Synthesis and characterization of impurities of barnidipine hydrochloride, an antihypertensive drug substance

Article abstract of DOI:10.3390/molecules19011344

Barnidipine hydrochloride is a long term dihydropyridine calcium channel blocker used for the treatment of hypertension. During the process development of barnidipine hydrochloride, four barnidipine impurities were detected by high-performance liquid chromatography (HPLC) with an ordinary column (Agilent ZORBAX Eclipse XDB-C18, 150 mm × 4.6 mm, 5 m). All these impurities were identified, synthesized, and subsequently characterized by their respective spectral data (MS, 1H-NMR, and 13C-NMR). The identification of these impurities should be useful for quality control in the manufacture of barnidipine.

Full text of DOI:10.3390/molecules19011344

Molecules 2014, 19, 1344-1352; doi:10.3390/molecules19011344
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Characterization of Impurities of Barnidipine
Hydrochloride, an Antihypertensive Drug Substance
Zhi-Gang Cheng 1,2, Xu-Yong Dai 2, Li-Wei Li 3, Qiong Wan 4, Xiang Ma 1,* and
Guang-Ya Xiang 1,*
1
School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology,
Wuhan 430030, China; E-Mail: champion88@163.com
2
Wuhan Biocause Pharmaceutical Development Co., Ltd, Wuhan 430056, Hubei, China;
E-Mail: daixuyong111@126.com
3
College of Chemical Engineering and Pharmacy, Jingchu University of Technology, Jingmen 448000,
Hubei, China; E-Mail: jmliliwei@163.com
4
Hubei Biocause Heilen Pharmaceutical Co., Ltd, Jingmen 448000, Hubei, China;
E-Mail: wanqiong@biocause.net
* Authors to whom correspondence should be addressed; E-Mails: maxwellcn@yahoo.com (X.M.);
gyxiang1968@hotmail.com (G.-Y.X.); Tel.: +86-27-8369-2793 (X.M. & G.-Y.X.);
Fax: +86-27-8369-2762 (X.M. & G.-Y.X.).
Received: 11 December 2013; in revised form: 15 January 2014 / Accepted: 15 January 2014 /
Published: 21 January 2014
Abstract: Barnidipine hydrochloride is a long term dihydropyridine calcium channel
blocker used for the treatment of hypertension. During the process development of
barnidipine hydrochloride, four barnidipine impurities were detected by high-performance
liquid chromatography (HPLC) with an ordinary column (Agilent ZORBAX Eclipse
XDB-C18, 150 mm × 4.6 mm, 5 µm). All these impurities were identified, synthesized,
1
and subsequently characterized by their respective spectral data (MS, H-NMR, and
13C-NMR). The identification of these impurities should be useful for quality control in the
manufacture of barnidipine.
Keywords: barnidipine hydrochloride; impurities; antihypertensive agent

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1. Introduction
Barnidipine hydrochloride (1, Figure 1), a long term dihydropyridine calcium channel blocker used
for the treatment of hypertension, is chemically known as (3'S,4S)-1-benzyl-3-pyrrolidinyl-methyl-1,4-
dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylate hydrochloride [1–4]. The product
was originally developed by Yamanouchi Pharmaceutical (Tokyo, Japan) and is currently marketed in
Japan under the trade name of Hypoca (Astellas Pharma Inc, Tokyo, Japan).
Figure 1. Structure of barnidipine hydrochloride (1).
NO2
HCl
O
O
N
O
O
CH3
N
CH3
H
1
The presence of impurities in a drug substance can have a significant impact on the quality and
safety of the drug product. According to the general guidelines on impurities in new drug substances
recommended by the International Conference on Harmonization (ICH), the acceptable level for all
impurities present should be less than 0.10% or 1.0 mg per day intake (whichever is lower) for drugs
with a maximum daily dose equal to or lesser than 2 g [5]. In order to meet these requirements, the
impurities present in the drug substance greater than above mentioned values must be identified and
characterized. These impurities are also required in pure form to check the analytical performance
characteristics, such as system suitability and relative correction factor. Hence, a comprehensive study
was undertaken in our current research to identify the impurities in a sample of the barnidipine
hydrochloride bulk drug substance. Their detection, synthesis, and characterization are described in
this article.
The four impurities identified in the barnidipine hydrochloride (1) manufacturing process were:
(3'S,4R)-1-benzyl-3-pyrrolidinyl methyl 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridine
dicarboxylate (2), 3-(R)-1-benzylpyrrolidin-3-yl 5-methyl 2,6-dimethyl-4-(3-nitrophenyl)pyridine-3,5-
dicarboxylate (3), (S)-3-ethyl 5-methyl 2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-
dicarboxylate (4), and (3'S,4S)-1-benzyl-3-pyrrolidinyl ethyl 1,4-dihydro-2,6-dimethyl-4-(3-
nitrophenyl)-3,5-pyridinedicarboxylate (5) (Figure 2). A literature survey revealed that only
compounds 2 [3,6] and 3 [7,8] were previously reported, but the studies did not involve the synthesis
and characterization of the compounds, so detailed synthetic processes have not been reported for the
four impurities, and compounds 4 and 5 are reported here for the first time.

