α-Chymotrypsin–catalyzed direct C (Sp3)–H functionalization reactions for synthesis of azaarene derivatives in water

Article abstract of DOI:10.1002/jhet.3712

α-Chymotrypsin from the bovine pancreas has been shown for the first time to display catalytic nonnatural catalytic ability toward the C (sp³)–H functionalization reaction of 2-methylquinoline and aromatic aldehydes in water. α-Chymotrypsin exhibited favorable catalytic activity with good adaptability to different substrates. The activity of the enzyme could be improved by adjusting the solvent, temperature, molar ratio of substrates, and protein loading. The products were obtained in moderate to excellent yields (51%-93%) in 24 samples. This process afforded a potential biocatalytic approach as an alternative to chemical synthesis for azaarene derivatives.

Full text of DOI:10.1002/jhet.3712

Received: 30 July 2019
DOI: 10.1002/jhet.3712
Revised: 18 August 2019
Accepted: 19 August 2019
A R T I C L E
α‐Chymotrypsin–catalyzed direct C (Sp3)–H
functionalization reactions for synthesis of azaarene
derivatives in water
Zhang‐Gao Le1,2 | Yue Lu1,2 | Guo‐Fang Jiang1,2 | Yi‐Shuai Liu1,2 | Jia Liu1,2
Zong‐Bo Xie1,2
|
1 State Key Laboratory of Nuclear
Abstract
Resources and Environment, East China
α‐Chymotrypsin from the bovine pancreas has been shown for the first
time to display catalytic nonnatural catalytic ability toward the C (sp3)–H
functionalization reaction of 2‐methylquinoline and aromatic aldehydes in
water. α‐Chymotrypsin exhibited favorable catalytic activity with good adapt-
ability to different substrates. The activity of the enzyme could be improved
by adjusting the solvent, temperature, molar ratio of substrates, and protein
loading. The products were obtained in moderate to excellent yields (51%‐93%)
in 24 samples. This process afforded a potential biocatalytic approach as an
alternative to chemical synthesis for azaarene derivatives.
University of Technology, Nanchang,
China
2 Department of Applied Chemistry, East
China University of Technology,
Nanchang, China
Correspondence
Guo‐Fang Jiang and Zong‐Bo Xie,
Department of Applied Chemistry, East
China University of Technology,
Nanchang 330013, China.
Email: gfjiang@ecut.edu.cn; zbxie@ecut.
edu.cn
Funding information
the National Natural Science Foundation
of China, Grant/Award Number:
21462001, 21465002 and 11765002;
National Natural Science Foundation of
Jiangxi, Grant/Award Number:
20181BAB203019; Science and Technol-
ogy Projects of Jiangxi, Grant/Award
Number: 20161BCB24006
1 | INTRODUCTION
Zhou et al developed a low‐loading cobalt nanocatalyst
using nitrogen‐silica–doped carbon as the support. The
Azaarene derivatives have gained attention because of
their broad range of potential biological and pharmaco-
logical activities,[1] eg, as anti‐inflammatory agents, anti-
cancer agents, anti‐HIV agents, and molecular probes.[2]
Therefore, the synthesis of azaarene derivatives continues
to receive attention from scientists. Azaarene derivatives
act as important intermediates in many industrial pro-
cesses, and one of the most efficient methods for their
synthesis is the direct C (sp3)–H functionalization of
alkyl‐substituted pyridines and quinolines. In 2018,[3]
combination of such a catalyst with 4‐nitrobenzoic acid
and molecular O2 exhibits excellent catalytic performance
toward the dehydrogenative coupling of (hetero)aryl‐
fused 2‐alkylcyclic amines with aldehydes to afford the
(E)‐2‐alkenylazaarene. In 2016,[4] Crisenza et al used
quinoline N‐oxide as the precursor and dimethyl sulfox-
ide as the solvent in the presence of TsOH·H2O at
120°C to generate azaarene derivatives. In 2016,[5]
Tan et al developed a novel iridium/acid cocatalyzed
transfer hydrogenative coupling strategy, enabling direct
J Heterocyclic Chem. 2019;1–10.
wileyonlinelibrary.com/journal/jhet
© 2019 Wiley Periodicals, Inc.
1

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LE ET AL.
alkylation of C (sp3)–H bonds and atom‐economic access
to alkyl chain–lengthened N‐heteroaromatics from six‐
membered 2‐alkyl cyclic amines and aldehydes. In
2015,[6] Wei's group reported the CuFe2O4 catalyzed
medium. We then investigated some hydrolases for the
catalysis of the model reaction (Table 1). The best yield
of 18% was achieved using α‐chymotrypsin from bovine
pancreas (Table 1, entry 1). In addition, lipase from
Candida rugosa (Table 1, entry 3) and trypsin from
bovine pancreas (Table 1, entry 4) both exhibited some
catalytic activity toward the model reaction. However,
the other enzymes tested demonstrated no catalytic abil-
ity. Next, inactivated α‐chymotrypsin (Table 1, entry 8)
and nonenzyme protein bovine serum albumin (BSA)
(Table 1, entry 7) were separately used in the reaction,
and neither showed catalytic activity, thus producing
results similar to those of the blank control reaction
(Table 1, entry 9). We performed the model reaction in
the absence of α‐chymotrypsin, and only a trace amount
of the desired product was observed after 84 hours
(Table 1, entry 9). These results confirmed that the catal-
ysis did not arise simply from the amino acid residues on
the surface of the protein. In view of the above‐
mentioned results, α‐chymotrypsin was chosen as the
catalyst.
