Design, Synthesis, and Antiviral Activities of Coumarin Derivatives Containing Dithioacetal Structures

Article abstract of DOI:10.1021/acs.jafc.9b06861

In this study, a series of coumarin derivatives containing dithioacetals were synthesized, characterized, and assessed for their anti-tobacco mosaic virus (TMV) activities. Biological tests showed that most of the title compounds exhibited significant anti-TMV biological activities; in particular, compound b21 showed good inactivating activity anti-TMV, with an EC₅₀ of 54.2 mg/L, superior to that of ribavirin (134.2 mg/L). Transmission electron microscopy analyses showed that compound 21 severely ruptured TMV particles. The interaction of compound b21 with TMV coat protein (TMV CP) was investigated using microscale thermophoresis and molecular docking. Compound b21 exhibited a strong binding ability to TMV CP, with a value of 2.9 μM, superior to ribavirin.

Full text of DOI:10.1021/acs.jafc.9b06861

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Agricultural and Environmental Chemistry
Design, Synthesis and Antiviral Activity of Coumarin
Derivatives Containing Dithioacetal Structures
Lei Zhao, Jian Zhang, Ting Liu, Honglan Mou, Chunle Wei, De-Yu Hu, and Baoan Song
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b06861 • Publication Date (Web): 31 Dec 2019
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Figure 1. Chemical structures of Cumarin, Ribavirin and Ningnanmycin.
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Figure 2. Design of title compounds
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Figure 3. Synthetic route for title compounds b1-b30.
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Figure 4. Microscale thermophoresis (MST) test results of compounds b20 (A), b24 (B), b21 (C), and
Ribavirin (D) with TMV CP.
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Figure 5. Molecule docking results of compounds b20 (A), b24 (B), b21 (C), and Ribavirin (D) with TMV CP
219x128mm (300 x 300 DPI)
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Figure 6. The effect on the morphology of TMV particles of compounds CK (A), Ribavirin (B) and b21 (C).
216x80mm (300 x 300 DPI)
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Design, Synthesis and Antiviral Activity of Coumarin
Derivatives Containing Dithioacetal Structures
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Lei Zhao, Jian Zhang, Ting Liu, Honglan Mou, Chunle Wei, Deyu Hu, Baoan Song*
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Current address: State Key Laboratory Breeding Base of Green Pesticide and
Agricultural Bioengineering, Key Laboratory of Green Pesticide and Agricultural
Bioengineering, Ministry of Education, Guizhou University, Huaxi District, Guiyang
550025, China.
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*Address of the corresponding author
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Fax: 0086-851-83622211; E-mail: songbaoan22@yahoo.com; basong@gzu.edu.cn.
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ABSTRACT: In this study, a series of coumarin derivatives containing dithioacetals
were synthesized, characterized, and assessed for their anti-tobacco mosaic virus
(TMV) activity. Biological tests showed that most of the title compounds exhibited
significant anti-TMV biological activity; in particular, compound b21 showed good
inactivating activity anti-TMV, with an EC50 of 54.2 mg/L, superior to that of
ribavirin (134.2 mg/L). Transmission electron microscopy analyses showed that
compound 21 severely ruptured TMV particles. The interaction of compound b21
with TMV coat protein (TMV CP) was investigated using microscale thermophoresis
(MST) and molecular docking. Compound b21 exhibited a strong binding ability (Kd)
to TMV CP, with a value of 2.9 μM, superior to ribavirin.
KEYWORDS: coumarin, dithioacetal, tobacco mosaic virus, transmission electron
microscopy, molecular docking
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INTRODUCTION
Tobacco mosaic virus (TMV) is known as plant cancer and is widely distributed in
nature. TMV is the most persistent plant virus, being able to survive in dry plants for
up to 100 years, and more than 885 species in 165 plant families can be infected by
the virus.1 It is difficult to effectively control TMV once a plant is infected by.
Ribavirin and ningnanmycin (Figure 1) are two current antiviral agents used to control
plant viral diseases, but the former has a poor effect, while the latter has an
unsatisfactory field control effect.2,3 Therefore, new, broad-spectrum, and highly
effective anti-TMV agents need to be developed.
