Discovery of coumarin-based selective aldehyde dehydrogenase 1A1 inhibitors with glucose metabolism improving activity

Article abstract of DOI:10.1016/j.ejmech.2019.111923

Overexpression of aldehyde dehydrogenase 1A1 (ALDH1A1) is associated with the occurrence and development of obesity and insulin resistance. Herein, a series of coumarin-based ALDH1A1 inhibitors were designed, synthesized and evaluated. Among them, compounds 10, 14 and 26 exhibited potent inhibitory activity against ALDH1A1 and high selectivity over ALDH1A2, ALDH1A3, ALDH2 and ALDH3A1. Optimized compound 10 showed markedly improved pharmacokinetic characters and ADME profiles comparing to the lead compound 1. In vitro study demonstrated that 10 alleviated palmitic acid-induced impairment of glucose consumption in HepG2 cells.

Full text of DOI:10.1016/j.ejmech.2019.111923

Journal Pre-proof
Discovery of coumarin-based selective aldehyde dehydrogenase 1A1 inhibitors with
glucose metabolism improving activity
Dailin Liang, Yazhou Fan, Zhou Yang, Zhenguo Zhang, Meiyang Liu, Li Liu, Cheng
Jiang
PII:
S0223-5234(19)31075-X
DOI:
https://doi.org/10.1016/j.ejmech.2019.111923
EJMECH 111923
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 10 August 2019
Revised Date: 28 November 2019
Accepted Date: 28 November 2019
Please cite this article as: D. Liang, Y. Fan, Z. Yang, Z. Zhang, M. Liu, L. Liu, C. Jiang, Discovery
of coumarin-based selective aldehyde dehydrogenase 1A1 inhibitors with glucose metabolism
improving activity, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.ejmech.2019.111923.
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Graphical Abstract
Discovery of coumarin-based selective aldehyde dehydrogenase 1A1 inhibitors with
glucose metabolism improving activity

Discovery of coumarin-based selective aldehyde dehydrogenase 1A1
inhibitors with glucose metabolism improving activity
a,b
c
a,b
a,b
b
Dailin Liang , Yazhou Fan , Zhou Yang , Zhenguo Zhang , Meiyang Liu , Li
c,
a,b,
Liu **, Cheng Jiang
*
a
Jiang Su Key Laboratory of Drug Design and Optimization, China Pharmaceutical
University, Tongjiaxiang 24, Nanjing 210009, China
b
Department of Medicinal Chemistry, China Pharmaceutical University, Tongjiaxiang
2
4, Nanjing 210009, China
c
Key Laboratory of Drug Metabolism and Pharmacokinetics, China Pharmaceutical
University, Tongjiaxiang 24, Nanjing 210009, China
AUTHOR INFORMATION
Corresponding Authors
*
*
*E-mail for L.L.: liulee@cpu.edu.cn
E-mail for C.J.: jc@cpu.edu.cn

Abstract: Overexpression of aldehyde dehydrogenase 1A1 (ALDH1A1) is associated
with the occurrence and development of obesity and insulin resistance. Herein, a
series of coumarin-based ALDH1A1 inhibitors were designed, synthesized and
evaluated. Among them, compounds 10, 14 and 26 exhibited potent inhibitory activity
against ALDH1A1 and high selectivity over ALDH1A2, ALDH1A3, ALDH2 and
ALDH3A1. Optimized compound 10 showed markedly improved pharmacokinetic
characters and ADME profiles comparing to the lead compound 1. In vitro study
demonstrated that 10 alleviated palmitic acid-induced impairment of glucose
consumption in HepG2 cells.
Key words: ALDH1A1 inhibitors; coumarin derivatives; synthesis; glucose
consumption

1
. Introduction
The human body meets with a variety of aldehydes from inner biomolecular
metabolism and outer environment [1]. When aldehydes are in overload quantities
within the human body, they can cause cytotoxicity [2,3]. Aldehyde dehydrogenases
(ALDHs) are used to relieve aldehyde pressure in the human body [4]. The human
genome encodes 19 NAD(P)+-dependent aldehyde dehydrogenases that metabolize
aldehydes into their respective carboxylic acid derivatives [5]. ALDHs play an
important physiological and toxicological role in cancers, metabolic disorders and
other diseases [6]. Changes of ALDHs biological activity and metabolic pathways in
vivo are associated with the occurrence and development of a series of diseases such
as inflammation, Parkinson’s disease and diabetes [7-10].
Several studies have demonstrated that the function and expression of aldehyde
dehydrogenase 1A1 (ALDH1A1, also known as RALDH1) was altered in obesity and
diabetes [11-13]. It has been reported that ALDH1A1 knockout can protect mice
against high-fat-diet (HFD) induced obesity and insulin resistance, accompanied by
remarkable reduction in hepatic gluconeogenesis and fatty acid biosynthesis [14, 15].
In addition, ALDH1A1 inhibitor citral treatment can reduce lipid accumulation and
improved glucose tolerance in ob/ob mice [16]. These results indicated involvement
of ALDH1A1 induction in the progress of obesity and insulin resistance. Our previous
research has confirmed that citral can improve glucose metabolism through inhibiting
ALDH1A1 activity [17]. Therefore, discovery of selective ALDH1A1 inhibitors can
be a novel treatment for ALDH1A1 related diseases.

Several small molecule ALDH1A1 inhibitors obtained from high throughput
screening (HTS) have been introduced (Fig. 1) in recent years [18-22].
Theophylline-based 1 (CM026), substituted tricyclic pyrimidinone 2 (CM37) and
pyrazolopyrimidinone 3 (CM39) reported by Hurley and co-workers are effective
ALDH1A1 inhibitors with IC50 values of 0.8, 0.02 and 4.6 µM respectively and
showed excellent selectivity against other ALDH isozymes, such as ALDH2 and
ALDH3A1 [18-20]. Quinoline-based compound 4 (NCT-505) was identified as a
potent and highly selective ALDH1A1 inhibitor (IC = 0.007 µM) and displayed
5
0
significant growth inhibition in MIA PaCa-2 cells [21,22]. It is noteworthy that all the
reported ALDH1A1 inhibitors contain a bicyclic or tricyclic hetero cyclic core
attached by two adjacent arms. It has also been considered that theophylline was not
active in inhibiting ALDH1A1 at concentrations up to 250 µM, suggesting that two
adjacent arms attached to the theophylline play an important role in ALDH1A1
inhibitory activity [18]. Therefore we focus on the modification of the two arms
aiming for improving the potency and selectivity of ALDH1A1 inhibitors [18].
Fig. 1. Representative small molecule ALDH1A1 inhibitors
Some coumarin derivatives were reported as inhibitors of ALDH1/2 family of

