Pyridazino[1,6-b]quinazolinones as new anticancer scaffold: Synthesis, DNA intercalation, topoisomerase I inhibition and antitumor evaluation in vitro and in vivo

Article abstract of DOI:10.1016/j.bioorg.2020.103814

A new anticancer N-containing heterocyclic scaffold was designed and 30 pyridazino[1,6-b]quinazolinone derivatives were synthesized and characterized. Antiproliferation evaluation in vitro against four human cancer cell lines including SK-OV-3(ovarian cell), CNE-2(nasopharyngeal cell), MGC-803(gastric cell) and NCI-H460(lung cell) indicated that most of them exhibited potent anticancer activity and the IC₅₀ value of the most potent compound lowered to sub-μM. DNA interaction assay indicated that compounds 4e, 4g, 6o, 6p, 8o can intercalate into DNA. Compounds 6 and 8 also demonstrated potent topoisomerase I (topo I) activity. Annexin V- FITC/propidium iodide dual staining assay and cell cycle analysis indicated that 2-(4-bromophenyl)-4-((3-(diethylamino)propyl)amino) ?10H-pyridazino [1,6-b]quinazolin- 10-one (8p) could induce arrest cell cycle at G2 phase and apoptosis in MGC-803 cells in a dose-dependent manner. The in vivo antitumor efficiency of compound 8p was also evaluated on MGC-803 xenograft nude mice, and the relative tumor growth inhibition was up to 55.9% at a dose of 20 mg/kg per two days. The results suggested that pyridazino[1,6-b]-quinazolinones might serve as a promising novel scaffold for the development of new antitumor agents.

Full text of DOI:10.1016/j.bioorg.2020.103814

BioorganicChemistry99(2020)103814
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Bioorganic Chemistry
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Pyridazino[1,6-b]quinazolinones as new anticancer scaffold: Synthesis, DNA
intercalation, topoisomerase I inhibition and antitumor evaluation in vitro
and in vivo
⁎
⁎
Wan-Yun Huanga, , Xiao-Rong Zhanga, Liang Lyub, , Shu-Qin Wanga, Xiao-Ting Zhanga
a Department of Pharmaceutical Chemistry, College of Pharmacy, Guilin Medical University, Guilin 541004, China
b Department of Pharmacology, College of Pharmacy, Guilin Medical University, Guilin 541004, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
A new anticancer N-containing heterocyclic scaffold was designed and 30 pyridazino[1,6-b]quinazolinone de-
rivatives were synthesized and characterized. Antiproliferation evaluation in vitro against four human cancer cell
lines including SK-OV-3(ovarian cell), CNE-2(nasopharyngeal cell), MGC-803(gastric cell) and NCI-H460(lung
cell) indicated that most of them exhibited potent anticancer activity and the IC50 value of the most potent
compound lowered to sub-μM. DNA interaction assay indicated that compounds 4e, 4g, 6o, 6p, 8o can inter-
calate into DNA. Compounds 6 and 8 also demonstrated potent topoisomerase I (topo I) activity. Annexin V-
FITC/propidium iodide dual staining assay and cell cycle analysis indicated that 2-(4-bromophenyl)-4-((3-
(diethylamino)propyl)amino) −10H-pyridazino [1,6-b]quinazolin- 10-one (8p) could induce arrest cell cycle at
G2 phase and apoptosis in MGC-803 cells in a dose-dependent manner. The in vivo antitumor efficiency of
compound 8p was also evaluated on MGC-803 xenograft nude mice, and the relative tumor growth inhibition
was up to 55.9% at a dose of 20 mg/kg per two days. The results suggested that pyridazino[1,6-b]-quinazoli-
nones might serve as a promising novel scaffold for the development of new antitumor agents.
Pyridazino[1,6-b]quinazolinones
Tricyclic quinazolinone
Topoisomerase I inhibition
Antitumor
Synthesis
1. Introduction
possess a poly(hetero)aromatic skeleton. Most of them also have a side
chain with ω-dialkyl-amino or nitrogen-containing heterocycle sub-
Quinazolinone and pyridazine are very important privileged struc-
tures. Many clinical drugs or bioactive compounds contain the quina-
zolinone [1–9] or pyridazine [10–15]. Tricyclic quinazolinone, as im-
portant kinds of fused quinazolin-4(3H)-one derivatives, present in
many natural products or bioactive compounds [16–21], re-
presentatives of them include mackinazolinone, vasicinone and their
aza- derivatives A and B (Fig. 1).
stituent [34–36]. The representative drugs and scaffolds are the CPTs
Topotecan and Belotecan [34], the indenoisoquinoline scaffold C
[37–39] and the aromathecins derivatives
D [40] developed by
Cushman’ group, as well as more recently the 3-benzazolyl quinoxaline
derivatives E reported by the PAN and SU [41] (Fig. 2). Inspired by
their work, herein, we report the synthesis of a novel tricyclic quina-
zolinone scaffold- pyridazino[1,6-b]quinazolinone derivatives and
evaluation of antitumor, DNA interaction and topo I inhibition of them.
DNA topoisomerase I (Topo I) is an effective and well-established
anticancer target [22–27]. Topo I inhibitor Camptothecin (CPT) and its
derivatives (CPTs) have been widely used in clinical therapy [28–30].
However, their unsatisfactory physicochemical and pharmacokinetic
properties originating from the intrinsic chemical instability and poor
solubility of CPT, as well as the emergence of drug resistance, limit their
clinical application [31–33]. To identify novel scaffolds of Topo I in-
hibitors endowed with improved physicochemical and pharmacokinetic
profiles has recently emerged as a promising strategy for the discovery
of new antitumor agents [34–36].
2. Results and discussion
2.1. Chemistry
The synthesis of compounds 4, 6–8 were outlined in Scheme 1. The
key intermediate 2a, readily prepared from methyl 2-aminobenzoate
following the literature procedure in two steps [42], was condensed
with ketone derivatives catalyzed by TsOH to give the acylhydrazones
compounds 3a-n. Then 3a-n undergo an intramolecular condensation
Structurally, DNA or topo I-targeting antitumor compounds usually
⁎ Corresponding authors.
E-mail addresses: hwygxnu@163.com (W.-Y. Huang), luliang998@163.com (L. Lyu).
https://doi.org/10.1016/j.bioorg.2020.103814
Received 17 December 2019; Received in revised form 26 February 2020; Accepted 31 March 2020
Availableonline04April2020
0045-2068/©2020ElsevierInc.Allrightsreserved.

W.-Y. Huang, et al.
BioorganicChemistry99(2020)103814
Fig. 1. The representatives bioactive tri-
cyclic quinazolinone derivatives.
Fig. 2. The design of novel DNA topo I-targeting scaffold.
Scheme 1. Reagents and conditions: (i) H2N4-H2O, reflux; (ii) (EtOCO)2, 180 °C; (iii) R1COCH2R2, toluene, TsOH, reflux; (iv) THF, NaH, reflux; (v) POCl3, reflux; (vi)
NH2(CH2)nR, toluene, reflux.
promoted by NaH in reflux THF to furnish the pyridazino-[1,6-b]qui-
nazolinones compounds 4a-n.
normal cell line (human hepatic LO2). CPT and Doxorubicin (Dox) were
used as the positive controls. The results are summarized in Table 1.
As shown in Table 1, most of the target compounds show moderate
to potent cytotoxicity against the tested cancer cell lines. Among them
the series of compound 8–10 with a side chain generally result in
stronger antitumor activity than the series of 4 which has no side chain.
The substituents on the other position of the pyridazino[1,6-b]quina-
zolinone nucleus also significantly affect the potency.
Compounds 6–8 with a side chain at the pyridazino[1,6-b]quina-
zolinone skeleton also can be readily prepared from compounds 4a, 4b
or 4g in two steps. Firstly compounds 4 were mixed with POCl3 and
refluxed to provide the intermediates compounds 5, which were further
reacted with different amine derivatives to provide the compounds 6–8
in high yields.
As to the series of compound 4, the data suggest that the kinds of R1
substituents on the pyridazino[1,6-b]quinazolinone nucleus played
significant roles in the antitumor activity. In general, an aryl (com-
pounds 4a-k) provide more potent anti-proliferative activity than an
alkyl (compounds 4l-n) and a common aromatic ring (4a-i) seems more
favorable than an aromatic heterocyclic motifs (4j-k). The exception is
that when the aryl group is a methoxyphenyl (compounds 4h-i), the
2.2. Evaluation of biological activity
2.2.1. Cytotoxicity
The cytotoxic activity of compounds 4a-n, 6–8 was evaluated via
MTT assay in four tumor cell lines (ovarian cell SK-OV-3, nasophar-
yngeal cell CNE-2, gastric cell MGC-803, lung cell NCI-H460) and one
2

W.-Y. Huang, et al.
