"On-Water" Facile Synthesis of Novel Pyrazolo[3,4-b]pyridinones Possessing Anti-influenza Virus Activity

Article abstract of DOI:10.1021/acscombsci.7b00016

A facile and versatile "on-water" protocol for the synthesis of pyrazolo[3,4-b]pyridinones was developed by the unprecedented construction of two rings and five new bonds in one-pot. It was proved that water was an important promoter of the reaction and P

Full text of DOI:10.1021/acscombsci.7b00016

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Article
“On-water” facile synthesis of novel pyrazolo[3,4-
b]pyridinones possessing anti-influenza virus activity
Li-Yan Zeng, Teng Liu, Jie Yang, Yueli Yang, Chun Cai, and Shu-Wen Liu
ACS Comb. Sci., Just Accepted Manuscript • Publication Date (Web): 05 Jun 2017
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“Onꢀwater” facile synthesis of novel
pyrazolo[3,4ꢀb]pyridinones possessing
antiꢀinfluenza virus activity
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LiꢀYan Zeng,† Teng Liu,† Jie Yang,† Yueli Yang,† Chun Cai, # Shuwen Liu*†‡
† Guangdong Provincial Key Laboratory of New Drug Screening, Guangzhou Key Laboratory of
Drug Research for Emerging Virus Prevention and Treatment, School of Pharmaceutical
Sciences, Southern Medical University, Guangzhou 510515, China.
‡
State Key Laboratory of Organ Failure Research, Guangdong Provincial Institute of
Nephrology, Southern Medical University, Guangzhou 510515, China.
#
Chemical Engineering College, Nanjing University of Science and Technology, Nanjing
210094, China.
KEYWORDS. Pyrazolo[3,4ꢀb]pyridinone, Oneꢀwater reaction, Catalystꢀfree, Green reaction
and separation, Antiꢀinfluenza virus.
ABSTRACT. A facile and versatile “onꢀwater” protocol for the synthesis of
pyrazolo[3,4ꢀb]pyridinones was developed by the unprecedented construction of two rings and
five new bonds in oneꢀpot. It was proved that water was an important promoter of the reaction
and PEG2000 was found to improve the reaction in terms of yield. 32 Derivatives were newly
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synthesized and most of them were prepared in an hour. The scope and limitation indicated that
electron withdrawing groups substituted on synthons, substituted benzoyl acetonitriles or aryl
aldehydes, were helpful to construct the pyrazolo[3,4ꢀb]pyridinones. The reaction media
PEG2000/H2O was successfully recycled and reused at least 5 times without any obvious
decrease in yield. The antiꢀinfluenza activities of the derivatives were evaluated and the
screening results highlighted two derivatives, which exhibited strong inhibitory activity against
H5N1 pseudovirus. These positive bioassay results implied that the library of potential
antiꢀinfluenza virus agent candidates could be rapidly prepared in an ecoꢀfriendly manner, and
provided a new insight into drug discovery for medicinal chemists.
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ꢀ INTRODUCTION
Proper modification of soꢀcalled ‘privileged structures’ allows for a rapid exploration of their
functional diversity space. Pyrazolopyridine is one of these privileged structures. When
appropriately modified, pyrazolopyridines can exert diverse biological activities1 or present
photophysical properties thanks to its DꢀπꢀA skeleton.2 The wellꢀknown example is Riociguat
(BAYꢀ63ꢀ2521) (Figure 1) ,3 which was launched in 2013 as an oral soluble guanylate cyclase
stimulator for the treatment of cardiopulmonary disease because of its vasodilatatory activity.
Other activities were also found in pyrazolopyridine or pyrazolo[3,4ꢀb]pyridinone derivatives,
such as antileishmania (Figure 1),4 antitumor, antimicrobial activities1c and so on. Nonetheless,
an underwhelming effort has been devoted to the efficient synthesis of the pyrazolopyridine
derivatives. To the best of our knowledge, the construction of the pyrazolopyridine skeleton
usually started from pyrazoles directly under heating or microwave conditions,1e, 2, 5 and a few
reports were involved in reacting the pyridine ring with hydrazine to get the pyrazolopyridines.1b,
1d, 1i, 6
However, existing substituted pyrazoles and pyridines were limited from commercial
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suppliers, and most of them have to be synthesized by chemists themselves. The resulting
lengthy syntheses increases the number of laboratory operations, the quantities of agents and
solvents used, regardless of the harsh reaction conditions in some cases. Such tedious synthetic
procedure thus limits the synthetic extension of the chemical space of pyrazolopyridine.
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R1
N
N
N
N
N
F
N
O
N
R2
O
NH
N
NH2
O
R3
H2N
N
O
R4
Riociguat BAY-63-2521
Anti-leishmania compounds
Figure 1. Structures of the functional pyrazolopyridines.
We found that, among the existing pyrazolopyridines, the aromatic substitution
rarely concurrently emerged at sites 6, 7 and 9 of pyrazolopyridine skeleton together
(Figure
2).
