Note: Descriptions are shown in the official language in which they were submitted.
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THERAPEUTIC COMPOUNDS AND METHODS
FIELD OF THE INVENTION
The present invention relates generally to compounds having a
cyclopentabenzofuran core. More particularly, the present invention relates to
cyclopentabenzofuran compounds having a bulky substituent at the 6-oxy
position, such as
where the cyclopentabenzofuran core is substituted by a dioxanyloxy moiety.
The
invention also relates to the use of these compounds in therapy and
compositions
comprising said compounds.
BACKGROUND
Aglaia is a large genus of the family Meliaceae comprising over 100 (mostly
1 S woody) species in Indo-Malaysia and the Western Pacific region. Uses
include treatment
of fever, fractures, parturition and inflammation. Extracts are also used as
bactericides,
insecticides, in perfumery, as an astringent, tonic, a refrigerant (Dr Duke's
PhytochenZical
ahd Ethhobotauical Databases) and for the treatment of abdominal tumours
(Pannel, et al,
1992, Kew Bull., (16) 273-283).
More recently, a number of 1H cyclopenta[b]benzofuran lignans have been
isolated
from Aglaia species (see for example, W097108161; JP 97171356; Ohse, et al,
JNat P~°od,
1996, 59(7):650-52; Lee et al, Clzem. Biol. Interact., 1998, 115(3):215-28; Wu
et al, J.
Nat. Prod., 1997, 60(6):606-08; Bohnenstengel et al, Z. Naturforsch., 1999,
54c (12):55-60
and Bohnenstengel et al, Z. Naturforsch, 1999, 54c (12):1075-83, Xu, Y. J., et
al, 2000, J.
Nat. Prod., 63, 4732-76, the entire contents of which are incorporated herein
by
reference). A number of these compounds have also been noted for their
insecticidal
activity (Janprasert, et al, 1993, Phytochemistry, 32 (1): 67-69; Ishibashi et
al, 1993,
Phytochemistry, 32 (2): 307-310; Hiort, et al, 1999, J. Nat. Prod., 62 (12):
1632-1635).
Insecticidal compounds with a closely related core structure were isolated
from Aglaia
roxburghiar~a and are described in WO 9604284 for use as active ingredients in
agrochemical formulations.
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New compounds (Compounds A and B, as described herein) have now been isolated
from
Aglaia leptantha, Miq. (Meliaceae) which uniquely possess a dioxanyloxy group
at the 6-
position of the cyclopenta[b]benzofuran core. Compounds A and B have been
shown to
exhibit potent cytotoxic and cytostatic effects on cancer cell growth and
viability and thus
the compounds of the invention and derivatives thereof, may be useful as
therapeutic
agents in the treatment of cancer and cancerous conditions or other diseases
associated
with cellular hyperproliferation.
SUMMARY OF THE INVENTION
Throughout this specification and the claims which follow, unless the context
requires otherwise, the word "comprise", and variations such as "comprises"
and
"comprising", will be understood to imply the inclusion of a stated integer or
step or group
of integers or steps but not the exclusion of any other integer or step or
group of integers or
steps.
In a first aspect, the invention relates to compounds of Formula (I) or a salt
or
prodrug thereof.
O R4 H
F''6~ ~ .~''~O R a (I )
R'
~~C(O)X
YO 6 / O _
H
I
I
~R~~
O R5
wherein
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each R4-R1° is independently selected from the group consisting of
hydrogen,
optionally substituted alkyl, optionally substituted acyl, optionally
substituted aryl,
optionally substituted arylalkyl, optionally substituted cycloalkylalkyl,
optionally
substituted arylacyl, optionally substituted cycloalkylacyl and a C-1 linked
saccharide;
X is OR8 or NR9RIO;
Rll and R12 are preferably each independently hydrogen or, alternatively, OR4
and
Rl l, and/or ORS and R12 together form a methylenedioxy group; and
Y is selected from the group consisting of optionally substituted phenyl,
optionally
substituted benzyl, optionally substituted benzoyl, optionally substituted C3-
C$ cycloalkyl,
(preferably optionally substituted CS-C6 cycloalkyl) optionally substituted
CHZ-(C3-C8
cycloalkyl) (preferably optionally substituted CH2-(CS-C6 cycloalkyl),
optionally
substituted 5-6 membered heterocyclyl, and optionally substituted CHZ-(5-6
membered
heterocyclyl).
In a preferred embodiment, the invention relates to compounds (including
stereoisomers within the dioxanyl group) of formula (i) or a salt or prodrug
thereof.
(i)
R~ O
wherein
and each RI-Rl° is independently selected from the group consisting of
hydrogen,
optionally substituted alkyl, optionally substituted acyl, optionally
substituted aryl,
optionally substituted arylalkyl, optionally substituted cycloalkylalkyl,
optionally
substituted arylacyl, optionally substituted cycloalkylacyl and a C-1 linked
saccharide; and
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X is OR8 or NR9Rlo;
RI1 and RI2 are each independently hydrogen or, OR4 and R11, and/or ORS and
Rlz
together form a methylenedioxy group. In one preferred embodiment, Rl l and
Rl2 are both
hydrogen.
In one preferred embodiment, compounds of the present invention have the
Formula (ii):
OCH3
or a salt or prodrug thereof.
Formula (ii) has 4 chiral centres in the dioxanyl moiety. Two isomers
(isomeric in
the dioxanyl group) of Formula (ii) have now been isolated - Compounds A and B
as
described in Example 1.
In another aspect, the invention provides a composition comprising a compound
of
Formula (I), such as Formula (i), or a salt or prodrug thereof, together with
a
pharmaceutically acceptable carrier, excipient or diluent.
In still a further aspect, the present invention provides a method for the
treatment of
cancer or a cancerous condition or a disease state or condition associated
with cellular
hyperproliferation comprising the administration of a treatment effective
amount of a
compound of Formula (I), such as Formula (i), or a salt, derivative or prodrug
thereof, to a
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subject in need thereof. Some particular cancerous conditions which may be
treated by the
compounds of the invention may include lung, prostate, colon, brain, melanoma,
ovarian,
renal and breast tumours and leukemia. Disease states or conditions associated
with
cellular hyperproliferation which may be treated by compounds of the invention
may
include atherosclerosis, restinosis, rheumatoid arthritis, osteoarthritis,
inflammatory
arthritis, psoriasis, peridontal disease or virally induced cellular
hyperproliferation.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1: Compound A promotes differentiation of THP-1 leukemic cells.
THP-1 cells were cultured for 4 days in the presence or absence of 10 nM
Compound A as
indicated. Where shown cells were also treated with IFNy (100ng/ml) (3 days)
or with
PMA (0.1 ~M) (4 days) in the presence or absence of Compound A. Images are of
cells
visualised by phase contrast microscopy (magnification x200).
Figure 2: Effects of Compound A on cell cycle progression and viability of THP-
1
cells.
THP-1 cells were cultured for 2 days with the indicated concentration of
Compound A or
1000 nM paclitaxel then collected and fixed in 70% ethanol prior to staining
with
propidium iodide and DNA content determined by flow cytometry. The numbers
indicate
the % of cells in the various cell cycle phases relative to all cells with
>_2N DNA content
and also the % dead cells (ie. subdiploid <_ 2N cells) to the left of the
marker (the vertical
line) that arose during the culture period.
Figure 3: Effects of Compound A on the proliferation of A549 cells.
A549 cells were seeded at 10,000 cells/well and cultured in the presence of
the indicted
concentrations of Compound A or paclitaxel. Cells were collected and the
viable cell
number determined by haemocytometer counting of trypan blue stained cells at
the various
times. The results are the averages ~SEM of triplicate cultures.
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Figure 4: Effects of Compound A on cell cycle progression and viability of
A549
cells.
A549 cells were cultured for 6 days with the indicated concentration of
Compound A or 1
~.M paclitaxel then collected and fixed in 70% ethanol prior to staining with
propidium
iodide and DNA content determined by flow cytometry. The numbers indicate the
% of
cells in the various cell cycle phases relative to all cells with >_2N DNA
content and also
the % dead cells (ie. subdiploid S 2N cells) to the left of the marker that
arose during the
culture period.
Figure 5: Compounds A and B induce G2/M phase accumulation of K562
leukemic cells
K562 cells were cultured for 3 days with the indicated concentration of
Compounds A or B
then collected and fixed in 70% ethanol prior to staining with propidium
iodide and DNA
content determined by flow cytometry. The numbers indicate the % of cells in
GO/Gl, S
and G2/M phases of the cell cycle respectively relative to all cells with >_2N
DNA content.
Figure 6: Cytostatic effects of Compound A on A549 cells are reversible
A549 cells were seeded at 10,000 cells/well and cultured in the presence of
the indicted
concentrations of Compound A or paclitaxel and the viable cell numbers
determined by
haemocytometer counting of trypan blue stained cells at the various times. On
day 5 some
of the cells were washed, resuspended in fresh medium lacking the various
treatments and
cultured for another 4 days prior to counting.
Figure 7: Compound A inhibits camptothecin- and paclitaxel-induced
cytotoxicity of A549 cells
A549 cells in 96 well plates were cultured for 3 days in the presence or
absence of 10 nM
Compound A together with the indicated concentrations of (A) camptothecin or
(B)
paclitaxel. Loss of membrane integrity was then assessed by the addition of
the
fluorescent DNA-binding dye YOYO-1 and the increased fluorescence accompanying
cell
death measured using a fluorescent plate reader.
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Figure 8: Compound A inhibits cell cycle arrest and cell death induced by anti-
cancer agents but not by staurosporine
A549 cells in 6 well plates were cultured for 3 days in the presence or
absence of 10 nM
Compound A together with 0.1 ~.M camptothecin, 10 ~,M vinblastin, 1 ~,M
paclitaxel or 1
~,M staurosporine as indicated. The cells were then collected and fixed in 70%
ethanol
prior to staining with propidium iodide and DNA content determined by flow
cytometry.
The numbers indicate the % of cells in the various cell cycle phases relative
to all cells
with >_2N DNA content and also the % dead cells (ie. subdiploid <- 2N cells)
to the left of
the dotted marker that arose during the culture period.
