Design, synthesis, and biological evaluation of novel 4H-chromen-4-one derivatives as antituberculosis agents against multidrug-resistant tuberculosis

Article abstract of DOI:10.1016/j.ejmech.2020.112075

A series of 4H-chromen-4-one derivatives obtained by scaffold morphing of the benzofuran compound, TAM16, were tested for antitubercular activity. Compound 8d was active against drug-sensitive and multidrug-resistant tuberculosis. A preliminary druggability evaluation showed that compound 8d displayed favorable mouse and human microsomal stability, low cytotoxicity, and acceptable oral bioavailability. An in vivo study indicated that compound 8d exhibited modest efficacy in an acute mouse model of TB after 3 weeks of treatment. Thus, 8d is a promising antituberculosis lead compound.

Full text of DOI:10.1016/j.ejmech.2020.112075

Journal Pre-proof
Design, synthesis, and biological evaluation of novel 4H-chromen-4-one derivatives
as antituberculosis agents against multidrug-resistant tuberculosis
Wenting Zhao, Bin Wang, Yuke Liu, Lei Fu, Li Sheng, Hongyi Zhao, Yu Lu, Dongfeng
Zhang
PII:
S0223-5234(20)30042-8
DOI:
https://doi.org/10.1016/j.ejmech.2020.112075
EJMECH 112075
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 9 December 2019
Revised Date: 14 January 2020
Accepted Date: 14 January 2020
Please cite this article as: W. Zhao, B. Wang, Y. Liu, L. Fu, L. Sheng, H. Zhao, Y. Lu, D. Zhang, Design,
synthesis, and biological evaluation of novel 4H-chromen-4-one derivatives as antituberculosis agents
against multidrug-resistant tuberculosis, European Journal of Medicinal Chemistry (2020), doi: https://
doi.org/10.1016/j.ejmech.2020.112075.
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Design, synthesis, and biological evaluation of novel 4H-chromen-4-one
derivatives as antituberculosis agents against multidrug-resistant tuberculosis
Wenting Zhaoa,b,c, Bin Wangd, Yuke Liua,c, Lei Fud, Li Shenga,c, Hongyi Zhaoa,b,c, Yu Lud,
Dongfeng Zhanga,b,c,*
aState Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of
Materia Medica, Peking Union Medical College and Chinese Academy of Medical Sciences, 1
Xian Nong Tan Street, Beijing 100050, P. R. China
bBeijing Key Laboratory of Active Substance Discovery and Druggability Evaluation, Institute of
Materia Medica, Peking Union Medical College and Chinese Academy of Medical Sciences, 1
Xian Nong Tan Street, Beijing 100050, P. R. China
cChinese Academy of Medical Sciences Key Laboratory of Anti-DR TB Innovative Drug Research,
Institute of Materia Medica, Peking Union Medical College and Chinese Academy of Medical
Sciences, 1 Xian Nong Tan Street, Beijing 100050, P. R. China
dBeijing Key Laboratory of Drug Resistance Tuberculosis Research, Department of Pharmacology,
Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing Chest Hospital, Capital
Medical University, 97 Ma Chang Street, Beijing 101149, P. R. China
Abstract
A series of 4H-chromen-4-one derivatives obtained by scaffold morphing of the
benzofuran compound, TAM16, were tested for antitubercular activity. Compound 8d
was active against drug-sensitive and multidrug-resistant tuberculosis. A preliminary
druggability evaluation showed that compound 8d displayed favorable mouse and
human microsomal stability, low cytotoxicity, and acceptable oral bioavailability. An
in vivo study indicated that compound 8d exhibited modest efficacy in an acute mouse
model of TB after 3 weeks of treatment. Thus, 8d is a promising antituberculosis lead
compound.
Keywords: 4H-Chromen-4-one, Scaffold morphing, Multidrug-resistant tuberculosis,
Polyketide synthase 13, Antitubercular agents
1

1. Introduction
Tuberculosis (TB) is one of the top 10 causes of death worldwide, and the lethality of
TB is higher than that of HIV/AIDS. According to the WHO [1], in 2018, there were
an estimated 10.0 million new cases of TB, around 1.2 million deaths due to TB, and
around 0.25 million more deaths associated with HIV coinfected TB patients.
Moreover, there were approximately 0.5 million new cases of rifampicin-resistant TB,
of which 78% were multidrug-resistant TB [1]. Therefore, there is an urgent need for
new anti-TB drugs.
The cell wall of M. tuberculosis are critical for its viability and virulence; therefore,
cell wall synthesizing enzymes are attractive targets for anti-TB drug development [2].
Mycolic acids are long-chain fatty acids that are important for the mycobacterial cell
wall permeability and integrity [3]. Disruption of the mycolic acids biosynthetic
pathway is a strategy for anti-TB drug discovery that has been validated by many of
the current first- and second-line anti-TB drugs, such as isoniazid, ethionamide,
ethambutol, and clinical MmpL3 inhibitor SQ109 [4].
Polyketide synthase 13 (Pks13) performs the final assembly step in mycolic acids
synthesis. Pks13 catalyzes a Claisen-type condensation between the carboxyacyl-CoA
and meromycoloyl-AMP to produce a mycolic β-ketoester, and which linked
through a thioester bond to the C-terminus (C-ACP) domain of Pks13. The
thioesterase (TE) domain of Pks13 cleaves this thioester bond and forms an ester bond
between the mycolic β-ketoester and Ser1533 of TE domain. Then, the TE domain
of Pks13 transfers the condensation product onto trehalose to synthesize precursor of
trehalose monomycolate (TMM). Therefore, inhibiting Pks13 interferes with the
critical pathway of mycolic acids synthesis and kills M. tuberculosis [5-7]. Recently, a
handful of Pks13 inhibitors have been reported (Fig. 1), and compounds TP2 (1) and
TP4 (2) have minimum inhibitory concentrations (MICs) of 1.0 and 0.5 µM,
respectively [8]. Compound 3 is a TP2 analogue that shows remarkable potency
against M. tuberculosis H37Rv (MIC: 0.23 µM) and an impressive potency (MIC:
0.20–0.44 µM) against M. tuberculosis strains resistant to isoniazid, rifampicin, and
fluoroquinolones [9]. β-Lactone compounds EZ120 (4) and orlistat (5) have been
2

identified as Pks13 inhibitors and EZ120 exhibited a MIC of 1.6 µM against M.
tuberculosis H37Rv [10,11]. Benzofuran derivatives have also been identified as
Pks13 inhibitors based on whole-cell screening and whole-genome sequencing of
resistant mutants [12]. TAM16 (6; MIC: 0.09 µM) was developed by structure-guided
optimization and is a promising candidate with potent efficacy in both acute and
chronic mouse TB infection models and has favorable druggability [13]. Recently, Yu
and coworkers reported coumestan compounds targeting Pks13, and compound 7
showed potent anti-TB activity in vitro (MIC: 0.0039 µg/mL) and in a serum
inhibition titration assay [14,15]. Therefore, Pks13 is a promising druggable target for
the development of new TB drugs.
Heterocycles containing N, O or S are valuable moieties for synthesizing many
antitubercular agents, such as imidazo[1,2-a]pyridine [16], quinolone [17], thiazole
[18], benzothiazinethione [19], pyridine [20], indole [21], 1,3,4-oxadiazole [22]. On
the basis of the essentiality of Pks13 for survival of M. tuberculosis and the
application of heterocyclic compounds in development of antitubercular agents, in
this work, we developed anti-TB compounds by scaffold morphing of the benzofuran
compound, TAM16. The heterocyclic compounds containing 4H-chromen-4-one
scaffold exhibited good activity against M. tuberculosis H37Rv and drug-resistant TB
isolates.
3