Since the reaction medium plays a significant role in
maintaining the catalytic activity and stability of an
enzyme,[15] other organic solvents were screened for the
α‐chymotrypsin–catalyzed model reaction (Figure 1).
The experimental results showed that solvents had an
obvious effect on the reaction. The best yield of 57% was
obtained when water was used as the solvent. Dimethyl
sulfoxide also afforded the product in good yields, while
low to moderate yields were obtained by other solvents.
Additionally, a blank experiment in water in the absence
of the catalyst gave only a trace amount of the desired
product. Thus, water was confirmed as the optimal
solvent.
direct
C
(sp3)–H functionalization reaction of 2‐
methylquinoline and aromatic aldehydes at 100°C. In
2010,[7] Huang's group reported the synthesis of azaarene
derivatives from imine and 2,6‐lutidine using Pd(OAc)2
as the catalyst. These synthetic methodologies are useful
to facilitate the synthesis of the desired compounds
in many instances. However, these synthetic strategies
have certain limitations such as serious pollution of the
environment, tedious processes, low yields of product,
expensive moisture‐sensitive catalysts, cumbersome prep-
aration processes for the required catalyst, and liberating
hazardous gas during recycling. If a direct synthetic
method with enzyme catalysis could be established for
the synthesis of azaarene derivatives, it would be greener
and more efficient, which will significantly reduce reac-
tion costs. Therefore, it is of great importance to develop
a green preparation method that is economical, environ-
ment friendly, and has a wide range of applications.
Enzymes have received considerable attention as effi-
cient, less toxic, mild, and selective catalysts for the agri-
cultural, industrial, and pharmaceutical industries.[8]
Among the various types of enzymes, hydrolases undoubt-
edly play an important role because of their high stability,
high selectivity, and nonnaturally catalytic activity.[9]
Recently, several enzymes have been used in multiple
types of organic reactions, such as Gewald,[10] aldol,[11]
retro‐Claisen reaction,[12] Mannich,[13] and Friedländer
reactions.[14] However, to the best of our knowledge, the
synthesis of azaarene derivatives by enzymatic catalysis
of the reaction between 2‐methylquinolines and aromatic
aldehydes has never been reported. Herein, we report
that α‐chymotrypsin can catalyze the reaction of 2‐
methylquinoline (I) with a series of aromatic aldehydes
(II) to afford the corresponding azaarenes in water as the
solvent (Scheme 1). The highest yield achieved was 93%,
and the reaction was a simple green synthesis.
The water content usually affects enzymatic reactions
in organic solvents because water can affect the confor-
mational flexibility of an enzyme.[16] Typically, enzymes
require a certain amount of water to maintain their
three‐dimensional conformation in an organic solvent.
Hence, the effect of water content on the model reaction
was investigated (Figure 2). A rise in the yield from 21%
to 57% was observed as the water content increased from
0% to 100% [water/(water + methanol), v/v]. The same
trend was observed in ethanol; however, the effect of eth-
anol content on the model reaction was lower than that
of water. Therefore, 100% water was selected as the opti-
mal solvent for the reaction.
2 | RESULTS AND DISCUSSION
Initial studies used the reaction between 2‐
methylquinoline and 4‐nitrobenzaldehyde as a model
reaction, and ethanol was chosen as the reaction
SCHEME 1 α‐Chymotrypsin–catalyzed
synthesis of azaarene derivatives [Color
figure can be viewed at wileyonlinelibrary.
com]

LE ET AL.
3
TABLE 1 Catalytic activities of different enzymesa
Entry
Enzyme
Yieldb/%
1
2
3
4
5
6
7
8
9
α‐Chymotrypsin
18
11
13
14
10
≤5
≤5
≤5
≤5
Protease from Aspergillus saitoi
Lipase from Candida rugosa
Trypsin from bovine pancreas
Lipase from Candida antarctica
Papain
Bovine serum albumin
Inactivated α‐chymotrypsinc
Blank
aReaction conditions: 2‐methylquinoline (0.3 mmol), 4‐nitrobenzaldehyde
(0.33 mmol), and catalyst (15 mg) in CH3CH2OH (2 mL) at 50°C for 84 h.
bIsolated yield after column chromatography.
cPretreated with urea solution (8 mol/L).
FIGURE 2 Catalytic effect of α‐chymotrypsin in different levels
of water content.a aReaction conditions: 2‐methylquinoline
(0.3 mmol), 4‐nitrobenzaldehyde (0.33 mmol), and α‐chymotrypsin
(15 mg) in ethanol or methanol aqueous solution (2 mL) with
b
different levels of water content at 50 °C for 84 h. Isolated yield
after column chromatography [Color figure can be viewed at
wileyonlinelibrary.com]
FIGURE 1 Catalytic effect of α‐chymotrypsin in different
solvents.a aReaction conditions: 2‐methylquinoline (0.3 mmol), 4‐
nitrobenzaldehyde (0.33 mmol), and α‐chymotrypsin (15 mg) in
b
different solvents (2 mL) at 50°C for 84 h. Isolated yield after
FIGURE
3
Catalytic effect of α‐chymotrypsin at different
temperatures.a aReaction conditions: 2‐methylquinoline
column chromatography [Color figure can be viewed at
wileyonlinelibrary.com]
(0.3 mmol), 4‐nitrobenzaldehyde (0.33 mmol), and α‐chymotrypsin
(15 mg) in H2O (2 mL) at different temperatures for 84 h. Isolated
b
yield after column chromatography [Color figure can be viewed at
wileyonlinelibrary.com]
Temperature is a crucial factor affecting the stability of
enzymes.[17] Increasing the temperature can accelerate
the chemical reaction rate; however, high temperatures
can inactivate the enzyme through protein denaturation.
