Palladium(II)-catalyzed efficient synthesis of wedelolactone and evaluation as potential tyrosinase inhibitor

Article abstract of DOI:10.3390/molecules24224130

Tyrosinase is an enzyme widely distributed in nature, which has multiple functions, especially in the melanin biosynthesis pathway. Despite the few clinically available tyrosinase inhibitors for whitening, a great demand remains for novel compounds with low side effects in terms of potential carcinogenicity and improved clinical efficacy. A natural product, wedelolactone (WEL), with a polyhydroxyl moiety, attracted our attention as a potential tyrosinase inhibitor. Before we studied the biological activity of the natural product, a synthetic methodological research was firstly carried to obtain enough raw material. WEL could be obtained efficiently through palladium-catalyzed boronation/coupling reactions and 2,3-dicyano-5,6-dichlorobenzoquinone (DDQ)-involved oxidative deprotection/annulation reactions. Immediately after, the natural product was proven to be an efficient tyrosinase inhibitor. In conclusion, we developed a mild and efficient approach for the preparation of WEL, and the natural product was disclosed to have anti-tyrosinase activity, which could be widely used in multiple fields.

Full text of DOI:10.3390/molecules24224130

molecules
Article
Palladium(II)-Catalyzed Efficient Synthesis of
Wedelolactone and Evaluation as Potential
Tyrosinase Inhibitor
Huidan Huang 1, Jianqiu Chen 1,*, Jie Ren 2, Chaofeng Zhang 2,* and Fei Ji 1,
*
1
College of Engineering, China Pharmaceutical University, #639 Longmian Avenue, Jiangning District,
Nanjing 211198, China; zgykdxbjl@163.com
State Key Laboratory of Natural Medicines, Research Department of Pharmacognosy, China Pharmaceutical
University, #639 Longmian Avenue, Jiangning District, Nanjing 211198, China; cpubjl@163.com
Correspondence: cjq_1980@163.com (J.C.); chaofengzhang@163.com (C.Z.); jifei672453142@126.com (F.J.);
Tel.: +86-1381-385-9949 (J.C. & C.Z. & F.J.)
2
*
Academic Editor: Marc C. Kimber
Received: 26 September 2019; Accepted: 13 November 2019; Published: 15 November 2019
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Tyrosinase is an enzyme widely distributed in nature, which has multiple functions,
especially in the melanin biosynthesis pathway. Despite the few clinically available tyrosinase
inhibitors for whitening, a great demand remains for novel compounds with low side effects in terms
of potential carcinogenicity and improved clinical efficacy. A natural product, wedelolactone (WEL),
with a polyhydroxyl moiety, attracted our attention as a potential tyrosinase inhibitor. Before we
studied the biological activity of the natural product, a synthetic methodological research was firstly
carried to obtain enough raw material. WEL could be obtained efficiently through palladium-catalyzed
boronation/coupling reactions and 2,3-dicyano-5,6-dichlorobenzoquinone (DDQ)-involved oxidative
deprotection/annulation reactions. Immediately after, the natural product was proven to be an efficient
tyrosinase inhibitor. In conclusion, we developed a mild and efficient approach for the preparation of
WEL, and the natural product was disclosed to have anti-tyrosinase activity, which could be widely
used in multiple fields.
Keywords: wedelolactone (WEL); tyrosinase; Suzuki–Miyaura reaction; inhibitors
1. Introduction
Tyrosinase (EC 1.14.18.1) is an enzyme widely distributed in nature, which has multiple
functions, especially in the melanin biosynthesis pathway [
l-3,4-dihydroxyphenylalanine (l-DOPA), with further oxidation to dopaquinone [
1
]. The enzyme can catalyze l-tyrosine to
]. Dopaquinone
2
transforms from brown to black through several reactions. Abnormal melanin production, including
melasma, freckles, lentigo, senilis, and other forms of melanin hyperpigmentation could be a serious
aesthetic problem [
2
,
3
]. In addition, tyrosinase is also involved in the defensive and developmental
]. Excessive dopaquinone was also reported to cause neurodegeneration related to
]. Thus, many tyrosinase inhibitors are applied in cosmetics and pharmaceutical
functions of pests [
4
Parkinson’s disease [
5
products [
remains for novel compounds with low side effects in terms of potential carcinogenicity and improved
clinical efficacy [ ]. Obviously, more efforts are still needed in that direction; therefore, we recently
6]. Despite the few clinically available tyrosinase inhibitors for whitening, a great demand
7,8
focused our interest on discovering novel tyrosinase inhibitors.
Compounds with the polyhydroxyl moiety were proven to be potential tyrosinase inhibitors [9–11].
Recently, our group undertook research on a natural product with a polyhydroxyl moiety named
wedelolactone (WEL), which is derived from the medical plant Eclipta prostrata [12]. Although a wide
Molecules 2019, 24, 4130; doi:10.3390/molecules24224130
www.mdpi.com/journal/molecules

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range of pharmacological activities of WEL were reported, there is less information on the inhibitory
effect and reversibility of WEL on tyrosinase. Thus, the inhibitory activity and mechanism of WEL
toward tyrosinase deserves deeper investigation; however, but the present knowledge on synthesis of
the natural product is limited. Although several groups invested substantial effort in the preparation of
WEL, these methods had several disadvantages, including a time-consuming nature with complicated
synthetic approaches [13–15].
