Novel tricyclic poly (ADP-ribose) polymerase-1/2 inhibitors with potent anticancer chemopotentiating activity: Design, synthesis and biological evaluation

Article abstract of DOI:10.1016/j.bmc.2016.08.016

8,9-Dihydro-2,4,7,9a-tetraazabenzo[cd]azulen-6(7H)-ones were designed and synthesized as a new class of PARP-1/2 inhibitors. The compounds displayed a variable pattern of PARP-1/2 enzymes inhibition profile that, in part, paralleled the antiproliferative

Full text of DOI:10.1016/j.bmc.2016.08.016

Accepted Manuscript
Novel Tricyclic Poly (ADP-ribose) Polymerase-1/2 inhibitors with Potent An-
ticancer Chemopotentiating Activity: Design, Synthesis and Biological evalu-
ation
Hui Li, Yan Hu, Xueyan Wang, Guangwei He, Yungen Xu, Qihua Zhu
PII:
S0968-0896(16)30623-X
http://dx.doi.org/10.1016/j.bmc.2016.08.016
BMC 13199
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
18 June 2016
10 August 2016
11 August 2016
Please cite this article as: Li, H., Hu, Y., Wang, X., He, G., Xu, Y., Zhu, Q., Novel Tricyclic Poly (ADP-ribose)
Polymerase-1/2 inhibitors with Potent Anticancer Chemopotentiating Activity: Design, Synthesis and Biological
evaluation, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.08.016
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Novel Tricyclic Poly (ADP-ribose) Polymerase-1/2 inhibitors with Potent
Anticancer Chemopotentiating Activity: Design, Synthesis and Biological
evaluation
a
b
a
c
a,b,*
a,b,*
Hui Li , Yan Hu , Xueyan Wang , Guangwei He , Yungen Xu
, Qihua Zhu
a
Jiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University,
Nanjing, 21009, China
b
c
Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing, 21009, China
Hefei YiGong Pharmaceutical Co. Ltd., Hefei, 230088, China
Keywords: PARP-1/2, inhibitors, Tricyclic, anticancer
Abstract 8,9-dihydro-2,4,7,9a-tetraazabenzo[cd]azulen-6(7H)-ones were designed and
synthesized as a new class of PARP-1/2 inhibitors. The compounds displayed a variable pattern
of PARP-1/2 enzymes inhibition profile that, in part, paralleled the antiproliferative activity in
cell lines. Among them, compound 9e exhibited not only the significant IC50 value of 28 nM in
the PARP-1 and 7.7 nM in PARP-2 enzyme assay, but also a profound synergic efficacy combined
with temozolomide with PF50 values of 2.6, 2.5, and 6.5 against MDA-MB-468, SW-620 and A549
and cell line, respectively.
1. Introduction
Poly (ADP-ribose) polymerases (PARPs) is a nuclear enzyme in nearly all eukaryotic cells which
is involved in the regulation of many cellular functions including chromatin remodeling,
transcription, DNA repair and protein degradation. To data, 18 members of the PARP family have
been identified and characterized with PARP-1 being the most abundant (>90%) and thoroughly
[
1-5]
studied and PARP-2 being its closest relative . It has been demonstrated that PARP-1 displays a
key role in the repair of single strand breaks (SSB) by the base excision repair (BER) pathway.
Given the biology of the enzyme, the inhibition of PARP-1 activity has the potential to augment
the cytotoxic drugs, involving DNA-methylating agent temozolomide, platinum-based drugs and
[
6-8]
topoisomerase inhibitors
. In addition, the PARP-1 inhibitor can induce synthetic lethality in
cells defective in homologous recombination (HR) repair, such as tumor cells with mutation or
[
9-11]
defective in BRCA1 or BRCA2
. The research found that while PARP-1 knockout mice are
viable, double PARP-1 and PARP-2 knockout mice are embryonically lethal. The results of these
research indicated that the importance of these two family members in genomic integrity and
cell survival. Similarly, PARP-1 and PARP-2 inhibition with small molecules has been demonstrated
to sensitize tumor cells to cytotoxic agents and ionizing radiation. Taken together, PARP-1/2 are
supposed to be a valuable pursued targets in the exploration of cancer therapy, as further
reinforced by the recognition of the clinical benefit arising from the multiply role in DNA repair
[
12-15]
.
A number of PARP-1 inhibitors have been disclosed over the past decade, either as
[
16-17]
monotherapy in specific or in combination with chemotherapy
. Several PARP-1 inhibitors

[
18]
[19]
[20]
are currently in different stages of clinical trials, including veliparib , niraparib , rucaparib
,
[
21]
[22]
and talazoparib
(Figure 1). Olaparib
(Figure 1), the first PARP-1 inhibitor for treatment in
patients with BRCA1/2 mutated platinum-sensitive relapsed ovarian cancer was approved by FDA
and EMA in 2014.
Figure 1. Structures of PARP inhibitors in mid to late stage clinical development
In an effort to search novel PARP-1 inhibitors and explore alternative chemical templates, our
group have disclosed a series of cyclic amine-substituted imidazo[4,5-c]pyridine carboxamide
derivatives as PARP-1 and PARP-2 inhibitors. Among them, compound 1 (Figure 2) was found to
be the most potent PARP-1/2 inhibitor with an IC50 value of 8.6 nM and 17.1 nM respectively, and
demonstrated good in vivo efficacy in lung cancer cell line A549 xenograft model of mouse in
[
23-24]
combination with cisplatin
. It has been confirmed that constraining the arylamide into
another ring would restrict the degrees of freedom for the amide moiety, and locking the
arylamide into lactam is beneficial for PARP-1 inhibitory potency. For example, rucaparib,
emerged from the series of [5,6,7]-tricyclic indole lactams, displayed good in vitro potency and in
vivo efficacy. More recently, rucaparib is the first PARP-1 inhibitor that be granted the
breakthrough drug from FDA for use against advanced ovarian cancer with BRCA mutations. In
order to obtain more potent PARP-1/2 inhibitor, based on our previous work, compound 1 was
chosen
as
lead
compound,
we
designed
a
series
of
8,9-dihydro-2,4,7,9a-tetraazabenzo[cd]azulen-6(7H)-ones 2 (Figure 2) in which the conformation
of the amide was locked in the s-trans via a lactam ring connection to the imidazole ring.
Modeling studies indicated that these tricyclic systems would allow maximum interaction (three
key hydrogen bonds) of the lactam carbonyl/N-H with Gly202 and Ser243 residues at the protein
active site. The tricyclic design allows for maximum incorporation of diversity by functionalization
at the C-2 position of the imidazole ring. In this paper, we described the synthesis, biological
evaluation and preliminary structure-activity relationship of variedly C-2 position substituted
8,9-dihydro-2,4,7,9a-tetraazabenzo[cd]azulen-6(7H)-ones derivatives.

Figure 2. Structures of compound 1 and scaffold 2
. Results and discussion
.1 Chemistry
-Piperidyl substituted compounds 9a-l
2
2
[
25]
4
were prepared according to the synthetic route
as outlined in Scheme 1. Starting from the commercial available 4-hydroxynicotinic acid 3,
nitration of 3 with mixed acid of fuming nitric acid and sulfuric acid to afford intermediate 4 by
[
26]
following a literature procedure . Treatment of 4 with thionyl chloride smoothly produced the
corresponding acid chloride, which was reacted with methanol to give methyl ester 5. The
formation of the seven-membered lactam ring was accomplished by refluxing 5 with ethylene
diamine in acetonitrile to afford 6. Diamine 7, obtained by catalytic hydrogenation of 6, was
condensed with N-Boc-4-piperidinecarboxaldehyde in the presence of 10% Pd/C to form the
tricyclic intermediate 8. Then, deprotection of Boc group using hydrochloric acid (4M in
tetrahydrofuran) provided the secondary amine hydrochloride 9a. N-alkylation of 9a with
aldehydes or ketones under sodium cyanborohydride reductive amination condition afforded
tertiary amines 9b-f. N-acylation of 9a with acyl chlorides under base condition provided target
compounds 9g-l.
Scheme 1. Synthetic route for compounds 9a-l. Reagents and conditions: (a) conc. H
HNO , 100 °C, 20h; (b) SOCl , DMF, CHCl , reflux, 24 h; (c) MeOH, TEA, -10 °C, 2h；(d) ethylene
diamine, CH CN, reflux, 24 h; (e) 10% Pd/C, MeOH, r.t., 16 h; (f)
2 4
SO , fuming
3
2
3
3
2
H ,

