U.S. patent application number 09/978324 was filed with the patent office on 2002-04-18 for family of canadensol taxanes, the semi-synthetic preparation and therapeutic use thereof.
This patent application is currently assigned to Institut National de la Recherche Scientifique. Invention is credited to Caron, Gaeton, Zamir, Lolita, Zheng, Yi Feng.
Application Number | 20020045771 09/978324 |
Document ID | / |
Family ID | 25678766 |
Filed Date | 2002-04-18 |
United States Patent
Application |
20020045771 |
Kind Code |
A1 |
Zamir, Lolita ; et
al. |
April 18, 2002 |
Family of canadensol taxanes, the semi-synthetic preparation and
therapeutic use thereof
Abstract
The present invention relates to a new taxane having the
structure as in formula I and its analogs as depicted in Formula II
and Formula III. The invention includes novel semisynthetic and
isolation methods for the preparation of these novel taxanes.
Specifically, the present invention relates to the new taxane
Canadensol isolated from plants of the Taxus genus, in particular,
Taxus canadensis. The present invention further relates to the use
of these new taxanes as anticancer agents.
Inventors: |
Zamir, Lolita; (Westmount,
CA) ; Caron, Gaeton; (Laval, CA) ; Zheng, Yi
Feng; (Salt Lake City, UT) |
Correspondence
Address: |
ELMAN & ASSOCIATES
P O BOX 1969
MEDIA
PA
19063-8969
US
|
Assignee: |
Institut National de la Recherche
Scientifique
Quebec
CA
|
Family ID: |
25678766 |
Appl. No.: |
09/978324 |
Filed: |
October 16, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09978324 |
Oct 16, 2001 |
|
|
|
08957138 |
Oct 24, 1997 |
|
|
|
Current U.S.
Class: |
549/510 |
Current CPC
Class: |
C07D 413/12 20130101;
C07D 409/12 20130101; C07D 305/14 20130101; C07D 407/12
20130101 |
Class at
Publication: |
549/510 |
International
Class: |
C07D 35/14 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 24, 1996 |
CA |
2188714 |
Feb 12, 1997 |
CA |
2197369 |
Claims
We claim:
1. A compound of formula III: 22wherein R.sub.3 is selected from
the group consisting of propyl, isopropyl and n-butyl.
2. The compound as in claim 1, wherein R.sub.3 is isopropyl.
3. A compound of formula IV: 23wherein R.sub.3 is selected from the
group consisting of propyl, isopropyl and n-butyl.
4. The compound as in claim 3, wherein R.sub.3 is isopropyl.
5. A compound of formula V: 24wherein R.sub.4 is SiEt.sub.3 or
CO.sub.2CH.sub.2CCl.sub.3.
6. A process for the preparation of a compund of formula V, which
comprises the steps of: (i) protecting a hydroxy group at the 7
position of baccatin III: 25to form a 7-OH-protected baccatin III;
and (ii) reacting the 7-OH-protected baccatin III with a compound
of formula VII. 26
7. The process of claim 6, wherein the hydroxy group at the 7
position is protected with SiEt.sub.3 or
CO.sub.2CH.sub.2CCl.sub.3.
8. A method of inhibiting tumours in a mammal in need of such
treatment, comprising administering to the mammal a therapeutically
effective amount of a compound of formula III: 27wherein R.sub.3 is
selected from the group consisting of propyl, isopropyl and
n-butyl.
9. The method of claim 8, wherein R.sub.3 is isopropyl.
10. A pharmaceutical formulation comprising one or more compounds
of formula III: 28and a pharmaceutically acceptable carrier;
wherein R.sub.3 is selected from the group consisting of propyl,
isopropyl and n-butyl.
11. The pharmaceutical formulation of claim 10, wherein R.sub.3 is
isopropyl.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of copending
U.S. patent application Ser. No. 08/957,138, filed Oct. 24, 1997,
which claims priority from Canadian Patent Application 2,188,714,
filed Oct. 24, 1996, and from Canadian Patent Application
2,197,369, filed Feb. 12, 1997. The entire disclosure of each of
the aforementioned patent applications is hereby incorporated
herein by reference.
FIELD OF THE INVENTION
[0002] The invention relates to novel taxanes, semi-synthetic and
isolation methods for their production and their use as anticancer
agents.
BACKGROUND OF THE INVENTION
[0003] The taxane family of terpenes are considered as an
exceptionally promising group of cancer chemotherapeutic agents.
Many taxane derivatives are highly cytotoxic and possess strong in
vivo activities in a number of leukemic and tumor systems.
[0004] The best known taxane is paclitaxel (Bristol-Myers Squibb's
Taxol .RTM.). Currently, paclitaxel and semisynthetic analog
docetaxel (Rhne Poulenc Rorer's Taxotere .RTM.) have been approved
for the treatment of breast and ovarian cancer, and many clinical
trials are underway for a number of other indications, including
lung cancer, Kaposi's sarcoma, and lymphoma.
[0005] A major problem with all of the clinical studies is the
limited availability of the compounds. Paclitaxel is a natural
product which can be isolated from the bark of Yew trees, but the
extraction is difficult, the process is expensive and the yield of
paclitaxel is low. Removal of the bark destroys Pacific or Western
yew, Taxus brevifolia, which is listed among the world's endangered
conifer species. The original method of obtaining Taxol.RTM.
involved cutting down and stripping tens of thousands of trees to
harvest small amounts of the compound (13,500 kg of Taxus
brevifolia bark yields about 1 kg of paclitaxel).
[0006] Alternative modes of obtaining paclitaxel have not solved
the supply problem. The total chemical synthesis of paclitaxel
requires more than thirty steps. Taxus plant cell culture only
supplies relatively low yields of taxanes, and biosynthesis by a
yew tree fungus, Taxomyces andreanae has not been sufficient to be
economically feasible (Nature Biotech., 14:1055, 1996).
[0007] Isolation of taxanes from the stems and needles of various
Taxus species offers hope that the supply of taxanes, which can be
used for semi-synthesis, will become more abundant. Because of the
structural complexity of paclitaxel, partial synthesis is a far
more viable approach to providing adequate supplies of paclitaxel
and paclitaxel precursors and derivatives than total synthesis. The
first successful partial synthesis of paclitaxel was developed by
Denis et al, (U.S. Pat No. 4,924,011 re-issued as 34,277) using the
starting material 10-deacetylbaccatin III which can be extracted in
relatively high yield from needles of specific species of taxus
baccata.
[0008] Many derivatives of paclitaxel have also been synthesized
since the realization of its utility as a therapeutic agent in the
treatment of cancer. Examples include 7-deoxy-taxol analogs (as
described in PCT Application No. WO 94/13655), which are useful for
breast, ovarian, lung, colonic and gastric cancers, malignant
melanoma and leukemia. A number of benzoate derivatives (Canadian
Patent Application No. 2,095,375), have also been synthesized.
Paclitaxel analogs with a ketone moiety (U.S. Pat. No. 5,440,047
and PCT Application No. WO 95/33736), have also been reported to
have remarkable antitumoral and antileukemic properties.
Derivatives with an epoxide residue have also been disclosed (PCT
Application No.WO /94/13654) which exhibit antitumour activity. The
preparation of a further group of taxane derivatives have been
described in PCT Application Nos. WO 95/02400 and WO 93/21173 which
are structurally very similar to paclitaxel but have somewhat
increased chemotherapeutic activity when compared to
paclitaxel.
[0009] Although the use of paclitaxel is successful against a
number of specific tumor types, it is not universally effective.
Different taxanes exhibit different efficacies to various tumor
types, hence there is an urgent need for novel compounds from the
taxane family that are closely related to paclitaxel in their
chemical structures but which have more potent chemotherapeutic
activities.
SUMMARY OF THE INVENTION
[0010] One embodiment of the present invention is to provide a new
taxane and its derivatives, which have chemotherapeutic
activities.
[0011] A further embodiment of the present invention is to provide
a compound, hereinafter referred to as Canadensol, having the
following structural Formula I: 1
[0012] a previously unknown constituent of the Canada yew, Taxus
canadensis.
[0013] It is another embodiment of the invention to provide analogs
having in common with Canadensol the following structural Formula
II: 2
[0014] as well as a process for preparing such compounds from the
starting material baccatin III.
[0015] Wherein: R.sub.1 in Formula II is selected from the group
consisting of:
[0016] C.sub.1-C.sub.6 alkyl including methyl, isopropyl,
isopropenyl, propenyl, butyl, cyclopropyl, substituted alkyl
including halo, di-(tri-halomethyl)methyl, 3'-trihalo-n-propyl, or
phenyl substituted with: one, 2 or 3 C.sub.1-C.sub.4 alkyl,
C.sub.1-C.sub.3 alkoxy, halo, C.sub.1-C.sub.3 alkylthio,
trifluoromethyl, C.sub.2-C.sub.6 dialkylamino, hydroxy or nitro,
2-furyl, 2-thienyl; 1-napthyl, 2-napthyl, isopropoxy, isopropenoxy,
propenoxy, cyclopropoxy, di-(trihalomethyl)methoxy,
3'-trihalo-n-propanoxy, dimethylamino, ethylamino, isopropylamino,
propylamino, isopropenamino, imidazol, acetyl, hydroxycarbonyl,
2-(hydroxy)ethyl, 2-(methyl)-propyl, benzyl.
[0017] Wherein: R.sub.2 in Formula II is selected from the group
consisting of: --OAc or --OH.
[0018] It is a further embodiment of the present invention to
provide analogs having in common with Canadensol the following
structural Formula III: 3
[0019] as well as a process for preparing such compounds from the
starting material baccatin III.
