U.S. patent number 5,059,699 [Application Number 07/573,731] was granted by the patent office on 1991-10-22 for water soluble derivatives of taxol.
This patent grant is currently assigned to Virginia Tech Intellectual Properties, Inc.. Invention is credited to David G. I. Kingston, Zhi-Yang Zhao.
United States Patent |
5,059,699 |
Kingston , et al. |
October 22, 1991 |
**Please see images for:
( Certificate of Correction ) ** |
Water soluble derivatives of taxol
Abstract
Sulfonated 2'-acryloyltaxol and sulfonated 2'-O-acyl acid taxol
derivatives are synthesized which have improved water solubility
and stability while maintaining bio-activity. In particular,
2'-[(3-sulfo-1-oxopropyl)oxy]taxol sodium salt is synthesized by
reacting taxol with acrylic acid, and subsequently reacting the
2'-acryloyltaxol with bisulfite in a Michael reaction.
2'-{[4-((2-sulfoethyl)amino)-1,4-dioxobutyl]oxy}taxol sodium salt
and 2'-{[4-((3-sulfopropyl)amino-1,4-dioxobutyl]oxy}taxol sodium
salt are synthesized by reacting 2'-succinyltaxol with the
tetrabutylammonium salts of taurine and 3-aminopropyl sulfonic
acid, respectively, and subsequently exchanging the ammonium with
sodium. Glycol derivatives of 2'-O-acyl acid taxols with improved
water solubility are synthesized by reaction of a glycol with
2'-O-acyl acid taxol.
Inventors: |
Kingston; David G. I.
(Blacksburg, VA), Zhao; Zhi-Yang (Blacksburg, VA) |
Assignee: |
Virginia Tech Intellectual
Properties, Inc. (Blacksburg, VA)
|
Family
ID: |
24293168 |
Appl.
No.: |
07/573,731 |
Filed: |
August 28, 1990 |
Current U.S.
Class: |
549/511 |
Current CPC
Class: |
A61P
35/00 (20180101); C07D 305/14 (20130101) |
Current International
Class: |
C07D
305/14 (20060101); C07D 305/00 (20060101); C07D
305/14 (); A61K 031/335 () |
Field of
Search: |
;549/511 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Wani et al., J. Am. Chem. Soc., 1971, 93, 2325. .
McGuire et al., Ann. Int. Med., 1989, 111, 273-279. .
Magri et al., J. Nat. Prod., vol. 51, No. 2, 298-306, Mar.-Apr.
1988. .
Duetsch et al., J. Med. Chem., 1989, 32, 788-792..
|
Primary Examiner: Lee; Mary C.
Assistant Examiner: McKane; Joseph K.
Attorney, Agent or Firm: Mason, Fenwick & Lawrence
Claims
We claim:
1. A taxol compound having the following structure: ##STR9##
wherein: X is selected from the group consisting of H, alkyls, and
aryls.
2. A compound according to claim 1, wherein:
X is H.
3. A water soluble taxol compound having the following structure:
##STR10## wherein: X is selected from the group consisting of H,
alkyls, and aryls; and
M is selected from the group consisting of H, alkaline metals, and
ammonio groups.
4. A compound according to claim 3, wherein:
X is H, and
M is Na.
5. A water soluble taxol compound having the following structure:
##STR11## wherein: R is selected from the group consisting of:
--(CH.sub.y).sub.n --CO--NH--(CH.sub.2).sub.z --SO.sub.2 O--M,
and
--(CH.sub.y).sub.n --CO--O--(CH.sub.2).sub.z --OH; wherein:
M is selected from the group consisting of H, alkaline metals, and
ammonio groups,
n is 1 to 3,
y is 1 to 2, provided y is not 1 when is 1, and
z is 2 to 3.
6. A compound according to claim 5, wherein:
Y is 2,
n is 2, and
z is 2.
7. A compound according to claim 5, wherein:
Y is 2,
n is 2, and
z is 3.
8. A compound according to claim 5, wherein:
M is a quaternary ammonium.
Description
FIELD OF THE INVENTION
The present invention relates to water soluble derivatives of taxol
with anti-neoplastic activity, and relates more particularly to
sulfonated 2'-acryloyltaxol derivatives, 2'-sulfoalkylamino-O-acyl
acid taxol derivatives, and 2'-ethylene glycol-O-acyl acid taxol
derivatives.
BACKGROUND OF THE INVENTION
Taxol is a naturally occurring diterpenoid which has great
potential as an anti-cancer drug, and which has shown activity in
several tumor systems. Taxol was first isolated and its structure
reported by Wani, et al., in "Plant Anti-Tumor Agents. VI. The
Isolation and Structure Of Taxol, A Novel Anti-Leukemic And
Anti-Tumor Agent From Taxus brevifolia, "J. Am. Chem. Soc., 1971,
93, 2325. Taxol is found in the stem bark of the Western Yew, Taxus
brevifolia, as well as in T. baccata and T. cuspidata.
The biological activity of taxol is related to its effect on cell
division. Taxol promotes formation of microtubules that form the
mitotic spindle during cell division. However, taxol prevents
depolymerization of the tubulin forming the microtubules of the
mitotic spindle, which is essential for cell division to take
place. Thus, taxol causes cell division to stop. Taxol's mechanism
is unique since it promotes the formation of tubulin polymers,
whereas other anti-cancer drugs, such as vinblastine and
colchicine, prevent microtubule formation.
Extensive testing of taxol has not been performed because taxol is
in short supply and has not yet been successfully synthesized.
Preliminary studies have shown that taxol may have marginal
activity in acute leukemia and melanoma, and some activity has been
noted in other tumors. Further, studies by McGuire et al. found
taxol to be an active agent against drug-refractory ovarian cancer.
See "Taxol: A Unique Antineoplastic Agent With Significant Activity
In Advanced Ovarian Epithelial Neoplasms," Ann. Int. Med., 1989,
111, 273-279, herein incorporated by reference. However, due to the
low water solubility of taxol, doses had to be delivered as
infusions diluted in aqueous dextrose solutions.
