Synthesis and acaricidal activities of scopoletin phenolic ether derivatives: Qsar, molecular docking study and in silico Adme predictions

Article abstract of DOI:10.3390/molecules23050995

Thirty phenolic ether derivatives of scopoletin modified at the 7-hydroxy position were synthesized, and their structures were confirmed by IR,¹H-NMR,¹³C-NMR, MS and elemental analysis. Preliminary acaricidal activities of these compounds against female adults of Tetranychus cinnabarinus (Boisduval) were evaluated using the slide-dip method. The results indicated that some of these compounds exhibit more pronounced acaricidal activity than scopoletin, especially compounds 32, 20, 28, 27 and 8 which exhibited about 8.41-, 7.32-, 7.23-, 6.76-, and 6.65-fold higher acaricidal potency. Compound 32 possessed the the most promising acaricidal activity and exhibited about 1.45-fold higher acaricidal potency against T. cinnabarinus than propargite. Statistically significant 2D-QSAR model supports the observed acaricidal activities and reveals that polarizability (HATS5p) was the most important parameter controlling bioactivity. 3D-QSAR (CoMFA: q² = 0.802, r² = 0.993; CoMSIA: q² = 0.735, r² = 0.965) results show that bulky substituents at R₄, R₁, R₂ and R₅ (C₆, C₃, C₄, and C₇) positions, electron positive groups at R₅ (C₇) position, hydrophobic groups at R₁ (C₃) and R₂ (C₄), H-bond donors groups at R₁ (C₃) and R₄ (C₆) will increase their acaricidal activity, which provide a good insight into the molecular features relevant to the acaricidal activity for further designing novel acaricidal agents. Molecular docking demonstrates that these selected derivatives display different bide modes with TcPMCA1 from lead compound and they interact with more key amino acid residues than scopoletin. In silico ADME properties of scopoletin and its phenolic ether derivatives were also analyzed and showed potential to develop as good acaricidal candidates.

Full text of DOI:10.3390/molecules23050995

molecules
Article
Synthesis and Acaricidal Activities of Scopoletin
Phenolic Ether Derivatives: QSAR, Molecular
Docking Study and in Silico ADME Predictions
Jinxiang Luo † ID , Ting Lai †, Tao Guo, Fei Chen, Linli Zhang, Wei Ding and Yongqiang Zhang *
ID
College of Plant Protection, Southwest University, Chongqing 400715, China;
xiangxiangnx@163.com (J.L.); laiting93@163.com (T. L.); 13994888326.guotao@163.com (T.G.);
cf759974605@126.com (F.C.); zhll87_9@163.com (L. Z.); dwing818@163.com (W.D.)
* Correspondence: zyqiang@swu.edu.cn; Tel./Fax: +86-023-6825-0218
† These two authors contributed equally to this work.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 3 April 2018; Accepted: 18 April 2018; Published: 24 April 2018
Abstract: Thirty phenolic ether derivatives of scopoletin modified at the 7-hydroxy position
were synthesized, and their structures were confirmed by IR, 1H-NMR, 13C-NMR, MS and
elemental analysis. Preliminary acaricidal activities of these compounds against female adults
of Tetranychus cinnabarinus (Boisduval) were evaluated using the slide-dip method. The results
indicated that some of these compounds exhibit more pronounced acaricidal activity than scopoletin,
especially compounds 32, 20, 28, 27 and 8 which exhibited about 8.41-, 7.32-, 7.23-, 6.76-, and 6.65-fold
higher acaricidal potency. Compound 32 possessed the the most promising acaricidal activity and
exhibited about 1.45-fold higher acaricidal potency against T. cinnabarinus than propargite. Statistically
significant 2D-QSAR model supports the observed acaricidal activities and reveals that polarizability
(HATS5p) was the most important parameter controlling bioactivity. 3D-QSAR (CoMFA: q2 = 0.802,
r2 = 0.993; CoMSIA: q2 = 0.735, r2 = 0.965) results show that bulky substituents at R4, R1, R2 and R5
(C6, C3, C4, and C7) positions, electron positive groups at R5 (C7) position, hydrophobic groups at R1
(C3) and R2 (C4), H-bond donors groups at R1 (C3) and R4 (C6) will increase their acaricidal activity,
which provide a good insight into the molecular features relevant to the acaricidal activity for further
designing novel acaricidal agents. Molecular docking demonstrates that these selected derivatives
display different bide modes with TcPMCA1 from lead compound and they interact with more key
amino acid residues than scopoletin. In silico ADME properties of scopoletin and its phenolic ether
derivatives were also analyzed and showed potential to develop as good acaricidal candidates.
Keywords: scopoletin; acaricidal activity; QSAR; molecular docking; ADME properties
1. Introduction
The carmine spider mite, Tetranychus cinnabarinus (Boisduval), is considered as one of the most
economically important arthropod pests [
grown in the field or greenhouse worldwide, especially cotton, beans, eggplants, tomatoes, peppers,
cucurbits and strawberries and so on [ ]. Spider mites usually feed through a piercing-sucking
process to remove cellular contents, resulting in reduction of photosynthesis and transpiration rates
in plants [ ]. The plants slightly infested by spider mite display discoloration of their leaves and
defoliation, bud and fruit dropping and reductions in fruit yield and quality; serious plants infestations
by this mite will cause whole plant death [ ]. It is recognized as one of the most difficult mites to
control mainly due to its small size, high reproductive potential, extremely short life cycle, and strong
1]. This mite has been reported to infest over 100 crops or plants
2–4
5,6
7
adaptability and ability to develop resistance [
8
,9]. The genetic system of spider mites is known as
Molecules 2018, 23, 995; doi:10.3390/molecules23050995
www.mdpi.com/journal/molecules

Molecules 2018, 23, 995
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arrhenotoky, wherein unfertilized haploid eggs develop into males and fertilized eggs develop into
females [ ]. This type of genetic system is highly vulnerable to mutations, conferring resistance to
6
acaricides [10]. In addition, for a long time, this pest mite was controlled mainly through frequent
applications of synthetic chemical acaricides, which have resulted in mite resistance to almost all major
classes of acaricides throughout the world as well as environmental problems [11–15]. Therefore, it is
necessary to develop novel, powerful, and environmentally-friendly acaricides from natural products,
which will be used as an alternative agent to control this pest mite.
Scopoletin is an important secondary metabolite found in many plant species, such as
Erycibe obtusifolia Benth [16], Aster tataricus [17], Foeniculum vulgare [18], Artemisia annua L. [19],
Sinomonium acutum [20], and Melia azedarach L. fruits [21]. Scopoletin is classified as a coumarin
and chemically known as 7-hydroxy-6-methoxy-2H-chromen-2-one [22]. Studies have shown that
scopoletin has a wide spectrum of biological activities, such as pronounced acaricidal [23
,24],
anti-inflammatory [25 26], antitumoral [27], antioxidative [20], hepatoprotective [28], insecticidal [29],
,
antifungal [30], and alleopathic properties [31]. Based on its pronounced acaricidal activities, our
research group further investigated the mechanism of action and found that Ca2+-ATPase, which is
vital in nervous signal conduction [32–
34], was inhibited [35] and TcPMCA1 from Ca2+-ATPase was
significantly upregulated after T. cinnabarlnus was exposed to scopoletin, and molecular docking also
showed that scopoletin inserts into the binding cavity and interacts with TcPMCA1 protein through
the driving forces of hydrogen bonds [36]. However, its acaricidal activity remain lower than that of
some registered synthetic chemical acaricides, such as pyridaben. To date, few studies have attempted
to improve the acaricidal effects of scopoletin by modifying its structure.
Quantitative structure-activity relationship (QSAR) and molecular docking are two important
computational approaches, which have been considered as effective facilitating tools in drug design
and discovery [37
,38]. Quantitative structure-activity relationship (QSAR) is a method that correlates
chemical structure of the compound with its biological activity [39
–41]. QSAR has also been widely
used to provide useful information for the design and discovery of insecticidal and acaricidal
agents [42,43]. Molecular docking is a computational method to identify targets or find possible
binding modes of the compound against its biological target, and has been successfully used to
investigate binding modes of many classes of pesticides [44,45].
Therefore, our interest now focused on the modification of scopoletin to increase its acaricidal
potency by using a molecular hybridization method. A series of scopoletin phenolic ether derivatives
1
were designed and synthesized. All the target compounds were characterized by IR, H-NMR,
13C-NMR, MS, and elemental analysis, and their acaricidal activities against female adults of
T. cinnabarlnus were evaluated. QSAR and molecular docking were also performed to provide useful
structure-activity relationship information for the discovery of novel acaricidal agents and insights
into the important interaction of compounds and TcPMCA1. An in silico study of scopoletin and its
synthetic phenolic ether derivatives was performed to predict their ADME properties.
2. Results and Discussion
2.1. Chemistry
The synthesis of scopoletin (
(scopoletin) followed literature methods [22
7-hydroxy-6-methoxy-2-oxo-2H-chromene-3-carboxylic acid (
and heating under microwave irradiation for 50 min to afford scopoletin (4).
The synthesis of target compounds 37 is outlined in Scheme 2. Target compounds 8–25 were
4
) is outlined in Scheme 1. The first two reaction steps of synthesizing
46], and the last step was altered by adding the
) to pyridine and ethylene glycol (1:1.1)
4
,
3
8–
obtained from scopoletin through a one-step reaction with alkyl or aromatic halides. To increase the
electronegativity of the oxygen atom of the hydroxyl from scopoletin, we first added scopoletin and
K2CO3 to acetone and stirred at reflux; subsequently alkyl or aromatic halides were added into the
mixed reaction solution to react, leading to the acceptable yields of the derivatives.

Molecules 2018, 23, 995
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Target compounds 26–37 were obtained through a three-step reaction. The intermediates 6a–l
were synthesized through the reaction of 2-chloroacetyl chloride with an alkylamine or substituted
benzylamine. To quickly remove the by-product hydrochloric acid from this reaction, we used
triethylamine as acid-binding agent, which was added before adding 2-chloroacetyl chloride into the
reaction mixture. Chlorinated intermediates 6a
–
l
were converted into iodine-substituted intermediates
(Scheme 3) to obtain high yields of the target compounds. Finally, the target compounds
37 were synthesized by reacting scopoletin with intermediates 7a in acetone. All of the target
7a
26
–
–
l
–
l
compounds provided satisfactory analytical and spectroscopic data, which were consistent with their
depicted structures.
OCH3
HOOC
O
OCH3
OH
H3CO
H3CO
CHO
H3CO
HO
CHO
OH
c
b
a
O
O
OH
O
OCH3
1
2
4 (Scopoletin)
3
Reagents and conditions: (a) anhydrous AlCl3, CTAB, CH2Cl2, reflux, 4 h; (b) malonic acid, pyridine,
phenylamine, rt, 24 h; (c) pyridine: ethylene glycol (1:1.1), microwave-assistance, 50 mins.
Scheme 1. Synthesis of scopoletin.
OCH3
RX
a
O
O
O R
8-25
OCH3
OH
O
O
O
I
OCH3
O
RHN
4(Scopoletin)
7a-l
NHR
O
O
a
O
26-37
Reagents and conditions: (a) K2CO3, CTAB, acetone, reflux, 6 h–24 h.
Scheme 2. Synthesis of scopoletin phenolic ether derivatives (8–37).
O
O
b
a
RNH2
5a-l
Cl
I
RHN
RHN
6a-l
7a-l
Reagents and conditions: (a) ClCH2COCl, triethylamine, CH2Cl2, 0 °C, then rt,0.5 h; (b) KI, acetone,
reflux, 2 h.
Scheme 3. Synthesis of iodoacetamide derivatives.
2.2. Acaricidal Activity
As shown in Table 1, all the tested compounds exhibited varying degrees of acaricidal potency
against female adults of T. cinnabarinus after treatment for 48 h, LC50 (mmol/L) and χ
2 values of all
tested compounds are less than 6.6 and 5.6, respectively, pLC50 (mol/L) and P values of all tested
compounds are more than 2.0 and 0.1, respectively. Except for compound 15, the rest of the target
compounds exhibited more pronounced acaricidal activities against T. cinnabarinus than scopoletin.
In particular, compounds 32, 20, 28, 27 and 8 exhibited about 8.41-, 7.32-, 7.23-, 6.76-, and 6.65-fold
higher acaricidal potency than the lead compound. Compound 32 possessed the the most promising