isoenzymes. These compounds could be further optimized to improve selectivity for
their respective isoenzymes [23]. However, most of the reported coumarin derivatives
were suffered with lack of selectivity against other ALDH isozymes [23]. Compound
1
showed potent ALDH1A1 inhibitory activity and excellent selectivity against other
ALDH isozymes, however, it was suffered from poor PK characters such as poor oral
bioavailability. Taking compound 1 as a lead, we replaced the theophylline core with a
more hydrophobic coumarin core. Two adjacent arms attached to nitrogen atom of the
lipophilic 7-amino-4-methyl-2H-chromen-2-one were modified (Fig. 2). A series of
coumarin-based derivatives were obtained and the optimized compound was
identified as potent and selective ALDH1A1 inhibitor with good pharmacokinetic
profiles, and showed glucose metabolism improving activity.
Fig. 2. Design of target compounds
2
. Results and discussion
2
.1. Chemistry
The synthesis of the designed compounds is shown in Scheme 1.
-Amino-4-methyl-2H-chromen-2-one (5) was reacted with appropriate halides or
7

aldehydes and sodium cyanoborohydride to give intermediates 6a-6m. Substitution of
a-6m with corresponding acyl chloride led to the intermediates 7a-7o, which were
6
reacted with 1-Boc-piperazine to generate compounds 8a-8o. The amines 9a-9o were
obtained by the deprotection of 8a-8o respectively in the presence of trifluoroacetic
acid. Acylation of 9 provided designed final compounds 10-37 in good yields.
1
Scheme 1. Reagents and conditions: (a) R -X (X=Br or I), K CO , DMF, 60℃ , 5-10 h, for 6a-6e
2
3
1
and 6j-6m; or R CHO, CH COOH, NaBH CN, DCM, 25℃ , 3-7 h, for 6f-6i; (b)
3
3
3
-Chloropropionyl chloride, Et N, DCM, 25℃ , 2-3 h, for 7a-7m; or Chloroacetyl chloride, Et N,
3 3
DCM, 25℃ , 2-3 h, for 7n; or 4-Chlorobutyryl chloride, Et N, DCM, 25℃ , 2-3 h, for 7o; (c)
3
1
-Boc-piperazine, K CO , KI, CH CN, 60℃ to reflux, overnight; (d) CF COOH, 25℃ , 1h; (e)
2 3 3 3
2
R COOH, EDCI, Et N, DCM, 25℃ , overnight.
3
2
.2. Biological evaluation
2
.2.1. Inhibitory activity against ALDH1A1
The inhibitory activity of the synthesized 7-amino-4-methyl-2H-chromen-2-one
derivatives were screened at a relatively high single concentration (10 µM) to identify
the most potent inhibitor. Compound 1 (CM26), which was synthesized according to
the literature, was used as a control [6]. The inhibition rate of compound 1 was 75.22%
1
at the concentration of 10 µM. Our initial process focused on the optimization of R

modification as shown in Table 1. Firstly, the linker between the nitrogen atom of the
core to piperazine was kept as three atoms and the terminal aromatic substitution was
furan-2-yl, same as compound 1. As shown in Table 1, benzyl (compound 19, 2.96 %)
1
or substituted benzyl (compounds 20-22, 6.43-13.23 %) as R substituents resulted in
notable loss of potency compared to compound 1 (75.22 %). Compounds with cyclic
1
aliphatic R substitution (compounds 15-18) also significantly dropped the potency. In
these four compounds, the inhibitory activity decreased accordingly with the
increasing size of the ring, indicating that bulky rings at this portion were not
favorable. Compounds with small alkyl substitutions, such as ethyl or propyl
(
compounds 10-11) showed no obvious improvement on inhibitory activity. The
inhibitory activity decreased about 3 times with bulky alkyl chains introduced
compounds 12-13). Interestingly, the inhibitory activity was comparable to that of
compound 1 when isopentenyl group was introduced (compound 14, 76.50 %). Data
(
1
1
obtained from R modification suggested that R group was critical to inhibitory
activity and isopentenyl is the optimal substitution.
1
Table 1. Structure-activity relationships for R substitution of coumarin derivatives based on
percent activity at 10 µM compound.
hALDH1A1
1
Compounds
R
a
%
inhibition at 10 µM

1
(CM26)
75.22 ± 5.28
61.82 ± 7.97
50.68 ± 3.23
26.52 ± 4.56
1
1
1
0
1
2
1
1
1
1
1
1
1
2
2
2
3
4
5
6
7
8
9
0
1
2
25.46 ± 1.97
76.50 ± 5.41
31.97 ± 6.21
35.10 ± 2.09
17.41 ± 2.56
26.19 ± 2.78
2.96 ± 0.54
13.23 ± 2.36
9.77 ± 1.25
6.43 ± 0.59
a
Values are the mean ± SD of three independent experiments (n = 3)
1
2
Then the isopentenyl group as R substitution and the terminal furan-2-yl R
substitution were kept and the length of the linker between the nitrogen atom of the
core to piperazine was explored. Compounds 23-24 were thus designed and evaluated
for their inhibitory activities (Table 2). Dramatically decreased inhibitory activity was
observed for compound 23 and 24 (33.65 % and 4.77 % for 23 and 24 respectively)
when the length of linker between nitrogen atom of the core to piperazine was
changed to 2 or 4 carbon atoms. The data suggested the length of the linker had an
important influence on the inhibitory activity and the optimal length is three atoms

(n=2 in Table 2).
Table 2. Structure-activity relationships for the length of linker between nitrogen atom to
piperazine of coumarin derivatives based on percent activity at 10 µM compound.
hALDH1A1
Compounds
n
a
%
inhibition at 10 µM
75.22 ± 5.28
1
(CM26)
1
2
2
4
3
4
2
1
3
76.50 ± 5.41
33.65 ± 2.95
4.77 ± 0.34
a
Values are the mean ± SD of three independent experiments (n = 3)
2
We then explored the substitutions of R for their effect to the inhibitory activity.
Compounds 25-37 (Table 3) were then designed and synthesized. As shown in Table 3,
2
compound 37 bearing an aliphatic ring as R substitution displayed significantly
2
decreased inhibitory activity. Compound 25 with a para-methyl phenyl group as R
substitution showed a weak variation in inhibition. Compounds 26 and 27, which
contained an electron-donating group at the para position of the phenyl ring
maintained the inhibitory activity and their inhibition rates were 77.28 % and 66.50 %
respectively at 10 µM. However, decreased activity was observed for compounds
2
8-33 bearing an electron-withdrawing group at the terminal phenyl group. These
2
results indicate that the electron density of the R group plays an important role in the

aspects of ALDH1A1 inhibitory activity. Similar results were also observed.
Compared with compounds 14 and 36, compounds 34 and 35 which contain a
2
pyridine ring on the R portion displayed 2-3 folds decrease in inhibitory activity. All
2
data obtained from R modifications suggested that compared with the aliphatic ring
and the electron-deficient aromatic ring, the electron-rich aromatic ring is beneficial
for the retention and improvement of the inhibitory activity. Among this series of
2
compounds, the preferred R group was 4-isopropylphenyl or furan-2-yl.
2
Table 3. Structure-activity relationships for R substitution of coumarin derivatives based on
percent activity at 10 µM compound.
hALDH1A1
2
Compounds
R
a
%
inhibition at 10 µM
1
(CM26)
75.22 ± 5.28
1
2
4
5
76.50 ± 5.41
49.69 ± 3.25
2
6
77.28 ± 4.78
2
2
2
3
7
8
9
0
65.20 ± 5.78
47.08 ± 6.12
25.07 ± 3.56
34.13 ± 4.79