However,
compound
)ꢀone with
3ꢀ(4ꢀchlorophenyl)ꢀ4,5ꢀdihydroꢀ1,4ꢀdiphenylꢀ1
H
ꢀpyrazolo[3,4ꢀ
b
]pyridinꢀ6(7
H
,
aromatic groups substituted at positions 6, 7 and 9 (Figure 2), was highlighted when
screening the molecular database for the potential antiꢀinfluenza virus agents targeting
hemagglutinin(HA), the receptorꢀbinding protein that plays an important role in the
process of influenza virus entry into host cells. The synthetic scheme for
3ꢀ(4ꢀchlorophenyl)ꢀ4,5ꢀdihydroꢀ1,4ꢀdiphenylꢀ1
Hꢀpyrazolo[3,4ꢀb]pyridinꢀ6(7H)ꢀone is not
reported. Therefore, we firstly have to synthesize the target compound, and secondly we
have
to
develop
an
efficient
protocol
for
the
synthesis
of
3ꢀ(4ꢀchlorophenyl)ꢀ4,5ꢀdihydroꢀ1,4ꢀdiphenylꢀ1
H
ꢀpyrazolo[3,4ꢀ
b
]pyridinꢀ6(7
H)ꢀone and
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derivatives, so that the diversity of the target compound could be readily expanded to
better establish a structureꢀactivity relationship. It is not difficult to construct target
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molecule from 3ꢀ(4ꢀchlorophenyl)ꢀ1ꢀphenylꢀ1
Hꢀpyrazolꢀ5ꢀamine, however, limited
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diversity of substituted 1,3ꢀdiphenylꢀ1 ꢀpyrazolꢀ5ꢀamines (Figure 2) was available from
H
commercial suppliers, and thus we had to conduct a series of experiments to synthesize
them. Fortunately, the preliminary experiments provided a green and facile “onꢀwater”
protocol by constructing pyrazole and pyridine directly to synthesize the target compound
in oneꢀpot. Compared to traditional methods, our newly developed protocol is much more
feasible and flexible, in addition to its ecoꢀfriendly methodology. Herein, we would like
to disclose our progress for the synthesis of pyrazolo[3,4ꢀ
any catalyst, and present the preliminary bioassay results.
b
]pyridines in PEG/H2O without
Cl
R
N
N
N
Ar
Ar
N
6
N
O
7
1
5
H
R'
8
N
2
4
N9
N
H
3
Substituted 1,3-diphenyl-1H-pyrazol-5-amine
Intermediates
Ar
Potential anti-virus compound
Figure 2. Structure of the target compound.
ꢀ RESULTS AND DISCUSSION
Preliminary Experiment
We
commenced
with
the
threeꢀcomponent
reaction
(Scheme
1)
of
3ꢀ(4ꢀchlorophenyl)ꢀ1ꢀphenylꢀ1Hꢀpyrazolꢀ5ꢀamine 3{1,1}, benzaldehyde 4{1} and Meldrum acid
5
in
refluxing
EtOH,
and
fortunately
got
the
expected
product
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3ꢀ(4ꢀchlorophenyl)ꢀ4,5ꢀdihydroꢀ1,4ꢀdiphenylꢀ1Hꢀpyrazolo[3,4ꢀb]pyridinꢀ6(7H)ꢀone 6{1,1,1} in
49% yield after 2.5 hrs. The intermediate 3{1,1} was also prepared by treatment of
4ꢀchlorobenzoyl acetonitrile 1{1} and 1ꢀphenylhydrazine 2{1} in refluxing EtOH for 8 hrs
(Scheme 1). With these preliminary results, we tried to combine the two steps into one. Directly
treating four starting materials 1{1}, 2{1}, 4{1} and 5 in refluxing EtOH failed to furnish the
expected compound, therefore, we conducted this reaction by adding the starting materials in two
batches. 4ꢀchlorobenzoyl acetonitrile 1{1} and 1ꢀphenylhydrazine 2{1} were stirred in refluxing
EtOH, and when the intermediate 3{1,1} was formed according to TLC, benzaldehyde 4{1} and
Meldrum acid 5 were added. After stirring an additional 2 hrs, the target compound 6{1,1,1} was
detected, but failed to be isolated from reaction mixture (Scheme 2). We then replaced
4ꢀchlorobenzoyl acetonitrile 1{1} with benzoyl acetonitrile 1{2} or 4ꢀflorobenzoyl acetonitrile
1{4}, and repeated this procedure. Only compound 1{4} successfully provided the
corresponding product 3ꢀ(4ꢀfluorophenyl)ꢀ4,5ꢀdihydroꢀ1,4ꢀdiphenylꢀ1Hꢀpyrazolo[3,4ꢀb]pyridinꢀ
6(7H)ꢀone 6{1,4,1} based on the analysis of NMR spectrums and HRMS.
