Identification of 2-substituted pyrrolo[1,2-b]pyridazine derivatives as new PARP-1 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2020.127710

A library of new 2-substituted pyrrolo[1,2-b]pyridazine derivatives were rapidly assembled and identified as PARP inhibitors. Structure-activity relationship for this class of inhibitor resulted in the discovery of most potent compounds 15a and 15b that e

Full text of DOI:10.1016/j.bmcl.2020.127710

Bioorg. Med. Chem. Lett. 31 (2021) 127710
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of 2-substituted pyrrolo[1,2-b]pyridazine derivatives as new
PARP-1 inhibitors
,
b
,
,
,
Hao-Yue Xianga e, Jian-Yang Chenb e, Xia-Juan Huanb e, Yi Chenc, Zhao-bing Gaod,
Jian Dingb, Ze-Hong Miaob *, Chun-Hao Yangb
,
,*
a College of Chemistry and Chemical Engineering, Central South University, Changsha, Hunan 410083, PR China
b State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, PR China
c Division of Anti-tumor Pharmacology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, PR China
d Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
A library of new 2-substituted pyrrolo[1,2-b]pyridazine derivatives were rapidly assembled and identified as
PARP inhibitors. Structure-activity relationship for this class of inhibitor resulted in the discovery of most potent
compounds 15a and 15b that exhibited about 29- and 5- fold selective activity against PARP-1 over PARP-2
respectively. The antiproliferative activity of the as-prepared compounds were demonstrated by further cel-
luar assay in BRCA2-deficient V-C8 and BRCA1-deficient MDA-MB-436 cell lines, displaying that compound 15b
could robustly reduce the corresponding cell proliferation and growth with CC50s of 340 and 106 nM respec-
tively. The PK property of 15b was also investigated here.
Antiproliferative
Cancer
PARP inhibitors
Pyrrolo[12-b]pyridazine
Poly(ADP-ribose)polymerase (PARP), as a nuclear enzyme, plays
critical roles in a variety of physiological functions related to cell sur-
vival, proliferation and apoptosis.1–4 Especially, DNA strand breaks can
selectively activate PARP, accompanied by catalyzing the poly-ADP
ribosylation of corresponding acceptor proteins with nicotinamide
adenine dinucleotide (NAD+).5–6 Drug resistance is frequently appeared
in cancer patients during the procedure of chemotherapeutics and ra-
diation by repair of DNA damages.7 Given its implication in processes
related to DNA damages in the base excision repair pathway, PARP has
emerged to be an efficient target for cancer treatment.8,9
(Fig. 1).14–24 Encouragingly, the concept that inhibition of PARP-1 as a
clinically efficacious cancer-therapeutic strategy have been well docu-
mented by the approval of olaparib (AZD2281).25,26 Afterwards, other
three PARP-1 inhibitors including rucaparib, talazoparib and niraparib
have also been approved.8 Currently a set of PARP-1 inhibitors25,26 such
as veliparib27 or our own mefuparib28 are active in a variety of clinical
trials. Not surprisingly, all these reported inhibitors share similar
structural and chemical features, including an aromatic ring and car-
boxamide moiety, which can interact with the catalytic domain of PARP-
1 through hydrogen bonds and
π
-stacking.13 Recently, a straightforward
There are at least 17 members consisted in PARP family and all of
them share a conserved domain. Between these members, DNA-binding
domain is only found in PARP-1 and PARP-2, with PARP-1 contributing
up to 95% of total activity for this family in response to DNA dam-
age.10,11 As a result, targeting PARP-1 is regarded as a rationale strategy
for cancer therapy and substantial breakthroughs in the realm of PARP-1
inhibitors have been achieved.12
methodology to 2-substituted pyrrolo[1,2-b]pyridazine derivatives was
reported by our lab.29 After analyzing the structural features of the
obtained pyrrolo[1,2-b]pyridazine products, we found that the structure
of these products possessed the common moiety in PARP-1 inhibitors
and thus rationalized that pyrrolo[1,2-b]pyridazine could be served as
novel scaffold for PARP-1 inhibitors. In this letter, we focused on
investigating the influence of the substituents at the 2-position of pyr-
rolo[1,2-b]pyridazine framework on the activity of the newly designed
compounds to inhibit PARP1/2 and cell proliferation, culminating in the
discovery of potent PARP-1 inhibitor 15b.
