Note: Descriptions are shown in the official language in which they were submitted.
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1
BENZAMIDE ANALOGUES
This application is a division of PCT International
Application No. PCT/GB95/00513 bearing Canadian
Application Serial No. 2,184,747 with the International
S Filing Date of March 9, 1995.
The present invention relates to benzamide
analogues, especially certain 3-substituted benzamide
derivatives that are of interest as being at least
potentially useful chemotherapeutic agents by virtue of
an ability to inhibit the activity of the enzyme poly
ADP-ribosyltransferase (EC 2.4.2.30), also known as
poly(ADP-ribose) polymerase, commonly referred to as
ADPRT or PARP. In general, the latter abbreviation,
PARP, will be used throughout the present specification.
BACKGROUND
At least in higher organisms, the enzyme poly ADP-
ribosyltransferase is known to catalyse a transfer of the
ADP-ribose moiety from the oxidized form NAD+ of
nicotinamide adenine dinucleotide to nuclear acceptor
proteins so as to form homo ADP-ribose polymers, and this
process has been implicated in a number of cellular
events such as, for example, repair of DNA damage,
development of cellular differentiation, transformation
of cells by oncogenes, and gene expression. A common
feature in a number of these processes is the formation
and repair of DNA strand breaks and the stage which
involves the PARP enzyme appears to be that of DNA ligase
II-mediated strand rejoining. In the majority of cases a
role for poly ADP-ribosylation has been implicated by the
use of inhibitors of the PARP enzyme, and this has led to
suggestions that such inhibitors, by interfering with the
intracellular DNA repair mechanism, may have a useful
chemotherapeutic role insofar as they should be able to
modify treatment resistance characteristics and
potentiate or enhance the effectiveness of cytotoxic
drugs in chemotherapy or of radiation in radiotherapy
where a primary effect of the treatment is that of
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2
causing DNA damage in target cells, as for example in
many forms of antit-umour therapy.
In this connection, several classes of PARP
inhibitors are already known, including benzamide itself
and various nicotinamide and benzamide analogues,
especially 3-substituted benzamides with small
substituent groups such as 3-amino, 3-hydroxy and 3-
methoxy. PARP inhibitory activity of certain N-
substituted benzamides has also been reported in EP-A-
0305008 wherein it has also been proposed to use these
compounds in medicine for increasing the cytotoxicity of
radiation or of chemotherapeutic drugs.
Regarding this use of benzamides as chemotherapeutic
agents, a number of studies on such compounds that are
known to exhibit PARP inhibitory activity have confirmed
that they can potentiate the cytoxicity of a range of
antitumour agents in vitro, for example, bleomycin and
methylating drugs. More limited data has further
indicated that such benzamides can also potentiate the
activity of cytotoxic drugs in vivo, although the dose
requirements have appeared to be rather high (e.g. in the
region of 0.5g kg-1 per dose for 3-aminobenzamide) and
there may be associated problems in preparing
satisfactory pharmaceutical formulations and in avoiding
toxicity limitations. Furthermore, a number of the known
benzamides have also been shown clearly to have potential
as radiosensitizers, increasing for example ionising
radiation-induced tumour cell kill both in vitro and in
vivo, and it is believed that in many cases this effect
is related to these compounds acting as PARP inhibitors
and interfering with DNA repair.
However, notwithstanding the existing data from in
vitro and in vivo studies suggesting that PARP inhibitors
have considerable potential as useful chemotherapeutic
35- agents which merit further clinical evaluation, for
instance in connection with cancer therapy, currently
available known PARP inhibitors are not considered as yet
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3
to be entirely suitable to represent candidate drugs.
Accordingly, there is a need to find and develop a
greater range of compounds having potentially useful PARP
inhibitory properties.
DISCLOSURE OF THE INVENTION
The present invention identifies a new range or
ranges of compounds of interest as PARP inhibitors that
can be useful in medicine, especially when administered
in conjunction with at least certain cytotoxic drugs or
with radiotherapy for increasing the cytotoxic
effectiveness thereof. In general, the compounds of this
invention as hereinbelow defined comprise benzamide
compounds, or analogues, which include 3-position
substituents linked in a ring structure with substituents
in the 2-position.
More specifically, from one aspect, the invention
resides in the use of a compound as herein defined for
the manufacture of a medical or veterinary preparation
for use in therapy for inhibiting activity of the enzyme
poly(ADP-ribose)polymerase or PARP (also known as ADP-
ribosyl transferase or ADPRT), such enzyme inhibition
constituting an element of a therapeutic treatment, said
compound providing an active PARP enzyme inhibiting agent
and being a 3-substituted oxybenzamide compound having
the general structural formula I
CONH2
I
,Y
-X
or a pharmaceutically acceptable salt thereof, and
characterised in that
30. Y and X together form a bridge -Y-X- that represents
the grouping -N=i- or -0-iH- or -S-iH-
Rs Rs Rs
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4
where Rs is H, alkyl or an optionally
substituted aralkyl or aryl group.
The invention also provides for use in therapy as
active pharmaceutical substances 3-substituted
oxybenzamide compounds having the general structural
formula I
CONH2
\ ~ I
,Y
-X
or a pharmaceutically acceptable salt thereof,
characterised in that
Y and X together form a bridge -Y-X- that represents
the grouping -N=C-
Rs
where Rs is H, alkyl or an optionally
substituted aralkyl or aryl group.
The invention further provides novel 3-substituted
oxybenzamide compounds having the general structural
formula I
CONH2
I
\ Y
X
or a pharmaceutically acceptable salt thereof,
characterised in that
Y and X together form a bridge -Y-X- that represents
the grouping -N=C-
Rs
where Rs is H, alkyl or' an optionally
substituted aralkyl or aryl group.
