A convenient and efficient approach to synthesize negletein from baicalin

Article abstract of DOI:10.1016/j.tetlet.2016.03.085

A simple two-step/one-pot and highly efficient strategy for the synthesis of negletein from naturally abundant and inexpensive baicalin has been developed. In this one-pot sequence, esterification of baicalin catalyzed by concentrated H₂SO

Full text of DOI:10.1016/j.tetlet.2016.03.085

Tetrahedron Letters 57 (2016) 2001–2005
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
A convenient and efficient approach to synthesize negletein
from baicalin
y
y
⇑
Guihua He , Yu Gao , Cailong Li, Guolin Wu, Yazhen Li, Longrong Dong, Changcang Huang, Haijun Chen
College of Chemistry, Fuzhou University, Fuzhou, Fujian 350116, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A simple two-step/one-pot and highly efficient strategy for the synthesis of negletein from naturally
abundant and inexpensive baicalin has been developed. In this one-pot sequence, esterification of baica-
lin catalyzed by concentrated H2SO4 in methanol followed by the treatment with excess NaBH4 afforded
negletein in moderate yield. This method provides an additional approach for the synthesis of negletein.
Ó 2016 Elsevier Ltd. All rights reserved.
Received 19 February 2016
Revised 23 March 2016
Accepted 28 March 2016
Available online 29 March 2016
Keywords:
Negletein
Baicalin
Reduction
One-pot
Methylation
Introduction
and then alkylation of HO-C(7) of compound 4 with methyl iodide
provided corresponding derivative 5. Deacetylation of compound 5
Flavonoids, which are ubiquitously distributed in many dietary
plants, have received increasing attention because most of them
display various beneficial effects on human health.1–3 Baicalin (1)
and its aglycone baicalein (2) (Scheme 1) are the major active
ingredients of Chinese herb Scutellaria baicalensis.4 Both of them
have been demonstrated to possess various clinically relevant
properties such as anti-oxidant, anti-cancer, anti-microbial, and
anti-allergy activities.4,5 However, both baicalin and baicalein suf-
fer from poor aqueous solubility and low bioavailability, limiting
their further potential clinical application.6 Negletein (3), the
methylated derivate at HO-C(7) position of baicalein, has similar
biological effects with more favorable pharmacokinetic parameters
and physicochemical properties including metabolic stability and
aqueous solubility (Scheme 1).7–9 Therefore, negletein represents
a suitable substitute of baicalin/baicalein and an ideal starting
point for further chemical optimization.10
afforded negletein.12 Kawabata et al. obtained negletein concisely
through methylation of baicalein with methyl iodide and lithium
carbonate in a decent yield of 72%.13 Waghmode et al. utilized
methylation of baicalein with MeI, K2CO3, and pyridine in acetone
to yield 5,6,7-trimethoxyflavone (6). Selective didemethylation of
6 was observed at the 5, 6-position in the presence of a protic acid
and a catalytic amount of phase-transfer catalyst (Aliquat-336) to
afford negletein.14 Righi et al. carried out the synthesis of negletein
outlined in Scheme 3.15 Methylation of HO-C(7) of crysin (7) pro-
vided compound 8. Bromination of 8 with tetrabutylammonium
tribromide (TBATB) afforded a mixture of 9 and 10. The mixture
was submitted to the usual methanolysis step to provide 11.
Demethylation of MeO-C(6) of 11 ran smoothly to give the desired
product negletein.15
Although these above mentioned approaches can be used to
successfully synthesize negletein, they have limited applicability
due to the complex synthetic routes, expensive starting
materials or difficult purification. For example, the first and the
fourth methods need additional protection and deprotection
manipulation steps. In addition, the intermediate 8 in the fourth
method and negletein in the third method are not easy to be
obtained because selective methylation is difficult to be manipu-
lated, resulting in potential complicated purification. Therefore,
development of an additional effective approach to synthesize
abundant negletein for clinical validation and chemical modification
is necessary.
However, the natural abundance of negletein is particularly
low.11 Toward this end, several approaches have been developed
in order to obtain sufficient amount of negletein for further drug dis-
covery. As shown in Scheme 2, starting from baicalein which was the
deglycosylated product of baicalin, peracetylization was performed
in Ac2O and AcONa to afford derivative 4. Selective deacetylation
⇑
Corresponding author. Tel.: +86 591 22866234; fax: +86 591 22866227.
