U.S. patent application number 15/142373 was filed with the patent office on 2017-11-02 for method of isolating phenanthroindolizidine alkaloids from tylophora atrofolliculata with hif-1 inhibitory activity, compositions comprising them and their use.
The applicant listed for this patent is Macau University of Science and Technology. Invention is credited to Cheng-Yu Chen, Zhi-Hong Jiang, Jing-Rong Wang, Guo-Yuan Zhu.
Application Number | 20170312266 15/142373 |
Document ID | / |
Family ID | 59981303 |
Filed Date | 2017-11-02 |
United States Patent
Application |
20170312266 |
Kind Code |
A1 |
Jiang; Zhi-Hong ; et
al. |
November 2, 2017 |
METHOD OF ISOLATING PHENANTHROINDOLIZIDINE ALKALOIDS FROM TYLOPHORA
ATROFOLLICULATA WITH HIF-1 INHIBITORY ACTIVITY, COMPOSITIONS
COMPRISING THEM AND THEIR USE
Abstract
A method of isolating at least one phenanthroindolizidine
alkaloid, in particular with HIF-1 inhibitory activity, from
Tylophora atrofolliculata is used to isolate and obtain for example
about 22 phenanthroindolizidine alkaloids, including at least 11
new phenanthroindolizidine alkaloids which have not been previously
isolated. Experimental tests confirmed an exceptional HIF-1
inhibitory activity of the phenanthroindolizidine alkaloids
isolated. A pharmaceutical composition includes at least one
phenanthroindolizidine alkaloid and at least one pharmaceutical
tolerable excipient. A method of treating a subject suffering from
cancer includes administering at least one phenanthroindolizidine
alkaloid isolated from Tylophora atrofolliculata. A method of
treating a subject suffering from cancer includes administering at
least one phenanthroindolizidine alkaloid to the subject.
Inventors: |
Jiang; Zhi-Hong; (Taipa,
MO) ; Wang; Jing-Rong; (Taipa, MO) ; Chen;
Cheng-Yu; (Taipa, MO) ; Zhu; Guo-Yuan; (Taipa,
MO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Macau University of Science and Technology |
Taipa |
|
MO |
|
|
Family ID: |
59981303 |
Appl. No.: |
15/142373 |
Filed: |
April 29, 2016 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 36/24 20130101;
A61K 31/4745 20130101 |
International
Class: |
A61K 31/4745 20060101
A61K031/4745; A61K 36/24 20060101 A61K036/24 |
Claims
1. A method of isolating at least one phenanthroindolizidine
alkaloid from Tylophora atrofolliculata comprising steps of: (i)
subjecting Tylophora atrofolliculata plant material to a solvent
extraction with an extraction solvent for obtaining a crude
extract, wherein the extraction solvent comprises an aliphatic
alcohol; (ii) contacting the crude extract with a first and a
second separation solvent for obtaining a first and a second layer,
wherein the first separation solvent comprises water and the second
separation solvent comprises an ester; (iii) contacting the first
layer with a third separation solvent comprising a halogenated
hydrocarbon for forming a third layer; (iv) subjecting the third
layer to at least a first chromatographic separation step.
2. The method of claim 1, wherein the phenanthroindolizidine
alkaloid is selected from a compound: having Formula (I):
##STR00039## wherein R.sub.1 is OH; having Formula (I) given above,
wherein R.sub.1 is OCH.sub.3; having Formula (II): ##STR00040##
having Formula (III): ##STR00041## wherein R.sub.1 is OH, R.sub.2
is OCH.sub.3, R.sub.3 is OH and R.sub.4 is H; having Formula (III)
given above, wherein R.sub.1 is OH, R.sub.2 is OCH.sub.3, R.sub.3
is OH and R.sub.4 is .alpha.-OH; having Formula (III) given above,
wherein R.sub.1 is H, R.sub.2 is OH, R.sub.3 is OH and R.sub.4 is
.alpha.-OH; having Formula (III) given above, wherein R.sub.1 is H,
R.sub.2 is OCH.sub.3, R.sub.3 is OH and R.sub.4 is .alpha.-OH;
having Formula (III) given above, wherein R.sub.1 is H, R.sub.2 is
OH, R.sub.3 is OCH.sub.3 and R.sub.4 is .alpha.-OH; having Formula
(III) given above, wherein R.sub.1 is H, R.sub.2 is OCH.sub.3,
R.sub.3 is OCH.sub.3 and R.sub.4 is .beta.-OH; having Formula (III)
given above, wherein R.sub.1 is OCH.sub.3, R.sub.2 is OCH.sub.3,
R.sub.3 is OCH.sub.3 and R.sub.4 is H; having Formula (III) given
above, wherein R.sub.1 is H, R.sub.2 is OCH.sub.3, R.sub.3 is
OCH.sub.3 and R.sub.4 is H; having Formula (III) given above,
wherein R.sub.1 is H, R.sub.2 is OCH.sub.3, R.sub.3 is OCH.sub.3
and R.sub.4 is .alpha.-OH; having Formula (IV): ##STR00042##
wherein R.sub.1 is H, R.sub.2 is OH, R.sub.3 is OH and R.sub.4 is
.beta.-OH; having Formula (IV) given above, wherein R.sub.1 is OH,
R.sub.2 is OCH.sub.3, R.sub.3 is OCH.sub.3 and R.sub.4 is
.beta.-OH; having Formula (IV) given above, wherein R.sub.1 is
OCH.sub.3, R.sub.2 is OCH.sub.3, R.sub.3 is OCH.sub.3 and R.sub.4
is .alpha.-OH; having Formula (IV) given above, wherein R.sub.1 is
OCH.sub.3, R.sub.2 is OCH.sub.3, R.sub.3 is OCH.sub.3 and R.sub.4
is H; having Formula (IV) given above, wherein R.sub.1 is H,
R.sub.2 is OCH.sub.3, R.sub.3 is OCH.sub.3 and R.sub.4 is H; having
Formula (V): ##STR00043## having Formula (VI): ##STR00044## having
Formula (VII): ##STR00045## having Formula (VIII): ##STR00046## or
a compound having Formula (IX): ##STR00047##
3. The method of claim 2, wherein the phenanthroindolizidine
alkaloid is selected from a compound: having Formula (I), wherein
R.sub.1 is OH; having Formula (II); having Formula (III), wherein
R.sub.1 is OH, R.sub.2 is OCH.sub.3, R.sub.3 is OH and R.sub.4 is
H; having Formula (III), wherein R.sub.1 is OH, R.sub.2 is
OCH.sub.3, R.sub.3 is OH and R.sub.4 is .alpha.-OH; having Formula
(IV), wherein R.sub.1 is H, R.sub.2 is OH, R.sub.3 is OH and
R.sub.4 is .beta.-OH; having Formula (IV), wherein R.sub.1 is OH,
R.sub.2 is OCH.sub.3, R.sub.3 is OCH.sub.3 and R.sub.4 is
.beta.-OH; having Formula (IV), wherein R.sub.1 is OCH.sub.3,
R.sub.2 is OCH.sub.3, R.sub.3 is OCH.sub.3 and R.sub.4 is
.alpha.-OH; having Formula (IV), wherein R.sub.1 is OCH.sub.3,
R.sub.2 is OCH.sub.3, R.sub.3 is OCH.sub.3 and R.sub.4 is H; having
Formula (IV), wherein R.sub.1 is H, R.sub.2 is OCH.sub.3, R.sub.3
is OCH.sub.3 and R.sub.4 is H; having Formula (V), or a compound
having Formula (VI).
4. The method of claim 1, wherein the Tylophora atrofolliculata
plant material comprises the whole plant.
5. The method of claim 1, wherein the extraction solvent in step
(i) comprises methanol and wherein the solvent extraction is
carried out at a temperature of at least 50.degree. C.
6. The method of claim 1, wherein the Tylophora atrofolliculata
plant material is refluxed with the extraction solvent and wherein
the solvent extraction is carried out three times with the
Tylophora atrofolliculata plant material with an amount of
Tylophora atrofolliculata plant material in relation to the total
amount of the extraction solvent of between 20 mg/ml and 60
mg/ml.
7. The method of claim 1, wherein contacting the crude extract with
the first and the second separation solvent in step (ii) is carried
out by: a) suspending the crude extract in the first separation
solvent; b) adjusting the pH of the suspension to less than 3 by
adding an inorganic acid; c) adding the second separation solvent
to the suspension obtained in step b) for forming the first and the
second layer and separating the first layer; and d) adjusting the
pH of the separated first layer to at least pH 8 by adding a base,
and wherein the second separation solvent comprises ethyl acetate,
the first separation solvent essentially consists of water and the
first separation solvent is mainly comprised in the first layer and
the second separation solvent is mainly comprised in the second
layer.
8. The method of claim 1, wherein the third separation solvent
comprises chloroform.
9. The method of claim 1, wherein the first chromatographic
separation step in step (iv) is carried out with a classical column
chromatography with a styrene-divinylbenzene polymer resin as
stationary phase and wherein methanol/water/diethyl amine and
subsequently acetone/methanol/diethyl amine are used as elution
solvents.
10. The method of claim 9, wherein at least a second
chromatographic separation step is carried out in step (iv) which
second and optionally further chromatographic separation step is
independently selected from the group consisting of classical
column chromatography or a high-performance liquid chromatography,
wherein the stationary phase is selected from unmodified silica gel
or a C18 reverse phase).
11. (canceled)
12. A method of treating a subject suffering from breast cancer
comprising administering to the subject an effective amount of at
least one phenanthroindolizidine alkaloid isolated from Tylophora
atrofolliculata, wherein the phenanthroindolizidine alkaloid is
selected from the group consisting of a phenanthroindolizidine
alkaloid: having Formula (III): ##STR00048## wherein R.sub.1 is OH,
R.sub.2 is OCH.sub.3, R.sub.3 is OH and R.sub.4 is H; having
Formula (III) given above, wherein R.sub.1 is OH, R.sub.2 is
OCH.sub.3, R.sub.3 is OH and R.sub.4 is .alpha.-OH; having Formula
(III) given above, wherein R.sub.1 is H, R.sub.2 is OH, R.sub.3 is
OH and R.sub.4 is .beta.-OH; having Formula (III) given above,
wherein R.sub.1 is H, R.sub.2 is OCH.sub.3, R.sub.3 is OH and
R.sub.4 is .beta.-OH; having Formula (III) given above, wherein
R.sub.1 is H, R.sub.2 is OH, R.sub.3 is OCH.sub.3 and R.sub.4 is
.beta.-OH; having Formula (III) given above, wherein R.sub.1 is H,
R.sub.2 is OCH.sub.3, R.sub.3 is OCH.sub.3 and R.sub.4 is
.beta.-OH; having Formula (III) given above, wherein R.sub.1 is
OCH.sub.3, R.sub.2 is OCH.sub.3, R.sub.3 is OCH.sub.3 and R.sub.4
is H; having Formula (III) given above, wherein R.sub.1 is H,
R.sub.2 is OCH.sub.3, R.sub.3 is OCH.sub.3 and R.sub.4 is H; having
Formula (III) given above, wherein R.sub.1 is H, R.sub.2 is
OCH.sub.3, R.sub.3 is OCH.sub.3 and R.sub.4 is .alpha.-OH; having
Formula (IV): ##STR00049## wherein R.sub.1 is H, R.sub.2 is OH,
R.sub.3 is OH and R.sub.4 is .beta.-OH; having Formula (IV) given
above, wherein R.sub.1 is OH, R.sub.2 is OCH.sub.3, R.sub.3 is
OCH.sub.3 and R.sub.4 is .beta.-OH; having Formula (IV) given
above, wherein R.sub.1 is H, R.sub.2 is OCH.sub.3, R.sub.3 is
OCH.sub.3 and R.sub.4 is H; having Formula (V): ##STR00050## having
Formula (VII): ##STR00051## having Formula (VIII): ##STR00052##
13. (canceled)
14. A method of treating a subject suffering from breast cancer
comprising administering an effective amount of a
phenanthroindolizidine alkaloid selected from the group consisting
of the following formulas to the subject: Formula (III):
##STR00053## wherein R.sub.1 is OH, R.sub.2 is OCH.sub.3, R.sub.3
is OH and R.sub.4 is H; Formula (III) given above, wherein R.sub.1
is OH, R.sub.2 is OCH.sub.3, R.sub.3 is OH and R.sub.4 is
.alpha.-OH; Formula (IV): ##STR00054## wherein R.sub.1 is H,
R.sub.2 is OH, R.sub.3 is OH and R.sub.4 is .beta.-OH; Formula (IV)
given above, wherein R.sub.1 is OH, R.sub.2 is OCH.sub.3, R.sub.3
is OCH.sub.3 and R.sub.4 is .beta.-OH; Formula (IV) given above,
wherein R.sub.1 is H, R.sub.2 is OCH.sub.3, R.sub.3 is OCH.sub.3
and R.sub.4 is H; and Formula (V): ##STR00055##
15. The method of claim 14, wherein the compound has a Formula
selected from the group consisting of the following formulas: a
compound having Formula (III), wherein R.sub.1 is OH, R.sub.2 is
OCH.sub.3, R.sub.3 is OH and R.sub.4 is H; a compound having
Formula (III), wherein R.sub.1 is OH, R.sub.2 is OCH.sub.3, R.sub.3
is OH and R.sub.4 is .alpha.-OH; a compound having Formula (IV),
wherein R.sub.1 is H, R.sub.2 is OH, R.sub.3 is OH and R.sub.4 is
.beta.-OH; and a compound having Formula (V).
16. (canceled)
Description
[0001] The present invention provides a method of isolating at
least one phenanthroindolizidine alkaloid in particular with HIF-1
inhibitory activity from Tylophora atrofolliculata. The present
invention further refers to a composition, in particular a
pharmaceutical composition, comprising the at least one
phenanthroindolizidine alkaloid and at least one excipient. Still
further, the present invention refers to a method of treating a
subject suffering from cancer by administering at least one
phenanthroindolizidine alkaloid isolated from Tylophora
atrofolliculata. In accordance with the invention is also a method
of treating a subject suffering from cancer by administering at
least one phenanthroindolizidine alkaloid of certain chemical
formula to the subject.
BACKGROUND OF THE INVENTION
[0002] Rapid tumor growth is accompanied by an unbalance between
oxygen supply and consumption resulting in low oxygen levels and
thus the existence of hypoxic regions. Tumor hypoxia is not only
the major problem for radiotherapy treatment failure and anticancer
drug resistance, but also an indicator for advanced disease stages,
disease relapse and poor prognosis (J. M. Brown, Cancer Res., 1999,
59, 5863-5870). The transcription factor HIF-1 (Hypoxia-inducible
factor-1) plays a significant role in cellular adaption and
survival under hypoxic condition (D. G. Nagle and Y. D. Zhou, Curr.
drug targets, 2006, 7, 355-369). Preclinical studies indicate that
the inhibition of HIF-1 activity has a remarkable impact on tumor
growth (G. L. Semenza, Nat. Rev. Cancer, 2003, 3, 721-732).
Besides, combination of HIF-1 inhibition with chemotherapeutic
agents/radiation gives rise to improved treatment outcomes (Li, L.
et al., Clin. Cancer Res., 2006, 12, 4747-4754, Moeller, B. J. et
al., Cancer Cell, 2005, 8, 99-110). Consequently, HIF-1 represents
a promising target for cancer therapy. Presently, early phase
clinical trials with topotecan, a natural product-derived
topoisomerase-1/HIF-1 inhibitor, have been completed. Moreover,
digoxin has entered phase two clinical trial as novel HIF-1
inhibitor, which exhibits the prospect of developing HIF-1 targeted
anticancer drugs.
[0003] There remains a strong need for therapeutically effective
compounds and improved ways for successfully treating cancer,
wherein inhibiting HIF-1 represents a highly promising approach as
explained above. As usual, it would generally be desirable to have
compounds with reduced risk for side effects, which can be prepared
in a cost-effective way and are directed only at tumor cells.
[0004] Recently, Traditional Chinese medicine as well as
complementary and alternative medicine has getting popular
providing a lot of treatment options. Traditional Chinese medicines
based on plant materials as well as plants or respective components
gained from plants usually allow for treatment of various diseases
and conditions while bearing a reduced risk for side effects. In
view of the rich medicinal plant resources, available respective
medicines can usually be produced in a cost-effective way.
Accordingly, there has been a lot of research with regard to plants
and respective ingredients for treatment of several diseases and
conditions.
[0005] For example, Tylophora atrofolliculata (Asclepiadaceae) is
already used as a traditional medicine. The roots of Tylophora
atrofolliculata which are mainly distributed in the Guangxi
Province in the Southwest of China have been used such as for the
treatment of rheumatism. Components isolated from said plant
include phenanthroindolizidine alkaloids (Huang, X. et al., Planta
Med., 2004, 70, 441-445, Abe, F. et al., Chem. Pharm. Bull, 1998,
46, 767-769, Abe, F. et al., Phytochemistry, 1995, 39, 695-699,
Ali, M. et al., J. Nat. Prod., 1991, 54, 1271-1278, M. Ali and K.
K. Bhutani, Phytochemistry, 1987, 26, 2089-2092, Ali, M. and
Bhutani, K. K., Phytochemistry, 1989, 28, 3513-3517, Bhutani, K. K.
et al., Phytochemistry, 1985, 24, 2778-2780, Dhiman, M. et al.,
Chem. Pap.-Chem. Zvesti, 2013, 67, 245-248), however, only
alkaloids such as tylophoridicine C-F, tylophorinine,
tylophorinidine have been isolated from this plant so far. Members
of the phenanthroindolizidine alkaloid class are generally well
known to possess multiple pharmacological effects, such as
anti-inflammatory, antifungal, antibacterial, and antiviral
activities. Besides, pronounced cytotoxicity of some
phenanthroindolizidine alkaloids against various cancer cell lines
attracted much attention in the discovery of anticancer drugs (Lee,
Y. Z. et al., Planta Med., 2011, 77, 1932-1938, Cai, X. F. et al.,
J. Nat. Prod., 2006, 69, 1095-1097, Damu, A. G. et al., Planta
Med., 2009, 75, 1152-1156, Damu, A. G. et al., J. Nat. Prod., 2005,
68, 1071-1075, Lykkeberg, A. K. et al., J. Nat. Prod., 2002, 65,
1299-1302).
[0006] In view of the presence of various different compounds in
plants usually with completely different mode of action and
therapeutic efficiency, there is a strong need for identifying and
providing components in isolated form with suitable therapeutic
efficiency such as with sufficient HIF-1 inhibitory activity for
treatment of cancer. Having those active ingredients in isolated
form could further reduce the risk of side effects or interactions
resulting from the presence of further compounds limiting the
therapeutic use.
SUMMARY OF THE INVENTION
[0007] The invention provides in a first aspect a method of
isolating at least one phenanthroindolizidine alkaloid in
particular with HIF-1 inhibitory activity from Tylophora
atrofolliculata which method comprises steps of:
[0008] (i) subjecting Tylophora atrofolliculata plant material to a
solvent extraction with an extraction solvent for obtaining a crude
extract, wherein the extraction solvent comprises an aliphatic
alcohol;
[0009] (ii) contacting the crude extract with a first and a second
separation solvent for obtaining a first and second layer, wherein
the first separation solvent comprises water and the second
separation solvent comprises an ester;
[0010] (iii) contacting the first layer with a third separation
solvent comprising a halogenated hydrocarbon for forming a third
layer;
[0011] (iv) subjecting the third layer to at least a first
chromatographic separation step, in particular carried out with
liquid column chromatography including separating by means of
fragmentation.