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Alkyl groups when present as such or as a moiety in
other groups such as alkoxy will generally be composed of
1-8 carbon atoms, preferably 1-6 carbon atoms, and more
usually 1-4 carbon atoms.
5 One very important group of compounds of special
interest from the point of view of PARP-inhibitory
activity comprises benzoxazole-4-carboxamide compounds,
i.e, compounds represented by the formula IV
CONH2
IV
N
~5
where R5, if not H, is preferably alkyl, phenyl or another
aryl group such as naphthyl or pyridyl. When RS is an
alkyl group this will generally be C1-6 alkyl, such as for
example methyl, ethyl, n-propyl, i-propyl, n-butyl, t-
butyl or cyclohexyl. However, it may in some cases be
larger, such as in adamantyl for instance . When RS is a
phenyl group this may be substituted, especially in the 4
(para) position but alternatively perhaps in the 2-
position or 3-position, by substituents such as alkoxy
for example.
Within this group of benzoxazole compounds preferred
members which are of particular interest include
2-methylbenzoxazole-4-carboxamide,
2-t-butylbenzoxazole-4-carboxamide,
2-phenylbenzoxazole-4-carboxamide,
2-(4-methoxyphenyl)benzoxazole-4-carboxamide.
In the above-mentioned compounds of formula IV,
wherein there is an electron-rich aromatic ring, it is
believed that the carboxamide group is constrained in a
fixed conformation, particularly favourable for
presenting the compound as an alternative substrate to
NAD+ for the, PARP enzyme, by an intramolecular hydrogen
bond between the ring nitrogen atom and one of the
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hydrogen atoms of the carboxamide group. A similar,
although probably somewhat weaker effect may also occur
in other compounds of formula I where the X and Y
substituents form a bridge, as defined above, containing
an oxygen or sulphur atom, i . a . where the ring N atom of
the benzoxazoles of formula IV is replaced by an 0 or S
atom.
The invention also embraces or extends to methods of
preparing compounds as hereinbefore defined (including
intermediates in some cases) and to the therapeutic use
of such compounds. This includes their use for making
medical or veterinary preparations or pharmaceutical
formulations containing an effective PARP inhibitory
amount of the active compound for administration to a
patient in conjunction with a cytotoxic drug or
radiotherapy in order to increase the cytotoxic
effectiveness of the latter. Such preparations or
formulations may be made up in accordance with any of the
methods well known in the art of pharmacy for
administration in any suitable manner, for example
orally, parenterally (including subcutaneously,
intramuscularly or intravenously), or topically, the mode
of administration, type of preparations or formulation
and the dosage being generally determined by the details
of the associated cytotoxic drug chemotherapy or
radiotherapy that is to be enhanced.
As indicated, the compounds according to this
invention have at least potential as PARP inhibitors, and
in vitro tests hereinafter described have demonstrated
positive pharmacological activity which it is believed
reflects the activity to be found in vivo in the course
of therapeutic clinical use.
It will be understood that where reference is made
in this specification to compounds of formula I (or
- formula IV) such reference should be construed as
extending also to their pharmaceutically acceptable salts
where relevant. Also, where any of the compounds
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7
referred to can exist in more than one enantiomeric form,
all such forms, mixtures thereof, and their preparation
and uses are within the scope of the invention.
DESCRIPTION OF EXAMPLES OF PREFERRED EMBODIMENTS
The following examples and descriptions of stages in
synthetic routes of preparation of various preferred
compounds of interest serve to further illustrate the
present invention, but should not be construed in any way
as a limitation thereof.
EXAMPLE 1
2-Methylbenzoxazole-4-carboxamide (Compound NU1056)
1st Stage - Preparing Methyl (3-Hydroxy-2-nitro)benzoate
3-hydroxy-2-nitrobenzoic acid (5g; 27.32mM) was
dissolved in anhydrous methanol (200m1). Anhydrous
hydrogen chloride gas was bubbled through the solution
until saturated. The mixture was then refluxed for 20
hours (reaction followed by T.L.C. . l0o methanol/900
dichloromethane). Next, the solvent was removed under
vacuum to yield a brown solid. The solid was dissolved
in water (100m1) and sodium bicarbonate was added until
effervescence stopped. Sodium chloride (15g) was added
to the aqueous solution, and the product was extracted
into ethyl acetate (3x50m1). The pooled aliquots were
dried over magnesium sulphate and the solvent removed
under vacuum to yield a malty brown solid.
T.L.C. (as before): r.f.: 0.53
NMR: 200MHz: d6 DMSO: b=3. 9 (s; 3H;OCH3) ; 7. 1 (m; 1H;H4) ;
7 . 35 (m; 1H; H6) ; 7 . 8 (t; 1H; HS)
Yield 92s
2nd Stage - Preparation of Methyl (2-Amino-3-hydroxy)-
Y, o ,., -n ., ~ + o
Under a nitrogen atmosphere a palladium/carbon
catalyst was suspended in anhydrous methanol (150m1). To
this suspension was added methyl (3-hydroxy-2-nitro)
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benzoate from Stage 1 (4g; 20.3mM). The mixture was left
under a hydrogen atmosphere for 4'~ hours. The catalyst
was removed by filtration through a "celite" pad, and the
solvent removed from the filtrate to yield an
orange/brown product.