E-mail address: chenhaij@gmail.com (H. Chen).
These authors contribute equally to this work.
y
http://dx.doi.org/10.1016/j.tetlet.2016.03.085
0040-4039/Ó 2016 Elsevier Ltd. All rights reserved.

2002
G. He et al. / Tetrahedron Letters 57 (2016) 2001–2005
OH
O
O
7
O
O
O
O
O
O
HO
HO
MeO
HO
HO
HO
O
6
OH
HO
Higher
Metabolic
Stability
5
OH
OH
OH
Sensitive
Metabolism
Position
Negletein (3)
20 mg/~$300
Baicalin (1)
Baicalein (2)
25 g/$100
1 kg/$60
Scheme 1. Chemical structures of baicalin (1), baicalein (2), and negletein (3).
Approach 1
O
O
OAc
O
OH
O
O
OAc
OAc
OH
OH
OAc
OAc
Ac2O, AcONa
80 oC, 2 h
92%
MeI, K2CO3
KI, acetone
60 oC, 8 h
58%
OMe
O
Baicalein (2)
5
4
Conc. HCl
EtOH, 60 oC
8 h, 95%
2011
Helv. Chim. Acta,
, 94, 2221-2230
Approach 2
O
O
OH
OH
, 55 oC, 17
MeI, Li C
O , DMF
2
h, 72%
3
conditions
2004
Biosci. Biotechnol. Biochem.,
, 68, 369-375
Approach 3
MeI, K2CO3, acetone, py
>90%
OMe
Negletein (3)
HO
O
O
OH
O
O
OMe
OH
OH
OH
OMe
OMe
47% HBr/H2O
O
Aliquat-336 (10 wt.%), 85%
O
O
Synthetic Commun., 2013, 43, 3272-3280
OH
Baicalin (1)
6
Scheme 2. Three approaches for synthesis of negletein from baicalein.
Approach 4
O
OH
8
O
O
OH
O
O
OH
8
Me2SO4
TBATB
CHCl3
6
6
K2CO3
acetone
O
OH
OMe
97%
OMe
Br
7
Crysin (7)
8
9 (position 6)/
10 (position 8) = 2:1
O
O
OH
O
O
OH
OH
OMe
OMe
MeONa, CuBr, DMF
85%
HBr, AcOH
96%
OMe
11
Negletein (3)
, 66, 1294-1298
2010
Tetrahedron
Scheme 3. Synthesis of negletein from crysin.
component was then purified (ꢀ11% yield) and structural charac-
terization revealed this side product to be negletein. The chemical
structure was further confirmed by single-crystal X-ray structural
analysis.17 To our best knowledge, this methylation is seldom
reported. Based on the preliminary result, we envisioned that it
might provide a new approach to synthesize negletein. In order
to obtain abundant negletein for further chemical modification,
various conditions were investigated.
As depicted in Table 1, we extended the reaction time from 12 h
to 24 h, and the reaction proceeded smoothly to give negletein in
32% yield (entry 3). Notably, oroxin A also can be detected in the
reaction mixture, indicating that we could extend the reaction time
Results and discussion
This new approach to synthesize negletein from baicalin was
found during our reported process about the synthesis of oroxin
A (13) which was shown in Scheme 4.16 Baicalin was converted
to baicalin methyl ester (12) in the presence of the catalytic
amount of concentrated H2SO4 in the solution of methanol. The
key intermediate (12) was then directly reacted with NaBH4 in
1 h to afford oroxin A in a very high yield without further purifica-
tion. To our surprise, when we extended the reaction time from 1 h
to 12 h, two major components including oroxin A were detected
by analytical TLC (thin-layer chromatography). The other major

G. He et al. / Tetrahedron Letters 57 (2016) 2001–2005
2003
OH
OMe
O
O
O
O
O
O
HO
HO
O
O
HO
HO
O
cat. H2SO4
OH
HO
OH
HO
MeOH, reflux, 2 h
OH
O
OH
.
aBH , r.t
N
4
Baicalin (1)
1kg/$60
Baicalin methyl ester (12)
Extending the
reaction time
NaBH4, r.t., 1h
Yield up to 80%
OH
O
O
O
HO
Extending the
reaction time
O
MeO
7
HO
OH
6
HO
HO
5
OH
O
OH
O
Oroxin A (13)
Negletein (3)
20 mg/~$300
1 g/$1500
RSC Adv., 2014, 4, 45151-45154
Scheme 4. Reagents and conditions: (a) cat. H2SO4, MeOH, reflux; (b) NaBH4, 0 °C to 25 °C.