BioorganicChemistry99(2020)103814
Table 1
Anticancer activity evaluation of compounds 4, 6–8.
IC50 (μM)a
Comp.
R1,R2 or n, R
SK-OV-3
CNE-2
MGC-803
NCI-H460
LO2
4a
4b
4c
4d
4e
4f
Ph, H
21.24
21.47
20.18
35.85
16.08
9.52
1.72
3.37
2.33
2.03
2.06
13.71
> 100
16.94
36.32
5.49
1.07
13.56
21.58
15.72
> 100
3.39
2.49
2.11
19.90
21.96
16.58
25.61
2.31
1.29
1.96
1.78
4.10
60.68
> 100
> 100
> 100
30.90
18.78
1.49
4.86
2-F-Ph, H
4-F-Ph, H
2-Cl-Ph, H
3-Cl-Ph, H
4-Cl-Ph, H
4-Br-Ph, H
3-MeO-Ph, H
4-MeO-Ph, H
2-furyl, H
2-thieny, H
Et, Me
0.89
2.81
1.96
7.53
1.32
1.02
1.28
0.16
1.36
1.07
1.04
0.93
4.10
0.73
0.11
9.63
18.56
6.13
2.36
4g
4h
4i
5.34
0.85
3.37
2.41
0.40
> 100
50.11
14.91
16.34
> 100
25.47
27.96
5.90
85.97
> 100
33.54
13.07
> 100
> 100
> 100
5.82
8.94
44.54
22.77
27.25
11.05
65.89
24.61
25.40
3.51
3.24
56.24
54.47
25.52
28.36
> 100
> 100
> 100
3.68
4.72
4.01
2.18
2.13
45.18
> 100
> 100
> 100
> 100
> 100
> 100
12.91
6.40
5.56
4.17
6.05
4.47
3.68
2.09
1.71
0.94
4j
1.09
1.73
4.60
4.35
4k
4l
4m
4n
6o
6p
6q
6r
e(CH2)3-
e(CH2)4-
3, N(Me)2
3, N(Et)2
3, morph.b
2, N(Me)2
2, N(Me)2
2, pyrrol.c
2, piper.d
3, N(Me)2
3, N(Et)2
3, morph.
2, N(Me)2
2, N(Me)2
2, pyrrol.
2, piper.
3, N(Me)2
3, N(Et)2
–
1.80
2.57
0.64
0.10
0.25
0.27
0.15
0.39
0.11
1.96
2.86
0.22
5.17
3.14
3.15
0.18
75.91
31.10
15.44
13.49
9.90
7.09
> 100
24.96
14.66
16.01
34.89
9.05
> 100
30.21
8.70
> 100
46.54
11.35
7.44
> 100
> 100
> 100
12.66
> 100
19.19
17.83
> 100
14.31
> 100
> 100
53.99
5.10
2.14
3.13
2.90
1.50
2.92
2.61
1.03
0.77
3.80
6s
2.05
3.21
2.51
0.94
0.40
6t
3.62
2.78
6u
7o
7p
7q
7r
2.69
11.50
9.20
1.98
42.76
7.01
10.75
7.61
1.34
1.67
1.25
2.24
1.88
1.22
3.60
3.13
10.17
> 100
23.31
> 100
47.76
42.07
1.66
2.67
3.91
9.54
10.40
> 100
31.38
43.15
> 100
62.87
2.74
3.18
> 100
19.38
23.21
> 100
15.48
3.31
> 100
15.27
20.19
30.58
16.51
5.06
3.8
8.8
3.18
6.00
5.64
3.16
7s
4.86
5.06
4.76
7t
9.86
6.00
7u
8o
8p
CPT
Dox
8.03
7.95
6.30
0.28
0.33
0.67
0.38
0.79
1.93
0.16
0.11
0.15
2.33
0.52
0.21
0.18
1.39
0.14
0.74
0.07
1.55
0.14
0.18
0.12
5.71
0.60
1.02
0.51
1.16
1.94
4.52
2.32
5.37
–
1.89
1.72
0.62
1.11
2.81
a
b
c
Results were expressed as means
morph. = morpholinyl.
pyrrol. = pyrrolidinyl.
piper = piperidinyl.
SD (standard deviation) from triplicate assay in a single experiment, P < 0.05.
d
cytotoxicity of the compounds will decrease significantly. As to the
heterocycle, a thienyl(compound 4k) seems more favorable than a furyl
(compound 4j).
ethylamino (compounds 6r-u) only give moderate cytotoxic activity.
The low cytotoxic activity of compounds 7o-u shown in Table 1 in-
dicate that an aryl with ortho-position substituent might give negative
effects on cytotoxicity, which suggest that the size of dihedral angle
formed by the pyridazino[1,6-b]quinazolinone nucleus with the aro-
matic ring, might also play significant effects on the activity of the
target compounds since a ortho-position substituent can significantly
affect the dihedral angle of the two units.
The substituent and its position on the phenyl of compounds 4a-i
also play important roles in the anti-proliferation activity. Br (com-
pound 4g) or Cl (compounds 4d-f) appear to be more favorable than H
(compound 4a), F(compounds 4b-c) or methoxyl (compounds 4h-i).
And substituent on the para- or meta-position usually result in higher
anti-proliferation activity than on the ortho-position. Among the series
of compounds 4, 4g and 4e display the most promising anti-pro-
liferative activity with IC50 values of 0.85–6.13 μM and 2.31–16.8 μM
against the four tested cancer cell lines, respectively. Though compound
4g exhibit higher potency than compound 4e, it also give the most
2.2.2. Interaction with DNA
Since many cytotoxic molecules with poly-aromatic systems usually
exert their antitumor properties via DNA intercalation [43]. To obtain
more detailed information about the cytotoxic properties of the com-
pounds, DNA binding properties of compounds 4e, 4g, 6o, 6p, 8o, 8p
were investigated via spectroscopic analyses. The UV–Vis spectra of
compounds 4e, 4g, 6o, 6p, 8o, 8p interaction with ct-DNA was shown
in Fig. 3(A–F), the dashed line is the absorption of ct-DNA. With the
addition of the compounds, the increase in the absorption intensity at
about 215 and 260 nm was observed, indicating the interaction of the
compounds with ct-DNA and all of the tested compounds can induce
hyperchromic effect when binding to ct-DNA.
toxicity to the normal cell LO2 with an IC50 value of 1.49
more than 20 fold higher than that of compound 4e
(30.90 1.36 μM).
0.40 μM,
To further investigation on how the bioactivity of compounds 4 is
affected when the hydroxyl group on pyridazino[1,6-b]quinazolinone
skeleton replaced by a side chain. Compounds 6–8 with different ω-
substituents of propylamino (compounds 6o-q, 7o-q, 8o-p) or ethyla-
mino (compounds 6r-u, 7r-u) were synthesized from compounds 4a, 4b
and 4g. The data in Table 1 indicate that the side chain can significantly
affect the cytotoxic activity. When the side chain is an ω-dialkyl-amino
substituted propylamino group (compounds 6o-p, 7o-p, 8o-p), the cy-
totoxicity of the compounds increased, especially when the ω-sub-
stituted group is diethylamino (compounds 6p, 8p). But when the ω-
substituted is a morpholinyl group (compounds 6q, 7q), the compound
will lose its cytotoxic activity. And the side chain with an ω-substituted
To obtain more information about the relationship between the
DNA binding properties and the cytotoxicity of compounds 4e, 4g, 6o,
6p, 8o and 8p, competitive binding assay was further performed using
the classic DNA intercalator ethidium bromide (EB) as a probe. As
shown in Fig. 3(G–L), the addition of compounds 4e, 4g, 6o, 6p, 8o, 8p
significantly quenched the ct-DNA–EB fluorescence intensity. The
quenching constant K was calculated to be 1.00–3.17 × 104, according
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W.-Y. Huang, et al.
BioorganicChemistry99(2020)103814
Fig. 3. Spectroscopic analyses of compounds 4e, 4g, 6o, 6p, 8o, 8p when interacting with DNA. (A-F) UV–Vis absorption spectra of ct-DNA in the absence (dashed
line) and presence (solid lines) of 4e, 4g, 6o, 6p, 8o, 8p. (G-L) Fluorescence quenching curves of EB bound to ct-DNA in the absence (dashed line) and presence (solid
lines) of 4e, 4g, 6o, 6p, 8o, 8p.
to the Stern–Volmer quenching equation, indicating that compounds
4e, 4g, 6o, 6p, 8o and 8p all can efficiently compete with EB to in-
tercalate into ct-DNA. Compound 4e (K = 3.17 × 104), with a higher
value of K while exhibited a less cytotoxicity than compounds 4g, and
6o, 8o, 8p which share almost the same value of K, displaying ob-
viously different in cytotoxic potency, suggesting beside the DNA
binding properties, other mechanism might be involve in their
anticancer activity.
