; the biological results revealed that N-phenylpyridazinone 5b, N-benzoylpyridazinones 8b and 8c showed promising activity as dual COX-2/15-LOX inhibitors along with high TNF-a inhibition activity. Thus, these compounds might be promising anti-inflammatory candidates and may need further studies to be used clinically.

Disclosure statement
Authors would like to declare that there are no relationships or interests that could have direct or potential influence or impart bias on the work.

Funding
Authors are grateful to Science and Technology Development Fund (STDF) for financial support of this research. 9a,9c have higher activity as LOX and TNF-α inhibitors 9b has higher activity against 3 enzymes Inactive compounds 6a,6b have higher activity against 3 enzymes 6c has higher activity as LOX and TNF-α inhibitors 4a,4b,4c have higher activity as TNF-α inhibitor and significant selective COX2 inhibitor 5a-c and 8a-c have higher activity against 3 enzymes 7a-c are inactive compounds 3a,3c have higher activity as LOX and TNF-α inhibitors 3b has higher activity as LOX inhibitor 2a-c Figure 2. Structure-activity relationship of the tested compounds against the tested enzymes.