U.S. patent application number 09/115016 was filed with the patent office on 2002-03-28 for hydroxylation activated drug release.
Invention is credited to BURKE, MICHAEL DANNY, PATTERSON, LAWRENCE HYLTON, POTTER, GERALD ANDREW.
Application Number | 20020037296 09/115016 |
Document ID | / |
Family ID | 10826848 |
Filed Date | 2002-03-28 |
United States Patent
Application |
20020037296 |
Kind Code |
A1 |
POTTER, GERALD ANDREW ; et
al. |
March 28, 2002 |
HYDROXYLATION ACTIVATED DRUG RELEASE
Abstract
The present invention concerns prodrugs whose aromatic
oxidation, particularly their enzymatic aromatic hydroxylation,
results in their activation by the release of a drug moiety. It
particularly concerns anti-tumour prodrugs and those which are
specifically activated by the hydroxylation activity of the P-450
enzyme CYP1B1.
Inventors: |
POTTER, GERALD ANDREW;
(LEICESTER, GB) ; PATTERSON, LAWRENCE HYLTON;
(LEICESTER, GB) ; BURKE, MICHAEL DANNY;
(LEICESTER, GB) |
Correspondence
Address: |
BAKER & BOTTS
30 ROCKEFELLER PLAZA
NEW YORK
NY
101120228
|
Family ID: |
10826848 |
Appl. No.: |
09/115016 |
Filed: |
July 14, 1998 |
Current U.S.
Class: |
424/400 |
Current CPC
Class: |
A61K 47/554 20170801;
A61K 47/54 20170801; A61K 47/555 20170801; A61P 35/00 20180101;
A61P 43/00 20180101 |
Class at
Publication: |
424/400 |
International
Class: |
A61K 009/00 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 12, 1998 |
GB |
9802597.2 |
Claims
1. A prodrug comprising a drug moiety bound to a carrier framework,
the prodrug being activated by aromatic oxidation of the carrier
framework to release the drug moiety.
2. A prodrug according to claim 1, being activated by aromatic
hydroxylation.
3. A prodrug according to claim 2, being activated by enzymatic
aromatic hydroxylation.
4. A prodrug according to claim 1, and having the formula (Z):
5wherein: X=H, OH, OMe or N(CH.sub.3).sub.2; and n=0-6; and:
R.sub.1=H, C.sub.1-4 lower alkyl, or together with R.sub.2 forms
part of a cycloalkyl group which may be further substituted to form
part of a polycyclic cycloalkyl group, or with R.sub.2 forms part
of a steroidal carbon framework; R.sub.2=H, C.sub.1-4 lower alkyl,
or together with R.sub.1 and/or R.sub.3 forms part of a cycloalkyl,
polycyclic cycloalkyl or steroidal carbon framework, or forms part
of a polycyclic aromatic group by linkage to R.sub.4; R.sub.3=H,
C.sub.1-4 lower alkyl or together wit R.sub.2 forms part of a
cycloalkyl, polycyclic cycloalkyl or steroidal carbon framework;
and R.sub.4=H or is fused directly to the aromatic position
designated by R.sub.2 and either: the drug moiety is derived from a
drug having a free amino, hydroxyl or thiol group and which links
it to the rest of the prodrug, such tat A represents NH, NR
(R=C.sub.1-4 lower alkyl), O or S; or the drug moiety is derived
from a drug having a carboxylate group, an ester linkage joining it
to the rest of the prodrug and A being absent
5. A prodrug according to claim 1, being an anti-tumour
prodrug.
6. A prodrug according to claim 1, the drug moiety being a
cytotoxic or cytostatic agent.
7. A prodrug according to claim 6, a cytotoxic drug moiety being
selected from the group of colchicine, esperimycin, taxol,
daunomycin, staurosporin, and nitrogen mustard.
8. A prodrug according to claim 1, being activated by hydroxylation
by CYP1B1.
9. A prodrug according to claim 1, the drug moiety being an
antimitotic agent, an alkylating agent, an antifolate, a DNA
damaging agent or an enzyme inhibitor.
10. A prodrug according to claim 4, the olefin linkage 6having a
cis- or trans-geometry.
11. A prodrug according to claim 4, the olefin linkage 7being
acyclic or cyclic.
