Design, synthesis and biological evaluation of novel molecules as potent PARP-1 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2021.128169

Two series of novel compounds with inhibition activity against PARP-1 were designed and synthesized. All target compounds were evaluated for their PARP-1 inhibition activity, and compounds with high PARP-1 inhibition activity were selected to assess for cellular assays in vitro. Among them, compound II-4 displayed impressive results in both PARP-1 enzyme inhibition with IC₅₀ value of 0.51 nM and anti-proliferation activity against HCT116 and HCC1937 cell lines with IC₅₀ values of 6.62 nM and 12.65 nM, respectively. Also, II-4 exhibited good metabolic stability in vitro with t_1/2 of 173.25 min and CL_int of 0.04 mL/min/mg. Prediction of molecular properties and protein docking were applied to structure design. Our study provides potential lead compounds and design directions for the development of PARP-1 inhibitors.

Full text of DOI:10.1016/j.bmcl.2021.128169

Bioorg. Med. Chem. Lett. 47 (2021) 128169
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and biological evaluation of novel molecules as potent
PARP-1 inhibitors
a,
†
b,
†
a
a
a,
b,
*
a,b,*
Hui Shen , Yiran Ge , Junwei Wang , Hui Li , Yungen Xu
, Qihua Zhu
a
Jiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Nanjing 211198, China
Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing 211198, China
b
A R T I C L E I N F O
A B S T R A C T
Keywords:
Two series of novel compounds with inhibition activity against PARP-1 were designed and synthesized. All target
compounds were evaluated for their PARP-1 inhibition activity, and compounds with high PARP-1 inhibition
activity were selected to assess for cellular assays in vitro. Among them, compound II-4 displayed impressive
results in both PARP-1 enzyme inhibition with IC50 value of 0.51 nM and anti-proliferation activity against
HCT116 and HCC1937 cell lines with IC50 values of 6.62 nM and 12.65 nM, respectively. Also, II-4 exhibited
good metabolic stability in vitro with t1/2 of 173.25 min and CLint of 0.04 mL/min/mg. Prediction of molecular
properties and protein docking were applied to structure design. Our study provides potential lead compounds
and design directions for the development of PARP-1 inhibitors.
Antitumor drug
DNA repair
PARP-1 inhibitors
Drug design
Poly (ADP-ribose) polymerase (PARP) is a ubiquitous DNA repair
enzyme in eukaryotic cells with a critical role in base-excision repair
Besides, various PARP inhibitors are under different clinical trial stages
as a single agent or combinatorial therapy with other antitumor agents.
With the success of PARP inhibitors as antitumor agents in clinical
practice, it is still a very active area of investigation.
(
BER) of single-strand breaks (SSB), which was first reported by
1
Chambon and his team in 1963. In 1980, Durkacz reported that
dimethyl sulfate’s cytotoxic activity could be potentiated by inhibiting
PARP, prompting the possibility to use PARP inhibitors and cytotoxic
drugs as a combination therapy for cancer treatment.2 The enzymes of
PARP family play essential roles in multiple cellular processes beyond
DNA repair, including cellular differentiation, gene transcription,
inflammation, mitosis, cell death, and metabolism.3 To date, 18 mem-
bers of PARP family have been identified and characterized. Among
them, PARP-1 shares the largest proportion of the family and is
responsible for about 90% of the function.4 Several studies confirmed
the involvement of PARP-1 in many pathological conditions, including
apoplexy, neurodegenerative disease, myocardial ischemia, inflamma-
tion, diabetes, cancer, and other diseases.5 It has validated that PARP
inhibition could achieve resultant killing cancer cells with BRCA1/2
mutation through synthetic lethality. Thus, PARP-1 is considered a
significant antitumor target and attracted much attention from aca-
demics and pharmaceutical companies.
To discover novel PARP inhibitors, our group has disclosed two novel
series of compounds with scaffolds of imidazole[4,5-c]pyridine car-
boxamide and 8,9-dihydro-2,4,7,9a-tetraazabenzo[cd]azulen-6(7H)-
one as PARP-1 inhibitors in our previous works and culminated in
obtaining two lead compounds XZ-1203121
2,13
and XZ-160811
14
(Fig. 1). However, they are still inferior to Olaparib and unsatisfied PK
properties also hinder further study. Hence, there is a continued need for
developing more potent PARP-1 inhibitors with improved druggable
properties. Herein, we described the discovery of novel potent com-
pounds as PARP-1 inhibitors and culminated in obtaining compound II-
4 which exhibited high potency against PARP-1 enzyme and cancer cell
lines in vitro.
Firstly, compounds of I series were prepared according to the pro-
cedures as shown in Scheme 1–4. Compounds I-1 ~ I-3 were synthesized
from key intermediates 1a ~ 1c and 2-fluoro-5-((4-oxo-3,4-dihydroph-
thalazin-1-yl)methyl)benzoic acid (2) by condensation reaction using
PyBOP as coupling agent (Scheme 1). In Scheme 2, the key intermediate
6, which was synthesized from starting material 2 via esterification,
chlorination, alkoxy substituted and hydrolysis reaction, condensed
To date, five PARP inhibitors, including Olaparib, Rucaparib,7
6
Niraparib,8,9 Talazoparib and Fluzoparib, have been approved to
10
11
treat ovarian or breast cancer harboring BRCA1 or BRCA2 mutations.
*
Corresponding authors at: Jiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Nanjing 211198, China.
E-mail addresses: xyg64@126.com (Y. Xu), zhuqihua@vip.126.com (Q. Zhu).
†
Both authors contributed equally to this work.
https://doi.org/10.1016/j.bmcl.2021.128169
Received 8 April 2021; Received in revised form 19 May 2021; Accepted 29 May 2021
Available online 6 June 2021
0
960-894X/© 2021 Elsevier Ltd. All rights reserved.

