Synthesis of 2,3-dihydroquinazolin-4(1H)-ones catalyzed by α-chymotrypsin

Article abstract of DOI:10.1016/j.cclet.2016.06.001

We discovered that α-chymotrypsin has a promiscuous ability to catalyze the cyclocondensation of aromatic and aliphatic aldehydes with 2-aminobenzamides to afford the corresponding 2,3-dihydroquinazolin-4(1H)-ones successfully in high yields (90%–98%) under alcohol solvent. The catalytic activity of α-chymotrypsin was evaluated through investigating the temperature, the enzyme loading and the ratio of substrates in the enzyme-catalyzed reactions. The present method proves to be efficient and environmentally friendly in terms of short reaction time, high yield, green catalyst and the clean products obtained without further purification processes.

Full text of DOI:10.1016/j.cclet.2016.06.001

Accepted Manuscript
Title: Synthesis of 2,3-dihydroquinazolin-4(1H)-ones
catalyzed by ␣-chymotrypsin
Author: Shi-Guo Zhang Zong-Bo Xie Lian-Sheng Liu Meng
Liang Zhang-Gao Le
PII:
S1001-8417(16)30157-7
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.cclet.2016.06.001
CCLET 3721
To appear in:
Chinese Chemical Letters
Received date:
Revised date:
Accepted date:
19-2-2016
3-5-2016
16-5-2016
Please cite this article as: S.-G. Zhang, Z.-B. Xie, L.-S. Liu, M. Liang, Z.-G. Le,
Synthesis of 2,3-dihydroquinazolin-4(1H)-ones catalyzed by rmalpha-chymotrypsin,
Chinese Chemical Letters (2016), http://dx.doi.org/10.1016/j.cclet.2016.06.001
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Graphical Abstract
Synthesis of 2,3-dihydroquinazolin-4(1H)-ones catalyzed by α-chymotrypsin
b
a, b
b
b
a,b
Shi-Guo Zhang , Zong-Bo Xie* , Lian-Sheng Liu , Meng Liang , Zhang-Gao Le*
a
Jiangxi 2011 Joint Center for the Innovative Mass Spectrometry and Instrumentation, East China University of Technology,
Nanchang 330013, China
b
School of Chemistry, Biology and Material Science, East China University of Technology, Nanchang 330013, China
O
C
O
C
O
α
-Chymotrypsin
Ethanol
NH2
NH
+ R
H
NH2
N
H
R
α-Chymotrypsin has a promiscuous ability to catalyze the cyclocondensation of aldehydes with 2-aminobenzamides to afford the
corresponding 2,3-dihydroquinazolin-4(1H)-ones.
Page 1 of 5