Fig. 1. Chemical structures of Pks13 inhibitors
2. Results and discussion
Our initial design was inspired by the Pks13 inhibitor TAM16. The crystal structure of
TAM16 bound to Pks13 confirmed that the ligand blocks the active site of the
Pks13-thioesterase (TE) domain [13]. Three hydrogen bonds were observed between
the carbonyl oxygen of TAM16 and the amino hydrogen of Asn1640, and between the
hydroxyls of TAM16 and the hydroxyl hydrogen of Asp1644 and the carbonyl oxygen
of Gln1633. The piperidine ring of TAM16 formed van der Waals stacking
interactions with residues such as Tyr1663. Guided by the crystal structure
information, we used scaffold morphing to explore potential anti-TB agents
containing the 4H-chromen-4-one frame (Fig. 2A). We made the key observation that
compound 8a adopts a similar conformation to TAM16 in the active pocket of the
Pks13-TE domain (Fig. 2B, 2C and 2D). Moreover, the docking result showed that
there were three hydrogen bond interactions, between 6-OH of 8a and the hydroxyl
hydrogen of Asp1644, and between 4’-OH of 8a and the carbonyl oxygen of Gln1633
4

and the hydroxyl hydrogen of Ser1636. The piperidine ring of compound 8a also
formed van der Waals stacking interactions with residues such as Tyr1663 (Fig. 2C).
Based on these results, we first synthesized and evaluated 4H-chromen-4-one 8a.
Compound 8a displayed a moderate MIC (3.84 µg/mL) against M. tuberculosis
H37Rv (Table 1), and this encouraged us to explore the systematic structure-activity
relationship (SAR) against M. tuberculosis further to demonstrate the potential of the
scaffold for hit-to-lead optimization.
N
O
O
N
O
Scaffold Morphing
NH
HO
HO
6
OH
O
OH
4'
8a
TAM16
A
B
C
5

D
Fig. 2. (A) Design strategy of 4H-chromen-4-one-based anti-TB agent; (B) interaction
of TAM16 bound to Pks13 (PDB code 5V3Y), TAM16 is shown in yellow, the
hydrogen bonds are shown as green lines, and π–π stacking interactions are shown as
orange lines; (C) predicted binding mode for 8a with Pks13 (PDB code 5V3Y),
compound 8a is shown in green, the hydrogen bonds are shown as green lines, and π–
π stacking interactions are shown as pink lines; (D) overlays of TAM16 and 8a in the
Pks13-TE domain, TAM16 is shown in gray, 8a is shown in green.
R1, R2, and R3 in the 4H-chromen-4-ones were identified as sites for diversification
(Table 1). When R1 was 4-methoxyphenyl, compound 13a displayed moderate
anti-TB activity (MIC: 7.30 µg/mL); however, compound 12a without substituent at
R2 did not show activity against M. tuberculosis (MIC: >32 µg/mL). This result
demonstrated that the substituent at R2 is an essential group for maintaining MIC and
that 4-hydroxyphenyl at R1 is preferred for cellular potency (compound 8a vs
compound 13a). Switching the 4-hydroxyphenyl to the 3-position in compound 20
resulted in no activity compared with reference compound 8a, indicating that the
position of the 4-hydroxyphenyl had an important effect on activity, and that the
2-position was favored. Next, we explored the effect of the R2 substituent on the
potency by keeping a 4-hydroxyphenyl substituent at R1. Azepan-1-yl-methyl (8b),
pyrrolidin-1-yl-methyl (8c), and azetidine-1-yl-methyl (8d) R2 substituents
maintained the activity, and compound 8d with a four-membered ring R2 substituent
showed the most potent activity (MIC: 0.45 µg/mL). In contrast, the ring-opened
diethylamine and dimethylamine analogues 8e and 8f, respectively, had no activity.
The addition of polar atoms to the piperidine ring (8g) removed the activity
(MIC: >32 µg/mL). Compounds with 2-thia-6-azaspiro[3.3]heptanyl-1-methyl (8h)
and cyclohexylaminomethyl (8i) groups showed weaker or no potency compared with
compound 8a. A small group at R2 was favorable for activity, and the SAR trends we
observed were different from those for benzofurans [13]. Next, we assessed the
effects of substituting the benzene ring at the 2-position of the 4H-chromen-4-ones
6

with H, an electron-donating Me group, and electron-withdrawing F and Cl groups.
Although the compounds with these substituents (13b, 13e, 13g, 13h and 13j) showed
modest activities, the hydroxyl substituent was the optimal group for anti-TB activity.
Because there was a hydrogen bond between the carbonyl oxygen of TAM16 and the
amino hydrogen of Asn1640, we investigated the introduction of ethyl ester and
amide groups at the 3-position. These groups were less tolerated with activities
ranging from 7.9 to >32 µg/mL. Small groups at R2 were also favorable for activity in
this series, which is consistent with the SAR trend above. As a result of their potency
against M. tuberculosis H37Rv, the activities of compounds 8c and 8d against
drug-resistant TB strains were investigated. All tested compounds displayed
equivalent potency against 13946 and 14862 strains (extensively drug-resistant-TB)
compared with the drug-sensitive strain (Table 2).
Table 1. Anti-TB activity of target compounds
MICa
R1
R2
H
R3
H
Compds.
(µg/mL)
12a
>32
13a
8a
H
H
7.30
3.84
>32
7.20
20
H
8b
H
7

8c
8d
8e
8f
H
H
H
H
H
H
0.94
0.45
29.89
>32
8g
8h
>32
15.34
8i
H
>32
13b
13e
13g
13o
13h
13i
H
H
H
H
H
H
H
3.12
3.68
3.22
31.11
3.55
28.54
6.52
13j
8

29a
29b
29c
29d
29e
32a
32b
32c
31.85
>32
>32
14.10
7.91
>32
>32
>32
TAM16
INH
0.047
0.039
a Minimum inhibitory concentration against M. tuberculosis H37Rv.
Table 2. In vitro activity of the selected compounds against drug-resistant TB strains
MIC (µg/mL)
MIC (µg/mL)
MIC (µg/mL)
Compds.
(H37Rv)
(13946a)
(14862b)
8c
8d
0.938
0.454
0.039
0.057
3.806
1.757
2.379
>10
3.818
1.644
>10
INH
RFP
>10
a
Resistance to isoniazid (INH), streptomycin (SM), rifampicin (RFP), ethambutol (EMB),
b
Rifabutin (RBT), p-aminosalicylate (PAS) and ofloxacin (OLFX). Resistant to INH, SM, RFP,
EMB, PAS, Prothionamide (1321) and Capreomycin (CPM).
The toxicities to Vero cells of compounds 8c and 8d (MIC: <1 µg/mL) were evaluated,
and the selectivity index (SI) values were calculated from the ratio of IC50/MIC (Table
9

3). Compound 8d possessed low toxicity with high SI values. Furthermore,
compounds 8c and 8d showed excellent metabolic stability against mouse and human
liver microsomes (Table 3). Pharmacokinetic (PK) studies for compound 8d were
performed in BALB/c mice, following administration of a single oral dose and of an
intravenous dose (Table 4). Compound 8d exhibited favorable PK performance and a
good PK profile with high plasma exposure (AUC0−∞ = 4286 ng·h/mL), and high
maximal plasma concentration (Cmax = 1656 ng/mL) after oral administration. Thus,
compound 8d had sufficiently promising PK properties to support in vivo efficacy
studies in an animal model. The in vivo efficacy of compound 8d was assessed in
BALB/c mice in an acute TB infection model. Compound 8d was orally administered
at 100 mg/kg, whereas the positive control drug isoniazid (INH) was given at 25
mg/kg. The same formulation of 0.5% carboxymethylcellulose in water was used for
both compounds. After three weeks of treatment, compound 8d showed modest
activity, reducing the bacterial burden in the lungs by 0.6 log10CFU compared with
the untreated control group (Table 5).
Table 3. Cell toxicity, ClogP, and mouse liver microsome stability of selected
compounds
Mouse microsome
stability
MIC
(µg/mL)
Cytotoxicity
IC50 (µg/mL)
Compds.
CLogPa
SIb
Substrate
Stabilityd
remaining (%)c
2.49
2.26
0.938
0.454
8.8
53.1
9
117
102.4
96
stable
stable
8c
8d
a
b
c
CLogP was calculated using ADMET Predictor (TM) version 9.0.0.10, SI = IC50/MIC,
Substrate concentrations were determined in incubations with NADPH after 30 min and
d
normalized to concentrations at time zero. Stability was determined without the NADPH
cofactor.
Table 4. Mouse PK properties of compound 8d
Parameters
unit
iv (5 mg/kg) po (100 mg/kg)
a
t1/2
h
h
1.28
-
1.42
0.25
1656
4223
Tmax
Cmax/C0
ng/mL
4459
1029
b
AUC(0-t) h*ng/mL
10