Figure 9: Compound A does not induce senescence-associated ~i-galactosidase
activity in A549 cells
A549 cells were seeded at 10,000 cells/well in 6 well plates in the presence
or absence of
varying concentrations of Compound A (10 -50 nM) or 250 nM doxorubicin for 10
days
prior to their processing and staining overnight for senescence-associated (3-
galactosidase
activity as described previously (Dimri et al., 1995, Proc Natl Acad Sci ZISA
1995
92(20):9363-7). For Compound A only the 10 nM treatment is shown but there was
no
detectable SA-[3 gal activity at any other concentrations tested. PC, phase
contrast
microscopy. BF, bright field microscopy. Magnification x200.
Figure 10: Compound A inhibits growth of human tumour cells in a mouse
xenograft model
Athymic Balb/c nude mice (Rygard and Povisen, 1969, Acta Pathol Micr~obiol
Scand, 77:
758) were inoculated subcutaneously in the dorsal flank with 2x 106 PC3 cells.
Compound
A was administered (3 mg/kg) after eight days when the tumours became palpable
by
intraperitoneal injection three times a week. Compound A was first solubilized
in ethanol
then mixed 1:1 with cremaphore and diluted in saline for injection. Control
animals were
treated in an analogous manner with the same vehicle but lacking Compound A.
(A)
Effect of Compound A on mean tumour volume. Tumour volumes were measured using
a
micrometer caliper at the indicated times. The data represents mean tumour
volume ~SEM
(B) Effect of Compound A on mean tumour mass. At the end of the experiment (29
days
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post inoculation of PC3 cells) the mice were sacrificed, the tumours excised
and then
weighed. The data represents mean tumour weight ~SEM.
DETAILED DESCRIPTION OF THE INVENTION
Cyclopenta[b]benzofurans previously reported carry a methoxy group or similar
small substituent (Greger et al, 2001, Phytochemist~y, 57, (1); 57-64) at the
6- or 8-
positions. In contrast, the compounds of the present invention carry a
sterically bulky
group at the 6-oxy-position, in particular, a dioxanyl group. The dioxanyl
group of
Formula (ii) (depicted below as sub-Formula (a)) has not previously been
reported from a
natural source. Without intending to limit the invention by theory, it is
believed that the
presence at the 6-oxy-position of a sterically bulky group, ie spatially
larger than a
methoxy group, may confer both cytotoxic and cytostatic properties on the
compounds
having a cyclopenta[b]benzofuran core.
(a)
H
The invention includes within its scope pharmaceutically acceptable salts,
derivatives, or prodrugs of compounds of Formula (I), particularly of Formula
(i), such as
Compounds A and B.
The term " salt, or prodrug" includes any pharmaceutically acceptable salt,
ester,
glycoside, solvate, hydrate or any other compound which, upon administration
to the
recipient subject is capable of providing (directly or indirectly, for
example, by chemical or
in vivo enzymatic or hydrolytic degradation) a compound of the invention as
described
herein.
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Suitable pharmaceutically acceptable salts include salts of pharmaceutically
acceptable inorganic acids such as hydrochloric, sulphuric, phosphoric,
nitric, carbonic,
boric, sulfamic, and hydrobromic acids, or salts of pharmaceutically
acceptable organic
acids such as acetic, propionic, butyric, tartaric, malefic, hydroxymaleic,
fumaric, citric,
lactic, mucic, gluconic, benzoic, succinic, oxalic, phenylacetic,
methanesulphonic,
toluenesulphonic, benzenesulphonic, salicyclic, sulphanilic, aspartic,
glutamic, edetic,
stearic, palmitic, oleic, lauric, pantothenic, tannic, ascorbic and valeric
acids. Base salts
include, but are not limited to those formed with pharmaceutically acceptable
canons, such
as sodium, potassium, lithium, calcium, magnesium, ammonium and alkylammonium.
The preparation of salts can be carried out by methods known in the art. It
will also
be appreciated that non-pharmaceutically acceptable salts also fall within the
scope of the
invention, since these may be useful as intermediates in the preparation of
pharmaceutically acceptable salts.
The compounds of the invention may be in crystalline form or as a solvate
(e.g.,
hydrates). Methods of solvation will be known to those skilled in the art.
Prodrugs of compounds of formula (I) are also within the scope of the
invention.
The teen "prodrug" includes derivatives that are converted i~ vivo to the
compounds of the
invention and include for example, ester (eg acetate) and glycoside
derivatives of free
hydroxy groups, which may undergo in vivo degradation to release a compound of
the
invention. Other suitable prodrugs may include esters of free carboxylic acid
groups. The
preparation of suitable prodrugs is further described in Design of Prodr~ugs,
H. Bundgaard,
Elseveir, 1985, the contents of which is incorporated by reference.
It will also be recognised that certain Y groups of Formula (I), in particular
the
dioxanyl groups of compounds as depicted in Formula (i) and (ii), may possess
asymmetric
centres and are therefore capable of existing in more than one stereoisomeric
form. The
invention thus also relates to compounds in substantially pure isomeric form
at one or
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more asymmetric (chiral) centres eg., greater than about 90% ee, such as about
95% or
97% ee, preferably greater than 99% ee, as well as mixtures, including racemic
mixtures,
thereof. Such isomers may be resolved by conventional methods, eg,
chromatography, or
use of a resolving agent. The present invention thus provides Compounds A and
B.
As used herein, the term "alkyl", when used alone or in compound words such as
"arylalkyl" refers to a straight chain, branched or cyclic hydrocarbon group,
preferably
C1_2o, such as C1_lo. The term "C1-C6 alkyl" refers to a straight chain,
branched or cyclic
alkyl group of 1 to 6 carbon atoms. Examples of "C1-6 alkyl" include methyl,
ethyl, iso-
propyl, n-propyl, n-butyl, sec-butyl, t-butyl, ~t-pentyl, isopentyl, 2,2-
dimethypropyl, n-
hexyl, 2-methylpentyl, 2,2-dimethylbutyl, 3-methylpentyl and 2,3-
dimethylbutyl.
Examples of cyclic C1_6 alkyl include cyclopropyl, cyclobutyl, cyclopentyl and
cyclohexyl.
Other examples of alkyl include: heptyl, 5-methylhexyl, 1-methylhexyl, 2,2-
dimethylpentyl, 3,3-dimethylpentyl, 4,4-dimethylpentyl, 1,2-dimethylpentyl,
1,3-
dimethylpentyl, 1,4-dimethyl-pentyl, 1,2,3-trimethylbutyl, 1,1,2-
trimethylbutyl, 1,1,3-
trimethylbutyl, octyl, 6-methylheptyl, 1-methylheptyl, 1,1,3,3-
tetramethylbutyl, nonyl, 1-,
2-, 3-, 4-, ~-, 6- or 7-methyl-octyl, 1-, 2-, 3-, 4- or 5-ethylheptyl, 1-, 2-
or 3-propylhexyl,
decyl, 1-, 2-, 3-, 4-, 5-, 6-, 7- and 8-methylnonyl, 1-, 2-, 3-, 4-, 5- or 6-
ethyloctyl, 1-, 2-, 3-
or 4-propylheptyl, undecyl, 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8- or 9-methyldecyl, 1-
, 2-, 3-, 4-, 5-, 6-
or 7-ethylnonyl, 1-, 2-, 3-, 4- or 5-propylocytl, 1-, 2- or 3-butylheptyl, 1-
pentylhexyl,
dodecyl, 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9- or 10-methylundecyl, 1-, 2-, 3-, 4-
, 5-, 6-, 7- or 8-
ethyldecyl, 1-, 2-, 3-, 4-, 5- or 6-propylnonyl, 1-, 2-, 3- or 4-butyloctyl, 1-
2-pentylheptyl
and the like. An alkyl group may be optionally substituted by one or more
optional
substituents as herein defined. Optionally, the straight, branched or cyclic
hydrocarbon
group (having at least 2 carbon atoms) may contain one, two or more degrees of
unsaturation so as to form an alkenyl or alkynyl group, preferably a CZ_2o
alkenyl, more
preferably a C2_6 alkenyl, or a CZ_zo alkynyl, more preferably a CZ_6 alkynyl.
Examples
thereof include a hydrocarbon residue containing one or two or more double
bonds, or one
or two or more triple bonds. Thus, "alkyl" is taken to include alkenyl and
alkynyl.
The term "aryl", when used alone or in compound words such as "arylalkyl",
denotes single, polynuclear, conjugated or fused residues of aromatic
hydrocarbons or
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aromatic heterocyclic (heteroaryl) ring systems, wherein one or more carbon
atoms of a
cyclic hydrocarbon residue is substituted with a heteroatom to provide an
aromatic
residue. Where two or more carbon atoms are replaced, this may be by two or
more of
the same heteroatom or by different heteroatoms. Suitable heteroatoms include
O, N, S
and Se.
Examples of "aryl" include phenyl, biphenyl, terphenyl, quaterphenyl,
naphthyl,
tetrahydronaphthyl, anthracenyl, dihydroanthracenyl, benzanthracenyl,
dibenzanthracenyl, phenanthrenyl, fluorenyl, pyrenyl, idenyl, azulenyl,
chrysenyl,
pyridyl, 4-phenylpyridyl, 3-phenylpyridyl, thienyl, fuxyl, pyrrolyl, indolyl,
pyridazinyl,
pyrazolyl, pyrazinyl, thiazolyl, pyrimidinyl, quinolinyl, isoquinolinyl,
benzofuranyl,
benzothienyl, purinyl, quinazolinyl, phenazinyl, acridinyl, benoxazolyl,
benzothiazolyl
and the like. Preferred hydrocarbon aryl groups include phenyl and naphthyl.
Preferred
heterocyclic aryl groups include pyridyl, thienyl, furyl, pyrrolyl. An aryl
group may be
optionally substituted by one or more optional substitutents as herein
defined.
The term "acyl" refers to a group -C(O)-R wherein R is any carbon containing
moiety such as an optionally alkyl or substituted aryl group. Examples of acyl
include
straight chain or branched alkanoyl such as, acetyl, propanoyl, butanoyl, 2-
methylpropanoyl, pentanoyl, 2,2-dimethylpropanoyl, hexanoyl, heptanoyl,
octanoyl,
nonanoyl, decanoyl, undecanoyl, dodecanoyl, tridecanoyl, tetradecanoyl,
pentadecanoyl,
hexadecanoyl, heptadecanoyl, octadecanoyl, nonadecanoyl and icosanoyl;
cycloalkylcarbonyl, such as cyclopropylcarbonyl cyclobutylcarbonyl,
cyclopentylcarbonyl
and cyclohexylcarbonyl; aroyl such as benzoyl, toluoyl and naphthoyl;
aralkanoyl such as
phenylalkanoyl (e.g. phenylacetyl, phenylpropanoyl, phenylbutanoyl,
phenylisobutylyl,
phenylpentanoyl and phenylhexanoyl) and naphthylalkanoyl (e.g. naphthylacetyl,
naphthylpropanoyl and naphthylbutanoyl]. Since the R group may be optionally
substituted as described above, "acyl" is taken to refer to optionally
substituted acyl.