12. A prodrug according to claim 4, the olefin linkage 8forming
part of an aromatic or polycyclic aromatic system.
13. A prodrug according to claim 1, the linkage to the drug moiety
from the carrier framework being from a hydroxyalkyl group in the
prodrug via a carbamate, carbonate or thiocarbonate linker to an
amino, hydroxy or thiol group in the drug moiety.
14. A prodrug according to claim 1, having a steroid carbon carrier
framework.
15. A prodrug according to claim 1, being derived from
estradiol.
16. A prodrug according to claim 4, having the formula of any one
of formulae (I)-(IX): 9wherein --OR=--OMe or --OH.
17. A prodrug according to claim 4 having the formula of any one of
Formulae (X)-(XV): 10
18. A prodrug according to any one of the preceding claims, its
aromatic oxidation being by hydroxylation and causing the release
of the drug moiety and carbon dioxide.
19. A prodrug according to claim 1 for use in a method of treatment
or diagnosis of the human or animal body.
20. The use of a prodrug according to claim 1 in the manufacture of
a medicament.
21. A method of manufacture of a medicament, comprising the use of
a prodrug according to claim 1.
22. A method of treatment of a patient, comprising administering to
the patient a prodrug according to claim 1.
Description
[0001] The present invention concerns prodrugs whose aromatic
oxidation, particularly their enzymatic aromatic hydroxylation,
results in their activation by the release of a drug moiety. It
particularly concerns anti-tumour prodrugs and those which are
specifically activated by the hydroxylation activity of the P-450
enzyme CYP1B1.
[0002] Many conventional cytotoxic drags are known (for example
colchicine, esperimycin, taxol, daunomycin and staurosporin) which
can be used for chemotherapeutic purposes. However, they typically
suffer from the problem that they are generally cytotoxic and
therefore may affect cells other than those which it is wished to
target. This can be alleviated somewhat by using targetted drug
delivery systems, for example direct injection to a site of
tumourous tissue, or by e.g. binding the cytotoxic agent to
antibody which specifically recognises an antigen displayed by
cancerous cells. Alternatively, electromagnetic radiation may be
used to cause chemical changes in an agent at a desired site in the
body such that it becomes cytotoxic. However, all of these
techniques have, to a greater or lesser extent, certain limitations
and disadvantages.
[0003] It has been reported (Murray, G. I. et al., 15 Jul. 1997,
Cancer Research, 57: 3026-3031) that the enzyme CYP1B1, a member of
the cytochrome P450 family of xenobiotic metabolizing enzymes, is
expressed at a high frequency in a range of human cancers including
cancers of the breast, colon, lung, oesophagas, skin, lymph node,
brain and testis, and that it is not detectable in normal tissues.
This led to the conclusion (p. 3030, final sentence) that ". . .
the expression of CYP1B1 in tumour cells provides a molecular
target for the development of new anticancer drugs that could be
selectively activated by the presence of CYP1B1 in tumour cells".
It was also reported (p.3030, column 1 lines 15-17) that CYP1B1 is
capable of 4-hydroxylation of estradiol. No specific anticancer
drugs were suggested.
[0004] The present inventors have now succeeded in creating a range
of prodrugs having a "carrier" framework with a drug moiety
conjugated to it (the prodrug other than the drug moiety is
referred to below as "the rest of the prodrug") which have little
or no cytotoxic effect when in their normal state, but whose
aromatic oxidation e.g. hydroxylation (for example by CYP1B1)
results in the release of the drug moiety. With CYP1B1 as a
hydroxylating enzyme, this provides for a self-targetting drug
delivery system in which a non-cytotoxic (or at least negligibly
cytotoxic) compound can be administered to a patient, for example
in a systemic manner, the compound then being hydroxylated at the
site of tumour cells (intratumoural hydroxylation) to release the
drug which acts to kill or otherwise affect the tumour cells. The
fact that CYP1B1 is not expressed by normal cells means that the
hydroxylation of the prodrug only occurs at the site of tumour
cells and therefore only tumour cells are affected, thus providing
a self-targetting drug delivery system.
[0005] The prodrugs of the present invention have the distinct
advantage of being useful in the treatment of tumours at any site
in the body, meaning that even tumours which have undergone
metastasis (which are not normally susceptible to site-specific
therapies) may be treated, as well of course as primary and
secondary tumours.