[0020] Wherein: R.sub.3 in Formula III is selected from the group
consisting of:
[0021] C.sub.1-C.sub.6 alkyl including methyl, isopropanoyl,
isopropenoyl, propenoyl, cyclopropanoyl, substituted alkyl
including halo, di-(tri-halomethyl)acetanoyl,
3'-trihalo-n-propanoyl, 3,4-methylene-dioxyphenyl or phenyl
substituted with one, 2 or 3 C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.3
alkoxy, halo, C.sub.1-C.sub.3 alkylthio, trifluoromethyl,
C.sub.2-C.sub.6 dialkylamino, hydroxy or nitro, 2-furyl, 2-thienyl,
1-napthyl, 2-napthyl, isopropoxycarbonyl, isopropenoxycarbonyl,
propenoxycarbonyl, cyclopropoxycarbonyl,
di-(trihalomethyl)methoxycarbonyl, 3'-trihalo-n-propanoxycarbonyl,
dimethylaminocarbonyl, ethylaminocarbonyl, isopropylaminocarbonyl,
propylaminocarbonyl, isopropenaminocarbonyl, imidazolcarbonyl,
pyruvyl, oxalyl, 2-(hydroxy)ethylcarbonyl,
2-(methyl)-propylcarbonyl, benzylcarbonyl.
[0022] Due to the immediate requirement for additional novel
taxanes, a further object of the present invention is to provide a
family of taxanes, represented by Formula II and Formula III. These
compounds are useful for the treatment of, or in the preparation of
paclitaxel derivatives for use in treatment of, cancers and
leukemias. The invention also provides for pharmaceutically
acceptable compositions compounds of Formula II and Formula III,
for use as therapeutic agents for use as anticancer agents in the
management of such disease.
[0023] It is a further embodiment of this invention to provide a
simple and inexpensive semi-synthetic method for the production of
these novel taxanes. Accordingly, it is an object of this invention
to provide a reproducible method for the isolation and
semi-synthesis of taxanes from plant matter derived from the Taxus
genus of plants.
[0024] Yet a further object of the present invention is to provide,
in particular a method for the isolation and semi-synthesis of a
novel taxane, named Canadensol, which exhibits higher
chemotherapeutic activity than paclitaxel.
[0025] It is a further object of this invention to provide a method
for the production of a number of protected intermediates, that are
useful in the semi-synthesis of novel taxanes.
[0026] It is yet a further embodiment of the present invention to
provide for the use of the compounds exhibited in Formula I, II,
and III in the treatment of various maladies, including various
forms of cancer.
[0027] These and other objectives, as well as the nature, scope and
utilization of this invention, will become readily apparent to
those skilled in the art from following the description, the
drawings and the appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] The present invention is disclosed in connection with the
appended drawings, in which:
[0029] FIG. 1 shows the results of a microtubule assay indicating
that Canadensol exhibits a higher antimitotic activity than
paclitaxel at the same concentration;
[0030] FIG. 2 shows the results of a microtubule assay
demonstrating a concentration dependent antimitotic effect of
Canadensol and that Canadensol is more active than paclitaxel at
the same concentration;
[0031] FIG. 3 demonstrates in a microtubule assay that, at the same
concentration, paclitaxel and taxcultine exhibit similar
antimitotic activity, but in the same experiment, Canadensol is
more effective;
[0032] FIG. 4 shows the activity of semi-synthetic Canadensol
versus that of a natural product prior to removing the taxcultine
in a microtubule assay;
[0033] FIG. 5 shows the NMR data of
N-debenzoyl-N-n-pentanoyl-paclitaxel;
[0034] FIG. 6 shows the in vitro cytotoxic effects of taxoids
(Paclitaxel, Canadensol, and Taxcultine) on A2780 cells versus
taxoid concentration;
[0035] FIG. 7 shows the in vitro cytotoxic effects of Paclitaxel on
A2780, MCF7 and DA3 cells verses Paclitaxel concentration;
[0036] FIG. 8A shows the perturbation of tumour volume with
differing doses of taxol.TM. versus time,
[0037] FIG. 8B shows the perturbation of body weight with differing
doses of taxol.TM. versus time; and
[0038] FIG. 9 shows the perturbation of tumour volume with
differing doses of Canadensol and a control sample versus time.
DETAILED DESCRIPTION OF THE INVENTION
[0039] The present invention comprises the compound Canadensol as
well as derivatives thereof, having the structure Formula II and
Formula III: 4
[0040] Wherein: R.sub.1 in Formula II is selected from the group
consisting of:
[0041] C.sub.1-C.sub.6 alkyl including methyl, isopropyl,
isopropenyl, propenyl, propyl, butyl, cyclopropyl, substituted
alkyl including halo, di-(tri-halomethyl)methyl,
3'-trihalo-n-propyl, or phenyl substituted with: one, 2 or 3
C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.3 alkoxy, halo,
C.sub.1-C.sub.3 alkylthio, trifluoromethyl, C.sub.2-C.sub.6
dialkylamino, hydroxy or nitro, 2-furyl, 2-thienyl; 1-napthyl,
2-napthyl, isopropoxy, isopropenoxy, propenoxy, cyclopropoxy,
di-(trihalomethyl)methoxy, 3'-trihalo-n-propanoxy, dimethylamino,
ethylamino, isopropylamino, propylamino, isopropenamino, imidazol,
acetyl, hydroxycarbonyl, 2-(hydroxy)ethyl, 2-(methyl)-propyl,
benzyl.
[0042] Wherein: R.sub.2 in Formula III is selected from the group
consisting of: --OAc or --OH.
[0043] It is also another object of the invention to provide
analogs having in common with Canadensol the following structural
Formula III: 5
[0044] as well as a process for preparing such compounds from the
starting material baccatin III.
[0045] Wherein: R.sub.3 in Formula III is selected from the group
consisting of:
[0046] C.sub.1-C.sub.6 alkyl including methyl, propyl, isopropyl,
butyl, 1'-methyl-propyl, 2'-methyl-propyl, pentyl and all possible
stereoisomers; isopropanoyl; isopropenoyl, propenoyl,
cyclopropanoyl, substituted alkyl including halo,
di-(tri-halomethyl)acetanoyl, 3'-trihalo-n-propanoyl,
3,4-methylene-dioxyphenyl or phenyl substituted with one, 2 or 3
C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.3 alkoxy, halo,
C.sub.1-C.sub.3 alkylthio, trifluoromethyl, C.sub.2-C.sub.6
dialkylamino, hydroxy or nitro, 2-furyl, 2-thienyl, 1-napthyl,
2-napthyl, isopropoxycarbonyl, isopropenoxycarbonyl,
propenoxycarbonyl, cyclopropoxycarbonyl,
di-(trihalomethyl)methoxycarbonyl, 3'-trihalo-n-propanoxycarbonyl,
dimethylaminocarbonyl, ethylaminocarbonyl, isopropylaminocarbonyl,
propylaminocarbonyl, isopropenaminocarbonyl, imidazolcarbonyl,
pyruvyl, oxalyl, 2-(hydroxy)ethylcarbonyl,
2-(methyl)-propylcarbonyl, benzylcarbonyl.
[0047] The following definitions apply to these compounds and
throughout the present disclosure.
[0048] The term "alkyl" as used herein refers to a monovalent group
derived by the removal of a single hydrogen atom from a straight-
or branched-chain saturated hydrocarbon containing one to six atoms
including, but not limited to, methyl, ethyl, n- and iso-propyl,
n-, sec-, iso- and tert-butyl, pentyl and hexyl.
[0049] The term "substituted alkyl" as used herein refers to an
alkyl group as defined above substituted with between one and three
groups such as hydroxyl, sulfhydryl, alkoxyl, carboxyl and
halogen.
[0050] The term "alkoxy" as used herein refers to an alkyl function
as defined above attached via an oxygen atom including, but not
limited to, methoxy, ethoxy, iso-propoxy, butoxy and
tert-butoxy.
[0051] The term "substituted alkoxy" as used herein refers to an
alkoxy group as defined above substituted with between one and
three groups such as hydroxyl, sulfhydryl, alkoxyl, thioalkoxyl,
carboxyl, amino and halogen.
[0052] The term "alkoxyalkyl" as used herein refers to an alkoxy
function as defined above attached to an alkyl group including, but
not limited to, methylpropionoyl and ethylbutanoyl.
[0053] The term "alkanoyl" as used herein refers to an alkyl
function as defined above attached via a carbonyl group including,
but not limited to, acetoyl, propionyl, butanoyl and
isobutanoyl.
[0054] The term "hydroxyalkyl" as used herein refers to an alkyl
function as defined above substituted with between one to three
hydroxyl groups including, but not limited to, hydroxyethyl and
hydroxypropyl.
[0055] The term "cycloalkyl" as used herein refers to an alkyl
function as defined above where some of the carbon atoms are joined
to form a ring including, but not limited to, cyclopropyl,
cyclobutyl and cyclopentyl.
[0056] The term "alkenyl" as used herein refers to an alkyl
function as defined above which contains between one and three
unsaturated bonds including, but not limited to, isopropenyl.
[0057] The term "phenoxy" as used herein refers to an phenyl group
as defined above attached via an oxygen atom.
[0058] The term "aminoalkyl" as used herein refers to an alkyl
function as defined above substituted with amino or substituted
amino, as defined elsewhere herein.
[0059] The term "aminoalkanoyl" as used herein refers to an
alkanoyl function as defined above substituted with between one and
three amino groups including, but not limited to, 2-aminopropanoyl,
4-aminobutanoyl, and 6-aminohexanoyl. Additionally, the amino
groups may optionally be substituted with peptidyl residues formed
therefrom.
[0060] The term "substituted alkoxyalkyl" as used herein refers to
an alkoxyalkyl group as defined above substituted with between one
and three groups such as hydroxyl, sulfhydryl, alkoxyl,
thioalkoxyl, carboxyl, amino and halogen.
[0061] The term "substituted alkanoyl" as used herein refers to an
alkanoyl group as defined above substituted with between one and
three groups such as hydroxyl, sulfhydryl, alkoxyl, carboxyl and
halogen.
[0062] The term "substituted hydroxyalkyl" as used herein refers to
an hydroxyalkyl group as defined above substituted with between one
and three groups such as sulphydryl, carbonyl and halogen.
[0063] The term "substituted cycloalkyl" as used herein refers to
an cycloalkyl group as defined above substituted with between one
and three groups such as hydroxyl, sulfhydryl, alkoxyl,
thioalkoxyl, carboxyl, amino and halogen.
[0064] The term "substituted alkenyl" as used herein refers to an
alkenyl group as defined above substituted with between one and
three groups such as hydroxyl, sulfhydryl, alkoxyl, thioalkoxyl,
carboxyl, amino and halogen.