Optional substituents for alkyl, aryl or acyl include halo (bromo, fluoro,
chloro,
iodo), hydroxy, C1_6alkyl (eg methyl, ethyl, propyl (~- and i- isomers)),
C1_6alkoxy (eg
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methoxy, ethoxy, propoxy (~- and i- isomers), butoxy (h-, see- and t-isomers),
nitro,
amino, C1_6alkylamino (eg methyl amino, ethyl amino, propyl (h- and i-
isomers)amino),
C1_6dialkylamino (eg dimethylamino, diethylamino, diisopropylamino),
halomethyl (eg
trifluoromethyl, tribromomethyl, trichloromethyl), halomethoxy (eg
trifluoromethoxy,
tribromomethoxy, trichloromethoxy) and acetyl. Furthermore, optional
substituents for Y
(phenyl, benzyl, benzoyl, C3-C$ cycloalkyl, CH2-(C3-C8 cycloalkyl), 5-6
membered
heterocyclyl and CH2-(5-6 membered-heterocyclyl)) include, as well as the
substituents
above, alkyl substituted with one or more of hydroxy C1_6alkyloxy,
C1_6acyloxy, aryloxy,
arylCl_6alkyloxy, C1_6cycloalkylCl_balkyloxy, arylCl_6acyloxy,
C1_6cycloalkylCl_6acyloxy
and C1-linked saccharidoxy.
The term "arylalkyl" and "cycloalkylalkyl" refer to an alkyl group (preferably
straight chain) substituted (preferably terminally) by an aryl and a
cycloalkyl group,
respectively. Similarly, the terms "arylacyl" and "cycloalkylacyl" refer to an
acyl group
(preferably where R is straight chain alkyl) substituted (for example,
terminally
substituted) by an aryl and a cycloalkyl group, respectively.
Preferred C-1 linked saccharides are a furanose or pyranose saccharide (sugar)
substituent which is linked to the backbone structure shown in Formula (I)
through the
saccharides's 1-carbon (conventional chemical numbering) to form an acetal at
any one of
positions Rl, RZ, R3, R~, R5, R6, or R~ or an ester linkage at the R$ or an
amide at R9 or Rio
position. Exemplary saccharide groups include reducing sugars such as glucose,
ribose,
arabinose, xylose, mannose and galactoses, each being linked to an oxygen atom
of the
structure of Formula (I) through the C-1 carbon of the saccharide group.
A 5-6 membered heterocyclyl group includes aromatic 5-6-membered heterocyclic
aryl groups (heteroaryl) as described above and non aromatic 5-6-membered
heterocyclic
groups containing one or more heteroatoms (preferably 1 or 2) independently
selected
from O, N, S and Se. Examples thereof include dioxanyl, pyranyl,
tetrahydrofuranyl,
piperidyl, morpholino, piperazinyl, thiomorpholino and saccharides, for
example, those
described above.
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In one embodiment of Formula (I) or Formula (i) of the invention, each of R4-
R'
and Rl-R~ respectively may independently be selected from the group consisting
of
hydrogen, methyl, ethyl, n-propyl, i-propyl, n-butyl, sec-butyl, t-butyl,
cyclopropylmethyl
(or cyclopropylethyl), cyclobutylmethyl (or -ethyl), cyclopentylmethyl (or -
ethyl),
cyclohexylmethyl (or -ethyl), phenyl, benzyl, acetyl and C-l .linked
saccharide.
In another embodiment of Formula (I), (i) or (ii) of the invention, R8 of
X=OR$ is
selected from the group of hydrogen, C1_6 alkyl, phenyl, benzyl and C-1 linked
saccharide.
In another embodiment of Formula (I), (i) or (ii) of the invention R9 and
R1° of
X=NR9R1° are independently selected from hydrogen, C1_6 alkyl, phenyl
or benzyl.
The derivatisation of hydroxy groups of Compounds A and B to form compounds
of Formula (i), (ie where any one of Rl-R~ is not hydrogen) can be carried out
by methods
known in the art for alkylating, arylating or acylating hydroxy groups, for
example as
described in Protective Groups in Of°ganic SyfZthesis T.W. Greene and
P.G.M. Wutz,
(1999) Wiley Interscience, New York, and Advanced Organic Chemisty, J. March,
(q.tn
Edition), Wiley-InterScience (the entire contents of which are incorporated
herein by
reference). For example, hydroxy groups can be alkylated using alkyl halides
such as
methyl iodide or dialkyl sulfates such as dimethyl and diethyl sulfate.
Acylation can be
effected by treatment with appropriate carboxylic acids, acid halides or acid
anhydrides in
the presence of a base or coupling agent. Benzylation may be effected by
treatment with a
benzyl halide compound such as benzyl bromide, chloride or iodide. De-
esterification of
the methyl ester can be effected by treatment of the ester with aqueous base.
Esterification
of a carboxylic acid can be achieved by conventional means including treatment
with an
appropriate alcohol in the presence of acid, or treatment with alkyl sulfates
or alkyl
halides.
Glycosidic formation may be effected chemically, eg by reacting the starting
compound with a protected sugar compound in which C-1 has been activated by
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halogenation for coupling with the hydroxyl or carboxyl groups and the sugar
hydroxyls
have been blocked by protecting groups. Alternatively, glycoside formation may
be
effected enzymatically using an appropriate glycosyltransferase such as UDP-
galactose
dependent galactocyltransferase and UDP-glucose dependent glycocyltransferase
(SIGMA).
The skilled person will recognise that in order to selectively install any one
or more
of the Rl-Rl° groups as defined herein (eg where Rl-R' are not
hydrogen), this may require
the judicious protection and/or deprotection, of one or more of the oxy andlor
carboxy
groups. Selective derivatisation of one or more hydroxy or carboxy groups may
be
achieved via conventional techniques by the use of protecting groups with
different
degrees of stability under appropriate conditions.
Methods for the conversion of a carboxylic acid or ester group; ie. where X is
OR$
to an amide (X is NR9R1°) are known to the skilled person and may
include treatment of a
carboxylic acid with an appropriate amine in the presence of a coupling
reagent such as
DCC or treatment of an acid halide with the appropriate amine. Other methods
which may
be suitable are described in Larock, R.E, Comp~ehe~rsive Orgatzic
Tt°a~csfo~naatio~s pp 963-
995, VCH Publishers (1989).
As used herein, the term "protecting group", refers to an introduced
functionality
which may temporarily render a particular functional group, eg hydroxy or
carboxylic acid,
inactive under certain conditions in which the group might otherwise be
reactive. Suitable
protecting groups are known to those skilled in the art, for example as
described in
Protective Groups i~ Organic Synthesis (supra). Suitable protecting groups for
hydroxy
include alkyl, (such as C1-C6alkyl), acyl (such as C(O)C1-C6alkyl, benzoyl and
the like),
benzyl, and silyl groups (such as trimethylsilyl, t-butyldimethyl silyl, t-
butyldiphenylsilyl
and the like). Other suitable groups for hydroxy substituents and a carboxy
substituent
(acid, amide etc) can be found within Greene supra. The stability of various
groups under
certain conditions is understood by the skilled person and is further
exemplified in
Protective Groups i~ Organic Syyzthesis (supra).
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It will be appreciated that these protected compounds may be useful as
intermediates in the preparation of certain compounds of Formula (I) and
therefore, these
also form a further aspect of the invention.
It will also be recognised that some groups, eg alkyl, acyl or arylalkyl,
(such as
methyl, ethyl, propyl, acetyl, benzyl etc) may serve as either a temporary
protecting group
or as a non-hydrogen Rl-R8 group in Formula (I).
The dioxanyl group may be cleaved from the 6-oxy position of the
cyclopentabenzofuran core using known methods to afford a dioxane compound.
The
resulting dioxane compound could be used to substitute other compounds, such
as oxy-
substituted compounds, including the corresponding 6-oxy position, or other
oxy positions,
on other cyclopentabenzofuran compounds such as those described in the
references
herein.
It will also be understood that cyclopentabenzofuran compounds, having a
methoxy
substituent at the 6-position, such as those described in the references cited
herein
(incorporated herein by reference) eg Reference Compounds 1-3 (as described in
Example
4), can, where appropriate be 6-demethylated, and the resulting 6-hydroxy
group reacted
with a suitable Y precursor to form an 6-OY group. Methods therefor are known
in the art,
for example, one method may involve reacting the 6-OH group with a Y-halogen
compound where halogen includes Cl, Br and I. Alternatively, access to the
cyclopentabenzofuran core, incorportation of the Y group can be achieved via
synthetic
methods analogous to that described in Trost et al, J. Am. Chem. Soc., 1990,
112, 9022-
9024. Such 6-OY compounds form a further aspect of the invention.
In some preferred embodiments of the present invention, one or more of the
following definitions apply:
-Ri and R2 are both hydrogen.
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-Rl and R2 are hydrogen, and R3 is methyl.
-at least one of R3-RS is methyl, ethyl or propyl, preferably methyl.
-at least two of R3-RS are methyl, ethyl or propyl, preferably methyl.
-all of R3-RS are methyl, ethyl or propyl, preferably methyl.
-R6 and R' are both hydrogen.
-at least one of Rl l and RI2 is hydrogen, preferably Rl l and R12 are both
hydrogen.
-X is OR8 where R$ is selected from hydrogen, methyl, ethyl or propyl,
preferably,
methyl.
-X is NR9R1° where R9 and Rl° are both hydrogen or methyl; or R9
and RI° are
different but at least one of R9 or Rl° is hydrogen and the other is
CI_6 alkyl, such as
methyl, ethyl or propyl.
-Y is an optionally substituted 5-6 membered heterocyclyl group or an
optionally
substituted CS-C6 cycloalkyl group.
Particularly preferred forms of Formula (ii) are Compounds A and B.
The compounds of the invention may have use in the treatment of cancerous
conditions, or other conditions associated with cellular hyperproliferation,
in a subject.