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Scheme 1. Synthesis of target compound in stepꢀwise
Cl
Cl
O
O
N
reflux
PhCHO
+
+
N
N
NH2
O
O
N
N
O
H
4{1}
5
6{1,1,1}
3{1,1}
Cl
O
CN
NHNH2
EtOH, reflux
Y. 53%
+
N
Cl
N
NH2
3{1,1}
1{1}
2{1}
Yield in 71% on water
Yield in 49% in EtOH
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Scheme 2. Oneꢀpot reaction for the synthesis of compounds 3ꢀ(4ꢀsubstituted
phenyl)ꢀ1ꢀphenylꢀ1Hꢀpyrazolꢀ5ꢀamine
7
8
9
R1
R1
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O
H
CN
N
PhCHO
NH2
N
N
+
N
O
O
N
N
O
R1
NH2
H
O
O
6
3
On-water reaction
R1 = Cl, H, no products, in EtOH
R1 = F, yielded in 19%, in EtOH
R1 = Cl, yielded in 34%
R1 = H, yielded in 29%
R1 = F, yielded in 46%
On the other hand, replacement of the hazard solvent with green solvents such as ionic liquids,
PEG and water has emerged as a commendable method to gratify the environmental issues.7
Additionally, the “onꢀwater” organic synthesis (a reaction where reactants are insoluble in
water), with many theoretical and practical advantages, has gained increasing attention.8
“Onꢀwater” synthesis provided not only a better reaction in terms of yield and rate but also a
green separation. So water was introduced into this reaction as a solvent. We firstly conducted an
“onꢀwater” reaction of intermediate 3ꢀ(4ꢀchlorophenyl)ꢀ1ꢀphenylꢀ1Hꢀpyrazolꢀ5ꢀamine 3{1,1},
benzaldehyde 4{1} and Meldrum acid 5. Surprisingly, a higher yield (71%) of target compound
was provided than in EtOH (49%) (Scheme 1). We subsequently carried out the oneꢀpot,
fourꢀcomponent reactions in refluxing water with the starting materials being added in two
batches (Scheme 2). Compared with the same reactions in EtOH, to our delight, all
4ꢀchlorobenzoyl acetonitrile 1{1}, benzoyl acetonitrile 1{2} and 4ꢀflorobenzoyl acetonitrile 1{4}
were successfully transformed to the corresponding products in 34%, 29% and 46% respectively
(Scheme 2). Water proved to be important to promote the oneꢀpot reaction for the synthesis of
target compound.
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Encouraged by these results, further optimization was conducted for the synthesis of
compound 6{1,1,1}, wherein compounds 4{1} and 5 were added into reaction after 1{1} reacted
with 2{1} for 40 minutes. The results were collected in Table 1. To improve the yield, iodine and
pꢀTsOH were tried to promote the reaction, unexpectedly, no product was detected from iodine
(Entry 2, Table 1), while TsOH slightly improved the yield of corresponding product 6{1,1,1} to
41% from 34% (Entry 3, Table 1). These two different results alert us that the solubility of the
organic compound in water should be considered, since compared to TsOH, iodine is hard to be
dissolved in water.
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Indeed, for many years water was seldom developed as reaction media for organic synthesis
8a, 8e, 8g,
9
because of the poor solubility for most reactants.7b,
As mentioned above,
PEG(polyethylene glycol) is also one of the green solvents with reduced flammability,
recyclability and a well documented toxicity and environmental burden data.10 With its special
properties, PEG/H2O provided a better reaction than water.7b, 11 To address the solubility issue,
we added PEGs to the oneꢀpot reaction of 1{1}, 2{1}, 4{1} and Meldrum acid 5.
The results collected in Table 1 indicated that 8 mg/mL of PEGs (from PEG200 to PEG2000,
Entries 4ꢀ8, Table 1) in water made less contribution to improve the yields, and the yield
decreased in PEG2000/H2O (8mg/mL). However, when the concentration was increased to 20
mg/mL, the yields were mainly improved. Surprisingly, PEG2000 gave the highest yield up to
76% (Entry 14, Table 1), and the total synthesis was completed in an hour. Further optimization
on PEGs (Entries 15ꢀ17, Table 1) did not provide better results. Then TsOH, iodine and TfOH
were applied to the reaction one by one. Iodine failed to form 6{1,1,1} again (Entry 19, Table 1),
while TsOH and TfOH made no improvement for the reaction (Entries 18 and 20, Table 1). We
next increased the amount of Meldrum acid 5 aiming to further improve the yield. The results
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collected in table 1 shows that 1.2 equivalent of Meldrum acid gave a slightly higher yield of
79% (Entry 21, Table 1). Therefore, treatment of 4ꢀchlorobenzoyl acenitrile 1{1},
1ꢀphenylhydrazine 2{1} in refluxing PEG2000/H2O (20mg/mL) for 40 minutes, followed by
adding benzaldehyde 4{1} and Meldrum acid 5 (1.2 equiv.) into the mixture will provide the
highest yield of product.