Molecules 2018, 23, 995
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acaricidal activity and exhibited about 1.45-fold higher acaricidal potency against T. cinnabarinus
than propargite.
Table 1. Contact activity of scopoletin and its phenolic ether derivatives (
T. cinnabarinus (48 h).
8–37) against female adults of
Compounds
R
LC50 (mmol/L)
pLC50 (mol/L)
χ2
P
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
methyl
ethyl
n-propyl
isopropyl
n-butyl
0.829 ± 0.169
0.840 ± 0.184
2.326 ± 0.522
1.767 ± 0.254
1.253 ± 0.292
3.021 ± 0.410
2.875 ± 0.579
6.588 ± 0.929
2.464 ± 0.549
3.586 ± 0.743
2.557 ± 0.363
3.196 ± 0.808
0.753 ± 0.105
1.686 ± 0.412
1.652 ± 0.222
4.208 ± 0.872
1.139 ± 0.218
2.853 ± 0.560
2.541 ± 0.467
0.815 ± 0.230
0.762 ± 0.0937
3.045 ± 0.799
1.015 ± 0.262
1.134 ± 0.1149
0.655 ± 0.2539
0.848 ± 0.1359
0.963 ± 0.2437
1.774 ± 0.3783
1.516 ± 0.2618
3.601 ± 0.4786
5.510 ± 1.553
0.953 ± 0.1093
3.0813
3.0757
2.6333
2.7527
2.9022
2.5199
2.5414
2.1812
2.6083
2.4454
2.5922
2.4954
3.1229
2.7730
2.7821
2.3759
2.9435
2.5447
2.5950
3.0888
3.1180
2.5165
2.9935
2.9452
3.1835
3.0716
3. 0165
2.7510
2.8193
2.4436
2.2588
3.0209
1.678
4.958
2.125
1.187
4.875
1.633
1.929
0.114
1.008
2.495
3.133
3.418
3.475
1.929
2.889
0.497
5.571
0.567
3.990
5.424
3.764
2.416
1.689
2.428
2.280
2.642
1.208
1.330
0.062
3.129
0.416
4.034
0.642
0.175
0.547
0.756
0.181
0.652
0.587
0.990
0.799
0.476
0.372
0.332
0.324
0.587
0.409
0.919
0.134
0.904
0.263
0.143
0.288
0.491
0.639
0.297
0.516
0.45
isobutyl
methyl cyclopropane
cyclopentyl
cyclohexyl
methyl cyclohexane
benzyl
4-CH3- benzyl
4-C(CH3)-benzyl
4-NO2-benzyl
3,4-2Cl-benzyl
4-Cl-benzyl
4-CF3-benzyl
4-OCF3-benzyl
methyl
ethyl
n-propyl
isopropyl
n-butyl
benzyl
3-Cl-benzyl
4-Cl-benzyl
3,4-2Cl-benzyl
4-CH3-benzyl
4-OCH3-benzyl
4-C(CH3)-benzyl
—
0.598
0.722
0.996
0.209
0.937
0.258
37
Scopoletin
Propargite
—
The acaricidal activity of compounds
groups increased. Compounds 14 17 with a naphthenic base displayed lower acaricidal activity.
When the hydrogen of a hydroxyl or amino from compounds 18 25 and 32 37 was substituted by
8–13 decreased as the carbon chain length of the substituent
–
–
–
a substituted phenyl group, different acaricidal potency was shown. This is due to the types, quantity
and position of substituents on the benzene rings of these compounds. However, all of them well
followed the Topliss tree rule [47]. Compound 23 with a para-chlorinated phenyl (pLC50 = 2.3759
exhibited low potency against T. cinnabarinus compared with compound 18 with a simple phenyl
pLC50 = 2.5922), therefore, both compound 19 with para-methylphenyl (pLC50 = 2.4954) and compound
)
(
22 with 3,4 dichlophenyl (pLC50 = 2.7821) don’t show excellent potency against T. cinnabarinus.
Compound 33 with a para-chlorinated phenyl containing an amide group (pLC50 = 3.0716) exhibited
equivalent potency against T. cinnabarinus compared with compound 31 with a phenyl-containing
amide group (pLC50 = 2.9452), compound 35 with a para-methylphenyl-containing amide group
exhibited low potency against T. cinnabarinus (pLC50 = 2.7510) compared with compound 33, therefore,
the compound 32 with a 3-chlorophenyl-containing amide group (pLC50 = 3.1835) show higher acaricidal
potency. Both compound 34 with a 3,4-dichlorophenyl-containing amide group (pLC50 = 3.0165) and

Molecules 2018, 23, 995
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compound 36 with a para-methoxyphenyl-containing amide group (pLC50 = 2.8193) don’t show good
potency against T. cinnabarinus.
The amino hydrogens of compounds 26–31 were substituted by alkyl groups, which also followed
the Topliss tree rule [47]. Compound 29 with isopropyl (pLC50 = 2.5165) show equivalent potency
against T. cinnabarinus compared with compound 26 with methyl (pLC50 = 2.5950), and compound 27
with an ethyl moiety shows prominent (pLC50 = 3.0888) acaricidal potency.
In addition, the acaricidal activity of the compounds 32–34 containing amide groups with benzene
rings substituted by electron-withdrawing groups (3-chloro-, 4-chloro-, and 3,4-dichloro-) was higher
than that of benzene rings substituted by electron-donating groups (4-methyl-, 4-methoxy- and
4-tert-butyl-) (compounds 35–37).
The 48 h LC50 value of scopoletin in the current study was different from our previous reports [23],
which may be attributed to the differences in scopoletin purity, the pesticide adjuvants, and the solvents
used to prepare the tested compounds.
Compound
activity against eggs, larval, and nymphal of T. cinnabarinus, basing on its higher acaricidal potency
against female adults. As shown in Table 2, compound exhibits excellent acaricidal potency against
8 was used as typical representative of all target compounds to evaluate acaricidal
8
larva, low activity against nymphs, and no ovicidal activity. The different acaricidal potency maybe due
to different expression of the possible target gene TcPMCA1 at different stages of T. cinnabarinus [36].
Table 2. Acaricidal activity of compound 8 against eggs, larvae, and nymphs of T. cinnabarinus (48 h).
Stages of T. cinnabarinus
LC50 (mmol/L)
pLC50 (mol/L)
χ2
P
Eggs
Larva
Nymph
Non-ovicidal
0.679 ± 0.2066
3.889 ± 1.059
—
3.1682
2.4101
—
3.268
5.177
—
0.195
0.075
2.3. QSAR Analysis
2.3.1. 2D-QSAR Analysis
The selected descriptors, their correlations and their values of the investigated compounds
are provided in Tables 3–5, respectively. The best performing 2D-QSAR models was successfully
constructed as shown in Equation (1):
pLC50 = 4.243 (± 0.704) − 1.045 (± 0.218) R8e + 12.920 (± 1.921) HATS5p + 0.313 (± 0.062) Depressant −
(1)
80 − 1.351 (±0.385) MATS6e − 1.274 (± 0.401) HNar
N = 25, n =5, R = 0.935, R2train = 0.875, R2
= 0.842, RMSEtrain = 0.1095, F = 26.527 > F0.005(5,25) = 4.43
adjusted
(the cut off value of F distribution)
R LOO = 0.876, R2 LOO = 0.768, R2 LOO adjusted = 0.758, and RMSE LOO = 0.1299, F = 75.998, R2
= 0.583
pred
The R2train value of this model reveals that it can explain 87.5% of the variances in activity. Root
mean square error (RMSEtrain = 0.1095) is also a measurable value for the attained model together with
the Fisher test value (F = 26.527) which reflects the ratio of the variance explained by the model and
the variance due to their errors. A high value of F-test compared with the RMSE is a validation of
the model.
To determine whether multicollinearity existed among the descriptors in the models or not,
a variable inflation factor (VIF) (VIF = 1/(1
−
Rj2), where Rj2 represents the multiple correlation
coefficient of one descriptor’s effect on the remaining molecular descriptors) was calculated for each
variable in the regression equation [48]. If VIF ranges from 1.0 to 5.0, the linked equation is suitable [49].
As shown in Table 3, the VIF of all descriptors were smaller than 2, indicating that the generated model
possessed statistical significance and good stability. Table 4 gives the correlation matrix of the selected
descriptors. From this table, it can be seen that the linear correlation coefficient value for each pair of

Molecules 2018, 23, 995
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descriptors was smaller than 0.6, suggesting that the selected descriptors were independent, meeting
the important criterion for the model selections [50].
The reliability and statistical relevance of the attained BMLR-QSAR model is examined by internal
and external validation procedures. Experimental and predicted activities (pLC50, mol/L) values of
the compounds are shown in Table 5 and Figure 1. The residual values obtained by calculating the
difference between the predicted and experimental pLC50 are below 0.35 logarithmic units for all
the compounds.
Internal validation is applied by the SPSS technique employing Leave One Out (LOO), which
involves developing a number of models with one example omitted at a time. The observed correlations
due to the internal validation techniques are R2
= 0.768. The R2
value was bigger than 0.5,
LOO
LOO
indicating that the developed model had good stability and predictive ability [48].
The synthesized thirty target compounds were randomly divided into a 25-molecule training set
with LC50 values range from 0.655 to 6.588 mmol/L and a 5-molecule (11, 16, 19, 27, and 33) test set
with LC50 values range from 0.815 to 3.196 mmol/L were used as an external test set for validating
the attained QSAR models. The predicted/estimated acaricidal properties of the test set compounds
are close to their experimentally observed values preserving their potencies. In addition, the value of
R2pred = 0.583 for the external prediction was an acceptable result, which conformed that the generated
MLR model was useful for meaningful predictions.
The QSAR model indicated that the descriptors representing polarizability (HATS5p) is main
property governing acaricidal active agent of the scopoletin phenolic ether derivatives as shown by
its high regression coefficient values of 12.920 (Equation (1)). The QSAR model demonstrated that
high values of HATS5p, and Depressant-80, but low value of R8e, MATS6e, and HNar are required for
potent activity of the compounds. Among these compounds
methyl and ethyl possessed high polarizability (HATS5p: 0.118 and 0.146) and low electronegativity
(R8e: 0.384 and 0.706) exhibited high acaricidal potency. Among compounds 25 37, compounds 28 and
32 containing an amide group substituted by n-propyl and 3-Cl-benzyl possessed high polarizability
8–25, compounds 8 and 9 substituted by
–
(HATS5p: 0.115 and 0.133) and low electronegativity (R8e: 0.609 and 0.567; MATS6e: 0.037 and
and also displayed high acaricidal potency.
−
0.063)
Table 3. Selected descriptors of multiple linear regression.
Descriptor
Chemical Meaning
Type
Sig.
t
VIF
constant
Intercept
0.000
6.026
R autocorrelation of lag 8/weighted by
Sanderson electronegativity
GETAWAY
descriptor
R8e
HATS5p
Depressant-80
MATS6e
HNar
0.000
0.000
0.000
0.002
0.005
−
4.787 1.314
Leverage-weighted autocorrelation of
lag5/weighted by polarizability
GETAWAY
descriptor
6.725
5.061
1.479
1.605
Ghose-Viswanadhan-Wendoloski
antidepressant-like index at 80%
Drug-like
indices
Moran autocorrelation of lag 6 weighted by
Sanderson electonegativity
2D
−
−
3.514 1.032
3.178 1.439
autocorrelations
Topological
indices
Narumi harmonic topological index
Table 4. The correlation matrix of descriptors.
R8e
HATS5p
Depressant-80 MATS6e
HNar
R8e
1
−0.314
0.165
−0.494
1
0.099
0.398
−0.023
−0.080
0.099
1
0.391
−0.141
0.398
−0.092
1
HATS5p
Depressant-80
MATS6e
HNar
−0.314
1
0.165
−0.494
−0.080
−0.141
−0.023
0.391
−0.092

Molecules 2018, 23, 995
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Table 5. Values of significant molecular descriptors, experimental and predicted acaricidal activities
(pLC50, mol/L) from 2D-QSAR of the compounds 8–37.
Experimental Prediced
Compounds
R8e
HATS-5p Depressant-80 MATS-6e HNar
Residual
Activity
Activity
8
9
0.384
0.706
0.749
0.919
0.81
0.821
0.807
0.955
0.99
0.118
0.146
0.116
0.117
0.117
0.075
0.098
0.099
0.097
0.103
0.097
0.1
0.112
0.102
0.117
0.098
0.097
0.114
0.118
0.117
0.115
0.092
0.109
0.108
0.133
0.113
0.124
0.103
0.113
0.105
0
0
0
0
1
1
1
1
1
1
1
1
1
1
1
1
1
0
0
1
1
1
1
1
1
1
0
1
1
0
−0.02
−0.03
−0.043
−0.043
0.004
−0.055
−0.055
−0.066
−0.034
0.025
1.837
1.846
1.855
1.789
1.862
1.8
2
2
2
2
3.0813
3.0757
2.6333
2.7527
2.9022
2.5199
2.5414
2.1812
2.6083
2.4454
2.5922
2.4954
3.1229
2.7730
2.7821
2.3759
2.9435
2.5447
2.5950
3.0888
3.1180
2.5165
2.9935
2.9452
3.1835
3.0716
3.0165
2.7510
2.8193
2.4436
3.0281
3.0818
2.6575
2.5732
2.8258
2.3743
2.4945
2.4751
2.2726
2.4343
2.4056
2.6896
2.9197
2.7490
2.7227
2.3697
2.9037
2.5984
2.5985
2.9454
3.0118
2.8367
2.9747
2.8549
3.4251
2.9827
2.9033
2.9068
2.9159
2.5076
0.0532
−0.0061
−0.0242
0.1795
0.0764
0.1456
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
0.0469
−0.2939
0.3357
0.83
0.0111
0.1866
0.747
0.678
0.66
0.695
0.755
0.747
0.674
0.74
0.761
0.668
0.609
0.59
0.604
0.586
0.567
0.589
0.591
0.568
0.625
0.693
0.025
2
−0.017
1.941
1.84
1.895
1.89
1.941
1.84
1.846
1.81
1.818
1.826
1.775
1.833
1.948
1.902
1.902
1.862
1.902
1.906
1.822
−0.1942
0.019
0.2032
−0.013
0.098
0.145
0.024
0.0594
0.0062
0.0398
−0.0537
−0.0035
0.1434
−0.115
0.001
0.05
0.08
0.037
0.106
0.1062
−0.3202
0.015
0.0188
−0.01
−0.063
−0.004
−0.052
−0.012
0.019
0.0903
−0.2416
0.0889
0.1132
−0.1558
−0.0966
−0.064
0.045
Training set
Test set
Experimental activity pLC50 (mol/L)
Figure 1. Plot of experimental versus predicted pLC50 (mol/L) values of training sets and test sets
against female T. cinnabarinus.
2.3.2. 3D-QSAR Analysis
Table 6 shows the PLS results of the CoMFA and CoMSIA models. The results showed that the
optimal CoMFA model yielded a cross-validated q2 = 0.802 with an optimal number of principal
components (ONC) of 6, non-cross-validated R2 of 0.993, SEE = 0.029 and F value of 422.047.
The contribution of steric and electrostatic fields is 70.8% and 29.2%, respectively. The best CoMSIA
model yielded a q2 of 0.735 with an ONC of 6, non-cross-validated R2 of 0.965, SEE = 0.059 and F value