Figure 1
Natural products have been increasingly used in the creation of new pesticides in
recent years because of their unique safety.4 Coumarin (Figure 1) are an important
organic heterocyclic compound that has pyranone as a parent ring structure. Natural
coumarins are present in many high-altitude plants in nature such as Umbelliferae,
Rutaceae, Leguminosae, Compositae, Orchidaceae, etc. Furthermore, coumarins have
a variety of unique physiological and biological activities, such as anti-cancer,5
anti-HIV,6,7 antibacterial,8,9 antioxidant and anti-inflammatory,10,11 anti-influenza,12
anti-tuberculosis,13 anti-Alzheimer's disease,14,15 antiviral16,17,18 and other biological
and medicinal activities.
Our group has reported a series of vanillin derivatives containing dithioacetal
(Figure 2) which are very active against cucumber mosaic virus (CMV) and potato
mosaic virus (PVY).19 By further derivatization, our group synthesized a series of
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dithioacetal derivatives with anti-CMV, TMV, PVY and tomato chlorosis virus (ToCV)
activities.20,21 However, no compounds with good curative, protective and passivation
activity against TMV have been obtained, especially compounds without very good
inactivating activity against TMV. Therefore, in the present study, a series of coumarin
derivatives containing dithioacetal (Figure 2) were designed and synthesized, and
their anti-TMV protective, curative and inactivating activities were tested. The results
of biological activity assays indicate that most of these compounds exhibit excellent
anti-TMV activity.
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Figure 2
MATERIALS AND METHODS
Chemicals and Instruments. Reagents and solvents were purchased from
commercial sources. Reaction progress was monitored by thin-layer chromatography
(TLC) on silica GF254, and the melting points of compounds were confirmed using a
WRX-4 monocular microscope (Shanghai Yice Apparatus & Equipment Co., Ltd,
China). 1H NMR and 13C NMR were conducted on a Bruker Ascend-400 spectrometer
(Bruker, Germany) with deuterated chloroform (CDCl3) and dimethyl sulfoxide
(DMSO-d6) as the solvent, while tetramethyl silane (TMS) was used as an internal
standard. High-resolution mass spectrometry (HRMS) data were obtained using a
Thermo Scientific Q Exactive instrument (Thermo, Missouri, USA).
General Procedure for the preparation of intermediates 2 and a1-a5. The
synthetic route of coumarin derivatives containing dithioacetal is shown in Figure 3.
Substituted 4-bromo-2H-chromen-2-one (intermediate 2) was synthesized according
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to Balalas’s method,22 where 4-hydroxycoumarin (1 g, 6.16 mmol) was added to
toluene (12.4 mL, 0.5 M), under Ar atmosphere and heated to 100 ℃. Subsequently,
Bu4NBr (2.98 g, 9.26 mmol) was added, heating was continued until the Bu4NBr was
dissolved, and then P2O5 (1.75 g, 12.34 mmol) was added, and the mixture was further
refluxed for 3 hours. The hot organic layer was then poured out, and the lower layer
was extracted with hot toluene (2×20 mL). The combined toluene layer was then
successively washed with 5% NaHCO3 (2 × 30 mL), water (50 mL) and saturated
brine (50 mL). Subsequently, the organic layer was dried with anhydrous Na2SO4 (10
g) and concentrated in vacuum to obtain the intermediate 2 (1.34 g, 96%), m.p.
87-89 ℃.
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Intermediates a1-a5 were prepared as described in the literature.23 K2CO3 (4 mmol)
was added to a solution containing intermediate 2 (2 mmol) and salicylaldehyde (2
mmol) or substituted 4-hydroxybenzaldehyde (2 mmol) in acetonitrile, after which the
mixture was heated at 80 °C, and the reaction was monitored by TLC until the
reaction was completed. The mixture then filtered and concentrated before being
recrystallized from ethyl acetate to afford intermediates a1-a5. The representative data
for compound a1 are shown below.
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2-((2-oxo-2H-chromen-4-yl)oxy)benzaldehyde (a1), light yellow solid; yield, 62%;
m.p. 145-147 ℃; 1H NMR (400 MHz, CDCl3) δ: 10.16 (s, 1H, -CHO), 8.08-8.01 (m,
2H, Ar-H), 7.77-7.73 (m, 1H, Ar-H), 7.68-7.63 (m, 1H, Ar-H), 7.52 (t, J = 7.6 Hz, 1H,
Ar-H), 7.46-7.33 (m, 2H, Ar-H), 7.32-7.25 (m, 1H, Ar-H), 5.37 (s, 1H, -CH-); 13C
NMR (101 MHz, CDCl3) δ: 187.54 (1C), 166.40 (1C), 162.16 (1C), 153.97 (1C),
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153.78 (1C), 136.32 (1C), 133.39 (1C), 130.88 (1C), 128.31 (1C), 127.64 (1C),
124.55 (1C), 123.03 (1C), 122.85 (1C), 117.16 (1C), 114.87 (1C), 94.48 (1C); HRMS
(ESI) m/z for C16H10O4Na [M+Na]+ calcd: 289.0471, found: 289.0466.