Among these methods, two routes listed in Figure 1 are commonly recognized by the industry.
However, both methods have several disadvantages. The first method (reported by Yang [14]) involves
a crucial intermediate, phenyl acetylene, which is difficult to prepare. The route has a low 15% overall
yield with a long linear sequence (total of 12 steps), and it is rarely applied to access a variety of WEL
analogues for structure transformations. The second method (reported by Lee et al. [13]) employs toxic
organotin and organomercurial reagents, which limit industrial production and increase operation
complexity. In addition, both methods can only obtain the natural products on a small scale. As the
present methods are imperfect and unsatisfactory for further investigation of WEL as an efficient
tyrosinase inhibitor, the development of a facile, versatile, and mild approach is urgently needed.
Figure 1. Reported synthesis routes of wedelolactone (WEL) and our proposal.
2. Results and Discussion
2.1. Palladium(II)-Catalyzed Efficient Synthesis of WEL
Retrosynthetically, WEL could be logically disconnected by the ring opening of furan to
afford the intermediate
to the intermediate 3-bromo-5-benzyloxy-7-acetoxyl-2-chromenone
4,5-dibenzyloxy-2-(4-methoxybenzyl)oxy-phenyl boronic ester (Scheme 1). This similar synthetic
strategy was ever used by Shen for the synthesis of hirtellanine A [16]. Synthetically, we expected
that polysubstituted coumarin could be obtained by Pd(II)-catalyzed Suzuki–Miyaura coupling
of 3-bromocoumarin and polysubstituted phenyl boronate ester which could be generated by a
Pd(II)-catalyzed boronation reaction of the polysubstituted bromobenzene . The coupling product
then underwent a DDQ-oxidation deprotection/annulation reaction to deliver the final product WEL
4
, which is further disconnected by C–C bond cleavage to trace back
3
and the readily prepared
2
4
3
2
1
4
5.

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Scheme 1. Synthesis of polysubstituted bromobenzene 1.
In the beginning of our synthesis, we focused on the generation of the polysubstituted
bromobenzene (Scheme 1). Selective protection of the three phenolic hydroxyl groups presented a
big synthetic challenge. After reviewing the literature [16 17], we chose the commercially available
3,4-dihydroxybenzaldehyde as the starting material to provide the polysubstituted bromobenzene
via the mCPBA-mediated Baeyer–Villiger oxidation strategy, which resolved the selective protection
of phenolic groups. Synthetically, protection of the phenol groups of 3,4-dihydroxy-benzaldehyde
with benzyl bromide afforded 3,4-bis(benzyloxy)benzaldehyde in 86% yield, which subsequently
underwent mCPBA-mediated oxidation and hydrolysis to deliver 3,4-bis(benzyloxy)phenol in 87%
yield. Next, was firstly protected with PMBCl and then selectively brominated with NBS to obtain
1
,
6
1
6
7
8
8
polysubstituted bromobenzene 1 in high yield.
Subsequently, we aimed to synthesize 3-bromo-5-benzyloxy-7-acetoxyl-2-chromenone 3 (Scheme 2).
According to reported methods [14], the commercially available phloroglucinol 10 and ethyl propiolate
as starting materials smoothly underwent ZnCl2-catalyzed esterification and cyclization reactions
to provide dihydroxycoumarin 11. Dihydroxycoumarin 11 was then treated with acetyl chloride to
deliver the corresponding diacetoxylcoumarin 12 in good yield. The following reaction involved the
dibromination and dehydrobromination of ketene 12 to afford 3-bromo-5,7-diacetoxyl-2-chromenone
13 in 75% yield. Reaction temperature is vital to the bromination reaction. It was found that maintaining
the reaction temperature at 10 ◦C could avoid the bromination of acetyl group of 12 and acquire the
optimal reaction yield. Finally, we ran the partial benzylation of 3-bromo-5,7-diacetoxyl-2-chromenone
13. The similar reactivity of the 5- and 7-hydroxy groups did not facilitate the selective 5-benzylation
of 13. According to Humbert’s selective alkylation reaction conditions [18], we tried similar normal
benzylation conditions (BnBr, K2CO3, acetone, reflux) and found that traces of water in acetone
chemoselectively hydrolyzed the 5-acetyl group as well, generating the intermediates 14 and 15
,
stabilized by the conjugating effect of the pyrone ring. Although the undesired deacetylation byproducts
still existed, the major benzylating product was 3-bromo-5-benzyloxy-7-acetoxyl-2-chromenone 3.
With the use of ethanol recrystallization, the crude product
used without further purification for the next step.
3 was obtained in 65% yield and directly
Scheme 2. Synthesis of 3-bromo-5-benzyloxy-7-acetoxyl-2-chromenone 3.

