Design, synthesis and biological evaluation of novel imidazo[4,5-c] pyridinecarboxamide derivatives as PARP-1 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2013.02.032

A series of novel cyclic amine-substituted imidazo[4,5-c] pyridinecarboxamide analogs were designed and synthesized. All the target compounds were evaluated for their PARP inhibition activity, and the result indicated that most of the compounds possessed

Full text of DOI:10.1016/j.bmcl.2013.02.032

Accepted Manuscript
Design, synthesis and biological evaluation of novel imidazo[4,5-c]pyridine‐
carboxamide derivatives as PARP-1 inhibitors
Qihua Zhu, Xuyang Wang, Zhaoxing Chu, Guangwei He, Guangping Dong,
Yungen Xu
PII:
S0960-894X(13)00208-4
DOI:
http://dx.doi.org/10.1016/j.bmcl.2013.02.032
BMCL 20150
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
26 October 2012
28 January 2013
6 February 2013
Please cite this article as: Zhu, Q., Wang, X., Chu, Z., He, G., Dong, G., Xu, Y., Design, synthesis and biological
evaluation of novel imidazo[4,5-c]pyridinecarboxamide derivatives as PARP-1 inhibitors, Bioorganic & Medicinal
Chemistry Letters (2013), doi: http://dx.doi.org/10.1016/j.bmcl.2013.02.032
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Graphical Abstract
Design, synthesis and biological evaluation of novel
imidazo[4,5-c]pyridinecarboxamide derivatives as PARP-1 inhibitors
Qihua Zhua, Xuyang Wanga, Zhaoxing Chub, Guangwei Heb,*, Guangping Donga, Yungen
Xua,*
A series of novel imidazo[4,5-c]pyridinecarboxamide derivatives as PARP inhibitors is described.

Design, synthesis and biological evaluation of novel
imidazo[4,5-c]pyridinecarboxamide derivatives as PARP-1
inhibitors
Qihua Zhua, Xuyang Wanga, Zhaoxing Chub, Guangwei Heb,*, Guangping Donga, Yungen
Xua,*
aJiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Nanjing 21009, China
bHefei YiGong Pharmaceutical Co. Ltd., Hefei, 230088, China
*Corresponding authors: YungenXu
Email: xu_yungen@hotmail.com
Tel.:86-25-83271244
Abstract
A series of novel cyclic amine-substituted imidazo[4,5-c]pyridinecarboxamide analogs were designed and
synthesized. All the target compounds were evaluated for their PARP inhibition activity, and the result indicated
that most of the compounds possessed inhibitory effect on PARP at the concentration of 1μM, among which
compound 8d (IC50=0.528μM) was selected for evaluating the antitumor effect in vivo. The result showed the
antitumor efficacy of the compound 8d and cisplatin combination group in a mouse A549 model is similar with
that of the ABT-888 and cisplatin combination group.
Keyword: Imidazo[4,5-c]pyridinecarboxamide; PARP-1; PARP Inhibitors; Antitumor
Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme with an important role in the process of genomic
repair. To date, 18 members of the PARP family have been identified and characterized. Among the PARP family,
PARP-1 is one of the most abundant proteins and has been believed the main isoform[1]. The PARP-1 enzyme has
three structural regions: the DNA binding domain containing two zinc fingers, the automodification domain, and
the catalytic domain[2]. The catalytic domain catalyze the transfer of ADP-ribose units from intracellular
nicotinamide adenine dinucleotide (NAD+) to nuclear acceptor proteins and is responsible for the formation of
ADP-ribose polymers[3]. It is generally accepted that the catalytic activity of PARP-1 is stimulated by DNA damage
caused by peroxidation, irradiation, and DNA-damaging chemicals, e.g., chemotherapeutic agents[4]. The major role
of PARP-1 is important in number of cellular processes, thus it is regarded as a target for treating diseases related to
ischemia-reperfusion injury and cancer. Indeed, PARP-1 inhibitors have been identified to be effective in animal
models of ischemic stroke, traumatic brain injury, Parkinson’s disease and cancer[5]. Moreover, recent studies have
shown that cells deficient in BRCA1 and BRCA2, proteins involved in DNA double strand break repair by
homologous recombination (HR) repair are highly sensitive to PARP-1 inhibitors[6].
Figure 1 is a schematic presentation of X-ray structure of NAD+ binding to PARP-1, and formation of multiple
hydrogen bond interactions between the carboxamide moiety and two critical amino acid residues in the PARP
active site, Gly-863 and Ser-904[7]. In addition, Tyr907 forms a planar surface which has a π-π-interaction with the
nicotinamide group and stabilizes the oxonuim ion. Early studies identified nicotinamide (IC50=210μM) and
3-aminobenzamide (IC50=30μM) as inhibitor of PARP-1[8]. However, these compounds show low potency and poor
specificity. The tremendous academic and industrial efforts have been made to find new inhibitors with good
potency against PARP-1. It is very pleasure that many groups have obtained good results. Over the past three
decades, a variety of PARP-1 inhibitors have been reported[9]. It is clear that the majority of PARP inhibitors are
based on the benzamide as pharmacophore, which mimics the nicotinamide moiety of NAD+binding mode, by

