Optical resolution and structure determination of new indolizidine alkaloids from Elaeocarpus sphaericus

Article abstract of DOI:10.1002/hlca.201000200

Two new indolizidine alkaloids, (±)-3-oxoisoelaeocarpine (1) and (±)-elaeocarpine N-oxide (2), along with three known alkaloids, (±)-isoelaeocarpine (3), (±)-elaeocarpine (4), and (-)-isoelaeocarpiline (5), were isolated from an EtOH extract of the branches and leaves of Elaeocarpus sphaericus. The structures of these compounds were determined by spectroscopic and chemical methods. Furthermore, enantiomers of compounds 1 and 3 were separated on a chiral CD-Ph column, and their absolute configurations were determined by TD-DFT (=time-dependent density-functional theory) quantumchemical calculations of their electronic circular dichroism (ECD) spectra.

Full text of DOI:10.1002/hlca.201000200

Helvetica Chimica Acta – Vol. 94 (2011)
347
Optical Resolution and Structure Determination of New Indolizidine
Alkaloids from Elaeocarpus sphaericus
by Chang-Xin Zhoua), Xue-Yao Wanga), Jian-Xia Moa), Jian Zhang*b), and Li-She Gan*a)
a) Institute of Modern Chinese Medicine (IMCM), College of Pharmaceutical Sciences, Zhejiang
University, Hangzhou 310058, P. R. China (phone: þ 86-571-88208456; fax: þ 86-571-88208457;
e-mail: lsgan@zju.edu.cn)
b) Institute of Medical Science, School of Medicine, Shanghai Jiaotong University, Shanghai 200025,
P. R. China (e-mail: jian.zhang@sjtu.edu.cn)
Two new indolizidine alkaloids, (ꢀ)-3-oxoisoelaeocarpine (1) and (ꢀ)-elaeocarpine N-oxide (2),
along with three known alkaloids, (ꢀ)-isoelaeocarpine (3), (ꢀ)-elaeocarpine (4), and (ꢁ)-isoelaeocarpi-
line (5), were isolated from an EtOH extract of the branches and leaves of Elaeocarpus sphaericus. The
structures of these compounds were determined by spectroscopic and chemical methods. Furthermore,
enantiomers of compounds 1 and 3 were separated on a chiral CD-Ph column, and their absolute
configurations were determined by TD-DFT (¼time-dependent density-functional theory) quantum-
chemical calculations of their electronic circular dichroism (ECD) spectra.
Introduction. – The genus Elaeocarpus (Elaeocarpaceae) comprises ca. 200 species
distributed over tropical and subtropical regions of East Asia, southwest Pacific, and
Oceania [1]. The earliest phytochemical investigations around 1970 have demonstrated
that indolizidine alkaloids are characteristic components of this genus [2]. Up to date,
besides minor triterpenoids [3], flavonoids [4], and phenolic glycosides [5], 28
structurally diversified alkaloids were reported from eight species of this genus [1][6].
Apart from one indole alkaloid [2f] and two pyrrolidine alkaloids [6d], most of these
alkaloids are indolizidine alkaloids which have demonstrated promising affinity for the
human d-opioid receptor [6b – 6e].
E. sphaericus is a tall tree growing in tropical areas of south China. Previous
pharmacological studies on the extracts have showed antimicrobial activity [7] and
efficacy against bronchial asthma [8]. Seven indolizidine alkaloids out of the 28
alkaloids mentioned above have been isolated from E. sphaericus [2g]. In the current
study, two new indolizidine alkaloids, (ꢀ)-3-oxoisoelaeocarpine (1) and (ꢀ)-elaeocar-
pine N-oxide (2), together with three known ones, (ꢀ)-isoelaeocarpine (3), (ꢀ)-
elaeocarpine (4), and (ꢁ)-isoelaeocarpiline (5), were isolated from an EtOH extract of
branches and leaves of E. sphaericus (see Fig. 1). The structures of the new compounds
were identified by a combination of IR, UV, MS, and NMR techniques, as well as by a
chemical correlation. Furthermore, enantiomeric mixtures of 1 and 3 were successfully
separated with a chiral CD-Ph column, and the respective absolute configurations were
determined by TD-DFT quantum-chemical calculations of their electronic circular
dichroism (ECD) spectra.
ꢀ 2011 Verlag Helvetica Chimica Acta AG, Zꢁrich