Molecules 2018, 23, 995
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of 83.553. The contribution of steric, electrostatic, hydrophobic, and hydrogen-bond acceptor are 21.5%,
28.5%, 44.9%, and 5.0%, respectively. Based on these field contributions, the steric field is the most
important field in the CoMFA model, whereas the hydrophobic field is the most important field in the
CoMSIA model. All the parameters in the Table 6 indicate that the CoMFA and CoMSIA models are
robust and stable.
Table 6. Summary of the results obtained from CoMFA and CoMSIA analyses.
Parameter
CoMFA
CoMSIA
R2 (correlation coefficient squared)
ONC (the optimal number of components)
(leave-one-out cross validation correlation coefficient squared)
0.993
6
0.802
422.047
0.029
SE
0.965
6
0.735
83.553
0.059
SEHDA
q2
LOO
F value
SEE
Fields
Field distribution (%)
Steric
Electrostatic
Hydrophobic
Donor
70.8
29.2
–
21.5
28.5
44.9
0
–
Acceptor
Testing set
–
5.0
R2
0.999
0.787
pred
The plot of experimental versus predicted acaricidal activities for CoMFA and CoMSIA models
are shown in Table 7, and Figures 2 and 3. The residual values obtained by calculating the difference
between the predicted and experimental pLC50 are below 0.3 logarithmic unit for all the compounds.
In addition, the values of R2
= 0.999 (CoMFA) and R2
= 0.787 (CoMSIA) for the external
pred
pred
prediction were acceptable results. The CoMFA R2pred is higher than its R2, which indicated CoMFA
model higher predictive ability. The prediced pLC50 values of five test compounds by CoMFA model
are very close to their experimental pLC50 values, and their residuals are less than 0.008 logarithmic
unit. These results indicate that the CoMFA and CoMSIA models are predictive.
Table 7. Experimental and predicted acaricidal activities (pLC50, mol/L) from 3D-QSAR of the
compounds 8–37.
CoMFA
Prediced pLC50
CoMSIA
Prediced pLC50
Compounds Experimental pLC50
Residual
Residual
8
9
3.0813
3.0757
2.6333
2.7527
2.9022
2.5199
2.5414
2.1812
2.6083
2.4454
2.5922
2.4954
3.1229
2.773
3.0973
3.0725
2.6274
2.7598
2.9078
2.4480
2.5493
2.2194
2.6112
2.4623
2.6016
2.4951
3.1170
2.7801
2.7970
2.3723
2.9283
2.5653
−0.016
3.0924
3.0575
2.5286
2.7593
2.9684
2.4733
2.5388
2.2796
2.8778
2.4475
2.5501
2.5121
3.1055
2.7715
2.8082
2.3906
2.9396
2.5621
−0.0111
0.0032
0.0182
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
0.0059
0.1047
−0.0071
−0.0056
0.0719
−0.0079
−0.0382
−0.0029
−0.0169
−0.0094
0.0003
−0.0066
−0.0662
0.0466
0.0026
−0.0984
−0.2695
−0.0021
0.0421
−0.0167
0.0059
0.0174
−0.0071
−0.0149
0.0036
0.0152
−0.0206
0.0015
2.7821
2.3759
2.9435
2.5447
−0.0261
−0.0147
0.0039
−0.0174

Molecules 2018, 23, 995
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Table 7. Cont.
CoMFA
CoMSIA
Prediced pLC50
Compounds Experimental pLC50
Prediced pLC50
Residual
Residual
26
27
28
29
30
31
32
33
34
35
36
37
2.595
3.0888
3.118
2.5165
2.9935
2.9452
3.1835
3.0716
3.0165
2.751
2.5766
3.0794
3.1368
2.5110
2.9990
2.9766
3.1459
3.0717
3.0186
2.7630
2.7858
2.4378
0.0184
0.0094
2.5922
3.0367
3.0989
2.5584
2.9795
2.9259
3.0742
3.1080
3.0850
2.8301
2.8629
2.3760
0.0029
0.0521
0.0191
−0.0188
0.0055
−0.0419
−0.0055
−0.0314
0.0376
−0.00005
−0.0021
−0.012
0.0335
0.0140
0.0193
0.1093
−0.0364
−0.0685
−0.0791
−0.0436
0.0676
2.8193
2.4436
0.0058
3.4
Training set
3.2
3
Test set
2.8
2.6
2.4
2.2
2
2
2.2
2.4
2.6
2.8
3
3.2
3.4
Experimental activity pLC50 (mol/L)
Figure 2. The plot of experimental versus calculated pLC50 values from CoMFA analyses for the
training and test set compounds.
3.4
Training set
3.2
Test set
3
2.8
2.6
2.4
2.2
2
2
2.2
2.4
2.6
2.8
3
3.2
3.4
Experimental activity pLC50 (mol/L)
Figure 3. The plot of experimental versus calculated pLC50 values from CoMSIA analyses for the
training and test set compounds.
Core structure of the studied scopoletin phenolic ether derivatives were shown in Figure 4A,
the compound 8 was employed as the template molecule for the analysis of contour maps (Figure 4B).

Molecules 2018, 23, 995
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B
A
H
H
H
O
R3
B
R2
H3CO
H
O
R4
R1
O
A
O
H3CO
R5
R6
R1, R2, R3, R6 = H
R5=-OR and
R4=OCH3,
NHR
O
O
Figure 4.
(A) The skeleton structure of the studied scopoletin phenolic ether derivatives and (B) the
chemical structure of compound 8.
The CoMFA steric and electrostatic contour maps are shown in Figure 5 with compound 8. Those
contours depict default contribution levels. In the CoMFA steric field shown in Figure 5A, A large-sized
and two medium-sized green contour near R4, R1, R2 and R5 (C6, C3, C4, and C7) indicate that bulky
substituents were preferred here. It can be explained that the most of synthesized 7-position scopoletin
phenolic ether derivatives have higher acaricidal activity than scopoletin. For CoMFA electrostatic
map (Figure 5B), there is one blue contour around the R5 (C7) position, which can be explain the fact
that compound 8 with the smallest electronegative OCH3 groups possesses higher acaricidal activity
among the synthesized target compounds.
B
A
Figure 5. CoMFA STDEV*COEFF contour maps around the compound
contours indicate regions where bulky groups increase activity, while yellow contours indicate regions
where bulky groups decrease activity; and ( ) electrostatic fields: blue contours indicate regions where
8. (A) Steric fields: green
B
electron positive groups increase activity, while red contours indicate regions where electron negative
groups increase activity. Compound 8 is displayed as a reference.
CoMSIA steric, electrostatic, hydrophobic, hydrogen-bond acceptor field contour maps are shown
in Figure 6 with compound
8 as an example. Those contours also depict the default contribution levels.
Since the steric and electrostatic contour are very similar with that of CoMFA, only hydrophobic,
hydrogen-bond acceptor will be described as follows: in the hydrophobic contour map (Figure 6C),
a large-sized yellow contour near R1 (C3) and R2 (C4) indicates that introducing hydrophobic groups
to that position could increase the acaricidal activity of the molecule. A large-sized white contour near
R6 (C8) suggests that hydrophilic substitutes preferentially localize at these positions. A medium-sized
white contour were found surrounding the R5 (C7) which indicates that introducing hydrophilic groups

Molecules 2018, 23, 995
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to this position could improve the acaricidal activity. Therefore, the synthesized target compouds
(e.g., 27, 28 and 32) containing amide groups show higher acaricidal activity.
A
B
D
C
Figure 6. CoMSIA STDEV*COEFF contour maps around the compound
contours and yellow contours show regions where an increase in bulky groups will increase and
decrease activity, respectively; ( ) electrostatic fields: blue contours and red contours show regions
where an increase in electron positive groups and electron negative groups will increase activity,
respectively; ( ) hydrophobic fields: yellow contours and white contours show regions where an
increase in lipophilicity and hydrophilicity will increase activity, respectively; ( ) H-bond acceptor
8. (A) Steric fields: green
B
C
D
contour map: magenta contour and red contour show regions where an increase in hydrogen-bond
acceptor groups and hydrogen-bond donor groups will increase activity.
In H-bond acceptor contour maps (Figure 6D), two large-sized red contours near R1 (C3) and
R4 (C6) indicate that introducing H-bond donors groups at those positions could increase the acaricidal
activity. A medium–sized magenta contour near R5 (C7) suggests that H-bond acceptor groups at
this position are favorable, and will increase the molecular activity. For example, several compounds
(e.g., 8, 9 and 12) with H-bond acceptor groups display higher acaricidal activity.
In this research, only R5 (C7)-position was be modified to investigate acaricidal activity, and the
contours maps of CoMFA and CoMSIA-derived models suggest that some favored group introduced
to other position of scopoletin could improve acaricidal activity, which need to be further study.
2.4. Molecular Docking
Some tested compounds exhibit higher acaricidal activity than scopoletin, which prompted us
to performed molecular docking study to understand the ligand-the target protein Ca2+-ATPase
interactions in detail. Scopoletin and the synthesized derivatives
8,
9,
12, 20 and 28 possessing the
higher acaricidal activity were selected for the docking study.
Docking results of scopoletin and its derivatives 8, 9, 12, 20 and 28 binding to TcPMCA1 are
listed in Table 8. Scopoletin and selected compound show low binding energies of much less than
5.0 kcal/mol, which can be generally considered as specific ligands of TcPMCA1. Figure 7 shows the
binding modes and orientations of scopoletin and its derivatives to TcPMCA1. The two dimensional

