Synthesis and in vitro antibacterial activity of quaternized 10-methoxycanthin-6-one derivatives

Article abstract of DOI:10.3390/molecules24081553

Background: Based on our previous work, we found that 10-methoxycanthin-6-one displayed potential antibacterial activity and quaternization was an available method for increasing the antibacterial activity. Here, we explored the antibacterial activity of quaternized 10-methoxy canthin-6-one derivatives. Methods and Results: Twenty-two new 3-N-benzylated 10-methoxy canthin-6-ones were designed and synthesized through quaternization reaction. The in vitro antibacterial activity against three bacteria was evaluated by the double dilution method. Moreover, the structure–activity relationships (SARs) were carefully summarized in order to guide the development of antibacterial canthin-6-one agents. Two highly active compounds (6p and 6t) displayed 8-fold superiority (MIC = 3.91 μg/mL) against agricultural pathogenic bacteria R. solanacearum and P. syringae compared to agrochemical streptomycin sulfate, and showed potential activity against B. cereus. Moreover, these two compounds exhibited good “drug-like” properties, low cytotoxicity, and no inhibition on seed germination. Conclusions: This work provides two new effective quaternized canthin-6-one derivatives as candidate bactericide, promoting the development of natural-sourced bactericides and preservatives.

Full text of DOI:10.3390/molecules24081553

molecules
Article
Synthesis and In Vitro Antibacterial Activity of
Quaternized 10-Methoxycanthin-6-one Derivatives
Na Li , Dan Liu , Jiang-Kun Dai , Jin-Yi Wang 1,2,* and Jun-Ru Wang 2,*
1
2
2
1
College of Veterinary Medicine, Northwest A&F University, Yangling 712100, Shaanxi, China;
lnuk@nwsuaf.edu.cn
2
College of Chemistry and Pharmacy, Northwest A&F University, Yangling 712100, Shaanxi, China;
ld127222@126.com (D.L.); daijkun@hotmail.com (J.-K.D.)
*
Correspondence: jywang@nwsuaf.edu.cn (J.-Y.W.); wangjunru@nwafu.edu.cn (J.-R.W.)
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 27 March 2019; Accepted: 16 April 2019; Published: 19 April 2019
Abstract: Background: Based on our previous work, we found that 10-methoxycanthin-6-one
displayed potential antibacterial activity and quaternization was an available method for increasing
the antibacterial activity. Here, we explored the antibacterial activity of quaternized 10-methoxy
canthin-6-one derivatives. Methods and Results: Twenty-two new 3-N-benzylated 10-methoxy
canthin-6-ones were designed and synthesized through quaternization reaction. The in vitro
antibacterial activity against three bacteria was evaluated by the double dilution method. Moreover, the
structure–activity relationships (SARs) were carefully summarized in order to guide the development
of antibacterial canthin-6-one agents. Two highly active compounds (6p and 6t) displayed 8-fold
superiority (MIC = 3.91 µg/mL) against agricultural pathogenic bacteria R. solanacearum and P. syringae
compared to agrochemical streptomycin sulfate, and showed potential activity against B. cereus.
Moreover, these two compounds exhibited good “drug-like” properties, low cytotoxicity, and no
inhibition on seed germination. Conclusions: This work provides two new effective quaternized
canthin-6-one derivatives as candidate bactericide, promoting the development of natural-sourced
bactericides and preservatives.
Keywords: 10-methoxycanthin-6-one; quaternization; antibacterial; SARs
1
. Introduction
Healthcare systems, farming, and the food production industry are the main sectors driving
antibiotic consumption [
1]. To maintain agricultural production and food safety, there are many
circumstances where antibiotics are required in the farming and food industry [2,3
]. Bacillus cereus
is pathogenic and is the common cause of spoilage of milk, cream and cooked rice. When people
eat B. cereus contaminated food, they will suffer from food poisoning with these symptoms’ nausea,
vomiting and diarrhea [
consumers are increasingly concerned about the safety of the pure synthetic chemicals [
the development of effective natural-sourced antibacterial agents for food preservation and food
safety is in great demand [ ]. Ralstonia solanacearum and Pseudomonas syringae, plant pathogens, could
infect the plant tissues during the whole plant growth cycle [ ]. Although chemical pesticides work
quickly, they cause serious environmental pollution, human and animal poisoning, and pesticide
residues [ ]. Recently, structural modifications of natural products for the discovery of agrochemical
candidates have received much attention because of their low toxicity, low residue and environmental
friendliness [ ]. Canthin-6-one 1, mainly from the Rutaceae and Simaroubaceae families, are a
subclass of the tryptophan-derived -carboline alkaloids [10 11]. In our previous work, we found
that quaternized modification could significantly improve the antibacterial activity (Figure 1) [12 13].
4]. The synthetic preservatives could fully retain food nutrients. However,
5
]. Therefore,
6
7
8
8,9
β
,
,
Molecules 2019, 24, 1553; doi:10.3390/molecules24081553
www.mdpi.com/journal/molecules

Molecules 2019, 24, 1553
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For example, the antibacterial activity of compounds
2
and
4
against B. cereus enhanced 2-fold
and 8-fold, respectively. Interestingly, 10-methoxycanthin-6-one
5, per se has good antibacterial
activity [14 15]. With the aim of finding more promising antibacterial canthin-6-one analogues,
,
we report here a simple idea and design to generate the bioactive compounds by quaternization
reaction of 10-methoxycanthin-6-one 5.
Note: MIC refers to minimum inhibitory concentration.
Figure 1. Design of the quaternized 10-methoxycanthin-6-one analogues.
2
. Results and Discussion
2
.1. Chemistry
The synthetic route of target compounds 6a–6v was outlined in Scheme 1. The N -benzyl-5-
b
methoxytryptamine
8
was prepared through Borch Reduction with 5-methoxytryptamine
7
as
was
obtained by the key Pictet–Spengler reaction (83% yield). The debenzylated compound 10
hexahydro-10-methoxycanthin-6-one) was sequentially obtained by catalytic hydrogen transfer
reaction (75% yield). Finally, the natural product 10-methoxycanthin-6-one was prepared by
oxidation reaction (90% yield) [15]. With this compound in hand, attention was focused on modifying
the 3-N position. A benzyl group in this position significantly increased the activity (Figure 1). Thus, we
synthesized a series of quaternized 10-methoxycanthin-6-one derivatives 6a 6v (42–72% yields) with
the starting material (98% yield). Then, N -benzylhexahydro-10-methoxycanthin-6-one
9
b
(
5
5
–
diverse substituted benzyl group which varied in electron-inducing ability and substitution position.