Hence, the effect of temperature on the model reaction
was investigated (Figure 3). Increasing the temperature
from 30°C to 70°C led to an increase in the yield from
22% to 75%. The best yield was achieved at 60°C. When
the temperature increased to 80°C, the reaction yield
decreased to 68%. Thus, 60°C was chosen as the optimum
temperature for the reaction.
Furthermore, the molar ratio of the substrates affects
the reaction. Thus, to further improve the yield of the α‐
chymotrypsin–catalyzed model reaction, the optimal
molar ratio of the substrates was determined (Figure 4).
When a molar ratio of 1:3 (2‐methylquinoline : 4‐
nitrobenzaldehyde) was used, the reaction gave a yield
of 76%. Increasing the molar ratio of 2‐methylquinoline : 4‐
nitrobenzaldehyde from 1.0:1.0 to 1.4:1.0 led to an

4
LE ET AL.
FIGURE 5 Effect of protein loading on the reaction.a aReaction
conditions: 2‐methylquinoline (0.42 mmol), 4‐nitrobenzaldehyde
(0.3 mmol), and α‐chymotrypsin (different quality) in H2O (2 mL) at
FIGURE 4 Effect of substrate molar ratio on reaction.a aReaction
conditions: α‐chymotrypsin (15 mg) in H2O (2 mL) at 60°C for 84 h.
b
different temperatures for 84 h. Isolated yield after column
c
b2‐Methylquinoline : 4‐nitrobenzaldehyde (mmol:mmol). Isolated
chromatography [Color figure can be viewed at wileyonlinelibrary.
com]
yield after column chromatography [Color figure can be viewed at
wileyonlinelibrary.com]
increase in yield (from 75% to 93%) of the desired prod-
uct; however, further increasing the molar ratio from
1.4:1.0 to 2.0:1.0 did not improve the yield. Thus, we
chose 1.4:1.0 as the optimal ratio of 2‐methylquinoline : 4‐
nitrobenzaldehyde for the reaction.
tolerant as a substrate to the reaction (Table 2, III s–III
x). Among these examples, the best yield (93%) was
obtained for the reaction of 4‐nitrobenzaldehyde and 2‐
methylquinoline (Table 2, III a).
According to previous reports, α‐chymotrypsin is a
serine protease of the peptide S1 family of 245 amino
acid residues, and the catalytic triad is composed of
His‐57, Asp‐102, and Ser‐195.[19] A possible mechanism
is proposed in Scheme 2. Firstly, in the presence of
Ser‐195, 2‐methylquinolinetends to isomerize and the
enamine intermediate is formed. Subsequently, the pro-
ton from the enamine tautomer may be abstracted and
effectively activated by His‐57. Then, the nucleophilic
addition of the enamine intermediate to the aromatic
aldehyde, which is activated by Ser‐195, occurs to afford
the desired adduct. Finally, the captured proton from the
intermediate is returned to Ser‐195, and the correspond-
ing alkyl azaarene derivatives are obtained, with the
regeneration of α‐chymotrypsin to complete the catalytic
cycle.
It is known that protein loading affects the yield and
kinetics of the reaction[18]; therefore, the effect of protein
loading on the α‐chymotrypsin–catalyzed model reaction
was investigated (Figure 5). The model reaction only gave
the product in a low yield of 38% in the absence of
enzyme. However, an obvious increase in the yield
(64%) was detected when 5 mg of α‐chymotrypsin was
added. After pepsin addition, a steady increase in yield
was observed as the catalyst dosage was increased from
5 to 15 mg; when 15 mg of the enzyme was used, the
highest yield of the product was obtained (93%). Further
increasing the protein loading from 15 to 20 mg did not
improve the results. Thus, 15 mg was chosen as the best
protein loading.
On the basis of the optimized conditions, we further
investigated the substrate scope and generality of the α‐
α‐Chymotrypsin from the bovine pancreas was used as
a biocatalyst in the C (sp3)–H functionalization reaction
for the synthesis of azaarene derivatives in water. Com-
pared with current chemical technologies, this enzymatic
reaction is more environment friendly and sustainable
because it uses a biocatalyst from inexpensive regenerable
resources. A wide range of substrates could be used with
α‐chymotrypsin. The activity of α‐chymotrypsin could be
improved by adjusting the solvent type, water content,
temperature, molar ratio of substrates, and protein
chymotrypsin–catalyzed
reaction. range of aromatic aldehydes and 2‐
C
(sp3)–H functionalization
A
methylquinoline derivatives were tested (Table 2). In
general, aromatic aldehydes with electron‐withdrawing
substituents showed better results than those with
electron‐donating or neutral substituents. In addition to
2‐methylquinoline, various substituted 2‐methylquinoline
derivatives were used as substrates (Table 2, III h–III r). It
was interesting that 1‐methylisoquinoline was also

LE ET AL.
5
TABLE 2 Substrate scope of the benzylic C (sp3)–H functionalization reaction of 2‐methylazaarenesa
aReaction conditions: 2‐methylquinoline derivatives (0.42 mmol), aromatic aldehyde (0.3 mmol), and α‐chymotrypsin (15 mg) in H2O (2 mL) at 60°C for 84 h.
Isolated yield after column chromatography.
loading. Yields of up to 93% were achieved. This study
offers a novel case of enzyme nonnaturally catalytic activ-
ity and a potential synthetic method for organic chemis-
try, helps to better understand metabolic pathways for
the biosynthesis of nitrogen‐containing compounds, and
extends the application of enzymatic nonnaturally cata-
lytic activity.