Table 1
Optimization of the NaBH4 reaction condition
OMe
O
entry 12
the product of
D3CO
O
O
O
O
MeO
HO
O
O
O
HO
HO
OH
HO
HO
OH
O
OH
OH
Baicalin methyl ester (12)
Negletein(D)
Negletein (3)
Entry
NaBH4 (equiv)
Solvent
T (°C)
Time (h)
Yielda (%)
1b
10.0
10.0
10.0
10.0
6.0
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
EtOH
0–25
25
25
25
25
25
25
25
25
25
25
25
1
12
24
48
48
48
48
48
48
48
48
48
0
2b
11
32
51
32
8
50
48
40
0
3b
4b
5b
6b
3.0
7c
10.0
10.0
10.0
10.0
10.0
10.0
8d
9e
10f
11g
12h
THF
CD3OD
0
52
a
b
c
Isolated yield.
12 (0.22 mmol), MeOH (10 mL).
Large scale, 1 (5.6 mmol), cat. H2SO4, and MeOH (200 mL) in two-steps/one-pot.
1 (11.2 mmol), cat. H2SO4, and MeOH (400 mL).
1 (45 mmol), cat. H2SO4, and MeOH (1000 mL).
12 (0.22 mmol), EtOH (10 mL).
d
e
f
g
h
12 (0.22 mmol), THF (tetrahydrofuran, 10 mL).
12 (0.10 mmol), CD3OD (2 mL).
to increase the conversion. Performing the reaction for 48 h
resulted in full conversion and the desired product was obtained
in 51% yield (entry 4). The optimization on the reaction condition
revealed that the decrease in the amount of NaBH4 had a dramatic
effect on the yield (entries 5 and 6). We next carried out the
reaction on gram scale from the starting material of baicalin and
the results demonstrated the practicability of this methodology
(entries 7–9). The desired product was obtained up to 40% yield
even performing the reaction on 20 g scale (see Figs. S1–S12).
Having the optimal reaction condition, we then began the study
to understand the reaction mechanism. First, we realized that with
EtOH or THF as solvent, the reaction did not afford the desired pro-
duct (entries 10 and 11). We then used K2CO3 or NaOMe as the
substitute of NaBH4 for this reaction. However, no desired product
was observed (Scheme 5). These results indicated that NaBH4 was
an important and enabling reagent for this reaction. Next we
intended to make clear whether the methoxyl moiety of negletein
originated from glucuronic acid methyl ester or methanol.18 To
address these issues, baicalin or oroxin A without methoxyl group
was directly subjected to NaBH4 in the solution of MeOH for 24 h.
These two starting materials smoothly generated negletein in the
yield of 24% and 38%, respectively. We further used baicalin ethyl
ester (14) with NaBH4 in the solvent of MeOH. Intriguingly, not
baicalein-7-ethylether but negletein was observed. We next used
CD3OD as the solvent and the desired product negletein(D) was
obtained as expected (entry 12). These results demonstrated that
the methoxy moiety of negletein came from methanol.
Conclusions
Herein, we present a simple two-step/one-pot chemical synthesis
of negletein (3) using cheap starting material baicalin (1 kg/$60).

2004
G. He et al. / Tetrahedron Letters 57 (2016) 2001–2005
OH
OMe
OH
O
O
O
O
O
O
O
O
O
O
O
O
HO
HO
O
O
HO
HO
HO
HO
OH
HO
OH
HO
OH
HO
OH
OH
12
OH
Baicalin (1)
Oroxin A (13)
NaOMe, MeOH
or K2CO3, MeOH
r.t, 48 h
10 eq. NaBH4, MeOH, 24 h
38% yield
10 eq. NaBH4, MeOH, 24 h
24% yield
Negletein (3)
Negletein (3)
Negletein (3)
O
O
O
O
O
O
MeO
HO
O
O
HO
HO
NaBH4
MeOH
32% yield
cat. H2SO4
Baicalin
OH
HO
EtOH
reflux
OH
14
OH
Negletein (3)
Scheme 5. Synthesis of negletein from baicalin or oroxin A or baicalin ethyl ester (14).
