A Practical and Efficient Approach to the Preparation of Bioactive Natural Product Wogonin

Article abstract of DOI:10.1021/acs.oprd.6b00249

A scalable and practical route to wogonin, a bioactive natural product with multiple pharmacological activities which is currently under phase I/II clinical studies, is described. Wogonin was obtained via a four-step process starting from commercially ava

Full text of DOI:10.1021/acs.oprd.6b00249

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Full Paper
A Practical and Efficient Approach to the
Preparation of Bioactive Natural Product Wogonin
Tinghan Li, Tianwei Weng, Jubo Wang, Zhihui Wei, Lu Zhao, and Zhiyu Li
Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.6b00249 • Publication Date (Web): 10 Jan 2017
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A Practical and Efficient Approach to
the Preparation of Bioactive Natural
Product Wogonin
Tinghan Li,†,‡ Tianwei Weng,†,‡ Jubo Wang,†,‡ Zhihui Wei,†,‡ Lu Zhao,†,‡ and
Zhiyu Li*,†,‡
†Jiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University,
Nanjing, 210009, China
‡Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University,
Nanjing, 210009, China
*Corresponding author:
Tel: +86 139 5167 8592
Eꢀmail: zhiyuli@cpu.edu.cn
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ABSTRACT: A scalable and practical route to wogonin, a bioactive natural product with multiple
pharmacological activities which is currently under phase I/II clinical studies, is described. Wogonin was
obtained via a fourꢀstep process starting from commercially available chrysin and including the Elbs
oxidation, benzylation, methylation and the final debenzylation. The whole procedure gives the target
product in a 38% overall yield with >99% purity. Key steps in this process including oxidation and
methylation are discussed in detail. The optimized process has been successfully demonstrated on kilogram
scale to support ongoing clinical development of wogonin.
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KEYWORDS: Natural product, Largeꢀscale synthesis, Elbs oxidation, Reaction condition optimization.
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INTRODUCTION
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Wogonin (1, Figure 1) is a natural phytochemical flavones isolated from the roots of Chinese herbal
HuangꢀQin (Scutellaria baicalensis Georgi).1 It has been shown to exhibit a wide range of significant
biological activities such as antiꢀoxidation,2 antiꢀinflammatory,3 neuroprotective,4 especially antitumor
activity.5 Substantial efforts from several research groups have been devoted to explore the antitumor
mechanisms of 1.6 Unlike other natural flavones, 1 has been reported to selectively kill cancer with little to
no toxicity to normal cells at concentrations that are lethal to tumor cells.7 Significantly, 1 has been
approved by the China Food and Drug Administration (CFDA) for phase I/II clinical studies since 2014.
The content of 1 in Scutellaria baicalensis Georgi is very low with the ratio of active to dry material about
1.3%.8 The compound supply for development cannot be met simply via isolation and purification. Thus, it
is not surprising that the synthesis of this pharmaceutically important natural product has triggered great
efforts in recent years.
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Figure 1. Structure of bioactive flavone wogonin
RESULTS AND DISCUSSION
Original and Revised Routes to 1. Three synthetic approaches toward 1 have been reported.9 Rao’s route
in 1947 selected chrysin (2) as starting material to obtain 1 through O7ꢀbenzylation, oxidation, methylation
and debenzylation (Scheme 1, Route A).9a Although the route is short and simple, the overall isolated yield
of 1 is 5% due to the low yielding oxidation step (less than 15% yield from 3 to 4). In 2003, Huang et al
reported that 1 could be synthesized by three steps from a relative expensive starting material
3,4,5ꢀtrimethoxyphenol (5) (Route B).9b Additionally, since the two hydroxyl groups of intermediate 6 were
competing for the ring closure reaction, this process was accompanied by the structurally similar
byꢀproduct oroxylin A (7) which was difficult to separate. In 2009, Li et al disclosed another method
(Route C) based on Route B by utilizing intermediate 9 which has only one reactive position in order to
avoid the competitive addition while forming the flavone nucleus.9c In this method, 9 was procured from
pyrogallic acid (8) sequentially via benzylation, oxidation, reduction, methylation, debenzylation and
FriedelꢀCrafts acylation. Subsequently, 9 was converted to 1 through cyclization and selective
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demethylation. Although this method can produce tenꢀgram scale of 1 in a single batch, there are two
shortcomings limiting the largeꢀscale synthesis: (1) long synthetic procedure and laborious workꢀups; (2)
the relatively low 14.9% overall yield.
