Identification and synthesis of impurities in pinocembrin-A new drug for the treatment of ischemic stroke

Article abstract of DOI:10.1002/cjoc.201200201

Four minor impurities in pinocembrin (1)-a new drug to treat ischemic stroke, were analysed and identified by means of HPLC-UV-MS analysis, spectroscopic evidences and chemical synthetic methods. Their chemical structures were identified as 5,7-dihydroxy-2-phenyl-4H-1-benzopyran-4-one (2), 3-phenyl-1-(2,4,6-trihydroxyphenyl)-1-propanone (3), 5,7-dihydroxy-2-cyclohexyl- 4H-1-benzopyran-4-one (4), and 2,3-dihydro-5,7-dihydroxy-2-cyclohexyl-4H-1- benzopyran-4-one (5), respectively. All of the impurities were side products of excessive hydrogenation of the target product 1 or the starting material 2 in the course of synthesis, and 5 was a new compound.

Full text of DOI:10.1002/cjoc.201200201

FULL PAPER
DOI: 10.1002/cjoc.201200201
Identification and Synthesis of Impurities in Pinocembrin—
A New Drug for the Treatment of Ischemic Stroke†
Yang, Qingyun(杨庆云)
Tong, Yuanfeng(童元峰)
Chen, Feng(陈锋)
Qi, Yan(戚燕)
Li, Wei(李薇) Wu, Song*(吴松)
State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Materia Medica,
Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China
Four minor impurities in pinocembrin (1)—a new drug to treat ischemic stroke, were analysed and identified by
means of HPLC-UV-MS analysis, spectroscopic evidences and chemical synthetic methods. Their chemical struc-
tures were identified as 5,7-dihydroxy-2-phenyl-4H-1-benzopyran-4-one (2), 3-phenyl-1-(2,4,6-trihydroxyphenyl)-
1-propanone (3), 5,7-dihydroxy-2-cyclohexyl-4H-1-benzopyran-4-one (4), and 2,3-dihydro-5,7-dihydroxy-2-
cyclohexyl-4H-1-benzopyran-4-one (5), respectively. All of the impurities were side products of excessive hydro-
genation of the target product 1 or the starting material 2 in the course of synthesis, and 5 was a new compound.
Keywords pinocembrin, impurity, ischemic stroke, side product, synthesis
Introduction
gation on the impurities in drug is an important and
meaningful research field.
Ischemic stroke has become an increasingly severe
medical and social problem with high attach and fatality
rate, due to rapid growth of aging people populations.[1]
It has been estimated that about six million people have
died from stroke in 2005 and that more than 90 percent
of these decedents will have occurred in less affluent
countries.[2] Stroke patients must not only survive the
acute stages of infarction, but must cope with significant
mental, physical and economic stresses associated with
neurological impairment. Considering the cost in loss of
life, physical and mental disability, the need for effec-
tive therapeutic interventions is obvious.[3]
2,3-Dihydro-5,7-dihydroxy-2-phenyl-4H-1-benzopy-
ran-4-one (1), well known under the generic name pi-
nocembrin, was isolated from propolis for the first time.
Previous investigations have demonstrated that pino-
cembrin exhibited antibiotic,[4] antioxidant and cyto-
toxic activities,[5-7] whereas our study found this com-
pound showed potent protective effect on neurovascular
unit. Pinocembrin induced relaxation of rat aortic rings
through an endothelium-dependent and independent
pathway, this compound also could improve rat cogni-
tive impairments induced by chronic cerebral hypoper-
fusion.[8] It is promising that pinocembrin could be de-
veloped as a new drug to treat ischemic stroke, and it is
now in phase I clinical trial. Nowadays impurities are
sure to affect the drug qualities seriously, which will
eventually reflect on people’s health. Therefore, investi-
Pinocembrin 1 was prepared generally from 5,7-di-
hydroxy-2-phenyl-4H-1-benzopyran-4-one (2) upon
treatment with hydrogen and palladium in ethanol
(Scheme 1).[9] As a part of our research on the drug im-
purities, we analyzed the pinocembrin sample on HPLC,
and four unconspicuous impurity peaks were found. On
the basis of HPLC-UV-MS analysis, we further ana-
lyzed their contents and possible structures. Their struc-
tures were eventually identified by the method of isola-
tion and synthesis. All of the impurities were deter-
mined as byproducts of excessive hydrogenation of pi-
nocembrin or the starting material in the course of the
synthesis, and impurity 5 was identified as a new chro-
mone derivative. Here we wish to report the analysis,
isolation, synthesis and structural elucidation of these
impurities in pinocembrin substances.