Figure 3
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General Synthesis Procedure for the Coumarin Derivatives Containing
Dithioacetal Moiety b1-b30. The synthetic route of the title compounds b1-b30 is
shown in Figure 3. The title compounds were synthesized from the intermediate a and
various types of thiols according to the methods described in the literature.24
Compounds a1-a5 (1 mmol) and thiol (2 mmol) were dissolved in dichloromethane at
room temperature, followed by the addition of the catalyst NaHSO4/SiO2 (0.01 mmol).
The reaction was monitored by TLC. After the material was completely converted, the
solid SiO2 was removed by filtration, and the filtrate was concentrated in vacuo to
obtain the crude product, which was recrystallized from ethyl acetate to afford title
compounds b1-b30 (Figure 3). The representative data for compound b1 are shown
below.
4-(2-(bis((2-hydroxyethyl)thio)methyl)phenoxy)-2H-chromen-2-one (b1), white
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solid, yield, 76.8%; m.p. 107-109 ℃; H NMR (400 MHz, CDCl3) δ: 8.11-8.01 (m,
1H, Ar-H), 7.83-7.81 (m, 1H, Ar-H), 7.68-7.61 (m, 1H, Ar-H), 7.42-7.36 (m, 4H,
Ar-H), 7.14-7.12 (m, 1H, Ar-H), 5.44 (s, 1H, -CH-), 5.27 (s, 1H, -SCHS-), 3.66-3.63
(m, 4H, -SCH2-), 2.83-2.76 (m, 2H, -CH2-), 2.65-2.59 (m, 2H, -CH2-), 2.45 (s, 2H,
-OH); 13C NMR (101 MHz, CDCl3) δ: 165.89 (1C), 162.69 (1C), 153.71 (1C), 149.14
(1C), 133.25 (1C), 132.92 (1C), 130.10 (1C), 129.99 (1C), 127.62 (1C), 124.56 (1C),
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123.07 (1C), 121.95 (1C), 117.13 (1C), 115.12 (1C), 94.01 (1C), 61.57 (1C), 46.49
(2C), 35.69 (2C); HRMS (ESI) m/z for C20H20O5NaS2 [M+Na]+ calcd: 427.0644,
found: 427.0644.
Antiviral activity assay. Extraction of TMV25. The TMV viruses were propagated
in Nicotiana. tabacum cv. K326, ground in phosphate buffer and filtered through
multilayer gauze. The extract was centrifuged at 10,000×g for 5 min, treated twice
with polyethylene glycol 6000, centrifuged again, and the extract was stored at 4 ℃.
The absorbance values were tested using an ultraviolet spectrophotometer at 260 nm.
Curative mode Anti-TMV26 activities of title compounds in vivo. Nicotiana.
tabacum L. plants were used to evaluate the anti-TMV activities. The whole leaf was
sprinkled with silicon carbide, inoculated with TMV virus at a concentration of 6 ×
10-3 mg mL-1, rinsed with water after half an hour, and then air dried. The drug
solution was evenly applied to the right side of the leaves, while the solvent was
applied to the left side of the leaves as a control. The plants were incubated at 28 ℃ in
a greenhouse under an illumination of 10 000 lx at 28±2 ℃. After 3-4 days, the
number of local lesions was counted. Measurements were performed in triplicate.
Protective mode Anti-TMV activities of title compounds in vivo. The drug solution
was evenly applied to the right side of the leaves, while the solvent was applied to the
left side of the leaves as a control. After air drying, the plants were placed in a
greenhouse for 24 hours. The whole leaves were sprinkled with diamond, inoculated
with TMV virus, and incubated for half an hour, and then rinsed with water and air
dried. The plants were incubated at 28 ℃ in a greenhouse under an illumination of 10
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000 lx at 28±2 ℃. After 3-4 days, the number of local lesions was counted.
Measurements were performed in triplicate.