348
Helvetica Chimica Acta – Vol. 94 (2011)
Fig. 1. Structures of compounds 1 – 5 from E. sphaericus
Results and Discussion. – Compound 1 was obtained as a white amorphous powder,
and the molecular formula was determined as C16H17NO3 according to its HR-ESI-MS
pseudo-molecular-ion peak at m/z 272.1286 ([M þ H]þ; calc. 272.1281). The IR
absorption band at 1683 cmꢁ1 and the UVabsorption bands at 326 and 258 nm indicated
the presence of an aryl ketone chromophore, similar to the phenyl indolizidine
Elaeocarpus alkaloids [2d]. The 1H-NMR spectrum of compound 1 (Table), similar to
that of (ꢀ)-isoelaeocarpine (3) and (ꢀ)-elaeocarpine (4), showed a Me singlet at d(H)
2.60, three aromatic H-atom signals between d(H) 6.87 and 7.40, and a narrow doublet
at d(H) 4.77 assignable to HꢁC(7). The 13C-NMR and HSQC spectra suggested the
presence of a benzene ring (d(C) 164.2, 143.8, 136.6, 126.3, 119.4, 117.3), two C ¼ O
groups (d(C) 195.1, 176.5), three CH groups (d(C) 75.3, 55.4, 54.7), four CH2 groups
(d(C) 23.4, 29.3, 30.9, 35.8), and one Me group (d(C) 23.3). Comparison of the 1H- and
13C-NMR data with those of (ꢀ)-isoelaeocarpine (3) revealed that compound 1 has an
additional CO group at the expense of a CH2 group unit in 3.
Table. NMR Data of Compounds 1 and 2 in CD3OD. d in ppm, J in Hz.
1
2
d(C)a) d(H)b)
d(C)c) d(H)d)
CH2(1)
CH2(2)
C(3) or CH2(3) 176.5
23.4
30.9
1.99 – 2.13 (m)
2.31 – 2.44 (m)
27.4
21.7
68.2
61.5
2.64 – 2.77 (m, Ha), 2.05 – 2.12 (m, Hb)
2.26 – 2.38 (m, Ha), 2.08 – 2.16 (m, Hb)
3.40 – 3.42 (m, Ha), 3.37 – 3.38 (m, Hb)
3.60 (ddd, J ¼ 12.1, 4.7, 2.1, Hb),
3.36 – 3.41 (m, Ha)
CH2(5)
35.8
4.03 (dd, J ¼ 13.3, 5.7, Hb),
3.22 (dd, J ¼ 13.3, 10.3, Ha)
2.18 (d, J ¼ 14.6, Ha),
1.74 – 1.88 (m, Hb)
CH2(6)
29.3
27.4
2.18 – 2.24 (m, Ha),
2.85 (tdd, J ¼ 13.2, 11.2, 4.7, Hb)
HꢁC(7)
HꢁC(8)
HꢁC(9)
C(10)
C(11)
C(12)
HꢁC(13)
HꢁC(14)
HꢁC(15)
C(16)
75.3
55.4
54.7
4.77 (d, J ¼ 2.4, Hb)
77.5
49.6
73.5
4.35 – 4.46 (m, Ha)
2.47 (dd, J ¼ 11.0, 2.4, Hb)
3.83 (dt, J ¼ 11.0, 6.9, Ha)
3.33 – 3.35 (m, Hb)
3.42 – 3.45 (m, Ha)
195.1
119.4
164.2
117.3
136.6
126.3
143.8
23.3
194.5
120.6
163.3
116.7
136
125.9
142.8
22.7
6.94 (d, J ¼ 7.9)
7.40 (t, J ¼ 7.9)
6.87 (d, J ¼ 7.9)
6.86 (d, J ¼ 8.1)
7.36 (dd, J ¼ 7.7, 8.1)
6.84 (d, J ¼ 7.7)
Me(17)
2.60 (s)
2.56 (s)
a) Recorded at 100 MHz. b) Recorded at 400 MHz. c) Recorded at 125 MHz. d) Recorded at 500 MHz.

