Highly selective carbamate-based butyrylcholinesterase inhibitors derived from a naturally occurring pyranoisoflavone

Article abstract of DOI:10.1016/j.bioorg.2019.102949

This current study described the design and synthesis of a series of derivatives based on a natural pyranoisaflavone, which was obtained from the seeds of Millettia pachycarpa and displayed attractive BChE inhibition and high selectivity in our previous study. The inhibitory potential of all derivatives against two cholinesterases was evaluated. Only a few compounds demonstrated AChE inhibitory activity at the tested concentrations, while 26 compounds showed significant inhibition on BChE (the IC₅₀ values varied from 9.34 μM to 0.093 μM), most of them presented promising selectivity to ward BChE. Prediction of ADME properties for 7 most active compounds was performed. Among them, 9g (IC₅₀ = 222 nM) and 9h (IC₅₀ = 93 nM) were found to be the most potent BChE inhibitors with excellent selectivity over AChE (SI ratio = 1339 and 836, respectively). The kinetic analysis demonstrated both of them acted as mixed-type BChE inhibitors, while the molecular docking results indicated that they interacted with both residues in the catalytic active site. A cytotoxicity test on PC12 cells showed that both 9g and 9h had a therapeutic safety range similar to tacrine. Overall, the results indicate that 9h could be a good candidate of BChE inhibitors.

Full text of DOI:10.1016/j.bioorg.2019.102949

BioorganicChemistry88(2019)102949
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Highly selective carbamate-based butyrylcholinesterase inhibitors derived
from a naturally occurring pyranoisoflavone
Chuanhai Wu, Yan-bei Tu, Ziyuan Li⁎, Yan-fang Li⁎
School of Chemical Engineering, Sichuan University, Chengdu 610065, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
This current study described the design and synthesis of a series of derivatives based on a natural pyranoisa-
flavone, which was obtained from the seeds of Millettia pachycarpa and displayed attractive BChE inhibition and
high selectivity in our previous study. The inhibitory potential of all derivatives against two cholinesterases was
evaluated. Only a few compounds demonstrated AChE inhibitory activity at the tested concentrations, while 26
compounds showed significant inhibition on BChE (the IC50 values varied from 9.34 μM to 0.093 μM), most of
them presented promising selectivity to ward BChE. Prediction of ADME properties for 7 most active compounds
was performed. Among them, 9g (IC50 = 222 nM) and 9h (IC50 = 93 nM) were found to be the most potent BChE
inhibitors with excellent selectivity over AChE (SI ratio = 1339 and 836, respectively). The kinetic analysis
demonstrated both of them acted as mixed-type BChE inhibitors, while the molecular docking results indicated
that they interacted with both residues in the catalytic active site. A cytotoxicity test on PC12 cells showed that
both 9g and 9h had a therapeutic safety range similar to tacrine. Overall, the results indicate that 9h could be a
good candidate of BChE inhibitors.
Butyrylcholinesterase inhibitor
Pyranoisoflavone
Carbamates
Kinetic study
Molecular docking
1. Introduction
level and improving cognition for the treatment of advanced AD
[8–11]. Furthermore, the key role of BChE is also emphasized in de-
Alzheimer's disease (AD) is a serious neurodegenerative disorder
characterized by progressive memory loss and central cognitive dys-
function. And the deficit of cholinergic neurons is one of the key pa-
thological hallmarks of AD [1]. According to cholinergic dysfunction
hypothesis, inadequate levels of the neurotransmitter acetylcholine is
the main cause of cognitive decline [2]. Acetylcholinesterase (AChE)
and butyrylcholinesterase (BChE), two metabolic serine hydrolases, can
catalyze the hydrolysis of acetylcholine [3]. AChE, as the predominant
cholinesterase in the brain, can improve cognitive function by enhan-
cing cholinergic neurotransmission. Thus, the AChE inhibitors have
been employed to treat AD. Moreover, most of current AChE inhibitors
to treat AD available in the clinic are related to natural products, such
as rivastigmine, galantamine, and huperzine A [4].
toxication and scavenging of polyproline-rich peptides [12], regulation
of the serum metabolism associated with obesity [13,14], diabetes
mellitus and insulin resistance [13,15], and cardiovascular risk factors
[16,17]. Thus, the development of selective BChE inhibitors becomes
an attractive topic for researchers in medicinal chemistry currently.
The seeds of Millettia pachycarpa Benth are widely used as anthel-
minthic drugs in China [18]. In our recent study, the 95% ethanol ex-
tract of these seeds demonstrated interesting BChE inhibitory activity in
vitro [19]. A potential natural BChE inhibitor 8 with significant in-
hibition (IC50 = 2.34 μM) and good selectivity (SI ratio = 56.1), which
possesses a 6″, 6″-dimethylpyranoisoflavone scaffold and demonstrates
apoptosis-inducing effects [20] and antiinflammatory activities pre-
viously [21], was obtained by subsequent bioassay-guided isolation
[19] (Fig. 1). More importantly, its occurrence in plant kingdom is
relatively rare, as this type of scaffold was detected only in M. pachy-
carpa and Spiranthes sinensis.
Compared with AChE, BChE plays a supportive role in the choli-
nergic neurotransmission. However, during the progression of AD, level
of AChE in the patient brain decreases while the level and activity of
BChE significantly increases, which suggests that the compensation of
BChE in the late AD stage is of great importance [5,6]. In addition,
selective BChE inhibitors do not display the adverse cholinergic effect
of AChE inhibitors [7]. Therefore, highly selective inhibition of BChE
presents a promising therapeutic strategy in promoting acetylcholine
Previously, only a few isoflavones were reported as cholinesterase
inhibitors with weak inhibition and poor selectivity towards BChE
[22–26] (Fig. 2). Additionally, structural modification of pyranoiso-
flavone core as potential site to improve cholinesterase inhibition were
not reported. Considering the importance of natural products as AD
⁎ Corresponding authors.
E-mail addresses: liziyuan@scu.edu.cn (Z. Li), lyf471@vip.163.com (Y.-f. Li).
https://doi.org/10.1016/j.bioorg.2019.102949
Received 31 January 2019; Received in revised form 21 April 2019; Accepted 22 April 2019
Availableonline26April2019
0045-2068/©2019ElsevierInc.Allrightsreserved.

C. Wu, et al.
BioorganicChemistry88(2019)102949
Fig. 1. Design strategy carried out starting from compound 8.
Fig. 2. Isoflavone-based structures which have been investigated as ChE inhibitors.
drugs, a series of structurally modified derivatives based on 6″,6″-di-
methylpyranoisoflavone were designed and synthesized in current
study. The strategy for the design of the inhibitors, including the in-
troduction of a series of substituents on B ring, replacing the B ring with
a pyridine ring, and the introduction of various carbamates on A ring
and B ring, is illustrated in Fig. 1. Some of derivatives presented more
potential (IC50 values varied from 0.093 μM to 1.47 μM) against BChE
than parent compound 8. Preliminary kinetic and modeling data are
also presented to investigate their inhibition mechanism.
respectively [27]. The synthetic route of 9a-9o is presented in Scheme
2. Treatment of 8f-8h with isocyanates and various types of acyl
chlorides yield 9a-9n [31–33], while 8t was converted into 9o by re-
acting with triethylamine and n-heptylisocyanate. The structure fea-
tures of the derivatives are presented in Tables 1 and 2. Twenty-nine
compounds were synthesized for the first time.
2.2. Inhibitory studies of AChE and BChE
The ChEs inhibitory capacity of all 41 derivatives was evaluated by
Ellman’s method using tacrine and galantamine as positive controls
[34]. Further IC50 values determination were performed for compounds
with higher inhibition rate than 50% at 10 μM (final concentration in
the reaction system). Their inhibitory activity and selectivity results are
listed in Tables 1 and 2. Most of the synthesized derivatives did not
present good inhibition toward AChE, while their inhibition potency on
BChE depended on structure features.
2. Results and discussion
2.1. Chemistry
The synthesis of 8a–8w, 11a and 11b are outlined in Scheme 1.
Compounds 2–5 were prepared using the methods reported previously
[27]. 1,1-Diethoxy-3-methyl-2-butene was obtained from 3-methylbut-
2-enal by following method [28]. Treatment of 5 with 3-picoline and
the crude 1,1-diethoxy-3-methyl-2-butene resulted in 7 [29], which was
treated with different boronic acids to give 8a–8w [27,30]. In addition,
the reaction of 5 and acyl chlorides give 10a and 10b, which coupled
with 3,4-dimethoxyphenylboronic acid to afford 11a and 11b,
Firstly, a series of substituents were introduced on the B ring to
provide derivatives 8a-8u. Introduction of three small substituents
(hydroxyl, methoxy, and fluorine) in different positions (ortho, meta and
para) of the B ring resulted in the decline of inhibitory activity (8a-8l).
On the whole, introduction these substituents on para position of the B
2

C. Wu, et al.
BioorganicChemistry88(2019)102949
Scheme 1. Synthesis of compounds 8a-8w and 11a-11b. Reagents and conditions: (i) DHP, PPTS, CH2Cl2, rt, 4 h; (ii) DMF-DMA, 95 °C, 3 h; (iii) I2, pyridine,
CHCl3, rt, 12 h (74.7% for three steps); (iv) pTsOH, CH3OH, THF, 60 °C, 1 h; (v) 1,1-diethoxy-3-methyl-2-butene, 3-picoline, xylene, reflux, 24 h; (vi) ArB(OH)2, 10%
Pd/C, Na2CO3, H2O, DME, 45 °C, 1 h; (vii) heteroaryl boronic acids, Pd[P(C6H5)3]4, Na2CO3, THF, H2O, 60 °C, 6 h; (viii) acyl chloride, K2CO3, DMF, CH3CN, 95 °C,
3 h; (ix) ArB(OH)2, 10% Pd/C, Na2CO3, H2O, DME, 45 °C, 1 h;
Scheme 2. Synthesis of compounds 9a-9o. Reagents and conditions: (i) acyl chloride, K2CO3, DMF, CH3CN, 95 °C, 3 h; (ii) isocyanate, NEt3, CH2Cl2, rt, 6 h; (iii)
acyl chloride, K2CO3, CH3CN, rt, 24 h.
3

C. Wu, et al.
BioorganicChemistry88(2019)102949
Table 1
The chemical structure of the tested compounds and their Inhibitory activities against eeAChE and eqBChE.
SEMb
Compd.
R1
Chain
IC50 (μM)
AChE
SEMb
a
a
Compd.
R1
Chain
IC50 (μM)
AChE
Position
BChE
5.27
SIc
Position
BChE
SIc
7
24.47%
0.13
> 1.9
8m
19.17%
21.24%
NDd
8
131.17
79.60
13.77
9.81
2.34
2.94
0.15
0.26
56.1
27.1
8n
8o
5.60%
17.04%
9.34
NDd
0.3
8a
ortho
2.71
0.11
0.49
0.25
8b
8c
meta
para
18.18%
8.26%
4.27
0.12
> 2.3
NDd
8p
8q
15.45%
13.28%
13.10%
4.59
NDd
13.65%
> 2.2
8d
8e
8f
ortho
meta
para
7.19%
6.04
8.20
6.47
0.31
0.16
0.23
> 1.7
> 1.2
> 1.6
8r
8s
8t
10.77%
14.50%
349.36
2.92
2.34
1.13
0.13
0.06
0.09
> 3.4
> 4.3
309
13.87%
15.05%
16.56
8g
ortho
8.27%
5.09
0.14
> 2.0
8u
8.36%
11.46
0.31
> 0.9
8h
8i
meta
para
130.27
17.73%
17.90
2.36
8.32
0.19
0.34
55.2
8v
16.74
8.67
0.23
0.18
0.04
0.006
8.26
3.39
0.10
0.13
2.0
2.6
> 1.2
8w
8j
15.72%
23.57%
3.11
4.96
0.12
0.13
> 3.2
> 2.0
Galantamine
Tacrine
3.75
44.07
0.91
0.0005
0.09
4.34
8k
0.0438
0.0101
8l
4.81%
9.34
0.51
> 1.1
a
b
The dash line represents connecting bond.