Subjects which may be treated by the compounds of the invention include
mammals, for
example, humans, primates, livestock animals (eg. sheep, cows, horses, goats,
pigs),
companion animals, (eg. dogs, cats, rabbits, guinea pigs), laboratory test
animals, (eg, rats,
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mice, guinea pigs, dogs, rabbits, primates) or captured wild animals. Most
preferably,
humans are the subj ects to be treated.
As used herein the term "treatment" is intended to include the prevention,
slowing,
interruption or halting of the growth of a cancer, tumour or
hyperproliferative cell, or a
reduction in the number of targeted cells (or size of the growth mass) or the
total
destruction of said cell, wherein said cells are cancer, tumour or
hyperproliferative cells.
Cancerous conditions which may be treated by the compounds of the present
invention include conditions wherein the cancers or tumours may be simple
(monoclonal,
ie composed of a single neoplastic cell type), mixed (polyclonal, ie. composed
of more
than one neoplastic cell type) or compound (ie. composed of more than one
neoplastic cell
type and derived from more than one germ layer) and may include benign and
malignant
neoplasia/hyperplasia. Some examples of cancerous conditions which may be
treated by
the present invention include leukemia and breast, colon, bladder, pancreatic,
endometrial,
head and neck, mesothelioma, myeloma, oesophagal/oral, testicular, thyroid,
uterine,
prostate, renal, lung, ovarian, cervical brain, skin, liver, bone, bowel and
stomach cancers,
sarcomas, tumours and melanomas. Examples of benign hyperplasias include those
of
vascular (eg hemangioma), prostate, renal, adrenal, hepatic, colon (eg colonic
crypt),
parathyroid gland and other tissues.
As the compounds of the invention may have cytostatic as well as cytotoxic
properties, they may also have potential use as therapeutic agents in the
suppression of the
growth of target populations of cells other than cancer or tumour cells, for
example disease
states or conditions associated with cellular hyperproliferation. Such
conditions may
include atherosclerosis and restinosis (neointimal hyperplasia) and
hyperproliferation due
to or accompanying an inflammatory response, eg arthritis, (including
rheumatoid arthritis,
osteoarthritis and inflammatory arthritis), psoriasis and periodontal disease,
or cellular
hyperproliferation due to the viral infection of cells such as human papilloma
virus.
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The compounds of the invention, eg Compounds A and B, may be used in therapy
in conjunction with other therapeutic compounds, such as anti-cancer
compounds,
including paclitaxel, camptothecin, vinblastin and doxorubicin.
Thus, another aspect of the invention relates to a method for the treatment of
cancer
or a cancerous condition comprising the administration of an effective amount
of a
compound of Formula (I) and a further therapeutic agent to a subject in need
thereof, and
the use of said compound in the manufacture of a medicament for use in
conjunction with
other therapeutic agents.
The compounds of the invention and the further therapeutic agent may be
administered simultaneously, as a single composition or as discrete
compositions, or may
be administered separately, ie, one after the other at suitable intervals as
determined by the
attending physician. Thus, the invention also provides a kit comprising a
compound of
Formula (I) together with a further therapeutic agent.
As used herein, the term "effective amount" of a compound relates to an amount
of
compound which, when administered according to a desired dosing regimen,
provides the
desired therapeutic activity. Dosing may occur at intervals of minutes, hours,
days, weeks,
months or years or continuously over any one of these periods. Suitable
dosages lie within
the range of about 0.1 ng per kg of body weight to 1 g per kg of body weight
per dosage.
The dosage is preferably in the range of 1 ~g to 1 g per kg of body weight per
dosage,
such as is in the range of 1 mg to 1 g per kg of body weight per dosage. In
one
embodiment, the dosage is in the range of 1 mg to 500 mg per kg of body weight
per
dosage. In another embodiment, the dosage is in the range of 1 mg to 250 mg
per kg of
body weight per dosage. In yet another embodiment, the dosage is in the range
of 1 ~g to
100 mg per kg of body weight per dosage, such as up to 50 mg per kg body
weight per
dosage. The dosing regime for each subject may be dependent upon the age,
weight,
health and medical history of the subject and the extent and progress of the
condition to be
treated, and can be determined by the attending physician.
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The active ingredient may be administered in a single dose or a series of
doses.
While it is possible for the active ingredient to be administered alone, it is
preferable to
present it as a composition, preferably as a pharmaceutical composition.
The carrier must be pharmaceutically acceptable in the sense of being
compatible
with the other ingredients of the composition and not injurious to the
subject.
Compositions include those suitable for oral, rectal, nasal, topical
(including buccal and
sublingual), vaginal or parental (including subcutaneous, intramuscular,
intravenous and
intradermal) administration. The compositions may conveniently be presented in
unit
dosage form and may be prepared by any methods well known in the art of
pharmacy.
Such methods include the step of bringing into association the active
ingredient with the
carrier which constitutes one or more accessory ingredients. In general, the
compositions
are prepared by uniformly and intimately bringing into association the active
ingredient
with liquid carriers or finely divided solid carriers or both, and then if
necessary shaping
the product.
Compositions of the present invention suitable for oral administration may be
presented as discrete units such as capsules, sachets or tablets each
containing a
predetermined amount of the active ingredient; as a powder or granules; as a
solution or a
suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid
emulsion or a
water-in-oil liquid emulsion. The active ingredient may also be presented as a
bolus,
electuary or paste.
A tablet may be made by compression or moulding, optionally with one or more
accessory ingredients. Compressed tablets may be prepared by compressing in a
suitable
machine the active ingredient in a free-flowing form such as a powder or
granules,
optionally mixed with a binder (e.g inert diluent, preservative disintegrant
such as sodium
starch glycolate, cross-linked polyvinyl pyrrolidone, cross-linked sodium
carboxymethyl
cellulose) surface-active or dispersing agent. Moulded tablets may be made by
moulding
in a suitable machine a mixture of the powdered compound moistened with an
inert liquid
diluent. The tablets may optionally be coated or scored and may be formulated
so as to
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provide slow or controlled release of the active ingredient therein using, for
example,
hydroxypropylmethyl cellulose in varying proportions to provide the desired
release
profile. Tablets may optionally be provided with an enteric coating, to
provide release in
parts of the gut other than the stomach.
Compositions suitable for topical administration in the mouth include lozenges
comprising the active ingredient in a flavoured base, usually sucrose and
acacia or
tragacanth gum; pastilles comprising the active ingredient in an inert basis
such as gelatin
and glycerin, or sucrose and acacia gum; and mouthwashes comprising the active
ingredient in a suitable liquid carrier.
Compositions for rectal administration may be presented as a suppository with
a
suitable base comprising, for example, cocoa butter, gelatin, polyethylene
glycol.
Compositions suitable for vaginal administration may be presented as
pessaries,
tampons, creams, gels, pastes, foams or spray formulations containing in
addition to the
active ingredient such carriers as are known in the art to be appropriate.
Compositions suitable for parenteral administration include aqueous and non-
aqueous isotonic sterile injection solutions which may contain anti-oxidants,
buffers,
bactericides and solutes which render the composition isotonic with the blood
of the
intended recipient; and aqueous and non-aqueous sterile suspensions which may
include
suspending agents and thickening agents. The compositions may be presented in
unit-dose
or multi-dose sealed containers, for example, ampoules and vials, and may be
stored in a
freeze-dried (lyophilised) condition requiring only the addition of the
sterile liquid carrier,
for example water for injections, immediately prior to use. Extemporaneous
injection
solutions and suspensions may be prepared from sterile powders, granules and
tablets of
the kind previously described.
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Preferred unit dosage compositions are those containing a daily dose or unit,
daily
sub-dose, as herein above described, or an appropriate fraction thereof, of
the active
ingredient.
It should be understood that in addition to the active ingredients
particularly
mentioned above, the compositions of this invention may include other agents
conventional in the art having regard to the type of composition in question,
for example,
those suitable for oral administration may include such further agents as
binders,
sweeteners, thickeners, flavouring agents disintegrating agents, coating
agents,
preservatives, lubricants and/or time delay agents. Suitable sweeteners
include sucrose,
lactose, glucose, aspartame or saccharine. Suitable disintegrating agents
include corn
starch, methylcellulose, polyvinylpyrrolidone, xanthan gum, bentonite, alginic
acid or agar.
Suitable flavouring agents include peppermint oil, oil of wintergreen, cherry,
orange or
raspberry flavouring. Suitable coating agents include polymers or copolymers
of acrylic
acid and/or methacrylic acid and/or their esters, waxes, fatty alcohols, zero,
shellac or
gluten. Suitable preservatives include sodium benzoate, vitamin E, alpha-
tocopherol,
ascorbic acid, methyl paraben, propyl paraben or sodium bisulphite. Suitable
lubricants
may include magnesium stearate, stearic acid, sodium oleate, sodium chloride
or talc.
Suitable time delay agents may include glyceryl monostearate or glyceryl
distearate.
One or more embodiments of the present invention may also provide methods,
compositions agents or compounds which have an advantage over (or avoid a
disadvantage) associated with known methods, compositions, agents or compounds
used
in the chemotherapeutic treatment of cancerous conditions or conditions
associated with
the hyperproliferation of cells. Such advantages may include one or more of:
increased
therapeutic activity, reduced side effects, reduced cytotoxicity to non-
cancerous or non-
proliferative cells, improved solubility or dispersibilty for formulation into
pharmaceutical
compositions, improved stability or a more readily available means of
obtaining said
compounds, eg. by simpler, cheaper or higher yielding synthetic or isolation
processes.
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Those skilled in the art will appreciate that the invention described herein
is
susceptible to variations and modifications other than those specifically
described. It is to
be understood that the invention includes all such variations and
modifications which fall
within the spirit and scope. The invention also includes all of the steps,
features,
compositions and compounds referred to or indicated in this specification,
individually or
collectively, and any and all combinations of any two or more of said steps or
features.
The references and citations disclosed within this specification are taken to
be
incorporated herein in their entirety.
The invention will now be described with reference to the following Examples
which are included for the purpose of illustrating embodiments of the
invention and not to
be construed as limiting the generality hereinbefore described.
EXAMPLES
Example 1
Isolation of Compounds A and B from A~laia leptantlta
Compounds A and B were isolated using the following procedure:
(a) Treat a sample of ground bark from the tree species Aglaia leptantlza with
methanol.
(b) Filter the extract and concentrate the resulting solution under vacuum.
(c) Fractionate the extract via solid-phase extraction on a C-18 Varian
extraction
column (10 g) using 0.1% formic acid in acetonitrile/water with increasing
acetonitrile concentrations.
(d) Collect the eluate obtained with an acetonitrile/water ratio of 7:20.