The purity of the compounds (>99%) has been provided using high performance liquid
chromatography (HPLC), which could be found in the Supporting Information. All the structures of the
target compounds (
NMR spectra of compound 6a, the signal of the methylene group was detected around
= 56.6 ppm, respectively, which indicated that the quaternized 10-methoxycanthin-6-one derivatives
were successfully synthesized. Moreover, the charactermatic signals of the methyl group were easily
5
and 6a
–
6v) were confirmed by NMR and high resolution MS (HRMS) spectra. In the
δ
= 6.39 ppm and
δ
1
13
detected around
δ
= 3.96 ppm ( H NMR) and
Br] could be found at 359.1182 Da in HRMS spectra of compound 6a (error = 2.2 ppm), which
confirmed to the theoretical value 359.1190 Da within the allowable error range (error < 5 ppm).
δ = 54.4 ppm ( C NMR). In addition, the signal of
+
[
M
−

Molecules 2019, 24, 1553
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Scheme 1. Synthetic route and chemical structures of compounds
) i: benzaldehyde, MeOH, r.t.; ii: NaBH , r.t., 98%; ( -ketoglutaric acid, p-TSA, dry toluene:dioxane
) HCOONH , 5% Pd/C, MeOH:toluene = 1:1, heated, 75%; ( ) 5% Pd/C,
5 and 6a–6v. Reagents and conditions:
(a
b) α
4
=
3:2, DST, reflux, 83%; (
c
d
4
xylene, reflux, 90%; (e) CH CN, reflux, r.t., 42–72%.
3
2
.2. Antibacterial Activity
The antibacterial activity in vitro of compounds
5 and 6a–6v were evaluated against bacteria
(
B
.
cereus, R. solanacearum and P. syringae) using the double dilution method giving the minimum
inhibitory concentration values (MICs) [16]. The concentration gradient was set according to our
previous work [12 15]. Broad spectrum antibiotic fosfomycin sodium and ampicillin sodium,
and agrochemical bactericide streptomycin sulfate were used as the positive controls [17 18].
,
,
The antibacterial results (Table 1) revealed that most of the quaternized 10-methoxycanthin-6-one
derivatives displayed good in vitro biological activity against R. solanacearum and P. syringae compared
with agrochemical streptomycin sulfate. Eight compounds (6e
-fold superiority (MIC = 3.91 g/mL) against R. solanacearum than lead compound
superiority than streptomycin sulfate. Five compounds (6f 6h 6i 6p and 6t) displayed 2-fold
superiority (MIC = 3.91 g/mL) against P. syringae compared to lead compound and 8-fold superiority
than streptomycin sulfate. Unfortunately, quaternized modification did not improve the activity for
B. cereus. It is worth mentioning that four compounds (6p 6q 6r and 6t) still have potential antibacterial
activity against B. cereus compared with natural canthin-6-one . Overall, compounds 6p and 6t were
considered to be the most highly active derivatives against these three bacteria.
,
6f
,
6i
,
6k
,
6l
,
6m
,
6p and 6t) displayed
4
µ
5
and 8-fold
,
,
,
µ
5
,
,
1

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Table 1. Antibacterial activity of compounds 5 and 6a–6v against three bacteria (MIC, µg/mL).
No.
R
X
B. cereus
R. solanacearum
P. syringae
6
a
-CH Ph(o-F)
Br
Br
Br
Cl
Cl
Cl
Br
Br
Br
Cl
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Cl
-
15.63
15.63
31.25
62.50
15.63
15.63
15.63
15.63
15.63
15.63
15.63
15.63
15.63
31.25
15.63
7.81
15.63
15.63
7.81
15.63
3.91
3.91
7.81
7.81
3.91
15.63
3.91
3.91
3.91
7.81
15.63
3.91
7.81
7.81
7.81
3.91
15.63
15.63
15.63
1.95
7.81
31.25
15.63
15.63
15.63
15.63
7.81
3.91
7.81
3.91
3.91
7.81
7.81
7.81
15.63
15.63
7.81
3.91
15.63
7.81
2
6
b
-CH Ph(p-F)
2
6
c
-CH Ph(m-F)
2
6
d
-CH Ph(m-CN)
2
6
e
-CH Ph(o-Cl)
2
6
f
-CH Ph(m-Cl)
2
6
g
-CH Ph(o-Me)
2
6
h
-CH Ph(p-Me)
2
6
6
i
j
-CH Ph(m-Me)
2
-CH Ph(m-OMe)
2
6
k
-CH Ph(o-CF )
2
3
6
l
-CH Ph(p-CF )
2
3
6
m
-CH Ph(m-CF )
2
3
6
n
-CH Ph(2,5-difluoro)
2
6
6
6
o
p
q
-CH Ph(2,4-difluoro)
2
-CH Ph(3,4-dichloro)
2
-CH Ph(o-Br)
7.81
7.81
2
6
6
6
r
s
t
-CH Ph(p-Br)
2
-CH Ph(m-Br)
15.63
7.81
15.63
15.63
3.91
3.91
3.91
15.63
7.81
3.91
2
-CH Ph(m-I)
2
6
u
-CH Ph
15.63
15.63
7.81
15.63
0.98
2
6
v
-CH Ph
2
5
-
-
-
-
a
F.S.
A.S.
S.S.
-
-
-
a
a
31.25
a
Fosfomycin sodium; Ampicillin sodium; Streptomycin sulfate.
2
.3. Structure–Activity Relationships
Based on the antibacterial activity data, the structure–activity relationships were carefully
investigated for R. solanacearum and P. syringae. Halogen anion did not affect the activity. For example,
the antibacterial activity of compounds 6u and 6v are equal. The substituted benzyl groups are
favorable for most of the quaternized derivatives. The detailed SARs of a diverse substituted benzyl
group which varied in electron-inducing ability and substitution position were summarized in Table 2.
In terms of halogen atom substitution, fluorine substituted benzyl groups are disadvantageous for
improving the activity. Chlorine and iodine substituents may be better. For example, compound 6t with
meta-iodine atom substituent displayed 4-fold superiority against P. syringae than compound 6c which
was replaced by a fluorine atom at meta-position. In most instances, large sterically hindered groups
such as methyl and trifluoromethyl are beneficial for activity enhancement. For example, compound 6k
displayed 8-fold superiority (MIC = 3.91 µg/mL) against R. solanacearum compared to positive control
streptomycin sulfate, which was replaced by the trifluoromethyl group at ortho-position. However,
cyano and methoxy groups were exceptional and complex, which need further study. Multiple
substitutions showed different activity trends. Compound 6p exhibited excellent antibacterial activity
compared with agrochemical streptomycin sulfate. Interestingly, multiple fluorine substituted benzyl
groups are still harmful, such as compounds 6n and 6o.