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The drawbacks of the above routes urged us to find an alternative synthetic route to 1 with simplified
procedure and improved yield ready for largeꢀscale manufacture. We envisioned that the most expeditious
route to 1 was to select a compound with 2ꢀphenylchromone as starting material and avoid the cumbersome
ring closure reaction. Therefore, Rao’s route was taken as reference, in which the Elbs oxidation was the
key step needed to be improved. A review of the literature revealed that the yields of the Elbs oxidations are
closely related to the starting materials, e.g. 5,7ꢀdihydroxyflavones always give higher yields than
5ꢀhydroxyꢀ7ꢀbenzyloxyflavones.10 Thus, in order to improve yield, chrysin (2) which is at the same time
inexpensive and commercially available was selected as starting material and the substrate of the Elbs
oxidation. Direct O7ꢀbenzylation of the sulfate 10 followed by hydrolysis, methylation and debenzylation
finally generated 1 (Scheme 2).
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Scheme 1. Initial Routes to Wogonin
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Scheme 2. An Improved Route to Wogonin
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Oxidation of Chrysin (2). The Elbs oxidation, generally applied in synthesis of flavone with low yield
and long reaction time,11 is a nucleophilic displacements reaction in which the nucleophile is a phenolate
anion and the product is an aromatic sulfate whose orientation relative to the phenolic group is
preferentially para. According to the literature procedure,12 the Elbs oxidation is typically carried out by
dissolving the substrate in an alkaline aqueous medium, and then adding a peroxydisulfate salt at room
temperature. Tetramethylammonium hydroxide was chosen as the alkali because the ammonium salt of
phenol was found to be much more soluble than the sodium and potassium salts, and potassium persulfate
was chosen as the oxidant because it is mild and cheap. The reaction concentration reported in the literature
was 120 L solvent mix/kg of starting material. Optimization of the reaction concentration showed that the
yield of oxidation was closely related to the solvent volume and the highest yield was obtained when the
reaction concentration being 20−30 L solvent mix/kg of starting material. 25 L solvent mix/kg of starting
material was eventually chosen as the reaction concentration.
The oxidation is known to be highly exothermic. Although potassium persulfate is a mild oxidant, an
○
exotherm from ~24 to ~36 C was observed during this reaction when potassium persulfate was added in
one portion. Subsequent experiments identified the addition rate of potassium persulfate as an important
parameter for achieving safety profile of this reaction. It was found that adding potassium persulfate in 12
equal portions (2 equiv in total) every 20 minutes exhibited very slight exotherm (temperature rise of ~6 ○C)
between each addition. The improved operation has been successfully applied to the kilogram manufacture
of compound 10.
Based on the literature report,13 after stirred for 3 hours, the pH of the reaction solution was neutralized to 6
by addition of monopotassium phosphate and extracted with nꢀbutanol. Considering that nꢀbutanol is
expensive with a high boiling point, the workup was improved with adding a large amount of sodium
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chloride to the neutral solution to precipitate the product relying on the salting out effect.14 The improved
workup procedure which was more environmentally friendly and costꢀefficient circumvented the
inconvenient extraction and purification, as well as increased the yield to a considerable extent.
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Benzylation and hydrolysis. Gratifyingly, a prior substitution on 7ꢀhydroxy of compound 10 was
observed during the benzylation and nearly no 5ꢀsubstitued product was obtained probably due to the
intraꢀmolecular hydrogen bond on this position. A brief screen of reaction conditions for O7ꢀbenzylation of
10 was performed. It was determined that N,NꢀDimethylformamide (DMF) was a suitable solvent because
of the good solubility of 10 in it and potassium carbonate (K2CO3) delivered the highest conversion as base.
It was also found that using fine powder K2CO3 was favourable for the rate of the benzylation.
Further optimization with varying amounts (1−2 equiv) of benzyl bromide (BnBr) was undertaken (Figure
2). The results showed that the conversion was low (~50%) when the amount of BnBr was below 1.5 equiv,
and a larger amount of BnBr (1.7−2 equiv) accelerated the formation of the undesired diꢀbenzylated
byproduct 13 (Scheme 3). As a result, 1.5 equiv of BnBr was adopted as a standard.
