Mushroom tyrosinase (EC 1.14.18.1), L-3,4-dihydroxy phenylalanine (l-DOPA), and kojic acid were obtained from Sigma (St. Louis, MO, United States). Dimethyl sulfoxide (DMSO) of molecular biological grade was purchased from J&K Chemical Co., Ltd. (Shanghai, China). Fetal bovine serum (FBS) and Dulbecco’s Modified Eagle’s Medium (DMEM) were obtained from Gibco (Grand Island, NY, United States). The 3-(4,5-Dimethythiazol-2-yl)-2,5-diphenyl-tetra-zoliumbromide (MTT) dye was supplied by Sigma-Aldrich (St. Louis, MO, United States). All other reagents were of analytical grade.

¹H and ¹³C NMR spectra were recorded in CDCl₃ on 400/300 MHz instruments, and spectral data were reported in ppm. High-resolution mass spectral analysis (HRMS) data were measured on Apex II by means of the ESI technique.

2-Cyanopyrrole derivatives A (1–17) were synthesized according to our previous article (Mou et al., 2016).

The general synthetic procedure for derivative A (1–12) is as follows. To a stirred mixture of aromatic olefin or alkyne (0.5 mmol, 1 equiv), DMF (15 equiv), TMSCN (trimethylsilanecarbonitrile, 5 equiv), and Cu(OTf)₂ (0.2 equiv), was added DDQ (2 equiv) through a batch-wise mode (the first equiv was added 0.2 equiv/2 h for 5 times, and the second equiv of DDQ was then introduced at once) at 80°C under an argon atmosphere. Then, the reaction mixture was stirred for an additional 14 h. Upon the completion of the reaction (monitored by TLC), the mixture was quenched by the addition of water. The aqueous layer was extracted three times with EtOAc, and the combined organic layers were washed with brine, dried over sodium sulfate, evaporated to dryness, and purified by column chromatography to afford desired products A (1–12).

The general synthetic procedure for derivative A (13–17) is as follows. To a mixed solvent of n-heptane and N,N-diethylacetamide (v/v = 6:1, 1 ml), aromatic olefin (0.5 mmol, 1 equiv), N,N-disubstituted formamide (3 equiv), TMSCN (6 equiv), and Cu(OTf)₂ (0.2 equiv) were successively added at 80°C under an argon atmosphere. Next, DDQ was added through a batch-wise mode (the first equiv was added 0.2 equiv/2 h five times, and the second equiv of DDQ was then introduced at once) at the same temperature. The reaction mixture was refluxed for an additional 14 h. Upon completion of the reaction (monitored by TLC), the reaction mixture was quenched by the addition of water. The aqueous layer was extracted three times with EtOAc, and the combined organic layers were washed with brine, dried over sodium sulfate, evaporated to dryness, and purified by column chromatography to afford desired products A (13–17).

The general synthetic procedure for derivative B (1–2) is as follows. To a stirred mixture of A5 or A6 (1 mmol) in DMSO (10 ml), K₂CO₃ (2 mmol) and hydrogen peroxide (30% aqueous, 30 ml) were added. The mixture was stirred at room temperature for 8 h. When the substrate was completely disappeared (monitored by TLC), the reaction mixture was extracted with ether and dichloromethane (2: 1) three times. The combined organic layers were washed with saturated brine (50 ml), dried over sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by column chromatography to give B (1–2).

The general synthetic procedure for derivative B (3–8) is as follows. To a stirred mixture of A6, A3, A5, A1, A9, or A2 (1 mmol) in ether (10 ml), lithium aluminum tetrahydride (1.2 mmol) was added slowly at 0°C. Then, the mixture was stirred at reflux for 3 h and quenched with water (10 ml). The mixture was extracted with ether three times. The combined organic layers were washed with brine (50 ml), dried over magnesium sulfate, and concentrated in vacuo to give crude primary amine 2. Then, the crude primary amine 2 (1 mmol), (Boc)₂O (1.3 mmol), and Et₃N (5 mmol) were added into methanol (10 ml) and refluxed for 20 min. The mixture was quenched with saturated aqueous ammonium chloride solution and extracted with diethyl ether three times. The combined organic layers were washed with brine (50 ml), dried over magnesium sulfate, and concentrated in vacuo. The crude residue was purified by column chromatography to afford B (3–8).