N-Boc-4-piperidinecarboxaldehyde, MeOH, 10% Pd/C, in a sealed tube, 16 h; (g) 4M HCl in THF,
r.t., 4 h; (h) NaBH CN, MeOH, r.t., overnight; (i) TEA, CH Cl , r.t., 2 h.
3
2
2
As outlined in Scheme 2, phenyl substituted compounds 11a-h were prepared by the direct
condensation of diamine 7 with benzaldehyde 10a or para-substituted benzaldehyde 10b-h.
Alternatively, phenyl substituted compounds 11i-j were synthesized as outlined in Scheme 3. The
diamine 7 and 4-(diethoxymethyl) benzaldehyde were coupled to give the tricyclic intermediate
12, and the acetal was then hydrolyzed to the corresponding aldehyde 13. Reductive amination
of 13 with pyrrolidine and piperidine gave the desired compounds 11i-j.
Scheme 2. Synthetic route for compounds 11a-h. Reagents and conditions: (a) MeOH, 10% Pd/C,
in a sealed tube, 24 h.
Scheme 3. Synthetic route for compounds 11i-j. Reagents and conditions: (a) 4-(diethoxymethyl)
benzaldehyde, MeOH, 10% Pd/C, in a sealed tube, 24 h; (b) 1M HCl, THF, 80 °C, 2 h; (c) NaBH
MeOH, r.t., overnight.
3
CN,
2.2 Enzymatic activity against PARP-1 and PARP-2
All the target compounds were evaluated for their PARP-1 enzyme inhibitory activity and the
results were summarized in Table 1. The result showed that most target compounds possessed
PARP-1 inhibitory activities. The 4-piperidyl substituted 9a exhibited potent inhibitory activity
against PARP-1 enzyme with an IC50 value of 54 nM, suggesting that the C-2 functional is
interacting favourably at the PARP-1 binding site. Encouraged by this result, further modification
of the nitrogen atom on the 4-piperidyl group with alkyls was conducted. 9b-f exhibited the same
order of magnitude in potency as 9a. Among them, i-butyl substituted 9e appeared to be slightly

less active than n-butyl 9d, and i-propyl 9c resulted in an approximately 4-fold loss of potency
compared to the n-propyl 9b, whilst more pronounced was the decrease in activity for the benzyl
9f, indicating that the propensity bulkier substitution was detrimental to inhibitory activity.
Prompted by the results of alkyl derivatives, we made further exploration to introduce acyls to
the 4-piperidyl. Disappointingly, the acetyl 9g and the cyclopropane carbonyl 9h failed to
demonstrate significant inhibitory effect up to the concentration of 100 nM, and little difference
was noted in benzoyl 9i. This also included both ortho-, meta- or para-chloro substituted benzoyl
9j-l.
In an effort to enhance potency and increase diversity of the structure, phenyl substituted
derivatives 11a-j were synthesized. The phenyl substituted 11a showed relatively weak PARP-1
inhibitory activity. While 11b, that piperazine substituted at the para position of the phenyl gave
good PARP-1 activity with an IC50 value of 20 nM, which was 5-fold more potent than the parent
11a (IC50 =110 nM). It was suggested that substitution at the para position of the phenyl ring
could be increase the potency. Therefore, the N-methyl substituted piperazine 11c was
synthesized, however, it displayed dramatic decrease in potency (IC50 = 139 nM). According to the
[
27]
previous literature reports , alkylamino substituted aryl tricyclic indole inhibitors not only
retained PARP-1 inhibitory activity, but also improved the physical properties. Successively, with
this idea further optimization was then undertaken to explore the effect of an alkylamino group
in the distal position via a methylene unit linked to the para position of the phenyl. Among them,
methylamino 11d, N,N-dimethylamino 11e and ethylamino 11f exhibited similar PARP-1
inhibitory activity in the low double-digit nanomolar range. Notable was the slight loss in activity
with i-propylamino 11g (IC50 = 58 nM) and t-butylamino 11h (IC50 = 75 nM) that indicated a steric
restriction at this end of the binding site. The cyclic amines 11i and 11j, derived from pyrrole and
piperidine respectively, were equipotent to 11d-f. It indicated that cyclic amine substituents at
the para position of phenyl were not tolerated. In general, the PARP-1 binding site might
accommodate the tricyclic scaffold and a variety of moieties in maintaining enzymatic inhibitory
activity.
In addition to PARP-1, PARP-2 is another enzyme that has been characterized to be involved
in DNA repair. In fact due to the high homology of the catalytic domain between PARP-1 and
PARP-2, PARP-1 inhibitors usually bind to PARP-2 as well. In this work, the potent PARP-1 inhibitor
9e, 11b, 11d and 11f were selected to test their inhibition effects on PARP-2 (Table 1). The result
showed all compounds exhibited similar PARP-2 inhibitory activity in the low nanomolar range.
a
Table 1. Chemical structures and PARP-1/2 inhibitory activity of compounds 9a-l and 11a-j.
IC50 (nM)
Compd
Substituents (R)
PARP-1
54
PARP-2
d
9
a
b
ND
9
19
ND

9
c
92
21
ND
ND
7.7
ND
ND
ND
ND
ND
ND
ND
ND
5.5
ND
7.2
ND
9.9
ND
ND
ND
9
d
e
9
28
9
f
103
b
9g
NA
9h
NA
NA
NA
NA
NA
110
20
9
i
9
j
9k
9
l
11a
11b
11c
139
13
11d
11e
28
11f
11
11g
58
11h
75
11i
20
1
1j
29
ND
1.3
e
veliparib
1.1(5.0 )
a
:
Values are means of two independent experiments.
b
:
:
The IC50 value is the concentration for 50% inhibition in PARP-1 enzyme assay.
NA: Not active (below 10% inhibition at the concentration of 100 nM).
C

d
e
:
ND: no determined.
Date from Ref 19
:
2.3 Cellular potency
Given the profile of PARP-1 inhibition, several compounds were evaluated in vitro for their
growth inhibitory activity as single agent at the concentration of 100 μM and compounds with
inhibitory ration >50% were selected to determine EC50 values, against human colorectal cancer
cell line SW-620 and breast cancer cell line MDA-MB-468. Veliparib was used as the positive
control and the results were summarized in Table 2. The results indicated that the 4-piperidyl
derivatives 9a, 9b, and 9d showed marginal activity in cells as 50% cell death was not observed
up to the concentration of 100 μM, whereas the most potent compound from the 4-piperidyls
appeared to be 9e, with EC50 values being below 100 μM in both the two cell lines (52.6 μM in
SW-620 and 18.9 μM in MDA-MB-468) and lower than that of veliparib in the MDA-MB-468 cell
line. Yet, in both cell lines, EC50 values being below 100 μM was noted in the selected phenyl
derivatives with the exception of 11b, which was less active in the MDA-MB-468 cell line.
Especially, the anti-proliferation effect of 11f was higher than veliparib in terms of EC50 values,
being 23.6 μM in SW-620 and 4.6 μM in MDA-MB-468 compared to veliparib which was 27.1 μM
and 43 μM respectively. In generally, the result showed that most compounds had the similar
cytotoxicity with veliparib on these tumor cell line as single agent.
Table 2. MDA-MB-468 and SW-620 cell inhibitory activities of some target compounds at 100 μM
a
compd
SW-620
MDA-MB-468
EC50 (μM)
b
c
inhibition(%)
EC50 (μM)
inhibition(%)
d
9
a
25
41
67
77
15
80
94
95
ND
32
30
47
53
57
71
82
69
ND
ND
9b
ND
95
9d
ND
18.9
94
9e
52.6
ND
1
1b
1d
1
89
81.4
4.6
11f
23.6
27.1
veliparib
43
a:
b
Values are means of two independent experiments.
:
Cell proliferative inhibition at the concentration of 100 μM.
c
:
The EC50 value is the concentration required to reduce cell proliferation by 50% in single agent cytotoxicity
assay.
d
:
ND: not determined.
In order to further evaluate these inhibitors as potentiating agents for chemotherapeutic

drugs, compounds 9e, 11b, 11d and 11f were selected to assessed the ability to enhance the
cytotoxic effect of chemotherapy agent temozolomide (TMZ) in vitro cellular growth inhibition
assays and the calculation of potentiation factors (PF50) in both SW-620, MDA-MB-468 and A549
cell lines. A standard concentration of 0.5 μM PARP-1/2 inhibitor, which is not intrinsically growth
inhibitory, was used. The results were summarized in Table 3. A value of PF50 greater than 1.0
indicates that the PARP-1 inhibitor is capable of enhancing the effects of cytotoxic agents. A PF50
value of 1.0 indicates no effect. Despite potent inhibition of the PARP-1 enzyme, the selected
compounds exhibited a broad array of potentiating efficacy. Compound 9e was capable of
enhancing the effect of TMZ in the SW-620, MDA-MB-468 and A549 cell lines by the PF50 values
of 2.5, 2.6 and 6.5 time respectively. Remarkably, compared to the veliparib, the potentiating
effect of 9e, was more potent in MDA-MB-468 (2-fold) and A549 (4-fold) cell lines, and has the
same potent in SW-620 cell line. Among of the phenyl derivatives 11b, 11d and 11f, only 11f was
capable of enhancing the effect of TMZ in the SW-620 cell line by PF50 values of 2.1. In contrast,
11f can enhance the effect of TMZ in both MDA-MB-468 and A549 cell lines by PF50 values of 2.0
and 3.2 respectively which was more potent than veliparib.
Table 3. The potentiation effect on temozolomide activity by selected PARP-1 inhibitors in
a
MDA-MB-468, SW-620 and A549 human tumor cell lines
MDA-MB-468
SW-620
EC50
A549
b
compound
EC50
EC50
(μM)
103.5
69.5
16.0
97.5
90.0
32.5
c
PF50
PF50
PF50
(
μM)
(μM)
37.0
12.0
14.8
33.6
25.3
18.0
Temozolomide
Temozolomide+veliparib
Temozolomide+9e
109.0
94
-
-
-
1.2
2.6
1.0
0.6
2.0
3.1
2.5
1.1
1.5
2.1
1.5
6.5
1.1
1.2
3.2
42.0
110.5
170.0
53.5
Temozolomide+11b
Temozolomide+11d
Temozolomide+11f
a:
b
Values are means of three independent experiments.
The EC50 value is the concentration required to reduce cell proliferation by 50% in cytotoxicity assay.
:
c
:
The PF50 value is the potentiation factor, calculated as the ratio of the EC50 for temozolomide divided by the EC50
of temozolomide + PARP-1 inhibitor, the test compounds were used at a fixed concentration of 0.5 μM.
2.4 Molecular modelling study
To gain an in-depth understanding of the binding mode of the new tricyclic PARP-1 inhibitors
with the PARP-1 protein, molecular modelling of the compounds from the 4-piperidyl (9b) and
the phenyl (11d) into the PARP-1 enzyme crystal structure (PDB code: 2RCW) was performed. The
binding mode was depicted in Figure 3. As we expected, in both the two compounds, the tricyclic
region was anchored to the nicotinamide binding site (NI site) while the long appendant side
chain extended to the adenine binding site (AD site) in a virtually identical way. The molecule of
9b sat inside the cavity with the planar bicyclic aromatic ring inserted between the Tyr246 and
Tyr235, thus forming the characteristic π-π stacking interactions (distance: 4.50Å for pyridine ring
and 4.26Å for imidazole ring with Tyr246, 6.10Å for pyridine ring and 4.83Å for imidazole ring
with Tyr235), and the amide group on the non-planar and anti-deposition of the
seven-membered ring gave rise to one hydrogen bond interaction with the Ser243 group
(distance: 1.99Å) and two with the Gly202 backbone (distance: 2.31Å and 2.39Å). The favorable
interaction distances established between the tricyclic ligand and the protein were observed in

