Chiral separation, configurational identification and antihypertensive evaluation of (±)-7,8-dihydroxy-3-methyl-isochromanone-4

Article abstract of DOI:10.1016/j.bmcl.2012.08.040

(±)-7,8-Dihydroxy-3-methyl-isochromanone-4 [(±)-XJP] is a natural antihypertensive product contained in banana (Musa sapientum L.) peel. (-)-XJP and (+)-XJP were first obtained by chiral resolution, meanwhile circular dichroism (CD) calculations and chiral synthesis were employed to investigate the absolute configuration. The results indicated that the absolute configuration of (+)-XJP is S-configured and the absolute configuration of (-)-XJP is R-configured. Furthermore, the evaluation of antihypertensive effects in vivo proved that R-(-)-XJP was more potent than S-(+)-XJP and [(±)-XJP].

Full text of DOI:10.1016/j.bmcl.2012.08.040

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6490–6493
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Chiral separation, configurational identification and antihypertensive
evaluation of ( )-7,8-dihydroxy-3-methyl-isochromanone-4
Renren Bai a,b, , Jie Liu a,b, , Yao Zhu a,b, Xue Yang a,b, Chen Yang a,b, Lingyi Kong b,c
Xiaobing Wang b,c, Hengyuan Zhang a,b, Hequan Yao a,b, Mingqin Shen d, Xiaoming Wu a,b,
,
⇑
,
Jinyi Xu a,b,
⇑
a Department of Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, PR China
b State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, PR China
c Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, PR China
d Jiangsu Provincial Institute of Traditional Chinese Medicine, Nanjing 210028, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
( )-7,8-Dihydroxy-3-methyl-isochromanone-4 [( )-XJP] is a natural antihypertensive product contained
in banana (Musa sapientum L.) peel. (ꢀ)-XJP and (+)-XJP were first obtained by chiral resolution, mean-
while circular dichroism (CD) calculations and chiral synthesis were employed to investigate the absolute
configuration. The results indicated that the absolute configuration of (+)-XJP is S-configured and the
absolute configuration of (ꢀ)-XJP is R-configured. Furthermore, the evaluation of antihypertensive effects
in vivo proved that R-(ꢀ)-XJP was more potent than S-(+)-XJP and [( )-XJP].
Received 13 July 2012
Revised 7 August 2012
Accepted 10 August 2012
Available online 16 August 2012
Keywords:
Ó 2012 Elsevier Ltd. All rights reserved.
Isochromanone
Chiral separation
Enantiomers
Antihypertensive activity
Circular dichroism
Life is heavily dependent on chirality, and chiral discrimination
is frequently encountered in biological systems.1 Although a num-
ber of chiral isomers of racemic drugs have almost identical phys-
ical and chemical properties, the enantiomers of many chiral
therapeutic agents exhibit differences in activity and toxicity. Re-
cently a great deal of attention has been focused on the separation
of stereochemically pure enantiomers for the purpose of marketing
drugs as single active compounds.2
the two enantiomers became interesting. Many attempts have
been made to investigate the separation of stereochemically pure
enantiomers for the purpose of obtaining single active moieties.
Herein, we wish to report the chiral separation, absolute configura-
tional analysis and evaluation of antihypertensive effects of XJP
enantiomers.
The general methods to obtain pure enantiomers are asymmet-
ric synthesis and resolution of racemates.8 Several methods have
been investigated to asymmetrically synthesize XJP.3,6 However,
due to keto-enol tautomerism of the carbonyl, the chiral carbon
at C-3 becomes racemic easily in the process of preparation, espe-
cially under vigorous reaction conditions. In order to obtain stereo-
chemically pure enantiomers, we tried to perform the chiral
synthesis and demonstrate the absolute configuration.