Original article
Synthesis of 2,3-dihydroquinazolin-4(1H)-ones catalyzed by α-chymotrypsin
b
a, b
b
b
a,b
Shi-Guo Zhang , Zong-Bo Xie* , Lian-Sheng Liu , Meng Liang , Zhang-Gao Le*
a
Jiangxi 2011 Joint Center for the Innovative Mass Spectrometry and Instrumentation, East China University of Technology, Nanchang 330013, China
School of Chemistry, Biology and Material Science, East China University of Technology, Nanchang 330013, China
b
ARTICLE INFO
ABSTRACT
Article history:
We discovered that α-chymotrypsin has a promiscuous ability to catalyze the cyclocondensation
of aromatic and aliphatic aldehydes with 2-aminobenzamides to afford the corresponding 2,3-
dihydroquinazolin-4(1H)-ones successfully in high yields (89%-98%) under alcohol solvent.
The catalytic activity of α-chymotrypsin was evaluated through investigating the temperature,
the enzyme loading and the ratio of substrates in the enzyme-catalyzed reactions. The present
method proves to be efficient and environmentally friendly in terms of short reaction time, high
yield, green catalyst and the clean products obtained without further purification processes.
Received 19 February 2016
Received in revised form 3 May 2016
Accepted 16 May 2016
Available online
Keywords:
2
α
,3-Dihydroquinazolin-4(1
-Chymotrypsin
H)-ones
Cyclocondensation
Biocatalysis
2
-Aminobenzamides
1. Introduction
2,3-Dihydroquinazolin-4(1H)-one derivatives acquired significance due to their broad range of potential biological and pharmacological
activities, as well as their crucial role in the preparation of drug molecules and natural products. 2,3-Dihydroquinazolinone derivatives act as
important intermediates that can be easily oxidized to their quinazolin-4(3H)-one analogues, which also include important biologically active
compounds. In recent years, the protocols for the synthesis of 2,3-dihydroquinazolin-4(1H)-ones have been developed in different ways using
iodine [1], succinimide-N-sulfonic acid [2], p-TSA [3], 2-morpholinoethanesulfonic acid [4], ZrCl
sulfuric acid [8], sulfamic acid [9], nanocrystalline sulfated zirconia [10], N-propylsulfamic acid on magnetic nanoparticles [11], metal-CNTs
12], β-cyclodextrin-SO H [13], PEG-400 [14] and ionic liquids [15] as catalysts. These synthetic methodologies are useful to facilitate the
4 4 4 2
[5], NH Cl [6], KAl(SO ) [7], silica
[
3
synthesis of the desired compounds in many instances. However some of the synthetic strategies have certain limitations such as tedious
processes, long reaction time, low yields of product, expensive moisture-sensitive catalysts, cumbersome preparation processes for the
required catalyst, and liberating hazardous HF during recycling. Therefore, there has been increasing effort on the design and use of
environmentally compatible catalysts for the preparation of 2,3-dihydroquinazolin-4(1H)-one derivatives owing to the growing demand for the
development of more sustainable and environmentally-friendly processes.
Enzymes have received great attention as sustainable, efficient, green and biodegradable catalysts for the synthesis of pharmaceutical,
industrial and agricultural chemicals and intermediates. Among the promiscuous enzymes, hydrolases beyond doubt play an important role
because of their high stability, availability, and broad range of substrates. Recently, several enzymes have been used in multiple types of
organic reactions such as asymmetric aldol reactions [16], asymmetric Michael additions [17] and asymmetric Henry reactions [18]. However,
to the best of our knowledge, enzyme-catalyzed cyclocondensation reaction for the preparation of 2,3-dihydroquinazolin-4(1H)-one
derivatives has never been reported. Herein, we wish to report the first discovery that α-chymotrypsin could catalyze the reaction of 2-
aminobenzamide with a series of aldehydes to afford the corresponding 2,3-dihydroquinazolinones in ethanol (Scheme 1), and the yields up to
9
8% were achieved, which was obviously a novel environmentally benign synthetic method.
O
O
C
O
C
α
-Chymotrypsin
Ethanol
NH2
NH
+ R
H
NH2
Scheme 1. The
N
H
R
α
-chymotrypsin-catalyzed cyclocondensation of 2-aminobenzamide with aldehydes.
2. Experimental
2.1. General
Alkaline protease (200 U/mg enzyme activity) and Papain (800 U/mg enzyme activity) were purchased from Jiangsu Ruiyang
Biotechnology Co., Ltd. (Jiangsu, PR China). Trypsin from bovin pancreas (≥ 2500 U/mg enzyme activity), α-chymotrypsin (800 U/mg
Page 2 of 5