AUC(0-∞) h*ng/mL
1040
0.61
0.69
4286
1.70
1.82
c
MRT(0-t)
MRT(0-∞)
h
h
V
mL/kg
mL/h/kg
-
8856
4806
-
-
-
CL
F%d
20.5%
a Plasma elimination half-life, b Plasma exposure, c Mean residence time, d Oral bioavailability.
Table 5. Efficacy of compound 8d after 21 days of treatment in BALB/c mice
infected with M. tuberculosis H37Rv (mean ± SD)
Compds. Dose (mg/kg) Weight of mice (g) Log10CFU/lung
Control
8d
21.32±1.03
20.57±1.16
20.90±0.95
5.35±0.17
4.77±0.25
1.76±0.49
100
25
INH
Lastly, the binding mode of compound 8d with PKs13 was investigated by using the
CDOCKER protocol in Discovery Studio 2018 (Fig. 3). There were three hydrogen
bond interactions, between the hydroxyl hydrogen of 8d and the hydroxyl oxygen of
ASP1644, the hydroxyl hydrogen of 8d and the carbonyl oxygen of Gln1633, and the
hydroxyl oxygen of compound 8d and the hydroxyl hydrogen of Ser1636. The
4H-chromen-4-one scaffold of 8d formed π-π interactions with Phe1670, and the
azetidine ring of 8d formed van der Waals stacking interactions with residues such as
Tyr1663. This is also consistent with the proposed binding mode of the piperidine in
TAM16. The interaction of the amide group oxygen of TAM16 with the side-chain
amine of Asn1640 is proposed to be a key interaction; however, Asn1640 in the
Pks13-TE domain had no obvious interactions with compound 8d, which may be key
for further structural modifications to increase the activity.
11

Fig. 3. Predicted binding mode of compound 8d with PKs13 obtained from
CDOCKER. The key amino acid residues are shown in purple in the active site of
Pks13 (PDB ID 5V3Y). Compound 8d is shown in cyan, the hydrogen bonds are
shown as green lines, and the π–π stacking interactions are shown as orange lines.
3. Chemistry
Compounds 13a–p and 8a–i were synthesized in three or four steps using the
synthetic routes shown in Scheme 1. Intermediates 11a–e were prepared starting from
2,5-dihydroxyacetophenone (9) and methyl benzoate derivatives 10a–e in the
presence of lithium hexamethyldisilazide (LiHMDS). Compounds 12a–e were
obtained via cyclization of 11a–e in the presence of concentrated sulfuric acid in
glacial acetic acid at room temperature [23]. Mannich reaction of 12a–e with 37%
aqueous formaldehyde and the corresponding amine analogues afforded 13a–p [24],
when the amine was piperidine, the yields of Mannich reaction (13a,b) were lower
than
benzofuran
system
[13].
Pyrrolidine,
azepane,
azetidine,
2-thia-6-azaspiro[3.3]heptane, morpholine, diethylamine and dimethylamine were
used as nucleophiles in Mannich reaction, the products were obtained in various
yields (12-64%). Then, the compounds containing an OMe group were reacted with
boron tribromide to give 8a–i.
12

O
O
COOCH3
OH
OH
O
R1
HO
b
a
+
HO
HO
O
R1
O
R1
9
10a-e
11a-e
12a-e
R2
O
O
R2
O
O
HO
HO
c
d
R1
OH
13a-p
8a-i
10a, 11a, 12a R1 = OMe
10b, 11b, 12b R1 = H
10c, 11c, 12c R1 = F
10d, 11d, 12d R1 = Cl
10e, 11e, 12e R1 = Me
R2 =
N
N
N
N
N
13f R1 = OMe
13g R1 = H
13h R1 = F
13i R1 = Cl
13d R1 = OMe
13e R1 = H
8c
13a R1 = OMe 13c R1 = OMe
13b R1 = H
8a
13k R1 = OMe
8e
8b
13j R1 = Me
8d
O
N
S
N
NH
N
13n R1 = OMe
13o R1 = H
8h
13p R1 = OMe
8i
13l R1 = OMe
8f
13m R1 = OMe
8g
Scheme 1. Synthesis of compounds 8a-i.
Reagent and conditions: (a) LiHMDS, THF, -78~10 °C, 3 h, -78 °C ~rt, overnight; (b)
HOAc, H2SO4, rt, 4 h, 45-58%; (c) EtOH, reflux, 12-48 h, 12-64%; (d) BBr3, CH2Cl2,
rt, 5 h, 41-92%.
Compound 20 was synthesized from 2,5-dihydroxyacetophenone (9) via the synthetic
route in Scheme 2. First, one hydroxyl in 9 was protected by tetrahydro-2H-pyran.
Compound 14 was reacted with N,N-dimethylformamide dimethyl acetal (DMF-DMA)
at 95 °C to afford compound 15. Compound 16 was formed via cyclization of
compound 15 in the presence of I2 and pyridine at room temperature. Compound 16
served as a Suzuki coupling partner with (4-(benzyloxy)phenyl)boronic acid.
Isoflavone derivative 17 was deprotected using p-toluenesulfonic acid to give
hydroxyl compound 18 [25], which then underwent a Mannich reaction with 37%
aqueous formaldehyde and piperidine to yield compound 19. The benzyl protection of
13

compound 19 was removed by catalytic hydrogenation to give target compound 20.
Scheme 2. Synthesis of compound 20.
Reagent and conditions: (a) p-TsOH-Py, CH2Cl2, rt, 5 h, 87%; (b) DMF-DMA, 95 °C,
5 h, 50%; (c) I2, Py, CHCl3, rt, 7 h, 79%; (d) Pd/C, NaCO3, DME-H2O, 45 °C, 4 h,
57%; (e) p-TsOH, MeOH-THF, 60 °C, 2 h, 64%; (f) HCHO, Piperidine, MeOH,
reflux, 48 h, 46%; (g) H2, Raney Ni, rt, 2 h, 37%.
Compounds 25a,b were prepared following the procedure described in the literature
from diethyl malonate and benzoyl chloride [26,27]. Compounds 26a,b were
constructed by cyclization of compounds 25a,b in the presence of polyphosphoric
acid [27]. Hydroxyl compounds 28a,b were formed from compounds 26a,b via
sequential demethylation and esterification using boron tribromide and EtOH/H2SO4,
respectively, and then 28a,b underwent a Mannich reaction with 37% aqueous
formaldehyde and corresponding amines to yield target compounds 29a–e. Amides
32a–c were synthesized from intermediates 27a,b (Scheme 3).
14