[0065] The term "substituted phenyl" as used herein refers to a
phenyl group substituted with between one and three substituents
independently selected from alkyl, halogen, haloalkyl, alkoxy,
benzyloxy, thioalkoxy, hydroxy, alkanoyl, carboxy, amino,
alkylamino, dialkylamino, nitro and --OSO.sub.3H.
[0066] The term "substituted phenoxy" as used herein refers to a
phenoxy group substituted with between one and three substituents
independently selected from alkyl, halogen, haloalkyl, alkoxy,
benzyloxy, thioalkoxy, hydroxy, alkanoyl, carboxy, amino,
alkylamino, dialkylamino, nitro and --OSO.sub.3H.
[0067] The term "substituted amino" as used herein refers to an
amino group substituted with one or two alkyl groups including, but
not limited to, t-butylamino, benzylamino and
N,N-dimethylamino.
[0068] The term "substituted aminoalkyl" as used herein refers to
an aminoalkyl group substituted with between one and three groups
including, but not limited to, hydroxyl, sulfhydryl, alkoxyl,
thioalkoxyl, carboxyl, amino and halogen.
[0069] The term "substituted aminoalkanoyl" as used herein refers
to an aminoalkanoyl group substituted with between one and three
groups including, but not limited to, hydroxyl, sulfhydryl,
alkoxyl, thioalkoxyl, carboxyl, amino and halogen.
[0070] The term "substituted imidazol" as used herein refers to an
imidazol group substituted with between one and three groups
including, but not limited to, hydroxyl, sulfhydryl, alkoxyl,
thioalkoxyl, carboxyl, amino and halogen.
[0071] The term "halogen" as used herein refers to a group selected
from bromo (Br), chloro (Cl), fluoro (F) and iodo (I).
[0072] The term "haloalkyl" as used herein refers to an alkyl group
as defined above substituted with between one and three halogen
atoms including, but not limited to, fluoromethyl, trifluoromethyl
and fluoroethyl.
[0073] The term "acylated" as used herein refers to an alkyl group
attached to a carbonyl.
[0074] The term "protecting group" as used herein is a term
well-known in the art and refers to substituents on functional
groups of compounds undergoing chemical transformation which
prevent undesirable reactions and degradations during a synthesis;
see, for example, T. H. Greene, "Protective Groups in Organic
Synthesis", John Wiley and Sons (1981).
[0075] The term "N-protected" and "N-protecting" as used herein
refer to the use of a group intended to protect an amino function
or the N-terminus of an amino acid or peptide against undesirable
reactions during a synthetic procedure or to prevent the attack of
exopeptidases on the compound or to increase the solubility of the
compound and includes, but is not limited to, such uses sulfonyl;
acyl, such as acetyl, pivaloyl and benzoyl; alkoxycarbonyl, such as
tert-butyloxycarbonyl (BOC), benzyloxycarbonyl (Cbz); and L- or
D-aminoacyl residues, which may themselves be N-protected. Other
examples may be found in `The Peptides` E. Gross and J.
Meinenhofer, Vol 3, Academic Press (1981).
[0076] It will be appreciated by those skilled in the art that the
compounds of Formula II and Formula III, depending on the
substituents, may contain one or more chiral centers and thus exist
in the form of many different isomers, optical isomers (i.e.
enantiomers) and mixtures thereof including racemic mixtures. All
such isomers, enantiomers and mixtures thereof including racemic
mixtures are included within the scope of the invention.
[0077] The compound of Formula I, Canadensol, has been isolated
from the needles of the Taxus genus. The vegetal material
preferably consists of the needles which is rapidly regenerated and
therefore in abundant supply, though other material such as the
roots or the bark of the Taxus bushes may be used.
[0078] Early attempts at the recovery of Canadensol from the
chromatographic fractions containing it, consisted of numerous
reverse phase HPLC steps resulting in low yields.
[0079] The compound of Formula I is extracted by means of
chlorinated solvents, for example dichloromethane, in admixture
with alcohols such as methyl alcohol. The purification of
Canadensol requires a purification through column chromatography,
wherein silica gel is preferably used as the stationary phase.
Solvent mixtures, consisting of an aliphatic hydrocarbon, for
example, hexane with a chlorinated solvent, such as
dichloromethane, together with a higher polarity solvent, such as
ethyl acetate or acetone are used as eluents.
[0080] The preferred compounds of the present invention can also be
synthesized using a new and unique combination of conventional
preparative steps and recovery methods known to those skilled in
the art of organic and bio-organic synthesis, while providing a new
and unique combinations for the overall synthesis of each compound.
Preferred synthetic routes for intermediates involved in the
synthesis as well as the resulting taxane derivative compounds of
the present invention follow. Successful preparation of these
compounds is possible by way of several synthetic routes: two of
which are outlined in Schemes I and II (demonstrated in Examples I
and II, correspondingly).
[0081] Briefly, the semi-synthesis methods use baccatin III as
starting material, which can be obtained from a number of its
derivatives, one of the preferred derivatives is
10-deacetylbaccatin III. The conversion of 10-deacetylbaccatin III
into baccatin III can be achieved according to the technique
described in Denis J N, et al., J. Am. Chem. Soc., 110:5917 (1988).
The conversion of baccatin III into taxol derivatives requires that
the hydroxyl moiety at C-7 be protected prior to derivatization
with the appropriate side chain at C-13. The appropriate side
chains can be synthesized from alkyl cinnamate. See Denis J N, et
al., J. Org. Chem. 55:1959 (1990) for an example of a suitable side
chain synthesised from methyl cinnamate.
[0082] Scheme I
[0083] Step A
[0084] Compound (i); baccatin III, is derivatized at C-7 by
reacting with Compound (ii); a suitable protecting group (X). It is
necessary to protect the hydroxyl group at position 7 of baccatin
III, to prevent coupling at C-7 and/or racemization at C-7 (through
retro-aldol reaction). This can be achieved through the use of
silyl chlorides (for example from triethyl, tri-isopropyl,
t-butyldimethyl or t-butyldiphenyl) or alkyl chlorides (for example
from benzyl chloride, methoxy-methyl chloride, allyl chloride or
methoxy-ethyl chloride) or by the use of dihydropyran. The above
reaction yields Compound (iii); a 7-protected baccatin III
intermediate.
[0085] Step B
[0086] The 7-protected baccatin III intermediate, Compound (iii),
is then coupled at C-13 by the use of an appropriate acid, Compound
(iv). The appropriate acid can be synthesized from alkyl cinnamate.
The ii above coupling procedure generates a taxol derivative with a
protecting group at C-7 as illustrated in Compound (v).
[0087] Step C
[0088] This step achieves two functions: the protecting group at
C-7 is removed, and the cyclized side chain at C-13 is opened-up
and the side chain amine is acylated. This transformation, into
Compound (vi), can be achieved through the use of an organic acid,
followed by acylation using bicarbonate salts and an acyl chloride.
In Compound (vi), R.sub.1 is selected from the group consisting:
C.sub.1-C.sub.6 alkyl including methyl, isopropyl, isopropenyl,
propenyl, butyl, cyclopropyl, substituted alkyl including halo,
di-(trihalomethyl)methyl, 3'-trihalo-n-propyl, or phenyl
substituted with: one, 2 or 3 C.sub.1-C.sub.4 alkyl,
C.sub.1-C.sub.3 alkoxy, halo, C.sub.1-C.sub.3 alkylthio,
trifluoromethyl, C.sub.2-C.sub.6 dialkylamino, hydroxy or nitro,
2-furyl, 2-thienyl; 1-napthyl, 2-napthyl, isopropoxy, isopropenoxy,
propenoxy, cyclopropoxy, di-(trihalomethyl)methoxy,
3'-trihalo-n-propanoxy, dimethylamino, ethylamino, isopropylamino,
propylamino, isopropenamino, imidazol, acetyl, hydroxycarbonyl,
2-(hydroxy)ethyl, 2-(methyl)propyl, benzyl.
[0089] Where R.sub.1 is isopropyl, this scheme can yield 0.86% (See
Example 1). 6
[0090] Scheme II
[0091] Step A
[0092] Compound (i); baccatin III, is derivatized at C-7 by
reacting with Compound (vii) a suitable protecting group (Y). It is
necessary to protect the hydroxyl group at position 7 of baccatin
III, to prevent coupling at C-7 and/or racemization at C-7 (through
a retro-aldol reaction). This can be achieved through the use of
chloroalkyl formates (for example from trichloroethyl
chloroformate, trichloromethylchloroform- ate,
dichloroethyl-chloroformate, dichloromethyl chloroformate, etc.).
The above reaction yields Compound (viii); a 7-protected baccatin
III intermediate. When Y is --CO.sub.2CH.sub.2CCl.sub.3, the yield
of Compound (viii) is 25.5%.
[0093] Step B
[0094] The 7-protected baccatin III intermediate, Compound (viii),
is then coupled at C-13 by the use of the acid, Compound (iv) which
can be synthesized from alkyl cinnamate. The above coupling
procedure generates a taxol derivative with a protecting group at
C-7 as illustrated in Compound (ix). When Y is
--CO.sub.2CH.sub.2CCl.sub.3, the coupling reaction with compound
(iv) affords Compound (ix) in 53.3% yield.
[0095] Step C
[0096] In Compound (x), R.sub.1 is selected from the group
consisting: C.sub.1-C.sub.6 alkyl including methyl, isopropyl,
isopropenyl, propenyl, butyl, cyclopropyl, substituted alkyl
including halo, di-(tri-halomethyl)methyl, 3'-trihalo-n-propyl, or
phenyl substituted with: one, 2 or 3 C.sub.1-C.sub.4 alkyl,
C.sub.1-C.sub.3 alkoxy, halo, C.sub.1-C.sub.3 alkylthio,
trifluoromethyl, C.sub.2-C.sub.6 dialkylamino, hydroxy or nitro,
2-furyl, 2-thienyl; 1-napthyl, 2-napthyl, isopropoxy, isopropenoxy,
propenoxy, cyclopropoxy, di-(trihalomethyl)methoxy,
3'-trihalo-n-propanoxy, dimethylamino, ethylamino, isopropylamino,
propylamino, isopropenamino, imidazol, acetyl, hydroxycarbonyl,
2-(hydroxy)ethyl, 2-(methyl)-propyl, benzyl.
