Pyrazolo[3,4-d]pyrimidine derivatives containing a Schiff base moiety as potential antiviral agents

Article abstract of DOI:10.1016/j.bmcl.2018.06.049

A series of pyrazolo[3,4-d]pyrimidine derivatives containing a Schiff base moiety were synthesized, characterised, and evaluated for their activity against tobacco mosaic virus (TMV). Biological assays indicated that several of the derivatives exhibited significant activity against TMV. In particularly, compounds 5y and 5aa displayed excellent inactivating activity against TMV, with half maximal effective concentration (EC₅₀) values of 70.3 and 53.65 μg/mL, respectively, which were much better than that of ribavirin (150.45 μg/mL), and 5aa was superior to ningnanmycin (EC₅₀ = 55.35 μg/mL). Interactions of compounds 5y and 5aa with TMV coat protein (TMV-CP) were investigated using microscale thermophoresis and molecular docking. Compounds 5y and 5aa displayed strong binding capability to TMV-CP with dissociation constant (K_d) values of 22.6 and 9.8 μM, respectively. These findings indicate that pyrazolo[3,4-d]pyrimidine derivatives containing a Schiff base may be potential antiviral agents.

Full text of DOI:10.1016/j.bmcl.2018.06.049

Accepted Manuscript
Pyrazolo[3,4-d]pyrimidine derivatives containing a Schiff base moiety as po-
tential antiviral agents
Yan-Yan Wang, Fang-Zhou Xu, Yun-Ying Zhu, Baoan Song, Dexia Luo, Gang
Yu, Shunhong Chen, Wei Xue, Jian Wu
PII:
S0960-894X(18)30550-X
https://doi.org/10.1016/j.bmcl.2018.06.049
BMCL 25930
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
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3 June 2018
27 June 2018
28 June 2018
Please cite this article as: Wang, Y-Y., Xu, F-Z., Zhu, Y-Y., Song, B., Luo, D., Yu, G., Chen, S., Xue, W., Wu, J.,
Pyrazolo[3,4-d]pyrimidine derivatives containing a Schiff base moiety as potential antiviral agents, Bioorganic &
Medicinal Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.06.049
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Pyrazolo[3,4-d]pyrimidine derivatives containing a Schiff base moiety as
potential antiviral agents
Yan-Yan Wanga,#, Fang-Zhou Xu a,#, Yun-Ying Zhu b, Baoan Song a,* , Dexia Luo a,
Gang Yu a, Shunhong Chen a, Wei Xue a, Jian Wu a, *
Corresponding author footnote
* To whom correspondence should be addressed. Tel.:/fax: +86(851)
+86(851)88292090. E-mails: basong@gzu.edu.cn, jwu6@gzu.edu.cn
#. The authors are equally contribution to this work.
Current address:
a. State Key Laboratory Breeding Base of Green Pesticide and Agricultural
Bioengineering, Key Laboratory of Green Pesticide and Agricultural Bioengineering,
Ministry of Education, Research and Development Center for Fine Chemicals,
Guizhou University, Guiyang 550025, China.
b. School of Chemical Engineering, Guizhou Institute of Technology, Guiyang
550001, China.
1

ABSTRACT
A series of pyrazolo[3,4-d]pyrimidine derivatives containing a Schiff base moiety
were synthesized, characterised, and evaluated for their activity against tobacco
mosaic virus (TMV). Biological assays indicated that several of the derivatives
exhibited significant activity against TMV. In particularly, compounds 5y and 5aa
displayed excellent inactivating activity against TMV, with half maximal effective
concentration (EC50) values of 70.3 and 53.65 µg/mL, respectively, which were much
better than that of ribavirin (150.45 µg/mL), and 5aa was superior to ningnanmycin
(EC50 = 55.35 µg/mL). Interactions of compounds 5y and 5aa with TMV coat protein
(TMV-CP) were investigated using microscale thermophoresis and molecular docking.
Compounds 5y and 5aa displayed strong binding capability to TMV-CP with
dissociation constant (Kd) values of 22.6 and 9.8 µM, respectively. These findings
indicate that pyrazolo[3,4-d]pyrimidine derivatives containing a Schiff base may be
potential antiviral agents.
Keywords: Pyrazolo[3,4-d]pyrimidine; Schiff base; Antiviral activity; Molecular
docking.
2