[0006] The prodrugs may be designed to be activated by other
oxidising agents, for example other enzymes (e.g. other members of
the cytochrome P-450 family of enzymes) which cause hydroxylation
of the prodrug.
[0007] CYP1B1 has not yet been fully characterised, and it is
therefore possible that tumour-specific isoforms of it may exist
which possess the same catalytic properties. The prodrugs of the
present invention may, of course, be used with such enzymes.
[0008] In the case of cytochrome P-450 activated prodrugs, the
therapeutic strategy achieved using them is referred to as SPEAR
(Specific P-450 Enzyme Activated drug Release).
[0009] According to the present invention there is provided a
prodrug comprising a drug moiety bound to a carrier framework, the
prodrug being activated by aromatic oxidation of the carrier
framework to release the drug moiety.
[0010] The prodrug may be activated by aromatic hydroxylation. It
may be activated by enzymatic aromatic hydroxylation.
[0011] Other enzymatically-activated prodrugs are known, for
example those which release a drug moiety as the result of cleavage
by a peptidase enzyme. However, nowhere has it been previously
suggested that a prodrug could be activated to release a drug
moiety by enzymatic hydroxylation.
[0012] A prodrug according to the present invention may have the
formula (Z): 1
[0013] wherein:
[0014] X=H, OH, OMe or N(CH.sub.3).sub.2; and
[0015] n=0-6;
[0016] and:
[0017] R.sub.1=H, C.sub.1-4 lower alkyl, or together with R.sub.2
forms part of a cycloalkyl group which may be further substituted
to form part of a polycyclic cycloalkyl group, or with R.sub.2
forms part of a steroidal carbon framework;
[0018] R.sub.2=H, C.sub.1-4 lower alkyl, or together with R.sub.1
and/or R.sub.3 forms part of a cycloalkyl, polycyclic cycloalkyl or
steroidal carbon framework, or forms part of a polycyclic aromatic
group by linkage to R.sub.4;
[0019] R.sub.3=H, C.sub.1-4 lower alkyl or together with R.sub.2
forms part of a cycloalkyl, polycyclic cycloalkyl or steroidal
carbon rework; and
[0020] R.sub.4=H or is fused directly to the aromatic position
designated by R.sub.2 and either:
[0021] the drug moiety is derived from a drug having a free amino,
hydroxyl or thiol group and which links it to the rest of the
prodrug, such that A represents NH, NR (R=C.sub.1-4 lower alkyl), O
or S; or
[0022] the drug moiety is derived from a drug having a carboxylate
group, an ester linkage joining it to the rest of the prodrug and A
being absent.
[0023] Enzymatic hydroxylation of the prodrugs of formula (Z)
results in the transfer of electrons from the site of hydroxylation
(for example the aromatic 4 position--see FIG. 1) to the drug
moiety, resulting in its release.
[0024] The prodrug may, for example, be an anti-tumour prodrug. The
drug moiety may be cytotoxic or cytostatic, although of course it
may be a moiety which has any other desired effect. Examples of
classes of drug moiety include antimitotic agents, alkylating
agents, antifolates, DNA-damaging agents and enzyme inhibitors.
Specific examples of possible cytotoxic drug moieties include
colchicine, esperimycin, taxol, daunomycin, staurosporin, and
nitrogen mustard. Alternatively, the drug moiety could be e.g. a
fluorescent organic molecule which would be released in an
intratumoural manner, aiding tumour detection by correlating
specific cell fluorescence with the presence of the drug moiety and
thus of the oxidising agent (e.g. CYP1B1) which caused its
release.
[0025] Thus the term "drug" also extends to moieties which may be
used for diagnostic purposes.
[0026] A possible nitrogen mustard is, for example, a para-hydroxy
aniline mustard that is linked through the para-hydroxy group to
the rest of the prodrug. In the case of nitrogen mustard prodrugs,
the mustard function is itself activated only when the drag moiety
is released from the prodrug.
[0027] The olefin linkage 2
[0028] may have a cis- or trans-geometry. It may be acyclic or
cyclic. It may form part of an aromatic or polycyclic aromatic
system.