conformational restriction of a primary amide or by transformation of an amide into a lactam.
Although a variety of PARP-1 inhibitors have been disclosed in the literature, many suffer from development
problems such as toxicity, poor solubility, or poor pharmacokinetic profiles. The effort of pursuing new inhibitors
with good potency against PARP-1 is still needed. The recent studies found that the benzimidazolecarboxamides
core is one of the most potent scaffold structures for PARP-1 inhibitors, and many benzimidazole-4-carboxamide
derivatives had been developed. For example, 2-[(R)-2-methyl-pyrrolidin-2-yl]-1H-benzimidazole-4-carboxamide
(ABT-888, veliparib)[10] is currently on-going phase II clinical trials. In an effort to search novel PARP-1 inhibitors
and explore alternative chemical templates, we took the pyridine ring instead of benzene ring in benzimidazole.
And the recent studies[11] also found that incorporation of a basic amine into the 2-substituent of the benzimidazole
ring system in many cases demonstrated good enzyme potency. On the basis of the above structure-activity
relationship of PARP-1 inhibitors, we designed
a
series of cyclic amine-substituted imidazo
[4,5-c]pyridinecarboxamide analogs as new PARP-1inhibtiors.
Figure 1. A schematic presentation of the X-ray structure of NAD+ binding PARP-1. The cycled part of
structure (red) showed critical hydrogen bond interactions of NAD+ with PARP-1[7].
Figure 2. The structure of ABT-888

Figure 3. Docking of compound 8d in the catalytic domain of human PARP-1 (PDB code: 2RCW)
Scheme 1. Synthesis route for target compounds. Reactions and conditions: (a) conc H2SO4, fuming HNO3; (b)
SOCl2; (c) NH3﹒H2O, CH3CN, 0°C to r.t, 24h, 80%; (d) 10% Pd-C, H2, MeOH, r.t., 12h, 90%; (e) i:
2-pyrrolidinecarboxylic acid, 4-pipecolinic acid, SOCl2, Pyridine, methylene chloride, reflux, 2h; ii: PyBOP,
3-pipecolinic acid, N,N-diisopropylethylamine, DMF, r.t, 12h; (f) AcOH, 160°C, in an autoclave, 24h,
42%-50.3%; (g) 10% Pd-C, H2, MeOH, r.t., 12h, 90%; (h) R1R2C(O), NaBH3CN, MeOH, r.t. 31%-65%;
Herein, we describe the design and synthesis of a new series of imidazo[4,5-c]pyridinecarboxamide derivatives.
Crystallographic structure of the human PARP-1 catalytic domain (PDB code: 2RCW) was obtained from the
Protein Data Bank (PDB) database, and a docking study was performed to approach the development of potent
inhibitors. Docking of compound 8d in the catalytic domain of human PARP-1 is shown in Figure 3. The key
interactions in the PARP-1 active site, consistent with previous literature reports, are highlighted. Both Ser-243 and