It should be noted that in phase 1 clinical trials, taxol itself
did not show excessive toxic effects, but severe allergic reactions
were caused by the emulsifiers administered in conjunction with
taxol to compensate for taxol's low water solubility. In fact, at
least one patient's death was caused by an allergic reaction
induced by the emulsifiers. Therefore, researchers have attempted
to create water soluble derivatives of taxol which retain their
anti-neoplastic and anti-cancer activity.
With reference to FIG. 1, the structure of taxol is illustrated
along with a .sup.1 H nuclear magnetic resonance (NMR) spectrum of
a taxol sample. The NMR signals are well separated and cover the
region from 1.0 to 8.2 ppm. For simplicity, the spectrum is divided
into three regions: a first region between 1.0 and 2.5 ppm formed
by strong 3-proton signals of the methyl and acetate groups as well
as complex multiplets caused by certain methylene groups; a second
region between 2.5 and 7.0 ppm represents the signals observed from
most of the protons on the taxane skeleton and the side chain; a
third region between 7.0 and 8.2 ppm is formed by the signals from
the aromatic protons of the C-2 benzoate, C-3' phenyl and C-3'
benzamide groups. The peaks of the NMR spectrum in FIG. 1 are
labeled according to the number of the carbon in the taxol
structure to which the protons including the signals are
attached.
Magri and Kingston reported on the biological activity of taxols
substituted at the C-2' and C-7 positions in order to make them
more water soluble. See "Modified Taxols, 4..sup.1 Synthesis And
Biological Activity Of Taxols Modified In The Side Chain," Journal
of Natural Products vol. 51, no. 2 pp. 298-306, March-April 1988,
herein incorporated by reference. A 2'-(t-butyldimethylsilyl)taxol
was synthesized and found to be essentially inactive; this was
taken as an indication of the need for a free hydroxyl group at the
2' position of the taxol side chain for biological activity.
Further, acyl substituents at the 2' position in 2'-acetyltaxol and
2',7-diacetyltaxol were readily hydrolyzed under in vivo
conditions, and both showed activity in a cell culture bioassay.
The lability of the acyl substituents at the 2' position suggested
that 2'-acetyltaxols could serve as pro-drug forms of taxol.
(Generally, a prodrug is a compound which exhibits pharmacologic
activity after biotransformation.)
Magri and Kingston reported that two taxols with increased water
solubility were prepared, 2'-(.beta.-alanyl)taxol: ##STR1## and
2'-succinyltaxol: ##STR2## The 2'-(.beta.-alanyl)taxol was found to
be active in vivo and in vitro, but was unstable. The
2'-succinyltaxol, prepared by the treatment of taxol with succinic
anhydride, had a much diminished P-388 in vivo activity as compared
with taxol. Thus, research efforts were concentrated on other
derivatives of taxol which did not suffer from instability, or
inactivity in vivo or in vitro.
Deutsch et al., in "Synthesis Of Congeners And Prodrugs. 3..sup.1
Water-Soluble Prodrugs Of Taxol With Potent Antitumor Activity," J.
Med. Chem. 1989, 32 788-792, herein incorporated by reference,
reported that salts of 2'-succinyltaxol and 2'-glutaryltaxol had
improved antitumor activities when compared to the free acids.
Since these researchers believed that salts prepared with different
counterions often have substantially different properties, a
variety of 2' substituted taxol salts were synthesized and tested.
Triethanolamine and N-methylglucamine salts of the 2' substituted
taxol derivatives showed greatly improved aqueous solubility and
had more activity than sodium salts. Further, a series of
2'-glutaryltaxol salts were found to have higher activity than
their 2'-succinyltaxol analogs. In particular, the taxol salt
resulting from the coupling of 2'-glutaryltaxol with
3-(dimethylamino)-1-propylamine using N,N'-carbonyldiimidazole
(CDI), demonstrated good solubility and bioactivity.
In addition to increasing the solubility and bioactivity of taxol,
it is desirable that the taxol derivatives formed have increased
stability to prolong their shelf life. It is believed that salts of
taxol esters are very susceptible to base hydrolysis, and
water-solubilizing groups, such as carboxylate salts or amine
salts, tend to be basic. Thus, it is desired that neutral,
water-soluble taxol derivatives be synthesized which also have
improved or the equivalent activity to taxol. Organic sulfonate
salts tend to be neutral or only slightly basic, and therefore,
sulfonate salts of taxol esters should have improved stability.
Further, due to the difficulties involved in synthesizing
carboxylic and amine salts of taxol esters, it is desirable to find
less expensive water-soluble taxol derivatives and processes for
forming them.
SUMMARY OF THE INVENTION
The present invention relates to the production of water soluble
taxol derivatives, and water soluble sulfonate salts of taxol. In a
preferred embodiment, 2'-[(3-sulfo-1-oxopropyl)-oxy] taxol sodium
salt is formed by reacting taxol with acrylic acid to form
2'-acryloyltaxol; the 2'-acryloyltaxol is then subjected to a
Michael reaction with sodium bisulfite to form the 2'-sulfoethyl
ester salt of taxol. In another preferred embodiment, 2'-O-acyl
acid taxols, such as 2'-succinyltaxol and 2'-glutaryltaxol, are
subjected to a novel reaction with the tetrabutylammonium salt of
taurine to form sulfoalkylamine salts of the 2'-O-acyl acid taxols.
Another preferred embodiment involves the reaction of amino
sulfonic acid salts with succinic or glutaric anhydride, and
reaction of the product with taxol to form sulfoalkylamine
2'-O-acyl acid taxol derivatives. In a further embodiment, ethylene
glycol derivatives of 2'-O-acyl acid taxols are formed. These
compounds exhibit high water solubility, and demonstrate
anti-leukemic, antineoplastic, and/or anti-cancer activity.
Thus, it is a primary object of this invention to produce
water-soluble derivatives of taxol with high bioactivity and
stability.
It is a further object of the present invention to provide a simple
and inexpensive process for forming 2'-acryloyltaxols and their
sulfonate salt derivatives.
It is yet another object of the present invention to produce
2'-O-acyl acid taxols and their sulfoalkylamine salts.
It is a still further object of the present invention to produce
sulfoalkylamine derivatives of 2'-O-acyl acid taxols by simple and
inexpensive processes.
It is yet a further object of the present invention to produce
hydroxyalkoxy derivatives of 2'-O-acyl acid taxol.