[0029] The prodrug may be activated by CYP1B1. Thus a prodrug which
releases a cytotoxic drug moiety upon hydroxylation by CYP1B1 may
be used as a self-targetting anti-tumour drug, being activated at
the site of a tumour by CYP1B1and having no (or negligible)
cytotoxicity in the rest of the body.
[0030] The linkage to the drug moiety from the carrier framework
may be from a hydroxyalkyl group in the prodrug via a carbamate,
carbonate or thiocarbonate linker to an amino, hydroxy or thiol
group in the drug moiety.
[0031] Using the strategy and prodrugs of the present invention, it
is possible to link any desired drug moiety through a free amino,
hydroxy or thiol group. The provision of a linker group comprising
a carbamate, carbonate or thiocarbonate linker joining the drug
moiety to the rest of the prodrug results in the release of carbon
dioxide upon release of the drug moiety, making the reaction
irreversible. Thus the hydroxylation (or other aromatic oxidation)
of the prodrug may cause the release of the drug moiety and carbon
dioxide.
[0032] A prodrug may have a steroid carbon carrier framework. For
example, it may be derived from estradiol.
[0033] An example of a prodrug according to the present invention
is the prodrug having the formula I, shown in FIG. 1. It is an
estradiol derivative and incorporates the drug moiety at the
steroid 6-position. In this position, the 3-hydroxy group of
estradiol does not provide the requisite electron release, but upon
4-hydroxylation the electron release from the 4-hydroxy group
triggers electron transfer within the prodrug, resulting in the
release of the drug moiety.
[0034] A prodrug according to the present invention may, for
example, have the formula of any one of formulae (I)-(IX): 3
[0035] wherein --OR=--OMe or --OH
[0036] The prodrug may have the formula of any one of formulae
(X)-(XV): 4
[0037] Formula (X) is a colchicine-estradiol prodrug; (XI) is a
combretastatin-estradiol prodrug; (XII) is a mustard-estradiol
prodrug; (XIII) is a fluorophore-estradiol conjugate; (XIV) is a
colchicine-naphthyl prodrug; and (XV) is a colchicine-benzyl
prodrug.
[0038] Also provided according to the present invention is a
prodrug according to the present invention for use in a method of
treatment or diagnosis of the human or animal body, particularly
the treatment or diagnosis of tumours.
[0039] Also provided according to the present invention is the use
of a prodrug according to the present invention in the manufacture
of a medicament, e.g. for the treatment of tumours.
[0040] Also provided according to the present invention is a method
of manufacture of a medicament, comprising the use of a prodrug
according to the present invention.
[0041] Also provided according to the present invention is a method
of treatment of a patient, comprising administering to the patient
a prodrug according to the present invention. The prodrug may be
administered to treat a medical condition e.g. an illness.
[0042] Methods of manufacture of medicaments are well known. For
example a medicament may additionally comprise a pharmaceutically
acceptable carrier, diluent or excipient (Remington's
Pharmaceutical Sciences and US Pharmacopeia, 1984, Mack Publishing
Company, Easton, Pa., USA)
[0043] The exact dose (i.e. a pharmaceutically acceptable dose) of
prodrug to be administered to a patient may be readily determined
by one skilled in the art, for example by the use of simple
dose-response experiments.
[0044] Since prodrugs of the present invention may be specific to
e.g. tumour cells, they may not only be used to treat tumours, but
may also be used to determine whether or not a patient (or a sample
taken from a patient) has tumour cells. For example, tumour cells
may be detected by using a SPEAR prodrug that is a fluorophore
conjugate which releases a fluorescent compound upon enzymatic
hydroxylation. An example of this type of fluorophore conjugate is
given by compound (XIII) below. Cell numbers in a sample may be
assayed, as may the presence and quantity of the oxidised e.g.
hydroxylated prodrug, thus providing for the diagnosis of the
presence of tumour cells.
[0045] The invention will be flier apparent from the following
description, with reference to the several figures of the
accompanying drawings, which show, by way of example only, forms of
prodrug.
[0046] Of the figures:
[0047] FIG. 1 shows the estradiol-derived prodrug having the
formula (I), together with its 4-hydroxylation; and
[0048] FIG. 2 shows the synthesis of an estradiol-colchicine
prodrug. R is designated as representing H or a protecting group,
for example an acetate group (COCH.sub.3) or a benzyl group
(CH.sub.2C.sub.6H.sub.5).