3
3
3
3
3
1
2
3
4
5
33.72 ± 2.79
55.36 ± 3.71
51.77 ± 4.75
23.54 ± 1.87
26.58 ± 2.53
3
3
6
7
50.82 ± 6.91
36.18 ± 2.76
a
Values are the mean ± SD of three independent experiments (n = 3)
2
.2.2. IC determination of selected analogs against ALDH1A1.
50
The IC50 values of the five compounds (10, 14, 26, 27, 32) with more than 55%
inhibition to the ALDH1A1 at 10 µM were examined with full concentration-response
curves as shown in Table 4. The results were consistent with the inhibition rate of
ALDH1A1 at 10 µM. Compound 10 (IC = 3.87 µM) was three folds less potent than
5
0
2
2
1
(IC50 = 1.21 µM), while compound 14 (R = 2-furanyl) and 26 (R =
p-isopropylphenyl) exhibited potent inhibitory activity to ALDH1A1 with IC values
50
2
of 1.96 µM and 2.75 µM, respectively. Compound 27 (R = p-methoxylphenyl, IC =
50
2
5
2
1
.86 µM) was 5 times less potent than 1. Compound 32 (R = m-nitrophenyl, IC =
50
2
9.34 µM) containing an electron-withdrawing R group was 24 times less potent than
2
in inhibitory activity. These results illustrated that the electron-withdrawing R
substituents were detrimental to the inhibitory activity of ALDH1A1.

Table 4. IC values for representative coumarin derivatives.
5
0
hALDH1A1
2
Compounds
R
a
IC50 ± SD [µM]
1
(CM26)
1.21 ± 0.18
3.87 ± 0.48
1.96 ± 0.31
1
1
0
4
2
6
2.75 ± 0.25
2
3
7
2
5.86 ± 0.67
29.34 ± 3.25
a
Values are the mean ± SD of three independent experiments (n = 3)
2
.2.3. Selectivity evaluation of selected analogs against other ALDH isozymes.
Structural research shows that ALDH1A1 shares more than 70 % sequence similarity
to both ALDH1A2 and ALDH1A3, nearly 70 % to the mitochondrial ALDH2 enzyme
and less than 50 % to ALDH3A1 [22]. Compounds with excellent ALDH1A1
inhibitory activity (10, 14, 26, 27) were selected to test their activity against a panel of
enzymes including ALDH1A subfamily (ALDH1A2, ALDH1A3) and ALDH
isozymes (ALDH2, ALDH3A1) as shown in Table 5. All of the tested compounds
showed high selectivity over other ALDH isozymes.

Table 5. Selectivity against other ALDH isozymes.
a
%
inhibition at 10 µM
hALDH1A1 IC50
Compounds
a
[
µM]
ALDH1A2
2.13 ± 0.75
1.95 ± 0.38
2.25 ± 0.98
1.98 ± 0.77
ALDH1A3
2.59 ± 0.24
1.34 ± 0.15
2.65 ± 0.12
1.04 ± 0.09
ALDH2
2.24 ± 0.32
1.87 ± 0.72
0.85 ± 0.15
0.62 ± 0.07
ALDH3A1
1.89 ± 0.19
0.78 ± 0.55
0.96 ± 0.31
0.82 ± 0.11
1
1
2
2
0
4
6
7
3.87 ± 0.48
1.96 ± 0.31
2.75 ± 0.25
5.86 ± 0.67
a
Values are the mean ± SD of three independent experiments (n=3).
2
.2.4. Metabolic stability in human liver microsomes of optimized compounds.
The most potent compound, 14, was then selected and evaluated for its metabolic
stability in human microsomes. However, compound 14 showed poor microsomal
stability (T1/2 = 7 min). This may be due to the isopentenyl substitution which was
easily be metabolized. Compound 10 containing an ethyl group was selected and
evaluated for its metabolic stability. Compound 10 exhibited acceptable human
microsomal stability (T = 38.5 min). While compound 1 showed higher human
1
/2
microsomal stability (T1/2 > 60 min).
2
.2.5. Pharmacokinetic (PK) results of compound 1and 10 in SD mice
With promising potency and cellular activity, as well as acceptable stability in human
liver microsomes, compound 10 was selected for further evaluation for

pharmacokinetics (PK) in male SD mice at 2.5 mg/kg and 25 mg/kg for intravenous
iv) and oral (po) administration, compared with compound 1. As shown in Table 6,
(
compound 10 showed dramatically improved oral bioavailability (41.9% for
compound 10 vs 1.9% for compound 1) and more favorable PK profile. The moderate
volume of distribution (Vss) of 0.8 L/kg suggested that compound 10 penetrated to
tissues well, and high systemic exposure (oral AUC =6475 h*ng/mL) indicated that
all
compound 10 was well absorbed after oral administration, while the oral AUC value
all
of compound 1 was 122 h*ng/mL. Compound 10 exhibited more reasonable plasma
clearance level (CL=27 mL/min/kg) compared to compound 1 (CL=63 mL/min/kg)
and longer half-life (T1/2 = 45 min and 108 min for iv and oral administration,
respectively).
Table 6. Pharmacokinetic (PK) results of compound 1 and 10.
admin
AUCall
V
ss
CL
compounds
sample
C
max (ng/mL)
T
1/2(min)
F(%)
(
dosage)
(min*ng/mL)
(mL/kg)
1773.76
(mL/min/kg)
a
b
iv (2.5 mg/kg)
plasma
plasma
plasma
plasma
2534.99
30.27
14.92
45.40
108.33
76937.22
7307.55
62.99
26.76
1
a
po (25 mg/kg)
235.75
1.9 %
a
b
iv (2.5 mg/kg)
2844.06
92676.01
388474.87
843.20
1
0
a
po (25mg/kg)
3316.41
41.9%
a
b
n=5. Cmax = C (t = 5 min) for iv administration.
5
2
.2.6. Compound 10 can alleviate palmitic acid-induced impairment of glucose
consumption in HepG2 cells
Effects of compound 10 and 1 on glucose consumption in palmitate-treated HepG2
were investigated according to the reported method [17,24]. It was found that palmitic

acid significantly impaired glucose metabolism in HepG2 cells. Compound 10 can
significantly alleviate palmitic acid-induced impairment of glucose metabolism in
HepG2 cells while compound 1 showed little alleviation (Fig. 3).
1.5
##
1.0
*#
**
**
**
0.5
0.0
0
0.2
2
5
2
5 µM
_
__ ___ _________ _________
Con PA Compound 10 Compound 1
Fig. 3. Effects of compound 10 and 1 on glucose consumption in palmitic acid (PA) treated
HepG2 cells. Data were expressed in mean ± S.D. (n = 4). *p < 0.05, **p < 0.01 versus untreated
#
##
cells; p < 0.05, p < 0.01 versus cells treated with PA alone.
3
. Conclusion
In summary, a series of coumarin-based ALDH1A1 inhibitors were designed,
synthesized and evaluated. Several of them exhibited potent inhibitory activity against
ALDH1A1 and high selectivity over other ALDH isozymes (ALDH1A2, ALDH1A3,
ALDH2 and ALDH3A1). The optimized compound 10 showed potent ALDH1A1
inhibitory activity and excellent selectivity against other ALDH isozymes, together
with markedly improved PK characters comparing to compound 1. Compound 10 can
effectively alleviate palmitic acid-induced impairment of glucose consumption in