Helvetica Chimica Acta – Vol. 94 (2011)
349
In the HMBC spectrum of 1, correlations from CH2(1) to C(3), from CH2(5) and
HꢁC(9) to C(3) and C(7), from HꢁC(7) to C(9), from HꢁC(8) to C(1), and from
CH2(1) to C(9) evidenced the structure of the indolizidine moiety and located the
additional CO at C(3) correctly (Fig. 2). The trisubstituted aromatic system was
established from HMBC correlations of Me(17) and HꢁC(13) to C(15), HꢁC(14) to
C(16) and C(12), and HꢁC(15) and Me(17) to C(11). The relative configuration of
compound 1 was deduced from the vicinal coupling constants (Table) and NOESY
correlations (Fig. 3). The H-atom HꢁC(7) resonating as a narrow multiplet with small
coupling constants indicated that HꢁC(7) was in an equatorial position and assigned b-
orientation, similar to that of (ꢀ)-isoelaeocarpine (3) and unlike that of (ꢀ)-
elaeocarpine [2d]. The NOESY correlation between HꢁC(7) and HꢁC(8), and large
coupling constant (J ¼ 11.0 Hz) between HꢁC(8) and HꢁC(9) revealed that HꢁC(8) is
b-configurated and HꢁC(9) a-oriented. The specific optical rotation of 1 is about zero,
indicating alkaloid 1 is a racemate. The structure of compound 1 was finally determined
as (ꢀ)-3-oxoisoelaeocarpine.
Fig. 2. Key HMBCs (H ! C) of compounds 1 and 2
Fig. 3. Optimized 3D structures and key NOESY correlations (H > H) of compounds 1 and 2
The molecular formula of alkaloid 2 was determined as C16H19NO3 on the basis of
HR-ESI-MS data (m/z 274.1435 ([M þ H]þ; calc. 274.1438)). The IR and UV spectra
1
also indicated an Elaeocarpus alkaloid with an aryl C¼O group for 2. The H-NMR
spectrum (Table) exhibited three aromatic characteristic H-atom signals between d(H)
6.86 and 7.36, and a sharp Me signal at d(H) 2.56. These data indicated a structure
similar to that of (ꢀ)-elaeocarpine (4) for compound 2. Compared to those of (ꢀ)-
elaeocarpine (4) [6e], the 13C-NMR data (with DEPT) showed similar C-atom signals
for six aryl C-atoms at d(C) 163.3, 142.8, 136.0, 125.9, 120.6, and 116.7, one CO C-atom
at d(C) 194.5, and one Me group at d(C) 22.7. Three strongly deshielded aliphatic C-