Compounds A
and B have a UV absorption maximum of 200, 273 nm (acetonitrile/water/0.1%
formic acid) and a HPLC retention times of approximately 30.67 (Compound A)
and 31.05 minutes (Compound B) under the following conditions:
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C-8 Symmetry column (WATERS), 4.6 x 250 mm, S~m, 1 mL/min, linear gradient
from 0% to 90% acetonitrile in water in 40 minutes with 0.1% formic acid.
(e) Concentrate fraction obtained under step (d).
(f) Chromatograph the concentrate obtained under step (e) on a C-18
preparative
column (WATERS, Nova-Pak C-18, 6 micron, 2.5 x 25 cm) at a flow rate of 20
mLlmin using a linear gradient from 25% to 45% acetonitrile in water in 30
minutes with 0.1 % formic acid.
(g) Collect and concentrate the eluates with the chromatographic and
spectroscopic
characteristics outlined in step (d) at approximately 22 minutes.
(h) Chromatograph each eluate obtained under (g) on a Sephadex LH20 column
using
methanol as a solvent. Collect and concentrate the fractions with spectral
characteristics outlined in (d). These samples were used for the structural
elucidation of Compounds A and B.
(i) Alternatively to steps (b), (c) and (d), the methanol extract obtained
under (a) may
be partitioned with equal volumes of water and dichloromethane. The
dichloromethane phase is then processed according to steps (e) to (h).
The compounds thus obtained have the following spectroscopic and physical
characteristics;
UV/Vis absorption maxima: 223, 275 nm (in MeCN/H20, 0.1% HCOOH).
MS: Mass spectra were obtained on a Finnigan LCQ iontrap mass spectrometer
using the
ESI source in the positive ion mode. The sample was dissolved in 0.1 %FA in
MeOH and
introduced into the source by infusion with a syringe pump at rate of 3
~.L/min. For
Compounds A, signals were observed at mlz 677 [M+Na]~; MS2 yielded nalz 659
[M+Na-
H20]+; MS3 yielded ynlz 627 (loss of 32 amu); MS4 yielded mlz 595 (loss of
another 32
amu) and m/z 451 (loss of 176 amu, equivalent to the dioxane sidechain). For
compound B
signals were observed in the positive ion mode at m/z 677.2 [M+Na]+; MSZ
yielded product
ions at mlz 627.2 and mlz 659.2. Further fragmentation of the signal at mlz
627.2 yielded a
product ion at na/z 595.3.
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Accurate mass spectra for Compound A were obtained on the Bruker 47e Fourier
Transform - Ion Cyclotron Resonance Mass Spectrometer (FTMS) fitted with an
Analytica
Electrospray Source (ESI). The sample was dissolved in MeOH and introduced in
to the
source by direct infusion with a syringe pump at a rate of 60 ~.L/min. The
source was
operated with capillary voltage of 100v. One signal was observed at rnlz
677.2194
[M+Na]+ meas.; C34H38013Na+ requires 677.2204.
NMR
The NMR spectra of Compounds A and B (see Formula (ii) below) were acquired on
400
and 500 MHz Varian INOVA NMR spectrometers, in CD30D and CDC13, respectively.
The following experiments were conducted: 1H, 13C, DEPT, HMQC, HMBC, COSY. The
1H NMR chemical shifts (obtained in CDC13) and the 13C NMR chemical shifts are
listed
in Table 1.
(i)
H
Compounds A and B
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Table 1: 1H and
13CNMR shifts for
Compounds A and
B
(Preliminary Pos ition
Assignments)
PositionAssignments Compound B
Compound
A
'H NMR '3C 'H NMR '~C
NMR NMR
m m m m
1 CH 5.03, d, 6.7 Hz, 79.6 5.04, d, 6.8 79.6
1 H Hz, 1 H
2 CH 3.89, dd, 14.2, 50.03 3.9, dd, 14, 50
6.7 Hz, 1 H 6.8 Hz, 1 H
COOCH3 170.6 170.7
COOCHa3.65, s, 3H 52.06 3.66, s, 3H 52
3 CH 4.28, d, 14.2 Hz, 55.03 4.28, d, 14 Hz, 55
1 H 1 H
3a C 101.9 101.8
4a C 160.6 160.2
CH 6.43, d, 2 Hz, 1 92.8 6.45, d, 2 Hz, 92.8
H 1 H
6 C 160 159.8
OCHa 3.87, s, 3H 55.9 3.86, s, 3H 55.8
7 CN 6.28 d, 2 Hz, 1 93.9 6.29 d, 2 Hz, 94.3
H 1 H
8 C 157.1 157.1
8a C 109.6 109.4
8b C 93.4
1' C 126.2 126.2
2', 2xCH 7.10, br d, 9 Hz, 128.9 7.10, br d, 9 128.9
6' 2H Hz, 2H
3', 2xCH 6.68, br d, 9 Hz, 112.7 6.69, br d, 9 112.8
5' 2H Hz, 2H
4' C 158.8 158.8
OCHs 3.71, s, 3H 55.05 3.72, s, 3H 55
1" C 136.7 136.6
2", 2xCH 6.84, m, 2H 127.8 6.86, m, 2H 127.5
6"
3", 2xCH 7.06, m, 2H 127.8 7.06, m, 2H 127.5
5"
4" CH 7.06, m, 1 H 126.6 7.06, m, 1 H 126.6
1 CH 5.28, s, 1 H 94 5.26, s, 1 H 93.4
"'
2"' CH 4.59, s, 1 H 95.2 4.60, s, 1 H 95.2
OCHa 3.49, s, 3H 55.1 3.5, s, 3H 55
3"' 4.13, t, 11.2 Hz, 59 4.02, f, 11.2 59.6
1 H Hz, 1 H
CHz 3.56, dd, 11.7, 3.78, dd, 11.7,
2 Hz, 1 H 2.4 Hz, 1 H
4"' CH 4.23, br t, 11.3 68.3 4.12, ddd, 11, 67.6
Hz, 1 H 6.8, 2.8 Hz,
1H
5"' CH 3.61, m, 1 H 70.6 3.66, m, 1 H 71.4
6"' CHz 3.61, m, 2H 63.3 3.61, dd, 10.4, 62.5
4.4 Hz, 1 H
3.72, m, 1 H
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Example 2
Determihatiou of the substitution position of the dioxanyl sidechaiu on the
cyclopeutabeuzofuraue core of Compoufids A and B (Acetylation of Co~zpouuds A
and
B)
The objective of this experiment was to unambiguously determine the attachment
position
of the dioxanyl sidechain to the cyclopentabenzofuran core in Compounds A and
B.
Compounds A and B were dissolved in anhydrous pyridine (A: 4.2 mg in 280 ~,L;
B: 3 mg
in 400 wL) and acetic anhydride was added (A: 140 ~,L, B: 200 ~.L). The
reaction mixtures
were stirred under an argon atmosphere for 14 (A) and 22 (B) hrs,
respectively. The
solvents were removed under reduced pressure to afford the diacetates as an
orange residue
(A: 5.8 mg; B: 3 mg). Purification of the crude residues was achieved by
silica gel
column chromatography eluting with 60% ethylacetate/petrol. The diacetate of
Compound
A, Compound A' (Formula (iii)), was obtained in 68% yield (3.2 mg), and the
diacetate of
Compound B, Compound B' (Formula (iii)), was obtained in 41% yield (1.4 mg).
The purity of the two reaction products was assessed by reversed phase HPLC
using the
same instrumentation as outlined in Example 1 (column: Xterra C-18, 1 mL/min,
gradient:
from 0 to 100% MeCN in 40 mins). The structures of compounds A' and B' were
elucidated by electrospray MS and 1D and 2D NMR experiments using the same
conditions as described in Example 1. NMR spectra of Compounds A' and B' were
obtained in CDC13 with 500 and 400 MHz Varian INOVA instruments.
Both compounds yielded a single peak in the HPLC analysis with retention times
of 26.3
wins for Compound A', and 27.7 mins for Compound B'. Compounds A' and B'
showed
positive molecular ions at m/z 761 [M+Na]+ and 1499 [2M+Na]+ indicative of a
molecular
formula C3gH42O15~ 1 and 2 D NMR experiments (IH, HMQC and HMBC, NOESY)
revealed that the two hydroxyl functions on the dioxanyl sidechain were
acetylated. The
1H and 13C NMR chemical shifts are summarized in Table 2, and the NOESY
spectra in
Table 3.
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The HMBC experiments of both diacetates show clear correlations of the proton
signals of
H-5, H-7 and H-1"' to the carbon 6 of the aromatic ring. The proton signals of
H-7 and a
methoxy group are correlated to the carbon 8. This clearly indicates that the
dioxanyl
sidechain is attached at the position C-6 of the cyclopentabenzofuran core.
Further support for the position of the dioxanyl sidechain was derived from
the NOESY
spectra of both compounds. The NOE signals are observed from both H-5 and H-7
to H-
1 "' of the dioxanyl side chain, and only H-7 shows a NOE signal to the C-8
methoxy
signal. The NOE signals observed in the dioxanyl ring systems of the two
compounds
clearly indicate that they differ in regard to the stereochemistry of the di-
hydroxyethane
sidechain. The NOE signals for the cyclopentabenzofuran core are in agreement
with
published data and confirm the stereochemistry depicted in Tables 1 and 2.
OCH3 H _OH
sl H
7 ~ ~ 8a 8b ~ ~~"'~~~~COOCH3 (ill
4a 3a
g z
H3COC0 O 5 O 1'
H
2...
