Design, synthesis and biological evaluation of novel 7-amino-[1,2,4]triazolo[4,3-f]pteridinone, and 7-aminotetrazolo[1,5-f]pteridinone derivative as potent antitumor agents

Article abstract of DOI:10.1016/j.ejmech.2018.12.009

To develop novel therapeutic agents with anticancer activities, two series of novel 7-amino-[1,2,4]triazolo[4,3-f]pteridinone, and 7-aminotetrazolo[1,5-f]pteridinone derivatives were designed and synthesized. All compounds were tested for anti-proliferative activities against five cancer cell lines. The structure-activity relationships (SARs) studies were conducted through the variation in two regions, the moiety of A ring and the terminal aniline B on pteridinone core. 1-Methyl-1,2,4-triazole derivative L₇ with 2,6-dimethylpiperazine showed the most potent antiproliferative activity against A549, PC-3, HCT116, MCF-7 and MDA-MB-231 cell lines with IC₅₀ values of 0.16 μM, 0.30 μM, 0.51 μM, 0.30 μM, and 0.70 μM, respectively. Combined with the results of the molecular docking and enzymatic studies, the PLK1 was very likely to be one of the drug targets of compound L₇. Furthermore, to clarify the anticancer mechanism of compound L₇, further explorations in the bioactivity were conducted. The results showed that compound L₇ obviously inhibited proliferation of A549 cell lines, induced a great decrease in mitochondrial membrane potential leading to apoptosis of cancer cells, suppressed the migration of tumor cells, and arrested G1 phase of A549 cells.

Full text of DOI:10.1016/j.ejmech.2018.12.009

Accepted Manuscript
Design, synthesis and biological evaluation of novel 7-amino-
[1,2,4]triazolo[4,3-f]pteridinone, and 7-aminotetrazolo[1,5-f]pteridinone derivative as
potent antitumor agents
Yunlei Hou, Liangyu Zhu, Zhiwei Li, Qi Shen, Qiaoling Xu, Wei Li, Yajing Liu, Ping
Gong
PII:
S0223-5234(18)31046-8
DOI:
https://doi.org/10.1016/j.ejmech.2018.12.009
EJMECH 10942
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 17 September 2018
Revised Date: 1 December 2018
Accepted Date: 3 December 2018
Please cite this article as: Y. Hou, L. Zhu, Z. Li, Q. Shen, Q. Xu, W. Li, Y. Liu, P. Gong, Design,
synthesis and biological evaluation of novel 7-amino-[1,2,4]triazolo[4,3-f]pteridinone, and 7-
aminotetrazolo[1,5-f]pteridinone derivative as potent antitumor agents, European Journal of Medicinal
Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2018.12.009.
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ACCEPTED MANUSCRIPT
Graphical abstract
Two
series
of
novel
7-amino-[1,2,4]triazolo[4,3-f]pteridinone,
and
7-aminotetrazolo[1,5-f]pteridinone derivatives were designed, synthesized and evaluated for their
biological activity.

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Design, synthesis and biological evaluation of novel 7-amino-[1,2,4]triazolo[4,3-f]pteridinone,
and 7-aminotetrazolo[1,5-f]pteridinone derivative as potent antitumor agents
Yunlei Hou, Liangyu Zhu, Zhiwei Li, Qi Shen, Qiaoling Xu, Wei Li, Yajing Liu , Ping Gong
Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education, Shenyang
Pharmaceutical University, 103 Wenhua Road, Shenhe District, Shenyang, 110016, China.
Abstract
To develop novel therapeutic agents with anticancer activities, two series of novel
7-amino-[1,2,4]triazolo[4,3-f]pteridinone, and 7-aminotetrazolo[1,5-f]pteridinone derivatives were
designed and synthesized. All compounds were tested for anti-proliferative activities against five
cancer cell lines. The structure-activity relationships (SARs) studies were conducted through the
variation in two regions, the moiety of A ring and the terminal aniline B on pteridinone core.
1-Methyl-1,2,4-triazole derivative L7 with 2,6-dimethylpiperazine showed the most potent
antiproliferative activity against A549, PC-3, HCT116, MCF-7 and MDA-MB-231 cell lines with
IC50 values of 0.16 µM, 0.30 µM, 0.51 µM, 0.30 µM, and 0.70 µM, respectively. Combined with
the results of the molecular docking and enzymatic studies, the PLK1 was very likely to be one of
the drug targets of compound L7. Furthermore, to clarify the anticancer mechanism of compound
L7, further explorations in the bioactivity were conducted. The results showed that compound L7
obviously inhibited proliferation of A549 cell lines, induced a great decrease in mitochondrial
membrane potential leading to apoptosis of cancer cells, suppressed the migration of tumor cells,
and arrested G1 phase of A549 cells.
1. Introduction
Despite continued research efforts, cancer remains the second leading disease after cardiovascular
and one of the major public health problems characterized by uncontrolled growth and spread of
the abnormal cells [1]. The Global Burden of Disease (GBD) 2015 studies stated that the most
common types of cancer are breast, colorectal, and lung cancer in females and prostate, colorectal,
and lung cancer in males [2-3]. To combat with cancer, lots of efforts have been concentrated on
the design and synthesis of new antitumor drugs with low toxicity and high efficiency to normal
cells and tissues. For these purposes, combination principle strategy has been extensively
employed to develop new antineoplastic drugs that acted synergistic on multiple targets, via fusing
two/more active pharmacophores covalently in a single-hybrid molecule with dual/multiple
anticancer activity [4].
Recently, accumulating evidences have illustrated that heterocyclic scaffolds are important tool
in the search for new active substances with a lot of potential applications [5]. Particularly,
substituted 2-aminopteridinone skeleton has been described as a privileged structure, which
appears extensively in many unrelated areas of biology and medicine. Especially, it could inhibit
several cancer related enzymes as well as modulate the activity of many receptors. For example,
PLK1 inhibitor I (currently in phase III clinical trials at Boehringer Ingelheim for the treatment of
acute myeloid leukemia), EGFR inhibitor II, beta-amyloid (Abeta) production inhibitor III, and
ALK inhibitor IV, et al. (Fig. 1) [6-9]. These characteristics indicated that the extent of ongoing
interests toward new 2-aminopteridinone derivatives and prompted us to develop this
pharmacophore as novel promising drugs.
Corresponding author. E-mail address: gongpinggp@126.com (P. Gong). lyjpharm@126.com (Y.J. Liu).

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Fig. 1. Structures of representative kinase inhibitors containing 2-aminopteridinone nuclei.
In recent years, 2-aminopyrido[2,3-d]pyrimidinone derivatives have received considerable
attention in anticancer agents due to their admirable inhibitory activity against CDK4/6 kinases,
FGFR kinases, PI3K kinases, and MAPK kinases, which play critical roles in the regulation of
tumor genesis [10-13]. Among these derivatives (Fig. 2), palbociclib (V), the first orally
bioavailable CDK4/6 kinase inhibitor, was approved by FDA for the treatment of
hormone-receptor positive breast cancer in Feburary 2015. The co-crystal structure of palbociclib
in complex with CDK6 kinase domains (PDB code: 5L2I) revealed that the
2-aminopyrido[2,3-d]pyrimidinone framework played key roles in the interaction with CDK6
kinase due to their binding modes and the presence of hydrogen bonds [14]. Thus, the
development of such small molecules with 2-aminopyrido[2,3-d]pyrimidinone framework, which
can readily bind with various enzymes and receptors through hydrogen bonds, is an effectual
process to develop new antineoplastic drugs.

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Fig. 2. Structures of representative kinase inhibitors containing 2-aminopyrido[2,3-d]pyrimidin-
one nuclei.
In addition, triazolo and tetrazolo heterocycles constitute a very important class of heterocyclic
compounds in the area of drug design, which exhibit a broad spectrum of biological activity,
including anti-inflammatory [15], central nervous system (CNS) dispersant [16], antimicrobial
[17], anti-AIDS [18], antifertility [19], anticancer [20], and anticonvulsant [21]. As continue of our
effort to discover new types of antitumor lead compounds and our interest in studying the
influence
of
fusing
triazolo
or
tetrazolo
to
the
2-aminopteridinone
or
2-aminopyrido[2,3-d]pyrimidinone frameworks, so these combined substructures could exhibit
synchronous antitumor effect. (Fig. 3) Furthermore, the hydrophilic “tail” piperidine motif
exposing to the solvent has been investigated extensively in recent years [22-24], indicating that
modification on the “tail” (B) could be tolerant to maintain efficacies.

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Fig. 3. Design strategy for the target compounds.
Murray et al. [14] and Adolf et al. [25] reported that the 2-aminopyrido[2,3-d]pyrimidinone
moiety of palbociclib and 2-aminopteridinone moiety of BI6727 were combined within the CDK6
and PLK1 active site, respectively. And the side chain anilines moiety were combined within
solvent region. Meanwhile, compound H2, Y2, L2 contained similar structural feature with
palbociclib and BI6727 including hydrophilic aniline side chain and pteridinone or
pyrido[2,3-d]pyrimidinone fragments. So in order to better understand the anti-tumor mechanisms,
molecular docking models of H2, Y2, L2 were performed based upon the cocrystal structure of
CDK6 with palbociclib (PDB code: 5L2I) and PLK1 with BI6727 (PDB code: 3FC2). As shown
in Fig. 4, compound H2, Y2, L2 occupied the kinase domain in a similar manner to palbociclib and
BI6727. Combined with the results of docking analysis, CDK6 or PLK1 was very likely to be one
of the potential drug targets of these 7-amino-pteridinone derivatives.
Fig. 4. (A) The predicted binding models of H2 (blue sticks), Y2 (red sticks), L2 (green sticks) with
PLK1 (PDB code: 3FC2); (B) The binding models of H2 (blue sticks), Y2 (red sticks), L2 (green
sticks) with CDK6 (PDB code: 5L2I).
In this study, two series of novel 7-amino-[1,2,4]triazolo[4,3-f]pteridinone, and
7-aminotetrazolo[1,5-f]pteridinone derivatives were firstly designed and synthesized. All
compounds were subsequently assayed for anti-proliferative activities in vitro against five cancer
cell lines A549, PC-3, HCT116, MCF-7 and MDA-MB-231. Based on the anti-proliferative
results, potent compounds were selected for further in vitro enzymatic inhibitory studies. To
further clarify the primary mechanism, L7 was taken forward and examined by in vitro AO/EB
dyeing, the migration of A549 cells and cell cycle analysis.
2. Results and discussion
2.1 Chemistry
The general synthetic routes of the title compounds are illustrated in Scheme 1-3.
Commercially available 2,4-dichloro-5-nitropyrimidine (1) as the starting material reacted with
cyclopentylamine to give compound 2, which was reduced using iron powder and catalytic
amounts of concentrated HCl in EtOH/H2O to obtain amide intermediate 3. The intermediate 4
was prepared from 3 by treatment with ethyl oxalyl monochloride in acetone. Intermediate 4 was
converted to chloro-pteridinone 5 by refluxing in SOCl2 and catalytic amounts of DMF.
Intermediate 5 was converted to tetrazole product 8 using NaN3 as a cyclization reagent in DMF at
0 °C. Subsequently, the intermediate 6 was available via hydrazinolysis of intermediate 5 with 80%
hydrazine monohydrate in EtOH at 40 °C. Intermediate 6 was treated with trimethyl orthoformate
or triethyl orthoformate under 80 °C to produce triazole intermediates 7 and 9, respectively. Side
chain anilines (12) were prepared in two steps as shown in Scheme 2. Aromatic nucleophilic
substitution of fluorine in 4-fluoronitrobenzene by secondary amines were readily achieved in
DMF at 40 °C. The resulting nitrobenzenes were reduced to anilines using Pd/C in EtOH and

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generally used without further purification. Subsequently, amination of 7, 8, and 9 by
corresponding aryl amines (12) with p-toluenesulfonic acid as catalyst furnished target compounds
H1-H14, Y1-Y14 and L1-L14 (Scheme 3).
Scheme 1. General scheme for the synthesis of key intermediates 7, 8, and 9; Reagents and
o
conditions: (a) Cyclopentylamine, NaHCO3, DCM, 25 C, 10 h; (b) Fe powder, HCl (cat.),
EtOH/H2O, reflux, 2 h; (c) i: Ethyl oxalyl monochloride, K2CO3, acetone, 2 h; ii: TEA, EtOH, 100
oC, 4 h; (d) SOCl2, DMF (cat.), reflux, 2 h; (e) NH2NH2H2O, EtOH, 40 oC, 2 h; (f) HC(OEt)3, 80
oC, 2 h; (g) NaN3, DMF, 0 oC, 10 h; (h) CH3HC(OEt)3, 80 oC, 2 h.
Scheme 2. Preparation of the side chain anilines; Reagents and conditions: (a) Amines R1R2NH,
K2CO3, DMF, 40 °C, 6 h; (b) H2, 10 % Pd/C, EtOH, rt, 5 h.

