Synthesis and SAR of novel, potent and orally bioavailable benzimidazole inhibitors of poly(ADP-ribose) polymerase (PARP) with a quaternary methylene-amino substituent

Article abstract of DOI:10.1016/j.bmcl.2008.06.023

Poly(ADP-ribose) polymerases (PARPs) play significant roles in various cellular functions including DNA repair and control of RNA transcription. PARP inhibitors have been demonstrated to potentiate the effect of cytotoxic agents or radiation in a number o

Full text of DOI:10.1016/j.bmcl.2008.06.023

Bioorganic & Medicinal Chemistry Letters 18 (2008) 3955–3958
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and SAR of novel, potent and orally bioavailable benzimidazole
inhibitors of poly(ADP-ribose) polymerase (PARP) with a quaternary
methylene-amino substituent
a
a
a
a
a
a
Gui-Dong Zhu a, , Viraj B. Gandhi , Jianchun Gong , Sheela Thomas , Yan Luo , Xuesong Liu , Yan Shi ,
Vered Klinghofer a, Eric F. Johnson a, David Frost a, Cherrie Donawho a, Ken Jarvis a, Jennifer Bouska a,
Kennan C. Marsh b, Saul H. Rosenberg a, Vincent L. Giranda a, Thomas D. Penning a
*
a Cancer Research, GPRD, Abbott Laboratories, Abbott Park, IL 60064, USA
b Preclinical Safety, GPRD, Abbott Laboratories, Abbott Park, IL 60064, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Poly(ADP-ribose) polymerases (PARPs) play significant roles in various cellular functions including DNA
repair and control of RNA transcription. PARP inhibitors have been demonstrated to potentiate the effect
of cytotoxic agents or radiation in a number of animal tumor models. Utilizing a benzimidazole carbox-
amide scaffold in which the amide forms a key intramolecular hydrogen bond for optimal interaction
with the enzyme, we have identified a novel series of PARP inhibitors containing a quaternary methy-
lene-amino substituent at the C-2 position of the benzimidazole. Geminal dimethyl analogs at the meth-
ylene-amino substituent were typically more potent than mono-methyl derivatives in both intrinsic and
cellular assays. Smaller cycloalkanes such as cyclopropyl or cyclobutyl were tolerated at the quaternary
carbon while larger rings were detrimental to potency. In vivo efficacy data in a B16F10 murine flank
melanoma model in combination with temozolomide (TMZ) are described for two optimized analogs.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 15 May 2008
Revised 5 June 2008
Accepted 5 June 2008
Available online 12 June 2008
Keywords:
PARP
Poly(ADP-ribose) polymerase
Inhibitor
Benzimidazole
Anticancer
Poly(ADP-ribose) polymerases (PARPs) are a family of DNA-
binding proteins found in nearly all eukaryotic cells.1 When acti-
vated by nicks in DNA occurring during inflammation, ischemia,
neurodegeneration, or cancer therapy, PARPs catalyze the transfer
of ADP-ribose units from nicotinamide adenine dinucleotide
(NAD+) to the acceptor proteins with NAD+ as substrate, leading
to formation of protein-bound ADP-ribose polymers and dramatic
cellular ATP depletion.2 This cellular ADP-ribose transfer process is
pivotal for DNA repair machinery and maintenance of genomic sta-
bility.1,2 PARP-1, the most abundant member of the PARP family, is
therefore regarded as a promising target for treating diseases re-
lated to inflammation and ischemia-reperfusion injury. This DNA
repair mechanism also contributes to the drug resistance that often
develops after cancer therapy.3 In addition, enhanced PARP expres-
sion and/or activation has been observed in a number of hemato-
logical and solid tumors as compared to normal cells, suggesting
a potential selectivity of PARP inhibitors against tumor cells. Inhi-
bition of PARP-1 would retard the intracellular DNA repair and
therefore sensitize tumor cells to cytotoxic agents or ionizing radi-
ation.4–8
There have been a plethora of PARP inhibitors developed in the
past two decades.9 The majority of the known inhibitors bind to
the nicotinamide binding site and structurally mimic the binding
mode of nicotinamide, where the amide functional group forms
multiple hydrogen bonds with Gly-863 and Ser-904.9 One struc-
tural class of inhibitors, namely benzimidazole carboxamide, was
first developed by researchers at the University of Newcastle.10
This series of compounds displayed relatively high intrinsic po-
tency against PARP-1, but suffered from poor activity in a whole
cell assay. Herein, utilizing the same benzimidazole carboxamide
scaffold, we incorporated a basic amino functionality to the C-2 po-
sition of the pharmacophore, and discovered a series of PARP
inhibitors with greater cellular activity. Two optimized analogs,
9b and 9k, with a quaternary methylene-amino substituent, dis-
played excellent intrinsic and cellular potency, adequate pharma-
ceutical properties, and potentiated the efficacy of cytotoxic
agent temozolomide (TMZ) in a B16F10 flank melanoma model.
Outlined in Scheme 1 are general syntheses of the PARP inhib-
itors with a representative structure 6 or 7. 2,3-Diaminobenzamide
dihydrochloride 111 was first coupled to an appropriately protected
amino acid 2 to provide mono-acylated 3. The 1,10-carbonyldiimid-
azole (CDI)-mediated amidation proceeded predominately at the
3-amino group. A 1:1 mixture of anhydrous DMF and pyridine
was typically used as solvent in which pyridine freed up the
* Corresponding author. Tel.: +1 847 935 1305; fax: +1 847 935 5165.
E-mail address: Gui-Dong.Zhu@abbott.com (G.-D. Zhu).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.06.023