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Inactivating mode Anti-TMV activities of title compounds in vivo. Nicotiana.
tabacum L. plants were used to evaluate the anti-TMV activities. After the whole leaf
was sprinkled with silicon carbide, the drug and TMV virus solutions were mixed for
30 min and then inoculated on the left leaf, while the right leaf was inoculated with
the TMV virus solution as a control. After incubating for half an hour, the leaves were
rinsed with water and incubated at 28 ℃ in a greenhouse under an illumination of 10
000 lx at 28±2 ℃. After 3-4 days, the number of local lesions was counted.
Measurements were performed in triplicate.
Effect of compounds on the morphology of TMV particles.27,28 After mixing the
compound solution with an equal volume of the TMV virus solution and incubating
for 30 min, the mixture was adsorbed on a 200-mesh copper mesh carbon support
membrane and counterstained with 1% phosphotungstic acid with a pH of 7.4. for 30
s. After fully drying, the morphologies of the TMV particles were observed via
transmission electron microscope (TEM) with an FEI Talos F200C instrument at 200
kV.
Molecular docking. The interaction model of compounds b20, b21 and b24 with
TMVCP (PDB code: 1EI7) was studied using Discovery Studio 4.5.29,30 The
molecular docking results were processed according to a previously described
method.31
Determination of binding capacity. The interaction of compounds with TMV CP
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was studied through MST. The Kd values of compounds with TMV CP were assessed
through MST using Monolith NT.115 software (Nano Temper Technologies, Germany)
at 25 ℃ .30,32
RESULTS AND DISCUSSION
Chemistry. The synthetic route of the coumarin derivatives containing dithioacetal
is shown in Figure 3. 4-Bromocoumarin (2) was synthesized by Appel Reaction,
acetonitrile was used as a solvent and K2CO3 was used as a catalyst. The intermediate
2 was refluxed with salicylaldehyde or the correspondingly substituted
4-hydroxybenzaldehyde for 5-10 hours to produce the intermediates a1-a5, and the
title compounds b1-b30 were synthesized using NaHSO4/SiO2 as a catalyst by
condensation of a1-a5 and a substituted thiol (Figure 3). The structures of
1
intermediates a1-a5 and title compounds b1-b30 have been identified by H NMR,
13C NMR and HRMS (Supporting Information).
Antiviral Activity. Anti-TMV Activity in Vivo. The anti-TMV activities of the target
compounds b1-b30 at a concentration of 500 mg/L were evaluated by the half-leaf
method, and ribavirin was used as a reference drug.33 The anti-TMV activity results
for these compounds are shown in Table 1. The target compounds showed excellent
activity against TMV. Compounds b15, b21 and b24 showed good curative effects
with values of 65.8, 62.1 and 67.3%, respectively, which were superior to ribavirin
(40.3%). Compounds b6, b16 and b18 showed good protective effects with values of
61.9, 68.5 and 64.6%, respectively, which were superior to ribavirin (51.2%).
Moreover, compounds b15, b21 and b24 showed good inactivating effects with values
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of 81.7, 94.2 and 84.2%, respectively, which were superior to that of ribavirin (71.2%).
In addition, the EC50 of inactivating activity of compounds b15, b21 and b24 (68.4,
54.2, and 65.3 mg/L, respectively) were superior to that of ribavirin (134.2 mg/L).
Interestingly, compounds b21 and b24 showed excellent activity in all three modes
(curative, protective, and inactivating), with compound b21 showing the best
inactivating activity.
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Structure-activity relationships (SARs). SARs were determined based on the
preliminary results. The type and position of the substituent of the title compounds
greatly influenced their anti-TMV curative, protective and inactivating activities.
When R2 was an aliphatic group (Et, n-Pr and n-butyl), it increases the anti-TMV
activity of the compound was increased. For example, in terms of anti-TMV curative
activity, b15, b16 and b18 > b14; b21 and b24 > b20; and b27, b20 and b30 > b26.
However, this was not followed by all the compounds. For example, b2 > b3, b4 and
b6. In terms of protective activity, b3, b4 and b6 > b2; b9 and b12 > b8; b16 and
b18 > b14; b21, b22 and b24 > b20; and b27, b28 and b30 > b26. However, this
effect was not observed in compound b10. When R2 was aliphatic, there was no
branching (Et, n-Pr and n-butyl group), which is better than i-Pr. For example, in
terms of protective activity, b16 >b17; b22 > b23; and b28 > b29, with the exception
of b10 < b11. In terms of inactivating activity, b10 > b11; b22 > b23; and b28 > b29.