Scheme 3. Synthesis of compounds 29a-e and 32a-c.
Reagent and conditions: (a) MgCl2, TEA, CH3CN, 0-rt, 5 h; (b) POCl3, TEA, 110 °C,
6 h; (c) K2CO3, DMF, 140, 6 h; (d) PPA, 100 °C, 4 h, four steps, 30-33%; (e) BBr3,
CH2Cl2, rt, 5 h; (f) H2SO4, EtOH, reflux, 12 h, two steps, 88-95%; (g) EtOH, reflux,
12-48 h, 34-57%; (h) (COCl)2, cat.DMF, CH2Cl2, rt, 4 h; (i) NH2CH3, THF, rt,
overnight, two steps, 24%.
Fig. 4. Structure-activity relationships of 4H-chromen-4-ones.
4. Conclusion
4H-Chromen-4-one derivatives were synthesized by scaffold morphing from
benzofurans and their anti-TB activities were tested. The SAR studies demonstrated
that the aryl group at the 2-position was essential for activity, and the
4-hydroxyphenyl substituent was favorable for activity (Fig. 4). At the 3-position of
the 4H-chromen-4-one frame, hydrogen gave the best activity. Compounds with a
15

small secondary-amine ring at the 5-position showed potent activities against M.
tuberculosis. The docking study revealed key hydrogen bond interactions of
compound 8d with Asp1644, Gln1633, and Ser1636 residues of Pks13. Importantly,
8d was active against multidrug-resistant TB isolates. Preliminary druggability
evaluation of this compound revealed favorable mouse and human microsomal
stability, low cytotoxicity, and acceptable oral bioavailability. Furthermore, an in vivo
study indicated that compound 8d exhibited moderate efficacy in an acute mouse
model of TB after 3 weeks of treatment. Hence, compound 8d is a promising anti-TB
lead compound warranting further optimization, which will be reported in due course.
5. Experimental
5.1. General information
All the solvents and chemical reagents were purchased from commercial sources and
used without further purification. TLC was performed on silica gel plates (GF254)
with visualization of components by UV light (254 nm). Column chromatography was
1
carried out on silica gel (300-400 mesh). H NMR spectra were obtained on Varian
Mercury at 400 MHz or 300 MHz. 13C NMR spectra were obtained on Varian
Mercury at 100 MHz or 125 MHz. CDCl3, DMSO-d6 or CD3OD were used as the
NMR solvents and tetramethylsilane (TMS) as an internal standard. Chemical shifts
(δ) values were referenced to the residual solvent peak and reported in ppm and all
coupling constant (J) values were given in Hz. The following multiplicity
abbreviations are used: (s) singlet, (d) doublet, (t) triplet, (q) quartet, (m) multiplet,
and (brs) broad. HRMS data were measured on Thermo Exactive Orbitrap plus
spectrometer. Melting points were determined on Yanaco MP-J3 microscope melting
point apparatus.
5.2. General procedure for the synthesis of 12a–e
A solution of LiHMDS in THF (1 M, 4 equiv) was added to a well-stirred solution of
2,5-dihydroxyacetophenone (9, 1 equiv) in anhydrous THF (20 mL) under argon at
16

-78 °C over 15 min. The reaction mixture was stirred at -78 °C for 1 h and at -10 °C
for 2 h and was cooled again to -78 °C, and a solution of 10a–e (1 equiv) in THF (5
mL) was added in one portion. The mixture was stirred at -78 °C for 1 h and at room
temperature overnight. The reaction mixture was poured into a mixture of ice and
neutralized with conc. HCl (6.5 mL) and extracted with dichloromethane. The
combined organic layer was washed with brine and dried over sodium sulfate, filtered,
and evaporated to give crude 11a–e. Crude 11a–e were dissolved in glacial acetic acid
(20 mL) and concentrated sulfuric acid (0.2 mL). The reaction was stirred at room
temperature for 4 h. H2O (20 mL) was added to the reaction mixture, and the resulting
solid was filtered, washed with water, and dried to afford 12a–e.
5.2.1. 6-Hydroxy-2-(4-methoxyphenyl)-4H-chromen-4-one (12a).
The title compound was synthesized from 2,5-dihydroxyacetophenone 9 (10 mmol,
1.52 g) and methyl p-methoxybenzoate 10a (10 mmol, 1.66 g) following the typical
1
procedure. Brown solid (1.47 g, 55%). mp: 136-138 °C. H NMR (400 MHz,
DMSO-d6) δ 8.04 (d, J = 9.2 Hz, 2H), 7.64 (d, J = 8.8 Hz, 1H), 7.32 (d, J = 3.2 Hz,
1H), 7.24 (dd, J = 8.8, 3.2 Hz, 1H), 7.12 (d, J = 8.8 Hz, 2H), 6.87 (s, 1H), 3.86 (s, 3H).
13C NMR (125 MHz, DMSO-d6) δ 177.51, 162.91, 162.63, 155.43, 149.92, 128.72,
124.84, 124.12, 123.49, 120.39, 115.18, 108.15, 105.14, 56.16. HR-MS (ESI): m/z
[M+H]+ calcd for C16H13O4: 269.0808, found: 269.0804.
5.2.2. 6-Hydroxy-2-phenyl-4H-chromen-4-one (12b).
The title compound was synthesized from 2,5-dihydroxyacetophenone 9 (10 mmol,
1.52 g) and methyl benzoate 10b (10 mmol, 1.26 mL) following the typical procedure.
1
Brown solid (1.07 g, 45%). H NMR (400 MHz, DMSO-d6) δ 10.03 (s, 1H),
8.10-8.08 (m, 2H), 7.67 (d, J = 9.2 Hz, 1H), 7.60-7.58 (m, 3H), 7.33 (d, J = 2.8 Hz,
13
1H), 7.27 (dd, J =8.8, 2.8 Hz, 1H), 6.96 (s, 1H). C NMR (125 MHz, DMSO-d6) δ
177.47, 162.65, 155.38, 149.86, 132.10, 131.86, 129.57, 126.71, 124.72, 123.57,
120.33, 107.96, 106.41.
17

5.2.3. 6-Hydroxy-2-(4-fluorophenyl)-4H-chromen-4-one (12c).
The title compound was synthesized from 2,5-dihydroxyacetophenone 9 (5 mmol, 761
mg) and methyl 4-fluorobenzoate 10c (5 mmol, 771 mg) following the typical
procedure. Gray solid (0.62 g, 48%). 1H NMR (400 MHz, DMSO-d6) δ 10.03 (s, 1H),
8.18-8.15 (m, 2H), 7.66 (d, J = 8.8 Hz, 1H), 7.45-7.40 (m, 2H), 7.32 (d, J = 3.2 Hz,
1H), 7.26 (dd, J = 8.8, 3.2 Hz, 1H), 6.96 (s, 1H). 13C NMR (100 MHz, DMSO-d6) δ
177.42, 164.56 (d, JC-F = 248.5), 161.71, 155.39, 149.80, 129.37 (d, JC-F = 8.9), 128.41
(d, JC-F = 2.9), 124.62, 123.56, 120.30, 116.65 (d, JC-F = 21.9), 107.96, 106.32.
5.2.4. 6-Hydroxy-2-(4-chlorophenyl)-4H-chromen-4-one (12d).
The title compound was synthesized from 2,5-dihydroxyacetophenone 9 (5 mmol, 761
mg) and methyl 4-chlorobenzoate 10d (5 mmol, 853 mg) following the typical
procedure. Gray solid (0.68 g, 50%). 1H NMR (400 MHz, DMSO-d6) δ 10.04 (s, 1H),
8.12 (d, J = 8.4 Hz, 2H), 7.67-7.64 (m, 3H), 7.32 (d, J = 2.8 Hz, 1H), 7.27 (dd, J = 8.8,
2.8 Hz, 1H), 7.00 (s, 1H). 13C NMR (100 MHz, DMSO-d6) δ 177.43, 161.48, 155.44,
149.80, 136.88, 130.76, 129.65, 128.55, 124.68, 123.67, 120.36, 107.94, 106.70.
5.2.5. 6-Hydroxy-2-(4-methylphenyl)-4H-chromen-4-one (12e).
The title compound was synthesized from 2,5-dihydroxyacetophenone 9 (5 mmol, 761
mg) and methyl 4-methylbenzoate 10e (5 mmol, 751 mg) following the typical
procedure. Brown solid (0.73 g, 58%). 1H NMR (400 MHz, DMSO-d6) δ 10.02 (s,
1H), 7.98 (d, J = 8.4 Hz, 2H), 7.65 (d, J = 8.8 Hz, 1H), 7.39 (d, J = 8.4 Hz, 2H), 7.32
13
(d, J = 3.2 Hz, 1H), 7.26 (dd, J = 8.8, 3.2 Hz, 1H), 6.91 (s, 1H), 2.40 (s, 3H). C
NMR (100 MHz, DMSO-d6) δ 177.41, 162.79, 155.32, 149.80, 142.27, 130.15,
129.03, 126.63, 124.72, 123.45, 120.26, 107.98, 105.77, 21.50.
5.3. General procedure for the synthesis of 13a–p
Formalin (37%, 1.5 equiv) and amine derivatives (1.5 equiv) were added to a solution
of 12a–e in ethanol (15 mL). The reaction mixture was stirred for 12–48 h under
argon at 80 °C. The reaction solution was then cooled to room temperature and
18