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Table 2. Structure–activity relationships (SARs) against R. solanacearum and P. syringae.
Location
Bacteria Strains
SARs
R. solanacearum
P. syringae
F < Br = Me < Cl = CF3
F = Br < Cl = Me = CF3
Ortho-position
Meta-position
Para-position
R. solanacearum
P. syringae
CN = OMe < F = Br < Cl = I = Me = CF3
F = CN = CF < OMe = Br < Cl = I = Me
3
R. solanacearum
P. syringae
F < Br = Me < CF3
F < Br = CF < Me
3
2
.4. Molecular Physicochemical Properties
In 1997, Lipinski et al. published what is widely regarded as the key paper defining
physicochemical and structural properties profiles for the optimal oral availability of drugs [19].
In 2003, Clarke and Delaney reported that most herbicides and fungicides also adhere to the Lipinski
rule [20]. The molecular properties of compounds 5, 6p and 6t are shown in Table 3. For all the preferred
compounds, the logP values were lower than 5, the nON values were lower than 10, the nONNH
values were lower than 10, the MW values were lower than 500 and the nrotb values were lower
than 5. Their physicochemical data conform to the Lipinski rule which demonstrate they may have
good “drug-like” properties. Further more, the topological polar surface area (TPSA), a parameter,
defined as the surface sum over all polar atoms and used extensively in medicinal chemistry to predict
absorption and optimize a compound’s membrane permeability, was also calculated. It is suggested
2
that compounds with a TPSA greater than 140 Å tend to be poor at permeating cell membranes [21].
Based on the predicted data mentioned above, the calculated TPSA values for these three compounds
would indicate a possible good bioavailability. Interestingly, the TPSA values of 6p and 6t are lower
than
5, which indicate that the modified molecules may pass through the bacteria cell membrane
more easily.
Table 3. Calculated molecular properties of some potential compounds.
TPSA b
nON c
nONNH d
Nrotb e
MW f
No.
LogP a
5
2.72
1.18
0.98
43.61
34.6
34.6
4
4
4
0
0
0
1
3
3
250.2
410.3
467.3
6
p
6t
a
LogP = octanol–water partition coefficient, b TPSA = topologic polar surface area, nON = number of hydrogen
c
bond acceptors, d nONNH = number of hydrogen bond donors, nrotb = number of rotatable bonds, f MW =
e
molecular weight.
2
.5. In Vitro Toxicity of the Highly Active Derivatives 6p and 6t
Many natural and synthetic agents with the ability to interact with DNA often have a relatively
low therapeutic index, most likely due to the unspecific manner of these agents in causing DNA
damage both in lesions and in highly proliferative normal tissues [22]. Similarly, the frequency of
utilization of
β-carbolines is greatly reduced because of their cytotoxic activity toward mammalian
cells (low selectivity) [14
,22]. We determined the cytotoxic effects of the highly bioactive quaternized
derivatives 6p and 6t on BHK-21 cells (Baby hamster Syrian kidney cells). As shown in Table 4, the cell
viabilities of all the tested compounds were more than 96% on BHK-21 cells even up to concentrations
of 15.62
µg/mL (2-fold or 4-fold MIC values). The results suggest that these molecules are of low
toxicity against mammalian cells when they act as antibacterial agents.

Molecules 2019, 24, 1553
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Table 4. The cell viability of highly active compounds 6p and 6t.
Cell Viability (%)
No.
3
.91 µg/mL
7.81 µg/mL
15.62 µg/mL
6
6
p
t
99.7 ± 2.9
98.7 ± 2.2
98.2 ± 2.7
98.2 ± 4.1
96.3 ± 4.4
96.1 ± 3.8
2
.6. Germination Rate Evaluation of the Highly Active Derivatives 6p and 6t
The disease caused by R. solanacearum could be controlled before planting [23]. Thus, the effect
of bactericides on seed germination rate is more important. Compounds 6p and 6t were evaluated
for allelopathic activity against dicotyledon turnip (Raphanus sativus) and monocotyledon wheat
(
Triticum aestivum L.) seeds by determining the germination rates with respect to the positive control,
glyphosate, a broad-spectrum systemic herbicide [14]. As shown in Table 5, compounds 6p and 6t
showed no inhibition on germination rates under an effective antibacterial dose (3.91 g/mL), which
µ
indicated that these molecules would not inhibit plant growth when it acts as bactericides.
Table 5. The germination rate on turnip (Raphanus sativus) and wheat (Triticum aestivum L.) of
compounds 6p and 6t (3.91 µg/mL).
No.
Turnip (%)
Wheat (%)
6
6
GP
CK
p
t
94 ± 0.33
91 ± 0.87
76 ± 0.33
93 ± 0.53
93 ± 0.89
96 ± 0.55
72 ± 0.83
95 ± 0.57
3
. Materials and Methods
3
.1. General Details
All the reagents and solvents were obtained locally or purified according to standard methods.
Melting points were determined using a digital melting-point apparatus and were uncorrected.
1
13
H NMR (500 MHz) and C NMR (125 MHz) spectra were recorded using a Bruker Avance III
00 MHz instrument (Bruker, Madison, WI, USA) with Tetramethylsilane(TMS) as the internal standard
5
and dimethylsulfoxide (DMSO-d ) as the solvent. High-resolution mass spectroscopy (HRMS) was
6
+
undertaken using an AB SCIEX Triple TOF 5600 spectrometer (Carlsbad, CA, USA). The purity of all
compounds was detected by HPLC (Shimadzu LC-20A, Tokyo, Japan) with the system equipped with
a C18 trap column (4.6 mm i.d.
50%) with the flow rate of 1.0 mL/min. The silica gel and GF254 silica gel of analytical thin-layer
chromatography (TLC) were produced by the Qingdao Haiyang Chemical Co., Ltd. (Qingdao, China).
×
250 mm, 5 µm). The mobile phase was an aqueous methanol solution
(
3
.2. Synthesis of Target Compounds 6a−6v
The 10-methoxy canthin-6-one could be easily obtained according to our previous work [15].
5
Compound
5
was dissolved in CH CN (50 mL) and the diverse substituted benzyl bromide or chloride
3
◦
(5 eq.) was added. The solution was then stirred at 80 C until the reaction is completed. The reaction
solution was concentrated under reduced pressure, and purified by flash column chromatography
using chloroform/methanol (30:1, v/v) as the eluent.