[0012] Preferably, the at least one phenanthroindolizidine alkaloid
is selected from a compound: [0013] having Formula (I):
[0013] ##STR00001## [0014] wherein R.sub.1 is OH (also referenced
as compound (1)); [0015] having Formula (I) above, wherein R.sub.1
is OCH.sub.3 (also referenced as compound (2)); [0016] having
Formula (II):
##STR00002##
[0016] (also referenced as compound (3)); [0017] having Formula
(III):
[0017] ##STR00003## [0018] wherein R.sub.1 is OH, R.sub.2 is
OCH.sub.3, R.sub.3 is OH and R.sub.4 is H (also referenced as
compound (4)); [0019] having Formula (III) above, wherein R.sub.1
is OH, R.sub.2 is OCH.sub.3, R.sub.3 is OH and R.sub.4 is
.alpha.-OH (also referenced as compound (5)); [0020] having Formula
(III) above, wherein R.sub.1 is H, R.sub.2 is OH, R.sub.3 is OH and
R.sub.4 is .alpha.-OH (also referenced as compound (13)); [0021]
having Formula (III) above, wherein R.sub.1 is H, R.sub.2 is
OCH.sub.3, R.sub.3 is OH and R.sub.4 is .alpha.-OH (also referenced
as compound (14)); [0022] having Formula (III) above, wherein
R.sub.1 is H, R.sub.2 is OH, R.sub.3 is OCH.sub.3 and R.sub.4 is
.alpha.-OH (also referenced as compound (15)); [0023] having
Formula (III) above, wherein R.sub.1 is H, R.sub.2 is OCH.sub.3,
R.sub.3 is OCH.sub.3 and R.sub.4 is .beta.-OH (also referenced as
compound (18)); [0024] having Formula (III) above, wherein R.sub.1
is OCH.sub.3, R.sub.2 is OCH.sub.3, R.sub.3 is OCH.sub.3 and
R.sub.4 is H (also referenced as compound (19)); [0025] having
Formula (III) above, wherein R.sub.1 is H, R.sub.2 is OCH.sub.3,
R.sub.3 is OCH.sub.3 and R.sub.4 is H (also referenced as compound
(20)); [0026] having Formula (III) above, wherein R.sub.1 is H,
R.sub.2 is OCH.sub.3, R.sub.3 is OCH.sub.3 and R.sub.4 is
.alpha.-OH (also referenced as compound (21)); [0027] having
Formula (IV):
[0027] ##STR00004## [0028] wherein R.sub.1 is H, R.sub.2 is OH,
R.sub.3 is OH and R.sub.4 is .beta.-OH (also referenced as compound
(6)); [0029] having Formula (IV) above, wherein R.sub.1 is OH,
R.sub.2 is OCH.sub.3, R.sub.3 is OCH.sub.3 and R.sub.4 is .beta.-OH
(also referenced as compound (8)); [0030] having Formula (IV)
above, wherein R.sub.1 is OCH.sub.3, R.sub.2 is OCH.sub.3, R.sub.3
is OCH.sub.3 and R.sub.4 is .alpha.-OH (also referenced as compound
(10)); [0031] having Formula (IV) above, wherein R.sub.1 is
OCH.sub.3, R.sub.2 is OCH.sub.3, R.sub.3 is OCH.sub.3 and R.sub.4
is H (also referenced as compound (11)); [0032] having Formula (IV)
above, wherein R.sub.1 is H, R.sub.2 is OCH.sub.3, R.sub.3 is
OCH.sub.3 and R.sub.4 is H (also referenced as compound (12));
[0033] having Formula (V):
##STR00005##
[0033] (also referenced as compound (7)); [0034] having Formula
(VI):
##STR00006##
[0034] (also referenced as compound (9)); [0035] having Formula
(VII):
##STR00007##
[0035] (also referenced as compound (16)); [0036] having Formula
(VIII):
##STR00008##
[0036] (also referenced as compound (17)); [0037] or a compound
having Formula (IX):
##STR00009##
[0037] (also referenced as compound (22)).
[0038] The present invention further refers to a method of treating
a subject suffering from cancer, in particular breast cancer,
comprising administering an effective amount of at least one
phenanthroindolizidine alkaloid isolated from Tylophora
atrofolliculata according to the method described before to the
subject. The phenanthroindolizidine alkaloid can be selected from
the group consisting of a phenanthroindolizidine alkaloid: [0039]
having Formula (III) given above, wherein R.sub.1 is OH, R.sub.2 is
OCH.sub.3, R.sub.3 is OH and R.sub.4 is H; [0040] having Formula
(III), wherein R.sub.1 is OH, R.sub.2 is OCH.sub.3, R.sub.3 is OH
and R.sub.4 is .alpha.-OH; [0041] having Formula (III), wherein
R.sub.1 is H, R.sub.2 is OH, R.sub.3 is OH and R.sub.4 is
.alpha.-OH; [0042] having Formula (III), wherein R.sub.1 is H,
R.sub.2 is OCH.sub.3, R.sub.3 is OH and R.sub.4 is .alpha.-OH;
[0043] having Formula (III), wherein R.sub.1 is H, R.sub.2 is OH,
R.sub.3 is OCH.sub.3 and R.sub.4 is .alpha.-OH; [0044] having
Formula (III), wherein R.sub.1 is H, R.sub.2 is OCH.sub.3, R.sub.3
is OCH.sub.3 and R.sub.4 is .beta.-OH; [0045] having Formula (III),
wherein R.sub.1 is H, R.sub.2 is OCH.sub.3, R.sub.3 is OCH.sub.3
and R.sub.4 is H; [0046] having Formula (III), wherein R.sub.1 is
H, R.sub.2 is OCH.sub.3, R.sub.3 is OCH.sub.3 and R.sub.4 is
.alpha.-OH; [0047] having Formula (IV) given above, wherein R.sub.1
is H, R.sub.2 is OH, R.sub.3 is OH and R.sub.4 is .beta.-OH; [0048]
having Formula (IV), wherein R.sub.1 is OH, R.sub.2 is OCH.sub.3,
R.sub.3 is OCH.sub.3 and R.sub.4 is .beta.-OH; [0049] having
Formula (IV), wherein R.sub.1 is H, R.sub.2 is OCH.sub.3, R.sub.3
is OCH.sub.3 and R.sub.4 is H; [0050] having Formula (V) given
above; [0051] having Formula (VII) given above; [0052] or having
Formula (VIII) given above.
[0053] The phenanthroindolizidine alkaloid administered in
particular has one of the following Formulas: [0054] Formula (III)
given above, wherein R.sub.1 is OH, R.sub.2 is OCH.sub.3, R.sub.3
is OH and R.sub.4 is H; [0055] Formula (III), wherein R.sub.1 is
OH, R.sub.2 is OCH.sub.3, R.sub.3 is OH and R.sub.4 is .alpha.-OH;
[0056] Formula (IV) given above, wherein R.sub.1 is H, R.sub.2 is
OH, R.sub.3 is OH and R.sub.4 is .beta.-OH; [0057] Formula (IV),
wherein R.sub.1 is OH, R.sub.2 is OCH.sub.3, R.sub.3 is OCH.sub.3
and R.sub.4 is .beta.-OH; [0058] Formula (IV), wherein R.sub.1 is
H, R.sub.2 is OCH.sub.3, R.sub.3 is OCH.sub.3 and R.sub.4 is H;
[0059] Formula (V) given above.
[0060] Still further, the present invention refers to a
composition, preferably a pharmaceutical composition, comprising
and in particular essentially consisting of: [0061] at least one,
in particular one phenanthroindolizidine alkaloid, in particular as
pharmaceutically effective ingredient, isolated from Tylophora
atrofolliculata according to the method described above, and [0062]
at least one pharmaceutically tolerable excipient such as one or
more of a diluent, a filler, a binder, a disintegrant, a lubricant,
a coloring agent, a surfactant and a preservative.
[0063] Another aspect of the present invention relates to a method
of treating a subject suffering from cancer comprising: [0064]
isolating a phenanthroindolizidine alkaloid from Tylophora
atrofolliculata by the method described above, in particular a
phenanthroindolizidine alkaloid: [0065] having Formula (III) given
above, wherein R.sub.1 is OH, R.sub.2 is OCH.sub.3, R.sub.3 is OH
and R.sub.4 is H; [0066] having Formula (III), wherein R.sub.1 is
OH, R.sub.2 is OCH.sub.3, R.sub.3 is OH and R.sub.4 is .alpha.-OH;
[0067] having Formula (IV) given above, wherein R.sub.1 is H,
R.sub.2 is OH, R.sub.3 is OH and R.sub.4 is .beta.-OH; [0068]
having Formula (IV), wherein R.sub.1 is OH, R.sub.2 is OCH.sub.3,
R.sub.3 is OCH.sub.3 and R.sub.4 is .beta.-OH; [0069] having
Formula (IV), wherein R.sub.1 is H, R.sub.2 is OCH.sub.3, R.sub.3
is OCH.sub.3 and R.sub.4 is H; [0070] or having Formula (V) given
above; [0071] formulating the phenanthroindolizidine alkaloid into
a pharmaceutically composition; and [0072] administering said
pharmaceutical composition to a subject suffering from cancer. The
subject is preferably a human. The cancer is preferably breast
cancer.
[0073] Further in accordance with the present invention is the at
least one phenanthroindolizidine alkaloid isolated from Tylophora
atrofolliculata for use in the treatment of cancer like breast
cancer and the use of the at least one phenanthroindolizidine
alkaloid isolated from Tylophora atrofolliculata for preparing a
medicament for the treatment of cancer like breast cancer.
[0074] Another aspect concerns a method of treating a subject
suffering from cancer comprising administering at least one
phenanthroindolizidine alkaloid isolated from Tylophora
atrofolliculata with the method described above in combination with
radiotherapy in particular with X-rays or chemotherapy, i.e. with
further active ingredients for treating cancer.
[0075] Still further, the present invention relates to a compound
selected from the group consisting of: [0076] a compound having
Formula (I) given above, wherein R.sub.1 is OH; [0077] a compound
having Formula (II) given above; [0078] a compound having Formula
(III) given above, wherein R.sub.1 is OH, R.sub.2 is OCH.sub.3,
R.sub.3 is OH and R.sub.4 is H; [0079] a compound having Formula
(III), wherein R.sub.1 is OH, R.sub.2 is OCH.sub.3, R.sub.3 is OH
and R.sub.4 is .alpha.-OH; [0080] a compound having Formula (IV)
given above, wherein R.sub.1 is H, R.sub.2 is OH, R.sub.3 is OH and
R.sub.4 is .beta.-OH; [0081] a compound having Formula (IV),
wherein R.sub.1 is OH, R.sub.2 is OCH.sub.3, R.sub.3 is OCH.sub.3
and R.sub.4 is .beta.-OH; [0082] a compound having Formula (IV),
wherein R.sub.1 is OCH.sub.3, R.sub.2 is OCH.sub.3, R.sub.3 is
OCH.sub.3 and R.sub.4 is .alpha.-OH; [0083] a compound having
Formula (IV), wherein R.sub.1 is OCH.sub.3, R.sub.2 is OCH.sub.3,
R.sub.3 is OCH.sub.3 and R.sub.4 is H; [0084] a compound having
Formula (IV), wherein R.sub.1 is H, R.sub.2 is OCH.sub.3, R.sub.3
is OCH.sub.3 and R.sub.4 is H; [0085] a compound having Formula (V)
given above; [0086] and a compound having Formula (VI).
[0087] In still another aspect, the present invention refers to a
method for targeting cancer cells in particular breast cancer
cells. Said method comprises the step of contacting a population of
cancer cells with at least one and preferably one
phenanthroindolizidine alkaloid isolated from Tylophora
atrofolliculata as described above.
[0088] The method of the present invention of isolating at least
one phenanthroindolizidine alkaloid from Tylophora atrofolliculata
can be used to isolate and obtain for example about 22
phenanthroindolizidine alkaloids. Among them are 11 new
phenanthroindolizidine alkaloids, namely compounds (1) and (3) to
(12), which have not been previously isolated and which include
phenanthroindolizidine alkaloids firstly identified in Tylophora
genus like compounds (4), (5), (7) to (9).
[0089] Most phenanthroindolizidine alkaloids isolated with the
method of the present invention exhibited extremely potent
inhibitory effects on HIF-1 with IC.sub.50 values in the low
nanomolar range without significant cytotoxicity. The potency of
several phenanthroindolizidine alkaloids isolated was even
comparable to Manassantin B (IC.sub.50 3 nM), the most potent
natural HIF-1 inhibitor identified so far. Finally, the HIF-1
inhibitory effects measured revealed the prerequisites for high
active alkaloids, including non-planarity at indolizidine moiety,
substitution types and patterns on the phenanthrene and
indolizidine moieties. Summing up, the present invention provides
plant-derived phenanthroindolizidine alkaloids with exceptional
HIF-1 inhibitory activity suitable to represent lead compounds for
the discovery of further HIF-1 inhibitors.
[0090] Other features and aspects of the invention will become
apparent by consideration of the following detailed description and
accompanying drawings.
BRIEF DESCRIPTION OF DRAWINGS
[0091] FIG. 1A and FIG. 1B show the key correlations observed in
.sup.1H-.sup.1H COSY, HMBC, and NOESY spectra of compound (1),
respectively.
[0092] FIG. 2A and FIG. 2B show the key correlations observed in
.sup.1H-.sup.1H COSY, HMBC, and NOESY spectra of compound (3),
respectively.
[0093] FIG. 3A and FIG. 3B show the key correlations observed in
.sup.1H-.sup.1H COSY, HMBC, and NOESY spectra of compound (4),
respectively.
DESCRIPTION OF THE EMBODIMENTS
[0094] The following preparations and examples are given to enable
those skilled in the art to more clearly understand and to practice
the present invention. They should not be considered as limiting
the scope of the invention, but merely as being illustrative and
for representing preferred embodiments thereof. The technical terms
used in the present patent application have the meaning as commonly
understood by a respective skilled person unless specifically
defined otherwise.
[0095] As used herein and in the claims, "comprising" means
including the following elements but not excluding others.
"Essentially consisting of" means that the material consists of the
respective element along with usually and unavoidable impurities
such as side products and components usually resulting from the
respective preparation or method for obtaining the material such as
traces of further components or solvents. "Consisting of" means
that the material solely consists of, i.e. is formed by the
respective element.
[0096] In a first aspect, the invention provides a method of
isolating at least one phenanthroindolizidine alkaloid in
particular with HIF-1 inhibitory activity from Tylophora
atrofolliculata. The method of the present invention comprises
steps of:
[0097] (i) subjecting Tylophora atrofolliculata plant material to a
solvent extraction with an extraction solvent for obtaining a crude
extract, wherein the extraction solvent comprises an aliphatic
alcohol;
[0098] (ii) contacting the crude extract with a first and a second
separation solvent for obtaining a first and second layer, wherein
the first separation solvent comprises water and the second
separation solvent comprises an ester;
[0099] (iii) contacting the first layer with a third separation
solvent comprising a halogenated hydrocarbon for forming a third
layer;
[0100] (iv) subjecting the third layer to at least a first
chromatographic separation step.
[0101] Optionally, the method includes further steps after step
(iv) of purifying the at least one phenanthroindolizidine
alkaloid.
[0102] The term "isolating" or "isolation" used herein means
separating a combination of two or more or one single
phenanthroindolizidine alkaloid from components present in the
Tylophora atrofolliculata plant material. In particular, the method
is for isolating a combination of at most 10, further preferred at
most 5, still further preferred at most two and in particular one
single phenanthroindolizidine alkaloid, in particular an
phenanthroindolizidine alkaloid with HIF-1 inhibitory activity,
from Tylophora atrofolliculata plant material.
[0103] The term "purifying" as used herein refers to methods
generally known to the skilled person for purifying compounds like
evaporation, lyophilization or (re-)crystallization for obtaining a
desired degree of purity, i.e. a desired degree of absence of
impurities.
[0104] The at least one ingredient isolated from Tylophora
atrofolliculata plant material is a phenanthroindolizidine
alkaloid. "Alkaloids" are known to the skilled person as a class of
components present in various plants characterized by a chemical
structure with at least one nitrogen atom, usually at least one
heterocyclic nitrogen atom. Alkaloids can be divided into several
subgroups depending on the specific nitrogen containing
heterocyclic ring system. Phenanthroindolizidine alkaloids
represent a small subgroup of alkaloids and the term generally
refers to compounds having a phenanthrene ring system fused with
that of an indolizidine.
[0105] Preferably, the at least one phenanthroindolizidine alkaloid
is selected from a compound: [0106] having Formula (I):
[0106] ##STR00010## [0107] wherein R.sub.1 is selected from OH or
OCH.sub.3; [0108] having Formula (II):
[0108] ##STR00011## [0109] having Formula (III):
[0109] ##STR00012## [0110] wherein R.sub.1 is selected from OH, H
or OCH.sub.3, R.sub.2 is selected from OCH.sub.3 or OH. R.sub.3 is
selected from OCH.sub.3 or OH and R.sub.4 is selected from
.alpha.-OH, .beta.-OH or H; [0111] having Formula (IV):
[0111] ##STR00013## [0112] wherein R.sub.1 is selected from OH, H
or OCH.sub.3, R.sub.2 is selected from OCH.sub.3 or OH, R.sub.3 is
selected from OCH.sub.3 or OH and R.sub.4 is selected from
.alpha.-OH, .beta.-OH or H; [0113] having Formula (V):
[0113] ##STR00014## [0114] having Formula (VI):
[0114] ##STR00015## [0115] having Formula (VII):
[0115] ##STR00016## [0116] having Formula (VIII):
[0116] ##STR00017## [0117] or a compound having Formula (IX):
##STR00018##
[0118] More preferably, the at least one phenanthroindolizidine
alkaloid is selected from a compound: [0119] having Formula (I)
given above, wherein R.sub.1 is OH (also referenced as compound
(1)); [0120] having Formula (I), wherein R.sub.1 is OCH.sub.3 (also
referenced as compound (2)); [0121] having Formula (II) given above
(also referenced as compound (3)); [0122] having Formula (III)
given above, wherein R.sub.1 is OH, R.sub.2 is OCH.sub.3, R.sub.3
is OH and R.sub.4 is H (also referenced as compound (4)); [0123]
having Formula (III), wherein R.sub.1 is OH, R.sub.2 is OCH.sub.3,
R.sub.3 is OH and R.sub.4 is .alpha.-OH (also referenced as
compound (5)); [0124] having Formula (III), wherein R.sub.1 is H,
R.sub.2 is OH, R.sub.3 is OH and R.sub.4 is .alpha.-OH (also
referenced as compound (13)); [0125] having Formula (III), wherein
R.sub.1 is H, R.sub.2 is OCH.sub.3, R.sub.3 is OH and R.sub.4 is
.alpha.-OH (also referenced as compound (14)); [0126] having
Formula (III), wherein R.sub.1 is H, R.sub.2 is OH, R.sub.3 is
OCH.sub.3 and R.sub.4 is .alpha.-OH (also referenced as compound
(15)); [0127] having Formula (III), wherein R.sub.1 is H, R.sub.2
is OCH.sub.3, R.sub.3 is OCH.sub.3 and R.sub.4 is .beta.-OH (also
referenced as compound (18)); [0128] having Formula (III), wherein
R.sub.1 is OCH.sub.3, R.sub.2 is OCH.sub.3, R.sub.3 is OCH.sub.3
and R.sub.4 is H (also referenced as compound (19)); [0129] having
Formula (III), wherein R.sub.1 is H, R.sub.2 is OCH.sub.3, R.sub.3
is OCH.sub.3 and R.sub.4 is H (also referenced as compound (20));
[0130] having Formula (III), wherein R.sub.1 is H, R.sub.2 is
OCH.sub.3, R.sub.3 is OCH.sub.3 and R.sub.4 is .alpha.-OH (also
referenced as compound (21)); [0131] having Formula (IV) given
above, wherein R.sub.1 is H, R.sub.2 is OH, R.sub.3 is OH and
R.sub.4 is .beta.-OH (also referenced as compound (6)); [0132]
having Formula (IV), wherein R.sub.1 is OH, R.sub.2 is OCH.sub.3,
R.sub.3 is OCH.sub.3 and R.sub.4 is .beta.-OH (also referenced as
compound (8)); [0133] having Formula (IV), wherein R.sub.1 is
OCH.sub.3, R.sub.2 is OCH.sub.3, R.sub.3 is OCH.sub.3 and R.sub.4
is .alpha.-OH (also referenced as compound (10)); [0134] having
Formula (IV), wherein R.sub.1 is OCH.sub.3, R.sub.2 is OCH.sub.3,
R.sub.3 is OCH.sub.3 and R.sub.4 is H (also referenced as compound
(11)); [0135] having Formula (IV), wherein R.sub.1 is H, R.sub.2 is
OCH.sub.3, R.sub.3 is OCH.sub.3 and R.sub.4 is H (also referenced
as compound (12)); [0136] having Formula (V) given above (also
referenced as compound (7)); [0137] having Formula (VI) given above
(also referenced as compound (9)); [0138] having Formula (VII)
given above (also referenced as compound (16)); [0139] having
Formula (VIII) given above (also referenced as compound (17));
[0140] or a compound having Formula (IX) given above (also
referenced as compound (22)).