concentrated in vacuo. The residue was purified by column chromatography to give
compounds 13a–p.
5.3.1. 6-Hydroxy-2-(4-methoxyphenyl)-5-(piperidin-1-ylmethyl)-4H-chromen-4-one
(13a).
The title compound was prepared from compound 12a (0.66 mmol, 178 mg), formalin
(37%, 1 mmol, 81 µL) and piperidine (1 mmol, 99 µL) following the typical
1
procedure. Pale yellow solid (90 mg, 37%). mp: 187-188 °C. H NMR (400 MHz,
DMSO-d6) δ 8.02 (d, J = 9.2 Hz, 2H), 7.57 (d, J = 8.8 Hz, 1H), 7.15-7.10 (m, 3H),
13
6.78 (s, 1H), 4.63 (s, 2H), 3.85 (s, 3H), 2.54 (brs, 4H), 1.60-1.46 (m, 6H). C NMR
(125 MHz, DMSO-d6) δ 180.01, 162.43, 161.11, 156.97, 150.67, 128.44, 123.52,
123.16, 122.35, 119.12, 118.58, 115.01, 106.29, 56.92, 55.98, 53.53, 25.60, 23.58.
HR-MS (ESI): m/z [M+H]+ calcd for C22H24O4N: 366.1700, found: 366.1708.
5.3.2. 6-Hydroxy-2-phenyl-5-(piperidin-1-ylmethyl)-4H-chromen-4-one (13b).
The title compound was synthesized from compound 12b (1.10 mmol, 261 mg),
formalin (37%, 1.64 mmol, 133 µL) and piperidine (1.64 mmol, 163 µL) following
the typical procedure. Yellow solid (104 mg, 28%). mp: 178-179 °C. 1H NMR (400
MHz, DMSO-d6) δ 8.07 (dd, J =8.0, 1.6 Hz, 2H), 7.60-7.57 (m, 4H), 7.16 (d, J = 9.2
Hz, 1H), 6.88 (s, 1H), 4.62 (s, 2H), 2.51 (brs, 4H), 1.58-1.46 (m, 6H). 13C NMR (125
MHz, DMSO-d6) δ 180.32, 161.09, 157.39, 150.95, 132.25, 131.61, 129.76, 126.79,
123.61, 122.54, 119.23, 119.07, 107.85, 57.47, 53.75, 25.98, 23.89. HR-MS (ESI):
m/z [M+H]+ calcd for C21H22O3N: 336.1594, found: 336.1604.
5.3.3. 6-Hydroxy-2-phenyl-5-(pyrrolidin-1-ylmethyl)-4H-chromen-4-one (13e).
The title compound was synthesized from compound 12b (0.91 mmol, 217 mg),
formalin (37%, 1.36 mmol, 110 µL) and pyrrolidine (1.36 mmol, 112 µL) following
the typical procedure. Yellow solid (101 mg, 23%). mp: 133-135 °C. 1H NMR (400
MHz, DMSO-d6) δ 8.08-8.06 (m, 2H), 7.61-7.57 (m, 4H), 7.17 (d, J = 9.2 Hz, 1H),
6.88 (s, 1H), 4.76 (s, 2H), 2.68-2.63 (m, 4H), 1.82-1.78 (m, 4H). 13C NMR (100 MHz,
19

DMSO-d6) δ 180.09, 160.95, 157.19, 150.65, 132.06, 131.46, 129.57, 126.61, 123.36,
122.00, 119.68, 119.07, 107.68, 54.06, 53.38, 23.73. HR-MS (ESI): m/z [M+H]+
calcd for C20H20O3N: 322.1438, found: 322.1448.
5.3.4. 5-(Azetidin-1-ylmethyl)-6-hydroxy-2-phenyl-4H-chromen-4-one (13g).
The title compound was synthesized from compound 12b (0.42 mmol, 100 mg),
formalin (37%, 0.63 mmol, 51 µL) and azetidine (0.63 mmol, 42 µL) following the
typical procedure. Brown solid (68 mg, 53%). mp: 130-133 °C. 1H NMR (500 MHz,
DMSO-d6) δ 8.07 (d, J = 6.5 Hz, 2H), 7.58 (brs, 4H), 7.17 (d, J = 9.0 Hz, 1H), 6.88 (s,
13
1H), 4.69 (s, 2H), 3.34 (t, J = 7.5 Hz, 4H), 2.14-2.08 (m, 2H). C NMR (100 MHz,
DMSO-d6) δ 180.01, 160.96, 156.85, 150.69, 132.03, 131.44, 129.56, 126.58, 123.47,
122.26, 119.19, 118.74, 107.66, 56.87, 54.61, 17.24. HR-MS (ESI): m/z [M+H]+
calcd for C19H18O3N: 308.1281, found: 308.1278.
5.3.5. 5-(Azetidin-1-ylmethyl)-2-(4-fluorophenyl)-6-hydroxy-4H-chromen-4-one (13h).
The title compound was synthesized from compound 12c (0.59 mmol, 150 mg),
formalin (37%, 0.88 mmol, 71 µL) and azetidine (0.88 mmol, 59 µL) following the
procedure. Pale yellow solid (75 mg, 39%). mp: 189-192 °C. 1H NMR (400 MHz,
DMSO-d6) δ 8.16-8.12 (m, 2H), 7.58 (d, J = 9.2 Hz, 1H), 7.43-7.39(m, 2H), 7.17 (d, J
= 9.2 Hz, 1H), 6.89 (s, 1H), 4.68 (s, 2H), 3.35 (t, J = 7.2 Hz, 4H), 2.15-2.08 (m, 2H).
13C NMR (100 MHz, DMSO-d6) δ 179.94, 164.47 (d, J = 248.4 Hz), 160.02, 156.88,
150.63, 129.23 (d, J = 8.9 Hz), 128.00 (d, J = 2.9 Hz), 123.44, 122.14, 119.12, 118.81,
116.63 (d, J = 21.9 Hz), 107.57, 56.96, 54.61, 17.25. HR-MS (ESI): m/z [M+H]+
calcd for C19H17O3NF: 326.1187, found: 326.1183.
5.3.6. 5-(Azetidin-1-ylmethyl)-2-(4-chlorophenyl)-6-hydroxy-4H-chromen-4-one (13i).
The title compound was synthesized from compound 12d (0.55 mmol, 150 mg),
formalin (37%, 0.83 mmol, 67 µL) and azetidine (0.83 mmol, 56 µL) following the
1
typical procedure. Pale yellow solid (22 mg, 12%). mp: 197-199 °C. H NMR (400
MHz, DMSO-d6) δ 8.10 (d, J = 8.8 Hz, 2H), 7.64 (d, J = 8.8 Hz, 2H), 7.58 (d, J = 8.8
20