enzyme activity) and albumin from bovine serum were purchased from Aladdin Chemistry Co., Ltd. (Shanghai, PR China). Protease from
Aspergillus saitoi (≥ 0.6 U/mg enzyme activity), proteinase from Aspergillus melleus (3.3 U/mg enzyme activity) and protease from Bacillus
licheniformis (2.4 U/g enzyme activity) were purchased from Sigma Aldrich Co., USA. All reagents were obtained from commercial suppliers
and used without further purification except that 2-aminobenzamide was recrystallized. Melting points were obtained on a WRS-1B Digital
6
Melting Point Apparatus. The NMR spectra were recorded on a Bruker 400 MHz instrument using DMSO-d as a solvent. Chemical shifts (δ)
were expressed in ppm using tetramethylsilane (TMS) as an internal standard, and coupling constants (J) were reported in Hz.
2.2 General procedure for the synthesis of 2,3-dihydroquinazilin-4(1H)-ones
α-Chymotrypsin (2 mg/mL) was added to a solution of 2-aminobenzamide (1 mmol) and aldehyde (1.8 mmol) in ethanol (10 mL) and the
mixture was stirred at 60 ºC in a constant temperature incubator shaker for a specified period of time. After the completion of the reaction,
water (20 mL) was added to the mixture to quench the reaction, the enzymes and the excess aldehydes were dissolved in an aqueous ethanol
solution, and the desired products precipitated out. The products were collected by filtration without further purification.
3. Results and discussion
Based on our previous research, initial efforts were undertaken using 2-aminobenzamide and 4-methylbenzaldehyde as a model reaction,
and ethanol was chosen as the reaction medium. In order to select the most efficient catalyst for the cyclocondensation, eight proteases were
screened using the model system. As shown in Table 1, the best yield of 62% was achieved using α-chymotrypsin (Table 1, entry 8) while
trypsin from bovin pancreas showed low activity in this reaction and only provided 13% yield (Table 1, entry 2). However, the other tested
enzymes demonstrated no catalytic ability. Non-enzyme protein BSA (Bovine serum albumin) (Table 1, entry 9) and denatured α-
chymotrypsin (Table 1, entry 10) were also used as controls to demonstrate the specific catalytic effect of the α-chymotrypsin, and both barely
showed catalytic activity, producing the similar results as the blank control reaction (Table 1, entry 11). All the results clearly indicated that the
catalytic ability of the α-chymotrypsin is responsible for the model reaction.
Table 1
a
The catalytic activities of different enzymes.
b
Entry Enzymes
Yield (%)
1
2
3
4
5
6
7
8
9
1
Alkaline protease
Trypsin
Papain
Proteinase from Aspergillus melleus
Papain from papayalatex
Protease from Aspergillus saitoi
Protease from Bacillus licheniformis
5
13
9
7
6
6
7
62
6
α
-Chymotrypsin
BSA
c
0
1
Denatured
α-chymotrypsin
6
5
1
No enzyme
a
Reaction conditions: 2-aminobenzamide 1 mmol, 4-methylbenzaldehyde 1 mmol, enzyme 2.0 mg/mL and ethanol 10 mL, stirred at 40 ºC for 60 min.
Isolated yield.
Pretreated with urea solution (8 mol/L).
b
c
To improve the catalytic ability of the enzyme, we carried out some experiments focusing on the effect of reaction temperature, which
plays a significant role in maintaining the stability and catalytic activity of enzymes. The range of temperature from 30 ºC to 70 ºC was
screened for the α-chymotrypsin-catalyzed cyclocondensation reactions and the results were shown in Table 2, entries 1-4. The best yield of
89% was achieved at 60 ºC (Table 2, entry 3) and higher temperature (70 ºC) gave lower yield probably due to the partial denaturation of the
enzyme at higher temperatures. So, we chose 60 ºC as an optimal temperature for the reaction. Next, the effect of enzyme loading on the α-
chymotrypsin-catalyzed model reaction was investigated in order to further optimize the reaction conditions. As shown in Table 2, entries 5-9,
the model reaction only gave the product in a low yield of 5% in the absence of enzyme. However, an obvious increase in the yield (77%) was
detected when 0.5 mg/mL of α-chymotrypsin was added. After that, only a slight rising trend was observed with the larger catalyst dosage
from 0.5 mg/mL to 2.5 mg/mL, thus, 2.0 mg/mL was chosen as the best enzyme loading.
Table 2
a
The effect of temperature and enzyme loading on the model reaction.
Entry
b
Temperature (ºC)
Enzyme loading
mg/mL)
Yield (%)
(
1
2
3
4
5
6
7
8
30
50
60
70
60
60
60
60
60
2.0
2.0
2.0
2.0
0
0.5
1.0
1.5
2.5
15
76
89
85
5
77
83
85
88
9
a
Reaction conditions: 2-aminobenzamide 1 mmol, 4-methylbenzaldehyde 1 mmol, α-chymotrypsin and ethanol 10 mL for 60 min.
Page 3 of 5