The inhibition activities are expressed as IC50 (μM) or as percentage of inhibition at 10 μM, the IC50 values are the mean of three independent
experiments
SEM.
c
d
SI represents selectivity index which is determined as ratio AChE IC50/BChE IC50
.
Not determined.
ring have a higher negative influence on BChE inhibition than on meta
and ortho position. Among them, 8h and 8a demonstrated similar po-
tency against BChE (IC50 = 2.36 μM and 2.94 μM, respectively)
No obvious improvements in BChE inhibition activity were ob-
served when a methylenedioxy or ethylenedioxy group was introduced
in ortho positions of the B ring (8j and 8k). Also, 8n-8p, obtained by the
introduction of trifluoromethyl acetyl and cyano in the para position of
B ring respectively, presented poor BChE inhibition. Considering the
slightly potency difference of 8a and 8h, 8q-8t, bearing two methoxy
and/or fluorine groups in meta and ortho position of the B ring, were
further synthesized, only 8t displayed a noticeable activity and sig-
nificant selectivity toward BChE (IC50 = 1.13 μM, SI = 309).
carbamates in the B ring result in the significantly increase of the BChE
inhibition activity. Four derivatives (9b, 9g, 9h, and 9k) showed sub-
micromolar potency, while most of them demonstrated low micromolar
activities. The BChE inhibition potency varied in the following se-
quence: heptyl > phenethyl > dimethyl > ethyl (methyl) > 4-iso-
propylphenyl. Notably, their BChE inhibitory activities were closely
associated with the substituted position in following pattern meta >
ortho > para (i.e., 9a-9n). This trend can be attributed to a more ad-
vantageous position in the gorge of BChE in the case of the meta-sub-
stituted compounds, which was partly confirmed by the docking results
of two most potent compounds among this series, 9g (IC50 = 93 nM,
SI = 836) and 9h (IC50 = 222 nM, SI = 1339). However, further in-
troduction of an ortho-fluoride in 9h significantly and unexpectedly
decreased the BChE inhibitory capacity (9o, IC50 = 4.84 μM).
Previously study revealed that selective BChE inhibitors can be
developed with a scaffold bearing carbamate to fit into the correct
position in the gorge of BChE [35]. Therefore, fifteen carbamate-based
derivatives (9a-9o) were synthesized. In general, introduction of
In order to investigate the contribution of pyran ring in the scaffold,
11a and 11b were synthesized and showed poor activity toward BChE,
4

C. Wu, et al.
BioorganicChemistry88(2019)102949
Table 2
The chemical structure of the carbamate-based compounds and their Inhibitory activities against eeAChE and eqBChE.
SEMb
Compd.
R1
Chain
IC50 (μM)
SEMb
a
a
Compd.
R1
Chain
IC50 (μM)
AChE
Position
BChE
SIc
Position
AChE
BChE
SIc
9a
9b
9c
ortho
meta
para
26.78%
33.55
8.72
0.46
0.019
0.52
> 1.1
49.3
9j
ortho
meta
para
49.71
7.26
17.97
1.09
0.09
0.009
45.6
449
NDd
1.71
0.681
11.79
9k
9l
164.80
5.54%
0.367
4.67%
27.60%
> 0.8
9d
9e
ortho
meta
6.27%
31.51
7.85
1.47
0.33
0.08
> 1.3
21.4
9m
9n
meta
para
14.21%
6.69%
13.44%
11.47%
NDd
NDd
1.52
0.22
9f
para
10.12
28.65%
< 1.0
9o
20.72%
4.84
0.13
> 2.1
9g
9h
9i
ortho
meta
para
297.28
77.79
11.28
6.74
0.222
0.014
0.001
1339
836
11a
11b
10.44%
6.33%
5.68%
6.42%
NDd
NDd
0.093
12.18%
32.07%
NDd
a
b
The dash line represents connecting bond.
The inhibition activities are expressed as IC50 (μM) or as percentage of inhibition at 10 μM, the IC50 values are the mean of three independent
experiments
SEM.
c
d
SI represents selectivity index which is determined as ratio AChE IC50/BChE IC50
.
Not determined.
which indicated that the pyran ring in the flavonoid nucleus played a
prominent role to keep BChE inhibition, It is also evident from the
moderate inhibition and selectivity of 7 (IC50 = 5.27 μM, 24.47% of
AChE inhibition at 10 μM).
2.4. Kinetic characterization of BChE inhibition
Two potent BChE inhibitors (9g and 9h) were selected for further
kinetic analysis to gain the information on their mechanisms of in-
hibition. The experiments were performed using the combination of six
concentrations of the substrate (from 0.05 to 0.8 mM) and four con-
centrations of the inhibitors. Double-reciprocal Lineweaver-Burk plots
were used to evaluate the type of inhibition. As shown in Fig. 3, in-
creasing the concentrations of 9g (Fig. 3A) and 9h (Fig. 3B) decreased
the Vmax and increased the Km, suggesting a mixed-type inhibition. The
dissociation constants Ki for 9g and 9h were estimated to be 0.158 μM
and 0.045 μM, respectively.
Considering that nitrogen of the pyridine ring may interact with
other residues as a result of slight changes in the scaffold. Replacement
of B ring of 8 with a pyridine ring bearing methoxy and fluoro increased
the AChE over BuChE inhibitory potency (8v and 8w), which suggested
the importance of pyridine ring for AChE inhibition. All other com-
pounds demonstrated weak to moderate AChE inhibition.
Due to their most potent activities and good selectivity toward
BChE, 9g and 9h were selected for following studies.
2.5. Molecular docking
2.3. ADME prediction
To help explain the higher potential of 9g and 9h than 8 and il-
lustrate their binding mode in the active sites of BChE enzyme, mole-
cular docking simulations were performed using the molecular docking
program GOLD 5.0 (The Cambridge Crystallographic Data Centre).
Hydrogen bonds were defined by Gold, and other interactions were
described by Discovery Studio 2016 (BIOVIA) based on the docking
data. The calculated GoldScores were 60.5219, 53.6077 and 40.6528
for the 9h-, 9g- and 8-hBChE complexes, respectively, which were
highly correlated with their inhibitory activity in vitro.
The ADME (absorption, distribution, metabolism and excretion)
properties are considered as critical parameters for drug candidates [36].
Hence, the ADME properties of seven potent inhibitors (IC50 < 2 μM)
were predicted in silico using the QikProp v. 5.5 (Schrodinger). Compared
to the characteristics of galantamine and tacrine, the data (Table S1, see
Supporting Information) indicated that all of seven compounds possessed
adequate clog P, good serum albumin binding and human absorption, low
levels of primary metabolites and good Caco-2 permeability. Among the
tested compounds, the solubility of three compounds (8t, 9b and 9e) were
within the limits. As an important parameter, LogBB is frequently used to
assess the permeability of compounds through the blood-brain barrier.
The calculated logBB of all these compounds ranged from −0.880 to
0.231, which were also within the reported limitation (−3 < logBB <
1.2), indicating that seven investigated inhibitors could be administered
orally as CNS-active compounds.
In the 8-hBChE complex (Fig. 4A and B), two hydrogen bonds were
observed between the oxygen atom of the pyran ring and Ser287
(2.6 Å), the oxygen atom of the carbonyl group and Gly117 (3.1 Å) in
the oxyanion cavity of BChE. Additionally, three carbon-hydrogen
bonds were formed between 4-OMe group and Tyr128 (2.7 Å), among
3-OMe group, Tyr128 (3.4 Å) and Gly115 (3.2 Å). Moreover, the B ring
of 8 was also involved in a T-shaped interaction with Trp82.
A further comparison between the 9g- and 9h-hBChE complexes
5

C. Wu, et al.
BioorganicChemistry88(2019)102949
Fig. 3. Lineweaver-Burk plots of BChE inhibition of 9g (A) and 9h (B).
Fig. 4. Binding mode of 8 (A and B), 9g (C and D) and 9h (E and F) in the BChE active site. Figures (B, D and F) were made by Discovery Studio 2016 Client software;
Figures (A, C and E) were created with PyMol 1.8.2.0.
6

C. Wu, et al.
BioorganicChemistry88(2019)102949
revealed additional interesting interaction modes. In the 9g-hBChE
complex (Fig. 4C and D), the oxygen atom of the carbamate moiety
contributed to the hydrogen bonds with Ser198 (2.1 Å) and His438
(2.9 Å), two key residues in the catalytic triad, and the carbonyl group
provided another hydrogen bond with Ser287. This complex was fur-
ther stabilized by the π-sigma interaction with Leu286, amide-π stacked
interaction with Gly116, and T-shaped π-π interaction with Trp231.
Furthermore, the heptyl moiety of 9g was involved in an alkyl-alkyl
interaction with Leu125 and a π-alkyl interaction with Tyr128.
Docking results for 9h (Fig. 4E and F) were similar to those observed
for 9g. Interestingly, one more hydrogen bond could be seen between
the carbonyl group of 9h with Gly117 (2.9 Å) of the oxyanion cavity,
one key residue which stabilized the negative charge in the process of
substrate hydrolysis by BChE. The terminal methyl of the heptyl moiety
formed π-alkyl interactions with Tyr332 and Trp430, and it was also
involved in an alkyl-alkyl interaction with Ala328, indicating the im-
portance of the heptyl moiety for the BChE affinity of 9h. Additionally,
this complex was further stabilized by two π-π T-shaped interactions
(among the B ring, Trp231 and Phe329) and an alkyl-alkyl interaction
(the methyl group of the pyran ring and Ala277).
than 8 (32.3%) at the concentrations of 25 μM. These results revealed
that 9g and 9h had a therapeutic safety range similar to tacrine [37].
3. Conclusion
In conclusion, 41 derivatives based on a naturally occurring pyr-
anoisoflavone have been designed and synthesized, and their inhibition
potency against two cholinesterases was evaluated. Twenty-six of them
demonstrated good inhibitory activities on BChE. Compared to the
parent compound 8, seven derivatives displayed higher inhibitory po-
tential. The loss of pyran ring led to a significant loss of BChE inhibition
activity, while the introduction of various substituents in B ring had
little impact on the activity. The introduction of carbamates in B ring
significantly promoted the BChE inhibitory activity while retaining
good selectivity. Among all active derivatives, 9g (IC50 = 222 nM) and
9h (IC50 = 93 nM) presented the strongest BChE inhibitory potential
with high selectivity (SI = 1339 and 836, respectively). Subsequent
kinetic analysis revealed that both compounds acted as mixed-type
BChE inhibitors. Docking simulation showed they directly interacted
with the catalytic triad of enzyme. The docking result helped to explain
the difference in their activity. A cytotoxicity test on PC12 cells de-
monstrated 9g and 9h had a therapeutic safety range similar to tacrine.