( )-7,8-Dihydroxy-3-methyl-isochromanone-4[( )-XJP],
iso-
lated from the banana (Musa sapientum L.) peel extract,3,4 is a
structurally unique polyphenolic compound possessing a chiral
center. It has been documented that ( )-XJP exerted a large variety
of biological activities including antioxidative, antiinflammatory,
antihypertensive and cardioprotective.5–7 Interestingly, in our pre-
vious investigations, ( )-XJP significantly decreased blood pressure
in a dose-dependent manner. In both acute and therapeutic antihy-
pertensive tests (RHRs), the maximum antihypertensive effect of
( )-XJP at the dose of 100 mg/kg was comparable to that of capto-
pril at the dose of 25 mg/kg.6
As shown in Scheme 1, alkylation of 1 with methyl S-(ꢀ)-lactate
in presence of NaH, followed by saponification of the methyl ester
gave compound 3. Intermediate 3 was treated with n-BuLi in THF
at ꢀ85 °C, and then the temperature was allowed to rise to room
temperature to give compound 4 in 58% yield.9 After deprotection
of phenolic methyl ether 4 by AlCl3 in CHCl3, target compound S-
(+)-XJP was obtained. However, the content percentage of the S-
configured intermediates and the optical rotation decreased with
every reaction step, while the ultimate optical rotation was only
However, all the above pharmacodynamic studies were based
on racemic XJP, the possible difference of the activities between
⇑
Corresponding authors.
E-mail address: jinyixu@china.com (J. Xu).
½
a 2D0
ꢁ
+31.58 (c, 0.202, MeOH). Using R-(+)-lactate as starting mate-
rial, R-(ꢀ)-XJP was synthesized following the above procedure, and
These authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.08.040

R. Bai et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6490–6493
6491
O
O
O
Br
Br
O
b
O
c
d
d
HO
O
H
O
O
O
O
O
O
O
O
O
OH
O
O
HO
O
O
O
O
OH
a
3
2
4
S -(+)-XJP
e.e. (%) 30.95
Br
Br
O
O
O
a
O
Br
Br
O
O
1
c
b
H
OH
O
O
O
O
O
O
OH
HO
O
O
OH
O
R -(-)-XJP
e.e. (%) 34.19
6
5
7
Scheme 1. Chiral synthetic routes to S-(+)-XJP and R-(ꢀ)-XJP. Reagents and conditions: (a) anhydrous DMF, NaH, 0 °C, 75–80%; (b) MeOH, 10% NaOH, rt, then 10% HCl, 90–
95%; (c) n-BuLi, anhydrous THF, ꢀ85 °C to rt, 58–60%; (d) AlCl3, NaI, CHCl3, reflux, 50–55%.
the optical rotation was ½a D20
ꢁ
ꢀ35.45 (c, 0.182, MeOH). Although
As illustrated in Figure 1, ( )-XJP could be separated success-
fully under the above condition, the retention times of the enanti-
omers were 3.99 and 4.43 min, respectively. Enantiomeric excess
of the two enantiomers were determined to be 98.09% and
99.84% by SFC using the same condition, respectively. Optical test
proved that the first elute peak is the dextro isomer and the optical
part of the aimed products changed into racemic mixtures during
the reaction procedures, it has been proved preliminarily that S-
XJP is the dextro isomer, and R-XJP is the levo isomer. In order to
get optically pure XJP enantiomers for further investigation, our
group has also attempted other methods.
Resolution of racemates by chromatographic techniques is an-
other available method for the development of isomerically pure
agents in industry.10 We performed the chiral separation of XJP
rotation is ½a 2D0
ꢁ
+104.93 (c, 0.209, MeOH), and the second elute
peak is the levo isomer and the optical rotation is ½a D20
ꢀ103.68
ꢁ
(c, 0.500, MeOH).
enantiomers on
a
chiral column CHIRALPAK AD-H (5 cm
The pure XJP enantiomers are white powder as their racemic
mixtures. In order to determine the absolute configuration by X-
ray diffraction analysis, although many attempts have been made,
the perfect crystals were not obtained. Therefore, circular dichro-
ism (CD) calculations, another mature method, was employed to
investigate the absolute configuration.
