New poly(ADP-ribose) polymerase-1 inhibitors with antioxidant activity based on 4-carboxamidobenzimidazole-2-ylpyrroline and-tetrahydropyridine nitroxides and their precursors

Article abstract of DOI:10.1021/jm801476y

4-Carboxamidobenzimidazoles were previously described as PARP inhibitor compounds. Here we report upon 4-carboxamido-1H-benzimidazoles substituted in the 2-position with nitroxides or their amine or hydroxylamine precursors. Among the new molecules, a hig

Full text of DOI:10.1021/jm801476y

J. Med. Chem. 2009, 52, 1619–1629
1619
New Poly(ADP-ribose) Polymerase-1 Inhibitors with Antioxidant Activity Based on
4-Carboxamidobenzimidazole-2-ylpyrroline and -tetrahydropyridine Nitroxides and Their
Precursors
Tama´s Ka´lai,† Ma´ria Balog,† Al´ız Szabo´,‡ Gergely Gulya´s,‡ Jo´zsef Jeko˝,§ Bala´zs Su¨megi,‡ and Ka´lma´n Hideg*,†
Department of Organic and Medicinal Chemistry and Department of Biochemistry and Medical Chemistry, UniVersity of Pe´cs, H-7624 Pe´cs,
Szigeti str. 12, Hungary, and Department of Chemistry, College of Ny´ıregyha´za, 4440 Ny´ıregyha´za, So´sto´i str. 31/B, Hungary
ReceiVed August 12, 2008
4-Carboxamidobenzimidazoles were previously described as PARP inhibitor compounds. Here we report
upon 4-carboxamido-1H-benzimidazoles substituted in the 2-position with nitroxides or their amine or
hydroxylamine precursors. Among the new molecules, a highly active PARP inhibitor 4h (IC50 ) 14 nM)
was identified with antioxidant/radical scavenger activity. We concluded that in most cases sterically hindered
amines are better PARP inhibitors than their oxidized form and structural changes in the 2-substituted
4-carboxamido-1H-benzimidazoles (such as N-substitution or changing the position of the carboxamide
group) were detrimental to PARP inhibition activity but not to antioxidant activity. These results indicate
the advantages of combining an antioxidant nitroxide or nitroxide precursor with a PARP inhibitor molecule
to decrease or eliminate the deleterious processes initiated by reactive oxygen and reactive nitrogen species
(ROS and RNS). The radical scavenging capability of 4h was demonstrated by EPR study of urine collected
after drug administration.
Introduction
Poly(ADP-ribose) polymerase-1 (PARP-1a) (EC 2.4.2.30) is
an abundant DNA-binding protein activated by damages to DNA
structure that occur during inflammation, ischemia, neurode-
generation, hemorrhagic shock, exposure to genotoxic agents
and other pathophysiological conditions affiliated with oxidative
stress.1,2 Activated PARP-1 consumes NAD+ which is cleaved
into nicotinamide and ADP-ribose and polymerizes later into
nuclear acceptor proteins such as caspases, topoisomerases,
histones, and PARP itself. This process plays a key role in the
genomic stability. Overactivation can lead to depletion of NAD+
Figure 1. Interactions of 4-carboxamidobenzimidazoles with PARP-1
enzyme active site.
and ATP which results in cell dysfunction and ultimately
necrotic cell death.3 A cellular suicide mechanism of necrosis
and apoptosis by PARP activation has been implicated in the
pathogenesis of brain injury and neurodegenerative disorders.
This observation is confirmed also by the fact that PARP-1
knockout mice were found to be fertile and normally developing
but resistant to cerebral ischemia. These findings suggest that
in the absence of DNA damage, PARP is not necessary for
survival.4 Therefore PARP-1 inhibition is a promising mecha-
nistic target for drug development in the context of various forms
of inflammation, ischemia, and cancer therapy. The protective
effect of PARP-1 inhibitors in a number of experimental models
of human diseases, caused by oxidative or nitrosative stress and
consequent PARP-1 activation, suggests that these compounds
can offer therapeutic advances either to prevent or to slow down
disease progression.5,6 The majority of PARP inhibitors bind
to the nicotinamide binding site; e.g., they are competitive
inhibitors of NAD+. It is well-known that nicotinamide forms
three hydrogen bonds in the catalytic region of PARP-1, one to
the hydroxyl group of Ser904 and two to the backbone amides
of Gly863. In addition, π-π interactions with Tyr 907 of the
aromatic moiety is also important (Figure 1).7 The proper
interaction requires an anti conformation of the amide carbonyl
group which is in equilibrium with the syn conformation. The
first PARP-1 inhibitors were nicotinamide and 3-aminobenza-
mide as benchmark inhibitors,8 but because of their low potency,
low aqueous solubility, and low specificity, new PARP inhibitors
were needed. The low potency was attributed to the flexibility
of the carboxamide group, and therefore, synthesis of confor-
mationally restricted PARP inhibitors, e.g., lactams or hetero-
cyclic compounds, resulted in more potent compounds by 2-4
orders of magnitude.1,9 The carboxamide group rotation can also
be restricted by intramolecular hydrogen bond as in the case of
benzoxazole-4-carboxamides and benzimidazole-4-carboxam-
ides.10
* To whom correspondence should be addressed. Phone: (36) (72)
536220. Fax: (36) (72) 536219. E-mail: kalman.hideg@aok.pte.hu.
†
Department of Organic and Medicinal Chemistry, University of Pe´cs.
Department of Biochemistry and Medical Chemistry, University of
‡
Pe´cs.