As discussed above, the binding of both 9g and 9h with the catalytic
triad had more conformation stabilizing interactions, which were ab-
sent from the 8-hBChE complex. These differences explained higher
BChE inhibitory potential of compounds 9g and 9h over 8. For 9h, the
interaction, formed by the methyl group of the pyran ring and Ala277
and disappeared in the 9g-hBChE complex, should be the key factor
contributing to its stronger potential over 9g. Overall, the docking re-
sults agree with the different inhibitory potency observed in experi-
ments.
4. Experimental section
4.1. Chemistry
4.1.1. General methods
All reagents were purchased from commercial sources without fur-
ther purification. Flash column chromatography was performed with
silica gel 60 (100–200 and 200–300 mesh) using petroleum ether and
ethyl acetate as the eluents. Precoated plates of silica gel F254 for TLC
(detected by UV light) were purchased from Qingdao Marine Chemical
Ltd. 1H NMR spectra and 13C NMR spectra were recorded on Bruker
AVANCE III-400 or III-600 spectrometers in DMSO‑d6 or CDCl3 with
tetramethylsilane as 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), dou-
ble doublet (dd), triplet (t), and multiplet (m). Mass spectra were ob-
tained by Waters Quattro Premier XE triquadrupole mass spectrometer
with an ESI Source (Waters Corporation, USA), while High Resolution
Mass spectra were obtained on a Q-TOF Priemier mass spectrometer
(Micromass, Manchester, UK) with electron spray ionization (ESI) as
the ion source. Melting points were measured by a YRT-3 device
(Xintianguang Corporation, Tianjin, China). The purity of the synthe-
sized compounds were determined by HPLC-ELSD and confirm ≥95%
purity. [HPLC-ELSD, Alltech Lab Alliance Model 201 ultraviolet de-
tector, equipped with Alltech ELSD 6000 detector and Cromasil C18
column (250 mm × 4.6 mm, 5 μm), mobile phase: methanol/0.1%
formic acid (70/30–100/0), 1 mL/min.]
2.6. Cytotoxicity
The promising compounds 9g and 9h were further evaluated for
their cytotoxic effect as well as 8, and tacrine was chosen as reference
drug. PC12 cells were treated with above compounds at three different
concentrations (1, 5, 25 μM, due to their solubility) for 48 h and the cell
viability was determined using MTT Assay. As shown in Fig. 5, no ob-
vious cytotoxicity was observed at a concentration of 1 μM for four
tested compounds. With increased concentration to 5 μM, 9g and 9h
induced a decrease of cell viability (64.2% and 66.2%, respectively)
similar to tacrine (64.2%) and 8 (66.4%). At the concentrations of
25 μM, 9g induced slightly decrease of cell viability (53.6%) while 9h
induced moderate decrease of cell viability (43.3%) than tacrine
(58.7%). it may be considered that the 9h exhibited little more cyto-
toxicity than 9g. On the other hand, both of them had higher safety
4.1.2. 3-Iodo-7-((tetrahydro-2H-pyran-2-yl)oxy)-4H-chromen-4-one (4)
DHP (12 mL, 155 mmol) was mixed with CH2Cl2 (64 mL); the so-
lution was dropwise added to a mixture of 1 (6.086 g, 40 mmol) and
PPTS (372 mg) in CH2Cl2 (128 mL). After 4 h stirring at room tem-
perature, the reaction mixture was washed with saturated NaHCO3
solution and extracted three times with CH2Cl2. After drying with an-
hydrous NaSO4, the filtered CH2Cl2 extract was concentrated in vacuo to
give compound 2 as a white solid. Compound 2 was dissolved in DMF/
DMA (6.13 mL, 46.2 mmol) and heated for 3 h under reflux; then, the
reaction mixture was concentrated to give compound 3 as a red solid.
The crude compound 3, pyridine (3.54 mL, 44 mmol), and I2 (20.3 g,
80 mmol) were added into CHCl3 (50 mL) and stirred for 12 h at room
temperature. The saturated Na2S2O3 solution was added and stirred for
30 min at room temperature. The reaction mixture was extracted with
CH2Cl2. After drying with anhydrous Na2SO4, the CH2Cl2 filtrate was
Fig. 5. In vitro cytotoxicity of compounds 8, 9g and 9h on PC12 cell line. Data
were expressed as mean
SD (n = 3). *p < 0.05, **p < 0.01 vs Ctrl.
7

C. Wu, et al.
BioorganicChemistry88(2019)102949
concentrated in vacuo. The resulting mixture was further purified by
flash chromatography to yield a white solid 4 (11.12 g, 74.70%). 1H
NMR (CDCl3, 400 MHz) δ: 1.55–1.78 (m, 3H), 1.89–2.03 (m, 3H),
3.63–3.67 (m, 1H), 3.80–3.86 (m, 1H), 5.54–5.55 (m, 1H), 7.09–7.12
(m, 2H), 8.14 (d, 1H, J = 9.6 Hz), 8.22 (s, 1H). 13C NMR (CDCl3,
100 MHz) δ: 18.4, 25.1, 30.1, 62.2, 87.1, 96.7, 103.3, 116.3, 116.7,
128, 157.5, 157.8, 161.9, 172.9.
boronic acid. White solid, yield, 42%; m.p. 162–163 °C; 1H NMR
(CDCl3, 400 MHz) δ: 1.51 (s, 6H), 5.73 (d, J = 10.0 Hz, 1H), 6.82 (d,
J = 10.0 Hz, 1H), 6.88 (d, J = 9.1 Hz, 1H), 7.14–7.19 (m, 1H),
7.19–7.23 (m, 1H), 7.34–7.40 (m, 1H), 7.47–7.51 (m, 1H), 8.00 (d,
J = 1.2 Hz, 1H), 8.06 (d, J = 8.8 Hz, 1H). 13C NMR (CDCl3, 100 MHz)
δ: 28.4, 78.0, 109.5, 115.4, 116.1, 118.5, 119.8, 120.1, 124.3, 127.0,
130.2, 130.6, 132.4, 152.6, 154.0, 157.7, 159.3, 161.8, 175.3. HR-TOF-
MS (positive mode): m/z calcd. C20H16FO3 [M+H]+ 323.1083, found
323.1082. HPLC purity: 98.3%.
4.1.3. 7-Hydroxy-3-iodo-4H-chromen-4-one (5)
Compound 4 (4.47 g, 12 mmol) was dissolved in a mixed solution of
CH3OH (120 mL) and THF (120 mL). Then, p-toluenesulfonic acid
(228 mg, 1.2 mmol) was added into the mixture at room temperature.
After 1 h stirring at 60 °C, the cooled mixture was concentrated in vacuo
and dissolved in ethyl acetate (500 mL), the resulting solution was
washed with saturated brine. The ethyl acetate extract was dried over
anhydrous Na2SO4 solution and concentrated in vacuo to provide
compound 5 as a white solid (3.21 g, 92.8%). 1H NMR (DMSO‑d6,
400 MHz) δ: 6.86 (t, J = 2 Hz, 1H), 6.93–6.96 (m, 1H), 7.90 (dd,
J = 1.2 Hz, 8.8 Hz, 1H), 8.68 (d, J = 2.4 Hz, 1H), 10.93 (s, 1H). 13C
NMR (DMSO‑d6, 100 MHz) δ: 18.4, 25.1, 30.1, 62.2, 87.1, 96.7, 103.3,
116.3, 116.7, 128.0, 157.5, 157.8, 161.9, 172.9.
4.1.5.3. 3-(3-Fluorophenyl)-8,8-dimethyl-4H,8H-pyrano[2,3-f]chromen-4-
one (8b). The corresponding aryl boronic acid was (3-fluorophenyl)
boronic acid. White solid, yield, 70%; m.p. 161–162 °C; 1H NMR
(CDCl3, 600 MHz) δ: 1.51 (s, 6H), 5.73 (d, J = 10.0 Hz, 1H), 6.81 (d,
J = 10.0 Hz, 1H), 6.88 (d, J = 8.6 Hz, 1H), 7.06–7.09 (m, 1H), 7.32–7.34
(m, 2H), 7.37–7.41 (m, 1H), 7.99 (s, 1H), 8.07 (d, J = 8.8 Hz 1H). 13C
NMR (CDCl3, 100 MHz) δ: 28.3, 78.0, 109.4, 115.0, 115.3, 115.6, 116.2,
118.4, 124.2, 124.6, 126.9, 130.0, 130.6, 134.2, 152.5, 157.6, 161.6,
164.1, 175.4. HR-TOF-MS (positive mode): m/z calcd. C20H16FO3 [M
+H]+ 323.1083, found 323.1079. HPLC purity: 96.7%.
4.1.5.4. 3-(4-Fluorophenyl)-8,8-dimethyl-4H,8H-pyrano[2,3-f]chromen-
4-one (8c). The corresponding aryl boronic acid was (4-fluorophenyl)
boronic acid. White solid, yield, 61%; m.p. 167–169 °C; 1H NMR
(CDCl3, 600 MHz) δ: 1.51 (s, 6H), 5.73 (d, J = 10.0 Hz), 6.81 (d,
J = 10.0 Hz, 1H), 6.87 (d, J = 8.8 Hz, 1H), 7.12 (t, J = 8.7 Hz, 2H),
7.53 (dd, J = 5.4, 8.4 Hz, 2H), 7.95 (s, 1H), 8.06 (d, J = 8.8 Hz, 1H).
13C NMR (CDCl3, 100 MHz) δ: 28.3, 77.9, 109.3, 115.0, 115.5, 115.7,
118.4, 124.4, 126.8, 128.0, 130.5, 130.8, 130.9, 152.2, 152.5, 157.6,
161.6, 164.1, 175.7. HR-TOF-MS (positive mode): m/z calcd.
4.1.4. 3-Iodo-8,8-dimethylpyrano[2,3-f]chromen-4(8H)-one (7)
3-Methylbut-2-enal (7.72 mL, 80 mmol), KHSO4 (0.545 g, 4 mmol),
and (EtO)3CH (13.3 mL, 80 mmol) were successively added to EtOH
(24 mL) at 0 °C, keep stirring for 30 min, and then stirred for another
30 min at room temperature. The resulting mixture was filtered and
washed with EtOH (5 mL). K2CO3 (1.106 g, 8 mmol) was added and
stirred for 2 h at room temperature. Then, the resulting mixture was
filtered and concentrated in vacuo to provide 6 as a colorless liquid. The
crude 6 was used in next step without further purification.
C
20H16FO3 [M+H]+ 323.1083, found 323.1080. HPLC purity: 99.5%.
Compound 5 (2.304 g, 8 mmol), the crude 6 (6 mL), and 3-picoline
(195 μL, 2 mmol) were successively added to xylene (46 mL) under
stirring at room temperature. Then the mixture was heated under reflux
for 24 h. After cooling to room temperature, the mixture was con-
centrated in vacuo. The resulting residue was purified by flash chro-
matography and washed with petroleum ether to provide a yellow solid
7. (1.565 g, 55.2%). 1H NMR (CDCl3, 400 MHz) δ: 1.49 (s, 6H), 5.72 (d,
J = 10.0 Hz, 1H), 6.73 (d, J = 10.0 Hz, 1H), 6.86 (d, J = 8.4 Hz, 1H),
8.00 (d, J = 8.4 Hz, 1H), 8.23 (s, 1H). 13C NMR (CDCl3, 100 MHz) δ:
28.3, 78.1, 87.3, 109.2, 114.8, 115.8, 116.1, 127.1, 130.8, 152.4,
157.0, 157.8, 172.8.