T.L.C.: 40o Ethyl acetate/60% petroleum ether 60/80.
r.f. 0.33
NMR 200mHz: doDMSO . b: 3.81(S;3H;OCH3);
6. 2 (broad; 2H; NHZ ) ; 6. 5 (t; 1H; HS) ; 6 . 9 (m; 1H; H6)
7. 3 (m; 1H; H4) 9. 8 (s; 1H; OH)
Yield 83%
3rd Staae - Preparation of Methyl 2-methylbenzoxazole-4-
carboxvlate
To a solution of the methyl (2-amino-3-hydroxy)-
benzoate (3g; l8.OlmM) in m-xylene (150m1) was added
acetyl chloride (1.518m1; 21.6mM). A precipitate was
formed; this was left to stir for 30 minutes. On the
addition of triethylamine (2.97m1; 21.6mM) the solution
became translucent. Pyridinium-p-toluene sulphonic acid
(1.2g; 21.6mM) was added, and the mixture refluxed for 34
hours. The solvent was removed by distillation (vacuum)
to yield a brown solid which was column chromatographed
(50o ethyl acetate/50o petrol 60/80) to give the desired
product as a yellow solid.
T.L.C. As above.
1HNMR :200MHz; CDC13; b: 2.69(3H;s;2-CH3)
3.99(3H;s;OMe); 7.33(3H;t;H6); 7.63(lH;dd;H7)
7 . 94 ( 1H; dd; HS )
4th Stage - Preparation of 2-methylbenzoxazole-4-
carboxylic acid
Methyl 2-methylbenzoxazole-4-carboxylate (0.1 g,
0.523 mmol) was dissolved in methanol (3 ml), and to this
was added aqueous sodium hydroxide solution (0.2 M, 3
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9
ml). The mixture was stirred at 40°C for 4 hours and
acidified with hydrochloric acid (6 M) until pH - 1Ø
The mixture was extracted with ethyl acetate (3 x 20 ml),
the combined organic layers were washed with water (2 x
20 ml), dried (MgS04) and the solvent was removed under
reduced pressure to afford the carboxylic acid (0.068 g,
730) .
5th Staqe - Preparation of 2-methylbenzoxazole-4-
carboxamide
A solution of 2-methylbenzoxazole-4-carboxylic acid
(0.1 g, 0.28 mmol) in anhydrous THF (10 ml) was stirred
under nitrogen, and thionyl chloride (0.022 ml, 0.31
mmol), and DMF (O. lm) were added, whereupon the mixture
was stirred for a further 5 hours at room temperature.
Aqueous ammonia (0.5 ml) was added and the mixture was
stirred for a further 30 minutes. The solvent was
removed under reduced pressure, the residual solid was
dissolved in water (20 ml), and the solution was
extracted with ethyl acetate (3 x 20 ml). The organic
layers were pooled, washed with water (2 x 20 ml) and
dried (MaSOa). The solvent was removed under reduced
pressure to furnish the carboxamide (0.083 g, 84%).
1H (200 MHz) CDC13 b = 6.0 (brs, 1H, NH) , 7. 4 (t, 1H, H6) ,
7. 6 (dd, 1H, H7) , 8. 15 (dd, 1H, H5) , 8. 8 (brs, 1H, NH) .
EXAMPLE 2
2-Methylbenzoxazole-4-carboxamide (Compound NU1056)
In a modification of the procedure described under
Example 1 above, the product of the 3rd stage, methyl 2-
methylbenzoxazole-4-carboxylate, was prepared directly
from the product of the 1st stage, as described below.
Methyl 3-hydroxy-2-nitrobenzoate (O.lg; 0.59mmo1)
from the 1st stage was dissolved in anhydrous ethanol
(20m1) and to this was added ethyl acetimidate hydro-
chloride (0.0678; 0.59mmo1). The reaction mixture was
then heated 'under reflux for 24 hours. The ethanol was
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removed under reduced pressure yielding a brown
crystalline solid. This was dissolved in ethyl acetate
(3 x 20m1) to produce a precipitate of excess ethyl
acetimidate hydrochloride. The excess imidate was
S filtered off and the solution washed with sodium
hydroxide solution (O.1N; 3 x 20m1), and water (3 x
50m1). The solvent was dried over magnesium sulphate and
removed under reduced pressure, leaving a orange
crystalline solid (0.1265g; 85%).
10 EXAMPLE 3
2-t-butylbenzoxazole-4-carboxamide (Compound NU1040)
(a) lst Stage - Preparation of Methyl 2-t-butyl-
benzoxazole-4-carboxylate
2-Amino-3-hydroxybenzoate (O.lg; 0.598mmo1) was
dissolved in m-xylene and warmed to 70°C. To this was
added pivaloyl chloride (0.117m1; 0.958mmo1), whereupon a
brown precipitate was observed to develop. The mixture
was stirred for 30 minutes before the addition of
triethylamine (0.099m1; 0.958mmo1) and pyridinium-4-
toluenesulphonate (0.048; 0.958mmo1). The mixture was
then heated under reflux for 26 hours. The m-xylene was
removed under reduced pressure to yield a sticky brown
solid. The solid was dissolved in water (50m1) and the
organics extracted into ethyl acetate (3 x 30m1), pooled,
dried over magnesium sulphate, filtered and the solvent
removed under reduced pressure.
The title compound was purified via silica column
chromatography, with 1:1 ethyl acetate:petrol as the
eluant to yield a yellow solid (69%).
IR cm-1: 3040 (3 x CH3) , 1709 (C=0) . M/Z; 233 (63%; M+) ;
218 (50%; -CH3) 202 (42%; -OCH3) ; 186 (100%) ; 173 (12%;
CH3); 160 (33%); 146 (62%); 117 (43%). 1H: d6 DMSO: b
1. 17 (9H; s; (CCH3) 3) ; 3. 99 (3H; s, OCH3) ; 7.31 (1H; t; H6
J=8Hz), 7.66 (1H; dd: H7; J=7.8, 1Hz), 7.95 (1H; dd; HS
J=7, 1Hz).