Designing structural analogues of NAD+ to compete with NAD itself
at the level of the catalytic domain is the most direct strategy thus to
inhibit the activity of PARP.13 By now, a number of PARP inhibitors
mimicking the nicotinamide moiety of NAD+ have been developed
As shown in Scheme 1, the commercially available starting materials
* Corresponding authors.
E-mail addresses: zhmiao@simm.ac.cn (Z.-H. Miao), chyang@simm.ac.cn (C.-H. Yang).
e
These authors contributed eaqually.
https://doi.org/10.1016/j.bmcl.2020.127710
Received 29 September 2020; Received in revised form 11 November 2020; Accepted 19 November 2020
Available online 24 November 2020
0960-894X/© 2020 Elsevier Ltd. All rights reserved.

H.-Y. Xiang et al.
Bioorganic & Medicinal Chemistry Letters 31 (2021) 127710
Fig. 1. Representative PARP-1 inhibitors and our design.
Scheme 3. Route for the synthesis of compounds 12. Reagents and conditions:
(a) HCl, CH2Cl2, reflux to remove Boc group; (b) NH3 in CH3OH, tube sealed,
90 ◦C; (c) RCOCl, NEt3, CH2Cl2, rt.
Scheme 1. Preparation of the key intermediates 4–6. Reagents and conditions:
(a) NaH, NH2Cl in ether, DMF 0 ◦C ~ rt, yield 85%; (b) KI, H2O2, AcOH, rt, yield
78% ; (c) Pd(OAc)2, CuI, DBU, toluene, MW, 90 ◦C.
Scheme 4. Route for the synthesis of compounds 15. Reagents and conditions:
(a) CF3COOH, CH2Cl2, rt; (b) 37% formaldehyde aqueous solution, AcOH,
NaBH3CN, CH3OH; (c) NH3 in CH3OH, tube sealed, 90 ◦C.
the corresponding intermediate 5e (yield 35%). Alternatively, acylation
of the deprotection product of intermediate 10 with appropriate acyl
chlorides produced the amide derivatives 12f-12i. Similarly, the third
series of products 15 could be conveniently prepared according to the
route outlined in Scheme 4 and the key intermediates 6 were prepared
according to our previous report.31
With these as-prepared pyrrolo[1,2-b]pyridazine derivatives in
hands, we next shifted our attention to evaluate their biological activity
at both enzymatic and cellular level with AZD2281 as reference com-
pound (Table 1). BRCA1-deficient human breast cancer cells MDA-MB-
436 and BRCA2-deficient Chinese hamster cell mutant V-C8 were
employed in cell assays to verify the antiproliferative activity. Most of
the tested compounds exhibited potent inhibition against PARP1 with
the IC50 values in the nanomolar range. Generally, the third series of
designed derivatives 15 bearing tetrahydrothienopyridinyl segments
showed more potent than the other two series of 2-substituted pyrrolo
[1,2-b]pyridazine derivatives 9 and 12 at both enzymatic and cellular
level. Interestingly, most of these compounds displayed stronger inhi-
bition toward PARP-1 over PARP-2. Specifically, as for the first series,
the substitutions on the nitrogen atom in the side chain of compounds 9
had tremendous effect on the inhibition potency and selectivity. The
inhibitory activity for PARP1/2 usually decreased with the increase
volume of basic side chain of compounds 9. Simplest methyl group
substituted on nitrogen atom led to compound 9b, resulting in a 47-fold
selectivity toward PARP-1 over PARP-2, however its enzymatic and
cellular potency against BRCA1-deficient MDA-MB-436 cells both
reduced compared to 9a. The introduction of piperazinyl moiety was
unable to improve their biological profiles. Obviously, the appendants
Scheme 2. Route for the synthesis of compounds 9. Reagents and conditions:
(a) 3 M HCl, dioxane, reflux, yield 80% ; (b) amines, NaBH3CN, AcOH, CH3OH,
rt ; (c) NH3 in CH3OH, sealed tube, 90 ◦C, yields for two steps.
methyl 4-chloro-1H-pyrrole-2-carboxylate 1 was able to converted to
methyl 1-amino-4-chloro-5-iodo-1H-pyrrole-2-carboxylate 3 smoothly
by sequential amination and iodization process. Following, the key in-
termediates 4–6 were easily prepared through
a domino cou-
pling–isomerization–condensation reaction between compound 3 and
corresponding (hetero)aryl propargyl alcohols. Subsequently, three se-
ries of designed target compounds were prepared according to the routes
displayed in Schemes 2–4 respectively.