Experimental
Chemicals, reagents and apparatus
Solvents and reagents (K2CO3, KOH) for reactions
and column chromatography (CC) were purchased from
Beijing Beihua Fine Chemicals Co., Ltd. Silica gel
(GF254 100—200 meshes) for CC were obtained from
Qingdao Marine Chemical Factory, Qingdao, Shandong
Province, China. 10% Pd/C, cyclohexanecarboxylic acid
chloride and 2',4',6'-trihydroxyacetophenone were pur-
chased from Acros Organics (New Jersey, USA). Pino-
*
E-mail: ws@imm.ac.cn; Tel.: 0086-010-83163542; Fax: 0086-010-63017757
Received February 28, 2012; accepted April 18, 2012.
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cjoc.201200201 or from the author.
Dedicated to Professor Jun Zhou on the occasion of his 80th birthday.
†
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Yang et al.
FULL PAPER
cembrin substances (20060901) were provided by Bei-
jing Collab Pharma Co. Ltd. IR spectra were recorded
on a IR-47 spectrometer. Melting points were deter-
mined with a YRT-3 apparatus (Tianjin University Pre-
cision Apparatus Factory, Tianjin, China). NMR spectra
were recorded on a Varian MERCURY-400 spectrome-
ter using tetramethylsilane (TMS) as internal standard
and deuterated dimethylsulfoxide (DMSO-d6) as solvent.
Chemical shifts were given on the δ-scale.
cal method mentioned above.
Synthesis of 3-phenyl-1-(2,4,6-trihydroxyphenyl)-1-
propanone (3)
Compound 2 (15.0 g, 0.059 mol) in ethyl acetate
(1400 mL) was hydrogenated with 10% Pd-C (2.5 g)
under H2 at 60 ℃ for 12 h. After cooling, the reation
mixture was filtered and concentrated in vacuo to give a
residue, which was recrystallized in hexane/ethyl ace-
tate (3∶1, V/V) to yield compound 3 (12.2 g) as a
1
High performance liquid chromatography
light-yellow solid in 80% yield, m.p. 160 ℃. Its H
NMR data (DMSO-d6, 400 MHz) see Table 1. ESI-MS
m/z: 259.2 [M＋H]＋. These data were in agreement
with those reported in the literature.[10]
HPLC analysis was performed on a Shimadzu liquid
chromatography LC-10ATvp equipped with SPD-
M10AT VP UV-Vis spectrophotometric detector (Shi-
madzu Co., Japan). The analysis was carried out on an
ODS column (Apollo C18, 150×4.6 mm I.D., 5 µm,
Grace) maintained at temperature 25 ℃. Mobile phase
A was a phosphate buffer, adjusted to pH 3.0 and
methanol used as mobile phase B. Mobile phase was A
and B mixed in the ratio of 64∶36 (V/V). Flow rate was
kept as 1.0 mL/min, injection volume was 20 µL, chro-
matographic data acquisition time for 45 min and UV
detection was carried out at 290 nm.