DESCRIPTION OF THE DRAWINGS
FIG. 1 is a representation of the taxol structure and its nuclear
magnetic resonance spectrum with peaks labeled according to the
part of the taxol structure to which they correspond.
DETAILED DESCRIPTION OF THE INVENTION
Taxol was obtained from the National Cancer Institute. .sup.1 H-NMR
and .sup.13 C-NMR spectra were made with a Bruker 270SY 270 MHz
spectrometer; 2D-NMR were obtained using a Bruker WP200 200 MHz
spectrometer. Chemical shifts are all recorded in parts per million
(ppm) downfield from TMS in .sup.1 H-NMR, and .sup.13 C-NMR
chemical shifts are based on chloroform's shift at 77.0 pm or on
the TMS shift at 0 ppm. Samples were generally recorded while in
CDCl.sub.3 or CD.sub.3 OD at ambient temperature. Mass spectra were
obtained using a Finnegan-MAT 112 gas chromatograph-mass
spectrometer and VG 7070 HF mass spectrometer equipped with data
system, FAB source, and EI/CI source. NMR and mass spectroscopy
data are most useful in studying taxol and its derivatives, with
other methods, such as IR and UV, providing additional structure
confirmation information.
Other analytical instruments used included Perkin-Elmer 710B
infrared and Perkin-Elmer 330 UV-visible spectrophotometers, and a
Perkin-Elmer polarimeter. HPLC was carried out on an apparatus
consisting of a Waters M6000 pump, a Rheodyne injection valve, a
Waters Radial-Pak RLM-100 RP-8 column, and a Waters 440 UV
detector.
2'-ACRYLOYLTAXOLS
2'-[(3-sulfo-1-oxopropyl)oxy]taxol sodium salt was prepared by
coupling taxol with acrylic acid followed by Michael addition of
bisulfite ion. Taxol was reacted with the acrylic acid using
isobutylchloroformate as the coupling agent. This produced
2'-acryloyltaxol in 94% yield after purification via flash
chromatography (silica gel, 1/1 dichloromethane/ethyl acetate).
Using TLC, the coupling of acrylic acid to taxol was found to be
90% complete in 15 hours at 60.degree. C. The disubstituted C-2',
C-7 product was not formed after extended reaction times. Proton
NMR spectra of the 2'-acryloyltaxol showed that the signal for the
C-2'proton was shifted downfield to 5.46 ppm (d, j=3), from the
4.73 ppm shift for the C-2'proton in unsubstituted taxol. The
downfield shift is consistent with acylation of the C-2'-hydroxyl
group. Since the signal for the C-7 proton at 4.43 ppm was
essentially unchanged when compared with the unsubstituted taxol
C-7 proton signal at 4.38 ppm, it was concluded that no reaction
had taken place at the C-7 position. Mass spectroscopy indicated a
molecular weight of 907 with peaks at m/z 930 (MNa.sup.+) and 908
(MH.sup.30).
The 2'-acryloyltaxol was then reacted with sodium bisulfite in a
Michael addition reaction. Sodium bisulfite was used because it is
a good nucleophile, and because it provides suitable pH conditions
for the reaction. Proton NMR spectra of the Michael addition
reaction product were contrasted with the spectra of the
2'-acryloyltaxol. The signals in the NMR spectra of the
2'-acryloyltaxol that are due to the presence of the vinyl protons
were not present in the spectra of the Michael addition product.
However, two triplets at 3.14 ppm and 2.93 ppm indicated the
presence of the two new methylene groups in the Michael addition
product. Mass spectroscopy of the Michael reaction product
indicated a molecular weight of 1011 with peaks present at m/z 1034
(MNa.sup.+) and 1012 (MH.sup.+).
The formation of 2'-[(3-sulfo-1-oxopropyl)oxy]taxol sodium salt was
attempted in a one-step reaction by combining taxol with
3-hydroxy-3-oxopropyl sulfonic acid in the presence of pyridine and
DCC (dicyclohexylcarbodiimide), but no product was obtained. This
is possibly due to inter-molecular attack by the sulfonyl group on
the reaction intermediate.
2'-O-ACYL ACID TAXOL DERIVATIVES
2'-{[4-((2-sulfoethyl)amino)-1,4-dioxobutyl]oxy}taxol sodium salt
and 2'-{[4-((3-sulfopropyl)amino-1,4-dioxobutyl]-oxy}taxol sodium
salt were produced in high yield by coupling 2'-succinyltaxol with
taurine (2-aminoethyl sulfonic acid) and 3-aminopropyl sulfonic
acid tetrabutylammonium salts, respectively. Note that other
quaternary ammonium salts may be used to make the aminoalkyl
sulfonic acids organic solvent soluble. 2'-succinyltaxol was formed
by the reaction of succinic anhydride with taxol for two hours at
room temperature in pyridine or DMP. In comparison with the NMR
spectrum of taxol, the NMR spectrum of 2'-succinyltaxol showed a
downfield shift of the C-2' proton signal to 5.51 ppm, and the
succinyl proton caused multiplets centered about 2.6 ppm.
The 2'-succinyltaxol was then reacted with taurine
tetrabutylammonium salt using isobutylchloroformate as the coupling
agent. 2'-{[4-((2-sulfoethyl)amino)-1,4-dioxobutyl]-oxy}taxol
tetrabutylammonium salt was produced in 100% yield after isolation
via flash chromatography on silica gel using 7/1
dichloromethane/methanol. The reaction was only 80% complete in two
hours as monitored by TLC; in order to obtain 100% yield, extended
reaction times were necessary. The NMR spectrum of the
sulfoalkylamine derivative of 2'-succinyltaxol showed new peaks at
3.6 ppm and 2.94 ppm for the two methylene groups. The sodium salt
of 2'-{[4-((2-sulfoethyl)amino)-1,4-dioxobutyl]oxy}taxol was
achieved by running
2'-{[4-((2-sulfoethyl)amino-1,4-dioxo-butyl]oxy}taxol
tetrabutylammonium salt through a Dowex 50 ion exchange column
(Na.sup.+ form). An NMR spectrum of the sodium salt showed the
absence of signals for the tetrabutyl group. Mass spectroscopy of
the sodium salt indicated a molecular weight of 1082 by the
presence of peaks at m/z 1105 (MNa.sup.+) and 1083 (MH.sup.+).