In order to streamline the synthetic procedure and simplify the workups, we envisaged that the hydrolysis
could be telescoped into the prior step. After the benzylation was deemed complete by HPLC, the reaction
slurry was filtered to remove K2CO3, and the stream of 12 in DMF was taken directly into the hydrolysis
step with 6 M hydrochloric acid (HCl) (Scheme 3). Precipitation from the hydrolysis mixture by addition of
water gave crude 4 in a 90% yield, contaminated with ~4 area % of 14 which was derived from the
byproduct 13 (Scheme 3). Subsequent experiments showed that recrystallization of crude 4 from
tetrahydrofuran (THF)/water could remove the undesired impurity 14 efficiently to a <1% level, providing
4 of 98% purity.
Scheme 3. Synthesis of 4 from 10 and Byproduct 13 and 14
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Figure 2. Benzylation conversion when using 1−2 equiv of BnBr (condition: 1 equiv. 10, 2 equiv. K2CO3,
0.25 M DMF).
Methylation of dihydric phenol 4. Methylation of phenol was typically carried out by reaction of phenol
and methylating agent in the presence of base in organic solvent. At the beginning, by using dimethyl
sulfate as methylating agent, the reaction was unsatisfactory due to moderate conversion and low isolated
yield of 11. Therefore, a lot of exploration with regard to base and reaction solvent was made to find the
best conditions for methylation.
Table 1. Screen of Base for Methylationa
entry
1
base
Et3N
solvent
Acetone
DMF
conversion (%)
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Et3N
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Pyridine
KHCO3
DMF
4
DMF
5
K2CO3
Acetone
DMF
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K2CO3
7
K2CO3 (2 equiv)
K2CO3 (3 equiv)
NaOH
DMF
8
DMF
9
Acetone
DMF
10
11
NaOH
NaOH (aq)
MeOH
a Standard condition: 4 (1 equiv), dimethyl sulfate (1.2 equiv), base (1.5 equiv), solvent (0.15 M).
b Conversions determined by HPLC.
As shown in Table 1, a low conversion was observed (<60%) when organic base such as triethylamine
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(Et3N) or pyridine or weak inorganic base such as potassium bicarbonate (KHCO3) was used (entry 1−4). It
was found that a stronger base such as sodium hydroxide (NaOH) (powder) produced more impurities, thus
perturbing the conversion to 11 (entry 9−10). We also attempted to use aqueous NaOH (28 wt %) as base
and methanol (MeOH) as solvent but only obtained poor conversion (entry 11). Pleasingly, K2CO3 (powder)
was found to be the optimal base affording high conversions when screened in acetone and DMF (entry
5−8). Meanwhile, a rapid screen of amounts of K2CO3 showed that conversion peaked at 92% for 2 equiv
of K2CO3 and remained unchanged when the amount of K2CO3 was further increased (entry 6−8).
Later, reactions in the presence of K2CO3 in different solvents, three nonpolar, four polar aprotic and one
polar protic were carried out at room temperature (Figure 3). The choice of solvent for the methylation
reaction proved to be critical. HPLC analysis indicated that the use of more polar solvent afforded higher
conversion and better reaction profile, the best being DMF with a conversion of 92%. However, an impurity
which was identified as the O5ꢀmethylated byproduct 15, was formed when polar especially protic solvent
MeOH was used. This was probably because MeOH could destroy the intraꢀmolecular hydrogen bond on
5ꢀhydroxyl and at the same time stabilize the nucleophilic phenoxyl ions, which promoted the
O5ꢀmethylation reaction. Utilizing DMF as solvent and K2CO3 as base, typical conversions of ~92% were
achieved on a laboratory scale (10 g) to produce several batches of 11 in 87−89% yield.
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OH
O
O
O
O
O
O
O
O
O
O
O
O
Me2SO4
+
base/solvent
OH
OH
4
15
11
Figure 3. Methylation conversion when using different solvents (condition: 1 equiv. 4, 1.2 equiv. dimethyl
sulfate, 2 equiv. K2CO3, 0.15 M reaction concentration, 25 ○C)
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The optimized procedure was further employed on the larger scale synthesis of 11. Unfortunately, a
dramatic drop in reaction conversion was observed and worse still, the more the input was, the lower the
conversion was. It was found that the use of DMF as a single solvent led to agglomeration over time even
when agitation was efficient, which did not appear at small scales, and presumably accounted for the
varying conversions. We postulated that adding a cosolvent with low boiling point may help to reduce the
viscosity of the reaction and improve the conversion. Hence, an assessment of single and mixed polar
solvents was undertaken at different scales (Figure 4). Conducting the reaction in the presence of DMF or
MeOH did not give fruitful results. Interestingly, THF was found to afford stable conversions around 80%
even at large scales, which was most likely due to the relatively good solubility of 4 in THF. Similar
stability was observed with better conversion (~90%) when a mixture of DMF and THF was used as
solvent. The optimum DMF/THF ratio was determined as 1:1 after further optimization.