3 | EXPERIMENTAL
3.1 | Materials
All major chemicals were obtained from commercial
sources and used without further purification. All reac-
tions were monitored by thin‐layer chromatography

6
LE ET AL.
SCHEME 2 Proposed mechanism for benzylic C (sp3)–H functionalization reaction of 2‐methylazaarenes catalyzed by α‐chymotrypsin in
aqueous media [Color figure can be viewed at wileyonlinelibrary.com]
(TLC) with silica gel plates. Yields given are isolated
yields after column chromatography.
and the solvent was removed under reduced pressure.
The filtrate was purified by column chromatography
using petroleum ether/ethyl acetate as an eluent to isolate
the corresponding product.
3.2 | General procedure
A sealable reaction tube, equipped with a magnetic stirrer
bar, was charged with 2‐methylazaarene (0.42 mmol),
aromatic aldehyde (0.3 mmol), α‐chymotrypsin (15 mg),
and H2O (2 mL). The reaction vessel was heated to 60°C
and stirred for 84 hours. TLC is used to follow the reac-
tion upon completion of the reaction, the enzyme was fil-
tered out, and ethyl acetate was added to wash the filter
cake and to make the product dissolved in the filtrate.
The organic layer was separated with a separatory funnel,
3.3 | NMR data
3.3.1 | 1‐(4‐Nitrophenyl)‐2‐(quinolin‐2‐yl)
ethanol (IIIa)
Yield, 93%; yellow solid, mp 150‐152°C; 1H NMR
(600 MHz, CDCl3): δ 8.20 (d, J = 8.6 Hz, 2H), 8.12
(d, J = 8.3 Hz, 1H), 8.06 (d, J = 8.4 Hz, 1H), 7.81
(d, J = 8.1 Hz, 1H), 7.75 (t, J = 7.7 Hz, 1H), 7.65

LE ET AL.
7
(d, J = 8.6 Hz, 2H), 7.55 (d, J = 7.8 Hz, 1H), 7.21
(d, J = 8.4 Hz, 1H), 6.80 (s, 1H), 5.45 (dd, J = 8.9,
2.2 Hz, 1H), 3.31 (ddd, J = 24.8, 15.7, 5.9 Hz, 2H); 13C
NMR (150 MHz, CDCl3): δ 159.64, 151.45, 147.19,
146.89, 137.22, 130.12, 128.61, 127.70, 126.95, 126.67,
123.64, 121.92, 72.17, 45.33.
3.3.5 | 4‐(1‐Hydroxy‐2‐(quinolin‐2‐yl)
ethyl)benzonitrile (IIIe)
Yield, 71%; white solid, mp 167‐171°C; 1H NMR
(600 MHz, CDCl3): δ 8.12 (d, J = 8.3 Hz, 1H), 8.05
(d, J = 8.4 Hz, 1H), 7.85‐7.77 (m, 2H), 7.77‐7.68
(m, 2H), 7.55 (t, J = 7.0 Hz, 2H), 7.45 (t, J = 7.7 Hz,
1H), 7.22 (d, J = 8.4 Hz, 1H), 6.57 (s, 1H), 5.36 (dd, J = 8.8,
3.2 Hz, 1H), 3.29 (qd, J = 15.7, 6.1 Hz, 2H); 13C NMR
(150 MHz, CDCl3): δ 157.5, 149.1, 145.1, 134.5, 130.0,
127.6, 126.3, 125.9, 125.0, 124.9, 124.1, 120.8, 116.9,
108.2, 70.2, 45.8.
3.3.2 | 1‐(3‐Nitrophenyl)‐2‐(quinolin‐2‐yl)
ethanol (IIIb)
Yield, 72%; yellow solid, mp 154‐156°C; 1H NMR
(600 MHz, CDCl3): δ 8.37 (s, 1H), 8.13 (dd, J = 9.5,
5.1 Hz, 2H), 8.07 (d, J = 8.4 Hz, 1H), 7.83 (t, J = 8.2 Hz,
2H), 7.75 (t, J = 7.7 Hz, 1H), 7.54 (dt, J = 16.1, 7.9 Hz,
2H), 7.31‐7.18 (m, 1H), 6.76 (s, 1H), 5.44 (dd, J = 8.8,
3.2 Hz, 1H), 3.34 (qd, J = 15.7, 6.1 Hz, 2H); 13C NMR
(150 MHz, CDCl3): δ 159.63, 148.34, 146.25, 137.34,
132.12, 130.16, 129.32, 128.46, 127.70, 126.96, 126.56,
122.28, 122.00, 121.02, 72.03, 45.44.
3.3.6 | 3‐(1‐Hydroxy‐2‐(quinolin‐2‐yl)
ethyl)benzonitrile (IIIf)
Yield, 66%; white solid, mp 171‐172°C; 1H NMR
(600 MHz, CDCl3): δ 8.12 (d, J = 8.4 Hz, 1H), 8.06
(d, J = 8.4 Hz, 1H), 7.85‐7.78 (m, 2H), 7.73 (dd, J = 16.7,
8.3 Hz, 2H), 7.55 (t, J = 7.0 Hz, 2H), 7.45 (t, J = 7.7 Hz,
1H), 7.22 (d, J = 8.4 Hz, 1H), 6.54 (s, 1H), 5.36 (dd, J = 8.8,
3.1 Hz, 1H), 3.29 (qd, J = 15.7, 6.0 Hz, 2H); 13C NMR
(150 MHz, CDCl3): δ 159.72, 146.83, 145.56, 137.23,
130.95, 130.39, 130.11, 129.65, 129.14, 128.56, 127.68,
126.94, 126.52, 121.93, 118.96, 112.40, 71.99, 45.45.
