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The present invention concerns enzymatic aromatic hydroxylation-activated
prodrugs, particularly anti-tumour prodrugs and those which are
specifically activated by the hydroxylation activity of the enzyme CYP1B1.
Many conventional cytotoxic drugs are known which can be used for
chemotherapeutic purposes. However, they typically suffer from the problem
that they are generally cytotoxic and therefore may effect cells other
than those which it is wished to destroy. 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
less extent, certain limitations and disadvantages.
It has been reported (Murray, G. I. et al., Jul. 15, 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, oesophagur, skin, lymph node, brain and testis, and that is
it 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". No specific anticancer drugs are suggested.
The present invention have now succeeded in creating a range of prodrugs
which have little or negligible cytotoxic effect when in their normal
state, but which are highly cytotoxic (i.e. have a substantially increased
cytotoxicity) when hydroxylated by CYP1B1. 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 form a highly
cytotoxic compound which acts to kill the tumour cells. The fact that
CYP1B1 is not expressed by normal cells means that the hydroxylation of
the compound only occurs at the site of tumour cells and therefore only
tumour cells are affected, thus providing a self-targetting drug delivery
system.
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.
According to the present invention there is provided a prodrug activated by
enzymatic aromatic hydroxylation and having the formula (I):
##STR1##
wherein:
X=H, OH or OMe;
R.sub.1 =H, C.sub.1-4 lower alkyl, CN or Ar;
R.sub.2 =H, CN, CONH.sub.2, CSNH.sub.2, COAr or Ar; and
Ar=phenyl, pyridyl or substituted aryl; and
R.sub.3 =H or C.sub.1-4 lower alkyl; and
R.sub.4 =H, OH or OMe; or;
R.sub.3, R.sub.4 =(CH.sub.2).sub.n, n=2, 3 or 4.
The prodrug may be an anti-tumour prodrug. Examples of tumours include
cancers (malignant neoplasms) as well as other neoplasms e.g. "innocent"
tumours. The prodrug may be activated by hydroxylation by CYP1B1.
These prodrugs are styrene-derivatives and their specific anti-tumour use
is neither suggested nor disclosed by Murray, G. I. et al. (supra), nor is
the fact that they are in fact prodrugs having an "activated" hydroxylated
form. Where compounds of formula (I) have been previously identified and
made, they have not been identified as anti-tumour agents due to their
poor (or negligible) cytotoxicity. Thus the intratumoural hydroxylation of
the prodrugs of the present invention provides them with a surprising and
unexpected efficacy.
The styrene sub-structure of the compounds of formula (I) is essential in
providing their efficacy. The Ar group may, for example, be a substituted
aryl comprising 4-methoxyphenyl, 4-nitrophenyl, 3,5-dihydroxyphenyl or
3,4,5-trimethoxyphenyl, although other substituted aryls are, of course,
also possible.
X may be hydroxy or methoxy.
As specified in formula (I) R.sub.3, and R.sub.4 may together form an alkyl
chain having 2-4 carbon atoms, and thus may form part of a cycloalkyl
group having 5, 6 or 7 carbon atoms.
The prodrug may have the formula of any one of formulae (II)-(V):
##STR2##
Alternatively, the prodrug may have the formula of either one of formulae
(VI) or (VII):
##STR3##
Hydroxylated forms of compounds (II)-(V) are potent tyrosine kinase
inhibitors, and hydroxylated forms of compounds (VI) and (VII) are potent
antimitotic agents. Previously, tyrosine kinase inhibitors have been of
little chemotherapeutic benefit since the tyrosine kinase enzymes are
ubiquitous in both normal and tumour cells and are thus not in themselves
tumour-specific. However, the targetted production of tyrosine kinase
inhibitor in tumour cells means that the inhibitory action will be
specific to tumour cells. Furthermore, since the inhibitory activity will
only be found in tumour cells, the tyrosine kinase inhibitor itself need
not be isoform specific for a particular tyrosine kinase enzyme since any
inhibition of tyrosine kinase activity will contribute to tumour
inhibition and cell destruction.
Similarly, the antimitotic prodrugs of formulae (VI) and (VII) are
particularly useful since present antimitotic agents are of limited use
due to the severe side-effects resulting from the poisoning of both normal
and tumour cells. The present invention however allows for the specific in
situ generation of the antimitotic agent at tumour cells, resulting in
their specific targetting.
Methods of synthesis of the prodrugs of the present invention will be
readily apparent to one skilled in the art, for example as exemplified
below. The compounds of the invention may be prepared in a variety of
different ways, for example by aldol condensation (Vogels Textbook of
Practical Organic Chemistry, 4th Edition, p. 146), by McMurry coupling
(McMurry and Fleming, 1974, J. Am. Chem. Soc., 96:4708-4709), or by the
Wittig reaction (1973, Org. Synth. Coll., 5:751).