Molecules 2014, 19
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Figure 2. Structures of the impurities.
NO2
NO
2
O
O
O
O
N
N
O
O
O
O
3
CH3
N
CH3
CH
N
CH
3
H
3
2
NO2
NO2
O
O
O
O
N
O
O
O
O
CH3
N
CH3
CH3
N
H
CH3
H
4
5
2. Results and Discussion
In our barnidipine hydrochloride manufacturing process of (Scheme 1) [9], a key intermediate,
2-cyanoethyl 2-(3-nitrobenzylidene)-3-oxobutanoate (8), was obtained with a yield of 80.8% by
reacting 2-cyanoethyl 3-oxobutanoate (6) with 3-nitrobenzaldehyde (7) for 15 h at room temperature.
Cyclization of compound 8 with methyl 3-aminobut-2-enoate (9) by refluxing for 2 h afforded
3-(2-cyanoethyl) 5-methyl 2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate (10,
yield 91.1%). Subsequent successive hydrolysis of 10 with sodium hydroxide and hydrochloric acid
gave 5-(methoxycarbonyl)-2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3-carboxylic acid
(11, yield 76.5%). (R)-5-(methoxycarbonyl)-2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3-
carboxylic acid (12, optical purity 99.2%, αD20 −25.9 (c 0.005 g/mL, acetone)) was obtained in 75.3%
yield by resolution of compound 11 using cinchonine as the resolving agent. Condensation of
compound 12 with (S)-1-benzylpyrrolidin-3-ol (14), and subsequent salt formation of the resulting
barnidipine (15), provided the desired barnidipine hydrochloride (1, optical purity 99.8%) in 65.5%
yield (α2D0 +116.5 (c 0.01 g/mL, MeOH)).
Compound 2 is the diastereoisomer of barnidipine (15). The presence of (S)-5-(methoxycarbonyl)-
2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3-carboxylic acid (13, the enantiomer of 12) in the
resolution product 12, leads to the formation of impurity 2. Impurity 2, which has the same 1H-NMR,
13C-NMR, and mass spectrum as barnidipine hydrochloride, but a totally different HPLC retention
1
time, was independently prepared using compound 13 as the starting material (Scheme 2). H-NMR,
13C-NMR, mass spectral (MS) data, and HPLC retention time of the prepared compound 2 were
identical with those of impurity 2 separated from crude barnidipine hydrochloride (1).
Compound 3 is the dehydrogenation product of barnidipine (15). The degradation of barnidipine
explains the formation of impurity 3. The same degradation product 3 was also reported in another
study, where the barnidipine was exposed to natural or stressing irradiation, and the aromatization of
the 1,4-dihydropyridine moiety in barnidine occurred [8]. This compound was prepared by reacting
barnidipine (15) with manganese dioxide (Scheme 2). The mass spectrum of 3 showed a molecular ion