350
Helvetica Chimica Acta – Vol. 94 (2011)
atom signals at d(C) 73.5, 68.2, and 61.5, and an additional O-atom indicated that 2 is
the N-oxide of (ꢀ)-elaeocarpine (4).
An HMBC experiment showed the same C-skeleton of 2 as that of (ꢀ)-
elaeocarpine (4). In the HMBC spectrum (Fig. 2), correlations of CH2(3) to C(1)
and C(5), CH2(1) to C(9) and C(8), HꢁC(9) to C(7), and CH2(6) to C(8) secured the
structure of the indolizidine moiety. The trisubstituted aromatic system moiety was
established from the correlations of Me(17) to C(11) and C(15), HꢁC(14) to C(16) and
C(12), and HꢁC(15) to C(11). These two moieties were connected by correlations from
HꢁC(7) to C(10).
The relative configuration of compound 2 was also deduced from the vicinal
coupling constants (Table), a NOESY experiment (Fig. 3), and previous findings [2d].
A broad multiplet at d(H) 4.35 – 4.46 for HꢁC(7) was similar to the signal observed for
(ꢀ)-elaeocarpine (4), which suggested a-orientation for HꢁC(7) and b-orientation for
HꢁC(8). NOESY Correlation between HꢁC(7) and HꢁC(9) indicated an a-
orientation for HꢁC(9). To fully confirm the above conclusion, a chemical trans-
formation of (ꢀ)-elaeocarpine (4) to its N-oxide 2 was accomplished by m-
chloroperbenzoic acid (m-CPBA) oxidation (see Exper. Part). Finally, lack of optical
activity suggested the structure of alkaloid 2 as (ꢀ)-elaeocarpine N-oxide.
The racemic mixture of 1 was separated on a Shiseido chiral CD-Ph column eluting
with 70% aqueous MeOH at a flow rate of 0.8 ml/min (Fig. 4). Two enantiomers, 1a
and 1b, were prepared and their ECD spectra were recorded. Simultaneously, a time-
dependent density-functional theory (TD-DFT) computational-chemistry study for the
two enantiomers was carried out following similar procedures we previously reported
[9]. Briefly, conformation searching at MMFF 94 molecular-mechanics force field by
using the Omega 2.1 program [10] showed only one dominating conformer for 1a and
1b, respectively. The resulting conformations were re-optimized using DFT at the
B3LYP/6-311 þþ G (2d, 2p) level using GAUSSIAN 09 software package [11]. The
B3LYP/6-311 þþ G (2d, 2p) harmonic vibrational frequencies were further calculated
to confirm their stability. ATD-DFT calculation of the ECD data was carried out at the
B3LYP/aug-cc-pVDZ level. To obtain the ECD spectrum, the Gaussian 09 calculation
results were processed with the Gausssum 2.2 software package (s ¼ 0.6) [12]. The
calculated ECD spectrum for (7S,8R,9R)-3-oxoisoelaeocarpine showed first negative
(348 nm) and second positive (303 nm) Cotton effects, similar to those of 1a (positive
at 353 and negative at 318 nm), which allowed the determination of the absolute
configuration of 1a as (7S,8R,9R). Similarly, the structure of 1b was determined as
(7R,8S,9S)-3-oxoisoelaeocarpine. (ꢀ)-Isoelaeocarpine (3) was also separated on the
chiral CD-Ph column eluted with 70% aqueous MeOH at a flow rate of 0.5 ml/min to
give 3a and 3b (Fig. 4). Following the same protocols as compound 1, 3a and 3b were
determined as (7S,8R,9R)- and (7R,8S,9S)-isoelaeocarpine, respectively (Fig. 5).
Experimental Part
General. Column chromatography (CC): commercial silica gel for TLC (SiO2; Qing Dao Hai Yang
Chemical Group Co., Ltd); C18 column (Phenomenex 00G-4324-N0; 10 mm, 10 mm (i.d.) ꢂ 25 cm);
MCI-GEL (MITSUBISHI, Japan); and chiral CD-Ph column (Shiseido, Japan). HPLC: Agilent1100,
American. TLC: precoated silica gel plates (HSGF 254, Yan Tai Jiang You Silica Gel Development Co.,

Helvetica Chimica Acta – Vol. 94 (2011)
351
Fig. 4. HPLC Spectrum for optical resolution of compound 1 (top) and 3 (bottom). Compound 1:
capacity factors, K1 ¼ 4.84, K2 ¼ 5.67; selectivity factor, a ¼ 1.17; resolution, R ¼ 2.27. Compound 3:
capacity factors, K1 ¼ 6.61, K2 ¼ 7.85; selectivity factor, a ¼ 1.19; resolution, R ¼ 2.34).
Ltd). Optical rotations: JASCO P-1010 polarimeter. ECD Spectra: JASCO J-815 CD spectrometer. UV
Spectra: BECKMAN DU-600 spectrometer; lmax (log e) in nm. IR Spectra: Bruker VECTOR-22
spectrophotometer; KBr pellets; in cmꢁ1. NMR Spectra: Varian Unity-INOVA-400/54 spectrometers; d
in ppm rel. to Me4Si as internal standard, J in Hz. ESI-MS: Micromass Quattro triple-quadrupole mass
spectrometer equipped with an ESI source (Micromass, Manchester, UK); in m/z (rel. %).
Plant Material. The branches and leaves of Elaeocarpus sphaericus were collected from Hainan
Province, P. R. China, in June 2008 and identified by Prof. Shi-Man Huang of Hainan University. A
voucher specimen has been deposited with the Institute of Modern Chinese Medicine, Zhejiang
University (accession No. ES2008).
Extraction and Isolation. Branches and leaves (10 kg) of Elaeocarpus sphaericus were dried,
powdered, and then extracted with 95% EtOH at r.t. After removal of the solvent under reduced