H. Shen et al.
Bioorganic & Medicinal Chemistry Letters 47 (2021) 128169
Fig. 1. Previous works for discovering novel PARP-1 inhibitors.
Scheme 1. Synthesis of compounds I-1 ~ I-3.
Scheme 2. Synthesis of compound I-4.
with compound 1a to provide the target compound I-4. Compounds I-5
and I-6 were synthesized by condensation reaction of compound 2 and
intermediates 7a ~ 7b (Scheme 3). The carboxyl acid of compound 2
diamine (9b ~ 9d). The structures of all target compounds were char-
1
13
acterized by H NMR, C NMR and HRMS (see in SI).
Analysis of the structure–activity relationships (SARs) of XZ-120312
indicated that the 2-(piperidin-4-yl)-3H-imidazo[4,5-c]pyridine-7-car-
boxamide (the blue portion of structure XZ-120312 in Fig. 2) is the
critical skeleton for PARP-1 inhibition, while the alkyl portion (the black
portion) is not necessary and that could be optimized to increase the
potency and improve ADMET properties, which was also found similar
SAR in Olaparib that cyclopropane carboxamide group is not essential
for PARP-1 inhibition. Hence, we initially designed compound I-1 by
merging the core structure of Olaparib (the red portion in Fig. 2) and XZ-
was reduced by (CH
3
)
2
S⋅BH
3
to alcohol, consequently oxidized by Dess-
Martin agent to afford intermediate 8 (Scheme 4). Compounds I-7 ~ I-8
were obtained by reductive amination of intermediates 8 and 7a ~ 7b.
Compounds II-1 ~ II-4 were prepared according to the synthetic
routes as shown in Scheme 5. Compounds II-1 ~ II-4 were obtained via
condensation reaction of intermediates 11a ~ 11d and 2. The key in-
1
5
termediates 11a ~ 11d were synthesized according to the literature
,
starting from benzene-1,2-diamine (9a) or 4-substituted benzene-1,2-
2