Virus in plants, known as plant cancer, are very difficult to control once plants
have been infected, and give rise to serious damage. As an example, tobacco mosaic
virus (TMV) is the most persistent plant virus and can survive in dry plant debris for
up to 100 years.1 More than 885 individual species in 65 plant families can be infected
by this virus,1,2 and U.S. $100 million is lost each year globally due to plant diseases
caused by TMV. Hence, the development of novel anti-viral molecules against TMV
has attracted an increasing amount of attention.
Pyrazolo[3,4-d]pyrimidine are fused heterocyclic ring systems that structurally
resemble purines and play a crucial roles in drug discovery due to their broad
spectrum of biological properties.3 And interestingly, the Schiff base is also a highly
efficient pharmacophore, which is an ideal sub-structure for developing
6d
agrochemicals and medicines,4-6 previous studies by our group also reported that
several Schiff bases contain quinazolinone derivatives that have excellent anti-TMV
activity.
Considering the above mentioned information and the purpose for discovery
novel antiviral agents for plant. In current study, we sought to incorporate the
sub-structure unit of a Schiff base with the backbone of pyrazolo[3,4-d]pyrimidine, by
replacing the quinazolinone backbone with pyrazolo[3,4-d]pyrimidine based on
previous works (Figure 1).6d Then we derived the structure by introducing different
types of moieties, which we hypothesised would result in pyrazolo[3,4-d]pyrimidines
containing a Schiff base substructure with good anti-plant virus activity. Based on this
hypothesis, a series of novel pyrazolo[3,4-d]pyrimidine derivatives containing a
Schiff base was designed and synthesized. Biological assay revealed that most of the
synthesized compounds exhibited excellent anti-TMV activity.
3

Figure 1. Design of pyrazolo[3,4-d]pyrimidine derivatives containing a Schiff base moiety
The synthesis protocol of pyrazolo[3,4-d]pyrimidines (5) is depicted in Scheme 1.
7-9
All intermediates (1–4) can be easily synthesized according to references,
and
compound 5 was prepared by the reaction of intermediate 4 with different aromatic
aldehyde (or hemiacetal) at reflux in glacial acetic acid with excellent yield. The
details of the protocols and spectroscopic data of the synthesized compounds are
included in the Supplementary Data.
O
O
R
NC
O
O
O
O
O
a
b
O
N
NC
+
O
O
R
O
R
N
NH2
O
R1
O
O
R
O
O
R
O
c
NH2
N
N
d
N
Ar
e
N
N
N
N
N
N
N
R1
R1
N
N
R
O
5a-5z, 5aa-5ah
4c: R=Me, R1=H;
4d: R=Me, R1=Me;
5d: R=H, R1=H, Ar =
5h: R=H, R1=Me, Ar =
4a: R=H, R1=H;
4b: R=H, R1=Me;
N
N
S
N
N
O
N
5a: R=H, R1=H, Ar =
5e: R=H, R1=H, Ar =
5b: R=H, R1=H, Ar =
5c: R=H, R1=H, Ar =
;
;
;
;
;
;
;
Cl
O
N
N
;
O
5f: R=H, R1=H, Ar =
5g: R=H, R1=Me, Ar =,
N
5i: R=H, R1= Me, Ar =
5m: R=Me, R1=H, Ar =
: R=H, R1=H, Ar =
5l: R=H, R1=H, Ar =
;
5j
;
;
: R=H, R1=H, Ar =
5k
;
OH
5n: R=Me, R1=Me, Ar =
5r: R=Me, R1=Me, Ar =
5o: R=Me, R1=H, Ar =
5s: R=Me, R1=Me, Ar =
;
N
S
N
5p: R=Me, R1=Me, Ar =
5t: R=Me, R1=H, Ar =
;
;
;
S
O
O
S
: R=Me, R1=H, Ar =
;
5q
;
;
;
S
: R=Me, R1=H, Ar = HN
5v: R=Me, R1=Me, Ar =
5x: R=Me, R1=Me, Ar =
HN
: R=Me, R1=H, Ar =
5w
5u
;
;
;
N
Br
S
: R=Me, R1=H, Ar =
5ab: R=Me, R1=Me, Ar =
5af: R=Me, R1=Me, Ar =
S
5z: R= H, R1=H, Ar =
5y: R=Me, R1=Me, Ar =
S
5aa
;
;
;
;
N
N
Br
Br
N
5ad: R=Me, R1=H, Ar =
5ae: R=Me, R1=H, Ar =
N
5ac: R=Me, R1=Me, Ar =
5ag: R=Me, R1=H, Ar =
Br
Br
;
;
;
;
5ah: R=Me, R1=Me, Ar =
N
N
;
.
Cl
Cl
Scheme 1 Reagents and conditions. (a) reflux, Ac2O; (b) 40% methylhydrazine, EtOH, 80oC;
(c) triethoxyethane/triethoxymethane, reflux; (d) 80%N2H4·H2O, EtOH, r.t; (e)
1,1-dimethoxy-N,N-dimethylmethanamine/aromatic aldehyde, Acetic acid, reflux.
The antiviral activity of pyrazolo[3,4-d]pyrimidine derivatives against TMV was
10
evaluated using the half-leaf spot method
(Table 1). Compound III-31,6d
4