352
Helvetica Chimica Acta – Vol. 94 (2011)
Fig. 5. Experimental CD spectra (top) and TD-DFT-calculated CD spectra (bottom) for enantiomers of 1
(dashed lines) and 3 (solid lines)
pressure, the crude extract (283 g) was dissolved in 1 l H2O and adjusted to pH 3 with 2m HCl, followed
by extraction with AcOEt. The aq. layer was then adjusted to pH 10 with Na2CO3, and extraction with
AcOEt gave a crude alkaloid fraction (45.5 g). The crude alkaloid fraction was then subjected to a MCI-
GEL column (H2O/EtOH 100 :0 – 95 :5) to give four fractions, Frs. A – D. Fr. A was further chromato-
graphed over SiO2 column (CHCl3/MeOH/Et2NH 50 :1:0.1) to give Frs. A1 and A2. Fr. A2 was
repurified through a C18 reversed-phase (RP) column (EtOH/H2O 1:9) to afford compound 2 (5 mg).
Fr. B was chromatographed over a SiO2 column (petroleum ether (PE)/AcOEt 4 :1) to yield 4 (100 mg),

Helvetica Chimica Acta – Vol. 94 (2011)
353
and Fr. B1 and Fr. B2. Fr. B1 was further purified by a C18 RP column (60% aq. MeOH) to give alkaloid
1 (5 mg). Fr. B2 was further chromatographed through a SiO2 column (PE/AcOEt/Et2NH 15 :1:0.1),
followed by HPLC purifications to give 3 (MeOH/H2O 90 :10; 14.3 mg) and 5 (MeOH/H2O 70 :30;
4.4 mg).
Preparation of (ꢀ)-Elaeocarpine N-Oxide (2) from (ꢀ)-Elaeocarpine (4). (ꢀ)-Elaeocarpine (4;
14.8 mg) was dissolved in 8 ml of CHCl3, and then 123.7 mg m-CPBA (m-chloroperbenzoic acid) in 5 ml
of CHCl3 was added in dropwise. The mixture was stirred for 1 h at r.t. After workup, the crude alkaloid
was subjected to CC (SiO2; AcOEt/MeOH 5 :1 (0.1% (v/v) Et2NH)) to give compound 2 (5 mg).
(ꢀ)-3-Oxoisoelaeocarpine (¼(6aS,12aR,12bR)-1,2,6,6a,12a,12b-Hexahydro-11-methyl-5H-chrome-
no[2,3-g]indolizine-3,12-dione; 1). White amorphous powder. UV (MeOH): 217 (4.25), 258 (3.90),
326 (3.51). IR (KBr): 2968, 2922, 2881, 1683, 1599, 1472, 1422, 793. 1H- and 13C-NMR: see the Table. ESI-
MS: 272 ([M þ H]þ). HR-ESI-MS (pos.): 272.1286 ([M þ H]þ, C16H18NOþ3 ; calc. 272.1281).
(ꢀ)-Elaeocarpine N-Oxide (¼(6aR,12aR,12bR)-1,2,3,5,6,6a,12a,12b-Octahydro-11-methyl-12H-
chromeno[2,3-g]indolizin-12-one 4-Oxide; 2). White amorphous powder. UV (MeOH): 217 (4.01),
1
254 (3.66), 322 (3.26). IR (KBr): 3442, 2972, 2820, 1688, 1602, 1470, 792. H- and 13C-NMR: see the
Table. ESI-MS: 274 ([M þ H]þ). HR-ESI-MS (pos.): 274.1435 ([M þ H]þ, C16H20NOþ3 ; calc. 274.1438).
This work was financially supported by the National Natural Science Foundation of China
(30801429), the Zhejiang Provincial Natural Science Foundation (Y2080602), and Science Foundation of
Chinese University (2009QNA7028).
REFERENCES
[1] An Editorial Committee of Chinese Academy of Sciences, in ꢂFlora of Chinaꢃ (ꢂZhongguo Zhiwu
Zhiꢃ), Science Press, Beijing, 1989, Vol. 49(1), pp. 1 – 7.
[2] a) S. R. Johns, J. A. Lamberton, A. A. Sioumis, J. A. Wunderlich, J. Chem. Soc., Chem. Commun.