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(b) 2nd Stage - Preparation of 2-t-Butylbenzoxazole-4-
carboxamide
Methyl 2-t-butyl-4-benzoxazole carboxylate (O.lg;
0.46mmo1) was dissolved in methanol (5ml), and to this
S was added aqueous ammonia ( 5m1 ) . The mixture was warmed
to 40°C and left to stir for 6 hours at ambient
temperature. Once the reaction was complete the solvent
was removed under reduced pressure and the product was
recrystallised from boiling ethyl acetate and petrol
(73a) .
IR cm-1: 3395; 3304 (amide NH); 3163 (3 x CH3), M/Z; 218
(96a; M+); 202 (43%; -NH2) 186 (850; -CONHZ); 175 (770);
160 (35%) 146 (790) ; 133 (230) ; 41 (1000) .
1H: CDC13: b - 1. 44 (9H; s; (CCH3) 3) ; 5. 95 (1H; br s; NH) ;
7.34 (1H; t; H6 J=8Hz), 7.6 (1H; dd: H7; J=7,& 2Hz) 8.07
(1H; dd; H5 J=6.7, 2Hz); 8.88 (1H; br s; NH)
13C 28. 416 (3xCH3) , 34 . 372 (CCH3) , 113. 905 (Ar) , 123.277
(Ar), 124.338 (Ar), 124.338 (Ar), 125.440 (Ar), 139.371
(0-Ar), 150.804 (N-Ar), 166.457 (Ar), 174.471 (C=C
amide).
CHN: Found: C 65.9150; H 6.390; N 12.480, Required: C
66.038%; H 6.465%; N 12.8350.
wTrrtnT ~ n
2-Phenylbenzoxazole-4-carboxamide (Compound NU1051)
(a) 1st Stage - Preparation of Ethyl benzimidate
Hydrochloride
Benzonitrile (0.514m1; 5mmo1) was added to anhydrous
ethanol (0.69g). Anhydrous hydrogen chloride gas was
bubbled through the solution until saturated. The
mixture was left to stir for 20 hours. The white
crystalline solid was collected and dried.
mpt: 125-130°C
IR . cm-1, 28,56, 1631. M/Z; 148 (M+, 30%) , 105 (1000) ;
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1H: d6 DMSO; b - 1.5 (3H; t, J=7Hz; CHZCH3), 4.75 (2H; q,
J=6.9Hz; CH~CH3), 8.0 (5H; m; aromatics).
Elemental Analysis:
ExpectedC: 58.25; H: 6.51; N: 7.54;
FoundC: 58.13; H: 6.43; N: 7.36.
b) 2nd Staqe - Preparation of Methyl 2-phenylbenzoxazole
-4-carboxylate
To Methyl 2-amino-3-hydroxybenzoate (O.lOg;
0.59mmo1) was added ethyl benzimidate hydrochloride
(0.1678; 0.998mmo1) in anhydrous ethanol (20m1). This
was refluxed for 20 hours, and the reaction followed by
TLC (1:4 Ethyl acetate: petrol).
The ethanol was removed under reduced pressure and
the solid dissolved in water (20m1), the organics were
extracted into ethyl acetate (3 x 20m1), pooled, and
washed with sodium hydroxide solution (2 x lOml; 0.2M),
water (2 x lOml) and then dried over magnesium sulfate.
The solvent was removed under reduced pressure to yield a
yellow solid. The title compound was isolated by flash
chromatography (eluant as for TLC), yielding an off-white
crystalline solid. (85%).
IR . cm-1, 1714 . 1H: d6 DMSO: b - 4. 1 (3H; s; OCH3) , 7. 39
(1H; t; J=6Hz; H6), 7.5 (3H; m; H3'; H4'; HS'), 7.78 (1H;
dd J=7.0, 1.0 Hz; H7;), 8.0 (1H; dd; J= 6.67 & 1.14Hz;
HS' ) , 8 . 3 (2H; m; Hz' ; H6' ) .
(c) 3rd Stage - Preparation of 2-phenylbenzoxazole-4-
carboxamide
To a solution of methyl 2-phenylbenzoxazole-4-
carboxylate (0.028; 0.0905mmo1) in methanol (3m1) was
added aqueous ammonia (3m1). This was warmed to 40°C and
left to stir, whilst the reaction was monitored by TLC
(1:4 EtOAc:petrol). Once all the starting material had
reacted a white precipitate was formed. The solvent was
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removed to yield a white solid, which was recrystallised
from ethyl acetate and petrol (70%).
MPt: 199-201°C.
1H: CDC13: b = 6.02 (1H; br s: NH); 7.43 (1H; t; H6;
J=8Hz ) , 7 . 57 ( 3H; m: H3' ; H4' ; HS' ) , 7 . 74 ( 1H; dd;
H7; J=lHz,& 7Hz); 8.23 (1H; dd; H5; J=1Hz & 7.3Hz);
8.27 (2H; m; HZ' ; H6' ) , 8. 97 (1H; br s; NH) .
13C ; M/Z; (EI ) ; 238 (m+; 100 % ) ; 222 (-NHZ 68 % ) ;
195 (-CONH2; 98 % ) . IR cm-1 3383; 3165.
Elemental Analysis:
ExpectedC:70.58, H:4.23, N:11.76.
Found C:70.41, H:4.24, N:11.77.