Molecules 2018, 23, 995
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interaction diagrams of scopoletin and its derivatives to TcPMCA1 are shown in Figure 8. Scopoletin
exhibits different binding poses compared to its derivatives. Five key amino acids (ASP222, ASP213,
GLU220, VAL224, and THR218) in the binding pocket interact with scopoletin via hydrogen bonding
and hydrophobic interaction. The H atom of hydroxyl at the 7-position in ring B forms a conventional
hydrogen bond (-H . . . OC-, 2.05 Å) with ASP222, the H atom of methoxy group at the 6-position
in ring B forms two nonconventional hydrogen bond (-H . . . OC-, 2.81 Å; -H . . . OC-, 3.33 Å) with
ASP213, and GLU220 respectively. The benzene and furan rings of scopoletin formed two pi–alkyl
(4.07 Å and 4.88 Å) interactions with the VAL224, and furan ring of scopoletin formed a pi–sigma
(
2.23 Å) interaction with the THR218. In addition, acid residues GLU214, SER215, HIS223, SER221, and
ARG781 in the binding pocket interact with scopoletin via Van der Waals interactions. Derivatives
12, and 20 display almost same binding mode, especially homologues and 12. The interactions
of these compounds with TcPMCA1 are analyzed using compound as an example. Six key amino
acids (GLU214, ASP222, ASP213, GLY723, ASP724, and VAL224) in the binding pocket interact with
compound via hydrogen bonding and hydrophobic interaction. The O atom of carbonyl at the
8, 9,
8, 9
8
8
2-position in ring A and O atom of furan ring form two conventional hydrogen bond (-O . . . NH-,
2.15 Å and -O . . . NH-, 1.97 Å) with GLU214, and ASP222, respectively. The H atom of the methoxy
group at the 6,7-position in ring B and the O atom of carbonyl at the 2-position in ring A form
three non-conventional hydrogen bonds (-H . . . OC-, 3.31, -H . . . OC-, 3.11, and -O . . . HC) with
GLY723, ASP724, and ASP213, respectively. The furan and benzene rings of compound
pi–alkyl (4.53 Å and 5.47 Å) interactions with the VAL224. Compound is also surrounded by
SER215, HIS223, THR218, ARG781, ASN725, GLU220, and SER221 through Van der Waals interactions.
There are few differences of specific biding poses between compounds 12 and 20. Compound
shows an analogous binding mode, except for some differences of binding bond length and two
non-conventional hydrogen bonds only form at 6-position instead of the 6,7-positions, compared
to compound . Compound 12 forms an alkyl–alkyl hydrophobic interaction at the 7-position with
MET490 instead of a non-conventional hydrogen bond, compared to compound . Compound 20 forms
pi–cation and pi–anion interactions with LYS481, ASP724, respectively, instead of non-conventional
hydrogen bonds, compared to compound . Compound 28 with an amide group displays very
different binding modes from compounds 12 and 20. Seven key amino acids (GLU214, VAL224,
8 form two
8
8, 9,
9
8
8
8
8, 9,
THR218, ASP213, GLY723, ARG781, and LEU217) in the binding pocket interact with compound 28 via
hydrogen bonding and hydrophobic interactions. The O atom of the carbonyl at the 2-position in ring
A, and the H atom of the amino group at the 7-position in ring B form three conventional hydrogen
bonds (-O . . . NH-, 1.77 Å, -O . . . NH-, 2.96 Å, -H . . . OC-, 2.15 Å) with GLU214, VAL224, and THR218,
respectively. The O atom of the carbonyl at the 2-position in ring A, the H atoms of the methoxy group
at the 6-position, the O atom of the acylamino at the 7-position, the H atom of the methylene at the
7-position in ring B form three non-conventional hydrogen bonds (-O . . . HC-, 2.53 Å, -H . . . OC-,
3.16 Å, -O . . . CH-, 2.21 Å, and -H . . . OC-,3.53 Å) with ASP 213, GLY 723, ARG 781, and GLU 214,
respectively. The furan and benzene rings of compound 28 form two pi–alkyl (3.78 Å and 4.45 Å)
interactions with the VAL224. The N-propyl connected to the amide at the 7-position forms alkyl–alkyl
interactions with the LEU217. Compound 28 interacts with ILE212, HIS223, ASP222, GLU220, ASN725,
ASP724, PRO784, SER783, and SER782 through Van der Waals interactions. These selected derivatives
display higher acaricidal activity that may be due to their different binding modes with TcPMCA1 from
the lead compound and they interact with more key amino acid residues. Three selected homologues
bind tighter with shortening of the side chain at the 7-position. The binding modes of scopoletin with
TcPMCA1 in the current study were different from our previous reports [32], which may be attributed
to our use of different software to find the active binding site interactions of TcPMCA1.

Molecules 2018, 23, 995
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Figure 7. The recognized binding modes and molecular interactions of the scopoletin (
A) and
compounds 8 (B), 9 (C), 12 (D), 20 (E), 28 (F) in the active site of TcPMCA1.

Molecules 2018, 23, 995
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Figure 8. Two dimensional interactions scheme of scopoletin (
20 (E), and 28 (F) to TcPMCA1.
A) and compounds 8 (B), 9 (C), 12 (D),

Molecules 2018, 23, 995
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Table 8. Binding energy and nonbonding interaction of scopoletin and its derivatives.
Hydrophobic Interaction
(Pi–Alkyl and Alkyl)
Electrostatic Interaction
(Pi–Anion and Pi–Cation)
H-Bond
Compounds
4
Binding Energy
Van der Waals
Amino Acid-Ligand
Atom
Amino Acid-Ligand
Atom
Amino
Acid-Ligand Atom
Distance (Å)
Distance (Å)
Distance (Å)
ASP222 [C-O . . . H]
ASP213 [C-O . . . H] *
GLU220 [C-O . . . H] *
2.05
2.81
3.33
GLU214, SER215, HIS223,
SER221, ARG781
VAL224 [Alkyl–Pi]
THR218 [Sigma–Pi]
4.07, 4.88
2.23
−5.25
GLU214 [N-H . . . O]
ASP222 [N-H . . . O]
ASP213 [C-H . . . O] *
GLY723 [C-O . . . H] *
ASP724 [C-O . . . H] *
2.15
1.97
2.75
3.31
3.11
SER215, HIS223, THR218,
ARG781, ASN725,
8
−5.15
VAL224 [Alkyl–Pi]
VAL224 [Alkyl–Pi]
4.53, 5.47
4.50, 5.39
GLU220, SER221
GLU214 [N-H . . . O]
ASP222 [N-H . . . O]
ASP213 [C-H . . . O] *
2.12
1.97
2.76
3.66
3.25
SER215, THR218,
ASN725, MET490,
9
−5.38
−5.40
GLU220, SER 221, HIS223 GLY723 [C-O . . . H] *
ASP724 [C-O . . . H] *
GLU214 [N-H . . . O]
SER215, HIS223, THR218,
ASP222 [N-H . . . O]
2.03
2.03
2.55
3.19
VAL224 [Alkyl–Pi]
MET490
[Alkyl–Alkyl]
4.17, 5.20
5.08
12
ASN725, GLY723, LYS481,
ASP213 [C-H . . . O]
LYS549, GLU220, SER221
*ASP724 [C-O . . . H] *
SER215, SER221, GLU220,
MET490, ILE732, ASP698, GLU214 [N-H . . . O]
1.98
2.05
2.48
3.16
THR728, GLU697,
GLY648, LEU649,
ARG781, ASN725,
THR218, HIS223
ASP222 [N-H . . . O]
ASP213 [C-H . . . O] *
GLY723 [C-O . . . H] *
LYS481[Cation–Pi]
ASP724[Anion–Pi]
4.54
4.45
20
28
−7.41
−6.22
VAL224 [Alkyl–Pi]
4.27, 5.47
GLU214 [N-H . . . O]
VAL224 [N-H . . . O]
THR218 [C-O . . . H]
ASP213 [C-H . . . O] *
GLY723 [C-O . . . H] *
ARG781 [C-H . . . O] *
GLU214 [C-O . . . H] *
1.77
2.96
2.15
2.53
3.16
2.21
3.53
ILE212, HIS223, ASP222,
GLU220, ASN725,
ASP724, PRO784, SER783,
SER782
VAL224
[Alkyl–Pi]LEU217
[Alkyl–Alkyl]
3.78, 4.45
4.78
* Nonconventional hydrogen bond.

Molecules 2018, 23, 995
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2.5. ADME Study
An in silico study of scopoletin and its semisynthesed derivatives (compounds
8–
37) was
performed for prediction of ADME properties [51] (Table 9). From all these parameters, it can be
observed that all tested compounds exhibited excellent % absorption (76.40–92.21%). It was also
observed that all of these compounds followed Lipinski’s rule of five and its extensions well. Four
typical Lipinski’s rule criteria are logP (octanol–water partition coefficient)
≤
5, molecular weight
5. Extension of
10, topological polar
≤
500,
number of hydrogen bond acceptors 10 and number of hydrogen bond donors
≤
≤
Lipinski’s rule of five includes the following criteria: number of rotatable bonds
≤
surface area ≤140 A2.
Table 9. Evaluation parameters of Lipinski’s rule of five and its extensions from scopoletin and its
phenolic ether derivatives (8–37).
TPSA
(A2)
n-ON
Acceptors
nOHNH
Donors
Lipinski’s
Violations
Entry
miLogp
%ABS
MW
n-ROTB
MV
Rule
≤5
—
≤140
≤500
≤10
≤5
≤1
≤10
—
4
8
9
1.33
1.64
2.01
2.52
2.38
3.07
2.76
2.51
3.04
3.54
3.92
3.23
3.68
4.94
3.19
4.51
3.91
4.13
4.20
0.74
1.12
1.62
1.42
2.18
2.14
2.79
2.82
3.42
2.59
2.20
3.85
88.41
92.21
92.21
92.21
92.21
92.21
92.21
92.21
92.21
92.21
92.21
92.21
92.21
92.21
76.40
92.21
92.21
92.21
89.02
82.17
82.17
82.17
82.17
82.17
82.17
82.17
82.17
82.17
82.17
78.98
82.17
59.67
48.68
48.68
48.68
48.68
48.68
48.68
48.68
48.68
48.68
48.68
48.68
48.68
48.68
94.50
48.68
48.68
48.68
57.91
77.78
77.78
77.78
77.78
77.78
77.78
77.78
77.78
77.78
77.78
87.01
77.78
192.17
206.20
220.22
234.25
234.25
248.28
248.28
246.26
260.29
274.32
288.34
282.30
296.32
338.40
327.29
351.19
316.74
350.29
366.29
263.25
277.28
291.30
291.30
305.33
339.35
373.79
373.79
408.24
353.37
369.37
395.45
4
4
4
4
4
4
4
4
4
4
4
4
4
4
7
4
4
4
5
6
6
6
6
6
6
6
6
6
6
7
6
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
1
1
1
1
1
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
2
3
4
3
5
4
4
3
3
4
4
4
5
5
4
4
5
6
4
5
6
5
7
6
6
6
6
6
7
7
162.15
179.68
196.48
213.28
213.07
230.08
229.87
219.51
236.31
253.11
269.91
251.33
267.89
317.51
274.66
278.40
264.86
282.62
291.61
227.87
244.67
261.47
261.25
278.27
299.51
313.05
313.05
326.59
316.08
325.06
365.70
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
Note: % ABS: percentage absorption, TPSA: topological polar surface area, n-ROTB: number of rotatable bonds,
MV: molecular volume, MW: molecular weight, milogP: logarithm of partition coefficient of compound between
n-octanol and water, n-ON acceptors: number of hydrogen bond acceptors, n-OHNH donors: number of hydrogen
bonds donors.
3. Materials and Methods
3.1. General Information
Microwave-assisted synthesis was performed on a CW-2000 Ultrasonic Microwave Assisted Extractor
(Xintuo Analytical Instruments Co., Ltd., Shanghai, China). Melting points were determined on a WRS-1B
Digital Melting-Point Apparatus (Shanghai Shenguang Instrument Co., Ltd., Shanghai, China) and were
uncorrected. IR spectra were obtained on a TENSOR 27 FT-IR spectrometer (Bruker Spectroscopic
Instruments Co., Rheinstetten, Germany) using KBr pellets and values were presented in cm−1
.

Molecules 2018, 23, 995
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1H- and 13C-NMR spectra were recorded on an Avance III 400 NMR spectrometer (Bruker Spectroscopic
Instruments Co., Rheinstetten, Germany) with CDCl3 or DMSO-d6 as solvent. Mass spectra were
carried out with a GCMS-QP2010 Ultra instrument (Shimadzu Corporation, Kyoto, Japan). Elemental
analyses were performed on a Vario EL III elemental analyzer (Elementar Analysensysteme GmbH,
Hanau, Germany)Propargite 90.00% TC was provided by Qingdao Hansen Biologic Science Co., Ltd.,
(Qingdao, China), and all other chemicals and solvents were of analytical grade and used as purchased.
Analytical thin-layer chromatography (TLC) was performed on a glass plate coated with silica gel GF-254
(Qingdao Haiyang Chemical Co., Ltd., Qingdao, China) and visualized under ZF-1 ultraviolet analyzer
(Shanghai Gucun Electro-optical Instrument Factory, Shanghai, China) under UV light (254 nm). Column
chromatography was performed on silica gel (200 to 300 mesh).
3.2. Chemistry
3.2.1. Procedure for the Synthesis of Scopoletin (4)
Preparation of 2,4-dihydroxy-5-methoxybenzaldehyde (2)
Following a literature method [22
8.8% mol) were added into dichloromethane (400 mL), and the reaction mixture was stirred at
room temperature for 30 min, then a solution of 2,4,5-trimethoxybenzaldehyde ( , 20 g, 0.1 mol)
,46], aluminum (III) chloride (40 g, 0.30 mol) and CTAB (3.2 g,
1
in dichloromethane (100 mL) was added dropwise, then the mixture was refluxed for 4 h (the reaction
progress was monitored by TLC with UV detection). The reaction mixture was cooled and poured
onto 500 g of ice to which 100 mL of concentrated hydrochloric acid was added. The organic layer
was separated and was washed with saturation salt solution, dried over anhydrous sodium sulfate,
evaporated under reduced pressure to give 2,4-dihydroxy-5-methoxybenzaldehyde (
2) as a light yellow
solid, 60.84% yeild, m.p. 152–153 ◦C.
Preparation 7-hydroxy-6-methoxy-2-oxo-2H-chromene-3-carboxylic acid (3)
2,4-Dihydroxy-5-methoxybenzaldehyde (2, 9.49 g, 56 mmol), malonic acid (13.5 g, 130 mmol), and
phenylamine (1 mL) were added into pyridine (30 mL), the resulting solution was stirred at room
temperature for over 24 h and then acidified to pH 4 using dilute HCl. The precipitate was collected
by suction filtration and followed by recrystallization from ethanol to give 7-Hydroxy-6-methoxy-2-
oxo-2H-chromene-3-carboxylic acid (3) as a yellow solid, 80.01% yeild, m.p. 231–232 ◦C.
Preparation of 7-hydroxy-6-methoxy-2H-chromen-2-one (scopoletin, 4)
7-Hydroxy-6-methoxy-2-oxo-2H-chromene-3-carboxylic acid (3, 3.5 g, 14.8 mmol) was refluxed in
ethylene glycol (17.6 mL) and pyridine (16 mL) for 50 min under microwave irradiation (115 W
)
and keep temperature above 110 ◦C. After cooling the reaction, the mixture was acidified to
about pH 5 using a solution of diluted HCl 30 mL, then 7-hydroxy-6-methoxy-2H-chromen-2-one
(scopoletin, 4) crystals were obtained after standing overnight, the filter liquor was extracted with
CH2Cl2. The CH2Cl2 layers were pooled and washed with saturation sodium bicarbonate, and
saturation salt solution, successively, dried over anhydrous sodium sulfate, evaporated under reduced
pressure and followed by recrystallization from acetone to give the target product
m.p. 201–202 ◦C.
4, 65.74% total yield,
3.2.2. General Procedure for the Synthesis of 8–13 and 18–25
K2CO3 (0.2073 g, 3 mmol) and CTAB (54.67 mg, 7.5% mmol) were added into a solution of
scopoletin ( , 0.3843 g, 2 mmol) in acetone (30 mL), and the reaction mixture was stirred at reflux
4
for 30 min. Then an alkyl or aromatic halide (3 mmol) was added into the mixture and maintained
at reflux for 6–24 h (the reaction progress was monitored by TLC with UV detection). After cooling
the reaction and filtration, the solvent was evaporated under reduced pressure, and the residue was
dissolved in ethyl acetate, washed with saturation sodium bicarbonate, and saturation salt solution,