I.D. ꢂ 25 cm L) by Supercritical Fluid Chromatography (SFC) and
desired results were obtained. The chiral column was made of
5 lm silica gel, which was coated with starch tri[(3,5-dimethyl-
phenyl)carbamate]. Methanol–CO2 (15:85, V/V) was applied as
the mobile phase and the detective wavelength was set at 212 nm.
Figure 1. Chiral separation of XJP enantiomers: (A) The chiral column used in the separation; (B) Chromatograms of the separation of ( )-XJP; (C) Chromatograms of (+)-XJP;
(D) Chromatograms of (ꢀ)-XJP.

6492
R. Bai et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6490–6493
Theoretical calculations of electronic circular dichroism (ECD)
spectra for compounds (+)-XJP and (ꢀ)-XJP were performed with
the Gaussian 09 program package.11,12 The ECD calculations of
compounds (+)-XJP and (ꢀ)-XJP were performed with time-
dependent density functional theory (TDDFT) calculations at the
b3pw91/6-311 + g(d, p) level of theory with the default solvent
model using methanol as solvent. The calculated excitation energy
(in nm) and rotatory strength, R, in dipole velocity (Rvel) and di-
pole length (Rlen) forms, were simulated into an ECD curve using
the:
X
1
E ꢀ
DE0a
D
2
D
e
ðEÞ ¼
D
E0aR0aexp½ꢀð
Þ ꢁ
pffiffiffi
2:296 ꢂ 10ꢀ39
D
p
D
a
Where
is half the bandwidth at 1/e height and expressed in
energy units. The parameters
DE0a and R0a are the excitation
energies and rotatory strengths for the transition from 0 to a,
respectively. The calculated CD spectrum for S-XJP showed an
unambiguously agreed behavior in comparison to the experimen-
tal CD data of (+)-XJP, and the calculated spectrum also matched
the experimental data perfectly (Fig. 2). A similar agreement ap-
plied between R-XJP and (ꢀ)-XJP. The calculated optical rotations
of S-XJP and R-XJP were +161.99° and ꢀ161.99°, respectively,
which were approximately consistent with the experimental data.
The results indicated that the absolute configuration of (+)-XJP is
S-configured and the absolute configuration of (ꢀ)-XJP is R-config-
ured, which could be drawn with the same conclusions as that of
chiral synthesis. So we confirmed that the XJP enantiomers are
S-(+)-XJP and R-(ꢀ)-XJP, respectively.13
Based on the results of chiral separation and configurational
identification, in vivo evaluation of antihypertensive effects in
spontaneously hypertensive rats (SHRs) was performed to provide
the information of possible different potency between XJP
enantiomers. After oral administration of control drug captopril
Figure 2. (A) Experimental CD spectra of (+)-XJP and comparison with the
experimental CD spectrum of S-XJP; (B) Experimental CD spectra of (ꢀ)-XJP and
comparison with the experimental CD spectrum of R-XJP.
Figure 3. (A) The acute antihypertensive activities of ( )-XJP, (ꢀ)-XJP, (+)-XJP and captopril in SHRs (SAP, systolic arterial pressure); B. The acute antihypertensive activities of
( )-XJP, (ꢀ)-XJP, (+)-XJP and captopril in SHRs (DAP, diastolic arterial pressure); C. The acute antihypertensive activities of ( )-XJP, (ꢀ)-XJP, (+)-XJP and captopril in SHRs
(MAP, mean artery pressure); D. The changes of heart rate (HR) of ( )-XJP, (ꢀ)-XJP, (+)-XJP and captopril in SHRs. Data are represented as mean SEM (n = 8). Significance
levels ⁄p <0.05 and ⁄⁄p <0.01 as compared with the respective control.