2'-{[4-((3-sulfopropyl)amino)-1,4-dioxobutyl]oxy}taxol sodium salt
was prepared by the same method used for the sulfoethylaminotaxol
sodium salt; however, the taurine was replaced with
3-amino-1-sulfopropionic acid tetrabutylammonium salt. An NMR
spectrum confirmed the synthesis of the 3-sulfopropylamino
derivative; new peaks were present at 3.28, 1.98, and 2.87 ppm,
representing the three additional methylene groups forming the
propyl moiety. The sodium salt form of the
sulfopropylamino-succinyltaxol derivative was formed by passing the
tetrabutyl-ammonium salt through a Dowex 50 ion exchange column
(Na.sup.+ form). Mass spectroscopy of the sodium salt of the
sulfopropylaminosuccinyltaxol derivative indicated a molecular
weight of 1096 by the presence of peaks at m/z 1119 (MNa.sup.+) and
1097 (MH.sup.+).
It is also contemplated that an amide linkage can be formed between
an amino sulfonic acid and an anhydride or diacid, and that the
product can be reacted with taxol to form water soluble 2'-O-acyl
acid taxol derivatives. Preferably, the amino sulfonic acid is an
organic solvent soluble salt.
Attempts to form
2'-}[4-((2-sulfoethyl)amino-1,4-dioxobutyl]oxy}taxol sodium salt
directly from 2'-succinyltaxol in a one-step reaction were
unsuccessful. 2'-succinyltaxol was combined with triethanolamine,
isobutylchloroformate, tetrahydrofuran (THF), taurine, DMF, and
water. However, water, necessary to solubilize taurine, hydrolyzed
the mixed anhydride intermediate back to the starting material.
When nonaqueous conditions were tried, the reaction still did not
succeed because the taurine did not dissolve in the organic
solvents.
2'-{[4-((2-ethanethiol)amino)-1,4-dioxobutyl]oxy}taxol was prepared
in low yield by combining 2'-succinyltaxol with triethylamine,
isobutylchloroformate, THF, 2-thioethylamine and dichloromethane.
Attempts to oxidize the thiol to the desired sulfonic acid with
meta-chloroperbenzoic acid, MCPBA, and dichloromethane did not
yield appreciable amounts of the desired sulfoalkylamine
succinyltaxol derivative.
ETHYLENE GLYCOL DERIVATIVES OF SUCCINYLTAXOL
2'-{[4-((hydroxylethyl)oxy)-1,4-dioxobutyl]oxy}taxol was prepared
by coupling succinyltaxol with ethylene glycol. The
hydroxyethyloxysuccinyltaxol derivative was formed in 83% yield
after a reaction time of 20 hours at room temperature. The
hydroxyethyloxysuccinyltaxol derivative was made in order to
convert the secondary hydroxyl group at the 2' position in taxol to
a primary hydroxyl group; it is hypothesized that the hydroxyl
group in the product is more reactive than that of the hydroxyl in
taxol, and that this will make it possible to make other taxol
derivatives under mild conditions. An NMR spectrum of the ethylene
glycol derivative showed the presence of new peaks at 3.7 ppm and
4.1 ppm, which are assigned to the two new methylene groups of the
hydroxyethyloxy derivative. Mass spectroscopy indicated a molecular
weight of 997 by the presence of peaks at m/z 1020 (MNa.sup.+) and
998 (MH.sup.+).
2'-.gamma.-AMINOBUTYRYLTAXOL FORMATE
2'-.gamma.-aminobutyryltaxol formate was synthesized by coupling
taxol with N-carbobenzyloxy(CBZ)-.gamma.-aminobutyric acid followed
by deprotection of the amine. Taxol was reacted with
N-CBZ-.gamma.-aminobutyric acid using dicyclohexylcarbodiimide
(DCC) as the coupling agent. The resulting
2'-NCBZ-.gamma.-aminobutyryl taxol was produced in 75% yield after
purification via preparative TLC with silica gel and 3/2
hexane/ethyl acetate. DCC decomposes to dicyclohexylurea with the
addition of water, so the excess reagents used to drive the
reaction did not present a problem; most of the dicyclohexylurea
and N-CBZ-.gamma.-aminobutyric acid were removed by filtration.
Deprotection of the 2'-N-CBZ-.gamma.-aminobutyryltaxol was effected
using 5% Pd/C as a catalyst and formic acid as a hydrogen source.
Formic acid provides an active form of hydrogen for removal of CBZ
protecting groups, and the reaction yields the
2'-.gamma.-aminobutyryltaxol derivative as a formate salt, which is
more water soluble than the neutral form. NMR confirmed the
synthesis of the 2'-.gamma.-aminobutyryl taxol formate. However,
the compound was unstable in methanol solution and decomposed back
to taxol after a few hours. This instability precluded further
consideration of 2'-.gamma.-aminobutyryltaxol formate as a prodrug
form of taxol.
WATER SOLUBILITY
Water solubilities for all compounds were determined by the
partition coefficient between 1-octanol and water. Octanol
saturated with distilled water and distilled water saturated with
octanol were used for the solubility determinations. Partition
experiment results showed that 2'-[(3-sulfo-1-oxopropyl)oxy]taxol
sodium salt is 210 times more water soluble than taxol,
2'-{[4-((2-sulfoethyl)amino)-1,4-dioxobutyl]oxy}taxol sodium salt
is 191 times more soluble than taxol, and
2'-{[4-((3-sulfopropyl)amino)-1,4-dioxobutyl]oxy}taxol sodium salt
is 118 times more water soluble than taxol.
EXAMPLES
The following nonlimiting examples provide specific synthesis
methods for preparing the water soluble taxol derivatives of the
present invention. All technical and scientific terms used herein
have the same meaning as commonly understood by one of ordinary
skill in the art. Other methods and materials similar or equivalent
to those described herein can be used in the practice or testing of
the present invention.