§
Although several classes of PARP inhibitors move toward
clinical development, new compounds are still needed.11 In our
previous studies we found that modification of cardiovascular
drugs, such as mexiletine,12 amiodarone,13 or trimetazidine,14
with pyrroline nitroxide precursors provided the parent com-
College of Ny´ıregyha´za.
a Abbrevations: DMF, dimethylformamide; DTT, dithiotreitol; EPR,
electron paramagnetic resonance; ip, intraperitoneally; MTT+, 3-(4,5-
dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; PARP, poly(ADP-
ribose) polymerase; PDB, Protein Data Bank; ROS, reactive oxygen species;
RNS, reactive nitrogen species.
10.1021/jm801476y CCC: $40.75
2009 American Chemical Society
Published on Web 02/26/2009

1620 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6
Ka´lai et al.
gation,22 and (3) the development of a similar PARP inhibitor
2-(1-propyl-4-piperidinyl)-1H-benzimidazol-4-carboxamide (ABT-
472)23 prompted us to synthesize and investigate the 4-car-
boxamidobenzimidazole series modified with nitroxides and
their precursors. Here, we report some structure-activity
relationships of these compounds as PARP inhibitors.
Chemistry
Benzimidazoles were achieved by heating a mixture of
paramagnetic alicyclic aldehyde or paramagnetic aromatic
aldehyde (2a-p) with 2,3-diaminobenzamide (1)10 in the
presence of a catalytic amount of p-toluenesulfonic acid in
toluene, followed by oxidation of the crude condensed product
with excess activated MnO2 in CHCl3 to give compounds
3a-n.24 This procedure, however, suffers from the low solubil-
ity of 2,3-diaminobenzamide in toluene and some aldehydes;
therefore, low yields were achieved (10-44%). The paramag-
netic benzimidazoles 3a-k were reduced by Fe powder in
AcOH to sterically hindered amines 4a-k.25 A very convenient
and alternative procedure for the above synthesis of diamagnetic
benzimidazoles is the heating of 2a-p aldehydes with 2,3-
diaminobenzamide (1) in the presence of an equivalent amount
of Na2S2O5 in DMF to yield 3l-n and 4a-m 2-substituted
benzimidazoles from moderate to good yield (55-81%) (Scheme
1 and Tables 1-4).26 Aldehydes 2a,27 2b,28 2c,29 2d,30 2g,30
2h,31 2i,32 2k,29 2l,33 2o34 were synthesized earlier, while
aldehyde 2f, 2n, and 2p were achieved by oxidation of alcohols
5,30 6,35 and 2636 with MnO2. The Suzuki cross-coupling
reaction30 of 2c vinyl bromide and 3-trifluoromethylphenylbo-
ronic acid in the presence of PdCl2(PPh3)2 and Ba(OH)2 in water/
dioxane mixture afforded aldehyde 2e. Acetylating of secondary
nitrogen of compound 733 with acetyl chloride gave aldehyde
2m, while alkylation of 8 vanillin in the presence of K2CO3 in
acetone yielded paramagnetic aldehyde 2j (Scheme 2). The
efficiency of 2-mercaptoquinazoline derivatives inspired us to
synthesize a series of S-alkylated 2-mercapto-4-carboxamido-
benzimidazoles as outlined in Scheme 3. The 2-mercapto-4-
carboxamidobenzimidazole 10 was synthesized by treatment of
compound 1 with CS2 in THF in the presence of a catalytic
amount of NaOMe. Alkylation of compound 10 with allylic
bromides 9 and 11 in methanol in the presence of KOH gave
compounds 12a and 12b. Reduction of 12a,b radicals with Fe
powder in AcOH yielded 13a,b sterically hidered amines.
Figure 2. Possible radical scavenging mechanisms and transformations
of nitroxides and prenitroxides.
pounds with additional antioxidant and radical scavenging
activity. For example, alkylation of trimetazidine secondary
amine with a 2,2,5,5-tetramethyl-2,5-dihydropyrrolin-3-ylmethyl
group provided protection from ischemia-reperfusion induced
contractile dysfunction. This approach well suits the new stream
of drug research,15 e.g., incorporation of two drug pharmacoph-
ores in a single molecule with the intention to exert dual
(cardiovascular and antioxidant) action. We considered the
combination of nitroxides and their sterically hindered amine
precursors with PARP inhibitors, realizing that most of the
deleterious processes resulting from PARP activation are initiated
by harmful ROS and RNS. These types of compounds would
inhibit not only poly-ADP-ribosylation but simultaneously would
suppress or decrease the harmful effect of initiator ROS and RNS
as well.
Cyclic nitroxides can be regarded as synthetic, multifunctional
antioxidant molecules, owing their unique features to either their
reduced forms, hydroxylamines, or their oxidized forms oxoam-
monium cations.16 It has been shown a decade ago17 that
nitroxides form superoxide dismutase mimetic oxoammonium
cations, followed by reduction of the latter species with
superoxide (eqs 1 and 2 of Figure 2). Hydroxylamines can act
as proton and electron donor molecules, reducing any radical
species (eq 3 of Figure 2). Nitroxides are also capable of
For further study of the structure-activity relationships,
additional structural modifications were made on the benzimi-
dazole part. In this new compound series, N-alkylation on
benzimidazole NH, removal of the carboxamide group, or
alteration of its position was attempted. N-Alkylation of
compound 1410 with allylic bromide 937 in acetonitrile in the
presence of K2CO3 gave 1,4-disubstituted benzimidazole 15.