3-(3-Bromophenyl)-1-methyl-1H-pyrrole-2-carboxamide (B1). Amorphous solid; 39% yield; ¹H NMR (400 MHz, CDCl₃) δ 7.53–7.52 (m, 1H), 7.52–7.49 (m, 1H), 7.31–7.30 (m, 1H), 7.29–7.28 (m, 1H), 6.74 (d, J = 2.4 Hz, 1H), 6.09 (d, J = 2.4 Hz, 1H), 5.43–5.29 (m, 2H), 3.93 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 163.45, 134.99, 131.81, 131.07, 127.98, 127.14, 121.46, 109.32, 109.28, 98.48, 37.31, and 37.27; HRMS (ESI) calcd for C₁₂H₁₁BrN₂O [M + H]⁺: 279.0128, found 279.0124.

3-(4-Bromophenyl)-1-methyl-1H-pyrrole-2-carboxamide (B2). White solid; mp 172–174°C; 33% yield; ¹H NMR (400 MHz, CDCl₃) δ 7.52 (d, J = 8.4 Hz, 2H), 7.30 (d, J = 8.4 Hz, 2H), 6.73 (d, J = 2.4 Hz, 1H), 6.09 (d, J = 2.4 Hz, 2H), 5.48 (d, J = 9.6 Hz, 2H), 3.92 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 163.52, 134.94, 131.79, 131.06, 127.95, 127.13, 121.44, 109.28, and 37.31; HRMS (ESI) calcd for C₁₂H₁₁BrN₂O [M + H]⁺:279.0128, found 279.0123.

tert-Butyl ((3-(4-bromophenyl)-1-methyl-1H-pyrrol-2-yl)methyl)carbamate (B3). White solid; mp 109–113°C; 29% yield; ¹H NMR (400 MHz, CDCl₃) δ 7.48 (d, J = 8.4 Hz, 2H), 7.19 (d, J = 8.4 Hz, 2H), 6.64 (d, J = 2.4 Hz, 1H), 6.19 (d, J = 2.8 Hz, 1H), 4.59 (s, 1H), 4.39 (d, J = 5.2 Hz, 2H), 3.65 (s, 3H), and 1.46 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 155.25, 135.28, 131.54, 129.55, 125.03, 123.82, 122.73, 119.67, 107.40, 79.65, 34.57, 34.10, and 28.33; HRMS (ESI) calcd for C₁₇H₂₁BrN₂O₂ [M + H]⁺: 365.0859, found 365.0853.

tert-Butyl ((3-(3-fluorophenyl)-1-methyl-1H-pyrrol-2-yl)methyl)carbamate (B4). White solid; mp 103–104°C; 44% yield; ¹H NMR (400 MHz, CDCl₃) δ 7.35–7.29 (m, 1H), 7.10–7.08 (m, 1H), 6.95–6.91 (m, 1H), 6.64 (d, J = 2.4 Hz, 1H), 6.21 (d, J = 2.4 Hz, 1H), 4.63 (s, 1H), 4.42–4.41 (d, J = 4.8 Hz, 2H), 3.66 (s, 3H), and 1.47 (s, 9H); ¹³C NMR (100 MHz,CDCl₃) δ 162.93 (d, J = 243 Hz), 155.28, 138.62 (d, J = 7.9 Hz), 129.85 (d, J = 8.6 Hz), 125.19, 123.89, 123.53, 122.71, 114.68 (d, J = 21.4 Hz), 112.54 (d, J = 21 Hz), 107.50, 79.64, 34.55, 34.07, and 28.32; HRMS (ESI) calcd for C₁₇H₂₁FN₂O₂ [M + Na]⁺: 327.1479, found 324.1472.

tert-Butyl ((3-(3-bromophenyl)-1-methyl-1H-pyrrol-2-yl)methyl)carbamate (B5). White solid; mp 124–127°C; 40% yield; ¹H NMR (400 MHz, CDCl₃) δ 7.46 (s, 1H), 7.35–7.32 (m, 1H), 7.23–7.20 (m, 2H), 6.61 (d, J = 2.8 Hz, 1H), 6.17 (d, J = 2.8 Hz, 1H), 4.59 (s, 1H), 4.37 (d, J = 5.2 Hz, 2H), 3.62 (s, 3H), and 1.44 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 155.25, 138.53, 130.89, 129.94, 128.70, 126.49, 125.23, 123.57, 122.75, 122.54, 107.50, 79.65, 34.52, 34.09, and 28.33; HRMS (ESI) calcd for C₁₇H₂₁BrN₂O₂ [M + H]⁺: 365.0859, found 365.0857.