2.2.3. Topo I inhibition assay
To determine whether the DNA intercalation properties of com-
pounds 4e, 4g, 6o, 6p, 8o and 8p might result in Topo I inhibition,
agarose gel electrophoresis assay was performed to evaluate the Topo I
inhibitory activity of them via agarose-gel electrophoresis and CPT was
4

W.-Y. Huang, et al.
BioorganicChemistry99(2020)103814
Fig. 4. (A) Topo I inhibitory activity of compounds 4e, 4g, 6o, 6p, 8p, 8o. Lane 1: DNA, lane 2: DNA + Topo I, lanes 3–4: 4e (50 or 100 µM) + DNA + Topo I, lanes
5–6: 4g (50, 100 µM) + DNA + Topo I, lanes 7–8: 6o (5, 50 µM) + DNA + Topo I, lanes 9–11: 6p (5, 25 or 50 µM) + DNA + Topo I, lanes 12–14: 8p (5, 25 or
50 µM) + DNA + Topo I, lanes 15–16: 8o (5, 50 µM) + DNA + Topo I, lanes 17–18: CPT (50, 100 µM) + DNA + Topo I. (B) Topo I inhibitory activity of 8p, 6p.
Lane 1: DNA, lane 2: DNA + Topo I, lanes 3–8: 8p (5, 15, 25, 35, 45, 50 µM) + DNA + Topo I, lanes 9–14: 6p (5, 15, 25, 35, 45, 50 µM) + DNA + Topo I, lanes
15–16: CPT (50, 100 µM) + DNA + Topo I.
used as the positive control. The results in Fig. 4(A) indicated that
compounds 6o, 6p, 8o, 8p exhibited potent topo I inhibition activity
while compounds 4e and 4g did not display obvious topo I activity at
concentrations up to 100 μM, suggesting that compounds 4e and 4g
might exert their cytotoxicity via a different mechanism to that of the
compounds 6o, 6p, 8o and 8p.
evaluated via MGC-803 xenograft tumors models. As shown in Fig. 7,
the tumor-bearing mice treated by 8p (10 or 20 mg/kg/2 days) showed
obvious tumor growth inhibition (TGI) during the treatment period in a
dose-dependent manner. The TGI was 42.8% (compound 8p, 10 mg/
kg/2 days), 55.9% (compound 8p, 20 mg/kg/2 days), and 74.8% (Dox,
10 mg/kg/2 days), respectively (p
< 0.01). It is noteworthy that
Since compounds 6p and 8p displayed the most promising cyto-
toxicity and topo I activity, the topo I activity were further tested at
concentrations from 5 to 50 μM. As shown in Fig. 4(B), compounds 6p
and 8p almost completely inhibited the Topo I activity at concentra-
tions of about 25–35 μM, which suggesting that their cytotoxicity might
closely related to their capability of topo I inhibition.
though the TGI of compound 8p was lower than that of the positive
drug Dox, they resulted in a less body weight loss of the treated mice
even in a dose of 20 mg/kg/ 2 days. And during the experiments, none
of the mice treated with compound 8p showed signs of morbidity,
suggesting that compound 8p possessed anticancer efficacy in vivo with
low toxicity.
2.2.4. Cell cycle analysis
2.2.7. Molecular docking studies
As compound 8p displayed the most promising activity in cyto-
toxicity and Topo I inhibition, the mechanism of its cytotoxic effect was
further studied. Whether compound 8p inhibited the proliferation of
cancer cells through cell cycle arrest was first examined. Flow cyto-
metry assay were performed to investigate the cell cycle arresting effect
of compound 8p on MGC-803 cells.
Molecular docking [41] using CDOCKER in Discovery Studio 2018
was performed to predict and better understand the binding of com-
pound 8p with DNA-topo I and the ternary complex of Topo I, DNA, and
CPT (PDB code 1T8I) for the docking study. As shown in Fig. 8, the
pyridazino[1,6-b]quinazolinone skeleton of compound 8p (navy blue
color) can interact via pi-pi stacking with DNA base pairs while the
carbonyl forms hydrogen bond with ARG364 in Topo I. The para-Br-
phenyl also can interact with ARG364 via pi-cation interaction. At the
same time, the hydrogen of amino group on the side chain can form
hydrogen bond with THR718 and base pair DG while the ω-substituent
of diethyl can interact with ILE535 and HIS632 via Van der Waals in-
teractions.
As shown in Fig. 5, treatment with compound 8p slightly increased
the proportion of cells in G2 phase (from 10.75% (0 μM), 12.68%
(1 μM) to 15.13% (5 μM), indicating that compound 8p could induce a
weak G2 phase cell cycle arrest in a dose-dependent manner.
2.2.5. Apoptosis induction studies
To further examine whether the cytotoxicity of compound 8p could
induce apoptosis, annexin V FITC/PI dual staining assay were per-
formed to evaluate the apoptosis inducing effect of compound 8p on
MGC-803 cells.
To validate the docking protocol and to elucidate the SAR more
clearly, the docking study also perform on compound 6p and 7p with
the same protocol. The results show that compound 6p, 7p and 8p all
share a same binding mode with the target, merely different in the
CDOCKER energy from −39.4348, −34.7381 to −30.6377 kcal/mol
for 8p, 6p and 7p, respectively (seen in the supporting information),
which is in good consistent with the potency in cytotoxicity and Topo I
inhibitory activity of the three compounds. The molecular docking not
only elucidated the binding mode of compound 8p with DNA-topo I,
but also explained the observed structure–activity relationship results
in Table 1.
As shown in Fig. 6, treatment with compound 8p could significantly
increase the percentage of apoptotic cells (from 5.3% (0 μM) to 13.8%
(1.0 μM), 18.1% (5.0 μM), 40.0% (10.0 μM)), demonstrating that
compound 8p could induce significant apoptosis in MGC-803 cells in a
dose-dependent manner.
2.2.6. In vivo anticancer evaluation of compound 8p on MGC-803
xenograft model
The anticancer efficiency of compound 8p in vivo was further
Fig. 5. Cell cycle analysis of compound 8p. MGC-803 cells were treated with 8p (0, 1.0, 5.0 μM, respectively) for 48 h.
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W.-Y. Huang, et al.
BioorganicChemistry99(2020)103814
Fig. 6. Compound 8p inducing apoptosis. MGC-803 cells were treated with 8p (0, 1.0, 5.0 and 10 μM, respectively) for 24 h.
Fig. 7. The in vivo antitumor efficacy of compound 8p. (A) Tumor volume curve of the mice over the administration period. (B) Photographs of the mice and the
tumor from them. (C) The tumor weight and the TGI of each group at the end of treatment. (D) Body weight curve of the tested mice after experiment. Results are
expressed as the mean
SD, error bars represented SD, n = 6, (∗) p < 0.05 and (∗∗) p < 0.01 vs vehicle.
Fig. 8. The 2D (A) and 3D (B) diagram of binding mode for compound 8p with DNA-Topo I.
6

W.-Y. Huang, et al.
BioorganicChemistry99(2020)103814
3. Conclusion
mixture was cooled, quenched by the addition of 10 mL water and then
5 mL 0.5 M of HCl. The precipitate was filtered, washed with water and
cool ethanol to afford compounds 4a-n as yellow solid, yield 48–85%.
In conclusion, 30 novel pyridazino[1,6-b]quinazolinone derivatives
were designed and synthesized. Cytotoxicity assay indicated that many
of them displayed potent antitumor activity in different cancer cell
lines. The drug-DNA interaction study revealed that most cytotoxic
compounds can intercalate into DNA. Agarose-gel electrophoresis assay
and molecular docking indicated that compound 8p demonstrated
strong inhibition of Topo I to cause G2 cell phase arrest and trigger
cancer cell apoptosis. Furthermore, the in vivo anticancer efficacy of
compound 8p was also carried out in MGC-803 xenograft models and
the results indicating that 8p displayed promising anticancer activity in
vivo with tumor growth inhibition (TGI) up to 55.9%, suggesting that
the novel pyridazino[1,6-b]quinazolinones reported here could be
served as a novel promising scaffold for the development of new che-
motherapeutic agent.