HepG2 cells. Compound 10 provided an excellent lead compounds for optimization
towards the development as a promising candidate for the treatment of obesity and
diabetes.
4
. Experimental section
4
.1. Chemistry
Chemical reagents and solvents were obtained from commercial sources and used
without further purification. All reactions were monitored by TLC on silica gel plates
1
and flash chromatography was performed using silica gel (300-400 mesh). H-NMR
1
3
and C-NMR spectra were measured on Bruker 300 MHz or 400 MHz spectrometer
using tetramethylsilane as the internal standard. Chemical shifts were reported in parts
per million (ppm, δ) downfield from tetramethylsilane. Proton coupling patterns were
described as singlet (s), doublet (d), doublet of doublets (dd), triplet (t) and multiplet
(m). The purity of the synthesized compounds were measured by analytical high
performance liquid chromatography (HPLC, Agilent) using the C18 reverse phase
column. The molecular masses of target compounds were determined using ESI-MS
(Thermo-Finnigan, San Jose, CA, USA).
4
.1.1. General procedure for preparation of compounds (6a-6d). To a solution of
7
-amino-4-methylcoumarin 5 (10.00 g, 57.08 mmol) in DMF (100 mL) was added
potassium carbonate (11.83 g, 85.62 mmol) and stirred at 0 °C for 15 min. Then

different halides (68.50 mmol) were added sequentially and stirred at 60 °C for 5-10 h
until the reaction was completed. After cooling to room temperature, the mixture was
poured into water (800 mL) and extracted with ethyl acetate (500 mL×3). The
combined organic layer was dried over anhydrous sodium sulfate and filtered. After
removal of solvent, the crude product was purified by silica gel chromatography to
give 6a-6d respectively.
4
.1.1.1. 7-Ethylamino-4-methyl-2H-chromen-2-one (6a). A yellow solid. Yield 62 %.
1
Mp 152-155 °C. H NMR (300 MHz, CDCl ) δ 7.31 (d, J = 8.6 Hz, 1H), 6.50-6.45 (m,
3
1
H), 6.41 (d, J = 1.5 Hz, 1H), 5.93 (s, 1H), 4.22 (s, 1H), 3.23-3.13 (m, 2H), 2.31 (s,
+
3
H), 1.26 (t, J = 7.2 Hz, 3H). ESI-MS m/z: 204.1 [M+H] .
4
.1.1.2. 7-Propylamino-4-methyl-2H-chromen-2-one (6b). A yellow solid. Yield 53 %.
1
Mp 164-167 °C. H NMR (300 MHz, CDCl ) δ 7.37 (d, J = 8.6 Hz, 1H), 6.52 (dd, J =
3
8
.6, 2.3 Hz, 1H), 6.46 (d, J = 2.2 Hz, 1H), 5.99 (s, 1H), 4.23 (s, 1H), 3.18-3.09 (m,
2
H), 2.36 (s, 3H), 1.72-1.63 (m, 2H), 1.04 (t, J = 7.4 Hz, 3H). ESI-MS m/z: 218.1
+
[
M+H] .
4
.1.1.3. 7-Butylamino-4-methyl-2H-chromen-2-one (6c). A yellow solid. Yield 63 %.
1
Mp 175-178 °C. H NMR (300 MHz, CDCl ) δ 7.36 (d, J = 8.6 Hz, 1H), 6.51 (dd, J =
3
8
.7, 2.3 Hz, 1H), 6.46 (d, J = 2.2 Hz, 1H), 5.99 (s, 1H), 4.21 (s, 1H), 3.23-3.15 (m,
2
H), 2.36 (s, 3H), 1.71-1.64 (m, 2H), 1.50-1.42 (m, 2H), 0.99 (t, J = 7.3 Hz, 3H).
+
ESI-MS m/z: 232.1 [M+H] .

4
.1.1.4. 7-Isopentylamino-4-methyl-2H-chromen-2-one (6d). A yellow solid. Yield
1
6
1 %. Mp 161-164 °C. H NMR (300 MHz, CDCl3) δ 7.33 (d, J = 8.4 Hz, 1H), 6.60
-6.51(m, 1H), 6.48 (d, J = 2.1 Hz, 1H), 6.01 (s, 1H), 4.23 (s, 1H), 3.29-3.22 (m, 2H),
2
.40 (s, 3H), 1.65-1.61 (m, 1H),1.48-1.42 (m, 2H), 0.91(d, J = 6.6 Hz, 6H). ESI-MS
+
m/z: 246.2 [M+H] .
4
.1.2. General procedure for preparation of compounds (6e, 6j-6m). To a solution of
7
-amino-4-methylcoumarin 5 (10.00 g, 57.08 mmol) in DMF (100 mL) was added
potassium carbonate (11.83 g, 85.62 mmol) and stirred at 0 °C for 15 min. Then
different halides (68.50 mmol) were added sequentially and stirred at room
temperature for 2-3 h until the reaction was completed. After cooling to room
temperature, the mixture was poured into water (800 mL) and extracted with ethyl
acetate (500 mL×3). The combined organic layer was dried over anhydrous sodium
sulfate and filtered. After removal of solvent, the crude product was purified by silica
gel chromatography to give 6e, 6j-6m respectively.
4
.1.2.1. 7-Isopentenylamino-4-methyl-2H-chromen-2-one (6e). A yellow solid. Yield
1
7
0 %. Mp 142-145 °C. H NMR (300 MHz, CDCl ) δ 7.35 (d, J = 8.6 Hz, 1H), 6.51
3
(dd, J = 8.7, 2.4 Hz, 1H), 6.43 (d, J = 2.3 Hz, 1H), 5.98-5.93 (m, 1H), 5.33-5.25 (m,
1
H), 4.21 (s, 1H), 3.78-3.69 (m, 2H), 2.33 (d, J = 1.2 Hz, 3H), 1.74 (d, J = 7.6 Hz,
+
6
H). ESI-MS m/z: 244.1 [M+H] .
4
.1.2.2. 7-Benzylamino-4-methyl-2H-chromen-2-one (6j). A yellow solid. Yield 65 %.
1
Mp 132-135 °C. H NMR (300 MHz, CDCl ) δ 7.39-7.35 (m, 5H), 7.34-7.30 (m, 1H),
3