Gly-202 are involved in key hydrogen-bond interactions with the carboxamide group of 8d. There is also a
π-stacking interaction between the imidazo[4,5-c]pyridine ring and Tyr-246 and Tyr-235. These same interactions
are also displayed in the binding of NAD+ (Fig. 1) to the PARP-1 active site.This result led us to design its analogs.
To verify this docking study, imidazo[4,5-c]pyridine-7-carboxamide derivativeswere synthesized, as outlined in
Scheme 1. Nitration of 1 with mixed acid of fuming nitric acid and sulfuric acid afforded nitro compound 2.
Treatment of 2 with thionyl chloride smoothly produced the corresponding acid chloride, which was reacted with
12% aqueous ammonia at 0°C for 30 min, then at room temperature overnight to give 4-amino-5-nitronicotinamide
3. 4,5-Diaminonicotinamide 4, obtained by catalytic hydrogenation of 3, was coupling with saturated
nitrogen-containing heterocyclic acid under PyBOP or SOCl2 conditions to form intermediates 5a-c, which were
heated in acetic acid to give imidazo[4,5-c]pyridinecarboxamide derivatives 6a-c. Deprotection of Cbz group using
catalytic hydrogenation provide a secondary amines 7a-c[12]. N-alkylation of 7a-cwithaldehydes or ketones under
sodium cyanoborohydride reductive amination condition afforded tertiary amines 8a-h[13]
.
Table1. The structures and PARP-1 enzyme inhibition activity of compounds 7a-c and 8a-h
Compd.
7a
Substituents (R)
Inhibition (%)a
32.6
IC50 (μM)
ND
7b
7c
76.2
60.3
0.825
0.636
8a
1.9
ND
8b
8c
4.7
ND
ND
17.4
8d
8e
79.0
56.6
0.528
1.452

8f
23.1
65.5
ND
8g
0.651
8h
67.1
0.938
3-AB
100b
97
ND
ABT-888
0.019
a: Inhibitory ratio % at 1μM; b: PARP-1 inhibition at the concentration of 100μM; ND, not determined.
600
400
200
0
4
3
2
1
0
Control
Cisplatin
Cisplatin+ABT-888
Cisplatin+8d
Control
Cisplatin
Cisplatin+ABT-888
Cisplatin+8d
3
6
9
12
Days
15
18
21
3
6
9
12
Days
15
18
21
Figure 4. Antitumor efficacy of 8d in mice bearing tumors derived from A549 cells when dosed orally at 25 mg/kg
for 19 days. For each group, n = 5.
All target compounds were evaluated in vitro for their PARP-1 enzyme inhibition activity (Table 1). The result
showed that some target compounds possessed PARP-1 inhibition activities at the concentration of 1μM.
Compounds with PARP-1inhibitory ratio >50% were selected to determine IC50 values, and the results were listed
in table 1. Among compounds 7a-c, compound7a, bearing a 2-pyrrolidinyl group at 2 position, showed low
inhibitory activity, while the 3- or 4-piperidyl analogs 7b and 7c showed relatively good potency. Among the
N-alkyl derivatives, compounds 8a-c, deriving from 7a, showed less active than 7a in the enzyme assay. Compared
to 7b, its N-alkyl derivatives 8d and 8e showed similar inhibitory effect on PARP-1. The N-methyl derivatives 8f
exhibited less inhibition activity against PARP-1 than its parent compound 7c and N-propyl or isopropyl analogues
(8g or 8h). Similar to 8f, N-methyl derivatives 8b and 8e displayed weaker activity in the enzyme assay compared