Reaction of o-phenylenediamine (16) and with aldehyde 2h in
the presence of an equivalent amount of Na2S2O5 in DMF
furnished 2-substituted benzimidazole 17 without a carboxamide
group. The N-alkylated carboxamide was achieved by acylation
of isopropylamine with imidazolide of 18 carboxylic acid10
followed by reduction of the nitro group of compound 19 to
give 2,3-diamino-N-isopropylbenzamide (20). Reaction of com-
pound 20 with 2a aldehyde in the presence of p-toluenesulfonic
acid in benzene followed by oxidation with activated MnO2
yielded compound 21. Reduction of nitroxide 21 with powdered
iron in glacial acetic acid furnished compound 22. Compounds
24 and 25, the isomers of compounds of 3h and 4h, respectively,
•
inhibiting OH formation by oxidizing Fe2+ to Fe3+ and hence
preventing its participation in Fenton reactions (eq 4 of Figure
2).18 The fully reduced form of the nitroxide, a sterically
hindered amine, is easily oxidized to nitroxide by various ROS
19
(eq 5 of Figure 2). This nitroxide is in an equilibrium with
the hydroxylamine depending upon the oxidative or reductive
nature of its environment (eq 6 of Figure 2).20
Although 2-substituted 4(3H)-quinazolinone and 4-substituted
2H-phthalazin-1-one derivatives with nitroxide ring substituents
were good antioxidants, these modifications resulted in reduced
PARP inhibitory activity.21 The fact that (1) the biological
activity of cyclic nitroxides is highly dependent on substituents
borne upon the ring, (2) in preliminary studies 2-(2,2,6,6-
tetramethyl-1,2,3,6-tetrahydro-pyridin-4-yl)-1H-benzimidazole-
4-carboxamide (4h) reduced the ADP-induced platelet aggre-

PARP-1 Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6 1621
Table 1. Data for Compounds 3a-j
were obtained by reaction of 3,4-diaminobenzamide (23)38 and
2h aldehyde analogously as described above (Scheme 4).
and N-acetyl (3m), N-methyl (3l), and N-O-alkyl (3n) substi-
tuents, and a six-membered ring with a secondary amine (NH)
(4h). Compounds with substituents (Br, Ph, CO2CH3, dibenzo-
furane) on the 4-position of the pyrroline ring are less effective.
In the PARP inhibition assay, longer spacers such as 4-phe-
noxymethylene (3i, 3j, 4i, 4j) or condensed ring spacers (3k,
4k, 4o, 4p) are not well tolerated; the IC50 for these compounds
is over 100 nM. The exception from the above tendencies are
compounds 3a and 4a without any substituent on the nitroxide
ring and without any spacer, featuring 721 and 345 nM IC50
values, respectively. Compounds 3h and 4h were found to be
the best-performing PARP inhibitors. Compound 3h was docked
into the PARP-1 enzyme’s active site and fits quite well. There
are five H-bonds between the enzyme and substrate: the
carboxamido groups form H-bonds with Ser904 and with
Gly863; water mediated H-bond between benzimidazole NH
and Glu988; nitroxide oxygen with Gln763; π-π stacking
interactions with Tyr907 (Figure 3). The docking was demon-
strated with the 3h nitroxide form because it is the metabolite
of compound 4h (see below). Compounds with a thiomethylene
Results and Discussion
Compounds in Tables 1-4 exhibited IC50 for PARP inhibition
in the range of 14 nM to 10 µM in the whole cell system assay
and IC50 values in the range from 9 nM to 60 µM. In the in
vitro antioxidant assay, all the compounds exhibited IC50 in the
range of 0.5 nM to 0.35 µM. During the structure-activity
relationship monitoring we changed the size of the nitroxide
ring (five- or six-membered), the saturation of the ring, the
substituents on the pyrroline ring, the spacer between the
nitroxide ring and the benzimidazole ring, and the oxidation
status of the ring nitrogen. The effect of substitution changes
in the benzimidazole ring also has been tested.
Regarding the PARP enzyme inhibition, the following
compounds were found to be the most effective: 4-carboxami-
dobenzimidazoles with 2-substituents as a five-membered ni-
troxide with a phenyl spacer (3f), six-membered nitroxide
without a spacer (3h), five-membered rings without a spacer

1622 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6
Table 2. Data for Compounds 3k-n and 4a-ga
Ka´lai et al.
a For definition of the R groups, see the top structure in Table 1.
spacer and five-membered ring (13a, 13b) or with six-membered
ring (14a, 14b) had weaker inhibitory activity. Alkylations of
benzimidazole NH or carboxamide nitrogen were detrimental
to PARP inhibitory potency, as we experienced for compounds
15, 21, 22. Compounds 17, 24, and 25 were prepared to examine
the influence of the carboxamide moiety on PARP inhibitory
activity. As we presumed, removal of carboxamide and changing
its position from 4 to 5 on the benzimidazole ring resulted in a
loss of PARP inhibitory activity.