Hz, 1H), 7.17 (d, J = 9.2 Hz, 1H), 6.92 (s, 1H), 4.67 (s, 2H), 3.33 (t, J = 7.2 Hz, 4H),
2.14-2.07 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ 179.95, 159.85, 156.86, 150.64,
136.82, 130.35, 129.64, 128.44, 123.55, 122.24, 119.27, 118.65, 107.97, 56.68, 54.62,
17.23. HR-MS (ESI): m/z [M+H]+ calcd for C19H17O3NCl: 342.0892, found:
342.0886.
5.3.7.
5-(Azetidin-1-ylmethyl)-2-(4-methylophenyl)-6-hydroxy-4H-chromen-4-one
(13j).
The title compound was synthesized from compound 12e (0.6 mmol, 150 mg),
formalin (37%, 0.89 mmol, 72 µL) and azetidine (0.89 mmol, 60 µL) following the
typical procedure. Yellow solid (65 mg, 34%). mp: 149-151 °C. 1H NMR (400 MHz,
DMSO-d6) δ 7.96 (d, J = 8.0 Hz, 2H), 7.59 (d, J =8.8 Hz, 1H), 7.38 (d, J = 8.0 Hz,
2H), 7.18 (d, J = 9.2 Hz, 1H), 6.86 (s, 1H), 4.69 (s, 2H), 3.38 (t, J = 7.2 Hz, 4H), 2.39
(s, 3H), 2.16-2.08 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ 179.96, 161.19, 156.68,
150.64, 142.23, 130.14, 128.59, 126.53, 123.35, 122.28, 119.30, 118.44, 107.06,
56.45, 54.61, 21.50, 17.20. HR-MS (ESI): m/z [M+H]+ calcd for C20H20O3N:
322.1438, found: 322.1433.
5.3.8. 5-((2-Thia-6-azaspiro[3.3]heptan-6-yl)methyl)-6-hydroxy-2-phenyl-4H-
chromen-4-one (13o).
The title compound was synthesized from compound 12b (0.94 mmol, 224 mg),
formalin (37%, 1.40 mmol, 114 µL) and 2-thia-6-azaspiro[3.3]heptane (1.40 mmol,
162 mg) following the typical procedure. Off-white solid (80 mg, 23%). mp:
207-210 °C. 1H NMR (400 MHz, DMSO-d6) δ 8.08-8.06 (m, 2H), 7.62-7.57 (m, 4H),
13
7.20 (d, J = 8.8 Hz, 1H), 6.88 (s, 1H), 4.63 (s, 2H), 3.39 (s, 4H), 3.30 (s, 4H). C
NMR (100 MHz, DMSO-d6) δ 179.97, 161.12, 156.13, 150.85, 132.12, 131.39,
129.59, 126.63, 123.40, 122.37, 119.54, 118.61, 107.71, 66.71, 54.99, 43.32, 36.13.
HR-MS (ESI): m/z [M+H]+ calcd for C21H20O3NS: 366.1158, found: 366.1153.
5.4. General procedure for the synthesis of 8a–i
21

Boron tribromide (3 equiv) was added to a solution of corresponding compound 13 (1
equiv) in dichloromethane (10 mL) at -78 °C under argon and the mixture was stirred
for 5 h at room temperature. The reaction was quenched with methanol and
evaporated under reduced pressure. The residue was purified by column
chromatography on silica gel to give compounds 8a–i.
5.4.1.
6-Hydroxy-2-(4-hydroxyphenyl)-5-(piperidin-1-ylmethyl)-4H-chromen-4-one
(8a).
The title compound was synthesized from compound 13a (0.25 mmol, 90 mg)
following the typical procedure. Yellow solid (80 mg, 92%). mp: >250 °C. 1H NMR
(400 MHz, DMSO-d6 ) δ 10.74 (s, 1H), 7.97 (d, J = 8.8 Hz, 2H), 7.81 (d, J = 9.2 Hz,
1H), 7.46 (d, J = 9.2 Hz, 1H), 6.96 (d, J = 8.8 Hz, 2H), 6.90 (s, 1H), 4.70 (d, J = 4.8
13
Hz , 2H), 3.21-3.12 (m, 4H), 1.87-1.64 (m, 6H). C NMR (125 MHz, DMSO-d6) δ
180.24, 162.91, 161.79, 154.87, 150.94, 129.02, 123.44, 123.26, 122.33, 121.68,
116.69, 113.07, 106.03, 53.23, 51.68, 22.76, 21.75. HR-MS (ESI): m/z [M+H]+ calcd
for C21H22O4N: 352.1543, found: 352.1550.
5.4.2. 5-(Azepan-1-ylmethyl)-6-hydroxy-2-(4-hydroxyphenyl)-4H-chromen-4-one (8b).
Compound 13c was synthesized from compound 12a (0.62 mmol, 167 mg), formalin
(37%, 0.93 mmol, 76 µL) and azepane (0.93 mmol, 105 µL) following the procedure
described in 5.3. Yellow solid (58 mg, 25%). 1H NMR (400 MHz, DMSO-d6) δ 8.03
(d, J = 9.2 Hz, 2H), 7.59 (d, J = 9.2 Hz, 1H), 7.17 (d, J = 9.2 Hz, 1H), 7.11 (d, J = 9.2
Hz, 2H), 6.79 (s, 1H), 4.76 (s, 2H), 3.86 (s, 3H), 2.78 (t, J = 5.6 Hz , 4H), 1.66-1.59
(m, 8H).
The title compound was synthesized from compound 13c（0.15 mmol, 58 mg）
following the typical procedure. Yellow solid (50 mg, 89%). mp: >250 °C. 1H NMR
(400 MHz, DMSO-d6) δ 10.36 (s, 1H), 7.97 (d, J = 8.8 Hz, 2H), 7.78 (d, J = 9.2 Hz,
1H), 7.42 (d, J = 9.2 Hz, 1H), 6.95 (d, J = 8.8 Hz, 2H), 6.89 (s, 1H), 4.71 (s, 2H), 3.26
13
(brs, 4H), 1.83 (brs, 4H), 1.63 (brs, 4H). C NMR (100 MHz, DMSO-d6) δ 180.12,
162.75, 161.62, 155.14, 150.80, 128.83, 123.26, 122.99, 121.74, 121.51, 116.49,
22

114.15, 105.76, 54.25, 51.94, 27.27, 23.38. HR-MS (ESI): m/z [M+H]+ calcd for
C22H24O4N: 366.1700, found: 366.1689.
5.4.3. 6-Hydroxy-2-(4-hydroxyphenyl)-5-(pyrrolidin-1-ylmethyl)-4H-chromen-4-one
(8c).
Compound 13d was synthesized from compound 12a (1.03 mmol, 275 mg), formalin
(37%, 1.54 mmol, 115 µL) and tetrahydropyrrole (1.54 mmol, 126 µL) following the
procedure described in 5.3. Yellow solid (180 mg, 50%). 1H NMR (400 MHz, CDCl3)
δ 7.85 (d, J = 8.8 Hz, 2H), 7.40 (d, J = 8.8 Hz, 1H), 7.16 (d, J = 8.8Hz, 1H), 7.01 (d, J
= 8.8 Hz, 2H), 6.60 (s, 1H), 4.91 (s, 2H), 3.89 (s, 3H), 2.78 (s, 4H), 1.91-1.88 (m,
4H).
The title compound was synthesized from compound 13d (0.51 mmol, 180 mg)
1
following the typical procedure. Yellow solid (122 mg, 71%). mp: 142-144 °C. H
NMR (400 MHz, DMSO-d6) δ 7.91 (d, J = 8.8 Hz, 2H), 7.54 (d, J =8.8 Hz, 1H), 7.12
(d, J = 9.2 Hz, 1H), 6.92 (d, J = 8.8 Hz, 2H), 6.69 (s, 1H), 4.76 (s, 2H), 2.64 (s, 4H),
13
1.80 (s, 4H). C NMR (125 MHz, DMSO-d6) δ 179.94, 161.54, 161.35, 157.08,
150.45, 128.52, 123.02, 121.89, 121.75, 119.50, 118.88, 116.40, 105.61, 53.97, 53.36,
23.70. HR-MS (ESI): m/z [M+H]+ calcd for C20H20O4N: 338.1387, found: 338.1394.
5.4.4.
5-(Azetidin-1-ylmethyl)-6-hydroxy-2-(4-hydroxyphenyl)-4H-chromen-4-one
(8d).
Compound 13f was synthesized from compound 12a (0.75 mmol, 200 mg), formalin
(37%, 1.12 mmol, 91 µL) and azetidine (1.12 mmol, 75 µL) following the procedure
described in 5.3. Pale yellow solid (85 mg, 34%). 1H NMR (500 MHz, DMSO-d6) δ
8.03 (d, J = 8.5 Hz, 2H), 7.58 (d, J = 9.0 Hz, 1H), 7.17 (d, J = 9.0 Hz, 1H), 7.11 (d, J
= 8.5 Hz, 2H), 6.80 (s, 1H), 4.70 (s, 2H), 3.85 (s, 3H), 3.40 (s, 4H), 2.18-2.07 (m,
2H).
The title compound was synthesized from compound 13f (0.25 mmol, 85 mg)
following the typical procedure. Yellow solid (64 mg, 79%). mp: >250 °C. 1H NMR
(400 MHz, DMSO-d6) δ 7.91 (d, J = 8.8 Hz, 2H), 7.54 (d, J = 8.8 Hz, 1H), 7.14 (d, J
23