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G.-D. Zhu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3955–3958
Table 1
R1
R2
NH
SAR of C-2 substituted benzimidazole carboxamide
O
NH2
NH2
HO2C
O
NH2
O
NH2
NH2
n
Cbz
2
N
HCl
HCl
NH
CDI/DMF/pyridine
R
n
45 oC/ 2 h
Cbz
NH2
N
O
N
8 H
H
R1 R2
3
1
PARP-1a (Ki, nM)
O
NH2
Compound
R
R1
100 oC
a
b
H
CH3
240 nM
99
10% Pd-C
H2
N
R2
NH
HOAc
3 h
N
n
H
Cbz
c
50
41
4
NH2
O
NH2
O
NH2
N
R1
N
R2
NH
R1
O=CR3R4
d
R2
NH2
NaBH3CN
MeOH
r.t.
HN Pr-n
HN Pr-i
HN
N
H
n
R4
N
H
n
R3
6
5
e
12
Br(CH2)nCl
Hunig's base
CH3CN/50 oC/3 days
60-70%
O
NH2
f
24
33
53
N
N
H
N
n= 0
m=1, 2
g
h
7
HN
Scheme 1.
HN
N
diamine from its hydrochloric salt form. The amides 3 were not
purified at this stage. Solvents were removed, and the residue
was directly heated in acetic acid, leading to benzimidazole prod-
uct 4 in good to excellent yields. Deprotection of compound 4 un-
der hydrogenation conditions provided the free amines 5 that were
converted to the corresponding alkyl analogs 6 through reductive
amination (Scheme 1). Alkylation of the free amines 5 with 1-bro-
mo-3-chloropropane or 1-bromo-4-chlorobutane in the presence
of Hunig’s base afforded cyclic amines 7.
i
j
61
35
As summarized in Table 1, benzimidazole carboxamide 8a is a
relatively potent scaffold with a Ki of 240 nM against PARP-1.
Installation of a methyl group at the C-2 position of the benzimid-
azole as exemplified by 8b improved its binding affinity by 2-fold.
An additional amino group (8c) is beneficial for both activity and
increased aqueous solubility. Attachment of an isopropyl group
onto the amino functionality (8e) improved the potency against
PARP-1 by 4-fold, whereas other alkylations (8d, 8f-j) appeared
to be less beneficial. Extension of the amino group with an ethyl-
ene spacer displayed no significant impact (8k and 8l). Dimethyla-
minosulfonylation on the amine was found to be detrimental to
potency.
In an attempt to make a bulkier C-2 substituent where the ami-
no functionality attaches to a quaternary carbon, an additional
methyl group was installed, and resulted in 8n with improved
intrinsic potency. Ring-formation at the quaternary carbon (8o–
8s) retained moderate activity. Smaller cycloalkanes such as cyclo-
propyl or cyclobutyl were tolerated at the quaternary carbon while
some other ring systems such as in 8q and 8s showed a small drop
in potency. Tolerance of a bulky group at the C-2 position of the
benzimidazole scaffold is consistent with the large binding pocket
of PARP in this area as indicated in a previous report.10
HN
k
l
32
45
NH2
HN
O
HN S N
O
m
n
140
19
NH2
NH2
o
25