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Figure 4. Methylation reaction profile in various polar solvent systems (condition: 1 equiv. 4, 1.2 equiv.
dimethyl sulfate, 2 equiv. K2CO3, 0.15 M reaction concentration, 25 ○C)
Synthesis and Isolation of 1. The final removal of the benzyl group was routinely achieved by
hydrogenation using THF as solvent and Pd/C as catalyst. The optimized catalyst loading was 10 wt % of
10% Pd/C (wet). The reaction mixture was maintained at room temperature for 4 hours until no further
hydrogen consumption was observed. After removal of the Pd/C by filtration through Celite, the filtrate was
concentrated to dryness, and the crude product was recrystallized with ethanol twice to reduce the Pd to an
acceptable level (< 5 ppm) and provide 1 as a yellow solid with 99.87% purity.
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Scale-up Results. Subsequently, with the optimal reaction conditions in hand, we intended to apply this
strategy to the kilogramꢀscale synthesis of 1. In several trials at this scale, the yield for the oxidation of 2
was ~60% (uncorrected), consistent with the yield of trials at small scales. However, the sulfate 10
precipitated from the reaction mixture was viscous and difficult to be dried off. MeOH could be used to
solve this problem and provide product of >90% purity. The crude intermediate 4 procured by benzylation
and hydrolysis showed a purity of ~88% but can be easily improved with an efficient recrystallization
(THF/H2O), which gave a combined yield of ~86%. The increasing impurities in methylation led to the
reduced purity of intermediate 11 at large scales. Several attempts were made to purify the material, and a
recrystallization from NMP/H2O was developed to remove impurities and afford the material with high
purity (>99%) effectively. The yield of eventual debenzylation was about 92−93% and a recrystallization
from ethanol can give pure 1 with >99% purity.
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CONCLUSION
In summary, we have demonstrated a practical and efficient process for the synthesis of wogonin, a
bioactive natural product with multiple pharmacological activities. Notable improvement was the
development of a telescoped fourꢀstep procedure that obviated building the flavone core with a relatively
high (38%) overall yield, comparing with the initial synthetic routes in literatures. The process from
commercially available chrysin to wogonin was significantly optimized with easy workups and high yields.
More importantly, the improved Elbs oxidation made it possible to be applied to largeꢀscale production. All
steps were run on more than 500 gram scale, and no chromatography was needed even at the final step.
This scalable process has allowed multiple batches of 1 with high purity (>99.8%) for clinical studies.
EXPERIMENTAL SECTION
General Information. All reagents and solvents purchased from commercial suppliers were used
without further purification. 1HNMR and 13CNMR spectra were collected on a Bruker AVANCE 300 MHz
spectrometer. Chemical shifts (δ) are reported in parts per million (ppm) from tetramethylsilane (TMS)
using the residual solvent resonance. Multiplicities are abbreviated as: s (singlet), d (doublet), t (triplet), q
(quartet), m (multiplet), br s (broad singlet). HRꢀMS was recorded on Agilent technologies 6520
AccurateꢀMass LC/MS QꢀTOF instruments. The procedures outlined in this section are those employed for
the reactions run at the largest scale. Unless specified otherwise, all compounds were ≥95% pure.
Preparation of tetramethylammonium 5,7-dihydroxy-4-oxo-2-phenyl-4H-chromen-8-yl sulfate
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(10). A 50 L reactor was charged with tetramethylammonium hydroxide (2184 g, 24 mol, 6 equiv) and
water (28.58 L). Chrysin (2, 1016 g, 4 mol, 1 equiv) was added over 20 minutes, and the mixture was
stirred at room temperature for an additional 20 minutes. Then, potassium persulfate (2160 g g, 8 mol, 2
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equiv) was added every 20 minutes in 12 equal portions. An exotherm from 24 to 30 C was observed
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between each addition. After the final portion was added, the resulting homogeneous dark solution was
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stirred at 25 ± 2 C for 2.5 hours, after which time HPLC showed that all starting material was consumed.