Molecules 2014, 19
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1
13
at m/z 489.0, which is 2 amu less than that of barnidipine (15). The H-NMR and C-NMR spectra
showed the transformation from the dihydropyridine ring to a pyridine ring. This compound was found
13
to be identical [1H-NMR, C-NMR, MS and HPLC retention time (RRT 1.68, compared with 1)] to
the impurity 3 separated from crude barnidipine hydrochloride (1).
Compound 4 is a derivative of compound 12. In the barnidipine hydrochloride manufacturing
process, the API was recrystallized in ethanol, which explains formation of impurity 4. This compound
1
13
was prepared by reacting compound 12 with ethanol (Scheme 2). H-NMR, C-NMR, MS data, and
HPLC retention time (RRT 3.14, compared with 1) of the prepared compound 4 are identical with
those of impurity 4 separated from crude barnidipine hydrochloride (1).
Scheme 1. Synthesis of barnidipine hydrochloride (1).
NH2
NO2
NO2
O
CH3
O
O
CH3COONH
4
9
O
O

CN
CH3CHOHCH
15h, r.t.
80.8%
CN
3
CHO
CH3OH
2h, reflux
91.1%
O
O
7
6
8
NO2
NO2
i) cinchonine, DMF, H2O
i) 4% NaOH, 1,2-dimethoxyethane
2h, r.t.
O
O
O
O
24h, 80～120℃ to r.t.
COOH
CH3
CN
O
O
ii) 35% NaOH
iii) Conc. HCl
75.3%
ii) 10% HCl
76.5%
CH3
N
H
CH3
N
CH3
H
10
11
NO2
NO2
i) PCl5, CH2Cl2
1h, 0～2℃
O
O
+
COOH
CH3
COOH
O
CH3
O
ii)
N
N
H
CH3
N
CH3
HO
14
H
13
12
3h, -15℃
NO2
NO2
O
O
HCl
N
CH2Cl2, HCl in ethanol
65.5% (2 steps)
O
O
N
O
O
O
O
CH3
N
CH3
H
CH3
N
CH3
H
15
1

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Compound 5 is the analog of barnidipine (15). The presence of ethyl 3-aminobut-2-enoate (16) in
compound 9 led to the formation of impurity 5. Compound 5 was independently prepared starting from
compound 8, following a synthetic process analogous to that of barnidipine (Scheme 2). The mass
spectrum of 5 showed a molecular ion at m/z 505.2, which is 14 amu greater than barnidipine (15).
The 1H-NMR and 13C-NMR spectra displayed the transformation from a methyl group to an ethyl
13
group. This compound was found to be identical [1H-NMR, C-NMR, MS and HPLC retention time
(RRT 1.84, compared with 1)] with the impurity 5 separated from crude barnidipine hydrochloride (1).
Both impurities 4 and 5 might be formed due to an ester interchange reaction when ethanolic
hydrochloride acid was used in the final step of the process.
Scheme 2. Synthesis of Barinidipine Hydrochloride Impurities.
NO2
NO2
O
O
(i) PCl5, CH2Cl2
O
N
COOH
CH3
O
O
O
(ii) (S)-1-benzylpyrrolidin-3-ol (14)
CH3
N
CH3
CH3
N
H
H
2
13
NO2
NO2
O
O
N
MnO2, CH2Cl2
O
O
N
O
O
O
O
CH3
N
CH3
CH3
N
CH3
H
3
15
NO2
NO2
O
O
(i) PCl5, CH2Cl2
(ii) ethanol
O
COOH
CH3
O
CH3
O
O
N
CH3
CH3
N
H
H
4
12
NO2
NO2
(i) 3.8% NaOH,
1,2-dimethoxyethane
NO2
O
Ethyl 3-aminobut-2-enoate (16)
O
O
COOH
CH3
ethanol
O
O
O
CN
(ii) 10% HCl
CN
O
O
CH3
N
H
CH3
N
H
CH3
O
18
8
17
NO2
NO2
O
O
O
O
N
(i) cinchonine, DMF
(i) PCl5, CH2Cl2
COOH
O
O
(ii) 35% NaOH
(iii) Conc. HCl
(S)-1-benzyl pyrrolidin-3-ol (14)
CH3
N
CH3
CH3
N
H
CH3
H
19
5