4.2.3.1. 4-Hydroxyl-2-pheny-10H-pyridazino [6,1-b] quinazolin-10-one
(4a). Yield 76%, m.p. 221–223 °C; 1H NMR (400 MHz, DMSO‑d6) δ:
8.35 (d, J = 8.0 Hz, 1H), 8.09–8.07 (m, 2H), 7.97–7.96 (m, 2H),
7.63–7.55 (m, 4H), 7.25 (s, 1H). 13C NMR (100 MHz, DMSO‑d6) δ:
158.65, 158.40, 152.82, 144.36, 144.00, 135.92, 135.48, 130.74,
129.36, 127.70, 127.36, 126.59, 125.72, 118.81, 102.93. HRMS
(ESI+
)
m/z calcd for C17H12O2N3 [M
+
H]+ 290.0931, found
290.0922.
4.2.3.2. 2-(2-Fluorophenyl)-4-hydroxy-10H-pyridazino [6,1-b] quinazolin-
10-one (4b). Yield 58%, m.p. 294–296 °C; 1H NMR (400 MHz,
DMSO‑d6) δ: 8.34 (d, J = 7.8 Hz, 1H), 8.01–7.94 (m, 2H), 7.86 (dd,
J = 7.7, 1.6 Hz, 1H), 7.65–7.57 (m, 2H), 7.45–7.38 (m, 2H), 6.94 (d,
J = 2.1 Hz, 1H). 13C NMR (100 MHz, DMSO‑d6) δ: 161.63, 159.15,
158.44, 158.32, 150.73, 144.29, 143.67, 135.73, 132.65, 132.57, 131.23,
131.20, 127.81, 126.74, 125.44, 125.41, 125.11, 124.72, 124.60, 118.58,
117.06, 116.84, 106.18, 19F NMR (376 MHz, DMSO‑d6) δ:-107.40. HRMS
(ESI+) m/z calcd for C17H11O2N3F [M + H]+ 308.0837, found 308.0822.
4. Experimental section
4.1. Materials and methods
All commercially available reagents and solvents were used as re-
ceived without further purification and all reactions were carried out
under an atmosphere of dry nitrogen. Flash column chromatography
was performed on silica gel (200–300 mesh). Melting points were re-
corded on WRSIA apparatus without correction. NMR spectra were
recorded in CDCl3 or CD3OD or DMSO‑d6 on a Bruker Advance (500 or
400 MHz) with TMS as the internal standard. HRMS were recorded on a
Thermo Scientific Exactive LC-MS Spectrometer measured in ESI mode
and the mass analyzer was TOF.
4.2.3.3. 2-(4-Fluorophenyl)-4-hydroxy-10H-pyridazino [6,1-b] quinazolin-
10-one (4c). Yield 49%, m.p. > 300 °C; 1H NMR (400 MHz, DMSO‑d6)
δ: 8.35 (d, J = 6.7 Hz, 1H), 8.17–8.12 (m, 2H), 7.97 (s, 2H), 7.60 (s, 1H),
7.41–7.39 (m, 2H), 7.21 (s, 1H), 19F NMR (376 MHz, DMSO‑d6) δ:-119.62.
The solubility of compound 4c was too low to be characterized by 13C
NMR. HRMS (ESI+) m/z calcd for C17H11O2N3F [M + H]+ 308.0837,
found 308.0820.
FITC Annexin V Apoptosis Detection kit was purchased from BD
Biosciences. Propidium iodide (PI), 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT), DNA and topo I were purchased
from Sigma (St. Louis, MO). Cell lines were purchased from ATCC
(Manassas, VA) and cultured in RPMI 1640 medium (GIBCO-BRL,
Grand Island, NY) supplemented with 10% fetal calf serum (FCS).
4.2.3.4. 2-(2-Chlorophenyl)-4-hydroxy-10H-pyridazino [6,1-b] quinazolin-
10-one (4d). Yield 85%, m.p. > 300 °C; 1H NMR (400 MHz, DMSO‑d6)
δ: 8.25–8.23 (m, 1H), 8.05 (d, J = 8.3 Hz, 1H), 7.86–7.76 (m, 1H),
7.59–7.53 (m, 2H), 7.50–7.43 (m, 3H), 5.99 (s, 1H). 13C NMR (100 MHz,
DMSO‑d6) δ: 166.89, 159.90, 155.18, 150.20, 146.94, 138.52, 133.87,
131.97, 131.52, 130.44, 130.04, 128.22, 127.67, 127.53, 126.94, 125.11,
118.63, 103.43. HRMS (ESI+) m/z calcd for C17H11O2N3Cl [M + H]+
324.0542, found 324.0531.
4.2. General procedure for the preparation of compounds
4.2.1. Synthesis of ethyl 3-amino-4-oxo-3,4-dihydroquinazoline-2-
carboxylate 2
4.2.3.5. 2-(3-Chlorophenyl)-4-hydroxy-10H-pyridazino [6,1-b] quinazolin-
Methyl anthranilate (6.08 g, 0.04 mol) was mixed with 30 mL
(0.75 mol) hydrazine monohydrate (80%) and was refluxed for 4 h. The
reaction mixture was cooled to 4–5 °C and the 2-amino-benzohydrazide
precipitate was filtered, washed with cool water and dried to give the 2-
amino-benzohydrazide in 72% yield. A mixture of the 2-amino-benzo-
hydrazide (3.00 g, 20 mmol) and diethyl oxalate (15 mL, 0.1 mol) was
stirred at 180 °C in an oil bath for 8 h. The excess diethyl oxalate was
removed under vacuum. The resulting crude product was further pur-
ified by column chromatography on silica gel eluted with ethyl acetate/
petroleum ether (V: V = 1:4) to afford 2.80 g of compound 2 as white
solid, yield 61%, mp 132–133 °C (lit. 131–133°) [42].
10-one (4e). Yield 85%
,
m.p. 295–296 °C; 1H NMR (400 MHz,
DMSO‑d6) δ: 8.34 (d, J = 8.0 Hz, 1H), 8.13–8.12 (m, 1H), 8.06–8.01
(m, 1H), 7.99–7.92 (m, 2H), 7.63–7.56 (m, 3H), 7.16 (s, 1H). 13C NMR
(100 MHz, DMSO‑d6) δ: 160.02, 158.50, 151.78, 144.92, 143.93, 138.37,
135.52, 134.17, 131.26, 130.40, 127.73, 127.42, 126.95, 126.55, 126.05,
125.37, 118.59, 102.67, 99.99. HRMS (ESI+) m/z calcd for C17H11O2N3Cl
[M + H]+ 324.0542, found 324.0527.
4.2.3.6. 2-(4-Chlorophenyl)-4-hydroxy-10H-pyridazino[6,1-b] quinazolin-
10-one (4f). Yield 72%, m.p. > 300 °C; 1H NMR (400 MHz, DMSO‑d6)
δ: 8.34 (d, J = 6.9 Hz, 1H), 8.12–8.10 (m, 2H), 7.97 (s, 2H), 7.64–7.58
(m, 3H), 7.18 (s, 1H). The solubility of compound 4f was too low to be
characterized by 13C NMR. HRMS (ESI+) m/z calcd for C17H11O2N3Cl
[M + H]+ 324.0542, found 324.0525.
4.2.2. General procedure for synthesis of compounds 3a-n
Compound 2 (1.17 g, 5.0 mmol) and 10.0 mmol ketone were dis-
solved in 20 mL toluene. Then 10 mg toluene-p-sulfonic acid was added
and the mixture was refluxed for about 5–6 h (monitored by TLC). After
completed, the solvent was removed under vacuum and the crude
product was purified by column chromatography on silica gel eluted
with ethyl acetate/petroleum ether (V: V = 1:6) to afford the com-
pounds 3a-n, yield 36–84%.
4.2.3.7. 2-(4-Bromophenyl)-4-hydroxy-10H-pyridazino[6,1-b]quinazolin-
10-one (4g). Yield 57%, m.p. > 300 °C; 1H NMR (400 MHz, DMSO‑d6)
δ: 8.34 (d, J = 8.0 Hz, 1H), 8.04–8.03 (m, 2H), 7.97 (d, J = 2.7 Hz,
2H), 7.77 (d, J = 8.6 Hz, 2H), 7.63–7.59 (m, 1H), 7.22 (s, 1H). 13C
NMR (125 MHz, DMSO‑d6) δ: 158.33, 151.90, 144.19, 135.61, 135.05,
132.37, 129.40, 127.87, 126.64, 125.22, 124.37, 118.48, 102.76.
HRMS (ESI+) m/z calcd for C17H11O2N3Br [M + H]+ 368.0036,
found 368.0031.
4.2.3. General procedure for synthesis of compounds 4a-n
Compound 3 (1.0 mmol) were dissolved in 5 mL dry THF. Then
0.08 g 60% NaH (2.0 mmol) was added and the mixture was refluxed
for about 1 h (monitored by TLC). After completed, the reaction
4.2.3.8. 4-Hydroxy-2-(3-methoxyphenyl)-10H-pyridazino [6,1-b] quinazolin-
7

W.-Y. Huang, et al.