[0141] In particular, the method of the present invention allows
for the isolation of at least one of the following
phenanthroindolizidine alkaloids, i.e. at least one of the
following phenanthroindolizidine alkaloids is isolated from
Tylophora atrofolliculata with the method of the present invention:
[0142] a phenanthroindolizidine alkaloid having Formula (I) given
above, wherein R.sub.1 is OH, [0143] a phenanthroindolizidine
alkaloid having Formula (II) given above; [0144] a
phenanthroindolizidine alkaloid having Formula (III) given above,
wherein R.sub.1 is OH, R.sub.2 is OCH.sub.3, R.sub.3 is OH and
R.sub.4 is H; [0145] a phenanthroindolizidine alkaloid having
Formula (III), wherein R.sub.1 is OH, R.sub.2 is OCH.sub.3, R.sub.3
is OH and R.sub.4 is .alpha.-OH; [0146] a phenanthroindolizidine
alkaloid having Formula (IV) given above, wherein R.sub.1 is H,
R.sub.2 is OH, R.sub.3 is OH and R.sub.4 is .beta.-OH; [0147] a
phenanthroindolizidine alkaloid having Formula (IV), wherein
R.sub.1 is OH, R.sub.2 is OCH.sub.3, R.sub.3 is OCH.sub.3 and
R.sub.4 is .beta.-OH; [0148] a phenanthroindolizidine alkaloid
having Formula (IV), wherein R.sub.1 is OCH.sub.3, R.sub.2 is
OCH.sub.3, R.sub.3 is OCH.sub.3 and R.sub.4 is .alpha.-OH; [0149] a
phenanthroindolizidine alkaloid having Formula (IV), wherein
R.sub.1 is OCH.sub.3, R.sub.2 is OCH.sub.3, R.sub.3 is OCH.sub.3
and R.sub.4 is H; [0150] a phenanthroindolizidine alkaloid having
Formula (IV), wherein R.sub.1 is H, R.sub.2 is OCH.sub.3, R.sub.3
is OCH.sub.3 and R.sub.4 is H; [0151] a phenanthroindolizidine
alkaloid having Formula (V) given above; [0152] or a
phenanthroindolizidine alkaloid having Formula (VI) given
above.
[0153] In still more preferred embodiments of the present
invention, the method of the present invention allows for the
isolation of at least one of the following phenanthroindolizidine
alkaloids, i.e. at least one of the following
phenanthroindolizidine alkaloids is more preferably isolated, in
particular one of the following phenanthroindolizidine alkaloids:
[0154] a phenanthroindolizidine alkaloid having Formula (III) given
above, wherein R.sub.1 is OH, R.sub.2 is OCH.sub.3, R.sub.3 is OH
and R.sub.4 is H; [0155] a phenanthroindolizidine alkaloid having
Formula (III), wherein R.sub.1 is OH, R.sub.2 is OCH.sub.3, R.sub.3
is OH and R.sub.4 is .alpha.-OH; [0156] a phenanthroindolizidine
alkaloid having Formula (IV) given above, wherein R.sub.1 is H,
R.sub.2 is OH, R.sub.3 is OH and R.sub.4 is .beta.-OH; [0157] a
phenanthroindolizidine alkaloid having Formula (IV), wherein
R.sub.1 is OH, R.sub.2 is OCH.sub.3, R.sub.3 is OCH.sub.3 and
R.sub.4 is .beta.-OH; [0158] a phenanthroindolizidine alkaloid
having Formula (IV), wherein R.sub.1 is H, R.sub.2 is OCH.sub.3,
R.sub.3 is OCH.sub.3 and R.sub.4 is H; [0159] or a
phenanthroindolizidine alkaloid having Formula (V) given above.
[0160] In most preferred embodiments of the present invention, the
method of the present invention allows for the isolation of one of
the following phenanthroindolizidine alkaloids, i.e. one of the
following phenanthroindolizidine alkaloids is most preferably
isolated with the method of the present invention: [0161] a
phenanthroindolizidine alkaloid having Formula (III) given above,
wherein R.sub.1 is OH, R.sub.2 is OCH.sub.3, R.sub.3 is OH and
R.sub.4 is H; [0162] a phenanthroindolizidine alkaloid having
Formula (III), wherein R.sub.1 is OH, R.sub.2 is OCH.sub.3, R.sub.3
is OH and R.sub.4 is .alpha.-OH; [0163] a phenanthroindolizidine
alkaloid having Formula (IV) given above, wherein R.sub.1 is H,
R.sub.2 is OH, R.sub.3 is OH and R.sub.4 is .beta.-OH; [0164] or a
phenanthroindolizidine alkaloid having Formula (V) given above.
[0165] In especially preferred embodiments of the present
invention, one of the phenanthroindolizidine alkaloids of Formula
(III) with R.sub.1 being OH, R.sub.2 being OCH.sub.3, R.sub.3 being
OH and R.sub.4 being H or of Formula (III) with R.sub.1 being OH,
R.sub.2 being OCH.sub.3, R.sub.3 being OH and R.sub.4 being
.alpha.-OH is isolated with the method of the present
invention.
[0166] The method of the present invention comprises a step (i) of
subjecting Tylophora atrofolliculata plant material to a solvent
extraction with an extraction solvent for obtaining a crude
extract, wherein the extraction solvent comprises an aliphatic
alcohol.
[0167] Preferably, the Tylophora atrofolliculata plant material
comprises the whole plant, i.e. it comprises roots and aerial parts
of Tylophora atrofolliculata. The method of the present invention
may further comprise steps before carrying out step (i) of
[0168] a) drying the Tylophora atrofolliculata plant material,
and/or
[0169] b) cutting, shredding, milling and/or pulverizing the
Tylophora atrofolliculata plant material.
[0170] For example, about 1 kg to 10 kg such as about 5.5 kg of the
Tylophora atrofolliculata plant material can be used in the method
of the present invention. The amount of Tylophora atrofolliculata
plant material in relation to the total amount of the extraction
solvent used in step (i) is preferably between 20 mg/ml and 60
mg/ml, further preferred about 42 mg/ml plant material relative to
the total amount of extraction solvent used in step (i). In
embodiments, in which the solvent extraction in step (i) is carried
out three times, the amount of Tylophora atrofolliculata plant
material in relation to the amount of extraction solvent in each of
the three solvent extractions is preferably of from 80 to 180
mg/ml, more preferably in the first solvent extraction about 100
mg/ml, in the second solvent extraction about 125 mg/ml and in the
third solvent extraction about 167 mg/ml, wherein the amount of
extraction solvent in the second and third solvent extraction is
calculated in relation to the starting weight of the Tylophora
atrofolliculata plant material used in the first solvent
extraction.
[0171] The extraction solvent comprises an aliphatic alcohol, which
means herein an aliphatic hydrocarbon, preferably a branched or
straight chain alkane, wherein at least one hydrogen atom of the
aliphatic hydrocarbon is substituted with a hydroxyl group,
preferably one hydrogen atom is substituted with a hydroxyl group
referenced as monohydric aliphatic alcohol. More preferably, the
aliphatic alcohol of the extracting solvent is a monohydric
aliphatic alcohol, still more preferably a monohydric alcohol with
1 to 4 carbon atoms, further preferably with 1 to 2 carbon atoms.
I.e. the aliphatic alcohol of the first extracting solvent is more
preferably selected from methanol, ethanol, propanol, isopropanol,
n-butanol, isobutanol, sec-butyl alcohol, tert-butyl alcohol or
mixtures thereof and further preferably from methanol, ethanol or
mixtures thereof. More preferably, the aliphatic alcohol of the
extraction solvent is methanol. The extraction solvent most
preferably essentially consists of methanol.
[0172] The solvent extraction in step (i) is preferably carried out
for 4 to 10 h in total. In embodiments, in which the solvent
extraction in step (i) is carried out three times, each of the
three solvent extractions is carried out for 2 to 4 h, more
preferably the first solvent extraction is carried out for about 4
h, the second solvent extraction for about 2 h and the third
solvent extraction for about 2 h.
[0173] The temperatures are preferably above 45.degree. C., in
particular at least 50.degree. C., and most preferably the
Tylophora atrofolliculata plant material is refluxed with the first
extraction solvent.
[0174] The solvent extraction in step (i) is preferably carried out
at least two, more preferably at least three times and in
particular three times, wherein the extracts obtained in each step
are combined for forming the crude extract. Thus, in especially
preferred embodiments of the present invention, the Tylophora
atrofolliculata plant material is refluxed with the extraction
solvent, in particular methanol, at least two times, in particular
three times. I.e. the solvent extraction is preferably carried out
three times with the Tylophora atrofolliculata plant material.
[0175] Preferably, the extraction solvent is removed before step
(ii) for forming the crude extract, i.e. step (i) preferably
further comprises removing the extraction solvent after the solvent
extraction and before step (ii). The extraction solvent is
preferably removed by evaporation under reduced pressure.
[0176] The method of the present invention further comprises a step
(ii) of contacting the crude extract obtained in step (i) with a
first and a second separation solvent for obtaining a first and a
second layer, wherein the first separation solvent comprises and
preferably essentially consists of water and the second separation
solvent comprises an ester. The ester is in particular a
C.sub.1-C.sub.6 aliphatic alcohol ester of a C.sub.1-C.sub.7 alkyl
carboxylic acid. Further preferably, the ester is a C.sub.3-C.sub.7
ester, in particular ethyl acetate or ethyl formate. In most
preferred embodiments of the present invention, the second
separation solvent comprises and preferably essentially consists of
ethyl acetate. In particular, the first separation solvent is
mainly comprised in the first layer and the second separation
solvent is mainly comprised in the second layer obtained. More
specifically, the first layer in particular comprises the at least
one phenanthroindolizidine alkaloid and the main part of the first
separation solvent. The second layer comprises the main part of the
second separation solvent. "Main part" usually means more than 90%
of the total amount of the separation solvent, preferably more than
95%. The term "layers" used herein and as generally understood by
the skilled person means separated phases resulting from contacting
at least two solvents which are substantially immiscible or
immiscible with each other, in the present invention for example
the first and the second separation solvent.
[0177] Preferably, contacting the crude extract with the first and
the second separation solvent in step (ii) means sequentially
adding the first and the second separation solvent to the crude
extract. In preferred embodiments of the present invention, the
crude extract is added, preferably suspended, in the first
separation solvent. Preferably, the pH of the suspension is
adjusted to less than 3, in particular to about 1 to 2 before
adding the second separation solvent, preferably by adding an
inorganic (mineral) acid, in particular by adding HCl. Then the
second separation solvent is preferably added accompanied by
shaking for forming the first and the second layer and the first
layer is then separated. Such procedure is especially suitable for
separating chlorophyll and neutral compounds from the
phenanthroindolizidine alkaloids.
[0178] Preferably, the pH of the first layer is adjusted to at
least pH 8 by adding a base before step (iii), in particular by
adding an alkali hydroxide. I.e. the base is preferably an alkali
hydroxide. Alkali hydroxides are a class of chemical compounds
which are composed of an alkali metal cation, i.e. on of lithium
(Li), sodium (Na), potassium (K), rubidium (Rb), caesium (Cs), and
the hydroxide anion (HO--). In particular, the alkali metal cation
is K or Na. More preferably, the base is NaOH. Most preferably, the
first layer is basified by adding 10% NaOH for obtaining a pH of
about 9 to 10 before step (iii).
[0179] The method of the present invention further comprises a step
(iii) of contacting the optionally basified first layer obtained
and in step (ii) with a third separation solvent for forming a
third layer, which third separation solvent comprises a halogenated
hydrocarbon. The third layer in particular comprises the main part
of the third separation solvent and the at least one
phenanthroindolizidine alkaloid.
[0180] The third separation solvent is added to the first layer
preferably accompanied by shaking for forming the third layer.
[0181] The term "halogenated hydrocarbon" as used herein refers to
a hydrocarbon, preferably an alkane, which hydrocarbon has at least
one hydrogen atom substituted with a halogen atom. Preferably, the
halogenated hydrocarbon in the first extracting solvent is a
hydrocarbon, preferably a branched or straight chain alkane, which
hydrocarbon has 1 to 4 carbon atoms and wherein at least one
hydrogen atom is substituted with a halogen selected from Br, Cl,
or F, in particular from Cl. Preferably, the halogenated
hydrocarbon is an alkane with 1 to 2 carbon atoms in which at least
one hydrogen atom is substituted with a Cl atom, in particular
selected from methyl chloride, dichloromethane or chloroform. Most
preferably, the halogenated hydrocarbon in the third separation
solvent is chloroform. In further preferred embodiments of the
present invention, the third separation solvent essentially
consists of chloroform.
[0182] The method of the present invention further comprises a step
(iv) of subjecting the third layer to at least a first
chromatographic separation step, preferably carried out with liquid
column chromatography including separating by means of
fragmentation.
[0183] Preferably, step (iv) further comprises a step of removing
the solvent portion of the third layer, i.e. in particular the
third separation solvent, before carrying out the first
chromatographic separation step. The solvent portion is preferably
removed by means of evaporation in vacuum.
[0184] In embodiments of the present invention, step (iv) comprises
a single, i.e. only a first chromatographic separation step. In
further embodiments of the present invention, a first and a second
chromatographic separation step are carried out in step (iv) and in
still further embodiments of the present invention, a first, a
second and a third separation step is carried out in step (iv). In
further embodiments of the present invention, at least a first, a
second, a third and a fourth chromatographic separation step is
carried out.
[0185] The first chromatographic separation step may be sufficient
and, thus, step (iv) consists of a single chromatographic
separation step in embodiments, in which a phenanthroindolizidine
alkaloid having Formula (III) given above with R.sub.1 being H,
R.sub.2 being OCH.sub.3, R.sub.3 being OCH.sub.3 and R.sub.4 being
.beta.-OH (compound (18)) is isolated.
[0186] The first and the second chromatographic separation step are
preferably carried out in step (iv) for isolating a
phenanthroindolizidine alkaloid selected from a compound: [0187]
having Formula (III) given above, wherein R.sub.1 is OH, R.sub.2 is
OCH.sub.3, R.sub.3 is OH and R.sub.4 is H (compound (4)); [0188]
having Formula (III), wherein R.sub.1 is OH, R.sub.2 is OCH.sub.3,
R.sub.3 is OH and R.sub.4 is .alpha.-OH (compound (5)); [0189]
having Formula (III), wherein R.sub.1 is H, R.sub.2 is OCH.sub.3,
R.sub.3 is OH and R.sub.4 is .alpha.-OH (compound (14)); [0190] or
a compound having Formula (III), wherein R.sub.1 is OCH.sub.3,
R.sub.2 is OCH.sub.3, R.sub.3 is OCH.sub.3 and R.sub.4 is H
(compound (19)).
[0191] At least the first, the second and the third chromatographic
separation step are preferably carried out in step (iv) for
isolating a phenanthroindolizidine alkaloid selected from a
compound: [0192] having Formula (I) given above, wherein R.sub.1 is
OCH.sub.3 (compound (2)); [0193] having Formula (II) given above
(compound (3)); [0194] having Formula (III) given above, wherein
R.sub.1 is H, R.sub.2 is OH, R.sub.3 is OH and R.sub.4 is
.alpha.-OH (compound (13)); [0195] having Formula (III), wherein
R.sub.1 is H, R.sub.2 is OCH.sub.3, R.sub.3 is OCH.sub.3 and
R.sub.4 is H (compound (20)); [0196] having Formula (III), wherein
R.sub.1 is H, R.sub.2 is OCH.sub.3, R.sub.3 is OCH.sub.3 and
R.sub.4 is .alpha.-OH (compound (21)); [0197] having Formula (IV)
given above, wherein R.sub.1 is OH, R.sub.2 is OCH.sub.3, R.sub.3
is OCH.sub.3 and R.sub.4 is .beta.-OH (compound (8)); [0198] having
Formula (IV), wherein R.sub.1 is OCH.sub.3, R.sub.2 is OCH.sub.3,
R.sub.3 is OCH.sub.3 and R.sub.4 is .alpha.-OH (compound (10));
[0199] having Formula (IV), wherein R.sub.1 is OCH.sub.3, R.sub.2
is OCH.sub.3, R.sub.3 is OCH.sub.3 and R.sub.4 is H (compound
(11)); [0200] having Formula (IV), wherein R.sub.1 is H, R.sub.2 is
OCH.sub.3, R.sub.3 is OCH.sub.3 and R.sub.4 is H (compound (12));
[0201] having Formula (VI) given above (compound (9)); [0202]
having Formula (VII) given above (compound (16)); [0203] having
Formula (VIII) given above (compound (17)); [0204] or a compound
having Formula (IX) given above (compound (22)).
[0205] At least the first, the second, the third and the fourth
chromatographic separation step are preferably carried out in step
(iv) for isolating a phenanthroindolizidine alkaloid selected from
a compound: [0206] having Formula (I) given above, wherein R.sub.1
is OH (compound (1)); [0207] having Formula (III) given above,
wherein R.sub.1 is H, R.sub.2 is OH, R.sub.3 is OCH.sub.3 and
R.sub.4 is .alpha.-OH (compound (15)); [0208] having Formula (IV)
given above, wherein R.sub.1 is H, R.sub.2 is OH, R.sub.3 is OH and
R.sub.4 is .beta.-OH (compound (6)); [0209] or a compound having
Formula (V) given above (compound (7)).