EXAMPLE 1
Triethylamine, 50 .mu.l and acrylic acid 30 .mu.l were dissolved in
5 ml dry THF in a 25 ml round-bottom flask under an argon gas
atmosphere. After cooling the solution to 0.degree. C. in an
icebath, 50 .mu.l of isobutylchloroformate were added, and the
reaction mixture was warmed to room temperature over a 15-minute
period. One hundred milligrams of taxol were added to the reaction
mixture, and the solution was stirred at 60.degree. C. for 15
hours, and monitored by TLC with dichloromethane/ethyl acetate
(2/1). Triethylamine hydrochloride precipitated during the
reaction, and was removed by filtration. The solvent was then
removed in vacuo, and the product was purified via flash
chromatography using silica gel and 1/1 dichloromethane/ethyl
acetate. This yielded 100 mg (94%) of 2'-acryloyltaxol:
##STR3##
The acryloyl moiety on the 2'-acryloyltaxol is a good Michael
acceptor due to the electrophilic .beta. alkene carbon atom which
is subject to nucleophilic attack. Thus reaction of
2'-acryloyltaxol with suitable nucleophiles will result in Michael
addition at the 2' position. An 85 mg quantity of the
2'-acryloyltaxol was dissolved in about 3 ml of distilled
isopropanol, and 84 mg of sodium meta-bisulfite were dissolved in
about 1 ml of distilled water. The two solutions were mixed
together, and the reaction mixture stirred at 60.degree. C. for
about 15 hours. TLC with 10/1 dichloromethane/methanol was used to
monitor the reaction. The solvents were then removed under vacuum,
and water was removed by azeotroping with acetonitrile. Flash
chromatography with 2/1 dichloromethane/isopropanol was used to
purify the product. A yield of 83.5 mg (83.5%) of
2'-[(3-sulfo-1-oxopropyl)oxy]taxol sodium salt resulted: ##STR4##
NMR, MS, UV, and IR (KBr) were performed on samples of the product,
and optical rotation, and melting point were determined, with the
characterization data and NMR data presented in Tables 1 and 2
below.
TABLE 1 ______________________________________ Characterization
Data For 2'-[(3-sulfo-1-oxopropyl)oxy]taxol sodium salt
______________________________________ m.p. 175-176.degree. C.
[.alpha.].sub.D.sup.20 -30.degree. (0.0012, MeOH) IR (KBr): 3500,
2950, 1760, 1730, 1660, 1380, 1250, 1190, 1100, 800 cm.sup.-1 UV
.lambda..sup.MeOH max : 279 nm (.epsilon. 579), 270 nm (.epsilon.
869), 228 nm (.epsilon. 15072) MS (FAB): 1034 (MNa.sup.+), 1012
(MH.sup.+) ______________________________________
TABLE 2 ______________________________________ NMR Data For
2'-[(3-sulfo-1-oxopropyl)oxy]taxol sodium salt .sup.1 H Shift (ppm
from TMS) .sup.13 C Shift Position Coupling (hertz) (ppm from TMS)
______________________________________ C-1 * C-2 6.2 (d,7) 75 C-3
3.82 (d,7) 45.8 C-4 80.5 C-5 5.0 (d,9) 84 C-6 2.48 m 35.2 C-7 4.35
m 76 C-8 57.9 C-9 203.8 C-10 6.45 s 70.8 C-11 131 C-12 141 C-13
6.09 (t,8) 75.4 C-14 2.48 m 35.8 C-15 43 C-16 1.15 s 25.9 C-17 1.17
s 19.8 C-18 1.95 s 13.8 C-19 1.67 s 9.4 C-20 4.21 s 70.8 C-1' 171
C-2' 5.45 (d,3) 74 C-3' 5.84 (d,7) 53.1 N--H 7.26 (t,9) CH.sub.3
(OAc) 2.2 s 21 CH.sub.3 (OAc) 2.4 s 21.9 Bz 7.4-8.1 m 126.8-138.1
CO(OAc) 168.4 CO(OAc) 169.9 CO(OBz) 166.2 CO(NBz) 168.2 C-1" 170.2
C-2" 2.93 (t 8) 29.2 C-3" 3.14 (t 8) 63.2
______________________________________ * under CHCl.sub.3
signal
Note that it is anticipated that the acrylic acid used may be
replaced with other members of the acrylic acid family which are
also good Michael acceptors, and that the salt-forming moiety may
be another alkaline metal, or an ammonio group, such as a
tetrabutylammonium group. It is also envisioned that the salt
forming moiety may be replaced with H. Biological testing of
2'-](3-sulfo-1-oxopropyl)oxy]taxol sodium salt demonstrated that
the compound is bioactive in addition to having improved water
solubility.
EXAMPLE 2
A 206 mg quantity of taxol was combined with 2.9 mg of
4-dimethylaminopyridine (DMAP) and 49 mg of succinic anhydride in a
25 ml flask equipped with a magnetic stirrer. A 2.0 ml quantity of
dry pyridine was added, and the solution was stirred at room
temperature for 2.5 hours. Several milliters of water were then
added to produce a white precipitate in an opaque suspension.
Several milliters of dichloromethane were then added to extract the
products. Addition of 1 ml of concentrated HCl caused the white
aqueous suspension to disappear. Sodium sulphate was used to dry
the dichloromethane layer, which was then filtered and evaporated.
TLC with 7/1 CH.sub.2 Cl.sub.2 MeOH indicated only a trace of
pyridine remaining. The remaining pyridine was removed by the
cyclical addition of heptane followed by evaporation; this yield
218 mg of succinyltaxol, representing a 96.6% yield. Proton NMR of
the product matched values given in the literature. The structure
was also confirmed using 2D-NMR HOMO COSY (homonuclear correlation
spectroscopy).
Taurine, H.sub.2 NCH.sub.2 CH.sub.2 SO.sub.3 H, is a highly polar
compound which is essentially insoluble in organic solvents such as
chloroform. Taurine derivatives of organic acids have been made in
the past by treating the acid chloride with taurine under
Schotten-Baumann conditions (i.e., in basic aqueous or
aqueous-ethanolic solution). This method was unacceptable for taxol
because it is readily hydrolyzed in base, and would thus decompose
under the reaction conditions. In order to overcome this problem, a
new method was developed which involved the addition of taurine to
tetrabutyl-ammonium hydroxide, followed by removal of unreacted
materials and evaporation. This yielded the tetrabutylammonium salt
of taurine instead of the sodium salt used in the prior art. The
tetrabutylammonium salt of taurine is soluble in organic solvents,
such as dichloromethane. Thus, 2'-succinyltaxol in THF and
triethylamine can be reacted with isobutylchloroformate and taurine
tetrabutylammonium salt to form the tetrabutylammonium salt of the
taxol taurine derivative. Note that the intermediate is a mixed
anhydride, which hydrolyzes back to the starting compound in the
presence of water.