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Scheme 3. General scheme for the synthesis of target compounds; Reagents and conditions: (a)
p-Toluenesulfonic acid, 1-butanol, 100 oC, 15 h.
Alternatively, the key Intermediate 5 existed two reaction sites in the pteridinone structure
(2-chlorine or 6-chlorine), which both could react with NaN3. So the tetrazole products 8 or 8a,
and the substitute products 8b or 8c could be obtained in theory (Fig. 5). In this sense, literature
precedent illustrating the possibility of using this behavior for the generation of tetrazole products
in which the methods of structure verification are rare. [26] Our efforts represent the first studies
on the structure verification by X-ray single crystal. To our delight, the single product was finally
achieved, and the X-ray single crystal diffraction analysis of this product (Fig. 6) permitted the
assignment of its absolute configuration as 6-cyclization product 8 (CCDC: 1848418). Meanwhile,
in the process of triazole product, the products 7 or 7a could be obtained in theory. But the same
result was achieved and the structure was confirmed by the X-ray single crystal diffraction as
6-cyclization product 7 (CCDC: 1848417). These results indicated that cyclization in 6-position
of pteridinone was more reactive than the 2-position.

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Fig. 5. The analysis of the cyclization products (tetrazole products 8, 8a, 8b, 8c and triazole
products 7, 7a) of intermediate 5 in theory.

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Fig. 6. X-ray crystal structure of compounds 7 and 8.
Later, we evaluated the role of NH proton at the C-7 position of L3 in cytotoxicity assays by
acylating L3 with acetic anhydride to obtain L15 (Scheme 4).
N
N
N
N
N
N
N
N
N
O
N
a
HN
N
O
N
N
O
N
N
N
N
L15
L3
Scheme 4. Acylation of NH group of L3; Reagents and conditions: (a) (Ac)2O, 100 oC, 4h.
2.2 Bioactivity and discussion
2.2.1 In vitro antiproliferative activity and SARs study
To evaluate in vitro antitumor activities, all synthesized compounds (H1-H14, Y1-Y14, and
L1-L15) were investigated against a panel of cancer cell lines, including A549 (human lung
adenocarcinoma), HCT116 (human colorectal cancer), PC-3 (human prostate cancer), MCF-7

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(human breast cancer), MDA-MB-231 (human breast cancer) cells by the MTT assay. Meanwhile,
palbociclib and BI6727 were served as positive controls. The results were expressed as
half-maximal inhibitory concentration (IC50) values and summarized in Table 1, Table 2, and
Table 3. As illustrated in Table 1, most of 7-amino-[1,2,4]triazolo[4,3-f]pteridinone derivatives
H1-H14 showed moderate to significant cytotoxic activities against the different cancer cell lines.
Several of these compounds were more potent than palbociclib against one or more cancer cell
lines, which suggested that the combination of 2-aminopteridinone framework and triazolo moiety
exhibit potent synergistic antitumor effect. Preliminary SARs indicated that the introduction of
different amino groups at the C-7 position of 7-amino-[1,2,4]triazolo[4,3-f]pteridinone moiety had
a significant influence on activity, which suggested that the hydrophilic B group contributed much
to their potency. Compounds bearing piperazine moiety (H2, H3, H7, and H9) displayed excellent
anti-tumor activities in the sigle-digit micromolar range against A549 and HCT116 cells. Notably,
the better promising compound H7 displayed stronger potency than palbociclib in A549, HCT116
and MCF-7 cells with IC50 values of 0.89 µM, 4.40 µM and 0.41 µM, respectively. However,
when the piperazine was substituted by cyclopentyl (H13), the cytotoxic activities decreased
significantly. Replacement of the nitrogen atom of piperazine group in H7 by oxygen, carbon or
sulfur atom (H1, H4 and H8) led to the diminished potency, indicating that the presence of cationic
nitrogen atom was a critical factor in anti-proliferative activities. In order to improve the polarity
of piperidine ring, hydrophilic hydroxyl group or N-methylpiperazinyl was embeded on the
piperidine ring. The results indicated that the hydrophilic N-methylpiperazinyl (H14) had more
positive impact on the activities than hydroxyl group (H10). Generally, five-membered pyrrolidine
analogs were less active than corresponding six-membered inhibitors (H6 vs. H4) despite
possessing equivalently basic nitrogen atoms, highlighting the importance of ring size. Besides, an
increase in potency was observed once the pyrrolidine group in H6 was "opened" to
dimethylamine
(H12).
Overall,
SARs
studies
identified
the
7-amino-[1,2,4]triazolo[4,3-f]pteridinone moiety bearing piperazine "tails" moieties harboring
cationic nitrogen atoms with favorable potency.
Further studies were performed to examine the effect of shifting the 1,2,3-triazole motif of
H1-H14 series compounds to the tetrazolo series compounds (Y1-Y14). The results displayed that
most of the tetrazolo series compounds had lower cytotoxicity than 1,2,3-triazole series.
Interestingly, Y7 was maintained approximately cytotoxicity in A549, HCT116, PC-3, MCF-7 and
MDA-MB-231cells with IC50 values of 0.44 µM, 0.60 µM, 1.00 µM, 0.50 µM, and 1.60 µM,
respectively.
The methyl group acts as an essential factor in the molecular recognition of endogenous and
exogenous substrates by means of bioreceptors. Although it only participates in London dispersion
interactions, methyl group has stereoelectronic effects on micromolecules and biomacromolecules.
Accordingly, the methyl group can lead to multiple biological effects, such as, selectivity among
bioreceptors, increased the binding affinity, and protection against enzyme metabolism.[27]
Further investigations were performed to study the introduction of a methyl group to the 1 position
of 7-amino-[1,2,4]triazolo[4,3-f]pteridinone core on the cytotoxic activity. Piperazine "tails"
moieties on the 7 positon of 7-amino-1-methyl-[1,2,4]triazolo[4,3-f]pteridinone core to a proper
degree was a key factor in improving inhibitory activity (L7 vs. H7, L3 vs. H3) suggesting that the
methyl group was a favorable motif to potency in these cell lines.
Obviously, all target compounds possessed selectivity for A549 and HCT116 cancer cell lines,

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and had the makings of good drugs for lung and colorectal cancer. Moreover, the examination of
SARs indicated that these analogs had similar SARs as summarized before.
In conclusion, the most potent compound L7 showed promising cytotoxicity against A549, PC-3,
HCT116, MCF-7 and MDA-MB-231cell lines with IC50 values of 0.16 µM, 0.30 µM, 0.51 µM,
0.30 µM, and 0.70 µM, respectively. The potency was much higher than palbociclib by 8.2-, 29.6-,
4.4-, 13.6-, and 5.0-fold (IC50 = 1.32 µM against A549, IC50 = 8.90 µM against HCT116, IC50
=
2.20 µM against PC-3, IC50 = 4.09 µM against MCF-7, IC50 = 3.50 µM against MDA-MB-231),
separately. However, the potency was slightly weaker than BI6727. These encouraging results
provided a valuable lead compound L7 and highlighted the potential for further development of
novel pteridinone derivatives as potent antitumor agents.
Table 1. Structures and cytotoxicity of compounds (H1-H14).
Compd.
-NR1R2
IC50 a (µM) ± SD b
A549
HCT116
PC-3
>50
MCF-7
>50
MDA-MB-231
3.50±0.04
>50
38.86±0.01 8.91±0.08
H1
6.96±0.03
1.07±0.02
3.11±0.03
3.31±0.04
5.63±0.01
0.89±0.06
2.12±0.09
9.80±0.03
8.00±0.2
26.51±0.03 >50
H2
7.50±0.03
20.10±0.2
12.00±0.2
3.48±0.02
>50
0.98±0.05 >50
H3
>50
>50
3.85±0.08
4.90±0.07
>50
H4
>50
H5
49.33±0.03 11.27±0.05 >50
H6
H7
4.40±0.07
12.00±0.1
9.71±0.08
3.26±0.01
0.41±0.03 4.80±0.02
>50 6.40±0.09
>50
H8
19.12±0.02 1.91±0.03 >50
H9
41.68±0.02 36.12±0.04 >50
>50
>50
8.10±0.02
>50
H10
H11
H12
H13
H14
Palbociclib
BI6727
>50
21.22±0.02 >50
19.13±0.03 >50
22.34±0.01 >50
3.89±0.07
>50
7.28±0.02 >50
>50 3.96±0.04
13.40±0.03 >50 >50
2.95±0.01
1.32±0.02
0.08±0.01
9.30±0.02
8.90±0.05
0.11±0.02
2.20±0.02
0.09±0.01
4.09±0.05 3.50±0.03
0.09±0.02 0.11±0.03
a Values are the means of at least three independent experiments. b SD: standard deviation.
Table 2. Structures and cytotoxicity of compounds (Y1-Y14).