L'VTMDT L'
2-(4-Nitrophenyl)benzoxazole-4-carboxamide (Compound
NU1053)
(a) 1st stage - Preparation of Methyl 3-hydroxy-2-(N-4-
nitrobenzoyl)aminobenzoate
Methyl 2-amino-3-hydroxybenzoate (0.5g; 2.99mmo1) was
dissolved in m-xylene (40m1) with warming to 60°C. 4-
Nitrobenzoyl chloride (0.556m1; 2.99mmol) was added
dropwise, and this was left to stir for 4 hours. The
solution was cooled to ambient temperature and the m-
xylene removed under reduced pressure. The solid was
dissolved in water (100m1) and the organics extracted
into ethyl acetate (3 x 50m1). The organic fractions
were pooled, dried over magnesium sulphate, filtered and
the solvent removed under reduced pressure.
The title product was purified via column chromatography
(1:4 ethyl acetate: petrol as eluent) to yield an orange
solid (49%).
_ IR . cm-1: 3443 (OH) , 2953, 1697, 1649, 1404. M/Z; 316
(15% M+) .
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14
1H: CDC13: b = 3.95 (3H; s; OCH3) , 7.25 (1H; t; HS
J=8Hz), 7.31 (IH; dd; H4,; J=6,& 2Hz), 7.67 (1H; dd;
H6,); 8.26 (2H; dd; HZ'; H6' J=2.3Hz); 8.30 (2H; dd;
H3' ; H5' J=2 . 2Hz ) ; 9 . 81 ( 1H; s; OH) ; 12 . 30 ( 1H; s;
S NH ) .
(b) 2nd Stage - Preparation of Methyl 2-(4-nitrophenyl)-
benzoxazole-4-carboxylate
Methyl 3-hydroxy-2-(N-4-nitrobenzoyl)aminobenzoate
(O.lg; 0.34 mmol) was dissolved in m-xylene (20m1), and
to this was added triethylamine (0.033m1; 0.45mmo1) and
pyridinium-4-toluene sulphonate (0.0708; 0.28mmo1). This
was refluxed for 32 hours. The m-xylene was removed
under reduced pressure and the~remaining solid dissolved
in water. The organics were extracted into ethyl acetate
(3 x 30m1), dried, filtered and the solvent removed under
reduced pressure to yield a brick red solid (74%).
IR . cm-1:, 1726; 1522; 1556, M/Z; 298 (84%, M+) 267
(100%, -OCH3) , 240 (-CO) 1H: CDC13: b = 4. O1 (3H; s; OCH3) ,
7.44 (1H; t; H6 J=8.lHz), 7.76 (1H; dd; H7,; J=7.2,& 1Hz),
8.04 (1H; dd; H5, ) , 8. 35 (2H; dd; HZ' ; H6' J=2.2Hz) ; 8.46
(2H; d; H3'; H5' J=2.2Hz).
(c) 3rd Stage - Preparation of 2-(4-nitrophenyl)
benzoxazole-4-carboxylic acid
Methyl 2-(4-nitrophenyl)benzoxazole-4-carboxylate
(0.1 g, 0.335 mmol) was dissolved in methanol (3 ml), and
to this was added aqueous sodium hydroxide solution (0.2
M, 3 ml). The mixture was stirred at 40°C for 4 hours
and acidified with hydrochloric acid (6 M) until pH -
1Ø The mixture was extracted with ethyl acetate (3 x
20 ml), the combined organic layers were washed with
water (2 x 20 ml), dried (MgS04) and the solvent was
removed under reduced pressure to afford the carboxylic
acid (0.084 g, 890).
(d) 4th Stage - Preparation of 2-(4-nitrophenyl)
benzoxazole-4-carboxamide
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A solution of 2-(4-nitrophenyl)benzoxazole-4-
carboxylic acid (0:084 g, 0.29 mmol) in anhydrous THF (10
ml) was stirred under nitrogen, and thionyl chloride
(0.022 ml, 0.31 mmol), and DMF (O.lml) were added,
5 whereupon the mixture was stirred for a further 5 hours
at room temperature . Aqueous ammonia ( 0 . 5 ml ) was added
and the mixture was stirred for a further 30 minutes.
The solvent was removed under reduced pressure, the
residual solid dissolved in water (20 ml), and the
10 solution was extracted with ethyl acetate (3 x 20 ml).
The organic layers were pooled, washed with water (2 x 20
ml) and dried (MgS04). The solvent was removed under
reduced pressure to furnish the carboxamide (0.07 g,
85%).
15 EXAMPLE 6
2-(4-Methoxyphenyl)benzoxazole-4-carboxamide (Compound
NU1054)
(a) 1st Stage - Preparation of Methyl 3-hydroxy-2-(N-4-
methoxvbenzoyl)aminobenzoate
Methyl 2-amino-3-hydroxybenzoate (0.58; 2.99mmo1)
was dissolved in m-xylene (40m1) with warming to 60°C.
4-Methoxybenzoyl chloride (0.5098; 2.99mmo1) was added
dropwise, and this was left to stir for 3 hours. The
solution was cooled to ambient temperature and the m-
xylene removed under reduced pressure. The solid was
dissolved in water (100m1) and the organics extracted
into ethyl acetate (3 x 50m1). The organic fractions
were pooled, dried over magnesium sulphate, filtered and
the solvent removed under reduced pressure.
The title product was purified via chromatography (1:4
ethyl acetate: petrol as eluent) and recrystallised from
boiling ethyl acetate/petrol to yield a brick red solid
(33%).