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Table 1. Optimization for reaction of 4ꢀchlorobenzoyl acenitrile 1{1}, 1ꢀphenylhydrazine 2{1},
benzaldehyde 4{1} and Meldrum acid 5.a
Entry
1
PEG/ H2O (mg/mL)
0
1{1}:2{1}:4{1}:5
1:1:1:1
1:1:1:1
1:1:1:1
1:1:1:1
1:1:1:1
1:1:1:1
1:1:1:1
1:1:1:1
1:1:1:1
1:1:1:1
1:1:1:1
1:1:1:1
1:1:1:1
1:1:1:1
1:1:1:1
1:1:1:1
1:1:1:1
1:1:1:1
Yield (%)b
34%
NRc
2
0
3
0
41%d
47%
44%
41%
37%
25%
44%
53%
66%
73%
76%
53%
67%
68%
73%d
NRc
4
PEG200 (8)
PEG400 (8)
PEG600 (8)
PEG1000 (8)
PEG2000 (8)
PEG200 (20)
PEG400 (20)
PEG600 (20)
PEG1000 (20)
PEG2000 (20)
PEG6000 (20)
PEG2000 (15)
PEG2000 (30)
PEG2000 (20)
PEG2000 (20)
5
6
7
8
9
10
12
13
14
15
16
17
18
19
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75%e
79%
75%
3
4
5
6
7
8
20
21
22
PEG2000 (20)
1:1:1:1
1:1:1:1.2
1:1:1:1.5
PEG2000 (20)
PEG2000 (20)
a Reaction conditions: 1{1} (0.5mmol) and 2{1} (0.5mmol), solvent (1.5mL),
refluxing for 40 minutes, 4{1} (0.5mmol) and 5 were added, refluxing them
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b
c
d
for 20 minutes. Isolated yield. 20mol% of iodine was added. 20mol% of
pꢀTsOH was added. e 20mol% of TfOH was added.
Substrate Scope
With the optimized conditions in hand, we investigated the scope and limitation of this
reaction. Under optimized conditions, a series of pyrazolo[3,4ꢀb]pyridinone derivatives 6 have
been obtained from substituted benzoyl acetonitriles 1, hydrazines 2 and aryl aldehydes 4 (Figure
3).
Substituted benzoyl acetonitriles 1
O
O
O
O
O
CN
CN
CN
CN
CN
O
F3C
Cl
F
1{1}
1{3}
1{4}
1{2}
1{5}
Hydrazines 2
H
N
H
N
N
NH2
N2H4 H2O
NH2
2{2}
2{1}
2{3}
Aryl aldehydes 4
CHO
CHO
CHO
Cl
CHO
OH
CHO
CHO
Cl
O
HO
3{1}
3{2}
3{3}
3{4}
3{5}
3{6}
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2
3
4
5
6
HO
O
CHO
CHO
CHO
HO
O
CHO
O
CHO
NC
Br
OH
3{7}
3{8}
3{9}
3{10}
3{11}
7
8
9
Figure 3. Building blocks for compound 6.
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Several paraꢀsubstituted benzoyl acetonitriles 1 were initially applied to the reaction. Most of
them successfully provided good yields of corresponding products 6{1ꢀ5,1,1} in 1 hour. The
results collected in Scheme 3 showed that electronꢀwithdraw groups 4ꢀCl, 4ꢀF, and 4ꢀCF3 on
benzoyl acetonitriles made a positive influence on the reaction in terms of yield, while benzoyl
acetonitrile 1{2} gave a lower yield of 6{2,1,1} in 63%, and we even failed to get pure
compound 6{3,1,1} from electronꢀdonor group 4ꢀMeO substituted benzoyl acetonitrile. Next, a
series of substituted benzylaldehydes 4{1ꢀ10} were tested, and most of them were smoothly
transformed into expected corresponding compounds. Generally, benzylaldehydes with
electronꢀwithdrawing groups were more active than those with electronꢀdonated groups. The
electronꢀwithdrawing group substituted benzylaldehydes including 2ꢀchlorobenzylaldehyde 4{4}
were all completely transformed in 1 h, and provided good yield up to 75% 6{1,8,1}, while we
got moderate yields from electronꢀdonated ones with prolonged reaction time up to 8 hrs. We
observed that substituent at orthoꢀposition resulted in lower yield because of steric hindrance, but
made no difference in reaction time, as product 6{1,4,1} (oꢀchloroꢀ) was generated after reaction
for 1 hour and yielded in 52%, much lower than 6{1,2,1} (pꢀchloroꢀ, yielded in 72%). However,
oꢀhydroxy benzylaldehyde 4{6} failed to provide the expected product 6{1,6,1}. We also get the
products 6{2,2,1} and 6{2,3,1} by reacting benzoyl acetonitrile 1{2} with 1ꢀphenylhydrazine
2{1}, Meldrum acid 5 and 4ꢀchlorobenzylaldehyde 4{1} or 4ꢀmethoxylbenzylaldehyde 4{3},
respectively. It is implied that electronꢀdonated groups substituted on synthons 1 and 4 made a
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negative contribution to the reactions in terms of yield and rate. To confirm this result, we then
undertook the reaction of 1{1}, 2{1}, 5 and 4ꢀhydroxyꢀ3,5ꢀdimethoxybenzaldehyde 4{9} or
4ꢀhydroxyꢀ3ꢀmethoxybenzaldehyde 4{10}, under standard conditions. With both –OH and ꢀOMe
groups on synthon 4, although pure product 6{1,1,9} and 6{1,1,10} were successfully obtained
in 45% and 47% respectively, but reaction time was extended to 8 hours (overnight).