3.3.3 | 1‐(2‐Nitrophenyl)‐2‐(quinolin‐2‐yl)
ethanol (IIIc)
Yield, 87%; yellow solid, mp 163‐164°C; 1H NMR
(600 MHz, CDCl3): δ 8.15 (d, J = 8.4 Hz, 1H), 8.06
(dd, J = 23.5, 8.1 Hz, 2H), 7.98 (d, J = 7.4 Hz, 1H), 7.83
(d, J = 8.1 Hz, 1H), 7.75 (t, J = 7.7 Hz, 1H), 7.66
(t, J = 7.6 Hz, 1H), 7.56 (t, J = 7.5 Hz, 1H), 7.45‐7.41
(m, 1H), 7.32 (d, J = 8.3 Hz, 1H), 5.81 (dd, J = 9.1,
1.9 Hz, 1H), 3.56 (dd, J = 15.3, 2.1 Hz, 1H), 3.26
(dd, J = 15.3, 9.2 Hz, 1H); 13C NMR (150 MHz, CDCl3):
δ 160.14, 147.57, 146.74, 139.99, 137.46, 133.63, 130.08,
128.68, 128.51, 128.01, 127.70, 127.05, 126.48, 124.31,
122.03, 68.89, 44.95.
3.3.7 | 1‐(4‐Trifluoromethylphenyl)‐2‐
(quinolin‐2‐yl)ethanol (IIIg)
Yield, 67%; yellow solid, mp 159‐161°C; 1H NMR
(600 MHz, DMSO‐d6): δ 8.09 (dd, J = 26.6, 8.3 Hz, 2H),
7.81 (d, J = 8.0 Hz, 1H), 7.74 (t, J = 7.5 Hz, 1H), 7.65‐
7.50 (m, 5H), 7.22 (d, J = 8.3 Hz, 1H), 5.39 (dd, J = 8.2,
3.0 Hz, 1H), 3.35‐3.22 (m, 2H); 13C NMR (150 MHz,
CDCl3): δ 159.94, 147.94, 146.81, 137.14, 130.04, 129.55,
129.34, 128.52, 127.64, 126.90, 126.44, 126.15, 125.12,
121.97, 72.37, 45.60.
3.3.4 | 1‐(4‐Chlorophenyl)‐2‐(quinolin‐2‐
yl)ethanol (IIId)
3.3.8 | 2‐(6‐Methylquinolin‐2‐yl)‐1‐(4‐
nitrophenyl)ethanol (IIIh)
Yield, 64%; yellow solid, mp 143‐145°C; 1H NMR
(600 MHz, CDCl3): δ 8.10 (d, J = 8.3 Hz, 1H), 8.06
(d, J = 8.3 Hz, 1H), 7.81 (d, J = 7.9 Hz, 1H), 7.76‐7.71
(m, 1H), 7.57‐7.52 (m, 1H), 7.41 (d, J = 8.2 Hz, 2H),
7.32 (d, J = 8.3 Hz, 2H), 7.21 (d, J = 8.3 Hz, 1H), 6.46
(s, 1H), 5.37‐5.27 (m, 1H), 3.27 (d, J = 5.8 Hz, 2H); 13C
NMR (150 MHz, CDCl3): δ 160.12, 146.88, 142.55,
137.07, 132.93, 130.00, 128.57, 128.48, 127.64, 127.30,
126.92, 126.40, 122.06, 72.34, 45.88.
Yield, 84%; yellow solid, mp 149‐151°C; 1H NMR
(600 MHz, CDCl3): δ 8.20 (d, J = 8.7 Hz, 2H), 8.02
(d, J = 8.4 Hz, 1H), 7.98‐7.90 (m, 1H), 7.60 (dd, J = 36.4,
7.6 Hz, 4H), 7.16 (d, J = 8.4 Hz, 1H), 5.43 (dd, J = 9.0,
2.8 Hz, 1H), 3.29 (ddd, J = 24.7, 15.7, 6.0 Hz, 2H), 2.55
(s, 3H); 13C NMR (150 MHz, CDCl3): δ 158.62, 151.53,
147.18, 145.47, 136.57, 136.48, 132.39, 128.26, 127.00,
126.67, 126.54, 123.62, 121.86, 72.25, 45.15, 21.54.

8
LE ET AL.
3.3.9 | 2‐(6‐Methylquinolin‐2‐yl)‐1‐(3‐
nitrophenyl)ethanol (IIIi)
3.3.13 | 2‐(6‐Methylquinolin‐2‐yl)‐1‐(4‐
(trifluoromethyl)phenyl)ethan‐1‐ol (IIIm)
Yield, 75%; yellow solid, mp 143‐144°C; 1H NMR
(600 MHz, CDCl3): δ 8.36 (s, 1H), 8.14‐8.09 (m, 1H),
8.03 (d, J = 8.3 Hz, 1H), 7.94 (d, J = 9.1 Hz, 1H), 7.82
(d, J = 7.7 Hz, 1H), 7.60‐7.47 (m, 3H), 7.19 (d,
J = 8.4 Hz, 1H), 5.41 (dd, J = 9.0, 3.0 Hz, 1H), 3.30 (qd,
J = 15.7, 6.1 Hz, 2H), 2.54 (s, 3H); 13C NMR (150 MHz,
CDCl3): δ 158.65, 148.37, 146.33, 145.48, 136.49, 136.38,
132.29, 132.03, 129.22, 128.25, 126.97, 126.48, 122.18,
121.84, 121.00, 72.06, 45.25, 21.47.