tert-Butyl ((1-methyl-3-phenyl-1H-pyrrol-2-yl)methyl)carbamate (B6). White solid; mp 87–90°C; 46% yield; ¹H NMR (400 MHz, CDCl₃) δ 7.37–7.30 (m, 4H), 7.24–7.20 (m, 1H), 6.61 (d, J = 2.4 Hz, 1H), 6.20 (d, J = 2.4 Hz, 1H), 4.67 (s, 1H), 4.40 (d, J = 4.8 Hz, 2H), 3.62 (s, 3H), and 1.45 (s, 9H); ¹³C NMR (100 MHz,CDCl₃) δ 155.26, 136.30, 128.41, 127.98, 125.73, 124.92, 122.43, 107.46, 79.40, 34.56, 33.91, and 28.24; HRMS (ESI) calcd for C₁₇H₂₂N₂O₂ [M + H]⁺: 287.1754, found 287.1750.

tert-Butyl ((1-methyl-3-(4-(trifluoromethyl)phenyl)-1H-pyrrol-2-yl)methyl)carbamate (B7). White solid; mp 120–122°C; 37% yield; ¹H NMR (400 MHz, CDCl₃) δ 7.62 (d, J = 8 Hz, 2H), 7.43 (d, J = 8.0 Hz, 2H), 6.67 (d, J = 2.8 Hz, 1H), 6.25 (d, J = 2.8 Hz, 1H), 4.66 (s, 1H), 4.42 (d, J = 4.8 Hz, 2H), 3.68 (s, 3H), and 1.47 (s, 9H); ¹³C NMR (100 MHz,CDCl₃) δ 155.27, 140.06, 127.99, 127.74 (q, J = 32.3 Hz), 125.56, 125.43 (q, J = 3.8 Hz), 124.41 (q, J = 270 Hz), 123.70, 122.98, 107.63, 79.75, 34.64, 34.12, and 28.34; HRMS (ESI) calcd for C₁₈H₂₁F₃N₂O₂ [M + H]⁺: 355.1628, found 355.1623.

tert-Butyl ((3-(2-fluorophenyl)-1-methyl-1H-pyrrol-2-yl)methyl)carbamate (B8). White solid; mp 81–84°C; 39% yield; ¹H NMR (400 MHz, CDCl₃) δ 7.31–7.27 (m, 1H), 7.25–7.21 (m, 1H), 7.17–7.08 (m, 2H), 6.68 (d, J = 2.4 Hz, 1H), 6.17 (d, J = 2.8 Hz, 1H), 4.81 (s, 1H), 4.29 (d, J = 5.6 Hz, 2H), 3.68 (s, 3H), and 1.45 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 159.50 (d, J = 241.7 Hz), 155.45, 131.63 (d, J = 3.7 Hz), 127.78 (d, J = 8.3 Hz), 126.72, 124.19 (d, J = 3.5 Hz), 124.05 (d, J = 15.3 Hz), 122.71, 117.68, 115.60 (d, J = 23.3 Hz), 108.39, 79.36, 34.81, 34.21, and 28.35; HRMS (ESI) calcd for C₁₇H₂₁FN₂O₂ [M + H]⁺: 305.1660, found 305.1657.

The mushroom tyrosinase activity assays of synthetic compounds were carried out according to the previously reported modification (Qin et al., 2015). l-DOPA was used as the substrate. Briefly, 800 μl of phosphate buffer (50 mM, pH 6.8), 50 μl of mushroom tyrosinase (666.67 U/ml, dissolved in PBS), and 50 μl of inhibitor (dissolved in DMSO) were placed in the plastic centrifuge tube. Then, 100 μl of l-DOPA (5 mM, dissolved in PBS) was added. Subsequently, its change in absorbance at 475 nm was measured in a time-dependent manner using a Beckman UV-650 spectrophotometer. The percentage inhibition was calculated by using [(OD₀ - OD₁)/OD₀] × 100%, where OD₀ was the absorbance without inhibitor, and OD₁ was the absorbance with the inhibitor. The IC₅₀ value of the potential compound was calculated from dose-response curves of percentage inhibition. Kojic acid was used as a standard control for comparison. All experiments were performed in duplicate.

Experiments were performed to analyze the inhibitory mechanism by the following method. For the text, the mushroom tyrosinase at a final concentration of 16.67–100.00 U/ml and l-DOPA at a final concentration of 0.5 mM were chosen. Then, the mushroom tyrosinase activity was measured in the presence of the inhibitor.

The inhibition type of the inhibitor on mushroom tyrosinase was evaluated by the Lineweaver–Burk plot, and the inhibition constants were obtained from the second plots of the apparent 1/V_max and K_m/V_max against the inhibitor concentration. In this assay, mushroom tyrosinase at a final concentration of 33.33 U/ml and l-DOPA at a final concentration of 0.25–2 mM were used. The equation for the Lineweaver–Burk plot can be written as follows: 1 v − K m V m ( 1 + [ I ] K I ) + 1 V m ( 1 + [ I ] K IS ) where v is the reaction velocity, K_m is the Michaelis constant, V_m is the maximal velocity, [I] is the concentration of the inhibitor, [S] is the concentration of the substrate, K_I is the constant for the inhibitor binding with the free enzyme, and K_IS is the constant for the inhibitor binding with the enzyme-substrate complex. K_I and K_IS were obtained from the slope or the vertical intercept vs. the inhibitor concentration, respectively.