Obviously, 4ꢀCF3 substituted benzoyl acetonitrile 1{5} gave the best yield. We then applied
4ꢀOMe benzylaldehyde 4{3}, 4ꢀCN benzylaldehyde 4{8} and furfural 4{11} to the reactions that
1{5} participated. Likewise, compound 6{5,1,3} was obtained in 66%, higher than 6{1,1,3}
(60%), while 6{5,1,8} was yielded in 77%, slightly higher than 6{1,1,8} (75%). With these
results, 4ꢀCF3 benzoyl acetonitrile 1{5} was confirmed to be more active than 4ꢀchlorobenzyl
acetonitrile 1{1}. Furfural was also transformed into the corresponding compound 6{5,1,11}
successfully.
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Scheme 3. The synthesis of 4,5ꢀdihydroꢀ1,3,4ꢀtriphenylꢀ1Hꢀpyrazolo[3,4ꢀb]pyridineꢀ6(7H)ꢀones
R1
R1
R3
4
O
R3CHO
CN
N
PhNHNH2
N
+
N
N
O
O
O
H
N
R1
NH2
O
O
1
2{1}
6{1-5,1,1-11}
5
3{1-5,1}
O
Cl
F
N
N
N
N
N
N
O
N
N
O
N
N
N
O
N
O
H
H
H
H
6{2,1,1}, 63%
6{3,1,1}, 00%a
6{4,1,1}, 77%
6{1,1,1}, 79%
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Cl
Cl
O
F3C
Cl
Cl
5
6
7
8
Cl
O
N
N
N
N
N
N
N
N
O
N
O
N
N
N
H
O
H
H
H
9
10
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6{5,1,1}, 83%
6{1,1,2}, 72%
6{1,1,3}, 60%b
6{1,1,4}, 52%
OH
Br
Cl
CN
Cl
Cl
Cl
OH
N
N
N
N
N
N
N
N
O
N
O
N
N
O
N
O
H
H
H
H
6{1,1,5}, 51%c
6{1,1, 6}, 00%d
6{1,1,7}, 69%
6{1,1,8}, 75%
OH
O
O
Cl
OH
Cl
Cl
O
O
O
N
N
N
N
N
N
N
N
O
N
O
N
N
N
O
H
H
H
H
6{1,1,9}, 45%e
6{2,1,2}, 64%
6{2,1,3}, 56%f
6{1,1,10}, 47%g
CN
O
F3C
F3C
F3C
O
N
N
N
N
N
N
N
O
N
O
N
H
O
H
H
6{5,1,8}, 77%
6{5,1,3}, 66%
6{5,1,11}, 42%
The reaction conditions: Refluxing 1 (0.5mmol) and 2{1} (0.5mmol) in PEG2000/H2O
(30mg/1.5mL) for 40 minutes, then compounds 4 (0.6mmol) and 5 (0.5mmol) were added.
Reactions were monitored by TLC, completed in 1 hour totally. a Reaction failed to provide pure
compound 6{3,1,1}. b Reaction was completed in 2 hours. c Reaction was completed in 7 hours. d
Reaction failed to provide comound 6{1,1,6}. e Reaction was completed after stirring overnight. f
Reaction was completed in 2 hours. g Reaction was completed after stirring overnight.
Next, we replaced 1ꢀphenylhydrazine 2{1} with hydrazine hydrate 2{2}, and conducted a
series of reactions of 1{1}, 2{2}, 5 and various benzyl aldehydes 4 (Scheme 4). Fortunately, a
series of bis(substituted phenyl)ꢀ4,5ꢀdihydroꢀ1Hꢀpyrazolo[3,4ꢀb]pyridineꢀ6(7H)ꢀones were
smoothly formed in 1 hr under optimized reaction conditions, with moderate to good yields. In
general, higher yields were provided compared with 1ꢀphenylhydrazine 2{1}. For example, we
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got compound 6{1,1,1} in 79%, but compound 6{1,2,1} was yielded in 82%. It is implied that
hydrazine hydrate 2b is more active than 2a. Consistent with 1ꢀphenylhydrazine 2a, the results
collected in scheme 4 also indicated that electronꢀwithdrawing group substituted benzyl
aldehydes are more active. As shown in scheme 4, 81% yield of compound 6{1,2,2} was
obtained from 4ꢀchlorobenzylaldehyde, while 4ꢀmethoxybenzylaldehyde gave compound 6{1,2,3}
in 71%, and 4ꢀhydroxybenzaldehyde and 4ꢀhydroxyꢀ3,5ꢀdimethoxybenzaldehyde provided much
lower yields of corresponding products 6{1,2,5} and 6{1,2,9}. We also observed that, compared
to 4ꢀchlorobenzylaldehyde, 57% yield of expected product 6{1,2,4} was produced from
2ꢀchlorobenzylaldehyde, which implied that steric hindrance affected the reaction in a certain
degree. In addition, 4ꢀmethoxylbenzoyl acetonitrile 1{3} was tried but provided the expected
compound 6{3,2,2} in moderated yield (Scheme 4) this time, since in previous experiment, we
failed to get compound 6{3,1,1} (Scheme 3). This result further confirmed that hydrazine
hydrate 2{2} was more active. Besides, we also get the compound 6{1,3,2} from
1ꢀ(pyridinꢀ2ꢀyl)hydrazine 2{3}, although in low yield 37% with prolonged reaction time (2 hrs).
Based on these results, it is apparent that hydrazine hydrate 2{2} is the most active for this
reaction among the three hydrazines 2{1}, 2{2} and 2{3}, while 1ꢀ(pyridinꢀ2ꢀyl)hydrazine 2{3}
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is less active than 1ꢀphenylhydrazine 2{1}.