IR . cm-1: 3100 (OH) 2571, 1691, 1643, 1606, M/Z; 301
(23 0, M+) , 2~70 (-OCH3) , 135 (100°s, COPhOCH3)
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16
1H: CDC13: b - 3.87 (3H; s; OCH3) ; 3. 93 (3H; s; COOCH3) ,
7. 02 (2H; dd; HS' ; H3' ) ; 7.14 (1H; t; HS J=8Hz) , 7.29 (1H;
dd; H4,; J=6.3,& l.7Hz), 7.63 (1H; dd; H6); 8.03 (2H;
dd; HZ' ; H6' ) ; 10. 38 ( 1H; s; OH) ; 11 . 98 ( 1H; s; NH) .
(b) 2nd Stage - Preparation of Methyl 2-(4-
methoxyphenyl) benzoxazole-4-carboxylate
Methyl 3-hydroxy-2-(N-4-methoxybenzoyl)aminobenzoate
(0.058; 0.166mmo1) was dissolved in m-xylene (20m1), and
to this was added triethylamine (0.016m1; 0.215mmo1) and
pyridinium-4-toluene sulphonate (0.034g; 0.13mmo1). This
was refluxed for 58 hours. The m-xylene was removed
under reduced pressure and the remaining solid dissolved
in water. The organics were extracted into ethyl acetate
(3 x 30m1), dried, filtered and the solvent removed under
reduced pressure to yield a solid (74%).
IR cm-1: 1718, 1614, 1502. M/Z; 283 (45%, M+) 252 (31%, -
OCH3) , 225, 63 (100%) .
1H: CDC13: b = 3. 88 (3H; s; COOCH3) ; 6. 9 (2H; d; H3.; H5. ) ;
7.35 (1H; t; H6), 7.74 (1H; dd; H7;), 7.96 (1H; dd;
HS) ; 8.2 (2H; d; Hz.; H6. ) .
(c) 3rd Stage - Preparation of 2-(4-methoxyphenyl)
benzoxazole-4-carboxamide
Methyl 2-(4-methoxyphenyl)benzoxazole-4-carboxylate
was dissolved in liquid ammonia (30m1) and sealed in an
autoclave. The reaction mixture was left at 55°C, 20bar
for >20 hours. Once the reaction was complete the
ammonia was removed and the resulting solid
recrystallised from boiling ethyl acetate and petrol.
ASSAY FOR PARP INHIBITORY ACTIVITY
Compounds of the present invention, particularly
those detailed in the preceding Examples, have been
tested in vitro for activity as PARP inhibitors using the
following methods and materials.
CA 02350941 2001-07-05
17
In principle, the PARP assay used relies upon
activating endogenous PARP (as hereinafter described) in
cells containing exogenous [32P]-NAD+ introduced therein
by suspending the cells in a solution of [32P]-NAD+ to
which they have been rendered permeable in an initial
pre-treatment step. The poly(ADP-ribose) which is then
synthesised by the enzyme can be precipitated by tri-
chloracetic acid (TCA) and the amount of radio-labelled
3zP incorporated therein measured, e.a. using a
scintillation counter, to give a measure of the activity
of the PARP under the particular conditions of the
experiment. By repeating the experiment following the
same procedure, and under the same conditions, in the
presence of each compound to be tested the reduction in
enzyme activity, represent-ative of the inhibitory effect
of the test compound, can then be ascertained from the
reduction, if any, of the amount of [32PJ measured in the
TCA precipitated poly(ADP-ribose).
The results of this assay may be expressed in terms
of percentage inhibition or reduction in activity for one
or more different concentrations of each compound tested,
or it may be expressed in terms of that concentration of
the tested compound which reduces the enzyme activity by
500, i.e. the ICSO value. Thus, with a range of different
compounds a set of comparative values for inhibitory
activity can be obtained.
In practice, L1210 murine leukaemia cells were used
as the source of the PARP enzyme after being rendered
permeable to exogenous [3zP]NAD by exposure to hypotonic
buffer and cold shock. In the preferred technique
developed, which has been found to give exact and
reproducible results, a defined amount of a small
synthetic oligonucleotide, in particular a single strand
oligo-nucleotide having the palindromic sequence
_ CGGAATTCCG, is introduced into the cell suspension for
activating the PARP enzyme. This oligonucleotide
sequence snaps back on itself to form a double-stranded
CA 02350941 2001-07-05
18
molecule with a single blunt end and provides an
effective substrate for activation of PARP. Its
behaviour as a potent activator of the enzyme was
confirmed in the tests carried out.
The experimental protocol adopted, in which a
synthetic oligonucleotide as mentioned above is
introduced as a specific activator of PARP, discriminates
between PARP and other mono-ADP-ribosyltransferases in
the cells. Thus, introduction of such synthetic
oligonucleotides causes a 5 to 6 fold stimulation in the
radioactive label incorporated and this is attributable
solely to PARP activity.
Further details of the assay are given below.
nn -, ~ 1. ,.. ., -, y.
The materials used included the following:
DTT (Dithiothreitol
A 100mM (15.4mg/ml) solution (for use as an anti-
oxidant) was made up, divided into 500u1 aliquots
and stored at -20°C.
Hypotonic buffer:
9mM Hepes (214mg/100m1)
4.5o Dextran (4.5g/100m1)
4.5mM MgCl2 (92mg/100m1)
The above ingredients were dissolved in about
80m1 distilled water, pH was adjusted to 7.8
(NaOH/HC1), the solution was then made up to 100m1
with distilled water, and stored in a refrigerator.
DTT was added to 5mM just before use (50u1/ml).
Isotonic buffer:
40mM Hepes (1.9g/200m1)
_ 130mM KC1 (1.94g/200m1)
4o Dextran (8g/200m1)
2mM EGTA (152mg/200m1)
CA 02350941 2001-07-05
19
2.3mM MgCl2 (94mg/200m1)
225mM Sucrose (15.39g/200m1)
The above ingredients were dissolved in about
150m1 distilled water, pH was adjusted to 7.8
(NaOH/HCl), the solution was then made up to 200m1
with distilled water and stored in a refrigerator.