However, b17 did not follow this rule.
Determination of binding capacity. TMV CP plays a very important role in tRNA
synthesis and mRNA translation in the self-assembly of anti-TMV agents.34 Since the
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target compound exhibited significant inactivating activity, with compound b21
showing the best inactivating activity, we speculated that compound b21 was highly
active against TMV CP. Therefore, the binding affinities of b20, b21, b24 and
ribavirin toward TMV CP were measured via MST. The results of the MST analysis
are shown in Table 2 and Figure 4. The results showed that compared with ribavirin
(128.7±34.7 μM), compound b21 and TMV CP had stronger binding activity, with a
dissociation constant (Kd) of 2.9±0.8 μM, which was better than that of b20
(527.7±210.9 μM) and b24 (123.4±54.7 μM). The results of the MST analysis were
consistent with the previously observed inactivating abilities of the compounds.
Figure 4
Molecular docking. To further investigate the combination modes of the target
compounds and TMV CP, we used Discovery Studio 4.5 for molecular docking
analysis. Compounds b20, b24 and b21 were selected for molecular docking with
TMV CP (PDB code: 1EI7).32 The docking results are shown in Figure 5, and the
binding activity of these compounds with TMV-CP can be explained by hydrogen
bonding and nonhydrogen bonding interactions. As shown in Figure 5A, Compound
b20 forms one conventional hydrogen bond with the active site of TYR139 at a
distance of 2.92 Å. In addition, it forms one halogen (2.79 Å) with GLU222, one
sulfur-X (2.66 Å) with PRO254, and VAL75 (5.21 Å & 5.28 Å), LYS253 (4.64 Å),
VAL260 (5.11 Å) and ARF134 (5.38 Å) form five pi-alkyl interactions. Compound
b24 forms two conventional hydrogen bonds with amino acids LYS268 (2.68 Å) and
ARG134 (1.82 Å), as shown in Figure 5B. In addition, it forms an unfavorable
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acceptor-acceptor interaction with PRO254 (2.77 Å), forming three pi-alkyl
interactions are formed with LYS253 (4.49 Å) and VAL75 (5.40 Å & 5.28 Å).
Compound b21 forms five conventional hydrogen bonds with amino acids GLN257
(2.82 Å), ASN73 (2.38 Å), TYR139 (2.05 Å), LYS268 (2.16 Å) and ARG134 (3.09
Å), one pi-donor hydrogen bond with GLY137 (2.57 Å), and form four alkyl or
pi-Alkyl bonds with amino acids VAL260 (4.44 Å), VAL75 (4.81 Å) and LYS253
(6.26 Å), as shown in Figure 5C. Molecular docking results of ribavirin with TMV CP
showed that ribavirin forms three conventional hydrogen bonds with amino acids
LYS268 (2.48 and 1.72 Å) and ARG134 (2.03 Å), one pi-alkyl interaction with
VAL75 (4.54 Å), and one unfavorable donor-donor interaction with ASP219 (1.08 Å),
as shown in Figure 5D. The docking results indicate that compound b21 and TMV-CP
have five conventional hydrogen bonds, which are more than ribavirin, compound b20
and compound b24, suggesting that hydrogen bonding between the compound and
TMV-CP may play a critical role in their interaction.
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Figure 5
Effect of the compounds on the morphology of TMV particles. Transmission
electron microscope (TEM) studies of the effects of b21 and ribavirin on the
morphology of TMV granules (Figure 6). Normal TMV particles are linear, rod-like
structures (Figure 6A) with almost no breaks. After 30 min of interaction between
ribavirin and TMV, partial fragmentation of TMV particles was observed (Figure 6B).
When b21 and TMV interacted for 30 min, the TMV particles were observably
broken, with different lengths and short rod-like structures (Figure 6C).