[0097] In this step the cyclized side chain at C-13 is opened-up.
This transformation, into Compound (x), can be achieved through the
use of an organic acid, followed by alkylation using bicarbonate
salts and an acyl chloride.
[0098] Step D
[0099] The protecting group at C-7 is then removed from Compound
(x) by the use of reducing agents such as zinc in the presence of
acetic acid and methanol to yield Compound (vi). When R.sub.1 is
isopropyl, this scheme can yield 74.7% (See Example 2). 7
[0100] One particular advantage of this invention is that the
starting material, baccatin III, can be obtained from
10-deacetylbaccatin III which is produced in relatively high
concentrations in the needles of Taxus canadensis. A further
important consideration when choosing starting materials is that
they must be available in relatively large quantities and be a
rapidly renewable resource. 10-deacetyl baccatin III satisfy this
criteria as they can be isolated by simple recrystallisations and
preparative reverse phase HPLC instead of many silica gel columns,
hence the yields are relatively high.
[0101] The starting material for use in this invention is vegetal
material, selected from a group of plants commonly referred to as
taxads. The most suitable plants of this group are the species
Taxus. The semi-synthetic method disclosed is effective when using
the roots, bark or leaves of the Taxus plant but we consider it
prudent to use a source that is rapidly regenerated (such as the
leaves i.e. needles) and therefore in abundant supply. Amongst the
Taxus species, Taxus canadensis is a preferred source for use in
the semi-synthesis of the novel taxanes claimed in this invention.
Thus one useful aspect of the current invention is largely
dependent upon an abundant supply of baccatin III which can be
derived from 10-deacetylbaccatin III.
[0102] Paclitaxel derivatives are useful for their antitumor
activity, particularly for the treatment of the same cancers for
which taxol has been shown active, including human lung tumors,
melanoma, leukemia, mammary tumors, and colon cancer. We anticipate
that the taxol derivatives of the current invention will have
utility in the treatment of the same cancers for which taxol has
been shown to be active.
[0103] Microtubules are an integral part of eukaryotic cells, and
microtubule assembly is importantly associated with cell division
and multiplication. Known antitumor compounds have been studied for
their effect on microtubule assembly. For example, vinca alkaloids,
such as vinblastine and vincristine have been shown to disrupt
cellular microtubules, i.e. in vitro they have been shown to
inhibit microtubule assembly and to depolymerize steady state
microtubules. Similarly, colchicine has been shown to depolymerize
microtubules in cells.
[0104] Paclitaxel has been shown to exhibit a very unique mechanism
of action, in that it promotes the assembly of microtubules, but
inhibits their disassembly, thereby interfering with the G2 and M
phases of cell cycles and division. In vitro studies have shown
that microtubules, once polymerized, in the presence of taxol
resists depolymerization by other agents such as CaCl.sub.2 or cold
temperature which normally depolymerize microtubules.
[0105] The present inventors have conducted studies to investigate
the effect of derivatives of taxol on the microtubule assembly. The
microtubule assembly study was conducted using Canadensol according
to the in vitro procedures disclosed, for example in Parness et
al., J.Cell Biol. 91:479 1981. In these studies, conditions can be
established whereby a dynamic steady state exists between tubulin
assembling into microtubules and the disassembly at the other end.
This dynamic steady state can be measured spectrophotometrically,
observing the absorbance of the solution at 350 nm. As stated
above, it has been shown that taxol binds specifically and
reversibly to the protein, stabilizing the microtubules in the
polymerized form. This effect can be visualized by following the
increase in absorbance of the solution at 350 nm. Cells treated
with taxol, or derivatives thereof which exhibit chemotherapeutic
activity, usually die as they are effectively blocked in
mitosis.
[0106] The results presented in FIGS. 1-4, indicate that compounds
of Formula II (eg. when R.sub.1 is isopropyl or propyl) exhibit
excellent tubulin stabilizing activity. These compounds, therefore,
are useful antitumor agents due to their biological activity. It is
therefore one embodiment of the present invention to claim the use
of the two compounds of Formula II, when R.sub.1 is isopropyl and
propyl, as anticancer agents.
[0107] The present invention also provides pharmaceutical
compositions containing a compounds as disclosed in the claims in
combination with one or more pharmaceutically acceptable, inert or
physiologically active, diluents or adjuvants. The compounds of the
invention can be freeze dried and, if desired, combined with other
pharmaceutically acceptable excipients to prepare formulations for
administration. These compositions may be presented in any form
appropriate for the administration route envisaged. The parenteral
and the intravenous route are the preferential routes for
administration.
[0108] Compounds of the general Formula II may be administered
orally, topically, parenterally, by inhalation or spray or rectally
in dosage unit formulations containing conventional non-toxic
pharmaceutically acceptable carriers, adjuvants and vehicles. The
term parenteral as used herein includes subcutaneous injections,
intravenous, intramuscular, intrasternal injection or infusion
techniques. In addition, there is provided a pharmaceutical
formulation comprising a compound of general Formula II and a
pharmaceutically acceptable carrier. One or more compounds of
general Formula II may be present in association with one or more
non-toxic pharmaceutically acceptable carriers and/or diluents
and/or adjuvants and if desired other active ingredients. The
pharmaceutical compositions containing compounds of general Formula
II may be in a form suitable for oral use, for example, as tablets,
troches, lozenges, aqueous or oily suspensions, dispersible powders
or granules, emulsion hard or soft capsules, or syrups or
elixirs.
[0109] Compositions intended for oral use may be prepared according
to any known to the art for the manufacture of pharmaceutical
compositions and such compositions may contain one or more agents
selected from the group consisting of sweetening agents, flavouring
agents, colouring agents and preserving agents in order to provide
pharmaceutically elegant and palatable preparations. Tablets
contain the active ingredient in admixture with non-toxic
pharmaceutically acceptable excipients which are suitable for the
manufacture of tablets. These excipients may be for example, inert
diluents, such as calcium carbonate, sodium carbonate, lactose,
calcium phosphate or sodium phosphate: granulating and
disintegrating agents for example, corn starch, or alginic acid:
binding agents, for example starch, gelatin or acacia, and
lubricating agents, for example magnesium stearate, stearic acid or
talc. The tablets may be uncoated or they may be coated by known
techniques to delay disintegration and absorption in the
gastrointestinal tract and thereby provide a sustained action over
a longer period. For example, a time delay material such as
glyceryl monosterate or glyceryl distearate may be employed.
[0110] Formulations for oral use may also be presented as hard
gelatin capsules wherein the active ingredient is mixed with an
inert solid diluent, for example, calcium carbonate, calcium
phosphate or kaolin, or as soft gelatin capsules wherein the active
ingredient is mixed with water or an oil medium, for example peanut
oil, liquid paraffin or olive oil.
[0111] Aqueous suspensions contain active materials in admixture
with excipients suitable for the manufacture of aqueous
suspensions. Such excipients are suspending agents, for example
sodium carboxymethylcellulose, methyl cellulose,
hydropropylmethylcellulose, sodium alginate, polyvinylpyrrolidone,
gum tragacanth and gum acacia: dispersing or wetting agents may be
a naturally-occurring phosphatide, for example, lecithin, or
condensation products of an alkylene oxide with fatty acids, for
example polyoxyethyene stearate, or condensation products of
ethylene oxide with long chain aliphatic alcohols, for example
hepta-decaethyleneoxycetanol, or condensation products of ethylene
oxide with partial esters derived from fatty acids and a hexitol
such as polyoxyethylene sorbitol monooleate, or condensation
products of ethylene oxide with partial esters derived from fatty
acids and hexitol anhydrides, for example polyethylene sorbitan
monooleate. The aqueous suspensions may also contain one or more
preservatives, for example ethyl, or n-propyl p-hydroxy-benzoate,
one or more coloring agents, one or more flavoring agents or one or
more sweetening agents, such as sucrose or saccharin.
[0112] Oily suspensions may be formulated by suspending the active
ingredients in a vegetable oil, for example arachis oil, olive oil,
sesame oil or coconut oil, or in a mineral oil such as liquid
paraffin. The oily suspensions may contain a thickening agent, for
example beeswax, hard paraffin or cetyl alcohol. Sweetening agents
such as those set forth above, and flavoring agents may be added to
provide palatable oral preparations. These compositions may be
preserved by the addition of an anti-oxidant such as ascorbic
acid.
[0113] Dispersible powders and granules suitable for preparation of
an aqueous suspension by the addition of water provide the active
ingredient in admixture with a dispersing or wetting agent,
suspending agent and one or more preservatives. Suitable dispersing
or wetting agents and suspending agents are exemplified by those
already mentioned above. Additional excipients, for example
sweetening, flavoring and coloring agents, may also be present.
[0114] Pharmaceutical compositions of the invention may also be in
the form of oil-in-water emulsions. The oils phase may be a
vegetable oil, for example olive oil or arachis oil, or a mineral
oil, for example liquid paraffin or mixtures of these. Suitable
emulsifying agents may be naturally-occurring gums, for example gum
acacia or gum tragacanth, naturally-occurring phosphatides, for
example soy bean, lecithin, and esters or partial esters derived
from fatty acids and hexitol, anhydrides, for example sorbitan
monoleate, and condensation products of the said partial esters
with ethylene oxide, for example polyoxyethylene sorbitan
monoleate. The emulsions may also contain sweetening and flavoring
agents.
[0115] Syrups and elixirs may be formulated with sweetening agents,
for example glycerol, propylene glycol, sorbitol or sucrose. Such
formulations may also contain a demulcent, a preservative and
flavoring and coloring agents. The pharmaceutical compositions may
be in the form of a sterile injectable aqueous or oleaginous
suspension. This suspension may be formulation according to known
art using those suitable dispersing or wetting agents and
suspending agents which have been mentioned above. The sterile
injectable preparation may also be sterile injectable solution or
suspension in a non-toxic parentally acceptable diluent or solvent,
for example as a solution in 1,3-butanediol. Among the acceptable
vehicles and solvents that may be employed are water, Ringer's
solution and isotonic sodium chloride solution. In addition,
sterile, fixed oils are conventionally employed as a solvent or
suspending medium. For this purpose any bland fixed oil may be
employed including synthetic mono- or diglycerides. In addition,
fatty acids such as oleic acid find use in the preparation of
injectables.
