Dialkylphosphorylhydrazones as Potent Tyrosinase Inhibitors

Dezenove dialquilfosforilidrazonas com diferentes substituintes no anel aromático foram avaliadas como inibidores potenciais de tirosinase, que poderiam ser usadas como agentes eficientes no controle de desordens de pigmentação. A atividade inibitória foi medida através de uma modificação do método de UV-Vis de Patil e Zucker. Os ensaios foram realizados com soluções contendo tampão fosfato, L-3,4-diidroxifenilalanina (L-DOPA), EDTA, tirosinase e diferentes concentrações de compostos organofosforados e a formação de dopacromona foi determinada através do monitoramento da absorvância a 475 nm. Três compostos foram os mais ativos da série (CI50 = 105 ± 20 mmol L), (CI50 = 127 ± 16 mmol L) e (CI50 = 188 ± 27 mmol L), sendo de 3 a 7 vezes mais ativos do que o ácido ascórbico (CI50 = 730 mmol L), utilizado como controle. O composto mais ativo é apenas 1,5 vezes menos ativo do que o ácido kójico (IC50 = 69,4 mmol L), que é utilizado comercialmente. Esta investigação pode levar à descoberta de agentes potentes contra importantes desordens de pigmentação inclusive a hiperpigmentação.


Introduction
][3][4] Tyrosinase belongs to a type-3 copper protein family harboring a catalytic center formed by dinuclear copper and catalyzes the o-hydroxylation of phenol and the subsequent oxidation of catechol to the corresponding quinone (Scheme 1). 5 The quinone product is a reactive precursor for the synthesis of melanin pigments.In mammals, this enzyme is responsible for skin pigmentation abnormalities, such as flecks and albinism. 6The development and screening of potent inhibitors of tyrosinase is therefore of particular interest to the cosmetics industry.
In plants, tyrosinase catalyzes the initial reactions in the formation of melanin from tyrosine but it also oxidizes various phenolic compounds, aminoacids and proteins that promote darkening of plant tissues when damaged. 7n insects, tyrosinase also has a fundamental role, both in their development and for defense, in processes such as melanogenesis, tissue healing, parasite encapsulation and sclerotization. 8,9ol. 23, No. 5, 2012   Although tyrosinase plays important roles in the metabolism of animals, plants and microorganisms, some disorders are also associated with this enzyme.Alterations on the distribution of melanins give rise to a series of dermatological disorders, among which there are those resulting from excessive accumulation of skin pigmentation, such as melasma, age related spots and actinic damages.][10] The excessive accumulation of dopamine, which is produced in the initial steps, leads to the formation of reactive oxygen species, such as o-quinones, which bring about cell death. 11][13] Dopamine and its oxidation metabolites are also associated with disorders that cause vitiligo. 11ecause of the undesirable effects either caused by or associated with tyrosinase, there has been a growing interest in compounds which may inhibit this enzyme, thus efficiently acting on pigmentation disorders.Also, cosmetic industries have shown their interest in the search for skin whiteners, food industries are interested in preventing darkening of foods and finally pesticide industries search for tyrosinase inhibitors as alternatives in the control of insect infestations. 8,14he interaction of organophosphorus compounds with tyrosinase has been investigated by some researchers who found that aminophosphonates are good inhibitors of that enzyme.Among these, (3,4-dihydroxyphenyl)methylamino phosphonic acid has shown the higher inhibitory potential. 15,16us, in continuation of our research dealing with the synthesis of new organophosphorus compounds and their potential biological activities, we have screened nineteen new dialkylphosphorylhydrazones, whose syntheses are described elsewhere, 17 (Figure 1) for their tyrosinase inhibitory potential.

Experimental
Reagents employed in the inhibitory investigation were obtained from Sigma-Aldrich.
Tyrosinase inhibition activity was measured by a modified Patil and Zucker UV-Vis method. 18The modifications consisted on the use of a different concentration of EDTA.Also, L-DOPA was employed as a substrate and finally, commercial tyrosinase was used whereas the authors isolated the enzyme.
All the assays were carried out with solutions containing phosphate buffer (50 mmol L -1 , pH 8), L-DOPA (0.17 mmol L -1 ), EDTA (0.022 mmol L -1 ), tyrosinase (50-100 units) and varying concentrations of organophosphorus compounds and were done in triplicate.The inhibitor solutions were prepared in DMSO with an initial concentration of 10 mmol L -1 .
Different aliquots were added to the solution containing buffer, L-DOPA and EDTA to reach a final concentration of 660 mmol L -1 , the enzyme being added in the end.Formation of dopachromone was determined by monitoring the absorbance at 475 nm with a Shimadzu (Kyoto, Japan) Mini UV-Vis 1240 spectrometer.Ascorbic acid was used as standard with an initial concentration of 1 mmol L -1 .Percent inhibition was calculated using the following equation,  where B 0 = absorbance of L-DOPA + tyrosinase at t = 0 min, B 30 = absorbance of L-DOPA + tyrosinase at t = 30 min, A 0 = absorbance of L-DOPA + tyrosinase + inhibitor at t = 0 min, and A 30 = absorbance of L-DOPA + tyrosinase + inhibitor at t = 30 min.
The IC 50 values were calculated from the equation generated by exponential fit of the experimental data.