the model of 11d bounding to the NI site as well. For the rather larger AD site, the strong π-π
stacking interaction (distance: 5.06Å) with Tyr228 seen in the phenyl 11d, appeared to be a fairly
weak π-σ interaction (distance: 2.54Å) in 9b, while another π-σ interaction (distance: 2.62Å) was
built up between one hydrogen atom of 4-piperidyl ring and Tyr235. As for 11d, the hydrogen
atom of secondary amine side chain has been an extra point of interaction with two hydrogen
bonds between the nearby Glu102 and Gly227 (distance: 3.78Å and 3.91Å), which may
somewhat account for improved binding affinity, resulting in a lower IC50 value, as anticipated.
This result led us to design its analogs
Figure 3. Molecular modelling of compound 9b (A) and 11d (B) in the binding site of PARP-1 (PDB code: 2RCW).
Amino acid residues were shown as stick model with the atoms colored as carbon-yellow, hydrogen-white,
nitrogen-blue and oxygen-red, and compound 9b and 11d were shown as ball and stick model with the same
color scheme as above except carbon atom colored green. The hydrogen bonds were displayed as dotted green
lines whereas the π-π interactions and π-σ interactions were shown as orange lines.
3. Conclusion
A potent new class of tricyclic lactam imidazole PARP-1/2 inhibitors have been designed and
synthesized. This new class of PARP-1/2 inhibitors, represented by compounds 9e and 11f
displays potent in vitro PARP-1/2 enzymatic inhibition and potent enhancement of cellular
growth inhibition. Compound 9e exhibited not only the significant IC50 value of 28 nM in the
PARP-1 and 7.7 nM in PARP-2 enzyme assay, but also a profound synergic efficacy combined with
temozolomide with PF50 values of 2.6, 2.5, and 6.5 against MDA-MB-468, SW-620 and A549 and
cell line respectively, considerable higher than the value obtained with veliparib in MDA-MB-468
and A549 cell lines, suggesting its potential to be further studied toward physicochemical and
pharmacokinetic properties, and ultimately, in vivo activity in clinical relevant models. The use of
this compound for potentiation of radiation and chemotherapy is being investigated in vivo
tumor models.
4. Experimental section
4.1 Materials and measurements
Unless otherwise stated, all solvents and materials were purchased from commercial sources
and used without further purification. Melting points were determined on RY-1 MP apparatus
1
13
and uncorrected. H-NMR spectra and C-NMR were recorded on a Bruker AV-300 spectrometer
using tetramethylsilane (TMS) as internal standard and chemical shifts were given in ppm (δ).The
mass spectra were recorded on an Agilent 1100 series LC/MSD Trap SL Mass spectrometer. The
HRMS spectra were acquired on a Waters Micros Q-TOF apparatus.

4.2 Synthesis
4.2.1 4-Hydroxy-5-nitronicotinic acid (4)
To an ice cooled solution of 4-hydroxynicotinic acid 3 (24.0 g, 172.5 mmol) in concentrated
sulfuric acid (210 mL) was added fuming nitric acid (48 mL) via dropwise over a period of 30min.
The reaction mixture was then allowed to come to room temperature and heated to 100 °C for
20 hours. After cooling to room temperature, the resulting solution was poured smoothly into a
beaker containing a liter volume of ice and the material was stirred vigorously for another 1 hour,
and the precipitate was collected by filtration, washed well with greater than 1 liter of water. The
solid was dried in vacuum and provided 4 as white solid (21.2 g), yield 67.7%. m.p. 246-248 °C.
1
H-NMR (300MHz, CDCl
3
), δ (ppm): 13.5 (br s, 1H, COOH), 9.13 (s, 1H, ArH), 8.75 (s, 1H, ArH). MS
+
(ESI+) m/z: 185 [M+H]
4.2.2 Methyl 4-chloro-5-nitronicotinate (5)
To a suspension of 4-hydroxy-5-nitronicotinic acid 4 (14.0 g, 76.0 mmol) in chloroform (200
mL) was added thionyl chloride (40 mL) and DMF (1 mL). The mixture was then heated at reflux
for 24 hours before the solvent was removed in vacuum. The residue dissolved in
dichloromethane (25 mL) was slowly added into a mixture of anhydrous methanol (200 mL) and
trimethylamine (2 mL) at the temperature below -10 °C. The mixture was stirred for another 2
hours before partitioned with dichloromethane (400 mL) and water (100 mL). The layers were
4
separated, and the organic layer was washed with water and brine, dried over anhydrous MgSO .
Filtration and evaporation gave 5 as pale yellow solid (12.8 g), yield 77.8%. m.p. 220-222 °C.
1
H-NMR (300MHz, CDCl
3
), δ (ppm): 9.13 (s, 1H, ArH), 9.06 (s, 1H, ArH), 4.03 (s, 3H, CH
3
). MS (ESI+)
+
m/z: 217 [M+H]
4
.2.3 9-Nitro-3,4-dihydro-1H-pyrido[4,3-e][1,4]diazepin-5(2H)-one (6)
Methyl 4-chloro-5-nitronicotinate 5 (8.0 g, 36.9 mmol) was dissolved in acetonitrile (140 mL).
Sodium carbonate (4.0 g, 37.7 mmol) was added to the solution followed by ethylene diamine
2.7 mL, 40.8 mmol). After heating at 90 °C for 24 hours yellow solid precipitated out of the
(
solution. The precipitate was collected by filtration, washed with acetonitrile for several times
1
and dried in vacuum to afford 6 as yellow solid (6.2 g), yield 80.7%. m.p. 180-182°C. H-NMR
(
300MHz, MeOD), δ (ppm): 8.83 (s, 1H, ArH), 8.54 (s, 1H, ArH), 3.77 (br s, 2H, CONHCH
2
CH
2
), 3.38
+
2 2
(s, 2H, CONHCH CH ). MS (ESI+) m/z: 209 [M+H]
4.2.4 9-Amino-3,4-dihydro-1H-pyrido[4,3-e][1,4]diazepin-5(2H)-one (7)
A suspension of 9-nitro-3,4-dihydro-1H-pyrido[4,3-e][1,4]diazepin-5(2H)-one 6 (3 g, 14.4
mmol) and 10% Pd/C (0.75 g) in methanol (400 mL) was hydrogenated under normal pressure at
ambient temperature for 16 hours. Filtration and evaporation afforded the crude product as
brown solid, which was purified by silica gel column chromatography using 10% methanol in
dichloromethane as eluent to afford 7 (2.3 g) as bright yellow solid, yield 89.6%. m.p. 194-196 °C.
1
H-NMR (300MHz, MeOD), δ (ppm): 8.33 (s, 1H, ArH), 8.24 (s, 1H, ArH), 3.77 (br s, 2H,
+
CONHCH
2 2 2 2
CH ), 3.38 (s, 2H, CONHCH CH ). MS (ESI+) m/z: 179 [M+H]
4.2.5
Tert-butyl-4-(6-oxo-6,7,8,9-tetrahydro-2,4,7,9a-tetraazabenzo[cd]azulen-1-yl)piperidine-1-carbo
xylate (8)
The 9-amino-3,4-dihydro-1H-pyrido[4,3-e][1,4]diazepin-5(2H)-one 7 (1.0 g, 5.62 mmol),
N-Boc-4-formylpiperidine (0.60 g, 5.62 mmol) and 10% Pd/C (130 mg) were dissolved in
methanol (60 mL) in a sealed tube and heated at 120 °C. After 16 hours, the reaction was