commercial ningnanmycin and ribavirin were selected as the positive controls.
Interestingly, the results indicated intermediate 4 showed good to excellent activity in
three models (curative, protection, and inactivation). In particular, the curative
activities of 4b and 4c were slightly better than that of ningnanmycin. And the
protective effects of the intermediates were equivalent to that of ningnanmycin, they
also showed moderate inactivation activity at 500 µg/mL. And it could conclude that
activities of intermediates 4a–4d could be decreased by introducing a methyl at the 6
position of pyrazolo[3,4-d]pyrimidine, but could be increased by a methyl at the 3
position; thus, the active order was 4b < 4a < 4d <4c. The above active data of
intermediates 4a–4d led us to conclude that pyrazolo[3,4-d]pyrimidine is an effective
pharmacophore. However, the activity of this pharmacophore could be impacted by
introducing different types of groups. Generally, compounds with a thiazole (5b),
pyrazole (5d), or pyrrole (compounds 5w and 5x) showed low activity, but
introduction of a pyridine ring (compounds 5g, 5i, 5ab, 5ad, 5ae, 5af, 5ag) and
5-methylthiophen-2-yl (5y, 5z, 5aa) resulted in excellent anti-TMV activity (curative
activity). For instance, compound 5ad showed 61.4% activity against TMV, and
curative activity of compounds 5y and 5aa was greater than 62%, which were at the
same levels as that of commercial ningnanmycin. Introduction of furan-2-yl (5h, 5q,
5r) and benzyl (5o, 5p) also led to good anti-TMV activity, the curative activity of
them was similar as that of positive control III-31. In addition, most of the
synthesized compounds had significant protective effects against TMV and were
much better than that of ribavirin (50.4%), as well as compounds 5a, 5g–5j, 5o, 5t, 5u,
5w, 5y, 5aa, and 5ab showed better inactivation activity than ribavirin (73.2%) and
compound III-31 (78.2%) at 500 mg L−1. In particularly, inactivation effects of
compounds 5y and 5aa approached the inhibition rate of ningnanmycin.
5