BioorganicChemistry99(2020)103814
10-one (4h). Yield 58%, m.p. 256–258 °C; 1H NMR (500 MHz, DMSO‑d6)
δ: 8.35 (d, J = 8.0 Hz, 1H), 7.97 (s, 2H), 7.75–7.54 (m, 3H), 7.48 (d,
J = 8.0 Hz, 1H), 7.24 (s, 1H), 7.14–7.12 (m, 1H), 3.86 (s, 3H). 13C NMR
(125 MHz, DMSO‑d6) δ: 160.11, 158.66, 158.38, 152.75, 144.14, 137.41,
135.55, 130.52, 127.76, 126.62, 125.51, 119.76, 118.77, 116.33, 112.79,
103.20, 55.79, 55.36. HRMS (ESI+) m/z calcd for C18H14O3N3 [M + H]+
320.1037, found 320.1029.
TLC), the excess POCl3 was removed under reduced pressure and ice-
water was poured in. The mixture was extracted with DCM and the
organic layer was washed with water and brine, dried over Na2SO4, and
concentrated in vacuum. The residue was chromatographed on silica gel
using hexane–EtOAc (1:1) as an eluent to give compounds 5 a (or 5b or
5 g) as pale yellow solid in 56–70% yield.
4.2.5. General procedure for synthesis of compounds 6–8
A solution 1 mmol of compounds 5a (or 5b or 5 g) and 5 mmol of
amine derivatives in toluene (5 mL) was heated in 90 °C for 1.5 h until
the reaction completed (monitored by TLC). Then the solvent was re-
moved under reduced pressure and the residue was purified by column
chromatography on silica gel eluted with ethyl acetate/petroleum ether
to provide compounds 6 (or 7 or 8) as pale yellow solid in 70–90%
yield.
4.2.3.9. 4-Hydroxy-2-(4-methoxyphenyl)-10H-pyridazino [6,1-b] quinazolin-
10-one (4i). Yield 53%, m.p. 263–265 °C; 1H NMR (500 MHz, DMSO‑d6)
δ: 8.33 (d, J = 8.1 Hz, 1H), 8.06–8.04 (m, 2H), 7.93–7.95 (m, 2H),
7.60–7.57 (m, 1H), 7.24 (s, 1H), 7.10 (d, J = 8.8 Hz, 2H), 3.84 (s, 3H). 13
C
NMR (125 MHz, DMSO‑d6) δ: 161.54, 158.34, 158.04, 152.33, 144.45,
143.70, 135.42, 128.89, 128.03, 127.71, 126.56, 125.77, 118.89, 114.76,
102.69, 55.83, 55.37. HRMS (ESI+) m/z calcd for C18H14O3N3 [M + H]+
320.1037, found 320.1028.
4.2.5.1. 2-Phenyl-4-((3-(dimethylamino)propyl)amino)-10H-pyridazino
[6,1-b] quinazolin-10-one (6o). Yield 78%, m.p. 152–154 °C; 1H NMR
(500 MHz, CDCl3) δ: 8.53–8.51 (m, 1H), 8.04–8.02 (m, 2H), 7.82–7.76
(m, 1H), 7.72 (d, J = 7.7 Hz, 2H), 7.53–7.41 (m, 4H), 6.50 (s, 1H), 3.47
(d, J = 6.4 Hz, 2H), 2.53 (t, J = 6.4 Hz, 2H), 2.34 (s, 6H), 2.01–1.93
(m, 2H). 13C NMR (125 MHz, CDCl3) δ: 159.16, 152.58, 146.25,
145.57, 140.50, 136.72, 134.22, 129.88, 128.69, 127.87, 127.35,
126.86, 125.71, 119.68, 92.16, 57.67, 45.49, 42.10, 25.93. HRMS
(ESI+) m/z calcd for C22H24ON5 [M + H]+ 374.1983, found 374.1985.
4.2.3.10. 2-(Furan-2-yl)-4-hydroxy-10H-pyridazino [6,1-b] quinazolin-
10-one (4j). Yield 59%, m.p. 285–287 °C; 1H NMR (500 MHz,
DMSO‑d6) δ: 8.32 (d, J = 8.0 Hz, 1H), 7.99–7.97 (m, 3H), 7.64–7.60
(m, 1H), 7.31–7.30 (m, 1H), 7.05 (s, 1H), 6.77–6.75 (m, 1H). 13C NMR
(125 MHz, DMSO‑d6) δ: 158.18, 149.66, 145.99, 145.46, 144.03,
135.50, 127.71, 126.70, 125.55, 118.81, 112.95, 112.36, 101.35,
55.35. HRMS (ESI+) m/z calcd for C15H10O3N3 [M + H]+ 280.0724,
found 280.0717.
4.2.3.11. 4-Hydroxy-2-(thiophen-2-yl)-10H-pyridazino [6,1-b] quinazolin-
10-one (4k). Yield 52%, m.p. 297–300 °C; 1H NMR (500 MHz,
DMSO‑d6) δ: 8.32 (d, J = 8.1 Hz, 1H), 7.95–7.93 (m, 2H), 7.91–7.90
(m, 1H), 7.79–7.77 (m, 1H), 7.63–7.57 (m, 1H), 7.23–7.21 (m, 2H). 13C
NMR (125 MHz, DMSO‑d6) δ: 158.26, 158.03, 149.14, 144.07, 143.99,
140.11, 135.46, 130.60, 128.97, 128.62, 127.68, 126.69, 125.54, 118.83,
101.95. HRMS (ESI+) m/z calcd for C15H10O2N3S [M + H]+ 296.0496,
found 296.0481.
4.2.5.2. 2-Phenyl-4-((3-(diethylamino)propyl)amino)-10H-pyridazino
[6,1-b] quinazolin-10-one (6p). Yield 80%, m.p. 105–108 °C; 1H NMR
(600 MHz, CDCl3) δ: 8.53–8.52 (m, 1H), 8.20 (s, 1H), 8.05–8.00 (m,
2H), 8.00–7.77 (m, 1H), 7.72 (d, J = 7.8 Hz, 1H), 7.51–7.43 (m, 4H),
6.42 (s, 1H), 3.45–3.43 (m, 2H), 2.64–2.63 (m, 6H), 1.97–1.90 (m, 2H),
1.12 (t, J = 7.1 Hz, 6H). 13C NMR (150 MHz, CDCl3) δ: 159.25, 152.63,
146.34, 145.74, 140.55, 136.83, 134.19, 129.84, 128.69, 127.86,
127.37, 126.83, 125.65, 119.70, 91.99, 51.97, 47.00, 43.06, 25.26,
11.77. HRMS (ESI+) m/z calcd for C24H28ON5 [M + H]+ 402.2296,
found 402.2278.
4.2.3.12. 3-Methyl-2-ethyl-4-hydroxy-10H-pyridazino [6,1-b] quinazolin-
10-one (4l). Yield 69%, m.p. 234–236 °C; 1H NMR (500 MHz,
DMSO‑d6) δ: 8.29 (d, J = 7.9 Hz, 1H), 7.91–7.90 (m, 2H), 7.58–7.49
(m, 1H), 2.83 (q, J = 7.4 Hz, 2H), 2.20 (s, 3H), 1.26 (t, J = 7.4 Hz, 3H).
13C NMR (125 MHz, DMSO‑d6) δ: 158.41, 158.21, 153.06, 145.34,
142.48, 135.30, 127.62, 125.97, 125.88, 118.50, 114.61, 27.24, 12.12,
10.97. HRMS (ESI+) m/z calcd for C14H14O2N3 [M + H]+ 256.1088,
found 256.1082.
4.2.5.3. 2-Phenyl-4-((3-morpholinopropyl)amino)-10H-pyridazino [6,1-b]
quinazolin- 10-one (6q). Yield 83%, m.p. 230–232 °C; 1H NMR
(600 MHz, CDCl3) δ: 8.53–8.52 (m, 1H), 8.04–7.99 (m, 2H), 7.84 (s,
1H), 7.81–7.78 (m, 1H), 7.74 (d, J = 7.8 Hz, 1H), 7.50–7.44 (m, 4H),
6.42 (s, 1H), 3.90–3.88 (m, 4H), 3.44 (d, J = 5.7 Hz, 2H), 2.59–2.57
(m, 6H), 2.01–1.94 (m, 2H). 13C NMR (150 MHz, CDCl3) δ: 159.15,
152.45, 146.27, 145.67, 140.52, 136.67, 134.43, 129.94, 128.73,
127.92, 127.33, 126.64, 125.77, 119.71, 92.29, 66.83, 57.73, 53.95,
43.05, 24.37. HRMS (ESI+) m/z calcd for C24H26O2N5 [M + H]+
416.2088, found 416.2070.