6
.57 (dd, J = 8.7, 2.3 Hz, 1H), 6.49 (d, J = 2.3 Hz, 1H), 5.98 (s, 1H), 4.76 (d, J = 5.2
+
Hz, 1H), 4.44-4.38 (m, 2H), 2.35 (s, 3H). ESI-MS m/z: 266.1 [M+H] .
4
.1.2.3. 7-(2-Methylbenzyl)amino-4-methyl-2H-chromen-2-one (6k). A yellow solid.
1
Yield 61 %. Mp 121-124 °C. H NMR (300 MHz, CDCl ) δ 7.37-7.33 (m, 4H),
3
7
.32-7.29 (m, 1H), 6.55 (dd, J = 8.6, 2.2 Hz, 1H), 6.47 (d, J = 2.1 Hz, 1H), 5.97 (s, 1
H), 4.74 (d, J = 5.1 Hz, 1H), 4.42-4.36 (m, 2H), 2.33 (s, 3H), 2.13(s, 3H). ESI-MS m/z:
+
2
80.1 [M+H] .
4
.1.2.4. 7-(3-Methylbenzyl)amino-4-methyl-2H-chromen-2-one (6l). A yellow solid.
1
Yield 59 %. Mp 112-115 °C. H NMR (300 MHz, CDCl ) δ 7.36-7.32 (m, 4H),
3
7
.31-7.28 (m, 1H), 6.53 (dd, J = 8.7, 2.3 Hz, 1H), 6.45 (d, J = 2.0 Hz, 1H), 5.98 (s, 1
H), 4.75 (d, J = 5.2 Hz, 1H), 4.42-4.35 (m, 2H), 2.34 (s, 3H), 2.15(s, 3H). ESI-MS m/z:
+
2
80.1 [M+H] .
4
.1.2.5. 7-(4-Methylbenzyl)amino-4-methyl-2H-chromen-2-one (6m). A yellow solid.
1
Yield 62 %. Mp 139-142 °C. H NMR (300 MHz, CDCl ) δ 7.35-7.30 (m, 4H),
3
7
.29-7.26 (m, 1H), 6.51 (dd, J = 8.6, 2.1 Hz, 1H), 6.42 (d, J = 2.1 Hz, 1H), 5.97 (s,
1
H), 4.73 (d, J = 5.1 Hz, 1H), 4.40-4.33 (m, 2H), 2.33 (s, 3H), 2.11(s, 3H). ESI-MS
+
m/z: 280.1 [M+H] .
4
.1.3. General procedure for preparation of compounds (6f-6i). To a solution of
-amino-4-methylcoumarin 5 (1.50 g, 8.56 mmol) in DCM (50 mL) was added acetic
7
acid (0.05 g, 0.86 mmol) and different aliphatic aldehydes (10.27 mmol). The mixture
was stirred at room temperature for 1 h. Then sodium cyanoborohydride (1.08 g,

1
7.12 mmol) was added sequentially and stirred at room temperature for 3-7h until the
reaction was completed. The reaction was quenched with a saturated aqueous solution
50 mL) and the mixture was extracted with DCM (50 mL×3). The combined organic
(
layer was dried over anhydrous sodium sulfate and filtered. After removal of solvent,
the crude product was purified by silica gel chromatography to give 6f-6i respectively.
4
.1.3.1. 7-Cyclopropylmethylamino-4-methyl-2H-chromen-2-one (6f). A yellow solid.
1
Yield 48 %. Mp 133-136 °C. H NMR (300 MHz, CDCl ) δ 7.34 (d, J = 8.5 Hz, 1H),
3
6
.51-6.47 (m, 1H), 6.44 (d, J = 2.1 Hz, 1H), 5.98 (s, 1H), 4.22 (s, 1H), 3.70-3.63 (m,
2
H), 2.36 (s, 3H), 1.02-0.92 (m, 1H), 0.49-0.42 (m, 2H), 0.14-0.09 (m, 2H). ESI-MS
+
m/z: 230.1 [M+H] .
4
.1.3.2. 7-Cyclobutylmethylamino-4-methyl-2H-chromen-2-one (6g). A yellow solid.
1
Yield 53 %. Mp 141-143 °C. H NMR (300 MHz, CDCl ) δ 7.32 (d, J = 8.7 Hz, 1H),
3
6
.50-6.45 (m, 1H), 6.43 (d, J = 2.0 Hz, 1H), 6.01 (s, 1H), 4.24 (s, 1H), 3.90-3.83 (m,
2
H), 2.43 (s, 3H), 2.05-1.90 (m, 3H), 1.90-1.80 (m, 2H), 1.75-1.65 (m, 2H). ESI-MS
+
m/z: 244.1 [M+H] .
4
.1.3.3. 7-Cyclopentylmethylamino-4-methyl-2H-chromen-2-one (6h). A yellow solid.
1
Yield 49 %. Mp155-158°C. H NMR (300 MHz, CDCl ) δ 7.35 (d, J = 8.7 Hz, 1H),
3
6
2
1
.51-6.45 (m, 1H), 6.45 (d, J = 2.1 Hz, 1H), 6.02 (s, 1H), 4.27 (s, 1H), 3.89-3.81 (m,
H), 2.45 (s, 3H), 2.09-1.98 (m, 1H), 1.74-1.59 (m, 4H), 1.55-1.46 (m, 2H),
+
.30-1.21(m, 2H). ESI-MS m/z: 258.1 [M+H] .
4
.1.3.4. 7-Cyclohexylmethylamino-4-methyl-2H-chromen-2-one (6i). A yellow solid.

1
Yield 51 %. Mp 164-167 °C. H NMR (300 MHz, CDCl ) δ 7.36 (d, J = 8.7 Hz, 1H),
3
6
.50-6.44 (m, 1H), 6.43 (d, J = 2.0 Hz, 1H), 6.00 (s, 1H), 4.27 (s, 1H), 3.90-3.83 (m,
2
H), 2.46 (s, 3 H), 1.77-1.69 (m, 4H), 1.68-1.61(m, 2H), 1.54-1.45 (m, 1H), 1.21-1.16
+
(
m, 2H), 1.06-0.94 (m,2H). ESI-MS m/z: 272.2 [M+H] .
4
.1.4. General procedure for preparation of compound (8a-8m). To a solution of 6
(14.76 mmol) in DCM (30 mL) was added triethylamine (2.39 g, 23.62 mmol) and
stirred at 0°C. Then 3-chloropropionyl chloride (2.44 g, 19.19 mmol) was added
dropwise and stirred at room temperature for 2-3 h until the reaction was completed.
The reaction was poured into water (30 mL) and the mixture was extracted with DCM
(
30 mL×3). The combined organic layer was dried over anhydrous sodium sulfate and
filtered. After removal of solvent, the crude product 7 was dissolved in acetonitrile
15 mL). Then 1-tert-butoxycarbonyl-piperazine (2.75 g, 14.76 mmol), potassium
(
carbonate (2.04 g, 14.76 mmol) and potassium iodide (0.25 g, 1.48 mmol) were added
and the mixture was stirred at room temperature for 10-20 h until the reaction was
completed. The reaction was diluted with water (30 mL) and extracted with ethyl
acetate (30 mL×3). The combined organic layer was dried over anhydrous sodium
sulfate and filtered. After removal of solvent, the crude product was purified by silica
gel chromatography to give 8a-8m respectively.
4
.1.4.1.
4-(3-(4-Methyl-2-oxo-2H-chromen-7-yl)
ethylamino)-3-oxopropyl)
1
piperazine-1-carboxylate tert-butyl (8a). A colourless oil. Yield 47%. H NMR (300
MHz, CDCl ) δ 7.77 (d, J = 8.4 Hz, 1H), 7.33-7.28 (m, 2H), 6.36 (s, 1H), 3.82 (q, J =
3