4.1.5.5. 3-(2-Hydroxyphenyl)-8,8-dimethyl-4H,8H-pyrano[2,3-f]chromen-4-
one (8d). The corresponding aryl boronic acid was (2-hydroxyphenyl)
boronic acid. Yellow solid, yield, 44%; m.p. 149–151 °C; 1H NMR (CDCl3,
400 MHz) δ: 1.50 (s, 6H), 5.76 (d, J = 10.0 Hz, 1H), 6.84 (d, J = 10.0 Hz,
1H), 6.93–6.99 (m, 2H), 7.10 (dd, J = 1.2, 8.0 Hz, 1H), 7.17 (dd, J = 1.6,
7.6 Hz, 1H), 7.33–7.37 (m, 1H), 8.11–8.14 (m, 2H). 13C NMR (CDCl3,
100 MHz) δ: 28.4, 78.3, 109.1, 114.7, 116.4, 117.2, 119.8, 120.8, 120.9,
125.0, 126.9, 129.7, 130.6, 130.8, 152.4, 155.2, 156.7, 158.3, 178.8. HR-
TOF-MS (positive mode): m/z calcd. C20H17O4 [M+H]+ 321.1127, found
321.1129. HPLC purity: 99.2%.
4.1.5. General procedure for the preparation of compounds 8, 8a–8u
Compound 7 (106 mg, 0.3 mmol) was dissolved in premixed solu-
tion of DME (1.8 mL) and H2O (1.8 mL). Then, Na2CO3 (95 mg,
0.9 mmol), aryl boronic acid (0.36 mmol), and Pd/C (16 mg, 5 mol %)
were added. After 1 h stirring at 45 °C, the reaction mixture was fil-
tered, and the cake was washed with H2O (4 mL) and CH2Cl2 (6 mL).
The aqueous phase was then extracted twice with CH2Cl2, 8a–8u were
obtained from the CH2Cl2 extracts by flash chromatography.
4.1.5.6. 3-(3-Hydroxyphenyl)-8,8-dimethyl-4H,8H-pyrano[2,3-f]
chromen-4-one (8e). The corresponding aryl boronic acid was (3-
hydroxyphenyl)boronic acid. Yellow solid, yield, 41%; m.p.
220–221 °C; 1H NMR (DMSO‑d6, 400 MHz) δ: 1.50 (s, 6H), 5.73 (d,
J = 10.0 Hz, 1H), 5.80 (s, 1H), 6.81 (d, J = 10.0 Hz, 1H), 6.85–6.89 (m,
2H), 7.02 (d, J = 7.6 Hz, 1H), 7.20 (dd, J = 2, 2.4 Hz, 1H), 7.29 (t,
J = 7.6 Hz, 1H), 7.99 (s, 1H), 8.08 (d, J = 8.8 Hz, 1H). 13C NMR
(CDCl3, 100 MHz) δ: 27.6, 77.9, 109.0, 114.1, 114.8, 115.0, 116.1,
117.9, 119.5, 123.7, 126.1, 129.1, 131.4, 133.0, 151.6, 153.7, 156.6,
157.0, 174.4. MS (ESI): m/z calcd. C20H17O4+ [M+H]+ 321.11, found
321.24. HPLC purity: 97.3%.
4.1.5.1. 3-(3,4-Dimethoxyphenyl)-8,8-dimethyl-4H,8H-pyrano[2,3-f]
chromen-4-one (8). The corresponding aryl boronic acid was (3,4-
dimethoxyphenyl) boronic acid. White solid, yield, 73.4%; m.p.
169–171 °C; 1H NMR (CDCl3, 400 MHz) δ: 1.51 (s, 6H), 5.73 (d,
J = 10.0 Hz, 1H), 6.82 (d, J = 10.0 Hz, 1H), 6.88 (d, J = 9.1 Hz, 1H),
7.14–7.19 (m, 1H), 7.19–7.23 (m, 1H), 7.34–7.40 (m, 1H), 7.47–7.51
(m, 1H), 8.00 (d, J = 1.2 Hz, 1H), 8.06 (d, J = 8.8 Hz, 1H). 13C NMR
(CDCl3, 100 MHz) δ: 28.4, 78.0, 109.5, 115.4, 116.1, 118.5, 119.8,
120.1, 124.3, 127.0, 130.2, 130.6, 132.4, 152.6, 154.0, 157.7, 159.3,
161.8, 175.3. MS (ESI): m/z calcd. C22H21O5 [M+H]+ 365.14, found
365.08. HPLC purity: 96.0%.
4.1.5.7. 3-(4-Hydroxyphenyl)-8,8-dimethyl-4H,8H-pyrano[2,3-f]
chromen-4-one (8f). The corresponding aryl boronic acid was (4-
hydroxyphenyl)boronic acid. Yellow solid, yield, 36%; m.p.
249–251 °C; 1H NMR (CDCl3, 400 MHz) δ: 1.50 (s, 6H), 5.72 (d,
J = 10.0 Hz, 1H), 6.81 (d, J = 10.0 Hz, 1H), 6.85–6.90 (m, 3H),
7.41–7.44 (m, 2H), 7.94 (s, 1H), 8.07 (d, J = 8.8 Hz, 1H). 13C NMR
(DMSO‑d6, 100 MHz) δ: 28.3, 64.5, 64.6, 77.4, 77.8, 109.3, 115.1,
115.3, 117.4, 118.1, 118.5, 122.3, 124.7, 125.3, 126.9, 130.4, 143.5,
+
4.1.5.2. 3-(2-Fluorophenyl)-8,8-dimethyl-4H,8H-pyrano[2,3-f]chromen-
143.8, 152.0, 152.4, 157.4, 175.9. MS (ESI): m/z calcd. C20H17O4 [M
4-one (8a). The corresponding aryl boronic acid was (2-fluorophenyl)
+H]+ 321.11, found 321.27. HPLC purity: 98.0%.
8

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BioorganicChemistry88(2019)102949
4.1.5.8. 3-(2-Methoxyphenyl)-8,8-dimethyl-4H,8H-pyrano[2,3-f]
chromen-4-one (8g). The corresponding aryl boronic acid was (2-
methoxyphenyl)boronic acid. White solid, yield, 53%; m.p.
159–160 °C; 1H NMR (CDCl3, 400 MHz) δ: 1.50 (s, 6H), 3.80 (s, 3H),
5.71 (d, J = 10.0 Hz, 1H), 6.82 (d, J = 10.0 Hz, 1H), 6.85 (d,
J = 8.8 Hz, 1H), 6.97–7.04 (m, 2H), 7.32 (dd, J = 1.6, 7.2 Hz, 1H),
7.34–7.39 (m, 1H), 7.93 (s, 1H), 8.05 (d, J = 8.8 Hz, 1H). 13C NMR
(CDCl3, 100 MHz) δ: 28.2, 55.9, 78.5, 109.4, 111.3, 115.2, 115.2,
118.6, 120.7, 121.1, 122.5, 126.9, 129.8, 130.3, 131.9, 152.5, 153.6,
157.3, 157.7, 175.6. MS (ESI): m/z calcd. C21H19O4 [M+H]+ 335.13,
found 335.23. HPLC purity: 98.0%.
100 MHz) δ: 28.3, 52.3, 78.0, 109.4, 115.0, 115.6, 118.4, 124.4,
126.8, 128.7, 129.4, 129.9, 130.6, 130.6, 132.4, 133.9, 152.5, 152.7,
157.6, 167.0, 175.5. HR-TOF-MS (positive mode): m/z calcd. C22H19O5
[M+H]+ 363.1232, found 363.1229. HPLC purity: 99.3%.
4.1.5.14. 3-(2,3-Dimethoxyphenyl)-8,8-dimethyl-4H,8H-pyrano[2,3-f]
chromen-4-one (8m). The corresponding aryl boronic acid was (2,3-
dimethoxyphenyl)boronic acid. White solid, yield, 39%; m.p.
129–131 °C; 1H NMR (CDCl3, 600 MHz) δ: 1.51 (s, 6H), 3.74 (s, 3H),
3.90 (s, 3H), 5.72 (d, J = 9.6 Hz, 1H), 6.83 (d, J = 10.2 Hz, 1H), 6.86
(d, J = 9.0 Hz, 1H), 6.95–6.98 (m, 2H), 7.11 (t, J = 7.8 Hz, 1H), 7.96
(s, 1H), 8.06 (d, J = 8.4 Hz, 1H). 13C NMR (CDCl3, 100 MHz) δ: 28.3,
56.0, 60.9, 77.8, 109.4, 112.8, 115.2, 115.2, 118.6, 121.9, 123.7,
124.0, 126.2, 126.8, 130.4, 147.4, 152.5, 153.0, 153.8, 157.4, 175.9.
4.1.5.9. 3-(3-Methoxyphenyl)-8,8-dimethyl-4H,8H-pyrano[2,3-f]
chromen-4-one (8h). The corresponding aryl boronic acid was (3-
methoxyphenyl)boronic acid. White solid, yield, 81%; m.p.
135–137 °C; 1H NMR (CDCl3, 400 MHz) δ: 1.50 (s, 6H), 3.85 (s, 3H),
5.73 (d, J = 10.0 Hz, 1H), 6.82 (d, J = 10.0 Hz, 1H), 6.87 (d,
J = 8.8 Hz, 1H), 6.92–6.95 (m, 1H), 7.11 (d, J = 7.6 Hz, 1H), 7.15
(dd, J = 1.6, 2.4 Hz, 1H), 7.34 (t, J = 7.6 Hz, 1H), 7.98 (s, 1H), 8.07 (d,
J = 8.4 Hz, 1H). 13C NMR (CDCl3, 100 MHz) δ: 28.3, 55.4, 77.9, 109.3,
114.2, 114.6, 115.0, 115.4, 118.5, 121.4, 125.0, 126.9, 129.6, 130.5,
133.4, 152.4, 152.5, 157.5, 159.7, 175.7. MS (ESI): m/z calcd C21H19O4
[M+H]+ 335.13, found 335.21. HPLC purity: 98.1%.
HR-TOF-MS (positive mode): m/z calcd.
C
22H21O5+[M+H]+
365.1389, found 365.1388. HPLC purity: 97.1%.
4.1.5.15. 8,8-Dimethyl-3-(4-(trifluoromethyl)phenyl)-4H,8H-pyrano[2,3-
f]chromen-4-one (8n). The corresponding aryl boronic acid was (2,3-
dimethoxyphenyl)boronic acid. White solid, yield, 47%; m.p.
193–194 °C; 1H NMR (CDCl3, 600 MHz) δ: 1.51 (s, 6H), 5.74 (d,
J = 9.6 Hz, 1H), 6.81 (d, J = 10.2 Hz, 1H), 6.89 (d, J = 9.0 Hz, 1H),
7.69 (s, 4H), 8.01 (s, 1H), 8.07 (d, J = 9.0 Hz, 1H). 13C NMR (CDCl3,
100 MHz) δ: 78.0, 114.9, 115.7, 118.4, 124.2, 125.5, 126.8, 129.4,
130.1, 130.4, 130.7, 135.8, 152.5, 28.3, 152.8, 157.8, 175.4. MS (ESI):
m/z calcd. C21H16F3O3 [M+H]+ 373.11, found 373.21. HPLC purity:
97.9%.
4.1.5.10. 3-(4-Methoxyphenyl)-8,8-dimethyl-4H,8H-pyrano[2,3-f]
chromen-4-one (8i). The corresponding aryl boronic acid was (4-
methoxyphenyl)boronic acid. White solid, yield, 68%; m.p.