Isolation and synthesis of 5,7-dihydroxy-2-cyclo-
hexyl-4H-1-benzopyran-4-one (4)
Isolation of compound 4 Compound 2 (100.0 g,
0.39 mol) was hydrogenated for 5 h using the same
method to synthesize crude product 1 (100.0 g), which
was recrystallized in ethyl acetate for five times. The
mother liquid was concentrated in vacuo to yield a solid,
which was analysed by HPLC and impurity 4 was ob-
served around 30%. Then, the solid was purified on sil-
ica gel column with a mixture of CH2Cl2/CH3OH
(200∶1, V/V) as eluent to obtain 4 (0.5 g) as a white
HPLC-ESI-MS and HPLC-UV
HPLC-UV and HPLC-ESI-MS analysis were carried
out on an Agilent 1100 series LC/MSD Trap equipped
with UV-VIS spectrophotometric detector (Agilent Co.,
Santaclara, CA, USA). Analyst software was used for
data acquisition and data processing. The turbo ion
spray voltage was maintained at 5.5 kV and temperature
was set at 350 ℃. The auxiliary gas and curtain gas
was high purity nitrogen. Zero air was used as the nebu-
lizer gas. LC-MS spectra were acquired from m/z 100—
600 in 0.1 amu steps with 2.0 s dwell time. The analysis
was carried out using a column (Apollo C18, 150
mm×4.6 mm I.D., 5 µm, Grace) and the same analyti-
1
amorphous powder in 0.5% yield, m.p. 214 ℃. Its H
NMR data (DMSO-d6, 400 MHz) see Table 1. ESI-MS
m/z: 261.2 [M＋H]＋. These data were in agreement
with those reported in the literature.[11]
Synthesis of compound 4 To a solution of
2',4',6'-trihydroxyacetophenone (6, 2.3 g, 0.013 mol) in
acetone (150 mL), anhydrous K2CO3 (20 g, 0.188 mol)
was added, and the solution was stirred for 1 h at room
temperature. Then cyclohexanecarboxylic acid chloride
(10 mL, 0.039 mol) was added dropwise, and the mix-
ture was refluxed for 10 h. The reaction mixture was
Table 1 Comparative 1H assignments for compounds 1, 2, 3, 4, and 5
No.
2
1
2
3
4
5
5.58 (dd, 1H, J＝2.8, 12.0)
2.78 (dd, 1H, J＝2.8, 16.0)
3.37 (dd, 1H, J＝4.0, 12.0)
—
—
—
—
4.18—4.23 (m, 1H)
3
6.97 (s, 1H)
6.08 (s, 1H)
2.53—2.80 (m, 2H)
—
5.80 (brs, 2H)
5
6
8
9
—
—
5.89 (d, 1H, J＝2.0)
5.92 (d, 1H, J＝2.0)
—
6.22 (brs, 1H)
6.52 (brs, 1H,)
—
—
6.15 (d, 1H, J＝2.0)
5.82 (brs, 2H)
3.28 (t, 2H, J＝7.2) 6.32 (d, 1H, J＝2.0)
2.87 (t, 2H, J＝7.2) —
—
1'
—
—
—
2.51—2.57 (m, 1H)
1.87—1.90 (m, 1H)
2', 6'
7.51 (d, 2H, J＝7.2)
8.05 (d, 2H, J=8.0)
7.57—7.59 (m, 3H)
—
7.16—7.26 (m, 5H) 1.18—1.92 (m, 10H) 1.02—1.74 (m, 10H)
3', 4', 5' 7.37—7.44 (m, 3H)
4-OH —
10.34 (s, 1H)
10.22 (s, 2H)
—
—
—
2,6-OH —
—
—
—
5-OH
12.12 (s, 1H)
12.84 (s, 1H)
12.80 (s, 1H)
10.79 (s, 1H)
12.10 (s, 1H)
10.71 (s, 1H)
7-OH
10.81 (s, 1H)
10.90 (s, 1H)
—
a Data were measured in DMSO-d6 at 400 MHz.
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Chin. J. Chem. 2012, 30, 1315—1319

Identification and Synthesis of Impurities in Pinocembrin
cooled to room temprature, filtered and concentrated in
vacuo to furnish a solid, which was dissolved in EtOAc
(100 mL) and washed with water (50 mL×3). The
combined organic layer was evaporated under vacuo to
obtain an orange solid. The solid was dissolved in 5%
KOH/EtOH (250 mL) and refluxed for 15 h. The reac-
tion mixture was cooled, evaporated and water (50 mL)
was added. The mixture was extracted with EtOAc (50
mL×3). The combined organic phase was dried over
anhydrous Na2SO4, filtered and evaporated under vacuo
to get a residue, which was purified by chromatography
on silica gel using a mixture of CH2Cl2/CH3OH (200∶1,
V/V) as eluent to give product 4 (2.3 g) as a white solid
found 263.1284.