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With
3-bromo-5-benzyloxy-7-acetoxyl-2-chromenone
two fragments via boronation and a subsequent coupling reaction (Scheme 3). Under strong
alkaline and low-temperature conditions [19], polysubstituted bromobenzene could be treated
with bis(pinacolato)diboron to afford the borate ester To simplify the operation procedure,
Pd(II)-catalyzed boronation was attempted, and the corresponding borate ester was obtained in
quantitative yield as well. The formed crude product could be utilized directly without further
the
key
intermediates
polysubstituted
bromobenzene
1
and
3
in hand, we focused on the combination of
1
2
.
2
2
purification for the following Suzuki–Miyaura reaction and afforded the deacetyl coupling product 16
in good yield. To avoid the stability issue of borate ester in column chromatography, the boronation
and subsequent coupling reaction were carried out conveniently in one pot and gave a 72% overall
yield. The one-pot reaction conditions were optimized by initiating an experiment to evaluate the
effect of the various parameters on the reaction yield (see Supplementary Materials). Methylation of
the deacetyl coupling product 16 in the standard treatment with methyl iodide gave polysubstituted
coumarin 4 in 85% yield.
Scheme 3. Synthesis of polysubstituted coumarin 4.
Finally, the completion of the remaining steps in WEL synthesis required PMB deprotection,
cyclization, and debenzylation.
Removing the PMB on the substituted coumarin 4 in
acidic conditions (HOAc, reflux) led to the decomposed mixture and gave an unsatisfactory
result [20]. It was reported that DDQ could also be used as a deprotecting reagent to remove
the PMB group [21]. Thus, we adopted this synthetic strategy and hoped that oxidation
cyclization could subsequently happen after the deprotection. Fortunately, DDQ-involved
deprotection and oxidative annulation proceeded well as anticipated, forming the desired product
1,8,9-tris(benzyloxy)-3-methoxy-6H-benzofuro[3,2-c]chromen-6-one 17 in 56% yield (Scheme 4).
Compound 17 was then treated with BCl3 as a debenzylating reagent, generating the final product
1
wedelolactone
5
in 81% yield. H- and 13C-NMR spectra of the synthetic product were in agreement
with reported data for WEL [13].
Scheme 4. Synthesis of wedelolactone 5.

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2.2. Biological Activity
With a sufficient amount of WEL in hand, we tested the in vitro tyrosinase activity. Kojic acid was
used as a positive control, as typically employed in the evaluation of tyrosinase inhibitors. As shown
in Figure 2, WEL caused strong tyrosinase inhibition in a concentration-dependent manner. The 50%
inhibitory concentration (IC50) values of WEL and kojic acid were determined to be 1.2
±
0.3 and 14.2
±
1.6 M, respectively. The strong tyrosinase inhibitory activity may be due to the multiple hydroxyl
µ
groups in the structure. However, this needs to be further confirmed by determining the derivatives of
WEL (data not shown). To confirm the inhibitory mechanism of WEL against tyrosinase, the plots of
initial velocity versus tyrosinase concentration at different concentrations of WEL were developed,
and a set of straight lines was obtained (as shown in Figure 2). All of the lines passed through the
origin, and an increase in the WEL concentration reduced the slopes of the lines, indicating that the
compound was a reversible inhibitor.
Figure 2.
(A) Inhibitory activity of WEL and the control Kojic acid on tyrosinase. (B) Inhibitory
mechanism of WEL on tyrosinase.
2.3. The Docking Studies
Computational docking studies were employed to determine the preferred binding sites of WEL
in tyrosinase using the GOLD5.1 software. Tyrosinase contains two copper ions, and each copper ion
is coordinated by three histidine residues. The first copper (Cu A) is coordinated by His61, His85,
and His94, and the ligands of the second copper ion (Cu B) are His259, His163, and His 296 [21].
WEL could insert into the active site with a copper domain and it was found to interact with various
amino-acid residues (Figure 3). The possible site of hydrogen-bonding interactions of WEL with
tyrosinase was Asn260. The hydrogen-bonding residues could affect the binding affinity considerably.
According to the molecular docking study, it was found that the His61, Val248, His259, Asn260,
and His263 amino-acid residues of tyrosinase interact with WEL. The enzyme kinetic analysis and
molecular docking studies confirmed that WEL binds to tyrosinase in the active site.

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Figure 3. Computational docking simulations between tyrosinase and WEL obtained using GOLD5.1.
The cyan stick structures indicate WEL and the white stick structures represent the amino-acid
residues of tyrosinase. The copper ions are depicted as yellow balls. The black dotted lines represent
hydrogen bonds.
3. Materials and Methods
3.1. Reagents and Materials
LC-2010 HPLC system (Shimadzu) equipped with a Phenomenex Luna C18 analytical column
(250 mm
×
4.6 mm, 5 µm), a 6520 Accurate-Mass Q-TOF LC/MS system (Agilent, Santa Clara, CA,
USA) were obtained for this study. The chemical reagents, TYR and L-tyrosine were purachased from
m Sigma-Aldrich (St. Louis, MO, USA). Koic acid was purchased from Shanghai Yuanye biological
technology Co., Ltd. (purity >98%; Shanghai, China). Ultrapure water was prepared using a Millipore
water purification system (Millipore, Bedford, MA, USA).
