One-pot construction of spirooxindole backbone via biocatalytic domino reaction

Article abstract of DOI:10.1016/j.tetlet.2017.06.031

A simple and environmentally friendly method to synthesize spiropyrazolo[3,4-b]pyridines derivatives starting from isatin, cyclic-1,3-diketone and 3-methyl-5-aminopyrazole was developed. With the optimized conditions for enzymes, solvents, enzyme loading and reaction time in hand, 9 compounds were obtained in acceptable yields. Moreover, the investigation in fluorescent properties of these products showed their potential application in the field of new fluorescent material.

Full text of DOI:10.1016/j.tetlet.2017.06.031

Accepted Manuscript
One-Pot Construction of Spirooxindole Backbone via Biocatalytic Domino Re-
action
Yi-Ru Liang, Yu-Jing Hu, Xue-Han Zhou, Qi Wu, Xian-Fu Lin
PII:
S0040-4039(17)30756-6
DOI:
http://dx.doi.org/10.1016/j.tetlet.2017.06.031
TETL 49023
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Accepted Date:
29 May 2017
12 June 2017
Please cite this article as: Liang, Y-R., Hu, Y-J., Zhou, X-H., Wu, Q., Lin, X-F., One-Pot Construction of
Spirooxindole Backbone via Biocatalytic Domino Reaction, Tetrahedron Letters (2017), doi: http://dx.doi.org/
10.1016/j.tetlet.2017.06.031
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One-Pot Construction of Spirooxindole
Backbone via Biocatalytic Domino Reaction
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Yi-Ru Liang a , Yu-Jing Hu a , Xue-Han Zhou a , Qi Wu a* and Xian-Fu Lin a*

1
Tetrahedron Letters
journal homepage: www.els evier.com
One-Pot Construction of Spirooxindole Backbone via Biocatalytic Domino Reaction
Yi-Ru Liang a , Yu-Jing Hu a , Xue-Han Zhou a, Qi Wu a ∗ and Xian-Fu Lin a ∗
aDepartment of Chemistry, Zhejiang University, Hangzhou 310027, People’s Republic of China
ARTICLE INFO
ABSTRACT
Article history:
Received
A simple and environmentally friendly method to synthesize spiro-pyrazolo[3,4-b]pyridines
derivatives starting from isatin, cyclic-1,3-diketone and 3-methyl-5-aminopyrazole was
developed. With the optimized conditions for enzymes, solvents, enzyme loading and reaction
time in hand, 9 compounds were obtained in acceptable yields. Moreover, the investigation in
fluorescent properties of these products showed their potential application in the field of new
fluorescent material.
Received in revised form
Accepted
Available online
.
Keywords:
Spirooxindole
Enzyme
One-pot
Fluorescent properties
1. Introduction
enzyme with new catalytic characters) makes it possible to
perform multicomponent reaction (MCRs) by one enzyme. Up to
Spirooxindole derivatives are widely found in natural
products, such as progesterone receptor modulators,1
now, a series of elegant works of enzymatic promiscuity have
been discovered, such as aldol reactions,14-16 Michael addition
antimicrobial,2-4 antimalarial5, and so on. Attracted by the
6
18
21
reactions,17,
Henry reactions,19 Mannich reactions20,
and
various one-pot domino reaction.22-25
specific structure and biological activities of spirooxindole
derivatives, much attention has been paid by chemists in this
field. In recent years, some spirooxindole scaffolds was
constructed via organometallic and organocatalytic methods. For
example, spirooxindoles containing [5.5]-fused heterocycle were
obtained by Jia and co-workers through a three-component
bicyclization reaction of isocyanide, substituted allenoate and
isatin.7 Praveen has reported microwave assisted synthesis of
spirooxindoles via tandem double condensation between isatins
and 4-hydroxycoumarin in the presence of gold catalysis in
2016.3 Tiwari and co-workers developed an efficient synthetic
method for the synthesis of new 2,3-dihydrooxazole-
spirooxindoles hybrids, which showed good antibacterial activity
and antifungal activity as antimicrobial agents.4 What is more,
Tin powder-promoted one-pot synthesis of 3-spirooxindole
derivatives or 3,3 ’ -disubstituted 2-oxindoles was reported by Hu
in the presence of a catalytic amount of Bronsted or Lewis acids.8
However, catalysts used in these methods always involve
environmental problem, high cost or complicated process.
Therefore, it is desirable to seek environmentally friendly and
economical catalysts in this field.
Spirocyclic skeletons catalyzed by various enzymes have been
reported in recent years. For example, Lipase from porcine
pancreas (PPL) could catalyze one-pot multiple reaction to
synthesize spirooxindole derivatives through the Knoevengel
condensation, Michael addition and cyclization in ethanol with
the presence of water.26 In 2015, α -Amylase from hog pancreas
was discovered the same catalytic activities as PPL for this
reaction and the same kind of spirooxindole pyrans were
obtained.27 Furthermore, Pepsin from porcine gastric mucosa
was used as a tool in the domino Knoevenagel/Michael/Michael
reaction for the synthesis of spirooxindole derivatives in
methanol.28 Aminoacylase displayed catalytic promiscuity in
both [5+1] double Michael additions and domino Knoevenagel
condensation-Michael addition-intramolecular cyclization to
obtain spiro[5.5]undecane derivatives and new spirooxindole
derivatives, correspondingly.29,
30
Besides, lipase could also
initiate multicomponent reaction for the one-pot synthesis of
spirooxazino derivatives starting from aldehydes, activated
olefins, cyclohexanone and acetamide.31, 32 However, in order to
increase the diversity of spirooxindoles, the further developments
of enzymatic methods are still highly desirable.
Enzymatic catalysis, featured as great environmental
acceptability, mild reaction conditions and high efficiency, is an
attractive alternative to conventional chemical catalysis during
Herein, we report a one-pot papain-catalyzed reaction to
synthesize a series of spiro-pyrazolo[3,4-b]pyridines starting
the past decades.9-13 Recently, enzymatic promiscuity (one
———
∗ Xian-Fu Lin. Tel.: +86-571-8795-3001; fax: +86-571-8795-2618; e-mail: llc123@zju.edu.cn
∗ Qi Wu. Tel.: +86-571-8795-3001; fax: +86-571-8795-2618; e-mail: wuqi1000@163.com