OCH3 \
OCH3
Compounds A' and B' (Diacetates of Compounds A and B)
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Table 2: 1H and 13C NMR chemical shifts for Compounds A' and B'
PositionAssignmentCompound A' Compound B'
(Diacetate of (Diacetate of
Compound A) Compound B)
H NMR C NMR H NMR C NMR
1 CH 5.06, d, 8 Hz 79.8 5.07, d, 8 Hz 79.8
2 CH 3.89, under 50.4 3.87, under 50
OCHs-8 OCHs-8
2 COOCHs 170.4 170.4
2 COOCHs 3.64, s 51.9 3.63, s 51.8
3 CH 4.26, d, 14 55 4.27, d, 14.4 54.8
Hz Hz
3a C 101.9 102
4a C 160.6 160.5
CH 6.43, d, 2 Hz 93.3 6.45, d, 2 Hz 93.2
6 C 159.6 159.7
7 CH 6.27 d, 2 Hz 93.2 6.29, d, 2 Hz 93
8 C 157 156.9
8 OCHs 3.89, s 56 3.88, s 55.8
8a C 109.8 109.6
8b C 93.5 93.4
8b OH 2.35, s 2.33
1' C 126.2 126.4
2', 2 x 7.10, br d, 129.1 7.10, br d, 129
6' CH 9 Hz 9 Hz
3', 2 x 6.67, br d, 112.7 6.68, br d, 112.6
5' CH 9 Hz 9 Hz
4' C 158.8 158.6
4' OCHs 3.72, s 55 3.72, s 55
1" C 136.8 136.6
2", 2 x 6.83, m 127.7 6.86, m 127.8
6" CH
3", 2 x 7.05, m 127.1 7.05, m 127.8
5" CH
4" CH 7.05, m 126.6 7.05, m 126.6
1 "' CH 5.38, s 93.7 , 5.31, s 93.2
2"' CH 4.61, s 95.2 4.62, s 95.2
2"' OCHs 3.50, s 55.2 3.5, s, 3H 55
3"' CHz 3.94, t, 11.2 58.8 3.93, t, 12 59.5
Hz Hz
3.54, dd, 11.2, 3.59, dd, 12,
3 Hz 2.5 Hz,
4"' CH 4.38, dt, 11, 66.3 4.37, td, 9, 64.9
3 Hz 2.5 Hz
5"' CH 5.12, td, 6, 69.1 5.00, ddd, 9, 70.2
3 Hz 4, 2.5 Hz
6"' CHz 4.22, dd, 11.2,61.3 4.21, dd, 12.4,61.5
6 Hz 4 Hz,
3.88, ? under 4.12, dd, 12.4,
OCHa-8 2.5 Hz
5"' COCHs 170.3 169.9
5"' COCHs 2.14, s 20.8 2.07 20.8
6"' COCHs 170.7 170.8
6"' COCHa 1.79 20.4 1.74 20.1
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H3COC0
Table 3: Comparison of NOESY Spectra of Compounds A' and B'
Compound A' Compound B'
1 H NMR NOEs 1 H NMR NOEs
6.43, d, H-5 5.38, s, H-1 "' 6.45, d, H-5 5.31, s, H-1
"'
4.38, dt, H-4"' 4.37, td, H-4"'
4.21, dd, H-6"'-1
1.74, s, COCH3-6"'
6.27, d, H-7 5.38, s, H-1 "' 6.29, d, H-7 5.31, s, H-1
"'
4.21, dd, H-6"-1
3.89, s, OCH3-8 3.88, s, OCH3-8
5.38, s, H-1 4.38, dt, H-4"' 5.31, s, H-1 4.37, td, H-4"'
"' "'
3.89, s, OCH3-8 3.88, s, OCH3-8
3.50, s, OCH3-2"' 3.5, s, OCH3-2"'
Examble 3
Compounds A and B are cytostatic and cytotoxic for human tumour cell lines
(a) Compounds A and B were identified from a bark sample of Aglaia lepta~ctha
through their ability to inhibit production of Tumour Necrosis Factor-a (TNF-
a) by THP-1
human promonocytic leukemia cells (Tsuchiya, et al, Iht. ,I. Cancer', 190,
26(2):171-6)
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activated with lipopolysaccharide (LPS). Table 4 summarises the results
comparing the
activity of Compounds A and B for inhibition of TNF-a production to their
effects on
general cell metabolism measured using WST-1 reduction, DNA synthesis and
protein
synthesis assays for THP-1 cells. Compounds A and B potently inhibited TNF-a
production at broadly similar concentrations that were active in the WST-1
reduction,
DNA and protein synthesis assays. For comparison, the effects of Compounds A
and B on
A549 lung epithelial carcinoma cells (Leiber et al, Int. J. Cancer, 1976,
17(1):62-70) were
also measured and the data is also included in Table 4. Compounds A and B are
significantly less potent for inhibition of interleukin-1 (IL-1)-induced
Intercellular
Adhesion Molecule-1 (ICAM-1) expression by A549 cells even though in these
cells the
protein and DNA synthesis inhibition occur at broadly similar concentrations
as for THP-1
cells.
Table 4 : Comparison of the effects of Compounds A and B in THP-1 and A549
Cells*
ICSO
~~M~
THP-1'cells A549
cells
'Compound TNF=a IrVST-1ProteinDN:4 ICAM-1 ProteinDNA
Prtx~'uet~nReductionSjmf~esisSyn~eslsProdc~SonSync Syn~resis
Compound 0. 06 0.03 0. 06 0. 015 2 0. 02 0. 007
A
Compound 0.015 0.04 0.003 0.003 5 0.01 0.004
B I
* Purified Compound A or Compound B solubilized in DMSO were tested over a
range of
concentrations in parallel for inhibitory activity in the various assays in
both THP-1 and
A549 cells. The concentration that resulted in a 50% inhibition of the
relevant response
(ICSO) is shown. Production of TNFa by THP-1 cells was measured as that
released into
the culture supernatant over 18 hours by sandwich enzyme-linked immunosorbent
assay
(ELISA) using the following mouse anti-TNFa monoclonal antibodies (capture
antibody,
MAB610; detection antibody, biotinylated MAB210; both from R & D Systems,
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Minneapolis MN, USA). Surface expression of ICAM-1 by A549 cells was assayed
after
24 hours of culture by direct antibody binding using a europium-labelled mouse
anti-
ICAM-1 monoclonal antibody (R&D Systems Cat No. BBA3) and measured by time-
resolved fluorescence using Delfia assay (EG&G Wallac, Turku, Finland).
Reduction of
WST-1 (Roche, Cat. No. 1644807) by THP-1 cells was measured after 18 hours of
culture
according to the manufacturer's instructions. Protein synthesis was measured
as the uptake
of [14C]-leucine (0.5 ~,Ci/mL) after 48 hours for THP-1 cells and 72 hours for
A549 cells
cultured in growth medium (RPMI-1640, 10% FBS) containing 10% the usual L-
leucine
concentration (5 mg/mL). DNA synthesis was measured as the uptake of [14C]-
thymidine
(0.5 ~Ci/mL) after 48 hours for THP-1 cells and 72 hours for A549 cells in
normal growth
medium.
(b) Compound A was assessed for cytotoxic and cytostatic activity against a
panel of
cell lines derived from a variety of human tumour types in addition to THP-1
and A549
cells (Table 5). These included K562 leukemic cells (Lozzio and Lozzio, 1975,
Blood
45:321-34), PC3 prostate tumour cells (I~aighn et al., 1979, hcvest. Urol.
17:16-23) and
SF268 glioblastoma cells (Westphal et al, 1985, Biochem. Biophys. Res.
Commun.,
132:284-9). Compound A exhibited potent cytostatic activity in nearly all cell
lines tested
with GISO values ranging between 1-7 nM. Compound A also exhibited potent
cytotoxic
effects against the various tumour cell lines. Interestingly, the THP-1 and
PC3 cells proved
the most rapidly killed with little difference in LCSO values obtained after 3
or 6 days of
culture. However, the cytotoxic potency of Compound A increased dramatically
after 6
days of culture for the K562, A549 and SF268 cells. It should be noted that
the
concentration of Compound A required to inhibit cell proliferation were
significantly lower
than those required to elicit a cytotoxic response. Hence, the cytostatic
effect of Compound
A is biochemically distinguishable from its ability to induce cell death.
Table 6 shows that
Compound B exhibited cytotoxic effects against the various tumour cell lines
with
comparable potency to that observed with Compound A.
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Table 5: Compound A has potent cytostatic and cytotoxic activity in various
human
tumour cell lines in vitro*
Tumour . < Tumour Compound A
Source Cell Line ; =Glso LCso (nN1) LC5o (nM)
(nM); ..
.
(3=.day (3 day , (6 day
~
culfures) cultures) cultures)
- .
Leukemia THP-1 - 36 24
K562 1 >1000 10
Luhg A549 7 914 21
Prostate PC3 5 18 12
Bsain SF268 3 461 29
* Purified Compound A was tested over a range of concentrations up to a
maximum of lx
10-6 M (1000 nM) for cytostatic and cytotoxic activity against a panel of cell
lines derived
from various human tumour types as indicated. The GISO value represents the
concentration
of compound that inhibited the cell number increase (relative to untreated
cells) by 50%
after 3 days of culture. Relative cell number was determined by measuring
cellular DNA
using a fluorescent DNA-binding dye (YOYO-1) after lysing the cells with
digitonin
(Becker et al., Anal Biochem,1994, 221(1):78-84). The LCSO value represents
the
concentration of compound that killed 50% of the cells. Cell death was
measured as the
proportion of dead cells exhibiting sub-diploid DNA content determined by flow
cytometry after staining with propidium iodide (Nicoletti et al., J. Immunol.
Methods,
1991, 139:271-79).
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Table 6: Compounds A and B exhibit similar cytotoxic activity*
~r TuriiourTumour ~LC5o(nM)s
r ;
Source $ ' Cell '~day cultures)
Line ~' '
(6
, ~
~
,
__
.a und Compound
Compo
x ~~ r t 4
~n z&"- ~~ . , x~ r
' x ~, ;ar W
B ' c
~
' t , ;
,s;g'~Pi
,
Leukemia THP-1 l l I S
K562 12 I S
Lung A549 15 12
Prostate PC3 12 12
Braun SF268 12 22
* The cytotoxic activity of Compounds A and B were compared for the various
tumour
cell lines as described in Table 5.
(c) Testing of Compound A against a much larger cell line panel in the NCI in
vitf°o
anticancer screen (Table 7) confirmed the results described above. Using a
different assay
methodology based on measurement of total cellular protein the results confirm
that
Compound A had broad and potent cytostatic effects with all of the cell lines
exhibiting
maximal inhibition of cell growth even at the lowest dose tested (lOnM).
Consistent with
the data in Table 5 the cytotoxic effects measured after 2 days of culture
were more varied
with LCSO values ranging from 10 nM for COLO-205 colon tumour cells to ~90 ~M
for
786-0 renal tumour cells. These data indicate that Compound A had potent in
vitro activity
against a wide range of tumour cell lines representing a variety of different
major types of
cancer including leukemia, lung, colon, brain, melanoma, ovarian, renal,
prostate and
breast tumours.
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Table 7: Activity of Compound A measured in the NCI ih vitro anticancer drug
discovery screen*
Tumour Source ~ Tumour Cell Lme GISO,(mM) LCSa (nM).