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Compd.
-NR1R2
IC50 a (µM) ± SD b
A549
HCT116
PC-3
MCF-7
>50
MDA-MB-231
48.45±0.09
15.81±0.08
>50
4.81±0.02
1.25±0.04
3.57±0.03
1.16±0.02
4.92±0.08
7.86±0.03
0.44±0.01
>50
43.23±0.02 >50
Y1
19.11±0.04 3.80±0.09
>50
Y2
>50
16.12±0.06 >50
Y3
>50
>50
>50
>50
>50
>50
Y4
>50
>50
>50
Y5
>50
>50
>50
Y6
Y7
0.60±0.01
>50
1.00±0.03
>50
0.50±0.03 1.60±0.04
>50 37.35±0.08
2.31±0.02 >50
Y8
3.85±0.04
>50
>50
>50
Y9
36.34±0.09 >50
43.22±0.08 >50
>50
>50
53.45±0.09
8.95±0.03
Y10
Y11
>50
10.06±0.09 >50
>50
2.58±0.05 >50
Y12
Y13
Y14
Palbociclib
BI6727
>50
2.12±0.04
>50
>50
>50
>50
1.28±0.01
1.32±0.02
0.08±0.01
7.62±0.03
8.90±0.05
0.11±0.02
>50
5.81±0.03
2.20±0.02
0.09±0.01
4.09±0.05 3.50±0.03
0.09±0.02 0.11±0.03
a Values are the means of at least three independent experiments. b SD: standard deviation.
Table 3. Structures and cytotoxicity of compounds (L1-L14).
Compd.
-NR1R2
IC50 a (µM) ± SD b
A549
HCT116
8.91±0.02
PC-3
>50
MCF-7
>50
MDA-MB-231
>50
4.29±0.02
1.92±0.05
0.68±0.03
5.24±0.07
>50
L1
L2
L3
L4
L5
L6
72.82±0.05 7.61±0.08 1.05±0.02 0.61±0.03
9.61±0.03 5.00±0.05 0.51±0.02 2.25±0.01
19.11±0.07 >50
11.23±0.08 >50
>50
>50
>50
6.26±0.05
>50
>50
>50
>50
>50

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L7
0.16±0.02
7.19±0.08
5.46±0.03
0.30±0.03
5.82±0.01
7.61±0.05
0.51±0.02 0.30±0.01 0.70±0.02
>50 >50 >50
8.44±0.01 0.75±0.01 3.35±0.04
L8
L9
21.69±0.09 18.12±0.03 >50
>50
>50
>50
>50
>50
L10
9.29±0.06
8.86±0.04
>50
29.11±0.02 >50
>50
12.12±0.03 >50
>50
>50
L11
>50
L12
11.36±0.04 >50
9.12±0.07
L13
2.38±0.05
1.32±0.02
0.08±0.01
3.21±0.03 5.85±0.09
L14
Palbociclib
8.90±0.05
2.20±0.02 4.09±0.05 3.50±0.03
BI6727
0.11±0.02
0.09±0.01 0.09±0.02 0.11±0.03
a Values are the means of at least three independent experiments. b SD: standard deviation.
Table 4. In vitro cytotoxicity of L3 and L15 Compounds
Compd.
R3
IC50 a (µM) ± SD b
A549
HCT116
PC-3
5.00±0.05 0.51±0.02 2.25±0.01
20.25±0.09 40.32±0.08 >50 >50 >50
MCF-7
MDA-MB-231
H
0.68±0.03
9.61±0.03
L3
CH3CO-
L15
a Values are the means of at least three independent experiments. b SD: standard deviation.
According to the literatures [14], the NH of the 7 positon at 7-aminopteridinone core acted as a
key hydrogen bond donor to interact with the corresponding amino acids in the kinase hinge
region. As hypothesized, amine-substituted anilines (L15) positioned at C7 of the
7-amino-1-methyl-[1,2,4]triazolo[4,3-f]pteridinone core led to an inactive compound. This was
not surprising for this bridging NH may act as a key hydrogen bond donor.
2.2.2 In vitro enzymatic assays

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Fig. 7. Enzymatic activities of the target compounds.
Based on the cellular assays and molecular docking results, potent compounds were selected for
further in vitro CDK6, and PLK1 % inhibition at 1 µM. The results were summarized in Fig. 7. As
shown in Fig. 7, all compounds poorly inhibited CDK6 kinases with % inhibition values ranging
from 8.9% to 17.6%. However, most of compounds displayed more highly inhibition against
PLK1 than CDK6 kinases with % inhibition values ranging from 19.3% to 86.4%. In parallel with
cellular results, inhibitors bearing the 1-methyl-1,2,4-triazole at A ring of pteridinone core were
found to more potent than corresponding 1,2,4-triazole or tetrazolo analogues, which further
proved 7-amino-1-methyl-triazolo[4,3-f]pteridin-4(5H)-one a favorable building block to increase
PLK1 potency. Even though it showed more selectivity against PLK1, the inhibitory activity of
compound L7 was still less than BI6727. These results indicated that two series of novel
compounds worth further studying as new potential anticancer agent for the treatment of human
cancers.
2.2.3 Cell apoptosis study
To investigate the molecular mechanisms of action preliminarily, cell apoptosis analysis of
A549 cells treated with the optimal compounds Y7 and L7 was performed using a biparametric
acridine orange (AO) and ethidium bromide (EB) staining. EB was able to penetrate through intact
membranes of live cells and colors DNA as green fluorescence, while AO was only taken up by
apoptotic cells with damaged membranes coloring DNA as orange fluorescence. Therefore,
normal live cells appeared uniformly stained green in color. Early apoptotic cells contained bright
green condensed bodies in their nuclei representing nuclear DNA fragmentation and later
apoptotic cells presented colored orange nuclei indicating that their membranes are broken.
As shown in Fig. 8, orange-red stained cell nucleus appeared after treatment with 1.0 µM
compound Y7 and L7 for 24 h, indicating the late stage apoptosis of A549 cells. Treatment with 5.0
µM compound Y7 and L7 led to a significant increase in the percentage of apoptotic cells, marked
by concentrated orange-red nucleus stained with EB. In addition, these results demonstrated that
compound L7 was more potent than Y7 in inducing cell apoptosis, indicating that compound L7
deserves further investigation.

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Fig. 8. Effect of compound Y7 and L7 on cell apoptosis in A549 cells.
2.2.4 Wound-healing assay.
As migration is an important characteristic for metastatic cancers, the effect of compound L7 on
migration of cancer cells was investigated by the wound healing assay. As shown in Fig. 9 and 10,
treatment of A549 cells with compound L7 at indicated concentrations markedly suppressed the
wound healing in a concentration-dependent manner.

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Fig. 9. In vitro wound healing assay on A549 cells. Phase contrast images were obtained by the
treatment of compounds L7 at indicated concentrations for 0, 12, 24 and 36 h.

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Fig. 10. In vitro wound healing on A549 cells of L7. Columnar graph represents the transferred
percentage distribution at different concentrations and at different time.
2.2.5 Cell cycle analysis
The cell cycle analysis was performed to investigate the prevention of proliferation in A549
cells with the most potent compound L7. After treatment of A549 cells with compound L7 for 24 h
at indicated concentrations (0.2, 1.0, 5.0 µM), the cells were fixed and stained with PI, the DNA
content was analyzed by flow cytometry. The obtained results were compared with non-treated
A549 cells, as control. As shown in Figs. 11 and 12, treatment of A549 cells with L7 at 0.2, 1.0,
and 5.0 µM concentrations increased the percentage of G1-phase cells from 63.34% (as control
group) to 74.99%, 79.82%, and 82.32%, respectively. These results confirmed that compound L7
significantly caused G1-phase arrest in A549 cells.
Fig. 11. Effect of compound L7 on the cell cycle distribution of A549 cells.

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Fig. 12. Quantitative analysis of cell cycle distributions; (A) Non-treated cells as control group; (B)
treated with L7 at 0.2 µM; (C) treated with L7 at 1.0 µM; (D) treated with L7 at 5.0 µM.
2.2.6 Molecular docking studies

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Fig. 13. The binding models of L7 or L15 with PLK1 and CDK6. (A) Predicted binding
conformation for L7 (blue sticks) in the binding site cavity of PLK1 (PDB code: 3FC2), (B) 2D
diagram of the interaction between L7 and the binding site cavity of PLK1, (C) L7 overlapping
with BI6727 (red sticks), (D) 2D diagram of the interaction between L7 (pink sticks) and the
binding site cavity of CDK6 (PDB code: 5L2I), (E) L7 overlapping with palbociclib, (F) 2D
diagram of the interaction between palbociclib (green sticks) and the binding site cavity of CDK6,
(G) 2D diagram of the interaction between L15 (dark green sticks) and the binding site cavity of
PLK1, and (H) 2D diagram of the interaction between L15 and the binding site cavity of CDK6.
In order to better understand the binding mode, molecular docking models of L7 or L15 were
performed based upon the cocrystal structure of PLK1 with BI6727 (PDB code: 3FC2) and CDK6
with palbociclib (PDB code: 5L2I), respectively.
As shown in Fig. 13, L7 occupied the kinase domain in a similar manner to BI6727. In contrast
to two hydrogen bonds observed in the cocrystal structure of BI6727 with PLK1 (Fig. 13A) at the
hinge area, L7 was found to form three hydrogen bonds via the 8-position nitrogen atom, the
7-position
NH
and
the
4-position
oxygen
atom
of
carbonyl
of
the
7-amino-1-methyl-[1,2,4]triazolo[4,3-f]pteridinone nuclei with Cys 133 and Cys 67, respectively
(Fig. 13A). In the meantime, the π-Sigma interaction between triazolo with Leu 130 and the π-π
stacked interaction between [1,2,4]triazolo[4,3-f]pteridinone with Phe 183 was also present.
Inspiringly, the hydrophilic "tail", 2,6-dimethylpiperazine moiety, was engaged in π-akyl
interactions and carbon hydrogen bonds with Leu 59, validating the rationality of our design and
the importance of the basic nitrogen atoms (Fig. 13B). Besides, its 1-position methyl group on the
triazolo moiety filled with active chamber which would be beneficial to increase the binding
affinity. All these interactions played an important role in stabilizing the conformation of
ligand-protein complex. Docking structure of compound L7 overlayed with BI6727 in PLK1
showed that the conformation and binding mode of compound L7 were well consistent with
BI6727 (Fig. 13C). Although L7 occupied the kinase domain in a similar manner to palbociclib
(Fig. 13E), palbociclib was found to form three hydrogen bonds via the 3-position nitrogen atom,
the 2-position NH and the 6-position oxygen atom of acetyl of the
pyridin-2-yl)amino)pyrido[2,3-d]pyrimidin-7(8H)-one nuclei with Val 101 and Asp 163,
respectively (Fig. 13F), compared to L7 was found to form only two hydrogen bonds with Val 101
(Fig. 13D).
On the other hand, the docking results of compound L15 suggested that the acetylated 7 position
N atom could not form hydrogen bond with Cys 133 of PLK1 protein, and with Val 101 of CDK6
protein. Meanwhile, the introduction of acetyl group resulted the forming of unfavorable bump.
Therefore, the NH of the 7 positon at 7-aminopteridinone core played an important role. This
result was also consisted with the in vitro antiproliferative activity, which the acetylated product
L15
displayed
lower
potency
than
compound
L3.
Therefore,
the
7-amino-1-methyl-[1,2,4]triazolo[4,3-f]pteridinone moiety could serve as a scaffold from which to
build a novel series of PLK1 inhibitors.
2.3 Physicochemical and ADME parameters
Furthermore, some physicochemical and ADME properties of the synthesized compounds and
positive controls were predicted using the SwissADME (a free web tool to evaluate
pharmacokinetics, drug likeness and medicinal chemistry friendliness of small molecules) for their
adaptability with Lipinski's rule of five [28-32]. Compounds obeying at least three of the four
criteria are considered to adhere to Lipinski Rule.
As demonstrated in Table 5, the most active compounds shown variable permeability based on