Results and Discussion
The ability of organophosphorus compounds to inhibit the conversion of L-DOPA into dopachromone, which is catalyzed by tyrosinase, was evaluated by monitoring the intensity of the absorption at 475 nm, characteristic of dopachromone.If a tyrosinase inhibitor is present, the amount of dopachromone decreases and so does the intensity of the absorption at 475 nm.A preliminary screening was performed at a maximum concentration of 660 μmol L -1 , which was the highest concentration where the inhibitors were soluble (Table 1).
At that concentration, compounds 5, 8 and 12 showed the highest inhibitory potencies and were screened at different concentrations in order to obtain their corresponding IC 50 values (Figure 2).
The IC 50 values for 5, 8 and 12 suggest that these dialkylphosphorylhydrazones are potent inhibitors of tyrosinase, with 5 being the most active.Comparison of the IC 50 value for 5 with those reported in the literature for commercial products shows that 5 (IC 50 = 105 ± 20 μmol L -1 ) is only less active than kojic acid (IC 50 = 69.4mmol L -1 ), 19 but is seven times more active than ascorbic acid (IC 50 = 730 mmol L -1 ).Compounds 8 (IC 50 = 127 ± 16 mmol L -1 ) and 12 (IC 50 = 188 ± 27 mmol L -1 ) have also been found to be more active than ascorbic acid.
Since compound 5 was found to be the most active, the kinetics of dopachromone formation was investigated in the presence of that compound.Thus, the absorbance was monitored at 10 min intervals until 30 min at four different concentrations.The results obtained were compared with those of solutions containing only L-DOPA and tyrosinase (Figure 3).It may be observed that, for all concentrations, after 30 min, the absorbance of the solution containing compound 5, L-DOPA and tyrosinase is lower than the solution containing only L-DOPA and enzyme and also, as the concentration of compound 5 increases, there is a decrease in the absorbance of this solution.These results indicate an inhibition effect of compound 5 on tyrosinase.
In order to evaluate the interaction between the dialkylphosphorylhydrazones 5, 8 and 12 with tyrosinase in the absence of L-DOPA, a set of UV-Vis curves were obtained for solutions containing only the organophosphorus compounds and another one for solutions containing both the enzyme and the organophosphorus compounds.Figure 4 shows these curves together with an experiment for L-DOPA.
As expected, the curve shows that L-DOPA interacts with tyrosinase, since the absorbance of the solution of L-DOPA plus enzyme is higher than that for only L-DOPA, evidencing a hyperchromic effect.Two new absorptions can be seen in the spectrum: one at 326 nm that can be attributed to the L-DOPA/tyrosinase complex 5 (Scheme 1) and another absorption that can be seen at 470 nm which is attributed to dopachromone that is being formed.It can be seen that both dialkylphosphorylhydrazones 5 and 8, which possess the CO 2 H and NO 2 groups respectively, interact with the enzyme in a similar way as L-DOPA, as shown in Figure 5.
On the other hand, compound 12, which has no substituents at the aromatic ring, and a lower inhibitory  ability, may interact with tyrosinase through both the phosphoryl moiety and the imidic nitrogen atom.Another possible way for inhibition to occur could be the interaction between the dialkylphosphorylhydrazones with L-DOPA, thus preventing the interaction between the oxidizing active site of the enzyme and the substrate.In order to investigate this possibility, the absorbance of solutions containing L-DOPA and the organophosphorus compounds 5, 8 and 12 was monitored at 10 min intervals for 30 min (Figure 6), using ascorbic acid as standard.No significant changes in absorbance were observed, indicating that these compounds do not interact with L-DOPA.
Compounds 1, 2, 4, 6, 7, 10, 11, 13, 14 and 17 showed low levels of inhibition while compounds 3 and 9 do not inhibit the enzyme.Compounds 15, 16, 18 and 19 potentiate the oxidative power of tyrosinase since the absorbance increased for these compounds.The relative increase for these compounds at a concentration of 660 mmol L -1 was calculated with equation 1 and the corresponding values of 59, 22, 32 and 75% were found.A possible explanation for this result may lie in the fact that these compounds have OH, OR and NR 2 groups and it has been reported that these compounds may be oxidized leading to dpachromone-like derivatives. 20In order to investigate if the introduction of the R 1 -phenylhydrazone moiety alters the enzyme activity, dipropylphosphorylhydrazine and diisopropylphosphorylhydrazine (Figure 7), precursors of the hydrazones, were also tested for inhibitory activity against tyrosinase.
Both hydrazines were initially tested at a concentration of 660 mmol L -1 .At this concentration, diisopropylphosphorylhydrazine showed an inhibition of 21% whereas dipropylphosphorylhydrazine showed 65% inhibition.In order to determine the IC 50 value for dipropylphosphorylhydrazine, this compound was assayed at different concentrations.The relative inhibition was calculated with equation 1 for each concentration.Figure 8 shows the tyrosinase inhibition curve for dipropylphosphorylhydrazine.The higher IC 50 value for    Compounds which possess the orto substituents, showed low inhibitory action, probably due to steric restrictions to binding to the enzyme.

Conclusions
Compounds 5, 8 and 12 have been found to be more active than ascorbic acid.However, the results described above indicate that although these compounds are significantly active, unfortunately, the mechanism of inhibition of tyrosinase by the dialkylphosphorylhydrazones is dependent on more than one factor and compounds with more substitutions are needed in order to carry out a quantitative structure/activity study.

Figure 4 .
Figure 4. Evaluation of the interaction between the dialkylphosphorylhydrazones 5, 8 and 12 with tyrosinase in the absence of L-DOPA.

Table 1 .
Percent inhibition of tyrosinase