142–144 °C; 1H NMR (CDCl3, 600 MHz) δ: 1.50 (s, 6H), 3.84 (s, 3H),
5.72 (d, J = 10.2 Hz, 1H), 6.81 (d, J = 9.6 Hz, 1H), 6.86 (d, J = 9.0 Hz,
1H), 6.96–6.98 (m, 2H), 7.48–7.51 (m, 2H), 7.94 (s, 1H), 8.07 (d,
J = 8.4 Hz, 1H). 13C NMR (CDCl3, 100 MHz) δ: 28.3, 55.5, 77.8, 109.3,
114.1, 115.1, 115.3, 118.5, 124.4, 124.8, 126.9, 130.3, 130.4, 151.9,
152.5, 157.4, 159.7, 176.0. MS (ESI): m/z calcd. C21H19O4 [M+H]+
335.13, found 335.21. HPLC purity: 99.1%.
4.1.5.16. 3-(4-Acetylphenyl)-8,8-dimethyl-4H,8H-pyrano[2,3-f]chromen-
4-one (8o). The corresponding aryl boronic acid was (4-acetylphenyl)
boronic acid. White solid, yield, 55.4%; m.p. 188–190 °C; 1H NMR
(CDCl3, 400 MHz) δ: 1.51 (s, 6H), 2.64 (s, 3H), 5.74 (d, J = 10.0 Hz,
1H), 6.82 (d, J = 10.0 Hz, 1H), 6.89 (d, J = 8.8 Hz, 1H), 7.69 (d,
J = 8.4 Hz, 2H), 8.02–8.04 (m, 3H); 8.07 (d, J = 8.8 Hz, 1H). 13C NMR
(CDCl3, 100 MHz) δ: 26.8, 28.3, 78.0, 109.4, 114.9, 115.6, 118.4,
124.3, 126.8, 128.6, 129.2, 130.6, 136.6, 137.1, 152.4, 152.9, 157.7,
175.3, 197.9. MS (ESI): m/z calcd. C22H19O4 [M+H]+ 347.13, found
347.22. HPLC purity: 99.6%.
4.1.5.11. 3-(Benzo[d][1,3]dioxol-5-yl)-8,8-dimethyl-4H,8H-pyrano[2,3-
f]chromen-4-one (8j). The corresponding aryl boronic acid was benzo
[d][1,3]dioxol-5-ylboronic acid. White solid, yield, 71%; m.p.
153–155 °C; 1H NMR (CDCl3, 600 MHz) δ: 1.50 (s, 6H), 5.72 (d,
J = 10.0 Hz, 1H), 6.00 (s, 2H), 6.81 (d, J = 10.0 Hz, 1H), 6.87 (dd,
J = 4.0, 8.0 Hz, 2H), 6.97 (dd, J = 1.8, 8.4 Hz, 1H), 7.09 (d, J = 1.2 Hz,
1H), 7.93 (s, 1H), 8.06 (d, J = 8.4 Hz, 1H). 13C NMR (CDCl3, 100 MHz)
δ: 28.3, 77.9, 101.3, 108.5, 109.3, 109.9, 115.1, 115.4, 118.4, 122.5,
125.0, 125.8, 126.9, 130.4, 147.8, 147.8, 152.0, 152.5, 157.5, 175.9.
MS (ESI): m/z calcd. C21H17O5 [M+H]+ 349.11, found 349.22. HPLC
purity: 98.0%.
4.1.5.17. 4-(8,8-Dimethyl-4-oxo-4H,8H-pyrano[2,3-f]chromen-3-yl)
benzonitrile (8p). The corresponding aryl boronic acid was (4-
cyanophenyl)boronic acid. White solid, yield, 60.8%; m.p.
205–206 °C; 1H NMR (CDCl3, 400 MHz) δ: 1.51 (s, 6H), 5.75 (d,
J = 10.0 Hz, 1H), 6.81 (d, J = 10.0 Hz, 1H), 6.90 (d, J = 8.8 Hz, 1H),
7.69–7.74 (m, 4H), 8.02 (s, 1H), 8.06 (d, J = 8.8 Hz, 1H). 13C NMR
(CDCl3, 100 MHz) δ: 28.3, 78.1, 109.4, 111.8, 114.8, 115.8, 118.2,
118.9, 123.7, 126.8, 129.6, 130.8, 132.3, 137.0, 152.4, 153.1, 157.9,
175.0. MS (ESI): m/z calcd. C22H19O4 [M+H]+ 330.11, found 330.15.
HPLC purity: 98.3%.
4.1.5.12. 3-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-8,8-dimethyl-4H,8H-
pyrano[2,3-f]chromen-4-one (8k). The corresponding aryl boronic acid
was (2,3-dihydrobenzo[b][1,4]dioxin-6-yl)boronic acid. White solid,
yield, 76%; m.p. 185–187 °C; 1H NMR (CDCl3, 600 MHz) δ: 1.52 (s, 6H),
4.28 (s, 4H), 5.72 (d, J = 10.0 Hz, 1H), 6.81 (d, J = 10.0 Hz, 1H), 6.86
(d, J = 9.0 Hz, 1H), 6.92 (d, J = 8.4 Hz, 1H), 7.03 (dd, J = 2.4, 8.4 Hz,
4.1.5.18. 3-(2,3-Difluorophenyl)-8,8-dimethyl-4H,8H-pyrano[2,3-f]
chromen-4-one (8q). The corresponding aryl boronic acid was (2,3-
difluorophenyl)boronic acid. White solid, yield, 61%; m.p. 137–139 °C;
1H NMR (CDCl3, 400 MHz) δ: 1.51 (s, 6H), 5.74 (d, J = 10.0 Hz, 1H),
6.82 (d, J = 10.0 Hz, 1H), 6.89 (d, J = 8.8 Hz, 1H), 7.11–7.25 (m, 3H),
8.01 (d, J = 1.6 Hz, 1H), 8.06 (d, J = 8.8 Hz, 1H). 13C NMR (CDCl3,
100 MHz) δ: 28.3, 78.0, 109.5, 114.9, 115.7, 117.3, 118.3, 119.2,
122.0, 124.0, 126.8, 126.9, 130.7, 147.5, 149.8, 152.5, 154.1, 157.8,
174.9. HR-TOF-MS (positive mode): m/z calcd. C20H15F2O3 [M+H]+
341.0989, found 341.0984. HPLC purity: 98.9%.
1H), 7.10 (d, J = 1.8 Hz, 1H), 7.92 (s, 1H), 8.06 (d, J = 8.4 Hz, 1H). 13
C
NMR (CDCl3, 100 MHz) δ: 28.3, 64.5, 64.6, 77.8, 109.3, 115.1, 115.3,
117.4, 118.1, 118.5, 122.3, 124.7, 125.3, 126.9, 130.4, 143.5, 143.8,
152.0, 152.4, 157.4, 175.9. MS (ESI): m/z calcd C22H19O5 [M+H]+
363.12, found 363.24. HPLC purity: 99.4%.
4.1.5.13. Methyl 3-(8,8-dimethyl-4-oxo-4H,8H-pyrano[2,3-f]chromen-3-
yl)benzoate (8l). The corresponding aryl boronic acid was (3-
(methoxycarbonyl)phenyl)boronic acid. White solid, yield, 72%; m.p.
169–170 °C; 1H NMR (CDCl3, 400 MHz) δ: 1.51 (s, 6H), 3.93 (s, 3H),
5.74 (d, J = 10.0 Hz, 1H), 6.82 (d, J = 10.0 Hz, 1H), 6.88 (dd, J = 0.4,
8.8 Hz, 1H), 7.52 (t, J = 8.0 Hz, 1H), 7.82–7.83 (m, 1H), 8.03 (s, 1H),
8.05–8.08 (m, 2H), 8.19 (t, J = 1.6 Hz, 1H). 13C NMR (CDCl3,
4.1.5.19. 3-(2,5-Difluorophenyl)-8,8-dimethyl-4H,8H-pyrano[2,3-f]
chromen-4-one (8r). The corresponding aryl boronic acid was (2,5-
difluorophenyl)boronic acid. White solid, yield, 46.3%; m.p.
182–183 °C; 1H NMR (CDCl3, 400 MHz) δ: 1.51 (s, 6H), 5.74 (d,
J = 10.0 Hz, 1H), 6.81 (d, J = 10.0 Hz, 1H), 6.88 (d, J = 8.4 Hz, 1H),
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BioorganicChemistry88(2019)102949
7.02–7.14 (m, 2H), 7.27–7.30 (m, 1H), 8.03 (d, J = 1.6 Hz, 1H), 8.05
(d, J = 8.8 Hz, 1H). 13C NMR (CDCl3, 100 MHz) δ: 28.3, 78.0, 109.5,
114.9, 115.7, 116.4, 116.9, 118.3, 118.6, 118.9, 119.0, 126.8, 130.7,
152.4, 154.3, 155.1, 157.7, 159.7, 174.8. HR-TOF-MS (positive mode):
m/z calcd. C20H15F2O3 [M+H]+ 341.0989, found 341.0990. HPLC
purity: 97.9%.
J = 8.8 Hz, 1H), 7.61 (t, J = 2.0 Hz, 1H), 8.04 (s, 1H), 8.06 (d,
J = 8.8 Hz, 1H), 8.27 (s, 1H), 8.32 (d, J = 2.4 Hz, 1H). 13C NMR
(CDCl3, 100 MHz) δ: 28.3, 55.8, 78.0, 109.4, 114.8, 115.8, 118.2,
121.3, 121.7, 126.7, 128.8, 130.7, 137.8, 140.7, 152.5, 152.5, 155.4,
157.8, 175.5. HR-TOF-MS (positive mode): m/z calcd. C20H18NO4 [M
+H]+ 336.1236, found 336.1234. HPLC purity: 99.4%.
4.1.5.20. 3-(2-Fluoro-5-methoxyphenyl)-8,8-dimethyl-4H,8H-pyrano[2,3-
f]chromen-4-one (8s). The corresponding aryl boronic acid was (2-
fluoro-5-methoxyphenyl)boronic acid. White solid, yield, 54%; m.p.
132–134 °C; 1H NMR (CDCl3, 400 MHz) δ: 1.51 (s, 6H), 3.81 (s, 3H),
5.73 (d, J = 10.0 Hz, 1H), 6.82 (d, J = 10.0 Hz, 1H), 6.85–6.89 (m,
2H), 8.01 (d, J = 1.6 Hz, 1H), 8.06 (d, J = 8.8 Hz, 1H). 13C NMR
(CDCl3, 100 MHz) δ: 28.3, 56.0, 77.9, 109.4, 115.0, 115.5, 115.5,
116.4, 116.7, 118.4, 119.9, 120.0, 126.8, 130.6, 152.4, 154.1, 155.6,
156.0, 157.6, 175.1. HR-TOF-MS (positive mode): m/z calcd.
4.1.7. General procedure for the preparation of compounds 9a-9e
Phenol (64.0 mg, 0.2 mmol) and potassium carbonate (44.0 mg,
0.32 mmol) were added into a mixture of dimethylformamide (1.5 mL)
and acetonitrile (0.6 mL). Then, carbamoyl chloride (0.22 mmol) in
acetonitrile (0.3 mL) was added dropwise. The reaction mixture was
stirred and refluxed at 95 °C for 3 h. After cooling to room temperature,
20 mL of water was added and the crude mixture was extracted three
times with ethyl acetate. The collected organic layer was washed three
times with saturated salt solution, dried over Na2SO4, concentrated in
vacuo, and purified by flash chromatography to provide 9a-9e.
C
21H18FO4 [M+H]+ 353.1189, found 353.1186. HPLC purity: 97.2%.