Table 1. Inhibitory effects of the test compounds against TMV at 500 µg/mL
Compd.
Curative activity (%)
Protection effect (%)
Inactivation effect (%)
4a
58.4±2.9
33.5±3.1
64.9±1.7
62.8±3.2
48.0±1.9
37.1±3.2
30.2±2.3
43.3±3.9
50.4±0.5
37.1±1.3
56.8 ±2.1
55.9±1.4
56.5±2.3
47.8±4.2
42.2±2.4
32.3±2.1
29.2±3.9
32.7±1.9
52.1±2.5
55.8±1.7
54.2±0.9
55.6±1.9
45.7±2.2
57.2±0.9
51.9±2.2
42.6±2.8
32.8±1.3
14.2±1.3
62.7±2.4
59.4±0.9
62.7±0.3
58.5±1.3
22.7±1.2
61.4±1.7
59.6±0.9
57.1±1.3
58.8±1.6
47.5±0.9
54.3 2.9
62.6 2.3
60.6±0.9
52.8±0.8
63.2±2.7
62.6±2.4
33.7 ±1.7
49.9±2.1
44.1±1.1
41.5±2.1
45.7±1.2
45.8±1.8
23.1±2.5
54.2±1.5
40.3±1.1
52.2±1.9
50.8±2.3
42.6±1.3
39.4±2.7
60.1±1.5
65.1±1.6
64.8±4.9
63.5±1.7
62.5±1.4
56.9±1.6
62.8±1.4
63.4±3.1
51.9±1.5
46.6±2.1
22.3±1.4
64.0±1.6
62.0±1.9
62.0±1.9
65.2±1.9
22.8±1.3
57.6±1.2
62.0±1.2
59.1±2.3
34.6±1.5
43.5±1.4
63.2 1.8
64.2 2.1
68.7±2.3
64.3±1.3
74.2±2.4
62.2±2.5
83.4 ±2.4
60.5±1.4
54.5±2.5
74.2±1.1
65.5±1.3
58.9±2.7
85.2±3.1
84.0±3.3
80.1±1.6
82.5±2.3
64.8±2.5
42.1±2.3
63.5±3.1
72.9±1.3
88.9±0.9
76.4±2.4
64.1±2.3
78.0±2.1
62.4±3.1
89.6±1.8
84.5 2.3
67.4±2.5
82.2±1.7
34.8±2.3
92.1±0.7
72.8±2.1
94.6±2.1
85.0±2.1
32.9±3.1
76.2±2.4
67.1±2.3
65.0±1.5
75.5±1.9
51.9±2.7
78.2 1.5
93.5±1.7
73.2±2.2
4b
4c
4d
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
5s
5t
5u
5v
5w
5x
5y
5z
5aa
5ab
5ac
5ad
5ae
5af
5ag
5ah
III-31a
ningnanmycin
ribavirin
37.9 1.9
50.4 2.3
a. Compound in reference 6d
The half maximal effective concentration (EC50) shown in Table 2 further
revealed that the synthesized compounds displayed good anti-TMV activity. In
6