Yield, 61%; yellow solid, mp 172‐173°C; 1H NMR
(600 MHz, CDCl3): δ 8.01 (d, J = 8.4 Hz, 1H), 7.97‐
7.92 (m, 1H), 7.58 (dt, J = 12.5, 7.6 Hz, 6H), 7.16
(d, J = 8.4 Hz, 1H), 5.37 (dd, J = 8.6, 3.4 Hz, 1H),
3.34‐3.21 (m, 2H), 2.54 (s, 3H); 13C NMR (150 MHz,
CDCl3): δ 159.03, 148.12, 145.57, 136.42, 136.33, 132.27,
129.57, 129.36, 128.34, 126.98, 126.51, 126.19, 125.32,
121.94, 72.48, 45.54, 21.53.
3.3.10 | 2‐(6‐Methylquinolin‐2‐yl)‐1‐(2‐
nitrophenyl)ethanol (IIIj)
3.3.14 | 2‐(6‐Phenylquinolin‐2‐yl)‐1‐(4‐
nitrophenyl)ethanol (IIIn)
Yield, 81%; yellow solid, mp 160‐162°C; 1H NMR
(600 MHz, CDCl3): δ 8.04 (t, J = 8.5 Hz, 2H), 8.00‐7.93
(m, 2H), 7.64 (td, J = 7.8, 0.9 Hz, 1H), 7.57 (dd, J = 10.5,
1.7 Hz, 2H), 7.46‐7.39 (m, 1H), 7.29‐7.24 (m, 1H), 5.79
(dd, J = 9.1, 2.2 Hz, 1H), 3.53 (dd, J = 15.3, 2.3 Hz, 1H),
3.21 (dd, J = 15.3, 9.1 Hz, 1H), 2.55 (s, 3H); 13C NMR
(150 MHz, CDCl3): δ 159.13, 147.58, 145.42, 139.57,
136.59, 136.25, 133.50, 132.20, 128.66, 128.23, 127.90,
127.04, 126.49, 124.22, 121.93, 68.90, 44.82, 21.50.
Yield, 51%; yellow solid, mp 138‐142°C; 1H NMR
(600 MHz, DMSO‐d6):
δ 8.27‐8.17 (m, 2H), 8.14
(dd, J = 23.9, 8.5 Hz, 2H), 8.03‐7.97 (m, 2H), 7.71
(dd, J = 9.3, 2.2 Hz, 2H), 7.68‐7.63 (m, 2H), 7.56‐7.47
(m, 2H), 7.45‐7.39 (m, 1H), 7.27‐7.21 (m, 1H), 5.46
(dd, J = 9.0, 2.9 Hz, 1H), 3.36 (dd, J = 15.7, 3.0 Hz, 1H),
3.29 (dd, J = 15.7, 9.0 Hz, 1H); 13C NMR (150 MHz,
CDCl3): δ 159.54, 151.39, 147.19, 146.25, 140.09, 139.39,
137.35, 130.88, 129.85, 129.01, 127.85, 127.41, 127.13,
126.65, 125.33, 123.63, 122.28, 72.17, 45.31.
3.3.11 | 4‐(1‐Hydroxy‐2‐(6‐methylquinolin‐
2‐yl)ethyl)benzonitrile (IIIk)
3.3.15 | 2‐(6‐(Trifluoromethyl)quinolin‐2‐
yl)‐1‐(4‐nitrophenyl)ethanol (IIIo)
Yield, 77%; yellow solid, mp 148‐150°C; 1H NMR
(600 MHz, CDCl3): δ 8.01 (d, J = 8.3 Hz, 1H), 7.95‐7.90
(m, 1H), 7.64‐7.59 (m, 2H), 7.59‐7.52 (m, 4H), 7.15
(d, J = 8.4 Hz, 1H), 5.35 (dd, J = 9.0, 3.0 Hz, 1H), 3.25
(ddd, J = 24.6, 15.6, 6.0 Hz, 2H), 2.54 (s, 3H); 13C NMR
(150 MHz, CDCl3): δ 158.67, 149.49, 145.47, 136.45,
136.38, 132.29, 132.16, 128.23, 126.95, 126.58, 126.50,
121.86, 118.93, 110.93, 72.35, 45.24, 21.49.
Yield, 71%; yellow solid, mp 134‐135°C; 1H NMR
(600 MHz, CDCl3): δ 8.24‐8.11 (m, 5H), 7.91 (dd, J = 8.8,
1.6 Hz, 1H), 7.65 (d, J = 8.6 Hz, 2H), 7.34 (d, J = 8.4 Hz,
1H), 5.48 (dd, J = 8.9, 2.8 Hz, 1H), 3.37 (ddd, J = 24.9,
15.9, 6.0 Hz, 2H); 13C NMR (150 MHz, CDCl3): δ 162.12,
151.07, 147.97, 147.32, 137.88, 129.87, 126.67, 125.96,
125.85, 125.83, 125.73, 125.71, 123.71, 123.29, 71.97, 45.73.