stopped and filtered off. The filtrate was concentrated under reduced pressure to give the crude
residue, which was purified by silica gel column chromatography using 3% methanol in
dichloromethane as eluent to afford 8 (0.90 g) as white crystal, yield 43.3%. m.p. 230-232 °C.
1
H-NMR (300MHz, DMSO-d
6
), δ (ppm): 8.99 (s, 1H, ArH), 8.79 (s, 1H, ArH), 8.48 (t, J = 4.4 Hz, 1H,
CH ), 4.10–3.99 (m, 2H, CONHCH CH ), 3.68–3.58 (m, 2H,
(2He)), 3.27–3.22 (m, 1H, CH), 3.02–2.85 (m, 2H, N-CH
.78–1.61 (m, 2H, CH (2Ha)), 1.42 (s, 9H, C(CH
CONH), 4.41 (br s, 2H, CONHCH
N-CH
2
2
2
2
2
2
(2Ha)), 2.02–1.90 (m, 2H, CH
2
(2He)),
+
1
2
3
)
3
). MS (ESI+) m/z: 372 [M+H] .
4.2.6 4-(6-Oxo-6,7,8,9-tetrahydro-2,4,7,9a-tetraazabenzo[cd]azulen-1-yl)piperidin-1-ium
chloride (9a)
-(6-Oxo-6,7,8,9-tetrahydro-2,4,7,9a-tetraazabenzo[cd]azulen-1-yl)piperidine-1-carboxylate
(0.2 g, 0.54 mmol) was suspended in tetrahydrofuran (5 mL). The white solid started to
4
8
precipitate followed by treating with hydrochloride acid (4M in tetrahydrofuran) via dropwise
addition at the temperature below 0 °C. After stirring for another 4 hours at room temperature,
the mixture was filtered out and the filter cake was dried in vacuum to give the hydrochloride
1
formation of 9a (0.13 g) as white solid, yield 78.2%. m.p. >250 °C (dec.). H-NMR (300MHz,
DMSO-d
.52 (br s, 2H, CONHCH
CH), 3.13–3.01 (m, 2H, N-CH
6
), δ (ppm): 9.47 (s, 1H, ArH), 9.16 (br s, 2H, NH, HCl), 8.97–8.93 (m, 2H, CONH, ArH),
CH ), 3.75–3.67 (m, 2H, CONHCH CH ), 3.58–3.38 (m, 3H, N-CH (2He),
(2Ha)), 2.18–1.92 (m, 4H, CH (2He), CH (2Ha)). C-NMR (75MHz,
4
2
2
2
2
2
1
3
2
2
2
D
2
O), δ (ppm): 164.52, 141.84, 139.48, 138.77, 136.52, 134.35, 115.49, 47.99, 43.05, 40.07, 31.55,
+
26.14. HRMS (ESI+): found 272.1501 (Calcd for C14
H
18
N
5
O [M+H] : 272.1506).
4.2.7 1-(1-Propylpiperidin-4-yl)-8,9-dihydro-2,4,7,9a-tetraazabenzo[cd]azulen-6(7H)-one (9b)
A solution of 9a (0.17 g, 0.55 mmol) in methanol (10 mL) was adjusted to pH 7 with
triethylamine. Then, the solution was stirred with n-propyl aldehyde (58 μl, 0.81 mmol) for 15
minutes before sodium cyanoborohydride (0.07 g, 1.08 mmol) was added. After stirring at room
temperature overnight, the solvent was removed under reduced pressure and the residue was
subject to silica gel column chromatography using 5% methanol in dichloromethane as eluent to
1
give 9b (90 mg) as white powder, yield 53.2%. m.p. >250 °C (dec.). H-NMR (300MHz, DMSO-d
6
),
δ (ppm): 8.99 (s, 1H, ArH), 8.78 (s, 1H, ArH), 8.51 (t, J = 5.3 Hz, 1H, CONH), 4.39 (br s, 2H,
CONHCH CH ), 3.65–3.56 (m, 2H, CONHCH CH ), 3.04–2.92 (m, 3H, N-CH (2He), CH), 2.32–2.27
m, 2H, N-CH (2Ha)), 2.10–2.02 (m, 2H, N-CH (2He)), 1.84 (br s, 2H,
(2Ha)), 1.51–1.41 (m, 2H, CH CH ), 0.87 (t, J = 7.3 Hz, 3H, CH
2
2
2
2
2
(
2
2
), 1.97–1.93 (m, 2H, CH
2
1
3
CH
2
2
3
2
CH
3
). C-NMR (75MHz,
DMSO-d
6
), δ (ppm): 166.49, 159.88, 143.63, 143.17, 139.52, 136.85, 112.55, 59.98, 52.80, 47.46,
+
4
3
4
0.23, 33.52, 29.94, 19.51, 11.81. HRMS (ESI+): found 314.1981 (Calcd for C17
H
24
N
5
O [M+H] :
14.1975).
.1.8 1-(1-Isopropylpiperidin-4-yl)-8,9-dihydro-2,4,7,9a-tetraazabenzo[cd]azulen-6(7H)-one (9c)
Compound 9c was prepared from 9a and acetone according to the procedure for
1
preparation of 9b. White powder, yield 59.2%. m.p. >250 °C (dec.). H-NMR (300MHz, DMSO-d
6
),
δ (ppm): 8.99 (s, 1H, ArH), 8.78 (s, 1H, ArH), 8.50 (t, J = 5.4 Hz, 1H, CONH), 4.38 (br s, 2H,
CONHCH CH ), 3.67–3.56 (m, 2H, CONHCH CH ), 3.01–2.89 (m, 3H, N-CH (2He), NCH(CH ),
.81–2.72 (m, 1H, CH), 2.34–2.27 (m, 2H, N-CH (2Ha)), 1.98–1.94 (m, 2H, CH (2He)), 1.80 (br s,
H, CH (2Ha)), 1.01 (d, J = 6.5 Hz, 6H, NCH(CH ), δ (ppm): 166.51,
2
2
2
2
2
3 2
)
2
2
1
2
2
1
3
2
3 2 6
) ). C-NMR (75MHz, DMSO-d
60.28, 143.62, 143.17, 139.33, 136.58, 112.54, 53.98, 47.81, 47.45, 40.23, 33.86, 30.26, 17.85.
+
HRMS (ESI+): found 314.1978 (Calcd for C17
H
24
N
5
O [M+H] : 314.1975).
4.1.9 1-(1-Butylpiperidin-4-yl)-8,9-dihydro-2,4,7,9a-tetraazabenzo[cd]azulen-6(7H)-one (9d)