7
2
.1 Hz, 2H), 3.76-3.67 (m, 4H), 3.32-3.24 (m, 2H), 3.08-2.97 (m, 4H), 2.74-2.63 (m,
+
H), 2.50 (s, 3H), 1.46 (s, 9H), 1.16 (t, J = 6.8 Hz, 3H). ESI-MS m/z: 443.2 [M+H] .
4
.1.4.2.
4-(3-(4-Methyl-2-oxo-2H-chromen-7-yl)
propylamino)-3-oxopropyl)
1
piperazine-1-carboxylate tert-butyl (8b). A colourless oil. Yield 50 %. H NMR (300
MHz, CDCl ) δ 7.75 (d, J = 8.3 Hz, 1H), 7.26-7.16 (m, 2H), 6.35 (s, 1H), 3.81 (t, J =
3
7
2
.0 Hz, 2H), 3.73-3.64 (m, 4H), 3.31-3.23 (m, 2H), 3.06-2.95 (m, 4H), 2.72-2.61 (m,
H), 2.48 (s, 3H), 1.44 (s, 9H), 1.34-1.28 (m, 2H), 1.14 (t, J = 7.1 Hz, 3H). ESI-MS
+
m/z: 458.3 [M+H] .
4
.1.4.3.
4-(3-(4-Methyl-2-oxo-2H-chromen-7-yl)
butylamino)-3-oxopropyl)
1
piperazine-1-carboxylate tert-butyl (8c). A colourless oil. Yield 46 %. H NMR (300
MHz, CDCl ) δ 7.74 (d, J = 8.3 Hz, 1H), 7.23-7.14 (m, 2H), 6.32 (s, 1H), 3.82 (t, J =
3
7
2
.1 Hz, 2H), 3.71-3.63 (m, 4H), 3.29-3.21 (m, 2H), 3.04-2.93 (m, 4H), 2.71-2.60 (m,
H), 2.47 (s, 3H), 1.69-1.61(m, 2H), 1.43 (s, 9H), 1.25-1.18 (m, 2H), 1.06 (t, J = 7.2
+
Hz, 3H). ESI-MS m/z: 472.3 [M+H] .
4
.1.4.4.
4-(3-(4-Methyl-2-oxo-2H-chromen-7-yl)
isopentylamino)-3-oxopropyl)
1
piperazine-1-carboxylate tert-butyl (8d). A colourless oil. Yield 52 %. H NMR (300
MHz, CDCl ) δ 7.78 (d, J = 8.4 Hz, 1H), 7.29-7.22 (m, 2H), 6.37 (s, 1H), 3.85 (t, J =
3
7
2
.0 Hz, 2H), 3.78-3.69 (m, 4H), 3.34-3.27 (m, 2H), 3.10-2.99 (m, 4H), 2.76-2.65 (m,
H), 2.51 (s, 3H), 1.66-1.57 (m, 1H), 1.46 (s, 9H), 1.31-1.24 (m, 2H), 0.91 (d, J = 6.6
+
Hz, 6H). ESI-MS m/z: 486.3 [M+H] .
4
.1.4.5. 4-(3-(4-Methyl-2-oxo-2H-chromen-7-yl) isopentenylamino)-3-oxopropyl)

1
piperazine-1-carboxylate tert-butyl (8e). A colourless oil. Yield 53 %. H NMR (300
MHz, CDCl ) δ 7.70 (d, J = 8.4 Hz, 1H), 7.25 (dd, J = 8.4, 2.1 Hz, 1H), 7.16 (d, J =
3
2
3
1
.1 Hz, 1H), 6.33 (s, 1H), 5.27-5.15 (m, 1H), 4.37-4.31 (m, 2H), 3.78-3.73 (m, 4H),
.25-3.18 (m, 2H), 2.97-2.90 (m, 4H), 2.81-2.69 (m, 2H), 2.47 (d, J = 1.4 Hz, 3H),
+
.69 (s, 3H), 1.49 (s, 3H), 1.45 (s, 9H). ESI-MS m/z: 484.3 [M+H] .
4
.1.4.6.
4-(3-(4-Methyl-2-oxo-2H-chromen-7-yl)
cyclopropylmethylamino)-3-
1
oxopropyl)piperazine-1-carboxylate tert-butyl (8f). A colourless oil. Yield 51 %. H
NMR (300 MHz, CDCl ) δ 7.73 (d, J = 8.3 Hz, 1H), 7.21-7.16 (m, 2H), 6.32 (s, 1H),
3
3
2
0
.84 (d, J = 6.5 Hz, 2H), 3.73-3.67 (m, 4H), 3.32-3.25 (m, 2H), 3.08-2.98 (m, 4H),
.74-2.64 (m, 2H), 2.50 (s, 3H), 1.46 (s, 9H), 1.22-1.14 (m, 1H), 0.89-0.78 (m, 2H),
+
.54-0.46 (m, 2H). ESI-MS m/z: 470.3 [M+H] .
4
.1.4.7.
4-(3-(4-Methyl-2-oxo-2H-chromen-7-yl)
cyclobutylmethylamino)-3-
1
oxopropyl) piperazine-1-carboxylate tert-butyl (8g). A colourless oil. Yield 49 %. H
NMR (300 MHz, CDCl ) δ 7.72 (d, J = 8.4 Hz, 1H), 7.22-7.17 (m, 2H), 6.31 (s, 1H),
3
3
2
2
.83 (d, J = 6.2 Hz, 2H), 3.72-3.68 (m, 4H), 3.31-3.24 (m, 2H), 3.09-2.99 (m, 4H),
.73- 2.65 (m, 2H), 2.48 (s, 3H), 2.03-1.92 (m, 3H), 1.87-1.79 (m, 2H), 1.71-1.63 (m,
+
H), 1.47 (s, 9H). ESI-MS m/z: 484.3 [M+H] .
4
.1.4.8.
4-(3-(4-Methyl-2-oxo-2H-chromen-7-yl)
cyclopentylmethylamino)-3-oxopropyl) piperazine-1-carboylate tert-butyl (8h). A
1
colourless oil. Yield 46 %. H NMR (300 MHz, CDCl ) δ 7.73 (d, J = 8.5 Hz, 1H),
3
7
.23-7.18 (m, 2H), 6.32 (s, 1H), 3.84 (d, J = 6.4 Hz, 2H), 3.73-3.69 (m, 4H),