DTT was added to 2.5mM just before use (25u1/ml).
NAD
NAD was stored as a solid in pre-weighed
aliquots at -20°C. From these, solutions of a
concentration of approximately 6mM (4-4.5mg/ml) were
freshly made up shortly before performing an assay,
and the molarity was checked by measuring the
optical density (O. D.) at 260nm. The stock solution
was then diluted with water to give a concentration
of 600~M and a small amount of 32P labelled NAD was
added (e. g. 2-5u1/ml).
Oliaonucleotide
The oligonucleotide having the palindromic
sequence CGGAATTCCG, synthesised by conventional
means, was vacuum dried and stored as pellets in a
freezer. Before use, it was made up to 200ug/ml in
lOmM Tris/HC1, pH 7.8, with each pellet being
dissolved completely in 50m1 of buffer. The
solution was then heated to 60°C in a water bath for
15 minutes, and allowed to cool slowly to ensure
correct reannealing. After adding 9.5m1 of buffer,
the concentration was checked by measuring the
optical density of a diluted sample at 260nm. The
main solution was then diluted to a concentration of
200ug/ml and stored in 500u1 aliquots in a freezer,
ready for use.
TCA
Solutions of TCA (Trichloroacetic acid) were
prepared at two concentrations. loo TCA + 10%
CA 02350941 2001-07-05
sodium pyrophosphate, and to TCA + 1% sodium
pyrophosphate:-
Cells
The L1210 cells used as the source of the PARP
5 enzyme were maintained as a suspension culture in
RPMI medium + 10% foetal bovine serum + glutamine
and antibiotics (penicillin and streptomycin).
HEPES and sodium bicarbonate were also added, and
the cells were seeded in 100m1 of medium such that
10 there would be a concentration of approximately 8 x
105/ml at the time of carrying out an assay.
Mot-hnrl
The compounds being tested were generally made up as
a concentrated solution in DMSO (Dimethyl sulphoxide).
15 The solubility of the compound was then checked by adding
a quantity of the DMSO solution to a quantity of the
isotonic buffer, in the required final proportions that
were to be used in carrying out the assay, and after an
interval the solution was examined under a microscope for
20 any signs of crystals forming.
A desired quantity of the cells, ascertained by
counting with a haemocytometer, was then centrifuged
(1500rpm in a "Europa" model 24M centrifuge for 5
minutes), the supernatant removed, and the pellets
obtained were resuspended in 20m1 Dul A at 4°C before
centrifuging again at 1500rpm and 4°C. After again
removing the supernatant, the cells were resuspended at a
concentration of 3 x 107 cells/ml in ice cold hypotonic
buffer and left for 30 minutes on ice. Nine volumes were
then added of ice cold isotonic buffer, and the cells,
now rendered permeable to exogenous NAD+, were then used
within the next hour for carrying out an assay. The
permeablisation of the cells may be checked at this stage
by adding duplicate aliquots of cells to an equal volume
of trypan blue, leaving for 5 minutes and then counting
on a haemocytometer.
CA 02350941 2001-07-05
21
The assay was then carried out using for convenience
plastic l5ml conical bottomed assay tubes set up in a
shaking water bath at 26°C which is the optimum
temperature for this enzyme. In a typical assay using
the oligonucleotide solution at a concentration of 5ug/ml
and the test compound/DMSO solution at a concentration of
2%, and carrying out the assay in quadruplicate, there
would then be placed in each assay tube 5ul of the
oligonucleotide solution, 50u1 of the 600um NAD + [3zP]-
NAD solution, 8ul of the test compound/DMSO solution, and
37u1 of water. Prior to the start of the experiment this
"cocktail" would be pre-warmed for 7 minutes at 26°C, as
would be also the cell suspension. The reaction would
then be started by adding 300u1 of the cell suspension.
The reaction would be stopped by adding 2m1 of the 10%
TCA + 10% sodium pyrophosphate solution.
In addition to the above, six assay tubes would
usually be set up as blanks, these containing the same
ingredients as above but, before adding the cell
suspension, TCA solution is added to prevent any reaction
from taking place. This enables corrections to be
applied for any non-specific binding of the labelled
material to the filter used (see below).
After adding the cell suspension at timed intervals
to each of the assay tubes, the 10% TCA + 10% sodium
pyrophosphate at 4°C was added to each assay tube exactly
5 minutes after addition of the cell suspension to that
tube. Then, after leaving the tubes on ice for a minimum
time of one hour, the contents of each individual tube
were filtered through an individual filter funnel of a
suction filter apparatus using GF/C filter elements
(rough side up) wetted with 10% TCA. After filtering the
contents of each tube and rinsing the filters several
times with 1% TCA + 1% sodium pyrophosphate solution, the
35_ filters were carefully removed and dried before being
placed in individual scintillation vials. Four
additional 'scintillation vials were also set up as
CA 02350941 2001-07-05
22
reference standards containing l0ul of the 600uM NAD +
[3zP]-NAD solution; lOml scintillant then being added to
each vial. Counting was carried out for 2 minutes on a
counter to obtain measures of the 3zP present, and thus
the amount of the poly(ADP-ribose) and activity of the
PARP enzyme.
RESULTS OF IN VITRO PARP INHIBITION STUDIES
Apart from applying the PARP enzyme assay in
accordance with the standard procedure outlined above to
a range of compounds which have been made in accordance
with the present invention, for comparison purposes it
was also applied to certain benzamide compounds, in
particular 3-hydroxybenzamide, 3-methoxybenzamide and 3-
aminobenzamide, that are already known to exhibit certain
PARP inhibitory activity. A full tabulated list of the
compounds which have been made and/or studied is
hereinafter presented in TABLE I, together with the PARP
inhibition assay results obtained in different
experiments for different concentrations of the compounds
when tested using the assay hereinabove described.