R. Bai et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6490–6493
6493
(40 mg/kg), ( )-XJP (80 mg/kg), (+)-XJP (80 mg/kg) and (ꢀ)-XJP
References and notes
(80 mg/kg), the blood pressure and heart rate of SHRs were de-
tected from 0 to 24 h. The evaluation results showed that (ꢀ)-XJP
exhibits more potent antihypertensive activity than ( )-XJP signif-
icantly, and the antihypertensive activity of ( )-XJP is stronger than
that of (+)-XJP, which demonstrate that (ꢀ)-XJP plays a more
important role in the antihypertensive effect of ( )-XJP (Fig. 3).
The diastolic arterial pressure (DAP) of SHRs treated with (ꢀ)-XJP
was reduced by almost 30% at 6 h, which is obviously superior to
that of control drug captopril. Meanwhile, the maximum antihy-
pertensive effect on the systolic arterial pressure (SAP) of (ꢀ)-XJP
is also somewhat more effective than that of captopril. Further-
more, ( )-XJP and its enantiomers produce no observable alter-
ation in the basal heart rate at these doses.
In summary, (ꢀ)-XJP and (+)-XJP was first obtained by chiral
resolution, and CD calculations as well as chiral synthesis were em-
ployed to investigate the absolute configuration. It has been dem-
onstrated that the absolute configuration of (+)-XJP is S-configured
and the absolute configuration of (ꢀ)-XJP is R-configured. Antihy-
pertensive effects of S-(+)-XJP and R-(ꢀ)-XJP in vivo were also eval-
uated. The results proved that ( )-XJP is a racemates with isomers
possessing similar pharmacodynamic effects, but of different po-
tency, and the R-(ꢀ)-XJP is more potent than S-(+)-XJP. Further
investigation involving with R-(ꢀ)-XJP and the mechanism are cur-
rently in progress and will be reported in due course.
1. Sun, Q.; Olesik, S. V. J. Chromatogr. B. 2000, 745, 159.
2. Matsuoka, Y.; Kanda, N.; Lee, Y. M.; Higuchi, A. J. Membrance. Sci. 2006, 280,
116.
3. Qian, H.; Huang, W. L.; Wu, X. M.; Zhang, H. B.; Zhou, J. P.; Ye, W. C. Chin. Chem.
Lett. 2007, 18, 1227.
4. Qian, H.; Huang, W. L.; Yao, G. L.; Ge, L.; Wu, X. M. J. China. Pharm. Univ. 2009,
40, 524.
5. Fu, R.; Chen, Z.; Wang, Q. J.; Guo, Q. L.; Xu, J. Y.; Wu, X. M. Atherosclerosis 2011,
219, 40.
6. Liu, J.; Ren, H.; Xu, J. Y.; Bai, R. R.; Yan, Q.; Huang, W. L.; Wu, X. M.; Fu, J. H.;
Wang, Q. J. W, Q.; Fu, R Bioorg. Med. Chem. Lett. 1822, 2009, 19.
7. Fu, R.; Yan, T. H.; Wang, Q. J.; Guo, Q. L.; Yao, H. Q.; Wu, X. M.; Li, Y. Vasc.
Pharmacol. (Available, online 17 May 2012).