3
1
.32-3.26 (m, 2H), 3.10-3.01 (m, 4H), 2.74-2.66 (m, 2H), 2.49 (s, 3H), 2.07-1.96 (m,
H), 1.72-1.58 (m, 4H), 1.57-1.49 (m, 2H), 1.45 (s, 9H), 1.29-1.22 (m, 2H). ESI-MS
+
m/z: 498.3 [M+H] .
4
.1.4.9. 4-(3-(4-Methyl-2-oxo-2H-chromen-7-yl) cyclohexylmethylamino)-3-oxopropyl)
1
piperazine-1-carboxylate tert-butyl (8i). A colourless oil. Yield 47 %. H NMR (300
MHz, CDCl ) δ 7.74 (d, J = 8.6 Hz, 1H), 7.21-7.16 (m, 2H), 6.31 (s, 1H), 3.83 (d, J =
3
6
2
1
.2 Hz, 2H), 3.72-3.67 (m, 4H), 3.31-3.25 (m, 2H), 3.11-3.03 (m, 4H), 2.72-2.65 (m,
H), 2.48 (s, 3H), 1.75-1.67 (m, 4H), 1.62-1.53 (m, 2H), 1.46 (s, 9H), 1.31-1.23 (m,
+
H), 1.15-1.04 (m, 2H), 0.96-0.85 (m,2H). ESI-MS m/z: 412.3 [M+H] .
4
.1.4.10.
4-(3-(4-Methyl-2-oxo-2H-chromen-7-yl)
benzylamino)-3-oxopropyl)
1
piperazine-1-carboxylate tert-butyl (8j). A colourless oil. Yield 45 %. H NMR (300
MHz, CDCl ) δ 7.79-7.75 (m, 5H), 7.74-7.70 (m, 1H), 6.97 (dd, J = 8.7, 2.3 Hz, 1H),
3
6
2
5
.89 (d, J = 2.3 Hz, 1H), 6.38 (s, 1H), 5.12 (s, 2H), 3.73-3.68 (m, 4H), 3.32-3.26 (m,
H), 3.12-3.05 (m, 4H), 2.73-2.64 (m, 2H), 2.49 (s, 3H), 1.44 (s, 9H). ESI-MS m/z:
+
06.2 [M+H] .
4
.1.4.11. 4-(3-(4-Methyl-2-oxo-2H-chromen-7-yl) (2-methybenzylamino)-3-oxopropyl)
1
piperazine-1-carboxylate tert-butyl (8k). A colourless oil. Yield 49 %. H NMR (300
MHz, CDCl ) δ 7.77-7.73 (m, 4H), 7.71-7.69 (m, 1H), 6.95 (dd, J = 8.5, 2.1 Hz, 1H),
3
6
.87 (d, J = 2.1 Hz, 1H), 6.37 (s, 1H), 5.14 (s, 2H), 3.72-3.67 (m, 4H), 3.31-3.25 (m,
2
H), 3.11-3.04 (m, 4H), 2.74-2.65 (m, 2H), 2.31 (s, 3H), 2.11 (s, 3H), 1.43 (s, 9H).
+
ESI-MS m/z: 520.3 [M+H] .

4
.1.4.12. 4-(3-(4-Methyl-2-oxo-2H-chromen-7-yl) (3-methybenzylamino)-3-oxopropyl)
1
piperazine-1-carboxylate tert-butyl (8l). A colourless oil. Yield 48 %. H NMR (300
MHz, CDCl ) δ 7.76-7.72 (m, 4H), 7.71-7.68 (m, 1H), 6.93 (dd, J = 8.6, 2.2 Hz, 1H),
3
6
.85 (d, J = 2.0 Hz, 1H), 6.38 (s, 1H), 5.15 (s, 2H), 3.73-3.68 (m, 4H), 3.32-3.26 (m,
2
H), 3.10-3.03 (m, 4H), 2.72-2.64 (m, 2H), 2.33 (s, 3H), 2.16 (s, 3H), 1.44 (s, 9H).
+
ESI-MS m/z: 520.3 [M+H] .
4
.1.4.13. 4-(3-(4-Methyl-2-oxo-2H-chromen-7-yl) (4-methybenzylamino)-3-oxopropyl)
1
piperazine-1-carboxylate tert-butyl (8m). A colourless oil. Yield 42 %. H NMR (300
MHz, CDCl ) δ 7.75-7.70 (m, 4H), 7.69-7.66 (m, 1H), 6.91 (dd, J = 8.7, 2.0 Hz, 1H),
3
6
.82 (d, J = 2.2 Hz, 1H), 6.37 (s, 1H), 5.13 (s, 2H), 3.75-3.69 (m, 4H), 3.33-3.25 (m,
2
H), 3.11-3.04 (m, 4H), 2.73-2.65 (m, 2H), 2.34 (s, 3H), 2.13 (s, 3H), 1.45 (s, 9H).
+
ESI-MS m/z: 520.3 [M+H] .
4
.1.5.
4-(2-(4-Methyl-2-oxo-2H-chromen-7-yl)
isopentenylamino)-2-oxoethyl)
piperazine-1-carboxylate tert-butyl (8n). To a solution of 6e (3 g, 12.33 mmol) in
DCM (30 mL) was added triethylamine (1.87 g, 18.5 mmol) and stirred at 0°C. Then
2
-chloroacetyl chloride (1.81 g, 16.03 mmol) was added dropwise and stirred at room
temperature for 2-3 h until the reaction was completed. The reaction was poured into
water (30 mL) and the mixture was extracted with DCM (30 mL×3). The combined
organic layer was dried over anhydrous sodium sulfate and filtered. After removal of
solvent, the crude product 7n was dissolved in acetonitrile (15 mL). Then
1
-tert-butoxycarbonylpiperazine (2.3 g, 12.33 mmol), potassium carbonate (1.7 g,