Molecules 2018, 23, 995
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successively, dried over anhydrous sodium sulfate, evaporated under reduced pressure to give the
target crude products. The crude products were purified by column chromatography using petroleum
ether/ethyl acetate from 10:1 to 7:1 as the gradient eluent system to yield the products 8–13 and 18–25.
6,7-Dimethoxy-2H-chromen-2-one (8)
White needle-like crystals; 70.83% yield; m.p. 145.2–146.9 ◦C; IR νmax (KBr) cm−1: 3082, 2938,
2877, 1713, 1619, 1559, 1513, 1467, 1424, 1379, 1275, 1248, 1169, 1027, 880; 1H-NMR (DMSO-d6,
ppm):
3.81 (s, 6H, 2 OCH3), 6.30 (d, 1H, J = 8Hz, C3-H), 7.05 (s, 1H, C8-H), 7.25 (s, 1H, C5-H), 7.96 (d, 1H,
δ
×
J = 12 Hz, C4-H); 13C-NMR (DMSO-d6,
δ ppm): 56.31, 70.56, 100.98, 109.39, 111.51, 112.99, 144.82,
146.36, 149.85, 152.32, 161.07; MS (m/z): [M]+ 206. Anal. Calcd. for C11H10O4: C, 64.08%; H, 4.89%.
Found: C, 64.22%; H, 4.94%.
7-Ethoxy-6-methoxy-2H-chromen-2-one (9)
◦
White granular crystals; 75.02% yield; m.p. 146.3–146.7 C; IR νmax (KBr) cm−1: 3064, 2982,
2916, 2882, 2830, 1705, 1616, 1600, 1510, 1465, 1424, 1388, 1274, 1247, 1147, 1023, 877. 1H-NMR
(DMSO-d6, δ ppm): 1.37 (t, 3H, J = 8 Hz, CH3), 3.81 (s, 3H, OCH3), 4.12 (q, 2H, J = 8 Hz, CH2), 6.30
(
d, 1H
, J = 12 Hz, C3-H), 7.04 (s, 1H, C8-H), 7.24 (s, 1H, C5-H), 7.95 (d, 1H, J = 8 Hz, C4-H) (Figure S1).
13C-NMR (DMSO-d6,
δ
ppm): 14.87, 56.24, 64.86, 100.95, 109.30, 111.51, 113.00, 144.81, 146.30, 149.84,
152.17, 161.06 (Figure S2). MS (m/z): [M]+ 220. Anal. Calcd. for C12H12O4: C, 65.45%; H, 5.49%. Found:
C, 65.62%; H, 5.52%.
6-Methoxy-7-propoxy-2H-chromen-2-one (10)
White granular crystals; 78.08% yield; m.p. 96.0–96.7 ◦C, IR νmax (KBr) cm−1: 3084, 2967, 2939,
1
2877, 1705, 1619, 1559, 1513, 1467, 1424, 1386, 1275, 1248, 1150, 1027, 880. H-NMR (DMSO-d6, δ ppm):
0.99 (t, 3H, J = 8Hz, CH3), 1.73–1.82 (m, 2H, C02-CH2), 3.82 (s, 3H, OCH3), 4.02 (t, 2H, J = 8 Hz,
C01-CH2), 6.30 (d, 1H, J = 12 Hz, C3-H), 7.04 (s, 1H, C8-H), 7.24 (s, 1H, C5-H), 7.95 (d, 1H, J = 8 Hz,
C4-H) (Figure S3). 13C-NMR (DMSO-d6,
δ ppm): 10.81, 22.26, 56.33, 70.57, 100.98, 109.42, 111.51, 112.98,
144.78, 146.38, 149.85, 152.34, 161.05 (Figure S4). MS (m/z): [M]+ 234. Anal. Calcd. for C13H14O4: C,
66.67%; H, 5.98%. Found: C, 66.99%; H, 6.08%.
7-Isopropoxy-6-methoxy-2H-chromen-2-one (11)
White needle crystals; 58.73% yield; m.p.108.4–108.5 ◦C; IR νmax (KBr) cm−1: 3098, 2979, 2939,
1
1706, 1613, 1560, 1513, 1464, 1424, 1383, 1269, 1245, 1148, 1025, 846. H-NMR (DMSO-d6,
δ
ppm): 1.31
(d, 6H, J = 8 Hz, CH3), 3.80 (s, 3H, OCH3), 4.71–4.80 (m, 1H, CH), 6.29 (d, 1H, J = 8 Hz, C3-H), 7.08
(
s, 1H, C8-H), 7.25 (s, 1H, C5-H), 7.95 (d, 1H, J = 8 Hz, C4-H) (Figure S5). 13C-NMR (DMSO-d6,
δ
ppm):
22.03, 56.24, 71.15, 102.02, 109.63, 111.48, 112.96, 144.78, 146.91, 149.84, 151.03, 161.09 (Figure S6). MS
(m/z): [M]+ 234. Anal. Calcd. for C13H14O4: C, 66.67%; H, 6.02%. Found: C, 66.82%; H, 6.21%.
7-Butoxy-6-methoxy-2H-chromen-2-one (12)
White granular crystals; 66.01% yield; m.p. 77.1–78.3 ◦C, IR νmax (KBr) cm−1: 3067, 2940, 2867,
1
1699, 1609, 1557, 1512, 1465, 1423, 1384, 1277, 1247, 1147, 1023, 873. H-NMR (DMSO-d6,
δ ppm): 0.95
(t, 3H, J = 6 Hz, CH3), 1.40–1.49 (m, 2H, C03-CH2), 1.70–1.79 (m, 2H, C02-CH2), 3.81 (s, 3H, OCH3),
4.06 (t, 2H, J = 6 Hz, C01-CH2), 6.29 (d, 1H, J = 8 Hz, C3-H), 7.05 (s, 1H, C8-H), 7.24 (s, 1H, C5-H), 7.95
(d, 1H, J = 12 Hz, C4-H). 13C-NMR (DMSO-d6,
δ ppm): 14.11, 19.18, 30.93, 56.32, 68.85, 100.98, 109.40,
111.51, 112.97, 144.77, 146.38, 149.86, 152.37, 161.05. MS (m/z): [M]+ 248. Anal. Calcd. for C14H16O4: C,
67.73%; H, 6.50%. Found: C, 68.05%; H, 6.51%.
7-Isobutoxy-6-methoxy-2H-chromen-2-one (13)
White granular crystals; 27.85% yield; m.p. 84.1–85.3 ◦C; IR νmax (KBr) cm−1: 3096, 2965, 2921,
1
2876, 1714, 1613, 1561, 1514, 1457, 1425, 1386, 1266, 1248, 1143, 1013, 864. H-NMR (DMSO-d6,
δ ppm):
1.00 (t, 3H, C02-CH3), 1.44 (d, 3H, J = 8 Hz, C01-CH3), 2.00–2.13 (m, 2H, CH2), 3.82 (s, 3H, OCH3),

Molecules 2018, 23, 995
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3.83–3.94 (m, 1H, CH), 6.29 (d, 1H, J = 8 Hz, C3-H), 7.04 (s, 1H, C8-H), 7.25 (s, 1H, C5-H), 7.95 (d, 1H
,
J = 8 Hz, C4-H). 13C-NMR (DMSO-d6,
δ ppm): 19.47, 28.02, 56.47, 75.21, 101.12, 109.61, 111.55, 113.02,
144.82, 146.45, 149.88, 152.45, 161.07. MS (m/z): [M]+ 248. Anal. Calcd. for C14H16O4: C, 67.73%; H,
6.50%. Found: C, 67.16%; H, 6.60%.
7-(Benzyloxy)-6-methoxy-2H-chromen-2-one (18)
White needle-like crystals; 72.41% yield; m.p. 124.6–125.6 ◦C; IR νmax (KBr) cm−1: 2959, 1715,
1611, 1561, 1510, 1462, 1427, 1379, 1274, 1247, 1145, 1027, 757. 1H-NMR (DMSO-d6,
δ ppm): 3.93
(
s, 3H, OCH3), 5.23 (s, 2H, CH2O), 6.27 (d, 1H, J = 8 Hz, C3-H), 6.87 (s, 1H, C8-H), 7.26 (s, 1H, C5-H),
7.33–7.45 (m, 5H, Ar-H), 7.61 (d, 1H, J = 8 Hz, C4-H) (Figure S9). 13C-NMR (DMSO-d6,
δ
ppm): 56.47,
71.13, 101.83, 108.36, 111.69, 113.59, 127.31, 128.37, 128.82, 135.63, 143.31, 146.77, 149.75, 151.83, 161.42
(
Figure S10). MS (m/z): [M]+ 282. Anal. Calcd. for C17H14O4: C, 72.33%; H, 5.00%. Found: C, 72.07%;
H, 4.98%.
6-Methoxy-7-(4-methylbenzyloxy)-2H-chromen-2-one (19)
White granular crystals; 80.17% yield; m.p. 126.1–127.0 ◦C; IR νmax (KBr) cm−1: 3075, 3011, 2944,
1
2929, 2861, 1705, 1616, 1563, 1518, 1460, 1425, 1392, 1279, 1249, 1145, 1020, 883. H-NMR (DMSO-d6,
δ ppm): 2.31 (s, 3H, CH3), 3.81 (s, 3H, OCH3), 5.15 (s, 2H, CH2O), 6.31 (d, 1H, J = 12Hz, C3-H), 7.15
(
s, 1H, C8-H), 7.21 (s, 1H, C5-H), 7.24, 7.36 (dd, 4H, J = 8 Hz, Ar-H), 7.95 (d, 1H, J = 8 Hz, C4-H).
13C-NMR (DMSO-d6,
δ ppm): 21.27, 56.32, 70.62, 101.60, 109.47, 111.78, 113.20, 128.69, 129.54, 133.55,
137.95, 144.77, 146.49, 149.65, 151.84, 161.02. MS (m/z): [M]+ 296. Anal. Calcd. for C18H16O4: C, 72.96%;
H, 5.44%. Found: C, 72.53%; H, 5.26%.
7-(4-tert-Butylbenzyloxy)-6-methoxy-2H-chromen-2-one (20)
◦
White sheet-like crystals; 83.23% yield; m.p. 149.4–149.7 C; IR νmax (KBr) cm−1: 3082, 2948, 2862,
1
1720, 1610, 1560, 1508, 1460, 1424, 1387, 1272, 1242, 1141, 1015, 858. H NMR (DMSO-d6,
δ
ppm): 1.28
(
s, 9H, 3
×
CH3), 3.81 (s, 3H, OCH3), 5.16 (s, 2H, CH2O), 6.30 (d, 1H, J = 12 Hz, C3-H), 7.17 (s, 1H,
C8-H), 7.27 (s, 1H, C5-H), 7.40, 7.43 (dd, 4H, J = 8 Hz, Ar-H), 7.95 (d, 1H, J = 8 Hz, C4-H). 13C-NMR
(DMSO-d6, δ ppm): 31.56, 34.80, 56.35, 70.55, 101.56, 109.54, 111.79, 113.21, 125.75, 128.52, 133.60, 144.77,
146.51, 149.69, 151.15, 151.92, 161.00. MS (m/z): [M]+ 338. Anal. Calcd. for C21H22O4: C, 74.54%; H,
6.55%. Found: C, 75.00%; H, 6.54%.
7-(4-Nitrobenzyloxy)-6-methoxy-2H-chromen-2-one (21)
White powder; 60.42% yield; m.p. 122.3–122.9 ◦C; IR νmax (KBr) cm−1: 2946, 1735, 1603, 1562,
1
1519, 1465, 1427, 1347, 1284, 1251, 1174, 1010, 853. H-NMR (DMSO-d6,
δ
ppm): 3.95 (s, 3H, OCH3),
5.30 (s, 2H, CH2O), 6.31 (d, 1H, J = 12 Hz, C3-H), 6.92 (s, 1H, C8-H), 7.27 (s, 1H, C5-H), 7.63 (d, 1H
,
J = 4Hz, C4-H), 7.65 (d, 2H, J = 4 Hz, Ar-H), 8.27 (d, 2H, J = 8 Hz, Ar-H). 13C-NMR (DMSO-d6,
δ
ppm):
56.45, 69.74, 101.84, 108.59, 112.26, 114.14, 124.04, 127.66, 143.13, 146.72, 147.82, 149.55, 151.01, 161.13.
MS (m/z): [M]+ 327. Anal. Calcd. for C17H13NO6: C, 62.39%; H: 4.00%; N: 4.28%. Found: C, 62.02%; H,
4.15%; N, 4.49%.
7-(3,4-Dichlorobenzyloxy)-6-methoxy-2H-chromen-2-one (22)
White granular crystals; 53.08% yield; m.p. 151.5–152.3 ◦C; IR νmax (KBr) cm−1: 2948, 1698, 1607,
1
1564, 1509, 1435, 1375, 1274, 1259, 1139, 1018, 860. H-NMR (DMSO-d6,
δ
ppm): 3.95 (s, 3H, OCH3),
5.14 (s, 2H, CH2O), 6.27 (d, 1H, J = 8 Hz, C3-H), 6.85 (s, 1H, C8-H), 7.27 (s, 1H, C5-H), 7.47 (d, 1H
,
J = 8 Hz, C4-H), 7.56–7.64 (m, 3H, Ar-H). 13C-NMR (DMSO-d6,
δ
ppm): 56.42, 69.67, 101.79, 108.48,
112.08, 113.94, 126.54, 129.22, 130.78, 133.02, 135.87, 143.36, 144.04, 146.71, 149.72, 151.21, 161.27. MS
(m/z): [M]+ 351. Anal. Calcd. for C17H12Cl2O4: C, 58.14%; H: 3.44%. Found: C, 58.95%; H, 3.91%.