Compound 9d was prepared from 9a and n-butyl aldehyde according to the procedure for
1
preparation of 9b. White powder, yield 49.1%. m.p. >250 °C (dec.). H-NMR (300MHz, DMSO-d
6
),
),
δ (ppm): 8.99 (s, 1H, ArH), 8.78 (s, 1H, ArH), 8.52(br s, 1H, CONH), 4.40 (br s, 2H, CONHCH
2
CH
2
3
.61 (br s, 2H, CONHCH
.08–1.83 (m, 6H, N-CH
CH CH ), 0.90 (t, J = 6.5 Hz, 3H, CH
2
CH
2
), 3.06–2.90 (m, 3H, N-CH
2
(2He), CH), 2.38–2.30 (m, 2H, N-CH
CH CH ), 1.34–1.24 (m, 2H,
). C-NMR (75MHz, DMSO-d ), δ (ppm): 166.48,
2
(2Ha)),
2
2
, CH
2
(2He), CH (2Ha)), 1.52–1.42 (m, 2H, CH
2
2
2
3
1
3
CH
2
2
3
2
CH
2
CH
3
6
1
2
4
59.90, 143.62, 143.16, 139.31, 136.66, 112.55, 57.80, 52.87, 47.45, 40.23, 33.55, 29.97, 28.57,
+
0.13, 13.92. HRMS (ESI+): found 328.2137 (Calcd for C18
H
26
N
5
O [M+H] : 328.2132).
.2.10 1-(1-Isobutylpiperidin-4-yl)-8,9-dihydro-2,4,7,9a-tetraazabenzo[cd]azulen-6(7H)-one (9e)
Compound 9e was prepared from 9a and i-butyl aldehyde according to the procedure for
1
preparation of 9b. White powder, yield 45.3%. m.p. >250 °C (dec.). H-NMR (300MHz, DMSO-d
6
),
δ (ppm): 8.98 (s, 1H, ArH), 8.78 (s, 1H, ArH), 8.52 (t, J = 5.3 Hz, 1H, CONH), 4.38 (br s, 2H,
CONHCH CH ), 3.65–3.57 (m, 2H, CONHCH CH ), 3.01–2.91 (m, 3H, N-CH (2He), CH), 2.08–2.01
m, 4H, N-CH (2Ha), N-CH ), 1.97–1.92 (m, 4H, CH (2He), CH (2Ha)), 1.86–1.75 (m, 1H, CH(CH )2),
.87 (d, J = 6.4 Hz, 6H, CH(CH ). C-NMR (75MHz, DMSO-d ), δ (ppm): 166.51, 160.07, 143.60,
2
2
2
2
2
(
2
2
2
2
3
1
3
0
1
3
)
2
6
43.11, 139.30, 136.58, 112.55, 66.49, 53.27, 47.43, 40.22, 33.58, 30.06, 24.98, 20.75. HRMS
+
(ESI+): found 328.2137 (Calcd for C18
H
26
N
5
O [M+H] : 328.2132).
4.2.11 1-(1-Benzylpiperidin-4-yl)-8,9-dihydro-2,4,7,9a-tetraazabenzo[cd]azulen-6(7H)-one (9f)
Compound 9f was prepared from 9a and benzaldehyde according to the procedure for
1
preparation of 9b. White powder, yield 76.9%. m.p. >250 °C (dec.). H-NMR (300MHz, DMSO-d
6
),
δ (ppm): 8.99 (s, 1H, ArH), 8.78 (s, 1H, ArH), 8.51 (t, J = 5.3 Hz, 1H, CONH), 7.33–7.27 (m, 5H, ArH),
.37 (br s, 2H, CONHCH CH ), 3.60 (s, 2H, CONHCH CH ), 3.53 (s, 2H, N-CH ), 3.03–2.91(m, 3H,
N-CH (2He), CH), 2.15–2.06 (m, 2H, N-CH (2Ha)), 1.96–1.83 (m, 4H, CH (2He), CH (2Ha)).
C-NMR (75MHz, DMSO-d ), δ (ppm): 166.48, 159.86, 143.63, 143.17, 139.31, 138.34, 128.76,
4
2
2
2
2
2
2
2
2
2
1
3
6
1
3
4
28.13, 126.83, 114.59, 112.63, 62.27, 52.60, 47.45, 40.21, 33.40, 30.00. HRMS (ESI+): found
+
62.1979 (Calcd for C21
H
24
N
5
O [M+H] : 362.1975).
.2.12 1-(1-Acetylpiperidin-4-yl)-8,9-dihydro-2,4,7,9a-tetraazabenzo[cd]azulen-6(7H)-one (9g)
A solution of 9a (0.25 g, 0.81 mmol) in methanol (10 mL) and tetrahydrofuran (10 mL) was
added several drops of triethylamine to adjust the pH to 7 before acetyl chloride (86.5 μl, 1.22
mmol) was added. After stirring for 2 hours at room temperature, the resulting solution was
concentrated in vacuum and the residue was further purified by silica gel column
chromatography using 5% methanol in dichloromethane as eluent to afford 9g (90 mg) as white
1
powder, yield 35.4%. m.p. >250 °C (dec.). H-NMR (300MHz, DMSO-d
6
), δ (ppm): 8.99 (s, 1H, ArH),
CH ), 3.96–3.92 (m, 1H, CH),
(2He, 1Ha), 2.81–2.73 (m, 1H, N-CH (1Ha)),
(2Ha)). C-NMR (75MHz, DMSO-d ), δ
8
3
2
.79 (s, 1H, ArH), 8.52 (s, 1H, CONH), 4.46–4.42 (m, 2H, CONHCH
2
2
.63 (br s, 2H, CONHCH
2
CH
2
), 3.37–3.09 (m, 3H, N-CH
2
2
1
3
.05 (s, 3H, COCH ), 1.99–1.61 (m, 4H, CH
3
2
(2He), CH
2
6
(
ppm): 168.03, 166.45, 159.36, 143.68, 143.18, 139.27, 136.55, 112.64, 47.48, 40.36, 33.02,
+
3
0.19, 29.62, 21.28. HRMS (ESI+): found 314.1619 (Calcd for C16
H
20
N
5
O
2
[M+H] : 314.1612).
4.2.13 1-(1-(Cyclopropanecarbonyl)piperidin-4-yl)-8,9-dihydro-2,4,7,9a-tetraazabenzo[cd]
azulen-6(7H)-one (9h)
Compound 9h was prepared from 9a and cyclopropyl formyl chloride according to the
1
procedure for preparation of 9g. White powder, yield 54.6%. m.p. 194-196 °C. H-NMR (300MHz,
DMSO-d
6
), δ (ppm): 9.00 (s, 1H, ArH), 8.80 (s, 1H, ArH), 8.54 (t, J = 5.5 Hz, 1H, CONH), 4.46–4.36
CH ), 3.64–3.59 (m, 2H, CONHCH CH ), 3.33–3.27 (m, 2H, N-CH (2He)),
(m, 2H, CONHCH
2
2
2
2
2

3
1
1
3
4
.11–3.03 (m, 2H, N-CH
2
(2Ha)), 2.86–2.78 (m, 1H, CH), 2.08–1.92 (m, 3H, CH
2
(2He), COCH),
1
3
.78–1.62 (m, 2H, CH (2Ha)), 0.75–0.71 (m, 4H, CH(CH
2
2
)
2
). C-NMR (75MHz, DMSO-d ), δ (ppm):
6
70.88, 166.43, 159.38, 143.65, 143.15, 139.28, 136.57, 112.66, 47.50, 44.52, 41.26, 40.19, 33.20,
+
0.51, 10.30, 6.82. HRMS (ESI+): found 340.1772 (Calcd for C18
H
22
N
5
O
2
[M+H] : 340.1768).
.2.14 1-(1-Benzoylpiperidin-4-yl)-8,9-dihydro-2,4,7,9a-tetraazabenzo[cd]azulen-6(7H)-one (9i)
Compound 9i was prepared from 9a and benzoyl chloride according to the procedure for
1
preparation of 9g. White powder, yield 72.7%. m.p. >250 °C (dec.). H-NMR (300MHz, DMSO-d
6
),
δ (ppm): 9.01 (s, 1H, ArH), 8.80 (s, 1H, ArH), 8.53 (t, J = 4.1Hz, 1H, CONH), 7.46 (s, 5H, ArH), 4.54
br s, 2H, CONHCH CH ), 3.63 (s, 2H, CONHCH CH ), 3.43–3.25(m, 2H, N-CH (2He)), 3.20–3.06 (m,
H, N-CH (2Ha), CH), 2.08–1.80 (m, 4H, CH (2He), CH (2Ha)). C-NMR (75MHz, DMSO-d ), δ
ppm): 169.03, 166.43, 159.21, 143.73, 143.21, 139.35, 136.60, 136.19, 129.38, 128.43, 126.66,
(
2
2
2
2
2
1
3
3
2
2
2
6
(
1
12.74, 47.51, 46.73, 40.19, 33.05, 30.00. HRMS (ESI+): found 376.1776 (Calcd for C21
H
22
N
5
O
2
+
[
M+H] : 376.1768).
4
1
.2.15
-(1-(2-Chlorobenzoyl)piperidin-4-yl)-8,9-dihydro-2,4,7,9a-tetraazabenzo[cd]azulen-6(7H)-one
(
9j)
Compound 9j was prepared from 9a and 2-chlorobenzoyl chloride according to the
1
procedure for preparation of 9g. White powder, yield 58.9%. m.p. 158-160 °C. H-NMR (300MHz,
DMSO-d
m, 4H, ArH), 4.60–4.41 (m, 2H, CONHCH
N-CH (2He), N-CH (2Ha), CH), 2.14–1.76 (m, 4H, CH
DMSO-d ), δ (ppm): 166.27, 165.47, 159.38, 149.32, 143.34, 142.84, 139.26, 136.77, 135.96,
6
), δ (ppm): 9.01 (s, 1H, ArH), 8.80 (s, 1H, ArH), 8.52 (t, J = 5.3 Hz, 1H, CONH), 7.57–7.38
CH ), 3.62 (s, 2H, CONHCH CH ), 3.41–3.02 (m, 5H,
(2He), CH (2Ha)). C-NMR (75MHz,
(
2
2
2
2
1
3
2
2
2
2
6
1
4
4
1
30.46, 129.44, 127.70, 119.20, 112.87, 47.53, 46.19, 40.29, 32.97, 29.61. HRMS (ESI+): found
+
10.1384 (Calcd for C21
H21ClN
5
O
2
[M+H] : 410.1378).
.2.16
-(1-(3-Chlorobenzoyl)piperidin-4-yl)-8,9-dihydro-2,4,7,9a-tetraazabenzo[cd]azulen-6(7H)-one
(
9k)
Compound 9k was prepared from 9a and 3-chlorobenzoyl chloride according to the
1
procedure for preparation of 9g. White powder, yield 58.9%. m.p. 135-137° C. H-NMR (300MHz,
DMSO-d
m, 4H, ArH), 4.52–4.37 (m, 2H, CONHCH
N-CH (2He), N-CH (2Ha), CH), 2.12–1.82 (m, 4H, CH
DMSO-d ), δ (ppm): 167.37, 166.43, 159.16, 143.73, 143.20, 139.35, 138.27, 136.55, 133.19,
6
), δ (ppm): 9.01 (s, 1H, ArH), 8.81 (s, 1H, ArH), 8.55 (t, J = 5.2 Hz, 1H, CONH), 7.56–7.40
CH ), 3.64 (s, 2H, CONHCH CH ), 3.41–3.17 (m, 5H,
(2He), CH (2Ha)). C-NMR (75MHz,
(
2
2
2
2
1
3
2
2
2
2
6
1
4
4
1
30.47, 129.31, 126.52, 125.28, 112.73, 47.52, 46.61, 40.18, 32.99, 30.03. HRMS (ESI+): found
+
10.1385 (Calcd for C21
H21ClN
5
O
2
[M+H] : 410.1378).
.2.17
-(1-(4-Chlorobenzoyl)piperidin-4-yl)-8,9-dihydro-2,4,7,9a-tetraazabenzo[cd]azulen-6(7H)-one
(
9l)
Compound 9l was prepared from 9a and 4-chlorobenzoyl chloride according to the
1
procedure for preparation of 9g. White powder, yield 58.9%. m.p. 146-148 °C. H-NMR (300MHz,
DMSO-d
.4 Hz, 4H, ArH), 4.51 (br s, 2H, CONHCH
N-CH (2He), N-CH (2Ha), CH), 2.06–1.82 (m, 4H, CH
DMSO-d ), δ (ppm): 167.97, 166.35, 159.35, 143.50, 142.95, 139.32, 136.68, 134.92, 134.05,
6
), δ (ppm): 9.01 (s, 1H, ArH), 8.80 (s, 1H, ArH), 8.52 (t, J = 4.3 Hz, 1H, CONH), 7.50 (q, J =
CH ), 3.63 (s, 2H, CONHCH CH ), 3.43–3.03(m, 5H,
(2He), CH (2Ha)). C-NMR (75MHz,
8
2
2
2
2
1
3
2
2
2
2
6