Cell death inhibition usually provides information on the
cellular potency of the compound investigated. Surprisingly,
there was a low correlation between cell death inhibition results
and PARP inhibition results. The cell death inhibitory IC50
values are below 100 nM, the most effective ones being 3c and
4c with a bromine substituent on pyrroline ring, 3h with a six-
membered-ring, 3k and 4k with a thienyl ring spacer, and 4j
with a 4-phenoxymethylene spacer. Compounds 13a, 13b, 14a,
14b with an -SCH2- spacer did not exhibit antinecrotic activity
as well as compounds without carboxamide function (17) or
with dispositional carboxamide function (24, 25). The alkylation
of carboxamide nitrogen with an isopropyl group did not
decrease cell death inhibitory activity, compared to compounds
3a and 4a. From these results we conclude that the cell death
inhibition, tested as protection of WRL-68 human liver cells
from H2O2 induced cell death, does not exclusively reflect on
PARP inhibitory activity but includes other protective mecha-
nisms of tested compounds as well. This observation inspired
us to study hydroxyl radical scavenging activity of the new
PARP inhibitors in a Fenton reaction. In this assay the water-
soluble hydroxylamines were used and may reduce hydroxyl
radicals to water while being oxidized to stable nitroxide free
radicals. The formed nitroxides can also oxidize Fe2+ to Fe3+
and hence prevent its participation in the reaction (Figure 2,
eqs 3 and 4). Sterically hindered amines can be oxidized to
nitroxides (Figure 2, eq 5). These multiple ways of preventing
•OH formation in the Fenton reaction justify the very low IC50
values measured for studied compounds, which were mostly
below 10 nM. This assumption confirmed that compounds 3l,
3m, 3n with higher IC50 values (over 70 nM) having blocked
NH and NOH functions were unable to react with hydroxyl
radicals or participate in electron transfers of Fenton reaction.
Hydroxyl radical scavenging activities of compounds with

PARP-1 Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6 1623
Table 3. Data for Compounds 4h-k,o,p, 13a,b, 14a,b, and 15a
a For definition of the R groups, see the top structure in Table 1.
-SCH2- spacer (13a, 13b, 14a, 14b) or with a condensed
aromatic ring spacer (3k, 4k, 4o, 4p) were found to be limited.
On the basis of nitrogen oxidative status, obvious structure-
activity relationships cannot be drawn yet; compound 4h appears
to be the best antioxidant and PARP inhibitor regarding the
PARP enzyme inhibition, cell death inhibition, and hydroxyl
radical-scavenging results. The toxicity of 4h was further
assessed on eight mice and in comparison with a five-membered
analogue (4a). The LD50 for 4a and 4h were 620 and 740 mg/
kg, respectively, both being higher than 500 mg/kg, the
acceptable therapy index.
Metabolism Study of Compounds 3h and 4h. During the
metabolism studies HCl salts of both compounds (3h, 4h) were
administered to rats intraperitoneally (ip) in 8 mg/kg doses, and
urine samples were collected before administration, for 4 h after
administration, and after 4 h till 18 h. An isotropic triplet in
the EPR spectra demonstrates the presence of nitroxide radicals
and thus the oxidative metabolism of sterically hindered amine
4h; i.e., it is metabolized to 3h in vivo. The concentration of
3h in the urine sample collected for 4 h is 0.7 nM, referenced
to 10 nM solution of 2h; however, the EPR absorption increased
almost 3-fold upon adding PbO2 to urine samples, indicating
the presence of not only nitroxide but also hydroxylamines
oxidizable to nitroxides. The final concentration of radical
species was 1.7 nM (Figure 4). The ip administered hydroxy-
lamine HCl salt of 3h to rats and collection of their urine showed
that hydroxylamine mostly (74%) oxidized to nitroxide after
4 h (the concentration was 87 nM, based on EPR measurements),
and only 26% remained in the hydroxylamine form. Further
EPR active substances were formed upon addition of PbO2
(Figure 5), and the final concentration of EPR active substance
was 116 nM. On the basis of these observations, we conclude
that sterically hindered secondary amine moiety of PARP
inhibitor 4h is oxidized to nitroxide 3h, while nitroxide 3h and
its hydroxylamine are in equilibrium in the rat model. Hydroxy-
lamines are rapidly ((0.2-1.2) × 104 M-1) oxidized by ROS
•-
(O2 or peroxynitnitrite or their decomposition products),
although autoxidation during sample collection cannot be
excluded either. More detailed studies demonstrated the oxida-
tion of hydroxylamines to nitroxide with rates influenced by
ring size and substituents of the nitroxide.39 Saito et al.
demonstrated that both hydroxylamine and nitroxide forms are

1624 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6
Table 4. Data for Compouds 17, 21, 22, 24, and 25a
Ka´lai et al.
a For definition of the R groups, see the top structure in Table 1.
Scheme 1. Synthesis of
partially reduced to hydroxylamine. It is interesting to note that
this structure is very similar to one that was synthesized
independently by an American research group in the 4-carboxa-
midobenzimidazole series, namely, 2-(1-propynylpiperidin-4-
yl)-1H-benzimidazole-4-carboxamide (ABT-472).42 Further bio-
logical studies of compound 4h are currently underway.
2-Substituted-4-carboxamidobenzimidazolesa
Experimental Procedures
Melting points were determined with a Boetius micro melting
point apparatus and are uncorrected. Elemental analyses (C, H, N,
S) were performed on Carlo Erba EA 1110 CHNS elemental
analyzer. Mass spectra were recorded on an Automass Multi
instrument in the EI mode (70 eV, direct inlet) or on a VG TRIO-2
instrument with thermospray technique. ESR spectra were obtained
from 10-5 molar solutions (CHCl3), using a Magnettech MS200
spectrometer, and all monoradicals gave triplet line aN ) 14.7-16.5
G. Preparative flash column chromatography was performed using
a Merck Kieselgel 60 (0.040-0.063 mm). Qualitative TLC was
carried out on commercially prepared plates (20 cm × 20 cm ×
a Reagents and conditions: (a) (i) TosOH (cat.), benzene, reflux, 8 h;
(ii) activated MnO2, CHCl3, reflux, 2 h; (b) Fe, AcOH, heated to reflux, 30
min, then K2CO3 to pH 8; (c) Na2S2O5, under N2, sealed tube, DMF, 120
°C, 4 h.
present, although hydroxylamine is more dominant from a
freshly prepared kidney.40 Urine collected between 4 and 22 h
after administration of 3h or 4h practically did not show triplet
EPR signal (data not shown), presumably because the loss of
the PARP-inhibitor metabolites.