4.1.5.21. 3-(2-Fluoro-3-methoxyphenyl)-8,8-dimethyl-4H,8H-pyrano[2,3-
f]chromen-4-one (8t). The corresponding aryl boronic acid was (2-
fluoro-3-methoxyphenyl)boronic acid. White solid, yield, 43%; m.p.
168–170 °C; 1H NMR (CDCl3, 400 MHz) δ: 1.50 (s, 6H), 3.92 (s, 3H),
5.73 (d, J = 10.0 Hz, 1H), 6.82 (d, J = 10.0 Hz, 1H), 6.87 (d,
J = 8.8 Hz, 1H), 6.98–7.97 (m, 2H), 7.11–7.15 (m, 1H), 7.99 (d,
J = 1.2 Hz, 1H), 8.06 (d, J = 8.8 Hz, 1H). 13C NMR (CDCl3, 100 MHz)
δ: 28.3, 56.5, 77.9, 109.4, 113.5, 115.0, 115.5, 118.4, 119.8, 120.5,
123.5, 123.8, 126.8, 130.5, 148.1, 148.2, 152.5, 154.0, 157.6, 175.1.
4.1.7.1. 2-(8,8-Dimethyl-4-oxo-4H,8H-pyrano[2,3-f]chromen-3-yl)phenyl
dimethylcarbamate (9a). The corresponding phenol was 8d, and the
corresponding carbamoyl chloride was N,N-dimethylcarbamoyl
chloride. Yellow solid, yield, 52%; m.p. 61–63 °C; 1H NMR (CDCl3,
600 MHz) δ: 1.51 (s, 6H), 2.89 (s, 6H), 5.73 (d, J = 10.2 Hz, 1H), 6.82
(d, J = 10.2 Hz, 1H), 6.86 (d, J = 8.4 Hz, 1H), 7.24–7.27 (m, 2H), 7.32
(dd, J = 1.2, 7.2 Hz, 1H), 7.39–7.42 (m, 1H), 7.91 (s, 1H), 8.04 (d,
J = 9.0 Hz, 1H). 13C NMR (CDCl3, 100 MHz) δ: 28.3, 36.5, 36.7, 77.9,
109.4, 115.0, 115.3, 118.3, 122.5, 123.2, 125.4, 125.5, 126.7, 129.7,
HR-TOF-MS (positive mode): m/z calcd.
C
21H18FO4 [M+H]+
130.4, 131.5, 150.0, 152.5, 153.4, 154.8, 157.4, 175.1. HR-TOF-MS
23H22NO5 (M+
)
392.1498, found
+
353.1189, found 353.1186. HPLC purity: 98.8%.
(positive mode): m/z calcd.
C
392.1503. HPLC purity: 98.9%.
4.1.5.22. 3-(2-Fluoro-3-hydroxyphenyl)-8,8-dimethyl-4H,8H-pyrano[2,3-
f]chromen-4-one (8u). The corresponding aryl boronic acid was (2-
fluoro-3-hydroxyphenyl)boronic acid. Yellow solid, yield, 60%; m.p.
214–216 °C; 1H NMR (DMSO‑d6, 400 MHz) δ: 1.51 (s, 6H), 5.73 (d,
J = 10.0 Hz, 1H), 5.89 (s, 1H), 6.81 (d, J = 10.0 Hz, 1H), 6.87–6.92 (m,
2H), 6.97–7.02 (m, 1H), 7.06 (t, J = 8.0 Hz, 1H), 7.96 (s, 1H), 8.07 (d,
J = 8.8 Hz, 1H). 13C NMR (DMSO‑d6, 100 MHz) δ: 27.6, 77.9, 109.1,
114.1, 115.2, 117.5, 117.8, 120.1, 120.6, 120.7, 121.4, 123.9, 125.9,
131.5, 145.0, 145.1, 151.7, 154.5, 156.8, 173.6. HR-TOF-MS (positive
mode): m/z calcd. C20H16FO4 [M+H]+ 339.1033, found 339.1028.
HPLC purity: 99.2%.
4.1.7.2. 3-(8,8-Dimethyl-4-oxo-4H,8H-pyrano[2,3-f]chromen-3-yl)phenyl
dimethylcarbamate (9b). The corresponding phenol was 8e, and the
corresponding carbamoyl chloride was N,N-dimethylcarbamoyl
chloride. White solid, yield, 65.0%; m.p. 234–236 °C; 1H NMR
(CDCl3, 600 MHz) δ: 1.51 (s, 6H), 3.01 (s, 3H), 3.11 (s, 3H), 5.73 (d,
J = 10.2 Hz, 1H), 6.81 (d, J = 9.6 Hz, 1H), 6.87 (dd, J = 0.6, 9.0 Hz,
1H), 7.13–7.15 (m, 1H), 7.36–7.38 (m, 2H), 7.41 (t, 7.8 Hz, 1H), 8.00
(s, 1H), 8.06 (d, J = 9.0 Hz, 1H). 13C NMR (CDCl3, 100 MHz) δ: 28.3,
36.6, 36.8, 77.9, 109.3, 115.0, 115.5, 118.4, 121.7, 122.6, 124.4,
125.7, 126.8, 129.4, 130.5, 133.3, 151.6, 152.4, 152.8, 155.0, 157.5,
+
175.6. HR-TOF-MS (positive mode): m/z calcd. C23H22NO5 (M+
)
4.1.6. General procedure for the preparation of compounds 8v and 8w
Na2CO3 (32 mg, 0.3 mmol), heteroaryl boronic acids (0.12 mmol),
and tetrakis(triphenylphosphine)palladium (6 mg, 5 mol %) were added
to a solution of 7 (35 mg, 0.1 mmol) in THF (3 mL) and H2O (0.4 mL).
After 6 h stirring at 60 °C, the mixture was concentrated in vacuo and
purified by flash chromatography to give 8v and 8w.
392.1498, found 392.1493. HPLC purity: 97.6%.
4.1.7.3. 4-(8,8-Dimethyl-4-oxo-4H,8H-pyrano[2,3-f]chromen-3-yl)phenyl
dimethylcarbamate (9c). The corresponding phenol was 8f, and the
corresponding carbamoyl chloride was N,N-dimethylcarbamoyl
chloride. White solid, yield, 62.7%; m.p. 212–213 °C; 1H NMR
(CDCl3, 600 MHz) δ: 1.50 (s, 6H), 3.03 (s, 3H), 3.12 (s, 3H), 5.73 (d,
J = 10.2 Hz), 6.81 (d, J = 10.2 Hz, 1H), 6.87 (d, J = 9.0 Hz, 1H), 7.18
(d, J = 8.4 Hz, 2H), 7.55 (d, J = 8.4 Hz, 2H), 7.96 (s, 1H), 8.06 (d,
J = 9.0 Hz, 1H). 13C NMR (CDCl3, 100 MHz) δ: 28.3, 36.6, 36.9, 77.9,
109.4, 115.1, 115.4, 118.5, 121.9, 124.6, 126.8, 129.0, 130.0, 130.5,
151.6, 152.4, 152.5, 155.0, 157.5, 175.8. HR-TOF-MS (positive mode):
m/z calcd. C23H22NO5 [M+H]+ 392.1498, found 392.1494. HPLC
purity: 98.9%.
4.1.6.1. 3-(5-Fluoropyridin-3-yl)-8,8-dimethyl-4H,8H-pyrano[2,3-f]
chromen-4-one (8v). The corresponding heteroaryl boronic acid was (5-
fluoropyridin-3-yl)boronic acid. Yellow solid, yield, 61.2%; m.p.
169–170 °C; 1H NMR (CDCl3, 400 MHz) δ: 1.51 (s, 6H), 5.75 (d,
J = 10.0 Hz, 1H), 6.8 (d, J = 10.0 Hz, 1H), 6.89 (d, J = 8.8 Hz, 1H),
7.84–7.87 (s, 1H), 8.05 (d, J = 8.8 Hz, 1H), 8.07 (s, 1H), 8.48 (d,
J = 2.8 Hz, 1H), 8.52 (s, 1H). 13C NMR (CDCl3, 100 MHz) δ: 28.3, 78.1,
109.5, 114.8, 115.9, 118.1, 120.8, 124.1, 126.8, 129.7, 130.8, 137.7,
144.4, 152.5, 152.7, 158.0, 160.5, 175.1. HR-TOF-MS (positive mode):
m/z calcd. C19H15FNO3 [M+H]+ 324.1036, found 324.1037. HPLC
purity: 99.1%.
4.1.7.4. 2-(8,8-Dimethyl-4-oxo-4H,8H-pyrano[2,3-f]chromen-3-yl)phenyl
ethyl(methyl)carbamate (9d). The corresponding phenol was 8d, and the
corresponding carbamoyl chloride was N-ethyl-N-methylcarbamoyl
chloride. Yellow solid, yield, 56.3%; m.p. 65–67 °C; 1H NMR (CDCl3,
600 MHz) δ: 0.88–0.92 (m, 3H), 1.50 (s, 6H), 2.78 (d, J = 16.8 Hz, 3H),
3.16–3.24 (m, 2H), 5.65 (d, J = 10.0 Hz, 1H), 6.73 (d, J = 10.0 Hz, 1H),
6.78 (d, J = 8.8 Hz, 1H), 7.15–7.20 (m, 2H), 7.25 (d, J = 7.6 Hz, 1H),
7.31–7.35 (m, 1H), 7.83 (s, 1H), 7.96 (d, J = 8.8 Hz, 1H). 13C NMR (CDCl3,
100 MHz) δ: 12.6, 28.3, 34.0, 44.0, 77.8, 109.4, 115.0, 115.3, 118.3, 122.5,
4.1.6.2. 3-(5-Methoxypyridin-3-yl)-8,8-dimethyl-4H,8H-pyrano[2,3-f]
chromen-4-one (8w). The corresponding heteroaryl boronic acid was
(5-fluoropyridin-3-yl)boronic acid. Yellow solid, yield, 40.8%; mp
159–161 °C; 1H NMR (CDCl3, 400 MHz) δ: 1.50 (s, 6H), 3.90 (s, 3H),
5.74 (d, J = 10.0 Hz, 1H), 6.80 (d, J = 10.0 Hz, 1H), 6.88 (d,
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BioorganicChemistry88(2019)102949
123.1, 125.4, 125.5, 126.6, 129.7, 130.5, 131.5, 150.0, 152.5, 153.5,
154.4, 157.4, 175.1. HR-TOF-MS (positive mode): m/z calcd. C24H23NO5Na
[M+Na]+ 428.1474, found 428.1472. HPLC purity: 98.2%.
(CDCl3, 100 MHz) δ: 14.2, 22.7, 26.9, 28.3, 29.1, 30.0, 31.9, 41.4, 77.9,
109.3, 115.0, 115.5, 118.4, 121.5, 122.4, 124.4, 125.9, 126.8, 129.4,
130.5, 133.3, 151.2, 152.4, 152.7, 154.7, 157.5, 175.5. HR-TOF-MS
(positive mode): m/z calcd. C28H32NO5 [M+H]+ 462.2280, found
462.2275. HPLC purity: 96.8%.
4.1.7.5. 3-(8,8-Dimethyl-4-oxo-4H,8H-pyrano[2,3-f]chromen-3-yl)phenyl
ethyl(methyl)carbamate (9e). The corresponding phenol was 8e, and the
corresponding carbamoyl chloride was N-ethyl-N-methylcarbamoyl
chloride. White solid, yield, 30.76%; m.p. 178–180 °C; 1H NMR
(CDCl3, 400 MHz) δ: 1.17–1.26 (m, 3H), 1.50 (s, 6H), 3.03 (d,
J = 34.4 Hz, 3H), 3.38–3.51 (m, 2H), 5.72 (d, J = 10.0 Hz, 1H), 6.8
(d, J = 10.0 Hz, 1H), 6.86 (d, J = 8.8 Hz, 1H), 7.14 (d, J = 8.0 Hz, 1H),
7.35–7.41 (m, 3H), 8.00 (s, 1H), 8.05 (d, J = 8.8 Hz, 1H). 13C NMR
(CDCl3, 100 MHz) δ: 13.0, 28.3, 34.2, 44.2, 77.9, 109.3, 115.0, 115.5,
118.4, 121.7, 122.7, 124.5, 125.7, 126.9, 129.4, 130.5, 133.3, 151.6,
152.5, 152.8, 154.6, 157.6, 175.7. HR-TOF-MS (positive mode): m/z
calcd. C24H23NO5Na [M+Na]+ 428.1474, found 428.1474. HPLC
purity: 99.3%.