H. Shen et al.
Bioorganic & Medicinal Chemistry Letters 47 (2021) 128169
Scheme 3. Synthesis of compounds I-5 ~ I-6.
Scheme 4. Synthesis of compounds I-7 ~ I-8.
20312. To our delight, compound I-1 exhibited remarkably high po-
1
Replacing the imidazo[4,5-c]pyridine with 2-(piperidin-4-yl) and 2-
(piperazin-1-yl) benzo[d]imidazole yielded compounds I-5 and I-6,
which retained high PARP-1 inhibition activity with IC50 values of 0.63
nM and 0.19 nM, respectively. Reduction of amide of compounds I-5 and
I-6 afforded corresponding amines I-7 and I-8, leading to much decrease
in enzymatic activity.
tency against PARP-1 with an IC50 value of 0.23 nM (Table 1) and was
more potent than compound XZ-120312 and Olaparib with 37-fold and
4
-fold, respectively. Encouraged by this result, we further explored the
PARP-1 inhibition activity affected by substituents on imidazo[4,5-c]
pyridine scaffold. Alkoxyl group, such as MeO and EtO, substituted on 4-
position of imidazo[4,5-c]pyridine framework yielded compounds I-2
and I-3, showing little reduced enzymatic activity with IC50 values of
To further explore more potent PARP-1 inhibitors, we merged the
benzimidazole and piperazine of compound I-6 into a tricyclic system
(Fig. 2). Unexpectedly, the newly designed compound II-1 also exhibited
excellent PARP-1 inhibition activity with IC50 value of 0.49 nM.
Considering the feasibility of compound synthesis, we preliminarily
explored different substitutes at 7-position of benzimidazole tricyclic
system, leading to compounds II-2 ~ II-4. As shown in Table 1, all newly
designed compounds exhibited excellent PARP-1 inhibition activity,
which are more potent than Olaparib. These results demonstrated that 7-
position substitutes have little effect on activity.
2
.4 nM and 1.7 nM, respectively. However, destructing the lactam of
pthalazin-1(2H)-one led to compound I-4, showing much reduced
enzymatic activity with only 40% inhibition rate at the concentration
1
00 nM. The above results indicated that pthalazin-1(2H)-one plays an
essential role in retaining inhibition activity as the pharmacophore of
PARP-1 inhibitors. In contrast, the imidazole[4,5-c]pyridine carbox-
amide has little effect on activity. Hence, it is reasonable to replace the
structure of imidazole[4,5-c]pyridine carboxamide with others.
3

H. Shen et al.
Bioorganic & Medicinal Chemistry Letters 47 (2021) 128169
Scheme 5. Synthesis of compounds II-1 ~ II-4.
Fig. 2. The process of designing target compounds.
Based on the profile of PARP-1 inhibition, compounds I-1, I-6 and II-
~ II-4 were selected to evaluate their anti-proliferation as single-agent
against primary cellular HCT116 (BRCA2-deficient colorectal cancer
cell line) and HCC1937 (BRCA1-deficient breast cancer cell line). As
shown in Table 2, although compound I-1 exhibited excellent PARP-1
enzyme inhibition activity, it has little effect on both human tumor
2
4