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3. Experimental
General Information
1
The H-NMR spectra were recorded on a Varian Mercury plus-400 MHz Fourier transform (FT)-
NMR spectrometer, and chemical shift values were reported as δ ppm relative to tetramethylsilane
13
(TMS). The C-NMR spectra were recorded on a Varian Mercury plus-400 MHz Fourier transform
(FT)–NMR spectrometer, and chemical shift values were reported on δ ppm relative to CDCl3 or
DMSO-d6. The mass spectra were recorded on an Agilent API 2000LC-MS/MS mass spectrometer.
The HPLC chromatograms were recorded on a Waters 1525 HPLC instrument.
(3'S,4R)-1-Benzyl-3-pyrrolidinyl methyl 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridine-
dicarboxylate (2) Phosphorus pentachloride (1.95 g, 0.02 mmol) was added slowly to a solution of
(S)-5-(methoxycarbonyl)-2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3-carboxylic acid (13,
2.47 g, 0.0074 mol) in dichloromethane (DCM, 32.5 mL) at −20 °C, and stirred for 1 h below −15 °C.
Then the mixture was cooled to −26 °C, and a solution of (S)-1-benzylpyrrolidin-3-ol (14, 1.35 g,
0.0076 mol) and dichloromethane (11.25 mL) were added to above mixture, and stirred for 2 h. The
reaction mixture was washed with 5% sodium carbonate solution (150 mL) and water (150 mL). The
organic layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure at
less than 35 °C. The residue was purified by flash column chromatography on silica gel, eluting with
ethyl acetate/petroleum ether, 3:2, to yield 2 as a light yellow solid (1.5g, 41%). HPLC purity 92.9%.
1H-NMR (CDCl3) δ: 7.27–8.10 (m, 9H), 5.90 (s, 1H), 5.10–5.14 (m, 1H), 5.08 (s, 1H), 3.64 (s, 3H),
3.51–3.58 (m, 2H), 2.74–2.79 (m, 1H), 2.66–2.70 (m, 1H), 2.48–2.51 (m, 1H), 2.35 (s, 6H), 2.10–2.28
13
(m, 2H), 1.85-1.86 (m, 1H); C-NMR (CDCl3) δ: 167.5, 166.8, 149.8, 148.4, 144.9, 144.8, 138.8,
134.5, 128.7, 128.6, 128.2, 127.0, 122.9, 121.3, 103.3, 73.8, 60.1, 59.8, 52.6, 51.1, 39.9, 29.7, 19.6.
MS (ESI−) m/z: 490.1 (M−H)−.
3-(R)-1-Benzylpyrrolidin-3-yl 5-methyl 2,6-dimethyl-4-(3-nitrophenyl) pyridine-3,5-dicarboxylate (3)
Manganese dioxide (3.5 g, 0.04 mol) was added to a solution of barnidipine (15, 2.0 g, 0.004 mol) in
dichloromethane (150 mL), exposed to UV light, and stirred for 30 days at room temperature. The
reaction mixture was filtered and concentrated. The residue was purified by flash column chromatography
on silica gel, eluting with dichloromethane/ethanol, 40:1, followed by ethyl acetate/petroleum ether, 3:2, to
afford 3 as a light yellow solid (0.6 g, 31%). HPLC purity 94.6%. 1H-NMR (CDCl3) δ: 7.13–8.10 (m,
9H), 4.96–4.99 (m, 1H), 3.34–3.47 (m, 5H), 2.46–2.51 (m, 8H), 2.21–2.27 (m, 1H), 2.11–2.14 (m,
13
1H), 1.92–2.03 (m, 1H), 1.37–1.43 (m, 1H). C-NMR (CDCl3) δ: 167.4, 166.7, 156.1, 156.0, 147.6,
143.4, 138.1, 137.8, 134.1, 128.1, 128.5, 128.0, 126.1, 126.1, 123.2, 123.0, 75.4, 59.6, 58.9, 52.2, 52.1,
31.1, 22.9. MS (ESI+) m/z: 490.0 (M+H)+.
(S)-3-Ethyl 5-methyl 2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate (4).
Phosphorus pentachloride (1.66 g, 0.017 mol) was added to the solution of compound 12 (2.1 g,
0.006 mol) in dichloromethane (28 mL) below −20 °C, and stirred for 1 h. The reaction mixture was
cooled to −30 °C, and was added ethanol (10 mL, 0.17 mol), and stirred for 4 h. The reaction mixture
was poured into saturated sodium carbonate solution (200 mL), and extracted with dichloromethane