[0210] Chromatographic separation steps in step (iv) are preferably
carried out with liquid chromatography including column
chromatography and can be carried out as classical (low pressure)
column chromatography usually operating with a low pressure up to
about 0.5 MPa, high-performance liquid chromatography (HPLC)
usually with operational pressures up to 5 MPa or higher. HPLC can
be carried out as semi-preparative or preparative HPLC.
[0211] Preferably, the first chromatographic separation step is
selected from classical (low pressure) column chromatography, in
particular with a polyaromatic adsorbent resin, preferably with a
styrene-divinylbenzene polymer resin as stationary phase like MCI
CH20P gel.
[0212] The optional further chromatographic separation steps, in
particular the second, the third and the fourth chromatographic
separation steps and any further chromatographic separation steps
preferably comprise liquid chromatography, which may be carried out
as classical column chromatography or HPLC. The stationary phase is
preferably selected from unmodified silica gel (further referenced
as "silica gel") or a reverse phase, in particular a C18 reverse
phase like octadecylsilyl groups.
[0213] Classical column chromatography can be carried out with
unmodified silica gel preferably with a particle size of about 40
.mu.m to about 63 .mu.m. Alternatively, classical column
chromatography can be carried out with a reverse phase, namely a
stationary phase having alkyl chains covalently bonded to a solid
support leading to a hydrophobic stationary phase, in particular
including C18 phases, i.e. with octadecyl-chains (C18 chains) in
particular with a particle size of about 55 .mu.m to 105 .mu.m and
preferably with a pore size of about 125 .ANG..
[0214] HPLC can be carried out with a reverse phase as stationary
phase, in particular a C18 reverse phase with a particle size of
about 5 .mu.m and preferably with column dimensions of about
250.times.10 mm.
[0215] In embodiments, in which the second chromatographic
separation step is carried out in step (iv), the second
chromatographic separation step is preferably carried out by means
of classical (low pressure) column chromatography with unmodified
silica gel as stationary phase. In embodiments, in which the third
chromatographic separation step is carried out in step (iv), the
third chromatographic separation step is preferably carried out by
means of classical (low pressure) column chromatography with a C18
reverse phase as stationary phase or by means of a HPLC with a C18
reverse phase as stationary phase. In embodiments, in which the
fourth or even more chromatographic separation steps are carried
out in step (iv), the fourth and any further chromatographic
separation step is preferably carried out by means of a HPLC with a
C18 reverse phase as stationary phase.
[0216] The first chromatographic separation step is preferably
carried out by means of classical column chromatography and
preferably includes fractionating the third layer and its
components, respectively, to obtain several fractions, in
particular at least 10 fractions, more preferably at least 15
fractions, i.e. including collecting individual eluate fractions
rich in the at least one phenanthroindolizidine alkaloid to be
isolated. The first chromatographic separation step is preferably
carried out as gradient elution, i.e. with a gradient of eluting
solvents.
[0217] More preferably, the first chromatographic separation step
is carried out with a styrene-divinylbenzene polymer resin as
stationary phase like MCI CH20P gel and preferably with eluting
solvents selected from at least three of C.sub.1 to C.sub.3
aliphatic alcohol, a C.sub.2 to C.sub.4 ketone, water, a C.sub.2 to
C.sub.6 aliphatic amine, preferably at least three of methanol,
acetone, water and/or diethyl amine.
[0218] The term "aliphatic amine" refers to an alkylamine in
particular having a formula NHyBx, wherein x and y are selected
from among x=1, y=2 and x=2, y=1, preferably x=2, y=1. Each B is a
straight chain or branched C1-C3 alkyl, i.e. the number of carbon
atoms in each B is 1 to 3 and preferably 2.
[0219] More preferably, methanol/water/diethyl amine and
subsequently acetone/methanol/diethyl amine are used as elution
solvents. Most preferably, the following elution solvents and
gradients are subsequently applied:
[0220] a) methanol/water/diethyl amine with a gradient of 7:3:0.05
to 10:0:0.05, and subsequently
[0221] b) acetone/methanol/diethyl amine with a gradient of
1:9:0.05 to 6:4:0.05.
[0222] Preferably, by subjecting the third layer in particular
after removing the solvent portion to a styrene-divinylbenzene
polymer resin as stationary phase and eluting with elution solvents
a) and b) phenanthroindolizidine alkaloid having Formula (III) with
R.sub.1 being H, R.sub.2 being OCH.sub.3, R.sub.3 being OCH.sub.3
and R.sub.4 being .beta.-OH (compound (18)) can be isolated, and
additionally at least 10 fractions are selected, more preferably
about 15 fractions are selected and most preferably 15 fractions
(i.e. fractions 1 to 15, further referenced as "fraction no. XX").
The fractions are preferably selected based on a thin layer
chromatography (TLC) monitoring which is usual practice in the art,
i.e. the number and size of each fraction is determined by the
specific composition and changes in the composition as well as the
presence of alkaloids. I.e. a change in the composition confirmed
with TLC means next fraction. For example, when a new compound
shows up in the eluted part compared with the already eluted parts
confirmed with TLC, this represents a new fraction until there is a
change in the composition, e.g. said new compound is no longer
eluted. The presence of alkaloids can be verified with usual and
well-known reagents. TLC is preferably carried out with silica gel,
in particular silica gel 60 F.sub.254.
[0223] For isolating the phenanthroindolizidine alkaloid of Formula
(IV) given above with R.sub.1 being H, R.sub.2 being OH, R.sub.3
being OH and R.sub.4 being .beta.-OH (compound (6)) and/or of
Formula (V) (compound (7)) given above: [0224] 15 fractions are
obtained as defined above, wherein fraction no. 3 is preferably
subjected to a classical column chromatography with silica gel and
with eluting solvents comprising a C.sub.1 to C.sub.2 halogenated
hydrocarbon, a C.sub.1 to C.sub.3 aliphatic alcohol and a C.sub.2
to C.sub.6 aliphatic amine, most preferably with
chloroform/methanol/diethyl amine and a gradient of 10:0:0.05 to
3:7:0.05 for obtaining about 17 subfractions (i.e. subfractions no.
3-1 to 3-17) as determined by TLC monitoring; and [0225] subjecting
subfraction no. 3-15 to classical column chromatography with a C18
reverse phase with eluting solvents comprising a C.sub.1 to C.sub.3
aliphatic alcohol and water, in particular methanol/water with a
gradient of 1:1 to 4:1 and subsequently to HPLC.
[0226] For isolating the phenanthroindolizidine alkaloid of Formula
(III) given above with R.sub.1 is OH, R.sub.2 is OCH.sub.3, R.sub.3
is OH and R.sub.4 is .alpha.-OH (compound (5)) and/or of Formula
(III) given above with R.sub.1 being H, R.sub.2 being OH, R.sub.3
being OH and R.sub.4 being .alpha.-OH (compound (13)): [0227]
fraction no. 4 is preferably subjected to a classical column
chromatography with silica gel and with eluting solvents comprising
a C.sub.1 to C.sub.2 halogenated hydrocarbon, a C.sub.1 to C.sub.3
aliphatic alcohol and a C.sub.2 to C.sub.6 aliphatic amine, most
preferably with chloroform/methanol/diethyl amine and a gradient of
10:0:0.05 to 3:1:0.05 for obtaining phenanthroindolizidine alkaloid
of Formula (III) given above with R.sub.1 is OH, R.sub.2 is
OCH.sub.3, R.sub.3 is OH and R.sub.4 is .alpha.-OH (compound (5))
and 6 subfractions (subfractions no. 4-1 to 4-6) based on the TCL
behavior, and [0228] subjecting subfraction no. 4-6 to classical
column chromatography with a C18 reverse phase with eluting
solvents comprising a C.sub.1 to C.sub.3 aliphatic alcohol and
water, in particular methanol/water 1:1 for obtaining a
phenanthroindolizidine alkaloid of Formula (III) given above with
R.sub.1 being H, R.sub.2 being OH, R.sub.3 being OH and R.sub.4
being .alpha.-OH (compound (13)).
[0229] For isolating the phenanthroindolizidine alkaloid of Formula
(III) given above with R.sub.1 is OH, R.sub.2 is OCH.sub.3, R.sub.3
is OH and R.sub.4 is H (compound (4)): [0230] fraction no. 5 is
preferably subjected to a classical column chromatography with
silica gel and with eluting solvents comprising a C.sub.1 to
C.sub.2 halogenated hydrocarbon, a C.sub.1 to C.sub.3 aliphatic
alcohol and a C.sub.2 to C.sub.6 aliphatic amine, most preferably
with chloroform/methanol/diethyl amine and a gradient of 10:0:0.05
to 5:2:0.05.
[0231] For isolating at least one of the phenanthroindolizidine
alkaloids of Formula (I) with R.sub.1 being OH (compound (1)), of
Formula (III) with R.sub.1 being H, R.sub.2 being OCH.sub.3,
R.sub.3 being OH and R.sub.4 being .alpha.-OH (compound (14)), of
Formula (III) with R.sub.1 being H, R.sub.2 being OH, R.sub.3 being
OCH.sub.3 and R.sub.4 being .alpha.-OH (compound (15)) and/or of
Formula (VII) (compound (16)): [0232] fraction no. 8 is preferably
subjected to a classical column chromatography with silica gel and
with eluting solvents comprising a C.sub.1 to C.sub.2 halogenated
hydrocarbon, a C.sub.1 to C.sub.3 aliphatic alcohol and a C.sub.2
to C.sub.6 aliphatic amine, most preferably with
chloroform/methanol/diethyl amine and a gradient of 10:0:0.05 to
7:3:0.05 for obtaining phenanthroindolizidine alkaloid of Formula
(III) with R.sub.1 being H, R.sub.2 being OCH.sub.3, R.sub.3 being
OH and R.sub.4 being .alpha.-OH (compound (14)) and 24 subfractions
(subfractions no. 8-1 to 8-24) determined by TLC monitoring; [0233]
subjecting subfraction no. 8-15 to classical column chromatography
with a C18 reverse phase with eluting solvents comprising a C.sub.1
to C.sub.3 aliphatic alcohol and water, in particular
methanol/water 1:1 and subsequently to HPLC for obtaining a
phenanthroindolizidine alkaloid of Formula (I) with R.sub.1 being
OH (compound (1)); [0234] subjecting subfraction no. 8-16 to
classical column chromatography with a C18 reverse phase with
eluting solvents comprising a C.sub.1 to C.sub.3 aliphatic alcohol
and water, in particular methanol/water 7:3 and subsequently to
HPLC for obtaining a phenanthroindolizidine alkaloid of Formula
(III) with R.sub.1 being H, R.sub.2 being OH, R.sub.3 being
OCH.sub.3 and R.sub.4 being .alpha.-OH (compound (15)); [0235]
subjecting subfraction no. 8-23 to classical column chromatography
with a C18 reverse phase with eluting solvents comprising a C.sub.1
to C.sub.3 aliphatic alcohol and water, in particular
methanol/water with a gradient 2:3 to 3:2 for obtaining a
phenanthroindolizidine alkaloid of Formula (VII) (compound
(16)).
[0236] For isolating the phenanthroindolizidine alkaloid of Formula
(IV) given above with R.sub.1 being OH, R.sub.2 being OCH.sub.3,
R.sub.3 being OCH.sub.3 and R.sub.4 being .beta.-OH (compound (8)):
[0237] fraction no. 9 is preferably subjected to a classical column
chromatography with silica gel and with eluting solvents comprising
a C.sub.1 to C.sub.2 halogenated hydrocarbon, a C.sub.1 to C.sub.3
aliphatic alcohol and a C.sub.2 to C.sub.6 aliphatic amine, most
preferably with chloroform/methanol/diethyl amine and a gradient of
10:0:0.05 to 5:5:0.05 and subsequently to repeated HPLC.
[0238] For isolating the phenanthroindolizidine alkaloid of Formula
(III) given above with R.sub.1 being OCH.sub.3, R.sub.2 being
OCH.sub.3, R.sub.3 being OCH.sub.3 and R.sub.4 being H (compound
(19)): [0239] fraction no. 11 is preferably subjected to a
classical column chromatography with silica gel and with eluting
solvents comprising a C.sub.1 to C.sub.2 halogenated hydrocarbon, a
C.sub.1 to C.sub.3 aliphatic alcohol and a C.sub.2 to C.sub.6
aliphatic amine, most preferably with chloroform/methanol/diethyl
amine and a gradient of 10:0:0.05 to 4:6:0.05.
[0240] For isolating the phenanthroindolizidine alkaloid of Formula
(VI) (compound (9)): [0241] fraction no. 12 is preferably subjected
to a classical column chromatography with silica gel and with
eluting solvents comprising a C.sub.1 to C.sub.2 halogenated
hydrocarbon, a C.sub.1 to C.sub.3 aliphatic alcohol and a C.sub.2
to C.sub.6 aliphatic amine, most preferably with
chloroform/methanol/diethyl amine and a gradient of 10:0:0.05 to
4:6:0.05 and subsequently to HPLC.
[0242] For isolating the phenanthroindolizidine alkaloid of Formula
(I) with R.sub.1 being OCH.sub.3 (compound (2)), of Formula (II)
(compound (3)), of Formula (III) with R.sub.1 being H, R.sub.2
being OCH.sub.3, R.sub.3 being OCH.sub.3 and R.sub.4 being H
(compound (20)), of Formula (III) with R.sub.1 being H, R.sub.2
being OCH.sub.3, R.sub.3 being OCH.sub.3 and R.sub.4 being
.alpha.-OH (compound (21)), of Formula (IV) with R.sub.1 being
OCH.sub.3, R.sub.2 being OCH.sub.3, R.sub.3 being OCH.sub.3 and
R.sub.4 being .alpha.-OH (compound (10)); of Formula (IV) with
R.sub.1 being OCH.sub.3, R.sub.2 being OCH.sub.3, R.sub.3 being
OCH.sub.3 and R.sub.4 being H (compound (11)), of Formula (IV) with
R.sub.1 being H, R.sub.2 being OCH.sub.3, R.sub.3 being OCH.sub.3
and R.sub.4 being H (compound (12)), of Formula (VIII) (compound
(17)) and/or of Formula (IX) (compound (22)): [0243] fraction no.
13 is preferably subjected to a classical column chromatography
with silica gel and with eluting solvents comprising a C.sub.1 to
C.sub.2 halogenated hydrocarbon, a C.sub.1 to C.sub.3 aliphatic
alcohol and a C.sub.2 to C.sub.6 aliphatic amine, most preferably
with chloroform/methanol/diethyl amine and a gradient of 10:0:0.05
to 4:6:0.05 and subsequently to HPLC.
[0244] In another aspect, the present invention refers to a
phenanthroindolizidine alkaloid, namely a compound selected from
the group consisting of: [0245] a compound having Formula (I):
[0245] ##STR00019## [0246] wherein R.sub.1 is OH; [0247] a compound
having Formula (II):
[0247] ##STR00020## [0248] a compound having Formula (III):
[0248] ##STR00021## [0249] wherein R.sub.1 is OH, R.sub.2 is
OCH.sub.3, R.sub.3 is OH and R.sub.4 is H; [0250] a compound having
Formula (III) above, wherein R.sub.1 is OH, R.sub.2 is OCH.sub.3,
R.sub.3 is OH and R.sub.4 is .alpha.-OH; [0251] a compound having
Formula (IV):
[0251] ##STR00022## [0252] wherein R.sub.1 is H, R.sub.2 is OH,
R.sub.3 is OH and R.sub.4 is .beta.-OH; [0253] a compound having
Formula (IV) above, wherein R.sub.1 is OH, R.sub.2 is OCH.sub.3,
R.sub.3 is OCH.sub.3 and R.sub.4 is .beta.-OH; [0254] a compound
having Formula (IV) above, wherein R.sub.1 is OCH.sub.3, R.sub.2 is
OCH.sub.3, R.sub.3 is OCH.sub.3 and R.sub.4 is .alpha.-OH; [0255] a
compound having Formula (IV) above, wherein R.sub.1 is OCH.sub.3,
R.sub.2 is OCH.sub.3, R.sub.3 is OCH.sub.3 and R.sub.4 is H; [0256]
a compound having Formula (IV) above, wherein R.sub.1 is H, R.sub.2
is OCH.sub.3, R.sub.3 is OCH.sub.3 and R.sub.4 is H; [0257] a
compound having Formula (V):
[0257] ##STR00023## [0258] and a compound having Formula (VI):
##STR00024##
[0259] which can be isolated from Tylophora atrofolliculata by the
method described above.
[0260] Further in accordance with the present invention is a
composition, preferably a pharmaceutical composition, comprising
and in particular essentially consisting of: [0261] at least one,
in particular one phenanthroindolizidine alkaloid, in particular as
pharmaceutically effective ingredient, isolated from Tylophora
atrofolliculata according to the method described above, and [0262]
at least one pharmaceutically tolerable excipient such as one or
more of a diluent, a filler, a binder, a disintegrant, a lubricant,
a coloring agent, a surfactant and a preservative.
[0263] The phenanthroindolizidine alkaloid comprised in the
composition, in particular in the pharmaceutical composition, is
preferably selected from at least one, more preferably one of:
[0264] a compound having Formula (I):
[0264] ##STR00025## [0265] wherein R.sub.1 is OH; [0266] a compound
having Formula (II):
[0266] ##STR00026## [0267] a compound having Formula (III):
[0267] ##STR00027## [0268] wherein R.sub.1 is OH, R.sub.2 is
OCH.sub.3, R.sub.3 is OH and R.sub.4 is H; [0269] a compound having
Formula (III) above, wherein R.sub.1 is OH, R.sub.2 is OCH.sub.3,
R.sub.3 is OH and R.sub.4 is .alpha.-OH; [0270] a compound having
Formula (IV):
[0270] ##STR00028## [0271] wherein R.sub.1 is H, R.sub.2 is OH,
R.sub.3 is OH and R.sub.4 is .beta.-OH; [0272] a compound having
Formula (IV) above, wherein R.sub.1 is OH, R.sub.2 is OCH.sub.3,
R.sub.3 is OCH.sub.3 and R.sub.4 is .beta.-OH; [0273] a compound
having Formula (IV) above, wherein R.sub.1 is OCH.sub.3, R.sub.2 is
OCH.sub.3, R.sub.3 is OCH.sub.3 and R.sub.4 is .alpha.-OH; [0274] a
compound having Formula (IV) above, wherein R.sub.1 is OCH.sub.3,
R.sub.2 is OCH.sub.3, R.sub.3 is OCH.sub.3 and R.sub.4 is H; [0275]
a compound having Formula (IV) above, wherein R.sub.1 is H, R.sub.2
is OCH.sub.3, R.sub.3 is OCH.sub.3 and R.sub.4 is H; [0276] a
compound having Formula (V):
[0276] ##STR00029## [0277] or a compound having Formula (VI):
##STR00030##
[0278] In more preferred embodiments of the present invention, the
phenanthroindolizidine alkaloid comprised in the composition is one
of: [0279] a compound having Formula (III) given above, wherein
R.sub.1 is OH, R.sub.2 is OCH.sub.3, R.sub.3 is OH and R.sub.4 is
H; [0280] a compound having Formula (III), wherein R.sub.1 is OH,
R.sub.2 is OCH.sub.3, R.sub.3 is OH and R.sub.4 is .alpha.-OH;
[0281] a compound having Formula (IV) given above, wherein R.sub.1
is H, R.sub.2 is OH, R.sub.3 is OH and R.sub.4 is .beta.-OH; [0282]
a compound having Formula (IV), wherein R.sub.1 is OH, R.sub.2 is
OCH.sub.3, R.sub.3 is OCH.sub.3 and R.sub.4 is .beta.-OH; [0283] a
compound having Formula (IV), wherein R.sub.1 is H, R.sub.2 is
OCH.sub.3, R.sub.3 is OCH.sub.3 and R.sub.4 is H; [0284] or a
compound having Formula (V) given above.