4.2.3.13. 12-Hydroxy-2,3-dihydrocyclopenta [3,4] pyridazino [6,1-b]
quinazolin- 6(1H)-one (4m). Yield 62%, m.p.
>
300 °C; 1H NMR
(400 MHz, DMSO‑d6) δ: 8.31 (d, J = 7.92 Hz, 1H), 7.94–7.92(m, 2H),
7.59–7.54 (m, 1H), 2.98 (t, J = 7.6 Hz, 2H), 2.89 (t, J = 7.6 Hz, 2H),
2.10–2.06 (m, 2H). 13C NMR (100 MHz, DMSO‑d6) δ: 164.02, 158.45,
151.80, 144.16, 143.69, 135.46, 127.71, 126.20, 125.07, 119.68,
118.05, 32.66, 27.66, 23.04. HRMS (ESI+) m/z calcd for C14H12O2N3
[M + H]+ 254.0931, found 254.0925.
4.2.5.4. 2-Phenyl-4-((2-(dimethylamino)ethyl)amino)-10H-pyridazino
[6,1-b] quinazolin-10-one (6r). Yield 76%, m.p. 190–193 °C; 1H NMR
(400 MHz, CDCl3) δ: 8.50 (d, J = 8.0 Hz, 1H), 8.05–7.98 (m, 2H),
7.80–7.74 (m, 2H), 7.50–7.45 (m, 4H), 7.15–7.13 (m, 1H), 6.45 (s, 1H),
3.42 (t, J = 6.2 Hz, 2H), 2.73 (t, J = 6.2 Hz, 2H), 2.36 (s, 6H). 13C NMR
(100 MHz, CDCl3) δ: 159.14, 152.49, 146.16, 145.23, 140.30, 136.59,
134.25, 129.95, 128.73, 127.79, 127.33, 127.07, 125.82, 119.68,
4.2.3.14. 13-Hydroxy-1,2,3,4-tetrahydro-7H-quinazolino [3,2-b] cinnolin-7-
one (4n). Yield 67%, m.p. 286–288 °C; 1H NMR (500 MHz,
CDCl3 + CD3OD) δ: 8.50 (d, J = 8.2 Hz, 1H), 7.89 (d, J = 8.2 Hz,
1H), 7.85 (d, J = 8.1 Hz, 1H), 7.55–7.54 (m, 1H), 3.01 (t, J = 6.0 Hz, 2H),
2.86 (t, J = 6.0 Hz, 2H), 1.93–1.88 (m, 4H). 13C NMR (125 MHz,
CDCl3 + CD3OD) δ: 158.15, 156.40, 141.97, 135.57, 128.27, 126.06,
123.41,117.7, 117.22, 30.49, 22.43, 22.01, 21.38. HRMS (ESI+) m/z calcd
for C15H14O2N3 [M + H]+ 268.1088, found 268.1085.
92.52, 57.04, 45.33, 40.44, 29.70. HRMS (ESI+
) m/z calcd for
C
21H22ON5 [M + H]+ 360.1826, found 360.1807.
4.2.5.5. 2-Phenyl-4((diethylamino)ethyl)amino)-10H-pyridazino [6,1-b]
quinazolin −10-one (6s). Yield 87%, m.p. 148–150 °C; 1H NMR
(600 MHz, CDCl3) δ: 8.51–8.50 (m, 1H), 8.04–8.00 (m, 2H),
7.79–7.71 (m, 2H), 7.50–7.42 (m, 4H), 7.31–7.29 (m, 1H), 6.46 (s,
1H), 3.38 (t, J = 6.2 Hz, 2H), 2.86 (t, J = 6.2 Hz, 2H), 2.66–2.64 (m,
4H), 1.13–1.11 (m, 6H). 13C NMR (150 MHz, CDCl3) δ: 159.19, 152.55,
146.24, 145.30, 140.40, 136.68, 134.22, 129.91, 128.72, 127.81,
127.34, 127.06, 125.76, 119.68, 92.43, 50.78, 47.05, 40.68, 12.10.
4.2.4. General procedure for synthesis of compounds 5
Redistilled POCl3 (5 mL, 50 mmol) was added to 2.0 mmol of
compound 4a (or 4b or 4g) and the mixture was heated in 90 °C for
about 2 h under nitrogen atmosphere. After completion (monitored by
8

W.-Y. Huang, et al.
BioorganicChemistry99(2020)103814
HRMS (ESI+) m/z calcd for C23H26ON5 [M + H]+ 388.2139, found
196–198 °C; 1H NMR (400 MHz, CDCl3) δ: 8.51 (d, J = 7.9 Hz, 1H),
8.02–7.98 (m, 1H), 7.84–7.76 (m, 2H), 7.53–7.39 (m, 2H), 7.28–7.24
(m, 1H), 7.18–7.13 (m, 2H), 6.50 (d, J = 2.3 Hz, 1H), 3.42 (t,
J = 6.1 Hz, 2H), 2.73 (t, J = 6.2 Hz, 2H), 2.36 (s, 6H). 13C NMR
(100 MHz, CDCl3) δ: 161.95, 159.46, 159.06, 149.86, 146.22, 144.79,
140.29, 134.33, 131.48, 131.39, 131.36, 131.34, 127.85, 127.09,
125.86, 125.09, 124.98, 124.73, 124.70, 119.63, 116.22, 116.00,
95.61, 95.54, 77.37, 77.05, 76.74, 57.04, 45.36, 40.43, 19F NMR
(376 MHz, CDCl3) δ:-108.23. HRMS (ESI+) m/z calcd for C21H21ON5F
[M + H]+ 378.1732, found 378.1717.
388.2121.
4.2.5.6. 2-Phenyl-4-((2-(pyrrolidin-1-yl)ethyl)amino)-10H-pyridazino
[6,1-b] quinazolin-10-one (6t). Yield 85%, m.p. 157–160 °C; 1H NMR
(400 MHz, CDCl3) δ: 8.52 (d, J = 7.9 Hz, 1H), 8.08–7.99 (m, 2H),
7.85–7.75 (m, 2H), 7.54–7.44 (m, 4H), 7.18 (s, 1H), 6.52 (s, 1H),
3.58–3.46 (m, 2H), 2.94 (t, J = 6.4 Hz, 2H), 2.67 (br, 4H), 1.86 (br,
4H). 13C NMR (100 MHz, CDCl3) δ: 159.13, 152.54, 146.18, 145.30,
140.39, 136.63, 134.28, 129.96, 128.74, 127.87, 127.36, 126.99,
125.83, 119.71, 92.56, 77.36, 77.04, 76.72, 54.15, 53.89, 41.77,
23.64. HRMS (ESI+) m/z calcd for C23H24ON5 [M + H]+ 386.1983,
found 386.1984.
4.2.5.12. 2-(2-Fluorophenyl)-4-((2-(diethylamino)ethyl)amino)-
10H-
pyridazino [6,1-b] quinazolin-10-one (7s). Yield 89%, m.p.
162–164 °C; 1H NMR (400 MHz, CDCl3) δ: 8.53–8.50 (m, 1H),
8.03–7.99 (m, 1H), 7.83–7.73 (m, 2H), 7.51–7.39 (m, 2H), 7.34 (t,
J = 4.7 Hz, 1H), 7.29–7.25 (m, 1H), 7.19–7.14 (m, 1H), 6.49 (d,
J = 2.4 Hz, 1H), 3.37 (d, J = 6.0 Hz, 2H), 2.85 (t, J = 6.0 Hz, 2H),
2.65 (t, J = 7.1 Hz, 4H), 1.12 (t, J = 7.1 Hz, 6H). 13C NMR (100 MHz,
CDCl3) δ: 161.96, 159.47, 159.11, 149.92, 146.29, 144.84, 140.39,
134.30, 131.42, 131.38, 131.35, 127.86, 127.07, 125.78, 125.18,
125.06, 124.71, 124.67, 119.63, 116.21, 115.98, 95.52, 95.44, 50.75,
47.07, 40.62, 12.12, 19F NMR (376 MHz, CDCl3) δ:-108.41. HRMS
4.2.5.7. 2-Phenyl-4-((2-(piperidin-1-yl)ethyl)amino)-10H-pyridazino
[6,1-b] quinazolin-10-one (6u). Yield 89%, m.p. 189–192 °C; 1H NMR
(600 MHz, CDCl3) δ: 8.53–8.51 (m, 1H), 8.02 (dd, J = 7.8, 1.5 Hz, 2H),
7.81–7.75 (m, 2H), 7.51–7.43 (m, 4H), 7.33 (t, J = 4.6 Hz, 1H), 6.47 (s,
1H), 3.43 (t, J = 6.3 Hz, 2H), 2.76 (t, J = 6.3 Hz, 2H), 2.51 (s, 4H),
1.71–1.62 (m, 4H), 1.50 (s, 2H). 13C NMR (150 MHz, CDCl3) δ: 159.17,
152.56, 146.22, 145.30, 140.42, 136.66, 134.26, 129.93, 128.72,
127.83, 127.35, 127.05, 125.79, 119.68, 92.55, 56.24, 54.41, 39.72,
26.13, 24.39. HRMS (ESI+) m/z calcd for C24H26ON5 [M + H]+
400.2139, found 400.2125.