-
Lung EKVX <10 23
Lung NCI-H226 <10 193
Lung NCI-H460 <10 38,019
Lung NCI-H522 <10 2,399
Colon COLD-205 <10 10
Colon HT29 <10 1,000
Brain SF-268 <10 1,000
Brain SF-295 <10 1,230
Brain SF-539 <10 1,096
Brain SNB-75 < 10 54
Melanoma LOX IMVI <10 1,000
Melanoma MALME-3M <10 23,988
Melanoma M14 <10 51
Melanoma SK-MEL-2 <10 19,055
Melanoma SK-MEL-28 <10 2,661
Melanoma SK-MEL-5 <10 67
Melanoma UACC-62 <10 30
Ovarian IGROV 1 <10 2,600
Ovarian 0 V CAR-4 < 10 11
Ovarian OVCAR-5 <10 1,000
Ovarian OVCAR-8 <10 21,878
Ovarian SK-OV-3 <10 82,224
Renal 786-0 <10 91,201
Renal A498 <10 18,621
Renal ACHN <10 31,623
Renal RXF 393 <10 1,641
Breast MCF7 <10 1,000
Breast MDA-MB-231/ATCC <10 25,410
Breast MDA-MB-43 5 < 10 45
Breast MDA-N <10 543
Breast BT-549 <10 32,734
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* Compound A was tested for activity in the National Cancer Institute in vitro
anticancer
drug discovery screen. For this Compound A was tested at five 10-fold
dilutions ranging
from 10'4M to 10-8M against a panel of different human tumour cell lines
representing maj or
types of cancer as described by Boyd and Paull, Drug Developme>zt Research,
1995, 34:91-
109. Briefly, this involved a 48 hr incubation of the cells with Compound A
prior to
measuring the relative cell number by staining with sulforhodamine B. GISO
values represent
the concentration of Compound A that inhibited net growth of the cells by 50%
compared to
untreated controls. LCSO values represent the concentration of Compound A that
resulted in a
net 50% loss (killing) of the cells relative to the start of the experiment.
The data represent
the average values from two such experiments conducted.
Example 4
Cytotoxic activity of Compound A is zzot shared by other knowzz related
compouzzds lacking
dioxanyloxy substitution. Compound A' displays cytotoxic activity.
(a) Table 8 compares the cytostatic and cytotoxic effects of Compound A to
three
previously identified 1H cyclopenta[b]benzofuran lignans that lack the
dioxanyloxy group at
the C6-position. The reference compounds are: Rocaglaol (Reference Compound 1)
(Ohse et
al., JNat Prod, 1996, 59(7):650-52); 4'-Demethoxy-3',4'-
methylenedioxyrocaglaol
(Reference Compound 2) and Methyl 4'-demethoxy-3',4'-methylenedioxyrocaglate
(Reference Compound 3) (hee et al., Chem Biol Iv~teract, 1998, 115(3):215-28).
All four
compounds exhibited detectable cytostatic activity in A549 cells with Compound
A being the
most potent followed in decreasing order by Reference Compounds 3, 2 and 1
respectively.
Importantly, of the compounds tested, other than Compound A none of the
Reference
Compounds exhibited any appreciable cytotoxicity in either THP-1 or A549 cells
at doses up
to 5000 nM over the 3 day assay. Without intending to limit the invention by
theory, it is
suggested that the novel dioxanyloxy substitution at the C6-position is
important for the
cytotoxic activity exhibited by Compound A and distinguishes it from any other
previously
identified 1H cyclopenta[b]benzofuran lignans.
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Reference Compound 1 Reference Compound 2 Reference Compound 3
Table ~: Related 1H cyclopenta[b]benzofuran lignans lacking the novel
dioxanyloxy
side chain do not exhibit cytotoxic activity*
~~ ~'~~ Compound ~'~' ,'~'~r4549~cNel~s ~THP 1 ccells
~ ' ~ ~ ~ *.-
,b ~
4
ti G~ (nMj L C (n ~ ' ,
' g ~ (nM~
~ ~ fi,
' LC
~ rt ,~, ~,., 50 ,!;, ~ 50_ . 50~
~ ~ ,
~ . ~ 4
Compound A 13 514 I S
Reference Compound390 >5000 >5000
1
Reference Compound3~9 >5000 >5000
2
Reference Compound56 >5000 >5000
3
* A549 and THP-1 cells were treated with increasing concentrations of the
various
compounds up to a maximum of 5 x 10'6 M (5000 nM) and the effects on cell
proliferation
and cell viability were determined after 3 days of culture. GIso values were
determined by
measuring relative changes in cell number using YOYO-1 as described for Table
5. L~CSo
values were determined by measuring cell death as a function of loss of
membrane integrity
using YOYO-1 uptake (Becker et al., Anal Biochern,1994, 221(1):78-84). The
structures of
the reference
compounds are also shown.
(b) Table 9 shows that acetylation of the dioxanyl side chain of Compounds A
and B did
not reduce their biological activity since Compounds A' and B' inhibited WST-1
reduction of
THP-1 leukemic with at least similar potencies to the unmodified compounds.
The lower
ICso values for all the compounds depicted in this WST-1 reduction experiment
compared to
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the values shown in Table 4 reflects the enhanced sensitivity of the cells
when treated for 3
days compared to the 18 hr treatment used in the latter assay.
Table 9: Acetylation of the dioxanyl side chain of Compounds A and B does not
inhibit
their biological activity*
Co'inpound ~ ., IC5o (nM)
~
y , ~t
y
f
Compound A 2.0
Compound A' 0.3
Compound 8 1.5
Compound 8' 0.7
* Purified compounds solubilized in DMSO were tested over a range of
concentrations for
their effects on the reduction of WST-1 by THP-1 leukemic cells as described
for Table 4
except that the cells were cultured in the presence of the various compounds
for 3 days prior
to measuring WST-1 reduction.
Example 5
CompoundA has acute protein synthesis inhibitory actirity
Compound A was also examined to determine whether it could rapidly iWibit
general protein
biosynthesis. Using [14C] leucine incorporation into insoluble cellular
material as an assay
for general protein biosynthesis, Table 9 shows that Compound A had an
inhibitory effect
evident within 3 hrs after addition to THP-1 cells with an ICSO of ~ 30 nM.
DNA synthesis
measured over the same time was also inhibited, but less potently (ICSO ~ 70
nM) and may be
secondary to protein synthesis inhibition. Cyloheximide, a known protein
synthesis inhibitor
(Obrig et al, 1971, J. Biol. Chem. 246(1): 174-181), also inhibited both
protein and DNA
synthesis with Compound A being significantly more potent than cycloheximide
in its
effects. Table 10 shows that Compound A also inhibited general protein
synthesis in A549
cells with an ICSO of ~30 nM which is similar to that observed in the THP-1
cells.
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Table 10: Compound A inhibits general protein biosynthesis*
IC5o (nM)
Compound THP-.1:.cells ~.A549 cells
Protein =_ ~ DNAP : ~ Profein~
synthesis Synfhes~s synthesis
'
Compound A 27 72 32
Cyclohexinzide 263 303 238
* THP-1 cells and A549 cells were pretreated with the indicated concentrations
of
Compound A for 1 hour prior to the addition of (1 ~.Ci/mL) [14C] leucine
(protein synthesis)
or [14C] thymidine (DNA synthesis) for a further 2 hours. The ICSO values
represent the
concentration of Compound A required to inhibit incorporation of isotope by
50% relative to
untreated control cell cultures.
Example 6
Compouzzd A induces differentiatiozz of human leukemic cell lines.
In the experiments with the THP-1 monocytic leukemia cells, which normally
grow
unattached in suspension, we noticed that prolonged exposure of the cells to
10 nM
Compound A resulted in accumulation of cells that adhered to the plastic and
exhibited
numerous pseudopodia (Figure 1). This is a morphology highly characteristic of
mature
macrophages and similar morphological effects were observed when the cells
were treated
with other known inducers of macrophage differentiation including interferon-y
(IFNy) or
phorbol 12-myristate 13-acetate (PMA). To investigate this further the effects
of Compound
A on HL60 human promyelocytic leukemic cells (Collins, et al, Nature, 1977,
270:347-9)
were examined (Table 11). This widely used cell line is well characterised as
a model of
human myelomonocytic differentiation (Collins, Blood, 1987, 70(5):1233-44). In
this
experiment monocytic differentiation was quantitated by measuring CD 14
surface antigen
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expression by flow cytometric analysis. CD14, an LPS-binding protein, is
expressed on the
surface of cells of the myelomonocytic lineage and is normally expressed at
very low levels
in undifferentiated HL60 cells (Ferrero et al., Blood,1983,61(1):171-9).
Consistent with the
THP-1 data above, Table 10 shows that Compound A at doses greater than 10 nM
significantly enhanced CD14 expression in the viable HL60 cells remaining
after 4 days of
culture. Taken together these data strongly indicate that Compound A has the
ability to
induce differentiation of human leukemic cell lines.
Table 11: Compound A promotes monocytic differentiation of HL60 leukemic
cells*
~~Compound A' % cells expressing
,
concentration (iiM) x ~ CD14 .
.. ~. a - .
0 1.3
S 3. 3 %
10 5. 7 %
25 46. 0 %
50 43. 0 %
* HL60 cells were cultured for 4 days with the indicated concentration of
Compound A then
collected and fixed in 70% ethanol. Cells were then stained with mouse
monoclonal anti
CD14 antibody (OKMl) and this was measured using FITC-conjugated goat anti-
mouse
IgGl as a secondary antibody. Stained cells were visualised by flow cytometry
and analysis
was restricted to cells judged viable at the time of fixing based on their
forward and side
light-scatter characteristics. Non specific staining of cells was controlled
for by incubating
with secondary antibody only.
Example 7
Cytostatic activity of Compound A is associated with a general inltibitioh of
cell cycle
progression iu A549 cells
DNA content analysis of THP-1 cells treated with varying concentrations of
Compound A
(Figure 2) demonstrated that at 10 nM it was only weakly cytotoxic (increased
accumulation
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of dead cells from 7% to 17%) and under these conditions caused cells to
accumulate in the
GO/G1 phases of the cell cycle. This indicates that Compound A also has
cytostatic activity
in THP-1 cells. For comparison, Figure 2 shows that the microtubule
destabilising drug
paclitaxel (Sorger et al., Cuf°f° Opi~ Cell Biol, 1997, 9(6):807-
14) which also induced THP-1
cell death, caused cells to accumulate in the G2/M phases of the cell cycle.
