Flurbiprofen–antioxidant mutual prodrugs as safer nonsteroidal anti-inflammatory drugs: synthesis, pharmacological investigation, and computational molecular modeling

Abstract

Flurbiprofen–antioxidant mutual prodrugs were synthesized to reduce the gastrointestinal (GI) effects associated with flurbiprofen. For reducing the GI toxicity, the free carboxylic group (–COOH) was temporarily masked by esterification with phenolic –OH of natural antioxidants vanillin, thymol, umbelliferone, and sesamol. The in vitro hydrolysis of synthesized prodrugs showed that they were stable in buffer solution at pH 1.2, indicating their stability in the stomach. The synthesized prodrugs undergo significant hydrolysis in 80% human plasma and thus release free flurbiprofen. The minimum reversion was observed at pH 1.2, suggesting that prodrugs are less irritating to the stomach than flurbiprofen. The anti-inflammatory, analgesic, antipyretic, and ulcerogenic activities of prodrugs were evaluated. All the synthesized prodrugs significantly (P<0.001) reduced the inflammation against carrageenan and egg albumin-induced paw edema at 4 hours of study. The reduction in the size of the inflamed paw showed that most of the compounds inhibited the later phase of inflammation. The prodrug 2-oxo-2H-chromen-7-yl-2-(2-fluorobiphenyl-4-yl)propanoate (4b) showed significant reduction in paw licking with percentage inhibition of 58%. It also exhibited higher analgesic activity, reducing the number of writhes with a percentage of 75%, whereas flurbiprofen showed 69% inhibition. Antipyretic activity was investigated using brewer’s yeast-induced pyrexia model, and significant (P<0.001) reduction in rectal temperature was shown by all prodrugs at all times of assessment. The results of ulcerogenic activity showed that all prodrugs produced less GI irritation than flurbiprofen. Molecular docking and simulation studies were carried out with cyclooxygenase (COX-1 and COX-2) proteins, and it was observed that our prodrugs have more potential to selectively bind to COX-2 than to COX-1. It is concluded that the synthesized prodrugs have promising pharmacological activities with reduced GI adverse effects than the parent drug.

Keywords:

• flurbiprofen prodrugs
• natural antioxidants
• molecular docking
• molecular dynamic simulation
• pharmacological investigation
• NSAIDs

Supplementary materials

Animal care and use

The proper care and use of laboratory animals in research, testing, teaching, and production (animal use) require scientific and professional judgment based on the animals’ needs and their intended use.

Temperature and humidity

The animals were housed within the temperature range of 26°C–34°C. The relative humidity was kept in a controlled range of 30%–70%.

Ventilation and air quality

The aim of proper ventilation is to provide the animals with quality air and stable environment. In the present experiment, animals were protected from direct exposure to high velocity air (drafts). In accordance with the guidelines provision, 10–15 fresh air changes per hour in the animal house were carried out to maintain macroenvironmental air quality by constant volume systems and may also ensure microenvironmental air quality. Modern heating, ventilation, and air conditioning systems were used to maintain the ventilation rates in accordance with heat load and other variables. Heating, ventilation, and air conditioning system were regularly evaluated for proper functioning.

Illumination

As light can affect the physiology, morphology, and behavior of various animals, light used in the animal house was between 130 lux and 325 lux in the room at the cage level. Lighting was diffused in the whole area used for animal handling and provided sufficient illumination for the animals’ well-being. To maintain the good housekeeping practices, the bottom-most cages in racks were inspected regularly.

Noise and vibration

Noise produced by animals and animal care activities is inherent in the operation of an animal facility. An environment was designed to accommodate animals’ exposure to sound louder than 85 dB, as it can have both auditory and nonauditory effects; hence, it is kept below this level.

Guidelines followed during experiments

1. Swiss albino mice with a weight of 22–25 g (mean weight, 23.1 g) were obtained from the animal house, University of Sargodha, Sargodha.

2. The animals were housed in stainless metabolic cages (each cage had four mice according to the National Institutes of Health (NIH) guidelines that recommends that animals occupy space according to their size) and observed under a 12-hour/12-hour light/dark cycle to maintain illumination, in a well-ventilated room at 26°C–27°C, because according to NIH guidelines this is a thermo neutral zone for mice.

3. Standard diet was given to these mice (Ladokun Feeds, Ibadan, Nigeria) and water ad libitum.

4. The experimental protocol was according to internationally approved guidelines for animal use and care (EEC Directive of 1986; 86/09/EEC; National Institutes of Health publication 85-23, revised 1985). The acclimatization period lasted for 7 days.

Figure S1 Superimposed structures of COX-1 and COX-2 with binding pocket.

Abbreviation: COX, cyclooxygenase.

Figure S2 The hydrophobic graphs for predicted model of COX-1, having row index (number of residues) at x-axis and hydrophobic value at y-axis.

Note: The blue lines show the hydrophobic intensity of COX-1.
Abbreviation: COX, cyclooxygenase.

Figure S3 The hydrophobic graphs for predicted model of COX-2, having row index (number of residues) at x-axis and hydrophobic value at y-axis.

Note: The blue lines show the hydrophobic intensity of COX-2.
Abbreviation: COX, cyclooxygenase.

Figure S4 Ramachandran plot of COX-1.

Notes: 90.7% (486/536) of all residues were in favored (98%) regions; 97.4% (522/536) of all residues were in allowed (>99.8%) regions.
Abbreviation: COX, cyclooxygenase.

Figure S5 Ramachandran plot of COX-2.

Notes: 94.7% (1,042/1,100) of all residues were in favored (98%) regions; 99.9% (1,099/1,100) of all residues were in allowed (>99.8%) regions.
Abbreviation: COX, cyclooxygenase.

Table S1 The clinical effects on animals during the experiments

Acknowledgments

This work was supported by a research grant from the Kongju National University in 2015.

Authors contributions

ZA, A, and SJK supervised the study, designed the experiment, and wrote the manuscript. MK performed the experiment to synthesize the title compounds and ZA characterized the compounds. MW and HA performed the in vivo studies. MH and SA designed the in silico experiment and helped in performing the docking studies of the title compounds. MH performed the molecular dynamic simulation studies. All authors contributed toward data analysis, drafting and critically revising the paper, gave final approval of the version to be published, and agree to be accountable for all aspects of the work.

Disclosure

The authors declare no conflicts of interest in this work.