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Figure 6
In summary, in this study, we synthesized a series of coumarin derivatives
containing dithioacetal moieties and tested their anti-TMV activities. The results of
the biological activity assay showed that the title compound exhibited good curative,
protective and inactivation activities. In particular, compounds b15, b21 and b24
exhibited excellent anti-TMV inactivation activity with EC50 values of 68.4, 54.2 and
65.3 mg/L, respectively. SAR analysis results showed that the introduction of
unbranched aliphatic groups (Et, n-Pr and n-butyl) into the compound enhances their
anti-TMV activity. With respect to the title compound, it has good anti-TMV
inactivation activity, and compound b21 exhibited good binding ability toward TMV
CP through molecular docking analysis. The results of MST experiments showed that
compound b21 has strong binding ability with TMV CP, which is consistent with the
results of molecular docking experiments. The TEM results showed that the
morphology of the TMV particles was severely disrupted and ruptured in the presence
of compound b21. These findings indicate that compound b21 has a strong binding
ability toward TMV and can disrupt its structure and cause its rupture, thereby
inactivating the virus and inhibiting the infection of plants. The results of the current
study provide reliable support for the use of the dithioacetal-containing coumarin
structure as a potential lead compound for anti-TMV virus agents.
CONTENT ASSOCIATED
Supporting Information
1
The synthesis, physical analysis, H NMR spectra, 13C NMR spectra and
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high-resolution mass spectrum (HRMS) of compounds a1-a5 and b1-b30 can be
found in the supplementary data.
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Corresponding Authors
*E-mail for Baoan Song: songbaoan22@yahoo.com. *Phone: 86-851-88292170. Fax:
86-851-83622211.
ORCID
Baoan Song: 0000-0002-4237-6167
ACKNOWLEDGMENT
This work has received the financial support of the National Natural Science
Foundation of China (No. 21732002) and Subsidy Project for Outstanding Key
Laboratory of Guizhou Province in China (20154004).
Notes
The authors declare no competing financial interest.
ABRREVIATIONS USED
1
1
EC50, half-maximal effective concentration; TMS, tetramethylsilane; H NMR, H
nuclear magnetic resonance; 13C NMR, 13C nuclear magnetic resonance; HRMS, high
resolution mass spectrum; CMV, cucumber mosaic virus; TMV, tobacco mosaic virus;
PVY, potato mosaic virus; ToCV, tomato chlorosis virus; THF, tetrahydrofuran; TEM,
transmission electron microscopy; MST, microscale thermophoresis; SARs,
Structure-activity relationships; Kd, dissociation constant.
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Information of FIGURE
Figure 1. Chemical structures of Cumarin, Ribavirin and Ningnanmycin.
Figure 2. Design of title compounds.
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Figure 3. Synthetic route for title compounds b1-b30.
Figure 4. Microscale thermophoresis (MST) test results of compounds b20 (A), b24
(B), b21 (C), and Ribavirin (D) with TMV CP.
Figure 5. Molecule docking results of compounds b20 (A), b24 (B), b21 (C), and
Ribavirin (D) with TMV CP
Figure 6. The effect on the morphology of TMV particles of compounds CK (A),
Ribavirin (B) and b21 (C).
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Table 1. Antiviral activities of target compounds b1-b30 against TMV in vivo at
500 mg/L
EC50 for TMV
Curative
Protective
Inactivating
Compound
inactivating activitya
(mg/L)
activitya (%)
activitya (%) activitya (%)
43.7±1.7
56.9±1.8
45.6±1.3
34.2±3.7
49.3±2.8
47.2±2.2
44.2±1.9
44.1±4.3
46.0±3.3
44.1±4.4
41.4±5.0
42.2±1.1
24.1±4.0
38.2±4.5
65.8±2.3
47.3±4.3
45.3±2.5
49.7±2.1
47.6±1.2
42.7±2.0
50.5±3.3
45.1±2.5
43.7±3.5
61.9±1.4
39.4±1.9
31.5±4.4
45.6±1.2
25.5±1.6
43.1±2.5
55.5±1.5
49.6±5.0
50.8±3.8
49.4±3.1
68.2±3.7
25.7±3.2
64.6±1.0
60.5±4.8
67.9±1.9
73.3±4.5
63.2±3.6
64.2±4.9
78.3±3.2
53.2±4.2
62.8±4.4
77.4±4.9
76.2±4.2
69.7±3.2
78.4±4.2
74.5±4.0
63.5±5.1
81.7±2.0
74.1±1.5
79.0±4.5
68.2±3.8
242.7±3.6
183.2±4.3
117.8±4.6
262.4±3.3
220.5±7.8
75.3±2.7
b1
b2
b3
b4
b5
b6
363.7±3.2
248.9±2.8
98.2±4.5
b7
b8
b9
104.3±4.3
162.3±5.3
102.2±2.8
132.4±6.4
216.4±3.1
68.4±4.4
b10
b11
b12
b13
b14
b15
b16
b17
b18
125.5±3.5
84.4±2.0
194.2±1.9
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