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.
Also provided according to the present invention is the use of a prodrug
according to the present invention in the manufacture of a medicament for
the treatment of tumours.
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. The medicament may be for the treatment of a tumour.
Also provided according to the present invention is a method of treatment
or diagnosis of a tumour in a patient, comprising administering to the
patient a prodrug according to the present invention.
Methods of manufacture of medicaments are well known. For example a
medicament may additionally comprise a pharmaceutically acceptable
carrier, diluant or excipient (Reminton's Pharmaceutical Sciences and US
Pharmacopoeia, 1984, Mack Publishing Company, Easton, Pa. USA).
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.
Since the prodrugs of the present invention are specific to 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, cell numbers in a sample may be assayed, as may
the presence and quantity of the hydroxylated prodrug, thus providing for
the diagnosis of the presence of tumour cells.
Also provided according to the present invention is the hydroxylated form
of a prodrug according to the present invention.
The invention will be further apparent from the following description,
which shows, by way of example only, forms of prodrugs.
Prodrugs according to the present invention were synthesised as described
below and the products of their hydroxylated metabolites assayed for the
presence of the desired hydroxylation products.
Microsomal Preparation of Resected Human Tumour Tissue
A microsomal preparation of human tumour tissue expressing the CYP1B1
enzyme was prepared essentially as described by the method of Barrie et
al., (1989, J. Steroid Biochem., 6: 1191-1195).
Metabolism Studies
Experiment were carried out at 37.degree. C., under yellow light.
An array of 1.5 ml centrifuge tubes were set up in a water bath shaker
under aerobic conditions. To each tube was then added 500 .mu.l of pH 7.6
buffer (0.1 M NaK.sub.2 PO.sub.4), followed by 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.
HPLC conditions: Spherisorb C18 (25 cm.times.4.6 mm id), used without guard
column. Flow rate 1 ml/min. Eluent 75% 0.1 M KH.sub.2 PO.sub.4 and 25%
acetonitrile.
The prodrugs were assayed as described above and were found to undergo
aromatic hydroxylation. The hydroxylated metabolite was detected by HPLC,
and confirmed by synthesis of the authentic hydroxylated metabolite.
Compound IIa (below), (Z)-1-Cyano-1-(3-pyridyl)-2-(4-methoxyphenyl)ethene,
was converted to the hydroxylated metabolite
(Z)-1-Cyano-1-(3-pyridyl)-2-(3-hydroxy-4-methoxyphenyl)ethene.
Compound IIIc, (E)-(3,4',5)-trihydroxystilbene was converted to the
hydroxylated metabolite (E)-(3,3',4,5')-tetrahydroxystilbene.
Compound VII
(E)-1-(4-Methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)prop-1-en-3-one was
converted to the hydroxylated metabolite
(E)-1-(3-Hydroxy-4-methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)prop-1-en-3-on
e.
Methods of Synthesis
Compound IIa-(Z)-1-Cyano-1-(3-pyridyl)-2-(4-methoxyphenyl)ethene
To a stirred mixture of 4-methoxybenzaldehyde (2 g, 14.69 mmol) and
3-pyridylacetonitrile (1.58 ml, 14.84 mmol) in methanol (30 ml) was added
50% w/v sodium hydroxide (1 ml). The reaction was stirred for 3 hours. The
reaction mixture was quenched with water (20 ml), acidified with 2N HCl,
then rebasified with dilute NaOH (aq), and the reaction product was
extracted successively into dichloromethene (3.times.20 ml). The organic
solutions were dried over anhydrous MgSO.sub.4 and the solvent removed.
Purification by column chromatography (SiO.sub.2, Hexane/Ethylacetate 8:2;
1:1) gave 2.01 g (58% yield) of compound 1 as a straw coloured solid:
1H-NMR (CDCl3): (8.80 (d, 1H), 8.50 (m, 1H), 7.80 (m, 3H), 7.45 (s, 1H),
7.25 (m, 1H), 6.90 (m, 2H), 3.80 (s, 3H). 13C NMR (CDCl3): (161.9, 157.6,
157.5, 149.5, 146.9, 143.3, 133.1, 131.4, 130.9, 126, 123.5, 117.7, 114.5,
105.2, 55.4. Mass Spectrum m/e (M+1) 237.