Molecules 2018, 23, 995
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7-(4-Chlorobenzyloxy)-6-methoxy-2H-chromen-2-one (23)
White powder; 76.13% yield; m.p. 171.8–172.3 ◦C; IR νmax (KBr) cm−1: 3074, 3014, 2949, 2927,
1
2875, 1705, 1615, 1562, 1515, 1461, 1426, 1393, 1279, 1249, 1145, 1015, 880. H-NMR (DMSO-d6,
δ ppm):
3.81 (s, 3H, OCH3), 5.21 (s, 2H, CH2O), 6.32 (d, 1H, J = 8 Hz, C3-H), 7.18 (s, 1H, C8-H), 7.30 (s, 1H,
C5-H), 7.48, 7.51 (dd, 4H, J = 8 Hz, Ar-H), 7.97 (d, 1H, J = 8 Hz, C4-H). 13C-NMR (DMSO-d6,
δ
ppm):
56.38, 69.80, 101.72, 109.60, 111.98, 113.38, 129.04, 130.34, 133.23, 135.69, 144.77, 146.48, 149.60, 151.57,
160.99. MS (m/z): [M]+ 316. Anal. Calcd. for C17H13ClO4: C: 64.47%; H: 4.14%. Found: C, 64.60%; H,
4.18%.
7-(4-(Trifluoromethyl)benzyloxy)-6-methoxy-2H-chromen-2-one (24)
White needle-like crystals; 83.56% yield; m.p. 192.8–193.3 ◦C; IR νmax (KBr) cm−1: 3073, 2938,
1
1713, 1616, 1565, 1515, 1463, 1427, 1393, 1281, 1250, 1146, 1019, 881. H-NMR (DMSO-d6,
δ ppm): 3.94
(s, 3H, OCH3), 5.26 (s, 2H, CH2O), 6.29 (d, 1H, J = 8 Hz, C3-H), 6.90 (s, 1H, C8-H), 7.27 (s, 1H, C5-H),
7.56-7.67 (m, 5H, Ar-H and C4-H). 13C-NMR (DMSO-d6,
δ ppm): 56.47, 70.22, 101.82, 108.53, 112.03,
113.95, 125.77, 125.81, 127.35, 130.39, 139.71, 143.19, 146.75, 149.65, 151.36, 161.23. MS (m/z): [M]+ 350.
Anal. Calcd. for C18H13F3O4: C, 61.72%; H, 3.74%. Found: C, 62.35%; H, 4.27%.
7-(4-(Trifluoromethoxy)benzyloxy)-6-methoxy-2H-chromen-2-one (25)
White powder; 84.32% yield; m.p. 127.0–127.2 ◦C; IR νmax (KBr) cm−1: 3056, 2960, 1727, 1615,
1
1565, 1514, 1466, 1426, 1382, 1248, 1147, 1023, 872. H-NMR (DMSO-d6,
δ
ppm): 3.92 (s, 3H, OCH3),
5.18 (s, 2H, CH2O), 6.27 (d, 1H, J = 8 Hz, C3-H), 6.89 (s, 1H, C8-H), 7.24 (d, 2H, J = 8 Hz, Ar-H), 7.28
(s, 1H, C5-H), 7.49 (d, 2H, J = 8 Hz, Ar-H), 7.62 (d, 1H, J = 12Hz, C4-H). 13C-NMR (DMSO-d6,
δ
ppm):
56.40, 70.17, 101.68, 108.48, 111.92, 113.74, 119.16, 121.26, 128.86, 134.40, 143.28, 146.73, 149.11, 149.65,
151.50, 161.28. MS (m/z): [M]+ 366. Anal. Calcd. for C18H13F3O5: C, 59.02%; H, 3.58%. Found: C,
59.87%; H, 4.26%.
3.2.3. General Procedure for the Synthesis of 14–17
K2CO3 (0.2073 g, 3 mmol) were added into a solution of scopoletin (4, 0.3843 g, 2 mmol) in DMF
(10 mL), and the mixture was reacted under microwave irradiation (80 W power) for 7 min. Then the
naphthenic halide (3 mmol) was added into the mixture and reacted under microwave irradiation
(120 W power) for 40 min (the reaction progress was monitored by TLC with UV detection). After
cooling the reaction and poured into 15 mL water, the mixture liquor was extracted with ethyl acetate.
The ethyl acetate layers were pooled and washed with saturation salt solution, dried over anhydrous
sodium sulfate, evaporated under reduced pressure to give the target crude products. The crude
products were purified by column chromatography using petroleum ether/ethyl acetate from 10:1 to
7:1 as the gradient eluent system to yield the products 14–17.
7-(Cyclopropylmethoxy)-6-methoxy-2H-chromen-2-one (14)
◦
Brown sheet-like crystals; 74.59% yield; m.p. 149.2–149.6 C; IR νmax (KBr) cm−1: 3076, 2963,
1
2937, 2880, 1715, 1615, 1563, 1462, 1426, 1387, 1276, 1248, 1146, 1024, 880. H-NMR (DMSO-d6,
δ ppm):
0.39 –0.43 (m, 2H, CH2), 0.68–0.73 (m, 2H CH2), 1.32–1.42 (m, 1H, CH), 3.91(d, 2H, J = 4 Hz, CH2),
3.92 (s, 3H, OCH3), 6.28 (d, 1H, J = 12 Hz, C3-H), 6.86 (s, 1H, C8-H), 7.27 (s, 1H, C5-H), 7.62 (d, 1H,
J = 8 Hz, C4-H). 13C-NMR (DMSO-d6,
δ ppm): 3.58, 9.89, 56.42, 74.19, 100.94, 108.23, 111.33, 113.32,
143.39, 146.58, 149.93, 152.35, 161.49; MS (m/z): [M]+ 246. Calcd. for C14H14O4: C, 68.26%; H, 5.69%.
Found: C, 68.38%; H, 5.81%.
7-(Cyclopentyloxy)-6-methoxy-2H-chromen-2-one (15)
White needle-like crystals; 72.89% yield; m.p. 80.7–81.2 ◦C; IR νmax (KBr) cm−1: 3075, 2944, 2867,
1718, 1611, 1558, 1511, 1467, 1425, 1387, 1277, 1248, 1144, 1024, 875. 1H-NMR (DMSO-d6,
δ
ppm):
1.64–1.70 (m, 2H, CH2), 1.82–1.87 (m, 2H, CH2), 1.89–1.94 (m, 2H, CH2), 1.98–2.07 (m, 2H, CH2), 3.88

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(
s, 3H, OCH3), 4.79-4.84(m, 1H, CH), 6.27 (d, 1H, J = 12 Hz, C3-H), 6.84 (s, 1H, C8-H), 7.27 (s, 1H, C5-H),
7.62 (d, 1H, J = 12 Hz, C4-H). 13C-NMR (DMSO-d6,
δ
ppm): 24.23, 32.78, 56.49, 81.03, 102.06, 108.51,
111.07, 113.07, 143.45, 146.99, 149.95, 151.76, 161.60. MS (m/z): [M]+ 260. Calcd. for C15H16O4: C,
69.23%; H, 6.15%. Found: C, 69.32%; H, 6.24%.
7-(Cyclohexyloxy)-6-methoxy-2H-chromen-2-one (16)
White granular crystals; 12.21% yield; m.p. 141.9–142.0 ◦C; IR νmax (KBr) cm−1: 3075, 2944, 2840,
1716, 1608, 1563, 1514, 1456, 1425, 1387, 1282, 1248, 1144, 1024, 873. 1H-NMR (DMSO-d6,
δ
ppm):
1.26–1.45 (m, 2H, CH2), 1.57–1.66 (m, 2H, CH2), 1.83–1.88 (m, 2H, CH2), 2.06–2.09 (m, 1H, CH), 3.89
s, 3H, OCH3), 6.27 (d, 1H, J = 8 Hz, C3-H), 6.86 (s, 1H, C8-H), 7.27 (s, 1H, C5-H),7.61 (d, 1H, J = 12 Hz,
(
C4-H) (Figure S7). 13C-NMR (DMSO-d6,
δ ppm): 23.91, 25.44, 31.50, 56.56, 77.03, 102.35, 108.81, 111.27,
113.21, 143.30, 147.26, 150.00, 151.32, 161.49 (Figure S8). MS (m/z): [M + H]+ 275. Calcd. for C16H18O4:
C, 70.07%; H, 6.57%. Found: C, 70.15%; H, 6.67%.
7-(Cyclohexylmethoxy)-6-methoxy-2H-chromen-2-one (17)
White granular crystals; 30.23% yield; m.p. 144.4–144.7 ◦C; IR νmax (KBr) cm−1: 3072, 2938, 2854,
1713, 1614, 1562, 1514, 1464, 1425, 1385, 1279, 1251, 1144, 1034, 882. 1H-NMR (DMSO-d6,
δ
ppm):
CH2), 1.89-1.97 (m, 1H, CH),
3.86 (d, 2H, J = 4 Hz, CH2), 3.90 (s, 3H, OCH3), 6.27 (d, 1H, J = 8 Hz, C3-H), 6.85 (s, 1H, C8-H), 7.28
1.02–1.11 (m, 2H, CH2), 1.20-1.32 (m, 4H, 2
×
CH2), 1.70–1.79 (m, 4H, 2
×
(
s, 1H, C5-H), 7.62 (d, 1H, J = 12 Hz, C4-H). 13C-NMR (DMSO-d6,
δ ppm): 25.66, 26.40, 29.81, 37.16,
56.54, 74.65, 100.80, 108.48, 111.17, 113.16, 143.41, 146.66, 150.04, 152.72, 161.53. MS (m/z): [M]+ 288.
Calcd. for C17H20O4: C, 70.83%; H, 6.94%. Found: C, 70.73%; H, 6.99%.
3.2.4. General Procedure for the Synthesis of 26–37
Triethylamine (0.3643 g, 3.6 mmol) was added to a solution of the appropriate alkylamine or
substituted benzylamine 5a–l (3 mmol) in dichloromethane (7.5 mL), and the reaction mixture was
stirred for 5 min at room temperature, then 2-chloroacetyl chloride (0.3857 g, 3.6 mmol) was added
◦
dropwise to this reaction mixture at 0 C and stirred for 15 min at room temperature. After completion
of the reaction, the solvent was evaporated under reduced pressure to afford 6a
–
l. KI (0.5976 g,
3.6 mmol) and CTAB (98.40 mg, 7.5% mmol) were added to a solution of the crude product 6a
–
l
in
acetone (30 mL) and maintained stirring at reflux for 2 h to afford 7a−l. K2CO3 (0.2073 g, 3 mmol)
was added to a solution of scopoletin (0.3843 g, 2 mmol) in acetone (30 mL), and the reaction mixture
was stirred at refluxed for 30 min. Then crude intermediates 7a–l were added into the mixture and
maintained reflux for 8–12 h (the reaction progress was monitored by TLC with UV detection). After
cooling the reaction and filtration, the solvent was evaporated under reduced pressure, and the residue
was dissolved in ethyl acetate, washed with saturation sodium bicarbonate, and saturation salt solution
successively, dried over anhydrous sodium sulfate, evaporated under reduced pressure to give the
target crude products. The crude products were purified by column chromatography using petroleum
ether/ethyl acetate from 6:1 to 2:1 as the gradient eluent system to yield the products 26–37.
2-(6-Methoxy-2-oxo-2H-chromen-7-yloxy)-N-methylacetamide (26)
White needle-like crystals; 21.22% yield; m.p. 155.8–156.40 ◦C; IR νmax (KBr) cm−1: 3327, 2982,
1
1750, 1704, 1609, 1566, 1515, 1425, 1394, 1280, 1251, 1144, 1019, 854. H-NMR (CDCl3,
δ ppm): 3.83
(
s, 3H, CH3), 3.94 (s, 3H, OCH3), 4.78 (s, 2H, CH2O), 6.32 (d, 1H, J = 12 Hz, C3-H), 6.91 (s, 1H, C8-H),
7.29 (s, 1H, C5-H), 7.64 (d, 1H, J = 8 Hz, C4-H). 13C-NMR (DMSO-d6,
δ ppm): 26.48, 52.61, 65.82, 101.45,
108.72, 112.56, 114.21, 143.23, 146.53, 149.40, 150.66, 161.21, 168.21. MS (m/z): [M + H]+ 264. Anal.
Calcd. for C13H13NO5: C, 59.31%; H, 4.98%; N, 5.32%. Found: C, 60.40%; H, 4.60%; N, 5.20%.
N-Ethyl-2-(6-methoxy-2-oxo-2H-chromen-7-yloxy)acetamide (27)
White granular crystals; 30.28% yield; m.p. 171.2–172.3 ◦C; IR νmax (KBr) cm−1: 3409, 2933, 1713,
1
1683, 1591, 1512, 1442, 1421, 1354, 1266, 1243, 1148, 1002, 881. H-NMR (DMSO-d6,
δ ppm): 1.19 (t, 3H,