3.2. Chemical Synthesis
Preparation of 3,4-bis(benzyloxy)benzaldehyde 7: A 250-mL reaction vessel with a magnetic stirring
bar was equipped with 3,4-dihydroxybenzaldehyde (5.13 g, 37.2 mmol), K2CO3 (25.70 g, 186.0 mmol),
BnBr (19.0 g, 111.6 mmol), and DMF (100 mL). The mixture was stirred at room temperature for 4 h.
After the reaction was completed, water (500 mL) was added, and the mixture was extracted with
ethyl acetate (500 mL). The organic layer was washed with water (250 mL
After drying over MgSO4, the extracts were filtered and concentrated. The residue was firstly stirred in
hexane (200 mL) overnight and then filtered to afford the intermediate without any purification for
×
2) and brine (250 mL).
7
the next step. White solid; 10.17 g, 86% yield; melting point (m.p.) 89–90 ◦C; Rf: 0.43 (EA:hexane = 1:4);
IR (cm−1): 3026, 2819, 2726, 1676, 1596, 1580, 1512, 1435, 1396, 1386, 1349, 1269, 1245, 1231, 1211,
1135, 1021; 1H-NMR (CDCl3, 300 MHz): 5.24 (s, 2H), 5.28 (s, 2H), 7.05 (d, J = 8.1 Hz, 1H), 7.34–7.52
(m, J = 12.0 Hz, 12 Hz), 9.84 (s, 1H); 13C-NMR (CDCl3, 75 MHz): 70.4, 70.5, 112.0, 112.7, 126.2, 126.6,
126.8, 127.5, 127.6, 128.1, 128.2, 129.9, 135.8, 136.1, 148.8, 153.9, 190.3 ppm; HR-MS (ESI) calculated for
C21H19O3 [M + H] 319.1334, found 319.1330.
Preparation of 3,4-bis(benzyloxy)phenol 8: To a solution of compound 7 (8.60 g, 27.0 mmol) in DCM
(135 mL) was added m-chloroperbenzoic acid (7.00 g, 40.5 mmol), and then the mixture was stirred
at room temperature for 15 h. The reaction was quenched with saturated aqueous Na2S2O3 solution
(25 mL) and saturated aqueous Na2CO3 solution (125 mL). The mixture was firstly extracted with DCM

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(125 mL) and then successively washed with saturated aqueous Na2CO3 solution (125 mL × 2) and
brine (75 mL). After drying over Na2SO4, the solvent was removed under vacuum. Next, the residue
was firstly dissolved in MeOH (135 mL) and then treated with K2CO3 (4.10 g, 29.7 mmol). The mixture
was stirred at room temperature for 30 min. After the reaction was completed, the solvent was removed
under vacuum, and water (100 mL) was added. HCl solution (3 M) was added to adjust the pH to
The mixture was extracted with ethyl acetate (250 mL 2). The combined organic layer was washed
with brine (200 mL) and dried over Na2SO4. Finally, the extracts were filtered and concentrated to
3.
×
afford the intermediate
8 without any purification for the next step. White solid; 7.19 g, 87% yield;
m.p. 105–106 ◦C; Rf: 0.25 (EA:hexane = 1:4); IR (cm−1): 3031, 2926, 1608, 1508, 1453, 1435, 1281, 1269,
1
1245, 1165, 1003; H-NMR (CDCl3, 300 MHz): 5.09 (s, 2H), 5.11 (s, 2H), 6.30–6.33 (m, 1H), 6.51 (s,
1H), 6.79–6.83 (m, 1H), 7.28–7.44 (m, 10H); 13C-NMR (CDCl3, 75 MHz): 70.6, 72.4, 102.9, 106.5, 117.2,
126.8, 127.2, 127.3, 127.4, 127.9, 128.0, 136.5, 137.1, 142.3, 149.8, 150.4 ppm; HR-MS (ESI) calculated for
C20H19O3 [M + H] 307.1334, found 307.1336.
Preparation of 1,2-dibenzyloxy-4-(p-methoxybenzyl)oxybenzene 9: To a solution of compound 8
(6.64 g, 21.7 mmol) in DMF (20 mL) was added NaH (60%, dispersion in paraffin liquid) (1.05 g,
26.1 mmol) at 0 ◦C. The mixture was stirred for 30 min, and PMBCl (3.22 mL, 23.9 mmol) was added.
The mixture was continually stirred at room temperature. After the reaction was finished according to
TLC monitoring, the saturated NH4Cl solution (100 mL) was added, and the mixture was extracted
with ethyl acetate (100 mL
(50 mL) and dried over Na2SO4. The filtrate was concentrated, and the residue was stirred in hexane
(75 mL) overnight. The mixture was filtered to provide the compound without any purification for
×
2). The organic layer was then washed with water (100 mL) and brine
9
the next step. White solid; 8.32 g, 90% yield; m.p. 80–81 ◦C; Rf: 0.62 (EA:hexane = 1:4); IR (cm−1): 3062,
3035, 2914, 2867, 1610, 1589, 1515, 1468, 1418, 1392, 1380, 1272, 1228, 1171, 1116, 1004; 1H-NMR (CDCl3,
300 MHz): 3.84 (s, 3H), 4.92 (s, 2H), 5.12 (s, 2H), 5.15 (s, 2H), 6.47–6.51 (m, 1H), 6.68 (d, J = 8.7 Hz, 1H),
6.88 (d, J = 8.7 Hz, 1H), 6.93 (s, 1H), 6.95 (s, 1H), 7.33–7.46 (m, 12H); 13C-NMR (CDCl3, 75 MHz): 54.8,
69.8, 70.6, 72.2, 103.4, 105.2, 113.5, 116.6, 126.9, 127.1, 127.2, 127.3, 127.9, 128.0, 128.6, 128.8, 136.6, 137.2,
142.7, 149.7, 153.7, 159.0 ppm; HR-MS (ESI) calculated for C28H27O4 [M + H] 427.1909, found 427.1909.