1
0.02 cm) coated with Merck Kieselgel GF254. H NMR spectra of
diamagnetic compounds were recorded with a Varian Unity Inova
400 WB spectrometer; chemical shifts were referenced to TMS.
Compounds 1,10 2a,27 2b,28 2c,29 2d,30 2g,30 2h,31 2i,32 2k,29 2l,33
2o,34 5,30 6,41 7,27 9,31 11,38 14,10 18,10 2338 were prepared as
described earlier, and all other reagents and compounds were
purchased from Aldrich or Fluka. All the compounds purchased or
synthesized exhibited g95% purity by combustion analysis.
Biological Activity Studies. For biological studies the com-
pounds were converted to water-soluble hydrochloride salts.
Compounds 3a-k, 13a, 14a, 16, 18, and 23 were refluxed for 15
min in ethanol, saturated previously with HCl gas. After evaporation
of the solvent the hydroxylamine salt was crystallized from acetone
or ether. Compounds 3l-n, 4a-p, 13b, 14b, 19, 31, 24 were
dissolved in ethanol, saturated previously with HCl gas. After
evaporation of the solvent the salt was crystallized from acetone
or ether.
Conclusions
In summary, we reported the synthesis and study of a series
of 4-carboxamidobenzimidazole PARP-1 inhibitors carrying
nitroxides and their precursors in the 2-position of the benz-
imidazole ring.41 In structure-activity studies we found that
five- or six-membered rings without substituents on the nitroxide
ring and without spacer between benzimidazole and nitroxide
moieties were the most efficient PARP-1 inhibitors. The results
of PARP inhibition and antioxidant studies did not correlate
because the cell death inhibition is based not only on the PARP
enzyme inhibition but probably on the ROS scavenging activity
also. Compound 4h was found as a potent PARP inhibitor (IC50
) 14 nM) with antiapoptotic (IC50 ) 98 nM) activity and an
acceptable therapy index LD50 > (500 mg/kg). The structure
of 4h is a six-membered, sterically hindered amine connected
directly to the 2-position of 4-carboxamidobenzimidazole and
lacking substituents on the cyclic amino moiety. On a rat model
we evaluated the metabolism of compound 4h and found that
during its oxidative metabolism a nitroxide is formed that is
Assay To Test Inhibitory Effects of Benzimidazole Deriva-
tives on PARP Enzyme in Vitro. Poly(ADP-ribose) polymerase
was isolated from rat liver based on a known method.43 The
potential inhibitory effect of benzimidazole derivatives was tested
using this assay system. The PARP activity was determined in 130
µL of reaction mixture containing 100 mM Tris-HCI buffer, (pH

PARP-1 Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6 1625
Scheme 2. Synthesis of New Paramagnetic Aldehydesa
a Reagents and conditions: (a) activated MnO2, CHCl3, reflux, 2 h; (b) 3-(CF3)C6H4B(OH)2, Ba(OH)2 ·8 H2O, PdCl2(PPh3)2, dioxane/water 4:1, reflux, 5 h;
(c) AcCl, Et3N, CH2Cl2, 0 °C to room temp; (d) acetone, K2CO3, reflux, 2 h.
Scheme 3. Synthesis of 2-S-Alkyl-4-carboxamidobenzimidazolesa
a Reagents and conditions: (a) THF, CS2, NaOMe, reflux 1 h, then 12 h, room temp; (b) MeOH, KOH, reflux, 2 h; (c) Fe, AcOH, heated to reflux, 30 min,
then K2CO3 to pH 8.
8.0), 10 mM MgCl2, 10% glycerol, 1.5 mM DTT, 1 mM [adenine-
2,8-3H] NAD+(4.500 cpm/nmol), 10 µg of activated DNA, and 10
µg of histones. The incubation time was l5 min, and the reaction
was stopped by addition of trichloroacetic acid (8%). After addition
of 0.5 mg of albumine, precipitation was allowed to proceed for at
least 20 min on ice, and the insoluble material was collected on
glass filters, washed with 5% perchloric acid. The protein-bound
radioactivity was determined using a LS-200 Beckman scincillation
counter.
Protecting Effect of Compounds against H2O2 Induced Cell
Death, Determined in WRL68-Human Liver Cell Line (Inhibi-
tory Cell Death). Cell Culture. WRL-68 human liver cells were
obtained from the American Type Culture Collection (ATCC,
Rockville, MD). Cell lines were grown in a humidified 5% CO2
atmosphere at 37 °C and maintained in culture as adherent
monolayers using Dulbecco’s modified Eagle’s medium (DMEM)
containing 1% antibiotic-antimycotic solution (Sigma, St. Louis,
MO) and 10% fetal calf serum. Cells were passaged at intervals of
3 days.
Detection of Cell Survival. Cells were seeded into 96-well plates
at a starting density of 2.5 × 104 cells/well and cultured overnight
in a humidified 5% CO2 atmosphere at 37 °C. The following day
0.3 mM H2O2 was added to the medium either alone or in the
presence of 10, 2, 1, 0.5, 0.1, and 0.02 µM of the protecting agents
(benzimidazole derivatives). Three hours later, the medium was
removed and 0.5% of the water soluble mitochondrial dye (3-(4,5-
dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT+) was
added. Incubation was continued for an additional 3 h, after which
the medium was removed and the metabolically reduced water-
insoluble blue formazan dye was solubilized by acidic isopropanol.