particularly, compounds 5y and 5aa showed the best curative activity, which were
much better than commercial ningnanmycin. And their EC50 values for inactivation
activity were 70.3 and 53.8 µg/mL, respectively, which were much lower than that of
ribavirin, and compound 5aa showed the best inactivation activity, which was
superior to that of ningnanmycin (EC50 = 55.35 µg/mL).
Table 2. The EC50 (µg/mL) of the title compounds against TMV
Compd.
4a
Curative activity
505.2 4.25
Protection activity
383.1 2.44
Inactivation activity
/
354.2 2.42
559.8 4.45
257.8 2.21
4c
315.6 2.33
481.4 4.25
480.7 5.34
355.5 3.12
/
/
4d
5g
5h
165.9 2.62
195.6 2.33
282.4 2.11
659.24 4.24
485.29 3.67
401.1 2.56
337.2 2.33
400.6 4.31
380.94 3.32
340.5 3.12
365.7 3.31
280.5 2.13
301.6 3.16
181.3 2.32
157.25 2.45
364.1 2.65
/
5o
5p
5q
5r
5t
109.5 3.11
402.4 3.14
272.8 2.31
392.9 4.12
283.5 2.23
400.6 5.12
317.2 1.34
400.4 4.13
353.5 3.23
306.1 3.41
367.6 2.12
296.8 2.15
313.2 2.12
412.7 2.15
274.4 3.32
144.3 2.46
70.3 2.43
301.1 3.18
53.8 1.43
155.5 2.15
167.2 1.34
329.7 2.15
5u
5y
5z
5aa
5ab
5ad
5ae
427.2 4.43
446.4 2.31
324.51 3.15
/
432.1 2.54
/
328.5 3.01
398.3 2.15
55.35 1.45
124.05 3.5
5af
5ag
ningnanmycin
ribavirin
165.95 2.15
435.99 4.23
TMV coat protein (TMV-CP) plays a vital role in the translation of mRNAs,
7

transcription of tRNA, and self-assembly of TMV particles, and may be a target
protein for the screening of anti-TMV agents.10 Due to the excellent inactivation
activity of compounds 5y and 5aa, we speculated that they would interact well with
TMV-CP. Hence, the binding interaction between 5y (or 5aa) and TMV-CP was
investigated using microscale thermophoresis (MST). As shown in Figure 3,
compound 5aa showed strong binding with TMV-CP. MST measurements showed
that 5aa bond to TMV-CP with a dissociation constant (Kd) of 9.8 2.6 µM, which
was much lower than that of ribavirin (Kd = 64.7 2.12 µM) and similar to that of
ningnanmycin (Kd = 9.75 0.55 µM). In addition, compound 5y displayed good
binding capability with TMV-CP (Kd = 22.6 2.7 µM), which was much better than
that of ribavirin. These results demonstrated that both 5y and 5aa interacted with
TMV-CP.
Figure 2. Microscale thermophoresis (MST) results of compounds 5y (A), 5aa (B), ribavirin (C),
and ninnanmycin (D).
11
Molecular docking studies for 5y and 5aa with TMV-CP (PDB code: 1EI7)
indicated that the two compounds were embedded well in the pocket (including
residues of TYR139, PHE12, ALA74, SER147, VAL75, THR136, ARG134, etc)
8

between the two subunits of TMV-CP (Figure. 3). Reports have demonstrated these
residues played a vital roles in the self-assembly of TMV particles.11-14 Such as
TYR139 and PHE12 are the components of hydrophobic cluster of aromatic
side-chains, and play an important role in a continuous belt of hydrophobic
interactions encircling each ring of the disk assembly.13 As well as Arg134, SER147,
ALA74, VAL75 showed strong interactions with another subunit of TMV-CP.11 Figure.
3 indicated the actions of the conventional hydrogen bond, π-Sulfur, π-Alkyl, and
Alkyl existed between both molecules and the above residues in TMV-CP (more
details can be found in Supporting information). For example, ALA-74 had strong
hydrogen bond with 5y (2.42 Å) and 5aa (2.41 Å), and pyrimidine showed an π-Alkyl
interaction with residue VAL-75, as well as an π-Alkyl interaction between the methyl
group and residue PHE-12. These interactions between small molecules and TMV-CP
are likely to weaken the interaction of two sub-unit of TMV-CP, thereby preventing
the self-assembly of TMV particle.
Figure 3. Molecular docking studies of compounds 5y (A, B, and C) and 5aa (D, E, F). A, B, D,
and E were depicted using PyMol soft, while 2D diagram C and F were drawn by Discovery
Studio 4.5.
In summary, a series of pyrazolo[3,4-d]pyrimidine derivatives containing a
9

