In the present paper, we report the synthesis and pharmacological evaluation of a new series of azo compounds with different groups (1-naphthol, 2-naphthol, and
Tyrosinase inhibitors are clinically useful for the treatment of skin diseases associated with melanin hyperpigmentation and applied in cosmetics for whitening and depigmentation after sunburn. Melanin is a heteropolymer of indole compounds and is produced inside melanosomes by the action of the tyrosinase enzyme on the tyrosinase precursor material in melanocytes. It has recently been discovered that some other factors such as metal ions and the TRP-1 and TRP-2 enzymes also contribute to the production of melanin. However, tyrosinase plays a critical role in the regulation of melanin biosynthesis. Therefore, many tyrosinase inhibitors that suppress melanogenesis have been widely studied with the aim of developing preparations for the treatment of hyperpigmentation [
It is well known that azo compounds are the most widely used class of industrial synthesized organic compounds because of their versatile usage in different fields, like dyeing textile fiber, biological–pharmacological activities, and advanced usage in organic synthesis [
In this research, we synthesized number of new azo compounds and studied chemical structures. Also we evaluated inhibitory effect on tyrosinase, melanin production inhibition and cytotoxicity of new compounds.
Diazonium salts could react readily with nucleophiles as aromatic compounds containing amino or hydroxyl group, which have been widely researched and applied for the preparation of molecules with importance for both academic and industrial applications. The solution of 4-amino hippuric acid and sodium nitrite in a 2.5% sodium carbonate was diazotized by slow addition of conc. HCl at 0°C. A yellow precipitate diazonium salt
Synthesis of compounds
Generally, variation in color of these dyes results from the alternation in the coupling components, since the synthesized dyes which were obtained varied in color from red to brown. Compounds (
Structures, UV-Vis absorption, yields, and melting points of new sulfanyl azo compounds
Compound | Structure | Yield (%)a |
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m.p. (°C) |
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63 | 498 | 293 (decom) |
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56 | 495 | 286 (decom) |
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59 | 505 | 308 (decom) |
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61 | 501 | 318 (decom) |
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71 | 499 | 325 (decom) |
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67 | 492 | 320 (decom) |
The compounds
Tyrosinase inhibitory activities of the compounds
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7 | Kojic acidb |
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Comparative graphical presentation of the tyrosinase inhibitory potentials of the compounds
Also inhibitions of the compounds
Melanin production and cytotoxicity.
Entry | Compound | Melanin production Inhibition (%) | Cytotoxicity cell viability (%) |
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7 | Kojic acida |
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Compounds
Comparing the activities with the structures of compounds, it turns out that the tyrosinase activity is mainly dependent on the substituent present at C-4 positions of aryl ring (-F or −CF3). When tyrosinase inhibitory activity of the most active compounds
A decrease in the activity of compounds
The least activity of compound
Compounds
All synthesized azo dyes exhibited high tyrosinase inhibitory behavior. The results of mushroom tyrosinase inhibition assays indicate that the 4-trifluoromethoxy derivatives have high degrees of inhibition and 1-naphthol and 2-naphthol derivatives are better for tyrosinase inhibition than
All the chemicals were obtained from Merck, Fluka, and Sigma-Aldrich and were used without further purification. Melting points were measured using Thermo Fisher Scientific. IR spectra were recorded by Bruker tensor 27, FT- IR spectrophotometer. All 1H NMR and 13C NMR spectra were recorded by a Bruker 400 MHz spectrophotometer. Chemical shifts are reported in parts per million (ppm) using tetramethylsilane (TMS) as an internal standard. The mass spectra were run on a Shimadzu Qp 5050 Ex spectrometer. The microanalyses for C, H, and N were performed on Perkin-Elmer elemental analyzer. Ultraviolet-visible (UV-vis) absorption spectra were recorded on a Perkin Elmer spectrophotometer at the wavelength of maximum absorption (
In a 125-mL erlenmeyer flask, 4-aminohippuric acid (0.01 mol) was added to 2% sodium carbonate solution (30 mL) until it was dissolved by boiling. The solution were then cooled down and sodium nitrite (0.01 mol) was added, with stirring, until it was dissolved. The solution was cooled down by placing in an ice bath, and then it was acidified by hydrochloric acid (2 mL), and then water (3 mL) was added. By acidifying the solution, a powdery yellow precipitate of the diazonium salt was separated.
2-Naphthol (0.01 mol) was dissolved in 5% sodium hydroxide solution (30 mL). The solution of 2-naphthol was added to suspension of hippuric acid diazonium salt with stirring, and base-stable form of the dye was separated. A stiff paste was formed in 5–10 min and then 10 mL of 10% acetic acid was added. The product was collected using saturated sodium chloride solution. The crude product was crystallized from water. The crude product was crystallized from water. Red powder, decomposed >236°C yield is 81%. IR (KBr):
1-Naphthol (0.01 mol) was dissolved in 5% sodium hydroxide solution (30 mL). The solution of 2-naphthol was added to suspension of hippuric acid diazonium salt with stirring and base-stable form of the dye was separated. A stiff paste was formed in 5–10 min, and then 10 mL of 10% acetic was added. The product was collected using saturated sodium chloride solution. The crude product was crystallized from water. Red powder, decomposed >259°C yield is 81%. IR (KBr):
A mixture of anhydrous sodium acetate (0.01 mol), 4-fluoro benzaldehyde or 4-trifluoromethoxy benzaldehyde (0.01 mol), sodium salt of azo dye
To a solution of compounds
Red powder; m.p. 293°C (decomposed); IR (KBr)
Brown powder; m.p. 286°C (decomposed); IR (KBr)
Brown powder; m.p. 308°C (decomposed); IR (KBr)
Red powder; m.p. 318°C (decomposed); IR (KBr)
Red powder; m.p. 325°C (decomposed); IR (KBr)
Red powder; m.p. 320°C (decomposed); IR (KBr)
The spectrophotometric assay for tyrosinase was performed according to the method Ref 15. Briefly, all the synthesized compounds were screened for the diphenolase inhibitory activity of tyrosinase using L-DOPA as substrate. All the compounds were dissolved in DMSO. The final concentration of DMSO in the test solution was 2.0%. Phosphate buffer, pH=6.8, was used to dilute the DMSO stock solution of test compounds. Thirty units of mushroom tyrosinase (0.5 mg/mL) were first preincubated with the compounds, in 50 mM phosphate buffer (pH 6.8), for 10 min at 25°C. Then the L-DOPA (0.5 mM) was added to the reaction mixture, and the enzyme reaction was monitored by measuring the change in absorbance at 475 nm of formation of the L-DOPA chrome for 10 min. The measurement was performed in triplicate for each concentration and averaged before further calculation. IC50 value, a concentration giving 50% inhibition of tyrosinase activity, was determined by interpolation of the dose-response curves. The percent of inhibition of tyrosinase reaction was calculated as the following:
Melanin production inhibition was ascertained by method of Wang et al. [
Cytotoxicity assays were performed using a microculture MTT method described by Han et al. [
All synthesized azo dyes exhibited high tyrosinase inhibitory behavior. The results of mushroom tyrosinase inhibition assays indicate that the 4-trifluoromethoxy derivatives have high degrees of inhibition and 1-naphthol and 2-naphthol derivatives are better for tyrosinase inhibition than
This work was supported by the Payame Noor University (PNU).