1968, 290; b) S. R. Johns, J. A. Lamberton, A. A. Sioumis, J. Chem. Soc., Chem. Commun. 1968,
1324; c) S. R. Johns, J. A. Lamberton, A. A. Sioumis, H. Suares, R. I. Willing, J. Chem. Soc., Chem.
Commun. 1970, 804; d) S. R. Johns, J. A. Lamberton, A. A. Sioumis, R. I. Willing, Aust. J. Chem.
1969, 22, 775; e) S. R. Johns, J. A. Lamberton, A. A. Sioumis, Aust. J. Chem. 1969, 22, 793; f) S. R.
Johns, J. A. Lamberton, A. A. Sioumis, Aust. J. Chem. 1969, 22, 801; g) S. R. Johns, J. A. Lamberton,
A. A. Sioumis, H. Suares, R. I. Willing, Aust. J. Chem. 1971, 24, 1679; h) N. K. Hart, S. R. Johns, J. A.
Lamberton, Aust. J. Chem. 1972, 25, 817.
[3] D. Meng, S. Qiang, L. Lou, W. Zhao, Planta Med. 2008, 74, 1741.
[4] S. Dasgupta, S. C. Dutta, A. B. Ray, Ind. J. Chem., Sect. B 1977, 15, 197.
[5] A. Ito, H.-B. Chai, D. Lee, L. B. S. Kardono, S. Riswan, N. R. Farnsworth, G. A. Cordell, J. M.
Pezzuto, A. D. Kinghorn, Phytochemistry 2002, 61, 171.
[6] a) A. B. Ray, L. Chand, V. B. Pandey, Phytochemistry 1979, 18, 700; b) A. R. Carroll, G. Arumugan,
R. J. Quinn, J. Redburn, G. Guymer, P. Grimshaw, J. Org. Chem. 2005, 70, 1889; c) P. L. Katavic,
D. A. Venables, P. I. Forster, G. Guymer, A. R. Carroll, J. Nat. Prod. 2006, 69, 1295; d) P. L. Katavic,
D. A. Venables, T. Rali, A. R. Carroll, J. Nat. Prod. 2007, 70, 866; e) P. L. Katavic, D. A. Venables, T.
Rali, A. R. Carroll, J. Nat. Prod. 2007, 70, 872.
[7] R. K. Singh, G. Nath, Phytother. Res. 1999, 13, 448.
[8] R. K. Singh, S. B. Acharya, S. K. Bhattacharya, Phytother. Res. 2000, 14, 36.
[9] L.-S. Gan, Y.-L. Zheng, J.-X. Mo, X. Liu, X.-H. Li, C.-X. Zhou, J. Nat. Prod. 2009, 72, 1497.
[10] J. Bostrçm, J. R. Greenwood, J. Gottfries, J. Mol. Graph. Mod. 2003, 21, 449.
[11] Gaussian 09, Revision A.1, M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb,
J. R. Cheeseman, G. Scalmani, V. Barone, B. Mennucci, G. A. Petersson, H. Nakatsuji, M. Caricato,
X. Li, H. P. Hratchian, A. F. Izmaylov, J. Bloino, G. Zheng, J. L. Sonnenberg, M. Hada, M. Ehara, K.
Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven,
J. A. Montgomery Jr., J. E. Peralta, F. Ogliaro, M. Bearpark, J. J. Heyd, E. Brothers, K. N. Kudin,
V. N. Staroverov, R. Kobayashi, J. Normand, K. Raghavachari, A. Rendell, J. C. Burant, S. S.
Iyengar, J. Tomasi, M. Cossi, N. Rega, J. M. Millam, M. Klene, J. E. Knox, J. B. Cross, V. Bakken, C.

354
Helvetica Chimica Acta – Vol. 94 (2011)
Adamo, J. Jaramillo, R. Gomperts, R. E. Stratmann, O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli,
J. W. Ochterski, R. L. Martin, K. Morokuma, V. G. Zakrzewski, G. A. Voth, P. Salvador, J. J.
Dannenberg, S. Dapprich, A. D. Daniels, ꢄ. Farkas, J. B. Foresman, J. V. Ortiz, J. Cioslowski, D. J.
Fox, Gaussian, Inc., Wallingford CT, 2009.
[12] N. M. OꢃBoyle, A. L. Tenderholt, K. M. Langner, J. Comp. Chem. 2008, 29, 839.
Received May 19, 2010