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gastrointestinal absorption (GI), according to the BOILED-Egg predictive model (Brain Or
IntestinaL EstimateD permeation method). All predicted compounds showed high gastrointestinal
absorption. With respect to oral bioavailability, it's expected 0.55 of probability of oral
bioavailability score >10% in the rat for all compounds, greater than BI6727 (0.17). Compound L7
exhibited potent in vitro antitumor activity, low toxicity and reasonable physicochemical
properties, because suitable flexible and size in the bioavailability radar map (Fig. 14). All these
data suggests that compound L7 could be considered as a promising candidate for further
development.
Table 5. Physicochemical properties and ADME properties of most active compounds.
MW
(g/mol)
<500
H-bond
acceptors
<10
H-bond
donors
<5
Log P
o/w
<5
Violation
Lipinski
Rule of 5
aPSA
(Ų)
<140
Rotatable
bonds
<10
bBBB
cGI
dBS
Compd.
459.55
460.53
473.57
459.55
460.53
473.57
489.57
542.68
447.53
618.81
6
7
6
6
7
6
7
7
6
7
2
2
2
1
1
1
2
1
2
2
2.06
2.11
2.40
2.10
2.16
2.46
1.78
2.47
2.24
3.59
0
1
0
0
1
0
1
2
0
2
105.27
118.16
105.27
96.48
4
4
NO
NO
NO
NO
NO
NO
NO
NO
NO
NO
High 0.55
High 0.55
High 0.55
High 0.55
High 0.55
High 0.55
High 0.55
High 0.17
High 0.55
H7
Y7
4
L7
5
H3
109.37
96.48
5
Y3
5
L3
116.71
99.72
6
L9
5
L14
Palbociclib
105.04
5
BI6727
106.17
11
High 0.17
b
c
d
aPSA - Polar surface area. BBB – blood-brain barrier. GI – Gastrointestinal absorption. BS –
Bioavailability Score.
Fig. 14. The bioavailability radar enables a first glance at the drug-likeness of a molecule (A: L7,
B: Palbociclib, C: BI6727). The pink area represents the optimal range for each properties
(lipophilicity: XLOGP3 between -0.7 and +5.0, size: MW between 150 and 500 g/mol, polarity:
TPSA between 20 and 130 Å2, solubility: log S not higher than 6, saturation: fraction of carbons in
the sp3 hybridization not less than 0.25, and flexibility: no more than 9 rotatable bonds.
3. Conclusion
In current investigation, two series of novel 7-amino-[1,2,4]triazolo[4,3-f]pteridinone, and
7-aminotetrazolo[1,5-f]pteridinone derivatives with hydrophilic "tails" (B) bearing aliphatic amine
groups, were firstly designed and synthesized. Exploration of SARs culminated in L7, which
contained a terminal 2,6-dimethylpiperazine at B and 1-methyl-1,2,4-triazole at A ring of
pteridinone core, showed the most potent antiproliferative activity against A549, PC-3, HCT116,
MCF-7 and MDA-MB-231cell lines with IC50 values of 0.16 µM,0.30 µM, 0.51 µM, 0.30 µM, and

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0.70 µM, respectively. Combined with the results of the molecular docking and enzymatic studies,
the PLK1 was very likely to be one of the drug targets of compound L7. Furthermore, to clarify
the mechanism of the anticancer activity of the pteridinone molecule, the AO/EB and
wound-healing assays confirmed that L7 induced cell apoptosis. Finally, cell cycle analysis of L7
by flow cytometry showed cell cycle arrest in G1 phase, DNA fragmentation and alteration in
mitochondrial membrane potential by compound L7 for the three concentrations tested. In
summary, the SARs studies together with the pharmacological assays on novel pteridin-4(5H)-one
analogues identified L7 as a promising anti-cancer agent, which will lead to the promising
development of new drugs.
4. Experimental procedures
4.1 Chemistry
All melting points were acquired on a Mettler Melting Point MP70 apparatus (Mettler, Toledo,
Switzerland) and uncorrected. Mass spectra (MS) were taken in ESI mode on Agilent 1100
LC-MS (Agilent, palo Alto, CA, USA). Reactions were monitored by thin-layer
chromatography (TLC) on silica plates (F-254) and visualized under UV light.1H NMR and
13C NMR spectra were performed using Bruker spectrometers (Bruker Bioscience, respectively,
Billerica, MA, USA) with TMS as an internal standard. Column chromatography was run on silica
gel (200-300 mesh) from Qingdao Ocean Chemicals (Qingdao, Shandong, China). X-Ray
diffraction studies where carried on an Bruker SMART Apex-IICCD-based X-ray diffractometer.
Unless otherwise noted, all materials were obtained from commercially available sources and used
without further purification.
4.1.1 General procedure for preparation of compounds (2-9)
4.1.1.1 2-chloro-N-cyclopentyl-5-nitropyrimidin-4-amine (2)
2,4-Dichloro-5-nitropyrimidine (50.0 g, 259 mmol) and NaHCO3 (43.5 g, 518 mmol) were
dissolved in DCM (200 mL) and cooled to 0 °C. Cyclopentylamine (26.4 g, 311 mmol) was
dissolved in DCM (50 mL) and added dropwise. After the completion of the dropwise addition,
the cooling bath was removed, and the reaction mixture was stirred at room temperature for about
10 h. Then organic was washed by water, and brine. The organic phase was dried over Na2SO4,
filtered, and the solvent evaporated to obtain 53.27 g of 2 as a yellow solid, m.p.:125.2 – 126.6 °C.
Yield: 85%; MS (ESI) m/z: 243.3 [M+H]+.
4.1.1.2 2-chloro-N4-cyclopentylpyrimidine-4,5-diamine (3)
To a suspension of compound 2 (50.0 g, 207 mmol) in 200 mL of 95% ethanol was added HCl (1
mL), and Fe Powder (57.8 g, 1035 mmol) in batches. The mixture was heated for 2 h at reflux.
After completion of the reaction as indicated by TLC, the mixture was filtered through a celite bed
and concentrated in a vacuum to afford pure product. The residue was purified by silica gel
column chromatographyto obtain 32.1 g of 3 as a white solid, m.p.: 128.2 – 130.0 °C. Yield:
73.3%; MS (ESI) m/z: 213.2 [M+H]+.
4.1.1.3 2-chloro-8-cyclopentyl-5,8-dihydropteridine-6,7-dione (4)
To a stirred solution of compound 3 (30.0 g, 124 mmol) and K2CO3 (34.2 g, 248 mmol) in acetone
(150 mL) was dropped ethyl oxalyl monochloride (28.9 g, 136 mmol). The mixture was stirred at
rt for 2 h. The reaction mixture was filtered and the filtrate was concentrated under reduced
pressure. The crude ketoester was dissolved in absolute EtOH (200 mL), placed in a pressure flask,
and TEA (15.1 g, 149 mmol) was added. The mixture was heated for 4 h at 100 °C. After
completion of the reaction as indicated by TLC, the mixture was filtered to obtain 27.4 g of 4 as a

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white solid, m.p.: 210.1 – 212.2 °C. Yield: 83.6%; MS (ESI) m/z: 264.9 [M-H]-.
4.1.1.4 2,6-dichloro-8-cyclopentylpteridin-7(8H)-one (5)
To a solution of compound 4 (25.0 g, 94 mmol) in SOCl2 (80 mL) at refulx was dropped DMF (1
mL), and the resulting mixture was kept at this temperature for 2 h. The solvent was concentrated
in vacuum and the residue was poured into stirring ice-water (200 mL), the resulting precipitate
was filtered and dried to obtain 24.3 g of 5 as a white solid, m.p.: 269.0 – 279.3 °C. Yield: 91.2%;
MS (ESI) m/z: 285.26 [M+H]+.
4.1.1.5 2-chloro-8-cyclopentyl-6-hydrazinylpteridin-7(8H)-one (6)
A mixture of 5 (18.0 g, 63.4 mmol) and 80% hydrazine monohydrate (9.5 g, 190 mmol) in EtOH
(200 mL) was stirred at 40 °C for 2h. After completion of the reaction as indicated by TLC, most
of the solvent was evaporated under reduced pressure when white solid appeared. The resulting
precipitate was filtered off, washed with water, and dried under vacuum to afford 15.6 g of 6 as a
white solid, m.p.: 210.7 – 212.1 °C. Yield: 88.4%; MS (ESI) m/z: 279.0 [M-H]-.
4.1.1.6 7-chloro-5-cyclopentyl-[1,2,4]triazolo[4,3-f]pteridin-4(5H)-one (7)
To a stirred solution of compound 6 (6.0 g, 21.4 mmol) in trimethyl orthoformate (20 mL). The
reaction mixture was stirred at 80 °C for 2h, cooled to room temperature. The resulting precipitate
was filtered off, washed with diethyl ether, and dried to afford 5.3 g of 7 as a white solid, m.p.:
251.8 – 253.1 °C. Yield: 86.1%; MS (ESI) m/z: 313.1 [M+Na]+. 1H NMR (400 MHz, DMSO-d6) δ
9.90 (s, 1H), 9.49 (s, 1H), 5.76 – 5.60 (m, 1H), 2.21-2.13 (m, 2H), 2.09 – 1.98 (m, 2H), 1.98 –
1.83 (m, 2H), 1.67-1.64 (m, 2H).
4.1.1.7 7-chloro-5-cyclopentyltetrazolo[1,5-f]pteridin-4(5H)-one (8)
NaN3 (1.14 g, 17.6 mmol) was added to suspension of 5 (5.0 g, 17.6 mmol) and DMF (20 mL).
The mixture was stirred at 0 °C for 10 h. After completion of the reaction as indicated by TLC, the
reaction mixture was cooled to room temperature. Water was then added, and the solid was filtered
and dried under reduced pressure to afford 4.2 g of 8 as a white solid, m.p.: 185.4 – 186.8 °C.
Yield: 80.2%; MS (ESI) m/z: 292.3 [M+H]+. 1H NMR (400 MHz, DMSO-d6) δ 9.71 (s, 1H), 5.78
– 5.70 (m, 1H), 2.21 – 2.13 (m, 2H), 2.08 – 2.02 (m, 2H), 2.00 – 1.88 (m, 2H), 1.73 – 1.62 (m,
2H).
4.1.1.8 7-chloro-5-cyclopentyl-1-methyl-[1,2,4]triazolo[4,3-f]pteridin-4(5H)-one (9)
To a stirred solution of compound 6 (6.0 g, 21.4 mmol) in triethyl orthoformate (20 mL). The
reaction mixture was stirred at 80 °C for 2h, cooled to room temperature. The resulting precipitate
was filtered off, washed with diethyl ether, and dried to afford 5.2 g of 9 as a white solid, m.p.:
255.3 – 257.1 °C. Yield: 79.1%; MS (ESI) m/z: 327.2 [M+Na]+.
4.1.2 General Procedure for Preparation of (11)
Substituted amine (1.2 equiv) was added to a mixture of 4-fluoronitrobenzene (1 equiv) and
K2CO3 (2.0 equiv) in DMF (7 mL/g). The reaction mixture was stirred at 40 °C and followed by
TLC. After completion of the reaction, the mixture was poured into stirring ice-water. The
resulting precipitate was filtered and dried to obtain compounds 11 as a yellow solid.
4.1.3 General Procedure for Preparation of (12)
The substituted nitro compound 11 (1 equiv in a mixture of EtOH-H2O, 95:5, 20 mL) was treated
with 10% Pd-carbon (5% w/w). The reaction was subjected to hydrogenation under hydrogen gas
at room temperature and the reaction was monitored by TLC. After completion of the reaction, the
mixture was filtered through a Celite bed and concentrated in a vacuum to afford product 12.
4.1.4 General procedure for preparation of compounds (H1-H14, Y1-Y14, L1-L14)