Scheme 4. The synthesis of 4,5ꢀdihydroꢀ3,4ꢀdiphenylꢀ1Hꢀpyrazolo[3,4ꢀb]pyridineꢀ6(7H)ꢀones
R1
R1
R3
4
O
R3CHO
CN
R2NHNH2
+
N
O
O
N
N
R1
N
O
H
N
O
O
NH2
R2
1
2
R2
5
6{1, 2-3,1-5}
6{1,2,9} and 6{3,2,2}
3{1-5,1-3}
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Cl
O
Cl
Cl
Cl
Cl
5
6
7
8
Cl
O
N
N
N
N
N
N
N
N
N
O
N
H
O
N
H
O
N
H
H
H
H
H
H
6{1,2,2}, 81%
6{1,2,4}, 57%
6{1,2,1}, 82%
6{1,2,3}, 73%
9
10
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Cl
Cl
OH
Cl
Cl
OH
O
Cl
O
O
O
N
N
N
O
H
N
N
N
N
N
N
N
N
O
N
O
N
H
H
H
H
H
H
6{1,2,5}, 58%
6{1,2,9}, 61%
6{3,2,2}, 53%
6{1,3,2}, 37%a
The reaction conditions: Refluxing 1 (0.5mmol) and 2{2} (0.5mmol) in PEG2000/H2O
(30mg/1.5mL) for 40 minutes, then compounds 4 (0.6mmol) and 5 (0.5mmol) were added.
Reactions were monitored by TLC, completed in 1 hour totally. starting material hydrazine
a
hydrate 2{2} was replaced by 1ꢀ(pyridinꢀ2ꢀyl)hydrazine 2{3}, and the reaction was completed in
2 hours.
To further broaden the scope of the reaction, we tried to explore more building blocks to
replace Meldrum acid 5, and thus carried out another series of reactions. The common used
building blocks containing active methylene, malononitrile, ethyl 2ꢀcyanoacetate,
pentaneꢀ2,4ꢀdione, ethyl 3ꢀoxobutanoate, 2ꢀoxopropanoic acid were firstly screened, but none of
them were transformed into the expected compound, not even ethyl 2ꢀoxobutanoate. We then
tried three diones (Entries 1ꢀ5, Table 2). To our delight, 5,5ꢀdimethylcyclohexaneꢀ1,3ꢀdione 7,
structurally similar with Meldrum acid 5, was successfully transformed into the expected
compound
8{1,1,1}
in
55%
yield
(Entry
1,
Table
2).
Focusing
on
5,5ꢀdimethylcyclohexaneꢀ1,3ꢀdione 7, 4ꢀmethoxybenzaldehyde 4{3} was firstly tested and gave
corresponding product 8{1,1,3} in 43% yield (Entry 2, Table 2), lower than 8{1,1,1} in
reasonable. Next, benzoylacetonitril 1{2} was applied to this reaction instead of 1{1} and
produced 8{2,1,2} in 57% yield (Entries 3, Table 2). From these results,
5,5ꢀdimethylcyclohexaneꢀ1,3ꢀdione 7 is less active than Meldrum acid in terms of yield for this
protocol. And the synthesis of compound 8{1,2,2} (Entry 4, Table 2) further verified this result,
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wherein hydrazine hydrate 2{2}, presented higher active than 1ꢀphenylhydrazine 2{1}, but gave
much lower yield (49%) than 6{1,2,2} (81%, Scheme 4). The last building block we tested is
cyclohexaneꢀ1,3ꢀdione 9, under optimized conditions, the reaction of 1{2}, 2{1}, 4{1} and 9
provided the anticipated results with expected product 10{2,1,1} and reasonable yield of 55%.
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Table 2. The screening of building block 7 and 9.a
R1
R1
4
R3
O
O
R3CHO
CN
R2NHNH2
O
O
N
+
R4
R4
N
N
N
H
R1
N
R2
NH2
R4
R4
1
2
R2
8 or 10{2,1,1}
7, R4 = CH3
9, R4 = H
3
Entry
1
1
2
3
Building block
Expected product
Yield (%)b
Cl
O
NHNH2
O
CN
CHO
55
N
Cl
N
N
H
4{1}
2{1}
1{1}
8{1,1,1}
O
Cl
O
CHO
NHNH2
O
CN
43
2
3
O
N
Cl
N
N
H
4{3}
2{1}
1{1}
O
O
8{1,1,3}
7
Cl
CHO
O
NHNH2
O
CN
57
Cl
N
N
2{1}
N
H
1{2}
4{2}
8{2,1,2}
8{1,2,2}
Cl
Cl
O
CHO
N2H4H2O
CN
O
49
55
4
5
Cl
Cl
N
4{2}
2{2}
1{1}
N
H
N
H
O
NHNH2
O
CHO
O
O
CN
N
N
9
N
4{1}
H
1{2}
2{1}
10{2,1,1}
a
Reaction conditions: 1 (0.5mmol) and 2 (0.5mmol) , PEG2000 (30mg) and H2O (1.5mL),
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refluxing for 40 minutes, 4 (0.5mmol) and 7 or 9 (0.6mmol) were added, refluxing for 20
minutes. b Isolated yield.