Preparation of 1,2-dibenzyloxy-4-bromo-5-(p-methoxybenzyl)oxybenzene 1: To a solution of compound
8
(7.32 g, 17.2 mmol) in DMF (185 mL) was added the solution of NBS (3.21 g, 18.0 mmol) in DMF
(35 mL) dropwise at 0 ◦C. After stirring for 30 min, the mixture was warmed to room temperature and
stirred for additional 4 h. When the reaction was completed, ice water (500 mL) was added and a white
solid appeared. After the suspension was filtered, the residue was washed with ice water, and dried
under reduced pressure to give the intermediate 1
. White solid; 7.54 g, 87% yield; m.p. 103–104 ◦C; Rf:
0.62 (EA:hexane = 1:4); IR (cm−1): 3058, 3012, 2910, 1589, 1520, 1420, 1395, 1375, 1225, 1180, 1123, 1001;
1H-NMR (CDCl3, 300 MHz): 3.84 (s, 3H), 4.96 (s, 2H), 5.09 (s, 2H), 5.10 (s, 2H), 6.63 (s, 1H), 6.92 (d,
J = 8.7 Hz, 2H), 7.17 (s, 1H), 7.33–7.46 (m, 12H); 13C-NMR (CDCl3, 75 MHz): 54.8, 71.4, 71.5, 72.1, 103.0,
104.4, 113.5, 120.1, 126.9, 127.1, 127.4, 127.5, 128.0, 128.1, 128.2, 128.5, 136.3, 136.5, 143.7, 148.5, 149.5,
158.9 ppm; HR-MS (ESI) calculated for C28H25BrKO4 [M + K] 543.0573, found 543.0559.
Preparation of 5,7-dihydroxy-2-chromenone 11: A stirred mixture of phloroglucinol dihydrate
(18.90 g, 150.0 mmol), ethyl propiolate (17.7 mL, 180.0 mmol), and ZnCl2 (1.00 g, 7.5 mmol) was heated
at 100 ◦C for 2 h. A flocculent precipitate formed during the reaction. After the mixture was filtered, the
solid was recrystallized from water to afford the intermediate 11. Yellow solid; 21.36 g, 80% yield; m.p.
263–264 ◦C; Rf: 0.50 (EA:hexane = 3:1); IR (cm−1): 3421, 3054, 1686, 1615, 1574, 1474, 1365, 1302, 1244,
1156, 1071; 1H-NMR (DMSO-d6, 300 MHz): 5.94 (d, J = 9.6 Hz, 1H), 6.09 (m, 1H), 6.17 (d, J = 2.1 Hz
,
1H), 7.86 (dd, J = 5.7, 9.6 Hz, 1H), 10.28 (s, 1H), 10.56 (s, 1H); 13C-NMR (DMSO-d6, 75 MHz): 94.0,
98.2, 101.6, 108.6, 139.5, 155.9, 156.4, 160.7, 162.0; HR-MS (ESI) calculated for C9H7O4 [M+H] 179.0344,
found 179.0330.
Preparation of 5,7-diacetoxyl-2-chromenone 12: A mixture of 11 (5.00 g, 28.0 mmol) and K2CO3
(19.40 g, 140.0 mmol) in dry acetone was heated to reflux (oil bath, 90 ◦C) for 1 h. Acetyl chloride
(8.0 mL, 112.3 mmol) was added dropwise with syringe. The reaction suspension was stirred for

Molecules 2019, 24, 4130
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another 1.5 h at 90 ◦C. The resulting solution was cooled down and filtrated out of the excess K2CO3.
The solvent was concentrated in vacuo, and the residue was recrystallized by MeOH to give compound
12. White solid; 6.24 g, 85% yield; m.p. 129–130 ◦C; Rf: 0.28 (EA:hexane = 1:2); IR (cm−1): 3075, 1767,
1745, 1630, 1575, 1433, 1365, 1309, 1242, 1188, 1131, 1066, 1027; 1H-NMR (DMSO-d6, 300 MHz): 2.31 (s,
3H), 2.40 (s, 3H), 6.51 (d, J = 9.6 Hz, 1H), 7.11 (d, J = 2.1 Hz, 1H), 7.24 (dd, J = 0.6, 2.1 Hz, 1H), 8.07
(dd, J = 0.6, 9.6 Hz, 1H); 13C-NMR (DMSO-d6, 75 MHz): 20.6, 20.8, 108.0, 110.6, 112.9, 115.9, 137.9,
147.4, 152.6, 154.3, 159.2, 168.5, 168.8 ppm; HR-MS (ESI) calculated for C13H11O6 [M + H] 263.0556,
found 263.0538.