2
Tetrahedron Letters
3-methyl-5-
from
isatins,
cyclic-1,3-diketones
and
of 50% was obtained in methanol and a similar yield of 48% was
aminopyrazoles for the first time. It is particularly exciting to
obtain most of products with strong fluorescence emission
ability, providing great potential application in the field of
fluorescent material.
achieved in ethanol (Table 2, entries 8 and 9). However, some
polar solvents, such as THF, water and ethylene glycol (EG) led
to lower yields (Table 2, entries 3, 10 and 11). To our surprise,
no product was obtained in dimethyl sulfoxide (DMSO) and
dimethylformamide (DMF) (Table 2, entries
1 and 2).
2. Results and discussion
Furthermore, trace of the desired compound was achieved in
acetonitrile, toluene, methyl tertiary butyl ether (MTBE) and
dioxane, which could be attributed to the poor solubility of
substrates in these solvents (Table 2, entries 4-7). In view of
environment protection, ethanol was finally chosen as the most
suitable medium for this enzymatic reaction.
As part of our program to develop a new enzymatic and
environmentally friendly method for spiro-heterocyclic
compounds, isatin (1a), 3-methyl-5-amino-1H-pyrazol (2a) and
1,3-cyclohexanedione (3a) were chosen as model substrates to
form 4a. In order to find the suitable enzyme for the model
reaction, various hydrolytic enzymes were investigated in our
initial research (Table 1).
Then the amount of papain was taken into account. It can be
observed that the yield increased from 23% to 56%, along with
the amount of papain increasing from 20 mg to 80 mg, (Table 2,
entries 9, 12 and 13). Nevertheless, higher enzyme loading
resulted in little increase of the product yield (Table 2, entries 14
and 15). Therefore, 80 mg papain was enough to catalyze the
reaction of isatin, 3-methyl-5-amino-1H-pyrazol and cyclic-1,3-
diketone. What is more, after the investigation of reaction time,
72 h was chosen as the optimal reaction time because the reaction
has already reached equilibrium during this time (Table 2, entries
16-19). The results of temperature screening showed that the
increase of reaction temperature from 20 oC to 50 oC will benefit
The results showed that only 11% yield of product was
obtained if the reaction was carried out in the absence of catalysts
(Table 1, entry 1). In contrast, the product (4a) was obtained in
48% yield after 48 h in EtOH when papain was added as catalyst
(Table 1, entry 4). Meanwhile, some other enzymes displayed
less activity for this reaction. For example, DA, AA and PPL
exhibited low activity in yields of 24%, 25% and 22%,
respectively (Table 1, entries 2, 3 and 5). However, CAL-B and
MJL showed higher catalytic activity (Table 1, entries 6 and 7)
compared with DA, AA and PPL. Next, urea-pretreated papain
was tested to confirm the role of enzyme in overall reaction
system. As a denaturation agent for enzymes, urea was employed
to pretreat the papain. When the urea-pretreated papain was used
in the model reaction, the desired product was obtained in a yield
of 14%, which indicated that the specific three-dimensional
structure of papain was crucial for its catalytic activity (Table1,
entries 8). The above investigations suggested that papain was
the most suitable enzyme for this reaction.
o
the reaction, and the best yield of 63% was obtained at 50 C
(Table 2, entries 18, 20-22). Besides reaction medium, enzyme
loading, reaction time and reaction temperature, sometimes water
content also affect enzymatic reactions by changing the
conformational flexibility of the enzyme.33 However, in our case,
no significant change of yields was observed when 0 to 30%
water was added into the reaction medium (Scheme S1).
Table 2. Screen of reaction conditions for one-pot reaction a
Table 1. One-pot reaction catalyzed by different enzymes a
Entry
Solvent
Amount
/mg
Time /h
Temperature/
oC
Yield b (%)
H
N
NH
O
O
1
DMSO
DMF
50
50
50
50
50
50
50
50
50
50
50
20
80
100
120
80
80
80
80
80
80
80
48
48
48
48
48
48
48
48
48
48
48
48
48
48
48
12
24
72
96
72
72
72
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
20
30
37
Trace
Trace
34
N
O
O
Enzyme
THF
O
N
NH2
N
H
O
2
N
H
N
H
3a
4a
1a
2a
3
THF
4
CH3CN
Toluene
MTBE
Dioxane
MeOH
EtOH
H2O
Trace
Trace
Trace
Trace
50
Entry
Catalyst
Blank
AA
Yield b (%)
5
1
2
3
4
5
6
7
8
11
25
24
48
22
36
37
14
6
7
DA
8
Papain
PPL
9
48
10
11
12
13
14
15
16
17
18
19
20
21
22
25
CALB
EG
19
MJL
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
23
Denatured papain c
56
a Experimental conditions: 1a (0.2 mmol), 2a (0.4 mmol), 3a (0.2 mmol),
Enzyme (50 mg), EtOH (2 ml), 50 oC, 48 h.
54
57
b All yields were determined by HPLC using Agilent 1100 series with
chiralpak AD-H (250×4.6 mm) (n-hexane: Isopropyl alcohol=65:35) and a
UV detector (210 nm).
14
39
c Denatured papain, incubated with urea at 100 oC for 24 h.
63
The reaction conditions, such as reaction medium, the amount
of enzyme, reaction time and reaction temperature, were screened
based on the model reaction to see whether it is possible to
improve the papain-catalyzed multi-component reaction (Table
2). First of all, different solvents were tested to obtain the
optimum medium for this reaction. As a result, the highest yield
61
14
29
52