Hydrolysis of acetal 4 gave the corresponding aldehyde 7. Further
reductive amination reaction of aldehyde 7 by being treated with a
range of diverse amines, provided the desired 2-(4-(aminomethyl)
phenyl)-pyrrolo[1,2-b]pyridazine analogs 8. Final aminolysis of com-
pounds 8 delivered the first series of target compounds 9 .
As shown in Scheme 3, the second series of designed 2-substituted
pyrrolo[1,2-b]pyridazine derivatives 12 could be obtained from com-
pounds 5. Direct aminolysis of intermediates 5a-5d yielded the desired
final products 12a-12d. Analogously, the products 11 and 12e were
formed through a two-step process of deprotection and aminolysis from
2

H.-Y. Xiang et al.
Bioorganic & Medicinal Chemistry Letters 31 (2021) 127710
Table 1
Table 2
Enzymatic and cellular assays of compounds 9, 12 and 15.a
PK properties of 15b⋅HCl.
Cpd.
PARP-1
IC50
PARP-2
IC50
V-C8
MDA-MB-436
Selectivityb
PARP ꢀ 1 vs
ꢀ 2
Dose
AUC0-∞
Cmax (ng/
mL)
MRT
(h)
CL (l/h/
kg)
T1/2
(h)
F
(BRCA2-/-
)
(BRCA1-/-
)
(mg/kg)
(ng⋅h/mL)
(%)
(nM)
(nM)
CC50
(
μ
M)
CC50 (nM)
5 (i.v.)
942
519
–
93.3
1.41
4.17
5.36
0.92
1.5
–
9a
93.27
248.69
433.50
432.39
818.27
626.68
1082.86
368.98
3328.75
361.84
3512.86
660.67
114.09
340.29
443.20
501.91
218.51
166.15
7.24
704.62
1118.31
2202.99
1118.78
1126.00
2325.82
997.49
6.20
2.00
3.51
2.15
2.78
n.d.
686.95
1284.32
3734.56
5398.52
5478.26
9668.86
75231.91
3546.08
8092.39
9644.52
>10
7.5
47.7
5.1
10 (p.o.)
–
23.5
9b
Abbreviations: i.v., intravenous injection; p.o., per oral; AUC, area under the
9c
concentration–time curve; Cmax, peak plama concentration of a drug after
9d
2.6
administratration; MRT, mean residence time; CL, plasma clearance; T1/2
,
9e
1.4
9f
3.7
elimination half-life; F, bioavailability.
9g
>10
2.70
>10
n.d.
0.92
3.9
9h
1444.72
445.06
IC50 = 11.31
= 1.31 M), and cisapride was used as the positive control (hERG IC50
0.27 M). As such, the compound 15b was further selected to investigate
μM) were much less toxic than compound 15a (hERG IC50
9i
0.13
4.74
1.28
2.4
μ
=
12a
12b
12c
12d
12e
12f
12g
12h
12i
15a
15b
15c
15d
15e
15f
15g
15h
15i
AZD
2281c
1714.13
4508.40
1594.00
1642.75
1005.03
1104.91
1010.30
2324.48
1923.75
209.38
4.42
n.d.
μ
its pharmacokinetic properties (PK).33 The mouse PK studies were per-
formed on 15b hydrochloride and the results of the behavior of 15b⋅HCl
in vivo were expressed in Table 2. Low oral exposure with high clearance
and short half-life was observed. Comparing to the clinical PARP in-
hibitor AZD2281, the oral availability of 15b⋅HCl were acceptable
(23%), which support itself for its anti-tumor efficacy in vivo. A pre-
liminary in vivo antitumor studies were carried out on BRAC-1-mutated
MAD-MB-436 xenograft model, revealing that 15b⋅HCl significantly
inhibited the growth of tumor with administrating orally once a day for
21 days in a dose-dependent manner (Table S1) with no obvious weight
loss (Table S2).33 Even so, further efforts to improve the PK and other
druggable profile of this series of compounds still need to be devoted.