3.3.12 | 3‐(1‐Hydroxy‐2‐(6‐methylquinolin‐
2‐yl)ethyl)benzonitrile (IIIl)
3.3.16 | 2‐(4‐Chloroquinolin‐2‐yl)‐1‐(4‐
Yield, 59%; white solid, mp 136‐137°C; 1H NMR
(600 MHz, CDCl3): δ 8.03 (d, J = 8.3 Hz, 1H), 7.98‐7.91
(m, 1H), 7.79 (s, 1H), 7.71 (d, J = 7.8 Hz, 1H), 7.61‐7.51
(m, 3H), 7.45 (t, J = 7.7 Hz, 1H), 7.17 (d, J = 8.4 Hz,
1H), 5.34 (dd, J = 8.8, 3.2 Hz, 1H), 3.26 (qd, J = 15.7,
6.0 Hz, 2H), 2.54 (s, 3H); 13C NMR (150 MHz, CDCl3): δ
158.70, 145.66, 145.50, 136.45, 136.37, 132.29, 130.87,
130.34, 129.64, 129.07, 128.27, 126.96, 126.48, 121.82,
118.91, 112.40, 72.04, 45.31, 21.47.
nitrophenyl)ethanol (IIIp)
Yield, 51%; yellow solid, mp 128‐129°C; 1H NMR
(600 MHz, CDCl3): δ 8.24‐8.18 (m, 3H), 8.06 (d, J = 8.4 Hz,
1H), 7.83‐7.76 (m, 1H), 7.65 (t, J = 8.4 Hz, 3H), 7.35 (s,
1H), 6.25 (s, 1H), 5.43 (dd, J = 8.9, 3.0 Hz, 1H), 3.28
(qd, J = 15.8, 6.0 Hz, 2H); 13C NMR (150 MHz, CDCl3):
δ 159.48, 151.04, 147.79, 147.31, 143.64, 131.04, 128.98,
127.53, 126.66, 125.27, 124.16, 123.71, 121.93, 71.99, 45.31.

LE ET AL.
9
3.3.17 | 2‐(8‐Chloroquinolin‐2‐yl)‐1‐(4‐
nitrophenyl)ethanol (IIIq)
3.3.21 | 2‐(Isoquinolin‐1‐yl)‐1‐(2‐nitrophe-
nyl)ethan‐1‐ol (IIIu)
Yield, 81%; yellow solid, mp 167‐169°C; 1H NMR
(600 MHz, CDCl3): δ 8.17 (dd, J = 27.9, 8.5 Hz, 3H),
7.85 (dd, J = 7.5, 1.1 Hz, 1H), 7.74 (dd, J = 8.1, 0.8 Hz,
1H), 7.71‐7.65 (m, 2H), 7.47 (t, J = 7.8 Hz, 1H), 7.30
(d, J = 8.4 Hz, 1H), 5.49 (dd, J = 8.8, 2.7 Hz, 1H), 3.40
(dd, J = 16.3, 2.8 Hz, 1H), 3.33 (dd, J = 16.3, 8.8 Hz,
1H); 13C NMR (150 MHz, CDCl3): δ 160.59, 151.38,
147.16, 142.94, 137.59, 132.79, 130.04, 128.14, 126.72,
126.71, 126.55, 123.59, 122.67, 71.98, 44.77.
Yield, 86%; yellow oil; 1H NMR (600 MHz, CDCl3): δ 8.46‐
8.37 (m, 2H), 8.13 (dd, J = 8.2, 1.4 Hz, 1H), 8.03
(d, J = 8.5 Hz, 1H), 7.85 (dd, J = 16.5, 7.9 Hz, 2H), 7.71
(t, J = 7.3 Hz, 1H), 7.60 (t, J = 6.4 Hz, 2H), 7.53
(t, J = 7.9 Hz, 1H), 5.53 (dd, J = 9.7, 2.1 Hz, 1H), 3.73
(dd, J = 16.5, 2.4 Hz, 1H), 3.48 (dd, J = 16.5, 9.8 Hz,
1H); 13C NMR (150 MHz, CDCl3): δ 159.06, 148.36,
146.32, 140.57, 136.16, 132.17, 130.53, 129.31, 127.69,
127.54, 127.13, 124.50, 122.27, 121.10, 120.16, 71.46, 41.15.
3.3.22 | 4‐(1‐Hydroxy‐2‐(isoquinolin‐1‐yl)
ethyl)benzonitrile (IIIv)
3.3.18 | 2‐(6‐Fluoroquinolin‐2‐yl)‐1‐(4‐
nitrophenyl)ethan‐1‐ol (IIIr)
1
Yield, 69%; yellow solid, mp 135‐137°C; 1H NMR
(600 MHz, CDCl3): δ 8.20 (d, J = 8.7 Hz, 2H), 8.12‐8.00
(m, 2H), 7.64 (d, J = 8.5 Hz, 2H), 7.51 (td, J = 8.7,
2.8 Hz, 1H), 7.43 (dd, J = 8.6, 2.7 Hz, 1H), 7.30‐7.21
(m, 1H), 5.44 (dd, J = 9.0, 2.9 Hz, 1H), 3.31 (ddd,
J = 24.8, 15.8, 6.0 Hz, 2H); 13C NMR (150 MHz, CDCl3):
δ 161.17, 159.52, 158.94, 151.25, 147.20, 143.99, 136.52,
130.99, 126.62, 123.62, 122.69, 120.33, 110.68, 72.08, 45.29.
Yield, 76%; white oil; H NMR (600 MHz, CDCl3): δ 8.42
(d, J = 5.7 Hz, 1H), 8.01 (d, J = 8.5 Hz, 1H), 7.84
(d, J = 8.2 Hz, 1H), 7.71 (t, J = 7.5 Hz, 1H), 7.68‐7.54
(m, 6H), 5.48 (dd, J = 9.7, 2.0 Hz, 1H), 3.68 (dd, J = 16.5,
2.2 Hz, 1H), 3.44 (dd, J = 16.5, 9.7 Hz, 1H); 13C NMR
(150 MHz, CDCl3): δ 159.09, 149.53, 140.58, 136.19,
132.19, 130.50, 127.65, 127.53, 127.17, 126.67, 124.48,
120.11, 118.88, 111.03, 71.72, 41.15.