Preparation of 3-bromo-5,7-diacetoxyl-2-chromenone 13: To a solution of 12 (2.00 g, 7.6 mmol) in
DCM (20 mL) was added Br2 (1.95 mL, 38.0 mmol) at 0 ◦C. The mixture was stirred at 10 ◦C for 8 h.
The reaction was quenched with a 2 M NaOH aqueous solution (25 mL). The brown solution was
extracted with DCM (50 mL). The organic layer was washed with water (25 mL
×
2) and brine (25 mL)
and then dried over Na2SO4. The filtrate was concentrated in vacuo to afford the dibromochromanone.
To the above dibromide in DCM (25 mL) was added Et3N (1.33 g, 13.2 mmol) at room temperature,
which was then stirred for 1 h. The resulting solution was removed from the solvent in vacuo, and the
residue was recrystallized by MeOH to give the desired product 13. White solid; 1.94 g, 75% yield;
m.p. 130–131 ◦C; Rf: 0.51(EA:hexane = 1:2); IR (cm−1): 3065, 1765, 1743, 1619, 1430, 1291, 1239, 1192,
1
1132, 1067, 1022; H-NMR (CDCl3, 300 MHz): 2.34 (s, 3H), 2.44 (s, 3H), 7.04–7.06 (m, 2H), 8.11 (s,
1H); 13C-NMR (CDCl3, 75 MHz): 20.4, 20.6, 107.3, 110.4, 111.1, 112.2, 137.6, 145.9, 152.4, 153.4, 167.5,
167.6 ppm; HR-MS (ESI) calculated for C13H9BrNaO6 [M + Na] 362.9480, found 362.9476.
Preparation of 3-bromo-5-benzyloxy-7-acetoxyl-2-chromenone 3: To a solution of 13 (1.53 g, 4.5 mmol)
and K2CO3 (1.88 g, 13.5 mmol) in acetone (30 mL) was added BnBr (0.81 mL, 6.8 mmol), and then the
mixture was stirred at 56 ◦C overnight. The suspension was concentrated in vacuo, and water (75 mL)
was added. The resulting solution was extracted with ethyl acetate (100 mL
×
2). The combined organic
layer was washed with brine (100 mL) and dried over Na2SO4. After the mixture was filtered, the
filtrate was removed from the solvent and the residue was◦recrystallized by EtOH to give the desired
product 3
. White solid; 1.13 g, 65% yield; m.p. 176–177 C; Rf: 0.60 (EA:hexane = 1:2); IR (cm−1):
3091, 2924, 2853, 1737, 1615, 1499, 1459, 1430, 1351, 1296, 1234, 1208, 1131, 1028; 1H-NMR (DMSO-d6,
300 MHz): 2.31 (s, 3H), 5.25 (s, 2H), 6.93 (d, J = 1.3 Hz, 1H), 6.98 (d, J = 1.6 Hz, 1H), 7.36–7.45 (m, 3H),
7.51–7.54 (m, 2H), 8.39 (s, 1H); 13C-NMR (DMSO-d6, 75 MHz): 20.8, 70.6, 102.8, 102.9, 107.7, 108.7, 127.8,
128.2, 128.5, 135.8, 138.9, 153.9, 154.0, 154.6, 156.3, 168.5 ppm; HR-MS (ESI) calculated for C18H14BrO5
[M + H] 389.0025, found 389.0022.
Preparation of 5-(benzyloxy)-3-(4,5-bis(benzyloxy)-2-((4-methoxybenzyl)oxy)phenyl)-7-hydroxy-
2-chromenone 16: To a solution of 1 (6.06 g, 12.0 mmol) and Pd(PPh3)2Cl2 (420 mg, 0.6 mmol, 5 mol%) in
THF (120 mL) was added TEA (14.4 mL, 103.0 mmol, 8.6 equivalents) and pinacoborane (13.0 mL,
◦
90.0 mmol, 7.5 equivalents) under Ar atmosphere. The mixture was heated overnight at 80 C.
After being cooled,
3 (3.18 g, 8.0 mmol), Pd(dppf)Cl2 (293 mg, 0.4 mmol, 5 mol.%), and a solution
of Na2CO3 (8.90 g, 84.0 mmol) in H2O (30 mL) was added under Ar atmosphere. The mixture was
stirred at 90 ◦C for another 12 h, and a 3 M HCl solution was added to adjust the pH to 3. The mixture
was filtered with diatomaceous earth, and the filtrate was extracted with ethyl acetate (200 mL
×
3). The combined organic layer was washed with brine (200 mL) and then dried over Na2SO4.
After drying and concentrating, the residue was purified by column chromatography with ethyl
acetate/petroleum ether (1:3–1:1) to afford the desired product 16. Yellow solid; 3.98 g, 72% yield; m.p.