[0285] In most preferred embodiments of the present invention, the
phenanthroindolizidine alkaloid comprised in the composition is one
of: [0286] a compound having Formula (III) given above, wherein
R.sub.1 is OH, R.sub.2 is OCH.sub.3, R.sub.3 is OH and R.sub.4 is
H; [0287] a compound having Formula (III), wherein R.sub.1 is OH,
R.sub.2 is OCH.sub.3, R.sub.3 is OH and R.sub.4 is .alpha.-OH;
[0288] a compound having Formula (IV) given above, wherein R.sub.1
is H, R.sub.2 is OH, R.sub.3 is OH and R.sub.4 is .beta.-OH; [0289]
or a compound having Formula (V) given above.
[0290] The phenanthroindolizidine alkaloid is contained in the
composition, in particular the pharmaceutical composition,
preferably in an effective amount, i.e. an amount suitable to treat
or prevent a disease in a subject, in particular a human, which
also depends on the frequency and number of compositions to be
administered. The phenanthroindolizidine alkaloid administered
preferably has an IC.sub.50 regarding the inhibition of HIF-1 which
is below 300 nM, more preferably below 100 nM and in particular
less than 50 nM such as at most 45 nM. The cancer may be a breast
cancer.
[0291] The skilled person is able to select suitable
pharmaceutically tolerable excipients depending on the form of the
pharmaceutical composition and is aware of methods for
manufacturing pharmaceutical compositions as well as able to select
a suitable method for preparing the pharmaceutical composition
depending on the kind of pharmaceutically tolerable excipients and
the form of the pharmaceutical composition.
[0292] The pharmaceutical composition according to the invention
can be present in solid, semisolid or liquid form to be
administered by an oral, rectal, topical, parenteral or transdermal
or inhalative route to a subject, preferably a human.
[0293] The pharmaceutical composition may comprise further
pharmaceutical effective ingredients such as therapeutic compounds
used for treating cancer. The pharmaceutical composition may be
provided in form of a kit comprising the pharmaceutical composition
described above and at least one further pharmaceutical composition
having another active ingredient for treating cancer such as a
cytotoxic ingredient or an angiogenesis inhibitor.
[0294] Further in accordance with the present invention is a method
of treating a subject suffering from cancer comprising
administering an effective amount of at least one
phenanthroindolizidine alkaloid, preferably one
phenanthroindolizidine alkaloid, isolated from Tylophora
atrofolliculata according to the method described above to the
subject.
[0295] In particular, the method comprises administering an
affective amount of at least one phenanthroindolizidine alkaloid
and preferably one phenanthroindolizidine alkaloid: [0296] having
Formula (III):
[0296] ##STR00031## [0297] wherein R.sub.1 is OH, R.sub.2 is
OCH.sub.3, R.sub.3 is OH and R.sub.4 is H; [0298] having Formula
(III) above, wherein R.sub.1 is OH, R.sub.2 is OCH.sub.3, R.sub.3
is OH and R.sub.4 is .alpha.-OH; [0299] having Formula (III) above,
wherein R.sub.1 is H, R.sub.2 is OH, R.sub.3 is OH and R.sub.4 is
.alpha.-OH; [0300] having Formula (III) above, wherein R.sub.1 is
H, R.sub.2 is OCH.sub.3, R.sub.3 is OH and R.sub.4 is .alpha.-OH;
[0301] having Formula (III) above, wherein R.sub.1 is H, R.sub.2 is
OH, R.sub.3 is OCH.sub.3 and R.sub.4 is .alpha.-OH; [0302] having
Formula (III) above, wherein R.sub.1 is H, R.sub.2 is OCH.sub.3,
R.sub.3 is OCH.sub.3 and R.sub.4 is .beta.-OH; [0303] having
Formula (III) above, wherein R.sub.1 is H, R.sub.2 is OCH.sub.3,
R.sub.3 is OCH.sub.3 and R.sub.4 is H; [0304] having Formula (III)
above, wherein R.sub.1 is H, R.sub.2 is OCH.sub.3, R.sub.3 is
OCH.sub.3 and R.sub.4 is .alpha.-OH; [0305] having Formula
(IV):
[0305] ##STR00032## [0306] wherein R.sub.1 is H, R.sub.2 is OH,
R.sub.3 is OH and R.sub.4 is .beta.-OH; [0307] having Formula (IV)
above, wherein R.sub.1 is OH, R.sub.2 is OCH.sub.3, R.sub.3 is
OCH.sub.3 and R.sub.4 is .beta.-OH; [0308] having Formula (IV)
above, wherein R.sub.1 is H, R.sub.2 is OCH.sub.3, R.sub.3 is
OCH.sub.3 and R.sub.4 is H; [0309] having Formula (V):
[0309] ##STR00033## [0310] having Formula (VII):
[0310] ##STR00034## [0311] or having Formula (VIII):
##STR00035##
[0312] which can be isolated from Tylophora atrofolliculata by the
method described above.
[0313] Most preferably, the method comprises administering an
affective amount of a phenanthroindolizidine alkaloid: [0314]
having Formula (III) given above, wherein R.sub.1 is OH, R.sub.2 is
OCH.sub.3, R.sub.3 is OH and R.sub.4 is H; [0315] having Formula
(III), wherein R.sub.1 is OH, R.sub.2 is OCH.sub.3, R.sub.3 is OH
and R.sub.4 is .alpha.-OH; [0316] having Formula (IV) given above,
wherein R.sub.1 is H, R.sub.2 is OH, R.sub.3 is OH and R.sub.4 is
.beta.-OH; [0317] having Formula (IV), wherein R.sub.1 is OH,
R.sub.2 is OCH.sub.3, R.sub.3 is OCH.sub.3 and R.sub.4 is
.beta.-OH; [0318] having Formula (IV), wherein R.sub.1 is H,
R.sub.2 is OCH.sub.3, R.sub.3 is OCH.sub.3 and R.sub.4 is H; [0319]
having Formula (V) given above.
[0320] The subject is an animal or human, preferably it is a mammal
and most preferably a human. The expression "effective amount"
generally denotes an amount sufficient to produce therapeutically
desirable results, wherein the exact nature of the result varies
depending on the specific disorder which is treated. When the
disorder is cancer, the result is usually an inhibition or
suppression of the proliferation of the cancer cells, a reduction
of cancerous cells or the amelioration of symptoms related to the
cancer cells.
[0321] The effective amount of the phenanthroindolizidine alkaloid
isolated from Tylophora atrofolliculata may depend on the
IC.sub.50, the species, body weight, age and individual conditions
of the subject and can be determined by standard procedures such as
with cell cultures or experimental animals. The
phenanthroindolizidine alkaloid preferably has an IC.sub.50
regarding the inhibition of HIF-1 which is below 300 nM, more
preferably below 100 nM and in particular less than 50 nM such as
at most 45 nM. The cancer may be a breast cancer.
[0322] Another aspect relates to a method of treating a subject
suffering from cancer comprising: [0323] isolating at least one and
preferably one phenanthroindolizidine alkaloid from Tylophora
atrofolliculata by the method described above, in particular a
phenanthroindolizidine alkaloid: [0324] having Formula (III):
[0324] ##STR00036## [0325] wherein R.sub.1 is OH, R.sub.2 is
OCH.sub.3, R.sub.3 is OH and R.sub.4 is H; [0326] having Formula
(III) above, wherein R.sub.1 is OH, R.sub.2 is OCH.sub.3, R.sub.3
is OH and R.sub.4 is .alpha.-OH; [0327] having Formula (IV):
[0327] ##STR00037## [0328] wherein R.sub.1 is H, R.sub.2 is OH,
R.sub.3 is OH and R.sub.4 is .beta.-OH; [0329] having Formula (IV)
above, wherein R.sub.1 is OH, R.sub.2 is OCH.sub.3, R.sub.3 is
OCH.sub.3 and R.sub.4 is .beta.-OH; [0330] having Formula (IV)
above, wherein R.sub.1 is H, R.sub.2 is OCH.sub.3, R.sub.3 is
OCH.sub.3 and R.sub.4 is H; [0331] or having Formula (V):
[0331] ##STR00038## [0332] formulating the at least one
phenanthroindolizidine alkaloid into a pharmaceutically
composition; and [0333] administering said pharmaceutical
composition to a subject suffering from cancer. The subject is
preferably a human. The cancer is in particular breast cancer.
[0334] Further in accordance with the present invention is an at
least one phenanthroindolizidine alkaloid isolated from Tylophora
atrofolliculata with the method described above for use in the
treatment of cancer like breast cancer and the use of the at least
one phenanthroindolizidine alkaloid isolated from Tylophora
atrofolliculata with the method described above for preparing a
medicament for the treatment of cancer like breast cancer. The
phenanthroindolizidine alkaloid in particular is at least one and
preferably one of: [0335] a compound having Formula (III) given
above, wherein R.sub.1 is OH, R.sub.2 is OCH.sub.3, R.sub.3 is OH
and R.sub.4 is H; [0336] a compound having Formula (III), wherein
R.sub.1 is OH, R.sub.2 is OCH.sub.3, R.sub.3 is OH and R.sub.4 is
.alpha.-OH; [0337] a compound having Formula (IV) given above,
wherein R.sub.1 is H, R.sub.2 is OH, R.sub.3 is OH and R.sub.4 is
.beta.-OH; [0338] a compound having Formula (IV), wherein R.sub.1
is OH, R.sub.2 is OCH.sub.3, R.sub.3 is OCH.sub.3 and R.sub.4 is
.beta.-OH; [0339] a compound having Formula (IV), wherein R.sub.1
is H, R.sub.2 is OCH.sub.3, R.sub.3 is OCH.sub.3 and R.sub.4 is H;
[0340] or a compound having Formula (V) given above.
[0341] In most preferred embodiments of the present invention, the
phenanthroindolizidine alkaloid is one of: [0342] a compound having
Formula (III) given above, wherein R.sub.1 is OH, R.sub.2 is
OCH.sub.3, R.sub.3 is OH and R.sub.4 is H; [0343] a compound having
Formula (III), wherein R.sub.1 is OH, R.sub.2 is OCH.sub.3, R.sub.3
is OH and R.sub.4 is .alpha.-OH; [0344] a compound having Formula
(IV) given above, wherein R.sub.1 is H, R.sub.2 is OH, R.sub.3 is
OH and R.sub.4 is .beta.-OH; [0345] or a compound having Formula
(V) given above.
[0346] Another aspect concerns a method of treating a subject
suffering from cancer such as breast cancer comprising
administering at least one and preferably one
phenanthroindolizidine alkaloid isolated from Tylophora
atrofolliculata with the method described above in particular one
of a phenanthroindolizidine alkaloid: [0347] having Formula (III)
given above, wherein R.sub.1 is OH, R.sub.2 is OCH.sub.3, R.sub.3
is OH and R.sub.4 is H; [0348] having Formula (III), wherein
R.sub.1 is OH, R.sub.2 is OCH.sub.3, R.sub.3 is OH and R.sub.4 is
.alpha.-OH; [0349] having Formula (IV) given above, wherein R.sub.1
is H, R.sub.2 is OH, R.sub.3 is OH and R.sub.4 is .beta.-OH; [0350]
having Formula (IV), wherein R.sub.1 is OH, R.sub.2 is OCH.sub.3,
R.sub.3 is OCH.sub.3 and R.sub.4 is .beta.-OH; [0351] having
Formula (IV), wherein R.sub.1 is H, R.sub.2 is OCH.sub.3, R.sub.3
is OCH.sub.3 and R.sub.4 is H; [0352] or having Formula (V) given
above,
[0353] in combination with radiotherapy in particular with X-rays
and/or in combination with chemotherapy with further active
ingredients for treating cancer, in particular cytotoxic drugs or
angiogenesis inhibitors. The expression "in combination with" means
a simultaneous or sequential administration or application. In an
embodiment of the present invention, the phenanthroindolizidine
alkaloid isolated from Tylophora atrofolliculata is administered in
combination with radiotherapy, wherein the phenanthroindolizidine
alkaloid is administered either before or after each radiation,
preferably after each radiation.
[0354] In still another aspect, the present invention refers to a
method for targeting cancer cells in particular breast cancer
cells. Said method comprises the step of contacting a population of
cancer cells with at least one and preferably one
phenanthroindolizidine alkaloid described above. The concentration
of the phenanthroindolizidine alkaloid for contacting the cancer
cells is preferably between 3 .mu.M and 300 .mu.M, more preferably
between 3 .mu.M and 100 .mu.M.
EXAMPLES
Example 1
Example 1A
[0355] Materials Used and Conditions Applied
[0356] Optical rotations were determined using a Rudolph Research
Analytical Autopol I Autometic polarimeter. CD spectra were
measured on a Chirascan Circular Dichroism spectrometer. UV data
were recorded using a Beckman DU 800 UV/vis spectrophotometer. IR
spectra were performed on a Perkin Elmer Spectrum One Fourier
transform infrared spectrometer. HRESIMS were recorded on an
Agilent 6230 ESI-TOF mass spectrometer. NMR spectral data were
obtained from Bruker Ascend.TM. 600 spectrometer equipped with a
cyro platform. Silica gel (Devisil.RTM., 40-63 micron), MCI CH20P
gel (Mitsubishi Chemical Corporation) and ODS (Waters, Preparative
C18 125 .ANG., 55.about.105 .mu.m) were used for column
chromatography. Silica gel plates (Merck, DC Kieselgel 60
F.sub.254) were used for TLC analysis. High-performance liquid
chromatography (HPLC) was carried out on Agilent 1100, Agilent 1200
and Waters 1525-2489 apparatus with a semi-preparative column
(Waters, XBridge.RTM. Prep C18, 5 .mu.m, 250.times.10 mm). Solvents
for HPLC separation analysis were HPLC grade.
[0357] The whole plant of T. atrofolliculata was collected in
Guangxi province, China in 2012 and identified by Dr. Zhifeng Zhang
(Faculty of Chinese Medicine, Macau University of Science and
Technology). A voucher specimen (No. MUST-TA201302) was deposited
at State Key Laboratory of Quality Research in Chinese Medicine,
Macau University of Science and Technology.
Example 1B
[0358] Isolation of Phenanthroindolizidine Alkaloids from T.
Atrofolliculata
[0359] The whole plant of T. atrofolliculata (5.5 kg) was refluxed
with methanol for three times to afford a crude extract, namely
with amounts of 55 L, 44 L and 33 L of methanol subsequently.
Totally 8 h were spent for the solvent extraction, which has been
carried out for three times following the order of 4 h, 2 h, and 2
h subsequently. The crude extract after methanol evaporation under
reduced pressure was suspended in water as first separation solvent
then adjusted to pH 1.about.2 by adding hydrochloric acid. After
being partitioned with ethyl acetate as second separation solvent,
the acidic aqueous phase as first layer was basified with 10%
sodium hydroxide to pH 9.about.10 then extracted with chloroform as
third separation solvent to afford the third layer with the crude
alkaloids (12.6 g).
[0360] The third layer after evaporation of chloroform in vacuum
was chromatographed over silica gel rendering thirteen fractions,
which were further separated through repeated column chromatography
(silica gel, ODS, HPLC) to afford twenty-two phenanthroindolizidine
alkaloids (compounds (1) to (22)).
[0361] The crude alkaloid extract was subjected to chromatography
on MCI CH20P gel eluting with methanol-H.sub.2O-diethyl amine
(7:3:0.05.about.10:0:0.05), followed by acetone-methanol-diethyl
amine (1:9:0.05.about.6:4:0.05) to yield compound (18) (451 mg) and
15 fractions based on the TLC behavior. Fr. 3 was separated by
silica gel using chloroform-methanol-diethyl amine
(10:0:0.05.about.3:7:0.05) to give 17 subfractions based on the TLC
behavior. Subfr. 3-15 was chromatographed over ODS
(methanol-H.sub.2O, 1:1.about.4:1), and then purified by HPLC to
give compound (6) (2 mg), compound (7) (1 mg). Fr. 4 was
chromatographed on silica gel column eluting with
chloroform-methanol-diethyl amine (10:0:0.05.about.3:1:0.05) to
afford compound (5) (14 mg) and (6) subfractions. Subfr. 4-6 was
subjected to chromatography on ODS (methanol-H.sub.2O, 1:1) to
afford compound (13) (6 mg). Fr. 5 was chromatographed on silica
gel column eluting with chloroform-methanol-diethyl amine
(10:0:0.05.about.5:2:0.05) to obtain compound (4) (111 mg). Fr. 8
was subjected to separation over silica gel using
chloroform-methanol-diethyl amine (10:0:0.05.about.7:3:0.05) to
give compound (14) (267 mg) and 24 subfractions based on the TLC
behavior. Subfr. 8-15 was chromatographed on ODS column
(methanol-H.sub.2O, 1:1) then purified by HPLC to afford compound
(1) (3 mg). Subfr. 8-16 was chromatographed on ODS column
(methanol-H.sub.2O, 7:3) then purified by HPLC to give compound
(15) (1 mg). Subfr. 8-23 was purified by ODS column
(methanol-H.sub.2O, 2:3.about.3:2) to give compound (16) (191 mg).
Fr. 9 was loaded on silica gel column eluting with
chloroform-methanol-diethyl amine (10:0:0.05.about.5:5:0.05), then
purified by repeated HPLC to give compound (8) (1 mg). Fr. 11 was
separated by silica gel eluting with chloroform-methanol-diethyl
amine (10:0:0.05.about.4:6:0.05) to give compound (19) (8 mg). Fr.
12 was subjected on silica gel eluting with
chloroform-methanol-diethyl amine (10:0:0.05.about.4:6:0.05), then
purified by HPLC to give compound (9) (1 mg). Fr. 13 was
chromatographed on silica gel eluting with
chloroform-methanol-diethyl amine (10:0:0.05.about.4:6:0.05) then
purified by HPLC to give compound (2) (1 mg), compound (3) (1 mg),
compound (10) (20 mg), compound (11) (5 mg), compound (12) (2 mg),
compound (17) (40 mg), compound (20) (3 mg), compound (21) (6 mg),
compound (22) (7 mg).
[0362] The chemical structures of compounds 1 and 3 to 12 were
elucidated by means of NMR methods including .sup.1H-.sup.1H COSY,
NOESY, HSQC and HMBC experiments, assisted by high-resolution MS
and CD spectral analysis.