G.-D. Zhu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3955–3958
3957
Table 1 (continued)
Compound 8n was then chosen as a lead for further optimiza-
tion. As shown in Table 2, installation of an isopropyl or cyclopro-
pylmethyl group on the amino functionality, led to compounds 9b
or 9c, with superior activity in both intrinsic and cellular assays.
Attachment of a straight chain alkyl group (9a) or a larger cyclic
substituent (9d) was slightly less beneficial. As indicated by com-
pound 9e, an additional phenyl group seems to be detrimental as
well. A replacement of the basic amino group with urea (9f) re-
sulted in significant loss in potency against PARP-1.
Compounds 9g–9j are methyl analogs of amine 8n with or with-
out an extra alkyl substituent, among which the less substituted 9g
showed superior activity in both enzyme and cellular assays. Teth-
ering of the two alkyl groups in the bis-alkylated amine derivatives
results in 9k and 9l with a cyclic structure as 7. Both compounds
displayed excellent potency against PARP, with the 5-membered
ring 9k more active.
Compound
R
PARP-1a (Ki, nM)
p
q
25
NH2
NH2
50
18
r
NH2
Listed in Table 3 are the pharmacokinetic properties of select
PARP inhibitors. All four compounds screened in the PK studies dis-
played a relatively short intravenous half-life, but demonstrated
respectable oral drug exposures with modest to excellent bioavail-
ability. With the excellent potency and acceptable PK property in
hand, we evaluated the efficacy of 9b and 9k in a B16F10 murine
flank melanoma model.12 Temozolomide, a cytotoxic alkylating
agent that is currently used to treat CNS malignancies and mela-
noma, was chosen as the DNA damaging agent of choice. The PARP
inhibitor was administered orally twice a day on days 6–10 at
doses of 3, 10, 30 mg/kg, while temozolomide was dosed daily at
50 mg/kg, which mimics the exposure in human at the clinically
relevant dose. As illustrated in Figure 1 and 2, both 9b and 9k in-
duced profound potentiation of the efficacy of temozolomide at
30 mg/kg as reflected by tumor volumes (bottom green curves),
whereas TMZ monotherapy had only modest tumor growth inhibi-
tion (bottom red curves). At lower doses (3 mg/kg for 9b and
10 mg/kg for 9k), less significant potentiation of the TMZ activity
was observed. The combination of PARP inhibitor with temozolo-
s
58
NH2
Assays follow the same protocols as previously described.5 Values are means of
a
two or more experiments, all assays generated data within 2-fold of mean.
Table 2
SAR of C-2 substituted benzimidazole carboxamide
O
NH2
N
9
N R1
R2
N
H
Compound
R1
R2
PARP-1a
Cellulara
(Ki, nM)
(EC50, nM)
a
b
n-Pr
i-Pr
H
H
10
7
3
2
Table 3
Mouse PK summary for selected PARP inhibitorsa
Compound
t1/2 (h)
Vd (L/kg)
CL (L/h/kg)
%F
82
100
85
Oral AUC (lM h)
c
H
7
2
9b
9c
9g
9k
0.6
0.8
0.6
0.4
2.1
13.3
3.5
4.0
11.5
4.2
5.78
3.38
8.64
6.33
d
e
H
H
9
4
9
1.8
3.0
52
a
Compounds were administered intravenously at 5 mg/kg or orally at 10 mg/kg.
37
O
f
H
870
NDb
4000
HN
9b /TMZ, 30/50 mkd
9b/ TMZ, 30/0 mkd
3500
9b /TMZ, 10/50 mkd
9b /TMZ, 3/50 mkd
9b /TMZ, 0/50 mkd
g
h
Me
Me
H
Me
4
9
1.3
1.8
3000
9b /TMZ, 0/0 mkd
2500
i
Me
19
NDb
2000
1500
1000
500
0
j
Me
17
3
NDb
0.9
O
k
0
5
10
15
20
l
15
3.1
9b administered PO d6-10, BID
Days
TMZ administered PO d6-10, QD
Assays follow the same protocols as previously described.5 Values are means of
a
Figure 1. Efficacy of compound 9b in combination with temozolomide in a B16F10
two or more experiments, all assays generated data within 2-fold of mean.
murine melanoma model.12
b
Not determined.