[0049] The synthesis of the estradiol-colchicine prodrug I is shown
in FIG. 2. The synthetic route uses estradiol as a starting
material. The 6-oxo group is introduced by oxidation of estradiol
with pyridinium chlorochromate to give 6-oxo estradiol. This is
then subjected to borohydride reduction to produce 6-hydroxy
estradiol. The desired cytotoxic agent is then coupled to the
6-hydroxy estradiol using triphosgene as coupling agent (Eckert and
Foster, 1987, Angew. Chem. Int. Ed Engl., 26: 894-895) to provide
the carbamate linked estradiol prodrug. In the synthesis of the
prodrug, the R group is initially a protecting group (for example
an acetate group). Once the final step (above) has been taken, the
protecting groups are substituted with hydrogen to give the final
prodrug product. The chemistry of protecting groups and their
substitution is well known and will be readily apparent to one
skilled in the art.
[0050] 4-hydroxylation of the prodrug (FIG. 1) results in electron
transfer from the 4-hydroxy group, causing release of the drug
moiety and carbon dioxide. The release of carbon dioxide makes the
reaction irreversible.
[0051] The invention is exemplified by the specific SPEAR prodrugs
given in the formulae (X) to (XV). Compound (X) is a carbamate
linked colchicine-estradiol prodrug, which releases the cytotoxic
agent des-acetyl colchicine upon enzymatic hydroxylation by CYP1B1.
Compound (XI) is a carbonate linked combretastatin-estradiol
prodrug. Compound (XII) is a SPEAR prodrug of a nitrogen mustard,
which generates the highly cytotoxic alkylating agent,
bis(chloroethyl)amine mustard, upon enzymatic hydroxylation.
Compound (XIII) is a SPEAR fluorophore conjugate which releases the
fluorescent compound 7-amino4-methylcoumarin upon enzymatic
hydroxylation. Compound (XIV) is an example of a non-steroidal
SPEAR prodrug of colchicine linked to
6-methoxy-1-naphthalenemethanol. Compound (XV) is an example of a
non-steroidal SPEAR prodrug derived from 3-methoxybenzyl
alcohol.
[0052] Prodrug Metabolism Studies
[0053] A microsomal preparation of resected human tumour tissue
expressing the cytochrome P-450 CYP1B1 enzyme was prepared
essentially as described by the method of Barrie et al. (1989, J.
Steroid Biochem., 6: 1191-1195). The prodrug metabolism experiment
was carried out under yellow light, at 37.degree. C.
[0054] An array of 1.5 ml centrifuge tubes were set up in a water
bath shaker under aerobic conditions. To each tube was added 500
.mu.l of pH 7.6 buffer (0.1 M NaK.sub.2PO.sub.4), followed by an
aqueous solution of NADPH (5 .mu.l of a 25 mM stock solution). The
microsomal preparation (80 .mu.l) was then added and the tubes
preincubated for 5 minutes at 37.degree. C. The prodrug substrate
was then added (10 .mu.l of a 5 mM stock solution) and incubated
for 1 hour at 37.degree. C. After 1 hour the tubes were transferred
to an ice/water cooling bath (0.degree. C.). The tubes were then
centrifuged at 15,000 rpm for 30 minutes. A sample of the
supernatant (100 .mu.l) was then taken and analysed by HPLC, using
the following BPLC conditions: Spherisorb C18 (25 cm.times.4.6 mm
id), used without guard column. Flow rate 1 ml/min. Eluent 75% 0.1
M. KH2PO4 and 25% acetonitrile. Metabolism of the prodrugs in this
way was found to result in release of the free drug moiety. For
example, tumour microsomal metabolism of the colchicine-estradiol
prodrug compound (X) liberated free N-desacetyl colchicine, which
was detected by HPLC analysis.