This method utilizes esterification of baicalin (1) catalyzed by con-
centrated H2SO4 in methanol and the consequent direct treatment
with NaBH4 to access negletein in a moderate yield. Our optimized
process for the second step was primarily achieved by using NaBH4
as reactant in the solvent of MeOH at 25 °C for 48 h to afford negle-
tein in 50% yield. Our mechanism of investigation revealed that
this procedure was accomplished by deglycosylation of baicalin
and concomitant methylation by methanol in the presence of
NaBH4. The synthetic strategy developed in this work provided
an additional facile approach for the synthesis of negletein for
clinical validation and further chemical modification.
3.92 (s, 3H). 13C NMR (100 MHz, DMSO-d6): 182.3, 163.1, 154.6,
149.8, 146.1, 131.9, 130.9, 130.1, 129.1, 126.3, 105.3, 104.7, 91.3,
56.3. HRMS (ESI) calcd for C16H11O5 283.0612 (MꢁH )ꢁ, found
283.0611. Data agreed with those reported in the literature.12–15
One-pot large-scale synthesis of negletein (3)
To the solution of baicalin (20.0 g, 44.8 mmol) in methanol
(400 mL) was added a catalytic amount of sulfuric acid (0.1 mL),
and the mixture was heated at reflux temperature for 2 h. The mix-
ture was added to methanol (600 mL) and cooled to 0 °C and then
NaBH4 (17.0 g, 448 mmol) was added portionwise in 2 h. After the
addition was complete, the mixture was stirred for an additional
48 h at 25 °C and then quenched with 600 mL of 1 N HCl (aq).
The solution was evaporated to obtain the brown suspension.
The residue was diluted with CH2Cl2/MeOH (10:1, 1000 mL) and
extracted with H2O (400 mL). The organic layer was then washed
with brine (200 mL), dried over anhydrous Na2SO4, and concen-
trated to give the crude product. This residue was purified with
silica gel column (CH2Cl2/MeOH = 20:1) to afford the crude
product, which was washed with EtOAc (50 mL) to give negletein
(5.1 g, 40%) as a yellow solid. The structural characterization data
are the same as those described above.
Experimental section
All commercially available starting materials and solvents were
reagent grade, and used without further purification. Reactions
were performed under a nitrogen atmosphere in dry glassware
with magnetic stirring. Preparative column chromatography was
performed using silica gel 60, particle size 0.063–0.200 mm
(70–230 mesh, flash). Analytical TLC was carried out employing
silica gel 60 F254 plates (Merck, Darmstadt). Visualization of the
developed chromatograms was performed with detection by UV
(254 nm). NMR spectra were recorded on a Bruker-400 (1H,
400 MHz; 13C, 100 MHz) spectrometer. 1H and 13C NMR spectra
were recorded with TMS as an internal reference. Chemical shifts
were expressed in ppm, and J values were given in Hz. High-
resolution mass spectra (HRMS) were obtained from Thermo Fisher
Scientific Exactive Plus mass spectrometer.
Acknowledgements
This work was supported by the National Natural Science Foun-
dation of China (No. 81402781), the Scientific Research Foundation
for the Returned Overseas Chinese Scholars, Technology Develop-
ment Foundation of Fuzhou University.
Synthesis of negletein (3)
Supplementary data
The key intermediate baicalin methyl ester (12)16 (0.1 g,
0.22 mmol) was suspended in methanol (10 mL) and cooled to
0 °C. Then NaBH4 (46 mg, 1.22 mmol) was added portionwise.
The reaction mixture was stirred at 25 °C for 48 h and then
quenched with 10 mL of 1 N HCl (aq). The solution was evaporated
to obtain the crude product. The residue was diluted with CH2Cl2/
MeOH (10:1, 40 mL) and extracted with H2O (40 mL). The organic
layer was then washed with brine (10 mL), dried over anhydrous
Na2SO4, and concentrated to give the crude product. This residue
was purified with silica gel column (CH2Cl2/MeOH = 20:1) to pro-
vide negletein (32 mg, 51%) as a yellow solid (mp 222–223 °C).