1
28.77, 128.52, 112.76, 47.53, 46.67, 40.17, 33.01, 29.95. HRMS (ESI+): found 410.1384 (Calcd
+
for C21
H21ClN
5
O
2
[M+H] : 410.1378).
4.2.18 1-Phenyl-8,9-dihydro-2,4,7,9a-tetraazabenzo[cd]azulen-6(7H)-one (11a)
The 9-amino-3,4-dihydro-1H-pyrido[4,3-e][1,4]diazepin-5(2H)-one 7 (0.20 g, 1.13 mmol),
benzaldehyde 10a (0.12 g, 1.13 mmol) and 10% Pd/C (100 mg) were dissolved in methanol (20
mL) in a sealed tube and heated at 120 °C. After 24 hours, the resulting solution was cooled to
room temperature and filtered off. The filtrate was concentrated under reduced pressure to give
the crude residue, which was purified by silica gel column chromatography using 2% methanol in
dichloromethane as eluent to give 11a (100 mg) as white solid, yield 33.5%. m.p. >250 °C (dec.).
1
H-NMR (300MHz, DMSO-d
6
), δ (ppm): 9.13(s, 1H, ArH), 8.84 (s, 1H, ArH), 8.59 (br s, 1H, CONH),
CH ), 3.57 (s, 2H, CONHCH CH ).
6
), δ (ppm): 166.45, 155.11, 143.97, 143.92, 139.68, 137.08, 130.53,
7
.90 (s, 2H, ArH), 7.62 (s, 3H, ArH), 4.49 (s, 2H, CONHCH
2
2
2
2
1
3
C-NMR (75MHz, DMSO-d
1
29.71, 128.75, 128.69, 113.24, 50.36, 40.25. HRMS (ESI+): found 265.1118 (Calcd for C15
H
13
N
4
O
+
[
M+H] : 265.1084).
4.2.19 1-(4-(Piperazin-1-yl)phenyl)-8,9-dihydro-2,4,7,9a-tetraazabenzo[cd]azulen-6(7H)-one
(
11b)
Compound 11b was prepared from 7 and 4-(piperazin-1-yl)benzaldehyde 10b according to
1
the procedure for preparation of 11a. Yellow solid, yield 92.6%. m.p. >250 °C (dec.). H-NMR
(
300MHz, MeOD), δ (ppm): 9.36 (s, 1H, ArH), 9.03 (s, 1H, ArH), 7.94 (d, J = 8.1 Hz, 2H, ArH), 7.30
2
(d, J = 8.1 Hz, 2H, ArH), 4.73 (br s, 2H, CONHCH
2
CH ), 3.83 (br s, 2H, CONHCH
2
CH
2
), 3.74–3.64 (m,
1
3
4
1
4
4
1
H, N-CH
2
, N-CH
2
), 3.50–3.37 (m, 4H, N-CH
2
, N-CH
2
). C-NMR (75MHz, D
2
O), δ (ppm): 165.43,
61.26, 151.57, 141.73, 139.25, 135.48, 133.35, 130.87, 116.71, 115.20, 114.83, 50.21, 44.26,
+
2.26, 39.71. HRMS (ESI+): found 349.1773 (Calcd for C19
H
21
N
6
O [M+H] : 349.1771).
.2.20
-(4-(4-Methylpiperazin-1-yl)phenyl)-8,9-dihydro-2,4,7,9a-tetraazabenzo[cd]azulen-6(7H)-one
(
11c)
Compound 11c was prepared from 7 and 4-(4-methylpiperazin-1-yl)benzaldehyde 10c
according to the procedure for preparation of 11a. Yellow solid, yield 37.4%. m.p. >250 °C (dec.).
1
H-NMR (300MHz, MeOD), δ (ppm): 8.89 (s, 1H, ArH), 8.80 (s, 1H, ArH), 7.65 (d, J = 8.6 Hz, 2H,
ArH), 7.04 (d, J = 8.5 Hz, 2H, ArH), 4.44 (br s, 2H, CONHCH
.33–3.27 (m, 4H, N-CH , N-CH ), 2.59–2.55 (m, 4H, N-CH , N-CH
75MHz, MeOD), δ (ppm): 167.49, 156.81, 152.39, 142.99, 141.98, 139.20, 137.32, 130.26,
2
CH
2
), 3.59 (br s, 2H, CONHCH
2
CH
2
),
1
3
3
2
2
2
), 2.28 (s, 3H, CH ). C-NMR
2 3
(
1
16.79, 114.08, 112.63, 53.81, 50.15, 46.44, 44.15, 40.01. HRMS (ESI+): found 363.1932 (Calcd
+
for C20
H
23
N
6
O [M+H] : 363.1928).
4
.2.21
-(4-((Methylamino)methyl)phenyl)-8,9-dihydro-2,4,7,9a-tetraazabenzo[cd]azulen-6(7H)-one
1
(
11d)
Compound 11d was prepared from 7 and 4-((methylamino)methyl)benzaldehyde 10d
according to the procedure for preparation of 11a. Yellow powder, yield 24.0%. m.p. 252-254 °C.
1
H-NMR (300MHz, DMSO-d
6
), δ (ppm): 9.12 (s, 1H, ArH), 8.83 (s, 1H, ArH), 8.61 (t, J = 5.4 Hz, 1H,
CH ),
). C-NMR (75MHz,
), δ (ppm): 166.50, 155.32, 143.87, 143.83, 143.31, 139.70, 137.15, 129.53, 128.22,
26.90, 113.21, 54.45, 50.38, 40.23, 35.43. HRMS (ESI+): found 308.1511 (Calcd for C17
CONH), 7.86 (d, J = 8.1 Hz, 2H, ArH), 7.56 (d, J = 8.1 Hz, 2H, ArH), 4.48 (br s, 2H, CONHCH
2
2
1
3
3
.76 (s, 2H, NHCH
2 2 2 3
), 3.58 (br s, 2H, CONHCH CH ), 2.30 (s, 3H, NHCH
DMSO-d
6
1
18 5
H N O