3
-(2-Fluorobenzyl)-10-methoxy-6-oxo-6H-indolo[3,2,1-de][1,5]naphthyridin-3-ium bromide 6a: yield 60%;
◦
1
yellow-green solid powder; m.p. 224.2–224.7 C; H NMR (500 MHz, DMSO-d ) δ 9.29 (d, J = 5.0 Hz,
6
1H), 8.96 (d, J = 5.0 Hz, 1H), 8.59 (d, J = 10.0 Hz, 1H), 8.43 (d, J = 10.0 Hz, 1H), 8.25 (d, J = 5.0 Hz, 1H),
7
.58–7.50 (m, 1H), 7.51–7.46 (m, 1H), 7.40 (d, J = 10.0 Hz, 1H), 7.36–7.32 (m, 2H), 7.24 (d, J = 10.0 Hz,
13
1H), 6.39 (s, 2H), 3.96 (s, 3H); C NMR (125 MHz, DMSO-d6) δ 158.7, 157.5, 142.6, 136.7, 136.3, 134.6,

Molecules 2019, 24, 1553
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1
34.5, 132.0, 131.9, 130.6, 130.2, 130.0, 125.7, 124.1, 122.8, 120.1, 117.9, 116.5, 116.3, 109.3, 56.6, 54.4.
+
HRMS (ESI) m/z calcd for C H BrFN O [M − Br] 359.1190, found 359.1182.
22
16
2
2
3
-(4-Fluorobenzyl)-10-methoxy-6-oxo-6H-indolo[3,2,1-de][1,5]naphthyridin-3-ium bromide 6b: yield 61%;
◦
1
yellow-green solid powder; m.p. 233.2–235.0 C; H NMR (500 MHz, DMSO-d )
δ
9.44 (d, J = 5.0 Hz,
H), 9.03 (d, J = 5.0 Hz, 1H), 8.67 (d, J = 10.0 Hz, 1H), 8.43 (d, J = 5.0 Hz, 1H), 8.31 (s, 1H), 7.58 7.54
m, 3H), 7.40 (d, J = 10.0 Hz, 1H), 7.32
7.28 (m, 2H), 6.35 (s, 2H), 3.98 (s, 3H); 13C NMR (125 MHz,
158.6, 157.5, 142.4, 136.5, 136.2, 134.6, 131.3, 130.6, 130.5, 130.3, 130.1, 124.1, 122.6, 120.3,
6
1
(
−
−
DMSO-d6)
δ
+
117.8, 116.5, 116.4, 109.4, 58.5, 56.6. HRMS (ESI) m/z calcd for C H BrFN O [M
−
Br] 359.1190,
22
16
2
2
found 359.1180.
3
-(3-Fluorobenzyl)-10-methoxy-6-oxo-6H-indolo[3,2,1-de][1,5]naphthyridin-3-ium bromide 6c: yield 65%;
◦
1
brick yellow solid powder; m.p. 241.6–241.9 C; H NMR (500 MHz, DMSO-d )
δ
9.40 (d, J = 5.0 Hz,
H), 9.00 (d, J = 5.0 Hz, 1H), 8.64 (d, J = 10.0 Hz, 1H), 8.40 (d, J = 10.0 Hz, 1H), 8.27 (d, J = 2.5 Hz,
H), 7.55–7.52 (m, 3H), 7.51 (d, J = 10.0 Hz, 1H), 7.39–7.25 (m, 2H), 6.31 (s, 2H), 3.95 (s, 3H); 13C NMR
163.7, 161.7, 158.6, 157.5, 142.4, 136.5, 136.2, 134.6, 134.6, 130.6, 130.5, 130.3,
30.1, 124.1, 122.6, 120.2, 117.8, 116.5, 116.4, 109.3, 58.5, 56.6. HRMS (ESI) m/z calcd for C H BrFN O
6
1
1
(125 MHz, DMSO-d6) δ
1
22
16
2
2
+
[M − Br] 359.1190, found 359.1184.
3
-(3-Cyanobenzyl)-10-methoxy-6-oxo-6H-indolo[3,2,1-de][1,5]naphthyridin-3-ium chloride 6d: yield 45%;
◦
1
brick red solid powder; m.p. 225.8–227.7 C; H NMR (500 MHz, DMSO-d )
δ
9.38 (d, J = 10.0 Hz,
H), 9.02 (d, J = 5.0 Hz, 1H), 8.53 (d, J = 10.0 Hz, 1H), 8.46 (d, J = 10.0 Hz, 1H), 8.30 (s, 1H), 7.94 (d,
J = 10.0 Hz, 2H), 7.60 (d, J = 10 Hz, 1H), 7.55 (d, J = 5.0 Hz, 2H), 7.39 (d, J = 10.0 Hz, 1H), 6.45 (s, 2H),
.99 (s, 3H); 13C NMR (125 MHz, DMSO-d6)
158.6, 157.5, 142.5, 136.5, 136.2, 135.1, 134.6, 134.5, 130.3,
30.1, 129.6, 129.4, 127.9, 124.1, 122.6, 120.2, 117.8, 112.6, 109.3, 59.3, 56.6. HRMS (ESI) m/z calcd for
6
1
3
1
δ
+
C H ClN O [M − Cl] 366.1237, found 366.1236.
23
16
3
2
3
-(2-Chlorobenzyl)-10-methoxy-6-oxo-6H-indolo[3,2,1-de][1,5]naphthyridin-3-ium chloride 6e: yield 44%;
◦
1
yellow solid powder; m.p. 231.2–232.9 C; H NMR (500 MHz, DMSO-d )
δ
9.44 (s, 1H), 9.00 (s, 1H),
.62 (d, J =10.0 Hz, 1H), 8.40 (d, J = 10.0 Hz, 1H), 8.26 (s, 1H), 7.54 (d, J = 5.0 Hz, 1H), 7.42–7.32 (m, 5H),
.36 (s, 2H), 3.95 (s, 3H); 13C NMR (125 MHz, DMSO-d6)
158.7, 157.5, 142.6, 136.6, 136.3, 136.2, 135.1,
35.1, 134.6, 130.3, 130.1, 130.1, 129.5, 129.4, 129.3, 127.9, 122.6, 120.2, 117.8, 109.5, 59.3, 56.6. HRMS
6
8
6
1
δ
+
(ESI) m/z calcd for C H Cl N O [M − Cl] 375.0894, found 375.0900.
