Alkaloids from Oxytropis ochrocephala and antiproliferative activity of sophoridine derivatives against cancer cell lines

Article abstract of DOI:10.1016/j.bmcl.2015.09.010

Ten alkaloids (1-10), with sophoridine (1) as the most abundant component, were obtained from the whole plants of Oxytropis ochrocephala Bunge. Furthermore, eight new sophoridine derivatives (11-16, 20, 21), with modification on the C-14 position of 1 wer

Full text of DOI:10.1016/j.bmcl.2015.09.010

Accepted Manuscript
Alkaloids from Oxytropis ochrocephala and Antiproliferative Activity of Soph-
oridine Derivatives Against Cancer Cell Lines
Cheng-jian Tan, Yu Zhao, Masuo Goto, Kan-Yen Hsieh, Xiao-ming Yang,
Susan L. Morris-Natschke, Li-na Liu, Bao-yu Zhao, Kuo-Hsiung Lee
PII:
S0960-894X(15)30044-5
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.09.010
BMCL 23091
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
28 July 2015
31 August 2015
4 September 2015
Please cite this article as: Tan, C-j., Zhao, Y., Goto, M., Hsieh, K-Y., Yang, X-m., Morris-Natschke, S.L., Liu, L-
n., Zhao, B-y., Lee, K-H., Alkaloids from Oxytropis ochrocephala and Antiproliferative Activity of Sophoridine
Derivatives Against Cancer Cell Lines, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/
10.1016/j.bmcl.2015.09.010
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Alkaloids from Oxytropis ochrocephala and Antiproliferative
Activity of Sophoridine Derivatives Against Cancer Cell Lines
Cheng-jian Tan a,b,॥,*, Yu Zhao a,॥, Masuo Goto a, Kan-Yen Hsieh a,c, Xiao-ming Yang a,
Susan L. Morris-Natschke a, Li-na Liud, Bao-yu Zhao e,*, and Kuo-Hsiung Lee a,f
*
aNatural Products Research Laboratories, UNC Eshelman School of Pharmacy, University of North
Carolina, Chapel Hill, NC 27599, United States
bCollege of Chemistry and Environment, Guizhou Minzu University, Guiyang 550025, People’s
Republic of China
cGraduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical University,
Kaohsiung, Taiwan
dDepartment of Medical Biotechnology, Guiyang Medical University, Guiyang 550004, People’s
Republic of China
eCollege of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, People’s
Republic of China
fChinese Medicine Research and Development Center, China Medical University and Hospital,
Taichung 40447, Taiwan

Abstract: Ten alkaloids (1−10), with sophoridine (1) as the most abundant component, were obtained
from the whole plants of Oxytropis ochrocephala Bunge. Furthermore, eight new sophoridine
derivatives (11−16, 20, 21), with modification on the C-14 position of 1 were synthesized. All
compounds (1−16, 20, 21) were evaluated for antiproliferative activity against five human tumor cell
lines. Among them, the newly synthesized derivative 20 exhibited the best inhibitory activity against
the tested cell lines. Its activity was increased by more than fourfold as compared with parent
compound 1.
Keywords: Oxytropis ochrocephala; Poisonous plant; Antiproliferative; Sophoridine derivatives
॥
These authors contributed equally to this work
* Corresponding authors. Tel./fax: +86 0851 3610313 (C.-J.T.); tel.: +1 919 9620066; fax: +1 919 966
3893 (K.-H.L.); Tel./fax: +86 029 87092429 (B.-Y.Z.)
E-mail addresses: tcj1229@163.com (C.-J. Tan), khlee@unc.edu, (K.-H. Lee),
zhaobaoyu2005@163.com (B.-Y. Zhao)

Locoweed is a common name for specific plants poisonous to livestock and belonging to
the genera Oxytropis and Astragalus, which are widely distributed in Eastern and Central
Asia, Western North and South America, and Australia1-2. Livestock ingesting locoweed
can develop a chronic neurological disease, characterized by a staggering gait, and
muscular incoordination, giving rise to the vivid name ‘locoweed’ for these poisonous
plants. 2-3. Today, locoweed is a main threat to rangelands worldwide1. In China,
locoweeds cover over 11 m ha, amounting to 3.3% of the total western grassland2.
The plant Oxytropis ochrocephala Bunge is one of the common locoweeds found on
the western grassland in China2, 4. It spreads 3 m ha across Qinghai, Ningxia, Gansu and
Xizang provinces of China. In recent years, due to overgrazing, salinization and damage
from drought and rodents, this poisonous plant has grown very so rapidly and even
become the dominant species in some places2. On the other hand, if the chemical
constituents in this plant had a therapeutic use, harm to the grassland could be mitigated.
To date, some efforts have been made to study locoism and poisonous alkaloid
constituents of O. ochrocephala. As a result, five alkaloids, including one indolizidine
and four quinolizidine alkaloids, were isolated previously from this plant5. Quinolizidine
alkaloids exhibit broad pharmacological effects, such as antibacterial, antipyrotic,
antipyretic, antiarrhythmic, antiasthmatic, antiulcerative, antivirus and antineoplastic
properties.6-7 However, the antiproliferative activities of the quinolizidine alkaloids
isolated from O. ochrocephala remain unclear.