+
[
M+H] : 308.1506).
4.2.22
1-(4-((Dimethylamino)methyl)phenyl)-8,9-dihydro-2,4,7,9a-tetraazabenzo[cd]azulen-6(7H)-one
(
11e)
Compound 11e was prepared from 7 and 4-((dimethylamino)methyl)benzaldehyde 10e
according to the procedure for preparation of 11a. Yellow powder, yield 27.5%. m.p. 220-222 °C.
1
H-NMR (300MHz, DMSO-d
6
), δ (ppm): 9.12 (s, 1H, ArH), 8.84 (s, 1H, ArH), 8.61 (t, J = 4.8 Hz, 1H,
CH ), 3.58
). C-NMR (75MHz, DMSO-d ), δ
CONH), 7.87 (d, J = 7.7 Hz, 2H, ArH), 7.53 (d, J = 7.7 Hz, 2H, ArH), 4.49 (s, 2H, CONHCH
2
2
1
3
(br s, 2H, NCH
2
), 3.51 (s, 2H, CONHCH
2
CH
2
), 2.20 (s, 6H, N(CH
3
)
2
6
(
ppm): 166.48, 155.12, 143.89, 143.84, 141.61, 139.69, 137.10, 129.59, 128.96, 127.23, 113.23,
+
6
3
4
1
2.84, 50.39, 44.96, 40.23. HRMS (ESI+): found 322.1671 (Calcd for C18
H
20
N
5
O [M+H] :
22.1662).
.2.23
-(4-((Ethylamino)methyl)phenyl)-8,9-dihydro-2,4,7,9a-tetraazabenzo[cd]azulen-6(7H)-one
(
11f)
Compound 11f was prepared from 7 and 4-((ethylamino)methyl)benzaldehyde 10f according
1
to the procedure for preparation of 11a. Yellow powder, yield 22.9%. m.p. 228-230 °C. H-NMR
(
300MHz, DMSO-d
.89 (d, J = 8.1 Hz, 2H, ArH), 7.61 (d, J = 8.1 Hz, 2H, ArH), 4.48 (br s, 2H, CONHCH
NHCH ), 3.59 (br s, 2H, CONHCH CH ), 2.66 (q, J = 7.1 Hz, 2H, CH CH ), 1.11 (t, J = 7.1 Hz, 3H,
CH2CH3). C-NMR (75MHz, DMSO-d ), δ (ppm): 166.49, 155.08, 143.92, 143.87, 141.83, 139.69,
37.10, 129.61, 128.59, 127.30, 113.30, 51.63, 50.38, 42.64, 40.28, 14.09. HRMS (ESI+): found
6
), δ (ppm): 9.13 (s, 1H, ArH), 8.84 (s, 1H, ArH), 8.63 (t, J = 5.4 Hz, 1H, CONH),
7
2
CH ), 3.91 (s, 2H,
2
2
2
2
2
3
1
3
6
1
3
4
1
+
20 5
22.1668 (Calcd for C18H N O [M+H] : 322.1662).
.2.24
-(4-((Isopropylamino)methyl)phenyl)-8,9-dihydro-2,4,7,9a-tetraazabenzo[cd]azulen-6(7H)-one
(
11g)
Compound 11g was prepared from 7 and 4-((isopropylamino)methyl)benzaldehyde 10g
according to the procedure for preparation of 11a. Yellow powder, yield 35.1%. m.p. 218-220 °C.
1
H-NMR (300MHz, DMSO-d
.82 (d, J = 6.0 Hz, 2H, ArH), 7.55 (d, J = 6.0 Hz, 2H, ArH), 4.46 (br s, 2H, CONHCH
NHCH ), 3.56 (br s, 2H, CONHCH CH ), 2.70 (m, 1H, CH(CH ), 1.00 (d, J = 6.0 Hz, 6H, CH(CH
C-NMR (75MHz, DMSO-d ), δ (ppm): 166.50, 155.25, 144.38, 143.95, 143.81, 139.67, 137.09,
6
), δ (ppm): 9.09 (s, 1H, ArH), 8.81 (s, 1H, ArH), 8.65 (br s, 1H, CONH),
CH ), 3.77 (s, 2H,
).
7
2
2
2
2
2
3
)
2
3 2
)
1
3
6
1
29.45, 128.10, 126.61, 113.19, 50.38, 49.97, 47.18, 40.21, 22.72. HRMS (ESI+): found 336.1814.
+
(Calcd for C19
H
22
N
5
O [M+H] : 336.1819).
4
.2.25
-(4-((Tert-butylamino)methyl)phenyl)-8,9-dihydro-2,4,7,9a-tetraazabenzo[cd]azulen-6(7H)-one
1
(
11h)
Compound 11h was prepared from 7 and 4-((tert-butylamino)methyl)benzaldehyde 10h
according to the procedure for preparation of 11a. Yellow powder, yield 25.2%. m.p. >250 °C
1
(
dec.). H-NMR (300MHz, DMSO-d
6
), δ (ppm): 9.09 (s, 1H, ArH), 8.82 (s, 1H, ArH), 8.61 (br s, 1H,
CH ),
). C-NMR (75MHz,
), δ (ppm): 166.49, 155.29, 145.32, 143.82, 139.70, 137.10, 129.40, 128.13, 126.51,
13.20, 50.38, 50.20, 45.91, 40.28, 28.89. HRMS (ESI+): found 350.1975 (Calcd for C20
CONH), 7.81 (d, J = 5.2 Hz, 2H, ArH), 7.57 (d, J = 4.8 Hz, 2H, ArH), 4.46 (br s, 2H, CONHCH
2
2
1
3
3
.75 (s, 2H, NHCH
2 2 2 3 3
), 3.57 (br s, 2H, CONHCH CH ), 1.10 (s, 9H, C(CH )
DMSO-d
6
1
H
24
N
5
O

+
[
M+H] : 350.1975).
4.2.26 1-(4-(Diethoxymethyl)phenyl)-8,9-dihydro-2,4,7,9a-tetraazabenzo[cd]azulen-6(7H)-one
(
12)
9
-Amino-3,4-dihydro-1H-pyrido[4,3-e][1,4] diazepin-5(2H)-one 7 (0.50 g, 2.8 mmol),
-(diethoxymethyl)benzaldehyde (0.58 g, 2. ) and 10% Pd/C (125 mg) were dissolved in methanol
30 mL) in a sealed tube and heated at 120 °C. After 24 hours, the resulting solution was cooled
4
(
to room temperature and filtered off. The filtrate was concentrated under reduced pressure to
give the crude residue, which was purified by silica gel column chromatography using 10%
methanol in dichloromethane as eluent to give 12 (0.62 g) as yellow powder, yield 60.5%.
1
H-NMR (300MHz, CDCl
3
), δ (ppm): 9.23 (s, 1H, ArH), 9.13 (s, 1H, ArH), 8.41 (br s, 1H, CONH), 7.82
CH ),
(d, J = 8.2 Hz, 2H, ArH), 7.72 (d, J = 8.2 Hz, 2H, ArH), 5.60 (s, 1H, CH), 4.55 (br s, 2H, CONHCH
2
2
3
.78 (br s, 2H, CONHCH
.2.27 4-(6-Oxo-6,7,8,9-tetrahydro-2,4,7,9a-tetraazabenzo[cd]azulen-1-yl)benzaldehyde (13)
The solution of 1-(4-(diethoxymethyl)phenyl)-8,9-dihydro-2,4,7,9a-
2 2 2 3 2 3
CH ), 3.72 (m, 4H, 2CH CH ), 1.30 (q, J = 7.02 Hz, 6H, 2CH CH ).
4
tetraazabenzo[cd]azulen-6(7H)-one 12 (0.10 g, 0.27 mmol) in tetrahydrofuran (15 mL) was
treated with 1M hydrochloride acid (1 mL) via dropwise addition at room temperature before
heating at 80 °C for 2 hours. After concentration, the residue was partitioned between saturated
sodium bicarbonate solution and EtOAc. The organic phase was washed with water, brine, dried
1
over anhydrous Na
DMSO-d
CONH), 8.18-8.06 (m, 4H, ArH), 4.50 (s, 2H, CONHCH
2
SO
4
to afford 13 (60 mg) as white powder, yield 75.9%. H-NMR (300MHz,
6
), δ (ppm): 10.14 (s, 1H, CHO), 9.15 (s, 1H, ArH), 8.85 (s, 1H, ArH), 8.58 (t, J =5.3 Hz, 1H,
CH ), 3.59 (s, 2H, CONHCH CH ).
2
2
2
2
4
.2.28
-(4-(Pyrrolidin-1-ylmethyl)phenyl)-8,9-dihydro-2,4,7,9a-tetraazabenzo[cd]azulen-6(7H)-one
1
(
11i)
A
solution
of
4-(6-oxo-6,7,8,9-tetrahydro-2,4,7,9a-tetraazabenzo[cd]azulen-1-yl)
benzaldehyde 13 (0.2 g, 0.68 mmol) and pyrrolidine (0.10 g, 1.36 mmol) in methanol (10 mL) was
stirred for 15min before sodium cyanoborohydride (0.10 g, 1.59 mmol) was added. After stirring
at room temperature overnight, the solvent was removed under reduced pressure and the
residue was subject to silica gel column chromatography using 20% methanol in dichloromethane
1
as eluent to give 11i as yellow powder, yield 42.4%. m.p. 118-120 °C. H-NMR (300MHz,
DMSO-d
6
), δ (ppm): 9.13 (s, 1H, ArH), 8.85 (s, 1H, ArH), 8.69 (br s, 1H, CONH), 7.93 (d, J = 8.1 Hz,
H, ArH), 7.73 (d, J = 7.5 Hz, 2H, ArH), 4.51 (s, 2H, CONHCH CH ), 4.05 (s, 2H, N-CH ), 3.62 (s,
H,CONHCH CH ), 2.83 (s, 4H, 2NCH CH ), 1.85 (s, 4H, 2NCH CH ). C-NMR (75MHz, DMSO-d ), δ
2
2
2
2
1
2
3
2
2
2
2
2
2
6
(
ppm): 166.93, 155.38, 144.47, 140.22, 137.6, 130.23, 129.78, 128.60, 113.78, 58.25, 53.57,
+
5
4
1
0.89, 23.43. HRMS (ESI+): found 348.1855 (Calcd for C20
H
22
N
5
O [M+H] : 348.1819).
.2.29
-(4-(Piperidin-1-ylmethyl)phenyl)-8,9-dihydro-2,4,7,9a-tetraazabenzo[cd]azulen-6(7H)-one
(
11j)
Compound 11j was prepared from 13 and piperidine according to the procedure for
1
preparation of 11i. Yellow powder, yield 40.7%. m.p. >250 °C (dec.). H-NMR (300MHz, DMSO-d
6
),
δ (ppm): 9.16 (s, 1H, ArH), 8.85 (s, 1H, ArH), 8.63 (t, J = 4.7 Hz, 1H, CONH), 7.94 (d, J = 8.0 Hz, 2H,
ArH), 7.81 (d, J = 7.6 Hz, 2H, ArH), 4.50 (br s, 2H, CONHCH CH ), 4.23–4.05 (m, 2H, CONHCH CH ),
(2He, 2Ha)), 1.82–1.64 (m, 4H, 2NCH CH (2He,
(1He, 1Ha)). C-NMR (75MHz, DMSO-d ), δ (ppm): 166.95, 155.26,
2
2
2
2
3
.59 (s, 2H, N-CH
2
), 3.03–2.70 (m, 4H, 2NCH
2
2
2
1
3
2Ha)), 1.58–1.41 (m, 2H, CH
2
6