[0116] The compound(s) of the general Formula I, Formula II and
Formula III may be administered, together or separately, in the
form of suppositories for rectal administration of the drug. These
compositions can be prepared by mixing the drug with a suitable
non-irritating excipient which is solid at ordinary temperatures
but liquid at the rectal temperature and will therefore melt in the
rectum to release the drug. Such materials are cocoa butter and
polyethylene glycols.
[0117] Compound(s) of general Formula I, Formula II, and Formula
III may be administered, together or separately, parenterally in
sterile medium. The drug, depending on the vehicle and
concentration used, can either be suspended or dissolved in the
vehicle. Advantageously, adjuvants such as local anaesthetics,
preservatives and buffering agents can be dissolved in the
vehicle.
[0118] The mode, dosage and schedule of administration of taxol in
human cancer patients has been studied extensively (see Ann. Int.
Med. 111:273 1989). For the compounds of this invention, the dose
to be administered, whether a single dose, multiple dose, or a
daily dose, will vary with the particular compound being used.
Factors to consider when deciding upon a dose regimen include
potency of the compound, route of administration, size of the
recipient and the nature of the patient's condition.
[0119] The dosage to be administered is not subject to defined
limits, but in will usually be an effective amount. It will usually
be the equivalent, on a molar basis of the pharmacologically active
free form produced from a dosage formulation upon the metabolic
release of the active free drug to achieve its desired
pharmacological and physiological effects.
[0120] An oncologist skilled in the art of cancer treatment will be
able to ascertain, without undue experimentations, appropriate
protocols for effective administration of the compounds of this
present invention by referring to the earlier studies of taxol and
its derivatives.
[0121] Particular reagents and conditions utilized in these
syntheses are described in detail in the Examples which follow.
Also, it will be appreciated by one skilled in the art that
selective protection and deprotection steps affecting the several
hydroxyl groups on the baccatin III structure may be carried out in
varying order or number of steps, as necessary, and that Schemes I
and II are intended to encompass such variations.
[0122] The Taxus canadensis plant was extracted as reported
previously (L. O. Zamir, M. E. Nedea, Z.-H. Zhou, S. Blair, G.
Caron, F. Sauriol, E. Jacqmain, F. I. Jean, F.-X. Garneau and O.
Mamer, Can. J. Chem., 73:655, 1995), the additional chromatographic
purification step leading to a new taxane with a side chain, never
isolated before in Taxus spp. is described below.
[0123] Isolation of Taxanes with Reverse Phase HPLC: Taxanes in the
brown solid are separated on a preparative HPLC using an ODS-2
reverse phase column (2.0.times.50 cm; Whatman) and a Waters Delta
Prep 3000 instrument coupled to a model 481 variable wavelength
detector at 227 nm. The products are eluted with a gradient over
140 minutes of acetonitrile:water (25:75) to 100% acetonitrile. At
55.5 mm, a peak comprising 10 hydroxyacetylbaccatin VI, among other
taxanes is collected.
[0124] Isolation of N-debenzoyl-N-n-pentanoylpaclitaxel with
Reverse Phase HPLC: The peak at 55.5 minutes was found to contain
the compound N-debenzoyl-N-n-pentanoylpaclitaxel. In order to
isolate this compound, the 55.5 minute peak was separated further
utilizing the same preparative HPLC system as described above.
However, this separation used a gradient over 50 minutes of
isopropanol:water (25:75) to 100% isopropanol at a flow rate of 6
mL per minute. The largest peak from this gradient was centered at
a retention time of 41 minutes. The shoulder of this 41
minute-centered peak, from 44 to 48 minutes, was collected and
purified further using the same HPLC system eluting with a gradient
over 100 minutes of isopropanol:water (25:75) to 100% isopropanol
at a flow rate of 7 mL per minute. The product eluting at 55
minutes was collected. Analysis of the product on analytical HPLC
yielded a single pure product eluting at 32.9 minutes when using a
gradient over 50 minutes of acetonitrile:water (25:75) to 100%
acetonitrile. The structure of N-debenzoyl-N-n-pentanoylpaclitaxel
was elucidated by NMR analysis, the results being listed in FIG.
5.
[0125] Further, to assist in understanding the current invention,
the following non-limiting examples are provided. The following
examples should not be construed as specifically limiting the
present invention, variations presently known or later developed,
which would be in the understanding of one skilled in the art and
considered to fall within the scope of the present invention as
described herein.
EXAMPLE 1
Semi-Synthesis of Canadensol (Scheme I)
[0126] The preparation of Compound 1 was carried out through eight
step reactions from methyl cinnamate (as described in Denis J. D.,
Correa A., Greene A. E., J. Org. Chem., 55:1959, 1990). The
preparation of Compound 2 was carried out through two step
reactions from 10-deacetyl baccatin III (as described in Denis J.
N., Greene A. E., Guenard D., Gueritte-Voegelelein F., Mangatal L.,
Potier P. J., Am. Chem. Soc., 110:5917, 1988). 8
[0127] The reaction conditions for the preparation of Compound 3
were determined experimentally to be as follows. A solution of
Compound 1 (32.8 mg, 0.102 mmol) and di-2-pyridyl carbonate (DPC,
22.2 mg, 0.102 mmol) in dry toluene (3.0 mL) was stirred at room
temperature under nitrogen for 15 mins. Dimethylaminopyridine
(DMAP, 4.2 mg, 0.034 mmol) and 7-triethyl silyl-10-deacetyl
baccatin III (Compound 2) (12.0 mg, 0.017 mmol) was added to the
mixture. The reaction mixture was heated at 72.degree. C. for 16
hours, then allowed to cool to room temperature. The organic
solvent was removed by rotary evaporation. The crude product was
dissolved in methanol (1.0 mL) and filtered. The solution of crude
product was purified by preparative reverse phase HPLC (50 cm)
using linear gradient of acetonitrile (25% to 100%) in water over
70 minutes to give pure Compound 3 (R.sub.t=77.6 min) as a white
solid (10.5 mg, 61.6% yield, R.sub.f=0.51, EtOAc/Hexane =3/7).
Analytical HPLC using linear gradient of acetonitrile (25% to 100%)
in water over 50 minutes for Compound 3 gave retention time
(R.sub.t=62.8 min). HRMS:MH.sup.+ requires: 1004.48277, Found
1004.48280; .sup.1H-NMR of Compound 3: (500 MHz, CDCl.sub.3)
.delta. 8.04 (d, J=7.3 Hz, 2H), 7.63 (t, J=7.3 Hz, 1H), 7.49 (t,
J=7.8 Hz, 2H), 7.40-7.30 (m, 5H, 3'-Ph), 6.44 (s, 1H, H-10), 6.23
(t, J=8.3 Hz, 1H, H-13), 5.65 (d, J=6.8 Hz, 1H, H-2), 5.04 (br.,
1H, H-3'), 4.87 (d, J=8.8 Hz, 1H, H-5), 4.46 (d, J=6.3 Hz, 1H,
H-2'), 4.44 (o.m, 1H, H-7), 4.24 (d, J=8.3 Hz, 1H, H-20a), 4.10 (d,
J=8.3 Hz, 1H, H-20b), 3.77 (d, J=6.8 Hz, 1H, H-3), 2.49 (ddd,
J=14.6, 9.8, 6.3 Hz, 1H, H-6a), 2.20 (s, 3H, OAc), 2.15 (d, J=9.3
Hz, 2H,1H-14), 2.04 (s, 3H, Me-18), 1.87 (s, 3H, OAc), 1.82 (o.m,
1H, H-6b), 1.65 (s, 3H Me-19), 1.60 (br, t-Bu), 1.22 (s, 3H, Me-16
or 17), 1.20 (s, 3H, Me-17 or 16), 1.11 (br, t-Bu), 0.57 (s, 6H,
Si--CH.sub.2), 0.91 (t, J=7.8 Hz, 9H, Si--CH.sub.2-Me). This
reaction was repeated another two times at the same scale. The
yield of the later two reactions were 51.2% and 68.0%. Thus, the
yield of this reaction varies from 51.2%-68.0%. 9
[0128] Compound 3 (4.0 mg, 0.004 mmol) was stirred in 0.3 mL of
95-97% formic acid at room temperature for 4 hours. The formic acid
was then removed by a flow of nitrogen and the crude product was
dried under vacuum. The residue was dissolved in EtOAc (0.5 mL),
and then NaHCO.sub.3 (10.0 mg) and isobutyryl chloride (5 .mu.L,
0.047 mmol) were added. After stirring for 1 hour, EtOAc (10 mL)
was added to the solution. The organic phase was washed with brine
(3.0 mL) and dried over anhydrous MgSO.sub.4. After filtration and
evaporation, the residue was purified by preparative HPLC
(R.sub.t=35.9 mins, acetonitrile from 25% to 100% in water over 70
minutes). Compound 4 (Canadensol) was obtained as a white solid (46
.mu.g, 1.4% yield based on Compound 3). Analytical HPLC using
linear gradient of acetonitrile (25% to 100%) in water over 50
minutes for Compound 4 gave retention time of 32.19 mins. HRMS:
MH.sup.+ requires: 820.35423, Found: 820.35443; .sup.1H-NMR of
combined samples of Compound 4: (500 MHz, CDCl.sub.3) .delta. 8.11
(d, J=7.8 Hz, 2H) 7.60 (t, J=7.3 Hz, 1H), 7.5 (t, J=7.6 Hz, 2H),
7.40-7.30 (m, 5H, 3'-Ph), 6.27 (s, 1H, H-10), 6.20 (t, J=9.5 Hz,
1H, H-13), 5.67 (d, J=6.5 Hz, 1H, H-2), 5.57 (d, J=8.8 Hz, 1H,
H-3'), 4.93 (d, J=9.1 Hz, 1H, H-5), 4.68 (o.m, 1H, H-2'), 4.39
(o.m, 1H, H-7), 4.24 (d, J=8.8 Hz, 1H, H-20a), 4.18 (d, J=8.8 Hz,
1H, H-20b), 3.78 (d, J=6.8 Hz, 1H, H-3), 3.40 (d, J=5.1 Hz, 1H,
2'--OH), 2.53 (ddd, J=15.4, 8.1, 7.2 Hz, 1H, H-6a), 2.38 (o.m, 1H
Me.sub.2CHCONH), 2.35 (s, 3H, OAc), 2.28 (o.m, 2H, H-14), 2.24 (s,
3H, OAc), 1.84 (o.m, 1H, 6b), 1.81 (s, 3H, Me-18), 1.67 (s, 3H,
Me-19), 1.26 (s, 3H, Me-16 or 17), 1.15 (s, 3H, Me-17 or 16), 1.11
(2d, J=6.5, 6.1 Hz, 6H Me.sub.2CHCONH). This reaction was repeated
two times. The yield of the later reaction was 1.8% and 12.8%. Thus
the yield of this reaction varies from 1.4%-12.4%.