A minimum volume of distilled water was used to dissolve 250 mg
taurine in a flask, and 1 ml of aqueous tetrabutylammonium
hydroxide was added to the solution. The solution was stirred at
room temperature for one hour, and then evaporated to dryness. The
dry product was dissolved in dry THF (about 15 ml), filtered, and
the filtrate was evaporated until dry. The dried product was then
redissolved in 2 ml of dried THF.
PREPARATION OF
2'-{[4-((2-SULFOETHYL)AMINO)-1,4-DIOXOBUTYL]OXY}TAXOL
TETRABUTYLAMMONIUM SALT
A solution of 2'-succinyltaxol, formed by dissolving 122 mg of
2'-succinyltaxol in about 4 ml of dried THF and 50 .mu.l of
triethylamine, was cooled to about 0.degree. C. The solution was
then combined with 50 .mu.l of isobutylchloroformate, the reaction
mixture was warmed to room temperature over a 15-minute period, and
0.5 ml of taurine tetrabutylammonium salt in THF solution
(equivalent to 91 mg of taurine tetrabutylammonium salt) were
added. Following the addition of the taurine tetrabutylammonium
salt, the reaction mixture was stirred at room temperature for 5
hours, and the reaction was monitored by TLC with 2/1 EtOAc/MeOH.
The reaction mixture was then filtered, and the solvents were
evaporated. Purification by flash chromatography using silica gel
(300.times.15 mm bed, 7/1 CH.sub.2 Cl.sub.2 /MeOH) yielded 168 mg
(100%) of 2'-{[4-((2-sulfoethyl)-amino)-1,4-dioxobutyl]oxy}taxol
tetrabutylammonium salt.
The tetrabutylammonium salt was converted to the sodium salt by
placing 160 mg of the tetrabutylammonium salt in a beaker with
Dowex 50 ion exchange resin in the Na.sup.+ form (about 3 ml of
resin in 3 ml of deionized water). After stirring the mixture at
room temperature for 1.5 hours, the mixture was then passed through
a small resin column which contained 2 ml of resin in the Na+ form,
using deionized water as the solvent.
The solution was azeotroped with acetonitrile to yield 122 mg
(91.7% of 2'-{[4-((2-sulfoethyl)amino)-1,4-dioxobutyl]oxy}taxol
sodium salt: ##STR5##
Characterization data are presented below in Table 3, and NMR
chemical shift data are presented in Table 4 below.
TABLE 3 ______________________________________ Characterization
Data For 2'-{[4-((2-Sulfoethyl)Amino)-1, 4-Dioxobutyl]Oxy}taxol
Sodium Salt ______________________________________ m.p.
174-175.degree. C. [.alpha.].sub.D.sup.20 -29.8.degree. (0.0055,
MeOH) IR (KBr): 3450, 3000, 1760, 1730, 1660, 1560, 1400, 1260,
1190, 1050 cm.sup.-1 UV .lambda..sup.MeOH max : 279 nm (.epsilon.
649), 271 nm (.epsilon. 8920), 228 nm (.epsilon. 12824) MS (FAB):
1105 (MNa.sup.+), 1083 (MH.sup.+)
______________________________________
TABLE 4 ______________________________________ NMR Data For
2'-((4-((2-Sulfoethyl)Amino)-1, 4-Dioxobutyl)Oxy)taxol Sodium Salt
.sup.1 H Shift (ppm from TMS) .sup.13 C Shift Position Coupling
(hertz) (ppm from TMS) ______________________________________ C-1
79 C-2 5.66 (d,7) 76.6 C-3 3.8 (d,7) 47.2 C-4 81.6 C-5 5.02 (d,9)
85.4 C-6 2.52 m 36 C-7 4.35 m 77.3 C-8 58.8 C-9 204.8 C-10 6.43 s
72.8 C-11 132.6 C-12 142.2 C-13 6.05 (t,8) 75.9 C-14 2.14 m 36.2
C-15 44.1 C-16 1.18 s 26.8 C-17 1.18 s 21 C-18 1.94 s 14.9 C-19
1.67 s 10.2 C-20 4.23 72 C-1' 173.4 C-2' 5.46 (d,7) 75.8 C-3' 5.8
(dd 7,7) 55 N--H 7.27 (t,7) CH.sub.3 (OAc) 2.2 s 22.2 CH.sub.3
(OAc) 2.4 s 23.3 Bz 7.4-8.1 m 126.8-138.1 CO(OAc) 170.2 CO(OAc)
170.2 CO(OBz) 167.2 CO(NBz) 171.2 C-1" 173.1 C-2" 2.72 m 30 C-3"
2.52 m 30 C-4" 173.1 C-1'" 3.58 m 47 C-2'" 2.96 m 51 N--H 3.58
(t,7) ______________________________________
Note that the tetrabutylammonium salt of taurine may easily be
reacted with other 2'-O-acyl acid taxols, such as 2'-glutaryltaxol.
2'-glutaryltaxol can be formed easily by substituting glutaric
anhydride for succinic anhydride. It is believed that other members
of the oxalic acid series and other anhydrides may react with taxol
more or less equivalently to the compounds specifically disclosed.
Note that, in some instances, 2'-glutaryltaxol may be preferred to
the use of other 2'-O-acyl acid taxols. Further, it is contemplated
that the salt forming moiety may be replaced with H or another
alkaline or alkaline earth metal.
EXAMPLE 3
A solution of 280 mg 3-amino-1-sulfopropionic acid in distilled
water was formed, and 1 ml tetrabutylammonium hydroxide was added.