166–167 ◦C; Rf: 0.32 (EA:hexane = 1:2); IR (cm−1): 3425, 3012, 2988, 1725, 1613, 1520, 1443, 1400, 1387,
1312, 1214, 1189, 1102, 1043, 1004; 1H-NMR (DMSO-d6, 300 MHz): 3.69 (s, 3H), 4.95 (s, 2H), 5.03 (s,
2H), 5.19 (s, 2H), 5.20 (s, 2H), 6.37 (s, 1H), 6.46 (s, 1H), 6.77 (s, 1H), 6.80 (s, 1H), 6.98 (s, 1H), 7.09 (s,
1H), 7.20–7.46 (m, 17H), 7.88 (s, 1H); 13C-NMR (DMSO-d6, 75 MHz): 55.0, 70.0, 70.4, 71.4, 93.7, 94.8,
96.6, 98.0, 102.2, 102.5, 113.6, 116.9, 118.5, 127.5, 127.6, 127.7, 127.8, 128.0, 128.2, 128.4, 128.5, 128.9,
129.1, 136.3, 136.6, 137.1, 137.4, 141.8, 149.2, 150.8, 155.5, 155.9, 158.8, 159.8, 161.8 ppm; HR-MS (ESI)
calculated for C44H37O8 [M + H] 693.2488, found 693.2484.

Molecules 2019, 24, 4130
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Preparation of 5-(benzyloxy)-3-(4,5-bis(benzyloxy)-2-((4-methoxybenzyl)oxy)phenyl)-7-methoxy-2-
chromenone : To a solution of 16 (1.38 g, 2.0 mmol) and K2CO3 (0.42 g, 3.0 mmol) in DMF (10 mL)
4
was added MeI (250 uL, 4.0 mmol) slowly, and then the mixture was stirred at the room temperature.
After TLC showed the reaction was completed, the mixture was diluted with ethyl acetate (50 mL),
and the organic layer was washed with H2O (40 mL
the filtrate was concentrated, and the residue was purified by column chromatography with ethyl
acetate/petroleum ether (1:3–1:2) to afford the desired product . Yellow solid; 1.20 g, 85% yield; m.p.
×
2) and brine (25 mL). After drying over Na2SO4,
4
130–132 ◦C; Rf: 0.46 (EA:hexane = 1:2); IR (cm−1): 3031, 3005, 2927, 2835, 1719, 1610, 1513, 1445, 1413,
1385, 1300, 1250, 1197, 1155, 1109, 1082, 1015; 1H-NMR (DMSO-d6, 300 MHz): 3.69 (s, 3H), 3.85 (s, 3H),
4.95 (s, 2H), 5.04 (s, 2H), 5.20 (s, 2H), 5.25 (s, 2H), 6.63 (d, J = 0.9 Hz, 2H), 6.76 (s, 1H), 6.79 (s, 1H), 7.00 (s,
1H), 7.11 (s, 1H), 7.20 (s, 1H), 7.23 (s, 1H), 7.30–7.44 (m, 11H), 7.46–7.49 (m, 4H), 7.90 (s, 1H); 13C-NMR
(DMSO-d6, 75 MHz): 55.0, 56.0, 70.2, 70.3, 70.4, 71.4, 93.0, 96.3, 102.2, 103.6, 113.6, 116.7, 118.4, 119.6,
127.6, 127.7, 127.8, 128.0, 128.2, 128.4, 128.5, 128.8, 129.1, 136.2, 136.4, 137.1, 137.4, 141.8, 149.4, 150.9,
155.5, 155.6, 158.8, 159.6, 163.0; HR-MS (ESI) calculated for C45H39O8 [M + H] 707.2645, found 707.2648.
Preparation of 1,8,9-tris(benzyloxy)-3-methoxy-6H-benzofuro[3,2-c]chromen-6-one 17: To a solution of
4
(0.71 g, 1.0 mmol) in toluene (20 mL) was added DDQ (0.45 g, 2.0 mmol), and then the mixture was
stirred under reflux for 24 h. After the reaction was finished, the mixture was concentrated and then
purified by column chromatography (DCM) to give the product 17. White solid; 327 mg, 56% yield;
m.p. 212–213 ◦C; Rf: 0.40 (EA:hexane = 1:2); IR (cm−1): 3031, 2930, 2850, 1736, 1604, 1450, 1401, 1352,
1301, 1273, 1197, 1161, 1137, 1081, 1049; 1H-NMR (CDCl3, 300 MHz): 3.88 (s, 3H), 5.25 (s, 2H), 5.27 (s,
2H), 5.31 (s, 2H), 6.48 (d, J = 2.1 Hz, 1H), 6.63 (d, J = 2.1 Hz, 1H), 7.20 (s, 1H), 7.37–7.63 (m, 15H), 7.68 (s,
1H); 13C-NMR (CDCl3, 75 MHz): 55.8, 70.8, 71.8, 72.0, 94.1, 97.1, 99.4, 105.5, 116.2, 126.8, 127.3, 127.5,
127.9, 128.0, 128.1, 128.5, 128.6, 128.7, 136.2, 136.8, 137.0, 148.0, 148.5, 150.1, 155.4, 155.9, 158.5, 159.7,
162.8; HR-MS (ESI) calculated for C37H28NaO7 [M + Na] 607.1733, found 607.1718.