EXAMPLE 2
Semi-Synthesis of Canadensol and a Structural Analogue
[0129] The preparation of Compound 1 was carried out through eight
step reactions from methyl cinnamate (as described in Denis J. D.,
Correa A., Greene A. E., J. Org. Chem., 55:1959, 1990). The
preparation of Compound 5 was carried out through three step
reactions from 10-deacetylbaccatin III (as described in Denis J.
N., Greene A. E., Guenard D., Gueritte-Voegelelein F., Mangatal L.,
Potier P. J., Am. Chem. Soc., 110:5917, 1988). The reaction
conditions for the preparation of Compound 6 were determined
experimentally to be as follows: 10
[0130] To a solution of Compound 5 (baccatin III) (78 mg, 0.133
mmol) in pyridine (1.5 mL) was added
2,2,2-trichloroethylchloroformate (62 .mu.L; 0.452 mmol) at
80.degree. C. This mixture was stirred at 80.degree. C. for 2.5
hours. Water (5 mL) was added and most of the pyridine was
evaporated. The aqueous phase was extracted with CH.sub.2Cl.sub.2
(3.times.20 mL). The organic phase was dried over MgSO.sub.4 and
evaporated. The crude product was dissolved in methanol (2.0 mL)
and filtered. The solution of crude product was purified by
preparative HPLC using linear gradient of acetonitrile (25% to
100%) in water over 70 minutes to give pure Compound 6 (R.sub.2
52.0 min) as a white solid (36 mg, 25.5% yield, R.sub.f=0.38,
EtOAc/CH.sub.2Cl.sub.2=1/4) and recovery of Compound 5 (baccatin
III). Analytical HPLC using linear gradient of acetonitrile (25% to
100%) in water over 50 minutes for Compound 6 gave a retention time
of 44.0 mins. .sup.1H-NMR of Compound 6: (500 MHz, CDCl.sub.3)
.delta. 8.09 (d, J=7.6 Hz, 2H) 7.61 (t, J=7.2 Hz, 1H), 7.48 (t,
J=7.6 Hz, 2H), 6.38 (s, 1H, H-10), 5.62 (d, J=7.5 Hz, 1H, H-2),
5.58 (m, 1H, H-7),5.02 and 4.63 (2d, J=12.5 Hz, 2H,
CH.sub.2CCl.sub.3),4.97 (d, J=9.3 Hz, 1H, H-5),4.87 (m, 1H, H-13),
4.32 (d, J=8.6 Hz, 1H, H-20a), 4.14 (d, J=8.6 Hz, 1H, H-20b), 4.01
(d, J=6.8 Hz, 1H, H-3), 2.63 (ddd, J=15.0, 8.5, 7.0 Hz, 1H, H-6a),
2.29 (s, 3H, OAc), 2.15 (d, J=9.3 Hz, 2H, H-14), 2.04 (s, 3H,
Me-18), 2.03 (m, 1H, H-6b) 1.82 (s, 3H, OAc), 1.58 (s, 3H, Me-19),
1.13 (s, 3H, Me-16 or 17), 1.09 (s, 3H, Me-17 or 16). 11
[0131] The reaction conditions for the preparation of Compound 7
were determined experimentally to be as follows: A solution of
Compound 1 (90.8 mg; 0.282 mmol) and di-2-pyridyl carbonate (DPC,
61.3 mg; 0.828 mmol) in dry toluene (5.0 mL) was stirred at room
temperature under nitrogen for 15 minutes. Dimethylaminopyridine
(DMAP, 11.7 mg; 0.0954 mmol) and
7-(2,2,2-trichloroethyloxycarbonyl)-baccatin III (Compound 6) (36.3
mg; 0.0476 mmol) was added to the mixture and stirred at 72.degree.
C. for 6 hours. The organic solvent was removed by rotary
evaporation. The crude product was dissolved in methanol (1.5 mL)
and filtered. The solution of crude product was purified by
preparative HPLC using linear gradient of acetonitrile (25% to
100%) in water over 70 minutes to give pure Compound 7
(R.sub.t=77.3 min) as a white solid (27.0 mg; 53.3% yield;
R.sub.f=0.43, EtOAc/hexane=1/1). Analytical HPLC using linear
gradient of acetonitrile (25% to 100%) in water over 50 minutes for
Compound 7 gave retention time (R.sub.t=56.2 min). .sup.1H-NMR of
Compound 7: (500 MHz, CDCl.sub.3) .delta. 8.04 (d, J=7.3 Hz, 2H),
7.64 (t, J=7.3 Hz, 1H), 7.50 (t, J=7.8 Hz, 2H), 7.40-7.30 (m, 5H,
3'-Ph), 6.36 (s, 1H H-10), 6.26 (br.t, J=8.5 Hz, 1H, H-13), 5.65
(d, J=7.1 Hz, 1H, H-2), 5.58 (dd, J=10.7, 7.1 Hz, 1H, H-7), 5.04
(br., 1H, H-3'), 5.03 and 4.64 (2d, J=12.0 Hz, 2H
CH.sub.2CCl.sub.3), 4.91 (dd, J=9.7, 1.5 Hz, 1H, H-5), 4.47 (d,
J=6.9 Hz, 1H, H-2'), 4.27 (d, J=8.5 Hz, 1H, H-20a), 4.11 (d, J=8.5
Hz, 1H, H-20b), 3.93 (d, J=7.1 Hz, 1H, H-3), 2.59, (ddd, J=14.6,
9.7, 7.1 Hz, 1H, H-6a), 2.18 (om, 2H, H-14), 2.17 (s, 6H, 2OAc),
2.03 (o.m, 1H, H-6b), 2.00 (d, J=1.0 Hz, 3H, Me-18), 1.91 and 1.80
(2s, 6H, Me.sub.2CNO), 1.76 (s, 3H Me-19), 1.24 (s, 3H, Me-16 or
17), 1.16 (s, 3H, Me-17 or 16), 1.10 (br, t-Bu). 12
[0132] The reaction conditions for the preparation of Compound 8
were determined experimentally to be as follows: Compound 7 (26.0
mg; 0.0244 mmol) was stirred in formic acid (2.0 mL) at room
temperature for 4 hours. Formic acid was then removed by a flow of
nitrogen and crude product was obtained under vacuum. The residue
was redissolved in EtOAc (3.0 mL). Then, NaHCO.sub.3 (61 mg; 0.726
mmol) and isobutyryl chloride (26 .mu.L; 0.0366 mmol) were added.
After being stirred for 1 hour EtOAc (20 mL) was added to the
mixture. The organic phase was washed with a saturated solution of
NaCl (5 mL) and dried over MgSO.sub.4. After filtration and
evaporation, the residue was purified by preparative HPLC using
linear gradient of acetonitrile (25% to 100%) in water over 70
minutes to give pure Compound 8 (R.sub.t=55.8 min) as a white solid
(12.0 mg; 50.0% yield; R.sub.f=1.19; EtOAc/Hexane=1/1). Analytical
HPLC using linear gradient of acetonitrile (25% to 100%) in water
over 50 mins for Compound 8 gave retention time (R.sub.f=46.1 min).