(ESI+
)
m/z calcd for C23H25ON5F [M
+
H]+ 406.2045, found
406.2029.
4.2.5.8. 2-(2-Fluorophenyl)-4-((3-(dimethylamino)propyl)amino)- 10H-
pyridazino [6,1-b] quinazolin-10-one (7o). Yield 70%, m.p. 91–93 °C;
1H NMR (400 MHz, CDCl3) δ: 8.43–8.41 (m, 1H), 7.96–7.84 (m, 1H),
7.74–7.66 (m, 2H), 7.64 (d, J = 7.6 Hz, 1H), 7.41–7.32 (m, 2H),
7.22–7.14 (m, 1H), 7.10–7.05 (m, 1H), 6.39–6.37 (d, J = 2.3 Hz, 1H),
3.35 (t, J = 6.5 Hz, 2H), 2.43 (t, J = 6.5 Hz, 2H), 2.25 (s, 6H), 1.87 (t,
J = 6.5 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ: 160.88, 158.40, 158.07,
148.95, 148.94, 145.24, 144.03, 139.39, 133.28, 130.37, 130.31,
130.29, 126.82, 125.86, 124.72, 124.19, 124.08, 123.65, 123.61,
118.55, 115.17, 114.95, 94.20, 94.13, 56.66, 44.41, 41.07, 24.72, 19F
4.2.5.13. 2-(2-Fluorophenyl)-4-((2-(pyrrolidin-1-yl)ethyl)amino)-10H-
pyridazino [6,1-b] quinazolin-10-one (7t). Yield 84%, m.p. 195–197 °C;
1H NMR (400 MHz, CDCl3) δ: 8.51 (d, J = 8.0 Hz, 1H), 8.02–7.98 (m,
1H), 7.83–7.72 (m, 2H), 7.53–7.38 (m, 2H), 7.31–7.24 (m, 1H),
7.23–7.09 (m, 2H), 6.51 (d, J = 2.3 Hz, 1H), 3.47–3.45 (m, 2H),
2.90 (t, J = 6.5 Hz, 2H), 2.64 (br, 4H), 1.91–1.78 (m, 4H). 13C NMR
(100 MHz, CDCl3) δ: 161.94, 159.45, 159.04, 149.89, 149.88, 146.18,
144.79, 140.31, 134.35, 131.47, 131.39, 131.35, 131.32, 127.84,
127.00, 125.84, 125.09, 124.97, 124.72, 124.68, 119.59, 116.23,
116.00, 95.61, 95.53, 77.40, 77.08, 76.76, 54.11, 53.81, 41.73,
23.62, 19F NMR (376 MHz, CDCl3) δ:-108.99. HRMS (ESI+) m/z calcd
for C23H23ON5F [M + H]+ 404.1888, found 404.1869.
NMR (376 MHz, CDCl3) δ:-108.20. HRMS (ESI+
) m/z calcd for
C
22H23ON5F [M + H]+ 392.1888, found 392.1873.
4.2.5.9. 2-(2-Fluorophenyl)-4-((3-(diethylamino)propyl)amino)-
10H-
4.2.5.14. 2-(2-Fluorophenyl)-4-((2-(piperidin-1-yl)ethyl)amino)-10H-
pyridazino [6,1-b] quinazolin-10-one (7u). Yield 88%, m.p. 171–173 °C;
1H NMR (400 MHz, CDCl3) δ: 8.54–8.47 (m, 1H), 8.02–7.98 (m, 1H),
7.83–7.75 (m, 2H), 7.52–7.32 (m, 3H), 7.29–7.25 (m, 1H), 7.19–7.13
(m, 1H), 6.49 (d, J = 2.3 Hz, 1H), 3.41 (t, J = 6.4 Hz, 2H), 2.75 (t,
J = 6.4 Hz, 2H), 2.51 (br, 4H), 1.73–1.60 (m, 4H), 1.52–149 (m, 2H).
13C NMR (100 MHz, CDCl3) δ: 161.91, 159.43, 159.06, 149.90, 146.23,
144.79, 140.34, 134.31, 131.42, 131.33, 131.30, 127.80, 127.03,
125.79, 125.12, 125.00, 124.68, 124.65, 119.58, 116.20, 115.98,
95.61, 95.54, 77.40, 77.08, 76.77, 56.16, 54.36, 39.62, 26.11, 24.37,
19F NMR (376 MHz, CDCl3) δ;-109.15. HRMS (ESI+) m/z calcd for
pyridazino [6,1-b] quinazolin-10-one (7p). Yield 83%, m.p.
101–103 °C; 1H NMR (400 MHz, CDCl3) δ: 8.53–8.51 (m, 1H), 8.22
(s, 1H), 8.02–7.97 (m, 1H), 7.82–7.71 (m, 2H), 7.52–7.38 (m, 2H),
7.30–7.24 (m, 1H), 7.20–7.11 (m, 1H), 6.45 (d, J = 2.3 Hz, 1H), 3.43
(t, J = 6.0 Hz, 2H), 2.69–2.57 (m, 6H), 1.98–1.88 (m, 6H), 1.13 (t,
J = 6.1 Hz, 6H). 13C NMR (100 MHz, CDCl3) δ: 161.93, 159.45, 159.17,
150.00, 146.38, 145.26, 140.52, 134.27, 131.38, 131.35, 131.34,
131.25, 127.90, 126.83, 125.68, 125.33, 125.21, 124.67, 124.64,
119.64, 116.18, 115.96, 95.09, 95.02, 51.96, 47.01, 43.01, 25.19,
11.76, 19F NMR (376 MHz, CDCl3) δ:-108.45. HRMS (ESI+) m/z calcd
for C24H27ON5F [M + H]+ 420.2201, found 420.2182.
C
24H25ON5F [M + H]+ 418.2045, found 418.2025.
4.2.5.10. 2-(2-Fluorophenyl)- 4-((3-morpholinopropyl)amino)- 10H-
pyridazino [6,1-b] quinazolin-10-one (7q). Yield 89%, m.p.
218–220 °C; 1H NMR (400 MHz, CDCl3) δ: 8.55–8.51 (m, 1H),
8.02–7.98 (m, 1H), 7.87–7.73 (m, 3H), 7.53–7.40 (m, 2H), 7.31–7.26
(m, 1H), 7.19–7.14 (m, 1H), 6.51–6.49 (m, 1H), 3.90 (t, J = 6.0 Hz,
4H), 3.47–3.45 (m, 2H), 2.67–2.47 (m, 6H), 2.02–1.90 (m, 2H). 13C
NMR (100 MHz, CDCl3) δ: 161.93, 159.45, 159.08, 149.90, 146.31,
145.22, 140.51, 134.54, 131.46, 131.37, 131.34, 128.00, 126.64,
125.83, 125.18, 125.07, 124.74, 124.71, 119.67, 116.21, 115.99,
95.50, 95.42, 77.36, 77.04, 76.72, 66.80, 57.60, 53.93, 42.88, 24.42,
19F NMR (376 MHz, CDCl3) δ:-109.13. HRMS (ESI+) m/z calcd for
4.2.5.15. 2-(4-Bromophenyl)-4-((3-(dimethylamino)propyl)amino)-10H-
pyridazino [6,1-b] quinazolin-10-one (8o). Yield 85%, m.p. 148–151 °C;
1H NMR (500 MHz, CDCl3) δ: 8.54–8.49 (m, 1H), 7.93 (d, J = 8.6 Hz,
2H), 7.87 (s, 1H), 7.83–7.77 (m, 1H), 7.73 (d, J = 8.2 Hz, 1H), 7.61 (d,
J = 8.6 Hz, 2H), 7.51–7.48 (m, 1H), 6.45 (s, 1H), 3.49 (t, J = 6.3 Hz,
2H), 2.55 (t, J = 6.2 Hz, 2H), 2.36 (s, 6H), 2.01–1.95 (m, 2H). 13C NMR
(125 MHz, CDCl3) δ: 159.10, 151.44, 146.18, 145.75, 140.33, 135.56,
134.35, 131.86, 128.86, 127.89, 126.91, 125.88, 124.44, 119.67,
91.49, 57.69, 45.51, 42.19, 25.82. HRMS (ESI+
) m/z calcd for
C22H23BrN5O [M + H]+ 452.1086, found 452.1078.