The reaction soluti8on was neutrallized to pH 6−7 by adding monopotassium phosphate (1360 g, 10 mol,
2.5 equiv) in 6 portions every 10 minutes, resulting in the formation of dark brown precipitate. Sodium
chloride (2340 g, 40 mol, 10 equiv) was added into the batch in 5 portions every 5 minutes and the
resulting mixture was stirred at room temperature for 4 hours. The precipitate was collected by filtration,
and the viscous cake was washed with 2 × 5 L of MeOH (manually stirring the viscous cake on the filter
then filtrating) and dried in vacuo at 45 ± 2 ○C until constant weight to give 10 as a brown solid (1032.3 g,
1
91.7 area% by HPLC purity, 56% corrected yield). H NMR (300 MHz, DMSOꢀd6):
δ = 12.61 (s, 1H,
5ꢀOH), 10.12 (s, 1H, 7ꢀOH), 8.18 (m, 2H, ArꢀH), 7.61 (m, 3H, ArꢀH), 7.01 (s, 1H, CHCO), 6.32 (s, 1H,
ArꢀH), 3.09 (s, 12H, N+(CH3)4) ppm. 13C NMR (75 MHz, DMSOꢀd6): 182.0, 163.3, 157.4, 157.0, 149.8,
132.1, 130.8, 129.0 (2 × C), 126.6 (2 × C), 121.5, 104.9, 104.1, 99.7, 54.4 (4 × C) ppm.
Preparation of 7-benzyloxy-5,8-dihydroxy-2-phenyl-4H-chromen-4-one (4). To a 20 L reactor
was added 10 (1057.7 g, 2.5 mol, 1 equiv), K2CO3 (690 g, 5 mol, 2 equiv) and anhydrous DMF (10 L).
After stirring for 20 minutes, benzyl bromide (641.3 g, 3.8 mol, 1.5 equiv) was added to the mixture. The
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resulting homogeneous dark solution was warmed to 50 C and held at this temperature for 8 hours, at
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which time HPLC analysis indicated reaction completion. The reaction mixture was then cooled to 20 C
and filtered. The filtrate was acidified to pH 2 by adding 1.5 L of 6 M HCl slowly (exothermic) and
agitated vigorously at room temperature overnight. Water (45 L) was added to the resulting mixture slowly
to precipitate the product. The suspension was filtered, and the filter cake was washed with 1 L of water and
dried in vacuum to get 840.6 g (93% yield, uncorrected) of crude 4 as an orange solid.
○
Recrystallization: Crude 4 (125 g, 0.35 mol) was suspended in THF (960 mL) and then heated to 40 C
○
until the solution was homogeneous. An equivalent amount of water (960 mL) was added slowly at 40 C
over 20 minutes, and the solution was naturally cooled down to room temperature. The suspension was
further chilled to 5 ○C for 1 hour and filtered. The filter cake was washed with 100 mL cold THF/water (1:5)
and dried in vacuum at ambient temperature, providing 118.8 g of pure 4 (90% yield, 97.6 area% by HPLC
1
purity). H NMR (300 MHz, CDCl3):
δ = 12.36 (s, 1H, 5ꢀOH), 7.96 (m, 2H, ArꢀH), 7.52 (m, 3H, ArꢀH),
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7.42 (m, 5H, ArꢀH), 6.66 (s, 1H, CHCO), 6.52 (s, 1H, ArꢀH), 5.20 (s, 2H, ArOCH2) ppm. 13C NMR (75
MHz, CDCl3): 182.9, 164.0, 154.1, 151.2, 143.4, 135.2, 131.9, 131.3, 129.1 (2 × C), 128.9 (2 × C), 127.9
(3 × C), 126.4 (2 × C), 105.3 (2 × C), 96.3, 71.6 ppm. HRMS (ESI), [M + H]+ calculated for C22H17O5
361.1071, found 361.1077.