Schiff base moiety was synthesized. The results of the bioactive tests for anti-TMV
activity revealed that several of the target compounds exhibited considerable antiviral
activity in vivo. In particular, compounds 5y and 5aa showed prominent inactivation
activity against TMV. Structure–activity relationship analysis showed that introduced
5-methylthiophen-2-yl enhanced the anti-TMV activity. Furthermore, the MST results
revealed that compound 5aa had strong binding capability with TMV-CP, and the
results of the molecular docking studies were consistent with the experimental results
(inactivation activity). These findings indicate that pyrazolo[3,4-d]pyrimidine with a
Schiff base may be a potential lead structure for developing novel anti-TMV agents.
Acknowledgements
This work was supported by the National Natural Science Foundation of China
(Nos. 21562012, No.21762012), the National Key Technologies R&D Project (No.
2017YFD0201302-03), the S&T Planning Project of Guizhou Province (No.
[2017]1402), and the Guizhou Province Governor Foundation for Outstanding Talents
(No. 2015-15#).
Supplementary Data
Supplementary data associated with this article can be found, in the online version,
at…..
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12

Table 1. Inhibitory effects of the test compounds against TMV at 500 µg/mL
Curative activity
(%)
Protection effect
(%)
Inactivation effect
(%)
Compd.
4a
58.4±2.9
33.5±3.1
64.9±1.7
62.8±3.2
48.0±1.9
37.1±3.2
30.2±2.3
43.3±3.9
50.4±0.5
37.1±1.3
56.8 ±2.1
55.9±1.4
56.5±2.3
47.8±4.2
42.2±2.4
32.3±2.1
29.2±3.9
32.7±1.9
52.1±2.5
55.8±1.7
54.2±0.9
55.6±1.9
45.7±2.2
57.2±0.9
51.9±2.2
42.6±2.8
32.8±1.3
14.2±1.3
62.7±2.4
59.4±0.9
62.7±0.3
58.5±1.3
22.7±1.2
61.4±1.7
59.6±0.9
57.1±1.3
58.8±1.6
47.5±0.9
54.3 2.9
62.6 2.3
60.6±0.9
52.8±0.8
63.2±2.7
62.6±2.4
33.7 ±1.7
49.9±2.1
44.1±1.1
41.5±2.1
45.7±1.2
45.8±1.8
23.1±2.5
54.2±1.5
40.3±1.1
52.2±1.9
50.8±2.3
42.6±1.3
39.4±2.7
60.1±1.5
65.1±1.6
64.8±4.9
63.5±1.7
62.5±1.4
56.9±1.6
62.8±1.4
63.4±3.1
51.9±1.5
46.6±2.1
22.3±1.4
64.0±1.6
62.0±1.9
62.0±1.9
65.2±1.9
22.8±1.3
57.6±1.2
62.0±1.2
59.1±2.3
34.6±1.5
43.5±1.4
63.2 1.8
64.2 2.1
68.7±2.3
64.3±1.3
74.2±2.4
62.2±2.5
83.4 ±2.4
60.5±1.4
54.5±2.5
74.2±1.1
65.5±1.3
58.9±2.7
85.2±3.1
84.0±3.3
80.1±1.6
82.5±2.3
64.8±2.5
42.1±2.3
63.5±3.1
72.9±1.3
88.9±0.9
76.4±2.4
64.1±2.3
78.0±2.1
62.4±3.1
89.6±1.8
84.5 2.3
67.4±2.5
82.2±1.7
34.8±2.3
92.1±0.7
72.8±2.1
94.6±2.1
85.0±2.1
32.9±3.1
76.2±2.4
67.1±2.3
65.0±1.5
75.5±1.9
51.9±2.7
78.3 1.5
4b
4c
4d
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
5s
5t
5u
5v
5w
5x
5y
5z
5aa
5ab
5ac
5ad
5ae
5af
5ag
5ah
III-31a
ningnanmycin
ribavirin
93.5±1.7
73.2±2.2
37.9 1.9
50.4 2.3
a. Compound in reference 6d
13