.sup.1H-NMR of Compound 8: (500 MHz, CDCl.sub.3) .delta. 8.10 (d,
J=7.8 Hz, 2H), 7.62 (t, J=7.3 Hz, 1H), 7.51 (t, J=7.6 Hz, 2H),
7.43-7.34 (m, 5H, 3'-Ph), 6.35 (s, 1H H-10), 6.26 (d, J=9.1 Hz, 1H,
NH-4'), 6.16 (br.td, J=9.0, 1.3 Hz, 1H, H-13), 5.69 (d, J=7.1 Hz,
1H, H-5), 5.58 (dd, J=9.0, 2.4 Hz, 1H, H-3'), 5.54 (dd, J=10.5, 7.3
Hz, 1H, H-7), 5.03 and 4.64 (2d, J=12.0, 12.0 Hz, 2H,
CH.sub.2CCl.sub.3), 4.95 (br.d, J=9.5 Hz, 1H, H-5), 4.70 (dd,
J=4.8, 2.4 Hz, 1H, H-2'), 4.31, (d, J=8.6 Hz, 1H, H-20a), 4.18 (d,
J=8.5 Hz, 1H, H-20b), 3.92 (d, J=7.0 Hz, 1H, H-3), 3.42 (d, J=4.9
Hz, 1H, OH-2'), 2.61 (ddd, J=14.4, 9.8, 7.1 Hz, 1H, H-6a), 2.40
(o.septet, J=6.8 Hz, 1H, Me.sub.2CHCONH), 2.36 (s, 3H, OAc), 2.33
(dd, J=9.0, 1.4 Hz, 2H, H-14),2.17 (s, 3H, OAc), 2.05 (m, 1H,
H-6b), 1.88 (s, 3H, Me-18), 1.84 (s, 3H, Me-19), 1.24 (s, 3H Me-16
or 17), 1.18 (s, 3H, Me-17 or 16), 1.13 and 1.12 (2d, J=6.8, 6.8
Hz, 6H, Me.sub.2CHCONH). 13
[0133] The reaction conditions for the preparation of Compound 4
were determined experimentally to be as follows: To a solution of
Compound 8 (11.7 mg; 0.0117 mmol) in acetic acid:methanol (1:1,
v/v, 2 mL) was added zinc powder (70.6 mg) at 60.degree. C. under
nitrogen. This mixture was stirred at 60.degree. C. under nitrogen
for 1 hour. Then CH.sub.2Cl.sub.2 (20 mL) was added and the mixture
was filtered through celite. The organic solvent was evaporated to
give crude product. The residue was purified by preparative HPLC
using linear gradient of acetonitrile (25% to 100%) in water over
70 minutes to give pure Compound 4 (R.sub.t=37.0 min) as a white
solid (7.2 mg; 74.7% yield). Analytical HPLC using linear gradient
of acetonitrile (25% to 100%) in water over 50 minutes for Compound
4 gave retention time (R.sub.t=33.0 min). HRMS: MH.sup.+ requires:
820.35423, Found: 820.35443; .sup.1H-NMR of Compound 4: (500 MHz,
CDCl.sub.3) .delta. 8.12 (d, J=6.8 Hz, 2H) 7.61 (t, J=7.3 Hz, 1H),
7.51 (t, J=7.6 Hz, 2H), 7.44-7.30 (m, 5H, 3'-Ph), 6.28 (s, 1H,
H-10), 6.24 (d, J=9.0 Hz, 1 h NH-4'), 6.20 (t, J=8.5 Hz, 1H, H-13),
5.68 (d, J=7.1 Hz, 1H, H-2), 5.57 (d, J=9.0, 2.5 Hz, 1H, H-3'),
4.94 (dd, J=9.8, 2.2 Hz, 1H, H-5), 4.69 (d, J=2.7 Hz, 1H, H-2'),
4.40 (dd, J=11.2, 5.8 Hz, 1H, H-7),4.29 (d, J=8.5 Hz, 1H, H-20a),
4.19 (d, J=8.5 Hz, 1H, H-20b), 3.78 (d, J=7.1 Hz, 1H, H-3), 2.54
(ddd, J=14.9, 9.8, 6.9 Hz, 1H, H-6a), 2.39 (o.septet, J=6.8 Hz, 1H,
Me.sub.2CHCONH), 2.35 (s, 3H, OAc), 2.28-2.32 (m, 2H, H-14), 2.24
(s, 3H, OAc), 1.88 (ddd, J=13.4, 11.0, 2.2 Hz, 1H, H-6b), 1.82 (s,
3H, Me-18), 1.68 (s, 3H, Me-19), 1.26 (s, 3H, Me-16 or 17), 1.15
(s, 3H, Me-17 or 16), 1.12 and 1.11 (2d, J=7.0, 6.8 Hz, 6H
Me.sub.2CHCONH).
EXAMPLE 3
Semi-Synthesis of N-debenzoyl-N-n-pentanoyl-paclitaxel
[0134] Following the same method of synthesis as described in
Example 2, alternatively, Compound 7 can be transformed into a
family of related taxanes by the use of different alkyl chlorides.
Specifically, Compound 7 can be transformed into a family of
related taxanes by the use of different acyl chlorides, for
example:
[0135] Compound 7 (12 mg; 0.011 mmol) was stirred in formic acid
(0.5 mL) at room temperature for 4 hours and the solution was then
evaporated with a stream of nitrogen. Ethyl acetate (1.5 mL),
sodium bicarbonate (15 mg; 0.18 mmol) and propanoyl chloride (14.5
.mu.L; 0.12 mmol) were added and the mixture stirred for 1 hour at
room temperature. Ethyl acetate (20 mL) was added and the solution
was washed with saturated sodium chloride, dried over anhydrous
magnesium sulphate and evaporated. The residue was purified on
preparative HPLC using a linear gradient of acetonitrile (25% to
100%) in water over 70 minutes to yield Compound 9 (Rt=57.5min) as
a white solid (8.5 mg; 77% yield). Analytical HPLC using a linear
gradient of acetonitrile (25% to 100%) in water over 50 min for
Compound 9 gave a retention time of 47.3 min. 14
[0136] Compound 9 (7.0 mg; 0.0072 mmol) was dissolved in 3.0 mL of
a solution of methanol:acetic acid (1:1; v/v) and zinc (30.0 mg;
0.46 mmol) was added. The mixture was stirred at 60.degree. C. for
1 hour. The mixture was then left to cool at room temperature and
then dichloromethane (20 mL) was added. Filtration over celite and
purification on preparative HPLC using a linear gradient of
acetonitrile (25% to 100%) in water over 70 minutes (Rt=42.5 min)
afforded Compound 10 as a white solid (2.6 mg; 44% yield). 15
[0137] Analytical HPLC using a linear gradient of acetonitrile (25%
to 100%) in water over 50 minutes for Compound 10 gave a retention
time of 34.8 min. HRMS with KI as an internal standard: expected
molecular weight; 872.32596 obtained 872.32574.
EXAMPLE 4
Semi-Synthesis of Canadensol (Scheme III)
[0138] The semi-synthesis of canadensol, taxcultine or any of the
other biological active compounds can be obtained using baccatin
III as starting material. The conversion of the natural product
10-deacetylbaccatin III into baccatin III can be achieved according
to the technique described in Denis J. N. et al., J. Am. Chem.
Soc., 110: 5917 (1988). The conversion of baccatin III into
canadensol requires that the hydroxyl at C-7 be protected prior to
derivatization with the appropriate side chain at C-13. An
appropriate side chain is the triethylsilyl derivative of the C-7
hydroxyl). For the supply of large amounts of canadensol,
taxcultine, etc, the addition of the side chains at C-13 is
achieved in higher yields with the use of a range of side chains by
Ojima's method (Ojima, I. et al., Tetrahedron, 48. 6985-7012, 1992;
and Ojima, I. et al., Tetrahedron Letters, 34, 4149-4152,
1992).
[0139] The .beta.-lactam (1) was obtained from (3R,4S)-3
-hydroxy-4-phenylazetidin-2-one (which was prepared according to
Ojima, I. et al Tetrahedron, 48, 6985-7012, 1992) by treatment with
tetrabutyl ammonium fluoride according to the following
conditions:
[0140] (3R,4S)-3-triisopropylsilyloxy-4-phenylazetidin-2-one (0.200
g; 0.626 mmol) was dissolved in dry tetrahydrofuran (4 mL) under
nitrogen at room temperature. To this was added a solution of a
n-Bu.sub.4N.sup.+F.sup.- (1.0M in dry tetrahydrofuran; 1.2 ml; 1.2
mmol; 1.9 eq.) and stirred for 2.5 hours at room temperature. The
reaction was complete as shown by thin layer chromatography with
35% ethylacetate/hexane which separated very well the starting
material from the product. The work up consisted of an extraction
with ethyl acetate, washing with brine, drying the ethyl acetate
layer with magnesium sulfate then evaporated. The product (0.12 g)
was obtained with almost 100% yield after flash chromatography.
16
[0141] The next step in the synthesis is protection of the hydroxy
group of the .beta.-lactam with triethylsily chloride according to
the following conditions:
[0142] The .beta.-lactam (1) (0.12 g; 0.735 mmol) was dissolved in
10 ml pyridine at room temperature under nitrogen. The
triethylsilyl chloride (0.40 ml; 2.38 mmol; 3.2 eq) was added at
room temperature and the reaction was complete after one hour at
room temperature. The work up consisted on quenching the reaction
with saturated ammonium chloride, extract with methylene chloride,
wash the methylene chloride solution with brine and dry the
methylene chloride layer with magnesium sulfate. After filtration
the solvent was evaporated. Most of the pyridine which was left
behind was evaporated with heptane before running a flash
chromatography with a 7.5" small column with 30%
ethylacetate/hexane and collected the fractions 24-45(0.129 g). The
yield of the combined two steps is 74%. The NMR was in accord with
structure 2. 17
[0143] Next step is specific to the side chain we want to add. Here
we will show the experimental for the side chain of canadensol and
in the next page the coupling reaction with the protected baccatin
derivative.
[0144] The triethyl-silyl-protected .beta.-lactam (2) (0.030 g:
0.108 mmol) was dissolved in 1.5 ml dry methylene chloride under
nitrogen at room temperature, triethylamine (41 .mu.l; 0.294 mmol;
2.7 eq.) a trace of 4-DMAP (4-dimmethylaminopyridine), the solution
was cooled in an ice water bath and isobutyryl chloride (0.025 ml;
0.239 mmol; 2.2 eq.) was stirred at room temperature for 3 hours
when the reaction was complete. The work up consisted on quenching
the reaction with saturated ammonium chloride, extract with
methylene chloride, wash the methylene chloride solution with brine
and dry the methylene chloride layer with magnesium sulfate. After
filtration the solvent was evaporated and flash chromatography with
25% ethyl acetate/hexane in a 6" chromatography column 0.034 g of
the required .beta.-lactam (90% yield) (3) for the semi synthesis
of canadensol was obtained. 18
[0145] Coupling reaction: the C-7 triethylsilylprotected baccatin
III (0.048 g: 0.0685 mmol) and the
triethylsily-isopropyl-.beta.-lactam (3) (0.043 g; 0.0978 mmol; 1.4
eq.) were dissolved in 3.6 ml dry tetrahydrofuran. The solution was
cooled to a temperature in the range -45.degree. C. to -50.degree.
C. (dry ice-acetonitrile bath) under nitrogen. The base sodium
bistrimethylsilylamide (NaHMDS) (1.0 M) solution in
tetrahydrofuran; 0.175 ml; 175 mmol; 2.6 eq) was added in one
portion at a temperature of -45.degree. C. and the reaction was
stirred for 30 min at this temperature. The solution became yellow
and was quenched at 30 min with an ammonium chloride saturated
solution. The work up consisted of quenching the reaction with
saturated ammonium chloride, extracting with ethyl acetate, washing
the organic phase with brine and drying the organic layer with
magnesium sulphate. After filtration the solvent was evaporated and
flash chromatography of the residue with 20% ethyl acetate/hexane
in a 6" chromatography column; 36 mg of the coupled protected
taxane (5) was obtained with 14 mg of starting material recovered
(4, uncoupled protected baccatin III). 19
[0146] Preparation of Canadensol
[0147] The deprotection of compound 5 was completed with 0.3 M HCl
in 95% ethanol at 4.degree. C. overnight. At this stage the
reaction was complete, the reaction mixture cooled in an ice-water
bath; ice added to reaction mixture and the pH adjusted to 5-6 with
the dropwise addition of a saturated sodium bicarbonate solution.