Molecules 2014, 19
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(200 mL). The organic phase was washed with water (300 mL), dried over anhydrous magnesium
sulfate and concentrated. The residue was purified by flash column chromatography on silica gel,
eluting with ethyl acetate/petroleum ether, 3:2, to yield 4 as a light yellow solid (1.2 g, 55%). HPLC
1
purity 99.7%. H-NMR (DMSO-d6) δ: 9.04 (s, 1H), 7.52-8.01 (m, 4H), 4.99 (s, 1H), 3.98–4.03 (m,
13
2H), 3.55 (s, 3H), 2.30 (s, 6H), 1.12–1.16 (m, 3H). C-NMR (DMSO-d6) δ: 167.7, 167.1, 150.7,
148.2, 147.2, 147.0, 134.7, 130.3, 122.3, 121.8, 101.7, 101.4, 59.9, 51.4, 41.0, 18.9 (2C), 14.7. MS
(ESI−) m/z: 359.1 (M−H)−.
3-(2-Cyanoethyl) 5-ethyl 2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydro-pyridine-3,5-dicarboxylate (17)
Ethyl 3-aminobut-2-enoate (16, 37.3 g, 0.29 mol) was added to the solution of compound 8 (85 g,
0.295 mol) in ethanol (216 mL), heated to reflux for 2 h. The reaction mixture was concentrated at
60 °C to about 100 mL, and cooled to 0–5 °C. The precipitate was collected by vacuum filtration and
1
dried at 60 °C for 10 h to provide 17 (45.4 g, 39%). H-NMR (DMSO-d6) δ: 9.09 (s, 1H), 7.48–7.99
(m, 4H), 4.97(s, 1H), 4.09–4.13 (m, 2H), 3.93–4.0 (m, 2H), 2.77–2.83 (m, 2H), 2.27–2.30 (d, 6H),
1.10–1.15 (m, 3H). 13C-NMR (DMSO-d6) δ: 167.1, 166.6, 150.7(2C), 148.3, 146.8, 134.9, 130.3, 122.6,
121.8, 119.2, 102.2, 100.9, 59.9, 59.2, 41.0, 19.1, 18.8, 18.0, 14.7. MS (ESI−) m/z: 398.2 (M−H)−.
5-(Ethoxycarbonyl)-2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3-carboxylic acid (18)
Compound 17 (45 g, 0.11 mol) was added to the mixture of 3.8% sodium hydroxide solution (350 g)
and 1,2-dimethoxyethane (170 mL) at 30 °C, stirred for 2 h. The reaction mixture was diluted with
water (115 mL), and extracted with dichloromethane (230 mL). The aqueous phase was acidized to
pH 3.0 by 10% hydrochloric acid. The precipitate was filtered and washed with water, and dried at 90 °C
for 2 h to afford 18 (31.8 g, 83.5%). 1H-NMR (DMSO-d6) δ:11.82 (s, 1H), 8.91 (s, 1H), 7.50–7.98 (m,
4H), 4.98 (s, 1H), 3.97–4.01 (m, 2H), 2.28 (s, 6H), 1.11–1.15 (m, 3H). 13C-NMR (DMSO-d6) δ: 169.1,
167.2, 150.9, 148.1, 147.2, 146.4, 134.8, 130.2, 122.4, 121.7, 102.3, 101.3, 59.8, 41.0, 18.9(2C), 14.7.
MS (ESI−) m/z: 345.1 (M−H)−.
(R)-5-(Ethoxycarbonyl)-2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3-carboxylic acid (19)
Cinchonine (29.4 g, 0.10 mol) was added to the solution of compound 18 (31.0 g, 0.09 mol) in
N,N-dimethylformamide (5.7 mL), and heated 80 °C to get a clear solution. Water (38.4 mL) was
added, and heated to 120 °C. The solution was cooled slowly to 20 °C and stirred for 12 h. The
precipitate was filtered and washed with N,N-dimethylformamide/water (3:2) solution (12 mL). The
filter mass was dissolved in 35% sodium hydroxide solution (92 mL), extracted with dichloromethane
(80 mL). The aqueous phase was acidized to pH 2.0 with concentrated hydrochloric acid. The
precipitate was collected and washed with water, and dried at 100 °C for 10 h to provide 19 (10.4 g,
33%). 1H-NMR (DMSO-d6) δ: 11.82 (s, 1H), 8.91 (s, 1H), 7.50–7.99 (m, 4H), 4.97 (s, 1H), 3.96–4.00
13
(m, 2H), 2.27 (s, 6H), 1.10–1.15 (m, 3H); C-NMR (DMSO-d6) δ: 169.1, 167.2, 150.9, 148.1, 147.2,
146.4, 134.8, 130.2, 122.4, 121.7, 102.3, 101.3, 59.8, 41.0, 18.9 (2C), 14.8. MS (ESI−) m/z: 345.1 (M−H)−.
(3'S,4S)-1-Benzyl-3-pyrrolidinyl
ethyl
1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridine-
dicarboxylate (5). Phosphorus pentachloride (3.5 g, 0.036 mol) was added to the solution of compound
19 (4.8 g, 0.014 mol) in dichloromethane (70 mL) below 0 °C, and stirred for 1 h. The reaction mixture
was cooled to −20 °C, and was added the solution of (S)-1-benzylpyrrolidin-3-ol (14, 2.5 g, 0.014 mol)