Sybyl 2.1.1 (Tripos, United States) was used for the docking simulations between mushroom tyrosinase and the inhibitor. First, compound A12 was prepared by the energy minimization using the MM2 program, with energy termination of 0.01 kcal/mol, iteration of 1,000 times, and charges of Gasteiger-Hückle. Second, the crystal structure of mushroom tyrosinase (PDB ID: 2Y9X) was prepared by extracting ligands, removing H₂O, termini treatment, and adding hydrogens. The active pocket of tyrosinase was generated using the ligand mode. Finally, the docking simulation between tyrosinase and compound A12 was carried out in the default format. PyMOL software was used to draw the view based on the Sybyl results.

B16 mouse melanoma cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C in a 5% CO₂ incubator.

B16 cells (100 µl) were plated in a 96-well plate (5 × 10³ cells/well) for 24 h and then treated with a 100 µl complete medium containing A12 or kojic acid for 24 h. MTT solution (0.5 mg/ml, final concentration) was added to each well, and plates were incubated at 37°C for another 3 h. The absorbance was measured at 490 nm using a multi-detection microplate reader.

The inhibitory activity of A12 on tyrosinase in B16 cells was evaluated. B16 cells (5 × 10⁴ cells/well) were plated in 96-well plates and incubated for 24 h. A measure of 100 µl of complete medium including A12 or kojic acid was added for further incubation of 48 h. Then, cells were added to 50 µl Triton X-100 (1%) and allowed for freezing for 1 h at −80°C. After thawing at room temperature, 10 μl l-DOPA solutions (0.1%, w/v) were added and co-incubated for 1 h at 37°C. The absorbance at 405 nm was measured.

Pyrrole derivatives A (1–17) and B (1–8) were synthesized according to the synthetic route shown in Scheme 1. A1–A12 were obtained using aromatic olefin or alkyne, DMF, and TMSCN as starting materials under the catalysis of Cu(OTf)₂ and DDQ. A (13–17) were synthesized from aromatic olefin, N,N-disubstituted formamide, and TMSCN with Cu(OTf)₂ as the catalyst and DDQ as additive. B 1–2 could be synthesized from compound A5 or A6 through a hydrolysis reaction. Meanwhile, the reduction of compounds A6, A3, A5, A1, A9, or A2 by LAH with subsequent amine derivatization by (Boc)₂O would produce B (3–8). ¹H NMR, ¹³C NMR, and HRMS were applied to confirm their structures.

Tyrosinase inhibitory assay of all synthesized pyrrole derivatives A (1–17) and B (1–8) was performed using l-DOPA as the substrate. As shown in Table 1, compounds A (1–17) exhibited moderate to excellent inhibitory activities against tyrosinase, with IC₅₀ values ranging from 0.97 to 89.15 μM. In particular, A12 (IC₅₀ = 0.97 μM) showed the strongest inhibitory activities, which were ∼30 times stronger than the reference inhibitor kojic acid (IC₅₀ = 28.72 μM).