3.3.23 | 3‐(1‐Hydroxy‐2‐(isoquinolin‐1‐yl)
3.3.19 | 2‐(Isoquinolin‐1‐yl)‐1‐(4‐nitrophe-
nyl)ethan‐1‐ol (IIIs)
ethyl)benzonitrile (IIIw)
1
Yield, 69%; white oil; H NMR (600 MHz, CDCl3): δ 8.43
Yield, 90%; yellow oil; 1H NMR (600 MHz, CDCl3): δ 8.43
(d, J = 5.8 Hz, 1H), 8.22 (d, J = 8.6 Hz, 2H), 8.02
(d, J = 8.5 Hz, 1H), 7.85 (d, J = 8.2 Hz, 1H), 7.75‐7.66
(m, 3H), 7.63‐7.58 (m, 2H), 5.54 (dd, J = 9.6, 2.0 Hz,
1H), 3.72 (dd, J = 16.6, 2.3 Hz, 1H), 3.47 (dd, J = 16.5,
9.7 Hz, 1H); 13C NMR (150 MHz, CDCl3): δ 159.02,
151.52, 147.17, 140.52, 136.18, 130.58, 127.73, 127.57,
127.13, 126.75, 124.45, 123.64, 120.20, 71.58, 41.06.
(d, J = 5.8 Hz, 1H), 8.03 (d, J = 8.5 Hz, 1H), 7.88‐7.81
(m, 2H), 7.79‐7.67 (m, 2H), 7.60 (ddd, J = 15.6, 9.3,
4.4 Hz, 3H), 7.47 (t, J = 7.7 Hz, 1H), 5.46 (dd, J = 9.8,
2.1 Hz, 1H), 3.69 (dd, J = 16.5, 2.3 Hz, 1H), 3.44
(dd, J = 16.5, 9.8 Hz, 1H); 13C NMR (150 MHz, CDCl3):
δ 159.12, 145.65, 140.54, 136.17, 130.96, 130.55, 130.47,
129.74, 129.16, 127.70, 127.55, 127.13, 124.51, 120.16,
118.95, 112.42, 71.43, 41.21.
3.3.20 | 2‐(Isoquinolin‐1‐yl)‐1‐(3‐nitrophe-
nyl)ethan‐1‐ol (IIIt)
3.3.24 | 2‐(Isoquinolin‐1‐yl)‐1‐(4‐
(trifluoromethyl)phenyl)ethan‐1‐ol (IIIx)
Yield, 81%; yellow oil; 1H NMR (600 MHz, CDCl3): δ 8.44
(d, J = 5.8 Hz, 1H), 8.11 (dd, J = 28.1, 8.1 Hz, 2H), 7.97
(dd, J = 8.2, 0.8 Hz, 1H), 7.85 (d, J = 8.2 Hz, 1H), 7.70
(ddd, J = 21.6, 11.3, 4.5 Hz, 3H), 7.65‐7.60 (m, 2H), 5.88
(dd, J = 9.5, 1.9 Hz, 1H), 4.06 (dd, J = 16.0, 2.1 Hz, 1H),
3.39 (dd, J = 16.0, 9.5 Hz, 1H); 13C NMR (150 MHz,
CDCl3): δ 159.42, 147.71, 140.37, 139.57, 136.33, 133.49,
130.83, 130.54, 128.84, 127.96, 127.69, 127.40, 124.89,
124.14, 120.14, 68.19, 40.58.
Yield, 90%; yellow oil; 1H NMR (600 MHz, CDCl3): δ 8.42
(d, J = 5.8 Hz, 1H), 8.02 (d, J = 8.5 Hz, 1H), 7.83
(d, J = 8.2 Hz, 1H), 7.69 (t, J = 7.6 Hz, 1H), 7.65‐7.61
(m, 4H), 7.58 (t, J = 6.9 Hz, 2H), 5.49 (dd, J = 9.8,
2.0 Hz, 1H), 3.69 (dd, J = 16.5, 2.3 Hz, 1H), 3.46
(dd, J = 16.5, 9.8 Hz, 1H); 13C NMR (150 MHz, CDCl3):
δ 159.39, 148.13, 140.61, 136.15, 130.45, 127.60, 127.51,
127.18, 126.27, 125.67, 125.34, 124.56, 120.04, 119.45,
71.80, 41.47.

10
LE ET AL.
[10] Y. Lu, G. F. Jiang, Z. B. Xie, G. Q. Chen, Z. G. Le, Chinese J.
Org. Chem. 2018, 38, 1837.
ACKNOWLEDGMENTS
We thank the National Natural Science Foundation of
China (nos. 21462001, 21465002, and 11765002), the
Science and Technology Projects of Jiangxi (no.
20161BCB24006), and the National Natural Science
Foundation of Jiangxi (no. 20181BAB203019) for the
financial support. We also appreciate the instrumentation
for the experimental testing provided by other research
groups in East China University of Technology.
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How to cite this article: Le Z‐G, Lu Y, Jiang G‐F,
Liu Y‐S, Liu J, Xie Z‐B. α‐Chymotrypsin–catalyzed
direct C (Sp3)–H functionalization reactions for
synthesis of azaarene derivatives in water. J
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