1
5
4
4
44.52, 140.29, 137.72, 131.43, 131.09, 130.26, 129.77, 129.26, 113.81, 72.55, 59.97, 52.79,
+
0.90, 23.67, 22.70. HRMS (ESI+): found 362.2022 (Calcd for C21
.2 Biological assays
H
24
N
5
O [M+H] : 362.2027).
.2.1 The PARP-1and PARP-2 enzyme inhibition assay
The inhibition of the target compounds on PARP-1/2 enzymatic activity was determined by
ELISA (Enzyme-Linked-Immunosorbent Assay) in 96-well plates. Each well was precoated with
histone (20 μg/mL) diluted in 100 μL of PBS buffer (10 mM NaH PO ,10 mM Na HPO , 150 mM
NaCl, pH 7.4) by incubation at 4 °C overnight. 100 μM NAD+, 25 μM biotinylated NAD+ and 200
nM slDNA diluted in 30 μL of reaction buffer (50 mM Tris, 2 mM MgCl , pH 8.0) were added into
2
4
2
4
2
each well, and then 5 μL of compound or solvent control was added at varying concentrations.
The reaction was initiated by the addition of 20 μL of PARP-1or PARP-2 (50 ng/well) at 30 °C for 1
hour. Then the reaction solution was added 50 µl streptavidin conjugated HRP. The assay was
2 2
performed at 30 °C for additional 30 min. Finally, 100 μL of solution (H O and luminol in citrate
buffer 0.1M, pH 5.4) was added and luminescent signal was measured using a multi-well
spectrophotometer (Molecular Devices SpectraMax M5 microplate reader). The inhibition rate of
PARP-1/2 enzymatic activity was calculated as (Lu control−Lu treated/Lu control) × 100%. The
concentration required for 50% inhibition of PARP-1/2 enzymatic activity (IC50 value) was
calculated using nonlinear regression with normalized dose−response fit using GraphPad Prism 5
software.
4.2.2 The cytotoxicity assay against SW-620 and MDA-MB-468 cell lines
In cytotoxicity assays, the cell proliferation of adherent cells was determined by SRB assay
(
sulforhodamine B assay). Briefly, cells were seeded in 96-well plates and then treated with
different concentrations of PARP-1 inhibitor. After incubation for 72 hours, cells were fixed with
0% trichloroacetic acid for 1 hour at 4 °C, washed five times with tap water and air-dried. Cells
1
that survived were stained with 0.4% (w/v) SRB for 20 min at room temperature and washed five
times with 1% acetic acid. Bound SRB was solubilized with 10 mM Tris and absorbance was
measured at 540 nm. EC50 values were calculated using GraphPad Prism 5 software.
4.2.3 The PF50 assay against SW-620, MDA-MB-468 and A549 cell lines
In PF50 assays, the cell proliferation of adherent cells was determined by SRB assay. Cells
were seeded in 96-well plates and then treated with PARP -1 inhibitor at a fixed concentration of
.5 μM and temozolomide at different concentrations. After incubation for 72 hours, cells were
0
fixed with 10% trichloroacetic acid for 1 hour at 4 °C, washed five times with tap water and
air-dried. Cells that survived were stained with 0.4% (w/v) SRB for 20 min at room temperature
and washed five times with 1% acetic acid. Bound SRB was solubilized with 10 mM Tris and
absorbance was measured at 540 nm. EC50 values were calculated using GraphPad Prism 5
software. Potentiation factor (PF50) was calculated as the ratio of the EC50 for temozolomide
divided by the EC50 of temozolomide + 0.5 μM PARP-1 inhibitor.
4.3 Molecular modelling
Molecular modelling of compound 9b and 11d into the three dimensional X-ray structure of
PARP-1 enzyme (PDB code: 2RCW) was carried out using the Discovery Studio (version 3.0) as
implemented through the graphical user interface DiscoveryStudio LibDock protocol.
The crystal structures of PARP-1 enzyme were retrieved from the RCSB Protein Data Bank
(http://www.rcsb.org/pdb/home/home.-do). All bound waters and ligands were eliminated from
the protein and the polar hydrogen was added. The whole 2RCW was defined as a receptor and

the site sphere was selected based on the active site of 2RCW. Compound 9b and 11d were
placed during the molecular docking procedure. Types of interactions of the docked protein with
ligand were analyzed after the end of molecular modelling.
Acknowledge
This work was supported by National Natural Science Foundation of China (Grant No.
81502928), Outstanding Scientific and Technological Innovation Team of Jiangsu Province of
China in 2015 and Fundamental Research Funds for the Central Universities (No. 2015PY015).
Reference:
1
2
3
4
5
1
6
7
8
8
9
2
1
Miwa, M.; Masutani, M. Cancer Sci. 2007, 98, 1528
Woodhouse, B. C; Dianov, G. L. DNA Repair (Amst). 2008, 7, 1077
Schreiber, V. ; Dantzer, F. ; Ame, J.-C.; de Murcia, G. Nat. Rev. Mol. Cell Biol. 2006, 7, 517
Gibson, B. ; Kraus, W. L. Nat. Rev. Mol. Cell Biol. 2012, 13, 411
Rouleau, M. ; Patel, A. ; Hendzel, M. J. ; Kaufmann, S. H. ; Poirier, G.G. Nat. Rev. Cancer. 2010,
0, 293
Hosoya, N. ; Miyagawa, K. Cancer Sci. 2014, 105, 370
Helleday, T.; Petermann, E.; Lundin, E. C.; Hodgson, B.; Sharma, R. A. Nat. Rev. Cancer. 2008,
, 193
Fauzee, N.J.S.; Pan, J.; Wang, Y. L. Pathol. Oncol. Res. 2010, 16, 469
Bryant, H.E.; Schultz, N.; Thomas, H. D.; Parker, K.M.; Flower, D.; Lopez, E.; et al. Nature.
005, 434, 913
Farmer, H.; McCabe, N.; Lord, C. J.; Tutt, A. N. J.; Johnson, D. A.; Richardson, T. B.; et al.
0
Nature. 2005, 434, 917
1
1
1
2
1
1
1
1
1
5
1
2
2
2
2
2
2
1
2
3
Amir, E.; Seruga, B.; Serrano, R.; Ocana, A. Cancer Treat. Rev. 2010, 36, 557
Ström, C. E.; Helleday, T. Biomolecules. 2012, 2, 635
Papeo, G.; Forte, B.; Orsini, P.; Perrera, C.; Posteri, H.; Scolaro, A.et al. Expert Opin. Ther. Pat.
009, 19, 1377
4
Sandhu, S.K.; a Yap, T.; de Bono, J. S. Eur. J. Cancer. 2010, 46, 9-20.
5
C. Schmidt, PARP inhibitors refocus for rebound., Cancer Discov. 2012, 2, 659
Ferraris, D. V. J. Med. Chem. 2010, 53, 4561
6
7
Underhill, C.; Toulmonde, M.; Bonnefoi, H. Ann. Oncol. 2011, 22, 268
Penning, T. D.; Zhu, G. D.; Gandhi, V. B.; J. Liu, Gong, X.; Shi, Y.et al. J. Med. Chem. 2009, 2,
8
14
9
Jones, P.; Altamura, S.; Boueres, J.; Ferrigno, F.; Fonsi, M.; Giomini, C. et al. J. Med. Chem.
009, 52, 7170
0
Plummer, R. ; Jones, C.; Middleton, M.; Wilson, R.; Evans, J.; Olsen, A. et al. Clin. Cancer. Res.
008, 14, 7917
1
Shen, Y.; Rehman, F. L.; Feng, Y.; Boshuizen, J.; Bajrami, I.; Elliott, R. et al. Clin. Cancer Res.
013, 19, 5003
2
Menear, K. A.; Adcock, C.; Boulter, R.; Cockcroft, X. L.; Copsey, L.; Cranston, A. et al. J. Med.
Chem. 2008, 51, 6581
23
24
25
Zhu, Q.; Wang, X.; Chu, Z.; He, G.; Dong, G.; Xu, Y. Bioorg. Med. Chem. Lett. 2013, 23, 1993
Zhu, Q.; Wang, X.; Hu, Y.; Gong, G.; Xu, Y. Bioorg. Med. Chem. 2015, 23, 6551
Ryabova, O. B.; Makarov, V. A.; Alekseeva, L. M.; Shashkov, A. S.; Chernyshev, V. V.; Granik, V.
G. Russ. Chem. Bull. Int. Ed. 2005, 54, 1907

2
2
2
6
Tikhe, J. G.; Webber, S. E.; Hostomsky, Z.; Maegley, K. A.; Ekkers, A.; Li, J. et al. J. Med. Chem.
004, 47, 5467
Zhu, Q. H.; Hu, Y.; Wang, X. Y.; Xu, Y. G.; Huang, Q. G. CN 104592232 A. Appl. 05. 06. 2015 (In
7
order to be beneficial to the follow-up development, we submitted the patent application before
publishing our work.)

Graphical Abstract
A series of novel Tricyclic Poly (ADP-ribose) Polymerase-1/2 inhibitors is described.