22
16
2
2
2
3
-(3-Chlorobenzyl)-10-methoxy-6-oxo-6H-indolo[3,2,1-de][1,5]naphthyridin-3-ium chloride 6f: yield 47%;
◦
1
brick yellow solid powder; m.p. 229.5–231.2 C; H NMR (500 MHz, DMSO-d ) δ 9.11 (d, J = 5.0 Hz,
6
1
5
H), 8.75 (d, J = 5.0 Hz, 1H), 8.38 (d, J = 10.0 Hz, 1H), 8.30 (d, J = 10.0 Hz, 1H), 8.03 (s, 1H), 7.61–7.32 (m,
H), 7.23 (d, J = 10.0 Hz, 1H), 6.18 (s, 2H), 3.90 (s, 3H); C NMR (125 MHz, DMSO-d6) δ 158.6, 157.5,
13
1
1
42.5, 136.6, 136.2, 134.7, 134.6, 134.1, 134.0, 132.4, 130.3, 130.2, 130.0, 130.0, 129.5, 124.1, 122.7, 120.2,
+
17.9, 109.3, 58.5, 56.6. HRMS (ESI) m/z calcd for C H Cl N O [M − Cl] 375.0894, found 375.0891.
22
16
2
2
2
1
0-Methoxy-3-(2-methylbenzyl)-6-oxo-6H-indolo[3,2,1-de][1,5]naphthyridin-3-ium bromide 6g: yield 68%;
◦
1
brick yellow solid powder; m.p. > 280 C; H NMR (500 MHz, DMSO-d )
δ
9.18 (d, J = 10.0 Hz, 1H),
.00 (d, J = 5.0 Hz, 1H), 8.44 (d, J = 10.0 Hz, 2H), 8.31 (d, J = 2.5 Hz, 1H), 7.58 7.56 (m, 1H), 7.37–7.34
m, 2H), 7.28 (t, J = 7.5 Hz, 1H), 7.09 (t, J = 7.5 Hz, 1H), 6.55 (d, J = 10.0 Hz, 1H), 6.33 (s, 2H), 3.96 (s,
H), 2.43 (s, 3H); 13C NMR (125 MHz, DMSO-d6)
158.6, 157.7, 142.4, 136.7, 136.3, 136.0, 134.7, 134.5,
6
9
(
3
−
δ
1
33.8, 130.9, 130.8, 130.3, 128.9, 127.0, 126.1, 124.2, 122.7, 120.3, 117.8, 109.4, 57.8, 56.6, 19.3. HRMS (ESI)
+
m/z calcd for C H BrN O [M − Br] 355.1441, found 355.1439.
23
19
2
2
1
0-Methoxy-3-(4-methylbenzyl)-6-oxo-6H-indolo[3,2,1-de][1,5]naphthyridin-3-ium bromide 6h: yield 71%;
◦
1
yellow solid powder; m.p. 228.6–228.9 C; H NMR (500 MHz, DMSO-d )
δ
9.40 (d, J = 5.0 Hz, 1H),
.00 (d, J = 2.5 Hz, 1H), 8.63 (d, J = 10.0 Hz, 1H), 8.42 (d, J = 10.0 Hz, 1H), 8.27 (d, J = 5.0 Hz, 1H), 7.56
d, J = 5.0 Hz, 1H), 7.39 7.34 (m, 3H), 7.26
7.25 (m, 2H), 6.29 (s, 2H), 3.97 (s, 3H), 2.31 (s, 3H); 13
NMR (125 MHz, DMSO-d ) 158.6, 157.5, 142.4, 139.0, 136.4, 136.2, 136.0, 134.6, 134.5, 132.1, 130.2,
6
9
(
−
−
C
δ
6

Molecules 2019, 24, 1553
8 of 12
1
30.1, 128.0, 124.1, 122.6, 120.2, 117.8, 109.3, 59.2, 56.6, 21.1. HRMS (ESI) m/z calcd for C H BrN O
23 19 2 2
+
[M − Br] 355.1441, found 355.1436.
1
0-Methoxy-3-(3-methylbenzyl)-6-oxo-6H-indolo[3,2,1-de][1,5]naphthyridin-3-ium bromide 6i: yield 70%;
◦
1
yellow solid powder; m.p. 222.4–222.7 C, H NMR (500 MHz, DMSO-d )
δ
9.38 (d, J = 5.0 Hz, 1H),
.98 (d, J = 10.0 Hz, 1H), 8.57 (d, J = 10.0 Hz, 1H), 8.35 (d, J = 10.0 Hz, 1H), 8.24 (d, J = 5.0 Hz, 1H),
.50–7.48 (m, 1H), 7.34 (d, J = 10.0 Hz, 1H), 7.28 (t, J = 10.0 Hz, 1H), 7.19–7.17 (m, 3H), 6.27 (s, 2H), 3.92
158.6, 157.5, 142.5, 139.0, 136.5, 136.2, 135.1, 134.6,
6
8
7
(
s, 3H), 2.25 (s, 3H); 13C NMR (125 MHz, DMSO-d6)
δ
1
34.5, 130.3, 130.1, 130.0, 129.5, 128.3, 125.0, 124.1, 122.6, 120.2, 117.8, 109.3, 59.2, 56.6, 21.4. HRMS (ESI)
+
m/z calcd for C H BrN O [M − Br] 355.1441, found 355.1449.
23
19
2
2
1
0-Methoxy-3-(3-methoxybenzyl)-6-oxo-6H-indolo[3,2,1-de][1,5]naphthyridin-3-ium chloride 6j: yield 43%;
◦
1
brick yellow solid powder; m.p. 225.3–226.0 C; H NMR (500 MHz, CD OD)
δ
9.11 (d, J = 5.0 Hz, 1H),
.74 (d, J = 10.0 Hz, 1H), 8.55 (d, J = 10.0 Hz, 1H), 8.30 (d, J = 10.0 Hz, 1H), 7.95 (d, J = 2.5 Hz, 1H),
.48 (d, J = 10.0 Hz, 2H), 7.37 7.35 (m, 1H), 7.30 (d, J = 10.0 Hz, 1H), 7.06 (d, J = 10.0 Hz, 2H), 6.20 (s,
3
8
7
2
−
1
3
H), 3.98 (s, 3H), 3.83 (s, 3H); C NMR (125 MHz, CD OD)
δ 160.5, 158.9, 157.5, 141.3, 137.0, 136.1,
3
1
34.0, 134.0, 132.0, 129.6, 129.6, 129.0, 125.3, 123.6, 122.4, 121.6, 119.2, 117.6, 114.8, 107.8, 59.6, 55.6, 54.9.
+
HRMS (ESI) m/z calcd for C H ClN O [M − Cl] 371.1390, found 371.1383.