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To a stirred solution of compound 7, 8, or 9 (1 equiv) in 1-butanol was added compounds 12(1.1
equiv) and p-toluenesulfonic acid (1 equiv). The mixture was placed in a pressure flask, and
heated to 100 °C for 15 h. The reaction mixture was quenched by saturated Na2CO3 aqueous
solution, and then was extracted with DCM and the organic phase was washed with water, dried
over anhydrous Na2SO4. The combined organic layer was concentrated under reduced pressure
and was further purified by flash column chromatography using dichloromethane/methanol as
eluent to afford product H1-H14, Y1-Y14, or L1-L14 as a pale yellow solid.
5-cyclopentyl-7-((4-morpholinophenyl)amino)-[1,2,4]triazolo[4,3-f]pteridin-4(5H)-one (H1)
1
Yield: 51.6%; m.p.: 315.3
–
316.5 °C; MS (ESI) m/z: 455.4 [M+Na]+; H NMR (400 MHz,
DMSO-d6) δ 9.73 (s, 2H), 9.18 (s, 1H), 7.51 (d, J = 8.9 Hz, 2H), 6.94 (d, J = 9.0 Hz, 2H), 5.78 –
5.70 (m, 1H), 3.75 – 3.73 (m, 4H), 3.07 – 3.05 (m, 4H), 2.29 2.21 (m, 2H), 1.97 1.89 (m, 2H),
1.87 1.79 (m, 2H), 1.64 – 1.56 (m, 2H); Anal. Calcd for C22H24N8O2: C, 61.10; H, 5.59; N, 25.91;
Found: C, 61.07; H, 5.51; N, 25.90.
–
–
–
5-cyclopentyl-7-((4-(4-methylpiperazin-1-yl)phenyl)amino)-[1,2,4]triazolo[4,3-f]pteridin-4(5H)-o
ne (H2)
Yield: 62.0%; m.p.: 304.5
DMSO-d6) δ 9.75 – 9.73 (m, 2H), 9.19 (s, 1H), 7.49 (d, J = 6.3 Hz, 2H), 6.93 (d, J = 7.8 Hz, 2H), 5.74
(s, 1H), 3.09 (s, 4H), 2.45 (s, 4H), 2.25 (s, 2H), 2.22 (s, 3H), 1.93 (s, 2H), 1.83 (s, 2H), 1.60 (s, 2H)
13C
–
305.7 °C; MS (ESI) m/z: 446.6 [M+H]+; 1H NMR (600 MHz,
;
NMR (101 MHz, DMSO-d6) δ 158.14, 153.79, 153.38, 149.23, 147.49, 142.52, 139.88, 137.87, 131.88,
121.74, 116.18, 55.14, 53.76, 49.15, 46.27, 40.64, 40.43, 40.22, 40.02, 39.81, 39.60, 39.39, 28.15,
25.58; Anal. Calcd for C23H27N9O: C, 62.01; H, 6.11; N, 28.30; Found: C, 61.98; H, 6.04; N,
28.28.
5-cyclopentyl-7-((4-(4-ethylpiperazin-1-yl)phenyl)amino)-[1,2,4]triazolo[4,3-f]pteridin-4(5H)-one
(H3)
1
Yield: 59.8%; m.p.: 318.7
–
320.0 °C; MS (ESI) m/z: 460.6 [M+H]+; H NMR (600 MHz,
DMSO-d6) δ 9.73 (s, 2H), 9.17 (s, 1H), 7.48 (d, J = 8.2 Hz, 2H), 6.93 (d, J = 8.6 Hz, 2H), 5.74 (s,
1H), 3.32 (s, 3H), 3.09 (s, 4H), 2.37 2.35 (m, 2H), 2.29 2.20 (s, 2H), 1.93 (s, 2H), 1.84 1.79
(m, 2H), 1.64
1.56 (m, 2H), 1.03 (t, J = 7.1 Hz, 3H); 13C NMR (101 MHz, DMSO-d6) δ 158.15,
–
–
–
–
153.38, 149.24, 147.55, 142.52, 137.84, 131.84, 121.79, 116.15, 108.64, 53.78, 52.86, 52.11, 49.28,
40.64, 40.43, 40.22, 40.01, 39.80, 39.59, 39.39, 28.14, 25.57, 12.48; Anal. Calcd for C24H29N9O: C,
62.73; H, 6.36; N, 27.43; Found: C, 62.70; H, 6.31; N, 27.41.
5-cyclopentyl-7-((4-(piperidin-1-yl)phenyl)amino)-[1,2,4]triazolo[4,3-f]pteridin-4(5H)-one (H4)
1
Yield: 48.3%; m.p.: 291.9
–
293.0 °C; MS (ESI) m/z: 429.6 [M-H]-; H NMR (600 MHz,
DMSO-d6) δ 9.53 (s, 1H), 8.94 (s, 1H), 7.41 (d, J = 8.2 Hz, 2H), 6.53 (d, J = 8.8 Hz, 2H), 5.75 (s,
1H), 3.21 (t, J = 6.3 Hz, 4H), 2.91 (s, 2H), 2.25 2.20 (m, 2H), 1.95 1.91 (m, 4H), 1.89 – 1.88
(m, 2H), 1.85 – 1.78 (m, 2H), 1.61 1.55 (s, 2H); Anal. Calcd for C23H26N8O: C, 64.17; H, 6.09;
–
–
–
N, 26.03; Found: C, 64.13; H, 6.02; N, 26.01.
5-cyclopentyl-7-((4-(4-methylpiperidin-1-yl)phenyl)amino)-[1,2,4]triazolo[4,3-f]pteridin-4(5H)-o
ne (H5)
1
Yield: 55.6%; m.p.: 329.8
–
331.3 °C; MS (ESI) m/z: 445.5 [M+H]+; H NMR (600 MHz,
DMSO-d6) δ 9.72 (s, 1H), 9.69 (s, 1H), 9.17 (s, 1H), 7.46 (d, J = 8.7 Hz, 2H), 6.92 (d, J = 9.0 Hz,
2H), 5.74 (s, 1H), 3.59 (d, J = 12.3 Hz, 2H), 2.67 – 2.55 (m, 2H), 2.28 – 2.22 (m, 2H), 1.97 1.87
(s, 1H), 1.85 – 1.80 (m, 2H), 1.70 1.68 (m, 2H), 1.61 1.56 (m, 2H), 1.50 – 1.46 (m, 2H), 1.27
1.21 (m, 2H), 0.94 (d, J = 6.5 Hz, 3H); Anal. Calcd for C24H28N8O: C, 64.85; H, 6.35; N, 25.21;
–
–
–
–

ACCEPTED MANUSCRIPT
Found: C, 64.79; H, 6.34; N, 25.19.
5-cyclopentyl-7-((4-(pyrrolidin-1-yl)phenyl)amino)-[1,2,4]triazolo[4,3-f]pteridin-4(5H)-one (H6)
1
Yield: 66.1%; m.p.: 327.1
–
329.2 °C; MS (ESI) m/z: 415.4 [M-H]-; H NMR (600 MHz,
DMSO-d6) δ 9.71 (s, 1H), 9.57 (s, 1H), 9.13 (s, 1H), 7.40 (d, J = 8.5 Hz, 2H), 6.53 (d, J = 8.9 Hz,
2H), 5.73 (s, 1H), 3.21 (t, J = 6.4 Hz, 4H), 2.28 3.22 (m, 2H), 1.96 1.94 (m, 4H), 1.93 – 1.88
(m, 2H), 1.82 – 1.81 (m, 2H), 1.59 1.55 (m, 2H); Anal. Calcd for C22H24N8O: C, 63.45; H, 5.81;
–
–
–
N, 26.90; Found: C, 63.44; H, 5.76; N, 26.88.
5-cyclopentyl-7-((4-(3,5-dimethylpiperazin-1-yl)phenyl)amino)-[1,2,4]triazolo[4,3-f]pteridin-4(5
H)-one (H7)
1
Yield: 63.2%; m.p.: 275.6
–
277.3 °C; MS (ESI) m/z: 460.5 [M+H]+; H NMR (400 MHz,
DMSO-d6) δ 9.73 (s, 1H), 9.68 (s, 1H), 9.17 (s, 1H), 7.47 (d, J = 8.8 Hz, 2H), 6.91 (d, J = 9.0 Hz,
2H), 5.78 – 5.69 (m, 1H), 3.48 (d, J = 9.7 Hz, 2H), 2.92 – 2.84 (m, 2H), 2.28 2.20 (m, 2H), 2.11
(t, J = 10.9 Hz, 2H), 1.99 – 1.90 (m, 2H), 1.87 1.78 (m, 2H), 1.64 – 1.56 (m, 2H), 1.04 (d, J =
–
–
6.3 Hz, 6H); Anal. Calcd for C24H29N9O: C, 62.73; H, 6.36; N, 27.43; Found: C, 62.71; H, 6.32; N,
27.40.
5-cyclopentyl-7-((4-thiomorpholinophenyl)amino)-[1,2,4]triazolo[4,3-f]pteridin-4(5H)-one (H8)
1
Yield: 56.2%; m.p.: 310.6
–
312.3 °C; MS (ESI) m/z: 447.5 [M-H]-; H NMR (400 MHz,
DMSO-d6) δ 9.72 (s, 2H), 9.17 (s, 1H), 7.50 (d, J = 8.9 Hz, 2H), 6.93 (d, J = 9.0 Hz, 2H), 5.76 –
5.72 (m, 1H), 3.46 3.43 (m, 4H), 2.70 – 2.68 (m, 4H), 2.28 2.23 (m, 2H), 1.94 1.90 (m, 2H),
1.85 1.79 (m, 2H), 1.64 – 1.58 (m, 2H); Anal. Calcd for C22H24N8OS: C, 58.91; H, 5.39; N,
–
–
–
–
24.98; Found: C, 58.84; H, 5.36; N, 24.96.
5-cyclopentyl-7-((4-(4-(2-hydroxyethyl)piperazin-1-yl)phenyl)amino)-[1,2,4]triazolo[4,3-f]pteridi
n-4(5H)-one (H9)
1
Yield: 44.6%; m.p.: 337.2
–
338.9 °C; MS (ESI) m/z: 476.6 [M+H]+; H NMR (400 MHz,
DMSO-d6) δ 9.73 (s, 1H), 9.70 (s, 1H), 9.17 (s, 1H), 7.48 (d, J = 8.8 Hz, 2H), 6.92 (d, J = 9.0 Hz,
2H), 5.76 5.72 (m, 1H), 3.55 3.52 (m, 2H), 3.10 – 3.07 (m, 4H), 2.57 – 2.55 (m, 4H), 2.43 (t, J
= 6.2 Hz, 2H), 2.45 – 2.42 (m, 2H), 1.92 (s, 2H), 1.85 (s, 2H), 1.64 1.56 (m, 2H); Anal. Calcd for
–
–
–
C24H29N9O2: C, 60.62; H, 6.15; N, 26.51; Found: C, 60.59; H, 6.12; N, 26.45.
5-cyclopentyl-7-((4-(4-hydroxypiperidin-1-yl)phenyl)amino)-[1,2,4]triazolo[4,3-f]pteridin-4(5H)-
one (H10)
1
Yield: 61.9%; m.p.: 307.2
–
308.5 °C; MS (ESI) m/z: 445.6 [M-H]-; H NMR (400 MHz,
DMSO-d6) δ 9.72 (s, 1H), 9.68 (s, 1H), 9.17 (s, 1H), 7.46 (d, J = 8.9 Hz, 2H), 6.92 (d, J = 9.0 Hz,
2H), 5.78 – 5.69 (m, 1H), 4.65 (d, J = 4.0 Hz, 1H), 3.63 3.58 (m, 1H), 3.51 – 3.45 (m, 2H), 3.17
(d, J = 4.7 Hz, 1H), 2.82 – 2.75 (m, 2H), 2.29 2.21 (m, 2H), 1.92 (s, 2H), 1.83 1.81 (m, 4H),
–
–
–
1.62 – 1.59 (m, 2H), 1.53 – 1.44 (m, 2H); Anal. Calcd for C22H25N9O2: C, 59.05; H, 5.63; N, 28.17;
Found: C, 59.03; H, 5.61; N, 28.13.
5-cyclopentyl-7-((4-(cyclopentylamino)phenyl)amino)-[1,2,4]triazolo[4,3-f]pteridin-4(5H)-one
(H11)
1
Yield: 62.3%; m.p.: 291.2
–
292.3 °C; MS (ESI) m/z: 429.5 [M-H]-; H NMR (400 MHz,
DMSO-d6) δ 9.71 (s, 1H), 9.48 (s, 1H), 9.13 (s, 1H), 7.27 (d, J = 8.6 Hz, 2H), 6.55 (d, J = 8.8 Hz,
2H), 5.72 5.68 (m, 1H), 5.38 (d, J = 6.5 Hz, 1H), 3.71 – 3.64 (m, 1H), 2.30 2.21 (m, 2H), 1.95
1.87 (m, 4H), 1.83 – 1.76 (m, 2H), 1.71 – 1.63 (m, 2H), 1.58 – 1.51 (m, 4H), 1.46 1.39 (m, 2H);
–
–
–
–
Anal. Calcd for C23H26N8O: C, 64.17; H, 6.09; N, 26.03; Found: C, 64.13; H, 6.05; N, 26.01.
5-cyclopentyl-7-((4-(dimethylamino)phenyl)amino)-[1,2,4]triazolo[4,3-f]pteridin-4(5H)-one (H12)