In reviewing this list, the known PARP inhibitors 3-
aminobenzamide, 3-methoxybenzamide and 3-hydroxybenzamide
may be regarded as reference compounds. Although there
is considerable variation in activity, and in some cases
at least the higher concentrations for aqueous solutions
of the test compounds could not be achieved because of
low solubility, in general the compounds of the present
invention which were tested showed a useful degree of
activity. Of especial interest were the benzoxazole
analogues, particularly those having the reference
numbers NU1056, NU1040, NU1051 and NU1054, which showed
relatively high inhibitory activity even at low
concentrations.
In contrast to the results obtained for the
compounds of the present invention, which have in many
CA 02350941 2001-07-05
23
cases showed PARP enzyme inhibitory properties that are
well above average-and at least comparable with, if not
considerably better than, those of other known benzamide
PARP inhibitors, various analogous nicotinamide compounds
studied showed no, or very poor, inhibitory activity when
tested in the same manner at similar concentrations.
FURTHER BIOLOGICAL ACTIVITY STUDIES
Again using cultures of the murine leukaemia L1210
cell line, growth inhibition experiments were carried out
to assess the cytostatic effects of the compounds and
clonogenic survival assays were performed to assess
cytotoxicity, especially in 'relation to use of the
compounds in conjunction with DNA damaging cytotoxic
agents such as cytotoxic antitumour drugs or high energy
radiation, e.g. gamma ray irradiation. DNA damage and
the effect of the PARP inhibitors on the process of DNA
strand break formation and repair has also been assessed
by carrying out DNA strand break assays and monitoring by
alkaline elution in accordance with published techniques.
In the growth inhibition assays, typically the L1210
cells would be seeded at 1 x 104/ml in triplicate in 24
well multidishes, and 24 hours later the compounds or
drugs being tested would be added in selected
combinations and concentrations. At this time one set of
replicates would be counted using a Coulter counter (No),
and 48 hours later the remaining samples would be counted
(N1). The percentage (%) growth inhibition of drug-
treated samples could then be estimated. In drug
combination experiments, where evidence of synergistic
effects on cell growth or clonogenicity was being sought,
a single, fixed concentration of a cytotoxic drug sample,
e.g. temozolomide, would be taken as the control value.
Overall, it is believed that the studies carried out
give clear evidence that the PARP inhibitory character-
istics of compounds of this invention reflects an ability
CA 02350941 2001-07-05
24
of these compounds to potentiate the cytotoxicity of DNA
damaging agents such as certain cytotoxic antitumour
drugs and radiation used in radiotherapy. Accordingly,
such compounds should be especially useful for
administration in conjunction with such cytotoxic drugs
or radiotherapy to potentiate their effect in the course
of medical treatment as hereinbefore indicated.
Summary
Although the present invention should be regarded
overall as comprising each and every novel feature or
combination of features disclosed herein, the main
aspects of the invention comprise, principally but not
exclusively, broadly the following:-
(i) Novel compounds of formula (I) or (IV) as
defined herein;
(ii) Compounds of formula (I) or (IV) with
substituents as hereinbefore defined (including
salts thereof) for therapy or for use in
medicine and in the manufacture of medical
preparations, useful for example as PARP
inhibitors to be administered in conjunction
with cytotoxic drugs or with radiotherapy to
potentiate the effectiveness of the
latter in treatment of cancer;
(iii) Processes for the preparation of novel
compounds of formula (I) or (IV) as defined
herein, including any novel intermediate
compounds produced in carrying out such
processes;
(iv) Pharmaceutical formulations comprising a
compound of formula (I) or (IV) as defined
herein together with a pharmaceutically
acceptable carrier therein;
(v) Processes for the preparation of a
pharmaceutical formulation as defined in (iv)
above, e.g. by methods referred to herein.
CA 02350941 2001-07-05
TABLE I
House Name Structure % Inhibition
Number
lOltM 301tM 1001.eM
Ref 3-hydroxybenzamide O 35 59 81
C~H7N0z / NHz
MW = 137 \
OH
Ref 3-methoxybenzamide O 55 78 89
CgH9N02 . I \ , NHZ
MW = 151 /
OCH3
Ref 3-aminobcnzarrtide O 36 63 79
C~HgN202 . / NHZ
MW= 136 . \ , ,
NH2
NU 1034 2,3-methylenedioxy ~ 66
benzam idc O 94
/ ~ ~2 ICsp = 53~M
CgH~N03
MW = 165 O
O-
NU 1040 2-t-butyibenzoxazote- O 87 88 93
4-carboxam ide
/ N~ ICsp = 8.4~M
C t2H t aN2~
MW = 218
gut
CA 02350941 2001-07-05
26
TABLE I (contd.)
House Name Structure % Inhibition
Number
tnmM Zn~~A,f tnn..~,~
_ _ _~ _ __
NU 1 OS 1 2-phenylbenzoxazole- O 82
4-carboxam ide
C H N O . / I NHz lCso ° 2.1 ~M .
I4 10 2 2
MW=238 N
. /
NU 1054 2-(4-methoxyphenyl)
benzoxazote-4- ICsp = 1.1 ~M
carboxam ide
C H N O
IS 12 2 2
MW = 252
y ~~.
NU 1056 2-methytbenzoxazote-4- O
carboxamide ~ ICso = 9.S~M
/ ~NH2
C9HgN202
MW = l76 N
CH3