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J = 8 Hz, CH3), 3.37–3.41 (m, 2H, CH2), 3.92 (s, 3H, OCH3), 4.53 (s, 2H, CH2O), 6.30 (d, 1H
,
J = 12 Hz
,
C3-H), 6.90 (s, 1H, C8-H), 7.29 (s, 1H, C5-H), 7.63 (d, 1H, J = 12 Hz, C4-H). 13C-NMR (DMSO-d6,
δ
ppm):
14.78, 34.05, 56.29, 68.59, 102.59, 108.58, 112.86, 114.50, 143.08, 146.56, 149.39, 150.22, 160.99, 166.94. MS
(m/z): [M]+ 277. Anal. Calcd. for C14H15NO5: C, 60.64%; H, 5.45%; N, 5.05%. Found: C, 60.86%; H,
5.45%; N, 4.65%.
2-(6-Methoxy-2-oxo-2H-chromen-7-yloxy)-N-propylacetamide (28)
White granular crystals; 34.42% yield; m.p. 173.2–173.8 ◦C; IR νmax (KBr) cm−1: 3422, 3053, 2962,
1
2876, 1717, 1673, 1611, 1563, 1510, 1427, 1390, 1278, 1251, 1143, 1023, 889. H-NMR (DMSO-d6,
δ ppm):
0.96 (t, 3H, J = 8 Hz, CH3), 1.55–1.64 (m, 2H, CH2), 3.31–3.36 (m, 2H, CH2), 3.95 (s, 3H, OCH3), 4.57
s, 2H, CH2O), 6.33 (d, 1H, J = 12 Hz, C3-H), 6.94 (s, 1H, C8-H), 7.31 (s, 1H, C5-H), 7.66 (d, 1H, J = 12Hz,
(
C4-H). 13C-NMR (DMSO-d6,
δ ppm): 11.30, 22.75, 40.77, 56.30, 68.63, 102.62, 108.60, 112.87, 114.51,
143.10, 146.58, 149.40, 150.25, 160.99, 167.03. MS (m/z): [M]+ 291. Anal. Calcd. for C15H17NO5: C,
61.85%; H, 5.88%; N, 4.81%. Found: C, 62.05%; H, 5.86%; N, 4.70%.
N-Isopropyl-2-(6-methoxy-2-oxo-2H-chromen-7-yloxy)acetamide (29)
White granular crystals; 19.52% yield; m.p. 197.2–197.5 ◦C; IR νmax (KBr) cm−1: 3305, 3077, 2967,
1
2875, 1722, 1663, 1613, 1558, 1507, 1463, 1422, 1396, 1275, 1248, 1151, 1034, 872. H-NMR (DMSO-d6,
δ ppm): 1.23 (d, 6H, J = 8 Hz, CH3), 3.95 (s, 3H, OCH3), 4.13–4.25 (m, 1H, CH), 4.55 (s, 2H, CH2O), 6.35
(d, 1H, J = 8 Hz, C3-H), 6.93 (s, 1H, C8-H), 7.28 (s, 1H, C5-H), 7.65 (d, 1H, J = 12Hz, C4-H). 13C-NMR
(DMSO-d6, δ ppm): 22.68, 41.24, 56.32, 69.00, 103.16, 108.68, 113.02, 114.74, 142.94, 146.69, 149.50, 150.40,
160.94, 166.25. MS (m/z): [M]+ 291. Anal. Calcd. for C15H17NO5: C, 61.85%; H, 5.88%; N, 4.81%.
Found: C, 60.90%; H, 5.82%; N, 4.65%.
N-Butyl-2-(6-methoxy-2-oxo-2H-chromen-7-yloxy)acetamide (30)
White powder; 19.59% yield; m.p. 168.4–168.5 ◦C; IR νmax (KBr) cm−1: 3426, 3055, 2959, 2934,
1
2862, 1721, 1675, 1614, 1565, 1511, 1464, 1427, 1391, 1280, 1252, 1146, 1027, 891. H-NMR (DMSO-d6,
δ ppm): 0.93-0.96 (t, 3H, CH3), 1.31–1.42 (m, 2H, CH2), 1.51–1.60 (m, 2H, CH2), 3.34-3.39 (m, 2H, CH2),
3.93 (s, 3H, OCH3), 4.56 (s, 2H, CH2O), 6.34 (d, 1H, J = 8 Hz, C3-H), 6.91 (s, 1H, C8-H), 7.27 (s, 1H,
C5-H), 7.63 (d, 1H, J = 8Hz, C4-H) (Figure S11). 13C-NMR (DMSO-d6,
δ ppm): 13.74, 20.01, 31.51, 38.86,
56.32, 69.70, 102.78, 108.63, 112.92, 114.64, 143.00, 146.61, 149.48, 150.28, 160.98, 167.02 (Figure S12). MS
(m/z): [M]+ 305. Anal. Calcd. for C16H19NO5: C, 62.95%; H, 6.23%; N, 4.59%. Found: C, 63.15%; H,
6.30%; N, 4.60%.
N-Benzyl-2-(6-methoxy-2-oxo-2H-chromen-7-yloxy)acetamide (31)
White powder; 38.02% yield; m.p. 84.50–86.20 ◦C; IR νmax (KBr) cm−1: 3420, 3053, 2931, 2872,
1
1717, 1671, 1612, 1565, 1508, 1460, 1423, 1389, 1276, 1249, 1143, 1018, 885. H-NMR (DMSO-d6,
δ ppm):
3.93 (s, 3H, OCH3), 4.09 (d, 2H, J = 8 Hz, CH2), 4.53 (s, 2H, CH2O), 6.29 (d, 1H, J = 12 Hz, C3-H),
6.91 (s, 1H, C8-H), 7.28 (s, 1H, C5-H), 7.49–7.66 (m, 5H, Ar-H), 8.20 (d, 1H, J = 8 Hz, C4-H). 13C-NMR
(DMSO-d6,
δ ppm): 44.58, 56.43, 66.63, 101.66, 108.39, 111.92, 113.73, 124.06, 127.70, 128.91, 131.00,
143.43, 146.72, 149.63, 151.50, 161.46, 167.79. MS (m/z): [M]+ 339. Anal. Calcd. for C19H17NO5: C,
67.25%; H, 5.05%; N, 4.13%. Found: C, 67.28%; H, 4.40%; N, 4.91%.
N-(3-Chlorobenzyl)-2-(6-methoxy-2-oxo-2H-chromen-7-yloxy)actamide (32)
White powder; 11.61% yield; m.p. 155.6–156.0 ◦C; IR νmax (KBr) cm−1: 3420, 3053, 2931, 2850,
1
1716, 1671, 1612, 1567, 1508, 1464, 1423, 1389, 1276, 1248, 1145, 1020, 881. H-NMR (DMSO-d6,
δ ppm):
3.88 (s, 3H, OCH3), 4.44 (d, 2H, J = 4 Hz, CH2), 4.53 (s, 2H, CH2O), 6.28 (d, 1H, J = 8 Hz, C3-H), 6.87
s, 1H, C8-H), 7.03 (s, 1H, C5-H), 7.29–7.40 (m, 3H, Ar-H), 7.46(s, 1H, Ar-H), 7.92 (d, 1H, J = 12 Hz,
(
C4-H). 13C-NMR (DMSO-d6,
δ ppm): 41.79, 56.77, 68.84, 103.20, 107.47, 111.51, 113.42, 126.32, 127.08,
127.68, 130.01, 133.66, 141.07, 143.36, 144.02, 149.70, 150.25, 161.51, 167.30. MS (m/z): [M]+ 373. Anal.
Calcd. for C19H16ClNO5: C, 61.13%; H, 4.29%; N, 3.75%. Found: C, 61.87%; H, 4.85%; N, 3.43%.

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N-(4-Chlorobenzyl)-2-(6-methoxy-2-oxo-2H-chromen-7-yloxy)acetamide (33)
◦
White sheet-like crystals; 30.12% yield; m.p. 178.3–179.2 C; IR νmax (KBr) cm−1: 3420, 3054, 2932,
1
1716, 1671, 1614, 1566, 1507, 1461, 1423, 1388, 1276, 1248, 1144, 1017, 884. H-NMR (DMSO-d6,
δ ppm):
3.83 (s, 3H, OCH3), 4.53 (d, 2H, J = 4Hz, CH2), 4.63 (s, 2H, CH2O), 6.35 (d, 1H, J = 12 Hz, C3-H), 6.88
(s, 1H, C8-H), 7.16 (s, 1H, C5-H), 7.23–7.32 (m, 4H, Ar-H), 7.63 (d, 1H, J = 12 Hz, C4-H). 13C-NMR
(DMSO-d6,
δ ppm): 42.42, 56.18, 68.92, 103.16, 108.60, 113.12, 114.84, 128.91, 129.12, 133.60, 136.21,
142.94, 146.60, 149.40, 150.11, 160.91, 167.28. MS (m/z): [M]+ 373. Anal. Calcd. for C19H16ClNO5: C,
61.05%; H, 4.31%; N, 3.75%. Found: C, 61.28%; H, 4.40%; N, 3.59%.
N-(3,4-Dichlorobenzyl)-2-(6-methoxy-2-oxo-2H-chromen-7-yloxy)acetamide (34)
White powder; 36.12% yield; m.p. 161.15–162.25 ◦C; IR νmax (KBr) cm−1: 3417, 3040, 2996, 1724,
1
1678, 1612, 1564, 1507, 1472, 1425, 1389, 1274, 1252, 1145, 1024, 878. H-NMR (DMSO-d6, δ ppm): 3.87
(s, 3H, OCH3), 4.36 (d, 2H, J = 8 Hz, CH2), 4.51 (s, 2H, CH2O), 6.35 (d, 1H, J = 12 Hz, C3-H), 6.93 (s, 1H,
C8-H), 7.30 (s, 1H, C5-H), 7.36–7.41 (m, 3H, Ar-H), 7.68 (d, 1H, J = 12 Hz, C4-H). 13C-NMR (DMSO-d6,
δ
ppm): 42.18, 56.32, 68.84, 103.04, 108.76, 113.16, 114.68, 127.61, 129.86, 130.63, 131.41, 132.41, 137.78,
143.19, 146.58, 149.33, 150.06, 162.90, 167.47. MS (m/z): [M
C, 55.90%; H, 3.70%; N, 3.43%. Found: C, 55.50%; H, 3.44%; N, 3.55%.
−
H]+ 407. Anal. Calcd. for C19H15Cl2NO5:
N-(4-Methylbenzyl)-2-(6-methoxy-2-oxo-2H-chromen-7-yloxy)acetamide (35)
◦
White sheet-like crystals; 65.36% yield; m.p. 171.4–171.6 C; IR νmax (KBr) cm−1: 3430, 3055, 2931,
1
1727, 1677, 1614, 1567, 1507, 1464, 1427, 1389, 1276, 1250, 1146, 1024, 881. H-NMR (DMSO-d6,
δ ppm):
2.34 (s, 3H, CH3), 3.80 (s, 3H, OCH3), 5.52 (d, 2H, J = 4 Hz, CH2), 4.62 (s, 2H, CH2O), 6.33 (d, 1H
,
,
J = 8 Hz, C3-H), 6.87 (s, 1H, C8-H), 7.15, 7.20 (dd, 4H, J = 8 Hz, Ar-H), 7.27 (s, 1H, C5-H), 7.63 (d, 1H
J = 12 Hz, C4-H). 13C-NMR (DMSO-d6,
δ ppm): 21.13, 42.88, 56.15, 69.01, 103.18, 108.61, 113.06, 114.73,
127.74, 129.43, 134.62, 137.41, 142.96, 146.67, 149.42, 150.29, 160.92, 167.11. MS (m/z): [M]+ 353. Anal.
Calcd. for C20H19NO5: C, 67.98%; H, 5.42%; N, 3.96%. Found: C, 67.69%; H, 5.46%; N, 3.85%.
N-(4-Methoxybenzyl)-2-(6-methoxy-2-oxo-2H-chromen-7-yloxy)acetamide (36)
White sheet crystal; 80.34% yield; m.p. 161.3–161.6 C; IR νmax (KBr) cm−1: 3430, 3054, 1727,
◦
1
1675, 1611, 1567, 1507, 1426, 1388, 1276, 1249, 1144, 1024, 880. H-NMR (DMSO-d6,
δ
ppm): 3.80 (s, 6H
,
OCH3), 4.48 (d, 2H, J = 4 Hz, CH2), 4.61 (s, 2H, CH2O), 6.32 (d, 1H, J = 12 Hz, C3-H), 6.84–6.88 (m, 2H
,
Ar-H), 7.11 (s, 1H, C8-H), 7.22 (d, 2H, J = 8 Hz, Ar-H), 7.28 (s, 1H, C5-H), 7.61 (d, 1H, J = 8 Hz, C4-H).
13C-NMR (DMSO-d6,
δ ppm): 42.60, 55.33, 56.16, 69.03, 103.18, 108.63, 113.05, 114.10, 114.68, 129.12,
129.74, 142.95, 146.66, 149.39, 150.29, 159.12, 160.89, 167.07. MS (m/z): [M]+ 369. Anal. Calcd. for
C20H19NO6: C, 65.03%; H, 5.19%; N, 3.79%. Found: C, 65.20%; H, 5.24%; N, 3.66%.
N-(4-tert-Butylbenzyl)-2-(6-methoxy-2-oxo-2H-chromen-7-yloxy)acetamide (37)
White powder; 50.34% yield; m.p.177.5–178.1 C; IR νmax (KBr) cm−1: 3430, 2961, 1730, 1682, 1615,
◦
1568, 1520, 1444, 1428, 1390, 1279, 1249, 1147, 1025, 880. 1H-NMR (DMSO-d6,
δ
ppm): 1.28 (s, 9H,
3 × CH3), 3.79 (s, 3H, OCH3), 4.39 (d, 2H, J = 8 Hz, CH2), 4.61 (s, 2H, CH2O), 6.32 (d, 1H, J = 8 Hz,
C3-H), 6.90 (s, 1H, C8-H), 7.24 (d, 2H, J = 8 Hz, Ar-H), 7.31 (s, 1H, C5-H), 7.37 (d, 2H, J = 8 Hz, Ar-H),
7.66 (d, 1H, J = 12 Hz, C4-H). 13C-NMR (DMSO-d6,
δ ppm): 31.34, 42.75, 42.88, 56.17, 69.04, 103.15,
108.71, 113.09, 114.62, 125.51, 127.66, 134.40, 143.13, 146.68, 149.38, 150.71, 150.29, 160.98, 167.15. MS
(m/z): [M]+ 395. Anal. Calcd. for C23H25NO5: C, 69.86%; H, 6.37%; N, 3.54%. Found: C, 69.91%; H,
6.91%; N, 3.59%.
3.3. Acaricidal Activity Assay
T. cinnabarlnus was reared on potted young cowpea plants in the laboratory at (26
70 ± 10) % relative humidity (R. H.) and a 14 h:10 h (light:dark) cycle with no acaricide exposure for at
±
1) ◦C and
(