[0363] Compound (1) was obtained as white amorphous solid. Its
molecular formula was determined as C.sub.22H.sub.21NO.sub.5 by
molecular ion at m/z 380.1488 ([M+H].sup.+, calcd 380.1492). The
.sup.1H NMR data (Table 1) indicated the presence of three aromatic
protons with a ABX system [.delta..sub.H 8.14 (d, J=9.0 Hz), 7.89
(d, J=2.4 Hz), 7.25 (dd, J=2.4, 9.0 Hz)], one 1, 2, 4,
5-tetra-substituted benzene ring (.delta..sub.H 8.10, 7.28, each
s), two methoxyl groups (.delta..sub.H 3.98, 4.02, each s), two
methylene doublets [.delta..sub.H 5.17 (d, J=17.4 Hz), 4.51 (d,
J=17.4 Hz)], four methylene multiplets (.delta..sub.H 2.44, 2.37,
2.36, 2.19, each m), one nitrogenated methine (.delta..sub.H 3.91,
m), one oxygenated methine (.delta..sub.H 5.09, dd, J=2.4, 7.2 Hz)
and one hydroxyl group (.delta..sub.H 5.41, d, J=7.2 Hz). The
.sup.13C NMR spectrum (Table 2) revealed twenty-two carbon
resonances, corresponding to fourteen aromatic (three oxygenated
and six quaternary carbons), three methylene (one nitrogenated
methylene group), two methine (one oxygenated and one nitrogenated
methine group), two methoxyl and one carbonyl carbons. The NMR data
of compound (1) (see Tables 1 and 2) closely resembled those of
compound (14) (Zhen, Y. et al., Acta Bot. Sin., 2002, 44, 349-353)
except for the presence of an additional carbonyl group
(.delta..sub.C 173.5) instead of the C-11 methylene group, which
was confirmed by HMBC correlations of H-9, H-12, H-13 and H-13a
with C-11 (FIGS. 1A and 1B). The methoxyl groups were determined to
be placed at C-3 and C-7 on the basis of HMBC correlations of
OCH.sub.3-3 with C-3, OCH.sub.3-7 with C-7 as well as the NOESY
correlations OCH.sub.3-3/H-2 and H-4, OCH.sub.3-7/H-8 (FIGS. 1A and
1B). The hydroxyl group was thus placed at C-6 due to the carbon
resonance at .delta..sub.C 146.9 (Table 2). Relative configuration
of compound (1) was assigned on the basis of the coupling constant
and NOE correlation. The small coupling constant (J=2.4 Hz) between
H-13a and H-14 and the strong NOE correlation H-13a/H-14 prompted
the cis orientation for both protons (FIGS. 1A and 1B). Absolute
configuration of compound (1) was determined through its CD
spectrum, where the negative Cotton effect was observed at 253 nM,
suggesting S configuration at C-13a (Damu, A. G. et al., Planta
Med., 2009, 75, 1152-1156, Zhen, Y. et al., Acta Bot. Sin., 2002,
44, 349-353, St.ae butted.rk, D. et al., J. Nat. Prod., 2000, 63,
1584-1586, S. F, J. F. Mi et al., Acta Pharm. Sin. B, 27, 197-203).
Thus, compound (1) was identified as 11-keto-tylophorinidine (i.e.
having Formula (I) with R.sub.1 being OH).
[0364] 11-keto-tylophorinidine (compound (1)). White powder;
[.alpha.].sub.D.sup.25 -66 (c 0.38 CHCl.sub.3-MeOH 3:1); UV
(CHCl.sub.3) .lamda..sub.max (log .epsilon.) 250 (4.56, sh), 259
(4.65), 286 (4.40), 313 (3.93) nm; CD (MeOH) 204 (.DELTA..epsilon.
-6.93), 253 (.DELTA..epsilon. -1.59) nm; .sup.1H and .sup.13C NMR
data, see Tables 1 and 2; HRESIMS m/z 380.1488 [M+H].sup.+ (calcd
for C.sub.22H.sub.21NO.sub.5, 380.1492).
[0365] Compound (3) was obtained as white powder. Its molecular
formula C.sub.26H.sub.29NO.sub.6 was deduced from HRESIMS peak at
m/z 452.2069 ([M+H].sup.+, calcd 452.2068). The .sup.1H and
.sup.13C NMR spectra (Table 3) collectively revealed three methoxyl
groups, one methyl group, four methylene groups (one nitrogenated),
three methine groups (two oxygenated, one nitrogenated), one
hydroxyl group and one carbonyl group. The NMR data of compound (3)
(Table 3) closely matched those of 14aS, 15S-hydroxyboehmeriasin A
(Z. Wang and Q. Wang, Tetrahedron Lett., 2010, 51, 1377-1379)
except for an additional oxazine instead of C-11 methylene, and a
2-oxopropyl group located at C-12. The formation of oxazine was
inferred from carbon resonance of C-12 at .delta..sub.C 74.1 (Table
3) (Labaziewicz, H. et al., J. Chem. Soc., 1977, 619-622). The HMBC
correlations of H-16 and H-18 with C-17, H-18 with C-16 established
the 2-oxopropyl group, which was connected to C-12 based on the
HMBC correlation of H-16 with C-12 along with .sup.1H-.sup.1H COSY
correlation H-16/H-12 (FIGS. 2A and 2B). The methoxyl groups were
assigned at C-3, C-6, C-7 by means of the HMBC correlations of
OCH.sub.3-3 with C-3, OCH.sub.3-6 with C-6, OCH.sub.3-7 with C-7,
as well as the NOE correlations OCH.sub.3-3/H-2 and H-4,
OCH.sub.3-6/H-5, OCH.sub.3-7/H-8 (FIGS. 2A and 2B). Relative
configuration of compound (3) was assigned on the basis of NOE
correlations and coupling constant. The strong NOE correlation
H-14a/H-15 and the coupling constant (J=6.0 Hz) between H-14a and
H-15 indicated that H-15 was cis to H-14a (Cai, X. F. et al., J.
Nat. Prod., 2006, 69, 1095-1097). The strong NOE correlation
H-12/H-14a suggested the cis position of both protons (FIGS. 2A and
2B). Absolute configuration of compound (3) was assigned through
comparing its optical rotation with known compounds. Since 14aS,
15S-hydroxyboehmeriasin A had a specific optical activity of
[.quadrature.].sub.D.sup.20 +57 (c 0.5 CHCl.sub.3) (Z. Wang and Q.
Wang, Tetrahedron Lett., 2010, 51, 1377-1379). Absolute
configuration of compound (3) at H-14a was deduced to be S
tentatively on the basis of its positive optical rotation value
(Cai, X. F. et al., J. Nat. Prod., 2006, 69, 1095-1097, Z. Wang and
Q. Wang, Tetrahedron Lett., 2010, 51, 1377-1379, T. F. Buckley and
R. Henry, J. Org. Chem., 1983, 48, 4222-4232). Hence, compound (3)
was established as 12S, 14aS,
15S-11-oxa-12-(2-oxopropyl)-hydroxyboehmeriasin A (i.e. having
Formula (II)).
[0366] 12S, 14aS, 15S-11-oxa-12-(2-oxopropyl)-hydroxyboehmeriasin A
(compound (3)). White powder; [.alpha.].sub.D.sup.23 +9 (c 0.1
CHCl.sub.3); UV (CHCl.sub.3) .lamda..sub.max (log .epsilon.) 257
(4.84), 285 (4.54), 310 (4.01) nm; .sup.1H and .sup.13C NMR data,
see Table 3; HRESIMS m/z 452.2069 [M+H].sup.+ (calcd for
C.sub.26H.sub.29NO.sub.6, 452.2068).
[0367] Compound (4) was isolated as white powder. HRESIMS gave a
protonated molecule at m/z 366.1692 [M+H].sup.+ (calcd 366.1700 for
C.sub.22H.sub.23NO.sub.4). The .sup.1H NMR data (Table 1) indicated
the presence of two 1, 2, 4, 5-tetra-substituted benzene rings
(.delta..sub.H 7.95, 7.82, 7.25, 7.14, each s), two methoxyl groups
(.delta..sub.H 3.93, 3.99, each s), two methylene doublets
[.delta..sub.H 4.52 (d, J=15.0 Hz), 3.47 (d, J=15.0 Hz)], eight
methylene multiplets (.delta..sub.H 3.30, 3.17, 2.65, 2.31, 2.13,
1.83, 1.82, 1.60, each m), one nitrogenated methine (.delta..sub.H
2.34, m). The .sup.13C NMR spectra (Table 2) revealed twenty-two
carbon resonances, corresponding to fourteen aromatic (four
oxygenated and six quaternary carbons), five methylene (two
nitrogenated methylene groups), two methoxyl and one nitrogenated
methine carbons. All the aforementioned information indicated the
presence of phenanthroindolizidine moiety (Huang, X. et al., Planta
Med., 2004, 70, 441-445). The two methoxyl groups were determined
to be placed at C-3 and C-7 via the strong HMBC correlations of
OCH.sub.3-3 with C-3, OCH.sub.3-7 with C-7, and weak correlations
of OCH.sub.3-3 with C-4, OCH.sub.3-7 with C-8 (FIGS. 3A and 3B).
This was further confirmed by the NOESY spectrum, which displayed
correlations OCH.sub.3-3/H-4 and OCH.sub.3-7/H-8 (FIGS. 3A and 3B).
The remaining functionalities were designated as two hydroxyl
groups and assigned to C-2 and C-6 due to the presence of carbon
resonances at .delta..sub.C 146.2 and 146.0 (Table 2). The absolute
stereochemistry of compound (4) was inferred from its and CD
spectrum. The negative Cotton effect at 257 nM in CD spectrum
inferred the S configuration at C-13a (Damu, A. G. et al., Planta
Med., 2009, 75, 1152-1156, Zhen, Y. et al., Acta Bot. Sin., 2002,
44, 349-353, St.ae butted.rk, D. et al., J. Nat. Prod., 2000, 63,
1584-1586). The data described above led to the assignment of the
structure of compound (4) as 2-hydroxyl deoxytylophorinidine (i.e.
having Formula (III) with R.sub.1 being OH, R.sub.2 being
OCH.sub.3, R.sub.3 being OH and R.sub.4 being H).
[0368] 2-hydroxyl deoxytylophorinidine (compound (4)). White
powder; [.alpha.].sub.D.sup.24 -156 (c 0.26, CHCl.sub.3-MeOH 4:1);
UV (MeOH) .lamda..sub.max (log .epsilon.) 257 (4.72), 248 (4.57,
sh), 291 (4.49), 304 (4.26, sh) nm; IR (KBr): v.sub.max=2960, 2936,
2831, 1620, 1516, 1465, 1261, 1203, 1155, 1039, 854, 785 cm.sup.-1;
CD (MeOH) 257 (.DELTA..epsilon. -45.00) nm; .sup.1H and .sup.13C
NMR data, see Tables 1 and 2; HRESIMS m/z 366.1692 [M+H].sup.+
(calcd for C.sub.22H.sub.23NO.sub.4, 366.1700).
[0369] Compound (5) was obtained as white powder. Its molecular
formula C.sub.22H.sub.23NO.sub.5 derived from protonated molecular
ion peak (m/z 382.1665 [M+H].sup.+, calcd 382.1649). Compound (5)
was clearly an analogue of compound (4), as it possessed multiple
same carbon and proton resonances (Table 2). A close inspection of
the NMR spectra revealed that C-14 was substituted with a hydroxyl
group, which was supported by .sup.1H-.sup.1H COSY correlation
OH-14/H-14 and HMBC correlation of OH-14 with C-14. The two
methoxyl groups were decided to be placed at C-3 and C-7 via the
HMBC correlations of OCH.sub.3-3 with C-3, OCH.sub.3-7 with C-7,
and the NOESY correlations OCH.sub.3-3/H-4 and OCH.sub.3-7/H-8.
Relative configuration of compound (5) was assigned on the basis of
NOE correlation and coupling constant. The small coupling constant
(J=1.8 Hz) between H-13a and H-14 and strong NOE correlation
H-13a/H-14 led to the cis configuration of both protons (Damu, A.
G. et al., Planta Med., 2009, 75, 1152-1156, St.ae butted.rk, D. et
al., J. Nat. Prod., 2000, 63, 1584-1586). The absolute
stereochemistry of compound (5) was inferred from its optical
rotation and CD spectrum. The positive optical rotation and the
negative Cotton effect at 261 nm in the CD spectrum indicated the S
configuration at C-13a (Damu, A. G. et al., Planta Med., 2009, 75,
1152-1156, St.ae butted.rk, D. et al., J. Nat. Prod., 2000, 63,
1584-1586, S. F, J. F. Mi, et al., Acta Pharm. Sin. B, 27,
197-203). Overall, the data above contributed to the
characterization of compound (5) as 2-hydroxyl tylophorinidine
(i.e. having Formula (III) above, wherein R.sub.1 is OH, R.sub.2 is
OCH.sub.3, R.sub.3 is OH and R.sub.4 is .alpha.-OH).
[0370] 2-hydroxyl tylophorinidine (compound (5)). White powder;
[.alpha.].sub.D.sup.24 +13 (c 0.18 CHCl.sub.3-MeOH 4:1); UV (MeOH)
.lamda..sub.max (log .epsilon.) 250 (4.60, sh), 258 (4.73), 290
(4.49), 304 (4.26, sh) nm; CD (MeOH) 261 (.DELTA..quadrature.
-0.26) nm; .sup.1H and .sup.13C NMR data, see Tables 1 and 2;
HRESIMS m/z 382.1665 [M+H].sup.+ (calcd for
C.sub.22H.sub.23NO.sub.5, 382.1649).
[0371] Compound (6) was obtained as brown amorphous solid. Its
molecular formula was determined as C.sub.21H.sub.21NO.sub.5 by
peak at m/z 368.1500 ([M+H].sup.+, calcd 368.1492). The NMR data of
compound (6) closely resembled those of compound (13) with
differences ascribed to the resonances at 9, 11, 13a, and 14
(Huang, X. et al., Planta Med., 2004, 70, 441-445, Damu, A. G. et
al., J. Nat. Prod., 2005, 68, 1071-1075) (Tables 1 and 2). In the
.sup.1H-NMR spectrum of 6 (Table 1), downfield shifts of H-9
(.delta..sub.H 5.13, 4.78), H-11 (.delta..sub.H 3.63, 3.57), H-13a
(.delta..sub.H 3.64), H-14 (.delta..sub.H 5.10) (Table 1) were
observed with reference to those of compound (13) (see Tables 4 to
6). Likewise, deshielding carbon resonances at C-9 (.delta..sub.C
65.9), C-11 (.delta..sub.C 69.3), C-13a (.delta..sub.C 69.4) (Table
2) were observed in .sup.13C-NMR spectrum. The aforementioned data
indicated that compound (6) was definitely a N-oxide analogue of
compound (13). The methoxyl group was thus connected to C-7 on the
basis of HMBC correlation of OCH.sub.3-7 with C-7, and NOESY
correlation OCH.sub.3-7/H-8. Neither coupling constant nor NOE was
observed between H-13a and H-14. Therefore, H-14 was assigned to be
trans to H-13a. The appearance of H-13a signal at .delta..sub.H
3.64 (Table 1) indicated no substituent effects of axial N-oxides,
prompting the trans configuration of N-oxide to H-13a (Damu, A. G.
et al., J. Nat. Prod., 2005, 68, 1071-1075, Lavault, M. et al.,
Pharm. Acta Helv., 1994, 68, 225-227). Compound (6) displayed a
positive optical rotation and a negative Cotton effect at 242 nm in
the CD spectrum, indicating S configuration at C-13a (Damu, A. G.
et al., Planta Med., 2009, 75, 1152-1156, St.ae butted.rk, D. et
al., J. Nat. Prod., 2000, 63, 1584-1586, S. F, J. F. Mi et al.,
Acta Pharm. Sin. B, 27, 197-203). As a result, compound (6) was
established as 10R, 14R-3-O-demethyl tylophorinidine N-oxide (i.e.
having Formula (IV), wherein R.sub.1 is H, R.sub.2 is OH, R.sub.3
is OH and R.sub.4 is .beta.-OH).
[0372] 10R, 14R-3-O-demethyl tylophorinidine N-oxide (compound
(6)). Brown amorphous solid; [.alpha.].sub.D.sup.30 +16 (c 0.32
DMSO-MeOH 1:1); UV (MeOH) .lamda..sub.max (log .epsilon.) 250
(4.43, sh), 258 (4.51), 285 (4.23), 315 (3.79) nm; CD (MeOH) 242
(.DELTA..epsilon. -5.37) nm; .sup.1H and .sup.13C NMR data, see
Tables 1 and 2; HRESIMS m/z 368.1500 [M+H].sup.+ (calcd for
C.sub.21H.sub.21NO.sub.5, 368.1492).
[0373] HRESIMS analysis of compound (7) gave a protonated molecule
at m/z 398.1604 [M+H].sup.+, which was consistent with the
molecular formula C.sub.22H.sub.23NO.sub.6 (calcd 398.1590).
Compound (7) was the N-oxide analogue of compound (5) on the basis
of the deshielding signals at 9, 11, 13a, and 14 (Huang, X. et al.,
Planta Med., 2004, 70, 441-445, Damu, A. G. et al., J. Nat. Prod.,
2005, 68, 1071-1075) (Tables 1 and 2). In the .sup.1H-NMR spectrum
of compound (7), downfield shifts of H-9 (.delta..sub.H 5.20,
4.86), H-11 (.delta..sub.H 3.71, 3.63), H-13a (.delta..sub.H 3.73),
H-14 (.delta..sub.H 5.03) were observed with reference to those of
5 (Table 1). Likewise, deshielding carbon resonances at C-9
(.delta..sub.C 66.0), C-11 (.delta..sub.C 69.3), C-13a
(.delta..sub.C 69.4) were observed in .sup.13C-NMR spectrum (Table
2). The methoxyl groups were thus connected to C-3 and C-7 on the
basis of HMBC correlation of OCH.sub.3-3 with C-3, OCH.sub.3-7 with
C-7, and NOESY correlations OCH.sub.3-3/H-4, OCH.sub.3-7/H-8. The
hydroxyl groups were determined to be placed at C-2 and C-6 based
on the carbon signals at .delta..sub.C 146.7 and 147.2 (Table 2).
The small coupling constant (J=1.8 Hz) between H-13a and H-14 and
strong NOE correlation H-13a/H-14 revealed cis orientation of both
protons (Damu, A. G. et al., Planta Med., 2009, 75, 1152-1156,
St.ae butted.rk, D. et al., J. Nat. Prod., 2000, 63, 1584-1586).
The appearance of H-13a signal at .delta..sub.H 3.73 (Table 1)
indicated no substituent effects of axial N-oxides. Hence, the
configuration of N-oxide was determined to be trans to H-13a (Damu,
A. G. et al., J. Nat. Prod., 2005, 68, 1071-1075, Lavault, M. et
al., Pharm. Acta Helv., 1994, 68, 225-227). Absolute configuration
of compound (7) was assigned via comparing its optical rotation
value with known compound. Since compound (5) had a specific
optical activity of [.alpha.].sub.D.sup.24 +13 (c 0.18
CHCl.sub.3-MeOH 4:1), the negative optical rotation of compound (7)
suggested the R configuration at C-13a (Lee, Y. Z. et al., Planta
Med., 2011, 77, 1932-1938, Lykkeberg, A. K. et al., J. Nat. Prod.,
2002, 65, 1299-1302) tentatively. Therefore, compound (7) was
established as 10S, 13aR, 14R-2-hydroxyl tylophorinidine (i.e.
having Formula (V)).
[0374] 10S, 13aR, 14R-2-hydroxyl tylophorinidine N-oxide (compound
(7)). White powder; [a].sub.D.sup.26 -307 (c 0.02 DMSO-MeOH 3:5);
UV (MeOH) .lamda..sub.max (log .epsilon.) 258 (4.54), 282 (4.18,
sh), 288 (4.23), 304 (3.97, sh) nm; .sup.1H and .sup.13C NMR data,
see Tables 1 and 2; HRESIMS m/z 398.1604 [M+H].sup.+ (calcd for
C.sub.22H.sub.24NO.sub.6, 398.1590).