[0055] Prodrug Synthesis
[0056] Estradiol 3,17-dipivaloate
[0057] Pivaloyl chloride (664 mg; 5.5 mmol) was added dropwise to a
solution of estradiol (250 mg; 0.9 mmol) in 1:1
pyridine/dichloromethane (3 ml) at 0.degree. C. After 15 hours the
reaction was quenched with water (10 ml) and the product was
extracted with ether (3.times.10 ml). The combined organic layers
were washed sequentially with 10% HCl (15 ml), saturated aqueous
copper sulfate (15 ml) and brine (15 ml), dried over MgSO.sub.4 and
finally concentrated in vacuo. The residue was recrystallised from
hot ethanol and isolated as a white crystalline solid (247 mg;
61%). IR(cm.sup.-1, KBr): (3000, CH) (1700, COO) (1500, ArC.dbd.C)
(1300, CH3); 1H-NM (250 MHz, CDCl3): (H (0.9, s; 3H) (1.2, s; 9H)
(1.3, s; 9H) (1.4, m; 6H) (1.8, m; 3H) (2.3, m; 3H) (2.9, t; 2H)
(4.7, t; 1H) (6.8, m; 2H) (7.1, d; 1H); 13C-NMR (250 MHz, CDCl3):
(c 12.0, 23.3, 26.1, 27.0, 27.1, 27.2, 27.6, 29.5, 36.9, 38.2,
38.8, 39.9, 43.1, 43.9, 49.8, 82.2, 118.4, 121.3, 126.3, 137.5,
138.0, 148.9, 157.8, 177.3, 178.5; Mass Spectrum (M+1) m/e=441.
[0058] 6-Oxoestradiol 3,17-dipivaloate
[0059] 3, 5-Dimethylpyrazole (545 mg; 5.7 mmol) was added to a
suspension of chromium trioxide (576 mg; 5.7 mmol) at -20.degree.
C. in dichloromethane (2 ml). After stirring for 15 minutes,
estradiol 3,17-dipivaloate (250 mg; 0.57 mmol) in dichloromethane
(1 ml) was added dropwise and the reaction mixture was stirred at
-15.degree. C. for 4 hours. The reaction was quenched with water
(15 ml) and the aqueous layer was extracted with ether (3.times.15
ml). The combined organic layers were washed with water (15 ml) and
brine (15 ml), dried over MgSO.sub.4 and concentrated in vacuo. The
residue was chromatographed on silica gel and the product was
isolated as a white solid (55 mg; 21%). IR (cm.sup.-1, KBr): (3000,
CH) (1700, COO) (1650, ArCO) (1500, ArC.dbd.C) (1300, CH3); 1H NMR
(CDCl3): (H(0.9, s; 3H) (1.2, s; 9H) (1.39, d; 9H) (1.41, m; ,6H)
(1.9, m; 2H) (2.2, m; 2H) (2.3, m; 1H) (2.4, m 1H) (2.7, d; 1in)
(4.7, t; 1H) (7.2, d; 1H) (7.4, d; 1H) (7.6, d; 1H); 13C NMR
(CDCl3): (c 11.9, 22.9, 25.3, 27.1, 27.2, 27.4, 36.5, 38.9, 39.1,
39.5, 42.9, 43.0, 43.8, 49.8, 81.7, 119.9, 126.6, 126.9, 133.6,
144.0, 149.8, 178.4, 196.9; Mass Spectrum (M+1) m/e=455.
[0060] 6-Hydroxyestradiol 3,17-dipivaloate
[0061] Sodium borohydride (104 mg; 2.8 mmol) was added to
6-oxoestradiol 3,17-dipivaloate (500 mg; 1.1 mmol) in ethanol (20
ml) at 25.degree. C. under nitrogen. The reaction was quenched with
water (100 ml) after 48 hours, and the aqueous layer was extracted
with dichloromethane (3.times.50 ml). The organic layers were
combined, dried over MgSO.sub.4 and concentrated in vacuo. The
product was purified on silica gel and isolated as an off-white
solid (150 mg; 30%). Mass Spectrum (M+1) m/e=457.
