U.S. patent application number 09/765861 was filed with the patent office on 2001-09-13 for hydroxylation activated prodrugs.
This patent application is currently assigned to De Montfort University. Invention is credited to Burke, Michael Danny, Patterson, Lawrence Hylton, Potter, Gerald Andrew.
Application Number | 20010021717 09/765861 |
Document ID | / |
Family ID | 26313066 |
Filed Date | 2001-09-13 |
United States Patent
Application |
20010021717 |
Kind Code |
A1 |
Potter, Gerald Andrew ; et
al. |
September 13, 2001 |
Hydroxylation activated prodrugs
Abstract
The present invention concerns enzymatic aromatic
hydroxylation-activated prodrugs, particularly anti-tumor prodrugs
and those which are specifically activated by the hydroxylation
activity of the enzyme CYP1B1.
Inventors: |
Potter, Gerald Andrew;
(Leicester, GB) ; Patterson, Lawrence Hylton;
(Leicester, GB) ; Burke, Michael Danny;
(Leicester, GB) |
Correspondence
Address: |
BAKER BOTTS L.L.P.
44TH FLOOR
30 ROCKEFELLER PLAZA
NEW YORK
NY
10112-4498
US
|
Assignee: |
De Montfort University
|
Family ID: |
26313066 |
Appl. No.: |
09/765861 |
Filed: |
January 19, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09765861 |
Jan 19, 2001 |
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09115015 |
Jul 14, 1998 |
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6214886 |
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Current U.S.
Class: |
514/354 ;
514/277; 514/520; 514/521; 514/599; 514/617; 546/314; 546/329;
546/331 |
Current CPC
Class: |
C07D 213/57 20130101;
C07C 2602/10 20170501; C07C 327/48 20130101; C07C 255/36 20130101;
C07C 49/84 20130101; A61P 43/00 20180101; C07C 43/215 20130101;
C07C 205/35 20130101; A61P 35/00 20180101 |
Class at
Publication: |
514/354 ;
514/277; 514/520; 514/521; 514/599; 514/617; 546/314; 546/329;
546/331 |
International
Class: |
A61K 031/44; C07D
211/82; C07D 211/70; A61K 031/277; A61K 031/165 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 6, 1998 |
GB |
9802552.4 |
Claims
1. A prodrug activated by enzymatic aromatic hydroxylation and
having the formula (I): 4wherein: 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
2. A prodrug according to claim 1, being an anti-tumour
prodrug.
3. A prodrug according to claim 1, being hydroxylated by
CYP1B1.
4. A prodrug according to claim 1, X being a hydroxy or methoxy
group.
5. A prodrug according to claim 1, having the formula of any one of
formulae (II)-(V): 5
6. A prodrug according to claim 1, having the formula of either one
of formulae (VI) or (VII): 6
7. A prodrug according to claim 1, being a tyrosine kinase
inhibitor in its hydroxylated state.
8. A prodrug according to claim 1 or 6, being an antimitotic agent
in its hydroxylated state.
9. A prodrug according to claim 1 for use in a method of treatment
or diagnosis of the human or animal body.
10. The use of a prodrug according to claim 1 in the manufacture of
a medicament for the treatment of a tumour.
11. A method of manufacture of a medicament for the treatment of a
tumour, comprising the use of a prodrug according to claim 1.
12. A method of treatment of a tumour in a patient, comprising
administering to the patient a prodrug according to claim 1.
13. The hydroxylated form of a prodrug according to claim 1.
Description
[0001] 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.
[0002] 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 affect 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 lesser extent, certain limitations and
disadvantages.
[0003] 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, oesophagus, 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".
No specific anticancer drugs are suggested.
[0004] The present inventors 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 leat 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.
[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] According to the present invention there is provided a
prodrug activated by enzymatic aromatic hydroxylation and having
the formula (I): 1
[0007] wherein
[0008] X=H, OH or OMe;
[0009] R.sub.1=H, C.sub.1-4lower alkyl, CN or Ar;
[0010] R.sub.2=H, CN, CONH.sub.2, CSNH.sub.2, COAr or Ar; and
[0011] Ar=phenyl, pyridyl or substituted aryl;
[0012] and:
[0013] R.sub.3=H or C.sub.1-4 lower alkyl; and
[0014] R.sub.4=H, OH or OMe;
[0015] or:
[0016] R.sub.3,R.sub.4=(CH.sub.2).sub.n, n=2, 3 or 4
[0017] 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.
[0018] 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.
[0019] 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.
[0020] X may be hydroxy or methoxy.
[0021] As specified in formula (I) R.sub.3 an 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.
[0022] The prodrug may have the formula of any one of formulae
(II)-(V): 2
[0023] Alternatively, the prodrug may have the formula of either
one of formulae (VI) or (VII): 3
[0024] 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.
[0025] 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.
[0026] 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).
[0027] 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.
[0028] 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.
[0029] 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.
[0030] 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.
[0031] 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 Pharmacopeia, 1984, Mack Publishing Company,
Easton, Pa., U.S.A.).
[0032] 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.
[0033] 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.
[0034] Also provided according to the present invention is the
hydroxylated form of a prodrug according to the present
invention.
[0035] The invention will be further apparent from the following
description, which shows, by way of example only, forms of
prodrugs.
[0036] 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.
[0037] Microsomal preparation of resected human tumour tissue
[0038] 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)
[0039] Metabolism Studies
[0040] Experiment were carried out at 37.degree. C., under yellow
light.
[0041] 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.2PO.sub.4), followed by
NADPH (5 .mu.l of a 25 mM stock solution). The microsomal
preparation (80 .mu.) 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.
[0042] 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.2PO.sub.4 and 25% acetonitrile.
[0043] 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.
[0044] 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.
[0045] Compound IIIc, (E)-(3,4',5)-trihydroxystilbene was converted
to the hydroxylated metabolite
(E)-(3,3',4,5')-tetrahydroxystilbene.
[0046] 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
-one.
Methods of Synthesis
Compound IIa
(Z)-1-Cyano-1-3-pyridyl)-2-(4-methoxyphenyl)ethene
[0047] 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 (CDCl.sub.3):(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
[0048] A mixture of 4-methoxy-3-hydroxybenzaldehyde (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.2Cl.sub.2 (5 ml) and dried under vacuum over P.sub.2O.sub.5
to yield 0.5 g (60%) of compound 3 as yellow solid: 1H-NMR
(CD3OD):(8.8(m,1H), 8.5(m,1H), 8.1(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
[0049] A mixture of 4-hydroxybenzaldehyde (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.2Cl.sub.2 (10 ml) and
dried under vacuum over P.sub.2O.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(m2H). 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
[0050] LiAlH.sub.d(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.2O (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
[0051] 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-(4Methoxyphenyl)-3-(3,4,5-trimethoxyphenyl)prop-1-en-3-one
[0052] To a stirred solution of 4-methoxybenzaldehyde (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-on-
e
[0053] 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.
* * * * *