As part of our ongoing research program on the identification of alkaloids from
Oxytropis ochrocephala, ten known alkaloids (Figure 1), sophoridine (1), isosophoridine
(2), matrine (3), sophoramine (4), 7,11-dehydromatrine (5), sophocarpine (6), lupanine
8-15
(7), (+)-9α-hydroxymatrine (8), (-)-9α-hydroxysophocarpine (9) and swainonine (10)
were obtained from the whole plants of Oxytropis ochrocephala. Among them,
compounds 1-9 are quinolizidine alkaloids, while compound 10 is an indolizidine
alkaloid. Moreover, in our study, sophoridine (1, Figure 1), obtained in 5 gram quantity,
provided an ideal starting material for further modifications. Compound 1 is one of three
main chemical ingredients of Fufang Kushen injection, which was approved by the
Chinese FDA (CFDA) in 1995 as an anticancer drug for treating non-small cell lung
carcinoma, liver cancer, and gastric cancer in combination with other anticancer
drugs.16-19 While some synthetic derivatives of 1 with an open D ring have exhibited
good antiproliferative activity 20-21, no research on the activity as well as structure-activity
relationship (SAR) correlations of derivatives of 1 with four intact rings has been
reported. In the present study, eight new derivatives of 1 with various substituents on the
C-14 position were synthesized.

Figure 1. The structures of alkaloids from Oxytropis ochrocephala
With 1 as starting material, eight new sophoridine derivatives were synthesized. As
shown in Scheme 1, aldol reaction of various aromatic aldehydes with 1 produced
compounds 11−16 (Scheme 1). Addition of a benzyl or methoxymethyl group on OH-4
of 17 produced 18 and 19, respectively (Scheme 2). Compounds 18 and 19 were further
reacted with 1 to yield 20 and 21, respectively.

Scheme 1. Synthesis of sophoridine derivations 11−16. (a) Aromatic aldehyde, NaH, THF, reflux, 6 h
Scheme 2. Synthesis of sophoridine derivations 20 and 21. (b) BnBr, 10% K2CO3, rt; (c) MomCl,
DIPEA, 0 C to rt
The anti-proliferative activities of the natural alkaloids (1-10) and newly synthesized
derivatives (11−16, 20, 21) against five tumor cell lines (A549, KB, KB-VIN,
MDA-MB-231, MCF7) were evaluated by the sulforhodamine B (SRB) colorimetric
assay. Paclitaxel was used as the positive control. The results are summarized in Table 1.
Table 1. Antiproliferative Activity of Natural (1–10) and Synthetic (11–16, 20, 21) Alkaloids

Compd
IC50 (µM)
KB-VIN
R1
A549
KB
MDA-MB-2 MCF7
31
>80
>40
>40
>80
>40
>40
>80
>40
>40
>80
>40
>40
>80
N/Da)
1
2−10
11
N/D
42 ± 0.94
74 ± 1.9
58 ± 2.1
74 ± 4.0
72 ± 1.6
>80
45 ± 7.8
67 ± 1.7
54 ± 21
>80
12
13
>80
71 ± 0.61
>40
>80
68 ± 4.7
>40
>80
14
15
16
20
>40
>40
N/D
>40
>40
>40
>40
N/D
17.6 ± 0.31
20.7 ± 0.56
21.6 ± 2.011
21.9 ± 0.54
20.5 ± 1.49
>40
>40
>40
>40
N/D
21
PXL (nM)b
0.03 ± 0.03
0.04 ± 0.03
2600 ± 160
18 ± 3.2
3.9 ± 1.3
a N/D: not determined. b: IC50 of paclitaxel (PXL) is represented in nM.

At 40 µM, none of the naturally occurring indolizidine and quinolizidine alkaloids
(1−10) from O. ochrocephala exhibited inhibitory activity against the five tested human
tumor cell lines. Compound 1 was inactive even at 80 µM. As compared with 1, neither a
conformation change at C-6 (2) or C-5 (3) nor the presence of unsaturation in the D or C
ring (4−6) resulted in significant antiproliferative activity. However, among the eight
newly synthesized derivatives with a substituted phenylmethylene group on C-14, some
beneficial effects on activity were seen. Regarding the substitution pattern on the phenyl
ring, 2-methyl-4-methoxy substituted 13 was less active than 3-methoxy-4-methyl
substituted 12. Notably, when the 3,4-substituted pattern was retained, with an ethoxy
group at position-3 and a benzyloxy group at position-4, the resulting compound, 20,
showed significantly increased antiproliferative activity, with IC50 values around 20 µM
against all five cancer cell lines, fourfold higher than those of 1. Interestingly, a change
from a benzyloxy (20) to methoxymethoxy (21) group on the same position of the phenyl
ring did not lead to increased antiproliferative activity. Thus, in this limited data set, the
benzyloxy moiety is crucial for activity. Notably, compound 20 was equipotent against
KB and MDR-subline KB-VIN, suggesting that this compounds is not a P-gp substrate.
Finally, compound 16 with a dimethylamino group at position-4 of the phenyl ring was
not active indicating that this particular basic group is not good for antiproliferative
activity.
Ten natural alkaloids from O. ochrocephala and eight synthetic sophoridine

analogues were evaluated for antiproliferative activity. Among them, synthetic derivative
20 exhibited the best inhibitory activity against five human tumor cell lines. The activity
results and SAR correlations of this compound series were reported. Our research
produced fundamental information for further study and exploitation of the poisonous
plant O. ochrocephala.
Acknowledgments
The work was financially supported by the Special Scientific Research Fund of
Agriculture Public Welfare Industry (Grant No. 201203062), the Chinese National
Natural Science Foundation (31360084), the Natural Science Foundation of Guizhou
Province (20102273), China Scholarship Council (CSC) to Cheng-jian Tan, and partial
support from NIH Grant CA177584 from the National Cancer Institute awarded to
Kuo-Hsiung Lee. K.Y. Hsieh was supported by the Teaching and Learning Excellence
Program from Kaohsiung Medical University
Supplementary Material
Supplementary data (experimental details and compound characterization for all
synthesized compounds) associated with this article can be found, in the online version,
at

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