1H NMR (400 MHz, DMSO-d6): 12.50 (s, 1H), 8.77 (s, 1H), 8.06 (d,
J = 7.1 Hz, 2H), 7.58 (t, J = 8.0 Hz, 3H), 6.96 (s, 1H), 6.92 (s, 1H),
Supplementary data (photographic guide for the synthesis of
negletein (20 g scale), copies of 1H and 13C NMR spectra of negle-
tein, copies of 1H NMR spectra of negletein(D), and HRMS spectra
of negletein(D)) associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.tetlet.2016.03.085.
References and notes
1. Havsteen, B. Biochem. Pharmacol. 1983, 32, 1141.
2. Romano, B.; Pagano, E.; Montanaro, V.; Fortunato, A. L.; Milic, N.; Borrelli, F.
Phytother. Res. 2013, 27, 1588.
3. Chen, A. Y.; Chen, Y. C. Food Chem. 2013, 138, 2099.
4. Li-Weber, M. Cancer Treat. Rev. 2009, 35, 57.

G. He et al. / Tetrahedron Letters 57 (2016) 2001–2005
2005
5. Chen, H.; Gao, Y.; Wu, J.; Chen, Y.; Chen, B.; Hu, J.; Zhou, J. Cancer Lett. 2014, 354,
5.
for about ꢀ$300/20 mg from BeiJing Hwrk Chemicals Limited, http://www.
hwrkchemical.com.
6. Lai, M. Y.; Hsiu, S. L.; Tsai, S. Y.; Hou, Y. C.; Chao, P. D. J. Pharm. Pharmacol. 2003,
55, 205.
12. Wang, J. F.; Ding, N.; Zhang, W.; Wang, P.; Li, Y. X. Helv. Chim. Acta 2011, 94,
2221.
7. Kiem, P. V.; Minh, C. V.; Huong, H. T.; Lee, J. J.; Lee, I. S.; Kim, Y. H. Arch. Pharm.
Res. 2005, 28, 1345.
13. Gao, H.; Nishioka, T.; Kawabata, J.; Kasai, T. Biosci. Biotechnol. Biochem. 2004, 68,
369.
8. Singh, B.; Sidiq, T.; Joshi, P.; Jain, S. K.; Lawaniya, Y.; Kichlu, S.; Khajuria, A.;
Vishwakarma, R. A.; Bharate, S. B. Nat. Prod. Res. 2013, 27, 2227.
9. Banerjee, P.; Sahoo, A.; Anand, S.; Ganguly, A.; Righi, G.; Bovicelli, P.; Saso, L.;
Chakrabarti, S. Neuromol. Med. 2014, 16, 787.
14. Waghmode, S. B.; Mahale, G.; Patil, V. P.; Renalson, K.; Singh, D. Synth. Commun.
2013, 43, 3272.
15. Righi, G.; Antonioletti, R.; Silvestri, I. P.; D’Antona, N.; Lambusta, D.; Bovicelli, P.
Tetrahedron 2010, 66, 1294.
10. Chen, H.; Mrazek, A. A.; Wang, X.; Ding, C.; Ding, Y.; Porro, L. J.; Liu, H.; Chao, C.;
Hellmich, M. R.; Zhou, J. Bioorg. Med. Chem. 2014, 22, 3393.
11. Baicalin (CAS: 21967-41-9) is available for about ꢀ$60/1 kg from Baoji
Haoxiang Bio-technology Co. Ltd, http://www.haoxiangbio.com. Baicalein
(CAS: 491-67-8) is available for about ꢀ$100/25 g from Energy Chemical,
http://www.energy-chemical.com.cn. Negletein (cas: 29550-13-8) is available
16. Chen, H.; He, G.; Li, C.; Dong, L.; Xie, X.; Wu, J.; Gao, Y.; Zhou, J. RSC Adv. 2014, 4,
45151.
17. The crystallographic data have been deposited in full at the Cambridge
Crystallographic Data Centre, reference CCDC 1453393, accessible at http://
www.ccdc.cam.ac.uk.
18. Suzuki, Y.; Kaneno, D.; Tomoda, S. J. Phys. Chem. A 2009, 113, 2578.