Synthesis of enhanced lipid solubility of indomethacin derivatives for topical formulations

2.1 Materials and chemical reagents

Indomethacin powder was given as a gift by Sama Pharmaceutical Company, Palestine. The other reagents that were used in the synthesis and the formulation were purchased from reliable sources: methanol and ethanol were obtained from Sun Pharm Ltd., Palestine; propanol was purchased from Avocado Ltd, Lancs; and isopropanol from Omega Raw Materials Drugstore, Palestine. Hexanol was obtained from Riedel DeHaen, Germany. Benzyl alcohol, sulfuric acid, hexane, octanol, and ethyl acetate were obtained from CS Ltd, and silica, anhydrous sodium sulfate, and diethyl ether was obtained from SDFCL, Mumbai.

2.2 Instruments

The following instruments were used throughout the research:

Infrared (IR) spectrophotometer (Nicolet iS5, Thermo-Scientific) was used to scan all the synthesized indomethacin pro-drug esters using attenuated total reflectance-fourier transform infrared at the range of 500–4,000 nm. Centrifuge (REF 1401, Hettich, Germany) was used to separate compounds. Rota evaporate (modal STONE, ST15 OSA, UK) was used to evaporate extracting solvent. UV/Vis spectrophotometer (Model 7315, Jenway, UK) was used to measure the absorbance. Franz diffusion cell using pump (modal PP-X-575) was used to measure transdermal penetration through mice skin of the synthesized drugs. Other instruments such as magnetic bar stirrer (modal LMS-1003) and Shakers (Model LSB-015S, Korea) were used.

2.3 Synthesis

Indomethacin esters were synthesized by esterification reactions with methanol, ethanol, propanol, and isopropanol reagents. The reaction progress was checked using thin-layer chromatography (TLC; Mineralight^® light, Model UVGL-58, USA). The TLC mobile phase used hexane and ethyl acetate at the ratio of 7:3.

The reaction started by weighing (500 mg) indomethacin in a round bottom flask. Subsequently, it was dissolved in 20 mL of reaction solvent (methanol, ethanol, propanol, and isopropanol). Then, four drops of concentrated sulfuric acid (H₂SO₄) were added to the reaction mixture. The reaction was refluxed for 48 h and was checked by TLC. The reaction was neutralized by adding 10 mL sodium bicarbonate. The ester was extracted using 30 mL dichloromethane by the separatory funnel. The organic layer was dried using sodium sulfate. The organic layer was evaporated using a rota evaporator. The dried residue was purified by silica gel column chromatography at the hexane:ethyl atetate (7:3) mobile phase. The synthesis reaction is shown in Figure 2.

Figure 2

Esterification reaction of Indomethacin.

Methyl-indomethacin (Comp 1): a pure white semisolid was obtained (280 mg, 54% yield). IR: ATR, υ _max (cm⁻¹): 3375.2 (stretch for carboxylic acid).

¹H NMR (dimethyl sulfoxide (DMSO)-d₆, 500 MHz) δ ppm: 2.31 (3H, s, CH₃), 3.35 (2H, s, –CH₂CO–), 3.59 (3H, s, –O–CH₃), 4.19 (3H, s, CO–O–CH₃), 6.59–7.13 (7H, m, Ar–H). ¹³C-NMR (DMSO-d₆, 125 MHz) δ ppm: 172.5, 153.5, 134.3, 133.1, 132.3, 130.5, 129.1, 112.3, 111.1, 111.5, 110.5, 100.4, 55.7, 39.9, 29.9, 11.8.

Ethyl-indomethacin (Comp 2): a pure yellow powder was obtained (310 mg, 57.4% yield). IR: ATR, υ_max (cm⁻¹): 3380.2 (stretch for carboxylic acid).

¹H NMR (DMSO-d₆, 500 MHz) δ ppm: 1.18 (3H, t, CH₃), 2.28 (3H, s, CH₃), 3.39 (2H, s, –CH₂CO–), 3.57 (3H, s, –O–CH₃), 4.17 (2H, q, CO–O–CH₂–), 6.59–7.13 (7H, m, Ar–H). ¹³C-NMR (DMSO-d₆, 125 MHz) δ ppm: 169.2, 167.7, 154.0, 140.1, 137.6, 135.1, 131.3, 131.1, 129.3, 128.7, 114.5, 113.4, 112.2, 100.6, 61.3, 55.8, 31.9, 14.1, 13.2.

Propyl-indomethacin (Comp 3): a pure yellow powder was obtained (350 mg, 62.5% yield). IR: ATR, υ_max (cm⁻¹): 3390.2 (stretch for carboxylic acid).

¹H NMR (DMSO-d₆, 500 MHz) δ ppm: 1.18 (3H, t, CH₃), 1.73 (2H, p, CH₃CH₂–), 2.28 (3H, s, CH₃), 3.39 (2H, s, –CH₂CO–), 3.57 (3H, s, –O–CH₃), 4.17 (2H, q, CO–O–CH₂–), 6.59–7.13 (7H, m, Ar–H). ¹³C-NMR (DMSO-d₆, 125 MHz) δ ppm: 170.2, 166.7, 153.0, 141.1, 136.6, 134.1, 131.3, 131.1, 129.1, 126.6, 114.2, 113.1, 111.1, 100.2, 65.2, 54.8, 31.7, 21.7, 12.1, 10.2.