4.1.9.3. 4-(8,8-Dimethyl-4-oxo-4H,8H-pyrano[2,3-f]chromen-3-yl)phenyl
heptylcarbamate (9i). The corresponding phenol was 8f, and the
corresponding isocyanate was 1-isocyanatoheptane. White solid,
yield, 70.4%; m.p. 140–142 °C; 1H NMR (CDCl3, 400 MHz) δ: 0.82 (t,
J = 6.8 Hz, 3H), 1.18–1.29 (m, 8H), 1.43 (s, 6H), 1.47–1.54 (m, 2H),
3.2 (q, J = 6.8 Hz, 2H), 4.99 (s, 1H), 5.66 (d, J = 10.0 Hz, 1H), 6.74 (d,
J = 10.0 Hz, 1H), 6.8 (d, J = 8.8 Hz, 1H), 7.12 (d, J = 8.8 Hz, 2H), 7.48
(d, J = 8.8 Hz, 2H),7.88 (s, 1H), 7.99 (d, J = 8.8 Hz, 1H). 13C NMR
(CDCl3, 100 MHz) δ: 14.2, 22.7, 26.9, 28.3, 29.1, 30.0, 31.9, 41.4, 77.9,
109.4, 115.0, 115.4, 118.5, 121.8, 124.6, 126.8, 129.0, 130.1, 130.5,
151.2, 152.4, 152.5, 154.6, 157.5, 175.8. HR-TOF-MS (positive mode):
m/z calcd. C28H32NO5 [M+H]+ 462.2280, found 462.2277. HPLC
purity: 99.5%.
4.1.8. 4-(8,8-Dimethyl-4-oxo-4H,8H-pyrano[2,3-f]chromen-3-yl)phenyl
ethyl(methyl)carbamate (9f)
64.0 mg (0.2 mmol) of 8f and potassium carbonate (83.0 mg,
0.32 mmol) were stirred in acetonitrile (3 mL). N-ethyl-N-methylcar-
bamoyl Chloride (29.0 mg, 26 μL) was added dropwise. After 12 h
stirring at room temperature, The reaction mixture was concentrated in
vacuo. The residue was purified by flash chromatography to provide a
white solid 9f (27 mg, 33.7%); m.p. 206–207 °C; 1H NMR (CDCl3,
400 MHz) δ: 1.17–1.26 (m, 3H), 1.49 (s, 6H), 3.04 (d, J = 2.4 Hz, 3H),
3.39–3.51 (m, 2H), 5.72 (d, J = 10.0 Hz, 1H), 6.8 (d, J = 10.0 Hz, 1H),
6.86 (d, J = 8.8 Hz, 1H), 7.17 (dd, J = 2.8, 8.0 Hz, 2H), 7.54 (d,
J = 8.4 Hz, 2H), 7.95 (s, 1H), 8.05 (d, J = 8.8 Hz, 1H). 13C NMR
(CDCl3, 100 MHz) δ: 13.0, 28.2, 34.2, 44.2, 77.9, 109.3, 115.0, 115.4,
118.4, 121.9, 124.5, 126.8, 128.9, 130.0, 130.4, 151.6, 152.4, 152.4,
154.5, 157.4, 175.8. HR-TOF-MS (positive mode): m/z calcd.
4.1.9.4. 2-(8,8-Dimethyl-4-oxo-4H,8H-pyrano[2,3-f]chromen-3-yl)phenyl
phenethylcarbamate (9j). The corresponding phenol was 8d, and the
corresponding isocyanate was (2-isocyanatoethyl)benzene. White solid,
yield, 60%; m.p. 123–125 °C; 1H NMR (CDCl3, 400 MHz) δ: 1.50 (s, 6H),
2.72 (t, J = 6.4 Hz, 2H), 3.41 (q, J = 6.4 Hz, 2H), 5.06 (s, 1H), 5.73 (d,
J = 10.0 Hz, 1H), 6.82 (d, J = 10.0 Hz, 1H), 6.88 (d, J = 8.4 Hz, 1H),
7.07 (d, J = 7.2 Hz, 2H), 7.16–7.22 (m, 4H), 7.26–7.34 (m, 2H), 7.41 (t,
J = 7.2 Hz, 1H), 7.91 (m, 1H), 8.04 (d, J = 8.4 Hz, 1H). 13C NMR
(CDCl3, 100 MHz) δ: 28.3, 36.1, 42.5, 77.9, 109.4, 115.0, 115.4, 118.3,
122.4, 123.1, 125.5, 125.8, 126.6, 126.8, 128.7, 128.8, 129.8, 130.6,
131.8, 138.6, 149.6, 152.6, 153.6, 154.6, 157.5, 175.3. HR-TOF-MS
(positive mode): m/z calcd. C29H26NO5 [M+H]+ 468.1811, found
468.1804. HPLC purity: 99.8%.
C
24H23NO5Na [M+Na]+ 428.1474, found 428.1476. HPLC purity:
99.5%.
4.1.9.5. 3-(8,8-Dimethyl-4-oxo-4H,8H-pyrano[2,3-f]chromen-3-yl)phenyl
phenethylcarbamate (9k). The corresponding phenol was 8e, and the
corresponding isocyanate was (2-isocyanatoethyl)benzene. White solid,
yield, 82%; m.p. 145–147 °C; 1H NMR (CDCl3, 400 MHz) δ: 1.51 (s, 6H),
2.89 (t, J = 6.8 Hz, 2H), 3.53 (q, J = 6.4 Hz, 2H), 5.11 (s, 1H), 5.73 (d,
J = 10.0 Hz, 2H), 6.81 (d, J = 10.0 Hz, 1H), 6.87 (d, J = 8.8 Hz, 1H),
7.13 (d, J = 6.4 Hz, 1H), 7.23–7.27 (m, 3H), 7.32–7.48 (m, 5H), 7.99
(s, 1H), 8.06 (d, J = 8.4 Hz, 1H). 13C NMR (CDCl3, 100 MHz) δ: 28.3,
36.1, 42.5, 77.9, 109.3, 115.0, 115.5, 118.4, 121.4, 122.4, 124.4,
125.9, 126.8, 126.9, 128.9, 129.0, 129.4, 130.5, 133.3, 138.7, 151.1,
152.4, 152.7, 154.6, 157.5, 175.5. HR-TOF-MS (positive mode): m/z
calcd. C29H26NO5 [M+H]+ 468.1811, found 468.1808. HPLC purity:
99.9%.
4.1.9. General procedure for the preparation of compounds 9g-9n
To a solution of phenol (0.2 mmol) in CH2Cl2 (3 mL), triethylamine
(31 μL, 0.22 mmol) was added at room temperature. After 15 min stir-
ring at room temperature, isocyanate (0.22 mmol) was added. The
mixture was stirred at room temperature for 6 h and concentrated in
vacuo. The resulting residue was purified by flash chromatography to
provide 9g-9n.
4.1.9.1. 2-(8,8-Dimethyl-4-oxo-4H,8H-pyrano[2,3-f]chromen-3-yl)phenyl
heptylcarbamate (9g). The corresponding phenol was 8d, and the
corresponding isocyanate was 1-isocyanatoheptane. White solid,
yield, 56%; m.p. 89–91 °C; 1H NMR (CDCl3, 400 MHz) δ: 0.78 (t,
J = 6.8 Hz, 3H), 1.11–1.31 (m, 10H), 1.43 (s, 6H), 3.03 (q, J = 6.8 Hz,
2H), 5.02 (t, J = 5.2 Hz, 1H), 5.65 (d, J = 10.0 Hz, 1H), 6.72 (d,
J = 10.0 Hz, 1H), 6.78 (d, J = 8.8 Hz, 1H), 7.14–7.20 (m, 2H), 7.24
(dd, J = 1.6, 7.2 Hz, 1H), 7.30–7.35 (m, 2H), 7.83 (s, 1H), 7.95 (d,
J = 8.8 Hz, 1H). 13C NMR (CDCl3, 100 MHz) δ: 14.2, 22.7, 26.6, 28.3,
28.9, 29.7, 31.8, 41.3, 77.9, 109.4, 115.0, 115.4, 118.2, 122.3, 123.0,
125.6, 125.7, 126.7, 129.8, 130.4, 131.7, 149.7, 152.5, 153.6, 154.7,
157.5, 175.3. HR-TOF-MS (positive mode): m/z calcd. C28H32NO5 [M
+H]+ 462.2280, found 462.2274. HPLC purity: 99.1%.
4.1.9.6. 4-(8,8-Dimethyl-4-oxo-4H,8H-pyrano[2,3-f]chromen-3-yl)phenyl
phenethylcarbamate (9l). The corresponding phenol was 8f, and the
corresponding isocyanate was (2-isocyanatoethyl)benzene. White solid,
yield, 83%; m.p. 174–176 °C; 1H NMR (CDCl3, 400 MHz) δ: 1.42 (s, 6H),
2.82 (t, J = 6.8 Hz, 2H), 3.47 (q, J = 6.8 Hz, 2H), 5.06 (t, J = 5.6 Hz,
1H), 5.65 (d, J = 10.0 Hz, 1H), 6.73 (d, J = 10.0 Hz, 1H), 6.79 (d,
J = 8.8 Hz, 1H), 7.09 (d, J = 8.8 Hz, 2H), 7.15–7.19 (m, 3H), 7.26 (t,
J = 7.2 Hz, 2H), 7.46 (d, J = 8.8 Hz, 2H), 7.87 (s, 1H), 7.98 (d,
J = 8.8 Hz, 1H). 13C NMR (CDCl3, 100 MHz) δ: 28.3, 36.1, 42.5, 77.9,
109.4, 115.0, 115.5, 118.5, 121.8, 124.6, 126.8, 126.9, 128.9, 129.0,
129.1, 130.1, 130.5, 138.7, 151.1, 152.4, 152.5, 154.6, 157.5, 175.8.
4.1.9.2. 3-(8,8-Dimethyl-4-oxo-4H,8H-pyrano[2,3-f]chromen-3-yl)phenyl
heptylcarbamate (9h). The corresponding phenol was 8e, and the
corresponding isocyanate was 1-isocyanatoheptane. White solid,
yield, 65%; m. p. 107–109 °C; 1H NMR (CDCl3, 400 MHz) δ: 0.89 (t,
J = 6.4 Hz, 3H), 1.29–1.33 (m, 8H), 1.50–1.58 (m, 8H), 3.25 (q,
J = 6.4 Hz, 2H), 5.01 (s, 1H), 5.72 (d, J = 10.0 Hz, 1H), 6.8 (d,
J = 10.0 Hz, 1H), 6.86 (d, J = 8.8 Hz, 1H), 7.14–7.15 (m, 1H),
7.36–7.42 (m, 3H), 7.98 (s, 1H), 8.06 (d, J = 8.8 Hz, 1H). 13C NMR
HR-TOF-MS (positive mode): m/z calcd.