Results and Discussion
Detection and identification of impurities
In the course of HPLC analysis on pinocembrin sub-
stances using analytical conditions, four inconspicuous
peaks due to impurities except for pinocembrin 1 (tR＝
9.3 min) were observed at the relative retention times of
10.4 min (3), 12.5 min (2), 21.7 min (4) and 28.1 min
(5). Their contents were analysed as 0.078% (3),
0.013% (2), 0.347% (4) and 0.288% (5) (HPLC-purity),
respectively (Figure 1). In order to clarify these impuri-
ties, we further researched the substances. Comparison
of retention time (tR) values on HPLC of these four im-
purities with that of the reactant in the preparation of
pinocembrin revealed that impurity 2 (tR＝12.5 min)
was the starting material. But compounds 3, 4, and 5
were unknown impurities, which did not exist in the
starting material.
1
in 67% yield, m.p. 212 ℃. H NMR (DMSO-d6, 400
MHz) δ: 1.22—1.93 (m, 10H, cyclohexyl-CH2), 2.53—
2.58 (m, 1H, H-1'), 6.08 (s, 1H, H-3), 6.16 (s, J＝2.0 Hz,
H-6), 6.32 (d, 1H, J＝2.0 Hz, H-8), 10.79 (s, 1H, OH-7),
12.80 (s, 1H, OH-5). ESI-MS m/z: 261.1 [M＋H]＋.
HR-ESI-MS calcd for C15H16O4 261.2924, found
261.2936 [M＋H]＋. These data were in agreement with
those isolated from crude product.
In order to determine the structures of these com-
pounds, the same samples were subjected to HPLC-ESI-
MS analysis using HPLC-ESI-MS conditions to identify
the mass of the impurities. In positive ion model of
HPLC-ESI-MS, compounds 1, 4, and 5 showed their ion
peaks at m/z 257.1 [M＋H]＋, m/z 261.2 [M＋H＋4H]＋,
and m/z 263.2 [M＋H＋6H]＋ respectively (Figures 2,
3), which suggested that these impurities could be side
products of excessive hydrogenation of pinocembrin or
its synthetic material in the course of the synthesis of
pinocembrin.
Synthesis of 2,3-dihydro-5,7-dihydroxy-2-cyclohexyl-
4H-1-benzopyran-4-one (5)
The solution of 4 (15.0 g, 0.057 mol) in anhydrous
ethanol (1400 mL) was stirred with 10% Pd-C (2.5 g)
under H2 (4 atm) at room temperature for 24 h. The re-
action mixture was filtered and concentrated in vacuo.
The obtained residues were subjected to dry flash col-
umn (petroleum ether/ethyl acetate＝25∶1, V/V) to get
compound 5 (12.2 g) as a white solid in 81% yield, m.p.
169.6—169.9 ℃. UV-vis (MeOH) λmax: 213, 226 (sh),
1
289, 322 (sh) nm. H NMR (DMSO-d6, 400 MHz) see
Table 1. 13C NMR (DMSO-d6, 400 MHz) δ: 196.7 (C-4),
166.4 (C-7), 163.3 (C-5), 163.0 (C-1a), 101.7 (C-4a),
95.5 (C-6), 94.7 (C-8), 80.9 (C-2), 40.9 (C-1'), 38.3
(C-3), 27.5 (C-2', C-6'), 25.8 (C-3'), 25.4 (C-5'), 25.3
(C-4'). IR (KBr) ν: 2926, 1629, 1584, 1488, 1302, 1146,
Isolation and synthesis of impurities
In order to confirm the above assumption, it is nec-
essary to obtain enough impurities. Therefore, we ex-
tended the catalytic hydrogenation time from 5 h to 12 h
in the course of the pinocembrin synthesis (Scheme 1).