8. Noyori, R. Angew. Chem., Int. Ed. 2008, 2002, 41.
9. Sudani, M.; Yoshio, T.; Eiichi, Y.; Tadashi, K. Tetrahedron. Lett. 1981, 22, 4253.
10. Tang, K.; Song, L.; Liu, Y. B.; Pan, Y.; Jiang, X. Y. Chem. Eng. J. 2010, 158, 411.
11. Frisch, M.J., Trucks, G.W., Schlegel, H.B., Scuseria, G.E., Robb, M.A., Cheeseman,
J.R., Scalmani, G., Barone, V., Mennucci, B., Petersson, G.A., Nakatsuji, H.,
Caricato, M., Li, X., Hratchian, H.P., Izmaylov, A.F., Bloino, J., Zheng, G.,
Sonnenberg, J.L., Hada, M., Ehara, M., Toyota, K., Fukuda, R., Hasegawa, J.,
Ishida, M., Nakajima, T., Honda, Y., Kitao, O., Nakai, H., Vreven, T., Montgomery,
J.A.Jr., Peralta, J.E., Ogliaro, F., Bearpark, M., Heyd, J.J., Brothers, E., Kudin, K.N.,
Staroverov, V.N., Keith, T., Kobayashi, R., Normand, J., Raghavachari, K., Rendell,
A., Burant, J.C., Iyengar, S.S., Tomasi, J., Cossi, M., Rega, N., Millam, J.M., Klene,
M., Knox, J.E., Cross, J.B., Bakken, V., Adamo, C., Jaramillo, J., Gomperts, R.,
Stratmann, R.E., Yazyev, O., Austin, A.J., Cammi, R., Pomelli, C., Ochterski, J.W.,
Martin, R.L., Morokuma, K., Zakrzewski, V.G., Voth, G.A., Salvador, P.,
Dannenberg, J.J., Dapprich, S., Daniels, A.D., Farkas, O., Foresman, J.B., Ortiz,
J.V., Cioslowski, J., and Fox, D.J., 2010. Gaussian 09, Revision B.01, Gaussian, Inc.,
Wallingford CT.
12. Bringmann, G.; Maksimenka, K.; Mutanyatta-Comar, J.; Knauer, M.; Bruhn,
Torsten Tetrahedron 2007, 63, 9810.
13. Analytical data for S-(+)-XJP: ½a D20
+104.93 (c, 0.209, MeOH); mp 163–164 °C
ꢁ
Acknowledgments
(dec.); 1H NMR (DMSO-d6, 300 MHz): d (ppm) 1.29 (d, J = 6.7 Hz, 3H, –CH3),
4.19 (q, J = 6.7 Hz, 1H, –CH–), 4.68 (d, J = 15.5 Hz, 1H, –CH2–), 4.97 (d,
J = 15.9 Hz, 1H, –CH2–), 6.82 (d, J = 8.7 Hz, 1H, Ar-H), 7.31 (d, J = 8.7 Hz, 1H,
Ar-H), 8.90 (1H, –OH), 10.40 (1H, -OH); 13C NMR (DMSO-d6, 75 MHz): d (ppm)
194.8, 150.8, 139.8, 130.3, 121.7, 119.0, 114.6, 76.8, 62.6, 15.9. HRMS (ESI)
calcd for C10H11O4 [M+H]+: 195.0652, found 195.0653. Analytical data for R-
This study was financially supported by grant from ‘Eleventh
Five-Year’ Major Innovation Projects for New Drug Candidates
(No. 2009ZX09103-128), Project for Research and Innovation of
Graduates in Colleges and Universities of Jiangsu Province
(CXZZ11-0798), Fundamental Research Funds for the Central Uni-
versities (JKY2011027) and Project Program of State Key Labora-
tory of Natural Medicines, China Pharmaceutical University
(JKGQ201115). Thanks for Daicel Chiral Technologies (China) Co.,
Ltd.
(ꢀ)-XJP:
½
a 2D0
ꢁ
ꢀ103.68 (c, 0.500, MeOH); mp 163–164 °C (dec.); 1H NMR
(DMSO-d6, 300 MHz): d (ppm) 1.26 (d, J = 6.7 Hz, 3H, –CH3), 4.19 (q, J = 6.7 Hz,
1H, –CH–), 4.68 (d, J = 15.5 Hz, 1H, –CH2–), 4.92 (d, J = 15.9 Hz, 1H, –CH2–), 6.82
(d, J = 8.7 Hz, 1H, Ar-H), 7.31 (d, J = 8.7 Hz, 1H, Ar-H), 8.92 (1H, –OH), 10.39 (1H,
–OH); 13C NMR (DMSO-d6, 75 MHz): d (ppm) 194.8, 150.8, 139.8, 130.3, 121.7,
119.0, 114.6, 76.8, 62.6, 15.9. HRMS (ESI) calcd for C10H11O4[M+H]+: 195.0652,
found 195.0650.