ACCEPTED MANUSCRIPT
1
Yield: 52.1%; m.p.: 311.2
DMSO-d6) δ 9.72 (s, 1H), 9.61 (s, 1H), 9.15 (s, 1H), 7.43 (d, J = 8.9 Hz, 2H), 6.74 (d, J = 9.0 Hz,
2H), 5.75 – 5.71 (m, 1H), 2.87 (s, 6H), 2.39 – 2.20 (m, 2H), 1.96 1.89 (m, 2H), 1.86 1.78 (m,
2H), 1.62 1.55 (m, 2H); Anal. Calcd for C20H22N8O: C, 61.52; H, 5.68; N, 28.70; Found: C,
61.46; H, 5.62; N, 28.67.
–
313.3 °C; MS (ESI) m/z: 389.5 [M-H]-; H NMR (400 MHz,
–
–
–
5-cyclopentyl-7-((4-(4-cyclopentylpiperazin-1-yl)phenyl)amino)-[1,2,4]triazolo[4,3-f]pteridin-4(5
H)-one (H13)
Yield: 66.2%; m.p.: 334.1
–
335.9 °C; MS (ESI) m/z: 500.7 [M+H]+; 1H NMR (400 MHz,
DMSO-d6) δ 9.72 (s, 2H), 9.17 (s, 1H), 7.48 (d, J = 7.9 Hz, 2H), 6.92 (d, J = 8.3 Hz, 2H), 5.78 – 5.70
(m, 1H), 3.08 (s, 4H), 2.55 (s, 4H), 2.29 – 2.21 (m, 2H), 1.96 – 1.88 (m, 2H), 1.68 – 1.76 (m, 4H), 1.67
– 1.57 (m, 4H), 1.53 – 1.48 (m, 2H), 1.41 – 1.31 (m, 2H); Anal. Calcd for C27H33N9O: C, 64.91; H,
6.66; N, 25.23; Found: C, 64.87; H, 6.62; N, 25.19.
5-cyclopentyl-7-((4-(4-cyclopentylpiperazin-1-yl)phenyl)amino)-[1,2,4]triazolo[4,3-f]pteridin-4(5
H)-one (H14)
1
Yield: 57.8%; m.p.: 320.0
–
322.1 °C; MS (ESI) m/z: 529.7 [M+H]+; H NMR (400 MHz,
DMSO-d6) δ 9.73 (s, 1H), 9.70 (s, 1H), 9.17 (s, 1H), 7.46 (d, J = 8.9 Hz, 2H), 6.92 (d, J = 9.0 Hz,
2H), 5.76 – 5.72 (m, 1H), 3.65 (d, J = 12.4 Hz, 2H), 3.51 (s, 1H), 2.62 (t, J = 11.5 Hz, 2H), 2.35 –
2.21 (m, 7H), 2.14 (s, 3H), 2.04
– 1.78 (m, 8H), 1.64 – 1.57 (m, 2H), 1.54 – 1.45 (m, 3H); Anal.
Calcd for C28H36N10O: C, 63.61; H, 6.86; N, 26.50; Found: C, 63.58; H, 6.82; N, 26.48.
5-cyclopentyl-7-((4-morpholinophenyl)amino)tetrazolo[1,5-f]pteridin-4(5H)-one (Y1)
Yield: 53.2%; m.p.: 303.1
1H), 7.52 (d, J = 8.4 Hz, 2H), 6.97 (d, J = 8.9 Hz, 2H), 5.77 (s, 1H), 3.76 – 3.74 (m, 4H), 3.08 –
3.07 (m, 4H), 2.29 – 2.21 (m, 2H), 2.00 1.92 (m, 2H), 1.88 1.82 (m, 2H), 1.67 1.57 (m, 2H);
–
306.2 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.05 (s, 1H), 9.29 (s,
–
–
–
Anal. Calcd for C21H23N9O2: C, 58.19; H, 5.35; N, 29.08; Found: C, 58.16; H, 5.30; N, 29.76.
5-cyclopentyl-7-((4-(4-methylpiperazin-1-yl)phenyl)amino)tetrazolo[1,5-f]pteridin-4(5H)-one (Y2)
1
Yield: 58.0%; m.p.: 311.2
–
312.5 °C; MS (ESI) m/z: 445.4 [M-H]-; H NMR (400 MHz,
DMSO-d6) δ 10.01 (s, 1H), 9.28 (s, 1H), 7.49 (d, J = 8.6 Hz, 2H), 6.95 (d, J = 9.0 Hz, 2H), 5.76 (s,
1H), 3.12 – 3.09 (m, 4H), 2.48 – 2.44 (m, 4H), 2.29 2.25 (m, 2H), 2.22 (s, 3H), 1.99 1.90 (m,
–
–
2H), 1.86 – 1.81 (m, 2H), 1.65 – 1.57 (m, 2H); Anal. Calcd for C22H26N10O: C, 59.18; H, 5.87; N,
31.37. Found: C, 59.14; H, 5.82; N, 31.35.
5-cyclopentyl-7-((4-(4-ethylpiperazin-1-yl)phenyl)amino)tetrazolo[1,5-f]pteridin-4(5H)-one (Y3)
325.9 °C; MS (ESI) m/z: 459.6 [M-H]-; 1H NMR (400 MHz, DMSO-d6
Yield: 58.0%; m.p.: 324.8 – )
δ 10.00 (s, 1H), 9.27 (s, 1H), 7.49 (d, J = 8.4 Hz, 2H), 6.95 (d, J = 9.0 Hz, 2H), 5.76 (s, 1H), 3.12 –
3.09 (m, 4H), 2.37 (q, J = 7.1 Hz, 2H), 2.28 – 2.20 (m, 2H), 1.98 – 1.81 (m, 4H), 1.64 – 1.58 (m, 2H),
1.04 (t, J = 7.2 Hz, 3H); Anal. Calcd for C22H26N10O: C, 59.18; H, 5.87; N, 31.37, Found: C, 59.14;
H, 5.82; N, 31.35.
5-cyclopentyl-7-((4-(piperidin-1-yl)phenyl)amino)tetrazolo[1,5-f]pteridin-4(5H)-one (Y4)
Yield: 61.2%; m.p.: 304.4
7.47 (d, J = 8.6 Hz, 2H), 6.94 (d, J = 9.0 Hz, 2H), 5.75 (s, 1H), 3.10 – 3.08 (m, 4H), 2.29 – 2.21
(m, 2H), 1.96 1.81 (m, 4H), 1.66 1.60 (m, 6H), 1.55 – 1.53 (m, 2H); Anal. Calcd for
–
305.9 °C; 1H NMR (400 MHz, DMSO-d6) δ 9.98 (s, 1H), 9.27 (s, 1H),
–
–
C22H25N9O: C, 61.24; H, 5.84; N, 29.21; Found: C, 61.21; H, 5.81; N, 29.16.
5-cyclopentyl-7-((4-(4-methylpiperidin-1-yl)phenyl)amino)tetrazolo[1,5-f]pteridin-4(5H)-one (Y5)
1
Yield: 56.7%; m.p.: 250.1
–
252.3 °C; MS (ESI) m/z: 468.4 [M+Na]+; H NMR (400 MHz,
DMSO-d6) δ 9.98 (s, 1H), 9.27 (s, 1H), 7.47 (d, J = 8.5 Hz, 2H), 6.94 (d, J = 9.0 Hz, 2H), 5.76 (s,

ACCEPTED MANUSCRIPT
1H), 3.62 (d, J = 12.2 Hz, 2H), 2.62 (dd, J = 12.2, 10.0 Hz, 2H), 2.29
– 2.21 (m, 2H), 1.97 – 1.84
(m, 4H), 1.71 – 1.67 (m, 2H), 1.62 – 1.60 (m, 2H), 1.51 – 1.46 (m, 1H), 1.29 – 1.23 (m, 2H), 0.94
(d, J = 6.5 Hz, 3H); Anal. Calcd for C23H27N9O: C, 62.01; H, 6.11; N, 28.30; Found: C, 61.93; H,
6.07; N, 28.29.
5-cyclopentyl-7-((4-(pyrrolidin-1-yl)phenyl)amino)tetrazolo[1,5-f]pteridin-4(5H)-one (Y6)
Yield: 65.2%; m.p.: 288.7
–
290.9 °C; 1H NMR (600 MHz, DMSO) δ 9.99 (s, 1H), 9.28 (s, 1H), 7.47
(d, J = 7.2 Hz, 2H), 6.94 (d, J = 8.9 Hz, 2H), 5.81 – 5.69 (m, 1H), 3.11 – 3.08 (m, 4H), 2.28 – 2.22 (m,
2H), 1.85 (s, 2H), 1.65 – 1.58 (m, 6H), 1.55 – 1.51 (m, 2H); Anal. Calcd for C21H23N9O: C, 60.42; H,
5.55; N, 30.20; Found: C, 60.40; H, 5.51; N, 30.17.
5-cyclopentyl-7-((4-(3,5-dimethylpiperazin-1-yl)phenyl)amino)tetrazolo[1,5-f]pteridin-4(5H)-one
(Y7)
1
Yield: 66.0%; m.p.: 297.4
–
298.1 °C; MS (ESI) m/z: 461.5 [M+H]+; H NMR (400 MHz,
DMSO-d6) δ 9.99 (s, 1H), 9.28 (s, 1H), 7.48 (d, J = 8.4 Hz, 2H), 6.94 (d, J = 9.0 Hz, 2H), 5.76 (s,
1H), 3.49 (d, J = 9.6 Hz, 2H), 2.89 – 2.84 (m, 2H), 2.27 2.20 (m, 2H), 2.10 (t, J = 10.9 Hz, 2H),
1.94 – 1.81 (m, 4H), 1.64
1.59 (m, 2H), 1.03 (d, J = 6.3 Hz, 6H); 13C NMR (101 MHz, DMSO) δ
–
–
159.11, 152.63, 149.67, 148.03, 147.74, 147.69, 143.33, 130.93, 122.34, 116.11, 56.03, 50.61, 40.63,
40.42, 40.21, 40.00, 39.79, 39.58, 39.37, 29.48, 28.03, 25.62, 19.79. Anal. Calcd for C23H28N10O: C,
59.98; H, 6.13; N, 30.41; Found: C, 59.96; H, 6.10; N, 30.38.
5-cyclopentyl-7-((4-(3,5-dimethylpiperazin-1-yl)phenyl)amino)tetrazolo[1,5-f]pteridin-4(5H)-one
(Y8)
1
Yield: 43.9%; m.p.: 296.1
–
297.4 °C; MS (ESI) m/z: 448.5 [M-H]-; H NMR (400 MHz,
DMSO-d6) δ 10.01 (s, 1H), 9.28 (s, 1H), 7.50 (d, J = 8.5 Hz, 2H), 6.95 (d, J = 8.9 Hz, 2H), 5.76 (s,
1H), 3.48 – 3.46 (m, 4H), 2.71 – 2.67 (m, 4H), 2.29 – 2.21 (m, 2H), 2.00 – 1.84 (m, 4H), 1.66 –
1.57 (m, 2H); Anal. Calcd for C21H23N9OS: C, 56.11; H, 5.16; N, 28.04; Found: C, 56.08; H, 5.14;
N, 28.03
5-cyclopentyl-7-((4-(4-(2-hydroxyethyl)piperazin-1-yl)phenyl)amino)tetrazolo[1,5-f]pteridin-4(5H
)-one (Y9)
1
Yield: 42.1%; m.p.: 344.6 – 345.6 °C; MS (ESI) m/z: 475.6 [M-H]-; H NMR (400 MHz,
DMSO-d6) δ 9.99 (s, 1H), 9.27 (s, 1H), 7.49 (d, J = 8.5 Hz, 2H), 6.94 (d, J = 9.0 Hz, 2H), 5.75 (s,
1H), 4.42 (s, 1H), 3.55
–
3.50 (m, 2H), 3.11 – 3.09 (m, 4H), 2.57 – 2.55 (m, 4H), 2.44 (t, J = 6.2
1.81 (m, 4H), 1.66 – 1.56 (m, 2H); Anal. Calcd for
Hz, 2H), 2.30 – 2.21 (m, 2H), 1.99
–
C23H28N10O2: C, 57.97; H, 5.92; N, 29.39; Found: C, 57.95; H, 5.89; N, 29.34
5-cyclopentyl-7-((4-(4-hydroxypiperidin-1-yl)phenyl)amino)tetrazolo[1,5-f]pteridin-4(5H)-one
(Y10)
Yield: 48.9%; m.p.: 312.5 – 313.8 °C; MS (ESI) m/z: 448.2 [M+H]+, 1H NMR (400 MHz,
DMSO-d6) δ 9.98 (s, 1H), 9.27 (s, 1H), 7.47 (d, J = 8.7 Hz, 2H), 6.95 (d, J = 9.0 Hz, 2H), 5.75 (s, 1H),
4.66 (d, J = 3.7 Hz, 1H), 3.62 (s, 1H), 3.51 (s, 2H), 2.83 – 2.78 (m, 2H), 2.28 – 2.20 (m, 2H), 2.00 –
1.78 (m, 2H), 1.83 – 1.78 (m, 4H), 1.65 – 1.57 (m, 2H), 1.53 – 1.45 (m, 2H); Anal. Calcd for
C21H24N10O2: C, 56.24; H, 5.39; N, 31.23; Found: C, 56.20; H, 5.36; N, 31.19
5-cyclopentyl-7-((4-(cyclopentylamino)phenyl)amino)tetrazolo[1,5-f]pteridin-4(5H)-one (Y11)
1
Yield: 37.6%; m.p.: 324.4 – 325.1 °C; MS (ESI) m/z: 430.3 [M-H]-; H NMR (400 MHz,
DMSO-d6) δ 9.79 (s, 1H), 9.22 (s, 1H), 7.28 (d, J = 8.1 Hz, 2H), 6.57 (d, J = 8.7 Hz, 2H), 5.75 (s,
1H), 5.45 (d, J = 5.6 Hz, 1H), 3.71
– 2.67 (m, 1H), 2.29 – 2.22 (m, 2H), 1.94 – 1.82 (m, 6H), 1.68
– 1.62 (m, 2H), 1.59 – 1.53 (m, 4H), 1.47 – 1.41 (m, 2H); Anal. Calcd for C22H25N9O: C, 61.24; H,