C
24H25O2N5F [M + H]+ 434.1994, found 434.1979.
4.2.5.16. 2-(4-Bromophenyl)-4-((3-(diethylamino)propyl)amino)-10H-
pyridazino [6,1-b] quinazolin-10-one (8p). Yield 91%, m.p. 113–115 °C;
1H NMR (500 MHz, CDCl3) δ: 8.51 (d, J = 8.0 Hz, 1H), 8.33 (s, 1H),
7.92 (d, J = 8.5 Hz, 2H), 7.80–7.77 (m, 1H), 7.74 (d, J = 8.1 Hz, 1H),
4.2.5.11. 2-(2-Fluorophenyl)-4-((2-(dimethylamino)ethyl)amino)- 10H-
pyridazino [6,1-b] quinazolin-10-one (7r). Yield 85%, m.p.
9

W.-Y. Huang, et al.
BioorganicChemistry99(2020)103814
7.60 (d, J = 8.5 Hz, 2H), 7.49 (t, J = 7.5 Hz, 1H), 6.38 (s, 1H),
3.47–3.45 (m, 2H), 2.66 (t, J = 7.1 Hz, 6H), 1.99–1.92 (m, 2H), 1.14 (t,
J = 7.1 Hz, 6H). 13C NMR (125 MHz, CDCl3) δ: 159.18, 151.47, 146.28,
145.90, 140.38, 135.67, 134.30, 131.84, 128.88, 127.87, 126.88,
125.80, 124.38, 119.69, 91.30, 52.01, 46.99, 43.17, 25.17, 11.73.
HRMS (ESI+) m/z calcd for C24H27BrN5O [M + H]+ 480.1399, found
480.1396.
4.3.4. Cell cycle analysis assay
MGC-803 cells were treated with different concentrations of com-
pound 8p and incubated for 48 h. Then the cells were centrifugated at
1200 rpm for 6 min, washed twice with PBS and then fixed with ice-
cold 70% ethanol in PBS at –20 °C overnight. Then the cells were fur-
ther treated with 100 μg/mL of RNase A at 37 °C in a water bath for
30 min in the dark before washing with PBS, and staining with 1 mg/
mL propidium iodide (PI) at 37 °C for 5 min. Analysis was performed
using the ModFit LT software.
4.3. Biological studies
4.3.5. Apoptosis induction assay
4.3.1. MTT assay
MGC-803 cells were seeded at a concentration of about 2 × 106
cells/mL in DMEM medium with 10% FBS in 6-well plates with a final
volume of 2 mL. The plates were treatment with different concentra-
tions of compound 8p and incubated for 24 h. Then the cells were
collected, washed with cold PBS three times, resuspended in Binding
Buffer at a density of 1 × 106 cells/mL. After stained with FITC
Annexin V and PI for 20 min at 25 °C in the dark, the compound 8p
treatment cells were analyzed on a flow cytometer equipped with a
488 nm argon laser (Becton-Dickinson). Analysis was performed using
the system software (Cell Quest).
The anticancer activities of the pyridazino[1,6-b]quinazolinone
derivatives 4, and 6–8 were determined using MTT assay. Cancer cells
(SK-OV-3, CNE-2, MGC-803, NCI-H460) and normal cell LO2 were used
in this assay. 1 × 104 cells/well were seeded in 180 μL RPMI 1640
supplemented with 10% FBS in 96-well microculture plates and in-
cubated in 5% CO2 incubator at 37 °C for 24 h. The tested compounds
were prepared in DMSO and added to the cells with indicated con-
centrations in culture medium. After incubation for 48 h, MTT was
added and the plates were further incubated for 4 h. Then the super-
natant from each well was carefully removed and DMSO was added to
dissolve the purple formazan crystals. The plates were swirled gently
for 10 min to dissolve the precipitate and quantified by measuring the
optical density (OD) of the plates at a wavelength of 570 nm on plate
reader (TECAN, infinite, M1000). Each concentration was repeated in
five wells and the same experimental conditions were provided for all
compounds and MTT analysis was repeated three times for each cell
line.
4.3.6. In vivo antitumor evaluation assay
The in vivo antitumor evaluation were performed as the method
described in literature [6] and all experiments were carried out ac-
cording to the NIH guidelines for the care and use of laboratory ani-
mals. The MGC-803 xenograft model was established by pathogen-free
male BALB/C nude mice aged six weeks. Tumors were introduced by
injection of 5 × 106 MGC-803 cells. When the tumor was grown to
about 80 mm3, the tumor-bearing mice were randomly divided into
four groups (n = 6/group). The vehicle group was intraperitoneally
injected with vehicle (5% DMSO in saline, v/v). The two treatment
groups were treated with compound 8p (dissolved in 0.5 mL DMSO,
0.5 mL PEG and diluted with 9.0 saline to form a solution of 1.0 mg/mL
or 2.0 mg/mL, respectively) in a dose of 10 or 20 mg·kg−1, respectively,
per 2 days while the positive control group were treated with Doxor-
ubicin (dissolved in 0.5 mL DMSO and diluted with 9.5 mL saline to
form a solution of 1.0 mg/mL) in a dose of 10 mg·kg−1, per 2 days. The
tumor size and body weight of the mice were measured every two days.
The tumor size was determined by measuring the length (l) and width
(w) and calculating the volume (V = lw2/2). The tumor growth in-
4.3.2. DNA interaction assay
The ct-DNA was dissolved in Tris–HCl buffer (pH 7.4) for DNA
binding studies, and its concentration was determined from the ab-
sorption intensity at 260 nm with a molar extinction coefficient of
6600 M−1 cm−1. The test compounds 4e, 4g, 6o, 6p, 8o and 8p was
prepared as 20 mM DMSO stock solution and then stored at 4 °C no
more than 3 days before use for all the experiments.
UV–Vis spectroscopy. For UV–Vis absorption spectral analysis, the
solutions ct-DNA were mixed with the tested of compound to reach the
indicating concentration. The mixed solutions were shaken and in-
cubated for 5 min at equilibrium before recording absorption spectrum,
and then the absorption spectrum were recorded in the range of
200–400 nm.
hibition (TGI) were calculated as follows: TGI
= (1 − TWt/
TWc) × 100%, where TWc and TWt are the tumor weight in the absence
and presence of test compound.
Fluorescence spectroscopy. The competitive binding research was
performed with a mixed solution of the ethidium bromide (EB, 10 μM)
and ct-DNA (100 μM) in Tris–HCl buffer (pH 7.4), while gradually
adding the solution of tested compounds. For every addition, the test
sample was shaken and equilibrated for 5 min, and then the fluores-
cence emission spectra were recorded. Further, the quenching constant
K of the compound has been calculated from classic Sterne–Volmer
equation: I0/I = K × [Q] + 1, where I0 and I are the emission intensity
of the EB–DNA system in the absence and presence of the tested com-
pounds as the quencher, and [Q] is the quencher concentration. The Kq
value is obtained as a slope from the plot of I0/I versus [Q].
4.3.7. Molecular docking
The crystal structures of the ternary complex of Topo I, DNA, and
CPT (PDB code 1T8I) was downloaded from the Protein Data Bank
(http://www.rcsb.org). Discovery Studio (vision 2018, BIOVIA, USA)
was employed to carry on molecular docking study. The structures of
1T8I were prepared and CHARMm force field was employed and de-
fined as a receptor. The 3D structures of compound 8p were generated
and minimized and docking into the receptor used the CDOCKER pro-
tocol.
4.3.3. Topo I inhibition assay
4.3.8. Statistical analysis
Topoisomerase I inhibitory activity assay was performed according
to the method described in the literature [41]. In brief, a mixture of 1 U
of Topo I, the indicated concentrations of CPT or the tested compounds
were incubated at 37 °C in 5 μL reaction buffer for 30 min. Then 250 ng
of pBR322 DNA was added and the total volume of the mixture was
adjusted to 10 μL by adding ddH2O and further incubated at 37 °C for
30 min. The reaction was terminated by the addition of 2 mL loading
buffer, and the mixture was electrophoresed on a 1% agarose gel in
buffer for 90 min at 80 V and stained with 0.5 μg/mL of EB. DNA bands
were visualized by transillumination with UV light.
Data are shown as means
SD. Statistical analysis was performed
using SPSS 17.0 software (Chicago, IL, USA), P < 0.05 were con-
sidered to be statistically significant.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
10

W.-Y. Huang, et al.
BioorganicChemistry99(2020)103814
Acknowledgments
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Appendix A. Supplementary material
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