Preparation of WEL
(3 mL, 1 M) slowly at 0 ◦C under Ar atmosphere. After the reaction was finished, the mixture was
concentrated and then purified by column chromatography (MeOH:DCM = 1:20) to give the product
5: To a solution of 17 (0.58 g, 1.0 mmol) in DCM (15 mL) was added BCl3
5.
Gray solid; 254 mg, 81% yield; m.p. >300 ◦C; Rf: 0.48 (MeOH:DCM = 1:10); IR (cm−1): 3397, 2955, 2923,
2853, 1710, 1670, 1612, 1447, 1324, 1284, 1206, 1151, 1070, 1047; 1H-NMR (DMSO-d6, 300 MHz): 3.82 (s,
3H), 6.45 (d, J = 1.8 Hz, 1H), 6.62 (d, J = 1.9 Hz, 1H), 7.16 (s, 1H), 7.24 (s, 1H); 13C-NMR (DMSO-d6,
75 MHz): 55.7, 93.2, 96.7, 98.1, 98.8, 101.7, 104.5, 113.7, 144.3, 145.4, 148.8, 154.8, 155.2, 157.7, 158.9,
162.2; HR-MS (ESI) calculated for C16H11O7 [M + H] 315.0505, found 315.0504.
1
Characterization data of WEL reported by Yang’s group were as follows: H-NMR (DMSO-d6,
300 MHz): 3.77 (s, 3H), 6.40 (d, J = 2.1 Hz, 1H), 6.55 (d, J = 1.8 Hz, 1H), 7.15 (s, 1H), 7.23 (s, 1H);
13C-NMR (DMSO-d6, 75 MHz): 55.7, 93.2, 96.7, 98.1, 98.9, 101.7, 104.6, 113.8, 144.3, 145.4, 148.9, 154.8,
155.3, 157.8, 158.9, 162.2.
All above chemicals were commercially available and used without further purification. Analytical
thin-layer chromatography was performed on glass plates precoated with silica gel impregnated
with a fluorescent indicator (254 nm). The plates were visualized by exposure to ultraviolet light.
1H-NMR spectra were recorded on a Bruker DRX (300 MHz), and 13C-NMR spectra were recorded on
a Bruker DRX (75 MHz). HR-MS spectra were taken on an Agilent Technologies Accurate-Mass Q-TQF
LC/MS 6520.
3.3. Biological Assays
The tyrosinase inhibition activity of the compounds was measured using l-DOPA as a substrate
according to a modified method of previous work [
at a concentration of 1.0 mM, and the final concentration of DMSO in the reaction mixture was
3%. In the investigation, the total volume of the reaction system was 300 L; l-DOPA was used
as the substrate for the determination of diphenolase activity. Briefly, l-DOPA (100 L, 0.5 mM),
phosphate buffer (180 L, pH 6.8, 50 mM), and different concentrations of inhibitors (10 L in DMSO)
4,10,11]. WEL was firstly dissolved in DMSO
µ
µ
µ
µ

Molecules 2019, 24, 4130
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◦
were mixed and incubated at 30 C. Then, an aqueous solution of mushroom tyrosinase (10 µL,
1000 U/mL) was added to the above solution and mixed quickly. Absorption and kinetic measurements
were carried out on a spectrophotometer from Thermo Fisher at 475 nm. Control experiments,
containing the same amount of DMSO without inhibitor, were routinely carried out. All measurements
were performed in triplicate. The enzyme activity was calculated according to Lambert–Beer’s law
(ε = 3700 L·mol−1·cm−1). The extent of inhibition by the inhibitor was expressed as the concentration
required to reduce tyrosinase activity to 50% (IC50). The percentage inhibition of tyrosinase activity
was calculated as follows:
% inhibition = [1 − (X − A1)/(A2 − A1)] × 100%,
where X is the absorbance at 475 nm of the inhibitor, A1 is the absorbance at 475 nm of the solution
without tyrosinase, and A2 is the absorbance at 475 nm of the bank (without inhibitor).
4. Conclusions
In summary, we developed an efficient synthetic route to synthesize wedelolactone, wherein
Pd(II)-catalyzed boronation/coupling reactions and DDQ-involved oxidative deprotection/annulation
are key reactions. The obtained WEL was determined as an efficient tyrosinase inhibitor which could
inhibit the enzyme in a reversible, competitive manner. Computational docking simulations showed
that WEL could bind with tyrosinase because its hydroxyl group on the scaffold bound residue Asn260
located in the active site. The study suggested that WEL may be efficient in restricting enzymatic
browning reactions, which could be widely used in multiple fields.
Supplementary Materials:
The following are available online.
Table S1:
Optimization of
boronation/Suzuki-Miyaura reactions; 1H NMR and 13C NMR spectra of the products.
Author Contributions: F.J. designed the experiments and wrote the manuscript; H.H. performed the in silico
molecular docking simulation and in vitro experiments, synthesized the compounds, and wrote the manuscript;
J.C. revised the manuscript; C.Z. advised on data interpretation; J.R. synthesized the compounds.
Funding: This work was supported by grants from the National Natural Science Foundation of China
(No. 81903498).
Conflicts of Interest: The authors declare no conflicts of interest.
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©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).