The solution was stirred at 60.degree. C. for one hour, and then
evaporated to dryness. The products were dissolved in about 15 ml
THF and excess 3-amino-1-sulfopropionic acid was removed by
filtration. The filtrate was evaporated, and redissolved in 2 ml
dried THF for subsequent reaction. A solution of 130 mg
2'-succinyltaxol and 50 .mu.l of triethylamine in 4 ml of dry THF
was formed, and the solution was cooled down to 0.degree. C. A 50
.mu.l aliquot of isobutylchloroformate was added to the reaction
mixture, and the solution was warmed to room temperature in about
15 minutes. This was followed by the addition of 0.6 ml of
3-amino-1-sulfopropionic acid tetrabutylammonium salt in THF
solution (equivalent to 108 mg of 3-amino-1-sulfopropionic acid
tetrabutylammonium salt). The reaction mixture was stirred at room
temperature for three hours, and reaction progress was monitored by
TLC with 4/1 ethyl acetate/methanol. The reaction solution was then
filtered and evaporated, with the product being purified by flash
chromatography using silica gel (300 mm.times.15 mm bed with a 10/1
dichloromethane/methanol eluent). A yield of 128 mg (71.2%) of the
homogenous tetrabutylammonium salt of taxol resulted.
The tetrabutylammonium salt was converted to the sodium salt by
placing 120 mg of
2'-{[4-((3-sulfopropyl)amino)-1,4-dioxobutyl]oxy}taxol
tetrabutylammonium salt in a beaker with Dowex 50 ion exchange
resin in the Na.sup.+ form (approximately 3 ml of resin per 3 ml
deionized water). The mixture was stirred at room temperature for
about 1.5 hours, and then passed through a resin column which
contained 2 ml of resin in the Na.sup.+ form, and using deionized
water as a solvent. The solution was azeotroped with acetonitrile
and yielded 84 mg (79.3%) of
2'-{[4-((3-sulfopropyl)-amino)-1,4-dioxobutyl]oxy}taxolsodium salt:
##STR6##
Characterization data for this compound is presented in Table 5,
and NMR chemical shift data is presented in Table 6
TABLE 5 ______________________________________ Characterization
Data For 2'-{[4-((3-Sulfopropyl)Amino)-1, 4-Dioxobutyl]oxy}taxol
Sodium Salt ______________________________________ m.p.
168-169.degree. C. [.alpha.].sub.D.sup.20 -29.degree. (0.001, MeOH)
IR (KBr): 3480, 3000, 1760, 1740, 1660, 1550, 1400, 1260, 1050
cm.sup.-1 UV .lambda..sup.MeOH max : 279 nm (.epsilon. 974), 271 nm
(.epsilon. 1240), 228 nm (.epsilon. 12719) MS (FAB): 1119
(MNa.sup.+), 1097 (MH.sup.+)
______________________________________
TABLE 6 ______________________________________ NMR Data For
2'-{[4-((3-Sulfopropyl)Amino)-1, 4-Dioxobutyl]oxy}taxol Sodium Salt
.sup.1 H Shift (ppm from TMS) .sup.13 C Shift Position Coupling
(hertz) (ppm from TMS) ______________________________________ C-1 *
C-2 5.63 (d,7) 74.8 C-3 3.8 (d,7) 46 C-4 80.8 C-5 4.99 (d,9) 84.2
C-6 2.5 m 34.7 C-7 4.34 m 75.9 C-8 57.5 C-9 204.2 C-10 6.44 s 71.3
C-11 131.6 C-12 141.2 C-13 6.05 (t,8) 75.2 C-14 2.14 m 35.7 C-15
42.8 C-16 1.16 s 25.6 C-17 1.16 s 19.4 C-18 1.93 s 13.6 C-19 1.67 s
8.9 C-20 4.21 s 70.8 C-1' 172 C-2' 5.44 (d,7) 74.2 C-3' 5.79 (dd
7,7) 53.6 N--H 7.25 (t,7) CH.sub.3 (OAc) 2.2 s 20.9 CH.sub.3 (OAc)
2.4 s 21.6 Bz 7.4-8.1 m 126.8-138.1 CO(OAc) 169 CO(OAc) 170.2
CO(OBz) 166.4 CO(NBz) 170.2 C-1" 171.9 C-2" 2.75 (t,7) 29 C-3" 2.54
(t,7) 29.8 C-4" 171.9 C-1'" 3.25 m 37.9 C-2' " 1.98 m 28.3 C-3'"
2.85 (t,7) ** ______________________________________ * under
CHCl.sub.3 signal ** under MeOH signal
Note, it is envisioned that sodium can be replaced with H or any
other salt forming moiety such as other alkaline or alkaline earth
metals, and ammonio groups.
EXAMPLE 4
A solution of 26 mg 2'-succinyltaxol and 20 .mu.l of triethylamine
in 2 ml of dried THF was prepared under argon gas atmosphere, and
the solution was cooled to 1.degree. C. A 10 .mu.l aliquot of
isobutylchloroformate was added to the solution, and the reaction
mixture was warmed to room temperature in about 15 minutes.
Following the warming step, 5 .mu.l ethylene glycol were added, and
the reaction mixture was stirred at room temperature for 15 hours,
with the reaction progress monitored by TLC with 1:1
dichloromethane/ethyl acetate. The reaction was stopped by
filtering the precipitate, and evaporating the solvent. Crude
products were purified by preparative TLC (1:3
dichloromethane/ethyl acetate), yielding 25 mg (83.3%) of
2'-{[4-((2-hydroxyethyl)oxy)-1,4-dioxobutyl]oxy}taxol: ##STR7##
Characterization data are presented in Table 7 and NMR chemical
shift data are presented in Table 8 below.
TABLE 7 ______________________________________ Characterization
Data For 2'-{[4-((2-hydroxyethyl)oxy)-1,4-dioxobutyl]oxy}taxol
______________________________________ m.p. 164-165.degree. C.
[.alpha.].sub.D.sup.20 -32.5.degree. (0.002, MeOH) IR (KBr): 3500,
2950, 1760, 1740, 1660, 1390, 1260, 1160, 1080, 1040 cm.sup.-1 UV
.lambda..sup.MeOH max : 279 nm (.epsilon. 609), 272 nm (.epsilon.