3
a Experimental conditions: 1a (0.2 mmol), 2a (0.4 mmol), 3a (0.2 mmol),
papain in organic solvent (2 ml).
addition product 6. Next, the electron-rich N-H performed the
nucleophilic addition to the carbonyl group of ketone, at the same
time, one molecular of water was lost from intermediate 7 to
form product 4a.
b All yields were determined by HPLC using Agilent 1100 series with
chiralpak AD-H (250×4.6 mm) (n-hexane: Isopropyl alcohol=65:35) and a
UV detector (210 nm).
Under the above optimized conditions, various substituted
isatins, 3-methyl-5-amino-pyrazoles, and cyclic-1,3-diketones
were examined as substrates to explore the scope of this protocol
(Table 3). An assembly of 9 compounds was synthesized in
moderate yields through this protocol. It was found that
substituted isatins bearing different halogen on the aromatic ring
led to obviously regular results. The reactions of 5-F isatins 1b
usually gave the highest yield compared with those of 5-Cl 1c
and 5-Br 1d (Table 3, entries 2-4) while the lowest yield was
obtained when 5-Br isatin 1d was used as the substrates (Table 3,
entry 4). The same phenomenon appeared when 5-F or 5-Cl
isatins reacted with 3-methyl-5-amino-1-phenylpyrazole 2b and
5,5-dimethyl-1,3-cyclohexanedione 3b (Table 3, entries 7 and 8).
These results implied that the different electron-withdrawing
property of F, Cl and Br influenced the yields. Regarding to the
effect of the structure of 2, it was found that 3-methyl-5-amino-1-
phenylpyrazole 2b always led to lower yields than 2a, which
could be attributed to the large steric hindrance of benzene ring
(Table 3, entries 1 and 5 or 7 and 9). What is more, it seemed
that 5,5-dimethyl-1,3-cyclohexanedione 3b had a similar activity
as 1,3-cyclohexanedione 3a in this reaction (Table 3, entry 9 vs.
2) and both products 4i and 4b were obtained in good yields
(71% vs. 67%).
Scheme 1. Proposed mechanism.
Considering the uniquely rigid structures of these prepared
spirooxindoles, we investigated their fluorescent properties. All
those products have maximum absorption peak at 320 nm in
methanol, so we chose the light of 305 nm as the excitation light.
Among all these compounds, six were studied in our research
because of their diverse substituent groups, which could
influence the maximum emission wavelength. The results
showed that products 4a, 4b and 4i starting from 2a had the same
maximum peak at 409 nm in violet light while 4e, 4f and 4g
using 2b as the substrate had the maximum peak at 439 nm in
blue light (Figure 1). According to the wavelength range of the
fluorescent, all these compounds can be used as a kind of
promising purple fluorescent compounds, such as fluorescent
whitening, labeling or probe.
Table 3. Substrate scope study of three-component reaction a
Entry
R1
R2
R3
Product
4a
Yield b (%)
1
2
3
4
5
6
7
8
9
H(1a)
H(2a)
H(2a)
H(2a)
H(2a)
Ph(2b)
Ph(2b)
Ph(2b)
Ph(2b)
H(2a)
H(3a)
63
67
49
34
29
32
35
27
71
5-F(1b)
5-Cl(1c)
5-Br(1d)
H(1a)
H(3a)
4b
H(3a)
4c
4a
4b
4e
4f
1.0
0.8
0.6
0.4
0.2
0.0
H(3a)
4d
H(3a)
4e
H(1a)
CH3(3b)
CH3(3b)
CH3(3b)
CH3(3b)
4f
4g
4i
5-F(1b)
5-Cl(1c)
5-F(1b)
4g
4h
4i
a Experimental conditions: 1 (0.2 mmol), 2 (0.4 mmol), 3 (0.2 mmol), papain
(80 mg) in ethanol (2 ml), 50 oC.
b Isolated yields.
300
350
400
450
500
550
600
650
Wavelength (nm)
To explain the papain-catalyzed process, we propose a
tentative mechanism (Scheme 1). According to the literature, the
active site of papain contains Cys-His-Asn “catalytic triad”.34
The reaction would start with the accommodation of 1a in the
active site. A proton of the nucleophile 3a was deprived by His
and nucleophile attack of 3a would lead to a zwitterionic
intermediate, which could be stabilized by the oxyanion hole.
One molecular water was lost and the Knoevenagel condensation
product 5 was obtained. Then Michael addition donor (2a) was
activated by His in the active site and the subsequent conjugated
addition of the nucleophile 2a to 5 would provide the Michael
Figure 1. Emission spectra of 6 products in methanol.
3. Conclusion
In summary, we have successfully established a simple and
environmentally friendly method for the preparation of spiro-
pyrazolo[3,4-b]pyridines derivatives, which starts from isatin,
cyclic-1,3-diketone and 3-methyl-5-aminopyrazole using the
papain as catalyst. The reaction conditions including solvents,
enzyme loading, water content and reaction time were