to 7a and 7b.
Compound 8d was selected for evaluating the in vivo antitumor effect, and the result showed the antitumor
efficacy of the compound 8d and cisplatin combination group in a mouse A549 model is similar with that of the
ABT-888 and cisplatin combination group (Figure 4)[16]. The cisplatin/ABT-888 and cisplatin/8d combination
group both showed significantly more effective than cisplatin alone in this model. Tumor regression was observed
with both combination group, and both were well tolerated, with no mortality. Less than 10% body weight loss was
seen during the experiment.
In summary, we reported the synthesis and biological evaluation of novel imidazo[4,5-c]pyridinecarboxamide
derivatives as potent PARP-1 inhibitors. Compound 8d (IC50=0.528μM) was found to be the most potent one in
vitro. In a mouse A549 model, the cisplatin/8d combination group is efficacious at well tolerated doses when
administered orally. Further biological evaluation of this class of derivatives is ongoing and will be reported in the
near future.
Acknowledgment
This work was supported by the enterprise innovation drug incubation base of the Ministry of Science and
Technology, China (No.2012ZX09401-006) and the Graduate Students' Innovation Personnel Training Plan of
China Pharmaceutical University (No. 2011IIBPY02).The authors are grateful to Institute of Pharmacology &
Toxicology and Biochemical Pharmaceutics at Zhejiang University for their help in the biological test.
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12. General Procedure for the synthesis of the target compounds 7a-7c: (i) A suspension of
2-pyrrolidinecarboxylic acid or 4-pipecolinic acid (10 mmol) in a mixture of dichloromethane (80 mL),
thionyl chloride (3.5 mL) was heated to reflux overnight. The resulting solution was concentrated and acid
chloride intermediate was used in the next step without additional purification. To a solution of
4,5-diaminonicotinamide 4 (6.5 mmol) in DMAc (20 mL) and pyridine (1.2 mL) was added the acid
chloride in anhydrous THF (10 mL) at 0 °C. After stirring at room temperature overnight, the reaction
mixture was concentrated under reduced pressure. The residue was poured into crushed ice. The
precipitate of that formed was filtered off and dried to give the intermediate 5 which used in next step
without purification. (ii) To a solution of 3-pipecolinic acid (10 mmol) and 5-diaminonicotinamide 4 (6.5
mmol) in DMF (10 mL) was treated with PyBOP (1.0mmol) and N,N-diisopropylethylamine (4.0 mmol).
The reaction mixture was stirred at ambient temperature overnight. The solvent was removed using high
vacuum. The residue was subjected to flash column chromatography using methylene chloride/methanol
(15:1) to give the intermediate 5 as a white solid. The intermediate 5 was dissolved in glacial acetic acid
(10 mL) in an autoclave and heated at 160 °C until the reaction was complete (monitoring by TLC). The
solvent was removed and the solid residue was purified by column chromatography using methylene
chloride/methanol (30:1) as eluentto give pure 6a-6c in 42%-50.3% yield. A solution of 6a-6c (2.7 mmol)
in methanol (50 mL) was reduced with hydrogen in the presence of palladium on carbon (10%, 0.1 g).
After stirring at room temperature for 12 h, the reaction mixture was filtered, and the filtrate was
concentrated to give pure target compounds 7a-7c in 90% yield.
13. General Procedure for the synthesis of the target compounds 8a-8h: A solution of 7a-7c in MeOH (10 mL)
was stirred with formaldehyde, acetone or propionaldehyde at ambient temperature for 5 min.Sodium
cyanoborohydride was added and the solution stirred at ambient temperature overnight. After

concentration, the residue was purified by column chromatography using methylene chloride/methanol
(30:1 to 15:1) as eluent to give the target compounds 8a-8h in 31-65% yield.
14. Analytical data for selected final compound 7b: white solid. M.P.: 96-98°C. 1H-NMR (300 MHz,
DMSO-d6) δ: 8.92 (s, 1H), 8.89 (s, 1H), 8.76 (s, 1H), 7.73 (s, 1H), 3.12 (m, 3H), 2.68 (m, 2H), 2.02 (m,
2H), 1.80 (m, 2H). 13C-NMR (DMSO, 75 MHz) δ: 166.9, 163.5, 144.1, 140.6, 138.6, 115.9, 44.8, 36.1,
30.7; ESI-MS (m/z): 246 [M+1]+. HRMS (m/z) for C12H16N5O: calcd 246.1349, found 246.1351.
15. Analytical data for selected final compound 8g: yellow solid. M.P.: 122-124°C.1H-NMR (300 MHz,
CD3OD) δ: 8.87(s, 1H), 8.82 (s, 1H), 3.31 (m, 1H), 3.22 (m, 1H), 2.91 (m, 2H), 2.78 (m, 1H), 2.47 (m, 1H),
2.14 (s, 1H), 1.80 (m, 3H), 1.14 (d, J=6.0 Hz, 6H); 13C-NMR (CD3OD, 75 MHz) δ: 168.9, 164.0, 145.3,
141.8, 139.5, 137.5, 117.4, 49.9, 56.5, 53.5, 37.9, 30.4, 25.2, 18.2, 18.0; ESI-MS (m/z): 288 [M+1]+, 310
[M+Na]+. HRMS (m/z) for C15H22N5O: calcd 288.1819, found 288.1823.
16. The antitumor efficacy of target compounds was conducted in a mouse A549 model. For A549 syngeneic
studies, 2×106 cells were injected subcutaneously in the flank of 5-week-old nude mice. When tumors
reached volume of 100 to 300 mm3, mice were randomized to form homogeneous groups and treatment
started, dosing orally. Mice were dosed orally with cisplatin (2mg/kg) once every three days, while treated
with ABT-888 (25mg/kg) or 8d (25mg/kg) twice a day respectively for 19 days, with tumor growth and
body weight measurement done once every three days.