[0062] Compound (X): Colchicine-Estradiol Prodrug
[0063] To a solution of triphosgene (0.25 mmol, 74 mg) in
dichloromethane (1 ml) was added a solution of 6-hydroxyestradiol
3,17-dipivaloate (0.3 mmol, 137 mg) and diisopropylamine (0.6 mmol,
0.1 ml) in dichloromethane (1.5 ml). The mixture was stirred for 30
minutes, then a solution of N-desacetyl colchicine (0.3 mmol, 107
mg) and diisopropylamine (0.6 mmol, 0.1 ml) in dichloromethane (1.5
ml) was added and the mixture stirred for 1 hour. The reaction
mixture was then evaporated to dryness, the residue redissolved in
ethyl acetate, and washed with 0.5 M NaCO.sub.3 (aq). The
ethylacetate solution was then dried over MgSO.sub.4, and
concentrated in vacuo to finish the title prodrug as its
3,17-dipivaloate ester.
[0064] The prodrug dipivaloate ester was then dissolved in methanol
(3 ml) and an aqueous solution of methylamine (40% w/w, 0.5 ml)
added, and the solution stirred for 1 hour. Dilute HCl (0.1 M) was
then added to neutralise the mixture to pH 7, and the product then
extracted with dichloromethane (3.times.10 ml). The solvent was
then evaporated in vacuo to give the title colchicine-estradiol
prodrug. IR (KBr) 1695 cm-1; MS (M+1) m/e=672.
[0065] Compound (XI): Combretastatin-Estradiol Prodrug
[0066] The procedure followed that described for compound (X)
above, but using combretastatin (0.3 mmol, 95 mg) in place of
N-desacetyl colchicine, to afford the title combretastatin prodrug.
IR (KBr) 1750 cm-1; MS (M+1) m/e=631.
[0067] Compound (XII): Mustard-Estradiol Prodrug
[0068] The procedure followed that described for compound (X), but
using bis(chloroethyl)amine hydrochloride (0.3 mmol, 53 mg) in
place of N-desacetyl colchicine together with an extra equivalent
of diisopropylamine (0.6 mmol, 0.1 ml). This gave the title mustard
prodrug as a white crystalline compound. IR (KBr) 1700 cm-1; MS
(M+1) m/e=456.
[0069] Compound (XIII): Fluorophore-Estradiol Conjugate
[0070] The procedure followed that described for compound (X) but
using 7-amino-4-methylcoumarin (0.3 mmol, 53 mg) in place of
N-desacetyl colchicine, to afford the title fluorophore conjugate.
IR (KBr) 1690 cm-1; MS (M+1) m/e=490.
[0071] Compound (XIV): Colchicine-Naphthyl Prodrug
[0072] To a solution of triphosgene (0.25 mmol, 74 mg) in
dichloromethane (1 ml) was added a solution of
6-methoxy-1-naphthalenemethanol (0.3 mmol, 56 mg) and
diisopropylamine (0.6 mmol, 0.1 ml) in dichloromethane (1.5 ml).
The mixture was stirred for 30 minutes, then a solution of
N-desacetyl colchicine (0.3 mmol, 107 mg) and diisopropylamine (0.6
mmol, 0.1 ml) in dichloromethane (1.5 ml) was added and the mixture
stirred for 1 hour. The reaction mixture was then evaporated to
dryness, the residue redissolved in ethylacetate, and washed with
0.5 M NaCO.sub.3 (aq). The ethylacetate solution was then dried
cover MgSO.sub.4, and concentrated in vacuo to finish the title
colchicine-naphthyl prodrug. IR (KBr) 1695 cm-1; MS (M+1)
m/e=572.
[0073] Compound (XV): Colchicine-Benzyl Prodrug
[0074] To a solution of triphosgene (0.25 mmol, 74 mg) in
dichloromethane (1 ml) was added a solution of 3-methoxybenzyl
alcohol (0.3 mmol, 41 mg) and diisopropylamine (0.6 mmol, 0.1 ml)
in dichloromethane (1.5 ml). The mixture was stirred for 30
minutes, then a solution of N-desacetyl colchicine (0.3 mmol, 107
mg) and diisopropylamine (0.6 mmol, 0.1 ml) in dichloromethane (1.5
ml) was added and the mixture stirred for 1 hour. The reaction
mixture was then evaporated to dryness, the residue redissolved in
ethylacetate, and washed with 0.5 M NaCO.sub.3 (aq). The
ethylacetate solution was then dried over MgSO.sub.4, and
concentrated in vacuo to finish the title colchicine-benzyl
prodrug. IR (KBr) 1695 cm-1; MS (M+1) m/e=522.
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