Molecules 2014, 19
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in dichloromethane (70 mL), and stirred for 2 h. The reaction mixture was poured into saturated
sodium carbonate solution (275 mL), and extracted with dichloromethane (275 mL). The organic phase
was washed with water (550 mL), dried over anhydrous magnesium sulfate and concentrated. The
residue was purified by flash column chromatography on silica gel, eluting with ethyl acetate/petroleum
1
ether, 3:2, to yield 5 as a light yellow solid (2.7 g, 38%). HPLC purity 98.2%. H-NMR (CDCl3) δ:
7.23–8.13 (m, 9H), 6.58 (br, 1H), 5.07–5.11 (m, 2H), 4.05–4.10 (m, 2H), 3.60–3.66 (m, 2H),
2.80–2.83 (m, 1H), 2.61–2.63 (m, 2H), 2.42–2.44 (m, 1H), 2.30 (s, 6H), 2.06–2.17 (m, 1H), 1.52–1.71
13
(m, 1H), 1.19–1.25 (m, 3H). C-NMR (CDCl3) δ: 167.1, 166.8, 150.0, 148.0, 145.2, 144.9, 138.7,
134.5, 128.5, 128.4, 128.1, 126.8, 122.9, 121.1, 103.0, 102.8, 77.0, 73.6, 60.0, 59.8, 52.4, 39.9, 31.7,
19.1, 14.1. MS (ESI−) m/z: 504.2 (M−H)−.
4. Conclusions
Information on different potential impurities and their synthetic routes is very important for better
understanding of the impurity formation pathways of the antihypertensive drug barnidipine
hydrochloride. All the impurities were identified, synthesized, and subsequently characterized by their
respective spectral data. Keeping in mind the regulatory importance of barnidipine hydrochloride
impurities, our efforts to synthesize and characterize them effectively should prove to be valuable.
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/19/1/1344/s1.
Acknowledgments
This study was partially supported by Natural Science Foundation of Hubei Province, China
(2012FFB02418), the Fundamental Research Funds for the Central Universities of China (HUST,
2012QN009) and Specialized Research Fund for the Doctoral Program of Higher Education of China
(20120142120095).
Author Contributions
G.-Y.X., X.M. and Z.-G.C. conceived and designed the study. Z.-G.C., X.-Y.D., and L.W.L.
performed the synthetic experiments. Q.W. performed HPLC and analysis work. Z.-G.C. and X.M.
wrote the paper. G.-Y.X. and X.M. reviewed and edited the manuscript. All authors read and approved
the manuscript.
Conflicts of Interest
The authors declare no conflict of interest

Molecules 2014, 19
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