Table 2. The EC50 (µg/mL) of the title compounds against TMV
Compd.
Curative activity
Protection activity
Inactivation activity
4a
4c
4d
5g
5h
505.2 4.25
354.2 2.42
315.6 2.33
481.4 4.25
480.7 5.34
383.1 2.44
559.8 4.45
355.5 3.12
/
/
257.8 2.21
/
165.9 2.62
195.6 2.33
282.4 2.11
5o
5p
5q
5r
5t
659.24 4.24
485.29 3.67
401.1 2.56
337.2 2.33
400.6 4.31
380.94 3.32
340.5 3.12
365.7 3.31
280.5 2.13
301.6 3.16
181.3 2.32
157.25 2.45
364.1 2.65
/
109.5 3.11
5u
5y
402.4 3.14
272.8 2.31
392.9 4.12
283.5 2.23
400.6 5.12
317.2 1.34
400.4 4.13
353.5 3.23
306.1 3.41
367.6 2.12
296.8 2.15
313.2 2.12
412.7 2.15
274.4 3.32
144.3 2.46
70.3 2.43
301.1 3.18
53.8 1.43
155.5 2.15
167.2 1.34
329.7 2.15
5z
5aa
5ab
5ad
5ae
5af
5ag
427.2 4.43
446.4 2.31
324.51 3.15
432.1 2.54
328.5 3.01
398.3 2.15
55.35 1.45
/
ningnanmycin
165.95 2.15
ribavirin
/
435.99 4.23
124.05 3.5
14

Figures/Scheme Captions
Figure. 1. Design of pyrazolo[3,4-d]pyrimidine derivatives containing a Schiff base
moiety
Scheme 1. Reagents and conditions. (a) reflux, Ac2O; (b) 40% methylhydrazine,
EtOH, 80 oC; (c) triethoxyethane/triethoxymethane, reflux; (d) 80%N2H4·H2O, EtOH,
r.t; (e) 1,1-dimethoxy-N,N-dimethylmethanamine/aromatic aldehyde, Acetic acid,
reflux.
Figure. 2. Microscale thermophoresis (MST) results of compounds 5y (A), 5aa (B),
ribavirin (C), and ninnanmycin (D).
Figure. 3. Molecular docking studies of compounds 5y (A, B, and C) and 5aa (D, E,
F). A, B, D, and E were depicted using PyMolsoft, while 2D diagram C and F were
drawn by Discovery Studio 4.5.
15

Figure 1 Design of pyrazolo[3,4-d]pyrimidine derivatives containing a Schiff base
moiety
16

Scheme 1 Reagents and conditions. (a) reflux, Ac2O; (b) 40% methylhydrazine, EtOH,
80 oC; (c) triethoxyethane/triethoxymethane, reflux; (d) 80%N2H4·H2O, EtOH, r.t; (e)
1,1-dimethoxy-N,N-dimethylmethanamine/aromatic aldehyde, Acetic acid, reflux.
17

Figure 2. Microscale thermophoresis (MST) results of compounds 5y (A), 5aa (B),
ribavirin (C), and ninnanmycin (D).
18

Figure 3. Molecular docking studies of compounds 5y (A, B, and C) and 5aa (D, E,
F). A, B, D, and E were depicted using PyMol soft, while 2D diagram C and F were
drawn by Discovery Studio 4.5.
19

A series of pyrazolo[3,4-d]pyrimidine derivatives containing Schiff base was synthesized.
Compounds 5y and 5aa exhibited excellent antiviral activity against TMV. The interaction mode
of 5y and 5aa with TMV-CP was conducted through molecular docking and microscale
thermophoresis (MST) techniques.
20

Highlights
1. Pyrazolo[3,4-d]pyrimidines with a Schiff base moiety were synthesized.
2. Compounds 5y and 5aa exhibited excellent antiviral activity against TMV.
3. Interactions of 5y (5aa) with TMV-CP were conducted using MST and docking.
4. Compound 5aa showed strong combining capability with TMV-CP.
21