[0375] The molecular formula of compound (8) was assigned as
C.sub.23H.sub.25NO.sub.6 by HRESIMS ([M+H].sup.+, m/z 412.1766;
calcd 412.1755), which showed one methylene more than that of
compound (7). The NMR data of compound (8) closely matched those of
compound (7) (Tables 1 and 2) except for the presence of an
additional methoxyl group instead of hydroxyl group at C-6, which
was further confirmed by HMBC correlation of OCH.sub.3-6 with C-6
together with NOESY correlation OCH.sub.3-6/H-5. The remaining two
methoxyl groups were placed at C-3 and C-7 based on HMBC
correlations of OCH.sub.3-3 with C-3, OCH.sub.3-7 with C-7, as well
as the NOESY correlations OCH.sub.3-3/H-4 and OCH.sub.3-7/H-8. The
hydroxyl group was connected to C-2 based on the carbon resonance
at .delta..sub.C 147.0 (Table 2). H-14 was assigned to be trans to
H-13 since no coupling constant nor NOE was observed between both.
The configuration of N-oxide was determined to be trans to H-13a
due to the appearance of H-13a signal at .delta..sub.H 3.76 (Table
1) without substituent effects of axial N-oxides (Damu, A. G. et
al., J. Nat. Prod., 2005, 68, 1071-1075, Lavault, M. et al., Pharm.
Acta Helv., 1994, 68, 225-227). The positive optical rotation value
and the negative Cotton effect at 259 nm in the CD spectrum
indicated the S configuration at C-13a (Damu, A. G. et al., Planta
Med., 2009, 75, 1152-1156, St.ae butted.rk, D. et al., J. Nat.
Prod., 2000, 63, 1584-1586, S. F, J. F. Mi et al., Acta Pharm. Sin.
B, 27, 197-203). Therefore, compound (8) was determined as
10R-2-hydroxyl tylophorinine N-oxide (i.e. having Formula (IV)
above, wherein R.sub.1 is OH, R.sub.2 is OCH.sub.3, R.sub.3 is
OCH.sub.3 and R.sub.4 is
[0376] 10R-2-hydroxyl tylophorinine N-oxide (compound (8)). White
powder; [.alpha.].sub.D.sup.24 +94 (c 0.19 CHCl.sub.3-MeOH 1:1); UV
(MeOH) .lamda..sub.max (log .epsilon.) 260 (4.22), 289 (3.97), 305
(3.72, sh) nm; CD (MeOH) 227 (.DELTA..epsilon. -0.88), 259
(.DELTA..quadrature. -2.56) nm; .sup.1H and .sup.13C NMR data, see
Tables 1 and 2. HRESIMS m/z 412.1766 [M+H].sup.+ (calcd for
C.sub.23H.sub.25NO.sub.6, 412.1755).
[0377] Compound (9) was obtained as white powder. The molecular
formula C.sub.23H.sub.25NO.sub.5 was deduced from HRESIMS peak at
m/z 396.1803 ([M+H].sup.+, calcd 396.1805 for
C.sub.23H.sub.26NO.sub.5). Compound (9) possessed similar NMR data
as those of compound (21) (Tables 1 and 2) (Zhen, Y. et al., Acta
Bot. Sin., 2002, 44, 349-353). The difference was attributed to the
additional hydroxyl group placed at C-2 on the basis of the carbon
signals at .delta..sub.C 146.7 (Table 2). The HMBC correlations of
OCH.sub.3-3 with C-3, OCH.sub.3-6 with C-6, OCH.sub.3-7 with C-7,
and NOESY correlations OCH.sub.3-3/H-4, OCH.sub.3-6/H-5,
OCH.sub.3-7/H-8 collectively indicated the placement of the
methoxyl groups at C-3, C-6, C-7. The strong NOE correlation
H-13a/H-14 indicated the cis configuration of both protons (Lee, Y.
Z. et al., Planta Med., 2011, 77, 1932-1938).
[0378] The negative optical rotation value and the positive Cotton
effect at 239 nm in the CD spectrum indicated the R stereochemistry
at C-13a (Damu, A. G. et al., Planta Med., 2009, 75, 1152-1156,
St.ae butted.rk, D. et al., J. Nat. Prod., 2000, 63, 1584-1586, S.
F, J. F. Mi et al., Acta Pharm. Sin. B, 27, 197-203). Thus compound
(9) was elucidated as 13aR-2-hydroxyl tylophorinine (i.e. having
Formula (VI)). Compound (9) was isolated as chloric salt form for
it was treated with hydrochloric acid before subjected to
chromatography for purification.
[0379] 13aR-2-hydroxyl tylophorinine (compound (9)). White powder;
[.alpha.].sub.D.sup.23 -23 (c 0.3 CHCl.sub.3); UV (CHCl.sub.3)
.lamda..sub.max (log .epsilon.) 249 (4.37, sh), 259 (4.53), 290
(4.30), 303 (4.08) nm; CD (MeOH) 239 (.DELTA..epsilon. +5.90) nm;
.sup.1H and .sup.13C NMR data, see Tables 1 and 2; HRESIMS m/z
396.1803 [M+H].sup.+ (calcd for C.sub.23H.sub.25NO.sub.5,
396.1805).
[0380] Compound (10) was obtained as white powder. The molecular
formula C.sub.24H.sub.27NO.sub.6 was deduced from HRESIMS peak at
m/z 426.1901 ([M+H].sup.+, calcd 426.1911). The NMR data of
compound (10) (Tables 1 and 2) corresponding to the phenanthrene
moiety closely resembled those of 14.beta.-hydroxytylophorine
N-oxide, suggesting it to be an isomer (Nakano, D. et al., J. Nat.
Med., 2015, 69, 397-401). The methoxyl groups inferred from .sup.1H
NMR spectrum were thus placed at C-2, C-3, C-6, C-7 based on HMBC
correlations of OCH.sub.3-2 with C-2, OCH.sub.3-3 with C-3,
OCH.sub.3-6 with C-6, OCH.sub.3-7 with C-7, as well as the NOESY
correlations OCH.sub.3-2/H-1, OCH.sub.3-3/H-4, OCH.sub.3-6/H-5,
OCH.sub.3-7/H-8. The small coupling constant (J=2.4 Hz) between
H-13a and H-14 and the strong NOE correlation H-13a/H-14 led to the
configuration of H-14, which was cis to H-13a (Damu, A. G. et al.,
Planta Med., 2009, 75, 1152-1156, St.ae butted.rk, D. et al., J.
Nat. Prod., 2000, 63, 1584-1586). The appearance of H-13a signal at
.delta..sub.H 3.73 (Table 1) indicated no substituent effects of
axial N-oxides. Hence, the configuration of N-oxide was determined
to be trans to H-13a (Damu, A. G. et al., J. Nat. Prod., 2005, 68,
1071-1075, Lavault, M. et al., Pharm. Acta Helv., 1994, 68,
225-227). The positive optical rotation value and the negative
Cotton effect at 279 nm in the CD spectrum suggest S configuration
at C-13a (Damu, A. G. et al., Planta Med., 2009, 75, 1152-1156,
St.ae butted.rk, D. et al., J. Nat. Prod., 2000, 63, 1584-1586, S.
F, J. F. Mi et al., Acta Pharm. Sin. B, 27, 197-203). Thus,
compound (10) was identified as 10R, 13aS,
14S-14-hydroxytylophorine N-oxide (i.e. having Formula (IV) above,
wherein R.sub.1 is OCH.sub.3, R.sub.2 is OCH.sub.3, R.sub.3 is
OCH.sub.3 and R.sub.4 is .alpha.-OH).
[0381] 10R, 13aS, 14S-14-hydroxytylophorine N-oxide (compound
(10)). White powder; [.alpha.].sub.D.sup.23 +25 (c 0.29
CHCl.sub.3); UV (CHCl.sub.3) .lamda..sub.max (log .epsilon.) 260
(4.73), 288 (4.48), 305 (4.19, sh), 313 (3.81, sh) nm; CD (MeOH)
279 (.DELTA..epsilon. -1.92) nm; IR (KBr): v.sub.max=3397, 2965,
1620, 1514, 1473, 1427, 1255, 1213, 1197, 1151, 1035, 1012, 846,
781 cm.sup.-1; .sup.1H and .sup.13C NMR data, see Tables 1 and 2;
HRESIMS m/z 426.1901 [M+H].sup.+ (calcd for
C.sub.24H.sub.27NO.sub.6, 426.1911).
[0382] Compound (11) was obtained as white powder. The molecular
formula C.sub.24H.sub.27NO.sub.5 was deduced from HRESIMS peak at
m/z 410.1956 ([M+H].sup.+, calcd 410.1962). The NMR data of
compound (11) (Tables 1 and 2) corresponding to the phenanthrene
moiety closely matched those of 10R, 13aR tylophorine N-oxide,
suggesting it to be an isomer (Damu, A. G. et al., J. Nat. Prod.,
2005, 68, 1071-1075). Consequently, the four methoxyl groups
deducing from .sup.1H NMR spectral data were placed at C-2, C-3,
C-6, and C-7. This was further confirmed by the observed HMBC
correlations of OCH.sub.3-2 with C-2, OCH.sub.3-3 with C-3,
OCH.sub.3-6 with C-6, OCH.sub.3-7 with C-7, and NOESY correlations
OCH.sub.3-2/H-1, OCH.sub.3-3/H-4, OCH.sub.3-6/H-5, OCH.sub.3-7/H-8.
The appearance of H-13a signal at .delta..sub.H 3.76 (Table 1)
indicated no substituent effects of axial N-oxides. Hence, the
configuration of N-oxide was determined to be trans to H-13a (Damu,
A. G. et al., J. Nat. Prod., 2005, 68, 1071-1075, Lavault, M. et
al., Pharm. Acta Helv., 1994, 68, 225-227). Compound (11) displayed
a positive optical rotation value and a negative Cotton effect at
238 nm in the CD spectrum, suggesting S configuration at C-13a
(Damu, A. G. et al., Planta Med., 2009, 75, 1152-1156, St.ae
butted.rk, D. et al., J. Nat. Prod., 2000, 63, 1584-1586, S. F, J.
F. Mi et al., Acta Pharm. Sin. B, 27, 197-203). Thus, compound (11)
was identified as 10R, 13aS-tylophorine N-oxide (i.e. having
Formula (IV) above, wherein R.sub.1 is OCH.sub.3, R.sub.2 is
OCH.sub.3, R.sub.3 is OCH.sub.3 and R.sub.4 is H).
[0383] 10R, 13aS-tylophorine N-oxide (compound (11)). White powder;
[.alpha.].sub.D.sup.24 +78 (c 0.67 CHCl.sub.3-MeOH 3:1); UV
(CHCl.sub.3) .lamda..sub.max (log .epsilon.) 258 (4.90), 289
(4.66), 304 (4.42) nm; CD (MeOH) 238 (.DELTA..epsilon. -2.04) nm;
.sup.1H and .sup.13C NMR data, see Tables 1 and 2; HRESIMS m/z
410.1956 [M+H].sup.+ (calcd for C.sub.24H.sub.27NO.sub.5,
410.1962).
[0384] Compound (12) was obtained as white powder. Compound (12)
was assigned a molecular formula C.sub.23H.sub.25NO.sub.4 from its
HRESIMS peak at m/z 380.1849 ([M+H].sup.+, calcd 380.1856), one
oxygen atom more than that of 20 (Lv, H. et al, PLoS One, 2012, 7,
e30342). Compound (12) possessed similar NMR data as those of
compound (20) (Tables 1 and 2). The distinctive signals were
attributed to the downfield shifts observed at H-9 (.delta..sub.H
5.67, 4.69), H-11 (.delta..sub.H 4.45, 3.68), H-13a (.delta..sub.H
3.45) in the .sup.1H NMR spectrum and C-9 (.delta..sub.C 64.4),
C-11 (.delta..sub.C 68.3), C-13a (.delta..sub.C 70.5) in the
.sup.13C NMR spectrum with respect to those of 20 (Lv, H. et al.,
PLoS One, 2012, 7, e30342) (Table 2). The aforementioned
information suggested the presence of N-oxide functionality. The
three methoxyl groups deduced from .sup.1H NMR spectrum were
determined to be placed at C-3, C-6, and C-7 by HMBC correlations
of OCH.sub.3-3 with C-3, OCH.sub.3-6 with C-6, OCH.sub.3-7 with
C-7, and NOESY correlations OCH.sub.3-3/H-4, OCH.sub.3-6/H-5,
OCH.sub.3-7/H-8.
[0385] The appearance of H-13a signal at .delta..sub.H 3.76 (Table
1) indicated no substituent effects of axial N-oxides. Hence, the
configuration of N-oxide was determined to be trans to H-13a (Damu,
A. G. et al., J. Nat. Prod., 2005, 68, 1071-1075, Lavault, M. et
al., Pharm. Acta Helv., 1994, 68, 225-227). The positive optical
rotation value and the negative Cotton effect at 234 nM in the CD
spectrum suggest S configuration at C-13a (Damu, A. G. et al.,
Planta Med., 2009, 75, 1152-1156, St.ae butted.rk, D. et al., J.
Nat. Prod., 2000, 63, 1584-1586, S. F, J. F. Mi et al., Acta Pharm.
Sin. B, 27, 197-203). Thus, compound (12) was identified as
10R-deoxytylophorinine N-oxide (i.e. having Formula (IV) above,
wherein R.sub.1 is H, R.sub.2 is OCH.sub.3, R.sub.3 is OCH.sub.3
and R.sub.4 is H).
[0386] 10R-deoxytylophorinine N-oxide (compound (12)). White
powder; [.alpha.].sub.D.sup.24 +54 (c 0.46 CHCl.sub.3); UV
(CHCl.sub.3) .lamda..sub.max (log .epsilon.) 260 (4.39), 286
(4.27), 313 (3.60) nm; CD (MeOH) 234 (.DELTA..epsilon. -4.15) nm;
.sup.1H and .sup.13C NMR data, see Tables 1 and 2; HRESIMS m/z
[M+H].sup.+ (calcd for C.sub.23H.sub.25NO.sub.4).
[0387] The known compounds were characterized as 11-keto-O-methyl
tylophorinidine (compound (2)) (Lv, H. et al., PLoS One, 2012, 7,
e30342), 3-O-demethyl tylophorinidine (compound (13)) (Dhiman, M.
et al., Chem. Pap.-Chem. Zvesti, 2013, 67, 245-248),
tylophorinidine (compound (14)) (Zhen, Y. et al., Acta Bot. Sin.,
2002, 44, 349-353),
trans-(+)-3,14a-dihydroxy-6,7-dimethoxyphenanthroindolizidine
(compound (15)) (Komatsu, H. et al., J. Med. Chem., 2001, 44,
1833-1836), tylophoridicine C (compound (16)) (Huang, X. et al.,
Planta Med., 2004, 70, 441-445), tylophorinine N-oxide (compound
(17)) (Abe, F. et al., Phytochemistry, 1995, 39, 695-699),
tylophorinine (compound (18)) (Y Zhen, Y. et al., Acta Bot. Sin.,
2002, 44, 349-353), 13aS-tylophorine (compound (19)) (Lee, Y. Z. et
al., Planta Med., 2011, 77, 1932-1938), deoxytylophorinine
(compound (20)) (Wang, Z. et al., PloS one, 2012, 7, e52933),
O-methyl-tylophorindine (compound (21)) (Y Zhen, Y. et al., Acta
Bot. Sin., 2002, 44, 349-353), tylophoridicine D (compound (22))
(Huang, X. et al., Planta Med., 2004, 70, 441-445) by comparing
their NMR and optical rotation data with those reported in the
literature (see Tables 4 to 6).
TABLE-US-00001 TABLE 1 .sup.1H-NMR (600 MHz) data for compounds (1)
and (4) to (12) (.delta. in ppm, J in Hz). NO. 1.sup.+ 4.sup.+
5.sup.+ 6.sup.+ 7.sup.+ 8.sup.+ 9.sup.+ 10.sup.+ 11* 12* 1 8.14, d,
9 7.25, s 7.61, s 7.95, d, 9 7.52, s 7.55, s 7.66, s 7.56, s 7.31,
s 7.85.sup.e, d 9.6 2 7.25, dd, 2.4, 9 7.05, dd, 7.23, dd, 2.4, 9
2.4, 9 4 7.89, d, 2.4 7.82, s 7.79, s 7.70, d, 2.4 7.82, s 8.02, s
7.98, s 8.04.sup.b, s 7.80.sup.c 7.84.sup.e 5 8.10, s 7.95, s 7.95,
s 7.85, s 8.00, s 8.04, s 8.00, s 8.06.sup.b, s 7.79.sup.c 7.82, s
8 7.28, s 7.14, s 7.17, s 7.11, s 7.16, s 7.18, s 7.19, s 7.19, s
6.95, s 6.85, s 9 5.17, d, 17.4 4.52, d, 15 4.55, d, 15.6 5.13, d,
15 5.20, d, 15.6 5.22, d, 15 4.57, d, 15 5.23, d, 15 5.30, d, 14.4
5.67, d, 11.4 4.51, d, 17.4 3.47, d, 15 3.43, d, 15.6 4.78, d, 15
4.86, d, 15.6 4.90, d, 15 3.48, d, 15 4.87, d, 15 4.71, d, 13.8
4.69, d, 15 11 3.30, m 3.30, m 3.63, m 3.73, m 3.71, m 3.32, m
3.71, m 4.14, m 4.45, m 2.31, m 2.38, m 3.57, m 2.61, m 3.63, m
2.38, m 3.63, m 3.61, m 3.68, m 12 2.44, m 1.82, m 1.82, m 2.27, m
2.33, m 2.33, m 1.83, m 2.34, m 2.63, m 2.52, m 2.37, m 1.83, m
1.82, m 2.02, m 2.14, m 2.07, m 1.83, m 2.09, m 2.09, m 2.15, m 13
2.36, m 2.13, m 2.17, m 2.64, m 2.70, m 2.71, m 2.20, m 2.72, m
2.44, m 2.30, m 2.19, m 1.60, m 1.83, m 2.05, m 2.14, m 2.13, m
1.84, m 2.12, m 2.27, m 2.30, m 13a 3.91, m 2.34, m 2.39, m 3.64, m
3.73, m 3.76, m 2.42, m 3.73, m 3.48, m 3.45, m 14 5.09, dd, 2.4,
3.17, m 4.76, dd, 5.10, brs 5.03, d, 1.8 5.06, brs 4.80, br 5.19,
d, 2.4 3.56, m 3.41, m 7.2 2.65, m 1.8, 9.6 3.24, m 3.30, m
OCH.sub.3-2 3.92, s 4.07, s OCH.sub.3-3 3.98, s 3.99, s 3.98, s
4.00, s 4.06.sup.a, s 4.04, s 4.06, s 4.15.sup.d, s 4.06, s
OCH.sub.3-6 4.05.sup.a, s 4.03, s 4.05, s 4.13.sup.d, s 4.11, s
OCH.sub.3-7 4.02, s 3.93, s 3.93, s 3.92, s 3.96, s 3.95, s 3.92, s
3.96, s 4.02, s 3.94, s OH-14 5.41, d, 7.2 4.46, d, 9.6
.sup.+Measured in DMSO-d.sub.6. *Measured in CDCl.sub.3.