1626 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6
Ka´lai et al.
Scheme 4. Synthesis of Isomers of 2-Substituted 4-Carboxamidobenzimidazolesa
a Reagents and conditions: (a) 9, K2CO3, acetonitrile, reflux, 3 h; (b) 2h, Na2S2O5, under N2, sealed tube, DMF, 120 °C, 4 h; (c) CDI, THF, reflux 15 min,
then i-Pr-NH2; (d) Pd/C, HCO2NH4, MeOH, under N2, 40 °C, 2 h; (e) (i) TosOH (cat.), benzene, reflux, 8 h; (ii) activated MnO2, CHCl3, reflux, 2 h; (f) Fe,
AcOH, heated to reflux, 30 min, then K2CO3 to pH 8.
Figure 4. EPR spectra of collected urine before administration of 4h
(· · ·), EPR spectra of collected urine (between 0-4 h) after administra-
tion of 4h (- - -), EPR spectra of collected urine (between 0-4 h) after
administration of 4h (-) and oxidation further with PbO2.
cence λex/λem ) 267/357 nm (Figure 6 in Supporting Information)
Figure 3. Compound 3h (yellow) docked in the active site of PARP1
has no influence on the determination of hydroxybenzoic acids.
Under Fenton reaction conditions in the presence of 4-carboxami-
dobenzimidazole (14), compounds 3h and 4h, and further aromatic
ring containing compounds 3f, 4f, 4g, 3j, 4j, no species were formed
with fluorescence at 407 nm (see Figures 7-14 in Supporting
Information).
Acute Toxicity Studies. C57BL/6 mice were purchased from
Charles River Hungary Breeding Ltd. The animals were kept under
standardized conditions; tap water and mouse chow were provided
ad libitum during the whole experimental procedure. Animals were
treated in compliance with the approved institutional animal care
guidelines. The experiments were conducted by distributing the
rodents into groups of eight mice each that were treated intraperi-
tonially by 100, 200, 300, and 500 mg/kg PARP inhibitors in a
single injection. Mice were observed for the presence of respiratory,
digestive, and neurological alterations. The number of deaths was
noted in each 24 h for 14 days.
enzyme.
Optical densities were determined by an Anthos Labtech 2010
ELISA reader (Wien, Austria) at 550 nm wavelength. All experi-
ments were run in at least 6 parallels and repeated 3 times. Data of
Tables 1-4 are the concentrations of benzimidazoles (in µM) at
which the rate of H2O2-induced cell death was inhibited by 50%.
Hydroxyl Radical Scavenging of Benzimidazole Derivates
(Antiox). Hydroxyl radical formation was detected using the
oxidant-sensitive nonfluorescent probe benzoic acid which is
hydroxylated to 2-, 3-, or 4-hydroxybenzoic acid.44 Hydroxylation
of benzoic acid results in the appearance of intense fluorescence,
which makes possible the fluorescence spectroscopic monitoring
of the hydroxylation reactions (excitation λ ) 305 nm; emission λ
) 407 nm). The reaction was studied in 2.5 mL reaction volumes
containing 20 mM potassium phosphate buffer (pH 6.8), 0.1 mM
benzoic acid, 0.1 mM H2O2, and 20 µM Fe2+-EDTA. Data of
Tables 1-4 show the concentration of benzimidazoles (in µM) at
which the rate of hydroxyl radical induced hydroxylation is inhibited
by 50%. The parent ring (4-carboxamidobenzimidazole) fluores-
Statistical Analysis. Data were presented as the mean ( SEM.
For multiple comparison of groups, ANOVA was used. Statistical
difference between groups was established by paired or unpaired

PARP-1 Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6 1627
reaction mixture was diluted with water (40 mL) and filtered. The
filtrate was basified with solid K2CO3 to pH 8 (intensive foaming).
The aqueous phase was extracted with CHCl3 containing 10%
MeOH (2 × 30 mL), and the combined organic phase was dried
(MgSO4), filtered, and evaporated. The residue was purified by flash
column chromatography (CHCl3/MeOH) to give the title amines
as white solids (48-65%).
2-(2,2,5,5-Tetramethyl-2,5-dihydro-1H-pyrrol-3-yl)-1H-ben-
zimidazol-4-carboxylic Acid Amide (4a). White solid (923 mg,
65%), mp 239-241 °C. Anal. (C16H20N4O) C, H, N. MS m/z (%):
284 (M+, 1), 269 (100), 252 (91), 224 (14). 1H NMR (399.9 MHz,
DMSO-d6): δ 1.27 (s, 6 H), 1.52 (s, 6 H), 6.65 (s, 1 H), 7.29 (t, J
) 7.7 Hz, 1 H), 7.62 (d, J ) 7.9 Hz, 1 H), 7.67 (s, 1 H), 7.80 (d,
J ) 7.4 Hz, 1 H), 9.18 (s, 1 H), 12.91 (s, 1 H). 13C NMR (100.5
MHz, DMSO-d6): δ 30.4, 30.5, 63.7, 66.4, 114.4, 122.3, 122.4,
122.5, 134.3, 136.5, 141.0, 141.1, 148.1, 166.1.