4
Tetrahedron Letters
15. Yuan, Y.; Guan, Z.; He, Y. H. Sci. China. Chem. 2013, 56, 939-
944.
investigated and optimized. An assembly of 9 compounds was
obtained in moderate yields. The fluorescent properties of these
products showed their potential application as a kind of
fluorescent material. What is more, this papain-catalyzed one-pot
reaction further extends the application of enzymes in organic
chemistry.
16. Feng, X. W.; Li, C.; Wang, N.; Li, K.; Zhang, W. W.; Wang, Z.;
Yu, X. Q. Green. Chem. 2009, 11, 1933-1936.
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The financial support from the National Natural Science
Foundation of China (No. 21072172, 21272208) and the
Zhejiang Provincial Natural Science Foundation (Project No.
2010-Z4090225) is gratefully acknowledged.
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5
Highlights
1. we report a one-pot papain-catalyzed reaction
to synthesize spiro-pyrazolo[3,4-b]pyridines
starting from isatins, cyclic-1,3-diketones and
3-methyl-5-aminopyrazoles in ethanol for the
first time;
2. 9 spiro-pyrazolo[3,4-b]pyridines are obtained
in acceptable yields;
3. We propose a tentative mechanism to explain
the papain-catalyzed process.
4. Most of the products show strong fluorescence
emission ability, providing great potential
application in the field of fluorescent material.