= 8.8 Hz, 1H), 6.92 (d, J = 8.8 Hz, 2H), 6.71 (s, 1H), 4.70 (s, 2H), 3.36 (t, J = 7.2 Hz,
13
4H), 2.15-2.08 (m, 2H). C NMR (100 MHz, DMSO-d6) δ 179.84, 161.77, 161.27,
156.28, 150.59, 128.60, 123.12, 122.28, 121.80, 119.60, 117.71, 116.38, 105.69,
55.35, 54.63, 17.08. HR-MS (ESI): m/z [M+H]+ calcd for C19H18O4N: 324.1230,
found: 324.1228.
5.4.5. 5-((Diethylamino)methyl)-6-hydroxy-2-(4-hydroxyphenyl)-4H-chromen-4-one
(8e).
Compound 13k was synthesized from compound 12a (0.78 mmol, 210 mg), formalin
(37%, 1.18 mmol, 95 µL) and diethylamine (1.18 mmol, 121 µL) following the
procedure described in 5.3. Pale yellow solid (44 mg, 16%). 1H NMR (400 MHz,
DMSO-d6) δ 8.02 (d, J = 8.8 Hz, 2H), 7.57 (d, J = 8.8 Hz, 1H), 7.13-7.10 (m, 3H),
6.78 (s, 1H), 4.72 (s, 2H), 3.86 (s, 3H), 2.64 (q, J = 7.2 Hz, 4H), 1.07 (t, J = 7.2 Hz,
6H).
The title compound was synthesized from compound 13k (0.12 mmol, 44 mg)
following the typical procedure. Yellow solid (30 mg, 71%). mp: >250 °C. 1H NMR
(400 MHz, DMSO-d6) δ 7.90 (d, J = 8.8 Hz, 2H), 7.52 (d, J = 8.8 Hz, 1H), 7.09 (d, J
= 8.8 Hz, 1H), 6.92 (d, J = 8.8 Hz, 2H), 6.68 (s, 1H), 4.71 (s, 2H), 2.61 (d, J = 7.2 Hz,
4H), 1.06 (t, J = 7.2 Hz, 6H). 13C NMR (100 MHz, DMSO-d6) δ 180.03, 161.45,
161.23, 157.52, 150.54, 128.54, 123.08, 122.16, 121.83, 119.39, 118.83, 116.36,
105.67, 52.36, 46.60, 11.30. HR-MS (ESI): m/z [M+H]+ calcd for C20H22O4N:
340.1543, found: 340.1552.
5.4.6. 5-((Dimethylamino)methyl)-6-hydroxy-2-(4-hydroxyphenyl)-4H-chromen-4-one
(8f).
Compound 13l was synthesized from compound 12a (0.29 mmol, 78 mg), formalin
(37%, 0.44 mmol, 35 µL) and dimethylamine (0.44 mmol, 218 µL) following the
1
procedure described in 5.3. Yellow solid (58 mg, 61%). H NMR (400 MHz,
DMSO-d6) δ 8.02 (d, J = 9.2 Hz, 2H), 7.58 (d, J = 9.2 Hz, 1H), 7.16 (d, J = 8.8 Hz,
1H), 7.11 (d, J = 9.2 Hz, 2H), 6.79 (s, 1H), 4.61 (s, 2H), 3.86 (s, 3H), 2.32 (s, 6H).
24

The title compound was synthesized from compound 13l (0.18 mmol, 58 mg)
following the typical procedure. Yellow solid (25 mg, 45%). mp: 184-187 °C. 1H
NMR (400 MHz, DMSO-d6) δ 7.91 (d, J = 8.8 Hz, 2H), 7.60 (d, J = 9.2 Hz, 1H), 7.20
(d, J = 9.2 Hz, 1H), 6.92 (d, J = 8.8 Hz, 2H), 6.73 (s, 1H), 4.61 (s, 2H), 2.42 (s, 6H).
13C NMR (125 MHz, DMSO-d6) δ 180.11, 161.97, 161.48, 156.77, 150.80, 128.81,
123.26, 122.52, 121.96, 119.89, 118.11, 116.57, 105.88, 56.82, 44.28. HR-MS (ESI):
m/z [M+H]+ calcd for C18H18O4N: 312.1230, found: 312.1237.
5.4.7. 6-Hydroxy-2-(4-hydroxyphenyl)-5-(morpholinomethyl)-4H-chromen-4-one (8g).
Compound 13m was synthesized from compound 12a (0.72 mmol, 192 mg), formalin
(37%, 1.08 mmol, 80 µL) and morpholine (1.08 mmol, 94 µL) following the
procedure described in 5.3. Off-white solid (168 mg, 64%). 1H NMR (500 MHz,
CDCl3) δ 7.85 (d, J = 8.5 Hz, 2H), 7.42 (d, J = 9.0Hz, 1H), 7.18 (d, J =9.0 Hz, 1H),
7.02 (d, J = 8.5 Hz, 2H), 6.61 (s, 1H), 4.81 (s, 2H), 3.89 (s, 3H), 3.80 (brs, 4H), 2.71
(brs, 4H).
The title compound was synthesized from compound 13m (0.46 mmol, 168 mg)
following the typical procedure. Yellow solid (49 mg, 34%). mp: >250 °C. 1H NMR
(400 MHz, DMSO-d6) δ 10.26 (s, 1H), 7.91 (d, J = 8.8 Hz, 2H), 7.57 (d, J =9.2 Hz,
1H), 7.17 (d, J = 8.8 Hz, 1H), 6.92 (d, J = 8.8 Hz, 2H), 6.70 (s, 1H), 4.62 (s, 2H), 3.62
(s, 4H), 2.53 (s, 4H). 13C NMR (100 MHz, DMSO-d6) δ 180.03, 161.64, 161.39,
156.24, 151.08, 128.75, 123.16, 122.62, 122.02, 119.33, 119.16, 116.54, 105.98,
66.68, 56.19, 53.14. HR-MS (ESI): m/z [M+H]+ calcd for C20H20O5N: 354.1336,
found: 354.1327.
5.4.8. 5-((2-Thia-6-azaspiro[3.3]heptan-6-yl)methyl)-6-hydroxy-2-(4-hydroxyphenyl)-
4H-chromen-4-one (8h).
Compound 13n was synthesized from compound 12a (1.23 mmol, 330 mg), formalin
(37%, 1.85 mmol, 150 µL) and 2-thia-6-azaspiro[3.3]heptane (1.85 mmol, 213 mg)
following the procedure described in 5.3. Yellow solid (150 mg, 31%). 1H NMR (500
MHz, DMSO-d6) δ 8.03 (d, J = 8.0 Hz, 2H), 7.62 (d, J = 9.0 Hz, 1H), 7.22 (d, J = 9.0
25