C
29H26NO5 [M+H]+
468.1811, found 468.1804. HPLC purity: 99.6%.
4.1.9.7. 3-(8,8-Dimethyl-4-oxo-4H,8H-pyrano[2,3-f]chromen-3-yl)phenyl
(4-isopropylphenyl)carbamate (9m). The corresponding phenol was 8e,
and
the
corresponding
isocyanate
was
1-isocyanato-4-
isopropylbenzene. White solid, yield, 51%; m.p. 184–186 °C; 1H NMR
11

C. Wu, et al.
BioorganicChemistry88(2019)102949
(DMSO‑d6, 400 MHz) δ: 1.24 (d, J = 6.8 Hz, 6H), 1.51 (s, 6H),
2.83–2.93 (m, 1H), 5.73 (d, J = 10.0 Hz, 1H), 6.81 (d, J = 10.0 Hz,
1H), 6.87 (d, J = 8.8 Hz, 1H), 6.99 (s, 1H), 7.18–7.20 (m, 3H),
7.36–7.43 (m, 5H), 8.0 (d, J = 2.8 Hz, 1H), 8.07 (d, J = 8.8 Hz, 1H).
13C NMR (CDCl3, 100 MHz) δ: 24.0, 27.7, 32.8, 77.9, 109.0, 114.1,
115.1, 117.8, 118.6, 121.5, 122.3, 122.7, 125.8, 126.1, 126.6, 129.1,
131.5, 133.2, 136.3, 143.1, 150.3, 151.6, 151.7, 154.3, 156.7, 174.3.
J = 2.0, 8.4 Hz, 1H), 7.18–7.21 (m, 2H), 7.33 (d, J = 2.4 Hz, 1H),7.99
(s, 1H), 8.29 (d, J = 8.8 Hz, 1H). 13C NMR (CDCl3, 100 MHz) δ: 36.7,
37.0, 56.1, 77.6, 110.8, 111.3, 112.5, 119.7, 121.2, 121.8, 124.5,
125.2, 127.6, 148.9, 149.3, 152.9, 153.8, 155.6, 156.8, 176.0. HR-TOF-
MS (positive mode): m/z calcd C20H20NO6 [M+H]+ 370.1291, found
370.1289. HPLC purity: 99.4%.
HR-TOF-MS (positive mode): m/z calcd.
C
30H28NO5 [M+H]+
4.1.10.2. 3-(3,4-Dimethoxyphenyl)-4-oxo-4H-chromen-7-yl ethyl(methyl)
carbamate (11b). The corresponding carbamoyl chloride was N-ethyl-
N-methylcarbamoyl chloride. White solid, yield over two steps, 56%;
m.p. 129–131 °C; 1H NMR (CDCl3, 400 MHz) δ: 1.20–1.29 (m, 3H), 3.02
(s, 3H), 3.10 (s, 3H), 3.41–3.53 (m, 2H), 3.91 (s, 3H), 3.93 (s, 3H), 6.93
(d, J = 8.0 Hz, 1H), 7.06 (dd, J = 2.0, 8.0 Hz, 1H), 7.18–7.21 (m, 2H),
482.1967, found 482.1969. HPLC purity: 98.7%.
4.1.9.8. 4-(8,8-Dimethyl-4-oxo-4H,8H-pyrano[2,3-f]chromen-3-yl)phenyl
(4-isopropylphenyl)carbamate (9n). The corresponding phenol was 8f,
and
the
corresponding
isocyanate
was
1-isocyanato-4-
isopropylbenzene. White solid, yield, 48%; m.p. 183–185 °C; 1H NMR
(CDCl3, 400 MHz) δ: 1.16 (d, J = 7.2 Hz, 6H), 1.43 (s, 6H), 2.77–2.84
(m, 1H), 5.65 (d, J = 10.0 Hz, 1H), 6.73 (d, J = 10.0 Hz, 1H), 6.79 (d,
J = 8.8 Hz, 1H), 7.10–7.18 (m, 5H), 7.31 (d, J = 7.6 Hz, 2H), 7.49 (d,
J = 7.6 Hz, 2H), 7.87 (s, 1H), 8.0 (d, J = 8.8 Hz, 1H). 13C NMR (CDCl3,
100 MHz) δ: 24.2, 28.3, 33.7, 77.9, 109.4, 115.0, 115.5, 118.4, 119.1,
121.8, 121.9, 124.5, 126.8, 127.1, 129.4, 130.2, 130.5, 135.2, 144.7,
150.7, 151.8, 152.5, 157.5, 175.9. HR-TOF-MS (positive mode): m/z
calcd. C30H28NO5 [M+H]+ 482.1967, found 482.1962. HPLC purity:
98.2%.
7.34 (dd, J = 2.4, 6 Hz, 1H), 8.00 (s, 1H), 8.29 (d, J = 8.8 Hz, 1H). 13
C
NMR (CDCl3, 100 MHz) δ: 13.0, 34.3, 44.4, 56.1, 110.8, 111.3, 112.5,
119.7, 121.2, 121.8, 124.5, 125.2, 127.6, 148.9, 149.3, 152.9, 153.4,
155.6, 156.8, 176.0. HR-TOF-MS (positive mode): m/z calcd.
C
21H22NO6 [M+H]+ 384.1447, found 384.1442. HPLC purity: 99.5%.
4.2. EeAChE and eqBChE inhibition assays
ChE inhibitory activities were determined according to the proce-
dure reported in our previous study [38].
4.1.9.9. 3-(8,8-Dimethyl-4-oxo-4H,8H-pyrano[2,3-f]chromen-3-yl)-2-
fluorophenyl heptylcarbamate (9o). The corresponding phenol was 8u,
and the corresponding isocyanate was 1-isocyanatoheptane. White
solid, yield, 74.7%; m.p. 113–115 °C; 1H NMR (CDCl3, 400 MHz) δ:
0.88 (t, J = 6.8 Hz, 3H), 1.27–1.34 (m, 8H), 1.50 (s, 6H), 1.53–1.59 (m,
2H), 3.26 (q, J = 6.4 Hz), 5.17 (s, 1H), 5.73 (d, J = 10.0 Hz, 1H), 6.81
(d, J = 10.0 Hz, 1H), 6.87 (d, J = 8.8 Hz, 1H), 7.15–7.24 (m, 2H),
7.31–7.35 (m, 1H), 8.02 (s, 1H), 8.05 (d, J = 8.4 Hz, 1H). 13C NMR
(CDCl3, 100 MHz) δ: 14.2, 22.7, 26.8, 28.3, 29.1, 29.9, 31.9, 41.6, 77.9,
109.4, 115.0, 115.5, 118.3, 119.4, 121.0, 123.8, 124.2, 126.8, 128.8,
130.5, 139.0, 151.3, 152.5, 153.8, 154.2, 157.6, 175.0. HR-TOF-MS
(positive mode): m/z calcd. C28H31FNO5 [M+H]+ 480.2186, found
480.2185. HPLC purity: 99.0%.
4.3. ADME prediction and molecular docking study
Seven compounds with potential BChE inhibitory activity
(IC50 < 2 μM) were further analyzed with QikProp v. 5.5
(Schrodinger) to predict their ADME properties.
The molecular docking program GOLD 5.0 was used. First, the
preprocessing of human butyrylcholinesterase (PDB code: 1P0I) and the
compounds was performed with Discovery Studio 2016 client software
because BChE from the equine serum has a 94% homology and 90%
sequence identity with BChE from the human serum [39]. The proce-
dure included removing H2O, adding hydrogens and assigning a
CHARMM-like force field. Subsequently, the binding site of the ligand
to the BChE protein was set as a sphere including the residues in the
active site of human butyrylcholinesterase and slight manual adjust-
ments were made. The “Number of dockings” parameter was set to 10
without using early termination option, and the “genetic algorithm
parameter” was set to “Automatic”. The “GoldScore” was selected to
evaluate the docking pose, and the settings for other parameters re-
mained default.
4.1.10. General procedure for the preparation of compounds 11a and 11b
Compound 5 (144 mg, 0.5 mmol) and potassium carbonate (111 mg,
0.8 mmol) were added into a mixture of dimethylformamide (1.5 mL)
and acetonitrile (0.6 mL). Then, carbamoyl chloride (0.68 mmol) in
acetonitrile (0.3 mL) was added dropwise. The reaction mixture was
refluxed at 95 °C for 3 h. After cooling to room temperature, the reac-
tion mixture was neutralized with hydrochloric acid. Excess reducing
agent was quenched by careful addition of water (20 mL), the mixture
was extracted three times with ethyl acetate. The organic layer was
washed three times with saturated salt solution, dried with Na2SO4,
filtered, and then concentrated. The residue was washed with 5 mL
methanol and filtered to give the crude compounds 10a and 10b. The
crude was used in the next step without further purification.
4.4. Kinetic studies
The kinetic studies were carried out under the same test conditions.
Five concentrations of the substrate (from 0.05 to 0.8 mM) were se-
lected for the test in combination with four concentrations of the in-
hibitors (0 ∼ 0.4 μM and 0 ∼ 0.15 μM for 9g and 9h, respectively). The
kinetic parameters and apparent inhibition constants were calculated
by the “Enzyme kinetics” module of Prism.
Na2CO3 (95 mg, 0.9 mmol), (3,4-dimethoxyphenyl) boronic acid
(0.36 mmol), and Pd/C (16 mg, 5 mol %) were added to a solution of
the crude compounds 10a/10b (0.3 mmol) in DME (1.8 mL) and H2O
(1.8 mL). The resulting mixture was stirred for 1 h at 45 °C and filtered.
The cake was successively washed with H2O (4 mL) and CH2Cl2 (6 mL).
Then, the aqueous phase was extracted twice with CH2Cl2. After drying
with anhydrous Na2SO4, the CH2Cl2 layer was filtered and concentrated
in vacuo. The residue was purified by flash chromatography to give 11a
and 11b.
4.5. Cytotoxicity assays
The cell viability of PC12 cells treated by 8, 9g, 9h and tacrine was
determined by the MTT cell proliferation assay. In brief, PC12 cells
(4–5 × 103) were added into 96-well plates and incubated for 24 h.
Subsequently, the cells were treated with various concentrations of the
compounds for 48 h. MTT solution (10 μL) was added into every well at
the concentration of 10 mg/mL. Then, the plate was incubated for an-
other 2.5 h at 37 °C. The supernatant was aspirated from each well, and
150 μL of dimethyl sulfoxide was added per well for 15–20 min. The
absorption was measured at 570 nm with a Spectra MAX M5 microplate
spectrophotometer (Molecular Devices).
4.1.10.1. 3-(3,4-Dimethoxyphenyl)-4-oxo-4H-chromen-7-yl
dimethylcarbamate (11a). The corresponding carbamoyl chloride was
N,N-dimethylcarbamoyl chloride. White solid, yield over two steps,
51%; m.p. 164–166 °C; 1H NMR (CDCl3, 400 MHz) δ: 3.05 (s, 3H), 3.14
(s, 3H), 3.91 (s, 3H), 3.93 (s, 3H), 6.93 (d, J = 8.0 Hz, 1H), 7.05 (dd,
12

C. Wu, et al.
BioorganicChemistry88(2019)102949
Acknowledgments
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We thank Assoc. Prof. HaoYu YE (State Key Laboratory of
Biotherapy, West China Hospital, West China Medical School, Sichuan
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13