23
19
2
3
1
0-Methoxy-6-oxo-3-(2-(trifluoromethyl)benzyl)-6H-indolo[3,2,1-de][1,5]naphthyridin-3-ium bromide 6k
:
◦
1
yield 70%; yellow solid powder; m.p. 243.6–244.1 C; H NMR (500 MHz, DMSO-d )
δ
9.22 (d,
6
J = 5.0 Hz, 1H), 9.00 (d, J = 10.0 Hz, 1H), 8.47 (d, J = 10.0 Hz, 1H), 8.30 8.28 (m, 2H), 7.96 (d, J = 7.5 Hz,
−
13
1
H), 7.69–7.55 (m, 3H), 7.36 (d, J = 10.0 Hz, 1H), 6.82 (d, J = 5.0 Hz, 1H), 6.51 (s, 2H), 3.98 (s, 3H);
C
NMR (125 MHz, DMSO-d6)
δ 158.7, 157.6, 143.0, 137.1, 136.4, 135.7, 134.8, 134.7, 134.0, 132.8, 130.8,
1
30.0, 129.8, 128.2, 127.3, 126.3, 124.1, 122.9, 120.3, 117.9, 109.4, 56.6, 56.5. HRMS (ESI) m/z calcd for
+
C H BrF N O [M − Br] 409.1158, found 409.1157.
23
16
3
2
2
1
6
0-Methoxy-6-oxo-3-(4-(trifluoromethyl)benzyl)-6H-indolo[3,2,1-de][1,5]naphthyridin-3-ium bromide 6l: yield
◦
8%; yellow solid powder; m.p. 238.5–238.9 C; H NMR (500 MHz, DMSO-d ) δ 9.43 (d, J = 10.0 Hz,
6
1
1H), 9.01 (d, J = 5.0 Hz, 1H), 8.63 (d, J = 10.0 Hz, 1H), 8.41 (d, J = 10.0 Hz, 1H), 8.28 (s, 1H), 7.92 (s, 1H),
7
3
1
.77 (d, J = 5.0 Hz, 1H), 7.64 (s, 2H), 7.54 (d, J = 10.0 Hz, 1H), 7.39 (d, J = 10.0 Hz, 1H), 6.43 (s, 2H),
.95 (s, 3H); 13C NMR (125 MHz, DMSO-d6)
158.6, 157.6, 142.7, 139.7, 136.7, 136.2, 134.8, 134.7, 130.5,
30.0, 128.6, 126.4, 125.5, 124.1, 123.3, 122.7, 120.3, 117.9, 109.3, 58.6, 56.6. HRMS (ESI) m/z calcd for
δ
+
C H BrF N O [M − Br] 409.1158, found 409.1162.
23
16
3
2
2
1
0-Methoxy-6-oxo-3-(3-(trifluoromethyl)benzyl)-6H-indolo[3,2,1-de][1,5]naphthyridin-3-ium bromide 6m:
◦
1
yield 72%; brick red solid powder; m.p. 223.3–223.7 C; H NMR (500 MHz, DMSO-d )
δ
9.38
d, J = 5.0 Hz, 1H), 8.99 (d, J = 5.0 Hz, 1H), 8.53 (d, J = 10.0 Hz, 1H), 8.40 (d, J = 5.0 Hz, 1H), 8.26 (d,
J = 2.5 Hz, 1H), 7.77 (d, J = 5.0 Hz, 2H), 7.57–7.54 (m, 3H), 7.35 (d, J = 10.0 Hz, 1H), 6.42 (s, 2H), 3.93 (s,
6
(
13
3
H); C NMR (125 MHz, DMSO-d6) δ 158.6, 157.6, 142.6, 136.6, 136.3, 136.2, 134.8, 134.7, 132.0, 130.7,
1
30.5, 130.2, 130.0, 126.1, 125.4, 125.0, 124.1, 122.7, 120.3, 117.8, 109.4, 58.5, 56.6. HRMS (ESI) m/z calcd
+
for C H BrF N O [M − Br] 409.1158, found 409.1165.
23
16
3
2
2
3
-(2,5-Difluorobenzyl)-10-methoxy-6-oxo-6H-indolo[3,2,1-de][1,5]naphthyridin-3-ium bromide 6n: yield 61%;
◦
1
yellow solid powder; m.p. 245.1–245.8 C; H NMR (500 MHz, DMSO-d )
8
7
δ
9.32 (d, J = 5.0 Hz, 1H),
.99 (d, J = 10.0 Hz, 1H), 8.58 (d, J = 10.0 Hz, 1H), 8.43 (d, J = 5.0 Hz, 1H), 8.29 (d, J = 5.0 Hz, 1H),
.58–7.56 (m, 1H), 7.50–7.33 (m, 3H), 7.21
7.19 (m, 1H), 6.39 (s, 2H), 3.95 (s, 3H); 13C NMR (125 MHz,
158.6, 157.6, 142.8, 136.8, 136.3, 134.7, 134.6, 130.7, 130.1, 124.2, 124.2, 124.1, 122.8, 120.2,
6
−
DMSO-d6)
δ
+
118.1, 117.9, 117.9, 116.6, 116.4, 109.4, 56.6, 53.9. HRMS (ESI) m/z calcd for C H BrF N O [M
−
Br]
22
15
2
2
2
3
77.1096, found 377.1103.
3
-(2,4-Difluorobenzyl)-10-methoxy-6-oxo-6H-indolo[3,2,1-de][1,5]naphthyridin-3-ium bromide 6o: yield 58%;
◦
1
yellow solid powder; m.p. 230.6–231.9 C; H NMR (500 MHz, DMSO-d )
δ
9.29 (d, J = 5.0 Hz, 1H),
6
8.98 (d, J = 5.0 Hz, 1H), 8.64 (d, J = 10.0 Hz, 1H), 8.45 (d, J = 10.0 Hz, 1H), 8.28 (d, J = 2.5 Hz, 1H),

Molecules 2019, 24, 1553
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7
.60
−
7.54 (m, 2H), 7.48–7.42 (m, 2H), 7.21
−
7.18 (m, 1H), 6.38 (s, 2H), 3.98 (s, 3H); 13C NMR (125 MHz,
DMSO-d )
δ
158.6, 157.5, 142.5, 136.7, 136.3, 134.6, 134.5, 132.0, 131.9, 131.2, 130.6, 130.0, 124.0, 122.8,
6
+
120.1, 118.7, 118.5, 117.9, 109.3, 105.2, 56.6, 53.8. HRMS (ESI) m/z calcd for C H BrF N O [M
−
Br]
22
15
2
2
2
3
77.1096, found 377.1099.