831), 228 nm (.epsilon. 14404) MS (FAB): 1020 (MNa.sup.+), 998
(MH.sup.+) ______________________________________
TABLE 8 ______________________________________ NMR Data For
2'-{[4-((2-hydroxyethyl)oxy)-1,4-dioxobutyl]oxy}taxol .sup.1 H
Shift (ppm from TMS) .sup.13 C Shift Position Coupling (hertz) (ppm
from TMS) ______________________________________ C-1 79.1 C-2 5.7
(d,7) 75.8 C-3 3.8 (d,7) 45.8 C-4 81 C-5 4.95 (d,9) 84.3 C-6 2.56 m
35.6 C-7 4.43 m 75.8 C-8 58.2 C-9 204 C-10 6.29 s 72.1 C-11 132
C-12 142.3 C-13 6.23 (t,8) 75.8 C-14 2.42 m 35.6 C-15 43.2 C-16
1.23 s 26.8 C-17 1.15 s 20.5 C-18 1.94 s 14.3 C-19 1.70 s 9.8 C-20
4.19 (d,8) 72.1 C-1' 172.2 C-2' 5.48 (d,3) 74.3 C-3' 5.97 (dd 3,9)
52.9 N--H 7.14 (d,9) CH.sub.3 (OAc) 2.25 s 22.1 CH.sub.3 (OAc) 2.45
s 22.8 Bz 7.4-8.1 m 126.8-138.1 CO(OAc) 168 CO(OAc) 169.9 CO(OBz)
167 CO(NBz) 167.3 C-1" 171 C-2" 2.65 m 29 C-3" 2.78 m 29 C-4" 171
C-1'" 3.7 (t,7) 66.2 C-2'" 4.1 m 61
______________________________________
EXAMPLE 5
To a 10 ml flask, 20 mg taxol, 40 mg of dicyclohexyl-carbodiimide,
and 20 mg of N-carbobenzyl-.gamma.-aminobutyric acid were added.
The reactants were dissolved in 4 ml of dry acetonitrile (dry
acetonitrile was obtained by passing acetonitrile through activated
alumina). After stirring the reaction mixture at room temperature
for 30 hours, the solution was filtered to remove precipitated
dicyclohexylurea. The solvent was then removed under vacuum, and
the crude products were separated by preparative TLC with 45:55
hexane/ethyl acetate. This yielded 19.1 mg (75.9%) of pure
2'-N-CBZ-.gamma.-aminobutyryltaxol.
2.degree.-.gamma.-aminobutyryltaxol formate was synthesized by the
addition of 6 mg of 2'-N-CBZ-.gamma.-aminobutyryltaxol to 1.5 ml of
methanol. Upon the dissolution of the CBZ-taxol derivative, 1 ml of
formic acid was added to form a 40% formic acid/methanol solution.
The reaction was carried out by adding 5 mg of 5% of Pd/C to the
solution, and stirring it at room temperature for 26 hours. The
reaction was stopped by filtering off the Pd/C, and drying the
filtrate under vacuum. This yielded 2'-.gamma.-aminobutyryltaxol
formate: ##STR8##
After a few hours, proton NMR and TLC with 2:1:0.02
dichloromethane/ethylacetate/methanol showed that the
2'-.gamma.-aminobutyryltaxol formate had decomposed back to
taxol.
EXAMPLE 6
Taxol's water solubility was determined by dissolving 1.6 mg of
taxol in 10 ml of distilled water saturated with 1-octanol in a 60
ml separatory funnel, and 10 ml of 1-octanol saturated with
distilled water was then added. The funnel was shaken, and allowed
to stand for about 30 minutes until the organic and aqueous phases
separated. UV absorption measurements at 228 nm were made of the
aqueous layer and/or octanol layer, with the octanol layer being
diluted 5 times before measurement.
Following the same procedure as above, 0.8 mg of
2'-[(3-sulfo-1-oxopropyl)oxy]taxol sodium salt, 0.8 mg of
2'-{[4-((2-sulfoethyl)amino)-1,4-dioxybutyl]oxy}taxol sodium salt
and 0.7 mg of 2'-{[40((3-sulfopropyl)amino)-1,4-dioxybutyl]oxy}
taxol sodium salt had their water solubilities determined relative
to taxol; the results are presented below in Table 9.
TABLE 9 ______________________________________ TAXOL DERIVATIVE
WATER SOLUBILITIES RELATIVE TO TAXOL Compound Relative Solubility
______________________________________ Taxol 1
2'-[(3-sulfo-1-oxopropyl)oxy]taxol 210 sodium salt
2'-{[4-((2-sulfoethyl)amino)-1,4-di- 191 oxybutyl]oxy}taxol sodium
salt 2'-{[4-((3-sulfopropyl)amino)-1,4-di- 118 oxybutyl]oxy}taxol
sodium salt ______________________________________
Table 9 indicates that the 2'-acryloyltaxol derivative had the
highest water solubility, and is 210 times more water soluble than
taxol. Note that the taurine 2'-succinyltaxol derivative has a much
greater water solubility than the 3-amino-1-sulfopropionic acid
derivative of 2'-succinyltaxol; however, both compounds have
solubilities more than 100 times greater than taxol. The decreased
solubility for the 3-amino-1-sulfopropionic acid derivative of
2'-succinyltaxol is probably due to the increased alkyl chain
length.
Thus, the present invention discloses new taxol derivatives with
increased water solubility in comparison to underivatized taxol,
and which are stable for longer periods of time than certain
previous derivatives of taxol which had increased water
solubilities. These compounds are produced by new processes that
result in high yields of essentially pure compounds.
Characterization data and NMR studies confirm the structure and
properties of the taxol derivatives of the present invention. In
addition to having high water solubilities and improved stability,
these compounds retain their bioactivity and usefulness as
antineoplastic, anti-leukemic and anti-cancer prodrugs.
Contemplated equivalents of the water soluble taxol derivatives of
the present invention include 2'-acryloyl and 2'-O-acyl acid
derivatives of taxol which have one or more side chain or ring
substituents substituted with a non-interfering group (e.g., a
substituted group which does not seriously alter the desirable
properties of the taxol derivatives of the present invention), such
as but not limited to, substitution of --H, --OH, --OR, --NR, --Ar,
(aryls) or .dbd.O for another non-interfering moiety.
From the above teachings, it is apparent that many modifications
and variations of the present invention are possible. It is
therefore to be understood that the invention may be practiced
otherwise than as specifically described.
* * * * *