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Preparation of 7-benzyloxy-5-hydroxy-8-methoxy-2-phenyl-4H-chromen-4-one (11). 4 (900 g,
2.5 mol, 1 equiv), K2CO3 (690 g, 5 mol, 2 equiv), anhydrous DMF (8 L) and THF (8 L) were charged to a
50 L reactor, and the mixture was stirred for 20 minutes. Dimethyl sulfate (378 g, 3 mol, 1.2 equiv) was
added to the solution at room temperature slowly over 30 minutes, maintaining the reaction temperature
○
between 25 and 30 C. The resulting slurry was then held at this temperature for an additional 3 hours.
After the reaction was deemed complete by HPLC analysis, 16 L water was added slowly to quench the
reaction (exothermic). The resulting mixture was stirred for 30 minutes and filtered. The filter cake was
washed with 1 L of water and dried in vacuum to give 841.5 g (90% yield, uncorrected) of crude 11 as a
yellow solid.
Recrystallization: Crude 11 (100 g, 0.27 mol) was dissolved in NMP (1.5 L), and then 500 mL water was
added to the solution slowly while stirring. The resulting suspension was stirred at room temperature for 12
hours and filtered. The filter cake was washed with 100 mL NMP/water (4:1) and dried in vacuum at
1
ambient temperature, providing 94.5 g of pure 11 (94.5% yield, 99.5 area% by HPLC purity). H NMR
(300 MHz, CDCl3):
δ = 12.53 (s, 1H, 5ꢀOH), 7.95 (m, 2H, ArꢀH), 7.54 (m, 3H, ArꢀH), 7.39 (m, 5H, ArꢀH),
6.68 (s, 1H, CHCO), 6.48 (s, 1H, ArꢀH), 5.23 (s, 2H, ArOCH2), 3.97 (s, 3H, ArOCH3) ppm. 13C NMR (75
MHz, CDCl3): 181.7, 162.9, 156.8, 156.3, 148.6, 134.8, 130.9, 130.3, 128.1 (2 × C), 127.7 (3 × C), 127.3,
126.2 (2 × C), 125.3 (2 × C), 104.4, 104.2, 96.2, 69.9, 60.7 ppm. HRMS (ESI), [M + H]+ calculated for
C23H19O5 375.1227, found 375.1233.
Preparation of 5,7-dihydroxy-8-methoxy-2-phenyl-4H-chromen-4-one (1). Compound 11 (748 g,
2 mol, 1 equiv) was dissolved in THF (12 L). The solution was charged into a 20 L flask containing Pd/C
(74.8 g, 10 wt % loading). The flask was first purged with nitrogen and then replaced by hydrogen. The
resulting solution was hydrogenated for 4 hours at ambient temperature and pressure. The mixture was
filtered through Celite to remove catalyst and the filter cake was washed with THF (2.4 L × 2). The
combined filtrates were evaporated to give 550 g (97% yield, uncorrected) of crude debenzylation product
1.
○
Recrystallization: Crude 1 (142 g, 0.5 mol) was suspended in ethanol (700 mL) and then heated to 70 C.
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The homogeneous mixture was filtered through Celite immediately without cooling. After the filtrate was
naturally cooled down for 15 minutes, seed crystals (1.7 g, 1.2 wt %) were added. The suspension was
further chilled to room temperature for 5 hours and filtered. The filter cake was washed with 200 mL cold
ethanol and once again recrystallized with the same procedure to provide 135 g of pure 1 as a yellow solid
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(95% yield, 99.8 area% by HPLC purity, Pd level < 5 ppm). 1H NMR (300 MHz, DMSOꢀd6):
δ = 12.52 (s,
1H, 5ꢀOH), 10.83 (s, 1H, 7ꢀOH), 8.07 (m, 2H, ArꢀH), 7.62 (m, 3H, ArꢀH), 6.99 (s, 1H, CHCO), 6.33 (s, 1H,
ArꢀH), 3.88 (s, 3H, ArOCH3) ppm. 13C NMR (75 MHz, DMSOꢀd6): 182.0, 162.9, 157.3, 156.2, 149.6,
132.0, 130.8, 129.2 (2 × C), 127.7, 126.2 (2 × C), 105.0, 103.7, 99.1, 61.0 ppm. HRMS (ESI), [M + H]+
calculated for C16H13O5 285.0757, found 285.0756.
ASSOCIATED CONTENT
Supporting Information
1HNMR and 13C NMR spectra for compounds 10, 4, 11 and 1, HPLC conditions and chromatograms.
ACKNOWLEDGMENTS
This work was supported by the National Natural Science Foundation of China (No. 21372260).
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