HPLC analysis of the reaction products showed the
content of 3 was above 95%, while 4 and 5 were not
－
1
1089, 835, 708, 553 cm ; ESI-MS m/z: 263.2 [M
＋H]＋. HR-ESI-MS calcd for C15H19O4 263.1283,
Figure 1 HPLC chromatogram of pinocembrin (1) (tR＝9.3 min), impurities 2 (tR＝12.5 min), 3 (tR＝10.4 min), 4 (tR＝21.7 min) and 5
(tR＝28.1 min). Mobile phase: methanol∶phosphate buffer, 64∶36 (V/V), pH 3.0; column temperature, 25 ℃.
Chin. J. Chem. 2012, 30, 1315—1319
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Yang et al.
FULL PAPER
Scheme 1
HO
O
HO
O
H2/Pd-C/EtOH
60 ℃, 5 h, Y = 84%
OH
O
OH
O
1
2
5
HO
OH
8
B
A
H2/Pd-C/EtOH
3
9
60 ℃, 5 h, Y = 84%
Figure 2 HPLC chromatogram and ionic current of pinocem-
brin (1) and impurities (4, 5)
OH
O
3
ESI-MS.
Unfortunately, neither excessive hydrogenation nor
recrystallization was helpful to get enough amount of
compound 5. So HPLC-MS and HPLC-UV analysis
were used to elucidate the structure. Its HPLC-ESI-MS
(Figure 3) in positive ionization mode showed the ion
peak at m/z 263.2 [M＋H]＋, suggesting the molecular
formula of C15H18O4. The UV spectrum (λmax 289 and
322 nm, sh) of 5 (Figure 4) in HPLC-UV was similar as
those of 1, suggesting the presence of a dihydrochro-
mone skeleton in 5.[7] Combined with the difference of
the HPLC-ESI-MS data between 4 (m/z 261.2 [M＋H]＋)
and 5 (m/z 263.2 [M＋H]＋), the structure of 5 was de-
termined as 2,3-dihydro-5,7-dihydroxy-2-cyclohexyl-
4H-1-benzopyran-4-one, which was identified as a new
compound.
Figure 3 HPLC-ESI-MS of pinocembrin (1) (m/z 257.1 [M＋
H]＋), impurities 4 (m/z 261.2 [M＋H]＋) and 5 (m/z 263.2 [M＋
H]＋).
detected. The reaction mixture was recrystallized in
Figure 4 UV apex spectrum peaks of compounds 1, 4, and 5.
hexane/ethyl acetate to obtain 3, which was identified as
In order to further confirm the structures, we em-
ployed Baker-Venkataraman transformation to synthe-
size 4 and 5 from the starting material 2',4',6'-trihy-
droxyacetophenone (6, Scheme 2).[13] 6 was treated with
cyclohexanecarboxylic acid chloride in acetone through
Baker-Venkataraman transformation to afford flavone
derivative 7, which was hydrolyzed in alkaline condi-
tion to furnish 4 in 67% yield. Eventually, 4 was further
hydrogenation in the presence of 10% pd/C to get com-
pound 5 in 81% yield successfully. Moreover, NMR
3-phenyl-1-(2,4,6-trihydroxyphenyl)-1-propanone
on
the basis of NMR spectral analysis. Thus, impurity 3
was determined as a side product of excessive hydro-
genation of pinocembrin.
Subsequently, the crude products reacted for 5 h
were recrystallized in ethyl acetate, the mother liquid
was observed to contain 32% impurity 4 in HPLC
analysis. The solution was concentrated and further pu-
rified by silica gel column chromatography to give
compound 4. Its HR-ESI-MS showed the ion peak at
m/z 261.2936 [M＋H]＋, indicating the molecular for-
mula of C15H16O41(calcd for C15H16O4, 260.2924). In
combination with H NMR spectral data, the structure
of 4 was elucidated as 5,7-dihydroxy-2-cyclohexyl-
4H-1-benzopyran-4-one. It could be a side product of
excessive hydrogenation of the starting material 2. The
results were in agreement with the deduce from HPLC-
1
experiments showed that the H NMR data of synthetic
compounds 3, 4, and 5 were in agreement with the iso-
lated ones. Co-injection of the synthetic compounds 3, 4,
and 5 into HPLC with the pinocembrin sample further
confirmed the above deduce. Accordingly, impurities 3
—5 were identified as side product of excessive hy-
drogenation during the synthesis of pinocembrin.