One-Pot Procedure for Synthesis of Amines 4h,o,p and 17
(Procedure C). A solution of compound 1 (750 mg, 5.0 mmol) or
o-phenylenediamine (16) (540 mg, 5.0 mmol) and paramagnetic
aldehyde 2h or 2o or 2p (5.0 mmol) in DMF (10 mL) in a tube
was deoxygenated with N2 for 10 min. Afterward Na2S2O5 (1.14
g, 6.0 mmol) was added, the tube was sealed, and the solution
heated for 4 h by immersing the tube into an oil bath heated to 120
°C. After cooling, the mixture was poured onto ice-water (200
mL) and the precipitated solid was filtered, air-dried, and purified
further by flash column chromatography for analysis. The crude
product was recrystallized from methanol/ether or methanol/acetone
and used for HCl salt formation. Yields ranged from 45% to 79%.
2-(2,2,6,6-Tetramethyl-1,2,3,6-tetrahydropyridin-4-yl)-1H-
benzimidazol-4-carboxylic Acid Amide (4h). White solid (1.17
g, 79%), mp 292-295 °C. Anal. (C17H22N4O) C, H, N. MS m/z
Figure 5. EPR spectra of collected urine before administration of 3h
(· · ·), EPR spectra of collected urine (between 0-4 h) after administra-
tion of 4h (- - -), EPR spectra of collected urine (between 0-4 h) after
administration of 4h and (-) and oxidation further with PbO2.
Student’s test, with Bonferroni correction. Differences with p-values
below 0.05 were considered to be significant.
Modeling PARP Inhibitor (3h) and PARP Enzyme Interac-
tions. The PARP enzyme catalytic fragment (3pax) was downloaded
from the Protein Data Bank, and the PDB crystal structure was
prepared by removing ligand and water. Structure is visualized by
Accelrys DS Visualizer, version 2.0.1.7347.
Oxidative Metabolism Studies with ESR. Male, 290-350 g
Wistar rats (Charles River, Budapest, Hungary) were used in our
study. All animals were housed in wire bottom individual metabolic
cages in order to collect urine. Rats were randomly divided into
two groups. One group was treated with 2 mg, ip, of 4h (HO-
3089) (group 1, n ) 3) one time. A single dose of 3h (HO-3088),
2 mg, ip, was administered to the other group (group 2, n ) 3).
Our agents were completely dissolved in 1.0 mL of 0.9% NaCl.
Urine was collected on three occasions. The first collection was
performed before the treatment. The second fraction was collected
right after the injection to 4 h. The third fraction of urine was
collected from 4 to 22 h after the treatment. The PbO2 treatment of
urine was exploited as follows: to 1 mL of urine, 50 mg of PbO2
was added and vortexed for 1 min, and then the sample was allowed
stay for 10 min and 50 µL from the supernatant was studied by
EPR. ESR analyses were performed on Magnettech MS200
spectrometer (X-band) in 50 µL glass capillaries. Modulation was
1400 mG, sweep was 59.5 G, sweep time was 60 s, gain was 500,
microwave attenuation was 10 dB, and urine was measured directly
after collection.
Synthesis of Compounds 3a-n, 21, 24 (Procedure A). To a
solution of compound 1 or 23 (1.51 g, 10.0 mmol) or 20 (1.93 g,
10.0 mmol) and aldehydes 2a-n (10.0 mmol) in toluene (70 mL)
p-toluenesulfonic acid (100 mg) was added, and the mixture was
heated to reflux under Dean-Stark apparatus to remove the water
formed for a period of 8 h. After the mixture was cooled, the toluene
was evaporated off and the residue was dissolved in CHCl3 (50
mL). Activated MnO2 (4.3 g, 50.0 mmol) was added, and the
mixture was stirred and refluxed for 2 h. After cooling, the mixture
was filtered through a Celite pad and the filtrate was washed with
water (20 mL). The organic phase was separated, dried (MgSO4),
and filtered. The residue was purified by flash column chromatog-
raphy (CHCl3/Et2O) to afford the title compounds (39-73%) as
yellow or white or off-white solids.
1
(%): 298 (M+, 23), 283 (81), 266 (29), 42 (100). H NMR (399.9
MHz, DMSO-d6): δ 1.13 (s, 6 H), 1.22 (s, 6 H), 2.39 (s, 2 H), 6.81
(s, 1 H), 7.26 (t, J ) 7.7 Hz, 1 H), 7.50-7.70 (m, 2 H), 7.79 (d,
J ) 7.3 Hz, 1 H), 9.29 (s, 1 H), 12.82 (s, 1 H). 13C NMR (100.5
MHz, DMSO-d6): δ 29.8, 31.0, 36.8, 48.7, 51.1, 114.4, 121.9, 122.1,
122.3, 123.2, 134.8, 138.3, 141.1, 153.3, 166.3.
2-Mercapto-4-carboxamidobenzimidazole (10). To a solution
of compound 1 (1.51 g 10.0 mmol) and CS2 (760 mg, 10.0 mmol)
in THF (20 mL), NaOMe solution (0.5 mL, 1.0 M stock solution
in MeOH) was added. After refluxing for 1 h, the reaction mixture
was allowed to stay at room temperature overnight. The precipitated
crystals were filtered, washed with Et2O (5 mL), and dried. Yellow
solid, 900 mg (46%), mp 354-356 °C. Anal. (C8H7N3OS) C, H,
N. MS m/z (%): 193 (M+, 90), 176 (100), 148 (33), 105 (20), 90
1
(33). H NMR (399.9 MHz, DMSO-d6): δ 7.13 (t, J ) 7.8 Hz, 1
H), 7.25 (d, J ) 7.8 Hz, 1 H), 7.55 (s, 1 H), 7.58 (d, J ) 7.8 Hz,
1 H), 8.15-8.35 (br s, 1 H), 10.80-12.30 (br s, 1H). 13C NMR
(100.5 MHz, DMSO-d6): δ 111.9, 115.9, 120.6, 121.4, 132.6 (br),
133.5, 167.4, 169.3.