Hz, 1H), 7.11 (d, J = 8.0 Hz, 2H), 6.81 (s, 1H), 4.67 (s, 2H), 3.86 (s, 3H), 3.53 (s, 4H),
3.38 (s, 4H).
The title compound was synthesized from compound 13n (0.38 mmol, 150 mg)
following the typical procedure. Yellow solid (60 mg, 41%). mp: >250 °C. 1H NMR
(300 MHz, DMSO-d6) δ 7.91 (d, J = 8.7 Hz, 2H), 7.55 (d, J = 9.0 Hz, 1H), 7.16 (d, J
= 9.0 Hz, 1H), 6.92 (d, J = 8.7 Hz, 2H), 6.70 (s, 1H), 4.63 (s, 2H), 3.37 (s, 4H), 3.30
13
(s, 4H). C NMR (100 MHz, DMSO-d6) δ 179.77, 161.74, 161.27, 155.84, 150.70,
128.60, 123.03, 122.32, 121.80, 119.60, 116.38, 105.73, 66.62, 54.49, 43.23, 36.11.
HR-MS (ESI): m/z [M+H]+ calcd for C21H20O4NS: 382.1108, found: 382.1118.
5.4.9. 5-((Cyclohexylamino)methyl)-6-hydroxy-2-(4-hydroxyphenyl)-4H-chromen-4-
one (8i).
Compound 13p was synthesized from compound 12a (0.75 mmol, 200 mg), formalin
(37%, 1.12 mmol, 91 µL) and cyclohexylamine (1.12 mmol, 128 µL) following the
procedure described in 5.3. Pale yellow solid (61 mg, 22%). 1H NMR (400 MHz,
DMSO-d6) δ 8.02 (d, J = 8.8 Hz, 2H), 7.55 (d, J =8.8 Hz, 1H), 7.15(s, 1H), 7.11 (d, J
= 8.8 Hz, 2H), 6.79 (s, 1H), 4.74 (s, 2H), 3.86 (s, 3H), 2.56-2.53 (m, 1H), 1.92-1.54
(m, 10H).
The title compound was synthesized from compound 13p (0.13 mmol, 52 mg)
following the typical procedure. Pale yellow solid (30 mg, 60%). mp: 222-225 °C. 1H
NMR (400 MHz, CD3OD) δ 7.91 (d, J = 8.8 Hz, 2H), 7.73 (d, J = 9.2 Hz, 1H), 7.42 (d,
J = 9.2 Hz, 1H), 6.95 (d, J = 8.8 Hz, 2H), 6.80 (s, 1H), 4.77 (s, 2H), 3.28 (brs, 1H),
2.26-2.23 (m, 2H), 1.94-1.73 (m, 3H), 1.53-1.26 (m, 5H). 13C NMR (100 MHz,
CD3OD) δ 181.02, 164.16, 161.53, 154.19, 151.17, 128.09, 122.51, 121.42, 121.08,
115.68, 113.71, 104.69, 57.57, 39.54, 29.07, 24.71, 24.15. HR-MS (ESI): m/z [M+H]+
calcd for C22H24O4N: 366.1700, found: 366.1708.
5.5. Synthesis of compound 20
5.5.1. 1-(2-Hydroxy-5-((tetrahydro-2H-pyran-2-yl)oxy)phenyl)ethan-1-one (14)
26

Compound 9 (3 mmol, 456 mg) was dissolved in dichloromethane (15 mL) and stirred
at room temperature. Pyridinium p-toluene sulfonate (1 mmol, 251 mg) was added
followed by the dropwise addition of 3,4-dihydro-2H-pyran (3 mmol, 273 µL) in
dichloromethane (2 mL). The reaction mixture was stirred at room temperature for 5 h.
The reaction mixture was washed with saturated aqueous sodium bicarbonate and
extracted with dichloromethane (15 mL × 3). The organic fractions were combined
and dried over anhydrous sodium sulfate, evaporated in vacuo, and purified by flash
chromatography eluting with n-hexanes and ethyl acetate to yield compound 14
(yellow oil, 584 mg, 87%). 1H NMR (500 MHz, CDCl3) δ 11.89 (s, 1H), 7.41 (s, 1H),
7.24 (s, 1H), 6.91 (d, J = 9.0 Hz, 1H), 5.31 (s, 1H), 3.93 (t, J = 11.0 Hz, 1H), 3.62 (d,
J = 11.0 Hz, 1H), 2.61 (s, 3H), 1.86–1.55 (m, 6H).
5.5.2.
(E)-3-(Dimethylamino)-1-(2-hydroxy-5-((tetrahydro-2H-pyran-2-yl)oxy)phenyl)prop-
2-en-1-one (15).
The
mixture
of
compound
14
(2.62
mmol,
584
mg)
and
N,N-dimethylformamide-dimethylacetal (3.83 mmol, 522 µL) was stirred at 95 °C for
4 h. The reaction mixture was evaporated in vacuo and purified by flash
chromatography eluting with n-hexanes and ethyl acetate to yield compound 15
(yellow solid, 382 mg, 50%). 1H NMR (500 MHz, CDCl3) δ 7.87 (d, J = 11.5 Hz, 1H),
7.37 (s, 1H), 7.14 (d, J = 8.5 Hz, 1H), 6.92-6.81 (m, 1H), 5.72 (d, J = 12.0 Hz, 1H),
5.28 (s, 1H), 3.98-3.89 (m, 1H), 3.67-3.55 (m, 1H), 3.18 (s, 3H), 2.97 (s, 3H),
1.86-1.54 (m, 6H).
5.5.3. 3-Iodo-6-((tetrahydro-2H-pyran-2-yl)oxy)-4H-chromen-4-one (16).
Compound 15 (1.31 mmol, 382 mg) was dissolved in chloroform (10 mL). Pyridine
(1.44 mmol, 114 mg) was added followed by iodine (2.62 mmol, 666 mg). The
reaction mixture was stirred at room temperature for 7 h. The mixture was poured into
saturated sodium thiosulphate pentahydrate and stirred at room temperature for 30
mins. The mixture was then extracted with dichloromethane (15 mL×3) and the
27

combined organic layer dried using anhydrous sodium sulphate. The solvent was
evaporated in vacuo and the residue was purified by flash chromatography eluted with
1
n-hexanes and ethyl acetate to give compound 16 (white solid, 384 mg, 79%). H
NMR (400 MHz, CDCl3) δ 8.27 (s, 1H), 7.82 (d, J = 1.6 Hz, 1H), 7.41-7.40 (m, 2H),
5.50 (t, J = 3.2 Hz, 1H), 3.90-3.84 (m, 1H), 3.65-3.60 (m, 1H), 1.90-1.53(m, 6H).
5.5.4. 3-(4-(Benzyloxy)phenyl)-6-((tetrahydro-2H-pyran-2-yl)oxy)-4H-chromen-4-one
(17).
Compound 16 (1.03 mmol, 384 mg) was suspended in dimethoxyethane/water (1:1,
10 mL) and stirred at room temperature. Sodium carbonate (3.09 mmol, 328 mg) was
added followed by the 4-benzyloxybenzeneboronic acid (1.24 mmol, 283 mg). 10%
Pd/C (0.05 mmol, 55 mg) was added and the reaction mixture was stirred at 45 °C for
4 h under Ar2. The reaction mixture was filtered through a short pad of celite. The
catalyst was washed using water, and then the filter was extracted with
dichloromethane (15 mL×3). The organic layer was dried using anhydrous sodium
sulphate and the solvent evaporated in vacuo. The residue was purified by flash
chromatography eluted using n-hexanes and ethyl acetate to give compound 17
1
(off-white solid, 253 mg, 57%). H NMR (500 MHz, CDCl3) δ 7.99-7.85 (m, 2H),
7.51-7.33 (m, 9H), 7.04 (d, J = 7.5 Hz, 2H), 5.51 (s, 1H), 5.11 (s, 2H), 4.01-3.89 (m,
1H), 3.65-3.55 (m, 1H), 1.69-1.54 (m, 6H).
5.5.5. 3-(4-(Benzyloxy)phenyl)-6-hydroxy-4H-chromen-4-one (18).
p-Toluenesulfonic acid (0.059 mmol, 10 mg) was added to a solution of compound 17
(0.59 mmol, 253 mg) in a 1:1 mixture of methanol and tetrahydrofuran (10 mL). The
reaction mixture was stirred at 60 °C for 1 h. The reaction was quenched with
triethylamine (0.59 mmol, 82 µL). Compound 18 was synthesized by filtration (pale
1
yellow solid, 130 mg, 64%). H NMR (500 MHz, DMSO-d6) δ 9.99 (s, 1H), 8.43 (s,
1H), 7.56-7.34 (m, 9H), 7.25 (d, J = 9.0 Hz, 1H), 7.07 (d, J = 7.5 Hz, 2H), 5.16 (s,
2H).
28