.sup.a-eOverlapped signals
TABLE-US-00002 TABLE 2 .sup.13C-NMR (150 MHz) data for compounds
(1) and (4) to (12) (.delta. in ppm). NO. 1.sup.+ 4.sup.+ 5.sup.+
6.sup.+ 7.sup.+ 8.sup.+ 9.sup.+ 10.sup.+ 11* 12* 1 125.9 107.6
109.2 127.0 109.5 109.5 109.7 105.8 104.0 125.1 2 115.9 146.2 146.1
116.9 146.7 147.0 146.7 149.0 149.0.sup.d 115.3 3 157.4 147.7 147.7
156.2 148.6 148.8 148.3 149.4 148.9.sup.d 158.1 4 103.5 103.7 103.4
106.1 103.8 104.5.sup.b 104.2 104.3.sup.c 103.4.sup.e 104.5 5 108.0
107.4 107.4 108.0 107.9 104.5.sup.b 104.4 104.6.sup.c 103.2.sup.e
103.9 6 146.9 146.0 146.6 147.4 147.2 149.4 149.2 149.2 148.8.sup.d
148.7 7 148.9 147.6 147.6 149.3 148.2 148.9 148.0 149.1 148.7.sup.d
149.7 8 103.8 103.4 103.9 104.6 104.5 104.4 104.5 104.3 102.6 102.5
9 40.3 53.5 53.8 65.9 66.0 66.0 54.1 66.0 66.4 64.4 11 173.5 54.6
55.0 69.3 69.3 69.4 55.3 69.4 69.9 68.3 12 30.3 21.3 21.7 20.3 20.4
20.4 22.1 20.4 20.1 19.8 13 19.2 30.9 24.0 22.3 22.3 22.3 24.4 22.4
27.3 27.1 13a 57.7 60.0 65.1 69.4 69.4 69.3 65.4 69.3 69.7 70.5 14
63.5 33.2 64.0 64.0 64.2 64.2 64.4 64.1 27.6 27.2 4a 130.2 125.4
122.7 130.9 123.3 123.6 126.4 124.3 123.7 130.8 4b 124.2 123.7
124.5 124.5 125.2.sup.a 124.8 123.4 123.2 123.9 123.7 8a 123.1
122.6 122.1 123.4 122.1 122.8 124.5 124.5 124.9.sup.f 124.4 8b
123.5 125.9 126.7 119.4 120.7 120.6 123.2 120.8 120.4 118.6 14a
127.5 124.6 128.0 128.7 127.9 128.6 127.0 128.9 124.7 124.5 14b
124.1 122.2 125.8 123.5 125.3.sup.a 125.5 129.2 125.0 124.0.sup.f
124.0 OCH.sub.3-2 55.8 56.0.sup.g OCH.sub.3-3 55.3 55.6 55.6 55.9
56.4.sup.d 56.4 56.3 55.9.sup.g 55.5 OCH.sub.3-6 56.3.sup.d 56.2
56.3 55.9.sup.g 55.9 OCH.sub.3-7 55.7 55.5 55.6 56.1 56.1 56.0 55.9
56.1 55.9.sup.g 56.0 .sup.+Measured in DMSO-d.sub.6. *Measured in
CDCl.sub.3. .sup.a-ioverlapped signals.
TABLE-US-00003 TABLE 3 .sup.1H-NMR (600 MHz) and .sup.13C-NMR (150
MHz) data for compound (3) (.delta. in ppm, J in Hz).sup.a.
position .delta..sub.H .delta..sub.C 1 8.16, d, 9 127.2 2 7.24, dd,
2.4, 9 116.0 3 157.9 4 8.07, d, 2.4 104.8 5 8.10, s 105.0 6 149.3 7
149.8 8 7.19, s 104.4 9 4.76, d, 15; 3.85, d, 15 56.0 12 4.24, m
74.1 13 1.81, m; 1.43, m 30.5 14 2.33, m; 1.82, m 24.9 14a 2.70, m
64.9 15 4.93, d, 6 67.3 16 2.63, m; 2.63, m 48.8 17 206.7 18 2.19,
s 30.7 4a 130.8 4b 124.4 8a 124.2 8b 123.8 15a 128.6 15b 124.3
OCH.sub.3-3 4.00, s 55.9 OCH.sub.3-6 4.04, s 56.3 OCH.sub.3-7 3.96,
s 55.9 OH-15 5.11, d, 9.6 .sup.aMeasured in DMSO-d.sub.6.
TABLE-US-00004 TABLE 4 .sup.1H-NMR (600 MHz) data for compounds
(2), (13) to (16) (.delta. in ppm, J in Hz). NO. 2.sup.+ 13.sup.+
14.sup.+ 15.sup.+ 16* 1 8.17, d, 9 8.10, d, 9 8.17, d, 9 8.15, d, 9
8.05, d, 9 2 7.28, dd, 2.4, 9 7.06, dd, 2, 9 7.18, dd, 9, 2.4 7.11,
dd, 9, 2.4 7.17, dd, 9, 2 4 8.10, d, 3 7.69, d, 2 7.83, d, 2.4
7.94, d, 2.4 7.73, s 5 8.13, s 7.83, s 8.02, s 7.93, s 7.97, s 8
7.29, s 7.19, s 7.11, s 7.21, s 7.10, s 9 5.18, d, 17.4 4.54, d, 15
4.48, d, 15.6 4.55, d, 15 5.21 4.53, d, 17.4 3.43, d, 15 3.38, d,
15.6 3.46 4.85, d, 15 11 3.33, m 2.30, m 3.32 3.71, m 2.37, m 3.25,
m 2.37, m 3.63, m 12 2.39, m 1.83, m 1.79-1.82, m 1.84, m 2.33, m
2.36, m 1.83, m 1.79-1.82, m 1.84, m 2.11, m 13 2.35, m 2.17, m
1.79-1.82, m 2.19, m 2.70, m 2.21, m 1.83, m 2.16, m 1.85, m 2.11,
m 13a 3.92, m 2.37, m 2.33, m 2.41, m 3.76, m 14 5.11, dd, 7.2, 1.8
4.89, dd, 1.8, 9.6 4.88, s 4.93, dd, 10.2, 1.8 5.18, d, 3
OCH.sub.3-2 OCH.sub.3-3 4.01, s 3.94, s 3.95, s OCH.sub.3-6 4.06, s
4.00, s OCH.sub.3-7 4.00, s 3.94, s 3.92, s 3.94, s 3.94, s OH-14
5.46, d, 7.2 4.50, d, 9.6 4.59, d, 10.2 .sup.+Measured in
DMSO-d.sub.6. *Measured in CDCl.sub.3. .sup.aOverlapped signals
TABLE-US-00005 TABLE 5 .sup.1H-NMR (600 MHz) data for compounds
(17) to (21) (.delta. in ppm, J in Hz). NO. 17.sup.+ 18* 19* 20*
21* 1 8.13, d, 9 8.43, d, 9 7.32, s 7.95, d, 9 8.44, d, 9 2 7.28,
dd, 9, 2.4 7.27, dd, 9, 2.4 7.24, dd, 2.4, 9 7.27 4 8.10, d, 3
7.78, d, 2.4 7.84, s 8.08, d, 2.4.sup.a 7.74, d, 1.8 5 8.12, s
7.65, s 7.83, s 8.09, s.sup.a 7.56, s 8 7.20, s 6.35, s 7.17, s
7.21, s 6.14 9 5.21, d, 15 3.59, d, 15.6 4.63, d, 14.4 4.56, d, 15
3.33, d, 14.4 4.87, d, 15 3.14, d, 15.6 3.68, d, 14.4 3.54, d, 15
2.99, d, 14.4 11 3.70, m 2.42, m 3.48, t, 8.1 3.35, m; 2.36, m
3.27, t, 8.4 3.62, m 3.33, m 2.48, m 2.36, m 2.18, q, 8.4 12 2.33,
m 2.03, m 2.04, m 1.82, m 2.02, m 2.08, m 1.93, m 1.94, m 1.82, m
1.89, m 13 2.70, m 1.93, m 2.25, m 2.17, m 2.40, m 2.11, m 2.27, m
1.78, m 1.60, m 1.86, m 13a 3.73, m 2.43, m 2.50, m 2.40, m 2.33, m
14 5.18, d, 3 4.98 brd 3.37, dd, 2.1, 3.37, m 4.91 15.6 2.92, t,
10.8, 15 2.79, m OCH.sub.3-2 4.06, s OCH.sub.3-3 4.02, s 4.07, s
4.12, s 3.98, s 4.05, s OCH.sub.3-6 4.05, s 4.13, s 4.12, s 4.02, s
4.10, s OCH.sub.3-7 3.97, s 3.88, s 4.05, s 3.94, s 3.80, s
OH-14
TABLE-US-00006 TABLE 6 .sup.13C-NMR (150 MHz) data for compounds
(2), (13) to (17), (19) to (21) (.delta. in ppm). position 2.sup.+
13.sup.+ 14.sup.+ 15.sup.+ 16* 17.sup.+ 19* 20* 21* 1 126.5 126.9
127.4 126.9 126.4 127.1 104.0 125.4 126.7 2 116.2 116.6 116.6 116.7
115.9 116.2 148.7 115.9 114.8 3 158.0 155.7 158.2 155.8 157.4 158.1
148.7 157.8 157.5 4 104.9 106.0 104.4 106.5 103.1 104.7 103.3 105.1
104.1 5 105.0 107.8 108.8 104.4 108.1 105.0 103.4 105.1 102.9 6
149.5 147.0 149.7 149.6 149.0 149.4 148.4 148.7 148.6 7 150.0 149.1
147.7 149.0 146.9 149.9 148.5 149.8 148.4 8 104.1 104.5 104.9 104.3
103.9 104.4 103.1 103.8 102.7 9 40.5 54.1 55.9 54.0 65.2 65.9 54.1
53.8 53.4 11 174.1 55.4 54.6 55.3 68.9 69.2 55.2 55.0 55.5 12 30.8
22.1 22.6 22.1 19.9 20.3 21.6 21.7 21.9 13 19.7 24.5 25.0 24.4 21.9
22.3 31.3 31.3 23.9 13a 58.1 65.4 65.9 65.4 69.0 69.3 60.2 60.3
65.4 14 64.0 64.1 64.6 64.1 63.5 64.0 33.8 33.5 64.5 OCH.sub.3-2
55.9 OCH.sub.3-3 56.0.sup.a 56.5 55.9 55.6 55.9 56.0 55.9 55.5
OCH.sub.3-6 56.3.sup.a 55.9 56.3 56.0 56.3 55.7 OCH.sub.3-7 55.9
56.0 56.3 55.1 56.9 55.9 55.9 55.5 C ring 131.0 130.7 124.6 131.0
119.8 131.0 124.4 125.5.sup.b 123.7 124.2 129.4 125.0 130.2 123.1
124.5 123.4 130.4 124.1 124.3 125.9 125.9 125.8 123.8 124.4 126.1
123.2 125.4 123.8 124.4 127.5 124.7 124.3 120.3 125.9 125.2.sup.b
128.8 128.8 124.2 129.7 124.4 128.1 129.5 123.6 125.9 130.6 124.7
124.0 131.1 123.8 130.4 124.3 126.3 127 126.2 .sup.+Measured in
DMSO-d.sub.6. *Measured in CDCl.sub.3. .sup.a-bOverlapped
signals
Example 2
[0388] Inhibition of HIF-1 Transcriptional Activity
[0389] HIF-1-mediated reporter gene assay in T47D cells was used to
evaluate the HIF-1 activation inhibitory effects of compounds (1)
and (3) to (22) on inhibiting HIF-1 activation induced by hypoxia.
Digoxin, a well-known HIF-1 inhibitor, was used as a positive
control (Zhang, H. et al., PNAS, 2008, 105, 19579-19586).
[0390] The HIF-1-mediated reporter gene assay in T47D cells was
performed according to a previous protocol with minor modification
(Parhira, S. et al., Sci. Rep., 2014, 4, 4748). T47D cells
(American Type Culture Collection) were cultured in DMEM medium
(Invitrogen), supplemented with 10% (v/v) fetal serum (FBS)
(Invitrogen), 100 U/mL penicillin, and 100 .mu.g/mL streptomycin
(Invitrogen) in a humidified atmosphere (5% CO.sub.2 and 95% air)
at 37.degree. C. Cells (5.times.10.sup.6) were co-transfected with
the HRE-luciferase (Addgene) and Renilla plasmids using
Lipofectamine 2000 (Invitrogen) according to the manufacturer's
protocol. The transfected cells were seeded in 96-well plates with
a density of 5.times.10.sup.4/well and cultured in DMEM with 10%
FBS overnight. After addition of the test compounds with different
concentrations, the cells were incubated for 1 h, and then exposed
to hypoxic (2% O.sub.2/5% CO.sub.2/93% N.sub.2) or normoxic (5%
CO.sub.2/95% air) conditions at 37.degree. C. for 20 h. Then the
cells were lysed, and luciferase assay was performed using a
Dual-Luciferase.RTM. reporter assay (Promega) kit according to the
instructions of the manufacturer. Luciferase activities of both HRE
and Renilla were determined by a multimode reader (Infinite 200
PRO, Teacan). HIF-1 transcriptional activity was shown by the ratio
of firefly/Renilla luciferase activity. IC.sub.50 values were
determined from the dose-response curves using Prism software. The
data were repeated by three independent experiments.
[0391] Most phenanthroindolizidine alkaloids exhibited extremely
potent inhibitory effects with IC.sub.50 values in the low
nanomolar range. The potency of compound (14) (IC.sub.50: 4 nM) and
compound (17) (IC.sub.50: 3 nM) were even comparable to Manassantin
B (IC.sub.50: 3 nM), the most potent natural HIF-1 inhibitor
identified so far.
[0392] As evident from Table 7, compounds (4), (5), (7), (13) to
(18) and (21) exhibited potent HIF-1 inhibitory activity in low
nanomole scale with IC.sub.50 values ranged from 3 to 37 nM, which
means 8-100 fold more potent than digoxin. Cytotoxicity, assessed
by MTT assay, proved that all compounds exert no significant
cytotoxicity against T47D cells at their effective concentrations
for the inhibition of HIF-1 activation (see Table 8). All
experiments were independently performed at least three times.
TABLE-US-00007 TABLE 7 HIF-1 activation inhibitory activity of
phenanthroindolizidine alkaloids (1) and (3) to (22) IC.sub.50 (nM)
compounds T47D.sup.a 1 >10000 3 2664 .+-. 1808 4 19 .+-. 2 5 10
.+-. 3 6 50 .+-. 35 7 37 .+-. 8 8 170 .+-. 106 9 489 .+-. 108 10
441 .+-. 138 11 930 .+-. 330 12 254 .+-. 75 13 10 .+-. 6 14 4 .+-.
1 15 6 .+-. 3 16 25 .+-. 7 17 3 .+-. 1 18 19 .+-. 6 19 438 .+-. 151
20 115 .+-. 53 21 13 .+-. 6 22 >10000 Digoxin 302 .+-. 21
.sup.ahuman breast tumor. Values are means .+-. SD, where SD =
standard deviation.
TABLE-US-00008 TABLE 8 Cytotoxicity of phenanthroindolizidine
alkaloids (3) to (22)against T47D cells under normoxic and hypoxic
conditions. IC.sub.50 (nM) compounds Normoxic Hypoxic 3 >10000
>20000 4 >500 >500 5 >250 >250 6 >200 >200 7
>1000 >1000 8 >1000 >1000 9 >2000 >2000 10
>2000 >2000 11 >2000 >2000 12 >1000 >2000 13
>250 >250 14 >10 >100 15 >250 >250 16 >100
>100 17 >100 >100 18 >500 >500 19 >1000 >2000
20 >600 >15000 21 >500 >12500
[0393] It follows that non-planarity at indolizidine moiety
enhanced the HIF-1 inhibitory activity. The inhibitory effect of
compound (20) (IC.sub.50 115 nM) was at least 8 times more potent
than its dehydrogenated product (22) (IC.sub.50>10000 nM), which
possessed extended conjugated system at indolizidine ring resulting
in higher planarity.
[0394] Furthermore, substitution at indolizidine ring exerted
crucial influence on HIF-1 inhibitory activity of the
phenanthroindolizidine alkaloids. Firstly, replacement of the C-11
methylene group with a keto group decreased the HIF-1 inhibitory
potency, which was exemplified by the observation that compound
(14) (IC.sub.50 4 nM) exhibited a remarkable increase in HIF-1
inhibitory activity comparing with its 11-keto analogue compound
(1) (IC.sub.50>10000 nM). Secondly, oxidation of the
indolizidine amine caused deleterious effect on HIF-1 inhibitory
activity. The N-oxide analogue compounds (11), (12), (16)
(IC.sub.50 930, 254, 25 nM, respectively) possessed 2-6 times in
inhibitory effects lower than their free amine counterparts (19),
(20), (14) (IC.sub.50 438, 115, 4 nM, respectively). Third,
hydroxylation at C-14 strengthened the HIF-1 inhibitory activity.
Compound (10), (18)/(21) (IC.sub.50 441, 19/13 nM) were 2-8 times
more active than their deoxygenated counterparts (11), (20)
(IC.sub.50 930, 115 nM, respectively).
[0395] Besides, substitution types and patterns on the phenanthrene
unit also played significant roles in HIF-1 inhibitory activity.
First, substitution of hydroxyl/methoxyl group at C-2 gave rise to
decrease in HIF-1 inhibitory activity. At least 2-fold loss was
observed between compound (14) (IC.sub.50 4 nM) and its
2-hydroxylated counterpart compound (5) (IC.sub.50 10 nM), while at
least 3-fold decrease were present between compounds (12), (20)
(IC.sub.50 254, 115 nM, respectively) and their 2-methoxylated
counterparts 11, 19 (IC.sub.50 930, 438 nM, respectively). Second,
methylation of hydroxyl group at C-3 favored higher inhibitory
activity. At least 2-fold increase was observed between compounds
(13) (IC.sub.5010 nM) and its 3-methylated analogue compound (14)
(IC.sub.50 4 nM).
[0396] HIF-1 does not only play a crucial role in response of
mammalian cells to hypoxia and a driving force in cancer
progression (G. Melillo, Mol. Cancer Res., 2006, 4, 601-605), but
does also represent a negative prognostic factor in cancer
treatment (G. L. Semenza, Nat. Rev. Cancer, 2003, 3, 721-73).
Numerous efforts had thus been undertaken to discover small
molecule HIF-1 inhibitors from natural products, and many natural
product based HIF-1 inhibitors had been characterized and
summarized previously (Nagle, D. G. and Zhou, Y. D., Curr. drug
targets, 2006, 7, 355-369). Among which, lignoid manassantins
(IC.sub.50 3.about.30 nM) (Hodges, T. W. et al., J. Nat. Prod.,
2004, 67, 767-771) and phenanthroquinolizidines alkaloids
(IC.sub.508.7-48.1 nM) (Cai, X. F. et al., J. Nat. Prod., 2006, 69,
1095-1097) were discovered to exhibit remarkable potency with
Manassantin B (IC.sub.50 3 nM) considered as the most potent
natural HIF-1 inhibitor so far (Hodges, T. W. et al., J. Nat.
Prod., 2004, 67, 767-771). In the above experiment, compounds (14)
(IC.sub.50 4 nM), (17) (IC.sub.50 3 nM) displayed comparable
potency to Manassantin B. Collectively, the data above indicate
that the isolated phenanthroindolizidine alkaloids are potent HIF-1
inhibitors.
* * * * *