Alkylation of 2-Mercapto-4-carboxamidobenzimidazole. Gen-
eral Procedure for 12a and 12b. To a solution of compound 10
(1.93 g, 10.0 mmol) and powdered KOH (560 mg, 10 mmol) in
methanol (20 mL) compound 9 (2.33 g, 10.0 mmol) or compound
11 (2.47 g, 10.0 mmol) was added, and the solution was refluxed
for 2 h. After the mixture was cooled, the inorganic salts were
filtered off, the filtrate was evaporated, and the residue was
dissolved in CHCl3 (30 mL). The organic phase was washed
with water (10 mL), and the organic phase was separated, dried
(MgSO4), filtered, and evaporated. The crude product was
purified by flash column chromatography (CHCl3/Et2O) to afford
compound 12a or 12b.
2-(1-Oxyl-2,2,5,5-Tetramethyl-2,5-dihydro-1H-pyrrol-3-ylm-
ethylsulfanyl)-1H-benzimidazole-4-carboxylic Acid Amide (12a).
Yellow solid, 1.55 g (45%), mp 249-251 °C. Anal. (C17H21N4O2S)
C, H, N. MS m/z (%): 345 (M+, 20), 315 (18), 300 (13), 193 (100).
2-(1-Oxyl-2,2,6,6-Tetramethyl-1,2,3,6-tetrahydropyridin-4-yl-
methylsulfanyl)-1H-benzimidazole-4-carboxylic Acid Amide
(12b). Pink solid, 1.36 g (38%), mp 102-104 °C. Anal.
(C18H23N4O2S) C, H, N. MS m/z (%): 359 (M+, 2), 329 (18), 196
(42), 41 (100).
2-(1-Oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl)-
1H-benzimidazol-4-carboxylic Acid Amide Radical (3a). Yellow
solid (1.52 g, 51%), mp 248-250 °C. Anal. (C16H19N4O2) C, H,
N. MS m/z (%): 299 (M+, 19), 269 (37), 223 (51), 41 (100).
Synthesis of Compounds 4a-p by Reduction of Nitroxide
with Fe in AcOH (4a-k, 13a,b, 22, 25) (Procedure B). To a
solution of nitroxide 3a-k or 12a or 12b or 21 or 24 (5.0 mmol)
in AcOH (15 mL) iron powder (1.4 g, 25.0 mmol) was added, and
the mixture was stirred at 60 °C for 30 min. After cooling, the

1628 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6
Ka´lai et al.
cardiovascular disease and cancer: an update. Drug News Perspect.
2007, 20, 171–181.
1-(1-Oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-ylm-
ethyl)-1H-benzimidazole-4-carboxamide (15). To a solution of
compound 14 (805 mg, 5.0 mmol) and powdered K2CO3 (690 mg,
5.0 mmol) in acetonitrile (20 mL), compound 9 (1.16 g, 5.0 mmol)
was added, and the solution was refluxed for 3 h. After the mixture
was cooled, the inorganic salts were filtered off, the filtrate was
evaporated, and the residue was dissolved in CHCl3 (30 mL). The
organic phase was washed with water (10 mL), and the organic
phase was separated, dried (MgSO4), filtered, and evaporated. The
crude product was purified by flash column chromatography
(CHCl3/Et2O) to afford compound 15 as a yellow solid, 970 mg
(62%), mp 247-249 °C. Anal. (C16H20N3O) C, H, N. MS m/z (%):
313 (M+, 48), 299 (15), 283 (24), 41 (100).
(10) White, A. W.; Almassy, R.; Calvert, A. H.; Curtin, N. J.; Griffin, R. J.;
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˝
B.; Osz, E.; Su¨megi, B.; Hideg, K. Synthesis and evaluation of the
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Acknowledgment. This work was supported by the Hungar-
ian National Research Fund, Grants OTKAT048334 and
OTKA-NKTH K67597. We thank to Prof. W. L. Hubbell for
help in PARP inhibitor docking into PARP1 enzyme. We thank
Noemi Lazsanyi for technical assistance, Krisztina Kis for
elemental analysis, and I. Pa´sztor for biological measurements.
´
A., Jr.; Gallyas, F.; Hideg, K.; Su¨megi, B.; Va´rb´ıro´, G. A novel SOD-
mimetic permeability transition inhibitor agent protects ischemic heart
by inhibiting both apoptotic and nectrotic cell death. Free Radical
Biol. Med. 2006, 41, 835–848.
(14) (a) Ka´lai, T.; Khan, M.; Balog, M.; Kutala, K. V.; Kuppusamy, P.;
Hideg, K. Structure-activity studies on the protection of trimetazidine
derivatives modified with nitroxides and their precursor from myo-
cardial ischemia-reperfusion injury. Bioorg. Med. Chem. 2006, 14,
5510–5516. (b) Kutala, V. K.; Khan, M.; Mandal, R.; Ganesan, L. P.;
Tridandapani, S.; Ka´lai, T.; Hideg, K.; Kuppusamy, P. Attenuation
of myocardial ischemia-reperfusion injury by trimetazidine derivatives
functionalized with antioxidant properties. J. Pharmacol. Exp. Ther.
2006, 317, 921–928.
Supporting Information Available: Table of micronalytical data
of compounds 2-25; physicochemical and spectral data of com-
pounds 2e,f,j,n,m,p, 3b-n, 4b-k,o,p, 13a,b, 17, 19, 20, 21, 22,
24, 25; blank fluorescence measurement under Fenton conditions.
This material is available free of charge via the Internet at http://
pubs.acs.org.
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