H. Shen et al.
Bioorganic & Medicinal Chemistry Letters 47 (2021) 128169
Table 1
respectively), and higher value of PlogPo/w (4.83) than Olaparib and I-
6 (2.79 and 4.15, respectively), which may contribute to good cell-
permeability.
PARP-1 inhibition activity of compounds I-1 ~ I-8, II-1 ~ II-4, XZ-120312 and
Olaparib.
Compd.
Inhibition
IC50
Compd.
Inhibition
IC50
To evaluate druggability, metabolic stability assessment was carried
out by incubating compound II-4 with human liver microsomes in vitro
for 50 min. The results showed that compound II-4 possessed stable
metabolic properties, with metabolic half-life of 173.25 min and
intrinsic clearance value of 0.04 mL/min/mg (Fig. S1). Besides, drug-
like molecular properties of these compounds predicted that II-4 has
the potential druggability for its low toxicity and considerable oral ab-
sorption (Table S1).
a
a
(
%)
(nM)
(%)
(nM)
b
I-1
I-2
I-3
I-4
I-5
I-6
–
98
0.23
2.40
1.70
I-8
II-1
II-2
II-3
II-4
XZ-
42
–
ND
0.49
0.47
0.40
0.51
8.60
100
40
–
b
ND
–
100
100
0.63
0.19
–
–
1
20312
b
I-7
47
ND
Olaparib
–
1.10
In order to certify the target role of the novel inhibitor, modeling
studies of compound II-4 in the binding pocket of PARP-1 (PDB code:
5DS3) and PARP-2 (PDB code: 4TVJ) were carried out. The results
a
Inhibition (%) at 10 nM.
ND, not determined.
b
(
Fig. 3) showed that the spatial orientation of II-4 is nearly consistent
with Olaparib, among which three key hydrogen bonding interactions
with Ser904 and Gly863 are reserved, and the 5-position nitrogen atom
on the tricyclic system could form a key hydrogen bond with the amino
of Arg878. These indicated that compound II-4 has the equal strength of
protein binding with Olaparib, which contributes to similar IC50 values
at the enzyme level.
Table 2
The anti-proliferation activity for PARP-1 inhibitors against HCT116 and
HCC1937 cell lines.
a
Compd.
IC50
HCT116
HCC1937
Unexpectedly, PARP-1 inhibitor II-4 displays much weaker interac-
tion with PARP-2 than Olaparib, a dual PARP1/2 inhibitor, lacking two
key hydrogen bonds (Fig. S2). Besides, referring to docking score, the
binding energy of II-4 and Olaparib with PARP-1, respectively, is
ꢀ 12.631 and ꢀ 12.114 kcal/mol. With PARP-2, the binding energy is
ꢀ 9.834 and ꢀ 8.781 kcal/mol, respectively. Lower values of docking
score are associated with stronger interaction, justifying that the
designed tricyclic ring of II-4 contributes to its better binding with
PARP-1 than PARP-2.
I-1
I-6
160.77 ± 7.30
μ
M
129.72 ± 8.03
μ
M
0.49 ± 0.04
μ
M
0.61 ± 0.13
μ
M
II-2
10.02 ± 0.83 nM
11.92 ± 0.38 nM
6.62 ± 0.51 nM
11.59 ± 1.60 nM
11.68 ± 2.30 nM
12.65 ± 1.90 nM
II-3
II-4
Olaparib
5.86 ± 0.28
μ
M
13.00 ± 1.05
μM
a
Values are represented as mean ± SD from three independent experiments.
cell lines. It may ascribe to poor membrane permeability of compound I-
In summary, two series of novel PARP-1 inhibitors have been
designed and synthesized. Most compounds exhibited excellent PARP-1
inhibition activity in vitro, even displayed picomolar-level, which were
more potent than compounds XZ-120312 and Olaparib. All II series
compounds demonstrated impressive results in vitro BRCA1/2-deficient
cell lines assay. Among them, compound II-4 showed excellent inhibi-
tion activity against both PARP-1 enzyme and tumor cell lines, also with
stable metabolic properties in vitro. A preliminary structure–activity
relationship was analyzed, providing further optimization directions for
the following works. To improve druggability of target compounds,
further optimization is still ongoing in our lab.
1
. Compound I-6 displayed moderate anti-proliferation activity against
both tumor cell lines with IC50 values of 0.49 and 0.61 M, respectively,
μ
which are more potent than positive compound Olaparib. To our sur-
prise, compounds II-2 ~ II-4 exhibited excellent anti-proliferation ac-
tivity with nanomolar levels. Especially, compound II-4 showed single-
nanomolar activity against HCT116, which were 885-time more potent
than Olaparib. We speculate that the differences in cellular anti-
proliferation activity of compounds II-4, I-6 and Olaparib may be
attributed to the following factors. Firstly, considering structural prop-
erties, the rigid tricyclic ring of II-4 has higher liposolubility than the
piperazine and benzimidazole ring of I-6, which is more conducive to
membrane penetration of II-4. Secondly, molecular properties calcu-
lated by QikProp (Table S1) predict that II-4 has lower value of PSA
2
2
(
100.444 Å ) than Olaparib and I-6 (112.73 and 115.022 Å ,
Fig. 3. Docking mode of Olaparib (A) and II-4 (B) with PARP-1 (PDB: 5DS3). The H-bonds were shown as green dot lines and the key residues in the active site of
PARP-1 were shown as purple red. (A) The docking pose of Olaparib in PARP-1 crystal structure, and the compound was shown as blue. (B) The docking pose of II-4
in PARP-1 crystal structure, and the compound was shown as orange.
5

H. Shen et al.
Bioorganic & Medicinal Chemistry Letters 47 (2021) 128169
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