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G.-D. Zhu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3955–3958
quaternary methylene-amino substituent at the C-2 position of
2000
the benzimidazole. Two optimized inhibitors in this series, 9b
and 9k, displayed an excellent intrinsic potency against PARP-1,
and showed good pharmaceutical properties, including aqueous
solubility and pharmacokinetics across multiple species. In a mur-
ine flank melanoma model in combination with temozolomide,
both compounds significantly potentiated the efficacy of temozol-
omide, without an observable increase in toxicity.
Vehicle
TMZ 50 N=9
9k
TMZ (30/50)
1500
1000
500
9k/
TMZ (10/50)
Acknowledgment
The authors are grateful to the Abbott analytical department for
acquisition of 1H NMR and MS.
References and notes
0
0
5
10
15
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2007, 13, 1383.
Figure 2. Efficacy of compound 9k in combination with temozolomide in a B16F10
murine melanoma model.12
Table 4
PK summary of compound 9b in different animal speciesa
5. Donawho, C. K.; Luo, Y.; Luo, Y.; Penning, T. D.; Bauch, J. L.; Bouska, J. J.;
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Ghoreishi-Haack, N. S.; Grimm, D. R.; Guan, R.; Han, E. K.; Holley-Shanks, R.;
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Lasko, L. M.; Liu, X.; Marsh, K. C.; McGonigal, T. P.; Meulbroek, J. A.; Olson, A.
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S.; Durkacz, B. W.; Hostomsky, Z.; Kumpf, R. A.; Kyle, S.; Li, J.; Maegley, K.;
Newell, D. R.; Notarianni, E.; Stratford, I. J.; Skalitsky, D.; Thomas, H. D.; Wang,
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7, 515; (b) Ratnam, K.; Low, J. Clin. Cancer Res. 2007, 13, 1383; (c) Peukert, S.;
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Chem. 2003, 10, 321.
10. White, A.; Almassy, R.; Calvert, A.; Curtin, N.; Griffin, R.; Hostomsky, Z.;
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11. Barkalow, J. H.; Breting, J.; Gaede, B. J.; Haight, A. R.; Henry, R.; Kotecki, B.; Mei,
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12. For B16F10 syngeneic studies, 6 ꢀ 104 cells were mixed with 50% matrigel (BD
Biosciences, Bedford, MA) and inoculated by sc injection into the flank of 6–8-
week old female C57BL/6 mice, 20 g (Charles River Laboratories, Wilmington,
MA), on day 1. Mice were injection-order allocated to treatment groups and
PARP inhibitor therapy was initiated on days 6 (for 9b) and 5 (for 9k) following
Animal
t1/2 (h)
Vd (L/kg)
CL (L/h/kg)
%F
Oral AUC (lM h)
Mouse
Rat
Dog
0.6
1.0
2.8
0.6
2.1
3.7
2.9
1.1
4.0
82
26
70.5
12.6
5.78
0.74
6.07
0.65
5.1
0.85
1.4
Monkey
a
Intravenous and oral doses for the above pharmacokinetic studies are as fol-
lows: 3 and 10 mg/kg for mouse, 5 and 5 mg/kg for rat, 2.5 and 2.5 mg/kg for dog,
and 2.5 and 2.5 mg/kg for monkey.
mide was well tolerated, with minimal loss of body weight at high-
est doses.
As shown in Table 4, 9b is orally bioavailable across all animals
species we tested, with significantly better bioavailability in mouse
and dog (82% and 70%, respectively), and modest bioavailability in
rat and monkey (26% and 12%, respectively). Largely due to a slow
rate of clearance, the IV half-life of this compound in dog is rela-
tively longer than that in other species. In addition, compound
9b displayed an excellent aqueous solubility (>5 mg/mL), showed
modest human plasma binding (60–70%), and demonstrated mini-
mal inhibition of several cytochrome p450s (<10% at 10 lM).
In summary, starting from a benzimidazole carboxamide scaf-
fold, we developed a novel series of PARP inhibitors containing a
inoculation, with temozolomide treatment starting on days
respectively.
6 and 5,