1
2.33 mmol) and potassium iodide (0.2 g, 1.23 mmol) were added and the mixture
was stirred at room temperature for 10-20 h until the reaction was completed. The
reaction was diluted with water (30 mL) and extracted with ethyl acetate (30 mL×3).
The combined organic layer was dried over anhydrous sodium sulfate and filtered.
After removal of solvent, the crude product was purified by silica gel chromatography
1
to give 8n as a colourless oil (3.24 g, 56 %). H NMR (300 MHz, CDCl ) δ 7.60 (d, J
3
=
8.3 Hz, 1H), 7.19-7.06 (m, 2H), 6.33 (s, 1H), 5.24-5.16 (m, 1H), 4.33 (d, J = 7.0 Hz,
2
1
H), 3.41-3.37 (m, 4H), 2.99 (s, 2H), 2.47 (s, 3H), 2.43-2.38 (m, 4H), 1.68 (s, 3H),
+
.49 (s, 3H), 1.44 (s, 9H). ESI-MS m/z: 470.3 [M+H] .
4
.1.6.
4-(4-(4-Methyl-2-oxo-2H-chromen-7-yl)
isopentenylamino)-4-oxobutyl)
piperazine-1-carboxylate tert-butyl (8o). To a solution of 6e (3 g, 12.33 mmol) in
DCM (30 mL) was added triethylamine (1.87 g, 18.5 mmol) and stirred at 0°C. Then
4
-chloroacetyl chloride (2.26 g, 16.03 mmol) was added dropwise and stirred at room
temperature for 2-3 h until the reaction was completed. The reaction was poured into
water (30 mL) and the mixture was extracted with DCM (30 mL×3). The combined
organic layer was dried over anhydrous sodium sulfate and filtered. After removal of
solvent, the crude product 7o was dissolved in acetonitrile (15 mL). Then
1
1
-tert-butoxycarbonylpiperazine (2.3 g, 12.33 mmol), potassium carbonate (1.7 g,
2.33 mmol) and potassium iodide (0.2 g, 1.23 mmol) was added and the mixture was
stirred at room temperature for 10-20 h until the reaction was completed. The reaction
was diluted with water (30 mL) and extracted with ethyl acetate (30 mL×3). The
combined organic layer was dried over anhydrous sodium sulfate and filtered. After

removal of solvent, the crude product was purified by silica gel chromatography to
1
give 8o as a colourless oil (3.75 g, 61 %). H NMR (300 MHz, CDCl ) δ 7.68 (d, J =
3
8
5
2
9
.3 Hz, 1H), 7.23 (dd, J = 8.2, 2.0 Hz, 1H), 7.14 (d, J = 1.6 Hz, 1H), 6.31 (s, 1H),
.25-5.16 (m, 1H), 4.31 (d, J = 6.9 Hz, 2H), 3.74-3.69 (m, 4H), 3.26-3.19 (m, 2H),
.95-2.89 (m, 4H), 2.79-2.68 (m, 2H), 2.45 (s, 3H), 1.67 (s, 3H), 1.46 (s, 3H), 1.43 (s,
+
H). ESI-MS m/z: 498.3 [M+H] .
4
.1.7. General procedure for preparation of compounds (10-24). To a solution of 8
(0.45 mmol) in DCM (2 mL) was added trifluoroacetic acid (0.10 g, 0.90 mmol) and
the mixture was stirred at room temperature for 1-2 h until the reaction was completed.
The solvent was evaporated under vacuum to give crude product 9. To a solution of 9
(0.45 mmol) in DCM (2 mL) was added triethylamine (0.09 g, 0.90 mmol), 2-furoic
acid (0.06 g, 0.54 mmol) and EDCI (0.11 g, 0.58 mmol). The mixture was stirred at
room temperature for 15-20 h until the reaction was completed. The reaction was
poured into water (5 mL) and the mixture was extracted with DCM (20 mL×3). The
combined organic layer was dried over anhydrous sodium sulfate and filtered. After
removal of solvent, the crude product was purified by silica gel chromatography to
give 10-24 respectively.
4
.1.7.1.
N-ethyl-N-(4-methyl-2-oxo-2H-chromen-7-yl)-3-(4-(furan-2-carbonyl)
1
piperazin-1-yl) propanamide (10). A colourless oil. Yield 61 %. H NMR (300 MHz,
CDCl ) δ 7.68 (d, J = 8.2 Hz, 1H), 7.47 (d, J = 1.8 Hz, 1H), 7.19-7.13 (m, 2H),
3
6
.99-6.95 (m, 1H), 6.49-6.45 (m, 1H), 6.36 (s, 1H), 3.84-3.79 (m, 2H), 3.78-3.70 (m,

4
H), 2.80-2.71 (m, 2H), 2.48 (s, 3H), 2.46-2.42 (m, 4H), 2.36-2.27 (m, 2H), 1.14 (t, J
13
=
7.1 Hz, 3H). C NMR (75 MHz, CDCl ) δ 169.73, 160.02, 158.90, 154.15, 151.63,
3
1
5
47.59, 144.85, 143.86, 126.02, 124.24, 119.71, 116.90, 116.61, 115.62, 111.41, 53.87,
3.01, 44.42, 31.44, 18.68, 13.16. HRMS (ESI-TOF) m/z calc’d for C H N O
5
24
28
3
+
[
M+H] 438.2029, found 438.2034.
4
.1.7.2.
N-propyl-N-(4-methyl-2-oxo-2H-chromen-7-yl)-3-(4-(furan-2-carbonyl)
1
piperazin-1-yl) propanamide (11). A colourless oil. Yield 53 %. H NMR (300 MHz,
MeOD) δ 7.88 (d, J = 8.4 Hz, 1H), 7.65 (d, J = 0.9 Hz, 1H), 7.39-7.30 (m, 2H), 7.00
(d, J = 2.9 Hz, 1H), 6.56 (dd, J = 3.4, 1.7 Hz, 1H), 6.38 (s, 1H), 3.79-3.68 (m, 6H),
2
.74-2.64 (m, 2H), 2.51 (s, 3H), 2.44-2.37 (m, 6H), 1.59-1.51 (m, 2H), 0.91 (t, J = 7.4
1
3
Hz, 3H). C NMR (75 MHz, MeOD) δ 171.25, 160.27, 158.99, 153.42, 152.66,
1
5
46.42, 144.82, 144.02, 125.75, 123.71, 119.56, 115.76, 115.71, 114.05, 110.50, 53.20,
2.28, 50.29, 31.08, 20.18, 16.74, 9.63. HRMS (ESI-TOF) m/z calc’d for C H N O
5
25
30
3
+
[
M+H] 452.2185, found 452.2190.
4
.1.7.3.
N-butyl-N-(4-methyl-2-oxo-2H-chromen-7-yl)-3-(4-(furan-2-carbonyl)
1
piperazin-1-yl) propanamide (12). A colourless oil. Yield 57 %. H NMR (300 MHz,
MeOD) δ 7.90 (d, J = 8.4 Hz, 1H), 7.67 (d, J = 0.9 Hz, 1H), 7.41-7.32 (m, 2H),
7
2
2
1
.03-6.99 (m, 1H), 6.57 (dd, J = 3.4, 1.8 Hz, 1H), 6.39 (s, 1H), 3.84-3.70 (m, 6H),
.75-2.66 (m, 2H), 2.52 (s, 3H), 2.46-2.38 (m, 6H), 1.58-1.48 (m, 2H), 1.39-1.30 (m,
1
3
H), 0.92 (t, J = 7.3 Hz, 3H). C NMR (75 MHz, Me OD) δ 171.16, 160.22, 158.95,
53.42, 152.64, 146.44, 144.83, 144.02, 125.75, 123.74, 119.06, 115.77, 115.72,