In an effort to develop novel PARP inhibitors, a series of 2-
substituted pyrrolo[1,2-b]pyridazine derivatives were synthesized and
evaluated. Noticeably, pyrrolo[1,2-b]pyridazine was widely encoun-
tered in the realm of kinase inhibitors, but it is the first time to be
identified as PARP inhibitors. Most of the newly prepared analogues
potently inhibits the target enzyme and the proliferation of BRCA1-
deficient MDA-MB-436 cells and BRCA2-deficient V-C8 cells. In some-
what, the selectivity of inhibition activity toward PARP-1 over PARP-2
was observed in several tested compounds. Intensive exploration of
the side chain on the 2-position of pyrrolo[1,2-b]pyridazine skeleton
delivered compound 15b with superior inhibitory against PARP1/2 and
antiproliferative activity. The results of the PK studies revealed that the
oral availability of 15b was acceptable. Preliminary antitumor efficacy
assays reinforced the lead-like performance of 15b, rendering it eligible
for further development or optimization.
>10
n.d,
>10
8.9
>10
2.48
5.75
2.31
7.03
1.33
0.34
5.52
6.65
n.d.
8092.39
9538.09
3836.46
2953.37
>10
3.0
2.5
2.0
10.7
11.6
29.0
4.7
213.99
106.31
2983.54
1846.58
n.d.
14.20
66.81
127.54
184.96
434.19
240.49
1033.81
156.53
140.83
1198.40
2756.47
811.39
9.4
14.9
1.9
501.60
2.68
2.04
2.64
3.62
1363.07
1461.94
3052.52
2579.66
2.1
999.67
0.97
2.3
363.35
382.13
2.7
5.22
1.87
0.40
11.38
0.36
a IC50 values were calculated by Logit method from the results of at least three
independent tests with six concentrations each (standard deviations were within
25% of the mean values); Cytotoxic effect (CC50) means the concentration
required to reduce cell proliferation and growth by 50%. Values were calculated
by Logit method from the results of at least three independent tests with six
concentrations each (standard deviations were within 25% of the mean val-
ues).bfold selectivity of PARP-1 vs PARP-2 (PARP-2 IC50/PARP-1 IC50).
cAZD2281 is olaparib.
on piperazinyl segment such as cyclopropyl, i-Pr and p-CF3Ph groups in
compounds 9g-9i were detrimental for their inhibition activity.
Subsequently, learning from the success of the discovery of the PARP
inhibitor olaparib,30 we introduced a range of benzamide side chains to
this scaffold and designed the corresponding compounds 12. These
modification maintained the PARP inhibition potency, but resulted in
decreased antiproliferative activity, which probably caused by their low
aqueous solubility. In our recent work,31 we demonstrated that the
tetrahydrothienopyridinyl motifs were well tolerated in a series of
benzimidazole carboxamides as PARP inhibitors. Therefore, various
tetrahydrothienopyridinyl segment was incorporated and evaluated.
Pleasingly, significantly increasing of the inhibition potency against
both PARP-1/2 and antiproliferative activity against both BRCA1-defi-
cient MDA-MB-436 cells and BRCA2-deficient V-C8 cells, were observed
in analogues 15. Especially, the compounds 15a and 15b exhibited the
highest PARP-1 (15a, IC50 = 7.24 nM; 15b, IC50 = 14.2 nM) and PARP-2
potency (15a, IC50 = 209.38 nM; 15b, IC50 = 66.81 nM), as well as
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
This work was supported by Institutes for Drug Discovery and
Development, Chinese Academy of Sciences (CASIMM0120185003) and
the State Key Laboratory of Drug Research Program (SIMM1903ZZ-03).
Appendix A. Supplementary data
cellular potency for BRCA2-deficient V-C8 cell (15a, CC50 = 1.33 μM;
15b, IC50 = 340 nM) and BRCA1-deficient MDA-MB-436 cell (15a, CC50
= 213.99 nM; 15b, IC50 = 106.31 nM), among all the investigated
compounds. Similar to the analogues 9g-9i, masking the nitrogen atom
in pyridinyl ring, resulted in decreased potency (compounds 15h-15i).
As a consequence, it could be concluded that a free NH group seems to be
beneficial for securing its high biological activity.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmcl.2020.127710.
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4