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least 15 years, which originally collected from field young cowpea plants in Beibei District, Chongqing
Municipality, China.
The slide-dip method [52] was adopted to evaluate the acaricidal activity of 8–37 against female
adults of T. cinnabarinus. The appropriate amounts of target compounds were dissolved in 0.2 mL
acetone and then diluted with water containing 0.1% Tween-80 to obtain the desired final concentration
of 1000 mg/L for the preliminary screening. Based on the preliminary test results, a series of five to
seven concentrations of the tested compounds were chosen to determine the median lethal concentration
(LC50) values of the compounds. Propargite 90.00% TC and scopoletin were used as positive controls,
and water containing 0.1% Tween-80 was used as a blank control. Acaricidal activity assays were
performed in triplicate and repeated thrice. The LC50 values of the tested compounds were calculated
using the probit analysis procedure of SPSS 17.0 for Windows (SPSS Inc., Chicago, IL, USA).
The leaf-dip method was used to evaluate the acaricidal activity of compound
8 against eggs,
larval, and nymphal of T. cinnabarinus. The test solutions of compound was prepared as above
8
slide-dip method. Leaf discs were prepared to obtain uniform individuals at different developmental
stages. Fresh cowpea leaves that had not been exposed to pesticides were washed thoroughly. Leaf
discs with 3 cm diameters were placed on a corresponding size water-saturated sponge in a Petri dish
(9 cm diameter) [53]. Adult females (20–30) were transferred to each leaf disc, allowed to lay eggs, and
removed after 12 h. The leaf disc with eggs, larvae, and nymphs were then dipped in the compound
8
solutions for 5 s, taken out, and then laid on sponge in Petri dish again. The observed results were
recorded after 48 h.
3.4. 2D- and 3D-QSAR Study
3.4.1. Data Set
The synthesized thirty target compounds and their acaricidal activities (LC50 values) were used
as data set for QSAR analysis. They were randomly divided into a 25-molecule training set for 2D-
and 3D-QSAR models development and 5-molecule test set (compounds 11, 16, 19, 27 and 33) for
external validation.
3.4.2. 2D-QSAR (Multiple Linear Regression Model) Method
2D structures of the 30 target synthesized compounds were generated by ChemDraw Ultra
(Cambridge Soft Corporation, Cambridge, MA, USA), and their energies were minimized using
MM2 of Chem3D Ultra. Then 1666 molecular descriptors were calculated for each compound using
DRAGON Web version 1.0 developed by the Milano Chemometrics and QSAR Research Group
(http://www.vcclab.org/lab/edragon/start.html). These descriptors included (i) 0D constitutional
(atom and group counts), (ii) 1D functional groups and atom-centred fragments, (iii) 2D topological,
counts, autocorrelations, connectivity indices, information indices, topological indices, and
eigenvalue-based indices, and (iv) 3D geometrical, WHIM, and GETAWAY descriptors, etc. [54].
1302 descriptors were utilized as input values for model construction after eliminating the descriptors
with constant values or mostly zero values (>90%) from the all the calculated descriptors.
2D-QSAR models were obtained using SPSS software (Version 17.0) that can run multiple linear
regression. Different mathematical transformations of the observed median lethal concentration (LC50)
of the training set analogs, including property LC50 (mg/L), LC50 (mol/L), 1/LC50 (mg/L), 1/LC50
(mol/L), log LC50 (mg/L), log LC50 (mol/L),
utilized in the present 2D-QSAR modeling to searching for the best model.
values were used as dependent variables. Stepwise method for variable selection along with multiple
linear regression was used to construct models.
−
log LC50 (mg/L) and
−
log LC50 (mol/L) values, were
log LC50 (mol/L) (pLC50)
−

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3.4.3. 3D-QSAR (CoMFA and CoMSIA) Methods
The molecular structures of synthesized compounds were generated and optimized using SYBYL
6.9 (Tripos Associates, St. Louis, MO, USA). The Gasteiger–Hückel charge, Tripos force field, and
Powell method were used for structure optimization. To guarantee the obtaining of the molecular
lowest energy conformation, conformation search was executed by using multisearch routin [55].
The most important component of a 3D-QSAR study is the alignment of the molecules based on the
scaffold they share [56]. In this paper, the 7-oxy-6-methoxy-2H-chromen-2-one structure was selected
as the common scaffold for molecular alignment. Compound
8 was used as the template molecule.
All other synthesized acaricidal agents were aligned with the 7-oxy-6-methoxy-2H-chromen-2-one core.
The comparative molecular field analysis (CoMFA) and comparative molecular similarity
indices analysis (CoMSIA) are commonly used 3D-QSAR methods [51]. In CoMFA, the steric and
electrostatic fields were calculated by setting the energy cutoff as the default value of 30 kcal .
mol−1
·
Five CoMSIA fields including the steric, electrostatic, hydrophobic, hydrogen-bond donor and
hydrogen-bond acceptor were calculated using the default attenuation factor of 0.3 for Gaussian
function. Field type “Stdev * Coeff” was used as the coefficient to analysis the contour map of each
field [36]. The partial least squares (PLS) [57] was used to quantify the relationships by setting the
biological activity (pLC50 values) as the dependent variables and the CoMFA/CoMSIA descriptors as
independent variables.
3.5. Molecular Docking
Molecular docking studies were performed using AutoDock 4.2 and AutoDock Tools version 1.5.6
(ADT). The 3D structure of TcPMCA1 (GenBank No. KP455490), and its binding pocket were obtained
from the I-TASSER server (Available online: http://zhanglab.ccmb.med.umich.edu/I-TASSER/),
then water molecules were removed, polar hydrogen atoms were added, Compute Gasteiger charges
were added, and AD 4 type atoms were assigned [41]. The 3D structure of ligands were constructed
and their energy minimization were performed using ChemOffice 2004. Following by the structural
optimization, all ligands were prepared for docking by merging non-polar hydrogen atoms, detecting
rotatable bonds and adding gasteiger charges [41]. The grid box size of 60
×
60
×
60 Å was generated
and allocated to center of binding cavity using x, y and z coordinates of 102.273, 100.115, and 118.080
for intend searching modality. Other parameters were set as the default. The Lamarckian genetic
algorithmwas applied to calculate the possible conformation of the ligand molecule and macromolecule.
Finally, the docking results were analyzed using the free version of Discovery Studio Visualizer 4.5
(Accelrys Software Inc., San Diego, CA, USA) [58].
3.6. In Silico ADME Prediction
On the basis of Lipinski’s rule of five and its extensions [59], we calculated molecular volume
(MV), molecular weight (MW), logarithm of partition coefficient (miLogP), number of hydrogen
bond acceptors (n-ON), number of hydrogen bonds donors (n-OHNH), topological polar surface area
(TPSA), number of rotatable bonds (n-ROTB) and Lipinski’s rule of five using Molinspiration online
property calculation toolkit [60]. Absorption (% ABS) was calculated as follows: % ABS = 109
× TPSA) [61].
−
(0.345
4. Conclusions
Thirty phenolic ether derivatives of scopoletin including twelve compounds with amide groups
were synthesized successfully using a molecular hybridization method. Their acaricidal activities,
QSAR, molecular docking and a silico ADME properties were investigated. Some of these compounds
exhibit more pronounced acaricidal activity than scopoletin, especially compounds 32, 20
exhibited about 8.41-, 7.32-, 7.23-, 6.76-, and 6.65-fold higher acaricidal potency than scopoletin.
Compound 32 possessed the the most promising acaricidal activity and exhibited about 1.45-fold
, 28, 27 and
8

Molecules 2018, 23, 995
26 of 29
higher acaricidal potency against T. cinnabarinus than propargite. Statistically significant 2D-QSAR
model supports the observed acaricidal activities and reveals that polarizability (HATS5p) was the
most important parameter controlling bioactivity. 3D-QSAR (CoMFA: q2 = 0.802, r2 = 0.993; CoMSIA:
q2 = 0.735, r2 = 0.965) results show that bulky substituents at R4, R1, R2 and R5 (C6, C3, C4, and
C7) positions, electron positive groups at the R5 (C7) position, hydrophobic groups at the R1 (C3)
and R2 (C4), H-bond donors groups at R1 (C3) and R4 (C6) will increase their acaricidal activity,
which provide a good insight into the molecular features relevant to the acaricidal activity for further
designing novel acaricidal agents. Molecular docking demonstrates that these selected derivatives
display different bide modes with TcPMCA1 from lead compound and they interact with more key
amino acid residues than scopoletin. In silico ADME properties study of scopoletin and its phenolic
ether derivatives were also analyzed and showed potential to develop these compounds as good
acaricidal candidates.
1
Supplementary Materials: H-NMR and 13C-NMR of representive compounds.
Author Contributions: Jinxiang Luo, Ting Lai, Wei Ding and Yongqiang Zhang conceived and designed the
experiments; Jinxiang Luo, Ting Lai, Tao Guo, Fei Chen performed the experiments and analyzed the data;
Jinxiang Luo wrote the paper; Jinxiang Luo, Linli Zhang, Wei Ding and Yongqiang Zhang revised the paper.
Acknowledgments: We are grateful to Yuwei Wang in School of Pharmacy, Lanzhou University for Molecular
Docking and 3D-QSAR analysis. This work was supported by the Chinese National Nature Science Foundation
(31272058, 31572041 and 31601674), Chongqing Municipal Natural Science Foundation of China (cstc2016jcyjA0501),
and the Fundamental Research Fund for the Central Universities of China (No. XDJK2014C183).
Conflicts of Interest: The authors declare that they have no conflict of interest.
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2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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