ACCEPTED MANUSCRIPT
5.84; N, 29.21; Found: C, 61.21; H, 5.80; N, 29.19.
5-cyclopentyl-7-((4-(dimethylamino)phenyl)amino)tetrazolo[1,5-f]pteridin-4(5H)-one (Y12)
1
Yield: 52.3%; m.p.: 295.7 – 296.8 °C; MS (ESI) m/z: 392.4 [M+H]+; H NMR (400 MHz,
DMSO-d6) δ 9.91 (s, 1H), 9.25 (s, 1H), 7.44 (d, J = 8.7 Hz, 2H), 6.75 (d, J = 9.0 Hz, 2H), 5.77
–
–
5.70 (m, 1H), 2.88 (s, 6H), 2.29 – 2.20 (m, 2H), 2.99 – 1.91 (m, 2H), 1.85 – 1.81 (m, 2H), 1.63
1.57 (m, 2H); Anal. Calcd for C19H21N9O: C, 58.30; H, 5.41; N, 32.21; Found: C, 58.25; H, 5.36;
N, 32.19.
5-cyclopentyl-7-((4-(4-cyclopentylpiperazin-1-yl)phenyl)amino)tetrazolo[1,5-f]pteridin-4(5H)-one
(Y13)
1
Yield: 55.6%; m.p.:298.4 – 299.1 °C; MS (ESI) m/z: 501.7 [M+H]+; H NMR (400 MHz,
DMSO-d6) δ 9.99 (s, 1H), 9.27 (s, 1H), 7.49 (d, J = 8.4 Hz, 2H), 6.94 (d, J = 8.9 Hz, 2H), 5.75 (s,
1H), 3.51 (s, 1H), 3.11
–
3.09 (m, 4H), 2.57
–
2.55 (m, 4H), 2.30 – 2.21 (m, 2H), 2.00
– 1.92 (m,
2H), 1.83 – 1.78 (m, 4H), 1.66
–
1.59 (m, 4H), 1.56 – 1.50 (m, 2H), 1.40
–
1.32 (m, 2H); Anal.
Calcd for C26H32N10O: C, 62.38; H, 6.44; N, 27.98; Found: C, 62.35; H, 6.41; N, 27.95
5-cyclopentyl-7-((4-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)amino)tetrazolo[1,5-f]pterid
in-4(5H)-one (Y14)
1
Yield: 61.3%; m.p.: 302.1 – 303.8 °C; MS (ESI) m/z: 530.7 [M+H]+; H NMR (400 MHz,
DMSO-d6) δ 9.99 (s, 1H), 9.27 (s, 1H), 7.47 (d, J = 8.4 Hz, 2H), 6.95 (d, J = 9.0 Hz, 2H), 5.76 (s,
1H), 3.67 (d, J = 12.1 Hz, 2H), 3.51 (s, 1H), 2.63 (t, J = 11.3 Hz, 2H), 2.36 – 2.21 (m, 7H), 2.14 (s,
3H), 2.00 – 1.79 (m, 8H), 1.66 – 1.57 (s, 2H), 1.54 – 1.45 (m, 3H); Anal. Calcd for C27H35N11O: C,
61.23; H, 6.66; N, 29.09; Found: C, 61.20; H, 6.63; N, 29.01
5-cyclopentyl-1-methyl-7-((4-morpholinophenyl)amino)-[1,2,4]triazolo[4,3-f]pteridin-4(5H)-one
(L1)
1
Yield: 49.8%; m.p.: 306.2
–
304.9 °C; MS (ESI) m/z: 447.7 [M+H]+; H NMR (400 MHz,
DMSO-d6) δ 9.70 (s, 1H), 8.98 (s, 1H), 7.53 (d, J = 8.8 Hz, 2H), 6.93 (d, J = 9.0 Hz, 2H), 5.79 –
5.74 (m, 1H), 3.76 – 3.73 (m, 4H), 3.07 – 3.05 (m, 4H), 2.92 (s, 3H), 2.29 2.20 (m, 2H), 1.99
–
–
1.91 (m, 2H), 1.88 – 1.80 (m, 2H), 1.64 – 1.57 (m, 2H); Anal. Calcd for C23H26N8O2: C, 61.87; H,
5.87; N, 25.10; Found: C, 61.84; H, 5.82; N, 25.07.
5-cyclopentyl-1-methyl-7-((4-(4-methylpiperazin-1-yl)phenyl)amino)-[1,2,4]triazolo[4,3-f]pteridi
n-4(5H)-one (L2)
1
Yield: 56.4%; m.p.: 296.6
–
297.3 °C; MS (ESI) m/z:460.6 [M+H]+; H NMR (400 MHz,
DMSO-d6) δ 9.67 (s, 1H), 8.98 (s, 1H), 7.50 (d, J = 8.7 Hz, 2H), 6.92 (d, J = 9.0 Hz, 2H), 5.79 –
5.74 (m, 1H), 3.09 – 3.07 (m, 4H), 2.92 (s, 3H), 2.47 – 2.44 (m, 4H), 2.28 2.24 (m, 2H), 2.22 (s,
3H), 1.98 1.91 (m, 2H), 1.87 – 1.80 (m, 2H), 1.64 1.56 (m, 2H); Anal. Calcd for C24H29N9O: C,
–
–
–
62.73; H, 6.36; N, 27.43; Found: C, 62.69; H, 6.31; N, 27.40.
5-cyclopentyl-7-((4-(4-ethylpiperazin-1-yl)phenyl)amino)-1-methyl-[1,2,4]triazolo[4,3-f]pteridin-
4(5H)-one (L3)
1
Yield: 53.0%; m.p.: 299.6
–
302.2 °C; MS (ESI) m/z: 474.7 [M+H]+; H NMR (400 MHz,
DMSO-d6) δ 9.68 (s, 1H), 8.97 (s, 1H), 7.50 (d, J = 8.7 Hz, 2H), 6.92 (d, J = 9.0 Hz, 2H), 5.78 –
5.75 (m, 1H), 3.10 – 3.08 (m, 4H), 2.92 (s, 3H), 2.37 (q, J = 7.1 Hz, 2H), 2.28 2.20 (m, 2H), 1.98
1.89 (m, 2H), 1.87 1.79 (m, 2H), 1.64
1.56 (m, 2H), 1.03 (t, J = 7.2 Hz, 3H); 13C NMR (101
–
–
–
–
MHz, DMSO-d6) δ 157.48, 153.26, 149.43, 148.66, 147.54, 146.75, 143.66, 131.86, 121.81, 116.13,
110.19, 53.89, 52.84, 52.11, 49.27, 40.61, 40.40, 40.19, 39.98, 39.78, 39.57, 39.36, 28.14, 25.59, 14.25,
12.45; Anal. Calcd for C25H31N9O: C, 63.40; H, 6.60; N, 26.62; Found: C, 63.37; H, 6.58; N,