Hydroxylated Metabolite of Compound IIa
-(Z)-1-Cyano-1-(3-pyridyl)-2-(3-hydroxy-4-methoxyphenyl)ethene
A mixture of 4-methoxy-3-hydroxylbenzaldehyde (0.5 g, 3.3 mmoles),
3-pyridylacetonitrile (0.35 ml, 3.3 mmoles) and 50% w/v of aqueous NaOH (3
ml) in methanol (10 ml) was stirred at room temperature for 30 minutes. A
yellow solid precipitated was filtered, washed with cooled methanol (1
ml), cooled CH.sub.2 Cl.sub.2 (5 ml) and dried under vacuum over P.sub.2
O.sub.5 to yield 0.5 g (60%) of compound 3 was yellow solid: 1H-NMR
(CD3OD): (8.8 (m, 1H), 8.5 (m, 1H), 7.7 (s, 1H), 7.5 (m, 1H), 7.3 (d, 1H),
7.2 (d, 1H), 6.8 (d, 1H); Mass Spectrum m/e (M+1) 253.
Compound IIb--(Z)-1-Cyano-1-(3-pyridyl)-2-(4-hydroxyphenyl)ethene
A mixture of 4-hydroxylbenzaldehyde (0.5 g, 4.1 mmoles),
3-pyridylacetonitrile (0.54 ml, 4.1 mmoles) and 50% w/v of aqueous NaOH
(3.3 ml) in methanol (10 ml) was stirred at room temperature for 30
minutes. A yellow precipitate formed was filtered, washed with cooled
methanol (1 ml), cooled CH.sub.2 Cl.sub.2 (10 ml) and dried under vacuum
over P.sub.2 O.sub.5 to yield 0.6 g (66%) of compound 2: 1H-NMR (CD3OD):
(8.8 (d, 1H), 8.4 (m, 1H), 8.05 (m, 1H), 7.8 (m, 2H), 7.6 (s, 1H), 7.4 (m,
1H), 6.6(m, 2H). 13C-NMR (DMSO): (177.52, 175.57, 146.59, 145.06, 144.78,
133.15, 132.73, 130.97, 123.8, 120.8, 120.3, 114.3, 88.5; Mass Spectrum
m/e (M+1) 223.
Compound IIIb (via McMurry Coupling)--(E)-(4,4')-Dimethoxystilbene
LiAlH.sub.4 (0.5 g, 13.18 mmoles) was added to a stirred slurry of
TiCl.sub.3 (3.13 g, 26.35 mmol) under N.sub.2 in dry THF (20 ml).
Instantaneous reaction occurred accompanied by the evolution of heat and
gas and by a rapid change of colour to deep black. A THF solution of
4-methoxybenzaldehyde (1.79, 13.18 mmoles) was added. The mixture was
refluxed for 4 hours. The reaction was quenched with cooled H.sub.2 O (2
ml), extracted into ethylacetate (5.times.20 ml) and purified by column
chromatography. Mass Spectrum m/e (M+1) 241.
Compound IIIb (via Wittig Reaction)--(E)-(4,4')-Dimethoxystilbene
4-methoxybenzyltriphenylphosphonium chloride (0.5 g, 1.19 mmoles) was added
over a 5 minute period to DMF (20 ml). The solution was stirred for 4
hours at room temperature. 4-methoxybenzaldehyde (0.16 g, 1.19 mmol) was
added and the mixture refluxed for 24 hours, solvent concentrated. The
crude compound was purified by column chromatography.
Compound
VII--(E)-1-(4-Methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)prop-1-en-3-one
To a stirred solution of 4-methoxylbenzaldehyde (1.0 g, 7.3 mmol) and
3,4,5-trimethoxyacetophenone (1.54 g, 7.3 mmol) in methanol (30 ml) was
added a 50% w/v solution of aqueous NaOH (1 ml). The mixture was stirred
for 24 hours at room temperature, acidified with 2N HCl and extracted with
chloroform (3.times.30 ml). The combined organic phase was dried over
anhydrous MgSO.sub.4, filtered, and the solvent concentrated under vacou.
The product was finally purified by column chromatography. Mass Spectrum
m/e (M+1) 329.
Hydroxylated Metabolite of Compound VII
(E)-1-(3-Hydroxy-4-methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)prop-1-en-3-one
To a stirred solution of 3-hydroxy-4-methoxybenzaldehyde (1.1 g, 7.3 mmol)
and 3,4,5-trimethoxyacetophenone (1.54 g, 7.3 mmol) in methanol (30 ml)
was added a 50% w/v solution of aqueous NaOH (1 ml). The mixture was
stirred for 24 hours at room temperature, acidified with 2N HCl and
extracted with chloroform (3.times.30 ml). The combined organic phase was
dried over anhydrous MgSO.sub.4, filtered, the solvent concentrated under
vacou. The product was purified by crystallisation from methanol. 1H-NMR
(CDCl3): 7.8 (d, 1H), 7.4 (d, 1H), 7.2-7.3 (m, 3H), 7.1 (dd, 1H), 6.9 (d,
1H), 5.8 (s, 1H) 4.0 (s, 9H) 3.9 (s, 3H); Mass Spectrum m/e (M+1) 329.
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