6
7
8
9
Recyclability of Reaction Media
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Finally, the recyclability and reusability were assessed by investigating the reactions starting
from Meldrum acid 5 (Figure 4, A). We commenced with the model reaction of 4ꢀchlorobenzoyl
acetonitrile 1{1}, 1ꢀphenylhydrazine 2{1}, benzaldehyde 4{1} and Meldrum acid 5 in refluxing
PEG2000/H2O (20mg/mL). After cooling the reaction mixture to room temperature, an inꢀtube
extraction with 3 X 1.5 mL of AcOEt was conducted (Figure 4, Ad), followed by the process of
purification to provide the product. The water phase (reaction media) was thus simply separated
and reused for the next cycle (Figure 4, Aa). With the same procedure, the reaction media was
recycled and reused over 8 times for the synthesis of compound 6{1,1,1} (Figure 4, B), and we
repeated this recycle experiments for 3 times. It was observed that the yield was always slightly
increased when the first recycled reaction media was used, and the substantial decrease in yield
occurred when the reaction media has been recycled for 6 or 7 times, and the reaction media
looks not clear as seen in Pict. Aa in Figure 4.
Thereafter, we carried out the crossꢀrecycling experiment in order to apply the recycled
reaction media to different reactions (Figure 4, C). The first recycled reaction media from model
reaction was reused for the reaction of benzoyl acetonitrile 1{2}, 1ꢀphenylhydrazine 2{1},
benzaldehyde 4{1} and Meldrum acid 5. To our delight, the starting material not only was
transformed into the right compound 6{2,1,1} but also provided a higher yield (68%) than the
corresponding yield (63%) obtained in fresh PEG2000/H2O. The second recycled reaction media
was subsequently applied to the model reaction again, and gave a 77% yield of compound
6{1,1,1}, then the reaction media was recycled the third time and reused for the synthesis of
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6{2,1,1} the second time. The recycling experiments continued like this, till the yield of 6{1,1,1}
decreased to 56% after the reaction media has been recycled for 7 times. In general, the reaction
media can be reused at least 5 times not only in the same reaction but also in other reactions in
crossꢀrecycling fashion without any obvious change in yields.
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11
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60
Figure 4. The recycle of the reaction media PEG2000/H2O. (A) procedure for the recycle and
reuse of reaction media PEG2000/H2O, (B) PEG2000/H2O recyclability study for the synthesis
of compound 6{1,1,1}, (C) crossꢀrecyclability of PEG2000/H2O for the synthesis of compound
6{1,1,1} and 6{2,1,1}.
Mechanism Discussion
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With these results, we tried to probe into the reaction mechanism. The synthesis of compound
6{2,1,1} was selected as model reaction. Although the intermediate 3{2,1} was confirmed to be
important for the promotion of the reaction, the reaction process after intermediate 3{2,1}
formed was unknown. We then isolated the main side product 11{2,1,1} (Figure 5), and the
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1
structure was confirmed by H NMR and MS. Notably, two molecules of intermediate 3{2,1}
instead of Meldrum acid 5 reacted with benzylaldehyde 4{1} to form the structure 11{2,1,1}.
Therefore, we can reasonably hypothesize that compared with Meldrum acid, intermediate 3{2,1}
is prior to react with aldehyde. From this point of view, the formation of intermediate 3{2,1} is
the key of this reaction, and also explained why we were failed to get compound 6{1,1,1} from
the reaction that four starting materials added in one batch in the preliminary experiment.
N
N
N
N
NH2 H2N
11{2,1,1}
Figure 5. The side product 11{2,1,1} obtained from the reaction for the synthesis of compound
6{2,1,1}
Technically, at a higher temperature 100 ℃, water can be ionized and provide high
concentration of H+. These almost bare protons can penetrate through the waterꢀoil phase
boundary as proposed by Marcus and Jung,12 and stay inside of the PEGꢀwater barrier to provide
catalytic sites under vigorous agitation, and thus promoted the formation of compounds 6. Based
on these knowledge, we proposed the mechanism of this onꢀwater reaction and showed in
Scheme 5. The penetration of proton provided catalytic H+, while PEG helped to increase the
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chance of collision for all the reactants as a coꢀsolvent. After the second batch of starting
materials (aldehyde and Meldrum acid) were added, the intermediate 3{2,1} attacked to the
aldehyde prior than Meldrum acid. Intermediate was supposed to be formed and react to
Meldrum acid or another molecule of 3{2,1}. Corresponding compounds 6{2,1,1} and 11{2,1,1}
were thus produced.