ACCEPTED MANUSCRIPT
26.59.
5-cyclopentyl-1-methyl-7-((4-(piperidin-1-yl)phenyl)amino)-[1,2,4]triazolo[4,3-f]pteridin-4(5H)-o
ne (L4)
Yield: 62.4%; m.p.: 325.8
–
327.9 °C; MS (ESI) m/z: 445.6 [M+H]+; 1H NMR (400 MHz,
DMSO-d6) δ 9.66 (s, 1H), 8.97 (s, 1H), 7.48 (d, J = 8.8 Hz, 2H), 6.91 (d, J = 9.0 Hz, 2H), 5.87 – 5.64
(m, 1H), 3.13 – 3.02 (m, 4H), 2.92 (s, 3H), 2.29 – 2.19 (m, 2H), 1.93 (s, 2H), 1.89 – 1.78 (m, 2H), 1.66
– 1.58 (m, 6H), 1.55 – 1.50 (m, 2H); Anal. Calcd for C24H28N8O: C, 64.85; H, 6.35; N, 25.21;
Found: C, 64.83; H, 6.33; N, 25.19.
5-cyclopentyl-1-methyl-7-((4-(4-methylpiperidin-1-yl)phenyl)amino)-[1,2,4]triazolo[4,3-f]pteridin
-4(5H)-one (L5)
1
Yield: 67.8%; m.p.: 308.7
–
309.6 °C; MS (ESI) m/z: 457.6 [M-H]-; H NMR (400 MHz,
DMSO-d6) δ 9.66 (s, 1H), 8.97 (s, 1H), 7.47 (d, J = 8.7 Hz, 2H), 6.91 (d, J = 9.0 Hz, 2H), 5.77 –
5.74 (m, 1H), 3.59 (d, J = 12.2 Hz, 2H), 2.92 (s, 3H), 2.60 (t, J = 11.2 Hz, 2H), 2.29 – 2.20 (m, 2H),
1.93 (s, 2H), 1.86 – 1.80 (m, 2H), 1.69 (d, J = 12.3 Hz, 2H), 1.61 – 1.57 (m, 2H), 1.52 – 1.44 (m, 1H),
1.28 – 1.23 (m, 2H), 0.94 (d, J = 6.5 Hz, 3H); Anal. Calcd for C25H30N8O: C, 65.48; H, 6.59; N,
24.44; Found: C, 65.46; H, 6.57; N, 24.42.
5-cyclopentyl-1-methyl-7-((4-(pyrrolidin-1-yl)phenyl)amino)-[1,2,4]triazolo[4,3-f]pteridin-4(5H)-
one (L6)
Yield: 55.7%; m.p.: 315.4
–
316.8 °C; MS (ESI) m/z: 431.5 [M+H]+; 1H NMR (400 MHz,
DMSO-d6) δ 9.54 (s, 1H), 8.94 (s, 1H), 7.41 (d, J = 8.4 Hz, 2H), 6.52 (d, J = 8.7 Hz, 2H), 5.79 – 5.72
(m, 1H), 3.21-3.19 (m, 4H), 2.91 (s, 3H), 2.28 – 2.18 (m, 2H), 1.99 – 1.88 (m, 6H), 1.85 – 1.77 (m, 2H),
1.62 – 1.54 (m, 2H).; Anal. Calcd for C23H26N8O: C, 64.17; H, 6.09; N, 26.03; Found: C, 64.15; H,
6.08; N, 26.01.
5-cyclopentyl-7-((4-(3,5-dimethylpiperazin-1-yl)phenyl)amino)-1-methyl-[1,2,4]triazolo[4,3-f]pter
idin-4(5H)-one (L7)
1
Yield: 53.2%; m.p.: 280.4
–
282.1 °C; MS (ESI) m/z: 474.5 [M+H]+; H NMR (400 MHz,
DMSO-d6) δ 9.64 (s, 1H), 8.97 (s, 1H), 7.48 (d, J = 8.7 Hz, 2H), 6.90 (d, J = 8.9 Hz, 2H), 5.78 –
5.74 (m, 1H), 3.46 (d, J = 9.6 Hz, 2H), 2.92 (s, 3H), 2.88 – 2.83 (m, 2H), 2.26 – 2.19 (m, 2H),
2.08 (t, J = 10.8 Hz, 2H), 1.96 – 1.89 (m, 2H), 1.87 – 1.79 (m, 2H), 1.64 – 1.56 (m, 2H), 1.02 (d, J
= 6.2 Hz, 6H); 13C NMR (101 MHz, DMSO) δ 157.51, 153.27, 149.43, 148.65, 147.68, 146.78, 143.67,
131.58, 121.88, 116.15, 110.15, 56.31, 53.87, 50.61, 40.63, 40.42, 40.22, 40.01, 39.80, 39.59, 39.38,
28.17, 25.63, 19.91, 14.25; Anal. Calcd for C25H31N9O: C, 63.40; H, 6.60; N, 26.62; Found: C,
63.38; H, 6.57; N, 26.56.
5-cyclopentyl-1-methyl-7-((4-thiomorpholinophenyl)amino)-[1,2,4]triazolo[4,3-f]pteridin-4(5H)-o
ne (L8)
1
Yield: 64.3%; m.p.: 312.0
–
314.6 °C; MS (ESI) m/z: 461.6 [M-H]-; H NMR (400 MHz,
DMSO-d6) δ 9.68 (s, 1H), 8.98 (s, 1H), 7.51 (d, J = 8.8 Hz, 2H), 6.92 (d, J = 9.0 Hz, 2H), 5.79
–
5.74 (m, 1H), 3.45 – 3.43 (m, 4H), 2.92 (s, 3H), 2.70 – 2.68 (m, 4H), 2.27 – 2.21 (m, 2H), 1.93 (s,
2H), 1.85 – 1.83 (m, 2H), 1.62 – 1.59 (m, 2H); Anal. Calcd for C23H26N8OS: C, 59.72; H, 5.67; N,
24.22; Found: C, 59.69; H, 5.62; N, 24.18.
5-cyclopentyl-7-((4-(4-(2-hydroxyethyl)piperazin-1-yl)phenyl)amino)-1-methyl-[1,2,4]triazolo[4,3
-f]pteridin-4(5H)-one (L9)
1
Yield: 58.5%; m.p.: 288.4
–
289.9 °C; MS (ESI) m/z: 490.7 [M+H]+; H NMR (400 MHz,
DMSO-d6) δ 9.66 (s, 1H), 8.97 (s, 1H), 7.49 (d, J = 8.8 Hz, 2H), 6.91 (d, J = 9.0 Hz, 2H), 5.81 –

ACCEPTED MANUSCRIPT
5.71 (m, 1H), 4.41 (s, 1H), 3.57
–
3.51 (s, 2H), 3.09 – 3.07 (m, 4H), 2.92 (s, 3H), 2.57 – 2.55 (m,
2.20 (m, 2H), 1.95 1.89 (m, 2H), 1.87 – 1.81 (m, 2H), 1.64 –
4H), 2.44 (t, J = 6.2 Hz, 2H), 2.28
–
–
1.56 (m, 2H); Anal. Calcd for C25H31N9O2: C, 61.33; H, 6.38; N, 25.75; Found: C, 61.29; H, 6.35;
N, 25.66.
5-cyclopentyl-7-((4-(4-hydroxypiperidin-1-yl)phenyl)amino)-1-methyl-[1,2,4]triazolo[4,3-f]pteridi
n-4(5H)-one (L10)
1
Yield: 56.1%; m.p.: 321.2
–
323.8 °C; MS (ESI) m/z: 461.5 [M+H]+; H NMR (400 MHz,
DMSO-d6) δ 9.65 (s, 1H), 8.97 (s, 1H), 7.48 (d, J = 8.8 Hz, 2H), 6.92 (d, J = 9.0 Hz, 2H), 5.78 –
5.74 (m, 1H), 4.65 (d, J = 4.1 Hz, 1H), 3.62 3.58 (m, 1H), 3.51-3.44 (m, 2H), 2.92 (s, 3H), 2.82
– 2.73 (m, 2H), 2.28-2.20 (m, 2H), 1.93 (s, 2H), 1.84 1.81 (m, 4H), 1.61 – 1.59 (m, 2H), 1.52 –
–
–
1.44 (m, 2H); Anal. Calcd for C23H27N9O2: C, 59.86; H, 5.90; N, 27.31; Found: C, 59.82; H, 5.87;
N, 27.26.
5-cyclopentyl-7-((4-(cyclopentylamino)phenyl)amino)-1-methyl-[1,2,4]triazolo[4,3-f]pteridin-4(5
H)-one (L11)
Yield: 49.2%; m.p.: 312.6
δ 9.45 (s, 1H), 8.93 (s, 1H), 7.28 (d, J = 8.5 Hz, 2H), 6.55 (d, J = 8.8 Hz, 2H), 5.73 (s, 1H), 5.38 (d,
J = 6.5 Hz, 1H), 3.68 (dd, J = 11.8, 5.6 Hz, 1H), 2.91 (s, 3H), 2.23 2.21 (m, 2H), 1.92 1.87 (m,
4H), 1.84 1.77 (m, 2H), 1.69 1.63 (m, 2H), 1.58 1.56 (m, 4H), 1.46 1.41 (m, 2H); Anal.
–
314.2°C; MS (ESI) m/z: 443.6 [M-H]-; 1H NMR (400 MHz, DMSO-d6)
–
–
–
–
–
–
Calcd for C24H28N8O: C, 64.85; H, 6.35; N, 25.21; Found: C, 64.83; H, 6.30; N, 25.18.
5-cyclopentyl-7-((4-(dimethylamino)phenyl)amino)-1-methyl-[1,2,4]triazolo[4,3-f]pteridin-4(5H)-
one (L12)
Yield: 64.3%; m.p.: 337.2 –
338.9 °C; MS (ESI) m/z: 403.5 [M-H]-; 1H NMR (400 MHz, DMSO-d6)
δ 9.79 – 9.36 (m, 1H), 8.96 (s, 1H), 7.45 (d, J = 8.8 Hz, 2H), 6.73 (d, J = 9.1 Hz, 2H), 5.82 – 5.71 (m,
1H), 2.92 (s, 3H), 2.87 (s, 6H), 2.28 – 2.20 (m, 2H), 1.96 – 1.89 (m, 2H), 1.86 – 1.78 (m, 2H), 1.62 –
1.55 (m, 2H); Anal. Calcd for C21H24N8O: C, 62.36; H, 5.98; N, 27.70; Found: C, 62.34; H, 5.93;
N, 27.65.
5-cyclopentyl-7-((4-(4-cyclopentylpiperazin-1-yl)phenyl)amino)-1-methyl-[1,2,4]triazolo[4,3-f]pte
ridin-4(5H)-one (L13)
1
Yield: 67.8%; m.p.: 331.4
–
334.6 °C; MS (ESI) m/z: 514.7 [M+H]+; H NMR (400 MHz,
DMSO-d6) δ 9.67 (s, 1H), 8.97 (s, 1H), 7.50 (d, J = 8.8 Hz, 2H), 6.91 (d, J = 9.0 Hz, 2H), 5.81
5.73 (m, 1H), 3.09 – 3.07 (m, 4H), 2.92 (s, 3H), 2.56 – 2.54 (m, 4H), 2.28 – 2.20 (m, 2H), 1.93 (s,
2H), 1.86 1.78 (m, 4H), 1.65 – 1.56 (m, 4H), 1.53 – 1.50 (m, 2H), 1.40 1.33 (m, 2H); Anal.
–
–
–
Calcd for C28H35N9O: C, 65.47; H, 6.87; N, 24.54; Found: C, 65.42; H, 6.86; N, 24.51.
5-cyclopentyl-1-methyl-7-((4-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)amino)-[1,2,4]triaz
olo[4,3-f]pteridin-4(5H)-one (L14)
1
Yield: 57.4%; m.p.: 327.9
–
329.5 °C; MS (ESI) m/z: 543.8 [M+H]+; H NMR (400 MHz,
DMSO-d6) δ 9.67 (s, 1H), 8.97 (s, 1H), 7.50 (d, J = 8.8 Hz, 2H), 6.91 (d, J = 9.0 Hz, 2H), 5.80
5.72 (m, 1H), δ 3.65 (d, J = 11.4 Hz, 1H), 3.51 (s, 1H), 2.92 (s, 3H), 2.64 – 2.59 (m, 2H), 2.35 –
2.20 (m, 7H), 2.14 (s, 3H), 1.99 1.78 (m, 8H), 1.64 – 1.57 (m, 2H), 1.55 – 1.43 (m, 3H); Anal.
–
–
Calcd for C29H38N10O: C, 64.18; H, 7.06; N, 25.81; Found: C, 64.13; H, 7.02; N, 25.77.
4.1.5
N-(5-cyclopentyl-1-methyl-4-oxo-4,5-dihydro-[1,2,4]triazolo[4,3-f]pteridin-7-yl)-N-(4-(4-ethylpip
erazin-1-yl)phenyl)acetamide (L15)
The compound L3 (0.5 g, 0.9 mmol) was taken into acetic anhydride and stirred at 100 °C for 4 h.