Based on the data displayed in Table 1, the SAR of the tested compounds against tyrosinase was analyzed. For compounds A (1–17), compound A1 (IC₅₀ = 7.24 μM) with no substitute group was selected as the template compound. It was clear that the introduction of a substituent on the phenyl ring at the 3-position of the pyrrole skeleton did show the clear influence of the corresponding inhibition potency. Among them, compound A2 with the 2-fluorine group on the benzene ring (IC₅₀ = 8.72 μM), A3 with the 3-fluorine group (IC₅₀ = 21.43 μM), A4 with the 2-bromine group (IC₅₀ = 8.47 μM), A5 with the 3-bromine group (IC₅₀ = 16.52 μM), A6 with the 4-bromine group (IC₅₀ = 8.17 μM), A7 with the 2-methoxy group (IC₅₀ = 23.57 μM), A8 with the 4-methoxy group (IC₅₀ = 89.15 μM), and A9 with the 4-trifluoromethyl group (IC₅₀ = 12.44 μM) all presented lower inhibitory activities than compound A1. These results indicated that the introduction of fluorine, bromine, methoxy and trifluoromethyl alone might cause a decrease in tyrosinase inhibitory activity. In contrast, two compounds A10 and A11 with dual substituents on the benzene ring provided better inhibitory results than compound A1 and showed an IC₅₀ value of 4.83 and 2.11 μM, respectively. Moreover, A12 with a 2-vinyl group on the benzene ring (IC₅₀ = 0.97 μM) also appeared to have much higher inhibitory activities than compound A1. This result revealed the special property of the 2-vinyl group during the interaction between A12 and tyrosinase. As for the substituent on the 1- and 5-positions of the pyrrole ring, additional five compounds A (13–17) were further tested. Based on the results, it is obvious that the presence of an untethered benzyl at 1-position (A14) or the phenyl group at 5-position (A15) would dramatically affect the inhibitory activities. On the contrary, A13 with the ethyl group on nitrogen of the pyrrole ring (IC₅₀ = 4.46 μM), A16, and A17 with the pyrrole as a part of a fused ring system showed higher inhibitory activities (IC₅₀ = 5.06 and 5.57 μM, respectively) than compound A1, which revealed that the alkyl group at 1-position or a planar type of the molecule might increase the inhibitory activity against tyrosinase. Therefore, based on the previous SAR results of all compounds, further derivatization of A1 with other ring systems at 3-position of the pyrrole ring or other fused ring systems will be designed and screened.

For investigating the effect of the cyano group on the inhibitory activities, it was transformed into an amide group (B1, B2) or carbamate group (B3 ∼ 8), and the inhibitory activities of corresponding derivatives were obviously reduced. These results indicated that the cyano group of compounds A1-A17 was crucial for tyrosinase inhibition.

Compound A12 with the best inhibition activity was selected as the lead compound to ascertain the inhibition mechanism of mushroom tyrosinase. Figure 1A shows the plots of initial velocity vs. tyrosinase concentrations at different concentrations of compound A12. As can be seen, the plots gave a family of straight lines, respectively, which passed through the origin point. Moreover, the slopes of the lines extended a descent with the increase of concentrations of compound A12. These results revealed that the presence of compound A12 did not bring down the amount of active enzyme but just resulted in the reduction of the enzyme activity. Hence, the tyrosinase inhibition of compound A12 was reversible.

To further explore the inhibition kinetic behavior of the most promising compound A12 on mushroom tyrosinase, the tyrosinase activity was determined under different concentrations of l-DOPA in the presence or absence of an inhibitor. The results were analyzed using Lineweaver–Burk double reciprocal plots. For compound A12 (Figure 2B), the results presented that the plots of 1/ν vs. 1/[S] gave straight lines with different slopes intersecting one another in the second quadrant. The values of V_m and K_m all descended with the increase in concentration of A12, which suggested that compound A12 induced mixed-type inhibition of mushroom tyrosinase. In other words, compound A12 could bind with both free enzyme and the enzyme-substrate complex, which was similar to the inhibition type of kojic acid. Hence, we determined the equilibrium constant for inhibitor binding with free enzyme (K_I) and the enzyme-substrate complex (K_IS) through the second plots of the apparent K_m/V_max and 1/V_max vs. inhibitor concentrations. The K_I and K_IS values of the compound A12 were calculated as 1.11 and 1.00 μM.

To better understand the inhibitory activity of compound A12, Sybyl and PyMOL software were conducted to understand the interaction pattern between the A12 and the active site of tyrosinase. Mushroom tyrosinase (PDB: 2Y9X) from the RCSB Protein Data Bank was selected as the target protein for simulation. The docking simulation results showed that A12 was well accommodated and bound in the active pocket of tyrosinase (Figure 3A) and had in-depth interactions with the active site residues (Figure 3B). The cyano group of A12 resided adjacent to the dicopper nucleus, indicating that there was a potential metal-ligand interaction, which was considered to be one of the key interactions between tyrosinase and the ligand compound. The pyrrole ring formed a π-π interaction with His263. Furthermore, A12 formed hydrophobic interactions with Ala286, Asn260, Gly281, His244, His259, Met280, Phe264, Ser282, Val248, and Val283.

For evaluating the effect of compound A12 on cellular tyrosinase activity, its cell cytotoxicity was first tested. The results showed that compound A12 and kojic acid showed no cytotoxicity on B16 melanoma cells under a concentration of 100 μM (Figure 4A). Thence, the concentration of A12 (under 100 μM) was selected for the assay of the cellular tyrosinase activity. A12 could effectively inhibit the tyrosinase activity in B16 melanoma cells in a dose-dependent manner (Figure 4B) and present an effective inhibitory of 33.48% at a concentration of 100 μM, which was equivalent to that of Kojic acid (39.81%).