3
-(3,4-Dichlorobenzyl)-10-methoxy-6-oxo-6H-indolo[3,2,1-de][1,5]naphthyridin-3-ium bromide 6p: yield 46%;
◦
1
orange solid powder; m.p. 223.7–224.5 C; H NMR (500 MHz, DMSO-d )
δ
9.40 (d, J = 5.0 Hz, 1H),
.02 (d, J = 10.0 Hz, 1H), 8.60 (d, J = 10.0 Hz, 1H), 8.42 (d, J = 10.0 Hz, 1H), 8.30 (d, J = 2.5 Hz, 1H), 7.77
d, J = 5.0 Hz, 1H), 7.73 (d, J = 10.0 Hz, 1H), 7.59 7.56 (m, 1H), 7.45 7.43 (m, 1H), 7.40 (d, J = 10.0 Hz,
H), 6.35 (s, 2H), 3.98 (s, 3H); C NMR (125 MHz, DMSO-d ) 158.7, 157.6, 142.6, 136.7, 136.2, 135.8,
34.7, 134.7, 132.2, 131.6, 130.4, 130.2, 130.0, 128.6, 128.5, 124.1, 122.7, 120.3, 117.8, 109.4, 57.9, 56.6.
6
9
(
1
1
−
−
13
δ
6
+
HRMS (ESI) m/z calcd for C H BrCl N O [M − Br] 409.0505, found 409.0504.
22
15
2
2
2
3
-(2-Bromobenzyl)-10-methoxy-6-oxo-6H-indolo[3,2,1-de][1,5]naphthyridin-3-ium bromide 6q: yield 62%;
◦
1
brick yellow solid powder; m.p. 235.5–236.0 C; H NMR (500 MHz, DMSO-d )
δ
9.20 (d, J = 10.0 Hz,
6
1
H), 8.97 (d, J = 5.0 Hz, 1H), 8.47
−
8.45 (m, 2H), 8.28 (d, J = 2.5 Hz, 1H), 7.81 (d, J = 5.0 Hz, 1H), 7.59 (d,
13
J = 10.0 Hz, 1H), 7.42–7.28 (m, 3H), 6.82–6.81 (m, 1H), 6.34 (s, 2H), 3.96 (s, 3H); C NMR (125 MHz,
DMSO-d6)
δ
158.7, 157.6, 142.6, 136.9, 136.4, 134.7, 134.6, 134.2, 133.6, 131.2, 130.9, 130.2, 129.1, 129.0,
+
1
24.1, 122.8, 122.4, 120.2, 117.9, 109.4, 59.6, 56.6. HRMS (ESI) m/z calcd for C H Br N O [M
−
Br]
22
16
2
2
2
4
19.0389, found 419.0355.
3
-(4-Bromobenzyl)-10-methoxy-6-oxo-6H-indolo[3,2,1-de][1,5]naphthyridin-3-ium bromide 6r: yield 65%;
◦
1
yellow solid powder; m.p. 234.5–235.2 C; H NMR (500 MHz, DMSO-d )
δ
9.39 (d, J = 10.0 Hz, 1H),
.01 (d, J = 5.0 Hz, 1H), 8.59 (d, J = 10.0 Hz, 1H), 8.44 (d, J = 10.0 Hz, 1H), 8.28 (d, J = 2.5 Hz, 1H), 7.65
d, J = 5.0 Hz, 2H), 7.59
7.57 (m, 1H), 7.41–7.38 (m, 3H), 6.32 (s, 2H), 3.98 (s, 3H); 13C NMR (125 MHz,
158.6, 157.5, 142.5, 136.6, 136.2, 134.7, 134.6, 134.4, 132.4, 130.3, 130.2, 130.0, 124.1, 122.7,
6
9
(
−
DMSO-d6)
δ
+
122.7, 120.2, 117.9, 109.3, 58.6, 56.6. HRMS (ESI) m/z calcd for C H Br N O [M
−
Br] 419.0389,
22
16
2
2
2
found 419.0381.
3
-(3-Bromobenzyl)-10-methoxy-6-oxo-6H-indolo[3,2,1-de][1,5]naphthyridin-3-ium bromide 6s: yield 64%;
◦
1
brick red solid powder; m.p. 238.7–239.1 C; H NMR (500 MHz, DMSO-d )
δ
9.41 (d, J = 10.0 Hz, 1H),
.01 (d, J = 5.0 Hz, 1H), 8.62 (d, J = 10.0 Hz, 1H), 8.43 (d, J = 10.0 Hz, 1H), 8.29 (d, J = 2.5 Hz, 1H), 7.71
s, 1H), 7.64
7.62 (m, 1H), 7.58–7.56 (m, 1H), 7.42–7.40 (m, 3H), 6.35 (s, 2H), 3.98 (s, 3H); 13C NMR
125 MHz, DMSO-d ) 158.6, 157.6, 142.6, 137.5, 136.6, 136.2, 134.7, 134.6, 132.3, 131.7, 130.7, 130.4,
30.0, 127.0, 124.1, 122.8, 122.7, 120.2, 117.8, 109.3, 58.4, 56.6. HRMS (ESI) m/z calcd for C H Br N O
6
9
(
(
−
δ
6
1
22
16
2
2
2
+
[M − Br] 419.0389, found 419.0386.
3
-(3-Iodobenzyl)-10-methoxy-6-oxo-6H-indolo[3,2,1-de][1,5]naphthyridin-3-ium bromide 6t: yield 42%; red
◦
1
solid powder; m.p. 245.6–246.3 C; H NMR (500 MHz, DMSO-d )
δ
9.38 (d, J = 10.0 Hz, 1H), 8.99
d, J = 5.0 Hz, 1H), 8.58 (d, J = 10.0 Hz, 1H), 8.44 (d, J = 5.0 Hz, 1H), 8.28 (d, J = 2.5 Hz, 1H), 7.81 (d,
J = 10.0 Hz, 2H), 7.60–7.57 (m, 1H), 7.39 (d, J = 10.0 Hz, 1H), 7.23 (d, J = 5.0 Hz, 2H), 6.29 (s, 2H), 3.98 (s,
6
(
13
3
H); C NMR (125 MHz, DMSO-d6)
δ 158.7, 157.6, 142.6, 136.9, 136.4, 134.7, 134.6, 134.2, 133.6, 131.2,
1
30.9, 130.2, 129.1, 129.0, 124.1, 122.8, 122.4, 120.2, 117.9, 109.4, 60.2, 56.6. HRMS (ESI) m/z calcd for
+
C H BrIN O [M − Br] 467.0251, found 467.0251.
22
16
2
2
3
-Benzyl-10-methoxy-6-oxo-6H-indolo[3,2,1-de][1,5]naphthyridin-3-ium bromide 6u: yield 65%; yellow solid
◦
1
powder; m.p. 224.1–224.5 C; H NMR (500 MHz, DMSO-d )
δ
9.43 (d, J = 5.0 Hz, 1H), 9.02 (d,
J = 10.0 Hz, 1H), 8.63 (d, J = 10.0 Hz, 1H), 8.40 (d, J = 10.0 Hz, 1H), 8.28 (d, J = 2.5 Hz, 1H), 7.56 7.53 (m,
158.6, 157.5, 142.5,
6
−
1
1
H), 7.44–7.37 (m, 6H), 6.36 (s, 2H), 3.97 (s, 3H); 13C NMR (125 MHz, DMSO-d6)
δ
36.5, 136.2, 135.1, 134.6, 134.5, 130.3, 130.1, 129.6, 129.4, 127.9, 124.1, 122.6, 120.2, 117.8, 109.3, 59.3, 56.6.