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Scheme 5. The proposed mechanism based on the “onꢀwater” synthesis of compound 6{2,1,1}
H
N
H
N
H2O
Ph
Ph
N
N
N
H
Ph
N
Ph
H
O
H
H
H
PEG
2{1}
H
4{1}
H
N
O
PEG
H
H
N
Ph
H
Ph
H
O
H
H
O
H
N
Ph
O
H
H
Ph
H2N
H
O
O
N
N
3{2,1}
1{2}
Ph
H
H
Water
H
O
Ph
PEG
H
Ph
H
N
N
N
N
N
N
O
N
O
Ph
H
O
NH
H
NH
Ph
Ph
N
OH
O
6{2,1,1}
N
N
N
N
H
O
Ph
O
Ph
NH2 H2N
Intermediate
PEG
N
O
O
Ph
H2N
O
Ph
O
O
O
Ph
O
5
3{2,1}
11{2,1,1}
O
N
N
O
H
H2N
CO2
Ph
Anti-influenza virus activity
The antiꢀinfluenza virus activities of the newly synthesized pyrazolo[3,4ꢀb]pyridinone
derivatives were evaluated by testing the inhibitory activity of 6, 8 and 10 on H5N1
pseudovirus infection. To our delight, 2 derivatives were highlighted with good inhibitory
activity against H5N1 pseudovirus and the results shown in Table 3 indicate that 6{5,1,1} and
6{5,1,11} exhibited strong inhibitory activity with IC50 value of 14.022 ꢁM and 2,68 ꢁM
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7
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9
respectively. Obviously, 6{5,1,11} provided even better result than 6{1,1,1} (IC50 value of
3.50 ꢁM)13 in terms of IC50, and comparable CC50 value. The preliminary pseudovirus
neutralization assay results imply this newly developed green synthesis methodology can provide
potential antiꢀinfluenza virus agents and encourage further investigations.
10
11
12
13
14
15
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60
Table 3. Highlighted results from screening the antiꢀinfluenza virus activity of compounds
6,8,10. a
IC50 (µM)
Mean ± SD
14.02±1.57
CC50 (µM)
Mean ± SD
153.23±7.31
Compounds
6{5,1,1}
6{5,1,11}
6{1,1,1}c
2.68±0.17
3.05±0.37
>200
>200
a The screening of the antiꢀinfluenza activity for compounds 6,8,10 were done in pseudovirus
neutralization assay against Influenza A/Thailand/Kan353/2004 H5N1. b 6{1,1,1} is used as a
positive control.
ꢀ CONCLUSION
In summary, we developed a novel protocol for the synthesis of 6,7ꢀ aromatic substituted
pyrazolopyridine derivatives 6 from substituted benzoyl acetonitrils 1, 1ꢀphenylhydrazine 2{1}
or hydrazine hydrate 2{2}, substituted benzaldehydes 4 and Meldrum acid 5 or
5,5ꢀdimethylcyclohexaneꢀ1,3ꢀdione 7 in a simple manner, involving a fourꢀcomponent oneꢀpot
reaction without any catalyst in PEG/H2O and the reaction can be completed in an hour. The
reaction was extremely expanded by using various benzoyl acetonitrils, benzaldehydes and
hydrazines, as well as by screening the building blocks that contained active methylene. It was
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concluded that electronꢀwithdrawing groups on benzoyl acetonitrils 1 and benzaldehydes 4 could
lead to better yield, hydrazine hydrate 2{2} was proved to be more active than
1ꢀphenylhydrazine 2{1} by comparing the yields. Above all, the reaction media PEG/H2O not
only promoted the reaction but also facilitate the separation. The organic phase can be easily
collected by an inꢀtube extraction. So that the reaction media (water phase) was successfully
recycled and reused more than 5 times without an obvious decrease of yield in different reactions
in crossꢀrecycling manner. The side product was isolated and the reaction mechanism was
discussed. All of these newly prepared derivatives were screened for the antiꢀinfluenza activities,
6{5,1,1} and 6{5,1,11} were found to exhibit strong inhibitory activity on H5N1 pseudoviruse.
This positive result indicates that a facile and flexible protocol has been developed to synthesize
of a library of potential antiꢀinfluenza agents rapidly in an ecoꢀfriendly manner, and further
proved our success of applying green synthesis to drug discovery. The further structure
modification and related antiꢀinfluenza virus testing are ongoing in our group.
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ꢀ ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publication website at DOI:
Experimental details and spectroscopic characterization of all the compounds, as well as the 1H
and 13C NMR spectrums for all products are compiled in Supporting Information.
ꢀ AUTHOR INFORMATION
Corresponding Author
*Eꢀmail: liusw@smu.edu.cn
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Author Contributions
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The manuscript was written through contributions of all authors. All authors have given approval
to the final version of the manuscript.
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Funding Sources
We thank the Natural Science Foundation of China (Nos. U1301224) to Shuwen Liu, and the
China Postdoctoral Science Foundation funded project (Nos. 2016M602496) to Liyan Zeng for
financial support.
Notes
The authors declare no competing financial interest.
ꢀ ACKNOWLEDGMENT
The authors are grateful to the platforms of Guangdong Provincial Key Laboratory of New Drug
Screening in Southern Medical University for providing financial and technical support.
ꢀ ABBREVIATIONS
PEG, polyethylene glycol; NMR, nuclear magnetic resonance; HRMS, high resolution mass
spectrometer; TLC, thin layer chromatography.
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“Onꢀwater” facile synthesis of novel
pyrazolo[3,4ꢀb]pyridinones possessing
antiꢀinfluenza virus activity
LiꢀYan Zeng,† Teng Liu,† Jie Yang,† Yueli Yang,† Chun Cai, # Shuwen Liu*†‡
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