Isopropyl-indomethacin (Comp 4): A pure yellow powder was obtained (290 mg, 51.8% yield). IR: ATR, υ_max (cm⁻¹): 3360.2 (stretch for carboxylic acid).

¹H NMR (DMSO-d₆, 125 MHz) δ ppm: 1.19 (6H, d, – (CH₃)₂), 2.28 (3H, s, CH₃), 3.39 (2H, s, –CH₂CO–), 3.57 (3H, s, –O–CH₃), 4.97 (1H, hept, CO–O–CH–), 6.59–7.13 (7H, m, Ar–H). ¹³C-NMR (DMSO-d₆, 500 MHz) δ ppm: 168.2, 166.7, 153.0, 136.6, 134.1, 131.3, 131.1, 129.4, 129.1, 128.6, 113.5, 112.4, 111.2, 68.4, 53.8, 31.2, 20.6, 12.2.

2.4 Log P determination

Log P determination was performed for the synthesized compounds to evaluate the lipophilicity of the synthesized pro-drugs and to judge its suitability for transdermal formulation. The test was done by measuring the concentration of the examined drug between two solvents, namely, octanol and water [21]. The concentrations of indomethacin esters were calculated using the absorbance at the predetermined λ max for each pro-drug determined by a spectrophotometer (Model 7315, Jenway, UK).

A serial standard solutions were prepared from stock solution (1 mg/mL) and were dissolved in octanol (0.1, 0.01, 0.02 mg/mL, and 0.04 mg/mL). The regression line equation from the calibration curve was used to demine the concentration of the pro-drug in octanol and consequently in the water.

To determine the log P of the compounds, 10 mg of indomethacin esters were added to 10 mL octanol and 10 mL distilled water in a test tube. The tube was placed on a shaker (Model LSB-015S, Korea) for 30 min at room temperature. Then, it was moved to a centrifuge (REF 1401, Hettich, Germany) for 10 min at 4,000 rounds per min. From the absorbance value of indomethacin esters, the concentration of the indomethacin derivative in both layers was determined, and consequently, the log P values were determined.

2.5 Dosage formulation

Ointment formulations were prepared for methyl indomethacin, ethyl indomethacin, propyl indomethacin, and isopropyl indomethacin (0.5% w/w). The ointment formulations were prepared by weighing 50 mg of the active ingredient and were dissolved in benzyl alcohol (0.5 g), which is used as a penetration enhancer. Vaseline (8.2 g) and paraffin (1.25 g) were dissolved at 75°C, and the two mixtures were added to each other and stirred until they cooled.

2.6 Transdermal testing using Franz cell diffusion

The dead animal abdomen skin used in the experimental part was provided by An-Najah National University animal house. The animal house provided the items after taking the required approval from the university ethical committee. All the animal experiments and methods were carried out in accordance with ARRIVE guidelines and regulations.

Franz cell diffusion test was carried out to measure the ability of Indomethacin and its esters to penetrate the skin and reach the blood circulation. The test was carried out by placing an ointment of 50 mg on rat skin. Diffusion cell apparatus was filled with absolute ethanol at 37°C, and the skin was placed above the solvent. Samples aliquots were taken every 30 min over 24 h, and the absorbance was measured by spectrophotometer. The drug concentration was calculated using the regression line equation of the previously generated calibration curve.

1. Ethical approval: The research related to animal use has been complied with all the relevant national regulations and institutional policies for the care and use of animals.

3.1 Synthesis

The proton and carbon NMR results show that all the targeted libraries of indomethacin derivatives were successfully synthesized. Moreover, the IR spectra of the compounds confirm the success of the synthesis of the targeted compound. The IR spectra show a strong stretch at around 1,700 cm⁻¹ and the absence of broad starch at 3,500 cm⁻¹, which was present in the nonesterified indomethacin. One of the synthesized compounds, the isopropyl derivative (comp 4), was tested for its mass spectrometry. The expected mass was found to be 400.88, and the result was 401.

3.2 Log P

Log P for the indomethacin esters was calculated using the calibration curve and the absorbance from the experiments. The results showed an increase in the log P with an increase in the alkyl chain. The highest log P was propyl indomethacin derivative compared (4.63) and was more than isopropyl (4.63). The detailed results are presented in Table 1.

Many researches showed that a good penetration occurs with higher log P. The log P, along with other factors, will affect the penetration, such as the molecular weight of the synthesized compound [22,23]. The results also show an increase in the log P value when compared to underivatized indomethacin, which has a value of 1.72 × 10⁻¹ [24].

3.3 Franz diffusion

All concentrations have been calculated from the absorption value of ethanol in the Franz cell released from the tested ointments. The results show the calculated concentration of each ointment formulation of the derivatized indomethacin using the predetermined regression line equation of the generated calibration curve for the synthesized compound. The result demonstrates the highest release of the drug after 3 hours (0.02073), which is almost equal to 10% of the total drug present in the ointment. However, underivatized indomethacin showed the lowest release (0.00941), which constitutes 3.76 release from the ointment. The detailed results are presented in Table 2. In the market, indomethacin topical preparation is used in a gel dosage. The absorption of this dosage from the gel in the lipophilic dermal is limited. Our novel derivatization approves the improvement of the skin penetration in an ointment formulations.

The concentrations were represented on a graph, which shows the differences between the concentrations as shown in Figure 3.

Figure 3

The concentration of indomethacin and its derivatives in the Franz diffusion cell after 3 h.