+
HRMS (ESI) m/z calcd for C H BrN O [M − Br] 341.1284, found 341.1275.
22
17
2
2
3
-Benzyl-10-methoxy-6-oxo-6H-indolo[3,2,1-de][1,5]naphthyridin-3-ium chloride 6v: yield 56%; yellow solid
◦
1
powder; m.p. 222.4–222.9 C; H NMR (500 MHz, DMSO-d ) δ 9.42 (d, J = 5.0 Hz, 1H), 9.01 (d, J = 5.0 Hz,
6

Molecules 2019, 24, 1553
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1
(
1
H), 8.62 (d, J = 10.0 Hz, 1H), 8.44 (d, J = 10.0 Hz, 1H), 8.29(s, 1H), 7.58 (d, J = 10.0 Hz, 1H), 7.44–7.38
m, 6H), 6.35 (s, 2H), 3.98 (s, 3H); 13C NMR (125 MHz, DMSO-d6)
158.6, 157.6, 142.5, 136.5, 136.2,
35.1, 134.6, 134.6, 130.3, 130.1, 129.6, 129.4, 127.9, 124.1, 122.6, 120.2, 117.9, 109.3, 59.3, 56.6. HRMS
δ
+
(ESI) m/z calcd for C H ClN O [M − Cl] 341.1284, found 341.1274.
22
17
2
2
3
.3. Antibacterial Assay
The MIC values were determined as described by the National Committee for Clinical Laboratory
Standards with minor modification [15 24 26]. B. cereus (CGMCC 1.1846) was purchased from the
,
–
China General Microbiological Culture Collection Center. R. solanacearum and P. syringae were provided
by the College of Plant Protection, Northwest A&F University [27]. The MIC was defined as the
minimum inhibitory concentration, each compound resulting in visible inhibition on bacteria growth
◦
5
(
incubation at 37 C for 12–14 h). Each bacterial suspension was adjusted to a concentration of 1
CFU/mL. All compounds were thoroughly dried before weighing. Initially, the compounds were
dissolved in dimethyl sulfoxide (DMSO) to prepare the stock solutions. The tested compounds (
and 6a 6v) and reference drugs were then prepared in liquid Luria–Bertani media. The required
×
10
5
–
concentrations were 125, 62.5, 31.25, 15.63, 7.81, 3.91 and 1.95
Triplicate experiments were conducted.
µg/mL, respectively (DMSO < 0.5%).
3
.4. Calculation of Molecular Physicochemical Properties
Molecular properties, including the octanol–water partition coefficient, the topological polar
surface area, the number of hydrogen bond acceptors, the number of hydrogen bond donors, the
number of rotatable bonds, and the molecular weight, were calculated using the Molinspiration tool
www.molinspiration.com.
3
.5. Cell Toxicity Evaluation
The cytotoxicity of highly active compounds 6p and 6t was assessed using the 3-(4,5-dimethyl-2-
thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, as described previously [13
,14]. Baby
hamster Syrian kidney cells BHK-21 were treated with compounds 6p and 6t at concentrations of 3.91,
7
.81 and 15.62
µ
g/mL. After incubation, 10
µ
L (5 mg/mL) of MTT in phosphate-buffered saline was
◦
added to each well, and the cells were incubated in 5% CO at 37 C for 4 h. The medium was removed.
2
Then, the DMSO (150 µL) was added to each well. The wells were shocked for 15 min in a flat shaker
to dissolve purple formazan crystals. Finally, the plate was tested at 540 nm. The untreated group
was considered as the control. Triplicate experiments were conducted. The data were analyzed by
GraphPad Prism 6.0 (GraphPad Software, San Diego, CA, USA). The formula used for cell viability
was (absorbance of compound-treated cells/absorbance of untreated cells) × 100%.
3
.6. Germination Rate Bioassay
The seeds of two herbaceous plants, turnip (Raphanus sativus) and wheat (Triticum aestivum L.),
were used for the bioassay. The procedure was conducted according to the reported protocol [14].
The plant seeds were washed with running water for 2 h, soaked in 0.3% KMnO for 15 min, and
4
flushed until they were colorless. The compounds 6p and 6t, positive control and blank solvent
acetone, were added to 12-well plates with filter paper to final concentration of 3.91 µg/mL. After the
evaporation of acetone, the plant seeds were sown in the microdishes of 12-well plates and irrigated
with sterile deionized water. Triplicate experiments were conducted. The plates were then incubated
◦
at 26 C for 48 h, and the germination rates were calculated according to Equation (1). Germination
rates were expressed as means ± the standard deviation (SD) for three replicates.
Germination rate (%) = number of germinated seeds/total number of seeds
(1)

Molecules 2019, 24, 1553
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4
. Conclusions
In this study, twenty-two new quaternarization 10-methoxycanthin-6-one derivatives were
designed and synthesized based on our previous work. Their antibacterial activity was evaluated
against three bacterial strains including two kinds of agricultural pathogenic bacteria. Four compounds
(6f, 6i, 6p and 6t) displayed 8-fold superiority (MIC = 3.91 µg/mL) against R. solanacearum and P. syringae
compared to agrochemical streptomycin sulfate. Simultaneously, the structure–activity relationships
were summarized, which provided some important guidance for the development of antibacterial
agents. The good “drug-like” properties, low cytotoxicity and no inhibition on seed germination of the
highly active compounds 6p and 6t further confirmed their potential as bactericides and preservatives,
which is of great meaning both to agricultural production and food industry.
Supplementary Materials: The following are available online, Figure S1: The NMR, HRMS and HPLC spectra of
compound 5, Figures S1–S23: The NMR, HRMS and HPLC spectra of compound 6a–6v.
Author Contributions: N.L. conceived and conducted this work; J.-K.D. reelucidated the compound structures
via their new spectroscopic data and prepared the discussion part of the manuscript; D.L. conducted the synthesis
work; J.-R.W. and J.-Y.W. designed and wrote the paper.
Funding: This work was funded by the National Natural Science Foundation of China (Grant No. 81773603).
Acknowledgments: We acknowledge the Life Science Large Research Core Services in the university, and also
acknowledge Hongli Zhang for providing the help in the NMR analysis.
Conflicts of Interest: The authors declare no conflict of interest.
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