b
Isolated yield.
Some further experiments were performed to investigate the effects of the molar ratio of substrates on the α-chymotrypsin catalyzed
model reaction. As shown in Table 3, increasing the molar ratio of 2-aminobenzamide to 4-methylbenzaldehyde from 1.0 : 1.2 to 1.0 : 1.8 led
to an growing yield (from 89% to 98%) of the desired product and further increasing the molar ratio from 1.0 : 1.8 to 1.0 : 2.0 hardly improved
the results. Thus, we chose the molar ratio of 2-aminobenzamide to 4-methylbenzaldehyde of 1.0 : 1.8 as the optimal proportion of the
substrate for the reaction.
Table 3
a
The effect of molar ratio on the model reaction.
Entry
1
2
3
4
b
c
Molarratio
1:1.2
1:1.4
1:1.6
1:1.8
Yield (%)
89
92
95
98
98
5
1:2.0
a
Reaction conditions:
α
-chymotrypsin 2.0 mg / mL and ethanol 10 mL, stirred at 60ºC for 30 min.
b
2
-Aminobenzamide / 4-methylbenzaldehyde (mmol / mmol).
c
Isolated yield.
Finally, we further investigated the substrate scope and the limitation of the α-chymotrypsin-catalyzed cyclocondensation reaction by
employing a wide range of aldehydes under the optimized conditions. The results are summarized in Table 4. It can be seen that the
corresponding dihydroquinazolin-4(1H)-ones were obtained with good to excellent yields in all cases. Furthermore, various types of aromatic
aldehydes with electron-donating groups reacted very well with 2-aminobenzamide under the optimized reaction conditions, as well as
aromatic aldehydes containing electron-withdrawing substituents, such as the methyl- and bromo-substituted benzaldehydes afforded the
corresponding products b and f in 98% and 96% yields, respectively. Generally, the reaction could proceed smoothly and was not influenced
by substituents in the aromatic ring. To our delight, aliphatic aldehydes such as caprylaldehyde were compatible with the reaction conditions to
give the desired product (o) in high yield of 95%. And hexaldehyde also afforded the corresponding product (n) in excellent yield although
another 30 minutes of reaction time is required.
Table 4
a
Investigation of the reactant scope of the cyclocondensation reaction for synthesis of 2,3-dihydroquinazolin-4(1H)-one derivatives.
O
O
C
O
C
NH
2
NH
+ R
H
NH
2
N
H
R
º
Entry
R
Time Compd Yield MP ( C)
b
(
min)
(%)
91
98
96
96
98
96
98
92
95
96
97
91
90
98
Found
Lit. (ref.)
224-226 [4]
225-227 [12]
1
2
3
4
5
6
7
8
9
C
4-CH
3-CH
4-Cl-C
2- Cl-C
3-Br-C
2-Br-C
4-OCH
3-OH-C
2-OH-C
4-OH-3OMe-C
(CH) -C
6
H
5
30
30
30
30
30
30
50
30
30
30
30
30
60
30
a
b
c
d
e
f
g
h
i
j
k
l
m
n
225-226
227-228
204-205
202-203
208-209
190-191
171-182
188-189
206-207
223-225
224-225
173-175
154-155
158-159
3
3
-C
-C
6
6
H
H
4
4
6
H
4
205-207 [19]
207-209 [19]
227-228 [19]
172-173 [12]
181-183 [12]
215-217 [20]
220-221 [4]
227-228 [4]
155-157 [20]
160-162 [20]
6
H
4
6 4
H
6
H
4
3
-C
6
H
4
6
H
H
4
1
1
1
1
0
1
2
3
4
6
4
6
H
4
2
6 4
H
C
C
5
H
H
11
1
7
15
a
Reaction conditions: 2-aminobenzamide 1 mmol, aldehyde 1.8 mmol,
α-chymotrypsin 2.0 mg / mL, ethanol 10 mL, stirred at 60 ºC.
b
Isolated yield.
4. Conclusion
In summary, we have succeeded in obtaining dihydroquinazolin-4(1H)-ones using α-chymotrypsin as a new biocatalyst. A wide range of
aromatic aldehydes and aliphatic aldehydes could be used to give the products in excellent yields. Notably, The unnatural ability of α-
chymotrypsin to catalyze a cyclocondensation reaction to prepare 2,3-dihydroquinazolin-4(1H)-one derivatives was discovered for the first
time. Compared with current chemical approaches, this enzymatic method is more environmental-friendly and sustainable using a biocatalyst
from bio-degradable and inexpensive sources. Moreover, the catalyst α-chymotrypsin accommodated broad substrates and produced the
desired products in high yields in a short period of time.
Acknowledgment
We gratefully acknowledge the financial support of the National Natural Science Foundation of China (Nos. 21262002, 21462001 and
21465002), the Program for Changjiang Scholars and Innovative Research Team in University (IRT13054), the Natural Science Foundation of
Jiangxi No. 20142BAB203008), the Science and Technology Foundation of the Jiangxi Education Department (Nos. KJLD12006, GJJ14466
Page 4 of 5

and KJLD14050).
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