The cytostatic effect of Compound A on the proliferation of A549 cells was
confirmed by
directly counting the number of cells at intervals over a nine day period
(Figure 3). When
compared to untreated cells 10 nM Compound A prevented the increase in cell
number by
more than 95% with fewer than 10% dead cells observed at this time (measured
by trypan
blue exclusion). Thus, under these conditions the decreased cell number can
not simply be
accounted for by increased cell death. A significant inhibition of cell number
was seen
within 2 days indicating that Compound A acts in a rapid manner. At the higher
concentrations of 50 nM and 250 nM Compound A had cytotoxic effects and
increased cell
death to 86% and 100% respectively after 9 days and accounts for the decline
in cell number
to levels below the original starting number at this time. At the non-
cytotoxic concentration
of 10 nM, Compound A has a rapid and potent cytostatic effect on A549 cells.
To help identify a potential mechanism for the effects of Compound A, DNA
content analysis
was performed to determine where in the cell cycle it exerted its effect
(Figure 4). Cell cycle
analysis of A549 cells treated with Compound A for 6 days showed that at 10
nM, where no
obvious cytotoxicity was evident, there was a minor decline in the proportion
of cells in the
GO/G1 phases of the cell cycle with a concomitant increase in cells in the
G2/M phases.
Taken together with the growth curve data in Figure 3 above, these data
indicate that 10 nM
Compound A results in a general lengthening of all phases of the cell cycle
with perhaps a
slightly more pronounced elongation of the G2/M phases. This contrasts to the
effects of
paclitaxel a drug known to act selectively at the G2/M phases of the cell
cycle (Figure 4). As
the concentration of Compound A was increased and its cytotoxic effects became
evident the
proportion of cells in the S and G2/M phases decreased with a corresponding
rise in cells in
GO/G1 phases. Although there was little difference in the number of dead cells
between 50
nM and 250 nM the higher dose resulted in a greater accumulation of cells in
the GO/G1
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phases of the cell cycle. Thus, compared to THP-1 cells (see Figure 2) higher
concentrations
of Compound A are required to inhibit progression through the GO/G1 phases of
the cell
cycle in A549 cells.
K562 leukemic cells treated with 10-15 nM Compounds A or B exhibited a
characteristic
accumulation of cells in G2/M phases of the cell cycle (Figure 5). This
occurred over a
narrow range of concentrations since Compounds A or B at less than 5-8 nM or
more than 25
nM did not cause a G2/M phase accumulation. These data indicate that different
cell lines
can vary in their sensitivity and responses to Compounds A and B for cell
cycle phase-
specific effects.
Example 8
The cytostatic effect of Compound A is reversible in A549 cells
The reversibility of the effects of Compound A was determined. For this, A549
cells
remained untreated or were cultured in the presence of various concentrations
of Compound
A or with paclitaxel for 5 days prior to removal of the compounds and the
cells cultured for a
further 4 days prior to determining cell number (Figure 6). 10 nM of Compound
A
significantly suppressed the increased cell number for up to 9 days without
significant
cytotoxicity. However, for these cultures when Compound A was removed after 5
days there
was over a five-fold increase in cell number over the subsequent 4 days of
culture,
representing 2 -3 population doublings. The effects of treatments which were
deleterious to
the cells, such as higher concentrations of Compound A or the presence of
paclitaxel, were
not reversed upon their removal.
Example 9
Conapound A inhibits cell cycle-dependent cytotoxicity elicited by various
anti-cancer
agents
To further examine the cell cycle effects of Compound A a cytostatic
concentration of this
compound was combined together with other anti-cancer agents known to act at
specific
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points in the cell cycle to see if Compound A could perturb their cell cycle-
dependent effects.
Cell viability was assayed after 3 days by measuring exclusion of the
fluorescent DNA-
binding dye YOYO-1. (Becker et al., Anal Biochem,1994, 221(1):78-84). A549
cells were
treated with 10 nM non-cytotoxic dose of Compound A in the presence of
increasing
concentrations of camptothecin and paclitaxel. Camptothecin is an inhibitor of
DNA
topoisomerase l, an enzyme required for DNA replication, and results in
pertubation of the S
phase of the cell cycle with subsequent cell death due to activation of an S
phase checkpoint
(Darzynkiewicz et al., Ahn N Y Acad Sci, 1996, 803:93-100). Paclitaxel, as
already
mentioned, inhibits microtubule function required for formation of the mitotic
spindle thereby
resulting in activation of an M phase checkpoint and subsequent cell death
(Sorger et al.,
Cuf°r Opi~ Cell Biol, 1997 9(6):807-14). Figure 7 shows that 10 nM
Compound A
significantly reduced the cytotoxic effects of both camptothecin and
paclitaxel even when
these drugs were added at up to a 2000-fold excess. Compound A may, in a
dominant
manner, prevent the cell cycle-dependent cytotoxic effects of camptothecin and
paclitaxel.
This was examined in more detail using DNA content analysis to specifically
measure cell
cycle progression and cell death. In this experiment in addition to
camptothecin and
paclitaxel cells were also treated with vinblastin (another microtubule
inhibitor) (Sorger et
al., 1997, supra) and staurosporine (a kinase inhibitor) (Gescher, Crit Rev
Oncol Hematol.,
2000, 34(2):127-35). As previously found, A549 cells treated with 10 nM
Compound A
showed a minor decrease in cells in GO/G1 with a slight increase in G2/M phase
cells with no
detectable increase in cell death over the three days of culture (Figure 8).
Consistent with its
known mechanism of action camptothecin resulted in accumulation of cells in S
phase of the
cell cycle and also increased the level of dead cells detected as those with a
sub-diploid DNA
content. Also as expected, both vinblastin and paclitaxel resulted in the
majority of cells
arresting in the G2/M phases of the cell cycle and increased appearance of sub-
diploid dead
cells. However, for all of these agents the presence of 10 nM Compound A
prevented their
characteristic cell cycle arrest and significantly inhibited their cytotoxic
effects, dramatically
reducing the appearance of sub-diploid dead cells. In contrast, Compound A had
little effect
on the cytotoxic effects of staurosporine, an agent which appears capable of
killing cells at all
active phases of the cell cycle.
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Example 10
Cytostatic effects of Compound A do hot correlate with a biomarker for
replicative
senescence.
The dramatically decreased growth rate of A549 cells cultured in the presence
of 10 nM
Compound A (see Figure 3) led to the consideration of the possibility that
this compound was
inducing replicative senescence of these immortal tumour cells. Consistent
with this
possibility under these conditions A549 cells with a morphology highly
suggestive of a
senescent phenotype were often observed, being highly flattened with an
enlarged surface
area compared to their usual appearance (compare for example Figures 9
subpanels a and b).
This was evaluated further by measuring senescence-associated ~3-galactosidase
(SA-~3-gal)
activity, a biomarker previously described to correlate well with senescence
of human cells
(Dimri et al., Proc Natl Acad Sci USA 1995 92(20):9363-7). Recently, it has
been found that
some anti-cancer agents that act by diverse mechanisms, including doxorubicin,
cisplatin,
cytarabine, etoposide and paclitaxel, can all induce SA-(3-gal activity in a
variety of tumour
cell lines (Chang et al., Cancer Res 1999, 59(15):3761-7). Therefore, in
addition to
Compound A A549 cells were also treated with doxorubicin as an experimental
control. This
drug acts by stabilising DNA/topoisomerase II complexes thereby causing DNA
damage
which results in subsequent S phase cell cycle arrest and/or cell death
(Froelich-Ammon and
Osheroff, 1995, J. Biol. ChenZ. 270(37):21429-21432). Figure 9 shows that
consistent with
the earlier report A549 cells treated with 250 nM doxorubicin displayed the
flattened
enlarged phenotype of senescent cells and exhibited SA-(3-gal activity. In
contrast,
Compound A at various doses from 10-50 nM failed to induce SA-~3-gal activity
even though
the cells exhibited the flattened enlarged morphology. Thus, in contrast to a
variety of other
anti-cancer drugs the cytostatic effects of Compound A do not correlate with
this particular
marker of cell senescence.
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Example 11
Compound A iulaibits cell proliferation but tZOt increased cell size
It is well known that cell proliferation and cell growth reflected as
increased mass of
individual cells are biochemically separable processes (Pardee, Science, 1989,
246:603-8).
Although at certain concentrations Compound A can inhibit cell proliferation
without overt
cytotoxicity it was also evaluated whether Compound A also affected cell
growth. For these
experiments A549 cells were treated with various non-cytotoxic doses of
Compound A up to
nM and the relative cell size determined after 6 days of culture by measuring
forward light
10 scatter using a flow cytometer. The data depicted in Table 11 show that in
the presence of
Compound A A549 cells exhibited an increase in the mean forward scatter by
over 20%.
This occurred only at concentrations which are cytostatic for this cell type.
Table 12: Compound A increases cell size*
Compounal A ~ % increase ya,nean
concentration (nM).cell ~olcii a
, "
0 -
2.5 10.4 %
5.0 10.7 %
10. 0 22. 4
* A549 cells cultured for six days with the various non-cytotoxic
concentrations of
Compound A as indicated were examined by flow cytometry for their forward
light scatter
characteristics which directly relates to cell size. The % increase in mean
cell volume
represents the relative change in the mean forward scatter value for the
treated versus
untreated cell populations.
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Example 12
Compound A inhibits growth of hunzau tumour cell lifaes iu a mouse xenograft
tumour
model.
The ability of Compound A to inhibit growth of human tumour cells ih vivo was
assessed
using male athymic mice injected subcutaneously in the dorsal flank region
with 2 x 106 PC3
human prostate tumour cells. Compound A administration (3 mglkg) by
intraperitoneal
injection commenced after eight days once the PC3 tumour was palpable and
continued three
times a week until 29 days after the initial inoculation of the tumour cells.
At this time all
mice were killed and tumours excised and weighed. Figure l0A shows that
compared to the
control animals treated with vehicle alone the mice treated with Compound A
displayed a
greatly reduced increase in mean tumour volume over the course of the
experiment. This was
confirmed at the end of the experiment when tumours were excised and weighed
it was found
that Compound A treatment reduced the mean tumour weight by ~ 60% (Figure
lOB). Body
weight was unaffected with both control and treated groups exhibiting a
similar ~12%
decrease in mean body weight over the duration of the experiment. Thus,
Compound A
exhibits in vivo antitumour activity.
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