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Chin. J. Chem. 2012, 30, 1315—1319

Identification and Synthesis of Impurities in Pinocembrin
Scheme 2
O
Cl
HO
OH
CH3
O
O
O
5% KOH/EtOH
O
K2CO3/Acetone
reflux,10 h
reflux,15 h
Y = 67% (two steps)
OH
O
OH
7
6
8
HO
O
O
1‘
HO
O
H2/10% Pd/C/EtOH
4 atm, r.t., 24 h
Y = 81%
3
6
OH
OH
O
5
4
Structural elucidation of impurities
gether with spectral analysis and classical synthetic
methods, we analyzed and identified the structures of
four impurities in a new drug—pinocembrin. All of
them were determined as side products of excessive
hydrogenation during the synthesis of pinocembrin.
These information will direct us to prepare enough
amount of impurities for pharmacological and toxico-
logical test subsequently, and will lay a foundation for
the new drug development. This investigation also
demonstrated that HPLC-MS and HPLC-UV are con-
venient methods for providing information of unknown
structures without isolation.
ESI mass spectrum of impurity 3 in positive ion
mode displayed a quasi molecular ion peak at m/z 259.2
[M＋H]＋, which was 2 amu more than that of 1 proto-
1
nated molecular ion. Compared with 1, the H NMR
spectrum of 3 is two protons more than that of 1 (Table
1), indicating that 3 should be a ring cleavage product of
1 through hydrogenation in the course of synthesis.
Thus, 3 was identified as 3-phenyl-1-(2,4,6-trihydroxy-
phenyl)-1-propanone.
HR-ESI-MS of impurity 4 gave the quasi molecular
ion peak at m/z 261.2936 [M＋H]＋, indicating the mo-
lecular formula of C15H16O4 (calcd for C15H16O4
260.2924), which was 6 amu more than that of the
starting material 2 (C15H10O4, 254.2). Comparing the 1H
NMR spectrum with 2 (Table 1), 5 aromatic proton sig-
nals (δH 7.58—8.05) in 2 were replaced by 11 aliphatic
hydrogens (δH 1.18—2.57) in 4, which suggested that 4
should be a product of full hydrogenation of an aromatic
ring in 2. Accordingly, the structure of 4 was deduced as
5,7-dihydroxy-2-cyclohexyl-4H-1-benzopyran-4-one.
HR-ESI-MS of impurity 5 showed the quasi ion
peak at m/z 263.1284, corresponding to the molecular
formula of C15H19O4, indicating that the structure of 5
Acknowledgement
We are grateful for financial support of the Research
Special Fund for Public Welfare Industry of Health (No.
200902008). We thank Dr. Yao Chunsuo of our institute
for constructive suggestions.
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1
was 2 protons more than that of 4. In H NMR spectra
of 4 and 5 (Table 1), an olefinic hydrogen in 4 (δH 6.08)
was substituted by 3 aliphatic hydrogens in 5 (δH 2.53—
4.20), indicating that 5 should be a product of hydro-
genation of double bond in 4. The 13C NMR, IR and UV
spectra of 5 further confirmed the above hypothesis.
Therefore, impurity 5 was determined as 2,3-dihydro-
5,7-dihydroxy-2-cyclohexyl-4H-1-benzopyran-4-one,
which was a new compound.
The structures of impurities 3, 4, and 5 were further
confirmed by chemical synthetic methods, and all of
them were determined as side products of excessive
hydrogenation of the target product 1 or the starting
material 2 in the course of synthesis.
Conclusions
[13] Jain, A. C.; Mathur, S. K.; Seshadri, T. R. J. Sci. Ind. Res. 1962, 21B,
214.
In summary, applying HPLC-UV-MS technique, to-
(Pan, B. Lu, Z.)
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