The reaction mixture is extracted four times with ethyl acetate,
the organic phase washed with brine, the organic layer dried with
MgSO.sub.4, filtered and the solvent evaporated. The residue was
subjected to flash chromatography with 75% ethyl acetate/hexane in
a 7" mini column and 0.024 g of canadensol (6) was obtained with
identical NMR and high resolution mass spectrometry as the
canadensol obtained by Denis et al. method. 20 21
EXAMPLE 5
In Vitro Microtubule Assay
[0148] Tubulin is obtained by the purification of calf brain
(Williams Jr, R. C. and J. C. Lee, Methods in Enzymology, 85:376,
1982). The taxanes to be tested are dissolved in dimethylsulfoxide
(DMSO) with a final concentration of 10 .mu.M. The methodologies
used in this microtubule assay are well known to those skilled in
the art. The method briefly consists of mixing the buffer and other
required ingredients in a cuvette in an ice bath, with the DMSO
solution containing the taxane to be analysed. Before the start of
the measurement, the tubulin is added and the cuvette is set in a
thermostatically controlled cell compartment of a UV-VIS
spectrophotometer. The temperature is set at 37.degree. C. to start
the polymerisation of the tubulin. The control is performed with
all the same ingredients just without the added taxane. Since
bio-active taxanes cause tubulin to polymerize to microtubules and
stabilize them, the turbidity of the mixture will rise and causing
a corresponding increase in the absorbance observed at 350 nm.
Theoretically, the faster the increase in absorbance, the more
active the taxane.
[0149] As shown in FIGS. 1-4, Canadensol is more active than
paclitaxel and taxcultine in these tests. FIG. 1 illustrates that,
at the same concentration, semi-synthetic Canadensol (ssC) causes
higher degree of tubulin polymerisation than Paclitaxel (P).
[0150] FIG. 2 illustrates that 5 .mu.M semi-synthetic Canadensol
(ssC) appears to cause a similar degree of tubulin polymerisation
as 10 .mu.M Paclitaxel (P), this figure also emphasizes the
dose-dependent response of tubulin polymerisation to semi-synthetic
Canadensol (ssC) at concentrations of 5 .mu.M and 10 .mu.M.
[0151] FIG. 3 illustrates that, at the same concentration (10
.mu.M), semi-synthetic Canadensol (ssC) exhibits greater
enhancement of tubulin polymerisation than semi-synthetic
Taxcultine (ssT) or paclitaxel (P).
[0152] FIG. 4 illustrates that semi-synthetic Canadensol (ssC)
obtained by the method described in the preferred embodiment of
this invention shows similar activity to a mixture of 13%
Canadensol and 87% Taxcultine purified from Taxus canadensis.
EXAMPLE 6
Preclinical Studies with Taxoids
[0153] Methods
[0154] 1--Cell lines and cell culture. The human ovarian
adenocarcinoma cell line A2780 was used to evaluate the
antiproliferative activity of taxoids. These cells were grown in
either RPMI medium supplemented with 10% fetal bovine serum and
penicillin-streptomycin antibiotics. Cells were maintained in
culture at 37.degree. C. in an atmosphere of 5% CO.sub.2.
[0155] 2--Cytotoxicity assay. Exponentially growing cells
(2-3.times.10.sup.3 cells/100 ml) were seeded in 96-well plates and
incubated for 16 h. Cells were then treated continuously with the
extracts. 72 h later, cell survival was evaluated by replacing the
culture media with 150 ml fresh medium containing 10 mM
4-(2-hydroxyethyl)-1 -piperazineethamesulfonic acid buffer, pH 7.4.
and 50 ml of 2.5 mg/ml of
3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
in PBS, pH 7.4, were then added. After as 3-4 h of incubation at
37.degree. C., the medium and MTT were removed, and 200 ml of DMSO
was added to dissolve the precipitate of reduced MTT, followed by
addition of 25 ml glycine buffer (0.1M glycine plus 0.1M NaCl, pH
10.5). The formazan crystals were then dissolved and the absorbance
was determined at 570 nm with a microplate reader (BIORAD, model
450). The MTT assay distinguished between viable and non-viable
cells on the basis for the requirement of physiologically active
mitochondria to metabolize the MTT only in viable cells. The
IC.sub.50 was calculated as the concentration of drug causing a 50%
inhibition in the absorbance compared to cells treated with solvent
alone, Table I.
[0156] In Vivo Study Using GA3 Cell Model
[0157] Origin: The DA3 cell line used in this study was derived
from a hyperplastic mammary outgrowth (preneoplastic lesion)
treated with 7,12-dimethylbenzanthracene (DMBA). DMBA-treated
explant was then transplanted into the mammary glands of a female
BALB/c mouse, and the resulting tumor was used to establish the DA3
cell line. DA3 is an immunogenic and non-metastatic mammary
adenocarcinoma.
[0158] Source: The cell line used in our study was originally
obtained from Dr. Medina in 1993. Stocks of these cells were
generated and stored during early passages in liquid nitrogen. One
vial from this early stock was cultured and cells generated were
used to confirm the absence of viral and mycoplasma infections.
Cells were then propagated and stocks of these cells were
established and stored in liquid nitrogen for further studies with
Dup compounds.
[0159] Cell culture: DA3 cells were maintained in RPMI 1640 medium
supplemented with 1M mercaptoethanol, 1M Hepes buffer solution, 100
nM sodium pyruvate, 200 mM L-glutamine, 10 mM non-essential amino
acids, 1M vitamins, 10% fetal bovine serum, 1%
penicillin-streptomycin. Cells were maintained at 37.degree. C.
under 5% CO.sub.2. Under these conditions, DA3 cells proliferate
but do not differentiate.
[0160] Animal. Female BALB/c mice (Charles River Inc.) were used to
grow DA3 tumors. Mice were maintained at the Lady Davis Institute
[LDI] Animal Care Facility. The animal facility is located in the
basement of the LDI and is completely isolated from the research
laboratories and administrative offices. Animal rooms are
segregated from surgery rooms, offices, autoclaves, incinerators,
and access to the animal facility is strictly limited to animal
users only. Animal room used in our studies has two doors, one
serving as the entrance, and the other door provides direct access
to washing/sterilization/incineration facilities. It permits
accurate adjustment of environmental parameters including
temperature, humidity, ventilation, and lighting. Cleaning and
sanitation practices are performed, on a daily basis, by personnel
with appropriate training.
[0161] Tumor cell inoculation and treatments. After one week
acclimatisation, mice were randomized into a group of 5 per cage.
Cages were randomly assigned to specific experimental groups. The
mice were then labeled by numbers using the "ear punching" method.
DA3 cells were transplanted subcutaneously to mice, as a suspension
of tumor cells [1.times.10.sup.6 viable cells per 0.1 ml], in the
right flank. All animals were inoculated at the same site.
[0162] For tumor induction, cells were grown to 70% confluence in
complete medium and then collected using trypsin-EDTA solution
[0.05% trypsin, 0.53 mM EDTA-4Na in HBSS Ca.sup.++, Mg.sup.++ and
NaHCO.sub.3 free]. cells were then centrifuged and washed three
times with phosphate buffer solution and resuspended at a dilution
of 0.1 to 1.times.10.sup.6 cells/0.1 ml. Viability was examined by
trypan blue staining and only flasks in which the viability was
.gtoreq.95% were used for in vivo studies.
[0163] Treatment was initiated when tumors become palpable. Drug
was given twice (day 1 and day 3) by iv. Control animals were given
the same volume of saline solution. The dose of each drug was
normalized to mouse body weight.
[0164] Tumor measurement. animals were examined every day but the
tumor growth was monitored every second or third day using
calipers. Parameters measured are: tumor measured along the longest
axis (length) and the perpendicular shortest axis (width) and the
relative tumor volume (in cm.sup.3) was calculated by the formula:
[Length (cm).times.(width cm).sup.2]/2. Tumors which reach the
limit size permitted by the Canadian regulation on the use of
laboratory animals, or showing any apparent distress or discomfort
were immediately sacrificed independently of the experimental
protocol [usually 2-3 cm.sup.3 but this depend on animal status and
the decision is taken by the animal room personnel]. Animals were
subjected, on a daily basis, to general examination.
[0165] Statistical analysis. The unpaired Student t-test was used
to compare statistical significance among various groups.
[0166] Results
[0167] In Vitro Studies
[0168] Canadensol was found to be as active as paclitaxel or
taxcultine, in inhibiting the growth of the human ovarian carcinoma
cell line, A2780, as shown in FIGS. 6, 7 and Table I.
[0169] In Vivo Studies
[0170] The mouse mammary adenocarcinoma tumors were resistant to
paclitaxel since no significant antitumor activity was observed
with tolerable doses (20-40 mg/Kg). Higher doses of paclitaxel were
found to be very toxic (body weight loss and mortality), see FIG.
8B. Canadensol, however, was found to induce antitumor activity
when given twice at 40 mg/kg, see FIG. 9. This effect was
significant from day 30 to day 40 of tumor growth. No toxic effect
was observed at this dose.
1 TABLE I A2780 Conc. (nM) Abs. % IC.sub.50 TAXOL 0 1.433 100.00%
0.2 1.455 101.54% 2 1.464 102.16% 10 1.162 81.09% 16.96 20 0.522
36.43% 100 0.021 1.47% 200 0.02 1.40% CANADENSOL 0 1.308 0.2 1.598
100.00% 2 1.504 94.12% 10 1.246 77.97% 20 0.812 50.81% 21.32 100
0.026 1.63% 200 0.018 1.13% TAXCULTINE 0 1.155 0.2 1.535 100.00% 2
1.536 100.07% 10 1.093 71.21% 16.04 20 0.554 36.09% 100 0.032 2.08%
200 0.022 1.43%
* * * * *